Delivery of Macromolecules into Live Cells by Simple Co‐incubation with a Peptide

Lee, Ya‐Jung; Erazo-Oliveras, Alfredo; Pellois, Jean‐Philippe

doi:10.1002/cbic.200900527

Cited by 37 publications

(43 citation statements)

References 43 publications

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“…(43) We have reported that E5-TAT, despite containing TAT, binds to a lesser extent than TAT to a negatively charged protein such as BSA. (21) This suggests that the TAT sequence in E5-TAT has a reduced affinity for negatively charged molecules because it might be involved in intramolecular interactions with E5. Yet, one would expect that these interactions would stabilize E5 in its deprotonated form and lead to a decrease in the pKas of the glutamate residues and in hemolysis pK50.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, E5-TAT can cause the release of macromolecules trapped inside endocytic organelles and this activity is dependent on the acidification of these organelles. (21) However the delivery efficiency associated with this peptide remains modest. Improving the properties of this delivery agent requires that one understand how this peptide functions.…”

Section: Discussionmentioning

confidence: 99%

“…This is consistent with results describing how the fluorescent protein HA2-TAT-mCherry, while being able to cause the release of a soluble dextran from endosomes into the cytosol of a cell, remains associated with endocytic organelles. (21) Finally, the composition of the endocytic organelles is also known to change during the transition from early to late endosomes. In particular, late endosomes are enriched in the anionic lipid lysobisphosphatidic acid (LBPA).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we have shown that this principle could be extended to macromolecules not labeled with TAT as peptides derived from HA2-TAT were able to stimulate both macropinocytosis and endosomal release of macromolecules present in the cell culture media. (21) Finally, HA2-TAT has also been used for the delivery of quantum dots into cardiac myocytes. (22) While these reports highlight the usefulness of HA2 conjugated to CPPs for cell culture applications, the potential value of this type of reagents has also been demonstrated in the context of therapeutic applications.…”

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confidence: 99%

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Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

2010

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HA2-TAT is a peptide-based delivery agent that combines the pH-sensitive HA2 fusion peptide from Influenza and the cell-penetrating peptide TAT from HIV. This chimeric peptide is engineered to induce the cellular uptake of macromolecules into endosomes via the TAT moiety and to respond to the acidifying lumen of endosomes to cause membrane leakage and release of macromolecules into cells via the HA2 moiety. The question of how HA2 and TAT affect the properties of one another remains, however, unanswered and the behavior of the peptide inside endosomes is mostly uncharacterized. To address these issues, the binding and membrane leakage activity of a glutamic acid-enriched analogue E5-TAT was assessed with red blood cells and giant unilamellar vesicles as membrane models for endosomes. Hemolysis and microscopy assays reveal that E5-TAT binds to membranes in a pH-dependent manner and causes membrane leakage by inducing the formation of pores through which macromolecules can escape. The TAT moiety contributes to this activity by causing a shift in the pH response of E5 and by binding to negatively charged phospholipids. On the other hand, TAT binding to glycosaminoglycans reduces the lytic activity of E5-TAT. Addition of TAT to the C-terminus of E5 can therefore either increase or inhibit the activity of E5 depending on the cellular components present at the membrane. Taken together, these results suggest a model for the endosomolytic activity of the peptide and provide the basis for the molecular design of future delivery agents.Cell-penetrating peptides (CPPs) provide general and useful tools to deliver macromolecular cargos into live cells. A prototypical CPP is the TAT peptide derived from HIV TAT.(1,2) TAT has been used to deliver peptides, proteins, DNA, liposomes and nanoparticles. (3,4) Multiple mechanisms are involved in cellular penetration mediated by TAT. TAT has been shown to directly translocate across the lipid bilayer of the plasma membrane when it is both labeled with a small molecule and present above a threshold concentration.(5) On the other hand, TAT is internalized by endocytosis at low concentration or when conjugated to large hydrophilic molecules.(6) Multiple endocytic mechanisms have been shown to be involved in the uptake of TAT-conjugates. TAT-conjugates can for instance enter cells by clathrin or caveolin-dependent endocytosis. In addition, TAT and other CPPs have also been shown to induce macropinocytosis, an effect that leads to the increase in uptake of fluid-phase endocytosis markers.(6-8) Induction of macropinocytosis appears to be mediated by the interaction of the positively charged peptide with membrane-associated heparan sulfate proteoglycans.(9) However, recent evidence suggests that HSPG do not act as receptors but † This work was supported by Award Number R01GM087227 from the National Institute of General Medical Sciences. G.J. was partially supported by the NIH molecular biophysics training grant (T32GM065088) * To whom correspondence should be addressed. Phone: 979 ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

2010

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“…TAT can escape the endocytic pathway, yet only at a very low efficiency (Gao et al, 2009; Gillmeister et al, 2011; Lee et al, 2010). Recently, we have identified that simply linking multiple copies of the HIV TAT peptide sequence (RKKRRQRRR) can greatly amplify this activity (endosomal escape) (Angeles-Boza et al, 2010; Erazo-Oliveras et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

The Late Endosome and Its Lipid BMP Act as Gateways for Efficient Cytosolic Access of the Delivery Agent dfTAT and Its Macromolecular Cargos

Erazo-Oliveras

Najjar

Truong

et al. 2016

Cell Chemical Biology

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Endosomal entrapment is a severely limiting bottleneck in the delivery of biologics into cells. The compound dfTAT was recently found to circumvent this problem by mediating endosomal leakage efficiently and without toxicity. Herein, we report on the mechanism of endosomal escape of this cell-penetrating peptide. By modulating the trafficking of the peptide within the endocytic pathway, we identify late endosomes as the organelles rendered leaky by dfTAT. We establish that dfTAT binds bis(monoacylglycero)phosphate (BMP), a lipid found in late endosomes, and that the peptide causes the fusion and leakage of bilayers containing BMP. Together, these data identify late endosomes as desirable gateways for cell penetration and BMP as a cellular factor that can be exploited for the development of future delivery agents.

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A Versatile Polymer‐Based Platform for Intracellular Delivery of Macromolecules

Kopytynski

Chen

Legg

et al. 2019

Advanced Therapeutics

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The plasma membrane barrier greatly restricts intracellular delivery of macromolecules. Currently available methods suffer from various limitations, including low delivery efficiency, high cytotoxicity, or incompatibility with a wider range of macromolecules or cell types. To overcome these issues, stimuli‐responsive polymers such as the bio‐inspired, pH‐responsive poly(l‐lysine isophthalamide) grafted with l‐phenylalanine at a stoichiometric ratio of 50% (PP50) can be used. In mildly acidic environments, the pseudopeptidic polymer can permeabilize the plasma membrane overcoming the problem of payload entrapment in the endosomes and allowing for efficient intracellular delivery. It is demonstrated that PP50 is capable of intracellular delivery by simple co‐incubation at pH 6.5 with various macromolecules, including different‐sized Dextrans, green fluorescent protein (GFP), and an apoptotic peptide. The delivery process is fast, nontoxic, and compatible with multiple cell types, including adherent and suspension cell lines, primary human mesenchymal stem cells, and cells grown as spheroids. In addition, apoptotic peptide delivery by co‐incubation with PP50 is over three times more effective than delivery using other common methods, including poly(ethyleneimine) (PEI), cell penetrating peptides (CPPs), and electroporation. The findings suggest that payload delivery by co‐incubation with PP50 is a flexible, controllable method allowing delivery of various payloads to many different cell types in vitro.

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Delivery of Macromolecules into Live Cells by Simple Co‐incubation with a Peptide

Cited by 37 publications

References 43 publications

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

The Late Endosome and Its Lipid BMP Act as Gateways for Efficient Cytosolic Access of the Delivery Agent dfTAT and Its Macromolecular Cargos

A Versatile Polymer‐Based Platform for Intracellular Delivery of Macromolecules

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