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WO2024178502A1 - Nanoparticules lipidiques modifiées couche par couche, utilisations correspondantes et leurs procédés de production - Google Patents

Nanoparticules lipidiques modifiées couche par couche, utilisations correspondantes et leurs procédés de production Download PDF

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Publication number
WO2024178502A1
WO2024178502A1 PCT/CA2024/050238 CA2024050238W WO2024178502A1 WO 2024178502 A1 WO2024178502 A1 WO 2024178502A1 CA 2024050238 W CA2024050238 W CA 2024050238W WO 2024178502 A1 WO2024178502 A1 WO 2024178502A1
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Prior art keywords
layer
lipid nanoparticle
lnp
coated
core
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Inventor
Victor PASSOS GIBSON
Houda TAHIRI
Chun Yang
Xavier BANQUY
Pierre Hardy
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Universite de Montreal
Valorisation HSJ LP
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Universite de Montreal
Valorisation HSJ LP
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    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
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    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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    • C12N2310/3517—Marker; Tag

Definitions

  • the present application is in the field of lipid nanoparticles. More specifically, the present application relates to layer-by-layer modified lipid nanoparticles, uses for gene and drug delivery and methods for producing the same.
  • RNA-based strategies have gained considerable attention.
  • LNP lipid nanoparticles
  • mRNA messenger RNA
  • SARS-CoV-2 virus responsible for the COVID-19 pandemic
  • the LNP-based vaccines produced by Pfizer and Moderna were approved in record time by the FDA bringing to light a seminal therapeutic intervention to a deadly global health crisis. In both situations, LNP allowed scientists to overcome the challenges of working with nucleic acids in therapeutics.
  • the present application includes a coated lipid nanoparticle comprising: a lipid nanoparticle core comprising a gene, a drug, a contrast agent or a mixture thereof; at least one first layer around the core comprising negatively charged nucleic acids; at least one second layer around the first layer comprising polycations; and a surface layer around the at least one second layer comprising polyelectrolytes negatively charged.
  • lipid nanoparticle of the present application Also included is a gene and/or drug delivery system comprising the coated lipid nanoparticle of the present application.
  • a medical imaging system comprising the coated lipid nanoparticle of the present application, wherein the core comprises the contrast agent.
  • a coated lipid nanoparticle of the present application for gene and/or drug delivery, for gene and/or drug delivery to target cells, in the manufacture of a medicament for gene and/or drug delivery to target cells, or where the core comprises the contrast agent, for medical imaging.
  • the present application also includes use of a layer-by-layer process for manufacturing a coated lipid nanoparticle comprising: a lipid nanoparticle core comprising a gene, a drug, a contrast agent or a mixture thereof; at least one first layer over the core comprising negatively charged nucleic acids; at least one second layer over the first layer comprising polycations; and a surface layer over the at least one second layer comprising polyelectrolytes negatively charged.
  • a method of producing a coated lipid nanoparticle for gene and/or drug delivery comprising: forming a lipid nanoparticle core comprising the gene, the drug or a mixture thereof; coating the lipid nanoparticle core with at least one first layer comprising negatively charged nucleic acids; coating the at least one first layer with at least one second layer comprising polycations; coating the at least one second layer with a surface layer comprising polyelectrolytes negatively charged; wherein the method comprises conducting a titration before each coating to determine a minimum amount of each layer to be used.
  • the present application also includes a method of producing a coated lipid nanoparticle for medical imaging, the method comprising: forming a lipid nanoparticle core comprising a contrast agent; coating the lipid nanoparticle core with at least one first layer comprising negatively charged nucleic acids; coating the at least one first layer with at least one second layer comprising polycations; coating the at least one second layer with a surface layer comprising polyelectrolytes negatively charged; wherein the method comprises conducting a titration before each coating to determine a minimum amount of each layer to be used.
  • Also provided is a method for treating a subject in need of a gene and/or a drug comprising administering the coated lipid nanoparticle of the present application to the subject in need thereof.
  • the present application also includes a method for treating a subject in need of a gene and/or a drug, the method comprising: forming a lipid nanoparticle core comprising the gene, the drug or a mixture thereof; coating the lipid nanoparticle core with at least one first layer comprising negatively charged nucleic acids; coating the at least one first layer with at least one second layer comprising polycations; coating the at least one second layer with a surface layer comprising polyelectrolytes negatively charged to provide a coated lipid nanoparticle; administering the coated lipid nanoparticle to the subject in need thereof.
  • a method to increase the contrast of cells for medical imaging the method comprising administering the coated lipid nanoparticle of the present application comprising a contrast agent to the cells.
  • FIGs.1 (A), 1 (B) and 1 (C) illustrate results of physicochemical characterization of hyaluronan Layer-by-Layer Lipid Nanoparticle (HA-LNP) according to exemplary embodiments of the application, where FIG.1 (A) is a schematic illustration of a process of preparation; FIG.1 (B) is DLS characterization; and FIG.1 (C) is images by atomic force microscopy of (starting clockwise from the top left): pure LNP, LNP after 1 st (Oligo), 2 nd (PLA), 3 rd (HA) coating, respectively.
  • FIGs. 2A and 2B illustrate results of the internalization of HA-LNP by U87 cells according to exemplary embodiments of the application, as fluorophore images after overnight incubation.
  • FIG. 2A represents HA-LNP tagged with siRNA-Cy5 in the layer compartment (first row), HA-LNP tagged with Rhodamine-PE in the core of LNP (second row), and HA-LNP doubled tagged with siRNA-Cy5 (layer) and Rhodmaine- PE (core) (third row), where stained nuclei (Hoechst) are shown in first column, stained siRNA (Cy5) in second column, stained lipid (Rhodamine-PE, 0.1 mol %) in the third column, and merged images of all three in the fourth column, where the scale is 40 pm for all pictures.
  • FIG. 2B is an emphasized and zoomed in image of the merged fluorophore for HA-LNP doubled tagged with siRNA-Cy5 (layer) where the scale is adjusted to 10 pm.
  • FIGs. 3(a), 3(b) and 3(c) illustrate results of the internalization of HA-NPs according to exemplary embodiments of the application, where FIG. 3(a) is a graph of flow cytometry analysis of internalization ratio of HA-LNP loaded with siRNA-Cy5 by GBM cells (U87 and 11251 ) and breast cancer cell line MCF-7; FIG. 3(b) shows CD44 expression measured by Western blot on U87, 11251 and MCF-7 cells FIG.
  • 3(c) shows median fluorescence intensity diagram showing blockage of CD44 with excess of ligand (HA) prior LNP incubation diminish their internalization (MCF-7, 1 h preincubated with HA-500KDa at final concentration of 5mg.mL-1 , 15 min incubation with HA-LNP or LNP I the presence of excess of ligand).
  • FIGs. 4(A), 4(B), 4(C) illustrate results of transfection of HeLa-GFP and U87 cells by HA-NPs in vitro according to exemplary embodiments of the application, where FIGs. 4(A) and FIG. 4(B) show the delivery of siRNA GFP (siGFP) promoted dose-dependent protein silencing 3 days after transfection; FIG.
  • 4(C) shows the transfection efficiency of HA-LNP by transfection of positive control siRNA All Stars (siAS) in U87 cells (3 days) - Mirus transfection reagent complexed with siRNA All Stars; for siGFP and siAS transfection, student t-test was performed where ns p > 0.05 (siSCR vs NT) and **** p ⁇ 0.0001 (siGFP vs NT, siAS vs siSCR, and Mirus siAs vs Mirus siSCR).
  • FIGs. 5(a) and 5(b) illustrate results of gene silencing promoted by HA- LNP/siGFP over time and using different ionizable lipid, according to exemplary embodiments of the application.
  • FIGs. 7(A) and 7(B) illustrate results of HA-NPs delivery of miR-181 a to U87 cells, according to exemplary embodiments of the application, where FIG. 7(A) shows upregulation of intracellular levels of miR after transfection (72h) with loading miR-181 a in the core or layer or both; and FIG. 7(B) shows comparative results for HA- deficient LNP (overnight incubation); student t-test, where ns p > 0.05, and *** p ⁇ 0.001.
  • FIGs.8A and 8B illustrate results of toxic effects induced by miR181 a in vitro according to exemplary embodiments of the application, where FIG.
  • FIG. 8(A) shows cellular proliferation in a cell line-dependent fashion impaired by HA-LNP/miR181 a (300 nM);
  • FIG. 8(B) shows cellular toxicity induced by delivery of miRNA 181 a on U87 cells at 64 nM compared to scramble treated cells, compared with strong cellular death in positive control siRNA All Stars (64 nM); student t-test, where ns p > 0.05 (siSCR vs NT), ** p ⁇ 0.005 (HA-LNP/miR181 -a vs HA-LNP/siSCR), and **** p ⁇ 0.0001 (HA- LNP/miR181 -a vs HA-LNP/siSCR, and HA-LNP/siSCR vs HA-LNP/siAS).
  • FIG.9 illustrates results of activation of caspase-3 in U87 cells after transfection of HA-LNP/miR-181 a (300 nM) for 72h, U87 cells treated with HA- LNP/miSCR, HA-LNP/miR-181 a, or buffer only for 72h, with addition of staurosporine condition one-hour prior imaging, according to exemplary embodiments of the application.
  • FIG.10 illustrates results of cellular death in U87 cells by transfection of miR-181 a by HA-LNP/miSCR, HA-LNP/miR-181 a, or HA-LNP/siAS (64 nM) after 3 days and stained with Live/Dead cell assay, according to exemplary embodiments of the application; with Calcein AM membrane permeable ester prodrug cleaved into fluorescent form only in alive cells (lighter contrast), and Ethidium Homodimer III cationic DNA-intercalating dye penetrating the damaged cellular membrane of dead cells (darker contrast).
  • FIGs.12A, 12B, 12C and 12D illustrates HA-LNP synthesized using different ionizable lipids, DODMA, MC3 and DODAP, according to exemplary embodiments of the application, and shows their respective FIG.12A intensity size distribution and correlogram for LNPs before LbL modification (dashed line) and after the addition of the three polyelectrolytes to yield HA-LNPs (full line); FIG.12B potential before and after each polyelectrolyte layer; FIG.12C encapsulation efficiency; and FIG.12D luciferase silencing 48h after transfection.
  • FIGs.13A and 13B show results of GL261 glioblastoma cells mediating gene silencing according to exemplary embodiments of the application, where FIG.13A: HA-LNP synthesized using an ionizable lipid (MC3) or a mix of ionizable and cationic lipid (MC3 and DDAB, and MC3 and DOTAP); and FIG.13B: HA-LNP synthesized using an ionizable lipid (MC3) or a mix of ionizable and cationic lipid (MC3 and DDAB) with or without the presence of hydrophilic stabilizing lipids (DSPE:PEG).
  • MC3 ionizable lipid
  • DDAB ionizable and cationic lipid
  • DSPE hydrophilic stabilizing lipids
  • FIG.14 shows a schematic representation of a five-layered hyaluronan decorated lipid nanoparticles (HA-LNPs), according to exemplary embodiments of the application.
  • FIG.15 shows physicochemical characterization of 5-layered HA-LNPs DODMA core according to exemplary embodiments of the application, where: left shows intensity distribution of LNPs DODMA core (top graph) and after each polyelectrolyte layer; middle shows graphs of zeta potential (middle top) and PDI (dot plot) of LNPs DODMA core before and after each polyelectrolyte layer; and right represents DLS measurements (mean and SD of z-average, PDI, and zeta potential) at each step of the layer-by-layer technique.
  • FIG. 16 shows physicochemical characterization of 5-layered HA-LNPs MC3 core according to exemplary embodiments of the application, where: where: left shows intensity distribution of LNPs MC3 core (top graph) and after each polyelectrolyte layer; middle shows graphs of zeta potential (middle top) and PDI (dot plot) of LNPs MC3 core before and after each polyelectrolyte layer; and right represents DLS measurements (mean and SD of z-average, PDI, and zeta potential) at each step of the layer-by-layer technique.
  • FIG.17 is a graph of the fluorescence of free siRNA and layered- encapsulated siRNA in HA-LNPs 5x MC3 and DODMA core, according to exemplary embodiments of the application.
  • FIG.18 shows internalization of three (3x) or five-layered (5x) HA-LNPs MC3 core by GL261 cells, according to exemplary embodiments of the application.
  • FIG.19 shows internalization five-layered (5x) HA-LNPs MC3 containing siRNA-Cyanine 3 at different layer location by GL261 cells, according to exemplary embodiments of the application.
  • FIG.20 shows luciferase silencing in GL261 -Luc cells (180 nM, 48h) mediated by siRNA-Luc encapsulated in the five-layered HA-LNPs formulated with either DODMA or MC3 ionizable lipid, according to exemplary embodiments of the application.
  • FIG.21 shows luciferase silencing in GL261 -Luc cells (60 nM, 48h) mediated by siRNA-Luc encapsulated solely in the core of MC3 LNPs and hyaluronan- decorated three and 5-layered HA-LNPs, according to exemplary embodiments of the application.
  • FIG.22 shows gene silencing in U251 GFP cells by layer-by-layer assembled LNP formulated with chitosan as positively charged layer, according to exemplary embodiments of the application.
  • FIGs. 23A, 23B, 23C and 23D show results of modulated surface layer of HA-LNP, according to exemplary embodiments of the application, where FIG. 23A top image is transfection efficiency of HA-LNP with 0, 5 or 10% PEGylated substitution and bottom image shows PEGylated Hyaluronic acid; FIG. 23B is the feasibility of constructing LbL LNP using fucoidan as surface layer; FIG. 23C shows that fucoidan- terminated LbL LNP targets macrophages to a superior extent than Hyaluronan- decorated LNP; and FIG. 23D shows that fucoidan-terminated LbL LNP induces superior protein expression (GFP) after transfection of mRNA.
  • FIG. 23A top image is transfection efficiency of HA-LNP with 0, 5 or 10% PEGylated substitution and bottom image shows PEGylated Hyaluronic acid
  • FIG. 23B is the feasibility of constructing LbL LNP using fucoidan as surface layer
  • FIG. 23C
  • FIG.24A shows uptake of HA-LNP where the outer hyaluronic acid has been modified with cyanine-5 (degree of substitution 5%); and FIG.24B illustrates the detection of nanoparticles across an in vitro BBB model, according to exemplary embodiments of the application.
  • FIGs. 25A, 25B, 25C, 25D and 25E show results of a lipid nanoparticles encapsulating mRNA in the lipidic core and siRNA control as first layer, according to exemplary embodiments of the application, where FIG. 25A is a physicochemical characterization of the nanoparticles; FIG. 25B is the expression of GFP in HeK293T cells transfected with HA-LNP encapsulating mRNA encoding the Green Fluorescence Protein (GFP); FIG. 25C shows the expression of Luciferase in Hek293T cells by HA- LNP encapsulating mRNA encoding the Luciferase protein; FIG.
  • FIG. 25A is a physicochemical characterization of the nanoparticles
  • FIG. 25B is the expression of GFP in HeK293T cells transfected with HA-LNP encapsulating mRNA encoding the Green Fluorescence Protein (GFP)
  • FIG. 25C shows the expression of Luci
  • FIG. 25D shows the expression of luciferase protein in pediatric glioblastoma cells SF188 by HA-LNP with lipid core composition of ALC:Cholesterol:DOPE encapsulating the mRNA Luciferase; and FIG. 25E shows the expression of luciferase in SF188 cells by HA-LNP containing a mix of ionizable and cationic lipid, DSPC, and PEG lipid (MC3:DDAB:DSPC:DMGPEG2k).
  • FIG.26 shows results of HA-LNP encapsulating small molecules, according to exemplary embodiments of the application, where left is cytotoxic effect of 5-azacytidine (5-AZA)-loaded HA-LNP containing siRNA negative control in core and layer; and right is cytotoxic effect of HA-LNP encapsulating both 5'- monophosphate sodium salt (5-FdU-MP) and siRNA negative control in the core on HeK293T cells.
  • 5-AZA 5-azacytidine
  • 5-FdU-MP 5'- monophosphate sodium salt
  • FIGs. 27A, 27B and 27C show results of HA-LNP encapsulating contrast agents, according to exemplary embodiments of the application, where FIGs. 27A and 27B shows physicochemical characterization of lipophilic Gadollinium-derivative (18:0 PE-DTPA-Gd) in the lipidic core of LNP surrounded by one layer of hyaluronic acid; and FIG. 27C shows T1 -weighted images under magnetic resonance imaging for HA- LNP containing Gadollinium (DOTA)-Gd.
  • DOTA Gadollinium
  • FIG.28 shows results of purification in a method according to exemplary embodiments of the application, where top shows removal of free polyelectrolytes by size exclusion chromatography; and bottom images shows layer-by-layer assembly of LNP achieved after purification.
  • the second component as used herein is chemically different from the other components or first component.
  • a “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
  • composition of the application or “composition of the present application” and the like as used herein refers to a composition comprising one or more components.
  • suitable means that the selection of the particular composition or conditions would depend on the specific steps to be performed, the identity of the components to be transformed and/or the specific use for the compositions, but the selection would be well within the skill of a person trained in the art.
  • cell refers to a single cell or a plurality of cells and includes a cell either in a cell culture or in a subject.
  • subject as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Thus the methods and uses of the present application are applicable to both human therapy and veterinary applications.
  • pharmaceutically acceptable means compatible with the treatment of subjects, for example humans.
  • pharmaceutically acceptable carrier means a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to a subject.
  • pharmaceutically acceptable salt means either an acid addition salt or a base addition salt which is suitable for, or compatible with the treatment of subjects.
  • solvate means a compound, or a salt and/or prodrug of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered.
  • treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treating” and “treatment” as used herein also include prophylactic treatment.
  • a subject with early cancer can be treated to prevent progression, or alternatively a subject in remission can be treated with a compound or composition of the application to prevent recurrence.
  • Treatment methods comprise administering to a subject a therapeutically effective amount of one or more of the compounds of the application and optionally consist of a single administration, or alternatively comprise a series of administrations.
  • “Palliating” a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
  • prevention or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a patient becoming afflicted with a disease, disorder or condition.
  • administered means administration of a therapeutically effective amount of a compound, or one or more compounds, or a composition of the application to a cell either in cell culture or in a subject.
  • the term “effective amount” means an amount effective, at dosages and for periods of time, necessary to achieve a desired result.
  • LbL layer-by-layer
  • LbL consists of alternatively adding polycharged molecules to surfaces or nanoparticles based on complimentary electrostatic interaction to form a self-assembled multi layered structure.
  • advantages conferred by LbL to nanoparticles include increased biomolecules loading capacity, staged release, protection of cargo by the multilayers assembly, modulation of targeting property by playing with the outer layer, enhanced shelf-life and in vivo stability [18-20],
  • the present application provides an improved core-shell system composed of an ionizable lipid-containing LNP core encapsulating therapeutic RNAs surrounded by alternating polyvalent electrolytes, specifically, a layer of nucleic acid, followed by poly-L-arginine and hyaluronic acid, to form a multilayered LbL structure named HA-LNP.
  • Hyaluronic acid is a natural target of CD44 receptors which are overexpressed in many cancer cell lines, including glioblastoma.
  • a wash-less method to coat LNP with discrete quantities of polyelectrolytes determined by titration was developed.
  • HA-LNP induced significant gene silencing in vitro regardless of the loading location of siRNA.
  • miR-181 a-loaded HA-LNPs as a promising strategy for the treatment of glioblastoma.
  • the resulting HA-LNP thus represents a novel platform for gene delivery against malignancies.
  • the present application includes a coated lipid nanoparticle comprising a lipid nanoparticle core comprising a gene, a drug, a contrast agent or a mixture thereof; at least one first layer over the core comprising negatively charged nucleic acids; at least one second layer over the first layer comprising polycations; and a surface layer over the at least one second layer comprising polyelectrolytes negatively charged.
  • the coated lipid nanoparticle further comprises at least one pair of a negatively charged layer and a positively charged layer between the second layer and the surface layer.
  • the total number of layers is selected from 3 or more, such as 5 or 7.
  • the total number of layer is selected from 3 or more layers, depending on the desired size, loading cargo, and release profile. In some embodiments, the total number of layers is 3.
  • the core comprises vesicles comprising lipid complexed with nucleic acids.
  • the core comprises both the gene and the drug encapsulated. Without being bound to theory, such co-delivery may provide a synergistic effect.
  • various combinations of drugs and genes to be delivered to a target may be encapsulated in the core.
  • the gene is a therapeutic RNA, including, but not limited to, same miRNA, siRNA, mRNA, any form of nucleic acids, such as gRNA, and mixtures thereof
  • the gene is overexpressed genes which downregulation through siRNA, miRNA, or a combinations thereof elicit a beneficial therapeutic response, or downregulated genes which transient expression through mRNA, saRNA, miRNA, regulatory RNAs or combinations thereof elicit a beneficial therapeutic response.
  • the beneficial therapeutic response may be measured in vitro by viability assay, western blot, cytokine expression, proliferation, migration or other molecular assays.
  • the beneficial therapeutic response may be measured in vivo by animal overall survival, tumour growth (if oncology), immune cells infiltration, metastasis, behavioral tests.
  • the gene is miR-181 a.
  • the drug is an anticancer agent, epigenetic modulators, an immunomodulatory drug, a cytokine, enzyme inhibitors, or an antibiotic.
  • the drug is an anticancer agent.
  • the drug is temozolomide and lipophilic derivatives thereof.
  • the contrast agent is selected from fluorophores, fluorescent lipids (e.g. 16:0 Liss Rhodamine PE), metal-chelated lipids (e.g. 18:00 PE- DTPA (Gd)), iron oxide nanoparticles (e.g. SPIONS), and combinations thereof.
  • fluorescent lipids e.g. 16:0 Liss Rhodamine PE
  • metal-chelated lipids e.g. 18:00 PE- DTPA (Gd)
  • iron oxide nanoparticles e.g. SPIONS
  • the lipid is a lipidic solution comprising one or more lipidic components diluted in an water-miscible organic solvent, wherein the one or more lipidic components are selected from: an ionizable lipid, 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE- PEG2k), cholesterol, distearoylphosphatidylcholine (DSPC) and combinations thereof.
  • DOPE 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine
  • DSPE- PEG2k 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt)
  • DSPC distea
  • ionizable lipid By ionizable lipid, a skilled person in the art would understand a lipid, lipidoid, multi-tail lipid, alkyl tail substituted polymer-lipids, that is positively charged at acidic pHs ( ⁇ 7), but not at neutral pHs.
  • the ionizable lipid is selected from, but not limited to, DODMA (1 ,2-Dioleyloxy-3-dimethylaminopropane), DOTMA (1 ,2-Dioleoyl-3-trimethylammonium propane), DOBAQ (N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleoyloxy)propan-1 -aminium), DLin-MC3-DMA
  • ALC-0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate), SM-102 (15eptadecane-9-yl 8-[2-hydroxyethyl-(6-oxo-6- undecoxyhexyl)amino]octanoate), and other gene transfection-enabling lipidoid structures.
  • the first layer comprises a second load of the gene, such as miRNA, siRNA, mRNA, any form of nucleic acids, such as gRNA, and mixtures thereof, same or different from the gene included in the core.
  • a second load of the same gene may provide for delivery of additional dose of the same to allow controlled, extended delivery.
  • a second load of a different gene or a mixture of genes may allow for combination of various treatments.
  • the at least one second layer comprises poly-L- arginine, poly-L-lysine, poly-L-histidine, positively charged peptides, chitosan, polyethylenimine, derivatives thereof or combinations thereof. Without being bound to theory, the second layer allows for protection of the first layer and boosts endosomal escape properties.
  • the surface layer comprises, but is not limited to, hyaluronic acid, dextran, chondroitin sulfate, aggrecans, mucins proteins, mucoadhesive polymers such as chitosan, cellulose-derived polymers, or combinations thereof.
  • the surface layer comprises, but is not limited to, poly-L-glutamate, fucoidan, PEG or functional PEG-modified polysaccharides, polyanions and polymers, negatively charged polypeptides, bottlebrush negatively charged copolymers, or combinations thereof.
  • the surface layer comprises, but is not limited to, hyaluronic acid, dextran, chondroitin sulfate, aggrecans, mucins proteins, chitosan, cellulose-derived polymers, poly-L- glutamate, fucoidan, PEG or functional PEG-modified polysaccharides, or combinations thereof.
  • the surface layer is configured to bind specific target cells. As such, it will be appreciated that the surface layer may be tunable to allow binding to different cells, depending on the needs and may be designed for selective targeting.
  • hyaluronic acid can be conjugated with targeting moieties, as antibodies or fragments thereof, nanobodies, proteins, protein A or G, cytokines, aptamers, cell penetrating peptides, mannose or other sugars, or any other component able to promote targeted delivery of cargo.
  • conjugation can be achieved by coupling targeting molecules to hyaluronic acid.
  • maleimide N-Succinimidyl 3-(2- Pyridyldithiojpropionate
  • NHS/EDC coupling chemistry or azide or any other click chemistry reagents.
  • Hyaluronate Maleimide MW 50k (Creative PEG Works, NC, USA).
  • the target cells are selected from a pool of cells under pharmacological investigation.
  • Target discovery techniques can be used to select an appropriate target.
  • Target discovery techniques comprise mass spectrometry, Systematic evolution of ligands by exponential enrichment (SELEX), flow cytometry, single-cell mass cytometry, or other single-cell techniques.
  • the target cells are selected from cancerous cells, cancer initiating stem cells (CSCs), and immune cells such as NK cells, cytotoxic lymphocytes, CD8+ cells, or macrophages.
  • coated lipid nanoparticle of the present application for gene and/or drug delivery.
  • lipid nanoparticle of the present application Also included is a gene and/or drug delivery system comprising the coated lipid nanoparticle of the present application.
  • a medical imaging system comprising a coated lipid nanoparticle of the present application.
  • the present application includes use of a coated lipid nanoparticle of the application for gene and/or drug delivery.
  • lipid nanoparticle of the present application for gene and/or drug delivery to target cells.
  • a coated lipid nanoparticle of the present application in the manufacture of a medicament for gene and/or drug delivery to target cells.
  • the present application includes a method for improving biodelivery of a gene and/or a drug to target cells, comprising administering the coated lipid nanoparticle of the application.
  • a method for treating a subject in need of a gene and/or a drug comprising administering the coated lipid nanoparticle of the present application to the subject in need thereof.
  • a method for treating a subject in need of a gene and/or a drug comprising: forming a lipid nanoparticle core comprising the gene, the drug or a mixture thereof coating the lipid nanoparticle core with at least one first layer comprising siRNA negatively charged; coating the at least one first layer with at least one second layer comprising polycations; coating the at least one second layer with a surface layer comprising polyelectrolytes negatively charged to provide a coated lipid nanoparticle; administering the coated lipid nanoparticle to the subject in need thereof.
  • a method of producing a coated lipid nanoparticle for gene and/or drug delivery comprising: forming a lipid nanoparticle core comprising the gene, the drug or a mixture thereof; coating the lipid nanoparticle core with at least one first layer comprising siRNA negatively charged; coating the at least one first layer with at least one second layer comprising polycations; coating the at least one second layer with a surface layer comprising polyelectrolytes negatively charged; wherein the method comprises conducting a titration before each coating to determine a minimum amount of each layer to be used.
  • the ratio of each polyelectrolyte to be added is determined by a titration curve observing paraments as particle size, polydispersity index, and surface zeta potential.
  • RNA, miRNA, and primers were obtained from Thermo Scientific, Qiagen (Hilden, Germany), and Horizon Discovery (Waterbeach UK). Sequences are provided on Table 1. Hoechst 33342 was purchased from Cayman Chemical (Ann Arbor, Ml, USA).
  • Hyaluran-decorated lipid nanoparticles were synthesized in two steps. Firstly, LNP were synthesized by ethanolic injection where 45 pL of sodium acetate buffer (10 mM, pH 4) containing 3.3 pL of siRNA or miRNA solution at 200 pM (N/P 4) was added to 15 pL ethanolic lipid solution (DODMA:Chol:DOPE 50:39:11 %mol, 0.22 pmol total lipids) by rapid pipetting. The colloidal suspension was let stand for 15 minutes at room temperature before proceeding to step 2. Then, 240 pL of sodium acetate buffer was added to the suspension to reach final volume of 300 pL.
  • sodium acetate buffer (10 mM, pH 4) containing 3.3 pL of siRNA or miRNA solution at 200 pM (N/P 4) was added to 15 pL ethanolic lipid solution (DODMA:Chol:DOPE 50:39:11 %mol, 0.22 pmol total lipid
  • the second step consisted of building the polyelectrolyte layers onto LNP using the layer-by-layer process.
  • 7 pL of siRNA or miRNA diluted to 20 pM in sodium acetate buffer (10 mM, pH 4) was added to 14.1 pL of LNP suspension.
  • 1 pL of poly-L-arginine (10-15 KDa) at 2 mg.mL-1 (Milli-QTM water) was added to form the second layer.
  • 5 pL of hyaluronic acid 60 KDa, 5 mg.mL-1 in Milli-Q water
  • the solution was further diluted in water for in vitro experiments.
  • HA-LNP For in vivo experiment, a scale up process was chosen where 4 batches of HA-LNP were produced in a single microtube. For example, 56.4 pL of LNP was mixed to 28 pL of siRNA/miRNA at 20 pM, followed by 4 pL of PLA at 2 mg.mL’ 1 , and 20 pL of HA at 5 mg.mL’ 1 . HA-LNP were then concentrate in an Amicon 100 KDa filter unit for 3 min at 15,000 g / 4 °C (Thermo Scientific SorvallTM Legend Micro 21 R). After concentration, HA-LNP were characterized (DLS and %EE) and no physicochemical difference was observed.
  • Physiochemical characterization of HA-LNP [00102] Particles’ hydrodynamic diameter, polydispersity index (PDI) and - potential were measured at 20°C using a Malvern Zetasizer Nano ZS (Malvern, Worcestershire, UK). Samples in sodium acetate buffer 10mM were diluted 25X in MilliCi water to a final volume up to 700 pL. Size measurements were performed with a scattered angle of 173° and reported as Z-average (intensity). The voltage for - potential was set at 150V. Measurements were performed at least in triplicate.
  • encapsulation efficiency of miR-181 a was indirectly calculated by quantifying the amount of free miRNA using the SYBR® Gold assay (Thermo Scientific, Waltham, MA, USA). Briefly, HA-LNP were prepared as previously stated and then diluted 40X up to 100 pL of Milli-Q water and added in triplicate to a black 96- well plate (Coming Inc., Coming, NY, USA) followed by 60 pL of SYBR® Gold 10X diluted in Milli-Q water.
  • Mt means total molar contribution from core and layer and Mf means molar concentration corresponding to non-encapsulated miR-181 a
  • the molar contribution from core and layer in 27.1 pL final formulation of HA-LNP is 1 .14 and 5.16 pM, respectively.
  • total molar contribution is 6.3 pM.
  • EE % is 84. Result obtained from three independent batches corresponds to EE % ⁇ standard deviation.
  • Samples were diluted 20 times in Milli-Q water. Samples were immobilized via electrostatic interactions between the surface charge of the nanoparticles and a substrate bearing the opposite electric charge. For positive samples, a 20 pL droplet of diluted sample was deposited on a silicon wafer freshly activated with piranha solution (H2S04:H202/70:30/v:v), which was done in duplicate.
  • a 20 pL droplet of diluted sample was deposited on silicon wafers first activated with piranha solution and then surface-functionalized with 11 -undecylaminotriethoxysilane (AUTES) which furnished the silicon wafer surface with a net positive charge, which was done in duplicate.
  • AUTES 11 -undecylaminotriethoxysilane
  • Sample droplets were left to evaporate overnight. All surfaces were gently rinsed with Milli-Q water and dried under a stream of nitrogen prior to imaging by using a Multimode AFM microscope equipped with a nanoscope V extended controller. Imaging was performed in dry air (30 % r.h.) at 25 °C in the PeakForce tapping-mode using an ACTA silicon probe from APP Nano (resonance frequency of 200-400 kHz).
  • HA-LNP containing siRNA-Cy5 at the layer compartment was added at final concentration of 100 nM (siRNA-Cy5) and incubated for 15 or 60 minutes, when cells were gently washed thrice with cold PBS, collected by trypsinization, then resuspended in FACS buffer (R&D Systems, Minneapolis, MN, USA) for immediate analysis on a BD FACS Canto II flow cytometer with data processed using the manufacture’s software (BD, Franklin Lakes, NJ USA).
  • MCF-7 cells were plated in a 96-well black plate at 10,000 cells/well density (clear flat bottom, VWR, Mississauga, Canada) 12h prior experiment in phenol- red-free media (DMEM, high glucose, HEPEs, no phenol red, Thermo Scientific). The next day, DMEM was gently replaced for fresh media containing HA (500 KDa) at final concentration of 25 mg.mL’ 1 for 1 h, before adding HA-LNP/siRNA-Cy5 at final concentration of 50 nM of siRNA-Cy5.
  • DMEM phenol- red-free media
  • siRNA-Cy5 reached 167 nM. Particles remained in contact with cells for 3 days, then the media was removed, cells were washed with cold PBS twice, and fixed with PFA 4% in PBS for 15 minutes. Cells were washed again with cold PBS thrice and maintained in a PBS solution with Hoescht 1 pg.mL’ 1 until imaging. Images were acquired on a Leica TCS SP8 (Wetzlar, Germany) and treated using Fiji software [21 ],
  • GBM cell lines (U87, U251 , T98G, and GL261 -Luc, 10,000 cells/well) were plated in 24-well plate and 50 pL of [3H]-thymidine from PerkinElmer (Waltham, MA, USA) was added immediately after transfection. After 72 h, the supernatant was removed, cells were rinsed with cold 5% trichloroacetic acid (Sigma-Aldrich, Oakville, Canada) thrice, rinsed with cold PBS, and lysed with 200 pL lysis solution (Triton X- 1000.1 % in 0.1 M NaOH). The lysates were transferred to 10 ml of scintillation cocktail (Research Product International, USA), and radio signals were detected with a Hidex 300SL scintillation counter (Yuseong-gu, South Korea).
  • Membranes were blocked with 5% skimmed for 1 hour and then incubated overnight at 4°C with rabbit anti-CD44 polyclonal antibody (1 :2000, #15675-1 -AP, Proteintech, Rosemont, IL, USA) or mouse anti-B-actin antibody (1 :5000, sc-8432, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
  • Membranes were washed thrice with TBST (0.15M NaCI, 0.02M Tris-HCI pH 7.4, 0.1 % TweenTM 20 (v/v)) and then incubated with either anti-rabbit (1 :5000, sc-2004, Santa Cruz Biotechnology) or anti-mouse (1 :5000, sc-2005, Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary horseradish peroxidase-conjugated antibodies at room temperature for 1 hour followed by washing with TBST and incubation with Clarity max ECL substrate (Bio-Rad Laboratories, Hercules, CA, USA) for 5 minutes before imaging on a ImageQuantTM LAS 500 chemiluminescent imaging system from GE Healthcare (Chicago, IL, USA). Blottings were treated using Imaged software. Chemicals for buffer preparation were obtained from Sigma-Aldrich (Oakville, ON, Canada).
  • tumors were weighted and fixed in 4% PFA overnight at 4 °C and submitted to sucrose gradient up to 30% (sucrose % w/w in PBS), then embedded in Optimal Cutting Temperature (OCT) Compound (Sakura, USA) and cut into 10-pm sections using CryoStarTM NX50 Cryostat (ThermoFisher Scientific, USA). Sections were stained for cleaved-caspase 3 (rabbit anti-cleaved caspase-3 (Asp 175), #9661 , cell signaling, Danvers, MA, USA, secondary antibody goat anti-rabbit AF594 #A11012, Thermo Scientific) according to manufacturer’s instructions. Sections were imaged on a DMi8 inverted microscope (Leica Biosystems).
  • OCT Optimal Cutting Temperature
  • Hyaluronic acid-coated lipid nanoparticles were prepared in two steps (FIG.1A) as indicated above. Firstly, the lipid nanoparticle core containing either siRNA or miRNA were synthesized by ethanol injection, followed by coating with alternating layers consisting of nucleic acids, poly-L-arginine (PLA), and hyaluronic acid (HA). The particles were characterized by DLS and Atomic Force Microscopy (AFM) (FIG.1 B and FIG.1 C). Starting from a LNP core of about 150 nm, an increase in particle size was observed after coating with miRNA/siRNA and PLA, but not after the HA outermost layer.
  • FAM Atomic Force Microscopy
  • FIG.2A illustrates this point as follows. Firstly, a strong internalization occurred when the siRNA was labelled at the core of HA-LNPs. As expected, similar results were obtained when the lipids, only located at the core of HA- LNP, are labelled.
  • HA-LNP enter cancer cells were further investigated in more detail. Specifically, it was considered to determine if the outer hyaluronic acid layer was playing a role on the internalization kinetics of LNP by directing the particles via CD44 receptors.
  • siRNA-Cy5-labelled HA-LNP was incubated with three different cell lines with varied expression of CD44. Binding was analyzed by flow cytometry (FIG.3A). At 15 minutes, strong binding is observed in U251 and U87 cells, both positive for CD44 (FIG.3B). At the same time point, little change in fluorescence was observed in the CD44 negative breast cancer cell line MCF7.
  • RNAi-mediate gene silencing was explored in three different cell lines. HeLa cells stably expressing GFP (HeLa/GFP) were incubated with increasing doses of siRNA-GFP encapsulated within core and layer of HA-LNP, using scramble siRNA as control. After 72h, GFP fluorescence was quantified and imaged by plate reader and microscopy, respectively (FIG.4A and FIG.4B).
  • siRNA All Stars siRNA All Stars
  • siRNA All Stars was encapsulated only at the layer of HA-LNP and evaluate cellular viability after 72h of incubation with U87 glioblastoma cells. Strong cellular death was noticed after incubation with doses as little as 50 nM, confirming transfection mediated by HA- LNP (FIG.4C). Transfection was also validated using the commercially available reagent Mirus which induced significant cellular death at 50 nM of siAII Stars but also at an important toxicity when complexed with scramble siRNA.
  • HA-LNP The internalization of miR-181 a by HA-LNP was further investigated, but this time to add more evidence on the role of CD44 on GBM internalization in vitro.
  • LNP were coated as described above, but without the HA outermost layer and then incubated the PLA- finished LNP with U87 cells overnight and the intracellular amount of miR-181 a was measured by qPCR (FIG.7B).
  • HA-deficient particles did not internalize miR-181 a as efficiently as HA-LNP.
  • fully coated HA-LNP enabled detection of miRNA at levels 3 times higher than PLA-LNP.
  • HA-LNP/miR-181 a was investigated.
  • three different human glioblastoma cells lines were transfected, namely U87, U251 , and T98G, and one murine-derived GBM cell line, GL261 -Luc, to evaluate the miRNA effect on cellular proliferation.
  • MicroRNA-181 a affected proliferation in a cell-line dependent fashion.
  • U87 cells responded better with 58% reduction in proliferation compared to non-treated and scramble transfected cells, followed by GL261 -Luc, T98G and 11251 cells with 27, 22 and 15% reduced proliferation, respectively (FIG.8A).
  • U87 cells was used as a model to investigate if miR-181 a also affected cellular viability. Indeed, transfecting U87 cells with HA-LNP loaded with miR-181 a reduced cell viability significantly as compared to scramble and non-treated cells. In this experiment, HA-LNP containing siRNA All Stars between layers was used as a positive control to confirm transfection (FIG.8B). Mechanistically, transfection of miR-181 a triggered caspase 3 activation and cellular death, the latter evidenced by the binding of propidium iodine to nuclear DNA (FIG.9 and FIG.10).
  • miR-181 a treated tumours were significantly smaller than scramble-treated ones (FIG.11 B and FIG.110) and lighter than non-treated and buffer control group (FIG.11 D), but not significantly different than scramble-treated mice.
  • Smaller tumors correlated with higher intracellular expression of miR-181 a mimic and activation of caspase 3 (FIG.11 E and FIG.11 F).
  • lipid nanoparticles for RNAi delivery in vitro and in vivo.
  • the LbL process has been used extensively to modify the surface of solid lipid nanoparticles [22, 23], liposomes [24], and polymeric nanoparticles[25], but the use of LbL assembly onto lipid nanoparticles encapsulating nucleic acids has never been reported.
  • Deng at al. (2013) [26] used the LbL strategy to coat negatively charged polystyrene latex nanoparticles (CML) with alternative layers of siRNA, Poly-L-Arginine (PLA), and Hyaluronic Acid (HA).
  • CML polystyrene latex nanoparticles
  • PPA Poly-L-Arginine
  • HA Hyaluronic Acid
  • Deng demonstrated a 50% in vitro silencing capacity of LbL coated CML loading siRNA targeting GFP in order of 1 pM.
  • Choi et al. (2019) [25] reported a dual targeted (CD44/CD20) LbL assembled system for the delivery of siRNA targeting the anti-apoptotic BCL-2 protein for the treatment of hematopoietic malignancies.
  • the PLA layer is the sole player contributing to the complexation and cytoplasmic delivery of siRNA or miRNA.
  • a system with multiple components contributing to siRNA delivery could potentialize those effects reported elsewhere.
  • the LNP core would exert a more pronounced effect due to the nucleic-acid core loading capacity, absent in bare liposomes or PLGA particles, thus increasing the overall amount of oligos delivered without further toxicity attributed higher amounts of carrier materials.
  • the limited encapsulation efficiency often described when using LbL assembly was also addressed.
  • microscopy and viability data confirm that siRNA loaded in between layers is internalized with the particle altogether and able to mediate gene silencing, adding more evidence that the presence of ionizable lipid is also necessary for cytoplasmic delivery of layer-encapsulated oligos.
  • HA-LNP would be able to deliver miR- 181a and mediate toxic effects on GBM cells.
  • miRNAs and siRNAs share structural similarities, it was prudent to confirm intracellular delivery of miR-181 a by qPCR after transfection. Having confirmed successful delivery, the effect of miR-181 a on proliferation and viability was assessed. A cell-line dependent response was observed, being U87 cells the most negatively affected by miR-181a, which was confirmed in the viability assay (FIG.8).
  • miR-181 a is inversely correlated to advanced grades of GBM [10, 32, 33].
  • re-expression of miR-181 a impaired viability and cellular proliferation [4], colony formation [8], and increased susceptibility to radio- and chemotherapy [5, 6, 9].
  • transfection of miR-181 a decreased tumor burden in an orthotopic immune-competent mice model implanted with murine-derived GL261 GBM cell line [29], and subcutaneous nude mice established with 11251 [34] and A172 human-derived GBM cells [32],
  • transfection of miR-181 a negatively affects tumor growth in vivo, but this time in a grade IV human-derived GBM cell line, U87.
  • HA-LNP successfully delivered miR-181 a to GBM cells in vitro and in vivo, triggering antitumor response in both conditions, corroborating to the choice of miR-181 a as an investigational intervention against GBM.
  • Hyaluronan-decorated Lipid Nanoparticles formed with different ionizable lipids in their core, namely DODMA, MC3 and DODAP, have been evaluated and results are shown in FIG.12. Specifically, structure of the three different ionizable lipids used to formulate LNPs by the ethanolic injection technique are shown in (a), from top to bottom: DODMA, MC3, and DODAP. Next to each structure is shown intensity size distribution and correlogram for each LNP before LbL modification (dashed lines) and after the addition of the three polyelectrolytes to yield HA-LNPs (full lines).
  • FIG.12 shows under (b) potential before and after each polyelectrolyte layer.
  • Under (c) is shown encapsulation efficiency measured by SYBR gold intercalating reagent of siRNA located at the layer compartment for the three different HA-LNPs.
  • Free siRNA (5.16 pM in a 27.1 pL solution for DODMA and MC3, 4.65 pM in 30.1 pL for DODAP containing HA-LNPs) was used as 100% fluorescence control against aliquots of HA-LNPs. All conditions were diluted equally in 10 mM Tris/I mM EDTA buffer pH 7.4 and mixed with SYBR gold 10X also diluted in TE Buffer.
  • FIG.12 under (d) also shows luciferase silencing 48h after transfection of the three different HA-LNPs containing siRNA-luciferase both in the core (N/P 4) and layer at a final concentration of 180 nM of siRNA/well in GL261 cells stably expressing firefly luciferase gene.
  • Scramble siRNA was used as a negative control.
  • Luciferase activity was measured using the Steady-Gio® Luciferase Assay and normalized to non-treated cells (filled bars). Cellular viability was measured using WST-8 assay reagent and also compared to non-treated cells (connected dots). All experiments were repeated at least three times.
  • Example 5 - HA-LNP with a mixture of ionizable and cationic lipids, and other helper lipids
  • HA-LNPs were also synthesized with different helper lipids (DMG-PEG for LNP core 1 and HA-LNP 1 , and DSPC and DMG-PEG for LNP core 2 and HA-LNP core 2) and mix of ionizable lipid and cationic lipid (MC3 + DDAB for LNP core 2 and HA-LNP-2), as shown in Table 2.
  • helper lipids DMG-PEG for LNP core 1 and HA-LNP 1
  • DSPC and DMG-PEG for LNP core 2 and HA-LNP core 2
  • MC3 + DDAB ionizable lipid and cationic lipid
  • HA-LNP formulated from LNP with different composition (presence of DSPC, DSPE-PEG and mix of ionizable lipid (MC3) and cationic lipid (DDAB). name / composition / lipid size (nm) PDI DCR ZP (mv) proportion LNP Core 1 129 ⁇ 1 0.1 ⁇ 0.01 5899 ⁇ 72 14 ⁇ 2 MC3:DOPE:Chol:PEG (47.5:38: 12:2.5J HA-LNP Core 1
  • Results from HA-LNPs formulated with mix of ionizable lipid and cationic lipid are shown in FIG.13A, which illustrates that these HA- LNPs efficiently delivered siRNA to GL261 glioblastoma cells mediating efficient gene silencing.
  • Results from HA-LNPs formulated with mix of ionizable lipid and cationic lipid (MC3 + DDAB) and different helper lipids (DSPC, DOPE or DMG-PEG) are shown in FIG.13B, which illustrates that these HA-LNPs efficiently delivered siRNA to GL261 glioblastoma cells mediating efficient gene silencing.
  • Example 6 five layers HA-LNP
  • Results presented above include HA-LNP formulated with three (3) layers.
  • the construction of five (5) layers on top of LNP is presented, as illustrated in FIG.14.
  • FIG.15 depicts the physicochemical characterization that confirms the deposition of 5 layers on top of LNP DODMA core using the layer-by-layer strategy.
  • FIG.15 shows intensity distribution of LNPs DODMA core (top graph) and after each polyelectrolyte layer.
  • Middle graphs in FIG.15 shows zeta potential (middle top) and PDI (dot plot) of LNPs DODMA core before and after each polyelectrolyte layer.
  • Right portion of FIG.15 represents the mean and SD of z- average, PDI, zeta potential at each step of the layer-by-layer technique.
  • Encapsulation efficiency was determined for LNPs DODMA core with and without disruption of vesicles with TritonTM (0.2%) and HA-LNPs 5x using SYBR gold as intercalating agent.
  • HA-LNPs 5x a solution of free siRNA was used for 100% fluorescence control.
  • AFM of HA-LNPs DODMA core 5x was used for 100% fluorescence control.
  • FIG.16 confirms the successful assembly of five (5) polyelectrolytes layers on LNP formulated with MC3 ionizable lipid, contrasting with FIG.15 (DODMA containing LNP). Adding five (5) layers on top of LNP has ben demonstrated not to impact the encapsulation efficiency of oligonucleotides, as can be seen on FIG.17.
  • FIG.16 shows intensity distribution of LNPs MC3 core (top graph) and after each polyelectrolyte layer.
  • Middle graphs in FIG.16 shows zeta potential (middle top) and PDI (dot plot) of LNPs MC3 core before and after each polyelectrolyte layer.
  • Right portion of FIG.16 represents the mean and SD of z-average, PDI, zeta potential at each step of the layer-by-layer technique.
  • Encapsulation efficiency was determined for LNPs MC3 core with and without disruption of vesicles with Triton (0.2%) and HA-LNPs 5x using SYBR gold as intercalating agent.
  • HA-LNPs 5x a solution of free siRNA was used for 100% fluorescence control.
  • AFM of HA-LNPs MC3 core 5x was used for 100% fluorescence control.
  • encapsulation efficiency was measured by SYBR gold intercalating reagent of siRNA located at the layer compartment for HA-LNPs DODMA and HA-LNPs MC3.
  • Free siRNA (9.92 pM in a 38.1 pL solution) was used as 100% fluorescence control against aliquots of HA-LNPs. All conditions were diluted equally (80x) in 10 mM Tris/1 mM EDTA buffer pH 7.4 and mixed with SYBR Gold 10X also diluted in TE Buffer.
  • HA-LNP constructed with three (3) or five (5) layers are efficiently internalized in vitro (FIG.18 and FIG.19).
  • HA-LNP 3x or 5x can deliver and promote siRNA transfection in vitro (FIG.20)
  • FIG.18 shows internalization of three (3x) or five-layered (5x) HA-LNPs MC3 core by GL261 cells.
  • Rhodamine-PE at 0,1 % mol ratio was used to formulate HA- LNPs MC3 core containing either with three (3) or five (5) layers.
  • Scramble siRNA was used for both core and layers. Particles were added at final concentration of 11 pM of total lipids per well and incubated overnight, followed by washing (PBS), fixation, a new round of washing and nuclei staining. Nuclei stained in are shown on the left (Hoechst 33,342) and particles are shown in the middle (Rhodamine-PE), with the merged images at the right. Scale bar was 20 pm for all images.
  • FIG.19 shows internalization five-layered (5x) HA-LNPs MC3 containing siRNA-Cyanine 3 at different layer location by GL261 cells.
  • Cyanine-3-conjugated siRNA siRNA-Cy3
  • Scramble siRNA was used in the core and appropriate layer where siRNA-Cy3 was not located.
  • Final concentration of siRNA-Cy3 was adjusted to 90 nM in all conditions. Particles were incubated overnight, washed, fixed, and stained with Hoechst 33,342 (blue, nuclei). Scale bar was 20 pm for all images.
  • FIG.20 shows luciferase silencing in GL261 -Luc cells (180 nM, 48h) mediated by siRNA-Luc encapsulated in the five-layered HA-LNPs formulated with either DODMA or MC3 ionizable lipid.
  • Five-layered HA-LNPs formulated with either DODMA or MC3 were used to transfect siRNA Luciferase located at either the first or third layers, as indicated by positive and negative signs.
  • the first sign indicates the presence (+) or absence (-) of designated siRNA (siCTL or siLuc) in the first layer.
  • the second sign after the dash represents the third layer.
  • Scramble siRNA was used in the core of all HA-LNPs and at the layer compartment when applicable. Two- way ANOVA multiple comparisons was used as statistical tool (* ⁇ 0,05, luciferase activity of HA-LNPs MC3 core with siLuc located at the first layer versus HA-LNPs MC3 core containing siLuc at the third layer; ns p > 0.05, viability difference between HA- LNPs MC3 core containing siLuc at the third layer versus HA-LNPs MC3 core containing siCTL at both first and third layers).
  • FIG.21 illustrates the situation where siRNA targeting the Luciferase protein is encapsulated in the core of MC3-containing LNP that were using as starting materials to formulate 3 or 5-layered HA-LNP.
  • LNP containing siRNA Luciferase without layers are not able to transfect cells, whereas HA-LNP 3x (3 layers) and HA-LNP 5x (5 layers) are able to transfect the model cell line chosen (GBM GL261 - Luc cells) and thus promote gene silencing. From an industrial perspective, it was thus demonstrated the use of a PEG-less solution for gene delivery overcoming the emerging concern of PEG immunogenicity.
  • FIG.21 shows luciferase silencing in GL261 -Luc cells (60 nM, 48h) mediated by siRNA-Luc encapsulated solely in the core of MC3 LNPs and hyaluronan- decorated three and 5-layered HA-LNPs. Filled bars and connected dots represent luciferase activity and viability compared to non-treated cells, respectively (not represented in the graph). The concentration was normalized across all conditions to 60 nM of siLuc per well.
  • Example 8 Chitosan as the positively charged layer
  • Hyaluronic acid terminated layer has been extensively exemplified above.
  • the present example demonstrate that modified HA can also be used to produce layer-by-layer assembled Lipid Nanoparticle with equal ability to transfect cells, and that the surface layer can be entirely replaced for other polyanions, i.e. fucoidan for macrophage targeting.
  • Potential benefits of modulating the chemical nature of the outer layer include achieving a desired stability, biodistribution and targeting in vivo.
  • FIG.23 The outer layer of HA-LNP was modulated and results are shown in FIG.23.
  • PEGylated hyaluronic acid with different degree of substitution (5 or 10%) was used as surface layer of LbL assembled LNP.
  • Top image is a direct comparison of transfection efficiency of HA-LNP with 0, 5 or 10% PEGylated substitution. All three formulations were equally capable of transfecting pediatric glioblastoma cells (SF188) with siRNA All Stars which induces strong decrease in viability.
  • Bottom image exemplifies one substitution to the surface layer which was the PEGylated Hyaluronic acid
  • B The nature of the surface layer was modulated by replacing the hyaluronic acid for other polyanions.
  • top pictures illustrate the feasibility of constructing LbL LNP using fucoidan as surface layer.
  • Bottom pictures C and D demonstrates that the outer layer can be replaced for achieving a specific purpose.
  • fucoidan- terminated LbL LNP target macrophages to a superior extent than Hyaluronan- decorated LNP (C) and induce superior protein expression (GFP) after transfection of mRNA (D).
  • FIG.24A shows uptake of HA-LNP where the outer hyaluronic acid (surface layer) has been modified with cyanine-5 (degree of substitution 5%), overnight incubation of HA-LNP at 25 nM final concentration of total siRNA by pediatric glioblastoma cells (SF188).
  • FIG.24B illustrates the detection of nanoparticles across an in vitro BBB model (bottom compartment of a transwell experiment).
  • Three different constructed LbL LNP were used.
  • Sample 1 LbL LNP containing MC3:Cholesterol:DOPE core and hyaluronic acid as outer layer
  • Sample 2 LbL LNP containing MC3:DDAB:Cholesterol:DSPC:DMGPEG2k core and hyaluronic acid as outer layer
  • Sample 3 LNP containing MC3:Cholesterol:DOPE core and fucoidan as outer layer.
  • Control represents particles detected across the BBB model not treated with LbL LNP.
  • Particles represent all types of vesicles that could be present in the solution, for example exosomes, micro vesicles, extracellular vesicles, and LbL LNP but not limited to these entities.
  • exosomes for example exosomes, micro vesicles, extracellular vesicles, and LbL LNP but not limited to these entities.
  • the higher number of particles in the LbL conditions indicate some degree of crossing.
  • Example 10 - mRNA core and siRNA first layer
  • lipid nanoparticle core where the gene encapsulated in the lipid core was mRNA and the gene in the first layer was a siRNA (structurally related to sgRNA) was prepared.
  • Lipid nanoparticle core composed of an ionizable lipid ALC- 0315 or MC3, Cholesterol, and DOPE (1 ,2-Dioleoyl-sn-glycero-3- phosphoethanolamine) (1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) with or without PEG-lipid at 1.5 or 2.5% (DMG-PEG 2000.
  • lipid nanoparticles core could also be formulated with DSPC (1 ,2- distearoyl-sn-glycero-3-phosphocholine) instead of DOPE.
  • Results are presented in FIG. 25, showing that layer-by-layer assembled lipid nanoparticles encapsulating mRNA in the lipidic core and siRNA control as first layer were able to mediate mRNA transfection in vitro.
  • A shows physicochemical characterization of nanoparticles demonstrating the layer-by-Layer assembly of HA-LNP composed of a lipid core containing ALC, Cholesterol and DOPE, siRNAs first layer, Poly-L-Arginine as second layer, and hyaluronic acid as third layer
  • B is the expression of GFP in HeK293T cells transfected with HA-LNP encapsulating mRNA encoding the Green Fluorescence Protein (GFP);
  • C shows the expression of Luciferase in Hek293T cells by HA-LNP encapsulating mRNA encoding the Luciferase protein ;
  • D shows the expression of luciferase protein in pediatric glioblastoma cells SF188
  • encapsulation of small molecules can be achieved by loading the agent in the lipidic core, as a polyelectrolyte layer or attached to the latter.
  • the encapsulation of two small molecules, 5-azacytidine and 5-Fluoro-2'-deoxyuridine 5'-monophosphate sodium salt (5-FdU-MP) within the lipidic core of HA-LNP were exemplified.
  • the encapsulated molecules induced cytotoxicity on HeK293T cells in vitro. This example also demonstrated the co-delivery of drug and genes by HA-LNP.
  • Results are presented in FIG.26, where the left graph shows cytotoxic effect of 5-azacytidine (5-AZA)-loaded HA-LNP containing siRNA negative control in both core and layer, and the graph at the right shows HA-LNP encapsulating both 5'- monophosphate sodium salt (5-Fdll-MP) and siRNA negative control in the LNP core induced cytotoxic effect on HeK293T cells. This effect is significantly different than when cells were treated with HA-LNP without the drug.
  • 5-AZA 5-azacytidine
  • 5-Fdll-MP 5'- monophosphate sodium salt
  • FIG.27 shows the obtained results where A and B show physicochemical characterization of lipophilic Gadollinium-derivative (18:0 PE-DTPA- Gd) in the lipidic core of LNP surrounded by one layer of hyaluronic acid; and C shows the encapsulation of contrast agent (Gadollinium (DOTA)-Gd) within LbL assembled LNP (HA-LNP) initiated a decrease in T1 -weighted images under magnetic resonance imaging.
  • a and B show physicochemical characterization of lipophilic Gadollinium-derivative (18:0 PE-DTPA- Gd) in the lipidic core of LNP surrounded by one layer of hyaluronic acid
  • C shows the encapsulation of contrast agent (Gadollinium (DOTA)-Gd) within LbL assembled LNP (HA-LNP) initiated a decrease in T1 -weighted images under magnetic resonance imaging.
  • DOTA contrast agent
  • Results are shown in FIG.28, where removal of free polyelectrolytes can be achieved by size exclusion chromatography as demonstrated in the top illustration.
  • the bottom images demonstrate that layer-by-layer assembly of LNP can be achieved after purification.
  • SephadexTM-G25 was used to purify unbound siRNA and lipids of LNP coated with first siRNA layer.

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Abstract

La présente demande concerne des nanoparticules lipidiques. Plus particulièrement, la présente invention concerne des nanoparticules lipidiques modifiées couche par couche, des utilisations pour le transfert de gènes et l'administration de médicaments et leurs procédés de production. La présente demande comprend une nanoparticule lipidique revêtue comprenant : un noyau de nanoparticule lipidique comprenant un gène, un médicament, un agent de contraste ou un mélange de ceux-ci; au moins une première couche sur/autour du noyau comprenant des acides nucléiques chargés négativement en ARNsi; au moins une seconde couche sur/autour de la première couche comprenant des polycations; et une couche de surface sur/autour de l'au moins une seconde couche comprenant des polyélectrolytes chargés négativement.
PCT/CA2024/050238 2023-02-27 2024-02-27 Nanoparticules lipidiques modifiées couche par couche, utilisations correspondantes et leurs procédés de production Ceased WO2024178502A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2694089A1 (fr) * 2007-07-16 2009-01-22 Northeastern University Nanoparticules therapeutiques stables
CA2739499A1 (fr) * 2008-10-02 2010-04-08 Capsulution Pharma Ag Compositions nanoparticulaires ameliorees de composes faiblement solubles
CA3087606A1 (fr) * 2018-01-22 2019-07-25 Beijing Inno Medicine Co., Ltd Systeme de distribution de nanovecteur de type liposome destine a cibler une molecule cd44 active, son procede de preparation et ses applications
CA3166934A1 (fr) * 2020-02-05 2021-08-12 Elias SAYOUR Nanoparticules chargees d'arn et leur utilisation pour le traitement du cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2694089A1 (fr) * 2007-07-16 2009-01-22 Northeastern University Nanoparticules therapeutiques stables
CA2739499A1 (fr) * 2008-10-02 2010-04-08 Capsulution Pharma Ag Compositions nanoparticulaires ameliorees de composes faiblement solubles
CA3087606A1 (fr) * 2018-01-22 2019-07-25 Beijing Inno Medicine Co., Ltd Systeme de distribution de nanovecteur de type liposome destine a cibler une molecule cd44 active, son procede de preparation et ses applications
CA3166934A1 (fr) * 2020-02-05 2021-08-12 Elias SAYOUR Nanoparticules chargees d'arn et leur utilisation pour le traitement du cancer

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