A co-drug-loaded micelle and its synergistic drug system, preparation method and application

Abstract

The present invention discloses a co-drug loaded micelle and its synergistic drug system, preparation method and application, specifically a gemcitabine prodrug and paclitaxel co-loaded micelle, wherein the gemcitabine prodrug, paclitaxel and oligoethylene glycol form a drug solution; the drug solution, polymer solution and oligoethylene glycol mixed solution are added into a buffer solution to obtain the gemcitabine prodrug and paclitaxel co-loaded micelle, which can be used in highly efficient synergistic treatment of secondary lung cancer metastasized from triple-negative breast cancer with a TLR9 nano agonist such as nano CpG, wherein CpG induces immune cells to produce type I interferon by activating the TLR9 pathway, thereby inducing T h1 Immune response: CpG can be administered through the intravenous system, reducing its potential immunogenicity and systemic toxicity.

Description

Technical Field

[0001] The present invention belongs to pharmaceutical technology, and specifically relates to a gemcitabine prodrug and paclitaxel co-loaded micelle and synergistic system and application, which can cooperate with nano CpG to effectively treat secondary lung cancer metastasized from triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) with negative expression of estrogen receptor, progesterone receptor and HER2 accounts for 24% of new breast cancers and is the most difficult and deadly breast tumor subtype. TNBC has a high recurrence rate, and about 36.9% of recurrent TNBC patients have lung metastasis. Local surgery combined with radiotherapy, systemic chemotherapy, targeted therapy and immunotherapy are currently the main means of preventing and treating secondary tumors, but the lack of drugs that specifically target metastatic cells and the "cold" TME make the immunotherapy response rate of TNBC low. Even ICB has a disease control rate of only 23.8% in the treatment of PD-L1-positive metastatic TNBC patients, a median survival of 18 months, and no significant improvement in overall survival compared with chemotherapy. Therefore, "heating" the "cold" TME has become a new strategy for the treatment of TNBC. In recent years, people have developed a variety of strategies to "heat" the TME, such as releasing tumor antigens through ICD of tumor cells induced by chemotherapy, radiotherapy and photodynamic therapy, through the activation and transport of immune cells, through the elimination or repolarization of immunosuppressive cells, and through the reconstruction of ECM barriers. The FDA has approved the combination therapy of PD-L1 inhibitors and albumin-paclitaxel (Nab-PTX) for the treatment of PD-L1-positive metastatic TNBC, but the disease response rate is only 25%, while the efficacy is minimal for patients with PD-L1-negative TNBC. Summary of the invention

[0003] The present invention discloses a co-loaded micelle and synergistic system of gemcitabine (Gem) prodrug (HPG) and paclitaxel (PTX), which can be used in synergistic combination with nano-CpG to effectively 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 T h1 Immune response: CpG can be administered through the intravenous system, reducing its potential immunogenicity and systemic toxicity.

[0004] The present invention adopts the following technical solution:

[0005] The invention discloses a co-drug-loaded micelle, which is a gemcitabine prodrug and paclitaxel co-loaded micelle, and comprises a polymer micelle, gemcitabine prodrug and paclitaxel.

[0006] A synergistic drug system comprises the above-mentioned gemcitabine prodrug and paclitaxel co-loaded micelles and nano-CpG. Preferably, the nano-CpG is a polymer vesicle loaded with CpG.

[0007] In the present invention, in the polymer micelle, the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC), or the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC), a targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC); in the polymer vesicle, the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC)-cationic segment. DTC is an existing cyclic carbonate monomer containing a disulfide five-membered ring functional group, which is polymerized to form a PDTC segment. The hydrophobic monomer includes a cyclic ester monomer and a cyclic carbonate monomer. Preferably, the cyclic carbonate monomer is other cyclic carbonate monomers, such as other cyclic carbonate monomers including trimethylene cyclocarbonate (TMC), and the cyclic ester monomer includes caprolactone (ε-CL), lactide (LA) or glycolide (GA); the hydrophobic monomer is polymerized to form other hydrophobic segments, such as PTMC segments and PCL segments. The PDTC segment and other hydrophobic segments constitute the hydrophobic segment of the polymer. Preferably, the hydrophilic segment is PEG; the cationic segment is a PEI segment or a spermine segment, and the targeting molecule is a polypeptide, such as ATN1 (Ac-PHSCNK-NH2), ATN2 (Ac-PhScNK-NH2), cRGD (c(RGDfC)), etc. 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.

[0008] In the present invention, in the polymer micelle, the molecular weight of the hydrophilic segment is 1 to 7.5 kg / mol, and the molecular weight of the hydrophobic segment is 1.5 to 7.5 kg / mol; preferably, the molecular weight of the hydrophilic segment is 1.5 to 5 kg / mol, and the molecular weight of the hydrophobic segment is 1.5 to 5 kg / mol. In the polymer vesicle, the molecular weight of the hydrophilic segment is 3 to 10 kg / mol, and the molecular weight of the hydrophobic segment is 10 to 20 kg / mol. The molecular weight of the cationic segment is 40% or less of the molecular weight of the hydrophilic segment, and the preferred molecular weight is 0.1 to 2 kg / mol.

[0009] In the present invention, in the polymer micelle, when the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC) and a targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC), the mass content of the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) is 0 to 20% excluding 0, preferably 1 to 15%, and more preferably 2 to 10%.

[0010] In the present invention, the molar ratio of gemcitabine prodrug to paclitaxel is (2-20):1, preferably (5-20):1, and more preferably (5-15):1.

[0011] In the present invention, the preparation method of gemcitabine prodrug and paclitaxel co-loaded micelles is to add a mixed solution of a drug solution, a polymer solution and oligoethylene glycol to a buffer solution to obtain gemcitabine prodrug and paclitaxel co-loaded micelles. Specifically, the mixed solution is added to a buffer solution, blown and then allowed to stand to obtain gemcitabine prodrug and paclitaxel co-loaded micelles. The drug solution consists of gemcitabine prodrug, paclitaxel, and oligoethylene glycol; the concentration of the drug solution is 5 to 100 mg / mL, preferably 10 to 70 mg / mL, and further preferably 20 to 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 to 500 mg / mL, preferably, the concentration of the hydrophilic segment-P (hydrophobic monomer-DTC) solution is 50 to 500 mg / mL, preferably 100 to 400 mg / mL; the concentration of the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) solution is 10 to 200 mg / mL.

[0012] In the present invention, the solvent in the drug solution and the polymer solution is oligoethylene glycol; the molecular weight of the oligoethylene glycol is 200-800, such as PEG200, PEG350, PEG400, PEG600, PEG800, etc. Preferably, the volume percentage of the oligoethylene glycol in the solution obtained by adding the mixed solution to the buffer solution is 2-10%, preferably 2.5-8%.

[0013] The invention discloses the application of gemcitabine prodrug and paclitaxel co-loaded micelle and synergistic drug system in preparing anti-tumor drugs, especially in preparing drugs for treating triple-negative breast cancer, and especially in preparing drugs for treating secondary lung cancer formed by metastasis of triple-negative breast cancer.

[0014] In the present invention, in order to heat up the immune microenvironment of the "cold" tumor TNBC and improve the immunotherapy effect of TNBC subcutaneous tumors and postoperative recurrent / secondary lung cancer, a disulfide cross-linked micelle ATN2-mG / P that targets α5β1 integrin and co-loads HPG and PTX is designed. The micelle can induce tumor cells to selectively produce ICD, effectively eliminate MDSC and stimulate DC maturation, resulting in a "warming up" of the immune microenvironment, which significantly improves the inhibition of 4T1 subcutaneous tumors and postoperative recurrence and metastasis to form secondary lung cancer. ATN2-mG / P can most effectively inhibit the recurrence and lung metastasis of 4T1 tumors when HPG / PTX = 10 / 1. When used in combination with NanoCpG, it can significantly inhibit the postoperative recurrence and secondary lung cancer of 4T1, without lung metastatic nodules and systemic toxic side effects, and significantly improves the survival of mice, with 60% of mice completely cured. This chemoimmunotherapy successfully stimulated the massive recruitment and activation of DCs, providing CD8 + and CD4+ T cells provide antigens, increase the secretion of IFN-γ and TNF-α, significantly reduce the infiltration of immunosuppressive MDSC and Treg, and improve the comprehensive treatment effect of postoperative TNBC. Both ATN2-mG / P and NanoCpG are simple to prepare, safe, and can effectively enhance T cell responses. Therefore, their combined therapy is expected to provide ideas for the treatment of "cold" tumors such as TNBC and the prevention and treatment of postoperative recurrent / secondary lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Characterization of mG / P: (A) particle size and particle size distribution of mHPG, mPTX, mG / P, ATN2-mG / P (HPG / PTX = 10 / 1) and (B) mG / P (HPG / PTX = 5 / 1, 10 / 1, 20 / 1); (C) DLS determination of particle size distribution of mHPG and HPG in PEG350 solution; (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; (F) UV absorption spectra of PEG350 solutions of mHPG, mPTX, mG / P and PEG-P (CL-DTC).

[0016] Figure 2 In vitro drug release of micellar drugs: (A) particle size of mG / P in the presence or absence of 10 mM GSH; (B) drug release of mG / P and (C) single micellar mHPG in the presence or absence of 10 mM GSH medium (pH 7.4) at 37 °C (n = 3); (D) drug release of mG / P at pH 5.0 or pH 7.4 without GSH (n = 3). (E) intact HPG content of free HPG, mHPG, and mG / P after incubation with CDA for 4 h in the presence or absence of 10% serum (n = 3). Each formulation was incubated with PB or PB solution containing THU (tetrahydrouridine, CDA inhibitor, 344 μM) as a control.

[0017] Figure 3 For cytotoxicity studies (n = 6): cell viability after incubation of (A) mG / P (HPG / PTX = 2 / 1, 5 / 1, 10 / 1, 20 / 1), (B) mPTX, and (C) ATN2-mG / P with 4T1-luc cells for 48 h.

[0018] Figure 44T1-luc cells were incubated with mG / P (HPG / PTX = 20 / 1, 10 / 1, 5 / 1), mHPG, mPTX, and ATN2-mG / P (HPG / PTX = 10 / 1) for 48 h to induce (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).

[0019] Figure 5 ICD of 4T1-luc cells induced by mG / P and ATN2-mG / P (HPG: 1 μg / mL (1.7 μM), PTX: 0.3 μg / mL (0.34 μM), 24 h, n = 3): (A, B) expression of cell surface CRT and (C) ATP concentration after treatment with mG / P (HPG / PTX = 20 / 1, 10 / 1, 5 / 1); (D, E) ATN2-mG / P induces cell surface CRT expression and (F) ATP secretion.

[0020] Figure 6 The maturation of BMDCs (CD80 + CD86 + mDC), free G / P, mHPG, mPTX, and PBS as controls (HPG: 1 μg / mL (1.7 μM), PTX: 0.3 μg / mL (0.34 μM), 24 h, n = 3).

[0021] Figure 7 Maturation of BMDCs co-incubated with 4T1 cells for mG / P and ATN2-mG / P stimulation: (A) Experimental design; (B) Representative flow cytometric histograms and semiquantitative analysis of mature BMDCs after mG / P (HPG / PTX = 20 / 1, 10 / 1, 5 / 1) and (C) ATN2-mG / P treatment (HPG: 1 μg / mL (1.7 μM), PTX: 0.3 μg / mL (0.34 μM), 24 h, n = 3).

[0022] Figure 8Mice bearing 4T1 subcutaneous tumors were treated with mG / P (n = 6). Mice were intravenously injected with mHPG, mG / P (HPG / PTX = 20 / 1, 10 / 1) (HPG: 15 mpk, 25.8 μmol / kg), and mPTX (PTX: 2.25 mpk, 2.58 μmol / kg) on ​​days 0, 2, 4, 6, 8, and 10: (A) treatment schedule, (B) tumor volume changes, (C) body weight changes, and (D) survival curves of mice.

[0023] Fig. 9 For ATN2-mG / P treatment of 4T1 subcutaneous tumor-bearing mice, ATN2-mHPG, mG / P, ATN2-mG / P (HPG / PTX = 10 / 1) (HPG: 10 mpk, 17.2 μmol / kg), and ATN2-mPTX (PTX: 1.5 mpk, 1.72 μmol / kg) were intravenously injected on days 0, 2, 4, 6, 8, and 10: (A) dosing schedule; (B) tumor volume and (C) body weight of mice (n = 6); the proportions of (D) mDCs and (E) MDSCs infiltrating the tumors on day 14 (n = 3).

[0024] Fig.10 ATN2-mG / P or mG / P combined with NanoCpG in 4T1-luc postoperative recurrent / secondary lung cancer mice (HPG / PTX = 10 / 1, HPG: 15 mpk, CpG: 1 mpk): (A) treatment regimen; (B) tumor volume and (C) body weight of mice (n = 6); (D) Kaplan-Meier survival curve of mice and (E) tumor growth curve of individual mice (n = 5).

[0025] Fig.11 Bioluminescence imaging and H&E staining images of lung tissues of treated mice on day 15. Scale bar: 1000 μm.

[0026] Fig.12 Representative flow cytometry plots of BMDC maturation stimulated by ATN2-mG / P or mG / P combined with NanoCpG and mDC (CD11c + CD80 + CD86 + ) ratio (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).

[0027] Fig.13Analysis of tumor microenvironment in mice with recurrent / secondary lung cancer after chemoimmunotherapy for TNBC (HPG / PTX = 10 / 1, HPG: 15 mpk, CpG: 1 mpk, n = 4): (A) treatment regimen; (B) tumor mass of mice; (C) lung weight; (D) number of lung metastatic nodules and (E) spleen mass.

[0028] Fig.14 Analysis of the tumor microenvironment of mice with recurrent / secondary lung cancer after TNBC surgery treated with chemoimmunotherapy (n = 4): (A) CD11c infiltrating in the tumor + DC and (B) CD11c + CD80 + CD86 + Proportion of mDCs; (C) tumor and (D) spleen infiltrating CD8 + The proportion of T; (E) tumor and (F) spleen infiltrating CD4 + The proportion of T cells and (G) Treg infiltrating in tumors; the proportion of MDSC infiltrating in (H) tumors and (I) spleens; the concentrations of (J) IFN-γ, (K) TNF-α, and (L) IL-10 in serum.

[0029] Fig.15 (A) Preparation of ATN2-mG / P, (B) its combination with NanoCpG to heat the tumor immune microenvironment, effectively inhibit the progression, recurrence and lung metastasis of 4T1 tumors, and (C) the molecular structure of the polymer. DETAILED DESCRIPTION

[0030] Paclitaxel (PTX, >98%, Shanghai Jinhe Biopharmaceutical Co., Ltd.) and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, ThermoFisher Scientific) were purchased and used directly. 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 is 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 previously reported method. CpG ODN 2018 (CpG) was provided by Jima Gene; Ac-PhScNK-NH2 (ATN2) with a purity greater than 98% was purchased from Shanghai Qiangyao Biotechnology. 4T1-luc cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and BMDCs were routinely extracted from healthy Balb / c mice. Balb / c (6 weeks, female, 20-22 g) mice were purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd. All animal experiments were approved by the Animal Care and Use Committee of Soochow University, and all protocols were in accordance with the Guide for the Care and Use of Laboratory Animals.

[0031] The drug loading capacity and drug loading efficiency of HPG and PTX were determined by HPLC under the following test conditions: acetonitrile / water (v / v) = 50 / 50, 1 mL / min, UV 220 nm. The drug loading capacity (DLC) and encapsulation efficiency (DLE) of CpG were determined by NanoDrop (NanoDrop 2000, Thermo).

[0032] Example 1 Preparation and Characterization of ATN2-mG / P

[0033] The present invention designs integrin-targeted micelles ATN2-mG / P that co-deliver HPG and PTX in a specific ratio. Both PTX and HPG can stimulate the ICD of tumor cells, and the activation of APCs by PTX and the elimination of MDSCs by HPG can further synergistically reverse the immunosuppressive TME. In addition, targeted delivery of the two drugs to tumor cells can reduce their systemic toxicity.

[0034] PEG-P(CL-DTC) was synthesized and characterized according to the existing method. 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. The chemical structure of the polymer can be found in Fig.15 C.

[0035] HPG and PTX were dissolved in PEG350 (25 mg / mL) and mixed into drug solutions at a molar ratio of 20 / 1, 10 / 1, 5 / 1 or 2 / 1 for later use; PEG-P(CL-DTC) (200 mg / mL) and ATN2-PEG-P(CL-DTC) (20 mg / mL) PEG350 stock solutions were prepared for later use. wt. For example, 4 μL HPG and 0.6 μL PTX drug solution were mixed with 5 μL PEG-P(CL-DTC) PEG350 solution and PEG350 to form a total volume of 50 μL solution, added to 950 μL PB (pH 7.4, 10 mM), pipetted 5 times, and placed at room temperature for 12 h to obtain mG / P with a PEG350 volume content of 5%. A similar method was used to prepare HPG / PTX co-loaded micelles with a concentration of 1 mg / mL, a theoretical HPG drug loading of 9.1 wt.%, and a PEG 350 volume content of 2% or 10% at a HPG / PTX ratio of 10 / 1. ATN2-mG / P was prepared similarly, and a mixed solution containing 4.75 μL PEG-P(CL-DTC), 2.5 μL ATN2-PEG-P(CL-DTC), HPG / PTX solution, and PEG350 in a total volume of 50 μL was added to 950 μL PB.

[0036] DLS was used to monitor the particle size and size distribution of mG / P and ATN2-mG / P, the storage stability of mG / P before and after dialysis, and the particle size and size change of micelles incubated with 10% serum or 10 mM GSH. The cross-linking of micelles with a polymer concentration of 1 mg / mL was determined by UV spectrophotometry. The DLC and DLE of HPG and PTX in mG / P and ATN2-mG / P were determined by HPLC. PB (pH 7.4, 10 mM) and acetic acid / sodium acetate buffer solution (pH 5.0, 5 mM) containing 10 mM GSH were prepared to simulate the reducing conditions of the tumor microenvironment and the weakly acidic conditions of tumor cell lysosomes, respectively. The formula of acetic acid / sodium acetate buffer solution was: 285.7 μL acetic acid and 262.43 mg sodium acetate dissolved in 1 L secondary water. Next, 1 mL of mG / P was placed overnight and transferred to a release bag (MWCO 14 kDa), and dialyzed in 25 mL tubes of two buffers, respectively, which were placed in a shaker at 37 °C and 200 rpm. At the set time point, 7 mL was taken and the same volume of fresh buffer was added to the centrifuge tube. The obtained release solution was freeze-dried and dissolved in 500 μL of a mixed solution of methanol and acetonitrile (1 / 1, v / v), filtered, and tested for the cumulative release of the two drugs by HPLC (n = 3).

[0037] ATN2-mG / P was self-assembled from 5% ATN2-PEG-P(CL-DTC), 95% PEG-P(CL-DTC) and PEG350 solutions with different HPG / PTX ratios (2 / 1, 5 / 1, 10 / 1 or 20 / 1). Single-loaded micelles ATN2-mHPG, ATN2-mPTX and non-targeted micelles mG / P, mHPG, and mPTX were prepared in the same way. All micelles obtained had a particle size of 19.8-23.5 nm and a narrow particle size distribution (PDI = 0.08-0.17) ( Figure 1 A, B). The drug loading efficiencies of HPG and PTX in ATN2-mG / P and mG / P were 94.0-100% and 86.7-97.0%, respectively. In contrast, the drug loading efficiencies of single HPG and PTX micelles were lower (Table 1); compared with the small particle size and clear solution of ATN2-mG / P, the mixture of HPG, PTX and PEG350 at the same concentration showed a wide particle size distribution, turbidity and PTX precipitation in water without copolymer ( Figure 1 C). ATN2-mG / P and mG / P can maintain colloidal stability for at least 8 days at room temperature, with clarity, unchanged particle size, and no drug precipitation ( Figure 1 D) and remained stable in PB buffer containing 10% FBS ( Figure 1E). The co-loaded micelles were dialyzed for 2 h in a dialysis bag with a molecular weight of 8-14 kDa and 10 volumes of PB solution (pH 7.4, 10 mM) (the buffer was changed every hour). After the PEG350 was removed by dialysis, the micelles could only be stored at room temperature for less than 5 days. In addition, the micelle particle size was affected by the volume content of PEG 350. The particle size of the HPG / PTX co-loaded micelles with a PEG 350 content of 10% increased to 27.5 nm, and the particle size distribution increased to above 0.2. These results demonstrate that PEG350 plays an important role in micelle assembly and stability. The superior stability of the micelles is also attributed to the disulfide cross-links in the ATN2-mG / P micelle core. The UV absorbance of the micelles was significantly reduced compared with that of the polymer solution ( Figure 1 F). The stability test of the micelles above takes HPG / PTX 10 / 1 as an example. When HPG / PTX is 5 / 1 and 20 / 1, the micelle stability is similar to that of 10 / 1. However, the particle size of the micelle solution with HPG / PTX of 2 / 1 is stable within 3 days at room temperature, and PTX precipitation will gradually appear after 4 days.

[0038] 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 were also used to prepare micelles loaded with HPG only. n : 5.0-(4.0-2.0) kg / mol, 50 mg / mL) and HPG (25 mg / mL) PEG350 mother solution for use, with the concentration of 1 mg / mL and the HPG theoretical drug loading of 9.1 wt. Taking the micellar mHPG with a molecular weight of 2.0-(1.0-1.2) and 2.0-(1.0-1.6) kg / mol as an example, 4 μL HPG, 20 μL PEG-P(CL-DTC) PEG350 solution and PEG350 were mixed into a total volume of 50 μL solution, added to 950 μL PB (pH 7.4, 10mM), and pipetted 5 times to obtain micelles with a particle size of 44.1 nm (PDI: 0.24). The particle sizes of mHPG prepared by polymers with molecular weights of 2.0-(1.0-1.2) and 2.0-(1.0-1.6) kg / mol were 36.4 and 32.5 nm, respectively. The particle size and particle size distribution of the prepared micelles became larger, and the stability became worse.

[0039]

[0040] In vitro, when the intracellular reducing environment was simulated at pH 7.4 and glutathione (GSH, 10 mM) was added, the particle size of mG / P increased rapidly and dramatically ( Figure 2 A), more than 95% of HPG and PTX are rapidly excreted from mG / P, while in the absence of GSH, HPG and PTX leakage is minimal ( Figure 2 B). However, the drug release of single HPG micelles mHPG in the absence of GSH exceeded 40% ( Figure 2 C); In addition, the drug in mG / P is rarely released under weak acidity, indicating that the micelles have the ability to stably escape from the endosomes and release the drug into the cytoplasm ( Figure 2 D).

[0041] Example 2 Cytotoxicity, apoptosis and cell cycle arrest experiments of ATN2-mG / P

[0042] 4T1-luc was seeded in 96-well plates (1×10 3 / well) for 24 h, then add 20 μL mG / P (HPG / PTX: 20 / 1, 10 / 1, 5 / 1 or 2 / 1), mHPG or mPTX, where the HPG concentration is 0.0017-68.8 μM and the PTX concentration is 0.005-12.8 μM. After incubation for 48 h, add 10 μL MTT solution (5 mg / mL) and incubate for 4 h, discard the supernatant, and add 150 μL DMSO to dissolve the living cells and the purple formazan crystals produced by MTT. The absorption of the cells at 570 nm was measured using a multifunctional microplate reader, with the medium wells with MTT added as the zero point and the wells with cells treated with PBS with MTT added as 100%. The cell viability (cell viability %) was the ratio of the absorbance value of the experimental group to the absorbance of the PBS group.

[0043] The combination index (CI) was calculated using the following formula to evaluate the synergistic effect of HPG and PTX:

[0044]

[0045] Where a and b represent the IC of each drug in mG / P respectively. 50 Values, A and B represent the IC values ​​of each drug in mHPG and mPTX, respectively. 50 The obtained CI>1 indicates that the two drugs have an antagonistic effect on each other, CI=1 is an additive effect, and CI<1 is a synergistic effect. The smaller the CI value, the stronger the synergistic effect between the drugs.

[0046] In order to study the targeting effect of ATN2-mG / P on 4T1 cells, 4T1 cells (1×10 3 / well) were incubated with ATN2-mG / P and mG / P (HPG / PTX = 10 / 1, HPG concentration: 0.0017-68.8 μM) for 4 h, and then incubated with drug-free fresh medium for 44 h. Subsequent sample processing and data analysis methods were as described above.

[0047] In the cell apoptosis assay, 4T1-luc cells (1×10 5 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 the cells. After 48 h of co-culture, routine cell treatment, staining, flow cytometry and FlowJo_V10 analysis were performed.

[0048] To investigate the ability of HPG and PTX to arrest 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 μg / mL (0.17 μM), PTX: 0.03 μg / mL (0.034 μM), n = 3) were added to 4T1-luc cells. After 48 h of incubation, the cells were trypsinized, washed with PBS, fixed with 95% ethanol overnight, stained with PI, and the proportion of different cell cycle phases was determined by flow cytometry and quantified by Modfit.

[0049] IC values ​​for mPTX (1.4 μM) and mHPG (3.8 μM) 50 Compared with the values, the IC values ​​of HPG and PTX at the HPG / PTX molar ratio of 2 / 1, 5 / 1, 10 / 1, and 20 / 1 were 50 The values ​​were significantly reduced, namely 0.3 / 0.14, 1.8 / 0.16, 0.4 / 0.074 and 2.3 / 0.10 ( Figure 3 A, B). The CI numbers of HPG and PTX were both less than 1 (Table 2), indicating that the two had a strong synergistic effect, and the CI of mG / P was the lowest when HPG / PTX = 10 / 1 or 2 / 1, indicating that low-dose PTX could also produce the same synergistic effect with HPG as when the PTX dose was higher.

[0050]

[0051] The cytotoxicity of ATN2-mG / P micelles containing 5% ATN2 was studied. When HPG / PTX = 10 / 1, the IC50 The value is 2.3 times lower than that of micelle mG / P ( Figure 3 C). Cell apoptosis experimental analysis showed that the proportion of cell apoptosis caused by mG / P at HPG / PTX = 10 / 1 was higher than that of other ratios (*p) and the drug loading group alone (**p). ATN2-mG / P further increased the cell apoptosis rate to 81.7% (*p), which was consistent with the MTT results ( Figure 4 A). Cell cycle studies have shown that mG / P can also seriously affect the cycle of 4T1 cells at very low drug concentrations (HPG: 0.1 μg / mL; PTX: 0.03 μg / mL), showing significant S phase and G2 / M phase arrest, which is in sharp contrast to the slight cell arrest of single micelles mHPG and mPTX at the same drug concentration, indicating that the two drugs in mG / P have a strong synergistic effect ( Figure 4 B). In addition to the significant enhancement of cell cycle arrest, the improvement of intracellular stability of GEM by reducing the expression of cytidine deaminase (CDA) by low-dose PTX is also one of the reasons for the strong synergistic effect of the two. Both prodrug modification (HPG) and micelle loading (mHPG) can enhance the resistance of Gem to enzymatic degradation, and the proportion of HPG drug that can maintain the intact structure after co-loading micelles mG / P with CDA and 10% FBS for 4 h is 7 times that of micelles mHPG loaded with HPG only ( Figure 2 E), further indicating that PTX can produce a strong synergistic effect with HPG by protecting it from degradation.

[0052] The ATN2-mG / P micelles used in subsequent studies all had an ATN2 content of 5% and HPG / PTX = 10 / 1.

[0053] Example 3 ATN2-mG / P induces immunogenic death (ICD) of tumor cells

[0054] 4T1-luc cells were plated in 12-well plates (1 × 10 5 After culturing for 24 h in 4% paraformaldehyde (P40 / 45 μg / mL), 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 24 h of incubation, the culture medium was collected and the ATP concentration in the culture medium was determined using an enhanced ATP detection kit; the cells were digested and stained, detected by flow cytometry, and the expression of CRT on the cell surface was analyzed using FlowJo_V10.

[0055] PTX and Gem can provide tumor antigens by inducing ICD of tumor cells and regulate the immune microenvironment of "cold" tumors by stimulating APCs to promote the cancer immune cycle. The two 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 was used to analyze the two main markers of ICD of 4T1-luc cells after treatment with each preparation: the expression of surface CRT and the secretion of ATP. The results showed that single-loaded micelles mPTX and mHPG had only a slight effect on CRT expression and ATP secretion, while the CRT and ATP induced by mG / P at HPG / PTX = 10 / 1 were significantly higher than those of mHPG and mPTX, and also higher than those of mG / P at HPG / PTX = 5 / 1, 20 / 1 ( Figure 5 AC). This indicates that the co-loading of HPG and PTX in micelles and their ratio are crucial. It is worth noting that ATN2-mG / P can further stimulate 4T1 cells to express more CRT (***p) and secrete more ATP (*p) compared with mG / P ( Figure 5 DF).

[0056] Low-dose PTX can regulate the proliferation and polarization of APCs through the TLR4 pathway to promote the maturation and proliferation of DCs. Gem can eliminate MDSCs and stimulate DC maturation. The experimental results show that both mPTX and mHPG can significantly stimulate BMDC maturation at low concentrations (CD80 + CD86 + mDC)(****p). It is worth mentioning that compared with mHPG, mG / P and ATN2-mG / P further promoted DC maturation by 58.6% and 59.6% (***p), ​​which was significantly higher than the mixture of free PTX and HPG (free G / P, ****p)( Figure 6 ), indicating that HPG and PTX in mG / P and ATN2-mG / P have a synergistic effect in stimulating DC maturation.

[0057] ICD-induced tumor antigens can release "eat me" signals to promote DC maturation and present antigens to T cells, leading to tumor-specific T cell responses. In order to simulate the tumor microenvironment, 4T1-luc tumor cells were co-cultured with BMDCs to study the ability of mG / P and ATN2-mG / P to stimulate BMDC maturation ( Figure 7A). The results showed that the proportion of mature DCs treated with mHPG and mPTX was significantly lower than that without 4T1 cells co-culture, indicating that they were preferentially internalized by 4T1 cells rather than DCs. mG / P (HPG / PTX = 10 / 1) had the highest proportion of DC maturation stimulation among other non-targeted micelles ( Figure 7 B); while ATN2-mG / P can further increase the proportion of DC maturation induced by the drug (40.3%, ***p) ( Figure 7 C).

[0058] Example 4 Study on the therapeutic efficacy of mG / P and ATN2-mG / P micelle drugs on mice bearing 4T1-luc subcutaneous tumors

[0059] The anti-tumor effect of 4T1-luc TNBC model was studied in mice. 4T1-luc cells (3×10 5 After 7 days, the average tumor volume grew to about 50 mm 3 Mice were randomly divided into 6 groups (n = 6), which was recorded as day 0 of the experiment. mG / P (HPG / PTX = 20 / 1 or 10 / 1, HPG: 15 mpk), mHPG (HPG: 15 mpk, 25.8 μmol / kg), and mPTX (PTX: 2.25 mpk, 2.58 μmol / kg) were injected intravenously every 2 days for a total of 6 times ( Figure 8 A) Monitor tumor volume and body weight when tumor volume exceeds 2000 mm 3 , weight loss of more than 15%, no response or no food intake were considered as death of mice. The results showed that the treatment of mG / P and mHPG effectively delayed tumor progression (****p)( Figure 8 B). In contrast, mPTX had no significant inhibitory effect ( Figure 8 B) Previous studies in 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). mG / P (HPG / PTX 10 / 1) had the best tumor inhibition effect, significantly better than mG / P (HPG / PTX 20 / 1) and mHPG (**p) ( Figure 8 B). Except for a slight decrease in the body weight of mice caused by mHPG during the administration period, the body weight of mice in other groups did not change much ( Figure 8 C). The results of mouse survival monitoring showed that the median survival time (MST) of mice in the mG / P (HPG / PTX = 10 / 1) group was significantly prolonged to 28.5 days compared with mHPG (*p) and mPTX (***p), ​​indicating the benefits of co-loading micelles and the synergy of the two drugs in tumor inhibition and survival extension in tumor-bearing mice.

[0060] To study the inhibitory effect of ATN2-mG / P on TNBC tumors, six injections of ATN2-mPTX, ATN2-mHPG, mG / P, and ATN2-mG / P (HPG / PTX = 10 / 1, HPG: 10 mpk (17.2 μmol / kg), PTX: 1.5 mpk (1.72 μmol / kg)) were injected into mice bearing 4T1 subcutaneous tumors via the tail vein ( Fig. 9 A). Four days after the last injection (day 14), three mice were randomly selected from each group for euthanasia, and the tumor tissue was extracted. The single cell suspension was obtained after grinding, centrifugation, and ACK lysis of red blood cells. FITC-αCD11c, APC-αCD80, PE-αCD86, FITC-αCD11b, and PE / Cy7-αGr-1 were then added and incubated at 4°C for 30 min. The mDC (CD11c + CD80 + CD86 + ) and MDSC (CD11b + Gr-1 + ) of the infiltration amount.

[0061] Fig. 9 B shows that the inhibitory effect of ATN2-mG / P on tumors was significantly better than that of mG / P and ATN2-mHPG (*p), while ATN2-mPTX had almost no inhibitory effect on 4T1 tumors. Treatment with all preparations did not cause weight loss in mice ( Fig. 9 C). As we all know, TNBC is a highly immunosuppressive tumor, with about 40% MDSC infiltration, which leads to a low response rate of TNBC patients to immunotherapy. In order to evaluate the effect of ATN2-mG / P treatment on the tumor immune microenvironment, three mice were randomly selected from each group on the 14th day and sacrificed to analyze the infiltration of MDSC and DC in their tumor tissues. Flow cytometry results showed that compared with the PBS group, the proportion of mature DC in the tumor of each micelle drug group increased by 2.2-2.5 times ( Fig. 9 D). In addition, the proportion of MDSC in the tumors of the PBS group was as high as 42%, confirming the high immunosuppression of TNBC tumors. The MDSC in the ATN2-mHPG, mG / P and ATN2-mG / P groups was significantly reduced (*p)( Fig. 9 E).

[0062] Example 5 Study on the chemoimmunotherapy of ATN2-mG / P and NanoCpG for the treatment of 4T1-luc secondary lung cancer

[0063] NanoCpG was obtained by self-assembly of CpG and terminally modified spermine copolymer PEG-P(TMC-DTC)-SP in aqueous solution. 100 μL of DMF solution (10 mg / mL) of PEG-P(TMC-DTC)-SP was added to 900 μL of HEPES buffer solution (5 mM, pH 6.8) containing CpG (100 μg), stirred for 10 min, dialyzed twice in HEPES, once in PB / HEPES (v / v, 1 / 1), and finally dialyzed twice in PB to obtain NanoCpG solution. DLS was used to determine its particle size and particle size distribution, and Nanodrop was used to determine the drug loading of CpG. Vesicle NanoCpG was self-assembled by terminally modified SP block copolymer PEG-P(TMC-DTC)-SP in 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) was obtained by amide reaction with SP after activation of the terminal hydroxyl group. δ 4.24, 2.05), PDTC ( δ 4.24, 3.02), SP ( δ 2.56-2.98) and the characteristic peak of PEG ( δ 3.64) integral ratio, it can be calculated that the functionalization degree of SP is about 95%. The prepared NanoCpG has a small particle size (50 nm), narrow distribution (PDI 0.10), high CpG loading efficiency, good stability and good reproducibility.

[0064] The method for establishing the 4T1-luc secondary lung cancer model is as follows: (1) 4T1-luc cells (3×10 5 (2) When the tumor volume grew to 200-300 mm, the 3 At 11 days after inoculation, 90-95% of the tumor was surgically removed and the wound was sutured. (3) Tumor recurrence, when its volume grew to about 100 mm 3 (18th day after inoculation), IVIS near-infrared imaging scans showed obvious lung metastasis in mice, and the secondary lung cancer mouse model was successfully established.

[0065] Tumor-bearing mice were divided into 6 groups (n = 6): PBS, free G / P, mG / P, mG / P+NanoCpG, ATN2-mG / P, ATN2-mG / P+NanoCpG, and this day was recorded as 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 into the tail vein on days 0, 2, 4, 6, 8, and 10, and NanoCpG (CpG: 1.0 mpk) was injected into the tail vein on days 1, 3, and 5. The body weight and tumor volume of mice were monitored every 3 days. On the 15th day, one mouse was randomly selected from each group for dissection. The biological development of the mouse lungs was observed using the IVIS imaging system, and the distribution of lung metastatic nodules was observed using H&E staining. The main organ sections of the mice were sliced ​​and H&E stained to study the systemic toxicity of the combined group. The remaining five mice were used to observe the survival period and draw the survival curve. During the observation period, when the mouse died or the tumor volume exceeded about 2000 mm 3 Or when the body weight decreases by more than 15%, it is considered death.

[0066] The recurrence and metastasis of TNBC after surgery need to be solved urgently, and the treatment of secondary lung cancer caused by it has attracted much attention. After 90-95% of the tumor was surgically removed on the 11th day after inoculation of 4T1 subcutaneous tumors, the tumor quickly recurred in situ; in addition, IVIS near-infrared imaging showed that mice had obvious metastasis and formed secondary lung cancer in the later stage (after 19 days), indicating that the TNBC postoperative recurrence / secondary lung cancer model was successfully established ( Fig.10 A). The therapeutic effects of mG / P and ATN2-mG / P on secondary lung cancer formed by recurrence and metastasis of TNBC after surgery were evaluated. The results showed that 100% of 4T1 tumors recurred after surgery, and the growth rate of the recurrent tumor was faster than that of the primary tumor ( Fig.10 B), although the mice did not lose significant weight during the administration period ( Fig.10 C), but its MST decreased from 15 days to 12 days ( Fig.10 A), which is consistent with the clinical manifestations of recurrent patients. ATN2-mG / P and mG / P had a significant inhibitory effect on the growth of 4T1 recurrent tumors, but the tumor growth curve of each mouse showed that the tumor recurred and grew rapidly in the late stage of treatment, and the MST of mice was only extended to 27 and 24 days, respectively ( Fig.10 D, E), which is directly related to lung metastasis in mice. Literature reports that 36.9% of recurrent TNBC patients have lung metastasis and a low five-year survival rate. Multiple large lung metastatic nodules were observed in the lung bioluminescence images and H&E sections of mice in the PBS group on day 15. There were also many metastatic nodules in the lungs of mice in the free G / P group, while the number of lung nodules in the mG / P and ATN2-mG / P groups was significantly less ( Fig.11 ), which reflects the inhibitory effect on secondary lung cancer.

[0067] The efficacy of chemoimmunotherapy with ATN2-mG / P or mG / P combined with NanoCpG on recurrent TNBC after surgery and secondary lung cancer was studied. Fig.10 A). The results showed that ATN2-mG / P or mG / P combined with intravenous NanoCpG (1 mpk) significantly enhanced the inhibitory effect on recurrent tumor growth and lung metastasis. The growth of recurrent tumors in the mG / P+NanoCpG group was stopped, and the tumors in the ATN2-mG / P+NanoCpG group even shrank ( Fig.10 B). The prior art believes that CpG alone has no therapeutic effect on 4T1 tumors. There was no significant change in the body weight of mice in each group during the treatment period ( Fig.10 C). After the combination of mG / P or ATN2-mG / P and NanoCpG, the median survival of mice was significantly prolonged (**p), ​​and 40% and 60% of the mice were completely cured, respectively, and no tumor recurrence was observed within 300 days of tumor-free survival ( Fig.10 D&E). In addition, the treatment of the two combined groups achieved the inhibition of TNBC lung metastasis, and the ATN2-mG / P+NanoCpG combined group even eliminated lung metastatic nodules ( Fig.11 ).

[0068] Example 6 Study on the combined therapy of ATN2-mG / P and NanoCpG to stimulate BMDC maturation

[0069] BMDCs (1 × 10 6 / well) were added 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 μg / mL (1.7 μM), PTX: 0.3 μg / mL (0.34 μM), n = 3. After incubation for 24 h, the cells were centrifuged and washed, and FITC-αCD11c, APC-αCD80 and PE-αCD86 were added and incubated for 30 min. Then, the cells were detected by flow cytometry, and the mBMDC (CD11c + CD80 + CD86 + mDCs).

[0070] To investigate the effects 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 BMDCs and incubated for 24 h. The procedure was the same as above, HPG: 1 μg / mL (1.7 μM), PTX: 0.15 μg / mL (0.17 μM), CpG: 0.4 μg / mL), n = 3.

[0071] To study the effects of drugs and 4T1-luc cells (1 × 10 5 BMDCs (1×10 / well) were co-incubated 6 / well), BMDCs and 4T1-luc cells were first cultured in 1640 medium in 12-well plates as before. After 24 h of culture, 4T1-luc cells were completely attached to the wall, the medium was removed, and BMDC suspension was added. Then ATN2-mG / P (HPG / PTX: 10 / 1), mG / P (HPG / PTX: 20 / 1, 10 / 1, or 5 / 1), mHPG, mPTX, or PBS were added, HPG: 1 μg / mL (1.7 μM), PTX: 0.3 μg / mL (0.34 μM). After 24 h of co-incubation, the cells were treated and measured as above (n = 3).

[0072] CpG ODN is a toll-like receptor 9 (TLR9) agonist, which is widely used as an adjuvant for tumor immunotherapy in preclinical studies and clinical trials. It is often administered intratumorally, which is not suitable for inaccessible tumors and has potential immunotoxicity. In addition, CpG has low cellular uptake efficiency and is easily degraded. The ability of NanoCpG to stimulate the maturation of BMDC was studied by flow cytometry. The results showed that the proportion of mature BMDC in the NanoCpG group was higher than that in the PBS group, and the combination of mG / P or ATN2-mG / P with NanoCpG could further increase the proportion of mature BMDC to 77.0% and 85.6% (****p)( Fig.12 ).

[0073] Example 7 Immune cell infiltration and cytokine secretion after combined treatment of 4T1-luc secondary lung cancer mice

[0074] The recurrent tumor volume of the 4T1-luc secondary lung cancer model after recurrence and metastasis was about 100 mm 3At day 0, 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). mG / P or ATN2-mG / P was injected on days 0, 2, and 4, and NanoCpG was injected on days 1, 3, and 5. 48 h after the last injection, mouse plasma was collected, and the concentrations of pro-inflammatory factors (TNF-α, IFN-γ) and anti-inflammatory factors (IL-10) were determined by ELISA kits. Mice were euthanized, and organs were obtained to study the inhibition and immune regulation of secondary lung cancer. After the mouse lung tissue was weighed, the sections were stained with H&E, and the number of lung metastatic nodules was observed under a microscope. Weigh the recurrent tumors and calculate the recurrent tumor inhibition rate (TIR). Weigh the spleen. Grind the lymph nodes, spleen, and tumors, centrifuge, and obtain a single cell suspension. After lysing the red blood cells with ACK, separate them into tubes and stain them with corresponding antibodies. Flow cytometry was used to detect the proportion of DC, T, Treg, macrophages, and MDSC in the tumor, and the infiltration of T, MDSC in the spleen, and DC in the lymph nodes.

[0075] The infiltration of immune cells and secretion of cytokines in tumors and major immune organs were analyzed ( Fig.13 A). The results showed that 48 hours after the third injection of NanoCpG, the weight of the recurrent tumors in the four treatment groups was significantly lower than that in the PBS group (0.5 g), and the tumor in the ATN2-mG / P+NanoCpG group was the lowest, only 0.05 g (****p) ( Fig.13 B); The mass trend of lung tissue containing metastases was the same, and the mass of the lungs of mice in the ATN2-mG / P+NanoCpG group was close to that of the lung tissues of healthy mice ( Fig.13 C). H&E staining analysis of the whole lung scan images showed that the number of lung metastatic nodules in the combined treatment group was significantly reduced compared with the PBS group (about 8 nodules). Fig.13 D). The spleen is the most important immune organ. The spleen of mice in the PBS group was the heaviest, which may be caused by the infiltration of immune cells that were not normally activated during the onset of TNBC. Such splenomegaly was significantly avoided in the ATN2-mG / P+NanoCpG group. The activated immune cells circulated to the lymph nodes, tumors and other parts. The spleen weight of mice in this group (0.18 g) was similar to that of healthy mice ( Fig.13 E).

[0076] The analysis results of tumor-infiltrating immune cells confirmed the strong anti-recurrence and metastasis of TNBC after surgery in the ATN2-mG / P+NanoCpG combination group. The total DC (CD11c+ DC) and mature mDC (CD11c + CD80 + CD86 + ) were significantly stronger than those in other groups ( Fig.14 A, B), promote the recruitment and maturation of DCs through TLR4 and TLR9 pathways respectively, promote the presentation of more antigens to T cells and recruit more T cells to the tumor site, and generate strong and lasting tumor-specific T cell responses; CD8 + Cytotoxic T cells and CD4 + Helper T cells can attack and kill tumor cells. Flow cytometry analysis showed that CD8 + T( Fig.14 C, D), CD4 + T( Fig.14 E, F) levels were significantly higher than those in the two single-drug groups. In addition, the immunosuppressive CD4 + The infiltration of regulatory T cells (Treg) decreased after treatment with each agent ( Fig.14 G). The infiltration rates of immunosuppressive MDSC in recurrent tumors and spleen were 40% and 12%, respectively, which directly led to the refractory nature of postoperative recurrent TNBC. All four agents significantly reduced the infiltration of MDSC in recurrent tumors and spleen ( Fig.14 H, I), where the infiltration rate of MDSC in the mouse tumors of the ATN2-mG / P+NanoCpG combined group was less than 4% on average. + T, CD8 + The number of T cells, NK cells or macrophages.

[0077] ELISA was used to detect the concentration of cytokines in mouse plasma. It was found that the concentrations of proinflammatory cytokines IFN-γ and TNF-α produced by the mG / P+NanoCpG and ATN2-mG / P+NanoCpG combined groups were higher than those of the mG / P and ATN2-mG / P groups alone (***p, **p) ( Fig.14 J, K). The secretion of IFN-γ and TNF-α can improve the activity of immune cells NK, DC and CTL. The secretion of IL-10 was significantly reduced in all four treatment groups ( Fig.14 L), of which two combination groups can reduce the IL-10 concentration to below the detection limit of the ELISA kit (8pg / mL). The above results all prove that the combination of ATN2-mG / P+NanoCpG can promote a favorable anti-tumor immune microenvironment, produce a strong immune response, and has an excellent therapeutic effect on secondary lung cancer after TNBC surgery, curing 60% of mice.

[0078] The data of the present invention are expressed as mean ± SD. One-way ANOVA was used to determine the significant differences between the groups. The survival analysis used the log-rank comparison in the Kaplan-Meier technique. *p < 0.05 represents a significant difference, **p < 0.01, ***p < 0.001, ****p < 0.0001 represent highly significant differences.

[0079] In view of the fact that TNBC is prone to metastasis and development into secondary lung cancer, this paper reports that integrin α5β1-targeted, co-delivered HPG and PTX micelles (ATN2-mG / P) combined with NanoCpG can effectively "heat" TME, efficiently treat postoperative TNBC tumors, and prevent secondary lung cancer ( Fig.15 Schematic diagram). In order to increase the drug loading of GEM, the present invention selects hydrophobic phosphorylated modified GEM prodrug (HPG). In addition to inducing ICD, PTX and HPG can also stimulate APC and reduce MDSCs respectively. Therefore, ATN2-mG / P can not only target and synergistically stimulate ICD, but also synergistically reverse the immunosuppressive TME with the help of PTX's activation of DC cells and GEM's elimination of MDSC. In the postoperative 4T1 TNBC model, ATN2-mG / P+NanoCpG evoked a strong anti-tumor immune response, completely inhibited tumor recurrence and lung metastasis, and 60% of mice had complete tumor regression and long-term survival. The combination therapy of HPG and PTX co-delivery with NanoCpG provides a unique strategy not only for "cold" tumors such as TNBC, but also for effective chemoimmunotherapy of secondary lung cancer.

Claims

1. A co-drug-loaded micelle, which is a gemcitabine prodrug and paclitaxel co-loaded micelle, comprising a polymer micelle, gemcitabine prodrug and paclitaxel, characterized in that: In the polymer micelle, the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC), or the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC) and a targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC); when the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC) and a targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC), the content of the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) is 0 to 20% excluding 0; The hydrophobic monomers include cyclic ester monomers and cyclic carbonate monomers; the hydrophilic segment is PEG; the targeting molecule is a polypeptide; the molecular weight of the hydrophilic segment is 1.0 to 7.5 kg / mol; the molecular weight of the hydrophobic segment is 1.5 to 7.5 kg / mol; The molar ratio of gemcitabine prodrug to paclitaxel is (2-20):1; The preparation method of gemcitabine prodrug and paclitaxel co-loaded micelles is as follows: adding a mixed solution of drug solution, polymer solution and oligoethylene glycol into a buffer solution to obtain co-loaded micelles; The drug solution is composed of gemcitabine prodrug, paclitaxel, and oligoethylene glycol; 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 drug solution is 5-100 mg / mL; the concentration of the polymer solution is 20-500 mg / mL; the molecular weight of the oligoethylene glycol is 200-800; the mixed solution is added with a buffer solution, and the volume percentage of the oligoethylene glycol in the obtained solution is 2-10%.

2. The method for preparing the co-drug loaded micelles according to claim 1, characterized in that: A mixed solution of drug solution, polymer solution and oligoethylene glycol is added into a buffer solution to obtain co-drug loaded micelles.

3. A co-drug-loaded micelle synergistic drug system, comprising the co-drug-loaded micelle according to claim 1 and nano-CpG.

4. The drug-co-loaded micelle synergistic drug system according to claim 3, characterized in that: Nano-CpG is a polymer vesicle loaded with CpG; in the polymer vesicle, the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC)-cationic segment; the cationic segment is a PEI segment or a spermine segment.

5. Use of the co-drug-loaded micelles according to claim 1 and the co-drug-loaded micelle synergistic drug system according to claim 3 in the preparation of anti-tumor drugs, characterized in that: Anti-tumor drugs are drugs used to treat triple-negative breast cancer or lung cancer.

6. The use according to claim 5, characterized in that: Anti-tumor effects include inducing selective production of ICD in tumor cells, effectively eliminating MDSCs, and stimulating DC maturation.

7. Use of the co-drug-loaded micelles according to claim 1 and the co-drug-loaded micelle synergistic drug system according to claim 3 in the preparation of anti-tumor drugs, characterized in that: Anti-tumor drugs are drugs used to treat secondary lung cancer.

8. The use according to claim 7, characterized in that: Anti-tumor effects include inducing selective production of ICD in tumor cells, effectively eliminating MDSCs, and stimulating DC maturation.

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