A dissolvable microneedle patch for promoting achilles tendon injury repair

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a soluble microneedle patch for promoting repair of achilles tendon injury. The soluble microneedle patch comprises a substrate and soluble microneedles arranged in an array on the surface of the substrate, wherein the soluble microneedles are loaded with biomimetic targeting particles of a core-shell structure, the shell of the biomimetic targeting particles is a cell membrane vesicle derived from achilles tendon cells, and the core is an active oxygen responsive nanoparticle loaded with Purmorphamine. In an achilles tendon injury mouse model, the biomimetic targeting nanoparticles in the microneedle patch can be effectively enriched and long-term retained at the injury site, and can respondively release drugs in a high active oxygen environment, thereby promoting synthesis of type I collagen, inhibiting deposition of type III collagen, reducing the degree of heterotopic ossification in the achilles tendon injury area, and finally promoting repair of the achilles tendon injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a soluble microneedle patch for promoting the repair of Achilles tendon injuries. Background Technology

[0002] Achilles tendon injury is a common musculoskeletal injury, and its repair process is often hindered by both oxidative stress and immune microenvironment imbalance. On the one hand, excessive accumulation of reactive oxygen species (ROS) accelerates tendon cell apoptosis and inhibits collagen synthesis; on the other hand, infiltration of pro-inflammatory M1 macrophages further exacerbates local inflammation, thus hindering normal tissue repair. In terms of treatment strategies, soluble microneedles have been introduced to overcome the problems of poor targeting, low bioavailability, and tissue damage risk associated with traditional local injections. However, existing microneedle systems rely primarily on passive diffusion after drug release, lacking active targeting capabilities to the Achilles tendon tissue. This results in most drugs being taken up by non-targeted tissues or entering systemic circulation, reducing local treatment efficiency and potentially causing systemic side effects.

[0003] The Hedgehog signaling pathway plays a crucial role in animal development, participating not only in the directed differentiation of cells and organogenesis during embryonic development but also in maintaining stem cell function and tissue homeostasis during adulthood. Studies have shown that activating this pathway can effectively promote the repair of Achilles tendon injuries. Its core mechanism of action lies in two aspects: firstly, activation significantly induces tendon cell proliferation and directed migration to the injury site, laying a sufficient cellular foundation for tissue regeneration of the damaged Achilles tendon; secondly, activation effectively promotes the synthesis and orderly deposition of key extracellular matrix components such as type I collagen and proteoglycans, ultimately achieving effective restoration of the Achilles tendon's structural strength and mechanical properties. However, current treatments often focus on single-faceted approaches such as anti-oxidation or immunomodulation, making it difficult to achieve perfect functional repair. Furthermore, platform-based treatment strategies that target and regulate the Hedgehog signaling pathway are still relatively scarce in existing technologies. Summary of the Invention

[0004] To address the two major problems of poor drug targeting and insufficient attention to the Hedgehog signaling pathway, which regulates endogenous tissue regeneration, in existing microneedle drug delivery systems for Achilles tendon injury treatment, this invention provides a soluble microneedle patch for promoting Achilles tendon injury repair. The soluble microneedle patch comprises a base and soluble microneedles arranged in an array on the base surface. Each soluble microneedle is loaded with core-shell biomimetic targeting particles. The outer shell of the biomimetic targeting particles is a cell membrane vesicle derived from Achilles tendon cells, and the core is a ROS-responsive nanoparticle loaded with Purmorphamine. In a mouse model of Achilles tendon injury, the biomimetic targeting nanoparticles in the microneedle patch can effectively accumulate and remain long-term at the injury site, and responsively release drugs in a high ROS environment, thereby promoting type I collagen synthesis, inhibiting type III collagen deposition, reducing the degree of ectopic ossification in the Achilles tendon injury area, and ultimately achieving Achilles tendon repair.

[0005] Based on the above findings, the present invention provides the following technical solution: In a first aspect, the present invention provides a soluble microneedle patch for promoting the repair of Achilles tendon injuries: The soluble microneedle patch includes a substrate and soluble microneedles arranged in an array on the surface of the substrate; The soluble microneedles are loaded with biomimetic targeting particles with a core-shell structure. The outer shell of the biomimetic targeting particles is a cell membrane vesicle derived from Achilles tendon cells, and the core of the biomimetic targeting particles is a reactive oxygen species responsive nanoparticle. The reactive oxygen species responsive nanoparticles include a support and Purmorphamine dispersed in the support. The support is a block copolymer with the following structural formula:

[0006] Where m is 150~160, n is 150~160, and x is 110~120.

[0007] In conjunction with the first aspect, in some embodiments, the soluble microneedles are designed with conical or pyramidal tips, the axis of which is perpendicular to the substrate, the tips are made of soluble biocompatible material, the height of a single needle is 550-650 μm, and the diameter of the bottom of a single needle is 300-800 μm.

[0008] In conjunction with the first aspect, in some implementations, the particle size of the biomimetic targeting particles is 100~600 nm.

[0009] In conjunction with the first aspect, in some implementations, Achilles tendon injury includes one or more of the following characteristics: (1) Heterotopic ossification of the Achilles tendon; (2) Disorders of collagen synthesis in the Achilles tendon tissue.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned soluble microneedle patch, comprising: Provide reactive oxygen species responsive nanoparticles loaded with Purmorphamine; Cell membrane vesicles derived from Achilles tendon cells are wrapped around the surface of reactive oxygen species-responsive nanoparticles to form biomimetic targeted particles. The biomimetic targeting particles were dispersed in a first hydrosol to obtain a precursor fluid. The precursor fluid is filled into the microcavity area of ​​the microneedle mold, and then the base area is filled with a second hydrosol. After drying, the mold is demolded to obtain a soluble microneedle patch. The first hydrosol includes hyaluronic acid; the second hydrosol includes hyaluronic acid.

[0011] In conjunction with the second aspect, in some embodiments, providing reactive oxygen species-responsive nanoparticles includes: A reactive oxygen species responsive carrier was prepared by means of methoxy polyethylene glycol-N-hydroxysuccinimide ester, dimethyl thioketal diamine, lactide and glycolide as raw materials, through amidation, ring-opening polymerization and end-group activation coupling reaction. Purmorphamine was encapsulated using this reactive oxygen species (ROS) responsive carrier to obtain ROS responsive nanoparticles.

[0012] In conjunction with the second aspect, in some embodiments, the concentration of Purmorphamine in the precursor fluid is 15-25 mg / mL.

[0013] Thirdly, the present invention provides the application of the above-mentioned soluble microneedle patch in the preparation of a drug for promoting the repair of Achilles tendon injuries.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention applies Purmorphamine to the treatment of Achilles tendon injuries. Western blot analysis confirmed that the smoothed receptor agonist Purmorphamine can effectively promote the synthesis of type I collagen in tendon cells under inflammatory stimulation and inhibit the overexpression of type III collagen, thereby promoting the transformation of the extracellular matrix from scar-related repair to functional repair.

[0015] 2. The microneedle patch provided by this invention uses "ROS-responsive nanoparticles encapsulated in cell membrane vesicles derived from Achilles tendon cells" as the treatment unit. The cell membrane vesicle shell derived from Achilles tendon cells endows the ROS-responsive nanoparticles with homologous targeting ability to Achilles tendon tissue, enabling them to actively accumulate at the lesion site. The nanoparticle core uses ROS-responsive material as a carrier, which can intelligently trigger the release of Purmorphamine in the high ROS microenvironment of Achilles tendon injury. The released Purmorphamine can promote the activation, proliferation, and functional differentiation of tendon stem cells by activating the Hedgehog pathway.

[0016] 3. In a mouse model of Achilles tendon injury, the biomimetic targeted nanoparticles in the microneedle patch provided by this invention can effectively accumulate and remain in the injury site for a long time, and accelerate drug release in a high ROS environment, thereby promoting type I collagen synthesis, inhibiting type III collagen deposition, and ultimately significantly reducing the degree of heterotopic ossification in the injury area. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The effect of Purmorphamine on the protein expression of type I and type III collagen in tendon cells under inflammatory stimulation: The control group consisted of primary rat tendon cells; the inflammatory stimulation group was treated with 10 ng / mL IL-1β and 10 ng / mL TNF-α in addition to the control group; and the Purmorphamine treatment group was treated with 1 μM Purmorphamine in addition to the inflammatory stimulation group.

[0019] Figure 2 Synthetic route and 1H NMR spectrum of ROS-responsive PLGA; where: (A) Synthetic route of ROS-responsive PLGA; (B) 1H NMR spectrum of ROS-responsive PLGA; where m is the average degree of polymerization of glycolide unit, n is the average degree of polymerization of lactide unit, and x is the average degree of polymerization of ethylene oxide unit.

[0020] Figure 3Characterization of the physicochemical properties and drug release performance of TM-RRP-Pur NPs; including: (A) Particle size detection of RRP-Pur NPs (left) and TM-RRP-Pur NPs (right); (B) Zeta potential detection diagram of RRP-Pur NPs, Achilles tendon cell-derived cell membrane vesicles and TM-RRP-Pur NPs; (C) Cumulative drug release of TM-RRP-Pur NPs under H2O2 treatment conditions; (D) Transmission electron microscopy images of RRP-Pur NPs (left) and TM-RRP-Pur NPs (right), scale bar: 100 nm; (E) Changes in particle size of RRP-Pur NPs and TM-RRP-Pur NPs at different time points.

[0021] Figure 4 Figure 1 shows the optimization and structural validation of the microneedle assembly, including: (A) the effect curve of different ROS-responsive PLGA / Purmorphamine mass ratios on the drug loading and encapsulation efficiency of RRP-Pur NPs; (B) the effect curve of different mass ratios of Achilles tendon cell-derived cell membrane vesicles and RRP-Pur NPs on the particle size and polydispersity index of TM-RRP-Pur NPs; (C) optical microscope image of the microneedle array; (D) confocal laser scanning microscopy image of the spatial distribution of DID-labeled TM-RRP-Pur NPs within the microneedle array; (E) optical images of the microneedles before and after compression testing; and (F) optical images of the microneedles before and after tissue insertion.

[0022] Figure 5 Scanning electron microscope (SEM) images of MN@TM-RRP-Pur; where: the top image is a cross-sectional image of the unloaded microneedle group; the bottom image is a cross-sectional image of the MN@TM-RRP-Pur group.

[0023] Figure 6 Fluorescent tracer images of the in vivo release effect of MN@TM-RRP-Pur at different time points.

[0024] Figure 7 Open field movement and Achilles tendon Micro-CT images of mice treated with MN@TM-RRP-Pur for Achilles tendon injury 4 weeks later; the upper part shows the movement trajectory of mice in the sham-operated group, the model group, and the MN@TM-RRP-Pur treatment group in the open field experiment; the lower part shows the Micro-CT three-dimensional reconstruction images of the lower limb bones of mice in the sham-operated group, the model group, and the MN@TM-RRP-Pur treatment group.

[0025] Figure 8Immunohistochemical staining results of type I and type III collagen in Achilles tendon tissue of mice treated with MN@TM-RRP-Pur for 4 weeks. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Achilles tendon injury is a common and difficult-to-treat disease in sports medicine and orthopedic clinics. Traditional treatments, such as oral medications and local injections, often have limitations such as significant systemic side effects, poor patient compliance, and the need for repeated invasive procedures. Against this backdrop, microneedle patches have shown groundbreaking application potential as a drug delivery method. This technology utilizes an array of soluble microneedles to painlessly penetrate the skin's stratum corneum barrier, delivering loaded therapeutic factors to the target area. Its core advantage lies in achieving an organic combination of minimally invasive transdermal delivery, targeted drug release at the lesion site, and long-lasting release, providing an unprecedented technical support platform for precisely regulating cell behavior in the complex pathological microenvironment.

[0028] This invention provides a soluble microneedle patch for promoting Achilles tendon injury repair. The soluble microneedle patch includes a substrate and soluble microneedles arranged in an array on the substrate surface. Each soluble microneedle is loaded with a core-shell structured biomimetic targeting particle. The shell of this biomimetic targeting particle is a cell membrane vesicle derived from Achilles tendon cells, and the core is a ROS-responsive nanoparticle. This ROS-responsive nanoparticle includes a carrier and Purmorphamine dispersed in the carrier. The carrier is a block copolymer, and its structural formula is shown below:

[0029] Where m is 150~160, n is 150~160, and x is 110~120.

[0030] This soluble microneedle patch is a multi-level drug delivery system integrating biomimetic targeting particles, ROS-responsive nanoparticles, and drugs. It utilizes soluble microneedles to penetrate the stratum corneum of the skin, avoiding the pain, trauma, and infection risks associated with traditional injections, thus improving patient compliance. Specifically, the biomimetic targeting particles, aided by homologous targeting molecules on the surface of cell membrane vesicles derived from Achilles tendon cells, can actively recognize and accumulate in damaged Achilles tendon tissue. In the high-ROS microenvironment of the damaged Achilles tendon tissue, the thioketal bonds in the ROS-responsive nanoparticles at its core are broken by oxidative attack, allowing the nanoparticles to responsively degrade in the inflammatory microenvironment of elevated ROS at the injury site. This precisely and rapidly releases the encapsulated Purmorphamine to the lesion site, achieving on-demand drug delivery. Purmorphamine, as a key activator of the Hedgehog signaling pathway, can promote the synthesis of type I collagen in tendon cells and inhibit the overexpression of type III collagen under inflammatory stimulation, driving the extracellular matrix from scar-related repair to functional repair. In summary, this system can simultaneously achieve the triple functions of skin penetration, targeted enrichment, and controllable drug release, thereby enabling the repair of Achilles tendon injuries.

[0031] In some embodiments, the soluble microneedles employ a conical or pyramidal tip design, with the axis of the soluble microneedle perpendicular to its base. The microneedle tip is made of a soluble, biocompatible material, with a single needle height of 550–650 μm and a single needle base diameter of 300–800 μm. This height is suitable for transdermal drug delivery using microneedles, and its structural design achieves a good balance between safety, efficacy, and practicality. It enables efficient transdermal drug delivery while ensuring safe and painless drug administration, which is beneficial for the treatment of tissue lesions such as Achilles tendon injuries.

[0032] In some implementations, the particle size of the biomimetic targeting particles is 100-600 nm. If the particle size is less than 100 nm, problems such as insufficient drug loading, drug burst release, incomplete membrane coating, and reduced targeting specificity may occur; if the particle size is greater than 600 nm, tissue penetration is difficult, it is easily cleared by macrophages, and the intracellular delivery efficiency is significantly reduced.

[0033] In some implementations, Achilles tendon injuries include one or more of the following characteristics: heterotopic ossification of the Achilles tendon, and disordered collagen synthesis in the Achilles tendon tissue. The unique pathological features of Achilles tendon injuries compared to injuries to other common tissues such as skin, muscle, and bone are the fundamental reasons for their difficult healing, prolonged recovery period, and high recurrence rate.

[0034] This invention provides a method for preparing the above-mentioned soluble microneedle patch, comprising: providing ROS-responsive nanoparticles loaded with Purmorphamine; encapsulating cell membrane vesicles derived from Achilles tendon cells on the surface of the ROS-responsive nanoparticles to form biomimetic targeting particles; dispersing the biomimetic targeting particles in a first hydrosol to obtain a precursor fluid; filling the microcavity region of a microneedle mold with the precursor fluid, then filling the base region with a second hydrosol, and demolding after drying to obtain a soluble microneedle patch, wherein the first hydrosol includes hyaluronic acid; and the second hydrosol includes hyaluronic acid.

[0035] Hyaluronic acid, a naturally occurring component of the human extracellular matrix, possesses excellent biocompatibility, biodegradability, and low immunogenicity. It can dissolve under mild conditions to form a stable hydrosol, maximizing the protection of biomimetic targeted nanoparticles. Simultaneously, hyaluronic acid exhibits good water solubility, tissue adhesion, and bioaffinity, improving the adhesion and local retention of microneedle patches. It can also participate in the regulation of the tissue microenvironment, contributing to the remodeling of the extracellular matrix. Furthermore, the membrane extrusion fusion method used in this preparation step can construct a highly uniform, structurally well-defined core-shell structure, maximizing the preservation of the natural orientation and function of membrane proteins and improving formulation stability through efficient removal of aggregates. It also boasts high reproducibility and ease of standardization. The method for preparing soluble microneedle patches provided by this invention has a clear process flow, with parameters independently controllable at each step, facilitating quality control and process scale-up.

[0036] In some embodiments, providing ROS-responsive nanoparticles includes: preparing a ROS-responsive carrier by using methoxy polyethylene glycol-N-hydroxysuccinimide, dimethyl thioketal diamine, lactide, and glycolide as raw materials, through amidation, ring-opening polymerization, and end-group activation coupling reactions; and encapsulating Purmorphamine with the ROS-responsive carrier to obtain ROS-responsive nanoparticles. Preferably, the ROS-responsive carrier is ROS-responsive PLGA, which uses methoxy polyethylene glycol as the hydrophilic segment and PLGA as the hydrophobic framework. It not only possesses good biocompatibility and degradability but also can efficiently encapsulate Purmorphamine through self-assembly, improving drug stability and local effective concentration.

[0037] In some embodiments, the concentration of Purmorphamine in the precursor fluid is 15-25 mg / mL. Preferably, the concentration of Purmorphamine in the precursor fluid is 20 mg / mL. In vitro cell experiments have confirmed that this concentration of Purmorphamine can effectively promote collagen synthesis in tendon cells without significantly adversely affecting cell viability.

[0038] The present invention also provides the application of the above-mentioned soluble microneedle patch in the preparation of a drug for promoting the repair of Achilles tendon injury.

[0039] The Purmorphamine used in the following examples is a smoothed receptor agonist, purchased from MCE, catalog number HY-15108; the hyaluronic acid was purchased from MCE, catalog number HY-B0633A.

[0040] Example 1: Purmorphamine can reverse inflammation-induced collagen synthesis disorders in tendon cells. (1) Establish an inflammatory stimulation model of tendon cells Primary rat tendon cells were isolated from healthy rat Achilles tendon tissue and cultured in vitro. They were then treated with complete culture medium containing 10 ng / mL IL-1β and 10 ng / mL TNF-α for 24 hours to obtain an inflammatory stimulation model of tendon cells.

[0041] (2) Verify the regulatory effect of Purmorphamine on tendon cells The isolated primary rat tendon cells were randomly divided into three groups, as follows: Control group: Rat primary tendon cells were treated with normal complete culture medium for 24 hours; Inflammation stimulation group: Rat primary tendon cells were treated with complete culture medium containing 10 ng / mL IL-1β and 10 ng / mL TNF-α for 24 hours; Purmorphamine treatment group: Rat primary tendon cells were treated for 24 hours with complete culture medium containing 10 ng / mL IL-1β, 10 ng / mL TNF-α and 1 μM Purmorphamine.

[0042] Subsequently, Western blot analysis was performed to detect the protein expression of type I and type III collagen in tendon cells in the control group, the inflammatory stimulation group, and the Purmorphamine treatment group. The results showed that, compared with the inflammatory stimulation group, the expression of type I collagen was significantly upregulated in the Purmorphamine treatment group, while the expression of type III collagen was significantly downregulated. Figure 1 This indicates that Purmorphamine can reverse collagen synthesis disorders caused by inflammatory stimulation and promote the production of functional extracellular matrix.

[0043] Example 2: Preparation of TM-RRP-Pur NPs (1) Preparation of ROS-responsive PLGA: 1.0 g of methoxy-PEG-NHS (molecular weight 2000, approximately 0.5 mmol) was dissolved in 5 mL of anhydrous DMF. Under nitrogen protection, 1.2 equivalents of dimethyl thioketal diamine were added, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. Deionized water was added and dialyzed for 48 hours to remove unreacted small molecules. The intermediate was obtained by lyophilization. Subsequently, lactide (LA) and glycolide (GA) were mixed at a molar ratio of 50:50 (the total molar amount of the two was approximately 10 mmol), and dissolved in 10 mL of anhydrous dichloromethane. 1.1 equivalents (approximately 0.22 mmol) of the obtained intermediate were added to the mixture, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by rotary evaporation, the residue was dissolved in chloroform and filtered, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 7000 Da. The solution was dialyzed against deionized water for 72 hours, and finally lyophilized to obtain the target product, ROS-responsive PLGA. Figure 2 A). The 1H NMR spectrum showed characteristic peaks of PLA, PEG, and thioketal bonds in the target product, confirming the successful synthesis of the target ROS-responsive PLGA. Calculations based on the hydrogen areas of the characteristic peaks of PLA, PGA, and PEG, combined with the number of hydrogen atoms in each unit, yielded an average degree of polymerization (m) of approximately 154 for the glycolide unit, approximately 154 for the lactide unit, and approximately 114 for the ethylene oxide unit. Figure 2 B).

[0044] (2) Preparation of RRP-Pur NPs: ROS-responsive PLGA was encapsulated with Purmorphamine using a single emulsion solvent evaporation method to obtain ROS-responsive nanoparticles, which were named RRP-Pur NPs. Specifically, 10 mg of ROS-responsive PLGA was dissolved in a mixed organic solvent consisting of 300 μL ethyl acetate and 700 μL dichloromethane to prepare an organic phase; Purmorphamine, pre-dissolved in DMSO, was added to the above PLGA organic phase solution at a PLGA to Purmorphamine mass ratio of 11.2:1, and the mixture was sonicated in a water bath for 5 minutes to obtain a mixed solution. Subsequently, under probe sonication (50W, 3-second on / 2-second off cycle), the above mixed solution was added dropwise to a 3% (w / v) polyvinyl alcohol (PVA) aqueous solution to form a stable oil-in-water (O / W) emulsion. The emulsion was immediately transferred to a 0.3% (w / v) PVA solution and magnetically stirred at room temperature for 4 hours to evaporate the organic solvent. The obtained RRP-Pur NPs were collected by centrifugation at 20,000 ×g for 20 minutes at 4°C. The precipitate was washed three times with deionized water to remove residual PVA and free drug. Finally, it was resuspended in PBS and stored at 4°C for later use.

[0045] (3) Extraction of cell membrane vesicles derived from Achilles tendon cells: Fresh healthy rat Achilles tendon tissue was collected, and tendon cells were isolated and cultured after collagenase digestion. The cells were expanded to P1-P2 generation, washed thoroughly with pre-cooled PBS to remove residual culture medium, and then centrifuged to collect the cell pellet. The cell pellet was then resuspended in hypotonic buffer containing protease inhibitors and incubated on ice for 30 minutes to allow the cells to swell. The cells were then gently homogenized using a Dounce homogenizer or broken up by freeze-thaw. Differential centrifugation was then performed to gradually remove the cell nucleus (500 ×g, 10 min), mitochondria (2,000 ×g, 20 min), and microsomal debris (10,000 ×g, 30 min). The supernatant rich in cell membrane debris was collected. The membrane fragment suspension was placed in an ice bath, and the ultrasonic power was set to 25%. The treatment was carried out in a pulse mode with a 2-second working time and a 3-second interval for 5 minutes to break up large cell membranes into nanoscale vesicles. At the same time, the sample temperature was strictly controlled below 10°C to protect the activity of membrane proteins. To further obtain vesicles with uniform particle size, the vesicles were extruded 13 times sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes using a liposome extruder, with the extrusion pressure controlled at 1.5 MPa. Finally, the vesicles were precipitated by ultracentrifugation (100,000 × g, 4 °C, 90 min), and the supernatant containing soluble protein impurities was discarded. The vesicles were resuspended and washed twice with pre-cooled PBS, and the final precipitate was resuspended in an appropriate amount of PBS to obtain cell membrane vesicles derived from Achilles tendon cells for later use.

[0046] (4) Preparation of TM-RRP-Pur NPs: Cell membrane vesicles derived from Achilles tendon cells were mixed with RRP-Pur NPs at a mass ratio of 3:1. The cell membrane vesicles derived from Achilles tendon cells were wrapped on the surface of RRP-Pur NPs by membrane extrusion fusion method to form biomimetic targeting particles, which were named TM-RRP-Pur NPs.

[0047] The particle sizes of the prepared RRP-Pur NPs and TM-RRP-Pur NPs were measured. The results showed that the particle size of RRP-Pur NPs was 198.35±5.89 nm, and the particle size of TM-RRP-Pur NPs was 219.16±7.57 nm. Figure 3 A). Zeta potential analysis showed that the surface potential of RRP-Pur NPs was approximately -10 mV, while the potential of cell membrane vesicles derived from Achilles tendon cells was approximately -25 mV. After being encapsulated by cell membrane vesicles derived from Achilles tendon cells, the zeta potential of the resulting TM-RRP-Pur NPs became approximately -20 mV, a value significantly close to that of the vesicles themselves. Figure 3 B). This indicates that the cell membrane vesicles derived from Achilles tendon cells effectively reduced the electrostatic repulsion between RRP-Pur NPs and cell membrane vesicles, thereby promoting their interaction and improving cellular uptake efficiency. Subsequently, transmission electron microscopy revealed that TM-RRP-Pur NPs possessed a clearer membrane-coated structure compared to RRP-Pur NPs. Figure 3 D). The drug release capacity of TM-RRP-PurNPs in a high ROS environment was tested using 50 μM H2O2 to simulate the ROS environment. The results showed that the drug release rate of TM-RRP-PurNPs was significantly faster under high ROS conditions compared with no H2O2 treatment. Figure 3 C). Furthermore, during the 10-day monitoring period, the particle size of TM-RRP-Pur NPs remained constant, while RRP-Pur NPs showed a significant increase in particle size. This indicates that cell membrane vesicles derived from Achilles tendon cells can effectively maintain the stability of TM-RRP-Pur NPs and prevent aggregation. Figure 3 E).

[0048] Example 3: Preparation and characterization of MN@TM-RRP-Pur (1) Preparation of matrix solution: Hyaluronic acid with a molecular weight of 200 kDa was dissolved in deionized water to prepare a solution with a total mass volume concentration of 30%, which was used as the microneedle substrate and needle matrix.

[0049] (2) Preparation of drug-loaded needle solution: The TM-RRP-Pur NPs prepared in Example 2 were uniformly dispersed in the matrix solution of (1), wherein the final concentration of Purmorphamine was 20 mg / mL.

[0050] (3) Microneedle mold forming: Polydimethylsiloxane conical microneedle mold (needle height 600 μm, bottom diameter 500 μm, 10×10 array) was used. First, the drug-loaded needle body solution was dropped onto the surface of the mold, centrifuged at 4000 rpm for 10 minutes to degas it so that it could fully fill the needle tip cavity; then the matrix solution was coated to form the substrate.

[0051] (4) Drying and demolding: After drying in a desiccator at room temperature for 24 hours, the soluble microneedles are demolded and named MN@TM-RRP-Pur.

[0052] (5) Optimization and structural validation of the microneedle assembly: First, by changing the mass ratio of PLGA to the drug Purmorphamine, the changes in drug loading and encapsulation efficiency of RRP-Pur NPs were evaluated. The optimal mass ratio of PLGA to Purmorphamine in the drug loading system was determined to be 11.2:1, under the condition that both drug loading and encapsulation efficiency are maximized. Figure 4 A). Subsequently, the effects of different mass ratios of Achilles tendon cell-derived cell membrane vesicles to RRP-Pur NPs on the particle size and polydispersity index of TM-RRP-Pur NPs were studied. Under conditions where the polydispersity index tended to stabilize, the optimal mass ratio of Achilles tendon cell-derived cell membrane vesicles to RRP-Pur NPs was determined to be 3:1. Based on this, the coating process was optimized to obtain TM-RRP-Pur NPs with uniform size and complete coating. Figure 4 B). Next, the optimized TM-RRP-Pur NPs were loaded into microneedles. Optical microscopy images showed that the microneedle array was complete and uniform, exhibiting a good macroscopic structure. Further observation using confocal laser scanning microscopy revealed that the TM-RRP-Pur NPs labeled with the red fluorescent dye DID were uniformly distributed within the microneedle matrix, confirming the successful loading of TM-RRP-Pur NPs. Figure 4 (C~D). A comparison of optical images of MN@TM-RRP-Pur before and after compression testing demonstrates that it possesses sufficient mechanical strength to maintain structural integrity; while images before and after microneedle insertion into tissue visually demonstrate its ability to effectively penetrate biological barriers while maintaining needle shape, meeting the basic mechanical requirements for practical applications. Figure 4E~F). Finally, the morphology of MN@TM-RRP-Pur was observed by scanning electron microscopy. The results showed that compared with the unloaded microneedle group, the morphology of MN@TM-RRP-Pur was significantly rougher, with visible uneven texture and porous structure, and the structural density was changed, further indicating that MN@TM-RRP-Pur had successfully loaded TM-RRP-Pur (E~F). Figure 5 ).

[0053] Example 4: Preparation of MN@Pur The preparation method is the same as in Example 3. Specifically, hyaluronic acid with a molecular weight of 200 kDa is dissolved in deionized water to prepare a solution with a total mass-volume concentration of 30%, which serves as the matrix solution for the microneedle substrate and needle body. Purmorphamine, which is pre-dissolved in DMSO, is added to the matrix solution, wherein the final concentration of Purmorphamine is 20 mg / mL. After being molded by a microneedle mold, dried and demolded, soluble microneedles are prepared and named MN@Pur.

[0054] Example 5: Evaluation of in vivo distribution of MN@TM-RRP-Pur To understand the in vivo release effect of MN@TM-RRP-Pur, seven 3-month-old male C57BL / 6 mice (purchased from Wuhan Beintech Co., Ltd.) were used. A mouse Achilles tendon injury model was successfully established by performing a hemitransection surgery on the Achilles tendon at a distance of 0.5 cm from the calcaneal fulcrum. Subsequently, MN@TM-RRP-Pur labeled with DID fluorescent markers was applied to the injury site. The distribution of fluorescence intensity in the mice at different time points was observed using an in vivo imaging system. The results showed that from day 1 to day 3, the fluorescence signal was strong and relatively concentrated in the injury area. At this time, the DID fluorescently labeled MN@TM-RRP-Pur had just been applied in vivo, and the drug had not yet been released in large quantities. From day 4 to day 14, the intensity of the fluorescence signal gradually decreased, and the distribution became more diffuse. At this time, the drug began to be released continuously and diffuse. Figure 6 The results above show that the fluorescent signal is specifically enriched and retained for a long time in the Achilles tendon area, which proves that MN@TM-RRP-Pur has the ability to slowly release drugs in vivo.

[0055] Example 6: In vivo efficacy evaluation of microneedle system for treating Achilles tendon injury Three-month-old male C57BL / 6 mice (purchased from Wuhan Beintech Co., Ltd.) were acclimatized for one week in an SPF environment with a normal diet, reaching a weight of approximately 25 g. The mice were then randomly divided into four groups of seven mice each, as follows: Sham surgery group: The skin on the surface of the Achilles tendon of mice was cut open, but the Achilles tendon was not partially severed and sutured. Model group: The skin on the surface of the Achilles tendon of the mouse was cut open, and a partial transection of the Achilles tendon was performed 0.5 cm away from the fulcrum of the Achilles tendon and then sutured. MN@Pur treatment group: The skin on the surface of the Achilles tendon of the mouse was cut open, and a semi-transverse transection of the Achilles tendon was performed 0.5 cm away from the Achilles tendon fulcrum. Then, an MN@Pur patch of the same size as the Achilles tendon area was pressed onto the Achilles tendon area of ​​the mouse and sutured. MN@TM-RRP-Pur treatment group: The skin on the surface of the Achilles tendon of the mouse was cut open, and a semi-transverse transection of the Achilles tendon was performed 0.5 cm away from the Achilles tendon fulcrum. Then, an MN@TM-RRP-Pur patch that conforms to the size of the Achilles tendon area was pressed onto the Achilles tendon area of ​​the mouse and sutured.

[0056] Open field movement trajectory analysis of mice revealed that: the sham-operated group exhibited a wide range of movement trajectories with complex and smooth lines, indicating normal activity and no motor dysfunction; the model group showed a significantly narrowed range of movement trajectories with simple lines and a rigid, repetitive pattern, confirming the successful establishment of the Achilles tendon injury model; the MN@Pur treatment group showed a slightly improved range of movement trajectories compared to the model group, demonstrating that the MN@Pur treatment group could partially restore the motor function of the mice; the MN@TM-RRP-Pur treatment group showed significantly better range and complexity of movement trajectories than both the model group and the MN@Pur treatment group, with more expansive and smooth trajectories, indicating that MN@TM-RRP-Pur treatment can effectively improve the motor function of Achilles tendon injury mice and alleviate their pain and movement disorders. Four weeks after treatment, all mice were euthanized, and Micro-CT was used to assess the heterotopic ossification of the Achilles tendon in these four groups of mice. The results showed that, compared to the model group and the MN@Pur treatment group, the heterotopic ossification in the MN@TM-RRP-Pur treatment group was significantly reduced (…). Figure 7 Furthermore, immunohistochemical staining results showed that, compared to the model group and the MN@Pur treatment group, the collagen fibers in the MN@TM-RRP-Pur treatment group were more orderly arranged. Specifically, in the model group, the expression level of type I collagen was significantly reduced and the expression level of type III collagen was significantly increased, indicating bone matrix destruction and fibrosis. MN@Pur could partially improve bone repair and fibrosis, but the effect was limited. The treatment advantage of the MN@TM-RRP-Pur treatment group was more significant, with significant recovery of type I collagen expression and effective inhibition of type III collagen expression. The overall expression level was close to that of the sham-operated group, indicating that it has a significant advantage in promoting tendon tissue regeneration and inhibiting fibrosis. The changes in the expression of type I and type III collagen further confirmed that MN@TM-RRP-Pur treatment can improve the disordered collagen synthesis after Achilles tendon injury and promote the transformation of damaged tissue from fragile scar repair to fully functional tendon tissue. Figure 8 ).

[0057] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0058] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0059] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. A soluble microneedle patch for promoting Achilles tendon injury repair, characterized in that: The soluble microneedle patch includes a substrate and soluble microneedles arranged in an array on the surface of the substrate. The soluble microneedles are loaded with biomimetic targeting particles with a core-shell structure. The outer shell of the biomimetic targeting particles is a cell membrane vesicle derived from Achilles tendon cells, and the core of the biomimetic targeting particles is a reactive oxygen species responsive nanoparticle. The reactive oxygen species responsive nanoparticles include a support and Purmorphamine dispersed in the support. The support is a block copolymer, and the structural formula of the support is shown below: Where m is 150~160, n is 150~160, and x is 110~120; The Achilles tendon injury includes one or more of the following characteristics: (1) Heterotopic ossification of the Achilles tendon; (2) Disorders of collagen synthesis in the Achilles tendon tissue.

2. The soluble microneedle patch according to claim 1, characterized in that: The soluble microneedles are designed with conical or pyramidal tips. The axis of the soluble microneedles is perpendicular to the substrate. The tips of the microneedles are made of soluble biocompatible materials. The height of a single needle is 550~650 μm and the diameter of the bottom of a single needle is 300~800 μm.

3. The soluble microneedle patch according to claim 1, characterized in that: The particle size of the biomimetic targeting particles is 100~600 nm.

4. The method for preparing the soluble microneedle patch according to claim 1, characterized in that: include: Provide reactive oxygen species responsive nanoparticles, wherein the reactive oxygen species responsive nanoparticles are loaded with Purmorphamine; Cell membrane vesicles derived from Achilles tendon cells are wrapped around the surface of the reactive oxygen species-responsive nanoparticles to form biomimetic targeting particles. The biomimetic targeting particles were dispersed in a first hydrosol to obtain a precursor fluid. The precursor fluid is filled into the microcavity area of ​​the microneedle mold, and then the base area is filled with a second hydrosol. After drying, the mold is demolded to obtain a soluble microneedle patch. The first hydrosol includes hyaluronic acid; the second hydrosol includes hyaluronic acid.

5. The preparation method according to claim 4, characterized in that: The reactive oxygen species responsive nanoparticles include: A reactive oxygen species responsive carrier was prepared by means of methoxy polyethylene glycol-N-hydroxysuccinimide ester, dimethyl thioketal diamine, lactide and glycolide as raw materials, through amidation, ring-opening polymerization and end-group activation coupling reaction. Purmorphamine was encapsulated using the aforementioned reactive oxygen species (ROS) responsive carrier to obtain ROS responsive nanoparticles.

6. The preparation method according to claim 4, characterized in that: The concentration of Purmorphamine in the precursor fluid is 15-25 mg / mL.

7. The use of the soluble microneedle patch according to any one of claims 1 to 3 in the preparation of a medicament for promoting Achilles tendon injury repair; The Achilles tendon injury includes one or more of the following characteristics: (1) Heterotopic ossification of the Achilles tendon; (2) Disorders of collagen synthesis in the Achilles tendon tissue.

Images