Preparation And Characterization Of Low Molecular Weight PEI-Based Vectors Via Acid-Labile Ortho Ester Linkage

Gene therapy, as an important component of current biotherapy, has drawn a lot of attention in the field of medicine research. Compared with traditional therapeutic methods, gene therapy has shown great potentials for the treatment of both inherited and acquired diseases by delivering therapeutic genes to diseased cells. However, developing an efficient and safe delivery system that delivers the therapeutic genes to a specific target tissue or organ is vital for the success of gene therapy. The present vectors used for gene therapy in basic studies and clinical trials are usually divided into two categories:viral vectors and non-viral vectors. Especially, non-viral vectors based on cationic polymers are receiving a tremendous amount of attention as gene delivery vehicles on account of their advantages, such as ease of manipulation and large scale manufacture, low immunogenicity, low cost and high flexibility regarding the size of the transgene delivered. Among the various synthetic polycations exploited, polyethylenimine (PEI) has become one of the most promising and widely studied gene vectors due in large part to high positive charge density and efficient escape from the endocytic pathway through the "proton-sponge" mechanism. As a matter of fact, branched PEI (25 kDa) is applied as golden standards for non-viral gene transfection. However, high molecular weight PEIs (HMW PEIs) are often associated with pronounced toxicity and poor biocompatibility in vitro and in vitro, which have restricted its clinical applications. To circumvent such dilemma, modifications of PEI to achieve high transfection efficiency as well as low toxicity have been investigated in recent years. One promising strategy has been developed by cross-linking low molecular weight PEIs (LMW PEIs) with stimuli-responsive linkages.In this paper, we have designed and developed a new type of acid-labile ortho ester linkage (OEAc) containing carbon-carbon double bonds through a simple and effective synthetic method. Then, a series of pH-sensitive LMW PEI-based gene vectors (POEI 1, POEI 2 and POEI 3) were synthesized via Michael addition between vinyl groups of ortho ester linkage (OEAc) and primary amines in LMW PEI (M.W. 600) by feed molar ratio of 1:1,1.75:1 and 3.5:1. The 1H and 13C NMR spectra confirmed OEAc was structurally correct and pure. The structure of obtained polycations (POEI 1-3) was also identified by’H NMR analysis, and the molecular weight of polymers was determined by gel permeation chromatography (GPC). To estimate the primary amine content of POEIs, the ninhydrin colorimetric method was carried out, and results revealed that POEI 1 had the highest primary amine content. Besides, the buffering capacities of polymers were assessed by acid-base titration, and all three polymers have slightly higher buffering capacity than 25 kDa PEI, and the buffering capacity was found to rise as the reaction molar ratio increased. Ortho ester structures are highly hydrolytically labile and acid-sensitive. Thus, we used 1H NMR to explore acid-triggered degradation and kinetics of ortho ester groups in main-chains of POEIs, confirming that POEI 1, POEI 2 and POEI 3 were highly sensitive to mildly acid pH (e.g. at endosomal pH) and follow an exocyclic cleavage mechanism.The polycations could efficiently bind and condense DNA into nanosized particles based on the electrostatic interaction with the negatively charged phosphate group of DNA under physiological condition. In this research, agarose gel retardation and heparin replacement assay were adopted to assess DNA condensation ability of POEIs. All POEIs showed effective DNA condensation ability from the weight ratio (POEI/DNA, w/w) of 0.5. And DNA condensation ability of POEI 1-3 was in the following order:POEI 1> POEI 2> POEI 3, suggesting that amide groups might benefit the interaction. The DNA protection by POEIs against DNase I was further studied, and the results indicated that POEI 1 and POEI 2 could protect DNA against enzymatic degradation by nucleases. Subsequently, Dynamic light scattering (DLS) assay was carried out to study the particle sizes and zeta potentials of all formed POEIs/DNA polyplexes, and results revealed that POEI 1 and POEI 2 could efficiently condense plasmid DNA into spherical nanoparticles with appropriate sizes around 200-300 nm and zeta-potentials about+15 mV, facilitating the interaction between the polyplexes and cell membrane and leading to more efficient cellular uptake. At the same time, the stability of POEIs/DNA polyplexes at mildly acid condition was investigated through analyzing particle size changes by DLS assay. The ortho ester group in main-chains of POEI hydrolyzed at pH 5.0, resulting in dissociation of POEIs/DNA polyplexes, which is expected to enhance endosomal escape of polyplexes and modulate release of DNA.In addition, using unmodified 25 kDa PEI as a contrast, the properties of newly synthesized polycations (POEI 1-3) as potential gene vectors were discussed, including cytotoxicity and in vitro transfection efficiency. Cell viability of POEI 1 to 3 was evaluated on 293 T, SH-SY5Y, and HeLa cells by using MTT assay at various concentrations, which cover the range used in gene transfection assays. MTT results demonstrated that cell viabilities of studied polymers (POEI 1-3) were distinctly higher than that of 25 kDa PEI at all concentrations after 24 h incubation, suggesting that these polycations have better biocompatibility. Especially, POEI 3 exhibited remarkably low cytotoxicity in all three cell lines, and above 80% cell viability was observed even at a high dose of 100 μg/mL. Finally, to inspect the transfection performance of the newly prepared polymers, the transfection efficiency of the POEIs/DNA polyplexes was quantitatively measured by flow cytometry, meanwhile, the transfection efficiency and the transgene expression level were directly visualized by observation of pEGFP-positive cells using the inverted fluorescence microscope. In 293T cells, POEI 1-3 showed lower transfection efficiencies in the absence of serum in comparison with 25 kDa PEI at its optimal N/P ratio of 10 (w/w= 1.4). And for these new cationic polymers, POEI 1 gave the highest transfection efficiency at w/w ratio of 4, reaching up to 7.63%. The transfections mediated by POEIs in SH-SY5Y cells were subsequently processed. We found that POEI 1 and POEI 2 showed much better EGFP expression in comparison with 25 kDa PEI, and they gave 2.42 and 1.15 times higher tansfection efficiency at w/w ratio of 8 than PEI, respectively. Meantime, POEI 3 exhibited poor transfection ability in all tested cell lines. This research thus provides an adoptable plan to address the toxicity-efficiency contradiction of non-viral gene delivery, and the results demonstrate that such acid-labile LMW PEI-based polymers (POEI 1 and POEI 2) with efficient DNA condensation ability might be promising candidates for improved gene therapy.