Synthesis and biological evaluation of methoxyphenyl porphyrin derivatives as potential photodynamic agents

A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 2',6'-dinitro-4'-trifluoromethylphenyl group by an amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamic activities of 5 and 6 were evaluated in vitro on Hep-2 cells. A considerable increase in the photocytotoxic effect was found for 6, which has higher singlet molecular oxygen, O(2)((1)Delta(g)), production.     

Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges

The photodynamic effect of meso-substituted cationic porphyrins, 5-[4-(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide 1, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were compared in N,N-dimethylformamide. Fluorescence quantum yields (varphiF) of 0.10, 0.06 and 0.08 were calculated for porphyrins 1, 2 and 3, respectively. The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene yielding values of 0.66, 0.36 and 0.42 for porphyrins 1, 2 and 3, respectively. Guanosine 5'-monophosphate was used as biological substrate model. Similar decomposition of guanosine 5'-monophosphate was obtained using these cationic porphyrins as sensitizer. In biological medium, photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. E. coli cultures were treated with sensitizer at 37 degrees C for 30 min in dark. In both procedures, a higher photoinactivation of cells (>99.999%) was found for cells treated with 10 microM of tricationic porphyrin 3 and irradiated for 5 min with visible light. Porphyrins 1 and 2 only show an important photodamage when the cells are irradiated without washing step. These results indicated that the tetracationic porphyrin 3 could be a promising sensitizer with potential applications in the photoinactivation of bacterial cells by photodynamic therapy.     

Photodynamic damages induced by a monocationic porphyrin derivative in a human carcinoma cell line

The photokilling activity of 5-(4-trimethylammoniumphenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin (CP) was evaluated on a Hep-2 human larynx-carcinoma cell line. Cell treatment was carried out with 5 μM CP incorporated into liposomal vesicles. Under violet-blue exciting light, the red fluorescence of CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. Similar pattern was also observed using rhodamine 123 in Hep-2 cells. No dark cytotoxicity was observed using 5 μM CP concentration and long incubation time (24 h). Using Hoechst-33258 and caspase-3 immunostaining methods, cell cultures treated for 24 h with CP and exposed to light for 7.5 min (27 J/cm²) showed a great amount of apoptotic cells (40%). In contrast, necrotic cells (58%) were observed using the same drug concentration but irradiated for 15 min (54 J/cm²). The results show that CP can induce different mechanisms of cell death depending on irradiation doses in the photodynamic treatments, which makes this agent an interesting sensitizer with potential application in photodynamic tumor therapy.     

Antibacterial effects of a monoporphyrinato ytterbium(III) complex and its free components on Staphylococcus aureus as determined by stop-flow microcalorimetry

The antibacterial effect of Yb3+, the free porphyrin base 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (H2TMP; 1), and the corresponding Yb3+ porphyrinato complex [Yb(III)(TMP)(H2O)3]+ Cl- (Yb(TMP); 2) towards Staphylococcus aureus was investigated by stop-flow microcalorimetry. By analyzing the obtained metabolic thermogenic curves, crucial parameters such as rate constant of bacterial growth (k), half inhibitory concentration (IC50), and generation time (t(G)) were determined. The antibacterial activities of the three compounds tested was 2>1>Yb3+, with an IC50 value of 273 mg/l for complex 2. The Yb3+ porphyrinato complex is proposed to benefit from synergetic effects of Yb3+ and the free porphyrin 1.     

Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

Photodynamic inactivation of Escherichia coli by novel meso-substituted porphyrins by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups.

The photodynamic effect of novel cationic porphyrins, with different pattern of meso-substitution by 4-(3-N,N,N-trimethylammoniumpropoxy)phenyl (A) and 4-(trifluoromethyl)phenyl (B) groups, have been studied in both solution bearing photooxidizable substrates and in vitro on a typical Gram-negative bacterium Escherichia coli. In these sensitizers, the cationic groups are separated from the macrocycle ring by a propoxy spacer. Thus, the charges have a high mobility and a minimal influence on photophysical properties of the porphyrin. These compounds produce singlet molecular oxygen, O2(1Delta(g)), with quantum yields of approximately 0.41-0.53 in N,N-dimethylformamide. In methanol, the l-tryptophan photodecomposition increases with the number of cationic charges in the sensitizer. In vitro investigations show that cationic porphyrins are rapidly bound to E. coli cells in approximately 5 min. A higher binding was found for A3B3+ porphyrin, which is tightly bound to cells still after three washing steps. Photosensitized inactivation of E. coli cellular suspensions follows the order: A3B3+ > A44+> ABAB2+ > AB3+. Under these conditions, a negligible effect was found for 5,10,15,20-tetra(4-sulfonatophenyl)porphyrin (TPPS4(4-)) that characterizes an anionic sensitizer. Also, the results obtained for these new cationic porphyrins were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammonium phenyl)porphyrin (TTAP4+), which is a standard active sensitizer established to eradicate E. coli. The photodynamic activity of TTAP4+ is quite similar to that produced by A4(4+). Studies in an anoxic condition indicate that oxygen is necessary for the mechanism of action of photodynamic inactivation of bacteria. The higher photodynamic activity of A3B3+ was confirmed by growth delay experiments. Photodynamic inactivation capacities of these sensitizers were also evaluated in E. coli cells immobilized on agar surfaces. Under these conditions, A3B3+ porphyrin retains a high activity to inactivate localized bacterial cells. Therefore, tricationic porphyrin A3B3+ is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria in liquid suspensions or on surfaces.     

Modulation of porphyrin binding to serum albumin by pH

In this study, we show that the difference in acidity of functional groups in porphyrin photosensitizers provides a meaningful avenue to achieve differential localization and retention of porphyrins in tissues and cells, and in the end could be a positive factor in the photodynamic treatment of cancer (PDT). We have demonstrated that meso-tetraphenylporphyrin derivative with four phosphonate (bond P(double bond O)(bond OH)(2)) moieties exists in aqueous solutions mainly in four forms that differ by a degree of protonation of the porphyrin ring and ionization of the phosphonate group. It is shown that each porphyrin form has different affinities toward the model protein (bovine serum albumin, BSA). Thus pH of the medium significantly modulates the affinity of the phosphonate porphyrin toward BSA. At lower pH (pH 6.0), the phosphonate porphyrin and BSA form a complex with affinity constant of K(b)=6.9 x 10(5) M(-1), while at pH 7.0 the K(b)=6.1 x 10(5) M(-1). At pH 8.0 the association is significantly lower. Because cancerous cells have generally lower pH (pH approximately 6.9) compared to healthy cells (pH approximately 7.4), the pH of such cells could be a decisive factor for cellular retention of the porphyrin in the form of an associate with intracellular proteins. Moreover, we have also demonstrated that the protonation/deprotonation equilibria do not negatively affect the photophysical properties or ability of phosphonate porphyrin to generate singlet oxygen.     

Photodynamic effects of porphyrin and chlorin photosensitizers in human colon adenocarcinoma cells

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate wavelength of light, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Photofrin, a complex mixture of porphyrin oligomers has recently received FDA approval for the photodynamic treatment of esophageal and endobronchial carcinoma, but its photodynamic and toxicity profiles are far from ideal. In the present study we evaluated a series of porphyrin-based PSs, some of which newly synthesized by our group, with the aim to identify agents with more favorable characteristics. For the most effective compounds in the porphyrin series, chlorin analogs were also synthesized; for comparison, the screening also included Photofrin. Cytotoxicity studies were performed by the MTT assay on a cultured human colon adenocarcinoma cell line (HCT116); the results indicate that the 3,4,5-trimethoxyphenyl, 3OH- and 4OH-phenyl, and the sulfonamidophenyl derivatives are significantly more potent than Photofrin. Flow cytometric studies and fluorescence microscopy indicate that in PDT-treated HCT116 cells death occurs mainly by apoptosis. In summary, novel PSs described in the present study, belonging both to the porphyrin and chlorin series, have proven more effective than Photofrin in killing colon cancer cells in vitro; extending these observation to in vivo models, particularly regarding the deeper reaching chlorin derivatives, might lead to significant advances in the development of tumor PDT.     

Synthesis and photodynamic activity of zinc(II) phthalocyanine derivatives bearing methoxy and trifluoromethylbenzyloxy substituents in homogeneous and biological media

Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents have been synthesized by a two-step procedure starting from 4-nitrophthalonitrile. Absorption and fluorescence spectroscopic studies were analyzed in different media. These compounds are essentially non-aggregated in the organic solvent. Fluorescence quantum yields (phi(F)) of 0.26 for ZnPc 3 and 0.25 for ZnPc 4 were calculated in tetrahydrofuran (THF). The photodynamic activity of these compounds was compared in both THF containing photooxidizable substrates and in vitro on Hep-2 human larynx-carcinoma cell line. The production of singlet molecular oxygen, O(2)((1)Delta(g)), was determined using 9,10-dimethylanthracene yielding values of approximately 0.56 for both sensitizers. Under these conditions, the addition of beta-carotene (Car) suppresses the O(2)((1)Delta(g))-mediated photooxidation. In biological medium, no dark cytotoxicity was found for cells incubated with 0.1 microM of phthalocyanines 3 and 4 for 24 h. However, under similar conditions 0.5 microM of ZnPc 4 was toxic (70% cell survival). The uptake into Hep-2 cells was evaluated using 0.1muM of sensitizer, reaching values of approximately 0.05 nmol/10(6) cells after 3h of incubation at 37 degrees C. The cell survival after irradiation of the cultures with visible light was dependent upon both light exposure level and intracellular sensitizer concentration. A higher photocytotoxic effect was found for ZnPc 3 with respect to 4 (32%/70% cell survival after 15 min of irradiation). Also, these studies were performed treating the cells with 0.5 microM of ZnPc 3. In this case, an increase in the uptake (approximately 0.28 nmol/10(6) cells) was observed, which is accompanied by a higher photocytotoxic activity (20% cell survival). These results show that even though both sensitizer present similar photophysical properties in homogeneous medium, the photodynamic behavior in cellular media can significantly be changed.     

Photodynamic inactivation of Escherichia coli immobilized on agar surfaces by a tricationic porphyrin

The photodynamic activity of 5,10,15-tris[4-(3-N,N,N-trimethylammoniumpropoxy)phenyl]-20-(4-trifluoromethylphenyl)porphyrin iodide (A3B3+) has been studied in vitro on a typical Gram-negative bacterium Escherichia coli immobilized on agar surfaces. The results obtained for the tricationic A3B3+ porphyrin were compared with those of 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin p-tosylate (TTAP4+), which is a standard active sensitizer established to eradicate E. coli in cellular suspension. The photobleaching of these porphyrins in solution was evaluated by decay in absorbance and in fluorescence. In both cases, a higher photostability was found for A3B3+ than for TTAP4+. Photodynamic inactivation capacities of these sensitizers were analyzed in E. coli cells immobilized on agar surfaces. Small colonies were treated with different amount of sensitizer (0-14 nmol) and irradiated with visible light for 3h. The light source used was either a projector or midday sun. The A3B3+ porphyrin produced a growth delay of E. coli colonies on agar surfaces. Similar result was obtained irradiating only one isolated colony through an optical fiber. Under these conditions, A3B3+ porphyrin shows a high activity to inactivate localized bacterial cells. The higher photodynamic activity of A3B3+ was confirmed by mechanical spreading of the colonies before treatment. This procedure produces complete inactivation of E. coli cells on the agar surface. Therefore, tricationic A3B3+ porphyrin is an interesting sensitizer with potential applications in photodynamic inactivation of bacteria growing as localized foci of infection.     

Carbohydrate-conjugated porphyrin dimers: Synthesis and photobiological evaluation for a potential application in one-photon and two-photon photodynamic therapy

We report the synthesis of bioconjugated zinc porphyrin dimers 1a–e designed as photosensitizers for one-photon and two-photon excited photodynamic therapy. These macrocycles are substituted with carbohydrate units (glucose, mannose, lactose) in order to target tumor cells over-expressing lectin membrane receptors. Polarity, singlet oxygen production and in vitro photocytotoxicity are studied to determine their photodynamic therapy potentiality.     

The Effect of a Cationic Porphyrin on Pseudomonas aeruginosa Biofilms

Current studies have indicated the utility of photodynamic therapy using porphyrins in the treatment of bacterial infections. Photoactivation of porphyrins results in the production of singlet oxygen (¹O₂) that damages biomolecules associated with cells and biofilms, e.g., proteins, polysaccharides, and DNA. The effect of a cationic porphryin on P. aeruginosa PAO1 biofilms was assessed by exposing static biofilms to 5,10,15,20-tetrakis(1-methyl-pyridino)-21H,23H-porphine, tetra-p-tosylate salt (TMP) followed by irradiation. Biofilms were visualized using confocal laser scanning microscopy (CLSM) and cell viability determined using the LIVE/DEAD BacLight viability assay and standard plate counts. At a concentration of 100 μM TMP, there was substantial killing of P. aeruginosa PAO1 wild-type and pqsA mutant biofilms with little disruption of the biofilm matrix or structure. Exposure to 225 μM TMP resulted in almost complete killing as well as the detachment of wild-type PAO1 biofilms. In contrast, pqsA mutant biofilms that contain less extracellular DNA remained intact. Standard plate counts of cells recovered from attached biofilms revealed a 4.1-log₁₀ and a 3.9-log₁₀ reduction in viable cells of wild-type PAO1 and pqsA mutant strains, respectively. Our results suggest that the action of photoactivated TMP on P. aeruginosa biofilms is two-fold: direct killing of individual cells within biofilms and detachment of the biofilm from the substratum. There was no evidence of porphyrin toxicity in the absence of light; however, biofilms pretreated with TMP without photoactivation were substantially more sensitive to tobramycin than untreated biofilms.     

Response to ALA-based PDT in an immortalised normal breast cell line and its counterpart transformed with the Ras oncogene.

Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has been successfully employed in the treatment of certain tumours. Porphyrins endogenously generated from ALA induce tumour regression after illumination with light of an appropriate wavelength. The aim of this work was to compare porphyrin production from ALA and sensitivity to photodynamic treatment in a tumour/normal cell line pair. We employed the HB4a cell line from normal mammary luminal epithelium and its counterpart transfected with the oncogen H-Ras (VAL/12 Ras). After 3 h of exposure to ALA, HB4a-Ras cells produce a maximum of 150 ng porphyrins per 10(5) cells whereas HB4a produce 95 ng porphyrins per 10(5) cells. In addition, HB4a-Ras cells show a plateau of porphyrin synthesis at 1 mM whereas HB4a porphyrins peak at the same concentration, and then decrease quickly. This higher porphyrin synthesis in the tumorigenic cell line does not lead to a higher response to the photodynamic treatment upon illumination. Lethal doses 50, LD(50), determined by MTT assay were 0.015 J cm(-2) and 0.039 J cm(-2) for HB4a and HB4a-Ras respectively after 3 h exposure to 1 mM ALA. The conclusion of this work is that a tumour cell line obtained by transfection of the Ras oncogene, although producing higher porphyrin synthesis from ALA, is more resistant to ALA-PDT than the parental non-tumour line, however the mechanism is not related to photosensitiser accumulation, but very likely to cell survival responses.     

Interest of RGD-containing linear or cyclic peptide targeted tetraphenylchlorin as novel photosensitizers for selective photodynamic activity

Destruction of the neovasculature is essential for tumor eradication by photodynamic therapy. Since the over-expression of integrins is correlated with tumor angiogenesis, we conjugated a photosensitizer (5-(4-carboxyphenyl)-10,15,20-triphenylchlorin or porphyrin) to the alpha(v)beta(3) integrin specific peptide RGD (H-Arg-Gly-Asp-OH) motif as a common sequence. We reported an efficient solid-phase synthesis of a new family of peptidic photosensitizers with linear or cyclic[RGDfK] RGD motif and compared conjugates in vitro selectivity and photodynamic activity. The conjugates were characterized by (1)H NMR, MALDI, UV-visible spectroscopy and singlet oxygen formation was performed. Chlorins containing linear and constrained RGD motif were incorporated up to 98- and 80-fold more, respectively, than the unconjugated photosensitizer over a 24-h exposure in human umbilical vein endothelial cells (HUVEC) over-expressing alpha(v)beta(3) integrin. Peptidic moiety also led to a non-specific increased cellular uptake by murine mammary carcinoma cells (EMT-6), lacking RGD binding receptors. Survival measurements demonstrated that HUVEC were greatly sensitive to conjugates-mediated photodynamic therapy.     

Comparative Studies of the Cellular Uptake, Subcellular Localization, and Cytotoxic and Phototoxic Antitumor Properties of Ruthenium(II)-Porphyrin Conjugates with Different Linkers

Six water-soluble free-base porphyrin-Ru(II) conjugates, 1-3, and Zn(II) porphyrin-Ru(II) conjugates, 4-6, with different linkers between the hydrophobic porphyrin moiety and the hydrophilic Ru(II)-polypyridyl complex, have been synthesized. The linear and two-photon-induced photophysical properties of these conjugates were measured and evaluated for their potential application as dual in vitro imaging and photodynamic therapeutic (PDT) agents. Conjugates 1-3, with their high luminescence and singlet oxygen quantum yields, were selected for further study of their cellular uptake, subcellular localization, and cytotoxic and photocytotoxic (under linear and two-photon excitation) properties using HeLa cells. Conjugate 2, with its hydrophobic phenylethynyl linker, was shown to be highly promising for further development as a bifunctional probe for two-photon (NIR) induced PDT and in vitro imaging. Cellular uptake and subcellular localization properties were shown to be crucial to its PDT efficacy.     

Peptide-protein interactions: photoinduced electron-transfer within the preformed and encounter complexes of a designed metallopeptide and cytochrome c

Photoinduced electron-transfer (ET) occurs between a negatively charged metallopeptide, [Ru(bpy)(2)(phen-am)-Cys-(Glu)(5)-Gly](3-) = RuCE(5)G, and ferricytochrome c = Cyt c. In the presence of Cyt c, the triplet state lifetime of the ruthenium metallopeptide is shortened, and the emission decays via biexponential kinetics, which indicates the existence of two excited-state populations of ruthenium peptides. The faster decay component displays concentration-independent kinetics demonstrating the presence of a preformed peptide-protein complex that undergoes intra-complex electron-transfer. Values of K(b) = (3.5 +/- 0.2) x 10(4) M(-1) and k(obs)(ET)= (2.7 +/- 0.4) x 10(6) s(-1) were observed at ambient temperatures. The magnitude of k(obs)(ET) decreases with increasing solvent viscosity, and the behavior can be fit to the expression k(obs)(ET) proportional to eta(-alpha) to give alpha = 0.59 +/- 0.05. The electron-transfer process occurring in the preformed complex is therefore gated by a rate-limiting configurational change of the complex. The slower decay component displays concentration-dependent kinetics that saturate at high concentrations of Cyt c. Analysis according to rapid equilibrium formation of an encounter complex that undergoes unimolecular electron-transfer yields K(b)' = (2.5 +/- 0.7) x 10(4) M(-1) and k(obs')(ET)= (7 +/- 3) x 10(5) s(-1). The different values of k(obs)(ET) and k(obs')(ET) suggest that the peptide lies farther from the heme when in the encounter complex. The value of k(obs')(ET) is viscosity dependent indicating that the reaction occurring within the encounter complex is also configurationally gated. A value of alpha = 0.98 +/- 0.14 is observed for k(obs')(ET), which suggests that the rate-limiting gating processes in the encounter complex is different from that in the preformed complex.     

Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants

The catalytic properties of the hammerhead ribozyme embedded in the (+) strand of the satellite tobacco ringspot viral genome are analyzed with the goal of obtaining the elemental rate constants of the cleavage (k(2)) and ligation (k(-)(2)) steps. Two different chimeras combining the sTRSV (+) hammerhead and the well-characterized hammerhead 16 were used to measure the cleavage rate constant (k(2)), the rate of approach to equilibrium (k(obs) = k(2) + k(-)(2)), and the fraction of full-length hammerhead at equilibrium (k(-)(2)/k(2) + k(-)(2)). When compared to minimal hammerheads that lack the recently discovered loop I-loop II interaction, an extended format hammerhead derived from sTRSV studied here shows at least a 20-fold faster k(2) and a 1300-fold faster k(-)(2) at 10 mM MgCl(2). However, the magnesium dependence of the cleavage rate is not significantly changed. Thus, the enhanced cleavage of this hammerhead observed in vivo is due to its higher intrinsic rate and not due to its tighter binding of magnesium ions. The faster k(-)(2) of this hammerhead suggests that ligation may be used to form circular RNA genomes. This in vitro system will be valuable for experiments directed at understanding the hammerhead mechanism and the role of the loop I-loop II interaction.     

In Vitro Differential Sensitivity of Head and Neck Squamous Cell Carcinoma to Cisplatin,Silver Nanoparticles,and Photodynamic Therapy

Background:The clinical effect of photodynamic therapy (PDT) may be correlated with the degree of dysplasia of cancer tissues. The aim of this study was to compare the effects of cisplatin, silver nanoparticles (AgNps), and photodynamic therapy (PDT) using methylene blue (MB) photosensitizer on Head and Neck squamous cell carcinoma - cell line (HNSCC), Hep-2, through genes expression.Methods:Hep-2 cells were divided into four groups: group I as control and without any treatment, group II and III were treated by cisplatin and AgNps, respectively, and group IV were incubated with MB for four minutes followed by PDT using laser irradiation at 650 nm for 8 minutes. The resulting toxicity was assessed in cell lines using MTT cytotoxicity assay. Further, apoptosis and the response to treatment was examined via RT-qPCR.Results:MB-PDT inhibited the proliferation of Hep-2 cells. Following PDT, compared with AgNps cells and via MTT assay, a highly significant decrease was observed in cell proliferation in Cancer cells treated with AgNps and MB- PDT groups compared to cancer group cells and cancer cells treated with Cisplatin (p value< 0.001). Mechanistically, both the mRNA and protein expression levels of Bcl-2, Caspase-3, Cyclin-D, HIF-1, IL-8, MAPK-38, and ROS were found to be down regulated in Hep-2 cell line after MB-PDT.DiscussionMB-PDT effectively killed Hep-2 cells in vitro, however, under the same conditions, the susceptibilities of the cell line to cisplatin, AgNps, and MB-PDT were different. Further studies are necessary to confirm whether this difference is present in clinical oral cancer lesions.Key Words: Head and Neck squamous cell carcinoma (HNSCC) , silver nanoparticles (AgNps), photodynamic therapy (PDT), Methylene Blue (MB), Photosensitizer (PS)     

Photochemical oxygen consumption sensitized by a porphyrin phosphorescent probe in two model systems

Phosphorescence quenching of certain metalloporphyrins is used to measure tissue and microvascular pO(2). Oxygen quenching of metalloporphyrin triplet states creates singlet oxygen, which is highly reactive in biological systems, and these oxygen-consuming reactions are capable of perturbing tissue oxygenation. Kinetics of photochemical oxygen consumption were measured for a Pd-porphyrin in two model systems in vitro over a range of irradiances (1.34-134 mW cm(-2)). For a given irradiance, and, after correction for differing porphyrin concentrations, rates of oxygen consumption were similar when the Pd-porphyrin was bound to bovine serum albumin and when it was taken up by tumor cells in spheroids. At irradiances comparable to those used in imaging superficial anatomy, rates of oxygen consumption were sufficiently low (2.5 microM s(-1)) that tissue oxygenation would be reduced by a maximum of 6%. An irradiance of 20 mW cm(-2), however, initiated a rate of oxygen consumption capable of reducing tissue pO(2) by at least 20-40%. These measured rates of consumption impose limitations on the use of phosphorescence quenching in thick tissues. The irreversible photobleaching of the Pd-porphyrin was also measured indirectly. The bleaching branching ratio, 23 M(-1), is significantly lower than that of porphyrin photodynamic agents.     

Determination of the triplet state energies of a series of conjugated porphyrin oligomers.

We report a systematic study of the photophysical parameters relevant to photodynamic therapy (PDT) by a new type of sensitizers, conjugated porphyrin oligomers. Due to the strong nonlinear properties of oligomers containing 2, 4 and 8 porphyrin units, these molecules are attractive candidates for PDT via multiphoton excitation. The triplet state energy levels for all molecules have been determined by the triplet quenching method, phosphorescence measurements and DFT calculations. We find that the triplet energies of all the oligomers are sufficient to generate singlet oxygen, >94 kJ mol(-1). However, low singlet oxygen quantum yields are observed for the tetramer and the octamer, as compared to the conjugated dimer and monomeric porphyrin, reflecting the decrease in triplet yield. Thus the conjugated porphyrin dimer is the most promising core structure for PDT applications via multiphoton excitation.