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.

The use of synthetic polymers for the delivery of nucleic acids holds considerable promise for understanding and treating disease at the molecular level. This work aims to decipher the cellular internalization mechanisms for a series of synthetic glycopolymer DNA delivery vehicles we have termed poly(glycoamidoamine)s (PGAAs). To this end, we have performed cellular delivery experiments in the presence of pharmacological endocytosis inhibitors. Confocal microscopy analysis showed colocalization of labeled pDNA in polyplexes with immunolabeled endocytic molecules to identify the cellular internalization pathways in HeLa cells. Direct membrane penetration was also investigated through various methods, including cellular energy depletion and leakage of a cytosolic enzyme from the cell. The data suggests that the cellular internalization of PGAA polyplexes occurs through a multifaceted internalization mechanism primarily involving caveolae, yet clathrin-coated vesicles and macropinosomes were also involved to a lesser degree. The primary mechanism that leads to efficient nuclear delivery and transgene expression appears to be caveolae/raft-mediated endocytosis. The cellular internalization pathways for PGAAs were not identical to those for polyethylenimine, illustrating that differences in the chemical structure of materials directly impacts the cellular internalization mechanisms.

Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.