Gradient-adapted ros-responsive exosome self-adaptive release microneedle dressing and preparation method thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DONGHUA UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-06-26
Smart Images
Figure CN121668088B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microneedle dressing technology, and relates to a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing and its preparation method. Background Technology
[0002] Exosomes carry important bioactive substances such as proteins, lipids, and nucleic acids from source cells, enabling them to precisely regulate the biological behavior of recipient cells by mediating intercellular communication. In the field of dermatology, exosomes exhibit multiple therapeutic advantages, possessing excellent anti-inflammatory and immunomodulatory functions, effectively alleviating symptoms of inflammatory skin diseases such as atopic dermatitis and psoriasis. Furthermore, exosomes have a natural nanoscale size and good biocompatibility, indicating low immunogenicity and superior tissue penetration potential. However, exosomes suffer from poor stability, easily agglomerating and becoming inactive in liquid environments. Systemic administration (such as subcutaneous injection) presents problems such as off-target effects, rapid clearance by the body, and difficulty in management. Traditional topical administration (such as topical application) is limited by the skin's stratum corneum barrier, making effective transdermal absorption of exosomes difficult and resulting in extremely low bioavailability. Emerging soluble microneedle transdermal drug delivery technology can directly bypass the skin barrier, delivering exosomes to skin tissues while efficiently loading and protecting their activity, representing a highly promising therapeutic strategy.
[0003] Several existing technologies utilize soluble microneedles loaded with exosomes for the treatment of skin-related diseases. For example, patent application CN202410067817.4 discloses an engineered exosome soluble microneedle dressing loaded with dimethyl fumarate for the treatment of psoriasis; patent application CN202411915286.6 discloses an engineered exosome soluble microneedle dressing loaded with cytokines for the treatment of skin-related diseases; and patent application CN202011148113.8 discloses soluble microneedles loaded with stem cell exosomes, antioxidants, and other components for the treatment of pigmentation and wrinkles. However, in these existing technologies, exosomes are released passively through diffusion, lacking intelligent responsiveness to the microenvironment, resulting in a large burst release in the early stages, which reduces the bioavailability and therapeutic effect of exosomes. Therefore, it is necessary to explore a microenvironment-responsive exosome adaptive release microneedle dressing for the treatment of skin-related diseases.
[0004] Since reactive oxygen species (ROS) are key signaling molecules in most skin-related diseases, and their overall levels are positively correlated with disease severity, they can serve as key biomarkers for disease assessment. Therefore, a ROS-responsive exosome adaptive release microneedle dressing has significant research value, aiming to enable the dressing to more precisely and autonomously regulate the exosome release rate on demand, thereby improving exosome bioavailability and therapeutic efficacy. For example, the literature (An ROS-Scavenging Treg-Recruiting Hydrogel Patch for Diabetic Wound Healing. Advanced Functional Materials, 34(26), 2314500.) discloses a hydrogel for treating diabetic wounds by anchoring therapeutic proteins to polymers using a ROS-responsive crosslinking agent. Under the influence of ROS in the tissue microenvironment, it can respond by releasing proteins while simultaneously clearing ROS. However, in vivo studies have shown that in inflammatory skin diseases such as psoriasis and atopic dermatitis, the disease center is often focused on the stratum corneum of the skin. Compared to normal skin tissue, the ROS content in the stratum corneum is significantly increased, and the ROS content gradually decreases from the outside to the inside of the skin tissue. Existing ROS-responsive drug-release hydrogel microneedles cannot accurately adapt to the natural gradient of response factors in different areas, resulting in uneven release of therapeutic drugs.
[0005] Therefore, it is of great significance to study a method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing to solve the problems existing in the prior art. Summary of the Invention
[0006] The purpose of this invention is to solve the problems existing in the prior art and provide a method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0008] A gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing includes a basal layer and an array of microneedles located on the basal layer.
[0009] Both the substrate layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels cross-linked with ROS-responsive cleaving cross-linking agents; the gelatin and carboxymethyl chitosan on the microneedles are also connected to exosomes through ROS-responsive cleaving bonds.
[0010] From the base layer to the tip of the microneedle, the degree of cross-linking of the microneedle dressing decreases in a gradient.
[0011] The microneedle dressing is entirely cross-linked by a ROS-responsive cross-linking agent. This agent reacts with amino groups on the molecular chains of gelatin and carboxymethyl chitosan, as well as on the exosome membrane. Exosomes within the microneedle dressing are directly anchored to the polymer chains of the microneedles via ROS-responsive cross-linking bonds (such as thioketal (TK) linkers and phenylboronic acid ester bonds). In the presence of ROS in the environment, the cross-linking agent cleaves, triggering the release of exosomes for therapeutic purposes. The entire exosome release process is mediated by ROS in the microenvironment. The cleavage of ROS-responsive cross-links can remove excess ROS from the environment, reducing oxidative stress in psoriasis, and decreasing inflammation and tissue damage; simultaneously, it can protect exosome activity from ROS degradation. Furthermore, because the degree of ROS-responsive gradient cross-linking in the microneedle dressing is adapted to the natural ROS gradient content in different areas of skin tissue, the ROS-responsive release of exosomes loaded in the microneedles is more balanced across different sites, exhibiting zoned, microenvironment-responsive intelligent exosome release.
[0012] As a preferred technical solution:
[0013] As described above, a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing has an equal degree of cross-linking in the base layer, which is not less than the degree of cross-linking at the bottom of the microneedle body; the degree of cross-linking gradually decreases from the bottom of the microneedle body to the tip.
[0014] The gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing described above has a base layer thickness of 0.1~5mm.
[0015] The gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing described above has microneedles shaped like a square pyramid or a cone, and the number of microneedles in the array is 4 to 400.
[0016] The height of the square pyramid or cone is 100~1000μm, the side length of the base of the square pyramid is 50~500μm, and the diameter of the base of the cone is 50~500μm; preferably, the height of the square pyramid or cone is 400~1000μm, the side length of the base of the square pyramid is 200~500μm, and the diameter of the base of the cone is 250~400μm.
[0017] As described above, a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing uses a ROS-responsive cleavage crosslinking agent that is a compound containing a ROS-responsive cleavage bond in the middle and N-hydroxysuccinimide ester groups at both ends, such as 2,2′-[propane-2,2-dimethylbis(thio)]diacetic acid diN-hydroxysuccinimide ester. The ROS-responsive cleavage bond is a ketethiolate bond, borate ester bond, disulfide bond, diselenide bond, thioether bond, or thioacetal bond.
[0018] This invention also provides a method for preparing a gradient-adapted ROS-responsive exosome adaptive release microneedle dressing as described in any of the preceding claims. First, a base layer precursor and a microneedle array precursor are prepared separately. Then, the microneedle array precursor is injected into a microneedle dressing mold. After vacuuming or centrifugation (using negative pressure to fill the mold with solution, or using centrifugal force to drive the liquid into the mold), the base layer precursor is injected into the microneedle dressing mold. After vacuuming or centrifugation, the microneedle dressing mold is pre-cooled. Finally, the pre-cooled ROS-responsive crosslinking agent solution is injected into the pre-cooled microneedle dressing mold. After the crosslinking reaction, the mold is dried and demolded to obtain the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing. To prevent the microneedles from shrinking and warping after crosslinking, thus altering their original size and shape, a weight is uniformly applied to the microneedle base layer during the microneedle crosslinking and subsequent drying and curing process. Pressure is used to ensure the microneedle matrix adheres tightly to the mold without separation.
[0019] The precursor for the basal layer is a mixed aqueous solution of gelatin and carboxymethyl chitosan, and the precursor for the microneedle array is a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes.
[0020] The concentration of the ROS-responsive crosslinking agent solution is 50~200 mg / mL;
[0021] The content of ROS-responsive crosslinking agent in microneedle dressings is 3~10wt%.
[0022] The pre-cooling temperature of the microneedle dressing mold is 4~10℃, and the pre-cooling temperature of the ROS-responsive crosslinking agent solution is 4~10℃. The substrate precursor and the microneedle array precursor need to be pre-cooled after mixing, and the crosslinking agent solution also needs to be pre-cooled. This is because low temperature is beneficial to the preservation of exosome activity; and secondly, it is to reduce the rate of crosslinking reaction, making the crosslinking more uniform. At the same time, the crosslinking agent will not react immediately, which is conducive to the free crosslinking agent diffusing downward according to the concentration difference to form a gradual crosslinking degree gradient.
[0023] The cross-linking reaction occurs at temperatures of 4~10℃.
[0024] As a preferred technical solution:
[0025] The preparation method of the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing described above, wherein the content of gelatin in the basal layer precursor is 1~10wt% and the content of carboxymethyl chitosan is 1~10wt%.
[0026] The preparation method of the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing described above, wherein the microneedle array precursor contains 1-10 wt% gelatin, 1-10 wt% carboxymethyl chitosan, and 0.1-10 mg / mL exosomes.
[0027] The preparation method of the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing described above involves a drying temperature of 4~37℃ and a drying time of 2~6h.
[0028] Invention principle:
[0029] This invention first injects the base layer precursor and the microneedle array precursor sequentially into a microneedle mold (first gelatin + carboxymethyl chitosan + exosomes to form microneedles, then gelatin + carboxymethyl chitosan to form the base layer). Then, a crosslinking agent is injected onto the surface (i.e., the bottom surface of the base layer). The crosslinking agent preferentially crosslinks with the bottom surface of the base layer, and the remainder passively diffuses towards the tip layer (i.e., the plane where the tips of the microneedle array are located) according to the concentration difference of the crosslinking agent, crosslinking the precursors as it diffuses. Ultimately, due to the passive diffusion of the crosslinking agent, the resulting microneedle dressing exhibits a gradient degree of crosslinking from the base layer to the tip layer.
[0030] The change in crosslinking degree is gradual. The crosslinking agent is injected from the base layer, crosslinking the surrounding macromolecular chains while diffusing downwards with the concentration gradient, thus forming a crosslinking degree gradient. Since the distribution of the crosslinking agent in the microneedle dressing is formed by passive diffusion due to the concentration gradient, the concentration of the crosslinking agent decreases from top to bottom, and the crosslinking degree also decreases from top to bottom. During this process, the crosslinking rate and the crosslinking agent dosage ratio need to be controlled to form a gradient crosslinking degree. First, the reaction temperature and crosslinking agent concentration must be reduced to decrease the reaction rate. If the crosslinking rate is too fast, the crosslinking agent reacts immediately with the surrounding macromolecules, failing to create a concentration gradient for downward diffusion; if the crosslinking rate is too slow, the crosslinking agent is completely and uniformly distributed in the dressing before crosslinking, also failing to form a gradient. Second, the dosage ratio of the crosslinking agent relative to the matrix macromolecules is crucial. If there is too little crosslinking agent, the needle tip will not be fully crosslinked; if there is too much crosslinking agent, all areas will be completely crosslinked, also failing to form a gradient. To adapt to the distribution of ROS in skin tissue and form a top-down crosslinking gradient, the needle body and the precursor of the basal layer must first be filled into the mold, and then the crosslinking agent is injected onto the surface (i.e., the bottom surface of the basal layer). The desired crosslinking gradient is formed by the diffusion behavior caused by the concentration difference. If the order of feeding is changed, such as mixing the crosslinking agent and the precursor before filling the mold, a top-down crosslinking gradient cannot be formed; instead, a uniform crosslinking is formed in the microneedles.
[0031] Exosomes are loaded onto the microneedles of a microneedle dressing and penetrate into the active epidermal layer and superficial dermis, such as... Figure 1As shown, the ROS content in skin tissue (green) decreases from the outside to the inside, and the cross-linking degree of the microneedle dressing (blue) decreases from top to bottom (from the basal layer to the microneedle tip). The release rate of exosomes on the microneedle body is related to the gradient cross-linking degree of the microneedle body, resulting in more removal in areas with high ROS content and less removal in areas with low ROS content. After ROS is released, it is taken up by cells to exert its therapeutic effect. The basal layer is adjacent to the stratum corneum and has a higher degree of cross-linking, which can remove more harmful ROS from the stratum corneum and reduce inflammation.
[0032] Beneficial effects:
[0033] (1) A gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing of the present invention can relieve oxidative stress and reduce inflammation by clearing ROS; and can release exosomes through environmental response to carry out immune regulation and anti-inflammation, thus having a synergistic effect in treating a variety of skin-related diseases, and with few side effects.
[0034] (2) The present invention provides a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing. The microneedle dressing has a gradient-adaptive ROS-responsive crosslinking degree. The overall crosslinking degree gradient of the dressing decreases from the basal layer to the needle tip. It can adapt to the ROS distribution of skin tissue in inflammatory skin diseases and reasonably regulate its level. That is, more ROS is removed in areas with high ROS content and less ROS is removed in areas with low ROS content, in order to restore ROS to normal physiological level as much as possible without excessive removal that would disrupt the normal immune balance. At the same time, because the ROS-responsive crosslinking degree of the microneedle dressing is adapted to the ROS content of skin tissue, the ROS-responsive release of exosomes loaded in the microneedle body is more balanced in various parts. It has a zone-adaptive microenvironment-responsive intelligent exosome release, which effectively improves the bioavailability of exosomes and improves treatment efficiency.
[0035] (3) A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing of the present invention. The microneedle dressing can remove ROS in the microenvironment while releasing exosomes, which is beneficial to protect the exosome activity from ROS damage and improve the therapeutic effect.
[0036] (4) The preparation method of the gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing of the present invention, through the regulation and optimization of key process flow and parameters, better maintains the exosome activity and has a high degree of exosome cross-linking, realizing more precise ROS-responsive exosome adaptive release, which is conducive to improving the treatment effect. Attached Figure Description
[0037] Figure 1 A schematic diagram of a ROS-responsive exosome adaptive release microneedle dressing;
[0038] Figure 2 This is a statistical graph showing the crosslinking agent density at different depths of the microneedle dressing in Example 1;
[0039] Figure 3 The image shows the morphology of the microneedle dressing in Example 1, where (a) is a photograph of the actual product and (b) is a SEM image.
[0040] Figure 4 The mechanical compression curve of the microneedle dressing in Example 1;
[0041] Figure 5 Images representing the penetration depth of the microneedle dressing in Example 1;
[0042] Figure 6 The cumulative exosome release curves of cross-linked and non-cross-linked microneedle dressings in PBS containing or without 5% hydrogen peroxide are shown in Example 1.
[0043] Figure 7 The images show the skin of mice in the treatment group, control group, and model group when the microneedle dressing was applied to psoriasis model mice in Example 1 and Comparative Example 1. The treatment group consisted of psoriasis model mice treated with the microneedle dressing; the control group consisted of normal mice treated with petroleum jelly; and the model group consisted of psoriasis model mice treated with petroleum jelly.
[0044] Figure 8 The H&E staining results of skin tissues from the treatment group, control group, and model group of mice when the microneedle dressing was applied to psoriasis model mice in Example 1 and Comparative Example 1;
[0045] Figure 9 This is a statistical graph showing the content of TNF-α (a) and IL-17A (b) in the skin homogenates of mice in the treatment group, control group and model group when the microneedle dressing was applied to a mouse model of psoriasis in Example 1. Detailed Implementation
[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0047] The test methods involved in the performance indicators in the embodiments and comparative examples of this invention are as follows:
[0048] Single needle breaking force: The microneedle dressing is placed at the fixed end of the electronic universal testing machine with the needle tip facing the moving end; during the compression test, pressure is applied at a displacement rate of 0.5 mm / min, and the relationship between the compression force and displacement is recorded.
[0049] Exosome release rate: Microneedle dressings were prepared using exosomes labeled with PKH26 fluorescent dye. The microneedle dressings were immersed in 5 mL of PBS buffer and kept at 37 °C to simulate physiological conditions. 200 μL of liquid was collected at predetermined time points for analysis, and 200 μL of fresh PBS was added to maintain a constant volume.
[0050] Crosslinking agent density: To characterize the gradient crosslinking degree of the microneedles, a crosslinking agent with N-hydroxysuccinimide ester groups at both ends was synthesized using cy3-bisCOOH. The specific steps are as follows: 100 mg of cy3-bisCOOH, 200 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 150 mg of NHS (N-hydroxysuccinimide) were dissolved in 2 mL of dimethyl sulfoxide (DMSO) and stirred overnight at room temperature. To remove unreacted EDC and NHS, the reaction product was washed with deionized water and freeze-dried to obtain a crosslinking agent powder with fluorescent properties. Microneedle dressings were prepared, and the fluorescence intensity of the microneedle dressings at different depths along the z-axis was observed and statistically analyzed under a laser confocal microscope. Dividing the fluorescence intensity by the corresponding cross-sectional area yielded the crosslinking agent density of the microneedle dressings at different depths along the z-axis, which directly corresponds to the degree of crosslinking. Fluorescence intensity was measured using a fluorescence spectrophotometer with an excitation wavelength of 551 nm and an emission wavelength of 567 nm.
[0051] Example 1
[0052] A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing, the specific steps of which are as follows:
[0053] (1) Preparation of microneedle array precursor;
[0054] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 7.4 with 1M hydrochloric acid, and MSC-Exo was added. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes was obtained, which is the precursor of microneedle array.
[0055] The precursor of the microneedle array contains 7 wt% gelatin, 3 wt% carboxymethyl chitosan, and 1 mg / mL MSC-Exo.
[0056] (2) Preparation of the basal layer precursor;
[0057] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 7.4 with 1M hydrochloric acid. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin and carboxymethyl chitosan was obtained, which is the base layer precursor.
[0058] The base layer precursor contains 7 wt% gelatin and 3 wt% carboxymethyl chitosan.
[0059] (3) Design and fabricate microneedle dressing molds;
[0060] The microneedle body is shaped like a square pyramid with a height of 650 μm and a base side length of 330 μm. The number of microneedle bodies in the array is 64.
[0061] (4) Inject the microneedle array precursor into the microneedle dressing mold, vacuum it 3 times, then inject the base layer precursor into the microneedle dressing mold, vacuum it 3 times, and then pre-cool the microneedle dressing mold at 4°C for 30 minutes.
[0062] (5) Synthesis of ROS-responsive crosslinking agents;
[0063] 100 mg of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, 200 mg of EDC, and 150 mg of NHS were dissolved in 2 mL of dimethyl sulfoxide (DMSO) and stirred overnight at room temperature. To remove unreacted EDC and NHS, the reaction product was washed with deionized water and freeze-dried to obtain a ROS-responsive cleavage crosslinking agent powder containing ketithiolide bonds, namely 2,2′-[propane-2,2-diylbis(thio)]diacetic acid diN-hydroxysuccinimide ester;
[0064] (6) Prepare a ROS-responsive crosslinking agent solution;
[0065] The ROS-responsive crosslinking agent powder from step (5) was dissolved in DMSO to obtain a ROS-responsive crosslinking agent solution with a concentration of 100 mg / mL, and then pre-cooled at 4 °C for 30 min.
[0066] (7) The pre-cooled ROS-responsive crosslinking agent solution was injected into the microneedle dressing mold after pre-cooling in step (4), and after crosslinking reaction at 4℃ for 12h, it was dried at 4℃ for 4h and demolded to obtain the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing; the ROS-responsive crosslinking agent content in the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing was 7wt%.
[0067] The final gradient-adapted ROS-responsive exosome adaptive release microneedle dressing comprises a 0.1 mm thick base layer and a microneedle array on the base layer; both the base layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels crosslinked with a ROS-responsive crosslinking agent; the gelatin and carboxymethyl chitosan on the microneedles are also connected to the exosomes through ROS-responsive crosslinking bonds; the degree of crosslinking of the microneedle dressing decreases gradually from the base layer to the tip of the microneedle; the degree of crosslinking in the base layer is equal; from the bottom of the microneedle to the tip, the degree of crosslinking gradually decreases, with the crosslinking agent density in the upper third (corresponding to the tip) being 35-61%, the middle third being 61-92%, and the lower third (corresponding to the bottom) being 92-100%; the average single-needle breaking force is 0.4 N / needle; containing 5% In the H2O2-containing PBS environment, the cumulative release rate of microneedle dressings was 53% at 6 hours and 100% at 24 hours; in the PBS environment, the cumulative release rate of microneedle dressings was 47% at 24 hours; while the non-crosslinked microneedle dressings achieved a cumulative release rate of 100% at 6 hours in both the PBS environment containing 5% H2O2 and the PBS environment.
[0068] like Figure 2 As shown, the crosslinking agent density of the microneedle dressing gradually decreases from 100% to 35% from the base layer to the microneedle tip, indicating that the microneedle dressing has a gradient of decreasing crosslinking degree from the base layer to the microneedle tip.
[0069] The morphology of the prepared microneedle dressings was characterized using a camera and field emission scanning electron microscopy, such as... Figure 3 (a) and Figure 3 As shown in (b) of the diagram.
[0070] The mechanical compressive properties of the microneedle dressing were tested using a universal testing machine, such as... Figure 4 As shown, the average breaking force of a single needle is 0.38 N / needle, which meets the mechanical strength required for skin penetration.
[0071] After a period of time following the insertion of a Rhodamine B-labeled microneedle dressing into pigskin, the penetration depth of the dressing contents was characterized using laser confocal fluorescence microscopy. Figure 5 As shown, the penetration depth is 240μm, which meets the treatment requirements.
[0072] The cumulative release rates of exosomes from cross-linked and non-cross-linked microneedles loaded with exosomes were measured in PBS and PBS containing 5% hydrogen peroxide, respectively. Figure 6 As shown, the exosome release behavior of the microneedle dressing of the present invention exhibits good microenvironment ROS response performance, and prolongs the sustained release time of exosomes compared with uncrosslinked microneedle dressings.
[0073] Comparative Example 1
[0074] A method for preparing a ROS-responsive exosome-releasing microneedle dressing is basically the same as in Example 1, except that: the ROS-responsive crosslinking agent solution is uniformly mixed with the microneedle array precursor and the base layer precursor, respectively, and then the mixed solution is filled into the microneedle mold by vacuuming. After the crosslinking reaction is carried out at 4°C for 12 hours, it is dried at 4°C for 4 hours and then demolded.
[0075] The prepared ROS-responsive exosome-releasing microneedle dressings had a crosslinking agent density of 70% from the base layer to the tip of the microneedle, meaning that the crosslinking degree of the microneedle dressings was the same.
[0076] To evaluate the therapeutic effect of microneedle dressings on skin-related diseases, a psoriasis mouse model was induced using IMQ. Skin condition photographs of mice in different groups were taken 7 days after treatment. Figure 7 As shown. The H&E staining results of skin tissues from different groups of mice are as follows. Figure 8 As shown, the use of the microneedle dressing in Example 1 of this invention significantly reduced the psoriasis symptoms in model mice. Scales and erythema disappeared from the skin surface, and the stratum corneum thickness thinned, essentially resembling the morphology of normal skin tissue. This demonstrates a good therapeutic effect on psoriasis. This is because the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing provides more precise regulation of ROS and release of exosomes in different regions, better meeting the needs of the pathological microenvironment.
[0077] The levels of psoriasis-specific inflammatory factors in skin homogenates from different groups of mice were tested using an enzyme-linked immunosorbent assay (ELISA) kit. Figure 9 As shown, the results indicate that the application of the microneedle dressing of the present invention significantly reduced the levels of TNF-α and IL-17A in the skin tissue of psoriasis model mice, and has a good therapeutic effect on psoriasis.
[0078] Example 2
[0079] A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing, the specific steps of which are as follows:
[0080] (1) Preparation of microneedle array precursor;
[0081] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 7 by 1M hydrochloric acid, and MSC-Exo was added. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes was obtained, which is the precursor of microneedle array.
[0082] The precursor for the microneedle array contains 1 wt% gelatin, 9 wt% carboxymethyl chitosan, and 0.1 mg / mL MSC-Exo.
[0083] (2) Preparation of the basal layer precursor;
[0084] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 7 with 1M hydrochloric acid. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin and carboxymethyl chitosan was obtained, which is the base layer precursor.
[0085] The base layer precursor contains 1 wt% gelatin and 9 wt% carboxymethyl chitosan.
[0086] (3) Design and fabricate microneedle dressing molds;
[0087] The microneedle body is shaped like a square pyramid with a height of 1000 μm and a base side length of 500 μm. The number of microneedle bodies in the array is 400.
[0088] (4) Inject the microneedle array precursor into the microneedle dressing mold, centrifuge for 5 min, then inject the base layer precursor into the microneedle dressing mold, centrifuge for 5 min, and then pre-cool the microneedle dressing mold at 5°C for 60 min.
[0089] (5) Synthesis of ROS-responsive crosslinking agents;
[0090] 100 mg of 3,3′-diselenodipropionic acid, 200 mg of EDC and 150 mg of NHS were dissolved in 2 mL of DMSO and stirred overnight at room temperature. To remove unreacted EDC and NHS, the reaction product was washed with deionized water and freeze-dried to obtain a ROS-responsive crosslinking agent powder containing diseleno bonds, namely 3,3′-diselenodipropionic acid diN-hydroxysuccinimide ester.
[0091] (6) Prepare a ROS-responsive crosslinking agent solution;
[0092] The ROS-responsive cleavage crosslinking agent powder from step (5) was dissolved in DMSO to obtain a ROS-responsive cleavage crosslinking agent solution with a concentration of 75 mg / mL, and then pre-cooled at 5 °C for 60 min.
[0093] (7) The pre-cooled ROS-responsive crosslinking agent solution was injected into the microneedle dressing mold after pre-cooling in step (4), and after crosslinking reaction at 5°C for 12 hours, it was dried at 10°C for 6 hours and demolded to obtain a gradient-adapted ROS-responsive exosome adaptive release microneedle dressing; the ROS-responsive crosslinking agent content in the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing was 10wt%.
[0094] The final gradient-adapted ROS-responsive exosome adaptive release microneedle dressing comprises a 1 mm thick base layer and a microneedle array on the base layer; both the base layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels crosslinked with a ROS-responsive crosslinking agent; the gelatin and carboxymethyl chitosan on the microneedles are also connected to the exosomes through ROS-responsive crosslinking bonds; the degree of crosslinking of the microneedle dressing decreases gradually from the base layer to the tip of the microneedle; the degree of crosslinking is equal in the base layer; from the bottom of the microneedle to the tip, the degree of crosslinking gradually decreases, and the degree of crosslinking on the three needles decreases. The cross-linking agent density is 28-53% in one-third of the needle body, 53-88% in the middle third, and 88-100% in the lower third; the average single needle breaking force is 0.07 N / needle; in PBS containing 5% H2O2, the cumulative release rate of the microneedle dressing is 41% after 6 hours and 82% after 24 hours; in PBS, the cumulative release rate of the microneedle dressing is 24% after 24 hours; while the non-cross-linked microneedle dressing achieves a cumulative release rate of 100% after 6 hours in both PBS containing 5% H2O2 and PBS.
[0095] Example 3
[0096] A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing, the specific steps of which are as follows:
[0097] (1) Preparation of microneedle array precursor;
[0098] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 7.5 with 1M hydrochloric acid, and MSC-Exo was added. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes was obtained, which is the precursor of microneedle array.
[0099] The precursor of the microneedle array contains 5 wt% gelatin, 5 wt% carboxymethyl chitosan, and 2 mg / mL MSC-Exo.
[0100] (2) Preparation of the basal layer precursor;
[0101] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 7.5 with 1M hydrochloric acid. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin and carboxymethyl chitosan was obtained, which is the base layer precursor.
[0102] The base layer precursor contains 5 wt% gelatin and 5 wt% carboxymethyl chitosan.
[0103] (3) Design and fabricate microneedle dressing molds;
[0104] The microneedle body is conical in shape, with a height of 800 μm and a base diameter of 400 μm. The array of microneedles consists of 200 microneedles.
[0105] (4) Inject the microneedle array precursor into the microneedle dressing mold, vacuum 10 times, then inject the base layer precursor into the microneedle dressing mold, vacuum 10 times, and then pre-cool the microneedle dressing mold at 6°C for 50 min.
[0106] (5) Synthesis of ROS-responsive crosslinking agents;
[0107] 100 mg of 3,3′-thiodipropionic acid, 200 mg of EDC and 150 mg of NHS were dissolved in 2 mL of DMSO and stirred overnight at room temperature. To remove unreacted EDC and NHS, the reaction product was washed with deionized water and freeze-dried to obtain a ROS-responsive cleavage crosslinking agent powder containing thioether bonds, namely 3,3′-thiodipropionic acid diN-hydroxysuccinimide ester.
[0108] (6) Prepare a ROS-responsive crosslinking agent solution;
[0109] The ROS-responsive crosslinking agent powder from step (5) was dissolved in ethanol to obtain a ROS-responsive crosslinking agent solution with a concentration of 50 mg / mL, and then pre-cooled at 6 °C for 50 min.
[0110] (7) The pre-cooled ROS-responsive crosslinking agent solution was injected into the microneedle dressing mold after pre-cooling in step (4), and after crosslinking reaction at 6°C for 4 hours, it was dried at 20°C for 5 hours and demolded to obtain a gradient-adapted ROS-responsive exosome adaptive release microneedle dressing; the ROS-responsive crosslinking agent content in the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing was 5wt%.
[0111] The final gradient-adapted ROS-responsive exosome adaptive release microneedle dressing comprises a 5 mm thick base layer and a microneedle array on the base layer; both the base layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels crosslinked with a ROS-responsive crosslinking agent; the gelatin and carboxymethyl chitosan on the microneedles are also connected to the exosomes through ROS-responsive crosslinking bonds; the degree of crosslinking of the microneedle dressing decreases gradually from the base layer to the tip of the microneedle; the degree of crosslinking is equal in the base layer; from the bottom of the microneedle to the tip, the degree of crosslinking gradually decreases, and the degree of crosslinking on the three needles decreases. The cross-linking agent density is 40-65% in one-third of the needle body, 65-86% in the middle third, and 86-100% in the lower third; the average single needle breaking force is 0.2 N / needle; in PBS containing 5% H2O2, the cumulative release rate of the microneedle dressing is 50% after 6 hours and 94% after 24 hours; in PBS, the cumulative release rate of the microneedle dressing is 33% after 24 hours; while the uncross-linked microneedle dressing achieves a cumulative release rate of 100% after 6 hours in both PBS containing 5% H2O2 and PBS.
[0112] Example 4
[0113] A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing, the specific steps of which are as follows:
[0114] (1) Preparation of microneedle array precursor;
[0115] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 8 by 1M hydrochloric acid, and MSC-Exo was added. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes was obtained, which is the precursor of microneedle array.
[0116] The precursor of the microneedle array contains 9 wt% gelatin, 1 wt% carboxymethyl chitosan, and 5 mg / mL MSC-Exo.
[0117] (2) Preparation of the basal layer precursor;
[0118] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 8 with 1M hydrochloric acid. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin and carboxymethyl chitosan was obtained, which is the base layer precursor.
[0119] The basal layer precursor contains 9 wt% gelatin and 1 wt% carboxymethyl chitosan.
[0120] (3) Design and fabricate microneedle dressing molds;
[0121] The microneedle body is shaped like a square pyramid with a height of 400 μm and a base side length of 200 μm. The number of microneedle bodies in the array is 300.
[0122] (4) Inject the microneedle array precursor into the microneedle dressing mold, evacuate once, then inject the base layer precursor into the microneedle dressing mold, evacuate once, and then pre-cool the microneedle dressing mold at 8°C for 20 min.
[0123] (5) Synthesis of ROS-responsive crosslinking agents;
[0124] 100 mg of 3,3′-dithiodipropionic acid, 200 mg of EDC and 150 mg of NHS were dissolved in 2 mL of DMSO and stirred overnight at room temperature. To remove unreacted EDC and NHS, the reaction product was washed with deionized water and freeze-dried to obtain a ROS-responsive crosslinking agent powder containing disulfide bonds, namely 3,3′-dithiodipropionic acid diN-hydroxysuccinimide ester.
[0125] (6) Prepare a ROS-responsive crosslinking agent solution;
[0126] The ROS-responsive crosslinking agent powder from step (5) was dissolved in water to obtain a ROS-responsive crosslinking agent solution with a concentration of 150 mg / mL, and then pre-cooled at 8°C for 20 min.
[0127] (7) The pre-cooled ROS-responsive crosslinking agent solution was injected into the microneedle dressing mold after pre-cooling in step (4), and after crosslinking reaction at 7°C for 8 hours, it was dried at 25°C for 3 hours and demolded to obtain a gradient-adapted ROS-responsive exosome adaptive release microneedle dressing; the ROS-responsive crosslinking agent content in the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing was 7wt%.
[0128] The final gradient-adapted ROS-responsive exosome adaptive release microneedle dressing consists of a 3 mm thick base layer and a microneedle array on the base layer. Both the base layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels crosslinked with a ROS-responsive crosslinking agent. The gelatin and carboxymethyl chitosan on the microneedles are also connected to the exosomes through ROS-responsive crosslinking bonds. The degree of crosslinking of the microneedle dressing decreases gradually from the base layer to the tip of the microneedle. The degree of crosslinking is equal in the base layer. From the bottom of the microneedle to the tip, the degree of crosslinking gradually decreases, with the crosslinking on the needle body decreasing to a certain extent. The cross-linking agent density was 43-72%, the cross-linking agent density in the middle third of the needle body was 72-97%, and the cross-linking agent density in the lower third of the needle body was 97-100%; the average single needle breaking force was 0.12 N / needle; in PBS containing 5% H2O2, the cumulative release rate of the microneedle dressing was 47% after 6 hours and 96% after 24 hours; in PBS, the cumulative release rate of the microneedle dressing was 29% after 24 hours; while the non-cross-linked microneedle dressing achieved a cumulative release rate of 100% after 6 hours in both PBS containing 5% H2O2 and PBS.
[0129] Example 5
[0130] A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing, the specific steps of which are as follows:
[0131] (1) Preparation of microneedle array precursor;
[0132] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 9 by 1M hydrochloric acid, and MSC-Exo was added. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes was obtained, which is the precursor of microneedle array.
[0133] The precursor for the microneedle array contains 10 wt% gelatin, 1 wt% carboxymethyl chitosan, and 10 mg / mL MSC-Exo.
[0134] (2) Preparation of the basal layer precursor;
[0135] Gelatin and carboxymethyl chitosan were mixed and dissolved in water. The pH of the solution was adjusted to 9 with 1M hydrochloric acid. The mixture was stirred at 4°C for 8 hours until the solution was homogeneous. After standing for 4 hours to eliminate bubbles, a mixed aqueous solution of gelatin and carboxymethyl chitosan was obtained, which is the base layer precursor.
[0136] The base layer precursor contains 10 wt% gelatin and 1 wt% carboxymethyl chitosan.
[0137] (3) Design and fabricate microneedle dressing molds;
[0138] The microneedle body is conical in shape, with a height of 500 μm and a base diameter of 250 μm. The array of microneedles consists of 100 microneedles.
[0139] (4) Inject the microneedle array precursor into the microneedle dressing mold, centrifuge for 30 min, then inject the base layer precursor into the microneedle dressing mold, centrifuge for 30 min, and then pre-cool the microneedle dressing mold at 10°C for 10 min.
[0140] (5) Synthesis of ROS-responsive crosslinking agents;
[0141] 100 mg of bis(2-carboxyethyl)thioacetal, 200 mg of EDC and 150 mg of NHS were dissolved in 2 mL of DMSO and stirred overnight at room temperature. To remove unreacted EDC and NHS, the reaction product was washed with deionized water and freeze-dried to obtain a ROS-responsive cleavage crosslinking agent powder containing thioacetal bonds, namely bis(2-carboxyethyl)thioacetal diN-hydroxysuccinimide ester.
[0142] (6) Prepare a ROS-responsive crosslinking agent solution;
[0143] The ROS-responsive crosslinking agent powder from step (5) was dissolved in DMSO to obtain a ROS-responsive crosslinking agent solution with a concentration of 200 mg / mL, and then pre-cooled at 10 °C for 10 min.
[0144] (7) The pre-cooled ROS-responsive crosslinking agent solution was injected into the microneedle dressing mold after pre-cooling in step (4), and after crosslinking reaction at 10°C for 24 hours, it was dried at 37°C for 2 hours and demolded to obtain a gradient-adapted ROS-responsive exosome adaptive release microneedle dressing; the ROS-responsive crosslinking agent content in the gradient-adapted ROS-responsive exosome adaptive release microneedle dressing was 3wt%.
[0145] The final gradient-adapted ROS-responsive exosome adaptive release microneedle dressing comprises a 0.1 mm thick base layer and a microneedle array on the base layer; both the base layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels crosslinked with a ROS-responsive crosslinking agent; the gelatin and carboxymethyl chitosan on the microneedles are also connected to the exosomes through ROS-responsive crosslinking bonds; the degree of crosslinking of the microneedle dressing decreases gradually from the base layer to the tip of the microneedle; the degree of crosslinking is equal in the base layer; from the bottom of the microneedle to the tip, the degree of crosslinking gradually decreases, and the degree of crosslinking on the three needles decreases. The cross-linking agent density is 54-78% in one-third of the needle body, 78-98% in the middle third, and 98-100% in the lower third; the average single needle breaking force is 0.15 N / needle; in PBS containing 5% H2O2, the cumulative release rate of the microneedle dressing is 60% after 6 hours and 100% after 24 hours; in PBS, the cumulative release rate of the microneedle dressing is 41% after 24 hours; while the uncross-linked microneedle dressing achieves a cumulative release rate of 100% after 6 hours in both PBS containing 5% H2O2 and PBS.
Claims
1. A gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing, characterized in that: Includes the basal layer and the microneedle array located on the basal layer; Both the substrate layer and the microneedle array are gelatin / carboxymethyl chitosan composite hydrogels cross-linked with ROS-responsive cleaving cross-linking agents; the gelatin and carboxymethyl chitosan on the microneedles are also connected to exosomes through ROS-responsive cleaving bonds. The ROS-responsive crosslinking agent is 2,2′-[propane-2,2-dimethylbis(thio)]diacetic acid diN-hydroxysuccinimide, 3,3′-diselenodipropionate diN-hydroxysuccinimide, 3,3′-thiodipropionate diN-hydroxysuccinimide, 3,3′-dithiodipropionate diN-hydroxysuccinimide or bis(2-carboxyethyl)thioformaldehyde diN-hydroxysuccinimide; The preparation method of the microneedle dressing is as follows: First, a base layer precursor and a microneedle array precursor are prepared separately. Then, the microneedle array precursor is injected into the microneedle dressing mold. After vacuuming or centrifugation, the base layer precursor is injected into the microneedle dressing mold. After vacuuming or centrifugation, the microneedle dressing mold is pre-cooled. Finally, the pre-cooled ROS-responsive crosslinking agent solution is injected into the pre-cooled microneedle dressing mold. After the crosslinking reaction, the mold is dried and demolded. The pre-cooling temperature of the microneedle dressing mold is 4~10℃, and the pre-cooling temperature of the ROS-responsive crosslinking agent solution is 4~10℃. From the base layer to the tip of the microneedle, the degree of cross-linking of the microneedle dressing gradually decreases.
2. The gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing according to claim 1, characterized in that, The degree of cross-linking in the base layer is equal and not less than that at the bottom of the microneedle body; the degree of cross-linking gradually decreases from the bottom of the microneedle body to the tip.
3. The gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing according to claim 1, characterized in that, The thickness of the base layer is 0.1~5mm.
4. The gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing according to claim 1, characterized in that, The microneedle body is shaped like a square pyramid or a cone, and the number of microneedle bodies in the array is 4 to 400. The height of a square pyramid or cone is 100~1000μm, the side length of the base of a square pyramid is 50~500μm, and the diameter of the base of a cone is 50~500μm.
5. A method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing as described in any one of claims 1 to 4, characterized in that: First, the base layer precursor and the microneedle array precursor are prepared separately. Then, the microneedle array precursor is injected into the microneedle dressing mold. After vacuuming or centrifugation, the base layer precursor is injected into the microneedle dressing mold. After vacuuming or centrifugation, the microneedle dressing mold is pre-cooled. Finally, the pre-cooled ROS-responsive crosslinking agent solution is injected into the pre-cooled microneedle dressing mold. After the crosslinking reaction, the mold is dried and demolded to obtain a gradient-adapted ROS-responsive exosome adaptive release microneedle dressing. The precursor for the basal layer is a mixed aqueous solution of gelatin and carboxymethyl chitosan, and the precursor for the microneedle array is a mixed aqueous solution of gelatin, carboxymethyl chitosan and exosomes. The concentration of the ROS-responsive crosslinking agent solution is 50~200 mg / mL; The content of ROS-responsive crosslinking agent in microneedle dressings is 3-10 wt%; The pre-cooling temperature of the microneedle dressing mold is 4~10℃, and the pre-cooling temperature of the ROS-responsive crosslinking agent solution is 4~10℃. The cross-linking reaction occurs at temperatures of 4~10℃.
6. The method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing according to claim 5, characterized in that, The base layer precursor contains 1-10 wt% gelatin and 1-10 wt% carboxymethyl chitosan.
7. The method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing according to claim 5, characterized in that, The precursor of the microneedle array contains 1-10 wt% gelatin, 1-10 wt% carboxymethyl chitosan, and 0.1-10 mg / mL exosomes.
8. The method for preparing a gradient-adaptive ROS-responsive exosome adaptive release microneedle dressing according to claim 5, characterized in that, The drying temperature is 4~37℃, and the drying time is 2~6h.