Orthogonal modification of M13 phages for highly selective photodynamic therapy and diagnostic applications

Photodynamic therapy (PDT) is an alternative minimally invasive approach for cancer treatment. PDT relies on the administration of photosensitizers (PSs) that, when exposed to specific light wavelengths in the presence of molecular oxygen, trigger the generation of cytotoxic reactive oxygen species (ROS) at the target site. However, PDT faces challenges related to the non-selectivity of current PSs towards target cells and their poor solubility in physiological environments. To address these drawbacks, M13 bacteriophages, non-toxic bacterial viruses, were selected as versatile carriers for attaching various types of PSs. Bacteriophages have a well-defined spaghetti-like structure and can be genetically refactored to exhibit tropism towards selected cellular receptors by expressing specific peptides and nanobodies on the phage tip. The approximately 2000 structural pVIII proteins of the M13 phage can be bioconjugated with hundreds to thousands of molecules using various methodologies documented in literature. This work presents an orthogonal approach developed for attaching PSs, imaging agents, and proteins to the phages while preserving their targeting capacity toward cells. The various techniques were mainly optimized for phages retargeted against receptors overexpressed by various tumor cells (EGFR and GD2) and components of the outer cell membrane of Gram-negative bacteria. This approach has led to the development of diverse, highly selective therapeutic, diagnostic, and theranostic agents and unveiled the potential of the phage platform in antitumor and antibacterial PDT. This potential is explored through investigations of photodynamic properties, penetration studies of 3D tumor cell structures (spheroids), and the ability to cross the blood-brain barrier and intestinal barrier. The results of the study hold great promise for developing new and innovative phage-based clinical applications in the field of nanomedicine.