Chloride Accumulation and Swelling in Endosomes Enhances DNA Transfer by Polyamine-DNA Polyplexes

Abstract

The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H ؉ buffering polyamines by enhanced endosomal Cl ؊ accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl ؊ concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl ؊ ] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl ؊ or pH indicators. [Cl ؊ ] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl ؊ accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl ؊ accumulation and swelling/lysis were accounted for by the greater H ؉ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H ؉ /pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.Although gene delivery using non-viral vectors offers potential advantages over virus-based delivery systems, the relatively low transfection efficiency of non-viral vectors has been their major limitation for in vivo applications (1-4). The archetypal non-viral gene delivery system is the cationic polymer-DNA complex (polyplex), 1 in which plasmid DNA and a cationic carrier are condensed into a tight complex suitable for cellular internalization by endocytosis (5, 6). Transgene delivery to the nucleus is thought to require escape of the polyplex from endosomes, DNA/polymer dissociation, cytoplasmic DNA diffusion, and nuclear uptake (5, 7, 8). The low efficiency of polyplex escape from endosomes is thought to be an important determinant of the overall efficiency of non-viral gene transfer. Polyamines are useful polycationic macromolecules for nonviral gene transfer because of their high density of positive charges, ease of synthesis, and efficient polyplex formation (8 -10). Cationic polyamines with fixed, non-titratable charges such as polylysine (POL) are substantially less efficient at gene transfer than polyamines with titratable amines such as polyamidoamine (PAM) (11) and polyethylenimine (PEI) (12, 13). The lower efficiency is not caused by differences in morphology of the complexes or cell association (14). To explain this observation it has been postulated without direct evidence that the high H ϩ buffer capacity of polyamines containing titratable amines results in endosomal Cl Ϫ accumulation during acidification with presumed ...

Chemistry specificity of DNA–polycation complex salt response: a simulation study of DNA, polylysine and polyethyleneimine

“…That said, the Cl − s do not notably penetrate between the DNA and PEI molecules, even when the complex is exposed to additional salt. Only little Cl − penetration is observed despite the fact that the Cl − concentration used here, 0.27 M, is considerably higher than the experimentally estimated maximum ∼ 0.1 M. 31 In contrast, the full detachment of one of the less protonated PEI25 molecules from the 5xPEI25-DNA complex occurred in our simulations upon addition of salt. However, the detachment is propelled by the positive ion (Na + ) and not the negative ion (Cl − ) entering the complex.…”

Intracellular trafficking of nonviral vectors

“…152 Evidence for Cl À accumulation and pH decrease induced by PEI and PAM was shown by using Cl À and pH indicators during polyplex uptake in CHO-K1 cells. 153 In this study, an increase in the volume of polyplex-containing vesicles was observed, followed by dissipation of fluorescentlytagged polyplexes, indicative of vesicle swelling and disruption. While the authors suggest that the polyplexes enter 'macropinocytic' endosomes, it is unclear whether these vesicles are indeed macropinosomes, as it has not been established whether these vesicles acidify or associate with vacuolar proton pumps.…”

A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery

“…12,32 This "proton sponge" mechanism was recently proven by Sonawane et al who showed that endosomes containing vectors with physiologically titratable amine groups like those formed from PEI but not vectors lacking these groups like vectors formed from polylysine buffered H + ions, increased the counterion Cl − concentration within the endosome, swelled the endosome in size, and led to lysis of the endosome. 8 This process is shown in Figure 1. The studies mentioned above highlight the importance of polymer structure including physiologically titratable amine groups in polymeric vector construction.…”