Intestinal Cgi-58 deficiency reduces postprandial lipid absorption.

Comparative Gene Identification-58 (CGI-58), a lipid droplet (LD)-associated protein, promotes intracellular triglyceride (TG) hydrolysis in vitro. Mutations in human CGI-58 cause TG accumulation in numerous tissues including intestine. Enterocytes are thought not to store TG-rich LDs, but a fatty meal does induce temporary cytosolic accumulation of LDs. Accumulated LDs are eventually cleared out, implying existence of TG hydrolytic machinery in enterocytes. However, identities of proteins responsible for LD-TG hydrolysis remain unknown. Here we report that intestine-specific inactivation of CGI-58 in mice significantly reduces postprandial plasma TG concentrations and intestinal TG hydrolase activity, which is associated with a 4-fold increase in intestinal TG content and large cytosolic LD accumulation in absorptive enterocytes during the fasting state. Intestine-specific CGI-58 knockout mice also display mild yet significant decreases in intestinal fatty acid absorption and oxidation. Surprisingly, inactivation of CGI-58 in intestine significantly raises plasma and intestinal cholesterol, and reduces hepatic cholesterol, without altering intestinal cholesterol absorption and fecal neutral sterol excretion. In conclusion, intestinal CGI-58 is required for efficient postprandial lipoprotein-TG secretion and for maintaining hepatic and plasma lipid homeostasis. Our animal model will serve as a valuable tool to further define how intestinal fat metabolism influences the pathogenesis of metabolic disorders, such as obesity and type 2 diabetes.

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro.

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.

Functionalization of iron oxide magnetic nanoparticles with targeting ligands: their physicochemical properties and in vivo behavior.

AIM: To develop and evaluate two tumor-specific nanoprobes by functionalization of a polyethylene glycol-immobilized nanoparticle with arginine-glycine-aspartic acid (RGD) or chlorotoxin ligand that targets α(v)ß(3) integrin and matrix metalloproteinase-2 receptors, respectively. MATERIALS & METHODS: The nanoprobes were made of iron oxide cores, biocompatible polymer coating, and surface-conjugated RGD or chlorotoxin peptide. The tumor-targeting specificity of the nanoprobes was evaluated both in vitro and in vivo. RESULTS & DISCUSSION: Both nanoprobes were highly dispersive and exhibited excellent long-term stability in cell culture media. The RGD-conjugated nanoprobe displayed a strong initial accumulation near neovasculatures in tumors followed by quick clearance. Conversely, the chlorotoxin-enabled nanoprobe exhibited sustained accumulation throughout the tumor. CONCLUSION: These findings revealed the influence of the targeting ligands on the intratumoral distribution of the ligand-enabled nanoprobes. With flexible surface chemistry, our nanoparticle platform can be used in a modular fashion to conjugate biomolecules for intended applications.

pH-Sensitive siRNA nanovector for targeted gene silencing and cytotoxic effect in cancer cells.

A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is composed of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ∼60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers.