Integrative Proteomic And Metabolomic Analysis Reveals The Toxicity Of Cationic Liposomes On Human Normal Hepatocyte Cells Line L02

In the process of gene therapy,viral vectors and non-viral vectors are widely used as the major transport tools.However,due to high cytotoxicity and other defects,vrial vectors have been increasingly limited by practical conditions.At this point,cationic liposomes（CLs）have been regarded as the most promising non-viral vector.CLs as the commonly non viral vectors can be used to transport genes or drugs into cells were also the nanoscale carriers riched in clinical.Nevertheless,some researchers also pointed out that although CLs has many advantages,it is still impossible to avoid the occurrence of cytotoxicity.Although researchers have been working hard to optimize the preparation and structural improvement of CLs for many years,there are unavoidable problems of cytotoxicity.Due to the small size,after entering the organism,the CLs are concentrated in the target organs,such as the liver,causing the adverse reactions and side effects of inflammation.Unfortunately,although the toxicity of CLs has been detected early,the mechanism for its toxicity has not been studied so far.Many documents have only reported the phenomenon of cytotoxicity and some mechanism of action of CLs,which is not very thorough for the deep study of the toxicity of CLs.Well.Therefore,to obtain full and accurate biological information in nanoscale toxicology is the biggest challenge we are facing at present,which also hinders the clinical application of CLs to a certain extent.Studies have shown that,CLs were smaller in the structure,but also have the cell membrane activity.Once the CLs entering the body,they can penetrate cell membranes,and affect the integrity and function of its structure then resulting in the toxic reactions.In this paper,we wish integrate various systems biology methods to establish the overall regulatory network of CLs,which leads to cytotoxic metabolism.We aim to elaborate the potential toxicity mechanism of CLs,comprehensively.It’s known that there were many models to study the toxicity mechanism of CLs,but the metabolism and removal of most of the compounds in the human body are mainly passed through the liver,and the liver is also recognized as the main site for the metabolism of drugs and nanoparticles;at the same time,the nanoparticles in the 100-200nm range are mainly deposited in the liver,and the liver cell lines are related to the carcinogenicity and liver toxicity of the body.The main models are widely used in various cytotoxicity tests,and are also the target cells for most nanoscale carriers.Therefore,human normal hepatocyte L02 was selected as the target analysis object for further study.Thin film dispersion is widely used in the preparation process because of its simple operation,low precision and easy operation.Based on the thin film dispersion method,a group of CLs with obvious physical characteristics and stable properties were prepared.We obtained the IC₅₀ of the CLs using the CCK-8examination,and took the 1/2 IC₅₀ as the dosage during the latter experiment.Then we used multi-omics technique to conduct a comprehensive analysis of the cell substances in the two groups.The method of exploring the expression level of all proteins in a specific subject or the process of translation is Proteomics.All proteins,specific time or conditions within a cell or tissue can be used as the research object of proteomics,and the qualitative,quantitative or functional analysis can be carried out by the technical platform of different characteristics.The relative and absolute quantification of isotopic markers（Isobaric Tags for Relative and Absolute Quantitation,iTRAQ）,which have been established in recent years,is a method of qualitative and quantitative detection of proteomics with many advantages.The selectivity of the iTRAQ method is low for the experimental samples.The protein identification and analysis of the complex samples,the single organic liquid or the special components have certain accuracy and accuracy.The metabolomics research object is mainly the metabolites of the total molecular weight less than 1000 in an organism at a given time.Through the analysis of these metabolites in the downstream of the gene and protein,it cannot only intuitively display the changes in the birth objects,but also can be used to explore external stimuli or drugs for each other.LC-MS is widely used in the study of metabonomics because of its good adaptability and high accuracy and sensitivity.Finally,65 differential metabolites and 368 proteins were obtained by digging out the experimental data.With the help of all kinds of omics data processing software,we integrate the differential metabolites and proteins to construct the whole network map of cytotoxicity mechanism caused by CLs.Subsequently,we used the protein immunoblotting method to verify the core differential proteins that were found in the proteomics.The results showed that the changes in the content of the 4 differential proteins in Western blot test were in accordance with the results of iTRAQ data.In conclusion,the proteomics method based on iTRAQ-based proteomics used in this experiment to analyze the protential toxicity mechanism of CLs with a great accuracy and applicability.Our study shown that,the single omics method only gives parts of the bio-information,and it is necessary and feasible to combine multiple omics analysis techniques.Therefore,the multi-omics integration strategy was used in this manuscript and will further explore the potential toxicity mechanism of CLs.