Intelligent delivery patch based on epidermal stem cell adhesion regulation and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to an intelligent delivery patch based on epidermal stem cell adhesion regulation and a preparation method of the intelligent delivery patch. The intelligent delivery patch is prepared from the following raw materials in parts by weight: 1-5 parts of a drug-loaded compound, 8-15 parts of methacrylated gelatin, 0.5-2 parts of an enzyme response cross-linking agent, 0.3-1.5 parts of a force sensitive linking ligand, 0.1-1 part of a cell adhesion ligand and 0.05-0.2 part of LAP, drug-loaded nanoparticles of which the surfaces are modified with beta-cyclodextrin are anchored through a hydrogel network containing an azobenzene group, MMP sensitive peptide and RGD peptide, and the drug-loaded nanoparticles and the RGD peptide are combined to form the intelligent delivery patch. Efficient capture and enzyme response desorption of epidermal stem cells are achieved through synergism of MMP sensitive peptide and RGD peptide, a force sensitive release system is constructed through a supramolecular subject and object formed by beta-cyclodextrin and azobenzene, long-acting delivery is achieved by responding to mechanical stimulation while burst release of drugs is inhibited, and wound repair is promoted synergistically.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an intelligent delivery patch based on the adhesion regulation of epidermal stem cells and its preparation method. Background Technology

[0002] The repair process of skin damage is highly dependent on the migration and proliferation of epidermal stem cells and the synergistic effect of growth factors. For large-area full-thickness skin defects or chronic, difficult-to-heal wounds such as diabetic ulcers, the body's own repair mechanisms often fail to achieve effective regeneration, leading to prolonged healing periods and increased risk of complications.

[0003] Currently, most dressings and tissue-engineered scaffolds used in clinical practice focus on providing physical barriers or a moist healing environment, lacking the ability to actively and intelligently regulate cell behavior. Although some materials are modified with RGD peptides to improve biocompatibility, this adhesion is usually static and irreversible. After stem cells are adsorbed onto the material surface, they often cannot detach from the scaffold and migrate to the wound area at the appropriate time. As a result, although the cells are recruited, they are permanently anchored to the dressing and cannot effectively participate in in-situ tissue regeneration. In addition, most existing materials lack the ability to specifically recognize and efficiently capture epidermal stem cells, resulting in insufficient cell recruitment in the early stages of repair, further weakening regeneration efficiency.

[0004] In terms of drug delivery, traditional hydrogel dressings rely on physical porous structures to encapsulate drugs, and the release behavior is diffusion-controlled, which can easily lead to initial drug burst release. This can not only cause excessively high local concentrations and adverse reactions, but also cause the drug concentration to drop rapidly in subsequent treatment phases, making it difficult to maintain a stable treatment window. In addition, the wound microenvironment has dynamic characteristics, including mechanical stress generated by fluid flow and cell migration, but existing drug delivery systems lack response mechanisms to these dynamic factors, making it impossible to achieve precise matching between drug release and wound physiological processes.

[0005] Therefore, developing a delivery system that can intelligently sense changes in the microenvironment and synergistically regulate cell migration and drug release has become a core requirement for breaking through the current bottlenecks in wound healing treatment. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a smart delivery patch based on epidermal stem cell adhesion regulation and its preparation method. This smart delivery patch utilizes the synergistic effect of MMP-sensitive peptides and RGD peptides to achieve efficient capture and enzyme-responsive desorption of epidermal stem cells. By constructing a force-sensitive release system through a supramolecular host-guest structure formed by β-cyclodextrin and azophenyl, it achieves long-term delivery in response to mechanical stimulation while inhibiting drug burst release, thus synergistically promoting wound repair.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a smart delivery patch based on epidermal stem cell adhesion regulation. The smart delivery patch comprises the following raw materials: 1-5 parts of drug-loaded complex, 8-15 parts of methacrylamide gelatin, 0.5-2 parts of enzyme-responsive crosslinking agent, 0.3-1.5 parts of force-sensitive linker ligand, 0.1-1 parts of cell adhesion ligand, and 0.05-0.2 parts of LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphite).

[0009] Further, the drug-loaded complex comprises raw materials in the following mass ratio: drug-loaded nanoparticles: mono-6-aldehyde-β-cyclodextrin: ethylenediamine: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide): NHS (N-hydroxysuccinimide): NaBH3CN (sodium cyanoborohydride) = 10:5:3:2:1:1.

[0010] Furthermore, the drug-loaded nanoparticles comprise raw materials in the following mass ratio: EGF (recombinant human epidermal growth factor): PLGA (polylactic acid-glycolic acid copolymer): PVA (polyvinyl alcohol) = 1:18:1.

[0011] The preparation method of the drug-loaded nanoparticles includes the following:

[0012] A1: Weigh EGF and dissolve it in PBS buffer to obtain the inner aqueous phase; weigh PLGA and dissolve it in dichloromethane to obtain the oil phase; weigh PVA and prepare a 2% PVA aqueous solution to obtain the outer aqueous phase; the EGF, PLGA and PVA are mixed in a mass ratio of 1:18:1.

[0013] A2: Under ice bath conditions, the inner aqueous phase is added to the oil phase and ultrasonically treated to obtain the primary emulsion. The primary emulsion is then added to the pre-cooled outer aqueous phase and emulsified to obtain the secondary emulsion.

[0014] A3: Transfer the emulsion to room temperature and stir continuously to allow the organic solvent DCM to evaporate completely and the nanoparticles to solidify. Centrifuge and collect the precipitate, wash the precipitate, freeze dry it, and obtain drug-loaded nanoparticles.

[0015] Furthermore, the preparation method of the drug-loaded complex includes the following steps:

[0016] S1: Weigh 10 parts of drug-loaded nanoparticles and disperse them in MES buffer (2-morpholinoethanesulfonic acid). After stirring evenly, add 2 parts of amide coupling agent EDC and 1 part of NHS to obtain an activation solution. After the reaction is complete, centrifuge, collect the precipitate and wash it to remove unreacted EDC / NHS and obtain activated nanoparticles.

[0017] S2: The activated nanoparticles were resuspended in PBS buffer, and 3 parts of ethylenediamine were added to obtain the reaction solution. After the reaction was completed, dialysis was performed to remove unreacted ethylenediamine and small molecule byproducts. The liquid in the dialysis bag was centrifuged and the precipitate was collected and freeze-dried to obtain aminated drug-loaded nanoparticles.

[0018] S3: Under room temperature and light-protected conditions, the aminated drug-loaded nanoparticles were dispersed in PBS buffer, and 5 parts of mono-6-aldehyde-β-cyclodextrin were added to obtain a mixture. After the reaction was completed, 1 part of reducing agent NaBH3CN was added to the mixture, and the reaction was continued. Finally, the drug-loaded complex was obtained by centrifugation and freeze-drying.

[0019] Furthermore, the enzyme-responsive crosslinking agent is prepared by modifying the N-terminus of the MMP-sensitive peptide with an acryloyl group and the C-terminus with an azide group, and its amino acid sequence is Acryl-GPLGYLWARK-N3, as shown in SEQ ID NO.1.

[0020] Furthermore, the cell adhesion ligand is prepared by modifying the N-terminus of the RGD peptide with a DBCO (dibenzocyclooctylene) group and the C-terminus with an acetyl group, and its amino acid sequence is DBCO-GRGDS-Ac, as shown in SEQ ID NO.2.

[0021] Furthermore, the force-sensitive linker ligand is prepared by linking azophenyl groups and acryloyl groups to both ends of polyethylene glycol, and its structure is Azo-PEG-Acryl.

[0022] This invention also provides a method for preparing a smart delivery patch based on epidermal stem cell adhesion regulation, specifically including the following steps:

[0023] Step 1: Under light-protected conditions, weigh 8-15 parts of methacrylamide gelatin, 0.5-2 parts of enzyme-responsive crosslinking agent, and 0.3-1.5 parts of force-sensitive linker ligand, and dissolve them together in PBS buffer. Stir until completely dissolved to obtain a prepolymer precursor. Add 1-5 parts of drug-loaded complex and 0.05-0.2 parts of photoinitiator LAP to the precursor and stir until evenly dispersed to obtain the prepolymer.

[0024] Step 2: In a light-protected environment, the prepolymer solution is injected into the groove of a PDMS (polydimethylsiloxane) mold with a circular groove. After irradiation under 365 nm ultraviolet light for 60 seconds, it is removed from the mold to obtain the hydrogel substrate.

[0025] Step 3: Weigh 0.1-1 part of cell adhesion ligand, dissolve it in PBS buffer to obtain a modification solution, immerse the hydrogel patch in the modification solution, and carry out a click chemical reaction at room temperature and in the dark to obtain an RGD peptide-grafted hydrogel patch. Immerse the patch in a large amount of fresh PBS buffer to wash it and remove unreacted cell adhesion ligands. Let it air dry to obtain a smart delivery patch based on epidermal stem cell adhesion regulation.

[0026] The beneficial effects achieved by this invention are as follows:

[0027] The intelligent delivery patch based on epidermal stem cell adhesion regulation prepared in this invention achieves intelligent regulation of epidermal stem cell adhesion behavior through the synergistic design of enzyme-responsive cross-linking agents and cell adhesion ligands. RGD peptides are covalently grafted onto the hydrogel surface via click chemistry, thereby efficiently capturing epidermal stem cells through integrin-mediated regeneration, providing an essential cell source for tissue regeneration. Furthermore, the introduced MMP-sensitive peptide responds specifically to the matrix metalloproteinase microenvironment highly expressed in the later stages of wound healing, causing RGD peptides and their attached stem cells to detach from the patch surface and migrate towards the wound. This sequential regulatory mechanism of capture followed by release ensures sufficient cell recruitment in the early stages of repair while overcoming the problem of delayed healing caused by permanently anchored cells.

[0028] In drug delivery, this invention constructs a force-sensitive drug release system based on supramolecular host-guest recognition. β-cyclodextrin on the surface of the drug-loaded complex forms a stable host-guest inclusion complex with azobenzene polyethylene glycol in the hydrogel network, locking nanoparticles within the hydrogel through physical anchoring. This structure effectively inhibits the burst release effect caused by simple diffusion of drugs in a static state. Simultaneously, under dynamic microenvironments (such as the traction force generated by body fluid flow or the adhesion and spreading of epidermal stem cells), the host-guest balance is disturbed, achieving long-term, controllable drug release through dynamic equilibrium dissociation.

[0029] The intelligent delivery patch designed in this invention integrates two core mechanisms: enzyme response regulation of cell adhesion and force response regulation of drug release. It has systematic advantages in epidermal stem cell recruitment, long-term sustained drug release, and wound microenvironment response, and has good potential for clinical translation. Attached Figure Description

[0030] Figure 1 The results of drug loading stability tests on the smart delivery patches prepared in Example 2 and Comparative Example 1;

[0031] Figure 2 The results of the in vitro release test of the smart delivery patches prepared in Example 2 and Comparative Example 2 under static conditions;

[0032] Figure 3The results of in vitro release tests under dynamic conditions for the smart delivery patches prepared in Example 2 and Comparative Example 2;

[0033] Figure 4 The results of rat pharmacodynamic tests on the smart delivery patches prepared in Example 2 and Comparative Examples 1-3 are shown. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0036] Unless otherwise specified, all methods used in the following examples are conventional. Unless otherwise specified, all materials used in the following examples are new materials purchased from the market and comply with the current edition of the Pharmacopoeia of the People's Republic of China or relevant drug / excipient standards issued by the National Medical Products Administration. The EGF is a white lyophilized powder with a specific activity of not less than 1.0 × 10⁻⁶. 6 The bacterial endotoxin content should be less than 1.0 EU / μg (IU / mg); the methacrylamide gelatin is a pale yellow to off-white solid or lyophilized powder, with a methacrylamide substitution degree typically between 60% and 90%; the PLGA has a lactic acid to glycolic acid molar ratio of 50:50 and an intrinsic viscosity of 0.55-0.75 dL / g; the LAP is a highly efficient water-soluble photoinitiator, suitable for polymerization initiated by ultraviolet light at wavelengths of 365 nm or 405 nm, with a purity ≥98%; the mono-6-aldehyde-β-cyclodextrin is a white powder with a purity ≥95%, and each β-cyclodextrin molecule is modified with approximately one aldehyde group on average; the ethylenediamine is a colorless and transparent liquid with a purity ≥99.0%, which is an amino group introduced onto the surface of nanoparticles; the EDC is in hydrochloride form; the NaBH3CN is a white powder with a purity ≥95.0%, which can specifically reduce imines to secondary amines.

[0037] Example 1: This example provides a smart delivery patch based on epidermal stem cell adhesion regulation. The smart delivery patch includes the following raw materials: 1 part of drug-loaded complex, 8 parts of methacrylamide gelatin, 0.8 parts of enzyme-responsive crosslinking agent, 0.3 parts of force-sensitive linker ligand, 0.1 parts of cell adhesion ligand, and 0.05 parts of LAP.

[0038] The drug-loaded complex comprises the following raw materials in parts by weight: 10 parts drug-loaded nanoparticles, 5 parts mono-6-aldehyde-β-cyclodextrin, 3 parts ethylenediamine, 2 parts EDC, 1 part NHS, and 1 part NaBH3CN.

[0039] The drug-loaded nanoparticles comprise the following raw materials in parts by weight: 5 parts EGF, 90 parts PLGA, and 5 parts PVA.

[0040] The preparation method of the drug-loaded nanoparticles includes the following:

[0041] A1: Weigh 5 parts of EGF and dissolve them in PBS buffer at pH 7.4 at a mass-to-volume ratio of 1 g: 20 mL to obtain the inner aqueous phase. Weigh 90 parts of PLGA and dissolve them in dichloromethane at a mass-to-volume ratio of 1 g: 25 mL to obtain the oil phase. Weigh 5 parts of PVA and dissolve them in 50 times the volume of ultrapure water to obtain the outer aqueous phase.

[0042] A2: Under ice bath conditions, the inner aqueous phase is added to the oil phase and ultrasonically treated for 60 seconds with a probe (power 100W, working time 3 seconds, interval 5 seconds) to obtain the primary emulsion. The primary emulsion is added to the pre-cooled outer aqueous phase and emulsified for 3 minutes at 10000 rpm under ice bath conditions to obtain the secondary emulsion.

[0043] A3: The double emulsion was transferred to room temperature and stirred continuously at 300 rpm for 6 hours. After centrifugation at 15,000 rpm at 4°C for 30 minutes, the precipitate was collected, washed three times with pre-cooled ultrapure water, and then freeze-dried to obtain drug-loaded nanoparticles.

[0044] The preparation method of the drug-loaded complex includes the following steps:

[0045] S1: Weigh 10 parts of drug-loaded nanoparticles and disperse them in 0.1 M, pH 6.0 MES buffer at a mass-to-volume ratio of 1 g: 100 mL. After stirring evenly, add 2 parts of EDC and 1 part of NHS to obtain the activation solution. After reacting at room temperature for 1 hour, centrifuge at 4℃ and 15000 rpm for 15 minutes, collect the precipitate, and wash it once quickly with pre-cooled MES buffer to obtain activated nanoparticles.

[0046] S2: The activated nanoparticles were resuspended in 50 times the volume of PBS buffer at pH 7.4, and 3 parts of ethylenediamine were added to obtain the reaction solution. After reacting at room temperature for 4 hours, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in a large volume of pre-cooled ultrapure water for 24 hours, with the water changed every 6 hours. The liquid in the dialysis bag was centrifuged at 4°C and 15,000 rpm for 30 minutes, the precipitate was collected, and the aminated drug-loaded nanoparticles were obtained by freeze drying.

[0047] S3: The aminated drug-loaded nanoparticles were dispersed in 50 times the volume of PBS buffer at pH 7.4. Five parts of mono-6-aldehyde-β-cyclodextrin were added to the mixture, and the mixture was reacted at room temperature in the dark for 12 hours to obtain a mixture. One part of NaBH3CN was added to the mixture, and the mixture was reacted at room temperature in the dark for another 6 hours. The mixture was then centrifuged at 4°C and 15,000 rpm for 30 minutes to collect the precipitate. The precipitate was resuspended in ultrapure water and washed three times by centrifugation. After freeze-drying, the drug-loaded complex was obtained.

[0048] This embodiment also provides a method for preparing a smart delivery patch based on epidermal stem cell adhesion regulation, which specifically includes the following steps:

[0049] Step 1: Weigh 8 parts of methacrylamide gelatin, 0.8 parts of enzyme-responsive crosslinking agent, and 0.3 parts of force-sensitive linker in a 40℃ light-protected water bath. Dissolve them together in 0.01 M, pH 7.4 PBS buffer at a mass-volume ratio of 1 g: 10 mL. Stir at 100 rpm in a 40℃ water bath for 10 minutes until completely dissolved to obtain the prepolymer precursor. Add 1 part of the drug-loaded complex and 0.05 parts of LAP to it and stir at room temperature and 100 rpm for 30 minutes to obtain the prepolymer.

[0050] Step 2: In a light-protected environment, quickly inject the PDMS (polydimethylsiloxane) into a mold groove with a diameter of 10 mm and a depth of 2 mm, and place it under 365 nm ultraviolet light at 25 mW / cm². 2 After being irradiated with strong ultraviolet light for 60 seconds, the sample was removed from the PDMS mold and immediately immersed in PBS buffer for storage in the dark to obtain the hydrogel substrate.

[0051] Step 3: Weigh 0.1 part of cell adhesion ligand and dissolve it in PBS buffer at pH 7.4 to prepare a 1 mM modification solution. Completely immerse the hydrogel patch in the modification solution and react it slowly at 80 rpm for 4 hours at room temperature and in the dark to obtain the RGD peptide-grafted hydrogel patch. Immerse it in a large amount of fresh PBS buffer and wash it on a shaker at 100 rpm for 30 minutes. Repeat the washing 3 times and air dry to obtain the smart delivery patch based on epidermal stem cell adhesion regulation.

[0052] Example 2: This example provides a smart delivery patch based on epidermal stem cell adhesion regulation. The smart delivery patch includes the following raw materials: 3 parts of drug-loaded complex, 10 parts of methacrylamide gelatin, 1 part of enzyme-responsive crosslinking agent, 0.5 parts of force-sensitive linker ligand, 0.2 parts of cell adhesion ligand, and 0.1 parts of LAP.

[0053] The drug-loaded complex comprises the following raw materials in parts by weight: 10 parts drug-loaded nanoparticles, 5 parts mono-6-aldehyde-β-cyclodextrin, 3 parts ethylenediamine, 2 parts EDC, 1 part NHS, and 1 part NaBH3CN.

[0054] The drug-loaded nanoparticles comprise the following raw materials in parts by weight: 5 parts EGF, 90 parts PLGA, and 5 parts PVA.

[0055] The preparation method of the drug-loaded nanoparticles is the same as in Example 1.

[0056] The preparation method of the drug-loaded complex is the same as in Example 1.

[0057] This embodiment also provides a method for preparing a smart delivery patch based on epidermal stem cell adhesion regulation, which specifically includes the following steps:

[0058] Step 1: Weigh 10 parts of methacrylamide gelatin, 1 part of enzyme-responsive crosslinking agent, and 0.5 parts of force-sensitive linker in a 40℃ light-protected water bath. Dissolve them together in 0.01 M, pH 7.4 PBS buffer at a mass-volume ratio of 1 g: 10 mL. Stir at 100 rpm in a 40℃ water bath for 10 minutes until completely dissolved to obtain the prepolymer precursor. Add 3 parts of drug-loaded complex and 0.1 parts of LAP to it and stir at room temperature and 100 rpm for 30 minutes to obtain the prepolymer.

[0059] Step 2: In a light-protected environment, quickly inject the PDMS (polydimethylsiloxane) into a mold groove with a diameter of 10 mm and a depth of 2 mm, and place it under 365 nm ultraviolet light at 25 mW / cm². 2 After being irradiated with strong ultraviolet light for 60 seconds, the sample was removed from the PDMS mold and immediately immersed in PBS buffer for storage in the dark to obtain the hydrogel substrate.

[0060] Step 3: Weigh 0.2 parts of cell adhesion ligand and dissolve it in PBS buffer at pH 7.4 to prepare a 1 mM modification solution. Completely immerse the hydrogel patch in the modification solution and react it slowly at 80 rpm for 4 hours at room temperature and in the dark to obtain the RGD peptide-grafted hydrogel patch. Immerse it in a large amount of fresh PBS buffer and wash it on a shaker at 100 rpm for 30 minutes. Repeat the washing 3 times and air dry to obtain the smart delivery patch based on epidermal stem cell adhesion regulation.

[0061] Example 3: This example provides a smart delivery patch based on epidermal stem cell adhesion regulation. The smart delivery patch includes the following raw materials: 5 parts of drug-loaded complex, 15 parts of methacrylamide gelatin, 2 parts of enzyme-responsive crosslinking agent, 1 part of force-sensitive linker ligand, 0.6 parts of cell adhesion ligand, and 0.2 parts of LAP.

[0062] The drug-loaded complex comprises the following raw materials in parts by weight: 10 parts drug-loaded nanoparticles, 5 parts mono-6-aldehyde-β-cyclodextrin, 3 parts ethylenediamine, 2 parts EDC, 1 part NHS, and 1 part NaBH3CN.

[0063] The drug-loaded nanoparticles comprise the following raw materials in parts by weight: 5 parts EGF, 90 parts PLGA, and 5 parts PVA.

[0064] The preparation method of the drug-loaded nanoparticles is the same as in Example 1.

[0065] The preparation method of the drug-loaded complex is the same as in Example 1.

[0066] This embodiment also provides a method for preparing a smart delivery patch based on epidermal stem cell adhesion regulation, which specifically includes the following steps:

[0067] Step 1: Weigh 15 parts of methacrylamide gelatin, 2 parts of enzyme-responsive crosslinking agent, and 1 part of force-sensitive linker in a 40℃ light-protected water bath. Dissolve them together in 0.01 M, pH 7.4 PBS buffer at a mass-volume ratio of 1 g: 10 mL. Stir at 100 rpm in a 40℃ water bath for 10 minutes until completely dissolved to obtain the prepolymer precursor. Add 5 parts of drug-loaded complex and 0.2 parts of LAP to it and stir at room temperature and 100 rpm for 30 minutes to obtain the prepolymer.

[0068] Step 2: In a light-protected environment, quickly inject the PDMS (polydimethylsiloxane) into a mold groove with a diameter of 10 mm and a depth of 2 mm, and place it under 365 nm ultraviolet light at 25 mW / cm². 2 After being irradiated with strong ultraviolet light for 60 seconds, the sample was removed from the PDMS mold and immediately immersed in PBS buffer for storage in the dark to obtain the hydrogel substrate.

[0069] Step 3: Weigh 0.6 parts of cell adhesion ligand and dissolve it in PBS buffer at pH 7.4 to prepare a 1 mM modification solution. Completely immerse the hydrogel patch in the modification solution and react it slowly at 80 rpm for 4 hours at room temperature and in the dark to obtain the RGD peptide-grafted hydrogel patch. Immerse it in a large amount of fresh PBS buffer and wash it on a shaker at 100 rpm for 30 minutes. Repeat the washing 3 times and air dry to obtain the smart delivery patch based on epidermal stem cell adhesion regulation.

[0070] The difference between Comparative Example 1 and Example 2 is that no enzyme-responsive cross-linking agent (Acryl-GPLGYLWARK-N3) was added, and an indestructible cross-linking agent (Acryl-PEG-N3) was used instead. The rest of the process is the same as in Example 2.

[0071] The difference between Comparative Example 2 and Example 2 is that no drug-loaded complex was prepared; instead, nanoparticles with the same theoretical drug loading capacity of EGF were used. The rest of the process is the same as in Example 2.

[0072] The difference between Comparative Example 3 and Example 2 is that no cell adhesion ligand was added; the rest of the parts are the same as Example 2.

[0073] Epidermal stem cell adhesion and detachment behavior test

[0074] The smart delivery patches prepared in Example 2, Comparative Example 1, and Comparative Example 3 were aseptically placed at the bottom of a 24-well plate (n=3), with 1×10⁶ patches inoculated into each well. 5 Pretreated HaCaT cells (1 mL / well) were used to simultaneously set up a blank group (no patch, only cell seeding) and a control group (only culture medium added). After incubation at 37℃ and 5% CO2 for 2 hours and 6 hours, the absorbance of each well at 570 nm was measured by crystal violet staining. The adhesion rate was calculated according to the formula: adhesion rate (%) = (OD570 value of experimental group - OD570 value of control group) / (OD570 value of blank group - OD570 value of control group) × 100%. The test results are shown in Table 1.

[0075] After the adhesion test, the remaining wells were divided into an enzyme-added group (medium medium supplemented with 50 μg / mL matrix metalloproteinase-13) and a non-enzyme-added group (normal medium), and cultured for 12 hours and 24 hours respectively. The culture medium was collected and centrifuged to obtain detached cells. The remaining cells on the patch surface were digested and counted. The detachment rate was calculated using the formula: Desorption rate (%) = (Number of detached cells / (Number of detached cells + Number of remaining cells) × 100%. The test results are shown below. Figure 1 .

[0076] Table 1. Results of cell adhesion test

[0077]

[0078] Drug loading stability and mechanical response release test

[0079] The smart delivery patches prepared in Example 2 and Comparative Example 2 were immersed in the same volume of PBS buffer (pH 7.4) (n=3) and incubated at 37°C. Samples were taken at 1, 6, 12, 24, 48, and 72 hours of incubation (with an equal volume of fresh PBS buffer added immediately). The concentration of EGF was detected by ELISA (enzyme-linked immunosorbent assay), and the cumulative release rate of EGF at each time point was calculated. The experimental results are shown in [Figure 1]. Figure 2 .

[0080] Two additional parallel samples were soaked in the same volume of pH 7.4 PBS buffer (n=3) and incubated on a 37°C constant-temperature shaker at 80 rpm (simulating the dynamic microenvironment of cell traction and fluid flow). Samples were taken and replenished with PBS at 1, 6, 12, 24, 48, and 72 hours of incubation, following the same method. EGF concentration was detected by ELISA, and the cumulative release rate of EGF at each time point was calculated. The experimental results are shown below. Figure 3 Cumulative release M n =C n ×V s +Σ(C i ×V s ×(V0-V s () / V0), cumulative release rate CR n (%)=(M n / M 总 )×100%, where C n V represents the EGF concentration at the nth time point. s V0 is the total volume of the released medium, and M is the volume of each sample taken. 总 This represents the total drug loading of EGF.

[0081] Rat pharmacodynamic testing

[0082] Healthy female SPF-grade SD rats were selected, and a full-thickness skin defect model was established on both sides of the spine on the back. The following experimental groups were set up: a blank control group (methacrylamide gelatin patch), a positive control group (commercially available EGF gel patch), and treatment groups (Examples 2 and Comparative Examples 1-3), with 8 rats in each group. The patch was changed every 3 days. On postoperative days 0, 3, 7, 10, and 14, digital photographs of the wound were taken. The wound area was calculated using ImageJ software. The wound healing rate (%) was calculated as (wound area on day 0 - wound area on day n) / wound area on day 0 × 100%. The experimental results are shown below. Figure 4Three rats were randomly selected and sacrificed on the 7th day after surgery. Wound tissue was collected and homogenized. The EGF concentration was detected by ELISA kit. The experimental results are shown in Table 2.

[0083] Table 2 Results of EGF retention concentration test in wound tissue on day 7

[0084]

[0085] Results Analysis

[0086] Table 1 shows that the smart delivery patches prepared in Example 2 and Comparative Example 1 exhibited significantly high adhesion rates at both 2 and 6 hours (adhesion rate >60% at 2 hours and >80% at 6 hours). In contrast, Comparative Example 3, which did not contain the cell adhesion ligand, showed an adhesion rate of only 18.7% at 6 hours, a highly significant difference compared to Examples 2 and 1. This indicates that the cell adhesion ligand (RGD peptide) is crucial for the initial capture of HaCaT cells; without this ligand, the smart delivery patch cannot actively recruit and adhere to cells.

[0087] Figure 1 The results showed that in Example 2, the desorption rate was 42.6% after 12 hours with enzyme addition, increasing to 71.3% after 24 hours, while the desorption rate remained below 25% without enzyme addition. Comparative Example 1 showed very low desorption rates under both enzyme-added and enzyme-free conditions. Comparative Example 3 had a very low and fluctuating desorption rate due to the extremely small initial number of adherent cells, making it not meaningful for comparison. This indicates that matrix metalloproteinase-13 specifically cleaves the enzyme-responsive cross-linking agent, promoting the detachment of RGD peptides, thereby achieving enzyme-responsive cell desorption. Without this mechanism, even in the presence of RGD ligands, cells cannot be effectively released.

[0088] Figure 2 The results showed that, under static conditions, the control group 2 exhibited significant drug burst release, with a cumulative EGF release rate of 25.5% at 1 hour and as high as 50.4% at 6 hours, while the cumulative release rate of the example group at 6 hours was only 13.0%, which was significantly lower than that of the control group. Figure 3 The results showed that, under dynamic conditions, Example 2 group exhibited a clear mechanically responsive release characteristic. As the oscillation time increased, the EGF release rate gradually increased, reaching a cumulative release rate of 85.8% after 48 hours and 92.8% after 72 hours, achieving long-term delivery. In contrast, Comparative Example 2 group showed rapid drug release without long-term effect, with a cumulative release rate approaching 80% after 12 hours. This indicates that the host-guest interaction formed between the mechanosensitive linker and the cyclodextrin on the surface of the drug-loaded complex can effectively inhibit drug burst release and achieve long-term, controllable drug delivery in response to mechanical stimuli.

[0089] Figure 4The results showed that the wound closure rate of Group 2 reached 76.9% on the 7th day after surgery and the wound healing rate was as high as 98.5% on the 14th day. In contrast, the healing rates of Group 1 (due to the lack of enzyme-responsive cross-linking agents) and Group 2 (due to the burst release of drugs) were the second lowest. Group 3 (due to the lack of cell adhesion ligands and insufficient stem cell recruitment) had a healing rate close to that of the blank control group. Similarly, the results in Table 2 showed that Group 2 had the highest EGF retention concentration in the wound tissue on the 7th day and the best local drug concentration maintenance. Group 2 (due to the burst release of drugs) had the lowest tissue concentration in the later stage.

[0090] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0091] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A smart delivery patch based on epidermal stem cell adhesion regulation, characterized in that, The smart delivery patch comprises the following raw materials in parts by weight: 1-5 parts of drug-loaded complex, 8-15 parts of methacrylamide gelatin, 0.5-2 parts of enzyme-responsive crosslinking agent, 0.3-1.5 parts of force-sensitive linker ligand, 0.1-1 parts of cell adhesion ligand, and 0.05-0.2 parts of LAP; The drug-loaded complex comprises raw materials in the following mass ratio: drug-loaded nanoparticles: mono-6-aldehyde-β-cyclodextrin: ethylenediamine: EDC: NHS: NaBH3CN = 10: 5: 3: 2: 1: 1; The preparation method of the drug-loaded complex includes the following steps: S1: Drug-loaded nanoparticles are reacted with EDC and NHS and activated to obtain activated nanoparticles; S2: The activated nanoparticles were resuspended, and ethylenediamine was added to them for reaction. After dialysis and drying, amination-loaded drug nanoparticles were obtained. S3: After dispersing the aminated drug-loaded nanoparticles, they were mixed with mono-6-aldehyde-β-cyclodextrin, reduced with NaBH3CN, centrifuged and dried to obtain the drug-loaded complex.

2. The intelligent delivery patch based on epidermal stem cell adhesion regulation according to claim 1, characterized in that, The drug-loaded nanoparticles comprise raw materials EGF:PLGA:PVA in the following mass ratio: 1:18:1; the preparation method of the drug-loaded nanoparticles includes the following: A1: Dissolve EGF to obtain the internal aqueous phase, dissolve PLGA to obtain the oil phase, and prepare an aqueous solution from PVA to obtain the external aqueous phase; A2: Add the inner aqueous phase to the oil phase, sonicate to obtain the primary emulsion, add the primary emulsion to the outer aqueous phase, emulsify to obtain the secondary emulsion; A3: Centrifuge the re-emulsion to collect the precipitate, wash and dry it to obtain drug-loaded nanoparticles.

3. The intelligent delivery patch based on epidermal stem cell adhesion regulation according to claim 2, characterized in that, The sequence of the enzyme-responsive crosslinking agent is Acryl-GPLGYLWARK-N3, as shown in SEQ ID NO.

1.

4. The intelligent delivery patch based on epidermal stem cell adhesion regulation according to claim 2, characterized in that, The sequence of the cell adhesion ligand is DBCO-GRGDS-Ac, as shown in SEQ ID NO.

2.

5. The intelligent delivery patch based on epidermal stem cell adhesion regulation according to claim 2, characterized in that, The force-sensitive linker ligand is prepared by linking azophenyl groups and acryloyl groups to both ends of polyethylene glycol, and its structure is Azo-PEG-Acryl.

6. A method for preparing a smart delivery patch based on epidermal stem cell adhesion regulation according to any one of claims 1-5, characterized in that, The specific preparation method is as follows: Step 1: Dissolve methacrylamide gelatin, enzyme-responsive crosslinking agent and force-sensitive linking ligand together to obtain a prepolymer precursor, add drug-loaded complex and LAP to it to obtain a prepolymer; Step 2: Inject the prepolymer liquid into the mold, irradiate it under ultraviolet light, and remove it to obtain the hydrogel substrate; Step 3: Dissolve the cell adhesion ligand to obtain a modification solution, immerse the hydrogel substrate in the modification solution to react, and obtain an RGD peptide-grafted hydrogel patch. Wash and dry the patch to obtain a smart delivery patch based on epidermal stem cell adhesion regulation.

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