Role And Mechanism Of VEGF-loaded ROS-responsive Nanoparticles In Type Ⅱ Diabetic Peripheral Neuropathy

Background:Diabetic peripheral neuropathy(DPN)is one of the most common complex and harmful complications of diabetes mellitus(DM),and DPN is one of the most prominent risk factors for non-traumatic amputations and foot ulcers,with a high rate of disability and death.The pathogenesis is not yet clear,but several mechanisms suggest that it is mainly related to oxidative stress.Reactive oxygen species(ROS)can lead to neurological and structural damage through direct damage,scorched death,and many other ways.However,available studies suggest that there is no effective treatment other than lifestyle improvement and strict glycemic control,but studies have shown that strict glycemic control only reduces the incidence of DPN in type I diabetes and has little effect on DPN in type II diabetes.More unfortunately,a single modality focused on antioxidant therapy is ineffective and limits the use of antioxidant drugs in the treatment of DPN due to low bioavailability,severe side effects,and high costs.Therefore,there is an urgent need for viable options that can effectively alleviate or treat DPN.In recent years,nanoparticles have attracted much attention due to their small size of gold nanoclusters,good biocompatibility,and unique physical and physiological properties,especially in the field of medical biology,and so far,they have achieved remarkable results in fluorescence imaging,antitumor,and anti-inflammation.For this reason,this study considers the development of nanoparticles with antioxidant effects for DPN applications.Materials and methods:1:DPN rat model:200-220 g Sprague-Dawley male rats were fed with a high-fat diet and STZ injection to construct a type 2 diabetic rat model,and after successful modeling,blood glucose,and body weight were tested regularly,and the heat pain threshold was tested regularly.The DPN model rats were identified as having a thermal pain threshold below 50%of normal at week 8 after successful diabetic modeling.2:Synthesis and characterization of ROS-responsive nanoparticles:The antioxidant AuNDs were synthesized by modifying sulfhydryl groups onto gold nanoparticle body arms through an amidation reaction,and the VEGF-laden ROS-responsive nanoparticles AuNDs-VEGF were synthesized by ligating VEGF onto AuNDs through electrostatic interactions.Meanwhile,the successful synthesis of AuNDs and AuNDs-VEGF nanoparticles and the detection of their stability were verified by electron microscopy,Zeta potential,infrared and ultraviolet spectroscopy,and X-ray photoelectron spectroscopy(XPS);and their antioxidant properties were examined by DPPH assay;in addition,the biocompatibility of AuNDs and AuNDs-VEGF nanoparticles were examined in vitro by CCK-8 and live-dead staining and other cellular assays.3:In vivo animal experiments:the DPN model rats were randomly divided into the DPN model control group(DPN),AuNDs nanoparticles treatment group(AuNDs),and VEGF-loaded AuNDs-VEGF nanoparticles treatment group(AuNDs-VEGF),4 rats in each group and 5 normal rats in the same period(Normol).The AuNDs and AuNDs-VEGF nanoparticles were injected intramuscularly every two weeks at 0.5 ml/kg into the gastrocnemius muscle,while the normal rats and the DPN model control rats were injected intramuscularly with the same amount of saline.Assessment of test indicators:(1)The improvement of neuropathy was assessed by measuring the thermal pain threshold 4 weeks and 8 weeks after the intervention;(2)The function of the sciatic nerve in each group of rats was assessed by measuring the conduction velocity,latency and amplitude of the sciatic nerve by electrophysiology at 8 weeks after the intervention;(3)HE staining of the middle segment of the sciatic nerve and the gastrocnemius muscle was performed immediately after the euthanasia of the rats at the end of the 8th week after the intervention to assess the sciatic nerve and the gastrocnemius muscle.The distribution and expression of the sciatic nerve were detected by NF200 and MBP immunofluorescence staining,the axon of the sciatic nerve was detected by TB staining,the myelin of the sciatic nerve was detected by LFB staining,and the vascularity of the sciatic nerve was assessed by CD31 immunofluorescence and VEGF immunohistochemistry.Results:(1)Diabetic rats exhibited typical "three more and one less" symptoms;the thermal pain threshold and nerve conduction velocity of DPN rats were significantly decreased,which were improved to a certain extent after the intervention of anti-oxidant AuNDs and AuNDs-VEGF.(2)AuNDs and AuNDs-VEGF nanoparticles have good stability and biocompatibility and have sound antioxidant effects.(3)ROS-responsive AuNDs and AuNDs-VEGF nanoparticles improved the conduction velocity of diabetic peripheral nerves,alleviated axonal and myelin lesions of the diabetic sciatic nerve,and improved the atrophy of gastrocnemius muscle and its structure.Conclusions:(1)ROS-responsive AuNDs and AuNDs-VEGF nanoparticles have antioxidant effects to improve the microenvironment of peroxidation and pro-neurotrophic vascular regeneration to improve microcirculation.(2)ROS-responsive AuNDs and AuNDs-VEGF nanoparticles can improve diabetic peripheral neuropathy,improve its function and structure,and alleviate the atrophy of gastrocnemius muscle;ROS-responsive nanoparticles loaded with VEGF are an effective measure to improve diabetic peripheral neuropathy,which opens up a new field of nanoparticle application and broadens a new way of diabetic neuropathy.