Simultaneous Two‐Photon Activation of Type‐I Photodynamic Therapy Agents

Abstract

The excitation and emission properties of several psoralen derivatives are compared using conventional single-photon excitation and simultaneous two-photon excitation (TPE). Two-photon excitation is effected using the output of a mode-locked titanium: sapphire laser, the near infrared output of which is used to promote nonresonant TPE directly. Specifically, the excitation spectra and excited-state properties of 8-methoxypsoralen and 4'-aminomethyl-4,5,8-trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two-photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two-photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamaging near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage.

New Approaches to Photodynamic Therapy from Types I, II and III to Type IV Using One or More Photons

“…Exposure of a PS to ultrashort (< 1 picosecond, ps) pulses of NIR light allows efficient "simultaneous" absorption of two photons by the chromophore in the ground state leading to the same excited state as commonly achieved by one-photon excitation (Figure 4B), as long as the overall energy provided is equivalent. Hence PS activation is also followed by the same photodynamic processes normally induced by one-photon excitation, but with improved treatment depth and selectivity to the treatment area [5,17,25,28,51,87]. Other advantages of 2PE are the highly localized light delivery restricting PS excitation to a very small (femtolitre) confined three dimensional volume and therefore reduction of damage to collateral healthy tissue or potential to target individual tumour blood vessels.…”

“…Other advantages of 2PE are the highly localized light delivery restricting PS excitation to a very small (femtolitre) confined three dimensional volume and therefore reduction of damage to collateral healthy tissue or potential to target individual tumour blood vessels. Compared with one-photon excitation for PS activation laser-induced hyperthermia and therefore phototoxicity is reduced when using 2PE, as NIR light scattering and absorption in tissues is lower compared with the absorption of UV or visible light by biological molecules [5,17,25,28,29,50,51,85,87,89,91]. Furthermore previously unsuitable PSs with excitation in the UV may now be explored by the 2P or multiphoton process using visible and NIR femtosecond light.…”

“…The main advantage of using 2PE in PDT is the significantly improved penetration depth of the excitation light in tissues compared with UV or visible light, which is strongly absorbed by endogenous chromophores such as haemoglobin or melanin [17,28,29,51,85,87,89]. In the life sciences two-photon and multiphoton excitation with NIR light has been successfully used for the imaging of thick biological specimens with reduced photo-damage and improved 3D sectioning compared to conventional scanning confocal microscopy [17,87,90]. Exposure of a PS to ultrashort (< 1 picosecond, ps) pulses of NIR light allows efficient "simultaneous" absorption of two photons by the chromophore in the ground state leading to the same excited state as commonly achieved by one-photon excitation (Figure 4B), as long as the overall energy provided is equivalent.…”

“…630 nm) and need to be carefully chosen depending on the PS being used. The development of laser diodes with multiple wavelengths is highly desired [10,28,32,68,87].…”

Use of coherent control methods through scattering biological tissue to achieve functional imaging

“…Coherent control schemes, for example, could enhance methods such as two-photon imaging (18,19), which has provided higher resolution, lower background scattering, and better sample penetration than traditional techniques. Selective two-photon excitation would be desirable for distinguishing healthy from cancerous tissue based on differences in their chemical properties, such as pH, or for activating a photodynamic therapy (PDT) agent only when it is absorbed by cancer cells by using twophoton activated PDT (20). These improvements would be possible only if shaped laser pulses maintain their unique properties as they transmit through scattering biological tissue.…”