Multifunctional microneedle patch with pH response sequential drug release function as well as preparation method and application of multifunctional microneedle patch

Abstract

The invention discloses a multifunctional microneedle patch with a pH response sequential drug release function and a preparation method and application thereof, the patch is of an inner-outer layer core-shell structure, the outer layer of a microneedle is made of a pH response type material, and an antibacterial peptide preparation with an instant and rapid sterilization effect is encapsulated in the outer layer of the microneedle; and the inner core layer uses photosensitive methacrylated hyaluronic acid and encapsulates the mesenchymal stem cell exosome rich in bioactive components. The intelligent microneedle system shows the treatment advantage of a sequential drug release function: in the initial stage, the antibacterial peptide component rapidly plays a pathogen removal role to form a therapeutic protective barrier; subsequent sustained release of the exosome is used for adjusting a local inflammation microenvironment and promoting repair of damaged cornea tissues. The preparation method is simple, high in universality and suitable for large-scale production, has good biocompatibility and has important economic and clinical application prospects in the field of corneal diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a multifunctional microneedle patch with pH-responsive sequential drug release function, its preparation method, and its application. Background Technology

[0002] Keratitis is a common ophthalmic disease with complex pathological features, characterized by persistent inflammation of the corneal tissue. Its pathogenesis is usually related to infection by microorganisms such as bacteria, viruses, or fungi, and it is prevalent in underdeveloped countries and regions. Typical clinical symptoms include progressive ulcerative lesions of the corneal epithelium and opacification of the stromal layer. Without timely clinical intervention, it can cause irreversible visual impairment. Currently, the standard treatment for keratitis involves broad-spectrum antibiotics combined with corneal repair drugs to promote corneal repair. However, keratitis-causing microorganisms may form biofilms, affecting the effective penetration of these drugs. This reduces the bactericidal efficacy of antibiotics and creates physical barriers that hinder drug molecule penetration. Residual biofilms can continuously interfere with the therapeutic effect of repair drugs, leading to persistent corneal inflammation and secondary complications. To address this treatment dilemma, developing novel drug delivery systems with biofilm-disrupting and sustained-release properties has become a key breakthrough in improving the treatment efficacy of infectious keratitis. Summary of the Invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a multifunctional microneedle patch with pH-responsive sequential drug release function and its preparation method, thereby optimizing the drug release strategy to achieve a dual mechanism of rapid antibacterial and long-term repair, and providing an optimized solution for the treatment of infectious keratitis.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A multifunctional microneedle patch with pH-responsive sequential drug release function has an inner and outer core-shell structure. The outer layer of the microneedle uses a pH-responsive material and encapsulates an antimicrobial peptide preparation with immediate and rapid bactericidal efficacy. The inner core layer uses photosensitive methacrylamide hyaluronic acid and encapsulates mesenchymal stem cell exosomes rich in bioactive components.

[0005] Preferably, the antimicrobial peptide is LL-37 antimicrobial peptide, which is an amphiphilic cathepsin-derived antimicrobial peptide with 37 residues; the mesenchymal stem cell exosomes are derived from the supernatant of human umbilical cord mesenchymal stem cells.

[0006] Furthermore, the present invention also provides a method for preparing the above-mentioned multifunctional microneedle patch with pH-responsive sequential drug release function, comprising the following steps: S1. Dissolve oxidized hyaluronic acid and carboxymethyl chitosan separately in deionized water, then mix them in a volume ratio of 1:1 to 1:3 and add antimicrobial peptides. Pour the mixture into a PDMS microneedle mold, centrifuge at low speed to fully fill the microneedle tip with the solution, and crosslink at room temperature to obtain an OHA-CMCS shell encapsulated with antimicrobial peptides. S2. Dissolve methacrylamide hyaluronic acid and photoinitiator in deionized water to prepare a solution, then add mesenchymal stem cell exosomes and mix. Add the mixture to the hollow OHA-CMCS shell containing antimicrobial peptides obtained in step S1, centrifuge to fill the microneedle cavity, and solidify by ultraviolet irradiation to form a microneedle core layer, thus obtaining the product.

[0007] Specifically, in step S1, the oxidized hyaluronic acid is dissolved in deionized water at a mass concentration of 2-4%; the carboxymethyl chitosan is dissolved in deionized water at a mass concentration of 2-4%.

[0008] Specifically, in step S1, the antimicrobial peptide is LL-37 antimicrobial peptide, which is added to the mixture at a mass concentration of 1-5%.

[0009] Specifically, in step S1, the volume of the mixture is 40-50% of the volume of the PDMS microneedle mold; the low-speed centrifugation speed is 2000-4000 rpm and the time is 5-15 min.

[0010] Specifically, in step S2, the methacrylamide hyaluronic acid is dissolved in deionized water at a mass concentration of 8-12%; the photoinitiator is dissolved in deionized water at a mass concentration of 0.2-0.3%.

[0011] Specifically, in step S2, the mesenchymal stem cell exosomes are derived from the supernatant of human umbilical cord mesenchymal stem cells, and their concentration in the mixture is 1-5%.

[0012] Specifically, in step S2, the centrifugation speed is 2000~4000 rpm and the time is 5~15 min; the UV curing time is 10~15 min.

[0013] Furthermore, the present invention also claims the use of the above-mentioned multifunctional microneedle patch with pH-responsive sequential drug release function in the preparation of drugs for treating keratitis.

[0014] This invention relates to a multifunctional microneedle patch with pH-responsive sequential drug release. Its unique layered controlled-release design precisely achieves the time-dependent synergistic release of antimicrobial peptides and mesenchymal stem cell exosomes. The outer layer of the system is coated with an antimicrobial peptide formulation with immediate and rapid bactericidal efficacy. These cationic peptide molecules specifically target and disrupt the cell membrane integrity of pathogens, triggering a bacterial lysis effect due to osmotic pressure imbalance, thereby efficiently eliminating pathogenic microorganisms at the site of infection. The core layer is rich in bioactive mesenchymal stem cell exosomes, which can promote corneal tissue repair through multiple mechanisms, including regulating the local inflammatory microenvironment and activating endogenous cell proliferation pathways. This drug release strategy achieves both rapid antibacterial action and long-term repair, providing an optimized solution for the treatment of infectious keratitis.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention designs a multifunctional microneedle patch with sequential drug release function. The core-shell structure can realize layered controlled release and sequentially release antimicrobial peptides and mesenchymal stem cell exosomes, so that active ingredients with different functions can play a role in specific treatment stages, which meets the treatment requirements of "killing bacteria first and then repairing" for infectious keratitis.

[0016] (2) The antimicrobial peptides in the outer layer of the microneedles of the present invention can kill bacteria and destroy microbial biofilms instantly and rapidly, and have a broad-spectrum antibacterial effect. They are not prone to drug resistance. They mainly rely on the specific destruction of pathogen cell membranes by cationic polypeptide molecules to induce bacterial lysis and quickly eliminate pathogenic microorganisms from the lesions of infection. The core layer of the microneedles can release bioactive components such as mesenchymal stem cell exosomes, which can promote corneal tissue repair by regulating the inflammatory microenvironment and activating cell proliferation pathways.

[0017] (3) The present invention prepares a multifunctional microneedle patch with sequential drug release function based on the template method. The method is simple, easy to operate, inexpensive and does not require high technical requirements. Attached Figure Description

[0018] 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.

[0019] Figure 1 This is a schematic diagram of the preparation process of the multifunctional microneedle patch with sequential drug release function of the present invention.

[0020] Figure 2 Light microscopy and fluorescence images of the multifunctional microneedle patch with sequential drug release function of the present invention.

[0021] Figure 3 This invention relates to the pH-responsive release function of a multifunctional microneedle patch with sequential drug release capabilities.

[0022] Figure 4 This invention relates to the in vivo sustained-release effect of a multifunctional microneedle patch with sequential drug release function.

[0023] Figure 5 The present invention provides a multifunctional microneedle patch with sequential drug release function for the treatment of a rat model of bacterial keratitis.

[0024] Figure 6 This is for staining corneal sections of animals after treatment with the microneedle patch of the present invention. Detailed Implementation

[0025] The present invention can be better understood from the following embodiments.

[0026] In the following examples, the antimicrobial peptide is LL37, which was purchased from Qiangyao Biotechnology Co., Ltd.

[0027] Mesenchymal stem cell exosomes were derived from the supernatant of human umbilical cord mesenchymal stem cells and prepared as follows: Human umbilical cord mesenchymal stem cells were purchased from the Jiangsu Provincial Stem Cell Center and cultured in F12 / DMEM complete medium. The stem cells used were within the 10th passage. When the cells reached approximately 80% confluence, they were passaged. Subsequently, the medium was replaced with exosome-free medium, and cultured for another 48 hours. The culture supernatant after 48 hours was collected, and exosomes were separated using gradient centrifugation. The culture supernatant was centrifuged at 300g for 10 minutes, and the supernatant was collected; then centrifuged at 2000g for 10 minutes to remove cells and cell debris. Cell residue was removed by centrifugation at 10,000g for 30 minutes, and finally centrifuged at 100,000g for 2 hours to obtain the exosome pellet. All centrifugation steps were performed at 4°C. Example 1

[0028] like Figure 1 As shown, the preparation method of the multifunctional microneedle patch with pH-responsive sequential drug release function of the present invention includes the following steps: S1. Dissolve oxidized hyaluronic acid and carboxymethyl chitosan separately in deionized water, then mix them in a volume ratio of 1:1 to 1:3 and add antimicrobial peptides. Pour the mixture into a PDMS microneedle mold, centrifuge at low speed to fully fill the microneedle tip with the solution, and crosslink at room temperature to obtain an OHA-CMCS shell encapsulated with antimicrobial peptides. S2. Dissolve methacrylamide hyaluronic acid and photoinitiator in deionized water to prepare a solution, then add mesenchymal stem cell exosomes and mix. Add the mixture to the hollow OHA-CMCS shell containing antimicrobial peptides obtained in step S1, centrifuge to fill the microneedle cavity, and solidify by ultraviolet irradiation to form a microneedle core layer, thus obtaining the product. Example 2

[0029] Preparation of a multifunctional microneedle patch with sequential drug release function.

[0030] To prepare a core-shell microneedle patch, OHA (3%) and CMCS (3%) were first dissolved in deionized water and mixed at a volume ratio of 1:1 to 1:3. Then, 2 mL of the mixed solution was poured into a PDMS microneedle mold, and centrifuged at low speed to ensure the solution fully filled the microneedle tips (3000 rpm, 10 min). After air drying at room temperature overnight, the cross-linked OHA-CMCS formed the shell layer of the multifunctional patch. Subsequently, a 10% HAMA solution dissolved in 0.5 mL of deionized water and a photoinitiator (LAP, 0.25%) were added to the cavity of the hollow MN patch, and centrifuged at low speed to fill the microneedle cavity with HAMA solution (3000 rpm, 10 min), forming the microneedle core layer. After 10 min of 365 nm ultraviolet (UV) irradiation to cure the HAMA, a blank core-shell microneedle patch was obtained.

[0031] The following method was used to prepare a multifunctional core-shell microneedle patch encapsulating a drug: Antimicrobial peptides of different concentrations were added to a mixture of OHA and CMCS. This mixture was then added to a PDMS mold, and centrifuged at low speed to ensure the solution fully filled the microneedle tips. After drying, the shell layer of the patch was formed. Exosome solution was mixed with an aqueous HAMA solution, and centrifuged to fill the microneedle cavity, forming the microneedle core layer. The HAMA was then cured by UV irradiation to obtain the dual-drug-loaded multifunctional core-shell microneedle patch. Figure 2 .like Figure 2 As shown in ab, the microneedles are neatly arranged with sharp tips. Figure 2 The image on the CD is a scanning electron microscope image, indicating that the surface of the microneedles is smooth. Figure 2 e represents the structure of the core-shell microneedle, indicating that the prepared microneedle has a height of 600 micrometers and a spacing of 500 micrometers. Figure 2 f is the fluorescence image of the microneedles, with the shell layer of the microneedles marked with green fluorescence and the core layer marked with red fluorescence. Example 3

[0032] Sustained-release effect of multifunctional microneedle patches with sequential drug release function.

[0033] To investigate the drug release performance of OHA-CMCS hydrogel under different acid and alkaline environments, this study used FITC-BSA as a model drug to evaluate the differences in drug release from the hydrogel under different pH conditions and at different time points. Figure 3 The results showed that the drug release rate was significantly faster in an acidic environment (pH=6.0), with a cumulative release rate of 50% after 12 hours; while in a neutral environment (pH=7.4), the drug release was slower, requiring 48 hours to reach a cumulative release rate of 50%.

[0034] The sustained release of microneedles on the rat cornea was evaluated using a small animal in vivo optical imaging system. Patients were divided into two groups based on administration route: microneedle administration with cell membrane far-infrared fluorescent probe labeling (microneedle group) and CM-DID ophthalmic drops administration (ophthalmic drops group). The amount of residual dye in the mouse cornea was examined on days 0, 1, and 2 post-administration using a small animal imaging system. All images from different mice were compared under the same exposure intensity. Figure 4 As shown in the small animal imaging results, the core-shell microneedles remained on the ocular surface of rats for at least 24 hours and gradually disappeared after 48 hours. In contrast, the eye drops (Drop group) were rapidly lost through the lacrimal duct, resulting in a shorter drug residence time and failing to achieve a prolonged local effect. The continuous residence time of the microneedles (MNs) not only prolonged the drug's residence time on the ocular surface but also significantly increased the local drug concentration, thereby contributing to enhanced therapeutic efficacy. Example 4

[0035] Multifunctional microneedle patches with sequential drug release function are used in the treatment of keratitis.

[0036] SD rats weighing approximately 200g were selected for animal experiments. The SD rats were anesthetized with isoflurane (RWD), and a drop of lidocaine hydrochloride was applied topically to the cornea. After removing the corneal epithelium, 20 μL of a Staphylococcus aureus bacterial suspension (1×10⁻⁶) was applied to the surface. 7 A bacterial keratitis model was established using CFU / mL. Multifunctional microneedle patches with sequential drug release were then applied to the cornea once daily for 3 consecutive days. Rats were treated according to their grouping: (1) Control group: No special treatment was given to the rats after modeling; (2) Blank microneedle group (MN): The microneedle patch was not encapsulated with the drug and applied to the cornea once daily for 3 consecutive days; (3) Encapsulated antimicrobial peptide microneedle group (AMP@MN): Only the microneedle shell was encapsulated with LL37 antimicrobial peptide, applied to the cornea once daily for 3 consecutive days; (4) Encapsulated antimicrobial peptide and MSC-Exo microneedle group (AMP / Exo@MN): The microneedle shell was encapsulated with LL37 antimicrobial peptide, and the core layer was encapsulated with MSC-Exo, applied to the cornea once daily for 3 consecutive days. The corneal condition of the rats was observed daily after treatment, and corneal inflammation and repair were observed using slit lamp and sodium fluorescein staining. Figure 5The images show corneal images 1, 3, 5, and 7 days after microneedling treatment. Image 5a indicates the degree of corneal opacity, and image 5b shows the degree of corneal defect represented by sodium fluorescein staining. Observation of the cornea revealed significant corneal pathological changes in both the control group and the blank microneedling group, manifested as persistent edema, extensive opacity, and large areas of positive sodium fluorescein staining; some samples even showed corneal perforation. Although most rats showed a tendency for spontaneous healing of the cornea on day 7, it remained in an active inflammatory state. In contrast, the AMP / Exo@MN treatment group showed the best efficacy, indicating that it effectively delayed the progression of corneal opacity and reduced the area of ​​corneal epithelial defect compared to the control group.

[0037] Figure 6 This is for staining corneal sections of animals treated with the microneedle patch of the present invention. Figure 6 a) is H&E staining of the cornea, used to assess tissue morphology. For example... Figure 6 As shown in b, significant corneal thickening was observed in both the control and blank microneedle groups (mean corneal thickness in the control group: 520.6±44.1 μm, in the blank microneedle group: 297.0±40.1 μm) accompanied by extensive inflammatory cell infiltration, indicating severe corneal inflammation. The AMP@MN group showed slight corneal thickening (170.0±28.2 μm), while the AMP / Exo@MN group had the lowest corneal thickness (105.8±10.8 μm) and no significant inflammatory cell infiltration, indicating that the microneedles have anti-inflammatory and repair-promoting effects. Compared to the control group, corneal thickness was significantly reduced in all treatment groups.

[0038] This invention provides a multifunctional microneedle patch with pH-responsive sequential drug release function, 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 multifunctional microneedle patch with pH-responsive sequential drug release function, characterized in that, It has an inner and outer core-shell structure. The outer layer of the microneedles uses a pH-responsive material to encapsulate antimicrobial peptides with immediate and rapid bactericidal efficacy. The inner core layer uses photosensitive methacrylamide hyaluronic acid and encapsulates mesenchymal stem cell exosomes rich in bioactive components.

2. The multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 1, characterized in that, The antimicrobial peptide is LL37 antimicrobial peptide; the mesenchymal stem cell exosomes are derived from the supernatant of human umbilical cord mesenchymal stem cells.

3. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function as described in claim 1, characterized in that, Includes the following steps: S1. Dissolve oxidized hyaluronic acid and carboxymethyl chitosan separately in deionized water, then mix them in a volume ratio of 1:1 to 1:3 and add antimicrobial peptides. Pour the mixture into a PDMS microneedle mold, centrifuge at low speed to fully fill the microneedle tip with the solution, and crosslink at room temperature to obtain an OHA-CMCS shell encapsulated with antimicrobial peptides. S2. Dissolve methacrylamide hyaluronic acid and photoinitiator in deionized water to prepare a solution, then add mesenchymal stem cell exosomes and mix. Add the mixture to the hollow OHA-CMCS shell containing antimicrobial peptides obtained in step S1, centrifuge to fill the microneedle cavity, and solidify by ultraviolet irradiation to form a microneedle core layer, thus obtaining the product.

4. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 3, characterized in that, In step S1, the oxidized hyaluronic acid is dissolved in deionized water at a mass concentration of 2-4%; the carboxymethyl chitosan is dissolved in deionized water at a mass concentration of 2-4%.

5. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 3, characterized in that, In step S1, the antimicrobial peptide is LL37 antimicrobial peptide, which is added to the mixture at a mass concentration of 1-5%.

6. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 3, characterized in that, In step S1, the volume of the mixture is poured into the PDMS microneedle mold at 40-50% of its volume, and the low-speed centrifugation is performed at 2000-4000 rpm for 5-15 minutes.

7. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 3, characterized in that, In step S2, the methacrylamide hyaluronic acid is dissolved in deionized water at a mass concentration of 8-12%; the photoinitiator is dissolved in deionized water at a mass concentration of 0.2-0.3%.

8. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 3, characterized in that, In step S2, the mesenchymal stem cell exosomes are derived from the supernatant of human umbilical cord mesenchymal stem cells, and their concentration in the mixture is 1-5%.

9. The method for preparing the multifunctional microneedle patch with pH-responsive sequential drug release function according to claim 3, characterized in that, In step S2, the centrifugation speed is 2000~4000 rpm and the time is 5~15 min; the UV curing time is 10~15 min.

10. The use of the multifunctional microneedle patch with pH-responsive sequential drug release function as described in claim 1 or 2 in the preparation of drugs for treating keratitis.

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