Certain human malignancies including prostate cancer overexpress sigma receptor, a membrane bound protein that binds haloperidol and various other neuroleptics with high affinity. An anisamide derivatized ligand possesses high affinity for sigma receptors and we hypothesized that its incorporation into the liposomes encapsulating doxorubicin (DOX) can specifically target and deliver the drug to prostate cancer cells that overexpress sigma receptors. A polyethylene glycol phospholipid was derivatized with an anisamide ligand, which was then incorporated into the DOX‐loaded liposome. The resulting anisamide‐conjugated liposomal DOX showed significantly higher toxicity to DU‐145 cells than non‐targeted liposomal DOX, the IC50 being 1.8 μM and 14 μM respectively. The cytotoxicity of the targeted liposomal DOX, however, was significantly blocked by haloperidol, suggesting that the enhanced cytotoxicity was specifically mediated by the sigma receptors. Fluorescence imaging studies after intravenous (i.v.) administration showed that incorporation of anisamide into liposomes significantly improved their accumulation into the tumor. A weekly injection of the targeted liposomal DOX for 4 weeks at a dose of 7.5 mg/kg led to a significant growth inhibition of established DU‐145 tumor in nude mice with minimal toxicity. Free DOX was effective, but associated with significant toxicities. The present study is the first to demonstrate the use of small molecular weight ligand for mediating efficient targeting of liposomal drugs to sigma receptor expressing prostate cancer cells both in vitro and in vivo.

In the present article, we report the in vitro and in vivo delivery of doxorubicin using biosynthesized gold nanoparticles (b-Au-PP). Gold nanoparticles were synthesized by a simple, fast, efficient, environmentally friendly and economical green chemistry approach using an extract of Peltophorum pterocarpum (PP) leaves. Because the biosynthesized b-Au-PP was highly stable in various physiological buffers for several weeks and biocompatible in both in vitro and in vivo systems, we designed and developed a biosynthesized gold nanoparticle (b-Au-PP)-based drug-delivery system (DDS) using doxorubicin (Dox) (b-Au-PP-Dox). Both b-Au-PP and b-Au-PP-Dox were thoroughly characterized using several analytical tools. Administration of doxorubicin-loaded DDS (b-Au-PP-Dox) resulted in a significant inhibition of the proliferation of cancer cells (A549, B16F10) in vitro and of tumor growth in an in vivo model compared to doxorubicin alone. Furthermore, we found that the cellular uptake and release of Dox in the nanoconjugated form (b-Au-PP-Dox) were faster than the uptake and release of unconjugated Dox. The in vivo toxicity study did not show any significant changes in the hematology, serum clinical biochemistry or histopathology in the C57BL6/J female mice after consecutive intraperitoneal (IP) injections over a period of seven days. To the best of our knowledge, our study is the first to report the application of a biosynthesized gold nanoparticle-based DDS for cancer therapy in an animal model, in addition to a detailed in vivo toxicity study. Together, the results demonstrate that a biosynthesized gold nanoparticle-based drug-delivery system (b-Au-PP-Dox) could be used in the near future as an alternative cost-effective treatment strategy for cancer therapy.

Multivalent binding allows high selectivity and affinity in a ligand–protein interaction. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which specific E3s, called N-recognins, mediate ubiquitylation through the recognition of types 1 and 2, destabilizing N-terminal residues of substrates. We recently identified a set of E3 Ub ligases (named UBR1–UBR7) containing the 70-residue UBR box, and we demonstrated that UBR1, UBR2, UBR4, and UBR5 can bind to destabilizing N-terminal residues. To explore a model of heterovalent interaction to the N-recognin family, we synthesized the small-molecule compound RF-C11, which bears two heterovalent ligands designed to target N-recognins, together with control molecules with two homovalent ligands. We demonstrate that heterovalent ligands of RF-C11 selectively and cooperatively bind cognate-binding sites of multiple N-recognins and thereby inhibit both types 1 and 2 N-end rule activities. Furthermore, the efficacy of heterovalent RF-C11 was substantially higher than homovalent inhibitors, which can target either a type 1 or type 2 site, providing the molecular basis of designing multivalent inhibitors for the control of specific intracellular pathways. In addition, RF-C11 exhibited higher efficacy and stability, compared with dipeptides bearing destabilizing N-terminal residues, which are known competitive inhibitors of the pathway. We also used the heterovalent compound to study the function of N-recognins in cardiac signaling. Using mouse and rat cardiomyocytes, we demonstrate that the N-end rule pathway has a cell-autonomous function in cardiac proliferation and hypertrophy, explaining our earlier results implicating the pathway in cardiac development and proteolysis of multiple cardiovascular regulators.