Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm) resonant laser scattering and Raman scattering cross-sections are too small to allow single nanoparticle observation. Nanodiamonds can however be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time-scales. In this work we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells . By blocking selectively different uptake processes we show that nanodiamonds enter cells mainly by endocytosis and converging data indicate that it is clathrin mediated. We also examine nanodiamonds intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule deliver

Cancer-selective, single agent chemoradiosensitising gold nanoparticles

Two nanometre gold nanoparticles (AuNPs), bearing sugar moieties and/or thiol-polyethylene glycol-amine (PEG-amine), were synthesised and evaluated for their in vitro toxicity and ability to radiosensitise cells with 220 kV and 6 MV X-rays, using four cell lines representing normal and cancerous skin and breast tissues. Acute 3 h exposure of cells to AuNPs, bearing PEG-amine only or a 50:50 ratio of alpha-galactose derivative and PEG-amine resulted in selective uptake and toxicity towards cancer cells at unprecedentedly low nanomolar concentrations. Chemotoxicity was prevented by co-administration of N-acetyl cysteine antioxidant, or partially prevented by the caspase inhibitor Z-VAD-FMK. In addition to their intrinsic cancer-selective chemotoxicity, these AuNPs acted as radiosensitisers in combination with 220 kV or 6 MV X-rays. The ability of AuNPs bearing simple ligands to act as cancer-selective chemoradiosensitisers at low concentrations is a novel discovery that holds great promise in developing low-cost cancer nanotherapeutics

Gold Nanoparticle-Based Surface-Enhanced Raman Scattering for Noninvasive Molecular Probing of Embryonic Stem Cell Differentiation

This study reports the use of gold nanoparticle-based surface-enhanced Raman scattering (SERS) for probing the differentiation of mouse embryonic stem (mES) cells, including undifferentiated single cells, embryoid bodies (EBs), and terminally differentiated cardiomyocytes. Gold nanoparticles (GNPs) were successfully delivered into all 3 mES cell differentiation stages without affecting cell viability or proliferation. Transmission electron microscopy (TEM) confirmed the localization of GNPs inside the following cell organelles: mitochondria, secondary lysosome, and endoplasmic reticulum. Using bright- and dark-field imaging, the bright scattering of GNPs and nanoaggregates in all 3 ES cell differentiation stages could be visualized. EB (an early differentiation stage) and terminally differentiated cardiomyocytes both showed SERS peaks specific to metabolic activity in the mitochondria and to protein translation (amide I, amide II, and amide III peaks). These peaks have been rarely identified in undifferentiated single ES cells. Spatiotemporal changes observed in the SERS spectra from terminally differentiated cardiomyocyte tissues revealed local and dynamic molecular interactions as well as transformations during ES cell differentiation

Decoupling the shape parameter to assess gold nanorod uptake by mammalian cells

The impact of nanoparticles (NPs) upon biological systems can be fundamentally associated with their physicochemical parameters. A further often-stated tenet is the importance of NP shape on rates of endocytosis. However, given the convoluted parameters concerning the NP–cell interaction, it is experimentally challenging to attribute any findings to shape alone. Herein we demonstrate that shape, below a certain limit, which is specific to nanomedicine, is not important for the endocytosis of spherocylinders by either epithelial or macrophage cells in vitro. Through a systematic approach, we reshaped a single batch of gold nanorods into different aspect ratios resulting in near-spheres and studied their cytotoxicity, (pro-)inflammatory status, and endocytosis/exocytosis. It was found that on a length scale of ∼10–90 nm and at aspect ratios less than 5, NP shape has little impact upon their entry into either macrophages or epithelial cells. Conversely, nanorods with an aspect ratio above 5 were preferentially endocytosed by epithelial cells, whereas there was a lack of shape dependent uptake following exposure to macrophages in vitro. These findings have implications both in the understanding of nanoparticle reshaping mechanisms, as well as in the future rational design of nanomaterials for biomedical applications

Intracellular SERS nanoprobes for distinction of different neuronal cell types.

Distinction between closely related and morphologically similar cells is difficult by conventional methods especially without labeling. Using nuclear-targeted gold nanoparticles (AuNPs) as intracellular probes we demonstrate the ability to distinguish between progenitor and differentiated cell types in a human neuroblastoma cell line using surface-enhanced Raman spectroscopy (SERS). SERS spectra from the whole cell area as well as only the nucleus were analyzed using principal component analysis that allowed unambiguous distinction of the different cell types. SERS spectra from the nuclear region showed the developments during cellular differentiation by identifying an increase in DNA/RNA ratio and proteins transcribed. Our approach using nuclear-targeted AuNPs and SERS imaging provides label-free and noninvasive characterization that can play a vital role in identifying cell types in biomedical stem cell research

Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems

The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level

Site-controlled quantum dots fabricated using an atomic-force microscope assisted technique

An atomic-force microscope assisted technique is developed to control the position and size of self-assembled semiconductor quantum dots (QDs). Presently, the site precision is as good as ± 1.5 nm and the size fluctuation is within ± 5% with the minimum controllable lateral diameter of 20 nm. With the ability of producing tightly packed and differently sized QDs, sophisticated QD arrays can be controllably fabricated for the application in quantum computing. The optical quality of such site-controlled QDs is found comparable to some conventionally self-assembled semiconductor QDs. The single dot photoluminescence of site-controlled InAs/InP QDs is studied in detail, presenting the prospect to utilize them in quantum communication as precisely controlled single photon emitters working at telecommunication bands

Comparative Toxicity of Nanoparticulate CuO and ZnO to Soil Bacterial Communities

The increasing industrial application of metal oxide Engineered Nano-Particles (ENPs) is likely to increase their environmental release to soils. While the potential of metal oxide ENPs as environmental toxicants has been shown, lack of suitable control treatments have compromised the power of many previous assessments. We evaluated the ecotoxicity of ENP (nano) forms of Zn and Cu oxides in two different soils by measuring their ability to inhibit bacterial growth. We could show a direct acute toxicity of nano-CuO acting on soil bacteria while the macroparticulate (bulk) form of CuO was not toxic. In comparison, CuSO4 was more toxic than either oxide form. Unlike Cu, all forms of Zn were toxic to soil bacteria, and the bulk-ZnO was more toxic than the nano-ZnO. The ZnSO4 addition was not consistently more toxic than the oxide forms. Consistently, we found a tight link between the dissolved concentration of metal in solution and the inhibition of bacterial growth. The inconsistent toxicological response between soils could be explained by different resulting concentrations of metals in soil solution. Our findings suggested that the principal mechanism of toxicity was dissolution of metal oxides and sulphates into a metal ion form known to be highly toxic to bacteria, and not a direct effect of nano-sized particles acting on bacteria. We propose that integrated efforts toward directly assessing bioavailable metal concentrations are more valuable than spending resources to reassess ecotoxicology of ENPs separately from general metal toxicity