Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells

Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled fibroblast growth factor 2 (FGF2) bound to heparan sulfate in the pericellular matrix of live fibroblast cells. The data are consistent with previous single nanoparticle tracking studies of the diffusion of FGF2 on these cells. Importantly, the data reveal faster FGF2 movement, previously inaccessible by photothermal tracking, and suggest that inhomogeneity in the distribution of bound FGF2 is dynamic

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

Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity

Cytokines and growth factors cross-link heparan sulfate

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors

Biocompatible Peptide-Coated Ultrasmall Superparamagnetic Iron Oxide Nanoparticles for In Vivo Contrast-Enhanced Magnetic Resonance Imaging

The biocompatibility and performance of reagents for in vivo contrast-enhanced magnetic resonance imag-ing are essential for their translation to the clinic. The quality of the surface coating of nanoparticle-based MRI contrast agents, such as ultra-small superparamagnetic iron oxide nanoparticles (USPIONs), is criti-cal to ensure high colloidal stability in biological environments, improved magnetic performance and dis-persion in circulatory fluids and tissues. Herein, we report the design of a library of 21 peptides and lig-ands and identify highly stable self-assembled monolayers on the USPIONs surface. A total of 86 differ-ent peptide coated USPIONs are prepared and selected using several stringent criteria, e.g., stability against electrolyte-induced aggregation in physiological conditions, prevention of non-specific binding to cells, absence of cellular toxicity and contrast-enhanced in vivo MRI. The bis-phosphorylated peptide 2PG-S∗VVVT-PEG4-ol provides highest biocompatibility and performance for USPIONs, with no de-tectable toxicity or adhesion to live cells. The 2PG-S∗VVVT-PEG4-ol coated USPIONs show enhanced magnetic resonance properties, r1 (2.4 mM-1.s-1) and r2 (217.8 mM-1.s-1) relaxivities, and greater r2/r1 relaxivity ratios (>90), when compared to commercially available MRI contrast agents. Furthermore, we demonstrate the utility of 2PG-S∗VVVT-PEG4-ol coated USPIONs as a T2 contrast agent for in vivo MRI applica-tions. High contrast enhancement of the liver is achieved as well as detection of liver tumors, with signifi-cant improvement of the contrast-to-noise ratio of tumor-to-liver contrast. It is envisaged that the reported peptide coated USPIONs have the potential to allow for the specific targeting of tumors, and hence early detection of cancer by MRI

Heparan sulfate determines the modes of diffusion of fibroblast growth factor 2 within the pericellular matrix

Heparan sulfate (HS) chains of proteoglycans (PG) are major components of pericellular and extracellular matrices. They regulate transport, gradient formation and effector functions of >250 proteins central to animal cell communication: contrasting mechanisms proposed for this range from cyclic protein-HS association/dissociation to protein sequestration by HS that requires enzymatic liberation. Previous measurements averaging across large numbers of protein molecules interacting with HS do not resolve these explanations. To address this, we stoichiometrically labelled single molecules of archetypal HS-binding morphogen, fibroblast growth factor 2 (FGF2) with gold nanoparticles and used photothermal heterodyne imaging to track individual molecules in the pericellular matrix. We show individual FGF2 molecules undergo mainly local motion in the matrix (~65 nm radius) from which they can escape by diffusion (slow, fast or directed). Similar molecular motion persists when membrane PG movement is impeded by cell fixation: this shows FGF2 can slide along >10 successive HS chains driven by thermal energy and the observed heterogeneously located binding sites on HS chains. We conclude morphogen transport in pericellular matrix involves multiple mechanisms: sliding along HS chains, local transfer between binding sites on neighbouring chains and diffusion of the PG core protein within the membrane, which can be cytoskeleton drive

Monovalent Maleimide Functionalization of Gold Nanoparticles Via Copper-Free Click Chemistry

A single maleimide was installed onto the self-assembled monolayer of gold nanoparticles by copper-free click chemistry. Simple covalent biofunctionalisation is demonstrated by coupling fibroblast growth factor 2 and an oligosaccharide in a 1 : 1 stoichiometry by thiol-Michael addition

Heparan sulfate determines the modes of diffusion of fibroblast growth factor 2 within the pericellular matrix

Heparan sulfate (HS) chains of proteoglycans (PG) are major components of pericellular and extracellular matrices. They regulate transport, gradient formation and effector functions of >250 proteins central to animal cell communication: contrasting mechanisms proposed for this range from cyclic protein-HS association/dissociation to protein sequestration by HS that requires enzymatic liberation. Previous measurements averaging across large numbers of protein molecules interacting with HS do not resolve these explanations. To address this, we stoichiometrically labelled single molecules of archetypal HS-binding morphogen, fibroblast growth factor 2 (FGF2) with gold nanoparticles and used photothermal heterodyne imaging to track individual molecules in the pericellular matrix. We show individual FGF2 molecules undergo mainly local motion in the matrix (~65 nm radius) from which they can escape by diffusion (slow, fast or directed). Similar molecular motion persists when membrane PG movement is impeded by cell fixation: this shows FGF2 can slide along >10 successive HS chains driven by thermal energy and the observed heterogeneously located binding sites on HS chains. We conclude morphogen transport in pericellular matrix involves multiple mechanisms: sliding along HS chains, local transfer between binding sites on neighbouring chains and diffusion of the PG core protein within the membrane, which can be cytoskeleton driven</p