Nanoparticle size and plasma binding profile contribute to a particle’s longevity in the bloodstream, which can have important consequences for therapeutic efficacy. In this study an approximate doubling in nanoparticle hydrodynamic size was observed upon in vitro incubation of 30- and 50-nm colloidal gold in human plasma. Plasma proteins that bind the surface of citrate-stabilized gold colloids have been identified. Effects of protein binding on the nanoparticle hydrodynamic size, elements of coagulation, and the complement system have been investigated. The difference in size measurements obtained from dynamic light scattering, electron microscopy, and scanning probe microscopy are also discussed.
Hemolysis (destruction of red blood cells) in vivo can lead to anemia, jaundice and other pathological conditions, therefore the hemolytic potential of all intravenously administered pharmaceuticals must be evaluated. Nanotechnology-derived devices and drug carriers are emerging as alternatives to conventional small-molecule drugs, and in vitro evaluation of their biocompatibility with blood components is a necessary part of early preclinical development. The small size and unique physicochemical properties of nanoparticles may cause their interactions with erythrocytes to differ from those observed for conventional pharmaceuticals, and may also cause interference with standardized in vitro tests. Separating true hemolytic responses from the false-positive or false-negative results caused by particle interference is important for correct interpretation of these tests. Here we describe validation of an in vitro assay for the analysis of nanoparticle hemolytic properties, and discuss observed nano-interferences with the assay. We propose alternative methods to avoid misleading results from nanoparticles, and discuss the potential relevance of nanoparticle in vitro hemolytic properties to in vivo systems.
Proteins bound to nanoparticle surfaces are known to affect particle clearance by influencing immune cell uptake and distribution to the organs of the mononuclear phagocytic system. The composition of the protein corona has been described for several types of nanomaterials, but the role of the corona in nanoparticle biocompatibility is not well established. In this study we investigate the role of nanoparticle surface properties (PEGylation) and incubation times on the protein coronas of colloidal gold nanoparticles. While neither incubation time nor PEG molecular weight affected the specific proteins in the protein corona, the total amount of protein binding was governed by the molecular weight of PEG coating. Furthermore, the composition of the protein corona did not correlate with nanoparticle hematocompatibility. Specialized hematological tests should be used to deduce nanoparticle hematotoxicity.
Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C60OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.
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