Chitosan-coated silver nanoparticles for adsorbing andrographolide and chlorogenic acid as well as preparation method and application of chitosan-coated silver nanoparticles

Abstract

The invention provides silver nanoparticles wrapped by chitosan and adsorbing andrographolide and chlorogenic acid and a preparation method and application of the silver nanoparticles, and relates to the technical field of biomedicine.The preparation method comprises the following steps that PEI-AgNPs is placed in a centrifugal tube to be centrifuged, supernatant is discarded, and obtained sediment is dried; adding the precipitate into a centrifugal tube, adding DMSO, adding AG and CA, dissolving in the centrifugal tube, stirring at room temperature in a dark place, centrifuging, and taking the supernatant after centrifuging; adding DMSO (dimethylsulfoxide), washing, collecting the obtained supernatant after washing, and drying the precipitate; dissolving CS in an acetic acid solution, and then adding the precipitate; according to the application, the characteristics of Ag < + >, andrographis paniculata and ginkgo leaves are integrated, andrographolide and chlorogenic acid are combined with Ag < + >, and a natural adhesive chitosan is used for wrapping, so that the growth of escherichia coli and staphylococcus aureus can be inhibited, and the antibacterial effect is achieved. The level of active oxygen and inflammatory factors of the wound can be reduced by inhibiting an NF-kappa B signal channel and activating a PI3K-AKT signal channel, and skin wound healing is promoted.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to chitosan-wrapped silver nanoparticles for absorbing andrographolide and chlorogenic acid, a preparation method and application thereof. Background Art

[0002] The skin is the largest organ in the human body and can protect the body from microbial infection, high temperature, radiation and other injuries. When the skin is injured by punctures, scratches and other injuries, it usually produces skin lesions of varying sizes. Some small and shallow skin lesions can usually heal on their own and do not require human intervention, but some relatively large wounds that penetrate the entire layer of the skin require additional treatment. At the same time, a large number of pathogenic pathogens live on the surface of the skin, such as Staphylococcus aureus and Escherichia coli. Normal skin has a certain barrier effect on pathogens. When the skin is damaged, the skin surface barrier is damaged, and these pathogens can quickly colonize the damaged area, which has a certain inhibitory effect on the healing of skin wounds. Therefore, for the treatment of skin injuries, it is necessary to focus on both promoting healing and its antibacterial properties.

[0003] Good antibacterial properties are the key to the development of materials for repairing skin injuries. Currently, a variety of antibacterial excipients have been used to inhibit clinical wound microbial infections. For example, silver sulfadiazine has been widely used in the treatment of skin burns. Summary of the invention

[0004] The purpose of the present invention is to explore the combination of silver nanoparticles with traditional Chinese medicine monomers, so as to achieve the dual effects of antibacterial and promoting wound healing.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid, the specific steps are as follows:

[0007] S1: Place PEI-AgNPs in a centrifuge tube, centrifuge, discard the supernatant, and dry the obtained precipitate;

[0008] S2: Take the precipitate obtained in S1 and add it to a centrifuge tube, add DMSO, then add AG and CA to dissolve in the centrifuge tube, stir at room temperature in the dark, and centrifuge. After the centrifugation is completed, take the supernatant;

[0009] S3: Place the supernatant obtained in S2 in a beaker, add DMSO, wash, collect the supernatant after completion, and dry the precipitate;

[0010] S4: Dissolve CS in acetic acid solution and adjust the pH to 6.5. After CS is completely dissolved in the acetic acid solution, add the precipitate obtained in S3.

[0011] Preferably, the centrifugation condition in S1 is 12000r for 15 min, and the precipitate drying condition is drying in the dark at room temperature for 10 min.

[0012] Preferably, the amount of PEI-AgNPs in S2 is 20000 mg, 10 mL of DMSO is added, and the amount of AG and CA added is 15 mg each.

[0013] Preferably, the S2 is stirred at room temperature in the dark for 24 hours, and the centrifugation condition is 12000r for 5min.

[0014] The present application also provides chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid, which are prepared using the preparation method described above.

[0015] The present application also provides the use of chitosan-encapsulated silver nanoparticles that adsorb andrographolide and chlorogenic acid in the preparation of a drug for promoting skin wound healing. The chitosan-encapsulated silver nanoparticles that adsorb andrographolide and chlorogenic acid are prepared by the preparation method described in any one of claims 1 to 4.

[0016] Preferably, the chitosan-encapsulated silver nanoparticles adsorbing andrographolide and chlorogenic acid promote skin wound healing by inhibiting the NF-κB signaling pathway and activating the PI3K / AKT signaling pathway.

[0017] This application integrates Ag + The characteristics of Andrographis paniculata and Ginkgo biloba leaves were studied by combining the monomers of andrographolide and chlorogenic acid from Andrographis paniculata and Ginkgo biloba leaves with Ag + It is combined with the natural adhesive chitosan for further wrapping. Through specific verification experiments, it is found that it can not only inhibit the growth of Escherichia coli and Staphylococcus aureus, but also reduce the levels of wound reactive oxygen and inflammatory factors by inhibiting the NF-κB signaling pathway and activating the PI3K-AKT signaling pathway. It promotes skin wound healing very well with significant effects, and is worthy of further promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The morphology and related performance verification analysis diagram of the material prepared in one embodiment of the present invention; wherein, A, electron microscopic photograph and particle size analysis of AgNPs; B, electron microscopic photograph and particle size analysis of AgNPs after drug loading; C, electron microscopic photograph and particle size analysis of AgNPs after being wrapped by CS; D, chemical structural formulas of AG and CA; E, comparison of cytotoxicity of different drug concentrations on Hacat cells; F, comparison of cytotoxicity of different drug pairs on Hacat cells; G, H, disc diffusion method for detecting the anti-Staphylococcus aureus and Escherichia coli activity of different solutions with an AgNPs concentration of 100 mg / ml.

[0019] Figure 2 The figure is a cell experiment analysis diagram in one embodiment of the present invention; wherein A, cell migration experiment, at 12 hours and 24 hours, CS-AG / CA-AgNPs can promote cell migration most significantly (p < 0.05), and the scratch area is almost completely covered at 24 hours; B, cell active oxygen level measurement, CS-AG / CA-AgNPs has the most obvious effect in reducing active oxygen (p < 0.05).

[0020] Figure 3 This is the detection of pathways and inflammatory factors by tissue WB and qPCR in one embodiment of the present invention. A, the inhibition of NF-κB in tissues on the third and sixth days, CS-AG / CA-AgNPs had the most obvious inhibitory effect (p < 0.05); B, D are the relative expression levels of TNF-α and IL-1β in tissues on the sixth day, and it can be seen that CS-AG / CA-AgNPs can significantly reduce the expression of both (p < 0.05); C, the activation of the PI3K / Akt signaling pathway in tissues on the third and sixth days, CS-AG / CA-AgNPs can significantly activate the PI3K / Akt signaling pathway compared with other groups (p < 0.05).

[0021] Figure 4 The degree of skin regeneration at 3, 6, and 9 days in one embodiment of the present invention. A, macroscopic images of wounds at different periods, scale bar 1 mm per grid; B, histological HE staining observation of wounds at different times; C, percentage of wound healing at different times compared to day 0, * indicates that the difference between the experimental group and the control group is statistically significant (p < 0.05); D, quantitative measurement of the thickness of the new layer of the skin at different times, the difference between the experimental group and the control group is statistically significant (p < 0.05). DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments.

[0023] The present application provides a method for preparing chitosan-encapsulated silver nanoparticles that adsorb andrographolide and chlorogenic acid as follows:

[0024] S1: Place 200 mL of PEI-AgNPs (100 mg / mL) in a 50 mL centrifuge tube, centrifuge at 12000 r for 15 min, discard the supernatant, keep the precipitate, and dry the precipitate at room temperature in the dark for 10 min;

[0025] S2: Take 20000 mg of PEI-AgNPs obtained in S1 and add them to a centrifuge tube. Then add 10 mL of DMSO to the centrifuge tube, and then take 15 mg of AG and 15 mg of CA and dissolve them in the centrifuge tube. Stir at room temperature in the dark for 24 h and then centrifuge at 12000 r and 5 min. After the centrifugation, take the supernatant.

[0026] S3: Place the supernatant in a beaker, add 10 mL of DMSO to the beaker, wash three times at 8000 r, 30 min each time, collect the supernatant and save for concentration measurement, and dry the precipitate at 37 °C for 30 min;

[0027] S4: Dissolve chitosan (CS) in 0.1% acetic acid solution, adjust the pH to 6.5, add the precipitate obtained in S3 after complete dissolution, and the final concentration is 200 mg / mL to obtain CS-AG / CA-AgNPs.

[0028] The present application also provides chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid, which are prepared using the above-mentioned preparation method.

[0029] In addition, the present application also provides the use of chitosan-encapsulated silver nanoparticles that adsorb andrographolide and chlorogenic acid in the preparation of a drug for promoting skin wound healing. The chitosan-encapsulated silver nanoparticles that adsorb andrographolide and chlorogenic acid promote skin wound healing by inhibiting the NF-κB signaling pathway and activating the PI3K / AKT signaling pathway.

[0030] The following is an explanation of the above content in combination with specific verification experiments:

[0031] Materials used in this application:

[0032] PEI-AgNPs (Nanjing Jike Nano-Biological Co., Ltd., China), AG, CA (Sigma), dimethyl sulfoxide (DMSO), CS (Shanghai Pharmaceutical Group, China), Staphylococcus aureus and Escherichia coli (Veterinary College of Yangzhou University, China), Bacterial Viability Detection Kit (Suzhou Youyi Landi Biotechnology Co., Ltd., China), CCK8 Cell Proliferation Detection Kit (NoviZan, China), ROS Detection Kit (Biyuntian, USA), Balb / C mice (Yangzhou University Comparative Medical College, China), fetal bovine serum was purchased from GIBCO (Cat. No.: 11960044, 12483020). MEM non-essential amino acid supplement (Yisheng, China).

[0033] Human skin keratinocytes (HaCat) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The temperature of the constant temperature cell incubator was set at 37°C and CO 2 The concentration was 5%.

[0034] Example 1: Preparation of CS-CA / AG-AgNPs

[0035] 200 mL PEI-AgNPs (100 mg / mL) was placed in a 50 mL centrifuge tube, centrifuged at 12000 r for 15 minutes, the supernatant was discarded, the precipitate was kept, and dried at room temperature in the dark for 10 minutes. 10 mL DMSO was added to three centrifuge tubes containing 20000 mg PEI-AgNPs, and 30 mg AG, 30 mg CA, 15 mg AG and 15 mg CA were dissolved in the above three centrifuge tubes respectively.

[0036] After stirring at room temperature in the dark for 24 hours, centrifuge at 12000r for 15 minutes. Place the supernatant in different beakers, add 10mL DMSO to the centrifuge tube, wash three times, 30 minutes each time, collect the supernatant for concentration measurement, and collect the precipitate and dry it at 37℃ for 30 minutes for subsequent preparation.

[0037] CS was dissolved in 0.1% acetic acid solution, adjusted to pH = 6.5, and PEI-AgNPs was added after complete dissolution, with a final concentration of 200 mg / mL to obtain CS-AgNPs, CS-AG-AgNPs, CS-CA-AgNPs, and CS-AG / CA-AgNPs.

[0038] Example 2: Drug loading rate and particle size analysis:

[0039] Particle size analysis: 10 transmission electron microscopy photos were randomly selected, and their diameters were measured using Image J, and particle size analysis was performed using Origin 2019b.

[0040] AgNPs can be optimized under certain conditions to nanoparticles with a clear size and morphology and a stable structure with a diameter below 100 nm. In this application, TEM was used to observe that the nanoparticles were uniform in size, regular in morphology, and nearly round. The average diameter ranged from 65.375 ± 3.639 nm ( Figure 1 , A). The diameter of AgNPs changed significantly after drug adsorption (p<0.05), with an average diameter range of 70.234±3.623nm ( Figure 1 , B). After the outer layer was coated with chitosan, the thickness of chitosan was included in its diameter, and the thickness increased, and the diameter changed slightly (p < 0.05). The average diameter range was 78.410 ± 2.138 nm ( Figure 1 , C).

[0041] Drug loading rate analysis: The supernatants of AG-AgNPs, CA-AgNPs, and AG / CA-AgNPs were thoroughly mixed, and the absorbance at 223 nm and 329 nm was measured using an ultraviolet spectrophotometer (Thermo Scientific, USA). The drug loading amount was calculated using the formula absorbance A = 0.0063 × concentration C (μg / mL) - 0.0006, and the drug loading rate was further calculated.

[0042] AgNPs are modified with PEI and have positive charges on their surfaces, while the structures of AG and CA both have hydroxyl groups ( Figure 1 D), carries a negative charge in aqueous solution, so AgNPs can adsorb drugs through the principle of attraction between positive and negative charges. The drug concentration in the supernatant is then determined using the ultraviolet absorption peaks of AG and CA to calculate the drug loading. In this application, the adsorption amount of AG in AG-AgNPs is calculated to be 15.82±0.63mg based on the OD value, and the adsorption rate is 52.73±0.21%. The adsorption amount of CA in CA-AgNPs is 15.96±0.21mg, and the adsorption rate is 53.18±0.41%. AG 11.58±0.06mg and CA 7.60±0.18mg are adsorbed in CA / AG-AgNPs, and the adsorption rates are 77.18±0.40% and 50.63±1.21%, respectively.

[0043] Example 3: Transmission electron microscopy:

[0044] AgNPs, CA / AG-AgNPs, CS-AgNPs, and CS-CA / AG-AgNPs were characterized by a Tecani G2F30 field emission transmission electron microscope from the United States.

[0045] Example 4: Antibacterial experiment:

[0046] Staphylococcus aureus and Escherichia coli were inoculated into LB liquid medium and cultured overnight at 37°C. Sterile drug-sensitive paper sheets were soaked in AG-AgNPs, CA-AgNPs, and AG / CA-AgNPs with an AgNPs concentration of 100 mg / mL. 5 The bacteria were inoculated into LB agar medium, and the soaked sterile drug-susceptibility paper was spread flat on the medium. The blank drug-susceptibility paper was used as a negative control. The culture was carried out at 37℃ for 24 hours, and the size of the inhibition ring was observed.

[0047] Ag was widely used as early as 2000 years ago, and the antibacterial property of silver has received widespread attention since the 19th century. With the deepening of research, it has been confirmed that AgNPs have a certain inhibitory effect on both Gram-negative and Gram-positive bacteria, and can affect the activity of more than 600 kinds of bacteria. Its main antibacterial mechanism is that AgNPs can attract the negative charge on the surface of bacteria through the positive charge on its surface, which is conducive to the entry of AgNPs into the bacteria through the cell membrane of bacteria, further promoting the production of active oxygen inside bacteria, thereby blocking the signal transduction effect of multiple signal pathways. Damage to the skin mucosal barrier is the main cause of infection, and the infected bacteria are mainly Staphylococcus aureus and Escherichia coli. Therefore, the present application uses the disc diffusion method to detect the anti-Staphylococcus aureus and Escherichia coli activity of different solutions with an AgNPs concentration of 100 mg / mL, among which CS-AG / CA-AgNPs show the best antibacterial activity against Staphylococcus aureus and Escherichia coli compared with other groups. The percentages of the sizes of the inhibition zones of CS-AgNPs, CS-AG-AgNPs, CS-CA-AgNPs, and CS-AG / CA-AgNPs relative to the size of the blank control group are shown in Table 1 ( Figure 1 G, H).

[0048] Table 1 Percentage of antibacterial zone compared with blank control group

[0049]

[0050] Example 5: In vitro biological toxicity test:

[0051] Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation and cytotoxicity. HaCat cells were trypsinized and counted, and a cell suspension containing about 1000 cells was inoculated in a 96-well plate. 100 μL of culture medium was added and cultured for 24 hours. 90 μL of serum-free culture medium was replaced, and 10 μL of different CS solutions with AgNPs concentrations of 200 mg / mL, 100 mg / mL, 50 mg / mL and 25 mg / mL were added and cultured for 24 hours. 90 μL of serum-free culture medium was replaced, and 10 μL of CCK-8 solution was added to each well. The cells were cultured in a 37°C incubator for 4 hours, and the absorbance at 450 nm was measured by a microplate reader.

[0052] Excess Ag +Andrographolide has certain toxicity to cells. If the dosage is too large, it is likely to put the cart before the horse. The results of CCK8 experiment showed that the above four nano drugs have certain cytotoxicity to HaCat cells, and the cell concentration is different at different concentrations. CS-AG / CA-AgNPs has the lowest cytotoxicity to HaCat cells compared with other groups (p < 0.05), and it shows a proliferation-promoting effect when the AgNPs concentration is 100mg / mL ( Figure 1 E). Among them, CS-AG / CA-AgNPs had the strongest effect in promoting proliferation when the AgNPs concentration was 100 mg / mL ( Figure 1 F).

[0053] Example 6: Cell migration assay

[0054] HaCat cells were trypsinized into single-cell suspension and then inoculated into 6-well plates at 37°C and 5% CO. 2 Culture in the incubator for 24 hours. After the cells completely cover the 6-well plate, scratch the 6-well plate with a 200μL sterile pipette tip vertically. Observe the center of the scratch under a microscope and wash it three times with sterile PBS. Add 1mL of serum-free culture medium to each well, and then add 100μL AgNPs with different CS solutions of 200mg / mL, 100mg / mL, and 50mg / mL for 12 hours and 24 hours. Take pictures under the microscope, and randomly draw 3 horizontal lines with Image J software to calculate the mean intercellular distance and cell migration rate. Cell migration rate = (mean of initial intercellular distance - mean of intercellular distance at time t) / mean of initial intercellular distance.

[0055] In vitro wound healing experiments can simulate the process of cell migration during wound healing. At 12 h, there was no significant difference between the Contral group and CS-AgNPs and CS-AG-AgNPs. At 24 h, CS-AG / CA-AgNPs showed significant differences compared with other groups. The scratched area was almost completely covered with cells, indicating that CS-AG / CA-AgNPs had a significant effect in promoting cell proliferation and migration. Figure 2 A).

[0056] Example 7: Cellular Reactive Oxygen Detection

[0057] The cells were seeded in a 6-well plate. After the cells completely covered the well plate, 100 μL of 0.2% hydrogen peroxide was added to induce cell damage. At the same time, 100 μL of different CS solutions with a concentration of 100 mg / mL of AgNPs were added. After 24 hours of co-culture, DCFH-DA was diluted with serum-free medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. The medium was removed and 1 mL of the diluted DCFH-DA was added. Incubate at 37°C for 20 minutes. Wash three times with serum-free cell culture medium and measure the fluorescence intensity with an ELISA reader.

[0058] Excessive ROS can directly or indirectly lead to the degradation of ECM proteins and promote the migration of inflammatory cells to the inner area of ​​the wound surface, which not only damages the function of dermal fibroblasts and keratinocytes, but also hinders wound angiogenesis. CS-AG / CA-AgNPs can significantly reduce the level of cellular reactive oxygen species, which is beneficial to wound healing ( Figure 2 B).

[0059] Example 8: In vivo experiment:

[0060] 1. Living wound model and surgical techniques

[0061] 6-8 week old Balb / c mice were anesthetized with 50mg / kg of Zotazone. After anesthesia, the back was shaved, and after disinfection with iodine, a full-thickness incision with a diameter of about 1cm was made with the spine as the center. The mice were randomly divided into groups. The treatment group was smeared with 100μL CS-AgNPs every two days after surgery, and the control group was not treated. The wound was photographed with a camera and a measuring scale on the 3rd, 6th, and 9th days after surgery. Image J was used to analyze the wound size and calculate the healing rate.

[0062] The immune response plays an important role in the acute wound healing process. The activation of immune cells and factors initiates the inflammatory process, promotes wound cleaning and promotes subsequent tissue healing. However, during the wound healing process, dysregulation of the immune system can lead to persistent inflammation and delayed healing, ultimately leading to chronic wounds. The chronic wound microenvironment is characterized by a large number of pro-inflammatory macrophages, overexpression of inflammatory mediators such as TNF-α and IL-1β, increased matrix metalloproteinase activity and abundant reactive oxygen species.

[0063] The NF-κB signaling pathway is one of the important pathways in cell signaling. The nuclear factor κB (NF-κB) transcription factor family plays an important role as a stressor in the cellular environment and controls the expression of important regulatory genes such as immunity, inflammation, death and cell proliferation. NF-kB protein is located in the cytoplasm and can be activated by various cell stimuli. After activation, it can induce the expression of IL-1β and TNF-α. After the release of the two, the action of fibroblasts can be inhibited, which further leads to a decrease in the production of growth factors. Therefore, IL-1β and TNF-α show certain negative effects in the process of skin wound healing. Chlorogenic acid has been shown to improve oxidative stress and inflammatory responses by regulating the NF-κB signaling pathway, thereby preventing the occurrence of diabetic nephropathy. In the present application, it was found that there was no significant difference in the inhibitory effect of the NF-κB signaling pathway on the third day of tissue growth, but the activation of the NF-κB signaling pathway could be significantly inhibited on the sixth day ( Figure 3 A), and can reduce the expression of IL-1β and TNF-α in tissues ( Figure 3B, D), among which CS-AG / CA-AgNPs were better than CS-AG-AgNPs and CS-CA-AgNPs (p < 0.05), which not only showed that CS-AG / CA-AgNPs could inhibit the activation of NF-κB signaling pathway and reduce the expression of inflammatory factors, but also showed that the combined use of AG and CA could better promote the healing of skin wounds.

[0064] (II) Extraction of tissue protein and RNA

[0065] Cells and tissues were lysed using RIPA lysis buffer containing protease inhibitors. After the protein concentration was consistent with that determined by BCA, SDS loading buffer was added, boiled at 100°C, denatured, separated by electrophoresis and transferred to a PVDF membrane (Merck, Germany). The membrane was blocked in 5% skim milk, incubated with specific primary antibodies at 4°C overnight, and then incubated with secondary antibodies (ABclonal, Wuhan, China) at room temperature for 2 hours. Signals were detected using the ChemiDocTM MP imaging system (BioRad Laboratories, California, USA). The antibodies used in Western blotting were purchased from Abcam. Total RNA was extracted using the FreeZolReagent kit (Nanjing, Novezan), and the isolated total RNA was quantified and transcribed into cDNA using a reverse transcription kit (TOLOBIO, Shanghai, China).

[0066] GAPDH was used as a control and 2 -ΔΔ The Cq method was used to calculate the mRNA expression level of the detected gene by quantitative RT-PCR. The primers were designed and synthesized by Sangon according to the NCBI gene information. The specific primer sequence information is shown in Table 2.

[0067] Table 2 Primer information

[0068]

[0069] (III) HE staining

[0070] Mice were treated with CO 2 After inhalation killing, cut about 0.5CM skin tissue around the wound, fix with 4% paraformaldehyde for 24 hours, dehydrate and embed, use a slicer to prepare 5um thick slices, dewax and HE stain, observe and take pictures under a microscope. The above experiments have passed the Yangzhou University Experimental Animal Welfare Ethics Review, No.: 202401014.

[0071] 4. Statistical methods

[0072] Each experiment was repeated at least three times. Continuous variables are expressed as mean and standard deviation (SD). Unpaired two-tailed Student's t-test or one-way analysis of variance was used for comparison of continuous variables. All analyses were performed using GraphPad Prism 8 software (GraphPad software, USA) and Origin 2019b 32Bit (ElectronicArts Inc, USA). P values ​​< 0.05 were considered statistically significant. Asterisks "*", "**", "***", and "****" represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.

[0073] The experiment of this application found that the combination of CS, AG, CA and AgNPs can promote the phosphorylation of PI3K and Akt at 3 days and 6 days, and activate the PI3K / Akt signaling pathway (p < 0.05). CS-AG / CA-AgNPs showed a significant activation effect on this pathway at 6 days (p < 0.05), which further confirmed that CS-AG / CA-AgNPs can better promote skin wound healing ( Figure 3 C).

[0074] In order to verify the efficiency of promoting wound healing, the present invention applies the nano drug to the whole layer of the skin wound surface of the experimental group mice every day, and applies normal saline to the control group. The skin defect area of ​​the mice skin on the 3rd, 6th and 9th days is recorded by camera ( Figure 4 A), calculate the wound area healing rate ( Figure 4 C). To avoid the effects of dry skin and open state of the wound in the control group, the physiological saline applied by the present application did not result in significant shrinkage in the control group on the third day, but CS-AG / CA-AgNPs showed the effect of promoting wound healing (p < 0.05). On the 6th day, CS-AG-AgNPs, CS-CA-AgNPs, and CS-AG / CA-AgNPs all showed the effect of promoting skin wound healing, and on the 9th day, the wounds of mice in the CS-AG / CA-AgNPs group were almost completely healed (p < 0.05) (Table 3).

[0075] Table 3 Wound healing rate

[0076]

[0077] The skin tissue undergoes three main phases in the repair process: inflammation, proliferation, and repair. Therefore, the wound surface specimens were stained with HE after surgery and at 3, 6, and 9 days after surgery, and tissue changes were observed under a microscope. Histological images showed that compared with the blank control group, the regenerated epithelial layer in the CS-AG-AgNPs, CS-CA-AgNPs, and CS-AG / CA-AgNPs groups was thicker and more complete during the proliferation phase ( Figure 4 B), the thickness of the newly generated epidermis in the CS-AG / CA-AgNPs-treated group was 95.965±2.163um, which was significantly higher than that in the control group (p less than 0.05). On the 9th day, the thickness of the epidermis in the wound treated with CS-AG / CA-AgNPs was no different from that on the 0th day ( Figure 4 D). This further demonstrates that CS-AG / CA-AgNPs can not only make the acute wound epithelium enter the proliferative phase earlier, but also accelerate the speed of wound repair. The key to successful wound healing is the regeneration of the epidermis. Histological results show that CS-AG / CA-AgNPs has the ability to significantly promote skin wound healing.

[0078] In summary, this application combines Ag + The characteristics of Andrographis paniculata and Ginkgo biloba leaves were studied by combining the monomers of andrographolide and chlorogenic acid from Andrographis paniculata and Ginkgo biloba leaves with Ag + The invention is combined with the natural adhesive chitosan for further encapsulation. It is found that the invention can not only inhibit the growth of Escherichia coli and Staphylococcus aureus, but also reduce the levels of reactive oxygen and inflammatory factors in wounds by inhibiting the NF-κB signaling pathway and activating the PI3K-AKT signaling pathway, thereby promoting skin wound healing.

[0079] In summary, CS-AG / CA-AgNPs have good antibacterial properties and have the potential to be developed into antibacterial dressings. CS-AG / CA-AgNPs can inhibit the NF-κB signaling pathway, activate the PI3K / AKT signaling pathway, reduce inflammation in skin damaged tissues, and promote skin wound healing.

Claims

1. A method for preparing chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid, characterized in that: The specific steps are as follows: S1: Place PEI-AgNPs in a centrifuge tube, centrifuge, discard the supernatant, and dry the obtained precipitate; S2: Take the precipitate obtained in S1 and add it to a centrifuge tube, add DMSO, then add AG and CA to dissolve in the centrifuge tube, stir at room temperature in the dark, and then centrifuge. After the centrifugation is completed, take the supernatant; S3: Place the supernatant obtained in S2 in a beaker, add DMSO, wash, collect the supernatant after completion, and dry the precipitate; S4: Dissolve CS in acetic acid solution and adjust the pH to 6.

5. After CS is completely dissolved in the acetic acid solution, add the precipitate obtained in S3.

2. The method for preparing chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid according to claim 1, characterized in that: The centrifugation condition in S1 is 12000r for 15min, and the precipitate drying condition is drying in the dark at room temperature for 10min.

3. The method for preparing chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid according to claim 2, characterized in that: The amount of PEI-AgNPs in S2 is 20000 mg, 10 mL of DMSO is added, and the amount of AG and CA added is 15 mg.

4. The method for preparing chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid according to claim 3, characterized in that: The S2 was stirred at room temperature in the dark for 24 h, and the centrifugation condition was 12000 r for 5 min.

5. Silver nanoparticles encapsulated with chitosan and adsorbing andrographolide and chlorogenic acid, characterized in that: The method is used to prepare the product according to any one of claims 1 to 4.

6. Use of chitosan-encapsulated silver nanoparticles absorbing andrographolide and chlorogenic acid in the preparation of a drug for promoting skin wound healing, characterized in that: The chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid are prepared by the preparation method according to any one of claims 1 to 4.

7. Use of the chitosan-coated silver nanoparticles that adsorb andrographolide and chlorogenic acid according to claim 6 in the preparation of a drug for promoting skin wound healing, characterized in that: The chitosan-encapsulated silver nanoparticles adsorbing andrographolide and chlorogenic acid promote skin wound healing by inhibiting the NF-κB signaling pathway and activating the PI3K / AKT signaling pathway.

Images