Plasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides

Serulla, Marc; Anees, Palapuravan; Hallaj, Ali; Trofimenko, Evgeniya; Kalia, Tara C.; Krishnan, Yamuna; Widmann, Christian

doi:10.1016/j.ejpb.2023.01.019

Cited by 11 publications

(11 citation statements)

References 62 publications

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“…86 Direct translocation across the membrane has also been proposed, and appears to occur most noticeably at higher peptide concentrations. 5,87–90 In this work, we obtained data across six different cell lines which show a linear correlation between clathrin-mediated endocytosis activity and cell penetration of ct-R9W and ct-Tat ( Figure 3C ). We observed a similar trend for macropinocytosis activity, especially for ct-R9W in all cell lines except Saos-2 ( Figure 3C ).…”

Section: Discussionmentioning

confidence: 91%

“…However, measuring cell penetration of biomolecules is not straightforward. In recent years, chemical biologists have developed a variety of strategies for quantifying passive penetration, total cellular uptake, and cytosolic/nuclear penetration with different detection methods including fluorescence microscopy, [4][5][6][7] mass-spectrometry, [8][9][10][11][12][13] NMR, 14 flow cytometry, [15][16][17] FACS, 18,19 qPCR, 20 luminescence, 21,22 or ELISA. 23 For cell-based assays, the choice of cell line is critical and measurement with different methods and/or in different cell lines can lead to different results.…”

Section: Introductionmentioning

confidence: 99%

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Comparing cell penetration of biotherapeutics across human cell lines

Batistatou,

Kritzer

2024

Preprint

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A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types, independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure data could be compared across cell lines. We compared the penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides, but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Comparing cell penetration of biotherapeutics across human cell lines

Batistatou,

Kritzer

2024

Preprint

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“…The U-2 OS cells were incubated with the FNPs-fR8 under PBS(K+) condition, which contained a high concentration of potassium ions to eliminate the membrane potential. 37,43 The results showed a decrease in cellular uptake after treatment of cells with PBS(K+) buffer, suggesting that FNPs-fR8 can also enter living cells by direct membrane translocation (Figure 3f,g). Taken together, our results suggest that the uptake of FNPs-fR8 involves not only endocytic pathways but also direct membrane translocation.…”

Section: Cellular Uptake Mechanism Of Fr8-modified Fnpsmentioning

confidence: 99%

Delivery of Fluorescent Nanoprobes into the Cytoplasm Using Cell-Penetrating Peptides for Live-Cell Super-Resolution Fluorescence Imaging

Xu,

Zhong,

et al. 2024

ACS Appl. Nano Mater.

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Vesicle entrapment remains a major bottleneck for the application of fluorescent nanoprobes (FNPs) with excellent optical properties in the field of super-resolution imaging of subcellular structures in living cells. Here, we developed a cellpenetrating peptide, fR8, which enables efficient cytoplasmic delivery of 3.5 nm FNPs instead of being trapped in vesicles, with a delivery efficiency of up to 75.6%. Further, we have successfully combined fusion protein technology with FNPs-fR8 to achieve high specificity and stable labeling of FNPs to various subcellular structures within live cells. Utilizing the advantages of FNPs, such as high brightness and resistance to photobleaching, FNPs modified with fR8 and the HaloTag ligand can be used for long-term super-resolution imaging of subcellular structures in living cells, allowing us to study the dynamic interactions between them. This work provides a practical tool for applying FNPs to live-cell super-resolution imaging and expands the application of super-resolution imaging technology in live-cell studies.

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“…Recent evidence indicates direct translocation rather than endocytosis mediates the transport of CPPs into the cytosol (Trofimenko et al, 2021;Serulla et al, 2023). The membrane potential provides the driving force for the direct translocation of CPPs (Trofimenko et al, 2021;Serulla et al, 2023).…”

Section: Direct Translocation Of Cpps Across the Plasma Membranementioning

confidence: 99%

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery

Novorolsky

Kasheke

Hakim

et al. 2023

Front. Cell. Neurosci.

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The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

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Plasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides

Cited by 11 publications

References 62 publications

Comparing cell penetration of biotherapeutics across human cell lines

Comparing cell penetration of biotherapeutics across human cell lines

Delivery of Fluorescent Nanoprobes into the Cytoplasm Using Cell-Penetrating Peptides for Live-Cell Super-Resolution Fluorescence Imaging

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery

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