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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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Flow cytometry analysis of cells for membrane permeability (7-AADpositive), apoptosis (Annexin V positive), and necrosis (both 7-AADand Annexin V positive). Data are plotted as 7-AAD (y-axis) versus Annexin V (x-axis). Pseudocolor representsthe density of 20,000 events plotted. Quadrant 1 (Q1) depicts cellsthat are only 7-AAD positive (seen as red bars in Figure 6), Q2 denotes cells that are 7-AAD and Annexin Vpositive (double positive gray bars in Figure 6), and Q3 denotes apoptotic cells that are only Annexin V positive(blue bars in Figure 6).
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fig5: Flow cytometry analysis of cells for membrane permeability (7-AADpositive), apoptosis (Annexin V positive), and necrosis (both 7-AADand Annexin V positive). Data are plotted as 7-AAD (y-axis) versus Annexin V (x-axis). Pseudocolor representsthe density of 20,000 events plotted. Quadrant 1 (Q1) depicts cellsthat are only 7-AAD positive (seen as red bars in Figure 6), Q2 denotes cells that are 7-AAD and Annexin Vpositive (double positive gray bars in Figure 6), and Q3 denotes apoptotic cells that are only Annexin V positive(blue bars in Figure 6).

Mentions: The flow cytometry data (Figures 5, S23,and S24, Supporting Information) showedvery compelling evidence that some cells did have destabilized membranes/smallholes (without causing a large population to be apoptotic), meaningthat 7-AAD could pass through the membrane but were not positive forAnnexin V. These populations came from cells treated with polymerscontaining tertiary amines: poly(G62-s-T23), poly(G46-b-P8-b-T9), poly(G46-b-P6-b-T17), and poly(G46-b-T26) (Figures 5, S23, and S24 (Supporting Information), and red bars in Figure 6). The tertiaryamine diblock [poly(G46-b-T26)] at 10 N/P showed the highest membrane disruption; only 6.6% ofthe cells were dead, but 65% of the cells had destabilized membranes,showing cellular internalization of 7AAD without cells being positivefor Annexin V. The amount of Annexin V positive (apoptotic) cellsindicates more information about the cytotoxicity of each polymer/polyplexformulation with HeLa cells. It was found that the two statisticalformulations poly(G45-s-P35) and poly(G62-s-T23) causeda portion of the cell population (about 20%) to undergo apoptosis(Figure 6). With respect to the block copolymers,the polyplex formulations were not toxic to cells (<7% dead); however,the polymer only samples of poly(G46-b-P8-b-T9), poly(G46-b-P6-b-T17), and poly(G46-b-T26) causedbetween 18 and 30% of the cells to die. A similar trend was noticedin the MTT and PI assays (Figure 4). It wasindeed evident from these data that the control polyplex formulationwith JetPEI caused over 60% of the cells analyzed to be dead and showsigns of apoptotic markers on their surface after only 4 h of polyplexexposure and that it was the most toxic formulation examined here(similar to the MTT results in Figure 4). Withthe exception of poly(G45-s-P35), all other formulations (polymers and polyplexes) with the highprimary amine content [poly(G32-s-P40-s-T21), poly(G47-s-P28-s-T18), poly(G46-b-P13), and poly(G46-b-P10-b-T2)] were found to be quite benign to the cells (Figure 6).


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

Sprouse D, Reineke TM - Biomacromolecules (2014)

Flow cytometry analysis of cells for membrane permeability (7-AADpositive), apoptosis (Annexin V positive), and necrosis (both 7-AADand Annexin V positive). Data are plotted as 7-AAD (y-axis) versus Annexin V (x-axis). Pseudocolor representsthe density of 20,000 events plotted. Quadrant 1 (Q1) depicts cellsthat are only 7-AAD positive (seen as red bars in Figure 6), Q2 denotes cells that are 7-AAD and Annexin Vpositive (double positive gray bars in Figure 6), and Q3 denotes apoptotic cells that are only Annexin V positive(blue bars in Figure 6).
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fig5: Flow cytometry analysis of cells for membrane permeability (7-AADpositive), apoptosis (Annexin V positive), and necrosis (both 7-AADand Annexin V positive). Data are plotted as 7-AAD (y-axis) versus Annexin V (x-axis). Pseudocolor representsthe density of 20,000 events plotted. Quadrant 1 (Q1) depicts cellsthat are only 7-AAD positive (seen as red bars in Figure 6), Q2 denotes cells that are 7-AAD and Annexin Vpositive (double positive gray bars in Figure 6), and Q3 denotes apoptotic cells that are only Annexin V positive(blue bars in Figure 6).
Mentions: The flow cytometry data (Figures 5, S23,and S24, Supporting Information) showedvery compelling evidence that some cells did have destabilized membranes/smallholes (without causing a large population to be apoptotic), meaningthat 7-AAD could pass through the membrane but were not positive forAnnexin V. These populations came from cells treated with polymerscontaining tertiary amines: poly(G62-s-T23), poly(G46-b-P8-b-T9), poly(G46-b-P6-b-T17), and poly(G46-b-T26) (Figures 5, S23, and S24 (Supporting Information), and red bars in Figure 6). The tertiaryamine diblock [poly(G46-b-T26)] at 10 N/P showed the highest membrane disruption; only 6.6% ofthe cells were dead, but 65% of the cells had destabilized membranes,showing cellular internalization of 7AAD without cells being positivefor Annexin V. The amount of Annexin V positive (apoptotic) cellsindicates more information about the cytotoxicity of each polymer/polyplexformulation with HeLa cells. It was found that the two statisticalformulations poly(G45-s-P35) and poly(G62-s-T23) causeda portion of the cell population (about 20%) to undergo apoptosis(Figure 6). With respect to the block copolymers,the polyplex formulations were not toxic to cells (<7% dead); however,the polymer only samples of poly(G46-b-P8-b-T9), poly(G46-b-P6-b-T17), and poly(G46-b-T26) causedbetween 18 and 30% of the cells to die. A similar trend was noticedin the MTT and PI assays (Figure 4). It wasindeed evident from these data that the control polyplex formulationwith JetPEI caused over 60% of the cells analyzed to be dead and showsigns of apoptotic markers on their surface after only 4 h of polyplexexposure and that it was the most toxic formulation examined here(similar to the MTT results in Figure 4). Withthe exception of poly(G45-s-P35), all other formulations (polymers and polyplexes) with the highprimary amine content [poly(G32-s-P40-s-T21), poly(G47-s-P28-s-T18), poly(G46-b-P13), and poly(G46-b-P10-b-T2)] were found to be quite benign to the cells (Figure 6).

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