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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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DLS measurements showthe hydrodynamic diameter of the polyplexesformed at 5 and 10 N/P with the statistical and block copolymers developedherein. Polyplex size was analyzed in water and Opti-MEM, and thesize was measured by dynamic light scattering (DLS) at 633 nm on aMalvern Instruments Zetasizer Nano ZS at 173° back angle scatter;time zero is when the polyplexes (formulated in water) were addedto Opti-MEM. Error bars are the standard deviation of all the datacollected, a minimum of three replicates. A table of this data canbe found in the Supporting Information (FigureS28).
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fig3: DLS measurements showthe hydrodynamic diameter of the polyplexesformed at 5 and 10 N/P with the statistical and block copolymers developedherein. Polyplex size was analyzed in water and Opti-MEM, and thesize was measured by dynamic light scattering (DLS) at 633 nm on aMalvern Instruments Zetasizer Nano ZS at 173° back angle scatter;time zero is when the polyplexes (formulated in water) were addedto Opti-MEM. Error bars are the standard deviation of all the datacollected, a minimum of three replicates. A table of this data canbe found in the Supporting Information (FigureS28).

Mentions: The stability of these polyplexeswas determined by monitoring the size/aggregation of particles inwater, Opti-MEM, and DMEM containing 10% FBS over the period of 6h. All of the polyplex types were stable in water as the size didnot change (generally around 100 nm) over the course of 6 h. Whenthe polyplexes were added to cell culture media (Opti-MEM; Figure 3 and Figure S28, SupportingInformation), some of the polyplex formulations aggregatedwith time, which was highly dependent on the polymer chemistry andtertiary polyplex structure. The perikinetic flocculation seen inOpti-MEM is most likely occurring because the increased concentrationof salts in solution decreases the Debye length.


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

Sprouse D, Reineke TM - Biomacromolecules (2014)

DLS measurements showthe hydrodynamic diameter of the polyplexesformed at 5 and 10 N/P with the statistical and block copolymers developedherein. Polyplex size was analyzed in water and Opti-MEM, and thesize was measured by dynamic light scattering (DLS) at 633 nm on aMalvern Instruments Zetasizer Nano ZS at 173° back angle scatter;time zero is when the polyplexes (formulated in water) were addedto Opti-MEM. Error bars are the standard deviation of all the datacollected, a minimum of three replicates. A table of this data canbe found in the Supporting Information (FigureS28).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: DLS measurements showthe hydrodynamic diameter of the polyplexesformed at 5 and 10 N/P with the statistical and block copolymers developedherein. Polyplex size was analyzed in water and Opti-MEM, and thesize was measured by dynamic light scattering (DLS) at 633 nm on aMalvern Instruments Zetasizer Nano ZS at 173° back angle scatter;time zero is when the polyplexes (formulated in water) were addedto Opti-MEM. Error bars are the standard deviation of all the datacollected, a minimum of three replicates. A table of this data canbe found in the Supporting Information (FigureS28).
Mentions: The stability of these polyplexeswas determined by monitoring the size/aggregation of particles inwater, Opti-MEM, and DMEM containing 10% FBS over the period of 6h. All of the polyplex types were stable in water as the size didnot change (generally around 100 nm) over the course of 6 h. Whenthe polyplexes were added to cell culture media (Opti-MEM; Figure 3 and Figure S28, SupportingInformation), some of the polyplex formulations aggregatedwith time, which was highly dependent on the polymer chemistry andtertiary polyplex structure. The perikinetic flocculation seen inOpti-MEM is most likely occurring because the increased concentrationof salts in solution decreases the Debye length.

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