Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

Teo, Serena L. Y.; Rennick, Joshua J.; Yuen, Daniel; Al-Wassiti, Hareth; Johnston, Angus P. R.; Pouton, Colin W.

doi:10.1038/s41467-021-23997-x

Cited by 103 publications

(91 citation statements)

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“…However, a punctuate fluorescence was also observed in both calcein and merged images (Fig. 4A ) suggesting that nanoparticles were sequestered within endosomal/lysosomal compartments ( 36 , 37 ). Although the fluorescence intensity was lower, the nanoparticles R 0 and R 25 also presented punctuated fluorescence inside the cells.…”

Section: Resultsmentioning

confidence: 91%

Defining Endocytic Pathways of Fucoidan-Coated PIBCA Nanoparticles from the Design of their Surface Architecture

et al. 2022

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Purpose This work investigated the endocytic pathways taken by poly(isobutylcyanoacrylate) (PIBCA) nanoparticles differing in their surface composition and architecture, assuming that this might determine their efficiency of intracellular drug delivery. Methods Nanoparticles (A0, A25, A100, R0, R25 ) were prepared by anionic or redox radical emulsion polymerization using mixtures of dextran and fucoidan (0, 25, 100 % in fucoidan). Cell uptake was evaluated by incubating J774A.1 macrophages with nanoparticles. Endocytic pathways were studied by incubating cells with endocytic pathway inhibitors (chlorpromazine, genistein, cytochalasin D, methyl-ß-cyclodextrin and nocodazole) and nanoparticle uptake was evaluated by flow cytometry and confocal microscopy. Results The fucoidan-coated PIBCA nanoparticles A 25 were internalized 3-fold more efficiently than R 25 due to the different architecture of the fucoidan chains presented on the surface. Different fucoidan density and architecture led to different internalization pathway preferred by the cells. Large A 100 nanoparticles with surface was covered with fucoidan chains in a loop and train configuration were internalized the most efficiently, 47-fold compared with A 0 , and 3-fold compared with R 0 and R 25 through non-endocytic energy-independent pathways and reached the cell cytoplasm. Conclusion Internalization pathways of PIBCA nanoparticles by J774A.1 macrophages could be determined by nanoparticle fucoidan surface composition and architecture. In turn, this influenced the extent of internalization and localization of accumulated nanoparticles within cells. The results are of interest for rationalizing the design of nanoparticles for potential cytoplamic drug delivery by controlling the nature of the nanoparticle surface. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11095-022-03202-4.

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Section: Resultsmentioning

confidence: 91%

Defining Endocytic Pathways of Fucoidan-Coated PIBCA Nanoparticles from the Design of their Surface Architecture

et al. 2022

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“…mApple was recombinantly expressed and purified using a method previously reported 44 , with the variation that expression was performed in the E. coli strain B-95.ΔA 45 and eluted mApple was buffer exchanged into pH 7.4 PBS. Protein concentration was determined by A568 with e = 82,000 M -1 cm -1 27 .…”

Section: Protein Expressionmentioning

confidence: 99%

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor

Rennick

Nowell

Pouton

et al. 2022

Preprint

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Changes in sub-cellular pH play a key role in metabolism, cell growth, membrane transport, and can also be exploited to control cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, we have developed a method to accurately quantify sub-cellular pH in individual vesicles using fluorescent lifetime imaging microscopy (pHLIM). pHLIM exploits the linear pH dependant lifetime of the fluorescent protein mApple and uses deep learning models to automatically identify and measure the pH of subcellular compartments. We have engineered mApple fusion proteins to measure the pH of the cytosol, endosomes, lysosomes and demonstrated the utility of pHLIM by measuring pH changes induced by drugs (bafilomycin A1) and polyethylenimine (a common transfection reagent). pHLIM is a simple and quantitative method to measure sub-cellular pH that has the potential to help with the design of the next generation of controlled drug release systems and to understand drug action and disease progression.

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“…Although data are very scarce, the reported EVs endosomal escape efficacies vary from 10% after 2 h to 24.5% after 12 h in Joshi et al [72] and about 20-30% in Bonsergent et al [71] Although direct fusion with the plasma membrane is possible, it is expected to represent a much smaller fraction of intra-cytosolic delivery when looking at the delivery kinetic. These numbers are to be compared with the natural endosomal escape that is reported to be about 2-7% depending on the cell type [73], about 0.1-2% for synthetic vectors [74] and about 40-50% for viral vectors like AAVs [75].…”

Section: A Very Interesting Intra-cytosolic Rna Delivery (Endosomal Escape)mentioning

confidence: 99%

Thinking Quantitatively of RNA-Based Information Transfer via Extracellular Vesicles: Lessons to Learn for the Design of RNA-Loaded EVs

Piffoux

Volatron²,

Silva

et al. 2021

Pharmaceutics

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Extracellular vesicles (EVs) are 50–1000 nm vesicles secreted by virtually any cell type in the body. They are expected to transfer information from one cell or tissue to another in a short- or long-distance way. RNA-based transfer of information via EVs at long distances is an interesting well-worn hypothesis which is ~15 years old. We review from a quantitative point of view the different facets of this hypothesis, ranging from natural RNA loading in EVs, EV pharmacokinetic modeling, EV targeting, endosomal escape and RNA delivery efficiency. Despite the unique intracellular delivery properties endowed by EVs, we show that the transfer of RNA naturally present in EVs might be limited in a physiological context and discuss the lessons we can learn from this example to design efficient RNA-loaded engineered EVs for biotherapies. We also discuss other potential EV mediated information transfer mechanisms, among which are ligand–receptor mechanisms.

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Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

Cited by 103 publications

References 50 publications

Defining Endocytic Pathways of Fucoidan-Coated PIBCA Nanoparticles from the Design of their Surface Architecture

Defining Endocytic Pathways of Fucoidan-Coated PIBCA Nanoparticles from the Design of their Surface Architecture

Resolving subcellular pH with a quantitative fluorescent lifetime biosensor

Thinking Quantitatively of RNA-Based Information Transfer via Extracellular Vesicles: Lessons to Learn for the Design of RNA-Loaded EVs

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