Stephan T. Stern

Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

Induction of Autophagy in Porcine Kidney Cells by Quantum Dots: A Common Cellular Response to Nanomaterials?

Quantum dots (QDs) are being investigated as novel in vivo imaging agents. The leaching of toxic metals from these QDs in biological systems is of great concern. This study compared the cytotoxic mechanisms of two QD species made of different core materials (cadmium selenide [CdSe] vs. indium gallium phosphide [InGaP]) but similar core sizes (5.1 vs. 3.7 nm) and surface compositions (both ZnS capped, lipid-coated and pegylated). The CdSe QD was found to be 10-fold more toxic to porcine renal proximal tubule cells (LLC-PK1) than the InGaP QD on a molar basis, as determined by MTT assay (48 h IC(50) 10nM for CdSe vs. 100nM for InGaP). Neither of the QD species induced appreciable oxidative stress, as determined by lipid peroxide and reduced glutathione content, suggesting that toxicity was not metal associated. In agreement, treatment of cells with CdSe QDs was not associated with changes in metallothionein-IA (MT-IA) gene expression or Cd-associated caspase 3 enzyme activation. By contrast, incubation of the LLC-PK1 cells with the InGaP QD resulted in a dramatic increase in MT-IA expression by 21- and 43-fold, at 8 and 24 h, respectively. The most remarkable finding was evidence of extensive autophagy in QD-treated cells, as determined by Lysotracker Red dye uptake, TEM, and LC3 immunobloting. Autophagy induction has also been described for other nanomaterials and may represent a common cellular response. These data suggest that QD cytotoxicity is dependent upon properties of the particle as a whole, and not exclusively the metal core materials.

Fullerenol Cytotoxicity in Kidney Cells is Associated with Cytoskeleton Disruption, Autophagic Vacuole Accumulation, and Mitochondrial Dysfunction

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.

Translational considerations for cancer nanomedicine

There are many important considerations during preclinical development of cancer nanomedicines, including: 1) unique aspects of animal study design; 2) the difficulties in evaluating biological potency, especially for complex formulations; 3) the importance of analytical methods that can determine platform stability in vivo, and differentiate bound and free active pharmaceutical ingredient (API) in biological matrices; and 4) the appropriateness of current dose scaling techniques for estimation of clinical first-in-man dose from preclinical data. Biologics share many commonalities with nanotechnology products with regard to complexity and biological attributes, and can, in some cases, provide context for dealing with these preclinical issues. In other instances, such as the case of in vivo stability analysis, new approaches are required. This paper will discuss the significance of these preclinical issues, and present examples of current methods and best practices for addressing them. Where possible, these recommendations are justified using the existing regulatory guidance literature.

A Novel Nanoparticulate Formulation of Arsenic Trioxide with Enhanced Therapeutic Efficacy in a Murine Model of Breast Cancer

Purpose: The clinical success of arsenic trioxide (As2O3) in hematologic malignancies has not been replicated in solid tumors due to poor pharmacokinetics and dose-limiting toxicity. We have developed a novel nanoparticulate formulation of As2O3 encapsulated in liposomal vesicles or “nanobins” [(NB(Ni,As)] to overcome these hurdles. We postulated that nanobin encapsulation of As2O3 would improve its therapeutic index against clinically aggressive solid tumors, such as triple-negative breast carcinomas. Experimental Design: The cytotoxicity of NB(Ni,As), the empty nanobin, and free As2O3 was evaluated against a panel of human breast cancer cell lines. The plasma pharmacokinetics of NB(Ni,As) and free As2O3 were compared in rats to measure drug exposure. In addition, the antitumor activity of these agents was evaluated in an orthotopic model of human triple-negative breast cancer. Results: The NB(Ni,As) agent was much less cytotoxic in vitro than free As2O3 against a panel of human breast cancer cell lines. In contrast, NB(Ni,As) dramatically potentiated the therapeutic efficacy of As2O3 in vivo in an orthotopic model of triple-negative breast cancer. Reduced plasma clearance, enhanced tumor uptake, and induction of tumor cell apoptosis were observed for NB(Ni,As). Conclusions: Nanobin encapsulation of As2O3 improves the pharmacokinetics and antitumor efficacy of this cytotoxic agent in vivo. Our findings demonstrate the therapeutic potential of this nanoscale agent and provide a foundation for future clinical studies in breast cancer and other solid tumors. Clin Cancer Res; 16(14); 3607–17. ©2010 AACR.

Synergistic Combination Therapy with Nanoliposomal C6-Ceramide and Vinblastine is Associated with Autophagy Dysfunction in Hepatocarcinoma and Colorectal Cancer Models

Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6-ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with C6-ceramide. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagy mechanism.

Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct.

The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.

Autophagy Monitoring Assay: Qualitative Analysis of MAP LC3-I to II Conversion by Immunoblot

Lysosomal dysfunction is a recognized toxic mechanism for xenobiotics, which can result in various pathological states. There is concern that nanoparticles, in particular, may cause lysosomal pathologies, since they are likely to accumulate within lysosomes. Dysregulation of the autophagy-lysosomal degradation pathway is an example of lysosomal dysfunction associated with exposure to some nanomaterials. Here, we present a method to monitor autophagy by measurement the autophagosome marker LC3-II, a phosphatidylethanolamine (PE)-conjugated form of microtubule-associated protein 1 light chain 3-I (MAP LC3-I). As other conditions could potentially result in LC3-II expression, treatment-related changes in expression should be further evaluated by morphological assessment, using techniques such as electron microscopy, to confirm autophagosome involvement.

Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics

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Monitoring lysosomal activity in nanoparticle-treated cells.

Certain nanoparticles have been shown to accumulate within lysosome and hence may cause lysosomal pathologies such as phospholipidosis, lysosomal overload, and autophagy. This chapter describes a method for evaluation of lysosomal activity in porcine kidney cells (LLC-PK1) after exposure to nanoparticles. This method uses the accumulation of a cationic fluorescent dye (LysoTracker Red) in acidic cellular compartments as an indicator of total lysosome content. The lysotracker signal is normalized to the signal from a thiol-reactive dye which is proportional to the total number of viable cells.