HK1261293A1 - Mesoporous silica nanoparticles with lipid bilayer coating for cargo delivery - Google Patents

Description

Mesoporous silica nanoparticles with lipid bilayer coating for cargo delivery

Cross Reference to Related Applications

The benefit and priority of USSN 62/276,634, filed on 8.1.2016, this application is hereby incorporated by reference in its entirety for all purposes.

Statement of government support

The invention was made with government support under fund numbers R01 CA133697 and UOl CAl98846 awarded by the National Institutes of health/National Cancer Institute. The government has certain rights in the invention.

Background

Pancreatic Ductal Adenocarcinoma (PDAC) is a fatal disease with a 5-year survival rate of less than 6% (Siegel et al (2014) CA Cancer J. Clin.64(1): 9-29). Currently, the primary treatment regimens for chemotherapy include single agent Gemcitabine (GEM) or four-drug regimens. Although FOLFIRINOX has a better response rate than GEM (31.6% compared to 9.4%), with improved survival (11 months compared to 6.8 months), the former combination is significantly more toxic and limited to a few PDAC patients with a well-behaved status (Conroy et al (2011) n.engl.j.med.364(19): 1817-1825). Irinotecan (irinotecan) significantly contributes to this toxicity, including severe effects on bone marrow (e.g., neutropenia), liver (e.g., necrosis and steatosis), and Gastrointestinal (GI) tract (e.g., vomiting, diarrhea) (Conroy et al (2011) n.engl.j.med.364(19): 1817-1825; Ueno et al (2007) Cancer chemother. pharmacol.59(4): 447-454; Loupakis et al (2013) br.j.cancer,108(12): 2549-2556). Therefore, there is a great need for therapeutic regimens that improve irinotecan toxicity with the aim of improving the available drugs for first line treatment of PDAC.

One way to reduce the toxicity of irinotecan while maintaining efficacy is to encapsulate the high dose drug in a nanocarrier, soThe nanocarriers have protected delivery to the cancer site while reducing systemic drug release. Different carrier types, including polymer particles and liposomes, have been used with some success for irinotecan delivery (Chou et al (2003) J.biosci.Bioeng.,95(4): 405-408; Onishi et al (2003) biol.Pharmaceut.Bull.26(1): 116-119; Messerer et al (2004) Clin.cancer Res,10(19): 6638-6649; Drummond et al (2006) Cancer Res, 66(6): 3271-3277; Valencia et al (2013) Nanomed.8(5): 687-698; Sadzuka et al (1998) Cancer Lett.,127(1): 99-106; Ramsay et al (2008) Eur.J.rm.Biopharm.68 (3): 607-617; Li et al (Fuv et al) (788) Mat.798). However, while polymeric nanoparticles show promising in vitro results, loading irinotecan (b)<1%, w/w) plus premature drug release (e.g., 40% in 5 hours), the nanoparticles did not achieve the desired reduction in toxicity while enhancing intratumoral drug delivery (Valencia et al (2013) nanomed.8(5): 687-698). Although liposomes can achieve high irinotecan loading capacities via the use of ammonium sulfate or proton encapsulants (Chou et al (2003) J.biosci.Bioeng.,95(4): 405-408; Messerer et al (2004) Clin.cancer Res,10(19): 6638-6649; Drummond et al (2006) Cancer Res, 66(6): 3271-3277; Sadzuka et al (1998) Cancer Lett, 127(1): 99-106; Ramsay et al (2008) Eur.J.Pharm.Biopharm.68(3):607-617), instability of the carrier under shear and osmotic stress and bilayer disruption by serum proteins can lead to premature drug release and toxicity (Liu et al (2000), Colloids and Surfaceces A: physiochemical and Engineering (172-4303): 2003-4223; Biomate et al (2003-4223);et al (2006) Eur.Phys.J.E.20(4): 401-408).

As noted above, many attempts to deliver Cancer drugs in clinical trials or in therapeutic settings have been based on liposomal systems (Messer et al (2004) Clin. Cancer Res,10(19): 6638-6649; Cancer Res.2006,66,3271) or polymer-based systems (Onishi et al (2003) biol. pharm. Bull.26(1): 116-119). Most of these carriers are spherical particles or supramolecular assemblies in the size range of 80nm-200nm, often containing a PEG coating on the surface to prolong the circulation half-life, and typically exhibit a loading capacity of about 5 w/w% (e.g. polymer-based nanoparticles) to about 50 w/w% (e.g. liposomal carriers). At the preclinical level, the potential benefits of these nanocarriers in animal studies, including murine PDAC models, have been demonstrated to include reduced toxicity in vivo, enhanced anti-tumor efficacy, and improved survival.

However, only a few nanocarriers have progressed into clinical trials in PDAC patients. Nanocarriers comprising ionophores (a23187, also known as calimycin (calcium magnesium salt mixture)) make it possible to deliver liposomal formulations of irinotecan (irinopophore C) and protonated irinotecan (MM-398) (Baker et al (2008) clin. Cancer res.14: 7260-7271; Drummond et al (2006) Cancer res.15(66): 3271-3277). Irinophore C formulation (Champions Biotechnology Inc.) is a liposomal carrier that utilizes active irinotecan loading by creating a transmembrane proton gradient using ionophore A23187 or ammonium sulfate (Ramsay et al (2008) Eur. J. pharm. Biopharm.68: 607-617). The iritophore C formulation was used in clinical studies beginning in 2011, but there has been no updated information on the outcome or outcome of the study.

Although the development of liposomal carriers for irinotecan (MM-398) by Merriack corporation in the latest phase 3 clinical trial showed improved survival benefits as a second-line treatment option in PDACs, the relatively high GI tract and bone marrow toxicity rates have led to the black box warning of severe and life-threatening diarrhea and neutropenia (Von Hoff et al (2013) Br. J. cancer,109(4): 920-925; www.fda.gov/newsevents/newfrom/presentations/ucm468654. htm). Human subjects involved in the MM-398 clinical trial also showed significant elevations of liver enzymes, including alanine Aminotransferase (ALT) (see, e.g., www.accessdata.fda.gov/drug atfdda _ docs/label/2015/207793lb. Nonetheless, MM-398 received FDA approval for PDAC for failure to respond to GEM treatmentAnd as a patient ofAre sold (see, e.g., www.fda.gov/newsevents/newfrom/presentations/ucm 468654. htm).

The MM-398 liposome formulation (Merrimack Pharmaceuticals company) contains irinotecan hydrochloride with the help of a polyanionic trapping agent (ESMOGl 2014, www.merrimackpharma.com). More specifically, irinotecan is loaded into MM-398 liposomes by the polyvalent anion trapping agent triethylammonium sucrose octasulfate (TEA)₈SOS) is administered. This chemical causes irinotecan to protonate and encapsulate at more than 10 times the loading that can be achieved via passive drug encapsulation (Drummond et al (2006) Cancer Res.66(6): 3271-3277). Intravenous administration of MM-398 liposomes has been shown to induce complete tumor regression in various PDAC tumor models in mice, including inhibition of metastatic tumor foci (Paz et al (2102) Cancer Res.72(12 suppl): Abstract A63). MM-398 is currently in phase 3 clinical trials and claims to provide improved tumor inhibition, pharmacokinetics, and efficacy compared to free irinotecan in animal and human studies (Kalra (2012) AACR meetting, abstract number 5622). This includes experimental data claiming complete PDAC regression using a dose of 20mg/kg MM-398 (human equivalent: 60mg/m2-120mg/m2) in murine xenograft studies (Paz et al (2102) Cancer Res.72(12 suppl.: Abstract A63). MM-398 also increased the Maximum Tolerated Dose (MTD) of free irinotecan from 80mg/kg to 324mg/kg in mice (Drummond et al (2006) Cancer Res.66(6): 3271-3277). Furthermore, in a phase 3 clinical trial involving 417 PDAC patients by Merripack Pharmaceuticals (Hoff et al ESMO GI 2014, www.merrimackpharma.com), the combination of MM-398, 5-FU and leucovorin (leucovorin) caused an Overall Survival (OS) of 6.1 months, which was 1.9 months longer than the control group receiving 5-FU and leucovorin. However, while active loading of irinotecan into MM-398 liposome formulations enhances drug loading capacity relative to passive encapsulation procedures, the synthetic techniques require multiple steps and are comparable to LB-MSNP platform provided hereinIn contrast, liposome carriers do not provide the same colloidal stability or the same amount of intracellular release. Nonetheless, MM-398 obtains FDA approval for PDACs, for patients who fail to respond to GEM treatment, and as suchAnd (5) selling. The use of polyanionic polymers to increase drug encapsulation in liposomes caused about 80nm drug precipitation (Zhu et al (1996)39(1): 138-142; Colbern et al (1998) Clin. cancer Res.4(12):3077-3082), which constitutes one of the reasons for the slow release of irinotecan from the liposome carrier compared to LB-MSNP pores.

Thus, there remains an unmet need for nanocarriers and delivery methods that enable effective drug delivery, including chemotherapy, such as irinotecan chemotherapy, with increased safety margins and reduced toxicity.

Disclosure of Invention

Urgent intervention is required to improve the 5-year survival rate of Pancreatic Ductal Adenocarcinoma (PDAC). Although the 4-dose regimen FOLFIRINOX (comprising irinotecan (IRIN), 5-fluorouracil (5-FU), Oxaliplatin (OX), and Leucovorin (LV)) has better survival outcomes than the more commonly used Gemcitabine (GEM), the former treatment regimen is highly toxic and limited for patients with good performance status. Since irinotecan significantly contributes to FOLFIRINOX toxicity (bone marrow and gastrointestinal tract), a specific object of the present invention is to reduce toxicity of the former drug with a custom designed Mesoporous Silica Nanoparticle (MSNP) platform that uses proton gradients for high dose irinotecan loading across the coated Lipid Bilayer (LB). In the Kras-derived in situ PDAC model of immunocompetent mice, the improved stability of LB-coated MSNP (LB-MSNP) vectors allows for better delivery of protected irinotecan and increased tumor drug concentrations when compared, for example, to liposome equivalents. The LB-MSNP nanocarriers are also more effective for treating tumor metastasis. Also importantly, the reduced leakage and slower drug release rate of LB-MSNP vectors significantly reduced bone marrow, gastrointestinal and hepatotoxicity rates compared to liposome vectors. The combination of high efficacy and reduced toxicity of the LB-MSNP vector helped to improve PDAC survival using irinotecan as a first-line therapeutic.

In general, in one aspect of the invention, there is provided a nanocarrier, the nanocarrier comprising a silica body having a surface and defining a plurality of pores adapted to contain molecules therein; a lipid bilayer (e.g., a phospholipid bilayer) coating the surface; and a cargo trap contained in the pores by the coated lipid bilayer, wherein the sub-micron structures have a largest dimension of less than 1 micron, and wherein the phospholipid bilayer stably seals the plurality of pores and can be used as a basis for additional packaging, targeting, imaging, and functionalization.

An important aspect of the present invention includes a novel process for the preparation of improved cargo/agent (e.g., irinotecan or one or more other molecules having the chemical structures described herein) nanocarriers. These methods generally include providing an unsupported nanocarrier comprising a silica body having a surface with a plurality of pores adapted to contain molecules therein; and encapsulating the cargo trapping agent, targeting agent, or imaging agent within the pores via a lipid bilayer (e.g., a phospholipid bilayer). As discussed in detail below, the working examples include, but are not limited to, a novel method of preparing concentrated irinotecan nanocarriers. In certain embodiments, the methods comprise selecting a nanocarrier comprising a silica body having a surface comprising a plurality of pores suitable for housing irinotecan therein. In these methods, an agent (e.g., triethylammonium sucrose octasulfate) having the ability to specifically affect diffusion and/or trapping of irinotecan molecules is also selected and disposed within the plurality of pores. The nanocarriers and pores are fully coated with a lipid bilayer (e.g., using sonication methods). Typically, the silica host nanocarriers are not dried and/or washed immediately prior to the coating step. Optionally, in certain embodiments, the lipid bilayer may comprise one or more bioactive molecules (such as polyethylene glycol, and/or a targeting ligand, and/or paclitaxel and/or activated irinotecan SN38) selected to promote nanocarrier function. After this coating step, irinotecan is then allowed to migrate through the lipid bilayer into the pores where it is encapsulated. This is also known as "remote drug loading". In this way, irinotecan nanocarriers with surprisingly high loading capacities (e.g., 40% or greater than 40% drug/MSNP) can be formed. In addition to these high drug loading capacities, irinotecan nanocarriers formed using these methods have a range of other desirable properties, such as exhibiting < 5% irinotecan leakage in 24 hours at 37 ℃ in biological buffers with pH of 7.4.

Various embodiments contemplated herein may include, but are not necessarily limited to, one or more of the following:

embodiment 1: a nanoparticle drug carrier, the nanoparticle drug carrier comprising:

a silica nanoparticle having a surface and defining a plurality of pores adapted to contain molecules therein;

a lipid bilayer coating the surface;

a cargo trapping agent within a pore comprising the plurality of pores; and

a cargo comprising a drug, wherein the cargo is associated with the cargo trap in the pores;

wherein the sub-micron structures have a largest dimension of less than 1 micron, and wherein the lipid bilayer stably seals the plurality of pores.

Embodiment 2: the nanoparticulate drug carrier of embodiment 1, wherein the lipid bilayer comprises phospholipids, Cholesterol (CHOL), and mPEG phospholipids.

Embodiment 3: the nanoparticulate drug carrier according to any one of embodiments 1 to 2, wherein the phospholipid comprises a natural lipid comprising a mixture of saturated fatty acids having a carbon chain of C14-C20, and/or unsaturated fatty acids having a carbon chain of C14-C20, and/or fatty acids having a carbon chain of C12-C20.

Embodiment 4: the nanoparticulate drug carrier of embodiment 3, wherein the phospholipid comprises a saturated fatty acid selected from the group consisting of: phosphatidylcholine (DPPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC) and Diacylphosphatidylcholine (DAPC).

Embodiment 5: the nanoparticulate drug carrier of embodiment 3, wherein the phospholipid comprises a natural lipid selected from the group consisting of: egg phosphatidylcholine (egg PC) and soybean phosphatidylcholine (soybean PC).

Embodiment 6: the nanoparticulate drug carrier of embodiment 3, wherein the phospholipid comprises an unsaturated fatty acid selected from the group consisting of: 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyloyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dieicosenoyl-sn-glycero-3-phosphocholine.

Embodiment 7: the nanoparticulate drug carrier according to any one of embodiments 1 to 6, wherein the lipid bilayer comprises mPEG phospholipids having phospholipid C14-C18 carbon chains and a PEG molecular weight in the range of about 350Da to 5000 Da.

Embodiment 8: the nanoparticulate drug carrier of embodiment 7, wherein the lipid bilayer comprises 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-PEG (DSPE-PEG).

Embodiment 9: the nanoparticle drug carrier of embodiment 2, wherein the lipid bilayer comprises DPPC/Chol/DSPE-PEG or DSPC/Chol/DSPE-PEG.

Embodiment 10: the nanoparticle drug carrier of embodiment 9, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG.

Embodiment 11: the nanoparticle drug carrier of embodiment 10, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG 2000.

Embodiment 12: the nanoparticle drug carrier according to any one of embodiments 1 to 11, wherein the lipid bilayer comprises phospholipids, cholesterol, and mPEG phospholipids in a ratio of 50-90 mol% phospholipids to 10-50 mol% CHOL to 1-10 mol% mPEG phospholipids.

Embodiment 13: the nanoparticle drug carrier of embodiment 10, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG at a molar ratio of about 3:2: 0.15.

Embodiment 14: the nanoparticle drug carrier according to any one of embodiments 1 to 13, wherein the lipid bilayer forms a substantially continuous bilayer surrounding the entire nanoparticle.

Embodiment 15: the nanoparticle drug carrier according to any one of embodiments 1 to 14, wherein the lipid bilayer forms a substantially uniform and intact bilayer surrounding the entire nanoparticle.

Embodiment 16: the nanoparticulate drug carrier according to any one of embodiments 1 to 15, wherein the silica nanoparticles are mesoporous silica nanoparticles.

Embodiment 17: the nanoparticulate drug carrier of embodiment 16, wherein the silica nanoparticles comprise sol-gel synthesized mesoporous silica nanoparticles.

Embodiment 18: the nanoparticulate drug carrier according to any one of embodiments 16 to 17, wherein the mesoporous silica nanoparticles are size-controlled.

Embodiment 19: the nanoparticulate drug carrier according to any one of embodiments 16 to 18, wherein the mesoporous silica nanoparticles are colloidally stable.

Embodiment 20: the nanoparticulate drug carrier according to any one of embodiments 16 to 19, wherein the mesoporous silica has an average pore size in the range of from about 1nm to about 20nm, or from about 1nm to about 10nm, or from about 2nm to about 8 nm.

Embodiment 21: the nanoparticulate drug carrier according to any one of embodiments 1 to 20, wherein the mesoporous silica nanoparticles have an average size within the following range: from about 50nm to about 300nm, or from about 50nm to about 200nm, or from about 50nm to about 150nm, or from about 50nm to about 100nm, or from about 50nm to about 80nm, or from about 50nm to about 70nm, or from about 60nm to about 70 nm.

Embodiment 22: the nanoparticulate drug carrier according to any one of embodiments 1 to 21, wherein the cargo trap is selected from triethylammonium sucrose octasulfate (TEA) prior to reaction with the drug₈SOS)、(NH₄)₂SO₄Ammonium, trimethylammonium, and triethylammonium salts.

Embodiment 23: the nanoparticle drug carrier of embodiment 22, wherein the cargo trap comprises (NH)₄)₂SO₄。

embodiment 24 the nanoparticle drug carrier of embodiment 22, wherein the cargo trap comprises an ammonium salt selected from the group consisting of ammonium sulfate, sucrose octasulfate, α -cyclodextrin ammonium sulfate, β -cyclodextrin ammonium sulfate, gamma-cyclodextrin ammonium sulfate, ammonium phosphate, α -cyclodextrin ammonium phosphate, β -cyclodextrin ammonium phosphate, gamma-cyclodextrin ammonium phosphate, ammonium citrate, and ammonium acetate.

embodiment 25 the nanoparticle drug carrier of embodiment 22 wherein the cargo trapping agent comprises a trimethylammonium salt selected from the group consisting of trimethylammonium sulfate, sucrose octasulfate trimethylammonium, α -cyclodextrin sulfate trimethylammonium, β -cyclodextrin sulfate trimethylammonium, gamma-cyclodextrin sulfate trimethylammonium, trimethylammonium trimethylphosphate, α -cyclodextrin phosphate trimethylammonium, β -cyclodextrin phosphate trimethylammonium, gamma-cyclodextrin phosphate trimethylammonium, trimethylammonium citrate, and trimethylammonium acetate.

embodiment 26 the nanoparticulate drug carrier of embodiment 22, wherein the cargo trap comprises a triethylammonium salt selected from triethylammonium sulfate, triethylammonium sucrose octasulfate, triethylammonium α -cyclodextrin sulfate, triethylammonium β -cyclodextrin sulfate, triethylammonium γ -cyclodextrin sulfate, triethylammonium phosphate, triethylammonium α -cyclodextrin phosphate, triethylammonium β -cyclodextrin phosphate, triethylammonium γ -cyclodextrin phosphate, triethylammonium citrate, and triethylammonium acetate.

Embodiment 27: the nanoparticle drug carrier of embodiment 22, wherein the cargo trapping agent is triethylammonium sucrose octasulfate (TEA) prior to reaction with the drug₈SOS)。

Embodiment 28: the nanoparticulate drug carrier of embodiment 27, wherein the drug is protonated and in contact with the SOS^8-Trapped in the pores as gelatinous precipitates upon association.

Embodiment 29: the nanoparticulate drug carrier according to any one of embodiments 1 to 28, wherein the drug comprises at least one weakly basic group capable of being protonated and the cargo trap comprises at least one anionic group.

Embodiment 30: the nanoparticulate drug carrier according to any one of embodiments 1 to 29, wherein the drug is selected to have a pKa greater than 7 and less than 11.

Embodiment 31: the nanoparticulate drug carrier according to any one of embodiments 1 to 30, the drug comprising a primary amine, a secondary amine, and a tertiary amine.

Embodiment 32: the nanoparticulate drug carrier according to any one of embodiments 1 to 31, wherein the drug is selected to have a water solubility index from about 5mg/mL to about 25 mg/mL.

Embodiment 33: the nanoparticulate drug carrier according to any one of embodiments 1 to 32, wherein the cargo is selected to have an octanol/water partition coefficient or logP value of from about-3.0 to about 3.0.

Embodiment 34: the nanoparticulate drug carrier according to any one of embodiments 1 to 33, wherein the cargo is selected to be 2nm to 8nm and smaller than the average or median size of the pores of the silica nanoparticles.

Embodiment 35: the nanoparticulate drug carrier according to any one of embodiments 29 to 34, wherein the cargo comprises an anticancer drug.

Embodiment 36: the nanoparticulate drug carrier of embodiment 35, wherein the cargo comprises irinotecan.

Embodiment 37: the nanoparticle drug carrier of embodiment 35, wherein the cargo comprises one or more drugs independently selected from the group consisting of: topoisomerase inhibitors, antineoplastic anthracyclines, mitotic inhibitors, alkaloids, basic alkylating agents, purine or pyrimidine derivatives, and protein kinase inhibitors.

Embodiment 38: the nanoparticulate pharmaceutical carrier of embodiment 37, wherein the carrier comprises a topoisomerase inhibitor comprising topotecan.

Embodiment 39: the nanoparticulate drug carrier of embodiment 37, wherein the carrier comprises an alkaloid selected from the group consisting of: topotecan, 10-hydroxycamptothecin (10-hydroxycamptothecin), belotecan (belotecan), rubitecan (rubitecan), vinorelbine, and LAQ 824.

Embodiment 40: the nanoparticulate drug carrier of embodiment 37, wherein the carrier comprises an antineoplastic anthracycline selected from the group consisting of doxorubicin and mitoxantrone.

Embodiment 41: the nanoparticulate drug carrier of embodiment 37, wherein the carrier comprises a mitotic inhibitor selected from vinblastine and vinorelbine.

Embodiment 42: the nanoparticle drug carrier of embodiment 37, wherein the carrier comprises a basic alkylating agent selected from the group consisting of cyclophosphamide, nitrogen mustard and temozolomide.

Embodiment 43: the nanoparticulate drug carrier of embodiment 37, wherein the carrier comprises a purine or pyrimidine derivative selected from the group consisting of: 5-fluorouracil, 5' -deoxy-5-fluorouridine and gemcitabine.

Embodiment 44: the nanoparticulate drug carrier of embodiment 37, wherein the carrier comprises a protein kinase inhibitor selected from the group consisting of: imatinib, oxitinib and sunitinib, pazopanib, enzastaurin, vandetanib, erlotinib, dasatinib and nilotinib.

Embodiment 45: the nanoparticulate drug carrier according to any one of embodiments 1 to 44, wherein the drug carrier is conjugated to a moiety selected from the group consisting of: targeting moieties, fusion peptides, and transit peptides.

Embodiment 46: the nanoparticulate drug carrier of embodiment 45, wherein the drug carrier is conjugated to a peptide that binds to a receptor on a cancer cell or tumor blood vessel.

Embodiment 47: the nanoparticle drug carrier of embodiment 46, wherein the drug carrier is conjugated to the iRGD peptide.

Embodiment 48: the nanoparticulate drug carrier of embodiment 46, wherein the drug carrier is conjugated to a targeting peptide shown in table 2.

Embodiment 49: the nanoparticulate drug carrier according to any one of embodiments 45 to 48, wherein the drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.

Embodiment 50: the nanoparticulate drug carrier according to any one of embodiments 45 to 49, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds a cancer marker.

Embodiment 51: the nanoparticulate drug carrier of embodiment 50, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds to a cancer marker set forth in table 1.

Embodiment 52: the nanoparticulate drug carrier according to any one of embodiments 29 to 34, wherein the cargo comprises an antibiotic, antiviral, or antifungal agent.

Embodiment 53: the nanoparticulate drug carrier of embodiment 52, wherein the cargo comprises an antibiotic selected from ciprofloxacin and levofloxacin.

Embodiment 54: the nanoparticle pharmaceutical carrier of embodiment 52, wherein the cargo comprises an HIV antiviral agent.

Embodiment 55: the nanoparticle pharmaceutical carrier of embodiment 54, wherein the cargo comprises an antiviral agent selected from the group consisting of: tenofovir (tenofovir), disoproxil (disoproxil), and fumarate.

Embodiment 56: the nanoparticulate drug carrier of embodiment 52, wherein the cargo comprises an antifungal agent selected from the group consisting of: amphotericin B, anidulafungin, caspofungin, fluconazole, flucytosine, Isavuconazole (Isavuconazole), itraconazole, micafungin, posaconazole and voriconazole.

Embodiment 57: the nanoparticulate drug carrier according to any one of embodiments 1 to 56, wherein the drug carrier has less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% leakage of the cargo within 24 hours at 37 ℃ in a biological buffer having a pH of 7.4.

Embodiment 58: the nanoparticulate drug carrier according to any one of embodiments 1 to 57, wherein the drug carrier has the following drug loading capacity: at least about 8% w/w, or at least about 10% w/w, or at least about 20% w/w, or at least about 30% w/w, or greater than about 40% w/w, or greater than about 50% w/w, or greater than about 60% w/w, or greater than about 70% w/w, or greater than about 80% w/w.

Embodiment 59: the nanoparticulate drug carrier according to any one of embodiments 1 to 57, wherein the drug carrier has a drug loading capacity of at least 80% w/w.

Embodiment 60: the nanoparticulate drug carrier according to any one of embodiments 1 to 59, wherein the lipid bilayer comprises a hydrophobic drug.

Embodiment 61: the nanoparticle drug carrier of embodiment 60, wherein the lipid bilayer comprises a hydrophobic drug selected from the group consisting of: paclitaxel, ellipticine (ellipticine), camptothecin, SN-38, and lipid prodrugs (e.g., acyclovir diphosphate dimyristoyl glycerol (acyclovir diphosphonate dimyristoyl glycerol), doxorubicin-conjugated phospholipid prodrugs, phospholipid derivatives of nucleoside analogs, phospholipid-linked chlorambucil, and the like).

Embodiment 62: the nanoparticle drug carrier of embodiment 60, wherein the lipid bilayer comprises paclitaxel.

Embodiment 63: the nanoparticulate pharmaceutical carrier according to any one of embodiments 1 to 61, wherein the pharmaceutical carrier is stable in suspension for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4 ℃.

Embodiment 64: the nanoparticulate drug carrier according to any one of embodiments 1 to 63, wherein a population of the drug carriers in suspension exhibits a size distribution with a width (full width at half maximum) in the following range: less than about 30nm, or less than about 20nm, or less than about 10nm, or less than about 5nm, or less than about 3nm, or less than about 2 nm.

Embodiment 65: the nanoparticulate drug carrier according to any one of embodiments 1 to 64, wherein a population of the drug carriers in suspension exhibits a substantially monomodal size distribution.

Embodiment 66: the nanoparticulate drug carrier according to any one of embodiments 1 to 65, wherein a population of the drug carriers in suspension exhibits a PDI of less than about 0.2, or less than about 0.1.

Embodiment 67: the nanoparticulate drug carrier according to any one of embodiments 1 to 66, wherein a population of the drug carriers in suspension exhibits a coefficient of size variation of less than about 0.1, or less than about 0.05, or less than about 1.7/120.

Embodiment 68: the nanoparticle drug carrier according to any one of embodiments 1-67, wherein about 3% or more of the nanoparticle drug carrier distributes to a developing tumor site upon intravenous injection.

Embodiment 69: the nanoparticulate drug carrier according to any one of embodiments 1 to 68, wherein the nanoparticulate drug carrier forms a stable suspension upon rehydration after lyophilization.

Embodiment 70: the nanoparticle drug carrier according to any one of embodiments 1 to 69, wherein the nanoparticle drug carrier provides more effective killing of cancer cells in an in situ PDAC model than free drug or liposomes containing the drug when loaded with an anti-cancer drug.

Embodiment 71: the nanoparticulate drug carrier according to any one of embodiments 1 to 70, wherein the nanoparticulate drug carrier exhibits reduced drug toxicity compared to the drug in the free drug and/or liposome when loaded with the anticancer drug.

Embodiment 72: the nanoparticulate drug carrier according to any one of embodiments 1 to 71, wherein the nanoparticulate drug carrier is colloidally stable in physiological fluids having a pH of 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site through vascular leakage (EPR effect) or transcytosis.

Embodiment 73: a pharmaceutical formulation, the formulation comprising:

a plurality of nanoparticulate drug carriers according to any one of embodiments 1 to 72; and

a pharmaceutically acceptable carrier.

Embodiment 74: the formulation of embodiment 73, wherein said formulation is an emulsion, dispersion, or suspension.

Embodiment 75: the formulation of embodiment 74, wherein said suspension, emulsion or dispersion is stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4 ℃.

Embodiment 76: the formulation according to any one of embodiments 73 to 75, wherein the nanoscale drug carrier in the formulation exhibits a substantially monomodal size distribution.

Embodiment 77: the formulation according to any one of embodiments 73 to 76, wherein said pharmaceutical carrier in said suspension, emulsion, or dispersion exhibits a PDI of less than about 0.2, or less than about 0.1.

Embodiment 78: a formulation according to any one of embodiments 73 to 77, wherein said formulation is formulated for administration via a route selected from the group consisting of: intravenous administration, intra-arterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery), intracranial administration via cannula, and subcutaneous or intramuscular depot deposition.

Embodiment 79: the formulation according to any one of embodiments 73 to 77, wherein the formulation is a sterile injectable formulation.

Embodiment 80: a formulation according to any one of embodiments 73 to 79, wherein said formulation is a unit dose formulation.

Embodiment 81: a method of treating cancer, the method comprising:

administering to a subject in need thereof an effective amount of a nanoparticle drug carrier according to any one of embodiments 1 to 51 or 57 to 72 or a pharmaceutical formulation according to any one of embodiments 73 to 80, wherein the nanoparticle drug carrier and/or the drug in the pharmaceutical formulation comprises an anti-cancer drug.

Embodiment 82: the method of embodiment 81, wherein said nanoparticulate drug carrier and/or said pharmaceutical formulation is the primary treatment in a chemotherapeutic regimen.

Embodiment 83: the method of embodiment 81, wherein said nanoparticulate drug carrier and/or said pharmaceutical formulation is a component of a multi-drug chemotherapy regimen.

Embodiment 84: the method of embodiment 83, wherein said multi-drug chemotherapy regimen comprises at least two drugs selected from the group consisting of: irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU) and Leucovorin (LV).

Embodiment 85: the method of embodiment 83, wherein said multi-drug chemotherapy regimen comprises at least three drugs selected from the group consisting of: irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU) and Leucovorin (LV).

Embodiment 86: the method of embodiment 83, wherein said multi-drug chemotherapy regimen comprises at least irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU) and Leucovorin (LV).

Embodiment 87: a method according to any one of embodiments 81 to 86, wherein said cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

Embodiment 88: the method according to any one of embodiments 81 to 86, wherein said cancer is selected from the group consisting ofCancer: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi sarcoma, lymphoma), anal carcinoma, appendiceal carcinoma, astrocytoma, atypical teratomas/rhabdoid tumors, cholangiocarcinoma, extrahepatic carcinoma, bladder carcinoma, bone cancers (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytoma, brain and spinal cord tumors, brain stem glioma, central nervous system atypical embryoid/rhabdoid tumors, central nervous system embryoid tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, breast carcinoma, bronchial tumors, Burkitt lymphoma (burkittlyhoma), carcinoid tumors (e.g., childhood, gastrointestinal tract), colon carcinoma, bladder carcinoma, malignant fibrous histiocytoma, glioma, colon carcinoma, bladder carcinoma, Cardiac tumors, cervical cancer, chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngeal carcinoma, cutaneous t-cell lymphoma, ductal cancers (e.g., cholangiocarcinoma, extrahepatic carcinoma), Ductal Carcinoma In Situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, ocular cancers (e.g., intraocular melanoma, retinoblastoma), malignant fibrous histiocytoma of bone, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancer, extragonadal cancer, central nervous system germ cell tumors), gestational trophoblastic tumors, malignant tumors, and multiple tumors, Brain stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, liver cell (liver) cancer, histiocytosis, langerhans cell cancer (langerhans cellcancer), Hodgkin lymphoma (Hodgkin lymphoma), hypopharynx cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma, Wilm's tumor, and other renal tumors), langerhans cell histiocytosis, laryngeal cancer, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), chronic bone Cancer (CLL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), chronic bone cancerMyeloid Leukemia (CML), hairy cell leukemia, lip and oral cancer, liver cancer (primary), Lobular Carcinoma In Situ (LCIS), lung cancer (e.g., childhood lung cancer, non-small cell lung cancer, small cell lung cancer), lymphoma (e.g., AIDS-related lymphoma, Burkitt ' S lymphoma (e.g., non-Hodgkin ' S lymphoma), cutaneous T-cell lymphoma (e.g., mycosis fungoides, Sezary syndrome), Hodgkin ' S lymphoma, non-Hodgkin ' S lymphoma, primary Central Nervous System (CNS) lymphoma), macroglobulinemia, Waldenstrom ' S macroglobulinemia (Cmacrogolulinemia), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood melanoma, intraocular (ocular) melanoma), merkel cell carcinoma (merkel cell carcinoma), mesothelioma, metastatic squamous neck cancer, midline carcinoma, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, Chronic Myelogenous Leukemia (CML), multiple myeloma, nasal and paranasal sinus cancers, nasopharyngeal cancers, neuroblastoma, oral cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic neuroendocrine tumor (islet cell tumor), papillomatosis, paragangliomas, paranasal and nasal cavity cancers, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacytoma, pleuropneumoblastoma, pleuroperitoma, and lymphomatosis, Primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, carcinoma of the renal pelvis and ureter, transitional cell carcinoma, rhabdomyosarcoma, salivary gland carcinoma, sarcomas (e.g. ewing's sarcoma, kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma), sezary syndrome, skin cancer (e.g. melanoma, merkel cell carcinoma, basal cell carcinoma, non-melanoma), small intestine cancer, squamous cell carcinoma, occult primary squamous neck cancer, stomach (stomach) cancer, testicular cancer, throat cancer, thymoma and thymus, thyroid cancer, trophoblastic tumors, ureter and renal pelvis cancer, urinary tract cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancerWaldenstrom macroglobulinemia and nephroblastoma.

Embodiment 89: the method according to any one of embodiments 81 to 88, wherein the nanoparticle drug carrier is not conjugated to the iRGD peptide and the nanoparticle drug carrier is administered in combination with the iRGD peptide.

Embodiment 90: a method of treating an infection, the method comprising:

administering to a subject in need thereof an effective amount of a nanoparticulate drug carrier according to any one of embodiments 1 to 34 or 52 to 56 or a pharmaceutical formulation according to any one of embodiments 73 to 80, wherein the nanoparticulate drug carrier and/or the drug in the pharmaceutical formulation comprises an antimicrobial drug.

Embodiment 91: the method of embodiment 81, wherein said infection comprises an infection with a drug resistant bacterium, virus, or fungus.

Embodiment 92: the method according to any one of embodiments 81 to 91, wherein the nanoparticle pharmaceutical carrier and/or pharmaceutical formulation is administered via a route selected from the group consisting of: intravenous administration, intra-arterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery), intracranial administration via cannula, and subcutaneous or intramuscular depot deposition.

Embodiment 93: the method according to any one of embodiments 81 to 91, wherein the nanoparticle drug carrier and/or drug formulation is administered as an injection, from an IV drip bag, or via a drug delivery cannula.

Embodiment 94: the method according to any one of embodiments 81 to 93, wherein the subject is a human.

Embodiment 95: a method according to any one of embodiments 81 to 93, wherein said subject is a non-human mammal.

Embodiment 96: a method of making a nanoparticulate drug carrier, the method comprising: providing a nanoparticle comprising silica having a surface and defining a plurality of pores adapted to contain drug molecules therein; disposing a trapping agent in a pore comprising the plurality of pores, wherein the trapping agent is selected for its ability to trap the drug within the pore; coating the pores of the nanoparticles with a lipid bilayer; and contacting or soaking the lipid bilayer coated nanoparticle with a drug that can pass through the bilayer, wherein the drug enters the pores, reacts with the trapping agent and remains within the bilayer.

Embodiment 97: the method of embodiment 96, wherein said lipid bilayer comprises phospholipids, Cholesterol (CHOL), and mPEG phospholipids.

Embodiment 98: the method according to any one of embodiments 96 to 97, wherein the phospholipid comprises a natural lipid comprising a mixture of saturated fatty acids having a carbon chain of C14-C20, and/or unsaturated fatty acids having a carbon chain of C14-C20, and/or fatty acids having a carbon chain of C12-C20.

Embodiment 99: the method of embodiment 98, wherein said phospholipid comprises a saturated fatty acid selected from the group consisting of: phosphatidylcholine (DPPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC) and Diacylphosphatidylcholine (DAPC).

Embodiment 100: the method of embodiment 98, wherein said phospholipid comprises a natural lipid selected from the group consisting of: egg phosphatidylcholine (egg PC) and soybean phosphatidylcholine (soybean PC).

Embodiment 101: the method of embodiment 98, wherein said phospholipid comprises an unsaturated fatty acid selected from the group consisting of: 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyloyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dieicosenoyl-sn-glycero-3-phosphocholine.

Embodiment 102: the method according to any one of embodiments 96 to 101, wherein the lipid bilayer comprises mPEG phospholipids having phospholipid C14-C18 carbon chains and a PEG molecular weight in the range of about 350Da to 5000 Da.

Embodiment 103: the nanoparticulate drug carrier of embodiment 102, wherein the lipid bilayer comprises 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-PEG (DSPE-PEG).

Embodiment 104: the method of embodiment 97, wherein said lipid bilayer comprises DPPC/Chol/DSPE-PEG or DSPC/Chol/DSPE-PEG.

Embodiment 105: the method of embodiment 104, wherein said lipid bilayer comprises DSPC/Chol/DSPE-PEG.

Embodiment 106: the method of embodiment 105, wherein said lipid bilayer comprises DSPC/Chol/DSPE-PEG 2000.

Embodiment 107: a method according to any one of embodiments 96 to 106, wherein the lipid bilayer comprises phospholipids, cholesterol, and mPEG phospholipids in a ratio of 50 mol% -90 mol% phospholipids to 10 mol% -50 mol% CHOL to 1 mol% -10 mol% mPEG phospholipids.

Embodiment 108: the method of embodiment 105, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG at a molar ratio of about 3:2: 0.15.

Embodiment 109: the method according to any one of embodiments 96 to 108, wherein the lipid comprising the lipid bilayer is combined with the nanoparticle in a ratio sufficient to form a continuous bilayer across the nanoparticle.

Embodiment 110: the method according to any one of embodiments 96 to 109, wherein the lipid comprising the lipid bilayer is combined with the nanoparticle at a particle to lipid ratio in a range starting from about 1.0: 3.0.

Embodiment 111: the method according to any one of embodiments 96 to 109, wherein the lipid comprising the lipid bilayer is combined with the nanoparticle at a particle to lipid ratio of about 1.0: 1.1.

Embodiment 112: the method according to any one of embodiments 96 to 111, wherein the lipid bilayer forms a substantially continuous bilayer surrounding the entire nanoparticle.

Embodiment 113: the method according to any one of embodiments 96 to 112, wherein the lipid bilayer forms a substantially uniform and intact bilayer surrounding the entire nanoparticle.

Embodiment 114: the method according to any one of embodiments 96 to 113, wherein the silica nanoparticles are mesoporous silica nanoparticles.

Embodiment 115: the method of embodiment 114, wherein the silica nanoparticles comprise sol-gel synthesized mesoporous silica nanoparticles.

Embodiment 116: the method according to any one of embodiments 96 to 115, wherein the mesoporous silica nanoparticles are size controlled.

Embodiment 117: the method according to any one of embodiments 96 to 116, wherein the mesoporous silica nanoparticles are colloidally stable.

Embodiment 118: the method according to any one of embodiments 96 to 117, wherein the mesoporous silica has an average pore size in the range of from about 1nm to about 20nm, or from about 1nm to about 10nm, or from about 2nm to about 8 nm.

Embodiment 119: the method according to any one of embodiments 96 to 118, wherein the mesoporous silica nanoparticles have an average size within the following range: from about 50nm to about 300nm, or from about 50nm to about 200nm, or from about 50nm to about 150nm, or from about 50nm to about 100nm, or from about 50nm to about 80nm, or from about 50nm to about 70nm, or from about 60nm to about 70 nm.

Embodiment 120: the method according to any one of embodiments 96 to 119, wherein the cargo trap is selected from triethylammonium sucrose octasulfate (TEA) prior to reacting with the drug₈SOS)、(NH₄)₂SO₄Ammonium, trimethylammonium, and triethylammonium salts.

Embodiment 121: the method of embodiment 120, wherein the cargo trap comprises (NH)₄)₂SO₄。

embodiment 122 the method of embodiment 120, wherein the cargo-trapping agent comprises an ammonium salt selected from the group consisting of ammonium sulfate, ammonium sucrose octasulfate, α -cyclodextrin sulfate, ammonium β -cyclodextrin sulfate, ammonium gamma-cyclodextrin sulfate, ammonium phosphate, α -cyclodextrin phosphate, ammonium β -cyclodextrin phosphate, ammonium gamma-cyclodextrin phosphate, ammonium citrate, and ammonium acetate.

embodiment 123 the method of embodiment 120, wherein the cargo trap comprises a trimethylammonium salt selected from the group consisting of trimethylammonium sulfate, sucrose octasulfate trimethylammonium, α -cyclodextrin sulfate trimethylammonium, β -cyclodextrin sulfate trimethylammonium, gamma-cyclodextrin sulfate trimethylammonium, trimethylammonium phosphate, α -cyclodextrin phosphate trimethylammonium, β -cyclodextrin phosphate trimethylammonium, gamma-cyclodextrin phosphate trimethylammonium, trimethylammonium citrate, and trimethylammonium acetate.

embodiment 124 the method of embodiment 120, wherein the cargo trap comprises a triethylammonium salt selected from the group consisting of triethylammonium sulfate, sucrose octasulfate triethylammonium, α -cyclodextrin sulfate triethylammonium, β -cyclodextrin sulfate triethylammonium, gamma-cyclodextrin sulfate triethylammonium, triethylammonium phosphate, α -cyclodextrin phosphate triethylammonium, β -cyclodextrin phosphate triethylammonium, gamma-cyclodextrin phosphate triethylammonium, triethylammonium citrate, and triethylammonium acetate.

Embodiment 125: the method of embodiment 120, wherein the cargo trap is triethylammonium sucrose octasulfate (TEA) prior to reacting with the drug₈SOS)。

Embodiment 126: the method of embodiment 125, wherein the drug is protonated and interacts with SOS^8-Trapped in the pores as gelatinous precipitates upon association.

Embodiment 127: a method according to any one of embodiments 96 to 126, wherein said drug comprises at least one weakly basic group capable of being protonated and said cargo trap comprises at least one anionic group.

Embodiment 128: the method according to any one of embodiments 96 to 127, wherein the drug is selected to have a pKa greater than 7 and less than 11.

Embodiment 129: the method according to any one of embodiments 96 to 128, wherein the drug comprises a primary, secondary, tertiary or quaternary amine.

Embodiment 130: the method according to any one of embodiments 96 to 129, wherein the drug is selected to have a water solubility index of about 5mg/mL to about 25 mg/mL.

Embodiment 131: the method according to any of embodiments 96-130, wherein the cargo is selected to have an octanol/water partition coefficient or logP value of about-3.0 to about 3.0.

Embodiment 132: the method according to any one of embodiments 96 to 131, wherein the cargo is selected to be 2nm-8nm and smaller than the average or median size of the pores of the silica nanoparticles.

Embodiment 133: the method according to any one of embodiments 127 to 132, wherein said cargo comprises an anti-cancer drug.

Embodiment 134: the method of embodiment 133, wherein the cargo comprises irinotecan.

Embodiment 135: the method of embodiment 133, wherein the cargo comprises one or more drugs independently selected from the group consisting of: topoisomerase inhibitors, antineoplastic anthracyclines, mitotic inhibitors, alkaloids, basic alkylating agents, purine or pyrimidine derivatives, protein kinase inhibitors.

Embodiment 136: the method of embodiment 135, wherein said carrier comprises a topoisomerase inhibitor comprising topotecan.

Embodiment 137: the method of embodiment 135, wherein the carrier comprises an alkaloid selected from the group consisting of: topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, and LAQ 824.

Embodiment 138: the method of embodiment 135, wherein said carrier comprises an anti-tumor anthracycline selected from the group consisting of doxorubicin and mitoxantrone.

Embodiment 139: the method of embodiment 135, wherein said carrier comprises a mitotic inhibitor selected from the group consisting of vinblastine and vinorelbine.

Embodiment 140: the method of embodiment 135, wherein said carrier comprises a basic alkylating agent selected from the group consisting of cyclophosphamide, nitrogen mustard and temozolomide.

Embodiment 141: the method of embodiment 135, wherein the carrier comprises a purine or pyrimidine derivative selected from the group consisting of: 5-fluorouracil, 5' -deoxy-5-fluorouridine and gemcitabine.

Embodiment 142: the method of embodiment 135, wherein the carrier comprises a protein kinase inhibitor selected from the group consisting of: imatinib, oxitinib and sunitinib, pazopanib, enzastalin, vandetanib, erlotinib, dasatinib and nilotinib.

Embodiment 143: the method according to any one of embodiments 96 to 142, wherein the drug carrier is conjugated to a moiety selected from the group consisting of: targeting moieties, fusion peptides, and transit peptides.

Embodiment 144: the method according to any one of embodiments 96 to 143, wherein the method produces a nanoparticulate drug carrier according to any one of embodiments 1 to 72.

Embodiment 145: the method of embodiment 143, wherein the drug carrier is conjugated to a peptide that binds to a receptor on a cancer cell.

Embodiment 146: the method of embodiment 145, wherein the drug carrier is conjugated to an iRGD peptide.

Embodiment 147: the method of embodiment 145, wherein the drug carrier is conjugated to a targeting peptide shown in table 2.

Embodiment 148: a method according to any one of embodiments 143 to 147, wherein said pharmaceutical carrier is conjugated to transferrin, and/or ApoE, and/or folate.

Embodiment 149: the method according to any one of embodiments 143 to 148, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds a cancer marker.

Embodiment 150: the method of embodiment 149, wherein said drug carrier is conjugated to a targeting moiety comprising an antibody that binds to a cancer marker set forth in table 1.

Embodiment 151: the method according to any one of embodiments 127-132, wherein the cargo comprises an antibiotic.

Embodiment 152: the method of embodiment 151, wherein said cargo comprises an antibiotic selected from the group consisting of: ciprofloxacin, levofloxacin, and HIV antiretroviral drugs (e.g., tenofovir disoproxil fumarate, and the like).

Embodiment 153: the method according to any one of embodiments 96 to 152, wherein the drug carrier is loaded to a capacity of: at least 30% w/w, or greater than about 40% w/w, or greater than about 50% w/w, or greater than about 60% w/w, or greater than about 70% w/w, or greater than about 80% w/w.

Embodiment 154: the method according to any one of embodiments 96 to 152, wherein the drug carrier is loaded to a capacity of at least 80% w/w.

Embodiment 155: the method according to any one of embodiments 96 to 154, wherein the lipid bilayer comprises a hydrophobic drug.

Embodiment 156: the method of embodiment 155, wherein the lipid bilayer comprises a hydrophobic drug selected from the group consisting of: paclitaxel, ellipticine, camptothecin, SN-38, and lipid prodrugs (e.g., acyclovir diphosphate dimyristoyl glycerol, doxorubicin-conjugated phospholipid prodrugs, phospholipid derivatives of nucleoside analogs, phospholipid-linked chlorambucil, etc.).

Embodiment 157: the method of embodiment 155, wherein the lipid bilayer comprises paclitaxel.

Embodiment 158: a method of preparing an irinotecan nanocarrier, the method comprising: providing a nanocarrier comprising a silica body having a surface comprising a plurality of pores suitable for housing irinotecan therein; providing an agent selected for its ability to trap irinotecan within the plurality of pores; coating the pores of the nanocarriers with a phospholipid bilayer (optionally using sonication methods); and introducing irinotecan into the phospholipid bilayer-coated pores to prepare a phospholipid bilayer-coated irinotecan nanocarrier.

Embodiment 159: the method of embodiment 158, wherein said silica body comprises a sol-gel synthesized, size-controlled, and colloidally-stabilized silica body.

Embodiment 160: the method of embodiment 158, wherein the irinotecan trapping agent is triethylammonium sucrose octasulfate (TEA)₈SOS)。

Embodiment 161: the method of embodiment 160, wherein the nanocarrier is: (a) having an irinotecan loading capacity of at least 20% (or 30% or 40%) w/w; and/or (b) exhibits < 5% (or < 10%) irinotecan leakage within 24 hours at 37 ℃ in a biological buffer having a pH of 7.4.

Embodiment 162: the method of embodiment 161, wherein the nanocarrier is colloidally stable in physiological fluids having a pH of 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering the disease site through vascular leakage (EPR effect) or transcytosis.

Embodiment 163: the method of embodiment 161, wherein the phospholipid bilayer comprises cholesterol and/or paclitaxel.

Embodiment 164: a method of making a nanocarrier, the method comprising: providing an unsupported nanocarrier, the unsupported nanocarrier comprising: a silica body having a surface and defining a plurality of pores adapted to contain molecules therein; and a phospholipid bilayer coating the surface; a cargo trapping agent is encapsulated within the phospholipid bilayer.

Embodiment 165: the method of embodiment 164, further comprising exposing said nanocarrier to a cargo selected to interact with said cargo trap.

Embodiment 166: the method of embodiment 165, wherein the cargo is selected to have a pKa greater than 7 and less than 11 and is capable of being protonated, and the cargo trap comprises at least one anionic group.

Embodiment 167: the method of embodiment 165, wherein the cargo is irinotecan and the cargo trap is sucrose octasulfate Triethylammonium (TEA)₈SOS)。

Embodiment 168: the method of embodiment 165, wherein the cargo is a topoisomerase I inhibitor: topotecan; one or more anti-tumor anthracycline antibiotics: doxorubicin and mitoxantrone; one or more mitotic inhibitors: vinblastine and vinorelbine; or one or more tyrosine kinase inhibitors: imatinib, oxitinib and sunitinib.

Embodiment 169: the method of embodiment 165, wherein said nanocarrier has a drug loading capacity of at least 30% w/w.

Embodiment 170: a nanocarrier, comprising: a silica body having a surface and defining a plurality of pores adapted to contain molecules therein; a phospholipid bilayer coating the surface; and a cargo trap within the phospholipid bilayer; wherein the sub-micron structures have a largest dimension of less than 1 micron, and wherein the phospholipid bilayer stably seals the plurality of pores.

Embodiment 171: the nanocarrier of embodiment 170, further comprising a cargo within the phospholipid bilayer.

Embodiment 172: the nanocarrier of embodiment 171, wherein the cargo is associated with the cargo trap.

Embodiment 173: the nanocarrier of embodiment 172, wherein the cargo comprises at least one weakly basic group capable of being protonated and the cargo trap comprises at least one anionic group.

Embodiment 174: the nanocarrier of embodiment 171, wherein the cargo is selected to have a pKa greater than 7 and less than 11.

Embodiment 175: the nanocarrier of embodiment 171, wherein the cargo comprises a primary, secondary, tertiary, or quaternary amine.

Embodiment 176: the nanocarrier of embodiment 171, wherein the cargo is selected to have a water solubility index of 5mg/mL to 25 mg/mL.

Embodiment 177: the nanocarrier of embodiment 171, wherein the cargo is selected to have an octanol/water partition coefficient or logP value of from-3.0 to 3.0.

Embodiment 178: the nanocarrier of embodiment 171, wherein the cargo is selected to be between 2nm and 8nm and is smaller than the size of the pores of the nanocarrier.

Embodiment 179: the nanocarrier of embodiment 171, wherein the cargo is irinotecan and the cargo trap is sucrose octasulfate Triethylammonium (TEA)₈SOS)。

Embodiment 180: the nanocarrier of embodiment 171, wherein the cargo is a topoisomerase I inhibitor: topotecan; one or more anti-tumor anthracycline antibiotics: doxorubicin and mitoxantrone; one or more mitotic inhibitors: vinblastine and vinorelbine; or one or more tyrosine kinase inhibitors: imatinib, oxitinib and sunitinib.

Embodiment 181: the nanocarrier of embodiment 171, wherein the nanocarrier has less than 5% leakage of the cargo within 24 hours at 37 ℃ in a biological buffer having a pH of 7.4.

Embodiment 182: the nanocarrier of embodiment 171, wherein the nanocarrier has a drug loading capacity of at least 30% w/w.

Embodiment 183: the nanocarrier of embodiment 171, wherein the nanocarrier has a drug loading capacity of at least 80% w/w.

Embodiment 184: the nanocarrier of embodiment 170, wherein the phospholipid bilayer comprises paclitaxel.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments of the invention, are given by way of illustration and not limitation. Many changes and modifications may be made which are within the scope of the invention without departing from the spirit thereof, and the invention includes all such modifications.

Definition of

The terms "subject," "individual," and "patient" may be used interchangeably and refer to humans as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, etc.). In various embodiments, the subject may be a human (e.g., adult male, adult female, juvenile male, juvenile female, male child, female child) in a hospital as an outpatient or in other clinical settings under the care of a physician or other health worker. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.

The phrase "subject in need thereof" as used herein refers to a subject having or at risk of having a lesion to which a nanoparticle drug carrier (silica body) as described herein is directed, as described below. Thus, for example, in certain embodiments, the subject is a subject having cancer (e.g., Pancreatic Ductal Adenocarcinoma (PDAC), breast cancer (e.g., drug-resistant breast cancer), colon cancer, brain cancer, etc.). In certain embodiments, the subject is a subject having a microbial infection, including but not limited to a drug-resistant microbial infection.

The term "treating" when used in reference to, for example, treating a disorder or disease, refers to reducing and/or eliminating one or more symptoms of the disorder or disease, and/or delaying the progression of and/or reducing the incidence or severity of one or more symptoms of the disorder or disease, and/or preventing the disorder or disease. The term treatment may refer to prophylactic treatment, which includes delaying the onset of a pathology or disease or preventing the onset of a pathology or disease.

The terms "co-administration" or "administration in combination … …" or "co-treatment", when used in reference to co-administration of a first compound (e.g., a silica body containing irinotecan) and a second compound (e.g., an iRGD peptide), mean that the first compound and the second compound are administered such that there is at least some temporal overlap in the biological activity of the first compound and the second compound in the organism receiving their administration. Co-administration may include simultaneous administration or sequential administration. In sequential administration, there may even be some substantial delay (e.g., minutes or even hours) between the administration of the first compound and the second compound, as long as their biological activities overlap. In certain embodiments, the co-administration is within a time frame that allows the first compound and the second compound to produce an enhanced therapeutic or prophylactic effect on the organism. In certain embodiments, the enhanced effect is a synergistic effect.

The terms "nanocarrier" and "nanoparticle drug carrier" and "silica body" are used interchangeably and refer to a nanostructure having a porous particle core that is interchangeable with the term "porous nanoparticle" as used herein; and a lipid bilayer encasing (or surrounding) the porous particle core. In certain embodiments, the silica nanoparticles are porous silica nanoparticles (e.g., Mesoporous Silica Nanoparticles (MSNPs)).

The term "lipid" as used herein refers to conventional lipids, phospholipids, cholesterol, chemically functionalized lipids for attachment of PEG and ligands, and the like.

The term "lipid bilayer" or "LB" as used herein refers to any bilayer of oriented amphiphilic lipid molecules with the hydrocarbon tails facing inward to form a continuous non-polar phase.

The term "liposome" as used herein refers to an aqueous compartment surrounded by a lipid bilayer, as conventionally defined (see, e.g., Stryer (1981) Biochemistry, 2 nd edition, w.h.freeman & co., page 213).

In contrast to the lipid bilayer defined in the silica body, the lipid bilayer in a liposome may be referred to as an "unsupported lipid bilayer" and the liposome itself (when unloaded) may be referred to as an "empty liposome". The lipid bilayer in the silica body may be referred to as a "supported lipid bilayer" because the lipid bilayer in the silica body is located on the surface and supported by the porous particle core. In certain embodiments, the lipid bilayer may have a thickness in the range of about 6nm to about 7nm, which includes a thickness of 3nm-4nm of the hydrophobic core, plus a layer of hydrated hydrophilic head groups (about 0.9nm each) plus two partially hydrated regions of about 0.3nm each.

The term "selective targeting" or "specific binding" as used herein refers to the use of targeting ligands on the surface of a silica body (empty or loaded), in particular on the surface of the lipid bilayer of a silica body, wherein the ligands specifically/selectively interact with a target, e.g. a receptor or other biomolecule component expressed on the surface of a cell of interest. Targeting ligands may include molecules and/or materials such as peptides, antibodies, aptamers, targeting peptides, polysaccharides, and the like.

Silica bodies with targeting ligands may be referred to as "targeting silica bodies".

The term "silica body" refers to a drug-containing (drug delivery) silica nanoparticle, wherein the silica nanoparticle is completely covered by a lipid bilayer (e.g., a phospholipid bilayer). In certain embodiments, the silica nanoparticles are porous silica nanoparticles (e.g., mesoporous silica nanoparticles).

The term "about" or "approximately" as used herein refers to within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of accuracy required for a particular purpose, such as a pharmaceutical formulation. For example, "about" can mean within 1 standard deviation or more than 1 standard deviation, as is customary in the art. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or methods, the term may mean within one order of magnitude, preferably within 5-fold, and more preferably within 2-fold of the value. Unless otherwise indicated, where a particular value is described in this application and the claims, it should be assumed that the term "about" means within an acceptable error range for the particular value.

The term "drug" as used herein refers to chemical entities of different molecular size (small and large), naturally occurring or synthetic, that exhibit therapeutic effects in animals and humans. Drugs may include, but are not limited to, organic molecules (e.g., small organic molecules), therapeutic proteins, peptides, antigens, or other biomolecules, oligonucleotides, sirnas, constructs encoding CRISPR cas9 components and optionally one or more guide RNAs, and the like.

A "pharmaceutically acceptable carrier" as used herein is defined as any one of the standard pharmaceutically acceptable carriers. The pharmaceutical compositions of the present invention may be formulated according to known methods for preparing pharmaceutically useful compositions. Pharmaceutically acceptable carriers may include diluents, adjuvants, and vehicles as well as carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating materials that do not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be a solvent or dispersion medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The formulations are described in a number of sources well known and readily available to those skilled in the art. For example, Remington's pharmaceutical sciences (Martin E W [1995] Easton (Easton Pa.) Pa., Pa.), Mach publishing Company (Mack publishing Company, 19 th edition) describe formulations that can be used in conjunction with the silica bodies described herein.

As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by or derived from an immunoglobulin gene or fragment of an immunoglobulin gene that is capable of binding (e.g., specifically binding) to a target (e.g., a target polypeptide).

Typical immunoglobulin (antibody) building blocks are known to comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50kDa-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The term variable light chain (V)_L) And a variable heavy chain (V)_H) These are referred to as the light chain and the heavy chain, respectively.

Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests the antibody under disulfide bonds in the hinge region to produce F (ab)'₂I.e. dimers of Fab which are themselves linked to V by disulfide bonds_H-C_H1 linked light chain. F (ab)'₂Can be reduced under mild conditions to disrupt disulfide bonds in the hinge region, thereby resulting in (Fab')₂The dimer is converted to Fab' monomer. The Fab' monomer essentially has a portion of the hinge region(iv) Fab (see, for a more detailed description of other antibody fragments, Fundamental Immunology, eds. W.E.Paul, Raven Press, N.Y. (1993)). Although various antibody fragments are defined using digestion of whole antibodies, those skilled in the art will appreciate that such Fab' fragments can be synthesized de novo by chemical means or by using recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods. Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv), in which a variable heavy chain and a variable light chain are linked together (either directly or via a peptide linker) to form a continuous polypeptide. The single-chain Fv antibody is covalently linked to V_H-V_LHeterodimers, which may be composed of V's including direct linkage or linked through peptide-encoding linkers_HCoding sequence and V_LNucleic acid expression of the coding sequence. Huston et al, (1988) Proc. nat. Acad. Sci. USA,85: 5879-5883. Albeit V_HAnd V_LInterconnected as a single polypeptide chain, except V_HDomains and V_LThe domains associate non-covalently. The first functional antibody molecule to be expressed on the surface of filamentous phage is single chain Fv (scFv), however, alternative expression strategies are also successful. For example, if one of the chains (heavy or light chain) is fused to the g3 capsid protein and the complementary chain is exported as a soluble molecule into the periplasm, a Fab molecule can be displayed on the phage. The two strands may be encoded on the same or different replicons; importantly, the two antibody chains in each Fab molecule are assembled post-translationally and the dimer is incorporated into the phage particle by linking one of the chains to, for example, g3p (see, e.g., U.S. Pat. No.: 5733743). Many other structures of scFv antibodies and molecules that convert naturally aggregated, but chemically separated, light and heavy chain polypeptides from antibody V regions into three-dimensional structures that fold into structures substantially similar to the structures of the antigen binding site are known to those of skill in the art (see, e.g., U.S. Pat. nos. 5,091,513, 5,132,405, and 4,956,778). In certain embodiments, the antibody should include already existing phageAll antibodies displayed above (e.g., scFv, Fv, Fab, and disulfide-linked Fv (see, e.g., Reiter et al (1995) Protein Eng.8:1323-1331), as well as affibodies, monoclonal antibodies, etc.).

The term "specific binding" as used herein in reference to a biomolecule (e.g., protein, nucleic acid, antibody, etc.) refers to a binding reaction that determines the presence of the biomolecule in a heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under specified conditions (e.g., immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), a specified ligand or antibody binds to its particular "target" molecule and does not bind in significant amounts to other molecules present in the sample.

Drawings

Referring now to the drawings in which like reference numbers represent corresponding parts throughout:

figures 1A-1E illustrate the preparation of drug-loaded LB-MSNP and liposomal irinotecan carriers using a protonating agent according to one or more embodiments described herein. Figure 1A shows a schematic depicting the synthesis of LB-MSNP and liposomes and loading of irinotecan (figure 1A, panel a 1). Then TEA is added₈After SOS immersion in MSNP particles, the pores were sealed with LB, which resulted from sonication of lipid biofilms (Lu et al (2007) Small,3: 1341-1346). Map a2 of fig. 1A: adding TEA₈The SOS-soaked particles were incubated in irinotecan solution, allowing the amphiphilic drug to diffuse through the lipid bilayer to remove TEA from the bilayer₈SOS protonationThe fat-soluble TEA leaves the particles, and H⁺Irinotecan is converted to a hydrophilic derivative that cannot pass through LB. Protonated drugs with SOS^8-Interact to form a gelatinous precipitate that remains in the pores. Map a3 of fig. 1A: production of a peptide for irinotecan using the same techniqueEncapsulated liposome equivalents (Drummond et al (2006) Cancer Res.,66(6): 3271-3277). FIG. 1B: evaluation of Drug Loading Capacity (DLC) of Ir-MSNP vectors and Ir-liposome vectors. Total amount of DLO [ irinotecan (m)₀) Unencapsulated irinotecan (m)₁)]/[ Total amount of particles (m)_MSNPOr m_Lipid)]X 100%. Adding TEA₈The effect of SOS inclusion on the hydrodynamic size and zeta potential of the particles is negligible. Hydrodynamic dimensions and zeta potential data are shown in table 3 (example 2). FIG. 1C: CryoEM images of empty, uncoated MSNP, Ir-MSNP, and Ir-liposome vectors. The technique is sensitive enough to visualize irinotecan precipitates in liposomes. FIG. 1D: vehicle stability was assessed by incubation in 100% serum at 37 ℃ for 24 hours and drug leakage determined by HPLC. Figure 1E) carrier stability as determined by change in hydrodynamic diameter and% drug leakage after lyophilization and water resuspension.

FIGS. 2A-2D are graphical representations of the biodistribution of Ir-LB-MSNP and Ir-liposomes according to one or more embodiments described herein. FIG. 2A: IVIS was imaged at intervals over 48 hours to compare the biodistribution of the intravenously injected NIR labelled vector to KPC derived in situ tumour sites (n-3). NIR fluorescence images in representative animals are shown after intravenous injection of 100mg/kg NIR-labeled LB-MSNP or liposomes. FIG. 2B: ex vivo imaging of an transplanted organ in the same experiment; animals were sacrificed after 24 hours. Confocal microscopy confirmed that NIR-labeled LB-MSNP was more abundant at the tumor site compared to liposomes. FIG. 2C: irinotecan tumor levels were determined in an orthotopic KPC-derived xenograft model (n ═ 3). For the different pharmaceutical formulations, animals received an intravenous injection of a dose equivalent of 60mg/kg irinotecan. The animals were sacrificed after 24 hours, and tumor tissues were subsequently collected to measure irinotecan content by HPLC. Irinotecan content was expressed as the total injected dose per gram of tumor tissue (% ID/g). Data represent mean ± SD,. p < 0.05. FIG. 2D: HPLC measurement of plasma irinotecan concentration in the same experiment. Data represent mean ± SD,. p < 0.05.

Figures 3A-3E show differential tumor inhibition of free drug and encapsulated irinotecan carrier in a KPC-derived in situ tumor model, according to one or more embodiments described herein. FIG. 3A: assessment of MTD in an acute dose exploration study using NCI protocol. FIG. 3B: inhibition of growth of KPC-derived orthotopic tumors in B6/129 mice after up to 8 administrations of 40mg/kg of free drug or encapsulated irinotecan administered intravenously every 4 days. Tumor growth was monitored using compartmental IVIS imaging, which is quantitatively expressed in terms of image intensity at the operator-defined ROI. FIG. 3C: quantitative analysis of apoptosis at primary tumor sites of animals (sacrificed at day 40-47) after treatment (IHC staining for cleaved caspase-3 was used). FIG. 3D: representative necropsy results and ex vivo imaging of bioluminescence intensity in moribund animals (sacrificed at day 40-day 47) to show the effect of treatment on peripheral metastasis. Metastatic spread can be seen in the stomach, intestine, liver, spleen, kidney, diaphragm and abdominal wall. There was no infiltration of the heart or lungs. FIG. 3E: the heat map shows comparative analysis to summarize tumor spread as determined by quantitative ex vivo imaging in (fig. 3D). Data represent mean ± SEM,. p < 0.05.

FIGS. 4A-4E are graphical representations of comparative analyses of toxicity reduction for Ir-LB-MSNP and Ir-liposomes according to one or more embodiments described herein. FIG. 4A: liver histology obtained from representative moribund animals (sacrificed at day 40-day 47) using tissues from the experiment shown in figure 3B. Arrows in H & E stained sections point to necrotic liver tissue, while the asterisked sites indicate steatosis. The scale bar is 200 μm. FIG. 4B: dual IHC staining of caspase-3 (apoptosis marker, red) and F4/80(KC marker, green) cleaved in the liver of animals receiving a dose equivalent of 60mg/kg of different irinotecan formulations followed by 24 hours of sacrifice. Nuclei were stained with Hoechst 33342 (blue). Scale bar 100 μm. FIG. 4C: IHC staining for cleaved caspase-3, counterstaining with H & E revealed the spread of apoptosis and blunting of intestinal villi in the same treated animal group studied in FIG. 3B. The scale bar represents 100 μm. FIG. 4D: separate experiments in which 40mg/kg dose equivalent of irinotecan was injected intravenously (3 times) every other day to study the effects on sternal bone marrow. Sternums were collected on day 7 for embedding, decalcification, and H & E staining. The scale bar represents 200 μm. FIG. 4E: schematic illustration to explain the differential hepatotoxicity of Ir-LB-MSNP and Ir-Liposome formulations in the liver. Without being bound by a particular theory, it is contemplated that the injected nanocarriers are initially taken up by KC, wherein carrier disintegration causes irinotecan to be released into bystander hepatocytes. The subsequent rate of carrier disintegration and drug release to the hepatocytes can determine the extent to which irinotecan is metabolized and rendered inactive. It is further hypothesized that the higher instability of the liposome carrier results in faster drug release compared to the more stable Ir-LB-MSNP, which explains the difference in apoptosis and necrosis.

FIG. 5 illustrates a synthesis procedure and TEA depicting lipid bilayer coated mesoporous silica nanoparticles (LB-MSNPs) according to one or more embodiments of the invention₈Schematic of the mechanism of SOS-mediated irinotecan (IRIN) loading. MSNP was synthesized using sol/gel chemistry. First TEA was added₈SOS (internal synthesis) was incubated with MSNP. After this, the LB coating was deposited using our biofilm technique (Meng et al (2015) ACS Nano,9(4): 3540-3557). Adding TEA₈SOS-soaked MSNP was added to a dry lipid biofilm (e.g., 3:2:0.15 molar ratio of DSPC/cholesterol/DSPE-PEG) followed by pore sealing upon sonication. After removal of free trapping agent, the TEA-loaded will be₈LB-MSNP to SOS was incubated with irinotecan. This allows free lipophilic irinotecan to diffuse into the particle to be protonated and trapped in the pores. The reaction proceeds as follows: due to the existence of the LB-MSNP internal trapping agent, the method has the advantages of low cost and high efficiencyCauses the lipid-permeable Triethylamine (TEA) to flow out into the particle incubation medium. Inside of the particle H⁺Release of (2) to produce a pH gradientThe pH gradient induces protonation of lipophilic irinotecan that diffuses across the LB. Lipid impermeable protonated IRIN and SOS^8-Interact to form a precipitate, resulting in efficient IRIN encapsulation in LB-MSNP.

Figures 6A and 6B show morphology and drug release profiles of irinotecan-loaded LB-MSNP (Ir-LB-MSNP (+ SOS)) with trapping reagent and irinotecan-loaded liposomes (Ir-Lipo (+ SOS)) using trapping reagent according to one or more embodiments described herein. FIG. 6A: cryoEM images (TF20, FET) of Ir-LB-MSNP (+ SOS) (upper panel) and Ir-Lipo (+ SOS) (lower panel). The lipid bilayer coating on the surface of MSNP can be clearly seen. The top magnified image shows MSNP with an intact lipid coating on the particle surface, while the porous interior shows TEO₈The presence of a high density complex between SOS and irinotecan. Similarly, in the liposome image (lower panel), the irinotecan-trapping agent complex can be seen as an internal high density precipitate (marked red arrow). FIG. 6B: drug release profiles of Ir-LB-MSNP (+ SOS) and Ir-Lipo (+ SOS) in PBS (pH 7.4) and phagolysosomal mock fluid (PSF, pH 4.5). For drug release measurements, NPs were prepared in PBS or PSF (1mL, irinotecan 0.1mg/mL) followed by shaking at 37 ℃. At the time points shown, the released irinotecan was separated from the NP by a centrifugal filter unit with a size cut-off of 30 kD. The irinotecan concentration in the filtrate was determined by plate reader at an OD of 360 nm. The experiment was repeated at least twice.

FIG. 7 illustrates cellular uptake and intracellular distribution of Ir-LB-MSNP (+ SOS) and Ir-Lipo (+ SOS) as determined by confocal microscopy according to one or more embodiments described herein. LB-MSNP and liposomes were fluorescently labeled with 0.1% w/w Texas Red (Texas red) -DHPE in the lipid bilayer. Both particles were incubated with PANC-1 cells for 24 hours and then washed three times in PBS. After the cells were fixed and washed with PBS, the cell membranes were stained with WGA 488 and the nuclei were stained with Hoechst dye. The slides were visualized using a confocal microscope.

FIGS. 8, Panels A-F, are pictorial representations of comparative cytotoxicity assays of Ir-LB-MSNP (+ SOS) and Ir-Lipo (+ SOS) or free drug, according to one or more embodiments of the present invention. Panels A-C of FIG. 8: cell viability of PANC-1 cells was determined by MTS assay after treatment with free IRIN, Ir-LB-MSNP (+ SOS), and Ir-Lipo (+ SOS) at the indicated concentrations. Experiments were also performed at different time intervals (24 hours, 48 hours and 72 hours). FIG. 8, panels D-F: cell viability of BxPC-3 cells after treatment with free IRIN, Ir-LB-MSNP (+ SOS), and Ir-LB-MSNP (+ SOS) at different drug concentrations and time points as described for PANC-1 cells.

Fig. 9 illustrates a histological analysis of bone marrow according to one or more embodiments described herein. Bone marrow histology after treatment with free IRIN, Ir-Lipo (+ SOS), and Ir-LB-MSNP (+ SOS). BALB/c male mice were injected intravenously with IRIN, Ir-Lipo (+ SOS) or Ir-LB-MSNP (+ SOS) at a drug dose of 60 mg/kg. After 24 hours, mice were sacrificed and sternums were collected and fixed in 10% formalin. Sections were stained with hematoxylin-eosin (H & E) and examined by light microscopy. The data indicate that both the LB-MSNP formulation and the liposome formulation reduced bone marrow ablation induced by irinotecan. Severe bone marrow damage and apoptosis can be seen in the free drug group.

Fig. 10 illustrates a histological analysis of a kidney according to one or more embodiments described herein. Histological analysis of kidneys after treatment with free IRIN, Ir-Lipo (+ SOS) and Ir-LB-MSNP (+ SOS). BALB/c male mice were injected intravenously with IRIN, Ir-Lipo (+ SOS) or Ir-LB-MSNP (+ SOS) at a drug dose of 60 mg/kg. After 24 hours, mice were sacrificed and kidneys were collected and fixed in 10% formalin followed by paraffin embedding. Tissue sections of 4 μm thickness were mounted on slides. Sections were stained with hematoxylin-eosin (H & E) and examined by light microscopy. Representative histological images showing swelling and edema of the renal capsule space (Bowman's) (arrowed) were observed in free IRIN or Ir-Lipo (+ SOS) treated animals. These lesions are indicative of acute glomerulonephritis. No significant renal abnormalities were found in the Ir-LB-MSNP (+ SOS) group.

Figure 11 shows a low resolution cyroEM image of a representative irinotecan-loaded LB-MSNP, according to one or more embodiments of the invention. The scale bar represents 100 nm. Visual inspection of about 500 particles confirmed the structural integrity of LB, > 99% of the particles were successfully coated.

Fig. 12, panels a-F, show H & E staining to show KPC-derived tumor infiltration into peripheral organs 5 weeks after implantation in B6/129 mice, according to one or more embodiments of the invention. Representative images were taken to show infiltration into the pancreas (panel a), liver (panel B), spleen (panel C), kidney (panel D), intestine (panel E), and stomach (panel F). The scale size is 200 μm.

Fig. 13 shows ex vivo IVIS images taken 48 hours after intravenous injection of NIR labeled particles in the KPC-derived model in fig. 2A, according to one or more embodiments described herein. This indicates that for equivalent particle doses, the abundance of LB-MSNP is increased at the tumor site compared to labeled liposomes.

Figure 14 shows the biodistribution of LB-MSNP in a KPC-derived in situ tumor model of B6/129 mice (n-3) after intravenous injection of LB-MSNP (100mg/kg) and sacrifice of animals at 24 hours, according to one or more embodiments of the invention. Tumor tissue and major organs were collected to measure Si content using inductively coupled plasma optical emission spectroscopy. Particle biodistribution is expressed as the total injected Si dose% (ID%) at each site. Data represent mean ± SD.

Figure 15A shows irinotecan tumor content determined in a subcutaneous PANC-1 xenograft model in nude mice (n-3). For the different pharmaceutical formulations, animals received an intravenous injection of a dose equivalent of 60mg/kg irinotecan. After sacrifice of the animals after 24 hours, tumor tissues were collected to measure irinotecan content by HPLC. Irinotecan content was expressed as the total injected dose per gram of tumor tissue (% ID/g). Data represent mean ± SD, MSNP p <0.05 compared to other groups. FIG. 15B shows NIR fluorescence images in representative animals 24 hours after intravenous injection of 100mg/kg NIR-labeled LB-MSNP.

Figure 16 shows HPLC quantification of irinotecan content in peripheral organs of B6/129 mice. For different pharmaceutical formulations, animals received an intravenous injection of a dose equivalent of irinotecan at 60mg/kg (n-3). After sacrifice of the animals after 24 hours, tissues were collected to measure irinotecan content by HPLC. Irinotecan content was expressed as the total injected dose per gram of tissue (ID%/g). Data represent mean ± SD,. p < 0.05.

Figure 17 shows representative images to show IHC staining for cleaved caspase-3 (an apoptotic marker) at primary tumor sites obtained from primary tumor sites in each animal group (sacrificed at day 40-47) in the experiment described in figure 3B. Apoptotic cells (brown) are indicated by red arrows. Scale bar 50 μm.

Figure 18 shows mortality rates of animals in each treatment group of the efficacy study described in figure 3B, according to one or more embodiments of the invention. The arrow on the x-axis indicates the day of injection. Mice were monitored frequently and sacrificed rather than spontaneously based on "moribund criteria" (Zucker et al (2009) j. control. release,139(1): 73-80). Irinotecan-loaded LB-MSNP induced a significant (p <0.01, compared by log rank test using SPSS 19.0(IBMSPSS staticistics, usa)) survival improvement compared to saline and free irinotecan. Better survival results were observed for Ir-LB-MSNP compared to Ir-liposomes.

FIG. 19 shows a complete blood count analysis of a sample obtained from the experiment described in FIG. 4D, according to one or more embodiments of the invention. Differential white blood cell counts showed a significant reduction in absolute neutrophil counts for the free drug or Ir-liposome treated groups. In contrast, treatment with Ir-LB-MSNP showed a small, but not significant, drop in neutrophil count. Data represent mean. + -. SEM, andcomparison of control group,. about.p<0.05, compared with the LB-MSNP group,^#p<0.05。

figure 20 shows HPLC quantification of irinotecan content in the liver for different drug carriers according to one or more embodiments described herein. For different pharmaceutical formulations, animals received an intravenous injection of a dose equivalent of irinotecan at 60mg/kg (n-3). After the animals were sacrificed after 0.5 hours, 4 hours, and 24 hours, respectively, liver tissues were collected to measure irinotecan content by HPLC. Irinotecan content was expressed as the total injected dose per gram of liver tissue (% ID/g). Data represent mean ± SD,. p < 0.05.

Fig. 21A and 21B illustrate synthesis and characterization of silica bodies for drug loading and visualization. FIG. 21A: the upper panel provides a schematic diagram showing the synthetic steps for constructing the silica body and remote drug loading (see example 1 and Liu et al (2016) ACS Nano.10(2): 2702-2715). Briefly, the MSNP core was synthesized by sol-gel chemistry and soaked in a solution containing the protonating agent TEA₈SOS solution. These particles were coated with LB in the presence of lipid biofilms using a sonication procedure (supra). After this, the passage is via TEA₈The proton gradient provided by SOS allows for remote irinotecan loading. Block 1: schematic representation of different silica body compositions. And 2, block 2: conjugation of iRGD peptides to LB Using a thiol-maleimide reaction to conjugate cysteine-modified iRGD peptides to DSPE-PEG₂₀₀₀Maleimide attachment. And a block 3: Cryo-EM images showing bare particles and silica bodies with and without the intercalation of Au cores at about 10nm (for TEM visualization). The synthetic procedure is described in example 3 supplementary materials and methods. Scale bar 50 nm. FIG. 21B: necropsy and IVIS images of KPC-derived in situ PDAC models of immunocompetent B6/129 mice. In situ implantation involves injection of 2X 10 in the tail of the pancreas⁶Minor surgery of individual KPC-luc cells (left panel). Necropsy and bioluminescence imaging revealed primary tumor growth after 1-2 weeks followed by tumor metastasis by 3-5 weeks. Macroscopic transfer is indicated by arrows.

Fig. 22A and 22B show that co-administration of iRGD enhances tumor biodistribution of intravenously injected silica bodies in KPC-derived in situ models. FIG. 22A: tumor-bearing mice received (i) intravenous 50mg/kg NIR-labeled silicalite IV co-administration with 8 μmol/kg free iRGD (n-3, referred to as "silicalite + iRGD"); (ii)50mg/kg of NIR-labeled silica conjugated with iRGD (n ═ 3, referred to as "silica-iRGD"); or (iii) 50mg/kg NIR-labeled silica bodies without iRGD. Animals were sacrificed 24 hours post injection followed by ex vivo NIR imaging using IVIS. FIG. 22B: NIR fluorescence intensity and Si content were used to quantify nanoparticle content in situ tumors. Data represent mean ± SD,. p < 0.05.

Figures 23A-23E show that iRGD co-administration enhances uptake and efficacy of Ir-silica bodies in KPC-derived in situ models. FIG. 23A: schedule of efficacy studies in a luciferase-expressing KPC-derived in situ tumor model (n ═ 6). The selected Ir-silica dose (40mg/kg irinotecan; 80mg/kg MSNP) was based on previous efficacy studies (Liu et al (2016) ACS Nano.10(2): 2702-2715). This dose of Ir-silica was injected intravenously with or without co-administration of 8. mu. mol/kg iRGD. Injections were repeated every 3 days for a total of 4 administrations. Control groups included animal groups that received only the same dose of free iRGD or Ir-silica. FIG. 23B: representative ex vivo imaging of bioluminescence intensity in mice prior to sacrifice to show primary tumor burden and metastasis. The images show that iRGD co-administration can enhance silica body efficacy. FIG. 23C: the heatmaps of the effect on tumor and tumor metastasis inhibition in fig. 23B are summarized. FIG. 23D: the viability impact of Ir-silica bodies was increased by iRGD co-administration as shown by Kaplan-Meier analysis (Kaplan-Meier analysis). The effect of silica bodies alone was very significant (p ═ 0.001) compared to PBS and free iRGD. iRGD co-administration further increased survival (p ═ 0.027). FIG. 23E: HPLC analysis of irinotecan content in tumors 24 hours after injection of one dose of Ir-silica (40mg/kg drug) with or without co-administration of 8. mu. mol/kg iRGD. Data represent mean ± SD (n ═ 3) ·, p < 0.05.

FIGS. 24A and 24C show that iRGD-mediated uptake of silicasomes requires NRP-1 expression on tumor vasculature. FIG. 24A: a schematic diagram illustrating the mechanism by which iRGD triggers nanoparticle transcytosis. FIG. 24B: multicolor IHC staining of NRP-1 (green) and CD31 (red) in KPC-derived tumor sections, plus nuclear staining (blue). IHC staining methods are described in the methods section. NRP-1 is expressed on both tumor tissue as well as blood vessels. The merged image shows a high co-localization of NRP-1 with CD31 (94.2%); the co-localization ratio (CR) was determined by Image J software. Scale bar 100 μm. FIG. 24C: interference of iRGD-mediated biodistribution of silica bodies by anti-NRP-1 antibodies. 50 μ g of blocking antibody or control IgG (n ═ 3) was injected intravenously 15 minutes before injection of 50mg/kg NIR-silica +8 μmol/kg free iRGD. An ex vivo NIR image of the nanoparticle biodistribution after 24 hours is shown. NIR intensity and Si content were used to quantify nanoparticle uptake at the in situ tumor site. Data represent mean ± SD,. p < 0.05.

FIGS. 25A-25C provide ultrastructural observations of the silica body transport system triggered by iRGD co-administration. FIG. 25A: mice bearing in situ tumors were injected with 50mg/kg of Au-silica with or without co-administration of 8. mu. mol/kg iRGD. Tumors were harvested at 24 hours and immediately fixed for TEM analysis. At least 10 regions of interest in each group were observed to quantitatively represent the abundance of interconnected vesicles grouped in vascular endothelial cells (yellow arrows). We calculated each 1 μm²Number of vesicles on the surface area of the cells (left panel). Data represent mean ± SD,. p<0.05. Representative TEM photographs with high and low magnification are shown. L ═ lumen; r ═ red blood cells. FIG. 25B: TEM visualization of silica transcytosis in tumor-bearing mice that received 50mg/kg of Au-silica, then were sacrificed 24 hours later. The electron micrograph shows that the silica: (i) in the lumen of tumor vessels (red arrows); (ii) transport in endothelial vesicles (pink arrow)) And deposition in the tumor stroma (blue arrows). High magnification images of regions "1" - "3" are provided in the right image. E ═ endothelial cells; l ═ lumen; p ═ pericytes; r ═ red blood cells. FIG. 25C: TEM images showing the presence of silica bodies in the perinuclear distribution inside cancer cells. N ═ core; m ═ mitochondria.

Figures 26A-26B are graphs illustrating iRGD-induced silica biodistribution in patient-derived xenografts in NSG mice. FIG. 26A: a pair of tumors (XWR #8 and XWR #187) with matching stromal abundance, but with different NRP-1 expression levels, were selected for biodistribution studies in the absence and presence of iRGD co-administration. Masson's trichrome staining showed equal levels of collagen expression in both tumors. Multicolor IHC staining (green fluorescent antibody to NRP-1, red fluorescent antibody to CD31) was used to determine the relative abundance of NRP-1 expression and the degree of overlap with endothelial cells using image J software. Data represent mean ± SD,. p < 0.05. FIG. 26B: tumor-bearing animals received an intravenous injection of 50mg/kg NIR-labeled silica bodies, with or without co-administration of 8. mu. mol/kg iRGD. Animals were sacrificed after 24 hours (n-3). Ex vivo assessment of uptake of silica bodies as determined by NIR fluorescence intensity and Si content. Data represent mean ± SD,. p < 0.05.

Fig. 27, panels a-C, illustrate successful covalent conjugation of iRGD to a lipid bilayer on a silica body using a thiol-maleimide reaction. To verify the success of covalent conjugation, a reactive Fluorescein (FAM) -labeled iRGD peptide supplied by doctor Ruoslahti (Sugahara et al (2009) Cancer Cell,16:510-520) was used in conjunction with DSPE-PEG-maleimide. The detailed procedure is described in the procedure section of example 3. FIG. A: fluorescence spectra of the original silica and FAM-iRGD-silica suspended at 100. mu.g/mL were obtained in a microplate reader (molecular sieve M5e) at an excitation wavelength of 488nm, and significant retention of the fluorescence signal on the washed and purified FAM-iRGD-silica confirmed successful conjugation reactions (Cancer Cell 2009,16: 510). FIGS. B and C: the effect of iRGD conjugation on silica body in terms of its effect on cellular uptake was tested. A batch of silica-iRGD was synthesized and the MSNP framework was NIR tagged (DyLight 680) as described in the methods section. KPC cells were treated at 37 ℃ for 2 hours at a particle concentration of 100 μ g/mL and uptake was determined by flow cytometry (panel B) and confocal microscopy (panel C). NIR-silica bodies without iRGD with similar labeling efficiency were used as controls. Data represent mean ± SD,. p < 0.05. Confocal microscopy confirmed the results (cell membrane stained green with wheat germ lectin and nucleus stained blue with Hoechst 33342). Scale bar 20 μm.

Figure 28, panels a-B, illustrate the evaluation of the effect of non-functional peptides (lacking the CendR motif) on intravenous silica body biodistribution. FIG. A: we repeated the biodistribution experiments performed in the in situ model shown in figure 22 using non-CendR peptide cyclic (RGDfK). Briefly, tumor-bearing animals received an intravenous injection of 50mg/kg NIR-labeled silica co-administered with PBS or 8 μmol/kg free cyclic (RGDfK), followed by sacrifice of the animals at 24 hours (n ═ 3). Representative ex vivo organ NIR fluorescence images were obtained to show nanoparticle biodistribution. And B: NIR fluorescence intensity analysis (by IVIS software) and determination of Si content (by ICP-OES) showed that non-CendR peptides did not increase the uptake of silica bodies at the tumor site. Data represent mean ± SD.

Fig. 29, panels a-B, illustrate assessment of silica body transport in tumors by confocal microscopy. FIG. A: tumor sections were obtained from the experiment in fig. 22. Representative confocal images were obtained using a 633nm laser under a SP2-1P-FCS Leica microscope to assess the intratumoral abundance of NIR labeled silica body abundance. IHC staining of the same sections to check for the presence of blood vessels (green CD31) and nuclei (blue DAPI staining) allowed us to determine the presence and migration of nanoparticles in tumor tissue using the method described by Sugahara et al (2010) Science,328: 1031-1035. Scale bar 20 μm. And B: the calculation of the penetration distance of the silica body from the nearest tumor vessel was estimated for about 15 vessels using Image J software. Box and whisker plots (Origin software) were developed to show median (horizontal line), 25 th-75 th percentile (box), mean (open square), and SD (whisker). P <0.05 compared to the silica body alone or silica body-iRGD.

Fig. 30 shows determination of Si content in liver (left side) and spleen (right side) by ICP-OES analysis using organs obtained from the animals used in fig. 22. Data represent mean ± SD, p <0.05 compared to the silica body group or "silica body + iRGD" group.

Fig. 31 shows representative TEM images of KPC-derived tumors from animals that received a 50mg/kg Au-embedded silica injection before (24 hours) without iRGD co-administration. The images show the absence of transcytosis vesicles, with particles deposited on the abluminal side. E ═ endothelial cells, L ═ lumens.

Fig. 32 illustrates the results of a comparison between the protocell (protocell) technique and the silica body technique.

Detailed Description

To address the high toxicity rate while maintaining or improving therapeutic efficacy, drug delivery nanocarriers are provided to enhance drug delivery (e.g., chemotherapeutic drugs, antimicrobial drugs, etc.) at target sites (e.g., tumor sites, infection sites, etc.) while limiting the amount of free drug that can cause systemic toxicity. It is desirable that the carrier itself maintain a stable drug load to prevent toxicity and improve efficacy. The use of nanocarriers to deliver chemotherapeutic agents in animal tumor models established from various Cancer cells has demonstrated the ability of such nanocarriers to extend the circulating half-life of the drug, deliver high drug concentrations to the tumor site with increased cytotoxic killing, and reduce systemic toxicity compared to the free drug equivalent (see, e.g., Messerer et al (2004) Clin. Cancer Res.10(19): 6638-6649; Drummond et al (2006) Cancer Res.66(6): 3271-3277; Ramsay et al (2008) Clin. Cancer Res.14(4): 1208-1217).

Given that many liposome carriers fail to improve the safety of highly toxic drugs, such as irinotecan, provided herein are novel nanoparticle drug carriers that use Lipid Bilayers (LB) applied to silica nanoparticles, such as Mesoporous Silica Nanoparticles (MSNPs), providing supported lipid bilayers. These lipid bilayer coated porous silica nanoparticles (also referred to as silica bodies) provide drug carriers with significantly less leakage than liposomes (Meng et al (2013) ACS Nano,7(2): 994-1005; Meng et al (2011) ACS Nano,5(5): 4131-4144; Meng et al (2013) ACS Nano,7(11): 10048-10065; Meng et al (2015) ACS Nano,9(4): 3540-3557). Due to the large internal surface area, tunable pore size, carrier stability and controlled drug release capacity that can be used for drug packaging, MSNP has been demonstrated to constitute a versatile and multifunctional nanocarrier platform for cancer treatment (Meng et al (2013) ACS Nano,7(2): 994-1005; Meng et al (2011) ACS Nano,5(5): 4131-4144; Meng et al (2013) Nano,7(11): 10048-10065; Meng et al (2015) ACS Nano,9(4): 3540-3557; Meng et al (2010) j.am.chem.soc.132(36): 12690-12697; Li et al (2012) chem.soc.rev.41(7): 2590-2605; Tang et al (2012) adv.mat.24(12): chem-1504; Tarn et al (3) res.2013): 2013-792-801). However, the methods of payload lipid bilayer coated silica particles are limited, typically providing a loading in the range of about 10 to 40 wt%.

In various embodiments, provided herein are novel loading methods that achieve significantly greater loading of lipid bilayer coated nanoparticle drug carriers. Generally, the new loading methods utilize a cargo trapping agent to retain a cargo (e.g., one or more drugs of interest) within the pores of the nanoparticle inside the lipid bilayer. More specifically, in certain embodiments, the method may comprise:

providing a nanocarrier comprising a porous silica body comprising a plurality of pores capable of holding a cargo (e.g., a drug of interest);

disposing a collector (cargo collector) within the plurality of pores, wherein the collector is selected for its ability to collect the cargo within the pores;

coating the surface pores of the nanocarriers with a lipid bilayer; and introducing the cargo into the pores coated by the lipid bilayer, wherein the cargo reacts with the trapping agent and remains within the pores.

In certain embodiments, the trapping agent comprises a protonating agent and the method comprises providing one or more drugs that can pass through a lipid bilayer into the bilayer-coated porous nanoparticle. The protonating agent in the porous nanoparticle converts the drug into a hydrophilic derivative that is unable to back-diffuse through the lipid bilayer.

In an illustrative, but non-limiting embodiment shown in example 1, the MSNP core is synthesized by sol-gel chemistry and soaked in a solution containing the protonating agent TEA₈SOS solution. These particles were coated with LB in the presence of lipid biofilm with an optimal composition for bilayer stability using sonication procedure. After that, by TEA₈The proton gradient provided by SOS allows for remote irinotecan loading. Irinotecan is a weakly basic and amphiphilic molecule that can diffuse through LB into the MSNP's internal packaging space, with the previously encapsulated triethylammonium sucrose octasulfate (TEA)₈SOS) converts the drug into a hydrophilic derivative that cannot diffuse back through the LB. Using this method, high loading capacity (e.g., greater than 40 wt.%, or greater than 45 wt.%, or greater than 50 wt.%, or greater than 60 wt.%, or greater than 70 wt.%, or greater than 80 wt.%, etc.) is achieved.

Lipid bilayer nanoparticle drug carriers (LB-MSNPs or "silica bodies") produced using the methods described herein offer a number of advantages. The existing biomembrane package for synthesizing LB-MSNPIs prepared from (A) a mixture of (B) and (B) a metal oxide and (C) a metal oxide such as TEA₈The protonating agent combination of SOS provides active and high dose loading of drugs such as irinotecan in a particulate carrier that exhibits many advantages over liposomes. These advantages include, but are not limited to, ease of synthesis, improved drug loading and release characteristics, increased stability, and improved biodistribution. In certain illustrative, but non-limiting embodiments, this remote loading approach increases the loading capacity of irinotecan by a factor of 4 or more, as compared to passive drug encapsulation followed by LB in MSNP. Using the methods described herein, unencapsulated irinotecan enters the MSNP pores by diffusing through the LB, whereupon protonation causes irinotecan to become hydrophilic and unable to escape.

Furthermore, the increased loading capacity of the nanocarriers allows for increased drug delivery at cancer sites, with less free drug available at sites such as bone marrow and gastrointestinal tract (GIT) where irinotecan would cause toxicity. In one example, LB-MSNP has an increased loading capacity (83.5 wt% irinotecan loading) compared to the internal liposome equivalent of MM-398 (42.5 wt% loading capacity). Embodiments of the LB-MSNP formulation have shown a 3-fold to 5-fold release capacity at acidic pH (4.5) than the liposomal formulation after optimization of loading time, trapping agent concentration, and the amount of irinotecan provided for loading. Concomitant dose adjustment allows smaller drug doses to be administered to achieve the same efficacy, resulting in further reduction of systemic drug toxicity. In one example, embodiments of LB-MSNP show a 5-fold increase in tolerated dose to free drug when calculating the Maximum Tolerated Dose (MTD). The increased MTD is similar to the liposome formulation. Histological analysis of bone marrow showed that irinotecan toxicity was greatly reduced by LB-MSNP.

In addition, the synthesis/loading methods described herein are easier to perform than the polymer-lipid techniques used to prepare conventional liposomes or by Zhang et al (2104) Biomaterials,35(11): 3560-3665. This is advantageous for scaling up to GMP manufacturing, including cost savings by reducing the amount of unencapsulated drug.

Although the method and the resulting nanoparticulate drug carrier (silica body) are described herein with respect to irinotecan, it will be appreciated that the method can be applied to many other payloads (e.g., payloads comprising one or more drugs), as explained below. Furthermore, in view of the teachings provided herein, it is believed that in the case of many other drugs, an increase in drug delivery (on the target), and/or a decrease in toxicity, and/or an improvement in biodistribution, and/or an improvement in release characteristics will be achieved using the methods and compositions described herein.

Thus, in certain embodiments, LB-MSNP based nanocarrier delivery systems are described that allow for stable and protected cargo loading at high loading levels with the help of cargo collectors. In some embodiments, the cargo may be a drug. Drug delivery through improved LB-MSNP encapsulation and entrapment allows for more frequent use of drug encapsulation, such as chemotherapeutic drugs, due to enhanced efficacy, high drug loading capacity, and reduced systemic toxicity. From the standpoint of nanocarrier design, various embodiments of such systems, such as polyanionic cargo traps within LB-MSNP, are used as an effective design principle for delivering a variety of additional weakly basic molecules and drugs to treat several different types of cancer and other disease processes.

In one example, for irinotecan, delivery by the improved LB-MSNP encapsulation and trapping procedure allowed irinotecan and FOLFIRINOX to be used more frequently in human PDAC patients due to enhanced efficacy, high drug loading capacity, and reduced systemic toxicity of irinotecan. This also allows more patients with PDAC to receive treatment with a more potent treatment regimen than gemcitabine, with the hope of improving survival. Finally, an intravenous injectable, efficacious, biocompatible, and transformingly competitive formulation of irinotecan (compared to MM398 liposomes) for PDAC treatment is provided. Particularly in view of the methods described herein and the resulting nanoparticle drug carriers, irinotecan and FOLFIRINOX treatments may also be useful for treating other cancers, including but not limited to colon, rectal, lung, and ovarian cancers.

The silica body delivery system disclosed herein is a multifunctional platform. Mesoporous silica drug carriers have been demonstrated to be able to deliver a wide range of cargo to cancer cells as well as to a variety of human cancer models in animals. These include co-delivery of gemcitabine and paclitaxel to mouse xenografts and orthotopic PDAC tumors. Furthermore, MSNP is biodegradable and proved safe in extensive animal testing (Meng et al (2013) ACS Nano,7(2): 994-1005; Meng et al (2011) ACS Nano,5(5): 4131-4144; Meng et al (2013) ACS Nano,7(11): 10048-10065; Meng et al (2015) ACS Nano,9(4): 3540-3557; Tang et al (2012) adv.mat.24(12): 1504-1534; tar et al (2013) acc.chem.res.46(3): 792-801; Zhang et al (2012) j.am.m.soc.134 (38): 15790-15804; Lu et al (2010, 6(16): 1794-1805).

In view of the enhanced loading, resulting pharmacokinetic profiles, etc., described herein, it is believed that the loading methods, lipid bilayer compositions, and stability of the resulting nanoparticle drug carriers described herein provide significant improvements and advantages over previous nanoparticle drug carriers.

Method for loading lipid bilayer coated porous nanoparticles

In various embodiments, improved methods of loading lipid bilayer coated porous nanoparticles (e.g., mesoporous silica particles) and drug delivery nanoparticles produced by such methods are provided. In certain embodiments, the methods described herein achieve very high levels of drug loading, e.g., greater than 40 wt.%, or greater than 45 wt.%, or greater than 50 wt.%, or greater than 60 wt.%, or greater than 70 wt.%, or greater than 80 wt.%, etc.

As described above, in certain embodiments, the method may comprise:

providing a nanocarrier comprising a porous silica body comprising a plurality of pores capable of holding a cargo (e.g., a drug of interest);

disposing a collector (cargo collector) within the plurality of pores, wherein the collector is selected for its ability to collect the cargo within the pores;

coating the surface pores of the nanocarriers with a lipid bilayer; and

introducing the cargo into the pores coated by the lipid bilayer, wherein the cargo reacts with the collector and remains within the pores.

Although the polyanionic Compound TEA₈SOS has never been used previously in the MSNP platform, but it was used in MM-398 to encapsulate irinotecan (Drummond et al (2006) Cancer Res.,66(6): 3271-3277). However, before the studies described herein, it was not clear that TEA was present₈Whether SOS can be used to efficiently perform remote loading of porous nanoparticles at significantly higher levels than other loading or immersion methods.

As described in the examples, TEA₈SOS is a proton generator that releases eight H + ions and an octavalent SOS upon hydrolysis^8-(FIG. 1A, FIG. A2). Production of TEA Using ion exchange chromatography₈SOS, immersed in MSNP as described below in the methods section (supra). The soaked particles were introduced into a round bottom flask coated with a lipid biofilm composed of DSPC/cholesterol/DSPE-PEG 2000 at a molar ratio of 3:2: 0.15. Sonication of the suspension resulted in LB-coated particles containing the encapsulant (fig. 1A, panel a 1). The particles were immediately incubated in irinotecan solution, allowing the drug to be introduced, protonated, and reacted with SOS^8-When associated, are encapsulated in the pores as gelatinous precipitates (fig. 1A, panels a1 and a2, fig. 1B). This allows us to achieve irinotecan loading capacities of up to 80% by weight or more (fig. 1B), which is approximately 850m²(ii) combined surface of/gProduct and sum of about 0.7cm³The theoretical maximum loading capacity of the porous support per gram of pore volume (about 100% by weight) was as previously demonstrated (Meng et al (2015) ACS Nano,9(4): 3540-3557).

While the method is described with respect to the use of the drug irinotecan and TEA8SOS as a trapping agent, it will be appreciated that where proof of principle has been demonstrated, many other drugs (particularly weakly basic drugs) can be incorporated into the silica body using the same method and many other trapping agents can be similarly utilized, for example as described herein.

Since drug loading capacity is an important manufacturing consideration, which is also critical to the limitations of therapeutic effectiveness and drug toxicity, much research has been devoted to optimizing the efficacy of drug encapsulation of MSNPs, leading to increased loading capacity and the ability to co-deliver synergistic drug combinations. While it is possible to introduce drugs such as irinotecan into MSNPs by traditional soaking methods, this loading method has proven to be relatively inefficient, achieving a loading level of about 10 wt% (see, e.g., He et al (2010) Biomaterials,31(12):3335-3346), and difficulty retaining the drug in the pores (see, e.g., Meng et al (2010) j.am. chem. soc.132(36): 12690-12697). Unlike traditional drug loading methods that rely on physical adsorption, electrostatic attachment, supramolecular assembly, or the use of encapsulation methods (e.g., plugs, nanomachines, or barrier entities), the use of an intact lipid bilayer coating disposed on a porous nanoparticle provides rapid and transient pore sealing for drug encapsulation. This simplifies synthesis and provides stable and high drug loading capacity. The methods described herein effectively load porous particles with high levels of cargo and are compatible with this rapid and transient pore sealing with lipid bilayers.

Biofilm technology has been developed for LB-coated MSNP platforms that can be used to rapidly encapsulate Gemcitabine (GEM) (e.g. water soluble nucleosides) via supported LB (Meng et al (2015) ACS Nano,9(4): 3540-3557). This not only allows LB-MSNP to achieve a loading capacity of up to 40 wt.% GEM, but also enables co-delivery of hydrophobic Paclitaxel (PTX) (Meng et al (2015) ACS Nano,9(4):3540-3557) that can be incorporated into the Lipid Bilayer (LB). This has provided a vehicle for synergistic and ratiometric design of PDAC treatment in the orthotopic human PDAC model of mice (supra).

This supported LB coating method has also proven to be far superior to similar liposome coating techniques for MSNP, which include several steps, including adhesion of the liposomes to the MSNP surface, disruption, partial coverage of the MSNP surface by an incomplete coating that is secondarily sealed by the addition of an additional liposome composition (Liu et al (2009) j.am.chem.soc.131: 7567-7569).

However, while the presence of the Lipid Bilayer (LB) provides an improvement over previous drug delivery nanoparticles, the bilayer still poses an obstacle to achieving even higher drug loading levels prior to utilizing the remote loading methods described herein.

As described above, in various embodiments, drug loading of the LB-MSNP platform has been further improved by using LB encapsulation of protonating agents as a first step followed by irinotecan loading, depending on the weakly basic nature of the drug (pKa of 8.1). As shown in the examples herein, a comparison was made between MSNP vectors and liposome equivalents in which triethylammonium sucrose octasulfate (TEA) was encapsulated₈SOS), an irinotecan load was performed using unsupported LB (Drummond et al (2006) Cancer res, 66(6): 3271-3277; von Hoff et al (2013) br.j. cancer,109(4): 920-925). The MSNP vector not only achieved higher irinotecan loading capacity and tumor killing than the liposome formulation in a robust in situ PDAC model, it also prevented drug toxicity due to increased vector stability and reduced leakage compared to liposomes. Thus, the LB-MSNP platform exhibits desirable properties for a first-line irinotecan vector for PDAC (or other cancer) treatment.

Recently, the polymer-lipid supported coating approach has been described by Zhang et al for irinotecan drug loading of MSNP to treat drug resistant breast cancer tumors in Balb/c nude mice (Zhang et al (2014) Biomaterials,35(11): 3650-3665). However, the methods described in this reference are different and do not achieve the same drug loading capacity as the methods and compositions described herein. First, Zhang et al did not use typical LB or trapping agents, resulting in a vector with a loading capacity of only 1/5 (about 15%, w/w) of the vector provided herein (about 83.5%, w/w).

The second major difference is in the drug loading procedure. The authors state that "CPT-11 @ MSN" was centrifuged and washed in PBS (pH 7.4) and then dried under vacuum at room temperature. In contrast, the methods described herein do not require drying of the unprotected MSNP. Drying MSNP can be problematic because it is difficult to obtain a dispersion when resuspended in an aqueous medium, which is a critical requirement for systemic drug delivery. In addition, the method of the present invention does not require washing of the drug-loaded (e.g., irinotecan) MSNP prior to pore sealing, which minimizes drug loss and facilitates high loading capacity (e.g., about 83.5% (w/w)).

The third major difference is the composition of the LB formulation. In one or more embodiments, a commercially available lipid plus cholesterol mixture (e.g., DSPC/cholesterol/DSPE-PEG) is used, while Zhang et al uses internally synthesized pH sensitive Pluronic P123 grafted DOPE. The use of Pluronic is apparently based on its ability to act as a drug efflux inhibitor (see, e.g., Batrakova et al (2004) pharm. res.21(12):2226-2233) and is therefore based on consideration of the treatment of drug resistant breast cancer. The absence of cholesterol in the coated bilayer reduces the fluidity and stability of the platform. In contrast, the presence of cholesterol in the silica bodies contributes to the superior stability of the LB-MSNP disclosed herein (see examples). Further, encapsulation of irinotecan in the provided vehicles showed < 5% leakage within 24 hours at 37 ℃ in biological buffer with pH of 7.4. This is 2.5 x lower than the premature leakage (about 16% leakage over 24 hours at pH 7.4 and 37 ℃) described by Zhang et al (2014) Biomaterials,35: 3650-3665.

Another difference from the drug delivery platform described by Zhang et al is that various embodiments of the present invention use probe sonication for biofilm rehydration and pore sealing followed by centrifugal purification or size exclusion chromatography. On the other hand, Zhang et al use membrane extrusion as for liposomes. In addition, CTAC was used as a template for MSNP synthesis, whereas Zhang et al utilized CTAB. Finally, the present invention addresses the development of therapies for PDAC and other cancers, and is not primarily designed to overcome drug resistance, as do the drug delivery particles described by Zhang et al for breast cancer studies. Taken together, and without being bound by a particular theory, it is believed that the lipid bilayer coated nanoparticle drug carriers described herein (e.g., LB-MSNP delivering irinotecan) provide a unique design over Zhang et al based on drug loading capacity, colloidal stability, ease of manufacture, and stable drug retention in blood and body fluids.

Nanoparticles

In various embodiments, the nanoparticle drug carriers described herein comprise a porous silica nanoparticle (e.g., a silica body having a surface and defining a plurality of pores suitable for containing molecules therein) coated with a lipid bilayer. For example, in certain embodiments, the silica nanoparticles may be mesoporous silica nanoparticles. The fact that the nanoparticles are referred to as silica nanoparticles does not exclude that materials other than silica are also incorporated within the silica nanoparticles. In some embodiments, the silica nanoparticles may be substantially spherical, having a plurality of pore openings through the surface, providing access to the pores. However, in various embodiments, the silica nanoparticles may have a shape other than substantially spherical. Thus, for example, in certain embodiments, the silica nanoparticles may be substantially ovoid, rod-shaped, substantially regular polygonal, irregular polygonal, and the like.

Generally, the silica nanoparticles include a silica body defining an outer surface between pore openings and sidewalls within the pores. The aperture may extend through the silica body to another aperture opening, or the aperture may extend only partially through the silica body such that it has a bottom surface defined by the silica body.

In some embodiments, the silica body is mesoporous. In other embodiments, the silica body is microporous. As used herein, "mesoporous" means having pores with diameters of about 2nm to about 50nm, while "microporous" means having pores with diameters of less than about 2 nm. In general, the pores may be of any size, but in typical embodiments are large enough to contain one or more therapeutic compounds therein. In such embodiments, the pores allow small molecules, e.g., therapeutic compounds, such as anticancer compounds, to adhere or bind to the inner surfaces of the pores and, when used for therapeutic purposes, to be released from the silica body. In some embodiments, the pores are substantially cylindrical.

In certain embodiments, the nanoparticle comprises pores having a pore size of about 1nm to about 10nm or about 2nm to about 8nm in diameter. In certain embodiments, the nanoparticle comprises pores having a pore size of about 1nm to about 6nm, or about 2nm to about 5 nm. Other embodiments include particles having a pore size of less than 2.5 nm. In other embodiments, the pore size is 1.5nm to 2.5 nm. Silica nanoparticles having other pore sizes can be prepared, for example, by using different surfactants or swelling agents during the preparation of the silica nanoparticles.

In various embodiments, the nanoparticles may include particles up to about 1000nm (e.g., average diameter or median diameter or other characteristic dimension). However, in various embodiments, the nanoparticles are typically less than 500nm or less than about 300nm, as in general, particles greater than 300nm may be less effective in entering living cells or vascular fenestrations. In certain embodiments, the size of the nanoparticles is in the range from about 40nm, or from about 50nm, or from about 60nm to about 100nm, or to about 90nm, or to about 80nm, or to about 70 nm. In certain embodiments, the nanoparticles have a size in the range of about 60nm to about 70 nm. Some embodiments include nanoparticles having an average largest dimension of about 50nm to about 1000 nm. Other embodiments include nanoparticles having an average largest dimension of about 50nm to about 500 nm. Other embodiments include nanoparticles having an average largest dimension of about 50nm to about 200 nm. In some embodiments, the average largest dimension is greater than about 20nm, greater than about 30nm, greater than 40nm, or greater than about 50 nm. Other embodiments include nanoparticles having an average largest dimension of less than about 500nm, less than about 300nm, less than about 200nm, less than about 100nm, or less than about 75 nm. The size of the nanoparticles as used herein refers to the average or median size of the primary particles as measured by Transmission Electron Microscopy (TEM) or similar visualization techniques.

Illustrative mesoporous silica nanoparticles include, but are not limited to, MCM-41, MCM-48, and SBA-15 (see, e.g., Katiyar et al (2006) J. chromatography.1122 (1-2): 13-20).

Methods for preparing porous silica nanoparticles are well known to those skilled in the art. In certain embodiments, mesoporous silica nanoparticles are synthesized by reacting Tetraethylorthosilicate (TEOS) with a template made from micellar rods. The result is a collection of nano-sized spheres or rods, which are filled with a regular arrangement of pores. The template can then be removed by washing with a solvent adjusted to an appropriate pH (see, e.g., Trewyn et al (2007) chem. eng.j.137(1): 23-29). In certain embodiments, Mesoporous particles may also be synthesized using simple sol-gel methods (see, e.g., Nandiiyanto et al, (2009) Microporous and Mesoporous Mat.120(3):447-453, etc.). In certain embodiments, tetraethyl orthosilicate may also be used with additional polymer monomers (as a template). In certain embodiments, (3-mercaptopropyl) trimethoxysilane (MPTMS) is used in place of TEOS.

In certain embodiments, the mesoporous silica nanoparticles are cores synthesized by a modification of the sol/gel procedure described by Meng et al (2015) ACSNano,9(4): 3540-3557. To synthesize a batch of about 500mg MSNP, 50mL of CTAC and 150mL of H were combined₂O is mixed in a flask (e.g., 500mL Erlenmeyer flask) followed by stirring (e.g., at 350rpm at 85 ℃ for 15 minutes). After this, 8mL of 10% triethanolamine was added at the same temperature for 30 minutes. Then, 7.5mL of silica precursor TEOS was added dropwise at a rate of 1mL/min using a peristaltic pump. The solution was stirred at 350rpm for 20 minutes at 85 ℃ to cause the formation of particles having a major dimension of about 65 nm. The surfactant can be removed by washing the particles with a mixture of methanol/HCl (500:19v/v) for 24 hours at room temperature. The particles can be centrifuged at 10000 rpm for 60 minutes and washed three times in methanol.

While the loading methods described herein have been described with respect to the loading of porous silica nanoparticles (e.g., mesoporous silica), it will be appreciated that similar loading methods may be used with other porous nanoparticles. Many other mesoporous materials that can be used in drug delivery nanoparticles are known to those skilled in the art. For example, in certain embodiments, mesoporous carbon nanoparticles may be utilized. Mesoporous Carbon nanoparticles are well known to those skilled in the art (see, e.g., Huang et al (2016) Carbon,101: 135-142; Zhu et al (2014) Asian J. pharm. Sci.,9(2): 82-91; etc.).

Similarly, in certain embodiments, mesoporous polymeric particles may be utilized. The synthesis of highly ordered mesoporous polymers and carbon frameworks from the organic-organic assembly of triblock copolymers with soluble low molecular weight phenolic resin precursors (resoles) by an evaporation-induced self-assembly strategy has been reported by Meng et al (2006) chem.Mat.6(18):4447-4464 and references cited therein.

The nanoparticles described herein are illustrative and not limiting. Many other lipid bilayer drug delivery nanoparticles will be available to those skilled in the art using the teachings provided herein.

A lipid bilayer.

The drug carrier nanoparticles described herein comprise porous nanoparticles (e.g., Mesoporous Silica Nanoparticles (MSNPs)) coated with a lipid bilayer. In certain embodiments, the bilayer composition is optimized to provide rapid and uniform particle coating, to provide colloidal and cyclic stability, and to provide effective cargo retention, while also allowing for desired cargo release characteristics.

In certain embodiments, the lipid bilayer comprises a combination of: phospholipids, cholesterol and, in certain embodiments, pegylated lipids (e.g., DSPE-PEG)₂₀₀₀) Or functionalized pegylated lipids (e.g. DSPE-PEG)₂₀₀₀Maleimide) to facilitate conjugation to a targeting or other moiety.

To attach surface LB coatings, a coated lipid membrane program was developed in which drugs or TEO were incorporated₈The SOS soaked MSNP suspension is added to the surface of a large lipid film coated on, for example, a round bottom flask. Using different lipid bilayer compositions, a series of experiments can be performed to find compositions and optimal lipid/particle ratios that provide rapid and uniform particle encapsulation, coating, and effective cargo retention and/or release upon sonication. It is believed that this lipid composition and encapsulation cannot be achieved by liposome fusion to the particle surface under low energy vortex conditions.

As described in example 1, in certain embodiments, 500mg of MSNP is soaked in 20mL of TEA₈SOS (80mM solution), added on top of a lipid biofilm comprising DSPC/Chol/DSPE-PEG₂₀₀₀550mg of the mixture (molar ratio 3:2:0.15) was coated onto the bottom of a round bottom flask (see example 1 and Liu et al (2016) ACS Nano.10(2): 2702-2715). If mol% is used to represent the ratio, then a ratio of "3: 2: 0.15" is equal to58.3 mol%, 38.8 mol%, 3.9 mol%. This provides a lipid to particle ratio of about 1.1: 1. After sonication to complete the coating and encapsulation of the particles by LB, free TEA was removed by size exclusion chromatography on an agarose gel CL-4B column₈And (4) SOS. The TEA-Supported solution was heated in a water bath at 65 deg.C₈The silica bodies of SOS were incubated in a 10mg/mL solution of irinotecan for drug loading. After 30 minutes the loading was stopped by quenching in an ice-water bath, after which the drug-loaded silica bodies were washed 3 times by centrifugation and resuspended in PBS.

The lipid bilayer formulations described above and in example 1 are illustrative and not limiting. In various embodiments, depending on the drug or drugs loaded into the silica body and the desired release provided, different lipid bilayer formulations may be used and the optimal formulation may be determined.

Thus, in certain embodiments, the lipid bilayer may comprise: 1) one or more saturated fatty acids having a carbon chain of C14-C20, such as Dimyristoylphosphatidylcholine (DMPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC) and Diacylphosphatidylcholine (DAPC); and/or 2) one or more unsaturated fatty acids having a carbon chain of from C14 to C20, such as 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-di-eicosenoyl-sn-glycero-3-phosphocholine; and/or 3) natural lipids comprising a mixture of fatty acids having carbon chains of C12-C20, such as egg PC as well as soy PC, sphingomyelin, and the like. These lipids are illustrative and not limiting and many other lipids are known and can be incorporated into lipid bilayers to form the silicasomes.

In certain embodiments, the silica body contains a lipid (e.g., a phospholipid), cholesterol, and a PEG-functionalized lipid (e.g., mPEG phospholipid). In certain embodiments, the mPEG phospholipids comprise C14-C18 phospholipid carbon chains and PEG molecular weights of 350-5000 (e.g., mPEG 5000, mPEG 3000, mPEG 2000, mPEG 1000, mPEG 750, mPEG 550, mPEG350, etc.). In certain embodiments, the mPEG phospholipid comprises DSPE-PEG5000, DSPE-PEG3000, DSPE-PEG2000, DSPE-PEG1000, DSPE-PEG750, DSPE-PEG550, or DSPE-PEG 350. MPEG is commercially available (see, e.g.,/avantiilipids. com/product-category/products/polymers-polymerizable-lipids/MPEG-phospholipids /).

In certain embodiments, the ratio of phospholipid: CHOL: PEG is about phospholipid (50 mol% -90 mol%), CHOL (10 mol% -50 mol%), PEG (1 mol% -10 mol%).

Such schemes provided above and in the examples are illustrative. In certain embodiments, the trapping agent can be varied, the lipid composition and molar ratio can be varied, and the drug or drugs can be varied to identify additional silica bodies optimized for one or more particular cargo.

It should be noted that, for example, an effective lipid formulation for gemcitabine-containing silica bodies contains DPPC/cholesterol/DSPE-PEG in a molar ratio of 77.5:20:2.5, while an effective lipid formulation for irinotecan-containing silica bodies contains DSPC/Chol/DSPE-PEG₂₀₀₀(molar ratio: 3:2:0.15, which equals 58.3 mol%: 38.8 mol%: 3.9 mol%).

In certain embodiments, these methods can be modified to increase drug loading capacity (weight of drug/total weight of carrier). In certain embodiments, the drug loading capacity is at least about 30% w/w, or at least about 40% w/w, at least about 50% w/w, at least about 60% w/w, at least about 70% w/w, or at least 80% w/w. In certain embodiments, the drug loading is greater than 40% w/w, or greater than 45% w/w, or greater than 50% w/w, or greater than 60% w/w, or greater than 70% w/w, or greater than 80% w/w.

The protocol described herein provides nanoparticle drug carriers (nanocarriers) that are superior to nanocarriers prepared by liposome-coating methods fused to the surface of MSNPs, as illustrated by the ease of synthesis and the description of improved loading capacity and release characteristics provided herein. In typical embodiments, the protonating agent is rapidly encapsulated using an LB coating program, such as TEA₈SOS, which subsequently provides irinotecan loading and encapsulation by protonating the incoming drug diffusing through the LB. This causes a high drug loading in the pores of the particles. Rapid and effective pore sealing to retain trapping agent (e.g., TEA)₈SOS) without leakage promotes carrier effectiveness and stability.

The cargo capture reagent.

The cargo capture reagent may be selected to interact with a desired cargo. In some embodiments, the interaction may be an ionic reaction or a protonation reaction, although other interaction means are contemplated. The cargo traps may have one or more ion sites, i.e. may be mono-ionic or multi-ionic. The ionic portion may be cationic, anionic, or in some cases, the cargo trapping agent may comprise a cationic portion and an anionic portion. The ionic sites may be in equilibrium with the corresponding uncharged forms; for example, anionic carboxylates (-COO)^-) Can equilibrate with its corresponding carboxylic acid (-COOH); or in another example, an amine (-NH)₂) May be associated with its corresponding protonated ammonium form (-NH)₃ ⁺) And (4) balancing. These balances are affected by the pH of the local environment.

Also, in certain embodiments, the cargo may include one or more ion sites. The cargo collectors and cargo may be selected to interact within the nanoparticles (e.g., mesoporous silica nanoparticles). This interaction may help to retain the cargo within the nanoparticles until release of the cargo is desired. In some embodiments, the cargo may exist in a pH-dependent balance between non-ionic and ionic forms. The nonionic form can diffuse through the lipid bilayer and into the pores of the MSNP. There, a cargo trapping agent (e.g., a multi-ion cargo trapping agent) may interact with the ionic form of the cargo and thereby retain the cargo within the nanocarrier, for exampleSuch as within the pores of MSNP (provided that the ionic form of the cargo and the cargo trap have opposite charges). The interaction may be an ionic interaction, and may include formation of a precipitate. Trapping the cargo within the nanocarrier may provide a higher level of cargo loading than similar systems, such as nanocarriers that omit cargo trapping agents, or liposomes that include trapping agents. Release of the cargo may be achieved by appropriate changes in pH to disrupt the interaction between the cargo and the cargo collector, for example by returning the cargo to its non-ionic state which may diffuse more readily through the lipid bilayer. In one embodiment, the cargo is irinotecan and the cargo trap is TEA₈SOS。

The cargo catchers need not be limited to TEA8 SOS. In certain embodiments, the cargo trapping agent comprises a small molecule, such as (NH)₄)₂SO₄other collectors include, but are not limited to, ammonium salts (e.g., ammonium sulfate, sucrose octasulfate, α -cyclodextrin ammonium sulfate, β -cyclodextrin ammonium sulfate, gamma-cyclodextrin ammonium sulfate, ammonium phosphate, β 0-cyclodextrin ammonium phosphate, β 1-cyclodextrin ammonium phosphate, gamma-cyclodextrin ammonium phosphate, ammonium citrate, ammonium acetate, etc.), trimethylammonium salts (e.g., trimethylammonium sulfate, sucrose octasulfate trimethylammonium, α -cyclodextrin trimethylammonium sulfate, β -cyclodextrin trimethylammonium sulfate, gamma-cyclodextrin trimethylammonium sulfate, trimethylammonium phosphate, α -cyclodextrin trimethylammonium phosphate, β -cyclodextrin trimethylammonium phosphate trimethylammonium, gamma-cyclodextrin trimethylammonium phosphate, trimethylammonium citrate, trimethylammonium acetate, etc.), triethylammonium salts (e.g., triethylammonium sulfate, sucrose octatriethylammonium sulfate, α -cyclodextrin triethylammonium sulfate, β -cyclodextrin triethylammonium, gamma-cyclodextrin triethylammonium sulfate, triethylammonium phosphate, α -cyclodextrin triethylammonium phosphate, β -cyclodextrin triethylammonium phosphate, gamma-cyclodextrin triethylammonium phosphate, triethylammonium acetate, etc.).

Except that TEA₈Besides SOS, also deservesIt is noted that transmembrane pH gradients can also be generated by: acidic buffers (e.g., citrate) (Chou et al (2003) J.biosci.bioengineerer, 95(4): 405-408; Nichols et al (1976) Biochimica et Biophysica Acta (BBA) -biomebranes, 455(1): 269-271); dissociable salts that produce protons (e.g., (NH)₄)₂SO₄) (Haran et al (1993) Biochimica et Biophysica Acta (BBA) -biomebranes, 1151(2): 201-215; Maurer-Spurej et al (1999) Biochimica et Biophysica Acta (BBA) -Biomembranes,1416(1): 1-10; fritze et al (2006) Biochimica et Biophysica Acta (BBA) -biomebranes, 1758(10): 1633-1640); or from metal salts (e.g. A23187 and MnSO)₄) Ionophore-mediated ion gradients (Messerr et al (2004) clinical cancer Res.10(19): 6638-6649; ramsay et al (2008) Eur.J.Pharmaceut.BioPharmaceut.68(3): 607-617; fenske et al (1998) Biochimica et Biophysica Acta (BBA) -biomebranes, 1414(1) L188-204). Furthermore, it is possible to create a reverse pH gradient for drug loading, such as using a calcium acetate gradient to increase amphipathic weak acid loading in LB-MSNP, a strategy that has been used for liposomes (Avnir et al (2008) Arthritis)&Rheumatism,58(1):119-129)。

Cargo/drugs.

In one or more embodiments, the cargo comprises an organic compound capable of being protonated comprising at least one primary amine group, or at least one secondary amine group, or at least one tertiary amine group, or at least one quaternary amine group, or any combination thereof. We have also identified a comprehensive list of weakly basic drugs that can be loaded into LB-MSNP via proton gradients. General characteristics of these cargo molecules include the following chemical properties:

(i) an organic molecular compound comprising one or more primary, secondary, tertiary or quaternary amines;

(ii) pKa <11 to allow protonation and encapsulation behind LB (Zucker et al (2009) j.control. release,139(1): 73-80; Cern et al (2012) j.control. release,160(2): 147-157; Xu et al (2014) pharmaceut. res.31(10): 2583-2592);

(iii) a water solubility index of 5mg/mL to 25mg/mL and an amphiphilicity characteristic that allows diffusion through LB;

(iv) -octanol/water partition coefficient or logP value of 3.0 to 3.0 (Zucker et al (2009) J.Control. Release,139(1): 73-80; Cern et al (2012) J.Control. Release,160(2): 147-157);

(v) suitable molecular weights and geometries smaller than the MSNP pore size (2nm-8nm) to allow access to the MSNP pores (Li et al (2012) chem.soc.rev.41(7): 2590-2605; Tang et al (2012) adv.mat.24(12): 1504-1534; tar et al (2013) acc.chem.res.46(3): 792-801).

Not all inclusive, in various embodiments, the list of potential chemotherapeutic agents may include irinotecan derivatives and metabolites, such as SN38 as well as other alkaloids (e.g., topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, vinblastine, vincristine, homoharringtonine, trabectedin), anthracyclines (e.g., doxorubicin, epirubicin, pirarubicin, daunorubicin, rubicin (rubidomycin), valrubicin, amrubicin), basic anthracenediones (e.g., mitoxantrone), basic alkylating agents (e.g., cyclophosphamide, mechlorethamine, temozolomide), purine or pyrimidine derivatives (e.g., 5-fluorouracil, 5' -deoxy-5-fluorouridine, gemcitabine, capecitabine), and protein kinase inhibitors (e.g., pazopanib, enzastarin, Vandetanib, erlotinib, dasatinib, nilotinib, sunitinib).

The ability to package and deliver one or a combination of the above agents will enhance the broader utility of the multifunctional LB-MSNP platform, including the therapeutic considerations of additional cancer types, such as colon cancer, breast cancer, lung cancer, liver cancer, glioma, melanoma, and the like.

It is also possible to co-package the drug combinations in the above list into a single carrier. For example, based on our success with our GEM/PTX co-delivery platform (see, e.g., Meng et al (2015) ACS Nano,9(4):3540-3557), it is possible to consider the use of the silica bodies described herein in combination with drugs in the FOLFIRINOX regimen (e.g., oxaliplatin and irinotecan) for synergistic and ratiometric delivery. In addition, drug loading by our LB-MSNP can be used for non-cancerous applications, such as encapsulating antibiotics for infectious disease applications, such as ciprofloxacin, levofloxacin, or HIV antiretroviral drugs (e.g. tenofovir disoproxil fumarate).

In addition to the cancer drugs described above, the capture reagent-facilitated LB-MSNP platform can be used for effective drug loading and delivery as long as the drug molecule is basic as described above. While capture reagents will provide limited help for non-basic drug molecules, the single-step biofilm techniques provided for MSNP pore closure remain effective for an even larger range of drug molecules, such as anticancer, antiviral, antifungal, and antibiotics.

in some embodiments, the effective drug encapsulation includes, but is not limited to, epirubicin, trabectedin, paclitaxel, TLK 286, AV-299, DN-101, Pazopanib, GSK690693, RTA 744, PEG 0910, AZD 6244 (ARE-142886), AMN-107, TKI-258, GSK461364, CERTA-PEG, CERTARE-1, CERTARE-D, CERTA-1, CERTARE-D, CERTARE-1, CERTARE-DENER, CERTARE-DENEX-DETACERTRIN, CERTARE-DEFICINOCERTRIN-1, CERTRICINOCERTRICINOLIDENOLIPTIDIN, CERTRIBECINOCERTRIN-CERTRIN-DENOCETACETACETACETACETACETACETACERACERACETACETACERACERACET-1, CERTRIN-DEFICINOCERTRIN-DEFICINOCETACETACETACETACETACETACETACETACETACETACETACETACETAX, DEFICINOLIPTIDA, DEFICINOCETACIN, DEPRECINOCETACETACETAX-1, CERTRIN-1, DEPRECINOCETACERTRIN-DEFICINOLIPTIDA, DEPRECINOCETACINOLIPTIDA, CERTRIN-1, DEPRECINOLIDEPRECINOCETACINOLIPTIDA, DEPRECINOCETACINOCETACINOLIPTIDA, DEPRECINOCERTRIN, DEFICINOCERTRIN, DEPRECINOCEPTIDS-DEPRECINOCERTRIN, DEPRECINOCETAX-DEFICINOLIPTIDA, DEPRECINOLIPTIDA, DEFICINOLIPTIDA, CETAX-DEFICINOLIPTIDA, DEFICATIN, DEFICINOLIPTIDA, CEPTIDA, DEFICINOTEX-DEFICATEPCINOTEX-DEFICINOTEX-DEFICAT, DEFICINOLIPTIDA, CETAX-DEFICINOTEX-DEFICATIN, CETAX-DEFICATE, CETAMINE, CETACINOBECINOTEX-DEFICATIN, DEFICATE, DEFICATEPCINOTEX-DEFICATE, DEFICATEPCINOCETAX-DEFICATE, DEFICATEPCINOTEX-DEFICATE, CETAX-DEFICATE, DEFICATEPCINOTEX-DEFICATE, CETAX-DEFICATE, CERTM-DEX-DEFICATE, CERACERACETAX-DEFICATE, CERTM-DEFICATEPCINOTEX-DEFICAT, DEFICAT-DEFICATE, CETAX-DEFICAT, DEX-DEFICATE, DEFICATE-DEX-DEFICATE, DEFICAT, DEFICATEPCIN-DEX-DEFICATE, DEFICAT, DEFICATE, DEFICAT-DEFICATE, DEFICAT, CERACETAX-DEFICAT, CERABECIN-DEFICAT, CERACERACERABECIN, CERABECIN, DEFICATE, DEFICAT-DEFICATE, DEX-DEFICATE, DEFICAT-DEX-DEFICAT-DEX-DEFICATE, DEX-DEFICAT-DEX-DEFICATE, DEX-DEFICATE, DEX-DEFICATA, CERACERAPTIDA, CERACERACERABECIN, DEFICAT-DEFICATE, CERACERASE-DEX-DEFICAT-DEFICATA, DEFICATEPIN, DEFICAT-DEFICATE, CERTM-DEFICATE, DEFICAT-DEFICATEPX-DEFICATE, DEX-DEFICAT-DEFICATEPCINOTEX-DEFICATE, CERAFICATE, CERACERACERACERACERABECIN, DEFICAT, CERACERTX-DEFICAT-1, DEX-DEFICATE, CERACERACERACERACERACERACERASE-DEX-DEFICATE, CERACERACERASE-DEX-1, DEX-DEFICAT, CERACERACERACERABECIN, DEFICAT, CERABECIN, CERACEX, DEFICAT, CERACERACERACERASE-DEX, DEFICATE, CERACERACERACERACERABECIN, PEPTIDA, CERABECIN, CERAFICAT-DEFICAT-DEFICATE, CERAPIX-DEFICAT-DEX-DEFICATE, CERACERACERACERACER-1, PEPTIDS-1, CERAFICATE, DEFICATE, CERABECIN-DEFICATE, DEFICAT-DEFICATE, CERACERACERACERACERACER-DEFICATE, CERACERACERACERACERACERACEX, DEX-DEX, CERAFICATE, DEX-DEFICATE, DEX-1, DEFICATE, DEX-DEFICATE, DEFICATIN, DEFICAT-DEFICATE, DEX-DEFICAT-DEX-DEFICAT-DEX-DEFICATE, CERABECIN, DEFICAT, DEX, DEFICAT, CERAFICATE, CERAFICAT, CERAFICATE, CEX-DEX-DEFICAT, DEX-DEFICAT, DEFICAT-DEFICAT, DEX, DEFICAT-DEFICATE, DEX, DEFICATE, DEFICAT, CERAFICATE, CERAFICAT-DEX, CERAFICAT, CERAFICATE, DEFICAT, CERAFICATE, CERAFICAT, CERAFICATE, CERACERAFICAT-DEFICATE, CERAFICATE, CERAFICAT-DEFICAT-DEFICATE, CERAFICATE, DEFICAT, DEFICATE, CERAFICAT, DEFICAT-DEFICAT, CERAFICATE, DEFICAT, CERAFICAT-DEFICAT, CERAFICAT, CERAFICATE, CEX, CERACERAFICATE, CEX-DEFICATE, CERAFICAT, DEFICATE, CERAFICAT-DEX-DEFICAT-DEX, DEFICATE, CERAFICATE, CERAFICAT-DEFICAT, CERAFICAT, CERAFICATE, CERAFICAT, CERAFICATE, CEX-DEX, CEX, CERAFICAT, CERAFICATE, CERAFICAT-DEFICATE, CERAFICATE, CERACERACERACERAFICATE, CERAFICAT-DEFICAT, CERAFICATE, CERAFICAT, CERAFICATE, CERAFICAT, CEX-DEFICATE, CERAFICATE, CEX, CERAFICATE, CERASE-DEX, CEX-D, CERAFICATE, CERAFICAT, CERAFICATE, CEX, CERAFICATE, CERAFICATIN, CERAFICATE, CERA.

In certain embodiments, the cargo comprises an antifungal agent. Illustrative antifungal agents include, but are not limited to, amphotericin B (e.g., for most fungal infections other than pseudoleishmania sp), anidulafungin (e.g., for candidiasis, including candidemia, etc.), caspofungin (e.g., for aspergillosis, candidiasis, including candidemia, etc.), fluconazole (e.g., for mucosal and systemic candidiasis, cryptococcal meningitis, coccidioidomycosis, etc.), flucytosine (e.g., for candidiasis (systemic), cryptococcosis, etc.), isaconazole (e.g., for aspergillosis, mucormycosis, etc.), itraconazole (e.g., for dermatophytosis, histoplasmosis, blastomycosis, coccidioidomycosis, sporotrichosis, etc.), micafungin (e.g., for candidiasis, including candidemia), posaconazole (e.g., for prevention of invasive aspergillosis and candidiasis), etc, Oral candidiasis, itraconazole-refractory oral candidiasis, etc.), voriconazole (e.g., for invasive aspergillosis, fusarium, spodoptera spongitis (Scedosporiosis), etc.), and the like.

Dual therapeutic silica bodies.

It will be appreciated that in certain embodiments, the nanoparticle drug carriers (silica bodies) described herein may comprise two or more therapeutic agents. Thus, for example, in certain embodiments, the pores in the silica body may be loaded with two, or three, or four, or more different therapeutic agents. In certain embodiments, this may allow for ratiometric delivery of these therapeutic agents. By way of non-limiting illustration, a number of multi-agent treatment regimens are known for the treatment of cancer. These include, but are not limited to, COMP (methotrexate, prednisone), LSA₂-L₂(Cyclophosphamide, Long)Vincristine, prednisone, daunomycin (daunomycin), methotrexate, cytarabine, thioguanine, asparaginase and carmustine, FOLFIRINOX (irinotecan, oxaliplatin, 5-fluorouracil, leucovorin) and the like. In certain embodiments, two or more agents meeting the requirements described herein for a drug to be loaded into a silica body using the methods described herein can be provided in a silica body. Where a multi-drug regimen includes agents incompatible with the loading methods described herein, some agents (e.g., irinotecan) may be provided in the silica body to provide improved tolerability and other components of the treatment regimen may be administered by conventional modes.

in certain embodiments, hydrophobic (e.g., lipophilic) drugs and other agents may be provided in the lipid bilayer component of the silica body, including, but not limited to, paclitaxel, ellipticine, camptothecin, L-asparaginase, doxorubicin, SN-38, etc. in certain embodiments, the lipid bilayer component of the silica body may contain one or more phospholipid prodrugs (e.g., drugs conjugated to lipids). illustrative lipid prodrugs include, but are not limited to, acyclovir diphosphate dimyristoyl glycerol (see, e.g., Hosteer et al, (1993) Proc.Natl.Acad.Sci.USA,90(24):11835-11839), phospholipid prodrugs of doxorubicin (see, e.g., Wang et al (2015) J.Mater.chem.B.,3:3297-3305), phospholipid derivatives of nucleoside analogs (e.g., 1- β -D-arabinofuranosyl cytosine (ara-C), 9- β -D-arabinofuranosyl adenine (ara-A), phospholipid derivatives of nucleoside analogs (see, e.g., Lipocalin-2. Columb. Thielaucine, et al, U.g., Lipocalin No. Ser. 5, No. 2, Leucosmin, No. (2015), Pasteosine.5), Lipocalin No. 2, see, et al, see, et al, Pendulcin, et al, see, Pendulcin, et al.

The multi-agent silica bodies described above are illustrative and not limiting. Using the teachings provided herein, many combinations of therapeutic agents for incorporation into (or onto) the silica bodies described herein will be available to those skilled in the art.

Targeting ligands and immunoconjugates.

In certain embodiments, the LB coated nanoparticles (silica bodies) may be conjugated with: one or more targeting ligands, e.g., to facilitate specific delivery in endothelial cells to cancer cells; fusion ligands, e.g., to facilitate endosomal escape; a ligand to facilitate transport across the blood brain barrier; and the like.

In an illustrative but nonlimiting embodiment, the silicasomes are conjugated to a fusion peptide, such as histidine-rich H5WYG (H)₂N-GLFHAIAHFIHGGWHGLIHGWYG-COOH, (SEQ ID NO:1)) (see, e.g., Midoux et al, (1998) bioconjugate. chem.9: 260-267).

In certain embodiments, the silica bodies are conjugated to targeting ligands, including antibodies and targeting peptides. Targeted antibodies include, but are not limited to, intact immunoglobulins, immunoglobulin fragments (e.g., F (ab))'₂Fab, etc.), single chain antibodies, diabodies, affibodies, monoantibodies, nanobodies, etc. In certain embodiments, antibodies that specifically bind to cancer markers (e.g., tumor associated antigens) will be used. A wide variety of cancer markers are known to those of skill in the art. The marker is not necessarily unique to the cancer cell, but may also be effective in cases where expression of the marker is elevated in cancer cells (as compared to normal healthy cells) or where the marker is not present at comparable levels in surrounding tissues, particularly where the chimeric moiety is delivered locally.

Illustrative cancer markers include, for example, tumor markers recognized by the ND4 monoclonal antibody. This marker is present on poorly differentiated colorectal cancers as well as on gastrointestinal neuroendocrine tumors (see, e.g., Tobi et al (1998) cancer and preservation, 22(2): 147-152). Other important targets for cancer immunotherapy are the membrane-bound complement regulatory glycoproteins CD46, CD55, and CD59, which have been found to be expressed on a large proportion of tumor cells in vivo and in vitro. Human mucins (e.g., MUC1) are known tumor markers, gp100, tyrosinase, and MAGE are also known tumor markers, and are present in melanoma. The wild-type wilms' tumor gene WT1 is expressed not only at high levels in most of acute myelogenous leukemia, acute lymphocytic leukemia, and chronic myelogenous leukemia, but also in various types of solid tumors including lung cancer.

Acute lymphocytic leukemia is characterized by TAA HLA-Dr, CD1, CD2, CD5, CD7, CD19 and CD 20. Acute myeloid leukemia is characterized by TAA HLA-Dr, CD7, CD13, CD14, CD15, CD33 and CD 34. Breast cancer is characterized by the markers EGFR, HER2, MUC1, Tag-72. Various carcinomas are characterized by the markers MUC1, TAG-72, and CEA. Chronic lymphocytic leukemia is characterized by the markers CD3, CD19, CD20, CD21, CD25 and HLA-DR. Hairy cell leukemia is characterized by the markers CD19, CD20, CD21, CD 25. Hodgkin's disease is characterized by the Leu-M1 marker. Various melanomas are characterized by HMB 45 markers. Non-hodgkin lymphoma was characterized by CD20, CD19, and Ia markers. And various prostate cancers are characterized by PSMA and SE10 markers.

Furthermore, many types of tumor cells display aberrant antigens that are inappropriate for the cell type and/or its environment or are usually present only during the development of the organism (e.g., embryonic antigens). Examples of such antigens include glycosphingolipid GD2, which is a disialoganglioside that is normally expressed only at significant levels on the outer surface membrane of neuronal cells, where its exposure to the immune system is limited by the blood-brain barrier. GD2 is expressed on the surface of a wide range of tumor cells including neuroblastoma, medulloblastoma, astrocytoma, melanoma, small cell lung carcinoma, osteosarcoma, and other soft tissue sarcomas. GD2 is therefore a suitable tumor-specific target for immunotherapy.

Other types of tumor cells exhibit cell surface receptors that are rare or absent on the surface of healthy cells and are responsible for activating cell signaling pathways that lead to unregulated growth and division of tumor cells. Examples include (ErbB2) HER2/neu, which is a constitutively active cell surface receptor produced at abnormally high levels on the surface of breast cancer tumor cells.

Other useful targets include, but are not limited to, CD20, CD52, CD33, epidermal growth factor receptor, and the like.

An illustrative, but non-limiting list of suitable tumor markers is provided in table 1. Antibodies to these and other cancer markers are known to those skilled in the art and can be obtained commercially or readily produced, for example using phage display technology. These antibodies can be readily conjugated to the silica bodies described herein, e.g., in the same manner as the iRGD peptide is conjugated in example 3.

Table 1: illustrative cancer markers and related references, all of which are incorporated herein by reference for the purpose of identifying the referenced tumor markers.

Any of the above markers can be used as targets for the targeting moieties that make up the silica body constructs described herein. In certain embodiments, the targets include, but are not limited to, members of the epidermal growth factor family (e.g., HER2, HER3, EGF, HER4), CD1, CD2, CD3, CD5, CD7, CD13, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD34, CD38, 5E10, CEA, HLA-DR, HM 1.24, HMB 45, 1a, Leu-M1, MUC1, PMSA, TAG-72, phosphatidylserine antigens, and the like.

The above markers are intended to be illustrative and not limiting. Other tumor-associated antigens will be known to those skilled in the art.

Where the tumour marker is a cell surface receptor, a ligand for that receptor may act as a targeting moiety. Similarly, mimetics of such ligands can also be used as targeting moieties. Thus, in certain embodiments, peptide ligands may be used in addition to or in place of various antibodies. An illustrative, but non-limiting list of suitable targeting peptides is shown in table 2. In certain embodiments, any one or more of these peptides can be conjugated to a silica body as described herein.

Table 2: illustrative, but not limiting, peptides that target membrane receptors expressed or overexpressed by various cancer cells.

In certain embodiments, the silica may be conjugated to a moiety that promotes stability in circulation and/or conceals the silica to avoid the reticuloendothelial system (REC) and/or promotes transport across barriers (e.g., matrix barriers, blood brain barrier, etc.) and/or into tissues. In certain embodiments, the silica body is conjugated to transferrin or ApoE to facilitate transport across the blood brain barrier. In certain embodiments, the silica body is conjugated to folic acid.

Methods of coupling silica bodies with targeting agents (or other agents) are well known to those skilled in the art. Examples include, but are not limited to, the use of biotin and avidin or streptavidin (see, e.g., U.S. Pat. No.: US 4,885,172A); by conventional chemical reactions using, for example, difunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides, and difunctional aryl halides such as 1, 5-difluoro-2, 4-dinitrobenzene; p, p '-difluorometh, m' -dinitrodiphenyl sulfone, mercapto-reactive maleimide, and the like. Suitable reactions which can be applied for such coupling are described in Williams et al Methods in Immunology and Immunology chemistry volume 1, Academic Press, New York, 1967. In one illustrative but non-limiting method described in example 3, by using DSPE-PEG₂₀₀₀Maleimide instead of DSPE-PEG₂₀₀₀To couple the peptide (iRGD in this example) to the silica body (see method section in example 3) to allow thiol-maleimide to be coupled to the cysteine-modified peptide. It will also be appreciated that in certain embodiments, the targeting moiety (and other moieties) may be conjugated to the lipids making up the lipid bilayer.

The former conjugates and coupling methods are illustrative and not limiting. Using the teachings provided herein, many other moieties can be conjugated to the silica bodies described herein by any of a variety of methods.

Pharmaceutical formulations, administration and treatment

Pharmaceutical preparation

In some embodiments, the nanoparticle pharmaceutical carriers described herein are administered alone or in admixture with a physiologically acceptable carrier (such as saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. For example, when used as an injection, the silica body can be formulated into a sterile suspension, dispersion, or emulsion with a pharmaceutically acceptable carrier. In certain embodiments, physiological saline may be used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like. In compositions comprising brine or other salt-containing carriers, the carrier is preferably added after formation of the silica body. Thus, after formation of the silica body and loading of one or more suitable drugs, the silica body may be diluted in a pharmaceutically acceptable carrier, such as physiological saline. These compositions can be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solution, suspension, dispersion, emulsion, etc., may be packaged for use or filtered under sterile conditions. In certain embodiments, the silica bodies are lyophilized, and the lyophilized formulation is combined with a sterile aqueous solution prior to administration. The compositions may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the like.

in addition, in certain embodiments, the pharmaceutical formulation may include a lipid protecting agent that protects the lipid from free radical and lipid peroxidation damage upon storage lipophilic free radical quenchers, such as α -tocopherol, and water soluble iron specific chelators, such as ferrioxamine (ferrioxamine), are suitable.

The concentration of silica bodies in the pharmaceutical formulation may vary widely, for example, from less than about 0.05% by weight, typically at least about 2% to 5% by weight up to 10% to 50% by weight, or to 40% by weight, or to 30% by weight and is selected primarily by fluid volume, viscosity, etc., depending on the particular mode of administration selected. For example, the concentration may be increased to reduce the fluid load associated with the treatment. This may be particularly desirable in patients with atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, the silica body composed of a stimulating lipid may be diluted to a low concentration to reduce inflammation at the site of application. The amount of silica body administered will depend on the particular drug used, the disease state being treated and the judgment of the clinician, but will generally be from about 0.01mg to about 50mg per kg of body weight, preferably from about 0.1mg to about 5mg per kg of body weight.

In some embodiments, for example, it may be desirable to include polyethylene glycol (PEG) modified phospholipids in the silica body. Alternatively or additionally, in certain embodiments, a PEG-ceramide, or ganglioside G may be added to the composition_MIThe modified lipid is incorporated into the silica body. The addition of such components helps prevent silica body aggregation and provides extended cycle life and increases delivery of the loaded silica body to the target tissue. In certain embodiments, the PEG-modified phospholipid, PEG-ceramide, or G in the silica body_MIThe concentration of modified lipid will be about 1% to 15%.

In some embodiments, the total silica body charge is an important determinant of silica body clearance from blood. It is believed that charged silica bodies will generally be taken up more rapidly by the reticuloendothelial system (see, e.g., Juliano (1975), biochem. biophysis. Res. Commun.63:651-658, which discusses clearance of liposomes by RES) and thus have a shorter half-life in the blood stream. Silica bodies with extended circulatory half-lives are often desirable for therapeutic use. For example, in certain embodiments, silica bodies that are maintained for 8 hours, or 12 hours, or 24 hours or longer are desirable.

In another example of their use, drug-loaded silica bodies may be incorporated into a wide range of topical dosage forms, including but not limited to gels, oils, emulsions, and the like, for example for the treatment of localized cancers. For example, in some embodiments, the suspension containing the drug-loaded silica bodies is formulated and administered as a topical cream, paste, ointment, gel, lotion, and the like.

In some embodiments, a pharmaceutical formulation comprising a silica body described herein further comprises a buffering agent. The buffer may be any pharmaceutically acceptable buffer. Buffering systems include, but are not limited to, citrate buffers, acetate buffers, borate buffers, and phosphate buffers. Examples of buffering agents include, but are not limited to, citric acid, sodium citrate, sodium acetate, acetic acid, sodium phosphate and phosphoric acid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate and carbonic acid, sodium succinate and succinic acid, histidine and sodium benzoate, benzoic acid, and the like.

In some embodiments, a pharmaceutical formulation comprising a silica body described herein further comprises a chelating agent. The chelating agent may be any pharmaceutically acceptable chelating agent. Chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (also synonymous with EDTA, edetic acid, Versene acid, and sequinsterene), and EDTA derivatives such as dipotassium edetate, disodium edetate, calcium disodium edetate, sodium edetate, trisodium edetate, and potassium edetate. Other chelating agents include citric acid (e.g. citric acid monohydrate) and derivatives thereof. The citric acid derivatives include anhydrous citric acid, trisodium citrate dihydrate, etc. Additional other chelating agents include, but are not limited to, niacinamide and its derivatives and sodium deoxycholate and its derivatives.

in some embodiments, the pharmaceutical formulations comprising the silica bodies described herein additionally comprise a bioactive agent comprising an antioxidant, which may be any pharmaceutically acceptable antioxidant, antioxidants are well known to those of ordinary skill in the art and include, but are not limited to, materials such as ascorbic acid, ascorbic acid derivatives (e.g., ascorbyl palmitate, ascorbyl stearate, sodium ascorbate, calcium ascorbate, etc.), butylated hydroxyanisole, butylated hydroxytoluene, alkyl gallate, sodium metabisulfate, sodium bisulfate, sodium hydrosulfite, sodium thioglycolate, sodium formaldehyde sulfoxylate, tocopherol and its derivatives (d- α tocopherol, d- α tocopherol acetate, dl- α tocopherol acetate, d- α tocopherol succinate, β tocopherol, delta tocopherol, gamma tocopherol, and d- α tocopherol polyoxyethylene glycol 1000 succinate), monothioglycerol, sodium sulfite, and N-acetylcysteine.

In some embodiments, a pharmaceutical formulation comprising a silica body described herein is formulated with a cryoprotectant. The cryoprotectant may be any pharmaceutically acceptable cryoprotectant. Common cryoprotectants include, but are not limited to, histidine, polyethylene glycol, polyvinyl pyrrolidine, lactose, sucrose, mannitol, polyols, and the like.

In some embodiments, a pharmaceutical formulation comprising a silica body described herein is formulated with an isotonic agent. The isotonic agent may be any pharmaceutically acceptable isotonic agent. The term is used interchangeably in the art with isotonic agents and is known as a compound that is added to a pharmaceutical formulation to increase the osmotic pressure, e.g., in some embodiments, to increase the osmotic pressure to that of a 0.9% sodium chloride solution (which is isotonic with human extracellular fluids, such as plasma). Illustrative isotonic agents include, but are not limited to, sodium chloride, mannitol, sorbitol, lactose, dextrose, and glycerol.

In certain embodiments, the pharmaceutical formulation of the silica body may optionally comprise a preservative. Common preservatives include, but are not limited to, those selected from the group consisting of: chlorobutanol, parabens, thimerosal, benzyl alcohol, and phenol. Suitable preservatives include, but are not limited to: chlorobutanol (e.g., 0.3% -0.9% w/v), parabens (e.g., 0.01% -5.0%), thimerosal (e.g., 0.004% -0.2%), benzyl alcohol (e.g., 0.5% -5%), phenol (e.g., 0.1% -1.0%), and the like.

In some embodiments, the pharmaceutical formulation comprising the silica body is formulated with a humectant to provide a pleasant mouth feel, e.g., upon oral administration. Humectants known in the art include, but are not limited to, cholesterol, fatty acids, glycerin, lauric acid, magnesium stearate, pentaerythritol, and propylene glycol.

In some embodiments, an emulsifier is included in the formulation, for example to ensure complete dissolution of all excipients, particularly hydrophobic components such as benzyl alcohol. Many emulsifiers are known in the art, such as polysorbate 60.

For some embodiments related to oral administration, it may be desirable to add pharmaceutically acceptable flavoring and/or sweetening agents. Compounds such as saccharin, glycerol, simple syrups, and sorbitol may be used as sweeteners.

Administration and treatment

The cargo (e.g., drug) -loaded silica bodies can be administered to a subject (e.g., a patient) by any of a variety of techniques.

In certain embodiments, the pharmaceutical formulation is administered parenterally, e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical composition is administered intravenously, intraarterially, or intraperitoneally by bolus injection (see, e.g., U.S. Pat. Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578, which describe administration of liposomes). Specific pharmaceutical formulations suitable for such administration are found in Remington's pharmaceutical Sciences, Mack publishing company, philiadelphia, Pa, 17 th edition (1985), Philadelphia, Pa. Typically, the formulation comprises a solution of the silica bodies suspended in an acceptable carrier, preferably an aqueous carrier. As noted above, suitable aqueous solutions include, but are not limited to, physiologically compatible buffers such as Hanks 'solution, Ringer's solution, or physiological (e.g., 0.9% isotonic) saline buffer and/or certain emulsion formulations. The one or more solutions may contain formulating agents, such as suspending, stabilizing and/or dispersing agents. In certain embodiments, the one or more active agents may be provided in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, prior to use. For transmucosal administration and/or for passage of the blood/brain barrier, penetrants appropriate to the barrier to be permeated are used in the formulation. These compositions may be sterilized by conventional, well-known sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged for use as is or lyophilized, the lyophilized formulation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate and the like, for example as described above.

In other methods, a pharmaceutical formulation containing a silica body described herein can be contacted with a target tissue by directly administering the formulation to the tissue. The administration may be by a topical, "open" or "closed" procedure. By "topical" is meant the direct application of a pharmaceutical formulation to tissue exposed to the environment, such as skin, oropharynx, external auditory canal, and the like. Open procedures are those that involve cutting the patient's skin and directly visualizing the underlying tissue receiving the application of the pharmaceutical agent. This is typically accomplished by a surgical procedure, such as a thoracotomy to access the lungs, an abdominal laparotomy to access the abdominal viscera, or other direct surgical approach to the target tissue. A sealing procedure is an invasive procedure in which the internal target tissue is not directly visualized, but is accessed through a small incision in the skin via an insertion instrument. For example, the formulation may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical formulation may be administered to the meninges or spinal cord by infusion during lumbar puncture, followed by appropriate positioning of the patient, as is typically done for spinal anesthesia or imaging of meglumine in the spinal cord. Alternatively, the formulation may be administered via an endoscopic device. In certain embodiments, the pharmaceutical formulation is introduced via a cannula.

In certain embodiments, a pharmaceutical formulation comprising a silica body described herein is administered via inhalation (e.g., as an aerosol). Inhalation can be a particularly effective delivery route for administration to the lung and/or brain. For administration by inhalation, the silica bodies are conveniently delivered in the form of an aerosol spray from a pressurised pack or nebuliser, with the aid of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

In certain embodiments, the silica bodies described herein are formulated for oral administration. For oral administration, suitable formulations can be readily formulated by combining one or more silica bodies with pharmaceutically acceptable carriers well known in the art for oral delivery. Such carriers enable one or more active agents described herein to be formulated as tablets, pills, dragees, caplets, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid preparations, such as, for example, powders, capsules and tablets, suitable excipients may include fillers, such as sugars (e.g. lactose, sucrose, mannitol and sorbitol), cellulose preparations (e.g. corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose), synthetic polymers (e.g. polyvinylpyrrolidone (PVP)), granulating agents; and a binder. If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. If desired, the solid dosage form may be sugar coated or enteric coated using standard techniques. The preparation of enteric coated particles is disclosed, for example, in U.S. Pat. nos. 4,786,505 and 4,853,230.

In various embodiments, the one or more silica bodies can be formulated in rectal or vaginal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. Methods of formulating active agents for rectal or vaginal delivery are well known to those skilled in the art (see, e.g., Allen (2007) Suppositories, Pharmaceutical Press) and generally involve combining the active agent with a suitable matrix (e.g., hydrophilic materials (PEG); lipophilic materials such as cocoa butter or Witepsol W45; amphiphilic materials such as Suppocire AP and pegylated glycerides, etc.). The matrix is selected and compounded for the desired melt/delivery characteristics.

The route of delivery of the silica bodies may also affect their distribution in the body. Passive delivery of the silica body involves the use of various routes of administration, e.g. parenteral, although other effective forms of administration are also envisaged, such as intra-articular injections, inhalation sprays, orally active formulations, transdermal iontophoresis, or suppositories. Each approach produces differences in the positioning of the silica bodies.

Since the dosage regimen for the medicament is well known to the practitioner, the amount of liposomal pharmaceutical preparation that is effective or therapeutic for treating a disease or condition in a mammal, particularly a human, will be apparent to those skilled in the art. The optimal amount and spacing of the individual doses of the formulations herein will be determined by the nature and extent of the condition being treated, the form, route and site of administration and the particular patient being treated, and such optimums can be determined by conventional techniques. One skilled in the art will also appreciate that the optimal course of treatment, e.g., the number of doses administered per day over a defined number of days, can be determined by one skilled in the art using conventional course of treatment determination tests.

In certain embodiments, the silica bodies and/or pharmaceutical forms thereof may be used therapeutically in animals (including humans) to treat various cancers or various infections, among others, including conditions requiring: (1) repeated application; (2) sustained delivery of the drug in its biologically active form; or (3) reduced toxicity compared to the free drug of interest with suitable efficacy. In various embodiments, the silica bodies and/or drug formers thereof are administered in a therapeutically effective dose. The term "therapeutically effective" as it relates to the silica bodies and formulations thereof described herein means that the biologically active substance present and/or in the silica body is provided/released in a manner sufficient to achieve the particular medical effect that the biologically active substance (therapeutic agent) is intended to achieve. Examples of desired medical effects that may be obtained are, but are not limited to, chemotherapy, antibiotic therapy, and metabolic regulation. Thus, for example, a therapeutically effective dose for cancer chemotherapy can be a dose (and/or dosing regimen) effective to slow growth and/or proliferation of cancer cells, and/or slow, stop growth of, or shrink or eliminate solid tumors, and/or slow, stop proliferation of metastatic cells, and the like. A therapeutically effective dose for treating an infection may be a dose (and/or dosing regimen) sufficient to inhibit the growth and/or proliferation of a pathogen, and/or to kill a pathogen, and/or to alleviate one or more symptoms caused by a pathogen.

The exact dosage will vary depending upon factors such as the particular therapeutic agent and the desired therapeutic effect, as well as patient factors such as age, sex, general condition, and the like. One skilled in the art can readily take these factors into account and use them to determine an effective therapeutic concentration without undue experimentation.

For administration to humans (or non-human mammals) for curative, palliative, delayed, or prophylactic treatment of a disease, the prescribing physician will ultimately determine the appropriate dosage of drug for a given human (or non-human) subject, and this expectation will vary according to the age, weight, and response of the individual, as well as the nature and severity of the patient's disease. In certain embodiments, the dose of drug provided by the one or more silica bodies may be about equal to the dose used for the free drug. However, as described above, the silica bodies described herein can significantly reduce the toxicity of one or more drugs administered therefrom and significantly increase the therapeutic window. Thus, in some cases, a dose in excess of the dose prescribed for the free drug will be utilized.

In certain embodiments, the dose of encapsulated drug administered at a particular time point will be within the following ranges: about 1mg/m²Daily to about 1,000mg/m²Daily, or to about 800mg/m²Daily, or to about 600mg/m²Daily, or to about 400mg/m²The day is. For example, in certain embodiments, dosages (dosage regimens) providing the following ranges are utilized: about 1mg/m²Daily to about 350mg/m²Day, 1mg/m²Daily to about 300mg/m²Day, 1mg/m²Daily to about 250mg/m²Day, 1mg/m²Daily to about 200mg/m²Day, 1mg/m²Daily to about 150mg/m²Day, 1mg/m²Daily to about 100mg/m²About 5 mg/m/day²Daily to about 80mg/m²About 5 mg/m/day²Daily to about 70mg/m²About 5 mg/m/day²Daily to about 60mg/m²About 5 mg/m/day²Daily to about 50mg/m²About 5 mg/m/day²Daily to about 40mg/m²About 5 mg/m/day²Daily to about 20mg/m²A day, about 10mg/m²Daily to about 80mg/m²A day, about 10mg/m²Daily to about 70mg/m²A day, about 10mg/m²Daily to about 60mg/m²A day, about 10mg/m²Daily to about 50mg/m²A day, about 10mg/m²Daily to about 40mg/m²A day, about 10mg/m²Daily to about 20mg/m²About 20 mg/m/day²Daily to about 40mg/m²About 20 mg/m/day²Daily to about 50mg/m²About 20 mg/m/day²Daily to about 90mg/m²About 30 mg/m/day²Daily to about 80mg/m²About 40 mg/m/day²Daily to about 90mg/m²About 40 mg/m/day²Daily to about 100mg/m²About 80 mg/m/day²Daily to about 150mg/m²About 80 mg/m/day²Daily to about 140mg/m²About 80 mg/m/day²Daily to about 135mg/m²About 80 mg/m/day²Daily to about 130mg/m²About 80 mg/m/day²Daily to about 120mg/m²A day, about 85mg/m²Day/dayTo about 140mg/m²A day, about 85mg/m²Daily to about 135mg/m²A day, about 85mg/m²Daily to about 135mg/m²A day, about 85mg/m²Daily to about 130mg/m²A day, or about 85mg/m²Daily to about 120mg/m²The day is. In certain embodiments, the dose administered at a particular time point may also be about 130mg/m²A day of about 120mg/m²A day, about 100mg/m²About 90 mg/m/day²A day, about 85mg/m²About 80 mg/m/day²About 70 mg/m/day²About 60 mg/m/day²About 50 mg/m/day²About 40 mg/m/day²About 30 mg/m/day²About 20 mg/m/day²A day, about 15mg/m²A day, or about 10mg/m²The day is.

The dose can also be estimated using in vivo animal models, as will be appreciated by those skilled in the art. In this regard, for the irinotecan-loaded silica described herein, it should be noted that the effective therapeutic dose of Ir-silica in KPC-derived in situ animal models is about 40mg/kg, which corresponds to 120mg/m in a 70 kg human subject²(Liu et al, (2016) ACS Nano,10: 2702-2715). Fibonacchialysine analysis (Fibonacchialysine) showed that the dose could pass 40mg/m²And 80mg/m²The initial dose and the intermediate dose of (a).

The dosage administered may be higher or lower than the dosage ranges described herein, depending on, among other factors, the bioavailability of the composition, the tolerance of the individual to adverse side effects, the mode of administration, and the various factors described above. The dosage and time interval may be adjusted individually at the discretion of the prescribing physician to provide plasma levels of the composition sufficient to maintain therapeutic effect. Given the teachings provided herein, one of skill in the art will be able to optimize effective topical dosages without undue experimentation.

Multiple doses (e.g., continuous or bolus) of a composition as described herein can also be administered to an individual in need thereof over the course of hours, days, weeks, or months. For example, but not limited to, 1,2, 3, 4,5, or 6 times per day, once every other day, once every 10 days, once a week, once a month, twice a week, three times a week, two times a month, three times a month, four times a month, five times a month, once every other month, etc.

Methods of treatment.

In various embodiments, methods of treatment using one or more nanoparticulate drug carriers and/or one or more drug formulations comprising nanoparticulate drug carriers described herein are provided. In certain embodiments, the one or more methods comprise a method of treating cancer. In certain embodiments, the method may comprise administering to a subject in need thereof an effective amount of a nanoparticle drug carrier and/or a pharmaceutical formulation comprising a nanoparticle drug carrier as described herein, wherein the nanoparticle drug carrier and/or a drug in the pharmaceutical formulation comprises an anti-cancer drug. In certain embodiments, the nanoparticle drug carrier and/or drug formulation is the primary treatment in a chemotherapeutic regimen. In certain embodiments, the nanoparticle drug carrier and/or drug formulation is a component of a multi-drug chemotherapy regimen. In certain embodiments, the multi-drug chemotherapy regimen comprises at least two drugs selected from the group consisting of: irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU) and Leucovorin (LV). In certain embodiments, the multi-drug chemotherapy regimen comprises at least three drugs selected from the group consisting of: irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU) and Leucovorin (LV). In certain embodiments, the multi-drug chemotherapy regimen comprises at least irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU), and Leucovorin (LV).

In various embodiments, one or more nanoparticle drug carriers and/or one or more pharmaceutical formulations comprising a nanoparticle drug carrier described herein are effective in treating any of a variety of cancers. In certain embodiments, the cancer is Pancreatic Ductal Adenocarcinoma (PDAC). In certain embodiments, the cancer is a cancer selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Karposi's sarcoma, lymphoma), anal cancer, appendiceal cancer, astrocytoma, atypical teratoma/rhabdoid tumor, cholangiocarcinoma, extrahepatic cancer, bladder cancer, bone cancers (e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytoma, glioblastoma, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoma/rhabdoid tumor, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal tract), cardiac tumors, cervical cancer, prostate cancer, bladder cancer of the head and neck, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal cancers (e.g., cholangiocarcinoma, extrahepatic carcinoma), Ductal Carcinoma In Situ (DCIS), embryonal tumors, endometrial cancers, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumors, extragonally germ cell tumors, extrahepatic bile duct cancers, ocular cancers (e.g., intraocular melanoma, retinoblastoma), malignant bone fibrohistiocytoma, and osteosarcoma, gall bladder cancer, stomach (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancer, extragonadal cancers, central nervous system germ cell tumors), gestational trophoblastic tumors, brain stem cancer, brain stem cell tumors, colon cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, hodgkin's lymphoma, hypopharynx cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kaposi's sarcoma, kidney cancer (e.g., renal cell carcinoma, wilms tumor and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), hairy cell leukemia, lip and oral cancer, liver cancer (primary), carcinoma of lobule in situ (LCIS), lung cancer (e.g., childhood lung cancer, non-small cell lung cancer, small cell lung cancer), lymphoma (e.g., AIDS-related lymphoma, burkitt lymphoma (e.g., non-hodgkin's lymphoma), Cutaneous T-cell lymphoma (e.g., mycosis fungoides, sezary syndrome), hodgkin's lymphoma, non-hodgkin's lymphoma, primary Central Nervous System (CNS) lymphoma), macroglobulinemia, waldenstrom's macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood melanoma, intraocular (eye) melanoma), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline carcinoma, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, Chronic Myelogenous Leukemia (CML), multiple myeloma, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, neuroendocrine tumor of the islet (pancreatic islet cell tumor), Papillomatosis, paraganglioma, cancer of the paranasal sinuses and nasal cavities, cancer of the parathyroid gland, cancer of the penis, cancer of the pharynx, pheochromocytoma, pituitary gland, plasma cell tumor, pleuropulmonoblastoma, primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, cancer of the kidney cells (kidneys), cancer of the renal pelvis and ureter, transitional cell carcinoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma (e.g. ewing's sarcoma, kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma), sezary syndrome, skin cancer (e.g. melanoma, merkel cell carcinoma, basal cell carcinoma, non-melanoma), small intestine cancer, squamous cell carcinoma, occult primary squamous neck cancer, stomach (stomach) cancer, testicular cancer, throat cancer, thymus and thymus cancer, thyroid cancer, trophoblastic cell tumor, ureter and renal pelvis cancer, urinary tract cancer, renal pelvis cancer, renal cell carcinoma, squamous cell carcinoma, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, waldenstrom's macroglobulinemia, and nephroblastoma.

In certain embodiments, the nanoparticle drug carrier is not conjugated to the iRGD peptide and the nanoparticle drug carrier is administered in combination with the iRGD peptide (e.g., the nanoparticle drug carrier and the iRGD peptide are co-administered as separate formulations).

In certain embodiments, the one or more methods comprise a method of treating an infection. In certain embodiments, the method may comprise administering to a subject in need thereof an effective amount of a nanoparticle drug carrier and/or a pharmaceutical formulation comprising a nanoparticle drug carrier as described herein, wherein the nanoparticle drug carrier and/or a drug in the pharmaceutical formulation comprises an antimicrobial or antiviral agent. In certain embodiments, the infection comprises a nosocomial infection. In certain embodiments, the infection is caused by a viral pathogen, a bacterial pathogen, or a fungal pathogen. In certain embodiments, the infection comprises a bloodstream infection (BSI), a pneumonia (e.g., Ventilator Associated Pneumonia (VAP)), a gastrointestinal tract infection, a Urinary Tract Infection (UTI), a Surgical Site Infection (SSI), or a skin infection. In certain embodiments, the infection is caused by a pathogen such as: staphylococcus aureus (e.g., blood infection), escherichia coli (e.g., UTI), enterococcus (e.g., blood, UTI, wound), pseudomonas aeruginosa (e.g., kidney or respiratory tract infection), Mycobacterium tuberculosis (e.g., lung), and the like. In certain embodiments, the infection is a viral infection (e.g., HIV, hepatitis b, hepatitis c, etc.).

In certain embodiments, the infection is caused by a drug-resistant pathogen. Illustrative drug resistant pathogens include, but are not limited to, Methicillin Resistant Staphylococcus Aureus (MRSA), Vancomycin Resistant Enterococci (VRE), and multidrug resistant mycobacterium tuberculosis (MDR-TB), as well as Klebsiella pneumoniae (Klebsiella pneumoniae) carbapenemase-producing bacteria (KPC).

In various embodiments of these methods of treatment, the nanoparticle pharmaceutical carrier and/or pharmaceutical formulation is administered via a route selected from the group consisting of: intravenous administration, intra-arterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery), intracranial administration via cannula, and subcutaneous or intramuscular depot deposition. In certain embodiments, the nanoparticle drug carrier and/or drug formulation is administered as an injection, from an IV drip bag, or via a drug delivery cannula. In various embodiments, the subject is a human and in other embodiments, the subject is a non-human mammal.

A kit.

In certain embodiments, the kit contains a lipid bilayer coated nanoparticle drug carrier as described herein for treating a pathology (e.g., cancer, microbial infection, viral infection, etc.). The kits typically include a drug-loaded silica body as described herein and/or an immunoconjugate comprising a drug-loaded silica body as described herein. In certain embodiments, the silica body contains irinotecan. In certain embodiments, the silica is attached to an iRGD peptide, while in other embodiments, the kit contains a separate iRGD peptide formulated for co-administration with a drug-loaded silica or a silica immunoconjugate (e.g., irinotecan).

Furthermore, in certain embodiments, the kits can include instructional materials disclosing the manner of use of the drug-loaded silica bodies or silica body immunoconjugates (e.g., as therapeutic agents for pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, etc.).

In addition, the kit optionally includes label materials and/or instructional materials that provide guidance (e.g., protocols) regarding the use of the nanoparticle drug carriers described herein, e.g., alone or in combination, for the treatment of various cancers. The instructional material may also include recommended dosages, instructions for contraindications, and the like.

Although the instructional materials in the various kits typically comprise written or printed materials, they are not limited thereto. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tape, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include an address of an internet website that provides such instructional material.

Examples

The following examples are provided to illustrate, but not to limit, the claimed invention.

Example 1

Irinotecan delivery of lipid-coated mesoporous silica nanoparticles showed to pancreatic cancer compared to liposomes

Improved efficacy and safety

Due to concerns that liposomal carriers may not improve the safety of highly toxic drugs, such as irinotecan, we focused on using a supported Lipid Bilayer (LB) applied to Mesoporous Silica Nanoparticles (MSNPs) to see if this would result in a more stable carrier (compared to liposomes). Due to the large internal surface area, tunable pore size, carrier stability and controlled drug release capacity that can be used for drug packaging, MSNP has proven to constitute a versatile and multifunctional Nano-carrier platform for cancer treatment (Lu et al (2007) Small,3: 1341-1346; slow et al (2008) adv. drug deliv. rev.60: 1278-1288; Meng et al (2010) j.am. chem.soc.132(36): 12690-12697; Lee et al (2010) angel w.chem.122: 8390-8395; Meng et al (2011) ACS Nano,5(5): 4131-4144; He and Shi (2011) j.mater.chem21: 5845-5855; Gao et al (2011) naacs, 5: 9788-9798; Li et al (song) son.2590: 2017-2013; Meng et al (2013) nac.31: 2013-2013; Meng et al (20123) nac.48: 2013-2013; Meng et al (20123-413; Meng et al (20123-2013), 7(11) 10048-10065; argyo et al (2014) chem.mater.26: 435-451; meng et al (2015) ACS Nano,9(4): 3540-3557). Furthermore, MSNP is biodegradable and proved safe in extensive animal testing (Meng et al (2011) ACS Nano,5(5): 4131-4144; Tang et al (2012) adv.mat.24(12): 1504-1534; Meng et al (2013) ACS Nano,7(2): 994-1005; tar et al (2013) acc.chem.res.46(3): 792-801; Meng et al (2013) ACS Nano,7(11): 10048-10065; Meng et al (2015) Nano,9(4): 3540-3557; Lu et al (2010) Small,6(16): 1794-18050). Although it is possible to introduce irinotecan or camptothecin into MSNPs by immersion, this approach is relatively inefficient (< 10% loading capacity by weight) (Lu et al (2007) Small,3: 1341-1346; He et al (2010) Biomaterials 31:3335-3346) and can lead to poor drug retention (Meng et al (2010) j.am. chem. soc.132(36): 12690-12697). However, we have developed a biofilm technology that can be used for rapid GEM encapsulation by supported LB (Meng et al (2015) ACS Nano,9(4): 3540-3557). Although a number of LB coating programs have been disclosed, including liposome interaction and fusion to the particle surface (Liu et al, (2009) j.am.chem.soc.,131(22): 7567-7569; Liu et al, (2009) j.am.chem.soc.,131(4):1354-1355) or solvent exchange using EtOH to disperse lipid solutions (Cauda et al (2010) Nano lett.10:2484-2492), the use of biofilm technology provides more reproducible and complete coating, rapid encapsulation and improved carrier stability (Meng et al (2015) ACS Nano,9(4): 3540-3557). This not only allows LB-MSNP to achieve loading capacities of up to 40 wt.% GEM, but it also enables co-delivery of Paclitaxel (PTX), which can be incorporated into LB (supra). This allowed us to develop synergistically and ratiometrically designed vectors for PDAC treatment in the orthotopic human PDAC model in mice (supra). Recently, Zhang et al reported polymer-lipid supported MSNPs for delivery of irinotecan, but the drug loading was lower (i.e., about 16 wt%) (Zhang et al (2014) Biomaterials,35: 3650-3665).

In this example, we introduced a novel design feature specifically for the LB-MSNP platform, where LB was used to encapsulate the protonating agent; this allows remote loading of high irinotecan drug loading, which takes advantage of the weakly basic properties of the drug (pKa 8.1). Furthermore, the increased capacity of supported LB also allowed us to conduct comparative analyses with liposome equivalents, where irinotecan was remotely loaded using unsupported LB vesicles via the trapping agent sucrose triethylammonium octasulfate (TEA8SOS) (Drummond et al (2006) Cancer Res.,66(6): 3271-3277; Von Hoff et al (2013) Br.J.cancer,109(4): 920-925). The MSNP vector not only achieved higher loading capacity and tumor killing than the liposome formulation in a robust in situ PDAC model, but it also prevented irinotecan toxicity due to improved vector stability and reduced leakage. This provides an innovative approach to reduce the toxicity of highly toxic chemotherapeutic agents through LB-coated vectors, which allows irinotecan to be used as a first-line treatment option for PDACs rather than being reserved for GEM treatment failure (current FDA-improved indications for the use of liposomal carriers).

Results

High irinotecan loading in MSNP was achieved via the use of coated LB and proton gradients.

Our first attempt was to produce MSNP vectors encapsulating irinotecan by an LB coating that provided rapid and uniform pore sealing (Meng et al (2015) ACS Nano,9(4): 3540-3557). This allowed passive encapsulation of 22 wt.% irinotecan (relative to MSNP weight). To further increase the loading capacity, we also determined whether encapsulation of the protonating agent converted amphiphilic irinotecan capable of diffusing through LB into hydrophilic derivatives that remained in the pores (fig. 1A, panel a 1). We and others have used this encapsulation procedure to efficiently introduce and encapsulate weakly basic drugs such as GEM and irinotecan in liposomes (Chou et al (2003) J.biosci.Bioeng.,95(4): 405-408; Meng et al (2013) ACS Nano,7(11): 10048-10065; Haran et al (1993) Biochim.Biophys.acta, biomembr.1151: 201-215). Although the polyanionic Compound TEA₈SOS has never been used previously in the MSNP platform, but it was used in MM-398 to encapsulate irinotecan (Drummond et al (2006) Cancer Res.,66(6): 3271-3277). TEA (TEA)₈SOS is a proton generating agent that releases eight upon hydrolysisH + ion and eight valent SOS^8-(FIG. 1A, FIG. A2). Production of TEA Using ion exchange chromatography₈SOS, immersed in MSNP as described below in the methods section (supra). The soaked particles were introduced into a round bottom flask coated with a lipid biofilm composed of DSPC/cholesterol/DSPE-PEG 2000 at a molar ratio of 3:2: 0.15. The optimal lipid to particle ratio was determined to be 1.1: 1.0. Sonication of the suspension resulted in LB-coated particles containing the encapsulant (fig. 1A, panel a 1). The particles were immediately incubated in irinotecan solution, allowing the drug to be introduced, protonated, and reacted with SOS^8-When associated, are encapsulated in the pores as gelatinous precipitates (fig. 1A, panels a1 and a2, fig. 1B). This allows us to achieve irinotecan loading capacities of up to 80 wt% (fig. 1B), which is approximately 850m²Combined surface area per g and about 0.7cm³The theoretical maximum loading capacity of the porous support per gram of pore volume (about 100% by weight) was as previously demonstrated (Meng et al (2015) ACS Nano,9(4): 3540-3557). Subsequent studies were performed using granules containing 50 wt% irinotecan.

To compare irinotecan-loaded LB-MSNP (Ir-LB-MSNP) with TEA₈Efficacy and safety of liposomal vectors for SOS liposomes (FIG. 1A, Panel A3) (Drummond et al (2006) Cancer Res.,66(6):3271-3277) were constructed by procedures similar to MM-398. This resulted in liposomes having a loading capacity of about 40 wt.% irinotecan (designated "Ir-liposomes"); this is significantly higher than the production of liposomes (about 5 wt%) in the absence of the encapsulant (fig. 1B). Cryoelectron microscopy (cryoEM) revealed that the particle sizes of liposomes and LB-MSNP were about 75nm and about 80nm, respectively (fig. 1C). High magnification cryoEM images confirmed that Ir-LB-MSNP was uniformly coated with a complete approximately 7nm thick bilayer. The cyroEM image with low magnification is displayed in>LB was intact in 99% of the particles (fig. 11). This confirms the reproducibility of the single-step encapsulation scheme, which is a significant improvement over other methods of double-layer coating of MSNP (Meng et al (2015) ACS Nano,9(4): 3540-3557).

Since our main hypothesis is that LB-MSNP can improve vector stability compared to liposomes, both vectors were incubated in serum and lyophilized to determine their drug retention capacity and irinotecan leakage. Both vectors were incubated in 100% serum at 37 ℃ for 24 hours with continuous and gentle shaking. Since adsorption of serum proteins interferes with cryoEM visualization, High Performance Liquid Chromatography (HPLC) analysis was used instead to determine drug release in the carrier suspension; this shows that LB-MSNP has < 6% of premature irinotecan release, compared to liposomes having about 22% of premature irinotecan release (fig. 1D). However, storage of the particles in serum-free suspension at 4 ℃ for 90 days showed that both carriers had high colloidal stability with less (< 5%) premature drug release or hydrodynamic size change (< 5%). In contrast, particle lyophilization in cryoprotective 5% dextrose solution 40 showed a significant difference in carrier stability upon resuspension in water (fig. 1E). Thus, while Ir-LB-MSNP did not change hydrodynamic size and only caused 2.7% of drug release, liposomes significantly aggregated and released 20.8% of their irinotecan content. This confirms that while LB-coated vectors appear morphologically similar, LB-MSNP is significantly more stable than liposomes.

LB-MSNP with improved biodistribution, Pharmacokinetics (PK), and irinotecan delivery compared to liposomes in Kras-derived in situ PDAC model

Biodistribution and PK studies were performed in immunocompetent B6/129 mice that were implanted in situ with a gene derived from the transgenic KrasLSL-G12D/+; trp53 LSL-R172H/+; luciferase-expressing Cell lines of spontaneous PDAC tumors in Pdx-1-Cre (KPC) animals (Hingorani et al (2005) Cancer Cell,7: 469-483). The KPC-derived in situ model mimics human PDAC in Kras mutations, expression of relatively large amounts of stroma, local tissue invasion, and metastatic development (Tseng et al (2010) Clin. cancer Res.16: 3684-3695; Provenzano et al (2013) Br. J. cancer,108: 1-8; Torres et al (2013) PLoS One,8: e 80580). Primary tumors develop within 2-3 weeks at the site of in situ implantation and metastasis occurs at about 5 weeks, which is in turn associated withThe literature is consistent (fig. 12) (supra). To follow the biodistribution of irinotecan vectors to the tumor site, animals were injected intravenously with Near Infrared (NIR) labeled (Dylight 680) liposomes or LB-MSNP 3 weeks after in situ implantation (fig. 2A). IVIS imaging was obtained at the indicated time points (columns 3-6, FIG. 2A) before (column 2) and after intravenous injection of 100mg/kg of labeled LB-MSNP or liposomes. This was combined with bioluminescent IVIS imaging to detect luciferase expression in developing tumors in animals receiving Intraperitoneal (IP) injection of D-luciferin (figure 2A, first column). Robust fluorescence intensity was observed at the tumor site within 2 hours of LB-MSNP injection, after which the signal lasted for at least 48 hours. In contrast, NIR signal intensity was darker and disappeared more rapidly in mice receiving liposome (with similar labeling efficiency) injections. This was also confirmed by ex vivo imaging of tumors and major organs collected from animals after sacrifice 24 hours after injection (fig. 2B). Ex vivo imaging was also performed after 48 hours, confirming that NIR signal intensity was stronger in the operator defined region of interest (ROI) in LB-MSNP compared to liposome treated animals (figure 13). In addition to the high particle uptake at the tumor site, the liver and spleen are also the major sites of particle distribution. Little signal conduction is obtained in the lungs, heart and kidneys. Inductively coupled plasma optical emission spectroscopy (ICP-OES) to quantify silicon (Si) abundance in tumor tissues and major organs showed that about 3% of the total administered elemental Si dose could be distributed to developing tumor sites (fig. 14). This is representative of liposomes, polymeric micelles, gelatin, or MoS₂/Fe₃O₄Nanoparticles compared to very good biodistribution, where the percentage of particles distributed to the PDAC site reached 0.2% -2.4% (Yoshida et al (2012) PLoS One,7: e 39545; Yu et al (2015) Theranostics,5: 931-945; Cabral et al (2011) nat. Nanotechnol.6: 815-823; Guo et al (2013) Biomaterials 34: 8323-8332; Xu et al (2013) mol. pharmaceuticals, 10: 2031-2044). We also obtained tumor sections for visualizing the intratumoral distribution of NIR-labeled particles by confocal microscopy; this showed higher particle abundance at the tumor site after 24 hours for LB-MSNP compared to liposomes (figure 2)B) In that respect To assess irinotecan content at the tumor site, 60mg/kg equivalent of drug was injected as a single dose in the form of free drug, Ir-LB-MSNP (60mg/kg drug; 120mg/kg pellet dose), or Ir-liposomes (60mg/kg drug; 150mg/kg pellet dose). Animals were sacrificed after 0.5 hours, 4 hours, 24 hours, and 48 hours for blood collection and tumor harvest. These tissues were used for HPLC analysis to quantify irinotecan tissue content, with Ir-LB-MSNP and Ir-liposome 5-fold and 2-fold higher than the free drug at 24 hours (fig. 2C). The circulating half-life of irinotecan was calculated by HPLC quantification using plasma drug concentrations, and LB-MSNP, liposomes and free drug were calculated as 11 hours, 8.7 hours and 1.0 hour, respectively (fig. 2D). The logistic limit on the volume of blood available from the mice makes it impossible to determine the blood residence time of the carrier. We also performed biodistribution and PK studies in the subcutaneous PANC-1 xenograft model, where we observed tumor drug content of Ir-LB-MSNP and Ir-liposome increased to 30-fold and 4-fold of the free drug, respectively (fig. 15). We also compared the drug content of individual organs (fig. 16). This shows that the drug content of Ir-liposomes in liver, spleen and intestinal tissues is 0.8 times, 0.2 times and 0.2 times that of Ir-LB-MSNP, respectively, at 24 hours after injection. No significant difference was seen for the kidneys.

Ir-LB-MSNP provided more effective killing than either free drug or Ir-liposomes in the in situ PDAC model.

To develop a dose exploration protocol for efficacy assessment, we used a protocol from the national Cancer institute to determine the Maximum Tolerated Dose (MTD) in healthy mice (Drummond et al (2006) Cancer res.,66(6): 3271-3277). The evaluation showed MTDs for free irinotecan, Ir-liposome and Ir-LB-MSNP were 60mg/kg, 295mg/kg and 295mg/kg, respectively (FIG. 3A). A drug dose of 40mg/kg (66.7% or 13.3% of MTD corresponding to the amount of free drug or encapsulated drug) was selected for the subsequent experiments. For Ir-LB-MSNP or Ir-liposomes, these doses correspond to nanoparticle doses of 80mg/kg or 100mg/kg, respectively. At 2X 10⁶First after in situ tumor implantation of individual KPC cellsIntravenous injection started on day 14; at this point, the average primary tumor size was about 5mm without macroscopic metastasis. Intravenous injections were repeated every 4 days for a maximum of 8 replicates (fig. 3B). The control group included animals that received intravenous saline only. In situ tumor growth was monitored by IVIS bioluminescence imaging at set time points (fig. 3B). Quantification of tumor growth by IVIS software determining imaging intensity in ROI showed that Ir-LB-MSNP treatment resulted in significantly slower tumor growth compared to saline, free drug, or Ir-liposome (fig. 3B). Anti-tumor efficacy was also measured as the rate of apoptosis in tumor explants collected from animals sacrificed at day 40-day 47. Using an Immunohistochemistry (IHC) staining protocol to detect activated (cleaved) caspase-3, we observed about 25% apoptotic cell death in Ir-LB-MSNP treated mice, compared to about 6% and about 11% apoptotic cell death for free irinotecan or Ir-liposome, respectively (fig. 3C).

A representative IHC image is shown in fig. 17. Although we did not proceed to formal survival studies, it was possible to compare the number of surviving animals at different stages of the study after 47 days using the kaplan-meier data display (fig. 18). This shows that the survival of both vectors is improved relative to the free drug, including that of Ir-LB-MSNP better than the liposome formulation (fig. 18).

Animal necropsies were performed to assess local tumor spread and the appearance of metastases (fig. 3D). In addition to direct tumor invasion of the stomach, intestine, liver, spleen, kidney, diaphragm, and abdominal wall, many macroscopic metastases can be seen in saline-treated animals, which also produce bloody ascites. Although free irinotecan did not affect the transfer, Ir-liposomes provided a modest decrease in bioluminescence intensity at the site of transfer (fig. 3D). In sharp contrast, Ir-LB-MSNP treatment exerted a strong inhibitory effect, as evidenced by a dim bioluminescence outside the primary tumor area during IVIS imaging (fig. 3D). This was particularly pronounced in the kidney region, which showed a higher, but not significant, content of irinotecan during HPLC analysis (fig. 16). The heat map in fig. 3E provides a quantitative display of the effect of treatment on tumor metastasis.

LB-MSNP improved the safety of irinotecan delivery in a mouse in situ model, but liposomes did not.

Since irinotecan causes FOLFIRINOX toxicity in a major manner, the possibility of reducing drug toxicity by encapsulation delivery is a major research goal. MTD evaluation showed that both vectors were able to increase the acute lethal dose by about 5-fold over the free drug (fig. 3A). To address irinotecan toxicity via encapsulated drug delivery (fig. 3B), liver, sternal bone marrow, and intestinal tissues were collected from animals sacrificed in efficacy studies. These are the major target organs for irinotecan toxicity in PDAC patients (Conroy et al (2011) n. engl. j. med.364(19): 1817-1825; Ueno et al (2007) cancer chemicother. pharmacol.59(4): 447-454; Loupakis et al (2013) br. j. cancer,108(12): 2549-2556). Histological examination of the liver revealed severe and extensive hepatocellular necrosis in animals receiving free drug treatment (fig. 4A). This reflects the presence of compact and fragmented nuclei in hepatocytes (Ziegler et al (2004) Newspysiol. Sci.19: 124-128). Although Ir-liposomes reduced the severity of liver injury to some extent, extensive liver necrosis was observed (fig. 4A). In contrast, Ir-LB-MSNP treated animals showed only slight steatosis, which is a reversible drug-induced stress response without necrosis (FIG. 4A) (King and Perry (2001) Oncoloist, 6: 162-176; Maor and Malnick (2013) int.J.Heatol.Art: 815105). Histological data were also confirmed by IHC analysis, in which liver tissue was used to stain Kuppfer Cells (KC) (anti-F4/80 antibody labeled with FITC), apoptotic cells were detected with RITC-labeled antibody (recognizing cleaved caspase-3) and the nuclei were stained with Hoechst 33342 (blue dye) (fig. 4B). Examination of the stained sections under a fluorescent microscope showed extensive apoptosis in the whole liver of animals receiving free drug treatment. Animals treated with Ir-liposomes showed less apoptosis, which often involved KC or adjacent liver tissue. In contrast, no apoptosis was seen in the liver of the animals treated with Ir-LB-MSNP.

Although H & E staining of the GI tract did not reveal microscopic evidence of toxicity in any of the treatment groups, IHC staining for cleaved caspase-3 showed the presence of apoptotic cells and blunting of intestinal villi caused by free drug (fig. 4C). While treatment with Ir-liposomes prevented blunting of villi, IHC staining revealed the presence of apoptosis in apical intestinal epithelial cells. This is in sharp contrast to treatment with Ir-LB-MSNP, which did not cause any apoptosis or villus damage (fig. 4C).

Bone marrow toxicity was studied in separate experiments in which animals received three injections of a 40mg/kg irinotecan dose equivalent, as described above. After sacrifice, sternal bone marrow was collected to assess bone marrow toxicity by H & E staining (fig. 4D) (Iusuf et al (2014) mol. cancer ther.13: 492-503). Although animals receiving free drug or Ir-liposome treatment showed extensive bone marrow damage, as evidenced by < 30% of the bone marrow cavities filled with hematopoietic cells (Travlos (2006) toxicol. pathol.34:566-598), bone marrow cell composition was unchanged in Ir-LB-MSNP treated animals (fig. 4D). Furthermore, bone marrow damage by free drug or Ir-liposomes was accompanied by peripheral blood neutropenia, whereas Ir-LB-MSNP treatment showed a non-significant drop in neutrophil count (fig. 19). Taken together, these data show a significant reduction in systemic toxicity and target organ toxicity during treatment with Ir-LB-MSNP, while the liposomal vector still exhibits considerable organ toxicity.

Discussion of the related Art

We developed a custom designed mesoporous silica nanoparticle platform using supported LB and proton-producing encapsulant for high-dose irinotecan loading and delivery and tested it in KPC-derived pancreatic cancer model in immunocompetent mice. The same loading procedure and collector (TEA) were used₈SOS) improved stability and loading capacity of the vector as compared to internally synthesized MM-398 liposome equivalentsThe biological distribution, the circulation half-life and the drug tumor content of the irinotecan are improved. Ir-LB-MSNP not only induces tumor killing more efficiently at the primary tumor site, it is also more active in treating metastases. Also importantly, the LB-MSNP vector did not induce significant systemic toxicity, in contrast to which liposomes had adverse effects on bone marrow, gastrointestinal tract, and liver. We attribute the reduced toxicity with maintained therapeutic efficacy to the increased carrier capacity of LB-MSNP compared to liposomes. Thus, the LB-MSNP vector provides an innovative approach for introducing irinotecan for the treatment of PDAC, potentially promoting this drug to serve as a first line of consideration rather than being reserved for patients with GEM treatment failure, as is currently the case with the recent FDA-improved liposomal vectors.

We have developed a robust MSNP vector for irinotecan with morphological similarity to the liposome equivalent. However, in addition to the superior drug encapsulation provided by intact supported LB, LB-MSNP also allows dense drug packaging against porous silica sidewalls due to bonding and/or electrostatic interactions (Tarn et al (2013) acc.chem.res.46(3): 792-801; Meng et al (2010) ACS Nano,4: 4539-4550; Ashley et al (2011) nat. mater.,10: 389-397). This allows us to achieve an increased irinotecan loading capacity compared to liposomes, which provides superior killing efficiency at the tumor site in situ. The increased loading capacity is further aided by the supported LB stability, which improves cargo protection during blood circulation and biodistribution to the tumor site compared to liposome bilayers. We propose that the reduced fluctuations in supported LB, presumably due to robust electrostatic interactions and van der Waals interactions with the particle surface, reduces lipid loss due to interaction of serum proteins with unsupported lipid bilayers (liposomes). Supported LB may also protect the vector from complement-mediated lysis and shear forces generated during blood flow (Liu et al (2000) In: Colloids and Surfaces A: physiochemical and engineering assays, 172(1-3): 57-67; Heurtault et al (2003) Biomaterials,24(23): 4283-4300; Ashley et al (2011) nat. mater, 10: 389-397; Liang et al (2004) J. Colloidi interface Sci.278: 53-62; Anderson et al (2009) Langmuir,25: 6997-7005; Michel et al (1162012.J.mol. Sci.13: 10-11642; Wang et al (3910: 201427-3931). For example, doxorubicin-loaded DOPC liposomes exhibit about 40% drug leakage within 24 hours at 37 ℃ in a simulated serum-containing biofluid environment (Ashley et al (2011) nat. mater.,10: 389-397). This is consistent with our findings, i.e., although Ir-liposomes were effective as nanocarriers, they were significantly less stable in whole serum or after particle lyophilization and resuspension than Ir-LB-MSNP (fig. 1D and 1E). The premature release of highly toxic topoisomerase inhibitors from Ir-liposomes in animal studies is also accompanied by high toxicity rates in bone marrow, liver and intestine, which is quite different from the protective effect of Ir-LB-MSNP. Thus, despite morphological similarities to liposomes, LB-MSNP vectors offer significant advantages over liposomal irinotecan delivery from both efficacy and drug toxicity perspectives.

The reduced bone marrow and gut toxicity of Ir-LB-MSNP is notable from the point of view of the recent FDA-approved Onevyde of the MM-398 liposomal formulation with a black box warning of possible severe neutropenia and diarrhea during treatment of PDAC patients (see, e.g., www.fda.gov/newsevents/newsrom/presentations/ucinancies/ucm468654. htm; www.accessdata.fda.gov/drugsatfda _ docs/label/2015/207793LB. pdf). The warning states that fatal neutropenic sepsis is observed in 0.8% of patients, while severe or life-threatening neutropenic fever occurs in 3% (www.accessdata.fda.gov/drug atfda _ docs/label/2015/207793lb. Life-threatening neutropenia was observed in 20% of patients receiving onivde in combination with 5-fluorouracil and leucovorin (supra). The same drug combination also caused severe diarrhea in 13% of treated subjects (supra). Although we did not use Onvyde for comparison, TEA was used₈Internal liposomal formulations of SOS for irinotecan loading were associated with significant bone marrow and intestinal toxicity, similar to free drug. In useSimilar effects were not seen during Ir-LB-MSNP platform treatment.

Interestingly, both MSNP and liposome carriers were sequestered by KC in the liver as confirmed by confocal microscopy using NIR-labeled particles and FITC-labeled anti-F4/80 antibody (not shown). Although difficult to provide direct in vivo validation, we propose that disintegration of LB and irinotecan release begin in the acidic endosomal/lysosomal compartment of KC (Hwang et al (1980) Proc. Natl. Acad. Sci. USA,77: 4030-4034; Derksen et al (1988)Biochim.Biophys.Acta,Bioenerg971: 127-136; kolter and Sandhoff (2010) FEBSLett.584: 1700-1712). The faster disintegration of the liposomes may explain why only rare NIR-labeled particles were seen in animals receiving Ir-liposome injection compared to the presence of large amounts of Ir-LB-MSNP (fig. 2B). The rate at which the carrier disintegrates can in turn determine the rate of drug release to bystander hepatocytes. This increases the likelihood that the burst release of irinotecan from the liposome carrier may exceed the liver metabolism of the drug by CYP 3A and uridine diphosphate glucuronosyltransferase (UGT1A1) (Mathijssen et al (2001) Clin. cancer Res.7: 2182-2194). The reduced metabolism may result in higher retention of non-metabolized drug, which may cause hepatocyte damage and necrosis, as shown in fig. 4A. This may explain that the content of unconjugated irinotecan in the liver tissue of the animals receiving Ir-liposome treatment was significantly higher compared to Ir-LB-MSNP 24 hours after intravenous injection, i.e. 26% ID/g compared to 15% ID/g (fig. 16). These findings were also compatible with IHC staining of KC and activated caspase-3, which showed high cell mortality in KC and neighboring hepatocytes during treatment with Ir-liposomes, in contrast to the absence of apoptosis in Ir-LB-MSNP treated animals (fig. 4B). The schematic In fig. 4E summarizes our hypothesis on the role of carrier stability and the rate of irinotecan release from KC In determining the extent of hepatocyte apoptosis and necrosis (Zhao et al (2015) In vivo. sci. fill., 60: 3-20). It is not possible to measure liver enzymes because of the limited amount of blood that can be obtained from the animal in a moribund state.

GEM is often used as a first-line treatment for PDAC with a survival outcome of 6.8 months (Burris et al (1997) j. clin. oncol.,15: 2403-2413; Teague et al (2014) the r.adv. med. oncol.7: 68-84). Although FOLFIRINOX can prolong survival to about 11 months, the frequent occurrence of severe drug toxicity (primarily due to irinotecan and oxaliplatin) prevents its use as a first-line therapy. Therefore, FOLFIRINOX is often reserved for patients with a well-behaved state. Unfortunately, this status did not change with the introduction of onivde, which is not approved as a first line therapy and includes a black box warning of severe diarrhea and neutropenia. Since about 80% of PDAC patients are in the advanced stages of the disease, it would be advantageous to use the drug in the FOLFIRINOX regimen as first-line treatment. Although we did not use the Onevyde for comparative analysis, the Ir-LB-MSNP platform was more potent and biocompatible with lower systemic toxicity than our equivalent internal Ir-liposome formulation. We therefore propose that the Ir-LB-MSNP platform can be developed for first line therapy of PDAC. We have recently demonstrated the feasibility of dual delivery of MSNP vectors for GEM and PTX in a PDAC model (Meng et al (2015) ACS Nano,9(4): 3540-3557). Each treatment should be carefully designed before implementation with regard to design complexity, cost, and impact on good manufacturing specifications (Sugahara et al (2010) Science,328: 1031-1035).

Although lipid-coated MSNPs have been shown to be effective for in vitro and/or in vivo cargo delivery (Meng et al (2015) ACS Nano,9(4): 3540-3557; Zhang et al (2014) Biomaterials,35:3650-3665 l; Ashley et al (2011) nat. mater, 10: 389-397; Ashley et al (2012) ACS Nano,6: 2174-2188; Dengler et al (2013) j.controlled Release,168:209-224), this is the first demonstration of how protonating agents can be used to increase the loading efficiency of the carrier platform. We have also identified a comprehensive list of weakly basic drugs that can be loaded into LB-MSNP via proton gradients. General characteristics of these cargo molecules include the following chemical properties:

(i) an organic molecular compound comprising one or more primary, secondary, tertiary or quaternary amines;

(ii) pKa <11 to allow protonation and encapsulation behind LB (Zucker et al (2009) j.control. release,139(1): 73-80; Cern et al (2012) j.control. release,160(2): 147-157; Xu et al (2014) pharmaceut. res.31(10): 2583-2592);

(iii) solubility in water in the range of about 5mg/mL to 25mg/mL and an amphiphilic character that allows diffusion through the LB;

(iv) -an octanol/water partition coefficient or log P value of 3.0 to 3.0; 71,72(v) suitable molecular weight and geometry smaller than the MSNP pore size (2nm-8nm) to allow access into the MSNP pores (Li et al (2012) chem.soc. rev.41: 2590-2605; Tang et al (2012) adv.mat.24(12): 1504-1534; tar et al (2013) acc.chem.res.46(3): 792-801).

Not all, but a list of potential chemotherapeutic agents include topoisomerase I inhibitors: topotecan; anti-tumor anthracycline antibiotics: doxorubicin and mitoxantrone; mitotic inhibitors: vinblastine and vinorelbine; tyrosine kinase inhibitors: imatinib, oxitinib, sunitinib, and the like. The ability to package and deliver one or a combination of the above agents will enhance the broader utility of our multifunctional LB-MSNP platform, including therapeutic considerations for additional cancer types, such as colon cancer, breast cancer, lung cancer, liver cancer, glioma, melanoma, and the like. It is also possible to co-package the drug combinations in the above list into a single carrier. For example, based on the success we have achieved with our GEM/PTX co-delivery platform (Meng et al (2015) ACS Nano,9(4):3540-3557), it is possible to consider combining drugs in the FOLFIRINOX regimen (e.g., oxaliplatin and irinotecan) for synergistic and ratiometric delivery. In addition, drug loading by our LB-MSNP can be used for non-cancerous applications, such as encapsulation of antibiotics for infectious diseases, such as ciprofloxacin, levofloxacin, or HIV antiretroviral drugs (e.g. tenofovir disoproxil fumarate). Except that TEA₈In addition to SOS, it is also worth noting that the transmembrane pH gradient may also be determined byGenerating: acidic buffers (e.g., citrate) (Chou et al (2003) J.biosci.Bioeng.,95(4): 405-408; Nichols et al (1976) Biochim.Biophys.acta, biomembr.455: 269-271); dissociable salts that produce protons (e.g., (NH)₄)₂SO₄) (Haran et al (1993) Biochim.Biophys.acta, biomembr.1151: 201-215; Maurer-Spurej et al (1999) Biochim. Biophys. acta, biomembr.1416: 1-10; fritze et al (2006) Biochim.Biophys.acta, Biomembr.1758: 1633-1640); or from metal salts (e.g. A23187 and MnSO)₄) Ionophore-mediated ion gradients (messererer et al (2004) clin. cancer Res,10(19): 6638-6649; ramsay et al (2008) Eur.J.pharm.Biopharm.68(3): 607-617; fenske et al (1998) Biochim. Biophys. acta, biomembr.1414: 188-204). Furthermore, it is possible to generate a reverse pH gradient for drug loading, such as using a calcium acetate gradient to increase amphipathic weak acid loading in LB-MSNP, a strategy that has been used for liposomes (Avnir et al (2008) Arthritis rheum.58: 119-129).

Conclusion

In summary, we have developed a MSNP delivery platform for irinotecan that, despite its morphological similarity to liposomes, provides biocompatibility and therapeutic efficacy superior to that of either equivalent liposomal formulations or free drug. The novel vectors can be used to elevate the drug combination in irinotecan delivery and FOLFIRINOX regimens to first line therapy positions for PDAC.

Method of producing a composite material

A material.

Tetraethylorthosilicate (TEOS), triethanolamine, cetyltrimethylammonium chloride solution (CTAC, 25 wt% in water), Triethylamine (TEA), Dowex 50WX8 resin, Sepharose CL-4B, and Sephadex G-25 were purchased from Sigma-Aldrich, USA. 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (ammonium salt) (DSPE-PEG2000), and cholesterol (Chol) were purchased from Avanti Polar Lipids, Inc. of the United states. Sucrose Octasulfate (SOS) sodium salt was purchased from Toronto Research Chemicals, Canada. Irinotecan hydrochloride trihydrate was purchased from LC Laboratories, inc. Penicillin (penicillin), streptomycin (streptomycin), and Dulbecco's Modified Eagle Medium (DMEM) were obtained from Invitrogen. Fetal Bovine Serum (FBS) was purchased from Gemini Bio Products, Inc. Rabbit mAb antibody (cat # 9664) to detect activated (cleaved) caspase-3 was purchased from Cell Signaling. Matrigel matrix basement membrane was purchased from BD Bioscience. Centrifugal filter units (cut-off size: 10K and 30K) were purchased from EMD Millipore. All chemicals were used directly without further purification.

Irinotecan-loaded LB-MSNP was prepared using the capture reagent.

MSNP was synthesized.

The MSNP core was synthesized by slightly modifying our previous sol/gel procedure (Meng et al (2015) ACSNano,9(4): 3540-3557). To synthesize a batch of about 500mg MSNP, 50mL of CTAC and 150mL of H were combined₂O was mixed in a 500mL Erlenmeyer flask, followed by stirring at 350rpm at 85 ℃ for 15 minutes. After this, 8mL of 10% triethanolamine was added at the same temperature for 30 minutes. Then, 7.5mL of silica precursor TEOS was added dropwise at a rate of 1mL/min using a peristaltic pump. The solution was stirred at 350rpm for 20 minutes at 85 ℃ to cause the formation of particles having a major dimension of about 65 nm. The surfactant can be removed by washing the particles with a mixture of methanol/HCl (500:19v/v) for 24 hours at room temperature. The particles can be centrifuged at 10000 rpm for 60 minutes and washed three times in methanol.

The collector agent TEA8SOS was synthesized.

Synthesis of TEA from commercially available SOS sodium salt by ion exchange chromatography₈SOS (Drummond et al (2006) Cancer Res.,66(6): 3271-3277). Briefly, 500mg of the salt was dissolved in 1mL Deionized (DI) water to give a 500mg/mL aqueous solution. The solution was passed through a 8cm by 1.5cm cation ion exchange resin (Do) prepared in deionized waterwex 50WX 8). The SOS salt was added to the column and eluted with water to convert to sucrose octasulfonic acid (SOS). The acidic SOS eluate was immediately titrated with pure triethylamine to reach a final pH of 5.8. This causes the formation of TEA8 SOS. The salt concentration was adjusted to about 80mM and stored in a refrigerator at 4 ℃ prior to use.

TEA8SOS was loaded and encapsulated by LB in MSNP.

We used a new approach to encapsulate TEA8SOS in MSNP via the use of an LB coating (Meng et al (2015) ACS Nano,9(4): 3540-3557). 100mg of empty MSNP was sonicated with a probe at 4mL of 80mM TEA₈The synthesis was started by soaking in an aqueous SOS solution for 5 minutes, using an on/off duty cycle of 15 seconds/15 seconds and a power output of 32.5W. The particle suspension was immediately added to the coated lipid biofilm obtained by adding 110mg of a mixture of DSPC/Chol/DSPE-PEG2000 (molar ratio: 3:2:0.15) suspended in chloroform at 10mg/mL to the bottom of a 6cm round bottom flask. This corresponds to a particle to lipid ratio of 1.0: 1.1. After evaporation of the solvent in a rotary evaporator at room temperature, we obtained a solution with about 30cm²The surface area of (a). After the particle suspension was added to the biofilm, the probe was sonicated for 30 minutes with a 15 second/15 second on/off duty cycle at a power output of 32.5W. Size exclusion chromatography was performed to remove unencapsulated TEA by elution on an agarose gel CL-4B column (1.5 cm. times.15 cm) using HEPES buffered dextrose solution (5mM HEPES, 5% dextrose, pH6.5)₈SOS。

₈Irinotecan loading was performed using TEASOS loaded LB-MSNP.

Irinotecan was dissolved at 10mg/mL in HEPES buffered dextrose (5mM HEPES, 5% dextrose, pH6.5) and reacted with TEA-loaded₈The particles of SOS were mixed to achieve an irinotecan/MSNP ratio of 1:1 (w/w). The mixture was incubated in a water bath at 65 ℃ for 30 minutes and then quenched in ice water for 10 minutes. The drug-loaded particles were washed and purified three times by centrifugation at 15000 rpm for 10 minutes. Washing the granules as describedShown resuspended in water, saline, or PBS.

Comparative liposomal formulations for irinotecan delivery were prepared.

Irinotecan encapsulation by LB-MSNP in the absence of a trapping agent.

Empty MSNP (40mg) was added to 4mL of a 10mg/mL solution of irinotecan (in water) under sonication, followed by stirring at room temperature for 4 hours. The drug was then encapsulated by LB using sonication of lipid biofilms with similar composition as described above. After probe sonication, the particles were purified by centrifugation and washing and resuspended as described above.

Irinotecan was liposome-encapsulated using TEA8 SOS.

To develop an internal analogue of the MM-398 formulation (Drummond et al (2006) Cancer Res.,66(6): 3271-3277; Ko et al (2013) Br. J. Cancer,109:920-925), 212mg of a lipid mixture containing DSPC/Chol/DSPE-PEG2000 (in a molar ratio of 3:2: 0.015) was dissolved in 0.4mL of ethanol at 65 ℃. At the same temperature, 4ml of TEA was added₈The SOS solution (80mM) was mixed with the lipid ethanol solution and then sonicated in a water bath for 2 minutes. The lipid suspension was extruded 15 times through a polycarbonate membrane with a pore size of 0.1 μm at 65 ℃. Removing unencapsulated TEA by size exclusion chromatography (Sepharose CL-4B)₈SOS, eluted with HEPES buffered dextrose solution (5mM HEPES, 5% dextrose, pH 6.5). Irinotecan solution prepared at 10mg/mL in HEPES buffered dextrose (5mM HEPES, 5% dextrose, pH6.5) was combined with TEA-loaded₈A liposomal suspension of SOS (irinotecan/lipid ═ 1:2.1w/w) was mixed and incubated in a water bath at 65 ℃ for 30 minutes. The sample was immediately quenched in ice water for 10 minutes. Free drug was removed using Sephadex G-25 column and eluted with HEPES buffered saline (5mM HEPES, 145mM NaCl, pH 6.5).

Irinotecan liposome vectors were synthesized in the absence of a trapping agent.

We have also synthesized irinotecan delivery liposomes using passive encapsulation methods rather than encapsulants. Briefly, the lipid mixture (53mg lipid, DSPC/Chol/DSPE-PEG2000 at a molar ratio of 3:2: 0.015) was dissolved in 0.1mL ethanol at 65 ℃.1mL of irinotecan solution (10mg/mL in water) was mixed with the lipid ethanol solution at 65 ℃ followed by sonication for 2 minutes. The lipid suspension was extruded 15 times through a polycarbonate membrane with a pore size of 0.1 μm at 65 ℃. The drug-loaded liposomes were purified using the same method as described above.

And (5) physical and chemical characterization.

The morphology, size distribution, shape and surface charge of all particles used in this example were broadly characterized. The uniformity of the LB coating and its integrity were characterized by TEM (JEOL 1200-EX) and cryoEM (TF20 FEI Tecnai-G2). Particle size and zeta potential were measured by a ZETAPALS instrument (Brookhaven Instruments Co.). These measurements were performed using nanoparticles suspended in water, PBS, and cell culture medium plus 10% FBS at a particle concentration of 100 μ g/mL.

The drug loading capacity of each vehicle was determined by a subtractive method as previously described (Meng et al (2015) ACSNano,9(4): 3540-3557). Briefly, the amount of unencapsulated irinotecan (M) detected during purification was determined by OD (360nm) in a microplate reader (M5e, Molecular Device, usa) or via the use of HPLC (Raytest, germany)₁). Drug loading capacity is defined as the total amount of DLC ═ irinotecan (m)₀) Unencapsulated irinotecan (m)₁)]/[ Total amount of particles (mMSNP or m lipids)]X 100%. Drug retention stability was tested in 100% FBS at 37 ℃ over 24 hours. Briefly, 40. mu.L of drug-loaded NP (irinotecan: 10mg/mL) was added to 1mL of serum at 37 ℃ with continuous shaking. The mixture was centrifuged at set time intervals using a centrifugal filter separation device (molecular weight cut-off: 10K). The released irinotecan was quantified in a plate reader or by HPLC. Serum filtrate without NP was used as a control. Both vehicles were also tested via the lyophilization procedureStability of the body. Briefly, Ir-MSNP was suspended in 5% dextrose. Samples were lyophilized overnight at-60 ℃ and stored in-80 ℃ refrigerator. The stored samples were resuspended in water by gentle vortexing, after which the suspension solutions were characterized for size, zeta potential, and drug release as previously described.

And (5) culturing the cells.

Derived from the transgene KrasLSL-G12D/+; trp53 LSL-R172H/+; immortalized cell lines of spontaneously occurring tumors in Pdx1-Cre mice were provided by Andrew Lowy doctor of San Diego, university of california, university (UC San Diego). PANC-1 cells were purchased from the American Type Culture Collection (ATCC). Both cell types were cultured in DMEM containing 10% FBS, 100U/mL penicillin, 100. mu.g/mL streptomycin, and 2 mML-glutamine. To allow imaging of bioluminescent tumors in the IVIS system, both cells were permanently transfected with a luciferase-based lentiviral vector in the UCLA vector core facility as previously described (Meng et al (2015) ACS Nano,9(4): 3540-3557). After performing limiting dilution protocols to select single cell clones, the PDAC model was developed using KPC-luc and PANC-1-luc cell populations.

Assessment of maximum tolerated dose.

The MTD of the free irinotecan formulation and the encapsulated irinotecan formulation was determined using a protocol from the Toxicology and pharmacology division of the national Cancer institute (Toxicology and pharmacology Branch) (dtp. nci. nih. gov/branches/tpb/default. htm) (Drummond et al (2006) Cancer res.,66(6): 3271-3277). Two healthy male BALB/C mice were injected intravenously with 50mg/kg (dose of C1) of an equivalent amount of free irinotecan or encapsulated irinotecan. After this, a dose escalation factor of 1.8 was used to obtain a Cn dose, which resulted in death of the animals within 24 hours after the last administration. The second round of dose exploration started with Cn-1 dose and utilized an increasing factor of 1.15 (n-2) to find the MTD, which was defined as the absence of mortality or morbidity. MTD was validated by injection into six mice, which were followed for 15 days to determine the absence of morbidity, mortality, or > 15% weight loss.

KPC-derived in situ tumor models were established in immunocompetent mice.

Female B6/129 mice (approximately 8 weeks) were purchased from The Jackson Laboratory and maintained under pathogen-free conditions. All Animal experiments were performed using protocols approved by the UCLA Animal Research Committee (UCLA Animal Research Committee). To grow the xenografts in situ, mice were anesthetized with isoflurane followed by intraperitoneal injection of 50mg/kg ketamine (ketamine) and 10mg/kg xylazine (xylazine). The surgical site was shaved to leave a 1cm margin around the incision site and sterilized by scrubbing with povidone iodine (betadine) and 70% ethanol. Mice were positioned on a heating pad for surgery and the incision site in the left abdomen was covered with sterile gauze. Making a 0.5cm-0.7cm surgical incision to expose the pancreas injection site, and then passing through a 27 gauge needle to contain 2X 10⁶50 μ L of DMEM/matrigel (1:1v/v) of individual KPC-luc cells was injected into the pancreatic tail. The fascia layer was closed with absorbable suture (PDS II, Ethicon) and the skin was closed with non-absorbable suture (PROLENE, Ethicon). Mice were kept on warming pads until recovery from anesthesia was complete, and then transferred to a clean cage. Efficacy studies were performed in tumor-bearing mice about 2 weeks after implantation, when the primary tumor had grown to about 0.5cm without evidence of macroscopic tumor metastasis. For biodistribution experiments, tumor-bearing mice were used 3 weeks after tumor implantation, at which time the primary tumor had grown to a size of about 0.8 cm.

Systemic and intratumoral biodistribution of NIR-labeled nanoparticles injected intravenously.

IVIS (Xenogen) in vivo imaging system was used to study the biodistribution of NIR-labeled MSNPs and liposomes in KPC-derived in situ models (n-3 mice). NIR labeling was performed by incorporating 0.1% w/w Dylight 680-DMPE in LB of both the drug-free liposome vector and the MSNP vector (Meng et al (2015) ACS Nano,9(4): 3540-3557). For bioluminescence imaging at the tumor site, mice were injected intraperitoneally with 75mg/kg of D-fluorescein. Reference fluorescence images of tumor-bearing mice were taken prior to particle injection. NIR images were obtained within 48 hours in animals receiving an intravenous injection of 100mg/kg of NIR labelled particles. After sacrifice of the animals, ex vivo images of excised tumors and major organs were obtained to quantitatively assess particle biodistribution. A small portion of tumor tissue was cryo-embedded using OCT reagents and used to prepare tumor sections. The intra-tumoral distribution of NIR-labeled particles was investigated by confocal microscopy (SP2-1P-FCS, Leica) using a NIR laser. The same sections were nuclear stained by using Hoechst 33342 dye (blue). In animals receiving LB-MSNP treatment, tumor tissues and major organs were also used to assess Si content by ICP-OES. Particle biodistribution is expressed as the total injected dose% (ID%), which can show distribution to individual organs. To measure irinotecan content in tumors or other normal tissues, animals were injected with 60mg/kg of free irinotecan or encapsulated irinotecan (particle dose: 120mg/kg of MSNP and 150mg/kg of liposomes) and then sacrificed after 24 hours. HPLC analysis was performed on the harvested tumor tissue, liver, spleen, kidney, intestine and blood to determine irinotecan content.

And (4) HPLC analysis.

Harvested tumor and organ samples were weighed and homogenized on ice. After 0.1mL of plasma or tissue homogenate was extracted with 0.4mL of acidic solution (0.1mol/L phosphoric acid/methanol, 1:4v/v), the extract was vortexed twice for 10 seconds and centrifuged at 13000 rpm for 10 minutes. The supernatant containing irinotecan was filtered through a 0.22 μm filter for HPLC analysis in a system containing a Knauer Smartline pneumatic pump, a C18 column, a K-2600 spectrophotometer, and Gina data collection software. The mobile phase delivered at a flow rate of 1.00 ml/min contained 3% triethylammonium acetate aqueous buffer (pH 5.5) and acetonitrile (73:27v/v) (Noble et al (2006) Cancer Res.,66(5): 2801-2806). A 20 microliter sample containing irinotecan was injected to measure drug absorption at 254nm, eluting typically within 4.4 minutes. Irinotecan standard curves were generated over a concentration range of 0.05. mu.g/mL to 100. mu.g/mL. Irinotecan content is expressed as the injected dose per gram of tissue (% ID/g).

Efficacy evaluation using KPC-derived in situ model.

Tumor-bearing B6/129 mice were randomly assigned to four groups, each group including eight animals. Mice were injected intravenously with either free irinotecan or encapsulated irinotecan to receive a 40mg/kg dose once every 4 days (i.e., a liposome dose of 100mg/kg or 80mg/kg of MSNP). A maximum of eight injections were administered over a 28 day observation period. Saline injection was used as a negative control. Tumor burden was monitored by IVIS imaging once every 8 days and quantified as bioluminescent imaging intensity in the operator defined ROI. Statistical analysis of the differences between groups was performed using the t-test (Excel software, Microsoft corporation). All surviving mice were sacrificed on day 47, at which time all animals in the saline and drug-free groups had died. Tumor tissues and major organs (gastrointestinal tract, liver, spleen, heart, lung, and kidney) were harvested for quantification of ex vivo bioluminescence image intensity.

The toxicity potential of different irinotecan formulations was compared.

The major tissues collected during the efficacy testing experiment (liver, kidney, spleen, stomach and intestine) were fixed in 10% formalin followed by paraffin embedding. Tissue sections were stained by H & E for histological analysis and also for IHC analysis of the expression of activated (cleaved) caspase-3. Slides were read blindly by an experienced veterinary pathologist. The second method for assessing bone marrow toxicity used three intravenous administrations (2 days apart) of free drug and carrier formulation at 40mg/kg in healthy mice. Animals were sacrificed on day 7 and sternums were used for fixation in 10% formalin, decalcification, paraffin embedding, and H & E staining. We also collected blood for differential white blood cell count by the UCLA Laboratory Animal Medicine (DLAM) diagnostic Laboratory service. To understand the mechanism of differential hepatotoxicity of free drug and vehicle, mice receiving a dose equivalent of 60mg/kg irinotecan were sacrificed after 24 hours in a single injection. Liver tissues were cryo-embedded for identification of KC and cleaved caspase-3 using FITC-labeled anti-F4/80 antibody or RITC-conjugated secondary antibody, respectively, for immunofluorescence staining for expression of KC and activated caspase-3. Hoechst 33342 dye was used to localize nuclei in the same sections. The stained slides were examined under a confocal microscope (Observer D1, Zeiss).

And (5) carrying out statistical analysis.

Comparative analysis of differences between groups was performed using a two-sided Student's t-test (Excel software, Microsoft corporation). Statistical significance differences were determined at p < 0.05. Values are expressed as mean ± SD or SEM of multiple determinations, as described in the figure legend.

Example 2

Pancreatic Ductal Adenocarcinoma (PDAC) is an incurable disease with a 5-year survival rate of less than 5%. Currently, first-line chemotherapy for PDAC includes treatment with Gemcitabine (GEM) (monotherapy) or using a four-drug regimen that includes leucovorin, 5-FU, oxaliplatin, and irinotecan, also known as FOLFIRINOX. Although FOLFIRINOX has an increased PDAC response rate compared to GEM, i.e. 31.6% compared to 9.4%, this regimen is much more toxic and therefore limited to a few advanced PDAC patients with good functional status (Conroy et al (2011) n.engl.j.med.364: 1817-1825). Irinotecan causes FOLFIRINOX toxicity in a major manner because it has adverse effects on bone marrow (e.g., incidence of neutropenia in about 60%) and gastrointestinal tract (e.g., vomiting, diarrhea, nausea, anorexia in about 50% of patients) (Ueno et al (2007) Cancer chemother. pharmacol.59(4): 447-454). There is an unmet need for the development of FOLFIRINOX regimens with lower toxicity, including reducing the toxicity of irinotecan.

Delivery of Ic Using nanoparticle CarriersCetikang provides a potential solution to reduce the toxicity of this drug, as demonstrated by the delivery of irinotecan with: liposomal nanocarriers (Messerr et al (2004) Clin. cancer Res,10(19): 6638-6649; Drummond et al (2006) cancer Res, 66(6):3271-3277) or polymer-based nanocarriers (Onishi et al (2003) biol. Pharmaceut. Bull.26(1): 116-119). At the preclinical level, the benefits of using these nanocarriers include reduced toxicity, increased anti-tumor efficacy, and improved survival in various murine PDAC models. To date, only a few irinotecan carriers have been tested in clinical trials, including passage through the ionophore A23187(Irinophore C) or the protonated encapsulating agent triethylammonium sucrose octasulfate (TEA)₈SOS) facilitate drug loading of the liposomal formulation. Therapeutic efficacy of MM-398 depends on TEA via the use of a polyvalent anion-trapping agent₈Encapsulation and entrapment of irinotecan by SOS in liposomes. TEA (TEA)₈SOS causes protonation of irinotecan that diffuses through the lipid bilayer, converting it to a hydrophilic component that cannot leave the lipid bilayer. The drug is thus trapped at a concentration that exceeds 10 times the passive encapsulation of the liposome bilayer (Drummond et al (2006) Cancer res, 66(6): 3271-3277). MM-398 is currently in phase 3 trials conducted by Merripack Pharmaceuticals (www.merrimackpharma.com) (Hoff et al, poster introduction to Society for Medical Oncology, 2014). The poster can be viewed at// merrimackpharma. com/sites/default/files/documents/esmogi2014mm398. pdf). In the AACR 2012 meeting, Merrimack corporation published a summary containing the following statements: "MM-398 induces tumor regression in various mouse pancreatic cancer models, including in situ metastasis models". Subcutaneous models include BxPC3, AsPC-1, Panc-1, and MiaPaCa. In situ implantation in the pancreas was performed using BxPC 3. No detailed data is provided. Intravenous administration of MM-398 has been shown to induce complete tumor regression in various PDAC tumor models in mice, including inhibition of metastatic tumor lesion formation (Drummond et al (2006) Cancer Res.15(66): 3271-3277). Treatment of 417 patients with a combination of MM-398, 5-FU and leucovorin in a phase 3 PDAC trial showed 1.9 months compared to a human control receiving 5-FU and leucovorinIn contrast, overall survival was extended (6.1 months) (Hoff et al, ESMO poster, www.merrimackpharma.com).

In this example, we describe a novel Mesoporous Silica Nanoparticle (MSNP) platform that enables active loading, encapsulation, and encapsulation of irinotecan by nanocarriers with superior capabilities compared to liposome equivalents. These particles were synthesized by coupling TEA to the lipid bilayer LB via a support₈SOS is rapidly encapsulated in the porous interior of the particles. Subsequent incubation of these particles in irinotecan solution allowed active encapsulation of irinotecan, which diffused through the bilayer and was then TEA₈The SOS is protonated. Comparative analysis was performed between irinotecan-loaded LB-MSNP and the internally synthesized liposome equivalent of MM-398. This analysis confirmed the superior loading capacity, loading efficiency and release profile of the MSNP vector. We also demonstrated similar in vitro efficacy and in vivo toxicity reduction compared to the internal MM-398 liposome. We are currently conducting animal efficacy studies to compare these vectors in subcutaneous and orthotopic human PDAC tumor models.

We believe that LB-MSNP-mediated nanocapsulation and targeted delivery of irinotecan would enhance the clinical use of FOLFIRINOX by reducing toxicity, including reducing irinotecan toxicity in the bone marrow and gastrointestinal tract. This discovery enables an intravenously injectable, efficacious, biocompatible, and commercially competitive formulation of irinotecan over liposomal carriers due to loading capacity and sufficient drug release at the cancer site. In certain embodiments, the invention can also be used as an efficient design principle for the delivery of other weakly basic molecules (pKa >7.0) that can be used for cancer therapy as well as for the treatment of other diseases.

The following description illustrates certain embodiments of vector design for irinotecan loading and comparative analysis of LB-MSNP vectors with internally synthesized liposome formulations similar to MM-398.

Irinotecan encapsulation by LB-MSNP further comprising a trapping agent to achieve additional active loading

To provide an effective loading of LB-MSNP on irinotecan, we encapsulated the trapping agent TEA first₈SOS to modulate our recently discovered biofilm program for drug loading, and TEA₈SOS is then responsible for irinotecan introduction and retention by protonating the drug diffusing through the supported LB into the pores. This results in stable encapsulation of irinotecan in the sealed MSNP pores, creating a gradient for further diffusion through the LB until equilibrium is reached. This actually allows the drug to accumulate in high concentrations with the aid of large surface area of the porous interior and trapping agent mediated drug retention. In certain embodiments, the synthesis procedure comprises four parts, i.e., MSNP core synthesis; synthesis of Supported TEA₈LB-MSNP to SOS; via TEA₈The action of SOS actively loads irinotecan into the particles; and finally purifying the vector.

Chemical products:

tetraethylorthosilicate (TEOS, ≧ 99.0%, lot: BCBK9540V), triethanolamine (. gtoreq.99.0%, lot: BCBH9036V), cetyltrimethylammonium chloride solution (CTAC, 25% by weight in water, lot: STBC7888V), triethylamine (TEA, > 99.0%, lot: SHBD9006V) were purchased from Sigma-Aldrich, USA. Distearoyl phosphatidylcholine (DSPC, > 99.0%, batch: 180PC-147), methoxypolyethylene glycol (PEG2000) -derivatized distearoyl phosphatidylethanolamine (DSPE-PEG2000, > 99.0%, batch: 180PEG2PE-122), and cholesterol (Chol, > 98.0%, batch: CH-94) were purchased from Avanti Polar Lipids, Inc., USA. Sucrose octasulfate sodium salt (lot number: 1-TMH-175-4) was purchased from Toronto Research Chemicals, Canada. Irinotecan hydrochloride trihydrate (IRIN, ≧ 99.0%, batch No. RCN-104) was purchased from LC Laboratories, Inc. of the United states. All chemicals were used directly without further purification.

Sol-gel synthesis and template removal of MSNPs

By slightly modifying our sol/gel procedureTo synthesize the MSNP core. The reaction was carried out in a 500mL Erlenmeyer flask. 50mL of CTAC (25%) and 150mL of H₂O was mixed while stirring at 350rpm for 15 minutes at 85 ℃. After this, 8mL of 10% triethanolamine was added at 85 ℃ for 30 minutes. Silica precursor TEOS (7.5mL) was added dropwise to the mixture at a rate of 1mL/min controlled by a peristaltic pump. To achieve synthesis of particles with a major dimension of about 60nm, the solution was stirred at 350rpm for 20 minutes at 85 ℃. To remove the surfactant, the particles were washed by menthol and HCl (500:19, v/v) at room temperature for 24 hours. The particles were centrifuged at 10,000rpm for 60 minutes and washed 3 times in menthol. To obtain high quality MSNP cores, frequent quality checks are performed, including DLS measurements to monitor particle aggregation and contamination; TEM for visualization of particle morphology; and infrared spectroscopy and cytotoxicity assays to check if the detergent was completely removed.

Using TEA₈Preparation of irinotecan-loaded LB-MSNP by using SOS as trapping agent

To establish the optimal procedure for irinotecan loading by LB-MSNP, we developed a new approach, where we trapped the protonating agent in the particles using a biofilm procedure (previously disclosed), before incubating the particles in irinotecan solution. This allows lipophilic irinotecan to diffuse across the LB membrane and subsequently protonate in the pores, thus reversing the concentration gradient for drug encapsulation (see fig. 5). Considering the loading capacity and drug release behavior, we optimized the irinotecan loading program by systematically varying the loading time, trapping agent concentration, LB-MSNP concentration, irinotecan concentration, and irinotecan/LB-MSNP ratio. This allowed us to achieve an irinotecan loading capacity of 85% (irinotecan/MSNP, w/w), which was about 4-fold greater than the drug loading capacity in the absence of trapping agent. This amounts to twice the loading capacity of the parallel synthesized liposomal version of MM-398. For further discussion, we will name irinotecan-loaded LB-MSNP as "Ir-LB-MSNP (+ SOS)".

Synthesis of Supported TEA₈LB-MSNP to SOS

₈The collector sucrose octasulfate Triethylammonium (TEASOS) was prepared from commercially available sucrose octasulfate sodium salt.

We used an ion exchange reaction to synthesize TEA from the precursor sucrose octasulfate sodium salt₈And (4) SOS. Briefly, 500mg of precursor salt was dissolved in 1mL of deionized water to give a 500mg/mL aqueous solution. The solution was passed through a cationic Dowex 50WX8 resin ion exchange column (15mm diameter and 8cm length from Sigma, lot: MKBH 8810V). After elution of the column with deionized water, the sodium sucrose is converted in the process to protonated octasulfate. Immediately titrating the collected sucrose octasulfonic acid with Trimethylamine (TEA) to a final pH of 5.5 to 6.0, thereby causing TEA₈Formation of SOS. Based on the titration of the amount of TEA, TEA was added₈The SOS concentration was adjusted to about 80 mM. The polyvalent anion TEA is added before use₈SOS was stored in a 4 ℃ refrigerator.

₈TEASOS-loaded LB-MSNP was prepared.

We adjusted our previously disclosed biofilm technology for LB coating (PCT/US2014/020857(UCLA publication 2013-534-2), which is incorporated herein by reference in its entirety) to encapsulate TEA prior to irinotecan loading₈And (4) SOS. Briefly, 100mg of empty MSNP were soaked in 4mL TEA using a probe sonication for 30 minutes at a power output of 32.5W with a 15 second/15 second on/off duty cycle₈SOS in aqueous solution (40 mM). The suspension was immediately added to the flask occupying about 30cm at the bottom of the round-bottom flask (6cm diameter)²On top of the surface area of the coated lipid biofilm. After conducting experiments to achieve optimal mixing of the lipid components (with the aim of stable coating without leakage), we decided to use a mixture containing 228mg dspc/Chol/DSPE-PEG (molar ratio: 3:2: 0.15). The mixture was dissolved in chloroform at 10 mg/mL. Biofilm formation was achieved by solvent evaporation at room temperature over about 1 hour using a rotary evaporator connected to a vacuum system. After 4mL of particle suspension was added to the biofilm, it was turned on/off for 15 sec/15 secCycling, sonication using the probe at a power output of 32.5W for 30 minutes. Since the sonicated suspension contains coated particles, liposomes and free TEA₈SOS, therefore TEA was purified using a chromatographic procedure₈SOS LB-MSNP, where an Sepharose CL-4B column (15mM diameter and 15cm length from Sigma, lot: MKBP0885V) was eluted with HEPES buffer (5mM HEPES, 5% dextrose, pH 6.5).

Using supported TEA₈LB-MSNP from SOS was actively loaded with irinotecan.

Irinotecan was dissolved at a concentration of 10mg/mL in HEPES buffer solution (5mM HEPES, 5% dextrose, ph 6.5). Mixing the drug solution with the TEA-loaded carrier₈The LB-MSNP suspension of SOS was mixed at a 1:1(w/w) irinotecan/particle ratio and incubated at 60 ℃ for 0.5 hours in a water bath. The reaction was terminated by placing the vessel in ice water for 10 minutes.

Particle purification

Free liposomes and free irinotecan were cleared by purifying Ir-LB-MSNP (+ SOS) by centrifuging the particles at 15,000rpm for 10 minutes followed by resuspension in saline or 5% dextrose. The washing procedure was repeated 3 times.

Control formulations were prepared using Ir-LB-MSNP (+ SOS) particles for comparative studies

To perform a comparative analysis reflecting the unique properties of Ir-LB-MSNP (+ SOS) particles, we synthesized LB-MSNP without trapping agent, allowing the soaked irinotecan to be LB-encapsulated. We also prepared the liposome equivalent of MM-398 prepared with a trapping agent and a control liposome encapsulating irinotecan without a trapping agent.

Synthesis of Captureless irinotecan LB-MSNP:

empty MSNP (40mg) was placed in a 4mL solution of irinotecan (in water, 10mg/mL) for 5 minutes under sonication, then stirred at room temperature for an additional 4 hours. Irinotecan-soaked MSNP suspension was added to dry lipid membranes (91mg lipid mixture containing DSPC/cholesterol/PE-PEG 2000 at a molar ratio of 3:2:0.15) followed by probe sonication for 30 minutes using a 15 second/15 second on/off duty cycle at a power output of 32.5W. Free liposomes and free drug were cleared as described above using centrifugation at 15,000rpm for 10 minutes followed by particle washing and resuspension to purify the particles. We refer to the particles without the trapping agent as "Ir-LB-MSNP (-SOS)".

Internal synthesis of liposome equivalents of the commercially developed liposome formulation MM-398.

The synthesis was performed according to the procedure disclosed for MM-398 by Drummond et al (Cancer Res.2006,66, 3271-3277). Briefly, the lipid mixture (212mg lipid, DSPC/Chol/DSPE-PEG at a molar ratio of 3:2: 0.015) was dissolved in 0.4mL ethanol at 60 deg.C-65 deg.C. At the same temperature, 4mL of TEA was added₈The SOS solution (80mM) was mixed with the lipid ethanol solution and then sonicated in a water bath for 2 minutes. The lipid suspension was extruded 15 times through a polycarbonate membrane with a pore size of 0.1 μm at 60-65 ℃. Removal of unencapsulated TEA by chromatography on agarose gel CL-4B column₈SOS, eluted using HEPES buffer (5mM HEPES, 5% dextrose, pH 6.5). Irinotecan solution (10mg/mL in water with HEPES buffer (5mM HEPES and 5% dextrose, pH 6.5)) and preloaded TEA₈A liposomal suspension of SOS (irinotecan: lipid ═ 1:2.1w/w) was mixed and then incubated in a water bath at 65 ℃ for 0.5 hours. The reaction was terminated by leaving the mixture in ice water for 10 minutes. The irinotecan-loaded liposomes were then purified by removing free irinotecan on a Sephadex G-25 column (15mm diameter and 15cm length, Sigma, lot: 019K1077), eluting with HEPES buffered saline (5mmol/L HEPES, 145mmol/L NaCl, pH 6.5). We refer to the internally synthesized liposome preparation as "Ir-Lipo (+ SOS)".

Liposomes encapsulating irinotecan were synthesized without the use of a trapping agent.

We also synthesized liposomes using passive encapsulation methods. Briefly, the lipid mixture (53mg lipid, DSPC/Chol/DSPE-PEG at a molar ratio of 3:2: 0.015) was dissolved in 0.1mL ethanol at 60 ℃ to 65 ℃.1mL of irinotecan solution (10mg/mL in water) was mixed with the lipid ethanol solution at 60 ℃ to 65 ℃ followed by water bath sonication for 2 minutes. The lipid suspension was extruded 15 times through a polycarbonate membrane with a pore size of 0.1um at 60 ℃ to 65 ℃. Drug-loaded liposomes were purified by removing free irinotecan using sephadex G-25 column, eluting with HEPES buffered saline (5mmol/L HEPES, 145mmol/L NaCl, pH 6.5). We refer to this liposome preparation as "Ir-Lipo (-SOS)".

Comparative analysis of Ir-LB-MSNP (+ SOS) with control irinotecan formulation.

Physicochemical characterization of Ir-LB-MSNP (+ SOS) and control formulations.

Table 3 shows the hydrodynamic size, size distribution, and surface charge of Ir-LB-MSNP (+ SOS) compared to the control formulation. Ir-LB-MSNP (+ SOS) has a hydrodynamic size of 110nm to 130nm, with a polydispersity index (PDI) of about 0.1. Such particles exhibit a negative zeta potential value of-24.7 mV in pure water. Zeta potential was changed to-2.95 mV in PBS and to-3.27 mV in complete DMEM medium. In different solutions, these physicochemical parameters were similar to control formulations, such as Ir-Lipo (-SOS), Ir-Lipo (+ SOS), and Ir-LB-MSNP (-SOS).

Table 3: the size and zeta potential of the Ir-loaded particles in the different incubation media.

Table 4 shows the loading capacity and loading efficiency of LB-MSNP with and without the trapping agent. The loading capacity (total irinotecan-mass of irinotecan in supernatant)/(MSNP or liposome) x 100%. The loading efficiency was defined as [ (total irinotecan-irinotecan in supernatant)/total irinotecan ] x 100%. For liposomes, the loading capacity was 4.12% w/w and 45.2% w/w in the absence or presence of 80mM trapping reagent. The loading efficiency of irinotecan in the liposome platform increased from 8.71% to 95.5% due to the action of the trapping agent. This is consistent with the data published for synthetic MM-398 liposomes (Cancer Res.2006,66,3271). After performing experiments to obtain the most stable particle formulation, we used 40mM trapping agent to synthesize LB-MSNP. After incubation with irinotecan, we achieved Ir-LB-MSNP (+ SOS) with a loading capacity of 83.5% w/w, compared to 22.5% w/w for LB-MSNP without capture reagent. Although Ir-LB-MSNP used less trapping agent (40mM), it actually produced a2 × loading capacity when compared to the internal MM-398 liposome containing 80mM trapping agent.

Table 4: in the absence and presence of TEO₈Loading capacity and efficiency of LB-MSNP and liposomes in the case of SOS collectors

We also performed cryoEM analysis to visualize the morphology of Ir-LB-MSNP (+ SOS). This confirmed that the MSNP surface was uniformly coated with the lipid bilayer (fig. 6A, top panel). High magnification cryoEM images show a primary particle size of about 80nm, with complete surface coating by a bilayer about 7.1nm thick. Magnifying the image confirms the presence of an intact lipid coating while also showing the presence of high density material in the pores; the material represents irinotecan-TEA₈An SOS complex. We observed occasional particles encapsulated by slightly larger liposomes that did not adhere tightly to the particle surface, however the LB layer remained intact. Images of Ir-Lipo (+ SOS) showed a liposome structure of about 75nm with a monolayer bilayer about 6.5nm thick (fig. 6A, bottom). High magnification cryoEM provides sufficient resolution to see high density IRIN-TEA inside liposomes₈SOS precipitate (red arrow).

To evaluate the stability of each formulation, we compared the drug stability in PBS (pH 7.4) at 37 ℃ (fig. 6B), with the intent of estimating the stability of irinotecan in the bloodstream. This shows that Ir-LB-MSNP (+ SOS) is very stable in PBS, causing < 5% release within 24 hours. Comparable stability in PBS was found in the internal MM-398 liposomes.

The pH of PDACs is acidic due to elevated glucose uptake, glycolysis, lactate production (Warburg effect), abnormal blood perfusion, and the presence of dense matrices (Wojtkowiak et al, (2012) Cancer Res.72(16): 3938-3947; Estralla et al (2013) Cancer Res.73(5): 1524-1535). Therefore, we also investigated the drug release profile under acidic pH conditions. The tumor pH typically drops to about 6.5, but can be as low as about 5.5: (b)Et al (1992) Cancer Res.52: 6209-6215). When the particles are taken up by PDAC cells, the pH may further drop to a value in the lysosomal compartment<5(Mindell et al (2012) Annu Rev physiol.74: 69-86). Therefore, we performed drug release experiments in phagolysosomal simulated fluid (PSF, pH 4.5). In PSF, a rapid irinotecan release (i.e., 20% release over 4 hours) was seen in LB-MSNP. However, in the internal MM-398 formulation, we found a significantly slower drug release profile in PSF, i.e. only about 4% of the drug was released within 24 hours.

Cellular uptake and killing efficiency of Ir-LB-MSNP (+ SOS) in cultured PDAC cells

Confocal microscopy using Texas Red-labeled LB-MSNP or liposomes showed massive cellular uptake in the perinuclear distribution in PANC-1 cells (FIG. 7). We also used the MTS cytotoxicity assay to determine the effect of Ir-LB-MSNP (+ SOS) on PANC-1 cells and BxPC3 cells during treatment with increasing irinotecan concentrations. We compared the effect of these particles with free drug and Ir-Lipo (+ SOS) control. While free drug was more effective at killing within 24 hours, both Ir-LB-MSNP (+ SOS) and liposomes showed comparable killing at 72 hours in both cell types (fig. 8). Empty nanoparticles did not induce toxicity at concentrations up to 500 μ g/mL within 48 hours.

Comparative analysis of in vivo toxicological effects of Ir-LB-MSNP (+ SOS) and Ir-Lipo (+ SOS) in mice.

Maximum Tolerated Dose (MTD).

We began an in vivo toxicity study by determining the MTD of various irinotecan formulations. This is using the NCI protocol from the toxicology and pharmacology divisions: (dtp.nci.nih.gov/branches/tpb/default.htm) And (4) completing. Two healthy male BALB/C mice were injected intravenously with 50mg/kg (C)₁Dose) free drug or encapsulated drug. We then used a dose escalation factor of 1.8 to determine C_nDose, wherein the two animals (n ═ 2) died within 24 hours after treatment with free irinotecan or encapsulated irinotecan. Second round dose exploration from C_n-1The dose was started and an increasing factor of 1.15 (n-2) was used to find the MTD, where there was no mortality or severe morbidity. By injecting 5 mice, and following them for 15 days to determine whether there is no morbidity, mortality or>The MTD was further verified by 20% weight loss. Our results show that the MTD dose values of Ir-LB-MSNP (+ SOS), Ir-Lipo (+ SOS), and free irinotecan were 295mg/kg, 350mg/kg, and 60mg/kg, respectively. The nano-formulations showed about 5-fold MTD dose compared to the free drug, clearly indicating the ability of reduced toxicity when using both LB-MSNP and liposome formulations.

And (4) histological analysis.

In separate experiments, healthy mice received a single intravenous administration of a drug dose of 60mg/kg IV of Ir-LB-MSNP (+ SOS), Ir-Lipo (+ SOS), and free irinotecan. At 24 hours post-treatment, mice were sacrificed for organ harvest. Appropriately sized sections of the sternum, gastrointestinal tract (GI, stomach and intestine), thymus, liver, kidney, spleen and lung were fixed in 10% formalin and then embedded in paraffin. Tissue sections of 4 μm thickness were mounted on slides. Sections were stained with hematoxylin-eosin (H & E) and examined by light microscopy. Slides were read by an experienced veterinary pathologist. The results showed that the myelotoxicity of Ir-LB-MSNP (+ SOS) and Ir-Lipo (+ SOS) was greatly reduced compared to free IRIN (FIG. 9). We are conducting a more detailed study of sternal bone marrow using immunohistochemistry to assess whether there is differential toxicity to erythroid, megakaryocyte or blood leukocyte precursors. Free irinotecan and liposome formulations caused nephrotoxicity, which was manifested as glomerular swelling. No significant renal abnormalities were found in mice treated with Ir-LB-MSNP (+ SOS) particles (fig. 10).

And (6) concluding.

The goal of this project was to develop an intravenously injectable irinotecan nano-delivery platform for effective PDAC killing and reduced toxicity. The LB-MSNP platform has shown promising results in PDAC cells and acute animal toxicity studies via the use of high drug loading capacity, nanocarrier biocompatibility, and effective drug encapsulation.

Example 3

iRGD-mediated transcytosis enhances irinotecan delivery of silicalite nanocarriers in murine and human pancreatic cancers

Delivery and efficacy of

Although Pancreatic Ductal Adenocarcinoma (PDAC) is almost uniformly fatal, a degree of improved overall survival has been achieved by the introduction of nanocarriers that deliver irinotecan or paclitaxel. We have further improved these results in an animal in situ model using an irinotecan silica support comprising Mesoporous Silica Nanoparticles (MSNPs) coated with a lipid bilayer. The silica body carrier also provides a substantial reduction in toxicity compared to liposome carriers for irinotecan. Although it is generally assumed that nanocarriers rely primarily on abnormally leaky vasculature (also known as enhanced permeability and retention effects) to enter tumors, transcytosis transport pathways modulated by neuropilin-1 (NRP-1) receptors have recently been reported. This unique transport pathway may be triggered by a cyclic iRGD peptide that binds to a tumor-associated integrin, wherein the peptide is processed for subsequent binding to NRP-1. Co-administration of iRGD in a robust Kras in situ PDAC model increased irinotecan-silica uptake 3-fold to 4-fold, resulting in significant reduction in survival benefit and metastasis. The silica bodies embedded with gold nanoparticle cores were used for ultrastructural observation of transcytosis pathways in vivo, showing that iRGD co-administration induces vesicle transport pathways that transport electron dense carriers from the vascular lumen to perinuclear sites in cancer cells. iRGD-mediated enhancement of silica uptake was also observed in patient-derived xenografts, which is commensurate with the expression level of NRP-1 on tumor vessels. These results show the utility of iRGD for a potentially personalized approach to PDAC treatment using irinotecan-silica body carriers.

In this example, we describe the development of lipid bilayer-coated mesoporous silica nanocarriers (silicasomes) for irinotecan delivery that outperform liposomes in a robust Pancreatic Ductal Adenocarcinoma (PDAC) animal model. We demonstrate that the uptake of silica at the in situ tumor site can be increased 3-fold to 4-fold by co-administration of iRGD peptides that trigger a new transcytosis pathway via attachment to neuropilin-1 receptors. iRGD also increases silica uptake in patient-derived xenografts expressing the same vascular receptor. We provide ultrastructural evidence for a vesicle-based transcytosis pathway that can complement the enhanced permeability and retention effects commonly used to explain the uptake of nanocarriers at tumor sites. The transcytosis pathway offers the possibility to enhance the benefit of irinotecan-silica carriers in PDAC patients.

Introduction to the design reside in

We have developed a multifunctional Mesoporous Silica Nanoparticle (MSNP) platform that has been adapted for drug encapsulation using a supported Lipid Bilayer (LB) to provide high dose PDAC chemotherapy (see examples)As in example 1). This vector has also been named "silica body" to distinguish it from morphologically similar liposomal vectors containing unsupported LB. The silica bodies described herein have been demonstrated to exhibit significantly higher loading capacity for this drug, improved circulatory stability (due to supported LB), and reduced drug leakage compared to liposomes comprising an internal liposome carrier for irinotecan. These features allow the pharmacokinetics and therapeutic efficacy of the silicasomes to be improved in a rigorous in situ Kras PDAC model compared to liposomes (see, e.g., example 1). In addition, the silica bodies also provide a substantial reduction in toxicity in the gastrointestinal tract, liver and bone marrow compared to the liposome equivalents (supra). In addition to the success of irinotecan, the silica body platform has also been adapted for the synergistic delivery of paclitaxel and gemcitabine, allowing it to be significant in the in situ PDAC model: (b)>10 times) better than the free gemcitabine andcombinations of (a) and (b). Notably, the above results of the silica body carrier can be achieved by "passive" delivery without the use of targeting ligands.

In view of this background, we focused on whether the PDAC therapeutic efficacy of irinotecan-loaded silica (Ir-silica) could be improved by transcytosis, and whether this could be best achieved by either conjugation of iRGD to the carrier or its co-administration. In this example, we demonstrate the feasibility of using co-administration of free iRGD peptides to enhance vector uptake and therapeutic efficacy in the Kras in situ model. Furthermore, we provide ultrastructural evidence of a grouped vesicle system that allows transport of Au-labeled silica bodies from the vascular lumen to the perinuclear sites in cancer cells. We also demonstrated in patient-derived PDAC xenografts that the relative abundance of NRP-1 expression on tumor vasculature determines the magnitude of response to the silica body via co-administration using iRGD.

Results

Synthesis and characterization of silica bodies for drug loading and visualization in PDAC tumors

We have demonstrated high irinotecan loading achieved by LB-coated MSNP (also known as silica bodies) using a remote loading technique that relies on protonating agents (see, e.g., the top panel of fig. 21A and Liu et al (2016) ACS nano.10(2): 2702-2715). Irinotecan is a weakly basic and amphiphilic molecule that can diffuse through LB into the MSNP's internal packaging space, with the previously encapsulated triethylammonium sucrose octasulfate (TEA)₈SOS) converts the drug into a hydrophilic derivative that cannot diffuse back through the LB (fig. 21A, top panel). The remote loading procedure was used to synthesize an Ir-silica bulk batch that achieved a drug loading capacity of 50 wt% (w/w, irinotecan/MSNP) (supra). To determine whether the conjugation of iRGD peptides to the surface of silica bodies could affect the biodistribution of the carriers to PDAC tumor sites, we also synthesized DSPE-PEG in which LB was used₂₀₀₀A batch of particles having components conjugated to cysteine residues in the peptide. This is achieved by using DSPE-PEG₂₀₀₀Maleimide instead of DSPE-PEG₂₀₀₀This was done (see method section) to allow thiol-maleimide to couple with the cysteine modified peptide Cys-c (CRGDKGPDC) (SEQ ID NO:11) (fig. 21A, box 2). To confirm the success of the conjugation reaction, we also used a fluorescein-labeled version of the peptide developed by Ruoslahti et al (FAM-iRGD) to perform fluorescence spectroscopy on the conjugated silica bodies after extensive washing (fig. 27, panel a) (Sugahara et al (2009) Cancer Cell,16(6): 510-520). This confirms a stable association of the fluorescent peptide with LB. The density of iRGD conjugation was limited to about 3 mol% (of all LB components) to prevent colloidal instability and interference with vector uptake. Uptake of the intact silica-iRGD vector was confirmed in KPC cells by flow cytometry and confocal microscopy (fig. 27, panels B and C).

To visualize the transcytosis process in PDAC tumors by TEM, we also synthesized a batch of core-shell MSNPs comprising about 10nm electron-dense Au-nanoparticles (fig. 1A, box 3). Similar to the bare particles, the core/shell particles can be effectively coated with LB as shown by CryoEM (fig. 21A, block 3). The detailed synthesis and characterization procedures for all carriers used in this information are discussed in the methods section supporting the information. The main physicochemical characteristics of the silica bodies are summarized in table 5 below.

Table 5: illustrative silica body physico-chemical properties.

Comparison of the effects of conjugated and unconjugated iRGD peptides on the biodistribution of silica bodies in an in situ PDAC model

Will be derived from a gene from transgenic Kras^LSL-G12D/+；Trp53^LSL-R172H/+(ii) a Luciferase-expressing KPC cells from spontaneous PDAC tumors of Pdx-1-Cre animals were implanted in situ into the pancreatic tail of immunocompetent B6/129 mice (see, e.g., the left panel of FIG. 21B and Liu et al (2016) ACS Nano.10(2): 2702-2715; Tseng et al (2010) Clin. cancer Res.16(14): 3684-3695). This rigorous PDAC tumor model mimics human PDAC in terms of oncogene expression, growth characteristics, metastasis, histological characteristics, and development of dysplastic stroma. FIG. 21B summarizes details of tumor growth characteristics and metastasis as seen during animal necropsy and IVIS imaging (FIG. 21B) (Tseng et al (2010) Clin. cancer Res.16(14): 3684-3695; Torres et al (2013) PloS One 8: e 80580). Biodistribution of the silicasomes to the in situ tumor sites was assessed by one Intravenous (IV) injection of 50mg/kg of Near Infrared (NIR) labeled (DyLight 680) particles either unconjugated (i.e. "no IRGD" state) or conjugated to peptides ("silicasomes-IRGD") (fig. 22A). The third group of animals received a co-administration of 8 μmol/kg of free peptide plus unconjugated particles ("silicasomes + iRGD"). IVIS imaging of explanted organs performed 24 hours after initial injection and animal sacrifice showed that "silicalite + iRGD" compared to the signaling intensity in the silicalite-iRGD group or the silicalite only groupThe NIR signaling intensity at the tumor site was significantly increased for the group (fig. 22A). Imaging intensity was quantified by IVIS lumine Living Image software. In contrast to their lack of in vivo effect, it can be seen that silica bodies conjugated to peptides enhance vector uptake in KPC cells (fig. 27, panels B and C). We interpret this as sufficient NRP-1 receptor density to initiate transmembrane uptake, while receptor abundance at the tumor vascular site may be limited in the number of conjugated particles that are allowed to pass, as previously reported by Ruoslahti et al for in vitro/in vivo comparisons (Sugahara et al (2009) Cancer Cell,16(6): 510-520; Teesalu et al (2009) proc.natl.acad.sci.usa,106(38): 16157-16162; Hussain et al (2014) Sci Rep 4: 5232). To confirm that NIR intensity (fig. 22B, left panel) reflects actual MSNP uptake, inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to quantify tumor silicon (Si) content (fig. 22B, right panel). This shows a significant (about 3-fold) increase in Si content in the "silica + iRGD" group compared to the silica-iRGD group or the silica only group. We also confirmed the importance of the C-terminal (CendR) motif on PDAC biodistribution by performing separate experiments to confirm that coadministration of silica with control non-CendR peptide cyclic (RGDfK) (Sugahara et al (2010) Science,328(5981):1031-1035) did not enhance silica uptake (fig. 28). The results were also confirmed by ICP-OES (FIG. 28).

Tumor tissue sections were used to assess the relative abundance of intratumoral biodistribution and distance traveled from tumor vessels of NIR-labeled silica bodies by anti-CD 31 Alexa488 staining (fig. 29). This analysis shows that iRGD co-administration is the most effective strategy to enhance uptake of silica at the in situ tumor site. In contrast, in our KPC in situ model, the vector conjugated with iRGD failed to have a significant effect on particle number and travel distance (fig. 29). The above findings allow subsequent efficacy studies using free iRGD.

Since all nanocarriers are also taken up in the reticuloendothelial system (RES), it is important to evaluate the observable uptake of silica bodies in the liver and spleen (with little apparent effect on lung, heart and kidney). Interestingly, IVIS imaging showed that the biodistribution of the conjugated vector to the RES organ could be increased (fig. 22A). This was confirmed by the increase in Si content in the liver and spleen during ICP-OES (fig. 30). Although we lack a clear explanation for this observation, it is possible that peptide conjugation directly or indirectly causes particle opsonization and increased uptake of scavenger receptors, regardless of NRP-1 (Li and Huang (2008) mol. pharm.5(4): 496-504).

iRGD co-administration enhances efficacy of irinotecan delivery of silica bodies

To demonstrate the possible therapeutic benefit of co-administration of iRGD in PDAC treatment with Ir-silica, efficacy experiments were performed in the same in situ tumor model. Animals received an intravenous injection of a drug dose of 40mg/kg (equivalent to a carrier dose of 80 mg/kg) of Ir-silica with or without co-administration of 8. mu. mol/kg iRGD. In situ implantation of 2X 10⁶Treatment was started 13 days after each KPC-luc cell (FIG. 23A), at which time the primary tumor size was about 3mm-5mm without the presence of macroscopic metastases (see above and Liu et al (2016) ACS Nano.10(2): 2702-2715). Injections were repeated every 3 days for a total of 4 administrations (fig. 23A). The control group consisted of animals receiving intravenous PBS, Ir-silica alone (same dose) or free iRGD alone (same dose). Kaplan-meier plots were used to show animal survival (supra) and animal necropsy was used to assess the presence of local tumor spread and metastasis. Numerous metastases were seen in spleen, intestine, stomach, liver and kidney of animals treated with saline or free iRGD peptide (fig. 23B). While Ir-silica significantly reduced tumor burden and number of metastases, iRGD co-administration further enhanced the shrinkage of primary tumors and inhibited spread to liver, stomach, and intestine (fig. 23B). The heatmap in fig. 23C provides a quantitative indication of the effect of co-administered peptides on metastatic disease. Logarithmic rank test (SPSS 19.0 software, IBM SPS in USASStatistics) also showed that treatment with Ir-silica alone increased survival by 28.6% compared to the PBS group, compared to 57.1% during co-administration of iRGD peptide (fig. 23D); this difference was statistically significant (p ═ 0.027). We also confirmed that the intratumoral content of irinotecan increased significantly (about 2-fold) at 24 hours in animals receiving one administration of iRGD (8 μmol/kg, i.v.) with Ir-silica (40mg/kg drug), as determined by High Performance Liquid Chromatography (HPLC) (fig. 23E).

iRGD-mediated uptake of silicasomes requires expression of NRP-1 on tumor vasculature

the mechanism of action of iRGD in mediating drug uptake depends on homing to α_vβ₃integrin or α_vβ₅Integrins, which are preferentially expressed on cancer vessels (Ruoslahti and Pierschbacher (1987) Science,238(4826): 491-497; Hanahan and Weinberg (2000) Cell,100(1): 57-70). The binding of the cyclic peptide to the integrin is followed by cleavage and release of the C-terminus of the peptide, which mediates interaction with NRP-1 (this is also referred to as the C-terminal rule). NRP-1 binding causes triggering of vesicular systems that can assist drug and nanoparticle transport (Sugahara et al (2009) cancer cell,16(6): 510-520; Pang et al (2014) nat. Commun.5: 4904). To determine the expression of NRP-1 at KPC tumor sites, by Alexa488-labeled anti-NRP-1 was subjected to IHC staining by Alexa594 marker anti-CD 31 and DAPI staining to locate endothelial cells and nuclei. Fluorescence microscopy and Image J analysis were performed to determine the% overlap of NRP-1 with CD 31; this showed 94.2% co-localization (fig. 24B). To verify the role of NRP-1 in the uptake of silica at the site of the orthotopic tumor, tumor-bearing mice were pre-injected with an antagonist antibody to the b1b2 domain of NRP-1 (Sugahara et al (2009) Cancer Cell,16(6): 510-520; Sugahara et al (2010) Science, 32)8(5981):1031-1035). Subsequent administration of iRGD plus NIR-labeled silica bodies showed a significant reduction in vector uptake compared to animals not receiving blocking antibody treatment (fig. 24C). The same interference was not seen in the case of control IgG (fig. 24C). These data confirm the role of NRP-1 in iRGD-mediated uptake of silica.

Ultrastructural validation of silica transport by transcytosis vesicles at KPC cancer sites

It has been demonstrated that binding of the C-terminus of iRGD to NRP-1 can trigger an overall transcytosis pathway involving a neovesicle transport mechanism important for nutrient delivery to Cancer (Sugahara et al (2009) Cancer Cell,16(6): 510-520; Sugahara et al (2010) Science,328(5981): 1031-1035; Pang et al (2014) nat. commun.5: 4904). To our knowledge, this transcytosis pathway has never been directly visualized during transport of the therapeutic nanocarriers to the tumor site. Ultrastructural analysis of iRGD-mediated transport pathways in KPC models was attempted using Electron Microscopy (EM), which can provide visual enhancement validation. Initially, we compared TEM images taken at different time intervals at harvested tumor sites after intravenous injection of silica in the presence or absence of iRGD co-administration (fig. 25A). At 24 hours, it can be clearly seen that iRGD co-administration induces the formation of vitiform vesicles with diameters of 110nm to 370nm that diffuse across endothelial cells, from luminal to abluminal side of the blood vessels (fig. 25A, regions "3" and "4"). These features are similar to vesicle-vacuolar organelles or VVO described by Dvorak et al (Feng et al (1996) J. exp. Med.183(5): 1981-1986). Semi-quantitative analysis of vesicle density, determined by counting the number of vesicles in at least 10 regions of interest (ROI) and representing the number of vesicles per square micron of internal surface area in the cells, showed that iRGD can increase vesicle density by about 3 fold over animals that did not receive co-administration of peptide (fig. 25A, left panel).

The low electron density of MSNP makes it difficult to resolve the presence of carriers in heterogeneous and complex PDAC microenvironments when attempting to visualize the transport of silica bodies by the vesicular system. To address this challenge, silica bodies were synthesized to include about 10nm Au-nanoparticles (FIG. 21A) (Liu et al 92013) ACS Nano.7(7):6244-6257) that can be easily visualized by TEM. Mice expressing in situ KPC tumors were injected intravenously with 50mg/kg of Au-silica in the absence or presence of 8. mu. mol/kg iRGD. Tumor tissue was harvested 24 hours after injection and fixed for TEM analysis. A representative electron micrograph showing in one image electron dense silica bodies in (i) the lumen of blood vessels of animals receiving iRGD co-administration is shown in fig. 25B; (ii) vesicle transport in endothelial cells; and (iii) deposition of particles in the tumor stroma. The higher magnification of regions 1-3 of the image confirms the presence of Au-containing particles. It was also possible to show the appearance of silicasomes in the perinuclear distribution in tumor cells undergoing apoptosis during iRGD co-administration (fig. 25C). This site was several hundred microns away from the nearest tumor vessel (fig. 25C). Although it is possible to observe lower particle density in the tumor stroma of animals not receiving iRGD treatment, we did not find any silica bodies being transported by the vesicle transport system, nor did we identify alternative entry mechanisms in the static images (fig. 31).

Differential effects of iRGD co-administration on silicalite uptake in patient-derived PDAC tumors phenotyped for NRP-1 expression

although it is clear that the NRP-1 pathway can be functionally involved by iRGD in KPC tumor models, we focused on observing whether the peptides can affect the silica uptake in patient-derived xenografts grown in NOD SCID IL2 α knockout (NSG) mice (huckert et al (2012). j.surg.res.172(1): 29-39). we wherein one person (TD) has established a library of 23 human PDAC-like tumors in NSG mice; these tumor samples were obtained from patients during whippler surgery (whipl's surgery). phenotypically characterize cancer features of metastatic tumor tissue, which are unique features of the corresponding human PDAC tumor, including the stroma abundance and expression of the cancer gene and signaling pathway components characteristic of PDAC (supra) of the respective PDAC tumor, which are selected to have an equivalent phenotype, but are determined by a pair of NRP-1 density and NRP-187, which are different as compared to the density of the tumor expression of the tumor # NRP-1 and the density of the tumor # NRP-187, which is determined by a comparison of the high NRP-1 density map of the mouse NRP-187, the mouse expressing the rat expressing the tumor # NRP-expressing the collagen #187, whereas the tumor #8 expressing the map shows the increased in vivo map when the rat is injected into the rat, the tumor model, the rat expressing the tumor expressing the rat expressing the tumor #8 expressing the tumor.

Discussion of the related Art

In this example, we demonstrate that the efficacy of irinotecan-silica vectors can be significantly improved by co-administering unconjugated iRGD peptides that do not require linkage to the vector to enhance tumor uptake. Co-administration of free iRGD peptides increased the uptake of silica bodies at the site of in situ KPC tumors 3-fold to 4-fold, resulting in enhanced killing of primary tumors and inhibition of metastasis. Overall, this resulted in a significant improvement in animal survival compared to Ir-silica alone. The iRGD effect is mediated by initial interaction with tumor-associated integrins, followed by peptide cleavage and release of the C-terminus conjugated to NRP-1. Although the physiological role of NRP-1 is to control transcytosis for nutritional purposes, the vesicular system may also be used to transport nanoparticles, as evidenced by a reduction in particle transport following injection of receptor blocking antibodies. In addition, EM imaging provides evidence of ultrastructure that iRGD can induce the appearance of grouped vesicles in endothelial cells with the ability to transport Au-labeled silica bodies from the lumen of the blood vessels to the tumor stroma. We also obtained evidence that NRP-1 regulates the transcytosis pathway in human pancreatic tumors that have been implanted in NSG mice. A pair of tumors selected to have differential NRP-1 expression on tumor vasculature showed differences in vector uptake and irinotecan delivery during iRGD treatment. Taken together, these data suggest that it is possible to use personalized PDAC chemotherapy approaches to improve the efficacy of irinotecan silica carrier by iRGD co-administration.

The utility of transcytosis pathway to enhance irinotecan delivery in PDACs is significant for a number of reasons. The first is a matrix that shows dysplasia, which, in addition to increasing tumor growth and metastasis, also leads to drug resistance at the tumor site (Feig et al (2012) clin. cancer res.18(16): 4266-4276; Dimou et al (2012) ther. adv. med. oncol.1758834012446008). Although it is often understood that abnormal vascular permeability is the cause of nanoparticle extravasation, this concept is known as the EPR effect, we know that pancreatic cancer stroma actively interferes with vascular permeability (Meng et al (2013) ACS nano.7(11): 10048-10065; Kano et al (2007) proc.natl.acad.sci.usa,104(9): 3460-3465; Cabral et al (2011) nat.nanotechnol.6(12): 815-823; Liu et al (2012) proc.natl.acad.sci.usa,109(41): 16618-16623). This includes the presence of pericytes that tightly adhere to vascular endothelial cells (supra). Thus, while the EPR effect may contribute to nanocarrier uptake in PDACs, it is important to consider the possibility that other vascular mechanisms may contribute to nanoparticle uptake at the tumor site, including the nutrient transport pathway and the vascular growth factors regulating this transport, as well as the possible contribution of vascular leakage (Jain and Stylianopoulos (2010) nat.Oncol.7(11): 653-664; Ruoslahti et al (2010) J.cell.biol.188(6): 759-768; Maeda et al (2000) J.control.Release 65(1): 271-284; Li and Huang (2008) mol.pharm.5(4): 496-504; Feng et al (1996) J.exp.Med.183(5): 1981-1986; Kobayashi et al (2014) Theranostics,4(1):81-89) Ruoslahti et al describe an endocytic pathway that plays a role in tumor nutrition and can also be therapeutically involved in the vascular system by tumor iRGD peptides (Pang et al (2014) nat.Commun.5:4904) furthermore, vascular growth factors such as VEGF, VEGF-A, VEGF-165A, beta- β and TGF-alpha-penetrators appear to allow for comparison with the vascular system_vβ₃integrin and alpha_vβ₅The integrin-bound RGD motif (Sugahara et al (2009) Cancer Cell,16(6): 510-520; Sugahara et al (2010) Science,328(5981): 1031-1035; Pang et al (2014) nat. Commun.5: 4904). Thus, in addition to the role of signal transduction pathways and vascular permeability associated with growth factor receptors (Kolodkin et al (1997) cell.90(4): 753-762; Ellis (2006) mol. cancer ther.5(5): 1099-1107; Glinka and Prud' homme (2008) J.Leukoc. biol.84(1):302-310), proteolytic cleavage and release of the CendR motif can also trigger NRP-1 mediated transcytosis (Pang et al (2014) nat. Commun.5: 4904). Thus, it is possible that the NRP-1 pathway may coexist with vascular leakage, including the EPR effect, but exhibit different temporal kinetics. Although response to the CendR motif can begin within minutes, the EPR effect typically takes 6-8 hours to peak (Sugahara et al (2009) Cancer Cell,16(6): 510-520; Maeda et al (2003) int. immunopharmacol.3(3): 319-328).

our data on silica transcytosis during iRGD co-administration complement previous attempts to overcome the stroma-vascular barrier during PDAC treatment (Feig et al (2012) clin. Cancer res.18(16): 4266-4276; Dimou et al (2012) the r.adv.med.o ncol. oncol.1758834012408; Meng et al (2013) No.7(11): 10048-10065; Meng et al (2015) ACS nano.9(4):3540-3557) several angiogenic drugs have been introduced to increase tumor entry of nanocarriers (Dimou et al (2012) the r.adv.med.1758846008; kobayashishi et al (2014) therapeutics, 4 (1-81-89) among these sites, VEGF can provide a transient increase of tumor permeability of endothelial cell receptor tyrosine kinase (TGF β) through percutaneous transdermal receptor kinase receptor.

iRGD-based co-delivery enhanced the validation of uptake of silica bodies at PDAC tumor sites (fig. 22), and it is important to emphasize that the co-administration approach overcomes the major limitation of alternative delivery mechanisms in which the peptide is conjugated to a nanocarrier. The difference is explained by the transport capacity of the vector system based on the number of NRP-1 receptors available (Sugahara et al (2010) Science,328(5981): 1031-1035; Ruoslahti (2012) adv.Mater.24(28): 3747-3756; Ruoslahti (2016) adv.drug Deliv.Rev.pii: S0169-409X (16)30094-1.doi: 10.1016/j.addr.2016.03.008). Thus, while transport of conjugated silica is limited by the relatively small and limited number of target receptors on the vascular system, injection of unconjugated peptide alone triggers massive metastasis (a greater number) of bystander silica at the tumor site. In addition, free iRGD also has anti-metastatic activity via modulation of integrin function, as evidenced by interference with the attachment and migration of cultured tumor cells on fibronectin matrices (Sugahara et al (2015) mol. cancer Ther.14(1): 120-128). This may explain in part the interference of the peptide on tumor metastasis in our study (fig. 23B and 23C). The use of free peptides is also more practical and affordable for clinical use than relying on conjugation mechanisms that increase the cost and complexity of vector synthesis.

Materials and methods

More detailed descriptions of materials and experimental procedures are found in the supplementary materials and methods below.

Preparation of silica bodies

Synthesis of irinotecan-supporting silica body:

the 65nm MSNP core was synthesized using a sol-gel procedure as shown above (see also Liu et al (2016) ACSNano.10(2): 2702-2715). As previously reported, lipid biofilms were used to generate silicasomes (Meng et al (2015) ACS Nano.9(4): 3540-3557; see example 1 and Liu et al (2016) ACS Nano.10(2): 2702-2715). Briefly, 500mg of MSNP was soaked in 20mL TEA₈SOS (80mM solution), added on top of a lipid biofilm comprising DSPC/Chol/DSPE-PEG₂₀₀₀550mg of the mixture (molar ratio 3:2:0.15) was coated onto the bottom of a round bottom flask (see example 1 and Liu et al (2016) ACS Nano.10(2): 2702-2715). After sonication to complete the LB coating of the particles, free TEA was removed by size exclusion chromatography on an agarose gel CL-4B column₈And (4) SOS. The TEA-Supported solution was heated in a water bath at 65 deg.C₈The silica bodies of SOS were incubated in a 10mg/mL solution of irinotecan for drug loading. After 30 minutes the loading was stopped by quenching in an ice-water bath, after which the drug-loaded silica bodies were washed 3 times by centrifugation and resuspended in PBS.

Synthesis of silica bodies conjugated with iRGD:

silica bodies conjugated with iRGD were synthesized by linking peptides to PEG chains included in LB. This is achieved by using commercially available DSPE-PEG₂₀₀₀Maleimide instead of DSPE-PEG₂₀₀₀While maintaining the lipid molar ratio as described above. An excess (0.15mL, 5mg/mL) of cysteine-modified iRGD peptide was reacted with DSPE-PEG using a thiol-maleimide reaction at room temperature for 4 hours₂₀₀₀Maleimide conjugation (Sugahara et al (2009) Cancer Cell,16(6): 510-520). The particles are washed to remove unreacted iRGD. The success of the conjugation reaction was confirmed by also preparing a batch of particles conjugated with Fluorescein (FAM) -labeled iRGD peptide, followed by extensive washing (supra) (fig. 27, panel a).

Synthesis of silica bodies with gold core labeling:

10nm Au nanoparticles were synthesized in citrate containing solution (see supplementary materials and materials below). To grow MSNP shells on Au nanoparticle cores, 36mL citrate-coated particles were rapidly injected into 12mL CTAC solution (25 wt% in H)₂O in). The particles were washed and resuspended in CTAC solution (6.25 wt% in H)₂O) while stirring at 350rpm for 5 minutes at 85 ℃. To this mixture we added 0.256mL of 10% (w/v) triethanolamine for 10 minutes followed by dropwise addition of 0.32mL of silica precursor TEOS. The solution was stirred at 350rpm for 20 minutes, causing the generation of Au core/MSNP shell particles with an average size of about 65 nm. The particles were purified by washing sequentially in methanol (w/v) containing 1% NaCl and pure methanol. The Au-labeled MSNPs were then coated with LB as before。

Biodistribution studies of intravenously injected silica bodies with or without iRGD co-administration Is especially suitable for the treatment of diabetes

IVIS (Xenogen) imaging was used to study the biodistribution of NIR-labeled silica bodies in KPC-derived in situ models (n 3 mice/group) (see example 1 and Liu et al (2016) ACS nano.10(2): 2702-2715). Animals were injected intravenously with 50mg/kg of conjugated silica bodies and unconjugated silica bodies with or without co-administration of 8 μmol/kg iRGD. Animals were sacrificed after 24 hours and excised tumors and major organs were subsequently imaged ex vivo. Tumor biodistribution was also confirmed by assessing Si content using the ICP-OES protocol (supra).

Assessment of the efficacy of Ir-silica bodies under iRGD co-administration in KPC-derived in situ tumor models

Tumor-bearing B6/129 mice were randomly assigned to 4 groups of 6 animals each. The first group was injected intravenously every 3 days with a dose of silica containing 40mg/kg irinotecan (80mg/kg MSNP) for a total of 4 administrations. The second group received the same dose of Ir-silica plus 8. mu. mol/kg iRGD co-administration. The third and fourth groups were treated with PBS or iRGD alone. Mice were monitored daily until spontaneous animal death or near moribund status (see example 1 and Liu et al (2016) ACS Nano.10(2): 2702-2715; Olive et al (2009) Science,324(5933): 1457-1461). Bioluminescent imaging of primary tumors and metastatic sites was performed by intraperitoneal injection of 75mg/kg D-fluorescein into the animals 10 minutes prior to sacrifice. Tumor tissues and major organs (gastrointestinal tract, liver, spleen, heart, lung, and kidney) were harvested to quantitatively assess bioluminescent image intensity.

Ultrastructural analysis of transcytosis pathway via TEM observation

With or without co-administration of 8. mu. mol/kg iRGD, by intravenous injection of 50mg/kg of Au-encapsulated silicaKPC orthotopic tumor mice were treated. Tumor biopsies were collected after 24 hours, washed in PBS, and immediately fixed with 2.5% glutaraldehyde at 4 ℃. Further sample preparation and sectioning was performed by the Electron microscopy services Center (Electron microscopy services Center) of UCLA. At 1% OsO₄After the fixation, the sample was dehydrated in propylene oxide and embedded in a resin. Tissue sections 60nm-80nm thick were placed on a copper mesh and observed under a JEOL 1200-EX electron microscope.

Biodistribution of silica bodies in patient-derived PDAC tumors

using phenotypic analysis data and IHC staining for NRP-1 expression, two patient samples (XWR #8 and XWR #187) were collected for growing fresh subcutaneous xenografts in the flank of 6-week old female NSG mice (rickert et al (2012) j.surg.res.172(1): 29-39.) when the tumor size was grown to a diameter of about 0.8cm, biodistribution to the tumor site was assessed using 3 animals in each group that received silica in the presence or absence of iRGD co-administration, similar to the procedure described above.

Statistical analysis

The differences between the groups were analyzed by comparison using a two-sided student's t-test (Excel software, Microsoft corporation). Statistical significance differences were determined at p < 0.05. Values are expressed as mean ± SD of multiple determinations, as described in the figure legend. Survival data were processed by log rank test (Mantel-Cox) using SPSS software.

Supplemental materials and methods

Material

Tetraethylorthosilicate (TEOS), triethanolamine, cetyltrimethylammonium chloride solution (CTAC, 25 wt% in water), (3-aminopropyl) triethoxysilane (APTES), Triethylamine (TEA), gold (III) chloride hydrate, trisodium citrate dehydrate, Dowex 50WX8 resin, and Sepharose CL-4B were purchased from Sigma-Aldrich, USA. 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000](ammonium salt) (DSPE-PEG₂₀₀₀) 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000](ammonium salt) (DSPE-PEG₂₀₀₀Maleimide) and cholesterol (Chol) from Avanti Polar Lipids, usa. Sucrose Octasulfate (SOS) sodium salt was purchased from Toronto Research Chemicals, Canada. Irinotecan hydrochloride trihydrate was purchased from LC Laboratories, inc. Penicillin, streptomycin and Du's Modified Eagle's Medium (DMEM) were obtained from Invitrogen corporation. Fetal Bovine Serum (FBS) was purchased from Gemini Bio Products, Inc. in the United states. iRGD (CRGDKGPDC, SEQ ID NO:11) was purchased from Biomatik, Inc., USA. Reactive iRGD modified with cysteine residues and blocking anti-NPR-1 antibodies (against the recombinant b1b2 domain of NRP-1) were provided by rooslahti friend. Circularity (-RGDfK) (SEQ ID NO:12) was purchased from ApexBio Technology, Inc. of the United states. anti-CD 31 antibody (Cat. No. 553708) was purchased from BD Pharmingen in the United states^TM. anti-NPR-1 antibody (ab81321) was purchased from Abcam, Inc. in the United states. Control Normal goat IgG (sc-2028) was purchased from Santa Cruz Biotechnology, Inc. of USA. Alexa488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (A11008), Alexa594 conjugated goat anti-rat IgG (H + L) secondary antibody (A11007) and DyLight 680NHS ester were purchased from Thermo Fisher Scientific, Inc. in the United states. Matrigel^TMThe substrate membrane was purchased from BD Bioscience, usa. All chemical(s)The product was used without further purification.

NIR labeling of MSNPs Using DyLight 680

MSNP labeling was performed using the NIR fluorescent dye DyLight 680NHS ester. First, the MSNP was coated with NH₂The groups were functionalized to conjugate NHS esters. Briefly, 10mg of MSNP was suspended in 1mL of ethanol and mixed with 1. mu.L of APTES. Under inert atmosphere of N₂The reaction was carried out under an atmosphere while stirring at 80 ℃ overnight. Subsequently, the mixture was centrifuged and washed 3 times with ethanol. Reacting NH₂Conjugated MSNP was suspended in 1mL DMF, mixed with 0.01mg DyLight 680NHS ester and stirred at room temperature for 2 hours. The labeled MSNP was washed with ethanol and deionized water. Silica bodies were prepared using NIR-labeled MSNP.

Synthesis of gold nanoparticles of about 10nm

By mixing 5mL of HAuCl₄(10mM) and 45mL Milli-Q water were added to a 100mL round-bottom flask equipped with a condenser to prepare gold nanoparticles at about 10 nm. After the boiling temperature was reached, 5.8mL of sodium citrate (38.8mM) was added to the boiled solution while stirring vigorously. This is accompanied by a change in color from pale yellow to dark red. The boiled solution was stirred at 160 ℃ for 10 minutes and then stirred without heating for another 15 minutes. Silica bodies were synthesized using gold particles as cores, as described in the method section of the manuscript.

Cell lines

Immortalized cell lines derived from transgenic Kras^LSL-G12D/+；Trp53^LSL-R172H/+(ii) a Spontaneous tumors of Pdx-1-Cre mice. To allow bioluminescent tumor imaging of growing tumors after in situ implantation, cells were permanently transfected with luciferase-based lentiviral vectors in the UCLA vector core facility. Representative cell clones were obtained after using limiting dilution protocol.

In situ tumor generation in immunocompetent mice using KPC-derived cell lines

All animal experiments were performed using protocols approved by the UCLA animal research council. Female B6/129 mice (approximately 8 weeks) were purchased from The Jackson Laboratory. For in situ xenograft growth, mice were anesthetized with isoflurane followed by an intraperitoneal injection of 50mg/kg ketamine and 10mg/kg xylazine. The surgical site was shaved to leave a 1cm margin around the incision site and sterilized by scrubbing with povidone iodine and 70% ethanol. Mice were positioned on a heating pad for surgery and the incision site in the left abdomen was covered with sterile gauze. An approximately 0.7cm surgical incision was made to expose the injection site, which in turn would contain 2X 10 through a 27 gauge needle⁶50 μ L of DMEM/matrigel (1:1v/v) of individual KPC-luc cells was injected into the pancreatic tail. The fascia layer was closed with absorbable suture (PDS II, Ethicon) and the skin was closed with non-absorbable suture (PROLENE, Ethicon). Mice were kept on warming pads until recovery from anesthesia was complete, and then transferred to a clean cage. The mouse eyes were protected during surgery using an artificial tear ointment.

Interference with iRGD action using anti-NRP-1 blocking antibodies

Mice bearing KPC-derived orthotopic tumors received 50mg/kg NIR-labeled silica plus 8 μmol/kg free iRGD intravenous injection of 50 μ g of blocking anti-NRP-1 antibody or control IgG 15 minutes prior to. Animals receiving only silica bodies were used as controls. Animals were sacrificed 24 hours post injection and the biodistribution of NIR-labeled silica bodies was studied using ex vivo NIR imaging. Ex vivo imaging data was quantified by NIR intensity analysis using IVIS software followed by Si content analysis using ICP-OES.

HPLC analysis

For HPLC analysis of irinotecan in tissues, harvested tumor and organ samples were weighed and homogenized on ice. After 0.1mL of tissue homogenate was extracted with 0.4mL of acidic solution (0.1mol/L phosphoric acid/methanol, 1:4v/v), the extract was vortexed twice for 10 seconds and centrifuged at 13,000rpm for 10 minutes. The supernatant containing irinotecan was filtered through a 0.22 μm filter for HPLC analysis in a system containing a Knauer Smartline pneumatic pump, a C18 column, a K-2600 spectrophotometer, and Gina data collection software. The mobile phase delivered at a flow rate of 1.0 ml/min contained 3% triethylammonium acetate aqueous buffer (pH 5.5) and acetonitrile (73:27 v/v). A 20 microliter sample containing irinotecan was injected to measure drug absorption at 254nm, eluting typically at about 4.4 minutes. Irinotecan standard curves were generated over a concentration range of 0.05. mu.g/mL to 100. mu.g/mL.

Immunofluorescence staining

NPR-1 positive vessels in KPC tumor tissues were determined using two-color immunofluorescence staining. Tumor tissues were cryo-embedded using OCT reagents and used to prepare tumor sections. Sections were first treated with anti-NRP 1 monoclonal antibody (1:250) overnight at 4 ℃. After removal of primary antibody and 3 washes in PBS, Alexa was added488 secondary antibody (1:500) and incubated at room temperature for 1 hour. The same sections were also stained with anti-CD 31 antibody, followed by Alexa594 conjugated secondary antibodies were treated to identify CD31 expression. DAPI was used to localize nuclei. The stained slides were examined under a fluorescent microscope (Observer D1, Zeiss). Determination of NRP-1 by Imaging J software⁺/CD31⁺Co-location ratio of blood vessels.

Example 4

Comparative analysis of protocytes and silica bodies

One method of making lipid bilayer coated nanoparticles involves the use of MSNPs synthesized by aerosol-assisted self-assembly methods. MSNPs are coated by using electrostatically charged liposomes that in turn adhere, rupture, and then fuse to the negatively charged MSNP surface (see, e.g., Liu et al (2009) j.am. chem. soc.131: 7567-7569). Such products, developed by the Brinker doctor group of Sandia (Sandia) and the University of New Mexico (University of New Mexico), are called "Protocells" and U.S. patents are obtained under the names Protocells and the use for targeted delivery of multi-component cars to cancer cells (US 8,992,984B 1). The patent specifically teaches that "the primary cells can be formed by mixing the cargo component and the porous particles with the liposomes or lipids, followed by fusing the lipid bilayers on the porous particles and synergistically loading the cargo component into one or more pores of the porous particles to form the primary cell compound. 8,992,984 patent, fig. 1C depicts that "positively charged porous particles can be fused to a negatively charged lipid bilayer, such as a DOPS lipid bilayer, where the positively charged porous particles can absorb negatively charged cargo components (e.g., calcein or DNA or siRNA)".

In developing the methods and silica bodies described herein, we are actively avoiding the procedures and components used in primary cells, as we believe this approach is unpredictable and not feasible for a number of reasons. Not only did we fail to achieve uniform MSNP coating using the liposome fusion method described in the 8,992,984 patent, but we also failed to achieve significant drug loading in a number of attempts. This prompted us to develop the biofilm technique, sonication procedure and loading method described herein to obtain a reproducible procedure for uniform particle coating using LB with a different lipid composition than the primary cells.

Figure 32 depicts data generated during our attempt to generate "primary cells" equivalent to those described by Ashley et al (2011) nat. mat.,10: 389-397. Briefly, 100 μ L of 25mg/mL MSNP at 2.5mg/mL was added to 100 μ L of liposomes followed by the sequence of steps described in the Ashley et al publication (supra). The final product is shown in the above figure. The product was evaluated for hydrodynamic size, size distribution, and colloidal stability. The photographic inset on the left shows the phase separation of the "coated" product and the middle DLS plot shows the small peak of the uncoated particles plus the large peak of the agglomerated particulate matter. The TEM image on the right confirms particle aggregation of the procell and colloidal stability of the silica body. The lack of colloidal stability of the primary cells renders the product ineligible for use by intravenous administration. In summary, unlike native cells, silica exhibits excellent colloidal stability, low PDI, and narrow size distribution.

Table 6 illustrates various differences between the silica body technique and the primary cell technique.

Table 6: comparison of certain characteristics of silica body technology with native cell technology.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (133)

1. A nanoparticulate drug carrier comprising:

a silica nanoparticle having a surface and defining a plurality of pores adapted to contain molecules therein;

a lipid bilayer coating the surface;

a cargo trapping agent within a pore comprising the plurality of pores; and

a cargo comprising a drug, wherein the cargo is associated with the cargo trap in the pores;

wherein the sub-micron structures have a largest dimension of less than 1 micron, and wherein the lipid bilayer stably seals the plurality of pores.

2. The nanoparticle drug carrier of claim 1, wherein the lipid bilayer comprises phospholipids, Cholesterol (CHOL), and mPEG phospholipids.

3. The nanoparticulate drug carrier of any one of claims 1 to 2, wherein the phospholipid comprises a natural lipid comprising a mixture of saturated fatty acids having carbon chains of C14-C20, and/or unsaturated fatty acids having carbon chains of C14-C20, and/or fatty acids having carbon chains of C12-C20.

4. The nanoparticulate drug carrier of claim 3, wherein the phospholipid comprises a saturated fatty acid selected from the group consisting of: phosphatidylcholine (DPPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC) and Diacylphosphatidylcholine (DAPC).

5. The nanoparticulate drug carrier of claim 3, wherein the phospholipid comprises a natural lipid selected from the group consisting of: egg phosphatidylcholine (egg PC) and soybean phosphatidylcholine (soybean PC).

6. The nanoparticulate drug carrier of claim 3, wherein the phospholipid comprises an unsaturated fatty acid selected from the group consisting of: 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyloyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dieicosenoyl-sn-glycero-3-phosphocholine.

7. The nanoparticulate drug carrier of any one of claims 1 to 6, wherein the lipid bilayer comprises mPEG phospholipids having phospholipid C14-C18 carbon chains and PEG molecular weights in the range of about 350Da to 5000 Da.

8. The nanoparticulate drug carrier of claim 7, wherein the lipid bilayer comprises 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-PEG (DSPE-PEG).

9. The nanoparticle drug carrier of claim 2, wherein the lipid bilayer comprises DPPC/Chol/DSPE-PEG or DSPC/Chol/DSPE-PEG.

10. The nanoparticulate drug carrier of claim 9, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG.

11. The nanoparticulate drug carrier of claim 10, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG 2000.

12. The nanoparticulate drug carrier of any one of claims 1 to 11, wherein the lipid bilayer comprises phospholipids, cholesterol, and mPEG phospholipids in a ratio of 50-90 mol% phospholipids to 10-50 mol% CHOL to 1-10 mol% mPEG phospholipids.

13. The nanoparticulate drug carrier of claim 10, wherein the lipid bilayer comprises a molar ratio of DSPC/Chol/DSPE-PEG of about 3:2: 0.15.

14. The nanoparticle drug carrier of any one of claims 1-13, wherein the lipid bilayer forms a substantially continuous bilayer surrounding the entire nanoparticle.

15. The nanoparticle drug carrier of any one of claims 1-14, wherein the lipid bilayer forms a substantially uniform and intact bilayer surrounding the entire nanoparticle.

16. The nanoparticulate drug carrier of any one of claims 1 to 15, wherein the silica nanoparticles are mesoporous silica nanoparticles.

17. The nanoparticulate drug carrier of claim 16, wherein the mesoporous silica nanoparticles are colloidally stable.

18. The nanoparticulate drug carrier of any one of claims 16 to 17, wherein the mesoporous silica has:

an average pore size in the range of from about 1nm to about 20nm, or from about 1nm to about 10nm, or from about 2nm to about 8 nm; and

average size within the following ranges: from about 50nm to about 300nm, or from about 50nm to about 200nm, or from about 50nm to about 150nm, or from about 50nm to about 100nm, or from about 50nm to about 80nm, or from about 50nm to about 70nm, or from about 60nm to about 70 nm.

19. The nanoparticulate drug carrier of any one of claims 1 to 18, wherein prior to reaction with the drug, the cargo trap is selected from sucrose triethylammonium octasulfate (TEA)₈SOS)、(NH₄)₂SO₄Ammonium, trimethylammonium, and triethylammonium salts.

20. According to claim19, wherein the cargo trap is triethylammonium sucrose octasulfate (TEA) prior to reaction with the drug₈SOS)。

21. The nanoparticulate drug carrier of claim 20, wherein the drug is protonated and interacts with SOS^8-Trapped in the pores as gelatinous precipitates upon association.

22. The nanoparticulate drug carrier of any one of claims 1 to 21, wherein:

the drug comprises at least one weakly basic group capable of being protonated and the cargo trap comprises at least one anionic group; and/or

The drug is selected to have a pKa greater than 7 and less than 11; and/or

The drug comprises a primary amine, a secondary amine, or a tertiary amine; and/or

The drug is selected to have a water solubility index of about 5mg/mL to about 25 mg/mL; and/or

The drug is selected to have an octanol/water partition coefficient or logP value of about-3.0 to about 3.0; and/or

The drug is selected to be less than the average or median size of the pores of the silica nanoparticles.

23. The nanoparticle medicament of claim 22, wherein the cargo comprises an anti-cancer drug.

24. The nanoparticulate drug carrier of claim 23, wherein the cargo comprises irinotecan.

25. The nanoparticulate drug carrier of claim 23, wherein the cargo comprises one or more drugs independently selected from the group consisting of: topoisomerase inhibitors, antineoplastic anthracyclines, mitotic inhibitors, alkaloids, basic alkylating agents, purine or pyrimidine derivatives, and protein kinase inhibitors.

26. The nanoparticulate drug carrier of claim 25, wherein the carrier comprises a drug selected from the group consisting of: topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, LAQ824, doxorubicin, mitoxantrone, vinblastine, vinorelbine, cyclophosphamide, mechlorethamine, temozolomide, 5-fluorouracil, 5' -deoxy-5-fluorouridine, gemcitabine, imatinib, oxitinib and sunitinib, pazopanib, enzastalin, vandetanib, erlotinib, dasatinib and nilotinib.

27. The nanoparticulate drug carrier of any one of claims 1 to 26, wherein the drug carrier is conjugated to a moiety selected from: targeting moieties, fusion peptides, and transit peptides.

28. The nanoparticulate drug carrier of claim 27, wherein the drug carrier is conjugated to a peptide that binds to a receptor on a cancer cell or tumor blood vessel.

29. The nanoparticulate drug carrier of claim 28, wherein the drug carrier is conjugated to an iRGD peptide.

30. The nanoparticulate drug carrier of claim 28, wherein the drug carrier is conjugated to a targeting peptide shown in table 2.

31. The nanoparticulate drug carrier of any one of claims 27 to 30, wherein the drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.

32. The nanoparticulate drug carrier of any one of claims 27 to 31, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds a cancer marker.

33. The nanoparticulate drug carrier of claim 32, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds a cancer marker set forth in table 1.

34. The nanoparticulate drug carrier of claim 22, wherein the cargo comprises an antibiotic, antiviral, or antifungal agent.

35. The nanoparticulate drug carrier of claim 34, wherein:

the cargo comprises an antibiotic selected from ciprofloxacin and levofloxacin; and/or

The cargo comprises an antiviral agent selected from tenofovir, disoproxil, and fumarate; and/or

The cargo comprises an antifungal agent selected from the group consisting of: amphotericin B, anidulafungin, caspofungin, fluconazole, flucytosine, isaconazole, itraconazole, micafungin, posaconazole and voriconazole.

36. The nanoparticulate drug carrier of any one of claims 1 to 35, wherein the drug carrier has less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% leakage of the cargo within 24 hours at 37 ℃ in a biological buffer having a pH of 7.4.

37. The nanoparticulate drug carrier of any one of claims 1 to 36, wherein the drug carrier has the following drug loading capacities: at least about 8% w/w, or at least about 10% w/w, or at least about 20% w/w, or at least about 30% w/w, or greater than about 40% w/w, or greater than about 50% w/w, or greater than about 60% w/w, or greater than about 70% w/w, or greater than about 80% w/w.

38. The nanoparticulate drug carrier of any one of claims 1 to 36, wherein the drug carrier has a drug loading capacity of at least 80% w/w.

39. The nanoparticulate drug carrier of any one of claims 1 to 38, wherein the lipid bilayer comprises a hydrophobic drug.

40. The nanoparticle drug carrier of claim 39, wherein the lipid bilayer comprises a hydrophobic drug selected from the group consisting of: paclitaxel, ellipticine, camptothecin, SN-38, and lipid prodrugs (e.g., acyclovir diphosphate dimyristoyl glycerol, doxorubicin-conjugated phospholipid prodrugs, phospholipid derivatives of nucleoside analogs, phospholipid-linked chlorambucil, etc.).

41. The nanoparticle drug carrier of claim 39, wherein the lipid bilayer comprises paclitaxel.

42. The nanoparticulate drug carrier of any one of claims 1 to 40, wherein the drug carrier is stable in suspension for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4 ℃.

43. The nanoparticulate drug carrier of any one of claims 1 to 42, wherein in suspension a population of the drug carrier:

shows a size distribution with a width (full width at half maximum) in the following range: less than about 30nm, or less than about 20nm, or less than about 10nm, or less than about 5nm, or less than about 3nm, or less than about 2 nm; and/or

Exhibits a substantially monomodal size distribution; and/or

Exhibit a PDI of less than about 0.2, or less than about 0.1; and/or

Exhibit a coefficient of dimensional variation of less than about 0.1, or less than about 0.05, or less than about 1.7/120.

44. The nanoparticulate drug carrier of any one of claims 1 to 43, wherein about 3% or more of the nanoparticulate drug carrier distributes to developing tumor sites upon intravenous injection.

45. The nanoparticulate drug carrier of any one of claims 1 to 44, wherein the nanoparticulate drug carrier forms a stable suspension upon rehydration after lyophilization.

46. The nanoparticulate drug carrier of any one of claims 1 to 45, wherein the nanoparticulate drug carrier, when loaded with an anticancer drug, provides more effective killing of cancer cells in an in situ PDAC model than free drug or liposomes containing the drug.

47. The nanoparticulate drug carrier of any one of claims 1 to 46, wherein the nanoparticulate drug carrier exhibits reduced drug toxicity when loaded with an anticancer drug as compared to the drug in free drug and/or liposomes.

48. The nanoparticulate drug carrier of any one of claims 1 to 47, wherein the nanoparticulate drug carrier is colloidally stable in physiological fluids having a pH of 7.4 and remains monodisperse to allow systemic biodistribution and is capable of entering a disease site through vascular leakage (EPR effect) or transcytosis.

49. A pharmaceutical formulation, the formulation comprising:

a plurality of nanoparticulate drug carriers according to any one of claims 1 to 48; and

a pharmaceutically acceptable carrier.

50. The formulation of claim 49, wherein the formulation is an emulsion, dispersion, or suspension.

51. The formulation of claim 50, wherein the suspension, emulsion or dispersion is stable for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months when stored at 4 ℃.

52. The formulation of any one of claims 49-51, wherein:

the nanoscale drug carrier in the formulation exhibits a substantially monomodal size distribution; and/or

The drug carrier in the suspension, emulsion, or dispersion exhibits a PDI of less than about 0.2, or less than about 0.1.

53. The formulation of any one of claims 49-52, wherein the formulation is formulated for administration via a route selected from the group consisting of: intravenous administration, intra-arterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery), intracranial administration via cannula, and subcutaneous or intramuscular depot deposition.

54. The formulation of any one of claims 49-52, wherein the formulation is a sterile injectable formulation.

55. The preparation of any one of claims 49-54, wherein the preparation is a unit dose preparation.

56. A method of treating cancer, the method comprising:

administering to a subject in need thereof an effective amount of the nanoparticulate drug carrier according to any one of claims 1 to 33 or 36 to 48 or the pharmaceutical formulation according to any one of claims 49 to 55, wherein the drug in the nanoparticulate drug carrier and/or the pharmaceutical formulation comprises an anticancer drug.

57. The method of claim 56, wherein the nanoparticle drug carrier and/or the pharmaceutical formulation is the primary treatment in a chemotherapeutic regimen.

58. The method of claim 56, wherein the nanoparticle drug carrier and/or the pharmaceutical formulation is a component in a multi-drug chemotherapy regimen.

59. The method of claim 58, wherein the multi-drug chemotherapy regimen comprises at least two drugs, or at least three drugs, or at least 4 drugs selected from: irinotecan (IRIN), Oxaliplatin (OX), 5-fluorouracil (5-FU) and Leucovorin (LV).

60. The method of any one of claims 56-59, wherein the cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

61. The method of any one of claims 56-59, wherein the cancer is a cancer selected from the group consisting of: acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Karposi's sarcoma, lymphoma), anal cancer, appendiceal cancer, astrocytoma, atypical teratomas/rhabdoid tumors, cholangiocarcinoma, extrahepatic cancer, bladder cancer, bone cancers (e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytoma, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratomas/rhabdoid tumors, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngiomas, ependymomas, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, cervical cancer, chordoma, neuroblastoma, lymphoma, melanoma, lymphoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal cancers (e.g., cholangiocarcinoma, extrahepatic carcinoma), Ductal Carcinoma In Situ (DCIS), embryonal tumors, endometrial cancers, ependymoma, esophageal cancer, olfactory neuroblastoma, extracranial germ cell tumors, extragonally germ cell tumors, extrahepatic cholangiocarcinoma, ocular cancers (e.g., intraocular melanoma, retinoblastoma), malignant bone fibrohistiocytoma, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancer, extragonadal cancer, central nervous system), gestational trophoblastic tumors, brain stem cancer, hairy cell leukemia, head and neck cancer, malignant bone cell tumors, and brain tumors, Cardiac cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kaposi's sarcoma, kidney cancer (e.g. renal cell cancer, wilms tumor and other renal tumors), langerhans cell histiocytosis, laryngeal cancer, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), hairy cell leukemia, lip and oral cancer, liver cancer (primary), Lobular Carcinoma In Situ (LCIS), lung cancer (e.g. childhood lung cancer, non-small cell lung cancer, small cell lung cancer), lymphoma (e.g. AIDS-related lymphoma, burkitt lymphoma (e.g. non-hodgkin's lymphoma), cutaneous T cell lymphoma (e.g. mycosis fungoides, mycosis coli, trichotheca, neuroendocrine tumor, pancreatic cancer, and other renal tumors), langerhans cell histiocytosis, Sezary syndrome), hodgkin lymphoma, non-hodgkin lymphoma, primary Central Nervous System (CNS) lymphoma), macroglobulinemia, waldenstrom's disease, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood melanoma, intraocular (eye) melanoma), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, mid-line cancer, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, Chronic Myelogenous Leukemia (CML), multiple myeloma, nasal and paranasal sinus cancers, nasopharyngeal cancer, neuroblastoma, oral cancer, lip and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic neuroendocrine tumor (islet cell tumor), papillomatosis, paraganglioma, Paranasal sinus and nasal cavity cancer, parathyroid gland cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, pleuropulmonoblastoma, primary Central Nervous System (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, transitional cell carcinoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma (e.g., ewing's sarcoma, kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma), sezary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, non-melanoma), small intestine cancer, squamous cell carcinoma, occult primary squamous neck cancer, stomach (stomach) cancer, testicular cancer, pharyngeal cancer, thymoma and thymus cancer, thyroid cancer, trophoblastic cell tumor, ureter and renal pelvis cancer, urinary tract cancer, uterine cancer, endometrial cancer, uterine sarcoma, cervical cancer, vaginal cancer, vulvar cancer, waldenstrom's macroglobulinemia, and nephroblastoma.

62. The method of any one of claims 56-61, wherein the nanoparticle drug carrier is not conjugated to an iRGD peptide and the nanoparticle drug carrier is administered in conjunction with an iRGD peptide.

63. A method of treating an infection, the method comprising:

administering to a subject in need thereof an effective amount of the nanoparticulate drug carrier according to any one of claims 1 to 22 or 34 to 35 or the pharmaceutical formulation according to any one of claims 49 to 55, wherein the drug in the nanoparticulate drug carrier and/or the pharmaceutical formulation comprises an antimicrobial drug.

64. The method of claim 56, wherein the infection comprises an infection with a drug-resistant bacterium, virus, or fungus.

65. The method of any one of claims 56 to 64, wherein the nanoparticle drug carrier and/or pharmaceutical formulation is administered via a route selected from: intravenous administration, intra-arterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation (including intranasal and intratracheal delivery), intracranial administration via cannula, and subcutaneous or intramuscular depot deposition.

66. The method of any one of claims 56-64, wherein the nanoparticle drug carrier and/or drug formulation is administered as an injection, from an IV drip bag, or via a drug delivery cannula.

67. The method of any one of claims 56-66, wherein the subject is a human.

68. The method of any one of claims 56-66, wherein the subject is a non-human mammal.

69. A method of making a nanoparticulate drug carrier, the method comprising:

providing a nanoparticle comprising silica having a surface and defining a plurality of pores adapted to contain drug molecules therein;

disposing a trapping agent in a pore comprising the plurality of pores, wherein the trapping agent is selected for its ability to trap the drug within the pore;

coating the pores of the nanoparticles with a lipid bilayer; and

contacting or soaking the lipid bilayer coated nanoparticle with a drug that can pass through the bilayer, wherein the drug enters the pores, reacts with the trapping agent and remains within the bilayer.

70. The method of claim 69, wherein the lipid bilayer comprises phospholipids, Cholesterol (CHOL), and mPEG phospholipids.

71. The method of any one of claims 69 to 70, wherein the phospholipid comprises a natural lipid comprising a mixture of saturated fatty acids having a carbon chain of C14-C20, and/or unsaturated fatty acids having a carbon chain of C14-C20, and/or fatty acids having a carbon chain of C12-C20.

72. The method of claim 71, wherein the phospholipid comprises a saturated fatty acid selected from the group consisting of: phosphatidylcholine (DPPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC) and Diacylphosphatidylcholine (DAPC).

73. The method of claim 71, wherein the phospholipid comprises a natural lipid selected from the group consisting of: egg phosphatidylcholine (egg PC) and soybean phosphatidylcholine (soybean PC).

74. The method of claim 71, wherein the phospholipid comprises an unsaturated fatty acid selected from the group consisting of: 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyloyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dieicosenoyl-sn-glycero-3-phosphocholine.

75. The method of any one of claims 69 to 74, wherein the lipid bilayer comprises mPEG phospholipids having phospholipid C14-C18 carbon chains and PEG molecular weights in the range of about 350Da to 5000 Da.

76. The nanoparticulate drug carrier of claim 75, wherein the lipid bilayer comprises 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-PEG (DSPE-PEG).

77. The method of claim 70, wherein the lipid bilayer comprises DPPC/Chol/DSPE-PEG or DSPC/Chol/DSPE-PEG.

78. The method of claim 77, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG.

79. The method of claim 78, wherein the lipid bilayer comprises DSPC/Chol/DSPE-PEG 2000.

80. The method of any one of claims 69 to 79, wherein the lipid bilayer comprises phospholipids, cholesterol, and mPEG phospholipids in a ratio of 50-90 mol% phospholipids to 10-50 mol% CHOL to 1-10 mol% mPEG phospholipids.

81. The method of claim 78, wherein the lipid bilayer comprises a DSPC/Chol/DSPE-PEG molar ratio of about 3:2: 0.15.

82. The method of any one of claims 69 to 81, wherein the lipid comprising the lipid bilayer is combined with the nanoparticle in a ratio sufficient to form a continuous bilayer over the entire nanoparticle.

83. The method of any one of claims 69-82, wherein the lipid comprising the lipid bilayer is combined with the nanoparticle at a particle-to-lipid ratio in a range starting from about 1.0: 3.0.

84. The method of any one of claims 69 to 82, wherein the lipid comprising the lipid bilayer is combined with the nanoparticle at a particle to lipid ratio of about 1.0: 1.1.

85. The method of any one of claims 69 to 84, wherein the lipid bilayer forms a substantially continuous bilayer surrounding the entire nanoparticle.

86. The method of any one of claims 69-85, wherein the lipid bilayer forms a substantially uniform and intact bilayer surrounding the entire nanoparticle.

87. The method of any one of claims 69 to 86, wherein the silica nanoparticles are mesoporous silica nanoparticles.

88. The method of claim 87, wherein the silica nanoparticles comprise sol-gel synthesized mesoporous silica nanoparticles.

89. The method of any one of claims 69-88, wherein:

the mesoporous silica nanoparticles have an average pore size in a range from about 1nm to about 20nm, or from about 1nm to about 10nm, or from about 2nm to about 8 nm; and/or

The mesoporous silica nanoparticles have an average size within the following ranges: from about 50nm to about 300nm, or from about 50nm to about 200nm, or from about 50nm to about 150nm, or from about 50nm to about 100nm, or from about 50nm to about 80nm, or from about 50nm to about 70nm, or from about 60nm to about 70 nm.

90. The method of any one of claims 69 to 89, wherein prior to reaction with the drug, the cargo trap is selected from triethylammonium sucrose octasulfate (TEA)₈SOS)、(NH₄)₂SO₄Ammonium, trimethylammonium, and triethylammonium salts.

91. The method of claim 90, wherein the cargo trap is triethylammonium sucrose octasulfate (TEA) prior to reaction with the drug₈SOS)。

92. The method of claim 91, wherein the drug is protonated and interacts with SOS^8-Trapped in the pores as gelatinous precipitates upon association.

93. The method of any one of claims 69-92, wherein:

the drug comprises at least one weakly basic group capable of being protonated and the cargo trap comprises at least one anionic group; and/or

The drug is selected to have a pKa greater than 7 and less than 11; and/or

The drug comprises a primary amine, a secondary amine, or a tertiary amine; and/or

The drug is selected to have a water solubility index of about 5mg/mL to about 25 mg/mL; and/or

The drug is selected to have an octanol/water partition coefficient or logP value of about-3.0 to about 3.0; and/or

The drug has a size less than the average or median size of the pores of the silica nanoparticles.

94. The method of claim 93, wherein the cargo comprises an anti-cancer drug.

95. The method of claim 94, wherein the cargo comprises irinotecan.

96. The method of claim 94, wherein the cargo comprises one or more drugs independently selected from the group consisting of: topoisomerase inhibitors, antineoplastic anthracyclines, mitotic inhibitors, alkaloids, basic alkylating agents, purine or pyrimidine derivatives, protein kinase inhibitors.

97. The method of claim 96, wherein the carrier comprises a drug selected from the group consisting of: topotecan, 10-hydroxycamptothecin, belotecan, rubitecan, vinorelbine, and LAQ824, doxorubicin, mitoxantrone, vinblastine, vinorelbine, cyclophosphamide, mechlorethamine, temozolomide, 5-fluorouracil, 5' -deoxy-5-fluorouridine, gemcitabine, imatinib, oxitinib and sunitinib, pazopanib, enzastalin, vandetanib, erlotinib, dasatinib, and nilotinib.

98. The method according to any one of claims 69 to 97, wherein the drug carrier is conjugated to a moiety selected from: targeting moieties, fusion peptides, and transit peptides.

99. The method according to any one of claims 69 to 98, wherein the method produces a nanoparticulate drug carrier according to any one of claims 1 to 48.

100. The method of claim 98, wherein the drug carrier is conjugated to a peptide that binds to a receptor on a cancer cell.

101. The method of claim 100, wherein the drug carrier is conjugated to an iRGD peptide.

102. The method of claim 100, wherein the drug carrier is conjugated to a targeting peptide shown in table 2.

103. The method of any one of claims 98 to 102, wherein the drug carrier is conjugated to transferrin, and/or ApoE, and/or folate.

104. The method of any one of claims 98 to 103, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds a cancer marker.

105. The method of claim 104, wherein the drug carrier is conjugated to a targeting moiety comprising an antibody that binds to a cancer marker set forth in table 1.

106. The method of claim 93, wherein the cargo comprises an antibiotic.

107. The method of claim 106, wherein the cargo comprises an antibiotic selected from the group consisting of: ciprofloxacin, levofloxacin, and HIV antiretroviral drugs (e.g., tenofovir disoproxil fumarate, and the like).

108. The method of any one of claims 69-107, wherein the drug carrier is loaded to a capacity of: at least 30% w/w, or greater than about 40% w/w, or greater than about 50% w/w, or greater than about 60% w/w, or greater than about 70% w/w, or greater than about 80% w/w.

109. The method of any one of claims 69 to 107, wherein the drug carrier is loaded to a capacity of at least 80% w/w.

110. The method of any one of claims 69-109, wherein the lipid bilayer comprises a hydrophobic drug.

111. The method of claim 110, wherein the lipid bilayer comprises a hydrophobic drug selected from the group consisting of: paclitaxel, ellipticine, camptothecin, SN-38, and lipid prodrugs (e.g., acyclovir diphosphate dimyristoyl glycerol, doxorubicin-conjugated phospholipid prodrugs, phospholipid derivatives of nucleoside analogs, phospholipid-linked chlorambucil, etc.).

112. The method of claim 110, wherein the lipid bilayer comprises paclitaxel.

113. A method of preparing an irinotecan nanocarrier, the method comprising:

providing a nanocarrier comprising a silica body having a surface comprising a plurality of pores suitable for housing irinotecan therein;

providing an agent selected for its ability to trap irinotecan within the plurality of pores;

coating the pores of the nanocarriers with a phospholipid bilayer (optionally using sonication methods); and

irinotecan is introduced into the pores of the phospholipid bilayer coating,

to prepare the irinotecan nano-carrier coated by the phospholipid bilayer.

114. The method of claim 113, wherein the silica body comprises a sol-gel synthesized, size-controlled, and colloidally-stabilized silica body.

115. The method of claim 113, wherein the irinotecan trapping agent is triethylammonium sucrose octasulfate (TEA)₈SOS)。

116. The method of claim 115, wherein the nanocarrier:

(a) having an irinotecan loading capacity of at least 20% (or 30% or 40%) w/w; and/or

(b) Irinotecan leakage of < 5% (or < 10%) was shown within 24 hours at 37 ℃ in biological buffer with pH of 7.4.

117. The method of claim 116, wherein the nanocarriers are colloidally stable in physiological fluids having a pH of 7.4 and remain monodisperse to allow systemic biodistribution and are capable of entering a disease site through vascular leakage (EPR effect) or transcytosis.

118. The method of claim 116, wherein the phospholipid bilayer comprises cholesterol and/or paclitaxel.

119. A method of making a nanocarrier, the method comprising:

providing an unsupported nanocarrier, the unsupported nanocarrier comprising: a silica body having a surface and defining a plurality of pores adapted to contain molecules therein; and a phospholipid bilayer coating the surface;

a cargo trapping agent is encapsulated within the phospholipid bilayer.

120. The method of claim 119, further comprising exposing the nanocarriers to a cargo selected to interact with the cargo trap.

121. The method of claim 120, wherein the cargo is selected to have a pKa greater than 7 and less than 11 and is capable of being protonated, and the cargo trap comprises at least one anionic group.

122. The method of claim 120, wherein the cargo is irinotecan and the cargo-trapping agent is triethylammonium sucrose octasulfate (TEA)₈SOS)。

123. The method of claim 120, wherein the cargo is a topoisomerase I inhibitor: topotecan; one or more anti-tumor anthracycline antibiotics: doxorubicin and mitoxantrone; one or more mitotic inhibitors: vinblastine and vinorelbine; or one or more tyrosine kinase inhibitors: imatinib, oxitinib and sunitinib.

124. The method of claim 120, wherein the nanocarrier has a drug loading capacity of at least 30% w/w.

125. A nanocarrier, comprising:

a silica body having a surface and defining a plurality of pores adapted to contain molecules therein;

a phospholipid bilayer coating the surface; and

a cargo trapping agent within the phospholipid bilayer;

wherein the sub-micron structures have a largest dimension of less than 1 micron, and wherein the phospholipid bilayer stably seals the plurality of pores.

126. The nanocarrier of claim 125, further comprising a cargo within the phospholipid bilayer.

127. The nanocarrier of claim 126, wherein the cargo is associated with the cargo trap.

128. The nanocarrier of claim 127, wherein:

the cargo comprises at least one weakly basic group capable of being protonated and the cargo trapping agent comprises at least one anionic group; and/or

The cargo is selected to have a pKa greater than 7 and less than 11; and/or

The cargo comprises a primary, secondary, tertiary or quaternary amine; and/or

The cargo is selected to have a water solubility index of 5mg/mL to 25 mg/mL; and/or

The cargo is selected to have an octanol/water partition coefficient or logP value of from-3.0 to 3.0; and/or

The cargo is selected to be 2nm-8nm and smaller than the size of the pores of the nanocarrier.

129. The nanocarrier of claim 126, wherein the cargo is irinotecan and the cargo-trapping agent is triethylammonium sucrose octasulfate (TEA)₈SOS)。

130. The nanocarrier of claim 126, wherein the cargo is a topoisomerase I inhibitor: topotecan; one or more anti-tumor anthracycline antibiotics: doxorubicin and mitoxantrone; one or more mitotic inhibitors: vinblastine and vinorelbine; or one or more tyrosine kinase inhibitors: imatinib, oxitinib and sunitinib.

131. The nanocarrier of claim 126, wherein the nanocarrier has less than 5% leakage of the cargo within 24 hours at 37 ℃ in a biological buffer having a pH of 7.4.

132. The nanocarrier of claim 126, wherein the nanocarrier has a drug loading capacity of at least 30% w/w, or at least 80% w/w.

133. The nanocarrier of claim 125, wherein the phospholipid bilayer comprises paclitaxel.