Lasers in Medical Science

, Volume 28, Issue 2, pp 465–471 | Cite as

Topical photodynamic treatment with poly-l-lysine–chlorin p6 conjugate improves wound healing by reducing hyperinflammatory response in Pseudomonas aeruginosa-infected wounds of mice

  • Khageswar Sahu
  • Mrinalini SharmaEmail author
  • Harsha Bansal
  • Alok Dube
  • Pradeep Kumar Gupta
Original Article


We report the results of our investigations on the effect of antimicrobial photodynamic treatment (APDT) with poly-lysine-conjugated chlorin p6 (pl–cp6) on proinflammatory cytokine expression and wound healing in a murine excisional wound model infected with Pseudomonas aeruginosa. Treatment of infected wounds with pl–cp6 and light doses of 60 and 120 J/cm2 reduced the bacterial load by ~1.5 and 2.0 log, respectively, after 24 h. The treated wounds healed ~5 days earlier as compared to untreated control and wound closure was not dependent on light dose. Interestingly, at 96 h post-treatment, drug-treated wounds irradiated at 60 J/cm2 showed considerable reduction of proinflammatory cytokines IL-6 (approximately five times) and TNF-α (approximately four times) compared to untreated control. Further, exposure of culture supernatants to similar light dose and pl–cp6 concentration under in vitro conditions reduced the protease activity by ~50 % as compared to the untreated control, suggesting inactivation of extracellular virulent factors. Additionally, histological analysis of treated infected wounds showed complete reepithelialization, ordered collagen fibers, and considerable decrease in inflammatory cell infiltration compared to untreated wounds. These results imply that pl–cp6-mediated PDT reduces hyperinflammatory response of infected wounds, leading to acceleration of wound healing.


Chlorin p6 Photodynamic treatment Pseudomonas aeruginosa Wound infection 


  1. 1.
    Ansermino M, Hemsley C (2004) Intensive care management and control of infection. BMJ 329:220–223PubMedCrossRefGoogle Scholar
  2. 2.
    Biffl WL, Moore EE, Moore FA, Peterson VM (1996) Interleukin-6 in the injured patient. Marker of injury or mediator of inflammation? Ann Surg 224:647–664PubMedCrossRefGoogle Scholar
  3. 3.
    Bjarnsholt T, Kirketerp-Møller K, Jensen PØ et al (2008) Why chronic wounds will not heal: a novel hypothesis. Wound Rep Regen 16:2–10CrossRefGoogle Scholar
  4. 4.
    Bryan D, Walker KB, Ferguson M, Thorpe R (2005) Cytokine gene expression in a murine wound healing model. Cytokine 31:429–438PubMedCrossRefGoogle Scholar
  5. 5.
    Caminos DA, Spesia MB, Durantini EN (2006) Photodynamic inactivation of Escherichia coli by novel meso-substituted porphyrins by 4-(3-N, N, N-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups. Photochem Photobiol Sci 5:56–65PubMedCrossRefGoogle Scholar
  6. 6.
    Dai T, Tegos GP, Zhiyentayev T, Mylonakis E, Hamblin MR (2010) Photodynamic therapy for methicillin-resistant Staphylococcus aureus infection in a mouse skin abrasion model. Lasers Surg Med 42:38–44PubMedCrossRefGoogle Scholar
  7. 7.
    Deng J, Younge BR, Olshen RA, Goronzy JJ, Weyand CM (2010) Th17 and Th1 T-cell responses in giant cell arthritis. Circulation 23:906–915CrossRefGoogle Scholar
  8. 8.
    Fajardo LF, Kwan HH, Kowalski J, Prionas SD, Allison AC (1992) Dual role of tumor necrosis factor-alpha in angiogenesis. Am J Pathol 140:539–554PubMedGoogle Scholar
  9. 9.
    Fogle MR, Griswold JA, Oliver JW, Hamood AN (2002) Anti-ETA IgG neutralizes the effects of Pseudomonas aeruginosa exotoxin. A J Surg Res 106:86–98CrossRefGoogle Scholar
  10. 10.
    Gollnick SO, Evans SS, Baumann H et al (2003) Role of cytokines in photodynamic therapy-induced local and systemic inflammation. Br J Cancer 88:1772–1779PubMedCrossRefGoogle Scholar
  11. 11.
    Granick M, Boykin J, Gamelli R, Schultz G, Tenenhaus M (2006) Towards a common language: surgical wound bed preparation and debridement. Wound Repair Regen 14:S1–S10PubMedCrossRefGoogle Scholar
  12. 12.
    Guo S, Dipietro LA (2010) Factors affecting wound healing. J Dent Res 89:219–229PubMedCrossRefGoogle Scholar
  13. 13.
    Hamblin MR, O’Donnell DA, Murthy N, Contag CH, Hasan T (2002) Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging. Photochem Photobiol 75:51–57PubMedCrossRefGoogle Scholar
  14. 14.
    Hamblin MR, Zahra T, Contag CH, McManus AT, Hasan T (2003) Optical monitoring and treatment of potentially lethal wound infections in vivo. J Infect Dis 187:1717–1725PubMedCrossRefGoogle Scholar
  15. 15.
    Hamblin MR, Hasan T (2004) Photodynamic therapy: a new antimicrobial approach to infectious disease. Photochem Photobiol Sci 3:436–450PubMedCrossRefGoogle Scholar
  16. 16.
    Han YP, Tuan TL, Wu H, Hughes M, Garner WL (2001) TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP. J Cell Sci 114:131–139PubMedGoogle Scholar
  17. 17.
    Heggers JP (2003) Assessing and controlling wound infection. Clin Plast Surg 30:25–35PubMedCrossRefGoogle Scholar
  18. 18.
    Hoober JK, Sery TW, Yamamoto N (1988) Photodynamic sensitizers from chlorophyll: purpurin-18 and chlorin p6. Photochem Photobiol 48:579–582PubMedCrossRefGoogle Scholar
  19. 19.
    Ipaktchi K, Mattar A, Niederbichler AD et al (2007) Topical p38 MAPK inhibition reduces bacterial growth in an in vivo burn wound model. Surgery 142:86–93PubMedCrossRefGoogle Scholar
  20. 20.
    Kanno E, Kawakami K, Ritsu M et al (2011) Wound healing in skin promoted by inoculation with Pseudomonas aeruginosa PAO1: the critical role of tumor necrosis factor-α secreted from infiltrating neutrophils. Wound Repair Regen 19:608–621PubMedCrossRefGoogle Scholar
  21. 21.
    Kjolseth D, Frank JM, Barker JH et al (1994) Comparison of the effects of commonly used wound agents on epithelialization and neovascularization. J Am Coll Surg 179:305–312PubMedGoogle Scholar
  22. 22.
    Kömerik N, Wilson M, Poole S (2000) The effect of photodynamic action on two virulence factors of gram-negative bacteria. Photochem Photobiol 72:676–680PubMedCrossRefGoogle Scholar
  23. 23.
    Kubler A, Finley RK 3rd, Born IA, Mang TS (1996) Effect of photodynamic therapy on the healing of a rat skin flap and its implication for head and neck reconstructive surgery. Lasers Surg Med 18:397–405PubMedCrossRefGoogle Scholar
  24. 24.
    Liu R, Bal HS, Desta T, Behl Y, Graves DT (2006) Tumor necrosis factor-alpha mediates diabetes-enhanced apoptosis of matrix-producing cells and impairs diabetic healing. Am J Pathol 168:757–764PubMedCrossRefGoogle Scholar
  25. 25.
    Lu Z, Dai T, Huang L, Kurup DB, Tegos GP, Jahnke A, Wharton T, Hamblin MR (2010) Photodynamic therapy with a cationic functionalized fullerene rescues mice from fatal wound infections. Nanomedicine 5:1525–1533PubMedCrossRefGoogle Scholar
  26. 26.
    Mc Ripley RJ, Whitney RR (1976) Characterization and quantitation of experimental surgical-wound infections used to evaluate topical antibacterial agents. Antimicrob Agents Chemother 10:38–44CrossRefGoogle Scholar
  27. 27.
    Meireles GC, Santos JN, Chagas PO, Moura AP, Pinheiro AL (2008) Effectiveness of laser photobiomodulation at 660 or 780 nanometers on the repair of third-degree burns in diabetic rats. Photomed Laser Surg 26:47–54PubMedCrossRefGoogle Scholar
  28. 28.
    Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N (2002) Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration. FASEB J 16:963–974PubMedCrossRefGoogle Scholar
  29. 29.
    Neely AN, Hoover DL, Holder IA, Cross AS (1996) Circulating levels of tumour necrosis factor, interleukin 6, and proteolytic activity in a murine model of burn and infection. Burns 22:524–530PubMedCrossRefGoogle Scholar
  30. 30.
    Omar GS, Wilson M, Nair SP (2008) Lethal photosensitization of wound-associated microbes using indocyanine green and near-infrared light. BMC Microbiol 8:111PubMedCrossRefGoogle Scholar
  31. 31.
    Parekh SG, Trauner KB, Zarins B, Foster TE, Anderson RR (1999) Photodynamic modulation of wound healing with BPD-MA and CASP. Lasers Surg Med 24:375–381PubMedCrossRefGoogle Scholar
  32. 32.
    Pruitt BA Jr, McManus AT, Kim SH, Goodwin CW (1998) Burn wound infections: current status. World J Surg 22:135–145PubMedCrossRefGoogle Scholar
  33. 33.
    Sharma M, Bansal H, Gupta PK (2005) Virulence of Pseudomonas aeruginosa cells surviving photodynamic treatment with toluidine blue. Curr Microbiol 50:277–280PubMedCrossRefGoogle Scholar
  34. 34.
    Simonetti O, Cirioni O, Orlando F et al (2011) Effectiveness of antimicrobial photodynamic therapy with a single treatment of RLP068/Cl in an experimental model of Staphylococcus aureus wound infection. Br J Dermatol 164:987–995PubMedCrossRefGoogle Scholar
  35. 35.
    Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85PubMedCrossRefGoogle Scholar
  36. 36.
    Smith RS, Harris SG, Phipps R, Iglewski B (2002) The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)homoserine lactone contributes to virulence and induces inflammation in vivo. J Bacteriol 184:1132–1139PubMedCrossRefGoogle Scholar
  37. 37.
    Sperandio FF, Simões A, Aranha AC, Corrêa L, Machado O, de Sousa SC (2010) Photodynamic therapy mediated by methylene blue dye in wound healing. Photomed Laser Surg 28:581–587PubMedCrossRefGoogle Scholar
  38. 38.
    Tanaka M, Kinoshita M, Yoshihara Y et al (2010) Influence of intra-articular neutrophils on the effects of photodynamic therapy for murine MRSA arthritis. Photochem Photobiol 86:403–409PubMedCrossRefGoogle Scholar
  39. 39.
    Wright JB, Lam K, Olson ME, Burrell RE (2003) Is antimicrobial efficacy sufficient? A question concerning the benefits of new dressings. Wounds 15:133–142Google Scholar

Copyright information

© Springer-Verlag London Ltd 2012

Authors and Affiliations

  • Khageswar Sahu
    • 1
  • Mrinalini Sharma
    • 1
    Email author
  • Harsha Bansal
    • 1
  • Alok Dube
    • 1
  • Pradeep Kumar Gupta
    • 1
  1. 1.Laser Biomedical Applications and Instrumentation DivisionRaja Ramanna Centre for Advanced TechnologyIndoreIndia

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