title: Combinatorial synthesis and screening of cancer cell-specific nanomedicines targeted via phage fusion proteins.
abstract: Active tumor targeting of nanomedicines has recently shown significant improvements in the therapeutic activity of currently existing drug delivery systems, such as liposomal doxorubicin (Doxil/Caelyx/Lipodox). Previously, we have shown that isolated pVIII major coat proteins of the fd-tet filamentous phage vector, containing cancer cell-specific peptide fusions at their N-terminus, can be used as active targeting ligands in a liposomal doxorubicin delivery system in vitro and in vivo. Here, we show a novel major coat protein isolation procedure in 2-propanol that allows spontaneous incorporation of the hydrophobic protein core into preformed liposomal doxorubicin with minimal damage or drug loss while still retaining the targeting ligand exposed for cell-specific targeting. Using a panel of 12 structurally unique ligands with specificity toward breast, lung, and/or pancreatic cancer, we showed the feasibility of pVIII major coat proteins to significantly increase the throughput of targeting ligand screening in a common nanomedicine core. Phage protein-modified Lipodox samples showed an average doxorubicin recovery of 82.8% across all samples with 100% of protein incorporation in the correct orientation (N-terminus exposed). Following cytotoxicity screening in a doxorubicin-sensitive breast cancer line (MCF-7), three major groups of ligands were identified. Ligands showing the most improved cytotoxicity included: DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD showing a 25-fold improvement (p < 0.05) in toxicity. Similarly DGQYLGSQ, ETYNQPYL, and GSSEQLYL ligands with specificity toward a doxorubicin-insensitive pancreatic cancer line (PANC-1) showed significant increases in toxicity (2-fold; p < 0.05). Thus, we demonstrated proof-of-concept that pVIII major coat proteins can be screened in significantly higher throughput to identify novel ligands displaying improved therapeutic activity in a desired cancer phenotype.
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University Auburn, AL, USA.
authors.forename: James W
authors.initials: JW
authors.lastname: Gillespie
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University Auburn, AL, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Gross
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University Auburn, AL, USA.
authors.forename: Anatoliy T
authors.initials: AT
authors.lastname: Puzyrev
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University Auburn, AL, USA.
authors.forename: Deepa
authors.initials: D
authors.lastname: Bedi
authors.affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University Auburn, AL, USA.
authors.forename: Valery A
authors.initials: VA
authors.lastname: Petrenko
keywords: breast cancer; doxorubicin; liposomal drug delivery; pancreatic cancer; phage display; targeted nanomedicines