A dual-module integrated threaded microneedle drug delivery system and a preparation method and application thereof

Abstract

The application provides a double-module integrated threaded microneedle drug delivery system and a preparation method and application thereof, and belongs to the technical field of drug delivery. The double-module integrated threaded microneedle drug delivery system and the preparation method and application thereof can realize layered drug delivery and programmable release from the epidermis to the subcutaneous layer. The surface hydrogel module (SRT1720@HA) releases a SIRT1 agonist immediately after insertion to improve dermal fibroblast aging; the deep LNP microsphere module (PGC-1alpha@LNP) releases PGC-1alpha mRNA through rotation after reaching the subcutaneous fat layer to restore adipocyte metabolic homeostasis. Spatial transcriptomics and clinical sample analysis reveal the hierarchical molecular expression profile in skin lesions. The application provides a spatially programmable hierarchical targeted treatment strategy, which provides a new idea for precise intervention of complex skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery technology, specifically relating to a dual-module integrated threaded microneedle drug delivery system, its preparation method, and its application. Background Technology

[0002] The occurrence and recurrence of skin diseases are closely related to abnormal remodeling of the epidermis, dermis, and subcutaneous layers, but current therapies lack effective layer-specific intervention strategies. At the physiological level, the epidermis is mainly composed of keratinocytes, which serve as a barrier and immune monitoring unit; the dermis is rich in fibroblasts and vascular networks, providing mechanical support and tissue repair; the subcutaneous layer is composed of fat and stromal cells, which are responsible for energy metabolism and regenerative reserves.

[0003] In recent years, single-cell omics and spatial transcriptomics have revealed a significant layer-specific pattern in skin pathological responses: the epidermal-dermal junction is characterized by inflammatory cell aggregation and barrier disruption; the reticular dermis is characterized by fibroblast activation, myofibrosis, and excessive ECM deposition leading to fibrosis; and the subcutaneous layer is characterized by fat atrophy, metabolic imbalance, and immune microenvironment remodeling. Therefore, simultaneous and precise specific interventions targeting different layers have become crucial for improving clinical efficacy and reducing recurrence.

[0004] Starting with multi-omics-driven target screening, coupled with the rapid validation and programmable expression of mRNA platforms, a disruptive new drug development pathway will be provided for skin diseases, achieving precision treatment targeting specific skin layers. Currently, mRNA therapy has been preliminarily explored in the field of skin wounds; for example, VEGF-A mRNA can promote angiogenesis and tissue repair in diabetic wounds. However, efficient and mature mRNA delivery technologies for the skin are still lacking: existing methods are difficult to effectively cross the stratum corneum and cannot achieve layer-specific differentiated intervention for the target layer. Traditional subcutaneous injection or electroporation methods cause mRNA to remain mostly in the superficial dermis, resulting in insufficient local expression and uncontrollable depth of action. We have also focused on the application of microneedle patches in percutaneous delivery. They have the potential for minimally invasive penetration of the stratum corneum and controllable penetration depth and spatial distribution. By modularly integrating the mRNA delivery carrier and polymer matrix into the microneedle system, it is expected to achieve efficient, localized, and programmable mRNA expression while achieving minimally invasive penetration. Based on this, further development of microneedle platforms with layer-specific differentiated intervention capabilities will better adapt to the hierarchical pathology of diverse skin diseases and promote precise and lasting in-situ treatment.

[0005] Currently, acupuncture microneedles can achieve precise control of the skin penetration depth of single needles / small cluster needles at the millimeter level through manual techniques or mechanical positioning. Compared with microneedle patches, they offer greater operability and target layer control in small to medium-sized lesions, especially in irregular areas such as curved surfaces, around joints, and folds. However, traditional acupuncture microneedles have limited drug delivery capabilities. They often rely on coating or impregnating the needle surface with drug-loaded substances, which tend to remain in the stratum corneum and superficial epidermis, making it difficult to stably and efficiently cross the epidermal-dermal barrier and deposit in the target dermal layer.

[0006] Skin fibrosis is a typical layer-specific remodeling skin disease, primarily affecting the activation of fibroblasts and excessive ECM deposition in the dermal reticular layer, and can extend across the epidermis and subcutaneous layer, leading to structural and functional imbalances. However, designing transdermal drug delivery systems specifically targeting layer-specific remodeling for better repair of layer-specific skin diseases remains a pressing technical challenge. Summary of the Invention

[0007] This invention aims to solve the aforementioned technical problems by providing a dual-module integrated threaded microneedle drug delivery system, its preparation method, and its applications. The technical objective of this invention is to provide a safe and minimally invasive treatment strategy for skin diseases involving deep tissue lesions, layer-specific pathology, and inconsistent disease progression, laying the foundation for future translational development.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing a dual-module integrated threaded microneedle drug delivery system, comprising the following steps: (1) Using ionizable lipids, cholesterol, DSPC and PEG-lipids as organic phases and sodium acetate buffer solution of PGC1α mRNA as aqueous phase, microfluidic methods were used to prepare PGC1α lipid nanoparticles. (2) SRT1720 was dissolved in dimethyl sulfoxide and then mixed with sodium hyaluronate solution under stirring to prepare SRT1720@HA hydrogel solution; (3) First, a layer of sodium hyaluronate solution is coated on the spiral groove of the threaded microneedle. Then, the PGC1α lipid nanoparticles obtained in step (1) are loaded in batches in the spiral groove. After a stable lipid layer is formed, the threaded microneedle is immersed in the SRT1720@HA hydrogel solution containing Rhodamine B, so that an outer coating is formed in the drug-loaded area of ​​the threaded groove.

[0009] The method described in this invention successfully constructed an mRNA delivery vector compatible with microneedle technology, ultimately forming an integrated layered approach: a rapidly release SIRT1 agonist hydrogel coating is constructed on the outer layer of the needle to inhibit SASP in senescent dermal fibroblasts; and PGC-1α mRNA LNPs are embedded in helical grooves in the form of microspheres, achieving targeted deposition and in situ expression into the subdermal fat layer as they rotate, thus achieving temporal and hierarchical synergistic intervention. Finally, in an in vivo bleomycin-induced skin fibrosis model, the treated group showed reduced dermal aging, downregulated SASP signaling, and restored skin fat structure, with a significant overall reduction in fibrosis. In summary, this invention constructs a spiral microneedle delivery system that achieves specific layer marker drug / mRNA delivery between the dermis and subcutaneous layer, providing a new strategy for precise intervention in skin diseases.

[0010] Furthermore, the molar ratio of ionizable lipids, cholesterol, DSPC and PEG-lipids in step (1) is 50:38.5:10:1.5.

[0011] Furthermore, the sodium acetate buffer solution in step (1) has a pH of 4.0 and a concentration of 25 mM.

[0012] Furthermore, the flow rate ratio of the organic phase to the aqueous phase in step (1) is 1:3.

[0013] Furthermore, the concentration of the sodium hyaluronate solution in step (2) is 2% w / v, and the concentration of the sodium hyaluronate solution applied in step (3) is 2% w / v.

[0014] Furthermore, the concentration of SRT1720@HA in step (2) is 10 mM.

[0015] Furthermore, the batch loading operation in step (3) is as follows: each batch of 100 microneedles is loaded with a total of 0.3 mg of lyophilized encapsulated PGC1α lipid nanoparticles.

[0016] Furthermore, the time for immersing the threaded microneedles in the SRT1720@HA hydrogel solution containing Rhodamine B in step (3) is 1 minute.

[0017] A second objective of this invention is to provide a dual-module integrated threaded microneedle drug delivery system prepared by the method described above.

[0018] A third objective of this invention is to provide the application of the dual-module integrated threaded microneedle drug delivery system described above in transdermal drug delivery.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention develops a dual-module integrated threaded microneedle system (denoted as HA-LNP@MS) that enables layered drug delivery and programmable release from the epidermis to the subcutaneous layer. The surface hydrogel module (SRT1720@HA) releases SIRT1 agonists immediately upon insertion to improve dermal fibroblast senescence; the deep LNP microsphere module (PGC-1α@LNP) releases PGC-1α mRNA through rotational delay after reaching the subcutaneous fat layer, restoring adipocyte metabolic homeostasis. Spatial transcriptomics and clinical sample analysis revealed a hierarchical molecular expression profile in skin lesions.

[0020] (2) In vitro experiments showed that SRT1720@HA significantly inhibited SASP expression in senescent dermal fibroblasts and enhanced their proliferation and migration abilities; PGC-1α@LNP effectively reversed the fibrotic phenotype of adipocytes. In vivo experiments showed that HA-LNP@MS reduced skin collagen deposition by about 30% and increased the thickness of the subcutaneous fat layer by nearly 3 times through layer-specific delivery, with the synergistic effect of the two drugs being the most significant. This study proposes a spatially programmable layered targeted therapy strategy, providing a new approach for the precise intervention of complex skin diseases. Attached Figure Description

[0021] Figure 1 Spatial and cytological characterization of lamellar-specific pathological features in dermal fibrosis; A) Spatial transcriptomic map of bleomycin-induced fibrotic skin tissue, showing the distribution of Fap4 and CDKN1A in the dermis and subcutaneous tissue; the dermis and subcutaneous tissue layers are labeled, and their boundaries are marked by red dashed lines; B) Semi-quantitative analysis of the spatial transcriptomic signals shown in Figure A, demonstrating the lamellar-specific expression intensity of Fap4 and CDKN1A; C) H&E staining of clinical dermal fibrosis specimens to show the dermal collagen structure and subcutaneous adipose tissue area; D) Immunofluorescence staining of Perilipin and α-SMA in subcutaneous tissue; cell nuclei were counterstained with DAPI; E) Immunofluorescence staining of CDKN1A and PDGFRα in dermal tissue; cell nuclei were counterstained with DAPI.

[0022] Figure 2Structural characterization of PGC1α@LNP and SRT1720@HA; A) Schematic diagram of PGC1α@LNP formulation; B) Schematic diagram of SRT1720@HA; C) Particle size distribution of blank LNP and PGC1α@LNP measured by dynamic light scattering (DLS); D) Zeta potential of blank LNP and PGC1α@LNP; E) TEM images show that both blank LNP and PGC1α@LNP have spherical morphology and uniform particle size; F) Confocal microscopy imaging shows that the colocalization degree of PGC1α@LNP with lysosomes is reduced at 4 h compared with 1 h, indicating that there is a time-dependent endosome escape phenomenon; G) The rheological curve of SRT1720@HA shows typical viscoelastic solid behavior, with Gʹ > Gʺ throughout the strain range; H) In vitro release curve of SRT1720 from HA hydrogel shows that it has a continuous cumulative release characteristic within 96 h.

[0023] Figure 3 Structural characterization of the spiral microneedle system: A) Optical microscopic image of the spiral microneedle, showing its spiral groove structure; B) Fluorescence image of the microneedle coated with LNPs (green) and HA hydrogel (red); C) Cross-sectional fluorescence image of the microneedle after it was inserted into a simulated skin model, showing the spatial distribution of LNPs (green) and HA hydrogel (red); D) In ​​vivo fluorescence image showing the distribution of PGC1α@LNP (green) and SRT1720@HA (red) in mouse skin.

[0024] Figure 4 For the in vitro biocompatibility assessment of SRT1720@HA and PGC1α@LNP: A) Calcein-AM / PI staining results of L929 fibroblasts and differentiated 3T3-L1 adipocytes after treatment with SRT1720@HA extract (100% v / v) or PGC1α@LNP (total lipid concentration of 100 µg / mL) for 12, 24, and 48 hours; B) Quantitative images corresponding to the Calcein-AM / PI staining results shown in Figure A; C) EdU staining results of L929 fibroblasts and differentiated 3T3-L1 adipocytes after treatment with SRT1720@HA extract or PGC1α@LNP for 24 hours; cell nuclei were counterstained with Hoechst; D) Quantitative analysis diagram corresponding to the EdU staining results shown in Figure C; E) SRT1720@HA extract or PGC1α@LNP staining results of L929 fibroblasts and differentiated 3T3-L1 adipocytes. CCK-8 activity was measured 24, 48 and 72 hours after treatment; statistical analysis was performed using an unpaired two-tailed t-test (mean ± standard deviation).

[0025] Figure 5 The effects of SRT1720@HA on fibroblast senescence and migration function are shown in Figure A. SA-β-gal staining results of senescent fibroblasts (Mock group) treated with HA or SRT1720@HA extract (100% v / v); B. Quantitative analysis diagram corresponding to the SA-β-gal staining results shown in Figure A; C. Scratch test images of senescent fibroblasts after treatment with HA or SRT1720@HA extract at 0, 12, 24 and 48 hours; D. Quantitative analysis diagram corresponding to the scratch closure status shown in Figure C; E. Schematic diagram of the mechanism by which SRT1720@HA reduces fibroblast senescence. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test, the same below.

[0026] Figure 6 The regulation of aging-related markers in senescent fibroblasts treated with SRT1720@HA is shown below: A) RT-qPCR analysis of genes related to aging and inflammation in senescent fibroblasts (Mock group) treated with HA or SRT1720@HA extract; B) Western blot analysis of the expression levels of CDKN2A and CDKN1A in senescent fibroblasts treated with HA or SRT1720@HA extract; C) Quantitative analysis corresponding to the Western blot bands shown in Figure B; D) Immunofluorescence staining of CDKN2A and F-actin in senescent fibroblasts treated with HA or SRT1720@HA extract; cell nuclei were counterstained with DAPI; E) Quantitative analysis corresponding to the immunofluorescence signals shown in Figure D; F) Immunofluorescence staining of CDKN1A and F-actin in senescent fibroblasts treated with HA or SRT1720@HA extract; cell nuclei were counterstained with DAPI; G) The quantitative analysis diagram corresponding to the immunofluorescence signal shown in Figure F.

[0027] Figure 7This study investigated the regulation of adipocyte-to-myofibroblast transformation in TGF-β1-induced 3T3-L1 adipocytes by PGC1α@LNP treatment. Differentiated 3T3-L1 adipocytes not stimulated by TGF-β1 were designated as the "untreated group." 3T3-L1 adipocytes stimulated by TGF-β1 were designated as the AMT model group (Mock). AMT model cells treated with empty LNPs or PGC1α@LNPs were defined as the empty LNP group and the PGC1α@LNP group, respectively. A) Oil Red O staining results of 3T3-L1 adipocytes in the untreated group, Mock group, empty LNP group, and PGC1α@LNP group; B) Quantitative analysis corresponding to the Oil Red O staining results shown in Figure A; C) RT-qPCR analysis of fibrosis-related and adipocyte-related genes in each experimental group; D) Schematic diagram of the mechanism by which PGC1α@LNP regulates adipocyte-to-myofibroblast transformation; E) Western blot analysis of α-SMA and Col1a1 protein expression levels in each experimental group; F) Quantitative analysis diagram corresponding to the Western blot bands shown in Figure E.

[0028] Figure 8 To investigate the in vivo anti-fibrotic effect of yarn-based microneedle-mediated drug delivery in a mouse model of bleomycin-induced cutaneous fibrosis; Experimental groups: Normal group – healthy mice not treated with bleomycin or microneedles; Mock group – fibrotic mice treated with uncoated microneedles; LNP@MS group – fibrotic mice treated with microneedles loaded with PGC1α@LNP; HA@MS group – fibrotic mice treated with microneedles coated with SRT1720@HA; HA / LNP@MS group – fibrotic mice treated with dual-drug-loaded microneedles; A) H&E staining results of dorsal skin; B) Masson's trichrome staining results of dorsal skin (collagen fibers appear blue, cytoplasm and myofibrils appear red); C) EVG staining results of dorsal skin (elastic fibers appear black; representative elastic fibers are marked with black arrows); D) α-SMA immunohistochemical staining results (positive myofibroblasts appear brown, marked with black arrows); E) COL1A1 immunohistochemical staining results (COL1A1 positive areas are brown); F) Bleomycin-induced skin fibrosis model and schematic diagram of microneedle-based intervention process; G) Quantitative analysis of skin thickness corresponding to Figure A; H) Quantitative analysis of subcutaneous fat layer thickness corresponding to Figure A; I) Quantitative analysis of collagen-positive areas corresponding to Figure B; J) Quantitative analysis of elastic fiber area corresponding to Figure C; K) Quantitative analysis of α-SMA-positive cells corresponding to Figure D; L) Quantitative analysis of COL1A1-positive areas corresponding to Figure E.

[0029] Figure 9To assess layer-specificity of skin aging and adipocyte-to-myofibrillar transformation after microneedling therapy; A) Dual immunofluorescence staining of CDKN2A (red), PDGFRα (green), and DAPI (blue) in back skin; B) Dual immunofluorescence staining of α-SMA (green), Perilipin (red), and DAPI (blue) in the subcutaneous layer; C) Immunohistochemical staining of CDKN2A in back skin, with positive staining shown in brown and marked by black arrows; D) Immunohistochemical staining of CDKN1A in back skin, with positive staining shown in brown and marked by black arrows; E) Quantitative analysis of CDKN2A⁺ / PDGFRα⁺ double-positive cells (expressed as the ratio to the total number of PDGFRα⁺ cells), corresponding to Figure A; F) Quantitative analysis of the α-SMA⁺ / Perilipin⁺ ratio, corresponding to Figure B; G) Quantitative analysis of the proportion of CDKN2A positive cells, corresponding to Figure C; H) Quantitative analysis of the proportion of CDKN1A positive cells, corresponding to Figure D.

[0030] Figure 10 Transcriptomic analysis of fibrotic skin after HA / LNP@MS treatment; Experimental groups: simulation group (bleomycin-induced skin fibrosis) and HA / LNP@MS group; A) Volcano plot showing differentially expressed genes between the two groups; B) KEGG pathway enrichment analysis of differentially expressed genes; C) GO enrichment analysis of differentially expressed genes; D) Comparison of specific pathways between the simulation group and the HA / LNP@MS group by GSEA analysis.

[0031] Figure 11 To investigate the in vitro release behavior and functional stability of PGC1α@LNP; A) The cumulative release curve of PGC1α mRNA within 96 h showed that it has a sustained release characteristic—the cumulative release amount was 65% at 96 h; B) RT-qPCR analysis was performed on the expression level of PGC1α mRNA after treating 3T3-L1 cells with freshly prepared PGC1α@LNP (Fresh), PGC1α@LNP (Lyo-0M) that was lyophilized and immediately reconstituted, or PGC1α@LNP (Lyo-1M) that was stored at −20°C for one month and then reconstituted; C) Western blot analysis was performed on the expression level of PGC1α protein after treating 3T3-L1 cells with the corresponding PGC1α@LNP formulation.

[0032] Figure 12To investigate the concentration-dependent cell compatibility of SRT1720@HA with PGC1α@LNP; A) L929 cells were incubated with SRT1720@HA hydrogel extract at concentrations of 25%, 50%, 75%, and 100% (v / v) for 24 hours, and cell viability was detected by the CCK-8 assay; B) 3T3-L1 cells were treated with PGC1α@LNP at total lipid concentrations of 25, 50, 100, and 200 μg / mL for 24 hours, and cell viability was detected by the CCK-8 assay; Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparison test.

[0033] Figure 13 To determine the biodistribution and systemic exposure of HA / LNP @MS after local administration; A) RT-qPCR analysis of exogenous PGC1α mRNA in plasma, treated back skin, draining lymph nodes, liver, and spleen at 1, 6, 24, and 48 hours after HA / LNP @MS treatment; B) LC-MS / MS analysis of SRT1720 in the corresponding samples and at different time points.

[0034] Figure 14 SRT1720@HA can inhibit the secretion of IL-6 and IL-1β in senescent L929 fibroblasts; A) The IL-6 level in the culture supernatant of L929 cells in the control group, Mock group, HA group and SRT1720@HA group was quantitatively analyzed by ELISA; B) The IL-1β level in the corresponding culture supernatant was quantitatively analyzed by ELISA.

[0035] Figure 15 To validate the bleomycin-induced skin fibrosis model and the effect of uncoated microneedle implantation; experimental groups: normal group (healthy mice, not treated with bleomycin or microneedles); scleroderma group (mice with bleomycin-induced skin fibrosis, not treated with microneedles); simulated group (fibrotic mice treated with uncoated microneedles); A) H&E staining of dorsal skin; B) Masson's trichrome staining of dorsal skin (collagen fibers stained blue, cytoplasm and myofibrils stained red); C) α-SMA immunohistochemical staining (positive myofibroblasts appear brown, marked with black arrows); D) COL1A1 immunohistochemical staining (COL1A1 positive areas appear brown); E) Quantitative analysis of skin thickness corresponding to Figure A; F) Quantitative analysis of subcutaneous fat layer thickness corresponding to Figure A; G) Quantitative analysis of collagen-positive areas corresponding to Figure B; H) Quantitative analysis of α-SMA-positive cells corresponding to Figure C; I) Quantitative analysis of COL1A1-positive areas corresponding to Figure D.

[0036] Figure 16To validate the representative differentially expressed genes identified by RNA sequencing using RT-qPCR, the figure shows the relative mRNA expression levels of Il6(A), Il1β(B), Lep(C), and Ppargc1α(D) in skin tissues of the Mock group and the HA / LNP@MS group. Statistical analysis was performed using an unpaired two-tailed t-test (mean ± standard deviation).

[0037] Figure 17 To achieve targeted mRNA delivery to different skin / subcutaneous tissues using LNP-hydrogel integrated spiral microneedles; when the microneedles advance through the dermis, the HA-based coating loaded with SRT1720@HA is released into the dermal layer, thereby delaying the skin aging process; when the microneedles reach the subcutaneous tissue, the rotational driving force generated by the spiral grooves promotes the targeted release of PGC1α@LNP, thereby restoring the fat structure of the subcutaneous tissue. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0039] 1. Preparation and characterization of PGC1α@LNP PGC1α mRNA was synthesized by Unigen Biotech Co. (Suzhou, China), and its nucleotide sequence is shown in Table 1. According to the manufacturer's quality control report, the mRNA has a purity of ≥90%, an A260 / A280 ratio of 1.9-2.2, and a concentration of 1.0 mg / mL (±3%).

[0040] Table 1 mRNA sequences ;

[0041] Lipid nanoparticles (LNPs) were prepared using a microfluidic mixing method, as follows: Ionizable lipids, cholesterol, DSPC, and PEG-lipids were dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5 to form the organic phase. A PGC1α mRNA solution was prepared in 25 mM sodium acetate buffer (pH 4.0) to form the aqueous phase. The organic and aqueous phases were mixed through a microfluidic chip at a flow rate of 1:3 to form PGC1α-encapsulated LNPs (PGC1α@LNP). The resulting suspension was dialyzed against PBS (pH 7.4) for 2 hours to remove ethanol and unencapsulated impurities. DiO was incorporated into the lipid phase for fluorescence tracking.

[0042] Particle size, polydispersity index (PDI), and zeta potential were determined using dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments, UK). Sample morphology was observed using transmission electron microscopy (TEM, JEOL JEM-2100) after negative staining with 2% phosphotungstic acid. Encapsulation efficiency and mRNA loading were determined using the Quant-iTRiboGreen RNA assay kit. Unencapsulated mRNA and total mRNA were measured before and after LNP lysis with 1% Triton X-100, respectively, and the calculation formulas are as follows: , ; Among them, C total and C free These represent the total mRNA concentration and the unencapsulated mRNA concentration, respectively. .

[0043] For storage and microneedle loading, PGC1α@LNP was frozen at −80°C for 2 hours, followed by overnight lyophilization, and then stored at −20°C under sealed, dry conditions for up to one month. To assess its functional stability, freshly prepared PGC1α@LNP, a lyophilized formulation immediately reconstituted, and a formulation reconstituted after one month of storage were compared. Intermediate doses of mRNA from each formulation were transfected into 3T3-L1 cells, and the expression levels of PGC1α mRNA and protein were subsequently detected using RT-qPCR and Western blotting, respectively.

[0044] 2. Preparation, rheological analysis, and drug release of SRT1720@HA hydrogel SRT1720 was dissolved in DMSO and then mixed with a 2% (w / v) sodium hyaluronate solution under gentle stirring to prepare a homogeneous SRT1720@HA hydrogel with a final concentration of 10 mM. In the fluorescence tracer experiment, Rhodamine B was added to the hydrogel to visualize the hydrogel coating.

[0045] Viscoelastic properties were measured using a rotational rheometer (MCR 302, Anton Paar, Austria) with 25 mm parallel plates at 25°C. Storage modulus (G′) and loss modulus (G″) were recorded as functions of oscillatory strain (0.1% to 100%). For in vitro release experiments, 1 mL of SRT1720@HA was placed in a dialysis bag (molecular weight cutoff 8–14 kDa) and incubated in 20 mL of PBS (pH 7.4, 37°C) with shaking at 100 rpm. At preset time points, 1 mL of culture medium was collected and replaced with fresh PBS. The concentration of SRT1720 in the hydrogel extract and release medium was determined by UV-Vis spectrophotometry (wavelength 320 nm), and the actual drug loading (mg / mL), drug loading efficiency (%), and cumulative release rate (%) were calculated accordingly.

[0046] 3. Preparation and multilayer coating of threaded microneedles This study employed single-threaded microneedles, which are fabricated by machining helical grooves of specific dimensions into a stainless steel needle body. The groove structure can be customized according to the target experimental model. To meet the skin drug delivery requirements of C57BL / 6 mice, each threaded microneedle used in this study had a total length of 4 mm and an outer diameter of 230 μm. The distal 2 mm section of the microneedle had a threaded groove structure, while the proximal 2 mm section had a non-threaded structure. The drug loading area was limited to the distal 1.5 mm section of the threaded area.

[0047] For layered loading, a 2% (w / v) sodium hyaluronate solution was first coated onto the spiral grooves to promote LNP deposition. Since the amount of drug retained by a single microneedle is below the reliable weighing range of a high-capacity microbalance (Cubis® II, Sartorius, Germany), batch loading and quantification of PGC1α@LNP were performed. Each batch of 100 microneedles was coated with a total of 0.3 mg of lyophilized PGC1α@LNP. After loading, unbound formulation was collected and quantified, and the average loading per microneedle was calculated by dividing the total retention by the number of microneedles. The resulting microneedles were lyophilized to stabilize the LNP layer defined within the grooves and stored at −20°C. Before application, the microneedles were immersed in a Rhodamine B-containing SRT1720@HA hydrogel for 1 minute to allow the drug-loaded hydrogel to form an outer coating in the drug-loaded region of the spiral. After extraction from the coated microneedles, the amount of SRT1720 retained on the microneedles was quantitatively analyzed using LC-MS / MS (SCIEX QTRAP 4500). This operation constructed a modular coating structure consisting of grooved PGC1α@LNPs and an outer SRT1720@HA hydrogel layer. Coating uniformity was observed using an optical microscope (Leica DVM6) and a fluorescence microscope (Zeiss LSM880).

[0048] 4. Clinical Sample Collection and Preparation Full-thickness skin samples were collected from fibrotic lesions in patients with localized scleroderma; skin samples from the same sites were collected concurrently from healthy controls during routine surgical procedures. All procedures were performed in accordance with the experimental protocol approved by the institution's ethics committee (ethics approval number: SH9H-2020-T138-2). All subjects signed written informed consent prior to sample collection. After sample retrieval, the samples were rinsed with cold phosphate-buffered saline (PBS) to remove blood; the tissues were then fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections.

[0049] 5. Biocompatibility assessment The cytocompatibility of SRT1720@HA with PGC1α@LNP was assessed using L929 and 3T3-L1 cell lines, respectively. Cells were cultured in high-glucose Dulbecco modified Eagle medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) under standard conditions (37°C, 5% CO2). Cytotoxicity was determined using a CCK-8 assay kit (Cell Counting Kit-8; catalog number C0038, Beyotime, Shanghai, China). Cells were cultured at 1 × 10⁶ cells / year. 4Seeded at a density of cells / well in 96-well plates, the samples were exposed to either hydrogel extract or nanoliposomes, and then incubated at 37°C with 10% (v / v) CCK-8 reagent for 1 hour. The absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3, MolecularDevices, USA).

[0050] To further assess cell proliferation, the EdU cell proliferation assay kit (catalog number: C0071S, Beoming Biotechnology, China) was used. After 24 hours of treatment, cells were co-incubated with EdU solution (50 μM, incubated for 2 hours), then fixed, permeabilized, and counterstained with Hoechst 33342. EdU-positive cell nuclei were observed using an inverted fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, USA), and quantitative analysis was performed using ImageJ software. In addition, cell viability was assessed using the Calcein-AM / propidium iodide (PI) double staining method (live / dead cell viability assay kit; catalog number: C2015S, Beoming Biotechnology, China): after incubation at 37°C for 30 minutes, green (Calcein-AM) and red (PI) fluorescence were used to distinguish live and dead cells, respectively, and the cells were observed under a fluorescence microscope and quantitatively analyzed using ImageJ.

[0051] 6. Assessment of systemic exposure To assess systemic exposure following local microneedle administration, plasma, treated back skin, draining lymph nodes, liver, and spleen samples were collected at 1, 6, 24, and 48 hours after HA / LNP @MS administration. Blood samples were centrifuged to obtain plasma. Tissue samples were washed with cold PBS, weighed, and homogenized. The delivered PGC1α mRNA level was analyzed by RT-qPCR using specific primers designed for the exogenous PGC1α mRNA sequence used in this study. Samples showing reproducible specific amplification in technical repeatability experiments were considered detectable. For samples with detectable amplification, the relative PGC1α mRNA level was calculated using a treated back skin sample collected 1 hour after administration as a calibrator. Samples without reproducible specific amplification were recorded as undetectable. The concentration of SRT1720 in plasma and tissue homogenates was quantified using LC-MS / MS (SCIEX QTRAP 4500). Calibration curves were prepared in blank plasma or corresponding blank tissue homogenates, and results are expressed as ng / mL plasma or ng / g tissue.

[0052] 7. Construction of cell experimental models and intervention measures This study established two cell models to evaluate the tissue-specific therapeutic effects of two delivery modules. To construct a cell senescence model, L929 cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μM H2O2 in six-well plates and then treated with 200 μM H2O2 for 6 hours to induce oxidative stress. After treatment, the culture medium was replaced with fresh complete culture medium. The cells were then cultured under normal conditions to allow cellular senescence-related changes to be observed.

[0053] To construct an adipocyte-to-myofibrillar (AMT) model, 3T3-L1 cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 g / mL in 0.1% gelatin-coated six-well plates and cultured in complete medium until 80% confluence. Subsequently, cells were induced to differentiate into mature adipocytes using OriCell® induction medium A for 3 days, followed by another day of induction using induction medium B (OriCell, MUXTL-90031). Medium A and medium B were then alternated until lipid droplet formation was observed. Finally, differentiated adipocytes were treated with TGF-β1 (10 ng / mL) for 24 hours to induce the AMT process.

[0054] In the interventional study, senescent L929 cells were co-incubated for 24 hours with 100% (v / v) hydrogel extracts prepared from unloaded HA hydrogels or HA hydrogels loaded with SRT1720 (SRT1720@HA; MedChemExpress, product number HY-10532). Myofibroblast-like cells derived from 3T3-L1 adipocytes were treated for 24 hours with either blank LNP or PGC1α@LNP (total lipid concentration 100 μg / mL) to assess their effect on AMT.

[0055] 8. Oil Red O staining After washing 3T3-L1 cells with PBS, fix them with 4% paraformaldehyde at room temperature for 20 minutes; then equilibrate them with 60% isopropanol for 5 minutes, and then incubate them with freshly prepared Oil Red O working solution (OriCell, catalog number OILR-10001) for 30 minutes; finally, gently rinse away excess staining agent with distilled water until the background is clear, and then acquire images using an optical microscope.

[0056] 9. Staining of aging-associated β-galactosidase (SA-β-Gal) L929 cells were seeded in 6-well plates and treated as specified. After treatment, the cells were washed with PBS and then fixed with 2% paraformaldehyde at room temperature for 10 minutes. After fixation, the cells were washed again with PBS and then incubated at 37°C for 2 hours in a CO2-free and light-protected environment with SA-β-Gal staining working solution (Beyotime, China). The stained cells were observed using a confocal laser scanning microscope (Leica, Germany), and the fluorescence intensity of SA-β-Gal was quantitatively analyzed using ImageJ software.

[0057] 10. Ulcer Healing Experiment L929 cells were seeded in 6-well plates and treated as specified. A linear scratch was then created on the cell monolayer using a sterile 200 μL pipette tip, followed by three washes with PBS to remove detached cells. The cell culture was then incubated under homogeneous conditions. Scratch closure was observed at 0, 12, 24, and 48 hours using an inverted fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, USA). Scratch width was measured in three randomly selected areas using ImageJ software to quantitatively analyze cell migration towards the scratch area.

[0058] 11. Western Blot Analysis Cells were placed in pre-chilled RIPA buffer (Beyotime, Beijing, China) and lysed on ice for 10 minutes. The lysis buffer was then centrifuged at 12,000 rpm (13,500 × g) at 4°C for 10 minutes to remove cell debris. The resulting supernatant was collected, and protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, USA). Equal volumes of protein (10 μg per sample) were subjected to 10% SDS-PAGE exercise electrophoresis at 150 V for 40 minutes, followed by protein transfer to a nitrocellulose membrane (Millipore, Bedford, Massachusetts, USA). The membrane was blocked with 5% bovine serum albumin (BSA) at room temperature for 1 hour, and then co-incubated with the following primary antibodies: α-SMA (Cell Signaling Technology [CST], catalog number 19245S), COL1A1 (CST, catalog number 72026S), CDKN1A (CST, catalog number 2947), CDKN2A (CST, catalog number 23200), PGC1α (Abcam, catalog number ab191838), and GAPDH (CST, catalog number 5174S). After washing, the membrane was co-incubated with horseradish peroxidase (HRP)-labeled secondary antibody (anti-rabbit IgG, CST, catalog number 7074S). Protein bands were visualized by chemiluminescence and quantified using ImageJ software.

[0059] 12. RT-qPCR Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Takara, Japan) with 1 μg of total RNA as a template. Quantitative PCR was performed using SYBR Green Master Mix (Takara, Japan) on a real-time quantitative PCR detection system (ABI7500, Applied Biosystems, USA). GAPDH was used as an internal control gene. −ΔΔCt The relative gene expression levels were calculated using a method. The primer sequences used in this study are listed in Table 2.

[0060] Table 2 Primer sequences used for PCR ;

[0061] 13. Immunofluorescence staining (cell culture) Cells seeded on sterile glass coverslips were fixed in PBS containing 4% paraformaldehyde at room temperature for 10 minutes, followed by permeabilization in PBS containing 0.5% Triton X-100 for 10 minutes. After blocking with PBS containing 1% BSA for 30 minutes, the cells were washed three times with PBS and then incubated with primary antibody at 4°C for 12 hours. After washing, the cells were incubated with FITC or Alexa Fluor-labeled secondary antibody at room temperature for 1 hour. F-actin was stained with phalloidin (green), and the cell nuclei were counterstained with DAPI (blue). Immunofluorescence images were acquired using a laser scanning confocal microscope (Leica, Germany).

[0062] 14. Enzyme-linked immunosorbent assay (ELISA) for IL-6 and IL-1β After inducing cell senescence and treating with the specified formulation, the cell culture supernatant was collected and cell debris was removed by centrifugation. The concentrations of IL-6 and IL-1β in the supernatant were quantified using a mouse IL-6 and mouse IL-1β ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA), and cytokine concentrations were calculated based on the corresponding standard curve.

[0063] 15. Animal experiments (1) Preparation of animal experimental models: All animal experiments were conducted in accordance with the relevant regulations of the institution and approved by the animal ethics committee. Male C57BL / 6 mice (6 weeks old) were purchased from the Shanghai Model Organisms Center (Shanghai, China) and underwent a one-week acclimatization period before the experiment. To establish a bleomycin-induced skin fibrosis model, a 1 cm × 1 cm area was marked on the midline of the back of each mouse. Mice received intradermal injections of bleomycin solution at the center of the marked area daily for 28 consecutive days. The concentration of bleomycin solution was 1 mg / mL (dissolved in PBS), and 100 μL was injected into each mouse daily; control mice received the same injection procedure, with 100 μL of PBS injected into the same site.

[0064] (2) Grouping and treatment in animal experiments: Before establishing the model, mice were randomly divided into six groups: (i) normal group; (ii) scleroderma group; (iii) sham treatment group (empty microneedles without drug loading); (iv) HA@MS group (microneedles loaded with SRT1720@HA); (v) LNP@MS group (microneedles loaded with PGC1α@LNP); (vi) HA / LNP@MS group (microneedles co-loaded with SRT1720@HA and PGC1α@LNP). Microneedle treatment was performed every two days for two weeks. At each treatment, the marked 1 cm × 1 cm dorsal fibrosis area was treated at five pre-set implantation sites (four corners and the center point). During drug administration, the unthreaded portion of the proximal end of each threaded microneedle was fixed in a sterile handheld microneedle holder to ensure stable implantation and rotation of the microneedle. The microneedle was inserted vertically into the dorsal skin until the distal 1.5 mm drug-loaded threaded segment was completely embedded in the skin. After implantation, the microneedle is gently rotated to remain in the skin for 30 seconds before being withdrawn. Throughout the study, all animals were monitored for signs of pain, skin ulceration, or infection, and appropriate care was provided as needed.

[0065] 16. Histological, immunohistochemical and immunofluorescence detection Tissue samples were fixed overnight in 4% paraformaldehyde solution, embedded in paraffin, and then cut into 5 μm thick sections. For histological evaluation, the sections were stained with hematoxylin and eosin (H&E) according to standard operating procedures; collagen deposition was assessed using the Masson trichrome staining kit (catalog number: G1340, Solarbio, Beijing, China); and elastic fiber distribution was detected using the Elastica van Gieson (EVG) staining kit (catalog number: G1590, Solarbio, Beijing, China). For immunohistochemical detection: After dewaxing and hydration, the sections were subjected to antigen retrieval in citrate buffer at 95°C for 20 minutes. Subsequently, the sections were blocked with 5% bovine serum albumin (BSA) and incubated overnight at 4°C with the following primary antibodies: anti-CDKN1A antibody (CST, catalog number: 2947, dilution: 1:200), anti-CDKN2A antibody (CST, catalog number: 23200, dilution: 1:200), anti-α-SMA antibody (CST, catalog number: 19245S, dilution: 1:200), and anti-COL1A1 antibody (CST, catalog number: 72026S, dilution: 1:200). The sections were then incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (Jackson Lab, catalog number: 138729, dilution: 1:200) at room temperature for 1 hour. Images of the stained sections were acquired using a bright-field microscope (Leica, Germany).

[0066] For immunofluorescence detection, tissue sections were incubated overnight at 4°C with primary antibodies against PDGFRα (Santa Cruz, sc-398206, 1:50 dilution) and CDKN2A (CST, 23200, 1:100 dilution), or with primary antibodies against α-SMA (CST, 19245S, 1:200 dilution) and Perilipin (Santa Cruz, sc-390169, 1:50 dilution). After washing, the sections were incubated in the dark for 1 hour with FITC-labeled goat anti-mouse IgG antibody (Servicebio, GB22301, 1:200 dilution) and Cy3-labeled goat anti-rabbit IgG antibody (Servicebio, GB21303, 1:200 dilution). Cell nuclei were counterstained with DAPI (Sigma, USA). Fluorescence images were acquired using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).

[0067] 17. RNA sequencing For RNA-Seq analysis, three full-thickness skin tissue bioreplicas were collected from each experimental group. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA), and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only samples with an RNA integrity score (RIN) ≥7 were selected for library construction. cDNA libraries were prepared using the TruSeq StrandedmRNA LT Sample Prep Kit (Illumina, San Diego, CA) and sequenced on the Illumina sequencing platform according to the manufacturer's instructions. Differentially expressed genes (DEGs) were identified using the R package DESeq2 (version v1.6.3); genes with a p-value <0.05 and a fold change in expression >2 or <0.5 were considered to have significant differential expression. Hierarchical cluster analysis was used to analyze the expression patterns of DEGs. Based on hypergeometric distribution, we performed Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in the R language environment.

[0068] 18. Spatial transcriptomics analysis The original counting matrix and spatial speckle information were downloaded from the original data repository and processed using the Seurat and SeuratData workflows in R. Low-quality specks were removed based on mitochondrial gene proportions and total feature counts. Data standardization, scaling, and dimensionality reduction were performed using the SCTransform workflow. Spatial clustering analysis was performed using the FindNeighbors and FindClusters functions, and the clustering results were annotated based on marker genes reported in the original literature. Tissue layer boundaries were determined based on speckle coordinates and histological images provided in the dataset. Differentially expressed genes between different tissue layers and under different experimental conditions were identified using FindMarkers combined with the Wilcoxon rank-sum test. All analyses followed standard operating procedures and were consistent with the experimental procedures reported in the original study.

[0069] 19. Statistical Analysis All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the Student's t-test for comparisons between two groups, followed by one-way ANOVA and Dunnett's multiple comparison test to compare each treatment group with a specified control group. A p-value < 0.05 was considered statistically significant.

[0070] Experimental Example 1 The experimental results and analysis for Example 1 are as follows: 1. Spatial and cellular analysis of specific pathological phenotypes in different dermal layers of fibrotic skin. Spatial transcriptomic analysis of bleomycin-induced dermal fibrosis tissue revealed a distinct layer-specific pathological pattern. With continued exposure to bleomycin, the adipocyte marker Fapp4 in subcutaneous tissue gradually decreased, while the aging marker Cdkn1a in the dermis gradually increased. Figure 1 (A, B). HE staining of clinically fibrotic skin specimens showed disordered arrangement of dermal collagen fibers and fibrotic replacement of the subcutaneous fat layer. Figure 1 (C). Immunofluorescence analysis further confirmed this stratified remodeling phenomenon: Perilipin / α-SMA co-staining indicated the presence of AMT in the subcutaneous layer, while CDKN1A / PDGFRA co-localization revealed the accumulation of senescent fibroblasts in the dermis (C). Figure 1 (D, E).

[0071] Similar layer-specific alterations have been reported in skin fibrosis models: adipocyte-derived cells participate in the formation of myofibroblast pools and lead to fat reduction, while senescent fibroblasts promote increased matrix stiffness through sustained SASP signaling and NF-κB / TGF-β pathway activation. Based on these observations, our spatial transcriptomics and histological data reveal a layer-specific pathological structure in skin fibrosis—localized scleroderma, a typical representative of fibrotic skin diseases, is characterized by dermal fibroblast senescence and subcutaneous adipocyte transdifferentiation. This spatial distribution pattern suggests that the dermis and subcutaneous tissue follow different pathogenic mechanisms: the former is driven by inflammation and cellular senescence, while the latter is driven by metabolic dysfunction and fibrosis, thus highlighting the necessity of employing layer-specific treatment strategies.

[0072] 2. Structural and functional characterization of PGC1α@LNP and SRT1720@HA DLS analysis showed that both the blank LNP and PGC1α@LNP exhibited uniform particle size distribution, with average particle sizes of 104.7 nm and 108.0 nm, respectively. Figure 2 (C). Zeta potential measurements showed that encapsulation with PGC1α slightly reduced the surface potential from –5.2 mV to –6.4 mV, indicating successful nucleic acid encapsulation and no aggregation. Figure 2 (D). TEM images further confirmed that both formulations maintained a clear spherical morphology, smooth surface, and uniform particle size distribution. Figure 2 The encapsulation efficiency of PGC1α@LNP was 92.6 ± 2.1%, and the mRNA loading was 18.4 ± 1.2 μg / mg lipid. This formulation exhibits sustained mRNA release characteristics, with a cumulative release rate approaching 65% at 96 hours. Figure 11 (A). Confocal imaging showed that the colocalization of PGC1α@LNP with lysosomes decreased at 4 hours compared to 1 hour, indicating a time-dependent endosome escape phenomenon. Figure 2 (F). In 3T3-L1 cells, freshly prepared, lyophilized and immediately reconstituted, and one-month-old PGC1α@LNP formulations all enhanced the mRNA and protein expression levels of PGC1α, confirming the effective intracellular delivery and translation of the encapsulated mRNA. The comparable expression levels among the three formulations further indicate that PGC1α@LNPs maintain their functional stability after lyophilization and one-month storage. Figure 11 (B, C)

[0073] For the hydrogel component, the actual drug loading of SRT1720 was 4.47 ± 0.17 mg / mL hydrogel, corresponding to a drug loading efficiency of 95.0 ± 3.5%. Rheological analysis showed that SRT1720@HA exhibited typical viscoelastic solid behavior—its storage modulus (G′) remained higher than its loss modulus (G″) with increasing oscillatory strain, indicating the formation of a stable viscoelastic hydrogel network (G in Figure 2). In vitro drug release curves showed that SRT1720 exhibited a sustained-release mode from the HA matrix, with a cumulative release of approximately 80% within 96 hours (H in Figure 2). In summary, these results indicate that PGC1α@LNP possesses suitable physicochemical properties and functional stability, while SRT1720@HA provides a stable matrix for sustained-release drug delivery.

[0074] Structural characterization of the threaded microneedle system As shown under an optical microscope, the prepared threaded microneedles exhibit a distinct helical groove structure with uniform pitch and consistent surface morphology. Figure 3 (A). Drug loading was quantified using hybrid microneedles. Batch-based quantitative analysis showed an average PGC1α@LNP loading of 2.40 ± 0.23 μg per microneedle, corresponding to a drug loading efficiency of 80.0 ± 7.5%, meaning each microneedle encapsulated approximately 44 ng of PGC1α mRNA. LC-MS / MS analysis of the hybrid-coated microneedles showed an average SRT1720 loading of 10.3 ± 1.3 ng per microneedle. Fluorescence imaging of the coated microneedles confirmed the distribution of green-labeled LNPs and red-labeled SRT1720@HA hydrogel within the designated drug loading area. Independent fluorescence channels further validated the spatial distribution of these two components along the microneedle surface. Figure 3 (B) Subsequently, threaded and non-threaded microneedles with the same fluorescent coating were inserted to the same depth and placed in a simulated skin model. The threaded microneedles produced deeper and more continuous LNP deposition along the insertion path, while the non-threaded microneedles mainly exhibited superficial and localized deposition. In both experiments, HA fluorescence was concentrated in the upper insertion region (B). Figure 3(C) In a mouse skin model, the spiral microneedles also promoted the distribution of PGC1α@LNP from the dermis to the subcutaneous layer, while the non-spiral microneedles resulted in a more limited distribution confined to the surface. SRT1720@HA was mainly retained in the dermis (D in Figure 3). This distribution pattern is attributed to the external localization of the HA hydrogel coating—which facilitates its clearance and retention within the dermis during implantation; while the spiral grooves protect the lyophilized PGC1α@LNP formulation from premature displacement and promote its delivery to deeper tissue layers. These results demonstrate that the spiral microneedle system enables differential spatial deposition of the two formulations: SRT1720@HA exhibits a retention characteristic preferential to the dermis, while PGC1α@LNP exhibits a distribution characteristic deeper throughout the treatment area.

[0075] 4. Biocompatibility evaluation of SRT1720@HA and PGC1α@LNP L929 fibroblasts were selected as a substitute model for primary skin fibroblasts, while adipocyte-differentiated 3T3-L1 cells served as a typical model for subcutaneous adipocytes. These cell lineage-related models are consistent with the skin-subcutaneous targeting strategy employed in this study. Cells were exposed to working concentrations of each carrier—SRT1720@HA extract (100% v / v) or PGC1α@LNP (total lipid concentration of 100 μg / mL)—and compared with a blank control group. CCK-8 levels were elevated in both treated and control cultures over 24 / 48 / 72 hours, with no significant difference between groups. Figure 4 The EdU incorporation level at 24 hours was unchanged compared to the control group. Figure 4 (C, D); while the Calcein-AM / PI staining results at 12, 24, and 48 hours showed that, under all experimental conditions, the cell monolayers were predominantly Calcein-positive and PI-negative (C, D). Figure 4 (A and B). To further evaluate concentration-dependent cell compatibility, cells were exposed to 25%, 50%, 75%, and 100% (v / v) SRT1720@HA extract, or 25, 50, 100, and 200 μg / mL PGC1α@LNP total lipids, for 24 hours. CCK-8 analysis showed that cell viability was not significantly reduced for either cell type across all tested concentration ranges. In summary, these results indicate that SRT1720@HA and PGC1α@LNP were well tolerated within the tested exposure concentration ranges and did not adversely affect cell viability or basal proliferation. Figure 12 ).

[0076] 5. Systemic exposure after local microneedle drug delivery To assess tissue distribution and systemic exposure after local microneedle administration, researchers measured the levels of PGC1α mRNA and SRT1720 in plasma, treated back skin, draining lymph nodes, liver, and spleen at 1, 6, 24, and 48 hours after HA / LNP @MS administration. Figure 13 In exogenous PGC1α mRNA, the substance was repeatedly detected in treated dorsal skin, and its concentration decreased in a time-dependent manner; in contrast, no reproducible specific amplification was observed in plasma, draining lymph nodes, liver, or spleen at any detection time point. Figure 13 (A). LC-MS / MS analysis showed that SRT1720 was mainly retained in the treated back skin, while its concentrations in plasma, draining lymph nodes, liver, and spleen were all below the limit of quantitation at all detection time points. Figure 13 (B). These results demonstrate that the linear microneedle system enables local delivery of both treatment modules without generating quantifiable systemic exposure.

[0077] 6. Evaluation of the effect of SRT1720@HA on reducing fibroblast senescence To investigate the effect of SRT1720-loaded hydrogel (SRT1720@HA) as the outer layer of a microneedle system on alleviating fibroblast senescence, we established a senescence model by exposing L929 cells to hydrogen peroxide (Mock), and then treated these cells with 100% (volume percentage) hydrogel extracts containing or without SRT1720.

[0078] β-galactosidase is a classic marker of cellular senescence, and its positivity rate directly reflects the degree of cellular senescence. Staining results showed that, compared with the Mock group, the proportion of β-galactosidase-positive cells in the SRT1720@HA group was significantly reduced. Figure 5 (A, B). The migration ability of fibroblasts is a key indicator of their functional status, and this ability is usually inhibited in cellular senescence. Scratch assay results showed that, at 24 hours, cells treated with SRT1720@HA exhibited significantly enhanced migration ability compared to the Mock group. Figure 5(C, D) MCP1, CCL5, and CXCL10 are typical inflammatory cytokines, often upregulated in senescent cells and are important components of the senescence-associated secretory phenotype (SASP). CDKN2A and CDKN1A are classic cell cycle repressors, widely considered markers of cellular senescence; while IL-6 and IL-1β are core SASP factors closely related to the pro-inflammatory microenvironment. RT-qPCR analysis showed that the expression levels of these genes were significantly increased in senescent L929 fibroblasts, while SRT1720@HA treatment significantly inhibited their upregulation. Figure 6 (A). Consistent with the transcriptomics results, ELISA analysis of the conditioned medium further confirmed that treatment with SRT1720@HA reduced the secretion of IL-6 and IL-1β. Figure 14 Western blot analysis consistently confirmed that SRT1720@HA significantly reduced the excessive expression levels of CDKN2A and CDKN1A in senescent fibroblasts, further validating its role in alleviating cellular senescence. Figure 6 (B, C). Immunofluorescence staining results also showed that the signal intensity of CDKN2A and CDKN1A was decreased in the SRT1720@HA group. Figure 6 (D–G), which provides direct cellular spatial evidence that SRT1720@HA can inhibit the expression of senescence-related proteins. In summary, these results indicate that SRT1720@HA can improve multiple characteristics of fibroblast senescence at the phenotypic, functional, and molecular levels, suggesting its potential role in partially reversing cellular senescence and promoting functional recovery.

[0079] 7. Evaluation of the effect of PGC1α@LNP on inhibiting the transformation of adipocytes into myofibroblasts Next, we investigated the effects of PGC1α-loaded lipid nanoparticles (PGC1α@LNP)—a functional component specifically designed for subcutaneous drug delivery in threaded microneedle systems—on adipocyte-to-myofibroblast (AMT) conversion. The conversion of skin fibroblasts to myofibroblasts is a core mechanism of skin fibrosis; therefore, we first targeted pathological fibroblast activation by reversing the senescent phenotype of skin fibroblasts using the SRT1720@HA module, as described above. Simultaneously, our spatial transcriptomics analysis revealed AMT-related tissue remodeling in the deep skin adipose tissue region and subcutaneous fat layer. Senescent fibroblasts and their secreted cytokines (SASP) may promote pathological adipose tissue remodeling and AMT, which may further enhance the local pro-fibrotic microenvironment, thus creating an interactive pathological cycle at the skin-adipose tissue interface. This finding is consistent with previous studies that indicate adiponectin-positive adipocyte precursors and skin adipocytes are direct contributors to myofibroblast formation and skin fibrotic remodeling. Accordingly, we used TGF-β1 to stimulate differentiated 3T3-L1 adipocytes to establish an AMT model (Mock group), followed by treatment with PGC1α@LNP (total lipid concentration of 100 µg / mL) to evaluate the therapeutic effect of subcutaneously administered PGC1α mRNA module.

[0080] Oil Red O staining (a method that reflects lipid accumulation and adipogenesis differentiation) showed that, compared with the Mock group, cells treated with PGC1α@LNP retained significantly more lipid droplets, indicating that TGFβ1-induced adipocyte loss was alleviated and adipocyte characteristics were maintained. Figure 7 (A, B). RT-qPCR analysis further confirmed that the expression levels of fibrosis-related markers were significantly downregulated after treatment with PGC1α@LNP ( ). Figure 7 (C). Among them, MMPs involved in extracellular matrix remodeling, α-SMA as a marker of myofibroblast activation, and Col1a1 reflecting collagen deposition all showed a significant decreasing trend; conversely, the expression of adipocyte-related genes was upregulated, including lipid metabolism markers Lipe, adipogenesis transcription factors PPARγ and C / EBPα, as well as Perilipin, Fapp4, and Adiponectin—these genes all suggest the process of adipocyte differentiation and functional maintenance. The above results indicate that PGC1α@LNP can effectively inhibit the fibrotic reprogramming process while restoring adipocyte characteristics. At the protein level, Western blotting further confirmed that PGC1α@LNP intervention can reduce the elevated α-SMA and Col1a1 protein levels in the AMT model, thus verifying its inhibitory effect on myofibroblast transformation. Figure 7 (E, F).

[0081] Overall, these findings indicate that PGC1α@LNP effectively inhibits TGFβ1-induced adipocyte-to-myofibroblast transformation at both the phenotypic and molecular levels, thereby maintaining adipocyte characteristics and mitigating fibrotic reprogramming.

[0082] 8. Therapeutic effect of the threaded microneedle system in a bleomycin-induced skin fibrosis model The ultimate goal of this study is to achieve precise drug delivery across different skin layers using a spiral microneedle system, thereby simultaneously targeting key pathological processes in the dermis and subcutaneous adipose tissue to alleviate fibrotic remodeling. To this end, we used a bleomycin-induced skin fibrosis model for in vivo validation experiments. After establishing the model, experimental animals were randomly assigned to designated groups: the untreated scleroderma group received no microneedle intervention; while the simulation group received uncoated microneedle implantation according to the same protocol and procedure as the treatment group. The simulation group was established to eliminate the potential influence of mechanical stimulation—because the acupuncture procedure itself can affect the local tissue microenvironment by inducing aseptic inflammation, altering connective tissue morphology, and activating mechanosensitive cells. Figure 15 As shown, compared with the normal group, the untreated scleroderma group exhibited significant dermal thickening, increased collagen deposition, and reduced subcutaneous adipose tissue, confirming the successful establishment of the model. No significant differences were observed between the untreated scleroderma group and the simulation group, indicating that uncoated microneedle implantation alone did not significantly alter the fibrotic phenotype. Each treatment group received microneedle-based drug delivery with an outer SRT1720@HA hydrogel coating (HA@MS), groove-loaded PGC1α@LNP (LNP@MS), or a combination of both (HA / LNP@MS) (Figure 8, F). During the treatment phase, microneedles were applied to the fibrotic skin every two days, ensuring continuous local intervention through evenly distributed puncture points.

[0083] This threaded microneedle system achieves differentiated spatial delivery through different loading configurations of its two treatment modules. Helical grooves on the distal drug loading area create recessed spaces that prevent premature displacement of the lyophilized PGC1α@LNP formulation during implantation and promote its delivery to deeper tissues. Following implantation and rotation, tissue hydration and mechanical shear forces jointly induce the release of the groove-constrained LNP formulation along the implantation path (including the skin and subcutaneous tissue areas). This conclusion is supported by simulated skin models and fluorescence tracing experiments in mouse skin—the results show that PGC1α@LNP exhibits a deeper distribution throughout the entire implantation path. Figure 3(C, D) In ​​contrast, SRT1720@HA is applied to the skin surface as a topical hydrogel coating. During implantation, tissue resistance causes this surface coating to be preferentially removed from and retained in the dermis, exhibiting delivery behavior similar to that of conventionally coated needles. After deposition, the HA matrix absorbs interstitial fluid, swells, and releases SRT1720 within the skin compartment. This distribution allows SRT1720 to target senescent skin fibroblasts and inhibit their pro-fibrotic secretory phenotype. Simultaneously, the PGC1α@LNP formulation distributes along the deep implantation channel, thereby protecting the encapsulated mRNA, supporting its sustained release, and enabling intracellular delivery and translation of SRT1720 within adipocytes. Delivering this module to deep skin and subcutaneous adipose tissue compartments aims to restore metabolic and differentiation homeostasis of adipocytes and inhibit the transformation of adipocytes into myofibroblasts. Therefore, the complementary distribution of these two modules provides a structural basis for simultaneously targeting senescent skin fibroblasts and pathological adipose tissue remodeling.

[0084] In vivo anti-fibrotic efficacy of the threaded microneedle system in a bleomycin-induced skin fibrosis model: After two weeks of routine intervention, histological and molecular biological analyses were performed on the fibrotic back skin. In the Mock group, the skin tissue showed significant dermal thickening with disordered collagen bundle arrangement and significant thinning of the subcutaneous fat layer (Figure 8A). Masson and EVG staining further revealed extensive and dense collagen accumulation in this area, accompanied by fragmented and sparse elastic fibers, resulting in near-complete disappearance of the elastic fiber network, suggesting severe fibrotic remodeling (Figure 8B, C). In contrast, all three microneedle-based treatment regimens alleviated the fibrotic manifestations, with the HA / LNP@MS group showing the most significant improvement: significantly reduced dermal thickness, significantly reduced collagen deposition, and substantial restoration of elastic fiber structure. Figure 8 (G–J). The specific effects on each tissue layer were also very obvious: HA@MS mainly acted on the dermis, reducing collagen accumulation and promoting the remodeling of elastic fibers; while LNP@MS was more effective in restoring subcutaneous fat thickness, but its effect on elastic fibers was limited when used alone. Immunohistochemical analysis results supported the above observations: the expression levels of COL1A1 and α-SMA were significantly upregulated in the Mock group, while all interventions significantly reduced their expression levels, with the HA / LNP@MS group showing the most significant inhibitory effect (D, E, K, L in Figure 8).

[0085] Laminar Regulation of Skin Aging and Adipocyte-Myofibroblast Transformation: To further explore the stratified mechanism of the spiral microneedle system, we assessed skin aging and adipocyte-myofibroblast transformation (AMT). Dual immunofluorescence staining with PDGFRα and p16 revealed a large accumulation of p16⁺ / PDGFRα⁺ fibroblasts in the Mock group, indicating enrichment of senescent fibroblasts. Figure 9 (A). Both HA@MS and HA / LNP@MS significantly reduced the proportion of senescent fibroblasts, while LNP@MS showed no significant effect. Figure 9 (Middle E). This trend is consistent with the immunohistochemical staining results of p16 and p21, further confirming the role of the hydrogel module in alleviating skin aging. Figure 9 C, D, G, H). Meanwhile, co-staining of α-SMA and Perilipin revealed active AMT in the Mock group, specifically manifested as an increase in α-SMA⁺ cells and a decrease in Perilipin⁺ adipocytes in the subcutaneous layer. Figure 9 Both LNP@MS and HA / LNP@MS significantly reduced the α-SMA⁺ / Perilipin⁺ ratio, indicating their effective inhibitory effect on AMT; while HA@MS showed no significant effect. Figure 9 (Middle F).

[0086] Transcriptome reprogramming mediating synergistic effects of translayer therapy—To further elucidate the molecular mechanisms behind its therapeutic effects, this study performed transcriptome sequencing analysis on fibrotic skin tissues from the Mock group and the HA / LNP@MS group. Figure 10Differential expression analysis revealed that HA / LNP @MS treatment induced extensive transcriptome reprogramming, characterized by downregulation of aging-related and inflammation-related pathways, such as the p53 signaling pathway. The expression of key inflammation and extracellular matrix remodeling-related genes, including Il1b, IL6, Mmp8, Vcam1, and Sgpp2, was significantly suppressed, consistent with the remission state of chronic inflammation and excessive extracellular matrix production. Correspondingly, signaling pathways involved in ECM-receptor interactions and fibrotic remodeling were also significantly weakened, consistent with histologically reduced collagen deposition. Conversely, HA / LNP @MS treatment reactivated adipogenesis and metabolic-related programs, as evidenced by upregulation of genes such as Lep, Acss2, Coq9, Ppargc1a, and Mogat2—genes crucial for lipid biosynthesis, mitochondrial electron transport chain, and PPAR-dependent metabolic regulation. RT-qPCR validation further confirmed that after HA / LNP@MS treatment, Il1β and Il6 expression were downregulated, while Ppargc1α and Lep expression were upregulated, a result consistent with RNA-seq analysis. Figure 16 Enrichment analysis further confirmed enhanced lipid biosynthesis and mitochondrial electron transport, indicating that mitochondrial function and adipocyte lineage stability in the subcutaneous tissue layer have been restored. Overall, these transcriptomic features suggest that this modular LNP-hydrogel integrated linear microneedle restores lipid metabolism and energy homeostasis while inhibiting the pro-fibrotic, pro-inflammatory, and pro-aging cascades, thus supporting its multi-faceted therapeutic effects in the skin and subcutaneous tissue.

[0087] Overall, these results indicate that the spiral microneedle system can exert a layered therapeutic effect in vivo: the hydrogel module mainly alleviates skin fibrosis by reducing fibroblast senescence, while the lipid nanoparticle module restores subcutaneous fat homeostasis by inhibiting AMT; when the two modules are used in combination, synergistic translayer regulation can be achieved, effectively blocking the pathological feedback loop, and ultimately producing a more comprehensive anti-fibrotic effect in fibrotic skin.

[0088] III. Conclusion This invention develops a simple, modular spiral microneedle system that combines a hydrogel coating with lipid nanoparticles for drug delivery to different skin layers. SRT1720@HA is coated on the needle tip and releases rapidly after implantation; while PGC1α@LNP is loaded in spiral grooves for a slower release rate. In this way, the system can simultaneously deliver drugs to both the dermis and subcutaneous fat layer in a single treatment. Figure 17Materials testing and biocompatibility assessments demonstrated that both components are suitable for dermal applications. SRT1720@HA alleviated senescence in dermal fibroblasts; PGC1α@LNP inhibited the transformation of adipocytes into myofibroblasts and improved adipocyte metabolic function. In animal studies, this threaded microneedle system improved collagen structure in the dermis and reduced subcutaneous fat loss. This treatment regimen synergistically repaired both layers of skin tissue. Transcriptomic analysis showed that the combined treatment downregulated gene expression associated with aging, inflammation, and fibrosis, while upregulating gene expression associated with lipid metabolism and mitochondrial function. These results reveal the molecular-level mechanism of action of this layered drug delivery approach. In summary, these findings demonstrate the therapeutic potential of this threaded microneedle system in mouse models.

Claims

1. A method for preparing a dual-module integrated threaded microneedle drug delivery system, characterized in that, Includes the following steps: (1) Using ionizable lipids, cholesterol, DSPC and PEG-lipids as organic phases and sodium acetate buffer solution of PGC1α mRNA as aqueous phase, microfluidic methods were used to prepare PGC1α lipid nanoparticles. (2) SRT1720 was dissolved in dimethyl sulfoxide and then mixed with sodium hyaluronate solution under stirring to prepare SRT1720@HA hydrogel solution; (3) First, a layer of sodium hyaluronate solution is coated on the spiral groove of the threaded microneedle. Then, the PGC1α lipid nanoparticles obtained in step (1) are loaded in batches in the spiral groove. After a stable lipid layer is formed, the threaded microneedle is immersed in the SRT1720@HA hydrogel solution containing Rhodamine B, so that an outer coating is formed in the drug-loaded area of ​​the threaded groove.

2. The method according to claim 1, characterized in that, The molar ratio of ionizable lipids, cholesterol, DSPC and PEG-lipids in step (1) is 50:38.5:10:1.

5.

3. The method according to claim 1, characterized in that, The sodium acetate buffer solution in step (1) has a pH of 4.0 and a concentration of 25 mM.

4. The method according to claim 1, characterized in that, The flow rate ratio of the organic phase to the aqueous phase in step (1) is 1:

3.

5. The method according to claim 1, characterized in that, The concentration of the sodium hyaluronate solution in step (2) is 2% w / v, and the concentration of the sodium hyaluronate solution applied in step (3) is 2% w / v.

6. The method according to claim 1, characterized in that, The concentration of SRT1720@HA in step (2) is 10 mM.

7. The method according to claim 1, characterized in that, In step (3), the batch loading operation is as follows: each batch of 100 microneedles is loaded with a total of 0.3 mg of lyophilized PGC1α lipid nanoparticles.

8. The method according to claim 1, characterized in that, The time for immersing the threaded microneedles in the SRT1720@HA hydrogel solution containing Rhodamine B in step (3) is 1 minute.

9. A dual-module integrated threaded microneedle drug delivery system prepared by the method of any one of claims 1-8.

10. The application of the dual-module integrated threaded microneedle drug delivery system of claim 9 in transdermal drug delivery.

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