Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers an unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF- 7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines.

Nucleic acids including antisense oligonucleotides, small interfering RNA (siRNA), aptamers and rybozymes, emerged as versatile therapeutics due to their ability to interfere in a well-planned manner with the flow of genetic information from DNA to protein. However, a systemic use of NAs is hindered by their instability in physiological liquids and inability of intracellular accumulation in the site of action. We first evaluated the potential of cancer specific phage fusion proteins as targeting ligands that provide encapsulation, protection, and navigation of siRNA to the target cell. The tumor-specific proteins were isolated from phages that were affinity selected from a landscape phage library against target breast cancer cells. It was found that fusion phage coat protein fpVIII displaying cancer-targeting peptides can effectively encapsulate siRNAs and deliver them into the cells leading to specific silencing of the model gene GAPDH. Complexes of siRNA and phage protein form nanoparticles (nanophages), that were characterized by atomic force microscopy and ELISA, and their stability was demonstrated by resistance of encapsulated siRNA to degradation by serum nucleases. The phage protein/siRNA complexes can make a new type of highly selective, stable, active and physiologically acceptable cancer nanomedicines.

A novel strategy to improve the therapeutic index of chemotherapy has been developed by the integration of nanotechnology with phage technique. The objective of this study was to combine phage display, identifying tumor-targeting ligands, with a liposomal nanocarrier for targeted delivery of doxorubicin. Following the proof of concept in cell-based experiments, this study focused on in vivo assessment of antitumor activity and potential side-effects of phage fusion protein-modified liposomal doxorubicin. MCF-7-targeted phage-Doxil treatments led to greater tumor remission and faster onset of antitumor activity than the treatments with non-targeted formulations. The enhanced anticancer effect induced by the targeted phage-Doxil correlated with an improved tumor accumulation of doxorubicin. Tumor sections consistently revealed enhanced apoptosis, reduced proliferation activity and extensive necrosis. Phage-Doxil-treated mice did not show any sign of hepatotoxicity and maintained overall health. Therefore, MCF-7-targeted phage-Doxil seems to be an active and tolerable chemotherapy for breast cancer treatment.

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumorspecific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long circulating liposomes targeted to PC3 prostate tumor cells via PCspecific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicinloaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor.

It is hypothesized that the use of targeted drug delivery systems can significantly improve the therapeutic index of small molecule chemotherapies by enhancing accumulation of the drugs at the site of disease. Phage display offers a high-throughput approach for selection of the targeting ligands. We have successfully isolated phage fusion proteins selective and specific for PANC-1 pancreatic cancer cells. Doxorubicin liposomes (Lipodox) modified with tumor-specific phage fusion proteins enhanced doxorubicin uptake specifically in PANC-1 cells as compared with unmodified Lipodox and also compared with normal breast epithelial cells. Phage protein-targeted Lipodox substantially increased the concentration of doxorubicin in the nuclei of PANC-1 cells in spite of P-glycoprotein-mediated drug efflux. The in vitro cytotoxic activity obtained with pancreatic cell-targeted Lipodox was greater than that of unmodified Lipodox. We present a novel and straightforward method for preparing pancreatic tumor-targeted nanomedicines by anchoring pancreatic cancer-specific phage proteins within the liposome bilayer.