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TAT and HA2 facilitate cellular uptake of gold nanoparticles but do not lead to cytosolic localisation.

Cesbron Y, Shaheen U, Free P, Lévy R - PLoS ONE (2015)

Bottom Line: However, conflicting results have been reported on the extent of the cytosolic delivery achieved.To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery.While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Liverpool, Liverpool, United Kingdom; Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom; CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Rennes, France; Université de Rennes 1, Université Européenne de Bretagne, Structure fédérative de recherche Biosit, Faculté de Médecine, Rennes, France.

ABSTRACT
The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

No MeSH data available.


Related in: MedlinePlus

Attachment of HA2 to gold cores via the C-terminus versus N-terminus.HeLa cells were incubated for 4h with 10nm gold nanoparticles (6nM) capped with peptide monolayers of different compositions, fixed, and imaged by electron microscopy. Nanoparticle peptide monolayer compositions were: (A) 20% CCALNN-HA2 and 80% CALNN; (B) 20% HA2-NNLACC and 80% CALNN; (C) 50% CCALNN-HA2 and 50% CALNN; (D) 50% HA2-NNLACC and 50% CALNN. Arrowheads indicate particles around or outside endosomes. Additional images are available on figshare (10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852).
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pone.0121683.g004: Attachment of HA2 to gold cores via the C-terminus versus N-terminus.HeLa cells were incubated for 4h with 10nm gold nanoparticles (6nM) capped with peptide monolayers of different compositions, fixed, and imaged by electron microscopy. Nanoparticle peptide monolayer compositions were: (A) 20% CCALNN-HA2 and 80% CALNN; (B) 20% HA2-NNLACC and 80% CALNN; (C) 50% CCALNN-HA2 and 50% CALNN; (D) 50% HA2-NNLACC and 50% CALNN. Arrowheads indicate particles around or outside endosomes. Additional images are available on figshare (10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852).

Mentions: The exact process by which HA2 destabilises membranes is not fully understood. This stems from a lack of precise structural data for the critical part of the HA2 sequence that undergoes conformational changes during the membrane fusion, due to failures at crystallising it (very high hydrophobicity at acidic pH) [41]. Han et al. identified the N–terminal domain of HA2 as critical for destabilisation of artificial model membranes. It was shown that all 20 residues of the fusion domain of HA2 were actively involved in the conformational changes necessary to insert deep into synthetic lipid bilayer membrane at acidic pH [42]. This process could be affected by the way the peptide is attached to the gold nanoparticle surface, e.g. attached to the gold core via either the N- or the C-terminal end. Two orientations of the HA2 peptide sequence (Table 1, sequences 2–3) were therefore compared to assess the influence of the direction of attachment to the gold core. These two sequences of HA2 peptide differ marginally from the sequence of HA2 peptide used previously (Table 1, sequence 1), but not enough to prevent the fusion process. Indeed, both sequences have been reported to show a consistent fusion activity (see Table A in S1 File for a comparison with other sequences from the literature) [38,39]. The HA2 fusion sequence was attached to the linker GGG by either its N-terminal end for the peptide designated as HA2-NNLACC, or via its C-terminal end for CCALNN-HA2 (Table 1, sequences 2,3). Nanoparticles were functionalised with a mixture of capping peptides CALNN and one of either version of the HA2 peptides. Two ratios (4:1 and 1:1) of the CALNN: CCALNN-HA2 or CALNN: HA2-NNLACC peptides were used to form the gold nanoparticles self-assembled monolayers. HeLa cells were then incubated separately with one of the resulting four types of nanoparticles (10nm diameter, 6nM) for 4h, fixed and imaged by TEM. The electron microscope images presented in Fig. 4A-D (datasets available on figshare [72–75]) do not show significantly different behaviours. Indeed, neither of the attachment orientations of the HA2 peptide showed a cytosolic release of the nanoparticles (compare Fig. 4A to Fig. 4B and Fig. 4C to Fig. 4D), with most of the particles inside endosomes or at their vicinity. For both orientations, varying the quantity of HA2 peptides present on the gold core did not show an improvement of the quantity of free cytosolic nanoparticles. Overall, attaching the HA2 peptides onto the gold nanoparticles surface by its N- or C-terminal end, with a larger (50%) or a smaller (20%) quantity of HA2 functions per nanoparticle, does not provoke any change in terms of cytosol localisation. The nanoparticles remain in intact endolysosomes or near ruptured ones.


TAT and HA2 facilitate cellular uptake of gold nanoparticles but do not lead to cytosolic localisation.

Cesbron Y, Shaheen U, Free P, Lévy R - PLoS ONE (2015)

Attachment of HA2 to gold cores via the C-terminus versus N-terminus.HeLa cells were incubated for 4h with 10nm gold nanoparticles (6nM) capped with peptide monolayers of different compositions, fixed, and imaged by electron microscopy. Nanoparticle peptide monolayer compositions were: (A) 20% CCALNN-HA2 and 80% CALNN; (B) 20% HA2-NNLACC and 80% CALNN; (C) 50% CCALNN-HA2 and 50% CALNN; (D) 50% HA2-NNLACC and 50% CALNN. Arrowheads indicate particles around or outside endosomes. Additional images are available on figshare (10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383524&req=5

pone.0121683.g004: Attachment of HA2 to gold cores via the C-terminus versus N-terminus.HeLa cells were incubated for 4h with 10nm gold nanoparticles (6nM) capped with peptide monolayers of different compositions, fixed, and imaged by electron microscopy. Nanoparticle peptide monolayer compositions were: (A) 20% CCALNN-HA2 and 80% CALNN; (B) 20% HA2-NNLACC and 80% CALNN; (C) 50% CCALNN-HA2 and 50% CALNN; (D) 50% HA2-NNLACC and 50% CALNN. Arrowheads indicate particles around or outside endosomes. Additional images are available on figshare (10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852).
Mentions: The exact process by which HA2 destabilises membranes is not fully understood. This stems from a lack of precise structural data for the critical part of the HA2 sequence that undergoes conformational changes during the membrane fusion, due to failures at crystallising it (very high hydrophobicity at acidic pH) [41]. Han et al. identified the N–terminal domain of HA2 as critical for destabilisation of artificial model membranes. It was shown that all 20 residues of the fusion domain of HA2 were actively involved in the conformational changes necessary to insert deep into synthetic lipid bilayer membrane at acidic pH [42]. This process could be affected by the way the peptide is attached to the gold nanoparticle surface, e.g. attached to the gold core via either the N- or the C-terminal end. Two orientations of the HA2 peptide sequence (Table 1, sequences 2–3) were therefore compared to assess the influence of the direction of attachment to the gold core. These two sequences of HA2 peptide differ marginally from the sequence of HA2 peptide used previously (Table 1, sequence 1), but not enough to prevent the fusion process. Indeed, both sequences have been reported to show a consistent fusion activity (see Table A in S1 File for a comparison with other sequences from the literature) [38,39]. The HA2 fusion sequence was attached to the linker GGG by either its N-terminal end for the peptide designated as HA2-NNLACC, or via its C-terminal end for CCALNN-HA2 (Table 1, sequences 2,3). Nanoparticles were functionalised with a mixture of capping peptides CALNN and one of either version of the HA2 peptides. Two ratios (4:1 and 1:1) of the CALNN: CCALNN-HA2 or CALNN: HA2-NNLACC peptides were used to form the gold nanoparticles self-assembled monolayers. HeLa cells were then incubated separately with one of the resulting four types of nanoparticles (10nm diameter, 6nM) for 4h, fixed and imaged by TEM. The electron microscope images presented in Fig. 4A-D (datasets available on figshare [72–75]) do not show significantly different behaviours. Indeed, neither of the attachment orientations of the HA2 peptide showed a cytosolic release of the nanoparticles (compare Fig. 4A to Fig. 4B and Fig. 4C to Fig. 4D), with most of the particles inside endosomes or at their vicinity. For both orientations, varying the quantity of HA2 peptides present on the gold core did not show an improvement of the quantity of free cytosolic nanoparticles. Overall, attaching the HA2 peptides onto the gold nanoparticles surface by its N- or C-terminal end, with a larger (50%) or a smaller (20%) quantity of HA2 functions per nanoparticle, does not provoke any change in terms of cytosol localisation. The nanoparticles remain in intact endolysosomes or near ruptured ones.

Bottom Line: However, conflicting results have been reported on the extent of the cytosolic delivery achieved.To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery.While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Liverpool, Liverpool, United Kingdom; Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom; CNRS, UMR 6290, Institute of Genetics and Development of Rennes, Rennes, France; Université de Rennes 1, Université Européenne de Bretagne, Structure fédérative de recherche Biosit, Faculté de Médecine, Rennes, France.

ABSTRACT
The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

No MeSH data available.


Related in: MedlinePlus