Herein, three new glycopolymers have been synthesized via "click polymerization" to promote nucleic acid delivery in the presence of biological media containing serum. These structures were designed to contain a trehalose moiety to promote biocompatibility, water solubility, and stability against aggregation, amide-triazole groups to enhance DNA binding affinity, and an oligoamine unit to facilitate DNA encapsulation, phosphate neutralization, and interactions with cell surfaces. A 2,3,4,2',3',4'-hexa-O-acetyl-6,6'-diazido-6,6'-dideoxy-D-trehalose (4) monomer was polymerized via copper(I)-catalyzed azide-alkyne cycloaddition with a series of dialkyne-amide comonomers that contain either one, two, or three Boc-protected secondary amines (7a, 7b, or 7c, respectively). After deprotection, three water-soluble polycations (9a, 9b, or 9c) were obtained with similar degrees of polymerization (n = 56-61) to elucidate the role of amine number on nucleic acid binding, complex formation, stability, and cellular delivery. Gel electrophoresis and ethidium bromide experiments showed that 9a-9c associated with plasmid DNA (pDNA) and formed complexes (polyplexes) at N/P ratios dependent on the amine number. TEM experiments revealed that 9a-9c polyplexes were small (50-120 nm) and had morphologies (spherical and rodlike) associated with the polymer chain stiffness. Dynamic light scattering studies in the presence of media containing serum demonstrated that 9c polyplexes had a low degree of flocculation, whereas 9a and 9b polyplexesd aggregate rapidly. Further biological studies revealed that these structures were biocompatible and deliver pDNA into HeLa cells. Particularly, 9c polyplexes promoted high delivery efficacy and gene expression profiles in the presence of serum.

Herein, a novel series of multivalent polycationic beta-cyclodextrin "click clusters" with discrete molecular weight have been synthesized, characterized, and examined as therapeutic pDNA carriers. The materials were creatively designed based on a beta-cyclodextrin core to impart a biocompatible multivalent architecture and oligoethyleneamine arms to facilitate pDNA binding, encapsulation, and cellular uptake. An acetylated-per-azido-beta-cyclodextrin (4) was reacted with series of alkyne dendrons (7a-e) (containing one to five ethyleneamine units) using copper-catalyzed 1,3-dipolar cycloaddition, to form a series of click clusters (9a-e) bearing 1,2,3-triazole linkers. Gel electrophoresis experiments, dynamic light scattering, and transmission electron microscopy revealed that the macromolecules bind and compact pDNA into spherical nanoparticles in the size range of 80-130 nm. The polycations protect pDNA against nuclease degradation, where structures 9c, 9d, and 9e did not allow pDNA degradation in the presence of serum for up to 48 h. The cellular uptake profiles were evaluated in Opti-MEM and demonstrate that all the click clusters efficiently deliver Cy5-labeled pDNA into HeLa and H9c2 (2-1) cells, and compounds 9d and 9e yielded efficacy similar to that of the positive controls, Jet-PEI and Superfect. Furthermore, the luciferase gene delivery experiments revealed that the level of reporter gene expression increased with an increase in oligoethyleneamine number within the cluster arms. The cytotoxicity profiles of these materials were evaluated by protein, MTT, and LDH assays, which demonstrate that all the click clusters remain nontoxic within the expected dosage range while the positive controls, Jet PEI and Superfect, were highly cytotoxic. In particular, 9d and 9e were the most effective and promising polycationic vehicles to be further optimized for future systemic delivery experiments.

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Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed © Springer-Verlag Berlin Heidelberg 2010 Correspondence to: Theresa M. Reineke, treineke@vt.edu. * both authors contributed equally to this review. NIH Public Access