CN113197860A

CN113197860A - Polymer vesicle nano STING agonist and preparation method and application thereof

Info

Publication number: CN113197860A
Application number: CN202110470598.0A
Authority: CN
Inventors: 孟凤华; 郑欢; 曲艳; 钟志远
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-08-03
Also published as: WO2022228469A1

Abstract

The invention discloses a polymer vesicle nano-STING agonist and a preparation method and application thereof. The STING agonist solution is added to the buffer solution, and then the polymer solution is added, and the polymer vesicle nano-STING agonist is obtained by dialysis after stirring. The polymer vesicle nano-STING agonist of the present invention is composed of polymer vesicles loaded with STING agonists; the polymer vesicles have small particle size, can efficiently load STING agonists, and have high biological safety, drug loading stability and Reduction-responsive, the first time vesicles have been loaded with STING agonists in combination with radiation for the treatment of malignant melanoma, the nanoscale properties of polymeric vesicles enhance antigen-presenting cell (APC) uptake of STING agonists, prolonging The retention of STING at the tumor site greatly enhanced the immune activity of STING agonists and enhanced the activation of the STING pathway.

Description

Polymer vesicle nano STING agonist and preparation method and application thereof

Technical Field

The invention belongs to a nano-drug technology, and particularly relates to a polymer vesicle nano-STING agonist, and a preparation method and application thereof.

Background

Activation of STING pathway enhances the body's immunity against tumors, and is a promising approach for tumor immunotherapy. Stinging agonists have been shown to be beneficial in promoting T cell infiltration in TME and in anti-tumor immunotherapy. In addition to natural cGAMP, viruses, liposomes, and Cyclic Dinucleotides (CDNs) are also agonists of the STING pathway, and CDNs such as synthetic cGAMP, cdiAMP, etc. are currently the major research. However, CDN is a small molecule with negative charge and strong hydrophilicity, which is not easy to pass through a cell membrane to enter APC, and is also easy to be enzymatically hydrolyzed, and in addition, CDN is easy to randomly diffuse and clear in vivo, pharmacokinetics is poor, and systemic diffusion thereof may also cause cytokine storm. The research and development of anti-tumor nano-drugs is underway. Cationic polymers, particularly Polyethyleneimine (PEI), have excellent capacity to compact/complex nucleic acids and endosomal/lysosomal escape capacity, and are commonly used for nucleic acid drug delivery. However, high molecular weight PEI has a strong transfection ability and high toxicity, which prevents its practical in vivo use. Low molecular weight PEI has low toxicity but also low transfection efficiency. Spermine (SP) is a human endogenous polypeptide micromolecular cationic compound, and has higher safety. How to efficiently load CDN with low toxicity and release it to APC cells with high efficiency, activate STING pathway to trigger high-efficiency anti-tumor immune response is a challenge.

Disclosure of Invention

The invention discloses a polymer vesicle nano STING agonist, a preparation method and application thereof, and solves the contradiction between high-efficiency nucleic acid drug delivery and carrier toxicity. The invention synthesizes and characterizes PEG-P (TMC-DTC) -PEI and PEG-P (TMC-DTC) -SP, which self-assemble into vesicles with asymmetric membrane structure in water phase containing CDN: the longer PEG chain segment (5 kDa) is a vesicle outer shell, the shorter PEI or SP is a vesicle inner shell, and the inner shell is compounded with the CDN with negative electricity through electrostatic interaction, so that the vesicle CPs-CDN with the high-efficiency CDN is obtained; the reduction-sensitive self-crosslinking structure endowed by the DTC contained in the vesicle hydrophobic membrane enables the loading of CDN to be more stable. The vesicle with the inner shell with positive charge and the asymmetric membrane structure can efficiently load and deliver CDN into tumor cells, thereby realizing the efficient treatment of tumors; the prior art has not studied and reported the vesicle loading of dinucleotides.

The invention designs and prepares two polymer vesicles with positively charged inner shells for loading CDN (CDN) to form vesicle STING agonists (CPs-CDN) based on reduction-sensitive reversibly crosslinked polymer vesicles by using low-toxic low-molecular-weight PEI (600 Da, 1200Da and the like) and SP as cationic fragments, and analyzes the effect of activating STING pathway to generate anti-tumor immunity. The invention adopts the following technical scheme:

a polymersome nano STING agonist consisting of a polymersome loaded STING agonist; the polymer comprises a hydrophilic chain segment, a hydrophobic chain segment and a cationic segment; and the side chain of the hydrophobic chain segment is dithiolane containing disulfide bonds.

Further, the chemical structural formula of the polymer constituting the carrier is as follows:

r is a hydrophilic segment, such as a polyethylene glycol segment; t chain segment and carbonate chain segment (DTC monomer unit) containing disulfide bond in the side chain form a hydrophobic chain segment, T is cyclic ester monomer or cyclic carbonate monomer unit, such as lactide, caprolactone, trimethylene carbonate; p is a cationic moiety, such as Polyethyleneimine (PEI), Spermine (SP); y and z represent repeating units.

In the invention, the molecular weight of the hydrophilic chain segment is 3000-10000 Da; the molecular weight of the hydrophobic chain segment is 2.1-8.0 times of that of the hydrophilic chain segment; the molecular weight of the carbonate chain segment with the disulfide bond in the side chain is 10 to 35 percent of the molecular weight of the T chain segment; the molecular weight of the cationic segment is 5-30% of the molecular weight of the hydrophilic segment.

In the present invention, the STING agonist is Cyclic Dinucleotide (CDN), including cyclic guanosine diphosphate (c-di-GMP), cyclic adenosine diphosphate (c-di-AMP), cyclic guanosine monophosphate (2 ', 3' -cGMAP), cyclic guanosine monophosphate (3 ', 3' -cGMAP), and substituted derivatives thereof, such as sulfur, fluorine, and nitrogen substituted cyclic dinucleotide derivatives.

In the invention, the polymer forms vesicles, the CPs-CDN is obtained by loading the STING agonist, the hydrophobic membrane of the vesicles contains a self-crosslinking structure with reduction sensitivity endowed by DTC (digital control transmitter), so that the CDN is loaded more stably, and the vesicles with the inner shells with positive charges and asymmetric membrane structures can efficiently load and deliver drugs into tumor cells, thereby realizing the efficient treatment of tumors. CPs-CDN is taken up by APC, escapes after destroying lysosome through proton sponge effect, releases CDN in cytoplasm, combines with STING protein on endoplasmic reticulum, starts STING pathway, promotes T cell recognition and kills tumor cells. The invention finds that vesicles based on two polymers have differences in physicochemical properties, endocytosis of cells, release of intracellular drugs, promotion of DC maturation, and anti-mouse melanoma activity. In addition, the X-ray can enable the tumor to release tumor-associated antigen TAA, induce the break of double-chain DNA of the tumor cells and further activate the cGAS-STING pathway, and the invention further explores the anti-tumor curative effect of synergistic enhancement of the combination of the CPs-CDN and the X-ray. The result shows that the combination of the two can improve the concentration of IFN-beta in the TME of the tumor microenvironment, promote the maturation of DC cells and improve the ratio of CD8+ T in the TME, thereby enhancing the inhibition on B16F10 tumors and prolonging the survival time of tumor-bearing mice. The invention discloses the application of the polymer vesicle nano STING agonist in the preparation of antitumor drugs; the tumor is preferably melanoma.

The invention discloses a preparation method of the polymer vesicle nano STING agonist, which comprises the steps of adding a STING agonist solution into a buffer solution, adding a polymer solution, stirring and dialyzing to obtain the polymer vesicle nano STING agonist. Further, the STING agonist solution is a STING agonist aqueous solution; the polymer solution is DMF solution of polymer; the buffer solution is HEPES buffer solution. Wherein the concentration of the STING agonist solution is 0.1-10 mg/mL; the concentration of the polymer solution is 1-100 mg/mL.

The invention designs a disulfide-crosslinked polymersome for delivering a STING agonist CDN, and researches on the enhancement of anti-tumor immune response by the disulfide-crosslinked polymersome in combination with radiotherapy. The particle size of the polymersome is 47 nm, CDN can be efficiently entrapped, and the polymersome has high biological safety, drug-loading stability and reduction responsiveness, and is used for treating melanoma by loading ADU-S100 with the polymersome and combining with radiation for the first time, and the influence of low-dose X-ray combined with nano STING agonist on TME change and anti-tumor treatment is compared for the first time. Compared with the presently reported STING agonist treatment, this new treatment strategy has the following advantages: 1) the polymer vesicle can realize the high-efficiency entrapment of the CDN, the disulfide cross-linking of the vesicle membrane enables the structure of the CDN to be stable, and the CDN is quickly released in an intracellular reduction environment; 2) the nanoscale characteristic of the polymer vesicle improves the cellular uptake of the STING agonist by APC cells, prolongs the retention of the APC cells at a tumor part, greatly improves the immunocompetence of the STING agonist, and enhances the activation of a STING pathway; 3) Combined with X-ray, the composition enhances the activation of STING pathway, promotes the secretion of type I IFN and other cytokines, activates the maturation of DC and the activation of T cells, improves TME, and induces strong anti-tumor immune response. 4) The combined strategy solves the defect that the low-dose X-ray can avoid tissue damage but has poor tumor inhibition effect.

Drawings

FIG. 1 is a schematic diagram of preparation of a polymersome nano-STING agonist CPs-CDN of the present invention and its use in conjunction with X-rays for anti-tumor immunotherapy of melanoma-bearing mice;

FIG. 2 shows PEG-P (TMC-DTC) -PEI¹H NMR Spectrum (600 MHz, CDCl)₃) And of PEG-P (TMC-DTC) -SP¹H NMR Spectrum (400 MHz, CDCl)₃）；

FIG. 3 is a physicochemical characterization of polymersome;

FIG. 4 shows the cell viability of vacuolar CPs/PEI (A) and CPs/SP (B) after incubation with three cells DC2.4, RAW 264.7 and B16F10 for 12 h;

FIG. 5 is a flow single parameter histogram of endocytosis after Cy5-CPs/PEI-diAMP and Cy5-CPs/SP-diAMP are incubated with three cells DC2.4, RAW 264.7 and B16F10 for 24 h;

FIG. 6 is a graph of the fluorescence semiquantitation of Cy3-diAMP (A) and (B) from CLSM pictures of DC2.4 cells incubated with CPs/PEI-Cy3-diAMP or CPs/SP-Cy3-diAMP for 2 h (Cy 3-diAMP concentration 300 nM). Nuclei were stained with DAPI (blue), lysozyme/endosome with Lysotracker deep red (red) and green fluorescence Cy 3-diAMP. The scale bar is 20 μm;

fig. 7 is activation of BMDCs in vitro. Flow cytometric pseudo-chromatograms (A) and quantitative statistics (B) of activated BMDCs after 24 h incubation of BMDCs with PBS, CPs/SP, CPs/PEI, CDN, CPs/SP-CDN or CPs/PEI-CDN (n = 3). BMDC cells are marked by FITC-alpha CD11c, and mature BMDC cells are marked by APC-alpha CD80 and PE-alpha CD 86; (C) detecting the concentration of secreted cytokine IFN-beta in the culture medium by using an ELISA kit;

FIG. 8 is a CLSM picture of tumor tissue sections showing the distribution of each fluorescence 24 h after intratumoral injection of Cy3-DiaMP or Cy5-CPs/PEI-Cy 3-DiaMP. DAPI (blue): nucleus, Cy3 (green): dinucleotide diAMP, Cy5 (red): a polymersome. The scale bar is 20 μm;

figure 9 is CPs/PEI-CDN and its immunotherapy in melanoma-bearing B16F10 mice with X-rays (n = 3). Dosing on

days

0, 3, 6, 9: 3Gy X-ray, CPs/PEI-CDN +3Gy X-ray or CPs/SP-CDN +3Gy X-ray, wherein the vesicle CDN is administrated intratumorally, the CDN dose is 20 mu g/tumor, the X-ray is irradiated on a subcutaneous tumor locally, and PBS is a control group. (A) B16F10 tumor model establishment and a dosing scheme; relative tumor volume (B) and body weight (C) changes; (D) relative tumor volume change per mouse;

FIG. 10 is a 3Gy X-ray treatment of B16F10 tumor-bearing mice in combination with CPs/PEI-CDN, CDN. (A) Establishing a tumor model and combining an intratumoral administration scheme; CPs/PEI-CDN treatment tumor volumes (B) and (C) relative body weight changes (n = 7) in B16F10 tumor-bearing mice; CPs/PEI-CDN in combination with X-ray treatment for tumor volume (D) and (E) weight change (n = 7) in tumor-bearing mice; effect of different doses of CDN treatment on mouse tumor volume (F) and (G) mouse body weight (n = 7); # indicates that 1 mouse survived thereafter, # indicates that 2 mice survived thereafter;

FIG. 11 is a survival curve of mice bearing tumors treated with 3Gy X-ray in combination with CPs/PEI-CDN, CDN B16F10 after treatment initiation (n = 7);

FIG. 12 is a graph of the change in tumor volume per mouse and the number of fully responsive mice treated with 3Gy X-ray in combination with CPs/PEI-CDN, CDN for B16F10 tumor bearing mice (n = 7);

figure 13 is the treatment of B16F10 tumor bearing mice (n = 7) with 5Gy X-rays in combination with CDN or CPs/PEI-CDN. (A) A dosing regimen; (B) photographs of mouse tumors at day 15; (C) change in tumor volume in mice; (D) survival curves of mice; (E) relative body weight change in mice; (F) the growth of the tumor after the mice are inoculated with B16F10 cells on the contralateral side is cured; (G) tumor volume change and number of fully responsive (CR) mice in a single mouse;

FIG. 14 shows that CPs/PEI-CDN combined with X-rays induced a specific anti-tumor immune effect in B16F 10-bearing mice. Flow cytometry analyses the proportion of mature DCs in mouse TDLN (a) and their statistical figures of quantitative data (n = 4) (B). (C) Content of CD8+ T cells in tumor TME (n = 4). Flow cytometry analyses of histograms positive for macrophage surface CD206 receptor expression in TME (D) and their quantitative data statistics (n = 4) (E). The final dose was 6 h mouse serum concentration of IFN- β (F) and TNF- α (G) (n = 3). A to E: TDLN or tumor analysis in the last 48 h mice dosed;

FIG. 15 is a histological section analysis of mouse tumors after combination treatment with CPs/PEI-CDN and X-ray. (A) H & E staining pattern; (B) immunofluorescent staining pattern of CRT; (C) immunofluorescent staining pattern of CD8+ T cells. A scale: 50 μm;

fig. 16 is an H & E staining pattern (40', 50 μm) of the major organs of B16F 10-bearing melanoma mice. PBS, 3Gy, 20 mug CDN, 20 mug CPs/PEI-CDN, 20 mug 3Gy + CPs/PEI-CDN, and 20 mug 5Gy + CPs/PEI-CDN;

FIG. 17 is a graph of the concentration of Cy7 in the plasma of mice over time after one intratumoral injection of CPs/PEI-Cy7-AMP or free Cy7-AMP (Cy 7: 10. mu.g/mouse, n = 3).

Detailed Description

High dose radiotherapy can cause systemic toxicity such as local tissue damage, and low dose can not effectively eliminate tumor; furthermore, local radiotherapy-induced STING signaling is transient and elicits an immune response insufficient to eliminate the tumor; the invention discloses a polymer vesicle nano STING agonist, which is used for further activating a STING pathway of APC (adenosine triphosphate) in a tumor subjected to low-dose radiotherapy, coordinating TAA (tumor necrosis factor) and TA (tumor necrosis factor) generated by radiotherapy, triggering specific CD8+ T cells, regulating TME (tumor necrosis factor), inducing strong anti-tumor immune response, stimulating secretion of cytokines and T cell recruitment factors, stimulating DC (dendritic cell) maturation and cross presentation of tumor antigens, further starting an anti-tumor T cell killing effect and enhancing inherent and adaptive immune response. The invention designs and prepares two polymer vesicles with positively charged inner shells for loading CDN (CDN) to form vesicle STING agonists (CPs-CDN) based on reduction-sensitive reversibly crosslinked polymer vesicles and using low-toxic low-molecular-weight PEI (600 Da, 1200Da and the like) and SP as cationic fragments, and explores the effect of activating STING pathway to generate anti-tumor immunity. The strategy of delivering CDN using nanocarriers may not only protect CDN from degradation, changing its biodistribution; can also promote the effective endocytosis of the antigen by APC, improve T cell response and anti-tumor immunity; the dosage can be reduced, and the potential risk caused by high dosage can be avoided.

The raw materials involved in the invention are conventional products or commercial products; the specific preparation procedures and test methods are conventional in the art. All data were analyzed using GraphPad Prism version 6.01. The significant differences of each group were determined using ANOVA one-way analysis of variance and t-test, and the mid-life was analyzed using Kaplan-Meier technique. Statistical significance was established at p < 0.05, p < 0.01, p < 0.001.

Referring to fig. 1, the present invention synthesizes and characterizes PEG-P (TMC-DTC) -PEI and PEG-P (TMC-DTC) -SP, which self-assemble into vesicles with asymmetric membrane structures in CDN-containing aqueous phase: the longer PEG chain segment (5 kDa) is a vesicle outer shell, the shorter PEI or SP is a vesicle inner shell, and the inner shell is compounded with the CDN with negative electricity through electrostatic interaction, so that the vesicle CPs-CDN with the high-efficiency CDN is obtained.

The cyclic dinucleotides ADU-S100 (99.23%) were purchased from MCE (Master of Small molecules); cy 3-labeled linear dinucleotides (Cy 3-diAMP, 90%) were purchased from Shanghai Bioworks. The amount of polyethyleneimine (PEI,M _n=0.6 kg/mol, 99%, Sigma), spermine (SP, Mn =0.2 kg/mol, 99%, Sigma), N' -carbonyldiimidazole (CDI, 98%, shanghai carbofuran) were purchased and used directly after purchase. Mouse IFN-. beta.ELISA test kit (InvivoGen) and mouse TNF-. alpha.ELISA test kit (Biolegend) were used according to the manufacturer's instructions. Mouse antibodies PerCP/Cy5.5- α 0CD45, APC- α 1CD3, FITC- α 2CD3, APC- α 3CD80, PE- α 4CD86, FITC- α CD11c, FITC- α CD11b, APC- α CD206, PE- α F4/80, APC- α FoxP3, PE- α CD4, FITC- α CD8a, PE/Cy7- α Gr-1 were all purchased from BioLegend.

Nuclear magnetic resonance hydrogen spectrum (¹H NMR) was measured at 600 MHz using a DD2-600 liquid superconducting nuclear magnetic resonance spectrometer (Agilent, USA) with deuterated chloroform (CDCl)₃) As solvent, chemical shifts are normalized to residual solvent signal. The particle size distribution and surface potential of the polymersome were measured by a nanometer particle sizer (Zetasizer Nano-ZS, Malvern instruments, UK) equipped with a 633 nm He-Ne laser. The concentration of ADU-S100 was determined using a NanoDrop 2000c MicroUV Analyzer (Thermo, USA). The morphology of the polymersomes was measured by transmission electron microscopy (TEM, FEI, usa) of Tecnai G220 type at an accelerating voltage of 120 kV. The uptake of vesicles by cells and the maturation of DC cells, the shift in macrophage phenotype and the activation of T cells in TME after stimulation with different formulations were analyzed using a BD FACSVerse flow cytometer. Confocal Laser (CLSM) images were taken by leiia TCS SP 5. Detection of live cells Using a multifunctional microplate reader (Thermo Fisher Scientific)The absorbance of formazan generated with MTT at 570 nm was quantitatively calculated to obtain the survival rate of the cells. Tumor-bearing mice were treated with local irradiation using a biological X-ray irradiator model RS 2000Pro (RAD Source, usa).

The mouse melanoma cell line (B16F 10) and mouse monocyte macrophage leukemia cell (RAW 264.7) were purchased from Shanghai reservoir of Chinese academy of sciences. Bone marrow-derived dendritic cells (BMDCs) were harvested from the leg bone and tibia of 6-week-old C57BL/6J female mice and cultured in 6-well plates with RPMI-1640 medium (HyClone, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin-streptomycin (Kino biomedical technology, China) and 20 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF, PeproTech, USA), all the medium was changed after 48 h, after which half of the medium was renewed every 48 h, DC cells that did not adhere to and loosely adhered to the culture dish were collected by day 9, and the phenotype was determined by expression of surface CD 11C. B16F10, RAW 264.7 and DC2.4 cells were cultured in DMEM medium (Gibco, USA) containing 10% FBS, 1% penicillin-streptomycin. All cells were incubated at 37 ℃ with 5% CO₂Type 3111 (Thermo Fisher Scientific, USA) in a single layer. When the cell confluence reached around 80%, B16F10 and DC2.4 cells were digested with 0.25% (w/v) trypsin containing 0.03% (w/v) ethylenediaminetetraacetic acid (EDTA), and the RAW 264.7 cells were detached with a spatula, followed by centrifugation with an L-420 low-speed centrifuge (Hunan instrument laboratory, China) at 1000 rpm for 3 minutes, followed by washing twice with PBS.

C57BL/6J female mice (6-8 weeks old, 18-20 g) were purchased from Witonglihua (Beijing, China). The culture was carried out at 25 ℃ and 55% humidity with sterile water and feed under programmed conditions. All animal experiments were performed strictly according to protocols approved by the university of suzhou animal laboratories center and the committee for animal protection and use.

Example one

The synthesis of PEG-P (TMC-DTC) -PEI and PEG-P (TMC-DTC) -SP can be found in the patents or articles already published by the applicant, and the specific preparation method is the prior art, such as:

in the first step, the ring-opening polymerization is used to prepare the polymer PEG-P (TMC-DTC). MeO-PEG-OH (0.50 g, M _n100. mu. mol, 5.0 kg/mol, DTC (0.23 g, 1.18 mmol), TMC (1.52 g, 14.55 mmol), dissolved in 7.0 mL of Dichloromethane (DCM), and stirred on a magnetic stirring table with diphenyl phosphate (DPP, DPP/OH molar ratio 10/1) added as catalyst. The sealed closed reactor was placed in an oil bath at 40 ℃ and stirred (600 rpm) to react for 48 hours, using triethylamine as a reaction terminator. According to the volume ratio of the polymer solution to the ethyl acetate of 1: 15 in glacial ethyl ether, centrifuged and dried in vacuo to give the polymer PEG-P (TMC-DTC).

In the second step, the hydroxyl group at the end of PEG-P (TMC-DTC) is activated by N ', N' -Carboxydiimidazole (CDI), and then reacted with primary amine of PEI. 1.1g of PEG-P (TMC-DTC) (hydroxyl 0.05 mmol) and 24 mg of CDI (0.15 mmol) were weighed out and dissolved in DCM and reacted at 30 ℃ for 24 h, then precipitated three times in ethyl glacial ether and dried in vacuo as before to give PEG-P (TMC-DTC) -CDI. Then, weighing PEI (M _n=0.6 kg/mol, 246 mg, 0.41 mmol) in a two-necked flask, 6 mL of anhydrous DCM is added to dissolve 150 mg/mL, and PEG-P (DTC-TMC) -CDI (D: (D) is weighedM _n= 22000 kg/mol, 900 mg, 0.041 mmol), add 9 mL anhydrous DCM to dissolve to 100 mg/mL. The PEG-P (DTC-co-TMC) -CDI polymer solution was transferred to a dry constant pressure dropping funnel (gauge: 10 mL) under nitrogen protection, sealed, and added dropwise to the PEI solution with stirring in an ice-water bath. After the dropwise addition, the mixture was transferred to a 30 ℃ oil bath and stirred by magnetic force to continue the reaction for 4 hours. The resulting product was transferred into a 25 mL dry single-neck round-bottom flask, rotary evaporated in vacuo to remove DCM (dichloromethane) and the polymer was concentrated (target concentration of polymer: 200 mg/mL). Precipitating in 30-fold excess (volume ratio) of mixed solvent of glacial ethanol and ether (v/v 1: 4) for three times, centrifuging, discarding supernatant, and drying in vacuum drying oven to constant weight to obtain final product PEG-P (TMC-DTC) -PEI with a final yield of 70.0%. Nuclear magnetism: PEG (delta 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。

the synthesis of the polymer PEG-P (TMC-DTC) -SP is also divided into two steps, the first step being the same as described above, i.e. the synthesis of PEG-P (TMC-DTC) is preceded by the activation of the terminal hydroxyl group with CDI; the second step is to react the activated polymer with the primary amine of SP. Briefly, PEG-P (TMC-DTC) (1 g, 45. mu. mol) was weighed into a two-necked flask, dissolved in 1mL DCM, and activated at 30 ℃ for 4 h by adding CDI (8.8 mg, 54. mu. mol) in DCM dropwise under a nitrogen blanket. The activated polymer was added dropwise to a pre-cooled solution of SP (98.2 mg, 0.45 mmol) in DMF and then left to react at room temperature for a further 4 h. After the reaction was completed, the reaction product was precipitated with ethyl acetate, centrifuged, and vacuum-dried to obtain a yield of 85.2%. Nuclear magnetism: PEG (delta 3.51); TMC (δ 4.24, 2.03); DTC (δ 4.19, 3.02); SP (. delta.2.84-2.92, 2.94, 3.29);M _n（¹H NMR）= 22.4 kg/mol。

PEG-P (TMC-DTC) -PEI and PEG-P (TMC-DTC) -SP are synthesized by a three-step method: the method comprises the steps of firstly taking methoxy PEG (5000 Da) as an initiator, obtaining a block polymer PEG-P (TMC-DTC) through controlled ring-opening polymerization TMC and DTC, then activating a terminal hydroxyl group with CDI, and reacting with primary amine of PEI or SP to obtain a final polymer, so that loading, protection and efficient delivery of the CDN into an APC activation STING pathway are realized. See fig. 2, by¹H NMR analyses of the structure of PEG-P (TMC-DTC) -PEI and PEG-PEG-P (TMC-DTC) -SP. The degree of polymerization and molecular weight of TMC and DTC, respectively, were calculated from the ratio of the integral of the characteristic peaks of TMC (δ 2.03, 4.24), DTC (δ 3.02, 4.19) to the integral of the characteristic peak of PEG (δ 3.65), and the results showed that the molecular weight and theoretical molecular weight of the polymer were very close, and the degree of functionalization of PEI or SP was 94.4% and 100.3% from the ratio of the integral of the characteristic peak of PEI (δ 2.59-2.80) or SP (δ 2.84-2.92) to the characteristic peak of DTC (δ 3.02), respectively, as shown in Table 1.

Preparation of empty vesicles and CDN-loaded polymersomes (CPs/PEI-CDN or CPs/SP-CDN). The specific process for preparing the empty vesicle by the nano precipitation method comprises the following steps: mu.L of PEG-P (TMC-DTC) -PEI or PEG-P (TMC-DTC) -SP in DMF (10 mg/mL) was aspirated by a pipette gun and added to 900. mu.L of HEPES buffer solution (5 mM, pH 6.8). Magnetic stirring was routinely performed for 10 minutes, and after dialysis for 6 hours with HEPES buffer (5 mM, pH 6.8), dialysis was performed for half an hour with PB (10 mM, pH 7.4) to obtain empty vesicles.

In order to study the preparation, properties and immune activation of the microvesicle STING agonist, a CDN (ADU-S100) which has been tested in phase II of clinical study was selected, and the antitumor therapy effect of the injected tumor for treating melanoma is better, unfortunately, the structure and properties of the CDN limit the uptake by APC, affecting the antitumor immune activity thereof. The structural formula of ADU-S100 (ML RR-S2 CDA) is as follows:

the specific process for preparing the drug-loaded vesicle by the nano-precipitation method comprises the following steps: taking ADU-S100 out of a-80 deg.C refrigerator, preparing into 2 mg/mL water solution, subpackaging and storing in-80 deg.C refrigerator, and avoiding repeated freeze thawing. The procedure for the preparation of CDN-loaded vesicles differed little from the above except that 80 μ L CDN solution (0.16 mg) was pumped into HEPES buffer solution, followed by polymer solution (10 mg/mL), as was the rest.

EXAMPLES characterization of dilaceous vesicles and CDN-loaded polymersomes (CPs/PEI-CDN or CPs/SP-CDN)

The vesicle particle size, particle size distribution and surface Zeta potential were determined by Dynamic Light Scattering (DLS) and electrophoretic techniques. CPs/PEI-CDN, CPs/SP-CDN were analyzed for stability in 10% FBS solution, high fold dilution and long term storage at 4 ℃ and reduction response behavior at 10 mM GSH by particle size tracking. The Drug Loading (DLC) and encapsulation efficiency (DLE) of ADU-S100 were determined by NanoDrop, and absorbance at 260 nm was recorded against a background of equal concentration and equal particle size empty carriers and calculated from a calibration curve drawn with absorbance for ADU-S100 at a known concentration. The characterization plots are shown in FIG. 3, (A) particle size distribution of CPs/PEI and (B) CPs/SP, TME electron microscopy (scale 50 nm), and SLS testing of (C) CPs/PEI and (D) CPs/SP; (E) CPs/PEI-CDN and (F) CPs/SP-CDN in 10% FBS, high dilution and a change in particle size distribution at 4 ℃ for 3 weeks; (G) both particle size changes after 12 h in PB containing 10 mM GSH; (H) both Cy3-diAMP loaded vesicles mimicked the cumulative release of Cy3-diAMP in vitro in both an intracellular reducing environment (PB, 10 mM, pH 7.4, 10 mM GSH) and a physiological environment (PB, 10 mM, pH 7.4) (n = 3).

The DMF solution of PEG-P (TMC-DTC) -PEI or PEG-P (TMC-DTC) -SP is added into HEPES buffer solution (pH 6.8, 5 mM), and can self-assemble to form vesicle CPs/PEI and CPs/SP with non-membrane-forming structures: the long PEG (5 kDa) array constituted the outer shell of the vesicle on the outside, while the short PEI and SP were masked to the inner shell of the vesicle. The DTC ring in the membrane is opened and self-crosslinked during the preparation process. The characterization results of CPs/PEI and CPs/SP are shown in Table 2. The TEM images show typical spherical hollow vesicle structures, see fig. 3A and B, and the ratios of the radius of gyration (Rg) to the hydrodynamic radius (Rh) of CPs/PEI and CPs/SP are 0.905 and 1.001, respectively, close to the theoretical value of 1.0, as determined by Static Light Scattering (SLS) measurement, confirming the hollow spherical vesicle structures of both, see fig. 3C and D.

PEG-P (TMC-DTC) -PEI or PEG-P (TMC-DTC) -SP can form highly efficient CDN-loaded vesicles in the CDN-containing aqueous phase, denoted as CPs/PEI-CDN and CPs/SP-CDN, respectively, e.g., at a theoretical drug load of 20 wt.%, the encapsulation efficiencies of the CDN are as high as 86.0% and 88.2%, respectively, corresponding to drug loads of 17.2 wt.% and 17.6 wt.%. Compared with an empty carrier, the particle size of the obtained vesicle is increased, the particle size of the CPs/SP-CDN is obviously larger than that of the CPs/SP, and the particle size distribution is maintained at a lower level. The drug loading of these two vesicles CDN is significantly better than that of liposomes, hydrogels, and DEAMEA-based polymersomes that have been reported so far. Such high drug loading is due to the complexation of CDN with PEI or SP of the vesicle inner shell and the protection of the cross-linked stable vesicle membrane. In contrast, vesicles prepared from PEG-P (TMC-DTC) loaded the same CDN with very low drug loading, see the CPs-CDN group of table 2. In experiments, the two vesicles have excellent colloidal stability, the particle size of the two drug-loaded vesicles does not change after being placed for 3 weeks in the storage process, and in addition, the vesicles are not dissociated or aggregated in a solution containing 10% of FBS, and the particle size distribution are not obviously changed; at very low concentrations, which mimic intravenous injection, vesicles also did not dissociate into single molecules, maintaining particle size and particle size distribution, fig. 3E, F. In contrast, in the solution containing 10 mM GSH, the particle size of the partial vesicles became significantly larger after 12 h due to reduction of disulfide crosslinks in the vesicle membrane by GSH, FIG. 3G.

^aMeasured in PB (10 mM, pH 7.4) buffer with Dynamic Light Scattering (DLS) at room temperature;^bmeasured by Zetasizer Nano-ZS in PB (10 mM, pH 7.4) buffer at room temperature;^cmeasured by Nanodrop.

The nanoparticles (PS-GMP) obtained by the same loading method (CPs/SP is a polymer) with ADU-S100 replaced by another CDN (c-di-GMP, cyclic diguanylic acid) were characterized as follows:

EXAMPLE triple blank vesicle cytotoxicity test

To study the in vitro toxicity of microvesicles (CPs/PEI, CPs/SP), B16F10, DC2.4 and RAW 264.7 cells were plated in 96-well plates at a density of 2000/well, 5000/well and 10000/well, respectively, after incubation for 24 h, microvesicles of different concentrations were added, after co-incubation for 24 h, 10. mu.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. The medium was slowly aspirated off with a pipette, 150. mu.L DMSO was added to each well, crystals of blue-violet formazan were dissolved, and the absorbance at 570 nm was measured for each well with a microplate reader. Cell viability was calculated by the ratio of absorbance per well to control.

Although PEI has shown great advantages in the efficient delivery of nucleic acid drugs and gene transfection, its systemic toxicity often limits its use in vivo. The invention evaluates the cytotoxicity of empty polymersome after being incubated with dendritic cells (DC 2.4), macrophages (RAW 264.7) and tumor cells (B16F 10) for 12 hours through MTT experiment. The viability of all three cells was above 85% at CPs/PEI and CPs/SP concentrations up to 400. mu.g/mL, FIG. 4. The results show that both CPs/PEI and CPs/SP have good biosafety.

Example in vitro endocytosis and endosome escape behavior study of four CPs-CDN

B16F10, RAW 264.7, DC2.4 cells according to 10⁵、2×10⁵、10⁶One well was plated in a 6-well plate and after 24 h incubation, 200. mu.L of Cy5-CPs/PEI-diAMP, Cy5-CPs/PEI-diAMP (16 nM concentration in Cy5 wells) was added, using PBS as a blank. After 24 h incubation, cells were trypsinized, centrifuged (1000 rpm, 5 min), washed with PBS, and finally suspended in 500. mu.L PBS for flow cytometry analysis using BD FACS Verse.

Cy 5-labeled vesicles Cy5-CPs/SP-diAMP and Cy5-CPs/PEI-diAMP were prepared according to a conventional method using a Cy 5-labeled polymer and linear diAMP as model dinucleotides, and the uptake of the two polymer drug-loaded vesicles after incubation with DC2.4, RAW 264.7, and B16F10 cells for 24 hours was measured by a flow cytometer. The results of the experiments show that there is a large difference in cellular uptake of these two vesicles, see fig. 5: in three cells of DC2.4, RAW 264.7 and B16F10, the fluorescence intensity of Cy5-CPs/PEI-diAMP is higher than that of Cy5-CPs/SP-diAMP, and the former is respectively 3.6 times, 4.6 times and 69 times of the latter; furthermore, the endocytosis capacity of DC2.4 cells was significantly higher for the other two cells.

To investigate the endosome escape behavior of drug-loaded vesicles, vesicles were used that were co-loaded with a fluorescently labeled dinucleotide (Cy 3-diAMP) and a linear model dinucleotide (diAMP). DC2.4 cells were plated at 1X 10⁵The density of individual cells/well was plated in 24-well plates containing round coverslips, incubated for 24 h and 100. mu.L of co-loaded Cy3-d was addedCPs/PEI-diAMP or CPs/SP-diAMP (Cy 3-diAMP at a concentration of 300 nM in wells) from iAMP were incubated at 37 ℃ for 1 h, the medium was aspirated with a pipette and washed 3 times with PBS. Endosome/lysosome were stained with Lysotracker Deep Red (100 nM) for 1 h, fixed with 4% paraformaldehyde for 15 min, nuclei were stained with DAPI for 5min, multiple washes with PBS were performed after each staining, finally mounted with glycerol, stored in dark conditions, and fluorescence images of cells were taken using CLSM.

The uptake of CDN in DC cells can be improved by the polymersome CPs/PEI and CPs/SP, but CDN must bind to STING protein on cytoplasmic endoplasmic reticulum to activate immune response, so that vesicle escape from endosome and release of CDN to cytoplasm is of great significance for enhancing CDN potency and enhancing anti-tumor immune response. According to the conventional method, the cell uptake and endosome escape of Cy3-DiaMP in drug-loaded vesicles CPs/PEI and CPs/SP are qualitatively researched by using Cy 3-labeled DiaMP (Cy 3-DiaMP) as a model, and the figure 6 shows that the cell uptake and endosome escape of Cy3-DiaMP are reduced. The results of the CLSM experiments showed that both Cy3-DiaMP were delivered to the DCs after 2 h incubation of the DC cells with CPs/PEI-Cy3-DiaMP and CPs/SP-Cy3-DiaMP, whereas the amount of Cy3-DiaMP delivered into the cells by CPs/PEI was significantly greater than that of CPs/SP, and ImageJ semi-quantitatively indicated a 2.35-fold increase, which is consistent with the results of flow cytometry measurements of cellular uptake. In addition, after superimposing the Cy3-diAMP green fluorescence with the endosome red fluorescence in DC2.4 cells, the two were found to be not completely coincident, indicating that both vesicles escaped from the endosome within 2 hours. Thus, Cy 3-diAMP-loaded vesicles were taken up by DC2.4 cells into the endosome, Cy3-diAMP escaped the endosome with the aid of PEI or SP, and then reduced in the cytoplasm to release the dinucleotide.

The CDN-loaded vesicle can activate BMDC cells of mice and activate a STING pathway, induces the generation of I-type IFN by activating the STING pathway, and promotes the activation of the DC cells and the maturation of functions. In anti-tumor immunity, maturation of DCs plays an important role in cross presentation of antigens and production of T cells. All BMDCs were labeled with FITC- α CD11c, and mature BMDCs were labeled with APC- α CD80 and PE- α CD 86. After 24 h incubation, mature DCs (CD 80+ CD86 +) accounted for 15.8% of the total cell number in the negative control PBS group, and vacuolar CPs/PEI and CPs/SP did not induce significant BMDC maturation. The literature reports that amine-containing compounds are capable of activating DCs, and that the defined vesicles of the invention are not activated. While free CDN, CPs/SP-CDN and CPs/PEI-CDN significantly increased the proportion of mature BMDCs (CD 80+ CD86 +) to 59.3%, 75.1% and 80.5%, respectively, FIG. 7, where CPs/PEI-CDN had the strongest ability to stimulate BMDC maturation. Compared with the CDN, the CPs/PEI-CDN and the CPs/SP-CDN enter the cytoplasm through endocytosis, which greatly increases the intracellular CDN concentration, greatly activates the STING signaling pathway, and significantly promotes the maturation of BMDCs (. about.p).

The concentration of IFN- β secreted by CDN-induced cells in BMDC medium was determined using an ELISA kit. As can be seen in FIG. 7C, PBS and vacuoles failed to induce IFN- β secretion, while there was a significant increase in IFN- β from the CDN group, indicating that IFN- β secretion was CDN-dependent; secondly, compared with CDN, CPs vesicle further promotes IFN-beta secretion, CPs/SP-CDN and CPs/PEI-CDN are respectively increased by 1.8 and 2.3 times (x p), which indicates that the vesicle enhances the capacity of CDN to activate STING pathway. Whereas CPs/PEI-CDN induced IFN- β secretion more significantly (p) than CPs/SP-CDN, due to the superior cellular uptake capacity and the ability of endosomal escape and efficient cytoplasmic release of CPs/PEI-CDN. STING-induced IFN- β not only coordinates the innate immune response, but also plays an important role in the adaptive immune response, including promoting maturation of APC functions, cross-presentation of antigens, and priming and activation of specific T cells; in addition to enhancing anti-tumor immune responses, type I IFNs may also be involved in tumor suppression by inducing apoptosis and anti-proliferative responses that act directly on tumors.

Example five CPs/PEI-CDN Retention experiments in tumor tissues

A polymer labeled with Cy5 (Cy 5-PEG-P (TMC-DTC)) was blended with PEG-P (TMC-DTC) -PEI to prepare fluorescently labeled polymer vesicles and loaded with Cy 3-DiaMP. When the tumor volume of the mouse is 100 mm³Then, 50. mu.L of Cy3-diAMP (Cy 3: 0.2. mu.g/mouse) and Cy5-CPs-Cy3-diAMP (Cy 5: 0.2. mu.g/mouse) were injected to the tumor site, respectively, and 24 hours later, the tumor tissue was taken out, sectioned (8 μm), nuclei were stained with DAPI, and pictures were taken with CLSM.

The antitumor application of CDN is intratumoral administration, and CDN may diffuse rapidly after local injection due to small molecular weight and strong water solubility, affecting CDN retention in tumor, and reducing bioavailability of CDN, which is a problem in the prior art. After the nano-drug enters the tumor, the lymphatic drainage at the tumor part is damaged, so that the nano-drug is locally accumulated in the tumor, the retention time of a carrier is prolonged, Cy3-DiaMP is used as a model, and Cy5 labeled vesicles are used for researching the distribution of Cy5-CPs/PEI-Cy3-DiaMP in the tumor tissue, so that the retention of CDN and CPs/PEI-CDN in the tumor is researched. In mice bearing subcutaneous melanoma B16F10, Cy3-diAMP or Cy5-CPs/PEI-Cy3-diAMP was injected intratumorally, respectively, and the mice were sacrificed 24 h later, tumors were isolated, sectioned, and stained. FIG. 8 shows fluorescence imaging that free Cy3-DiaMP had only a weak green fluorescence signal in tumor sections, indicating that Cy3-DiaMP has a small molecular weight and is strongly water soluble and rapidly diffuses in vivo. And the green fluorescence of the tumor of the Cy5-CPs/PEI-Cy3-DiaMP group is strong, the red fluorescence of the vesicle is also strong, and the two are uniformly distributed in the tumor. In addition, 24 hours after injection, the red fluorescence of Cy5 and the green fluorescence of Cy3 were both in tumor tissue, but the overlap ratio was low, demonstrating in vivo that the delivery of vesicular CPs/PEI can prolong the retention of dinucleotides at the injection site, and release CDN from the vesicle.

Example anti-tumor treatment of B16F10 tumor-bearing mice with six-nanometer STING agonists

50 μ L of PBS suspension containing 40% Matrigel (Corning, USA) of B16F10 cells was mixed as 10⁵Density/density was injected laterally into the mouse subcutaneously above the hind legs. Tumor volume was measured every three days with a vernier caliper (formula V = L × W)²2, L and W represent the longest and shortest sides of the tumor, respectively). On day 8 post inoculation, treatment was performed according to the schedule.

The CPs/PEI-CDN intratumoral injection disclosed by the invention can be retained in a tumor for a long time, is favorable for the uptake of APC infiltrated by the tumor, the activation of a STING pathway and immune response, can improve the anti-tumor effect of radiotherapy by enhancing the activation of the STING pathway through the addition of the CPs/PEI-CDN, can directly damage DNA, kill tumor cells, change the Tumor Microenvironment (TME) and release Tumor Associated Antigens (TAA), and can also enhance the induction of cytoplasmic DNA through a cGAS/STING pathway and induce I-type IFN, thereby promoting the maturation of DC and the activation of T cells and inducing adaptive immune response. In this preliminary experiment, mice were given the following treatment regimens on

days

0, 3, 6, and 9, with the day of treatment dosing being day 0: 3Gy X-ray, CPs/PEI-CDN +3Gy X-ray or CPs/SP-CDN +3Gy X-ray, wherein the vesicle CDN is administrated intratumorally, the CDN dose is 20 mu g/tumor, the X-ray is irradiated on a subcutaneous tumor locally, and PBS is a control group. Referring to fig. 9, from the preliminary treatment results, it was found that the local X-ray treatment had a certain effect on the alleviation of the disease progression, but the effect was not good when used alone, and the combination of X-rays with CPs/PEI-CDN could greatly inhibit tumor growth and reduce tumor size. In contrast, the tumor suppression ability of the combination of the CPs/SP-CDN and X-ray is obviously weaker than that of the CPs/PEI-CDN (p = 0.056), which indicates that the CPs/PEI-CDN is remarkably superior to the CPs/SP-CDN in promoting endocytosis of cells, stimulating maturation of DC cells and secreting cytokines. In addition, the tail vein was less effective than intratumoral administration for CPs/PEI-CDN; furthermore, the combination of the PD-1 antibody, the X-ray and the CPs-CDN does not show obvious treatment advantages, so the anti-tumor effect of the combination of the X-ray and the STING agonist is intensively researched in subsequent experiments.

In a further study of systemic treatment, each mouse was vaccinated 1.5X 10⁵B16F10 cells, every two days, the tumor volume was measured with a vernier caliper, when the volume reached 50 mm³And (4) treating according to a plan scheme. Recording as day 0 on the day of radiotherapy, and performing X-ray irradiation with dose of 3Gy or 5Gy without irradiation every 3 days for three cycles; intratumoral injection of 50 μ L PBS, free CDN, empty vesicles (CPs/PEI) or CPs/PEI-CDN was done on

days

1, 4, and 8. Measuring the volume and body weight of the tumor every two days, when the volume reaches 2000 mm³The animals were euthanized.

The invention researches the treatment of melanoma-bearing B16F10 mice (n = 7) by using low-dose X-rays (3 Gy or 5 Gy) and a CPs/PEI-CDN combined system, and avoids the increase of DNA shearing enzyme and the damage of tissues caused by high-dose X-rays. Referring to FIGS. 10 to 12, when mice were treatedThe tumor volume of the tumor reaches 50 mm³Treatment was given with PBS as control. Treatment results showed that intratumoral CDN injection treatment significantly inhibited tumor growth (×) and significantly extended mouse survival. Compared to free CDN, CPs/PEI-CDN then more effectively enhanced the immune activity of CDN, further inhibited melanoma growth (p), significantly extended median survival (p). X-ray treatment alone was found to slow tumor growth only initially, with rapid rebound after drug withdrawal, ultimately limiting melanoma growth and improving mouse survival (23 days vs 15 days), while X-ray combined with intratumoral injection of free CDN delayed tumor growth and prolonged mouse survival was better (xp). Furthermore, 3Gy X-ray combined with CPs/PEI-CDN showed significant effects on tumor suppression in tumor-bearing mice, prolongation of survival time (42 days) and survival benefit (. about.p). However, the empty vesicle CPs/PEI cannot achieve the effect of further synergistic tumor treatment by using 3Gy X-ray. Furthermore, 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 and X-ray combination group had no solid tumors, at a dose of 10 μ g/group of 0/21 and at a dose of 20 μ g/group of 3/21. At a dose of 30 μ g/mouse, solid tumors disappeared in all mice of this group. There was no significant weight loss in the mice for a long period of time after treatment, again demonstrating that 3Gy X-ray treatment alone or in combination with CPs, CDN, CPs/PEI-CDN did not cause severe body toxicity.

Further, 5Gy X-ray was combined with CDN or CPs/PEI-CDN in the same dosing regimen as before. Referring to fig. 13, the results show that the effect of inhibiting primary growth of melanoma was superior to that of 3Gy X-ray treatment with 5Gy X-ray alone, but the antitumor immune response caused by local X-ray irradiation was transient, failed to activate a strong antitumor immune response, and the effect of inhibiting long-term tumor and extending the overall survival of mice was not significant. However, when 5Gy X-rays were administered in combination with CDN or CPs/PEI-CDN, the tumor suppression effect (p) was significantly enhanced, extending the overall survival of the mice. After 48 hours of first injection treatment by 5Gy X-ray and CDN or CPs/PEI-CDN, the tumor part of an individual mouse has red and inflammatory crusting, after 48 hours of second injection, most mice (5/7) in the CPs/PEI-CDN treatment group have large-area and cautery black crusting, the individual mice (2/7) have light brown crusting, the tumor part is regressed, and only two mice in the CDN group have crusting. Acute necrosis at the injection site was associated with STING-induced TNF- α production. On days 8-12 of dosing, solid tumors of the CPs/PEI-CDN group all regressed, but one mouse tumor in each case recurred in situ (scab had gone) on

days

14, 16, 18, and 34. Furthermore, 5Gy X-ray and CPs/PEI-CDN combinations were similar in tumor suppression compared to the CDN combinations, but median survival was extended from 39 days to 54 days in mice, with 3/7 mice eventually tumor-free. From the change of the body weight of the mice, it can be found that the single treatment or the combined treatment of the 5Gy X-ray has little influence on the body weight of the mice and does not cause serious toxic and side effects.

To evaluate the local 5Gy X-ray in combination with CDN or CPs/PEI-CDN treatment of B16F10 tumor mice and the induction of tumor immunological memory after tumor regression, on day 176 of primary treatment, two groups of mice (n = 3) with complete cure, contralateral to the original tumor, were inoculated subcutaneously with 1.5X 10⁵B16F10 cells, mice were monitored for changes in tumor volume and survival. In the absence of additional treatment, tumor growth was reduced in the two groups of mice previously treated, compared to the control group of PBS, and was less evident in the mice treated with CPs/PEI-CDN, and the mice that still had 1/3 in the group previously treated with 5Gy X-ray in combination with CPs/PEI-CDN were completely resistant to tumor growth and survived for 131 days by now, whereas the mice previously treated with 5Gy X-ray in combination with CDN survived for 0% of 25 days. Resistance to secondary tumor cell vaccination thus confirms that an adaptive immune response against the tumor antigen is induced after treatment of the primary tumor.

Example seven flow cytometry analysis of modulation of immune microenvironment of tumors

The tumor volume of a B16F10 tumor-bearing mouse is 100 mm³The drug was administered according to the protocol of the systemic therapeutic study. 48 h after the last administration, the tumor, Tumor Draining Lymph Node (TDLN) of the mice was injectedAnd removing spleen, washing tissue with PBS, placing into a surface dish containing 1% FBS PBS buffer solution, obtaining an original suspension by using a mechanical dissociation method, and filtering by using a 200-mesh nylon net to obtain a single-cell suspension. The single cell suspension obtained was centrifuged (1200 rpm, 5 min) and resuspended in 1mL PBS buffer containing 1% FBS. 1mL of ACK lysis buffer was added to the tumor cells to lyse the erythrocytes at 4 ℃ and 10 mL of PBS was added after 10 minutes, followed by centrifugation. Counting with cell counting plate, 10⁶The cells/tube were incubated with the corresponding antibody at 4 ℃ for 20 min in the dark, and the cell suspension was gently tapped every 5min to prevent the cells from bottoming. Mouse antibodies PerCP/Cy5.5- α CD45, FITC- α CD11c, APC- α 0CD80, PE- α CD86 for DC maturation analysis; mouse antibodies PerCP-alpha CD45, FITC-alpha CD11b, APC-alpha CD206, PE-alpha F4/80 were used for the phenotypic analysis of macrophages; mouse antibodies APC-alpha CD3, FITC-alpha CD8 alpha labeled CD8+ T; detection was performed using a BD FACS Verse flow cytometer and data analysis was performed using FlowJo v 10.

To assess whether the combination of X-ray and CPs/PEI-CDN could activate DCs in B16F10 melanoma TME to trigger T cell immune responses, blood was taken 6 h after the last intratumoral injection of CPs/PEI-CDN to determine cytokine concentrations, 48 h after sacrifice of mice, dissected of tumors and Tumor Draining Lymph Nodes (TDLNs) and analyzed by flow cytometry. Referring to fig. 14A, B, treatment with 3Gy X-rays alone had limited ability to promote DC maturation compared to DC cells matured in the PBS group TDLN (CD 11c + CD80+ CD86 +) (39.6%); while administration of free CDN and CPs/PEI-CDN both significantly increased the maturation of DCs (× p), with the vesicular CPs/PEI-CDN group being more significant than the free CDN group (67.7% vs. 56.1%, × p), consistent with in vitro BMDC cell experimental results. Furthermore, the effect of CPs/PEI-CDN or CDN in combination with X-ray treatment on DC maturation was significant (. p) compared to STING agonists alone, but the increase in CDN dose did not improve the maturation of DCs.

Cytotoxic T lymphocytes (CTL, CD3+ CD4-CD8 +) play a key role in killing tumor cells and are considered as key effector cells in cancer immunotherapy. The invention researches the change of the proportion of CD8+ T cells infiltrated in tumors after CPs/PEI-CDN and combined X-ray treatment by using the CPs/PEI-CDN, and the change is shown in FIG. 14C, the content of CD8+ T cells in tumor TME of a PBS group, an X-ray group and a free CDN group injected in tumors is less than 0.4%, and the CPs/PEI-CDN greatly promotes infiltration (p) of CD8+ T cells, thereby showing that the vesicle nano agonist is favorable for recruiting CTL to the TME. Further increase in tumor infiltration of CD8+ T cells was induced when it was treated in combination with X-ray. The dose of CPs/PEI-CDN and the increase in X-ray dose did not greatly increase the proportion of CD8+ T cells in the TME. The polymer vesicle loading improves the retention of the CDN in the tumor, promotes the endocytosis of the CDN, and further promotes the activation of the STING pathway and the activation of T cells; the combination of the CPs/PEI-CDN and the X-ray further activates the STING pathway of the APC, obviously improves the proportion of effective T cells in the TME, and enhances the anti-tumor immune response.

Tumors contain different subtypes of macrophages, including immunosuppressive type M2 (CD 11b + F4/80+ CD206 +) and pro-inflammatory type M1, so immunotherapy, if macrophages are reprogrammed to shift immunosuppressive type M2 macrophages to pro-inflammatory type M1, may shift immunosuppressive tumors to immunogenic, hot tumors. The present invention investigates the phenotype of tumor-associated macrophages (TAM) in TME following CPs/PEI-CDN and its combination X-ray treatment. Figure 14D, E shows a greater decrease in free CDN group M2 compared to M2 type macrophages in the PBS group (CD 11b + F4/80+ CD206 +) whereas M2 is significantly decreased in the vesicular CPs/PEI-CDN group (p), indicating decreased immune suppression by repolarization or recruitment of macrophages treated with CPs/PEI-CDN. According to the report of the literature, the STING agonist and the nano preparation thereof can reprogram macrophages, convert immunosuppressive M2 type into M1 type, and convert immunosuppressive cold tumor into immunogenic hot tumor; however, further combinations of X-rays did not significantly contribute to the conversion of macrophage phenotype.

And (3) ELISA detection: at 6 h after the last administration, blood samples were collected from the orbits of the eyes, and serum was isolated using a serum separator tube and the expression levels of TNF-. alpha.and IFN-. beta.in the mouse serum were measured according to the instructions of the ELISA kit. Readings were taken using a multifunctional microplate reader.

The anti-tumor immune response is highly dependent on the signaling of type I IFNs, which promote DC processing and antigen presentation, facilitating their migration to lymph nodes, and is critical for a functional CD8+ T cell-mediated anti-tumor immune response. Therefore, at 6 h of the last administration, blood samples were taken and the concentrations of IFN-. beta.and TNF-. alpha.in the mouse sera were measured using the ELISA kit. FIG. 14F, G shows that intratumoral injection of CPs/PEI-CDN resulted in a significant increase in IFN- β, TNF- α secretion, while 3Gy X-ray induced production of low levels of endogenous cytokines, indicating that radiotherapy alone had limited efficacy in the initiation of an anti-tumor immune response. The combination of the two can further improve the expression of the cytokine IFN-beta in serum (p); IFN- β secretion is directly proportional to the dose administered to the CDN. Increasing the combined X-ray dose to 5Gy, both IFN- β and TNF- α secretion increased very significantly (. beta. times.p). The control of tumors by intratumoral CDN treatment is divided into two phases, the first phase being early stage hemorrhagic necrosis of tumors caused by TNF- α secretion and the second phase being CD8+ T cell-dependent relapse control.

In summary, the combination of X-rays with CPs/PEI-CDN produced a strong immune response at the tumor site.

Example eight tissue sections and immunohistochemical analysis

To assess the tissue toxicity resulting from the treatment and T cell infiltration of tumor tissue, 48H after the last dose, the heart, liver, spleen, lung, kidney and tumor were removed from the mice, washed, fixed, embedded, sectioned, stained with hematoxylin and eosin (H & E) for pathological analysis. For immunohistochemical staining of tumor tissue, paraffin sections (8 μm) of tumor tissue were deparaffinized, antigen repaired, serum blocked, incubated with rabbit α CD8 (1: 500; Abcam) or rabbit α CRT antibody (1: 200; Abcam) for 1 h at 37 ℃, washed 3 times with PBS, incubated with Alexa Fluor 633-coupled goat-anti-rabbit secondary antibody (1: 500; Invitrogen) for 1 h at 37 ℃, washed with PBS and stained for cell nuclei with DAPI, and fluorescence pictures were taken using an Olympus BX41 upright fluorescence microscope (Olympus BX 41).

And dissecting a mouse 48 h after the last injection of the CPs/PEI-CDN, taking a tumor as a section, and performing immunohistochemical analysis. FIG. 15 shows that H & E staining of tumor tissue sections demonstrates the therapeutic effect of X-rays in combination with CPs/PEI-CDN. The combination of 5Gy X-ray and CPs/PEI-CDN resulted in the greatest area of necrosis of tumor cells across the section, with massive infiltration of inflammatory cells, consistent with the previous results. In contrast, tumor cells in free CDN, X-ray and combined X-ray and free CDN groups also show necrosis and melanin deposition, however, the loading of CPs/PEI enhances the anti-tumor effect of CDN, the apoptosis degree of tumor cell nuclei in the combined CPs/PEI-CDN and 3Gy X-ray and CPs/PEI-CDN groups is obviously increased, a large amount of tumor cells are shrunk and cracked, the number of tumor cells is obviously reduced, and a small amount of immune cells are infiltrated. Exposure of CRT on the surface of tumor cells is a prerequisite to cause immunogenic killing. As can be seen in fig. 15, neither the PBS group nor the CDN preparation group that was not X-rayed triggered CRT exposure, and little red fluorescence was observed. And the X-ray can induce tumor cells to secrete CRT, and obvious red fluorescence is observed in X-ray and X-ray combination treatment groups, so that the secretion of the CRT is increased, the ICD effect is obvious, and the ICD can be used as an 'eat-me' signal, promote the endocytosis and antigen presentation of DC, recruit CTL, induce immune response and inhibit the growth of tumor. In addition, the immunofluorescent staining result of CD8+ T cells shows that the vesicle CPs/PEI-CDN group and the free CDN have red fluorescence to obviously increase the CD8+ T cells in the tumor tissue, and the CPs/PEI-CDN and the X-ray combined group have more infiltration of the CD8+ T cells in the tumor tissue, so that the anti-tumor immune effect is further enhanced. X-ray alone has limited ability to increase tumor infiltration of CD8+ T cells, which is consistent with flow results.

Mouse major organs (heart, liver, spleen, lung, kidney) were collected 48 h at the end of treatment for histological analysis. FIG. 16 shows that no significant toxic effects were observed with H & E staining, indicating that the CPs/PEI-CDN and its combination with X-ray treatment are biocompatible.

EXAMPLE pharmacokinetics of nine CPs/PEI-CDN in tumor tissue

To study the pharmacokinetics of intratumorally injected vesicles, CPs-PEI-Cy7-AMP or Cy7-AMP (Cy 7 concentration: 10. mu.g/mouse) was added to the tumor in tumor-bearing mice, blood was taken from the mouse orbit at set time points, and then the concentration of Cy7 in plasma was measured using a multifunctional plate reader. The results of the experiment (FIG. 17) show that the plasma concentration of Cy7 was higher in the Cy7-AMP group mice than in the CPs/PEI-Cy7-AMP group half an hour after intratumoral injection. After intratumoral injection of Cy7-AMP, it rapidly diffused into the blood due to its small molecular weight and strong water solubility and reached C within 5 minutes_maxAnd cleared rapidly, the concentration of Cy7 in the blood dropped rapidly to 162.6 ng/mL after 1 hour. Whereas the concentration of Cy7 in blood was significantly lower than the free Cy7-AMP group at 5 and 10 minutes in the initial phase after intratumoral injection of CPs/PEI-Cy7-AMP, and the concentration of Cy7 in plasma reached C at 0.5 h_maxHowever, this value is much lower than the highest plasma concentration of Cy7 of the free Cy7-AMP group (367.5 ng/mL vs 904.1 ng/mL). The lower total amount of Cy7 in the plasma of the CPs/PEI-Cy7-AMP group compared to Cy7-AMP suggests that the loading of the polymersomes is beneficial to alleviate the problem of rapid diffusion of Cy7-AMP and its rapid clearance by blood, and to enhance the enrichment of Cy7-AMP at the tumor site and activation of the STING pathway.

The invention designs and researches a disulfide-crosslinked inner shell positively-charged polymersome (CPs/PEI-CDN) for efficiently delivering small-molecule STING agonist Cyclic Dinucleotide (CDN) and application of the disulfide-crosslinked inner shell positively-charged polymersome to immunotherapy of mouse melanoma by combining low-dose X-rays. The vesicle is formed by self-assembling and self-crosslinking PEG-P (TMC-DTC) -PEI in an aqueous phase, has small and uniform particle size (45 nm, PDI 0.16) and is stable in circulation; the positively charged inner shell composed of low molecular weight PEI is compounded with the negatively charged CDN through electrostatic interaction, so that the contradiction between the high-efficiency delivery of small nucleic acid drugs and the toxicity of carriers is solved, and the escape of endosomes is facilitated through the proton sponge effect. The CPs/PEI-CDN prolongs the tumor retention time of the CDN, enhances the cytoplasmic delivery of the CDN, enhances the activation of the STING pathway in the tumor microenvironment and the Tumor Draining Lymph Node (TDLN), and improves the curative effect of the CDN. In B16F10 tumor-bearing mice, low-dose X-rays (3 Gy × 3 and 5Gy × 3) are combined with CPs/PEI-CDN to further activate the STING pathway of tumor-infiltrating APC, promote the migration of mature DC to TDLN, recruit and activate CD8+ T cells, induce adaptive immune response, effectively inhibit the growth of tumor and prolong the median survival period of the mice.

Claims

1. A polymer vesicle nano-STING agonist, wherein the polymer vesicle nano-STING agonist is composed of a polymer vesicle carrier loaded with a STING agonist; the polymer comprises a hydrophilic segment, a hydrophobic segment and a cationic segment; the side chain of the hydrophobic segment is a disulfide bond.

2. The polymer vesicle nano-STING agonist according to claim 1, wherein the chemical structural formula of the polymer is as follows:

R is a hydrophilic segment; the T segment and the carbonate segment containing disulfide bonds in the side chain form a hydrophobic segment; T is a unit formed by a cyclic ester monomer or a cyclic carbonate monomer; P is a cationic segment; y, z represents a repeating unit.

3. The polymer vesicle nano-STING agonist according to claim 2, wherein 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 10% to 35% of the molecular weight of the T segment; the molecular weight of the cationic segment is 5% to 30% of the molecular weight of the hydrophilic segment.

4. The polymer vesicle nano-STING agonist according to claim 2, wherein the hydrophilic segment is polyethylene glycol.

5 . The polymer vesicle nano-STING agonist according to claim 1 , wherein the STING agonist is a cyclic dinucleotide. 6 .

6 . The polymer vesicle nano-STING agonist according to claim 1 , wherein the cationic fragment is polyethyleneimine or spermine. 7 .

7. The application of the polymer vesicle nano-STING agonist of claim 1 in the preparation of antitumor drugs.

8. The preparation method of the polymer vesicle nano-STING agonist according to claim 1, wherein the STING agonist solution is added to the buffer solution, and then the polymer solution is added, and then dialyzed after stirring to obtain the polymer vesicle nano-STING agonist.

9. Use of the polymersomes of claim 1 as nanocarriers for STING agonists.

10. The application of the polymer of claim 1 in the preparation of STING agonist nanomedicine.