Effect Of Surface Chemistry And Lipid Structure Of Lipid-assisted Polymeric Nanoparticles On Nucleic Acid Delivery

Over the past few years,gene therapy is considered to have "returned to centre stage" with remarkable expansion of its application for diagnosis and treatment of various cancers,inflammation and other disorders.This has led to numerous candidates in clinical trials demonstrating significant therapeutic benefits and safety.With vigorous development of nanotechnology,increased attention has been paid towards materials based nanocarriers for gene delivery.However,their success has been hindered by physicochemical,biological and translational barriers.There has been no comprehensive study on the impact of surface chemistry of gene delivery carriers on their biological performance.In this dissertation,we focused on studying the influence of surface chemistry and lipid structure of lipid assisted polymeric nanoparticles on nucleic acid drug delivery.The main contents and conclusions are divided into two parts as described below:1.Nanoparticulate delivery systems have been successfully designed and implemented to orally deliver small interfering RNA(siRNA)for inflammatory disorders.However,the influence of surface charge on orally administered siRNA nanocarriers has not been investigated.In this study,we prepared structurally related poly(ethylene glycol)-block-poly(lactic-co-glycolic acid)(PEG5K-b-PLGA10K)nanoparticles(NPs)with the assistance of a synthesized lipid featuring surface amine groups for subsequent charge tuning.NPs were formulated by double emulsion method and their surface charge can be tuned and well controlled by succinylation reaction to yield NPs with different surface charge while maintained their size and composition.The prepared NPs were termed as aminated(ANPs),plain(PNPs)or carboxylated(CNPs)based on their surface charge.All NPs showed anticipated structural stability and siRNA integrity from enzymatic degradation.In vitro cellular uptake and gene silencing was influenced by surface charge.ANPs showed better uptake and silencing efficiency in Raw 264.7 cells among all other nanoparticles.In vivo studies showed that ANPs significantly accumulated in inflamed colons and decreased TNF-a secretions and mRNA expression levels while maintaining colonic histology in a murine model of acute ulcerative colitis(UC).This work described a methodology to modify the surface charge of siRNA encapsulating polymeric NPs and highlighted the influence of surface charge on oral delivery of siRNA for localized inflammatory disorders.2.Lipid-based nanocarriers are amongst the prominent strategies for non-viral gene delivery.Inclusion of lipids allows efficient encapsulation,cytoplasmic delivery and endosomal escape.However,they show lower transfection efficiency,toxicity and colloidal instability.In this work,we focused on the synthesis of a library of lipids for preparing lipid assisted nanoparticles.The library includes two class of lipids i.e.cholesterol and alkyl chain based lipids.Cholesterol based lipids with different linking groups(amide,ester)and cationic head groups(aliphatic,aromatic)were synthesized and well characterized(1H NMR and ESI-MS).Lipidoids like materials with either three or five dodecyl chains were synthesized for generation of alkyl chain lipids.A two-stage microfluidic device was fabricated to implement single step,size controlled and mono-disperse synthesis of nanoparticles.This platform allowed effective entrapment of hydrophilic nucleic acid inside the core of nanocarrier without complicated synthesis used in conventional batch methods requiring emulsifiers,stabilizers and laborious purification procedures.The nanoparticles were well characterized by their size,zeta potential and recovery percentage.