Study On The Treatment Of Acute Lung Injury Based On Membrane Fusion Liposome And Bioorthogonal Reaction

Acute lung injury（ALI）is a common respiratory emergency and critical illness with high mortality,which currently lacks effective treatment.Endothelial glycocalyx is a polysaccharide complex structure located on the surface of vascular endothelial cells,which not only serves as a selective permeability barrier of the vascular wall,but also limits the contact of blood cells with endothelial cells and maintains the normal microenvironment of vascular endothelium.Disruption of endothelial glycocalyx increases vascular permeability,accelerates pulmonary edema,and promotes the initiation and progression of ALI.In the study,we innovatively proposed a strategy to repair endothelial glycocalyx by using heparan sulfate（HS）as repair material,seeded on the endothelial cell membrane at the site of endothelial glycocalyx defect.First,preparation of membrane-fusion liposomes（FLs）of E-selectin-binding peptides（EBP）modified and loaded with tetrazines（Tz）,and the targeting of EBP to the highly expressed E-selectin on the surface of inflame-stimulated endothelial cells was used to anchor Tz to the endothelial cell membrane,and then trans cyclooctene（TCO）-modified HS is then injected.Subsequently,bioorthogonal reactions between Tz and TCO can fix HS on endothelial cell membranes,thereby repairing endothelial glycocalyxes.In this study,ALI was used as a disease model to elucidate how the drug delivery system can repair the endothelial glycocalyx,and to provide a theoretical and experimental basis for the research and clinical application of novel pharmaceutical agents targeting the repair of the endothelial glycocalyx.This study mainly includes the following 6 parts:1.Material synthesis and structural identification The targeted lipid material EBP-PEG-DMPE,which can specifically bind to E-selectin,was synthesized through the addition reaction between the sulfhydryl group on E-selectin binding peptide（EBP）and the MAL group on DMPE-PEG-MAL.Second,Tz was covalently linked to dimyristoylphosphatidylethanolamine（DMPE）via an acylation reaction to synthesize the anchoring material Tz-DMPE.Finally,through the acylation reaction of N-hydroxysuccinimide active ester（NHS）,TCO was modified on HS to synthesize the glycocalyx repair material TCO-HS.2.EBP-Tz-FLs formulation process optimization and physicochemical properties characterization The membrane fusion liposome EBP-Tz-FLs was prepared by thin film dispersion method.The effects of（2,3-dioleyl-propyl）-trimethylammonium chloride（DOTAP）content,mass ratio of EBP-PEG-DMPE/Tz-DMPE,ultrasonic time,ultrasonic power and hydration volume on the size,potential and cell uptake of EBP-Tz-FLs were investigated.The optimal formulation of EBP-Tz-FLs was 20%DOTAP and 5:4 EBP-PEG-DMPE/Tz-DMPE mass ratio.The optimal formulation of EBP-Tz-FLs was 390 W ultrasonic power,7 min ultrasonic time and 10 m L hydration volume.EBP-Tz-FLs was prepared with a particle size of122.80±5.00 nm and a uniform distribution.The zeta potential was 42.25±3.67 m V.Transmission electron microscopy showed that EBP-Tz-FLs had a round liposome-like structure.Stability experiments showed that EBP-Tz-FLs had good stability at 4℃.3.In vitro cytological study of EBP-Tz-FLs and TCO-Cy5 Firstly,the effects of temperature and energy inhibitor sodium azide（Na N₃）on the uptake of EBP-Tz-FLs cells were investigated,and the distribution of EBP-Tz-FLs in cells incubated with HUVECs for different time was observed by fluorescence microscope.The experiment showed that EBP-Tz-FLs had better membrane fusion ability.Lipopolysaccharide（LPS）was used to construct a cell inflammation model,and E-selectin binding peptide was given in advance to competitively bind E-selectin sites.The results showed that EBP-Tz-FLs showed E-selectin targeting by modification of E-selectin binding peptide.The modification of EBP could target the delivery of Tz-containing liposomes to the site of broken glycocalyx in lung endothelial cells,and then give TCO-Cy5.The fluorescence intensity of TCO-Cy5 was determined by laser confocal and cytometry,and it was found that more TCO-Cy5 could be modified into HUVECs after administration of EBP-Tz-FLs.Furthermore,the fluorescence intensity of TCO-Cy5 on the cell membrane was detected by multifunctional enzyme marker,and the experimental results showed that EBP-Tz-FLs+TCO-Cy5 group had the most distribution on the cell membrane.4.Tissue distribution of EBP-Tz-FLs and TCO-Cy5 in vivo Firstly,a mouse model of acute lung injury was successfully established by intranasal instillation of LPS.When DID labeled EBP-Tz-FLs（EBP-Tz-FLs@DID）was injected intravenously into mice with acute lung injury,its fluorescence intensity in the lung was stronger than that of membrane fusion liposomes without EBP-peptide modification at each time point.The results showed that the modification of EBP peptide could target the membrane fusion liposomes to the lung tissue of mice with acute lung injury.Subsequently,EBP-Tz-FLs was first injected into the blood through the tail vein,and TCO-Cy5 was injected into the mice 1 h later.The results showed that EBP-Tz-FLs+TCO-Cy5 had more and longer distribution in the lung tissue than other groups during the whole experiment.5.The effects of EBP-Tz-FLs+TCO-HS on glycocalyx repair and acute lung injury were evaluated LPS was used to construct an inflammatory endothelial cell model,and FITC labelled wheat germ agglutinin（FITC-WGA）was used after administration.EBP-Tz-FLs+TCO-HS group had the strongest fluorescence intensity of FITC-WGA,indicating that EBP-Tz-FLs+TCO-HS could better repair endothelial glycocalyx.Then,FITC labelled bovine serum albumin（FITC-BSA）were used to detect the effects of the system on the cell permeability of HUVECs.EBP-Tz-FLs+TCO-HS could improve the permeability of endothelial cells.Finally,leukocytes were labeled with Carboxyfluorescein diacetate-succinimidyl ester（CFDA-SE）,and their adhesion to endothelial cells was investigated.It was found that EBP-Tz-FLs+TCO-HS could reduce the adhesion of leukocyte.In vivo,AF680-CD31 antibody and FITC-WGA were used to label the endothelial vessels and glycocalyx,respectively.Fluorescence imaging showed that EBP-Tz-FLs+TCO-HS could repair the pulmonary endothelial glycocalyx.EBP-Tz-FLs+TCO-HS could improve pulmonary vascular permeability.In the study of the therapeutic effect of acute lung injury,whether it is the edema of isolated lung tissue,the wet/dry weight ratio of lung tissue,the TNF-αlevel in lung tissue homogenate,the H&E staining results of lung tissue,the survival rate of acute lung injury mice,or the total protein concentration and cytological analysis of bronchoalveolar lavage fluid.EBP-Tz-FLs+TCO-HS has a good ability to treat acute lung injury.6.Safety of EBP-Tz-FLs and TCO-HS in vitro and in vivo EBP-Tz-FLs preparation had no solvent residue,and the percentage of hemolysis in vitro of EBP-Tz-FLs and TCO-HS met the clinical requirements.All three materials,EBP-PEG-DMPE,Tz-DMPE and TCO-HS,showed greater than 70%cell viability at the concentration of 0-160μg/m L.Normal mice were injected with EBP-Tz-FLs+TCO-HS for four consecutive days.During the whole administration cycle,there was no significant change in body weight and pathological changes in various organs,and the blood routine and biochemical indexes were similar to those in the Saline group.These results indicated that EBP-Tz-FLs+TCO-HS had higher safety in vitro and in vivo.The EBP modified fusion liposomes can achieve good lung targeting.The fusion liposomes loaded with Tz can have a bioorthogonal reaction with TCO,and further deliver HS to the damaged part of the sugar calyx,playing a better therapeutic effect.In addition,EBP-Tz-FLs+TCO-HS has good security.These results indicate that this two-step targeting delivery system can effectively increase the content of HS modified to the damaged part of the calyx,and can be used as a promising tool to effectively regulate the behavior of nano carriers in vitro and in vivo.