Application of metformin in the preparation of anti-photoaging drugs or medical devices for skin.

Abstract

The application of metformin in the preparation of anti-photoaging drugs or medical devices for the skin. Metformin has been demonstrated to promote autophagosome formation and restore autophagic flux patency, reshaping the autophagy-ECM metabolic regulatory network. When applied to the skin (e.g., the dermis), it exerts an anti-photoaging effect by regulating the AMPK signaling pathway. Using metformin as the active ingredient in drugs or medical devices facilitates clinical applications for anti-photoaging of the skin.

Description

Technical Field

[0001] This invention relates to a novel use of a known compound, and more particularly to the application of metformin in skin photoprotection, using it as an active ingredient in the preparation of pharmaceuticals or medical devices. Background Technology

[0002] As the largest organ in the human body, the skin is the first to be affected by various internal and external environmental factors, making it the earliest and most obvious sign of aging. Skin aging and other skin diseases account for approximately 1.79% of the global disease burden, making it the fourth leading cause of disability worldwide. Therefore, the prevention and treatment of skin aging has become an urgent clinical problem. Among various factors, ultraviolet radiation (UVR), as an environmental oxidant and mutagen, has a particularly profound impact. It can accelerate skin aging through oxidative stress, DNA damage, and activation of matrix metalloproteinases (MMPs). This process is known as photoaging, and its clinical characteristics include deep wrinkles, dryness, roughness, uneven pigmentation, reduced repair capacity, and an increased risk of malignant transformation. More importantly, photoaging accounts for approximately 80% of facial skin aging, significantly affecting the physical and mental health of patients.

[0003] In the process of skin photoaging, ultraviolet radiation (UVR) induces premature aging of fibroblasts, keratinocytes, and melanocytes through multiple mechanisms, including DNA damage, oxidative stress, and mitochondrial dysfunction. These senescent cells express p16... INK4a P21 Waf-1 Autophagy, as a key stress response system, maintains cellular homeostasis by clearing damaged proteins, lipids, and mitochondria. It secretes age-associated secretory phenotypes (SASPs) containing IL-6, MMPs, and extracellular vesicles, creating a pro-inflammatory microenvironment that accelerates epidermal atrophy and barrier function impairment. However, autophagy and the aging process are in a dynamic interplay: autophagy receptors inhibit SASP activation by degrading the transcription factor GATA4, while autophagy inhibition leads to GATA4 accumulation and triggers inflammatory signaling pathways. Simultaneously, mitophagy precisely clears photodamaged mitochondria, while autophagy dysfunction exacerbates oxidative damage and premature aging phenotypes. For example, autophagy defects in fibroblasts from Cockayne syndrome patients can be reversed through functional recovery. Studies have shown that autophagy flux imbalance in photoaged skin not only weakens the cell's ability to clear toxic substances but also amplifies the aging cascade through mechanisms such as escaping GATA4 degradation and mitochondrial quality control failure.

[0004] Photoaging is an important manifestation of skin aging. Therefore, the dynamic regulation of autophagy has become a key strategy to improve skin photoaging and delay the skin aging process by rebuilding the autophagy-senescence balance network. Summary of the Invention

[0005] One object of the present invention is to provide the application of metformin in the preparation of skin anti-photoaging drugs.

[0006] Another objective of this invention is to provide an application of metformin in the preparation of medical devices for skin anti-photoaging.

[0007] Another object of the present invention is to provide a medicine containing metformin for the treatment of skin photoaging.

[0008] Another object of the present invention is to provide a material composition containing metformin as an active ingredient for skin anti-photoaging.

[0009] Another object of the present invention is to provide a medical device containing metformin for the treatment of skin photoaging.

[0010] Metformin is a biguanide hypoglycemic agent containing two linked guanidine rings and is widely used to treat diabetes. This invention has verified that metformin promotes the formation of autophagosomes and restores the patency of autophagic flux, reshaping the autophagy-extracellular matrix (ECM) metabolism regulatory network. When applied to the skin (e.g., the dermis), it exerts an anti-photoaging effect by regulating the adenosine monophosphate-activated protein kinase signaling pathway (AMPK signaling pathway), for example, at a dose of 6–8 mg / kg by weight. Metformin can be used as an active ingredient in pharmaceutical or medical device formulations, facilitating its clinical application in anti-photoaging of the skin.

[0011] Metal-organic frameworks (MOFs) are porous materials with a periodic network structure formed by the coordination between organic ligands and metal ions. Due to their structural diversity, high porosity, and ability to transport important metal ions, they have been widely used in regenerative medicine.

[0012] A material composition comprising metformin and MOFs, wherein metformin is loaded within the MOFs to provide sustained-release properties of metformin and effectively address the short in vivo half-life of metformin.

[0013] Zeolitic imidazolate framework-8 (ZIF-8), an important member of MOFs, possesses a single-crystal structure synthesized with zinc ions and can continuously release Zn. 2+It possesses positive biological effects such as reducing reactive oxygen species levels, promoting angiogenesis, and antibacterial activity. Compared with other bioactive molecules, the synthesis of nanoscale ZIF-8 has advantages such as good biosafety, ultra-high porosity, modifiable size, thermochemical stability, and ease of synthesis.

[0014] Another material composition contains metformin and ZIF-8, denoted as Met@ZIF-8, with metformin loaded within ZIF-8 to form nanoparticles with a particle size of 30 nm to 300 nm.

[0015] The Met@ZIF-8 of the present invention is prepared by the following method:

[0016] Zinc nitrate hexahydrate (0.5 mmol) was used as the metal core, and metformin hydrochloride powder (e.g., 25 mg, 50 mg, 100 mg and 150 mg) was added to methanol (e.g., 5 mL). The mixed solution was sonicated (e.g., 5 min) to make the solute evenly dispersed.

[0017] Next, stir the mixture at room temperature until it becomes clear (approximately 1 hour), and label it as: Solution A;

[0018] Then, add 4 mmol of 2-methylimidazole to methanol (e.g., 10 mL) and mix well at room temperature (for about 1 hour), and record this as solution B.

[0019] Mix solution A with solution B to form a milky white suspension, and stir at room temperature (e.g., for 24 hours) to allow the reaction to proceed fully.

[0020] Then, the synthesized nanoparticles are precipitated by high-speed centrifugation (e.g., centrifugation at 12000 rpm for 30 min), and the precipitate is resuspended (e.g., in methanol solution). High-speed centrifugation is then continued (e.g., centrifugation at 12000 rpm for 30 min) to clean the impurities on the precipitate surface. The supernatant is then aspirated and stored (or repeated multiple times).

[0021] Finally, collect the white precipitate, dry it, and crush it to obtain the final product.

[0022] This invention has verified that Met@ZIF-8, when applied to the skin (e.g., the dermis), has an anti-photoaging effect. For example, treatment of cells with a concentration of 20 μg / mL of Met@ZIF-8 significantly improves the proliferation, migration, and ECM synthesis metabolism of HDFs, while reducing cellular senescence phenotypes. Transcriptome analysis, Western blotting, TEM, and dual-fluorescence autophagy flux detection further reveal that Met@ZIF-8 can significantly promote autophagosome formation and restore autophagy flux patency, reshaping the autophagy-ECM metabolism regulatory network.

[0023] Microneedles (MNs), as a minimally invasive and efficient drug delivery system, have the advantages of controlled drug release and high drug loading capacity, and have been widely used in tissue regeneration, wound healing and skin rejuvenation. Their materials include metals, silicon or biocompatible and / or biodegradable materials.

[0024] Biocompatible and / or biodegradable materials, such as, but not limited to, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid copolymer, polycaprolactone, hydroxyapatite, hyaluronic acid, gelatin, chondroitin sulfate, collagen, chitosan, cellulose, extracellular matrix, and one or more of their cross-linked derivatives. Cross-linked derivatives include, for example, methacrylamide chitosan, methacrylamide gelatin, methacrylamide hyaluronic acid, and methacrylamide chondrocyte extracellular matrix.

[0025] This invention incorporates metformin into a material to create microneedles (patches) that facilitate transdermal delivery of metformin. As a medical device, it can be used to combat photoaging and promote skin repair, such as wrinkle reduction or restoration of dermal thickness.

[0026] Another medical device includes Met@ZIF-8 and hyaluronic acid, with hyaluronic acid serving as the substrate for manufacturing microneedles. Met@ZIF-8 is distributed within the hyaluronic acid microneedles to form a microneedle patch.

[0027] The microneedle-based transdermal delivery system successfully achieved targeted accumulation of Met@ZIF-8 in the dermis of photoaged mice. After 28 days of intervention, the Met@ZIF-8 microneedle group showed more significant anti-wrinkle effects and dermal thickness recovery compared to retinoic acid cream and the blank microneedle group. Histological analysis showed that its mechanism of action was closely related to autophagy activation, enhanced ECM synthesis, and inhibition of MMPs. Furthermore, the system exhibited good biocompatibility, without inducing local inflammation or systemic toxicity.

[0028] The technical solution provided by this invention, based on comprehensive macroscopic observation, histological staining, and biosafety assessment, demonstrates that the Met@ZIF-8 functionalized microneedles exhibit good biosafety. It alleviates excessive ECM degradation by rescuing autophagy dysfunction in photoaged dermal fibroblasts, promoting new collagen synthesis, and reducing the local inflammatory microenvironment. This not only effectively restores the dermal volume loss and appendage degenerative changes caused by UVB, but also significantly reduces skin wrinkles macroscopically. Attached Figure Description

[0029] Figure 1 The images show the microstructures of ZIF-8 and various types of Met@ZIF-8; where A is a transmission electron microscope (TEM) field of view and B is a scanning electron microscope (SEM) field of view.

[0030] Figure 2 XRD patterns of various substances;

[0031] Figure 3 Fourier transform infrared spectra of various substances;

[0032] Figure 4 TEM-EDS elemental distribution diagrams for ZIF-8 and various Met@ZIF-8 samples;

[0033] Figure 5 The graphs show the yield, encapsulation efficiency, and drug loading of MOF products; where A is a line graph of the yield of ZIF-8 and various Met@ZIF-8, and B is a graph of the encapsulation efficiency and drug loading of various Met@ZIF-8.

[0034] Figure 6 This is the in vitro release curve; where A is Zn. 2+ Release curves, B is the metformin release curve;

[0035] Figure 7 Figure showing the effect of different concentrations of Met@ZIF-8 on the proliferation capacity of HDFs;

[0036] Figure 8 The results show the effects of different concentrations of Met@ZIF-8 on the migration ability of HDFs; where A is the result of the scratch assay, B is the result of the Transwell cell migration assay, C is the quantitative statistical graph of cell migration rate in the scratch assay, and D is the quantitative statistical graph of the number of migrating cells in the Transwell assay.

[0037] Figure 9 The effects of different concentrations of Met@ZIF-8 on the senescence phenotype of HDFs are shown in Figure A, Figure B shows the results of SA-β-gal staining, Figure C shows the results of intracellular ROS expression levels, Figure D shows the quantitative statistics of the number of SA-β-gal positive cells, and Figure D shows the quantitative statistics of the number of ROS positive cells.

[0038] Figure 10 Statistical graph showing the effects of each experimental group on the proliferation capacity of HDFs;

[0039] Figure 11 The figures show the experimental results of the effects of each experimental group on the migration ability of HDFs; where A is the result of the scratch assay, B is the result of the Transwell migration assay, C is the quantitative statistical graph of cell migration rate in the scratch assay, and D is the quantitative statistical graph of the number of migrating cells in the Transwell assay.

[0040] Figure 12Figure 1 shows the experimental results of each experimental group on the effect of HDFs on the aging phenotype; where A is the result of SA-β-gal staining, B is the result of intracellular ROS expression level, C is the quantitative statistical graph of the number of SA-β-gal positive cells, and D is the quantitative statistical graph of the number of ROS positive cells.

[0041] Figure 13 Heatmaps of ECM metabolism-related gene expression in each experimental group;

[0042] Figure 14 The diagram shows the transcriptomic analysis of HDFs cells before and after Met@ZIF-8 treatment; where A is the PCA diagram and B is the volcano diagram.

[0043] Figure 15 The diagram shows the results of the enrichment experiment for differentially expressed genes; where A is the GO enrichment result diagram, B is the network diagram of the GO enrichment pathway, C is the KEGG enrichment result diagram, and D is the chord diagram of the KEGG enrichment pathway.

[0044] Figure 16 The graphs show the expression results of autophagy-related genes in HDFs before and after Met@ZIF-8 treatment; where A is the GSEA enrichment result of autophagy, B is the heatmap of differential expression of autophagy-related genes, and C is the heatmap of differential expression of genes related to the AMPK signaling pathway.

[0045] Figure 17 The image shows the results of Met@ZIF-8 on the expression of autophagy-related proteins in HDFs; where A is the electrophoresis image of autophagy-related proteins and B is the electrophoresis image of proteins related to the AMPK signaling pathway.

[0046] Figure 18 Representative transmission electron microscopy images of the effects of Met@ZIF-8 on mitochondrial morphology and autophagy in HDFs; yellow arrows indicate mitochondria, white arrows indicate autolysosomes, and red arrows indicate autophagosomes.

[0047] Figure 19 The figure shows the experimental results of detecting autophagic flux based on dual-fluorescently labeled autophagic lentivirus;

[0048] Figure 20 The images show the surface morphology of functionalized HA microneedles loaded with Met@ZIF-8; where A is a bright-field image and B is a SEM image.

[0049] Figure 21 The image shows the loading verification of Met@ZIF-8 component in functionalized microneedles; where A is the Fourier transform infrared spectrum and B is the fluorescence image of Met@ZIF-8 distribution in the microneedles (Met@ZIF-8 is marked in red);

[0050] Figure 22Figures show the mechanical and puncture performance results of functionalized microneedles; where A is an H&E staining image of microneedles inserted into mouse dorsal skin tissue, B is a bright-field photograph of a micropore array formed after trypan blue-loaded microneedles are inserted into pigskin, and C is a mechanical-displacement curve.

[0051] Figure 23 Photographs of the needle tip morphology of the functionalized microneedles at various sampling time points after insertion into pigskin;

[0052] Figure 24 Photographs of the skin appearance of each group of test animals on days 0, 4, 7, 14, 21 and 28;

[0053] Figure 25 The images show the histological staining and quantitative results of the skin of the test animals in each group; where A is the Masson trichrome staining image (the area circled in yellow is the dermis), B is the H&E staining image, and C is the quantitative statistical chart of dermal thickness.

[0054] Figure 26 The images show the histological staining results related to collagen synthesis; where A is the immunohistochemical staining image of type I collagen, B is the immunohistochemical staining image of type III collagen, C is the Sirius red staining image, D is the quantitative statistical chart of type I collagen staining intensity, E is the quantitative statistical chart of type III collagen staining intensity, and F is the quantitative statistical chart of the ratio of type III to type I collagen.

[0055] Figure 27 Images and statistical graphs of LC3B immunohistochemical staining in the skin of each group of test animals;

[0056] Figure 28 The images show the expression results of MMPs in the skin of the test animals in each group; where A is the immunofluorescence staining image of MMP-1, B is the immunofluorescence staining image of MMP-9, C is the quantitative statistical graph of MMP-1 staining intensity, and D is the quantitative statistical graph of MMP-9 staining intensity.

[0057] Figure 29 A quantitative statistical graph showing the hair follicle count in the skin tissue of each group of test animals;

[0058] Figure 30 The chart shows the statistical results of the main serum biochemical indicators (pink dashed line area: normal reference range); among them, A is the statistical chart of alanine aminotransferase (ALT), B is the statistical chart of alkaline phosphatase (ALKP), C is the statistical chart of total protein (TP), D is the statistical chart of total bilirubin (TB), E is the statistical chart of creatinine (CR), and F is the statistical chart of blood urea nitrogen (BUN).

[0059] Figure 31 Representative H&E staining images of the major internal organs of mice in each experimental group. Detailed Implementation

[0060] The technical solution 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 solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0061] The specific experimental methods used in the following embodiments of the present invention are described below:

[0062] 1) Preparation of Met@ZIF-8

[0063] The Met@ZIF-8 nanomedicine delivery system was synthesized using a one-pot method. The specific experimental steps are as follows:

[0064] (1) Weigh 148.7 mg of zinc nitrate hexahydrate (0.5 mmol) as the metal core, and weigh a gradient of metformin hydrochloride (Met) powder (0, 25, 50, 100 and 150 mg). Add the powder to 5 mL of methanol and sonicate the mixture for 5 min to ensure uniform dispersion of the solute.

[0065] (2) Stir the mixture at room temperature for 1 hour until a clear solution (solution A) is formed;

[0066] (3) Weigh 328.5 mg of 2-methylimidazole (4 mmol) and add it to 10 mL of methanol. Stir at room temperature for 1 h (solution B);

[0067] (4) Quickly pour solution A into solution B to form a milky white suspension. Stir at room temperature for 24 hours to allow the reaction to proceed fully.

[0068] (5) After the reaction is complete, the mixed solution is transferred to a centrifuge tube and centrifuged at 12,000 rpm for 30 min in an ultra-low temperature centrifuge to allow the synthesized nanoparticles to precipitate to the bottom of the centrifuge tube. The supernatant is then aspirated and saved, and the precipitate is collected.

[0069] (6) Resuspend the precipitate with methanol solution, centrifuge the mixture in an ultra-low temperature centrifuge at 12000 rpm for 30 min to clean the impurities on the surface of the precipitate, aspirate and save the supernatant, and repeat the process 3 times.

[0070] (7) Collect the white precipitate and put it into a vacuum drying oven. Dry it at 60°C for 24 hours. Crush the dried product with an agate mortar and store it in a vacuum dryer.

[0071] 2) Characterization of Met@ZIF-8

[0072] The product obtained is named according to the amount of metformin hydrochloride in the synthesis system. For example, if the amount of Met in the system is 25 mg, the crystal is named Met 25@ZIF-8, and so on.

[0073] To clarify the correctness of the synthesized nanoparticle structure, determine the successful drug loading into the nanoparticles, and screen suitable synthesis systems, a series of characterizations were performed on ZIF-8 and Met@ZIF-8 obtained under different synthesis systems. The crystal morphology and size characteristics of the synthesized particles were observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM); the structure and composition of the particles were determined using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR); the drug loading was confirmed using TEM-based energy-dispersive X-ray spectroscopy (EDS); and the drug and ion content in the solution was detected using high-performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS) to help calculate the encapsulation efficiency (EE), loading efficiency (LE), yield, and active ingredient release rate of different systems.

[0074] 3) Screening of the optimal biological effect concentration of Met@ZIF-8 in vitro

[0075] Human dermal fibroblasts (HDFs) were used to verify the in vitro biosafety and biological effects of Met@ZIF-8. The optimal in vitro concentration of Met@ZIF-8 was determined by cell proliferation, cell migration, senescence-related galactosidase activity staining (SA-β-gal staining), and reactive oxygen species detection experiments.

[0076] 4) Investigation on the in vitro anti-photoaging effect of Met@ZIF-8

[0077] After determining the optimal in vitro concentration of Met@ZIF-8, HDFs were used to evaluate the biological effects of the Met@ZIF-8 system. Cell proliferation, cell migration, SA-β-gal staining, and reactive oxygen species detection experiments were conducted to compare the in vitro biological effects of empty nanoparticles (ZIF-8), pure metformin hydrochloride (Met), and drug-loaded nanoparticles (Met@ZIF-8) under the same system.

[0078] To further explore the biological mechanism of this anti-photoaging effect, HDFs cells were subjected to transcriptome sequencing, analysis, and corresponding verification after treatment with photoaged cells Met@ZIF-8.

[0079] 5) Preparation and characterization of Met@ZIF-8 functionalized microneedles

[0080] Hyaluronic acid (HA) was selected as the material for microneedles, and Met@ZIF-8 was added to soluble microneedles to construct a soluble microneedle system. The surface morphology, main components, transdermal properties, mechanical properties, and solubility of the soluble microneedles were characterized.

[0081] 6) Evaluation of the in vivo photoaging and skin rejuvenation effects of Met@ZIF-8 functionalized microneedles

[0082] Microneedling intervention was performed on a mouse model of photoaged skin. After treatment, skin tissue samples were taken, and the effects of Met@ZIF-8 functionalized microneedles on promoting photoaged skin rejuvenation were comprehensively evaluated through histological staining (including H&E, Masson's trichrome, Sirius red, immunohistochemistry, and immunofluorescence staining) and in vivo biosafety assessment.

[0083] Mice were randomly divided into 5 groups of 5 mice each for intervention. Before each intervention, the macroscopic morphology of the skin on the back of the mice was recorded using a camera. The groupings are as follows:

[0084] (1) Control group: The skin on the back of the mice was dehaired only during the modeling period and was not exposed to ultraviolet radiation;

[0085] (2) Negative control group (UVB group): The skin on the back of the mice was routinely dehaired and exposed to ultraviolet light, but no treatment was given;

[0086] (3) Positive control group (RA group): On days 0, 4, 7, 14 and 21 after modeling, 0.05% retinoic acid (RA) cream was applied to the skin on the back of the mice.

[0087] (4) Blank microneedle group (HA group): On days 0, 4, 7, 14 and 21 after modeling, two prepared blank microneedles were vertically pressed into the skin tissue on the back of the mice, and the microneedle base was removed after 10 minutes.

[0088] (5) Met@ZIF-8 functionalized microneedle group (HA / Met@ZIF-8 group): On days 0, 4, 7, 14 and 21 after modeling, two prepared functionalized microneedles were vertically pressed into the skin tissue on the back of mice, and the microneedle base was removed after 10 minutes.

[0089] 7) Statistical Analysis

[0090] Data analysis was performed using GraphPad Prism 8 statistical software. Quantitative results are expressed as mean ± standard deviation (SD). T-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons of three or more groups. The statistical significance threshold was set at 0.05. Significance levels are expressed as follows: ns represents no statistical difference compared to the control group. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; compared with the UVB group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001.

[0091] Example 1: Characterization of Met@ZIF-8

[0092] ZIF-8 prepared by the one-pot method and Met@ZIF-8 with different dosing systems all maintained the regular characteristic dodecahedral morphology. TEM Figure 1 A) and SEM ( Figure 1 Figure B) shows that the blank ZIF-8 particles have a uniform size distribution, with diameters ranging from 30 to 50 nm. With increasing dosage, the size of the Met@ZIF-8 particles gradually increases, indicating that metformin hydrochloride molecules may influence Zn through coordination. 2+ Nucleation kinetics with 2-methylimidazole.

[0093] XRD patterns show ( Figure 2 All samples maintained the characteristic diffraction peaks of ZIF-8, and the peaks were sharp and the baselines were stable, indicating that the drug loading process did not disrupt the crystal structure of the main framework. Even in samples with high drug loading, no crystallization peaks of free metformin hydrochloride (2θ = 17.5° and 25.1°) were detected, proving that the drug was dispersed in an amorphous state or co-loaded within the framework through chemical action.

[0094] FTIR analysis showed that pure ZIF-8 contains Zn-N coordination bonds (420-450 cm⁻¹). -1 ) and imidazole ring vibration (1574.2cm) -1 1455.7cm -1 and 1141.8cm -1 Characteristic peaks such as ) were observed. After the introduction of metformin hydrochloride, the Zn-N vibrational peak shifted to lower wavenumbers, and the peak at 1574.2 cm⁻¹... -1 The decrease in C=N peak intensity accompanied by an increase in peak width suggests that the drug molecule is co-loaded within the ZIF-8 framework through hydrogen bonding with the N atom of the imidazole ring. Figure 3 ).

[0095] TEM-EDS analysis showed that ( Figure 4 The uniform distribution of C, N, and Zn elements in the Met@ZIF-8 particles indicates the integrity of the ZIF-8 framework. Furthermore, the gradient distribution of Cl within the crystal, partially overlapping with the distribution trajectory of Zn, suggests a possible interaction between Cl and Zn, indicating that metformin hydrochloride was successfully loaded onto the ZIF-8 support.

[0096] like Figure 5 As shown in Figure A, under fixed ZIF-8 synthesis conditions, the yield of Met@ZIF-8 gradually decreased with increasing metformin hydrochloride dosage (below 65% when dosage exceeded 100 mg). Despite the decreased yield, the drug loading and encapsulation efficiency still increased with increasing dosage. Figure 5 B) indicates that excessive drug promotes preferential adsorption of the drug by the carrier.

[0097] Met 50@ZIF-8, which exhibits the best overall performance, was selected for subsequent experiments. ICP-MS and HPLC techniques were used to analyze its ion and drug release behavior under simulated physiological conditions (pH = 7.4, 37℃). The results showed that Zn in ZIF-8 and Met50@ZIF-8... 2+ It releases rapidly within the initial 24 hours, followed by a slow release trend, and the Zn content of Met 50@ZIF-8... 2+ The cumulative release was significantly lower than that of pure ZIF-8, possibly due to the enhanced stability of the carrier structure caused by the drug loading. Figure 6 A). Meanwhile, metformin hydrochloride in Met 50@ZIF-8 also showed a stable sustained-release trend after 24 hours, further confirming that the drug was successfully loaded into the ZIF-8 nanodelivery system. Figure 6 B).

[0098] The above results demonstrate that this embodiment successfully constructed a metformin hydrochloride-loaded nanodrug delivery system (Met@ZIF-8). This system not only possesses uniform nanoscale size but also ensures high yield, drug loading rate, and encapsulation efficiency. Furthermore, Met@ZIF-8 enables the slow release of the active ingredient, effectively improving the problems of poor stability and short half-life of metformin hydrochloride, providing a potential solution for its clinical application.

[0099] Example 2: Validation of the in vitro biological effects of Met@ZIF-8

[0100] After the photoaging cell model was constructed, HDFs were treated with different concentrations of Met@ZIF-8 in this embodiment. By analyzing cell proliferation, migration and aging-related markers, the optimal in vitro concentration was screened.

[0101] like Figure 7 As shown, 10-30 μg / mL of Met@ZIF-8 can significantly improve the proliferation capacity of photoaged cells, with 20 μg / mL showing the most significant effect.

[0102] The migratory ability of fibroblasts is crucial for tissue regeneration and rejuvenation. Scratch assay ( Figure 8 A and Figure 8 C) and Transwell cell migration assays ( Figure 8 B and Figure 8 D) shows that UVB irradiation significantly reduced the migration ability of HDFs, but Met@ZIF-8 could improve this situation in the range of 10-30 μg / mL, with the 20 μg / mL group showing the most significant improvement.

[0103] UVB induces DNA damage and mitochondrial dysfunction, leading to cellular senescence (increased SA-β-gal activity) and ROS accumulation. SA-β-gal staining results show ( Figure 9 A and Figure 9 C) Low concentrations of Met@ZIF-8 significantly improved the aging phenotype of photo-aged HDFs. Furthermore, the ROS detection results were consistent with other in vitro experiments, further validating the antioxidant effect of Met@ZIF-8. Figure 9 B and Figure 9 D).

[0104] Example 3: In vitro anti-photoaging effect of Met@ZIF-8

[0105] Normal and photoaged HDFs were treated with 20 μg / mL Met@ZIF-8 (the validated optimal concentration), free metformin hydrochloride (Met), and blank ZIF-8 at the same carrier dose, and an untreated group was set up as a control.

[0106] The results of the CCK-8 cell proliferation experiment showed that in photoaged HDFs, the cell viability of the Met@ZIF-8 group and the Met group was significantly higher than that of the ZIF-8 group, while the cell viability of the ZIF-8 group was significantly higher than that of the untreated group on day 5. The Met group showed a rapid effect on day 3 (cell viability was higher than that of the Met@ZIF-8 group), but there was no significant difference between the two by day 5, which may be related to the sustained-release properties of Met@ZIF-8. Figure 10 In subsequent cell migration experiments, Met@ZIF-8 and Met showed similar improvement effects on photoaged HDFs. Figure 11 ).

[0107] Both Met@ZIF-8 and Met significantly reduced the levels of aging-related biomarkers and ROS in photoaged HDFs, with no statistically significant difference between the two groups. Figure 12 It is worth noting that, in Transwell cell migration assays and SA-β-gal staining assays, the blank vector ZIF-8 also showed some anti-photoaging effect. Figure 12 A, Figure 12 B Figure 12 C and Figure 12 D).

[0108] Metabolic balance in the ECM is crucial for maintaining the homeostasis of the skin microenvironment, and its dynamic balance is regulated by multiple factors. To investigate the effect of Met@ZIF-8 on ECM metabolism, this study also examined the expression of ECM metabolism-related genes in HDFs.

[0109] qRT-PCR results showed ( Figure 13 Met@ZIF-8 treatment significantly downregulated MMP-1 expression in photoaged HDFs and effectively reversed the degradation trend of ECM by reducing the MMP-1 / TIMP-1 ratio to normal levels. Furthermore, the expression of collagen synthesis-related genes COL1A1, COL3A1, and TGFβ was significantly upregulated, suggesting that Met@ZIF-8 can inhibit ECM degradation while promoting collagen regeneration, thereby reversing photoaging damage.

[0110] By detecting cell proliferation, migration, aging markers, and ROS levels, and analyzing ECM remodeling-related genes, we found that Met@ZIF-8 and an equal dose of free metformin hydrochloride both have proliferative, anti-aging, and pro-migration effects on photoaged HDFs. Since the sustained-release properties of Met@ZIF-8 may improve drug stability and cellular uptake, these results suggest that Met@ZIF-8 delays photoaging by regulating fibroblast function.

[0111] Example 4: Verification of the cellular mechanism of photoaging after Met@ZIF-8 treatment

[0112] Transcriptome sequencing and analysis were performed on cells before and after treatment. PCA results showed a significant separation in gene expression profiles between the Met@ZIF-8 treated group and the untreated group. Using |log2(Fold Change)|>1 and P.adj<0.05 as thresholds, a total of 130 DEGs were screened, of which 80 were upregulated and 50 were downregulated. Figure 14 ).

[0113] Functional enrichment analysis showed that DEGs upregulated after Met@ZIF-8 treatment were significantly enriched in the ECM, cell-ECM interactions, and autophagy-related GO entries. Figure 15 A). The GO entry interaction network diagram shows a high correlation between ECM remodeling and autophagy entries (such as "Collagen metabolic process", "Extracellular matrix organization", "Macroautophagy", and "Autolysosome"). Figure 15 B). KEGG pathway analysis further confirmed that DEGs were significantly enriched in the "Mitophagy", "Autophagy", and "AMPK signaling pathway" (B). Figure 15 C). Chord diagrams visually demonstrate that "autophagy"-related genes (such as IRS2 and PIK3R2) have a direct regulatory relationship with "Cellular senescence" and the "AMPK signaling pathway." Figure 15 D).

[0114] GSEA analysis showed that the "Autophagy" pathway was significantly enriched after Met@ZIF-8 treatment (NES = 1.751, P.adj = 0.043). Figure 16 A). The differential gene heatmap further revealed significant clustering differences in the expression patterns of the AMPK signaling pathway and autophagy-related genes between the treatment and control groups. Figure 16 B). Based on this, it is believed that Met@ZIF-8 activates the AMPK signaling pathway through drug sustained release, thereby improving UVB-induced autophagy abnormalities.

[0115] Western blot (WB) was used to detect the expression of proteins related to autophagy and the AMPK signaling pathway. The results showed that the protein levels of Beclin1, LC3B, and p62 in HDFs increased after UVB irradiation, suggesting that UVB irradiation may inhibit autophagosome-lysosome fusion or impair lysosomal function, thereby suppressing autophagy flux. After Met@ZIF-8 treatment, Beclin1 expression was further upregulated, indicating that autophagy initiation was promoted. Combined with the upregulation of the LC3BII / LC3B I ratio and the downregulation of p62 expression, this indicates that autophagosome degradation was restored and autophagy flux was significantly enhanced. Figure 17 A). Further analysis revealed that Met@ZIF-8 treatment upregulated the expression of p-AMPK and p-ULK1. Figure 17 B). The above results suggest that Met@ZIF-8 enhances autophagy flux by promoting autophagosome formation and degradation, an effect that may be related to the activation of the AMPK-ULK1 signaling pathway.

[0116] TEM observations showed that in HDFs irradiated with UVB, mitochondria exhibited significant swelling, increased vacuolation, blurred inner membrane cristae, and reduced fusion of autophagosomes and lysosomes. However, after treatment with Met@ZIF-8, the morphology of mitochondria was significantly improved, and the number of autophagosomes and lysosomes was significantly increased under the microscope. Figure 18 These results indicate that Met@ZIF-8 treatment can significantly promote the fusion of autophagosomes and lysosomes, thereby accelerating the degradation of damaged organelles and protein aggregates.

[0117] A dual-fluorescent lentivirus system was also employed to monitor autophagy flux in real time by observing the fluorescence changes of acid-sensitive green fluorescent protein (GFP) and acid-stable red fluorescent protein (mRFP), thus overcoming the limitations of traditional static methods (such as WB and TEM) in comprehensively reflecting changes in autophagy flux.

[0118] like Figure 19 As shown, in the Control group, the fluorescently labeled LC3 protein was uniformly diffused in the cytoplasm. In the UVB group, the LC3 protein aggregated, forming punctate fluorescent colocalizations with mRFP and GFP (appearing as yellow fluorescent spots), indicating that autophagosomes had not yet fused with lysosomes. In contrast, in cells treated with Met@ZIF-8, the number of GFP fluorescent spots was significantly reduced compared to mRFP, indicating enhanced fusion of autophagosomes with lysosomes, leading to GFP fluorescence quenching.

[0119] The above results indicate that the Met@ZIF-8 system improves photoaging phenotype through an AMPK-dependent autophagy regulation mechanism.

[0120] Example 5: Characterization of HA microneedles loaded with Met@ZIF-8

[0121] The prepared microneedles consist of a pyramidal array of needle tips with a high aspect ratio. These needle tips are neatly arranged, structurally intact, and no obvious defects were observed. Figure 20 A and Figure 20 B).

[0122] In FTIR spectroscopy, the Met@ZIF-8 functionalized microneedles were observed at 691.8 cm⁻¹. -1 and 758.6cm -1 The characteristic peak of ZIF-8 is shown at the (imidazolium ring vibration) location. Figure 21 A). Confocal microscopy observations showed that PKH26-labeled Met@ZIF-8 was uniformly distributed in the microneedles, with consistent fluorescence signals throughout the microneedles and no obvious aggregation or missing areas, indicating that Met@ZIF-8 had been successfully loaded into the microneedles. Figure 21 B).

[0123] Mechanical performance test results show that a single needle can withstand a mechanical force exceeding 0.1N, reaching the skin penetration threshold of human skin. Figure 22 C). In the H&E staining image of mouse skin after microneedle penetration, the continuity of the stratum corneum is disrupted, indicating that the needle tip successfully penetrated the dermis. Figure 22 A). Furthermore, the microneedles loaded with trypan blue successfully left an array of blue pinholes on pigskin. Figure 22 B) further verified that the microneedles possess suitable mechanical properties.

[0124] Soluble microneedles should dissolve rapidly after penetration into the skin to ensure efficient delivery of the active ingredient. Light microscopic observations at different time points after insertion into pigskin showed that the microneedles completely dissolved within 10 minutes. Figure 23 This indicates that it has rapid dissolution properties and can effectively release the loaded active ingredients.

[0125] Example 6: Met@ZIF-8 Functionalized HA Microneedles for Photoaging Skin Rejuvenation

[0126] like Figure 24 As shown, the UVB group mice developed numerous deep wrinkles on their backs. In contrast, after 28 days of intervention, the wrinkles on the backs of mice in the RA, HA, and HA / Met@ZIF-8 groups were all improved, with varying degrees of reduction in the severity of wrinkles.

[0127] Masson trichrome staining of mouse skin ( Figure 25 A) and H&E staining ( Figure 25 B), and based on the staining results, a quantitative analysis of dermal thickness was performed ( Figure 25C). The results showed that the dermal volume of all three treatment groups was improved compared with the UVB group. The dermal volume of the HA / Met@ZIF-8 group increased the most significantly, even higher than that of the Control group, indicating that metformin has a very significant effect on skin photoaging and has a repair function.

[0128] The dermal ECM is mainly composed of type I and type III collagen. Therefore, this embodiment analyzes the type I collagen in each group of skin tissue (…). Figure 26 A and Figure 26 D) and type III collagen ( Figure 26 B and Figure 26 E) Immunohistochemical staining was performed, and the staining intensity was quantitatively analyzed. Results showed that the content of type I and type III collagen in the RA and HA groups was not significantly different from that in the UVB group. However, after Met@ZIF-8 functionalized microneedling intervention, the content of both types of collagen was significantly upregulated, with the increase in type III collagen being more significant, even significantly higher than in the Control group. This indicates that metformin has a highly significant effect on skin photoaging and possesses repair functions.

[0129] The changes in the type III / type I collagen ratio were further assessed using Sirius red staining. Figure 26 C and Figure 26 The results showed that the type III / type I collagen ratio was significantly increased in the UVB group, while the HA / Met@ZIF-8 group restored this ratio to the control group level. The data also indicated that the significant increase in type III collagen content in the HA / Met@ZIF-8 group did not lead to an abnormality in the type III / type I collagen ratio, suggesting that the synchronous increase in type I collagen maintained the homeostasis of collagen composition.

[0130] Immunohistochemical staining and quantitative analysis of LC3B were performed on the tissue. Figure 27 To verify whether it also has the same biological effect of regulating autophagy in vivo, tissue section staining results showed that the HA / Met@ZIF-8 group showed strong positive signals of LC3B in both the epidermis and dermis, indicating that it has an autophagy-regulating effect on cells throughout the entire skin layer. Figure 27 In contrast, although LC3B expression was increased in the skin tissues of the RA and HA groups, the difference was not statistically significant compared with the UVB group.

[0131] Overexpression of MMPs is one of the characteristic pathological changes in photoaged HDFs and a major cause of excessive ECM degradation in skin tissue. Therefore, this study performed immunofluorescence staining and quantitative analysis of MMPs in skin tissue to evaluate the regulatory effects of different interventions on dermal microenvironment homeostasis. Figure 28The results showed that the expression of MMP-1 and MMP-9 was significantly upregulated in photoaged skin. Met@ZIF-8 functionalized microneedles significantly reduced the expression of these MMPs, restoring them to normal levels.

[0132] In addition to cellular dysfunction and ECM degradation, photoaged skin tissue is also accompanied by abnormalities in the structure and function of appendages. Therefore, cytokeratin 14 immunofluorescence staining was used for visualization and quantitative analysis of hair follicles. Figure 29 The results showed a significant reduction in the number of hair follicles in photoaged skin, indicating impaired hair regeneration. However, the introduction of Met@ZIF-8 significantly increased the number of hair follicles, suggesting that Met@ZIF-8 can not only improve fibroblast function but may also rescue UV-induced skin dysfunction through regulating hair follicle stem cell activity or other mechanisms.

[0133] Serum biochemical analysis of mice ( Figure 30 ) and histological staining of major internal organs ( Figure 31 The biosafety of different in vivo intervention methods was evaluated. The results showed that all interventions had good biosafety: serum biochemical indicators were all within the normal reference range, and no obvious organic changes were observed in internal organs.

Claims

1. The application of a microneedle in the preparation of medical devices for anti-photoaging and wrinkle reduction or restoration of dermal thickness, characterized in that... The microneedles are made of hyaluronic acid, and metformin is distributed within the microneedles. The metformin is loaded onto ZIF-8 to form nanoparticles. After the microneedles are inserted into the skin, metformin is released into the dermis, allowing cells to be exposed to a concentration of 20 μg / mL of Met@ZIF-8.

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