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.
Highlights
Cancer remains the second leading cause of death in the United States across all age groups for both genders and continues to produce a significant burden on the healthcare system for improved patient outcomes with an improved quality of life in cancer survivors (Siegel et al, 2015)
UV/Vis spectroscopy of the phage protein supernatant (Figure 2A) revealed a spectrum commonly observed for a pure protein sample with a corresponding 260/280 ratio of 0.559, which is close to the theoretical 260/280 ratio of 0.5 expected for pure pVIII major coat protein
We hypothesized that there would be a significant increase in intracellular doxorubicin over time in DMPGTVLPmodified Liposomal Doxorubicin (Lipodox) compared to unmodified Lipodox
Summary
Cancer remains the second leading cause of death in the United States across all age groups for both genders and continues to produce a significant burden on the healthcare system for improved patient outcomes with an improved quality of life in cancer survivors (Siegel et al, 2015). Liposomal doxorubicin is the first nanomedicine approved for use in the clinic for a variety of cancer types It showed significant improvements in circulation and bioavailability, and leads to overall reduction of tumor volume while significantly increasing the long-term survival of patients (Gabizon et al, 2003). Nanomedicines provide an increase in therapeutic activity by passively targeting their therapeutic payloads and using the leaky vasculature commonly associated with many tumors, a pathophysiological phenotype known as the enhanced permeability and retention (EPR) effect (Fang et al, 2011). It has been suggested extensively from pre-clinical models that active targeting of nanomedicines can provide an additional improvement in the therapeutic activity compared to solely relying on the EPR effect and other passive targeting strategies. The homing molecule would provide cell-specific accumulation of the therapeutic molecule to the desired cells, thereby reducing major side effects and reducing doselimiting toxicities commonly seen with highly active molecules like doxorubicin
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