Relating Chemical and Biological Diversity Space: A Tunable System for Efficient Gene Transfection

Abstract

Polyethyleneimine (PEI), a well-established nonviral transfection reagent, was combinatorially modified with varying proportions of methyl, benzyl, and n-dodecyl groups to create a library of 435 derivatized polymers. Screening of this library for transfection, DNA binding, and toxicity allows systematic correlation of the biological properties of our polymers to their derivatizations. Combinations of derivatizations bring about a 100-fold variation in transfection efficiency between library members. The best PEI derivatives exhibit increases in transfection efficiency of more than 80-fold over unmodified PEI (up to 28+/-7 % of cells transfected) and rival commercial reagents such as Lipofectamine 2000 (21+/-10 %) and JetPEI (32+/-5.0 %). In addition, we can identify compounds that are specifically tuned for efficient transfection in CHO-K1 over Ishikawa cells and vice versa, demonstrating that the approach can lead to cell-type selectivity of at least one order of magnitude. This work demonstrates that multivalent derivatization of a polymeric framework can create functional diversity substantially greater than the structural diversity of the derivatization building blocks and suggests an approach to a better understanding of the molecular underpinnings of transfection as well as their exploitation.

Peptide Dendrimer/Lipid Hybrid Systems Are Efficient DNA Transfection Reagents: Structure–Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations

“…79,80 It has been shown that good transfection is achieved as a trade-off between low toxicity (including nonspecific binding to nucleic acids) and sufficient affinity for DNA. 45 …”

“…45,82 To address the binding and dissociation properties of the lipid-dendrimer-DNA complexes, PicoGreen-labeled DNA was used to detect binding by the fluorescence decrease of encapsulated DNA. Addition of negatively charged heparin leads to DNA release and restores PicoGreen fluorescence.…”

“…However, there are relatively few instances in which it was possible to correlate combinatorial or systematic changes in the molecular structure of the transfection reagents to a change in their properties. 14,45,61−63 This is partly due to the limits of the synthetic protocols: variation of the backbone to allow incorporation of heterogeneous building blocks is difficult in dendrimers, so that the character of the interior and surface of the dendrimers is difficult to vary in a differential manner. The limits of the synthetic degrees of freedom had the consequence that previous analyses of transfection ability focused mainly on surface modifications.…”

Discovery of Cationic Polymers for Non-Viral Gene Delivery Using Combinatorial Approaches

“…Retroorbital administration of the polyplexes, generated from the five most effective polymers and DNA containing luciferase gene, into 6–8 week old Black Swiss male mice resulted in preferential accumulation of the polyplexes in the lungs (1–5×10 5 RLU/mg) followed by spleen, liver, heart and kidney, indicating the potential use of these polymers in therapeutic gene delivery for lung cancer. Van Vliet et al investigated functional modalities of branched 25kDa pEI (pEI-25) by functionalizing the primary, secondary or tertiary amines of the polymer by methylation, benzylation and n-dodecylations [29]. A library of 435 polymers was evaluated in a high-throughput microwell format for DNA binding activity, toxicity and transgene expression efficacy; CHO-K1 (chinese hamster ovary) cells were transfected with an enhanced green fluorescent protein (EGFP) reporter gene in order to evaluate transgene expression.…”

Nonviral gene delivery to the liver

“…Assaying these large libraries becomes cumbersome and expensive without miniaturization to save time, materials, and cost. Several reports have utilized 384 well plates for screening of transfection agents 205,210,[215][216][217][218][219][220][221][222][223] , but did not report optimization parameters for cell culture and transfection. Even fewer reports are available for gene transfection assays in 1536 well plates [224][225][226][227] .…”