Endosomal escape pathways for delivery of biologicals

Varkouhi, Amir K.; Scholte, Marije; Storm, Gert; Haisma, Hidde J.

doi:10.1016/j.jconrel.2010.11.004

Cited by 1,352 publications

(1,222 citation statements)

References 141 publications

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“…3,4 Other examples of methods developed to mediate endosomal escape that disrupt the membrane barrier include 'fusogenic' peptides or proteins that can mediate membrane fusion or transient pore formation in the phospholipid bilayer. 5 Homopolymers of anionic alkyl acrylic acids such as poly(propylacrylic acid) are another well-studied approach, and in these polymers, the protonation state of pendant carboxylic acid dictates transition into a hydrophobic, membrane-disruptive state in endo-lysosomal pH ranges. 6,7 One useful model system for screening endosomolytic behavior is the ex vivo pH-dependent hemolysis assay.…”

Section: Introductionmentioning

confidence: 99%

<em>Ex Vivo</em> 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

274

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: Introductionmentioning

confidence: 99%

<em>Ex Vivo</em> 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

274

245

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“…This hypothesis is supported by experimental results from multiple groups demonstrating interaction of polymer directly with cellular membranes, because charged cationic polyplexes have been demonstrated to lead to membrane disruption directly. 14,18 Further, the spacing of cationic charges in polymer structure has been demonstrated to affect both transfection efficacy and cellular viability, as observed in the odd-even effect with polyaspartamides. 44 Coupled with results from groups demonstrating efficacy of amphiphilic polymers for gene delivery that have relatively low charge densities, this balance between ability to possibly generate osmotic pressure because of buffering capability and ability to disrupt membranes by physical means may be critical for polymeric vector cytosolic delivery.…”

Section: Ymthe 4338mentioning

confidence: 99%

“…The latter contain hydrogen pump V-ATPases and digestive enzymes that result in acidification and degradation of the nucleic acid contents of polymeric nanoparticles. 17,18 To escape the endosome and avoid lysosomal degradation, polymeric nanoparticles have been designed specifically with either moieties that facilitate membrane pore formation or amine groups designed to enable them to buffer vesicle acidification and consequently escape the endosome via the hypothesized proton sponge mechanism. 18 Beginning with branched polyethylenimine (bPEI), many polymeric nanoparticles have been engineered to take advantage of endosome acidification as a means to protect their nucleic acid cargo and enable endosomal escape.…”

Section: Introductionmentioning

confidence: 99%

“…17,18 To escape the endosome and avoid lysosomal degradation, polymeric nanoparticles have been designed specifically with either moieties that facilitate membrane pore formation or amine groups designed to enable them to buffer vesicle acidification and consequently escape the endosome via the hypothesized proton sponge mechanism. 18 Beginning with branched polyethylenimine (bPEI), many polymeric nanoparticles have been engineered to take advantage of endosome acidification as a means to protect their nucleic acid cargo and enable endosomal escape. 19 Despite extensive study in the two decades since the first use of bPEI, the mechanism of endosomal escape for cationic nanoparticles is still not universally agreed upon.…”

Section: Introductionmentioning

confidence: 99%

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A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

et al. 2017

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There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.

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“…the integrity and viability of cells. Beside cellular uptake efficiency, the general applicability of MSNs requires that delivered proteins escape the endosomes and retain their function[20]. …”

Section: Introductionmentioning

confidence: 99%

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

Chiu

Bates

Helma

et al. 2018

Nucleus

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Protein transfection is a versatile tool to study or manipulate cellular processes and also shows great therapeutic potential. However, the repertoire of cost effective techniques for efficient and minimally cytotoxic delivery remains limited. Mesoporous silica nanoparticles (MSNs) are multifunctional nanocarriers for cellular delivery of a wide range of molecules, they are simple and economical to synthesize and have shown great promise for protein delivery. In this work we present a general strategy to optimize the delivery of active protein to the nucleus. We generated a bimolecular Venus based optical sensor that exclusively detects active and bioavailable protein for the performance of multi-parameter optimization of protein delivery. In conjunction with cell viability tests we maximized MSN protein delivery and biocompatibility and achieved highly efficient protein transfection rates of 80%. Using the sensor to measure live-cell protein delivery kinetics, we observed heterogeneous timings within cell populations which could have a confounding effect on function studies. To address this problem we fused a split or dimerization dependent protein of interest to chemically induced dimerization (CID) components, permitting control over its activity following cellular delivery. Using the split Venus protein we directly show that addition of a small molecule dimerizer causes synchronous activation of the delivered protein across the entire cell population. This combination of cellular delivery and triggered activation provides a defined starting point for functional studies and could be applied to other protein transfection methods.

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Endosomal escape pathways for delivery of biologicals

Cited by 1,352 publications

References 141 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

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

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