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

Michael R. Hamblin, David A. O'Donnell, Naveen Murthy, Krishnan Rajagopalan, Norman Michaud, Margaret E. Sherwood, Tayyaba Hasan

Research output: Contribution to journalArticlepeer-review

252 Citations (Scopus)

Abstract

Objectives: We have shown previously that a polycationic conjugate between poly-L-lysine and the photosensitizer chlorine6 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 chlorine6 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 chlorine6 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 chlorine6 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 chlorine6 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 chlorine6 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 chlorine6 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.

Original languageEnglish
Pages (from-to)941-951
Number of pages11
JournalJournal of Antimicrobial Chemotherapy
Volume49
Issue number6
DOIs
Publication statusPublished - 2002
Externally publishedYes

Keywords

  • Electron microscopy
  • Escherichia coli
  • Photodynamic therapy
  • Polylysine
  • Staphylococcus aureus

ASJC Scopus subject areas

  • Pharmacology
  • Microbiology (medical)
  • Infectious Diseases
  • Pharmacology (medical)

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