Sliver Nanoparticles Modified HFGF2-linking Oil Bodies For Accelerating Bacterial Infection Wound Healing

In recent years,by using plant oil bodies(OB)to express exogenous protein as a safe lipid delivery system,human basic fibroblast growth factor(hFGF2)has been successfully expressed on camelina oil bodies(hFGF2-OB)which improves the stability and transdermal absorption effect of hFGF2 protein and promoting wound healing.However,the antibacterial activity of hFGF2-OB is not significant.The ideal antimicrobial wound dressing should not only have antimicrobial activity,but also release substances that accelerate tissue repair.Therefore,this project aims to remould hFGF2-OB,and obtain Ag NPs-hFGF2-OB by modifying silver nanoparticles(Ag NPs)with broad-spectrum antibacterial activity.The prepared Ag NPs-hFGF2-OB can not only promote the bactericidal effect in the inflammatory stage,but also release hFGF2 protein to promote proliferation and migration of fibroblasts,which is expected to provide a research strategy for the development of new wound healing preparations and an experimental basis for the design of materials to accelerate wound healing in bacterial infection.The specific results of the study are as follows:1.Preparation and characterization of Ag NPs-hFGF2-OB: Ag NPs were synthesized on the surface of hFGF2-OB by in-situ reduction method.The results of X-ray diffraction patterns,UV visible absorption spectra,and transmission electron microscopy(TEM)photos indicated the successful synthesis of Ag NPs on the hFGF2-OB surface.The average diameters of Ag NPs,hFGF2-OB and Ag NPs-hFGF2-OB were 11.8 ± 4.7 nm,259.3 ± 58.4 nm and 244.9 ± 66.5 nm,respectively.The protein content was detected by Western Blot method,and the results showed that the expression levels of hFGF2 protein in hFGF2-OB and Ag NPs-hFGF2-OB were 3.23 ±0.31 μg/m L and 2.75 ± 0.30 μg/m L,respectively.The stability analysis results showed that OB improved the stability of hFGF2 protein,while the modification of Ag NPs did not affect the stability of hFGF2 protein.2.Study on the Antibacterial Activity of Ag NPs-hFGF2-OB: The antibacterial activity of Ag NPs-hFGF2-OB was evaluated by the antibacterial zone experiment,plate counting experiment and bacterial live death staining experiment against Gram negative Escherichia coli(E.coli)and Gram positive Staphylococcus aureus(S.aureus).The results showed that Ag NPs-hFGF2-OB exhibited good antibacterial activity against two bacteria and the bacteriostatic rate reached 70%.By detecting the levels of reactive oxygen species(ROS)and glutathione(GSH)in bacteria,the antibacterial mechanism of Ag NPs hFGF2-OB was explored.The results showed that Ag NPs-hFGF2-OB could induce bacteria to produce ROS,causing severe loss of GSH in the bacterial body,causing oxidative stress damage in the bacterial body,and leading to bacterial death.3.Study on the Effect of Ag NPs-hFGF2-OB on Promoting Fibroblast Proliferation and Migration: The cell compatibility of Ag NPs hFGF2 OB was detected through MTT method,blood compatibility experiment,and cell viability staining experiment.The results showed that when the concentration of hFGF2 protein was 20 ng/m L,Ag NPs-hFGF2-OB had no significant toxicity,and this concentration was used for subsequent experiments.Ed U cell proliferation demonstrated that Ag NPs hFGF2 OB could effectively promote the proliferation of NIH/3T3 cells.Cell scratch test showed that the migration rate of NIH/3T3 cell migration treated with Ag NPs-hFGF2-OB was the most significant.4.Study on the ability of Ag NPs-hFGF2-OB to promote wound healing: Established a mouse model of E.coli infected wound,observed the healing of infected wound after skin administration,calculated the wound healing rate,and analyzed the wound skin with histopathology HE staining,Masson staining,and immunohistochemical staining.The results showed that the wound healing rate of Ag NPs-hFGF2-OB treatment group was much higher than that of other treatment groups,especially on the 7th day after treatment,the wound healing rate reached 71.71 ± 2.38%,while the wound healing rate of PBS 、 Ag NPs-WT-OB and hFGF2-OB treatment groups was only 34.54 ± 1.10%,37.08 ± 2.85% and 47.99 ± 2.01%,and the wound of Ag NPs-hFGF2-OB treatment group was completely closed on the 12 th day after treatment.Histopathology results showed that the Ag NPs-hFGF2-OB treatment group had obvious collagen deposition,granulation tissue regeneration and neovascularization.The expression of endogenous factors(Cytokinetin 5,Cytokinetin 19,Collagen I,Collagen III,α-SMA,MMP-2)in the newly formed skin tissue of wounds treated with Ag NPs-hFGF2-OB was significantly higher than that of other groups,and the significant decrease in levels of related inflammatory factors(TNF-α)indicated that Ag NPs-hFGF2-OB had better ability to promote tissue repair.In summary,we successfully prepared silver nanoparticle-modified hFGF2-OB.Ag NPs-hFGF2-OB had significant antibacterial activity against gram-negative E.coli and gram-positive S.aureus,could effectively promote the proliferation and migration of fibroblasts,reducing the inflammatory response of infected wounds,thus as a tissue repair agent to accelerate the tissue regeneration process of bacteria-infected wounds.