The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol‐based vehicles

Moore, Nicole M.; Sheppard, Clayton; Barbour, Tiffany R.; Sakiyama‐Elbert, Shelly E.

doi:10.1002/jgm.1234

Cited by 59 publications

(42 citation statements)

References 55 publications

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“…In studies that employ this method, the quantification of unquenched fluorophores has been found to correlate with the ability of a vehicle to mediate endosomal escape of its payload. 21,23 Microscopy-based measurements can provide a more robust but lower-throughput method that is complementary to the hemolysis assay. For example, it is common to assess colocalization of the carrier or the drug itself with dye-labeled lysosomes (e.g.…”

Section: Discussionmentioning

confidence: 99%

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Evans

Nelson

et al. 2013

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271

245

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Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems.In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Evans

Nelson

et al. 2013

JoVE

271

245

View full text Add to dashboard Cite

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“…We used a hemolysis assay with the erythrocyte membrane as a model of the endosomal membrane (Chen et al 2009;Wang et al 2007). Early endosomal compartments have a pH from 6.5 to 6.8, while the lumen of late endosomes has a lower pH, of about 5.5 (Moore et al 2008;Stayton et al 2000). The lysosome has a pH as low as 4.6 to 5.0 (Mellman et al 1996).…”

Section: Hemolysis Assaymentioning

confidence: 99%

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

et al. 2011

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“…At pH 5.4, the hemolytic activity of the surfactants increased considerably (p<0.05), reaching maxima of between 65.31% and 84.69%, with the following ranking for the HC 50 values: 77KT < 77KK < 77KS < 77KP < 77KL. Endosomal compartments have a pH of between 5.5 to 6.8, while the pH in the lysosome is about 5.0 [2,21,36]. Therefore, this pH-responsive membrane disruption around pH 5.4, which is in the pH range characteristic of late endosomes, is favorable for intracellular trafficking of therapeutic compounds.…”

Section: Ph-dependent Hemolysismentioning

confidence: 99%

“…Lytic activity at the level of late endosomes should facilitate membrane destabilization, and allow surfactant-drug complexes to escape to the cytoplasm for efficient intracellular drug delivery. Gene delivery vehicles that exhibit specific membrane lytic activity at pH 5.5 have higher transfection efficiency than vehicles that destabilize membranes at pH 6.0-6.5 [21]. Moreover, surfactants with high hemolytic activity at pH 5.4 gave the best siRNA delivery efficiency in U87-luc cells [8].…”

Section: Ph-dependent Hemolysismentioning

confidence: 99%

“…Cationic and anionic polymers with pH-sensitive activity, including nonbiodegradable polymers and biodegradable poly(amino acid)s and pseudo-peptides [2,7,[12][13][14][15][16][17][18], have also been developed to promote endosomal escape. Cationic compounds have commonly been used to form stable cationic complexes; however, they show cytotoxicity and non-specifically adsorb serum proteins, thereby leading to rapid blood clearance as a result of the strong cationic surface charge [19][20][21]. In contrast, anionic pHresponsive polymers are considered of interest as drug carriers because they mimic the structure and pH-dependent membrane-lytic behavior of endosomolytic viral peptides [1].…”

Section: Introductionmentioning

confidence: 99%

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The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants

et al. 2011

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Surfactants are among the most versatile and widely used excipients in pharmaceuticals.This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Here we studied five anionic lysine-based surfactants that differ in the nature of their counterion.We examined their capacity to disrupt the cell membrane under a range of pH values, concentrations and incubation times using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that at high concentrations surfactant behavior is independent of pH. We addressed the mechanism of surfactant-mediated membrane destabilization, and also performed scanning electron microscopy studies to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications.

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The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol‐based vehicles

Abstract: Endosomal escape is a key step in nonviral cellular trafficking and thus the transfection efficiency of nonviral vehicles can be increased by targeting release of DNA from the endosome with EEPs.

Cited by 59 publications

References 55 publications

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length

The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants

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