Materials, Microneedles and Medical Devices for Alleviating Laser-Induced Thermal Damage and Pain

Abstract

A material for alleviating laser-induced thermal damage and pain, comprising copper ions and asiatic acid, further comprising lidocaine and a biodegradable polymer for making microneedles. The copper ions and asiatic acid are heated under alkaline conditions and self-assembled into a MOF structure. It has been verified that the material of the present invention for making CuMOF@AA@Lidocaine microneedles not only significantly improves the pain response of animals, but also effectively alleviates the inflammatory response caused by thermal damage, reduces the damage to tissues caused by high fever, and is suitable as a medical device for alleviating pain during laser treatment and skin thermal damage caused.

Description

Technical Field

[0001] The present invention relates to an organic microparticle material, in particular to a metal-organic framework material, its application in alleviating thermal damage and pain caused by laser treatment, and its microneedles and medical devices. Background Art

[0002] Ablative lasers represented by carbon dioxide fractional lasers are widely used in dermatology and plastic surgery. Its main principle is fractional photothermolysis. The laser beams arranged in an array act on the skin surface, especially the dermis layer. By stimulating the generation of dermal collagen and the rearrangement of collagen fibers, as well as promoting the repair of skin tissue and pigment decomposition, multiple effects such as improving skin quality, repairing scars, removing wrinkles, removing freckles and beautifying the skin, and shrinking pores can be achieved.

[0003] Skin burns are common adverse reactions of ablative lasers. Taking carbon dioxide fractional lasers as an example, when it acts on the skin, it will produce thermal damage such as thermal effects, thermal ablation and thermal coagulation, and the highest temperature can reach 100°C. Improper operation or improper parameter settings will cause skin thermal damage of different depths, leading to skin inflammation, and may seriously cause pigmentation and scars.

[0004] In traditional Chinese medicine, the umbelliferous plant Centella asiatica (L.) Urban is considered to have anti-inflammatory and anti-scar effects (Int J Mol Sci, 2024, 25(2)). Modern research has found that the organic component asiatic acid (AA) extracted from Centella asiatica can effectively inhibit the activation and proliferation of inflammatory cells, and inhibit macrophages from expressing inflammatory factors such as IL-6, TNF-α and PGE2. Therefore, it can effectively reduce tissue damage and inflammatory responses (FASEB J, 2024, 38(9):e23645, Int Immunopharmacol, 2017, 50:313-318). However, due to the extremely low solubility of AA in water, it directly limits its bioavailability in vivo (Photochem Photobiol, 2020, 96(1):124-138). Summary of the Invention

[0005] One object of the present invention is to provide a composite material, which is beneficial to improving the solubility of asiatic acid, and thus improving the bioavailability.

[0006] Another object of the present invention is to provide a composite material that acts on the skin to achieve alleviating thermal damage caused by laser treatment.

[0007] Another object of the present invention is to provide an application of a composite material in the preparation of a medical device for alleviating thermal damage caused by laser treatment.

[0008] Another object of the present invention is to provide a microneedle comprising lidocaine for relieving heat damage caused by laser treatment.

[0009] Copper ions (Cu 2+ ) can participate in the cross-linking process of collagen and elastin in the extracellular matrix by regulating enzyme activity as a coenzyme, affecting collagen maturation (Am J Clin Nutr, 1979, 32(4): 856-71). It has been found that Cu 2+ also has a certain anti-inflammatory effect and can reduce the damage caused by oxidative stress to cells (Int Immunopharmacol, 2017, 50: 313-318). In the present invention, it combines with AA to produce a synergistic effect in relieving heat damage.

[0010] A composite material comprising copper ions and asiatic acid, with a weight ratio of 1:0.9 to 1.

[0011] Another composite material is a MOF structure composed of copper ions and asiatic acid, with a weight ratio of copper ions to asiatic acid of 1:0.9 to 1.

[0012] The composite material of the present invention is self-assembled by heating copper ions and asiatic acid under alkaline conditions (such as pH>11) (such as 70°C±2°C) for 24 hours.

[0013] The composite material of the present invention is in the form of flaky microparticles with an average particle size of 236.8 nm.

[0014] In the composite material of the present invention, copper and oxygen are crystal-bonded.

[0015] The composite material of the present invention further comprises lidocaine to facilitate the application of the composite material to heat damage caused by laser treatment, with a content of 8.6 wt%±0.4 wt%.

[0016] The composite material of the present invention is mixed with a biodegradable polymer capable of forming a gel (such as, but not limited to: PLGA, PLA, PGA, PLL, etc.) as a substrate to form a microneedle patch. The patch includes an array formed by arranging a plurality of microneedles, and the height of the microneedles is 400 μm±20 μm.

[0017] The present invention synthesizes a novel MOF structural material (CuMOF@AA) from Cu ions and AA. This material can make full use of the biological properties of Cu ions and AA to reduce the inflammatory reaction in the wound microenvironment. At the same time, it realizes the controlled release of AA by using the degradation process of the MOF material in the body, and can also avoid the safety risks that may be caused by directly orally taking or injecting copper as a heavy metal.

[0018] In addition, due to the photothermal effect, patients also experience obvious pain during the use of ablative lasers. Clinically, topical anesthetic drugs such as lidocaine cream are usually applied before the operation to relieve pain. However, cream-based anesthetic drugs have the defects of long onset time and limited subcutaneous penetration depth during use, and the actual analgesic effect for laser treatment is not good as observed clinically.

[0019] The microneedles provided by the present invention, especially the microneedle patch made of a soluble substrate, achieve transdermal drug delivery. The needle body contains functional drug components. According to the different lengths of the needle tips, it can penetrate into different depths of the skin and release the drug as the needle body dissolves, realizing efficient penetration and absorption of the subcutaneous tissue and the human body, and will not leave obvious damage on the epidermis, effectively avoiding the pain, bleeding and infection risks of traditional injection methods, and is also simple and comfortable to use.

[0020] It has been verified that in vitro, the CuMOF@AA@Lidocaine microneedle solution can reduce the stress changes generated by cells after thermal injury, and there is no obvious increase in the content and mRNA expression of HSP70 protein. It has also been confirmed that the number of cells dead under the action of CuMOF@AA@Lidocaine microneedles has no obvious change, effectively protecting cells and reducing apoptosis caused by thermal injury.

[0021] Experiments in experimental animals show that CuMOF@AA@Lidocaine microneedles not only significantly improve the pain response of animals, but also can effectively relieve the inflammatory response caused by thermal injury, reduce the damage to tissues caused by high fever, and are suitable as medical devices for relieving pain during laser treatment and skin thermal injury caused. Description of the Drawings

[0022] Figure 1 It is the bright-field photograph of the prepared CuMOF@AA powder;

[0023] Figure 2 It is the bright-field photograph of the CuMOF@AA microneedle array sheet;

[0024] Figure 3 It is the electron microscope image of the CuMOF@AA powder under the FE-SEM field of view;

[0025] Figure 4 It is the DLS particle size result graph of the CuMOF@AA powder;

[0026] Figure 5 It is the elemental analysis result graph of C, O, Cu, etc. of the CuMOF@AA powder;

[0027] Figure 6 It is the XRD detection graph of the CuMOF@AA powder;

[0028] Figure 7It is the ultraviolet spectrophotometric detection diagram of the CuMOF@AA solution;

[0029] Figure 8 It is the electron micrograph of the CuMOF@AA-Lidocaine microneedle patch under the FE-SEM field of view;

[0030] Figure 9 It is the histological section diagram after inserting the CuMOF@AA-Lidocaine microneedle patch; among them, A is the puncture effect diagram presented by the paraffin section of the mouse skin, and B is the puncture effect diagram presented by the frozen section of the human scar skin;

[0031] Figure 10 It is the result diagram of HSP70 in each group of cells after thermal injury; among them, A is the protein electrophoresis result of HSP70 and its statistical chart, B is the statistical chart of the change of HSP70-related gene expression mRNA, * indicates that compared with the Ctrl-neg group, p<0.05; # indicates that compared with the Ctrl-pos group, p<0.05;

[0032] Figure 11 It is the result diagram of dead cell staining in each group after thermal injury; among them, A is the staining diagram under the field of view, B is the statistical chart of the staining count result, * indicates: compared with the Ctrl-neg group, p<0.05; # It indicates: compared with the Ctrl-pos group, p<0.05;

[0033] Figure 12 It is the analgesic and anti-thermal injury effect diagram of evaluating the CuMOF@AA-Lidocaine microneedle patch by the skin scald model; among them, A is the bright-field photos of the skin of each experimental group of animals before treatment (Pre), after treatment (Post) and after modeling (Post-burn) with lidocaine cream (Ctrl group) or CuMOF@AA-Lidocaine microneedle (MN group), B is the retraction frequency of the rabbit during the dot scald process, *p<0.05; C is the result diagram of HE staining in each experimental group at 3 days (3day) and 7 days (7day) respectively. Specific implementation manners

[0034] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

[0035] Example 1 Preparation and characterization of CuMOF@AA microneedles

[0036] Dissolve 120 mg of CuCl 2 ·2H 2 O and 50 mg of asiatic acid in 20 mL of double-distilled water with pH > 11 (adjusted with 10 mol / L NaOH). After stirring well, heat the mixture in a dark water bath at 70 °C for 24 h. Collect the turbid liquid, centrifuge at 12000 rpm for 15 min at 4 °C, discard the supernatant, and resuspend and wash twice with absolute ethanol and double-distilled water respectively. After washing, centrifuge at 20000 rpm for 10 min. Collect the dark gray solid at the bottom and freeze-dry to obtain a brown powder (as Figure 1 ).

[0037] Use FE-SEM to photograph the synthesized brown powder, and it can be seen that Cu and AA self-assembled into uniformly sized flake-like particles under alkaline conditions (as Figure 3 ).

[0038] DLS detection shows that the average particle size of the brown powder is 236.8 nm (as Figure 4 ).

[0039] According to energy-dispersive X-ray spectroscopy analysis, carbon (C), oxygen (O), and copper (Cu) are uniformly distributed in the brown powder particles (as Figure 5 ), and their contents (w%) are approximately 28.16%, 29.10%, and 42.74%.

[0040] XRD analysis shows that the crystal structure peaks of the brown powder particles conform to the characteristic peaks of CuO nanocompounds (as Figure 6 ), indicating that in this example, a material with a complete crystal structure was successfully synthesized.

[0041] In addition, ultraviolet spectrophotometry detected that the brown powder solution showed an absorption peak stably at 250 nm ( Figure 7 ), which is also consistent with the absorption spectrum of asiaticoside in the literature at 276 nm.

[0042] These results prove that in this example, a MOF-structured nanoparticle composed of Cu and asiatic acid stably was obtained, denoted as: CuMOF@AA.

[0043] Example 2 Preparation and Characterization of CuMOF@AA Microneedle Patches

[0044] Dissolve the CuMOF@AA powder in 20 mg / mL lidocaine hydrochloride and dilute to 4 mg / mL. Add 600 μL of the diluted CuMOF@AA-lidocaine solution to 350 mg of γ-PGA. Ultrasonically vibrate at room temperature for 20 min to disperse well. Centrifuge at 12000 rpm for 15 min at 4 °C to remove air bubbles to prepare the CuMOF@AA gel.

[0045] The CuMOF@AA gel was filled into the PDMS microneedle mold and centrifuged at 3000 rpm at room temperature to remove air bubbles. Then, the excess gel around was wiped with a cotton swab, and the filled mold was placed in an oven at 40 °C overnight. After the microneedles were dried, they were demolded for use, and the CuMOF@AA microneedle array sheet was obtained (as Figure 2 ).

[0046] As Figure 8 , it can be seen from the FE-SEM images that the pyramid-shaped microneedles in the CuMOF@AA-Lidocaine microneedle patch are arranged in a matrix pattern, with the bottom side about 260 μm long and the height about 400 μm. They can penetrate the epidermis in mouse skin ( Figure 9 A) and human scar skin ( Figure 9 B), and the depths are >300 μm and >100 μm, respectively.

[0047] Detected by HPLC liquid chromatography, the lidocaine content in the CuMOF@AA-Lidocaine microneedle patch is about 8.6% (w / w).

[0048] Example 3 In vitro experiment on the anti-thermal damage of CuMOF@AA-Lidocaine microneedles

[0049] The ethylene oxide-sterilized CuMOF@AA-Lidocaine microneedle patch was prepared into a solution at a ratio of 2 pieces / 40 mL of DMEM cell culture medium. At the same time, γ-PGA / DMEM solution and lidocaine / DMEM solution with the same concentration were respectively prepared. After treating skin fibroblasts with DMEM medium, CuMOF@AA-Lidocaine solution, γ-PGA / DMEM solution and lidocaine / DMEM solution for 15 minutes, the culture dishes were placed in a 45 °C water bath for heat treatment for 10 minutes, and then the cells in each group were collected to detect the expression of heat shock protein 70 (HSP70). Under normal physiological conditions, HSP70 is constitutively expressed in cells, but under in vitro environmental stress conditions such as high temperature, cold, and hypoxia, the synthesis rate of HSP70 increases significantly. As Figure 10 shown, compared with the skin fibroblasts before heat treatment (Ctrl-neg), the content of HSP70 in the skin fibroblasts without special treatment (Ctrl-pos), treated with γ-PGA solution (γ-PGA) and treated with lidocaine solution (Lidocaine) increased limitedly after heat damage, while the content of HSP70 protein ( Figure 10 A) and mRNA expression ( Figure 10B) There was no significant increase, proving that the CuMOF@AA@Lidocaine microneedle solution can reduce the stress changes in cells after thermal injury in vitro.

[0050] In another group of cell experiments, we stained the cells in each group after heat treatment with Propidium Iodide (PI). PI cannot pass through the live cell membrane but can penetrate the damaged cell membrane to stain the cell nucleus (red fluorescence), so it is used to detect cell apoptosis. As Figure 11 shown, compared with Ctrl-neg, the number of dead cells in the Ctrl-pos, γ-PGA, and Lidocaine groups increased significantly after thermal injury, while the number of dead cells in the CuMOF@AA@Lido group showed no obvious change. This further proves that CuMOF@AA-Lidocaine can effectively protect cells in vitro and reduce cell apoptosis caused by thermal injury.

[0051] Example 4 In vivo experiments on the analgesic effect and anti-thermal injury effect of CuMOF@AA-Lidocaine microneedles

[0052] As Figure 12 shown in A, in the back of male New Zealand white rabbits aged 3 - 6 months, the hair was thoroughly removed with a razor and depilatory cream. In the control group (Ctrl group), compound lidocaine cream (containing 25 mg of lidocaine per g) was applied, and in the experimental group (MN group), the thumb was perpendicular to the skin and the CuMOF@AA-Lidocaine microneedle was continuously pressed for 1 minute to make the needle tip penetrate into the subcutaneous tissue and dissolve completely, repeating 3 times. After wiping the surface drug with a 75% alcohol cotton ball, it could be seen that the skin after microneedle puncture in the experimental group was grayish black, indicating that the needle tip components could effectively penetrate into the subcutaneous tissue.

[0053] Then, referring to the literature, the laser temperature range for skin treatment is 60 - 100 °C (Adv Wound Care (New Rochelle), 2021, 11(4): 163–178), and similar to conventional scalds, it can cause pathological changes such as epidermal damage, skin inflammatory reactions, and collagen production (Cell Biochem Biophys, 2013, 67(3): 1005–1014). Therefore, a 100 °C pointed soldering iron was used to simulate the laser to create 9 thermal injury points in a 3×3 matrix within the drug treatment range, and the above experiment was repeated 4 groups.

[0054] The retraction frequency of the rabbits during the spot scalding process ( = total number of retractions / total number of times) was used to reflect the pain degree of the skin under different treatment methods. As Figure 12As shown in Figure B, the average retraction frequency of the experimental group was 0.1, and that of the control group was 0.36, indicating that the CuMOF@AA-Lidocaine experimental group had a better analgesic effect.

[0055] Three days later, as Figure 12 shown in Figure A, the skin lesion area of the experimental group (MN group) showed a light pink color, with clear edges, slightly rough surface but overall flat. The skin lesion area of the control group (Ctrl group) was darker in color, showing a brownish color, with an obviously rough surface, irregular edges, and obvious blood scabs formed, indicating that the experimental group had a better repair effect after skin thermal injury. Take the skin at the modeling site for HE staining, and the results are as Figure 12 shown in Figure C. At 3 days, compared with the control group, the experimental group had a smaller epidermal injury depth and a lighter degree of inflammatory cell infiltration (single arrow). At 7 days, the experimental group still did not shed the scab (black five-pointed star), and the epidermal thickening was more obvious in the experimental group (double arrow). It shows that although the experimental group using the CuMOF@AA-Lidocaine microneedles of this embodiment was thermally damaged, the damage shown by its epidermis was more minor, the skin inflammatory reaction was smaller, and the epithelialization was more mature.

Claims

1. A composite material, characterized in that The MOF structure includes copper ions and Centella asiatica acid, and the weight ratio of copper ions to Centella asiatica acid is 1:0.9~1.

2. The composite material according to claim 1, characterized in that It is self-assembled by heating copper ions and Centella asiatica acid under alkaline conditions.

3. The composite material according to claim 1, characterized in that It is a flake-like particle.

4. The composite material according to claim 3, characterized in that The average particle size is 236.8 nm.

5. The composite material according to claim 1, characterized in that Copper is crystalline bonded with oxygen.

6. The composite material according to claim 1, characterized in that Also includes lidocaine.

7. The composite material according to claim 1, characterized in that Also included are biodegradable polymers for making the microneedles.

8. Use of the composite material according to any one of claims 1 to 7 in the preparation of medical devices for alleviating laser-induced thermal damage and pain.

9. A microneedle, characterized in that The invention comprises the combined material according to any one of claims 1 to 7.

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