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

Abstract

The spatial control of optical absorption provided by twophoton excitation (TPE) has led to tremendous advances in microscopy 1 and microfabrication 2 . Medical applications of TPE in photodynamic therapy (PDT) 3,4 have often been suggested 5-18 , but have been made impractical by the low twophoton cross-sections of photosensitiser drugs (i.e. compounds taken up by living tissues that become toxic on absorption of light). The invention of efficient two-photon activated drugs will allow precise manipulation of treatment volumes in three dimensions, to a level unattainable with current techniques. Here we present a new family of PDT drugs designed for efficient TPE, and use one of them to demonstrate selective closure of blood vessels via TPE-PDT in vivo. These conjugated porphyrin dimers have two-photon cross-sections that are more than two orders of magnitude greater than those of clinical photosensitisers 17 . This is the first demonstration of in vivo PDT using a photosensitiser engineered for efficient two-photon excitation.Photodynamic therapy is used to treat diseases characterised by neoplastic growth including various cancers, age-related macular degeneration (AMD) and actinic keratosis 3,4 . Cell death is induced by photoexcitation of a sensitiser, generally via production of singlet oxygen. In the absence of light the photosensitiser is benign, so systemic toxicity is rare and treatment may be repeated without acquired resistance. Two-photon excitation of the photosensitiser should allow greater precision than is attainable by conventional one-photon excitation, as a consequence of the quadratic dependence of TPE on the local light intensity -the amount of TPE is inversely proportional to the fourth power of the distance from the focus. In addition, the longer wavelengths associated with TPE allow treatment deeper into tissue, by minimising absorption from endogenous chromophores.High instantaneous photon densities are essential for two-photon excitation. Early TPE-PDT studies used nanosecond lasers, but the dominant effect was photothermal damage [5][6][7] . The advent of commercial femtosecond tuneable Ti:sapphire lasers has greatly facilitated the investigation of TPE-PDT, and the limiting factor has become the availability of suitable photosensitisers. The majority of chromophores possess low two-photon cross-sections, of the order of 1-100 Goeppert-Mayer units (1 GM = 10 -50 cm 4 s photon -1 ). For example, the two FDA-approved PDT photosensitisers, verteporfin and Photofrin (cross sections 50 GM and 10 GM respectively) 17 , are unlikely to be suitable for TPE-PDT, as the high light intensities needed to achieve a therapeutic effect are close to the thresholds for photothermal or photomechanical damage 18 .Several design strategies for TPE-PDT photosensitisers have been reported recently [11][12][13][14][15][16] , but few of these compounds have yet been studied in vitro 15 , and, to date, none have progressed to in vivo testing. Porphyrin derivatives are often effective PDT agents, as exemplified ...

Multi-photon excitation in uncaging the small molecule bioregulator nitric oxide

“…The latter has been demonstrated in vivo by using TPE and PDT dyes to seal blood vessels in mice [42]. Nonetheless, there is some concern about the difficulty of achieving appropriate focusing of lasers given the scattering inherent to a nonhomogeneous material such as mammalian tissue.…”

Imaging intracellular viscosity of a single cell during photoinduced cell death

“…1b) and initiates cell death. 17,20 The series of emission spectra obtained upon irradiation of 1 in a single cell, Fig. 2c, correspond to a significant increase in the viscosity of the intracellular environment surrounding 1.…”

“…Recently, the remarkable non-linear optical properties of 1, together with its high intracellular uptake, photostability and favorable photophysical properties, have been exploited to create an efficient photosensitizer for one-photon and two-photon excited photodynamic therapy (PDT) of cancer. [17][18][19][20] In PDT, cell death and tissue eradication is achieved upon irradiation of the photosensitizer and the subsequent production of cytotoxic species, in particular 4 singlet molecular oxygen, O 2 (a 1 Δ g ), Fig. 1b.…”

Photophysical properties and intracellular imaging of water-soluble porphyrin dimers for two-photon excited photodynamic therapy

“…28,29 This photosensitiser was used successfully to close 40 ± 5 µm blood vessels in the mouse window chamber model by applying two-photon excitation. 28 This article explores the photophysical properties and intracellular 100 uptake of a series of amphiphilic conjugated porphyrin dimers, related to P 2 C 2 -NMeI, see Fig. 1.…”