A cryo-microneedle patch with infrared thermal response separation and its preparation method and application

Abstract

The invention discloses a cryo-microneedle patch separated by infrared thermal response and its preparation method and application, including a hydrogel needle tip with ultra-low temperature biocompatibility and a backing separated by thermal response, wherein the hydrogel needle tip material is methacrylated sodium alginate and methacrylated gelatin, and contains fat peptide-mesoporous polydopamine nanoparticles carrying active small molecule drugs, and the backing is composed of a poly (N-isopropylacrylamide) layer, a methacrylated sodium alginate hydrogel layer and a photoinitiator. The microneedle patch can be used to treat obesity and metabolic diseases induced by it, and treat obesity by activating a mechanosensitive channel (TRPV1) and inducing lipid droplet dissolution.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an infrared thermal response separation cryo-microneedle patch and a preparation method and application thereof. Background Art

[0002] Obesity and overweight seriously increase the risk of metabolic diseases, which plague about 40% of the world's population. As the active ingredient in red pepper, capsaicin shows positive effects in combating obesity in regulating lipid metabolism and inducing browning of white adipose tissue. The latest research shows that microneedle patches are a minimally invasive drug delivery strategy that can reduce local drug use and alleviate skin irritation. Although some progress has been made in microneedle delivery systems, most microneedle patches composed of hydrogels generally lack sufficient mechanical strength to effectively penetrate the skin barrier and enter the adipose tissue for anti-obesity treatment. In addition, existing microneedle patches need to adhere to the treatment site for a relatively long time, which may cause inconvenience and discomfort to patients. In addition, the significant hydrophobicity and extremely low bioavailability of naked capsaicin hinder its effective uptake by adipocytes, which in turn affects its therapeutic effect and wider application. Therefore, further efforts are still needed to construct new detachable cryo-microneedle patches with safety and effectiveness. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a safe and effective new type of detachable cryo-microneedle patch in view of the deficiencies in the prior art.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A cryo-microneedle patch with infrared thermal response separation comprises an ultra-low temperature biocompatible hydrogel needle tip and a thermal response separation backing, wherein the hydrogel needle tip material is methacrylated sodium alginate and methacrylated gelatin, and contains fat peptide-mesoporous polydopamine nanoparticles carrying active small molecule drugs, and the backing is composed of a poly (N-isopropylacrylamide) layer, a methacrylated sodium alginate hydrogel layer and a photoinitiator.

[0005] Preferably, the fat peptide is CKGGRAKDC, and the mass ratio of mesoporous polydopamine nanoparticles to the fat peptide is 1:1-5.

[0006] Preferably, the active small molecule drug is selected from at least one small molecule fat-reducing drug selected from capsaicin (Cap), rosiglitazone, and 2,4-dinitrophenol, and the concentration of the active small molecule drug in the hydrogel needle tip is 100-1000 μg / ml.

[0007] Preferably, the photoinitiator is 2-hydroxy-2-methylpropiophenone (HMPP).

[0008] Furthermore, the present invention also claims a method for preparing the above-mentioned infrared thermal response separation cryo-microneedle patch, comprising the following steps: (1) mixing mesoporous polydopamine nanoparticles with active small molecule drugs to obtain drug-loaded mesoporous polydopamine drug-loaded nanoparticles; (2) mixing the mesoporous polydopamine drug-loaded particles prepared in step (1) with adipose peptide, and freeze-drying the prepared mixture to obtain adipose-targeted mesoporous polydopamine drug-loaded nanoparticles; (3) dissolving methacrylated gelatin, methacrylated sodium alginate and a photoinitiator in water, stirring in the dark, adding the fat-targeted mesoporous polydopamine drug-loaded nanoparticles of step (2), mixing evenly and pouring into a microneedle mold, centrifuging to allow the mixed solution to fill the microneedle mold, scraping off the liquid in the backing layer, curing by ultraviolet irradiation, and obtaining a needle tip array; (4) Preparation of detachable microneedle backing: Add a poly(N-isopropylacrylamide) solution containing a photoinitiator to the above-mentioned needle tip array, add a methacrylated sodium alginate solution containing a photoinitiator after UV curing, and UV cure again. Finally, freeze-dry the UV-cured detachable microneedles and demold them.

[0009] Specifically, in step (1), Pluronic F127 and TMB are used as organic templates to prepare mesoporous polydopamine nanoparticles by a one-step synthesis method.

[0010] Specifically, in step (1), the mesoporous polydopamine nanoparticles and the active small molecule drug are mixed at a mass ratio of 1 to 20:1, incubated for 1 to 3 hours at a temperature of 20 to 25°C, and then dialyzed with double distilled water to remove excess active small molecule drugs.

[0011] Specifically, in step (2), the mesoporous polydopamine drug-loaded particles and the fat peptide are mixed at a mass ratio of 1:1-5 and incubated at room temperature for 1-12 h, and then dialyzed with double distilled water to remove excess fat peptide.

[0012] Specifically, in step (3), methacrylated gelatin, methacrylated sodium alginate and a photoinitiator are dissolved in water in a mass ratio of 1:1:0.01-0.001 to prepare a solution.

[0013] Specifically, in step (3), the content of fat-targeted mesoporous polydopamine drug-loaded nanoparticles in the obtained needle tip array is 100-1000 μg / ml.

[0014] Specifically, in step (4), in the poly(N-isopropylacrylamide) solution containing a photoinitiator, the photoinitiator concentration is 0.1-0.2% (w / v), the poly(N-isopropylacrylamide) concentration is 1-10% (w / v), and in the methacrylated sodium alginate solution containing a photoinitiator, the photoinitiator concentration is 0.05-0.2% (w / v), and the methacrylated sodium alginate concentration is 2-10% (w / v). The above concentrations are all expressed in g / ml.

[0015] Specifically, in step (4), a poly(N-isopropylacrylamide) solution containing a photoinitiator is added and then UV-cured for 10 to 30 seconds, and then a methacrylated sodium alginate solution containing a photoinitiator is added and UV-cured again for 10 to 30 seconds; the freeze-drying temperature is below -80 degrees.

[0016] Furthermore, the present invention also claims to protect the use of the above-mentioned infrared thermal response separation cryo-microneedle patch in the preparation of drugs for treating obesity and metabolic diseases induced by obesity. Beneficial Effects

[0017] (1) The detachable cryo-microneedle patch of the present invention has strong penetrating power and can directly deliver drugs through the skin, achieving precise weight loss in specific areas; the needle of the microneedle patch does not reach the dermis and causes almost no pain; the microneedle patch has excellent light responsiveness under the stimulation of near-infrared light and can quickly heat up and release drugs.

[0018] (2) The detachable cryo-microneedle patch of the present invention has both sustained-release and stimulated rapid-release properties of drugs, and the drug release is controllable.

[0019] (3) The detachable microneedle patch of the present invention can be used to treat obesity and metabolic diseases induced by it, and treat obesity by activating mechanosensitive channels (TRPV1) and inducing lipid droplet dissolution. The method is simple and easy to operate. Biocompatible hydrogel is used as the microneedle patch matrix, combined with fat-targeted mesoporous polydopamine (which gives the drug adipocyte targeting and constructs a heating system), and loaded capsaicin as a weight-loss drug. The drug is released through the needle tip to induce white fat browning and lipid droplet dissolution, thereby achieving the purpose of precise fat reduction. The detachable frozen microneedle patch is attached to the abdomen, and the drug's excellent white fat browning induction is used to eliminate fat through metabolic pathways. Mesoporous polydopamine nanoparticles have excellent photothermal properties and have a high sensitivity to near-infrared light excitation, so that the microneedle backing can be separated by photothermal response. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 Schematic diagram of the preparation of the detachable cryo-microneedles provided by the present invention.

[0022] Figure 2 Macroscopic schematic diagram of the detachable cryo-microneedle patch.

[0023] Figure 3 This is a transmission electron micrograph of the frozen microneedles in Example 1.

[0024] Figure 4 The detachable cryo-microneedle patch has excellent photothermal stability under the excitation of near-infrared light.

[0025] Figure 5 Photo of obese mice and abdominal fat treated with the detachable cryo-microneedle patch.

[0026] Figure 6 H&E staining of the liver of obese mice treated with detachable cryo-microneedle patches. DETAILED DESCRIPTION

[0027] The present invention can be better understood with reference to the following examples.

[0028] The preparation process of the infrared thermal response separation cryo-microneedle patch of the present invention is as follows: Figure 1 As shown, the following steps are included: (1) mixing mesoporous polydopamine nanoparticles with active small molecule drugs to obtain drug-loaded mesoporous polydopamine drug-loaded nanoparticles; (2) mixing the mesoporous polydopamine drug-loaded particles prepared in step (1) with adipose peptide, and freeze-drying the prepared mixture to obtain adipose-targeted mesoporous polydopamine drug-loaded nanoparticles; (3) dissolving methacrylated gelatin, methacrylated sodium alginate and a photoinitiator in water, stirring in the dark, adding the fat-targeted mesoporous polydopamine drug-loaded nanoparticles of step (2), mixing evenly and pouring into a microneedle mold, centrifuging to allow the mixed solution to fill the microneedle mold, scraping off the liquid in the backing layer, curing by ultraviolet irradiation, and obtaining a needle tip array; (4) Preparation of detachable microneedle backing: Add a poly(N-isopropylacrylamide) solution containing a photoinitiator to the above-mentioned needle tip array, add a methacrylated sodium alginate solution containing a photoinitiator after UV curing, and UV cure again. Finally, freeze-dry the UV-cured detachable microneedles and demold them.

[0029] The prepared infrared thermal response separation cryo-microneedle patch comprises an ultra-low temperature biocompatible hydrogel needle tip and a thermal response separation backing, wherein the hydrogel needle tip material is methacrylated sodium alginate and methacrylated gelatin, and contains fat peptide-mesoporous polydopamine nanoparticles carrying active small molecule drugs, and the backing is composed of a poly (N-isopropylacrylamide) layer, a methacrylated sodium alginate hydrogel layer and a photoinitiator. Example 1

[0030] Preparation and characterization of CKGGRAKDC&mPDA@Cap (TmCNPs) 1. Preparation of mPDA: Pluronic F127 was used as a template, ethanol and water were used as organic cosolvents, and dopamine (DA) was used as a raw material. 1.0 g F127 and 0.5 g DA were dissolved in a mixture of 100 mL of water and ethanol (1:1), and stirred vigorously at room temperature to obtain a clear solution. Then 2.0 mL of TMB was slowly injected at a stirring speed of 500 rpm to form a nanoemulsion system. After stirring for 30 min, 5.0 mL of concentrated ammonia (NH4OH) was dripped into the above mixture to induce self-polymerization of dopamine oligomers. After another 30 min of continuous reaction, the dendritic mesoporous TMB / F127 / PDA polymer nanoparticles were washed with water and ethanol at least three times in sequence. The polymerized TMB / F127 / PDA nanoparticles were preheated at 350 °C for 4 h and heated at 800 °C for 2 h, with a heating rate of 1 °C min -1 , mesoporous polydopamine was prepared by N2 pyrolysis, with a particle size of 80~100 nm.

[0031] 2. mPDA adsorbs Cap: The prepared mPDA is dissolved in phosphate buffer, and Cap powder is added in a mass ratio of 1-10:1. The mixture is incubated at room temperature for 1-12 h, centrifuged at 5000-12000 rpm at 4 °C for 10-30 min, and washed three times with phosphate buffer to obtain drug-loaded mPDA@Cap nanoparticles.

[0032] 3. Connecting mPDA@Cap with adipose peptide CKGGRAKDC: The prepared mPDA@Cap and adipose peptide were dissolved in aqueous solution at a certain mass ratio of 1-10:1, stirred at room temperature for 2-4 hours, and freeze-dried after dialysis to obtain TmCNPs.

[0033] 4. Characterization of TmCNPs: The particle size and polydispersity coefficient of the mPDA@Cap nanoparticle aqueous solution were determined by dynamic light scattering. The results showed that the average particle size of the nanoparticles was 86.12±7.26 nm. The encapsulation efficiency of Cap measured in Example 1 was 649.34±19.76 μg / mg. Example 2

[0034] Preparation and characterization of detachable cryo-microneedles TmCNPs@AGMN 1. Preparation of separable microneedle array: The microneedle matrix is ​​composed of methacrylated gelatin, methacrylated sodium alginate and a photoinitiator. The three materials are dissolved in water at a mass ratio of 1:1:0.01-0.001, stirred in the dark, and then the fat-targeted mesoporous polydopamine nanoparticles (TmCNPs) are added and mixed evenly; the mixture is poured into the microneedle mold, centrifuged at 2000-3000 rpm for 5 min to completely fill the microneedle mold, the liquid in the backing layer is scraped off, and the needle tip array is obtained by UV curing.

[0035] 2. Preparation of detachable microneedle backing: Add poly (N-isopropylacrylamide) solution containing photoinitiator and UV cure on ice for 10-30 s; then add methacrylated sodium alginate solution containing photoinitiator and UV cure again for 10 s.

[0036] 3. Preparation of frozen microneedles: Place the UV-cured detachable microneedles in a -80 degree freezer for 1 to 3 hours, and demold to obtain detached frozen microneedles TmCNPs@AGMN. Figure 2 As shown, it is demonstrated that the microneedle prepared by the present invention has a smooth and dense surface, a bright texture, no cracks, and a complete shape.

[0037] 4. Characterization of detachable frozen microneedles: Scanning electron microscopy was used to detect freeze-dried microneedles. The results showed that there were obvious pores at the tip of the microneedle, with a size of tens to hundreds of microns, indicating that the microneedle matrix can successfully carry TmCNPs. Figure 3 As shown in Figure 2, SEM can clearly show the shape, size, sharpness of the needle tip, and surface roughness of the microneedles. In the SEM image, the surface of the microneedle may show a certain degree of microscopic roughness, which helps to improve the adhesion and skin penetration of the drug. The rough surface can increase the contact area between the drug and the skin, thereby improving the drug release efficiency. Example 3

[0038] In vitro NIR thermal stability study of separable cryo-microneedle TmCNPs@AGMN.

[0039] In order to evaluate whether the prepared detachable microneedles can have thermal conversion ability and thermal stability in vitro, we used a near-infrared light source to irradiate the microneedles and monitor the changes in the temperature of the microneedles. By measuring the temperature changes of the microneedles under NIR light irradiation, the thermal conversion efficiency was evaluated, the local temperature of the microneedles at different time points was recorded, and the stability and efficiency of the photothermal effect were analyzed. The microneedles were irradiated with NIR multiple times to test their performance stability in repeated photothermal conversion, observe whether there are significant changes in drug release and structural changes, and verify their feasibility for long-term use. Figure 4 As shown, it was shown that mesoporous dopamine nanoparticles have good thermal stability, demonstrating their potential in drug delivery and photothermal therapy. Example 4

[0040] Evaluation of the therapeutic effect of TmCNPs@AGMN on high-fat-induced obese mice Obesity model establishment: All animal experiments involved in this paper were in compliance with the legal requirements of the People's Republic of China and the Animal Care and Use Regulations of Nanjing University. All mice were housed in an SPF animal room with a lighting cycle controlled to a 12-h (light) / 12-h (dark) day-night cycle, a humidity of 50-60%, and a temperature of 20-22 °C.

[0041] Construction of a disease model of nutritional obesity (HFD) induced by high-fat diet in mice: After one week of adaptive feeding, 5-week-old male C57BL / 6J mice were randomly divided into two groups. The model group was fed with a high-fat diet (60% kJ, D12492), and the control group was fed with a standard diet (LFD, 10% kJ, D12450B). Five mice were placed in each cage. From left to right, the toes were cut in sequence as marks. All mice had free access to water and food. The weights were recorded at the same time period every week. The weight trend line, biochemical indicators (blood sugar and blood lipid levels) and GTT and ITT curves of the obesity model were used to measure and evaluate whether the model was successful. After the model was established, the weight of the mice was weighed and recorded every week.

[0042] Treatments in the HFD model After 1 week of adaptive feeding, 5-week-old C57BL / 6J male mice were randomly divided into cages, 5 mice per cage, and divided into the following groups (8-12 mice per group): Group I (LFD): fed with standard diet when the model was established; Group II (HFD): fed with a high-fat diet when the model was established; Group III (HFD + TmCNPs@AGMN): 8 weeks after the model was established, the mice were depilated on their abdomens using a depilatory device and the cryo-microneedle patch was applied once every two weeks; Group IV (HFD + NIR-TmCNPs@AGMN): 8 weeks after the model was established, the mice were depilated on their abdomens with a depilatory device, and cryo-microneedle patches were applied. They were then irradiated with infrared light for 5 minutes every three days and applied once every two weeks. Tissue samples were collected.

[0043] The mice were fasted for 8-12 hours before sampling. The mice were weighed and photographed, and the eyeballs were removed to obtain blood samples and separate the serum. The tissues that needed to be retained were quickly dissected and separated, including the liver and white adipose tissue (subcutaneous inguinal white adipose tissue, subWAT; epididymal white adipose tissue, eWAT), weighed and recorded. The samples for protein level detection were quickly placed in liquid nitrogen, frozen and stored in a -80 ℃ refrigerator. The samples for histopathological analysis were fixed and embedded in 4% paraformaldehyde, and HE stained and photographed. Figure 5 and Figure 6 As shown, in Figure 5 Compared with the model group, the treatment group showed a significant reduction in body size and adipose tissue. Figure 6 It can be seen that after treatment with infrared irradiated cryopreservation patches, the macrovesicular fatty degeneration of hepatocytes was significantly improved, as manifested by a decrease in the appearance of lipid droplets in the cytoplasm; the abdominal adipose tissue showed atrophy of single-chamber white adipocytes, and a significant decrease in the volume of adipocytes.

[0044] The present invention provides an infrared heat response separation cryo-microneedle patch and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A cryo-microneedle patch with infrared thermal response separation, characterized in that: The invention comprises an ultra-low temperature biocompatible hydrogel needle tip and a thermally responsive separation backing, wherein the hydrogel needle tip material is methacrylated sodium alginate and methacrylated gelatin, and contains fat peptide-mesoporous polydopamine nanoparticles carrying active small molecule drugs, and the backing is composed of a poly (N-isopropylacrylamide) layer, a methacrylated sodium alginate hydrogel layer and a photoinitiator.

2. The infrared thermal response separation cryo-microneedle patch according to claim 1, characterized in that: The fat peptide is CKGGRAKDC, and the mass ratio of mesoporous polydopamine nanoparticles to the fat peptide is 1:1-5.

3. The infrared thermal response separation cryo-microneedle patch according to claim 1, characterized in that: The active small molecule drug is selected from at least one small molecule fat-reducing drug selected from capsaicin, rosiglitazone, and 2,4-dinitrophenol, and the concentration of the active small molecule drug in the hydrogel needle tip is 100-1000 μg / ml.

4. The infrared thermal response separation cryo-microneedle patch according to claim 1, characterized in that: The photoinitiator is 2-hydroxy-2-methylpropiophenone.

5. The method for preparing the infrared thermal response separation cryo-microneedle patch according to claim 1, characterized in that: The steps include: (1) mixing mesoporous polydopamine nanoparticles with active small molecule drugs to obtain drug-loaded mesoporous polydopamine drug-loaded nanoparticles; (2) mixing the mesoporous polydopamine drug-loaded particles prepared in step (1) with adipose peptide, and freeze-drying the prepared mixture to obtain adipose-targeted mesoporous polydopamine drug-loaded nanoparticles; (3) dissolving methacrylated gelatin, methacrylated sodium alginate and a photoinitiator in water, stirring in the dark, adding the fat-targeted mesoporous polydopamine drug-loaded nanoparticles of step (2), mixing evenly and pouring into a microneedle mold, centrifuging to allow the mixed solution to fill the microneedle mold, scraping off the liquid in the backing layer, curing by ultraviolet irradiation, and obtaining a needle tip array; (4) Preparation of detachable microneedle backing: Add a poly(N-isopropylacrylamide) solution containing a photoinitiator to the above-mentioned needle tip array, add a methacrylated sodium alginate solution containing a photoinitiator after UV curing, and UV cure again. Finally, freeze-dry the UV-cured detachable microneedles and demold them.

6. The method for preparing the infrared thermal response separation cryo-microneedle patch according to claim 5, characterized in that: In step (1), mesoporous polydopamine nanoparticles are prepared by a one-step synthesis using Pluronic F127 and TMB as organic templates; the mesoporous polydopamine nanoparticles are mixed with active small molecule drugs at a mass ratio of 1 to 20:1, incubated for 1 to 3 hours at a temperature of 20 to 25°C, and then dialyzed with double distilled water to remove excess active small molecule drugs.

7. The method for preparing the infrared thermal response separation cryo-microneedle patch according to claim 5, characterized in that: In step (2), the mesoporous polydopamine drug-loaded particles and the fat peptide are mixed at a mass ratio of 1:1-5 and incubated at room temperature for 1-12 h, and then dialyzed with double distilled water to remove excess fat peptide.

8. The method for preparing the infrared thermal response separation cryo-microneedle patch according to claim 5, characterized in that: In step (3), methacrylated gelatin, methacrylated sodium alginate and photoinitiator are dissolved in water at a mass ratio of 1:1:0.01-0.001 to prepare a solution; in the obtained needle tip array, the content of fat-targeted mesoporous polydopamine drug-loaded nanoparticles is 100-1000 μg / ml.

9. The method for preparing the infrared thermal response separation cryo-microneedle patch according to claim 5, characterized in that: In step (4), in the poly(N-isopropylacrylamide) solution containing a photoinitiator, the photoinitiator concentration is 0.1-0.2% (w / v), and the poly(N-isopropylacrylamide) concentration is 1-10% (w / v); in the methacrylated sodium alginate solution containing a photoinitiator, the photoinitiator concentration is 0.05-0.2% (w / v), and the methacrylated sodium alginate concentration is 2-10% (w / v).

10. Use of the infrared thermal response separation cryo-microneedle patch according to claim 1 in the preparation of drugs for treating obesity and metabolic diseases induced therefrom.

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