CN116059167B - Co-drug-loaded micelle and synergistic drug system thereof, and preparation method and application thereof - Google Patents
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Abstract
本发明公开了一种共载药胶束及其协同药物体系与制备方法和应用，具体为一种吉西他滨前药与紫杉醇共载胶束，由吉西他滨前药、紫杉醇、寡聚乙二醇组成药物溶液；将药物溶液、聚合物溶液以及寡聚乙二醇的混合液加入缓冲液中，得到吉西他滨前药与紫杉醇共载胶束，可与TLR9纳米激动剂比如纳米CpG高效协同治疗三阴乳腺癌转移的继发性肺癌，其中，CpG通过激活TLR9通路诱导免疫细胞产生I型干扰素，从而诱导Th1免疫应答；可通过静脉系统给药，降低了CpG潜在的免疫原性和系统毒性。
The invention discloses a co-drug loaded micelle and a synergistic drug system, a preparation method and an application thereof, specifically a gemcitabine prodrug and paclitaxel co-loaded micelle, wherein the gemcitabine prodrug, paclitaxel and oligoethylene glycol form a drug solution; the drug solution, the polymer solution and the mixed solution of the oligoethylene glycol are added into a buffer solution to obtain the gemcitabine prodrug and paclitaxel co-loaded micelle, which can be used in a highly efficient synergistic manner with a TLR9 nano agonist such as nano CpG to treat secondary lung cancer metastasized from triple-negative breast cancer, wherein CpG induces immune cells to produce type I interferon by activating the TLR9 pathway, thereby inducing a Th1 immune response; and the micelle can be administered through the intravenous system, thereby reducing the potential immunogenicity and systemic toxicity of CpG.
Description
Technical Field
The invention belongs to the medicine technology, and in particular relates to a co-carried micelle of gemcitabine prodrug and paclitaxel, a synergistic system and application thereof, which can be used for efficiently treating secondary lung cancer of triple negative breast cancer metastasis in cooperation with nano CpG.
Background
Triple Negative Breast Cancers (TNBC), negative for estrogen receptor, progestin receptor and HER2 expression, account for 24% of new breast cancers, the most troublesome and fatal breast tumor subtype. The recurrence rate of TNBC is high, and about 36.9% of recurrent TNBC patients undergo lung metastasis. Local surgery combined with radiotherapy, systemic chemotherapy, targeted therapy and immunotherapy are currently the main means for preventing and treating secondary tumors, but the immune therapy response rate of TNBC is low due to lack of drugs specifically targeting metastatic cells and the very "cold" of TME. Even if ICB had only 23.8% disease control in treatment of PD-L1 positive metastatic TNBC patients, the median survival was 18 months and the overall survival was not significantly improved compared to chemotherapy, thus, heating "cold" TME "became a new strategy for TNBC treatment. In recent years, various strategies have been developed for "heating" TMEs, such as tumor cell ICD induced by chemotherapy, radiation therapy, and photodynamic therapy, release of tumor antigens, activation and transport by immune cells, elimination or repolarization by immunosuppressive cells, and by reconstitution of ECM barriers, etc. The FDA approved combination therapy of PD-L1 inhibitors and albumin paclitaxel (Nab-PTX) was used for the treatment of PD-L1 positive metastatic TNBC with only 25% disease remission rate, with minimal efficacy in PD-L1 negative TNBC patients.
Disclosure of Invention
The invention discloses a gemcitabine (Gem) prodrug (HPG) and Paclitaxel (PTX) co-loaded micelle, a synergistic system and application, which can be used for efficiently treating the secondary lung cancer of triple negative breast cancer metastasis in a synergistic way with nano CpG, wherein CpG induces immune cells to generate I-type interferon by activating TLR9 channels so as to induce T h1 immune response, and potential immunogenicity and systemic toxicity of CpG can be reduced by intravenous system administration.
The invention adopts the following technical scheme:
A drug-loaded micelle is a drug-loaded micelle of gemcitabine prodrug and paclitaxel, and comprises a polymer micelle, the gemcitabine prodrug and the paclitaxel.
A synergistic medicine system comprises the gemcitabine prodrug, paclitaxel co-loaded micelle and nano CpG. Preferably, the nano-CpG is a CpG-loaded polymeric vesicle.
In the invention, in the polymer micelle, the polymer is hydrophilic segment-P (hydrophobic monomer-DTC), or the polymer is hydrophilic segment-P (hydrophobic monomer-DTC) and targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC), and in the polymer vesicle, the polymer is hydrophilic segment-P (hydrophobic monomer-DTC) and cation fragment. DTC is a prior cyclic carbonate monomer containing disulfide five-membered ring functional groups, and is polymerized to form PDTC chain segments. The hydrophobic monomers include cyclic ester monomers, cyclic carbonate monomers, preferably the cyclic carbonate monomers are other cyclic carbonate monomers, such as other cyclic carbonate monomers including trimethylene cyclic carbonate (TMC), including caprolactone (. Epsilon. -CL), lactide (LA) or Glycolide (GA), and the hydrophobic monomers polymerize to form other hydrophobic segments, such as PTMC segments, PCL segments. The PDTC segment and other hydrophobic segments constitute the hydrophobic segments of the polymer. Preferably, the hydrophilic segment is PEG, the cationic segment is PEI segment or spermine segment, and the targeting molecule is a polypeptide such as ATN1 (Ac-PHSCNK-NH 2）、ATN2（Ac-PhScNK-NH2), cRGD (c (RGDfC)), and the like. As an example, the hydrophilic segment-P (hydrophobic monomer-DTC) is PEG-P (CL-DTC), the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) is ATN2-PEG-P (CL-DTC), and the hydrophilic segment-P (hydrophobic monomer-DTC) -cationic segment is PEG-P (TMC-DTC) -SP.
In the polymer micelle, the molecular weight of a hydrophilic segment is 1-7.5 kg/mol, the molecular weight of a hydrophobic segment is 1.5-7.5 kg/mol, and preferably, the molecular weight of the hydrophilic segment is 1.5-5 kg/mol, and the molecular weight of the hydrophobic segment is 1.5-5 kg/mol. In the polymer vesicle, the molecular weight of the hydrophilic segment is 3-10 kg/mol, and the molecular weight of the hydrophobic segment is 10-20 kg/mol. The molecular weight of the cationic fragment is 40% or less of the molecular weight of the hydrophilic segment, preferably 0.1 to 2 kg/mol.
In the invention, when the polymer is hydrophilic segment-P (hydrophobic monomer-DTC) and targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC), the mass content of the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) is 0-20% excluding 0, preferably 1-15%, and more preferably 2-10%.
In the present invention, the molar ratio of the gemcitabine prodrug to paclitaxel is (2-20) to 1, preferably (5-20) to 1, and more preferably (5-15) to 1.
The preparation method of the gemcitabine prodrug and paclitaxel co-supported micelle comprises the step of adding a mixed solution of a drug solution, a polymer solution and oligomeric ethylene glycol into a buffer solution to obtain the gemcitabine prodrug and paclitaxel co-supported micelle. Specifically, the mixed solution is added into a buffer solution, and is kept stand after being blown to obtain the gemcitabine prodrug and paclitaxel co-loaded micelle. The drug solution consists of gemcitabine prodrug, paclitaxel and oligomeric ethylene glycol, and the concentration of the drug solution is 5-100 mg/mL, preferably 10-70 mg/mL, and more preferably 20-50 mg/mL. The polymer solution is a hydrophilic segment-P (hydrophobic monomer-DTC) solution, or the polymer solution is a hydrophilic segment-P (hydrophobic monomer-DTC) solution and a targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) solution. The concentration of the polymer solution is 20-500 mg/mL, preferably, the concentration of the hydrophilic segment-P (hydrophobic monomer-DTC) solution is 50-500 mg/mL, preferably 100-400 mg/mL, and the concentration of the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) solution is 10-200 mg/mL.
In the invention, the solvent in the drug solution and the polymer solution is oligomeric ethylene glycol, and the molecular weight of the oligomeric ethylene glycol is 200-800, such as PEG200, PEG350, PEG400, PEG600, PEG800 and the like. Preferably, the mixed solution is added into a solution obtained by adding a buffer solution, and the volume percentage of the oligoethylene glycol is 2-10%, preferably 2.5-8%.
The invention discloses an application of a gemcitabine prodrug and paclitaxel co-supported micelle and a synergistic drug system in preparing an anti-tumor drug, in particular to an application in preparing a drug for treating triple-negative breast cancer, and in particular to an application in preparing a drug for treating secondary lung cancer formed by triple-negative breast cancer metastasis.
In the invention, in order to heat the immune microenvironment of 'cold' tumor TNBC and improve the immune treatment effect of TNBC subcutaneous tumor and postoperative recurrent/secondary lung cancer, alpha 5β1 integrin targeting and HPG and PTX co-loading disulfide cross-linked micelle ATN2-mG/P are designed. The micelle can induce tumor cells to selectively generate ICD, effectively eliminate MDSC and stimulate DC maturation, so that the immune microenvironment is heated, and the inhibition of secondary lung cancer formed by 4T1 subcutaneous tumor and postoperative recurrence and metastasis is obviously improved. ATN2-mG/P can most effectively inhibit recurrence and pulmonary metastasis of 4T1 tumor when HPG/PTX=10/1, and can obviously inhibit recurrence and secondary lung cancer after 4T1 operation when being combined with NanoCpG, and has no pulmonary metastasis nodule and systemic toxic and side effects, so that the survival time of mice is obviously improved, and 60% of mice are completely cured. The chemotherapy successfully stimulates the recruitment and activation of a large amount of DC, provides antigens for CD8 + and CD4 + T cells, increases secretion of IFN-gamma and TNF-alpha, obviously reduces infiltration of immunosuppressive MDSC and Treg, and improves comprehensive treatment effect of postoperative TNBC. ATN2-mG/P and NanoCpG are simple to prepare, good in safety and capable of effectively enhancing T cell response, so that the combined therapy is expected to provide thought for the treatment of cold tumors such as TNBC and the prevention and treatment of postoperative recurrence/secondary lung cancer.
Drawings
FIG. 1 is a graph showing the characteristics of mG/P (A) mHPG, mPTX, mG/P, ATN2-mG/P (HPG/PTX=10/1) and (B) mG/P (HPG/PTX=5/1, 10/1, 20/1) particle size and particle size distribution, (C) DLS assay mHPG and particle size distribution of a PEG350 solution of HPG, (D) change in particle size of mG/P stored at room temperature before and after dialysis to remove PEG350, (E) change in particle size of mG/P incubated with 10% FBS, and (F) ultraviolet absorbance spectra of a PEG350 solution of mHPG, mPTX, mG/P and PEG-P (CL-DTC).
Fig. 2 shows in vitro drug release of micellar drugs, (a) particle size of mgp in the presence or absence of 10 mM GSH, (B) drug release in the presence or absence of 10 mM GSH medium (pH 7.4) at 37 ℃ of mgp and (C) single-carrier micelle mHPG (n=3), and (D) drug release in the absence of GSH at pH 5.0 or pH 7.4 (n=3). (E) Intact HPG content (n=3) when free HPG, mHPG and mgp were incubated with CDA with or without 10% serum at 4h, each formulation was incubated with PB or a PB solution containing tha (tetrahydrouridine, CDA inhibitor, 344 μm) as a control.
FIG. 3 shows the cell viability of (A) mG/P (HPG/PTX=2/1, 5/1, 10/1, 20/1), (B) mPTX and (C) ATN2-mG/P incubated with 4T1-luc cells 48 h for cytotoxicity studies (n=6).
FIG. 4 shows that mGs/P (HPG/PTX=20/1, 10/1, 5/1), mHPGs, mPTX, and ATN 2-mGs/P (HPG/PTX=10/1) incubated with 4T1-luc cells 48 h induced (A) apoptosis (HPG: 0.5 μg/mL (0.85 μM), PTX:0.15 μg/mL (0.17 μM), n=3), and (B) cell cycle arrest (HPG: 0.1 μg/mL (0.17 μM), PTX:0.03 μg/mL (0.034 μM), n=3).
FIG. 5 shows the induction of ICD (HPG: 1. Mu.g/mL (1.7. Mu.M), PTX: 0.3. Mu.g/mL (0.34. Mu.M), 24 h, n=3) by mG/P and ATN2-mG/P of 4T1-luc cells after treatment of (A, B) mG/P (HPG/PTX=20/1, 10/1, 5/1) with respect to cell surface CRT expression and (C) ATP concentration, (D, E) ATN2-mG/P of cell surface CRT expression and (F) ATP secretion.
FIG. 6 shows the stimulation of maturation of BMDC (CD 80 +CD86+ mDC) with mG/P (HPG/PTX=20/1, 10/1, 5/1) and ATN2-mG/P (HPG/PTX=10/1), free G/P, mHPG, mPTX and PBS as controls (HPG: 1. Mu.g/mL (1.7. Mu.M), PTX: 0.3. Mu.g/mL (0.34. Mu.M), 24 h, n=3).
FIG. 7 shows representative flow histograms and semi-quantitative analysis (HPG: 1. Mu.g/mL (1.7. Mu.M), PTX: 0.3. Mu.g/mL (0.34. Mu.M), 24 h, n=3) of (A) experimental design, (B) mG/P (HPG/PTX=20/1, 10/1, 5/1) and (C) ATN2-mG/P post-treatment maturation BMDC following mG/P and ATN2-mG/P stimulation.
FIG. 8 shows treatment of 4T 1-bearing subcutaneous tumor mice with mG/P (n=6), intravenous injection of mHPG, mG/P (HPG/PTX=20/1, 10/1) (HPG: 15 mpk, 25.8. Mu. Mol/kg), mPTX (PTX: 2.25 mpk, 2.58. Mu. Mol/kg) on days 0, 2, 4, 6, 8, 10, (A) treatment schedule, (B) tumor volume change, (C) weight change, and (D) survival curves.
FIG. 9 shows treatment of ATN2-mG/P on 4T 1-bearing subcutaneous tumor mice, intravenous injection of ATN2-mHPG, mG/P, ATN-mG/P (HPG/PTX=10/1) (HPG: 10 mpk, 17.2. Mu. Mol/kg) and ATN2-mPTX (PTX: 1.5 mpk, 1.72. Mu. Mol/kg) on days 0, 2,4,6, 8, 10 on (A) with dosing schedule, (B) tumor volume (C) body weight (n=6) of mice, (D) mDC and (E) MDSC ratio (n=3) infiltrated in tumors on day 14.
FIG. 10 shows (A) treatment regimen, (B) tumor volume and (C) body weight (n=6) of mice, (D) Kaplan-Meier survival curve and (E) tumor growth curve (n=5) of single mice of ATN2-mG/P or mG/P combination NanoCpG treatment of 4T1-luc post-operative recurrent/secondary lung cancer mice.
FIG. 11 is a bioluminescence image and H & E stained image of lung tissue of treated mice at day 15, scale bar 1000 μm.
FIG. 12 is a representative flow chart of ATN2-mG/P or mG/P in combination NanoCpG to stimulate BMDC maturation and semi-quantitative analysis of the proportion of mDC (CD 11c +CD80+CD86+) (HPG/PTX=10/1, HPG:1 μg/mL (1.7 μM), PTX:0.15 μg/mL (0.17 μM), cpG:0.4 μg/mL, n=3).
Fig. 13 is a tumor microenvironment analysis (HPG/ptx=10/1,HPG:15 mpk,CpG:1 mpk,n =4) of a chemotherapeutic immunotherapy TNBC post-operative relapsed/secondary lung cancer mouse (a) treatment regimen, (B) tumor mass, (C) lung weight, (D) lung metastasis node number and (E) spleen mass of the mouse.
FIG. 14 shows tumor microenvironment analysis of tumor (n=4) in tumor-infiltrated (A) CD11C + DC and (B) CD11C +CD80+CD86+ mDC, tumor-infiltrated (C) CD8 + T in tumor and (D) spleen, tumor-infiltrated CD4 + T in (E) spleen and Treg-infiltrated in (G) tumor, tumor-infiltrated MDSC in (H) spleen, and serum concentration of (J) IFN-gamma, (K) TNF-alpha and (L) IL-10 in serum in mice with recurrent/secondary lung cancer after TNBC surgery by chemotherapy immunotherapy.
FIG. 15 shows (A) preparation of ATN2-mG/P, (B) its combination with NanoCpG can heat tumor immune microenvironment, effectively inhibiting 4T1 tumor progression, recurrence and pulmonary metastasis, and (C) polymer molecular structure.
Detailed Description
Paclitaxel (PTX, >98%, shanghai gold and Bio-pharmaceutical Co., ltd.) was used directly after purchase of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, thermoFisher Scientific). PEG-P (CL-DTC) n = 2.0-(1.1-0.9) kg/mol)、ATN2-PEG-P(CL-DTC)( n =3.4- (1.1-1.1) Kg/mol), ATN2 functionalization degree of 71.2%), PEG-P (TMC-DTC) -SP n =5.0- (14.5-2.0) -0.2 Kg/mol, SP functionalization degree 95.0%) was synthesized according to the method reported previously. CpG ODN 2018 (CpG) is provided by Ji Ma gene, ac-PhScNK-NH2 (ATN 2) purity is higher than 98%, and is purchased from Shanghai blaze. 4T1-luc cells were purchased from the Shanghai cell Bank of the national academy of sciences and BMDC were routinely extracted from healthy Balb/c mice. Balb/c (6 weeks, female, 20-22 g) mice were purchased from Peking Vitre Liwa laboratory animal Co. All animal experiments were approved by the committee for animal care and use at university of su, and all protocols were in compliance with the guidelines for laboratory animal care and use.
The drug loading and drug loading rates of HPG and PTX were determined by high performance liquid chromatography under conditions of acetonitrile/water (v/v) =50/50, 1 mL/min, UV 220 nm. The Drug Loading (DLC) and the encapsulation efficiency (DLE) of CpG were measured using NanoDrop (NanoDrop 2000, thermo).
Example preparation and characterization of ATN2-mG/P
The present invention contemplates integrin-targeted micelle ATN2-mG/P that co-delivers HPG and PTX in specific ratios. Both PTX and HPG stimulate ICD in tumor cells, and activation of APCs by PTX and elimination of MDSCs by HPG can further synergistically reverse immunosuppressive TME. In addition, targeted delivery of both drugs to tumor cells can reduce their systemic toxicity.
Synthesis and characterization of PEG-P (CL-DTC) according to the existing methods n = 2.0-(1.1-0.9) kg/mol)、ATN2-PEG-P(CL-DTC) ( n = 3.4-(1.1-1.1) kg/mol)、PEG-P(TMC-DTC)-SP( n =5.0- (14.5-2.0) -0.2 Kg/mol, see fig. 15C for chemical structural formula of the polymer.
Dissolving HPG and PTX in PEG350 (25 mg/mL), mixing to obtain medicinal solution at molar ratio of 20/1, 10/1, 5/1 or 2/1, and preparing PEG350 mother liquor of PEG-P (CL-DTC) (200 mg/mL) and ATN2-PEG-P (CL-DTC) (20 mg/mL). Taking the preparation of a drug solution of 4. Mu.L HPG and 0.6. Mu.L PTX with a total volume of 50. Mu.L PEG-P (CL-DTC) in PEG350 and PEG350 as an example of a co-loaded micelle mG/P with a concentration of 1 mG/mL, a theoretical drug loading of 9.1 wt% HPG and 10/1 HPG/PTX, adding to 950. Mu.L PB (pH 7.4,10 mM), blowing 5 times with a pipette, and standing at room temperature for 12 h to obtain mG/P with a PEG350 volume content of 5%. HPG/PTX co-supported micelles with a concentration of 1 mg/mL, a theoretical drug loading of 9.1 wt% of HPG and a volume content of 2% or 10% of PEG350 with an HPG/PTX of 10/1 were prepared in a similar manner. ATN2-mG/P was prepared in a similar manner by adding a total volume of 50. Mu.L of a mixed solution containing 4.75. Mu.L of PEG-P (CL-DTC), 2.5. Mu.L of ATN2-PEG-P (CL-DTC), HPG/PTX solution, PEG350 to 950. Mu.L of PB.
DLS monitors particle size and particle size distribution of mG/P and ATN2-mG/P, storage stability of mG/P before and after dialysis, and particle size variation of micelles incubated with 10% serum or 10 mM GSH. The UV spectrophotometer measures the cross-linking of micelles with a polymer concentration of 1 mg/mL. DLC and DLE of HPG and PTX in mG/P and ATN2-mG/P were measured by HPLC. PB (pH 7.4, 10 mM) and acetic acid/sodium acetate buffer (pH 5.0,5 mM) containing 10 mM GSH were formulated to mimic the reducing conditions of tumor microenvironment and the slightly acidic conditions of tumor cell lysosomes, respectively. The acetic acid/sodium acetate buffer solution was formulated by 285.7 μl of acetic acid and 262.43 mg sodium acetate dissolved in 1L secondary water. Next, 1 mL mG/P was left overnight and transferred to a release bag (MWCO 14 kDa), dialyzed in 25mL buffer tubes, respectively, placed in a 37℃and 200 rpm shaker. At the set time point 7 mL was taken and the same volume of fresh buffer was replenished into the centrifuge tube. The release solution obtained was freeze-dried and then dissolved in 500 μl of a mixed solution (1/1, v/v) of methanol and acetonitrile, and after filtration, the cumulative release amounts of the two drugs were tested by HPLC (n=3).
ATN2-mG/P was self-assembled from 5% ATN2-PEG-P (CL-DTC), 95% PEG-P (CL-DTC) and PEG350 solutions of different HPG/PTX ratios (2/1, 5/1, 10/1 or 20/1). Single-carrier micelles ATN2-mHPG, ATN2-mPTX and non-targeted micelle mG/P, mHPG, mPTX were prepared in the same manner. All the resulting micelles had particle sizes of 19.8-23.5 nm and a narrow particle size distribution (pdi=0.08-0.17) (fig. 1a, b). The loading rates of HPG and PTX in ATN2-mG/P and mG/P are 94.0-100% and 86.7-97.0%, respectively. In contrast, HPG and PTX micelles alone were less efficient in drug delivery (Table 1), and a mixture of HPG, PTX and PEG350 at the same concentration showed a broad particle size distribution, turbidity and PTX precipitation in water without copolymer compared to the small particle size and clear solution of ATN2-mG/P (FIG. 1C). ATN2-mG/P and mG/P remained stable for at least 8 days at room temperature, clear, transparent, unchanged in particle size, without drug precipitation (FIG. 1D), and stable in PB buffer with 10% FBS (FIG. 1E). The co-supported micelles were dialyzed against 2 h (buffer change per hour) in dialysis bags with molecular weights of 8-14 kDa and 10 volumes of PB solution (pH 7.4,10 mM) to remove PEG350, and then stored at room temperature for less than 5 days. In addition, the micelle particle size is affected by the volume content of PEG350, the particle size of HPG/PTX co-supported micelle of PEG350 with a content of 10% increases to 27.5 nm, and the particle size distribution increases to over 0.2. These results demonstrate that PEG350 plays an important role in micelle assembly and stability. The superior stability of micelles is also due to disulfide cross-linking in the ATN2-mG/P micelle core, the ultraviolet absorbance of the micelles is significantly reduced compared to the polymer solution (FIG. 1F). In the stability test of the micelle, taking HPG/PTX 10/1 as an example, when the HPG/PTX is 5/1 and 20/1, the stability of the micelle is similar to 10/1, but a micelle solution with the HPG/PTX of 2/1 is stable in particle size after being placed at room temperature for 3 days, and PTX precipitation can gradually occur in more than 4 days.
PEG-P (CL-DTC) with different molecular weights n 2.0- (1.0-1.2), 2.0- (1.0-1.6) Or 5.0- (4.0-2.0) kg/mol) polymers are also used to prepare micelles loaded with HPG alone. The preparation method is similar, and PEG-P (CL-DTC) is prepared n 5.0- (4.0-2.0) Kg/mol,50 mg/mL) and HPG (25. 25 mg/mL) were used, and 4. Mu.L of HPG, 20. Mu.L of PEG350 solution of PEG-P (CL-DTC) and PEG350 were mixed to a total volume of 50. Mu.L of solution, and added to 950. Mu.L of PB (pH 7.4,10 mM), and the mixture was blown 5 times with a pipette to give a micelle having a particle size of 44.1 nm (PDI: 0.24), as exemplified by the prepared micelle mHPG having a concentration of 1 mg/mL and a theoretical drug loading of HPG of 9.1. 9.1 wt%. The particle sizes of mHPG prepared from polymers with molecular weights of 2.0- (1.0-1.2) and 2.0- (1.0-1.6) kg/mol are 36.4 and 32.5 nm respectively, and the particle sizes and particle size distribution of the prepared micelles are increased and the stability is poor.
In vitro mimicking the intracellular reducing environment, i.e. the particle size of mgs/P rapidly and sharply increased after addition of glutathione (GSH, 10 mM) at pH 7.4 (fig. 2A), more than 95% of HPG and PTX were rapidly expelled from mgs/P, with minimal leakage of HPG and PTX without GSH (fig. 2B). However, single-carrier HPG micelles mHPG released more than 40% of the drug in the absence of GSH (FIG. 2C), and in addition, the drug release in mG/P was very small in weak acidity, indicating that the micelles had the ability to stably escape from the endosome and release the drug to the cytoplasm (FIG. 2D).
Example cytotoxicity, apoptosis and cell cycle arrest assay of Diatn 2-mgp
After inoculating 4T1-luc in a 96-well plate (1X 10 3/well) at 24: 24h, 20. Mu.L of mG/P (HPG/PTX: 20/1, 10/1, 5/1 or 2/1), mHPG or mPTX, wherein HPG concentration is 0.0017-68.8. Mu.M and PTX concentration is 0.005-12.8. Mu.M, is added. After incubation 48 h, 10 μl of MTT solution (5 mg/mL) was added to incubate 4h, the supernatant was discarded, and 150 μl DMSO was added to lyse the living cells and the purple formazan crystals produced by MTT. The absorption of cells at 570 nm was measured using a multifunctional microplate reader, and the cell viability (cell viability%) was the ratio of the absorbance value of the experimental group to the absorbance value of the PBS group, taking the wells of the MTT-added medium as zero points and the wells of the MTT-added PBS-treated cells as 100%.
The synergy of HPG and PTX was evaluated by calculating the joint index (CI) using the following formula:
Wherein a, b represent the IC 50 values in mgs/ps for each drug, respectively, and A, B represent the IC 50 values in mHPG and mPTX, respectively, for each drug alone. The obtained CI >1 shows that the two medicines have antagonistic effect, CI=1 is superposition effect, CI <1 is synergistic effect, and the smaller the CI value is, the stronger the synergistic effect between medicines is.
To investigate the targeting of ATN 2-mgp to 4T1 cells, 4T1 cells (1×10 3/well) were incubated with ATN 2-mgp and mgp (HPG/ptx=10/1, HPG concentration: 0.0017-68.8 μm) 4 h, then 44 h with fresh medium without drugs. Subsequent sample processing and data analysis methods are described above.
In an apoptosis experiment, ATN2-mG/P（HPG/PTX：10/1）、mG/P（HPG/PTX：20/1、10/1、5/1）、mHPG、mPTX（HPG：0.5 μg/mL（0.85 μM）,PTX：0.15 μg/mL（0.17 μM）,n = 3） and PBS were added to 4T1-luc cells (1X 10 5/well) cultured in 12-well plates. Co-culture 48 h was followed by conventional cell handling, staining, flow cytometry and FlowJo_V10 analysis.
To investigate the ability of HPG and PTX to block the cell cycle, ATN2-mG/P (HPG/PTX: 10/1), mG/P (HPG/PTX: 20/1, 10/1 or 5/1), mHPG and mPTX (HPG: 0.1. Mu.g/mL (0.17. Mu.M), PTX: 0.03. Mu.g/mL (0.034. Mu.M), n=3) were added to 4T1-luc cells. After 48h incubation, cells were trypsinized, washed with PBS, fixed with 95% ethanol overnight, PI stained, and the ratio of the different phases of the cell cycle was quantified by flow cytometry.
The IC 50 values for HPG and PTX were significantly reduced at molar ratios of HPG/PTX of 2/1, 5/1, 10/1, 20/1 compared to IC 50 values for mPTX (1.4. Mu.M) and mHPG (3.8. Mu.M), respectively, of 0.3/0.14, 1.8/0.16, 0.4/0.074 and 2.3/0.10 (FIGS. 3A, B). The CI numbers of HPG and PTX were both less than 1 (table 2), indicating a strong synergy with mgp/P having the lowest CI at HPG/ptx=10/1 or 2/1, indicating that low doses of PTX could also produce the same synergy with HPG at higher doses of PTX.
Cytotoxicity of ATN2-mG/P micelles containing 5% ATN2 was studied. ATN 2-mgp at HPG/ptx=10/1, where the IC 50 value of HPG is 2.3 times lower than that of micelle mgp (fig. 3C). Apoptosis experimental analysis showed that mgp induced higher proportion of apoptosis than other proportion (×p) and drug alone group (×p) at HPG/ptx=10/1, ATN 2-mgp further increased apoptosis rate to 81.7% (×p), consistent with MTT results (fig. 4A). Cell cycle studies showed that mG/P also severely affected the cycle of 4T1 cells at very low drug concentrations (HPG: 0.1. Mu.g/mL; PTX: 0.03. Mu.g/mL), presenting significant S-phase and G2/M-phase arrest, in sharp contrast to slight cell arrest of singly loaded micelles mHPG and mPTX, which were the same as the respective drug concentrations, showing strong synergy of both drugs in mG/P (FIG. 4B). In addition to the significant enhancement of cell cycle arrest, low dose PTX reduces the increase in intracellular stability of GEM by Cytidine Deaminase (CDA) expression, which is also one of the reasons for the strong synergistic effect of both. Both prodrug modification (HPG) and micelle loading (mHPG) enhanced resistance of Gem to enzymatic degradation, whereas co-loaded micelle mgp/P maintained the intact structure after incubation with CDA and 10% fbs for 4 h at a drug ratio of 7 times that of HPG-only loaded micelle mHPG (fig. 2E), further demonstrating that PTX could exert a strong synergistic effect on HPG by its degradation protection.
The ATN2-mG/P micelles used in the subsequent study were all ATN2 content 5%, HPG/PTX=10/1.
Example three ATN2-mG/P induces immunogenic death (ICD) of tumor cells
After 4T1-luc cells were cultured in 12-well plates (1X 10 5/well) for 24: 24h, ATN2-mG/P （HPG/PTX：10/1）、mG/P（HPG/PTX：20/1、10/1、5/1）、mHPG、mPTX（HPG：1 μg/mL（1.7 μM）,PTX：0.3 μg/mL（0.34 μM）,n = 3） or PBS was added. After incubation 24h, the medium was collected and assayed for ATP concentration in the medium using an enhanced ATP detection kit, stained after cell digestion, flow cytometer detected, and analyzed for cell surface CRT expression using FlowJo_V10.
PTX and Gem can provide tumor antigens by inducing tumor cells ICD, modulate the immune microenvironment of "cold" tumors by stimulating APCs to promote the cancer immune cycle, and have a synergistic effect when co-loaded into the same micelle, mG/P and ATN2-mG/P induce ICD and stimulate BMDC maturation at low concentrations (HPG: 1 μg/mL (1.7 μM), PTX:0.3 μg/mL (0.34 μM)). Flow cytometry analyzed ICD of 4T1-luc cells after each preparation treatment for expression of surface CRT and secretion of ATP. The results show that single-loaded micelles mPTX and mHPG only have a slight effect on CRT expression and ATP secretion, whereas mgp induces CRT and ATP significantly higher than mHPG and mPTX at HPG/ptx=10/1, and also higher than mgp at HPG/ptx=5/1, 20/1 (fig. 5A-C). Indicating that HPG and PTX co-load in micelles and their ratio are critical. Notably, ATN 2-mgp was able to stimulate further 4T1 cells to express more CRT (P) and secrete more ATP (P) than mgp (fig. 5D-F).
Low doses of PTX modulate proliferation and polarization of APCs through TLR4 pathways to promote maturation and proliferation of DCs, gem eliminates MDSCs and also stimulates DC maturation, and experimental results show that mPTX and mHPG both significantly stimulate BMDC maturation (CD 80 +CD86+ mDC) at low concentrations (p). It is worth mentioning that mgp and ATN 2-mgp further promoted DC maturation to 58.6% and 59.6% (. P) compared to mHPG, significantly higher than the mixture of free PTX and HPG (free G/P, (. P) (fig. 6), indicating that HPG and PTX in mgp and ATN 2-mgp have a synergistic effect in stimulating DC maturation.
ICD-induced tumor antigens can release a "eat me" signal to promote DC maturation and present the antigen to T cells, resulting in a tumor-specific T cell response. To mimic the tumor microenvironment, 4T1-luc tumor cells were co-cultured with BMDC, and the ability of mGs/P and ATN 2-mGs/P to stimulate BMDC maturation was studied (FIG. 7A). The results showed that the proportion of mHPG and mPTX treated mature DCs was significantly lower than that without co-culture of 4T1 cells, indicating preferential endocytosis by 4T1 cells rather than DCs. mgp (HPG/ptx=10/1) stimulated the highest proportion of DC maturation in other non-targeted micelles (fig. 7B), whereas ATN 2-mgp induced a further increase in the proportion of DC maturation (40.3%,/P) (fig. 7C).
Example four mG/P and ATN2-mG/P micelle drug efficacy study on 4T1-luc subcutaneously tumor mice
The antitumor effect was studied on the mouse 4T1-luc TNBC model. After 7 days of subcutaneous inoculation with 4T1-luc cells (3×10 5/dose), the tumor average volume grew to about 50mm 3, and the mice were randomly divided into 6 groups (n=6), which was recorded as day 0 of the experiment. Mice were judged to die by intravenous injection of mgp every 2 days (HPG/ptx=20/1 or 10/1, HPG:15 mpk), mHPG (HPG: 15 mpk,25.8 μmol/kg), mPTX (PTX: 2.25 mpk,2.58 μmol/kg), 6 total times (fig. 8A), monitoring tumor volume and body weight, tumor volume exceeding 2000 mm 3, body weight loss exceeding 15%, no response or no feeding. The results indicate that treatment with mgs/P and mHPG effectively delayed tumor progression (fig. 8B). In contrast, mPTX had no significant inhibition (fig. 8B), and previous studies on the same mouse model found that mPTX showed the ability to inhibit 4T1 tumor growth at 7.5 mpk (3.3 times the current dose). The tumor inhibition effect of mgp (HPG/PTX 10/1) was best, significantly better than mgp (HPG/PTX 20/1) and mHPG (P) (fig. 8B). The body weight of each group of mice did not change much during the administration period except for mHPG causing a slight decrease in body weight of mice (fig. 8C). The results of the mice survival monitoring showed that the Median Survival (MST) of mice in the mgp (HPG/ptx=10/1) group was significantly prolonged to 28.5 days compared to mHPG (P) and mPTX (P), showing the benefit of co-loaded micelles and synergy of both for tumor suppression and prolonged survival in tumor bearing mice.
To investigate the inhibition of TNBC tumors by ATN2-mG/P, six needles ATN2-mPTX、ATN2-mHPG、mG/P、ATN2-mG/P（HPG/PTX = 10/1,HPG：10 mpk（17.2 μmol/kg）,PTX：1.5 mpk（1.72 μmol/kg））（ were injected in vivo through the tail vein into 4T 1-bearing subcutaneous tumor mice FIG. 9A). After the last injection for 4 days (day 14), 3 mice were euthanized at random for each group, tumor tissue was extracted from the tumor, and single cell suspensions were obtained after grinding, centrifugation, and ACK lysis of the erythrocytes. FITC- αCD11c, APC- αCD80, PE- αCD86, FITC- αCD11b, and PE/Cy7- αGr-1 were then added and incubated at 4℃for 30 min, and the infiltration amounts of mDC (CD 11C +CD80+CD86+) and MDSC (CD 11b +Gr-1+) were determined by flow cytometry.
FIG. 9B shows that ATN2-mG/P has significantly better tumor inhibition than mG/P and ATN2-mHPG (P), while ATN2-mPTX has little inhibition on 4T1 tumors. Treatment with all formulations did not result in weight loss in mice (fig. 9C). TNBC is well known to be a highly immunosuppressive tumor, with about 40% infiltration of MDSCs, resulting in lower response rates of TNBC patients to immunotherapy. To assess the effect of ATN2-mG/P treatment on tumor immune microenvironment, three mice were randomly selected from each group for sacrifice on day 14, and tumor tissues were analyzed for infiltration of MDSC and DC. Flow cytometry results showed 2.2-2.5 fold increase in the proportion of mature DCs in tumors of each micelle drug group compared to PBS group (fig. 9D). In addition, the proportion of MDSCs in PBS group tumors was up to 42%, confirming the highly immunosuppressive nature of TNBC tumors, with significantly reduced MDSCs in ATN2-mHPG, mgp and ATN 2-mgp groups (P) (fig. 9E).
EXAMPLES five ATN2-mG/P and NanoCpG chemotherapy treatment of 4T1-luc secondary Lung cancer study
NanoCpG is obtained by self-assembly of the copolymer PEG-P (TMC-DTC) -SP of CpG and terminally modified spermine (spermine) in aqueous solution. 100. Mu.L of PEG-P (TMC-DTC) -SP in DMF (10 mg/mL) was added to 900. Mu.L of CpG (100. Mu.g) containing HEPES buffer (5 mM, pH 6.8), stirred conventionally for 10min, dialyzed 2 times in HEPES, dialyzed 1 time in PB/HEPES (v/v, 1/1), and finally dialyzed 2 times in PB to give NanoCpG solution. DLS measures particle size and particle size distribution, and Nanodrop measures CpG drug loading. The vesicles NanoCpG are self-assembled from the end-modified SP block copolymer PEG-P (TMC-DTC) -SP in an aqueous solution containing CpG. PEG-P (TMC-DTC) -SP was prepared by PEG-P (DTC-TMC) (5.0- (14.5-2.0) kg/mol, w/ n =1.10) After terminal hydroxyl activation amidation with SP. The functionalization degree of the SP can be calculated to be about 95% by integrating the characteristic peaks (delta 3.64) of the PTMC (delta 4.24,2.05), the PDTC (delta 4.24,3.02) and the SP (delta 2.56-2.98) in the nuclear magnetic resonance hydrogen spectrogram with the PEG. The grain diameter of NanoCpG is small (50 nm), the distribution is narrow (PDI 0.10), the CpG loading efficiency is high, the stability is good, and the reproducibility is good.
The secondary lung cancer model of 4T1-luc was established by (1) inoculating 4T1-luc cells (3X 10 5/mouse) subcutaneously above the hind limbs of Balb/c mice (female, 6 weeks). (2) When the tumor volume is as large as 200-300 mm 3 (day 11 of inoculation), 90-95% of the tumor is surgically excised and the wound is sutured. (3) Tumor recurrences, when the volume of the tumor grows to about 100mm 3 (day 18 of inoculation), IVIS near infrared imaging scanning mice already have obvious lung metastasis, and the establishment of a secondary lung cancer mouse model is successful.
Tumor-bearing mice were divided into 6 groups (n=6) of PBS, free G/P, mG/P, mG/P+NanoCpG, ATN2-mG/P, ATN2-mG/P+ NanoCpG, this day being day 0. Free G/P, mG/P and ATN2-mG/P (HPG/ptx=10/1, HPG:15 mpk (25.8 μmol/kg), PTX:2.25 mpk (2.58 μmol/kg)) were injected caudally intravenously on days 0, 2,4, 6, 8, 10, day 1, 3, 5, and NanoCpG (CpG: 1.0 mpk). Mice body weight and tumor volume were monitored every 3 days. On day 15, 1 mouse was randomly dissected, the biological development of the mouse lung was observed with the IVIS imaging system, the distribution of lung metastasis nodules was observed with H & E staining, and the main organ sections of the mice were taken and the systemic toxicity of the combination group was studied with H & E staining. The remaining five mice were used to observe survival and to plot survival curves. During observation, death was judged when mice died, tumor volumes exceeded about 2000 mm 3, or body weight was reduced by more than 15%.
The problem of recurrence and metastasis after TNBC surgery is urgently to be solved, and the treatment of secondary lung cancer caused by the problem is of great concern. In addition, IVIS near infrared imaging showed that mice had significant metastasis to secondary lung cancer at a later stage (19 days later), indicating successful establishment of a TNBC postoperative recurrence/secondary lung cancer model (FIG. 10A). The therapeutic effect of mG/P and ATN2-mG/P on secondary lung cancer formed by recurrent metastasis after TNBC surgery was evaluated. As a result, it was found that the 4T1 tumor recurred 100% post-operatively, with the growth rate of the recurred tumor being faster than that of the primary tumor (fig. 10B), and the MST was reduced from 15 days to 12 days (fig. 10A) although the mouse body weight was not significantly reduced during the administration period (fig. 10C), which is consistent with the clinical manifestations of the recurred patient. ATN2-mG/P and mG/P showed significant inhibition of growth of 4T1 recurrent tumor, but the tumor growth curve of each mouse showed that tumor recurred and grew rapidly in the late treatment period, and MST of the mice was prolonged to only 27 and 24 days, respectively (FIGS. 10D, E), which was directly related to lung metastasis of the mice. The literature reports that 36.9% of recurrent TNBC patients develop lung metastasis with low five-year survival. Multiple large-area lung metastasis nodules were observed in both the lung bioluminescence pictures and H & E sections of PBS group mice on day 15, and metastasis nodules were also seen in the lungs of free G/P group mice, whereas the lung nodules were significantly fewer in the mG/P and ATN2-mG/P group mice (FIG. 11), indicating suppression of secondary lung cancer.
The efficacy of chemotherapy of ATN2-mG/P or mG/P in combination NanoCpG on TNBC postoperative recurrence and secondary lung cancer was studied (FIG. 10A). The results showed that the inhibition of recurrent tumor growth and lung metastasis by ATN2-mG/P or by means of intravenous NanoCpG (1 mpk) in combination with mG/P was significantly enhanced, while the recurrent tumor growth stopped in group mG/P+ NanoCpG and even the tumors in group ATN2-mG/P+ NanoCpG were reduced (FIG. 10B). The prior art considers CpG alone to have no therapeutic effect on 4T1 tumors. There was no significant change in body weight of each group of mice during treatment (fig. 10C). After the combination of mgp or ATN 2-mgp with NanoCpG, median survival of mice was significantly prolonged (×p), 40% and 60% of mice were completely cured, respectively, and no tumor recurrence was seen within 300 days of tumor-free survival (fig. 10d & e). Furthermore, treatment of both combination groups achieved inhibition of TNBC lung metastasis, and the ATN2-mG/P+ NanoCpG combination group even eliminated lung metastasis nodules (FIG. 11).
Example six combination therapies for ATN2-mG/P and NanoCpG stimulate BMDC maturation studies
BMDCs (1X 10 6/well) seeded in 12-well plates were supplemented with ATN2-mG/P (HPG/PTX: 10/1), mG/P (HPG/PTX: 20/1, 10/1 or 5/1), mHPG, mPTX or PBS, HPG: 1. Mu.g/mL (1.7. Mu.M), PTX: 0.3. Mu.g/mL (0.34. Mu.M), n=3. After incubation 24 h, cells were centrifuged, washed, incubated for 30min with FITC- αCD11c, APC- αCD80 and PE- αCD86, detected by flow cytometry, and the ratio of mBMDC (CD 11c +CD80+CD86+ mDC) was quantified using FlowJo_V10 software.
To investigate the effect of NanoCpG and its combination with ATN2-mG/P on BMDC maturation, nanoCpG, free G/P, mG/P, ATN2-mG/P, mG/P+NanoCpG, ATN2-mG/P+ NanoCpG (HPG/PTX=10/1) or PBS were added to BMDC and incubated for 24: 24h as described above with HPG 1 μg/mL (1.7 μM), PTX 0.15 μg/mL (0.17 μM), cpG 0.4 μg/mL), n=3.
To investigate the effect of drug on maturation of BMDC (1X 10 6/well) incubated with 4T1-luc cells (1X 10 5/well), BMDC and 4T1-luc cells were first cultured in the same manner as before in 1640 medium in 12-well plates, respectively. After culturing 24h, 4T1-luc cells were completely adherent, the medium was removed, and BMDC suspension was added thereto. ATN2-mG/P (HPG/PTX: 10/1), mG/P (HPG/PTX: 20/1, 10/1 or 5/1), mHPG, mPTX or PBS, HPG: 1. Mu.g/mL (1.7. Mu.M), PTX: 0.3. Mu.g/mL (0.34. Mu.M) were then added. After 24h co-incubations, the treatment and assay of the cells were as above (n=3).
CpG ODN is a toll-like receptor 9 (TLR 9) agonist and is widely used as an adjuvant in tumor immunotherapy in preclinical studies and clinical trials, often by intratumoral administration, and is not suitable for untouchable tumors and has potential for immunotoxicity. In addition, cpG is inefficient in cellular uptake and is susceptible to degradation. The maturation ability of NanoCpG to stimulate BMDC was studied using flow cytometry and showed that NanoCpG constituted a higher proportion of mature BMDC than PBS, whereas mgp or ATN 2-mgp in combination with NanoCpG further increased the maturation rate of BMDC to 77.0% and 85.6% (P) (fig. 12).
Example seven combination treatment of immune cell infiltration and cytokine secretion following 4T1-luc secondary lung cancer mice
Mice were divided into 5 groups （n = 4）：PBS、mG/P、ATN2-mG/P、mG/P+NanoCpG、ATN2-mG/P+NanoCpG（HPG/PTX = 10/1, HPG：15 mpk（25.8 μmol/kg）,PTX：2.25 mpk（2.58 μmol/kg）,CpG：1.0 mpk）., day 0, 2, 4, and NanoCpG, injected with either mG/P or ATN2-mG/P, at about 100 mm 3 (day 0) of the recurrent tumor volume of the postoperative recurrent metastatic 4T1-luc secondary lung cancer model. After the last injection 48 h, mouse plasma was collected and ELISA kits were used to determine the concentrations of pro-inflammatory factors (TNF-. Alpha., IFN-. Gamma.) and anti-inflammatory factors (IL-10). Mice were euthanized and organs were taken to study suppression and immunomodulation of secondary lung cancer. After weighing the mouse lung tissue, sections were stained with H & E and the lung metastasis node numbers were observed microscopically. Recurrent tumors were weighed and the recurrent Tumor Inhibition (TIR) was calculated. Spleen was weighed. Lymph node, spleen and tumor were ground, centrifuged to obtain single cell suspension, red blood cells were lysed with ACK, the tubes were split, stained with the corresponding antibodies, and flow cytometry examined the proportion of DC, T, treg, macrophages and MDSC in tumor, infiltration of T, MDSC in spleen and DC in lymph node.
Infiltration of immune cells and secretion of cytokines in tumors and major immune organs were analyzed (fig. 13A). The results showed that 48 h after the third injection NanoCpG, the quality of recurrent tumors in the four treatment groups were significantly lower than that in the PBS group (0.5 g), and that the ATN2-mG/P+ NanoCpG group had the lowest tumor, with only 0.05 g (P) (FIG. 13B), and that the lung tissue containing metastases had the same tendency to approach that of healthy mice (FIG. 13C). From the H & E staining analysis of the whole lung scan images, the number of lung metastasis nodules in the combination treatment group was significantly reduced compared to PBS group (about 8 nodules) (fig. 13D). Spleen as the primary immune organ, the spleen of PBS group mice was the heaviest, probably due to infiltration of immune cells not normally activated at the time of TNBC onset. Such splenomegaly was markedly avoided in ATN2-mG/P+ NanoCpG mice with spleen quality (0.18 g) similar to healthy mice, with activated immune cells circulating to lymph nodes, tumors, etc. (FIG. 13E).
Analysis of tumor-infiltrated immune cells demonstrated that the potent anti-TNBC post-operative recurrent metastasis of the ATN 2-mgp+ NanoCpG combination group was significantly more invasive than the other groups (fig. 14a, b) with both total DCs (CD 11c + DC) and mature mdcs (CD 11c +CD80+CD86+), which together promote recruitment and maturation of DCs through TLR4 and TLR9 pathways, respectively, promoting presentation of more antigen to T cells and recruitment of more T cells to the tumor site, resulting in a strongly durable tumor-specific T cell response, and that CD8 + cytotoxic T cells and CD4 + helper T cells were able to attack and kill tumor cells. Flow cytometry analysis results showed that the levels of infiltrated CD8 + T (fig. 14c, d) and CD4 + T (fig. 14e, f) in spleens and tumors of ATN 2-mgp+ NanoCpG mice were significantly higher than those of both single drug groups. In addition, infiltration of immunosuppressive CD4 + regulatory T cells (tregs) in PBS group TME was reduced after each formulation treatment (fig. 14G). The infiltration rates of immunosuppressive MDSCs in recurrent tumor and spleen were 40% and 12%, respectively, directly resulting in the intractability of postoperative recurrent TNBC. While each of the four formulations significantly reduced the infiltration of MDSCs in recurrent tumors and spleen (fig. 14h, i), with an average of less than 4% of MDSCs in the tumors of mice in the ATN 2-mgp+ NanoCpG combination group. The prior art considers that Gem does not affect the number of CD4 + T、CD8+ T, NK cells or macrophages.
The concentrations of pro-inflammatory cytokines IFN-gamma and TNF-alpha produced by the combined mG/P+ NanoCpG and ATN2-mG/P+ NanoCpG groups were found to be higher than those produced by the individual mG/P and ATN2-mG/P groups (& gtp ) using ELISA in mice plasma (FIGS. 14J, K). Secretion of IFN-gamma and TNF-alpha may improve the activity of immune cells NK, DC and CTL. Four treatment groups were able to significantly reduce IL-10 secretion (FIG. 14L), two of which reduced IL-10 concentrations below the ELISA kit limit of detection (8 pg/mL). The results prove that the ATN2-mG/P+ NanoCpG can promote favorable anti-tumor immune microenvironment, generate strong immune response, have excellent treatment effect on the lung cancer after TNBC operation, and cure 60 percent of mice.
The data of the present invention are expressed as mean.+ -. SD. Significant differences between groups were determined using GRAPHPAD PRISM 9.0.0 One-way ANOVA. The lifetime analysis uses log-rank comparisons in Kaplan-Meier technology. * p <0.05 represents a significant difference, p <0.01, < p <0.001, < p <0.0001 represents a highly significant difference.
Aiming at TNBC easy metastasis and development into secondary lung cancer, the invention reports that integrin alpha 5β1 targeted micelle (ATN 2-mG/P) co-delivering HPG and PTX is combined with NanoCpG, and can effectively heat TME and treat postoperative TNBC tumor with high efficiency, and prevent secondary lung cancer (figure 15 is a schematic diagram). In order to increase the drug loading of GEM, the invention selects hydrophobic phosphorylation modified GEM prodrug (HPG), and PTX and HPG can stimulate APC and reduce MDSCs respectively besides inducing ICD. Thus, ATN2-mG/P can not only target co-stimulatory ICDs, but also synergistically reverse immunosuppressive TMEs by PTX activation of DC cells and GEM elimination of MDSCs. In the post-operative 4T1 TNBC model, ATN2-mG/P+ NanoCpG elicits a strong anti-tumor immune response, completely inhibits tumor recurrence and lung metastasis, and 60% of mice tumors completely regress and survive for a long period of time. The combination therapy of HPG and PTX co-delivery with NanoCpG provides a unique strategy for not only "cold" tumors such as TNBC, but also for effective chemo-immunotherapy of secondary lung cancer.
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