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.
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Sriram
authors.initials: S
authors.lastname: Vaidyanathan
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Kevin B
authors.initials: KB
authors.lastname: Anderson
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Merzel
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Binyamin
authors.initials: B
authors.lastname: Jacobovitz
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Milan P
authors.initials: MP
authors.lastname: Kaushik
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Christina N
authors.initials: CN
authors.lastname: Kelly
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Mallory A
authors.initials: MA
authors.lastname: van Dongen
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Casey A
authors.initials: CA
authors.lastname: Dougherty
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Bradford G
authors.initials: BG
authors.lastname: Orr
authors.affiliation: †Departments of Biomedical Engineering, ‡Chemistry, and §Physics, and ∥the Programs in Applied Physics and ⊥Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Banaszak Holl
mesh_terms: D002412:Cations / Q000032:analysis / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004247:DNA / Q000737:chemistry*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000032:analysis* / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007364:Intercalating Agents / Q000032:analysis* / Q000494:pharmacology; D010316:Particle Size; D011108:Polymers / Q000032:analysis* / Q000494:pharmacology; D016062:Porosity; D013499:Surface Properties
chemical_list: D002412:Cations; D007364:Intercalating Agents; D011108:Polymers; D004247:DNA
keywords: gene delivery; gene therapy; polymer-membrane partition; polymer−cell membrane interactions; polyplex−membrane partition; stable pore model; whole cell patch clamp