Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane

Sriram Vaidyanathan, Kevin B. Anderson, Rachel L. Merzel, Binyamin Jacobovitz, Milan P. Kaushik, Christina N. Kelly, Mallory A. van Dongen, Casey A. Dougherty, Bradford G. Orr, Mark M. Banaszak Holl

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

Original languageEnglish
Pages (from-to)6097-6109
Number of pages13
JournalACS Nano
Volume9
Issue number6
DOIs
Publication statusPublished - 23 Jun 2015
Externally publishedYes

Keywords

  • gene delivery
  • gene therapy
  • polymer cell membrane interactions
  • polymer-membrane partition
  • polyplex membrane partition
  • stable pore model
  • whole cell patch clamp

Cite this

Vaidyanathan, Sriram ; Anderson, Kevin B. ; Merzel, Rachel L. ; Jacobovitz, Binyamin ; Kaushik, Milan P. ; Kelly, Christina N. ; van Dongen, Mallory A. ; Dougherty, Casey A. ; Orr, Bradford G. ; Banaszak Holl, Mark M. / Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane. In: ACS Nano. 2015 ; Vol. 9, No. 6. pp. 6097-6109.
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title = "Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane",
abstract = "Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.",
keywords = "gene delivery, gene therapy, polymer cell membrane interactions, polymer-membrane partition, polyplex membrane partition, stable pore model, whole cell patch clamp",
author = "Sriram Vaidyanathan and Anderson, {Kevin B.} and Merzel, {Rachel L.} and Binyamin Jacobovitz and Kaushik, {Milan P.} and Kelly, {Christina N.} and {van Dongen}, {Mallory A.} and Dougherty, {Casey A.} and Orr, {Bradford G.} and {Banaszak Holl}, {Mark M.}",
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Vaidyanathan, S, Anderson, KB, Merzel, RL, Jacobovitz, B, Kaushik, MP, Kelly, CN, van Dongen, MA, Dougherty, CA, Orr, BG & Banaszak Holl, MM 2015, 'Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane' ACS Nano, vol. 9, no. 6, pp. 6097-6109. https://doi.org/10.1021/acsnano.5b01263

Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane. / Vaidyanathan, Sriram; Anderson, Kevin B.; Merzel, Rachel L.; Jacobovitz, Binyamin; Kaushik, Milan P.; Kelly, Christina N.; van Dongen, Mallory A.; Dougherty, Casey A.; Orr, Bradford G.; Banaszak Holl, Mark M.

In: ACS Nano, Vol. 9, No. 6, 23.06.2015, p. 6097-6109.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Vaidyanathan, Sriram

AU - Anderson, Kevin B.

AU - Merzel, Rachel L.

AU - Jacobovitz, Binyamin

AU - Kaushik, Milan P.

AU - Kelly, Christina N.

AU - van Dongen, Mallory A.

AU - Dougherty, Casey A.

AU - Orr, Bradford G.

AU - Banaszak Holl, Mark M.

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N2 - Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

AB - Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40- 50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

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KW - gene therapy

KW - polymer cell membrane interactions

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KW - polyplex membrane partition

KW - stable pore model

KW - whole cell patch clamp

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Vaidyanathan S, Anderson KB, Merzel RL, Jacobovitz B, Kaushik MP, Kelly CN et al. Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane. ACS Nano. 2015 Jun 23;9(6):6097-6109. https://doi.org/10.1021/acsnano.5b01263