Neutrophil-Hitchhiking Nucleic Acid/Peptide Nanocomplexes For The Treatment Of Spinal Cord Injury

Spinal cord injury(SCI)is a disease of the central nervous system caused by external physical trauma.The microenvironment of the spinal cord is imbalanced after SCI and the infiltrating macrophages continue to polarization towards the pro-inflammatory phenotype(M1 macrophage),which secretes pro-inflammatory factors and leads to apoptosis of surrounding normal cells.At the same time,neural stem cells(NSCs)differentiate into astrocytes and form astroglial scars to inhibit the spread of inflammation.However,the long-term presence of the scars severely inhibits nerve and axon regeneration.The imbalance of the spinal cord microenvironment is an essential cause of poor recovery of SCI.Therefore,regulating the microenvironment of the injured spinal cord,reversing the phenotype of M1 macrophages and enhancing neuronal differentiation of NSCs are crucial for the treatment of SCI.Interferon regulatory factor 5(IRF5),a member of the interferon regulatory factor family,is one of the major regulators of macrophages polarization.In response to inflammatory stimuli,IRF5 induces inflammatory gene expression,which leads to the polarization of macrophages towards the M1 phenotype.IRF5 siRNA(siIRF5)can downregulate IRF5 expression and promote the conversion of M1 macrophages to an inflammatory phenotype(M2 macrophages)and contribute to the regression of inflammation.Stromal cell derived factor 1α-derived peptide(SDF-1α peptide)with good chemotactic properties for mesenchymal stem cells(MSCs).SDF-1α peptide can recruit MSCs to SCI sites and thus promote neuronal differentiation of NSCs.Therefore,siIRF5/SDF-1α peptide co-delivery for improving the damaged spinal cord microenvironment may be a promising therapeutic approach.However,siIRF5/SDF-1αpeptide co-delivery is challenging because of the multiple biological barriers in vivo that severely limit the targeted delivery of siIRF5/SDF-1α peptide.In addition,siIRF5 and SDF-1α peptide need to function intracellularly and extracellularly,respectively.Therefore,based on the in vivo physiological barrier and the microenvironmental characteristics of the lesion site,a safe and efficient nucleic acid/peptide co-delivery system with the ability to target the injured spinal cord was developed to achieve intracellular/extracellular co-delivery of siIRF5/SDF-1α peptide and efficient treatment of SCI.The main research of this thesis is summarized as follows:In Chapter 1,SCI,RNA interference techniques are described.Then,delivery barriers and types of non-viral gene delivery vectors,nano-delivery systems based on cell-hitchhiking are reviewed.Finally,stem cell therapies for SCI are introduced.In Chapter 2,neutrophil-specific internalized,microthrombus-responsive separated nanocomplexes were designed and synthesized for neutrophil-hitchhiking,efficient encapsulation of nucleic acid/peptide drugs,and SCI site-targeted delivery.Firstly,linearα-helical cationic polypeptide with glucose in the side chain(Glucose-LPP,GP)were synthesized by ring-opening polymerization of γ-(4-propyloxybenzyl)-L-glutamic acidN-carboxylic anhydride monomers and click chemistry,and GP condensed siRNA by electrostatic interactions to form GP/siRNA binary nanocomplexes.Subsequently,phenylboronic acid/SDF-lα peptide was modified on the surface of denatured bovine serum albumin(PBA-SDF-1α peptide-dBSA,PSdB)by an amidation reaction.PSdB and GP/siRNA binary nanocomplexes form a PSdB/GP/siRNA ternary nanocomplex with a particle size of approximately 160 nm and a potential of approximately-30 mV through reversible borate ester bonding and electrostatic interactions.The PSdB/GP/siRNA nanocomplex has good in vitro and serum stability,maintains its structural integrity after neutrophil-hitchhiking,and achieves responsive separation of microthrombus from SCI sites.In Chapter 3,the ternary nanocomplexes constructed in Chapter 2 were loaded with siIRF5/SDF-1α peptide(PSdB/GP/siIRF5)and used to modulate the spinal microenvironment for the treatment of SCI.In vitro experiments showed that GP/siIRF5 nanocomplex induced efficient IRF5 gene silencing(～72%)and promoted the conversion of RAW 264.7 cells from M1 to M2 type,while PSdB successfully induced the migration of MSCs(～3.2-fold)and promoted the differentiation of NSCs to neurons.After systemic administration,the PSdB/GP/siIRF5 nanocomplex successfully targeted to SCI sites,with a 71%IRF5 gene silencing efficiency,a 7.4-fold increase in MSCs migrating to SCI sites and a much higher Basso Mouse Scale score(5 points)than PSdB/GP/siScr(3.2 points)and PdB/GP/siIRF5 nanocomplex treatment group(3.7 points).siIRF5-mediated macrophages polarization combined with SDF-1α peptide-mediated neuronal differentiation synergistically improved the microenvironment of spinal cord injury and promoted SCI repair.In chapter 4,the thesis is summarized and the future work that needed to be improved is discussed.In conclusion,this paper designed and constructed a neutrophil-hitchhiking nucleic acid/peptide nanocomplex.This paper explored its application in SCI therapy and showed good efficacy in SCI mice,providing a new idea for in vivo trans-barrier and targeted delivery of nucleic acid and peptide and a new protocol for SCI therapy.