Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities

A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exhibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application.     

Aminosquaraines as potential photodynamic agents: Synthesis and evaluation of in vitro cytotoxicity

The synthesis of several aminosquaraine cationic dyes displaying strong absorption within the so-called phototherapeutic window (650–850 nm) is described. Their cytotoxicity, under dark and illuminated conditions, was tested against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and non-tumor porcine liver primary cells. All compounds showed to inhibit the growth of the tumor cells upon irradiation more than in the absence of light, in more or less extension, clearly exhibiting photodynamic activity. The photosensitizing ability against some cell lines, together with the low toxicity for the non-tumor primary PLP2 cells displayed by some of the compounds synthetized, turns them into potential candidates as photosensitizers for PDT.     

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.     

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.     

Tumor microenvironment-activated nanosystems with selenophenol substituted BODIPYs as photosensitizers for photodynamic therapy

NIR-light-absorbing photosensitizers with the capability of selective localization and activation in tumor regions are of great importance for practical photodynamic therapy (PDT). Here, selenophenol substituted BODIPYs were designed and synthesized as new photosensitizers for PDT. One of these obtained BODIPYs, IBSeOV, possesses an intense and low energy absorption with a high singlet oxygen quantum yield (Φ_Δ = 60%). Considering manganese dioxide (MnO₂) nanosheets as versatile nanocarriers in cancer theranostics, nanosystem IBSeOV/MnO₂ was then fabricated to furnish tumor environment selective activation. Such designed nanoplatform allowed for GSH-controllable ¹O₂ production and exhibited low cytotoxicity in dark but good photocytotoxicity to cancer cells. The in vivo antitumor outcome suggested the high treatment efficiency of IBSeOV/MnO₂ for tumor therapy.     

Synthesis and biological evaluation of 173-dicarboxylethyl-pyropheophorbide-a amide derivatives for photodynamic therapy

Three novel 17³-dicarboxylethyl-pyropheophorbide-a amide derivatives as photosensitizers for photodynamic therapy (PDT) were synthesized from pyropheophorbide-a (Ppa). Their photophysical and photochemical properties, intracellular localization, photocytotoxicity in vitro and in vivo were investigated. All target compounds exhibited low cytotoxicity in the dark and remarkable photocytotoxicity against human esophageal cancer cells. Among them, 1a showed highest singlet oxygen quantum yield. Upon light activation, 1a exhibited significant photocytotoxicity. After PDT treatment, the growth of Eca-109 tumor in nude mice was significantly inhibited. Therefore, 1a is a powerful and promising antitumor photosensitizer for PDT.     

Compromising the plasma membrane as a secondary target in photodynamic therapy-induced necrosis

Photodynamic therapy (PDT) is a non-invasive treatment widely applied to different cancers. The goal of PDT is the photo-induced destruction of cancer cells by the activation of different cell death mechanisms, including apoptosis and/or necrosis. Recent efforts focusing on understanding the mechanisms of cell death activated by PDT find that it depends on the type of photosensitizer (PS), targeted organelles, and nature of the light used. It is generally accepted that very short incubation times are required to direct the PS to the plasma membrane (PM), while longer periods result in the accumulation of the PS in internal compartments such as the endoplasmic reticulum or mitochondria. Glycosylation of the PS targets cancer via saccharide receptors on the cell surface, and is generally assumed that these compounds rapidly internalize and accumulate, e.g. in the endoplasmic reticulum. Herein we demonstrate that a minor fraction of a glycosylated chlorin compound residing at the PM of cancer cells can activate necrosis upon illumination by compromising the PM independently of the length of the incubation period. The results presented here show that the PM can also be targeted by glycosylated PS designed to accumulate in internal organelles. PS activation to induce necrosis by compromising the plasma membrane has the benefits of fast cell death and shorter irradiation times. The findings described here expand our understanding of the cellular damage induced by phototherapies, presenting the possibility of activating another cell death mechanism based on the incubation time and type of light used.     

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.     

Zn phthalocyanines loaded into liposomes: Characterization and enhanced performance of photodynamic activity on glioblastoma cells

Photodynamic therapy (PDT) is considered a promising strategy for cancer treatment. PDT utilizes light in combination with a photosensitizer (PS) to induce several phototoxic reactions. Phthalocyanines (Pcs), a second generation of photosensitizers, have been studied in several cancer models. Among these, Pcs, have become of interest for the treatment of glioblastomas which are one of the most common and aggressive forms of tumors of the central nervous system. Due to the lipophilic nature of Pcs and their limited solubility in water, Pcs can be loaded in liposomes. In this work, we characterized liposomes of ZnPc and TAZnPc and their effectiveness to photoinactivate glioblastoma cells, was evaluated. Both Pcs show an increase in their photosensitizing activity when they were administrated in Dipalmitoylphosphatidylcholine-cholesterol liposomes compared to Pcs administrated in dimethylformamide.     

A cationic chalcogenoxanthylium photosensitizer effective in vitro in chemosensitive and multidrug-resistant cells

Pentacyclic thio- (1) and seleno- (2) analogues of tetramethylrosamine (TMR) were prepared with a julolidyl fragment replacing the 3-dimethylamino substituent in the xanthylium core. The pentacylic structure increases the lipophilicity of 1 and 2 relative to TMR-S and TMR-Se and locks the lone-pair of electrons on the julolidyl N atom into conjugation with the xanthylium core. This conformational rigidization leads to longer wavelengths of absorption, but has little impact on other photophysical properties such as quantum yields for fluorescence and singlet-oxygen generation and fluorescence lifetimes in 1 and 2 relative to TMR-S and TMR-Se. Both 1 and 2 are effective photosensitizers against chemosensitive AUXB1 cells in vitro at 1 × 10⁻⁷ M and compound 2 is an effective photosensitizer against multidrug-resistant CR1R12 cells in vitro at 1 × 10⁻⁷ M. While the uptake TMR-S into CR1R12 cells as measured by fluorescence is significantly lower than uptake into chemosensitive AUXB1 cells, there is no significant difference in the uptake of 1 into either AUXB1 or CR1R12 cells. The addition of 2 × 10⁻⁴ M verapamil to the cells prior to treatment with 1 had no significant effect on the uptake of 1 into either AUXB1 or CR1R12 cells. Treating lipid-activated, purified Pgp with 2 and light gave complete inhibition of Pgp ATPase activity.     

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     

Synthesis, electrochemistry and spectroscopic properties of ruthenium phthalocyanine and naphthalocyanine complexes with triphenylarsine ligands

The synthesis, electrochemistry and spectroscopic properties of [PcRu(AsPh₃)₂] (1) and [{(tBu)₄Nc}Ru(AsPh₃)₂] (2), where Pc = phthalocyanine and Nc = naphthalocyanine are reported. These complexes are the first examples of metal phthalocyanine and naphthalocyanine complexes with axially-coordinated arsine ligands. The AsPh₃ ligands readily dissociate in non-coordinating solvents with 2 showing more rapid dissociation. In cyclic voltammetry experiments, 1 displayed three macrocycle-centred redox processes; one reduction and two oxidation processes. One reduction and three oxidation processes were observed for 2. The reduction and first oxidation are assigned to macrocycle-centred processes. The UV-Vis spectra of both complexes recorded over time showed macrocycle-centred oxidation. The oxidation was hindered by removing dioxygen from the solvent or adding excess AsPh₃.     

Water-soluble cationic gallium(III) and indium(III) phthalocyanines for photodynamic therapy

The new tetra-non-peripheral and peripheral 2-mercaptopyridine substituted gallium(III) and indium(III) phthalocyanine complexes (np-GaPc, p-GaPc, np-InPc and p-InPc) and their quaternized derivatives (Qnp-GaPc, Qp-GaPc, Qnp-InPc and Qp-InPc) have been synthesized and characterized. The quaternized complexes show excellent solubility in water, which makes them potential photosensitizer for use in photodynamic therapy (PDT) of cancer. Photophysical and photochemical properties of these phthalocyanines were investigated. General trends are described for quantum yields of photodegradation, fluorescence and fluorescence lifetimes as well as singlet oxygen quantum yields of these compounds. In this study, the effects of the position of the substituents, the nature of the metal ion and quaternization of the substituents on the photophysical and photochemical parameters of the gallium(III) and indium(III) phthalocyanines are also reported. This study also presented the ionic gallium(III) and indium(III) phthalocyanines strongly bind to bovine serum albumin (BSA).     

A Ru(II) polypyridyl complex bearing aldehyde functions as a versatile synthetic precursor for long-wavelength absorbing photodynamic therapy photosensitizers

The use of Photodynamic Therapy (PDT) for the treatment of several kinds of cancer as well as bacterial, fungal or viral infections has received increasing attention during the last decade. However, the currently clinically approved photosensitizers (PSs) have several drawbacks, including photobleaching, slow clearance from the organism and poor water solubility. To overcome these shortcomings, many efforts have been made in the development of new types of PSs, such as Ru(II) polypyridyl complexes. Nevertheless, most studied Ru(II) polypyridyl complexes have a low absorbance in the spectral therapeutic window. In this work, we show that, by carefully selecting substituents on the polypyridyl complex, it is possible to prepare a complex absorbing at a much higher wavelength. Specifically, we report on the synthesis as well as in-depth experimental and theoretical characterisation of a Ru(II) polypyridyl complex (complex 3) combining a shift in absorbance towards the spectral therapeutic window with a high ¹O₂ production. To overcome the absence or poor selectivity of most approved PSs into targeted cells/bacteria, they can be linked to targeting moieties. In this line, compound 3 was designed with reactive aldehyde groups, which can be used as a highly versatile synthetic precursor for further conjugation. As a proof of concept, 3 was reacted with benzylamine and the stability of the resulting conjugate 4 was investigated in DMSO, PBS and cell media. 4 showed an impressive ability to act as a PDT PS with no measurable dark cytotoxicity and photocytotoxicity in the low micromolar range against cancerous HeLa cells from 450 nm up to 540 nm.     

A molecular hybrid producing simultaneously singlet oxygen and nitric oxide by single photon excitation with green light

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on “conventional” drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate ¹O₂ and NO with single photon excitation of biocompatible visible light, (ii) excellent ¹O₂ quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic ¹O₂ and NO.     

Pyridone-containing phenalenone-based photosensitizer working both under light and in the dark for photodynamic therapy

Photosensitizer attracts great attentions and has potential applications in cancer treatment. We developed here a novel pyridone-containing phenalenone-based (PPN-PYR) photosensitizer with excellent singlet oxygen generating ability. Upon light irradiation, PPN-PYR can produce singlet oxygen and transform to its endoperoxide form which in turn release singlet oxygen via thermal cycloreversion at dark. The ability of PPN-PYR to generate reactive oxygen species (ROS) in cell culture and induce corresponding apoptosis both at dark and under light was demonstrated. The efficient PDT performance of PPN-PYR was further verified on cancer cell in vitro. Our study indicate that PPN-PYR can alleviate tumor hypoxia problem and enhance the availability of intermittent 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.