Angela O. Choi

Quantum dot-induced epigenetic and genotoxic changes in human breast cancer cells

The staggering array of nanotechnological products, found in our environment and those applicable in medicine, has stimulated a growing interest in examining their long-term impact on genetic and epigenetic processes. We examined here the epigenomic and genotoxic response to cadmium telluride quantum dots (QDs) in human breast carcinoma cells. QD treatment induced global hypoacetylation implying a global epigenomic response. The ubiquitous responder to genotoxic stress, p53, was activated by QD challenge resulting in translocation of p53, with subsequent upregulation of downstream targets Puma and Noxa. Consequential decrease in cell viability was in part prevented by the p53 inhibitor pifithrin-α, suggesting that p53 translocation contributes to QD-induced cytotoxicity. These findings suggest three levels of nanoparticle-induced cellular changes: non-genomic, genomic and epigenetic. Epigenetic changes may have long-term effects on gene expression programming long after the initial signal has been removed, and if these changes remain undetected, it could lead to long-term untoward effects in biological systems. These studies suggest that aside from genotoxic effects, nanoparticles could cause more subtle epigenetic changes which merit thorough examination of environmental nanoparticles and novel candidate nanomaterials for medical applications.

Quantum dot-induced cell death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells.

BackgroundNeuroblastoma, a frequently occurring solid tumour in children, remains a therapeutic challenge as existing imaging tools are inadequate for proper and accurate diagnosis, resulting in treatment failures. Nanoparticles have recently been introduced to the field of cancer research and promise remarkable improvements in diagnostics, targeting and drug delivery. Among these nanoparticles, quantum dots (QDs) are highly appealing due to their manipulatable surfaces, yielding multifunctional QDs applicable in different biological models. The biocompatibility of these QDs, however, remains questionable.ResultsWe show here that QD surface modifications with N-acetylcysteine (NAC) alter QD physical and biological properties. In human neuroblastoma (SH-SY5Y) cells, NAC modified QDs were internalized to a lesser extent and were less cytotoxic than unmodified QDs. Cytotoxicity was correlated with Fas upregulation on the surface of treated cells. Alongside the increased expression of Fas, QD treated cells had increased membrane lipid peroxidation, as measured by the fluorescent BODIPY-C11 dye. Moreover, peroxidized lipids were detected at the mitochondrial level, contributing to the impairment of mitochondrial functions as shown by the MTT reduction assay and imaged with confocal microscopy using the fluorescent JC-1 dye.ConclusionQD core and surface compositions, as well as QD stability, all influence nanoparticle internalization and the consequent cytotoxicity. Cadmium telluride QD-induced toxicity involves the upregulation of the Fas receptor and lipid peroxidation, leading to impaired neuroblastoma cell functions. Further improvements of nanoparticles and our understanding of the underlying mechanisms of QD-toxicity are critical for the development of new nanotherapeutics or diagnostics in nano-oncology.

Lipopolysaccharide-QD micelles induce marked induction of TLR2 and lipid droplet accumulation in olfactory bulb microglia.

The intranasal entry of biological and artificial nanoparticles can induce inflammatory responses both locally and more widely in surrounding tissues. The aim of this study was to assess the microglia activation induced by nanoparticles with different surfaces in (i) a transgenic mouse (Toll-like receptor (TLR)-2-luciferase (Luc) reporter) which allowed the biophotonic imaging of microglial activation/innate immune response after intranasal delivery of nanoparticles and (ii) in microglial dispersed cells in vitro. Cadmium selenide nanoparticles (quantum dots, QD), surface-exchanged with lipopolysaccharide (LPS) to form micelles, were tested to assess microglia activation and lipid droplet formation in both model systems. In vivo imaging revealed a robust increase in the extent of microglial activation/TLR2 response, initially in the olfactory bulb, but also in other more caudal brain regions. The increased TLR2 expression was complemented with enhanced CD68 expression in activated microglia in the same regions. Intense in vitro microglial activation by LPS-QD micelles was accompanied by a significant enhancement of nitric oxide production and formation of large lipid droplets, suggesting the possibility of this organelle acting as an inflammatory biomarker in response to nanoparticles, and not simply as a storage site in fat tissues.

Caspase-1 Activity in Microglia Stimulated by Pro-Inflammagen Nanocrystals

Although caspase-1 is a key participant in inflammation, there is no sensitive assay to measure its enzymatic activity in real time in cells or animals. Here we describe a nanosensor for caspase-1 ratiometric measurements, consisting of a rhodamine-labeled, caspase-1 cleavable peptide linked to quantum dots (QDs). Microglia cells were stimulated by lipopolysaccharide (LPS) and by hybrid nanoparticles LPS-QDs. These stimuli activated caspase-1 in microglia monolayers and in the mouse brain, while a selected caspase inhibitor markedly reduced it. LPS-QDs entered into the lysosomal compartment and led to an enlargement of these cellular organelles in the exposed microglia. Both lysosomal swelling and mitochondrial impairment contributed to caspase-1 activation and to the consequent interleukin-1β release. The results from these studies highlight how the unique properties of QDs can be used to create versatile biotools in the study of inflammation in real time in vivo.

Probing and preventing quantum dot-induced cytotoxicity with multimodal α-lipoic acid in multiple dimensions of the peripheral nervous system

AIM Toxicity of nanoparticles developed for biomedical applications is extensively debated as no uniform guidelines are available for studying nanomaterial safety, resulting in conflicting data obtained from different cell types. This study demonstrates the varied toxicity of a selected type of nanoparticle, cadmium telluride quantum dots (QDs), in three increasingly complex cell models of the peripheral nervous system. MATERIALS & METHODS QD-induced cytotoxicity was assessed via cell viability assays and biomarkers of subcellular damage in PC12 cells and mixed primary dispersed dorsal root ganglia (DRG) cultures. Morphological analysis of neurite outgrowth was used to determine the viability of axotomized DRG explant cultures. RESULTS & DISCUSSION Cadmium telluride QDs and their core metals exert different degrees of toxicity in the three cell models, the primary dispersed DRGs being the most susceptible. alpha-lipoic acid is an effective, multimodal, cytoprotective agent that can act as an antioxidant, metal chelator and QD-surface modifier in these cell systems. CONCLUSION Complex multicellular model systems, along with homogenous cell models, should be utilized in standard screening and monitoring procedures for evaluating nanomaterial safety.

Minocycline Block Copolymer Micelles and their Anti-Inflammatory Effects on Microglia

MH, a semisynthetic tetracycline antibiotic with promising neuroprotective properties, was encapsulated into PIC micelles of CMD-PEG as a potential new formulation of MH for the treatment of neuroinflammatory diseases. PIC micelles were prepared by mixing solutions of a Ca(2+)/MH chelate and CMD-PEG copolymer in a Tris-HCl buffer. Light scattering and (1)H NMR studies confirmed that Ca(2+)/MH/CMD-PEG core-corona micelles form at charge neutrality having a hydrodynamic radius approximately 100 nm and incorporating approximately 50 wt.-% MH. MH entrapment in the micelles core sustained its release for up to 24 h under physiological conditions. The micelles protected the drug against degradation in aqueous solutions at room temperature and at 37 degrees C in the presence of FBS. The micelles were stable in aqueous solution for up to one month, after freeze drying and in the presence of FBS and BSA. CMD-PEG copolymers did not induce cytotoxicity in human hepatocytes and murine microglia (N9) in concentrations as high as 15 mg x mL(-1) after incubation for 24 h. MH micelles were able to reduce the inflammation in murine microglia (N9) activated by LPS. These results strongly suggest that MH PIC micelles can be useful in the treatment of neuroinflammatory disorders.

Intranasal Fluorescent Nanocrystals for Longitudinal In Vivo Evaluation of Cerebral Microlesions

Current neuroimaging techniques in experimental medicine and clinical diagnosis are limited by low resolution and restricted image depth. Fluorescence in vivo imaging using near-infrared-emitting nanostructures, including nanocrys- tal quantum dots (QDs) can overcome these limitations. The objective of the present study was to establish if nanocrystals are suitable for repeated live imaging of deep structures (500 �m) in the intact animal. Intranasal instillation of QDs (5 �L) in mice with unilateral cortical microlesions resulted in marked QD accumulation at the microlesion site in the brain. Glial cell activation in response to the local devascularization played a key role in the uptake of QDs. The majority of QDs were taken up by activated microglia, whereas astroglia played a smaller role in this process. Progression and re- gression of the lesion upon therapeutic interventions were determined in real-time. Intranasal administration of anti- inflammatory nanotherapeutics (micelle-incorporated nimodipine or minocycline) was effective in preventing lesion pro- gression as evidenced by the smaller lesion volumes compared to the untreated controls. Moreover, lesion reduction was accompanied by significantly improved motor function. Near-infrared fluorescence imaging using nanocrystals is a valu- able addition to current neuropathological methods for the diagnosis of cerebral microlesions and eventually other neu- rodegenerative diseases, both in experimental models and eventually in humans.

Applications of quantum dots in biomedicine

Research and development in nanotechnology has become an increasingly popular trend in the last 5 years as the demand and production of nanometer-sized materials continue to grow. Nanotechnology is an area of research encompassing multidisciplinary studies (including chemistry, physics, engineering, and biotechnology), and has diverse applications in agriculture, automobile, clothing, defense and more recently, biology and biomedicine [1], [2]. Among many different nanotechnological products, quantum dots (QD) have gained a lot of popularity as imaging probes in biology due to their very special physico-chemical and optical properties [3], [4]. They are stable, highly fluorescent, tunable and can be functionalized via surface modifications. Despite the numerous ongoing studies on QD synthesis to improve their physical properties, the biological effects of QDs are poorly investigated. Thus far, it is known that QD biocompatibility is largely dependent on their size, surface charge, core and surface materials [2]. Currently, extensive studies on the interactions (or interference) of QDs with cellular processes are under investigation in many scientific centers.

Quantum Dots for Imaging Neural Cells In Vitro and In Vivo

Quantum dots (QDs) have been used for optical imaging of neural cells in vitro and in vivo. This chapter lists the basic materials, instrumentation and step-by-step procedures to image live microglia cells and to show the functional and biochemical changes in microglia exposed to QDs. Details are also provided for the real-time imaging of cerebral ischemic lesions in animals and for the assessment of lesion reduction after therapeutic interventions. Microglia are brain cells which detect, internalize, and eliminate particulate matter, thereby maintaining homeostasis in the central nervous system. Although the protocols for imaging microglia shown here are developed for QDs without specific ligands or antibodies, the principles are the same for imaging other QDs.