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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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Potentiometric titration curves of AEMA and DMAEMA monomersandhomopolymers. The solutions were acidified to pH 1 with 1 M HCl andtitrated with 0.20 mol L–1 NaOH. ThepKa of the AEMA and DMAEMA were 9.32 and8.62, respectively, while the pKa of theprimary and tertiary amine homopolymers were 8.46 and 7.84, respectively.
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fig2: Potentiometric titration curves of AEMA and DMAEMA monomersandhomopolymers. The solutions were acidified to pH 1 with 1 M HCl andtitrated with 0.20 mol L–1 NaOH. ThepKa of the AEMA and DMAEMA were 9.32 and8.62, respectively, while the pKa of theprimary and tertiary amine homopolymers were 8.46 and 7.84, respectively.

Mentions: To measure the pKa of the monomers, 0.10 M solutions of the AEMAand DMAEMA monomerswere made in Millipore water. The solution was first acidified topH 1.0 with 1.00 M hydrochloric acid, and then 0.20 M NaOH was addedin known increments at 25 °C, and the pH was monitored with aAB15 digital pH electrode (Accumet Basic, Fisher Scientific, Pittsburgh,PA). The potentiometer was standardized with buffers at pH 4, pH 7,and pH 10. Solutions of the same concentration (0.10 M, on a per monomerbasis) of the homopolymers containing AEMA and DMAEMA were also madeand similarly analyzed for pKa and bufferingcapability in the same manner as that described above (Figure 2).


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

Sprouse D, Reineke TM - Biomacromolecules (2014)

Potentiometric titration curves of AEMA and DMAEMA monomersandhomopolymers. The solutions were acidified to pH 1 with 1 M HCl andtitrated with 0.20 mol L–1 NaOH. ThepKa of the AEMA and DMAEMA were 9.32 and8.62, respectively, while the pKa of theprimary and tertiary amine homopolymers were 8.46 and 7.84, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Potentiometric titration curves of AEMA and DMAEMA monomersandhomopolymers. The solutions were acidified to pH 1 with 1 M HCl andtitrated with 0.20 mol L–1 NaOH. ThepKa of the AEMA and DMAEMA were 9.32 and8.62, respectively, while the pKa of theprimary and tertiary amine homopolymers were 8.46 and 7.84, respectively.
Mentions: To measure the pKa of the monomers, 0.10 M solutions of the AEMAand DMAEMA monomerswere made in Millipore water. The solution was first acidified topH 1.0 with 1.00 M hydrochloric acid, and then 0.20 M NaOH was addedin known increments at 25 °C, and the pH was monitored with aAB15 digital pH electrode (Accumet Basic, Fisher Scientific, Pittsburgh,PA). The potentiometer was standardized with buffers at pH 4, pH 7,and pH 10. Solutions of the same concentration (0.10 M, on a per monomerbasis) of the homopolymers containing AEMA and DMAEMA were also madeand similarly analyzed for pKa and bufferingcapability in the same manner as that described above (Figure 2).

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