Application of Cryopreserved Human Hepatocytes in Trichloroethylene Risk Assessment: Relative Disposition of Chloral Hydrate to Trichloroacetate and Trichloroethanol

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common ground-water contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver by cytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by aldehyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) by alcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymorphisms are known to affect the elimination of ethanol. It is therefore possible that polymorphisms in CH metabolism will yield subpopulations with greater than expected TCA formation with associated enhanced risk of liver tumors after TCE exposure. METHODS: The present studies were undertaken to determine the feasibility of using commercially available, cryogenically preserved human hepatocytes to determine simultaneously the kinetics of CH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined. Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations. RESULTS: There was large interindividual variation in the V(max) values for both TCOH and TCA formation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent. Despite the large variation in V(max) values among individuals, disposition of CH into the two competing pathways was relatively constant. CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimental system to generate metabolic and genomic information for incorporation into TCE cancer risk assessment models. The data are discussed with regard to cellular factors, other than genotype, that may contribute to the observed variability in metabolism of CH in human liver

Enabling nanomaterial, nanofabrication and cellular technologies for nanoneuromedicines

Nanoparticulate delivery systems represent an area of particular promise for nanoneuromedicines. They possess significant potential for desperately needed therapies designed to combat a range of disorders associated with aging. As such, the field was selected as the focus for the 2014 meeting of the American Society for Nanomedicine. Regenerative, protective, immune modulatory, anti-microbial and anti-inflammatory products, or imaging agents are readily encapsulated in or conjugated to nanoparticles and as such facilitate the delivery of drug payloads to specific action sites across the blood-brain barrier. Diagnostic imaging serves to precisely monitor disease onset and progression while neural stem cell replacement can regenerate damaged tissue through control of stem cell fates. These, taken together, can improve disease burden and limit systemic toxicities. Such enabling technologies serve to protect the nervous system against a broad range of degenerative, traumatic, metabolic, infectious and immune disorders

Bench-to-bedside translation of magnetic nanoparticles

Magnetic nanoparticles (MNPs) are a new and promising addition to the spectrum of biomedicines. Their promise revolves around the broad versatility and biocompatibility of the MNPs and their unique physicochemical properties. Guided by applied external magnetic fields, MNPs represent a cutting-edge tool designed to improve diagnosis and therapy of a broad range of inflammatory, infectious, genetic and degenerative diseases. Magnetic hyperthermia, targeted drug and gene delivery, cell tracking, protein bioseparation and tissue engineering are but a few applications being developed for MNPs. MNPs toxicities linked to shape, size and surface chemistry are real and must be addressed before clinical use is realized. This article presents both the promise and perils of this new nanotechnology, with an eye towards opportunity in translational medical science

Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy.

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection

Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy.

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection

Formulation design facilitates magnetic nanoparticle delivery to diseased cells and tissues

Magnetic nanoparticles (MNPs) accumulate at disease sites with the aid of magnetic fields; biodegradable MNPs can be designed to facilitate drug delivery, influence disease diagnostics, facilitate tissue regeneration and permit protein purification. Because of their limited toxicity, MNPs are widely used in theranostics, simultaneously facilitating diagnostics and therapeutics. To realize therapeutic end points, iron oxide nanoparticle cores (5–30 nm) are encapsulated in a biocompatible polymer shell with drug cargos. Although limited, the toxic potential of MNPs parallels magnetite composition, along with shape, size and surface chemistry. Clearance is hastened by the reticuloendothelial system. To surmount translational barriers, the crystal structure, particle surface and magnetic properties of MNPs need to be optimized. With this in mind, we provide a comprehensive evaluation of advancements in MNP synthesis, functionalization and design, with an eye towards bench-to-bedside translation

Multimodal Theranostic Nanoformulations Permit Magnetic Resonance Bioimaging of Antiretroviral Drug Particle Tissue-Cell Biodistribution

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, “multimodal imaging theranostic nanoprobes” were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models. METHODS: Europium (Eu3+)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution. RESULTS: Folic acid (FA) decoration of EuCF-DTG (FA-EuCF-DTG) nanoparticles facilitated macrophage targeting and sped drug entry across cell barriers. Macrophage uptake was higher for FA-EuCF-DTG than EuCF-DTG nanoparticles with relaxivities of r2 = 546 mM-1s-1 and r2 = 564 mM-1s-1 in saline, and r2 = 850 mM-1s-1 and r2 = 876 mM-1s-1 in cells, respectively. The values were ten or more times higher than what was observed for ultrasmall superparamagnetic iron oxide particles (r2 = 31.15 mM-1s-1 in saline) using identical iron concentrations. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses. After parenteral injection of FA-EuCF-DTG and EuCF-DTG into rats and rhesus macaques, drug, iron and cobalt levels, measured by LC-MS/MS, magnetic resonance imaging, and ICP-MS were coordinate. CONCLUSION: We posit that these theranostic nanoprobes can assess LASER ART drug delivery and be used as part of a precision nanomedicine therapeutic strategy

Europium sulfide nanoprobes predict antiretroviral drug delivery into HIV-1 cell and tissue reservoirs

Background: Delivery of long-acting nanoformulated antiretroviral drugs (ARVs) to human immunodeficiency virus type one cell and tissue reservoirs underlies next generation antiretroviral therapeutics. Nanotheranostics, comprised of trackable nanoparticle adjuncts, can facilitate ARV delivery through real-time drug tracking made possible through bioimaging platforms. Methods: To model HIV-1 therapeutic delivery, europium sulfide (EuS) nanoprobes were developed, characterized and then deployed to cells, tissues, and rodents. Tests were performed with nanoformulated rilpivirine (NRPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used clinically to suppress or prevent HIV-1 infection. First, CD4+ T cells and monocyte-derived macrophages were EuS-treated with and without endocytic blockers to identify nanoprobe uptake into cells. Second, Balb/c mice were co-dosed with NRPV and EuS or lutetium177-doped EuS (177LuEuS) theranostic nanoparticles to assess NRPV biodistribution via mass spectrometry. Third, single photon emission computed tomography (SPECT-CT) and magnetic resonance imaging (MRI) bioimaging were used to determine nanotheranostic and NRPV anatomic redistribution over time. Results: EuS nanoprobes and NRPV entered cells through dynamin-dependent pathways. SPECT-CT and MRI identified biodistribution patterns within the reticuloendothelial system for EuS that was coordinate with NRPV trafficking. Conclusions: EuS nanoprobes parallel the uptake and biodistribution of NRPV. These data support their use in modeling NRPV delivery to improve treatment strategies