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Blood-vessel closure using photosensitizers engineered for two-photon excitation


The spatial control of optical absorption provided by two-photon excitation has led to tremendous advances in microscopy1 and microfabrication2. Medical applications of two-photon excitation in photodynamic therapy3,4 have been widely suggested5,6,7,8,9,10,11,12,13,14,15,16,17,18, but thus far have been rendered impractical by the low two-photon cross-sections of photosensitizer drugs (which are compounds taken up by living tissues that become toxic on absorption of light). The invention of efficient two-photon activated drugs will allow precise three-dimensional manipulation of treatment volumes, providing a level of targeting unattainable with current therapeutic techniques. Here we present a new family of photodynamic therapy drugs designed for efficient two-photon excitation and use one of them to demonstrate selective closure of blood vessels through two-photon excitation photodynamic therapy in vivo. These conjugated porphyrin dimers have two-photon cross-sections that are more than two orders of magnitude greater than those of standard clinical photosensitizers17. This is the first demonstration of in vivo photodynamic therapy using a photosensitizer engineered for efficient two-photon excitation.

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Figure 1: Structures and absorption spectra of conjugated porphyrin dimers 1–5, and the clinical photosensitizer verteporfin, 6.
Figure 2: In vitro photodynamic therapy.
Figure 3: Fluorescence from photosensitizer 1 in vivo.
Figure 4: In vivo two-photon blood-vessel closure with photosensitizer 1.

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  1. Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: multiphoton microscopy in the biosciences. Nature Biotechnol. 21, 1369–1377 (2003).

    Article  Google Scholar 

  2. Cumpston, B. H. et al. Two-photon polymerization initiators for three-dimensional optical data storage and microfabrication. Nature 398, 51–54 (1999).

    Article  ADS  Google Scholar 

  3. Castano, A. P., Mroz, P. & Hamblin, M. R. Photodynamic therapy and anti-tumour immunity. Nature Rev. Cancer 6, 535–545 (2006).

    Article  Google Scholar 

  4. Macdonald, I. J. & Dougherty, T. J. Basic principles of photodynamic therapy. J. Porphyrins Phthalocyanines 5, 105–129 (2001).

    Article  Google Scholar 

  5. Marchesini, R. et al. A study on the possible involvement of nonlinear mechanism of light absorption by HpD with Nd:YAG laser. Lasers Surg. Med. 6, 323–327 (1986).

    Article  Google Scholar 

  6. Patrice, T. M. et al. Neodymium-yttrium aluminium garnet laser destruction of nonsensitized and hematoporphyrin derivative-sensitised tumors. Cancer Res. 43, 2876–2879 (1983).

    Google Scholar 

  7. Lenz, P. In vivo excitation of photosensitizers by infrared light. Photochem. Photobiol. 62, 333–338 (1995).

    Article  Google Scholar 

  8. Fisher, W. G., Partridge, W. P., Dees, C. & Wachter, E. A. Simultaneous two-photon activation of type-I photodynamic therapy agents. Photochem. Photobiol. 66, 141–155 (1997).

    Article  Google Scholar 

  9. Bhawalkar, J. D., Kumar, N. D., Zhao, C. F. & Prasad, P. N. Two-photon photodynamic therapy. Lasers Surg. Med. 15, 201–204 (1997).

    Article  Google Scholar 

  10. Samkoe, K. S. & Cramb, D. T. Application of an ex ovo chicken choriallantoic membrane model for two-photon excitation photodynamic therapy of age-related macular degeneration. J. Biomed. Optics 8, 410–417 (2003).

    Article  ADS  Google Scholar 

  11. Karotki, A. et al. Efficient singlet oxygen generation upon two-photon excitation of a new porphyrin with enhanced nonlinear absorption. IEEE J. Select. Top. Quant. Electron. 7, 971–975 (2001).

    Article  ADS  Google Scholar 

  12. Spangler, C. W. et al. Synthesis, characterisation and preclinical studies of two-photon-activated targeted PDT therapeutic triads. Proc. SPIE 6139, 61390X (2006).

    Article  Google Scholar 

  13. Oar, M. A. et al. Light-harvesting chromophores with metalated porphyrin cores for tuned photosensitization of singlet oxygen via two-photon excited FRET. Chem. Mater. 18, 3682–3692 (2006).

    Article  Google Scholar 

  14. Dy, J. T., Ogawa, K., Satake, A., Ishizumi, A. & Kobuke, Y. Water-soluble self-assembled butadiyne-bridged bisporphyrin: a potential two-photon-absorbing photosensitizer for photodynamic therapy. Chem. Eur. J. 13, 3491–3500 (2007).

    Article  Google Scholar 

  15. Kim, S., Ohulchanskyy, T. Y., Pudavar, H. E., Pandey, R. K. & Prasad, P. N. Organically modified silica nanoparticles co-encapsulating photosensitizing drug and aggregation-enhanced two-photon absorbing fluorescent dye aggregates for two-photon photodynamic therapy. J. Am. Chem. Soc. 129, 2669–2675 (2007).

    Article  Google Scholar 

  16. Arnbjerg, J. et al. Two-photon absorption in tetraphenylporphycenes: are porphycenes better candidates than porphyrins for providing optimal optical properties for two-photon photodynamic therapy? J. Am. Chem. Soc. 129, 5188–5199 (2007).

    Article  Google Scholar 

  17. Khurana, M. et al. Quantitative in vitro demonstration of two-photon photodynamic therapy using Photofrin® and Visudyne®. Photochem. Photobiol. 83, 1441–1448 (2007).

    Article  Google Scholar 

  18. Samkoe, K. S., Clancy, A. A., Karotki, A., Wilson, B. C. & Cramb, D. T. Complete blood vessel occlusion in chick chorioallantoic membrane using two-photon excitation photodynamic therapy: implications for treatment of wet age-related macular degeneration. J. Biomed. Opt. 12, 034025 (2007).

    Article  ADS  Google Scholar 

  19. Drobizhev, M. et al. Understanding strong two-photon absorption in π-conjugated porphyrin dimers via double-resonance enhancement in a three-level model. J. Am. Chem. Soc. 126, 15352–15353 (2004).

    Article  Google Scholar 

  20. Drobizhev, M. et al. Extremely strong near-IR two-photon absorption in conjugated porphyrin dimers: quantitative description with three essential states model. J. Phys. Chem. B. 109, 7223–7236 (2005).

    Article  Google Scholar 

  21. Ogawa, K. et al. Strong two-photon absorption of self-assembled butadiyne-linked bisporphyrin. J. Am. Chem. Soc. 125, 13356–13357 (2003).

    Article  Google Scholar 

  22. Kuimova, M. K. et al. Determination of the triplet state energies of a series of conjugated porphyrin oligomers. Photochem. Photobiol. Sci. 6, 675–682 (2007).

    Article  Google Scholar 

  23. Cory, A. H., Owen, T. C., Barltrop, J. A. & Cory, J. G. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 3, 207–212 (1991).

    Article  Google Scholar 

  24. Algire, G. H. & Legallais, F. Y. Recent developments in the transparent-chamber technique as adapted to the mouse. J. Natl Cancer Inst. 10, 225–253 (1949).

    Google Scholar 

  25. Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with verteporfin: one-year results of 2 randomized clinical trials — TAP report 1. Arch. Ophthalmol. 117, 1329–1345 (1999).

  26. Huang, D. et al. Optical coherence tomography. Science 22, 1178–1181 (1991).

    Article  ADS  Google Scholar 

  27. Mariampillai, A. et al. Optical cardiogram gated 2D Doppler flow imaging at 1000 fps and 4D imaging at 36 fps on a swept source OCT system. Opt. Express 15, 1627–1638 (2007).

    Article  ADS  Google Scholar 

  28. Yang, V. X. D. et al. Improved phase-resolved optical Doppler tomography using the Kasai velocity estimator and histogram segmentation. Opt. Commun. 208, 209–214 (2002).

    Article  ADS  Google Scholar 

  29. Karotki, A. et al. Enhancement of two-photon absorption in tetrapyrrolic compounds. J. Opt. Soc. Am. B 20, 321–332 (2003).

    Article  ADS  Google Scholar 

  30. Wilkinson, F., Helman, W. P. & Ross, A. B. Rate constants for the decay and reactions of the lowest electronically excited singlet-state of molecular-oxygen in solution — an expanded and revised compilation, J. Phys. Chem. Ref. Data 24, 663–1021 (1995).

    Article  ADS  Google Scholar 

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We thank M.K. Akens, T.D. McKee and K. Patel for assistance with tail vein injections, J. Jonkman and G. Netchev for microscopy assistance, the EPSRC (Engineering and Physical Sciences Research Council) Mass Spectrometry Service (Swansea) for mass spectra and Thorlabs for support with OCT imaging. This work was supported by grants from EPSRC (to H.L.A. and D.P.), the Canadian Institute for Photonic Innovations (to B.C.W.), Wenner-Gren Foundation (to E.D.) and the European Commission (Marie Curie Fellowship to M.B., MEIT-CT-2006-041522).

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H.A.C., E.D. and M.B. synthesized the porphyrin dimers. H.A.C. and E.D. screened the compounds in vitro. M.D. and A.R. measured the two-photon absorption spectra. M.K.K. measured singlet oxygen yields. E.H.M. and M.K. implanted the window chambers. A.M. imaged vessels by OCT, M.K. by stereomicroscopy and H.A.C. by hyperspectral and laser scanning techniques. All authors discussed the results and contributed to the manuscript.

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Correspondence to Brian C. Wilson or Harry L. Anderson.

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Collins, H., Khurana, M., Moriyama, E. et al. Blood-vessel closure using photosensitizers engineered for two-photon excitation. Nature Photon 2, 420–424 (2008).

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