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Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

van Bracht E, Versteegden LR, Stolle S, Verdurmen WP, Woestenenk R, Raavé R, Hafmans T, Oosterwijk E, Brock R, van Kuppevelt TH, Daamen WF - PLoS ONE (2014)

Bottom Line: Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker.HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy.These results were confirmed by confocal microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.

ABSTRACT
Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

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Related in: MedlinePlus

Quenching of FITC fluorescent lyophilisomes by trypan blue in the absence of cells.Results show decreased fluorescence (a) and efficient quenching (b) up to three washings steps of FITC fluorescent lyophilisomes by trypan blue.
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pone-0110813-g004: Quenching of FITC fluorescent lyophilisomes by trypan blue in the absence of cells.Results show decreased fluorescence (a) and efficient quenching (b) up to three washings steps of FITC fluorescent lyophilisomes by trypan blue.

Mentions: To determine the internalization efficiency of sorted lyophilisomes with and without TAT peptide, a trypan blue quenching assay was used in order to distinguish between internalized and non-internalized (but plasma membrane-associated) lyophilisomes. This assay is based on the quenching of fluorescence of FITC-labeled lyophilisomes by the vital stain trypan blue (which does not penetrate plasma membranes). To validate that trypan blue also quenches fluorescence of FITC labeled lyophilisomes, lyophilisomes were incubated with trypan blue in the absence of cells and evaluated by FACS (Fig. 4). Lyophilisomes that were not incubated with trypan blue showed a mean fluorescence intensity of 1339±252. Lyophilisomes incubated in a 0.5% trypan blue solution gave a mean fluorescent intensity of 85±31, corresponding to a quenching efficacy of 94±2% (Fig. 4b). After one, two and three washings after the trypan blue incubation, the measured mean fluorescence was 161±25, 176±22, and 192±19 or 88±1%, 87±1% and 85±1% quenching efficacy, respectively. This indicates that trypan blue was not easily washed out of the lyophilisomes and fluorescence remained quenched. This is important as three washings steps were used prior to FACS analysis.


Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

van Bracht E, Versteegden LR, Stolle S, Verdurmen WP, Woestenenk R, Raavé R, Hafmans T, Oosterwijk E, Brock R, van Kuppevelt TH, Daamen WF - PLoS ONE (2014)

Quenching of FITC fluorescent lyophilisomes by trypan blue in the absence of cells.Results show decreased fluorescence (a) and efficient quenching (b) up to three washings steps of FITC fluorescent lyophilisomes by trypan blue.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219704&req=5

pone-0110813-g004: Quenching of FITC fluorescent lyophilisomes by trypan blue in the absence of cells.Results show decreased fluorescence (a) and efficient quenching (b) up to three washings steps of FITC fluorescent lyophilisomes by trypan blue.
Mentions: To determine the internalization efficiency of sorted lyophilisomes with and without TAT peptide, a trypan blue quenching assay was used in order to distinguish between internalized and non-internalized (but plasma membrane-associated) lyophilisomes. This assay is based on the quenching of fluorescence of FITC-labeled lyophilisomes by the vital stain trypan blue (which does not penetrate plasma membranes). To validate that trypan blue also quenches fluorescence of FITC labeled lyophilisomes, lyophilisomes were incubated with trypan blue in the absence of cells and evaluated by FACS (Fig. 4). Lyophilisomes that were not incubated with trypan blue showed a mean fluorescence intensity of 1339±252. Lyophilisomes incubated in a 0.5% trypan blue solution gave a mean fluorescent intensity of 85±31, corresponding to a quenching efficacy of 94±2% (Fig. 4b). After one, two and three washings after the trypan blue incubation, the measured mean fluorescence was 161±25, 176±22, and 192±19 or 88±1%, 87±1% and 85±1% quenching efficacy, respectively. This indicates that trypan blue was not easily washed out of the lyophilisomes and fluorescence remained quenched. This is important as three washings steps were used prior to FACS analysis.

Bottom Line: Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker.HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy.These results were confirmed by confocal microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical centre, Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands.

ABSTRACT
Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

Show MeSH
Related in: MedlinePlus