WO2021155631A1

WO2021155631A1 - Erythrocyte gel delivery system, and preparation method therefor and application thereof

Info

Publication number: WO2021155631A1
Application number: PCT/CN2020/080992
Authority: WO
Inventors: 汪超; 范亲
Original assignee: 苏州大学
Priority date: 2020-02-08
Filing date: 2020-03-25
Publication date: 2021-08-12
Also published as: CN111184684A; CN111184684B

Abstract

Provided are an erythrocyte gel delivery system, and a preparation method therefor and application thereof, relating to the technical field of biomaterials. The erythrocyte gel delivery system is prepared by uniformly mixing an active ingredient or a delivery vector containing the active ingredient with fresh blood, and after preliminary coagulation, performing mild drying. The preparation method for the erythrocyte gel delivery system is simple and quick, and the erythrocyte gel delivery system is high in drug loading, and high in biological safety and biodegradability. Erythrocyte gel has the functions of immune stimulation and immune cell recruitment, and may form an immune niche at an implantation site. After being implanted, an immune modulator and a tumor-related antigen contained in a prepared erythrocyte gel vaccine stimulate recruited immune cells to differentiate into immune cells having specificity for a tumor antigen, and induce the production of a highly effective anti-tumor immune response.

Description

Red blood cell gel delivery system and preparation method and application thereof

Technical field

The invention relates to a red blood cell gel delivery system and a preparation method and application thereof, and belongs to the technical field of biological materials.

Background technique

Conventional treatment of diseases often requires repeated high-dose administration to patients in order to achieve a good prognostic effect. However, the overall efficiency produced by the conventional administration method is low, the patient's compliance is poor, and it is easy to cause serious toxic and side effects. Intravenously injected drugs can enter the blood circulation without crossing any barriers, there is a certain risk of systemic toxicity, and they cannot be relieved and eliminated in time. Oral administration has a slower onset time, lower bioavailability, and usually lacks targeting.

In recent years, the rapid development of materials engineering has provided new opportunities for the field of biomedicine. Reasonably designed biological materials can be used to construct nano/micron size delivery systems and drug storage systems for drug delivery. Common drug carriers include: organic/inorganic nanoparticles, liposomes, cell membranes, and hydrogels. The new delivery system based on biomaterials can increase the long circulation time of drugs in the body, continuously release a variety of active drugs, target specific lesions, and greatly increase the concentration of drugs in lesion tissues and diseased cells. At present, the new drug delivery system has shown excellent therapeutic effects in clinical practice. Among them, hydrogels have been used in many medical fields, including heart disease treatment, tumor treatment, immunotherapy, wound repair and pain treatment. Traditional hydrogels are composed of a large amount of water and a cross-linked polymer network. Hydrogels with high water content (water content of about 70-99%) are similar to tissues, have good biocompatibility, and are easy to load multiple drug. In addition, hydrogels are usually prepared in aqueous solutions, which can avoid the risk of denaturation and aggregation caused by exposure to organic agents. The hydrogel formed by crosslinking of traditional polymers has excellent mechanical properties. Some hydrogel delivery systems have entered clinical applications. For example, the sales of INFUSE collagen gel (for bone repair) developed by Medtronic has reached 750 million US dollars.

However, compared with the hydrogel materials that have been reported, the hydrogel delivery systems that have actually entered clinical applications are very limited. The hydrogel delivery system still faces the problem of clinical transformation. The preparation process of traditional polymer hydrogel carriers is complicated and often requires multiple synthesis steps. The high water content of hydrogels makes terminal sterilization difficult. Therefore, all raw materials and manufacturing processes must be tested for sterility. At the same time, the storage process of hydrogels needs to be investigated in detail. Materials or drugs that are easily hydrolyzed need to be dehydrated after the gel is prepared, while gels that need to be stored in a hydrated state should avoid evaporation of water to prevent the loss of drugs. Therefore, the regulatory issues and commercialization costs of hydrogels also limit their clinical conversion. In addition, hydrogels constructed from synthetic materials have certain potential risks in terms of biocompatibility and biosafety, and their degradation behavior, in vivo transport behavior, and metabolites are unclear. Therefore, seeking high biosafety biomaterials to construct hydrogel delivery systems, enhance the therapeutic effect on diseases, and reduce safety risks is one of the current research focuses of drug delivery systems.

After the blood is isolated, an agglutination reaction can be triggered to form a red blood cell gel, which has the advantages of abundant sources, simple preparation, good biocompatibility, and the like. Red blood cell gel can be completely metabolized in the body without producing toxic by-products. In addition, the red blood cells contained in the gel also play a role in immune regulation and maintain body homeostasis. Under normal circumstances, the most common function of red blood cells is to carry oxygen. However, when inflammation or oxidative stress occurs, red blood cells can promote immune activation. Contrary to the function of healthy red blood cells, oxidized or senescent red blood cells promote lipopolysaccharide (LPS)-induced dendritic cells (DCs) to show a fully mature phenotype. Aging or damaged red blood cells can be completely eliminated by tissue-resident macrophages. Hemoglobin and hemoglobin released from damaged red blood cells or stored red blood cells will induce macrophages to polarize to a pro-inflammatory (M1) state and secrete pro-inflammatory cytokines. In addition, oxidized/aged red blood cells can also promote the proliferation and activation of T cells. There are no reports about the use of red blood cell gels based on blood in the biological field.

Summary of the invention

In order to solve the related problems of traditional hydrogels, the present invention provides a red blood cell gel delivery system with high biological safety. After the blood is isolated, the soluble fibrinogen in the plasma is gradually transformed into insoluble fibrin, which attracts a large number of red blood cells, causing the flowing blood to turn into a gel state. This process can be used to load active ingredients to form a red blood cell gel delivery system. The red blood cell gel is not only simple to prepare, has better biosafety and biocompatibility, but also the damaged red blood cells inside the gel can participate in immune regulation.

The first object of the present invention is to provide an erythrocyte gel delivery system, which includes an erythrocyte gel; and, an active ingredient loaded on the erythrocyte gel, or a delivery carrier containing an active ingredient loaded on the erythrocyte gel.

Further, the active ingredients include one or a combination of excipients, chemotherapeutic drugs, radiosensitizers, immunomodulatory drugs, photosensitizer molecules, magnetic molecules, and antigens.

Further, the delivery carrier includes one or more combinations of polymer microspheres, organic nanoparticles, inorganic nanoparticles, micelles, and polypeptide derivatives.

Further, the drug loading rate of the red blood cell gel delivery system is 90-99%.

Further, the administration mode of the red blood cell gel delivery system is injection, implantation or directly filling the lesion site.

The second object of the present invention is to provide a method for preparing the red blood cell gel delivery system, which includes the following steps: by mixing the active ingredient or a delivery vehicle containing the active ingredient with fresh blood, and performing gentle drying after preliminary coagulation The erythrocyte gel delivery system is prepared.

Further, the preparation method specifically includes the following steps:

(1) Mix the active ingredient or the delivery vehicle containing the active ingredient into fresh blood to obtain a mixed solution;

(2) Place the mixed solution of step (1) at 20-30°C for 5-15 minutes for preliminary coagulation;

(3) Drying the preliminarily coagulated mixed blood in step (2) at 35-40° C. until dehydrated by 50-90% to obtain the red blood cell gel delivery system.

The third object of the present invention is to provide the application of the red blood cell gel delivery system in the preparation of products for preventing, treating and diagnosing diseases.

Further, the disease is an infectious disease, a parasitic disease, a blood disease, an endocrine system disorder, a nutritional and metabolic disease, a mental and behavioral disorder, a respiratory system disease, a digestive system disease, or cancer.

The fourth objective of the present invention is to provide an anti-tumor gel vaccine, which is prepared by mixing tumor neoantigens and immunomodulators with fresh blood, and then performing gentle drying after preliminary coagulation. .

Further, the tumor neoantigen is obtained by repeatedly freezing and thawing 4-6 times with the aid of ultrasonic lysis.

Further, the immunomodulator contained in the anti-tumor gel vaccine includes one or more combinations of immunogenic adjuvants, immunosuppressive agents, and immune enhancers.

Further, the anti-tumor gel vaccine can be administered by injection or implantation.

Further, the anti-tumor gel vaccine can treat a variety of tumors, including but not limited to bladder cancer, bone cancer, brain cancer, colon cancer, prostate cancer, skin cancer, stomach cancer, breast cancer, and ovarian cancer.

In order to further improve the therapeutic effect of tumor vaccines, the present invention provides an anti-tumor combination therapy based on red blood cell gel vaccine. Combining the red blood cell gel tumor vaccine with immune checkpoint inhibitors enhances the specific anti-tumor immune response. , Produce long-lasting immune memory effect, effectively prevent and treat a variety of tumors.

The fifth object of the present invention is to provide an anti-tumor combined medicine composition, which includes an anti-tumor gel vaccine and an immune checkpoint inhibitor.

Further, the immune checkpoint inhibitor includes one or more combinations of CTLA-4 antibody, PD-1 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, and VISTA antibody.

Furthermore, immune checkpoint inhibitors can exert a combined immune response by loading red blood cell gel vaccine or by intravenous injection.

The beneficial effects of the present invention:

(1) The preparation method of the red blood cell gel delivery system of the present invention is simple and quick, has a high drug loading, and has high biological safety and biodegradability.

(2) The red blood cell gel itself has immune stimulation and immune cell recruitment, and can form an immune niche at the implantation site.

(3) The immunomodulators and tumor-associated antigens contained in the red blood cell gel vaccine prepared by the present invention, after being implanted, stimulate the recruitment of immune cells to differentiate into immune cells specific for tumor antigens, and induce efficient anti-tumor immunity reaction.

(4) The composition of the red blood cell gel tumor vaccine and immune checkpoint inhibitor of the present invention effectively enhances the anti-tumor effect, produces a durable immune memory effect, and prevents tumor recurrence and metastasis.

Description of the drawings

Figure 1 is the appearance and morphology of the red blood cell gel in the present invention;

Figure 2 shows the colloidal properties of the red blood cell gel verified by the rheometer of the present invention;

Figure 3 shows the SEM of the present invention showing that the red blood cell gel has a regular internal structure;

Figure 4 shows the degradation of red blood cell gel in vitro in the present invention;

Figure 5 shows the gel volume change of the red blood cell gel of the present invention when it is degraded in vivo;

Figure 6 shows the degradation of the red blood cell gel in mice in the present invention;

Figure 7 shows the volume change of the red blood cell gel implanted in the body on the 0th, 1, 3, 5, and 7 days of the present invention;

Fig. 8 shows the appearance and morphology changes of the red blood cell gel implanted in the body on the 0th, 1, 3, 5, and 7 days of the present invention;

Figure 9 shows the internal microscopic changes of the SEM characterization of the red blood cell gel implanted in the body on the 0th, 1, 3, 5, and 7 days in the present invention;

Fig. 10 is the HE section of the red blood cell gel implanted on the 0th, 3rd, and 7th days of the present invention;

Figure 11 is the immunofluorescence section of the red blood cell gel implanted in the present invention on the 0th, 3rd and 7th days;

Fig. 12 is the fluorescence intensity of the red line marked part on the

immunofluorescence section

0, 3, and 7 days after the red blood cell gel implanted in the present invention;

Fig. 13 is an overall quantitative analysis of immunofluorescence slices of the present invention on the 0th, 3rd, and 7th days after implantation of the red blood cell gel;

Fig. 14 is an analysis of immune cells infiltrated in the skeleton after the red blood cell gel implanted in the present invention on the 0th, 3rd, and 7th days;

Figure 15 is a study on the drug-loading efficiency of OVA and CpG of the red blood cell gel in the present invention;

Figure 16 is the distribution of FITC-labeled OVA in the red blood cell gel of the present invention;

Figure 17 shows the release of OVA and CpG controlled by red blood cell gel in the present invention;

Figure 18 shows the stimulating effect of the red blood cell gel vaccine on dendritic cells in the present invention;

Figure 19 shows the stimulating effect of the red blood cell gel vaccine on macrophages in the present invention;

Fig. 20 is the HE section of the red blood cell gel vaccine implanted in the present invention on the 0th, 3rd, and 7th days;

Fig. 21 is an immunofluorescence section of the red blood cell gel vaccine of the present invention after implantation on the 0th, 1, 3rd, and 7th days;

Figure 22 is an analysis of the phenotype of immune cells infiltrated 3 days after implantation of the red blood cell gel vaccine in the present invention;

Figure 23 shows the effect of red blood cell gel vaccine and its combination with immune checkpoint blocker in preventing tumors in the present invention

Figure 24 is an analysis of the therapeutic effect of the red blood cell gel vaccine and its combination therapy with immune checkpoint blocker on B16-OVA melanoma after intervention in the present invention;

Figure 25 is an analysis of the therapeutic effect of the red blood cell gel vaccine and its combination therapy with immune checkpoint blocker in the present invention on 4T1 tumors;

Figure 26 is the red blood cell gel vaccine of the present invention and its combination therapy with immune checkpoint blocker on the tumor fluorescence imaging of the mouse B16-luc melanoma recurrence model after resection;

Figure 27 is an analysis of the therapeutic effect of the red blood cell gel vaccine and its combination therapy with immune checkpoint blocker on the recurrence model of mouse B16-luc melanoma after resection.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the examples cited are not intended to limit the present invention.

The source of materials in each embodiment of the present invention is:

The immune adjuvant CpG-1826 (5'-TCC ATG ACG TTC CTG ACG TT-3', tlrl-1826-5) was purchased from Invivogen. Mouse granulocyte-macrophage colony stimulating factor GM-CSF (315-03-50) was purchased from PeproTech. FITC fluorescently labeled ovalbumin (FITC-OVA) was purchased from Soleibao. Cy5.5-labeled CpG was synthesized and purified by Shanghai Shenggong Co., Ltd. Mouse breast cancer cells 4T1 were purchased from the American Type Culture Collection (ATCC for short). B16 cells expressing OVA (B16-OVA) were donated by Dr. Liu Haiyan from Soochow University. Luciferase-labeled black B16-luc cells were donated by Dr. Wenjun Zhu from Soochow University. 4T1 was cultured in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100U/ml streptomycin. B16-OVA and B16-luc were cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100U/ml streptomycin.

BALB/c and C57BL/6 mice aged 6-10 weeks were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. The mice were processed in accordance with the instructions of the Laboratory Animal Management Committee (IACUC) of the Institute of Biochemistry and Cell, Chinese Academy of Sciences.

Anti-PD-1 antibody (Anti-PD-1) was purchased from Bioxcell, (antibody number is BE0033-2-5MG).

Example 1: Synthesis and characterization of red blood cell gel

(1) Quickly take out 200μL of blood from the vein of the organism;

(2) Immediately inject the fresh blood obtained in step (1) into a sterile mold, and place it at room temperature for 10 minutes;

(3) Transfer the preliminarily coagulated blood in step (2) to a sterile vacuum drying box, and gently dry it at 37°C to obtain a red blood cell gel, as shown in Figure 1;

(4) Use a rheometer to prove the rheological properties of the gel obtained in step (3), and prove that the red blood cell gel of the present invention has good mechanical properties (Figure 2);

(5) Use scanning electron microscope (Scanning Electron Microscope, SEM) to microscopically image the red blood cell gel (Figure 3), which proves that the red blood cell gel has a regular internal structure;

(6) Record the degradation of the red blood cell gel carrier in vivo and in vitro. Figure 4 shows that the red blood cell gel is slowly degraded in a neutral PBS solution. Figure 5 shows that the red blood cell gel degrades slowly in the first three days of implantation in the mouse, and is completely degraded at about 15 days (Figure 6), which proves that the red blood cell gel has excellent biodegradability and biocompatibility.

(7) In order to further study the degradation process of the red blood cell gel in vivo, the red blood cell gel was taken out, weighed and photographed on the 0th, 1, 3, 5, and 7 days after implantation. Figure 7 shows that the quality of the red blood cell gel gradually decreases, while the appearance and morphology also show a decreasing trend (Figure 8). In addition, SEM was used to directly analyze the internal degradation behavior of the blood gel. It can be seen from Figure 9 that the red blood cell gel is gradually engulfed by white blood cells over time.

Example 2: Immune stimulation and immune cell recruitment of red blood cell gel

(1) After C57BL/6J mice were anesthetized with 2.5% isoflurane, the red blood cell gel prepared in Example (1) was implanted under the skin of the mice;

(2) The red blood cell gel implanted in the body for 0, 1, 3, and 7 days was taken out at the same time, and embedded in paraffin for HE tissue section and embedded in OCT gel for fluorescence staining analysis. It can be seen that the red blood cell gel of the present invention has an immune cell recruitment effect (Figure 10), a large number of white blood cells infiltrate from the edge of the red blood cell gel to the inside (Figure 11), and the fluorescent signal of the white blood cell gradually increases from the edge of the red blood cell gel to the inside (Figure 12). The overall fluorescence intensity increases with time (Figure 13);

(3) Further use flow cytometry to analyze the immune cells and their subpopulations recruited by the red blood cell gel (Figure 14). The results showed that after the red blood cell gel was implanted in the body, a large number of white blood cells were recruited, including macrophages, dendritic cells, T cells, B cells and NK cells.

Example 3: Analysis of the slow-release effect of red blood cell gel

(1) Put Cy5.5-CpG-OND and FITC-OVA into the corresponding molds in step (2) of Example 1, and mix them with fresh isolated blood according to the steps in Example 1, and then gently dry them to prepare the drug-loaded Red blood cell gel

(2) Aspirate the small amount of liquid precipitated by the drug-loaded red blood cell gel, detect the unloaded drug content by a microplate reader, and calculate the drug-loading efficiency of the red blood cell gel. The erythrocyte gel loading efficiency of OVA and CpG can reach 95% and 83% (Figure 15);

(3) The prepared drug-loaded red blood cell gel was embedded in OCT gel, and after sectioning, the drug loading was observed through a confocal microscope. The results showed that OVA can be effectively loaded into the red blood cell gel (Figure 16);

(4) Put the drug-loaded red blood cell gel in 2mL 37℃ Phosphate Buffered Solution (PBS), take out 100μL at the corresponding time point and supplement with 100μL isothermal PBS solution, and use a microplate reader to detect Cy5 in it. The fluorescence content of 5-CpG-OND and FITC-OVA is used to make the drug release curve. Figure 17 shows that CpG-OND and FITC-OVA can be released continuously for about 7 days and have a sustained release effect.

Example 4: Construction of erythrocyte gel vaccine and its immunostimulatory effect at the cellular level in vitro

(1) Put 5μg CpG-OND, 0.05μg GM-CSF and 5μg OVA into the sterile mold corresponding to step (2) of Example 1, and mix with fresh isolated blood according to the steps in Example 1. Dry preparation of drug-loaded red blood cell gel vaccine;

(2) To study the immunostimulatory effect of red blood cell gel vaccine on dendritic cells (DC), 1×10 ⁶ myeloid-derived dendritic cells (DC) were combined with PBS, blank red blood cell gel, free vaccine group or red blood cell coagulation. Co-cultivation of glue vaccine. After about 20 hours, immediately collect the cell culture supernatant and store it at -80°C for testing. Flow cytometry was used to analyze the maturity level of DCs, and the DCs were stained with FITC-CD11c, APC-CD80 and PE-CD86. The cytokine concentration (TNF-α, IL-6 and IL-12) in the supernatant was tested according to Elisa standard operation manual. The results show that compared with the control group, the immunomodulatory drugs released from the red blood cell gel vaccine can effectively stimulate the maturation of DC cells and promote the secretion of large amounts of immune-related cytokines by DC cells (Figure 18);

(3) In order to study the immunostimulatory effect of red blood cell gel vaccine on macrophages, 5×10 ⁵ peritoneal macrophages were co-cultured with blank red blood cell gel, free vaccine group or red blood cell gel vaccine. After about 24 hours, immediately collect the cell culture supernatant and store it at -80°C for testing. The polarization level of macrophages was analyzed by flow cytometry, and the macrophages were stained with FITC-F4/80, PE-CD206, and APC-CD80. The cytokine concentration (TNF-α and IL-6) in the supernatant was tested according to Elisa standard operating manual. The results showed that the erythrocyte gel vaccine can promote the polarization of macrophages to the M1 pro-inflammatory state, and make the macrophages secrete pro-inflammatory cytokines (Figure 19).

Example 5: In vivo immune stimulation effect of red blood cell gel vaccine

(1) Put 10μg CpG-OND, 0.1μg GM-CSF and 10μg OVA into the sterile mold corresponding to step (2) of Example 1, and mix with fresh isolated blood according to the steps in Example 1. Dry preparation of drug-loaded red blood cell gel vaccine;

(2) After C57BL/6J mice were anesthetized with 2.5% isoflurane, the red blood cell gel prepared in step (1) of Example 5 was implanted under the skin of the mice;

(3) The red blood cell gel vaccines implanted in the body for 0, 1, 3, and 7 days were taken out at the same time, respectively embedded in paraffin for HE tissue section analysis and embedded in OCT gel for fluorescent staining. It can be seen that the red blood cell gel vaccine loaded with immunomodulators further expands the recruitment of immune cells (Figure 20), and more immune cells infiltrate the inside of the blood gel skeleton (Figure 21);

(4) Use flow cytometry to further analyze the changes and phenotypes of immune cells recruited by the red blood cell gel vaccine. The results showed that the red blood cell gel vaccine not only significantly increased the number of recruited immune cells, but also could effectively activate the recruited immune cells (Figure 22).

Example 6: Effect analysis of red blood cell gel vaccine and anti-tumor combination therapy constructed therefrom in tumor prevention

(1) Put 100μg CpG-OND, 1μg GM-CSF and 100μg OVA into the sterile mold corresponding to step (2) of Example 1, and mix with fresh isolated blood according to the steps in Example 1. Dry preparation of drug-loaded red blood cell gel vaccine;

(2) Weigh C57BL/6J mice and randomly divide them into 6 groups, namely PBS group, erythrocyte gel group, free vaccine group, erythrocyte gel vaccine group, Anti-PD-1 group and erythrocyte gel vaccine and Anti-PD-1 combination treatment group. On day -7, the erythrocyte gel vaccine prepared in step (1) and the blank erythrocyte gel prepared in Example 1 were implanted subcutaneously in mice, and the PBS group was injected with the same volume of neutral PBS of about 200μl. In the free vaccine group, the same dose of immunomodulators and antigens (100μg CpG-OND, 1μg GM-CSF and 100μg OVA) were diluted with PBS to 200μl, and then injected directly into the mouse subcutaneously. On

days

5, 7, 9, and 11, mice in the Anti-PD-1 group and the combined treatment group were injected with 20 μg PD-1 antibody through the tail vein. 3.5×10 ⁵ B16-OVA melanoma cells were injected subcutaneously into the right side of the mouse on day 0. The tumor size and mouse body weight were measured every two days. The tumor volume calculation formula is: short diameter ² × long diameter × 0.5. When the tumor volume exceeds 1.5 cm ³ or the tumor ruptures or hemorrhages, the animal is euthanized. The results show that the red blood cell gel vaccine has a significant preventive effect and effectively inhibits tumor growth. After combined with immune checkpoint inhibitor PD-1 antibody, the anti-tumor effect was significantly enhanced and the survival time of mice was prolonged (Figure 23).

Example 7: Research on red blood cell gel vaccine and its anti-tumor combination therapy in tumor treatment

(1) Establish a B16-OVA melanoma model in C57BL/6J mice

A mouse B16-OVA tumor model was established by injecting B16-OVA tumor cells (5×10 ⁵ /mouse) into the back side of C57BL/6J mice. Three days after tumor injection, the mice were treated according to the method in step (2) of Example 6, and the PBS group, red blood cell gel group, free vaccine group, red blood cell gel vaccine group, Anti-PD-1 group and Red blood cell gel vaccine and Anti-PD-1 combined treatment group. The body weight and tumor size of the mice were then monitored. It can be seen from Figure 24 that the erythrocyte gel vaccine of the present invention and the anti-tumor combination therapy constructed therefrom also have an inhibitory effect on the growth of B16-OVA melanoma, the combination therapy has the best effect, and the tumor tissue volume increases slowly (Figure 24 ).

(2) Establish a 4T1 breast cancer model in Balb/c mice

A mouse 4T1 breast cancer tumor model was established by injecting 4T1 tumor cells (1×10 ⁶ /mouse) into the back side of Balb/c mice. Three days after tumor injection, the mice were treated according to the method in step (2) of Example 6, and the PBS group, red blood cell gel group, free vaccine group, red blood cell gel vaccine group, Anti-PD-1 group and Red blood cell gel vaccine and Anti-PD-1 combined treatment group. The treatment effect was then monitored and the survival rate of the mice was recorded. The results showed that the red blood cell gel vaccine of the present invention and the anti-tumor combination therapy constructed therefrom also showed a good anti-tumor effect on the mouse 4T1 tumor model, and the survival time of the mice was prolonged (Figure 25).

Example 8: Analysis of therapeutic effect of red blood cell gel vaccine and anti-tumor combination therapy constructed therefrom on tumor recurrence models after surgery

(1) B16-luc tumor cells (5×10 ⁵ per mouse) were injected into the back side of C57BL/6J mice to establish a mouse B16-luc tumor model. After 10 days, the mouse tumor was completely removed by surgery. Subcutaneous injection or implantation of PBS, free vaccine, red blood cell gel, red blood cell gel vaccine. At the same time, 1×10 ⁶ B16-luc cells were injected into the other side of the back of the mouse. From the 3rd day after surgical resection, mice in the PD-1 antibody group and the combined treatment group were injected with 20 μg of PD-1 antibody through the tail vein. After that, the treated C57BL/6J black mice were evaluated by fluorescence imaging on the 17, 20, and 23 days. The tumor size was measured every two days, and the tumor volume calculation formula was: short diameter ² × long diameter × 0.5. When the tumor volume exceeds 1.5 cm ³ or the tumor ruptures or hemorrhages, the animal is euthanized. Figure 26 shows that the tumor tissue treated with the red blood cell gel vaccine and the anti-tumor combination therapy constructed from it has a significantly smaller fluorescent imaging area than other controls after 23 days. It can be seen that the anti-tumor method of the present invention can also be used for melanoma recurrence models. Inhibition. In addition, the therapeutic effect of the combination therapy group constructed by the red blood cell gel vaccine is better than that of the single red blood cell gel vaccine group, which further proves the anti-tumor superiority of the immune combination therapy of the present invention (Figure 27).

The above-mentioned embodiments are only preferred embodiments for fully explaining the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or alterations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

A red blood cell gel delivery system, which is characterized in that it comprises a red blood cell gel; and, an active ingredient loaded on the red blood cell gel, or a delivery carrier containing an active ingredient loaded on the red blood cell gel.
The erythrocyte gel delivery system according to claim 1, wherein the active ingredients include excipients, chemotherapeutic drugs, radiosensitizers, immunomodulatory drugs, photosensitizer molecules, magnetic molecules, antigens A combination of one or more ingredients.
The erythrocyte gel delivery system according to claim 1, wherein the delivery carrier comprises one or more of polymer microspheres, organic nanoparticles, inorganic nanoparticles, micelles, and polypeptide derivatives combination.
The erythrocyte gel delivery system of claim 1, wherein the drug loading rate of the erythrocyte gel delivery system is 90-99%.
The erythrocyte gel delivery system of claim 1, wherein the administration mode of the erythrocyte gel delivery system is injection, implantation or directly filling the lesion site.
A method for preparing an erythrocyte gel delivery system according to any one of claims 1 to 5, characterized in that it comprises the following steps: by mixing the active ingredient or the delivery carrier containing the active ingredient with fresh blood, the initial After coagulation, gentle drying is performed to prepare the red blood cell gel delivery system.
The use of the red blood cell gel delivery system according to any one of claims 1 to 5 in the preparation of products for preventing, treating and diagnosing diseases.
An anti-tumor gel vaccine, which is characterized in that the anti-tumor gel vaccine is prepared by mixing tumor neoantigens and immunomodulators with fresh blood, and performing gentle drying after preliminary coagulation.
An anti-tumor combined medicine composition, which is characterized by comprising the anti-tumor gel vaccine according to claim 8 and an immune checkpoint inhibitor.
The anti-tumor combination drug composition according to claim 9, wherein the immune checkpoint inhibitor comprises CTLA-4 antibody, PD-1 antibody, PD-L1 antibody, LAG-3 antibody, TIM-3 antibody, One or more combinations of TIGIT antibody and VISTA antibody.