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.

There is considerable interest in the binding and condensation of DNA with polycations to form polyplexes because of their possible application to cellular nucleic acid delivery. This work focuses on studying the binding of plasmid DNA (pDNA) with a series of poly(glycoamidoamine)s (PGAAs) that have previously been shown to deliver pDNA in vitro in an efficient and nontoxic manner. Herein, we examine the PGAA-pDNA binding energetics, binding-linked protonation, and electrostatic contribution to the free energy with isothermal titration calorimetry (ITC). The size and charge of the polyplexes at various ITC injection points were then investigated by light scattering and zeta-potential measurements to provide comprehensive insight into the formation of these polyplexes. An analysis of the calorimetric data revealed a three-step process consisting of two different endothermic contributions followed by the condensation/aggregation of polyplexes. The strength of binding and the point of charge neutralization were found to be dependent upon the hydroxyl stereochemistry of the carbohydrate moiety within each polymer repeat unit. Circular dichroism spectra reveal that the PGAAs induce pDNA secondary structure changes upon binding, which suggest a direct interaction between the polymers and the DNA base pairs. Infrared spectroscopy experiments confirmed both base pair and phosphate group interactions and, more specifically, showed that the stronger-binding PGAAs had more pronounced interactions at both sites. Thus, we conclude that the mechanism of poly(glycoamidoamine)-pDNA binding is most likely a combination of electrostatics and hydrogen bonding in which long-range Coulombic forces initiate the attraction and hydroxyl groups in the carbohydrate comonomer, depending on their stereochemistry, further enhance the association through hydrogen bonding to the DNA base pairs.

No abstract

The transfer of genetic material into cells using non-viral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, linewidth, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR timescale (< millisecond). The dendrimers retain a high degree of mobility in the polyplex, whereas the DNA shows restrained mobility, suggesting that the polyplex is a highly dynamic complex with a rapidly exchanging dendrimer atmosphere around a more rigid DNA framework.

Glycopolymers with repeat units comprised of the disaccharide trehalose and an oligoamine of increasing amine have been previously synthesized by our group and shown to efficiently deliver pDNA (plasmid DNA) to HeLa cells while remaining relatively nontoxic. Complexes formed between the most amine-dense of these polycations and pDNA were also found to be relatively stable in serum and have low aggregation, which is desirable for in vivo gene delivery. To lend insight into these interesting results, this study was aimed at investigating the binding strength and mechanism of interaction between these macromolecules, via isothermal titration calorimetry (ITC) and ethidium bromide exclusion assays. The size of these pDNA-polymer complexes, or polyplexes, at various states of formation was determined through light scattering and zeta-potential measurements. Varying degrees of pDNA secondary structure change occurred upon interaction with the polymers, as evidenced by circular dichroism spectra through increasing molar ratios of polymer amine to DNA phosphate, and Fourier transform infrared (FT-IR) results demonstrated stronger electrostatic binding with the phosphate backbone with the least amine-dense of the series. It was concluded that, depending on the number of secondary amines in the repeat unit, these polymers interact with pDNA via different mechanisms with varying extents of electrostatic interaction and hydrogen bonding. These differing mechanisms may affect the ability of trehalose to serve as a deterrent against aggregation in serum conditions and lend insight into the roles of polymer-pDNA binding during the complex transfection process.