Preparation Of A Plasmin-sensitive Hydrogel Microsphere And The Effect On Peripheral Nerve Repairation

Objective:In this paper,plasmin-sensitive microspheres containing NGF are designed by the change of plasmin concentration,and the nerve comduitis is used as a scaffold to construct a good peripheral nerve repair environment,which provided experimental basis for the repairation of peripheral nervous system.Methods:The plasmin-sensitive peptide and ACRL-PEG-NHS were used to synthesize plasmin-sensitive peptide hydrogel monomer,and verified by MALDI-TOF.The PEGDA hydrogel monomer was synthesized by PEG and acryloyl chloride.The structure and esterification rate were analyzed by 1H-NMR spectroscopy.The distribution coefficient of NGF in the two phases(hydrogel phase/dextran phase)was measured.Two kinds of hydrogel microspheres were prepared by aqueous two-phase emulsion polymerization.PLGA sustained-release microspheres were prepared by W/O/W emulsification method.Microscope was applied to observe the morphology,Particle size and distribution of the microspheres were evaluated by particle size analyzer.The drug loading and encapsulation efficiency of three kinds of microspheres were determined by ELISA.PLA nerve conduits were prepared by solvent evaporation method.PC 12 cells were co-cultured with NGF microspheresrelease medium,and the biological activity of NGF was evaluated according to the differentiation rate of PC 12 cells.PLA nerve conduits were prepared by solvent evaporation.A model of rat sciatic nerve was established.The sciatic nerve was bridged by conduits with microspheres.Results:The molecular weight of ACRL-PEG-peptide-PEG-ACRL was mainly concentrated in 8194 by MALDI-TOF,with a yield of 94.82%.The result of 1H-NMR spectrum proved that the product was PEGDA and the average yield and esterification rate were(76.02± 1.22)%and(77.05±3.44)%.The two-phase partition coefficient of the plasmin-sensitive hydrogel material was 28.7%,while the two-phase partition coefficient of the PEGDA hydrogel material was 30.7%.The plasmin-sensitive microspheres obtained were round and smooth with the average particle size being 38.9?m.The drug loading and encapsulation efficiency of plasmin-sensitive microspheres were 0.0029%and 25.73%.The average particle size of PEGDA microspheres was 32.5?m.The drug loading and encapsulation efficiency of PEGDA microspheres were 0.0028%and 19.21%.The average particle size of PLGA microspheres was 15?m.The drug loading and encapsulation efficiency of PLGA microspheres were 0.012%and 86.23%.The release curve in vitroaccorded with Higuchi equation(Q = 0.0228t1/2 + 0.0138,R2= 0.9984).The cumulative release rate of plasmin-sensitive microspheres in vitro showed that the cumulative release rate was 55.90%in 1 days,existing a certain burst effectand the cumulative release rate reached 88.20%on the fifth day.The sustained release curve was in accordance with Higuchi equation,Q=0.0926t1/2-0.0629,R2=0.9914.The rate of differentiation of PC12 cells was 74.21%in NGF releasing medium(at 4h)of plasmin-sensitive microspheres,of which the differentiation rate of in 7d was 45.68%.The rate of differentiation of PC12 cells was 71.25%in NGF releasing medium(at 3h)of plasmin-sensitive microspheres,while the differentiation rate of that in 7d was 45.68%.The tensile strength of the nerve conduit was 2.08±0.36MPa,and the tensile elongation was(257.6±53.22)%.One month after the operation,in vivo animal experiments showed that the sciatic nerves in each group were regenerated to different degrees,all of which were not completely anastomosis.There were some cell tissue migrations in the middle of the conduits.Nerve regeneration is starting.Conclusion:The esterification rate of PEGDA hydrogel monomer synthesized by acid chloride esterification method is high,and the plasmin-sensitive hydrogel with high purity is synthesized.The two hydrogels have good swelling ability;Theplasmin-sensitive microspheres and PEGDA hydrogel microspheres have better sustained release properties.Compared with PEGDA hydrogel microspheres,the former has better plasmin sensitivity and can control the release dose according to the concentration of plasmin.Preparation of NGF-loaded microspheres by aqueous two-phase synthesis method is easier to maintain the activity of NGF than by W/O/W emulsion solvent evaporation method.