The Study Of The Conjugation Mechanisms Of Gellan Gum With Microbial Antimicrobial Peptides And The Properties Of Resultant Conjugates

Microbial antimicrobial peptides（MAMPs）with several advantages,such as wide antibacterial spectrum,low susceptibility to causing drug resistance and high productivity,are regarded as promising substitutes for traditional antibiotics.However,the applications of MAMPs in medicine and biomaterials have been limited due to their unstable antimicrobial activity,nonexistent biofunctions,or high cytotoxicity.Gellan is a microbial polysaccharide with advantages of good water solubility and excellent biocompatibility.Gellan has been widely used in food industry but the applications of gellan in biomaterials are still limited.Gellan can be used to optimize the properties of MAMPs.In this study,gellan was conjugated with three typical MAMPs and the bioactivities of resultant products were analyzed.The application of MAMPs in anti-infection material,neuronal anti-inflammable material,suture and endotoxin affliation hemoperfusion material were realized,and these investigations were important for the broad use of MAMPs in biomedicines and biomaterials.（1）By conjugation with gellan,the stability of the antibacterial activity of nisin toward heat,alkali and chymotrysion was remarkably improved.Fourier transform infrared spectra（FT-IR）of the conjugates showed that the carboxyl groups of gellan formed new amide bonds with the amino groups of nisin and stable conjugates were achieved.Nuclear magnetic resonance spectra indicated that the binding sites of nisin with gellan were on the N terminal,while C terminal of nisin was not influenced and the intermolecular ring structure of nisin was undamaged.Zeta potential results showed that the conjugates could form stable clusters and nisin could be protected inside the cluster.In antibacterial tests,the conjugate with the nisin load of 405.6±2.5 mg·g^-1（GNc）showed good inhibitory activity against Staphylococcus epidermidis and Bacillus subtilis.Meanwhile,at different p H values（4～10）,the antibacterial duration of the conjugate was at least twice that of nisin.GNc could also maintain antibacterial activity against S.epidermidis after heat（60℃ and 80℃ for 30 min）and chymotrypsin chymotrypsin(400 and 800 mg·L^-1 for 30 min)treatment in weak alkali environment（p H 7.5）,whereas nisin could not.Moreover,the conjugate had no cytotoxic/hemolytic effect and could prevent S.epidermidis cells from infecting 3T3 cells.（2）The cytotoxicity of polymyxin B was effectively decreased after conjugated with gellan,and the neuronal anti-inflammation mechanism of corresponding conjugates was preliminarily revealed.FT-IR spectra of the conjugates showed that gellan formed new conjugates with polymyxin B.via amide reaction.According to the polymyxin utilization ratio results,the utilization ratio of polymyxin B in the conjugate with the polymyxin B load of 490.8±1.6 mg×g^-1 was relatively high.The polymyxin B in GPC could be slowly released in simulated body fluid（SBF）and the released rate was 25μg×m L^-1 in the steady release section（60～132 h）.GPC showed good antibacterial activity against Escheria coli and Pseudomonas aeruginosa in the antimicrobial test.For BV-2 cells,the cell viability and proliferation were not influenced until the concentration of GPC reached 2048 and 512μg×m L^-1（polymyxin B effective content）,which were much higher than the medium lethal concentration of polymyxin B(16μg×m L^-1).Moreover,GPC(1μg×m L^-1)could effectively decrease the NO production of endotoxin-excited BV-2 cells(<3μmol·L^-1),thereby inhibit neuronal inflammation.（3）Gellan-polylysine complex fibers were prepared through wet-spinning via in-situ polymerization and the fibers with good mechanical properties were successfully applied as sutures.The wet spinning process was single factor optimized and achieved fibers were collected,twisted,and elongated to obtain GPF.The tensile strengths and rupture modulus of GPF were 4.29±0.48 N and 195.53±29.83 N×m^-1,respectively.Decomposition test results showed that GPF was nondegradable in normal saline and slightly degradable（residual ratio of83.2%±1.2%）in SBF.Antibacterial tests showed that GPF exerted antibacterial activity against S.epidermidis/E.coli and antibiofilm activity against S.epidermidis.Moreover,GPF showed no significant cytotoxicity and hemolysis.Its use as a suture material was evaluated in a dorsal wound model.Wounds treated with GPF healed well within 5 d,and the results of routine blood tests showed no inflammation in tested mice.（4）Polymyxin B loaded gellan–polylysine yarns（PLGP）with good endotoxin bonding properties were obtained by using wet spinning via in-situ polymerization.Considering the viscoscity and binding rate of gellan with polylysine,the addition amount of polymyxin B was set as 10 mg×L^-1.Under wet,dry,and rewet conditions,the tensile strengths of the obtained PLGP were 1.54±0.12,14.59±1.08,and 1.50±0.13 N,respectively,and the rupture moduli of PLGP were 346.21±50.26,4738.87±571.68 and 333.27±53.36 N×m^-1,respectively.Fluorescein isothiocyanate-marked endotoxin molecules were observed on the surfaces of PLGP by using a fluorescent microscope,and the binding capabilities of PLGP with endotoxins from E.coli in SBF and fresh mouse blood were 2.78±0.04 and 2.45±0.11 EU×mg^-1,respectively.Moreover,PLGP did not exhibit cytotoxic,hemolytic,and cell-adhesive properties.Blood clotting test results showed that PLGP had no significant influence on the clotting properties of C57 mice blood.In the blood filtration test,the PLGP fabric did not retain blood cells,and blood cells were not massively broken during filtration.Therefore,PLGP is a potential hemoperfusion material with high endotoxin affinity.