Paul Free

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation.

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.

Cathepsin L Digestion of Nanobioconjugates upon Endocytosis

Understanding the dynamic fate and interactions of bioconjugated nanoparticles within living cells and organisms is a prerequisite for their use as in situ sensors or actuators. While recent research has provided indications on the effect of size, shape, and surface properties of nanoparticles on their internalization by living cells, the biochemical fate of the nanoparticles after internalization has been essentially unknown. Here we show that, upon internalization in a wide range of mammalian cells, biological molecules attached to the nanoparticles are degraded within the endosomal compartments through peptide cleavage by the protease cathepsin L. Importantly, using bioinformatics tools, we show that cathepsin L is able to cleave more than a third of the human proteome, indicating that this degradation process is likely to happen to most nanoparticles conjugated with peptides and proteins and cannot be ignored in the design of nanomaterials for intracellular applications. Preservation of the bioconjugates can be achieved by a combination of cathepsin inhibition and endosome disruption.

Identification of Heparin-binding Sites in Proteins by Selective Labeling

Heparan sulfate proteoglycans are key regulators of complex molecular networks due to the interaction of their sugar chains with a large number of partner proteins, which in humans number more than 200 (Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008) The heparanome and regulation of cell function: structures, functions and challenges. Front. Biosci. 13, 4309–4338). We developed a method to selectively label residues involved in heparin binding that matches the requirements for medium/high throughput applications called the “Protect and Label” strategy. This is based on the protection against chemical modification given by heparin/heparan sulfate to the residues located in the heparin-binding site. Thus, analysis of fibroblast growth factor-2 bound to heparin and incubated with N-hydroxysuccinimide acetate showed that lysines involved in the sugar binding are protected against chemical modification. Moreover following release from heparin, the protected lysine side chains may be specifically labeled with N-hydroxysuccinimide biotin. After protein digestion, the biotinylated peptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptides obtained from three well characterized heparin-binding proteins with very different heparin-binding sites, fibroblast growth factor-2, platelet factor-4, and pleiotrophin demonstrates the success of this new approach, which thus provides a rapid and reliable procedure to identify heparin-binding sites.

Stripy nanoparticles revisited

In May 2004, Jackson et al. published an article entitled “Spontaneous assembly of subnanometre-ordered domains in the ligand shell of monolayer-protected nanoparticles”.1 This was to become the first of a series which now counts over ten research articles.1, 2 All of these are based on the existence of “stripy” nanoparticles, where the stripes are constituted by the self-organization of two different thiolated ligands. A number of unusual and exciting properties are attributed to the nanoscale organization of the ligands. Thus, stripy nanoparticles are reported as being “extremely effective in avoiding non-specific adsorption of a variety of proteins”,1 having the ability to “penetrate the plasma membrane without bilayer disruption”2j and having poles which are particularly reactive and can be selectively addressed to obtain divalent nanoparticles.2c This series of articles and the corresponding structure–property relationships are important because of their direct impact on our understanding of several of the key contemporary problems in the field of nanoscience. The latter include the characterization of nanomaterials with sub-nanometer resolution,3 the possibility of controlling the self-organization of ligands on gold nanoparticles,4 the understanding of nanoparticle–biomolecule and nanoparticle–cell interactions,5 and the intracellular delivery of nanoparticles.6 The proposed stripy structure is based on scanning tunneling microscopy (STM) images which have not yet been reproduced by other groups to date. Our interest lies in nanoparticle surface engineering7 and the interaction of nanoparticles with living cells.6b Carefully following the published results for producing stripy nanoparticles, we failed to substantiate a number of the claims made about their properties, so in the first part of this paper we critically revisit the published evidence for stripiness and in the second part we present our own results regarding their physicochemical properties.

PEGylation modulates the interfacial kinetics of proteases on peptide-capped gold nanoparticles

Fluorescence unquenching measurements of protease-dependent release of fluorescent biomolecules from peptide-capped gold nanoparticles reveal the effect of the monolayer composition on enzyme kinetics.

Features of Thiolated Ligands Promoting Resistance to Ligand Exchange in Self-Assembled Monolayers on Gold Nanoparticles

An important feature necessary for biological stability of gold nanoparticles is resistance to ligand exchange. Here, we design and synthesize self-assembled monolayers of mixtures of small ligands on gold nanoparticles promoting high resistance to ligand exchange. We use as ligands short thiolated peptidols, e.g. H-CVVVT-ol, and ethylene glycol terminated alkane thiols (HS-C11-EG4). We present a straightforward method to evaluate the relative stability of each ligand shell against ligand exchange with small thiolated molecules. The results show that a ligand with a ‘thin’ stem, such as HS-C11-EG4, is an important feature to build a highly packed self-assembled monolayer and provide high resistance to ligand exchange. The greatest resistance to ligand exchange was found for the mixed ligand shells of the pentapeptidols H-CAVLT-ol or H-CAVYT-ol and the ligand HS-C11-EG4 at 30:70 (mole/mole). Mixtures of ligands of very different diameters, such as the peptidol H-CFFFY-ol and the ligand HS-C11-EG4, provide only a slightly lower stability against ligand exchange. These ligand shells are thus likely to be suitable for long-term use in biological environments. The method developed here provides a rapid screening tool to identify nanoparticles likely to be suitable for use in biological and biomedical applications.

Synthesis of Silver Nanoparticles with Monovalently Functionalized Self-Assembled Monolayers

The importance of having nanoparticles that are soluble, stable, and that have no non-specific binding is often overlooked, but essential for their use in biology. This is particularly prominent with silver nanoparticles that are susceptible to the effects of aggregation and metal-surface reactivity. Here we use a combination of several small peptidols and short alkanethiol ethylene glycol ligands to develop a ligand shell that is reasonably resistant to ligand exchange and non-specific binding to groups common in biological molecules. The stability of the nanoparticles is not affected by the inclusion of a functional ligand, which is done in the same preparative step. The stoichiometry of the nanoparticles is controlled, such that monofunctional silver nanoparticles can be obtained. Two different sets of nanoparticles, functionalized with either Tris-nitrilotriacetic acid or a hexa-histidine peptide sequence, readily form dimers/oligomers, depending on their stoichiometry of functionalization.

Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently available approaches and provides a new route to design tailored and well-controlled hybrid nanoparticles.

High colloidal stability of gold nanorods coated with a peptide-ethylene glycol: Analysis by cyanide-mediated etching and nanoparticle tracking analysis.

The stability of gold nanorods was assessed following coating with various charged or uncharged ligands, mostly peptides. Highly stable monodispersed gold nanorods were obtained by coating CTAB-stabilized gold nanorods with a pentapeptide with C-terminal ethylene glycol units (peptide-EG). UV-vis spectroscopy of these nanorods suspended in saline solutions indicated no signs of aggregation, and they were easily purified using size-exclusion chromatography. A more stringent measure of nanorod stability involved observing changes in the UV-vis absorbance of gold nanorods subjected to etching with cyanide. The λmax absorbance of peptide-EG coated nanorods red-shifted in etchant solution. The hypothesis that changes in the nanorod aspect ratio led to this red-shift was confirmed by TEM analysis, which showed pit formation along the transverse axis. The etching process was followed in solution using nanoparticle tracking analysis. The red-shift was shown to occur while the particles remained mono-dispersed, and so was not due to aggregation. Adding both etchant solution and peptide-EG to the nanorods was further shown to allow modulation of the Δλmax red-shift and increase the etchant resistance of peptide-EG nanorods. Thus, very stable gold nanorods can be produced using the peptide-EG coating approach and their optical properties modulated with etchant.

Photothermal Laser Material Interactions - From the Sledgehammer to Nano-GPS

In this chapter we will summarize the main photothermal, photoacoustic and photomechanical effects of coupling a laser beam into a material from the absorption of the laser light to the deactivation of vibrationally and electronically excited states. Some methods to estimate the resulting temperature rise will be discussed and the resulting pressure increase in the heated area explained. The relaxation of both pressure and thermal transients will be explored and several methods described, such as pump-probe spectroscopy and imaging techniques, which can be used to investigate the dynamics of the relaxation pathways. We will explain how photothermal effects can manifest as optical effects. Finally, we will describe how we can harness photothermally induced optical changes to provide a new methodology in bioimaging involving indestructible 5-10 nm noble metal nanoparticles that can be observed using photothermal tracking microscopy for unprecedented periods of time in live cell imaging.