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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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Luciferase gene expression measured 48 h post-transfectionin HeLacells. Luminescence measured by a BioTek plate reader. RLU is therelative light units. Error bars are the standard deviation of threereplicates.
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fig7: Luciferase gene expression measured 48 h post-transfectionin HeLacells. Luminescence measured by a BioTek plate reader. RLU is therelative light units. Error bars are the standard deviation of threereplicates.

Mentions: It is generally thought thatgenetic cargo mostly enters the nucleusduring mitosis when the nuclear membrane disassembles; however, ithas been shown in previous research that polymers that induce membranepermeability also have higher expression efficiencies.49 Previous work by our group demonstrated thatPEI polyplexes induced plasma membrane permeabilization within halfan hour of transfection and nuclear membrane permeabilization by 4h post-transfection; this led to apoptosis and an increase in cellulartoxicity/death but also appears to increase gene expression.49 Knowing that the tertiary amine causes the plasmamembrane to destabilize (and here it is also linked to toxicity),it was thought that the polymers containing the tertiary amines mayalso have higher delivery efficiency/gene expression. To test thishypothesis, HeLa cells were transfected with polyplexes formulatedwith pDNA containing the firefly luciferase gene. It was observed(Figure 7) that the poly(G45-s-P35), poly(G47-s-P28-s-T18), poly(G46-b-P8-b-T9), and poly(G46-b-P6-b-T17) all revealed relatively high gene expression.Because of high membrane permeability, it was expected that poly(G46-b-T26) would have revealed higherexpression; however, only half of the cells survived the transfectionassay (MTT assay, Figure 4a). The two triblockpolymers, poly(G46-b-P8-b-T9) and poly(G46-b-P6-b-T17), had high geneexpression, likely because these polymers are nontoxic and may containa slightly lower fraction of tertiary amines (to aid in permeabilizingcell membranes). It was surprising to find that poly(G45-s-P35) had a much higher expressionprofile than poly(G62-s-T23). While we currently do not understand this trend, we speculatethat the statistical copolymer composed of only tertiary amine charges,poly(G62-s-T23), may dissociatebefore the polyplex can traffic to the nucleus. For a similar reason,this could be why the statistical copolymer made with only primaryamines, poly(G45-s-P35), hadhigher gene expression than the block copolymer analogue, poly(G46-b-P13). Similar to previouswork by Ahmed and Narain,19 we have foundthat spreading the charge throughout the polymer in a statisticalfashion can lead to increased gene expression (particularly with primaryamine charges). We conclude that the incorporation of tertiary aminesin cationic polymer vehicles does promote higher gene expression,due to their ability to permeabilize cell membranes. However, incorporatinga large fraction of tertiary amines leads to an increase in cytotoxicity,apoptosis, and cell death. Thus, the composition of amine types onthis vehicle class should be balanced by including mostly primaryamines that facilitate stable polyplex formation and are more benignto the cell.


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

Sprouse D, Reineke TM - Biomacromolecules (2014)

Luciferase gene expression measured 48 h post-transfectionin HeLacells. Luminescence measured by a BioTek plate reader. RLU is therelative light units. Error bars are the standard deviation of threereplicates.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Luciferase gene expression measured 48 h post-transfectionin HeLacells. Luminescence measured by a BioTek plate reader. RLU is therelative light units. Error bars are the standard deviation of threereplicates.
Mentions: It is generally thought thatgenetic cargo mostly enters the nucleusduring mitosis when the nuclear membrane disassembles; however, ithas been shown in previous research that polymers that induce membranepermeability also have higher expression efficiencies.49 Previous work by our group demonstrated thatPEI polyplexes induced plasma membrane permeabilization within halfan hour of transfection and nuclear membrane permeabilization by 4h post-transfection; this led to apoptosis and an increase in cellulartoxicity/death but also appears to increase gene expression.49 Knowing that the tertiary amine causes the plasmamembrane to destabilize (and here it is also linked to toxicity),it was thought that the polymers containing the tertiary amines mayalso have higher delivery efficiency/gene expression. To test thishypothesis, HeLa cells were transfected with polyplexes formulatedwith pDNA containing the firefly luciferase gene. It was observed(Figure 7) that the poly(G45-s-P35), poly(G47-s-P28-s-T18), poly(G46-b-P8-b-T9), and poly(G46-b-P6-b-T17) all revealed relatively high gene expression.Because of high membrane permeability, it was expected that poly(G46-b-T26) would have revealed higherexpression; however, only half of the cells survived the transfectionassay (MTT assay, Figure 4a). The two triblockpolymers, poly(G46-b-P8-b-T9) and poly(G46-b-P6-b-T17), had high geneexpression, likely because these polymers are nontoxic and may containa slightly lower fraction of tertiary amines (to aid in permeabilizingcell membranes). It was surprising to find that poly(G45-s-P35) had a much higher expressionprofile than poly(G62-s-T23). While we currently do not understand this trend, we speculatethat the statistical copolymer composed of only tertiary amine charges,poly(G62-s-T23), may dissociatebefore the polyplex can traffic to the nucleus. For a similar reason,this could be why the statistical copolymer made with only primaryamines, poly(G45-s-P35), hadhigher gene expression than the block copolymer analogue, poly(G46-b-P13). Similar to previouswork by Ahmed and Narain,19 we have foundthat spreading the charge throughout the polymer in a statisticalfashion can lead to increased gene expression (particularly with primaryamine charges). We conclude that the incorporation of tertiary aminesin cationic polymer vehicles does promote higher gene expression,due to their ability to permeabilize cell membranes. However, incorporatinga large fraction of tertiary amines leads to an increase in cytotoxicity,apoptosis, and cell death. Thus, the composition of amine types onthis vehicle class should be balanced by including mostly primaryamines that facilitate stable polyplex formation and are more benignto the cell.

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