| Acute lung injury(ALI)is one of the most common complications occured in sepsis patients,its worsening stage is acute respiratory distress syndrome(ARDS),which showed poor prognosis and high mortality.Until now,there is no safe and effective early intervention drug for ALI,which needs to be studied and further solved.Pulmonary vascular endothelial cells are the most important target cells for effector cell damage in sepsis ALI,and also are an important drug intervention target of ALI therapy.Our research group has constructed the intercellular adhesion molecule-1(ICAM-1)antibody-conjugated simvastatin nano-lipid carrier(ICAM-NLC)targeted to pulmonary vascular endothelial cells in previous study,which has a certain early anti-inflammatory ability of ALI,but the overall effect remains to be improved.Therefore,gene transfection technology based on nano-carriers was introduced in this study,the ICAM-1 antibody-conjugated simvastatin nanostructured lipid carriers/Protamine/Ang-1 gene ternary nanoparticles(ICAM-NLC/Pro/Ang)were prepared through electrostatic adsorption method by using cationic protamine(Pro)as DNA contact materials,Ang-1 gene as model gene drug.The purposes were to co-delivered simvastatin and Ang-1 gene to pathological pulmonary vascular endothelium mediated by anti-ICAM-1 antibody through NLC-based ternary nanoparticles.We hypothesized that the combination of gene therapy and chemotherapy may further improve anti-inflammation efficacy of ALI.The ICAM-NLC/Pro/Ang ternary nanoparticles were constructed by electrostatic adsorption method,its particle size increased with the increase of NLC ratio in ternary nanoparticles,and is shaped with spherical particles.When the mass ratio of ICAM-NLC/Pro/Ang increased to 30:4:1,the particle size of ternary nanoparticles reached up to about 351.7nm,and Zeta potential is 12.1 mV.The gel block test showed that when the ratio of NLC to DNA in ternary nanoparticles was reached between 10 and 30,the DNA in the ternary complex could be completely blocked,reflecting the good protective effect of the prepared ternary nanoparticles on the Ang-1 gene.The results of the MTT cytotoxicity test showed that after 48h co-incubation of the ternary nanoparticles with EAhy926 cells,the cell survival rate was up to over 90%,indicating that the low cytotoxicity of the ternary nanoparticles,and was a potential nanoparticle drug carrier for the combination of ALI gene therapy and chemotherapy.Human vascular endothelial cell line EAhy926 cells were induced by lipopolysaccharide(LPS)for 24h to construct ALI vascular endothelial cell model.Compared with normal EAhy926 cells,the proportion of cells expressing ICAM-1 adherent molecules detected by flow cytometry increased from 17.2%to 50.0%after LPS activation,indicating the successful construction of ALI vascular endothelial cell model.The rox-labeled ICAM-NLC/Pro/Ang ternary nanoparticles was prepared by replacing the target gene Ang-1 with DNA labeled ROX dye.Cell uptake studies showed that cell uptake of ternary nanoparticles was decreased with increasing the ratio of NLC in nanoparticles,which was related to the bigger particle size of higher NLC ratio in nanoparticles.Compared with the non-ICAM-1 antibody-conjugated ternary nanoparticles group,the transport capacity of ICAM-NLC/Pro/Ang to cells modified by ICAM-1 was significantly improved,indicating the targeting characteristics of ICAM-NLC/Pro/Ang to EAhy926 cells activated by LPS via ICAM-1 mono-antibody.ROX-DNA and isothiocyanin(FITC)-NLC labeled ICAM-NLC/Pro/DNA ternary nanoparticles were prepared,and the co-localization of gene and NLC cell uptake in the ternary nanoparticles was investigated.After the ternary nanoparticles incubating with EAhy926 cells for 6h,NLC was mainly distributed in the cytoplasm,while DNA is partially transferred to the nucleus,indicating that the NLC-based drug delivery system has potential DNA nuclear import capacity.After blocking the ICAM-1 adhesion molecules on the cell surface with ICAM-1 monoclonal antibody,the flow cytometer quantitative detection showed that the MFI of EAhy926 cell uptaking the ICAM-NLC/Pro/DNA(30:4:1,w/w/w)ternary nanoparticles decreased from 32 to 23,indicating that the cell adhesion molecule(CAM)mediated cell endocytosis was one of the main cell transport pathways of the ternary nanoparticles.DiR dye was used to label ICAM-NLC/Pro/DNA(30:4:1,w/w/w)ternary nanoparticles,and the ALI Balb/c mice model was constructed with LPS.After 6h of administration of the ternary nanoparticles,the in vivo distribution study showed the strongest fluorescence intensity in lungs,indicating the lung-targeted characteristics of ICAM-NLC/Pro/DNA ternary nanoparticles.To investigate the gene transfection efficiency of ternary nanoparticles in EAhy926 model cells,the green fluorescent protein labeled ICAM-NLC/Pro/EGFP ternary nanoparticles were prepared by replacing the Ang-1 gene with the green fluorescent protein expression plasmid pEGFP.The results showed that the ternary nanoparticles showed strong expression of green fluorescent protein(over 20%)in EAhy926 cells-As the weight ratio of ICAM-NLC and DNA increases from 10/1 to 30/1,the cell transfection efficiency of the ternary nanoparticles showed a slight downward trend,which might be related to the larger size of the ternary nanoparticles with a higher proportion of NLC,thus making it more difficult for cells to uptake.Compared with the non-ICAM-1 antibody-conjugated ternary nanoparticles group,the gene transfection efficiency of the ICAM-NLC/Pro/EGFP group was significantly improved.Considering dosage need of simvastatin administration and the larger particles size in favour of lung-targeting,ICAM-NLC/Pro/Ang(30:4:1,w/w/w)was use for Ang-1 protein expression and animal pharmacodynamics study.The expression of Ang-1 protein in ALI cell model and animal model were determined by Western Blot.The results showed that after being incubated with EAhy926 cells for 48h,the expression of Ang-1 protein in the ICAM-NLC/Pro/Ang group was significantly higher than that in the resting cells,LPS stimulated cells and the ICAM-NLC group(P<0.001).The ICAM-NLC/Pro/Ang group was also significantly higher than non-ICAM-1 antibody-conjugated ternary nanoparticles group(P<0.05),which may be based on the targeting of EAhy926 cells with high expression of ICAM-1.After 48h administration of ICAM-NLC/Pro/Ang ternary nanoparticles in ALI mice,the expression trend of Ang-1 protein in lung tissue was basically consistent with that of EAhy926 cells.The results indicated that the ICAM-NLC/Pro/Ang(30:4:1,w/w/w)ternary nanoparticles had the effect of enhancing the expression level of Ang-1 protein in ALI cells model and animal model,and had the potential effect of improving the endothelial injury of AI J blood vessels.Balb/c mice were selected,and LPS was injected into the trachea to construct ALI mouse model,and the pharmacodynamic study of the protective effect of ternary nanoparticles on ALI mice model was conducted.The study found that after treatment with ICAM-NLC/Pro/Ang(30:4:1,w/w/w)for 24 h,48 h,the ICAM-NLC/Pro/Ang group showed significantly improving TNF-a,IL-6 levels,inflammatory cells,polymorphonuclear leukocytesin(PMNs)in mice bronchoalveolar lavage fluid,compared that with the ICAM-NLC group,the non-ICAM-1 antibody-conjugated ternary nanoparticles group.Moreover,the levels of inflammatory cytokines and inflammatory cells at 48h were similar to that at 24h.The histopathologic sections of HE staining also suggested that ICAM-NLC/Pro/Ang(30:4:1,w/w/w)had the most significant improvement on inflammatory cells infiltrate and alveolar septum thicken in lung tissue of ALI model mice.The results suggested that the Ang-1 gene and simvastatin can be effectively targeted to ALI lung tissue by ICAM-NLC/Pro/Ang(30:4:l,w/w/w)ternary nanoparticles.Through the combination of gene therapy and chemotherapy,the early inflammatory level of ALI was improved and the early intervention ability of sepsis ALI was further improved. |
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