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Investigating the effects of block versus statistical glycopolycations containing primary and tertiary amines for plasmid DNA delivery.

Sprouse D, Reineke TM - Biomacromolecules (2014)

Bottom Line: 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.Moreover, it was found that increasing the content of tertiary amines imparted higher membrane disruption/destabilization.Overall, the triblock terpolymers offer an attractive composition profile that exhibited interesting properties as pDNA delivery vehicles.

View Article: PubMed Central - PubMed

Affiliation: University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.

ABSTRACT
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, N-(2-aminoethyl) methacrylamide (AEMA), and/or a tertiary amine moiety, N,N-(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.

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(a) MTT assay (percent cell survival) of cellstreated with polymeronly or polyplexes formulated at N/P ratios of 5 or 10. Samples wereanalyzed 48 h post-transfection. (b) The percent of cells whose membranesare intact and not permeable to propidium iodide stain as determinedvia flow cytometry. Cells were treated with polymer only or polyplexesat N/P ratios of 5 and 10. Samples were analyzed 4 h post-transfection.All data are standardized to cells only, and control and error barsare the standard deviation of three replicates.
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fig4: (a) MTT assay (percent cell survival) of cellstreated with polymeronly or polyplexes formulated at N/P ratios of 5 or 10. Samples wereanalyzed 48 h post-transfection. (b) The percent of cells whose membranesare intact and not permeable to propidium iodide stain as determinedvia flow cytometry. Cells were treated with polymer only or polyplexesat N/P ratios of 5 and 10. Samples were analyzed 4 h post-transfection.All data are standardized to cells only, and control and error barsare the standard deviation of three replicates.

Mentions: Maintaining cellviability is one important component to obtaininghigher delivery efficiency. To investigate the cytotoxicity of polyplexesat N/P ratios of 5 and 10, MTT assays were performed with HeLa cells.The cell viability was measured 48 h post-transfection (Figure 4a). A clear toxicity trend was observed; polyplextoxicity increased as the amount of tertiary amine in the polymerincreased, particularly with the block copolymers. Rawlinson et al.reported that the cytotoxicity of pDMAEMA is cell type and molecularweight dependent.43 With the statisticalcopolymers, a similar trend was noticed but was not as pronounced.Although the composition of poly(G62-s-T23) and poly(G46-b-T26) are similar, the poly(G62-s-T23) model is likely slightly less toxic possibly dueto the cationic amine being spread throughout the polymer backbonewith the glucose moiety. Ahmed and Narain have examined similar polymerscreated with AEMA and monomers containing glucose and showed thata trend exists similar to that which we have observed; statisticalcopolymers are less toxic than their block copolymer counterparts.17,19 When considering the effect of free polymer on toxicity, it wasinteresting to note that both the statistical [poly(G62-s-T23)] and block [poly(G46-b-T26)] analogues containing only thetertiary amine charged groups caused a large portion of the cellsto die (when not complexed with pDNA into polyplexes). Indeed, thetoxicity of free polymer was higher than that when the same concentrationof polymer was contained in a polyplex. Thus, free polymer in solutioninteracts strongly with cells, which may be internalized, and theseinteractions/pathways may be different from those when the polymeris complexed with pDNA in a polyplex. This high toxicity was not observedin the MTT assays with the polymer only samples that contained theprimary amine moieties.


Investigating the effects of block versus statistical glycopolycations containing primary and tertiary amines for plasmid DNA delivery.

Sprouse D, Reineke TM - Biomacromolecules (2014)

(a) MTT assay (percent cell survival) of cellstreated with polymeronly or polyplexes formulated at N/P ratios of 5 or 10. Samples wereanalyzed 48 h post-transfection. (b) The percent of cells whose membranesare intact and not permeable to propidium iodide stain as determinedvia flow cytometry. Cells were treated with polymer only or polyplexesat N/P ratios of 5 and 10. Samples were analyzed 4 h post-transfection.All data are standardized to cells only, and control and error barsare the standard deviation of three replicates.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215899&req=5

fig4: (a) MTT assay (percent cell survival) of cellstreated with polymeronly or polyplexes formulated at N/P ratios of 5 or 10. Samples wereanalyzed 48 h post-transfection. (b) The percent of cells whose membranesare intact and not permeable to propidium iodide stain as determinedvia flow cytometry. Cells were treated with polymer only or polyplexesat N/P ratios of 5 and 10. Samples were analyzed 4 h post-transfection.All data are standardized to cells only, and control and error barsare the standard deviation of three replicates.
Mentions: Maintaining cellviability is one important component to obtaininghigher delivery efficiency. To investigate the cytotoxicity of polyplexesat N/P ratios of 5 and 10, MTT assays were performed with HeLa cells.The cell viability was measured 48 h post-transfection (Figure 4a). A clear toxicity trend was observed; polyplextoxicity increased as the amount of tertiary amine in the polymerincreased, particularly with the block copolymers. Rawlinson et al.reported that the cytotoxicity of pDMAEMA is cell type and molecularweight dependent.43 With the statisticalcopolymers, a similar trend was noticed but was not as pronounced.Although the composition of poly(G62-s-T23) and poly(G46-b-T26) are similar, the poly(G62-s-T23) model is likely slightly less toxic possibly dueto the cationic amine being spread throughout the polymer backbonewith the glucose moiety. Ahmed and Narain have examined similar polymerscreated with AEMA and monomers containing glucose and showed thata trend exists similar to that which we have observed; statisticalcopolymers are less toxic than their block copolymer counterparts.17,19 When considering the effect of free polymer on toxicity, it wasinteresting to note that both the statistical [poly(G62-s-T23)] and block [poly(G46-b-T26)] analogues containing only thetertiary amine charged groups caused a large portion of the cellsto die (when not complexed with pDNA into polyplexes). Indeed, thetoxicity of free polymer was higher than that when the same concentrationof polymer was contained in a polyplex. Thus, free polymer in solutioninteracts strongly with cells, which may be internalized, and theseinteractions/pathways may be different from those when the polymeris complexed with pDNA in a polyplex. This high toxicity was not observedin the MTT assays with the polymer only samples that contained theprimary amine moieties.

Bottom Line: 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.Moreover, it was found that increasing the content of tertiary amines imparted higher membrane disruption/destabilization.Overall, the triblock terpolymers offer an attractive composition profile that exhibited interesting properties as pDNA delivery vehicles.

View Article: PubMed Central - PubMed

Affiliation: University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.

ABSTRACT
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, N-(2-aminoethyl) methacrylamide (AEMA), and/or a tertiary amine moiety, N,N-(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.

Show MeSH
Related in: MedlinePlus