A hydrogel microneedle patch based on bletilla striata polysaccharide composite engineered exosome and a preparation method and application thereof
By cross-linking hydrogel microneedles with Bletilla striata polysaccharide and methacrylated hyaluronic acid, and using caffeic acid-modified bone marrow mesenchymal stem cell-engineered exosomes, the biocompatibility and multiple barriers of hydrogel microneedles in the repair of diabetic wounds were solved, and a highly efficient repair effect with synergistic effects of multiple pharmacological activities was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING DRUM TOWER HOSPITAL
- Filing Date
- 2026-06-18
- Publication Date
- 2026-08-04
AI Technical Summary
Existing hydrogel microneedles have poor biocompatibility in the repair of diabetic wounds and are unable to simultaneously address multiple obstacles such as high glucose, high oxidative stress and persistent inflammation. Traditional single-drug loading methods have limited effectiveness.
A multi-mechanism synergistic intervention system was constructed by using a physically cross-linked hydrogel microneedle substrate composed of Bletilla striata polysaccharide and methacrylated hyaluronic acid, with caffeic acid-modified bone marrow mesenchymal stem cell-engineered exosomes loaded in the needle tip.
It provides a biocompatible and synergistic repair environment with multiple pharmacological activities, significantly improving drug penetration efficiency and utilization, and promoting comprehensive repair of diabetic wounds.
Smart Images
Figure CN122499095A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedical materials and transdermal drug delivery technology, specifically to a hydrogel microneedle patch for repairing diabetic wounds, and more particularly to a hydrogel microneedle patch based on Bletilla striata polysaccharide composite engineered exosomes, its preparation method and application. Background Technology
[0002] Chronic diabetic wounds are one of the most common and serious complications of diabetes, characterized by long healing periods, frequent recurrence of infections, and high rates of disability. The high-glucose microenvironment continuously induces oxidative stress imbalance, excessive inflammatory activation, abnormal bacterial proliferation, and fibroblast dysfunction in the wound, leading to stagnation of the wound healing process. Traditional wound dressings such as gauze and ointments only provide simple physical coverage and cannot effectively address the aforementioned multiple pathological problems, resulting in very limited therapeutic effects.
[0003] In recent years, microneedling technology has received widespread attention in the field of wound repair due to its advantages of being minimally invasive, painless, and capable of efficiently penetrating the stratum corneum to deliver drugs. Among them, hydrogel microneedles, which combine the moisturizing and repairing properties of hydrogels with the transdermal delivery capabilities of microneedles, have become a research hotspot. However, most existing hydrogel microneedles are made of chemically synthesized polymer materials, which have drawbacks such as poor biocompatibility and the risk of toxic degradation products. More importantly, traditional microneedles usually only load a single drug or growth factor, making it difficult to simultaneously address the multiple challenges of high glucose, high oxidative stress, and persistent inflammation in diabetic wounds. This results in a single dimension of repair, and the therapeutic effect falls far short of clinical needs.
[0004] Bone marrow mesenchymal stem cell exosomes are rich in various bioactive molecules that promote repair and possess natural advantages such as immunomodulation and angiogenesis. However, natural exosomes exhibit poor stability and insufficient antioxidant capacity in the high-glucose oxidative stress environment of wounds, making them highly susceptible to inactivation and limiting their repair efficacy. Therefore, effectively modifying natural exosomes to enhance their activity in harsh microenvironments and constructing a biocompatible delivery system capable of multi-mechanism synergistic intervention is a pressing technical challenge in this field. Summary of the Invention
[0005] Purpose of the invention: The present invention aims to solve the above-mentioned problems existing in the prior art and provide a hydrogel microneedle patch based on Bletilla striata polysaccharide composite engineered exosomes, its preparation method and application.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] A hydrogel microneedle patch based on Bletilla striata polysaccharide-engineered exosomes, the microneedle patch comprising a hydrogel microneedle substrate and an array of microneedle tips; the hydrogel microneedle substrate is a physically cross-linked hydrogel structure composed of Bletilla striata polysaccharide and methacrylated hyaluronic acid, and the microneedle tips are enriched with bone marrow mesenchymal stem cell-engineered exosomes loaded with caffeic acid.
[0008] Furthermore, the present invention also claims protection for the preparation method of the above-mentioned hydrogel microneedle patch based on Bletilla striata polysaccharide complex engineered exosomes, comprising the following steps:
[0009] S1. Preparation of composite hydrogel precursor solution: Disperse Bletilla striata polysaccharide and methacrylated hyaluronic acid in deionized water and stir until dissolved to obtain composite hydrogel precursor solution;
[0010] S2. Preparation of drug-loaded engineered exosome suspension: Exosomes derived from bone marrow mesenchymal stem cells are extracted and an exosome suspension is prepared. Caffeic acid is mixed with the exosome suspension and incubated. The exosome suspension is loaded by physical osmosis to obtain an engineered exosome suspension loaded with caffeic acid.
[0011] S3. Preparation of exosome-pregel mixture: Add the drug-loaded engineered exosome suspension obtained in step S2 to the composite hydrogel precursor solution obtained in step S1, mix evenly to obtain exosome-pregel mixture.
[0012] S4. Centrifugation and needle tip enrichment: The exosome-pregel mixture obtained in step S3 is filled into the microneedle mold and centrifuged to make the exosomes in the mixture directionally enriched in the needle tip area of the mold, followed by pre-curing.
[0013] S5. Curing and Demolding: The microneedles pre-cured in step S4 are subjected to light-shielded ultraviolet irradiation to allow the hydrogel to fully cross-link and cure. After demolding, the hydrogel microneedle patch based on Bletilla striata polysaccharide composite engineered exosomes is obtained.
[0014] As a preferred technical solution, in step S1, the mass ratio of Bletilla striata polysaccharide to methacrylated hyaluronic acid is (4-6):(2-4), more preferably 5:3; the stirring temperature is 30-40℃ and the stirring time is 2-4h.
[0015] As a preferred technical solution, in step S2, the mass ratio of caffeic acid to bone marrow mesenchymal stem cell exosomes is (0.5-2):(0.5-2), more preferably 1:1; the incubation temperature is 37°C, and the incubation time is 1-4 hours, more preferably 4 hours.
[0016] As a preferred technical solution, in step S4, the centrifugation speed is 3000-6000 r / min and the time is 5-15 min; the pre-curing condition is standing at room temperature for 30-60 min.
[0017] As a preferred technical solution, in step S5, the intensity of the ultraviolet irradiation is 10-20 mW / cm². 2 The duration is 20-40 seconds.
[0018] As a preferred technical solution, the microneedle mold is a conical needle hole mold made of polydimethylsiloxane, with a needle hole length of 0.8-1.2 mm, a bottom radius of 500-800 μm, and a needle array density of 80-120 needles / cm². 2 .
[0019] As a preferred technical solution, in step S1, a photoinitiator is also added to the composite hydrogel precursor solution. The photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone, and the amount added is 0.5%-1.5% of the volume of the precursor solution.
[0020] As a preferred technical solution, in step S2, the method for preparing bone marrow mesenchymal stem cell exosomes includes: culturing bone marrow mesenchymal stem cells in serum-free culture medium, collecting the supernatant, and purifying it by ultracentrifugation and ultrafiltration, wherein the exosomes have a particle size of 30-150 nm.
[0021] Furthermore, the present invention claims protection for the use of the above-described microneedle patch in the preparation of a drug for treating chronic diabetic wounds.
[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0023] (1) This invention uses natural Bletilla striata polysaccharide as the main matrix material of hydrogel microneedles, replacing traditional chemically synthesized polymers. Bletilla striata polysaccharide itself has excellent biocompatibility and biodegradability, and has multiple pharmacological activities such as hemostasis, antibacterial and anti-inflammatory, providing a safe, mild matrix environment with active repair function for wound repair.
[0024] (2) The core innovation of this invention lies in the engineered modification of bone marrow mesenchymal stem cell exosomes using natural polyphenol caffeic acid. Caffeic acid not only possesses potent antioxidant and anti-inflammatory activities, but also significantly enhances the stability of exosomes in a high-glucose oxidative stress microenvironment, protecting their structural integrity and biological activity. The resulting dual synergistic system of "exosomes promoting tissue repair + caffeic acid's antioxidant / anti-inflammatory effects" completely solves the problem of limited efficacy of natural exosomes in diabetic refractory wounds.
[0025] (3) This invention constructs a composite structure combining the inherent function of the matrix with precise needle-tip targeted therapy. The Bletilla striata polysaccharide matrix provides a basic antibacterial wound-protecting environment, while the caffeic acid-engineered exosomes delivered by the microneedle tip precisely remove oxidative damage, inhibit inflammation, and promote angiogenesis and collagen remodeling. The synergistic effect of the three can comprehensively improve the disordered microenvironment of diabetic wounds, and the repair effect is significantly better than that of traditional single modes.
[0026] (4) The present invention adopts centrifugal filling and fixed-point loading process, which is simple and mild, and preserves the activity of exosomes to the greatest extent. At the same time, the microneedles can penetrate the thickened keratin barrier of diabetic wounds in a minimally invasive manner, and deliver highly active engineered exosomes to the deep layers of the wound, which greatly improves the penetration efficiency and utilization rate of drugs.
[0027] (5) The composite microneedle system of the present invention has strong functional adjustability. By adjusting parameters such as the caffeic acid modification ratio and exosome loading, it can be adapted to diabetic wounds of different severity, and has extremely high clinical translational value and broad prospects for medical application. Attached Figure Description
[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.
[0029] Figure 1 This is a flowchart illustrating the preparation process of a hydrogel microneedle patch based on Bletilla striata polysaccharide-engineered exosomes according to the present invention.
[0030] Figure 2 This is a transmission electron microscopy (TEM) comparison of the morphology of natural exosomes derived from bone marrow mesenchymal stem cells and caffeic acid-engineered exosomes in an embodiment of the present invention.
[0031] Figure 3 The images shown are scanning electron microscope (SEM) images of Bletilla striata polysaccharide microneedles loaded with caffeic acid-engineered exosomes prepared according to an embodiment of the present invention; wherein, Figure a is an overall structural diagram of the microneedle array, and Figure b is a diagram of the morphology of exosome loading on the needle tip surface.
[0032] Figure 4 The image shows the fluorescence characterization of exosomes loaded with Bletilla striata polysaccharide microneedles prepared in an embodiment of the present invention.
[0033] Figure 5 This is a fluorescence characterization comparison of the antioxidant properties of caffeic acid-modified exosomes and unmodified natural exosomes under oxidative stress in embodiments of the present invention.
[0034] Figure 6 This image shows the effect of masson staining on diabetic wound tissue in mice after intervention with engineered exosomes encapsulated in Bletilla striata polysaccharide microneedles, as described in this embodiment of the invention. Detailed Implementation
[0035] The present invention can be better understood from the following embodiments.
[0036] Example 1: Preparation of a hydrogel microneedle patch based on Bletilla striata polysaccharide-engineered exosomes
[0037] This embodiment provides a specific method for preparing microneedle patches, the process of which is as follows: Figure 1 As shown, it includes the following steps:
[0038] (1) Extraction and purification of exosomes from bone marrow mesenchymal stem cells: Rat bone marrow mesenchymal stem cells in good growth condition were cultured in exosome-free fetal bovine serum DMEM / F12 medium. After 48 hours of culture, the cell supernatant was collected. Exosomes were purified by gradient ultracentrifugation: first, the cells and broken impurities were removed by low-speed centrifugation at 3000 rpm, and then the exosomes were enriched by high-speed centrifugation at 55000 rpm for 90 minutes. After resuspending in sterile PBS buffer and filtering with a 0.22 μm microporous membrane for sterilization, the purified rat bone marrow mesenchymal stem cell exosomes were obtained. Under transmission electron microscopy, they showed a typical cup-shaped double membrane structure and were stored at low temperature for later use.
[0039] (2) Preparation of caffeic acid-engineered exosomes: The purified exosome suspension was mixed with caffeic acid solution at a mass ratio of 1:1 and incubated at 37°C in the dark for 4 hours, allowing caffeic acid to be loaded onto the surface and interior of the exosomes through physical osmosis and non-covalent bonding. After incubation, unbound free caffeic acid was removed by ultracentrifugation at 55000 g for 90 minutes. The precipitate was collected, resuspended in PBS, and the caffeic acid-loaded engineered exosome suspension was obtained and stored at 4°C in the dark for later use.
[0040] (3) Preparation of composite hydrogel precursor solution: Bletilla striata polysaccharide (BSP) and methacrylated hyaluronic acid (HAMA) were mixed at a mass ratio of 5:3 and added to deionized water. 1% of the total volume of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone was also added. The mixture was stirred at 35°C for 3 hours until completely dissolved, forming a uniform and transparent composite hydrogel precursor solution. The solution was then allowed to stand to remove bubbles before use.
[0041] (4) Preparation of exosome-pregel mixture: Take the caffeic acid engineered exosome suspension obtained in step (2) and slowly add it to the composite hydrogel precursor solution obtained in step (3). Stir gently to mix evenly and ensure that the exosomes are uniformly dispersed in the pregel.
[0042] (5) Centrifugal filling and tip enrichment: The mixture obtained in step (4) was carefully filled into a polydimethylsiloxane (PDMS) microneedle mold. The mold needle holes were conical, with a needle length of 1.0 mm, a bottom radius of 750 μm, and an array density of 100 needles / cm.2 The filled mold was placed in a centrifuge and centrifuged at 4000 rpm for 8 minutes. This step utilizes centrifugal force to remove air bubbles from the mixture and, on the other hand, uses density differences to directionally enrich exosomes in the needle tip region.
[0043] (6) Curing and Demolding: After centrifugation, remove the mold and allow it to pre-cur at room temperature for 45 minutes. Subsequently, under light-protected conditions, use ultraviolet light (light intensity 15mW / cm²) to cure. 2 Irradiation for 30 seconds allows HAMA to fully cross-link, forming stable hydrogel microneedles. Finally, the cured microneedle patch is carefully peeled from the mold to obtain the finished product. The overall thickness of the finished patch is approximately 1.5 mm, and the needle tips are uniformly enriched with a large number of caffeic acid-engineered exosomes.
[0044] Example 2: Characterization of material microstructure and exosome loading effect
[0045] To verify the structural integrity and exosome loading effect of the microneedle patch prepared by the present invention, the sample obtained in Example 1 was systematically characterized in this embodiment.
[0046] (1) Morphological characterization of exosomes: Natural exosomes (unmodified) and caffeic acid-engineered exosomes prepared in step (2) of Example 1 were taken and observed by transmission electron microscopy. The results are as follows: Figure 2 As shown in the figure, the natural exosomes (left image) exhibit a typical round or elliptical cup-shaped vesicle structure. After modification with caffeic acid (right image), the exosomes remained intact, without any damage, collapse, or abnormal aggregation, indicating that the caffeic acid modification process did not damage the microstructure of the exosomes.
[0047] (2) Microstructure of microneedle patches: The microstructure of the finished microneedle patches was observed using a scanning electron microscope. For example... Figure 3 As shown in Figure a, the prepared Bletilla striata polysaccharide / HAMA composite microneedle array is neatly arranged, with intact needle morphology and no breakage or collapse, indicating that it has good mechanical strength. Figure b is a high-magnification magnified image of the needle tip surface, which clearly shows that the needle tip surface has a rough and porous structure, with a large number of spherical particles (i.e., engineered exosomes) uniformly attached and stably loaded in the needle tip area, without obvious detachment or aggregation. Figure 4 The fluorescence characterization image further confirmed that the fluorescently labeled exosomes were successfully enriched and mainly distributed at the tip of the microneedles.
[0048] The above results collectively demonstrate that the preparation process proposed in this invention is mature and controllable, and can successfully construct composite microneedle patches with regular structure, excellent mechanical properties, and precise and efficient exosome loading.
[0049] Example 3: Evaluation of the in vitro antioxidant properties of engineered caffeic acid exosomes
[0050] To verify the effect of caffeic acid modification on enhancing the antioxidant capacity of exosomes, this embodiment designed an in vitro oxidative stress experiment to compare the scavenging capacity of natural exosomes and caffeic acid-engineered exosomes for reactive oxygen species.
[0051] Two groups of exosomes were cultured in a simulated hydrogen peroxide stress environment at a constant temperature. Specifically, exosome samples were added to basal cell culture medium with a final concentration of 100–400 μmol / L hydrogen peroxide and incubated at 37°C in a 5% CO2 incubator for 2–4 h in the dark to construct an in vitro cellular oxidative stress model. Subsequently, reactive oxygen species (ROS) in the samples were specifically labeled using the specific fluorescent staining reagent DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate). The antioxidant effects of the two groups of exosomes were observed using a laser confocal microscope. Stronger fluorescence indicated higher levels of ROS in the cells and weaker antioxidant capacity.
[0052] The results are as follows Figure 5 As shown, the natural exosome group (left figure) exhibits strong green fluorescence, indicating that it accumulates a large amount of reactive oxygen species in an oxidative stress environment, making its structure extremely vulnerable to damage. In contrast, the fluorescence signal of the caffeic acid-engineered exosome group (right figure) is significantly weakened, indicating that it effectively scavenges reactive oxygen species in the environment and exhibits excellent antioxidant properties.
[0053] This experiment demonstrates that caffeic acid modification can endow exosomes with a strong reactive oxygen species scavenging ability and significantly enhance their stability in harsh oxidative environments, providing a key advantage for application in diabetic wounds with high glucose and high oxidative stress.
[0054] Example 4: Verification of the in vivo repair effect of composite micro-targets on chronic diabetic wounds
[0055] This embodiment establishes a rat model of chronic diabetic wounds to evaluate the in vivo repair effect of the microneedle patch prepared in Example 1.
[0056] Six-week-old healthy SPF-grade SD rats were selected. A diabetic model was first established by injecting STZ (streptozotocin). Then, a full-thickness skin defect was constructed. The specific construction method was as follows: SD rats were continuously anesthetized with isoflurane gas. After the rats were fully in a stable anesthetized state, their limbs were fixed, and their dorsal skin was fully exposed. Hair was shaved from the midline of the back. The shaved skin was then sterilely disinfected with iodine and 75% ethanol, and allowed to air dry. At the pre-designed modeling area on the rat's back, the epidermis and dermis were precisely removed using surgical scissors, preserving the underlying subcutaneous fascia layer, thus constructing a standardized full-thickness skin defect. After modeling, any remaining small tissue fragments at the wound edges were removed, and the wound was rinsed with sterile saline. The animal skin defect model was then completed. Mice with successful modeling were randomly divided into a blank control group and a microneedling treatment group, with several parallel samples in each group. The blank control group's wounds healed naturally without any treatment; the treatment group's wounds were treated with engineered exosome microneedle patches of Bletilla striata polysaccharide loaded with caffeic acid prepared in this invention, with dressings changed regularly and a uniform feeding environment.
[0057] Fourteen days after intervention, skin tissue from the wounds of mice in each group was fixed, embedded, and sectioned for HE staining to observe wound healing, wound width, and inflammatory infiltration status. The staining results are as follows: Figure 6 As shown, HE staining can clearly display the tissue structure of skin wounds, the distance between wound edges, and the degree of inflammatory cell infiltration. It is a core indicator for directly evaluating the healing rate and repair status of skin wounds.
[0058] The control group (left image) showed significant tissue defects in the wound, with sparse and disordered distribution of collagen fibers (blue area), and numerous immature collagen and gaps, indicating poor wound healing. In contrast, the microneedle treatment group (right image) showed a significant increase in collagen fiber deposition in the wound, with a denser and more orderly arrangement, forming a continuous epithelial structure. The overall tissue remodeling effect was far superior to that of the control group.
[0059] The results confirm that the Bletilla striata polysaccharide-caffeic acid engineered exosome microneedle patch prepared in this invention can significantly promote the orderly healing of diabetic chronic wounds by synergistically regulating processes such as oxidative stress, inflammation and tissue remodeling, and has excellent in vivo repair effects and application potential.
[0060] This invention provides a hydrogel microneedle patch based on Bletilla striata polysaccharide composite engineered exosomes, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. A hydrogel microneedle patch based on Bletilla striata polysaccharide-engineered exosomes, characterized in that, It includes a hydrogel microneedle base and an array of microneedle tips; the hydrogel microneedle base is a physically cross-linked hydrogel structure composed of Bletilla striata polysaccharide and methacrylated hyaluronic acid, and the microneedle tips are enriched with bone marrow mesenchymal stem cell-engineered exosomes loaded with caffeic acid.
2. The method for preparing the hydrogel microneedle patch based on Bletilla striata polysaccharide-engineered exosomes as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of composite hydrogel precursor solution: Disperse Bletilla striata polysaccharide and methacrylated hyaluronic acid in deionized water and stir until dissolved to obtain composite hydrogel precursor solution; S2. Preparation of drug-loaded engineered exosome suspension: Exosomes derived from bone marrow mesenchymal stem cells are extracted and an exosome suspension is prepared. Caffeic acid is mixed with the exosome suspension and incubated. The exosome suspension is loaded by physical osmosis to obtain an engineered exosome suspension loaded with caffeic acid. S3. Preparation of exosome-pregel mixture: Add the drug-loaded engineered exosome suspension obtained in step S2 to the composite hydrogel precursor solution obtained in step S1, mix evenly to obtain exosome-pregel mixture. S4. Centrifugation and needle tip enrichment: The exosome-pregel mixture obtained in step S3 is filled into the microneedle mold and centrifuged to make the exosomes in the mixture directionally enriched in the needle tip area of the mold, followed by pre-curing. S5. Curing and Demolding: The microneedles pre-cured in step S4 are subjected to light-protected ultraviolet irradiation to allow the hydrogel to fully cross-link and cure. After demolding, the product is obtained.
3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of Bletilla striata polysaccharide to methacrylated hyaluronic acid is (4-6):(2-4); the stirring temperature is 30-40℃ and the stirring time is 2-4h.
4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of caffeic acid to bone marrow mesenchymal stem cell exosomes is (0.5-2):(0.5-2); the incubation temperature is 37±2℃ and the incubation time is 1-4h.
5. The preparation method according to claim 2, characterized in that, In step S4, the centrifugation speed is 3000-6000 r / min and the time is 5-15 min; the pre-curing condition is to stand at room temperature for 30-60 min.
6. The preparation method according to claim 2, characterized in that, In step S5, the intensity of the ultraviolet irradiation is 10-20 mW / cm². 2 The duration is 20-40 seconds.
7. The preparation method according to claim 2, characterized in that, The microneedle mold is a conical needle mold made of polydimethylsiloxane, with a needle length of 0.8-1.2 mm, a bottom radius of 500-800 μm, and a needle array density of 80-120 needles / cm². 2 .
8. The preparation method according to claim 2, characterized in that, In step S1, a photoinitiator is also added to the composite hydrogel precursor solution. The photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone, and the amount added is 0.5%-1.5% of the volume of the precursor solution.
9. The preparation method according to claim 2, characterized in that, In step S2, the method for preparing bone marrow mesenchymal stem cell exosomes includes: culturing bone marrow mesenchymal stem cells in serum-free culture medium, collecting the supernatant, and purifying it by ultracentrifugation and ultrafiltration, wherein the exosomes have a particle size of 30-150 nm.
10. The application of the hydrogel microneedle patch based on Bletilla striata polysaccharide-engineered exosomes as described in claim 1 in the preparation of a drug for treating chronic diabetic wounds.