Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes

Kilchrist, Kameron V.; Evans, Brian C.; Brophy, Colleen M.; Duvall, Craig L.

doi:10.1007/s12195-016-0446-7

“…37–39 Our in vitro decoration approach preserved the favorable endogenous composition of EVs as delivery vectors, thus eliminating the need to build artificial endosome-escape strategies into the EV vectors compared to using other synthetic nanovectors for siRNA delivery. 44, 45 …”

Section: Inhibition Of Tumor Growth By Ligand-3wj-displaying Evsmentioning

confidence: 99%

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Pi

¹

,

Binzel

²

,

Lee

³

et al. 2017

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Nanotechnology holds many advantages. Here we report another advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand-display on extracellular vesicle (EV) membranes for specific cell targeting, or to regulate intracellular trafficking of siRNA/miRNA. Placing membrane-anchoring cholesterol at the arrow-tail results in display of RNA aptamer or folate on EV outer surface. In contrast, placing the cholesterol at the arrow-head results in partial loading of RNA nanoparticles into the EVs. Taking advantage of the RNA ligand for specific targeting and EVs for efficient membrane fusion, the resulting ligand-displaying EVs were competent for specific delivery of siRNA to cells, and efficiently block tumor growth in three cancer models. PSMA aptamer-displaying EVs loaded with survivin siRNA inhibited prostate cancer xenograft. The same EV but displaying EGFR aptamer inhibited orthotopic breast cancer models. Likewise, survivin-loaded and folate-displaying EVs inhibited patient derived colorectal cancer xenograft.

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“…Concentrations. A characteristic feature of damaged endosomes (20)(21)(22)(23)(24)(25)(26) is the presence of cytosolic β-galactosides that are ordinarily found on the luminal side of endolysosomal compartments (27).…”

Section: Cpmps and Cpps Do Not Induce Gal Recruitment At Submicromolarmentioning

confidence: 99%

“…In particular, Gal3 and Gal8 are recruited to damaged Rab7 + and Lamp1 + endosomes that form along the degradative branch of the endocytic pathway (20,31). Previous work has shown that endosomal damage can be detected by monitoring the translocation of eGFP fusions of Gal3 or Gal8 from the cytosol to endosome surfaces (20)(21)(22)(23)(24)(25)(26). However, the effects of CPPs or CPMPs on the extent of Gal-recruitment to potentially damaged endosomes have not previously been studied.…”

Section: Cpmps and Cpps Do Not Induce Gal Recruitment At Submicromolarmentioning

confidence: 99%

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Steinauer

¹

,

LaRochelle

²

,

Knox

³

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cellbased assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7 + and Lamp1 + endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1 + late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.cell-penetrating peptides | peptidomimetics | enzyme replacement therapy | endocytosis | protein therapeutics

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“…A characteristic feature of endosomes that have been damaged by invading viruses (29, 30), bacteria-triggered autophagy (31)(32)(33)(34), or endosomolytic nanoparticles (35) is the cytosolic display of β-galactosides linked to proteoglycans that are ordinarily found on the luminal side of endolysosomal compartments (36). Once displayed to the cytosol, β-galactosides recruit cytosolic galectin (Gal) proteins (37,38), such as Gal1, 3, 4, 8, and 9, that share a conserved βgalactoside binding site ( Figure S2A) (39,40).…”

Section: Cpmps and Cpps Do Not Induce Galectin Recruitment At Sub-micmentioning

confidence: 99%

“…Previous work has shown that endosomal damage can be detected by monitoring the translocation of eGFP fusions of Gal3 or Gal8 from the cytosol to endosome surfaces (29-34). Fusions of Gal3 or Gal8 and eGFP have also been used to visualize late endosome damage upon siRNA release from endosomes (40), screen for small molecules that induce lysosomal rupture (41), visualize endosomal leakage induced by osmocytosis and propanebetaine (iTOP) (42), and evaluate cell-permeant peptide-polymer complexes (35). However, the effects of CPPs or CPMPs on the extent of galectin-recruitment to damaged endosomes have not previously been studied.…”

Section: Cpmps and Cpps Do Not Induce Galectin Recruitment At Sub-micmentioning

confidence: 99%

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Steinauer

¹

,

LaRochelle

²

,

Wissner

³

et al. 2018

Preprint

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Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7 + and Lamp1 + endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs-an environment that serves as a prerequisite for efficient endosomal escape. 4 conclude that HOPS allows CPMPs to traffic into intraluminal vesicles (ILVs), a favorable environment for endosomal escape. The identification of ILVs as a portal for passing proteins into the cytosol will aid the development of next-generation biologics that overcome the limitations imposed by cellular membranes. RESULTS Evaluating endosomal damageThe simplest way for a CPMP to escape from an endosome is if the endosome ruptures, in part or in full (25). Although there has been limited work on the effects of certain CPPs on the integrity of large unilamellar vesicles (LUVs) in vitro (26), the concentration-dependent effects of CPMPs or more traditional cell-penetrating peptides (CPPs) on endosomal integrity in cultured cells have not been thoroughly evaluated. Thus, we began our analysis with two complementary assays that together detect both subtle and severe endosomal damage in cells treated with a CPMP or CPP. One assay exploits a set of eGFP-labeled galectins to fluorescently tag damaged endosomes to enable their visualization using confocal microscopy, while the other employs a fluorescently tagged version of the nonalysine (Lys9) peptide to quantify the extent of endosome rupture in cells treated with a CPMP or CPP. In both cases, the effects of the two most efficient ...

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Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes

Cited by 29 publications

References 43 publications

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

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