Timothy M. Potter

Dendrimer-induced leukocyte procoagulant activity depends on particle size and surface charge

AIMS Thrombogenicity associated with the induction of leukocyte procoagulant activity (PCA) is a common complication in sepsis and cancer. Since nanoparticles are increasingly used for drug delivery, their interaction with coagulation systems is an important part of the safety assessment. The purpose of this study was to investigate the effects of nanoparticle physicochemical properties on leukocyte PCA, and to get insight into the mechanism of PCA induction. MATERIALS & METHODS A total of 12 formulations of polyamidoamine (PAMAM) dendrimers, varying in size and surface charge, were studied in vitro using recalcification time assay. RESULTS Irrespective of their size, anionic and neutral dendrimers did not induce leukocyte PCA in vitro. Cationic particles induced PCA in a size- and charge-dependent manner. The mechanism of PCA induction was similar to that of doxorubicin. Cationic dendrimers were also found to exacerbate endotoxin-induced PCA. CONCLUSION PAMAM dendrimer-induced leukocyte PCA depends on particle size, charge and density of surface groups.

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.

Assay to Detect Lipid Peroxidation upon Exposure to Nanoparticles

This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered to be an important component of biocompatibility screens. The products of lipid peroxidation, lipid hydroperoxides, and aldehydes, such as MDA, can be measured via a thiobarbituric acid reactive substances (TBARS) assay. In this assay, which can be performed in cell culture or in cell lysate, MDA combines with thiobarbituric acid (TBA) to form a fluorescent adduct that can be detected at an excitation wavelength of 530 nm and an emission wavelength of 550 nm. The results are then expressed as MDA equivalents, normalized to total cellular protein (determined by Bradford assay).

In Vitro Analysis of Nanoparticle Uptake by Macrophages Using Chemiluminescence

This chapter provides a protocol for qualitative evaluation of nanoparticle internalization by phagocytic cells such as macrophages. This protocol uses luminol chemiluminescence to detect nanoparticle uptake. This protocol provides a preliminary qualitative look at phagocytosis which should be confirmed by other techniques such as electron microscopy, confocal microscopy, or as applicable to a given nanoparticle sample.

Evaluation of cytotoxicity of nanoparticulate materials in porcine kidney cells and human hepatocarcinoma cells.

This chapter describes method for evaluation of nanomaterial cytotoxicity by examining effects on porcine kidney (LLC-PK1) and human cancerous liver cells (Hep G2). Several studies indicate that many nanoparticles are cleared from the body through the kidney or liver, making these organs good choices for target organ toxicity evaluation. In this standard, two separate metrics (MTT and LDH) provide complementary data, that can be used to identify interference.

Method for analysis of nanoparticle effects on cellular chemotaxis.

Chemotaxis is the phenomenon in which cells direct their movements in the presence of certain chemicals (chemoattractants or chemorepellents). Leukocyte recruitment (via chemotaxis) is an important component of the inflammatory response, both in physiological host defense and in a range of prevalent disorders that include an inflammatory component. Circulating leukocytes in the bloodstream migrate towards the site of inflammation in response to a complex network of proinflammatory signaling molecules (including cytokines, chemokines and prostaglandins). This chapter describes a method for rapid measure of the chemoattractant capacity of nanoparticulate materials. This method is an in vitro model for chemotaxis, in which promyelocytic leukemia cell migration through a filter is monitored using a fluorescent dye.

Detecting reactive oxygen species in primary hepatocytes treated with nanoparticles.

This chapter describes a protocol for testing nanoparticle formulations for reactive oxygen species generation in male Sprague-Dawley (SD) primary hepatocytes. The protocol utilizes the fluorescent redox active probe, dichlorofluorescein diacetate (DCFH-DA). Primary hepatocytes were chosen for this assay since they have greater metabolic activity than hepatocyte cell lines. This method extends previous standardized cytotoxicity methods for particulates by evaluating mechanisms of toxicity in potential target organ cells. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered an important component of biocompatibility screens.

Analysis of microbial contamination in nanoparticle formulations.

This chapter describes a procedure for quantitative determination of microbial contamination of a nanoparticle formulation. The protocol includes tests for yeast, mold, and bacteria using Millipore sampler devices. This approach is primarily intended to avoid contamination of cell cultures and transmitting potential microbial contaminants to animals in preclinical studies of efficacy, biodistribution, and toxicity. Other methods common to microbiology will likely work equally well.

Monitoring Glutathione Homeostasis in Nanoparticle-Treated Hepatocytes

This chapter describes a method for the analysis of human hepatocarcinoma cells (Hep G2 cells) for reduced and oxidized glutathione, following treatment with nanoparticle formulations. Glutathione is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) present intracellularly in millimolar concentrations and one of the primary cellular antioxidant defenses against oxidative stress. An increase in the relative amount of oxidized to reduced glutathione may be indicative of oxidative stress, while a decrease in the overall glutathione pool may be indicative of conjugative metabolism or impaired synthesis. The method presented in this chapter utilizes a colorimetric method for detection of reduced and oxidized glutathione.

Monitoring nanoparticle-treated hepatocarcinoma cells for apoptosis.

This chapter describes a method for monitoring nanoparticle treated human hepatocarcinoma cells (Hep G2) for apoptosis. The protocol utilizes a fluorescent method to determine the degree of caspase-3 activation.