Preparation method and application of soluble microneedle patch for delivering biomineralized nano vaccine

Abstract

The invention discloses a preparation method and application of a soluble microneedle patch for delivering a biomineralized nano vaccine, and relates to the technical field of biological medicines. The soluble microneedle patch comprises a backing layer, a separation layer and microneedles, the microneedle is loaded with a biomineralized nano vaccine; the preparation method of the biomineralized nano vaccine comprises the following steps: adding an antigen and a MnCl2. 4H2O solution into a sugar-free DMEM culture medium, carrying out a first mixing reaction, then adding a photosensitizer solution and an anhydrous CaCl2 solution, and carrying out a second mixing reaction to obtain the biomineralized nano vaccine. The antigen, the manganese ion adjuvant and the photosensitizer are prepared into the biomineralized nano vaccine through a safe, simple and convenient biomineralization method, the biomineralized nano vaccine is locally delivered to a tumor site by using a soluble microneedle, and superficial malignant tumors can be ablated and metastasis and recurrence of the superficial malignant tumors can be effectively inhibited by combining photothermal / photodynamic therapy and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of a soluble microneedle patch for delivering biomineralized nano-vaccines. Background Art

[0002] The incidence and mortality of superficial malignant tumors (melanoma, basal cell carcinoma, squamous cell carcinoma of the skin, etc.) continue to rise, seriously threatening human life and health. Photothermal / photodynamic therapy is an innovative tumor treatment method that is beneficial to superficial malignant tumors. It activates photosensitizers through light sources of specific wavelengths to increase local temperature and ablate tumors. At the same time, it produces reactive oxygen to cause tumor cell damage, effectively controlling the rapid growth of primary tumors. Near-infrared fluorophores are the focus of extensive research in phototherapy. Small molecule photosensitizers have good optical and thermal generation properties, and have significant advantages such as high biocompatibility, rapid metabolism, and high safety. However, free small molecule photosensitizers have poor stability in vivo and in vitro, and are prone to photobleaching. The excited fluorescence intensity gradually weakens or even disappears over time. Encapsulating photosensitizers with nanoparticles can enhance their stability and make them easier to penetrate tissue gaps and biological barriers, significantly improving absorption and utilization.

[0003] Although single phototherapy can effectively ablate in situ tumors, it usually cannot prevent the metastasis and recurrence of tumor cells, and the prognosis of tumors is closely related to it. Therefore, further combination with immunotherapy is crucial for the treatment of tumors that are prone to metastasis and recurrence. At present, immunotherapy focuses on several aspects such as monoclonal antibodies, cytokine therapy, adoptive cell therapy, immune checkpoint blockade therapy, and tumor vaccines. Among them, tumor vaccines use antigens and adjuvants to stimulate the maturation and activation of antigen-presenting cells, further activate specific immune responses and immune memory, effectively prevent tumor recurrence and metastasis, and show good application prospects. In the selection of immune adjuvants, manganese ions promote cGAS to recognize cytoplasmic dsDNA, produce cGAMPs, bind to STING in tumor cells and DCs, induce STING protein transformation, activate TBK1 and downstream signal cascade IRF3, upregulate IFN-β expression, promote DCs maturation, and stimulate a strong anti-tumor immune response. However, free antigens and adjuvants are easily degraded in the body and have low bioavailability. The use of nanocarriers can improve the internalization efficiency of tumor vaccines by cells. Under special physical and chemical conditions that simulate the biological environment, the biomineralization method, which is controlled by biological organic matter to convert ions in solution into solid-phase minerals, has become the research frontier of green synthetic nanomaterials. The biomineralization reaction conditions are mild and have the advantages of simple operation, safety, economy, and environmental friendliness. The obtained nano-products usually have good dispersibility and stability, as well as high biosafety and degradability, and are suitable for the synthesis of integrated tumor vaccines with antigen proteins and adjuvants.

[0004] In the process of tumor treatment, in order to achieve a good therapeutic effect, it is necessary to effectively deliver the therapeutic drugs to the tumor site to improve the anti-tumor efficiency. There are many disadvantages in systemic administration, such as low oral bioavailability, poor patient adaptability of subcutaneous / intravenous administration, and easy off-target effects that cause systemic toxicity. Therefore, it is necessary to develop a safe and efficient anti-tumor drug delivery system. Local drug delivery can bypass the challenges faced by systemic administration, has enhanced therapeutic effects and lower toxicity, and is more conducive to the treatment of superficial malignant tumors. Microneedles are painless, multi-point drug delivery, and can penetrate the stratum corneum of the skin. They are a new type of transdermal drug delivery system that is better than patches. After loading anti-tumor preparations, they can be used for efficient superficial tumor treatment. The present invention intends to develop a tumor nanovaccine and use soluble microneedles to deliver it locally to the tumor site, combined with photothermal / photodynamic therapy and immunotherapy to completely ablate superficial malignant tumors, and effectively inhibit their metastasis and recurrence. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a soluble microneedle patch for delivering biomineralized nano-vaccines to solve the problems existing in the above-mentioned prior art. The soluble microneedle patch provided by the present invention is suitable for the photothermal / photodynamic-immunotherapy of tumors. After administration, 808nm laser irradiation can rapidly heat up and ablate the tumor site, while releasing reactive oxygen, effectively controlling the growth of the primary tumor; the nano-vaccine composed of antigens and manganese ion adjuvants promotes antigen uptake, activates the cGAS-STING pathway, promotes the maturation of antigen-presenting cells, activates effector T cells, activates the body's specific immune response to tumors, and induces immune memory to avoid tumor metastasis and recurrence.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a biomineralized nano-vaccine, comprising the following steps:

[0008] The antigen and MnCl 2 ·4H 2 O solution was added to the sugar-free DMEM medium for the first mixing reaction, followed by the addition of the photosensitizer solution and anhydrous CaCl 2 The solution is subjected to a second mixing reaction to obtain the biomineralized nano-vaccine.

[0009] Furthermore, the photosensitizer is IR820.

[0010] Furthermore, the MnCl 2 ·4H 2 The concentration of the O solution is 0.1 M, and the anhydrous CaCl 2 The concentration of the solution is 1M;

[0011] The antigen, the sugar-free DMEM medium, the MnCl 2 ·4H 2 O solution and the anhydrous CaCl 2 The mass-to-volume ratio of the solution is (0.5-4) mg:1 mL:(5-20) μL:(5-20) μL.

[0012] Furthermore, the temperature of the first mixing reaction and the second mixing reaction are both 37° C., and the time is both 24 hours.

[0013] The present invention also provides a biomineralization nano-vaccine prepared according to the above preparation method.

[0014] The present invention also provides the use of the above-mentioned biomineralized nano-vaccine in the preparation of a soluble microneedle patch.

[0015] The present invention also provides a soluble microneedle patch for delivering biomineralized nano-vaccines, comprising a backing layer, a separation layer and microneedles;

[0016] The microneedles are loaded with the above-mentioned biomineralized nano-vaccine.

[0017] Furthermore, the backing layer is an adhesive tape.

[0018] Furthermore, the shape of the microneedle is a cone, a triangular pyramid, a quadrangular pyramid, a pentagonal pyramid, a hexagonal pyramid, a heptagonal pyramid or an octagonal pyramid.

[0019] The tip height of the microneedle is 600-1000 μm, the side length of the base of the microneedle is 50-500 μm, and the center distance of the microneedle is 100-900 μm.

[0020] The separation layer is one of the soluble or temperature-sensitive materials such as low molecular weight sodium hyaluronate (HA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), poly (N-isopropylacrylamide-co-butyl acrylate), PNIPAM-B, etc., with a concentration of 100-1000 mg / mL;

[0021] The microneedle is one of the high molecular weight polymer materials such as sodium hyaluronate, gelatin, PVP, chitosan, etc., with a concentration of 20 to 200 mg / mL.

[0022] The present invention also provides a method for preparing the above-mentioned soluble microneedle patch, comprising the following steps:

[0023] Adding a high molecular weight soluble polymer containing the biomineralized nano-vaccine into a mold having microneedle holes, centrifuging to remove excess material on the mold surface, and vacuum drying and solidifying to obtain a microneedle body;

[0024] Adding a low molecular weight soluble polymer to the mold, centrifuging, removing excess material on the mold surface, and drying at room temperature overnight to obtain a microneedle separation layer;

[0025] After attaching the backing layer, the microneedles are demoulded to obtain the soluble microneedle patch;

[0026] The high molecular weight refers to a molecular weight ≥300 kDa; the low molecular weight refers to a molecular weight ≤10 kDa.

[0027] The present invention discloses the following technical effects:

[0028] The biomineralization nano vaccine provided by the invention is prepared from antigens, manganese ions and photosensitizers through a biomineralization method, with mild preparation conditions and a simple method.

[0029] The soluble microneedle patch provided by the present invention is suitable for the combined photothermal / photodynamic-immunotherapy of tumors. After administration, 808nm laser irradiation can rapidly heat up and ablate the tumor site, while releasing reactive oxygen species to effectively control the growth of the primary tumor. The nanovaccine composed of antigens and manganese ion adjuvants promotes the maturation of antigen-presenting cells by promoting antigen uptake and activating the cGAS-STING pathway, activates effector T cells, and activates the body's specific immune response against tumors, while inducing immune memory to avoid tumor metastasis and recurrence.

[0030] The soluble microneedle patch of the present invention is significantly suitable for the combined photothermal / photodynamic-immunotherapy of superficial malignant tumors. Compared with systemic administration, local administration has higher bioavailability and lower systemic phototoxicity; compared with patches, it is easier to penetrate the skin barrier; compared with intratumoral / peritumoral injection, microneedles are painless, have adjustable shapes, and can be evenly administered at multiple points, and can be used as a drug reservoir to continuously release drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 UV spectra of free IR820, OCMPNPs, and OCMPI NPs;

[0033] Figure 2The photothermal and photodynamic effects of biomineralized nanoparticles and their microneedle patches; A is the temperature rise of OCMPI MNs within 5 minutes under 808nm laser irradiation of different powers; B is the temperature rise of OCMPI MNs and Blank MNs under 0.35W / cm 2 Temperature rise within 5 min of irradiation under different power levels; C is the temperature rise of OCMPI MNs and Blank MNs at 0.35 W / cm 2 Thermal images taken at different time points within 5 minutes of irradiation under power of 0.35 W / cm 2 Figure 1 shows the temperature rise in three cycles of 5 min on / 5 min off under 808 nm laser irradiation; E shows the test results of the ability of different preparations to induce intracellular ROS production under conditions of laser irradiation or not;

[0034] Figure 3 Figure 1 is a graph showing the results of the ability of biomineralized nanoparticles to induce ICD in tumor cells under laser irradiation or not; A is the expression of CRT on the surface of B16-OVA cells; B is the expression of HMGB1 in B16-OVA cells; C is a statistical graph of ATP content in the supernatant of different groups;

[0035] Figure 4 The figures are the detection diagrams of the toxicity of biomineralized nanoparticles to dendritic cells, the ability of dendritic cells to take up free antigens and nanoparticles, and the ability of biomineralized nanovaccines to promote the maturation of BMDCs in vitro; A is a representative flow cytometry scatter plot of the toxicity of biomineralized nanoparticles to dendritic cells; B is a detection diagram of the free antigens and the antigens loaded in the nanoparticles being taken up by DCs at 1h, 4h and 12h; C is a statistical diagram of the data of B; DF are statistical diagrams of the expression ratios of the co-stimulatory molecules CD80, CD86 and CD40 on the surface of BMDCs, respectively;

[0036] Figure 5 Characterization diagram of the microneedle patch loaded with biomineralized nanovaccine; A is an optical microscope image of MN loaded with OCMPI NPs; B is an image of neatly arranged micropores remaining after the microneedle is removed after piercing the skin; C is a compression load-displacement curve;

[0037] Figure 6 The figures are the growth curves of the orthotopic tumor (A), the growth curves of the distal tumor (B) and the statistical chart of the survival period (C) of each group of mice;

[0038] Figure 7 The lung images of mice in each group;

[0039] Figure 8The results of the test on the ability of the microneedle patch loaded with biomineralized nano-vaccine to activate the body's immune system; A is a representative FACS test of DCs maturation in the lymph nodes of each group of mice; B and C are CD4 + T cell ratio and CD8 + T cell ratio statistics; D and E are CD4 + T cells and CD8 + T cell proliferation ability statistics; F is the M2 TAMs ratio in the spleen; G and H are the CD4 + T cell ratio and CD8 + T cell proportion statistics; I is the M2 TAMs proportion statistics in tumors;

[0040] Fig. 9 To characterize the CD4 + and CD8 + Central memory T cells (CD44 + CD62L + ) ratio was detected; A is the FACS detection diagram of each group of mice; B and C are the CD4 + and CD8 + Central memory T cells (CD44 + CD62L + ) ratio. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The microneedle template used in the PDMS mold used in the following examples or comparative examples is set to a 5×5 microneedle array, the microneedle is a regular tetrahedron with a height of 1000 μm and a bottom side length of 500 μm, and the center distance is 900 μm. The preparation method is: polydimethylsiloxane (PDMS) prepolymer and curing agent (STLGARD TM 184Silicone Elastomer Curing Agent) was mixed evenly in a mass ratio of 10:1, and after vacuuming to remove bubbles, the mixture was poured into a container containing a microneedle template, and the bubbles were removed again, followed by curing at 60°C for 5h, and the PDMS mold was obtained after peeling.

[0047] Example 1

[0048] A method for preparing a soluble microneedle patch for delivering biomineralized nanovaccines:

[0049] (a) Preparation of biomineralized nanoparticles OCMPI NPs:

[0050] Dissolve 2 mg (0.5-4 mg can achieve the same effect) of model antigen chicken ovalbumin OVA in 1 mL of sugar-free DMEM medium, add 10 μL (5-20 μL can achieve the same effect) of MnCl 2 ·4H 2 O solution (0.1 M) and placed in 37°C, CO 2 After 24 h of standing in a cell culture incubator with a concentration of 5%, 100 μL (50-200 μL can achieve the same effect) IR820 (1 mg / mL) and 10 μL (5-20 μL can achieve the same effect) anhydrous CaCl were added.2 The solution (1 M) was returned to the incubator and allowed to stand for 24 h, centrifuged, washed three times with deionized water, and the product was collected to obtain biomineralized nanoparticles, which were recorded as OCMPI nanoparticles.

[0051] (b) Preparation of microneedle patch:

[0052] 150 μL of high molecular weight sodium hyaluronate (300 kDa) solution containing OCMPI nanoparticles (OVA concentration of 6.65 mg / mL) was added to the PDMS mold, excess material on the mold surface was removed, vacuum dried and solidified, and the process was repeated to fill the microneedle cavity to obtain the microneedle body; then, low molecular weight sodium hyaluronate solution (5 kDa) was added to the PDMS mold, centrifuged, excess material on the mold surface was removed, and a separation layer was obtained after drying at room temperature overnight; the microneedles were demolded using tape as a backing layer to obtain the microneedle patch OCMPI MNs.

[0053] Comparative Example 1

[0054] This comparative example is the preparation of microneedle patch OCPMNs:

[0055] (a) Preparation of biomineralized nanoparticles OCPNPs:

[0056] The model antigen chicken ovalbumin OVA (2 mg) was dissolved in 1 mL of sugar-free DMEM medium and placed in a 37°C, CO 2 After 24 h of stabilization in a cell culture incubator with a concentration of 5%, 10 μL of anhydrous CaCl 2 The solution (1 M) was returned to the incubator and allowed to stand for 24 h, centrifuged, washed three times with deionized water, and the product was collected and recorded as OCP nanoparticles.

[0057] (b) Preparation of microneedle patch:

[0058] 150 μL of high molecular weight sodium hyaluronate (300 kDa) solution containing OCP nanoparticles (OVA concentration of 6.65 mg / mL) was added to the PDMS mold, excess material on the mold surface was removed, vacuum dried and solidified, and the process was repeated to fill the microneedle cavity to obtain the microneedle body; then, low molecular weight sodium hyaluronate solution (5 kDa) was added to the PDMS mold, centrifuged, excess material on the mold surface was removed, and a separation layer was obtained after drying at room temperature overnight; the microneedle was demolded using tape as a backing layer to obtain a microneedle patch OCPMNs.

[0059] Comparative Example 2

[0060] This comparative example is the preparation of OCMPMNs:

[0061] (a) Preparation of biomineralized nanoparticles OCMPNPs:

[0062] The model antigen chicken ovalbumin OVA (2 mg) was dissolved in 1 mL of sugar-free DMEM medium and 10 μL MnCl was added. 2 ·4H 2 O solution (0.1 M) and placed in 37°C, CO 2 After 24 h of stabilization in a cell culture incubator with a concentration of 5%, 10 μL of anhydrous CaCl 2 The solution (1 M) was returned to the incubator and allowed to stand for 24 h, centrifuged, washed three times with deionized water, and the product was collected and recorded as OCMP nanoparticles.

[0063] (b) Preparation of microneedle patch:

[0064] 150 μL of high molecular weight sodium hyaluronate (300 kDa) solution containing OCMP nanoparticles (OVA concentration of 6.65 mg / mL) was added to the PDMS mold, excess material on the mold surface was removed, vacuum dried and solidified, and the process was repeated to fill the microneedle cavity to obtain the microneedle body; then, low molecular weight sodium hyaluronate solution (5 kDa) was added to the PDMS mold, centrifuged, excess material on the mold surface was removed, and a separation layer was obtained after drying at room temperature overnight; the microneedles were demolded using tape as a backing layer to obtain OCMPMNs.

[0065] Comparative Example 3

[0066] This comparative example is the preparation of microneedle patch Blank MNs:

[0067] A high molecular weight sodium hyaluronate solution (300 kDa) without nanoparticles was added to the PDMS mold, excess material on the mold surface was removed, vacuum dried and solidified, and the process was repeated to fill the microneedle cavity to obtain the microneedle body; then a low molecular weight sodium hyaluronate solution (5 kDa) was added to the PDMS mold, centrifuged, excess material on the mold surface was removed, and a separation layer was obtained after drying at room temperature overnight; the microneedles were demolded using tape as a backing layer to obtain a microneedle patch Blank MNs.

[0068] Experimental Example 1

[0069] The particle size and distribution of the OCMPI NPs loaded with photosensitizer, antigen and immune adjuvant prepared in step (a) of Example 1 were measured using a Nano ZS particle size analyzer. The OVA loading amount was detected using a BCA kit; the elemental content of metal ions in the nanoparticles was quantified using ICP-MS; and the successful loading of IR820 was confirmed using an ultraviolet spectrophotometer.

[0070] Table 1 shows the particle size and polydispersity coefficient, Zeta potential and OVA encapsulation efficiency of OCMPI NPs, OCPNPs and OCMP NPs prepared in step (a) of Example 1, Comparative Example 1 and Comparative Example 2 of the present invention. The data show that the particle sizes of OCMPI NPs, OCPNPs and OCMP NPs are 242.57±3.38nm, 217.13±1.94nm and 209.67±0.80nm, respectively, with good dispersibility; the Zeta potentials are all negative, namely -17.87±0.24mV, -19.40±0.08mV and -20.47±0.12mV, respectively. The Mn content in OCMPI NPs was measured to be 13.53% by ICP-MS. Figure 1 Figure 2 shows the UV spectra of free IR820, OCMP NPs, and OCMPI NPs. Compared with OCMP NPs, the UV absorption peak corresponding to free IR820 was observed in OCMPI NPs, and a slight red shift was observed.

[0071] Table 1 Particle size, polydispersity coefficient, Zeta potential and OVA encapsulation efficiency of biomineralized nanoparticles in each group

[0072]

[0073] Experimental Example 2

[0074] The OCMPI NPs and OCMPI MNs prepared in Example 1 of the present invention were used to study the effects of photothermal / photodynamic therapy in vitro.

[0075] (a) In vitro temperature rise and photostability studies:

[0076] A near-infrared thermal imager was used to observe and record the 5×5 OCMPI MNs microneedle array at different powers (0.25 W / cm 2 , 0.35W / cm 2 , 0.45W / cm 2 ) Temperature rise within 5 min under 808 nm laser irradiation; Different preparations (Blank MNs, OCMPINPs, OCMPI MNs) at a power of 0.35 W / cm 2 The temperature rise of OCMPI MNs within 5 minutes under 808 nm laser irradiation; and the temperature rise of OCMPI MNs under 0.35 W / cm 2 The photostability was evaluated by three cycles of heating with 5 min on / 5 min off under 808 nm laser irradiation.

[0077] Figure 2 A is the OCMPI MNs prepared in Example 1 of the present invention at different powers (0.25 W / cm 2, 0.35W / cm 2 , 0.45W / cm 2 The temperature rises within 5 minutes under 808nm laser irradiation. The temperature rises rapidly within 30 seconds of laser irradiation. When the power is 0.35W / cm 2 The temperature can reach 53.7℃ within 1 minute and maintain high temperature within 5 minutes. Figure 2 B is the OCMPI MNs and Blank MNs prepared in Example 1 and Comparative Example 3 at 0.35 W / cm 2 The temperature of Blank MNs remained almost unchanged within 5 minutes of irradiation at high power, while the temperature of OCMPI MNs increased significantly. Figure 2 C is OCMPI MNs and BlankMNs at 0.35W / cm 2 The thermal imaging photos taken at different time points within 5 minutes of irradiation at high power more intuitively demonstrate the heating ability of OCMPIMNs after laser irradiation. Figure 2 D is OCMPI MNs at a power of 0.35 W / cm 2 The temperature rise in three cycles of 5min on / 5min off under 808nm laser irradiation is shown in the figure. As shown in the figure, the temperature in the third irradiation cycle is still above 50°C, proving that the photosensitizer carried by the biomineralized nanoparticles loaded in the microneedles has good photostability.

[0078] (b) In vitro photodynamic therapy efficacy assessment:

[0079] DCFH-DA probe was used to quantitatively detect the generation of intracellular reactive oxygen species (ROS). Mouse melanoma cells (B16-OVA) were seeded in 12-well plates, with 5×10 5 After overnight culture, the cells were replaced with fresh culture medium containing different sample solutions (OVA equivalent concentration was 20 μg / mL). After 12 h of culture, the cells were gently washed once with PBS to remove free nanoparticles. 1 mL of DCFH-DA (D6883, Sigma) with a final concentration of 10 μM (4.87 μg / mL) was added to the serum-free culture medium. The cells were incubated at 37°C in the dark for 30 min, and the cells were washed once with serum-free culture medium to remove excess probes. 1 mL of serum-free culture medium was added to each well. The laser group was irradiated with 808 nm laser, and the cells were washed once with PBS. The cells were collected by centrifugation and detected by FACS.

[0080] Figure 2 E in the figure shows the ability of different preparations to induce intracellular ROS production under conditions of laser irradiation or not. Figure 2 As shown in Figure E, after laser irradiation of biomineralized nanoparticles containing photosensitizer IR820, the proportion of cells containing ROS increased significantly, proving that ROS can be produced for photodynamic therapy.

[0081] (c) In vitro immunogenic cell death (ICD) induction studies:

[0082] The ability of photothermal-induced ICD was evaluated by studying the extracellular release of calreticulin (CRT), high-mobility group box 1 (HMGB1) and adenosine triphosphate (ATP) secretion levels. B16-OVA cells were seeded in 12-well plates with 5×10 cells per well. 5 After overnight culture, fresh culture medium containing different sample solutions (OVA equivalent concentration is 20 μg / mL) was replaced. After 2 hours of culture, the laser group was irradiated with 808 nm laser and incubated for 10 hours; the non-laser group was incubated for 12 hours. The supernatant was collected and ATP was quantitatively detected using an ATP detection kit, and the cells were collected for staining and detected using FACS.

[0083] Figure 3 Middle A shows the expression of CRT on the surface of B16-OVA cells. The CRT expression ratio in the OCMPI+L group was significantly increased; Figure 3 Middle B shows the expression of HMGB1 in B16-OVA cells. The negative proportion of HMGB1 in the OCMPI+L group increased significantly, indicating that HMGB1 was secreted outside the cells; Figure 3 Middle C shows that the ATP content in the supernatant of the OCMPI+L group was significantly increased. In summary, the OCMPI+L group significantly induced tumor ICD and released damage-associated molecular patterns (DAMPs).

[0084] Experimental Example 3

[0085] The OCMPI NPs prepared in step (a) of Example 1 of the present invention were used to study their in vitro cytotoxicity, cellular uptake and ability to promote BMDCs maturation and activation.

[0086] (a) In vitro cytotoxicity studies:

[0087] After incubating the preparations in different groups with BMDCs for 24 h, the cells were collected and incubated and stained with APC-Annexin V and 7-AAD viability staining solution, and the survival of BMDCs was detected by FACS analysis.

[0088] Figure 4 A in the figure is a representative flow cytometry plot, and the cell viability is higher than 90%, indicating that free OVA, OCP, OCMP and OCMPI have no obvious toxicity to BMDCs.

[0089] (b) Cellular uptake studies:

[0090] OVA was labeled with FITC to prepare OCMPI NPs, which were co-cultured with DC2.4 cells and FITC was detected by FACS. +A free OVA-FITC group was set up to investigate the effects of nanoparticles and incubation time on the antigen uptake by DCs.

[0091] Figure 4 Figures B and C are the percentages of free antigen and antigen loaded in nanoparticles taken up by DCs at 1h, 4h and 12h. The results show that with the increase of time, the uptake of antigen by DCs gradually increased, and the antigen uptake of nanoparticles by cells was significantly higher than that of free antigen. At 12h, the uptake of OCMPI NPs group was close to 100%.

[0092] (c) Study on the ability to promote BMDCs maturation and activation:

[0093] B16-OVA cells were inoculated in the chamber of the Transwell plate and co-incubated with PBS, Free OVA, OCP NPs, OCMP NPs, and OCMPI NPs. The laser group was irradiated with 808 nm laser for 5 min. Immature BMDCs were inoculated in the Transwell plate, and BMDCs were collected after co-culture. They were stained with fluorescently labeled antibodies CD11c, CD40, CD80, and CD86, and the expression of co-stimulatory molecules on the surface of BMDCs was detected by FACS.

[0094] Figure 4 DF in the middle is the expression ratio of co-stimulatory molecules CD80, CD86 and CD40 on the surface of BMDCs obtained by FACS analysis. As shown in the figure, compared with the PBS group, the presence of manganese ions helps promote the maturation and activation of BMDCs. In addition, due to the light-induced ICD of tumor cells, DAMPs are released, further increasing the proportion of mature BMDCs.

[0095] Experimental Example 4

[0096] The morphology of the OCMPI MNs prepared in Example 1 of the present invention was characterized:

[0097] The overall morphology was observed using an ordinary optical microscope. The mouse back skin was depilated, and the microneedle patch was vertically inserted into the mouse back skin for a period of time, and photos were taken to record the skin condition after insertion. A universal tensile testing machine was used to characterize the mechanical strength of the microneedle.

[0098] like Figure 5 As shown in Figure A, the MN loaded with OCMPI NPs appears light green under an optical microscope, and the microneedles are regular tetrahedral structures with complete structures. Figure 5 As shown in B, the microneedles penetrate the skin and are removed, leaving neatly arranged micropores. Figure 5 C in the middle is the compressive load-displacement curve, which proves that the prepared OCMPI MNs have the ability to pierce the skin.

[0099] Experimental Example 5

[0100] 1. Experimental Methods

[0101] A B16-OVA subcutaneous tumor model was established on the right back of C57BL / 6 mice. 3 At about 14:00, B16-OVA cells were subcutaneously inoculated on the left back to establish a distal tumor model. After inoculation, the mice were randomly divided into groups, and the orthotopic tumors were treated with OCMPI MNs and each control group, and the laser group was irradiated with 808nm laser for 5 minutes.

[0102] 1. The in vivo anti-tumor effect of OCMPI MNs prepared in Example 1 of the present invention was studied based on photothermal / photodynamic-immunotherapy synergistic therapy:

[0103] (a) Evaluation of therapeutic effects on orthotopic and distal tumors in mice:

[0104] Monitor the weight of the mice, the size of the orthotopic tumor, whether the distal tumor grows and its size. The tumor volume is calculated according to the following formula: Tumor volume = long diameter × short diameter 2 / 2. For tumors up to 2000mm 3 The mice were euthanized and the survival curves were drawn.

[0105] Figure 6 Figures A and B show the growth curves of the orthotopic tumor and distal tumor. As shown in the figure, 6 mice in the OCMPI MNs+L group were completely cured, while OCMPI NPs+L had limited inhibitory effect on tumors, and only a small number of mice were cured. Figure 6 As shown in C, the survival time of mice in the OCMPI MNs+L group and the OCMPI NPs+L group was prolonged.

[0106] (b) Evaluation of the effect of tumor re-challenge on cured mice:

[0107] On day 50 of treatment, the tumor-free cured mice with complete ablation of the in situ tumor and no growth of the distal tumor were re-challenged with tumor cells. That is, B16-OVA cells were subcutaneously inoculated again near the in situ tumor to observe whether the tumor would grow, in order to study whether they had acquired specific immune memory protection.

[0108] like Figure 6 As shown in C, the cured mice were re-challenged with tumor cells on the 50th day. The results showed that there was still no tumor growth in the mice in the OCMPI MNs+L group, and the cured mice were able to survive for more than 300 days.

[0109] (c) Anti-tumor lung metastasis study in mice:

[0110] After 7 days of treatment, the uncured mice underwent surgery and were injected with B16-OVA cells (1×10 5 The mice were euthanized 28 days later, and the lung tissues were collected and photographed.

[0111] like Figure 7 As shown, except for the OCMPI MNs+L group, the other groups of mice had tumor nodules of different sizes and numbers in the lungs, indicating that the OCMPI MNs+L group could effectively control tumor lung metastasis.

[0112] 2. Study the anti-tumor mechanism of OCMPI MNs prepared in Example 1 of the present invention based on photothermal / photodynamic-immunotherapy synergistic therapy:

[0113] On the 9th day of treatment, the mice were killed, and the bilateral tumor-draining lymph nodes, spleen and primary tumor were separated and removed, and single cell suspensions were prepared by grinding, cell sieve filtration, red blood cell lysis and centrifugation purification. For the lymph node single cell suspension, after staining with fluorescently labeled antibodies CD11c, CD40 and CD86, FACS was used to detect the ability of the microneedle patch of the present invention to promote DCs maturation and activation. For the spleen and tumor single cell suspensions, FACS was used to detect cytotoxic T lymphocytes (CTLs, CD3 + CD8 + ), helper T cells (Ths, CD3 + CD4 + ) and M2 macrophages (CD11b + F4 / 80 + CD206 + ) ratio, and use Ki67 + The proliferation capacity of effector T cells in the spleen was detected to evaluate the recruitment and activation levels of immune cells.

[0114] like Figure 8 As shown in A, DCs (CD11c + ) Surface CD86 + CD40 + The proportion was 30.4%, which was significantly higher than that of other groups, indicating that the prepared biomineralized nanovaccine can promote the maturation of DCs in TDLNs through phototherapy-induced ICD, effective delivery of antigens and activation of the STING pathway. The use of microneedle delivery achieved multi-point drug administration and sustained drug release, which is more conducive to the anti-tumor immune effect of tumor vaccines than direct intratumoral injection. Figure 8 B and C are CD4 + T cell ratio and CD8 +Compared with the groups without manganese adjuvant (PBS, FreeOVAMNs, OCP MNs, Blank MNs), the OCMPI MNs+L group significantly increased the proportion of effector T cells in the spleen and effectively promoted T cell proliferation ( Figure 8 (D and E) CD11b in spleen + The results of further analysis of the cell population showed that compared with the other groups, the F4 / 80 + CD206 + The proportion of M2 macrophages is the smallest. The proportion of immune cells in the primary tumor microenvironment is analyzed, such as Figure 8 As shown in Figure FI, OCMPI MNs+L effectively increased the proportion of effector T cells in the tumor microenvironment, reduced the proportion of immunosuppressive M2 tumor-associated macrophages, and improved the immunosuppressive tumor microenvironment.

[0115] 3. The long-term memory effect of OCMPI MNs prepared in Example 1 of the present invention based on photothermal / photodynamic-immunotherapy was studied:

[0116] After 30 days of administration, the spleen was removed and ground, filtered through a cell sieve, lysed, and centrifuged to prepare a single cell suspension. The CD4 + and CD8 + Central memory T cells (CD44 + CD62L + ) ratio was detected and the immune memory function was analyzed.

[0117] like Fig. 9 As shown, FACS results showed that central memory T cells (CD44 + CD62L + ) ratio was significantly different from that in the control group, proving that photothermal combined with immunotherapy can induce the body to produce specific immune memory to prevent tumor recurrence and metastasis.

[0118] In summary, the soluble microneedle patch prepared by the present invention can have a direct killing effect on tumors through photothermal / photodynamic therapy, induce immunogenic death of tumor cells and release damage-related molecular patterns such as CRT, HMGB1, ATP, etc. to recruit and activate immune cells; deliver antigens and enhance their uptake and processing by antigen-presenting cells, and effectively induce the body's specific anti-tumor immune response; manganese ions activate the cGAS-STING pathway, significantly improve the ability of cGAS to synthesize the second messenger cGAMP and STING to bind to cGAMP, activate the natural immune response of immune cells, and promote the activation of effector T cells; through phototherapy combined with immunotherapy, an excellent anti-tumor effect was produced in a superficial malignant melanoma tumor model.

[0119] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a biomineralized nanovaccine, characterized in that: The following steps are involved: The antigen and the MnCl2·4H2O solution are added to the sugar-free DMEM culture medium to carry out the first mixing reaction, and then the photosensitizer solution and the anhydrous CaCl2 solution are added to carry out the second mixing reaction to obtain the biomineralization nano vaccine.

2. The preparation method according to claim 1, characterized in that: The photosensitizer is IR820.

3. The preparation method according to claim 1, characterized in that: The concentration of the MnCl2·4H2O solution is 0.1M, and the concentration of the anhydrous CaCl2 solution is 1M; The mass volume ratio of the antigen, the sugar-free DMEM culture medium, the MnCl2·4H2O solution and the anhydrous CaCl2 solution is (0.5-4) mg:1 mL:(5-20) μL:(5-20) μL.

4. The preparation method according to claim 1, characterized in that: The temperature of the first mixing reaction and the second mixing reaction were both 37° C., and the time was both 24 h.

5. A biomineralized nanovaccine prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the biomineralization nanovaccine as claimed in claim 5 in the preparation of a soluble microneedle patch.

7. A soluble microneedle patch for delivering biomineralized nano-vaccines, characterized in that: including a backing layer, a separation layer and microneedles; The microneedles are loaded with the biomineralized nanovaccine according to claim 5.

8. The soluble microneedle patch according to claim 7, characterized in that: The backing layer is an adhesive tape.

9. The soluble microneedle patch according to claim 7, characterized in that: The shape of the microneedle is a cone, a triangular pyramid, a quadrangular pyramid, a pentagonal pyramid, a hexagonal pyramid, a heptagonal pyramid or an octagonal pyramid.

10. A method for preparing a soluble microneedle patch according to any one of claims 7 to 9, characterized in that: The following steps are involved: Adding a high molecular weight soluble polymer containing the biomineralized nano-vaccine into a mold having microneedle holes, centrifuging to remove excess material on the mold surface, and vacuum drying and solidifying to obtain a microneedle body; Adding a soluble polymer to the mold, centrifuging to remove excess material on the mold surface, and drying at room temperature overnight to obtain a microneedle separation layer; After attaching the backing layer, the microneedles are demoulded to obtain the soluble microneedle patch; The high molecular weight refers to a molecular weight ≥300 kDa; the low molecular weight refers to a molecular weight ≤10 kDa.

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