A multi-layer detachable microneedle, a preparation method and application thereof, and a treatment and synergistic monitoring platform for skin superficial diseases

Abstract

A multi-layer detachable microneedle and its preparation method and application and a treatment synergistic monitoring platform for skin superficial diseases, which can realize light-controlled drug delivery treatment through dPCR based on real-time in-situ monitoring conditions, the detachable microneedle is composed of the innermost polyethylene diacrylate extraction layer, the outer gelatin methacrylate drug carrier layer and the polyvinyl alcohol connecting layer designed for thermal separation. The outer layer realizes the light-responsive release of the drug through the photothermal characteristics of BP, and the release rate reaches 78% within 24 hours. Subsequently, the significant mechanical strength and swelling characteristics promote the effective extraction of about 26 muL of interstitial fluid within 10 minutes. Melanoma studies in vitro and in vivo show that the platform can improve the therapeutic effect while minimizing systemic toxicity. It can monitor the MCAM and BRAF genes based on dPCR, and detect protein markers through digital proximity ligation assay, and the carefully designed biological system highlights its ability to interact with the pathophysiological environment, paving the way for real-time monitoring and personalized management of skin diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a multi-layer detachable microneedle, a preparation method and application thereof, and a collaborative monitoring platform for the treatment of superficial skin diseases. Background Art

[0002] Cutaneous melanoma is a highly aggressive skin tumor that arises from melanocytes. Its incidence and mortality continue to rise, with cases projected to double by 2040. Approximately 50% of melanomas harbor the BRAF V600E mutation, which drives aberrant cell proliferation and survival. Vemurafenib, a selective BRAF inhibitor, has demonstrated remarkable antitumor efficacy in these patients, significantly improving overall survival and median progression-free survival by six months; however, its long-term oral use is often associated with systemic toxicity and patient compliance issues. While chemotherapy and radiotherapy can eliminate tumor cells, they lack specificity and often damage surrounding healthy tissue, leading to adverse side effects. Standard therapies, including surgery and chemotherapy, can achieve durable responses, but approximately 3–13% of patients experience recurrence. These findings highlight the need for alternative or precision treatments, such as intelligent localized drug delivery platforms, to improve efficacy and reduce systemic toxicity. In addition to standard clinical diagnostic methods—such as biopsy followed by histopathological examination—emerging biomolecular technologies offer promising adjunctive tools. These molecular diagnostics not only improve detection efficiency but also have significant potential for predicting tumorigenesis, monitoring cancer progression, and assessing treatment efficacy. Microneedles (MNs) have attracted widespread attention for their applications in drug therapy, rapid diagnosis, and personalized health monitoring. These micrometer-sized needle arrays penetrate the stratum corneum and penetrate deep into the dermis, forming microchannels that enable painless sampling or drug delivery with minimal invasiveness. Recent studies have demonstrated that 3D-printed, self-locking, dissolvable microneedles loaded with microdoses of drugs are more effective at delivering anti-cancer drugs directly into melanoma tumors compared to intratumoral injection. Wearable microneedles based on plasmon- and nanozyme-catalyzed Au@Ag-Pt nanoparticles have achieved in situ noninvasive monitoring of melanoma treatment using surface-enhanced Raman scattering and colorimetric readout. However, current microneedle systems using dissolvable structures and visible phase-change polymers face difficulties in providing quantitative feedback on treatment progress. Despite the rapid advancement of microneedle technology, high-resolution systems combining therapy and real-time monitoring on a single microneedle platform remain a challenge, primarily due to customization complexity, synchronization issues with therapy and quantitative melanoma tracking, and the need for appropriate technology. While multilayered microneedles with on-demand detachment capabilities can be loaded with a variety of drugs and bioactive substances, perform interstitial fluid (ISF) extraction, and monitor therapy, a fully functionalized system of this type has yet to be realized. Summary of the Invention

[0003] In order to address the technical deficiencies of existing microneedle systems, such as difficulties in providing quantitative feedback on treatment progress and the need for high-resolution systems combining treatment and real-time monitoring on a single microneedle platform due to customization complexity, synchronization issues between treatment and quantitative tracking of melanoma, and the need for applicable technologies, the present invention provides a multi-layer detachable microneedle, a preparation method and application thereof, and a treatment collaborative monitoring platform for superficial skin diseases.

[0004] The technical solution adopted by the present invention is: a multi-layer detachable microneedle, which includes an inner rigid layer of polyethylene diacrylate (PEGDA) hydrogel for ISF sampling and dPCR detection, a middle thin layer of polyvinyl alcohol (PVA) hydrogel, and an outermost drug-loaded outer layer of gelatin methacryloyl (GelMA) hydrogel. The drug-loaded outer layer, the middle thin layer, and the rigid inner layer are all conical structures, and the three are nested in sequence to form a multi-layer microneedle structure. The drug-loaded outer layer is loaded with vemurafenib and a responsive material.

[0005] Preferably, the responsive material is black phosphorus nanosheets (BP).

[0006] Preferably, the mass percentages of polyethylene diacrylate (PEGDA) hydrogel, polyvinyl alcohol (PVA) hydrogel and gelatin methacryloyl (GelMA) hydrogel in the multi-layer detachable microneedle are 60%:15%:25%.

[0007] Preferably, the concentration of the black phosphorus nanosheets (BP) is 0.20 mg / mL.

[0008] A method for preparing multi-layer detachable microneedles, comprising the following steps:

[0009] (1) Preparation of a polyethylene diacrylate (PEGDA) hydrogel rigid inner layer: PEGDA gel prepolymer with excellent swelling properties was filled into a pyramid-shaped negative mold and then cured with UV light to form the first layer of the MN patch;

[0010] (2) Preparation of a two-layer MN patch: PVA solution was introduced into one of the pyramid-shaped negative molds, followed by placing a hollow heightening pad and the PEGDA-based MN patch prepared in step (1). The assembly was dried overnight to form a two-layer MN patch.

[0011] (3) Preparation of three-layer detachable MN: The GelMA prepolymer solution containing vemurafenib and black phosphorus nanosheets was filled into a pyramid-shaped negative mold. After removing the excess solution, the mold was combined with the hollow height-enhancing pad and two layers of MN patches, and then cured by UV light to form a three-layer detachable MN patch.

[0012] Preferably, the needle lengths of the pyramid-shaped negative mold in step (1), step (2) and step (3) are 1000 µm, 450 µm and 650 µm, respectively.

[0013] An application of the multi-layer detachable microneedle in preparing a material for controlling treatment and monitoring melanoma biomarkers.

[0014] The monitoring of melanoma biomarkers is achieved by extracting interstitial fluid (ISF) using the multi-layer detachable microneedles and combining it with digital polymerase chain reaction (dPCR).

[0015] A therapeutic collaborative monitoring platform for superficial skin diseases, comprising the multi-layer detachable microneedles, is provided. The therapeutic collaborative monitoring platform realizes drug-controlled treatment and real-time monitoring of nucleic acids and proteins through the multi-layer detachable microneedles.

[0016] Real-time monitoring of nucleic acids and proteins was achieved by ISF extraction using multilayer detachable microneedles combined with digital polymerase chain reaction (dPCR) and digital proximity ligation assay (dPLA).

[0017] The present invention provides a multilayered, detachable microneedle, its preparation method, and application, as well as a platform for collaborative monitoring of superficial skin diseases. This platform enables light-controlled drug delivery therapy via droplet PCR (dPCR) under real-time in situ monitoring conditions. The detachable microneedle consists of an innermost polyethylene diacrylate (PEGDA) extraction layer, an outer gelatin methacryloyl drug carrier layer containing vemurafenib and black phosphorus (BP), and a polyvinyl alcohol (PVA) linker layer designed for thermal release. The outer layer utilizes the photothermal properties of BP to achieve photoresponsive drug release, achieving a 78% release rate within 24 hours. Subsequently, its remarkable mechanical strength and expansion properties enable the efficient extraction of approximately 26 μL of interstitial fluid within 10 minutes. In vitro and in vivo melanoma studies have demonstrated that this platform can enhance therapeutic efficacy while minimizing systemic toxicity. It can monitor MCAM and BRAF genes, including the drug-resistant V600E polymorphism, based on dPCR with a detection limit of 223 copies / μL, and simultaneously detect protein markers IL-6, VEGF, and Ki-67 via digital proximity ligation assay (dPLA) with a detection limit of 0.64 pg / mL. This carefully designed biological system highlights its ability to interact with the pathophysiological environment, paving the way for practical real-time monitoring and personalized management of skin diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the multifunctional, detachable microneedle assembly.

[0019] Figure 2The fabrication process and morphological characterization of multilayered detachable microneedles. (A) Schematic diagram of the fabrication process of multilayered detachable microneedles. (B-C) Optical microscopy images of multilayered detachable microneedles. SEM images showing (D) a multilayered MN array and (E) a magnified view of the nested structure. (F) Confocal microscopy images of multilayered MNs, characterizing their nested structure. (G) Statistical quantification of the total height of assembled MNs. (H) Force-displacement measurements and photographs of microneedles at the critical fracture point. (I) H&E-stained image showing a MN patch inserted into the back of a BALB / c mouse. Scale bars: 200 μm in (B-D, F, H, I) and 100 μm in (E).

[0020] Figure 3 In vitro performance of a multilayer detachable microneedle (MN) patch. (A) Optical and fluorescence images at different angles show the multilayer detachable MNs before and after insertion into agarose. (B) Photothermal response curves of the MNs at 1.5 W and 2.75 W near-infrared (NIR) powers. (C) Temperature changes of the MNs at 1.5 W NIR power during five photothermal cycles. (D) Effects of BP loading and NIR on drug release from the MNs. (E) Enzymatic degradation rate of 25% GelMA hydrogels. (F) Swelling rates of PEGDA hydrogels with different concentrations. (G) Time-dependent changes in the extract quality after 60% PEGDA MNs were inserted into agarose. (H) CCK8 assay to determine the viability of NIH-3T3 cells co-cultured with PVA microneedles, PEGDA microneedles, multilayer detachable MNs, and BP-loaded MNs. Experiments were performed on days 1, 2, and 3. (I) Cytotoxicity of different concentrations of Vemurafenib on A375 cells (0.5% DMSO). Scale bar in (A) is 400 μm.

[0021] Figure 4The performance of multilayered detachable microneedles (MNs) for real-time cellular detection by combining dPCR and dPLA. (A) Diagram illustrating in vitro dPCR / dPLA analysis based on microneedle extraction. (B) Schematic diagram of the microfluidic channel structure and droplet generation process. (C) Monodisperse droplet arrays generated by varying the flow rate. (D) Fluorescence images of the designed microneedles after treatment at different time points (0, 2, 3, 4, 5, and 7 days). (E-F) Quantitative analysis based on Poisson statistics, inferring DNA concentrations (black dots) at different time points from the percentage of bright droplets (bar graph). (G) Fluorescence images of a serial dilution of IL-6 ranging from 80 pg / mL to 0.64 pg / mL detected by dPLA. (H) Percentage of bright droplets observed in serial dilutions of IL-6, VEGF, and Ki-67 in dPLA experiments. Error bars represent mean ± standard error (n = 3). (I) Linear regression analysis between extracted analyte concentrations and estimated copy numbers was used to generate standard curves for each target. Scale bars: 500 μm in panel B and 100 μm in panels D and G.

[0022] Figure 5 Multilayered, detachable microneedles used to treat mouse melanoma and extract ISF for in situ monitoring. (A) Melanoma model establishment and treatment monitoring process. (B) Tumor volume, (C) tumor images, and (D) mouse body weight changes after 14 days of various treatments. (E) H&E, TUNEL, and Ki67 staining of treated tumor tissue. (F) Monitoring curves showing changes in BRAFV600E expression over 14 days. (G) P-value significance heatmap for 14 days of continuous monitoring. Six mice per group (n = 6). Scale bar: 100 μm in panel E.

[0023] Figure 6 (A) Optical microscope image of a two-layer microneedle. (B) A three-layer microneedle with red fluorescent nanoparticles added to the middle layer, imaged using a stereofluorescence microscope. Scale bars: 400 μm in both (A) and (B).

[0024] Figure 7 Scanning electron microscope (SEM) images of the various layers of a multilayered, detachable microneedle. (A) SEM image of a microneedle with a polyethylene glycol diacrylate (PEGDA) layer. (B) SEM image of a microneedle with a glycidyl methacrylate-modified gelatin (GelMA) layer. (C) SEM image of a microneedle with a polyvinyl alcohol (PVA) layer. Scale bars: 10 μm in (A), (B), and (C).

[0025] Figure 8(A) Step-by-step preparation of separable microneedles using a mold-assisted method: (i) Customized negative molds for each of the three microneedle layers and spacers for height adjustment; (ii) Sequential addition of prepolymer solution followed by vacuum degassing; (iii) UV curing. (B) Representative images of multiple batches of microneedles with identical dimensions, along with statistical quantification of the height of the innermost microneedle layer. (C) Incomplete morphology of the inner layer due to insufficient PEGDA concentration or insufficient UV curing time. (D) Blurred morphology of the outer layer, resembling melted ice cream, is caused by low polymer concentration or inadequate curing conditions.

[0026] Figure 9 Hematoxylin and eosin (H&E)-stained images of a cross-section of the microneedle patch inserted into the back of a mouse. Scale bar: 2 mm.

[0027] Figure 10 The temperature change curves of microneedles loaded with different concentrations of BP under 1.5 W near-infrared light.

[0028] Figure 11 (a) UV-Vis-NIR spectroscopy of vemurafenib, showing absorbance over a concentration range of 4–19 μg / mL. (b) The established standard curve correlating drug concentration with absorbance exhibits a good linear relationship.

[0029] Figure 12 Agarose gel electrophoresis images of melanoma cell adhesion molecule (MCAM). A375 and 3T3 cells represent human and mouse cell lines, respectively. N is a negative control without DNA template.

[0030] Figure 13 Figure 3 shows the Sanger sequencing results of A375 and human umbilical vein endothelial cells (HUVEC). The mutation points are marked with red boxes.

[0031] Figure 14 (A) Fluorescence images of BRAF V600E DNA serially diluted from 10-fold to 104-fold using droplet digital polymerase chain reaction (dPCR). (B) Fluorescence intensity readings of droplets of BRAF V600E DNA serially diluted from 10-fold to 104-fold using a commercial dPCR instrument (QuantStudio, Thermo Fisher Scientific). (C) Back-to-back comparison of dPCR results using a commercial dPCR instrument and a dPCR protocol developed in our laboratory, and (D) linear fit curves. N represents a negative control using water as the template. Scale bar: 100 μm in (A).

[0032] Figure 15Thermal images of multi-layer detachable microneedles applied to the back of a mouse before and after 2 minutes of 1.5-watt near-infrared light irradiation.

[0033] Figure 16 Optical images of the mouse back at different time points after removal of the microneedle patch. Scale bar: 5 mm.

[0034] Figure 17 (A) Photographs of mouse red blood cell suspensions (i) and hemolysis rates (ii) after microneedling with (1) positive control, (2) negative control, (3) polyvinyl alcohol (PVA), (4) glycidyl methacrylate-modified gelatin (GelMA), and (5) polyethylene glycol diacrylate (PEGDA) + polyvinyl alcohol (PVA) + glycidyl methacrylate-modified gelatin (GelMA). (B) Hematoxylin-eosin (H&E)-stained images of major mouse organs after different treatments. Scale bar: 100 μm in (B). DETAILED DESCRIPTION

[0035] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0036] Example 1 Preparation, morphology and mechanical properties of multilayer detachable microneedles

[0037] Multilayer detachable microneedles (MNs) were fabricated using layer-by-layer replication technology and different templates ( Figure 2 A). First, a PEGDA gel prepolymer with excellent expansion properties was filled into a pyramid-shaped negative mold with a needle length of approximately 1000 µm, and then cured with ultraviolet light (UV) to form the first layer of the MN patch. Next, a PVA solution was introduced into a similar mold with a needle length of 450 µm, followed by a hollow heightening pad and a prefabricated PEGDA-based MN patch. The assembly was dried at 37°C overnight to form a two-layer MN patch. Finally, a GelMA prepolymer solution containing Vemurafenib and BP was filled into a pyramid-shaped negative mold with a needle length of 650 µm. After removing the excess solution, the mold was combined with the hollow heightening pad and two layers of MN, and then cured by UV light to form a three-layer detachable MN patch ( Figure 2 B, 2C and Figure 6 ).

[0038] Scanning electron microscopy (SEM) images confirmed the successful preparation of a three-layer MN structure, with each layer consisting of a different hydrogel matrix and having different properties ( Figure 2 D, 2E and Figure 7 During the fabrication process, confocal microscopy measurements using fluorescent nanoparticles of different colors revealed a uniform three-layer MN structure with consistent interlayer spacing between the PEGDA and PVA layers and uniform nesting between the PEGDA and GelMA layers ( Figure 2 Figure S3 shows bright-field microscopy images of the step-by-step preparation process and samples from different batches. The inter-batch variation in MN height and layer thickness was quantitatively analyzed ( Figure 2 G and Figure 8 The average total and tip lengths of the MNs were 1279 μm and 1059 μm, respectively, with standard deviations ranging from 24 to 32 μm. Consistent and high-quality MN patches were successfully obtained in all replicate preparations, demonstrating the reproducibility and scalability of the fabrication process.

[0039] This three-layer design can effectively and continuously release drugs into the deep layers of the skin while effectively extracting interstitial fluid (ISF) for monitoring. To ensure successful percutaneous puncture, the mechanical strength of the multilayer MN patch was evaluated using an electronic universal testing machine. During the test, the needle tip was facing upward and the pressure sensor moved vertically downward. Analysis of the force-displacement curve revealed two platform structures: the first platform corresponds to the rupture of the 25% GelMA and 15% PVA layers, and the second platform corresponds to the rupture of the 60% PEGDA layer ( Figure 2 H). These results confirm that MNs have sufficient mechanical strength and puncture ability to effectively penetrate the skin. H&E-stained images of sagittal and cross-sectional sections of mouse dorsal skin further demonstrate that the patch successfully formed microchannels within the skin ( Figure 2 I and Figure 9 Compared to traditional sagittal sections, cross-sections of the loose connective tissue beneath the stratum corneum are more difficult to prepare, resulting in a localized tear pattern. Each MN was tested as an independent unit, and the high reproducibility observed was statistically significant.

[0040] Example 2: In vitro evaluation of microneedle performance

[0041] The effective detachability of multilayer microneedles is crucial for the proper function of each layer. To prevent the problems of adhesion and delayed detachment in the double-layer MN design, we developed a three-layer structure and selected polyvinyl alcohol (PVA) as the intermediate connecting layer. This choice was mainly based on the biocompatibility, water solubility and mechanical strength of PVA. PVA is a highly hydrophilic thermosensitive polymer that has been widely studied and applied in the biomedical field, especially in the development of dissolving microneedle drug delivery systems. Through literature review and preliminary experiments, we evaluated the hardness and dissolution rate of PVA and other thermosensitive matrices (such as gelatin-based microneedles) in aqueous solution. The results showed that PVA has excellent hardness and rapid solubility, making it suitable as a connecting layer material. To evaluate this, three-layer MNs containing green fluorescent nanoparticles were inserted into 1.4% agarose gel and removed after 10 minutes. Fluorescence microscopy confirmed that the GelMA layer remained intact in the agarose ( Figure 3 A). The PVA hydrogel component dissolves rapidly upon contact with interstitial fluid due to its low content and temperature-sensitive phase transition properties. In addition, the viscosity of a 15 wt% PVA solution is suitable for maintaining the sharpness of the needle tip, while higher concentrations of PVA hinder the formation of the needle tip. Through near-infrared (NIR) irradiation, BP increases the local temperature, accelerates the separation between the PEGDA and GelMA layers, and triggers the controlled release of Vemurafenib. At a BP concentration of 0.20 mg / mL and an NIR power of 1.5 W, the patch reached approximately 48°C within 2 minutes, remaining below the thermal damage threshold ( Figure 3 B and Figure 10 The short exposure time was not sufficient to cause photothermal damage or skin burns, as temperatures exceeding 53°C are usually required to produce these effects. The photothermal activation and cooling performance of the MN patch remained consistent over five repeated cycles, demonstrating its stability and reusability under NIR stimulation ( Figure 3 C).

[0042] The cumulative release of Vemurafenib was quantitatively determined by UV-vis-NIR spectroscopy. Based on its characteristic absorption peak at approximately 270 nm and the established standard curve, the relationship between drug concentration and absorbance was ( Figure 11 In the NIR irradiation group, which was irradiated for 10 minutes per hour, 78% of the drug was released after 24 hours, compared to 63% in the non-irradiated group ( Figure 3D). In vitro release studies further confirmed that the presence or absence of BP had no significant effect on drug loading efficiency or cumulative release. In addition, the GelMA-based drug-loaded layer remained within the skin for several days, providing controlled and sustained drug release, with drug release reaching almost 100% because the layer completely degraded within the skin and did not need to be removed. This design also allows for the customization of drug loading according to specific therapeutic needs for future clinical applications. In degradation studies, the biocompatible GelMA hydrogel showed a degradation rate of over 80% after incubation with type II collagenase in PBS for 15 days ( Figure 3 E), indicating that enzymatic degradation and NIR irradiation are key regulators of drug release.

[0043] A significant limitation of existing ISF sampling platforms is their low sampling volume in a short period of time, which affects the accuracy of detection. To address this issue, the ISF extraction substrate must strike a balance between high swelling capacity and sufficient mechanical strength to maximize fluid absorption and maintain structural integrity during insertion and removal from the skin. PEGDA is a promising candidate material that has been widely reported in previous studies given its hardness and swelling properties. Hydrogels with significant swelling properties, such as gelatin, or lower concentrations of PEGDA, although they can provide enhanced swelling, often lack similar mechanical support or are fragile. To determine the optimal formulation, the present invention studied the swelling behavior and morphological stability of PEGDA hydrogels with different concentrations when incubated with type II collagenase in PBS and found that the PEGDA concentration was inversely proportional to the swelling rate ( Figure 3 F). 50% PEGDA swells excessively, causing deformation and cracking, making it unsuitable for the extraction process. Therefore, the present invention selected 60% PEGDA as the extraction layer. When the 60% PEGDA MN patch was tested in a 1.4% agarose gel at a physiological temperature of 37°C, it was found that it extracted 26.3 μL of liquid ( Figure 3 G). This volume is sufficient for downstream molecular analysis, and sampling can be further increased by expanding the effective application area of ​​the MN. Taken together, these findings demonstrate the great potential of PEGDA hydrogels for minimally invasive ISF sampling and highlight the overall utility of multilayered MN patches as integrated diagnostic and therapeutic platforms.

[0044] Dispersing BP into nanosheets significantly reduced its toxicity. Experiments using NIH-3T3 cells in the present invention also confirmed that BP nanosheets are less toxic than BP powder ( Figure 3H). The cells showed normal proliferation and morphology, as confirmed by CCK-8 assays and live / dead staining. The BP loading in the drug layer at the microneedle tip was in the microgram range, sufficient to ensure responsiveness without detectable toxicity. There was no significant difference in cell viability compared to the untreated control group, further supporting the biosafety of the MN patch. The present invention also evaluated the cytotoxicity of the patch against the human melanoma cell line A375 ( Figure 3 I). Vemurafenib concentrations ranging from 0 to 30 μg / mL were tested to determine the half-maximal inhibitory concentration (IC50), and the results support a drug loading capacity of 20 μg / mL (equivalent to 20 mg / kg) for MNs. Based on preliminary experiments, this dose demonstrated significant tumor killing.

[0045] Example 3: Cellular level monitoring

[0046] To achieve real-time monitoring of drug response in A375 cells, drug-loaded multilayer microneedles were introduced into the cell culture wells at intervals of 0, 2, 3, 4, 5, and 7 days, each lasting 0.5 hours. This experiment was designed to evaluate the real-time monitoring capability of microneedles combined with dPCR and to characterize the detection limit and linearity in the cell model ( Figure 4 A). The BRAF V600E gene mutation is a key biomarker for malignant melanoma and has attracted widespread attention due to its role in the MAPK signaling pathway. At the same time, the high expression of melanoma cell adhesion molecule (MCAM) in melanoma cells is also an important marker. These two biomarkers are quantitatively detected by dPCR, which can sensitively measure tumor progression and reflect changes in the number of extracted tumor cells by analyzing the abundance of genomic DNA (gDNA) fragments. In order to accurately simulate the changes in the active tumor cell population during treatment, the nucleic acid extraction protocol was designed to exclude the contribution of lysed or apoptotic cells. The protocol involves using DNAse to degrade free DNA, using proteinase K to inactivate DNAse, and then lysing living cells to purify intracellular nucleic acids. This sequential approach ensures that dPCR results only reflect nucleic acids associated with living cells, thereby accurately quantifying the abundance of tumor cells during treatment. Two TaqMan probes carrying FAM and Cy5 fluorescent groups, respectively, were designed to detect target gDNA and related single nucleotide polymorphisms (SNPs) ( Figure 12 The SNP TaqMan probe is complementary to the BRAF V600E mutation, and the Cy5 fluorophore will emit light only when fully hybridized to the mutant allele, but not in the presence of the wild-type sequence ( Figure 13 ).

[0047] Sensitive dPCR technology monitors melanoma biomarkers by quantitatively detecting changes in target tumor DNA copy number. A microfluidic chip with a flow focusing structure is used to prepare uniform microdroplets ( Figure 4 B). The water-oil emulsion system allows precise control of the droplet diameter by adjusting the flow rate ratio, resulting in droplets of 66, 90, and 107 μm ( Figure 4 C). For this study, 66 μm droplets were selected, containing approximately 165,000 droplets per 25 μL reaction. Total DNA from cells in the PEGDA layer was extracted, purified, and mixed with PCR reagents to form the inner aqueous phase of the emulsion. The specific primers and TaqMan probe sequences used in this study are detailed in Table S1 below. After PCR thermal cycling, the proportion of fluorescent droplets was quantified. At different time points during microneedle treatment, the droplets were imaged using confocal microscopy ( Figure 4 D). Fluorescent droplets (PCR (+)) indicate successful amplification of the target DNA, while non-fluorescent droplets (PCR (-)) indicate that the target template was not detected. The absolute copy number is calculated using Poisson statistics based on the proportion of PCR (+) droplets to determine the concentration of tumor cells. By knowing the population size of the emulsion droplets, the gDNA copy number can be directly calculated without the need for a standard curve or control gene reference, providing a simple and immediate readout. For example, on day 0 of microneedle treatment, red fluorescent droplets targeting the BRAF V600E mutation accounted for 61.5% of the total droplets, corresponding to 1.58 × 10^5 copies and 0.96 copies per droplet. By day 5, only 25.6% of the droplets were fluorescent, reflecting 4.79 × 10^4 copies and 0.29 copies per droplet ( Figure 4 D). These results indicate that nucleic acid copy number and tumor cell concentration gradually decreased during treatment ( Figure 4 E and 4F). Although dPCR can achieve absolute quantification, standard curves can still serve as a useful reference for tracking specific biomarkers, especially when combined with microneedle-based sampling platforms.

[0048] Table S1. Primer and molecular probe design for detecting MCAM and BRAF V600E

[0049]

[0050] In addition to nucleic acid biomarkers, protein indicators such as IL-6, VEGF, and Ki-67 were also tested. IL-6, as an immunoregulatory cytokine, is a key regulator of angiogenesis and cell proliferation, respectively, along with VEGF and Ki-67, and is closely related to tumorigenesis. It is an important marker for the treatment of melanoma. dPLA quantifies protein targets by amplifying oligonucleotides from DNA proximity probes that bind to the target. The pretreatment modifications of protein molecules and linker oligonucleotide sequences followed established protocols. The eluate extracted from the microneedle-treated cell model was serially diluted from 80 to 0.64 pg / mL and subjected to dPLA analysis. Confocal fluorescence microscopy showed that the proportion of bright droplets increased with increasing eluate concentration ( Figure 4 G). Notably, compared with IL-6, the expression levels of VEGF and Ki-67 were 8.5-fold and 13.1-fold lower, respectively ( Figure 4 H). Standard curves for all three dPLA assays, constructed using fluorescent droplet counting and Poisson statistics, showed excellent linearity ( Figure 4 I). These results contribute to the comprehensive molecular characterization of tumor response. The combination of multi-layer detachable microneedles and dPCR technology is particularly suitable for in situ micro-sample detection. This method meets the needs of small-volume sampling, convenience and high sensitivity, providing a painless and minimally invasive method for monitoring superficial tumors. This integration goes beyond traditional PCR technology, and the research in this invention demonstrates its feasibility and adaptability in practical applications. The overall detection limit of the MN system is 223 copies / μL, which includes not only the nucleic acid quantification process based on dPCR technology, but also covers ISF extraction, cell lysis and nucleic acid purification steps performed by the detachable microneedle system. Therefore, this value represents the effective detection limit of tumor-derived nucleic acids using our complete microneedle workflow. To verify this result, we performed a DNA template dilution experiment to directly compare the performance of dPCR with quantitative real-time PCR (qPCR). At 104-fold dilution, qPCR failed to distinguish the target from the negative control, while dPCR accurately quantified the DNA and clearly distinguished it from the blank ( Figure 14 A).

[0051] Regarding dPCR technology alone, whether using commercial instruments or laboratory-developed workflows, the detection limits are generally comparable, improving by 1-2 orders of magnitude compared to qPCR. We performed a series of template dilution experiments, comparing our proprietary dPCR workflow to commercial dPCR methods ( Figure 14). Both successfully detected 29-44 copies / μL of DNA, demonstrating similar performance. However, the overall detection limit of the microneedle-based system was higher than that of pure dPCR. This difference was attributed to losses incurred during steps such as cell lysis and nucleic acid purification. To further evaluate detection consistency, we generated three commonly used dPCR droplet sizes—56 μm, 78 μm, and 100 μm. Based on Poisson distribution calculations, template quantification values ​​inferred from the proportion of fluorescent droplets were consistent for samples with the same copy number.

[0052] Example 4: In vivo theranostics via established microneedles

[0053] To verify the in vivo sensitivity of therapeutic monitoring, a subcutaneous xenograft model of A375 melanoma cells was established to evaluate the dual functionality of the multilayer microneedle system for both therapeutic and biomarker detection. Figure 5 As shown in A. Model mice were randomly divided into five groups: control group, microneedle-only group, microneedle plus near-infrared radiation group (MN+NIR), microneedle loaded with vemurafenib group (MN+VE), and MN+VE+NIR group. Multilayer microneedles were successfully inserted into the back skin, and under near-infrared irradiation, the temperature of the application site reached approximately 48°C ( Figure 15 Notably, the patch could be worn for hours without causing significant discomfort or requiring auxiliary equipment (such as an indwelling needle). The insertion site healed within 30 minutes, demonstrating excellent biocompatibility and approximately 90% needle penetration efficiency ( Figure 16 Vemurafenib alone or in combination with NIR resulted in varying degrees of tumor inhibition. The MN+VE+NIR group showed the most significant tumor inhibition compared to the MN+VE group ( Figure 5 B), which is attributed to the enhanced release of drugs under near-infrared stimulation. After 14 days, the tumor volume and weight of the MN+VE+NIR group were the smallest ( Figure 5 C). The mice in all groups gained weight normally, and H&E staining showed no significant histopathological abnormalities in major organs, further confirming the biocompatibility of the microneedles ( Figure 5 D and Figure 17 Histological analysis of H&E, TUNEL, and Ki67 staining showed that the MN+VE+NIR group exhibited the most significant tissue necrosis, the highest apoptotic activity, and the lowest proliferation rate ( Figure 5 E), indicating that it has excellent therapeutic effects.

[0054] The relative biomarker expression was quantitatively analyzed by dPCR. The dPCR detection curve showed that the Cy5-labeled BRAF V600E level in the MN+VE+NIR group fluctuated minimally throughout the treatment period. In contrast, the BRAF V600E level in the control group and the microneedle-only group increased significantly, demonstrating the high precision and sensitivity of the multilayer microneedle drug delivery system ( Figure 5 F). P-value significance heatmap shows no significant difference in melanoma progression between the MN-only and NIR-only groups. However, on the sixth day of treatment, the MN+VE+NIR group showed a significant tumor inhibition effect compared to the control group ( Figure 5 G). These results are consistent with studies of the efficacy of vemurafenib in melanoma and highlight the use of genetic markers in guiding treatment decisions.

[0055] Furthermore, the results highlight the capabilities of the engineered microneedles for spatiotemporally controlled drug release and real-time therapeutic monitoring. By utilizing the sampling layer for continuous biomarker monitoring, drug dosage, release timing, and frequency can be dynamically adjusted, thereby establishing a closed-loop system for drug administration and long-term monitoring. This system has strong translational potential and could advance personalized melanoma management.

[0056] Summarize

[0057] The present invention provides a multifunctional multi-layer detachable microneedle platform that integrates real-time monitoring of nucleic acids and proteins with light-controlled personalized drug therapy (see Figure 1Digital polymerase chain reaction (dPCR) is a cutting-edge detection method that partitions a reaction solution into millions of monodisperse reaction chambers. It offers significant advantages over traditional qPCR, including absolute quantification, high throughput, high sensitivity, ease of multiplexing, high signal-to-noise ratio, and compatibility with miniaturized integration. As a portable trace tracking method, dPCR has shown promise for monitoring DNA levels after melanoma treatment and can be extended to protein detection via digital proximity ligation assays (dPLA), enabling precise quantification and a streamlined "sample-to-digital result" workflow. This study demonstrates the first integration of a custom microneedle platform with dPCR for real-time, in situ monitoring of molecular profiling and therapeutic response. To our knowledge, this approach is most relevant to previous studies utilizing nanostructure-assisted microneedles for nucleic acid sequence recognition. Black phosphorus (BP) nanosheets are versatile photothermal materials with excellent near-infrared (NIR) responsiveness and biocompatibility, making them ideal for tumor photothermal therapy, phosphorus-mediated therapy, and drug delivery. NIR lasers are widely used in clinical practice due to their safety at low power, particularly for photothermal therapy of superficial tumors. The present invention incorporates BP into the drug loading layer of a multi-layer microneedle structure, enabling precise controlled-release therapy, and the release schedule can be dynamically adjusted based on the molecular diagnostic results obtained from the extracted sample. The small sample size and real-time monitoring capabilities of the microneedles, combined with the ability of dPCR to capture trace markers in microsamples, can construct a comprehensive melanoma diagnosis and treatment system. Here, the present invention adopts a template replication strategy to prepare multi-layer detachable hydrogel microneedles for light-controlled drug delivery and subsequent ISF sampling, and integrates dPCR technology to monitor the progress of melanoma treatment (see Figure 1The outermost layer, composed of gelatin methacryloyl (GelMA), is loaded with vemurafenib and a responsive material for controlled therapy. A thin middle layer, composed of polyvinyl alcohol (PVA), rapidly dissolves upon contact with interstitial fluid, while the innermost layer, composed of polyethylene glycol diacrylate (PEGDA), is a rigid material and allows for on-demand sampling and dPCR detection. Due to the degradability of GelMA and the swelling properties of PEGDA, the dual-functional microneedles exhibit sustained drug release and efficient ISF extraction. Cell-based studies demonstrated that incorporation of BP into the GelMA layer enabled controlled drug delivery. These microneedles promoted apoptosis in melanoma (A375) cells and, under NIR irradiation, simultaneously monitored BRAF V600E levels using dPCR and interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and Ki-67 levels using a digital proximity ligation assay (dPLA). Furthermore, in vivo experiments confirmed that drug-loaded microneedles under NIR irradiation selectively targeted melanoma tumors, reduced systemic toxicity, promoted cell apoptosis, and simultaneously monitored melanoma growth inhibition. These findings highlight the potential of multilayered detachable microneedles combined with dPCR, supporting the advancement of intelligent clinical treatment platforms for skin diseases.

[0058] This invention provides a versatile, multi-layered, detachable microneedle platform that combines light-controlled therapy with dPCR and dPLA monitoring, advancing the development of personalized microneedle-based melanoma management. The invention provides a smart, three-layered, detachable microneedle structure with the dual functions of controlled therapy and ISF extraction for follow-up monitoring. Vemurafenib, controlled by near-infrared light, has successfully treated cutaneous melanoma with superior targeting and minimal toxicity. The microneedles extracted 26.3 µL of ISF within 10 minutes, enabling dPCR-based quantitative monitoring of MCAM and BRAF V600E during treatment. Microneedle-based, in situ, real-time detection is ideally suited for portable point-of-care diagnostic systems, particularly when combined with biochemical techniques like dPCR, which can analyze trace samples collected from microliter volumes. By using highly sensitive molecular techniques to detect trace substances in microvolume samples and expanding validation of protein markers, dPLA detection limits as low as 0.64 pg / mL were achieved for IL-6, VEGF, and Ki-67.

[0059] This multilayered, detachable microneedle platform, combined with a dPCR platform, effectively monitored melanoma treatment in a mouse model, providing precise quantitative assessment of BRAF V600E and other targets with higher resolution than tumor volume changes and other macroscopic phenotypes. Compared to other microneedle platforms, this invention not only solves the problem of preparing single-function microneedles and microneedles with complex functions, significantly reducing production costs, but also allows for diversification and functional customization based on the purpose of detection. Its needle tip size, penetration depth, drug dosage, and release profile can be tailored to the characteristics of the tumor or wound. This research provides valuable insights into the design of integrated microneedles for diagnosis and treatment.

[0060] The present invention does not consider analyzing serum samples because IL-6 levels in serum primarily reflect systemic inflammation, which may not accurately represent local immune activity at the melanoma site. In contrast, liquid samples collected directly from the tumor site via microneedles could provide a more relevant and accurate indicator of treatment progress. ELISA is known to lack sensitivity when detecting low-abundance targets and is limited by nonspecific binding, complex wash steps, and labor-intensive procedures. In contrast, numerous studies have demonstrated that PLA improves detection limits by at least two orders of magnitude compared to ELISA. Furthermore, ELISA relies on standard curves and is incompatible with small-volume or in situ sampling, making it unsuitable for minimally invasive, milliliter-scale sampling applications like the microneedle system of the present invention. The droplet dPLA demonstrated in the present invention is a uniform, wash-free immunoassay with high sensitivity, resolution, and quantitative capabilities. It is not only well-suited for detecting low-concentration biomarkers in microscale samples, but can also capture subtle changes in copy number variation—up to fivefold. In summary, dPLA is highly compatible with the microneedle system of the present invention and offers analytical advantages that cannot be replaced by ELISA.

Claims

1. A multi-layer detachable microneedle, characterized in that: The multi-layer detachable microneedle includes an inner rigid layer of polyethylene diacrylate (PEGDA) hydrogel for ISF sampling and dPCR detection, an intermediate thin layer of polyvinyl alcohol (PVA) hydrogel, and an outermost drug-loaded outer layer of gelatin methacryloyl (GelMA) hydrogel. The drug-loaded outer layer, the intermediate thin layer, and the rigid inner layer are all conical structures, and the three are nested in sequence to form a multi-layer microneedle structure. The drug-loaded outer layer is loaded with vemurafenib and a responsive material. The responsive material is black phosphorus nanosheets (BP). The mass percentages of polyethylene diacrylate (PEGDA) hydrogel, polyvinyl alcohol (PVA) hydrogel, and gelatin methacryloyl (GelMA) hydrogel in the multi-layer detachable microneedle are 60%:15%:25%.

2. The multi-layer detachable microneedle according to claim 1, wherein The concentration of the black phosphorus nanosheets (BP) is 0.20 mg / mL.

3. A method for preparing the multi-layer detachable microneedle according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of a polyethylene diacrylate (PEGDA) hydrogel rigid inner layer: PEGDA gel prepolymer with excellent swelling properties was filled into a pyramid-shaped negative mold and then cured with UV light to form the first layer of the MN patch; (2) Preparation of two-layer MN patch: PVA solution was introduced into one of the pyramid-shaped negative molds, followed by placing a hollow heightening pad and the PEGDA-based MN patch prepared in step (1) and drying overnight to form a two-layer MN patch; (3) Preparation of three-layer detachable MN: The GelMA prepolymer solution containing vemurafenib and black phosphorus nanosheets was filled into a pyramid-shaped negative mold. After removing the excess solution, the mold was combined with the hollow height-enhancing pad and two layers of MN patches, and then cured by UV light to form a three-layer detachable MN patch.

4. The preparation method according to claim 3, wherein The needle lengths of the pyramid-shaped negative mold in steps (1), (2) and (3) are 1000 µm, 450 µm and 650 µm respectively.

5. Use of the multi-layer detachable microneedle according to claim 1 in preparing a material for controlling the treatment of skin melanoma and monitoring biomarkers of skin melanoma.

6. The use according to claim 5, characterized in that The monitoring of melanoma biomarkers is achieved by extracting interstitial fluid (ISF) using the multi-layer detachable microneedles and combining it with digital polymerase chain reaction (dPCR).

7. A collaborative monitoring platform for the treatment of superficial skin diseases, characterized by: The superficial skin disease is skin melanoma, and the treatment collaborative monitoring platform includes the multi-layer detachable microneedle according to claim 1. The treatment collaborative monitoring platform realizes drug-controlled treatment and real-time monitoring of nucleic acids and proteins through the multi-layer detachable microneedle.

8. The treatment collaborative monitoring platform for superficial skin diseases according to claim 7, characterized in that: Real-time monitoring of nucleic acids and proteins was achieved by ISF extraction using multilayer detachable microneedles combined with digital polymerase chain reaction (dPCR) and digital proximity ligation assay (dPLA).

Images