Preparation Of Antimicrobial Peptide Hydrogel ProSP-B And Its Effect On Wound Healing

Wound infections caused by bacteria such as methicillin-resistant Staphylococcus aureus are difficult to treat because they can develop resistance to antibiotics by forming biofilms.Therefore,a new antibacterial drug should be found for the treatment of methicillin-resistant Staphylococcus aureus infection on the wound surface.Antimicrobial peptides(AMPs)can destroy bacterial cell membranes through physical electrostatic interactions to kill bacteria.They will not cause bacteria to develop drug resistance and their own safety(no cytotoxicity or low cytotoxicity)has attracted widespread attention.The precursor protein of lung surfactant protein B(proSP-B)belongs to the SAPLIPs(saponsin-like-proteins)family.Relevant studies have confirmed that proSP-B has antibacterial activity.In the previous laboratory research,it was found that recombinant proSP-B has a significant antibacterial effect.It has antibacterial effect on Gram-positive(G~+)and Gram-negative bacteria(G~-),Candida albicans,and methicillin-resistant Staphylococcus aureus(MRSA),etc.,and has an antibacterial spectrum.More extensive.Hydrogel dressings are considered to be an important means of wound treatment and care,and have been widely used in clinical research.Hydrogel can maintain the water balance on the surface of the wound and is essential for the moisturizing and healing of the wound.Polyvinyl alcohol(PVA),polyethylene glycol(PEG),chitosan(CS),etc.are a class of polymers with good water solubility,stable chemical properties and no cytotoxicity.Based on the above reasons,the subject prepared an absorbent and controlled-release antimicrobial peptide hydrogel double-layer dressing.The inner layer of the absorbent membrane was obtained by freeze-drying the blend of chitosan and polyethylene glycol,and the outer layer of the antimicrobial peptide controlled-release hydrogel It is prepared from polyvinyl alcohol,proSP-B protein and polyethylene glycol by freezing and thawing.The research content of this subject is mainly divided into five parts.First,the structure of the antimicrobial peptide proSP-B is analyzed by bioinformatics,and its physical and chemical properties and antimicrobial structure are analyzed.The second part uses affinity chromatography to purify proSP-B protein.The third part is to prepare the double-layer dressing of imbibition and controlled release antimicrobial peptide hydrogel and test its performance.The fourth part is to evaluate the biological safety of the double-layer dressing of the imbibition and controlled release antimicrobial peptide hydrogel.The fifth part is mainly to construct SD rat full-thickness wounds,and use MRSA invasion model to evaluate the healing effect of imbibition controlled-release antimicrobial peptide hydrogel double-layer antimicrobial peptide dressing on MRSA-infected wounds.The main conclusions are as follows:the theoretical molecular weight of proSP-B is9.19 kDa,and the theoretical isoelectric point is 5.55.The number of negatively charged(Asp+Glu)residues is 11,the number of positively charged(Arg+Lys)residues is 9,the instability index is 38.56,proSP-B is a relatively stable protein peptide,and the total GRAVY value is 0.130,indicating proSP-B is a hydrophobic polypeptide.The aliphatic amino acid index is 113.09,which makes proSP-B have better heat resistance.The500μg/m L proSP-B protein can significantly inhibit methicillin-resistant Staphylococcus aureus,and the diameter of the inhibition zone is 11mm±0.74 mm.Orthogonal experiments show that 3%CS/0.6%PEG/1%GL,as the best combination of absorbent membranes,can maintain good swelling properties and have superior tensile properties.The antimicrobial peptide hydrogel prepared with 6%PVA/1%PEG can release the protein slowly.In vitro evaluation of the antibacterial activity of the imbibition controlled-release antimicrobial peptide hydrogel double-layer dressing on MRSA was found,and it was found that the double-layer dressing had a good inhibitory effect on MRSA.When the concentration of the antimicrobial peptide proSP-B in the imbibition controlled release antimicrobial peptide hydrogel double-layer dressing reached 1000μg/m L,the in vitro cell survival rate was 80.8±3.1%,and there was no cytotoxicity;the average primary irritation index was 0,no Skin irritation;blood compatibility index<1%,no hemolysis.In vitro cytotoxicity,skin irritation and blood compatibility are in line with national biological evaluation safety standards.In addition,the imbibition controlled-release antimicrobial peptide hydrogel double-layer dressing can significantly accelerate the healing rate of MRSA-infected wounds.After 11 days,the wound healing rate is 94.8±5.4%,which is significantly higher than the control’s 85.5±4.5%,P<0.05 And the increased content of collagen in the wound was significantly higher than that of the control,P<0.05.The above shows that the imbibition controlled release antimicrobial peptide hydrogel double-layer dressing can be used for wound treatment and can accelerate the healing of MRSA wound infection wounds,and has certain potential in the fields of wound infection and wound healing.