WO2022253080A1 - 一种化疗免疫联合药物及其制备方法与应用 - Google Patents

一种化疗免疫联合药物及其制备方法与应用 Download PDF

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WO2022253080A1
WO2022253080A1 PCT/CN2022/095048 CN2022095048W WO2022253080A1 WO 2022253080 A1 WO2022253080 A1 WO 2022253080A1 CN 2022095048 W CN2022095048 W CN 2022095048W WO 2022253080 A1 WO2022253080 A1 WO 2022253080A1
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ptx
cdn
drug
cps
chemotherapy
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French (fr)
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孟凤华
邱欣昀
郭贝贝
钟志远
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention belongs to nano-medicine technology, and in particular relates to a chemotherapy-immunity combination medicine and a preparation method and application thereof.
  • the invention discloses a combined chemotherapy and immunization drug as well as its preparation method and application, respectively preparing nano-medicines ATN1-Ms-PTX and ATN2-Ms-PTX coupled with PHSCNK polypeptide (ATN1), PhScNK polypeptide (ATN2) or cRGD polypeptide and cRGD-Ms-PTX; and prepared PEG-P(TMC-DTC)-Sp-based polymersomes with asymmetric membrane structure to load, protect and deliver cyclic dinucleotide (CDN) ADU-S100 ( CPs-CDN) to APCs to better activate anti-tumor immune responses.
  • PHSCNK polypeptide ATN1
  • PhScNK polypeptide ATN2
  • cRGD polypeptide and cRGD-Ms-PTX prepared PEG-P(TMC-DTC)-Sp-based polymersomes with asymmetric membrane structure to load, protect and deliver cyclic dinucleotide
  • ATN2-Ms-PTX can efficiently target and enrich mouse tumors, increase the concentration of PTX in tumor cells, and then sequentially deliver CPs-CDN to activate the STING pathway, which significantly delays the growth of mouse tumors. Inhibited the lung metastasis of breast cancer and prolonged the survival of mice.
  • a combined chemotherapeutic-immune drug which is composed of a chemotherapeutic drug micelle and a vesicular nano-STING agonist
  • oligoethylene glycol a mixed solution is obtained, and then the mixed solution is added to the buffer solution to obtain chemotherapy drug micelles; or small molecule drugs, amphiphilic polymers, and targeted amphiphilic polymers are added to oligomerization ethylene glycol to obtain a mixed solution, and then add the mixed solution to the buffer solution to obtain chemotherapy drug micelles;
  • the molecular weight ( M n ) of the oligoethylene glycol is 200-600;
  • the vesicle nano STING Agonists consisted of polymersome-loaded STING agonists.
  • the mixed solution under standing, the mixed solution is squeezed into the buffer solution, and then stirred, left standing, vortexed, blown or inverted; the punching is a prior art, such as using a syringe or a syringe pump; the stirring speed is 100 ⁇ 1000rpm.
  • the concentration of the small molecule drug is 0.1 to 10 mg/mL, and the concentration of the polymer is 1 to 100 mg/mL; the polymer is an amphiphilic polymer, or the polymer is an amphiphilic polymer and targets two A mixture of hydrophilic polymers.
  • the dosage of the targeted amphiphilic polymer is 1-30% of the weight of the amphiphilic polymer, preferably 2-10%.
  • the amphiphilic polymer is not coupled with targeting molecules, and the targeting amphiphilic polymer is coupled with targeting molecules.
  • the volume ratio of oligoethylene glycol and buffer solution is 1: (5-40), preferably 1: (10-30).
  • the invention prepares micelles with high PTX drug loading, and the prepared micelles with small particle size (20-40 nm) can penetrate deep into the tumor.
  • the invention discloses a preparation method of the above-mentioned vesicle nano STING agonist.
  • the STING agonist solution is added to a buffer solution, and then a polymer solution is added, stirred and then dialyzed to obtain the vesicle nano STING agonist.
  • the STING agonist solution is the STING agonist aqueous solution;
  • the polymer solution is the DMF solution of the polymer;
  • the buffer solution is the HEPES buffer solution.
  • the concentration of the STING agonist solution is 1-30 mg/mL; the concentration of the polymer solution is 1-100 mg/mL.
  • the small molecule drug is paclitaxel PTX; the molecular weight ( M n ) of the oligoethylene glycol is 200-600.
  • the molecular weight of the amphiphilic polymer is 3000-15000, and the molecular weight of the targeting amphiphilic polymer is 2000-15000.
  • the molecular weight of the polymer of the present invention is the number average molecular weight ( M n ) determined by NMR, and the unit is Da.
  • the STING agonist is cyclic dinucleotide (CDN), including cyclic diguanosine monophosphate (c-di-GMP), cyclic dimonophosphate adenosine (c-di-AMP), 2 ⁇ ,3 ⁇ Cyclic guanine monophosphate monophosphate (2 ⁇ ,3 ⁇ -cGMAP), 3 ⁇ ,3 ⁇ cyclic guanine monophosphate monophosphate (3 ⁇ ,3 ⁇ -cGMAP) and their substituted derivatives , such as sulfur, fluorine, nitrogen substituted cyclic dinucleotide derivatives.
  • CDN cyclic dinucleotide
  • CDN cyclic dinucleotide
  • CDN cyclic dinucleotide
  • CDN cyclic diguanosine monophosphate
  • c-di-AMP cyclic dimonophosphate adenosine
  • 2 ⁇ ,3 ⁇ Cyclic guanine monophosphate monophosphate 2 ⁇ ,3 ⁇
  • the amphiphilic polymer is PEG-P (CL-DTC), PEG-P (TMC-DTC), PEG-P (LA-DTC), etc.;
  • the targeted amphiphilic polymer is the amphiphilic polymer
  • the targeting molecule is coupled to the hydrophilic polymer, preferably, the targeting molecule is a polypeptide, such as PHSCNK polypeptide, PhScNK polypeptide or cRGD polypeptide; in the amphiphilic polymer, the molecular weight ( M n ) of PEG is 1000-5000 Da.
  • the polymer when preparing the vesicle nano-STING agonist, includes a hydrophilic segment, a hydrophobic segment and a cationic segment; the side chain of the hydrophobic segment is a disulfide bond-containing dithiolane, and its chemical
  • the structural formula is as follows: .
  • R is a hydrophilic segment, such as a polyethylene glycol segment; a T segment and a side chain containing a disulfide bond-containing carbonate segment (units formed by DTC monomers) form a hydrophobic segment, and T is a cyclic ester monomer or Units formed from cyclic carbonate monomers, such as lactide, caprolactone, trimethylene carbonate; P is a cationic segment, such as polyethyleneimine (PEI), spermine (SP); y, z represent repeating units
  • the molecular weight of the hydrophilic segment is 2000 ⁇ 9000Da; the molecular weight of the hydrophobic segment is 2.1 ⁇ 8.0 times of the molecular weight of the hydrophilic segment; 10% to 35%; the molecular weight of the cationic segment is 5% to 30% of the molecular weight of the hydrophilic segment; for example, the polymer is PEG-P(TMC-DTC)-Sp.
  • the invention prepares micelles with high drug loading of PTX, and the prepared micelles with small particle size (20-40 nm) can penetrate deep into the tumor, and the sequence of CPs-CDN activating STING pathway, activating and recruiting APC gives The drug strategy can activate the immune system more strongly, produce more pro-inflammatory cytokines, significantly increase the proportion of CD4 + and CD8 + T cells in the tumor and spleen, reduce the inhibitory T reg , and better inhibit the growth of TNBC in mice. Growth and occurrence of lung metastases.
  • the invention discloses the application of the above chemotherapy-immunity combination drug in the preparation of anti-tumor drugs, preferably the tumor is triple-negative breast cancer (TNBC).
  • TNBC triple-negative breast cancer
  • the present invention first designs and prepares high-efficiency PTX-loaded, disulfide-crosslinked micellar Ms-PTX and three kinds of micellar nanomedicines actively targeting TNBC.
  • the drug-loading capacity is as high as 23.1 wt. It has good stability and reduction-responsive drug release performance.
  • ATN2-Ms-PTX has the strongest targeted endocytosis effect on 4T1 cells, the lowest IC 50 value, and the highest biodistribution in tumors.
  • ATN2-Ms-PTX can induce ICD, promote the proliferation and maturation of BMDC, promote the polarization of BMDM to M1 macrophages, and create a favorable immune microenvironment.
  • disulfide cross-linked vesicle CPs-CDN loaded with STING agonist CDN was prepared to activate the STING pathway of APC and induce a stronger immune response.
  • the combination of ATN2-Ms-PTX and CPs-CDN can promote the maturation of BMDC and release IFN- ⁇ and TNF- and other cytokines.
  • ATN2-Ms-PTX was injected intravenously to induce ICD to release tumor antigens, followed by intratumoral injection of CPs-CDN 8-24 h later to activate the STING pathway, activate and recruit APC sequentially.
  • mice can activate the immune system more strongly, produce more proinflammatory cytokines, significantly increase the proportion of CD4 + and CD8 + T cells in the tumor and spleen, reduce the inhibitory T reg , and better inhibit the growth of TNBC and lung cancer in mice.
  • the occurrence of metastasis significantly prolongs the survival period of mice, which reflects the advantages of chemotherapy combined with immunotherapy, and brings hope for the treatment of highly metastatic tumors.
  • Figure 1 shows the preparation and physicochemical properties of Ms-PTX and ATN2-Ms-PTX micelles (drug loading 4.8 wt. %).
  • A Schematic diagram of the preparation process of micelles;
  • B The particle size distribution of Ms-PTX and ATN2-Ms-PTX and the TEM schematic diagram of MS-PTX;
  • C MS-PTX with a drug loading of 23.1 wt.
  • PB physiological environment
  • PB containing 10 mM DTT intracellular reducing environment
  • Figure 4 shows the endocytosis of Cy5-labeled Ms-PTX, AT1-Ms-PTX, ATN2-Ms-PTX and cRGD-Ms-PTX by 4T1 cells by flow cytometry (A) and CLSM (B&C) (incubated for 4 h )Case.
  • B Semi-quantitative fluorescence intensity for Cy5.
  • PhScNK polypeptide (ATN2) was used to incubate 4T1 cells for 2 h before adding Cy5/ATN2-Ms-PTX.
  • the Cy5 polymer concentration was 2 ⁇ g/mL. Scale bar is 25 ⁇ m.
  • Figure 5 shows the preparation of CPs-CDN (A) and the particle size change of 1 mg/mL CPs-CDN at 4°C for 3 weeks and in PB containing 10% FBS (B).
  • A Flow cytometry false-color plots of representative samples of BMDC (within the CD11c + total BMDC gate) and their statistical analysis (B). Concentrations of (C) IFN- ⁇ , (D) TNF- ⁇ and (E) IL-6 in the medium.
  • Ratio of mature DC CD45 + CD80 + CD86 +
  • A and M1M (CD11b + F4/80 + CD206 ⁇ )
  • B Representative flow cytometry of CD3 + CD4 + and CD3 + CD8 + T cells
  • C CD3 + CD4 +
  • D CD3 + CD8 +
  • E T cell ratios.
  • D The content of Tregs (CD3 + CD4 + FoxP3 + , within the CD45 + gate) in CD4 + T
  • E the ratio of CD8+ T/Tregs.
  • H Concentrations of cytokines in serum.
  • Figure 10 is the flow cytometry analysis of mouse spleen 24 hours after the administration of ATN2-Ms-PTX and its combination with CPs-CDN.
  • A Representative flow cytometry false-color plots of CD3 + CD4 + , CD3 + CD8 + T cells (within the CD45 + total immune cell gate) and
  • B CD3 + CD4 + and
  • C CD3 + CD8 + T cells Cell ratio statistics.
  • n 4.
  • Figure 12 is H&E of mouse heart, liver, kidney and spleen slices after PBS, free PTX, Ms-PTX, free CDN, CPs-CDN, ATN2-Ms-PTX and their combination with CPs-CDN treated tumor-bearing mice for 22 days Staining map (40 ⁇ , the scale bar is 50 ⁇ m).
  • WP white pulp
  • RP red pulp.
  • Figure 13 is the pictures (A) and mass (B) of the spleens of mice administered free PTX, Ms-PTX, free CDN, CPs-CDN, ATN2-Ms-PTX and their combination with CPs-CDN on day 22.
  • n 3.
  • Figure 14 is the in vitro imaging (A), semi-quantitative fluorescence value (B) and mass (C) of the lungs of tumor-bearing mice 22 days after administration.
  • ⁇ -caprolactone ( ⁇ -CL, 99%, Alfa Aesar) was dried over calcium hydride and distilled under reduced pressure for use.
  • 1,2-Dithiolane trimethylene carbonate (DTC) was synthesized and purified by our laboratory.
  • Cyclic peptide c(RGDfC) (cRGD), Ac-PHSCNK-NH 2 (ATN-1) and Ac-PhScNK-NH 2 (ATN-2) were all more than 98% pure and were purchased from Shanghai Qiangyao Biotech. Reagents for high performance liquid chromatography (HPLC) were purchased from Sigma Aldrich (USA).
  • Micro BCA Kit (Pierce, Thermo Scientific), Enhanced ATP Detection Kit (S0027, Beyond), mouse high mobility group box B1 (HMGB-1) (E-EL-M0676c, Elabscience), mouse white blood cells Interferon-6 (IL-6) ELISA kit (JN-029562-S, Shanghai Research Field), mouse interferon- , Interferon- (IFN- , IFN- ) and cell necrosis factor- (TNF- ) ELISA detection kit (Invivogen), calreticulin (CRT) antibody (Ab2907, Abcam), and mouse fluorescently labeled various antibodies (Biolegend) CD45-PerCP/Cyanine5.5, CD80-APC, CD86-PE, Kits and antibodies such as CD11c-FITC, CD11b-FITC, CD206-Alexa Fluor 647, F4/80-PE, CD4-PE, CD8-FITC, FoxP3-Alexa Fluor 647 and goat anti-rabbit-Alexa
  • the proton nuclear magnetic resonance spectrum ( 1 H NMR) of the polymer was detected by a 400 MHz superconducting NMR spectrometer (Unity Inova, Agilent) or a 600 MHz superconducting NMR spectrometer (DirectDrive2, Varian) with deuterated chloroform (CDCl 3 ) or deuterated dimethyl sulfoxide (DMSO-d 6 ) as the solvent, and the chemical shift takes the solvent peak as the reference standard.
  • Polymer molecular weight and molecular weight distribution were determined by Waters 1515 gel permeation chromatography (GPC), with DMF as the mobile phase (flow rate 0.8 mL/min), the test temperature was 40 °C, and a series of monodisperse linear polymethacrylic acid Methyl esters were used as molecular weight standards.
  • the particle size and particle size distribution of polymer micelles and vesicles were determined by a ZetaSizer Nano-ZS nanoparticle sizer (Malvern Instruments) equipped with a He/Ne laser light source (wavelength 633 nm) and a 173 ° back-refraction detector, temperature at 25oC.
  • micellar samples The microscopic morphology of the micellar samples was determined by a Tecnai G220 transmission electron microscope (TEM) at an accelerating voltage of 120 kV, and the samples were stained with 1% phosphotungstic acid solution.
  • HPLC high-performance liquid chromatograph
  • TCS SP5 confocal laser scanning microscope (CLSM, Leica) was used to study the endocytic behavior of micelles.
  • Flow cytometry FACS Calibur, BD Biosciences was used to study micellar endocytic behavior and qualitative and quantitative analysis of immune cells in vivo.
  • a microplate reader (Thermo Multiskan FC) was used to measure the absorbance at 570 nm of purple formazan formed by living cells and MTT.
  • a multifunctional microplate reader (Varioskan LUX, Thermo Scientific) was used for ELISA detection of cytokines.
  • the present invention designs and prepares PTX and CDN immune adjuvant in nano dosage form, and combines them in TNBC with mouse 4T1 as a model for chemical immunotherapy.
  • targeted, disulfide-crosslinked PTX micellar nanomedicine based on PEG-P (CL-DTC) was prepared to increase the concentration of PTX in tumor cells, improve curative effect, and reduce toxic and side effects.
  • CDN is difficult to enter antigen-presenting cells (APCs) and endoplasmic reticulum due to its small molecular weight, easy degradation, and strong water-solubility in negatively charged STING protein binding to activate the STING pathway.
  • APCs antigen-presenting cells
  • the present invention prepared a PEG-P(TMC-DTC)-Sp polymersomes with asymmetric membrane structure to load, protect and deliver STING agonist cyclic dinucleotide ADU-S100 (CPs-CDN) to APCs for better Activate anti-tumor immune response.
  • CPs-CDN STING agonist cyclic dinucleotide ADU-S100
  • PTX micelles targeting TNBC are used to efficiently deliver PTX to the tumor, kill some tumor cells and induce immunogenic cell death (ICD), produce tumor-associated antigens and be effectively endocytosed by APC, thereby increasing the activation of CTL; Then intratumoral injection of CPs-CDN activates the STING pathway, further activates APCs including DCs in the tumor, enhances their ability to present antigens, recruits and activates T cells to generate anti-tumor immune responses.
  • ICD immunogenic cell death
  • mice The experimental results show that ATN2-Ms-PTX can be efficiently targeted and enriched in mouse 4T1 tumors, which increases the concentration of PTX in tumor cells, and the sequential delivery of CPs-CDN activates the STING pathway, which significantly delays the growth of mouse tumors , Inhibit lung metastasis and prolong the survival period of mice.
  • the raw materials of the present invention are all existing products, and the specific operation method and test method are conventional methods in this field. All data of the present invention are all presented with average value, and intergroup difference is assessed by ANOVA single-factor analysis of variance, and *p ⁇ 0.05 is considered to have significant difference, **p ⁇ 0.01 and ***p ⁇ 0.001 were considered highly significant differences.
  • MeO-PEG-OH 800 mg, 0.4 mmol
  • DPP 1.0 g, 4 mmol
  • DTC 400 mg, 2.1 mmol
  • DTC 400 mg, 2.1 mmol
  • CL 400 mg, 3.5 mmol
  • anhydrous DCM 3.2 mL
  • ATN-PEG-P(CL-DTC) is obtained by amidation reaction between PHSCNK or PhScNK polypeptide and NHS-PEG-P(CL-DTC). Briefly, NHS-PEG-P(CL-DTC) (132 mg, 0.024 mmol) was dissolved in 1 mL of anhydrous DMSO, and PHSCNK peptide or PhScNK peptide (20 mg, 0.028 mmol) was dissolved in 0.5 mL of anhydrous DMSO.
  • the polymer solution was added dropwise to the polypeptide solution at 30 °C, and after 30 min of dropping, 7 ⁇ L of triethylamine was added to adjust the pH of the system to 8.0, and the reactor was sealed and then reacted at 30 °C for 48 h.
  • the reaction solution was put into a dialysis bag (MWCO 3500 Da) and dialyzed in DMSO for 4 h, DCM for 2 h, and then precipitated twice in 30 times volume of ice ether/absolute ethanol (9/1, v/v) , centrifuged and vacuum-dried for 24 h to obtain ATN-PEG-P(CL-DTC).
  • cRGD-PEG-P(CL-DTC) is prepared from the sulfhydryl group of c(RGDfC) polypeptide and Mal-PEG-P(CL-DTC) through Michael addition reaction. The steps, purification and characterization of the polymer are the same as above. The response is as follows: .
  • PTX and PEG 2k -P(CL 1k -DTC 1k ) were dissolved in PEG 350 according to the mass ratio (1/20), and the polymer concentration was kept at 1 mg/mL.
  • 100 ⁇ L of the mixed solution was poured into the bottom of 900 ⁇ L of PB solution (pH 7.4, 10 mM) at 37 °C, and stood without stirring during the process.
  • PB solution pH 7.4, 10 mM
  • Example 1 Chemotherapy-immunization combination drug preparation of PTX micelles: PTX and PEG 2k -P(CL 1k -DTC 1k ) in different mass ratios (5/100, 10/100, 20/100, 30/100) Soluble in PEG 350 with a polymer concentration of 50 mg/mL. After conventional ultrasonication for 5 min, 100 mL of the mixture was poured into the bottom of 900 mL of PB solution (pH 7.4, 10 mM) at 37 °C, and stood without stirring during the process. After injection, use a pipette gun to stick to the liquid surface and pipette 5 times to obtain micellar MS-PTX with a PTX drug loading of 4.8 wt.
  • PB solution pH 7.4, 10 mM
  • % to 23.1 wt. % which is a non-targeting small micellar nano drug.
  • the particle size and particle size distribution (PDI) were determined by DLS, and the micelle morphology was determined by TEM.
  • MS-PTX polymer concentration: 0.1 mg/mL
  • MS-PTX polymer concentration: 0.1 mg/mL
  • acetonitrile containing 20 mM DTT was dissolved in acetonitrile containing 20 mM DTT, and then the PTX concentration was tested by HPLC, and the drug loading and drug loading efficiency were calculated.
  • DLS was used to monitor the particle size and particle size distribution of PTX micelles stored at 25 and 37°C, diluted to a low concentration (20 mg/mL) and in a solution containing 10% FBS at different time points, see Table 1.
  • PEG-PCL-based PTX-loaded micelles were also prepared by the same method.
  • the drug loading capacity of PTX nanocarriers reported in most literatures in the prior art is relatively low.
  • the present invention utilizes a small amount of PEG350 to dissolve the polymer PEG-P (CL-DTC) and PTX, and the mixture is added to the water phase (the final volume content of PEG350 is 10%), and the polymer forms the micelles of the core-shell structure through the hydrophobic interaction.
  • PTX is wrapped in the hydrophobic core, and DTC in the core can quickly self-crosslink, which can fix PTX in the micelle core more stably, and obtain the PTX micellar nanomedicine MS-PTX ( Figure 1A).
  • the PEG350 used here is non-toxic and FDA-approved for use.
  • the present invention prepared micellar MS-PTX with drug loading from 4.8 wt. % to 23.1 wt. % by this method, with a particle size range of 30-38 nm and a particle size distribution of 0.13-0.17 (Table 1).
  • DLS test results showed that MS-PTX loaded with 4.8 wt. % had a particle size of 32.7 nm and a PDI of 0.16, and the TEM image showed that it had a more regular shape of solid spheres (Fig. 1B).
  • MS-PTX micelles remained clear and transparent, with a particle size of 38.1 nm and a PDI of 0.13 (Table 1).
  • micellar nanomedicine Due to the low water solubility of PTX (water solubility is 5.56 mg/L), the same amount of PTX was dissolved in PEG350 and directly added to water, PTX precipitated rapidly, while MS-PTX loaded with 23.1 wt. % PTX remained clear, indicating that PTX is fully loaded inside the micelles (Fig. 1C).
  • Using this method to prepare micellar nanomedicine is simple, reproducible, and the stability of the micelles is good. After three weeks of storage at room temperature, the particle size and particle size distribution of the micelles are basically unchanged ( Figure 1 D), and there is no precipitation.
  • the 330 nm ultraviolet absorption value of the dithiolane of DTC in the nanoparticles decreased a lot, indicating that the sulfur-sulfur cross-linking reaction occurred to obtain cross-linked nanoparticles.
  • PEG350 since there is no dialysis in the preparation of the micelles, PEG350 still exists, and it is unpredictable whether cross-links can be formed in the micelles core after preparation.
  • the present invention firstly measures the ultraviolet absorption of two kinds of micelles and corresponding polymer solution (5 mg/mL) with the same concentration, and finds that the PEG-PCL polymer solution and its micellar ncMs have no obvious absorption peak.
  • the DTC in Ms showed the ultraviolet absorption peak of dithiolane at 326 nm, but its intensity was lower than that of DTC in the corresponding polymer solution (Fig. 1 E), which decreased by 40.7%. This result indicated that some dithiolane rings in the Ms micelles were ring-opened to form intranuclear self-crosslinks.
  • pyrene as a fluorescent dye to determine the critical micelle concentration (CMC) of the two micelles, it was found that the CMC of the ncMs was 15.6 mg/mL, but the Ms ranged from 1.2 mg/mL to 2.5 mg/mL in the polymer concentration No obvious transition was found for I 1 /I 3 (Fig. 1F), i.e.
  • micellar MS-PTX based on PEG-P(CL-DTC) of the present invention is much more stable than Genexol-PM based on PEG-PDLLA with much higher Tg .
  • Preparation of polymer vesicle CPs-CDN loaded with STING agonist CDN The synthesis of PEG-P(TMC-DTC)-SP can refer to the applicant’s published patents or articles, and the specific preparation method is the prior art.
  • Use polyethylene glycol-poly(trimethylene carbonate-dithiolantrimethylene carbonate)-spermine (PEG-P(TMC-DTC)-Sp, M n 5.0-(15-2)- 0.2 kg/mol)
  • CDN-loaded polymersomes were prepared by the solvent replacement method, and the CDN was selected from ADU-S100.
  • the characterization methods of CPs-CDN particle size, PDI and stability are the same as those of PTX micelles.
  • the drug loading capacity (DLC) and drug loading efficiency (DLE) of CDN were calculated based on the standard curve by measuring the absorbance value at 260 nm of NanoDrop 2000 in the oligo DNA mode.
  • PTX micelles and polymer vesicles CPs-CDN loaded with STING agonist CDN constitute a chemotherapy-immune combination drug.
  • Ms-PTX remained stable in PB buffer containing 10% FBS (simulating in vivo blood environment), and the particle size remained unchanged for 24 h (Fig. 2 A); however, in PB solution containing 10 mM DTT (simulating cytoplasmic In reducing environment), large particles of 500-1000 nm appear quickly in 2 h, and small particles of more than ten nanometers appear in 4 h (Fig. 2 B). This is because the disulfide bond in the hydrophobic core is reducing The cross-linking is gradually decomposed under the conditions, the micelles swell, and they will dissociate into single molecules at low polymer concentrations. This indicates that the micelles have fast reduction response properties.
  • Example 2 The targeting micelles loaded with PTX are composed of amphiphilic block polymer PEG 2k -P(CL 1k -DTC 1k ) and polymer Ta-PEG-P(CL-DTC) coupled with targeting molecules (Ta is the polypeptide cRGD, ATN1 or ATN2) self-assembled in the aqueous phase.
  • ATN1-PEG-P (CL-DTC), ATN2-PEG-P (CL -DTC) or cRGD-PEG-P (CL-DTC) as the initial polymer solution, then mixed with the PTX solution, all the other steps are the same as in Example 1, to obtain micelles ATN1-MS-PTX, ATN2 with different polypeptide surface densities -MS-PTX and cRGD-MS-PTX, for targeting small micellar nanomedicine.
  • targeting micelles were prepared, and the same method as in Example 1 was used to keep the PTX drug loading at 4.8 wt. %.
  • Three series of micellar cRGD-MS-PTX, ATN1-MS-PTX and ATN2-MS-PTX with different targeting molecule densities were prepared respectively. DLS test results showed that when the surface peptides did not exceed 5%, the micellar particle size remained basically unchanged with the increase of peptides, and the PDI remained around 0.2 between 31 and 35 nm (Table 3).
  • ATN2-MS-PTX with a drug loading of 4.8 wt. % and a peptide density of 5% as an example, the effects of PEG350 and DTC on micellar stability, FBS stability, and freeze/lyophilization reconstitution were studied. The effect on particle size was found to be the same as for non-targeted MS-PTX.
  • the drug release behavior of PTX micelles in the reduction response environment was studied in simulated cells, using PB solution (containing 0.1% Tween 80) with or without 10 mM DTT as the release medium, taking ATN2-MS-PTX as an example The cumulative amount of PTX released into the medium at different time points was shown.
  • amphiphilic block polymer PEG 2k -P (CL 1k -DTC 1k ) and the polymer Ta-PEG 2k -P (CL 1k -DTC 1k ) coupled with targeting molecules (weight percent content of 5%) self-assembled in the aqueous phase to form PTX-loaded targeting micelles;
  • Ta is the polypeptide cRGD, ATN1 or ATN2 corresponding to particle sizes of 27 nm, 29 nm and 28 nm, respectively.
  • the invention prepares micelles with high drug loading of PTX, and the prepared micelles with small particle size (20-40 nm) can effectively penetrate deep into the tumor, especially during the drug loading process of the invention, there is no drug precipitation, static There is no drug leakage even after three weeks, and the small molecular weight PGE used does not need to be removed.
  • Example 3 MTT test to evaluate the cytotoxicity of PTX micelles the specific test method is an existing method, and you can refer to the invention application titled "a small micellar nanomedicine and its preparation method and application" submitted by the applicant on the same day.
  • the present invention is consistent with it.
  • the cells were selected from mouse highly metastatic triple-negative breast cancer cell line 4T1, and the PTX concentration ranged from 0.002 to 5 ⁇ g/mL.
  • the present invention takes murine triple-negative breast cancer 4T1 cells as the research object, and uses the MTT method to study the cytotoxicity of three kinds of micelles with different targeting molecule densities.
  • ATN2-MS-PTX is slightly more toxic than cRGD-MS-PTX, much higher than ATN1-MS-PTX; 5% cRGD-MS-PTX, 20% ATN1-MS-PTX and 5%ATN2-MS-PTX had the lowest IC 50 values relative to other polypeptide density micelles in each group, which were 0.27, 0.46, and 0.21 ⁇ g/mL (Table 4), among which 5%ATN2- The IC 50 value of MS-PTX was the lowest, even lower than the IC 50 value (0.55 ⁇ g/mL) of free PTX co-incubated with 4T1 cells for 48 h.
  • cytotoxicity of 5% ATN2-MS-PTX co-incubated with 4T1 cells for 24 h and 48 h was further investigated (IC 50 values were 0.54 and 0.020 ⁇ g/mL, respectively), and it was found that the IC 50 values were higher than those of micelles without target (respectively 2-3 times lower than 1.19 and 0.064 ⁇ g/mL).
  • co-incubation of empty micellar Ms and ATN2-Ms at a concentration of 1 mg/mL for 48 h did not show toxicity to 4T1 cells, which indicated that the nanocarriers prepared by this method had good biocompatibility.
  • Example 4 Study on the endocytic behavior of PTX micelles use flow cytometry FACS and CLSM to investigate the uptake of PTX micelles by 4T1 cells, through the amide of NHS-PEG-P (CL-DTC) and Cy5-NH 2 Cy5-labeled PEG-P (CL-DTC) was obtained through the reaction, and it was doped into the original polymer at 1% to obtain Cy5-labeled micelles.
  • the specific test method is an existing method, and you can refer to the invention application titled "a small micellar nanomedicine and its preparation method and application" submitted by the applicant on the same day, and the present invention is consistent with it.
  • the Cy5-labeled micelles were prepared legally with water, and the optimal targeting densities in Table 4 were selected for different targeting densities.
  • M n 5.0-(15-2)-0.2 kg/mol).
  • Example 5 Characterization of STING agonist-loaded polymersome CPs-CDN Since STING proteins generally reside in APCs, to activate the STING pathway, its agonists such as cyclic dinucleotides (CDNs) must be delivered to APCs Inside. However, CDN has small molecular weight, negative charge, strong hydrophilicity, poor ability to enter the cytoplasm, and is easy to degrade, resulting in low bioavailability and greatly reduced effect.
  • CDNs cyclic dinucleotides
  • the present invention designed DTC-based reduction-sensitive, disulfide-crosslinked polymersomes to achieve this goal, using the polymer PEG-P(TMC-DTC)- Polymeric vesicles (CPs) with asymmetric membrane structure formed by Sp to deliver CDN.
  • PEG-P(TMC-DTC)- Polymeric vesicles CPs
  • ADU-S100 which is currently in phase II clinical trials, was selected as the model CDN for research.
  • the CDN-loaded vesicle CPs-CDN was prepared by solvent replacement method (Figure 5 A), and the loading efficiency of CDN was determined by NanoDrop 2000.
  • the chemotherapy-immune combination drug was composed of 5% ATN2-Ms-PTX and CPs-CDN (the theoretical drug loading of CDN was 13.0 wt. %), and the cell and animal experiments were carried out.
  • BMDC (1 ⁇ 10 6 /well) were cultured in a 12-well plate for 24 h and then added with PBS, PTX, Ms-PTX, ATN2-Ms- PTX, CDNs, and CPs-CDN were incubated for 24 h.
  • ATN2-Ms-PTX was first added for 8 h, and then CPs-CDN was added for 16 h.
  • STING agonist CDN enters DC and other APCs and binds to STING protein on the endoplasmic reticulum, inducing the production of cytokines such as type I interferon (IFN- ⁇ ), thereby activating the maturation of related immune cells and presenting antigens to T cells, recruiting more Many immune cells enter the tumor area; but it is unpredictable whether the CDN in CPs-CDN can still achieve such a function because it is installed in the vesicle.
  • the present invention detects the CD11c + DC of CPs-CDN and free CDN and BMDC after incubation for 16 h cells and the proportion of mature BMDCs that were CD11c + CD86 + CD80 + .
  • ATN2-Ms-PTX can induce ICD, produce a series of tumor antigens, and enable BMDC proliferation and maturation. Therefore, treatment with ATN2-Ms-PTX can make BMDC proliferate, and then combined with CPs-CDN can also increase the amount of CDN entering DC, which will enhance the activation of the STING pathway, thereby increasing the maturation of BMDC; on the other hand, ATN2-Ms -PTX-induced TAAs are more efficiently presented to T cells by these activated APCs, thereby recruiting more T cells.
  • the combination of ATN2-Ms-PTX can further increase the maturation of BMDC, and the BMDC of CD11c + CD80 + CD86 + is as high as 90.2%, which is significantly higher than that of any one alone (*p/***p).
  • IFN- ⁇ , TNF- and IL-6 concentrations The increase of cytokines indicates that BMDCs are mature and have enhanced antigen presentation ability, which can strongly activate T cells.
  • IFN- ⁇ is a marked cytokine produced after the combination of CDN and STING, which plays a key role in the activation of T cells induced by tumors.
  • the cells were treated with the combined group of ATN2-Ms-PTX and then CPs-CDN, the secreted IFN- ⁇ , TNF- and IL-6 concentrations were significantly higher than those in CPs-CDN group, ATN2-Ms-PTX group (*p/***p) and free CDN group (**p/***p) (Fig. 6C, D&E) .
  • CPs-CDN improves the function of CDN on DC activation, and the combination with ATN2-Ms-PTX can more significantly promote the proliferation and activation of BMDC, activate the STING pathway, induce IFN- ⁇ and other cytokines, and more effectively promote BMDC mature and present antigens to T cells.
  • Example 7 PTX micelles combined with CPs-CDN for chemoimmunotherapy of mouse TNBC: Balb/c mice bearing 4T1 subcutaneous tumors were used as a mouse model of triple-negative breast cancer to investigate the efficacy and administration of Ms-PTX Methods and the effect of combined use with CPs-CDN on tumor suppression, administration was started when the tumor volume of 15 tumor-bearing mice was 200-250 mm 3 , and the day of administration was defined as day 0. Divided into 5 groups, 3 mice in each group, administered every 2 days, 3 times in total.
  • Group 1 Ms-PTX tail vein (iv 10 mg PTX/kg, 200 ⁇ L);
  • Group 2 Ms-PTX intratumoral injection (it 5 mg PTX/kg, 50 ⁇ L);
  • Group 3 Ms-PTX tail vein (iv iv 10 mg PTX/kg, 200 ⁇ L) 2 hours after intratumoral injection of CPs-CDN (1 mg CDN/kg);
  • Group 4 intratumoral injection of Ms-PTX (it 5 mg PTX/kg, 50 ⁇ L) After 2 hours, CPs-CDN (1 mg CDN/kg) was injected into the tumor; group 5: PBS.
  • the relative body weight of mice is the percentage of the body weight at the time of measurement to the body weight on day 0 (M/M 0 ). It was found that whether Ms-PTX was injected intravenously or intratumorally, the tumor volume continued to increase, which was similar to the growth curve of the PBS group, which was related to the large initial tumor volume and the high degree of malignancy and invasiveness of the tumor.
  • both methods When administered in combination with CPs-CDN, both methods have significantly more tumor-inhibitory effects than Ms-PTX alone, and the Ms-PTX iv + CPs-CDN it group has the most significant tumor inhibition (**p/***p ), the tumor volume did not increase in the first 10 days, and began to grow slowly after 10 days (Fig. 7A). The tumors in the Ms-PTX it + CPs-CDN it group had been growing slowly. This is related to the high concentration of PTX injected into the tumor of Ms-PTX, which kills tumor cells and immune cells at the same time.
  • CPs-CDN cannot be endocytosed by many APCs, the overall STING activation degree is poor, and the resulting immune response weaker.
  • the amount of Ms-PTX enriched in the tumor in a short period of time is relatively less, the concentration of PTX is low, and it is non-toxic to immune cells, but it can induce tumor cell ICD and make DC proliferate; After that, a strong immune response can be elicited in a large number of DCs.
  • the body weight of the mice in the Ms-PTX iv + CPs-CDN it group decreased during the administration period and then recovered (Fig. 7B).
  • the nine groups are: PBS, free PTX (iv), Ms-PTX (iv), ATN2-Ms-PTX (iv), free CDN (iv), CPs-CDN (it), three ATN2-Ms-PTX ( iv) + CPs-CDN (it) combination group, ATN2-Ms-PTX (iv) was given 2 h, 8 h or 24 h after administration of CPs-CDN (it). The mouse body weight, tumor volume, and mouse status were monitored every 2 days during the experiment. Mice died or the tumor volume was greater than 2000 mm 3 were judged to be dead, and the survival curve was drawn.
  • ATN2-Ms-PTX Inject tumor-targeted ATN2-Ms-PTX into the tail vein of tumor-bearing mice (tumor volume about 50 mm 3 ) every 2 days, and reduce the drug dose to 7.5 mg/kg, and administer 4 times in total; - After Ms-PTX administration, intratumoral injection of CPs-CDN (1 mg/kg) was performed to study the effect of this regimen on the tumor size, body weight and survival period of the mice. When the tumor volume was greater than 2000 mm 3 , the mice were judged to be dead (indicated by the # sign). The antitumor effect of this regimen was compared with single administration of ATN2-Ms-PTX, Ms-PTX, free CDN or CPs-CDN. In addition, the effect of the dosing interval (2, 8 or 24 h) between ATN2-Ms-PTX and CPs-CDN on the antitumor effect was also investigated (Fig. 8A).
  • the free PTX group the tumors grew slowly during the period of administration, and began to grow rapidly after stopping the administration.
  • ATN2-Ms-PTX, Ms-PTX, free CDN, CPs-CDN and combined administration groups can significantly inhibit the growth of tumors.
  • the tumors basically did not grow within 10 days, but the tumors began to grow slowly after 10-12 days.
  • Ms-PTX may have a significant EPR effect due to its small particle size, and it can inhibit tumor growth more effectively than PTX (*p).
  • ATN2-Ms-PTX can further restrict tumor growth (***p) than PTX and Ms-PTX (Fig. 8 B), and prolong the survival time of mice, reflecting the targeted anti-tumor effect.
  • intratumoral administration of CDN or CPs-CDN can also slow down the tumor growth rate, the degree of CDN slowing down is similar to that of MS-PTX, CPs-CDN is similar to ATN2-Ms-PTX, but CPs-CDN has more significant advantages compared with free CDN ( **p), again indicating that the vesicles delivered CDN more efficiently, improving its bioavailability and ability to activate STING channels (Fig. 8B).
  • the median survival period of the mice in the combination group with an interval of 2, 8, and 24 hours was 30, 32, and 33 days, respectively (18 days in the PBS group) (Fig. 8C).
  • the effect of combined administration with an interval of 2 h was poor, which may be due to the short interval, the small release of PTX and/or the insufficient degree of induction of ICD and promotion of DC proliferation, resulting in weak antigen presentation by APC.
  • mice Inhibition or insufficiency of a certain item in the circulation process will prevent the anti-tumor immunity from reaching its optimal state.
  • body weight of the mice changed little during the treatment period, and the tail vein was given 7.5 mg PTX/kg and/or intratumoral administration 1 None of the mg/kg CPs-CDN produced toxic side effects on mice (Fig. 8D).
  • Example 8 Regulation of ATN2-Ms-PTX and its combination with CPs-CDN on the immune system of tumor-bearing mice: As described in Example 7, tumor-bearing mice were randomly divided into 7 groups, 7 in each group. The seven groups are: PBS (iv), free PTX (iv), Ms-PTX (iv), ATN-Ms-PTX (iv), free CDN (iv), CPs-CDN (it), ATN-Ms-PTX (iv) + CPs-CDN (it) (interval 8 h), administered 4 times at 7.5 mg PTX/kg and/or 1 mg CDN/kg, once every 2 days.
  • mice in each group were sacrificed, and the whole blood of the mice was collected to separate the serum for the determination of IFN- ⁇ and IFN- , IL-6 and TNF- content.
  • the spleen and tumor were collected, ground into a single cell suspension, and counted after cracking.
  • 6 x 106 cells per sample were stained for T cells.
  • each sample was divided into 4 parts, each with 6 ⁇ 10 6 cells, stained for DC, macrophages, and T cells, and analyzed by FACS test.
  • the corresponding fluorescently labeled antibodies of immune cells are: DC: anti-CD11c-FITC, anti-CD80-APC, anti-CD86-PE; macrophage: anti-CD11b-FITC, anti-F4/80-PE, anti- CD206- Alexa Fluor 647; T cells: anti-CD3-APC, anti-CD4-PE, anti-CD8-FITC; Tregs: anti-CD3-FITC, anti-CD4-PE, anti-Foxp3-Alexa Fluor 647.
  • the tumor immune microenvironment is an environment composed of malignant cells, immune cells, blood vessels, extracellular matrix and signaling molecules, which can act alone or jointly affect the sensitivity of immunotherapy. It has been proved above that ATN2-Ms-PTX and CPs-CDN combined drugs can enter tumor cells to induce ICD, release tumor-associated antigens, create a favorable immune microenvironment, enter APC and activate STING channel to make DC mature. Excellent immune effect has been demonstrated.
  • ATN2-Ms-PTX, CPs-CDN and their combination on the TME of mice and T cells in the spleen after 24 hours of administration according to the scheme in Figure 8.
  • Figure 9A shows that the proportion of mature DC in PTX micelles is higher than that in PBS, and the proportion of CDN micelles is higher.
  • the mature DC in CPs-CDN group is 52.5% was higher than that of free CDN (48.7%); while mature DC in the combined group (62.9%) was significantly higher than that in the single-drug group (*p).
  • PTX can convert macrophages from M2M to M1M.
  • ATN2-Ms-PTX, CPs-CDN and combined administration all promoted the polarization of M2M to M1M ( Figure 9B).
  • T cells are immune cells that attack and kill tumors.
  • the T cell subsets involved in anti-tumor immunity are divided into two categories: CD8 + cytotoxic T cells (CTL) and CD4 + helper T cells (T h cells), while Th can also produce lymphokines to enhance the function of CTL, activate macrophages, DCs or other APCs, and produce tumor necrosis factor to play an oncolytic effect.
  • CTL cytotoxic T cells
  • T h cells helper T cells
  • FIG. 9 CE the results show that the CD4 + T and CD8 + T cells in the ATN2-Ms-PTX+CPs-CDN combination group are significantly higher than those of ATN2-Ms-PTX and CPs-CDN (CD4 + T: ***p and **p; CD8 + T: *p).
  • T reg regulatory T cells in T h are the main type of T cells that attenuate T cell activity and make TME immunosuppressive.
  • the CD8 + T/T reg in the CPs-CDN group was significantly higher than that in the free group CDN (***p).
  • the combination group can strongly stimulate the killing ability of T cells, and its CD8 + T/T reg is further enhanced, which is 14 times that of ATN2-Ms-PTX (***p) and 3.4 times that of CPs-CDN (*p).
  • ELISA test showed that the pro-inflammatory cytokine IFN- , IFN- ⁇ , TNF- ⁇ , and IL-6 levels were all increased (Fig. 9H), these results confirm that it can induce a strong anti-tumor immune response.
  • the spleen is the most important immune organ of the human body. It is rich in immune cells and has 25% of the body's lymphocytes. The immunoregulatory effect of the spleen on T lymphocytes is an important part of anti-tumor immunity.
  • the ratio of CD4 + and CD8 + T cells to CD45 + total immune cells in the spleen of mice after ATN2-Ms-PTX and its combined administration with CPs-CDN was analyzed.
  • Figure 10 shows that the change trends of CD4 + T and CD8 + T cells in the spleen of the mice on day 8 after drug administration in each group were consistent with those in the tumor, and the combination group accounted for as high as 18.7% and 6.3%, respectively.
  • Example 9 Toxicity study of the combination of ATN2-Ms-PTX and CPs-CDN on tumor-bearing mice: With the development of 4T1 breast cancer, the blood system and organs of mice will be affected; and long-term medication will also affect mice Bring systemic cumulative toxicity.
  • the PTX micelles and CDN vesicle preparations designed in the present invention and their combination on the systemic cumulative impact or the improvement of toxic and side effects on tumor-bearing mice were studied.
  • 3 mice were sacrificed in each group. Whole blood was collected for blood routine testing, serum was separated for blood biochemical testing, major organs were dissected for observation and sectioned, and H&E staining was used for histological analysis.
  • PLT platelet
  • PCT platelet volume
  • MPV mean platelet concentration
  • the blood routine indicators of the mice treated with free PTX or free CDN group were basically not relieved, and the blood routine indicators of the mice treated with ATN2-Ms-PTX or CPs-CDN single preparation were all relieved; while ATN2 -The total amount of WBC in the blood of mice treated with Ms-PTX combined with CPs-CDN, as well as Neut and Lymph, RBC, HGB, HCT, PLT, PCT and MPV in WBC were all close to those of healthy mice, further illustrating the effect of combined treatment on The tumor inhibition effect is obvious, the tumor metastasis is reduced, and anemia and thrombus formation in mice are avoided.
  • the H&E stained sections of the main organs of the tumor-bearing mice showed that the hearts, livers, and kidneys of the mice in each group had no obvious tissue damage.
  • the hearts of the mice in the PBS group, CDN and CPs-CDN groups had a little inflammation, while the hearts of the mice in the PTX preparation groups and the combination group were normal; Inflammation occurs.
  • the spleen of the mice in the PBS group was significantly enlarged and its mass increased (Figure 13). Yong et al. also found that the Balb/c mice bearing 4T1 breast cancer had splenomegaly.
  • the quality of the spleen of the mice in the treatment group was positively correlated with the size of the tumor, that is, the smaller the tumor, the smaller the spleen.
  • the spleen of the combination group was the smallest (about 0.15 g, which was close to that of healthy mice), which was significantly different from that of ATN2-Ms-PTX and CPs-CDN single preparation groups (***p and *p). From the H&E staining of the spleen (Figure 12), it can be seen that the white pulp was significantly reduced and the red pulp was increased in the PBS group, which is one of the signs of the development of the disease in 4T1 mice.
  • the treatment group especially the combination group, had abundant white pulp, which indicated that the development of the course of breast cancer was prevented.
  • the above results show that the tail vein to 7.5 mg PTX/kg and intratumoral injection of 1 mg CDN/kg and their combined administration, administered every 2 days for a total of 4 times, had no obvious toxicity to mice.
  • Example 10 ATN2-Ms-PTX combined with CPs-CDN inhibits lung metastasis of 4T1 breast cancer: On the 22nd day of administration, the mice were intraperitoneally injected with fluorescein potassium salt, and the remaining 3 mice in each group were killed within 10 minutes , Whole blood was taken for blood routine analysis, and serum was separated for blood biochemical analysis. In addition, mice were dissected, and organs such as heart, liver, spleen, lung, and kidney were taken, fixed with tissue fixative, sectioned, and sealed with paraffin. The sections were stained with hematoxylin and eosin (H&E), and the sections were observed under a microscope for histological analysis and judgment of lung metastasis.
  • H&E hematoxylin and eosin
  • the present invention also studies the inhibition of PTX micelles and CDN vesicles and the combination of ATN2-Ms-PTX and CPs-CDN on lung metastasis of breast cancer bearing 4T1-luc. Twenty-two days after the administration of the scheme in Figure 8, the lungs were dissected, weighed, imaged in vitro, and sectioned for histological analysis by H&E staining. The results of lung imaging and fluorescence quantification (Fig.
  • ATN2-Ms-PTX also has obvious lung metastasis inhibition (*p), indicating that ATN2 can be precisely targeted to 4T1.
  • mice in the ATN2-Ms-PTX and CPs-CDN combined group had almost no fluorescent signal (8.9 ⁇ 10 5 p/s); the lung mass of this group was the smallest, about 0.10 g, which was close to that of healthy mice; while the other The more lung metastases in the group, the larger the mass (Fig. 14C), proving that the combined treatment of ATN2-Ms-PTX and CPs-CDN can most effectively inhibit the lung metastasis of 4T1.
  • the H&E staining pictures of the lungs showed that there were a large number of tumor cells and inflammatory cell infiltration in the lungs of the PBS group and free PTX mice, and there was almost no alveolar structure; the Ms-PTX group was improved, and there was no obvious difference compared with ATN2-Ms-PTX There are tumor cell groups, but there are also infiltration of tumor cells and inflammatory cells, and the alveolar structure is not obvious. Compared with the free CDN group, the CPs-CDN group could see a large area of alveolar structure, with less tumor infiltration, but there was also inflammation.

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Abstract

本发明公开了一种化疗免疫联合药物及其制备方法与应用，设计了基于PEG-P(CL-DTC)的PTX胶束，研究了其与载干扰素基因刺激蛋白（STING）通路激动剂环二核苷酸（CDN）的具有不对称膜结构的囊泡（CPs-CDN）联用在治疗乳腺癌（TNBC）中的技术效果。本发明制备的PTX胶束方法简便、粒径小、载药量高，具有良好的稳定性及还原响应性，实验表明，在和CPs-CDN联用后，可促进更多BMDC成熟，肿瘤和脾脏内的CD4 + T和CD8 + T细胞显著增多，而抑制性T reg减少，使得肿瘤生长和肺转移受到明显抑制，小鼠生存期得以延长，体现了化疗免疫治疗的优势。

Description

一种化疗免疫联合药物及其制备方法与应用 技术领域
本发明属于纳米药物技术，具体涉及一种化疗免疫联合药物及其制备方法与应用。
 背景技术
化疗后期很多病人会出现耐药、癌细胞转移、肿瘤复发等不良预后反应，对于高度转移性肿瘤，如孕激素受体、雌激素受体和人表皮生长受体都不表达的三阴性乳腺癌（TNBC）患者几乎是束手无策。最近兴起的免疫疗法在肿瘤治疗上显示了巨大潜力，尤其是基于PD-1单抗、PD-L1单抗、或CTLA-4单抗的免疫检查点疗法，通过唤醒或恢复免疫细胞对肿瘤的免疫学监控来实现肿瘤治疗。但是单一免疫疗法如PD-1/PD-L1单抗只对20%-25%的患者有效，而对棘手的TNBC病人的客观响应率（ORR）只有12％-19％，是因为TNBC肿瘤细胞突变率低、免疫微环境弱、肿瘤浸润淋巴细胞水平低。
 技术问题
本发明公开了一种化疗免疫联合药物及其制备方法与应用，分别制备了偶联PHSCNK多肽（ATN1）、PhScNK 多肽（ATN2）或cRGD多肽的纳米药物ATN1-Ms-PTX、ATN2-Ms-PTX和cRGD-Ms-PTX；并且制备了基于PEG-P(TMC-DTC)-Sp的具有不对称膜结构的聚合物囊泡来装载、保护和递送环二核苷酸（CDN）ADU-S100（CPs-CDN）到APC，以更好地激活抗肿瘤免疫应答。实验结果表明，ATN2-Ms-PTX能高效靶向富集到小鼠肿瘤，增加了PTX在肿瘤细胞的浓度，其后顺序递送CPs-CDN激活了STING通路，明显延缓了小鼠肿瘤的生长、抑制了乳腺癌的肺转移，延长了小鼠的生存期。
 技术解决方案
本发明采用如下技术方案：一种化疗免疫联合药物，由化疗药物胶束与囊泡纳米STING激动剂组成；所述化疗药物胶束的制备方法为，将小分子药物、两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到化疗药物胶束；或者将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到化疗药物胶束；所述低聚乙二醇的分子量（ M n ）为200～600；所述囊泡纳米STING激动剂由聚合物囊泡装载STING激动剂组成。
本发明中，静置下，将所述混合溶液打入缓冲溶液中，然后搅拌、静置、涡旋、吹打或者倒置；打入为现有技术，比如用注射器或者注射泵；搅拌的转速为100～1000rpm。混合溶液中，小分子药物的浓度为0.1至10 mg/mL，聚合物浓度为1～100 mg/mL；聚合物为两亲性聚合物，或者聚合物为两亲性聚合物和靶向两亲性聚合物的混合物。
上述技术方案中，混合溶液中含有靶向两亲性聚合物时，靶向两亲性聚合物的用量为两亲性聚合物重量的1～30%，优选2～10%。本发明中，两亲性聚合物不偶联靶向分子，靶向两亲性聚合物偶联靶向分子。低聚乙二醇、缓冲溶液的体积比为1∶（5～40），优选1∶（10～30）。本发明制备了PTX载药量高的胶束，且制备的小粒径胶束（20-40 nm）能够穿透至肿瘤深处。
本发明公开了上述囊泡纳米STING激动剂的制备方法，将STING激动剂溶液加入缓冲溶液中，再加入聚合物溶液，搅拌后透析，得到囊泡纳米STING激动剂。STING激动剂溶液为STING激动剂水溶液；聚合物溶液为聚合物的DMF溶液；缓冲溶液为HEPES缓冲溶液。STING激动剂溶液的浓度为1～30 mg/mL；聚合物溶液的浓度为1～100 mg/mL。
上述技术方案中，小分子药物为紫杉醇PTX；所述低聚乙二醇的分子量（ M n ）为200～600。两亲性聚合物的分子量为3000～15000，靶向两亲性聚合物的分子量为2000～15000。本发明聚合物的分子量为核磁测定的数均分子量（ M n ），单位为Da。
本发明中，STING激动剂为环二核苷酸（CDN），包括环二单磷酸鸟苷（c-di-GMP）、环二单磷酸腺苷（c-di-AMP）、2`,3`环单磷酸鸟-单磷酸腺苷酸（2`,3`-cGMAP）、3`,3`环单磷酸鸟-单磷酸腺苷酸（3`,3`-cGMAP）及其取代衍生物，比如硫、氟、氮取代环二核苷酸衍生物。
上述技术方案中，两亲性聚合物为PEG-P(CL-DTC)、PEG-P(TMC-DTC)、PEG-P(LA-DTC)等；靶向两亲性聚合物为所述两亲性聚合物偶联靶向分子，优选的，靶向分子为多肽，比如PHSCNK多肽、PhScNK 多肽或cRGD多肽；两亲性聚合物中，PEG的分子量（ M n ）为1000～5000 Da。
上述技术方案中，制备囊泡纳米STING激动剂时，聚合物包括亲水链段、疏水链段以及阳离子片段；所述疏水链段的侧链为含双硫键的二硫戊环，其化学结构式如下：
Figure 61855dest_path_image002
。
R为亲水链段，比如聚乙二醇链段；T链段与侧链含双硫键的碳酸酯链段（DTC单体形成的单元）组成疏水链段，T为环酯单体或者环碳酸酯单体形成的单元，比如丙交酯、己内酯、三亚甲基碳酸酯；P为阳离子片段，比如聚乙烯亚胺（PEI）、精胺（SP）；y、z表示重复单元；所述亲水链段的分子量为2000～9000Da；疏水链段的分子量为亲水链段分子量的2.1～8.0倍；侧链含双硫键的碳酸酯链段的分子量为T链段分子量的10%～35%；阳离子片段的分子量为亲水链段分子量的5%～30%；比如所述聚合物为PEG-P(TMC-DTC)-Sp。
本发明制备了PTX载药量高的胶束，且制备的小粒径胶束（20-40 nm）能够穿透至肿瘤深处，并且CPs-CDN激活STING通路、激活并招募APC的顺序给药策略，可以更强地激活免疫系统，产生更多促炎细胞因子，使肿瘤和脾脏内的CD4 +、CD8 + T细胞比例显著增多、抑制性T reg减少，更好地抑制小鼠TNBC的生长和肺转移的发生。本发明公开了上述化疗免疫联合药物在制备抗肿瘤药物中的应用，优选肿瘤为三阴性乳腺癌（TNBC）。
有益效果
本发明首先设计制备了高效载PTX、双硫交联的胶束Ms-PTX以及三种TNBC主动靶向的胶束纳米药物，载药量高达23.1 wt.%，粒径在30～38 nm之间，具有良好的稳定性、还原响应的药物释放性能。细胞层面和动物层面的实验证实，ATN2-Ms-PTX对4T1细胞的靶向内吞作用最强，IC 50值最低，在肿瘤的生物分布最高。此外，ATN2-Ms-PTX可诱导ICD、促进BMDC增值以及成熟、促进BMDM向M1型巨噬细胞极化，产生有利的免疫微环境。此外，为探索进一步加强靶向PTX胶束的免疫治疗效果，制备了载STING激动剂CDN的双硫交联的囊泡CPs-CDN，以激活APC的STING通路，诱导更强的免疫应答。ATN2-Ms-PTX和CPs-CDN联合可促进BMDC的成熟，释放IFN-β和TNF-
Figure 320274dest_path_image003
等细胞因子。在荷4T1乳腺癌小鼠中，先静脉注射ATN2-Ms-PTX、诱导ICD释放肿瘤抗原，8-24 h后再瘤内注射CPs-CDN激活STING通路、激活并招募APC的顺序给药策略，可以更强地激活免疫系统，产生更多促炎细胞因子，使肿瘤和脾脏内的CD4 +、CD8 + T细胞比例显著增多、抑制性T reg减少，更好地抑制小鼠TNBC的生长和肺转移的发生，显著延长小鼠生存期，体现了化疗联合免疫治疗的优势，为治疗高转移性肿瘤带来了希望。
附图说明
图1为Ms-PTX和ATN2-Ms-PTX胶束的制备和理化性质（载药量4.8 wt.%）。（A）胶束的制备过程示意图；（B）Ms-PTX和ATN2-Ms-PTX的粒径分布以及MS-PTX的TEM示意图；（C）载药量为23.1 wt.%的MS-PTX（I）及相同PTX浓度的PB溶液（II）照片；（D）胶束在室温存放3周、制备后再透析后室温放置1 h的粒径变化；（E）空Ms、ATN2-Ms和ncMs以及相同浓度的聚合物溶液（5 mg/mL）的紫外吸收谱图；（F）胶束中芘的荧光吸收谱中I 372/I 383（I 1/I 3）比值随浓度的变化；（G）Ms-PTX和ATN2-Ms-PTX浓度为20 μg/mL的粒径，以及ncMs-PTX浓度为1 mg/mL、50 μg/mL的粒径及其在室温下放置2天的粒径。
图2为Ms-PTX和ATN2-Ms-PTX在37 度10%FBS中孵育24 h后的粒径分布（A），在10 mM DTT中孵育2 h后的粒径分布（B）；ATN2-Ms-PTX在模拟生理环境（PB，pH 7.4）和细胞内还原环境（含10 mM DTT 的PB）下PTX的累积释放（n=3）（C）。
图3为MTT法（n = 5）测定含不同多肽密度的（A）cRGD-Ms-PTX、（B）ATN1-Ms-PTX和（C）ATN2-Ms-PTX对4T1细胞的毒性；（D）自由PTX先孵育4 h、换新鲜培养基再孵育44 h（4+44 h）及共孵育48 h的细胞毒性；（E）空胶束Ms和ATN2-Ms共孵育48 h的细胞毒性。
图4为流式细胞仪（A）和CLSM（B&C）测试了标记Cy5的Ms-PTX、AT1-Ms-PTX、ATN2-Ms-PTX和cRGD-Ms-PTX被4T1细胞内吞（孵育4 h）的情况。（B）为Cy5半定量荧光强度。受体抑制实验用PhScNK多肽（ATN2）预先和4T1细胞孵育2 h再加Cy5/ATN2-Ms-PTX。Cy5聚合物浓度为2 μg/mL。标尺为25 μm。
图5为CPs-CDN的制备（A）以及1 mg/mL CPs-CDN在4℃放置3周和在含10%的FBS的PB中粒径变化（B）。
图6为BMDC的成熟（CD11c +CD86 +CD80 +）和培养基中细胞因子的浓度（n = 3）。自由CDN、ATN2-Ms-PTX、CPs-CDN及后两者联合和BMDC孵育24 h。（A）BMDC的代表性样品流式细胞仪伪色图（在CD11c +总BMDC圈门内）及其统计学分析（B）。培养基中（C）IFN-β、（D）TNF-α和（E）IL-6的浓度。
图7为Ms-PTX（i.v. 10 mg PTX/kg，i.t. 5 mg PTX/kg）及其给药后2 h联合CPs-CDN（i.t. 1 mg CDN/kg）治疗荷4T1小鼠（n = 3）的肿瘤体积（A）和相对体重（B）。
图8为ATN2-Ms-PTX胶束及其与CPs-CDN联合治疗荷4T1乳腺癌小鼠（7.5 mg PTX/kg，1 mg CDN/kg）的（A）抗肿瘤治疗方案；（B）肿瘤体积（#号代表肿瘤体积大于2000 mm 3死亡或自然死亡）；（C）小鼠的生存曲线；（D）小鼠体重。n = 7。
图9为ATN2-Ms-PTX及其与CPs-CDN联合给药结束24 h后小鼠肿瘤的流式细胞仪分析（n = 4）。成熟DC（CD45 +CD80 +CD86 +）（A）和M1M（CD11b +F4/80 +CD206 -）（B）的比例。CD3 +CD4 +和CD3 +CD8 + T细胞代表性流式细胞图（C）。CD3 +CD4 +（D）和CD3 +CD8 +（E）T细胞比例。（D）Tregs（CD3 +CD4 +FoxP3 +，CD45 +圈门内）在CD4 + T中的含量，（E）CD8+ T/Tregs比例。（H）血清中细胞因子的浓度。
图10为ATN2-Ms-PTX及其与CPs-CDN联合给药结束24 h后小鼠脾脏的流式细胞仪分析。（A）CD3 +CD4 +、CD3 +CD8 + T细胞代表性流式细胞伪色图（在CD45 +总免疫细胞圈门内）以及（B）CD3 +CD4 + 和（C）CD3 +CD8 + T细胞比例统计。n = 4。
图11为自由PTX、Ms-PTX、自由CDN、CPs-CDN、ATN2-Ms-PTX及其与CPs-CDN联合治疗后第22天小鼠的血常规（A）和血生化（B）分析（n = 3）。
图12为PBS、自由PTX、Ms-PTX、自由CDN、CPs-CDN、ATN2-Ms-PTX及其与CPs-CDN联合治疗荷瘤小鼠22天后小鼠心、肝、肾、脾切片的H&E染色图（40×，标尺为50 μm）。WP：白髓，RP：红髓。
图13为自由PTX、Ms-PTX、自由CDN、CPs-CDN、ATN2-Ms-PTX及其与CPs-CDN联合给药第22天小鼠的脾脏图片（A）和质量（B）。n = 3。
图14为荷瘤小鼠在给药22天后肺部离体成像（A）、荧光值半定量（B）和质量（C）。（D）肺H&E染色图。左图为10×，标尺为500μm；右图为40×，标尺为50μm。
 本发明的实施方式
甲氧基聚乙二醇（MeO-PEG-OH， M n = 2.0 kg/mol）、马来酰亚胺和琥珀酰亚胺酯官能化的聚乙二醇（Mal/NHS-PEG-OH， M n = 3.5 kg/mol）均购自北京键凯科技有限公司，经甲苯共沸蒸馏后使用。ε-己内酯（ε- CL, 99%，Alfa Aesar）经氢化钙干燥、减压蒸馏后使用。1,2-二硫戊环三亚甲基碳酸酯（DTC）由本实验室合成并提纯。磷酸二苯酯（DPP，> 99%，TCI）使用前真空干燥30分钟。环肽c(RGDfC)（cRGD）、Ac-PHSCNK-NH 2（ATN-1）和Ac-PhScNK-NH 2（ATN-2）的纯度都大于 98%，均购自上海强耀生物。高效液相色谱（HPLC）用试剂均从Sigma Aldrich (USA) 购买得到。紫杉醇（PTX，> 99%，上海金和生物制药有限公司）、甲氧基低聚乙二醇（PEG 350， M n = 350 g/mol，Sigma）、STING激动剂ADU-S100（> 99%，MCE）、谷胱甘肽（GSH，> 99%，Roche）和Cy5-NH 2（Lumiprobe）等试剂均是购买后直接使用。Micro BCA试剂盒（Pierce，Thermo Scientific）、增强型ATP检测试剂盒（S0027，碧云天）、小鼠高迁移率族蛋白B1（HMGB-1）（E-EL-M0676c， Elabscience）、小鼠白细胞介素-6（IL-6）ELISA检测试剂盒（JN-029562-S，上海研域）、小鼠干扰素-
Figure 631170dest_path_image004
、干扰素-
Figure 432904dest_path_image005
（IFN-
Figure 778434dest_path_image004
、IFN-
Figure 7421dest_path_image005
）和细胞坏死因子-（TNF-
Figure 805613dest_path_image003
）ELISA检测试剂盒（Invivogen）、钙网蛋白（CRT）抗体（Ab2907，Abcam）、以及小鼠荧光标记的各种抗体（Biolegend）CD45-PerCP/Cyanine5.5、CD80-APC、CD86-PE、CD11c-FITC、CD11b-FITC、CD206- Alexa Fluor 647、F4/80-PE、CD4-PE、CD8-FITC、FoxP3- Alexa Fluor 647和山羊抗兔-Alexa Fluor 633等试剂盒和抗体均购买后按说明书使用。小鼠乳腺癌细胞株4T1是从中科院上海细胞库购买。
聚合物的核磁共振氢谱（ 1H NMR）由400 MHz超导核磁共振谱仪（Unity Inova，安捷伦）或600 MHz 超导核磁共振谱仪（DirectDrive2，瓦里安）以氘代氯仿（CDCl 3）或氘代二甲亚砜（DMSO-d 6）为溶剂测定，化学位移以溶剂峰为参考标准。聚合物分子量以及分子量分布由Waters 1515凝胶渗透色谱仪（GPC）测定，以DMF为流动相（流速为0.8 mL/min），测试温度为40℃，以一系列单分散的线性聚甲基丙烯酸甲酯作为分子量标准品。聚合物胶束和囊泡的粒径和粒径分布通过配有He/Ne激光光源（波长为633 nm）和173 o背折射检测器的ZetaSizer Nano-ZS纳米粒度仪（Malvern Instruments）测定，温度为25 ºC。胶束样品的微观形貌由Tecnai G220透射电镜（TEM）在120 kV加速电压下测定，样品用1% 磷钨酸溶液染色。PTX的浓度由配有反相C18色谱柱（4.6×150 mm，5μm）的高效液相色谱仪（HPLC）测试，流动相为 0.1%三氟乙酸的水/乙腈（v/v =45/55），流速为 1 mL/min，检测温度为40℃，检测波长为 227 nm。TCS SP5共聚焦激光扫描显微镜（CLSM，Leica）用来研究胶束的细胞内吞行为。流式细胞仪（FACS Calibur, BD Biosciences）用来研究胶束内吞行为以及体内免疫细胞的定性和定量分析。酶标仪（Thermo Multiskan FC）用来测定活细胞与MTT形成的紫色甲瓒在570 nm处的吸光度值。多功能酶标仪（Varioskan LUX，Thermo Scientific）用来进行细胞因子的ELISA检测。
本发明设计制备了纳米剂型的PTX 和CDN免疫佐剂，将其联合在以小鼠4T1为模型的TNBC中进行化学免疫治疗。首先制备了靶向、基于PEG-P(CL-DTC)的双硫交联的PTX胶束纳米药物，提高PTX在肿瘤细胞里的浓度、提高疗效、降低毒副作用，分别制备了偶联PHSCNK、PhScNK 多肽或cRGD多肽的纳米药物ATN1-Ms-PTX、ATN2-Ms-PTX和cRGD-Ms-PTX。另一方面，CDN由于分子量小、易被降解、带负电水溶性强，难以进入抗原递呈细胞（APC）和内质网的STING蛋白结合激活STING通路，为了克服这些问题，本发明制备了基于PEG-P(TMC-DTC)-Sp的具有不对称膜结构的聚合物囊泡来装载、保护和递送STING激动剂环二核苷酸ADU-S100（CPs-CDN）到APC，以更好地激活抗肿瘤免疫应答。研究发现，化学免疫疗法中化药和免疫调节剂的递送顺序会影响总体疗效。本发明首先用靶向TNBC的PTX胶束高效输送PTX至肿瘤，杀死部分肿瘤细胞并诱导免疫原性细胞死亡（ICD），产生肿瘤相关抗原并被APC有效内吞，从而增加CTL的激活；然后瘤内注射CPs-CDN激活STING通路，并进一步活化肿瘤里包括DC在内的APC，增强其递呈抗原的能力，招募并激活T细胞产生抗肿瘤免疫应答。实验结果表明，ATN2-Ms-PTX能高效靶向富集到小鼠4T1肿瘤，增加了PTX在肿瘤细胞的浓度，其后顺序递送CPs-CDN激活了STING通路，显著延缓了小鼠肿瘤的生长、抑制了肺转移，延长了小鼠的生存期。
本发明的原料都是现有产品，具体操作方法以及测试方法为本领域常规方法。本发明所有数据均是以平均值呈现，组间差异性由ANOVA单因素方差分析评估，*p < 0.05视为具有显著性差异，将**p < 0.01和***p < 0.001视为高显著性差异。
制备例：PEG-P(CL-DTC) 嵌段共聚物的合成是以MeO-PEG-OH ( M n = 2.0 kg/mol) 为大分子引发剂、磷酸二苯酯（DPP）为催化剂引发DTC和CL的开环聚合而得。具体地，在惰性气体手套箱里，向25 mL密闭反应器中依次加入MeO-PEG-OH (800 mg, 0.4 mmol)、DPP (1.0 g，4 mmol)、DTC (400 mg，2.1 mmol) 、CL (400 mg，3.5 mmol) 和无水DCM（3.2 mL），搅拌溶解后密封，移出手套箱，置于40 ºC 油浴中磁力搅拌反应48 h。反应结束后，加入冰醋酸终止反应，在30倍体积的冰乙醚中沉淀2次，抽滤、真空干燥24 h后得到PEG 2k-P(CL 1k-DTC 1k)，制备路线如下所示：
Figure 145459dest_path_image006
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用分子量为5000 Da的MeO-PEG-OH为引发剂、DPP为催化剂，根据上述方法可控开环聚合CL和DTC得到PEG-P(CL-DTC)，核磁分子量为5k-4k-3k或者5k-4k-2k。
用上述相同方法：不加DTC合成了PEG-PCL；以Mal-PEG-OH或NHS-PEG-OH（ M n = 3.5 kg/mol）替换MeO-PEG-OH ( M n = 2.0 kg/mol)作为引发剂合成了Mal-PEG-P(CL-DTC)或NHS-PEG-P(CL-DTC)。
ATN-PEG-P(CL-DTC) 通过PHSCNK或PhScNK多肽与NHS-PEG-P(CL-DTC)通过酰胺化反应得到。简言之，在氮气保护下，将NHS-PEG-P(CL-DTC)（132 mg，0.024 mmol）溶解在1 mL无水DMSO中，PHSCNK多肽或PhScNK多肽（20 mg，0.028 mmol）溶解在0.5 mL无水DMSO中。将聚合物溶液滴加至30℃的多肽溶液中，30 min滴加结束后再加入7 μL的三乙胺调节体系pH在8.0，密闭反应器后再30度反应48 h。将反应液装入透析袋（MWCO 3500 Da）在DMSO中透析4 h、DCM中透析2 h，然后在30倍体积的冰乙醚/无水乙醇（9/1，v/v）中沉淀2次，离心、真空干燥24 h后得到ATN-PEG-P(CL-DTC)。 1H NMR（600 MHz，DMSO- d 6 ）测试得到谱图可进行聚合物结构和分子量分析，用Micro BCA蛋白试剂盒测试可计算多肽的官能化度，计算出PhScNK和PHSCNK的官能化度分别为71.2%和79.6%。反应如下：
Figure 345496dest_path_image008
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cRGD-PEG-P(CL-DTC) 由c(RGDfC) 多肽的巯基和Mal-PEG-P(CL-DTC) 通过迈克尔加成反应制得，其步骤、聚合物的提纯和表征同上。反应如下：
Figure 745384dest_path_image010
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上述聚合物及表征请参见申请人同日提交的发明名称为“一种小胶束纳米药物及其制备方法与应用”的发明申请，本发明与其一致。
将PTX和PEG 2k-P(CL 1k-DTC 1k) 按照质量比（1/20）溶于PEG 350中，其中聚合物浓度保持1 mg/mL。常规超声5 min后，在37℃取100 μL的该混合溶液打入900 μL的PB溶液底部（pH 7.4，10 mM），过程中静置不搅拌。打入之后用移液枪贴液面吹打5次，即得到PTX载药胶束，为非靶向小胶束纳米药物，DLS测定其粒径为27.8nm，粒径分布（PDI）为0.18。在上述方法基础上：将聚合物更换为PEG 5k-P(CL 4k-DTC 3k)，同样方法得到的PTX载药胶束，DLS测定其粒径为55.1nm，粒径分布（PDI）为0.27。如果混液过程中常规搅拌（600 rpm），其余不变，得到的PTX载药胶束粒径达到168 nm，且存放1小时即出现药物析出。将PEG 350更换为DMF或者DMSO，同样方法得到的PTX载药胶束，粒径较小，但是稳定性差，常规环境下存放4小时后，出现大量沉淀，说明药物明显析出，较本发明存放20天以上无药物析出差得多。
实施例一 化疗-免疫联合药物：PTX胶束的制备：将PTX和PEG 2k-P(CL 1k-DTC 1k) 按照不同质量比（5/100、10/100、20/100、30/100）溶于PEG 350，其中聚合物浓度在50 mg/mL。常规超声5 min后，在37℃取100 mL的该混合液打入900 mL的PB溶液底部（pH 7.4，10 mM），过程中静置不搅拌。打入之后用移液枪贴液面吹打5次，即得到PTX载药量为4.8 wt.%～23.1 wt.%的胶束MS-PTX，为非靶向小胶束纳米药物。DLS测定其粒径及粒径分布（PDI），TEM测定胶束形貌。用含20 mM DTT的乙腈溶解MS-PTX（聚合物浓度为0.1 mg/mL），再用HPLC测试其中PTX浓度，计算载药量和载药效率。用DLS在不同时间点监测PTX胶束在25和37℃存放、稀释至低浓度（20 mg/mL）及在含10%FBS溶液中粒径和粒径分布的变化，见表1。
作为对照，用相同方法也制备了基于PEG-PCL的载PTX胶束（ncMS-PTX）。
现有技术多数文献中报道的PTX纳米载体载药量均较低。本发明利用少量PEG350来溶解聚合物PEG-P(CL-DTC)和PTX，将混合物加入到水相中（PEG350的最终体积含量10%），通过疏水作用聚合物形成核壳结构的胶束把PTX包裹在疏水核中，而核中DTC可快速自交联，能更稳定地把PTX固定在胶束核中，得到PTX胶束纳米药物MS-PTX（图1 A）。这里使用的PEG350无毒，获FDA批准使用。本发明通过该方法制备了载药量从4.8 wt.%到23.1 wt.%的胶束MS-PTX，粒径范围在30-38 nm，粒径分布0.13-0.17（表1）。例如，DLS测试结果表明，载4.8 wt.%的MS-PTX粒径为32.7 nm，PDI为0.16，TEM图显示出其具有较规则的实心小球的形状（图1B）。此外，即使是理论载药量达23.1 wt.%时，MS-PTX胶束依然保持澄清透明，粒径为38.1 nm，PDI为0.13（表1）。由于PTX的水溶性很低（水中溶解度为5.56 mg/L），将相同量PTX用PEG350 溶解后直接加入到水中，PTX迅速析出，而载23.1 wt.% PTX的MS-PTX能保持澄清，说明PTX全装载在胶束里面（图1 C）。使用该方法制备胶束纳米药物过程简单、重现性好、胶束的稳定性好，在室温存放三周后胶束的粒径和粒径分布也基本不变（图1 D），没有沉淀；这比基于PEG-PDLLA聚合物制备的Genexol-PM稳定得多，Genexol-PM在2～4 h内粒径会增大并且产生很多晶状沉淀。本发明制备的MS-PTX比文献中报道的脂质体和多数纳米载体及Genexol-PM（PTX载药量10 wt.%）具有更高的载药量。
Figure 30872dest_path_image012
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研究了制剂中的PEG350对MS-PTX载药量和稳定性的影响，将1 mL新制的载药量为4.8 wt.%的MS-PTX（1 mg/mL）的一半室温存储，另一半装入透析袋（MWCO 3500 Da）中，放入50 mL PB溶液（pH 7.4，10 mM）中透析3 h（每30分钟换一次缓冲液）。测定透析后粒径及PDI变化和PTX载药量的变化。结果发现，除去PEG350后胶束的PTX载药量稍微降低，但包封率保持在87%以上，说明该方法能把PTX紧密包在胶束里；但是透析后胶束粒径减小6-8 nm，PDI变大（表2），室温下放置1 h就逐渐出现浑浊和PTX析出，在200-1000 nm出现聚集体（图1 D），这与没有除去PEG350样品的稳定形成鲜明对比。
研究了聚合物中DTC对形成的PTX胶束载药量和稳定性的影响，相同条件制备的基于PEG-PCL载PTX不交联胶束ncMS-PTX和载4.8 wt.% PTX的PEG-P(CL-DTC)（MS-PTX），一半室温存放、一半透析，测定两组各种透析前后粒径及PDI变化和PTX载药量的变化，研究比较了ncMs和Ms两种胶束的紫外吸收、临界胶束浓度（CMC）以及两种载药胶束的稳定性。纳米粒中DTC的二硫戊环的330 nm的紫外吸收值降低很多，说明发生硫硫交联反应得到交联纳米粒。而本胶束在制备中由于没有透析，PEG350仍然存在，是否在制备后也能在胶束核内形成交联是不可预知的。本发明首先测定了两种胶束和相同浓度的对应聚合物溶液（5 mg/mL）的紫外吸收，发现PEG-PCL聚合物溶液及其胶束ncMs无明显的吸收峰。而Ms中DTC在326 nm出现二硫戊环的紫外吸收峰，但是其强度低于对应的聚合物溶液中DTC的强度（图1 E），下降了40.7%。这结果说明Ms胶束中部分二硫戊环开环，形成核内自交联。其次，用芘为荧光染料来确定两种胶束的临界胶束浓度（CMC），发现ncMs的CMC为15.6 mg/mL，但Ms在聚合物浓度为1.2 mg/mL至 2.5 mg/mL范围内I 1/I 3没有发现明显的转变（图1 F），即没有CMC，胶束在稀释到CMC以下时也不会解离成单分子。在实验中也发现将MS-PTX胶束浓度稀释到20 mg/mL测DLS时，胶束仍然完整，粒径呈正态分布，而ncMS-PTX胶束稀释至50 mg/mL时测试就出现聚集现象，PDI也迅速变大（图1 G）。此外，ncMS-PTX在室温下放置2天后变得不稳定，出现PTX析出以及大分子聚集（图1 G）。这些实验结果都证实了本发明制备的MS-PTX胶束是双硫交联的，这有助于进一步稳定PTX。综上，本发明基于PEG-P(CL-DTC) 的胶束MS-PTX比基于T g高得多的PEG-PDLLA的Genexol-PM还稳定得多。
Figure 174409dest_path_image014
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载STING激动剂CDN的聚合物囊泡CPs-CDN的制备：PEG-P(TMC-DTC)-SP的合成可参见申请人已经公开的专利或者文章，具体制备方法为现有技术。使用聚乙二醇-聚（三亚甲基碳酸酯-二硫戊环三亚甲基碳酸酯）-精胺（PEG-P(TMC-DTC)-Sp， M n = 5.0-(15-2)-0.2 kg/mol）、通过溶剂置换法制备载CDN聚合物囊泡，CDN选用ADU-S100。简要步骤如下：配制PEG-P(TMC-DTC)-Sp的DMF溶液（10 mg/mL）和CDN水溶液（不含核糖核酸酶的DEPC水，2 mg/mL）。将100 µL聚合物溶液加入900 µL含CDN的4℃的HEPES缓冲溶液（pH 6.8），CDN理论载药量为13.0 wt.%。200 rpm搅拌10分钟后形成载药囊泡，先用HEPES在4度透析2 h，再用pH 7.4的PB透析1 h得到CPs-CDN（1 mg/mL），研究表征见实施例五。CPs-CDN的粒径、PDI及稳定性等表征方法同PTX胶束。CDN的载药量（DLC）和载药效率（DLE）通过测定NanoDrop 2000在寡DNA模式下260 nm处的吸光度值、基于标准曲线计算得来。
Figure 228952dest_path_image015
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PTX胶束与载STING激动剂CDN的聚合物囊泡CPs-CDN组成化疗免疫联合药物。
此外，Ms-PTX在含10%FBS的PB缓冲液中（模拟体内血液环境）保持稳定，24 h粒径保持不变（图2 A）；然而在含10 mM DTT的PB溶液（模拟细胞质内还原环境）中其粒径在2 h时很快出现500-1000 nm的大颗粒，4 h时出现十几纳米的小颗粒（图2 B），这是由于疏水核中的二硫键在还原条件下逐渐解交联、胶束溶胀、在低聚合物浓度下会解离成单分子的缘故。这表明该胶束具有快速还原响应性能。
实施例二：装载PTX的靶向胶束是由两亲性嵌段聚合物PEG 2k-P(CL 1k-DTC 1k)和偶联靶向分子的聚合物Ta-PEG-P(CL-DTC)（Ta为多肽cRGD、ATN1或ATN2）在水相中自组装而成。在PEG-P(CL-DTC) 中分别混合重量百分含量为2.5%、5%、7.5%、10%或20%的ATN1-PEG-P(CL-DTC)、ATN2-PEG-P(CL-DTC)或cRGD-PEG-P(CL-DTC) 作为起始的聚合物溶液，然后和PTX溶液混合，其余步骤同实施例一，得到不同多肽表面密度的胶束ATN1-MS-PTX、ATN2-MS-PTX和cRGD-MS-PTX，为靶向小胶束纳米药物。
PEG-(CL-DTC)和不同比例的偶联cRGD、PHSCNK或PhScNK的靶向聚合物混合后，制备靶向胶束，采用实施例一相同方法、保持PTX载药量为4.8 wt.%，分别制备了三个系列具有不同靶向分子密度的胶束cRGD-MS-PTX、ATN1-MS-PTX和ATN2-MS-PTX。DLS测试结果表明，在表面多肽不超过5%时，随着多肽的增加，胶束粒径基本不变，在31到35 nm之间，PDI保持在0.2左右（表3）。此外，以PTX载药量为4.8 wt.%、多肽密度为5%的 ATN2-MS-PTX为例，研究了其中PEG350及DTC对胶束稳定性、FBS稳定性，以及冷冻/冻干复溶对粒径的影响，发现结果与非靶向MS-PTX相同。研究了PTX胶束在模拟细胞中还原响应环境的药物释放行为，分别用含或不含10 mM DTT的PB溶液（含0.1%吐温80）作为释放介质，以ATN2-MS-PTX为例研究了在不同时间点释放到介质中的PTX的累积量。发现浓度为0.5 mg/mL的胶束在10 mM DTT中能够快速释放PTX，24 h的释放PTX累积量达79.4%；而在不含DTT环境中PTX 24 h仅泄漏17.0%（图2C），结果表明胶束结构因为DTC交联而保持相对稳定、药物始终释放缓慢；而在还原条件下，双硫键断裂生成的巯基有一定的亲水性，胶束在较稀浓度下很难维持胶束结构，药物快速释放，呈现出明显的还原响应性药物控释行为。
Figure 65321dest_path_image017
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采用同样的步骤，由两亲性嵌段聚合物PEG 2k-P(CL 1k-DTC 1k)和偶联靶向分子的聚合物Ta-PEG 2k-P(CL 1k-DTC 1k)（重量百分含量为5%）在水相中自组装而成装载PTX的靶向胶束；Ta为多肽cRGD、ATN1或ATN2对应的粒径分别为27 nm、29 nm 和28nm。
本发明制备了PTX载药量高的胶束，且制备的小粒径胶束（20～40 nm），能够有效穿透至肿瘤深处，尤其是本发明载药过程中无药物析出，静置三周也无药物泄露，采用的小分子量PGE无需去除。
实施例三 MTT实验评估PTX胶束的细胞毒性：具体测试方法为现有方法，可以参见申请人同日提交的发明名称为“一种小胶束纳米药物及其制备方法与应用”的发明申请，本发明与其一致。细胞选用小鼠高转移性三阴乳腺癌细胞株4T1，PTX浓度范围为0.002至5 μg/mL。本发明以鼠源三阴乳腺癌4Tl细胞为研究对象，用MTT法研究了不同靶向分子密度的三种胶束的细胞毒性。结果表明，MS-PTX对4T1的毒性具有浓度依赖性，IC 50为0.6 μg/mL，明显低于PTX（IC 50为1.12 μg/mL），偶联靶向分子的胶束均表现了比MS-PTX更高的毒性（图3）。三种胶束对4T1的毒性不尽相同，整体来看，ATN2-MS-PTX比cRGD-MS-PTX毒性略高，远高于ATN1-MS-PTX；5%cRGD-MS-PTX、20%ATN1-MS-PTX、5%ATN2-MS-PTX在各组中相对其他多肽密度胶束具有最低的IC 50值，分别为0.27、0.46、0.21 μg/mL（表4），其中5%ATN2-MS-PTX的IC 50值最低，甚至低于自由PTX与4T1细胞共孵育48 h的IC 50值（0.55 μg/mL）。
Figure 838105dest_path_image019
。
进一步考察了5%ATN2-MS-PTX与4T1细胞共孵育24 h和48 h的细胞毒性（IC 50值分别为0.54 和0.020 μg/mL），发现该IC 50值均比无靶胶束（分别为1.19和0.064μg/mL）的低2-3倍。此外，空胶束Ms和ATN2-Ms在浓度1 mg/mL时共孵育48 h对4T1细胞均没有显示出毒性，这说明该方法制备的纳米载体具有良好的生物相容性。
实施例四 PTX胶束的细胞内吞行为研究：用流式细胞术FACS和CLSM来考察4T1细胞对PTX胶束的摄取，通过NHS-PEG-P(CL-DTC)与Cy5-NH 2的酰胺化反应得到Cy5标记的PEG-P(CL-DTC)，并将其按照1% 掺杂到原聚合物中得到Cy5标记的胶束。具体测试方法为现有方法，可以参见申请人同日提交的发明名称为“一种小胶束纳米药物及其制备方法与应用”的发明申请，本发明与其一致。用水合法制备即得到Cy5标记的胶束，不同靶向密度均选用表4中的最佳靶向密度。 M n = 5.0-(15-2)-0.2 kg/mol）。
FACS测试结果显示（图4 A），和Cy5/cRGD-MS-PTX、Cy5/ATN2-MS-PTX孵育4 h后的4T1细胞的荧光强度是对照无靶Cy5/MS-PTX组细胞的2.1倍和2倍，而Cy5/ATN1-MS-PTX与无靶组相比只有1.2倍。通过CLSM观察到，4T1细胞经含Cy5胶束孵育4 h后，各靶向胶束的荧光强度均高于无靶组，其中Cy5/ATN2-MS-PTX组最高，与PBS 和Cy5/cRGD-MS-PTX组都存在显著性差异（图4 B，*p），且Cy5/ATN2-MS-PTX组Cy5荧光出现在每个细胞的细胞质中。而Cy5/cRGD-MS-PTX和Cy5/ATN1-MS-PTX组中均有部分细胞的细胞质中没有Cy5荧光（图4 C）。
实施例五 载STING激动剂的聚合物囊泡CPs-CDN的表征：由于STING蛋白一般驻留在APC中，所以要激活STING通路，其激动剂如环二核苷酸（CDNs）必须递送到APC内。但CDN分子量小、带负电荷、亲水性强，进入胞质能力差，易降解，导致其生物利用率不高，效果大打折扣。为了将CDN有效递送至APC胞质中激活STING通路，本发明设计基于DTC的还原敏感、双硫交联的聚合物囊泡来实现这一目标，利用聚合物PEG-P(TMC-DTC)-Sp形成的具有不对称膜结构的聚合物囊泡（CPs）来递送CDN。为了验证该CPs是否能稳定装载分子量小的CDN并递送到APC中，选用目前临床II期试验中的ADU-S100为模型CDN展开研究。采取溶剂置换法制备装载CDN的囊泡CPs-CDN（图5 A），CDN的装载效率用NanoDrop 2000测定。实验结果表明，在CDN理论载药量为16.7 wt.%时，其包封率和载药量分别能达86.4%和14.4 wt.%（表5）。考虑到CDN的小分子量和强亲水性，这样高的载药量还是令人意想不到的，CPs-CDN的粒径为55.3-56.8 nm，PDI为0.12-0.18，具有良好的胶体稳定性，在4度冰箱中能储存3周且在含有10%胎牛血清（FBS）中能保持粒径不变（图5B）。
Figure 528543dest_path_image021
。
 以5%ATN2-Ms-PTX、CPs-CDN（CDN理论载药量为13.0 wt.%）组成化疗免疫联合药物，进行细胞、动物实验。
实施例六PTX胶束、CPs-CDN及其联合激活BMDC的研究：BMDC（1×10 6个/孔）在12孔板中培养24 h后加入PBS、PTX、Ms-PTX、ATN2-Ms-PTX、CDNs和CPs-CDN孵育24 h，联合组是先加ATN2-Ms-PTX孵育8 h后再加CPs-CDN共孵育16 h，最终PTX浓度为5 µg/mL，CDN浓度为0.2 µg/mL（n = 3）。孵育结束后离心（1500 rpm，5 min），分离出上层培养基，收集细胞后用荧光标记的CD11c、CD80和CD86抗体按说明书来染细胞，最后用FACS测试各组中总CD11c + BMDC的量和CD11c +CD80 +CD86 +成熟BMDC的含量。分离的培养基用ELISA试剂盒测定IFN-β、TNF-α和IL-6浓度。
STING激动剂CDN进入DC等APC中与内质网上的STING蛋白结合，诱导I型干扰素（IFN-β）等细胞因子的产生，从而激活相关免疫细胞的成熟递呈抗原给T细胞，招募更多的免疫细胞进入肿瘤区域；但是不可预知CPs-CDN中CDN由于装在囊泡里面是否还能实现这样的功能，本发明检测了CPs-CDN及自由CDN和BMDC孵育16 h后CD11c +的DC细胞以及CD11c + CD86 +CD80 +的成熟BMDC的比例。此外，结合ATN2-Ms-PTX可诱导ICD，产生一系列肿瘤抗原，并能使BMDC增殖与成熟。因此，用ATN2-Ms-PTX处理使BMDC增殖，之后再联用CPs-CDN也能增加CDN进入DC的量，会增强STING通路的激活，从而增加BMDC的成熟量；另一方面，ATN2-Ms-PTX诱导的TAA能更有效地被这些激活的APC呈递给T细胞，从而招募更多的T细胞。检测了两者与BMDC孵育后其表达CD11c +的BMDC的量以及成熟BMDC比例的变化，来研究联合是否对BMDC的增殖、成熟有更强劲的提升。FACS测试结果表明，相比于PBS组，CDN制剂和联合组都能在促进总CD11c +的BMDC含量，而CPs-CDN组中成熟 BMDC（81.7%）远高于自由CDN组（66.9%）（***p）（图6 A&B），表明CPs的装载不但没有影响CDN的细胞内释放和功能，反而具有更高的刺激BMDC成熟的能力。此外，联用ATN2-Ms-PTX能进一步增加BMDC成熟，CD11c +CD80 +CD86 +的BMDC高达90.2%，比单用任何一种要显著高（*p/***p）。此外，也测定了BMDC培养基中IFN-β、TNF-
Figure 375276dest_path_image022
和IL-6的浓度。细胞因子的增多表明BMDC成熟，抗原呈递能力增强，可极强地激活T细胞，其中IFN-β是CDN与STING结合后产生的标志性细胞因子，对肿瘤引发T细胞启动具有关键作用。结果显示，三个CDN制剂组诱导的IFN-β是PBS和ATN2-Ms-PTX组的两个数量级以上，体现了CDN的强烈激活STING通路的功能。先用ATN2-Ms-PTX再用CPs-CDN的联合组处理细胞，分泌的IFN-β、TNF-
Figure 913705dest_path_image022
和IL-6的浓度显著高于CPs-CDN组和ATN2-Ms-PTX组（*p/***p）和自由CDN组（**p/***p）（图6 C、D&E）。这些结果表明，CPs-CDN提升了CDN对DC激活的功能，和ATN2-Ms-PTX联用能更显著促进BMDC的增殖和激活、激活STING通路、诱导IFN-β等细胞因子，更有效促进BMDC成熟并递呈抗原给T细胞。
实施例七 PTX胶束联合CPs-CDN用于小鼠TNBC的化学免疫治疗：采用荷4T1皮下瘤的Balb/c小鼠作为小鼠三阴乳腺癌模型，考察Ms-PTX的药效、给药方式以及和CPs-CDN联用对肿瘤抑制的影响，在15只荷瘤小鼠的肿瘤体积为200-250 mm 3时开始给药，给药当天为第0天。共分5组，每组3只小鼠，每2天给药、共3次。组1：Ms-PTX尾静脉（i.v. 10 mg PTX/kg，200 µL）；组2：Ms-PTX瘤内注射（i.t. 5 mg PTX/kg，50 µL）；组3：Ms-PTX尾静脉（i.v. 10 mg PTX/kg，200 µL）给药2h后瘤内注射CPs-CDN（1 mg CDN/kg）；组4：Ms-PTX瘤内注射（i.t. 5 mg PTX/kg，50 µL）给药2h后瘤内注射CPs-CDN（1 mg CDN/kg）；组5：PBS。每2天监测肿瘤体积和小鼠体重，肿瘤体积通过公式V = 0.5×L×W 2来计算（L、W分别为肿瘤最宽和最窄处的长度）。小鼠相对体重是测量时体重占第0天时体重的百分比（M/M 0）。发现无论是静脉或瘤内注射Ms-PTX，肿瘤体积持续增加，和PBS组生长曲线相似，这和起始肿瘤体积过大、该肿瘤的恶性程度和侵袭性很高有关。而联合CPs-CDN给药时，两种方式均比单给Ms-PTX有显著的肿瘤抑制作用，其中Ms-PTX i.v. + CPs-CDN i.t. 组肿瘤抑制最显著（**p/***p），前10天肿瘤体积没有增长，10天后开始缓慢增长（图7 A）。Ms-PTX i.t. + CPs-CDN i.t. 组肿瘤一直有缓慢的增长。这和Ms-PTX瘤内注射PTX浓度过高，杀死肿瘤细胞的同时也杀死免疫细胞有关，导致再给CPs-CDN也不能被很多APC内吞，整体STING激活程度差，产生的免疫应答较弱。而Ms-PTX尾静脉注射后，短时间富集至肿瘤的Ms-PTX相比要少、PTX浓度低，对免疫细胞无毒，却可诱导肿瘤细胞ICD、使DC增殖；在给CPs-CDN后就能在大量DC中激起强免疫应答。Ms-PTX i.v. + CPs-CDN i.t. 组小鼠体重在给药期间有下降，而后又恢复（图7 B）。
系统研究PTX胶束以及其联合CPs-CDN的化学免疫治疗。在接种4T1（3×10 5/只）的荷瘤小鼠的肿瘤体积在50-100 mm 3（接种后第6天）开始给药，给药当天为第0天。小鼠随机分9组，每组7只，每两天给药，共4次；给药剂量为7.5 mg PTX/kg和/或1 mg CDN/kg。九组分别为：PBS、自由PTX（i.v.）、Ms-PTX（i.v.）、ATN2-Ms-PTX（i.v.）、自由CDN（i.v.）、CPs-CDN（i.t.）、三个ATN2-Ms-PTX（i.v.）+ CPs-CDN（i.t.）联合组，分别是ATN2-Ms-PTX（i.v.）给药2 h、8 h或24 h后再给CPs-CDN（i.t.）。实验过程中每2天监测小鼠体重、肿瘤体积和小鼠状态。小鼠死亡或肿瘤体积大于2000 mm 3判定死亡，绘制生存曲线。给荷瘤（肿瘤体积约50 mm 3）小鼠每2天尾静脉注射肿瘤靶向的ATN2-Ms-PTX，并把药量降低到7.5 mg/kg，共给药4次；在每次ATN2-Ms-PTX给药之后再瘤内注射CPs-CDN（1 mg/kg），研究该方案对小鼠肿瘤尺寸、体重和生存期的影响，当肿瘤体积大于2000 mm 3时，判定小鼠死亡（#号表示）。系统比较该方案与单给ATN2-Ms-PTX、Ms-PTX、自由CDN或CPs-CDN的抗肿瘤效果。此外，还研究了ATN2-Ms-PTX和CPs-CDN之间的给药间隔时间（2、8或24 h）对抗肿瘤效果的影响（图8 A）。
结果显示，4T1肿瘤的恶性程度极高，PBS组肿瘤快速增长（图8 B），到第20天肿瘤体积均大于2000 mm 3，中位生存期为18天。自由PTX组在给药期间肿瘤缓慢生长，停药后开始快速生长。而ATN2-Ms-PTX、Ms-PTX、自由CDN、CPs-CDN与联合给药各组均能显著抑制肿瘤的生长，10天内肿瘤基本不长，但10-12天开始肿瘤都开始缓慢生长，其中联合组的肿瘤生长最慢。比较第22天肿瘤体积可知，Ms-PTX可能由于小粒径而使其EPR效应显著，比PTX能更有效抑制肿瘤生长（*p）。而ATN2-Ms-PTX则能比PTX和Ms-PTX更进一步限制肿瘤生长（***p）（图8 B），延长小鼠的生存时间，体现了靶向抗肿瘤疗效。此外，瘤内给CDN或CPs-CDN也能减缓肿瘤生长速度，CDN减缓程度与MS-PTX相似，CPs-CDN和ATN2-Ms-PTX类似，但CPs-CDN与自由CDN相比优势更显著（**p），再次表明囊泡能更高效地递送CDN，提高其生物利用率和激活STING通道的能力（图8 B）。
令人兴奋的是，ATN2-Ms-PTX和CPs-CDN联用的抑瘤效果比单药组都更强，且二者间隔8或24 h比间隔2 h的疗效更佳（ATN2-Ms-PTX+CPs-CDN（2或8或24 h）vs ATN2-Ms-PTX，p = 0.1656（ns）或p = 0.0042（**）或0.0068（**）；ATN2-Ms-PTX+CPs-CDN（2或8或24 h）vs CPs-CDN，p = 0.2872（ns）或p = 0.0099（**）或0.0158（*））。联合组间隔2、8、24h组的小鼠中位生存期分别30、32、33天（PBS组为18天）（图8 C）。联合给药间隔2 h效果差些，可能是由于间隔太短，PTX释放量不多和/或诱导ICD及促使DC增殖的程度还不足，导致APC的抗原递呈作用弱。研究发现，在“癌症-免疫周期”的循环中，不仅需要TAA的释放，同时需要APC的加工和递呈，激活T细胞，募集更多T细胞至肿瘤，从而识别杀伤肿瘤，每一环节都很重要。循环过程某一项被抑制或不足，都将使抗肿瘤免疫无法达到最佳状态。此外，小鼠在治疗期间体重变化很小，尾静脉给7.5 mg PTX/kg和/或瘤内给1 mg/kg的CPs-CDN都没有对小鼠产生毒副作用（图8 D）。
实施例八 ATN2-Ms-PTX及其与CPs-CDN联合对荷瘤小鼠免疫系统的调节：同实施例七描述，荷瘤小鼠随机分7组，每组7只。七组分别为：PBS（i.v.）、自由PTX（i.v.）、Ms-PTX（i.v.）、ATN-Ms-PTX（i.v.）、自由CDN（i.v.）、CPs-CDN（i.t.）、ATN-Ms-PTX（i.v.）+ CPs-CDN（i.t.）（间隔8 h），按照7.5 mg PTX/kg和/或1 mg CDN/kg给药4次，每2天一次。最后一次给药后24 h每组处死4只小鼠，取小鼠全血分离血清测定IFN-β、IFN-
Figure dest_path_image023
、IL-6和TNF-
Figure 580309dest_path_image022
的含量。取脾脏和肿瘤，研磨成单细胞悬液，裂红后计数。对脾脏，每个样品6×10 6个细胞进行T细胞染色。对肿瘤，每个样品分为4份，每份6×10 6个细胞，进行DC、巨噬细胞、T细胞染色，通过FACS测试分析。免疫细胞相应的荧光标记的抗体为：DC：anti-CD11c-FITC、anti-CD80-APC、anti-CD86-PE；巨噬细胞：anti-CD11b-FITC、anti- F4/80-PE、anti-CD206- Alexa Fluor 647；T细胞：anti-CD3-APC、anti-CD4-PE、anti-CD8-FITC；Tregs：anti-CD3-FITC、anti-CD4-PE、anti-Foxp3- Alexa Fluor 647。
肿瘤免疫微环境（TME）是由恶性细胞、免疫细胞、血管、细胞外基质和信号分子组成的环境，可单独发挥作用，也可共同影响免疫疗法的敏感性。上文已证明，ATN2-Ms-PTX、CPs-CDN联合药物可进入肿瘤细胞诱导ICD、释放肿瘤相关抗原，产生有利的免疫微环境，进入APC激活STING通道使DC成熟，两者联合在细胞层面已经展现出了优异的免疫效果。这里将分析ATN2-Ms-PTX、CPs-CDN及其联合按图8方案给药24 h后小鼠TME以及脾脏中T细胞的影响。首先分析了TME中CD80 +CD86 + 成熟DC和CD206 -的M1M的含量，图9 A显示，PTX胶束成熟DC比例都要比PBS高，CDN胶束更高些，CPs-CDN组成熟DC 为52.5%高于自由CDN（48.7%）；而联合组成熟DC（62.9%），显著高于分别单药组（*p）。前面细胞实验中，PTX可使巨噬细胞从M2M转化为M1M。在TME中，ATN2-Ms-PTX、CPs-CDN及联合给药均能促进了M2M极化为M1M（图9 B）。
T细胞是人体攻击和杀灭肿瘤的免疫细胞，参与抗肿瘤免疫的T细胞亚群分为两类：CD8 +细胞毒性T细胞（CTL）和CD4 +辅助性T细胞（T h细胞），而T h又可产生淋巴因子增强CTL功能，激活巨噬细胞、DC或其他APC，产生肿瘤坏死因子发挥溶瘤作用。见图9 C-E，结果显示，ATN2-Ms-PTX+CPs-CDN联合组的CD4 + T和CD8 + T细胞比ATN2-Ms-PTX和CPs-CDN都显著提升（CD4 + T：***p和**p；CD8 + T：*p）。另外，T h中的调节性T细胞（T reg）是减弱T细胞活性、使TME免疫抑制的T细胞的主要类型。分析了T regs在CD4 + T中的占比及CD8 + T/T reg，结果表明（图9 F&G），CPs-CDN能急剧降低T reg比例，与联合组相当。此外，CPs-CDN组的CD8 + T/T reg显著高于自由组CDN（***p）。STING激活程度越大，CD8 + T细胞介导的抗肿瘤免疫力越强，也正因为CPs-CDN高效的CDN利用率，使其单独使用也能有效激活抗肿瘤免疫。联合组能强效激发T细胞的杀伤能力，其CD8 + T/T reg进一步增强，是ATN2-Ms-PTX的14倍（***p）、CPs-CDN的3.4倍（*p）。ELISA测试表明，ATN2-Ms-PTX和CPs-CDN联用组的促炎细胞因子IFN-
Figure dest_path_image024
、IFN-γ、TNF-α和IL-6含量均增加（图9 H），这些结果证实了其能诱导强烈的抗肿瘤免疫反应。
脾脏是人体最重要的免疫器官，含有丰富的免疫细胞，拥有全身25%的淋巴细胞。脾脏对T淋巴细胞的免疫调节作用是抗肿瘤免疫的一个重要环节。接着，分析了ATN2-Ms-PTX及其与CPs-CDN联合给药后小鼠脾脏中CD4 +和CD8 + T细胞占CD45 +总免疫细胞的比例。图10显示，各组药物给药后第8天小鼠脾中的CD4 + T和CD8 + T细胞的变化趋势和肿瘤中的一致，联合组占二者比分别高达18.7%和6.3%，分别是单制剂ATN2-Ms-PTX和CPs-CDN组的1.3/1.4倍（CD4+：**p/*p）和2.1/1.5倍（CD8+ ***p/*p）。因此，联合组有效促进了脾脏T淋巴细胞的免疫调节作用，增加了CD4 + T和CD8 + T细胞的比例，有效增强抗肿瘤免疫应答。
实施例九 ATN2-Ms-PTX与CPs-CDN联合对荷瘤小鼠的毒性研究： 随着4T1乳腺癌病情的发展，小鼠的血液系统以及器官会受到影响；而长期用药也会给小鼠带来全身累计毒性。研究了本发明设计的PTX胶束和CDN囊泡制剂及其联合对荷瘤小鼠的全身累计影响或毒副作用的改善，按图8方案给药第22天，每组牺牲3只小鼠，取全血进行血常规检测、分离血清进行血生化检测，并解剖取主要脏器观察并切片，用H&E染色进行组织学分析。
血常规结果显示（图11 A），荷瘤小鼠血液（PBS组）中白细胞（WBC）数大量增多，是正常Balb/c小鼠的91倍（**p）。其中的中性粒细胞数（Neut）（*p）和淋巴细胞数（Lymph）（**p）升高最显著，这是由于肿瘤的生长和转移，以及出现了一些炎症导致的。红细胞数（RBC）、血红蛋白浓度（HGB）、红细胞压积（HCT）较正常小鼠有所下降，这主要是由于荷瘤小鼠出现贫血。此外，PBS组小鼠血液中的血小板数（PLT）、血小板压积（PCT）和平均血小板浓度（MPV）均有增加，这将增加血栓发生的概率。有研究表明，血小板有助于肿瘤细胞的存活和转移，因此血小板的增加也促使了4T1乳腺癌发生转移。和PBS组小鼠相比，自由PTX或自由CDN组治疗小鼠的血常规指标基本无缓解，ATN2-Ms-PTX或CPs-CDN单制剂治疗的小鼠血常规指标均有所缓解；而ATN2-Ms-PTX与CPs-CDN联合给药的小鼠血液中的WBC总量以及WBC中的Neut和Lymph、RBC、HGB、HCT、PLT、PCT和MPV均接近健康小鼠，进一步说明联合治疗对肿瘤的抑制效果明显、减少了肿瘤转移、避免小鼠发生贫血以及形成血栓。血生化检测表明，主要肝（碱性磷酸酶ALP、谷氨酰基转移酶GGT、天门冬氨酸氨基转移酶AST、丙氨酸氨基转移酶ALT）、肾（肌酐CRE、尿素URE）功能的各项指标与正常Balb/c小鼠的无显著性差异（图11 B）。
荷瘤小鼠主要脏器的H&E染色切片（图12）显示，各组小鼠心、肝、肾没有出现明显的组织损伤。PBS组、CDN和CPs-CDN组小鼠心脏有少许炎症，而PTX制剂各组和联合组小鼠心脏正常；除ATN2-Ms-PTX与CPs-CDN联合组，其他组小鼠肝脏均有轻微炎症发生。此外，PBS组小鼠脾脏显著增大、质量增加（图13），Yong等人也发现荷4T1乳腺癌Balb/c小鼠有脾肿大现象。治疗组小鼠脾脏的质量和肿瘤大小呈现正相关，即肿瘤越小，脾越小。其中联合组脾最小（约0.15 g，和健康小鼠接近），和ATN2-Ms-PTX和CPs-CDN单制剂组相比具有显著性差异（***p和*p）。从脾的H&E染色（图12）可以看到，PBS组白髓明显减少，红髓变多，这是4T1小鼠病程发展的标志之一。治疗组尤其是联合组具有丰富的白髓，说明阻止了乳腺癌病程的发展。以上结果说明，尾静脉给7.5 mg PTX/kg及瘤内注射1 mg CDN/kg及其联合给药，每2天给药、共4次的方式对小鼠没有明显毒性。
实施例十 ATN2-Ms-PTX与CPs-CDN联合抑制4T1乳腺癌的肺转移：在给药第22天，给小鼠腹腔注射荧光素钾盐，10分钟内处死每组剩余的3只小鼠，取全血进行血常规分析、分离出血清进行血生化分析。此外，解剖小鼠，取和心、肝、脾、肺、肾等器官，用组织固定液固定、切片、石蜡封切。切片用苏木精和伊红（H&E）染色，显微镜观察切片进行组织学分析和肺转移判断。由于TNBC具有高浸润性及高转移性，本发明也研究了PTX胶束和CDN囊泡及ATN2-Ms-PTX与CPs-CDN联合对荷4T1-luc乳腺癌肺转移的抑制。同图8方案给药后22天，解剖取肺部、称重以及离体成像、切片H&E染色做组织学分析。肺部成像及荧光定量结果显示（图14 A&B），PBS和自由PTX组小鼠发生了严重的肺转移，自由CDN和Ms-PTX组肺部荧光有显著下降（***p），而ATN2-Ms-PTX、CPs-CDN及联合联合能更有效抑制肺转移，分别为PBS组的22.8、19.3和107.7倍（***p）。其中ATN2-Ms-PTX较Ms-PTX也具有明显的肺转移抑制（*p），说明了ATN2能精准靶向到4T1上。ATN2-Ms-PTX和CPs-CDN联合组小鼠肺部几乎检测不到荧光信号（ 8.9×10 5 p/s）；该组肺的质量最小，约0.10 g，和健康小鼠接近；而其他组肺转移瘤越多，质量也大（图14 C），证明了ATN2-Ms-PTX和CPs-CDN联合治疗能最有效抑制4T1的肺转移。此外，肺部H&E染色图片显示，PBS组和自由PTX小鼠肺部存在大量肿瘤细胞和炎症细胞的浸润，几乎没有肺泡结构；Ms-PTX组有所改善，ATN2-Ms-PTX相比没有明显的肿瘤细胞群，但也存在肿瘤细胞和炎症细胞的浸润，肺泡结构也不明显。CPs-CDN组比自由CDN组能看到大片的肺泡结构，肿瘤浸润少，但也存在炎症。形成鲜明对比的是，ATN2-Ms-PTX和CPs-CDN联合组肺部切片看不到肿瘤细胞和炎症细胞，生理结构和正常小鼠无异，肺泡多且都有明显的中空结构（图14 D），结果和生物发光图吻合。结果证明本发明化疗与免疫药物相结合不仅能有效抑制原发性TNBC肿瘤，也能显著抑制其肺转移，尤其是，联合组显著延长了小鼠的中位生存期（33天）。

Claims (10)

  1. 一种化疗免疫联合药物，由化疗药物胶束与囊泡纳米STING激动剂组成，其特征在于，所述化疗药物胶束的制备方法为，将小分子药物、两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到化疗药物胶束；或者将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到化疗药物胶束；所述低聚乙二醇的分子量为200～600；所述囊泡纳米STING激动剂由聚合物囊泡装载STING激动剂组成。
  2. 根据权利要求1所述化疗免疫联合药物，其特征在于，小分子药物包括紫杉醇；所述低聚乙二醇的分子量为200～600；STING激动剂为环二核苷酸。
  3. 根据权利要求1所述化疗免疫联合药物，其特征在于，两亲性聚合物为PEG-P(CL-DTC)、PEG-P(TMC-DTC)、PEG-P(LA-DTC)中的一种或几种；靶向两亲性聚合物为所述两亲性聚合物偶联靶向分子；聚合物囊泡中，聚合物包括亲水链段、疏水链段以及阳离子片段，所述疏水链段的侧链为含双硫键的二硫戊环。
  4. 权利要求1所述化疗免疫联合药物在制备抗肿瘤药物中的应用。
  5. 权利要求1所述化疗免疫联合药物的制备方法，其特征在于，将小分子药物、两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到化疗药物胶束；或者将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到化疗药物胶束；将STING激动剂溶液加入缓冲溶液中，再加入聚合物溶液，搅拌后透析，得到囊泡纳米STING激动剂；由化疗药物胶束与囊泡纳米STING激动剂组成化疗免疫联合药物。
  6. 根据权利要求5所述化疗免疫联合药物的制备方法，其特征在于，将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液中，靶向两亲性聚合物的用量为聚合物总重量的1～30%。
  7. 根据权利要求5所述化疗免疫联合药物的制备方法，其特征在于，制备囊泡纳米STING激动剂时，所述聚合物的化学结构式如下：
    Figure dest_path_image001
    R为亲水链段；T链段与侧链含二硫戊环的碳酸酯链段组成疏水链段；T为环酯单体或者环碳酸酯单体形成的单元；P为阳离子片段；y、z表示重复单元。
  8. 根据权利要求7所述化疗免疫联合药物的制备方法，其特征在于，所述阳离子片段为聚乙烯亚胺或者精胺。
  9. 权利要求1所述化疗药物胶束与囊泡纳米STING激动剂在制备化疗免疫联合药物中的应用。
  10. PTX与STING激动剂在制备化疗免疫联合药物中的应用。
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