Two-photon Photodynamic Therapy Research Articles

Water-Soluble Conjugated Polymers for Amplification of One- and Two-photon Properties of Photosensitizers

Two new water-soluble polyfluorene-based conjugated polymers have been designed and synthesized to act as two-photon light harvesting complexes to amplify the two-photon property of photosensitizers and organic dyes through fluorescence resonance energy transfer (FRET). The two-photon absorption cross sections of the conjugated polymers at 800 nm have been significantly improved by inserting ethynylene and vinylene groups as the bridges, which extend the conjugation length and electron delocalization. By using these conjugated polymers as two-photon light harvesting complexes, the two-photon excitation emission of the photosensitizer, Rose Bengal, was found to be significantly enhanced (by up to 85 fold) through FRET. These studies are expected to provide insight on designing systems with further improved performance for potential applications in two-photon photodynamic therapy, sensing, and imaging.

Carboxylate modified benzylidene cyclopentanone dyes for one- and two-photon excited photodynamic therapy

Benzylidene cyclopentanone dyes have high photosensitization activity and large two-photon absorption (TPA) cross-section in near infrared region. To explore their application potentials in two-photon excited photodynamic therapy (TPE-PDT), a series of carboxylate modified benzylidene cyclopentanone dyes were synthesized by varying number of carboxylate groups. Their linear and nonlinear photophysical properties, reactive oxygen yields and in vitro PDT activities were investigated systematically. The results showed that the water solubility of these dyes was significantly enhanced after introducing carboxylate groups. All dyes exhibited large TPA cross-section around 1000–1400 GM at 840 nm. In addition, they could generate both superoxide anion radical and singlet oxygen species under illumination. However, only the dye Y1 modified by one carboxylate group presented strong PDT activity to human rectal cancer 1116 cells, which indicated that moderate water–lipid amphipathy was a very important factor for PDT dyes. Finally, Y1 was proved to have large potential in TPE-PDT by in vitro experiments.

Influence of Carbohydrate Biological Vectors on the Two-Photon Resonance of Porphyrin Oligomers

We propose a spectroscopic study of π-conjugated porphyrin dimers and a triphenylamine centered trimer bearing monoethyleneglycol-peracetylated α-mannose targeting moieties, which were synthesized for application to two-photon absorption photodynamic therapy (2PA-PDT). The collected spectroscopic data are explained through comparison with previous results obtained on similar nonvectorized analogues. It appears that the different shifts and variations in linear and nonlinear absorption and emission bands are mainly due to an increased twist between the tetrapyrrolic units. The 2PA properties of the triphenylamine-centered trimer have been studied, and two fluorescence emission bands are observed that seem to originate from two different 2PA excitation pathways. These results have important implication for the design of new vectorized photosensitizers for 2PA-PDT.

Potential sensitizers for two-photon photodynamic therapy

Potential sensitizers for two-photon photodynamic therapy

Functionalized mesoporous silica nanoparticles for two-photon photodynamic therapy

Functionalized mesoporous silica nanoparticles for two-photon photodynamic therapy

Enhanced Two-Photon Singlet Oxygen Generation by Photosensitizer-Doped Conjugated Polymer Nanoparticles

We have prepared photosensitizer-doped conjugated polymer nanoparticles by using a reprecipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP). These TPP-doped PFEMO nanoparticles are stable and have a uniform size of ∼50 nm. Efficient intraparticle energy transfer from PFEMO to TPP has been observed. The TPP emission of the nanoparticles was found to be enhanced by 21-fold by PFEMO under two-photon excitation. Enhanced two-photon excitation singlet oxygen generation efficiency in the TPP-doped PFEMO nanoparticles has been demonstrated. Our results suggest that these photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.

Photosensitizer-doped conjugated polymer nanoparticles for simultaneous two-photon imaging and two-photon photodynamic therapy in living cells

Photosensitizer doped conjugated polymer nanoparticles have been prepared by incorporating polyoxyethylene nonylphenylether (CO-520) into the nanoparticles using a re-precipitation method. The conjugated polymer, poly[9,9-dibromohexylfluorene-2,7-ylenethylene-alt-1,4-(2,5-dimethoxy)phenylene] (PFEMO), was used as the host matrix to disperse tetraphenylporphyrin (TPP) and an energy donor to enhance the two-photon excitation properties of TPP. These CO-520 incorporated, TPP-doped PFEMO nanoparticles are stable and have low cytotoxicity in the dark. The TPP emission of the nanoparticles was found to be enhanced by about 20 times by PFEMO under two-photon excitation. The nanoparticles showed significantly enhanced two-photon excitation singlet oxygen generation efficiency and two-photon photodynamic therapy activity in cancer cells. These composite nanoparticles display features required for ideal photosensitizers, such as low cytotoxicity in the dark and efficient two-photon photodynamic activity under laser radiation. In addition, these novel nano-photosensitizers allow simultaneous in vivo monitoring by two-photon fluorescence imaging during two-photon photodynamic treatment. These photosensitizer-doped conjugated polymer nanoparticles can act as novel photosensitizing agents for two-photon photodynamic therapy and related applications.

Polyethylene glycol-functionalized benzylidene cyclopentanone dyes for two-photon excited photodynamic therapy

A series of polyethylene glycol-functionalized benzylidene cyclopentanone dyes with varying lipid/water partition coefficients were synthesized in high yields by a simple process. Detailed characterization and systematic studies of these molecules, including linear and nonlinear photophysical properties, reactive oxygen yields, and in vitro photodynamic therapy (PDT) activities, were conducted. Four of these dyes exhibited good solubility in PBS (>2 mg ml(-1), which is sufficient for clinical venous injection), high reactive oxygen yields, large two-photon absorption and low dark toxicity, under the therapy dosage. Among them, two dyes could be absorbed efficiently by human rectal cancer 1116 cells, and presented strong two-photon excited PDT activity in in vitro cell experiments.

Banana-shaped biphotonic quadrupolar chromophores: from fluorophores to biphotonic photosensitizers

This study aims at designing dual-role biphotonic chromophores that could be used for photodynamic therapy (PDT) while maintaining some fluorescence in order to locate them, thus allowing selective irradiation of cancer cells when combined with targeting. Quadrupolar two-photon absorbing fluorophores were synthesized from the symmetrical functionalization of a fluorene core bearing elongated conjugated rods made from arylene–vinylene or arylene–ethynylene building blocks in order to test modifications which could increase singlet oxygen production ability while retaining some fluorescence and high two-photon absorption (TPA) cross-sections in the biological spectral range of interest. All chromophores show a polar emissive excited state whose dipole moment is highly dependent on the nature of the conjugated linker. Interestingly, the largest TPA responses in the NIR region as well as singlet oxygen quantum yield are correlated with the smallest dipole moment of the emissive excited-state. The molecular optimization study led to a multifunctional biphotonic chromophore combining high TPA cross-sections in the whole spectral range of interest (700–900 nm), reasonable singlet oxygen production efficiency, significant remaining fluorescence, and alcohol end-groups for further covalent grafting. This compound offers thus interesting potentialities for highly selective spatially-resolved two-photon PDT by incorporation in nanostructures.

Near-Unity Quantum Yields for Intersystem Crossing and Singlet Oxygen Generation in Polymethine-like Molecules: Design and Experimental Realization

The photophysical properties of two pairs of squaraine molecules where two oxygen atoms in a squaraine bridge are replaced with sulfur atoms are investigated. This substitution leads to an inversion of the lowest singlet π−π* electronic transition by an n−π* transition, effectively reducing the energy difference between singlet and triplet states and significantly increasing the intersystem crossing efficiency without the use of “heavy atoms”. Experimental results, in agreement with quantum chemical calculations, show near-unity values for triplet quantum yield and singlet oxygen generation quantum yield for sulfur-containing squaraines, which are potentially useful for two-photon photodynamic therapy.

Indirect activation of porphyrin by semiconductor quantum dots with two-photon excitation

Steady-state and nanosecond time-resolved spectroscopies are performed to investigate the interaction between CdSe quantum dots (QDs) and mesotetraphenylporphyrin (TPP) photosensitizers under two-photon excitation with 800-nm femtosecond laser pulses. The luminescent intensity and lifetime of the QDs decrease intensively in the QD-porphyrin mixture, whereas those of the TPP increase, suggesting occurence of a nonradiative fluorescence resonance energy transfer (FRET) process between the excited states of the donors and acceptors. An energy transfer efficiency of 0.27 is estimated from quenching of the excited-state radiative lifetime of the QDs. A 4-nm red shift in the emission spectrum of the CdSe QDs is observed after mixing with TPP, which is speculated to be caused by the energy transfer from QDs of small size to large. This study could be valuable for developing a QD-based drug system for application in two-photon excitation photodynamic therapy.

Silica-based nanoparticles for photodynamic therapy applications

Silica-based nanoparticles for applications in photodynamic therapy (PDT) have emerged as a promising field for the treatment of cancer. In this review, based on the pathway the photosensitizer is entrapped inside the silica matrix, the different methods for the synthesis of silica-based nanoparticles are described from the pioneering works to the latest achievements which concern multifunctional nanoplatforms, up-converting nanoparticles, two-photon PDT, vectorization and in vivo applications.

Activation of porphyrin photosensitizers by semiconductor quantum dots via two-photon excitation

Energy transfer from water-soluble quantum dots (QDs) to porphyrinlike sensitizers is studied by time-resolved spectroscopy of two-photon excitation with femtosecond laser pulses. Evident transfer results are observed. Electron exchange is found to be the dominant transfer mechanism. Relative intensity change between excitonic and trapping emission implies that nonradiative energy transfer occurs through the trapping state of QDs, which presents a way of raising energy transfer efficiency in this type of donor-acceptor pairs. This study underlines the potential of QD-porphyrin model system for applications in two-photon excitation photodynamic therapy.

Fluorescence, Cyto-, And Photo-toxicity, And Structural Studies Of Substituted Piperidones: Potential Sensitizers For Two-photon Photodynamic Therapy

Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones, with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and x-ray crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence lifetimes are less than 1 ns, suggesting that there may be a significant energy transfer to the triplet state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cyto-toxicity studies with a number of human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that increasing the number of double bonds, from one to two, in the piperidone “wings” increases the photo-toxicity with little corresponding change in the dark cyto-toxicity. Two-photon photo-toxicity studies are also underway (exposure in the range of 740 - 860 nm) as well as singlet oxygen detection studies(detection at about 1270 nm).

One- and two-photon activated phototoxicity of conjugated porphyrin dimers with high two-photon absorption cross sections

Two-photon excited photodynamic therapy (PDT) has the potential to provide a highly targeted treatment for neoplastic diseases, as excitation can be pin-pointed to small volumes at the laser focus. In addition, two-photon PDT offers deeper penetration into mammalian tissue due to the longer wavelength of irradiation. Here we report the one-photon and two-photon excited PDT results for a collection of conjugated porphyrin dimers with high two-photon absorption cross sections. These dimers demonstrate high one-photon PDT efficacy against a human ovarian adenocarcinoma cell line (SK-OV-3) and exhibit no significant dark-toxicity at concentrations of up to 20 microM. Their one-photon excited PDT efficiencies, following irradiation at 657 nm, approach that of Visudyne, a drug used clinically for PDT. We investigated and optimised the effect of the photosensitizer concentration, incubation time and the light dose on the PDT efficacy of these dimers. These studies led to the selection of P2C2-NMeI as the most effective porphyrin dimer. We have demonstrated that P2C2-NMeI undergoes a two-photon activated process following excitation at 920 nm (3.6-6.8 mW, 300 fs, 90 MHz) and compared it to Visudyne. We conclude that the in vitro two-photon PDT efficacy of P2C2-NMeI is about twice that of Visudyne. This result highlights the potential of this series of porphyrin dimers for two-photon PDT.

Synthesis of hydrophilic conjugated porphyrin dimers for one-photon and two-photon photodynamic therapy at NIR wavelengths

We report the synthesis of a series of hydrophilic butadiyne-linked conjugated zinc porphyrin dimers, designed as photodynamic therapy (PDT) agents. These porphyrin dimers exhibit exceptionally high two-photon absorption cross sections (delta(max) approximately 8,000-17,000 GM) and red-shifted linear absorption spectra (lambda(max) approximately 700-800 nm) making them ideal candidates for one-photon and two-photon excited photodynamic therapy. Four polar triethyleneglycol substituents are positioned along the sides of each dimer, but, on their own, these TEG chains do not confer sufficient solubility in aqueous physiological media for reproducible delivery into live cells. Charged cationic (methylpyridinium and trimethylammonium) and anionic (sulfonate and carboxylate) substituents have been appended to the meso-positions of porphyrin dimers using three synthetic strategies: 1) Suzuki coupling, 2) Sonogashira coupling, and 3) nucleophilic Senge arylation. Approaches 1 and 3 both allow attachment of aromatic substituents directly to the meso-positions of porphyrins. Approach 2 provides a route to hydrophilic porphyrin dimers with an ethyne link between the porphyrin and the polar aromatic substituent. The palladium-catalysed approaches 1 and 2 allow the synthesis of a broader range of meso-capped porphyrins, as many aryl halides are available. However the synthesis of the intermediate required for these routes necessitates a statistical reaction step, which decreases the overall yield. On the other hand, Senge-arylation provides highly regioselective nucleophilic aromatic substitution, and offers higher overall yield than the other routes. All these charged dimers exhibit good solubility in polar solvents (e.g. methanol) and aqueous solvent mixtures (aqueous DMSO or DMF).

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

We have investigated the photophysical properties and intracellular behaviour of a series of hydrophilic conjugated porphyrin dimers. All the dimers exhibit intense linear absorption at 650-800 nm and high singlet oxygen quantum yields (0.5-0.9 in methanol), as required for an efficient sensitiser for photodynamic therapy (PDT). They also exhibit fluorescence at 700-800 nm, with fluorescence quantum yields of up to 0.13 in methanol, and show extremely large two-photon absorption maxima of 8,000-17,000 GM in the near-IR. The dimers aggregate in aqueous solution, but aggregation is reduced by binding to bovine serum albumin (BSA), as manifested by an increase in fluorescence intensity and a sharpening in the emission bands. This process can be regarded as a model for the interaction with proteins under physiological conditions. Confocal fluorescence microscopy of live cells was used to monitor the rate of cellular uptake, intracellular localisation and photostability. Porphyrin dimers with positively charged substituents partition into cells more efficiently than the negatively charged dimers. The photostability of these dimers, in living cells, is significantly better than that of the clinical photosensitiser verteporfin. Analysis of the photophysical parameters and intracellular imaging data indicates that these dimers are promising candidates for one-photon and two-photon excited PDT.

Drug and light dose responses to focal photodynamic therapy of single blood vessels in vivo

As part of an ongoing program to develop two-photon (2-gamma) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-gamma and 2-gamma activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-gamma cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-gamma PDT of AMD.

An ethylene-glycol decorated ruthenium(ii) complex for two-photon photodynamic therapy

A novel water-soluble Ru(ii) complex has been prepared, which represents a promising new class of selective two-photon sensitizers for use in photodynamic therapy within a confined space.

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. Two-photon excitation is attractive for photodynamic therapy as it potentially allows deeper penetration within biological tissue and targeting with better precision. However, two-photon cross-sections of light-sensitive drugs are typically small, which has until now limited their practical utility. Now Anderson and colleagues have come up with a new family of light-sensitive drugs that are designed for efficient two-photon excitation. They demonstrate selective closure of blood vessels in mice using one of their new drugs.