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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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Cellular binding and internalization of unmodified lyophilisomes and TAT-conjugated lyophilisomes.HeLa, OVCAR-3, Caco-2 and SKOV-3 cells incubated with TAT-conjugated and unmodified lyophilisomes resulted in 86±3% and 12±4%, 87±3% and 16±8%, 97±3% and 19±3%, and 95±10% and 67±20% lyophilisome-positive cells, respectively. TAT  =  trans-activating transcriptional activator. *p<0.01 ***p<0.0001.
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pone-0110813-g002: Cellular binding and internalization of unmodified lyophilisomes and TAT-conjugated lyophilisomes.HeLa, OVCAR-3, Caco-2 and SKOV-3 cells incubated with TAT-conjugated and unmodified lyophilisomes resulted in 86±3% and 12±4%, 87±3% and 16±8%, 97±3% and 19±3%, and 95±10% and 67±20% lyophilisome-positive cells, respectively. TAT  =  trans-activating transcriptional activator. *p<0.01 ***p<0.0001.

Mentions: To address the presence of the TAT peptide on TAT-conjugated lyophilisomes, unmodified lyophilisomes and TAT-conjugated lyophilisomes were administered to HeLa, OVCAR-3, Caco-2 and OVCAR-3 cells. Using standard FACS as a functional assay, it is not possible to discriminate between cellular attachment and internalization. Instead, the total of cell binding and internalization is measured (Fig. 2). For HeLa cells, TAT-conjugated lyophilisomes showed an about 8-fold increase in lyophilisome-positive cells compared to lyophilisomes without the TAT peptide (86±3% and 12±4% lyophilisome-positive cells for TAT-conjugated and unmodified lyophilisomes, respectively. OVCAR-3 and Caco-2 cells showed about a 5-fold increase in lyophilisome-positive cells compared to lyophilisomes without the TAT peptide (lyophilisome-positive cells: 97±3% and 19±3% for OVCAR-3; 87±3% and 16±8% for Caco-2) for TAT-conjugated and unmodified lyophilisomes, respectively. SKOV-3 cells gave a high background value when incubated with lyophilisomes without TAT (67±20%), but still showed a 1.6 fold statistically significant increase with the presence of TAT peptide (95±10%).


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)

Cellular binding and internalization of unmodified lyophilisomes and TAT-conjugated lyophilisomes.HeLa, OVCAR-3, Caco-2 and SKOV-3 cells incubated with TAT-conjugated and unmodified lyophilisomes resulted in 86±3% and 12±4%, 87±3% and 16±8%, 97±3% and 19±3%, and 95±10% and 67±20% lyophilisome-positive cells, respectively. TAT  =  trans-activating transcriptional activator. *p<0.01 ***p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110813-g002: Cellular binding and internalization of unmodified lyophilisomes and TAT-conjugated lyophilisomes.HeLa, OVCAR-3, Caco-2 and SKOV-3 cells incubated with TAT-conjugated and unmodified lyophilisomes resulted in 86±3% and 12±4%, 87±3% and 16±8%, 97±3% and 19±3%, and 95±10% and 67±20% lyophilisome-positive cells, respectively. TAT  =  trans-activating transcriptional activator. *p<0.01 ***p<0.0001.
Mentions: To address the presence of the TAT peptide on TAT-conjugated lyophilisomes, unmodified lyophilisomes and TAT-conjugated lyophilisomes were administered to HeLa, OVCAR-3, Caco-2 and OVCAR-3 cells. Using standard FACS as a functional assay, it is not possible to discriminate between cellular attachment and internalization. Instead, the total of cell binding and internalization is measured (Fig. 2). For HeLa cells, TAT-conjugated lyophilisomes showed an about 8-fold increase in lyophilisome-positive cells compared to lyophilisomes without the TAT peptide (86±3% and 12±4% lyophilisome-positive cells for TAT-conjugated and unmodified lyophilisomes, respectively. OVCAR-3 and Caco-2 cells showed about a 5-fold increase in lyophilisome-positive cells compared to lyophilisomes without the TAT peptide (lyophilisome-positive cells: 97±3% and 19±3% for OVCAR-3; 87±3% and 16±8% for Caco-2) for TAT-conjugated and unmodified lyophilisomes, respectively. SKOV-3 cells gave a high background value when incubated with lyophilisomes without TAT (67±20%), but still showed a 1.6 fold statistically significant increase with the presence of TAT peptide (95±10%).

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