Nerve Growth Factor Delivery By Ultrasound-mediated Nanobubble Destruction As A Treatment For Acute Spinal Cord Injury In Rats

The clinical treatment of SCI poses difficult problems.In SCI,the force of the primary and secondary injury destroys neural tissues,which may lead to permanent loss of function.When the spinal cord is injured,cells and molecules associated with scar formation are activated to form a glial scar,which can limit axon regeneration.Although many promising molecular strategies have emerged to reduce secondary injury and promote axonal regrowth,there is still no effective cure and functional recovery remains limited.In recent years,the use of therapeutic exogenous genes to promote neuronal regeneration has gained much interest and clinical trials have been initiated.Nevertheless,the safety and continuous expression of these exogenous genes must be addressed before they can be implemented in clinical practice.This research is to explore the effect of ultrasound(US)-mediated destruction of poly(lactic-co-glycolic acid)(PLGA)nanobubbles(NBs)expressing nerve growth factor(NGF)(NGF/PLGA NBs)on nerve regeneration in rats following SCI.Part ? : Optimization of preparation and characteristics for NGF-loaded PLGA nanobubbles: small-sized nanobubbles improve gene encapsulation and releaseBackground: Nanobubbles formulated from poly(lactic acid)and poly(D,L-lactide-co-glycolide)(PLGA)are being extensively investigated as non-viral gene delivery systems due to their biological characteristics.Purpose: The purpose of our study was to compare two kinds of preparation procedures of coexpression vector(p CMV6-green fluorescent protein [GFP]/nerve growth factor [NGF])-loaded PLGA nanobubbles and explore the optimized parameters for this gene-PLGA system.Materials and methods: In this study,NGF/PLGA nanobubbles were prepared by a modified double-emulsion method,and the morphological characterization including mean diameter,size distribution and Zeta potential,gene encapsulation efficiency and loading efficiency,gene release behavior,US imaging,and experiment of cell transfection in vitro were calculated and statistically analyzed.Results: The results from the morphological observation revealed that NGF/PLGA nanobubbles which were fabricated via a modified process had smaller size,high dispersity and well-defined spherical morphology.Furthermore,these smaller-sized nanobubbles had higher encapsulation efficiency,better gene-releasing rate.Conclusions: In conclusion,our modified formulation optimizes the characteristics of NGF-loaded PLGA nanobubbles,and the smaller-sized fraction of the nanoparticle formulation predominantly contributes to their encapsulation efficiency,gene-releasing effect.Part ? : Nerve growth factor delivery by ultrasound-mediated nanobubble destruction as a treatment for acute spinal cord injury in ratsBackground: Spinal cord injuries(SCI)can cause severe disability or death.Treatment options include surgical intervention,drug therapy,and stem cell transplantation.However,the efficacy of these methods for functional recovery remains unsatisfactory.Purpose: To explore the effect of ultrasound(US)-mediated destruction of poly(lactic-co-glycolic acid)(PLGA)nanobubbles(NBs)expressing nerve growth factor(NGF)(NGF/PLGA NBs)on nerve regeneration in rats following SCI.Materials and methods: Adult male Sprague-Dawley rats were randomly divided into four treatment groups after Allen hit models of SCI were established.The groups were: normal saline(NS)group,NGF and NBs group,NGF and US group,and NGF/PLGA NBs and US group. Histological changes after SCI were observed by hematoxylin and eosin staining.Neuron viability was determined by Nissl staining.Terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL)staining was used to examine cell apoptosis.NGF gene and protein expression were detected by quantitative reverse-transcription PCR and western blotting.Green fluorescent protein(GFP)expression in the spinal cord was examined using an inverted fluorescence microscope.The recovery of neural function was determined using the Basso,Beattie,and Bresnahan(BBB)test.Results: NGF therapy using US-mediated NGF/PLGA NBs destruction significantly increased NGF expression,attenuated histological injury,decreased neuron loss,inhibited neuronal apoptosis in injured spinal cords,and increased BBB scores in SCI rats.Conclusions: US-mediated NGF/PLGA NBs destruction effectively transfects the NGF gene into target tissues and has a significant effect on the injured spinal cord.The combination of US irradiation and gene therapy through NGF/PLGA NBs holds great promise for the future of nanomedicine and the development of non-invasive treatment options for SCI and other diseases.