Pirazosin-loaded hybrid liposome nano-vesicle vaccine as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a lipidosome hybridized nano-vesicle vaccine loaded with piperazosin, a preparation method of the lipidosome hybridized nano-vesicle vaccine and application of the lipidosome hybridized nano-vesicle vaccine in preparation of anti-tumor drugs. The lipidosome hybrid nano-vesicle vaccine loaded with the prazosin, disclosed by the invention, can be used for inhibiting autophagy and inducing the death of immunogenic cells of tumors. The pirazosin-loaded liposome hybrid nano-vesicle vaccine can activate and promote the maturation of bone marrow-derived dendritic cells, the expression of costimulatory factors CD80 and CD86 is obviously increased, and the antigen presentation is better promoted. The lipidosome hybrid nano-vesicle vaccine loaded with the prazosin can promote infiltration of mature dendritic cells and T cells in tumor tissues, can remarkably inhibit tumor growth, can remodel a tumor microenvironment as a tumor vaccine, and effectively improves the immunotherapy effect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a hybrid liposome nanovesicle vaccine loaded with prazosin, and a preparation method and application thereof. Background Art

[0002] Tumor-derived nanovesilces (TNVs) are nanoscale tumor cell-derived vesicles obtained by extrusion. They are rich in tumor antigens and endogenous danger signals and have innate tumor targeting properties, which enable them to enter lymph nodes through tumor-draining lymphatic vessels and further induce adaptive immune responses mediated by antigen-presenting cells. Therefore, nanovesicle-based formulated vaccines, namely nanovesicle vaccines, play an important role in the field of cancer immunotherapy.

[0003] Prazosin (Prz) is a potential anti-tumor agent and autophagy inhibitor. It can not only directly kill tumor cells and induce immunogenic cell death (ICD) of tumor cells to improve immunogenicity, but also improve the antigen presentation function of DCs by inhibiting autophagy, thereby enhancing the tumor immune response mediated by cytotoxic T lymphocytes. Prazosin improves the efficiency of antigen cross-presentation through its potential autophagy inhibition effect. Its combined use with TNVs is expected to make up for the defect of insufficient antigen presentation ability of nanovesicle vaccines. However, when TNVs are used as nanocarriers to load prazosin drugs, it is difficult to achieve sufficient drug delivery to improve the tumor immune microenvironment due to its poor colloidal stability and low drug loading efficiency.

[0004] Liposome hybrid nanovesicle vaccine is a complex formed by fusing TNVs and liposomes (LIP) with similar lipid bilayers by extrusion. Liposome preparations such as Doxil® have successfully transitioned to clinical applications and obtained regulatory approval. They are widely used in the field of biology due to their standardized production process yield, easy surface modification and high drug loading efficiency. More importantly, given the amphiphilic properties of prazosin, prazosin drugs can be encapsulated in liposomes through active loading technology to improve the efficiency of drug delivery. Liposome hybrid nanovesicle vaccine can inherit the advantages of liposomes and cell-derived nanovesicles: prevent the loss of immunogenicity caused by the degradation of a single antigen; reduce the strong toxicity produced by the whole tumor lysate vaccine; have good drug loading capacity, and can improve the therapeutic effect by combining drugs and immune adjuvants. Liposome hybrid nanovesicle vaccine performs well in encapsulating drugs and actively improving the tumor microenvironment. It is suitable as a prazosin drug delivery platform to achieve synergistic effects with immunotherapy and improve tumor treatment. It is a very promising chemotherapy-cooperative immunotherapy platform.

[0005] Chinese patent document CN 108403658 A discloses the application of a cell vesicle preparation combined with low-dose radiotherapy in the preparation of a drug for treating tumors, wherein the cell vesicle preparation is derived from apoptotic tumor cells and encapsulates chemotherapeutic drugs, and the low-dose radiotherapy is a radiotherapy with a lower total dose of radiation for treating the tumor. Chinese patent document CN119215154 A discloses an engineered bionic nano dendritic cell vesicle, a melanoma vaccine, a preparation method and an application, which specifically belong to the field of biomedical technology. The engineered bionic nano dendritic cell vesicle of the present invention is loaded with an interferon gene stimulating factor agonist and an oxidized melanoma cell-derived full antigen engineered vesicle. However, there is no report on a hybrid liposome nano vesicle vaccine loaded with prazosin and its preparation method and application. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a liposome hybrid nanovesicle vaccine (Prz@LINV) loaded with prazosin (Prz), a preparation method thereof, and the application of the hybrid nanovesicle vaccine in anti-tumor treatment. Liposomes loaded with prazosin are prepared by ammonium sulfate gradient active drug loading technology, and further fused with TNVs by continuous extrusion. The prepared Prz@LINV nanovaccine has both efficient drug loading and delivery capabilities, excellent tumor targeting and immune activation properties.

[0007] The first aspect of the present invention provides a hybrid liposome nanovesicle vaccine loaded with prazosin, wherein the hybrid liposome nanovesicle vaccine loaded with prazosin is prepared by encapsulating prazosin in liposomes through an ammonium sulfate gradient active drug loading method to obtain liposomes loaded with prazosin, and then the liposomes loaded with prazosin are mixed and incubated with nanocell vesicles and then co-extruded to obtain a liposome hybrid nanovesicle vaccine loaded with prazosin.

[0008] Furthermore, the particle size of the liposome hybrid nanovesicle vaccine loaded with prazosin is 225 nm.

[0009] Furthermore, the drug loading rate of the prazosin-loaded liposome hybrid nanovesicle vaccine for prazosin is 60.41% (200 µg / ml).

[0010] The second aspect of the present invention provides a method for preparing the liposome hybrid nanovesicle vaccine loaded with prazosin as described above, comprising the following steps:

[0011] Step 1: The tumor cells are squeezed out through an extruder to remove cell debris and microcapsules, and finally purified and concentrated using a centrifugal filter to obtain nanocellular vesicles;

[0012] Furthermore, the tumor cells described in step one are colon cancer cells.

[0013] Step 2: preparing liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol and mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) by thin film hydration method;

[0014] Step 3, encapsulating prazosin in liposomes by an ammonium sulfate gradient active drug loading method to obtain prazosin-loaded liposomes;

[0015] Step 4: The liposomes loaded with prazosin are mixed with the nanocell vesicles, incubated, and then co-extruded to obtain the liposome hybrid nanovesicle vaccine loaded with prazosin.

[0016] The third aspect of the present invention provides a use of the liposome hybrid nanovesicle vaccine loaded with prazosin as described above in the preparation of anti-tumor drugs.

[0017] The liposome hybrid nanovesicle vaccine loaded with prazosin of the present invention can induce immunogenic cell death of tumors, activate dendritic cells and reshape the tumor immune microenvironment, thereby producing a good anti-tumor effect.

[0018] Furthermore, the tumor is colon cancer.

[0019] The advantages and beneficial effects of the present invention are:

[0020] 1. The present invention constructs liposome hybrid nanovesicles composed of liposomes and tumor cell vesicles, and loads prazosin drugs.

[0021] 2. The liposome hybrid nanovesicle vaccine loaded with prazosin described in the present invention can inhibit autophagy and induce immunogenic cell death of tumors.

[0022] 3. The liposome hybrid nanovesicle vaccine loaded with prazosin described in the present invention can activate and promote the maturation of bone marrow-derived dendritic cells, significantly increase the expression of co-stimulatory factors CD80 and CD86, and better promote antigen presentation.

[0023] 4. The liposome hybrid nanovesicle vaccine loaded with prazosin described in the present invention can promote the infiltration of mature dendritic cells and T cells in tumor tissues and can significantly inhibit tumor growth. As a tumor vaccine, it can reshape the tumor microenvironment and effectively improve the immunotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The structure, preparation process and mechanism of action of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 of the present invention are shown in Figure 1. Part A is the structure and preparation process, and Part B is the mechanism of action.

[0025] Figure 2 The scanning electron microscope images of comparative example 1, comparative example 2 and embodiment 1 in test example 1 of the present invention are shown. The left side shows the scanning electron microscope image of LIP, the middle shows the scanning electron microscope image of TNV, and the right side shows the scanning electron microscope image of LINV.

[0026] Figure 3 The particle size, Zeta potential distribution and polydispersity index (PDI) of Comparative Example 1, Comparative Example 2 and Example 1 in Test Example 2 of the present invention.

[0027] Figure 4 It is the drug loading amount of Example 1 and Comparative Example 3 in Test Example 3 of the present invention.

[0028] Figure 5 This is the in vitro release of Example 1 and Comparative Example 3 in Test Example 4 of the present invention under simulated normal physiological conditions.

[0029] Figure 6 The results are shown in Figure 5 for the in vitro uptake of tumor cells by Example 1 and Comparative Example 3. A is dye-labeled immunofluorescence staining, and B is the quantitative result of immunofluorescence staining.

[0030] Figure 7 This is an evaluation of the autophagy inhibition effect of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 in Test Example 6 of the present invention. Part A is an immunofluorescence staining of the alkalinity of lysosomes (scale bar: 100 μm). Part B is an immunoblot result analyzing the levels of p62 and LC3II / I in colon cancer cells after 24 h of drug treatment. Parts C and D are quantitative analyses of the expression of p62 and LC3II / I proteins using Image J software.

[0031] Figure 8 This is an evaluation of the effect of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 in Test Example 7 of the present invention on promoting dendritic cell maturation in vitro. The evaluation indicators are the cytokines TNF-α and IL-6 secreted by dendritic cells detected by the Elisa kit.

[0032] Fig. 9 This is an evaluation of the effect of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 in Test Example 7 of the present invention on promoting dendritic cell maturation in vitro. The evaluation index is the expression level of CD80 and CD86 on the surface of dendritic cells. Among them, Part A is the expression level of CD80 and CD86 on the surface of dendritic cells measured by flow cytometry, and the LPS group is the positive control group. Part B is a quantitative analysis of the flow cytometry analysis results using Flowjo software. Part C is a proportional analysis of dendritic cells expressing SIINFEKL-MHC I in mouse lymph nodes.

[0033] Fig.10 This is an evaluation of the effect of inducing tumor immunogenic cell death (ICD) in vivo by the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 of Test Example 8 of the present invention. Part A is the immunofluorescence staining of the ICD indicator Calreticulin (CRT) on the surface of tumor cells (scale bar: 100 μm), Part B is the quantitative result of immunofluorescence staining, Part C is the quantitative result of the ICD indicator Adenosine Triphosphate (ATP) detected by the Elisa kit, and Part D is the quantitative result of the ICD indicator High-mobility group box 1 (HMGB1) detected by the Elisa kit.

[0034] Fig.11 The figure is a schematic diagram of the process of establishing a tumor-bearing mouse model according to the present invention.

[0035] Fig.12 This is an evaluation of the immunotherapy effect of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 of Test Example 9 of the present invention on tumor-bearing mice. Part A is the result of the complete removal of the tumor after the immunotherapy animal experiment of the tumor-bearing mice. Part B is the change curve of the tumor weight after the tumor was inoculated in the mice of different treatment groups, and Part C is the change curve of the tumor volume after the tumor was inoculated in the mice of different treatment groups.

[0036] Fig.13 This is an evaluation of the effect of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Example 1 of Test Example 10 of the present invention on activating dendritic cell antigen presentation and regulating the immune microenvironment in tumor-bearing mice. Part A is an analysis of the proportion of dendritic cells expressing CD11c, CD80, and CD86 in mouse lymph nodes detected by flow cytometry, and Part C is an analysis of the proportion of dendritic cells expressing CD3 in mouse tumor tissue detected by flow cytometry. + CD8 + Analysis of the proportion of T lymphocytes in mice. Part D is the detection of CD8 in mouse tumor tissues by flow cytometry. + IFN-γ + Parts B, E, and F are quantitative analyses of the above flow cytometry analysis results using Flowjo software.

[0037] Fig.14 This is the biosafety evaluation of the hybrid liposome nanovesicle vaccine (Prz@LINV) loaded with prazosin in Test Example 11 of the present invention on tumor-bearing mice. The organ status of the heart, liver, spleen, lung, and kidney was evaluated by hematoxylin-eosin (H&E) staining to evaluate the in vivo safety. DETAILED DESCRIPTION

[0038] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0039] Embodiment 1:

[0040] A liposome hybrid nanovesicle vaccine loaded with prazosin and a preparation method thereof, specifically comprising the following steps:

[0041] (1) Step 1: Preparation of tumor nanocellular vesicles: Culture colon cancer cells MC38 to reach 70% - 90% confluence, digest the cells in a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant; resuspend the cells in phosphate-buffered saline (PBS) (pH 7.4) and adjust the cell density to 1 × 10 6 cells; the cell suspension was sequentially extruded 15 times through 5 and 1 μm polycarbonate membrane filters using a micro-extruder; the final extruded sample (1 mL) was centrifuged at 10000 g for 10 min at room temperature to remove cell debris and microcapsules; the extruded sample was concentrated using a 100 kDa centrifugal filter at 1000 g for 15 min at room temperature with PBS (pH 7.4), and the purified exosome mimics were separated and purified, and then concentrated using a 100 kDa centrifugal filter at room temperature with PBS (pH 7.4) for 15 min to obtain tumor nanocellular vesicles (TNVs).

[0042] (2) Step 2, preparation of liposomes: Purchase various lipids required for the experiment and prepare lipid solutions at the following concentrations: prepare DOPC (MW=786) in chloroform to make a 10 mg / ml solution; prepare DC-Chol (MW=386.65) in chloroform to make a 10 mg / ml solution; prepare mPEG2000-DSPE (MW=2000) in chloroform to make a 10 mg / ml solution. DOPC, cholesterol and mPEG2000-DSPE were prepared by thin film dispersion method at a molar ratio of 4:4:1, i.e. 1.56 mg, 0.77 mg and 1.00 mg of DOPC, cholesterol and mPEG2000-DSPE were weighed and dissolved in 3 mL of chloroform, and then the solvent was evaporated in a rotary evaporator at 45°C using a round flask; then it was hydrated with 2 mL of 300 mM ammonium sulfate solution, and then vortexed and ultrasonicated for 10 min; then, the lipid suspension was extruded 15 times through a 0.2 μm polycarbonate membrane filter using a micro extruder; finally, free ammonium sulfate was removed using a dialysis bag (1000MWCO) in a pH 7.4 10 mM HEPES solution to obtain uniform liposomes (LIP).

[0043] (3) Step 3, preparation of prazosin-loaded liposomes: Weigh 1000 μg of Prz and dissolve it in 1 mL of PBS by ultrasonic dissolution. Then, add 1000 μg / mL of Prz solution to the prepared LIP solution to make the final concentration of 500 μg / mL. Incubate on a shaker at 45°C for 30 min. The unbound Prz is dialyzed through a dialysis bag (3500 MWCO) against a pH 7.4 10 mM HEPES buffer solution to obtain prazosin-loaded liposomes (Prz@LIP).

[0044] (4) Step 4: Preparation of prazosin-loaded liposome hybrid nanovesicle vaccine: The synthesized prazosin-loaded liposome (Prz@LIP) was mixed with tumor nanovesicles (TNV), incubated at 37°C for 1 h, and the resulting mixture was co-extruded 12 times through a 0.2 μm polycarbonate membrane to obtain a prazosin-loaded liposome hybrid nanovesicle vaccine (Prz@LINV).

[0045] Comparative Example 1:

[0046] Comparative Example 1 is to prepare tumor nanocellular vesicles (TNV) according to the same process as step 1 of Example 1.

[0047] Comparative Example 2:

[0048] Comparative Example 2 is to prepare liposomes (LIP) according to the same process as step 2 of Example 1.

[0049] Comparative Example 3:

[0050] Comparative Example 3 is to prepare prazosin-loaded liposomes (Prz@LIP) according to the same process as step 3 of Example 1.

[0051] Comparative Example 4:

[0052] Comparative Example 4 is to prepare a free prazosin drug solution (F-Prz) by weighing 1000 μg of Prz and dissolving it in 1 mL of PBS by ultrasonic dissolution.

[0053] Test Example 1:

[0054] The specific steps for morphological detection of nanomaterials are as follows:

[0055] The materials of Comparative Example 1, Comparative Example 2 and Example 1 were dissolved in deionized water with a concentration of 0.5 mg / ml; the samples were dripped onto the copper mesh, dried at room temperature, and 2% phosphotungstic acid solution was dripped, and dyed for 2 min. The excess dye solution was absorbed by absorbent paper, and the excess phosphotungstic acid dye solution was absorbed, and then observed with a transmission electron microscope. The results are as follows: Figure 1 shown.

[0056] Depend on Figure 2 It can be seen that transmission electron microscopy shows that the nanovaccine prepared by the present invention is spherical, evenly distributed, regular in morphology, and has a particle size of about 225 nm.

[0057] Test Example 2:

[0058] The specific steps for testing the particle size, Zeta potential distribution and polydispersity index (PDI) of nanomaterials are as follows:

[0059] The materials prepared in Example 1 and Comparative Examples 1 and 2 were diluted with 1 ml of deionized water, with a volume ratio of 1:4 to the appropriate concentration, and ultrasonically bathed for 30 min to increase the dispersibility of the nanoparticles in water. Then, the nanoparticles were placed in a sample cell and their particle size, Zeta potential distribution, and polydispersity index (PDI) were measured using a Malvern light scattering instrument. The results are shown in Figure 2. Figure 3 shown.

[0060] Depend on Figure 3 It can be seen that the particle size of the nanovaccine prepared by the present invention is 225.0 ± 4.9 nm, the zeta potential is -19.57 ± 0.65 mV, and the polydispersity index (PDI) is 0.16 ± 0.019.

[0061] Test Example 3:

[0062] The specific steps for drug loading detection of nanomaterials are as follows:

[0063] Take 10 μL of the materials prepared in Example 1 and Comparative Example 3, 80 μL of cell lysis buffer (CLB), add 10 μL of 1% Triton X-100, and then shake to mix thoroughly. Take 60 μL of the mixed solution, mix it with 240 μL of CLB, and then prepare 3 5-fold dilution samples. Add the above 4 concentration gradient samples (200 μL per well) to a 96-well microplate. The CLB solution was used as a blank control, and the fluorescence intensity was measured at an excitation wavelength of 340 nm and an emission wavelength of 380 nm. The drug loading was calculated based on the measured fluorescence intensity and the linear regression equation. The results are as follows: Figure 4 shown.

[0064] Depend on Figure 4 It can be seen that the drug loading of the nanovaccine prepared by the present invention is 65.68% ± 2.27% (200 μg / mL).

[0065] Test Example 4:

[0066] The specific steps for in vitro release testing of nanomaterials are as follows:

[0067] 1 mL of the materials prepared in Example 1 and Comparative Example 3 were respectively placed in dialysis bags (1000 MWCO), and the dialysis bags were placed in 30 mL of pH 7.4 PBS and 30 mL of pH 6.5 PBS release media, respectively, and incubated in a constant temperature shaker (37°C, 120 rpm). Samples were taken at fixed time points of 1h, 2h, 4h, 6h, 8h, 12h, and 24h (three parallel samples at each time point), and the sampling volume was 1 mL. Then 1 mL of fresh release medium was added, and the measurement was performed on an ELISA instrument, and the cumulative release amount of the two was calculated by fluorescence intensity. The results are shown in Figure 2. Figure 5 shown.

[0068] Depend on Figure 5 It can be seen that the nanovaccine prepared by the present invention has a good and stable sustained release effect under normal physiological conditions, and weakly acidic conditions will increase its release efficiency.

[0069] Test Example 5:

[0070] The specific steps for testing the in vitro uptake effect of nanomaterials are as follows:

[0071] Colon cancer cells in the logarithmic growth phase were inoculated into a 6-well culture plate and the cell density was adjusted to 3×10 5 / well, and then put the culture plate into a constant temperature incubator at 37℃ for 24h. After 24h, replace with fresh culture medium, and then add appropriate concentrations of red fluorescent dye-stained drug-loaded liposomes (DiI-Prz@LIP) and drug-loaded liposome hybrid nanovesicles (DiI-Prz@LINV) according to the cytotoxicity experiment. Then put the culture plate into a CO2 constant temperature incubator and incubate for 6h, discard the old culture medium containing the drug, wash the cells three times with PBS buffer, and then use 4% paraformaldehyde at room temperature for 10 min to fix the cells, then discard the paraformaldehyde and wash twice with PBS buffer, and use DAPI staining solution at room temperature in the dark for 10 min to stain the cell nucleus. Finally, wash twice with PBS buffer and observe under a fluorescence microscope. The results are as follows Figure 6 shown.

[0072] Depend on Figure 6 It can be seen that colon cancer cells take up both drug-loaded liposomes and drug-loaded liposome hybrid nanovesicles, and the uptake of drug-loaded liposome hybrid nanovesicles is significantly greater than that of drug-loaded liposomes. This shows that the nanovaccine prepared by the present invention has tumor targeting ability, and the targeting ability is significantly enhanced.

[0073] Test Example 6:

[0074] The specific steps for evaluating the in vitro cell autophagy inhibition effect of nanomaterials are as follows:

[0075] (1) Fluorescent probe staining method to detect lysosomal alkalinization level of tumor cells

[0076] Colon cancer cells in the logarithmic growth phase were inoculated into a 6-well culture plate, and the cell density was adjusted to 3×105 cells / well. The culture plate was then placed in a constant temperature incubator at 37°C for 24 h. When the cells grew to an appropriate abundance, they were washed once with PBS and grouped into blank control (PBS), comparative example 1 (TNV), comparative example 3 (Prz@LIP), comparative example 4 (F-Prz), and Example 1 (Prz@LINV). Each group was dosed with a drug-containing culture medium concentration of 6.3 μg / mL. 24 hours after administration, the drug-containing culture medium was removed, and 1 μM 37°C preheated lysosomal green fluorescent probe (LysoSensor Green DND-189) working solution was added and incubated for 30 min. The probe working solution was then replaced with fresh culture medium, fixed with 4% paraformaldehyde, and observed under a fluorescence microscope. The results are as follows. Figure 7 As shown in A.

[0077] (2) Western blotting method to detect the expression of LC3-I / II and p62 autophagy-related proteins in tumor cells

[0078] Colon cancer cells in the logarithmic growth phase were inoculated into a 6-well culture plate and the cell density was adjusted to 3×10 5 / well, and then put the culture plate into a constant temperature incubator at 37°C for 24 h. When the cells grow to an appropriate abundance, wash them once with PBS and group them into blank control (PBS), comparative example 1 (TNV), comparative example 3 (Prz@LIP), comparative example 4 (F-Prz), and Example 1 (Prz@LINV). Each group was dosed with a drug-containing culture medium concentration of 6.3 μg / mL. 24 hours after administration, the drug-containing culture medium was removed, and protein lysis buffer was added at 4°C to extract cell proteins. After quantification by BCA, Western blot experiments were performed to detect the expression of LC3-I / II and p62 autophagy-related proteins. The results are shown in the following table. Figure 7 As shown in BD.

[0079] Depend on Figure 7 It can be seen that the nanovaccine prepared by the present invention can effectively inhibit the autophagy level of tumor cells by alkalizing lysosomes and increasing the permeability of lysosomal membranes.

[0080] Test Example 7:

[0081] The specific steps for evaluating the effect of nanomaterials on promoting dendritic cell maturation in vitro are as follows:

[0082] (1) Elisa method to detect the levels of cytokines TNF-α and IL-6 secreted by dendritic cells

[0083] Dendritic cells in the logarithmic growth phase were inoculated into 6-well culture plates and the cell density was adjusted to 3×105 The culture plate was then placed in a constant temperature incubator at 37°C for 24 h. The dosing grouping, concentration and time were the same as those in Test Example 6. After 24 h, the supernatant was collected and the concentrations of TNF-α and IL-6 were measured using a TNF-α detection kit and an IL-6 detection kit, respectively. The results are shown in Figure 8 shown.

[0084] (2) Detection of CD80 and CD86 expression levels on the surface of dendritic cells by flow cytometry

[0085] Dendritic cells in the logarithmic growth phase were inoculated into 6-well culture plates and the cell density was adjusted to 3×10 5 / well, and then place the culture plate in a constant temperature incubator at 37°C for 24 hours. The dosing group, concentration and time are consistent with those in Test Example 6. After 24 hours, the cells are digested, and the digested single cell suspension is washed with phosphate buffer, and then the antibodies against CD80 and CD86 molecules and dendritic cells are incubated, and then the anti-CD16 / 32 antibody and DC are incubated and Fc blocked, and finally the expression of CD80 and CD86 molecules of dendritic cells is detected by flow cytometry.

[0086] The results are as follows Fig. 9 shown.

[0087] Depend on Figure 8 , Fig. 9 It can be seen that the nanovaccine prepared by the present invention significantly upregulated the expression levels of CD80 and CD86 on the surface of dendritic cells, increased the secretion of IL-6 and TNF-α, and can effectively promote the maturation of dendritic cells and activate the anti-tumor immune system.

[0088] Test Example 8:

[0089] The specific steps for evaluating the in vivo induction of tumor immunogenic cell death (ICD) by nanomaterials are as follows:

[0090] (1) Immunofluorescence staining method to detect ICD indicator CRT

[0091] Colon cancer cells in the logarithmic growth phase were inoculated into a 6-well culture plate and the cell density was adjusted to 3×10 5 / well, and then put the culture plate into a constant temperature incubator at 37℃ and culture for 24h. The dosing group, concentration and time are consistent with test example 6. After incubation for 24h, wash once with PBS, fix with 4% paraformaldehyde for 30 min, treat with 0.5% Triton-100 + 1% normal goat serum (Normal Goat Serum, NGS) diluted with PBS on ice for 10min, block with 1% NGS for 10min, add ICD marker CRT antibody and incubate at 4℃ overnight. The next day, add secondary antibody in the dark at room temperature for 1h. After washing with PBS, stain with DAPI for 10 min and observe under a fluorescence microscope. The results are as follows Fig.10 As shown in AB.

[0092] (2) Elisa method to detect ICD indicators ATP and HMGB1

[0093] Colon cancer cells in the logarithmic growth phase were inoculated into a 6-well culture plate and the cell density was adjusted to 3×10 5 Each well was incubated at 37°C for 24 hours. The dosing group, concentration and time were the same as those in Test Example 6. After 24 hours of incubation, the supernatant was collected and the concentrations of ATP and HMGB1 were determined by Elisa method using ATP detection kit and HMGB1 kit, respectively. The results are shown in Table 1. Fig.10 CD shown.

[0094] Depend on Fig.10 It can be seen that the nanovaccine prepared by the present invention can stimulate tumor cells to express CRT, secrete HMGB1 and ATP, and induce the ICD effect of tumor cells. Compared with Comparative Examples 3 and 4, the nanovaccine prepared by the present invention has a stronger ICD induction effect. This may be attributed to the stronger uptake ability of tumor cells for Example 1 (Prz@LINV), thereby achieving effective internalization of prazosin (F-Prz).

[0095] Test Example 9:

[0096] The specific steps for evaluating the immunotherapy effect of nanomaterials on tumor-bearing mice are as follows:

[0097] (1) Establishment of tumor-bearing mouse model

[0098] Colon cancer cells MC38 were digested and resuspended at 5×10 5 / mouse subcutaneously injected into the right back of 6-8 week old C57BL6 mice and incubated for about 7 days (dose: 0.1 mL / mouse). When the tumor volume reached 50 mm 3The mice were randomly divided into 5 groups (5 mice / group), including blank control (PBS), comparative example 1 (TNV), comparative example 3 (Prz@LIP), comparative example 4 (F-Prz), and example 1 (Prz@LINV) to start the experiment. Fig.11 shown.

[0099] (2) Tumor inhibition experiment of Prz@LINV in tumor-bearing mice

[0100] The drug was administered by intratumoral injection. Comparative Example 1 was administered at a protein concentration of 25 μg / mouse. Comparative Example 3, Comparative Example 4 and Example 1 were administered at a dose of 1.5 mg / kg. The negative control group was injected with PBS. Each mouse was administered 0.1 mL. The drug was administered once every 3 days for 5 consecutive times. The weight and tumor volume of the mice were measured every 2 days. The mice were killed after 15 days, the tumors were removed and weighed, and the tumor size of each group was observed. Tumor volume = (a 2 b) / 2. (Note: a represents the width of the tumor; b represents the length of the tumor). The results are as follows Fig.12 shown.

[0101] Depend on Fig.11 , Fig.12 It can be seen that compared with the blank control and comparative example 1 group, the tumor volumes of comparative example 3, comparative example 4 and example 1 groups were reduced, and the tumor volume of example 1 group was the smallest among all groups. This shows that the nano vaccine prepared by the present invention has a good anti-tumor effect, which may be due to the improvement of antigen presentation ability to T cells by inducing ICD, improving the immunogenicity of tumors, and exerting T cell immune response.

[0102] Test Example 10:

[0103] The specific steps for evaluating the in vivo activation of dendritic cell antigen presentation and regulation of immune microenvironment by nanomaterials are as follows:

[0104] Weigh each group of tumors and tumor-draining lymph nodes, cut them into small pieces and place them in a 15 mL centrifuge tube. Add 5-10 mL of type IV collagenase digestion solution (collagenase concentration 0.5-2.5 mg / mL), place in a 37°C water bath, and shake continuously until the fragments are dispersed. Collect the cell suspension, pass the cell suspension through a 70-micron cell sieve, grind large cell clumps, rinse with PBS, allow single cells to pass through the filter, and collect in a 50 mL centrifuge tube; wash with ice-cold PBS and centrifuge to remove the supernatant; lyse the red blood cells with preheated red blood cell lysis buffer and resuspend, centrifuge and discard the supernatant; resuspend with PBS and adjust the cell concentration to 1×10 6 ; Add CD3 + , CD4 + and CD8 +Antibodies were used to detect the expression levels by flow cytometry.

[0105] Depend on Fig.13 AB shows that compared with the control group, the nanovaccine Example 1 prepared by the present invention has a stronger ability to activate dendritic cell maturation, which is consistent with the in vitro immune stimulation effect. Fig.13 CF shows that compared with the control group, the nanovaccine Example 1 group prepared by the present invention produced more CD8 + IFN-γ + T cells, which means that the nano vaccine prepared by the present invention induces the body to produce a stronger immune response and can better play an anti-tumor effect.

[0106] Test Example 11:

[0107] The specific steps for biosafety evaluation of nanomaterials are as follows:

[0108] A tumor-bearing mouse model was constructed according to the method of the aforementioned test example 10 (1), and a tumor inhibition experiment was performed according to the method of the aforementioned test example 10 (2). After reaching the experimental endpoint, the heart, liver, spleen, lung, and kidney of each group of mice were taken, fixed with 4% paraformaldehyde at 4°C overnight, dehydrated, embedded in paraffin, and sliced. The status of each organ was evaluated by hematoxylin-eosin (H&E) staining to evaluate in vivo safety. The results are as follows Fig.14 shown.

[0109] Depend on Fig.14 It can be seen that compared with the blank control group, no obvious tissue damage was found in the heart, liver, spleen, lung and kidney of the nanovaccine prepared by the present invention, the muscle fibers were arranged neatly, the edges were clearly defined, and the structure was relatively complete, which illustrates the good safety of the nanomaterial prepared by the present invention.

[0110] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A hybrid liposome nanovesicle vaccine loaded with prazosin, characterized in that: The hybrid liposome nanovesicle vaccine loaded with prazosin is prepared by encapsulating prazosin in liposomes through an ammonium sulfate gradient active drug loading method to obtain liposomes loaded with prazosin, and then mixing and incubating the liposomes loaded with prazosin with nanocell vesicles and co-extruding them to obtain a liposome hybrid nanovesicle vaccine loaded with prazosin.

2. The liposome hybrid nanovesicle vaccine loaded with prazosin according to claim 1, characterized in that: The particle size of the liposome hybrid nanovesicle vaccine loaded with prazosin is 225 nm.

3. The liposome hybrid nanovesicle vaccine loaded with prazosin according to claim 1, characterized in that: The concentration of prazosin in the liposome hybrid nanovesicle vaccine loaded with prazosin is 200 μg / ml.

4. A method for preparing the liposome hybrid nanovesicle vaccine loaded with prazosin as claimed in claim 1, characterized in that: The following steps are involved: Step 1: The tumor cells are squeezed out through an extruder to remove cell debris and microcapsules, and finally purified and concentrated using a centrifugal filter to obtain nanocellular vesicles; Step 2: preparing liposomes from dioleoyl phosphatidylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 by thin film hydration method; Step 3, encapsulating prazosin in liposomes by an ammonium sulfate gradient active drug loading method to obtain prazosin-loaded liposomes; Step 4: The liposomes loaded with prazosin are mixed with the nanocell vesicles, incubated, and then co-extruded to obtain the liposome hybrid nanovesicle vaccine loaded with prazosin.

5. The method for preparing the liposome hybrid nanovesicle vaccine loaded with prazosin according to claim 4, characterized in that: The tumor cells described in step 1 are colon cancer cells.

6. Use of the liposome hybrid nanovesicle vaccine loaded with prazosin as claimed in any one of claims 1 to 3 in the preparation of anti-tumor drugs.

7. Use of the liposome hybrid nanovesicle vaccine loaded with prazosin according to claim 6 in the preparation of anti-tumor drugs, characterized in that: The tumor is colon cancer.

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