Development of a novel endosomolytic diblock copolymer for siRNA delivery

Convertine, Anthony J.; Benoit, Danielle S. W.; Duvall, Craig L.; Hoffman, Allan S.; Stayton, Patrick S.

doi:10.1016/j.jconrel.2008.10.004

Cited by 373 publications

(540 citation statements)

References 79 publications

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“…peptides, siRNA, ODNs, proteins), and results of this assay can be predictive of performance as an intracellular drug delivery vehicle. 3,4,8,18,19 Thus, this assay represents an effective screen to gauge the ability of polymeric drug carriers to mediate intracellular drug delivery based on their pH-dependent membrane disruption.…”

Section: Discussionmentioning

confidence: 99%

“…This activity has been shown to promote the endosomal escape of peptide and nucleic acid-based therapeutics, allowing them to access their cytosolic targets. 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.…”

Section: Introductionmentioning

confidence: 99%

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<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

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274

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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%

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

Self Cite

274

245

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“…(TPMMA) [92] (Continued) MMA [94] St [51] 122 [293] S S CN CO 2 H C 2 H 5 S DMAEMA [293] DMAEMA-b-AA/BMA/ DMAEMA [293] 123 [294] MMA [27] MAA/CMA [295] 126 [294] PEO macro-RAFT agent…”

Section: S S Cn C 2 H 5 Smentioning

confidence: 99%

Living Radical Polymerization by the RAFT Process - A Second Update

Moad¹,

Rizzardo²,

Thang³

2009

Aust. J. Chem.

906

720

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This paper provides a second update to the review of reversible deactivation radical polymerization achieved with thiocarbonylthio compounds (ZC(=S)SR) by a mechanism of reversible addition-fragmentation chain transfer (RAFT) that was published in June 2005 (Aust. J. Chem. 2005, 58, 379-410). The first update was published in November 2006 (Aust. J. Chem. 2006, 59, 669-692). This review cites over 500 papers that appeared during the period mid-2006 to mid-2009 covering various aspects of RAFT polymerization ranging from reagent synthesis and properties, kinetics and mechanism of polymerization, novel polymer syntheses and a diverse range of applications. Significant developments have occurred, particularly in the areas of novel RAFT agents, techniques for end-group removal and transformation, the production of micro/nanoparticles and modified surfaces, and biopolymer conjugates both for therapeutic and diagnostic applications.

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“…Many groups interested in the delivery of siRNA have investigated various tools for overcoming this barrier, including endosome-disrupting polymers (49). Our measurements of the number of toxin molecules required to achieve a single translocation event should be a useful assay for investigating the potential of these tools to enhance intracellular delivery in a quantitative way.…”

mentioning

confidence: 99%

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

Pirie¹,

Hackel²,

Rosenblum

et al. 2011

Journal of Biological Chemistry

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Gelonin-based immunotoxins vary widely in their cytotoxic. Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ϳ5 ؋ 10 6 toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.Immunotoxins are a promising approach to the targeted delivery of highly potent, cancer-specific, cytotoxic agents. Immunotoxins are frequently composed of a targeting moiety (derived from antibodies or other cell-binding proteins) either chemically conjugated or genetically fused to highly cytotoxic plant or bacterial protein toxins. Clinical success for immunotoxins has been mostly limited to hematological malignancies due to transport limitations in solid tumors (1). Such limitations have been extensively studied experimentally (2) and with several computational models (3, 4).The potency of a particular immunotoxin is dependent on the ability to deliver the toxin to the cytoplasm, which is commonly considered to be the rate-limiting step. For some native toxins such as ricin, intracellular delivery is achieved through lectin binding, followed by internalization and toxin release with membrane fusion or retrograde trafficking (5). Immunotoxins attempt to recreate this scenario by replacing the indiscriminate lectin binding with cancer-specific antigen binding as a means of targeting and internalization (6). Subsequent intracellular trafficking, release, and endosomal escape are often achieved using existing toxin characteristics, translocation domains, protease cleavage sites, disulfide bonds, and/or signaling peptides (7-10). However, the inclusion of toxins with domains facilitating cytoplasmic access can also lead to increased nonspecific toxicity in vivo (11,12).Gelonin is a plant toxin and classified as a type I ribosomeinactivating protein because it lacks any cell-binding or cytoplasmic delivery domains. Recombinant gelonin (rGel) 2 is an ϳ30-kDa N-glycosidase with activity similar to the ricin A chain but exhibiting better stability and lower immunogenicity (13,14). The use of rGel in tumor-targeted cytotoxic agents has been well studied (15, 16). Furthermore, rGel has been shown to be active without cleavage from the binding domain and without negative impact on the targeting agent's pharmacokinetics (...

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Development of a novel endosomolytic diblock copolymer for siRNA delivery

Cited by 373 publications

References 79 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

Living Radical Polymerization by the RAFT Process - A Second Update

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

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