A Preliminary Study On The Targeted Treatment Of Spinal Cord Injury With Macrophage Membrane Coated Liposomes

Spinal cord injury(SCI)is a common traumatic spine disease,which often leads to motor,sensory and autonomic nervous system dysfunctions of the limbs below the injured site,so that patients have to receive long-term or even life-long treatment and care.This not only seriously affects the quality of life of patients,but also brings huge challenges to the social healthcare system.Currently,there are very limited therapeutics for SCI.Although intravenous administration of methylprednisolone(MP)has been recommended as the standard clinical treatment for SCI,this method has poor clinical effects,and is often accompanied by side effects such as respiratory infection and gastrointestinal bleeding.Therefore,there is an urgent need for a safer and more effective method for the treatment of SCI.In recent years,biomimetic nano-delivery systems based on natural cell membranes have attracted widespread attention.By combining natural cell membranes and synthetic nanocarriers,the delivery systems not only retain the drug-loading capacity and nanosize advantages of nanoparticles,but have inherent “self-recognition” characteristics of natural cells to evade the reticuloendothelial system(RES)clearance.At the same time,some functional proteins present on the cell membrane can also give the biomimetic vector the ability to target delivery.After SCI,a large number of macrophages will be recruited and infiltrate into the spinal cord injury site.This process is mainly mediated by receptors on the surface of the macrophage membrane,indicating that the macrophage membrane has the potential to become a targeted delivery material for SCI.Based on this,this project used minocycline hydrochloride(MC·HCl)as a model drug,and constructed a macrophage membrane-coating liposome biomimetic delivery system for targeted delivery therapy of SCI.This delivery system has three advantages:(1)high biocompatibility and no immunogenicity;(2)escaping the recognition and clearance of the RES and prolonging circulation time;(3)active targeting of SCI.Furthermore,considering that macrophages have a variety of polarization phenotypes,this project also preliminarily investigated the effect of macrophage polarization on the ability of macrophage membrane-coating liposomes to target SCI.This paper is divided into three parts.In the first part,a biomimetic drug delivery system with macrophage membrane-coating liposomes was constructed,and its physicochemical properties and biological characteristics were characterized.The macrophage membrane was extracted by gradient centrifugation,and coated on the surface of the liposome by physical extrusion to prepare the macrophage membranecoated liposome(RM-LIP).Transmission electron microscope(TEM)observation displayed that RM-LIP had clear core-shell structure.The laser granulometry analysis showed that the hydrated particle size of RM-LIP was about 110.1 nm,which was higher than that of the inner layer liposome(PEG-LIP)(77.6 nm).Meanwhile,the Zeta potential of RM-LIP changed from-15.3 m V to-21.0 m V,which was similar to the potential of the macrophage membrane(-22.3 m V).In the fluorescence co-localization experiment,the red fluorescence of the macrophage membrane was highly overlapped with the green fluorescence of the liposome,further confirming that the macrophage membrane was successfully coated on the surface of the nanoparticles.Content analysis using high performance liquid chromatography(HPLC)showed the encapsulation efficiency(EE)of minocycline hydrochloride liposomes encapsulated by macrophage membrane(RMLIP/MC)was about 91.2%.The EE and particle size of RM-LIP/MC did not change significantly for 72 h,and remained clear and translucent,which confirmed RM-LIP/MC had good stability.The in vitro release results showed that cumulative release of RMLIP/MC in 72 h was about 62.6%,exhibiting sustained-release capability compared with LIP/MC(86.4%)and PEG-LIP/MC(85.3%).Western blot analysis revealed that two important functional proteins,Integrin α4 and Mac-1,remained intact on RM-LIP.In addition,the influence of macrophage polarization on the targeted delivery ability of macrophage membrane-coating liposomes was further analyzed.Classically activated M1 macrophages and alternatively activated M2 macrophages were induced,and used to prepare corresponding membrane-coating liposomes RM1-LIP and RM2-LIP.The experimental results revealed that RM-LIP,RM1-LIP and RM2-LIP had similar physicochemical properties,but there are differences in biological characteristics.The second part of this paper evaluated the long circulation and targeting performance of RM-LIP.The results of fluorescence microscopy and flow cytometry both demonstrated that the camouflage of macrophage membrane significantly reduced the uptake of liposomes by RAW264.7 cells.Pharmacokinetic studies showed that the halflife of RM-LIP/MC was about 12.12 h,which was about 2.5 times that of LIP/MC,indicating that macrophage membrane encapsulation can significantly prolong the circulation time of liposomes in vivo.In addition,three types of macrophage membranecoating liposomes had been confirmed to have similar long-circulation effects.HUVECs were induced by LPS to simulate the inflamed blood vessels in the spinal cord injury site to investigate the in vitro targeting of RM-LIP.The results revealed that the binding of RM-LIP to LPS induced HUVECs was significantly higher than that of PEG-LIP and Blocked RM-LIP(using antibodies to block Mac-1 and Integrin α4 on RM-LIP),which proved that RM-LIP can achieve specific binding to inflamed vascular endothelium through Mac-1 and Integrin α4.In order to investigate the in vivo targeting of RM-LIP,a mouse spinal cord contusion model was constructed.The distribution of preparations in the spinal cord and various tissues of SCI mice was investigated through ex vivo fluorescence imaging and tissue distribution experiments.The results displayed that the accumulation of RM-LIP in the injured spinal cord of SCI mice was significantly higher than that of PEG-LIP and Blocked RM-LIP,further confirming that RM-LIP had active targeting to SCI.Fluorescence sections revealed that RM-LIP can penetrate into the spinal cord tissue.In addition,in vivo and in vitro experiments also revealed that RMLIP,RM1-LIP and RM2-LIP had similar active targeting to SCI,indicating that macrophage polarization has no significant effect on the active targeting of macrophage membrane-coating liposomes.The third part of this paper examined the in vitro and in vivo efficacy and safety of RM-LIP/MC.LPS stimulated BV2 cells to construct an in vitro neuroinflammation model.ELISA results showed that the release of TNF-α and IL-6 was significantly reduced after the LPS treated BV2 cells were incubated with RM-LIP/MC.In vivo pharmacodynamic results indicated that after 24 h of administration,TNF-α and IL-6levels in the injured spinal cord of RM-LIP/MC-treated mice were significantly reduced.the pyrolysis was significantly decreased,and the secondary injury was effectively suppressed.After 49 days of SCI,the motor function of RM-LIP/MC-treated mice recovered significantly,the hind limbs of the mice could achieve mostly coordinated and consistent plantar stepping.The tissue section results showed that the cavities in the injured sites of the RM-LIP/MC-treated mice were reduced,the formation of glial scars was inhibited,and the nerve axons were relatively intact.After 7 days of continuous administration,levels of creatinine(CREA)and urea(UREA)in the MC·HCl group increased significantly,and the kidneys appeared lesions,while the RM-LIP/MC group had no significant difference compared with the normal group.These results indicated that the RM-LIP encapsulation can reduce the renal toxicity caused by high-dose MC·HCl,and RM-LIP carrier itself also had good biological safety.In summary,based on the pathological phenomenon that macrophages can infiltrate into the spinal cord damaged site after SCI,this paper designed a biomimetic delivery system with macrophage membrane-coating liposomes for targeted delivery therapy of SCI.It not only retains the good drug-loading capacity of liposomes,but also delays the clearance of RES and actively targets SCI,showing excellent SCI therapeutic effects.In addition,the paper also preliminarily investigated the effect of macrophage polarization on the targeted delivery ability of macrophage membrane-coating liposomes.