Advisor(s)

Vladimir P. Torchilin

Contributor(s)

Mansoor M. Amiji, Ferris Craig, John S. Gatley, Srinivas Sridhar

Date of Award

2012

Date Accepted

1-2012

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

Bouvé College of Health Sciences, Department of Pharmaceutical sciences

Keywords

pharmaceutical sciences, nanotechnology, breast cancer, chemotherapeutics, liposome and micelle, phage display and landscape phage fusion protein, pharmaceutical nanocarriers, tumor targeting and cytoplasmic delivery

Disciplines

Pharmaceutics and Drug Design | Pharmacy and Pharmaceutical Sciences

Abstract

Objective : Conventional chemotherapy is often accompanied by severe side-effects in cancer treatment owing to its inability to kill tumor cells specifically. The use of targeted pharmaceutical nanocarriers has improved the pharmacokinetic and pharmacodynamic properties of chemotherapy. Ongoing challenges for targeted delivery include the search for alternative ligands (e.g. "substitute antibodies") and development of methods for their conjugation with nanomedicines. The phage display technique is emerging as a powerful tool to identify tumor recognition molecules. 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. The traditional phage display approach for targeted delivery assumes the necessity of a chemical synthesis of identified tumor-selective peptides and their conjugation with pharmaceuticals or pharmaceutical nanocarriers. Here, 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. Using fluorescence microscopy, fluorescence spectrometry, and FACS, the cellular uptake and intracellular trafficking of phage protein-modified nanoparticles were determined. Cytotoxicity and apoptosis were evaluated using the Cell-Titer-Blue assay, Caspase-3 assay and TUNEL assay. The fluorescence resonance energy transfer (FRET) technique was employed to assess the fusogenic property of the phage protein. The factorial design methodology was used to evaluate the effect of the lipid composition on the specific binding of phage-liposomes to target cells in a co-culture model. Antitumor activity and potential toxicity of MCF-7-specific phage-Doxil were evaluated using MCF-7 tumor-bearing xenografted nude mice.

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. By examination of the mechanism of targeting of phage fusion protein-modified nanocarriers, we have demonstrated that only the binding peptide motif was involved in the targeting specificity of phage-liposomes, while the presence of phage pVIII coat protein motif did not interfere with the targeting specificity. With the use of factor design methodology, we identified the effect of lipid species and protein density made of phage liposomes on their targeting efficiency.

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.

The in vitro results represent a proof of concept, showing the ability of phage fusion protein displaying cancer cell-targeting peptides to self-assemble with pharmaceutical nanocarriers to make them cancer cell-targeted and capable of cytoplasmic delivery of chemotherapeutics to breast cancer cells to induce effective tumor cell killing. The in vivo results further demonstrate the efficacy and safety of phage-derived protein-based targeted drug delivery systems.

We believe that the integration of nanotechnology with a combinatorial phage technique represents a paradigm shift in the development of efficient targeted nanomedicines. This method of preparation of targeted liposomes and micelles is simple, requires no chemical modification, and opens the way for the use of inexpensive and easy-to-prepare fused phage proteins as "substitute antibodies" for site-specific targeted delivery of drugs and drug-loaded pharmaceutical nanocarriers to disease sites.

Document Type

Dissertation

Rights Information

copyright 2012

Rights Holder

Tao Wang


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