Reactive Oxygen Species Responsive Polymers and Multifunctional Injectable Hydrogels for Treatment of Spinal Cord Injury
Author:Zhang Tian Hui
Supervisor:zhuang xiu li
Spinal cord injury(SCI)can cause severe neurological impairment or permanent disability.Research on spinal cord injury can be divided into two main areas:neuroprotection and nerve regeneration.Neuroprotective therapy focuses on preventing the further development of secondary injury,while nerve regeneration therapy focuses on restoring damaged or lost function by repairing the broken spinal cord nerve circuit.A large number of studies have shown that reactive oxygen species(ROS)are overproduced after spinal cord injury and play a key role in cascade reaction of secondary injury.Removal of ROS can prevent or reduce secondary injury after SCI.In this paper,a series of ROS-responsive polymer materials were designed and synthesized,and their oxidation-responsive properties and potential as ROS-responsive drug carriers were studied.One of the ROS-responsive materials was prepared into lipid-polymer nanoparticles to explore the neuroprotective effect after spinal cord injury.At the same time,aiming at the multiple regeneration obstacles in the process of spinal cord injury repair,a multifunctional injectable hydrogel containing neural stem cells,cetuximab and FTY720 was designed and prepared,and its nerve regeneration effect after spinal cord injury was systematically studied.Specific research contents and main conclusions are as follows:(1)An oxidation-responsive PEG-lipid conjugate(mPEG2k-PBPE-DSA)was designed and synthesized.An alkynyl functionalized methoxy polyethylene glycol)with a phenylboronic pinacol ester linker was firstly prepared via the Passerini reaction and then click coupled with 3-azido-1,2-propanediol distearate(N3-DSA)to form the H2O2-responsive mPEG2k-PBPE-DSA.The oxidationresponsive mechanism of the phenylboronic pinacol ester linker was proposed based on the in situ 1H NMR and mass spectra characterization.Furthermore,the obtained mPEG2k-PBPE-DSA appeared to self-assemble into micelles in aqueous media.Nile red was used as the model drug and was loaded into the micelles.The oxidation-responsive drug release behaviors of the micelles were further investigated.(2)Here,we report the design and synthesis of a new class of ROS-responsive polypeptide via ROP of 1,4-dithian-substituted L-glutamate NCA(DTG-NCA)monomer.By using an amino-terminated methoxy poly(ethylene glycol)(mPEG-NH2)as the mcromolecular initiator,an amphiphilic block copolymer,mPEG-b-PDTG,was synthesized,which could self-assemble into micelles in aqueous media.The resultant mPEG-b-PDTG micelles exhibited structural disintegration upon treatment with H2O2 and the oxidation mechanism was further studied by 1H NMR and FT-IR spectrum.Finally,the potential use of mPEG-b-PDTG micelles for ROS-responsive drug delivery was also evaluated by using Nile Red as a model drug.(3)Herein,we report on the development of lipid-polymer nanoparticles(denoted as PELPNPs)with a high ROS-scavenging ability that effectively eliminated ROS and thus reduced long-term secondary injury in a clinically relevant rat SCI model.In vitro tests demonstrated that PELPNPs readily scavenged overproduced ROS,reduced inflammation,and protected glial cells and neurons against H2O2-induced oxidative damage.After intravenous administration,PELPNPs significantly improved the recovery of locomotor function and reduced the lesion area through effective protection of neurons and myelin sheaths.Mechanism studies proved that the superior therapeutic effect of PELPNPs was attributed to their high ROS scavenging and anti-inflammatory abilities.Furthermore,PELPNPs displayed good biocompatibility and biosafety both in vitro and in vivo.Thus,the proposed ROS-scavenging nanoparticle system shows promise for the safe and effective treatment of clinical SCI.(4)Injectable covalent adaptive hydrogel Gel4P-hy-D was prepared by using o-Dex and 4-arm-PEG-NHNH2 with hydrazine as crosslinking points.The hydrogel has rapid gelation,similar modulus to that of spinal cord tissue,and has certain expansibility and adhesion It is suitable for filling the injured spinal cord.Ge14P-hy-D carries nerve stem cells(NSCs),and adds cetuximab,which promotes the differentiation ofNSCs into neurons,and FTY720,a drug that inhibits glial scar,to prepare a multifunctional injectable hydrogel scaffold(G-N-C-F).The results of in vitro and in vivo experiments showed that cetuximab and FTY720 had synergistic effects on nerve regeneration.G-N-C-F stent was injected into the injured area of rats with acute spinal cord transection injury to verify its combined therapeutic effect on spinal cord regeneration and functional recovery.The experimental results show that the implantation of G-N-C-F stent in SCI site of rats can increase the number of neurons in the injured center,reduce the occurrence of glial scar,promote axon regeneration,thus better reconstruct the injured neural network,and contribute to the recovery of motor function in rats with spinal cord injury.The comparative experiment shows that the tissue repair effect of multi-functional injectable hydrogel scaffold is better than that of single-function hydrogel scaffold.The multi-functional injectable hydrogel scaffold provides a new idea for nerve regeneration therapy of SCI and is expected to be used in clinical research of SCI.