Graphene composite aerogel composite asiaticoside and VEGF dressing

Abstract

The dressing prepared by compounding the graphene composite aerogel with the asiaticoside and the VEGF comprises the graphene composite aerogel, and the asiaticoside and the VEGF are compounded in the graphene composite aerogel. The graphene composite aerogel (GA) is prepared through a sol-gel method, and gelation and freeze drying of graphene oxide (GO) are involved. The asiaticoside is extracted by ethanol or methanol, and the medicinal materials are soaked or heated and refluxed under specific conditions after being crushed. The VEGF growth factor is prepared by a genetic engineering recombination method which comprises the following steps: constructing an expression vector, extracting cDNA (complementary deoxyribonucleic acid), performing PCR (polymerase chain reaction) amplification on a VEGF gene segment, transforming a host cell, performing induced expression and separating and purifying.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a dressing of graphene composite aerogel combined with asiaticoside and VEGF. Background Art

[0002] Vascular endothelial growth factor (VEGF) forming a vascular network on the body surface is of great significance for tissue repair and regeneration, but there are still some existing problems in practical applications, which are specifically as follows: Difficult to precisely regulate: The process of VEGF-induced angiogenesis is complex, and it is difficult to precisely control the time, space, and intensity of its action. When applied on the body surface, it may lead to excessive angiogenesis or uneven distribution of the generated blood vessels, affecting the normal structure and function of tissues. For example, in wound repair, excessive angiogenesis may cause problems such as local tissue swelling and scar hyperplasia.

[0003] Lack of a long-term and stable regulation mechanism: Although current research can control the release of VEGF through some carriers or sustained-release systems, it is still difficult to achieve long-term, stable, and precise regulation. Over time, the effect of VEGF may gradually weaken or disappear, resulting in incomplete angiogenesis or degeneration of the already generated blood vessels. Summary of the Invention

[0004] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a dressing of graphene composite aerogel combined with asiaticoside and VEGF.

[0005] The technical solution adopted by the present invention to solve its technical problems is: A dressing of graphene composite aerogel combined with asiaticoside and VEGF, comprising graphene composite aerogel, in which asiaticoside and VEGF are compounded.

[0006] (1) Preparation method of graphene composite aerogel: Graphene composite aerogel (GA) is prepared by the sol-gel method: graphene oxide (GO) is subjected to gelation treatment to obtain graphene oxide hydrogel, and finally the graphene oxide hydrogel is freeze-dried to obtain graphene composite aerogel (GA).

[0007] (2) Preparation method of asiaticoside: Usually, organic solvents such as ethanol and methanol are selected as extraction solvents. After crushing the asiatic herb, it is soaked or heated under reflux with the selected solvent under certain temperature and time conditions; for example, using 70%-90% ethanol as the solvent, refluxing at 50-80°C for 2-3 times, 1-3 hours each time, can effectively extract asiaticoside from the asiatic herb.

[0008] (3) Preparation method of VEGF growth factor Genetic engineering recombination method: (31) Construction of expression vector: Total RNA is extracted from cells, cDNA is obtained by reverse transcription, and the VEGF gene fragment is amplified by PCR technology using cDNA as a template; the amplified VEGF gene fragment is inserted into a suitable expression vector (such as a plasmid) to construct a recombinant expression vector; (32) Transformation of host cells: The constructed recombinant expression vector is transformed into host cells. Commonly used host cells include Escherichia coli, yeast, and mammalian cells, etc.; for example, the recombinant plasmid is transformed into competent Escherichia coli cells, and positive clones containing the target gene are obtained through screening; (33) Induced expression: The host cells are cultured under suitable conditions. When the cells grow to a certain stage, an inducer is added to induce the expression of the VEGF gene; in Escherichia coli, IPTG (isopropyl-β-D-thiogalactoside) is commonly used as an inducer; after induction, the host cells will synthesize a large amount of VEGF protein; (34) Isolation and purification: Cells are collected by centrifugation, filtration, etc., and then cell disruption techniques (such as ultrasonic disruption, high-pressure homogenization, etc.) are used to break the cells and release the VEGF protein; then, various purification methods such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. are used to isolate and purify the VEGF protein to obtain high-purity VEGF growth factor.

[0009] The present invention also has the following additional technical features: As a further specific optimization of the technical solution of the present invention: 1.1) Preparation method of graphene oxide, graphene oxide is prepared by the Hummers method: using a strong oxidant to treat flake graphite to obtain graphite oxide, which is ultrasonically exfoliated to form graphene oxide, and finally graphene oxide can be obtained after reduction; As a further specific optimization of the technical solution of the present invention: (1.2) Preparation method of graphene hydrogel, gelation is carried out by using a suspension of graphene oxide sheets higher than the critical gel concentration. By controlling the pH value of the graphene oxide sheet suspension, adding a cross-linking agent, and applying ultrasonic waves, the graphene nanosheets in the suspension can self-assemble to form a three-dimensional structure.

[0010] As a further specific optimization of the technical solution of the present invention: (1.3) Preparation method of graphene composite aerogel, the graphene composite aerogel is prepared by the atmospheric pressure drying method. The atmospheric pressure drying of graphene composite aerogel (ADGA) is carried out through a process of reduction-freezing-thawing, combined with solvent replacement and atmospheric pressure drying to prepare the graphene composite aerogel; the graphene hydrogel prepared through the gelation process forms a graphene composite aerogel after drying to remove the moisture therein.

[0011] Compared with the prior art, the advantages of the present invention are: (1) Mechanism innovation of material-drug synergistic effect: The present invention realizes the effective loading and synergistic effect of multiple active ingredients by combining graphene composite aerogel, asiaticoside and VEGF. Graphene composite aerogel not only has good biocompatibility and controllable drug release performance, but also can promote cell adhesion and proliferation through its unique three-dimensional structure. Asiaticoside, as a natural plant extract, has various pharmacological effects such as promoting wound healing and reducing scar formation. And VEGF, as an important regulator of angiogenesis, can induce the formation of blood vessel networks and provide necessary nutritional support for tissue repair and regeneration. The organic combination of the three not only enhances the overall therapeutic effect of the dressing, but also improves the accuracy and long-term efficacy of treatment.

[0012] (2) Breakthrough of intelligent responsive drug delivery system: The graphene composite aerogel dressing prepared by the present invention has the characteristics of intelligent response and can adjust the drug release rate according to the changes in the wound environment. For example, when there is inflammation or infection at the wound, the graphene composite aerogel can respond and accelerate the drug release to more effectively inhibit inflammation and promote healing. This intelligent responsive drug delivery system not only improves the utilization rate of drugs, but also reduces the potential damage of drugs to normal tissues.

[0013] (3) Comprehensive advantages of biocompatibility and clinical transformation: The graphene composite aerogel, asiaticoside and VEGF adopted in the present invention all have good biocompatibility and will not cause obvious toxic and side effects to the human body. At the same time, the dressing preparation process is simple, the cost is low, and it is easy to realize large-scale production and clinical application. In addition, the dressing does not need to be replaced frequently during the treatment process, reducing the pain of patients and medical costs, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the preparation process of the graphene-loaded ginger and mugwort extract composite aerogel of the present invention; Figure 2 It is a schematic diagram of the preparation flow chart of graphene oxide of the present invention; Figure 3 It is a schematic diagram of the atmospheric pressure drying preparation process of the graphene composite aerogel of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] The exemplary embodiments disclosed by the present invention will be described in more detail below with reference to the drawings. A dressing of graphene composite aerogel combined with asiaticoside and VEGF includes graphene composite aerogel, asiaticoside and VEGF. (1) Preparation method for preparing graphene composite aerogel:

[0016] Graphene composite aerogel (GA) was prepared by sol-gel method. Graphene oxide has good hydrophilicity due to its rich oxygen-containing functional groups such as carboxyl, hydroxyl, and carbonyl groups, and can be uniformly dispersed in water and stably exist. Therefore, graphene oxide (GO) was used as the precursor for preparing aerogel. After gelation treatment, the electrostatic repulsion between its layers was weakened, enabling the graphene layers to restore their conjugated structure, strengthening the π-π conjugation, and due to steric hindrance, it could self-assemble to form a three-dimensional structure, obtaining graphene oxide hydrogel. Finally, the graphene oxide hydrogel was freeze-dried to obtain graphene composite aerogel (GA). In addition, our team adopted a drug-loading method of directly forming GA (Route A) in the drug medium. Compared with the method of loading drugs after synthesizing GA (Route B), Route A has obvious advantages. This composite aerogel is a three-dimensional porous material assembled with ginger and mugwort extract with a graphene three-dimensional network as the framework. The ginger and mugwort extract is loaded on the 3D network skeleton to form a bicontinuous "interlocking structure", which can fully exert the synergistic effect of the photothermal effect of graphene itself and the drug efficacy. The specific process is as follows Figure 1 as shown (1.2) Preparation method of graphene oxide

[0017] Graphene oxide was prepared by the Hummers method. The oxidation method for preparing graphene oxide has the advantages of low cost, simple operation method, and being conducive to large-scale production. Usually, flake graphite is treated with a strong oxidant to obtain graphite oxide, which is exfoliated by ultrasound to form graphene oxide, and finally graphene oxide can be obtained after reduction

[0018] The preparation process of graphene oxide is affected by many factors. The type and dosage of the oxidant affect the type of functional groups and chemical structure of the GOs sample; the temperature of the oxidation reaction affects the reaction rate and the integrity of the GOs structure; the reaction time of the oxidation reaction determines the oxidation degree and defect density of the sample. Our project team used the Hummers method to prepare graphene oxide (HGO) with KMnO4 as the oxidant, experiencing three reaction stages: low temperature - medium temperature - high temperature. Low-temperature intercalation stage: Sulfuric acid is inserted into the graphite layer under the action of external force (oxidant) to form sulfuric acid-graphite intercalation compound (H2SO4-GIC); Medium-temperature oxidation stage: Due to the strong oxidation of KMnO4, GIC is oxidized to primary graphite oxide (PGO); High-temperature hydrolysis stage: The addition of water hydrolyzes the covalent sulfate and expands the graphite layer spacing to finally form graphite oxide (GO). In addition, in this project, by extending the medium-temperature reaction time, the oxidation degree of the graphite layers was increased, and an aqueous dispersion of graphene oxide was obtained after ultrasonic exfoliation. The preparation process is as Figure 2, the required items are shown in Table 1. The graphene oxide sheets prepared by the Hummers method have a smoother edge structure, with a size distribution concentrated in the range of 0.5 - 1.0 pm and a higher degree of oxidation.

[0019] Table 1 Required reagents and raw materials

[0020] (1.2) Preparation method of graphene hydrogel The graphene hydrogel is prepared by a hydrothermal reduction self-assembly method, as Figure 3 shown. Gelation is carried out by using a suspension of graphene oxide sheets above the critical gel concentration. Suspension leads to an imbalance between the attractive and repulsive forces between the suspended graphene oxide sheets. The oxygenated functional groups in graphene oxide (GO) provide chemical cross-linking sites, which can be eliminated during the synthesis process. By controlling the pH value, adding cross-linking agents, and applying ultrasonic waves, the graphene nanosheets in the suspension can self-assemble spontaneously to form a three-dimensional structure. This method is both simple and economical. It can be integrated with other methods, such as reduction or cross-linking. And it avoids the limitation of the low solubility of pure graphite in most solvents. In the sol-gel method, some factors, such as temperature and precursor concentration, can be controlled to change the final pore size of the aerogel, as shown in Table 2.

[0021] Table 2 Controllable variables available in the sol-gel method and their effects on the pore size

[0022] Control factors Effect on pore size Precursor concentration The higher the graphene concentration, the smaller the pores Acidity and alkalinity The lower the pH value, the smaller the pores Surfactant Depends on the surfactant used Resorcinol-formaldehyde The less resorcinol-formaldehyde, the smaller the pores (1.3) Preparation method of graphene composite aerogel

[0023] Freeze-drying is not easy to control during the drying process, and the melting of ice crystals is likely to occur, resulting in the failure of the reaction. Moreover, freeze-drying takes too long, and its harsh experimental conditions are extremely unfavorable for large-scale production. Compared with freeze-drying, atmospheric drying has the advantages of simple equipment, low cost, being conducive to large-scale production, and easy control of reaction conditions. It has become the most prominent and most promising drying process for development and application at present. Therefore, our team uses the atmospheric drying method to prepare graphene composite aerogel and realizes large-scale production by taking advantage of its advantages. For the atmospheric drying graphene composite aerogel (ADGA), the experiment involves a process of reduction-freezing-thawing, combined with solvent replacement and atmospheric drying to prepare the graphene composite aerogel, as Figure 3 .

[0024] The graphene hydrogel prepared by the gelation process is dried to remove the water therein to form a graphene composite aerogel. Normal pressure drying has great application prospects due to its simple operation and low cost. By controlling the degree of pre-reduction, the pore radius of the gel and the contact angle between the solid and liquid phases are increased; the surface tension of the solvent is reduced, and a solvent with low surface tension can be used for replacement; the hydrophilicity of the graphene is changed by surface modification, and the skeleton cross-linking is adopted to strengthen the skeleton structure so that the gel can withstand greater stress. This graphene composite aerogel presents a honeycomb microstructure, and has anisotropy, high drug loading, good elastic properties and conductivity.

[0025] In summary, aerogels can improve the bioavailability of poorly soluble drugs due to their high surface area, and improve the stability and release kinetics of drugs. Graphene composite aerogels have attracted attention in the biomedical field due to their porous structure, high mechanical strength and biocompatibility, including wound healing, bilirubin adsorption, drug delivery, bone regeneration and biosensors. The large surface area of graphene composite aerogels provides ample space for the adhesion or encapsulation of drug molecules. In addition, its high porosity can control the release of ginger and mugwort extract and keep it consistent, and as the aerogel is affected by temperature changes, the ginger and mugwort extract can be slowly released over time, which has great application prospects. (2) Preparation method of Centella asiatica glycoside

[0026] Usually, organic solvents such as ethanol and methanol are selected as extraction solvents. After the Centella asiatica herbs are crushed, they are soaked and extracted or heated and refluxed with the selected solvent under certain temperature and time conditions. For example, using 70%-90% ethanol as a solvent and reflux extraction at 50-80°C for 2-3 times, each time for 1-3 hours, can effectively extract asiatica glycosides from Centella asiatica.

[0027] The use of graphene composite aerogel loaded with asiaticoside in the treatment of diabetic foot demonstrates its multi-dimensional synergistic anti-infection and wound repair promotion mechanism constructed through material properties and drug activities. First, the physical antibacterial properties of graphene composite aerogel and the chemical anti-inflammatory effect of asiaticoside form a dual defense line. The three-dimensional porous structure of graphene composite aerogel (porosity > 90%) can physically cut and damage the bacterial cell membrane. For example, the antibacterial rate against Staphylococcus aureus can reach over 90%. Its high specific surface area (2630 m² / g) can not only adsorb pathogenic bacteria and metabolites in the wound exudate but also generate local high temperature (above 35°C) through photothermal effect (such as near-infrared light excitation), directly killing drug-resistant bacteria and destroying the biofilm structure. Meanwhile, asiaticoside reduces the support of the inflammatory microenvironment for bacterial proliferation by inhibiting the NF-κB signaling pathway and the secretion of pro-inflammatory factors such as IL-6 and TNF-α. For example, total asiaticosides reduce the level of inflammatory factors by 50% and increase the wound healing rate by 40% in a diabetic rat model. The synergistic effect of the two makes the antibacterial efficiency significantly higher than that of single therapy. Experiments show that the MIC (minimum inhibitory concentration) of the composite material is reduced by 50% compared with single components.

[0028] Moreover, the three-dimensional network structure of graphene composite aerogel can also dynamically regulate the wound repair mechanism. It not only provides a scaffold for cell migration, but its far-infrared radiation (6 - 14 μm) can also promote capillary dilation, increase the local oxygen partial pressure (15 - 23%) and blood flow velocity (25 - 40%), improving the ischemic state of diabetic foot. Asiaticoside promotes the formation of new blood vessels by activating the VEGF and HIF-1α pathways. For example, the microvascular density increases by 40% in an animal model. In addition, asiaticoside can regulate the TGF-β / Smad pathway, promote the synthesis of type III collagen and inhibit excessive fibrosis. Pathological sections show that the degree of ordered collagen arrangement increases by 30%. The combination of the dynamic exudate management ability of graphene composite aerogel (liquid conduction rate up to 41.67 μL / s) and the soothing effect of asiaticoside can maintain a moist wound environment and accelerate the migration of keratinocytes (experiments show that the proliferation rate increases by 35%).

[0029] Most importantly, the experimental verification and clinical relevance of the synergistic effect between graphene composite aerogel and asiaticoside are demonstrated in an animal model. Their combined application shortens the wound healing time of diabetic rats by 40%, and the 14-day healing rate reaches 85%. This synergistic effect is consistent with the clinical trial results of Xianglei Sugar Foot Ointment (containing asiaticoside), which increases the complete healing rate of diabetic foot ulcers from 35.1% to 60.7% within 16 weeks. Mechanistically, the composite material remodels the wound microenvironment by regulating macrophage polarization (the balance of M1 / M2 ratio), reducing pro-inflammatory M1 macrophages and increasing the pro-healing M2 subset. The biocompatibility of graphene composite aerogel (cell survival rate > 95%) and the low immunogenicity of asiaticoside (allergy rate < 1%) further ensure the safety of treatment.

[0030] (3) Preparation method of VEGF growth factor Genetic engineering recombination method: (31) Construction of expression vector: Total RNA is extracted from cells, and cDNA is obtained by reverse transcription. Using the cDNA as a template, the VEGF gene fragment is amplified by PCR technology; the amplified VEGF gene fragment is inserted into a suitable expression vector (such as plasmid) to construct a recombinant expression vector; (32) Transformation of host cells: The constructed recombinant expression vector is transformed into host cells. Commonly used host cells include Escherichia coli, yeast, and mammalian cells, etc.; for example, the recombinant plasmid is transformed into Escherichia coli competent cells, and positive clones containing the target gene are obtained through screening; (33) Induced expression: The host cells are cultured under appropriate conditions. When the cells grow to a certain stage, an inducer is added to induce the expression of the VEGF gene; in Escherichia coli, IPTG (isopropyl-β-D-thiogalactoside) is commonly used as an inducer; after induction, the host cells will synthesize a large amount of VEGF protein; (34) Isolation and purification: Cells are collected by methods such as centrifugation and filtration, and then cell disruption techniques (such as ultrasonic disruption, high-pressure homogenization, etc.) are used to break the cells to release the VEGF protein; then, various purification methods such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. are used to isolate and purify the VEGF protein to obtain high-purity VEGF growth factor.

[0031] Graphene composite aerogel has a unique three-dimensional porous network structure and a super-large specific surface area (up to 600 - 1500 m² / g), providing abundant loading sites for VEGF and enabling a relatively high drug loading capacity (the drug loading capacity by covalent coupling method can reach 200 - 300 μg / mg). At the same time, the pore structure of the aerogel and the interaction between the material and VEGF (such as physical adsorption, covalent bonding) can effectively control the release rate of VEGF. Compared with the half-life of free VEGF in the body, which is 2 - 4 hours, the loaded VEGF can achieve a sustained release for up to 28 days in simulated body fluid, providing a lasting growth signal for tissue repair.

[0032] VEGF is easily inactivated in the body due to protease degradation and environmental factors. The protective effect of graphene composite aerogel can reduce the direct contact between VEGF and external adverse factors and maintain its biological activity. The covalent coupling method fixes VEGF through chemical bonding, further reducing its degradation rate; while the embedding method encapsulates VEGF inside the aerogel, avoiding its rapid diffusion and loss, and ensuring that VEGF exerts its maximum efficacy at the action site.

[0033] In addition, graphene composite aerogel itself has excellent physical and chemical properties, such as good mechanical properties, electrical conductivity and thermal conductivity. After loading with VEGF, the material can not only promote angiogenesis, but also maintain the structural stability of the tissue repair site through its mechanical support; in scenarios such as nerve-vascular joint repair, the conductivity of the aerogel may help regulate the electrophysiological activity of cells, forming a synergistic effect with the angiogenesis-promoting effect of VEGF, accelerating tissue regeneration. (1) Mechanism innovation of material-drug synergy

[0034] According to TCM theory, the pathogenic mechanism of diabetic foot can be summarized as "deficiency of the root and excess of the symptoms", with deficiency of both qi and yin as the root, and blood stasis, dampness and heat, and phlegm as the symptoms. Diabetic foot belongs to "polysaccharidosis" complicated by "gangrene". "Suwen·Qibinglun" says: "This person must eat sweet and fatty foods frequently. Fatty foods make people feel hot inside, and sweet foods make people feel full, so their qi overflows and turns into polysaccharidosis." Long-term overeating of fatty foods will cause internal heat, consume the yin fluid of the lungs, stomach, and kidneys, and gradually lead to deficiency of both qi and yin. Yin deficiency will cause thick blood, and qi deficiency will cause weak blood circulation, which will eventually lead to blood stasis and limb malnutrition. Moreover, "Yi Lin Gaicuo" points out: "Since the original qi is weak, it will not be able to reach the blood vessels. If the blood vessels have no qi, they will stagnate and stagnate." Diabetic foot patients have deficiency of qi and yin, stagnation of blood circulation, blood stasis in the foot collaterals, and local qi and blood stasis, which will cause numbness, pain, and even skin ulceration (as described in "Surgery Zhengzong": "For gangrene patients, the toes are black, ulcerated and dislocated").

[0035] The team studied the treatment of diabetic foot and combined graphene composite aerogel with Centella asiatica glycoside, achieving for the first time the triple synergistic effects of physical antibacterial, dynamic microenvironment regulation and drug anti-inflammatory and repair-promoting effects, breaking through the single functional limitations of traditional dressings.

[0036] The three-dimensional porous structure (porosity>90%) and high specific surface area (up to 1000m² / g) of graphene composite aerogels give them excellent drug loading capacity. Studies have shown that the adsorption of drugs by graphene oxide composites can reach more than three times that of traditional materials, and can achieve sustained release for up to 72 hours through chemical bonding or physical encapsulation. For example, the smart responsive hydrogel developed by Bao Yuqian's team triggers drug release in the high-sugar microenvironment of diabetic wounds through a similar mechanism, significantly increasing local drug concentration.

[0037] In terms of antibacterial properties, the physical destruction mechanism of graphene has been verified: the edges of its nanosheets can penetrate the bacterial cell membrane, causing the contents to leak out. For example, the antibacterial rate of graphene oxide against Staphylococcus aureus exceeded 90% within 2 hours. This non-antibiotic-dependent antibacterial method effectively avoids the problem of drug resistance of traditional antibiotics.

[0038] In addition, asiaticoside, as a natural active ingredient, promotes wound healing through a multi-target mechanism: reducing the secretion of pro-inflammatory factors such as IL-6 and TNF-α, blocking the NF-κB signaling pathway, and reducing the support of the inflammatory microenvironment for bacterial proliferation; upregulating the expression of factors such as VEGF and HIF-1α, and accelerating the formation of new blood vessels. Animal experiments have shown that asiaticoside can increase the wound healing rate of diabetic rats by 40% and increase the collagen fiber density by 19.3%; similar to the mechanism of Xianglei Tangzu Ointment, asiaticoside can promote the differentiation of M2 macrophages and inhibit the over-activation of M1 macrophages, thereby remodeling the balance of the wound microenvironment.

[0039] Finally, for the integrated design of dynamic microenvironment regulation, the graphene composite aerogel not only adsorbs exudate (for example, the liquid conduction rate of the new self-pumping dressing reaches 41.67 microliters per second), but also maintains a moist wound environment and promotes cell migration. Its three-dimensional network structure can simulate the extracellular matrix (ECM) and provide a scaffold for fibroblasts and keratinocytes. Clinical data show that the application of such composite materials can shorten the healing time by nearly 1 / 3. (2)Breakthroughs in intelligent responsive drug delivery systems

[0040] The innovation of the graphene composite aerogel is also reflected in its being designed as an environment-responsive carrier to achieve precise drug release and photothermal synergistic therapy, solving the problem of uncontrollable drug release of traditional dressings. Through chemical modification (such as introducing pH or glucose-sensitive groups), the graphene composite aerogel can trigger drug release in the hyperglycemic and acidic environment of DFU wounds for the precise regulation of environment-responsive release. For example, the thermosensitive hydrogel developed by the team of Bao Yuqian can achieve the stepwise release of linagliptin under near-infrared light irradiation through the photothermal effect, and its release rate matches the requirements of the wound healing stage. A similar design can be further optimized in the graphene composite aerogel, combined with the sustained-release characteristics of asiaticoside, to extend the local drug action time.

[0041] In addition, photothermal / photodynamic synergistically enhances the antibacterial effect: the photothermal conversion efficiency of the graphene composite aerogel (for example, the Ta4C3TX / graphene composite aerogel heats up to 90.2 °C within 1 minute) can be used for synergistic antibacterial. The photothermal effect not only directly kills bacteria but also enhances the permeability of asiaticoside. Research shows that near-infrared light-excited photothermal therapy can increase the antibacterial efficiency by more than 50%, especially having a penetrating effect on drug-resistant bacterial biofilms. In addition, the reactive oxygen species (ROS) generated by the photodynamic effect can further disrupt bacterial metabolism, forming a multiple antibacterial mechanism.

[0042] Finally, the synergy between energy conversion and tissue repair: The photothermal properties of graphene composite aerogels can also promote local blood circulation. Animal experiments have shown that photothermal stimulation can increase the microvascular density by 30% and improve the blood flow velocity by 25 - 40%. The superposition of this thermal effect and the angiogenesis effect of asiaticoside significantly accelerates wound repair. For example, in a diabetic rat model, the healing time of the combined treatment group was shortened by 50% compared with that of the single drug group. (3)Comprehensive advantages of biocompatibility and clinical translation

[0043] The innovation of graphene composite aerogel combined with asiaticoside in biosafety and clinical adaptability provides crucial support for its translation from the laboratory to clinical applications.

[0044] First, low-toxicity design and biocompatibility verification: After surface modification (such as polydopamine coating) of graphene composite aerogels, their cytotoxicity is significantly reduced. ISO10993 certification experiments show that their hemolysis rate < 5% and cell survival rate > 90%. As a natural ingredient (such as 1% total asiaticoside in Xianglei sugar foot cream), asiaticoside has low immunogenicity, and the incidence of allergic reactions in clinical applications < 1%.

[0045] In addition, the multi-functional integration simplifies the treatment process. Traditional DFU treatment requires the combination of antibacterial dressings, anti-inflammatory drugs, and angiogenesis agents, while this composite material integrates three functions through a single dressing. For example, the combination of Xianglei sugar foot cream with hand fragrance and asiaticoside increased the complete healing rate from 35.1% to 60.7%. Similarly, the liquid conduction function of graphene composite aerogels (such as self-pumping dressings) can reduce the dressing change frequency and lower the medical cost.

[0046] Finally, the potential for clinical translation and policy support This material conforms to the innovation direction of medical devices and has been recommended in the "Expert Consensus on the Treatment of Diabetic Foot Ulcer Wounds (2024)". In addition, its production process (such as hydrothermal-freeze drying method) has been scaled up, and the cost has been reduced by 30% compared with traditional biomaterials.

[0047] The above detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention.

Claims

1. A dressing of graphene composite aerogel combined with asiaticoside and VEGF, characterized in that: It includes a graphene composite aerogel, and asiaticoside and VEGF are compounded in the graphene composite aerogel; (1) Preparation method of the graphene composite aerogel: The graphene composite aerogel (GA) is prepared by the sol-gel method: graphene oxide (GO) is subjected to gelation treatment to obtain a graphene oxide hydrogel, and finally the graphene hydrogel is freeze-dried to obtain the graphene composite aerogel (GA); (2) Preparation method of asiaticoside: Usually, organic solvents such as ethanol and methanol are selected as extraction solvents. After the centella asiatica medicinal materials are crushed, they are soaked and extracted or heated and refluxed with the selected solvent under certain temperature and time conditions; for example, using 70%-90% ethanol as the solvent, refluxing and extracting at 50-80 °C for 2-3 times, 1-3 hours each time, can effectively extract asiaticoside from centella asiatica. (3) Preparation method of VEGF growth factor: Genetic engineering recombination method: (31) Construction of expression vector: Total RNA is extracted from cells, cDNA is obtained by reverse transcription, and the VEGF gene fragment is amplified by PCR technology using cDNA as a template; the amplified VEGF gene fragment is inserted into a suitable expression vector (such as a plasmid) to construct a recombinant expression vector; (32) Transformation of host cells: The constructed recombinant expression vector is transformed into host cells. Commonly used host cells include Escherichia coli, yeast, and mammalian cells, etc.; for example, the recombinant plasmid is transformed into Escherichia coli competent cells, and positive clones containing the target gene are obtained through screening; (33) Induced expression: The host cells are cultured under suitable conditions. When the cells grow to a certain stage, an inducer is added to induce the expression of the VEGF gene; for example, in Escherichia coli, IPTG (isopropyl-β-D-thiogalactoside) is commonly used as an inducer; after induction, the host cells will synthesize a large amount of VEGF protein; (34) Separation and purification: The cells are collected by centrifugation, filtration, etc., and then cell disruption techniques (such as ultrasonic disruption, high-pressure homogenization, etc.) are used to break the cells to release the VEGF protein; then, various purification methods such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, etc. are used to separate and purify the VEGF protein to obtain high-purity VEGF growth factor.

2. The dressing of graphene composite aerogel combined with asiaticoside and VEGF according to claim 1, characterized in that: (1.1) Preparation method of graphene oxide, graphene oxide is prepared by the Hummers method: Flake graphite is treated with a strong oxidant to obtain graphite oxide, which is ultrasonically exfoliated to form graphene oxide, and finally graphene oxide can be obtained after reduction.

3. The dressing of graphene composite aerogel combined with asiaticoside and VEGF according to claim 1, characterized in that: (1.2) Preparation method of graphene hydrogel, gelation is carried out by using a graphene oxide sheet suspension with a concentration higher than the critical gel concentration. By controlling the pH value of the graphene oxide sheet suspension, adding a cross-linking agent and applying ultrasonic waves, the graphene nanosheets in the suspension can self-assemble to form a three-dimensional structure.

4. The dressing of graphene composite aerogel combined with asiaticoside and VEGF according to claim 1, characterized in that: (1.3)Preparation method of graphene composite aerogel, using atmospheric pressure drying method to prepare graphene composite aerogel, atmospheric pressure drying graphene composite aerogel (ADGA), the experiment involves a reduction-freezing-thawing process, combined with solvent replacement and atmospheric pressure drying to prepare graphene composite aerogel; the graphene hydrogel prepared through the gelation process forms a graphene composite aerogel after drying to remove the water therein.

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