CN119367521A

CN119367521A - A multifunctional autologous bionic nanovaccine for treating breast cancer and its preparation method and application

Info

Publication number: CN119367521A
Application number: CN202410469071.XA
Authority: CN
Inventors: 朱康顺; 李锬; 林立腾; 蔡明岳; 王晓彬
Original assignee: Second Affiliated Hospital of Guangzhou Medical University
Current assignee: Second Affiliated Hospital of Guangzhou Medical University
Priority date: 2024-04-18
Filing date: 2024-04-18
Publication date: 2025-01-28

Abstract

本发明提供了一种治疗乳腺癌的多功能自体仿生纳米疫苗，所述多功能自体仿生纳米疫苗包括作为内核的MnO_x纳米颗粒，以及包裹在所述MnO_x纳米颗粒内核表面的融合细胞膜；所述融合细胞膜是由重塑后的4T1细胞膜和重塑后的DC细胞膜融合得到的。本发明在应用上实现纳米疫苗在皮下注射的情况下发挥免疫治疗作用治疗乳腺癌。本发明多功能自体仿生纳米疫苗经皮下瘤周注射后可靶向肿瘤组织发挥原位化学动力治疗作用，并趋化至引流淋巴结实现高效地T细胞免疫应答发挥免疫治疗作用，且全身主要脏器聚集较少，安全可靠；而在现有技术制备的SPNE经静脉注射后会引起一定的肝脏聚集效应，产生较大的毒副作用。The present invention provides a multifunctional autologous bionic nanovaccine for treating breast cancer, the multifunctional autologous bionic nanovaccine comprises MnO _x nanoparticles as a core, and a fused cell membrane wrapped on the surface of the core of the MnO _x nanoparticles; the fused cell membrane is obtained by fusion of a reshaped 4T1 cell membrane and a reshaped DC cell membrane. The present invention realizes the use of nanovaccines to play an immunotherapeutic role in treating breast cancer under the condition of subcutaneous injection. The multifunctional autologous bionic nanovaccine of the present invention can target tumor tissues to play an in situ chemodynamic therapy role after subcutaneous peritumoral injection, and chemotaxis to the draining lymph nodes to achieve efficient T cell immune response to play an immunotherapeutic role, and the main organs of the whole body are less aggregated, which is safe and reliable; while the SPNE prepared by the prior art will cause a certain liver aggregation effect after intravenous injection, resulting in greater toxic side effects.

Description

Multifunctional self-bionic nano vaccine for treating breast cancer and preparation method and application thereof

Technical Field

The invention relates to a multifunctional self-bionic nano vaccine for treating breast cancer, and a preparation method and application thereof.

Background

The bionic nanometer vaccine prepared by combining personalized immunotherapy with other traditional therapies (including chemotherapy, radiotherapy, photodynamic therapy and chemo-dynamic therapy) by utilizing a cell membrane wrapping technology is a most promising approach for treating solid tumors. However, the limited efficacy of nanovaccines in eliciting immune responses remains a significant challenge for tumor therapy. In general, the nanovaccine-mediated immune response involves a five-step cascade of in vivo 1) entrapment of antigen 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 have been explored to optimize T cell immunity. Unlike the traditional method of only initiating DC maturation or T cell activation, kim et al designed a nanoparticle CCM-MPLA-aCD28 modified by cancer cell membrane, which contained monophosphoryl lipid A adjuvant for DC maturation and aCD28 antibody for T cell activation, as a biomimetic nanovaccine, with the function of DC and T cell dual activation. In addition, zhang et al developed a nanoadapter (SPNE) for multicellular binding between tumor cells, DC cells and T cells, promoting dual activation of DC cells and T cells, and exerting photothermal therapeutic effects. They not only remodel 4T1 cells with doxorubicin to enhance damage-associated molecular patterns (DAMPs), but also remodel DC cells with R848 and 4T1 cell lysates to enhance antigen presentation. Subsequently, they coated the reshaped fusion cell membrane of 4T1 cells and DC cells on the surface of poly (benzobisthiadiazole-alt-thiophene) (pBBTT) with NIR-II absorbing capacity to form a "core-shell" structured nanovaccine for synergistic photothermal-immunotherapy. Although these nanosystems, which construct synergistic immunotherapy, can achieve systemic immune responses, it is urgent to seek simpler, efficient methods to develop next generation biomimetic nanovaccines based on DC/T cell dual activation functions.

The prior art has more needed basic substances in the process of preparing the nanometer vaccine SPNE. For example, the chemotherapeutic DOX is needed for tumor cell remodeling, the tumor cell lysate and R848 are needed for tumor cell remodeling, the additional coating of photosensitizer poly (benzobisthiadiazole-alt-thiophene) (pBBTT) is needed to exert the photothermal therapeutic effect of NIR-II, and PEG-b-PPG-bPEG is needed to coat pBBTT to form nanokernels.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a multifunctional self-bionic nano vaccine for treating breast cancer, and a preparation method and application thereof. The multifunctional self-bionic nano vaccine has the double activation function of DC cells/T cells and the chemodynamic treatment function.

In order to achieve the above purpose, the following technical scheme is adopted:

The invention provides a multifunctional self-bionic nano vaccine for treating breast cancer, which comprises MnO _x nano particles serving as inner cores and fusion cell membranes wrapping the surfaces of the inner cores of the MnO _x nano particles, wherein the fusion cell membranes are obtained by fusing remodeled 4T1 cell membranes and remodeled DC cell membranes.

Preferably, the MnOx nanoparticle is obtained by wrapping MnO _x in PDPA-PEG, wherein the MnOx nanoparticle comprises PDPA and MnOx positioned in the inner core and PEG positioned in the outer layer. The preparation method comprises the steps of mixing PDPA-PEG and MnO _x, dissolving in chloroform, then adding into deionized water, carrying out ultrasonic oscillation on the mixture to form milky liquid, and then removing chloroform to obtain the MnOx nano-particles. MnO _x is a hydrophobic nanoparticle of about 8nm in size. PDPA-PEG (PDPA is a hydrophobic polymer and PEG is a hydrophilic polymer) is a high molecular polymer with a molecular weight of about 7 kDa. When PDPA-PEG and hydrophobic MnOx are blended with chloroform and then are dispersed in pure water, the chloroform is removed by ultrasonic rotary evaporation, PDPA and MnOx with hydrophobic cores are formed through hydrophilic-hydrophobic interaction, and PEG is exposed to the outermost layer to form a hydrophilic layer, so that nano-particles MP with the size of about 102nm and containing MnO _x are finally obtained. The PDPA-PEG mainly plays a role in coating the ultra-small MnO _x on the inner core of the nano-particle in the process of preparing the nano-particle.

Preferably, the preparation method of MnO _x comprises the steps of dissolving Mn (acac) ₃, oleic acid, oleylamine and 1, 2-hexadecanediol in benzyl ether, then removing water, then heating the solution at 200 ℃ for 3 hours, then heating to 300 ℃ for 1 hour at 300 ℃ to obtain nanocrystals, then cooling the obtained nanocrystals to room temperature under the protection of argon, precipitating with ethanol, and centrifuging to obtain MnO _x with ultra-small particle size. Preferably, the molar ratio of Mn (acac) ₃, oleic acid, oleylamine, and 1, 2-hexadecanediol is 3:9:9:10.

Preferably, the weight ratio of the diisopropylamine-polyethylene glycol acrylate to the MnO _x is 20:1.

Preferably, the weight ratio of the remodeled 4T1 cell membrane to the remodeled DC cell membrane is 1:1.

Preferably, the remodelled 4T1 cell membrane is obtained by incubating MnO _x nanoparticles with 4T1 cells and then lysing, preferably the concentration of MnO _x is 400 μg/mL during the incubation.

Preferably, the remodeled DC cell membrane is obtained by incubating 4T1 cell lysate and DC cells and then lysing, and preferably, the protein concentration in the 4T1 cell lysate is 200 mug/mL.

Preferably, the 4T1 cell lysate is obtained by lysing MnO _x nanoparticles after incubation with 4T1 cells, and preferably the concentration of MnO _x is 400 μg/mL during the incubation.

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

The invention provides a preparation method of the multifunctional self-bionic nano vaccine, which comprises the following steps:

(1) Incubating MnO _x nano-particles and 4T1 cells to obtain remodeled 4T1 cells;

(2) Lysing the remodeled 4T1 cells obtained in the step (1) to obtain remodeled 4T1 cell lysate, and incubating the remodeled 4T1 cell lysate with DC cells to obtain remodeled DC cells;

(3) Respectively lysing the remodeled 4T1 cells obtained in the step (1) and the remodeled DC cells obtained in the step (2) to obtain remodeled 4T1 cell membranes and remodeled DC cell membranes, and mixing and extruding the remodeled 4T1 cell membranes and the remodeled DC cell membranes to obtain fusion cell membranes;

(4) And (3) mixing and extruding the MnO _x nano-particles and the fusion cell membrane obtained in the step (3) to enable the fusion cell membrane to wrap the surface of the inner core of the MnO _x nano-particles, thus obtaining the multifunctional self-bionic nano-vaccine.

The invention provides application of the multifunctional self-bionic nano vaccine in preparation of a medicament for treating breast cancer.

The beneficial effects are that:

1. The invention utilizes MnO _x nano particles to realize the preparation of the multifunctional bionic nano vaccine with the 'core-shell' structure in one-stop mode. Firstly, remodelling tumor cells by utilizing MnO _x nano particles, collecting remodelled tumor cell lysate, continuing remodelling DC cells without adding an additional immunological adjuvant, secondly, fusing the remodelled tumor cell membrane and the DC cell membrane to obtain a nano shell fusion membrane RHM, wherein the nano inner core is taken from the MnO _x nano particles, and in the process of preparing the SPNE nano preparation in the prior art, the DC cells are remodelled by adding an additional adjuvant immunological adjuvant R848 and an additional pBBTT photo-thermal agent serving as the nano inner core.

The invention realizes that the nanometer vaccine plays an immune treatment role to treat breast cancer under the condition of subcutaneous injection. The MP@RHM can target tumor tissues to exert an in-situ chemomotive treatment effect after being injected subcutaneously, and chemotaxis to drainage lymph nodes to realize an efficient T cell immune response to exert an immune treatment effect, and main organs of the whole body are less in aggregation, so that the preparation method is safe and reliable, and the SPNE prepared in the prior art can cause a certain liver aggregation effect after being injected intravenously, so that a large toxic and side effect is generated.

Drawings

FIG. 1 is a diagram showing the preparation of a multifunctional biomimetic nano-vaccine using MnO _x nanoparticles (MP) in one-stop mode. (iDCs: immature DC cells; mDCs: mature DC cells; RCM: remodeled 4T1 cell membrane; RDM: remodeled DC cell membrane; CD80/86: B7 costimulatory molecules; MHC-I-Ag: major histocompatibility complex class I presentation antigen; DAMPs: damaging molecular pattern; sonication: sonication).

FIG. 2 shows a Transmission Electron Microscope (TEM) of ultra-small particle size MnO _x nanoparticles. The prepared MnO _x drops were dried on a 5. Mu.L copper mesh and subjected to transmission electron microscopy imaging at 120kV voltage. Scale 50nm.

FIG. 3 (A) changes in viability of 4T1 cells with increasing MP concentration. (B) Flow assay as MP treated 4T1 cell lysate protein concentration increases, the proportion of mature DC cells (CD80+CD86+) varies and the corresponding flow statistics (C). Wherein 400. Mu.g/mL MP is the optimal concentration for remodelling 4T1 cells, and 200. Mu.g/mL MP treated 4T1 cell lysate is the optimal concentration for remodelling DC cells. Cell viability (%) as percentage of Cell viability, concentration: concentration, matured DCs: mature DC cells, amount of protein: protein mass.

FIG. 4 (A) Transmission Electron Microscopy (TEM) of PDPA-PEG, MP and MP@RHM nanoparticles. (B) The particle size potentiometer detects the size and Zeta potential of PDPA-PEG, MP and MP@RHM nano particles. Wherein the presence of a complete membranous structure on the surface of MP@RHM confirms the decoration of RHM. In addition, the size and Zeta potential of PDPA-PEG NPs were 95.6nm and-8.1 mV, MP NPs were 103.3nm and-8.6 mV, MP@RHM was 117.2nm and-32.3 mV, respectively, further indicating that RHM was successfully coated on MP surfaces. Number (%) as a percentage of the Number, diameter, zeta potential.

FIG. 5 shows protein gel electrophoresis experiments showing the CRT protein bands (calreticulin, a marker protein for DAMPs) on CM, RCM, RDM and MP@RHM. Of these, the RCM group exhibited significant CRT upregulation. CM, non-remodeled 4T1 cell membrane, RCM, remodeled 4T1 cell membrane, RDM, remodeled DC cell membrane.

FIG. 6 (A) schematic diagram of the mechanism of tumor and LNs targeting by injecting MP@RHM nanovaccine around tumor. (B) Fluorescence images of tumors and LNs in and out of mice after peritumor injection of DiR-labeled MP, MP@RDM, MP@RCM and mp@rhm nanoparticles. Red dotted circles LNs, green dotted circles tumor. (C, D) fluorescence intensity of tumors and LNs in vivo (n=3). (E) fluorescence intensity of isolated major organs (n=3). Peritumoral injection injection around Tumor, targeting, homing homing, tumor Tumor, LNs lymph node, CTLs toxic T lymphocytes, ex vivo, he, heart, li, liver Liver, sp, spleen Spleen, lu, lung Lung, ki, kidney Kidney, fluorescence intensity (a.u.) fluorescence intensity, time, tumors in vivo Tumor in vivo, LNs in vivo, lymph node in vivo, organs Ex vivo organ Ex vivo.

FIG. 7 (A) is a schematic illustration of a processing schedule. (B) Growth curve of primary tumor of mice after different treatments (n=5). (C, E) volumes and photographs of LNs (n=4). Scale bar 5.0mm. (D) H & E staining of tumors and TUNEL immunohistochemical staining. Scale 100 μm. PREPARING NANOVACCINE preparation of nanovaccine, 4T1 subcutaneous incubation subcutaneous inoculation of 4T1 cells, immune analysis, day, peritumoral injection of nanovaccine tumor week injection of nanovaccine, analysis, tumor size, primary tumor, LNs size, lymph node size, H & E hematoxylin-eosin staining, TUNEL in situ terminal transferase labeling.

FIG. 8 biodistribution of SPNU and SPNE in 4T1 tumor-bearing mice. a) Ex vivo fluorescence images of organs and tumors of mice were treated with SPNU or SPNE. H, heart. Li is liver. S, spleen. Lu, lung. K, kidney. T is tumor. b) Fluorescence intensity quantification. c) SPNU or SPNE treated mice fluorescence image of lymph nodes. NL cervical lymph node. LT is the lateral thoracic lymph node. IN, inguinal lymph node. d) In vitro fluorescence image of lymph nodes. Radiance fluorescence intensity.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.

In the invention, a multifunctional bionic nano vaccine is developed by utilizing MnO _x nano particles in a one-stop preparation mode, and has high-efficiency chemical power treatment capability and DC cell/T cell dual activation capability, so that a personalized treatment scheme is provided for chemical power treatment-immunotherapy of solid breast cancer. Firstly, remodelling 4T1 tumor cells with MnO _x nano-particles promotes the cell membrane (RCM) of the 4T1 tumor cells to express more damaged molecular patterns DAMPs, and secondly, continuing remodelling DC cells with remodelled tumor cell lysates promotes the cell membrane (RDM) of the DC cells to express more antigen presentation complexes. And then fusing RCM and RDM to form a fusion membrane RHM, and wrapping the fusion membrane RHM on the surface of MnO _x nano particles (MP) serving as a nano inner core to form the bionic nano vaccine MP@RHM with a 'core-shell' structure (figure 1). In a mouse model for treating breast cancer, the mode of utilizing the ternary combination of MnO _x, RCM and RDM can be realized by on one hand chemotactic to lymphocyte activation of DC cells and T cells to improve the proliferation and activation level of tumor specific toxic T lymphocytes (CTL) and on the other hand targeting tumor tissues to generate Fenton reaction in an acidic tumor microenvironment to catalyze H ₂O₂ to release hydroxyl free radicals (OH) to play a role of Chemical Dynamic Therapy (CDT). Wherein the released O ₂ can reverse the immunosuppression induced by tumor hypoxia, and Mn ²⁺ can be used as an immunoadjuvant to amplify immune response. The invention aims to construct the multifunctional bionic nano vaccine MP@RHM by utilizing MnO _x nano particles through a one-stop strategy, and inhibit the growth of tumors in a mode of cooperative chemodynamic therapy-immunotherapy in a mouse breast cancer tumor model.

Example 1

1. Synthesis of ultra-small particle size manganese oxide nanoparticles (MnO _x).

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 30mL benzyl ether and heated at 110℃for 30 min in vacuo to remove water. Next, the solution was heated at 200 ℃ for 3 hours, then rapidly warmed to 300 ℃ and heated at 300 ℃ for 1 hour. Finally, the resulting nanocrystals were cooled to room temperature under argon and precipitated with ethanol and centrifuged at 6000rpm to give ultra-small particle size MnO _x (about 8 nm) (fig. 2).

Assembly of mp nanoparticles.

Diisopropylamine-polyethylene glycol (PDPA-PEG) acrylate is a pH sensitive block polymer, which is commercially available directly from commercial companies (Sesamini, inc., PDPA _5k-PEG_2k). First, 10mg of PDPA-PEG and 0.5mg of MnO _x were mixed and dissolved in 1mL of chloroform, followed by slow addition to 10mL of deionized water and ultrasonic shaking of the mixture with an ultrasonic shaker to form a milky liquid. Then, chloroform was removed by evaporation with a rotary evaporator heated to 40 ℃. Finally, the larger precipitate was removed by filtration using 220nm polyvinylidene fluoride (PVDF) to obtain MP nanoparticles entrapped with ultra-small particle size MnO _x (fig. 4A).

3. The 4T1 cells and DC cells were remodelled using MnO _x.

First, 4T1 cells were isolated from 4T1 breast cancer tumor mice, added to RPMI-1640 medium containing 10% fetal bovine serum, and cultured at 5% CO ₂ and 37℃for 24 hours. Next, to remodel 4T1 cells using MnO _x for immunogenic death, we incubated 4T1 cells (1X 10 ⁵) with varying concentrations of MP nanoparticles (final concentration of MnO _x: 100, 200, 400, 800, 1200. Mu.g/mL) for 24h, assayed 4T1 cell viability and tested for IC ₅₀ concentration (FIG. 3A). In addition, to assess the ability of cell lysates to remodel DC cells, we first induced 4T1 cells to undergo immunogenic death with MP at IC ₅₀ concentration (MnO _x concentration: 400 μg/mL). The remodeled 4T1 cells were then sonicated using a sonicator (40W power for 5 minutes) to give 4T1 cell lysates, and protein concentration was determined using the BCA method. The 4T1 cell lysate (protein concentration: 50, 100, 150, 200, 300. Mu.g/mL) was then incubated with immature DC cells (1X 10 ⁵) for 24h, and the degree of activation (CD80+CD86+ expressing cells) of each group of DC cells was examined by flow cytometry (FIGS. 3B and C) and it was found that CD80+CD86+ DC cells stopped increasing at a protein concentration of 200. Mu.g/mL of tumor cell lysate. Taken together, our analysis showed that 400 μg/mL MP and 200 μg/mL MP treated 4T1 cell lysates were optimal concentrations of remodeled 4T1 cells and DC cells, respectively. The remodeled 4T1 cells, remodeled DC cells were subsequently prepared at the optimal concentrations herein.

Preparation of MP@RHM nanovaccine.

Remodelled 4T1 cell membranes (RCM) and DC cell membranes (RDM) are obtained by the following convenient methods. First, the remodeled 4T1 cells, the remodeled DC cells (4X 10 ⁵) were each resuspended in hypotonic lysis buffer (Xinfan organism, cat# XF 1512) and incubated for 2 hours. Then, the cells were centrifuged (3000 g centrifugal force) and precipitated, and then the resuspended PBS was transferred to a sonicator, and after 10 times (5 seconds each) of sonication, the supernatant was collected by centrifugation for 5 minutes (3000 g centrifugal force). Finally, RCM and RDM pellets were obtained after centrifugation at 12000g for 30 minutes. RCM and RDM contained protein concentrations of about 560. Mu.g/mL and about 478. Mu.g/mL, respectively, as determined by BCA method. To prepare the cell membrane nanovesicles, RCM and RDM pellet were separately suspended in PBS buffer and then sonicated, and then extruded 15 times through a 400nm polycarbonate membrane using an Avanti mini-extruder, the RCM and RDM nanovesicles were collected and mass calculated by lyophilization for subsequent use.

To prepare a fused cell membrane (RHM), RCM nanovesicles, RDM nanovesicles were prepared in PBS solution to obtain 1.2mg/mL of RCM nanovesicle solution and 1.2mg/mL of RDM nanovesicle solution, respectively, we mixed RCM nanovesicle solution (1 mL,1.2 mg/mL) and RDM nanovesicle solution (1 mL,1.2 mg/mL) in a weight ratio of RCM nanovesicle to RDM nanovesicle=1:1, and then sonicated at 37 ℃ for 10 minutes, and extruded 15 times through 200nm polycarbonate membrane to obtain RHM vesicles. Subsequently, MP solution (2 mL,0.1 mg/mL) was added to RHM solution (2 mL,0.2 mg/mL) (MP solution: MP nanoparticles were formulated with PBS to 0.1mg/mL (PDPA-PEG concentration). RHM solution: the RHM vesicles prepared above were lyophilized, then weighed to a mass, and quantified with PBS to 0.2 mg/mL), and extruded 15 times through a 100nm polycarbonate film, and RHM was wrapped around the surface of nanokernel MP. Finally, the mixed solution was centrifuged for 5 minutes (14000 rpm) to remove the excess cell membrane, and the resulting biomimetic nanovaccine MP@RHM was resuspended in PBS for use, and in addition, RCM nanovesicle solution or RDM nanovesicle solution (1 mL,0.2 mg/mL) was mixed with MP solution (2 mL,0.1 mg/mL) according to the same procedure as above, and then extruded 15 times through 100nm polycarbonate membrane to encapsulate RCM or RDM on the surface of nanokernel MP. Finally, the mixed solution was centrifuged for 5 minutes (14000 rpm) to remove the excess cell membrane, and the resulting biomimetic nanovaccine MP@RCM or MP@RDM was resuspended in PBS for use (FIG. 4). RCM and mp@rhm showed up-regulated Calreticulin (CRT), a marked DAMPs, compared to the non-remodeled 4T1 cell membrane CM, indicating that remodelling 4T1 cells with MnO _x can highly express DAMPs (fig. 5).

Tumor targeting and Lymph Node (LNs) homing effects of mp@rhm nanovaccines.

For tumor targeting and LNs homing studies, 2×10 ⁶ T1 cells were resuspended in 0.1mL of 1640 medium and injected subcutaneously into the thigh of Balb/c mice. When the tumor size reached 200mm ³, a peri-tumor injection of MP, MP@RDM, MP@RCM or MP@RHM (DiR labeled MP,1.0mg/mL,100 μl) was performed (FIG. 6A). Fluorescence intensities were measured at different time points using an IVIS imaging system. In vivo images of 1h, 4h, 12h, 36h, 60h, 84h and 108h after mouse dosing were captured and fluorescence intensities of tumors and LNs were quantified using in vivo image software. After 108h, tumors and major organs including LNs were taken and analyzed by IVIS imaging system.

From fig. 6B and C, it is shown that the fluorescence intensities of the tumors in both the mp@rcm and mp@rhm groups are comparable within 108h, demonstrating the targeting ability of the RCM homologous tumors. Notably, the LNs of the mp@rhm group showed the highest fluorescence intensity compared to the mp@rcm and mp@rdm groups (fig. 6B and D). Similar trends were observed in ex vivo examination of tumors and LNs (fig. 6B and E). Importantly, the mp@rhm profile showed minimal aggregation of heart, liver, spleen, lung and kidney, indicating that delivery of mp@rhm nanovaccine was safe.

Anti-tumor effect of mp@rhm nanovaccine.

Subcutaneous tumor models were established by subcutaneously injecting 4T1 cells into the right side of Balb/c mice. After the tumor volume reached 50mm ³, the mice were divided into 5 groups. We then injected various therapeutic drugs to the left of mice via tumor circumference on days 1,4 and 7 (fig. 7A). As shown in fig. 7B, the volume of the primary tumor increases significantly over time in the PBS-treated group, while the MP and mp@rcm treated groups exhibited moderate inhibition of tumor size. Interestingly, the mp@rdm treated group exhibited a significant inhibitory effect on subcutaneous tumors within 32 days, despite the lack of tumor targeting ability. In contrast, the mp@rhm treated group exhibited a superior tumor growth inhibition effect and promoted an increase in the volume of LNs (fig. 7C and E). These results indicate that MP@RHM has an extremely remarkable tumor inhibiting effect compared with other components.

In addition, histological staining with hematoxylin and eosin (H & E) showed high cell density in tumor sections of PBS-treated animals (fig. 7D). In contrast, various other nanovaccines exhibited significantly different degrees of low cell density and increased necrotic areas, with the mp@rhm treatment group exhibiting the most significant effects. In addition, to assess cell proliferation and apoptosis, a terminal deoxynucleotidyl transferase biotin-DUTP notch end-marker (TUNEL) assay was performed. FIG. 7D shows that TUNEL positive cells were the most in the MP@RHM group. These data indicate that the mp@rhm system formed by only RHM encapsulating MnO _x nanoparticles shows significant anti-cancer activity in 4T1 tumor bearing mice.

Comparative example 1

Comparison of experimental protocol:

1. SPNE nanosystems were prepared.

1) PBBTT synthesis.

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

(4,8-bis((2-ethylhexyl)oxy)benzo[1,2-b:4,5-b']dithiophene-2,6-diyl)bis(trimethylstan nane)(OT)(20mg,0.03mmol),Pd₂(dba)₃(1.5mg,0.0015mmol),tri(o-tolyl)phosphine(4.0mg,0.012mmol) And chlorobenzene (4 mL) was added to the 100mL flask. After three freeze pump-thawing cycles and degassing, the reaction was carried out for 2 hours under the protection of N ₂ at 100 ℃, and then the reaction was carried out by adding into cold methanol for precipitation, thus obtaining a black precipitate. Subsequently, the black precipitate was washed three times with cold methanol and collected by centrifugation (3500 rpm), and finally dried under vacuum overnight to give poly

(benzobisthiadiazole-alt-thiophene)pBBTT。

2) SPNU preparation. pBBTT (0.25 mg) and 2,3-naphthalocyanine bis (trihexylsilyloxide) (NCBS, 0.005 mg) were dissolved in tetrahydrofuran (THF, 2 mL), and then PEG-b-PPG-bPEG (2.5 mg) dissolved in THF (1 mL) was added. The mixture was then added drop wise to deionized water (10 mL) and phacoemulsified with a sonicator. Tetrahydrofuran was then removed by evaporation under reflux of N ₂. Finally, the larger precipitate was removed by filtration with 220nm pore size polyvinylidene fluoride (PVDF) and isolated to give semiconducting polymer nanoparticles SPNU (uncoated semiconducting polymer nanoparticle) (3500 rpm,25 min) that did not encapsulate the cell membrane.

3) DC cell extraction and remodeling. Balb/c mice were sacrificed, femur and tibia were harvested, and then muscle and tissue were excised. The removed bones were sterilized with 70% ethanol and then placed in sterile RPMI-1640 medium. Both ends of the bone were cut and the bone marrow was flushed out with RPMI and larger cell clusters were removed by 50 μm cell strainer. The bone marrow-containing cell culture medium was then centrifuged at 2000rpm for 10 minutes. Then 1mL of erythrocyte lysis buffer was added, after 5 minutes incubation at room temperature, 10mL of sterile PBS was added to stop lysis and centrifugation was carried out at 2000rpm for 10 minutes. Cells were resuspended in RPMI medium containing 10% fetal bovine serum and 20ng/mL macrophage colony stimulating factor (GM-CSF). Immature DC cells were collected after day 3. On day 6, DC cells were remodelled with 4T1 cell lysate and 40ng/mL of R848 for 48h. And finally collecting the remodeled mature DC cells for subsequent use.

4) 4T1 cell culture and remodeling. 4T1 cells were cultured at 37℃and 5% CO ₂ in DMEM medium containing 10% fetal bovine serum. To remodel tumor cells by means of immunogenic cell death, 4T1 cells (1X 10 ⁵) were treated with 100ng/mL doxorubicin DOX for 48h. Finally, the remodeled 4T1 cells are collected for subsequent use.

5) Preparation of SPNE. The 4T1 cells and DC cells after the pre-remodelling were respectively dispersed in PBS, then sonicated for 5 minutes (on/off 2s, 20W of power supply) with a sonicator probe, and then centrifuged for 10 minutes (700 g centrifugal force) to obtain 4T1 cell membrane pellet and DC cell membrane pellet. The cell membrane was then assayed for protein concentration using the BCA method. Then, after mixing the DC cell membrane and the 4T1 cell membrane, the mixture was extruded 15 times through a polycarbonate membrane of 400nm and a polycarbonate membrane of 200nm in order by using an Avanti micro extruder (Avanti Polar Lipids, USA) (the mixture of the DC membrane and the 4T1 membrane was extruded 15 times through a polycarbonate membrane of 400nm to fuse the DC membrane and the 4T1 membrane, and the fused membrane was reduced by 15 times through the extrusion of 200 nm), so as to obtain fused cell membrane nanovesicles with uniform sizes. To encapsulate this fused cell membrane on the surface of nanokernel SPNU, the fused cell membrane (100 μg/mL) was then mixed with SPNU solution (100 μg/mL) and extruded 15 times through a 200nm polycarbonate membrane. And finally removing redundant vesicles by centrifugation to obtain the semiconductor polymer nano adhesive SPNE (semiconducting polymer nanoengager) wrapped by the cell membrane.

Experimental results show that SPNE prepared by the prior art can cause a certain liver aggregation effect (shown in figures 8a and b) after intravenous injection, and has larger toxic and side effects.

According to the invention, the manganese oxide nano-particles MnO _x are utilized to remodel tumor cells, the remolded tumor cell lysate is utilized to remodel DC cells continuously, and simultaneously, the two remolded cell membranes are utilized to fuse and then are wrapped on the surfaces of MnO _x particles.

Specifically, the invention remodels tumor cells by utilizing manganese oxide nano-particles MnO _x to highly express DAMPs, remodels DC cells by utilizing remodelled tumor cell lysate to highly express antigen presentation complex, fuses cell membranes (RHM) of the two cells, wraps the surfaces of nano-particles (MP) of PDPA-PEG loaded with MnO _x, and forms the self-bionic nano vaccine MP@RHM.

In the prior art, (1) remodelling tumor cells by using a chemotherapeutic drug doxorubicin DOX to highly express DAMPs, (2) remodelling DC cells by using a non-remodelled tumor cell lysate and an immunoadjuvant R848 to highly express antigen presentation complex, and (3) fusing cell membranes of the two and wrapping the cell membranes on the surface of a nanoparticle (SP) loaded with a photothermal agent pBBTT to form the bionic nano vaccine SPNE.

The two are different in (1) remodelling tumor cell medicines, namely MnO _x used in the invention and DOX used in the prior art. (2) The remodelling of DC cell material is different from that of cell lysate obtained by treating tumor cells with MnO _x as the source of the material used in the invention, that of cell lysate obtained by treating tumor cells with untreated source of the material used in the prior art, and that of adding an additional immunoadjuvant R848. (3) The nanometer vaccine has different kernel, and the nanometer kernel is MnO _x nanometer particle with chemical dynamic effect and pBBTT nanometer particle with photothermal effect.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit 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 nanovaccine comprises MnO _x nanoparticles as an inner core, and a fused cell membrane wrapped on the surface of the inner core of the MnO _x nanoparticles; the fused cell membrane is obtained by fusion of a reshaped 4T1 cell membrane and a reshaped DC cell membrane.

2. The multifunctional autologous bionic nanovaccine according to claim 1, characterized in that the MnOx nanoparticles are obtained by encapsulating _MnOx in PDPA-PEG, and the _MnOx nanoparticles include PDPA and MnOx in the inner core and PEG in the outer layer.

3. The multifunctional autologous bionic nanovaccine as described in claim 2, characterized in that the weight ratio of PDPA-PEG to MnO _x 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 obtained by incubating MnO _x nanoparticles and 4T1 cells and then lysing them; preferably, the concentration of MnO _x during the incubation process is 400 μg/mL.

6. The multifunctional autologous bionic nanovaccine according to claim 1, characterized in that the reshaped DC cell membrane is obtained by lysing 4T1 cell lysate and DC cells after incubation; 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 obtained by incubating MnO _x nanoparticles and 4T1 cells and then lysing them; preferably, the concentration of MnO _x during the incubation process is 400 μg/mL.

8. The multifunctional autologous bionic nanovaccine as described in claim 2 is 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 it comprises the following steps:

(1) incubating MnO _x nanoparticles and 4T1 cells to obtain remodeled 4T1 cells;

(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) Mixing and extruding the MnO _x nanoparticles and the fused cell membrane obtained in step (3) so that the fused cell membrane wraps the surface of the core of the MnO _x nanoparticles, thereby obtaining 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.