Polycationic photosensitizer conjugates: Effects of chain length and Gram classification on the photodynamic inactivation of bacteria

Objectives: We have shown previously that a polycationic conjugate between poly-L-lysine and the photosensitizer chlorin_e6 was effective in photodynamic inactivation (PDI) of both Gram-positive and Gram-negative bacteria. In this report we explore the relationship between the size of the polylysine chain and its effectiveness for mediating the killing of Gram-negative and Gram-positive bacteria. Methods: Conjugates were prepared by attaching precisely one chlorin_e6 molecule to the α-amino group of poly-(ε-benzyloxycarbonyl)lysines of average length eight and 37 lysine residues, followed by deprotection of the ε-amino groups, and were characterized by iso-electric focusing. The uptake of these conjugates and free chlorin_e6 by Gram-positive Staphylococcus aureus (ATCC 27659) and Gram-negative Escherichia coli(ATCC 29181) after washing was measured as a function of photosensitizer concentration (0-4 μM chlorin_e6 equivalent) and incubation time. After incubation the bacteria were exposed to low fluences (10-40 J/cm²) of 660 nm light delivered from a diode laser, and viability was assessed after serial dilutions by a colony-forming assay. Results: S. aureus and E. coli took up comparable amounts of the two conjugates, but free chlorin_e6 was only taken up by S. aureus. After illumination S. aureus was killed in a fluence-dependent fashion when loaded with the 8-lysine conjugate and free chlorin_e6 but somewhat less so with the 37-lysine conjugate. In contrast, PDI of E. coli was only effective with the 37-lysine conjugate at concentrations up to 4 μM. PDI using the 8-lysine conjugate and free chlorin_e6 on E. coli was observed at a concentration of 100 μM. Transmission electron micrographs showed internal electron-lucent areas consistent with chromosomal damage. Conclusion: These results can be explained by the necessity of a large polycation to penetrate the impermeable outer membrane of Gram-negative E. coli, while Gram-positive S. aureus is more easily penetrated by small molecules. However, because S. aureus is more sensitive overall than E. coli the 37-lysine conjugate can effectively kill both bacteria.