CN113663060A

CN113663060A - Whole-cell tumor nano vaccine, preparation method and application thereof

Info

Publication number: CN113663060A
Application number: CN202010360667.8A
Authority: CN
Inventors: 于海军; 李天亮; 祝奇文; 宋润迪
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2021-11-19
Anticipated expiration: 2040-04-30
Also published as: CN113663060B

Abstract

The invention relates to a whole-cell tumor nano vaccine, a preparation method and application thereof. The whole-cell tumor nano vaccine is a nano vaccine with engineered tumor cell membranes coated on the surfaces of acid-sensitive polymer immune adjuvant-loaded nanoparticles, or a nano vaccine with engineered tumor cell membranes coated on the surfaces of acid-sensitive polymer immune adjuvant conjugates, or a combination of the two. The whole-cell tumor nano vaccine can enhance the antigen presentation efficiency of dendritic cells, activate various specific cytotoxic T lymphocytes, realize high-efficiency anti-tumor recurrence and metastasis effects, and is beneficial to realizing the personalized treatment of malignant tumors.

Description

Whole-cell tumor nano vaccine, preparation method and application thereof

Technical Field

The invention relates to the fields of biological medicine and tumor treatment, in particular to a personalized tumor nano vaccine designed based on an engineered tumor cell membrane, a preparation method thereof and application in tumor treatment and prevention of tumor recurrence and metastasis.

Background

Treatment of malignancies remains a global challenge, with over 80% of tumor patients dying from postoperative recurrence and metastasis, and combating them remains a significant challenge in clinical treatment. In recent years, tumor immunotherapy has shown good potential in combating recurrence and metastasis of malignant tumors, among which tumor vaccines play an important role. The conventional tumor vaccine activates the autoimmune system of a patient by introducing a single tumor-associated antigen or antigen peptide into the body of the patient, generates specific anti-tumor immune response, induces or amplifies cellular immunity and humoral immunity which are pre-stored in the body and aim at the target antigen, and can form long-term immune memory reaction, thereby achieving the purpose of controlling or eliminating the tumor. However, the widely existing tumor heterogeneity is one of the biggest difficulties at present, and not only in the tumor tissues, even if the tumor is of the same type, the tumor vaccines have great difference among different patients, the clinical treatment effect of the tumor vaccine has obvious difference, and the acquisition process of the new antigen is long, so that the optimal treatment time after the operation of the patient is easily delayed, which also provides a challenge for quickly implementing the personalized treatment of the tumor.

Multiple antigens usually exist on the surface of the tumor cell, and the cell membrane of the tumor cell extracted by an engineering technology is targeted to the lymph node, so that Dendritic Cells (DCs) in the lymph node can recognize the antigens on the surface of the tumor cell, the tumor cell can be comprehensively eliminated by immune cells of an organism, and the escape of the tumor cell can be prevented; the generation of memory cells in the tumor immunization process is beneficial to inhibiting the relapse and metastasis processes of tumors. Considering the difference of the abundance of the surface antigen of the tumor cells, how to effectively improve the presentation efficiency of the tumor antigen and activate the generation of tumor specific T lymph is an important guarantee for enhancing the treatment effect of the tumor vaccine. Immune adjuvants such as interferon gene Stimulant (STING) agonist and Toll-like receptor (TLR) agonist, and can promote the cross presentation process of dendritic cells to antigens to activate tumor-specific T lymphocytes by acting on the dendritic cells; by promoting the secretion of inflammatory cytokines by dendritic cells, the activity of cytotoxic T lymphocytes can be improved, and the clearing capacity of an organism on tumor cells is enhanced.

Disclosure of Invention

In order to establish a personalized tumor treatment system and prevent postoperative tumor recurrence and metastasis, the invention aims to provide a whole-cell nano vaccine for personalized tumor treatment, a preparation method thereof and application thereof in anti-tumor immunotherapy.

In order to achieve the above objects, in one aspect, the present invention provides a whole-cell tumor nano-vaccine comprising a whole series of tumor-associated antigens, which is a nano-vaccine comprising an acid-sensitive polymer-immunoadjuvant-loaded nanoparticle surface coated with an engineered tumor cell membrane, or a nano-vaccine comprising an acid-sensitive polymer-immunoadjuvant conjugate nanoparticle surface coated with an engineered tumor cell membrane, or a combination of the two,

wherein the acid-sensitive polymer has a structure represented by the following formula 1:

the acid-sensitive polymer-immunoadjuvant conjugate has a structure as shown in the following formula 2:

in the above-mentioned formula 1, the,

R₁selected from hydroxyl group,

m is an integer of 10-145, preferably m is an integer of 100-140, more preferably m is 113;

R₂selected from phenyl or

n is an integer of 3 to 12;

R₃any one selected from N, N-diethylamino, N-dibutylamino, N-diisopropylamino, pentamethylene-amino and hexamethylene-amino;

x is an integer from 20 to 60, preferably from 40 to 50,

in the above-mentioned formula 2, the,

R₁、R₂and R₃And x is the same as defined in formula 1,

R₄selected from the group derived from immunological adjuvants;

linker is R₄A linking group with a carbonyl group, the linking group forming a p, pi-conjugated linkage with the carbonyl group;

y is an integer of 1 to 20, preferably 1 to 10.

In an embodiment, R₃Selected from the group consisting of N, N-diisopropylamino, hexamethyleneamino; r₂Is composed of

n is 12; x is 40, 42, 50; y is 0, 3, 4 or 5.

In embodiments, the Linker may be selected from any one of the following structures:

in embodiments, the immunoadjuvant comprises: any one selected from the group consisting of STING agonists and TLR agonists, for example any one selected from the group consisting of,

in an embodiment, said R₄Can be selected from any one of the following groups:

in embodiments, the tumor cells include, but are not limited to, 4T-1 breast cancer cells, MCF-7 breast cancer cells, CT-26 colon cancer cells, HCT116 colon cancer cells, B16-F10 melanoma cells, or Panc02 pancreatic cancer cells.

In embodiments, the chemotherapeutic agent includes, but is not limited to, oxaliplatin, doxorubicin, epirubicin, idarubicin, paclitaxel, docetaxel, cyclophosphamide; photosensitizers used in such photodynamic therapy include, but are not limited to, chlorin e6, pyropheophorbide a, indocyanine green; the laser wavelength required for photodynamic therapy is the optimal wavelength range for the photosensitizer to achieve photodynamic therapy, for example, the laser wavelength required for photodynamic therapy of pyropheophorbide a is 671 nm.

In embodiments, the loading of the immunoadjuvant in the nanoparticle is 130 wt%.

In another aspect, the present invention provides a method for preparing the whole-cell tumor nano vaccine, which comprises the following steps:

1) dissolving an acid-sensitive polymer and an immunologic adjuvant in an organic solvent, mixing the acid-sensitive polymer and the immunologic adjuvant with an emulsifier aqueous solution, performing ultrasonic treatment on the mixed solution, and removing the organic solvent by rotary evaporation to obtain polymer nanoparticles physically encapsulating the immunologic adjuvant;

2) co-extruding and fusing the prepared polymer nanoparticles and the engineered tumor cell membrane through a nano extruder to obtain the acid-sensitive polymer immunoadjuvant-loaded nanoparticle vaccine with the surface of the engineered tumor cell membrane coated with the surface of the nanoparticles, or

1) Dissolving the acid-sensitive polymer-immunologic adjuvant conjugate in an organic solvent, mixing the solution with an emulsifier aqueous solution, performing ultrasonic treatment on the mixed solution, and removing the organic solvent by rotary evaporation to obtain acid-sensitive polymer-immunologic adjuvant conjugate nanoparticles;

2) and co-extruding and fusing the prepared acid-sensitive polymer-immune adjuvant conjugate nano-particles and the engineered tumor cell membrane through a nano extruder to obtain the whole-cell tumor nano-vaccine with the surface of the acid-sensitive polymer-immune adjuvant conjugate nano-particles coated with the engineered tumor cell membrane.

In the above preparation methods, the acid-sensitive polymer, the immunoadjuvant, the acid-sensitive polymer-immunoadjuvant conjugate, and the engineered tumor cell membrane are defined as above, and are not described herein again.

In embodiments, the organic solvent includes, but is not limited to, chloroform, dichloromethane, ethyl acetate, acetone.

In embodiments, the emulsifier includes, but is not limited to, polyvinyl alcohol, and the aqueous emulsifier solution is preferably a 2 to 10 wt% aqueous polyvinyl alcohol solution.

In embodiments, in step 1), the weight ratio of the acid-sensitive polymer to the immunoadjuvant is 1-50: 1.

in embodiments, in step 2), the weight ratio of the nanoparticles to the tumor cell membrane is between 0.1 and 10: 1.

in still another aspect, the invention also provides an application of the whole-cell tumor nano-vaccine in preparing a medicament for treating malignant tumor and resisting tumor recurrence and metastasis.

In embodiments, the malignancy includes, but is not limited to: breast cancer, colorectal cancer, melanoma, and pancreatic cancer.

Advantageous effects

The whole-cell tumor nano vaccine can be targeted to lymph nodes through a nanoscale effect and is taken up by DCs, calreticulin which is everted on a cell membrane of an engineered tumor cell under the stimulation of chemotherapy or photodynamic therapy acts on dendritic cells in the lymph nodes, the taking up of the dendritic cells to the vaccine can be increased, and by virtue of the acid sensitivity of a polymer, the whole-cell tumor nano vaccine is beneficial to the cytoplasm release of tumor antigens and immune adjuvants in the nano vaccine, the antigen presentation efficiency of the dendritic cells is enhanced, various specific cytotoxic T lymphocytes are activated, and the efficient anti-tumor recurrence and transfer effects are realized. The nano whole-cell tumor vaccine is beneficial to realizing the personalized treatment of malignant tumors.

Drawings

FIG. 1 is a transmission electron micrograph of the engineered cell membrane prepared in preparation example 1 of the present invention, with a scale of 100 nm.

FIG. 2: a) a transmission electron microscope image of the mPEG-PC7A nano-particles coated with Racemate prepared in preparation example 4 of the invention, and b) a transmission electron microscope image of the whole-cell tumor nano-vaccine prepared in preparation example 7 of the invention, wherein the scale in the image is 100 nm.

FIG. 3 shows the particle size variation of the whole cell tumor vaccine of preparation example 7 under different pH conditions.

FIG. 4 shows the mouse lymph node targeting effect of the fluorescence-labeled whole-cell tumor nano-vaccine in preparation example 8 of the present invention.

Fig. 5 shows the growth inhibition curve of the whole-cell tumor nano-vaccine of the present invention against CT-26 mouse transplanted tumor, wherein a: a PBS control group; b: the mPEG-PC7A nano vaccine group without entrapped Racemide; c: the mPEG-PC7A nano vaccine group coated with Racemate; and D: mPEG₁₁₃-b-P(DPA₄₂-co-R848₃) And (4) a nano vaccine group.

Detailed Description

The present invention will be described below with reference to specific embodiments, but the present invention is not limited to these specific embodiments.

The reagents and equipment used in the following examples are as follows:

methoxy-terminated polyethylene glycol amine 5000, polyethylene glycol diamine 5000, 2- (diisopropylamino) ethyl methacrylate, ranisimethide (R848), 4-cyano-4- [ [ (dodecylthio) thiolmethyl ] thio ] pentanoic acid were purchased from sigma aldrich (china). The remaining reagents and solvents were purchased from the national pharmaceutical group (Shanghai) Chemicals, Inc., unless otherwise specified.

Culture of CT-26 colon cancer cells and B16-F10 melanoma cells were performed in DMEM medium and 1640 medium, respectively, and fetal bovine serum was purchased from Gibco.

Balb/C white mice, C57BL/6 black mice, 4-5 weeks old were purchased from Shanghai Si Laike laboratory animals, Inc., and tumor-bearing mouse models with B16 tumors were constructed by inoculating the right back of the mice with B16 melanoma cancer cells cultured in vitro. The procedure of the whole animal experiment strictly followed the relevant regulations of the Shanghai pharmaceutical research institute animal Care and use Committee.

Sample data were determined by the following instrument: nuclear magnetic resonance hydrogen spectrum (¹H-NMR) using a Varian-MERCURY Plus-400 NMR spectrometer with TMS as an internal standard and chemical shift units in ppm-1; the hydrodynamic particle size and surface potential of the cationic particles were determined by a MALVERN NANO size type particle size meter; transmission electron micrographs were obtained with a transmission electron microscope model Tecnai G2F 20S-TWIN.

In the present invention, the equipment and the test method are those which are conventional in the art, unless otherwise specified.

Preparation example 1: preparation of engineered cell membranes

The CT-26 cells treated by the oxaliplatin are collected and washed by Tris-HCl pH 7.0, sucrose and D-mannitol, then the cells are mechanically destroyed by a homogenizer in the presence of a mixture of phosphatase inhibitor and protease inhibitor, and cell membranes are obtained by ultracentrifugation. The obtained cell membrane is washed by ethylene diamine tetraacetic acid aqueous solution and then is preserved at the temperature of minus 20 ℃. Total membrane protein content was quantified by BCA protein assay kit.

FIG. 1 is a transmission electron micrograph of the prepared engineered cell membrane extruded through a nano-extruder, which shows a good membrane structure after negative dyeing with uranium acetate, and the thickness is about 10 nm.

Preparation example 2: synthesis of polymers

Reacting 4-cyano-4- [ [ (dodecylthio) thione methyl]Sulfur based radicals]Pentanoic acid (CTA) (19.3mg,0.048mmol) was dissolved in 5mL of N, N-dimethylformamide, and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) (27.5mg,0.144mmol), 1-Hydroxybenzotriazole (HOBT) (19.4mg,0.144mmol), Triethylamine (TEA) (16.16mg,0.160mmol) were added thereto and reacted at room temperature for 1.5 h. Subsequently, mPEG was added thereto₁₁₃-NH₂(200.8mg,0.040mmol), reacting at room temperature for 24h, dialyzing the reaction solution with ethanol and deionized water, carrying out dialysis bag with molecular weight cut-off of 3500D, and freeze-drying to obtain 182mg of pale yellow powdery solid with yield of 84.3%.¹H NMR(400MHz,CDCl₃)δ3.84–3.80(m,3H),3.69–3.62(m,456H),3.56(dd,J＝9.9,5.1Hz,6H),3.47(dd,J＝9.2,4.7Hz,6H),3.39(s,3H),3.35–3.30(m,2H),2.51(d,J＝3.9Hz,3H),1.69(dt,J＝15.0,7.5Hz,2H),1.45–1.36(m,4H),1.35–1.28(m,8H),1.26(s，12H)，0.88(t，J＝6.8Hz，3H).

Mixing mPEG₁₁₃CTA (100mg, 18.6. mu. mol),2- (azepan-1-yl) ethyl methacrylate (PC7A) (235mg,1.11mmol) and Azobisisobutyronitrile (AIBN) (0.30mg, 1.9. mu. mol) were dissolved in 1mL anhydrous dioxane, oxygen was removed from the system by three freeze-thaw cycles, and then reacted at 70 ℃ for 24 h. After the reaction is finished, the reaction solution is dialyzed by ethanol and deionized water and then is freeze-dried, and the molecular weight cut-off of a dialysis bag is 3500D, so that 210mg of light yellow white solid mPEG-PC7A is obtained, and the yield is 79.9%.

Preparation example 3: preparation of acid-sensitive polymer-immunoadjuvant conjugates

Compound 1(6.0g,0.04mol), TEA (8.08g,0.08mol) were dissolved in 10mL of dichloromethane, and 10mL of a solution of methacryloyl chloride (2.08g,0.08mol) in dichloromethane was slowly added dropwise thereto under an ice-water bath, and after completion of the dropwise addition, the reaction was carried out at room temperature for 12 hours. The reaction mixture was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and subjected to column separation to obtain 3.8g of compound 2, which was 87.2% in yield.¹H NMR(400MHz,CDCl₃)δ6.15(s,1H),5.64–5.55(m,1H),4.35–4.30(m,2H),3.79–3.75(m,2H),3.75–3.71(m,2H),3.69(s,4H),3.64–3.60(m,2H),1.96(s,3H).

Compound 2(0.20g,0.91mmol), N, N-Diisopropylethylamine (DIEA) (354mg,2.75mmol) was dissolved in 10mL of tetrahydrofuran, and di (p-nitrophenyl) carbonate (NPC) (418mg,1.37mmol) was added thereto under ice-water bath and reacted at room temperature for 12 hours. After the reaction, the reaction solution was washed with water and then with a saturated ammonium chloride solution, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and then subjected to column separation to obtain 285mg of compound 3, with a yield of 81.8%.¹H NMR(400MHz,CDCl₃)δ8.29(d，J＝9.2Hz，2H)，7.40(d，J＝9.2Hz，2H)，6.14(s，1H)，5.59(s，1H)，4.47–4.43(m，2H)，4.35–4.31(m，2H)，3.85–3.81(m，2H)，3.80–3.76(m,2H),3.72(s,4H)，1.96(s,3H).

Compound 3(80mg,0.21mmol), Rasimotent (55mg,0.17mmol) was dissolved in 2mL of N, N-Dimethylformamide (DMF), and 1-Hydroxybenzotriazole (HOBT) (35.1mg,0.26mmol) was added thereto and reacted at room temperature for 12 hours. After the reaction, the solvent was removed by rotary evaporation, the reaction solution was washed with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and then column-separated to obtain 72mg of compound 4, with a yield of 77.7%.¹H NMR(400MHz,CDCl₃)δ8.47(s,1H)，8.14(d,J＝8.2Hz,2H),7.58(dd,J＝11.3,4.1Hz，1H)，7.49–7.43(m，1H)，6.12(s，1H)，5.59–5.53(m，1H),4.90(s,2H),4.78(s，2H)，4.46–4.40(m，2H)，4.30–4.24(m，2H)，3.84–3.79(m,2H),3.77(dd,J＝5.5,4.2Hz，2H)，3.71(s，4H)，3.64(q，J＝7.0Hz，2H)，3.35(s，1H)，1.93(s,3H),1.33(s,6H),1.25(t,J＝7.0Hz,3H).

Compound 4(65mg,0.12mmol), mPEG₁₁₃CTA (89.5mg, 16.6. mu. mol),2- (diisopropylamino) methacrylic acid (DPA) (177mg,0.83mmol) and Azobisisobutyronitrile (AIBN) (0.27mg, 1.7. mu. mol) were dissolved in 1mL anhydrous dioxane, oxygen was removed from the system by three freeze-thaw cycles, and then reacted at 70 ℃ for 24 h. After the reaction is finished, dialyzing the reaction solution by using ethanol and deionized water, freeze-drying, and obtaining 210mg of light yellow white solid mPEG with the cut-off molecular weight of 3500D by using a dialysis bag₁₁₃-b-P(DPA₄₂-co-R848₃) The yield was 75.8%.

Preparation example 4: preparation of Racemate-coated or non-coated Racemate nanoparticles

5mg of mPEG-PC7A prepared in preparation example 2 and 0.5mg of Racemide are dissolved in 0.5mL of chloroform, the obtained solution is added into 5mL of 2 wt% polyvinyl alcohol aqueous solution to form a water phase and an oil phase after being fully dissolved, an ultrasonic probe is immersed into the liquid surface in an ice water bath for 5min by 80w of ultrasound, residual chloroform is removed by rotary evaporation at room temperature after the ultrasound is finished, and precipitates obtained after the mixed solution is centrifuged by 20000g for 60min are dispersed by phosphate buffer solution to obtain the Racemide-encapsulated polymer nanoparticles.

In addition, mPEG-PC7A nanoparticles that were not loaded with ranitidine were prepared in the same manner as described above, except that no ranitidine was added.

Preparation example 5: preparation of Polymer-Racemate conjugate nanoparticles

5mg of mPEG prepared in preparation example 3₁₁₃-b-P(DPA₄₂-co-R848₃) Dissolving in 0.5mL of chloroform, adding into 5mL of 2% polyvinyl alcohol aqueous solution after fully dissolving to form water-oil two phases, immersing an ultrasonic probe in an ice-water bath to the liquid level, carrying out 80w ultrasonic treatment for 5min, carrying out rotary evaporation at room temperature after the ultrasonic treatment is finished to remove residual chloroform, centrifuging the mixed solution for 60min by 20000g, and dispersing the obtained precipitate in phosphate buffer solution to obtain the polymer-Rasimoter conjugate nanoparticles.

Preparation example 6: preparation of fluorescently labeled nanoparticles

5mg are carried outmPEG-PC7A prepared in example 2, 0.5mg of Rasimotent and 0.05mg of IR-780 dye were dissolved in 0.5mL of chloroform or 5mg of mPEG prepared in example 3₁₁₃-b-P(DPA₄₂-co-R848₃) And 0.05mg of IR-780 dye is dissolved in 0.5mL of chloroform, the mixture is added into 5mL of 2 wt% polyvinyl alcohol aqueous solution to form water and oil phases after the mixture is fully dissolved, an ultrasonic probe is immersed into the liquid surface in an ice water bath, 80w of ultrasound is carried out for 5min, residual chloroform is removed by rotary evaporation at room temperature after the ultrasound is finished, and precipitates obtained after the mixed solution is centrifuged by 20000g for 60min are dispersed by phosphate buffer salt solution to obtain the IR-780 dye marked nano-particles.

Preparation example 7: preparation of whole cell tumor nano vaccine

Mixing the tumor cell membrane of preparation example 1 containing 1mg of membrane protein with 3mg of the polymer nanoparticles of preparation example 4 or 5 in deionized water, shaking the mixture in a shaker for 60min, and repeatedly extruding 200nm polycarbonate filter membrane for 21 times by a nano extruder to obtain the nano vaccine (separately, the nano vaccine of the uncoated Racemot-loaded mPEG-PC7A, the nano vaccine of the uncoated Racemot-loaded mPEG-PC7A, and the mPEG)₁₁₃-b-P(DPA₄₂-co-R848₃) Nano-vaccines).

Preparation example 8: preparation of fluorescence labeled whole-cell tumor nano vaccine

Mixing tumor cell membrane containing 1mg of membrane protein prepared in preparation example 1 and 3mg of nanoparticles prepared in preparation example 6 in deionized water, placing the mixture in a shaking table to shake for 60min, and repeatedly extruding a 200nm polycarbonate filter membrane for 21 times through a nano extruder to obtain the whole-cell tumor nano vaccine.

Test example 1: acid sensitivity testing of nano-vaccines

mu.L of the nano-vaccine prepared in preparation example 7 at a concentration of 2mg/mL was added to 900. mu.L of citric acid-phosphate buffer solution having pH values of 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2 and 7.4, respectively, and after standing for 20min, the change in DLS particle size was recorded, and the test results are shown in FIG. 3.

The test result shows that the particle structure of the nano vaccine is dissociated when the pH value is 6.2, which indicates that the nano particles still have good acid sensitivity after being coated with the engineered cell membrane.

Test example 2: small animal imaging test

The fluorescence-labeled whole-cell nano-vaccine prepared in preparation example 8 was administered to Balb/c mice by footpad injection, and the distribution of the nano-vaccine in the mice was observed by a small animal imager at 2h,5h,8h,12h,24h, and 36h, respectively, and the test results are shown in FIG. 4.

Test results show that the nano vaccine can be targeted to lymph nodes at the inguinal position of a mouse after being administered for 2 hours, and is retained in the lymph nodes for a long time until the fluorescence intensity is gradually reduced after 24 hours, and the nano vaccine is gradually decomposed and metabolized by an organism.

Test example 3: tumor inhibition experiment of nano vaccine on subcutaneous tumor-bearing mice

After inoculating CT-26 tumor cells on the back of Balb/c white mouse, when the tumor volume reaches 50mm³On the left and right, the mice are randomly divided into 4 groups, each group is provided with 5 mice, and the groups are PBS groups respectively; the mPEG-PC7A nano-vaccine group of the rapamycin-unencapsulated vaccine of example 7 was prepared; the mPEG-PC7A nano vaccine group coated with Racemate; mPEG₁₁₃-b-P(DPA₄₂-co-R848₃) And (4) a nano vaccine group. Administering 1 time every seven days, wherein the administration volume is 100 μ L and the concentration is 30 mg/kg; the tumor volume was recorded every three days with two administrations and the results of the test are shown in figure 5.

The test results show that the entrapped or coupled ranimuster nano-vaccine group (see fig. 5C and 5D) significantly inhibited tumor growth and extended the survival cycle of mice.

Claims

1. A whole-cell tumor nano-vaccine containing a whole series of tumor-associated antigens is a nano-vaccine of acid-sensitive polymer loaded immune adjuvant nanoparticles with engineered tumor cell membranes coated on the surfaces, or a nano-vaccine of acid-sensitive polymer-immune adjuvant conjugates with engineered tumor cell membranes coated on the surfaces, or a combination of the two,

wherein the engineered tumor cell membrane is a cell membrane of a tumor cell treated by a chemotherapeutic drug or photodynamic therapy;

in the above-mentioned formula 1, the,

R₁selected from hydroxyl group,

R₂selected from phenyl or

n is an integer of 3 to 12;

x is an integer from 20 to 60, preferably from 40 to 50,

in the above-mentioned formula 2, the,

R₁、R₂and R₃And x are each as defined in the above formula 1,

R₄selected from the group derived from immunological adjuvants;

y is an integer of 1 to 20, preferably 1 to 10.

2. The whole cell tumor nano-vaccine of claim 1,

R₂is composed of

n is 12; r₃Selected from the group consisting of N, N-diisopropylamino, hexamethyleneamino; x is 40, 42, 50; y is 0, 3, 4 or 5.

3. The whole-cell tumor nano-vaccine according to claim 1, wherein the Linker is selected from any one of the following structures:

4. the whole-cell tumor nano-vaccine according to claim 1, wherein the immune adjuvant is selected from any one of the following,

alternatively, the group R derived from an immunoadjuvant in formula 2₄Any one selected from the following groups:

5. the whole-cell tumor nano-vaccine according to claim 1,

wherein the tumor cell is selected from 4T-1 breast cancer cell, MCF-7 breast cancer cell, CT-26 colon cancer cell, HCT116 colon cancer cell, B16-F10 melanoma cell or Panc02 pancreatic cancer cell;

the chemotherapeutic drug is selected from oxaliplatin, doxorubicin, epirubicin, idarubicin, paclitaxel, docetaxel, cyclophosphamide; and

the photosensitizer used in the photodynamic therapy is selected from chlorin e6, pyropheophorbide a and indocyanine green.

6. The whole-cell tumor nano-vaccine according to claim 1, wherein the loading amount of the immune adjuvant in the nanoparticles is 1-30 wt%.

7. A method of preparing the whole cell tumor nano-vaccine of any one of claims 1 to 6, the method comprising the steps of:

2) co-extruding and fusing the prepared polymer nanoparticles and the engineered tumor cell membrane through a nano extruder to obtain the whole-cell tumor nano vaccine with the surface of the nanoparticles of the acid-sensitive polymer loaded with the immunoadjuvant and the engineered tumor cell membrane coated with the engineered tumor cell membrane,

or,

the method comprises the following steps:

2) and co-extruding and fusing the prepared acid-sensitive polymer-immune adjuvant conjugate nano-particles and the engineered tumor cell membrane through a nano extruder to obtain the whole-cell tumor nano-vaccine with the surface of the nano-particles of the acid-sensitive polymer-immune adjuvant conjugate coated with the engineered tumor cell membrane.

8. The method of claim 7, wherein,

in the step 1) of the process,

the organic solvent is selected from chloroform, dichloromethane, ethyl acetate or acetone; and is

The emulsifier comprises polyvinyl alcohol.

9. The method of claim 7, wherein,

in the step 1), the weight ratio of the acid-sensitive polymer to the immunoadjuvant is 1-50: 1, and

in the step 2), the weight ratio of the nanoparticles to the tumor cell membrane is 0.1-10: 1.

10. use of a whole-cell tumor nano-vaccine according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of malignant tumors, preferably selected from the group consisting of breast cancer, colorectal cancer, melanoma and pancreatic cancer, and for the prevention of tumor recurrence and metastasis.