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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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Microscopy images taken at the time polyplexes were addedand 4h post-transfection for selected formulations. Purple overlay on thesecond column is fluorescence microscopy taken at 628 nm. The scalebar represents 100 μm.
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fig8: Microscopy images taken at the time polyplexes were addedand 4h post-transfection for selected formulations. Purple overlay on thesecond column is fluorescence microscopy taken at 628 nm. The scalebar represents 100 μm.

Mentions: To monitor cells duringthe transfection process, selected polyplexesformulated with Cy5-pDNA were added to cultured HeLa cells and imagedfor 4 h (with the exception of Jet-PEI transfection, which was imagedfor 1.5 h due to severe toxicity and cell death by this time). Theimages were compiled into time-lapse videos to visualize cell behaviorand morphology during this time period (Figure 8 shows the DIC image at t = 0 and an overlay ofthe DIC and Cy-5 channel images at 4 h; time-lapse movie files areavailable in the Supporting Information). In Figure 8 and the movie files, the toxicityof some formulations was clearly evident. For cells exposed to JetPEIpolyplexes, all cells appeared to be under severe stress as earlyas 30 min post-transfection (the cells start blebbing, and the cellsshrink/shrivel up; movie S1, Supporting Information).50 When the Cy5 channel was observed,the polyplexes appeared to interact with the cell membrane, the cytoplasm,and the nucleus (Figure S26, Supporting Information). Of the polymer vehicles synthesized for this study, the most toxicformulation appeared to be poly(G46-b-T26), which agrees with the MTT (Figure 4), 7-AAD, and Annexin V assays (Figures 5, 6, S23, and S24, Supporting Information). After 4 h, almost all of the cells appeared to have polyplexeswithin or on the cell surface, and most of the cells appear dead (severelyshriveled/lysed; Figure 8 and movie S2, Supporting Information). Similarly, poly(G62-s-T23) also caused the cellsto bleb and shrivel (Figure 8 and movie S3, Supporting Information). Cells exposed to poly(G46-b-P13) polyplexes did not appearto bleb (similar to previous toxicity studies); however, a slightdecrease in cell volume was noted (Figure 8 and movie S4, Supporting Information).The formulation with poly(G45-s-P35) did not appear toxic to cells over the 4 h time courseof this experiment (Figure 8 and movie S5 (Supporting Information); no blebbing or decreasein cell volume was noticed even though polyplexes were clearly internalizedwithin cells).


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

Sprouse D, Reineke TM - Biomacromolecules (2014)

Microscopy images taken at the time polyplexes were addedand 4h post-transfection for selected formulations. Purple overlay on thesecond column is fluorescence microscopy taken at 628 nm. The scalebar represents 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Microscopy images taken at the time polyplexes were addedand 4h post-transfection for selected formulations. Purple overlay on thesecond column is fluorescence microscopy taken at 628 nm. The scalebar represents 100 μm.
Mentions: To monitor cells duringthe transfection process, selected polyplexesformulated with Cy5-pDNA were added to cultured HeLa cells and imagedfor 4 h (with the exception of Jet-PEI transfection, which was imagedfor 1.5 h due to severe toxicity and cell death by this time). Theimages were compiled into time-lapse videos to visualize cell behaviorand morphology during this time period (Figure 8 shows the DIC image at t = 0 and an overlay ofthe DIC and Cy-5 channel images at 4 h; time-lapse movie files areavailable in the Supporting Information). In Figure 8 and the movie files, the toxicityof some formulations was clearly evident. For cells exposed to JetPEIpolyplexes, all cells appeared to be under severe stress as earlyas 30 min post-transfection (the cells start blebbing, and the cellsshrink/shrivel up; movie S1, Supporting Information).50 When the Cy5 channel was observed,the polyplexes appeared to interact with the cell membrane, the cytoplasm,and the nucleus (Figure S26, Supporting Information). Of the polymer vehicles synthesized for this study, the most toxicformulation appeared to be poly(G46-b-T26), which agrees with the MTT (Figure 4), 7-AAD, and Annexin V assays (Figures 5, 6, S23, and S24, Supporting Information). After 4 h, almost all of the cells appeared to have polyplexeswithin or on the cell surface, and most of the cells appear dead (severelyshriveled/lysed; Figure 8 and movie S2, Supporting Information). Similarly, poly(G62-s-T23) also caused the cellsto bleb and shrivel (Figure 8 and movie S3, Supporting Information). Cells exposed to poly(G46-b-P13) polyplexes did not appearto bleb (similar to previous toxicity studies); however, a slightdecrease in cell volume was noted (Figure 8 and movie S4, Supporting Information).The formulation with poly(G45-s-P35) did not appear toxic to cells over the 4 h time courseof this experiment (Figure 8 and movie S5 (Supporting Information); no blebbing or decreasein cell volume was noticed even though polyplexes were clearly internalizedwithin 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