Synthesis and in vitro evaluation of a PDT active BODIPY–NLS conjugate

Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an NLS peptide, which is linked to the BODIPY’s core using the copper catalysed azide–alkyne click reaction. The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization experiments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER (for the peptide-bearing BODIPY).     

A naphthalocyanine based near-infrared photosensitizer: Synthesis and in vitro photodynamic activities

A hydrophilic near-infrared (NIR) photosensitizer featuring a naphthalocyanine core and peripheral carboxylate acid groups was synthesized and characterized. Its photophysical and photochemical properties were studied and compared with phthalocyanine. Due to the extended π-conjugation, both the Q band and fluorescence emit of this naphthalocyanine bathochromically shift to NIR region. It also exhibits superior NIR photodynamic efficiency to phthalocyanine as evidenced by high efficiency in generating singlet oxygen (Φ_Δ = 0.66) and in vitro phototoxicity toward Hela human cervical cancer cells. Therefore, this novel naphthalocyanine could potentially be a NIR photosensitizer for photodynamic therapy.     

Evaluation of oxygen dependence on in vitro and in vivo cytotoxicity of photoimmunotherapy using IR-700–antibody conjugates

Photoimmunotherapy (PIT) using the near-infrared-absorbing photosensitizing phthalocyanine dye, IRDye 700DX (IR-700), conjugated with a tumor-targeting antibody such as panitumumab (Pan) has shown efficacy in in vitro studies and several preclinical models in mice with promise for clinical translation. PIT results in rapid necrotic cell death in vitro and tumor shrinkage in vivo. Photochemical studies with the Pan-IR-700 conjugate showed that this agent can support generation of singlet oxygen and also generate reactive oxygen species after exposure to near-infrared (NIR) light. Moreover, in vitro studies using A431 cells, singlet oxygen scavengers abrogated the efficacy of PIT with Pan-IR-700, while oxygen depletion to undetectable levels in the exposure chamber almost completely inhibited the cellular cytotoxicity of PIT. Survival of tumor bearing mice was prolonged in PIT-treated animals but mice whose tumors were made transiently hypoxic prior to PIT had no benefit from the treatment. The results from this study support a central role for molecular oxygen-derived species in cell death caused by PIT.     

Investigation into the influence of an acrylic acid acceptor in organic D-π-A sensitizers against phototoxicity

Photodynamic therapy (PDT) is a non-invasive, selective, and cost-effective cancer therapy. We previously reported that thiophene-based organic D-π-A sensitizers consist of an electron-donating (D) moiety, a π-conjugated bridge (π) moiety, and an electron-accepting (A) moiety, and are readily accessible and stable templates for photosensitizers that could be used in PDT. In addition, acrylic acid acceptor-containing photosensitizers exert a high level of phototoxicity. This study was an investigation into 1) the possibility of increasing phototoxicity by introducing another carboxyl group or by replacing a carboxyl group with a pyridinium group, and 2) the importance of an alkene in the acrylic acid acceptor for phototoxicity. A review of the design, synthesis, and evaluation of sensitizers revealed that neither dicarboxylic acid nor pyridinium photosensitizers enhance phototoxicity. An evaluation of a photosensitizer without an alkene in the acrylic acid moiety revealed that the alkene was not indispensable in the pursuit of phototoxicity. The obtained results provided new insight into the design of ideal D-π-A photosensitizers for PDT.     

Elucidating the mode of action for thiophene-based organic D-π-A sensitizers for use in photodynamic therapy

Photodynamic therapy (PDT) is a non-invasive, selective, and cost-effective cancer therapy. The development of readily accessible templates that allow rapid structural modification for further improvement of PDT remains important. We previously reported thiophene-based organic D-π-A sensitizers consisted of an electron-donating (D) moiety, a π-conjugated bridge (π) moiety, and an electron-accepting (A) moiety as valuable templates for a photosensitizer that can be used in PDT. Our preliminary structure-activity relationship study revealed that the structure of the A moiety significantly influences its phototoxicity. In this study, we evaluated the photoabsorptive, cellular uptake, and photo-oxidizing abilities of D-π-A sensitizers that contained different A moieties. The level of phototoxicity of the D-π-A sensitizers was rationalized by considering those three abilities. In addition, we observed the ability of amphiphilic sensitizers containing either a carboxylic acid or an amide in an A moiety to form aggregates that penetrate cells mainly via endocytosis.     

Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are over-expressed on the surface of many tumour cells. In this study, different folic acid–OEG-conjugated photosensitizers were synthesized, characterized and their photophysical properties were evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the FA–porphyrin. All the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and singlet oxygen quantum yields were determined and were compared to the corresponding photosensitizer alone.     

Synthesis,spectroscopic studies and biological evaluation of acridine derivatives: The role of aggregation on the photodynamic efficiency

Two new photoactive compounds (1 and 2) derived from the 9-amidoacridine chromophore have been synthesized and fully characterized. Their abilities to produce singlet oxygen upon irradiation have been compared. The synthesized compounds show very different self-aggregating properties since only 1 present a strong tendency to aggregate in water. Biological assays were conducted with two cell types: hepatoma cells (Hep3B) and human umbilical vein endothelial cells (HUVEC). Photodynamic therapy (PDT) studies carried out with Hep3B cells showed that non-aggregating compound 2 showed photoxicity, ascribed to the production of singlet oxygen, being aggregating compound 1 photochemically inactive. On the other hand suspensions of 1, characterized as nano-sized aggregates, have notable antiproliferative activity towards this cell line in the dark.     

Online electrochemical monitoring of nitric oxide during photodynamic therapy

Photodynamic therapy (PDT), as a novel treatment modality, is based on the use of a photosensitizing agent with an excitation light source for the treatment of various malignancies. Its effect is mediated through reactive oxygen species and nitric oxide (NO), which are shown to be present in apoptosis. Individual differences among patients and even in different areas of the same tumor in one patient may cause a major problem with PDT: dose calculation during application of the light. An electrochemical sensor is proposed for online monitoring of NO generation as a solution of this problem. 5-Aminolevulinic acid (ALA) was administered as the photosensitizer in rat cerebellum. An amperometric sensor, selective to NO, was designed and tested both in vitro and in vivo during PDT. ALA-mediated PDT resulted in rapid generation of NO, starting as early as the application of light on the tissue. Simultaneous amperometric recordings have been carried out for 5 min during PDT. The progressive increase in NO concentration peaked at 1.10 min and then the response current began to decrease until it reached a plateau at around 70% of its peak value. This study, for the first time, electrochemically demonstrates the generation of NO during PDT. Rapid and stable responses obtained by the experimental setup confirmed that this method could be used as an online monitoring system for PDT-mediated apoptosis.     

New photodynamic molecular beacons (PMB) as potential cancer-targeted agents in PDT

Further improvements in Photodynamic therapy (PDT) necessitate that the dye targets more selectively tumour tissues or neovascularization than healthy cells. Different enzymes such as matrix metalloproteinases (MMPs) are overexpressed in tumour areas. Among these MMPs, gelatinases (MMP-2 and MMP-9) and its activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of many cancers such as glioblastoma multiforme (GBM), an aggressive malignant tumour of the brain. These last years, the concept of photodynamic molecular beacons (PMB) became interesting for controlling the photosensitizer’s ability to generate singlet oxygen (¹O₂) close to target biomolecules as MMPs. We report herein novel PMBs triggered by MMP-2 and/or MMP-9 and/or MMP-14, comprising a photosensitizer and a singlet oxygen quencher linked by MMP cleavable peptide linker (H-GRIGFLRTAKGG-OH). First of all, we focused on the synthesis and the photophysical study of different derivatives photosensitizer-peptide. This preliminary work concluded on an influence of the nature and the distance from the peptide, but not of the position of the photosensitizer in these derivatives on the proteolytic enzymatic action. The nature of the quencher used (a blackberry quencher (BBQ-650) or a black hole quencher (BHQ3)) does not influence the enzymatic action. We also studied the influence of an additional PEG spacer. Finally, the synthesis, the singlet oxygen quenching efficiency and the enzymatic activation of these new MMP- cleavable-PMBs were compared.     

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.     

Effect of intermittency factor on singlet oxygen and PGE2 formation in azulene-mediated photodynamic therapy: A preliminary study

In photodynamic therapy, intermittent irradiation modes that incorporate an interval between pulses are believed to decrease the effect of hypoxia by permitting an interval of re-oxygenation. The effect of the irradiation intermittency factor (the ratio of the irradiation pulse time to the total irradiation time) on singlet oxygen formation and inflammatory cytokine production was examined using azulene as a photosensitizer. Effects of difference intermittency factor on singlet oxygen formation and inflammatory cytokine were examined. Azulene solutions (1/10 μM) were irradiated with a 638-nm 500 mW diode laser in fractionation (intermittency factor of 5 or 9) or continuous mode using 50 mW/cm² at 4 or 8 J/cm². Singlet oxygen measurement was performed using a dimethyl anthracene probe. Peripheral blood mononuclear cells (PBMC) were stimulated by 10 ng/ml rhTNF-α for 6 h, before addition of 1 and 10 μM azulene solutions and irradiation. PGE₂ measurement was undertaken using a human PGE₂ ELISA kit. Kruskal-Wallis with Dunn Bonferroni test was used for statistical analyses at p < 0.05.Irradiation of 1 μM azulene+4 J/cm²+intermittency factor of 9 increased singlet oxygen 3-fold (p < 0.0001). Irradiation of 10 μM azulene at either 4 J/cm²+intermittency of 9 or 8 J/cm²+intermittency factor of 5 reduced PGE₂ expression in PBMCs to non-inflamed levels. Thus, at 50 mW/cm², 10 μM azulene-mediated photodynamic therapy with a high intermittency factor and a low energy density generated sufficient singlet oxygen to suppress PGE₂ in Inflamed PBMCs.     

Harmful singlet oxygen can be helpful

Highly reactive harmful singlet oxygen O2(1delta(g)) can be helpful while relaxing to its triplet ground state O2(3sigma(g)-). The energy emitted during this relaxation from the excited energy state is discernable at 634 nm. We report here on the effect of this energy as photon illumination and as energy transfer in air on the production of reactive oxygen species (ROS) by human monocytes, measured as isoluminol-enhanced chemiluminescence. We demonstrate up to 60% decrease in the secretion of ROS after 2-min illumination of the monocytes stimulated with phorbol myristate acetate (PMA). The results provide in vitro documentation of the utility of singlet oxygen energy in modifying cellular behaviour.     

Photodynamic biofilm inactivation by SAPYR—An exclusive singlet oxygen photosensitizer

Prevention and control of biofilm-growing microorganisms are serious problems in public health due to increasing resistances of some pathogens against antimicrobial drugs and the potential of these microorganisms to cause severe infections in patients. Therefore, alternative approaches that are capable of killing pathogens are needed to supplement standard treatment modalities. One alternative is the photodynamic inactivation of bacteria (PIB). The lethal effect of PIB is based on the principle that visible light activates a photosensitizer, leading to the formation of reactive oxygen species, e.g., singlet oxygen, which induces phototoxicity immediately during illumination. SAPYR is a new generation of photosensitizers. Based on a 7-perinaphthenone structure, it shows a singlet oxygen quantum yield Φ_Δ of 99% and is water soluble and photostable. Moreover, it contains a positive charge for good adherence to cell walls of pathogens. In this study, the PIB properties of SAPYR were investigated against monospecies and polyspecies biofilms formed in vitro by oral key pathogens. SAPYR showed a dual mechanism of action against biofilms: (I) it disrupts the structure of the biofilm even without illumination; (II) when irradiated, it inactivates bacteria in a polymicrobial biofilm after one single treatment with an efficacy of ≥99.99%. These results encourage further investigation on the potential of PIB using SAPYR for the treatment of localized infectious diseases.     

Synthesis, photophysical and photochemical properties of novel soluble tetra[4-(thiophen-3yl)-phenoxy]phthalocyaninato zinc(II) and Ti(IV)O complexes

The synthesis, photophysical and photochemical properties of zinc and oxo-titanium phthalocyanine derivatives 4-(tetra[4-(thiophen-3yl)-phenoxy]phthalocyaninato)zinc(II), (2); and 4-(tetra[4-(thiophen-3yl)-phenoxy]phthalocyaninato)oxo-titanium(IV), (3), are described for the first time. These peripherally substituted complexes (2 and 3) have been synthesized and characterized by elemental analysis, IR, ¹H NMR and electronic spectroscopy. The compounds (2 and 3) have good solubility in organic solvents such as CHCl₃, DCM, DMSO, DMF, THF and toluene and are not aggregated within a wide concentration range. General trends are described for singlet oxygen, photodegradation, fluorescence quantum yields, triplet quantum yields and triplet life times of these complexes in DMSO, DMF and THF. Compound 2 has higher fluorescence quantum yields, triplet quantum yields and triplet life times than 3, however, the former has lower singlet oxygen quantum yields and photodegradation quantum yields than the latter.     

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs). The 30-nm polyacrylamide-based NPs provide protection for the embedded MB against reduction by diaphorase enzymes. Furthermore, our data shows the matrix-protected MB efficiently induces photodynamic damage to tumor cells. The unprecedented results demonstrate the significant in vitro photodynamic effectiveness of MB when encapsulated within NPs, which promises to open new opportunities for MB in its in vivo and clinical studies.     

Influence of the pH on the photodynamic effect in lysozyme A comparative kinetic study with the sensitized photooxidation of isolated amino acids

Summary The kinetics of the eosin-sensitized photooxidation ([O₂(¹_g)]-mediated) of the protein lysozyme (Lyso) was investigated under two different pH conditions (pH 7 and pH 11). Rates of oxygen consumption and the fade in the protein fluorescence spectrum upon sensitized irradiation were monitored. Parallel studies on both denatured Lyso (absence of the four-S-S- bridges in the protein) and different mixtures of the photooxidizable amino acids of Lyso were also carried out. The mixtures maintained the same molar ratio as in the native protein, and were selected just in order to throw into relief the preferential amino acids that were being photooxidized at both pH values.Under work conditions Lyso was only photooxidizable at pH 7, whereas the opposite accounted for the denatured protein: only measurable oxygen consumption was detected at pH 11. Nevertheless, Lyso at pH 11, evidenced an important physical quenching of O₂(¹_g) due to the Tyr and Trp residues.The results for the native protein were interpreted on the basis of a previously described dark complex Eosin-Lyso, which selectively favours the photooxidation of the bounded protein. The Trp residues were the main reactive entities in the native protein. The photodinamic effect in denatured Lyso was characterized by the prevalence of Tyr residues as photooxidizable targets.In the discussion of the results, a comparisson with the photooxidation kinetics of the mixtures of free amino acids was made.Abbreviations O₂(³_g ^–) ground state triplet oxygen - O₂(¹_g) singlet molecular oxygen - Lyso lysozyme - LysoD denatured lysozyme - Eos eosin - FFA furfuryl alcohol - Trp tryptophan - Tyr tyrosine - Cys cysteine - Cis cystine - Met methionine - His histidine - AA amino acid - a.u. arbitrary units     

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications.