Multifunctional self-bionic nano vaccine for treating breast cancer as well as preparation method and application of multifunctional self-bionic nano vaccine

Abstract

The invention provides a multifunctional self-bionic nano vaccine for treating breast cancer. The multifunctional self-bionic nano vaccine comprises MnOx nano particles serving as inner cores and fusion cell membranes wrapping the surfaces of the inner cores of the MnOx nano particles, the fused cell membrane is obtained by fusing a remodeled 4T1 cell membrane and a remodeled DC cell membrane. According to the invention, the nano vaccine can play an immunotherapy role in treating breast cancer under the condition of subcutaneous injection. After subcutaneous peritumoral injection, the multifunctional autologous bionic nano vaccine can target tumor tissues to play an in-situ chemical power treatment role, chemotactic to drainage lymph nodes to realize efficient T cell immune response to play an immunotherapy role, and main organs of the whole body are less gathered, so that the vaccine is safe and reliable; the SPNE prepared in the prior art can cause a certain liver aggregation effect after intravenous injection, and great toxic and side effects are generated.

Description

Technical Field

[0001] The invention relates to a multifunctional autologous bionic nano vaccine for treating breast cancer and a preparation method and application thereof. Background Art

[0002] The use of cell membrane encapsulation technology to prepare biomimetic nanovaccines that synergistically combine personalized immunotherapy with other traditional therapies (including chemotherapy, radiotherapy, photodynamic therapy, and chemodynamic therapy) is one of the most promising approaches for treating solid tumors. However, the limited efficacy of nanovaccines in eliciting immune responses remains a major challenge in tumor treatment. Generally speaking, the immune response mediated by nanovaccines involves a cascade of five steps in vivo: 1) encapsulation of antigens and immune adjuvants; 2) lymph node drainage; 3) dendritic cell (DC) uptake; 4) DC cell maturation and 5) presentation of specific antigens to T cells. To date, various strategies for optimizing T cell immunity have been explored. Unlike traditional methods that only initiate DC maturation or T cell activation, Kim et al. designed a nanoparticle CCM-MPLA-aCD28 modified by cancer cell membranes, which contains a monophosphoryl lipid A adjuvant for DC maturation and an aCD28 antibody for T cell activation. As a biomimetic nanovaccine, it has the function of dual activation of DC and T cells. In addition, Zhang et al. developed a nano-adapter (SPNE) for multicellular binding between tumor cells, DC cells and T cells, promoting the dual activation of DC cells and T cells and exerting photothermal therapy. They not only reshaped 4T1 cells with doxorubicin to enhance damage-associated molecular patterns (DAMPs), but also reshaped DC cells with R848 and 4T1 cell lysates to enhance antigen presentation ability. Subsequently, they wrapped the fusion cell membrane of the reshaped 4T1 cells and DC cells on the surface of poly(benzobisthiadiazole-alt-thiophene) (pBBTT) with NIR-II absorption ability to form a "core-shell" structure nanovaccine for synergistic photothermal therapy-immunotherapy. Although these nanosystems for constructing synergistic immunotherapy can achieve systemic immune responses, it is urgent to seek simpler and more efficient methods to develop the next generation of biomimetic nanovaccines based on the dual activation function of DC cells / T cells.

[0003] The existing technology requires a lot of basic materials in the preparation of nano-vaccine SPNE. For example, the chemotherapy drug DOX is needed to reshape tumor cells; tumor cell lysate and R848 as immune adjuvant are needed to reshape DC cells; additional encapsulation of photosensitizer poly (benzobisthiadiazole-alt-thiophene) (pBBTT) is required to exert the photothermal therapy effect of NIR-II; PEG-b-PPG-bPEG is needed to encapsulate pBBTT to form a nano-core. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a multifunctional autologous bionic nano-vaccine for treating breast cancer and its preparation method and application. The multifunctional autologous bionic nano-vaccine of the present invention has both DC cell / T cell dual activation function and chemodynamic therapy function.

[0005] To achieve the above objectives, the technical solutions adopted are:

[0006] The present invention provides a multifunctional autologous bionic nano vaccine for treating breast cancer, wherein the multifunctional autologous bionic nano vaccine comprises MnO as a core. x Nanoparticles, as well as those encapsulated in the MnO x The fused cell membrane on the surface of the nanoparticle core is obtained by fusion of the reshaped 4T1 cell membrane and the reshaped DC cell membrane.

[0007] Preferably, the MnOx nanoparticles are x The MnOx nanoparticles are encapsulated in PDPA-PEG; the MnOx nanoparticles include PDPA and MnOx in the inner core and PEG in the outer layer. x The mixture is mixed and dissolved in chloroform, and then added to deionized water, and the mixture is ultrasonically vibrated to form a milky white liquid, and then the chloroform is removed to obtain the MnOx nanoparticles. x It is a hydrophobic nanoparticle with a size of about 8nm. PDPA-PEG (PDPA is a hydrophobic polymer, and PEG is a hydrophilic polymer) is a high molecular weight polymer with a molecular weight of about 7kDa. When PDPA-PEG is mixed with hydrophobic MnOx in chloroform and then dispersed in pure water, the chloroform is removed by ultrasonic rotary evaporation. At this time, PDPA and MnOx with a hydrophobic core are formed through hydrophilic-hydrophobic interaction, and PEG is exposed to the outermost layer to form a hydrophilic layer, and finally a MnO-encapsulated nanoparticle is obtained. x The size of the nanoparticles is about 102nm. PDPA-PEG is mainly used to bind ultra-small MnO xThe role of encapsulation in the nanoparticle core.

[0008] Preferably, the MnO x The preparation method comprises the following steps: Mn(acac) 3 , oleic acid, oleylamine and 1,2-hexadecanediol were dissolved in benzyl ether, and then the water was removed; then, the solution was heated at 200 ° C for 3 h, and then the temperature was raised to 300 ° C and heated at 300 ° C for 1 h to obtain nanocrystals; then the obtained nanocrystals were cooled to room temperature under argon protection, precipitated with ethanol, and centrifuged to obtain ultra-small particle size MnO x Preferably, the Mn(acac) 3 The molar ratio of oleic acid, oleylamine and 1,2-hexadecanediol is 3:9:9:10.

[0009] Preferably, the diisopropyl acrylate-polyethylene glycol and MnO x The weight ratio is 20:1.

[0010] Preferably, the weight ratio of the reshaped 4T1 cell membrane to the reshaped DC cell membrane is 1:1.

[0011] Preferably, the remodeled 4T1 cell membrane is made by adding MnO x The nanoparticles are incubated with 4T1 cells and then lysed; preferably, during the incubation process, MnO x The concentration is 400μg / mL.

[0012] Preferably, the reshaped DC cell membrane is obtained by incubating 4T1 cell lysate and DC cells and then lysing them; preferably, the protein concentration in the 4T1 cell lysate is 200 μg / mL.

[0013] Preferably, the 4T1 cell lysate is a solution containing MnO x The nanoparticles are incubated with 4T1 cells and then lysed; preferably, during the incubation process, MnO x The concentration is 400μg / mL.

[0014] Preferably, the weight ratio of the fusion cell membrane to PDPA-PEG is 2:1.

[0015] The present invention provides a method for preparing the multifunctional autologous bionic nano-vaccine described above, comprising the following steps:

[0016] (1) MnO x After incubation of nanoparticles and 4T1 cells, remodeled 4T1 cells were obtained;

[0017] (2) lysing the reshaped 4T1 cells obtained in step (1) to obtain a reshaped 4T1 cell lysate, and incubating the reshaped 4T1 cell lysate with DC cells to obtain reshaped DC cells;

[0018] (3) Lysing the reshaped 4T1 cells obtained in step (1) and the reshaped DC cells obtained in step (2) to obtain reshaped 4T1 cell membranes and reshaped DC cell membranes, respectively, and mixing and extruding the reshaped 4T1 cell membranes and the reshaped DC cell membranes to obtain fused cell membranes;

[0019] (4) MnO x The nanoparticles and the fused cell membrane obtained in step (3) are mixed and extruded so that the fused cell membrane wraps the MnO x The surface of the nanoparticle core is obtained to obtain the multifunctional autologous bionic nanovaccine.

[0020] The present invention provides the use of the multifunctional autologous bionic nano-vaccine described above in the preparation of a medicine for treating breast cancer.

[0021] Beneficial effects:

[0022] 1. The present invention utilizes MnO x Nanoparticles realize the "one-stop" preparation of "core-shell" structure multifunctional bionic nanovaccine. First, MnO x Nanoparticles reshape tumor cells, and collect the reshaped tumor cell lysate to continue to reshape DC cells without adding additional immune adjuvants; secondly, the reshaped tumor cell membrane and DC cell membrane are fused to obtain the nanoshell fusion membrane RHM, while the nanocore is taken from MnO x Nanoparticles themselves; in the existing technology for preparing SPNE nanoformulations, additional auxiliary materials, immune adjuvant R848, need to be added to reshape DC cells, and additional pBBTT photothermal agent needs to be added as the nanocore.

[0023] 2 The present invention realizes the immunotherapy of nano vaccines in the treatment of breast cancer under subcutaneous injection. After subcutaneous peritumoral injection, MP@RHM can target tumor tissue to exert in situ chemodynamic therapy, and chemotaxis to draining lymph nodes to achieve efficient T cell immune response to exert immunotherapy, and less accumulation in major organs of the whole body, safe and reliable; while SPNE prepared by the prior art will cause a certain liver aggregation effect after intravenous injection, resulting in greater toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Using MnO xNanoparticles (MP) are used to prepare a multifunctional biomimetic nanovaccine in a "one-stop" manner. (iDCs: immature DC cells; mDCs: mature DC cells; RCM: remodeled 4T1 cell membrane; RDM: remodeled DC cell membrane; CD80 / 86: B7 co-stimulatory molecules; MHC-I-Ag: major histocompatibility complex class I antigen presentation; DAMPs: damage molecular patterns; Sonication: ultrasonic treatment).

[0025] Figure 2 :Ultra-small particle size MnO x Transmission electron microscopy (TEM) of nanoparticles. x The drop was dried on a 5 μL copper grid and imaged by transmission electron microscopy at 120 kV. Scale bar: 50 nm.

[0026] Figure 3 :(A) Changes in the viability of 4T1 cells with increasing MP concentration. (B) Flow cytometry detection of changes in the proportion of mature DC cells (CD80+CD86+) with increasing protein concentration of 4T1 cell lysate treated with MP and the corresponding flow cytometry statistics (C). Among them, 400μg / mL of MP is the optimal concentration for remodeling 4T1 cells; 200μg / mL of MP-treated 4T1 cell lysate is the optimal concentration for remodeling DC cells. Cell viability (%): percentage of cell viability; Concentration: concentration; Matured DCs: mature DC cells; Amount of protein: protein amount.

[0027] Figure 4 :(A) Transmission electron microscopy (TEM) images of PDPA-PEG, MP and MP@RHM nanoparticles. (B) The size and Zeta potential of PDPA-PEG, MP and MP@RHM nanoparticles were detected by particle size potentiostat. Among them, the presence of a complete membrane structure on the surface of MP@RHM confirmed the decoration of RHM. In addition, the size and Zeta potential of PDPA-PEG NPs were 95.6nm and -8.1mV, MP NPs were 103.3nm and -8.6mV, and MP@RHM were 117.2nm and -32.3mV, further indicating that RHM was successfully wrapped on the surface of MP. Number(%): number percentage; Diameter: diameter; Zeta potential: Zeta potential.

[0028] Figure 5: Protein gel electrophoresis experiments showed CRT protein bands (calreticulin, a marker protein of DAMPs) on CM, RCM, RDM and MP@RHM. Among them, the RCM group showed obvious upregulation of CRT. CM: unremodeled 4T1 cell membrane; RCM: remodeled 4T1 cell membrane; RDM: remodeled DC cell membrane.

[0029] Figure 6 : (A) Schematic diagram of the mechanism of peritumoral injection of MP@RHM nanovaccine targeting tumors and LNs. (B) Fluorescence images of tumors and LNs in mice in vivo and in vitro after peritumoral injection of DiR-labeled MP, MP@RDM, MP@RCM, and MP@RHM nanoparticles. Red dotted circle: LNs; green dotted circle: tumor. (C,D) Fluorescence intensity of tumors and LNs in vivo (n=3). (E) Fluorescence intensity of major organs in vitro (n=3). Peritumoral injection: peritumoral injection; Targeting: targeting; Homing: homing; Tumor: tumor; LNs: lymph nodes; CTLs: cytotoxic T lymphocytes; Ex vivo: ex vivo; He, Heart: heart; Li, Liver: liver; Sp, Spleen: spleen; Lu, Lung: lung; Ki, Kidney: kidney; Fluorescence intensity (au): fluorescence intensity; Time: time; Tumors in vivo: tumors in vivo; LNs in vivo; lymph nodes in vivo; Organs ex vivo: organs ex vivo.

[0030] Figure 7 :(A) Description of the treatment schedule. (B) Growth curves of primary tumors in mice after different treatments (n=5). (C, E) Volumes and photos of LNs (n=4). Scale bar: 5.0 mm. (D) H&E staining and TUNEL immunohistochemical staining of tumors. Scale bar: 100 μm. In the figure, Preparing nanovaccine: Preparation of nanovaccine; 4T1 subcutaneous incubation: subcutaneous incubation of 4T1 cells; immune analysis: immune analysis; day: day; peritumoral injection of nanovaccine: peritumoral injection of nanovaccine; analysis: analysis; tumor size: tumor size; primary tumor: primary tumor; LNs size: lymph node size; H&E: hematoxylin-eosin staining; TUNEL: in situ terminal transferase labeling.

[0031] Figure 8: Biodistribution of SPNU and SPNE in 4T1 tumor-bearing mice. a) Ex vivo fluorescence images of organs and tumors of mice treated with SPNU or SPNE. H: Heart. Li: Liver. S: Spleen. Lu: Lung. K: Kidney. T: Tumor. b) Fluorescence intensity quantification graph. c) Fluorescence images of lymph nodes of mice treated with SPNU or SPNE. NL: Cervical lymph node. LT: Lateral thoracic lymph node. IN: Inguinal lymph node. d) Ex vivo fluorescence images of lymph nodes. Radiance: Fluorescence intensity. DETAILED DESCRIPTION

[0032] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0033] In the present invention, we use MnO in a "one-stop" preparation method. x Nanoparticles have developed a multifunctional bionic nanovaccine that has both efficient chemodynamic therapy and DC / T cell dual activation capabilities, which provides a personalized treatment plan for chemodynamic therapy-immunotherapy of solid breast cancer. x Nanoparticles remodeled 4T1 tumor cells to cause the cell membrane (RCM) of the 4T1 tumor cells to express more damage molecular patterns (DAMPs); secondly, the remodeled tumor cell lysate was used to continue remodeling DC cells to cause the cell membrane (RDM) of the DC cells to express more antigen presenting complexes. RCM and RDM were then fused to form a fusion membrane RHM, and at the same time, the RCM was wrapped in a MnO x Nanoparticles (MP) are bionic nano-vaccines with a "core-shell" structure formed on the surface of the nano-core. Figure 1 ). In a mouse model of breast cancer, this approach using MnO x The ternary combination of RCM and RDM can achieve the following: on the one hand, it can chemotaxis to lymphoid tissues to activate DC cells and T cells to increase the proliferation and activation level of tumor-specific cytotoxic T lymphocytes (CTLs); on the other hand, it can target tumor tissues to undergo Fenton reaction in the acidic tumor microenvironment to catalyze H 2 O 2 The release of hydroxyl radicals (·OH) exerts a chemodynamic therapeutic (CDT) effect. 2 Mn can reverse tumor hypoxia-induced immunosuppression 2+ It can be used as an immune adjuvant to amplify the immune response. x The nanoparticles constructed a multifunctional bionic nanovaccine MP@RHM through a "one-stop" strategy, which synergized chemodynamic therapy-immunotherapy to inhibit tumor growth in a mouse breast cancer tumor model.

[0034] Example 1

[0035] 1. Ultra-small size manganese oxide nanoparticles (MnO x ) synthesis.

[0036] First, Mn(acac) 3 (1.06 g, 3.0 mmol), oleic acid (3.3 mL, 9.0 mmol), oleylamine (3 mL, 9.0 mmol) and 1,2-hexadecanediol (2.8 g, 10 mmol) were dissolved in 30 mL of benzyl ether and heated at 110 °C for 30 minutes in vacuum to remove water. Then, the solution was heated at 200 °C for 3 h, then quickly heated to 300 °C and heated at 300 °C for 1 h. Finally, the obtained nanocrystals were cooled to room temperature under argon protection, precipitated with ethanol, and centrifuged at 6000 rpm to obtain ultra-small particle size MnO x (about 8nm)( Figure 2 ).

[0037] 2. Assembly of MP nanoparticles.

[0038] Diisopropylamino acrylate-polyethylene glycol (PDPA-PEG) is a pH-sensitive block polymer that can be purchased directly from commercial companies (Xi'an Ruixi Biotechnology, PDPA 5k -PEG 2k First, 10 mg of PDPA-PEG and 0.5 mg of MnO x The mixture was mixed and dissolved in 1 mL of chloroform, then slowly added to 10 mL of deionized water and ultrasonically vibrated the mixture using an ultrasonic oscillator to form a milky white liquid. Then, a rotary evaporator was heated to 40 °C to evaporate and remove the chloroform. Finally, 220 nm polyvinyl fluoride (PVDF) was used to filter and remove the larger precipitate to obtain the ultra-small particle size MnO x MP nanoparticles ( Figure 4 A).

[0039] 3. Utilize MnO x Remodeling of 4T1 cells and DC cells.

[0040] First, 4T1 cells were isolated from mice bearing 4T1 breast cancer tumors and added to RPMI-1640 medium containing 10% fetal bovine serum and incubated in 5% CO. 2 and 37°C for 24 h. x To reshape 4T1 cells to induce immunogenic death, we injected 4T1 cells (1×10 5 ) with different concentrations of MP nanoparticles (MnO xFinal concentration: 100, 200, 400, 800, 1200 μg / mL) and incubated for 24 h to determine the survival rate of 4T1 cells and detect IC 50 concentration( Figure 3 A) In addition, to evaluate the ability of cell lysates to remodel DCs, we first used IC 50 Concentration of MP(MnO x The remodeled 4T1 cells were then ultrasonically disrupted with an ultrasonic disruptor (power 40W, 5 minutes) to obtain a 4T1 cell lysate, and the protein concentration was determined by the BCA method. The 4T1 cell lysate (protein concentration: 50, 100, 150, 200, 300 μg / mL) was then mixed with immature DC cells (1×10 5 ) were incubated for 24 h, and the activation degree of DC cells in each group (expressing CD80+CD86+ cells) was detected by flow cytometry ( Figure 3 B and C), it was found that when the protein concentration was 200 μg / mL tumor cell lysate, CD80+CD86+DC cells stopped increasing. In summary, our analysis showed that 400 μg / mL MP and 200 μg / mL MP-treated 4T1 cell lysate were the optimal concentrations for remodeling 4T1 cells and DC cells, respectively. The remodeled 4T1 cells and remodeled DC cells were subsequently prepared according to the optimal concentrations here.

[0041] 4. Preparation of MP@RHM nanovaccine.

[0042] Remodeled 4T1 cell membrane (RCM) and DC cell membrane (RDM) were obtained by the following simple method. First, remodeled 4T1 cells and remodeled DC cells (4×10 5 ) were resuspended in hypotonic lysis buffer (Xinfan Bio, Catalog No.: XF1512) and incubated for 2 hours. Then, the cells were centrifuged (3000g centrifugal force) and precipitated, then resuspended in PBS and transferred to an ultrasonic disruptor. After 10 ultrasonic times (5 seconds each time), the supernatant was collected by centrifugation for 5 minutes (3000g centrifugal force). Finally, RCM and RDM precipitates were obtained after centrifugation at 12000g centrifugal force for 30 minutes. The BCA method was used to determine that RCM and RDM contained protein concentrations of approximately 560μg / mL and approximately 478μg / mL, respectively. In order to prepare cell membrane nanovesicles, the RCM and RDM precipitates were suspended in PBS buffer and then ultrasonically dispersed, and then extruded 15 times through a 400nm polycarbonate membrane using an Avanti microextruder, RCM and RDM nanovesicles were collected, and the mass was calculated by freeze-drying for subsequent use.

[0043] In order to prepare hygroscopic membrane (RHM), RCM nanovesicles and RDM nanovesicles were dissolved in PBS solution to obtain 1.2 mg / mL RCM nanovesicle solution and 1.2 mg / mL RDM nanovesicle solution. We mixed RCM nanovesicle solution (1 mL, 1.2 mg / mL) and RDM nanovesicle solution (1 mL, 1.2 mg / mL) at a weight ratio of RCM nanovesicle: RDM nanovesicle = 1:1, then sonicated at 37°C for 10 min, and extruded through a 200 nm polycarbonate membrane 15 times to obtain RHM vesicles. Subsequently, the MP solution (2 mL, 0.1 mg / mL) was added to the RHM solution (2 mL, 0.2 mg / mL) (MP solution: the MP nanoparticles were prepared with PBS to 0.1 mg / mL (PDPA-PEG concentration). RHM solution: the RHM vesicles prepared above were freeze-dried and weighed, and quantified with PBS to 0.2 mg / mL), and extruded through a 100 nm polycarbonate membrane 15 times, and RHM was wrapped on the surface of the nanocore MP. Finally, the mixed solution was centrifuged for 5 minutes (14000 rpm) to remove excess cell membranes, and the obtained biomimetic nanovaccine MP@RHM was resuspended in PBS for later use; in addition, according to the same steps above, the RCM nanovesicle solution or the RDM nanovesicle solution (1 mL, 0.2 mg / mL) was mixed with the MP solution (2 mL, 0.1 mg / mL) and extruded through a 100 nm polycarbonate membrane 15 times to wrap RCM or RDM on the surface of the nanocore MP. Finally, the mixed solution was centrifuged for 5 minutes (14000 rpm) to remove excess cell membranes, and the obtained biomimetic nanovaccine MP@RCM or MP@RDM was resuspended in PBS for later use ( Figure 4 Compared with unremodeled 4T1 cell membrane CM, RCM and MP@RHM showed upregulated calreticulin (CRT), a hallmark DAMP, indicating that the utilization of MnO x Remodeling 4T1 cells can highly express DAMPs ( Figure 5 ).

[0044] 5. Tumor targeting and lymph node (LNs) homing effects of MP@RHM nanovaccine.

[0045] In order to conduct tumor targeting and LNs homing studies, 2×10 6 4T1 cells were resuspended in 0.1 mL of 1640 medium and injected subcutaneously into the thigh of Balb / c mice. When the tumor size reached 200 mm 3 MP, MP@RDM, MP@RCM or MP@RHM (DiR-labeled MP, 1.0 mg / mL, 100 μL) were injected peritumorally ( Figure 6A). Fluorescence intensity was detected at different time points using the IVIS imaging system. Live images of mice were captured at 1h, 4h, 12h, 36h, 60h, 84h, and 108h after administration, and the fluorescence intensity of tumors and LNs was quantified using the live imaging software. After 108h, tumors and major organs including LNs were harvested and analyzed using the IVIS imaging system.

[0046] from Figure 6 B and C show that the fluorescence intensity of tumors in the MP@RCM and MP@RHM groups was comparable within 108 h, demonstrating the targeting ability of RCM homologous tumors. It is worth noting that the fluorescence intensity of LNs in the MP@RHM group was the highest compared with the MP@RCM and MP@RDM groups ( Figure 6 B and D). Similar trends were observed in ex vivo examinations of tumors and LNs ( Figure 6 B and E). Importantly, the MP@RHM distribution showed minimal aggregation in the heart, liver, spleen, lung, and kidney, indicating that the delivery of the MP@RHM nanovaccine was safe.

[0047] 6. Anti-tumor effect of MP@RHM nanovaccine.

[0048] A subcutaneous tumor model was established by subcutaneously injecting 4T1 cells into the right side of Balb / c mice. 3 After that, the mice were divided into 5 groups. We then injected various therapeutic drugs into the left side of the mice through the tumor on days 1, 4, and 7 ( Figure 7 A). Figure 7 B shows that the volume of primary tumors in the PBS-treated group increased significantly over time, while the MP and MP@RCM-treated groups showed moderate inhibition of tumor size. Interestingly, despite the lack of tumor targeting ability, the MP@RDM-treated group showed a significant inhibitory effect on subcutaneous tumors within 32 days. In contrast, the MP@RHM-treated group showed a strong tumor growth inhibitory effect and promoted the increase in the volume of LNs ( Figure 7 C and E). These results indicate that MP@RHM has a significantly more significant anti-tumor effect compared with other components.

[0049] In addition, histological staining with hematoxylin and eosin (H&E) showed a high cell density in tumor sections from PBS-treated animals ( Figure 7 D). In contrast, the other various nanovaccines significantly reduced cell density and increased necrotic areas to varying degrees, with the MP@RHM-treated group showing the most significant effect. In addition, to evaluate cell proliferation and apoptosis, terminal deoxynucleotidyl transferase-biotin-DUTP nick end labeling (TUNEL) assay was performed. Figure 7D shows that the number of TUNEL-positive cells in the MP@RHM group is the highest. These data indicate that only RHM encapsulated MnO x The MP@RHM system formed by nanoparticles exhibited significant anticancer activity in 4T1 tumor-bearing mice.

[0050] Comparative Example 1

[0051] Comparative experimental scheme:

[0052] 1. Preparation of SPNE nanosystem.

[0053] 1) Synthesis of pBBTT.

[0054] 4,8-Bis[5-bromo-4-(2-octyldodecyl)-2-thienyl]-benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole(BBT)(32mg,0.0375mmol),

[0055] (4,8-bis((2-ethylhexyl)oxy)benzo[1,2-b:4,5-b']dithiophene-2,6-diyl)bis(trimethylstan nane)(OT)(20mg,0.03mmol), Pd 2 (dba) 3 (1.5 mg, 0.0015 mmol), tri(o-tolyl)phosphine (4.0 mg, 0.012 mmol) and chlorobenzene (4 mL) were added to a 100 mL flask. After three freeze-pump-thaw cycles of degassing, the mixture was heated at 100 °C under N 2 The reaction was carried out under protection for 2 hours, and then cold methanol was added to precipitate to obtain a black precipitate. Subsequently, the black precipitate was washed three times with cold methanol and collected by centrifugation (3500 rpm), and finally dried overnight under vacuum conditions to obtain poly

[0056] (benzobisthiadiazole-alt-thiophene)pBBTT.

[0057] 2) Preparation of SPNU. pBBTT (0.25 mg) and 2,3-naphthalocyanine bis(trihexylsilyloxide) (NCBS, 0.005 mg) were dissolved in tetrahydrofuran (THF, 2 mL); then PEG-b-PPG-bPEG (2.5 mg) dissolved in THF (1 mL) was added. The mixture was then added dropwise to deionized water (10 mL) and ultrasonically emulsified using an ultrasonic disruptor. Then, N2 The tetrahydrofuran was evaporated under reflux. Finally, the large precipitate was removed by filtration using polyvinylidene fluoride (PVDF) with a pore size of 220 nm, and the semiconductor polymer nanoparticles SPNU (uncoated semiconducting polymer nanoparticles) not wrapped with cell membranes were separated (3500 rpm, 25 minutes).

[0058] 3) DC cell extraction and remodeling. Balb / c mice were sacrificed, and the femur and tibia were collected, and then the muscles and tissues were removed. The removed bones were disinfected with 70% ethanol and then placed in sterile RPMI-1640 culture medium. Both ends of the bones were cut open and the bone marrow was flushed out with RPMI, and larger cell clumps were removed through a 50μm cell strainer. The culture medium containing bone marrow cells was then centrifuged at 2000rpm for 10 minutes. Then 1mL of red blood cell lysis buffer was added, incubated at room temperature for 5 minutes, and then 10mL of sterile PBS was added to stop lysis, and centrifuged at 2000rpm for 10 minutes. The cells were resuspended in RPMI culture medium containing 10% fetal bovine serum and 20ng / mL of macrophage colony stimulating factor (GM-CSF). Immature DC cells were collected after the 3rd day. On the 6th day, DC cells were remodeled with 4T1 cell lysate and 40ng / mL of R848 for 48h. Finally, the remodeled mature DC cells were collected for subsequent use.

[0059] 4) 4T1 cell culture and remodeling. 4T1 cells were cultured at 37°C and 5% CO 2 Conditions were set up and cultured in DMEM medium containing 10% fetal bovine serum. In order to reshape tumor cells by immunogenic cell death, 4T1 cells (1×10 5 ) were treated with 100 ng / mL doxorubicin (DOX) for 48 h. Finally, the remodeled 4T1 cells were collected for subsequent use.

[0060] 5) Preparation of SPNE. The pre-reshaped 4T1 cells and DC cells were dispersed in PBS, and then ultrasonicated for 5 minutes (on / off 2s, power supply 20W) using an ultrasonic disruptor probe, and then centrifuged for 10 minutes (700g centrifugal force) to obtain 4T1 cell membrane precipitation and DC cell membrane precipitation. The cell membrane was then measured for protein concentration using the BCA method. The DC cell membrane and 4T1 cell membrane were then mixed and extruded 15 times through 400nm and 200nm polycarbonate membranes using an Avanti micro-extruder (Avanti Polar Lipids, USA) (first, the DC membrane and 4T1 membrane were mixed and then extruded 15 times through a 400nm polycarbonate membrane to fuse them, and then the 200nm extrusion was used 15 times to make the fusion membrane smaller) to obtain uniformly sized fusion cell membrane nanovesicles. In order to wrap the fusion cell membrane on the surface of the nanocore SPNU, the fusion cell membrane (100 μg / mL) was then mixed with SPNU solution (100 μg / mL) and extruded through a 200 nm polycarbonate membrane for 15 times. Finally, the excess vesicles were removed by centrifugation to obtain the cell membrane-wrapped semiconductor polymer nanoadhesive SPNE (semiconducting polymer nanoengager).

[0061] The experimental results show that the SPNE prepared by the prior art can cause a certain liver aggregation effect after intravenous injection (such as Figure 8 a, b), resulting in significant toxic side effects.

[0062] The present invention utilizes manganese oxide nanoparticles MnO x Remodeling tumor cells, and using the remodeled tumor cell lysate to continue remodeling DC cells, while using the two remodeled cell membranes to fuse and wrap in MnO x Particle surface.

[0063] Specifically, the present invention utilizes manganese oxide nanoparticles MnO x Tumor cells were reshaped to highly express DAMPs; DC cells were further reshaped using the reshaped tumor cell lysate to highly express antigen presenting complexes; the cell membranes of the two were fused (RHM) and then wrapped in MnO x The surface of PDPA-PEG nanoparticles (MP) was formed to form the autologous bionic nanovaccine MP@RHM.

[0064] The existing technologies: (1) use the chemotherapy drug doxorubicin DOX to reshape tumor cells to highly express DAMPs; (2) use non-reshaped tumor cell lysate and immune adjuvant R848 to reshape DC cells to highly express antigen presenting complexes; (3) fuse the cell membranes of the two and wrap them on the surface of pBBTT nanoparticles (SP) loaded with photothermal agents to form a bionic nanovaccine SPNE.

[0065] The difference between the two is: (1) The drugs used to reshape tumor cells are different: the MnO x ; DOX used in the prior art. (2) Different materials for remodeling DC cells: The material used in the present invention is derived from MnO x The cell lysate after treating tumor cells; the material used in the prior art is derived from the cell lysate after untreated tumor cells, and the additional immune adjuvant R848 is added. (3) The core of the nano vaccine composition is different: the nano core used in the present invention is MnO with chemical kinetics. x Nanoparticles; the nanocore used in the prior art is pBBTT nanoparticles with photothermal effect.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A multifunctional autologous bionic nanovaccine for treating breast cancer, characterized in that: The multifunctional autologous bionic nano-vaccine comprises MnO as the core x Nanoparticles, as well as those encapsulated in the MnO x The fused cell membrane on the surface of the nanoparticle core is obtained by fusion of the reshaped 4T1 cell membrane and the reshaped DC cell membrane.

2. The multifunctional autologous bionic nanovaccine according to claim 1, characterized in that: The MnOx nanoparticles are MnO x The MnO encapsulated in PDPA-PEG x The nanoparticles consisted of PDPA and MnOx in the inner core and PEG in the outer layer.

3. The multifunctional autologous bionic nanovaccine according to claim 2, characterized in that: The PDPA-PEG and MnO x The weight ratio is 20:

1.

4. The multifunctional autologous bionic nanovaccine according to claim 1, characterized in that: The weight ratio of the reshaped 4T1 cell membrane to the reshaped DC cell membrane is 1:

1.

5. The multifunctional autologous bionic nanovaccine according to claim 1, characterized in that: The reshaped 4T1 cell membrane is MnO x The nanoparticles are incubated with 4T1 cells and then lysed; preferably, during the incubation process, MnO x The concentration is 400μg / mL.

6. The multifunctional autologous bionic nanovaccine according to claim 1, characterized in that: The reshaped DC cell membrane is obtained by incubating 4T1 cell lysate and DC cells and then lysing them; preferably, the protein concentration in the 4T1 cell lysate is 200 μg / mL.

7. The multifunctional autologous bionic nanovaccine according to claim 6, characterized in that: The 4T1 cell lysate is MnO x The nanoparticles are incubated with 4T1 cells and then lysed; preferably, during the incubation process, MnO x The concentration is 400μg / mL.

8. The multifunctional autologous bionic nanovaccine according to claim 2, characterized in that: The weight ratio of the fusion cell membrane to PDPA-PEG is 2:

1.

9. A method for preparing the multifunctional autologous bionic nanovaccine according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) MnO x After incubation of nanoparticles and 4T1 cells, remodeled 4T1 cells were obtained; (2) lysing the reshaped 4T1 cells obtained in step (1) to obtain a reshaped 4T1 cell lysate, and incubating the reshaped 4T1 cell lysate with DC cells to obtain reshaped DC cells; (3) Lysing the reshaped 4T1 cells obtained in step (1) and the reshaped DC cells obtained in step (2) to obtain reshaped 4T1 cell membranes and reshaped DC cell membranes, respectively, and mixing and extruding the reshaped 4T1 cell membranes and the reshaped DC cell membranes to obtain fused cell membranes; (4) MnO x The nanoparticles and the fused cell membrane obtained in step (3) are mixed and extruded so that the fused cell membrane wraps the MnO x The surface of the nanoparticle core is obtained to obtain the multifunctional autologous bionic nanovaccine.

10. Use of the multifunctional autologous bionic nanovaccine according to claim 1 in preparing a drug for treating breast cancer.

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