An "immune co-activation" recombinant biomimetic anti-tumor nanovaccine and its preparation method and application

Abstract

The present invention discloses a "immune co-activation" recombinant biomimetic anti-tumor nanovaccine and its preparation method and application. The nanovaccine is composed of a recombinant exosome carrier shell modified with an immune-activating functional hybrid peptide and a nano-form drug core. The recombinant exosomes are formed by recombinant treatment of ginsenosides and exosomes. The preparation method of the present invention has simple and mild conditions, low cost, and simple operation. The prepared "immune co-activation" recombinant biomimetic anti-tumor nanovaccine has the advantages of high endogenous, lesion site targeting, efficient drug loading, strong immune activation, and high biosafety. The recombinant biomimetic anti-tumor nanovaccine can simultaneously achieve direct tumor killing and strong co-activation of innate immunity and adaptive immunity, and has considerable clinical application prospects in inhibiting the growth and recurrence of various malignant tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-preparations, and specifically relates to an "immune co-activation" recombinant biomimetic anti-tumor nano-vaccine and its preparation method and application. Background Art

[0002] Chemotherapy, radiotherapy, and surgery are conventional treatments for cancer, but their overall effectiveness is limited and they are prone to drug resistance or treatment resistance. Malignant tumors are characterized by their highly invasive nature and prone to recurrence. Furthermore, tumor tissues harbor an extremely complex immunosuppressive microenvironment. Therefore, personalized treatment strategies with enhanced targeting, minimal side effects, and the ability to improve the immune microenvironment are needed. With the rapid advancement of cancer genomics and immunology, immunotherapy has become one of the most promising approaches for cancer treatment. Current cancer immunotherapy approaches primarily include adoptive cell immunotherapy, immune checkpoint blockade, chimeric antigen receptor T cell therapy, and tumor vaccines. Tumor vaccines, by directly introducing tumor-associated antigens such as tumor cell lysates, tumor-associated proteins or antigenic peptides, and genes expressing tumor antigens, along with immune adjuvants, can effectively overcome the immunosuppressive state of the tumor microenvironment, enhance immunogenicity, activate the patient's own immune system, and induce a more robust immune response, thereby achieving tumor control or clearance. Currently, tumor vaccines have become a research hotspot in the field of cancer treatment.

[0003] Dendritic cells (DCs), known as the most powerful professional antigen-presenting cells, are key cells in initiating anti-tumor immune responses. Research and development of DC vaccines has been underway since before 2005. Currently, most tumor vaccines focus on enhancing specific T cell activation through the use of antigen-loaded, adjuvant-enhancing, and engineered DCs to induce robust, specific immunity. However, innate immunity may also play a crucial role in tumor immunotherapy. The innate immune system is the body's first line of defense against pathogen invasion and infection. It recognizes pathogen-associated molecular patterns (PAMPs) shared by different pathogens, such as flagellin, lipopolysaccharide, and viral nucleic acids, through a variety of pattern recognition receptors (PRRs). PRRs primarily include Toll-like receptors (TLRs), RIG-I-like helicase receptors (RLHs), NOD-like receptors (NLRs), and cGAS-STING. Secondly, innate immune cells such as natural killer cells (NKs) can be activated to directly lyse tumor cells or secrete cytokines and chemokines to inhibit tumor cell proliferation. Therefore, designing innate + specific "immune co-activation" tumor vaccines is of great significance for inducing a strong immune response.

[0004] Although DCs, as the most important and powerful professional antigen-presenting cells, play a key role in inducing immunity, in reality, DC infiltration in the tumor microenvironment is low and most are non-functional. Therefore, cultivating and expanding autologous DCs loaded with tumor antigens in vitro to produce large quantities of DC tumor vaccines is an effective way to enhance the body's anti-tumor immune response. However, DC cells themselves are complex in composition, contain a large amount of content, have a very limited loading capacity, and are difficult to store, which seriously restricts their application development as carriers and clinical translation.

[0005] Exosomes (Exos) are lipid bilayer vesicles with a diameter of 40 nm to 160 nm. They are widely distributed in various body fluids and are secreted by almost all living cells, including stem cells, immune cells, and tumor cells. Exos have the same topological structure as cells and are rich in proteins, lipids, nucleic acids, and sugar complexes. Exos contain a series of membrane-associated high-order oligomeric protein complexes that show significant molecular heterogeneity and are produced by budding on the plasma membrane and endosomal membranes. After release, Exos can be taken up by distal or neighboring cells, thereby exerting intercellular communication functions. This intercellular vesicle trafficking pathway plays an important role in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. Exos have a stable phospholipid bilayer structure, which protects their contents in the extracellular environment for a long time without degradation or dilution. DC-derived exosomes (DE) present a variety of DC-related cytoplasmic proteins on their outer surface, participating in a variety of immune-related biological effects and also possessing efficient immune activation functions: (1) HSP70 protein family members on the DE membrane surface together with the HSP90 family on the membrane surface form the immunogenicity of DE and participate in the activation of tumor-related immune cells; (2) DE surface is rich in ICAM-1, MHC-I class molecules, and CD86, which can induce T cell activation and immune response. As a "cell-free" carrier, DE not only inherits the immune activation ability of parent DCs, but also has higher safety and stability, is more conducive to drug delivery, and can be stored for a long time without losing immunotherapy activity; and as an inert vesicle, DE is resistant to the regulation of tumor-related factors compared to DC, and can overcome tumor-mediated immunosuppression. However, the construction of vaccines using DE as a carrier alone still has problems such as short systemic circulation time and limited in vivo stability, which is not conducive to the occurrence of long-term immune activation of vaccines.

[0006] Therefore, there are currently no reports on how to utilize exosomes derived from dendritic cells and recombinantly construct innate + specific "immune co-activation" tumor vaccines to induce potent immunity. Summary of the Invention

[0007] Purpose of the Invention: To address the above-mentioned technical problems, the present invention aims to provide a recombinant biomimetic anti-tumor nanovaccine with "immune co-activation," as well as its preparation method and application. This biomimetic anti-tumor nanovaccine retains all the physical and chemical properties of exosomes derived from endogenous immune cells and contains a large number of hallmark immune signature proteins derived from parental cells. It can be used as a cell-free alternative to DC vaccines for the initial construction of tumor vaccine systems. Furthermore, antigen hybrid peptides are loaded onto the surface of the exosomes to further enhance their specific immune activation strength; ginsenosides are selected to reconstitute the exosomes and further extend their in vivo circulation time. Furthermore, the hydrophilic space within the carrier is fully utilized to load innate immune-activating components. This addresses the issues of existing dendritic vaccines, such as high cost, difficult storage, and limited immune activation, achieving the dual efficacy of directly killing tumors and inducing a powerful immune response.

[0008] Technical solution: In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows:

[0009] A "immune co-activation" recombinant biomimetic anti-tumor nanovaccine, which is composed of a recombinant exosome carrier shell modified with an immune-activating functional hybrid peptide and a nano-form drug core. The recombinant exosomes are formed by recombinant processing of ginsenosides and exosomes.

[0010] Preferably, the immune activation functional hybrid peptide is composed of an antigenic peptide and a biological peptide, and the antigenic peptide is selected from OVA 257-264 、OVA 323-339 、gp 10025-33 , MUC1 glycosylated peptides, but not limited to these substances; the biological peptide is selected from one or more of α-Mel, iRGD, NAP, Angiopep-2, Tf, but not limited to these substances.

[0011] Preferably, the exosomes are derived from the patient's autologous cells, and are selected from one or more of tumor cells, dendritic cells, macrophages, neutrophils, and mesenchymal stem cells, but are not limited to these substances.

[0012] Preferably, the ginsenoside is selected from one or more of 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rh2, and 20(R)-Rh2.

[0013] Preferably, the recombinant exosomes are obtained by ultrasonically fragmenting the extracted exosomes and then co-incubating with ginsenosides; preferably, the exosomes are calculated based on protein content, the ginsenosides are calculated based on mass, and the mass ratio of the recombinant exosomes to the ginsenosides is 1:(1-2); preferably, the ultrasonic fragmentation conditions are as follows: the ultrasonic fragmentation time is 5-10 min, the power is 250-350 W, and the ultrasonic frequency is 1-2 s / 4-6 s on / off; the co-incubation is incubated at 32-40 ° C for 0.5-2 h.

[0014] Preferably, the drug contained in the nano-form drug core is an innate immune activating substance, and the nano-form drug core is selected from nanoparticles formed by one or more of manganese-albumin complexes, manganese colloids, CpG, and aluminum hydroxide, but is not limited to these substances.

[0015] Preferably, the particle size of the "immune co-activation" biomimetic anti-tumor nanovaccine is 100-140 nm.

[0016] The preparation method of the "immune co-activation" biomimetic anti-tumor nanovaccine comprises the following steps:

[0017] (1) Extracting and isolating exosomes, ultrasonically disrupting them, and then incubating them with ginsenosides to obtain recombinant exosomes;

[0018] (2) preparing drug cores in nanoform;

[0019] (3) mixing the recombinant exosomes obtained in step (1) with the drug core obtained in step (2), and incubating them together to obtain a drug-loaded exosome solution;

[0020] (4) Add the biopeptide solution to the drug-loaded exosome solution under stirring and emulsify for 1-2 hours, then add the immune activation functional hybrid peptide solution and emulsify for 1-2 hours;

[0021] (5) Filter to remove free drugs.

[0022] Preferably, in step (1), exosomes are extracted and separated by combining iodixanol density gradient centrifugation and ultracentrifugation.

[0023] Preferably, in step (3), the drug core is calculated based on the drug mass, and the exosomes are calculated based on the protein content, and the mass ratio of the drug to the recombinant exosomes is 1:(1-4).

[0024] Preferably, in step (3), the recombinant exosomes obtained in step (1) are mixed with the drug core obtained in step (2), and an electroporation medium solution is first added, followed by electric shock, and then incubated at 32-40° C. for 0.5-2 h.

[0025] Preferably, in step (4), when the solid mass is in mg, the liquid mass is in mL, the drug-loaded exosome solution is 1 part, and the immune-activating functional hybrid peptide is 1 to 2 parts.

[0026] Preferably, the incubation time in step (4) is 60 to 120 minutes.

[0027] The present invention also provides the use of the aforementioned "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in the preparation of anti-tumor drugs. Tumors include lung cancer, breast cancer, glioma, ovarian cancer, gastric cancer, colorectal cancer, osteosarcoma, prostate cancer, cervical cancer, malignant pleural effusion, or melanoma. During use, the nanovaccine is dissolved in physiological saline, phosphate buffer, or 5% glucose solution and administered intravenously, intramuscularly, or orally. The nanovaccine self-assembles to load antigenic peptides and efficiently load innate immune activators, increasing the bioavailability and in vivo transport efficiency of both substances and improving immunogenicity, thereby awakening the innate and specific immune systems to jointly inhibit tumor growth.

[0028] The present invention extracts DC cells from diseased animals and induces the differentiation of exosomes, thereby retaining the immune activation function of DC as antigen-presenting cells. Exosomes, as cell-free biomimetic carriers, overcome key problems of traditional DC vaccines, such as complicated production processes, harsh storage conditions, and difficulty in large-scale production.

[0029] This invention uses DC-derived exosomes as vaccine carriers and reconstitutes them, replacing their original cholesterol component with ginsenosides to further prolong their systemic circulation time. Furthermore, leveraging their bilayer lipid structure, they can load hydrophilic drugs while also modifying their surface with amphiphilic hybrid peptides, fully utilizing their drug-carrying capacity for efficient drug loading. The amphiphilic hybrid peptides impart direct tumor-killing and specific immune-activating properties to the nanovaccine, while the hydrophilic drugs impart innate immune-activating properties to the nanovaccine, effectively overcoming the shortcomings of traditional vaccines, such as poor in vivo stability, short in vivo circulation, low in vivo activity, and limited immune response.

[0030] This invention utilizes exosomes derived from the patient's own cells, antigenic peptides, and biological peptides to construct a potent immune-activating biomimetic nanovaccine through electroporation and ultrasonic fragmentation. Simultaneously, it loads innate immune-activating components, potentially overcoming the problems of traditional nanovaccines, such as poor in vivo stability, effective bioavailability, low response efficiency, and insufficient immune activation. It offers the following advantages:

[0031] (1) Highly endogenous: Exosomes are produced by autologous cells and can completely retain the physical and chemical characteristics and biological properties of the parent cells, which is highly endogenous;

[0032] (2) Biosafety: The high endogenous nature gives the biomimetic nanovaccine high biocompatibility, is easily biodegradable, has low toxic side effects, and has good safety effects;

[0033] (3) Strong drug loading capacity: The double-layer phospholipid structure of exosomes is fully utilized. The amphiphilic phospholipid layer is modified with amphiphilic functional peptides, and the hydrophilic space of the core is loaded with hydrophilic drugs, which fully utilizes the drug loading space of the carrier and improves the drug loading capacity and encapsulation efficiency;

[0034] (4) Strong penetrating ability: Nanovaccine particles are in the nanometer range and can easily penetrate multiple layers of tissue and various physiological barriers to reach the lesion area. The degree of tumor infiltration is high, laying the foundation for its full efficacy;

[0035] (5) High immunogenicity: Exosomes derived from the autologous cells of diseased animals are used as carriers, which endow the exosomes with high immunogenicity. After modification with antigen hybrid peptides, their immune activation ability is further improved, laying the foundation for activating strong in vivo immunity;

[0036] (6) High in vivo activity: The use of nanocarriers to deliver immune-activating substances overcomes the problems of easy decomposition and poor efficacy of traditional vaccine preparations. The use of recombinant biomimetic nanocarriers to deliver immune-activating substances further overcomes the problem of traditional nanoformulations being easily recognized and quickly cleared by the endothelial reticular system, thereby improving the bioavailability of drugs and enhancing the in vivo activity and utilization efficiency of vaccines.

[0037] (7) Complementary therapeutic mechanisms and multiple treatment methods: By modifying antigen hybrid peptides and delivering immune activating substances, the effects of direct tumor killing and co-activation of innate immunity and specific immunity "double immunity" can be achieved, completely breaking the bottleneck problems of limited efficacy of nano vaccines, low patient responsiveness, and poor protection efficiency, fully improving the tumor treatment effect and producing long-term protection.

[0038] The "immune co-activation" recombinant biomimetic anti-tumor nanovaccine provided by the present invention can achieve efficient in vivo delivery of specific immune-activating polypeptide antigen peptides and innate immune activators. The nanoformulation is highly endogenous, biosafe, strongly penetrable, and highly immunogenic. The efficient loading and delivery of two physical and chemical property drugs further fully utilizes the advantages of the diversified drug loading methods of such biomimetic drug carriers, and successfully constructs an innate + specific "immune co-activation" recombinant biomimetic nanovaccine, which has high in vivo activity, in vivo stability, long in vivo circulation time, and tumor treatment efficiency. It is in line with the development trend of tumor treatment, meets the clinical needs of tumor treatment, and provides a model for the efficient and precise treatment of tumors and the construction of a long-term protection platform. It has broad application prospects and clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The expression of surface characteristic proteins of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3 1.1;

[0040] Figure 2 This is a graph showing the particle size and zeta potential of the recombinant biomimetic anti-tumor nanovaccine "Immune Co-activation" in Example 3, 1.2;

[0041] Figure 3 This is an investigation on the storage stability of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.3;

[0042] Figure 4 This is an in vitro hemolytic study of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.4;

[0043] Figure 5 This is an investigation of DC cell uptake of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.5.1;

[0044] Figure 6 This is an investigation of tumor cell uptake of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.5.2;

[0045] Figure 7 This is an investigation of the cellular uptake dependence of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.5.3;

[0046] Figure 8 This is an investigation of the cytotoxicity of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.5.4;

[0047] Figure 9 This is an investigation of the DC activation ability of the recombinant biomimetic anti-tumor nanovaccine "immune co-activation" in Example 3, 1.6;

[0048] Figure 10 This is an investigation of the T cell activation ability of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.7;

[0049] Figure 11 This is a serum pharmacokinetics study of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine in Example 3, 1.8. DETAILED DESCRIPTION

[0050] The present invention will be further described by the following examples. These examples are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention. The invention will be further described below in conjunction with the accompanying drawings and examples.

[0051] Example 1: Preparation of BSA / Mn nanocores

[0052] Weigh 20 mg of bovine serum albumin (BSA), add 2 mL of ultrapure water, and vortex to dissolve to prepare a 10 mg / mL BSA solution. Transfer the solution to a vial. Weigh 15 mg of MnCl2·4H2O, add 1 mL of ultrapure water, and vortex to dissolve to prepare a 15 mg / mL stock solution. Pipette 100 μL of MnCl2·4H2O solution dropwise into the vial under magnetic stirring. After 5 minutes, add an appropriate amount of 1 M NaOH to adjust the pH to 11. Stir magnetically at room temperature for 2 hours. Remove free drug by centrifugation using a 30 kDa ultrafiltration tube (4500 rpm, 8 minutes). Resuspend the diluted BSA / Mn core solution in PBS to volume and store at 4°C until ready for use.

[0053] Example 2: Preparation of DC-derived exosomes loaded with BSA / Mn and modified with antigen hybrid peptides

[0054] GL261 cells in the logarithmic growth phase were resuspended in PBS buffer (pH 7.4) (6×10 7 / mL) and stored on ice. C57BL / 6 mice were anesthetized by intraperitoneal injection of 1% pentobarbital (40mg / kg) and fixed with a small animal brain stereotaxic instrument. The mouse head was disinfected with 75% alcohol, and a longitudinal incision was made along the intersection of the medial canthus line and the sagittal midline of the head. A small amount of 3% hydrogen peroxide was applied to a cotton swab to destroy the surface tissue of the skull and separate and expose the skull. The periosteum was peeled off to expose the anterior fontanelle. The needle insertion site was located 1.5mm behind the anterior fontanelle and 2mm to the right, and a bone drill was used to drill a hole at this point. A microsyringe was used to draw up the GL261 cell suspension (30×10 4 The needle was inserted to a depth of 3 mm and injected slowly for 5 minutes. After the cell suspension was completely injected into the mouse brain, the injection was allowed to rest for 5 minutes. The syringe was then slowly withdrawn and the scalp was sutured with medical sutures. After 8 days of tumor growth, the mouse was sacrificed by cervical dislocation and the brain was dissected to confirm the success of the tumor implantation.

[0055] Sterilize surgical instruments with 75% alcohol and sterilize them under ultraviolet irradiation in a clean bench for 30 minutes. Soak the mice in 75% ethanol for 5 minutes to disinfect them. Pour PBS into a sterile 12-well plate. Aseptically remove the femur and tibia from the mouse in a clean bench, transfer them to a PBS plate, rinse to remove hair, and use a 1 mL needle to aspirate the culture medium. Repeatedly flush the bone marrow cavity until the bone turns white. Centrifuge the obtained bone marrow cells (1200 rpm, 5 minutes) and discard the supernatant. Add red blood cell lysis buffer and let stand for 3 minutes. Add 1640 complete culture medium to terminate digestion and centrifuge (1200 rpm, 5 minutes). Discard the supernatant. Adjust the cell concentration to 106 / mL and resuspend in fresh culture medium for culture. Change half the culture medium every two days. After 6 days, gently blow on the wall to collect the loosely attached cells as dendritic cells. Centrifuge (1200 rpm, 5 minutes), discard the supernatant, and resuspend in RPMI 1640 serum-free culture medium. After 48 h, the supernatant was collected by centrifugation (1200 r / min, 5 min), and an exosome extraction reagent was added to collect exosomes to obtain DE solution.

[0056] Weigh 1.5 mg of Rh2 and dissolve it in 100 μL of DMSO. The DE solution obtained in "Example 2" was ultrasonically disrupted in an ice bath, and then the Rh2 solution was added dropwise (exosomes were calculated by protein content, Rh2 was calculated by mass, and the mass ratio of exosomes to ginsenosides was 1:1). Incubate at 37°C for 1 hour to obtain RDE. The BSA / Mn solution prepared in "Example 1" was added dropwise to RDE. After electroporation, the mixture was incubated (37°C for 1 hour) to prepare RDE / Mn (drug core calculated by drug mass, exosomes calculated by protein content, and the mass ratio of Mn to recombinant exosomes was 1:2). α-Mel solution was added under magnetic stirring and emulsified for 1 hour. Then, α-Mel-OVA solution (purchased from Guoping Pharmaceuticals) was added and emulsified for 1 hour (RDE / Mn = 1 part, α-Mel = 1 part, α-Mel = 1 part, α-Mel-OVA = 1 part). The free drug was removed by centrifugation through a 0.8 μm filter and 30 kDa ultrafiltration tube (5000 rpm for 10 minutes). The product was then resuspended in PBS to obtain MORDE / Mn. MODE / Mn (DE not recombined with Rh2), MDE / Mn (DE not recombined with Rh2 and the hybrid peptide did not contain an antigenic peptide), and DE / Mn (DE not recombined with Rh2 and not modified with a hybrid peptide) were prepared using the same method.

[0057] Example 3: Investigation of the properties of “immune co-activation” biomimetic anti-tumor nanovaccine

[0058] 1.1 Expression of MORDE / Mn surface characteristic proteins

[0059] Take the DE and MORDE obtained in Example 2, resuspend the exosomes in PBS, add CD81, CD63, CD9 and negative control in order according to the amount of protein, incubate at room temperature in the dark for 15 minutes, transfer to flow tube, and measure by flow cytometry. The results are as follows Figure 1 As shown, CD9, CD63 and CD81 in DE and MORDE were all positively expressed, proving that the extracted vesicles were exosomes and that peptide modification did not affect the expression of exosome characteristic proteins.

[0060] 1.2 Investigation of MORDE / Mn particle size and potential

[0061] The particle size and Zeta potential of the preparation were measured using a Malvern laser particle size analyzer. Figure 2 The zeta potentials of DE / Mn, MDE / Mn, MODE / Mn, and MORDE / Mn were all lower than that of BSA / Mn, and the particle sizes gradually increased, indicating that the antigen hybrid peptide was successfully modified and Mn was successfully loaded.

[0062] 1.3 Investigation of the storage stability of MORDE / Mn

[0063] MORDE / Mn was prepared and stored in PBS at 4°C in the dark. After preparation, the particle size, PDI and Zeta potential were measured every day to investigate its storage stability. MODE / Mn was prepared and stored in PBS at 4°C in the dark. After preparation, the particle size, PDI and Zeta potential were measured every day to investigate its storage stability. Figure 3 The experimental results show that compared with MODE / Mn, MORDE / Mn has better stability after the introduction of Rh2, and both PDI and particle size show significant changes within 5 days.

[0064] 1.4 In vitro hemolytic activity of MORDE / Mn

[0065] Mel, α-Mel, and α-Mel-OVA solutions were dissolved in ultrapure water to prepare a maximum concentration of 50 μM. The prepared MORDE solution was diluted with PBS buffer to a maximum α-Mel concentration of 50 μM. The three sample solutions were serially diluted to seven concentrations: 50 μM, 25 μM, 12.5 μM, 7.5 μM, 3.75 μM, 1.875 μM, 0.9375 μM, and 0.46875 μM. Fresh mouse blood was collected and centrifuged at 5000 rpm for 6 minutes to remove plasma. The lower layer of blood cells was gently resuspended in saline. This process was repeated until the upper layer of liquid no longer showed red color. The blood cells were diluted to a 2% concentration and added to the drug-containing EP tube. The solution was incubated at 37°C for 3 hours. The supernatant was then centrifuged at 5000 rpm / min for 6 minutes. The supernatant was collected and added to a 96-well plate. The absorbance was measured at 540 nm.

[0066] Hemolysis rate (%) = (As-Ab) / (Ac-Ab) × 100%

[0067] As is the average absorbance value of the experimental wells, Ac is the average absorbance value of the Triton wells, and Ab is the absorbance value of the saline wells.

[0068] The experimental results are as follows Figure 4 As shown in the figure, after constructing MORDE, α-Mel was deeply buried in the phospholipid layer, shielding its positive charge and reducing its hemolytic activity, thus proving its hemolytic safety.

[0069] Investigation of cellular uptake of 1.5MORDE / Mn

[0070] 1.5.1 Investigation of DC Uptake of MORDE / Mn

[0071] Dissolve DiI in DMSO to prepare a 10 mM stock solution. Pipette 5 μL of DiI and mix with MORDE, and ultrasonicate for 5 minutes to obtain MORDE / DiI. Set up control groups: Mel Lipos / DiI, DE / DiI, MDE / DiI, MODE / DiI, and MORDE / DiI. Resuspend DC2.4 cells in RPMI 1640 complete medium (1×10 4 Cells were seeded in 96-well plates (100 μL per well) in triplicate, and incubated overnight at 37°C, 5% CO2. After cell attachment, the culture medium was removed and Mel Lipos / DiI, DE / DiI, MDE / DiI, MODE / DiI, and MORDE / DiI were added to the treatment wells, respectively. Serum-free medium was added to the blank control wells. After a 2-hour incubation, the drug solution was removed and the plates were washed three times with PBS. The PBS was aspirated and Hoechst staining solution was added for 15 minutes. The stain was then aspirated and the plates were washed three times with PBS. The plates were photographed under an inverted microscope (DiI: λEx / λEm = 549 / 565 nm).

[0072] Dissolve DiI in DMSO to prepare a 10 mM stock solution. Pipette 5 μL of DiI and mix with MORDE, and ultrasonicate for 5 minutes to obtain MORDE / DiI. Set up control groups: Mel Lipos / DiI, DE / DiI, MDE / DiI, MODE / DiI, and MORDE / DiI. Resuspend DC2.4 cells in RPMI 1640 complete medium (1×10 4Cells were seeded in 96-well plates (100 μL per well) in triplicate, and incubated overnight at 37°C, 5% CO2. After cell attachment, the plates were removed and the cells were treated with Mel Lipos / DiI, DE / DiI, MDE / DiI, MODE / DiI, and MORDE / DiI, respectively. Serum-free medium was added to the blank control wells. After a 2-hour incubation, the cells were removed and washed three times with PBS. DMSO was added to lyse the cells, and fluorescence intensity was measured using a microplate reader (DiI:DiI:λEx / λEm=549 / 565nm).

[0073] The experimental results are as follows Figure 5 As shown in the results, the exosomes DE secreted by BMDCs have the ability to target parental cells. After modification with α-Mel, the uptake ability of BMDCs is further improved.

[0074] 1.5.2 Investigation of GL261 Uptake Capacity and Mechanism of MORDE / Mn

[0075] Dissolve DiI in DMSO to prepare a 10mM stock solution. Pipette 5μL of DiI and mix it with MORDE. Ultrasonicate for 5 minutes to obtain MORDE / DiI. Take DC cells in the logarithmic growth phase and resuspend them in DMEM complete medium (1×10 4 GL261 cells were seeded with 100 μL of the solution per well (cells / mL) in 96-well plates, with three replicates per group. The cells were incubated at 37°C, 5% CO2 overnight. After the cells attached, the culture medium was removed and the dosing wells were added with DE / DiI, D4F-DE / DiI, MDE / DiI, MDE(-) / DiI, Rh2-DE / DiI, Rh2-DE(-) / DiI, MODE / DiI, and MORDE / DiI, respectively. MDE(-) / DiI was treated with D4F (0.5 μM) solution for 12 hours to saturate the SR-BI receptor; Rh2-DE(-) / DiI was treated with WZB-117 solution (40 μM) for 12 hours to saturate the glucose transporter. Serum-free medium was added to the blank control wells. After a 2-hour co-incubation, the drug solution was removed and the cells were washed three times with PBS buffer. PBS was removed by aspiration, and Hoechst staining solution was added for 15 min. The staining solution was removed by aspiration, and the sample was washed three times with PBS buffer. The sample was then photographed under an inverted microscope (DiI: λEx / λEm=549 / 565 nm).

[0076] Dissolve DiI in DMSO to prepare a 10mM stock solution. Pipette 5μL of DiI and mix it with MORDE. Ultrasonicate for 5 minutes to obtain MORDE / DiI. Take DC cells in the logarithmic growth phase and resuspend them in DMEM complete medium (1×10 4GL261 cells were seeded with 100 μL of the solution per well (cells / mL) in 96-well plates, with three replicates per group. The cells were incubated at 37°C, 5% CO2 overnight. After the cells attached, the culture medium was removed and the dosing wells were added with DE / DiI, D4F-DE / DiI, MDE / DiI, MDE(-) / DiI, Rh2-DE / DiI, Rh2-DE(-) / DiI, MODE / DiI, and MORDE / DiI, respectively. MDE(-) / DiI was treated with D4F (0.5 μM) solution for 12 hours to saturate the SR-BI receptor; Rh2-DE(-) / DiI was treated with WZB-117 solution (40 μM) for 12 hours to saturate the glucose transporter. Serum-free medium was added to the blank control wells. After a 2-hour co-incubation, the drug solution was removed and the cells were washed three times with PBS buffer. DMSO was added to dissolve the cells, and the fluorescence intensity was measured using a microplate reader (DiI: λEx / λEm=549 / 565 nm).

[0077] The experimental results are as follows Figure 6 As shown, uptake in the D4F-DE group was significantly higher than in the DE group, demonstrating that D4F-mediated SR-BI receptor binding can promote tumor cell uptake of DE. Uptake in the MDE group was higher than in the D4F-DE group. However, after SR-BI receptor saturation, uptake in the MDE(-) group decreased compared to the MDE group. This suggests that the membrane potential-mediated cell binding of melittin in α-Mel can significantly enhance tumor cell uptake of the agent. After saturation of glucose transporters, uptake of Rh2-DE significantly decreased, suggesting that glucose transporter-mediated endocytosis is also an important pathway for enhancing tumor cell uptake. In summary, the significantly higher cellular uptake of MORDE than that of DE is believed to be the result of the combined effects of membrane potential-mediated cell binding, SR-BI-mediated receptor binding, and glucose transporter-mediated endocytosis. Membrane potential-mediated cell binding may play a major role.

[0078] 1.5.3 Investigation of MORDE / Mn Uptake Dependence

[0079] GL261 cells and DC2.4 cells in the logarithmic growth phase were taken and resuspended in complete culture medium (1×10 4 Cells were seeded in 96-well plates (100 μL / well) in triplicate, with 100 μL per well. The cells were incubated overnight at 37°C, 5% CO2. After cell attachment, the medium was removed and the cells were incubated with MORDE solution. Cell uptake was monitored at 30, 60, 90, 120, and 150 min. At these time points, the solution was aspirated, the cells were washed three times with PBS, and DMSO was added to lyse the cells. Fluorescence intensity (DiI: λEx / λEm = 549 / 565 nm) was measured using a microplate reader.

[0080] The experimental results are as follows Figure 7As shown, the uptake of MODE by tumor cells was higher than that by BMDCs, and the uptake was time-dependent.

[0081] 1.5.4 Cytotoxicity study of MORDE / Mn

[0082] GL261 cells in the logarithmic growth phase were taken and resuspended in DMEM complete medium (1×10 4 Cells (cells / well) were seeded in a 96-well plate, with 100 μL per well. The cells were cultured overnight in a 37°C, 5% CO2 incubator. After cell attachment, the medium was removed by aspiration. 100 μL of serum-free medium was added to the control group, while Mel, Mel Lipos, DE / Mn, MDE, MODE, and MORDE were added to the drug-treated groups. After 12 hours of culture, the medium was removed by aspiration, and 100 μL of a LIVE / DEAD dye mixture (Calcium AM concentration: 2 μM, EthD-1 concentration: 4 μM) diluted in PBS was added to each well. The cells were incubated at 37°C for 20 minutes and observed under a fluorescence microscope.

[0083] DC2.4 cells were resuspended in RPMI 1640 complete medium (1×10 4 Cells were plated in 96-well plates (cells / mL) in triplicate, with 100 μL per well, and incubated overnight at 37°C, 5% CO2. After cell attachment, the medium was removed by aspiration, and 100 μL of serum-free medium was added to the control group. Mel, Mel Lipos, DE / Mn, MDE, MODE, and MORDE were added to the dosing wells. After 12 hours of incubation, the medium was removed by aspiration, and a LIVE / DEAD dye mixture (Calcium AM concentration: 2 μM, EthD-1 concentration: 4 μM) diluted in PBS was added to each well at 100 μL. The plates were incubated at 37°C for 20 minutes and observed under a fluorescence microscope.

[0084] The experimental results are as follows Figure 8 As shown, free Mel has a strong cell-lethal effect, while the positive charge of modified Mel is shielded, greatly reducing its cell-killing effect. Since GL261 cells take up a significantly higher amount of the preparation than DCs, their apoptosis degree is significantly greater than that of DCs.

[0085] 1.6 Investigation of DC cell activation ability of MORDE / Mn

[0086] GL261 cells in the logarithmic growth phase were taken and resuspended in complete culture medium (20×10 4The cells were seeded into 24-well plates (500 μL per well) and incubated in a 37°C, 5% CO2 incubator overnight. The culture medium was removed, 300 μL of serum-free culture medium was added to the control group, and BSA / Mn, DE / Mn, MDE / Mn, MODE / Mn, and MORDE / Mn were added to the dosing wells. The cells were incubated in a 37°C, 5% CO2 incubator for 24 hours, and the supernatant and cells were collected. Dendritic cells were extracted and resuspended in complete culture medium (20 × 10 4 Cells (cells / well) were seeded into 24-well plates, 500 μL per well. Cultured overnight in a 37°C, 5% CO2 incubator. Previously collected tumor supernatant and cells were added, cultured for 36 hours, and cells were harvested by centrifugation. CD11c, CD80, and CD86 markers were added and incubated in the dark for 15 minutes before flow cytometry analysis.

[0087] The experimental results are as follows Figure 9 As shown, after administration, the expression of CD80 and CD86 increased significantly. Compared to DE / Mn, MDE showed significantly higher expression, demonstrating that α-Mel has the ability to activate DCs. Furthermore, after the introduction of the antigen peptide OVA, DC maturation increased further, demonstrating that tumor-associated antigen peptides are crucial for DC activation and maturation.

[0088] 1.7 Investigation of T cell activation ability of MORDE / Mn

[0089] Following the procedure under "1.6", drug-stimulated DCs were co-incubated with mouse spleen total lymphocytes (DCs: total lymphocytes = 1:10) and the co-incubation continued for 36 hours. The cells in each well were then blown down and centrifuged (1000r / min, 5min), and the supernatant was collected for enzyme-linked immunosorbent assay to measure the expression of TNF-α, IL-6, IL-10, IFN-γ, and IL-12. The well plate was washed twice with PBS, the cell pellet was resuspended in 500μL PBS, 5μL flow cytometry antibodies (CD3, CD4, CD8a) were added, and the cells were stained in the dark for 15 minutes. The proportion of CD3+, CD4+, and CD8a+ cell subsets was analyzed by flow cytometry, and the results were analyzed using Kaluza software.

[0090] The experimental results are as follows Figure 10 As shown in the results, after administration, the expression of CD8+T cells increased significantly, among which the degree of T cell activation increased significantly after the introduction of tumor-associated antigen peptide OVA, proving that strong specific immunity was successfully induced; the expression levels of TNF-α, IL-6, IL-12 and IFN-γ increased significantly, and IL-10 decreased significantly, proving that the preparation has a significant immune activation effect and improves the immunosuppressive microenvironment.

[0091] 1.8 Serum pharmacokinetics of MORDE / Mn

[0092] C57BL / 6 mice were randomly divided into four groups: Lipos / DiI, Rh2-Lipos / DiI, DE / DiI, and MORDE / DiI. Drugs were administered via tail vein injection. Eyeballs were enucleated and blood was collected 5, 10, 15, 30, 1, 2, 4, 6, 8, 12, and 24 hours after administration. Blood was collected by centrifugation at 6000 rpm for 6 minutes, and serum was collected. Fluorescence intensity (DiI: λEx / λEm = 549 / 565 nm) was measured using a microplate reader.

[0093] The experimental results are as follows Figure 11 As shown, the experimental results showed no significant difference between MODE / DiI and DE / DiI. Compared with Lipos / DiI, Rh2-Lipos / DiI had a longer blood circulation time. The blood circulation time of MODE / DiI and DE / DiI was longer than that of Rh2-Lipos / DiI, suggesting that the endogenous carrier can further evade recognition by the endothelial reticulophagic system, extending the drug half-life. After modification with Rh2, MORDE / DiI had a longer half-life, which is believed to be the result of the combined action of Rh2 and DE.

Claims

1. An "immune co-activation" recombinant biomimetic anti-tumor nanovaccine, characterized in that: The nano vaccine is composed of a recombinant exosome carrier shell modified with an immune-activating functional hybrid peptide and a nano-sized drug core. The recombinant exosomes are obtained by ultrasonically fragmenting the extracted exosomes and then incubating them with ginsenosides. The exosomes are derived from dendritic cells, and the ginsenosides are selected from ginsenoside Rh2. The immune-activating functional hybrid peptide is composed of an antigenic peptide and a biological peptide, and the antigenic peptide is selected from OVA. 257-264 , the biological peptide is selected from α-Mel; the nano-form drug core is a BSA / Mn nano-core.

2. The "immune co-activation" recombinant biomimetic anti-tumor nanovaccine according to claim 1, characterized in that: The exosomes are calculated based on protein content, ginsenosides are calculated based on mass, and the mass ratio of the recombinant exosomes to ginsenosides is 1:(1-2); the ultrasonic fragmentation conditions are as follows: the ultrasonic fragmentation time is 5-10 minutes, the power is 250-350W, and the ultrasonic frequency is 1-2 seconds / 4-6 seconds on / off; the co-incubation is incubated at 32-40°C for 0.5-2 hours.

3. The method for preparing the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine according to claim 1, characterized in that: The steps include: (1) Extract and separate exosomes, disrupt them with ultrasound, and then incubate them with ginsenosides to obtain recombinant exosomes; (2) Preparation of nanoparticle drug cores; (3) mixing the recombinant exosomes obtained in step (1) with the drug core obtained in step (2), and incubating them together to obtain a drug-loaded exosome solution; (4) Under stirring, add the biopeptide solution to the drug-loaded exosome solution and emulsify for 1-2 h, then add the immune activation functional hybrid peptide solution and emulsify for 1-2 h; (5) Filter and remove free drugs to obtain the product.

4. The method for preparing the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine according to claim 3, characterized in that: In step (3), the drug core is measured by drug mass, the exosomes are measured by protein content, and the mass ratio of the drug to the recombinant exosomes is 1:(1-4); in step (4), when the solid mass is mg, the liquid mass is mL, 1 part of the drug-loaded exosome solution, and 1 to 2 parts of the immune-activating functional hybrid peptide; the emulsification time is 60 to 120 min.

5. Use of the "immune co-activation" recombinant biomimetic anti-tumor nanovaccine described in claim 1 in the preparation of anti-glioma drugs.

Images