Study On Biological Safety Of Cationic Lipid Complex Carrying Nucleic Acid

Background and Objective:The emergence of nanomedicine has significantly altered the landscape of precision medicine,offering a novel approach to disease diagnosis,treatment,and vaccine development.Cationic lipids,among various nano-drug delivery systems,have surfaced as key players in gene therapy,serving as a promising non-viral vector for drug administration.Ever since the discovery of liposomes in 1965,substantial advancements have been made in the realm of carrying nucleic acid drugs,particularly in the creation of preventive m RNA vaccines(e.g.,vaccines targeting the Novel Coronavirus).The positively charged surface of cationic drug carriers enhances their interaction with cell membranes,facilitating phagocytosis and increasing the likelihood of cytotoxicity.These adverse effects have raised concerns,particularly in systemic administration,posing a significant hurdle to their clinical application.The noticeable toxic and side effects arising from the combination of cationic lipids and plasmid DNA to generate complexes are attributed to a synergistic interaction,rather than the individual components acting alone.Upon systemic administration,the complex formed by cationic lipids and plasmid DNA engages with diverse cells in vivo,resulting in a range of toxicities and side effects that impact its overall safety profile.Nonetheless,there remains a paucity of research elucidating the underlying mechanisms of cytotoxicity and inflammatory responses incited by this complex.Over an extended period,the issues of cytotoxicity,inflammatory reactions,and biosafety concerns stemming from these complexes,particularly in the context of systemic administration,have significantly impeded the advancement of gene therapy.Addressing these challenges necessitates a comprehensive investigation.It has been demonstrated that the association between cationic lipids and plasmid DNA can result in a persistent inflammatory response.Nonetheless,the potential of inducing an acute inflammatory reaction remains unproven.The present study utilized a cationic lipid nanoparticle consisting of cationic lipid and lecithin,along with its complex with plasmid DNA(CLN/DNA),to comprehensively assess the cytotoxicity and inflammatory response of CLN/DNA both in vivo and in vitro.Additionally,the investigation aimed to elucidate the specific mechanism underlying the varying toxic effects elicited by the complex of cationic liposome/DNA and cationic liposome alone.The primary objective is to identify the crucial determinants of toxicity and offer recommendations for the development of safer gene delivery vectors.Methods:We first synthesized and characterized the cationic lipid nanoparticles(CLN)and their complexes with plasmid DNA(CLN/DNA).Then we detected the distribution of CLN and CLN/DNA nanoparticles in mice and their interaction with vascular endothelial cells by in vivo imaging system(IVIS),flow cytometry(FCM),laser confocal fluorescence microscopy(LSCM),and immunofluorescence staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect vascular endothelial injury induced by CLN/DNA.Moreover,we detected the uptake of CLN and CLN/DNA nanoparticles by vascular endothelial cells,lysosomal escape,and mitochondrial damage by LSCM,FCM,and immunofluorescence staining in vitro.The effect of nanoparticles on the structure and quantity of mitochondria in vascular endothelial cells by transmission electron microscope(TEM).We detected the DamageAssociated Molecular Patterns(DAMP)and cytokine release by ELISA,LSCM,and immunofluorescence staining.The effect of CLN/DNA on immune cells and hematopoietic stem cells was detected by flow cytometry.Finally,we examined the impact of CLN/DNA treatment on the ability of mouse bone marrow to reconstruct blood cells.Results:1.Intravenous administration of a cationic lipid complex(CLN/DNA)containing plasmid DNA can promptly influence the peripheral blood,predominantly manifesting as a reduction in the count of platelets,leukocytes,and lymphocytes;conversely,no comparable alterations were observed in subjects from the experimental group who were administered either the cationic lipid or the plasmid DNA.2.CLN/DNA nanoparticles can be concentrated within immune cells and hematopoietic stem cells(LSK).3.Intravenous administration of CLN and CLN/DNA results in notable accumulation primarily in the liver,spleen,and lungs.The presence of CLN and CLN/DNA was notably observed in vascular endothelial cells located in the aorta,lungs,and liver,with a more pronounced association detected between CLN/DNA and vascular endothelial cells.It was evident that CLN/DNA administration led to a significant elevation in serum thrombomodulin levels,suggesting an induction of vascular endothelial cell impairment by CLN/DNA.This particular effect was not observed with the administration of CLN.4.In the course of in vitro experimentation,it was determined that CLN exhibited a higher propensity for ingestion by EOMA cells due to its pronounced positive charge.Nevertheless,the capacity for lysosomal evasion was more robust in the case of CLN/DNA.The impact of CLN/DNA on EOMA cells encompassed a reduction in mitochondrial membrane potential and ATP synthesis,culminating in a decline in both mitochondrial quantity and structural integrity.Moreover,there was a notable elevation in the presence of calreticulin(CRT)on the surface of EOMA cells upon exposure to CLN/DNA,suggesting the potential of CLN/DNA to elicit immunogenic cell death in endothelial cells.5.In the course of in vivo experiments,it was observed that intravenous administration of CLN/DNA nanoparticles led to an elevation in HMGB1 and ATP levels,as well as an enhancement in CRT expression on the surface of lung and liver tissue cells in mice.In comparison to CLN alone,CLN/DNA notably amplified the secretion of cytokines(IFN-γ,IL-6,and TNF-α),particularly exhibiting a nearly 100-fold surge in IFN-γ concentration in the bloodstream,with activated NK cells being identified as the primary source of IFN-γ.Furthermore,CLN/DNA was shown to diminish the count of platelets,leukocytes,and lymphocytes in the blood,a phenomenon not observed with CLN administration.6.Comparatively to CLN,the administration of CLN/DNA resulted in an augmentation of hematopoietic stem cells and precursor cells,consequently fostering the expansion of HSC.Nonetheless,these expanded HSCs exhibited an incapacity to regenerate blood cells within the bone marrow.Conclusion:This investigation unveiled the specific mechanism underlying vascular endothelial cell damage triggered by CLN/DNA and established the correlation between mitochondrial dysfunction induced by CLN/DNA and vascular endothelial cell demise.After systemic delivery,CLN/DNA prompted the secretion of cytokines and inflammation-linked molecules,leading to the expansion of hematopoietic stem cells and impacting the bone marrow’s capacity to regenerate blood cells.Particularly,cytokines like IFN-γ,IL-6,and TNF-α generated by immune cells such as NK cells assume a crucial role in stress hematopoiesis induced by CLN/DNA.Hence,our study offers a comprehensive comprehension of the safety profile of cationic lipid and plasmid DNA complexes,serving as a significant point of reference for assessing vascular endothelial damage and hematopoietic stem cell proliferation triggered by such nano-drugs and laying a foundation for enhancing their safe and efficient utilization.