Two-photon Photodynamic Therapy Research Articles

Two-photon spectroscopic behaviors and photodynamic effect on the BEL-7402 cancer cells of the new chlorophyll photosensitizer

The spectroscopic properties of a new chlorophyll derivate photosensitizer (CDP) are studied under the excitation wavelengths at 800 and 400 nm using femtosecond pulses from a Ti: sapphire laser. The damaging effect of CDP on the BEL-7402 cancer cells is also investigated upon two-photon illumination at 800 nm. The normalized fluorescence spectra of CDP in tetrahydrofuran (THF) show that two-photon and one-photon spectra have the same distributions and the same emission bands (675 nm). The lifetimes of two-and one-photon induced fluorescence of this molecule are of the order of 5.0 ns. By comparing the data it is shown that there is some difference between the two lifetimes, but the difference is less than one nanosecond. The two-photon absorption cross section of the molecule is also measured at 800 nm and estimated as about σ′2 ≈ 31.5×10−50 cm4·s·photon−1. The results of two-photon photodynamic therapy (TPPDT) tests show that CDP can kill all of the tested cancer cells according to the usual Eosine assessment. Our results indicate that the two-photon-induced photophysical, photo-chemical and photosensitizing processes of CDP may be basically similar to those of one-photon excitation. These behaviors of the sample suggest that one may find other possible methods to estimate some photosensitizers’ effects in details such as their distribution in cells and the reactive targets of the sub-cellular parts of some tumor cells via two-photon excitation techniques.

Recent Advances in Two-Photon Photodynamic Therapy

Photodynamic therapy (PDT) is a treatment for tumors and accepted in several countries in the world. Introduction of two-photon absortion (2PA) into PDT allows spatially selective treatment of cancers. Possibilities and limitations of the use of two-photon excitation in PDT are discussed, and many literatures in this area are reviewed. The conclusions are that 2PA-PDT has an advantage for higher selectivity than one-photon absorption PDT, and femtosecond pulsed laser is more suitable for 2PA-PDT than pico- and nanosecond pulses. However, most of photosensitizers used in the past studies had low 2PA cross section values less than 50 GM, and resulted in a low PDT efficiency under two-photon irradiation conditions. To realize 2PA-PDT, much larger 2PA cross sections must be required.

Quantitative In Vitro Demonstration of Two‐Photon Photodynamic Therapy Using Photofrin® and Visudyne®

Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne and Photofrin are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed two-photon photosensitizers before proceeding to in vivo studies in preclinical animal models.

Syntheses and properties of acetylene-linked bis- and trisporphyrins toward two-photon photodynamic therapy

Acetylene-bridged bisporphyrins and trisporphyrins having branched bulky bis(carboxylethyl)methyl meso-substituents were synthesized. These compounds showed large effective two-photon absorption cross-section values at 890 nm measured by using a nanosecond Z-scan method. Sodium salt of hydrolyzed trisporphyrins showed broad and red-shifted Q-bands over 900 nm. Two-photon absorption cross-section values of water-soluble dimers in water were similar to, or slightly larger than, those of ester forms evaluated in toluene. Furthermore, the generation of singlet oxygen upon one-photon irradiation for dimers in water was confirmed.

Two-photon photodynamic therapy of C6 cells by means of 5-aminolevulinic acid induced protoporphyrin IX

Photodynamic therapy (PDT) has received increased attention as a treatment modality for malignant tumors as well as non-oncologic diseases such as age-related macular degeneration (AMD). An alternative to excite the photosensitizer by the common one-photon absorption is the method of two-photon excitation (TPE). This two-photon photodynamic therapy has the potential of improving the therapeutic outcome due to a highly localized photodynamic effect. The present study investigated the two-photon excited PDT performing in vitro experiments where C6 rat glioma cells were irradiated with a pulsed and focused fs Ti:sapphire laser emitting light at 800 nm. The irradiance distribution of the laser beam was carefully analyzed before the experiment and the applied irradiance was known for each position within the irradiated cell layer. Cells were divided into four groups and one group was incubated with 5-ALA and irradiated 4–5 h later. The survival of this group was tested after irradiation by means of ethidium bromide and acridine orange staining and compared to a control group, which was irradiated under the same conditions, but not incubated with 5-ALA before. Both groups showed necrotic areas depending on the applied irradiance, the value of which at the margin of the necrotic area could be deduced from its size. 5-ALA incubated cells became necrotic after irradiation with a mean irradiance above 6.1 × 10 10 W/cm 2, while non-incubated cells remained viable. Cells of both groups became necrotic when treated with an irradiance above 10.9 × 10 10 W/cm 2. The observed affected area of the cell layers was between 0.13 mm 2 and 1.10 mm 2. Since the irradiation of non-incubated cells below the mean power density of 10.9 × 10 10 W/cm 2 induced no necrosis, apparently no thermal damage was induced in the cells and necrosis of the 5-ALA incubated cells can be ascribed to the photodynamic effect induced by two-photon excitation. The successful photodynamic treatment of a large area of a monolayer cell culture induced by two-photon excitation offers new perspectives for photodynamic treatment modalities.

Two-Photon Absorption in Tetraphenylporphycenes: Are Porphycenes Better Candidates than Porphyrins for Providing Optimal Optical Properties for Two-Photon Photodynamic Therapy?

Porphycenes are structural isomers of porphyrins that have many unique properties and features. In the present work, the resonant two-photon absorption of 2,7,12,17-tetraphenylporphycene (TPPo) and its palladium(II) complex (PdTPPo) has been investigated. The data obtained are compared to those from the isomeric compound, meso-tetraphenylporphyrin (TPP). Detection of phosphorescence from singlet molecular oxygen, O2(a(1)Delta(g)), produced upon irradiation of these compounds, was used to obtain two-photon excitation spectra and to quantify two-photon absorption cross sections, delta. In the spectral region of 750-850 nm, the two-photon absorption cross sections at the band maxima for both TPPo and PdTPPo, delta = 2280 and 1750 GM, respectively, are significantly larger than that for TPP. This difference is attributed to the phenomenon of so-called resonance enhancement; for the porphycenes, the two-photon transition is nearly resonant with a comparatively intense one-photon Q-band transition. The results of quantum mechanical calculations using density functional quadratic response theory are in excellent agreement with the experimental data and, as such, demonstrate that comparatively high-level quantum chemical methods can be used to interpret and predict nonlinear optical properties from such large molecular systems. One important point realized through these experiments and calculations is that one must exercise caution when using qualitative molecular-symmetry-derived arguments to predict the expected spectral relationship between allowed one- and two-photon transitions. From a practical perspective, this study establishes that, in comparison to porphyrins and other tetrapyrrolic macrocyclic systems, porphycenes exhibit many desirable attributes for use as sensitizers in two-photon initiated photodynamic therapy.

Organically Modified Silica Nanoparticles Co-encapsulating Photosensitizing Drug and Aggregation-Enhanced Two-Photon Absorbing Fluorescent Dye Aggregates for Two-Photon Photodynamic Therapy

We report energy-transferring organically modified silica nanoparticles for two-photon photodynamic therapy. These nanoparticles co-encapsulate two-photon fluorescent dye nanoaggregates as an energy up-converting donor and a photosensitizing PDT drug as an acceptor. They combine two features: (i) aggregation-enhanced two-photon absorption and emission properties of a novel two-photon dye and (ii) nanoscopic fluorescence resonance energy transfer between this nanoaggregate and a photosensitizer, 2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide. Stable aqueous dispersions of the co-encapsulating nanoparticles (diameter < or = 30 nm) have been prepared in the nonpolar interior of micelles by coprecipitating an organically modified silica sol with the photosensitizer and an excess amount of the two-photon dye which forms fluorescent aggregates by phase separation from the particle matrix. Using a multidisciplinary nanophotonic approach, we show: (i) indirect excitation of the photosensitizer through efficient two-photon excited intraparticle energy transfer from the dye aggregates in the intracellular environment of tumor cells and (ii) generation of singlet oxygen and in vitro cytotoxic effect in tumor cells by photosensitization under two-photon irradiation.

Complete blood vessel occlusion in the chick chorioallantoic membrane using two-photon excitation photodynamic therapy: implications for treatment of wet age-related macular degeneration

Complete blood vessel occlusion is required for the treatment of age-related macular degeneration (AMD). AMD is the leading cause of blindness in developed countries and current treatment regimes have potential to cause collateral damage, or do not remove pre-existing unwanted vasculature. It has been proposed that two-photon excitation (TPE) photodynamic therapy (PDT) can be applied to cause local blood vessel occlusion without damaging surrounding retinal tissues. The in ovo chicken chorioallantoic membrane (CAM) is used as the model for vascularization in the wet form of AMD; novel techniques for the utilization of the CAM are reported. Complete occlusion of CAM vessels approximately 15 microm in diameter is achieved using the clinically approved photosensitizer Verteporfin (Visudyne, QLT, Incorporated, Vancouver, British Columbia, Canada) and TPE activation. The average and peak irradiances used for treatment are 3.3x10(6) Wcm(2) and 3.7x10(11) Wcm(2), respectively. A total fluence of 1.1x10(8) Jcm(2) is the dosage required for successful occlusion, and it is expected that for optimal conditions it will be much less. These results are the first proof-of-principle evidence in the literature that indicate TPE-PDT can be used to occlude small blood vessels. Further investigation will help determine the utility of TPE-PDT for treating wet AMD, perhaps through targeting feeder vessels.

Nanoparticles for two-photon photodynamic therapy in living cells.

We describe here a nontoxic two-photon photodynamic nanoparticle platform and its cellular application. We demonstrate that the dye's potential toxicity can be circumvented by its permanent encapsulation into a biocompatible nanoparticle polymer matrix; this was examined by dye leaching experiments and confirmed by cell uptake experiments. Infrared two-photon nanoplatform phototoxicity was demonstrated for rat C6 glioma cells, while the controls showed no dark toxicity for these living cells.

Synthesis and Characterization of New Fluorene-Based Singlet Oxygen Sensitizers

The synthesis, photophysical characterization, and determination of singlet oxygen quantum yields (Phi(Delta)) for a class of fluorene derivatives with potential application in two-photon photodynamic therapy (PDT) is reported. It has been demonstrated that these compounds possess the ability to generate singlet oxygen (1O2) upon excitation. A photochemical method, using 1,3-diphenylisobenzofuran (DPBF) as 1O2 chemical quencher, was employed to determine the singlet oxygen quantum yields (Phi(Delta)) of the fluorene-based photosensitizers in ethanol. Phi(Delta) values ranged from 0.35 to 0.75. These derivatives may have potential application as two-photon photosensitizers when pumped via two-photon excitation in the near-IR spectral region.

Singlet oxygen generation via two-photon excited FRET.

A new approach to two-photon excited photodynamic therapy has been developed. A dendritic array of eight donor chromophores capable of two-photon absorption (TPA) was covalently attached to a central porphyrin acceptor. Steady-state fluorescence measurements demonstrated that the donor chromophores transfer excited-state energy to the porphyrin with 97% efficiency. Two-photon excitation of the donor chromophores at 780 nm resulted in a dramatic increase in porphyrin fluorescence relative to a porphyrin model compound. Enhanced singlet oxygen luminescence was observed from oxygen-saturated solutions of the target compound under two-photon excitation conditions.

Ultrafast studies of the excited-state dynamics of copper and nickel phthalocyanine tetrasulfonates: potential sensitizers for the two-photon photodynamic therapy of tumors.

In order to evaluate the potential of copper and nickel phthalocyanine tetrasulfonates as sensitizers for two-photon photodynamic therapy, we conducted kinetic femtosecond measurements of transient absorption and bleaching of their excited state dynamics in aqueous solution. Samples were pumped with 620 nm and 310 nm laser light, which allowed us to study relaxation processes from both the first and second singlet (or doublet for the copper phthalocyanine) excited states. A second excitation from the first excited triplet state, approximately 685 and 105 ps after the first excitation for copper and nickel phthalocyanine tetrasulfonate respectively, was the most efficient way to bring the molecules to an upper triplet state. Presumably this highest triplet state can inflict molecular damage on adjacent biomolecules int eh absence of oxygen, resulting in the desired cytotoxic cellular response. Transient absorption spectra at different fixed delays indicate that optimum efficiency would require that the second photon has a wavelength of approximately 750 nm.

Two-photon excitation studies of hypocrellins for photodynamic therapy

The photophysical and photochemical properties of hypocrellins (HA and HB) are examined with two-photon excitations at 800 nm using femtosecond pulses from a Ti:sapphire laser. The two-photon excited fluorescence spectra of HA and HB are very similar to those obtained by one-photon excitation, which may indicate that the two-photon induced photodynamic processes of hypocrellins are similar to one-photon induced photodynamic processes. The two-photon excitation cross sections of HA and HB are measured at 800 nm as about 34.8×10 −50 cm 4 s/photon and 21.3×10 −50 cm 4 s/photon, respectively. The large two-photon cross sections of both HA and HB, suggest that the hypocrellins can be potential two-photon phototherapeutic agents. As an example for two-photon photodynamic therapy of hypocrellins, we also further examine the cell-damaging effects of HA upon two-photon illumination. Our preliminary results of cell viability test indicate hypocrellins can effectively damage the Hela cells under two-photon illumination.

Simultaneous two-photon activation of type-I photodynamic therapy agents.

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.