Facially Amphiphilic Polyionene Biocidal Polymers Derived From Lithocholic Acid

Bacterial infections have become a global issue that requires urgent attention, particularly regarding to emergence of multidrug resistant bacteria. We developed quaternary amine-containing antimicrobial poly(bile acid)s that contain a hydrophobic core of lithocholic acid in the main-chain. Interestingly, by choosing appropriate monomers, these cationic polymers can form core-shell micelles. These polymers exhibited biocidal activity against both Gram-positive and Gram-negative bacterial species. It is demonstrated that the micelles can deliver hydrophobic antibiotics that functionally have dual antimicrobial activities. Cytotoxicity assays against HeLa cells showed dosage-dependent toxicity for polymers with longer linkers

Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 1. Carbohydrate Size and Its Distance from Charge Centers

Cationic polymers have the ability to bind plasmid DNA (pDNA) through electrostatic interactions and condense it into particles that can be readily endocytosed by cultured cells. The effects that polycation structure has on toxicity and gene delivery efficiency are investigated here by synthesizing a series of amidine-based polycations that contain the carbohydrates d-trehalose and β-cyclodextrin (CD) within the polycation backbone. The carbohydrate size (trehalose vs CD) and its distance from the charge centers affect the gene delivery behavior in BHK-21 cells. It is found that as the charge center is further removed from the carbohydrate unit, the toxicity is increased. Also, as the size of the carbohydrate moiety is enlarged from trehalose to β-cyclodextrin, the toxicity is reduced. The absence of a carbohydrate in the polycation produces high toxicity. All carbohydrate polycations transfect BHK-21 cells to approximately the same level of gene expression

Structural Effects of Carbohydrate-Containing Polycations on Gene Delivery. 1. Carbohydrate Size and Its Distance from Charge Centers

Cationic polymers have the ability to bind plasmid DNA (pDNA) through electrostatic interactions and condense it into particles that can be readily endocytosed by cultured cells. The effects that polycation structure has on toxicity and gene delivery efficiency are investigated here by synthesizing a series of amidine-based polycations that contain the carbohydrates d-trehalose and β-cyclodextrin (CD) within the polycation backbone. The carbohydrate size (trehalose vs CD) and its distance from the charge centers affect the gene delivery behavior in BHK-21 cells. It is found that as the charge center is further removed from the carbohydrate unit, the toxicity is increased. Also, as the size of the carbohydrate moiety is enlarged from trehalose to β-cyclodextrin, the toxicity is reduced. The absence of a carbohydrate in the polycation produces high toxicity. All carbohydrate polycations transfect BHK-21 cells to approximately the same level of gene expression

Polycationic β-Cyclodextrin “Click Clusters”: Monodisperse and Versatile Scaffolds for Nucleic Acid Delivery

Herein, a novel series of multivalent polycationic β-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 β-cyclodextrin core to impart a biocompatible multivalent architecture and oligoethyleneamine arms to facilitate pDNA binding, encapsulation, and cellular uptake. An acetylated-per-azido-β-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

Exploring the Mechanism of Plasmid DNA Nuclear Internalization with Polymer-Based Vehicles

Cationic polymers are commonly used to transfect mammalian cells, but their mechanisms of DNA delivery are unknown. This study seeks to decipher the mechanism by which plasmid DNA delivered by a class of cationic polymers traffics to and enters the nucleus. While studies have been performed to elucidate the mechanism of naked plasmid DNA (pDNA) import into the nuclei of mammalian cells, our objectives were to determine the effects of polymer complexation on pDNA nuclear import and the impact of polymer structure on that import. We have performed studies in whole cells and in isolated nuclei using flow cytometry and confocal microscopy to characterize how polymer–DNA complexes (polyplexes) are able to deliver their pDNA cargo to the nuclei of their target cells. The polymers tested herein include (i) linear poly­(ethylenimine) (JetPEI), a polyamine, and (ii) two poly­(glycoamidoamine)­s (PGAAs), polyamines that contain carbohydrate moieties (meso-galactarate, Glycofect (G4), and l-tartarate, T4) within their repeat units. Our results indicate that, when complexed with the PGAAs, pDNA association with the nuclei was severely hampered in isolated nuclei compared to whole cells. When the pDNA was complexed with JetPEI, there was slight inhibition of pDNA–nuclear interaction in isolated nuclei compared to whole cells. However, even in the case of PEI, the amount of pDNA imported into the nucleus increases in the presence of cytosolic extract, thus indicating that intracellular components also play a role in pDNA nuclear import for all polymers tested. Interestingly, PEI and G4 exhibit the highest reporter gene expression as well as inducing higher envelope permeability compared to T4, suggesting that the ability to directly permeabilize the nuclear envelope may play a role in increasing expression efficiency. In addition, both free T4 and G4 polymers are able to cross the nuclear membrane without their pDNA cargo in isolated nuclei, indicating the possibility of different modes of nuclear association for free polymers vs polyplexes. These results yield insight to how the incorporation of carbohydrate moieties influences intracellular mechanisms and will prove useful in the rational design of safe and effective polymer-based gene delivery vehicles for clinical use

Investigating the Effects of Block versus Statistical Glycopolycations Containing Primary and Tertiary Amines for Plasmid DNA Delivery

Polymer composition and morphology can affect the way polymers interact with biomolecules, cell membranes, and intracellular components. Herein, diblock, triblock, and statistical polymers that varied in charge center type (primary and/or tertiary amines) were synthesized to elucidate the role of polymer composition on plasmid DNA complexation, delivery, and cellular toxicity of the resultant polyplexes. The polymers were synthesized via RAFT polymerization and were composed of a carbohydrate moiety, 2-deoxy-2-methacrylamido glucopyranose (MAG), a primary amine group, <i>N</i>-(2-aminoethyl) methacrylamide (AEMA), and/or a tertiary amine moiety, <i><i>N,N</i></i>-(2-dimethylamino)­ethyl methacrylamide (DMAEMA). The lengths of both the carbohydrate and cationic blocks were kept constant while the primary amine to tertiary amine ratio was varied within the polymers. The polymers were characterized via nuclear magnetic resonance (NMR) and size exclusion chromatography (SEC), and the polyplex formulations with pDNA were characterized in various media using dynamic light scattering (DLS). Polyplexes formed with the block copolymers were found to be more colloidally stable than statistical copolymers with similar composition, which rapidly aggregated to micrometer sized particles. Also, polymers composed of a higher primary amine content were more colloidally stable than polymers consisting of the tertiary amine charge centers. Plasmid DNA internalization, transgene expression, and toxicity were examined with each polymer. As the amount of tertiary amine in the triblock copolymers increased, both gene expression and toxicity were found to increase. Moreover, it was found that increasing the content of tertiary amines imparted higher membrane disruption/destabilization. While both block and statistical copolymers had high transfection efficiencies, some of the statistical systems exhibited both higher transfection and toxicity than the analogous block polymers, potentially due to the lack of a hydrophilic block to screen membrane interaction/disruption. Overall, the triblock terpolymers offer an attractive composition profile that exhibited interesting properties as pDNA delivery vehicles

Investigating the Effects of Block versus Statistical Glycopolycations Containing Primary and Tertiary Amines for Plasmid DNA Delivery

Polymer composition and morphology can affect the way polymers interact with biomolecules, cell membranes, and intracellular components. Herein, diblock, triblock, and statistical polymers that varied in charge center type (primary and/or tertiary amines) were synthesized to elucidate the role of polymer composition on plasmid DNA complexation, delivery, and cellular toxicity of the resultant polyplexes. The polymers were synthesized via RAFT polymerization and were composed of a carbohydrate moiety, 2-deoxy-2-methacrylamido glucopyranose (MAG), a primary amine group, <i>N</i>-(2-aminoethyl) methacrylamide (AEMA), and/or a tertiary amine moiety, <i><i>N,N</i></i>-(2-dimethylamino)­ethyl methacrylamide (DMAEMA). The lengths of both the carbohydrate and cationic blocks were kept constant while the primary amine to tertiary amine ratio was varied within the polymers. The polymers were characterized via nuclear magnetic resonance (NMR) and size exclusion chromatography (SEC), and the polyplex formulations with pDNA were characterized in various media using dynamic light scattering (DLS). Polyplexes formed with the block copolymers were found to be more colloidally stable than statistical copolymers with similar composition, which rapidly aggregated to micrometer sized particles. Also, polymers composed of a higher primary amine content were more colloidally stable than polymers consisting of the tertiary amine charge centers. Plasmid DNA internalization, transgene expression, and toxicity were examined with each polymer. As the amount of tertiary amine in the triblock copolymers increased, both gene expression and toxicity were found to increase. Moreover, it was found that increasing the content of tertiary amines imparted higher membrane disruption/destabilization. While both block and statistical copolymers had high transfection efficiencies, some of the statistical systems exhibited both higher transfection and toxicity than the analogous block polymers, potentially due to the lack of a hydrophilic block to screen membrane interaction/disruption. Overall, the triblock terpolymers offer an attractive composition profile that exhibited interesting properties as pDNA delivery vehicles