Preparation method and application of nano-drug loaded injectable composite hydrogel

Abstract

The invention discloses a preparation method and application of nano-drug loaded injectable composite hydrogel, and belongs to the field of biomedical materials. The composition is prepared from lysine, F127, lecithin and a sinomenine-gallic acid nano-drug. The preparation method provided by the invention has the characteristics of simple formula and easy process operation, not only has good biocompatibility and tissue adhesion, but also can effectively package and protect the nanoparticles, prolong the residence time of the drug at the wound part and realize more stable and controllable local release, thereby obviously enhancing the treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a preparation method and application of an injectable composite hydrogel loaded with sinomenine-gallic acid nanomedicine. Background Art

[0002] Wound healing is a crucial physiological process in which skin tissue regenerates after mechanical, chemical, or pathogenic damage, relying on the body's own repair mechanisms. Wound types, including acute trauma, surgical incisions, and burns, are often accompanied by inflammatory responses and an imbalance between cell migration and proliferation. In chronic or infected wounds, local immune dysfunction and inadequate extracellular matrix remodeling can significantly prolong the wound repair cycle, leading to the development of refractory ulcers, severely impacting patients' quality of life and increasing the medical burden. Currently, methods for promoting wound healing primarily include topical antimicrobial agents, dressings, growth factor sprays, and tissue engineering scaffolds. However, these approaches still have limitations in clinical application, such as short local drug residence time, low bioavailability, difficulty achieving sustained release, and poor tissue adhesion, resulting in limited therapeutic efficacy and high recurrence rates. Therefore, the development of novel local wound delivery systems with controlled release, strong adhesion, ease of operation, and synergistic therapeutic effects has become a research hotspot.

[0003] Nano-drug delivery systems, due to their excellent drug loading capacity, biopenetration, and sustained-release properties, show promising application prospects in wound treatment. Sinomenine and gallic acid, natural molecules with anti-inflammatory and antioxidant activities, can achieve synergistic therapeutic effects through self-assembly into nanoparticles, potentially improving local wound inflammation and promoting epithelial regeneration and collagen deposition. However, direct application of nanoparticles to wound surfaces presents challenges such as easy particle aggregation, short clearance times, and insufficient local concentration, limiting the sustainability and stability of their therapeutic effects.

[0004] Hydrogel materials are widely used in wound dressings and drug delivery due to their high water content, good biocompatibility, and tissue adhesion. F127 (Poloxamer 407) exhibits excellent thermosensitive properties and rapidly gels at body temperature, forming a three-dimensional network structure that provides a foundation for sustained drug release and mechanical support for the system. However, hydrogels constructed solely from F127 still suffer from weak mechanical properties, poor tissue adhesion, unstable drug release, and lack of bioactivity, making them difficult to meet the clinical needs of complex wound repair and precise drug delivery. Therefore, there is an urgent need to develop a novel composite hydrogel system that combines structural support, drug loading, synergistic release, and repair-promoting functions to achieve efficient delivery and repair of sinomenine-gallic acid nanomedicines at the wound site. Summary of the Invention

[0005] In order to solve the above problems, the object of the present invention is to provide a method for preparing an injectable composite hydrogel loaded with sinomenine-gallic acid nanomedicine for promoting wound healing.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preparing a composite hydrogel loaded with sinomenine-gallic acid nanomedicine comprises the following steps: (1) Weigh 1-10 parts by weight of sinomenine-gallic acid nanomedicine and add it to 10-100 parts by weight of PBS buffer to obtain a mixture; (2) Further, the mixture in (1) was stirred and dissolved at 40°C; (3) Further, 0.1% to 1% by weight of lecithin was gradually added to the mixture solution obtained in (2), and stirred at 40° C. for 2 hours; (4) Further, 0.1% to 1% by weight of lysine is gradually added to the mixture solution obtained in (3), and stirred at 25° C. until dissolved; (5) Further, 15% to 30% by weight of F127 was gradually added to the mixture solution obtained in (4), and stirred at 4°C overnight to obtain an injectable composite hydrogel loaded with sinomenine-gallic acid nanomedicine.

[0007] (6) The present invention uses a C57BL / 6 mouse wound model to evaluate the bioactivity of the composite hydrogel loaded with sinomenine-gallic acid nanomedicine.

[0008] Furthermore, in step (3), lecithin is added in the form of an aqueous solution of lecithin.

[0009] Furthermore, the preparation method of the sinomenine-gallic acid nanomedicine is based on the patent (a kind of orally administrable and injectable sinomenine-gallic acid nanoparticles and their preparation method and application - CN119770433A).

[0010] The present invention provides a composite hydrogel loaded with sinomenine-gallic acid nanomedicine (abbreviated as: F127-Lys-PC@SGNPs), which is prepared by the above method.

[0011] Preferably, the F127-Lys-PC@SGNPs are administered at concentrations of 50 μM and 100 μM in a mouse wound model, calculated as sinomenine concentration.

[0012] The present invention has the following beneficial effects: (1) The present invention constructs an injectable composite hydrogel system based on lysine, F127 and lecithin, which can efficiently encapsulate sinomenine-gallic acid self-assembled nanomedicine and form a sustained-release environment locally in the wound. It has the advantages of rapid gelation, good adhesion and stable sustained release.

[0013] (2) The present invention uses sinomenine and gallic acid drugs with anti-inflammatory and antioxidant functions, and is suitable for the treatment of various wounds such as chronic or infectious wounds.

[0014] (3) The hydrogel formula of the present invention is simple in composition and mild in preparation conditions. It does not require a cross-linking agent or a complex synthesis process. The process is controllable and easy to promote and apply.

[0015] (4) The raw materials lysine, F127 and lecithin used in the present invention all have good biocompatibility and degradability. The constructed hydrogel system can quickly gel at body temperature and has good injectability and tissue adaptability.

[0016] (5) The nano-drug-hydrogel composite system constructed by the present invention can form a three-dimensional network tubular structure in the local tissue, control the drug release rate, prolong the drug action time, slow down the inflammatory response, and help accelerate the wound healing process.

[0017] (6) The composite hydrogel prepared in the present invention showed good wound closure rate and tissue repair effect in the C57BL / 6 mouse wound model, has a clear biological activity basis, and is suitable for further development into a wound treatment material with clinical translation potential.

[0018] (7) In addition to carrying sinomenine-gallic acid nanoparticles, the composite hydrogel prepared by the present invention can also carry other poorly soluble drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The gel properties of F127-Lys-PC@SGNPs prepared in this invention; Figure 2 This is a scanning electron micrograph of F127-Lys-PC@SGNPs prepared in the present invention; Figure 3 The particle size diagram of F127-Lys-PC@SGNPs prepared in the present invention; Figure 4The stability experiment of F127-Lys-PC@SGNPs prepared by the present invention; Figure 5 The release characteristics of F127-Lys-PC@SGNPs prepared in this invention; Figure 6 Hemolysis experiment of F127-Lys-PC@SGNPs prepared in the present invention; Figure 7 The cytotoxicity of F127-Lys-PC@SGNPs prepared in the present invention; Figure 8 The effect of the F127-Lys-PC@SGNPs prepared by the present invention on the cell scratch model; Figure 9 The therapeutic effect of F127-Lys-PC@SGNPs prepared by the present invention on the mouse wound model; Figure 10 This is the process in which the F127-Lys-PC@SGNPs prepared in the present invention quickly form a gel in PBS at 37°C by injection. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods in the examples are conventional methods. Unless otherwise specified, the reagents used are conventional commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered a limitation of the present invention, but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The preparation method of sinomenine-gallic acid nanomedicine comprises the following steps: (1) Weigh 1 part by weight of sinomenine and 1 part by weight of gallic acid, add them to 3 parts by weight of PBS buffer (0.01 M, pH = 7.2-7.4), and stir vigorously to obtain a mixture; (2) Further, the mixture in (1) was placed in a water bath at 60°C and heated for 2 hours; (3) Further, the mixture solution in (2) was gradually added into 10 parts by weight of PBS buffer (0.01 M, pH = 7.2-7.4) at 60°C, and the mixture was heated and stirred for 4 hours; (4) Further, the mixture solution in (3) was placed in a dialysis bag (MWCO = 100 Da); (5) Furthermore, the dialysis bag was placed in ultrapure water for 12 h and freeze-dried to obtain sinomenine-gallic acid nanomedicine.

[0027] Sinomenine and gallic acid in a weight ratio of 1:1 can completely self-assemble to form nanomedicines.

[0028] The present invention provides a method for preparing an injectable composite hydrogel loaded with sinomenine-gallic acid nanomedicine for treating wound healing, comprising the following steps: (1) Weigh 1 part by weight of sinomenine-gallic acid nanomedicine and add it to 10 parts by weight of PBS buffer (0.01 M, pH = 7.2-7.4), stir, and obtain a mixture solution; (2) Further, 0.1% by weight of lecithin was gradually added to the mixture solution obtained in (1), and stirred at 40° C. for 2 hours; (3) Further, 0.3% by weight of lysine was gradually added to the mixture solution obtained in (2), and stirred at 25° C. until dissolved; (4) Further, 25% by weight of F127 was gradually added to the mixture solution obtained in (4), and the mixture was stirred at 4°C overnight to obtain F127-Lys-PC@SGNPs.

[0029] The F127-Lys-PC@SGNPs provided by the present invention have been experimentally verified to be able to effectively promote cell migration and promote wound healing in a mouse skin wound model, and have great research significance and application prospects.

[0030] In the above preferred embodiment, the sinomenine-gallic acid nanoparticles SGNPs were independently developed by our laboratory.

[0031] In the above preferred embodiment, the lecithin is soybean lecithin with a purity of >98%.

[0032] In the above preferred embodiment, the lysine is L-lysine with a purity of 98.5%.

[0033] The technical solution of the present invention will be further described below with reference to embodiments.

[0034] Example 1 Stability experiment: Prepare F127-Lys-PC@SGNPs solution with the same concentration, and measure its particle size on the 1st, 3rd, 5th, 7th and 14th day respectively.

[0035] Example 2 Drug release experiments: SGNPs, F127-SGNPs, and F127-Lys-PC@SGNPs were prepared and 1 mL was placed in a dialysis bag (molecular weight cutoff, 3.5 kDa). The bag was then immersed in 50 mL of PBS at 37°C. At predetermined time points (1, 2, 4, 6, 8, 10, 12, 24, and 48 hours), the dialysate (0.5 mL) was collected and supplemented with the same volume of PBS to maintain the total volume in the beaker. The peak area of each sample solution was measured using high-performance liquid chromatography (HPLC) with a mobile phase of 40% acetonitrile / 60% triethylamine-phosphoric acid solution. The release rate of each sample was calculated and plotted.

[0036] Example 3 Cell hemolysis experiment: Blood was collected from mouse eyeballs and washed with PBS until the supernatant was colorless to obtain mouse erythrocytes. The erythrocytes were diluted 30 times. The positive control was 0.1% Triton solution. The concentrations of the hydrogel extract were set to 3.1%, 6.3%, 12.5%, 25%, and 50%, respectively. The cells were incubated at 37°C for 2 h. After centrifugation for 5 min, 100 μL of the supernatant was transferred to a 96-well plate and the absorbance was measured at 450 nm using a UV spectrophotometer.

[0037] Example 4 Cell viability and cell scratch: Human immortalized keratinocytes (HaCaT) were used for the experiment. A cell viability assay (CCK-8) was used to examine the effects of different concentrations of F127, F127-Lys-PC, and F127-Lys-PC@SGNPs (5 µM, based on sinomenine concentration) on HaCaT cell growth. A cell scratch assay was used to observe and evaluate the effect of F127-Lys-PC@SGNPs on HaCaT migration ability, verifying its potential to promote wound healing. Example 5 To establish the experimental animal model, 8-week-old male C57BL / 6 mice weighing 20-25g were anesthetized with isoflurane and their back hair was shaved. A 1-cm diameter skin wound model was then created on the back of the mice. The mice were divided into five groups (n=6): model group, F127 group, F127-Lys-PC group, F127-Lys-PC@SGNPs (50 μM, based on sinomenine concentration), and F127-Lys-PC@SGNPs (100 μM, based on sinomenine concentration). Postoperatively, the wounds were photographed daily, and wound changes were recorded. Wound area was measured using ImageJ software (V1.8.0), and wound closure rates were calculated for each group.

[0038] Wound close rate( %) = (A0 − A n ) / A0 × 100% Among them, A0, A n represents the original wound area and the wound area on the nth day after wounding.

[0039] The specific experimental results are as follows: Figure 1 Figure 3 shows the changes in the F127 (left), F127-Lys-PC (center), and F127-Lys-PC@SGNPs (right) hydrogels after being inverted for seven days. Due to its low viscosity, the F127 hydrogel collapsed and settled under gravity, demonstrating poor shape retention. However, the F127-Lys-PC and F127-Lys-PC@SGNPs hydrogels showed improved stability, maintaining their shape even under inversion. This is likely due to the nanoparticles forming a secondary physical support within the three-dimensional network, enhancing viscosity and shape retention.

[0040] Figure 2Scanning electron microscopy images of F127 (left), F127-Lys-PC (center), and F127-Lys-PC@SGNPs (right) from this example show a lack of apparent pores in the F127 sample, indicating that F127 alone is unable to form a stable network structure. F127-Lys-PC exhibits more porous structures with a denser arrangement, indicating that the introduction of lysine and phosphatidylcholine enhances inter-micellar interactions and improves structural stability. The number of pores in F127-Lys-PC@SGNPs is further increased and more evenly distributed, suggesting that the SGNPs, acting as a supporting filler, enhance the hydrogel's spatial structure. This demonstrates that the composite system described herein can significantly improve the microstructural integrity and porosity of the hydrogel.

[0041] Figure 3 Dynamic light scattering (DLS) was used to measure the particle size of F127-Lys-PC and F127-Lys-PC@SGNPs in this example. The results showed that both particle sizes remained stable at approximately 300 nm, demonstrating that the system can form a stable nanomicelle structure. Further analysis showed that the introduction of SGNPs did not significantly increase the particle size, indicating that they are well embedded or adsorbed into the F127-Lys-PC micelle structure, without causing significant aggregation or disrupting the micelle network, and that the system is stable.

[0042] Figure 4 The particle size of the F127-Lys-PC@SGNPs provided in this embodiment is stable within two weeks.

[0043] Figure 5 The results showed that compared with SGNPs and F127-SGNPs, the F127-Lys-PC@SGNPs composite hydrogel provided in this embodiment has a more significant sustained-release effect, a more stable drug release rate, and can be retained in the wound site for a longer time, thereby helping to improve the stability and local efficacy of treatment.

[0044] Figure 6 The following table shows the hemolysis test results for the three hydrogel samples used in this example: F127, F127-Lys-PC, and F127-Lys-PC@SGNPs. Hemolysis rates were determined using a direct contact method, and the results showed that all three samples had hemolysis rates below 5%, meeting the international biosafety requirement for biomaterials with a hemolysis rate of less than 5%. This demonstrates that these materials exhibit excellent hemocompatibility and biosafety, making them suitable for in vivo applications.

[0045] Figures 1-6 The concentration of the composite hydrogel raw material used in the experiment was 500 ug / mL, calculated as sinomenine concentration.

[0046] Figure 7The effects of the three hydrogel samples of F127, F127-Lys-PC, and F127-Lys-PC@SGNPs on the growth of HaCaT cells in this example are shown. The results show that F127 at a concentration of 50% has a certain toxicity to HaCaT cells, and the cell survival rate drops to 88%. In contrast, F127-Lys-PC and F127-Lys-PC@SGNPs do not show obvious cytotoxicity at the same concentration, indicating good cell compatibility. In addition, within the concentration range of 6.3% and 12.5%, both F127-Lys-PC and F127-Lys-PC@SGNPs hydrogels have a promoting effect on HaCaT cell proliferation, suggesting that they have certain biological activity and are beneficial to promoting tissue repair.

[0047] Figure 8 The results show the efficacy of the three hydrogel samples (F127, F127-Lys-PC, and F127-Lys-PC@SGNPs) in a HaCaT cell scratch model. The results indicate that compared with the model group and the F127 group, F127-Lys-PC@SGNPs significantly promoted the migration of HaCaT cells in a concentration-dependent manner, demonstrating the potential of this composite hydrogel in promoting cell migration and accelerating wound repair.

[0048] Figure 9 The repair effects of the three hydrogel samples described in this example—F127, F127-Lys-PC, and F127-Lys-PC@SGNPs—in a mouse skin wound model were demonstrated. The results showed that compared with the model and F127 groups, the F127-Lys-PC group promoted wound healing to a certain extent, while the F127-Lys-PC@SGNPs group exhibited a more significant healing-promoting effect at various concentrations, with a concentration-dependent trend. These results demonstrate that the composite hydrogels provided by this invention have excellent tissue repair potential and are suitable for promoting the rapid healing of skin wounds.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing an injectable composite hydrogel loaded with nano-drugs for wound healing, characterized in that: The following steps are involved: (1) Preparation of sinomenine-gallic acid nanoparticles; (2) Dissolving the nanomedicine prepared in step (1) in PBS buffer by stirring; (3) adding the lecithin solution to the mixture solution obtained in step (2) and continuing stirring; (4) adding lysine to the mixture solution obtained in step (3) and continuing stirring; (5) F127 is added to the mixture solution obtained in step (4) and the stirring is continued; an injectable composite hydrogel loaded with sinomenine-gallic acid nanomedicine is obtained.

2. The preparation method according to claim 1, characterized in that The amount of PBS buffer used in step (2) is 10-100 parts by weight.

3. The preparation method according to claim 1, characterized in that The concentration of PBS buffer in step (2) is 0.01 M and the pH is 7.2-7.

4.

4. The preparation method according to claim 1, characterized in that In step (2), the sinomenine-gallic acid nanoparticles account for 1% to 10% by weight of the mixed solution obtained in step (2); and the stirring temperature is 40°C.

5. The preparation method according to claim 1, characterized in that In step (3), the lecithin accounts for 0.1% to 1% by weight of the mixed solution obtained in step (3); and the stirring temperature is 40°C.

6. The preparation method according to claim 1, characterized in that In step (4), lysine accounts for 0.1% to 1% by weight of the mixed solution obtained in step (4); and the stirring temperature is 25°C.

7. The preparation method according to claim 1, characterized in that In step (5), F127 accounts for 15% to 30% by weight of the mixed solution obtained in step (5); and the stirring temperature is 4°C.

8. The preparation method according to claim 1, characterized in that The lecithin solution is an aqueous solution of lecithin.

9. An injectable composite hydrogel loaded with sinomenine-gallic acid nanomedicine prepared according to the preparation method according to any one of claims 1 to 8.

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