Nucleic acid drugs have great potential to treat many devastating aliments, but their application has been hindered by the lack of efficacious and nontoxic delivery vehicles. Here, a new library of poly(glycoamidoamine)s (D1-D4, G1-G4, and M1-M4) has been synthesized by polycondensation of esterified d-glucaric acid (D), dimethyl-meso-galactarate (G), and d-mannaro-1,4:6,3-dilactone (M) with diethylenetriamine (1), triethylenetetramine (2), tetraethylenepentamine (3), and pentaethylenehexamine (4). The stereochemistry of the carbohydrate hydroxyl groups and the number of amine units have been systematically changed in an effort to examine how the polymer chemistry affects the plasmid DNA (pDNA) binding affinity, the compaction of pDNA into nanoparticles (polyplexes), the material cytotoxicity, and the efficacy of nucleic acid delivery. The polymers with four secondary amines (D4, G4, and M4) between the carbohydrates were found to have the highest pDNA binding affinity and the galactarate polymers generally yielded the smallest polyplexes. Delivery studies with pDNA containing the firefly luciferase or beta-galactosidase reporter genes in BHK-21, HeLa, and HepG2 cells demonstrated that all of the poly(glycoamidoamine)s deliver pDNA without cytotoxicity. Polymers D4, G4, and M4 displayed the highest delivery efficiency, where G4 was found to be a particularly effective delivery vehicle. Heparin competition assays indicated that this may be a result of the higher pDNA binding affinity displayed by G4 as compared to D4 and M4. Polyplexes formed by polymers with weaker pDNA affinities may dissociate at the cell surface due to interactions with negatively charged glycosaminoglycans, which would cause a decrease in the number of polyplexes that are endocytosed.

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.

In this report, four new poly(d-glucaramidoamine)s (1-4) have been designed to lower the toxicity of conventional polymeric nucleic acid delivery vehicles by incorporating a carbohydrate comonomer within a polyethylenimine (PEI)-like backbone. Polymers 1-4 were synthesized via polycondensation of esterified d-glucaric acid and four different amine-containing comonomers [diethylenetriamine (1), triethylenetetramine (2), tetraethylenepentamine (3), and pentaethylenehexamine (4)] in methanol. Viscometry and NMR studies suggest that the polymers are mostly linear (for 1-4, the alpha value in the Mark-Houwink-Sakurada equation = 0.6-0.7), thus indicating that polymerization occurs predominantly through the primary amines with a low degree of branching off the secondary amines. Results of gel electrophoresis shift assays show that polymers 1-4 bind pDNA at N/P ratios of 5, 3, 2, and 2, respectively. Also, dynamic light scattering and TEM experiments indicate that 1-4 compact DNA into nanoparticles (polyplexes) between 140 and 440 nm at an N/P ratio of 30. Furthermore, polyplexes formed with 1-4 deliver pDNA (plasmid DNA) containing the firefly luciferase reporter gene to BHK-21 cells in a nontoxic and highly efficient manner (as determined by luciferase gene expression). In particular, polymer 4 reveals very high delivery efficiency (equivalent to linear PEI). This result may be due in part to the "proton sponge" hypothesis proposed by Behr et al. Polymers containing amines that are protonated in the endosomal pH range (between about 7.4-5.0) reveal enhanced gene delivery profiles.

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