WO2022228469A1 - 聚合物囊泡纳米sting激动剂及其制备方法与应用 - Google Patents

聚合物囊泡纳米sting激动剂及其制备方法与应用 Download PDF

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WO2022228469A1
WO2022228469A1 PCT/CN2022/089592 CN2022089592W WO2022228469A1 WO 2022228469 A1 WO2022228469 A1 WO 2022228469A1 CN 2022089592 W CN2022089592 W CN 2022089592W WO 2022228469 A1 WO2022228469 A1 WO 2022228469A1
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cdn
cps
sting agonist
tumor
pei
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孟凤华
郑欢
曲艳
钟志远
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苏州大学
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6915—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the form being a liposome with polymerisable or polymerized bilayer-forming substances, e.g. polymersomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to nano-medicine technology, and specifically relates to a polymer vesicle nano-STING agonist and a preparation method and application thereof.
  • STING Activation of the STING pathway can enhance the body's anti-tumor immunity and is a promising approach for tumor immunotherapy.
  • viruses, liposomes, and cyclic dinucleotides (CDNs) are also agonists of the STING pathway.
  • CDNs such as synthetic cGAMP and cdiAMP, are mainly studied.
  • CDN is not easy to pass through the cell membrane to enter APC, and it is easy to be enzymatically hydrolyzed.
  • CDN is easy to diffuse and clear randomly in the body, and its pharmacokinetics is poor, and its systemic diffusion is also difficult. May cause cytokine storm.
  • Cationic polymers especially polyethyleneimine (PEI)
  • PEI polyethyleneimine
  • the high transfection ability and high toxicity of high-molecular-weight PEI hinder its practical in vivo application.
  • Low molecular weight PEI has low toxicity but low transfection efficiency.
  • STING is a small molecule cationic compound of human endogenous polypeptide with higher safety. How to efficiently load CDN with low toxicity and release it into APC cells efficiently and activate the STING pathway to trigger an efficient anti-tumor immune response is a challenge.
  • the invention discloses a polymer vesicle nano-STING agonist, a preparation method and application thereof, and solves the contradiction between efficient nucleic acid drug delivery and carrier toxicity.
  • the present inventors synthesized and characterized PEG-P(TMC-DTC)-PEI and PEG-P(TMC-DTC)-SP, which self-assembled into vesicles with asymmetric membrane structures in CDN-containing aqueous phases: longer
  • the PEG segment (5 kDa) is the outer shell of the vesicle, and the shorter PEI or SP is the inner shell of the vesicle.
  • the inner shell and the negatively charged CDN are combined by electrostatic interaction, thereby obtaining the vesicle CPs-CDN with high CDN loading;
  • the reduction-sensitive self-crosslinking structure conferred by DTC in the vesicle hydrophobic membrane makes the loading of CDN more stable.
  • Such a vesicle with a positively charged inner shell and an asymmetric membrane structure can efficiently load and deliver CDNs into tumor cells, and achieve efficient tumor treatment; there is no research and study on the loading of dinucleotides into vesicles in the prior art. report.
  • the present invention is based on reduction-sensitive reversibly cross-linked polymer vesicles, using low-toxic low-molecular-weight PEI (600 Da, 1200Da, etc.) and SP are cationic fragments.
  • PEI 600 Da, 1200Da, etc.
  • SP cationic fragments.
  • Two kinds of polymer vesicles with positively charged inner shells were designed and prepared for loading CDNs to form vesicular STING agonists (CPs-CDN).
  • CPs-CDN vesicular STING agonists
  • the polymer vesicle nano-STING agonist is composed of a polymer vesicle loaded with a STING agonist; the polymer includes a hydrophilic segment, a hydrophobic segment and a cationic segment; the side chain of the hydrophobic segment is a disulfide bond-containing side chain the dithiolane.
  • R is a hydrophilic segment, such as a polyethylene glycol segment; the T segment and the carbonate segment (unit formed by the DTC monomer) containing a disulfide bond in the side chain form a hydrophobic segment, and T is a cyclic ester monomer or Units formed by cyclic carbonate monomers, such as lactide, caprolactone, trimethylene carbonate; P is a cationic fragment, such as polyethyleneimine (PEI), spermine (SP); y, z represent repeating units .
  • PEI polyethyleneimine
  • SP spermine
  • the molecular weight of the hydrophilic segment is 3000-10000 Da; the molecular weight of the hydrophobic segment is 2.1-8.0 times the molecular weight of the hydrophilic segment; the molecular weight of the carbonate segment containing disulfide bonds in the side chain is the T chain The molecular weight of the cationic segment is 5% to 30% of the molecular weight of the hydrophilic segment.
  • the STING agonist is a cyclic dinucleotide (CDN), including cyclic guanosine dimonophosphate (c-di-GMP), cyclic adenosine dimonophosphate (c-di-AMP), 2 ⁇ ,3 ⁇ Cyclic monophosphate guano-monophosphate adenylate (2 ⁇ ,3 ⁇ -cGMAP), 3 ⁇ ,3 ⁇ cyclic monophosphate guano-monophosphate (3 ⁇ ,3 ⁇ -cGMAP) and their substituted derivatives , such as sulfur, fluorine, nitrogen substituted cyclic dinucleotide derivatives.
  • CDN cyclic dinucleotide
  • CDN cyclic dinucleotide
  • c-di-GMP cyclic guanosine dimonophosphate
  • c-di-AMP cyclic adenosine dimonophosphate
  • the polymer forms vesicles, and STING agonists are loaded to obtain CPs-CDN.
  • the hydrophobic membrane of vesicles contains a reduction-sensitive self-crosslinking structure endowed by DTC, which makes the loading of CDN more stable, and such an inner shell is positively charged.
  • the vesicles with asymmetric membrane structure can efficiently load and deliver drugs into tumor cells, and achieve efficient tumor treatment. There is no research and report on the loading of cyclic dinucleotides in the vesicles in the prior art.
  • the CPs-CDN is taken up by APC, and escapes after destroying the lysosome through the proton sponge effect.
  • the CDN is released in the cytoplasm and binds to the STING protein in the endoplasmic reticulum to initiate the STING pathway, which promotes the recognition and killing of tumor cells by T cells.
  • the present invention found that the vesicles based on the two polymers are different in physicochemical properties, endocytosis, intracellular drug release, promotion of DC maturation, and anti-mouse melanoma activity.
  • X-rays can make tumors release tumor-associated antigen TAA, induce double-stranded DNA breakage in tumor cells and activate the cGAS-STING pathway, the present invention further explores the synergistically enhanced anti-tumor efficacy of CPs-CDN combined with X-rays.
  • the invention discloses the application of the above-mentioned polymer vesicle nano-STING agonist in the preparation of anti-tumor drugs; the tumor is preferably melanoma.
  • the invention discloses a preparation method of the above polymer vesicle nano STING agonist.
  • the STING agonist solution is added into a buffer solution, then a polymer solution is added, and the polymer vesicle nano STING agonist is obtained by dialysis after stirring.
  • the STING agonist solution is an aqueous STING agonist solution;
  • the polymer solution is a DMF solution of the polymer;
  • the buffer solution is a HEPES buffer solution.
  • the concentration of the STING agonist solution is 0.1-10 mg/mL; the concentration of the polymer solution is 1-100 mg/mL.
  • disulfide cross-linked polymer vesicles are designed for the delivery of STING agonist CDN, and combined with radiotherapy to enhance anti-tumor immune response.
  • the particle size of polymer vesicles is 47 nm, which can efficiently encapsulate CDN, and has high biosafety, drug loading stability and reduction responsiveness. This is the first time that ADU-S100 is loaded with vesicles and combined with radiation for melanoma treatment, and compared the effects of low-dose X-ray combined with nano-STING agonist on TME changes and anti-tumor therapy for the first time.
  • this new therapeutic strategy has the following advantages: 1) The polymer vesicles can realize the efficient encapsulation of CDNs, and the disulfide cross-linking of the vesicle membrane makes it structurally stable and stable in the vesicle membrane.
  • Figure 1 is a schematic diagram of the preparation of the polymer vesicle nano-STING agonist CPs-CDN of the present invention and its combined X-ray anti-tumor immunotherapy in melanoma-bearing mice.
  • Figure 2 shows the 1 H NMR spectrum of PEG-P(TMC-DTC)-PEI (600 MHz, CDCl 3 ) and the 1 H NMR spectrum of PEG-P(TMC-DTC)-SP (400 MHz, CDCl 3 ) .
  • Figure 3 shows the characterization of the physicochemical properties of the polymersomes.
  • Figure 4 shows the cell viability of empty vesicles CPs/PEI ( A ) and CPs/SP ( B ) incubated with DC 2.4, RAW 264.7, and B16F10 cells for 12 h.
  • Figure 5 is a flow-through single-parameter histogram of the endocytosis of Cy5-CPs/PEI-diAMP and Cy5 - CPs/SP-diAMP co-incubated with DC 2.4, RAW 264.7, and B16F10 cells for 24 h.
  • Figure 6 shows the CLSM images of DC 2.4 cells co-incubated with CPs/PEI-Cy3-diAMP or CPs/SP-Cy3-diAMP for 2 h ( A ) and ( B ) fluorescence semi-quantitative images of Cy3-diAMP (the concentration of Cy3-diAMP is 300 nM). Nuclei were stained with DAPI (blue), lysosomes/endosomes were stained with Lysotracker deep red (red), and green fluorescence was Cy3-diAMP. The scale bar is 20 ⁇ m.
  • Figure 7 is the activation of BMDC in vitro.
  • Flow cytometry iso-pseudo-color plots ( A ) and quantitative statistics of activated BMDCs ( B ) after co-incubating BMDCs with PBS, CPs/SP, CPs/PEI, CDN, CPs/SP-CDN or CPs/PEI-CDN for 24 h ) (n 3).
  • C ELISA kit was used to detect the secreted cytokine IFN- ⁇ concentration in the medium.
  • Figure 8 is a CLSM image of tumor tissue sections showing the distribution of each fluorescence 24 h after intratumoral injection of Cy3-diAMP or Cy5-CPs/PEI-Cy3-diAMP.
  • DAPI blue: nucleus
  • Cy3 green: dinucleotide diAMP
  • Cy5 red: polymeric vesicles.
  • the scale bar is 20 ⁇ m.
  • A B16F10 tumor model establishment and dosing schedule; relative tumor volume ( B ) and body weight ( C ) changes;
  • Figure 10 shows the treatment of B16F10 tumor-bearing mice with 3 Gy X-ray combined with CPs/PEI-CDN and CDN.
  • A Dosing schedule;
  • B photo of mouse tumor on day 15;
  • C mouse tumor volume change;
  • D mouse survival curve;
  • E mouse relative body weight change;
  • F Tumor growth after contralateral inoculation of B16F10 cells in cured mice;
  • G Changes in tumor volume in a single mouse and the number of complete response (CR) mice.
  • Figure 14 shows that CPs/PEI-CDN combined with X-ray induces specific anti-tumor immune effects in B16F10-bearing mice.
  • Flow cytometry analysis of the proportion of mature DCs in mouse TDLN ( A ) and their quantitative data statistics (n 4) ( B ) .
  • ( C ) Content of CD8+ T cells in tumor TME (n 4).
  • Flow cytometry analysis of histogram ( D ) of positive expression of CD206 receptor on the surface of macrophages in TME and histogram of quantitative data (n 4)
  • AE TDLN or tumor analysis of mice at 48 h of last dose.
  • Figure 15 shows the tissue section analysis of mouse tumors after CPs/PEI-CDN and X-ray combined treatment.
  • A H&E staining
  • B CRT immunofluorescence staining
  • C CD8+ T cell immunofluorescence staining. Scale bar: 50 ⁇ m.
  • Figure 16 is an H&E staining image (40 ⁇ , 50 ⁇ m) of major organs of B16F10 melanoma-bearing mice.
  • a, b, c, d, e, f PBS, 3Gy, CDN 20 ⁇ g, CPs/PEI-CDN 20 ⁇ g, 3Gy+CDN 20 ⁇ g, 3Gy+CPs/PEI-CDN 20 ⁇ g, 5Gy+CPs/PEI-CDN 20 ⁇ g.
  • High-dose radiotherapy can cause systemic toxicity such as local tissue damage, while low-dose radiotherapy cannot effectively eliminate tumors; in addition, the STING signal transduction induced by local radiotherapy is short-lived, and the induced immune response is not enough to eliminate tumors; the invention discloses a polymer A vesicular nano-STING agonist, using STING agonists to further activate the STING pathway of APCs in low-dose radiotherapy-treated tumors, synergistically generate TAA and TA generated by radiotherapy, elicit specific CD8+ T cells, modulate TME, and induce robust anti-tumor immunity In response, it stimulates the secretion of cytokines and T cell recruitment factors, stimulates DC maturation and cross-presentation of tumor antigens, which in turn initiates anti-tumor T cell killing and enhances innate and adaptive immune responses.
  • the present invention uses low-toxicity low-molecular-weight PEI (600 Da, 1200 Da, etc.) and SP as cationic fragments, and designs and prepares two kinds of polymer vesicles with positively charged inner shells for use in
  • the vesicular STING agonist (CPs-CDN) was used to load CDNs and explored its effect on activating the STING pathway to generate anti-tumor immunity.
  • the strategy of using nanocarriers to deliver CDNs can not only protect CDNs from degradation and change their biodistribution, but also promote their efficient endocytosis by APCs, improve T cell responses and antitumor immunity, and reduce dosage and avoid high-dose effects. potential risks.
  • the raw materials involved in the present invention are all conventional products or commercially available products; the specific preparation operations and testing methods are all conventional methods in the art. All data were analyzed using GraphPad Prism version 6.01. Using ANOVA One-way ANOVA and t-test to determine significant differences in each group, Kaplan-Meier for mid-survival technical analysis. Statistical significance was established at *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the present invention synthesized and characterized PEG-P(TMC-DTC)-PEI and PEG-P(TMC-DTC)-SP, which self-assembled into vesicles with asymmetric membrane structures in CDN-containing aqueous phase Vesicles:
  • the longer PEG segment (5 kDa) is the outer shell of the vesicle, and the shorter PEI or SP is the inner shell of the vesicle.
  • the inner shell and the negatively charged CDN are recombined through electrostatic interaction, thus obtaining the vesicle CPs with high CDN loading.
  • CDN the longer PEG segment
  • Cyclic dinucleotide ADU-S100 (99.23%) was purchased from MCE (Master of Small Molecules); Cy3-labeled linear dinucleotide (Cy3-diAMP, 90%) was purchased from Shanghai Sangon Bioengineering.
  • Use directly after purchase Use directly after purchase.
  • Mouse IFN- ⁇ ELISA detection kit InvivoGen
  • mouse TNF- ⁇ ELISA detection kit Biolegend
  • Mouse Antibody PerCP/Cy5.5- ⁇ CD45, APC- ⁇ CD3, FITC- ⁇ CD3, APC- ⁇ CD80, PE- ⁇ CD86, FITC- ⁇ CD11c, FITC- ⁇ CD11b, APC- ⁇ CD206, PE- ⁇ F4/80, APC- ⁇ FoxP3, PE - ⁇ CD4, FITC- ⁇ CD8a, PE/Cy7- ⁇ Gr-1 were purchased from BioLegend.
  • Hydrogen nuclear magnetic resonance ( 1 H NMR) was measured by a DD2-600 liquid superconducting nuclear magnetic resonance spectrometer (Agilent, USA) at 600 MHz, using deuterated chloroform (CDCl 3 ) as the solvent, and the chemical shifts were determined by the residual solvent signal as standard.
  • the particle size distribution and surface potential of the polymersomes were determined by a nanoparticle size analyzer (Zetasizer Nano-ZS, Malvern Instruments, UK) equipped with a 633 nm He-Ne laser.
  • the concentration of ADU-S100 was determined with a NanoDrop 2000c Micro UV Analyzer (Thermo, USA).
  • the morphology of the polymersomes was measured by a Tecnai G220 transmission electron microscope (TEM, FEI, USA) at an accelerating voltage of 120 kV.
  • TEM transmission electron microscope
  • FEI transmission electron microscope
  • the cellular uptake of vesicles and the maturation of DCs, phenotypic shift of macrophages and activation of T cells in the TME after stimulation with different formulations were analyzed using a BD FACSVerse flow cytometer.
  • Confocal laser (CLSM) images were taken by Lecia TCS SP5.
  • the absorbance at 570 nm of formazan generated by live cells and MTT was detected by a multi-function microplate reader (Thermo Fisher Scientific), and the cell viability was calculated by quantitative calculation.
  • Tumor-bearing mice were subjected to local irradiation treatment using a biological X-ray irradiator model RS 2000Pro (RAD Source, USA).
  • BMDC Bone marrow-derived dendritic cells
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • B16F10, RAW 264.7, and DC 2.4 cells were cultured in DMEM medium (Gibco, USA) containing 10% FBS, 1% penicillin-streptomycin. All cells were cultured in monolayer in a 37°C, 5% CO 2 incubator Model 3111 (Thermo Fisher Scientific, USA).
  • B16F10 and DC 2.4 cells were trypsinized with 0.25% (w/v) containing 0.03% (w/v) ethylenediaminetetraacetic acid (EDTA), and RAW 264.7 cells were detached with a spatula , followed by centrifugation at 1000 rpm for 3 min with an L-420 low-speed centrifuge (Hunan Xiangyi Laboratory Instrument, China), followed by two washes with PBS.
  • EDTA ethylenediaminetetraacetic acid
  • mice C57BL/6J female mice (6-8 weeks old, 18-20 g) were purchased from Viton Lihua (Beijing, China) company. Incubate with sterile water and feed at 25°C, 55% humidity and programmed light and dark conditions. All animal experiments were carried out in strict accordance with the protocols approved by the Animal Experiment Center of Soochow University and the Animal Care and Use Committee.
  • DCM dichloromethane
  • the sealed closed reactor was placed in an oil bath at 40 °C, stirred (600 rpm) for 48 h, and triethylamine was used as the reaction terminator. According to the volume ratio of the polymer solution/ice ether of 1:15, precipitation in ice ether twice, centrifugation, and vacuum drying to obtain polymer PEG-P (TMC-DTC).
  • the hydroxyl group at the end of PEG-P(TMC-DTC) was activated with N′,N′-carboxydiimidazole (CDI), and then reacted with the primary amine of PEI.
  • CDI N′,N′-carboxydiimidazole
  • the synthesis of the polymer PEG-P(TMC-DTC)-SP is also divided into two steps.
  • the first step is the same as the above description, that is, PEG-P(TMC-DTC) is synthesized first, and then the terminal hydroxyl group is activated with CDI; the second step is to react the activated polymer with the primary amine of SP.
  • PEG-P(TMC-DTC) (1 g, 45 ⁇ mol) was weighed into a two-necked flask, dissolved in 1 mL of DCM, and CDI (8.8 mg, 54 ⁇ mol) in DCM was added dropwise under nitrogen protection. solution (88 mg/mL), activated at 30 °C for 4 h.
  • the activated polymer was added dropwise to the pre-cooled SP (98.2 mg, 0.45 mmol) solution in DMF, and then the reaction was continued for 4 h at room temperature. After the reaction was completed, the reactant was precipitated with ice ether, centrifuged, and dried in vacuo. The yield was 85.2%.
  • NMR: PEG: ( ⁇ 3.51); TMC ( ⁇ 4.24, 2.03); DTC ( ⁇ 4.19, 3.02); SP ( ⁇ 2.84-2.92, 2.94, 3.29); Mn ( 1 H NMR) 22.4 kg/mol.
  • PEG-P(TMC-DTC)-PEI and PEG-P(TMC-DTC)-SP were synthesized by a three-step method: firstly, TMC and DTC were polymerized by controlled ring opening using methoxyPEG (5000 Da) as an initiator. The block polymer PEG-P (TMC-DTC) was obtained, and then the terminal hydroxyl group was activated with CDI, and then reacted with the primary amine of PEI or SP to obtain the final polymer, which realized the loading, protection and efficient delivery of the CDN into APC to activate the STING pathway .
  • Preparation of empty vesicles and CDN-loaded polymeric vesicles CPs/PEI-CDN or CPs/SP-CDN.
  • the specific process of preparing empty vesicles by nanoprecipitation is as follows: Pipette 100 ⁇ L of PEG-P(TMC-DTC)-PEI or PEG-P(TMC-DTC)-SP in DMF solution (10 mg/mL ), added to 900 ⁇ L of HEPES buffer solution (5 mM, pH 6.8). Routine magnetic stirring for 10 minutes, then dialyzed against HEPES buffer (5 mM, pH 6.8) for 6 h, and then dialyzed against PB (10 mM, pH 7.4) for half an hour to obtain empty vesicles.
  • ADU-S100 ML The structural formula of RR-S2 CDA is as follows:
  • the specific process of preparing drug-loaded vesicles by nanoprecipitation method is as follows: ADU-S100 is taken out from the -80°C refrigerator, prepared into a 2 mg/mL aqueous solution, and stored in a -80°C refrigerator to avoid repeated freezing and thawing.
  • the preparation process of CDN-loaded vesicles is not much different from the above, except that 80 ⁇ L of CDN solution (0.16 mg) is poured into HEPES buffer solution, and then into the polymer solution (10 mg/mL), and the rest of the steps were the same.
  • Example 2 Characterization of empty vesicles and CDN-loaded polymeric vesicles (CPs/PEI-CDN or CPs/SP-CDN)
  • the particle size, particle size distribution, and surface Zeta potential of vesicles were determined by dynamic light scattering (DLS) and electrophoresis techniques. Through particle size tracking, the stability of CPs/PEI-CDN and CPs/SP-CDN in 10% FBS solution, high dilution and long-term storage at 4 °C, as well as the reduction response behavior under 10 mM GSH, were analyzed.
  • the drug loading (DLC) and encapsulation efficiency (DLE) of ADU-S100 were determined by NanoDrop, and the empty carrier of equal concentration and particle size was used as the background, and the absorption value at 260 nm was recorded. According to the known concentration of ADU-S100 A standard curve of absorbance values was drawn for calculation.
  • the TEM image shows a typical spherical hollow vesicle structure, see Figure 3A, B.
  • the static light scattering (SLS) measurement results show that the radius of gyration (Rg) and hydrodynamic radius (Rh) of CPs/PEI and CPs/SP The ratios were 0.905 and 1.001, respectively, close to the theoretical value of 1.0, confirming the hollow spherical vesicle structure of the two, see Figure 3C,D.
  • PEG-P(TMC-DTC)-PEI or PEG-P(TMC-DTC)-SP can form CDN-loaded vesicles with high efficiency in CDN-containing aqueous phase, denoted as CPs/PEI-CDN and CPs/SP- CDN, as in the theoretical drug load of 20 wt.%, the encapsulation efficiency of CDN was as high as 86.0% and 88.2%, corresponding to 17.2 wt.% and 17.6 wt.% of drug loading.
  • the particle size of the obtained vesicles increased.
  • the particle size of CPs/SP-CDN was significantly larger than that of CPs/SP, and the particle size distribution maintained a low level.
  • the drug-loading capacity of the CDN of these two vesicles is significantly better than that of liposomes, hydrogels, and DEAMEA-based polymer vesicles that have been reported so far.
  • Such high drug loading is attributed to the complexation of CDN with PEI or SP of the inner vesicle shell and the protection of the cross-linked stable vesicle membrane.
  • vesicles prepared from PEG-P(TMC-DTC) loaded with the same CDN had very low drug loadings, see Table 2 for the CPs-CDN group. In the experiment, it has been found that both vesicles have excellent colloidal stability, and the particle size of these two drug-loaded vesicles did not change during storage for 3 weeks.
  • ADU-S100 was replaced with another CDN (c-di-GMP, cyclic diguanylic acid), and the nanoparticles (PS-GMP) obtained by the same drug loading method (CPs/SP as polymers) were characterized as follows:
  • B16F10, DC 2.4 and RAW 264.7 cells were plated in 96-well plates at densities of 2000/well, 5000/well and 10000/well, respectively After incubation for 24 h, blank vesicles with different concentrations were added, and after co-cultivation for 24 h, 10 ⁇ L of sterile MTT solution (5 mg/mL) was added to each well, and the cells were placed in an incubator for further incubation for 4 h.
  • PEI has shown great advantages in the efficient delivery of nucleic acid drugs and gene transfection, its application in vivo is often limited due to its systemic toxicity.
  • the present invention evaluated the relationship between empty polymer vesicles and dendritic cells (DC 2.4), macrophages (RAW) by MTT assay. 264.7) and tumor cells (B16F10) after co-incubation for 12 h.
  • DC 2.4 dendritic cells
  • RAW macrophages
  • B16F10 tumor cells
  • B16F10, RAW 264.7, and DC 2.4 cells were plated in 6-well plates at 10 5 , 2 ⁇ 10 5 , and 10 6 cells/well. After incubation for 24 h, 200 ⁇ L Cy5-CPs/PEI-diAMP, Cy5-CPs/PEI were added. -diAMP (16 nM in the well of Cy5) with PBS as blank control. After co-incubating for 24 h, the cells were digested with trypsin, centrifuged (1000 rpm, 5 min), and washed with PBS. Finally, the cells were suspended in 500 ⁇ L of PBS and analyzed by flow cytometry with BD FACS Verse.
  • Cy5-labeled vesicles Cy5-CPs/SP-diAMP and Cy5-CPs/PEI-diAMP were prepared according to conventional methods using Cy5-labeled polymer and linear diAMP as model dinucleotide, and measured by flow cytometry The two polymer drug-loaded vesicles were respectively associated with DC 2.4. The uptake of RAW 264.7 and B16F10 cells after co-incubating for 24 h.
  • the vesicles were co-loaded with a fluorescently labeled dinucleotide (Cy3-diAMP) and a linear model dinucleotide (diAMP).
  • DC 2.4 cells were plated in 24-well plates containing round coverslips at a density of 1 ⁇ 10 5 cells/well, and 100 ⁇ L of CPs/PEI-diAMP or CPs/ SP-diAMP (the in-well concentration of Cy3-diAMP is 300 nM), after incubation at 37 °C for 1 h, the medium was aspirated with a pipette and washed three times with PBS.
  • Endosomes/lysosomes were stained with Lysotracker Deep Red (100 nM) for 1 h, fixed with 4% paraformaldehyde for 15 min, nuclei were stained with DAPI for 5 min, and washed several times with PBS after each staining. , and finally mounted with glycerol, stored in the dark, and used CLSM to take fluorescence images of cells.
  • Polymer vesicles CPs/PEI and CPs/SP can enhance the uptake of CDN in DC cells, but CDN must bind to STING protein in the cytoplasmic endoplasmic reticulum to activate the immune response, so the vesicle escapes from the endosome and releases the CDN to the cytoplasmic ER. Cytoplasm is important for enhancing the efficacy of CDNs and enhancing anti-tumor immune responses.
  • the present invention uses Cy3-labeled diAMP (Cy3-diAMP) as a model to qualitatively study the cellular uptake and endosomal escape of Cy3-diAMP in drug-loaded vesicles CPs/PEI and CPs/SP, as shown in Figure 6.
  • Cy3-diAMP Cy3-labeled diAMP
  • the results of CLSM experiments showed that after DC cells were incubated with CPs/PEI-Cy3-diAMP and CPs/SP-Cy3-diAMP for 2 h, Cy3-diAMP was delivered into DCs, while CPs/PEI delivered Cy3-diAMP into cells.
  • Cy3-diAMP-loaded vesicles are blocked by DCs 2.4 Cells uptake into endosomes, Cy3-diAMP escapes from endosomes with the help of PEI or SP, and then is reduced in the cytoplasm to release dinucleotides.
  • the CDN-loaded vesicles of the invention can activate the BMDC cells of mice and activate the STING pathway, induce the production of type I IFN by activating the STING pathway, and promote the activation and functional maturation of DC cells.
  • the maturation of DCs plays an important role in the cross-presentation of antigens and the generation of T cells.
  • All BMDCs were labeled with FITC- ⁇ CD11c and mature BMDCs were labeled with APC- ⁇ CD80 and PE- ⁇ CD86.
  • CPs/PEI-CDN and CPs/SP-CDN enter the cytoplasm through endocytosis, which greatly increases the concentration of intracellular CDN, greatly activates the STING signaling pathway, and significantly promotes the maturation of BMDCs (***p) .
  • the concentration of IFN- ⁇ secreted by CDN preparation-induced cells in BMDC medium was detected by ELISA kit.
  • PBS and empty vesicles could not induce the secretion of IFN- ⁇ , while the free CDN group had significantly increased IFN- ⁇ , indicating that the secretion of IFN- ⁇ was CDN-dependent;
  • CPs vesicles further promoted the secretion of IFN- ⁇ , and the CPs/SP-CDN and CPs/PEI-CDN increased by 1.8 and 2.3 times, respectively (***p), indicating that the vesicles enhanced the ability of CDN to activate the STING pathway.
  • CPs/PEI-CDN induced IFN- ⁇ secretion more significantly (**p), which was attributed to the superior cellular uptake capacity and endosomal escape and cytoplasmic escape of CPs/PEI-CDN The ability to effectively release.
  • STING-induced IFN- ⁇ not only coordinates the innate immune response, but also plays an important role in the adaptive immune response, including promoting the maturation of APC function, the cross-presentation of antigens, and the priming and activation of specific T cells; in addition to enhancing antitumor
  • type I IFNs can also participate in tumor suppression by inducing apoptosis and directly acting on tumor antiproliferative responses.
  • Cy5-labeled polymer (Cy5-PEG-P(TMC-DTC)) was blended with PEG-P(TMC-DTC)-PEI to prepare fluorescently labeled polymersomes and loaded with Cy3-diAMP.
  • Cy3-diAMP (Cy3: 0.2 ⁇ g/mouse)
  • Cy5-CPs-Cy3-diAMP (Cy5: 0.2 ⁇ g/mouse) were injected into the tumor site, respectively, 24 h later Tumor tissue was removed, sectioned (8 ⁇ m), nuclei were stained with DAPI, and pictures were taken with CLSM.
  • CDN may diffuse rapidly after local injection due to its small molecular weight and strong water solubility, which affects the retention of CDN in tumors and reduces the bioavailability of CDN.
  • the problem After the nanomedicine of the present invention enters the tumor, due to the damaged lymphatic drainage at the tumor site, it accumulates locally in the tumor and prolongs the retention time of the carrier.
  • Cy3-diAMP is used as a model and Cy5-labeled vesicle is used to study Cy5-CPs/PEI- The distribution of Cy3-diAMP in tumor tissue, to explore the retention of CDN and CPs/PEI-CDN in tumor.
  • Cy3-diAMP or Cy5-CPs/PEI-Cy3-diAMP was injected intratumorally, respectively.
  • the mice were sacrificed 24 hours later, and the tumors were isolated, sectioned and stained.
  • Figure 8 Fluorescence imaging shows that free Cy3-diAMP has only weak green fluorescence signal in tumor sections, indicating that Cy3-diAMP has small molecular weight, strong water solubility and rapid in vivo diffusion.
  • the green fluorescence in the tumor was very strong, and the red fluorescence of the vesicles was also very strong, and the two were evenly distributed in the tumor.
  • the intratumoral injection of the CPs/PEI-CDN of the present invention can remain in the tumor for a long time, which is beneficial for the APC infiltrated by the tumor to uptake, activate the STING pathway and immune response, and enhance the activation of the STING pathway by adding CPs/PEI-CDN.
  • Its anti-tumor effect can not only directly damage DNA, kill tumor cells, change the tumor microenvironment (TME), and release tumor-associated antigens (TAA), but also enhance cytoplasmic DNA induction through the cGAS/STING pathway and induce the production of type I IFN. Thereby promoting DC maturation and activation of T cells, inducing an adaptive immune response.
  • the day of administration was taken as the 0th day, and the mice were given the following treatment regimens on the 0th, 3rd, 6th, and 9th days: 3Gy X-ray, CPs/PEI-CDN+3Gy X-ray, or CPs/SP-CDN+3Gy X-ray, vesicular CDN was administered intratumorally, and the dose of CDN was 20 ⁇ g/vesicle.
  • X-ray was irradiated locally in the subcutaneous tumor, and PBS was the control group.
  • each mouse was inoculated with 1.5 x 105 B16F10 cells, the tumor volume was measured with a vernier caliper every two days, and when the volume reached 50 mm3 , the treatment was performed as planned.
  • the day of radiotherapy was recorded as day 0, and X-ray irradiation with a dose of 3 Gy or 5 Gy was performed every 3 days for a total of three cycles; 50 ⁇ L of PBS and free CDN were injected into the tumor on days 1, 4, and 8. , empty vesicles (CPs/PEI) or CPs/PEI-CDN.
  • the tumor volume and body weight were measured every two days, and when the volume reached 2000 mm 3 , the animals were euthanized.
  • the mice were administered when the tumor volume reached 50 mm3 , with PBS as the control group.
  • the treatment results showed that intratumoral injection of CDN therapy significantly inhibited tumor growth (***P) and significantly prolonged the survival of mice.
  • CPs/PEI-CDN more effectively enhanced the immune activity of CDN, further inhibited the growth of melanoma (**p), and significantly prolonged the median survival time (*p). It was found that X-ray therapy alone delayed tumor growth only initially, rebounded rapidly after drug withdrawal, and ultimately had limited ability to inhibit melanoma growth and improve survival in mice (23 days vs 15 days), while X-ray and intratumoral injections were free
  • the combination of CDN is better at delaying tumor growth and prolonging the survival of mice (***p).
  • the combination of 3Gy X-rays and CPs/PEI-CDN had significant effects on tumor inhibition, prolongation of mouse survival (42 days) and survival benefit in tumor-bearing mice (***p).
  • the combination of empty vesicle CPs/PEI with 3Gy X-ray could not achieve the effect of further enhancing tumor therapy.
  • tumor suppression was positively correlated with the injected CDN dose 10 days before treatment, and on day 10, 10/21 mice in the CPs/PEI-CDN combined with X-ray group were free of solid tumors at a dose of 10 ⁇ g/ The only group accounted for 0/21, and the dose of 20 ⁇ g/only group accounted for 3/21.
  • mice At a dose of 30 ⁇ g/mouse, all the solid tumors in this group disappeared. For a long time after the treatment, the body weight of the mice did not decrease significantly, which once again proved that 3Gy X-ray treatment alone or in combination with CPs, CDN, CPs/PEI-CDN did not cause serious body toxicity.
  • mice 48 h after the first injection of 5 Gy X-ray and CDN or CPs/PEI-CDN, red, inflamed crusts appeared in the tumor sites of individual mice, and 48 h after the second injection, CPs/PEI-CDN
  • the vast majority of mice (5/7) in the treatment group developed large, burning black scabs, and a few mice (2/7) had light brown scabs with partial tumor regression, while only two mice in the CDN group developed Scab condition. Acute necrosis at the injection site was associated with STING-induced production of TNF- ⁇ .
  • mice On the 8th to 12th day of administration, all solid tumors in the CPs/PEI-CDN group regressed, but on the 14th, 16th, 18th, and 34th days, one mouse tumor recurred in situ (the scab had fallen off) .
  • the combination of 5 Gy X-ray and CPs/PEI-CDN was similar in tumor suppression to the combination with CDN, but the median survival of mice was extended from 39 days to 54 days, and 3/7 mice ended up tumor-free. From the body weight changes of mice, it can be found that single or combined treatment with 5 Gy X-rays has little effect on the body weight of mice, and does not cause serious toxic side effects.
  • mice in the group treated with 5 Gy X-ray combined with CPs/PEI-CDN still completely resisted tumor growth, and have survived for 131 days so far, while the mice previously treated with 5 Gy X-ray combined with CDN survived less.
  • the 25-day survival rate of mice was 0%. Resistance to secondary tumor cell vaccination thus confirms the induction of an adaptive immune response against tumor antigens following treatment of the primary tumor.
  • Example 7 Analysis of the regulation of tumor immune microenvironment by flow cytometry
  • mice When the tumor volume of B16F10 tumor-bearing mice was ⁇ 100 mm3 , they were administered according to the protocol of the systemic therapy study. 48 h after the last administration, the tumor, tumor-draining lymph node (TDLN), and spleen of the mice were dissected, the tissues were washed with PBS, and placed in a watch dish containing 1% FBS in PBS buffer, using mechanical lysis The original suspension was obtained by the separation method, and the single-cell suspension was obtained after filtering through a 200-mesh nylon mesh. The obtained single-cell suspension was centrifuged (1200 rpm, 5 min), and 1 mL of PBS buffer containing 1% FBS was added to resuspend.
  • TDLN tumor-draining lymph node
  • spleen of the mice 48 h after the last administration, the tumor, tumor-draining lymph node (TDLN), and spleen of the mice were dissected, the tissues were washed with PBS, and placed
  • Tumor cells were added with 1 mL of ACK lysis buffer to lyse red blood cells at 4 °C, 10 mL of PBS was added after 10 minutes, and centrifuged. Count using a cell counting plate, incubate 10 6 cells/tube with the corresponding antibody at 4° C. in the dark for 20 minutes, and gently tap the cell suspension every 5 minutes to prevent cells from sinking to the bottom.
  • Mouse antibodies PerCP/Cy5.5- ⁇ CD45, FITC- ⁇ CD11c, APC- ⁇ CD80, PE- ⁇ CD86 were used for DC maturation analysis; mouse antibodies PerCP- ⁇ CD45, FITC- ⁇ CD11b, APC- ⁇ CD206, PE- ⁇ F4/80 were used for Phenotypic analysis of macrophages; mouse antibodies APC- ⁇ CD3, FITC- ⁇ CD8 ⁇ labeled CD8+T; BD FACS Verse flow cytometer was used for detection, and FlowJo v10 was used for data analysis.
  • Cytotoxic T lymphocytes (CTL, CD3+CD4-CD8+) play a key role in the killing of tumor cells and are considered to be key effector cells in cancer immunotherapy.
  • flow cytometry was used to study the changes in the proportion of CD8+ T cells infiltrated in the tumor after CPs/PEI-CDN and its combined X-ray treatment.
  • the PBS group, the X-ray group and the intratumoral injection The content of CD8+ T cells in the tumor TME of the free CDN group was less than 0.4%, while the CPs/PEI-CDN greatly promoted the infiltration of CD8+ T cells (*p), showing that vesicular nanoagonists are beneficial to recruit CTLs to TME.
  • the induction of tumor-infiltrating CD8+ T cells was further enhanced when it was combined with X-ray therapy.
  • the increase of CPs/PEI-CDN dose and X-ray dose did not increase the proportion of CD8+ T cells in TME.
  • Polymer vesicle loading enhanced the retention of CDN in tumors, promoted CDN endocytosis, and then promoted the activation of STING pathway and T cell activation;
  • CPs/PEI-CDN combined with X-ray further activated the STING pathway of APC , significantly increased the proportion of effector T cells in the TME and enhanced the antitumor immune response.
  • Figure 14D and E show that compared with M2-type macrophages (CD11b+F4/80+CD206+) in the PBS group, the M2 of the free CDN group decreased significantly, while the M2 of the vesicular CPs/PEI-CDN group was significantly decreased ( ***p), indicating that macrophages were repolarized or recruited after CPs/PEI-CDN treatment, and immunosuppression was reduced.
  • STING agonists and their nanoformulations can reprogram macrophages to switch from immunosuppressive M2 to M1 types and from immunosuppressive cold tumors to immunogenic hot tumors; however, further combined X-ray did not significantly promote the transformation of macrophage phenotype.
  • ELISA detection 6 hours after the last administration, the orbital blood was collected to collect the blood samples of the mice, the serum was separated using a serum separation tube, and the expression levels of TNF- ⁇ and IFN- ⁇ in the serum of the mice were detected according to the instructions of the ELISA kit. Read using a multi-plate reader.
  • the antitumor immune response is highly dependent on the signaling of type I IFNs, which promote DC processing and antigen presentation, and promote their migration to lymph nodes, which are critical for functional CD8+ T cell-mediated antitumor immune responses. Therefore, 6 h after the last administration, blood samples were taken to detect the concentrations of IFN- ⁇ and TNF- ⁇ in mouse serum using ELISA kit.
  • Figure 14F and G show that intratumoral injection of CPs/PEI-CDN resulted in a significant increase in the secretion of IFN- ⁇ and TNF- ⁇ , while the content of endogenous cytokines induced by 3 Gy X-ray was low, indicating that radiotherapy alone responded to the anti-tumor immune response. Startup has limited effect.
  • the combination of the two can further increase the expression of cytokine IFN- ⁇ in serum (**p); the secretion of IFN- ⁇ is proportional to the dose of CDN. Increasing the combined X-ray dose to 5 Gy resulted in a very significant increase in both IFN- ⁇ and TNF- ⁇ secretion (***p).
  • the control of tumor by intratumoral injection of CDN therapy is divided into two stages. The first stage is the early hemorrhagic necrosis of the tumor caused by the secretion of TNF- ⁇ , and the second stage is CD8+T cell-dependent recurrence control.
  • the combination of X-ray and CPs/PEI-CDN can generate a powerful immune response at the tumor site.
  • Example 8 Tissue section and immunohistochemical analysis: In order to evaluate the tissue toxicity caused by the treatment and the infiltration of T cells in the tumor tissue, 48 h after the last administration, the heart, liver, spleen, lung, kidney and tumor tissues of the mice were removed. Tumors were washed, fixed, embedded, sectioned, and stained with hematoxylin and eosin (H&E) for pathological analysis.
  • H&E hematoxylin and eosin
  • paraffin sections (8 ⁇ m) of tumor tissue were deparaffinized, antigen retrieved, and serum blocked with rabbit ⁇ CD8 (1:500; Abcam) or rabbit ⁇ CRT antibody (1:200; Abcam) Incubate at 37 °C for 1 h, wash 3 times with PBS, and incubate with Alexa Fluor 633-conjugated goat anti-rabbit secondary antibody (1:500; Invitrogen) for 1 h at 37 °C. Fluorescence images were taken with a Bath BX41 upright fluorescence microscope (Olympus BX41).
  • FIG. 15 shows H&E of tumor tissue sections Staining confirmed the therapeutic effect of X-ray in combination with CPs/PEI-CDN.
  • the combination of 5 Gy X-ray and CPs/PEI-CDN resulted in the largest area of necrosis of tumor cells on the whole section, and a large number of inflammatory cells infiltrated, which was consistent with the previous results.
  • tumor cells in the free CDN, X-ray, and X-ray and free CDN combined groups also exhibited necrosis and melanin deposition, however, the loading of CPs/PEI enhanced the antitumor effect of CDN, and CPs/PEI-CDN and 3
  • the degree of apoptosis of tumor cell nuclei was significantly increased, a large number of tumor cells shrank and ruptured, the number of tumor cells was significantly reduced, and there was a small amount of immune cell infiltration. Exposure of tumor cell surface CRT is necessary for immunogenic killing. As can be seen from FIG.
  • the PBS group and the CDN preparation group without X-ray irradiation failed to trigger CRT exposure, and almost no red fluorescence was observed.
  • X-rays can induce tumor cells to secrete CRT, and obvious red fluorescence was observed in both the X-ray and X-ray combined treatment groups, indicating that the secretion of CRT increased, and the ICD effect was obvious, which could be used as an "eat-me" signal to promote the internalization of DC Endocytosis and antigen presentation, recruitment of CTLs, induction of immune responses, and inhibition of tumor growth.
  • the results of immunofluorescence staining of CD8+ T cells showed that the red fluorescence in the vesicular CPs/PEI-CDN group and free CDN significantly increased, and the CD8+ T cells in the tumor tissue increased, while the CPs/PEI-CDN combined with X-rays
  • the infiltration of CD8+ T cells in the tumor tissue of the group was more, which further enhanced the anti-tumor immune effect.
  • the limited ability of X-ray alone to increase tumor-infiltrating CD8+ T cells is consistent with the flow cytometry results.
  • Example 9 Pharmacokinetics of CPs/PEI-CDN in tumor tissue:
  • Cy7-labeled oligonucleotide AMP was used as a model nucleotide, one-time Injected CPs/PEI-Cy7-AMP or Cy7-AMP (Cy7 concentration: 10 ⁇ g/mice) into the tumor of tumor-bearing mice, blood was collected from the mouse orbit at the set time point, and then measured using a multi-plate reader Cy7 concentration in plasma.
  • the present invention designs and studies disulfide cross-linked inner shell positively charged polymer vesicles (CPs/PEI-CDN) for efficient delivery of small molecule STING agonist cyclic dinucleotides (CDN) and their combination with low-dose X-rays Immunotherapy for mouse melanoma.
  • CPs/PEI-CDN disulfide cross-linked inner shell positively charged polymer vesicles
  • CDN small molecule STING agonist cyclic dinucleotides
  • the vesicles are formed by self-assembly and self-crosslinking of PEG-P(TMC-DTC)-PEI in the aqueous phase, and the particle size is small and uniform (45 nm, PDI 0.16), the cycle is stable; its positively charged inner shell composed of low molecular weight PEI composites the negatively charged CDN through electrostatic interaction, which not only solves the contradiction between the efficient delivery of small nucleic acid drugs and the toxicity of the carrier, but also through the proton sponge. The effect helps it escape the endosome.
  • CPs/PEI-CDN prolonged the tumor retention time of CDN, enhanced its cytoplasmic transmission, enhanced the activation of STING pathway in the tumor microenvironment and tumor draining lymph nodes (TDLN), and improved the efficacy of CDN.
  • low-dose X-rays (3 Gy ⁇ 3, 5 Gy ⁇ 3) combined with CPs/PEI-CDN further activated the STING pathway of tumor-infiltrating APCs, promoted the migration, recruitment and activation of mature DCs to TDLN CD8+ T cells induce adaptive immune responses, effectively inhibit tumor growth, and prolong the median survival of mice.

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Abstract

聚合物囊泡纳米STING激动剂及其制备方法与应用。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，得到聚合物囊泡纳米STING激动剂。聚合物囊泡纳米STING激动剂由聚合物囊泡装载STING激动剂组成；聚合物囊泡的粒径小，可高效装载STING激动剂，并且具有高生物安全性、载药稳定性以及还原响应性，这是首次用囊泡装载STING激动剂并联合放射用于恶性黑色素瘤的治疗，聚合物囊泡的纳米级特性提高了抗原递呈细胞(APC)对STING激动剂的摄取，延长了其在肿瘤部位的滞留，大大提高了STING激动剂的免疫活性，增强STING通路的激活。

Description

聚合物囊泡纳米STING激动剂及其制备方法与应用 技术领域
本发明属于纳米药物技术，具体涉及聚合物囊泡纳米STING激动剂及其制备方法与应用。
 背景技术
STING通路的激活可增强机体抗肿瘤的免疫能力，是肿瘤免疫治疗的一种有前途的方法。有研究表明，STING激动剂有利于促进TME中T细胞的浸润以及抗肿瘤免疫治疗。除了天然的cGAMP，病毒、脂质体和环状二核苷酸（CDN）也是STING通路的激动剂，目前主要研究的是CDN，如合成的cGAMP、cdiAMP等。但CDN作为带负电荷、亲水性强的小分子，不容易穿过细胞膜进入APC，还容易被酶解，此外，CDN在体内容易随机扩散和清除，药代动力学差，其全身扩散还可能引起细胞因子风暴。抗肿瘤纳米药物的研发方兴未艾。阳离子聚合物尤其是聚乙烯亚胺（PEI）具有出色的压缩/复合核酸的能力和内涵体/溶酶体逃逸能力，常用于核酸类药物的递送。然而，高分子量PEI的转染能力强，毒性也高，阻碍了其实际体内应用。低分子量PEI毒性虽低但转染效率也低。精胺（SP）是人体内源性多肽小分子阳离子化合物，安全性更高。如何高效低毒地装载CDN并高效释放到APC细胞、激活STING通路引发高效抗肿瘤免疫应答是一个挑战落。
 技术问题
本发明公开了聚合物囊泡纳米STING激动剂及其制备方法与应用，解决了高效核酸药物递送与载体毒性间的矛盾。本发明合成和表征了PEG-P(TMC-DTC)-PEI和PEG-P(TMC-DTC)-SP，它们在含有CDN的水相中自组装成具有不对称膜结构的囊泡：较长PEG链段（5 kDa）为囊泡外壳、较短PEI或SP为囊泡内壳，内壳与带负电的CDN通过静电相互作用复合，从而得到了高效载CDN的囊泡CPs-CDN；囊泡疏水膜中含有DTC所赋予的还原敏感的自交联结构使得CDN的装载更稳定。这样的内壳带正电荷的具有不对称膜结构的囊泡能高效装载和递送CDN到肿瘤细胞内，实现了肿瘤的高效治疗；现有技术没有过该囊泡装载二核苷酸的研究和报道。
 技术解决方案
本发明基于还原敏感可逆交联的聚合物囊泡，使用低毒的低分子量PEI（600 Da、1200Da等）和SP为阳离子片段，设计制备了两种内壳带正电的聚合物囊泡用于装载CDN形成囊泡STING激动剂（CPs-CDN），分析了其激活STING通路产生抗肿瘤免疫的效果。本发明采用如下技术方案：
聚合物囊泡纳米STING激动剂，由聚合物囊泡装载STING激动剂组成；所述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。
进一步的，组成载体的聚合物的化学结构式如下。
Figure 826046dest_path_image001
。
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。
本发明中，所述亲水链段的分子量为3000～10000Da；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%。
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。
本发明中，聚合物形成囊泡，装载STING激动剂得到CPs-CDN，囊泡疏水膜中含有DTC所赋予的还原敏感的自交联结构使得CDN的装载更稳定，这样的内壳带正电荷的具有不对称膜结构的囊泡能高效装载和递送药物到肿瘤细胞内，实现了肿瘤的高效治疗，现有技术还没有过该囊泡装载环二核苷酸的研究和报道。CPs-CDN被APC摄取，通过质子海绵效应破坏溶酶体后逃离，细胞质中释放CDN，与内质网上的STING蛋白结合，启动STING通路，促进T细胞识别和杀死肿瘤细胞。本发明发现基于两种聚合物的囊泡在理化性质、细胞内吞、胞内药物释放、促进DC成熟，以及抗小鼠黑色素瘤活性方面存在差异。此外，由于X射线能使肿瘤释放肿瘤相关抗原TAA，诱导肿瘤细胞双链DNA的断裂进而激活cGAS-STING通路，本发明进一步探究了CPs-CDN与X射线联用协同增强的抗肿瘤疗效。结果表明，二者联用提高了肿瘤微环境TME中IFN-β的浓度，促进了DC细胞的成熟，提高了TME中CD8+T的比率，从而增强了对B16F10肿瘤的抑制，延长了荷瘤小鼠的生存时间。本发明公开了上述聚合物囊泡纳米STING激动剂在制备抗肿瘤药物中的应用；肿瘤优选为黑色素瘤。
本发明公开了上述聚合物囊泡纳米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，得到聚合物囊泡纳米STING激动剂。进一步的，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。其中，STING激动剂溶液的浓度为0.1～10 mg/mL；聚合物溶液的浓度为1～100 mg/mL。
 有益效果
本发明设计了双硫交联的聚合物囊泡用于STING激动剂CDN的递送，并研究了其联合放疗增强抗肿瘤免疫反应。聚合物囊泡的粒径为47 nm，可高效包载CDN，并且具有高生物安全性、载药稳定性以及还原响应性，这是首次用囊泡装载ADU-S100并联合放射用于黑色素瘤的治疗，并首次比较了低剂量的X射线联合纳米STING激动剂对TME的改变以及抗肿瘤治疗的影响。与目前报道的STING激动剂治疗相比，这种新的治疗策略有以下优点：1）聚合物囊泡可实现CDN的高效包载，囊泡膜的二硫交联使其结构稳定，并在细胞内还原环境下快速释放CDN；2）聚合物囊泡的纳米级特性提高了APC细胞对STING激动剂的细胞摄取，延长了其在肿瘤部位的滞留，大大提高了STING激动剂的免疫活性，增强STING通路的激活； 3）与X射线联用，增强了STING通路的激活，促进I型IFN以及其他细胞因子的分泌，激活DC的成熟以及T细胞的活化，改善TME，诱导了强大的抗肿瘤免疫反应。4）联用的策略解决了低剂量的X射线可以避免造成组织的损伤但肿瘤抑制效果不佳的缺陷。
 附图说明
图1为本发明聚合物囊泡纳米STING激动剂CPs-CDN的制备及其联合X射线用于荷黑色素瘤小鼠的抗肿瘤免疫治疗的示意图。
图2为PEG-P(TMC-DTC)-PEI 的 1H NMR谱图（600 MHz，CDCl 3）以及PEG-P(TMC-DTC)-SP 的 1H NMR谱图（400 MHz，CDCl 3）。
图3为聚合物囊泡的理化性质表征。
图4为空囊泡CPs/PEI （ A ）和CPs/SP （ B ）与DC 2.4、RAW 264.7、B16F10三种细胞共孵育12 h后的细胞存活率。
图5为Cy5-CPs/PEI-diAMP和Cy5 -CPs/SP-diAMP与DC 2.4、RAW 264.7、B16F10三种细胞共孵育24 h后的细胞内吞的流式单参数直方图。
图6为DC 2.4细胞与CPs/PEI-Cy3-diAMP或CPs/SP-Cy3-diAMP共孵育2 h的CLSM图片 （ A ）和 （ B ）Cy3-diAMP的荧光半定量图（Cy3-diAMP浓度为300 nM）。细胞核用DAPI（蓝色）染色，溶酶/内涵体用Lysotracker deep red（红色）染色，绿色荧光为Cy3-diAMP。标尺为20 μm。
图7为体外BMDC的活化。BMDC与PBS、CPs/SP、CPs/PEI、CDN、CPs/SP-CDN或CPs/PEI-CDN共孵育24 h后的流式细胞等伪色图 （ A ）和被激活BMDC的定量统计 （ B ）（n = 3）。FITC-αCD11c标记BMDC细胞，APC-αCD80和PE-αCD86标记成熟的BMDC细胞； （ C ）使用ELISA试剂盒检测培养基中分泌的细胞因子IFN-β浓度。
图8为肿瘤组织切片CLSM图片显示瘤内注射Cy3-diAMP或Cy5-CPs/PEI-Cy3-diAMP后24 h的各荧光的分布。DAPI（蓝色）：细胞核，Cy3（绿色）：二核苷酸diAMP，Cy5（红色）：聚合物囊泡。标尺为20 μm。
图9为CPs/PEI-CDN及其与X射线联用在荷黑色素瘤B16F10小鼠中的免疫治疗（n = 3）。在第0、3、6、9天给药： 3Gy X射线、CPs/PEI-CDN+3Gy X射线、或CPs/SP-CDN+3Gy X射线，囊泡CDN通过瘤内给药，CDN剂量为20 μg /只，X射线在皮下瘤局部照射，PBS为对照组。 （ A ）B16F10肿瘤模型的建立及给药方案；相对肿瘤体积 （ B ）和体重 （ C ）变化； （ D ）每只老鼠的相对肿瘤体积变化。
图10为3 Gy X射线与CPs/PEI-CDN、CDN联合治疗B16F10荷瘤小鼠。 （ A ）肿瘤模型的建立以及联合瘤内给药方案；CPs/PEI-CDN治疗B16F10荷瘤小鼠的肿瘤体积 （ B ）和 （ C ）相对体重变化（n=7）；CPs/PEI-CDN与X 射线联合治疗荷瘤小鼠的肿瘤体积 （ D ）和 （ E ）体重变化（n = 7）；不同剂量的CDN治疗对小鼠肿瘤体积 （ F ）和 （ G ）小鼠体重的影响（n = 7）；#表示此后有1只老鼠存活，##表示此后有2只老鼠存活。
图11为3 Gy X射线与CPs/PEI-CDN、CDN联合治疗B16F10荷瘤小鼠，开始治疗后小鼠的生存曲线（n=7）。
图12为3 Gy X射线与CPs/PEI-CDN、CDN联合治疗B16F10荷瘤小鼠，每只老鼠的肿瘤体积变化图以及完全响应的小鼠数量（n=7）。
图13为使用5 Gy X射线与CDN或CPs/PEI-CDN联合治疗B16F10荷瘤小鼠（n = 7）。 （ A ）给药方案； （ B ）第15天的小鼠肿瘤照片； （ C ）小鼠肿瘤体积变化； （ D ）小鼠的生存曲线； （ E ）小鼠的相对体重变化； （ F ）治愈小鼠对侧接种B16F10细胞后肿瘤的生长情况； （ G ）单只老鼠的肿瘤体积变化以及完全响应（CR）小鼠数量。
图14为CPs/PEI-CDN与X射线联合诱导荷B16F10小鼠的特异性的抗肿瘤免疫效应。流式细胞仪分析小鼠TDLN中成熟DC的比例 （ A ）及其定量数据统计图（n = 4） （ B ）。 （ C ）肿瘤TME中CD8+T细胞的含量（n = 4）。流式细胞术分析TME中巨噬细胞表面CD206受体表达阳性的直方图 （ D ）及其定量数据统计图（n = 4） （ E ）。最后一次给药6 h小鼠血清中IFN-β （ F ）和TNF-α （ G ）的浓度（n = 3）。 A-E：在最后一次给药的48 h小鼠的TDLN或肿瘤分析。
图15为CPs/PEI-CDN和X射线联合治疗后小鼠肿瘤的组织切片分析。 （ A ）H&E染色图； （ B ）CRT的免疫荧光染色图； （ C ）CD8+T细胞免疫荧光染色图。标尺：50 μm。
图16为荷B16F10黑色素瘤小鼠主要器官的H&E染色图（40´，50 μm）。a, b, c, d, e, f: PBS, 3Gy, CDN 20μg, CPs/PEI-CDN 20μg, 3Gy+CDN 20 μg, 3Gy+CPs/PEI-CDN 20 μg, 5Gy+CPs/PEI-CDN 20 μg。
图17为一次瘤内注射CPs/PEI-Cy7-AMP或自由Cy7-AMP后小鼠血浆中Cy7的浓度随时间的变化（Cy7: 10 μg/只，n = 3）。
 本发明的实施方式
高剂量的放疗会造成局部组织损伤等系统毒性，而低剂量又不能有效消除肿瘤；此外，局部放疗诱导的STING信号传导是短暂的，引起的免疫应答不足以消除肿瘤；本发明公开了聚合物囊泡纳米STING激动剂，使用STING激动剂来进一步激活低剂量放疗的肿瘤中APC的STING通路，协同放疗产生的TAA和TA，引发特异性CD8+T细胞，调节TME，诱导强大的抗肿瘤免疫应答，刺激细胞因子、T细胞募集因子的分泌，刺激DC成熟和肿瘤抗原的交叉呈递，进而启动抗肿瘤T细胞杀伤作用，增强固有和适应性免疫应答。本发明基于还原敏感可逆交联的聚合物囊泡、使用低毒的低分子量PEI（600 Da、1200Da等）和SP为阳离子片段，设计制备了两种内壳带正电的聚合物囊泡用于装载CDN形成囊泡STING激动剂（CPs-CDN），探索了其激活STING通路产生抗肿瘤免疫的效果。使用纳米载体递送CDN的策略不仅可以保护CDN免受降解，改变其生物分布；还能促进其被APC有效内吞，提高T细胞应答和抗肿瘤免疫力；还能减少用量，避免高剂量带来的潜在风险。
本发明涉及的原料都是常规产品或者市售产品；具体制备操作以及测试方法都是本领域常规方法。所有数据使用6.01版本的GraphPad Prism进行分析。使用ANOVA 单因素方差分析和t-test来确定每组的显著性差异，生存中期采用Kaplan-Meier 技术进行分析。统计学显著性建立在*p＜0.05，**p＜0.01，***p＜0.001。
参见图1，本发明合成和表征了PEG-P(TMC-DTC)-PEI和PEG-P(TMC-DTC)-SP，它们在含有CDN的水相中自组装成具有不对称膜结构的囊泡：较长PEG链段（5 kDa）为囊泡外壳、较短PEI或SP为囊泡内壳，内壳与带负电的CDN通过静电相互作用复合，从而得到了高效载CDN的囊泡CPs-CDN。
环二核苷酸ADU-S100（99.23%）购自MCE（Master of Small Molecules）；Cy3标记的线形二核苷酸（Cy3-diAMP，90%）购自上海生工生物工程。聚乙烯亚胺（PEI， M n =0.6 kg/mol，99％，Sigma）、精胺（SP，Mn =0.2 kg/mol，99%，Sigma）、N，N'-羰基二咪唑（CDI，98%，上海百灵威）购买后直接使用购买后直接使用。小鼠IFN-β ELISA检测试剂盒（InvivoGen）、小鼠TNF-α ELISA检测试剂盒（Biolegend）按照生产商提供的说明书进行使用。小鼠抗体PerCP/Cy5.5-αCD45、APC-αCD3、FITC-αCD3、APC-αCD80、PE-αCD86、FITC-αCD11c、FITC-αCD11b、APC-αCD206、PE-αF4/80、APC-αFoxP3、PE-αCD4、FITC-αCD8a、PE/Cy7-αGr-1均购自BioLegend。
核磁共振氢谱（ 1H NMR）由DD2-600液体超导核磁共振谱仪（安捷伦，美国）在600 MHz 下测得，以氘代氯仿（CDCl 3）为溶剂，化学位移以残留溶剂信号为标准。聚合物囊泡的粒径分布及表面电位通过纳米粒度仪（Zetasizer Nano-ZS，马尔文仪器，英国）测定，并配备 633 nm He-Ne激光器。ADU-S100的浓度用 NanoDrop 2000c 微量紫外分析仪（Thermo，美国）测定。聚合物囊泡的形貌通过 Tecnai G220 型透射电子显微镜（TEM，FEI，美国）在120 kV加速电压下测得。使用BD FACSVerse流式细胞仪分析细胞对囊泡的摄取以及经不同制剂刺激后DC细胞的成熟、巨噬细胞表型的转变以及TME中T细胞的激活。激光共聚焦（CLSM）图像由Lecia TCS SP5拍摄。使用多功能酶标仪（Thermo Fisher Scientific）检测活细胞与MTT生成的甲瓚在570 nm处的吸光值，通过定量计算得到细胞的存活率。使用型号为RS 2000Pro的生物学X射线辐照仪（RAD Source，美国）对荷瘤小鼠进行局部辐照治疗。
小鼠黑色素瘤细胞系（B16F10）、小鼠单核巨噬细胞白血病细胞（RAW 264.7）均从中科院上海库购买。骨髓来源的树突状细胞（BMDC）从6周龄的C57BL/6J雌性小鼠的腿骨和胫骨中获取，并用含10% 胎牛血清（FBS，Gibco，美国）、1%青-链霉素（基诺生物医学技术公司，中国）以及20 ng/mL的粒细胞-巨噬细胞集落刺激因子（GM-CSF，PeproTech，美国）的RPMI-1640培养基（HyClone，美国）于6孔板中培养，48 h后更换全部培养基，之后每48 h更新一半的培养基，培养至第9天后收集未贴壁以及松散粘附在培养皿上的DC细胞，通过表面CD11c的表达确定表型。B16F10、RAW 264.7以及DC 2.4细胞均用含10% FBS、 1% 青-链霉素的DMEM培养基（Gibco，美国）培养。所有的细胞放在 37 ℃、 5％ CO 2的3111型培养箱（Thermo Fisher Scientific，美国）中单层培养。当细胞汇合率达80%左右时，使用含 0.03％（w/v）乙二胺四乙酸（EDTA）的 0.25％（w/v）胰蛋白酶消化B16F10和DC 2.4细胞，用刮刀分离 RAW 264.7细胞，接着用L-420 低速离心机（湖南湘仪实验室仪器，中国），1000 rpm 离心3分钟，随后用 PBS 洗涤两次。
C57BL/6J雌鼠（6-8周龄，18-20 g）从维通利华（北京，中国）公司购买。在25 ℃，55%湿度以及程序明暗的条件下，用无菌水和饲料培养。所有的动物实验均严格按照苏州大学动物实验中心和动物保护和使用委员会批准的方案进行。
实施例一
PEG-P(TMC-DTC)-PEI和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，比如：
第一步，开环聚合制备聚合物PEG-P(TMC-DTC)。在氮气保护的手套箱内称取MeO-PEG-OH（0.50 g， M n=5.0 kg/mol，100 μmol），DTC（0.23 g，1.18 mmol），TMC（1.52 g，14.55 mmol），将它们溶解在7.0 mL的二氯甲烷（DCM）中，放在磁力搅拌台上边搅拌边加入磷酸二苯酯（DPP，DPP/OH摩尔比为10/1）作为催化剂。将密封好的密闭反应器放于40 ℃油浴中，搅拌（600 rpm）反应48 h，使用三乙胺作为反应终止剂。按照聚合物溶液/冰乙醚的体积比为1：15的比例，在冰乙醚中沉淀两次，离心，真空干燥得到聚合物PEG-P(TMC-DTC)。
第二步，先用N′，N′-羧基二咪唑（CDI）对PEG-P(TMC-DTC)末端的羟基进行活化，然后与PEI的伯胺进行反应。称取1.1g PEG-P(TMC-DTC)（羟基0.05 mmol）和24 mg CDI（0.15 mmol）溶于DCM中，30 ℃反应24 h，然后在冰乙醚中沉淀三次，按之前方法真空干燥得到PEG-P(TMC-DTC)-CDI。接着，称取PEI（ M n= 0.6 kg/mol，246 mg，0.41 mmol）于两颈瓶中，加入 6 mL无水DCM溶解为150 mg/mL，另称取PEG-P(DTC-TMC)-CDI（ M n= 22000 kg/mol，900 mg，0.041 mmol），加入9 mL无水DCM溶解为100 mg/mL。在氮气保护条件下，将该PEG-P(DTC-co-TMC)-CDI聚合物溶液转移至干燥的恒压滴液漏斗（规格: 10 mL），密封，在冰水浴搅拌下，滴加至PEI溶液中。滴加完成后，转移至30 ℃油浴磁力搅拌下继续反应4 h。所得产物转移入25 mL干燥的单颈圆底烧瓶中，真空旋蒸，蒸出DCM（二氯甲烷），浓缩聚合物（聚合物目标浓度：200 mg/mL）。在30倍过量（体积比）的冰乙醇/乙醚混合溶剂（v/v 1:4）中沉淀三次，离心弃上清，放置真空干燥箱干燥至恒重，得到最终产物PEG-P(TMC-DTC)-PEI，最终产率为70.0%。核磁：PEG:（δ 3.51）；TMC（δ 4.24，2.03）；DTC（δ 4.19，3.02）；PEI（δ 2.51-3.15）； M n（ 1H NMR）= 23.2 kg/mol。
聚合物PEG-P(TMC-DTC)-SP的合成也分为两步，第一步与上述描述相同，即先合成PEG-P(TMC-DTC)，然后用CDI活化末端羟基；第二步是将活化的聚合物与SP的伯胺进行反应。简而言之，称取PEG-P(TMC-DTC)（1g，45 μmol）置于两颈烧瓶，用1mL DCM溶解，在氮气保护的环境下滴加CDI（8.8 mg，54 μmol）的DCM溶液（88 mg/mL），30 ℃活化4 h。将活化好的聚合物滴加到预冷的SP（98.2 mg，0.45 mmol）的DMF溶液中，然后置于室温继续反应4 h。反应结束后，使用冰乙醚沉淀反应物，离心，真空干燥，产率为85.2%。核磁：PEG:（δ 3.51）；TMC（δ 4.24，2.03）；DTC（δ 4.19，3.02）；SP（δ 2.84-2.92，2.94，3.29）； M n（ 1H NMR）= 22.4 kg/mol。
三步法合成了PEG-P(TMC-DTC)-PEI和PEG-P(TMC-DTC)-SP：首先以甲氧基PEG（5000 Da）为引发剂、通过可控开环聚合TMC和DTC得到嵌段聚合物PEG-P(TMC-DTC)，然后用CDI活化末端羟基、再和PEI或SP的伯胺反应得到最终聚合物，实现了装载、保护和高效递送该CDN进入APC激活STING通路。参见图2，通过 1H NMR对PEG-P(TMC-DTC)-PEI以及PEG-PEG-P(TMC-DTC)-SP的结构进行分析。通过TMC（δ 2.03，4.24）、DTC（δ 3.02，4.19）特征峰的积分与PEG特征峰（δ 3.65）的积分的比值，可分别计算出TMC和DTC的聚合度和分子量，结果表明聚合物的分子量和理论分子量很接近，见表1，通过PEI（δ 2.59-2.80）或SP（δ 2.84-2.92）的特征峰积分和DTC特征峰（δ 3.02）的比值，可得到的PEI或SP的官能化度分别为94.4%和100.3%。
Figure 216621dest_path_image002
空囊泡和装载CDN的聚合物囊泡（CPs/PEI-CDN或CPs/SP-CDN）的制备。通过纳米沉淀法制备空囊泡的具体过程如下：用移液枪吸取100 μL的PEG-P(TMC-DTC)-PEI或PEG-P(TMC-DTC)-SP的DMF溶液（10 mg/mL），加入到900 μL的HEPES缓冲溶液（5 mM，pH 6.8）中。常规磁力搅拌10分钟，再用HEPES缓冲液（5 mM，pH 6.8）透析6 h后，再用PB（10 mM，pH 7.4）透析半小时，得到空囊泡。
为了研究聚合物囊泡STING激动剂的制备、性质和免疫激活作用，选用已在临床II期试验的一种CDN（ADU-S100）来研究，其瘤内注射治疗黑色素瘤的抗肿瘤治疗效果更佳，遗憾的是，该CDN的结构和性质都会限制其被APC的摄取，影响其抗肿瘤免疫活性。ADU-S100（ML RR-S2 CDA）的结构式如下：
Figure 934041dest_path_image003
通过纳米沉淀法制备载药囊泡的具体过程如下：将ADU-S100从-80℃冰箱取出，配置成2 mg/mL的水溶液，分装保存于-80℃冰箱，避免反复冻融。装载CDN的囊泡的制作过程与上述差别不大，区别在于将80 μL CDN溶液（0.16 mg）打入到HEPES缓冲溶液中，然后打入聚合物溶液（10 mg/mL），其余步骤一样。
实施例二 空囊泡和装载CDN的聚合物囊泡（CPs/PEI-CDN或CPs/SP-CDN）的表征
囊泡的粒径、粒径分布以及表面Zeta电位通过动态光散射（DLS）和电泳技术进行测定。通过粒径追踪，分析CPs/PEI-CDN、CPs/SP-CDN在10% FBS溶液、高倍稀释以及在4 ℃条件下长期储存的稳定性以及在10 mM GSH条件下的还原响应行为。ADU-S100的载药量（DLC）和包封率（DLE）通过NanoDrop进行测定，以等浓度、等粒径的空载体作为背景，记录260 nm处的吸收值，根据已知浓度ADU-S100的吸收值绘制的标准曲线进行计算。表征图见图3， （ A ）CPs/PEI和 （ B ）CPs/SP的粒径分布、TME电镜图（标尺为50 nm），以及 （ C ）CPs/PEI和 （ D ）CPs/SP的SLS测试； （ E ）CPs/PEI-CDN和 （ F ）CPs/SP-CDN在10% FBS中、高倍稀释以及4℃放置3周的粒径分布的变化；（G）二者在含10 mM GSH 的PB中12 h后粒径的变化； （ H ）装载Cy3-diAMP的两种囊泡在体外模拟细胞内还原环境（PB，10 mM，pH 7.4，10 mM GSH）和生理环境（PB，10 mM，pH 7.4）下Cy3-diAMP的累积释放（n = 3）。
PEG-P(TMC-DTC)-PEI或PEG-P(TMC-DTC)-SP的DMF溶液加入到HEPES缓冲溶液（pH 6.8，5 mM）中，可自组装形成具有不对成膜结构的囊泡CPs/PEI和CPs/SP：长的PEG（5 kDa）排列在外部组成囊泡的外壳，而短PEI和SP被屏蔽到囊泡内壳。囊泡在制备过程中膜中的DTC开环自交联。CPs/PEI、CPs/SP的表征结果见表2。其TEM图显示典型的球形中空囊泡结构，参见图3A,B，由静态光散射仪（SLS）测定结果可知，CPs/PEI和CPs/SP的回旋半径（Rg）与流体力学半径（Rh）的比值分别为0.905和1.001，接近理论值1.0，证实了二者的中空的球形囊泡结构，参见图3C,D。
PEG-P(TMC-DTC)-PEI或PEG-P(TMC-DTC)-SP在含有CDN的水相中可形成高效装载CDN的囊泡，分别表示为CPs/PEI-CDN和CPs/SP-CDN，如在理论载药量为20 wt.%时，CDN的包封率分别高达86.0%和88.2%，对应载药量17.2 wt.%和17.6 wt.%。得到囊泡的粒径和空载体相比有增大，CPs/SP-CDN的粒径显著大于CPs/SP粒径，粒径分布维持较低的水平。这两个囊泡的CDN的载药量明显优于目前已报道的脂质体、水凝胶、以及基于DEAMEA的聚合物囊泡。这样高载药量要归因于CDN与囊泡内壳的PEI或SP的复合以及交联稳定的囊泡膜的保护。相比之下，由PEG-P(TMC-DTC)制备得到的囊泡装载相同CDN的载药量非常低，见表2的CPs-CDN组。实验中已经发现两种囊泡均具备优异的胶体稳定性，这两种载药囊泡在储存过程中放置3周粒径也没有变化，不仅如此，在含10%的FBS溶液中，也没有造成囊泡解离或是团聚，粒径及粒径分布变化不明显；在模拟静脉注射的极低浓度下，囊泡也没有解离成单分子，保持了粒径及粒径分布，图3E,F。而在含有10 mM GSH的溶液中，由于GSH还原了囊泡膜中的双硫交联，12 h后部分囊泡粒径明显变大，图3G。
Figure 283245dest_path_image004
a在PB（10 mM, pH 7.4）缓冲液中用动态光散射（DLS）在室温下测得； b在PB（10 mM，pH 7.4）缓冲液中通过Zetasizer Nano-ZS在室温下测得； c通过 Nanodrop测得。
将ADU-S100更换为另一种CDN（c-di-GMP, 环二鸟苷酸），同样的载药方法（CPs/SP为聚合物）得到的纳米粒（PS-GMP）表征如下：
Figure 35301dest_path_image005
实施例三 空白囊泡的细胞毒性实验
为了研究空白囊泡（CPs/PEI、CPs/SP）的体外毒性，将B16F10、DC 2.4和RAW 264.7细胞分别按2000个/孔、5000个/孔和10000个/孔的密度铺于96孔板中，孵育24 h后，加入不同浓度的空白囊泡，共同培养24 h后，每孔加入10 μL无菌的MTT溶液（5 mg/mL），放入培养箱继续孵育4 h。用移液枪慢慢地吸走培养基，每孔加入150 μL DMSO，溶解蓝紫色甲瓚结晶，用酶标仪测定每孔在570 nm 处的吸收值。通过每孔与对照组吸光度的比值计算细胞存活率。
虽然PEI在核酸药物的高效递送以及基因转染方面表现出了极大的优势，但往往因为其系统毒性限制了在活体中的应用。本发明通过MTT实验评估了空聚合物囊泡与树突状细胞（DC 2.4）、巨噬细胞（RAW 264.7）、肿瘤细胞（B16F10）共孵育12 h后的细胞毒性。在CPs/PEI和CPs/SP浓度高达400 μg/mL时，三种细胞的存活率均高于85%，图4。结果说明，CPs/PEI和CPs/SP均具有良好的生物安全性。
实施例四 CPs-CDN的体外细胞内吞以及内涵体逃逸行为研究
B16F10、RAW 264.7、DC 2.4细胞按10 5、2×10 5、10 6个/孔铺于6孔板中，孵育24 h 后，加入200 μL Cy5-CPs/PEI-diAMP、Cy5-CPs/PEI-diAMP （Cy5的孔内浓度为16 nM），以PBS作为空白对照。共孵育24 h后用胰酶消化、离心（1000 rpm，5 min）、PBS清洗，最后将细胞悬浮在500 μL的PBS中，用BD FACS Verse进行流式细胞术分析。
使用Cy5标记的聚合物、以线性的diAMP作为模型二核苷酸，根据常规方法分别制备Cy5标记的囊泡Cy5-CPs/SP-diAMP和Cy5-CPs/PEI-diAMP，用流式细胞仪测定了这两种聚合物载药囊泡分别与DC 2.4、RAW 264.7、B16F10细胞共孵育24 h后被摄取的情况。实验结果显示，这两种囊泡的细胞摄取存在巨大差异，参见图5：在DC 2.4、RAW 264.7、B16F10三种细胞中，Cy5-CPs/PEI-diAMP的荧光强度均高于Cy5-CPs/SP-diAMP，前者分别是后者的3.6倍、4.6倍和69倍；此外，DC2.4细胞的内吞能力显著高另外两种细胞。 
为了考察载药囊泡的内涵体逃逸行为，使用囊泡共同载荧光标记的二核苷酸（Cy3-diAMP）和线性的模型二核苷酸（diAMP）。将DC 2.4细胞以1×10 5个细胞/孔的密度铺在含有圆形盖玻片的24孔板中，培养24 h后加入100 μL共载Cy3-diAMP的CPs/PEI-diAMP或CPs/SP-diAMP（Cy3-diAMP的孔内浓度为300 nM），37℃孵育1 h后，用移液枪吸走培养基，用PBS洗涤3次。用 Lysotracker Deep Red（100 nM）对内涵体/溶酶体染色1 h，用4％多聚甲醛固定15 min，用DAPI对细胞核染色5分钟，每次染色结束后都要用PBS进行多次洗涤，最后用甘油封片，保存在黑暗条件中，使用CLSM拍摄细胞荧光图像。
聚合物囊泡CPs/PEI和CPs/SP可以提高CDN在DC细胞中的摄取，但CDN必须与细胞质内质网上的STING蛋白结合才能激活免疫反应，因此囊泡从内涵体逃逸、并释放CDN到细胞质对于提高CDN的效能以及增强抗肿瘤免疫反应具有重要意义。本发明根据常规方法，用Cy3标记的diAMP（Cy3-diAMP）为模型，定性地研究载药囊泡CPs/PEI和CPs/SP中Cy3-diAMP的细胞摄取和内涵体逃逸情况，图6。CLSM实验结果显示，DC细胞在和CPs/PEI-Cy3-diAMP和CPs/SP-Cy3-diAMP孵育2 h后，均有Cy3-diAMP被递送到DC中，而CPs/PEI递送进入细胞的Cy3-diAMP的量要比CPs/SP的明显要多，ImageJ半定量表明高2.35倍，这和流式细胞仪测定的细胞摄取的结果相吻合。此外，将在DC 2.4细胞中Cy3-diAMP绿色荧光与内涵体红色荧光叠加后，发现二者不完全重合，表明两种囊泡在2小时就从内涵体逃逸。因此，载Cy3-diAMP囊泡被DC 2.4细胞摄取进入内涵体，在PEI或SP的辅助下Cy3-diAMP逃离内涵体，然后在细胞质中被还原释放出二核苷酸。
本发明的载CDN囊泡能活化小鼠的BMDC细胞和激活STING通路，通过激活STING通路，诱导I型IFN的产生，促进DC细胞的激活和功能的成熟。在抗肿瘤免疫中，DC的成熟对于抗原的交叉呈递以及T细胞的产生具有重要作用。使用FITC-αCD11c标记所有的BMDC，用APC-αCD80和PE-αCD86标记成熟的BMDC。孵育24 h后，阴性对照PBS组中成熟DC（CD80+CD86+）占总细胞数的15.8%，空囊泡CPs/PEI、CPs/SP并未诱导明显的BMDC成熟。文献报道含有胺的化合物能够激活DC，本发明的限定的囊泡没有激活。而自由CDN、CPs/SP-CDN以及CPs/PEI-CDN则显著增加了成熟BMDC（CD80+CD86+）的比例分别到59.3%、75.1%和80.5%，图7，其中CPs/PEI-CDN刺激BMDC成熟的能力最强。相比于CDN，CPs/PEI-CDN及CPs/SP-CDN通过内吞进入细胞质，大大增加了细胞内CDN的浓度，极大激活STING信号通路，显著促进了BMDC的成熟（***p）。
用ELISA试剂盒检测了BMDC培养基中CDN制剂诱导细胞分泌的IFN-β的浓度。由图7C可知，PBS和空囊泡无法诱导IFN-β的分泌，而自由CDN组有显著提升的IFN-β，说明IFN-β的分泌是CDN依赖的；其次，相比于CDN，CPs囊泡进一步促进了IFN-β的分泌，CPs/SP-CDN和CPs/PEI-CDN分别提高了1.8和2.3倍（***p），说明了囊泡增强了CDN的激活STING通路的能力。而相比于CPs/SP-CDN，CPs/PEI-CDN更显著地诱导IFN-β分泌（**p），这要归因于CPs/PEI-CDN优越的细胞摄取能力和内涵体逃逸以及细胞质有效释放的能力。STING的诱导的IFN-β不仅可以协调先天免疫应答，还在适应性免疫应答中发挥重要作用，包括促进APC功能的成熟、抗原的交叉呈递以及特异性T细胞的引发和激活；除了增强抗肿瘤免疫应答外，I型IFN还可以通过诱导凋亡和直接作用于肿瘤的抗增殖反应参与肿瘤抑制。
实施例五 CPs/PEI-CDN在肿瘤组织中的滞留实验
用Cy5标记的聚合物（Cy5-PEG-P(TMC-DTC)）与PEG-P(TMC-DTC)-PEI共混制备荧光标记的聚合物囊泡并载Cy3-diAMP。当小鼠肿瘤体积~100 mm 3时，将50 μL Cy3-diAMP（Cy3:0.2 μg/鼠）和Cy5-CPs-Cy3-diAMP（Cy5：0.2 μg/鼠）分别注射到肿瘤部位，24 h后将肿瘤组织取出、切片（8 μm），用DAPI对细胞核进行染色，用CLSM拍摄图片。
CDN的抗肿瘤应用为瘤内给药，而CDN因分子量小以及水溶性强导致可能在局部注射后快速扩散，影响CDN在肿瘤中的滞留，降低CDN的生物利用度，这是现有技术存在的问题。本发明的纳米药物在进入肿瘤后，由于肿瘤部位淋巴引流受损，使其在肿瘤局部积累，延长了载体的滞留时间，用Cy3-diAMP为模型、Cy5标记囊泡研究Cy5-CPs/PEI-Cy3-diAMP在肿瘤组织中的分布，探究CDN和CPs/PEI-CDN在肿瘤的滞留。在荷皮下黑色素瘤B16F10小鼠中，分别瘤内注射Cy3-diAMP或Cy5-CPs/PEI- Cy3-diAMP，24 h后牺牲小鼠，分离肿瘤，并切片、染色。图8荧光成像显示，自由Cy3-diAMP在肿瘤切片中仅有微弱的绿色荧光信号，显示了Cy3-diAMP分子量小、水溶性强而快速发生了体内扩散。而Cy5-CPs/PEI-Cy3-diAMP组肿瘤处的绿色荧光很强，囊泡的红色荧光也很强，并且二者在肿瘤中分布均匀。此外，注射24小时后，Cy5的红色荧光与Cy3的绿色荧光均在肿瘤组织中，但是重叠的比例很低，在活体上证明了囊泡CPs/PEI 的递送可以延长二核苷酸在注射部位的保留，能把CDN从囊泡中释放出来。
实施例六 纳米STING激动剂对B16F10荷瘤小鼠的抗肿瘤治疗
将50 μL含有40 % Matrigel（康宁，美国）的B16F10细胞的PBS悬浮液按照10 5/只的密度从侧面注射到小鼠后腿上方的皮下。每三天用游标卡尺测量肿瘤的体积大小（公式V=L*W 2/2，L和W分别代表肿瘤的最长边和最短边）。接种后第8天，按照计划方案进行治疗。
本发明的CPs/PEI-CDN瘤内注射能在肿瘤保留较长时间，对于被肿瘤浸润的APC摄取、激活STING通路和免疫应答有利，通过外加CPs/PEI-CDN增强STING通路的激活可以提高放疗的抗肿瘤作用，不但能直接损伤DNA、杀死肿瘤细胞，改变肿瘤微环境（TME），释放肿瘤相关抗原（TAA），还能通过cGAS/STING途径增强细胞质DNA感应，诱导产生I型IFN，从而促进DC成熟和T细胞的激活，诱导适应性免疫应答。在本次初步实验中，以给药治疗当天为第0天，在第0、3、6、9天给小鼠以下治疗方案：3Gy X射线、CPs/PEI-CDN+3Gy X射线、或CPs/SP-CDN+3Gy X射线，囊泡CDN通过瘤内给药，CDN剂量为20 μg /只，X射线在皮下瘤局部照射，PBS为对照组。参见图9，从初步治疗结果发现，局部X射线治疗对于缓解疾病的进展有一定的作用，但单独使用的效果不佳，而X射线与CPs/PEI-CDN的联用可以极大程度抑制肿瘤生长、降低肿瘤尺寸。相比之下，CPs/SP-CDN联用X射线的肿瘤抑制能力明显弱于 CPs/PEI-CDN（p=0.056），说明CPs/PEI-CDN在促进细胞内吞、刺激DC细胞的成熟以及细胞因子的分泌方面显著优于CPs/SP-CDN。此外，尾静脉给CPs/PEI-CDN的疗效比瘤内给药要差；进一步的，PD-1抗体与X射线、CPs-CDN三者联用未显示出明显的治疗优势，所以在后续的实验中将重点研究X射线与STING激动剂二者联用的抗肿瘤效果。
在进一步的系统治疗研究中，每只老鼠接种1.5×10 5 个B16F10细胞，每隔两天用游标卡尺测量肿瘤体积，当体积到达50 mm 3时，按照计划方案治疗。放疗当天记为第0天，每3天进行不辐照、剂量为3 Gy或5 Gy的X射线光照，共三个循环；在第1、4、8天瘤内注射50 μL PBS、自由CDN、空囊泡（CPs/PEI）或CPs/PEI-CDN。每隔两天测肿瘤的体积和体重，当体积达到2000 mm 3，动物安乐死。
本发明使用低剂量的X射线（3Gy或5Gy）与CPs/PEI-CDN联合系统研究了荷黑色素瘤B16F10小鼠的治疗（n = 7），避免高剂量的X射线造成DNA剪切酶的增加以及组织的损伤。参见图10至图12，当小鼠的肿瘤体积达50 mm 3时给药治疗，以PBS为对照组。治疗结果显示，瘤内注射CDN治疗显著抑制了肿瘤的生长（***P），明显延长小鼠生存期。相比于自由CDN，CPs/PEI-CDN则更有效地增强了CDN的免疫活性，进一步抑制了黑色素瘤的生长（**p），显著延长了中位生存期（*p）。发现X射线单独治疗仅在初期延缓肿瘤的生长，停药之后迅速反弹，最终抑制黑色素瘤的生长以及改善小鼠生存期的能力有限（23天vs 15天），而X射线与瘤内注射自由CDN联用在延缓肿瘤生长，延长小鼠生存期更好些（***p）。而且3Gy X射线与CPs/PEI-CDN联用对荷瘤小鼠肿瘤的抑制、小鼠生存期的延长（42天）以及生存受益均有明显作用（***p）。而空囊泡CPs/PEI联用3Gy X射线并不能达到进一步增效肿瘤治疗的作用。此外，在治疗前10天，肿瘤抑制与注射的CDN剂量呈正相关，在第10天，CPs/PEI-CDN与X射线联用组中有10/21的老鼠无实体瘤，剂量为10 μg/只组占0/21，剂量为20 μg/只组占3/21。在给药剂量为30 μg/只时，该组所有小鼠的实体肿瘤均消失。在治疗后的很长一段时间里，小鼠的体重无明显下降，再次证明3Gy X射线单独治疗或与CPs、CDN、CPs/PEI-CDN联用并未造成严重的机体毒性。    
进一步的，将5 Gy X 射线与CDN或CPs/PEI-CDN联合，和之前的给药方案相同。参见图13，结果显示，单独使用5 Gy X射线治疗对黑色素瘤初期生长的抑制效果优于3 Gy X射线，然而这种由局部X射线照射引起的抗肿瘤免疫反应比较短暂，无法激活强大的抗肿瘤免疫反应，对于肿瘤的长期抑制以及小鼠总生存期的延长效果不显著。但是当5 Gy X射线联合CDN或CPs/PEI-CDN给药时，可显著增强肿瘤抑制效果（**p），延长小鼠的总生存期（*** p）。经5 Gy X射线和CDN或CPs/PEI-CDN首次注射治疗的48 h后，个别小鼠的肿瘤部位出现红色、发炎性的结痂，第二次注射的48 h后，CPs/PEI-CDN治疗组的绝大多数老鼠（5/7）出现了大面积、烧灼性的黑色结痂，个别老鼠（2/7）为浅褐色的痂，肿瘤部分消退，而CDN组仅有两只老鼠出现结痂情况。注射部位的急性坏死与STING诱导产生的TNF-α有关。在给药的第8-12天，CPs/PEI-CDN组的实体肿瘤全部消退，但在第14、16、18、34天，各有一只小老鼠肿瘤在原位复发（结痂已掉）。此外，5 Gy X射线和CPs/PEI-CDN联合比其和CDN联合在肿瘤抑制方面类似，但是小鼠中位生存期由39天延长到54天，有3/7只小鼠最后无肿瘤。从小鼠的体重变化可发现，用5 Gy X射线单独治疗或联合治疗对小鼠体重影响不大，并未引起严重的毒副作用。
为评估局部5 Gy X射线联合CDN或CPs/PEI-CDN治疗B16F10肿瘤小鼠，并消退肿瘤后对肿瘤免疫记忆的诱导作用，在原发的第176天，在完全治愈的两组小鼠（n = 3）原先有肿瘤的对侧，皮下接种1.5×10 5个B16F10细胞，监测小鼠的肿瘤体积变化以及生存情况。与对照组PBS相比，在未接受额外治疗的情况下，先前接受过治疗的两组小鼠的肿瘤生长减缓，接受过CPs/PEI-CDN治疗的小鼠的肿瘤生长很不明显，先前接受过5 Gy X射线联合CPs/PEI-CDN的治疗组仍有1/3的老鼠完全抵制肿瘤的生长，截止目前已存活了131天，而先前接受5 Gy X射线联合CDN治疗的小鼠存活小鼠25天存活率为0 %。因此对继发性肿瘤细胞接种的抗性证实了在治疗原发性肿瘤后诱导了针对肿瘤抗原的适应性免疫应答。
Figure 44714dest_path_image006
实施例七 流式细胞术分析肿瘤的免疫微环境的调节
B16F10荷瘤小鼠肿瘤体积~100 mm 3时，按系统治疗研究的方案给药。最后一次给药的48 h后，将小鼠的肿瘤、肿瘤引流淋巴结（TDLN）以及脾脏剥离，用PBS清洗组织，将其放入含有1% FBS的PBS缓冲液的表面皿中，使用机械解离法获取原始悬液，经200目的尼龙 网过滤后即得到单细胞悬液。将获得的单细胞悬液离心（1200 rpm，5min）, 加入1 mL含有1% FBS的PBS缓冲液重悬。肿瘤细胞加入1 mL ACK裂解缓冲液4 ℃裂解红细胞，10分钟后加入10 mL PBS，离心。使用细胞计数板计数，将10 6个/管的细胞与相应的抗体在4℃ 避光孵育20分钟，每隔5分钟轻轻拍打细胞悬液，防止细胞沉底。小鼠抗体PerCP/Cy5.5-αCD45、FITC-αCD11c、APC-αCD80、PE-αCD86用于DC成熟分析；小鼠抗体PerCP-αCD45、FITC-αCD11b、APC-αCD206、PE-αF4/80用于巨噬细胞的表型分析；小鼠抗体APC-αCD3、FITC-αCD8α标记CD8+T；使用BD FACS Verse 流式细胞仪进行检测，并使用FlowJo v10进行数据分析。
为了评估X射线和CPs/PEI-CDN联合治疗是否能在B16F10黑色素瘤TME中激活DC触发T细胞免疫反应，在最后一次瘤内注射CPs/PEI-CDN 后6 h取血液测定细胞因子浓度，后48 h牺牲小鼠，解剖出肿瘤和肿瘤引流淋巴结（TDLN）用流式细胞术进行分析。参见图14A、B，与PBS组TDLN中成熟的DC细胞（CD11c+CD80+CD86+）相比（39.6%），仅采用3Gy X射线治疗对促进DC成熟的能力有限；而给予自由CDN以及CPs/PEI-CDN 均显著提高了DC的成熟度（***p），其中囊泡CPs/PEI-CDN组比自由CDN组更加显著（67.7% vs. 56.1%,**p），这与体外BMDC细胞实验结果一致。此外，与单独使用STING激动剂相比，CPs/PEI-CDN或CDN联合X射线的治疗诱导DC成熟的作用明显（* p），但CDN剂量的增加并没有对DC的成熟度有提高。
细胞毒性T淋巴细胞（CTL，CD3+CD4-CD8+）对肿瘤细胞的杀伤起关键作用，被认为是癌症免疫疗法中的关键效应细胞。本发明通过流式细胞术研究了CPs/PEI-CDN及其联用 X射线治疗后肿瘤中浸润的CD8+T细胞比例的变化，图14C可看出，PBS组、X射线组以及瘤内注射的自由CDN组的肿瘤TME中CD8+T细胞的含量小于0.4%，而CPs/PEI-CDN大大促进了CD8+T细胞的浸润（*p），显示了囊泡纳米激动剂有利于招募CTL到TME。当其与X射线联合治疗时诱导的肿瘤浸润CD8+T细胞的进一步提高。CPs/PEI-CDN的剂量以及X射线剂量的增加对于提高TME中CD8+T细胞比例的增幅不大。聚合物囊泡装载提高了CDN在肿瘤中的滞留，促进了CDN的细胞内吞，进而促进STING通路的激活以及T细胞的活化；CPs/PEI-CDN与X射线联合进一步激活了APC的STING通路，显著提高了TME中效应T细胞的比例，增强了抗肿瘤免疫应答。
肿瘤中有不同亚型的巨噬细胞，有免疫抑制的M2型（CD11b+F4/80+CD206+）和促炎性的M1型，所以免疫治疗如果对巨噬细胞进行重新编程，使免疫抑制的M2型巨噬细胞向促炎性的M1型转变，就可能使免疫抑制性肿瘤向免疫原性的热肿瘤转变。本发明研究了CPs/PEI-CDN及其联用 X射线治疗后TME中的肿瘤相关巨噬细胞（TAM）的表型。图14D、E显示，与PBS组中M2型巨噬细胞（CD11b+F4/80+CD206+）相比，自由CDN组M2有较大下降，而囊泡CPs/PEI-CDN组的M2显著降低（*** p），说明经CPs/PEI-CDN治疗巨噬细胞重新极化或募集，免疫抑制降低。根据文献报道，STING激动剂及其纳米制剂可以对巨噬细胞进行重新编程，使免疫抑制的M2型向M1型转变，使免疫抑制性冷肿瘤向免疫原性的热肿瘤转变；然而，进一步联合X射线对巨噬细胞表型的转变无明显的促进作用。
ELISA检测：最后一次给药的6 h，眼眶取血收集小鼠血样，使用血清分离管分离血清，按照ELISA试剂盒的说明书检测小鼠血清中TNF-α以及IFN-β的表达水平。使用多功能酶标仪进行读数。
抗肿瘤免疫应答高度依赖于I型IFN的信号传导，I型IFN促进DC加工和呈递抗原，促使其向淋巴结迁移，对功能性CD8+T细胞介导的抗肿瘤免疫应答至关重要。因此，在最后一次给药6 h，取血样使用ELISA试剂盒检测小鼠血清中IFN-β和TNF-α的浓度。图14F、G显示，瘤内注射CPs/PEI-CDN导致IFN-β、TNF-α的分泌增加显著，而3 Gy X射线诱导产生的内源性细胞因子含量低，说明单独放疗对抗肿瘤免疫应答启动的效果有限。二者联合可进一步提高血清里细胞因子IFN-β的表达（** p）；IFN-β的分泌与CDN的给药剂量呈正比。增加联合的X射线剂量到5 Gy，IFN-β和TNF-α的分泌都增加非常显著（*** p）。瘤内注射CDN治疗对肿瘤的控制分为两阶段，第一阶段是由TNF-α分泌引起的肿瘤早期出血性坏死，第二阶段是CD8+T细胞依赖性的复发控制。
综上所述，X射线与CPs/PEI-CDN联用可以在肿瘤部位产生强大的免疫反应。
实施例八 组织切片和免疫组化分析：为了评估治疗造成的组织毒性以及肿瘤组织的T细胞浸润情况，在最后一次给药48 h后，取出小鼠的心、肝、脾、肺、肾和肿瘤，洗涤、固定、包埋、切片，用苏木精和伊红（H&E）染色，用于病理学分析。为了对肿瘤组织进行免疫组织化学染色，肿瘤组织石蜡切片（8 μm）经脱蜡、抗原修复、血清封闭处理后用兔αCD8（1:500；Abcam）或兔αCRT抗体（1：200；Abcam）37 ℃ 孵育1 h，PBS清洗3次，用Alexa Fluor 633偶联的山羊抗兔二抗（1：500；Invitrogen）37 ℃孵育1 h，用PBS清洗后使用DAPI对细胞核进行染色，使用奥林巴斯BX41正置荧光显微镜（Olympus BX41）拍摄荧光图片。
在最后一次注射CPs/PEI-CDN 后48 h解剖小鼠取肿瘤做切片，进行免疫组化分析。图15显示，肿瘤组织切片的H&E 染色证实了X 射线与CPs/PEI-CDN联用的治疗效果。5 Gy X射线和CPs/PEI-CDN联用导致整个切片上肿瘤细胞出现最大面积的坏死，有大量的炎症细胞的浸润，和前面的结果吻合。相比之下，自由CDN、X射线以及X射线和自由CDN联合组的肿瘤细胞也出现了坏死和黑色素的沉积，然而CPs/PEI的装载增强了CDN抗肿瘤效果，CPs/PEI-CDN以及3 Gy X射线和CPs/PEI-CDN联合组的肿瘤细胞核发生凋亡的程度显著增大，大量肿瘤细胞皱缩和破裂，肿瘤细胞数量明显减少，有少量的免疫细胞的浸润现象。肿瘤细胞表面CRT的暴露是造成免疫原性杀伤的必要条件。由图15可知，PBS组以及未经X射线照射的CDN制剂组均未能触发CRT的暴露，几乎观察不到红色荧光。而X射线可以诱导肿瘤细胞分泌CRT，在X射线以及X射线联合治疗组均观察到明显的红色荧光，表明CRT的分泌增加，ICD效应明显，可作为“eat-me”信号，促进DC的内吞和抗原呈递，招募CTL，诱导免疫反应，抑制肿瘤生长。此外，CD8+T细胞的免疫荧光染色结果显示，囊泡CPs/PEI-CDN组和自由CDN相比红色荧光明显增多肿瘤组织中的CD8+T细胞增多，而CPs/PEI-CDN与X射线联合组肿瘤组织中的CD8+T细胞的浸润更多，进一步增强了抗肿瘤免疫效应。单独X射线对增加肿瘤浸润CD8+T细胞的能力有限，这与流式结果相符。
在治疗结束48 h收集小鼠主要器官（心、肝、脾、肺、肾）以进行组织学分析。图16显示，H＆E染色未观察到明显的毒性作用，表明该CPs/PEI-CDN及其与X射线联合治疗具有生物相容性。
实施例九 CPs/PEI-CDN在肿瘤组织中的药代动力学：为了研究瘤内注射的囊泡的药代动力学，用Cy7标记的寡聚核苷酸AMP作为模型核苷酸，一次性注射CPs/PEI-Cy7-AMP或Cy7-AMP（Cy7浓度：10 μg/只）到荷瘤小鼠的肿瘤内，在设定的时间点从小鼠眼眶取血，然后使用多功能酶标仪测定血浆中Cy7的浓度。实验结果（图17）显示，在瘤内注射后的半小时内，Cy7-AMP组小鼠血浆中Cy7的浓度均高于CPs/PEI-Cy7-AMP组。瘤内注射Cy7-AMP后，因其分子量小、水溶性强而迅速扩散到血液中，并在5分钟内达到C max，且被快速清除，1小时后血液中Cy7的浓度快速降至162.6 ng/mL。而瘤内注射CPs/PEI-Cy7-AMP后的起始阶段的5分钟和10分钟，血液中Cy7的浓度显著低于自由Cy7-AMP组浓度，在0.5 h时，血浆中Cy7的浓度达到C max，但是该值远低于自由Cy7-AMP组的Cy7的最高血浆浓度（367.5 ng/mL vs 904.1 ng/mL）。相比于Cy7-AMP，CPs/PEI-Cy7-AMP组血浆中Cy7的总量较低，这说明聚合物囊泡的装载有利于缓解Cy7-AMP快速扩散以及其被血液迅速的清除的问题，有利于增强Cy7-AMP在肿瘤处的富集和STING通路的激活。
本发明设计研究了高效递送小分子STING激动剂环二核苷酸（CDN）的双硫交联的内壳带正电的聚合物囊泡（CPs/PEI-CDN）及其联合低剂量X射线用于小鼠黑色素瘤的免疫治疗。囊泡由PEG-P(TMC-DTC)-PEI在水相中自组装、自交联而成，粒径小而均匀（45 nm，PDI 0.16），循环稳定；其由低分子量PEI组成的带正电内壳通过静电相互作用复合带负电的CDN，不仅解决了小的核酸药物高效递送与载体毒性之间的矛盾，还可通过质子海绵效应帮助其逃逸内涵体。CPs/PEI-CDN延长了CDN的肿瘤滞留时间，增强了其胞质传递，增强了肿瘤微环境和肿瘤引流淋巴结（TDLN）中STING通路的激活，提高了CDN的疗效。在荷B16F10瘤小鼠中，低剂量X射线（3 Gy×3、5 Gy×3）与CPs/PEI-CDN联用进一步激活肿瘤浸润APC的STING通路，促进成熟DC向TDLN迁移，招募和激活CD8+T细胞，诱导适应性免疫应答，有效抑制肿瘤的生长，延长小鼠的中位生存期。

Claims (10)

  1. 聚合物囊泡纳米STING激动剂，其特征在于，所述聚合物囊泡纳米STING激动剂由聚合物囊泡载体装载STING激动剂组成；所述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双硫键。
  2. 根据权利要求1所述聚合物囊泡纳米STING激动剂，其特征在于，所述聚合物的化学结构式如下：
    Figure dest_path_image001
    R为亲水链段；T链段与侧链含双硫键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。
  3. 根据权利要求2所述聚合物囊泡纳米STING激动剂，其特征在于，所述亲水链段的分子量为3000～10000Da；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%。
  4. 根据权利要求2所述聚合物囊泡纳米STING激动剂，其特征在于，所述亲水链段为聚乙二醇。
  5. 根据权利要求1所述聚合物囊泡纳米STING激动剂，其特征在于，所述STING激动剂为环二核苷酸。
  6. 根据权利要求1所述聚合物囊泡纳米STING激动剂，其特征在于，所述阳离子片段为聚乙烯亚胺或者精胺。
  7. 权利要求1所述聚合物囊泡纳米STING激动剂在制备抗肿瘤药物中的应用。
  8. 权利要求1所述聚合物囊泡纳米STING激动剂的制备方法，其特征在于，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，得到聚合物囊泡纳米STING激动剂。
  9. 权利要求1所述聚合物囊泡作为STING激动剂纳米载体的应用。
  10. 权利要求1所述聚合物在制备STING激动剂纳米药物中的应用。
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