<title>Photophysics of photosensitizing dyes in living cell suspensions</title>

Abstract

Photophysics of photosensitising dyes in living cell suspensionsTimothy C Oldham, Ilya V Eigenbrot, Ben Crystall and David PhillipsDepartment of Chemistry, Imperial College of Science, Technology and MedicineExhibition Road, London 5W7 2AY, UKABSTRACTThe use of photosensitisers to generate cytotoxic species in situ followingexposure to light is finding a wide range ofapplications. The photophysics of these sensitisers have been well characterised in homogeneous solution and insomemicroheterogeneous phases, however there have only been limited studies of the photophysics of the sensitisers insideliving cells. Such studies are crucial to the unambiguous elucidation of the photodynamic mechanism within the celland to the development of improved sensitisers and treatment modalities. We have constructeda laser flash photolysissystem designed specifically to probe the fate of the triplet state of excited sensitiser molecules in living cellsuspensions. Steady state and time resolved fluorescence techniques have also been applied to characterise thephotophysics of aluminium disulphonated phthalocyanine (A1PcS2) in aqueous suspensions of bacteria,yeast andmurine cell lines. We have observed considerable aggregation of AlPcS2 in all cell lines. Wereport triplet lifetimes inthe absence of oxygen which suggest that sensitiser triplet state deactivation isprimarily oxygen dependent.Fluorescence lifetime measurements suggest that there is no quenching of the singlet state. We have also madeprogresstowards the reduction of early time spikes which render singlet oxygen luminescence detection inaqueous solutiondifficult.Keywords: Photodynamic therapy, aluminium disulfonated phthalocyanine, cell suspensions, triplet states, singletoxygen, absorption spectroscopy.1 PDT MECHANISMS IN CELLS: A BLACK BOXThe applications of photodynamic therapy (PDT) are growing rapidly and nowencompass not only the treatment ofcancer, but also inactivation of bacteria,' viruses,2 yeasts,3 fungi and even insects.4 Similarly the number of sensitisersbeing screened is enormous,5 although relatively few of these have reached the clinical testing stage. Although it isobvious that PDT sensitisers are effective and that PDT is a viable method of inactivating a widerange of undesirablecell types, the chemical mechanism by which this is achieved remains unknown.In its initialstages it must bedetermined by the photophysics of the sensitiser. It is probable that each system of sensitiser plustarget cell represents adifferent black box.The PDT mechanism is generally believed to be mediated by the triplet state of the sensitiser. Recent studies havetended to support the view that no single or even predominant pathway is applicable to allsystems. Both Type I(electron transfer between sensitiser triplet state and biological substrates or even native free radicals15) andType II(energy transfer to form singlet oxygen) processes can have an impact.1619 The products oftheseprocesses ultimatelylead to cell death. The wide acceptance of the Type II singlet oxygen pathway as the dominant mode of actionstems inpart from the observation of efficient singlet oxygen generation in homogeneous and microheterogeneous solution, buteffective sensitisers incapable of formIng singlet oxygen have been reported.2° Further, thecompetition between Type Iand Type II processes is likely to be altered in vivo with respect to in vitro studies because the close associationbetween sensitiser and biological subsirate may enhance the former (and in principle could even lead to contributionsfrom excited singlet state electron transfer). The indirect quenching and trapping experiments used toimplicate singletoxygen can also be ambiguous due to the lack of specificity of many probes for singlet oxygen21 ,22 and the need forthese probes to be present in high concentrations.Notwithstanding a number of excellent indirect studies (e.g.614) it is our belief that we cannot begin to understand theexact processes by which excitation of sensitisers translates into the observed physiological effects without directspectroscopic studies of sensitiser photophysics on location inside cells. An understanding of the initial darkprocesses which occur following sensitiser excitation has obvious implications for the design of improved sensitisers inany given application. Such measurements can also be revealing about biochemical pathways and mechanisms of cell266