Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell

Lönn, Peter; Dowdy, Steven F.

doi:10.1517/17425247.2015.1046431

Cited by 116 publications

(121 citation statements)

References 83 publications

(72 reference statements)

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“…Even non-hydrophobic CPPs are likely to be tightly bound to membrane proteins involved in endocytosis or transmembrane translocation, which would be expected to greatly hinder endosomal escape by covalently attached cargo proteins. One group estimates that less than 1% of TAT-fused cargos escape endosomes (Lonn and Dowdy, 2015). Our strategy of high-affinity but reversible non-covalent attachment of cargos overcomes trapping effects via Ca 2+ -dependent dissociation, allowing rapid and efficient cargo distribution to the cytoplasm even though TAT might remain trapped in the endosome.…”

Section: Resultsmentioning

confidence: 97%

“…More than 25 CPP clinical trials are underway, including a Phase III trial (Glogau et al, 2012;Lonn and Dowdy, 2015). However, CPPs have largely disappointed (Palm-Apergi et al, 2012) for a variety of reasons including non-penetration (Lundberg et al, 2003), limited endosomal escape (Erazo-Oliveras et al, 2012) and requirements for hydrophobic cargos (Hirose et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos

Salerno

Ngwa

Nowak

et al. 2016

Journal of Cell Science

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Fig. 2. Confocal imaging of cell penetration. BHK cells were treated for 1 h with DyLight 550 fluorescently labeled cargo proteins β-Gal (A), HRP (B) and myoglobin (C) (rendered as white in left panels, red in center and right panels), in either the absence or presence of TAT-CaM, washed and imaged live. Center images are optical sections set at a similar depth of the nucleus (NucBlue staining, white, center and right panels), as determined by position within the Z-stack. Orthogonal projections are shown at the right (boxed in red) and top (boxed in green) sides of each panel. Cytoplasmic compartments in live cells were visualized using CellTracker Green CMFDA dye (green in right panels). Comparison of TAT-CaM-treated versus untreated cells indicates that cargo proteins are entering the cell, and are localized primarily to the cytoplasm. Scale bars in all panels, 20 μm. Each experiment was replicated at least twice with the same results. 2474 ABSTRACTThe use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos

Salerno

Ngwa

Nowak

et al. 2016

Journal of Cell Science

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“…These biomaterials are able to readily conjugate nucleic acids and to deliver DNA molecules into a wide variety of mammalian somatic cells [19][20][21].…”

Section: Introductionmentioning

confidence: 99%

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Campelo

Canel

Bevacqua

et al. 2016

J Assist Reprod Genet

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Purpose Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes. Methods PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid. Results Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgeneexpressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05). Conclusions Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.

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“…It has been used to deliver a wide variety of cargo into cells and has shown efficacy in preclinical models in vivo (38,59,60). In addition, several clinical trials are currently underway using TAT-mediated delivery (57,(60)(61)(62)(63)(64), although no therapy as of yet has received FDA approval, as issues associated with efficacy, bioavailablilty, and targeting remain primary challenges.…”

Section: Discussionmentioning

confidence: 99%

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

Kang¹,

Jung²,

Yin³

et al. 2017

Journal of Clinical Investigation

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ResultsA TAT-SNX9 peptide specifically blocks pSMAD3 nuclear import and profibrotic TGF-β signaling. We previously determined that SNX9 has an obligate role in mediating profibrotic TGF-β signaling dependent upon SMAD3 (24). In order to investigate colony formation, and cell migration; and (b) fibrotic changes associated with the bleomycin (BLM) and adenoviral models of lung fibrosis. Moreover, a 3-amino acid point mutant of the TAT-SNX9 peptide unable to bind pSMAD3 was ineffective in analogous in vitro and in vivo assays. Lysates from AKR-2B cells untreated (-) or stimulated (+) for 45 minutes with 5 ng/ml TGF-β were incubated with GST beads or the indicated fusion proteins immobilized on GST beads. Bound proteins were eluted and assessed by Western blot analysis for pSMAD3 or pSMAD2. Cell lysate reflects signal obtained from 10 μg total protein; the slower migrating band in the pSMAD3 lane is a nonspecific protein (right, representative of 3 separate experiments). (B) AKR-2B cells were transduced for 90 minutes with the indicated concentration of TAT peptide. Following washing and 1 hour TGF-β (5 ng/ml) treatment, nuclear fractions were isolated and assessed by Western blot analysis for pSMAD2, pSMAD3, or histone deacetylase 1 (HDAC) (left, representative of 3 separate experiments). Quantitation of nuclear pSMADs was performed with ImageJ software (NIH) and represents the mean ± SEM of 3 experiments (right). (C) Left panels: AKR-2B cells were incubated with vehicle (top panels) or transduced (bottom panels) as in B with TAT-SH3 or TAT-LC (1.8 μM). Following treatment with or without TGF-β (5 ng/ml) for 1 hour, immunofluorescence for SMAD3 or the HA-tagged TAT peptide was performed as described in Methods and nuclei were stained with DAPI. Upper and lower panels show 2 distinct microscopic fields for each condition. Original magnification in C: ×100. Right: quantitation of nuclear SMAD3 from 30 cells in each of 3 experiments. *P < 0.05; **P < 0.005; ***P < 0.0005, 1-way ANOVA followed by Dunnett's multiple comparisons test. The Journal of Clinical Investigation R E S E A R C H A R T I C L E2 5 4 3 jci.orgVolume 127 Number 7 July 2017 sion of this fragment could act in trans as a dominant inhibitor of pSMAD3 nuclear uptake. To address this issue, constructs were prepared expressing either the SNX9 SH3 or LC domains fused to the cell-penetrating TAT peptide from HIV (39). Subsequent to TAT peptide transduction, cultures were treated with TGF-β and nuclear accumulation of pSMAD2 or pSMAD3 determined. As shown in Figure 1B and Supplemental Figure 2, while the TAT-SH3 peptide inhibited nuclear import of pSMAD3 in a dose-dependent manner and increased cytoplasmic pSMAD3, it had no effect on pSMAD2. Furthermore, consistent with the inability of the LC domain to bind receptorregulated SMADs (R-SMADs) ( Figure 1A), it was similarly ineffective in modulating nuclear translocation ( Figure 1B). These biochemical findings were independently confirmed using immunofluorescence ( Figure 1C and Supplemental Figure 3). While t...

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Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell

Cited by 116 publications

References 83 publications

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

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