Double-time-phase microneedle based on molecular hydrogen / photo-thermal triggering as well as preparation method and application of double-time-phase microneedle

Abstract

The invention provides a double-time-phase microneedle based on molecular hydrogen / photo-thermal triggering and a preparation method of the double-time-phase microneedle. The double-time-phase microneedle comprises a soluble microneedle body matrix, PDA (at) ZIF-8 (at) AB nanoparticles and PCL (at) QX-314 microspheres, wherein the PDA (at) ZIF-8 (at) AB nanoparticles and the PCL (at) QX-314 microspheres are dispersed in the matrix; the PDA-ZIF-8 (at) AB nanoparticles are obtained by carrying out a self-polymerization reaction on ZIF-8 nanoparticles loaded with ammonia borane and dopamine hydrochloride; the PCL (at) QX-314 microspheres are prepared by a water-in-oil-in-water type multiple emulsion-solvent evaporation method. The AB nano particles are applied to the postoperative incision part for the first time, the AB nano particles have good mechanical pain and heat pain treatment effects and wound healing promotion effects, meanwhile, controllable release of the PCL-coated QX-314 microspheres is combined, and the multifunctional postoperative incision management system which has a long-acting analgesia mode and a patient on-demand analgesia mode and effectively promotes wound healing is provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a dual-phase microneedle based on molecular hydrogen / photothermal triggering, and a preparation method and application thereof. Background Art

[0002] Postoperative pain management is an important topic in the field of anesthesia. Despite advances in pain control technology and drugs, many surgical patients still face the problem of insufficient postoperative pain relief. Studies have shown that more than 80% of surgical patients experience acute postoperative pain, of which more than 50% are moderate to extremely severe pain. At present, opioids are still the most commonly used analgesics after surgery. In my country, 63.7% of patients use opioid analgesics as the main treatment for postoperative analgesia. Local anesthesia is also a way to manage postoperative pain, which can avoid the risk of opioid-related adverse events. However, the duration of action of local anesthetics is generally within 2 to 4 hours, which severely limits its application in postoperative analgesia. In addition, pain perception is inherently subjective, and existing anesthesia regimens cannot fully consider the differences in pain sensitivity among patients, resulting in poor individualized treatment effects. In addition, surgical incision complications are not uncommon in clinical practice, such as infection and incision dehiscence. Postoperative incision management is a key link in optimizing patient prognosis and helps reduce the risk of infection and related complications.

[0003] As a physiologically inert gas, molecular hydrogen (H2) has been widely studied to show its potential protective effects on a variety of diseases, especially in the treatment of neurological diseases such as acute postoperative pain, neuropathic pain, and cognitive dysfunction caused by surgery. At the same time, studies have confirmed that H2 therapy has significant effects in anti-inflammation, cell proliferation and migration, angiogenesis, and tissue regeneration, and can improve wound healing. However, the existing H2 administration methods are mainly intraperitoneal injection or oral administration of hydrogen-rich water, which have problems such as high frequency of administration, poor patient compliance, and uncontrollable hydrogen gas escape.

[0004] Therefore, in response to the above problems, it is necessary to develop a microneedle that can achieve continuous and long-lasting analgesia at the postoperative incision, and at the same time, when the pain worsens, it can self-control the release of drugs through external photothermal stimulation to meet the patient's personalized analgesia needs while promoting wound healing. Summary of the invention

[0005] The purpose of the present invention is to provide a dual-phase microneedle based on molecular hydrogen / photothermal triggering and its preparation method and application, which can provide sustained and stable pain relief and meet individualized treatment needs, and effectively promote postoperative incision healing.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In the first aspect of the present invention, a dual-phase microneedle based on molecular hydrogen / photothermal triggering is provided, wherein the dual-phase microneedle comprises a soluble microneedle body matrix and PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres dispersed in the matrix;

[0008] The PDA-ZIF-8@AB nanoparticles are PDA-coated nanoparticles obtained by self-polymerization of ZIF-8 nanoparticles loaded with ammonia borane and dopamine hydrochloride;

[0009] The PCL@QX-314 microspheres are QX-314-loaded PCL microspheres prepared by a water-in-oil-in-water type emulsion-solvent evaporation method.

[0010] Furthermore, the particle size of the PDA-ZIF-8@AB nanoparticles is 160-220 nm; the particle size of the PCL@QX-314 microspheres is 3-15 μm.

[0011] Furthermore, in the PDA-ZIF-8@AB nanoparticles, the mass content of ammonia borane is 4-14%, and the mass content of polydopamine is 5-13%; in the PCL@QX-314 microspheres, the mass content of QX-314 is 1-8%.

[0012] In a second aspect of the present invention, a method for preparing a dual-phase microneedle based on molecular hydrogen / photothermal triggering is provided, the method comprising:

[0013] The ZIF-8 nanoparticles loaded with ammonia borane were subjected to self-polymerization reaction with dopamine hydrochloride to obtain PDA-ZIF-8@AB nanoparticle powder;

[0014] PCL@QX-314 microsphere powder prepared by water-in-oil-in-water emulsion-solvent evaporation method;

[0015] Add the PDA@ZIF-8@AB nanoparticle powder and PCL@QX-314 microsphere powder to the soluble matrix solution and mix well to obtain a needle tip matrix solution;

[0016] The needle tip matrix solution is added to a microneedle mold, centrifuged and vacuumed, and the microneedle is demoulded after drying to obtain a dual-phase microneedle based on molecular hydrogen / photothermal triggering.

[0017] In a third aspect of the present invention, a method for preparing PDA-ZIF-8@AB nanoparticles is provided, the method comprising:

[0018] Ammonia borane, water, soluble zinc salt and 2-methylimidazole are mixed for coordination reaction to obtain ZIF-8 nanoparticles loaded with ammonia borane;

[0019] The ZIF-8 nanoparticles loaded with ammonia borane are mixed with dopamine hydrochloride and a buffer solution to undergo a self-polymerization reaction to obtain PDA-ZIF-8@AB nanoparticle powder.

[0020] In the fourth aspect of the present invention, PDA-ZIF-8@AB nanoparticles prepared by the method are provided.

[0021] In a fifth aspect of the present invention, a method for preparing PCL@QX-314 microspheres is provided, the method comprising:

[0022] The aqueous solution containing the raw material drug QX-314 and the viscosity enhancer as the inner aqueous phase is mixed with the dichloromethane solution of the organic high molecular weight polymer polycaprolactone as the oil phase, and the first emulsification is performed under ice water bath conditions to obtain W / O type colostrum;

[0023] The W / O type colostrum is quickly poured into an external aqueous phase consisting of an aqueous solution containing sodium chloride and an emulsifier polyvinyl alcohol for a second emulsification to obtain a W / O / W type multiple emulsion;

[0024] The W / O / W type emulsion was quickly poured into the aqueous solution containing PVA, and stirred at a uniform speed at room temperature to solidify the polymer carrier with the volatile oil phase solvent DCM to form PCL microspheres loaded with QX-314.

[0025] In the sixth aspect of the present invention, PCL@QX-314 microspheres prepared by the method described are provided.

[0026] In the seventh aspect of the present invention, there is provided the use of the dual-phase microneedle based on molecular hydrogen / photothermal triggering in the preparation of a drug for postoperative wound management.

[0027] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] 1. Innovative application of dual-phase analgesia mode: The present invention adopts a dual-phase drug release mode, combining pH-responsive hydrogen release and photothermal-triggered local anesthetic drug release. Through this design, the microneedle system can achieve continuous and long-lasting analgesia at the postoperative incision. At the same time, when the pain worsens, the drug is released automatically through external photothermal stimulation to meet the patient's personalized analgesia needs. This dual-phase mode significantly reduces the individual differences in analgesic effects and improves the patient's comfort. In addition, the analgesic microneedles of the present invention can intelligently regulate the release of drugs according to the actual pain situation of the patient's postoperative incision site. This self-controlled analgesia mechanism enables patients to adjust the intensity of analgesia according to changes in pain, greatly improving the flexibility and effectiveness of analgesia, further reducing patients' dependence on analgesics, and improving the comfort and compliance of postoperative rehabilitation.

[0029] 2. Comprehensive wound management: The present invention not only provides a biphasic analgesic effect, but also significantly promotes wound healing at the postoperative incision through the synergistic effect of nanomaterials and drugs in the microneedles. The release of hydrogen has anti-inflammatory and antioxidant effects, which can reduce the inflammatory response around the wound and accelerate the wound healing process.

[0030] 3. Reduce the complexity of postoperative care: Since the microneedles of the present invention can quickly dissolve and release drugs after use, there is no need for frequent nursing operations, which reduces the complexity of postoperative care and the risk of postoperative complications such as infection, further improving the safety and efficiency of patients' postoperative recovery, and patients have higher compliance.

[0031] 4. Simple structure and low production cost: The microneedle system of the present invention has a simple structure design, good mechanical properties and stability, and is easy to store. The production process is mature and easy to manufacture on a large scale, with low production cost, and is suitable for wide promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0033] Figure 1 This is a flow chart of the method for preparing dual-phase microneedles based on molecular hydrogen / photothermal triggering in Example 1 of the present invention;

[0034] Figure 2 It is a transmission electron microscopy image of PDA@ZIF-8@AB nanoparticles in Example 1 of the present invention;

[0035] Figure 3 This is a near-infrared thermal imaging image of PDA@ZIF-8@AB nanoparticles in Example 2 of the present invention;

[0036] Figure 4 The in vitro hydrogen release curve of PDA@ZIF-8@AB nanoparticles at different pH values ​​in Experimental Example 1 of the present invention;

[0037] Figure 5 This is a scanning electron microscope image of PCL@QX-314 microspheres in Example 1 of the present invention;

[0038] Figure 6 This is a scanning electron microscope image of PCL@QX-314 microspheres after photothermal treatment in Experimental Example 2 of the present invention;

[0039] Figure 7The in vitro QX-314 release curves of PCL@QX-314 microspheres at different pH values ​​after photothermal treatment in Experimental Example 2 of the present invention;

[0040] Figure 8 This is an optical microscope image of the dual-phase microneedle based on molecular hydrogen / photothermal triggering prepared in Example 1 of the present invention;

[0041] Fig. 9 for Figure 8 Scanning electron micrograph of a single microneedle;

[0042] Fig.10 The test results of mechanical pain of the microneedles prepared in Experimental Example 3;

[0043] Fig.11 The test results of thermal pain of the microneedles prepared in Experimental Example 3;

[0044] Fig.12 Optical images of the wound healing effects of the microneedles prepared in Experimental Example 4 on different groups;

[0045] Fig.13 HE staining results of the wound healing effect of the microneedles prepared in Experimental Example 4 on different groups. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0047] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0048] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0049] The relevant terms of the present invention are explained as follows:

[0050] Ammonia borane (AB) is used to provide hydrogen release function.

[0051] Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was used as a source of zinc ions.

[0052] 2-Methylimidazole (2-MI) was used as a ligand to form the ZIF-8 framework.

[0053] Polydopamine (PDA), used for modification of photothermal materials.

[0054] QX-314, as a local anesthetic.

[0055] Polycaprolactone (PCL) is used as a polymer carrier for microspheres.

[0056] Hyaluronic acid (HA), used to increase the viscosity of the internal aqueous phase.

[0057] Sodium chloride (NaCl) is used to adjust the osmotic pressure of the external aqueous phase.

[0058] Polyvinyl alcohol (PVA) is used as an emulsifier for the external aqueous phase.

[0059] Dichloromethane (DCM) was used as the oil phase solvent.

[0060] PDA@ZIF-8@AB nanoparticles: PDA-coated nanoparticles obtained by self-polymerization of ZIF-8 nanoparticles loaded with ammonia borane and dopamine hydrochloride.

[0061] PCL@QX-314 microspheres: PCL microspheres loaded with QX-314.

[0062] The general idea of ​​the present invention is as follows:

[0063] According to a typical embodiment of the present invention, a dual-phase microneedle based on molecular hydrogen / photothermal triggering is provided, wherein the dual-phase microneedle comprises a soluble microneedle body matrix and PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres dispersed in the matrix;

[0064] The PDA-ZIF-8@AB nanoparticles are PDA-coated nanoparticles obtained by self-polymerization of ZIF-8 nanoparticles loaded with ammonia borane and dopamine hydrochloride;

[0065] The PCL@QX-314 microspheres are QX-314-loaded PCL microspheres prepared by a water-in-oil-in-water type emulsion-solvent evaporation method.

[0066] The present invention provides a dual-phase microneedle based on molecular hydrogen / photothermal triggering, which can dissolve quickly after puncturing into the skin, releasing PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres in the needle body, wherein the PDA-ZIF-8@AB nanoparticles have a persistent acid-responsive hydrogen release function, and the PCL@QX-314 microspheres can release QX-314 on demand through the photothermal effect of PDA. The operating temperature of the PCL@QX-314 microspheres that can release QX-314 on demand through the photothermal effect is 48 to 60°C.

[0067] The PDA@ZIF-8@AB nanoparticles achieve acid-responsive decomposition and release hydrogen in the local acidic microenvironment of the postoperative incision, thereby producing a lasting pain relief effect and effectively promoting wound healing. At the same time, the PCL@QX-314 microspheres combine the characteristics of near-infrared light and utilize the photothermal effect of PDA to release QX-314 on demand and activate TRPV1 channels, prompting QX-314 to enter neurons and achieve self-controlled analgesia to meet individualized treatment needs. The present invention applies AB nanoparticles to the postoperative incision site for the first time, and has good mechanical pain, thermal pain treatment effects and wound healing effects. At the same time, combined with the controlled release of PCL@QX-314 microspheres, it provides a multifunctional postoperative incision management system that has both long-term analgesia and patient-on-demand analgesia mode, and effectively promotes wound healing.

[0068] The present invention combines PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres to achieve a synergistic technical effect, which is specifically manifested in: in the analgesic effect test of the dual-phase microneedle based on molecular hydrogen / photothermal triggering, when using a single-phase microneedle (a single-phase microneedle is a Tris-HCl solution of HA containing PDA@ZIF-8@AB nanoparticles as a needle tip matrix solution (HA-PZA When the rats were treated with the dual-phase microneedles (MN, without microspheres) for analgesia, regardless of whether NIR irradiation was given, the mechanical pain threshold and thermal pain threshold of the rats were higher than those of the rats in the simple incision group without treatment and the rats treated with blank microneedles within 120 hours. When the dual-phase microneedles were used for analgesia and NIR irradiation was given at the same time, the mechanical pain threshold and thermal pain threshold of the rats could be restored to almost 100% within 120 hours, which shows that the PCL@QX-314 microspheres provided by the present invention have good controlled release properties, which can supplement and assist hydrogen analgesia, reduce the individual differences in pain between different individuals, and make the dual-phase microneedles based on molecular hydrogen / photothermal triggering have excellent analgesic effect. In addition, compared with blank microneedles, the microneedles provided by the present invention can significantly promote wound healing.

[0069] According to another typical embodiment of the present invention, a method for preparing a dual-phase microneedle based on molecular hydrogen / photothermal triggering is provided, the method comprising:

[0070] Step S1, subjecting ZIF-8 nanoparticles loaded with ammonia borane to a self-polymerization reaction with dopamine hydrochloride to obtain PDA-ZIF-8@AB nanoparticle powder;

[0071] The step S1 specifically includes:

[0072] Step S101, mixing ammonia borane, water, a soluble zinc salt and 2-methylimidazole for coordination reaction to obtain ZIF-8 nanoparticles loaded with ammonia borane;

[0073] Step S102, mixing the ZIF-8 nanoparticles loaded with ammonia borane, dopamine hydrochloride and a buffer solution to perform a self-polymerization reaction to obtain PDA-ZIF-8@AB nanoparticle powder.

[0074] The mass ratio of the soluble zinc salt to 2-MI is 0.05-0.15:1; the mass ratio of AB to 2-MI is 0.01-0.04:1; and the mass ratio of dopamine hydrochloride to ZIF-8 nanoparticles loaded with AB is 0.05-0.15:1.

[0075] Step S2, preparing PCL@QX-314 microsphere powder by water-in-oil-in-water type double emulsion-solvent evaporation method;

[0076] The step S2 specifically includes:

[0077] Step S201, mixing an aqueous solution containing the raw material drug QX-314 and the thickener hyaluronic acid (HA) (inner aqueous phase W1) with a dichloromethane (DCM) solution (oil phase O) of an organic high molecular weight polymer polycaprolactone (PCL), and performing a first emulsification with an ultrasonic cell disruptor under ice-water bath conditions to obtain W / O type colostrum;

[0078] Step S202, quickly pouring the W / O type colostrum into an aqueous solution (external aqueous phase W2) containing sodium chloride and an emulsifier polyvinyl alcohol (PVA), and performing a second emulsification by hand shaking or ultrasound to obtain a W / O / W type double emulsion;

[0079] Step S203, quickly pouring the W / O / W type emulsion into the aqueous solution containing PVA, stirring at a uniform speed at room temperature with volatile oil phase solvent DCM, so as to solidify the polymer carrier and form PCL microspheres loaded with QX-314.

[0080] The mass concentration of QX-314 in the inner aqueous phase is 1%-6%; the mass concentration of HA in the inner aqueous phase is 1%-8%; the mass concentration of PCL in the oil phase is 3%-10%; the mass concentration of PVA in the outer aqueous phase is 0.5%-6%; the mass concentration of sodium chloride in the outer aqueous phase is 0-1%; and the mass concentration of PVA in the final dispersant is 0.1%-1%.

[0081] The volume ratio of W1 to O is 1:1.25-10.

[0082] The power of the first ultrasonic emulsification is 90-150W, and the time is 30-120s; the power of the second emulsification is 60-120W of ultrasound or 100 times / min of manual shaking, and the time is 30-120s; the speed of the uniform stirring at room temperature is 100-800rpm, and the time is 4-24h.

[0083] Step S3, adding the PDA@ZIF-8@AB nanoparticle powder and PCL@QX-314 microsphere powder into the soluble matrix solution and mixing them to obtain a needle tip matrix solution;

[0084] In the step S3, the mass ratio of the PDA@ZIF-8@AB nanoparticle powder to the PCL@QX-314 microsphere powder is in the range of 1-3:1.

[0085] Step S4, adding the needle tip matrix solution into the microneedle mold, centrifuging and evacuating, and demolding the microneedle after drying to obtain a dual-phase microneedle based on molecular hydrogen / photothermal triggering.

[0086] The step S4 specifically includes:

[0087] Step S401, dispersing a water-soluble biocompatible polymer in a Tris-HCl solution to dissolve it, to obtain a soluble matrix solution;

[0088] Step S402, adding the PDA@ZIF-8@AB nanoparticle powder and PCL@QX-314 microsphere powder to the soluble matrix solution and mixing them to obtain a needle tip matrix solution;

[0089] Step S403, adding the needle tip matrix solution into the microneedle mold, centrifuging and evacuating, and demolding the microneedle after drying to obtain a dual-phase microneedle based on molecular hydrogen / photothermal triggering.

[0090] Furthermore, the water-soluble biocompatible polymer is one or more of HA, PVA and sucrose, with a mass concentration of 14%-20%.

[0091] Furthermore, the centrifugation time of the matrix solution is 10-30 min, the centrifugal speed is 4200 rpm; the vacuum time is 1-3 h; and the drying time is 24-48 h.

[0092] The present application will be described in detail below with reference to embodiments and experimental data.

[0093] Example 1

[0094] This embodiment provides a typical preparation method of dual-phase microneedles based on molecular hydrogen / photothermal triggering:

[0095] Step 1, preparation of PDA@ZIF-8@AB nanoparticles: zinc nitrate hexahydrate (104.125 mg) was dissolved in 700 μL sterile deionized water and ultrasonicated for 5 min to fully dissolve it, 2-MI (2.05 g) and AB (50 mg) were dissolved in 10 mL sterile deionized water and stirred at 500 rpm for 5 min to obtain a mixed solution, and then the zinc nitrate solution was added to the mixed solution very slowly, stirred at 1500 rpm at room temperature for 30 min, centrifuged and washed once with sterile deionized water to obtain a milky precipitate, resuspended the precipitate with 10 mL sterile deionized water and added Tris-HCl buffer to adjust the pH to 8.5, added 10 mg dopamine hydrochloride, stirred at 1500 rpm at room temperature for 2 h, centrifuged and washed 3 times with sterile deionized water, and then dried under vacuum for 48 h to obtain PDA@ZIF-8@AB nanoparticles;

[0096] Step 2: Preparation of PCL@QX-314 microspheres: 1 mL of aqueous solution containing 30 mg QX-314 and 20 mg HA (inner aqueous phase W1) was mixed with 5 mL of aqueous solution containing 250 mg The mixture was mixed with a dichloromethane solution of PCL (oil phase O), and the first emulsification was performed using an ultrasonic cell disruptor under ice-water bath conditions (ultrasonic power 150 W, ultrasonic time 1 min) to obtain W / O type colostrum; the colostrum was then quickly poured into a 10 mL aqueous solution containing 2% (w:v) PVA and 1% (w:v) NaCl (external aqueous phase W2), and the second emulsification was performed by hand shaking (hand shaking at a speed of 100 times / min for 1 min); the double emulsion was quickly poured into a 20 mL aqueous solution containing 0.2% (w:v) PVA, and stirred at 300 rpm for 4 h at room temperature to volatilize the oil phase solvent DCM, thereby solidifying the polymer carrier to form microspheres; the precipitate was separated by centrifugation at a speed of 7000 rpm for 15 min and washed 3 times with sterile deionized water, and dried under vacuum for 48 h to obtain PCL@QX-314 microspheres;

[0097] Step 3: PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres [mass ratio 1.18:1, 3% (w:v)] were dispersed in 18% (w:v) HA Tris-HCl solution to obtain a needle tip matrix solution, which was injected into a customized polydimethylsiloxane (PDMS) microneedle array mold and centrifuged at 4200 rpm for 15 min. The excess polymer solution was scraped off and the needle tip matrix solution was continued to be injected into the PDMS mold. The solution was treated under vacuum for 4 h and then transferred to a 40°C oven and dried for 48 h. The solution was then peeled off from the PDMS mold to obtain a dual-phase microneedle (HA-PPMN) based on molecular hydrogen / photothermal triggering.

[0098] The schematic diagram of the preparation process of the above-mentioned dual-phase microneedle based on molecular hydrogen / photothermal triggering is as follows Figure 1 The actual transmission electron microscopy image of PDA@ZIF-8@AB nanoparticles is shown in Figure 2 The actual scanning electron microscope image of PCL@QX-314 microspheres is shown in Figure 5 As shown, the actual optical microscope image of the dual-phase microneedle based on molecular hydrogen / photothermal triggering is shown in Figure 8 As shown, the scanning electron microscope image of a single microneedle based on molecular hydrogen / photothermal triggering dual-phase microneedle is shown Fig. 9 The results show that the preparation method provided in this embodiment can prepare PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres with good morphology, and the particle sizes are 176.5±1.22nm and 6.60±0.30μm respectively; the preparation method provided in this embodiment can prepare a microneedle patch that can be completely demolded, and the nanoparticles and microspheres are evenly dispersed in the needle body.

[0099] Comparative Example 1: Blank Microneedle

[0100] The comparative example is a blank microneedle, which has a Tris-HCl solution containing only HA as the needle tip matrix solution (HA MN) and does not contain nanoparticles and microspheres.

[0101] Comparative Example 2: Single-phase microneedle

[0102] The comparative example is a single-phase microneedle, that is, a Tris-HCl solution of HA containing PDA@ZIF-8@AB nanoparticles is used as the needle tip matrix solution (HA-PZA MN), without microspheres, and the other preparation steps are the same as Example 1.

[0103] Experimental Example 1: Performance test of PDA@ZIF-8@AB nanoparticles

[0104] 1. Photothermal effect of PDA@ZIF-8@AB nanoparticles

[0105] In order to study the photothermal effect of PDA@ZIF-8@AB nanoparticles, PBS solutions containing different concentrations of PDA@ZIF-8@AB (250, 500, and 800 μg / mL) were illuminated by 808 nm NIR (laser power of 1.0 W / cm 2 ) for 8 min. In every 1 min interval, near infrared images of solutions of different concentrations were taken using a near infrared thermal imager. Sterile deionized water was used as a control group under the same conditions. The results are shown in Figure 3As shown in the figure, when the concentration of PDA@ZIF-8@AB is 800μg / mL, the temperature can reach about 58°C within 8 minutes. When the concentration is 500μg / mL, the temperature reaches about 51°C within 8 minutes. When the concentration is 250μg / mL, the temperature reaches about 40°C. However, the temperature of the solution in the control group hardly changes under light irradiation. This proves that the PDA@ZIF-8@AB nanomaterial has good photothermal conversion performance.

[0106] 2. Hydrogen release of PDA@ZIF-8@AB nanoparticles in solutions with different pH values

[0107] The hydrogen release test of the PDA@ZIF-8@AB nanoparticles prepared in step 2 of Example 1 in solutions with different pH values ​​was conducted as follows: First, a methylene blue (MB) probe solution with a concentration of 300 μg / mL and a platinum (Pt) nanocrystal probe solution with a concentration of 13.9 μg / mL was prepared, and the MB concentration was ensured to be within the detection range of the UV spectrophotometer and to be able to completely absorb all hydrogen released by the PDA@ZIF-8@AB nanoparticles. PDA@ZIF-8@AB nanoparticles (2.5 mg / mL) were dispersed in MB-Pt probe solutions (4 mL) with different pH values ​​(7.4, 6.8) and incubated on a 37°C shaker at 150 rpm. Under different pH conditions, the hydrogen released by the nanoparticles catalytically will reduce MB to colorless, and the amount of hydrogen released is quantified by monitoring the absorbance changes of the solution at predetermined time points (0, 1, 2, 4, 6, 12, 24, 48, 72, 96, 120, 144 and 168 h). An in vitro release curve was drawn, as shown in FIG. Figure 4 The results show that in a release system with a pH of 7.4, the PDA@ZIF-8@AB nanoparticles prepared in step 2 of Example 1 only release a small amount of hydrogen before 6 hours, but in a release system with a pH of 6.8, a slow release of hydrogen can be achieved, and the release time exceeds 120 hours, indicating that the microneedles can release hydrogen for a long time in a weakly acidic environment.

[0108] Experimental Example 2: Thermal response characteristics of PCL@QX-314 microspheres

[0109] 1. Stability test of PCL@QX-314 microspheres with and without NIR irradiation

[0110] PCL@QX-314 microsphere powder was dispersed in PBS solution, and the PDA@ZIF-8@AB nanoparticles prepared in step 2 of Example 1 were added. For the NIR irradiation group, NIR irradiation (1 W / cm 2, 5min), and the control group was not subjected to NIR irradiation. For the NIR irradiation group, samples were taken after 0, 2, 4, and 6 light cycles, and for the non-NIR irradiation group, samples were taken after 0, 3, 5, and 7 days. The scanning electron microscopy images of the samples are shown in Figure 6 shown.

[0111] Figure 6 The results show that with the increase of the number of light cycles, the surface of the PCL@QX-314 microspheres provided by the present invention has obvious collapse and wrinkles, and the spherical structure begins to gradually break, showing significant morphological degradation. The microspheres that have not been irradiated by NIR can maintain a stable morphology within 7 days. The microspheres have a regular shape, a smooth surface, and a complete structure, showing a more typical spherical structure. This shows that the PCL@QX-314 microspheres provided by the present invention have good thermal response characteristics. When PDA is present, they can be deformed due to the increase in temperature when irradiated by NIR.

[0112] 2. In vitro release test of PCL@QX-314 microspheres with or without NIR irradiation and in different pH solutions:

[0113] PBS solutions with pH values ​​of 6.8 and 7.4 were prepared. 10 mg PCL@QX-314 microsphere powder was placed in each solution. The PDA@ZIF-8@AB nanoparticles prepared in step 2 of Example 1 were added and incubated on a shaker at 37°C at 150 rpm. For the NIR irradiation group, NIR irradiation (1 W / cm 2 , 5min). At the predetermined time point, 200 μL of supernatant was collected from each release system and stored in a 4°C refrigerator, and 200 μL of fresh PBS solution was added to each system. The collected samples were analyzed using a UV spectrophotometer, and the content of QX-314 in the samples at each time point was counted to draw an in vitro release curve, such as Figure 7 shown.

[0114] Figure 7 The results showed that PCL@QX-314 microspheres had a transient burst release at the beginning of the release. Subsequently, when the release system was exposed to NIR irradiation, the microspheres released QX-314. When there was no NIR irradiation, the microspheres did not release QX-314. Different pH values ​​had no obvious effect on the release of QX-314. This indicates that the PCL@QX-314 microspheres provided by the present invention have good thermal response characteristics. When PDA is present, NIR irradiation can be used as a switch for PCL@QX-314 microspheres to control drug release.

[0115] Experimental Example 3: Analgesic effect test of dual-phase microneedle based on molecular hydrogen / photothermal triggering

[0116] S1: 6-8 week old SPF healthy male rats were randomly divided into 7 groups, namely control group (Control), plantar incision group (S), blank microneedle treatment group after plantar incision (S+HA MN), single-phase microneedle treatment group after plantar incision (S+HA-PZA MN), single-phase microneedle treatment and NIR irradiation group after plantar incision (S+HA-PZA MN+NIR), dual-phase microneedle treatment group based on molecular hydrogen / photothermal triggering after plantar incision (S+HA-PPMN), and dual-phase microneedle treatment and NIR irradiation group after plantar incision (S+HA-PP MN+NIR).

[0117] S2: Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital combined with isoflurane inhalation. After disinfection, a longitudinal incision of about 1 cm was made on the left hind paw of the rat, about 2 mm away from the heel. The incision penetrated the skin, fascia and muscle, while maintaining the integrity of the muscle attachments at both ends. The skin was sutured with 5-0 sutures, and a small amount of antibiotic ointment was applied to the wound. The rats in the control group were given anesthesia, disinfection and antibiotic treatment, and no surgical operation was performed. After the righting reflex was restored, the rats in each group were immediately given the corresponding treatment measures. For rats that needed NIR treatment, NIR irradiation (1W / cm 2 , 5min).

[0118] S3: Pain behavior testing was performed at specific time points to assess the development and duration of postoperative pain, including the day before surgery and designated time intervals after surgery (basal value, 0, 2, 4, 6, 12, 24, 48, 72, 96, 120, 144, and 168 h). The mechanical pain sensitivity of rats was assessed using the von Frey fiber analgesia instrument. Before each test, the rats were placed in the test environment for 30 min to adapt. Fibers with different bending forces (1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, and 15.0 g) were applied sequentially near the plantar incision. Each fiber was pushed into the tissue 3 times, with an interval of 5 minutes between each time. Starting from the fiber with the smallest bending force, until a withdrawal reaction was observed. The force that produced a withdrawal reaction was recorded as the mechanical paw withdrawal threshold.

[0119] S4: The hot plate test was used to evaluate the thermal pain sensitivity of rats. The test was also performed one day before surgery and at specified time intervals after surgery (basal value, 0, 2, 4, 6, 12, 24, 48, 72, 96, 120, 144 and 168h). Three days before the formal test, the rats were placed in the test environment for 30 minutes every day to adapt. During the experiment, the rats were placed on the surface of the heating plate and maintained at a constant temperature of 52.5±0.5℃. The latency of the rats to withdraw or lick their paws was recorded, and each rat was tested three times, with an interval of 10 minutes each time. The blocking time was set to 30s to prevent tissue damage.

[0120] S5: Statistic the test results of mechanical pain and thermal pain respectively, and draw the pain behavior test curve, as shown in Fig.10 and Fig.11 shown.

[0121] The results showed that when single-phase microneedle analgesia was used, regardless of whether NIR irradiation was given, the mechanical pain threshold and thermal pain threshold of the rats were higher than those of the rats in the simple incision group without treatment and the rats treated with blank microneedles within 120 hours, indicating that the PDA@ZIF-8@AB nanoparticles provided by the present invention have good pH responsiveness, and the slowly released hydrogen can exert a long-lasting analgesic effect, and the analgesic effect can be maintained for more than 120 hours, but the analgesic effect can reach about 75%, and 100% analgesia was not achieved; when double-phase microneedle analgesia was used alone, the analgesic effect was 75%, and 100% analgesia was not achieved. When dual-phase microneedles are used for analgesia without NIR irradiation, the analgesic effect is close to that of single-phase microneedles; when dual-phase microneedles are used for analgesia and NIR irradiation is given at the same time, the mechanical pain threshold and thermal pain threshold of rats can be restored to almost 100% within 120 hours, which shows that the PCL@QX-314 microspheres provided by the present invention have good controlled release properties, which can supplement and assist hydrogen analgesia, reduce the individual differences in pain between different individuals, and make the dual-phase microneedles based on molecular hydrogen / photothermal triggering have excellent analgesic effect.

[0122] Experimental Example 4: Testing the effect of molecular hydrogen / photothermal-triggered dual-phase microneedling on promoting wound healing

[0123] S1: 6-8 week old SPF healthy male rats were randomly divided into 7 groups, namely control group (Control), plantar incision group (S), blank microneedle treatment group after plantar incision (S+HA MN), single-phase microneedle treatment group after plantar incision (S+HA-PZA MN), single-phase microneedle treatment and NIR irradiation group after plantar incision (S+HA-PZA MN+NIR), dual-phase microneedle treatment group based on molecular hydrogen / photothermal triggering after plantar incision (S+HA-PPMN), and dual-phase microneedle treatment and NIR irradiation group after plantar incision (S+HA-PP MN+NIR).

[0124] S2: Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital combined with isoflurane inhalation. After disinfection, a longitudinal incision of about 1 cm was made on the left hind paw of the rat, about 2 mm away from the heel. The incision penetrated the skin, fascia and muscle, while maintaining the integrity of the muscle attachments at both ends. The skin was sutured with 5-0 sutures, and a small amount of antibiotic ointment was applied to the wound. The rats in the control group were given anesthesia, disinfection and antibiotic treatment, and no surgical operation was performed. After the righting reflex was restored, the rats in each group were immediately given the corresponding treatment measures. For rats that needed NIR treatment, NIR irradiation (1W / cm 2 , 5min).

[0125] S3: On the fifth day after surgery, the recovery of the plantar incision of each group of rats was recorded by photographing. The rats were then euthanized, and the skin samples around the plantar incision were collected and fixed with 4% paraformaldehyde. The samples were embedded in paraffin and sliced ​​for H&E staining. The results were as follows: Fig.12 , 13 shown.

[0126] The results show that the microneedles provided by the present invention can significantly promote wound healing.

[0127] In summary, the dual-phase microneedle based on molecular hydrogen / photothermal triggering provided by the present invention can not only achieve continuous and long-lasting analgesia at the postoperative incision, but also significantly promote wound healing. At the same time, when the pain worsens, it can self-control the release of drugs through external photothermal stimulation to meet the patient's personalized analgesia needs.

[0128] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0129] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0130] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A dual-phase microneedle based on molecular hydrogen / photothermal triggering, characterized in that: The dual-phase microneedle based on molecular hydrogen / photothermal triggering includes a soluble microneedle needle body matrix and PDA@ZIF-8@AB nanoparticles and PCL@QX-314 microspheres dispersed in the matrix; The PDA-ZIF-8@AB nanoparticles are PDA-coated nanoparticles obtained by self-polymerization of ZIF-8 nanoparticles loaded with ammonia borane and dopamine hydrochloride; The PCL@QX-314 microspheres are PCL microspheres loaded with QX-314 prepared by a water-in-oil-in-water type emulsion-solvent evaporation method.

2. A dual-phase microneedle based on molecular hydrogen / photothermal triggering according to claim 1, characterized in that: The particle size of the PDA-ZIF-8@AB nanoparticles is 160-220 nm; the particle size of the PCL@QX-314 microspheres is 3-15 μm; in the PDA-ZIF-8@AB nanoparticles, the mass content of ammonia borane is 4-14%, and the mass content of polydopamine is 5-13%; in the PCL@QX-314 microspheres, the mass content of QX-314 is 1-8%.

3. A method for preparing a dual-phase microneedle based on molecular hydrogen / photothermal triggering according to claim 1-2, characterized in that: The method comprises: The ZIF-8 nanoparticles loaded with ammonia borane were subjected to self-polymerization reaction with dopamine hydrochloride to obtain PDA-ZIF-8@AB nanoparticle powder; PCL@QX-314 microsphere powder prepared by water-in-oil-in-water emulsion-solvent evaporation method; Add the PDA@ZIF-8@AB nanoparticle powder and PCL@QX-314 microsphere powder to the soluble matrix solution and mix well to obtain a needle tip matrix solution; The needle tip matrix solution is added to a microneedle mold, centrifuged and vacuumed, and the microneedle is demoulded after drying to obtain a dual-phase microneedle based on molecular hydrogen / photothermal triggering.

4. A method for preparing PDA-ZIF-8@AB nanoparticles, characterized in that: The method comprises: Ammonia borane, water, soluble zinc salt and 2-methylimidazole are mixed for coordination reaction to obtain ZIF-8 nanoparticles loaded with ammonia borane; The ZIF-8 nanoparticles loaded with ammonia borane are mixed with dopamine hydrochloride and a buffer solution to undergo a self-polymerization reaction to obtain PDA-ZIF-8@AB nanoparticle powder.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the soluble zinc salt to 2-MI is 0.05-0.15:1; the mass ratio of AB to 2-MI is 0.01-0.04:1; and the mass ratio of dopamine hydrochloride to ZIF-8 nanoparticles loaded with AB is 0.05-0.15:

1.

6. PDA-ZIF-8@AB nanoparticles prepared by the method described in claims 4-5.

7. A method for preparing PCL@QX-314 microspheres, characterized in that: The method comprises: The aqueous solution containing the raw material drug QX-314 and the viscosity enhancer as the inner aqueous phase is mixed with the dichloromethane solution of the organic high molecular weight polymer polycaprolactone as the oil phase, and the first emulsification is performed under ice water bath conditions to obtain W / O type colostrum; The W / O type colostrum is quickly poured into an external aqueous phase consisting of an aqueous solution containing sodium chloride and an emulsifier polyvinyl alcohol for a second emulsification to obtain a W / O / W type double emulsion; The W / O / W type emulsion was quickly poured into the aqueous solution containing PVA, and stirred at a uniform speed at room temperature to solidify the polymer carrier with the volatile oil phase solvent DCM to form PCL microspheres loaded with QX-314.

8. The preparation method according to claim 7, characterized in that: The mass concentration of QX-314 in the inner aqueous phase is 1%-6%; the mass concentration of the viscosity enhancer in the inner aqueous phase is 1%-8%; the mass concentration of PCL in the oil phase is 3%-10%; the mass concentration of PVA in the outer aqueous phase is 0.5%-6%; the mass concentration of sodium chloride in the outer aqueous phase is 0-1%; the mass concentration of PVA in the final dispersant is 0.1%-1%, the volume ratio of the inner aqueous phase to the oil phase is 1:1.25-10, the power of the first ultrasonic emulsification is 90-150W, and the time is 30-120s; the power of the second emulsification is ultrasonic 60-120W or hand shaking 100 times / min, and the time is 30-120s; the speed of uniform stirring at room temperature is 100-800rpm, and the time is 4-24h.

9. PCL@QX-314 microspheres prepared by the method described in claims 7-8.

10. Use of the molecular hydrogen / photothermal triggered dual-phase microneedle according to any one of claims 1 to 3 in the preparation of a drug for postoperative wound management.

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