Construction Of Non-alcoholic Fatty Liver Disease Cell Mode, Selection Of Its Specific Aptamer And Targeted Drug Delivery

Non-alcoholic Fatty Liver Disease (NAFLD) is a clinical pathological syndrome occurring when excessive triglycerides is deposited in the liver (>5%of liver tissue) not due to excessive alcohol and other factors may damage liver. NAFLD is associated not only with type2diabetes, obesity,but also with non-alcoholic steatohepatitis (NASH), liver cirrhosis or even liver cancer.In recent years,the incidence of NAFLD increased and has become a global public health problem. Current drug treatment for NAFLD is mainly for insulin resistance and oxidative stress. However, the effects of treatment is uncertain and there may be some rebound in risk of treatment interruption. In recent years, nanotechnology and aptamer technology have been increasingly used in drug research and become a hot topic in the biomedical field. In this paper, to aim at some difficulties and prospects faced by NAFLD therapy, we designed and constructed drug carrier based on gold nanoparticle and aptamer, and applied its on targeted drug delivery, with the goal of exploring the potential application of NAFLD therapy and providing new ideas for NAFLD research through studies across muiti-disciplines.Given the subject of design, this paper includes three parts:(1)Construstion of Non-alcoholic Fatty Liver Disease Cell mode, Selection of its Specific Aptamer and study the characteristics of aptamer NFD-1Objective:To construct differential model of non-alcoholic fatty Liver disease cell line (NAFLD) induced from HepG2cell line and select aptamers with high affility and specificity to the membrane proteins of NAFLD cell by the Cell-based systematic evolution of ligands by exponential enrichment Cell-SELEX).Analysis of the features of aptamers NFD-1,including specificity and affinity at4℃and37℃,as well as membrane protein binding affinity.Methods:According to protocol, we induced HepG2cells into NAFLD cells with100uM oleic acid and50uM palmitic acid for one week. We designed and synthesized random DNA library sequences flanked on both sides with fixed16bases primer sequences as well as25random bases in the middle. The two primers were labeled with fluorescein isothiocyanate (FITC) and biotin, respectively. We used differentiated NAFLD cells as positive cells and HepG2cells as negative cells to perform cell-SELEX. After repeated incubation,separation steps with positive and negative cells,the enriched pools which binding to the positive cells while not binding to the negative cells were amplified by polymerase chain amplification and then used for the next round of selection. The pool enrichment was monitored by flow cytometry every three to four rounds. Last, the enriched pools were then sequenced with454sequencing technology and their binding ability was identified by performing binding test with flow cytometry. At4℃and37℃,aptamer NFD-1was synthesized and tested the binding ability to a varity of cells from different tissues by using flow cytometry; Aptamer NFD-1was diluted to different concentrations and incubated with differentiated NAFLD cells. The binding fluorescence was obtained at each point by flow cytometry used for Kd determination. Trypsin and Proteinase K were used to detect the target of NFD-1.Results:According to the protocol, we have successfully constructed the NAFLD cell mode and selected two aptamers after twenty-four rounds of positive and negative selection processes.By sequencing analysis,one aptamer named NFD-1was selected capable of binding to NAFLD cells with high affility and specificity.At4℃and37℃, aptamer NFD-1can bind to differentiated NAFLD cells with high affility and specificity while no fluorescence can be observed when incubated with cells from other tissues. Dissociation constant determination showed that NFD-1can bind to NAFLD cell with Kd values of21.5±5.6nM. Protease detection confirmed that the target of NFD-1is a membrane protein.Conclusion:Applying Cell-SELEX technology to differentiated NAFLD cells, we successfully selected one aptamer named NFD-1with high affinity and specificity to NAFLD cells. At4℃and37℃, aptamer NFD-1can bind to differentiated NAFLD cells with high affinity and specificity.(2) Construction of gold nanoparticle based drug carrierObjective:Constructed the gold nanoparticle based drug carrier according to the protocol.Method:Gold nanoparticles with diameter of13nm were synthesized and decorated with selected NFD-1and GC sequences which can hold doxorubicin (Dox). Last, we test the affinity and specificity of drug carrier based on gold nanoparticle and aptemer to differentiated NAFLD cells.Results:Gold nanoparticles with13diameter were successfully synthesized and decorated with NFD-1and GC DNA sequences.Results shown that the drug carrier based on gold nanoparticle can bind to NAFLD cells with high affinity and specificity. Conclusion:The drug carrier based on gold nanoparticle also showed binding ability to differentiated NAFLD cells with high affinity and specificity.(3) Load of Dox and the study of targeted delivery of aptamerconjugated gold nanoparticle and dox to differentiated non-alcoholic fatty liver disease cellObjective:Load Dox on gold nanoparticle drug carrier and test whether the carrier decorated with aptamer NFD-1and Dox can specifically be endocytosed to the differentiated NAFLD cell.Method:The confocal was used to check whether the gold nanoparticle drug carrier can be endocytosed to the differentiated NAFLD cells.Results:The results showed that gold nanoparticle drug carrier decorated with aptamer NFD-1can hold more than three hundred times Dox and can specifically be endocytosed to the differentiated NAFLD cells whereas not to the HepG2cells.Conclusion:The gold nanoparticle drug carrier hold high carry Dox ability and can be targeted endocytosed to the differentiated NAFLD cells compared with HepG2cells.