Experimental Study Of ROS-Responsive PAMAM Composite Nanoparticles For The Treatment Of Traumatic Spinal Cord Injury

Objectives:Traumatic spinal cord injury（SCI）often results in loss of motor,sensory,and autonomic functions,which has not been yet cured in medicine.Herein,we constructed a phenylboric acid（PBA）modified dendritic polyamidoamine（PAMAM）nanoparticle（PPN-NPs）loaded with nerve growth factor（NGF）.The PPN-NPs decomposed due to the reactive oxygen species（ROS）in the SCI niche:PAMAM inhibited inflammation and iron overload by absorbing nucleic acid debris（NAD）and iron ions,meanwhile,the NGF provided nutrients for e NSC neurogenesis.We further explored the treatment effect of PPN-NPs on SCI rat models,with the aim to lay the foundation for its clinical application.Methods:This study mainly included the following three works:（1）Preparation and physicochemical characterization of PPN-NPs:PBA modified PAMAM nanoparticles（PP-NPs）were prepared by mixing PAMAM and PBA under heating condition,and then the PP-NPs were incubated with NGF at room temperature to construct PPN-NPs;The ¹H nuclear magnetic resonance（¹H NMR）and Fluorescence resonance energy transfer（FRET）test was used to verify the successful loading of NGF with PP-NPs.The size,ζ-potential and morphology of PPN-NPs were characterized by dynamic light scattering（DLS）,potentiometric,and transmission electron microscopy（TEM）.The adsorption efficiency of PP-NPs for NAD was tested by competitive binding of ethidium bromide（EB）.The ROS sensitivity and clearance rate were determined by ¹H NMR and 2,2-Diphenyl-1-picrylhydrazyl（DPPH）.The ability for chelating iron ions of PP-NPs was detected by inductively coupled plasma mass spectrometer（ICP-MS）.（2）In vitro evaluation of the neuroprotective,inflammatory and neurogenesis promotion abilities of PPN-NPs.The CCK-8 experiments were performed to test the biocompatibility,oxidative damage protection and iron overload protection abilities of PPN-NPs on PC-12 neurons.The effects of PPN-NPs on microglia BV2 polarization（CD86,M1 polarization;CD206,M2 polarized）and neural stem cells NE4C differentiation（Tuj-1,neuron;GFAP,astrocytes）after NAD stimulation were demonstrated by immunofluorescence staining results.（3）In vivo evaluation of the therapeutic effect of PPN-NPs on SCI rats.A contusion SCI rat model was established,and three groups of samples（saline,PP-NPs and PPN-NPs）were locally injected at the injured site.Immunofluorescence staining（iba-1,activated microglia cells;GFAP;Tuj-1;nestin,neural stem cells）was conducted to evaluate the early anti-inflammatory and promote e NSC neurogenesis effects of nanoparticles in vivo.The lower limb motor ability of rats was evaluated weekly by BBB score for 6 consecutive weeks,and the effect of nanoparticles on motor function and spinal cord histological recovery of SCI rats was evaluated by spinal H&E and immunofluorescence staining（Tuj-1&GFAP）.Results:（1）PPN-NPs were successfully prepared.Under TEM,the particle size was about 110 nm,while the hydrodynamic size was 222.2 nm.The zeta potential of nanoparticle solution was 1.1 m V,indicating a weak positive electricity.The precursor particle,PP-NP,reached saturation adsorption at the mass ratio of nanoparticle/nucleic acid 5:1.The surface modified PBA decomposed in 100μmol/L H₂O₂ environment.ROS clearance happened in a dose-dependent manner;iron chelation efficiency can reach 88.2%.（2）In vitro,CCK-8 results showed that PPN-NPs had no obvious cytotoxicity,increased survival of PC-12 in oxidative damaged environments（65%to 90%）;and improved the survival rate of PC-12 under iron overload conditions（73%to 89%）;Immunofluorescence results showed that PPN-NPs induced the expression of M2-type polarization marker CD206 in NAD-stimulated BV2 and promoted the expression of NE4C neuron marker Tuj-1.（3）In vivo,immunofluorescence results of SCI rats 5 days after surgery showed that PPN-NPs inhibited the expression of iba-1 and promoted the expression of Tuj-1.BBB score showed that the score of PPN-NPs group was significantly higher than that of PP-NPs group and saline group.H&E and immunofluorescence results at 6 weeks after surgery showed that compared with PP-NPs and saline groups,PPN-NPs group achieved better tissue repair and nerve regeneration outcomes.Conclusions:In summary,the PPN-NPs we designed can remove danger signal factors,including adsorption of NAD,removal of ROS and chelated iron ions,which can reverse NAD-mediated pro-inflammatory activation of microglial cells,and promote the differentiation of neural stem cells into neurons.Through local injection,PPN-NPs inhibited inflammatory responses,increased e NSC neurogenesis,and promoted motor function and histological recovery in SCI rats.PPN-NPs provides valuable reference for subsequent combined therapies designing to treat SCI.