Nanohydrogel microneedle of nuclide and preparation method and application thereof

Abstract

The application provides a radionuclide nanohydrogel microneedle and a preparation method and application thereof, and belongs to the field of nanobiomedicine. 32 P-labeled calcium phosphate, which is formed by a biomimetic reaction of Ag2S@Ca 32 P nanoparticles, and a base layer which is a base matrix and comprises hyaluronic acid 32 P nanohydrogel microneedle can release Ag2S nanoparticles in response to a weakly acidic tumor environment, and under the irradiation of near-infrared light, the photothermal conversion efficiency can effectively kill bacteria, effectively improve the tumor hypoxic environment, enhance the treatment effect of radionuclides, play an antibacterial and wound healing promoting role in melanoma defect repair, and maximize the inhibition of tumor growth and tumor recurrence.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine, specifically relating to a radionuclide nanohydrogel microneedle, its preparation method, and its application. Background Technology

[0002] Melanoma is a highly malignant tumor, accounting for only 4% of skin cancer cases, but causing more than 75% of skin cancer deaths. The onset of melanoma is mainly due to the uncontrolled proliferation of melanocytes in the basal layer of the epidermis, and its development is the result of multiple factors, including genes and environment. The Chinese Society of Clinical Oncology guidelines state that patients with localized melanoma (stage 0-II) can be cured by surgical resection of the primary lesion. However, surgical treatment still carries a certain risk of postoperative recurrence. The recurrence rates for stage IB and II melanoma patients are 8% and 29%, respectively, and the recurrence rate is positively correlated with mortality. The main risk factors for local tumor recurrence are: 1) positive surgical margins; 2) the depth of tumor invasion into the skin; if the tumor infiltration depth is greater than 4 mm, even after tumor removal, the patient may experience recurrence within a short period; 3) melanoma often occurs in the extremities and mucous membranes, making complete removal of the tumor lesion difficult during surgery; and the presence of residual microsatellite lesions and microinfiltration of tumor cells after surgery can also lead to local recurrence of melanoma. Fourth, intraoperative bleeding can also cause tumor cells to spread into the bloodstream, thereby increasing the level of circulating tumor cells and increasing the risk factors for postoperative recurrence. This indicates that postoperative recurrence in patients with localized melanoma is a problem that urgently needs to be addressed. Furthermore, the difficulty in wound healing after surgery for melanoma is also a challenging issue in treatment. The main factors affecting postoperative wound healing in melanoma patients are: bacterial infection, circulatory disorders, and flap transplantation issues. Bacterial infection is the most common pathogenic factor during wound healing, and the overuse of antibiotics can lead to the development of drug-resistant bacteria, further exacerbating the degree of wound infection. In addition, whether a patient experiences postoperative recurrence is also related to the presence or absence of wound infection. Patients with postoperative wound infection have a significantly increased tumor recurrence rate, while patients receiving neoadjuvant therapy can, to some extent, prolong recurrence-free survival and reduce the risk of recurrence. Therefore, in order to improve the survival rate of patients with malignant melanoma, it is urgent to establish a treatment strategy that effectively removes residual infiltrating tumor cells, controls infection, and accelerates wound repair.

[0003] With the rapid development of nuclear medicine, the application of radionuclide therapy for tumors has become a safer and more efficient new treatment method. Radionuclide therapy refers to the use of radionuclides with appropriate half-lives, radiation distances, and radiation energies (e.g., radionuclides with suitable half-lives, radiation distances, and radiation energies). 32 P, 90 (e.g., Y) emits rays with high linear energy to kill tumors. Among them, 32P is widely used for the treatment of superficial capillary hemangiomas and scars due to its advantages such as long range (up to 8 mm), high energy (1.7 MeV), and suitable half-life (14 days). Furthermore, studies have shown that... 32 The β rays emitted by P are greater than those emitted by an equal dose of high-energy extracellular electrons (such as...). 90 Y) is more likely to cause double-strand breaks in tumor cell DNA, preventing replication, and is a potential novel anti-tumor drug. Therefore, we hypothesize whether it is possible to... 32 P is used in the treatment of melanoma and has an anti-tumor effect against postoperative recurrence of melanoma.

[0004] To address the problems of postoperative infection and poor wound healing in melanoma patients, several emerging treatment methods have emerged, including photothermal therapy (PTT). PTT is a minimally invasive treatment that converts near-infrared (NIR) radiation into heat energy (45°C) using a photothermal agent, ablating bacteria or tumor cells without damaging surrounding tissue. Compared to traditional antibacterial methods, it is non-invasive, does not induce drug resistance, has fewer side effects, and boasts a high bactericidal rate. To date, a series of photothermal nanomaterials with excellent properties have been developed.

[0005] However, how to effectively and locally deliver these drugs to the tumor site has always been one of the bottlenecks in biomedicine. Developing a radionuclide nanomaterial that can resist postoperative recurrence and infection of melanoma has good clinical application value. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a radionuclide nanohydrogel microneedle, its preparation method, and its application. It constructs a radionuclide nanomedicine delivery system capable of resisting postoperative recurrence and infection in melanoma patients. Specifically targeting the problem of postoperative recurrence in melanoma, the microneedles carry... 32 P can cause double-strand DNA breaks in tumor cells, thus playing a role in preventing tumor recurrence; regarding postoperative infection issues in melanoma, Ag2S@Ca within the microneedle tip... 32 P nanoparticles can release Ag2S nanoparticles in response to the acidic environment of the tumor, and the heat generated by near-infrared light irradiation exerts an antibacterial effect; while the methacrylamide gelatin (GelMA) component of the microneedle tip has antibacterial, hemostatic, and cell adhesion-promoting effects, further promoting wound healing. This radionuclide nanomedicine hydrogel microneedle, through internal irradiation with radionuclide combined with photothermal therapy, can inhibit postoperative recurrence and infection of melanoma and effectively promote wound healing.

[0007] To achieve the above objectives, this solution first provides a radionuclide nanohydrogel microneedle, which includes a base layer and a tip layer. The tip layer is composed of methacrylamide gelatin loaded with the radionuclide nanodrug Ag2S@Ca. 32 P composition, the radionuclide nanomedicine Ag2S@Ca 32 P includes core Ag2S nanoparticles and radioactive nuclides. 32 P-labeled calcium phosphate and bovine serum albumin shell.

[0008] Preferably, the nuclide nanohydrogel microneedles have a needle length of 550 μm and the base layer has a diameter of 9 mm.

[0009] Based on a general inventive concept, this solution also provides a method for preparing radionuclide nanohydrogel microneedles, including the following steps:

[0010] S1. Bovine serum albumin was dissolved in ultrapure water, sonicated, and AgNO3 was added and stirred to mix. After adjusting the pH to 12, Na2S·9H2O was added, heated and stirred, and incubated overnight. Ag2S nanoparticles were obtained by dialysis.

[0011] S2. Disperse bovine serum albumin and Ag2S nanoparticles obtained in step S1 into a mixture of ultrapure water and sugar-free DMEM, and then add phosphorus [ 32 Incubate with sodium phosphate solution and CaCl2 overnight, then centrifuge, collect the precipitate, and sonicate to reconstitute. 32 p-labeled Ag2S@Ca 32 P nanoparticles;

[0012] S3. Add methacrylated gelatin to the photoinitiator to prepare a methacrylated gelatin solution, dissolve it by heating in a light-protected water bath, and then add the Ag2S@Ca obtained in S2. 32 P nanoparticles were dissolved in a methacrylamide gelatin solution, injected into a microneedle mold, centrifuged, vacuumed, dried, and then UV-cured to cure the needle tips.

[0013] S4. The base matrix is ​​injected into the microneedle mold with the needle tip already solidified as the base layer. After centrifugation and vacuuming, it is dried and demolded to obtain the radionuclide nanohydrogel microneedles.

[0014] Preferably, the molar ratio of AgNO3, Na2S·9H2O, and CaCl2 is 1:2:1; the phosphorus [ 32 The radioactivity concentration of the sodium salt solution of P] acid is 1 mCi / mL.

[0015] Preferably, the heating and stirring temperature in S1 is 55 °C for 4 h, and the overnight incubation temperature is 37 °C.

[0016] Preferably, the incubation temperature in S2 is 37 °C.

[0017] Preferably, the photoinitiator in S3 is LAP with a concentration of 0.25%, the mass concentration of the methacrylamide gelatin solution is 20%, the water bath heating temperature is 60 °C for 30 min, and the drying time is 24 h.

[0018] Preferably, the substrate matrix in S4 comprises a hyaluronic acid solution with a mass concentration of 50%.

[0019] Preferably, the hyaluronic acid has a molecular weight of 10 kDa.

[0020] Based on a general inventive concept, this solution also provides the application of radionuclide nanohydrogel microneedles in the preparation of drugs to inhibit melanoma growth and postoperative recurrence, wherein the drugs need to be combined with near-infrared light irradiation.

[0021] Based on a general inventive concept, this solution also provides the application of radionuclide nanohydrogel microneedles in the preparation of drugs for treating wound infection and promoting wound healing, wherein the drugs require irradiation with near-infrared light.

[0022] The mechanism of action of the radionuclide nanohydrogel microneedles prepared by this method is as follows:

[0023] To address the issue of melanoma recurrence after surgery, microneedles are equipped with... 32 P can cause double-strand DNA breaks in tumor cells, thus playing a role in preventing tumor recurrence; regarding postoperative infection issues in melanoma, Ag2S@Ca within the microneedle tip... 32 P nanoparticles can release Ag2S nanoparticles in response to the acidic environment of the tumor, and the heat generated by near-infrared light irradiation exerts an antibacterial effect; while the methacrylamide gelatin (GelMA) component of the microneedle tip has antibacterial, hemostatic, and cell adhesion-promoting effects, further promoting wound healing. This radionuclide nanomedicine hydrogel microneedle, through internal irradiation with radionuclide combined with photothermal therapy, can inhibit postoperative recurrence and infection of melanoma and effectively promote wound healing.

[0024] Under near-infrared light irradiation, Ag2S releases Ag + The inherent antibacterial activity and the high temperature generated by PTT therapy work together to kill bacteria. Based on this, Ag2S is used as a photothermal agent to produce an antibacterial effect through near-infrared light irradiation, which synergistically inhibits melanoma recurrence and postoperative infection with radionuclide therapy. In addition, PTT therapy can also enhance the sensitization of radionuclide therapy by increasing local blood flow to the tumor and improving tissue oxygenation.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The radionuclide nanomedicine hydrogel microneedles provided in this scheme utilize radionuclides 32 P with 32 PO4 3− In the form of Ca, it participates in biomineralization reactions to form Ca 32 P nanoparticles deliver Ca via efficient local delivery through microneedles. 32 P nanoparticles are delivered in situ to the tumor site, effectively exerting an anti-tumor recurrence effect.

[0027] (2) The radionuclide nanomedicine hydrogel microneedles in Ca 32 Ag2S nanoparticles were introduced during the mineralization process of P nanoparticles to prepare Ag2S@Ca 32 P can release Ag2S nanoparticles in response to the weakly acidic environment of tumors. Under near-infrared light irradiation, its photothermal conversion efficiency can effectively kill bacteria, while also effectively improving the hypoxic environment of tumors, enhancing the therapeutic effect of radionuclides, and playing an antibacterial and wound-healing role in the repair of melanoma defects.

[0028] (3) The radionuclide nanomedicine hydrogel microneedles Ag2S@Ca prepared in this scheme 32 PMN's combination of radionuclide irradiation and photothermal effects can maximize its ability to inhibit tumor growth and recurrence.

[0029] (4) The radionuclide nanomedicine hydrogel microneedles Ag2S@Ca prepared by this scheme 32 PMN possesses excellent photothermal conversion efficiency, and the resulting photothermal effect has good antibacterial properties. When combined with photothermal therapy in vivo, it has a significant in vivo antibacterial effect.

[0030] (5) Microneedles carrying Ag2S@Ca 32 The drug delivery mode of P nanoparticles can effectively reduce damage to surrounding normal tissues or organs, and the carrier components are all non-toxic substances with good biocompatibility and biodegradability. In addition, the methacrylamide gelatin component of the microneedle tip has antibacterial, hemostatic, and cell adhesion-promoting effects, which further promote wound healing.

[0031] (6) The preparation process of this scheme is simple and controllable, effectively avoiding the toxicity of the carrier, and has high drug loading and modification rate; the radionuclide nanomedicine prepared by biomineralization method has uniform particle size, with an average particle size of 162.5±7.2 nm and good dispersibility. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The Ag2S@Ca obtained in Experiment Example 1 32 Transmission electron microscopy image and particle size distribution of P, where A is Ag2S@Ca. 32 Transmission electron microscopy images of P at 50 nm and 200 nm scales, B being Ag₂S@Ca 32 Particle size distribution diagram of P;

[0034] Figure 2 Different concentrations of Ag2S@Ca obtained in Experiment Example 1 32 Photothermal conversion efficiency and photothermal stability of P under near-infrared light irradiation; A is Ag2S@Ca 32 Photothermal conversion efficiency of P under near-infrared light irradiation, where B represents Ag2S@Ca. 32 Photothermal stability of P under near-infrared light irradiation;

[0035] Figure 3 The Ag2S@Ca obtained in Experiment Example 1 32 Photothermal imaging of P;

[0036] Figure 4 The image shows the in vitro antitumor effects of nanoparticles with different components obtained in Experiment 1 on B16F10 melanoma cells.

[0037] Figure 5 The Ag2S@Ca obtained in Experiment Example 2 32 Array diagram and optical microscope image of PMN; A represents Ag2S@Ca 32 Array diagram of PMN, B is Ag2S@Ca 32 Optical microscope image of P MN;

[0038] Figure 6 The Ag2S@Ca obtained in Experiment Example 2 32 Stress test diagram of P MN;

[0039] Figure 7 The Ag2S@Ca obtained in Experiment Example 2 32 Light micrograph of PMN skin penetration depth stained with hematoxylin and eosin;

[0040] Figure 8 The MN and Ag2S@Ca obtained in Experiment Example 2 were detected32 Photothermal image of PMN under near-infrared light illumination;

[0041] Figure 9 The graph shows the inhibition of Staphylococcus aureus by different MNs obtained in Experiment Example 2.

[0042] Figure 10 The image shows the therapeutic effects of different MNs obtained in Experiment 3 on the C57 mouse melanoma model.

[0043] Figure 11 The image shows the therapeutic effects of different MNs obtained in Experiment 4 on a C57 mouse melanoma recurrence model.

[0044] Figure 12 The images show the therapeutic effects and bacterial infection patterns of different MNs on the C57 mouse wound infection model obtained in Experiment Example 5. A shows the therapeutic effects of different MNs on the C57 mouse wound infection model, and B shows the bacterial infection patterns of different MNs on the C57 mouse wound infection model. Detailed Implementation

[0045] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0046] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0047] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0048] Example 1

[0049] Preparation of radionuclide nanomedicine Ag2S@Ca 32 P

[0050] S1. Dissolve 500 mg bovine serum albumin (BSA) in 14 mL of ultrapure water, sonicate, add 2 mL of 100 mmol / L silver nitrate (AgNO3), stir well, and adjust the pH to 12; then add 4 mL of 100 mmol / L sodium sulfide nonahydrate (Na2S·9H2O) to the above solution, heat and stir at 55℃ for 4 h, incubate overnight at 37℃, and dialyze to obtain Ag2S nanoparticles;

[0051] S2. Take an equal volume of ultrapure water and sugar-free DMEM in a beaker, add 200 mg of BSA, and add 2 mL of Ag2S nanoparticles prepared in step S1. Stir well. Add phosphorus [concentration 1 mCi / mL]. 32 [P] sodium salt solution, sonicated, then 200 μL of 1 mol / L CaCl2 was added, incubated overnight at 37 ℃, centrifuged, and the precipitate was collected and reconstituted to obtain Ag2S@Ca 32 P nanoparticles.

[0052] Based on the nanomaterials obtained in Example 1, the Ag2S@Ca nanomaterials were tested. 32 The physicochemical properties and in vivo and in vitro pharmacokinetic and pharmacodynamic characteristics of P nanoparticles are specifically described, including micromorphology, particle size, photothermal conversion characteristics, and in vitro cytotoxicity, with the nanomedicine obtained in Example 1 being used as a specific example.

[0053] Example 2

[0054] Preparation of radionuclide nanomedicine hydrogel microneedles Ag2S@Ca 32 PMN

[0055] S1. Microneedle patches were prepared using a polydimethylsiloxane (PDMS) mold with a conical array of holes. The microneedles were 9 mm × 9 mm in size, and the entire array included 100 needle tips with a bottom diameter of 250 μm and a length of 550 μm.

[0056] S2. Add 1 g of GelMA to 0.25% LAP to prepare a 20% (w / w) methacrylamide gelatin solution, and heat in a 60 ℃ light-protected water bath for 30 min to dissolve it.

[0057] S3. Mix Ag2S@Ca at a volume ratio of 1:4. 32 P MN nanoparticles were dissolved in the solution from step S2, and the mixture was stirred to obtain a solution containing Ag2S@Ca. 32 The GelMA solution of P nanoparticles was injected into a microneedle mold, centrifuged, and then vacuumed at 0.1 kPa for 10 min. The centrifugation and vacuuming steps were repeated 3 times. After discarding the excess liquid in the mold, the mold was dried in an oven at 37 ℃ for 24 h and then UV cured to form microneedle tips.

[0058] (4) Take 100 μL of hyaluronic acid solution with a mass concentration of 50% and inject it into the microneedle mold as the base layer. After centrifugation and vacuuming, dry it overnight in an oven at 37 ℃. After demolding, obtain the radionuclide nanomedicine hydrogel microneedles.

[0059] Based on the radionuclide nanomedicine hydrogel microneedles obtained in Example 2, the Ag2S@Ca was detected. 32The apparent characteristics and antitumor and antibacterial properties of PMN are specifically described, including its micromorphology, photothermal conversion properties, in vitro and in vivo antibacterial effects, and in vitro antitumor recurrence effects. The hydrogel microneedles of related radionuclide nanomedicines obtained in Example 2 are used as a specific example.

[0060] Experimental Example 1

[0061] Investigating the radionuclide nanomedicine Ag2S@Ca 32 P-characteristics

[0062] (1) Observe Ag2S@Ca 32 morphology of P nanoparticles

[0063] A small amount of sample was dropped onto a 400-mesh zinc grid covered with a carbon film, placed in a desiccator to dry, and after it dried naturally, the morphology of the nanoparticles was observed under a transmission electron microscope (TItan G2-F20).

[0064] The result is as follows Figure 1 As shown in Figure A, the Ag2S@Ca of the present invention 32 P nanoparticles appear as amorphous polymers under a transmission electron microscope.

[0065] (2) The particle size of nanoparticles was determined using a dynamic light scattering particle size analyzer.

[0066] Take Ag2S@Ca 32 The P sample solution was placed in a Marlven Nano ZS instrument to detect the nanoparticle size.

[0067] The result is as follows Figure 1 As shown in B, Ag2S@Ca 32 The P particles have a diameter of approximately 162.5 ± 7.2 nm and are spherical nanostructures.

[0068] (3) Detection of different concentrations of Ag2S@Ca 32 Photothermal conversion efficiency of P at the same power

[0069] Ag₂S@Ca was irradiated with an 808 nm laser at concentrations of 0 μg / mL, 100 μg / mL, 250 μg / mL, and 500 μg / mL, respectively. 32 P nanoparticle solution, temperature change of nanoparticle solution recorded every 20 s.

[0070] The result is as follows Figure 2 As shown in A, with Ag2S@Ca 32 The photothermal conversion efficiency is further enhanced by increasing the concentration of P nanoparticles.

[0071] (4) Detection of Ag2S@Ca 32 Photothermal stability of P

[0072] Ag₂S@Ca was irradiated cyclically with an 808 nm laser at a concentration of 500 μg / mL. 32 P nanoparticle solution, recording nanoparticles

[0073] Changes in solution temperature.

[0074] The result is as follows Figure 2 As shown in B, Ag2S@Ca 32 The P nanoparticles can maintain photothermal stability during continuous light irradiation cycles, indicating that they can also maintain good stability during treatment.

[0075] (5) Detection of Ag2S@Ca 32 P's photothermal imaging

[0076] Ultrapure water and Ag2S@Ca were irradiated with an 808 nm laser, respectively. 32 The temperature change of the P nanoparticle solution was recorded every 20 seconds; and photothermal images of different nanoparticles were captured every 1 minute using an infrared thermal imager.

[0077] The result is as follows Figure 3 As shown, compared with ultrapure water, Ag2S@Ca 32 P nanoparticles can be heated to the 40 ℃~50 ℃ range more quickly under fixed power.

[0078] (6) Investigation of radionuclide nanomedicine Ag2S@Ca 32 The killing effect of P on tumor cells

[0079] B16F10 cells were used as model cells, and the experimental groups were Ag2S and Ag2S@Ca. 32 P, Ag2S@Ca 32 P+NIR (808nm near-infrared radiation). After incubation for 48 h, the supernatant was discarded, and 5 mg / mL MTT solution was added to each well and incubated in a cell culture incubator for 4 h. Subsequently, the MTT solution was discarded, and 100 μL DMSO solution was added to each well. The absorbance (OD) of each well was measured at 570 nm using a microplate reader.

[0080] The result is as follows Figure 4 As shown, Ag2S and Ag2S@Ca 32 P, Ag2S@Ca 32 Both P+NIR exhibited concentration-dependent cytotoxicity, due to Ag2S@Ca 32 The P+NIR group of nanomedicines, combined with radionuclides and photothermal therapy, works synergistically, thus exhibiting the strongest tumor cell killing effect.

[0081] Experimental Example 2

[0082] Investigating the use of radionuclide nanomedicine hydrogel microneedles Ag2S@Ca 32 PMN characteristics

[0083] (1) Observation of Ag2S@Ca with a digital camera 32 Macroscopic morphology of PMN microneedles: Ag2S@Ca 32 PMN was placed on a table to observe the morphology of the microneedles.

[0084] The result is as follows Figure 5 As shown in Figure A, Ag2S@Ca 32 PMN needle tips are arranged in an orderly manner in the basal layer, forming a regular and orderly microneedle array.

[0085] (2) Observation of Ag2S@Ca under an optical microscope 32 P MN microneedle morphology: Ag2S@Ca 32 PMN was placed under an upright microscope to observe the morphology of the microneedles.

[0086] The result is as follows Figure 5 As shown in B, Ag2S@Ca 32 The PMN needle tip is 10 μm wide and the needle length is 550 μm.

[0087] (3) Detection of Ag2S@Ca by electronic universal testing machine 32 PMN mechanical properties

[0088] Place the microneedle (tip side up) on the test platform. The force sensor probe automatically compresses the microneedle at a speed of 0.5 mm / min. After reaching the maximum load (set to 55 N), it automatically stops running and records the applied force to plot the force-displacement curve.

[0089] The result is as follows Figure 6 As shown, the drug-loaded microneedle did not break under a pressure of 0.55 N / needle, indicating that the microneedle has excellent mechanical strength, exceeding the 0.2 N / needle required for skin penetration, and can penetrate the stratum corneum to achieve effective drug delivery.

[0090] (4) Observe Ag2S@Ca 32 PMN skin puncture depth

[0091] Ag2S@Ca 32 PMN was inserted into the skin on the back of hairless C57 mice. The thumb was pressed continuously for 2 minutes, the microneedles were removed, and the skin tissue at the insertion site was collected, fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin, and the micropores formed after the microneedles were observed under a light microscope.

[0092] The results are as follows Figure 7As shown, the results of section staining showed that the microneedles could form pinholes of approximately 263.5 μm in length on mouse skin, indicating that the prepared microneedles could successfully penetrate the stratum corneum and enter the subcutaneous layer.

[0093] (5) Detection of photothermal conversion efficiency of microneedles containing nanoparticles of different formulations in vitro: MN, Ag2S@Ca 32 PMN was applied to the skin on the back of C57 mice. After continuous thumb pressure for 2 minutes, each group of microneedle patches was irradiated with an 808 nm laser. Infrared thermal imagers were used to capture in vivo photothermal images of different microneedles every 1 minute.

[0094] The result is as follows Figure 8 As shown, compared to MN, Ag2S@Ca 32 PMN can heat up to the 40℃~50℃ range more quickly under fixed power, and has better photothermal conversion efficiency.

[0095] (6) Ag2S@Ca 32 In vitro antibacterial efficacy assay of PMN: Microneedles loaded with different formulations were immersed in 100 μL of PBS solution and 100 μL of Staphylococcus aureus suspension (1×10⁻⁶). 8 Incubate the bacterial suspension ( / mL) together for 3 h. After incubation, dilute the bacterial suspension and plate it.

[0096] The results are as follows Figure 9 As shown, Ag2S@Ca 32 The number of colonies was significantly reduced after PMN+NIR treatment, indicating that Ag2S@Ca 32 The photothermal effect produced by PMN has good antibacterial properties.

[0097] Experimental Example 3

[0098] Constructing a mouse melanoma model to investigate the effects of radionuclide nanomedicine hydrogel microneedles Ag2S@Ca 32 PMN treatment efficacy.

[0099] (1) Establishment of mouse melanoma model: 1×10 6 A melanoma model was established by inoculating male C57 mice with a B16F10 cell suspension at a concentration of 1 cell / mL on the right back.

[0100] (2) Ag2S@Ca 32 Evaluation of the efficacy of PMN in treating tumors: When the tumor volume in mice reached 100 mm 3 They were randomly divided into 3 groups for microneedling: Control, Ag2S@Ca 32 PMN, Ag2S@Ca 32P MN+NIR. The long and short diameters of the tumor were measured with vernier calipers over 15 days, and a tumor volume-time change graph was plotted.

[0101] The result is as follows Figure 10 As shown, compared with the control group, Ag2S@Ca 32 PMN, Ag2S@Ca 32 The PMN+NIR groups all exhibited excellent tumor suppression effects, among which Ag2S@Ca 32 The PMN+NIR effect was the most significant, indicating that the combination of radionuclide irradiation and photothermal effect of this microneedle can maximize the inhibition of tumor growth.

[0102] Experiment Example 4

[0103] Constructing a mouse melanoma recurrence model to investigate the effects of radionuclide nanomedicine hydrogel microneedles Ag2S@Ca 32 PMN treatment efficacy.

[0104] (1) Construction of a mouse melanoma recurrence model: 1×10 6 A B16F10 cell suspension at a concentration of [number] cells / mL was inoculated into the right back of male C57 mice to establish a melanoma model. The tumors were cultured until they reached a size of 100 mm. 3 95% of the tumor tissue was removed and sutured.

[0105] (2) Ag2S@Ca 32 Efficacy evaluation of P in treating recurrent tumors: C57 mice with 95% tumor tissue resection were randomly divided into 3 groups and treated with different microneedles: Control, Ag2S@Ca 32 PMN, Ag2S@Ca 32 P MN+NIR. Measure the long and short diameters of the tumor over 27 days using vernier calipers, calculate the tumor volume, and plot the tumor volume-time change graph.

[0106] The result is as follows Figure 11 As shown, compared with the control group, Ag2S@Ca 32 PMN, Ag2S@Ca 32 The PMN+NIR groups all showed excellent anti-tumor recurrence effects, among which Ag2S@Ca 32 The PMN+NIR effect was the most significant, indicating that the combined application of radionuclide irradiation and photothermal effects of this microneedle can maximize the inhibition of tumor recurrence.

[0107] Experimental Example 5

[0108] Constructing a mouse wound infection model to investigate the effects of radionuclide nanomedicine hydrogel microneedles Ag2S@Ca 32 PMN antibacterial effect.

[0109] (1) Establishment of mouse wound infection model: A circular full-thickness skin lesion with a diameter of 10 mm was established on the back of C57 mice, and 20 μL of Staphylococcus aureus suspension (1×10⁻⁶) was aspirated. 8 A C57 mouse wound infection model was established by applying a 100 mL agar solution to the wound.

[0110] (2) Ag2S@Ca 32 Evaluation of PMN antibacterial efficacy: One hour after bacterial inoculation, microneedles of different formulation groups were applied to the wound. The groups were: Control, Ag2S@Ca 32 PMN, Ag2S@Ca 32 P MN+NIR. Before microneedle treatment and on days 0, 3, 7, and 12 after treatment, sterile cotton swabs were used to collect exudate from the wound, which was then diluted and coated onto a plaster.

[0111] The result is as follows Figure 12 As shown in A and 12B, compared with the control group, Ag2S@Ca 32 The PMN+NIR group showed significant reduction in wound infection, a significant decrease in bacterial count, and accelerated wound healing, indicating that the combined radionuclide and photothermal therapy has a significant in vivo antibacterial effect.

[0112] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, any improvements and modifications obtained without departing from the technical concept of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A radionuclide nanohydrogel microneedle, characterized in that, The radionuclide nanohydrogel microneedles comprise a base layer and a tip layer, wherein the tip layer is composed of methacrylamide gelatin loaded with the radionuclide nanodrug Ag2S@Ca. 32 P composition, the radionuclide nanomedicine Ag2S@Ca 32 P includes core Ag2S nanoparticles and radioactive nuclides. 32 The shell consists of P-labeled calcium phosphate and bovine serum albumin; the base layer is hyaluronic acid.

2. The radionuclide nanohydrogel microneedles according to claim 1, characterized in that, The nuclide nanohydrogel microneedles have a needle length of 550 μm and the base layer has a diameter of 9 mm.

3. A method for preparing radionuclide nanohydrogel microneedles as described in any one of claims 1-2, characterized in that, Includes the following steps: S1. Bovine serum albumin was dissolved in ultrapure water, sonicated, and AgNO3 was added and stirred to mix. After adjusting the pH to 12, Na2S·9H2O was added, heated and stirred, and incubated overnight. Ag2S nanoparticles were obtained by dialysis. S2. Disperse bovine serum albumin and Ag2S nanoparticles obtained in step S1 into a mixture of ultrapure water and sugar-free DMEM, and then add phosphorus [ 32 Incubate with sodium phosphate solution and CaCl2 overnight, then centrifuge, collect the precipitate, and sonicate to reconstitute. 32 p-labeled Ag2S@Ca 32 P nanoparticles; S3. Add methacrylated gelatin to the photoinitiator to prepare a methacrylated gelatin solution, dissolve it by heating in a light-protected water bath, and then add the Ag2S@Ca obtained in S2. 32 P nanoparticles were dissolved in a methacrylamide gelatin solution, injected into a microneedle mold, centrifuged, vacuumed, dried, and then UV-cured to cure the needle tips. S4. The base matrix is ​​injected into the microneedle mold with the needle tip already solidified as the base layer. After centrifugation and vacuuming, it is dried and demolded to obtain the radionuclide nanohydrogel microneedles. The base matrix is ​​a hyaluronic acid solution with a mass concentration of 50%.

4. The preparation method according to claim 3, characterized in that, The molar ratio of AgNO3, Na2S·9H2O, and CaCl2 is 1:2:1; the phosphorus [ 32 The radioactivity concentration of the sodium salt solution of P] acid is 1 mCi / mL.

5. The preparation method according to claim 3, characterized in that, The heating and stirring temperature in S1 is 55 ℃ for 4 h, and the overnight incubation temperature is 37 ℃.

6. The preparation method according to claim 3, characterized in that, The incubation temperature in S2 is 37 ℃.

7. The preparation method according to claim 3, characterized in that, The photoinitiator in S3 is LAP with a concentration of 0.25%, the mass concentration of the methacrylamide gelatin solution is 20%, the water bath heating temperature is 60 °C for 30 min, and the drying time is 24 h.

8. The application of a radionuclide nanohydrogel microneedle as described in any one of claims 1-2 or a radionuclide nanohydrogel microneedle prepared by any one of claims 3-7 in the preparation of drugs against melanoma growth and postoperative recurrence, characterized in that, The drug requires combined irradiation with near-infrared light.

9. The application of a radionuclide nanohydrogel microneedle as described in any one of claims 1-2 or a radionuclide nanohydrogel microneedle prepared by any one of claims 3-7 in the preparation of drugs for treating wound infection and promoting wound healing, characterized in that, The drug requires combined irradiation with near-infrared light.

Images