Landscape Phage Fusion Protein-Modified Pharmaceutical Nanocarriers for Targeted and Cytoplasmic Delivery of Chemotherapeutics to Breast Cancer Cells in Vitro and in Vivo

Objective : The objective of the project is to integrate phage display technology with nanocarrier-based drug delivery systems (liposomes and micelles) for advanced targeted drug delivery. We have proposed a new approach using a phage fusion protein (a tumor specific peptide fused to the phage pVIII coat protein) to avoid drawbacks associated with chemical modifications, and improve the therapeutic index of anti-tumor agents by the integration of nanomedicines and phage display techniques.;Methodologies: After an 8-mer phage library f8/8 and a biopanning protocol identified a MCF-7 cancer cell-specific phage fusion protein, the detergent dialysis methods were developed for self-assembly of the phage fusion protein with liposomes or micelles. The formed nanoparticles were characterized by dynamic light scattering and electron microscopy. Drug-loading efficiency was determined by fluorescence spectrometry or HPLC. Western-blotting was used to examine the orientation of the phage fusion protein within the liposomes. A co-culture model was developed for the assessment of targeting selectivity.;Results : A phage fusion protein specific for MCF-7 cells screened from a phage peptide library was incorporated directly into the bilayer of doxorubicin-loaded PEGylated liposomes (phage-Doxil), as well as self-assembled with the micelle-forming PEG-PE conjugate to form mixed micelles loaded with paclitaxel (PTX) (phage-micelles). The formed nanoparticles maintained either liposomal or micellar morphology and had a substantial drug retention after phage protein incorporation. Both phage-Doxil and phage-micelles showed specific targeting to MCF-7 cells and significantly higher cytotoxicity towards target cells than non-targeted formulations, but this effect was absent with non-target cells. Furthermore, the FRET technique demonstrated the pH-dependent membrane destabilization activity of the phage fusion protein which in turn enhanced the endosomal escape and cytosolic delivery of phage-liposomes as observed under fluorescence microscopy. Endosome acidification inhibition by bafilomycin A1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, along with cytosol localization of phage liposomes, further confirming its endosomal escape. Animal studies showed that phage-Doxil led to greater tumor remission compared to non-targeted formulations, indicating an enhanced anticancer effect. Consistently, tumor sections after treatment group of phage-Doxil revealed much more apoptotic cells compared to those from treatment with non-targeted formulations. Meanwhile, treated mice showed normal body weight gain, no sign of discomfort and normal Serum Alanine Transaminase (ALT) and Serum Lactate Dehydrogenase (LDH) values, indicating overall health and well-being of mice during the treatment with phage-Doxil.;Discussion and Conclusions: We have developed an innovative approach for cancer cell targeting which relies on the use of a phage-derived fusion protein (e.g. the tumor-specific peptides fused to the N-terminus of the phage major PVIII coat protein) as a targeting moiety for drug-loaded liposomes or micelles. This innovative approach avoids drawbacks associated with chemical modifications. The novelty of the approach involves its exploration of intrinsic properties of the phage fusion protein. First, we have utilized the unique propensity of phage coat proteins to incorporate spontaneously into lipid bilayers to form liposomal particles mimicking the structure of phage proteins in bacterial membranes. Furthermore, the amphiphilic nature of phage fusion protein also allows the construction of a mixed micelle self-assembled with the phage fusion protein and micellar-forming material, such as PEG-PE, for targeted delivery of a hydrophobic drug. Last but not least, pH-sensitive carboxylic group, appended to acidic amino acid residues within N-terminus of a MCF-7 specific landscape phage protein add another layer of value to the phage protein-mediated delivery system that facilitate endosome escape and cytoplasmic delivery of liposomal drugs. The innovative landscape phage approach combined with drug-loaded nanocarriers for ligand-mediated tumor targeting and pH-sensitive controlled release is expected to enhance the efficacy of chemotherapeutics. (Abstract shortened by UMI.).