Bacteriophage bionanowire as a carrier for both cancer-targeting peptides and photosensitizers and its use in selective cancer cell killing by photodynamic therapy

Abstract

A photosensitizer, pyropheophorbid-a (PPa), is conjugated to SKBR-3 breast cancer cell-specific biological nanowire phage, to form a novel PPa-phage complex, which is further successfully used in selectively killing SKBR-3 breast cancer cells by the mechanism of photodynamic therapy (PDT).

Figure 1

Use of spectroscopy to confirm the conjugation of PPa and phage and the production of singlet oxygen from the PPa-phage complex upon light irradiation. (a) UV/Vis absorption spectra of phage-PPa complex at 10%, 50%, and 100% of PPa-phage conjugation. (b) Fluorescence spectra of PPa-phage complex at 10%, 50%, and 100% of PPa-phage conjugation collected around PPa fluorescence maxima at 701 nm and phage fluorescence maxima at 300 nm (inset) to observe the energy hoping and to estimate the percentage conjugation of PPa by tryptophan fluorescence resonance energy transfer (FRET). (c) Time resolved ¹O₂ phosphorescence signal of PPa(black), p-PPPa (50%)(red), and f-PPPa (100%)(green) in DMSO at 1270 nm using Nd-YAG laser at 532 nm of excitation wavelength.

Figure 2

High resolution transmission electron microscopy (TEM) images of SKBR-3 specific bacteriophage without any modification (A), with major coat proteins fully modified with PPa (B, i.e., f-PPPa) and with major coat proteins partially modified with PPa (C, i.e., p-PPPa).

Figure 3

Detection of p-PPPa complex binding affinity to SKBR-3 cells using immunofluorescence microscopy (A–C). (A) SKBR-3 cells treated with p-PPPa complex that were labeled with anti-phage antibody conjugated to a fluorescent green dye, showing fluorescence associated with phage. (B) DAPI-stained SKBR-3 cells treated with p-PPPa complex, showing fluorescence associated with nuclei. (C) is a merged image of (A) and (B).

Figure 4

Cytotoxicity studies of porphyrin and phage conjugated porphyrin (p-PPPa) on SKBR-3 and MCF-7 cells in the absence (A) and presence of 658 nm laser (B). Time course quantitative estimation of PPa and p-PPPa on SKBR-3 cells viability after PDT (C). (Note: The bars indicated in blue, red, green and orange are corresponding to control (cells without any treatment), phage, PPa and p-PPPa, respectively).

Figure 5

In vitro evaluation of PDT on control MCF-7 cells (A–C) and target SKBR3 cells (D–F). A,D) Light microscope images of MCF-7 and SKBR-3 cells respectively. B,C,E,F) Fluorescent images of MCF-7 and SKBR-3 cells using Live/dead cell viability kit. B,E) upon incubation with only porphyrin and laser irradiation. C,F) Upon incubation with phage-porphyrin complex. (All images were captured at 10× objectives).

Scheme 1

Schematic diagram of photosensitizer conjugation onto biological nanowire and its targeted photodynamic therapy. (A) Side view of biological nanowire (filamentous phage) with major coat protein (pVIII) fully modified with PPa (no singlet oxygen is produced due to excitonic coupling as a result of close packing of porphyrin residues on the sidewalls). (B) Side view of biological nanowire with major coat protein (pVIII) partially modified with PPa (singlet oxygen is produced due to absence of excitonic coupling) (red arrows) owing to distinct PPa, (C) Illustration of PPa-phage conjugation and in vitro photo-dynamic activity in SKBR-3 cells using 658 nm of laser. Green color cells represent the live cells and red represents the dead cells after PDT. In (A) and (B), pink line is the SKBR-3 cell-targeting peptide fused to the N-terminal of pVIII (green).