Programmed nanocarrier microneedle and preparation method and application thereof

Abstract

The application provides a programmed nano-particle-loaded microneedle and a preparation method and application thereof, and belongs to the technical field of biological medicine of tumor photothermal and immunotherapy combination therapy.The programmed nano-particle-loaded microneedle is obtained by assembling polylysine, L-arginine and CpG, connecting a mannose ligand to obtain MPLAC nanoparticles, and then obtaining PDA nanoparticles through dopamine self-polymerization under heating conditions, and then using a layer-by-layer assembly method to prepare the programmed nano-particle-loaded microneedle by taking polyvinylpyrrolidone with different molecular weights as a matrix material.The programmed nano-particle-loaded microneedle can first heat ablate a tumor to generate an in-situ nano-vaccine, and then further inhibit tumor growth, metastasis and recurrence through immunotherapy, and has the time sequence of realizing photothermal and immunotherapy combination.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of tumor photothermal immunotherapy combined therapy biological medicine, and particularly relates to a programmed nano-particle-loaded microneedle and a preparation method and application thereof. BACKGROUND

[0002] Melanoma has high invasiveness and metastasis. At present, different treatment methods are adopted for patients in different disease development periods in clinical practice, such as surgery, chemotherapy, phototherapy, radiotherapy, immunotherapy and gene therapy. However, the high morbidity and mortality of melanoma make the treatment effect and prognosis of single treatment method for patients to be improved, and therefore, a reasonable combined treatment strategy needs to be designed urgently. Photothermal therapy (PTT) is a treatment method for using near-infrared light (NIR) absorbed by a photothermal agent to raise the temperature through photothermal conversion to cause tumor cell death. Polydopamine (PDA) is a commonly used photothermal agent, which has been widely used in tumor photothermal therapy due to its advantages of simple preparation, light stability, biodegradability, good biocompatibility and strong photothermal conversion capacity. However, due to the limited laser penetration depth and uneven distribution of the photothermal agent, it is difficult to remove residual marginal tumor cells, and it is impossible to treat distal and metastatic tumors. Therefore, current tumor treatment research focuses more on the field of combined therapy. Tumor immunotherapy aims to activate immune cells in the host and tumor microenvironment (TME) to recognize and destroy tumor cells, promote systemic immune surveillance and prevent tumor metastasis and recurrence. However, due to the heterogeneity of tumors, individual differences of patients and inhibitory immune microenvironment, single immunotherapy cannot activate effective immune effects. Therefore, the combination of photothermal therapy and immunotherapy has the potential to eradicate primary tumors and metastatic tumors.

[0003] Currently, PTT can achieve combination therapy by combining with adjuvants, immune checkpoint inhibitors, immune activators, small molecule inhibitors, etc., among which the strategy of combining photothermal therapy with adjuvants to form tumor vaccines has significant efficacy in inhibiting tumor growth, metastasis and recurrence. However, the preparation of tumor-specific antigens in vitro is complex and costly, and the same antigen is difficult to activate the immune system of different patients, so the in situ tumor vaccine loaded with tumor-associated antigens (TAAs) is selected. In addition, in addition to antigens, the selection of adjuvants in tumor vaccines also directly affects the final effect of immune activation, among which Toll-like receptor (TLRs) agonists have shown significant therapeutic effect in cancer treatment, and TLR9 agonist unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) up-regulates NF-κB activity to reverse macrophage phenotype, activate NK cells and effector T cells, and improve the immunosuppressive microenvironment.

[0004] In order to realize the combination of photothermal and immune therapy, the tumor vaccine based on nanomaterials can protect the vaccine from premature degradation, co-deliver antigens and adjuvants to the same antigen-presenting cells (APCs), promote antigen cross-presentation, induce lymph node homing, and thus enhance the tumor immune cascade. However, the currently studied "integrated" nanoparticle drug delivery system still has problems such as difficult preparation, low bioavailability, off-target effect, etc. SUMMARY

[0005] The nano-drug delivery system combines PTT and adjuvant to form a tumor vaccine treatment strategy, which achieves good therapeutic effect through the following mechanism: first, PTT directly kills tumor cells, and at the same time triggers tumor immunogenic cell death (ICD), which can release damaged-associated molecular patterns (DAMPs) and TAAs that maintain normal physiological structure inside the cell to participate in immunotherapy, the DAMPs induced by PTT include adenosine triphosphate (ATP), calreticulin (CRT), etc., ATP acts as a "find me" signal to recruit antigen presenting cells (APCs), and CRT acts as an "eat me" signal for APCs and phagocytes, and the TAAs released by the dying tumor cells together provide sufficient antigens, after the nano-adjuvant captures the TAAs, it promotes the uptake of dendritic cells (DCs), the antigens and the adjuvant induce the maturation of DCs together, the antigen presentation related protein molecules MHC I and MHC II and co-stimulating expression molecules are up-regulated, the antigen cross-presentation is promoted, the lymphocyte migration ability is enhanced, the antigen specific T lymphocytes are activated in the lymph nodes, and the residual tumor cells are recognized and removed.

[0006] The application provides a programmed nano-particle-loaded microneedle and a preparation method and application thereof, the microneedle has a double-layer structure, an outer shell loaded with a photothermal agent PDA-NPs, and tumor cells are ablated by heat under the action of 808 nm laser, TAAs and DAMPs are induced to be released by ICD, the TAAs are captured by subsequently released inner core MPLAC-NPs to form a personalized vaccine to activate the immune system, and the microneedle realizes photothermal- immune combined treatment by programmed release of nanoparticles.

[0007] To achieve the above object, the application provides a preparation method of a programmed nano-particle-loaded microneedle, comprising the following steps:

[0008] Poly-(L-lysine) (PLL), L-arginine (L-Arg) and CpG are assembled into PLAC nanoparticles (PLAC-NPs), and then a mannose ligand is connected to obtain MPLAC nanoparticles (MPLAC-NPs);

[0009] PDA nanoparticles (PDA-NPs) are obtained by self-polymerization of dopamine under heating conditions;

[0010] The programmed nanocarrier microneedle is prepared by using layer-by-layer assembly method, and different molecular weight polyvinylpyrrolidone (PVP) is used as a matrix material.

[0011] Further, the preparation method of the MPLAC nanoparticle comprises the following steps:

[0012] The L-arginine aqueous solution and the CpG aqueous solution are added dropwise into the polylysine aqueous solution, and shaken at room temperature, centrifuged and washed to obtain the PLAC nanoparticle (PLAC-NPs), the PLAC nanoparticle is resuspended with deionized water, and then the active ester polyethylene glycol mannose (NHS-PEG-Mannose) aqueous solution is added dropwise, shaken at room temperature, centrifuged and washed to obtain the MPLAC nanoparticle (MPLAC-NPs).

[0013] Further, the concentration of the L-arginine aqueous solution is 0.2 mg / mL, the concentration of the CpG aqueous solution is 1 mg / mL, the concentration of the polylysine aqueous solution is 0.4 mg / mL, and the concentration of the active ester polyethylene glycol mannose aqueous solution is 1 mg / mL.

[0014] Further, the volume ratio of the L-arginine aqueous solution, the CpG aqueous solution, the polylysine aqueous solution and the active ester polyethylene glycol mannose aqueous solution is 250:75:250:7.5.

[0015] Further, the preparation method of the PDA nanoparticle comprises the following steps:

[0016] The dopamine hydrochloride is dissolved in water to obtain a dopamine hydrochloride aqueous solution, and then the sodium hydroxide aqueous solution is added, stirred, centrifuged and separated to obtain the PDA nanoparticle (PDA-NPs).

[0017] Further, the concentration of the sodium hydroxide aqueous solution is 1 mol / L, and the ratio of the dopamine hydrochloride to the sodium hydroxide aqueous solution is 18 mg:76.5 μL.

[0018] Further, the matrix material for the shell is polyvinylpyrrolidone with a molecular weight of 360 kDa and 55 kDa, the matrix material for the core is polyvinylpyrrolidone with a molecular weight of 360 kDa, and the matrix material for the backing layer is polyvinylpyrrolidone with a molecular weight of 55 kDa.

[0019] Further, the layer-by-layer assembly method for preparing the programmed nanocarrier microneedle comprises the following steps:

[0020] The polyvinylpyrrolidone solution containing PDA nanoparticles is added to the surface of the polydimethylsiloxane mold, centrifuged, dried, and the above operation is repeated once to obtain the microneedle shell, the polyvinylpyrrolidone solution containing MPLAC nanoparticles is added dropwise to the surface of the mold, centrifuged, dried to obtain the microneedle core, then the polyvinylpyrrolidone solution is continuously added dropwise, centrifuged to obtain the backing layer, and the programmed nanoparticle-loaded microneedle is obtained after demolding.

[0021] The application further provides the programmed nanoparticle-loaded microneedle prepared by the preparation method.

[0022] The application further provides application of the programmed nanoparticle-loaded microneedle in preparation of a product for inhibiting growth or metastasis of tumors in an animal body by photothermal cascade personalized immunotherapy.

[0023] The application further provides application of the programmed nanoparticle-loaded microneedle in preparation of a product for activating a systemic immune response.

[0024] The application further provides application of the programmed nanoparticle-loaded microneedle in preparation of a product for improving an immunosuppressive microenvironment.

[0025] Compared with the prior art, the application has the following advantages and technical effects:

[0026] (1) The programmed nanoparticle-loaded microneedle is formed by a simple layer-by-layer assembly method, can generate an in-situ nano vaccine by first heat ablating a tumor, and then further inhibit tumor growth, metastasis and recurrence by immunotherapy, and has the time sequence of realizing photothermal combined immunotherapy.

[0027] (2) The immunoadjuvant nanoparticles (MPLAC-NPs) contained in the microneedle core prepared by the application can capture TAAs generated by photothermal therapy, enhance the uptake of TAAs and adjuvant CpG by DCs, promote the activation and maturation of BMDCs, promote antigen lysosome escape, realize antigen cross-presentation, and induce the activation and proliferation of Ths and CTLs cells.

[0028] (3) The programmed nanoparticle-loaded microneedle of the application can promote the activation and proliferation of antigen-specific T cells in the secondary lymphoid organs, activate NK cells, induce TNF-α secretion, trigger a systemic immune response, increase the number and activity of antigen-specific T lymphocytes at the tumor site, and reduce the proportion of regulatory T cells (Treg) and M2 type tumor-associated macrophages (TAM), thereby improving the immunosuppressive tumor microenvironment; in addition, the programmed nanoparticle-loaded microneedle patch prepared by the application can inhibit primary tumors, produce an immunological memory effect, and prevent tumor lung metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings, which form a part of the present application, are intended to provide further understanding of the present application and are incorporated herein in conjunction with the description of the present application to explain the present application. In the drawings:

[0030] Figure 1 Particle size distribution, atomic force microscope image of the adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting function prepared in Example 1 of the present application, and UV scan spectrum of MPLAC-NPs and CpG between 200-400 nm;

[0031] Figure 2 Columnar statistics and representative flow histogram of DC2.4 cell and RAW264.7 cell uptake of CpG under different incubation times of the adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting function prepared in Example 1 of the present application;

[0032] Figure 3 Columnar statistics of immunocytotoxicity of the adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting function prepared in Example 1 of the present application;

[0033] Figure 4 Laser confocal microscope image and colocalization coefficient of the adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting function prepared in Example 1 of the present application promoting antigen lysosome escape in immune cells;

[0034] Figure 5 Representative histogram of the level of MHC I, MHC II, CD86, CD40, and CD80 expressed by BMDCs detected by flow cytometry after incubation of the adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting function prepared in Example 1 of the present application with BMDCs, and the corresponding columnar statistics;

[0035] Figure 6 Representative histogram of the proportion of M2 type macrophages detected by flow cytometry after incubation of the adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting function prepared in Example 1 of the present application with RAW264.7 cells, and columnar statistics;

[0036] Figure 7 Particle size distribution and atomic force microscope image of PDA-NPs prepared in Example 2 of the present application;

[0037] Figure 8 The photothermal heating curve of PDA-NPs prepared in embodiment 2 of the present application under 808nm laser irradiation of different concentrations and different powers, and the temperature change curve and thermal imaging diagram of H2O and PDA-NPs under 808nm laser irradiation when the highest temperature;

[0038] Figure 9 The cell survival rate column chart of B16F10 cells after co-incubation with PDA-NPs prepared in embodiment 2 of the present application under the action of laser or not, and the B16F10 dead and live chart under laser irradiation or not;

[0039] Figure 10 The column chart of ATP contained in the supernatant after co-incubation of B16F10 cells with PDA-NPs prepared in embodiment 2 of the present application under the action of laser or not, and the laser confocal microscope image of CRT expression on the surface of B16F10;

[0040] Figure 11 The morphology characterization, mechanical strength, skin insertion and in-vitro photothermal image of the programmed nano-loaded microneedle patch PDA@MPLAC-MNs prepared in embodiment 3 of the present application;

[0041] Figure 12 The cumulative release curve of Cy5-CpG of PDA@Cy5-CpG(PVP360)-MNs and PDA@Cy5-CpG(PVP360 / 55)-MNs prepared in comparative example 1 and 2 of the present application under the action of laser or not;

[0042] Figure 13 The thermal imaging diagram and temperature change curve of B16F10 tumor-bearing mice treated by the programmed nano-loaded microneedle prepared in embodiment 3, comparative example 4, comparative example 5 and comparative example 6 of the present application when the highest temperature within 5min of 808nm laser irradiation;

[0043] Figure 14 The growth curve of primary tumor, body weight curve, survival curve diagram and tumor growth curve diagram of each mouse of B16F10 tumor-bearing mice treated by the programmed nano-loaded microneedle prepared in embodiment 3, comparative example 3, comparative example 4, comparative example 5 and comparative example 6 of the present application;

[0044] Figure 15 The expression of co-stimulatory molecules CD86 and CD80 on the surface of DCs in the tumor-draining lymph nodes of B16F10 tumor-bearing mice treated by the programmed nano-loaded microneedle prepared in embodiment 3, comparative example 3, comparative example 4, comparative example 5 and comparative example 6 of the present application after 3 days of treatment;

[0045] Figure 16Representative flow plots and bar graphs of CD4 + T and CD8 + T cell ratio representative flow plots and bar graphs;

[0046] Figure 17 Representative flow plots and bar graphs of NK cell ratio in spleen cells of B16F10 tumor-bearing mice treated with programmed nanometer-loaded microneedles prepared by the present application Example 3, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 for 3 days;

[0047] Figure 18 Representative flow plots and bar graphs of M2 macrophage cell ratio in spleen cells of B16F10 tumor-bearing mice treated with programmed nanometer-loaded microneedles prepared by the present application Example 3, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 for 3 days;

[0048] Figure 19 Representative flow plots and bar graphs of TNF-α secreting effector T cell ratio in spleen cells of B16F10 tumor-bearing mice treated with programmed nanometer-loaded microneedles prepared by the present application Example 3, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 for 3 days;

[0049] Figure 20 Representative flow plots and bar graphs of CD4 + T, CD8 + T cell ratio representative flow plots and bar graphs;

[0050] Figure 21 Representative flow plots, bar graphs and immunofluorescence images of Tregs ratio in tumor cells of B16F10 tumor-bearing mice treated with programmed nanometer-loaded microneedles prepared by the present application Example 3, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 for 3 days;

[0051] Figure 22 Representative flow plots and bar graphs of M2 TAMs ratio in tumor cells of B16F10 tumor-bearing mice treated with programmed nanometer-loaded microneedle patches prepared by the present application Example 3, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 for 3 days;

[0052] Figure 23Representative flow cytometry plots and columnar statistics of the proportion of effector memory T cells in the spleen cells of B16F10 tumor-bearing mice treated with the programmed nanometer-loaded microneedle prepared in Example 3, Comparative Example 6 of the present application for 3 days, and a photograph of tumor nodules in lung tissue;

[0053] Figure 24 H&E staining images of the heart, liver, spleen, lung, and kidney of B16F10 tumor-bearing mice treated with the programmed nanometer-loaded microneedle prepared in Example 3, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 of the present application. DETAILED DESCRIPTION

[0054] The various illustrative embodiments of the present application will now be described in detail below. This detailed description is merely intended to teach a person skilled in the art further details about the various aspects and features of the present application and is not intended to limit the scope of the application. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and for teaching one skilled in the art to various embodiments of the present application.

[0055] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is also encompassed within the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0057] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0058] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0059] The present application provides a preparation method of a programmed nanometer-loaded microneedle, comprising the following steps:

[0060] Poly-(L-lysine) (PLL, MW 30000-70000), L-arginine (L-Arg) and CpG are assembled into PLAC nanoparticles (PLAC-NPs), and then connected with a mannose ligand to obtain MPLAC nanoparticles (MPLAC-NPs);

[0061] PDA nanoparticles (PDA-NPs) are obtained by self-polymerization of dopamine under heating conditions;

[0062] The programmed nanoparticle-loaded microneedle is prepared by using polyvinylpyrrolidone (PVP) with different molecular weights as a matrix material, the matrix material used for the shell is used for loading the PDA nanoparticles, and the matrix material used for the core is used for loading the MPLAC nanoparticles, and then the matrix material is used as a backing layer to obtain the programmed nanoparticle-loaded microneedle (PDA@MPLAC-MNs).

[0063] In the embodiment of the present application, the preparation method of the MPLAC nanoparticles comprises the following steps:

[0064] The L-arginine aqueous solution and the CpG aqueous solution are added dropwise into the polylysine aqueous solution, and the mixture is shaken vigorously at room temperature for 30 min, centrifuged (10000 rpm, 10 min), and washed with deionized water to obtain the PLAC nanoparticles (PLAC-NPs), the PLAC nanoparticles are resuspended with deionized water, and then the active ester polyethylene glycol mannose (NHS-PEG-Mannose) aqueous solution is added dropwise, the mixture is shaken at room temperature for 2 h, centrifuged (10000 rpm, 10 min), and washed with deionized water to obtain the MPLAC nanoparticles (MPLAC-NPs).

[0065] In the preferred embodiment of the present application, the concentration of the L-arginine aqueous solution is 0.2 mg / mL, the concentration of the CpG aqueous solution is 1 mg / mL, the concentration of the polylysine aqueous solution is 0.4 mg / mL, and the concentration of the active ester polyethylene glycol mannose aqueous solution is 1 mg / mL.

[0066] In the preferred embodiment of the present application, the volume ratio of the L-arginine aqueous solution, the CpG aqueous solution, the polylysine aqueous solution and the active ester polyethylene glycol mannose aqueous solution is 250:75:250:7.5.

[0067] In the embodiment of the present application, the preparation method of the PDA nanoparticles comprises the following steps:

[0068] Dopamine hydrochloride was dissolved in water to obtain a dopamine hydrochloride aqueous solution (the ratio of the amount of dopamine hydrochloride to the amount of water was 18 mg:10 mL) at 50℃ for 15 min, and then sodium hydroxide aqueous solution was added, and stirred at 50℃ for 5 h. After centrifugation (10000 rpm, 30 min), the PDA nanoparticles (PDA-NPs) were separated.

[0069] In a preferred embodiment of the present application, the concentration of the sodium hydroxide aqueous solution is 1 mol / L, and the ratio of the amount of dopamine hydrochloride to the amount of sodium hydroxide aqueous solution is 18 mg:76.5 μL.

[0070] In a preferred embodiment of the present application, the matrix material for the shell is polyvinylpyrrolidone with a molecular weight of 360 kDa and 55 kDa, the matrix material for the inner core is polyvinylpyrrolidone with a molecular weight of 360 kDa, and the matrix material for the backing layer is polyvinylpyrrolidone with a molecular weight of 55 kDa.

[0071] In an embodiment of the present application, the preparation procedure of the programmed nanoparticle-loaded microneedle by layer-by-layer assembly method comprises the following steps:

[0072] The polyvinylpyrrolidone solution containing PDA nanoparticles was added to the surface of the polydimethylsiloxane mold, centrifuged at 3500 rpm to remove the excess solution on the surface of the mold, and then dried. The above operation was repeated once again to obtain the shell of the microneedle. The polyvinylpyrrolidone solution containing MPLAC nanoparticles was added dropwise to the surface of the mold, centrifuged, dried, and the above steps were repeated three times to obtain the inner core of the microneedle. Then, the polyvinylpyrrolidone solution was continuously added dropwise, centrifuged to obtain the backing layer, and dried to obtain the programmed nanoparticle-loaded microneedle.

[0073] In a preferred embodiment of the present application, the concentration of CpG in the polyvinylpyrrolidone solution (PVP360 / 55) containing PDA nanoparticles is 3 mg / mL, and the concentrations of polyvinylpyrrolidone with a molecular weight of 360 kDa and polyvinylpyrrolidone with a molecular weight of 55 kDa are both 65 mg / mL.

[0074] In a preferred embodiment of the present application, the concentration of MPLAC nanoparticles in the polyvinylpyrrolidone solution containing MPLAC nanoparticles is 1.13 mg / mL, and the concentration of polyvinylpyrrolidone with a molecular weight of 360 kDa (PVP360) is 200 mg / mL.

[0075] In a preferred embodiment of the present application, the concentration of the polyvinylpyrrolidone solution (PVP55) for preparing the backing layer is 750 mg / mL.

[0076] The present application also provides a programmed nanoparticle-loaded microneedle prepared by the preparation method.

[0077] The raw materials used in the embodiments of the application are commercially available.

[0078] Room temperature in the embodiments of the application refers to 25±3 DEG C.

[0079] The technical solutions of the application are further described below through examples.

[0080] Example 1

[0081] A preparation method of an immunoadjuvant nanoparticle (MPLAC-NPs) with a targeting function, comprising the following steps:

[0082] (a) L-arginine (L-Arg) aqueous solution (250 μL, 0.2 mg / mL) and CpG aqueous solution (75 μL, 1 mg / mL) are added dropwise into poly-(L-lysine) (PLL, MW 30000-70000) aqueous solution (250 μL, 0.4 mg / mL) in sequence, and the solution is shaken vigorously at room temperature for 30 min, then the solution is centrifuged (10000 rpm, 10 min) and the obtained precipitate is washed with deionized water to obtain PLAC-NPs;

[0083] (b) The obtained PLAC-NPs are resuspended with 800 μL of deionized water, and then NHS-PEG-Mannose aqueous solution (7.5 μL, 1 mg / mL) is added dropwise, and the solution is shaken and reacted at room temperature for 2 h, then the solution is centrifuged (10000 rpm, 10 min) and the obtained precipitate is washed with deionized water to obtain MPLAC-NPs.

[0084] Example 2

[0085] A preparation method of a nano-photothermal agent (PDA-NPs), comprising the following steps:

[0086] (a) 18 mg of dopamine hydrochloride is dissolved in 10 mL of deionized water, and a dopamine hydrochloride aqueous solution is obtained by water bath at 50 DEG C for 15 min;

[0087] (b) 76.5 μL of 1 mol / L sodium hydroxide aqueous solution is quickly added to the obtained dopamine hydrochloride aqueous solution, and the solution is stirred at 50 DEG C for 5 h, and then PDA-NPs are obtained by centrifugation (11000 rpm, 30 min).

[0088] Example 3

[0089] A preparation method of a programmed nanoparticle-loaded microneedle (PDA@MPLAC-MNs), comprising the following steps:

[0090] (a) PVP360 / 55 aqueous solution (360 kDa and 55 kDa PVP 65 mg / mL each, noted as PVP360 / 55) containing PDA-NPs (3 mg / mL) prepared in Example 2 was added to the mold surface, then centrifuged at 3500 rpm to remove the excess solution on the mold surface, and dried in the fume hood, and the above operation was repeated once to obtain the microneedle shell;

[0091] (b) PVP360 / 55 aqueous solution (360 kDa and 55 kDa PVP 65 mg / mL each, noted as PVP360 / 55) containing MPLAC-NPs (CpG concentration 1.13 mg / mL) prepared in Example 1 was added dropwise to the mold surface, centrifuged at 3000 rpm to remove the excess solution on the mold surface, and dried in the fume hood for 1.5 h, and the above operation was repeated three times to obtain the microneedle core;

[0092] (c) PVP55 solution (55 kDa, 750 mg / mL, noted as PVP55) was taken and centrifuged at 3000 rpm to obtain the backing layer, which was dried in the fume hood;

[0093] (d) After drying, the PDA@MPLAC-MNs were demolded.

[0094] Comparative Example 1

[0095] A method for preparing a core-shell structure microneedle, comprising the following steps:

[0096] (a) PVP360 / 55 aqueous solution (360 kDa and 55 kDa PVP 65 mg / mL each, noted as PVP360 / 55) containing PDA-NPs (3 mg / mL) prepared in Example 2 was added to the mold surface, then centrifuged at 3500 rpm to remove the excess solution on the mold surface, and dried in the fume hood, and the above operation was repeated once to obtain the microneedle shell;

[0097] (b) PVP360 aqueous solution containing Cy5-labeled free CpG (Cy5-CpG, purchased from Shengong Bioengineering (Shanghai) Co., Ltd.) (1.13 mg / mL) was added dropwise to the mold surface, centrifuged at 3000 rpm to remove the excess solution on the mold surface, and dried in the fume hood for 1.5 h, and the above operation was repeated three times to obtain the microneedle core;

[0098] (c) PVP55 aqueous solution was taken and centrifuged at 3000 rpm to obtain the backing layer, which was dried in the fume hood;

[0099] (d) After drying, the PDA@Cy5-CpG (PVP360)-MNs were demolded.

[0100] Comparative Example 2

[0101] A method for preparing a core-shell structure microneedle, comprising the following steps:

[0102] (a) PVP360 / 55 aqueous solution (65 mg / mL of PVP of 360 kDa and 55 kDa, denoted as PVP360 / 55) containing PDA-NPs (3 mg / mL) prepared in Example 2 was added to the surface of a polydimethylsiloxane mold, then centrifuged at 3500 rpm to remove the excess solution on the surface of the mold, blown dry in a fume hood, and the above operation was repeated once to obtain the outer shell of the microneedle;

[0103] (b) PVP360 / 55 aqueous solution (65 mg / mL of PVP of 360 kDa and 55 kDa, denoted as PVP360 / 55) containing Cy5-CpG (1.13 mg / mL) was added dropwise to the surface of the mold, centrifuged at 3000 rpm to remove the excess solution on the surface of the mold, dried in a fume hood for 1.5 h, and the above steps were repeated three times to obtain the inner core of the microneedle;

[0104] (c) PVP55 aqueous solution was aspirated and centrifuged at 3000 rpm to obtain the backing layer, which was dried in a fume hood;

[0105] (d) After drying, PDA@Cy5-CpG (PVP360 / 55)-MNs were obtained by demolding.

[0106] Comparative Example 3

[0107] A method for preparing a microneedle loaded with free adjuvant CpG (CpG-MNs), comprising the following steps:

[0108] (a) PVP360 / 55 aqueous solution (65 mg / mL of PVP of 360 kDa and 55 kDa, denoted as PVP360 / 55) was added to the surface of a polydimethylsiloxane mold, then centrifuged at 3500 rpm to remove the excess solution on the surface of the mold, blown dry in a fume hood, and the above operation was repeated once to obtain the outer shell of the microneedle;

[0109] (b) PVP360 aqueous solution containing CpG (1.13 mg / mL) was added dropwise to the surface of the mold, centrifuged at 3000 rpm to remove the excess solution on the surface of the mold, dried in a fume hood for 1.5 h, and the above steps were repeated three times to obtain the inner core of the microneedle;

[0110] (c) PVP55 aqueous solution was aspirated and centrifuged at 3000 rpm to obtain the backing layer, which was dried in a fume hood;

[0111] (d) After drying is completed, the MPLAC-MNs are released from the mold.

[0112] Comparative Example 4

[0113] A method for preparing MPLAC-NPs-loaded microneedles (MPLAC-MNs) comprises the following steps:

[0114] (a) PVP360 / 55 aqueous solution (65 mg / mL of PVP of 360 kDa and 55 kDa, denoted as PVP360 / 55) containing MPLAC-NPs prepared in Example 1 (CpG concentration of 1.13 mg / mL) is added to the mold surface, and then centrifuged at 3500 rpm to remove the excess solution on the mold surface. The mold is placed in a fume hood to dry, and the above operation is repeated once to obtain the microneedle shell;

[0115] (b) PVP360 aqueous solution is added dropwise to the mold surface, centrifuged at 3000 rpm to remove the excess solution on the mold surface, and placed in a fume hood to dry for 1.5 h. The above step is repeated three times to obtain the microneedle core;

[0116] (c) PVP55 aqueous solution is aspirated and centrifuged at 3000 rpm to obtain the backing layer, which is placed in a fume hood to dry;

[0117] (d) After drying is completed, the MPLAC-MNs are released from the mold.

[0118] Comparative Example 5

[0119] A method for preparing PDA-NPs-loaded microneedles (PDA-MNs) comprises the following steps:

[0120] (a) PVP360 / 55 aqueous solution (65 mg / mL of PVP of 360 kDa and 55 kDa, denoted as PVP360 / 55) containing PDA-NPs (3 mg / mL) of Example 2 is added to the mold surface, and then centrifuged at 3500 rpm to remove the excess solution on the mold surface. The mold is placed in a fume hood to dry, and the above operation is repeated once to obtain the microneedle shell;

[0121] (b) PVP360 aqueous solution is added dropwise to the mold surface, centrifuged at 3000 rpm to remove the excess solution on the mold surface, and placed in a fume hood to dry for 1.5 h. The above step is repeated three times to obtain the microneedle core;

[0122] (c) PVP55 aqueous solution is aspirated and centrifuged at 3000 rpm to obtain the backing layer, which is placed in a fume hood to dry;

[0123] (d) After drying is completed, the PDA-MNs are released from the mold.

[0124] Comparative Example 6

[0125] A method for preparing a microneedle loaded with PDA-NPs and PLAC-NPs (PDA@PLAC-MNs) includes the following steps:

[0126] (a) A PVP360 / 55 aqueous solution (360 kDa and 55 kDa PVP, 65 mg / mL each, denoted as PVP360 / 55) containing PDA-NPs (3 mg / mL) prepared in Example 2 was added to the surface of a polydimethylsiloxane mold, then centrifuged at 3500 rpm to remove excess solution on the surface of the mold, dried in a fume hood, and the above operation was repeated once to obtain a microneedle shell;

[0127] (b) A PVP360 / 55 aqueous solution (360 kDa and 55 kDa PVP, 65 mg / mL each, denoted as PVP360 / 55) containing PLAC-NPs (CpG concentration of 1.13 mg / mL, prepared according to the method of Example 1, except that no NHS-PEG-Mannose aqueous solution was added) was added dropwise to the surface of the mold, centrifuged at 3000 rpm to remove excess solution on the surface of the mold, dried in a fume hood for 1.5 h, and the above steps were repeated three times to obtain a microneedle core;

[0128] (c) A PVP55 solution (55 kDa, 750 mg / mL, denoted as PVP55) was centrifuged at 3000 rpm to obtain a backing layer, which was dried in a fume hood;

[0129] (d) After drying, the PDA@PLAC-MNs were removed from the mold.

[0130] In order to better characterize the effect of the programmed nano-particle-loaded microneedle, the following experiments were performed:

[0131] Experimental Example 1

[0132] (1) Particle size, zeta potential determination and morphology characterization of the immunoadjuvant nanoparticles (MPLAC-NPs) with targeting function prepared in Example 1 of the present application: MPLAC-NPs were dispersed into an aqueous solution of 0.12 mg / mL, and the particle size and zeta potential were analyzed by a particle size analyzer. Atomic force microscopy was used to characterize the morphology of the nanoparticles. The successful encapsulation of CpG was verified by scanning the aqueous solutions of CpG and MPLAC-NPs in the wavelength range of 200-400 nm by ultraviolet-visible spectrophotometry.

[0133] The particle size distribution (A), atomic force microscopy (B), and UV-Vis spectra (C) of the targeted immunoadjuvant nanoparticles (MPLAC-NPs) prepared in Example 1 of this invention are shown in the figure. Figure 1 .like Figure 1 As shown in Figure A, the average particle size of MPLAC-NPs is 162.93 ± 6.13 nm, the PDI (aggregation density distribution) is 0.0427, and the particle size exhibits a normal distribution. The morphology of the nanoparticles was observed using atomic force microscopy. Figure 1 (B) The results were consistent with those measured by the particle size analyzer, indicating that the MPLAC-NPs were uniformly spherical with suitable size and uniform particle size. Further measurement of their Zeta potential using the particle size analyzer showed it to be 36.64 ± 1.09 mV.

[0134] (2) Determination of CpG encapsulation efficiency in the targeted immunoadjuvant nanoparticles (MPLAC-NPs) prepared in Example 1 of this invention: The measurement results of the MPLAC-NPs solution at 265 nm were measured by a UV-Vis spectrophotometer. The formula for calculating the CpG encapsulation efficiency is as follows:

[0135] CpG encapsulation efficiency (%) = (Mass of CpG in MPLAC-NPs / Mass of CpG added) × 100%

[0136] The results showed that when the CpG dosage was 75 μg, the CpG encapsulation efficiency in MPLAC-NPs was 64.24 ± 1.43%, indicating that the nanoparticles had a high CpG loading capacity.

[0137] Experimental Example 2

[0138] Example 1: Determination of antigen capture capacity of targeted immune adjuvant nanoparticles (MPLAC-NPs) prepared in Example 1

[0139] A method for preparing nanoparticles with a protein-capturing component includes the following steps:

[0140] Digested B16F10 cells were washed three times with pre-chilled PBS and resuspended in PBS to a density of 1 × 10⁻⁶ cells. 7The cell suspension was quickly frozen in liquid nitrogen for 5 min, and then thawed at 37 °C for 5 min, and the freezing and thawing was repeated 5 times. Subsequently, the cell lysate was collected and centrifuged at 1000 g for 10 min to remove insoluble cell debris to obtain TAAs. The supernatant after centrifugation (containing 220 μg of TAAs) was incubated with MPLAC-NPs (containing 55 μg of CpG) prepared in Example 1 at room temperature for 20 h, and ultrafiltration centrifugation (3500 rpm, 15 min) was performed using an ultrafiltration tube with an Ultracel membrane (100000 NMWL), and the nanoparticles were washed twice with PBS to obtain MPLAC-NPs@TAAs.

[0141] The obtained MPLAC-NPs@TAAs were subjected to capture protein amount and particle size, potential determination:

[0142] The particle size distribution and potential of MPLAC-NPs@TAAs were determined using a particle size analyzer; the antigen capture capacity of MPLAC-NPs was evaluated using the BCA method.

[0143] After capturing the antigen, the particle size of MPLAC-NPs@TAAs increased to 1634.33 ± 47.65 nm, and the Zeta potential changed from positive to negative (-11.65 ± 0.58 mV). The antigen capture amount was 55.79 ± 5.23% of the input antigen amount, and the above results showed that MPLAC-NPs had strong antigen capture capacity.

[0144] Experimental Example 3

[0145] The immune adjuvant nanoparticles (MPLAC-NPs) prepared in Example 1 with targeting function were determined for immune cell uptake:

[0146] The DC2.4 cells / RAW264.7 cells were digested and centrifuged, and then resuspended at a density of 5 × 10 5 The cells were seeded in a 12-well plate at a density of 5 × 10

[0147] The columnar chart of statistics and the representative flow histogram of the CpG uptake of DC2.4 cells and RAW264.7 cells at different incubation times of the adjuvant nanoparticles MPLAC-NPs prepared in Example 1 with DC2.4 cell and RAW264.7 cell targeting functions are shown in Figures 1A and 1B and Figures 1C and 1D, respectively. Figure 2 It can be seen that after incubation of the different preparations with DC2.4 cells for 1 h, the uptake ratios of CpG, PLAC-NPs and MPLAC-NPs were 0.58%, 7.09% and 8.96%, respectively, and after incubation for 4 h, the uptake ratios increased to 34.40%, 63.37% and 68.1%, respectively. After incubation of the different preparations with RAW264.7 cells for 1 h, the uptake ratios of CpG, PLAC-NPs and MPLAC-NPs were 7.03%, 77.37% and 89.93%, respectively, and after incubation for 4 h, the uptake ratios increased to 51.6%, 97.43% and 98.23%, respectively, indicating that the nanoparticles promote the uptake of immune cells by virtue of the appropriate size and surface potential and protect CpG from acidic enzyme degradation, thereby increasing the uptake ratio.

[0148] Experimental Example 4

[0149] Immune cell cytotoxicity assay of the immune adjuvant nanoparticles (MPLAC-NPs) prepared in Example 1 with targeting functions:

[0150] The DC2.4 cells / RAW264.7 cells cultured in the culture flask were trypsinized and diluted with complete culture medium to 1 x 10 5 The diluted cell suspension was inoculated into a 96-well plate at a volume of 100 μL, and after complete adhesion, the original culture medium was removed and 100 μL of different concentrations of CpG, PLAC-NPs and MPLAC-NPs (preparation method as described above, wherein the concentration of CpG was 1.25, 2.5, 5, 10, 20 and 30 μg / mL) were added, and the same volume of fresh culture medium was added to the negative control group. Then, the 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 h, after which the drug-containing culture medium was removed and washed once with PBS, and 100 μL of MTS-containing 1640 culture medium (MTS: 1640 culture medium = 1:5) was incubated with the cells for 30 min, and the absorbance value of each well of cells at 490 nm was measured on a microplate reader, and the cell survival rate was calculated using the following formula: cell survival rate (%) = (absorbance value of experimental group - absorbance value of blank group) / (absorbance value of negative control group - absorbance value of blank group) x 100%.

[0151] The immune cell cytotoxicity columnar chart of the adjuvant nanoparticles MPLAC-NPs prepared in Example 1 with DC2.4 cell (A) and RAW264.7 cell (B) targeting functions is shown in Figure 2. Figure 3It can be seen that when the concentration of CpG is in the range of 1.25-30 μg / mL, the survival rates of the two kinds of immune cells incubated with CpG, PLAC-NPs and MPLAC-NPs are all above 90%, which indicates that the adjuvant itself and the polymer carrier can be ignored in terms of cytotoxicity in the concentration range, and have good biocompatibility.

[0152] Experimental Example 5

[0153] The antigen protein with a fluorescent group obtained from the immune adjuvant nanoparticles (MPLAC-NPs) with targeting function prepared in Example 1 and the lysosome escape of the captured antigen nanoparticles were determined.

[0154] The MPLAC-NPs@TAAs obtained in Experimental Example 2 were diluted with PBS to 1 mg / mL to obtain a TAAs solution, FITC (fluorescent dye fluorescein isothiocyanate) was dissolved in DMSO to prepare a FITC solution of 1 mg / mL, and the TAAs solution and the FITC solution were mixed at a mass ratio of 10:1 at 4°C and incubated with stirring for 16 h, and the reaction mixture was dialyzed (MWCO 1000) with deionized water to obtain MPLAC-NPs@FITC-TAAs.

[0155] In the same way, the MPLAC-NPs prepared in Example 1 were replaced with the PLAC-NPs of Comparative Example 6 to prepare PLAC-NPs@FITC-TAAs.

[0156] DC2.4 cells were inoculated in a confocal dish at a density of 2×10 5 The DC2.4 cells were inoculated in a confocal dish at a density of 2×10

[0157] The laser confocal microscope images and colocalization coefficients of the prepared adjuvant nanoparticles MPLAC-NPs with DC2.4 cell and RAW264.7 cell targeting functions in Example 1 promoting antigen lysosome escape in immune cells are shown in Figure 4 As shown in Figure 4 the green fluorescence of free FITC-TAAs is mostly overlapped with the red fluorescence (97.1%), while the green fluorescence of PLAC-NPs@FITC-TAAs and MPLAC-NPs@FITC-TAAs mostly appears in the cytoplasm, and a small part is overlapped with the red fluorescence of lysosomes, with colocalization coefficients of 31.4% and 16.6%, respectively, indicating that the two groups of constructed nanovaccines successfully promote antigen lysosome escape, which is mainly due to the proton sponge effect induced by PLL and L-Arg, and secondly the mannose receptor-mediated endocytosis allows the rapid and continuous accumulation of mannosylated antigens to DCs, and finally the constitutive recycling of MHC-antigen peptide presentation to the DC cell membrane surface further enhances lysosome escape.

[0158] Experimental Example 6

[0159] The prepared immune adjuvant nanoparticles (MPLAC-NPs) with targeting functions in Example 1 promote the maturation and activation ability of bone marrow-derived dendritic cells (BMDCs) to be determined:

[0160] The BMDCs cultured for 6 days were collected and plated in a 12-well plate (5×10 5 After 6h, the BMDCs special medium containing PBS, free CpG, PLAC-NPs and MPLAC-NPs was added, ensuring that the concentration of CpG contained in each well was 10μg / mL, and the cells were cultured for another 24h, and then the cells were collected and washed once with PBS. CD86-FITC, CD40-PE, CD11c-PerCP-Cy5.5, CD80-APC, MHCClassII(I-Ab)-SuperBright780 and MHCClassI(H-2Kd)-eFluor450 antibodies were used to stain the cells at 4℃ for 30min, and then centrifuged after adding 1mL PBS, the supernatant was discarded, and the cells were resuspended with 300μL 4% paraformaldehyde, and then screened and determined by flow cytometry for the expression levels of CD86, CD40, CD80, MHCII and MHCI on the surface of BMDCs. The preparation method of PLAC-NPs used in the experiment is as described above, and the same applies here.

[0161] Representative histograms of flow cytometry detection of BMDCs expression of MHC I, MHC II, CD86, CD40, CD80 levels and corresponding column charts after incubation of MPLAC-NPs with DC2.4 cells and RAW264.7 cells target function prepared in Example 1 are shown in Figure 5 As shown in Figure 5 As shown in A-B, compared with the control group (containing PBS group), free CpG significantly enhanced the expression of MHC I (15.3%) and MHC II molecules (36.3%). After incubation with BMDCs, the expression levels of MHC I and MHC II of PLAC-NPs increased to 21.2% and 58.0%, because CpG needs to enter the cell to act on TLR9 on the lysosome membrane, and the uptake ability of DCs for free CpG is limited, and PLAC-NPs can enhance the uptake of DCs for CpG. Under the targeting effect of mannose ligand and the induction of structure circulation mechanism, MPLAC-NPs further enhance the internalization of CpG in DCs, and the expression levels of MHC I and MHC II are 5 times and 3.5 times of the control group, which shows that the adjuvant nanoparticles significantly improve the antigen presentation ability of BMDCs. As shown in Figure 5 As shown in C-E, the expression of costimulatory molecules in the CpG group using the immune adjuvant alone was significantly increased compared with the PBS group, and the expression levels of CD86, CD40, and CD80 increased from 16.4%, 13.0%, and 10.5% to 44.0%, 35.4%, and 30.5%, respectively. Because the cationic polymer PLL enhances the interaction of nanoparticles with cell membranes, thereby enhancing the internalization of adjuvants, the proportions of CD86+, CD40+, and CD80+ cells in the PLAC-NPs group increased to 66.8%, 64.6%, and 61.3%, respectively, and MPLAC-NPs further increased the internalization under the action of mannose ligand, and the expression of costimulatory molecules on the surface of BMDCs was further improved to 71.1%, 66.1%, and 66.8%. This shows that MPLAC-NPs have good antigen presentation and the ability to induce the activation and maturation of BMDCs.

[0162] Experimental Example 7

[0163] Determination of the ability of the immune adjuvant nanoparticles (MPLAC-NPs) prepared in Example 1 to promote macrophage polarization:

[0164] The density of 5×10 5RAW264.7 cell suspension at 1 x 105cells / mL was inoculated in 12-well plates, and after adhering, PBS, CpG, PLAC-NPs and MPLAC-NPs (CpG concentration 10 μg / mL) were added to RAW264.7 cells, and incubated for 24 h. The cells were collected with PBS and centrifuged to discard the supernatant. 100 μL of diluted CD11b-PE and F4 / 80-PerCP-Cy5.5 antibodies were added to the cells and incubated at 4°C for 30 min, washed once with PBS, vortexed with 100 μL of fixation buffer, and incubated at room temperature for 60 min in the dark. 1 mL of 1x permeabilization solution was added, and after centrifugation, the CD206-APC antibody was diluted with 1x permeabilization solution, and incubated at room temperature for 60 min in the dark. 1 mL of 1x permeabilization solution was added for washing once, 300 μL of PBS was added for resuspension, and the proportion of M2 macrophages was determined using a flow cytometer.

[0165] The representative histogram (A) and columnar statistical chart (B) of the proportion of M2 macrophages detected by flow cytometry after incubation of the adjuvant nanoparticles MPLAC-NPs prepared in Example 1 with DC2.4 cells and RAW264.7 cells are shown in Figure 6 As shown in Figure 6 , after incubation of PBS, CpG, PLAC-NPs and MPLAC-NPs with RAW264.7 cells for 24 h, the proportions of CD11b + F4 / 80 + CD206 + cells were 22.2% and 16.3%, respectively, and compared with the PBS group, the proportion of M2 macrophages in the CpG group was only slightly reduced. However, when CpG was loaded into PLAC-NPs and MPLAC-NPs, both nanoparticles could significantly reduce the proportion of M2 macrophages to 13.1% and 12.4%, respectively. The mannose ligand further enhanced the internalization of TAMs to CpG, and induced RAW264.7 cells to polarize to M1 type.

[0166] Experimental Example 8

[0167] The particle size, zeta potential and morphology of the nanophotothermal agent (PDA-NPs) prepared in Example 2 were characterized.

[0168] The particle size distribution chart (A) and atomic force microscope chart (B) of the PDA-NPs prepared in Example 2 are shown in Figure 7 The particle size and zeta potential of the prepared PDA-NPs were determined by a particle size analyzer, and the particle size was 142.80 ± 2.98 nm Figure 7A), PDI is 0.0658, and the particle size distribution is normal distribution. The potential is measured as -29.17 ± 0.68 mV, which is related to the catechol structure on dopamine. Under atomic force microscopy Figure 7 B) PDA-NPs present nearly spherical shape, uniform size, consistent with the results of particle size analyzer.

[0169] Experimental Example 9

[0170] The in vitro photothermal efficiency of the nano-photothermal agent (PDA-NPs) prepared in Example 2 was determined:

[0171] First, different concentrations of PDA-NPs (5, 10, 20, 30, 40 μg / mL, respectively) were added to 200 μL centrifuge tubes, and PBS group was set as control. Second, 808 nm laser with power of 1.5 W / cm2was used to irradiate for 5 min, and the temperature was monitored and recorded using thermal imager, and the thermal imaging images of PBS and PDA-NPs (20 μg / mL) at the highest temperature were taken. 2

[0172] The photothermal heating curve of the nano-photothermal agent (PDA-NPs) prepared in Example 2 under different concentrations and different powers of 808 nm laser irradiation, and the temperature change curve of H2O and PDA-NPs groups under 808 nm laser irradiation and the thermal imaging images at the highest temperature are shown in Figure 8 As shown in Figure 8 A, at concentrations of 5, 10, 20, 30, 40 μg / mL, the highest temperature reached by PDA-NPs solution within 5 min was 34.6, 42.9, 52.1, 66.1, 68.1 ℃, respectively, and it can be seen that the temperature rise is concentration-dependent, and PDA-NPs show photostability. Then the temperature change of PDA-NPs with concentration of 20 μg / mL under different powers was determined, and with the increase of power, the highest temperature gradually increased to 43.1, 47, 52.1, 58.8, 64.6 ℃ Figure 8 B). Water was selected as control, and the temperature change curve of PDA-NPs with concentration of 20 μg / mL under 1.5 W / cm2was determined, and the infrared thermal imager was used to take a photo at the highest temperature, as shown in Figure 8 C, D, PDA-NPs can reach a relatively suitable temperature for killing tumors (52.1 ℃) under this concentration and power, confirming that PDA-NPs have good heating effect, and this concentration and power can be used as experimental conditions for subsequent cell experiments.

[0173] Experimental Example 10

[0174] (1) The tumor cell toxicity of the nano-photothermal agent (PDA-NPs) prepared in Example 2 was determined: ​

[0175] B16F10 tumor cells were evenly seeded in 96-well plates at a density of 1×10⁶ cells / well. 4 Cells / well were incubated overnight until cell adhesion was achieved. The culture medium was then removed, and culture medium containing 5, 10, 20, 30, or 40 μg / mL PDA-NPs was added. The PBS and PDA-NPs groups were incubated for 24 h, while the PBS+laser and PDA-NPs+laser groups were incubated for 2 h. Afterward, each well was irradiated with an 808 nm laser (1.5 W / cm²). 2 The cells were incubated for another 24 hours. Cell viability was then calculated using the MTS assay.

[0176] Example 2: Bar graph showing cell viability after co-incubation of B16F10 cells with different concentrations of photothermal agent PDA-NPs under laser irradiation. Figure 9 A and B in the example, Figure 9 As shown in Figure A, without laser irradiation, PDA-NPs exhibited almost no toxicity to B16F10 cells within a concentration range of 5-40 μg / mL, with cell viability remaining close to 100%. However, under 808 nm laser irradiation (1.5 W / cm²), the cell viability was significantly reduced. 2 PDA-NPs convert light energy into heat energy; at concentrations greater than or equal to 20 μg / mL, the high temperature induces tumor cell apoptosis and coagulative necrosis. Figure 9 As shown in Figure B, the cell survival rate was 87.8% when the PDA-NPs concentration was 10 μg / mL. As the concentration increased, the heat generated increased. When the PDA-NPs concentration reached 20 μg / mL, the cell survival rate was only 25.5%, indicating that most cells had died. When the concentration reached 30 and 40 μg / mL, the cell survival rates were only 24.4% and 24.0%, respectively. This shows that PDA-NPs concentrations of 20 μg / mL or higher, under laser irradiation, can achieve a powerful killing effect on tumor cells.

[0177] (2) Assay of tumor cell viability of the photothermal nanoparticles (PDA-NPs) prepared in Example 2:

[0178] Inoculate 5×10 in each small dish 5 B16F10 cells were cultured overnight, then the culture medium was aspirated, and drug-containing medium was added. Cells were divided into four groups: PBS, PDA-NPs, PBS + laser, and PDA-NPs + laser (PDA-NPs concentration was 20 μg / mL for all groups). Two hours after drug addition, the PBS + laser and PDA + laser groups were treated with 1.5 W / cm² water. 2808nm laser irradiation for 5min, and then continued to culture for 12h. The medium was aspirated, and staining was performed according to the instructions of the dead and live double staining kit: 1mL of pre-cooled PBS was added for washing twice, the cells were collected, and the cell precipitate was obtained by centrifugation; first, a staining working solution was prepared (according to a total volume of 1mL, 1mL of 1x buffer, 1μL of 2mM calcein, and 3μL of 1.5mM propidium iodide dye solution were mixed), then 100μL of the staining working solution was added to each tube of cells, and the mixture was mixed, incubated in an incubator for 15min, and then centrifuged to remove the supernatant, washed with PBS for 3 times, resuspended with 300μL of 4% paraformaldehyde, and then added dropwise to a small dish, and observed under a laser confocal microscope.

[0179] Example 2 B16F10 dead and live map of different concentrations of photothermal agent PDA-NPs with or without laser irradiation Figure 9 C, as shown in Figure 9 C, green represents live cells, and red represents dead cells. Without laser irradiation, PBS and PDA-NPs had no obvious cytotoxicity to B16F10 cells. Under 1.5W / cm2 irradiation, PBS did not generate heat, so it had almost no cytotoxicity, while PDA-NPs generated heat, which killed B16F10 cells by protein denaturation, enzyme inactivation, and prevention of DNA replication, resulting in the death of most cells. This result was consistent with the cytotoxicity experiment.

[0180] Experimental Example 11

[0181] (1) ATP secretion assay induced by the nano-photothermal agent (PDA-NPs) prepared in Example 2:

[0182] B16F10 cells were uniformly plated in a 6-well plate at a density of 5×10 5 After the cells adhered, the medium was aspirated, and 1mL of drug-containing medium was added to each well. The groups were PBS, PDA-NPs, PBS+laser, and PDA-NPs+laser (PDA-NPs concentration was 20μg / mL). The PBS group and the PDA-NPs group were cultured for 12h, and the PBS+laser group and the PDA-NPs+laser group were added with the medium for 2h, and then irradiated with 1.5W / cm 2The 808 nm laser irradiation of power for 5 min, continue to culture to 12 h. The supernatant was collected in 2 mL EP tube, and centrifuged once. Before testing ATP, ATP standard solution of different concentrations was prepared in advance, and standard reaction solution was prepared according to a total volume of 5 mL (4.45 mL water, 0.25 mL 20x buffer, 0.05 mL 100 mM DTT, 0.25 mL 10 mM fluorescein and 1.25 μL 5 mg / mL firefly luciferase). White 96-well plates were selected, and the test wells and standard wells were set. 90 μL of standard reaction solution was added to each well, the bioluminescence of the background plate at 560 nm was measured, then 10 μL of test / standard solution was added, and the bioluminescence of the test wells and standard wells at 560 nm was measured after incubation at room temperature for 15 min in the dark. The bioluminescence of the background plate was deducted, and the ATP content in the test sample was calculated by the standard curve method.

[0183] The columnar statistical chart of ATP contained in the supernatant after the nano-photothermal agent (PDA-NPs) prepared in Example 2 was co-incubated with B16F10 cells with or without laser action is shown in Figure 10 A in FIG. 2, as shown in Figure 10 A, PBS as a control group, without laser irradiation, the ATP secreted by the PBS group and the PDA group was 14.07 nM and 15.71 nM, and after laser irradiation, the ATP secreted by the PDA group rose to 36.77 nM, which was significantly different from the other three groups, proving that PTT mediated by PDA-NPs can trigger ICD and promote the release of ATP.

[0184] (2) Measurement of CRT expression on the surface of cell membrane induced by the nano-photothermal agent (PDA-NPs) prepared in Example 2:

[0185] 10 6 μg / mL) was added to each dish. The PBS group and the PDA-NPs group were cultured for 12 h, and the PBS+laser group and the PDA-NPs+laser group were added with culture medium for 2 h, and then irradiated with 1.5 W / cm 2The 808 nm laser power irradiation was continued for 5 min, and the culture was continued for 4 h. The drug-containing culture medium was aspirated, and the cells were incubated with 4% paraformaldehyde at room temperature for 10 min. The cells were washed with ice PBS for 3 times. The cells were incubated with 5% BSA dissolved in PBS for 30 min to block the non-specific binding of antibodies. The cells were incubated with Alexa Fluor 488 fluorescent Anti-Calreticulin antibody diluted 500 times with 1% BSA at room temperature for 1 h. The solution was aspirated, and the cells were washed with PBS for 3 times, each for 5 min. The cells were incubated with DAPI at room temperature for 10 min, and then the DAPI was aspirated, and the cells were washed with PBS for 3 times. 300 μL of PBS was added dropwise, and then the observation was performed under a laser confocal microscope.

[0186] The laser confocal microscope images of the CRT expression on the surface of B16F10 under the action of the laser with or without the nano-photothermal agent (PDA-NPs) prepared in Example 2 are shown in Figure 10 B, as shown in Figure 10 B, the PBS group, the PDA group and the PBS+laser group have almost no CRT green fluorescence, and the green fluorescence of the PDA-NPs+laser group is obviously enhanced, indicating that the expression amount of CRT is obviously increased, and further indicating that the PDA-NPs can induce ICD under the action of the laser as a photothermal agent, and increase the apoptosis of tumor cells.

[0187] Experimental Example 12

[0188] (1) The surface morphology of the PDA@MPLAC-MNs prepared in Example 3 was determined:

[0189] The overall morphology of the microneedle patch was photographed by a general optical microscope, and the result is shown in Figure 11 A, it can be seen that the microneedle is a 5×5 quadrangular pyramid microneedle array, each needle has consistent size and a sharp right quadrangular pyramid tip, and is arranged in order on the base. The outer layer is a PVP360 / 55 layer carrying black PDA-NPs.

[0190] (2) The mechanical strength of the PDA@MPLAC-MNs prepared in Example 3 was determined:

[0191] The mechanical strength of the microneedle was characterized by using a universal tensile testing machine. The 5×5 microneedle patch of the PDA@MPLAC-MNs prepared in Example 3 was flatly pasted on the central part of the lower end pressure plate of the tensile testing machine by using double-sided tape. The upper end pressure plate was adjusted to be infinitely close to the tip of the microneedle. The load load was adjusted to be zero, the trigger force was set to be 0.5 N, and the compression speed was 100 μm / s. The compression load-compression displacement curve was drawn. The result is shown in Figure 11 B, it can be seen that the compression load of the microneedle can reach 0.97 N per needle, which meets the minimum force 0.045 N required for penetrating the stratum corneum, indicating that the PDA@MPLAC-MNs are sufficient to penetrate the skin and subcutaneous tissue.

[0192] (3) The skin insertion ability of PDA@MPLAC-MNs prepared in Example 3 was determined:

[0193] After the back of a 6-week-old female C57BL / 6 mouse was depilated, the mouse was dislocated and killed, the back skin was cut and laid on a glass slide, the excess connective tissue on the skin was cleaned with a cotton ball dipped in normal saline, PDA@MPLAC-MNs prepared in Example 3 were placed on the isolated skin of the mouse, and then a flat and smooth hard object was used to apply pressure for 2.5 min. After pressing, the microneedle patch was removed. Then, an appropriate amount of 4% trypan blue solution was added to the area where the microneedle patch was pressed, and the staining was performed for 10 min. Then, the residual trypan blue solution on the surface of the skin was absorbed with an absorbent paper, and observed and photographed under an optical microscope. The results are shown in FIG. 8C. Figure 11

[0194] As shown in FIG. 8C, after pressing for 2.5 min, the microneedle was removed, and the surface of the skin showed obvious puncture marks. Then, the trypan blue solution was added, and the surface excess solution was absorbed after 10 min of standing, showing blue pinhole marks (FIG. 8C, right), indicating that the microneedle could insert the skin and deliver drugs to the subcutaneous tissue. Figure 11 Figure 11

[0195] (4) The photothermal efficiency of PDA@MPLAC-MNs prepared in Example 3 was determined in vitro:

[0196] An 808 nm laser was used to irradiate for 5 min under the condition of 0.25 W / cm 2 The temperature rise curve of the microneedle was observed using a near-infrared imager, and the data was recorded every 30 s. The temperature change under 4 laser irradiation / cooling cycles was recorded to evaluate the light stability. The results are shown in FIG. 9D. Figure 11

[0197] As shown in FIG. 9D, the temperature of the microneedle could quickly reach 46.2°C after 0.5 min of irradiation, and then the temperature slowly rose to 49.2°C after 4.5 min of continuous irradiation. It can be seen that the photothermal effect of the microneedle is sufficient to kill tumor cells. After the microneedle was subjected to 4 cycles of cooling and heating, it was found that the photothermal performance remained good after the cycle, proving that it has good light stability. Figure 11

[0198] (5) The drug loading of PDA@MPLAC-MNs prepared in Example 3 was determined:

[0199] The PDA@MPLAC-MN microneedles prepared in Example 3 were placed in a centrifuge tube containing PBS buffer. After the microneedles were completely dissolved, the CpG drug loading of the microneedles was calculated according to the fluorescence intensity of the solution.​​​​​

[0200] The drug loading measured by fluorescence spectrophotometer was 0.677 ± 0.008 μg / 100 needles. For subsequent in vivo animal experiments, 5 × 5 microneedle patches were used for each mouse, i.e. 25 microneedles, and the CpG drug dosage for each mouse was 0.169 ± 0.002 μg.

[0201] Experimental Example 13

[0202] In vitro drug release test of PDA@Cy5-CpG(PVP360)-MNs and PDA@Cy5-CpG(PVP360 / 55)-MNs prepared in Comparative Examples 1 and 2:

[0203] Four groups of PDA@Cy5-CpG(PVP360)-MNs, PDA@Cy5-CpG(PVP360 / 55)-MNs, PDA@Cy5-CpG(PVP360)-MNs+laser, and PDA@Cy5-CpG(PVP360 / 55)-MNs+laser were set up, and the microneedle arrays in each group were stuck in a 50 mL centrifuge tube, 20 mL of PBS (pH 7.4) was added, and the laser group was first irradiated with an 808 nm laser at a power of 0.25 W / cm 2 After 5 min of irradiation at a power of 0.25 W / cm Figure 12 .

[0204] As Figure 12As shown, without the effect of laser irradiation, PDA@Cy5-CpG(PVP360)-MNs and PDA@Cy5-CpG(PVP360 / 55)-MNs accumulated 77% and 87% of Cy5-CpG, respectively, after 30 min of release medium immersion, while the PDA@Cy5-CpG(PVP360)-MNs+laser group released 96% at 10 min, and the PDA@Cy5-CpG(PVP360 / 55)-MNs+laser group released 100% at 5 min, which indicates that the release rates of the two matrix materials are relatively close without laser effect, and when PDA-NPs generate heat under the action of laser, the skin transfer coefficient is improved and the molecular heat motion is accelerated, and the PVP360 / 55 with relatively lower molecular weight exhibits faster drug release characteristics, which can realize the programmed release of the PDA-NPs in the shell layer first, followed by the further release and penetration of the MPLAC-NPs in the core in the actual application in vivo.

[0205] Experimental Example 14

[0206] In vivo photothermal effect test of MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, PDA@MPLAC-MNs prepared in Comparative Example 4, Comparative Example 5, Comparative Example 6 and Example 3, respectively:

[0207] Each 6-8 week old female C57BL / 6 mouse was subcutaneously injected with 106 B16F10 cells in the right hind limb, and when the tumor volume of the mouse grew to 50 mm3, the mouse was randomly divided into four groups: MPLAC-MNs+laser, PDA-MNs+laser, PDA@PLAC-MNs+laser, PDA@MPLAC-MNs+Laser, and each group was treated with different microneedles. Subsequently, the mouse was irradiated with a laser with a wavelength of 808 nm at a power of 0.25 W / cm2 for 5 min. At the same time, the temperature of the tumor area was observed and recorded with a thermal imager, and a thermal imaging image was taken when the highest temperature was reached. The results are shown in Figure 13 .

[0208] As shown in Figure 13 , the near-infrared light image shows that the highest temperature of the MPLAC-MNs+laser group reaches 41.8°C, and this group does not contain a photothermal agent, so it mainly relies on the heat absorption and temperature rise of melanin itself, and the temperature is not high enough to kill the tumor. The highest temperatures of the PDA-MNs+laser, PDA@PLAC-MNs+laser, PDA@MPLAC-MNs+laser groups can reach 54.7, 55.1, 55.2°C, Figure 13Figure B shows the specific temperature at each time point. All three groups can quickly reach about 50℃ within 0.5 minutes, indicating that the PDA-NPs in the microneedle shell can generate a good photothermal effect in a short time and remain stable, causing irreversible tumor damage.

[0209] Experimental Example 15

[0210] Evaluation of the tumor growth inhibition effects of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Examples 3, 4, 5, 6, and Example 3:

[0211] Skin was removed from the back of 6-8 week old female C57BL / 6 mice near the right hind limb, and 106 B16F10 cells were subcutaneously inoculated at this location. When the tumor volume reached 50-60 mm, 3 At approximately 10:00 AM, the tumor cells were randomly divided into the following 8 groups: PBS, CpG-MNs, MPLAC-MNs, MPLAC-MNs+laser, PDA-MNs+laser, PDA@PLAC-MNs+laser, PDA@MPLAC-MNs, and PDA@MPLAC-MNs+laser. The microneedle patches were pressed into the tumor tissue for 5 minutes to allow the tissue fluid to fully dissolve the microneedles. Afterwards, the laser group was irradiated with an 808nm laser for 5 minutes (0.25W / cm²). 2 The day of drug administration was recorded as day 0. The long and short diameters of the tumor and the mouse's body weight were recorded daily, and the tumor volume (tumor volume (mm)) was calculated. 3 = Tumor long diameter × Tumor short diameter 2 / 2). When the tumor volume exceeds 800 mm 3 At that time, the mice were euthanized, and mouse survival curves were plotted.

[0212] The growth curves, body weight curves, and survival curves of the primary tumor after treatment, as well as the tumor growth curves of each mouse, are shown in the figure. Figure 14 ,like Figure 14A, C, D, compared with the PBS group, the other groups receiving microneedle treatment showed a significant slowdown in tumor growth, the average tumor volume of the CpG-MNs group reached 450 mm3 on the 6th day, and all mice died within 11 days, the MPLAC-MNs group, the MPLAC-MNs+laser group, and the PDA@MPLAC-MNs group reached the same tumor volume on the 7th and 8th days, and all mice died on the 12th day after administration, the nanoparticles had the effect of protecting CpG from degradation and promoting the uptake of immune cells, but the treatment effect was not obvious because the tumor immunosuppressive microenvironment was not alleviated, and the immunogenicity was low and the effect was poor. However, the laser group containing the photothermal agent showed a particularly obvious effect in slowing down tumor growth, and all laser groups regressed after irradiation, but the local photothermal effect alone released antigens to form an in situ tumor vaccine, and the natural antigen immunogenicity was low enough to induce an effective anti-tumor immune response, and the uneven distribution of the photothermal agent led to the possibility of residual live tumor cells at the tumor edge, so the PDA-MNs+laser group recurred after the 6th day, and all mice died within 18 days of administration. The PDA@PLAC-MNs+laser group and the PDA@MPLAC-MNs+laser group co-delivered TAAs and the immunoadjuvant CpG to the same DCs through nanoparticles after photothermal therapy, then enhanced antigen cross-presentation, activated DCs, induced T cell immune activation, and improved the insufficient activation of the innate immune system, thus achieving better therapeutic effect, and the mice with tumor recurrence appeared on the 13th day after administration, and until the 30th day after administration, the PDA@PLAC-MNs+laser group and the PDA@MPLAC-MNs+laser group had 2 and 3 cured mice, respectively, indicating that the photothermal- immunotherapy microneedle patch can inhibit tumor growth and prolong survival. Figure 14 The 21-day weight change curve measured in B showed that the mice did not experience abnormal weight loss, indicating that the microneedles prepared in Example 3 of the present application had good biosafety.

[0213] Experimental Example 16

[0214] The proportion of DCs promoting tumor-draining lymph node maturation was evaluated for CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Examples 3, 4, 5, 6, and Example 3, respectively.

[0215] C57BL / 6 female mice of 6-8 weeks were selected, after depilation on the back, 106 B16F10 cells were inoculated on the right side of each mouse, and the mice were randomly divided into 8 groups and treated with corresponding drugs: PBS, CpG-MNs, MPLAC-MNs, MPLAC-MNs+laser, PDA-MNs+laser, PDA@PLAC-MNs+laser, PDA@MPLAC-MNs and PDA@MPLAC-MNs+laser. The mice were dislocated and sacrificed on the third day of treatment, and the inguinal lymph nodes were collected. After grinding and filtering of the tumor-draining lymph nodes, the lymphocyte suspension was obtained by red blood cell lysis treatment, centrifuged at 450 g for 5 min, and the supernatant was discarded. Fluorescently labeled antibodies CD86-FITC, CD11c-PerCP-Cy5.5 and CD80-APC were added to the cell precipitate and stained at 4°C for 30 min. After centrifugal washing with 1 mL of PBS, 4% paraformaldehyde was added, and the expression levels of CD86 and CD80 in CD11c+ cells were quantitatively detected using a flow cytometer. The results are shown in Figure 15 .

[0216] As shown in Figure 15 , the expression of CD86 and CD80 in the CpG-MNs group was slightly increased compared with the PBS group (the proportions were 18.4% and 19.6%, respectively). Since free CpG is easily degraded by nucleases in the body and has limited uptake by immune cells, the use of nanoparticle delivery of CpG can increase stability and DC uptake and induce DC activation. The expression of CD86 and CD80 in the MPLAC-MNs group, MPLAC-MNs+laser group and PDA@MPLAC-MNs group was increased compared with the CpG-MNs group. The expression of CD86 and CD80 in the PDA-MNs+laser group was 26.0% and 24.7%, respectively, indicating that the local delivery of photothermal agents under laser irradiation induced the release of TAAs from dying tumor cells, activated part of the DCs, but due to the clearance of TAAs by the innate immune system in the body and the weak immunogenicity of natural antigens, the resulting tumor immune response was limited. In the PDA@PLAC-MNs+laser group, the addition of immunoadjuvant nanoparticles to the photothermal therapy formed a nanovaccine, which co-delivered antigens and adjuvants to the same DCs, promoted antigen cross-presentation, and the expression proportions of CD86 and CD80 increased to 33.8% and 27.0%, respectively. The mannose connected on the surface of the nanoparticles significantly promoted the migration of the nanovaccine to the TDLNs, and the expression of CD86 and CD80 in the PDA@MPLAC-MNs+laser group reached 38.9% and 30.9%, respectively. The expression of CD86 in this group was significantly different from that in the other groups.

[0217] Experimental Example 17

[0218] The CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, PDA@MPLAC-MNs prepared in Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Example 3, respectively, affect the spleen antigen-specific T cells:

[0219] The 6-8 week old C57BL / 6 female mice were selected, and after the back depilation treatment, 106 B16F10 cells were inoculated in the right side of each mouse. The mice were randomly divided into 8 groups and subjected to corresponding drug treatment: PBS, CpG-MNs, MPLAC-MNs, MPLAC-MNs+laser, PDA-MNs+laser, PDA@PLAC-MNs+laser, PDA@MPLAC-MNs and PDA@MPLAC-MNs+laser. The mice were dislocated and sacrificed on the third day of treatment, and the spleen was collected. In the clean bench, a six-well plate containing 1% penicillin-streptomycin-containing RPMI1640 medium was prepared in advance, and the spleen was placed on a cell screen with sterilized tweezers. The spleen was uniformly ground with a 5 mL syringe piston, and the spleen cell suspension was treated with red blood cell lysis solution for 2 min. After stopping lysis, centrifugation, PBS resuspension and centrifugation again, the cell pellet was stained with fluorescently labeled antibodies CD3e-FITC, CD4-PE, CD8a-PerCP-Cy5.5, and the cells were stained at 4°C for 30 min. PBS was washed once, 300 μL of 4% paraformaldehyde was added, and the cells were resuspended and screened. The proportion of Ths and CTLs cells was quantitatively detected by flow cytometry, and the results are shown in Figure 16 .

[0220] As shown in Figure 16 A, the proportion of Th cells in the PDA@MPLAC-MNs+laser group was significantly higher than that in the PBS group (20.0%), the CpG-MNs group (25.6%), the MPLAC-MNs group (25.9%), the MPLAC-MNs+laser group (26.6%), the PDA-MNs+laser group (29.2%), the PDA@PLAC-MNs+laser group (35.4%) and the PDA@MPLAC-MNs+laser group (40.1%). The proportion of Th cells in the PDA@MPLAC-MNs+laser group was twice that of the PBS group. The proportion of CTLs was determined by measuring the proportion of CD8 expression in CD3+T cells, and the results are shown in Figure 16As shown in Figure C, the proportions of CD3+CD8+ T cells in the PDA@PLAC-MNs+laser group and the PDA@MPLAC-MNs+laser group reached 30.6% and 32.0%, respectively, which were significantly higher than those in the PBS group (22.0%) and the CpG-MNs group (25.7%). This demonstrates that the microneedle patch of photothermal combined with immunotherapy in Example 3, after activating DCs, further activated antigen-specific T cells, initiating a powerful adaptive immune response.

[0221] Experimental Example 18

[0222] Effects of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Examples 3, 4, 5, and 6, and Example 3, on spleen NK cells:

[0223] Spleens were collected and processed into spleen cell suspension according to the steps in Experiment Example 17. The suspension was stained with fluorescently labeled antibodies CD3e-FITC and NK1.1-APC antibody at 4℃ for 30 min, washed once with PBS, resuspended in 300 μL of 4% paraformaldehyde, and sieved. The proportion of NK cells was quantitatively detected by flow cytometry. The results are shown in [Figure 1]. Figure 17 .

[0224] like Figure 17 As shown, the proportions of NK cells in the PBS group and the CpG-MNs group were 0.69% and 0.83%, respectively. Due to the enhanced CpG uptake by the nanoparticles, the proportions of NK cells in the MPLAC-MNs group (1.05%), MPLAC-MNs+laser group (1.01%), PDA-MNs+laser group (1.20%), and PDA@MPLAC-MNs group (1.12%) were slightly increased. The enhanced immune response following PTT (Pulsed Therapy) improved immunotherapy sensitivity, resulting in the NK cell proportions in the PDA@PLAC-MNs+laser group and the PDA@MPLAC-MNs+laser group increasing to 2.56 times and 3.38 times that of the PBS group, respectively. Therefore, it can be seen that PDA@MPLAC-MNs in Example 3 can also induce an innate immune response under laser irradiation.

[0225] Experimental Example 19

[0226] Effects of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Examples 3, 4, 5, and 6, and Example 3, on spleen M2 macrophages:

[0227] Spleens were collected and processed into spleen cell suspensions according to the steps in Experiment Example 17. Fluorescently labeled antibodies CD11b-PE and F4 / 80-PerCP-Cy5.5 were added and stained at 4°C for 30 min. After adding 1 mL of PBS and centrifuging to remove the supernatant, 100 μL of IC fixation buffer was added to each sample and stained for 60 min at room temperature in the dark. After the incubation time, each sample was washed once with 1× permeabilization buffer and the supernatant was discarded. CD206-APC antibody diluted with 1× permeabilization buffer was added to each sample and stained at room temperature in the dark for 60 min. After washing once with 1× permeabilization buffer, the sample was resuspended in 300 μL of PBS and sieved. The proportion of M2 macrophages was detected by flow cytometry. The results are shown in 18.

[0228] like Figure 18 As shown, the proportion of M2 macrophages in the control group was 12.2%, while the proportions in the CpG-MNs group (9.06%), MPLAC-MNs group (7.03%), MPLAC-MNs+laser group (7.20%), PDA-MNs+laser group (6.18%), and PDA@MPLAC-MNs group (6.35%) were all slightly reduced. This indicates that the effect of the immune adjuvant CpG alone is insufficient to reverse the immunosuppressive microenvironment in vivo. After PDA-NPs induced ICD under laser treatment, the immunogenicity was enhanced, and the nanovaccine targeted APCs to induce a strong immune response, significantly reducing the proportion of M2 macrophages. The proportion of M2 macrophages in the PDA@MPLAC-MNs+laser group decreased to 2.74%, indicating that the photothermal combined immunotherapy strategy of Example 3 can trigger a strong innate immune response.

[0229] Experimental Example 20

[0230] Effects of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Examples 3, 4, 5, 6, and Example 3 on TNF-α secretion by splenic T cells:

[0231] Spleen was collected and treated into spleen cell suspension according to the procedure in Experimental Example 17, and the cell precipitate was centrifuged, resuspended with complete medium containing cell lysate, cultured in a cell incubator to 56 h, PMA and calcimycin were added, and the culture was continued for 10 h, BFA was added, and the cells were collected after 6 h of further culture, washed once with PBS, incubated with CD3e-FITC and CD4-PE at 4 ℃ for 30 min, washed once with 1 mL of PBS, added 100 μL per tube, vortexed, incubated at room temperature for 30-60 min in the dark, added 1 mL of 1x permeabilization solution per tube, centrifuged at 450 g for 5 min, diluted TNF-α-APC with 1x permeabilization solution, incubated at room temperature for 30-60 min, washed once with 1x permeabilization solution, resuspended with 300 μL of PBS, screened, and the TNF-α secretion of T cells was detected by flow cytometry. The results are shown in 19.

[0232] As shown in Figure 19 , compared with the control group, the TNF-α produced by the treatment group was increased to a certain extent. The proportion of TNF-α produced by Ths and CTLs in the PBS group was 10.5% and 21.6%, respectively. For the group treated only with immunoadjuvant and the group treated only with photothermal treatment, the TNF-α produced by Ths and CTLs was only slightly increased. The TNF-α produced by Ths and CTLs in the PDA@PLAC-MNs+laser group of photothermal combined immunotherapy reached 2.50 times and 1.93 times that of the PBS group, respectively. Under the action of the enhanced targeting internalization of mannose, the proportion of TNF-α produced by Ths and CTLs in the PDA@MPLAC-MNs+laser group reached 28.6% and 45.3%, respectively, indicating that the microneedle patch mediated photothermal immunotherapy of Example 3 effectively promoted the secretion of cytokine TNF-α and activated cellular immunity.

[0233] Experimental Example 21

[0234] Effect of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs and PDA@MPLAC-MNs prepared in Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Example 3, respectively, on the proportion of tumor antigen specific T cells:

[0235] Select 6-8 weeks of C57BL / 6 female mice, after depilation on the back, each right side near the hind leg part of 106 B16F10 cells, the mice were randomly divided into 8 groups and do the corresponding drug treatment: PBS, CpG-MNs, MPLAC-MNs, MPLAC-MNs+laser, PDA-MNs+laser, PDA@PLAC-MNs+laser, PDA@MPLAC-MNs and PDA@MPLAC-MNs+laser, the third day of administration dislocation to death mice, tumor collection. First use of paraformaldehyde fixed tumor 24h, then use 40% sucrose solution dehydration 48h or more, the tissue is placed in OCT embedding, freeze sectioning after blow dry again with PBS rinse twice, each 5min, wipe the water around the tissue, drop CD4-PE, CD8-PE antibody (diluted with PBS 1:200), in the dark wet box incubation 2h, with PBS wash 3 times, each 5min, dry, with DAPI containing mounting medium mounting, finally use inverted microscope observation.

[0236] By immunofluorescence staining and then observed under the inverted microscope CD4 + T cells and CD8 + T cell infiltration, such as Figure 20 As shown in PBS group, CD4 + T cells and CD8 + T cell ratio is very low, CpG-MNs group, MPLAC-MNs group, MPLAC-MNs+laser group, PDA-MNs+laser group, PDA@MPLAC-MNs group tumor tissue CD4 + T cells and CD8 + T cell ratio is low, especially CD4 + T cell content is very little, it is not just immunotherapy or photothermal treatment can not cause effective antigen-specific T cell activation, low immunogenicity of TME makes the delivery of nanoparticles loaded with immune adjuvant also can not play a therapeutic effect, photothermal induced tumor cell apoptosis release antigen, immune effect has not been activated antigen has been rapidly cleared in vivo, compared with the light and heat and immune combined treatment of PDA@PLAC-MNs+laser group and PDA@MPLAC-MNs+laser group effective start of antigen-specific T cell activation, CD4 + T cells and CD8 + T cell infiltration, sufficient to see the effectiveness and necessity of combination therapy.

[0237] Experimental example 22

[0238] The effects of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, and Example 3, respectively, on the proportion of intratumoral regulatory T cells were investigated.

[0239] The tumor was collected from the mouse treated in Experimental Example 21, and the tumor removed from the mouse was ground in 5 mL of RPMI1640 medium containing 1% penicillin-streptomycin using a 5 mL syringe. The obtained mixture was filtered through a 70 μm sieve to obtain a uniform cell suspension. The cell suspension was centrifuged (450 g, 5 min), and the cell pellet was dispersed and lysed with red blood cell lysis solution for 2 min. Subsequently, 10 mL of PBS was quickly added and centrifuged. The tumor single cell suspension was obtained by resuspension with PBS, and the supernatant was discarded by centrifugation. Each tube of the cell pellet was stained with CD3e-FITC and CD4-PE antibodies for 30 min (4°C). After centrifugation, 100 μL of Foxp3 fixation / permeabilization working solution was added to each tube of the cells, and the cells were fixed and permeabilized for 30-60 min at room temperature in the dark. Each tube of the cells was centrifuged after the addition of 1 mL of 1x permeabilization solution. The supernatant was discarded, and each tube of the cell pellet was stained with Foxp3-eflour450 antibody diluted in 1x permeabilization solution for 60 min. Each tube was centrifuged after the addition of 1 mL of 1x permeabilization solution. The cells were resuspended with 300 μL of PBS, transferred to a flow tube through a sieve, and analyzed by flow cytometry to determine the proportion of Tregs.

[0240] The tumor tissue frozen section was obtained according to the procedure in Experimental Example 21. The section was washed twice with PBS for 5 min each time, incubated with 0.3% Triton X-100 at room temperature for 15 min, washed twice with PBS for 5 min each time, and dried. The sample was added dropwise with Foxp3-PE antibody (diluted 1:200 with PBS), and incubated in a dark wet box at room temperature for 2 h. The section was washed three times with PBS for 5 min each time, dried, and mounted with a mounting medium containing DAPI. The section was observed under an inverted microscope, and the results are shown in Figure 21 .

[0241] As shown in Figure 21 , the use of an immune adjuvant alone or photothermal therapy alone can reduce the proportion of Tregs in the TME, and the combination of photothermal therapy and immunotherapy can more significantly reduce the proportion of Tregs. The proportions of Tregs in the PDA@PLACs-MNs group and the PDA@MPLAC-MNs group were 0.089% and 0.075%, respectively, and the proportion of Tregs in the PBS group was 8 times that in the PDA@MPLAC group. The results of immunofluorescence staining Figure 21 C) further confirmed that the PBS group was infiltrated with a large number of Tregs, and that immunotherapy or photothermal therapy alone could only weaken the proliferation of a part of Tregs. Only the combination therapy can significantly reduce the number of Tregs and relieve the immunosuppressive microenvironment of the tumor.

[0242] Experimental Example 23

[0243] Effects of CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs, and PDA@MPLAC-MNs prepared in Comparative Examples 3, 4, 5, and 6 and Example 3, respectively, on the proportion of intratumoral M2 TAMs:

[0244] After the tumor cell suspension was collected, the tumor cells were centrifuged to obtain a cell pellet, and fluorescently labeled antibodies CD11b-PE and F4 / 80-PerCP-Cy5.5 were added to stain the cells at 4°C for 30 min. After centrifugation to remove the supernatant, 1 mL of PBS was added to each sample, followed by the addition of 100 μL of IC fixation buffer. The staining time was 60 min at room temperature in the dark. After the incubation time, each sample was washed once with 1x permeabilization buffer, and the supernatant was discarded. Each sample was then added with 1x permeabilization buffer-diluted CD206-APC antibody, and the staining was performed at room temperature in the dark for 60 min. Each sample was washed once with 1x permeabilization buffer, resuspended in 300 μL of PBS, and sieved. The proportion of M2 TAMs was detected by flow cytometry. The results are shown in Figure 22 .

[0245] As shown in Figure 22 , the proportion of M2 TAMs in the PDA@MPLAC-MNs+laser group and the PDA@PLAC-MNs+laser group was lower than that in the other groups, being 20.1% and 16.9%, respectively. The difference between the two groups was mainly due to the combination of the mannose on the MPLAC-NPs in the microneedle core and the high expression of CD206 on TAMs, which promoted the uptake and enhanced the immune effect. The proportion of M2 TAMs in the CpG-MNs group, the MPLAC-MNs group, the MPLAC-MNs+laser group, the PDA-MNs+laser group, and the PDA@MPLAC-MNs group was 28.3%, 26.3%, 26.1%, 23.5%, and 26.1%, respectively. The proportion of M2 TAMs in the PBS group was as high as 44.0%. It is obvious that the microneedle patch based on the combination of photothermal and immunotherapy reduced the proportion of M2 TAMs in the TME, reshaped the immunosuppressive TME, prevented immune tolerance, and enhanced the anti-tumor immune effect.

[0246] Experimental Example 24

[0247] Effects of PDA@PLAC-MNs and PDA@MPLAC-MNs prepared in Comparative Example 6 and Example 3, respectively, on the proportion of effector memory T cells in tumors and determination of the ability to inhibit lung metastasis:

[0248] (1) Immunological memory assay: On the 3rd day after drug administration, spleens were collected, and spleen cells were treated according to the steps in Experiment Example 17. Then, CD8a-PE, CD62L-PerCP-Cy5.5, CD44-APC, and CD3e-eflour450 antibodies were added and incubated at 4°C for 30 min. The cells were washed once with PBS and finally resuspended with 4% paraformaldehyde. The proportion of effector memory T cells was detected by flow cytometry.

[0249] The results are as follows Figure 23 As shown in Figures A and B, in the control group, CD8 + The TEM proportion was 8.43%, and the CD8 concentrations in the two treatment groups, the PDA@PLAC-MNs+laser group and the PDA@MPLAC-MNs+laser group, were significantly higher. + The proportion of TEM was significantly higher than that of the control group, at 13.7% and 15.2%, respectively, indicating that the microneedle patch of Example 3 can induce an effective immune memory effect under laser treatment.

[0250] (2) Anti-tumor lung metastasis study: Hair was removed from the skin near the right hind limb on the back of 6-8 week old C57BL / 6 female mice, and B16F10 cells (1×10⁻⁶) were subcutaneously injected into this area. 6 (cells / each), when the measured tumor volume grows to 50-60 mm 3 Mice were divided into two groups: PDA@PLAC-MNs+laser and PDA@MPLAC-MNs+laser. After microneedle drug administration, the mice were irradiated with an 808nm laser for 5 minutes (0.25W / cm²). 2 The day of treatment was recorded as day 0. Completely cured mice and female C57BL / 6 mice of the same age were used as the PBS control group. On day 30 of microneedle treatment, each mouse was injected with 2 × 10⁻⁶ ppm via the tail vein. 5 Fourteen days after injection of B16F10 cells, lung tissue samples were collected from mice to observe lung nodules and take photographs.

[0251] The results are as follows Figure 23 As shown in Figure C, the control group exhibited numerous obvious tumor metastases, the PDA@PLAC-MNs+laser group showed only a few smaller tumor metastases, and the PDA@MPLAC-MNs+laser group showed no visible metastases. This demonstrates that treatment with PDA@MPLAC-MNs+laser in mice induces memory effector T cells and inhibits lung tumor metastasis.

[0252] Experimental Example 25

[0253] The CpG-MNs, MPLAC-MNs, PDA-MNs, PDA@PLAC-MNs and PDA@MPLAC-MNs prepared in Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Example 3, respectively, were evaluated for biological safety:

[0254] On the 8th day after microneedle treatment, the mice were euthanized, and the heart, liver, spleen, lung and kidney were collected and immersed in 10 times the volume of 4% paraformaldehyde for 24 h of fixation. The tissues were trimmed before dehydration, and different dehydration procedures were performed according to different organs. The tissues were paraffin-embedded and sectioned at a thickness of 5 μm. The tissue sections were stained with H&E, and observed and photographed under a 20x objective lens using an upright microscope. The results are shown in Figure 24 .

[0255] As shown in Figure 24 , the main organs of the mice treated with different formulations were dehydrated, embedded and sectioned, and stained with an H&E kit. No obvious lesions were observed in the heart, liver, spleen, lung and kidney of the mice under different treatments, indicating that the microneedle patch has good biological safety and no obvious toxic side effects.

[0256] The above merely describes the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or replacements within the technical scope disclosed in the present application can be easily thought of by those skilled in the art, and should be encompassed within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing programmed nanoparticle-loaded microneedles, characterized in that, Includes the following steps: L-arginine aqueous solution and CpG aqueous solution were added dropwise to polylysine aqueous solution, shaken at room temperature, centrifuged and washed to obtain PLAC nanoparticles. The PLAC nanoparticles were resuspended in deionized water, and then active ester polyethylene glycol mannose aqueous solution was added dropwise. The mixture was shaken at room temperature, centrifuged and washed to obtain MPLAC nanoparticles. PDA nanoparticles were obtained by dopamine self-polymerization under heating conditions; A layer-by-layer assembly method was used to prepare programmed nanoparticle-loaded microneedles using polyvinylpyrrolidone (PVP) of different molecular weights as matrix materials. The outer shell matrix materials were PPVP with molecular weights of 360 kDa and 55 kDa, used to load the PDA nanoparticles. The core matrix material was PPVP with a molecular weight of 360 kDa, used to load the MPLAC nanoparticles. Then, a PPVP matrix material with a molecular weight of 55 kDa was used as a backing layer to obtain the programmed nanoparticle-loaded microneedles.

2. The method for preparing programmed nanoparticle-loaded microneedles according to claim 1, characterized in that, The concentration of the L-arginine aqueous solution is 0.2 mg / mL, the concentration of the CpG aqueous solution is 1 mg / mL, the concentration of the polylysine aqueous solution is 0.4 mg / mL, and the concentration of the active ester polyethylene glycol mannose aqueous solution is 1 mg / mL. The volume ratio of the L-arginine aqueous solution, CpG aqueous solution, polylysine aqueous solution, and active ester polyethylene glycol mannose aqueous solution is 250:75:250:7.

5.

3. The method for preparing programmed nanoparticle-loaded microneedles according to claim 1, characterized in that, The preparation method of the PDA nanoparticles includes the following steps: Dopamine hydrochloride was dissolved in water to obtain an aqueous solution of dopamine hydrochloride. Then, an aqueous solution of sodium hydroxide was added, and the mixture was stirred, centrifuged, and separated to obtain the PDA nanoparticles. The concentration of the sodium hydroxide aqueous solution is 1 mol / L, and the ratio of dopamine hydrochloride to sodium hydroxide aqueous solution is 18 mg: 76.5 μL.

4. The method for preparing programmed nanoparticle-loaded microneedles according to claim 1, characterized in that, The layer-by-layer assembly method for preparing programmed nanoparticle-loaded microneedles includes the following steps: A polyvinylpyrrolidone solution containing PDA nanoparticles was added to the surface of a polydimethylsiloxane mold. After centrifugation and drying, a microneedle shell was obtained. A polyvinylpyrrolidone solution containing MPLAC nanoparticles was dropped onto the mold surface. After centrifugation and drying, a microneedle core was obtained. Then, a polyvinylpyrrolidone solution was added dropwise, and after centrifugation, a backing layer was obtained. After drying, the microneedles were demolded to obtain the programmed nanoparticle-loaded microneedles.

5. A programmed nanoparticle-loaded microneedle prepared by the preparation method according to any one of claims 1-4.

6. The application of the programmed nanoparticle-loaded microneedles as described in claim 5 in the preparation of products for inhibiting the growth or metastasis of tumors in animal tissues through photothermal cascade personalized immunotherapy.

7. The application of the programmed nanoparticle-loaded microneedles as described in claim 5 in the preparation of products that activate systemic immune responses.

8. The application of the programmed nanoparticle-loaded microneedles as described in claim 5 in the preparation of products that improve the immunosuppressive microenvironment.

Images