Journal of Antimicrobial Chemotherapy (2002) 49, 941-951
© 2002 The British Society for Antimicrobial Chemotherapy
Polycationic photosensitizer conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria
Wellman Laboratories of Photomedicine, Massachusetts General Hospital and Department of Dermatology, Harvard Medical School, Boston, MA 02114-2698, USA
Received 5 November 2001; returned 24 January 2002; revised 25 February 2002; accepted 8 March 2002.
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 
-amino group of poly-(
-benzyloxycarbonyl)lysines of average length eight and 37 lysine residues, followed by deprotection of the -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.
Keywords: photodynamic therapy, polylysine, Escherichia coli, Staphylococcus aureus, electron microscopy
* Correspondence address. Department of Dermatology, Massachusetts General Hospital, 50 Blossom Street WEL 224, Boston, MA 02114-2698, USA. Tel: +1-617-726-6182; Fax: +1-617-726-8566; E-mail: hamblin{at}helix.mgh.harvard.edu
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