Potential Photosensitizers For Photodynamic Therapy Research Articles

Diamino acid derivatives of PpIX as potential photosensitizers for photodynamic therapy of squamous cell carcinoma and prostate cancer: In vitro studies

Photodynamic therapy (PDT) is an alternative treatment modality involving light activated drugs, called photosensitizers (PSs), to treat cancer and non-cancerous conditions. The search for new compounds which might become effective PSs is the major direction for PDT development. In the present work we have studied the dark toxicity, intracellular localization and photodynamic properties of four potential, water soluble, second generation PSs – PP(Arg) 2, PP(Ser) 2Arg 2, PP(Ala) 2Arg 2, PP(Phe) 2Arg 2, all diamino acid derivatives of protoporphyrin IX. Human prostate cancer (DU-145) and squamous carcinoma (A431) cells were used as experimental model. Among investigated compounds PP(Ser) 2Arg 2 exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines. Fluorescence microscopy revealed the time-dependent changes in intracellular localization of the PS which were related to the phototoxicity. The results show that PP(Ser) 2Arg 2 may be a potential PS for PDT.

Photosensitizer effects on cancerous cells: A combined study using synchrotron infrared and fluorescence microscopies

Hypocrellin A (HA), a lipid-soluble peryloquinone derivative, isolated from natural fungus sacs of Hypocrella bambusae, has been reported to be a highly potential photosensitizer in photodynamic therapy (PDT). It has been studied increasingly because of its anticancer activities when irradiated with light. We have studied the interaction mechanisms of HA with HeLa cells as a function of incubation time. Fluorescence microscopy confirmed that HA localisation is limited in the cytoplasm before eventually concentrating in clusters around the nucleus. The IR spectra of HA-treated, PDT-treated and control HeLa cells were recorded at the ESRF Infrared beamline (ID21). Principal component analysis has been used to assess the IR spectral changes between the various HeLa cells spectral data sets (The Unscrambler software, CAMO). PCA revealed that there is a frequency shift of protein amide I and amide II vibrational bands, indicating changes in the protein secondary structures of the HA-treated and PDT-treated cancer cells compared to the control cells. In addition, the relative DNA intensity in HA-treated cells decreases gradually along the incubation time. The use of synchrotron infrared microscopy is shown to be of paramount importance for targeting specifically the biochemical modification induced in the cell nucleus.

Absorption and emission spectral shifts of rose bengal associated with DMPC liposomes

Rose bengal is a water-soluble xanthene dye that is currently used in ophthalmology for the diagnosis of dry eyes. Although the dye is also a potential photosensitizer for photodynamic therapy of tumors, owing to insufficient lipophilicity and tumor accumulation, the clinical application of rose bengal in photodynamic therapy has been hampered. Liposomal encapsulation was seen as a promising approach to overcome these disadvantages, to which end, the spectral properties of the dye in the presence of materials for liposome preparation were studied. The presence of phospholipid influenced the spectral properties of the dye, probably due to the establishment of an equilibrium between monomeric and dimeric forms of the dye, since the photophysical properties of rose bengal depend strongly on its environment. The liposomal encapsulation of the dye generates stronger emission than the free form of the colorant; increased lipid:dye ratio further enhances this emission.

Dithiaporphyrin Derivatives as Photosensitizers in Membranes and Cells

We synthesized a series of analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin (I) as potential photosensitizers for photodynamic therapy (PDT). The photosensitizers differ in the length of the side chains that bind the carboxyl to the phenol at positions 10 and 15 of the thiaporphyrin. The spectroscopic, photophysical, and biophysical properties of these photosensitizers are reported. The structural changes have almost no effect on the excitation/emission spectra with respect to I's spectra or on singlet oxygen generation in MeOH. All of the photosensitizers have a very high, close to 1.00, singlet oxygen quantum yield in MeOH. On the contrary, singlet oxygen generation in liposomes was considerably affected by the structural change in the photosensitizers. The photosensitizers possessing short side chains (one and three carbons) showed high quantum yields of around 0.7, whereas the photosensitizers possessing longer side chains showed smaller quantum yield, down to 0.14 for compound X (possessing side-chain length of 10 carbons), all at 1 microM. Moreover a self-quenching process of singlet oxygen was observed, and the quantum yield decreased as the photosensitizer's concentration increased. We measured the binding constant of I to liposomes and found Kb = 23.3 +/- 1.6 (mg/mL)-1. All the other photosensitizers with longer side chains exhibited very slow binding to liposomes, which prevented us from assessing their Kb's. We carried out fluorescence resonance energy transfer (FRET) measurements to determine the relative depth in which each photosensitizer is intercalated in the liposome bilayer. We found that the longer the side chain the deeper the photosensitizer core is embedded in the bilayer. This finding suggests that the photosensitizers are bound to the bilayer with their acid ends close to the aqueous medium interface and their core inside the bilayer. We performed PDT with the dithiaporphyrins on U937 cells and R3230AC cells. We found that the dark toxicity of the photosensitizers with the longer side chain (X, VI, V) is significantly higher than the dark toxicity of sensitizers with shorter side chains (I, III, IV). Phototoxicity measurements showed the opposite direction; the photosensitizers with shorter side chains were found to be more phototoxic than those with longer side chains. These differences are attributed to the relationship between diffusion and endocytosis in each photosensitizer, which determines the location of the photosensitizer in the cell and hence its phototoxicity.

The synthesis and characterization of titanium dioxide nanoparticles as potential photosensitizer in photodynamic therapy

TiO2 nano-photosensitizer particles less than 10nm have been synthesized and characterized. They were prepared by hydrolysing Titanium (IV) isopropoxide in presence of ultrasonic waves and further characterized by absorption and fluorescence spectroscopy, XRD, FTIR, TGA, DSC, SEM, and HRTEM techniques. The XRD patterns revealed exclusive formation of anatase without contamination by rutile form. High resolution transmission electron microscopic measurements revealed their size below 10nm. The photohemolysis induced by TiO2 NPs reveals that the percent hemolysis increased with the increase in concentration and light dose. The study of effect of scavengers, GSH and NaN3 showed the formation of considerable amount of superoxide anion and singlet oxygen that caused cell death. The mechanism has been discussed. TiO2 nano-photosensitizer being non-toxic, serves as proper substitute for the classical photosensitizers (organic dyes).

Dendrimer Generation Effects on Photodynamic Efficacy of Dendrimer Porphyrins and Dendrimer-Loaded Supramolecular Nanocarriers

A series of poly(benzyl ether) dendrimer porphyrins (DPs) (Gn = n-generation dendrimer, n = 1−3) was examined as potential photosensitizers for photodynamic therapy (PDT). Polyion complexes (PICs) between the DPs and poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) were formed via an electrostatic interaction between the positively charged poly(l-lysine) (PLL) segment and negatively charged periphery of the DPs. Dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) showed that G3 formed a core−shell-type nanocarrier micelle, whereas G1 and G2 formed irregular-shaped nanoparticles with a relatively high polydispersity. The photophysical properties of the DP-loaded PIC nanocarriers strongly depend on the generation of the DPs. In the case of G1 and G2, their fluorescence lifetime and oxygen consumption ability were significantly reduced by the formation of the PIC nanocarriers, whereas the G3-loaded PIC nanocarrier exhibited almost comparable fluorescence lifetimes and oxygen consumption abilities to the free G3. The incorporation of DPs into PIC nanocarriers resulted in an appreciable increase in the cellular uptake, yet inversely correlated with the generation. Alternatively, the photocytotoxicity of the DPs within the nanocarriers increased with an increase in the generation despite a decrease in the cellular uptake. By correlating the effects of the uptake amount with the photocytotoxicity, the PIC nanocarriers showed remarkable enhancement of the PDT efficacy dependent on the generation of DPs.

Influence of Aggregation on Interaction of Lipophilic, Water‐Insoluble Azaphthalocyanines with DOPC Vesicles

Dioleoylphosphatidylcholine unilamellar vesicles made by extrusion technique (LUVETs) were studied as the delivery system for lipophilic water-insoluble potential photosensitizers for photodynamic therapy (PDT). Two azaphthalocyanines (AzaPcs) with hydrophobic substituents only and two also possessing two charged amino groups were introduced into the study. All compounds are insoluble in water and form aggregates in PBS with tetrahydrofuran as cosolvent. The size of these aggregates depends on the concentration of AzaPc in solution. AzaPcs with tert-butyl substituents were found to be incorporated into the lipid bilayer of vesicles in the monomeric form even at high concentrations. The stability of LUVETs with incorporated AzaPc was excellent for at least 4 weeks. Therefore, they are suitable for use as a delivery system for these water-insoluble photosensitizers. Very low amount of AzaPc with n-octyl substituents incorporated into LUVETs due to its stronger self-aggregation. Values of binding constants determined for all AzaPcs showed inverse order than expected from their lipophilicities. However, the binding constants followed the order of the strength of aggregation forces. Aggregation of AzaPcs in water medium plays a very important role in the interaction of AzaPcs with LUVETs.

Combinatorial Synthesis and Characterization of New Asymmetric Porphyrins as Potential Photosensitizers in Photodynamic Therapy

Porphyrins play a major role as active photosensitizers in noninvasive optical photodynamic therapy (PDT). In a modular approach, this paper presents a short review of the recent developments of porphyrin structures and materials with improved photosensitizing properties and then presents the synthesis and characterization of a series of new second generation asymmetrical meso-tetraphenylporphyrins varied by substituent in the meta positions of the phenyl rings with either -OH or -OCH3 groups, whereas in the para positions only with -OCH3 groups. The new series of differentially functionalized porphyrins were obtained by a combinatorial multicomponent synthesis (Adler-Longo method) by simultaneously using two different aldehydes: 3,4-dimethoxybenzaldehyde and 3-hydroxybenzaldehyde. The porphyrins were isolated, purified and characterized by HPLC, TLC, UV-vis, fluorescence, MS, 1H-NMR, and 13C-NMR analysis, accompanied by DEPT 135 experiments. Because of the fact that the medium in cancerous tissues is often more acidic than in normal tissues, the capacity of these porphyrins to exist simultaneously in aggregated and protonated forms was also investigated, in tetrahydrofuran (THF) and acid THF-water systems, underlying the changes in the photophysical behaviour. The relative fluorescence quantum yields (Phif) were calculated in comparison with meso-tetraphenylporphyrin (TPP), and the values between 0.14-0.26 were found to be promising for further trials. The series of asymmetrically substituted tetraphenylporphyrins, as the new class of supramolecular materials, are suitable for further functionalization in order to improve their photophysical properties, and they could represent interesting potential PDT photosensitizers.

Spectroscopic Studies and Photodynamic Actions of Hypocrellin B in Liposomes¶

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg l-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of 1O2 generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB·−) to oxygen. The disproportionation of O2·− can generate H2O2 and ultimately the highly reactive ·OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O2·− directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O2·−) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.

Part 2. meso-Tetraphenylporphyrin Dimer Derivatives as Potential Photosensitizers in Photodynamic Therapy¶

Part 2. meso-Tetraphenylporphyrin Dimer Derivatives as Potential Photosensitizers in Photodynamic Therapy¶

The potential application of chlorin e6—polyvinylpyrrolidone formulation in photodynamic therapy

Much research has been focused on developing effective drug delivery systems for the preparation of chlorins as potential photosensitizers for PDT. This report describes the evaluation of a new water-soluble formulation of chlorin e6 consisting of a complex of trisodium salt chlorin e6 and polyvinylpyrrolidone (Ce6-PVP) for application in photodynamic therapy (PDT) with 2 specific aims: (i) to investigate its fluorescence kinetics in skin, normal and tumor tissue after intravenous administration, and (ii) to investigate its PDT efficacy. Our results demonstrate that this new formulation possesses photosensitizing properties with rapid accumulation in tumor tissue observed within 1 h after intravenous administration. Although high selectivity in tumor tissue was found between the period of 3 and 6 h, the efficacy of Ce6-PVP mediated PDT was best at 1 h drug-light interval. It is suggested that, the extent of tumor necrosis post PDT is dependent on the plasma concentration of Ce6-PVP, implying a vascular mediated cell death mechanism. A faster clearance rate of Ce6-PVP from the skin of nude mice was observed compared to Ce6. The new formulation of Ce6-PVP seems to show promise as an effective therapeutic agent.

Photo-irradiated Titanium Dioxide Catalyzes Site Specific DNA Damage via Generation of Hydrogen Peroxide

Titanium dioxide (TiO2) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO2 was examined using [32P]-5′-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO2 (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2′ -deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO2 particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO2.

PH-sensitive Assembly of Light-Harvesting Dendrimer Zinc Porphyrin Bearing Peripheral Groups of Primary Amine with Poly(ethylene glycol)-b-poly(aspartic acid) in Aqueous Solution

The light-harvesting dendrimer zinc porphyrin [NH2CH2CH2NHCO]32DPZn, a potential photosensitizer in photodynamic therapy, was synthesized and successfully assembled with poly(ethylene glycol)-b-poly(aspartic acid) to form micelles in PBS buffer (10 mM) following the concept of polyion complex micelle. The coupling reaction between the [CO2H]32DPZn and N-trifluoroacetylethylene-1,2-diamine using the DCC/HOBt method afforded the protected dendrimer [CF3CONHCH2CH2NHCO]32DPZn, which was then mildly hydrolyzed to remove all the trifluoroacetyl protecting groups. Light-scattering measurements showed that the [NH2CH2CH2NHCO]32DPZn has the ability to assemble with poly(ethylene glycol)-b-poly(aspartic acid) to form a spherical micelle with a diameter of about 55 nm, having a narrow size polydispersity in PBS. The micelles were stable both in size and composition up to a 0.90 M (NaCl) salt concentration. The high salt stability is associated with a hydrogen-bonding network formed in the micellar core among the ami...

Efficacy of hypocrellin pharmacokinetics in phototherapy

Hypocrellins A and B are pigments which are isolated from the parasitic fungi Hypocrella bambuase sacc and Shiraia bambusicola P. Heen found in the People's Republic of China and other parts of Asia including Sri Lanka. These agents, which belong to the general class of perylene quinonoid pigments, have a long history of traditional medicinal agents especially in Asia. Hypocrellins are under extensive investigation as photosensitizing agents for photodynamic therapy (PDT). Hypocrellin compounds were selected as potential photosensitizers for PDT owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. The cellular uptake, evaluated by spectrofluorimetry and confocal laser scanning microscopy (CLSM) demonstrated that both HA and HB exhibited high and fast uptake and rapid internalization as revealed by their bio-distribution pattern. In addition, the present study employed both immunocytochemical and Western blot techniques to explore the photo-induced expression of apoptosis related proteins in NPC as well as other human carcinoma cells. Using spectrofluorimetry and CLSM we have determined the cellular fluorescence as a marker for the uptake of HA and HB. Co-staining with either HA or HB and fluorescent dyes specific for cell organelles revealed an intracellular localization of HA and HB in lysosomes other than mitochondria.

Investigation of a novel triazatetrabenzcorrole photosensitizer

A novel phthalocyanine-like photosensitizer, oxophosphorus(V) tetrasulfotriaza-tetrabenzcorrole ( POTBCS 4), has been synthesized. Its structure and absorption spectrum are unique. POTBCS 4 has an axial P = O group and peripheral sulfo groups. The fluorescence emission spectra, fluorescence quantum yield and quantum yield of singlet oxygen generation have been studied. The uptake and the photodynamic activities against HeLa cells were measured. The results indicated that POTBCS 4 was a potential photosensitizer for photodynamic therapy (PDT).

Magnetic Resonance Studies and Molecular Orbital Calculations on the Doublet and Triplet States of Bacteriopurpurin: a Potential Second-Generation Photosensitizer for Photodynamic Therapy

Bacteriopurpurin is a macrocyclic compound at the saturation level of bacteriochlorins that was specifically synthesized as a potential photosensitizer in photodynamic therapy. It exhibits a long-wavelength Qy absorption band at 843 nm, which is a necessary prerequisite for a deep tissue penetration. Magnetic resonance spectroscopic properties of bacteriopurpurin have been examined by fluorescence-detected magnetic resonance (FDMR) and electron paramagnetic resonance (EPR) in conjunction with molecular orbital calculations. The electronic structure of the radical cation state obtained by oxidation of bacteriopurpurin with iodine is mapped by the determination of isotropic proton hyperfine coupling constants from electron−nuclear double-resonance spectroscopy at 298 K. A tentative assignment of the couplings to the various proton positions in the molecule is achieved by comparison of the experimental values with simulated ones based on semiempirical intermediate neglect of differential overlap calculations...

Photophysical properties of Zn(II) phthalocyaninates incorporated into liposomes

Amphiphilic phthalocyanines are used as potential photosensitizers in photodynamic therapy. The low water solubility of most of these dyes is the reason that different kinds of vehicles are employed as endogenous carriers. Due to the photophysical inactivity of the phthalocyanine aggregates, it is important to improve their solubility in order to study dyes in solid tumor models. On the other hand, the photodynamic therapy activity depends on the location of phthalocyanines in the cell and the interaction of cellular components. In this work, the photophysical properties of tetrasubstituted Zn ( II ) phthalocyanines with different lipophilic properties are incorporated into liposomes of L-α-dimyristoylphosphatidylcholine (DMPC). The fluorescence quantum yields, the association constants and their localization in the membranes are reported.

Effect of aggregation on bacteriochlorin a triplet-state formation: a laser flash photolysis study.

Bacteriochlorin a (BCA) is a potential photosensitizer for photodynamic therapy of cancer. It has been shown previously that the photoefficiency of the dye is mainly dependent on singlet oxygen (1O2) generation. Nanosecond laser flash photolysis was used to produce and to investigate the excited triplet state of the dye in methanol, phosphate buffer and dimiristoyl-L-alpha-phosphatidylcholine (DMPC) liposomes. The transients were characterized in terms of their absorption spectra, decay kinetics, molar absorption coefficients and formation quantum yield of singlet-triplet intercrossing. The lifetime of the BCA triplet state was measured at room temperature. The triplet-state quantum yield is quite high in methanol (0.7) and in DMPC (0.4) but only 0.095 in phosphate buffer. In the last case, BCA is in a monomer-dimer equilibrium, and the low value of the quantum yield observed was ascribed to the fact the triplet state is only formed by the monomers.

In-vitro activity and tissue distribution of new fluorinated meso-tetrahydroxyphenylporphyrin photosensitizers.

Tetra(hydroxyphenyl)porphyrins started to attract interest as potential photosensitizers for photodynamic therapy in the early eighties. Subsequently, a number of derivatives of these compounds have been studied. In 1997 we reported the synthesis of the fluorinated derivatives 5,10,15,20-tetrakis(2-fluoro-3-hydroxyphenyl)porphyrin (8), 5,10,15,20-tetrakis(2,4-difluoro-3-hydroxyphenyl)porphyrin (9), and 5,10,15,20-tetrakis(3,5-difluoro-4-hydroxyphenyl)porphyrin (10). We have measured their biological activity, using the MTT test, against cancer cell cultures in-vitro. The test showed that these compounds were as potent as 5,10,15,20-tetrakis(3-hydroxyphenyl)chlorin (5), one of the leading photosensitizers in photodynamic therapy. The highest photoactivity was shown by the meta-hydroxy compounds 8 and 9. The para-compound showed high toxicity in the dark. Distribution of these compounds between normal and cancer tissue was studied using 19F NMR spectroscopy. The highest cancer tissue localization was also shown by the meta-hydroxy compounds 8 and 9. The para compound showed poor localization in tumour tissue. This study has shown that 19F NMR spectroscopy can be used to estimate the tissue distribution of fluorinated tetrahydroxyphenylporphyrins in-vivo.

Spectroscopic Studies and Photodynamic Actions of Hypocrellin B in Liposomes¶

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg L-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of 1O2 generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB.-) to oxygen. The disproportionation of O2.- can generate H2O2 and ultimately the highly reactive .OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O2.- directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O2.-) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.