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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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Related in: MedlinePlus

Microscopyimages taken at 48 h post-transfection of HeLa cellsthat were transfected with a plasmid encoding GFP with selected polymers.The fluorescence microscopy images were taken at 350 nm (DAPI) and470 nm (GFP expression). The scale bar represents 100 μm.
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fig9: Microscopyimages taken at 48 h post-transfection of HeLa cellsthat were transfected with a plasmid encoding GFP with selected polymers.The fluorescence microscopy images were taken at 350 nm (DAPI) and470 nm (GFP expression). The scale bar represents 100 μm.

Mentions: Cells were also transfected with a plasmid encoding GFP andimaged48 h post-transfection at 350 and 470 nm to visualize cells positivefor gene expression (Figure 9) with selectedpolyplex formulations. Transfected cells were viewed under 470 nmwavelength light 48 h post-transfection (Figure 8). It can clearly be seen that cells were positive for GFP expressionin all cases and that a fraction of the population did not exhibitGFP expression. From the image, it can also be noticed that cellstransfected with poly(G46-b-P13) appeared to have a lower intensity of GFP, whereas poly(G46-b-T26) promoted higher expression levels.However, the difference in cell morphology supported the toxicitytrend noticed in Figure 8; cells transfectedwith poly(G46-b-T26) appearedlarger/swollen with blebs and vesicles surrounding the cells.


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

Sprouse D, Reineke TM - Biomacromolecules (2014)

Microscopyimages taken at 48 h post-transfection of HeLa cellsthat were transfected with a plasmid encoding GFP with selected polymers.The fluorescence microscopy images were taken at 350 nm (DAPI) and470 nm (GFP expression). The scale bar represents 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Microscopyimages taken at 48 h post-transfection of HeLa cellsthat were transfected with a plasmid encoding GFP with selected polymers.The fluorescence microscopy images were taken at 350 nm (DAPI) and470 nm (GFP expression). The scale bar represents 100 μm.
Mentions: Cells were also transfected with a plasmid encoding GFP andimaged48 h post-transfection at 350 and 470 nm to visualize cells positivefor gene expression (Figure 9) with selectedpolyplex formulations. Transfected cells were viewed under 470 nmwavelength light 48 h post-transfection (Figure 8). It can clearly be seen that cells were positive for GFP expressionin all cases and that a fraction of the population did not exhibitGFP expression. From the image, it can also be noticed that cellstransfected with poly(G46-b-P13) appeared to have a lower intensity of GFP, whereas poly(G46-b-T26) promoted higher expression levels.However, the difference in cell morphology supported the toxicitytrend noticed in Figure 8; cells transfectedwith poly(G46-b-T26) appearedlarger/swollen with blebs and vesicles surrounding the cells.

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