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Progenitor and pluripotent cell types offer promise as regenerative therapies but transfecting these sensitive cells has proven difficult. Herein, a series of linear trehalose-oligoethyleneamine "click" copolymers were synthesized and examined for their ability to deliver plasmid DNA (pDNA) to two progenitor cell types, human dermal fibroblasts (HDFn) and rat mesenchymal stem cells (RMSC). Seven polymer vehicle analogs were synthesized in which three parameters were systematically varied: the number of secondary amines (4-6) within the polymer repeat unit (Tr4(33), Tr5(30), and Tr6(32)), the end group functionalities [PEG (Tr4(128)PEG-a, Tr4(118)PEG-b), triphenyl (Tr4(107)-c), or azido (Tr4(99)-d)], and the molecular weight (degree of polymerization of about 30 or about 100) and the biological efficacy of these vehicles was compared to three controls: Lipofectamine 2000, JetPEI, and Glycofect. The trehalose polymers were all able to bind and compact pDNA polyplexes, and promote pDNA uptake and gene expression [luciferase and enhanced green fluorescent protein (EGFP)] with these primary cell types and the results varied significantly depending on the polymer structure. Interestingly, in both cell types, Tr4(33) and Tr5(30) yielded the highest luciferase gene expression. However, when comparing the number of cells transfected with a reporter plasmid encoding enhanced green fluorescent protein, Tr4(33) and Tr4(107)-c yielded the highest number of HDFn cells positive for EGFP. Interestingly, with RMSCs, all of the higher molecular weight analogs (Tr4(128)PEG-a, Tr4(118)PEG-b, Tr4(107)-c, Tr4(99)-d) yielded high percentages of cells positive for EGFP (30-40%)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Herein, we report the efficient synthesis of high molecular weight polymers (up to 331 kDa) that contain beta-cyclodextrin within the polymer backbone and the examination of these structures for pDNA delivery within cultured mammalian cells. Two series of polymers were synthesized, one with variation in oligoethyleneamine stoichiometry, Cd1(46), Cd2(44), Cd3(49), and Cd4(47) (1-4 oligoethyleneamines in the repeat unit, respectively and similar degree of polymerization, n(w)=44-49) and another with variation in polymer length (four ethyleneamines in the repeat unit), Cd4(27), Cd4(47), Cd4(93), and Cd4(200) [n(w)=27, 47, 93, 200] via the "click reaction". The two series of polymers revealed efficient pDNA binding and compaction through gel electrophoresis, dynamic light scattering, and transmission electron microscopy experiments. The DNase protection assay showed a decrease in pDNA degradation with an increase in the polymer amine stoichiometry, where polymer Cd3(49) and all of the Cd4 analogs completely protected pDNA for up to 8 h in serum. The cellular uptake and gene expression profiles were examined in HeLa cells, which similarly demonstrated that both the series of polymers had high pDNA delivery where, Cd3(49) and Cd4(93) had the most effective luciferase gene expression. In addition, the cell viability profiles were quite high with all of the structures.

Cationic polymers are currently being studied as non-viral vectors to deliver therapeutic DNA into cells. In this study, a series of trehalose-based glycopolymers containing four secondary amines in the repeat unit were synthesized via the 'click reaction' [degrees of polymerization (n(w))=35, 53, 75, or 100] to elucidate how the polymer length affects the bioactivity. The four structures bound and charge-neutralized pDNA with similar affinity that was independent of the length, as determined through gel electrophoresis, heparin competitive displacement, and isothermal titration calorimetric assays. Dynamic light scattering measurements revealed that the polyplexes formed with the longer polymers (n(w)=53, 75, or 100) inhibited flocculation in media containing serum, whereas the polyplexes formed with the shorter polymer (n(w)=35) aggregated rapidly. Similar results were observed via transmission electron microscopy studies, where the nanoparticles formed with the polymers having longer degrees of polymerization showed discrete particles in media containing 10% serum. Transfection experiments revealed that the polymers exhibited low cytotoxicity at low N/P ratios and could facilitate high cellular uptake and gene expression in HeLa and H9c2(2-1) cells, and the results were dependent on the degrees of polymerization (longer polymers yielded higher transfection and toxicity).