Nano vaccine for treating HPV (human papilloma virus) infection related cervical lesions as well as preparation method and application of nano vaccine

Abstract

The invention discloses a nano vaccine for treating HPV (human papilloma virus) infection related cervical lesions as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. In order to solve the problem that the response rate of an existing cervical lesion treatment vaccine does not meet clinical requirements, the hybrid membrane with enhanced immunogenicity and improved safety is prepared by applying a membrane fusion technology and combining an autologous tumor cell membrane and an escherichia coli cytoplasmic membrane; meanwhile, PLGA nano particles loaded with HPV16 E5 / E6 / E7 specific CTL epitope long peptide are used as a delivery carrier, and the HM-NPs-LP nano vaccine co-delivering the hybrid membrane and the HPV16 specific antigen peptide is developed. According to the nano vaccine, tumor cells and HPV16 E5 / E6 / E7 specific CTL epitope long peptide are taken as antigens for immunotherapy, dual immune activation of broad spectrum and specific antigens can be realized, personalized multi-effect treatment capacity for HPV infection related cervical diseases is exerted, targeted killing of HPV infection and pathological cells is enhanced, pathological relapse is prevented, and the nano vaccine has good clinical application prospects. And a new strategy is provided for individualized precise immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanovaccine for treating HPV-related cervical lesions, its preparation method, and its application. Background Technology

[0002] Cervical cancer is the fourth leading cause of cancer death among women worldwide, and it is showing a significant trend towards affecting younger women. Persistent infection with high-risk human papillomavirus (HPV) is the core driving factor for the malignant transformation of cervical lesions. HPV infection in the female reproductive tract is relatively common, but about 90% of HPV infections can be cleared by the host's innate immunity within 1-2 years. However, 10% of women will experience persistent HPV infection, of which 1-4% may progress to cervical squamous intraepithelial lesions or invasive cervical carcinoma. The 10-15 years from HPV infection to precancerous lesions and finally to cervical cancer provides a good window for early intervention and treatment. Current treatments for HPV-related cervical lesions have many shortcomings. HPV prophylactic vaccines have extremely low viral clearance rates in infected individuals; patients with precancerous lesions face risks of recurrence, cervical insufficiency, and premature birth after surgery or ablation; the treatment strategy for cervical cancer is mainly surgery, supplemented by radiotherapy, chemotherapy, targeted therapy, and immunotherapy, but the prognosis for advanced cervical cancer is poor, with a 5-year survival rate of less than 20%, and it has varying degrees of side effects on surrounding healthy tissues. How to develop a comprehensive treatment strategy that covers all stages during this window of opportunity, and how to target and prevent key pathogenic factors of persistent HPV infection and cervical lesions to effectively improve patient prognosis, is an important area of ​​current research.

[0003] Immunotherapy, as an emerging cancer treatment approach, has brought new hope to the treatment of HPV-related cervical lesions. Immunotherapy aims to activate or enhance the body's immune response against tumors through cytokines, antibodies, vaccines, etc., achieving specific killing of tumor cells. This includes adoptive cell therapy, immune checkpoint blockade therapy, and tumor vaccines. HPV early proteins E6 and E7 are two commonly used targets for therapeutic vaccines. E6 and E7 proteins affect the host's tumor suppressor genes p53 and Rb, thereby leading to carcinogenesis. Currently, therapeutic vaccines under development (such as VGX-3100) are DNA vaccines targeting HPV16 / 18 E6 / E7. Their phase III clinical trial (NCT03721978) showed that it could achieve lesion regression and viral clearance in 27.6% of HSIL patients, but its response rate is still far from meeting clinical needs. The reasons may be as follows: most studies focus on enhancing specific T-cell immunity against HPV E6 and E7 antigens. However, other HPV viral antigens (such as E1, E2, and E5) are expressed in persistent HPV infection and precancerous lesions, so targeting only the two "classic" antigens E6 and E7 cannot completely inhibit the progression of cervical lesions. The high heterogeneity of advanced cervical cancer tumors means that vaccines based on single antigens may show different therapeutic effects in different cervical cancer patients, failing to achieve the expected anti-tumor efficacy. The immunogenicity of vaccines is insufficient. The dense matrix of solid tumors limits the penetration of immune cells, antibodies, and drugs, and the immunosuppressive microenvironment further weakens the effect of immunotherapy. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide a nanovaccine for treating HPV-related cervical lesions, its preparation method, and its application. Utilizing membrane fusion technology, a hybrid membrane with enhanced immunogenicity and improved safety is prepared by combining autologous tumor cell membranes and E. coli DH5α cytoplasmic membranes. Simultaneously, using PLGA nanoparticles loaded with HPV16 E5 / E6 / E7 specific CTL epitope long peptides as delivery carriers, an HM-NPs@LP nanovaccine co-delivering the hybrid membrane and HPV16 specific antigenic peptides was developed. To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides a method for preparing a nano-vaccine for treating HPV-related cervical lesions, comprising the following steps: Preparation of tumor cell membrane™ using autologous tumor cells; Preparation of bacterial cytoplasmic membrane EM; Hybrid membranes (HM) were synthesized using tumor cell membrane™ and bacterial cytoplasmic membrane EM. Preparation of PLGA nanoparticles NPs@LP loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes; The nanovaccine HM-NPs@LP was synthesized using hybrid membrane HM and PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes.

[0005] Furthermore, the tumor cells include HPV16-positive cervical precancerous lesion cells or cervical cancer cell lines.

[0006] Furthermore, the bacterial cytoplasmic membrane EM was prepared using Escherichia coli DH5α.

[0007] Furthermore, the specific process for preparing bacterial cytoplasmic membrane EM includes: Escherichia coli DH5α was cultured in LB medium until the OD600 absorbance of the bacterial suspension reached approximately 0.8-1.1. The culture was then terminated and the bacterial cells were collected as a precipitate. The cell walls of Escherichia coli DH5α bacteria were digested using bacterial cytoplasmic membrane extraction buffer I, and the precipitate was collected by centrifugation to obtain protoplasts. Protoplasts were digested using bacterial cytoplasmic membrane extraction buffer II, the supernatant was collected, and the precipitate was collected by centrifugation, which is the bacterial cytoplasmic membrane EM.

[0008] Furthermore, in the hybrid membrane HM, the protein mass ratio of tumor cell membrane TM to bacterial cytoplasmic membrane EM is 1:3.

[0009] Furthermore, the specific steps for preparing PLGA nanoparticles NPs@LP loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes include the following: HPV16 E5, E6 and E7 were linked together to form HPV16 E5 / E6 / E7 specific CTL epitope long peptides. PLGA powder was dissolved in dichloromethane as the oil phase; HPV16 specific antigen peptides were dissolved in deionized water as the aqueous phase. PLGA nanoparticles NPs@LP loaded with HPV16 E5 / E6 / E7 specific CTL epitopes were prepared by mixing the oil phase and the water phase at a volume ratio of 5:1 and then using a double emulsification method.

[0010] Furthermore, in the nanovaccine HM-NPs@LP, the mass ratio of PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes to hybrid membrane HM is 5:1.

[0011] On the other hand, the present invention provides a nanovaccine HM-NPs@LP for treating HPV infection-related cervical lesions prepared using the preparation method described above.

[0012] In another aspect, the present invention also provides the use of the aforementioned nanovaccine HM-NPs@LP in the preparation of a medicament for treating HPV infection-related cervical lesions.

[0013] Specifically, the HPV infection-related cervical lesions include cervical intraepithelial neoplasia, cervical carcinoma in situ, and invasive cervical cancer.

[0014] The beneficial effects of this invention are: 1. The nanovaccine HM-NPs@LP of the present invention utilizes membrane fusion technology to combine autologous tumor cell membranes and Escherichia coli DH5α cytoplasmic membranes to prepare a hybrid membrane HM with enhanced immunogenicity and improved safety. Simultaneously, PLGA nanoparticles loaded with HPV16 E5 / E6 / E7 specific CTL epitope long peptides (HPV16 specific antigenic peptides) are used as delivery carriers to synthesize HM-NPs@LP nanovaccine that simultaneously delivers the hybrid membrane HM and HPV16 specific antigenic peptides. Tumor cell membranes provide a personalized tumor antigen library, while bacterial cytoplasmic membranes act as natural immune adjuvants, removing pathogenic bacterial components and improving vaccine immunogenicity. HPV16-specific antigenic peptides cover the key E5 / E6 / E7 epitopes in cervical lesion progression, generating specific immune responses and tumor-killing effects against precancerous cervical lesions and cervical cancer, achieving dual immune activation with broad-spectrum and specific antigens. This more effectively stimulates specific cellular immune responses against persistent HPV infection and cervical lesions. PLGA nanoparticles, used as drug delivery carriers, enhance lymph node targeting effects, effectively activating the body's ability to kill diseased cells. The nanovaccine in this invention uses biodegradable PLGA nanoparticles, reducing potential systemic toxicity and making it suitable for the treatment of patients with persistent HPV infection and cervical intraepithelial neoplasia, as well as postoperative treatment and prevention of lesion recurrence in cervical cancer.

[0015] 2. Tumor cell membranes have low immunogenicity and contain immunosuppressive factors that cannot yet be quantified. The ratio of tumor-specific antigens and tumor-associated antigens that can truly generate an effective immune response is difficult to determine and enrich. Providing antigens solely through tumor cell membranes carries certain therapeutic risks. Therefore, this invention uses tumor cell membranes and HPV16-specific antigenic peptides as antigens for immunotherapy. The tumor cell membrane retains a broad spectrum of individualized tumor-associated antigens, while the HPV16-specific antigenic peptides generate specific immune responses and tumor-killing effects against precancerous cervical lesions and cervical cancer. The combination of these two methods can improve treatment efficacy more individually for different patients.

[0016] 3. Due to the heterogeneity of tumor cells, vaccines based on a single antigen may exhibit different therapeutic effects in different patients. Therefore, this invention synthesizes HPV16 E5 / E6 / E7 long peptides as HPV16 specific antigenic peptides, which can predict human leukocyte antigen (HLA) typing and more effectively stimulate specific cellular immune responses against persistent HPV infection and cervical lesions. This expands the application of nanovaccines to the treatment of HPV infection-related cervical lesions in different populations.

[0017] 4. This invention uses the Escherichia coli DH5α cytoplasmic membrane as an immune adjuvant, activating the body's innate immune response to exogenous danger signals. Furthermore, the bacterial cytoplasmic membrane is structurally separate from the cell wall, making it easier to obtain from bacteria, and it does not contain lipopolysaccharides or other cell wall components, reducing the risk of acute toxicity. Tumor cell membranes possess relative stability and a certain degree of fluidity. Forming a thermodynamically stable hybrid membrane by conformationally transforming tumor cell membranes and bacterial cytoplasmic membranes allows for the integration of properties from multiple biological membranes, providing a new research platform for tumor immunotherapy. Attached Figure Description

[0018] Figure 1 The results show the preparation and characterization of the nanovaccine HM-NPs@LP in this invention; In this diagram, a shows the preparation of the HM-NPs@LP nanovaccine; b shows the transmission electron microscopy (TEM) imaging results of PLGA NPs, TM-NPs, EM-NPs, and HM-NPs@LP nanovaccines; c shows the particle size distribution of PLGA NPs, TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP nanovaccines; d shows a statistical graph of particle sizes for each type of nanovaccine (sample size: 5); e shows the zeta potential of each type of nanovaccine; f shows a statistical graph of the zeta potential of each type of nanovaccine (sample size: 5); g shows the full-wavelength absorbance values ​​of PLGA NPs, long peptides, and NPs@LP (abbreviation: au, Absorbance unit); h shows the gel electrophoresis and Coomassie brilliant blue staining analysis results of EM-NPs, TM-NPs, and HM-NPs@LP nanovaccines; and i shows the Western blotting results of EM-NPs, TM-NPs, and HM-NPs@LP nanovaccines (abbreviation: ATP1A1). (Sodium-potassium ATPase); FtsZ, Filamentous temperature-sensitive protein Z.

[0019] Figure 2 The results show the optimized composition ratio of tumor cell membrane TM and bacterial cytoplasmic membrane EM components in the nanovaccine HM-NPs@LP of the present invention. Wherein, ac represents the expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the supernatant after mouse BMDCs were co-incubated with EM and TM membrane protein mixtures at specific protein ratios (3:1, 1:1, 1:3) and EM and TM for 24 hours; df represents the preparation of hybrid membranes HM using EM and TM at specific protein ratios (3:1, 1:1, 1:3), and the preparation of different HM-NPs nanovaccines. Various nanovaccines were co-cultured with BMDCs for 8 hours. The nanovaccines were labeled by adding Rhodamine B dye during the preparation of PLGA nanoparticles. The ability of BMDCs to take up nanovaccines was detected by flow cytometry. Wherein, d is a schematic diagram of flow cytometry, e is a schematic diagram of the uptake results, and f is a statistical graph of the percentage of BMDC uptake.

[0020] Figure 3 The results of stability testing of the HM-NPs@LP nanovaccine in this invention are shown. Wherein, a represents the particle size of HM-NPs@LP nanovaccine before and after freeze-drying and resuspended in PBS buffer; b represents the zeta potential of HM-NPs@LP nanovaccine before and after freeze-drying and resuspended in PBS buffer; cd represents the ability of BMDC to take up the nanovaccine by flow cytometry after co-culturing HM-NPs@LP nanovaccine with BMDC for 8 hours after freeze-drying and resuspending in PBS buffer; c is a schematic diagram of the uptake results; d is a statistical graph; eg represents the expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the supernatant after co-culturing HM-NPs@LP nanovaccine with BMDC for 24 hours after freeze-drying and resuspending in PBS buffer; h represents the particle size of TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP nanovaccine at different time points in PBS buffer; i represents the zeta potential of TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP nanovaccine at different time points in PBS buffer.

[0021] Figure 4 The results of biosafety testing of the HM-NPs@LP nanovaccine in this invention; Wherein, a represents the concentration of lipopolysaccharide in TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP nanovaccines; b and c represent the procedures and results of adding TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP nanovaccines to mouse erythrocytes, with PBS buffer as a negative control and Triton X-100 as a positive control, and evaluating the hemolysis ratio after 3 hours of treatment; d and e represent the procedures and results of adding HM-NPs@LP nanovaccines of different concentration gradients to mouse erythrocytes, with PBS buffer as a negative control and Triton X-100 as a positive control, and evaluating the hemolysis ratio after 3 hours of treatment.

[0022] Figure 5 The results of the study on the promotion of antigen presentation and activation of in vitro BMDCs by the nano-vaccine HM-NPs@LP in this invention; In this diagram, a is the flowchart of the in vitro immune activation experiment of the nanovaccine; b and c are the flow cytometry analysis of the rhodamine B fluorescence intensity of BMDCs and the percentage of each nanovaccine taken up by BMDCs after 24 hours of co-culturing of various rhodamine B fluorescently labeled nanovaccines with mouse BMDCs; d is the Western blot analysis of the expression levels of Toll-like receptors (including TLR1, TLR2, TLR4, and TLR6) and NF-κB after 24 hours of co-culturing of various nanovaccines with BMDCs; e is the immunofluorescence image of various rhodamine B fluorescently labeled nanovaccines after 24 hours of co-culturing with mouse BMDCs, with blue representing Hoechst 33342 staining of BMDC nuclei and red representing rhodamine B-labeled nanoparticles (NPs@RhB). B (Rhodamine B); fh represents the expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the supernatant after 24 hours of co-culturing various nanovaccines with BMDCs; ik represents the expression of co-stimulatory factors CD80 and CD86 and MHC-II of BMDCs detected by flow cytometry after 24 hours of co-culturing of various nanovaccines with BMDCs; l represents the co-localization immunofluorescence imaging of Mix NPs+LP and HM-NPs@LP nanovaccines after 24 hours of co-culturing with BMDCs: blue fluorescence (nucleus, DAPI); red fluorescence (EM); green fluorescence (TM); m represents the co-localization correlation analysis of TM and EM components in the immunofluorescence imaging in l.

[0023] Figure 6 The results of this study show that the HM-NPs@LP nanovaccine in this invention accumulates in the inguinal lymph nodes of mice, promotes DC maturation and activates splenic T cells; In the figures, a is a schematic diagram of the in vivo experiment, where i represents obtaining tumor tissue from mice through surgery and then preparing tumor cell membrane TM, and ii represents a schematic diagram of small animal in vivo imaging after the nano-vaccines are injected into mice; b and c are schematic diagrams and statistical graphs of the distribution of the nano-vaccines in mice after subcutaneous injection of DiR fluorescent dye-labeled nano-vaccines or saline into the back of mice, respectively; d and e are the results of in vitro small animal imaging and statistical graphs of fluorescence intensity after 24 hours of injection of various fluorescent dye-labeled nano-vaccines into mice and separation of the inguinal lymph nodes; f and g are the results of in vivo microscopy of mice after inoculation with DiO fluorescent dye-labeled nano-vaccines. The diagram shows the imaging results of the draining lymph nodes 48 hours after vaccination, captured by in vivo microscopy. Blue represents cell nuclei. h and i represent the expression of co-stimulatory factors CD80 and CD86 in DCs cells after mice were immunized subcutaneously three times with various nano-vaccines (once on days 1, 2, and 7, for a total of three immunizations). jl represents the expression of pro-inflammatory cytokines in the peripheral blood of mice after mice were immunized subcutaneously three times with various nano-vaccines (once on days 1, 2, and 7, for a total of three immunizations). m represents the number of immune spots in the spleen cells of mice after mice were immunized subcutaneously three times with various nano-vaccines (once on days 1, 2, and 7, for a total of three immunizations). IFN-γ ELISPOT was used to detect the number of immune spots after stimulation with long peptides or TM. Mock indicates no stimulation, PMA indicates the addition of PMA (phorbol ester) to positively stimulate T cells, and n is a statistical graph of the number of positive spots in m.

[0024] Figure 7 The T cells of the spleen of immunized mice in this invention and Results of cytotoxicity studies on tumor targets; Among them, a, b, and c represent TC-1 cells obtained by isolating immune cells from the spleen of mice after three consecutive subcutaneous immunizations with various nano-vaccines (on days 1, 2, and 7 respectively), co-culturing these immune cells with various tumor cells, and detecting them using the CCK-8 assay. Tumor cells), MC38 cells ( Mouse colon cancer cells) and U14 cells ( Changes in absorbance values ​​after co-culturing mouse cervical cancer cells.

[0025] Figure 8 This is the research result of the HM-NPs@LP nanovaccine in this invention inducing tumor regression in mice and improving the immunosuppressive tumor microenvironment; In this diagram, a is a flowchart of the animal experimental design; b is a macroscopic image of subcutaneous tumors in mice from each nanovaccine treatment group, obtained by dissection on day 31; c is representative in vivo images of mice from each nanovaccine treatment group at different time points using a small animal in vivo imaging system; d is the average growth curve of subcutaneous tumors in each nanovaccine treatment group; e is the individual tumor growth curve of mice in each nanovaccine group; f is the weight of subcutaneous tumors in each nanovaccine treatment group; g is the TUNEL apoptosis fluorescence detection and Ki67 immunohistochemical analysis results of subcutaneous tumors in each nanovaccine treatment group; h is a statistical graph of TUNEL apoptosis fluorescence detection in g; i is a statistical graph of Ki67 immunohistochemistry in g; j is the multicolor immunohistochemical staining results of subcutaneous tumors in each nanovaccine treatment group, labeling CD4 (shown as red fluorescence), CD8 (shown as pink fluorescence), FoxP3 (shown as yellow fluorescence), and PD-1 (shown as green fluorescence), while using DAPI dye to label cell nuclei with blue fluorescence, specifically showing Tex (CD8+). + PD-1 + ) and Treg (CD4) + FoxP3 + The staining results of ). Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0027] Example 1: Example 1 provides a method for preparing a nanovaccine for treating HPV-related cervical lesions. The reagents and materials used in this preparation method include: The hydroxyl-terminated poly(lactic-co-glycolic acid) copolymer OH-PLGA-OH 75 / 25 (Poly(Lactic-Co-Glycolic Acid), PLGA) was purchased from Jinan Daigang Bioengineering Co., Ltd. Escherichia coli (E. coli) DH5α chemocompetent cells were purchased from Qingke Biotechnology Co., Ltd. Neutral protease II, deoxyribonuclease I (Dnase I), and hyaluronidase were purchased from Beijing Solarbio Co., Ltd. Protein phosphatase inhibitor mixture, 1M Tris-HCl buffer (pH 8.0), 1M Tris-HCl buffer (pH 7.4), and Bis-Tris powder were purchased from Beijing Solarbio Co., Ltd. HBSS buffer (containing Ca+ and Mg+), collagenase I, and Coomassie brilliant blue ultrafast staining solution were purchased from Wuhan Saiwei Company. The E. coli LPS detection kit was purchased from Chondrex, Inc., USA. Lysozyme was purchased from Sigma-Aldrich, USA. Sodium cholate was purchased from Shanghai Yisheng Biotechnology Co., Ltd. The BCA rapid protein quantification kit was purchased from Wuhan Yacoin Biotechnology Co., Ltd. Triton X-100 and HPLC-grade dichloromethane were purchased from Shanghai Aladdin Company.

[0028] See attached document Figure 1 As shown in Figure a, in an embodiment of this application, the preparation method includes the following steps: Step 1: Prepare tumor cell membrane™ using autologous tumor cells; Female C57BL / 6 mice or HLA-A*02:01 transgenic mice aged 6-8 weeks were subcutaneously inoculated with mouse tumor cells until the tumor volume reached 300-500 mm. 3 Subsequently, the tumor tissue is separated. It should be noted that the tumor tissue in this invention can also be obtained directly from isolated mouse tumor cells or directly from the tumor tissue of a patient (human). The process of obtaining the patient's tumor tissue adopts the methods in the prior art. Any method that can obtain mouse tumor tissue or human tumor tissue is applicable to this invention.

[0029] Prepare tumor tissue lysis buffer (containing 1 mg / mL collagenase I, 2 mg / mL neutral protease II, 0.1 mg / mL hyaluronidase, and 0.25 mg / mL DNase I, dissolved in a solution containing Ca...) 2+ and Mg 2+ In HBSS buffer), and cell membrane separation buffer (containing 225 mM / L mannitol, 75 mM / L sucrose, 0.5% (wt / vol) BSA, 0.5 mM / L EGTA and 30 mM / L Tris-HCl buffer (pH 7.4), 1% (vol / vol) protease and phosphatase inhibitors).

[0030] The isolated tumor tissue was added to tumor tissue lysis buffer and digested into a single-cell suspension. Red blood cells were then lysed. The single cells were then sonicated in cell membrane separation buffer under ice bath conditions using an ultrasonic cell disruptor with 35% power for 5 minutes (3 seconds on, 5 seconds off). The cells were then centrifuged at 10,000 g for 20 minutes at 4°C, and the supernatant containing the cell membrane was collected. Further ultracentrifugation was performed at 120,000 g for 2 hours at 4°C. The final precipitate was the purified tumor cell membrane™. The membrane was washed and resuspended in 5 mM / L Bis-Tris buffer (pH 6.0), aliquoted, and stored at -80°C for later use. The protein content of the tumor cell membrane™ was determined using a BCA protein quantification kit to ensure the accuracy and reproducibility of subsequent experiments.

[0031] Step 2: Prepare bacterial cytoplasmic membrane EM; This invention uses *Escherichia coli* DH5α to prepare bacterial cytoplasmic membranes. Specifically, *E. coli* DH5α is inoculated into LB liquid medium (Lysogeny broth, used for culturing *E. coli*) and cultured at 200 rpm for 12 hours in a 37°C constant-temperature shaker. A small amount of the bacterial suspension is then spread onto LB solid medium and cultured again at 37°C in a biochemical incubator until colonies grow. Single colonies are picked and cultured again in 5 mL of LB liquid medium at 200 rpm for 12 hours. The bacterial suspension is collected and, after a 1:100 dilution, re-inoculated into fresh LB liquid medium and cultured at 37°C with shaking for 4-6 hours until the OD600 absorbance of the bacterial suspension reaches approximately 0.8-1.1. At this point, the culture is terminated, and the bacterial cell pellet is collected.

[0032] Prepare bacterial cytoplasmic membrane extraction buffer I (composed of 1 M / L sucrose, 0.2 M / L Tris-HCl buffer (pH 8.0), 5 mg / ml lysozyme, and deionized water) to digest the cell wall of Escherichia coli DH5α bacteria. Centrifuge at 3000 g for 20 min at 4℃ and collect the precipitate to obtain protoplasts (bacteria after removing the cell wall).

[0033] Prepare bacterial cytoplasmic membrane extraction buffer II (composed of 2% (vol / vol) Triton X-100, 50 mM Tris-HCl buffer (pH 8.0), 10 mM magnesium chloride, 10 μg / mL DNase I enzyme, and deionized water). Resuspend the protoplasts obtained in the previous step, incubate on a shaker at 4°C for 30 min, centrifuge at 3000 g at 4°C for 20 min, and collect the supernatant. Further centrifuge at 60000 g at 4°C for 1 hour, and collect the precipitate as the bacterial cytoplasmic membrane EM. Resuspend the obtained bacterial cytoplasmic membrane EM in PBS buffer, aliquot and store at -80°C for later use. The protein content in the bacterial cytoplasmic membrane EM was determined using a BCA protein quantification kit.

[0034] Step 3: Synthesize the hybrid membrane HM using tumor cell membrane TM and bacterial cytoplasmic membrane EM; Tumor cell membrane (TM) and bacterial cytoplasmic membrane (EM) were mixed at a protein ratio of 1:3, and then shaken and mixed in a constant temperature shaker at 37°C and 120 rpm for 15 min. The mixture of TM and EM was then sonicated in an ice bath using an ultrasonic cell disruptor with ultrasonic parameters of 30% power for 2 min (3s on, 5s off) to promote the mixing of HM. Using a nanoextruder with a filter membrane of 400 nm pore size, the mixture was repeatedly extruded through the filter membrane more than 13 times to obtain hybrid membrane (HM). The concentration of total protein in the hybrid membrane (HM) was determined using a BCA protein quantification kit.

[0035] Step 4: Prepare PLGA nanoparticles NPs@LP loaded with HPV16 E5 / E6 / E7 specific CTL epitopes and fluorescently labeled; This invention uses a double-emulsion method to prepare PLGA nanoparticles loaded with HPV16 E5 / E6 / E7 specific CTL epitope long peptides (hereinafter referred to as HPV16 specific antigen peptides).

[0036] Specifically, HPV16 E5 (LSVSTYTSL), E6 (NKPLCDLLI), and E7 (RAHYNIVTF) are linked together to form a long peptide of HPV16 E5 / E6 / E7 specific CTL epitopes (LSVSTYTSLAAYNKPLCDLLIAAYRAHYNIVTF).

[0037] Take 20 mg of PLGA powder and dissolve it in 1 mL of dichloromethane at a concentration of 10 mg / mL as the oil phase; dissolve the HPV16 specific antigen peptide in deionized water at a concentration of 1 mg / mL as the aqueous phase; during the dissolution process, sonication can be used to aid dissolution, with sonication conditions of 20% power (3s on, 5s off), and the time adjusted according to the dissolution effect.

[0038] Take 2 mL of the oil phase and mix it with 0.4 mL of the aqueous phase. Perform a first emulsification using an ultrasonic homogenizer in an ice bath at an amplitude of 30% for 3 min (3 s on, 5 s off). Add the mixture after the first emulsification to 4 mL of 1% (wt / vol) sodium cholate solution and perform a second emulsification using ultrasound in an ice bath at an amplitude of 35% for 6 min (3 s on, 5 s off). Collect the second emulsion and slowly add it dropwise to 20 mL of 0.5% (wt / vol) sodium cholate solution. Stir at room temperature for 30 min until the solution is relatively clear. Use a rotary evaporator for 15 min to remove dichloromethane from the solution. Centrifuge the resulting nanoparticle solution at 10,000 g for 15 min at 4 °C. The precipitate is the nanoparticle. Wash the nanoparticle once with deionized water and centrifuge a second time at 10,000 g for 15 min at 4 °C. Resuspend the nanoparticle in 200 μL of deionized water, aliquot, and store at -80 °C for later use.

[0039] Step 5: Synthesize the nanovaccine HM-NPs@LP using hybrid membrane HM and PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes.

[0040] The hybrid membrane HM was mixed with PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes, wherein the mass ratio of the PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes to the total protein in the hybrid membrane HM was 5:1. After mixing, the mixture was treated with an ultrasonic homogenizer in an ice bath at an amplitude of 30% for 10 min. Using a nanoextruder equipped with a filter membrane with a pore size of 200 nm, the mixture of the hybrid membrane HM and the PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes was repeatedly extruded through the filter membrane more than 13 times to prepare the nanovaccine HM-NPs@LP loaded with the hybrid membrane HM by physical means. The nanovaccine HM-NPs@LP was finally aliquoted and stored at -80 ℃ for later use.

[0041] Comparative example: In this embodiment, to compare and analyze with the nanovaccine HM-NPs@LP prepared in this invention, nanovaccines TM-NPs, EM-NPs, Mix NPs+LP, HM-NPs, TM-NPs@LP, and EM-NPs@LP were also prepared. The preparation methods were similar to those for HM-NPs@LP in Example 1. Tumor cell membrane TM, bacterial cytoplasmic membrane EM, or hybrid membrane HM were mixed with PLGA nanoparticles without HPV16 E5 / E6 / E7 specific CTL epitope long peptides. The mass ratio of nanoparticles to TM, EM, or HM protein was 5:1. After mixing, the mixture was treated with an ultrasonic disruptor in an ice bath at 30% amplitude for 10 min. Using a nanoextruder equipped with a 200 nm pore size filter membrane, the mixture was repeatedly extruded through the filter membrane more than 13 times, thereby preparing nanovaccines TM-NPs, EM-NPs, and HM-NPs respectively.

[0042] Nanovaccine TM-NPs and nanovaccine EM-NPs are mixed at a protein ratio of 1:3, and HPV16E5 / E6 / E7 specific CTL epitope long peptides are added to obtain nanovaccine Mix NPs+LP.

[0043] Tumor cell membrane TM or bacterial cytoplasmic membrane EM were mixed with PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes. The mass ratio of PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes to TM or EM protein was 5:1. After mixing, the mixture was treated with an ultrasonic homogenizer in an ice bath at an amplitude of 30% for 10 min. Using a nanoextruder equipped with a 200 nm pore size filter membrane, the mixture was repeatedly extruded through the filter membrane more than 13 times to prepare nanovaccines TM-NPs@LP and EM-NPs@LP. Finally, the various nanovaccines were aliquoted and stored at -80 °C for later use.

[0044] In order to perform fluorescent labeling on the various nanovaccines prepared in Example 1 and the comparative example, when preparing the oil phase in step 4, the fluorescent dye (such as Rhodamine B, IR780, DiO or DiR fluorescent dye) was dissolved in dichloromethane with PLGA at a concentration of 0.2 mg / mL as the oil phase (all other operations remain unchanged). In subsequent processes, attention should be paid to avoiding light throughout the process, and dark glass bottles should be used for preparation and dark EP tubes for storage.

[0045] Example 2: Example 2 characterizes the performance of various nanovaccines prepared in Example 1 and the comparative example.

[0046] The different nanovaccines prepared in Example 1 and the comparative example were characterized by transmission electron microscopy (TEM), DLS particle size analysis, zeta potential measurement, Western blotting, and Coomassie brilliant blue staining. The results are attached. Figure 1 As shown in bf. From the attached Figure 1 As can be seen from the TEM, various nano-vaccines exhibit a bilayer spherical structure (see attached image). Figure 1 (b) DLS particle size analysis showed that the average particle size of the nanovaccine HM-NPs@LP was approximately 177.33 ± 4.76 nm (see attached image). Figure 1 (c and d); ζ-potential measurement results show that the mean ζ-potential of the HM-NPs@LP nanovaccine is -20.2mV (see attached figures). Figure 1 (e and f).

[0047] To further demonstrate that the hybrid membrane HM in the HM-NPs@LP nanovaccine contains two membrane structures: TM and EM, this embodiment also investigated the characteristic proteins in the nanovaccines TM-NPs, EM-NPs, and HM-NPs@LP using Coomassie brilliant blue staining on SDS-PAGE gels and Western blotting experiments. The Coomassie brilliant blue staining results are attached. Figure 1 As shown in Figure h, the nano-vaccines EM-NPs and HM-NPs@LP exhibited similar bands in the 15-25 kDa range (see Figure h). Figure 1 (The yellow circle and yellow arrow in h) The nano-vaccine TM-NPs did not have this characteristic band, but in the 40-55 kDa range, the nano-vaccine TM-NPs and the nano-vaccine HM-NPs@LP showed similar bands.

[0048] The results of the Western blot experiment are attached. Figure 1 As shown in Figure i, filamentous temperature-sensitive protein Z (FtsZ) is a homolog of tubulin in prokaryotes and a key protein in bacterial cell division. Figure 1 As can be seen from i, the FtsZ protein is expressed on both the nanovaccines EM-NPs and HM-NPs@LP; while sodium-potassium ATPase (Na... + / K + –adenosine triphosphate (ATP1A1), an enzyme present in mammalian cell membranes, showed distinct bands in both the nanovaccine TM-NPs and the nanovaccine HM-NPs@LP.

[0049] Example 3: Example 3 compares the hybrid membranes (HM) synthesized from tumor cell membranes (TM) and bacterial cytoplasmic membranes (EM) with different protein mass ratios.

[0050] Tumor cell membrane (TM) and bacterial cytoplasmic membrane (EM) were mixed at protein ratios of 3:1, 1:1, and 1:3 to prepare HMs with different TM / EM protein ratios. These HMs with different protein ratios were co-incubated with primary mouse bone marrow-derived dendritic cells (BMDCs) for 24 hours. The expression levels of cytokines IL-6, IL-1β, and TNF-α in the cell supernatant were detected by ELISA to assess the activation level of BMDCs. The results are shown in the attached figures. Figure 2 As shown in a, b, and c, the comparison reveals that the activation of BMDCs is strongest when the protein mass ratio of EM to TM is 3:1.

[0051] Furthermore, hybrid membrane HM nanovaccines with different protein ratios were prepared using the preparation method in Example 1. These nanovaccines were then loaded with rhodamine B fluorescent dye using the method described in the comparative example. After incubation with mouse bone marrow-derived dendritic cells (BMDCs) for 8 hours, the uptake of BMDCs was detected by flow cytometry. The results are shown in the attached figure. Figure 2 As shown in df. The results showed that the nanovaccine constructed when the protein mass ratio of EM to TM was 3:1 had the highest proportion of uptake by BMDC.

[0052] Example 4: Example 4 investigates the stability of the nanovaccine HM-NPs@LP prepared in Example 1 of this invention.

[0053] The particle size and zeta potential of the HM-NPs@LP nanovaccine were measured before and after freeze-drying and resuspended in PBS buffer. The results are shown in the attached figure. Figure 3 As shown in Figures a and b, the results indicate that the re-dissolution and resuspension process before and after freeze-drying had no effect on the particle size and zeta potential of the HM-NPs@LP nanovaccine.

[0054] Furthermore, this embodiment also constructed nanovaccines loaded with rhodamine B fluorescent dye, both before and after freeze-drying and resuspended in PBS buffer. BMDC uptake was detected by flow cytometry, and the results are shown in the attached figure. Figure 3 As shown in Figures c and d, the levels of cytokines IL-6, IL-1β, and TNF-α in the cell supernatant were detected by ELISA to assess the activation level of BMDCs. The results are attached. Figure 3 As shown in Figure 1, the results indicate that the freeze-drying and resuspending processes had no effect on the function of the HM-NPs@LP nanovaccine.

[0055] In addition, this embodiment also continuously monitored the particle size and zeta potential of nano-vaccines TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP after resuspending in PBS buffer for 8 days. The results are shown in the attached figure. Figure 3 As shown in h and i, the results indicate that the particle size and potential of various nano-vaccines remained relatively stable during the observation period.

[0056] The above experimental results show that the nanovaccine HM-NPs@LP prepared in this invention has good stability, can withstand freeze-thaw processes, and can be stored in room temperature solution for a long time without significant degradation. The potential transformation of the nanovaccine HM-NPs@LP into clinical applications has good prospects.

[0057] Example 5: The HM-NPs@LP nanovaccine contains endothelial membrane (EM) derived from bacteria. During EM preparation, bacterial cell wall components, such as lipopolysaccharide (LPS), may have been contaminated. LPS is a component of the cell walls of many Gram-negative bacteria and can be released during bacterial lysis, acting as an endotoxin to exert toxic effects on the host. Therefore, this embodiment measured the LPS concentration in the TM-NPs, EM-NPs, HM-NPs, and HM-NPs@LP nanovaccines, and the results are attached. Figure 4 As shown in Figure a. The results showed that, compared with the control group (physiological saline without LPS) and the nano-vaccine TM-NPs without bacterial components, the LPS concentration of nano-vaccines EM-NPs, HM-NPs, and HM-NPs@LP did not show a significant increase.

[0058] Hemolysis experiments were conducted on mouse erythrocytes using different nano-vaccines; the results are shown in the attached figure. Figure 4 In comparison with the negative control group treated with PBS and the positive control group treated with Triton X-100, no hemolysis was observed in any of the various nano-vaccines, and no hemolysis was observed in the HM-NPs@LP nano-vaccines at different concentrations.

[0059] In summary, the HM-NPs@LP nanovaccine exhibits low endotoxin content, demonstrating good biocompatibility and safety, laying a foundation for its further clinical development.

[0060] Example 6: To further investigate the ability of the nanovaccine HM-NPs@LP prepared in this invention to present antigens and activate mouse BMDCs in vitro, this embodiment designed a corresponding in vitro immune activation experiment, as shown in the attached figure. Figure 5 As shown in Figure a. Five experimental groups and one control group were set up in the in vitro experiment. The experimental groups included nano-vaccines TM-NPs, EM-NPs, Mix NPs+LP, HM-NPs, and HM-NPs@LP.

[0061] First, this embodiment investigated the efficiency of various nanovaccines in uptake and antigen presentation by mouse BMDCs. Rhodamine B-labeled nanovaccines were co-cultured with mouse BMDCs for 24 hours, and BMDC uptake was detected by flow cytometry. The results are shown in the appendix. Figure 5 As shown in Figures b and c, the results indicate that the nanovaccine TM-NP was taken up by BMDCs with low efficiency, while the nanovaccines EM-NPs, Mix NPs+LP, HM-NPs, and HM-NPs@LP containing bacterial-derived components were taken up by BMDCs with significantly increased efficiency.

[0062] Furthermore, this embodiment also investigated pattern recognition receptor signaling in BMDCs using Western blot and immunofluorescence analysis. The results of the Western blot analysis are attached. Figure 5 As shown in d, from the appendix Figure 5 As shown in the figure, the HM-NPs@LP nanovaccine significantly upregulated the expression of TLR1, TLR2, TLR4, TLR6, and NF-κB. Immunofluorescence results are attached. Figure 5 As shown in e, from the appendix Figure 5 As can be seen from Figure e, the fluorescence intensity of co-localized BMDCs in the HM-NPs and HM-NPs@LP nanovaccines was significantly higher, while the in vitro uptake efficiency of the two hybrid membrane vaccines showed no significant difference. These results indicate that the HM-NPs@LP nanovaccines can be efficiently uptaken by mouse BMDCs in vitro and presented with antigens, providing a research basis for subsequent immune activation and tumor cell killing.

[0063] Next, this embodiment further investigated the ability of the HM-NPs@LP nanovaccine to promote the activation and maturation of BMDCs. After co-culturing various nanovaccines with mouse BMDCs for 24 hours, the concentration of pro-inflammatory cytokines in the supernatant was detected by ELISA, and the results are shown in the attached figure. Figure 5 As shown in fh, the results indicate that the HM-NPs@LP nanovaccine significantly promoted the secretion of pro-inflammatory factors IL-6, IL-1β, and TNF-α compared to other vaccine groups (except the HM-NPs group) (P<0.0001). Flow cytometry was used to detect CD80, CD86, and MHC-II positive CD11c. + Cell percentages, results are attached. Figure 5As shown in Figure ik, the results indicate that, compared with other nanovaccine groups, the HM-NPs@LP nanovaccine group showed CD80, CD86, and MHC-II positive CD11c. + The higher proportion of cells showed a stronger ability to promote the maturation of BMDCs (P<0.0001).

[0064] Furthermore, in this embodiment, the nanovaccines HM-NPs@LP and Mix NPs+LP were co-cultured with BMDCs to study the co-localization of TM and EM components in the HM-NPs@LP nanovaccines on BMDCs. The results are shown in the appendix. Figure 5 As shown in l, m. In the appendix Figure 5 In l and m, sodium-potassium ATPase labeled with green fluorescence was used as the marker protein of TM, and FtsZ labeled with red fluorescence was used to label EM components. The results showed that the colocalization ratio of the nanovaccine HM-NPs@LP was significantly higher than that of the nanovaccine MixNPs+LP group (P<0.0001).

[0065] Example 7: In this embodiment, the nanovaccine HM-NPs@LP was prepared using TM of mouse solid TC-1 tumors, and an in vivo imaging method for fluorescence tracking of the nanovaccine in mice was designed. The specific implementation process is shown in the attached figure. Figure 6 As shown in a. The DiR fluorescent dye-labeled nanovaccine HM-NPs@LP was injected subcutaneously into the back of mice. In vivo imaging results at different time points after injection are shown in the attached figure. Figure 6 As shown in Figures b and c, DiR signals gradually accumulated in the bilateral inguinal lymph nodes of mice over time, and quantitative results showed (see attached figures). Figure 6 (c) Peak intensity occurs approximately 48 hours after injection.

[0066] Furthermore, in this embodiment, nanovaccines TM-NPs (prepared using TM of mouse solid TC-1 tumors), EM-NPs, and HM-NPs@LP (prepared using TM of mouse solid TC-1 tumors) were subcutaneously injected into mice. The mice were then sacrificed, and the draining lymph nodes were rapidly separated and analyzed using in vivo fluorescence imaging. The results are shown in the attached figure. Figure 6 As shown in d and e, the results indicate that almost no fluorescence was observed in the lymph nodes of the nano-vaccine TM-NPs and nano-vaccine EM-NPs treatment groups, while the fluorescence intensity of the nano-vaccine HM-NPs@LP group was significantly higher than that of nano-vaccine TM-NPs, nano-vaccine EM-NPs and the control group (P<0.0001).

[0067] To more intuitively observe the accumulation of the HM-NPs@LP nanovaccine in lymph nodes, this example used DiO-labeled HM-NPs@LP nanovaccine to subcutaneously immunize mice. Forty-eight hours later, in vivo fluorescence microscopy was performed on the left inguinal lymph nodes. The results are shown in the attached figure. Figure 6 As shown in f and g, the results indicate that the nanovaccine HM-NPs@LP aggregates extensively around the cell nucleus within the lymph node.

[0068] To further verify the efficacy of the HM-NPs@LP nanovaccine in promoting DC maturation and activating antigen-specific immune responses in draining lymph nodes of mice, this embodiment also administered various nanovaccines derived from mouse solid tumors subcutaneously to the abdomen of mice three times consecutively on days 1, 3, and 7. Mice were sacrificed 24 hours after the last immunization, and single-cell suspensions were prepared from the inguinal draining lymph nodes. CD11c of CD80 and CD86 was detected by flow cytometry. + Cell percentages, results are attached. Figure 6 As shown in h and i, the results indicate that the proportions of CD80+ and CD86+ DCs were highest in the HM-NPs and HM-NPs@LP nanovaccines. ELISA was used to detect pro-inflammatory factors IL-6, IL-1β, and TNF-α in mouse serum; the results are shown in the attached figure. Figure 6 As shown in Figure 1, the results indicate that the nanovaccine HM-NPs@LP can significantly promote the expression of various pro-inflammatory cytokines.

[0069] To investigate the ability of the HM-NPs@LP nanovaccine to activate antigen-specific T cells in mice, spleens were isolated and splenic lymphocytes were extracted simultaneously with the sacrifice of mice. To assess the immune response of HPV16-specific antigen peptide-specific T cells, splenic lymphocytes were co-incubated with HPV16-specific antigen peptide and TM for 24 hours, respectively. The antigen-specific T cell immune level was then detected using an ELISApot assay. The results are attached. Figure 6 The values ​​of m, n, and o are shown in the figure. The results indicate that, under HPV16-specific antigen peptide stimulation, the nanovaccines HM-NPs@LP and EM-NPs@LP induced similarly high-frequency IFN-γ secretory T cells, while the HPV16-specific antigen peptide alone, TM-NPs@LP, and Mix NPs+LP showed only weak activity, and the peptide-free group maintained basal levels. When TM vesicles were used as the stimulation source, the HM-NPs and HM-NPs@LP nanovaccines showed strong responses, while TM-NPs and Mix NPs+LP only produced weak activation.

[0070] To further verify functional cytotoxicity, this embodiment also uses Cells and Cytotoxicity assays were performed on spleen cells from immunized mice using control cells. The results are shown in the attached figure. Figure 7 As shown in the figure. The results indicate that the T cells of mice in the HM-NPs@LP group exhibited the strongest antigen-specific killing activity and could selectively eliminate... Cells, and for It had no effect on MC38 cell targets.

[0071] In summary, the nanovaccine HM-NPs@LP effectively promoted the maturation of dendritic cells (DCs) and the activation of splenic lymphocytes by co-delivering tumor antigens (TM and long peptides) and EM adjuvants to lymph nodes, and induced the generation of a large number of HPV16 E5 / E6 / E7 and tumor cell membrane antigen-specific T cells.

[0072] Example 8: This embodiment also utilizes mouse solid TC-1 tumor TM to prepare nanovaccines HM-NPs@LP, TM-NPs, EM-NPs, HM-NPs, Mix NPs+LP, TM-NPs@LP, and EM-NPs@LP. (See attached...) Figure 8 As shown in a, 2 × 10⁶ cells were subcutaneously injected into the abdomen of 6-week-old female C57BL / 6 mice. 5 TC-1-luciferase cells in terms of cell volume, when the tumor volume reaches 50 mm. 3 Mice were immunized three times consecutively with 100 μL of various vaccines at 1 and 3-day intervals (days 10, 12, and 16). Each mouse received 100 μg of the vaccine, administered subcutaneously to the medial aspect of both hind limbs. Control mice received 100 μL of saline. After tumor cell inoculation, tumor size was measured and calculated using calipers, and tumor growth was continuously monitored using small animal IVIS. Results are shown in the attached figure. Figure 8As shown in the figure (bf). The results showed that the tumor volume in the control group continued to grow until it exceeded the ethically permissible maximum; the tumor volume in the peptide group (simple HPV16 E5 / E6 / E7 CTL long peptide) was significantly larger than that in the other nanovaccine groups; the nanovaccine Mix NPs+LP group showed poor tumor growth inhibition, and the tumor continued to grow rapidly; the nanovaccine HM-NPs group performed relatively well in inhibiting tumor growth, but the tumor-suppressing effect was unstable, and the tumor volume began to increase after approximately 26 days; finally, the nanovaccine HM-NPs@LP group exhibited the most significant tumor inhibition effect, with the tumor gradually shrinking or even disappearing. These results indicate that nanovaccines carrying TM, EM, and HPV16 long peptides can effectively elicit a strong tumor-suppressing response in mice, and compared to the nanovaccine group lacking the long peptide HM-NPs, the addition of the HPV16 long peptide enhanced the anti-tumor efficacy of the HM-NPs@LP nanovaccine.

[0073] The results of TUNEL apoptosis fluorescence detection are attached. Figure 8 As shown in the figure, the results indicate that the DNA damage rate was significantly increased in the HM-NPs@LP nanovaccine group. Figure 8 In the middle g, h). Ki67 immunohistochemical experiments, which are related to the proliferative activity, invasiveness, and poor prognosis of malignant tumors, confirmed ( Figure 8 In the HM-NPs@LP nanovaccine group (g, i), Ki67 expression was relatively low, reflecting a lower malignant proliferation capacity of tumor cells. Several vaccine groups that performed poorly in previous tumor suppression experiments showed lower TUNEL apoptosis fluorescence and higher Ki67 expression.

[0074] To visually demonstrate the local infiltration of various lymphocytes in the tumor microenvironment, this embodiment employs multicolor immunohistochemical staining to study CD4+ in the tumor microenvironment. + T, CD8 + The infiltration ratios of T, Tex, and Treg were determined. Tumor tissue sections from the control group and different nanovaccine treatment groups were labeled and stained with CD4, CD8, FoxP3, and PD-1, and stained with fluorescence. Tex (CD8+) was also stained. + PD-1 + (cells) and Treg (CD4) + FoxP3 + Cells were individually labeled and displayed, and the results are shown in the attached figure. Figure 8 As shown in j in the appendix. Figure 8 As can be seen from the data, the infiltration of various types of lymphocytes was generally less in the control group, especially CD8. +Low T cell infiltration, while PD-1 expression was relatively high; in the peptide group with poor tumor suppression ability, the nanovaccine Mix NPs+LP group, and the nanovaccine HM-NPs group with unstable inhibitory effect, although a higher proportion of CD8 was observed. + T cell infiltration was observed, but the expression levels of PD-1 and FoxP3 were also significantly increased, particularly Treg cells, which maintain immune tolerance. Figure 8 (white dashed arrow in j) and Tex (related to tumor immunotherapy resistance and T cell dysfunction) Figure 8 The proportion of CD8 (the solid white arrow in j) also increased significantly. In contrast, the proportion of CD8 in the HM-NPs@LP nanovaccine group was significantly higher. + T cell infiltration was significantly increased, and PD-1 and FoxP3 expression was low, while Tex and Treg were also difficult to observe.

[0075] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a nano-vaccine for treating cervical lesions associated with HPV infection, characterized in that, Includes the following steps: Preparation of tumor cell membrane™ using autologous tumor cells; Preparation of bacterial cytoplasmic membrane EM; Hybrid membranes (HM) were synthesized using tumor cell membrane™ and bacterial cytoplasmic membrane EM. Preparation of PLGA nanoparticles NPs@LP loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes; The nanovaccine HM-NPs@LP was synthesized using hybrid membrane HM and PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes.

2. The method of claim 1, wherein the method of preparing a nano-vaccine for treating cervical lesions associated with HPV infection is characterized by, The tumor cells include HPV16-positive cervical precancerous lesion cells or cervical cancer cell lines.

3. The method for preparing a nanovaccine for treating HPV-related cervical lesions according to claim 2, characterized in that, The bacterial cytoplasmic membrane EM was prepared using Escherichia coli DH5α.

4. The method for preparing a nanovaccine for treating HPV-related cervical lesions according to claim 3, characterized in that, The specific process for preparing bacterial cytoplasmic membrane EM includes: Escherichia coli DH5α was cultured in LB medium until the OD600 absorbance of the bacterial suspension reached approximately 0.8-1.

1. The culture was then terminated and the bacterial cells were collected as a precipitate. The cell walls of Escherichia coli DH5α bacteria were digested using bacterial cytoplasmic membrane extraction buffer I, and the precipitate was collected by centrifugation to obtain protoplasts. Protoplasts were digested using bacterial cytoplasmic membrane extraction buffer II, the supernatant was collected, and the precipitate was collected by centrifugation, which is the bacterial cytoplasmic membrane EM.

5. The method for preparing a nanovaccine for treating HPV-related cervical lesions according to claim 4, characterized in that, In the hybrid membrane HM, the protein mass ratio of tumor cell membrane TM to bacterial cytoplasmic membrane EM is 1:

3.

6. The method of claim 5, wherein the method is for preparing a nano-vaccine for treating cervical lesions associated with HPV infection. The specific steps for preparing PLGA nanoparticles NPs@LP loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes include the following: HPV16 E5, E6 and E7 were linked together to form HPV16 E5 / E6 / E7 specific CTL epitope long peptides. PLGA powder was dissolved in dichloromethane as the oil phase; HPV16 specific antigen peptides were dissolved in deionized water as the aqueous phase. PLGA nanoparticles NPs@LP loaded with HPV16 E5 / E6 / E7 specific CTL epitopes were prepared by mixing the oil phase and the water phase at a volume ratio of 5:1 and then using a double emulsification method.

7. The method for preparing a nanovaccine for treating HPV-related cervical lesions according to claim 6, characterized in that, In the nanovaccine HM-NPs@LP, the mass ratio of PLGA nanoparticles loaded with long peptides of HPV16 E5 / E6 / E7 specific CTL epitopes to hybrid membrane HM is 5:

1.

8. The HM-NPs@LP nanovaccine for treating HPV infection-related cervical lesions prepared by the preparation method according to any one of claims 1-7.

9. The use of the nanovaccine HM-NPs@LP as described in claim 8 in the preparation of a medicament for treating HPV infection-related cervical lesions.

10. The application according to claim 9, characterized in that: The HPV-related cervical lesions include cervical intraepithelial neoplasia, cervical carcinoma in situ, and invasive cervical cancer.

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