Chitosan-heparin hydrogel composite gelatin microsphere drug delivery system and its preparation method

Abstract

This invention discloses a chitosan-heparin hydrogel composite gelatin microsphere drug delivery system and its preparation method, belonging to the field of biomedical engineering technology. The system comprises a chitosan-heparin hydrogel matrix and gelatin microspheres loaded with asiaticoside; the asiaticoside-loaded gelatin microspheres are uniformly dispersed in the chitosan-heparin hydrogel matrix, forming a dual-drug-loaded composite structure. The beneficial effects of this invention are: the system combines the synergistic effects of rapid drug release from the hydrogel and slow, sustained release from the microspheres; it exhibits good biocompatibility and a mild preparation process; it can precisely match the needs of different stages of diabetic wound healing; it effectively inhibits inflammation, promotes angiogenesis and collagen remodeling; and significantly improves the healing effect of diabetic wounds, making it suitable for the preparation of dressings for diabetic wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a chitosan-heparin hydrogel composite gelatin microsphere drug delivery system and its preparation method. Background Technology

[0002] China has the largest number of diabetes patients in the world, with an incidence of diabetic wounds (such as diabetic foot ulcers) of approximately 15% and an amputation rate as high as 19.03%, making it a significant public health issue. Previous studies have shown that hyperglycemia inhibits wound healing through the accumulation of AGEs (advanced glycation end products), excessive activation of the NF-κB inflammatory pathway, and dysfunction of EPCs (endothelial progenitor cells). Wounds are characterized by long healing cycles, susceptibility to infection, persistent inflammatory responses, insufficient angiogenesis, and impaired collagen remodeling. Traditional treatments focus on exploring the mechanisms of wound healing impairment in the hyperglycemic microenvironment (such as oxidative stress, inflammatory disorders, insufficient angiogenesis, and neuropathy), combining traditional Chinese medicine or natural active ingredients, while also focusing on low-cost, easily disseminated wound care techniques. Centella asiatica glycoside, as a natural active ingredient, has anti-inflammatory effects and promotes fibroblast proliferation, angiogenesis, and collagen synthesis. However, its poor water solubility and short half-life mean that direct topical application is easily and rapidly eliminated, making it difficult to maintain an effective therapeutic concentration at the wound site. Hydrogel dressings commonly utilize naturally derived polymers such as chitosan. Chitosan is widely available, and its structure is similar to glycosaminoglycans in the human extracellular matrix, thus causing minimal irritation and reducing the likelihood of immune rejection. Heparin, as an endogenous substance, possesses excellent biocompatibility. The hydrogel formed by these two components can be gradually degraded into smaller molecules in vivo through enzymatic hydrolysis, which are then absorbed and utilized by the body. In its mechanism of action as a dressing for wound treatment, chitosan, carrying a positive charge, interacts with negatively charged red blood cells and platelets, effectively promoting blood clotting and rapidly controlling wound bleeding. Heparin, at a certain concentration, can indirectly inhibit thrombin by activating antithrombin III. By designing a specific chitosan / heparin ratio in the hydrogel system, its synergistic effect with chitosan avoids the risk of thrombosis due to excessive clotting and creates a suitable coagulation microenvironment at the wound site, achieving highly efficient and safe hemostasis. Furthermore, it exhibits excellent antibacterial activity. Under acidic conditions, the amino groups in chitosan molecules are protonated to form positively charged cationic groups. These groups can adsorb and disrupt bacterial cell membranes, causing leakage of bacterial contents, thus exerting a broad-spectrum antibacterial effect, showing good inhibitory effects against both Gram-positive and Gram-negative bacteria. Simultaneously, chitosan can stimulate the activity of the body's immune cells, enhance local immunity, and further reduce the chance of wound infection.

[0003] To control the release performance of encapsulated drugs, hydrogel / microsphere composite structures are a widely used approach. Gelatin microspheres, as drug-release carriers, allow for control of drug release rates by adjusting microsphere particle size and cross-linking degree. However, in existing technologies, single hydrogel or microsphere carriers struggle to achieve "programmed drug release" (i.e., dynamically adjusting the release rate according to the needs of different stages of wound healing). For example, rapid drug release is needed to suppress inflammation during the inflammatory phase, continuous release is needed to promote repair during the proliferative phase, and stable release is needed to maintain the effect during the remodeling phase. Therefore, constructing a programmed drug delivery system that matches the stage-specific needs of diabetic wound healing, while also incorporating antibacterial and hemostatic effects, is crucial for improving the treatment efficacy of chronic wounds.

[0004] A prior art technique discloses a hydrogel / nonwoven composite dressing loaded with Centella asiatica extract (publication number CN113842492A). This dressing uses chitosan-gelatin hydrogel to load the active ingredients of Centella asiatica, achieving passive sustained drug release through hydrogel swelling, and can be used for the repair of ordinary wounds. However, this technology has significant shortcomings: it uses a single hydrogel carrier, lacks environmental responsiveness, and cannot achieve programmed drug release; it does not incorporate heparin or specific modified structures, and therefore lacks targeted anti-inflammatory and angiogenesis-promoting capabilities; it is only suitable for conventional wounds and cannot address the healing bottlenecks in diabetic wounds, such as persistent inflammation, insufficient angiogenesis, and impaired collagen remodeling in the high-glucose microenvironment, thus failing to meet the clinical treatment needs of chronic diabetic wounds. Summary of the Invention

[0005] To address the limitations of existing drug delivery systems for diabetic wound treatment, which cannot achieve programmed release of asiaticoside and are difficult to match the needs of different stages of wound healing, this invention provides a chitosan-heparin hydrogel composite gelatin microsphere structure. The hydrogel formed by this structure, based on its excellent antibacterial, hemostatic, and self-healing properties, and its chemical bond responsiveness to high sugar and inflammatory environments, achieves programmed delivery of asiaticoside through the synergistic effect of the hydrogel's moisturizing and antibacterial properties and the slow, sustained release of the microspheres, thereby improving the healing rate of diabetic wounds.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: Chitosan-heparin hydrogel composite gelatin microsphere drug delivery system: The drug delivery system of this invention is composed of a chitosan-heparin hydrogel matrix and gelatin microspheres loaded with asiaticoside. The gelatin microspheres loaded with asiaticoside are uniformly dispersed in the chitosan-heparin hydrogel matrix to form a dual-drug-loaded composite structure, which can realize the programmed release of asiaticoside in response to the high sugar and inflammatory microenvironment of diabetic wounds.

[0007] Chitosan-heparin hydrogel: Chitosan-heparin hydrogel (CS-Hep) is a three-dimensional network formed by self-crosslinking of 3-aminophenylboronic acid-modified chitosan and heparin-modified polyvinyl alcohol after mixing. It contains borate ester bonds and has rapid swelling and initial drug release capabilities. The gelatin microspheres (GMs) loaded with asiaticoside were prepared by emulsification cross-linking method, using glutaraldehyde as the cross-linking agent, which can achieve continuous and stable release of asiaticoside (AA) by controlling the degree of cross-linking.

[0008] Preferably, the mass fraction of the gelatin microspheres loaded with asiaticoside in the composite system is 5% to 15%.

[0009] The mass ratio of 3-aminophenylboronic acid modified chitosan to heparin modified polyvinyl alcohol is 1:1; the mass percentage of heparin in the chitosan-heparin hydrogel is 1%.

[0010] The mass ratio of asiaticoside to gelatin in the gelatin microspheres loaded with asiaticoside is 1-10%.

[0011] Preparation method of chitosan-heparin hydrogel composite gelatin microsphere drug delivery system: Step 1: Preparation of gelatin microspheres loaded with asiaticoside; Step 2: Preparation of 3-aminophenylboronic acid modified chitosan and heparin modified polyvinyl alcohol; Step 3: Disperse the gelatin microspheres loaded with asiaticoside in 3-aminophenylboronic acid modified chitosan and heparin modified polyvinyl alcohol respectively, and then mix them to obtain a composite drug delivery system.

[0012] Specifically, it includes: Step 1: Preparation of gelatin microspheres loaded with asiaticoside (AA-GMs) Dissolve 5g of gelatin in 100mL of deionized water and stir at 55℃ until completely dissolved. Add 0.05-0.5g of asiaticoside and mix well to obtain the aqueous phase. The aqueous phase was added dropwise to liquid paraffin containing Tween-20 at a constant flow rate of 1 mL / min to 3 mL / min from 10 cm above the liquid surface. The concentration of Tween-20 was 0.5% to 1%. The mixture was stirred at 300 rpm to 500 rpm for 30 min to 60 min to form a W / O emulsion. Add glutaraldehyde crosslinking agent with a volume fraction of 0.5% v / v to 1% v / v, crosslink at 30℃ to 40℃ for 2h to 4h, allow to stand and separate into layers, collect microspheres by centrifugation, wash successively with petroleum ether and anhydrous ethanol, and freeze dry to obtain AA-GMs.

[0013] Step 2: Preparation of 3-aminophenylboronic acid modified chitosan (PABA-CS) and heparin modified polyvinyl alcohol (PVA-Hep) 4g of chitosan with a molecular weight of 100kd was dissolved in 100 mL of 1% glacial acetic acid solution and magnetically stirred until completely dissolved. 6g of EDS and 8g of NHS were added and stirred for 30 min. 4g of 3-aminophenylboronic acid (PABA) was added and the pH was adjusted to 8-10. After reacting for 6-8 h, unreacted small molecules were washed away with a 20kd-30kd ultrafiltration membrane and freeze-dried to obtain PABA-CS. 1 g of polyvinyl alcohol with a molecular weight of 10 kDa was dissolved in 100 mL of deionized water, heated to 90 °C, and magnetically stirred until completely dissolved. 3 g of EDS and 4 g of NHS were added and stirred for 30 min. Then, 0.5 g of sodium heparin (Hep) was added, and the pH was adjusted to 8–10. After reacting for 6–8 h, unreacted small molecules were washed away using a 3 kDa–5 kDa ultrafiltration membrane. PVA-Hep was obtained by freeze-drying.

[0014] Step 3: Preparation of the combined drug delivery system (AA-GMs / CS-Hep Gel) The AA-GMs obtained in step 1 were dispersed in PABA-CS and PVA-Hep at a mass fraction of 5% to 15%, respectively. After being mixed evenly, they were then mixed together to obtain AA-GMs / CS-Hep Gel.

[0015] The procedural delivery mechanism: Programmed release of asiaticoside for diabetic wound healing achieves the following: Initial rapid release (days 0-3, inflammatory phase): CS-Hep hydrogel swells rapidly upon contact with wound exudate, and the borate ester bonds in the hydrogel network respond to ROS and other factors to rapidly release asiaticoside, reaching peak local drug concentration (release amount accounts for 30%-40% of the total), inhibiting the release of inflammatory factors (such as TNF-α, IL-6) and reducing the inflammatory response. Mid-term sustained release (4 to 14 days, proliferation phase): As the hydrogel swelling stabilizes, AA-GMs continuously release asiaticoside (40% to 50% of the total release amount) due to the pores on the surface of the microspheres and slow degradation, promoting fibroblast migration and proliferation, vascular endothelial cell differentiation (upregulating VEGF expression), and accelerating granulation tissue formation. Stable release in the later stage (15-22 days, remodeling period): Gelatin microspheres further degrade, and residual asiaticoside is slowly released (the release amount accounts for 10%-20% of the total amount), which regulates the balance between collagen synthesis and degradation (reduces MMP-2 / 9 activity) and reduces scar formation.

[0016] Preferably, the drug delivery system is used in the preparation of dressings for diabetic wound treatment. The drug delivery system can maintain a long-lasting therapeutic concentration locally on diabetic wounds, shorten the wound healing cycle, reduce the risk of infection, and improve the healing quality of chronic wounds such as diabetic foot ulcers.

[0017] The beneficial effects of this invention are as follows: 1. Programmed release matching the wound healing stage: Through the synergistic effect of rapid release from hydrogel and slow sustained release from microspheres, the dynamic release of asiaticoside is achieved during the inflammatory, proliferative, and remodeling phases, thereby improving the precision of treatment; 2. Biocompatibility and safety: Chitosan, heparin, and gelatin are all natural biomaterials that are biodegradable, non-immunogenic, and reduce the risk of wound irritation. 3. Promotes wound healing: The anti-inflammatory, angiogenic and collagen-regulating effects of asiaticoside, combined with the moist environment protection of hydrogel, significantly shorten the healing time of diabetic wounds; 4. Simple preparation process: Microspheres are prepared by emulsification crosslinking and hydrogels are prepared by chemical crosslinking. The conditions are mild and easy to scale up. Attached Figure Description

[0018] Figure 1 The images are scanning electron microscope (SEM) images of the blank hydrogel group and the composite drug delivery system in Example 1.

[0019] Figure 2 The diagram shows the cumulative release of drugs in vivo and in vitro in Example 8, where (a) is the cumulative release of drugs in vitro and (b) is the cumulative release of drugs in vivo.

[0020] Figure 3 The graph shows the antibacterial activity of the composite drug delivery system at different mass concentrations in Example 9.

[0021] Figure 4 The bar chart shows the coagulation performance test results of the composite drug delivery system in Example 10.

[0022] Figure 5 The figure shows the evaluation results of the combined drug delivery system on HaCaT cell proliferation and toxicity in Example 11.

[0023] Figure 6 This is a comparison of the gross morphology of wound healing in diabetic mice treated with the combined drug delivery system in Example 12. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0025] Example 1: Preparation of a chitosan-heparin hydrogel composite gelatin microsphere drug delivery system Step 1: Preparation of gelatin microspheres loaded with asiaticoside (AA-GMs) Dissolve 5g of gelatin in 100mL of deionized water and stir at 55℃ until completely dissolved. Add 0.5g of asiaticoside and mix well to obtain the aqueous phase. The aqueous phase was added dropwise to liquid paraffin containing Tween-20 at a constant flow rate of 1 mL / min from a height of 10 cm above the liquid surface. The concentration of Tween-20 was 0.5%. The mixture was stirred at 300 rpm for 30 min to form a W / O emulsion. Add 0.5% v / v glutaraldehyde crosslinking agent, crosslink at 30℃ for 2 h, allow to stand and separate into layers, collect microspheres by centrifugation, wash successively with petroleum ether and anhydrous ethanol, freeze dry to obtain AA-GMs.

[0026] Step 2: Preparation of chitosan-heparin hydrogel (CS-Hep Gel) 4g of chitosan with a molecular weight of 100kd was dissolved in 100 mL of 1% glacial acetic acid solution and magnetically stirred until completely dissolved. 6g of EDS and 8g of NHS were added and stirred for 30 min. Then, 4g of 3-aminophenylboronic acid (PABA) was added and the pH was adjusted to 8. After reacting for 6 h, unreacted small molecules were removed by ultrafiltration with a 20kd membrane and freeze-dried to obtain PABA-CS.

[0027] 1 g of polyvinyl alcohol with a molecular weight of 10 kDa was dissolved in 100 mL of deionized water, heated to 90 °C, and magnetically stirred until completely dissolved. 3 g of EDS and 4 g of NHS were added and stirred for 30 min. Then, 0.5 g of sodium heparin (Hep) was added, the pH was adjusted to 8, and the reaction was carried out for 6 h. Unreacted small molecules were removed by ultrafiltration with a 3 kDa membrane, and PVA-Hep was obtained by freeze drying.

[0028] The above PABA-CS and PVA-Hep were mixed in a mass ratio of 1:1 to obtain CS-Hep Gel hydrogel.

[0029] Step 3: Preparation of the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system (AA-GMs / CS-Hep Gel) The AA-GMs obtained in step 1 were dispersed at a mass fraction of 5% in PABA-CS and PVA-Hep, respectively, with a mass ratio of 1:1. After thorough mixing, they were then combined to obtain AA-GMs / CS-Hep Gel. As a control, AA-GMs were replaced with GMs to obtain GMs / CS-Hep Gel (blank hydrogel group).

[0030] like Figure 1 The image shown is a scanning electron microscope image of a chitosan-heparin hydrogel composite gelatin microsphere drug delivery system. Figure 1 The image above is a scanning electron microscope image of GMs / CS-HepGel. Figure 1 The image below is a scanning electron microscope image of the AA-GMs / CS-HepGel hydrogel, showing that the microspheres are uniformly dispersed in the hydrogel, with an intact structure and uniform pores.

[0031] Example 2: Based on Example 1, the remaining steps are exactly the same as in Example 1, except that the drop rate of the aqueous phase is adjusted to 2 mL / min, the concentration of Tween-20 is 0.75%, and the mixture is stirred at 400 rpm for 45 min to form a W / O emulsion. Glutaraldehyde crosslinking agent with a volume fraction of 0.75% v / v is added, and the mixture is crosslinked at 35°C for 3 h. The resulting gelatin microspheres have uniform particle size, and the drug loading rate and sustained-release performance are comparable to those in Example 1.

[0032] Example 3: Based on Example 1, the remaining steps are exactly the same as in Example 1, except that the drop rate of the aqueous phase is adjusted to 3 mL / min, the concentration of Tween-20 is 1%, and the mixture is stirred at 500 rpm for 60 min to form a W / O emulsion. Glutaraldehyde crosslinking agent with a volume fraction of 1% v / v is added, and the mixture is crosslinked at 40°C for 4 h. The resulting gelatin microspheres have uniform particle size, and the drug loading rate and sustained-release performance are comparable to those in Example 1.

[0033] Example 4: Based on Example 1, the remaining steps are exactly the same as in Example 1, except that the pH of the modified reaction is adjusted to 9 and the reaction time is extended to 7 hours. The modified products are purified by ultrafiltration membranes of 25kd and 4kd respectively. The purity of the obtained modified products is high, and the hydrogel formability and performance are comparable to those of Example 1.

[0034] Example 5: Based on Example 1, the remaining steps are exactly the same as in Example 1, except that the pH of the modified reaction is adjusted to 10 and the reaction time is extended to 8 hours. The modified products are purified by ultrafiltration membranes of 30kd and 5kd respectively. The purity of the obtained modified products is high, and the hydrogel formability and performance are comparable to those of Example 1.

[0035] Example 6: Based on Example 1, the remaining steps are exactly the same as in Example 1, except that the mass fraction of gelatin microspheres loaded with asiaticoside is adjusted to 10%. The resulting composite drug delivery system has a uniform structure and drug release performance comparable to that of Example 1.

[0036] Example 7: Based on Example 1, the remaining steps are exactly the same as in Example 1, except that the mass fraction of gelatin microspheres loaded with asiaticoside is adjusted to 15%. The resulting composite drug delivery system has a uniform structure and drug release performance comparable to that of Example 1.

[0037] Example 8: This example is based on Example 1, and the in vivo and in vitro release assays were performed on the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system. In vitro drug release: Sample preparation: Accurately weigh the drug-loaded chitosan-heparin hydrogel composite gelatin microsphere delivery system (containing 100 mg of asiaticoside), place it in a dialysis bag (molecular weight cutoff 3500 Da), add a small amount of release medium to swell, and then seal.

[0038] Release apparatus: A dialysis bag-constant temperature magnetic stirring method was used, with the dialysis bag immersed in 200 mL of release medium. Medium 1 simulated a diabetic environment with a glucose concentration of 30 mM and an H2O2 concentration of 0.01%. Medium 2 was a standard PBS solution. The temperature was maintained at 37 ± 0.5℃, and the stirring speed was 50 rpm to 100 rpm (to ensure uniformity of the medium).

[0039] Sampling and replenishment: Take 5 mL samples at preset time points (0.5, 1, 4, 8, 12, 24, 48, 72, 120 h), and simultaneously add an equal volume of fresh medium at 37℃. After lyophilization, dissolve the samples in the mobile phase, filter them through a 0.45 μm filter membrane, and prepare them for testing.

[0040] Drug release in the body: The same dose of drug (drug-loaded chitosan-heparin hydrogel composite gelatin microsphere drug delivery system) was implanted subcutaneously at a dose of 200 mg. The implanted material was removed at 1 h, 8 h, 12 h, 24 h, 72 h, 7 d, 14 d, and 21 d after implantation. The material was destroyed in 5 ml of methanol and the liquid was reserved for subsequent testing.

[0041] Detection Method: The content of asiaticoside was determined by HPLC. Chromatographic conditions were as follows: column: C18 (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-water (70:30, v / v); flow rate: 0.8 mL / min; detection wavelength: 205 nm; column temperature: 25℃; injection volume: 10 μL. 10.00 mg of asiaticoside was weighed, dissolved, and diluted to 100 mL to prepare a stock solution. This stock solution was then diluted to 0, 1, 2, 4, 8, 16, 32, 64, and 128 μm. A standard curve was plotted using peak area (Y) against concentration (X), and a regression equation was obtained. The concentration of the sample was calculated, and the in vitro release process of asiaticoside gelatin microsphere hydrogel was determined. The calculation formula is as follows:

[0042] (V) represents the concentration of the nth sample; (V) represents the total volume of the released medium. (W): Sampling volume per sample; Total loading of asiaticoside in microsphere hydrogel.

[0043] The cumulative release of asiaticoside in vitro is as follows Figure 2As shown in (a), the horizontal axis represents release time (in hours), and the vertical axis represents the cumulative percentage of asiaticoside release. The figure contains two curves: one for the blank hydrogel group (GMs / CS-HepGel) and the other for the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system (AA-GMs / CS-HepGel). The results showed that the blank group had no significant drug release; the drug-loaded group experienced rapid release from 0h to 12h, entered a sustained and stable release phase from 12h to 48h, and after 48h, the release curve showed no abrupt jumps or bursts, exhibiting an overall programmed release characteristic of "rapid release in the early stage and stable release in the middle stage." This highly matches the drug requirements during the inflammatory and proliferative phases of diabetic wounds, demonstrating that this composite drug delivery system can achieve time-controlled release of asiaticoside.

[0044] The cumulative release of asiaticoside in the body is as follows Figure 2 As shown in (b), the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system (AA-GMs / CS-HepGel) initially releases 0-3 days (inflammatory phase). The CS-Hep hydrogel rapidly swells upon contact with wound exudate, and the borate ester bonds in the hydrogel network respond to ROS and other stimuli to rapidly release asiaticoside, accounting for 30%-40% of the total release. This inhibits the release of inflammatory factors (such as TNF-α and IL-6) and reduces the inflammatory response. In the middle phase, release continues for 4-14 days (proliferative phase), and the hydrogel swelling tends to... Due to the pores on the surface of the microspheres and slow degradation, AA-GMs continuously release asiaticoside, accounting for 40% to 50% of the total release. This promotes fibroblast migration and proliferation, vascular endothelial cell differentiation (upregulates VEGF expression), and accelerates granulation tissue formation. In the later stage, stable release lasts for 15 to 22 days (remodeling period). During this period, the gelatin microspheres further degrade, and the remaining asiaticoside is slowly released (accounting for 10% to 20% of the total release), which regulates the balance between collagen synthesis and degradation (reduces MMP-2 / 9 activity) and reduces scar formation.

[0045] Example 9: This example, based on Example 1, tests the antibacterial properties of a chitosan-heparin hydrogel composite gelatin microsphere drug delivery system. 1. Take the bacterial culture in the logarithmic growth phase and dilute it with sterile physiological saline to a concentration of 1×10⁻⁶. 6 CFU / mL, take 10 mL of the bacterial suspension and add it to five sterile conical flasks containing a chitosan-heparin hydrogel composite gelatin microsphere drug delivery system with mass concentrations of 0.2%, 0.4%, 1%, 1.5%, and 2%.

[0046] 2. Place the conical flask in a constant temperature shaker at 37℃ and 150r / min and incubate for 72h. Take out 1mL of bacterial culture and serially dilute it 10 times with sterile physiological saline. Take 0.1mL of the diluted solution and spread it on a nutrient agar plate.

[0047] 3. After culturing for 24 hours, photograph and record the bacterial colonies.

[0048] like Figure 3 As shown, Figure 3 Plate images showing the colony growth of Staphylococcus aureus after treatment with the chitosan-heparin hydrogel-gelatin microsphere drug delivery system at different mass fractions, from left to right: 0.2%, 0.4%, 1%, 1.5%, and 2% concentration groups. 0.2%, 0.4%, 1%, and 1.5% concentration groups: A large number of white circular colonies were observed growing on the plates, and the number of colonies did not decrease significantly, indicating that the hydrogel in this concentration range had no significant antibacterial effect on Staphylococcus aureus. 2% concentration group: There was almost no visible colony growth on the plate, and only a very small number of residual colonies were found at the edge of the culture medium. This indicates that the 2% mass fraction chitosan-heparin hydrogel composite gelatin microsphere drug delivery system has excellent in vitro antibacterial activity against Staphylococcus aureus and can effectively inhibit the proliferation of common pathogenic bacteria in wounds.

[0049] The chitosan-heparin hydrogel composite gelatin microsphere drug delivery system prepared by this invention has concentration-dependent antibacterial activity against Staphylococcus aureus. When the mass fraction reaches 2%, it can achieve highly efficient inhibition of Staphylococcus aureus, effectively reduce the risk of bacterial infection in diabetic wounds, provide anti-infection protection for the healing of chronic wounds, and meet the antibacterial performance requirements of medical wound dressings.

[0050] Example 10: This example is based on Example 1 and tests the coagulation performance of the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system. 1. PABA-CS and PVA-Hep were mixed in different proportions to obtain CS-Hep Gel hydrogels with heparin mass contents of 0.2%, 0.5%, 1%, 2% and 3%, respectively. The remaining steps were the same as in Example 1 to obtain the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system. 1g was added to 10mL PBS to obtain a solution.

[0051] 2. Take 1 mL of anticoagulated rat blood, centrifuge at 3000 rpm for 10 min to obtain plasma, add 50 μL of plasma to a 96-well plate, add 50 μL of hydrogel extract, incubate at 37℃ for 3 min, add 50 μL of APTT reagent, incubate at 37℃ for 5 min, then add 50 μL of 0.25 mol / L calcium chloride solution, start a stopwatch and record the plasma coagulation time.

[0052] Figure 4The bar chart shows the changes in rat plasma APTT values ​​under the action of chitosan-heparin hydrogels with different heparin concentrations. The results showed that the APTT value in the blank control group was approximately 35 seconds; the APTT values ​​in the 0.2% and 0.5% heparin groups were not significantly different from the control group; the APTT value in the 1% heparin group remained at a normal physiological level, without excessive anticoagulation; when the heparin concentration was greater than 1% (2% and 3%), the APTT value was significantly prolonged, the clotting time increased significantly, and there was a risk of bleeding. Based on these results, this invention preferably uses 1% heparin as the addition amount in the hydrogel, balancing anticoagulation safety and wound repair effect.

[0053] Based on APTT test results, when the heparin content in the hydrogel is greater than 1%, the plasma clotting time is significantly prolonged, which can easily lead to excessive anticoagulation and bleeding risks. However, the hydrogel with 1% heparin content can maintain normal physiological clotting time without obvious anticoagulation side effects, while retaining the wound repair advantages of heparin such as anti-inflammatory and angiogenesis. Therefore, this invention preferably uses 1% as the heparin addition amount for chitosan heparin hydrogel, taking into account both biocompatibility and wound treatment effect.

[0054] Example 11: This example is based on Example 1 and assesses the in vitro safety of the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system. L929 mouse fibroblasts or HaCaT human keratinocytes were passaged to the logarithmic growth phase at 37°C, 5% CO2, DMEM + 10% FBS medium, and after digestion and centrifugation, 1×10⁶ cells were seeded per well in 96-well plates. 4 100 μl / well of cells were pre-cultured for 24 h to allow the cells to adhere.

[0055] Hydrogel materials with solvent weight fractions of 0, 1, 2, 4, and 8 (g / v, %) were added to Transwell chambers. The chambers were co-cultured for 24 h. Five replicates were set up.

[0056] After 24 hours, 10 μL of CCK8 working solution was added to each well. After 30 minutes, the absorbance at 450 nm was measured, and the relative cell viability was calculated using the following formula: Survival rate (%) = (OD of experimental group - OD of blank well) / (OD of negative control group - OD of blank well) × 100%.

[0057] Figure 5This image illustrates the biocompatibility and cell proliferation-promoting effect of the composite drug delivery system. Using HaCaT human keratinocytes as a model, Transwell co-culture and the CCK-8 assay were employed to detect the relative cell viability. Results showed that under different concentration gradients of material extracts, cell viability remained above 90%, with no significant cytotoxicity, indicating that the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system possesses excellent biocompatibility. Furthermore, compared to the control group, the drug-loaded composite system group showed a significantly increased cell number, intact cell morphology, and good spreading, demonstrating that the drug delivery system is not only safe and harmless but also significantly promotes keratinocyte proliferation, providing cellular evidence for accelerating wound re-epithelialization.

[0058] Example 12: Verification of cell compatibility and wound healing based on the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system of Example 1. A diabetic wound model was established using db / db mice, which were divided into a control group, a hydrogel microsphere group, and a asiaticoside-loaded gelatin microsphere hydrogel group. Six mice were in each group. Drug fixation was achieved using 3M dressings.

[0059] The size of the wound was recorded every 3 days. After 21 days, recording was stopped, and the group results were analyzed.

[0060] Figure 6 This image shows a gross morphological comparison of how the composite drug delivery system promotes chronic wound healing in diabetic mice. A full-thickness skin defect model was constructed using db / db diabetic mice, and three groups were set up: a blank control group, a blank hydrogel group (GMs / CS-HepGel), and a drug-loaded composite hydrogel group (AA-GMs / CS-HepGel). Wound healing morphology was recorded at 0, 3, 7, and 21 days. Results showed that the control group and the blank hydrogel group exhibited slow wound healing, with little granulation tissue formation, and were not completely closed at 21 days. The drug-loaded composite hydrogel group showed significant wound contraction, abundant granulation tissue, and rapid re-epithelialization, with near-complete wound healing at 21 days. The healing quality was significantly better than the other two groups, demonstrating that the drug delivery system of this invention can effectively promote the healing of chronic diabetic wounds and has good clinical application potential.

Claims

1. A chitosan-heparin hydrogel composite gelatin microsphere drug delivery system, characterized in that, It is composed of a chitosan heparin hydrogel matrix and gelatin microspheres loaded with asiaticoside; the gelatin microspheres loaded with asiaticoside are uniformly dispersed in the chitosan heparin hydrogel matrix to form a dual drug-loaded composite structure.

2. The drug delivery system according to claim 1, characterized in that, The chitosan-heparin hydrogel is formed by self-crosslinking of 3-aminophenylboronic acid-modified chitosan and heparin-modified polyvinyl alcohol to form a three-dimensional network containing borate ester bonds, which can rapidly swell and achieve rapid initial drug release.

3. The drug delivery system according to claim 1, characterized in that, The gelatin microspheres loaded with asiaticoside were prepared by emulsification cross-linking method, using glutaraldehyde as the cross-linking agent, which can achieve continuous and stable release of asiaticoside by controlling the degree of cross-linking.

4. The drug delivery system according to claim 1, characterized in that, The mass fraction of the gelatin microspheres loaded with asiaticoside in the composite system is 5% to 15%.

5. The drug delivery system according to claim 2, characterized in that, The mass ratio of 3-aminophenylboronic acid modified chitosan to heparin modified polyvinyl alcohol is 1:1; the mass percentage of heparin in the chitosan-heparin hydrogel is 1%.

6. The method for preparing the chitosan-heparin hydrogel composite gelatin microsphere drug delivery system according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Preparation of gelatin microspheres loaded with asiaticoside; Step 2: Preparation of 3-aminophenylboronic acid modified chitosan and heparin modified polyvinyl alcohol; Step 3: Disperse the gelatin microspheres loaded with asiaticoside in 3-aminophenylboronic acid modified chitosan and heparin modified polyvinyl alcohol respectively, and then mix and self-crosslink to obtain a composite drug delivery system.

7. The preparation method according to claim 6, characterized in that, In step 1, liquid paraffin containing 0.5% to 1% Tween-20 is added to the aqueous phase at a dropping rate of 1 mL / min to 3 mL / min, and the mixture is stirred at 300 rpm to 500 rpm for 30 min to 60 min to form a W / O emulsion. Then, 0.5% v / v to 1% v / v glutaraldehyde is added and the mixture is crosslinked at 30℃ to 40℃ for 2 h to 4 h.

8. The preparation method according to claim 6, characterized in that, The gelatin microspheres prepared in step 1 were washed with petroleum ether and anhydrous ethanol in sequence and then freeze-dried.

9. The preparation method according to claim 6, characterized in that, In step 2, the modification reaction is carried out using EDS / NHS as an activator at pH 8 to pH 10 for 6 to 8 hours. After purification by 20kd to 30kd and 3kd to 5kd ultrafiltration membranes, respectively, the mixture is freeze-dried to obtain 3-aminophenylboronic acid modified chitosan and heparin modified polyvinyl alcohol.

10. The use of the drug delivery system according to any one of claims 1-5 in the preparation of a dressing for the treatment of diabetic wounds.

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