Codelivery Of Minocycline Hydrochloride And Dextran Sulfate Via Bionic Liposomes For The Treatment Of Spinal Cord Injury

ObjectiveThe aim of this study was to design macrophage membrane-mimetic nanoliposomes（M-Lips）with a spinal cord injury（SCI）targeting function,which can be used as a drug carrier for minocycline hydrochloride（MH）and dextran sulphate（DS）to target drug delivery across the blood-spinal cord barrier（BSCB）to the injured tissue,exerting anti-inflammatory and neuroprotective effects to treat SCI and promote recovery of motor function after SCI.MethodsPrimary macrophages were extracted from the peritoneal cavity of mice and validated using immunofluorescence and flow cytometry.Primary macrophage membranes were extracted from primary macrophages by gradient centrifugation and observed using transmission electron microscopy（TEM）.Drug-loaded nanoliposomes（MH-DS@M-Lips）with mimetic modification of macrophage membranes were prepared in one step by thin film hydration.The MH-DS@M-Lips were characterised by TEM,confocal laser scanning microscope（CLSM）co-localisation analysis,western blotting,dynamic light scattering（DLS）,drug loading（DL）determination,encapsulation rate（EE）determination,assay of drug release,and stability assay.In in vitro experiments,the biosafety of MH-DS@M-Lips was investigated by thiazole blue colorimetric assay（MTT）.The ability of MH-DS@M-Lips to cross BSCB was verified by simulating BSCB through the Transwell model,while the uptake of MH-DS@M-Lips by BV2 cells was investigated using this model.A model of LPS-induced inflammation in RAW264.7 cells was established to study the anti-inflammatory effects of MH-DS@M-Lips by immunofluorescence and flow cytometry.A model of H2O2-induced apoptosis in VSC4.1 cells was established,and the neuroprotective effects of MH-DS@M-Lips were verified by immunofluorescence and flow cytometry.In animal experiments,the SCI model of C57BL/6J mice was constructed using Allen’s percussion method,and the spinal cord injury targeting ability of MH-DS@M-Lips was verified using multifunctional in vivo fluorescence imaging of small animals and fluorescent labelling of tissue sections.The calcium-lowering,anti-inflammatory and neuroprotective effects of MH-DS@M-Lips were verified by chemical fluorescent labelling,immunofluorescence,kit assay and western blotting,while the recovery of motor function in SCI mice after MH-DS@M-Lips treatment was assessed by immunofluorescence,Nissl staining and kinematic evaluation.ResultsThe successful extraction of primary macrophage membranes was verified by immunofluorescence and flow cytometry,and the extracted cell membrane vesicles were observed using TEM.MH-DS@M-Lips were observed as monodisperse spherical nanoparticles with a particle size of around 140 nm under TEM.CLSM co-localisation analysis showed that the macrophage membranes were successfully incorporated into liposomes and western blotting indicated that MH-DS@M-Lips inherited functional proteins from the macrophage membranes.The drug loading,encapsulation rate,and drug release assays demonstrated the successful preparation of MH-DS@M-Lips and the slow release of MH-DS@M-Lips.DLS showed that MH-DS@M-Lips has good stability.In in vitro assays,MH-DS@M-Lips efficiently crossed the blood-spinal cord barrier simulated by the Transwell model and could be taken up by BV2 cells.Chemiluminescent labelling,immunofluorescence assays and flow cytometry results showed that MH-DS@M-Lips had a good inflammatory inhibitory effect on LPS-induced RAW264.7 cells,and that MH-DS@M-Lips significantly reduced the H₂O₂-induced calcium surge in VSC4.1 cells,while reducing the intensity of reactive oxygen species and achieving neuroprotective effects.In the in vivo experiments in mice,small animal multifunctional in vivo fluorescence imaging results showed that MH-DS@M-Lips could cross the BSCB and enrich in spinal cord injury tissue.Immunofluorescence,kit assays,chemical fluorescent labelling and western blotting showed that MH-DS@M-Lips effectively inhibited the inflammatory burst,reduced calcium surge at the injury site,inhibited oxidative stress at the injury site and achieved neuroprotective effects.Behavioural evaluation of mice,Nissl staining and immunofluorescence results showed that MH-DS@M-Lips effectively promoted the recovery of motor function in SCI mice.ConclusionsThe results show that drug-loaded nanoparticles MH-DS@M-Lips,which inherit functional proteins from macrophage membranes,can be successfully prepared in one step using thin film hydration.MH-DS@M-Lips can cross the blood-brain barrier for targeted delivery of MH and DS to injured tissues of SCI mice.Upon reaching the injured tissues,MH-DS@M-Lips reduced the calcium surge in situ and sustained the inflammatory inhibitory effect,ultimately providing neuroprotection and restoring motor function in SCI mice.Thus,MH-DS@M-Lips is a potentially clinically effective strategy for the treatment of spinal cord injury.