No abstract

Small interfering RNA (siRNA) has been chemically conjugated to a variety of bioactive molecules, lipids, polymers, peptides, and inorganic nanostructured materials to enhance their pharmacokinetic behavior, cellular uptake, target specificity, and safety. To efficiently deliver siRNAs to the target cells and tissues, many different siRNA bioconjugates were synthesized and characterized, and their gene silencing efficiencies were tested in vitro and in vivo. In this review, recent developments of siRNA bioconjugates are summarized.

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 DOXresistant 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. DOXconjugated 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.

Small interfering RNA (siRNA) with 19-21 base pairs has been recently recognized as a new therapeutic agent for effectively silencing a specific gene on a post-transcription level. For siRNA therapeutics, safe and efficient delivery issues are significant hurdles to clinical applications. Here we present a new class of biologically active siRNA structure based on chemically self-crosslinked and multimerized siRNA through cleavable disulphide linkages. The multimerized siRNA can produce more stable and compact polyelectrolyte complexes with less cytotoxic cationic carriers than naked siRNA because of substantially increased charge densities and the presence of flexible chemical linkers in the backbone. The cleavable and multimerized siRNA shows greatly enhanced gene-silencing efficiencies in vitro and in vivo through a target-messenger-RNA-specific RNA interference processing without significantly eliciting immune induction. This study demonstrates that the multimerized siRNA structure complexed with selected cationic condensing agents can serve as potential gene-silencing therapeutics for treating various diseases.

Ribonucleic acid interference (RNAi) is a powerful molecular tool that has potential to revolutionize the treatment of cancer. One major challenge of applying this technology for clinical application is the lack of site-specific carriers that can effectively deliver short interfering RNA (siRNA) to cancer cells. Here we report the development and assessment of a cancer-cell specific magnetic nanovector construct for efficient siRNA delivery and non-invasive monitoring through magnetic resonance imaging (MRI). The base of the nanovector construct is comprised of a superparamagnetic iron oxide nanoparticle core coated with polyethylene glycol (PEG)-grafted chitosan, and polyethylenimine (PEI). The construct was then further functionalized with siRNA and a tumor-targeting peptide, chlorotoxin (CTX), to improve tumor specificity and potency. Flow cytometry, quantitative RT-PCR, and fluorescence microscopy analyses confirmed receptor-mediated cellular internalization of nanovectors and enhanced gene knockdown through targeted siRNA delivery. The ability of this nanovector construct to generate specific contrast enhancement of brain tumor cells was demonstrated through MR imaging. These findings suggest that this CTX enabled nanoparticle carrier may be well suited for delivery of RNAi therapeutics to 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 comprised 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 tumorspecific 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 6 0 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.

Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A). Little is known, however, about how a defective KIF1B gene leads to CMT2A. Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90). KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments. KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain. In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM. These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

Xanthorrhizol, a natural sesquiterpenoid isolated from the rhizome of Curcuma xanthorrhiza Roxb (Zingiberaceae), has antibacterial activities and protective effects against cisplatin-induced hepatotoxicity. In this study, we investigated the activities of xanthorrhizol as an antioxidant or antiinflammatory agent using neuronal and microglial cells. Xanthorrhizol had potent neuroprotective effects on glutamate-induced neurotoxicity and reactive oxygen species (ROS) generation in the murine hippocampal HT22 cell line. Also, xanthorrhizol inhibited H(2)O(2)-induced lipid peroxidation in rat brain homogenates. The properties of xanthorrhizol as an antiinflammatory agent were investigated in microglial activation by lipopolysaccharide. It reduced the expression of cyclooxygenase-2 and the inducible nitric oxide synthase, which consequently resulted in the reduction of nitric oxide. The production of proinflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha in activated microglial cells, was reduced by xanthorrhizol. These results suggest that xanthorrhizol could be an effective candidate for the treatment of Alzheimer's disease- and other neurological disease-related ROS and inflammation.