Low Toxic And Efficient Non-Viral Gene Carriers Based On Low Molecular Weight Polyethylenimine

Gene therapy is promising for the effective treatments of genetic diseases such ashaemophilia, muscular dystrophy and cystic fibrosis as well as acquired diseases includingcardiovascular, infectious diseases, wound healing and cancers. However, lack of safe andefficient gene delivery system is the bottleneck of gene therapy. Cationic polymer genedelivery systems have attracted a tremendous amount of attention for gene transfer becausethey can be prepared reproducibly and inexpensively in pharmaceutical grade at alarge-scale, may be administered repeatedly due to low immune response, and may furtherbe modified to achieve long circulation time and targeted delivery of therapeutic genes invivo. Polyethylenimine (PEI) owing to its unique combination of high charge density andproton sponge effect is, undoubtedly, among the most efficient synthetic gene carriers.Currently,25kDa branched PEI (denoted as25kDa PEI) and22kDa linear PEI are widelyemployed as golden standards for nonviral gene transfer. Both PEI reagents, however,show varying levels of cytotoxicities in vitro, as well as acute and/or long-term toxicity invivo due to their high molecular weight, excessive positive charge and lack ofbiodegradability. Low molecular weight PEIs (e.g.,1.8kDa PEI) with favorablecytotoxicity profiles, on the other hand, display minimum transfection activities as a resultof inadequate DNA complexation and protection. The aim of this thesis was to developstructurally well-defined, low toxic and efficient gene carriers based on low molecularweight PEI. To this end, we have designed and synthesized a series of novelstimuli-sensitive hydrophobe modified low molecular weight PEI and poly(ethyleneoxide)-graft-low molecular weight PEI (PEO-g-PEI) derivatives.In chapter1, a literature overview is presented to give a brief introduction about genetherapy, cationic polymer gene delivery systems and development of PEI derivatives forgene delivery.In chapter2, we reported on novel reduction-sensitive hydrophobic modification of 1.8kDa PEI with lipoic acid (LA) for nontoxic and superior nonviral gene delivery. The1.8kDa PEI-LA conjugates could condense DNA into nano-scale particles and the particlesize was much smaller than unmodified1.8kDa PEI.1.8kDa PEI-LA polyplexes weremuch more stable against exchange of negatively charged dextran sodium sulfate (DSS)than1.8kDa PEI controls, indicating that lipoylation of1.8kDa PEI resulted in strongerbinding with DNA. Importantly, DNA was released from1.8kDa PEI-LA polyplexes uponaddition of10mM dithiothreitol (DTT). MTT assays demonstrated that all PEI-LAconjugates were essentially non-toxic to HeLa and293T cells up to a tested concentrationof50μg/mL. The in vitro gene transfection studies in HeLa and293T cells showed thatlipoylation of1.8kDa PEI markedly boosted its transfection activity. For example,1.8kDaPEI-LA2polyplexes displayed400-fold and500-fold higher levels of gene expression thanunmodified1.8kDa PEI controls, which were ca.2-fold and3-fold higher than25kDa PEIcontrols in293T cells, in serum-free and10%serum media, respectively. These nontoxic1.8kDa PEI-LA conjugates form a superb basis for the development of targeting,biocompatible and highly efficient carriers for specific in vivo delivery of therapeuticgenes.In chapter3,1.8kDa PEI modified with acid-degradable acetal containinghydrophobe,2,4,6-trimethoxybenzylidene-tris(hydroxymethyl)ethane (TMB-THME), wasused as non-toxic and effective gene delivery system. Dynamic light scatteringmeasurements (DLS) revealed that1.8kDa PEI-(TMB-THME) conjugates condensedDNA into smaller sizes (189-97nm) than the parent1.8kDa PEI (444-130nm) at N/Pratios of20/1to80/1. The acetal degradation assays as well as acid-triggered unpackinganalyses displayed that1.8kDa PEI-(TMB-THME) polyplexes were highly pH dependentand give rise to unpacking under mildly acidic condition mimicking that of endosome.MTT assays demonstrated that1.8kDa PEI-(TMB-THME) polyplexes displayed lowcytotoxicity (>80%) to293T and HeLa cells at N/P ratios ranging from20/1to60/1. Thein vitro gene transfection studies showed that transfection activity of1.8kDa PEI wassignificantly enhanced by modifications with TMB-THME, in which transfectionefficiencies increased with increasing DS, and1.8kDa PEI-(TMB-THME)10polyplexeswere approximately4-fold and2-fold exceeding that of25kDa PEI control in293T and HeLa cells, respectively. These pH sensitive hydrophobe modified1.8kDa PEI are highlypromising for safe and efficient gene delivery.In chapter4, Poly(ethylene oxide) grafted with1.8kDa branched polyethylenimine(PEO-g-PEI) copolymers were prepared and investigated for in vitro non-viral genetransfer. Dynamic light scattering (DLS) and zeta potential measurements revealed thatPEO-g-PEI graft copolymers were able to effectively condense DNA into small-sized (80~245nm) particles with moderate positive surface charges (+7.2~+24.1mV) at N/P ratiosranging from5/1to40/1. The polyplex sizes and zeta-potentials intimately depended onPEO molecular weights and PEI graft densities. Notably, unlike25kDa PEI control,PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as20%serum conditions due to the shielding effect of PEO. MTT assays in293T cellsdemonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEOmolecular weights and decreasing PEI graft densities. Interestingly, in vitro transfectionresults showed that PEO-g-PEI polyplexes showed more than4-fold GFP expressionrelative to25kDa PEI at their optimal condition.In chapter5, based on the work of chapter4, we introduced ε-caprolactone oligomer(OCL) linker between PEI and PEO main chain (PEO-g-OCL-PEI). The introduction ofdegradable OCL would on one hand decrease carrier-associated cytotoxicity and on theother hand trigger DNA release inside cells. PEO-g-OCL-PEI displayed good DNAcondensation ability, forming polyplexes with enhanced colloidal stability against150mMNaCl or serum. MTT results showed that PEO-g-OCL-PEI had low cytotoxicities. Thetransfection efficiency of PEO-g-OCL-PEI copolymer polyplexes at their optimalcondition was2~4fold higher than those of25k Da PEI.In chapter6, we present a summary of this thesis work and give a future perspective.