title: Polycationic photosensitizer conjugates: effects of chain length and Gram classification on the photodynamic inactivation of bacteria.
abstract: OBJECTIVE
  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 alpha-amino group of poly-(epsilon-benzyloxycarbonyl)lysines of average length eight and 37 lysine residues, followed by deprotection of the epsilon-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 microM chlorin(e6) equivalent) and incubation time. After incubation the bacteria were exposed to low fluences (10-40 J/cm(2)) 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 microM. PDI using the 8-lysine conjugate and free chlorin(e6) on E. coli was observed at a concentration of 100 microM. Transmission electron micrographs showed internal electron-lucent areas consistent with chromosomal damage.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Wellman Laboratories of Photomedicine, Massachusetts General Hospital and Department of Dermatology, Harvard Medical School, Boston, MA 02114-2698, USA. hamblin@helix.mgh.harvard.edu
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hamblin
authors.forename: David A
authors.initials: DA
authors.lastname: O'Donnell
authors.forename: Naveen
authors.initials: N
authors.lastname: Murthy
authors.forename: Krishnan
authors.initials: K
authors.lastname: Rajagopalan
authors.forename: Norman
authors.initials: N
authors.lastname: Michaud
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Sherwood
authors.forename: Tayyaba
authors.initials: T
authors.lastname: Hasan
mesh_terms: D002735:Chlorophyllides; D004926:Escherichia coli / Q000187:drug effects; D006090:Gram-Negative Bacteria / Q000145:classification / Q000187:drug effects*; D006094:Gram-Positive Bacteria / Q000145:classification / Q000187:drug effects*; D010778:Photochemotherapy / Q000379:methods; D017319:Photosensitizing Agents / Q000737:chemistry* / Q000494:pharmacology*; D011073:Polyamines / Q000737:chemistry / Q000494:pharmacology; D000071228:Polyelectrolytes; D011107:Polylysine / Q000737:chemistry / Q000494:pharmacology; D011166:Porphyrins / Q000737:chemistry / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects
chemical_list: D002735:Chlorophyllides; D017319:Photosensitizing Agents; D011073:Polyamines; D000071228:Polyelectrolytes; D011107:Polylysine; D011166:Porphyrins; C062985:phytochlorin