Applied Microbiology and Biotechnology

, Volume 101, Issue 11, pp 4691–4700 | Cite as

Rapid killing of bacteria by a new type of photosensitizer

  • Yaxin Zhang
  • Ke Zheng
  • Zhuo Chen
  • Jincan Chen
  • Ping Hu
  • Linrong Cai
  • Zafar Iqbal
  • Mingdong Huang
Applied microbial and cell physiology


Photodynamic antimicrobial chemotherapy (PACT) uses non-traditional mechanisms (free radicals) and is a highly advocated method with promise of inactivating drug-resistance bacteria for local infections. However, there is no related drug used in clinical practice yet. Therefore, new photosensitizers for PACT are under active development. Here, we report the synthesis of a series of photosensitizers with variable positive charges (ZnPc(TAP)4 n+, n = 0, 4, 8, 12) and their inactivation against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The binding kinetics of ZnPc(TAP)4 n+ to bacteria were measured by flow cytometer. Reactive oxygen species (ROS) generation mechanism of the photosensitizers was studied. The toxicity of these compounds to human blood cells was also evaluated. These compounds showed negligible toxicity against human erythocytes but potent bactericidal effects. The compound with 8 positive charges, ZnPc(TAP)4 8+, turned out to have the strongest antibacterial effect among this series of compounds, giving IC50 value of 59 nM at a light dosage of 5 J/cm2 toward E. coli. For a multi-resistant E. coli strain, ZnPc(TAP)4 8+ decreased the bacteria load by 1000-fold at a concentration of 1 μM. Interestingly, ZnPc(TAP)4 12+, instead of ZnPc(TAP)4 8+, exhibited the highest amount of binding to bacteria. Flow cytometry studies showed that all PSs have fast binding onto bacteria, reaching saturated binding within 5 min. Mechanistically, ZnPc(TAP)4 12+ generated ROS primarily via Type I mechanism, while ZnPc(TAP)4 4+ or ZnPc(TAP)4 8+ created ROS by both type I and type II mechanisms. ZnPc(TAP)4 n+ are highly potent, rapid-acting and non-toxic photosensitizers capable of inactivating bacteria.


Photodynamic antimicrobial chemotherapy (PACT) Mechanistic study ROS measurement Phthalocyanine Photosensitizer 



We thank The Second Municipal Hospital of Fuzhou for providing us the strain of multi-resistant E. coli. This work was supported by grants from National Natural Science Foundation of China (81171634), Natural Science Foundation of Fujian Province (2013J01066).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


This work is supported by grants from Natural Science Foundation of China (31,370,737, 31,400,637, 31,570,745, 31,670,739, 81,572,944, and U1405229), and the CAS/SAFEA International Partnership Program for Creative Research Teams.

Supplementary material

253_2017_8133_MOESM1_ESM.pdf (498 kb)
ESM 1 (PDF 497 kb).


  1. Alves E, Costa L, Carvalho CM, Tome JP, Faustino MA, Neves MG, Tome AC, Cavaleiro JA, Cunha A, Almeida A (2009) Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins. BMC Microbiol 9:70. doi: 10.1186/1471-2180-9-70 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bai H, Yuan H, Nie C, Wang B, Lv F, Liu L, Wang S (2015) A supramolecular antibiotic switch for antibacterial regulation. Angew Chem Int Ed Engl. doi: 10.1002/anie.201504566 PubMedCentralGoogle Scholar
  3. Banfi S, Caruso E, Buccafurni L, Battini V, Zazzaron S, Barbieri P, Orlandi V (2006) Antibacterial activity of tetraaryl-porphyrin photosensitizers: an in vitro study on Gram negative and Gram positive bacteria. J Photochem Photobiol B 85(1):28–38. doi: 10.1016/j.jphotobiol.2006.04.003 CrossRefPubMedGoogle Scholar
  4. Chen Z, Zhou S, Chen J, Li L, Hu P, Chen S, Huang M (2011) An effective zinc phthalocyanine derivative for photodynamic antimicrobial chemotherapy. J Lumin 152:103–107. doi: 10.1016/j.jlumin.2013.10.067 CrossRefGoogle Scholar
  5. Chen Z, Zhou S, Chen J, Li L, Hu P, Chen S, Huang M (2014) An effective zinc phthalocyanine derivative for photodynamic antimicrobial chemotherapy. J Lumin 152:103–107. doi: 10.1016/j.jlumin.2013.10.067 CrossRefGoogle Scholar
  6. Dumoulin F, Durmuş M, Ahsen V, Nyokong T (2010) Synthetic pathways to water-soluble phthalocyanines and close analogs. Coord Chem Rev 254(23–24):2792–2847. doi: 10.1016/j.ccr.2010.05.002 CrossRefGoogle Scholar
  7. Dwyer DJ, Belenky PA, Yang JH (2014) Antibiotics induce redox-related physiological alterations as part of their lethality. 111(20):E2100–9 doi: 10.1073/pnas.1401876111
  8. Eichner A, Gonzales FP, Felgentrager A, Regensburger J, Holzmann T, Schneider-Brachert W, Baumler W, Maisch T (2013) Dirty hands: photodynamic killing of human pathogens like EHEC, MRSA and Candida within seconds. Photochem Photobiol Sci 12(1):135–147. doi: 10.1039/c2pp25164g CrossRefPubMedGoogle Scholar
  9. Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel T (2003) In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24(7):1121–1131. doi: 10.1016/s0142-9612(02)00445-3 CrossRefPubMedGoogle Scholar
  10. Gümüştaş MK, Sesalan BS, Atukeren P, Yavuz B, Gül A (2010) The photodegradation of a zinc phthalocyanine. J Coord Chem 63(24):4319–4331. doi: 10.1080/00958972.2010.534987 CrossRefGoogle Scholar
  11. Guo Q, Yue Q, Zhao J, Wang L, Wang H, Wei X, Liu J, Jia J (2011) How far can hydroxyl radicals travel? An electrochemical study based on a DNA mediated electron transfer process. Chem Commun (Camb) 47(43):11906–11908. doi: 10.1039/c1cc14699h CrossRefGoogle Scholar
  12. Hamblin MR (2016) Antimicrobial photodynamic inactivation: a bright new technique to kill resistant microbes. Curr Opin Microbiol 33:67–73. doi: 10.1016/j.mib.2016.06.008 CrossRefPubMedGoogle Scholar
  13. Hemmerling A, Harrison W, Schroeder A, Park J, Korn A, Shiboski S, Foster-Rosales A, Cohen CR (2010) Phase 2a study assessing colonization efficiency, safety, and acceptability of Lactobacillus crispatus CTV-05 in women with bacterial vaginosis. Sex Transm Dis 37(12):745–750. doi: 10.1097/Olq.0b013e3181e50026 CrossRefPubMedGoogle Scholar
  14. Huang L, Xuan Y, Koide Y, Zhiyentayev T, Tanaka M, Hamblin MR (2012) Type I and Type II mechanisms of antimicrobial photodynamic therapy: An in vitro study on gram-negative and gram-positive bacteria. Lasers Surg Med 44(6):490–499. doi: 10.1002/lsm.22045 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jiang Z, Shao J, Yang T, Wang J, Jia L (2014) Pharmaceutical development, composition and quantitative analysis of phthalocyanine as the photosensitizer for cancer photodynamic therapy. J Pharm Biomed Anal 87:98–104. doi: 10.1016/j.jpba.2013.05.014 CrossRefPubMedGoogle Scholar
  16. Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, Fantetti L, Chiti G, Roncucci G (2006) Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers Surg Med 38(5):468–481. doi: 10.1002/lsm.20361 CrossRefPubMedGoogle Scholar
  17. Karsi A, Lawrence ML (2007) Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria. Plasmid 57(3):286–295. doi: 10.1016/j.plasmid.2006.11.002
  18. Kiesslich T, Gollmer A, Maisch T, Berneburg M, Plaetzer K (2013) A comprehensive tutorial on in vitro characterization of new photosensitizers for photodynamic antitumor therapy and photodynamic inactivation of microorganisms. Biomed Res Int 2013:840417. doi: 10.1155/2013/840417 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Li K, Lei W, Jiang G, Hou Y, Zhang B, Zhou Q, Wang X (2014) Selective photodynamic inactivation of bacterial cells over mammalian cells by new triarylmethanes. Langmuir 30(48):14573–14580. doi: 10.1021/la5028724 CrossRefPubMedGoogle Scholar
  20. Li R, Xiao F, Zheng X, Yang H, Wang L, Yin D, Yin T, Xin Q, Chen B (2016) Antibiotic misuse among children with diarrhea in China: results from a national survey. PeerJ 4:e2668. doi: 10.7717/peerj.2668 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li XS, Guo J, Zhuang JJ, Zheng BY, Ke MR, Huang JD (2015) Highly positive-charged zinc(II) phthalocyanine as non-aggregated and efficient antifungal photosensitizer. Bioorg Med Chem Lett 25(11):2386–2389. doi: 10.1016/j.bmcl.2015.04.004 CrossRefPubMedGoogle Scholar
  22. Maisch T (2015) Resistance in antimicrobial photodynamic inactivation of bacteria. Photochem Photobiol Sci 14(8):1518–1526. doi: 10.1039/c5pp00037h CrossRefPubMedGoogle Scholar
  23. Maisch T, Spannberger F, Regensburger J, Felgentrager A, Baumler W (2012) Fast and effective: intense pulse light photodynamic inactivation of bacteria. J Ind Microbiol Biotechnol 39(7):1013–1021. doi: 10.1007/s10295-012-1103-3 CrossRefPubMedGoogle Scholar
  24. Maisch T, Szeimies RM, Jori G, Abels C (2004) Antibacterial photodynamic therapy in dermatology. Photochem Photobiol Sci 3(10):907–917. doi: 10.1039/b407622b CrossRefPubMedGoogle Scholar
  25. Mantareva VN, Angelov I, Wöhrle D, Borisova E, Kussovski V (2013) Metallophthalocyanines for antimicrobial photodynamic therapy: an overview of our experience. J Porphyrins Phthalocyanines 17(06n07):399–416. doi: 10.1142/s1088424613300024 CrossRefGoogle Scholar
  26. Marinho CM, Santos T, Goncalves A, Poeta P, Igrejas G (2016) A decade-long commitment to antimicrobial resistance surveillance in Portugal. Front Microbiol 7:1650. doi: 10.3389/fmicb.2016.01650 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Mikula P, Kalhotka L, Jancula D, Zezulka S, Korinkova R, Cerny J, Marsalek B, Toman P (2014) Evaluation of antibacterial properties of novel phthalocyanines against Escherichia coli–comparison of analytical methods. J Photochem Photobiol B 138:230–239. doi: 10.1016/j.jphotobiol.2014.04.014 CrossRefPubMedGoogle Scholar
  28. Morley S, Griffiths J, Philips G, Moseley H, O'Grady C, Mellish K, Lankester CL, Faris B, Young RJ, Brown SB, Rhodes LE (2013) Phase IIa randomized, placebo-controlled study of antimicrobial photodynamic therapy in bacterially colonized, chronic leg ulcers and diabetic foot ulcers: a new approach to antimicrobial therapy. The British journal of dermatology 168(3):617–624. doi: 10.1111/bjd.12098 CrossRefPubMedGoogle Scholar
  29. Osifeko OL, Durmuş M, Nyokong T (2015) Physicochemical and photodynamic antimicrobial chemotherapy studies of mono- and tetra-pyridyloxy substituted indium(III) phthalocyanines. J Photochem Photobiol A Chem 301:47–54. doi: 10.1016/j.jphotochem.2014.12.011 CrossRefGoogle Scholar
  30. Redmond RW, Kochevar IE (2006) Spatially resolved cellular responses to singled oxygen. Photochem and Photobiology 82:1178–1186. doi: 10.1562/2006-04-14-1R-874 CrossRefGoogle Scholar
  31. Scalise I, Durantini EN (2005) Synthesis, properties, and photodynamic inactivation of Escherichia coli using a cationic and a noncharged Zn(II) pyridyloxyphthalocyanine derivatives. Bioorg Med Chem 13(8):3037–3045. doi: 10.1016/j.bmc.2005.01.063 CrossRefPubMedGoogle Scholar
  32. Sesalan BŞ, Koca A, Gül A (2008) Water soluble novel phthalocyanines containing dodeca-amino groups. Dyes Pigments 79(3):259–264. doi: 10.1016/j.dyepig.2008.03.006 CrossRefGoogle Scholar
  33. Smirnova ZS, Oborotova NA, Makarova OA, Orlova OL, Polozkova AP, Kubasova IY, Luk'yanets EA, Meerovich GA, Zimakova NI, Kuz'min SG, Vorozhtsov GN, Baryshnikov AY (2005) Efficiency and pharmacokinetics of photosense: a new liposomal photosensitizer formulation based on aluminum sulfophthalocyanine. Pharm Chem J 39(7):341–344. doi: 10.1007/s11094-005-0150-8 CrossRefGoogle Scholar
  34. World Health Assembly (2015) Sixty-eighth World Health Assembly. Publisher.
  35. Wright PM, Seiple IB, Myers AG (2014) The evolving role of chemical synthesis in antibacterial drug discovery. Angew Chem Int Ed Engl 53(34):8840–8869. doi: 10.1002/anie.201310843 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Yin R, Hamblin M (2015) Antimicrobial photosensitizers: drug discovery under the spotlight. Curr Med Chem 22(18):2159–2185. doi: 10.2174/0929867322666150319120134 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Yaxin Zhang
    • 1
  • Ke Zheng
    • 2
  • Zhuo Chen
    • 1
  • Jincan Chen
    • 1
  • Ping Hu
    • 1
  • Linrong Cai
    • 1
  • Zafar Iqbal
    • 1
    • 3
  • Mingdong Huang
    • 1
    • 4
  1. 1.State Key Laboratory of Structural ChemistryFujian Institute of Research on the Structure of Matter, Chinese Academy of SciencesFuzhouChina
  2. 2.College of Chemical EngineeringQingdao University of Science and TechnologyQingdaoChina
  3. 3.Department of ChemistryCOMSATS Institute of Information Technology (CIIT)AbbottabadPakistan
  4. 4.College of ChemistryFuzhou UniversityFuzhouChina

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