Experimental Study On The Effect Of Photothermal Therapy Of PDA NPs@HAMA On Peripheral Nerve Adhesion In Rats

Background and purpose:Peripheral nerve adhesion is a common complication of peripheral nerve injury and peripheral nerve surgery,and its postoperative recurrence rate is as high as 20%.In severe cases,it can cause limb dysfunction and paresthesia,and increase social and economic burden.Adhesion is mainly caused by excessive aggregation of fibrous tissue around the nerve after injury,resulting in local nerve compression,affecting local blood circulation and substance exchange,which causes neurodegenerative changes at the distal end of adhesion,and ultimately affects the function of target organs.At present,peripheral nerve adhesion is mainly treated by surgical methods,but the postoperative recurrence has always puzzled surgeons.Complications or failure of neurolysis occur in 3% to 25% of patients with carpal tunnel syndrome.Therefore,solving the dysfunction caused by peripheral nerve adhesion is still a great challenge for surgeons.As a kind of physical therapy,photothermal therapy has the advantages of noninvasive,mild and repeatable operation,and has been widely used in the treatment of a variety of diseases.Studies have shown that overexpression of heat shock protein(HSP)72 in local tissues can reduce the occurrence of adhesion.Therefore,the aim of this study is to design and develop a photothermal material,polydopamine nanoparticles @hyaluronic acid methacryloyl hydrogel(PDA NPs@HAMA),which is suitable for peripheral nerve adhesion,and to verify its biocompatibility through in vitro and in vivo experiments.A rat sciatic nerve adhesion model was used to evaluate the preventive effect of its controlled photothermal therapy on nerve adhesion.Furthermore,the mechanism of PDA NPs@HAMA photothermal action in preventing nerve adhesion was investigated.To provide a new treatment strategy for clinical peripheral nerve adhesion.Materials and Methods:1.PDA NPs@HAMA was prepared and characterized for its physical and chemical properties.HAMA was prepared by esterification,and PDA NPs were prepared by classical methods,which were dispersed into HAMA precursor solution to obtain PDA NPs@HAMA.Nuclear magnetic resonance spectroscopy(1H NMR)and Fourier transformed infrared spectroscopy(FTIR)were used to determine the synthesis of HAMA.Scanning electron microscopy and transmission electron microscopy were used to observe the morphology and internal structure of PDA NPs@HAMA.A spectrophotometer was used to measure the UV-absorbable curve,and an 808 nm laser was used to measure the photothermal conversion ability.The biosafety of PDA NPs@HAMA was evaluated by in vitro and in vivo experiments,and its degradation in vivo was observed.2.72 rats were randomly divided into 4 groups: control group;Hyaluronic acid(HA)group;Polydopamine nanoparticles(PDA)group and PDA NPs@HAMA group(n = 18 in each group).The rat sciatic nerve adhesion model was established.At 6 weeks after operation,scar formation was evaluated by adhesion score,biomechanical evaluation,and histological examination.Neurological function was assessed by electrophysiological examination,sensorimotor analysis,and gastrocnemius muscle weight and histological examination.3.In order to study the mechanism of photothermal therapy inhibiting peripheral nerve adhesion,the expression of HSP72 and inflammation-related indicators were detected at 2 weeks after photothermal therapy(the end point of photothermal therapy).The expression of HSP72 and α-SMA was detected at 6 weeks after operation(the end of the experiment),and the expression and translation of related genes were detected by immunohistochemistry,western blot and RT-PCR.Cell proliferation was used to detect the effect of HSP72 on the proliferation of fibroblasts.Results: 1.PDA NPs@HAMA with photo-curing and photothermal conversion properties was successfully prepared.The results of CCK8 assay and cell viability/cytotoxicity assay showed no difference between groups.The results of peripheral blood test and H&E staining of tissues and organs showed no difference between groups.2.There were significant differences in the score on nerve adhesion between the groups(p < 0.001).Tukey multiple comparisons showed that the PDA NPs@HAMA group(95%CI: 0.83,1.42)was significantly higher than the control group(95%CI: 1.86,2.64;P = 0.001).Motor nerve conduction velocity and muscle compound potential were higher in PDA NPs@HAMA group than in control group.3.Immunohistochemical analysis showed that the PDA NPs@HAMA group had higher expression of HSP72,more M2-type macrophages activation and lower expression of IL-1β and TNF-α than the control group at 2 weeks after operation.At 6 weeks after operation,the expression of α-smooth muscle actin(α-SMA)and phosphorylation of STAT3 in PDA NPs@HAMA group were lower than those in control group.The proliferation of fibroblasts decreased after the application of HSP72 agonist,and increased after the application of HSP72 inhibitor.Conclusion: In this study,a new type of photo-cured material with a photothermic effect was designed and synthesized-PDA NPs@HAMA.PDA NPs@HAMA has stable photothermal conversion and good biocompatibility.The photothermic effect of PDA NPs@HAMA protected the nerve from adhesion to preserv the nerve function in the rat sciatic nerve adhesion model.This effectively prevented the generation of nerve adhesions and adhesion-related damage.HSP72 produced by photothermal therapy may inhibit the proliferation and activation of fibroblasts by inhibiting the phosphorylation of STAT3.Application of hyaluronic acid reduced the release of proinflammatory cytokines IL-1β and TNF-α and promoted the polarization of M1 macrophages to M2 macrophages.