Effects of Trehalose Polycation End-Group Functionalization on Plasmid DNA Uptake and Transfection

Abstract

In this study, we have synthesized six analogs of a trehalose-pentaethylenehexamine glycopolymer (Tr4) that contain (1A) adamantane, (1B) carboxy, (1C) alkynyl-oligoethyleneamine, (1D) azido trehalose, (1E) octyl, or (1F) oligoethyleneamine end groups and evaluated the effects of polymer end group chemistry on the ability of these systems to bind, compact, and deliver pDNA in cultured HeLa cells. The polymers were synthesized in one-pot azide-alkyne cycloaddition reactions with an adaptation of the Carothers equation for step-growth polymerization to produce a series of polymers with similar degrees of polymerization. An excess of end-capping monomer was added at the end of the polymerizations to maximize functionalization efficiency, which was evaluated with GPC, NMR and MALDI-TOF. The polymers were all found to bind and compact pDNA at similarly low N/P ratios and form polyplexes with plasmid DNA. The effects of the different end group structures were most evident in the polyplex internalization and transfection assays completed in the presence of serum, as determined by flow cytometry and luciferase gene expression respectively. The Tr4 polymers end-capped with carboxyl groups (1B) (N/P = 7), octyne (1E) (N/P = 7), and oligoethyleneamine (1F) (N/P = 7), were taken into cells as polyplex and exhibited the highest levels of fluorescence, resulting from labeled reporter plasmid. Similarly, the polymers end-functionalized with the carboxyl groups (1E at N/P = 7), octyl groups (1E at N/P = 15) and, in particular, the oligoethyleneamine groups (F at N/P = 15) yielded dramatically higher reporter gene expression in the presence of serum. This study yields insight into how very subtle structural changes in the polymer chemistry such as end groups can yield very significant differences in the biological delivery efficiency and transgene expression of polymers used for pDNA delivery.

Fibroblast cell proliferation on photo-cured trehalose cinnamoyl ester thin films

“…3 Due to the chemical stability and the symmetry of the structure, trehalose is useful as a building block of polymers; 3 and our research group 4–8 and other groups 9–16 have synthesized various types of polymers from trehalose. Srinivasachari et al 13 and Anderson et al 14 developed low toxic trehalose-based cationic polymers for effective gene delivery. Miura et al 15,16 reported the suppression of Alzheimer amyloid aggregation by a glycopolymer carrying trehalose as pendant groups.…”

Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery

“…Step-growth polymers have been synthesized using a modified trehalose as one of the monomers. Polyplexes formed with these polymers have been found to display lower toxicity, high cellular uptake, and similar gene expression when compared to the commercially available gene delivery polymer jetPEI. − Polytrehalose has been shown to impart thermal stability to proteins: lysozyme enzymatic activity was retained upon extreme heating and cooling when polytrehalose was covalently attached . Our lab has reported polyplexes formulated with short interfering RNA (siRNA) and a trehalose block copolycation 6-methacrylamido-6-deoxy trehalose- co - N -(2-aminoethyl) methacrylamide (pMAT- b -AEMA) .…”

Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells

“…Tr4 polymers were synthesized and characterized as described previously by Anderson et al and Boyle et al , In brief, an alkyne–azide cycloaddition click reaction was performed to yield a polymer with alternating charged and neutral trehalose groups. Boc-protected dialkynyl oligoethyleneamine and acetylated diazido trehalose were dissolved in dimethylformamide (DMF).…”

“…8,15,23−25 A particular variant termed "Tr4", a glycopolymer containing four cationic ethylenimine units alternating with trehalose, has shown the ability to efficiently deliver pDNA, especially when end-functionalized with cationic groups. 23 Further work has shown that the addition of heparin to Tr4 polyplexes after complexation can lead to increased transfection efficiency by altering the mechanism through which polyplexes are trafficked within the cells after uptake. 26 The addition of heparin leads to accumulation of negatively charged glycosaminoglycan on the surface of the polyplex that does not inhibit endocytosis of the particles at low concentrations.…”

“…Our previous work in the field of nucleic acid delivery has resulted in the development of cationic glycopolymers that have been created through the copolymerization of amines (providing a cationic charge at physiological pH) and monomers derived from naturally occurring carbohydrates such as d -glucarate, glucose, , N -acetyl galactosamine, cyclodextrin, trehalose, and many others. ,− The incorporation of carbohydrate-derived moieties into polycations has been shown to mitigate the cytotoxicity inherent to many polycationic delivery vehicles. ,,, These moieties can also promote stealth properties and colloidal stability due to their ability to form a hydrophilic corona around polyplexes . Glycopolymers containing trehalose, a nonreducing disaccharide known for its lyoprotective properties, have shown particular promise due to their low in vitro toxicity and high gene delivery efficiency in a variety of cell types. ,,− A particular variant termed “Tr4”, a glycopolymer containing four cationic ethylenimine units alternating with trehalose, has shown the ability to efficiently deliver pDNA, especially when end-functionalized with cationic groups . Further work has shown that the addition of heparin to Tr4 polyplexes after complexation can lead to increased transfection efficiency by altering the mechanism through which polyplexes are trafficked within the cells after uptake .…”