CN116059167A - Co-drug-loaded micelle and synergistic drug system thereof, and preparation method and application thereof - Google Patents
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- CN116059167A CN116059167A CN202310018542.0A CN202310018542A CN116059167A CN 116059167 A CN116059167 A CN 116059167A CN 202310018542 A CN202310018542 A CN 202310018542A CN 116059167 A CN116059167 A CN 116059167A
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Abstract
The invention discloses a co-drug-carrying micelle, a synergistic drug system, a preparation method and application thereof, in particular to a gemcitabineThe drug solution is composed of the gemcitabine prodrug, paclitaxel and oligomeric ethylene glycol; adding the mixed solution of the drug solution, the polymer solution and the oligoethylene glycol into a buffer solution to obtain a gemcitabine prodrug and paclitaxel co-loaded micelle which can be used for efficiently and synergistically treating the secondary lung cancer of the triple negative breast cancer metastasis with a TLR9 nano agonist such as nano CpG, wherein the CpG induces immune cells to generate I-type interferon by activating a TLR9 channel, thereby inducing T h1 An immune response; can be administered by intravenous system, reducing 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 co-carried micelle of a prodrug (HPG) of gemcitabine (Gem) and Paclitaxel (PTX), a synergistic system and application, which can be cooperated with nano CpGSecondary lung cancer with high efficacy for the treatment of triple negative breast cancer metastasis, wherein CpG induces immune cells to produce type I interferon by activating TLR9 pathway, thereby inducing T h1 An immune response; can be administered by intravenous system, reducing potential immunogenicity and systemic toxicity of CpG.
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); in the polymer vesicles, the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC) -cationic segment. DTC is a prior cyclic carbonate monomer containing disulfide five-membered ring functional groups, and is polymerized to form PDTC chain segments. The hydrophobic monomer includes a cyclic ester monomer, a cyclic carbonate monomer, preferably the cyclic carbonate monomer is other cyclic carbonate monomer, such as other cyclic carbonate monomer including trimethylene cyclic carbonate (TMC), including caprolactone (epsilon-CL), lactide (LA) or Glycolide (GA); the hydrophobic monomer is polymerized 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 cation fragment is PEI fragment or spermine fragment, and the targeting molecule is polypeptide, such as ATN1 (Ac-PHSCNK-NH) 2 ）、ATN2（Ac-PhScNK-NH 2 ) 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 invention, in the polymer micelle, the molecular weight of a hydrophilic segment is 1-7.5 kg/mol, and the molecular weight of a hydrophobic segment is 1.5-7.5 kg/mol; preferably, the hydrophilic segment has a molecular weight of 1.5 to 5kg/mol and the hydrophobic segment has a molecular weight of 1.5 to 5kg/mol. In the polymer vesicle, the molecular weight of the hydrophilic segment is 3-10 kg/mol, the molecular weight of the hydrophobic chain 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, and preferably the molecular weight is 0.1 to 2 kg/mol.
In the present 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 to 20% excluding 0, preferably 1 to 15%, and more preferably 2 to 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 medicinal solution consists of gemcitabine prodrug, taxol and oligomeric ethylene glycol; the concentration of the drug solution is 5 to 100 mg/mL, preferably 10 to 70 mg/mL, and more 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 20-500 mg/mL, preferably, the concentration of the hydrophilic segment-P (hydrophobic monomer-DTC) solution is 50-500 mg/mL, preferably 100-400 mg/mL; 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 polyethylene glycol; the molecular weight of the oligoethylene glycol is 200-800, such as PEG200, PEG350, PEG400, PEG600, PEG800, etc. Preferably, the mixed solution is added into a solution obtained by adding 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 is designed 5 β 1 Integrin targets, co-loads HPG and PTX disulfide cross-linked micelles ATN2-mG/P. 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. This chemotherapy successfully stimulated DC massive recruitment and activation, CD8 + And CD4 + The T cells provide antigen, increase secretion of IFN-gamma and TNF-alpha, obviously reduce infiltration of immunosuppressive MDSC and Treg, and improve comprehensive treatment effect of postoperative TNBC. ATN2-mG/P and NanoCpG are simple to prepare, have good safety and can efficiently enhance 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 representation 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); (C) DLS determining particle size distribution of PEG350 solutions of mHPG and HPG; (D) Dialysis to remove changes in particle size of mgp/P stored at room temperature before and after PEG 350; (E) particle size change of mgp incubated with 10% FBS; (F) UV absorption spectra of PEG350 solutions of mHPG, mPTX, mG/P and PEG-P (CL-DTC).
Fig. 2 is an in vitro drug release of micelle drug: (a) particle size of mgp in the presence or absence of 10 mM GSH; (B) Drug release (n=3) in 10 mM GSH medium (pH 7.4) with or without mgp and (C) single-carrier micelles mHPG at 37 ℃; (D) Drug release of mgp at pH 5.0 or pH 7.4 without GSH (n=3). (E) Intact HPG content (n=3) when free HPG, mHPG and mgp were incubated with CDA with or without 10% serum for 4h, each formulation was incubated with PB or a PB solution containing tha (tetrahydrouridine, CDA inhibitor, 344 μm) as a control.
Fig. 3 is a cytotoxicity study (n=6): cell viability of (a) mgp (HPG/ptx=2/1, 5/1, 10/1, 20/1), (B) mPTX, and (C) ATN 2-mgp incubated with 4T1-luc cells 48 h.
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) were incubated with 4T1-luc cells 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).
FIG. 5 shows that mG/P and ATN2-mG/P induce 4T1-luc cells to produce ICD (HPG: 1. Mu.g/mL (1.7. Mu.M), PTX: 0.3. Mu.g/mL (0.34. Mu.M), 24 h, n=3): (a, B) expression of cell surface CRT and (C) ATP concentration after mgp (HPG/ptx=20/1, 10/1, 5/1) treatment; (D, E) ATN2-mG/P induces cell surface CRT expression and secretion of (F) ATP.
FIG. 6 shows that mG/P (HPG/PTX=20/1, 10/1, 5/1) and ATN2-mG/P (HPG/PTX=10/1) stimulate maturation of BMDC (CD 80) + CD86 + mDC), free G/P, mHPG, mPTX and PBS as controls (HPG: 1 μg/mL (1.7 μΜ), PTX:0.3 μg/mL (0.34 μM), 24 h, n=3).
FIG. 7 shows BMDC maturation with mG/P and ATN2-mG/P stimulation incubated with 4T1 cells: (A) experimental design; (B) 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 mature BMDC after mG/P (HPG/PTX=20/1, 10/1, 5/1) and (C) ATN2-mG/P treatment.
FIG. 8 shows treatment of 4T 1-bearing subcutaneous tumor mice with mGs/P (n=6), mHPG, mGs/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) were intravenously injected on days 0, 2, 4, 6, 8, 10: (A) treatment schedule, (B) tumor volume change, (C) body weight change and (D) survival curve of mice.
FIG. 9 shows treatment of ATN2-mG/P in 4T 1-bearing subcutaneous tumor mice by intravenous injection of ATN2-mHPG, mG/P, ATN2-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: (a) a dosing regimen; tumor volume (B) and body weight (C) of mice (n=6); the ratio of (D) mDC and (E) MDSC infiltrated in the tumor on day 14 (n=3).
Fig. 10 is a graph of treatment of 4T1-luc post-operative recurrent/secondary lung cancer mice (HPG/ptx=10/1,HPG:15 mpk,CpG:1 mpk) with ATN 2-mgp or mgp in combination with nano cpg: (a) a treatment regimen; tumor volume (B) and body weight (C) of mice (n=6); mice (D) Kaplan-Meier survival curve and (E) tumor growth curve of single mice (n=5).
Fig. 11 is a bioluminescence imaging and H & E staining 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 combined with NanoCpG to stimulate BMDC maturation and mDC (CD 11c + CD80 + CD86 + ) Semi-quantitative analysis of the 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).
Fig. 13 is tumor microenvironment analysis of chemotherapy immunotherapy TNBC post-operative recurrence/secondary lung cancer mice (HPG/ptx=10/1,HPG:15 mpk,CpG:1 mpk,n =4): (a) a treatment regimen; tumor mass of mice (B); (C) lung weight; (D) number of lung metastasis nodes and (E) spleen quality.
Fig. 14 is tumor microenvironment analysis (n=4) of chemotherapy immunotherapy TNBC post-operative recurrence/secondary lung cancer mice: invasive (A) CD11c in tumors + DC and (B) CD11c + CD80 + CD86 + The ratio of mDC; (C) Tumor and (D) CD8 infiltrated in the spleen + The ratio of T; (E) Tumor and (F) CD4 infiltrated in the spleen + T proportion and (G) proportion of infiltrated Treg in tumor; proportion of (H) tumor and (I) infiltrated MDSC in spleen; concentration of (J) IFN-gamma, (K) TNF-alpha and (L) IL-10 in serum.
FIG. 15 shows (A) preparation of ATN2-mG/P, (B) its combination with NanoCpG can heat tumor immune microenvironment, effectively inhibit 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; the Ac-PhScNK-NH2 (ATN 2) had a purity of greater than 98% and was purchased from Shanghai blaze organisms. 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 the following test conditions: 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) respectively, and mixing into pharmaceutical solution according to the molar ratio of 20/1, 10/1,5/1 or 2/1 for standby; PEG350 stock solutions of PEG-P (CL-DTC) (200 mg/mL) and ATN2-PEG-P (CL-DTC) (20 mg/mL) were prepared for use. To prepare the drug with the concentration of 1 mg/mL and the theoretical drug loading rate of 9.1 of HPGwt.For example, 4. Mu.L of HPG and 0.6. Mu.L of PTX drug solution and 5. Mu.L of PEG-P (CL-DTC) PEG350 solution and PEG350 were mixed to a total volume of 50. Mu.L of solution, added to 950. Mu.L of PB (pH 7.4, 10 mM), and blown 5 times with a pipette gun and placed 12 h at room temperature to give 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 10/1 of PEG350 of HPG/PTX of 2% or 10% respectively were prepared in a similar manner. The preparation method of ATN2-mG/P is similar, and the totalA50. Mu.L volume 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 was added 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 formulation of acetic acid/sodium acetate buffer solution is: 285.7 mu.L of acetic acid and 262.43 mg sodium acetate were dissolved in 1L secondary water. Next, 1 mL of mG/P was allowed to stand overnight and transferred to a release bag (MWCO 14 kDa), and dialyzed in 25 mL buffer tubes, respectively, placed in a shaker at 37℃and 200 rpm. 7 mL was taken at the set time point 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, with 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, single-loading HPG and PTX micelles were less efficient in drug delivery (table 1); the same concentration of the mixture of HPG, PTX and PEG350 in water without copolymer resulted in a broad particle size distribution, turbidity and PTX precipitation 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 only 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. 27.5 nm, and the particle size distribution increases to 0.2 or more. 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) (. About.>
n :5.0- (4.0-2.0) kg/mol,50 mg/mL) and HPG (25 mg/mL) to prepare a PEG350 mother liquor with a concentration of 1 mg/mL and a theoretical drug loading of 9.1 HPGwt.% micelle mPPG as an example, 4. Mu.L of HPG, 20. Mu.L of PEG-P (CL-DTC) in PEG350 and PEG350 were mixed to a total volume of 50. Mu.L, added to 950. Mu.L of PB (pH 7.4, 10 mM) and blown 5 times with a pipette to give a micelle having a particle size of 44.1 nm (PDI: 0.24). The particle sizes of the mHPGs prepared by the 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 and nm respectively, and the particle sizes and the 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, the single-carrier HPG micelle mHPG released more than 40% of the drug in the absence of GSH (fig. 2C); in addition, little drug release occurs in mgs/ps with weak acidity, suggesting that micelles have the ability to stably escape endosomes, releasing the drug into the cytoplasm (fig. 2D).
Example cytotoxicity, apoptosis and cell cycle arrest assay of Diatn 2-mgp
4T1-luc was seeded in 96-well plates (1X 10) 3 /well) 24. 24 h, 20 μl mG/P (HPG/PTX: 20/1, 10/1,5/1, or 2/1), mHPG, or mPTX, wherein the HPG concentration is 0.0017-68.8 μm and the PTX concentration is 0.005-12.8 μm. After incubation of 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 absorbance of cells was measured at 570 nm using a multifunctional microplate reader, with the MTT-added medium wells as zero points, the MTT-added PBS-treated cells as 100% and the cell viability (% cell viability) as the ratio of the absorbance values of the experimental group to the absorbance values of the PBS group.
The synergy of HPG and PTX was evaluated by calculating the joint index (CI) using the following formula:
wherein a and b respectively represent the IC of each drug in mG/P 50 Values A, B represent the IC of each drug alone in mHPG and mPTX, respectively 50 Values. 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 among medicines is.
To investigate the targeting of ATN2-mG/P to 4T1 cells, 4T1 cells (1X 10 3 /well) with ATN 2-mgp and mgp (HPG/ptx=10/1, HPG concentration: 0.0017-68.8 μm) was incubated with 4h, then 44 h with fresh medium without drug. Subsequent sample processing and data analysis methods are described above.
In the apoptosis experiments, 4T1-luc cells (1X 10) 5 Well) to ATN2-mG/P (HPG/PTX: 10/1), mgs/ps (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. 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 48 h 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.
IC with mPTX (1.4. Mu.M) and mPPG (3.8. Mu.M) 50 IC of HPG and PTX at molar ratios of HPG/PTX of 2/1, 5/1, 10/1, 20/1 50 The values were significantly reduced, 0.3/0.14, 1.8/0.16, 0.4/0.074 and 2.3/0.10, respectively (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. ATN2-mG/P at HPG/PTX=10/1, wherein the IC of HPG 50 The value was 2.3 times lower than that of micelle mG/P (FIG. 3C). Apoptosis experimental analysis shows that mgp causes higher apoptosis ratio than other ratio (P) and single drug-loaded (P) group at HPG/ptx=10/1, ATN 2-mgpThe apoptosis rate was further increased 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 single-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 maintained the intact structure of the HPG drug at a ratio of 7 times that of HPG-only loaded micelle mHPG after incubation of 4h with CDA and 10% fbs (fig. 2E), further demonstrating that PTX could produce strong synergy with HPG through 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
4T1-luc cells in 12-well plates (1X 10) 5 /well) 24-mG/P (HPG/PTX) was added after incubation for 24 h: 10/1), mgs/ps (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. After incubation at 24 h, the medium was collected and the concentration of ATP in the medium was determined using the enhanced ATP assay kit; cell digestion followed by staining, flow cytometry detection, and analysis of 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 analysis of ICD two main markers of 4T1-luc cells after treatment with each preparation: expression of surface CRT and secretion of ATP. The results show that the 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 regulate proliferation and polarization of APCs via TLR4 pathway to promote maturation and proliferation of DCs, gem eliminates MDSCs and also stimulates DC maturation, experimental results show that both mPTX and mPEG significantly stimulate BMDC maturation at low concentrations (CD 80 + CD86 + mDC) (×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 mature DCs treated with mHPG and mPTX was significantly lower than without 4T1 cell co-culture, 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 the proportion of ATN2-mG/P that induced DC maturation was further increased (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. Subcutaneous inoculation of 4T1-luc cells (3X 10) 5 After 7 days/only) the tumor average volume grew to about 50 mm 3 Mice were randomly divided into 6 groups (n=6), and scored as day 0 of the experiment. mG was intravenously injected every 2 daysPer P (HPG/PTX=20/1 or 10/1, HPG:15 mpk), mHPG (HPG: 15 mpk, 25.8. Mu. Mol/kg), mPTX (PTX: 2.25 mpk, 2.58. Mu. Mol/kg) 6 times total (FIG. 8A), tumor volume and body weight were monitored, tumor volume exceeded 2000 mm 3 Mice were judged to die by a weight loss of more than 15%, either unresponsive or non-feeding. The results indicate that treatment with mgp and mHPG effectively delayed tumor progression (P) (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 (fig. 8B). The body weight of each group of mice did not change much during the dosing period except that mHPG caused 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 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 prolongation of survival in tumor bearing mice.
To investigate the inhibition of TNBC tumors by ATN2-mG/P six needles of ATN2-mPTX, ATN2-mHPG, mG/P, ATN2-mG/P (HPG/PTX=10/1, HPG:10 mpk (17.2. Mu. Mol/kg), PTX:1.5 mpk (1.72. Mu. Mol/kg)) were injected into 4T1 subcutaneous tumor mice via the tail vein (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 added and incubated at 4deg.C for 30 min, and mDC (CD 11 c) was measured by flow cytometry + CD80 + CD86 + ) And MDSC (CD 11 b) + Gr-1 + ) Is used as a wetting agent.
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).
EXAMPLE five ATN2-mG/P and NanoCpG chemotherapy treatment of 4T1-luc secondary Lung cancer study
The NanoCpG is obtained by self-assembling a copolymer PEG-P (TMC-DTC) -SP of CpG and terminal modified spermine (spimine) in an aqueous solution. 100. Mu.L of PEG-P (TMC-DTC) -SP in DMF (10 mg/mL) was added to 900. Mu.L of CpG-containing (100. Mu.g) HEPES buffer (5 mM, pH 6.8), stirred conventionally for 10 min, dialyzed 2 times in HEPES, dialyzed 1 time in PB/HEPES (v/v, 1/1), and finally dialyzed 2 times in PB to give a NanoCpG solution. DLS measures particle size and particle size distribution, and Nanodrop measures CpG drug loading. The vesicle NanoCpG is formed by self-assembling a segmented copolymer PEG-P (TMC-DTC) -SP of end modified 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) with SP. PTMC in hydrogen spectrogram through nuclear magnetic resonanceδ4.24，2.05）、PDTC（δ4.24，3.02）、SP（δ2.56-2.98) and PEG characteristic peakδ3.64 The integrated ratio, the degree of functionalization of the SP can be calculated to be about 95%. The prepared nano CpG has small particle size (50 nm), narrow distribution (PDI 0.10), high CpG loading efficiency, good stability and good reproducibility.
4T1-lucThe method for establishing the secondary lung cancer model comprises the following steps: (1) 4T1-luc cells (3X 10) 5 /only) Balb/c mice (female, 6 weeks) were vaccinated subcutaneously above hind limbs. (2) In the tumor volume as long as 200-300 mm 3 At this time (day 11 of inoculation), 90-95% of the tumor was surgically excised and the wound was sutured. (3) Tumor recurrences when its volume grows to about 100 mm 3 (day 18 of inoculation), IVIS near infrared imaging scan mice had developed significant lung metastasis, and secondary lung cancer mice model was successfully established.
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, this day was identified 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 caudally intravenously on days 0, 2, 4, 6, 8, 10, and nanogps (CpG: 1.0 mpk) were injected caudally intravenously on days 1, 3, 5. Mice body weight and tumor volume were monitored every 3 days. On day 15, 1 mouse was randomly dissected from each group, the biological development of the mouse lung was observed with an IVIS imaging system, and H was used&E, staining to observe the distribution of lung metastasis nodules; taking main organ slices of mice, H&The E staining study was combined for systemic toxicity. The remaining five mice were used to observe survival and to plot survival curves. During observation, when mice die, tumor volume exceeded about 2000 mm 3 Or death is judged when the weight is reduced by more than 15 percent.
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. After surgical excision of 90-95% of the tumors on day 11 post inoculation of 4T1 subcutaneous tumors, the tumors quickly recur in situ; in addition, IVIS near infrared imaging showed that mice had significant metastasis to secondary lung cancer at later stages (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 ATN2-mG/P or mG/P combined with NanoCpG chemotherapy on TNBC postoperative recurrence and secondary lung cancer was studied (FIG. 10A). The results show that ATN2-mG/P or the combined intravenous injection of mG/P and NanoCpG (1 mpk) significantly enhanced the inhibition of recurrent tumor growth and lung metastasis, and that the recurrent tumor growth of mG/P+NanoCpG group stopped, while ATN2-mG/P+NanoCpG group tumors even shrunken (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 combined use of mgp or ATN 2-mgp and nano cpg, the 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 suppression of TNBC lung metastasis, and the ATN2-mG/P+NanoCpG combination group even eliminated lung metastasis nodules (FIG. 11).
EXAMPLE six combination therapy of ATN2-mG/P and NanoCpG stimulated BMDC maturation study
BMDC (1X 10) seeded in 12-well plates 6 /well) to add ATN2-mG/P (HPG/PTX: 10/1), mgs/ps (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 μg/mL (0.34 μm), n=3. After incubation of 24 h, the cells were centrifuged and washed, incubated for 30 min with FITC- αCD11c, APC- αCD80 and PE- αCD86, detected by flow cytometry, and analyzed quantitatively for mBMDC (CD 11 c) by FlowJo_V10 software + CD80 + CD86 + mDC).
To investigate the effect of NanoCpG and its combination with ATN 2-mgp on BMDC maturation, the procedure after 24 h incubation with NanoCpG, free G/P, mG/P, ATN 2-mgg/P, mG/p+nanocpg, ATN 2-mgp+nanocpg (HPG/ptx=10/1) or PBS was followed as above for HPG: 1.μg/mL (1.7 μM), PTX:0.15 μg/mL (0.17 μM), cpG:0.4 μg/mL), n=3.
To study drug pairs and 4T1-luc cells (1X 10) 5 BMDC (1X 10) co-incubated per well 6 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 24 h, 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.μg/mL (1.7 μM), PTX:0.3 mu.g/mL (0.34. Mu.M). After 24 h co-incubation, 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 the NanoCpG to stimulate BMDC was studied using a flow cytometer and showed that the ratio of the NanoCpG to the mature BMDC was higher than that of the PBS group, and that the mgp or ATN 2-mgp combined with the NanoCpG further increased the maturation ratio 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
Recurrent tumor volume in postoperative recurrent metastatic 4T1-luc secondary lung cancer model was about 100 mm 3 On 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. Mu. Mol/kg), PTX:2.25 mpk (2.58. Mu. Mol/kg), cpG:1.0 mpk). mG/P or ATN2-mG/P are injected on days 0, 2, 4, 1, 3, 5And injecting NanoCpG. After the last injection 48 h, mouse plasma was collected and the ELISA kit was 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 the lung tissue of the mice is weighed, the mice are sliced with H&E staining, and observing the lung metastasis node number by a microscope. 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 of the nano-cpg, the quality of recurrent tumors in all four treatment groups was significantly lower than that in the PBS group (0.5, g), and ATN 2-mG/p+nano-cpg group had the lowest tumor, with only 0.05, g (fig. 13B); the tendency of the mass of lung tissue containing metastases was the same, and the lung mass of ATN2-mG/P+NanoCpG group mice was close to 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 group 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-infiltrating immune cells confirmed that the potent anti-TNBC post-operative recurrence and metastasis of the ATN2-mG/P+NanoCpG combination group was confirmed by total DC (CD 11c + DC) and mature mDC (CD 11c + CD80 + CD86 + ) Is significantly more invasive than the other groups (FIGS. 14A, B), and promotes recruitment and maturation of DCs via TLR4 and TLR9 pathways, respectively, contributing to the presentation of more antigenTo T cells and recruiting more T cells to the tumor site, producing a strong and durable tumor-specific T cell response; CD8 + Cytotoxic T cells and CD4 + Helper T cells can attack and kill tumor cells. Flow cytometry analysis results prove that ATN2-mG/P+NanoCpG group mice spleen and CD8 infiltrated in tumor + T (FIGS. 14C, D), CD4 + The T (fig. 14e, f) content was significantly higher than for both single drug groups. Furthermore, immunosuppressive CD4 in PBS group TME + Infiltration of regulatory T cells (tregs) 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 MDSC infiltration in recurrent tumors and spleen (fig. 14h, i), with an average of less than 4% MDSC infiltration in mice tumors of the ATN 2-mgp+nanogp combination group. The prior art considers that Gem does not affect CD4 + T、CD8 + T, NK number of cells or macrophages.
The concentration of cytokines in plasma of mice was found by ELISA to be higher in the combined mG/P+NanoCpG and ATN2-mG/P+NanoCpG groups than in the individual mG/P and ATN2-mG/P groups (P, P) (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 combination can promote favorable anti-tumor immune microenvironment, generate strong immune response, has excellent treatment effect on the secondary lung cancer after TNBC operation, and enables 60 percent of mice to be healed.
The data of the present invention are expressed as mean.+ -. SD. Significant differences between groups were determined using GraphPad Prism 9.0.0 single factor analysis of variance (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 of secondary lung cancer, the inventionIntegrin alpha is reported 5 β 1 Targeted, co-delivered micelles of HPG and PTX (ATN 2-mG/P) in combination with NanoCpG are effective in "heating" TME, and in treating post-operative TNBC tumors with high efficacy, preventing secondary lung cancer (FIG. 15 is a schematic). 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. Combination therapies of HPG and PTX co-delivery with nanomcpg provide a unique strategy for not only "cold" tumors such as TNBC, but also for effective chemoimmunotherapy of secondary lung cancer.
Claims (10)
1. A drug co-loaded micelle comprising a polymeric micelle, a gemcitabine prodrug and paclitaxel.
2. The drug co-loaded micelle of claim 1, wherein in the polymer micelle, the polymer is hydrophilic segment-P (hydrophobic monomer-DTC), or the polymer is hydrophilic segment-P (hydrophobic monomer-DTC), targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC); the molar ratio of the gemcitabine prodrug to the paclitaxel is (2-20) to 1.
3. The drug co-loaded micelle of claim 2, wherein the hydrophobic monomer comprises a cyclic ester monomer, a cyclic carbonate monomer; the hydrophilic segment is PEG; the targeting molecule is a polypeptide; the molecular weight of the hydrophilic section is 1.0-7.5 kg/mol; the molecular weight of the hydrophobic chain segment is 1.5-7.5 kg/mol; in the polymer micelle, when the polymer is hydrophilic segment-P (hydrophobic monomer-DTC) and targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC), the content of the targeting molecule-hydrophilic segment-P (hydrophobic monomer-DTC) is 0-20% and not including 0.
4. The method for preparing the drug-loaded micelle of claim 1, wherein the drug solution, the polymer solution and the mixed solution of the oligomeric ethylene glycol are added into a buffer solution to obtain the drug-loaded micelle; the medicinal solution consists of gemcitabine prodrug, taxol and oligomeric ethylene 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.
5. The method for preparing the drug-loaded micelle according to claim 4, wherein 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; adding the mixed solution into a buffer solution to obtain a solution, wherein the volume percentage of the oligoethylene glycol is 2-10%.
6. A co-drug-loaded micelle synergistic drug system, comprising the co-drug-loaded micelle and nano CpG according to claim 1.
7. The co-drug-loaded micelle synergistic drug system of claim 6, in which nano CpG is a CpG-loaded polymeric vesicle; in the polymer vesicles, the polymer is a hydrophilic segment-P (hydrophobic monomer-DTC) -cationic segment; the cationic fragment is a PEI fragment or a spermine fragment.
8. The co-drug-carrying micelle of claim 1 and the co-drug-carrying micelle synergistic drug system of claim 6 are applied to the preparation of antitumor drugs.
9. The use according to claim 8, wherein the antineoplastic agent is a medicament for the treatment of triple negative breast cancer, lung cancer or secondary lung cancer.
10. Use of the co-drug-loaded micelle, co-drug system of claim 1 in the manufacture of a medicament for inducing tumor cell selective production of ICD, effective elimination of MDSC and stimulation of DC maturation.
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