Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3 h after intravenous injection and rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.     

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.     

Self-assembled chlorin e6 conjugated chondroitin sulfate nanodrug for photodynamic therapy

Acetylated-chondroitin sulfate/chlorin e6 conjugates (Ac-CS/Ce6 1, 2, 3) were synthesized via the formation of an ester linkage between CS and Ce6 and evaluated as nanoscale drugs for photodynamic therapy. Ac-CS/Ce6 2 and 3 with higher Ce6 contents of 11.7 and 17.6%, respectively, had average diameters of <150 nm and were very stable in phosphate-buffered saline (PBS) for 1 month. The critical self-quenching concentration (CQC) of Ac-CS/Ce6 decreased as the conjugated-amount of Ce6 increased. All samples displayed autophotoquenching properties in aqueous solution, whereas their fluorescence intensity strongly correlated with the amount of Ce6 in the organic solvent dimethyl sulfoxide (DMSO). Compared with free Ce6, Ac-CS/Ce6 nanodrug photoactivity was maintained in terms of fluorescence properties and singlet oxygen ((1)O(2)) generation. In a HeLa cell culture system, we observed rapid cellular uptake of the Ac-CS/Ce6 nanodrug without any other ligands using confocal imaging and fluorescence-activated cell sorting (FACS) analysis. Upon light irradiation following cellular uptake, phototoxicity was detected via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The self-quenching effect and fluorescence recovery of Ac-CS/Ce6 were also determined both in vitro and in vivo. Taken together, our results indicate that Ac-CS/Ce6 has potential as an effective photodynamic therapy (PDT) prodrug for clinical application.     

Butylcycloheptylprodigiosin and undecylprodigiosin are potential photosensitizer candidates for photodynamic cancer therapy

Molecular Biology Reports - Prodiginines are bacterial red polypyrrole pigments and multifaceted secondary metabolites. These agents have anti-proliferative, immunosuppressive, antimicrobial, and...     

Diacetylcurcumin: a new photosensitizer for antimicrobial photodynamic therapy in Streptococcus mutans biofilms

This study evaluated the effect of antimicrobial photodynamic therapy (aPDT) on S. mutans using diacetylcurcumin (DAC) and verified DAC toxicity. In vitro, S. mutans biofilms were exposed to curcumin (CUR) and DAC and were light-irradiated. Biofilms were collected, plated and incubated for colony counts. DAC and CUR toxicity assays were conducted with Human Gingival Fibroblast cells (HGF). In vivo, G. mellonella larvae were injected with S. mutans and treated with DAC, CUR and aPDT. The hemolymph was plated and incubated for colony counts. Significant reductions were observed when DAC and CUR alone were used and when aPDT was applied. HGF assays demonstrated no differences in cell viability for most groups. DAC and CUR reduced the S. mutans load in G. mellonella larvae both alone and with aPDT. Systematic toxicity assays on G. mellonella demonstrated no effect of DAC and CUR or aPDT on the survival curve.     

Novel hydrophilic galactose-conjugated chlorin e6 derivatives for photodynamic therapy and fluorescence imaging

We synthesized new hydrophilic chlorin e₆ derivatives with two and four galactose fragments conjugated to the macrocycle via carbon atom in position 6 of the galactose fragment. Galactose fragments were inserted by alkylation of the amino groups of chlorin e₆ amides with one and two ethylene diamine fragments on the macrocycle periphery with triflate of diacetone galactose, followed by removal of diisopropylidene protection by 70% aqueous trifluoroacetic acid. The synthesized compounds were shown to be capable of penetrating the membrane of HeLa cells; they have intense red fluorescence inside the cell and have phototoxic properties towards HeLa cells (upon LED irradiation at 660 nm and light exposure value of 12 J/cm²). These properties, along with water solubility, allow us to consider the synthesized compounds to be promising as potential antitumor PSs and diagnostic compounds for visualizing malignant tumors and creating on their basis preparations for simultaneous diagnostics and therapy of oncological diseases.     

Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer

New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and l-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer.     

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (¹O₂) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect ¹O₂ phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of ¹O₂ phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with ¹O₂ phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time ¹O₂ dosimetry during PDT treatment using CW light.     

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.     

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.     

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.     

Insights into photodynamic therapy dosimetry: simultaneous singlet oxygen luminescence and photosensitizer photobleaching measurements

Photodynamic therapy (PDT) is generally based on the generation of highly reactive singlet oxygen (¹O₂) through interactions of photosensitizer, light, and oxygen (³O₂). These three components are highly interdependent and dynamic, resulting in variable temporal and spatial ¹O₂ dose deposition. Robust dosimetry that accounts for this complexity could improve treatment outcomes. Although the 1270 nm luminescence emission from ¹O₂ provides a direct and predictive PDT dose metric, it may not be clinically practical. We used ¹O₂ luminescence (or singlet oxygen luminescence (SOL)) as a gold-standard metric to evaluate potentially more clinically feasible dosimetry based on photosensitizer bleaching. We performed in vitro dose-response studies with simultaneous SOL and photosensitizer fluorescence measurements under various conditions, including variable ³O₂, using the photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC). The results show that SOL was always predictive of cytotoxicity and immune to PDT's complex dynamics, whereas photobleaching-based dosimetry failed under hypoxic conditions. However, we identified a previously unreported 613 nm emission from mTHPC that indicates critically low ³O₂ levels and can be used to salvage photobleaching-based dosimetry. These studies improve our understanding of PDT processes, demonstrate that SOL is a valuable gold-standard dose metric, and show that when used judiciously, photobleaching can serve as a surrogate for ¹O₂ dose.     

Real-time molecular recognition between protein and photosensitizer of photodynamic therapy by quartz crystal microbalance sensor

Real-time investigation of molecular recognition between protein and the photosensitizer of photodynamic therapy (PDT) was carried out by a quartz crystal microbalance (QCM) sensor integrated into a flow injection analysis (FIA) system. The photosensitizer meso-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) was immobilized on the gold electrode of the QCM chip by combining the sol-gel and self-assembly methods. Such a rapid screen analysis of molecular recognition showed that the p-THPP-immobilized sensor exhibited sensitive and specific interaction only with hemoglobin (Hb). The kinetic rate constants (k_ass and k_diss) and the equilibrium association constant (K_A) for p-THPP-Hb interaction were calculated by linear regression. The sensing performance characteristics of the proposed sensor were investigated. The sensor showed excellent selectivity, reproducibility, and repeatability for the detection of Hb. A linear calibration plot was obtained over a range from 0.2 to 1.0 μM with a detection limit (signal/noise ratio = 3) of 0.15 μM. The response mechanism of the sensor is discussed in detail. Due to its low cost and simple manipulation, this QCM-FIA system was shown to be a highly effective method for the investigation of interaction between biomacromolecules and the PDT photosensitizer. It also provides a potential strategy for screening an efficient and less harmful photosensitizer for PDT application.     

Uptake of photofrin II,a photosensitizer used in photodynamic therapy,by tumour cells in vitro

Photosensitizing dyes are used in fluorescence diagnostics and photodynamic therapy (PDT). These usually hematoporphyrin derivatives (HpD) accumulate preferentially within neoplastic tissues. HpD is a mixture of ether and ester linked porphyrins. Its partially purified form is known under the commercial name of photofrin II (PII). PII emission spectra were studied in a hydrophilic (PBS) and a lipophilic (PC liposomes) environment. Red shift was observed in their emission maxima from 615 nm in buffer solution to 635 nm in lipid. Identical red shift was obtained when the intracellular fluorescence of two cancer cell lines, MCF 7 and Jurkat, incubated with PII was investigated. Thus, intramembrane localization of PII may be suggested. As determined from the total fluorescence intensity, the uptake of PII was only slightly higher for Jurkat than for MCF 7 cells. Nevertheless the kinetics of the uptake was found to be different for both cell lines.     

Investigation of a novel dendritic derivative of 5-aminolaevulinic acid for photodynamic therapy

Photodynamic therapy is a treatment for malignant and certain non-malignant lesions that involves administration of a photosensitising drug. The use of 5-aminolaevulinic acid-induced porphyrins has become one of the most active fields of photodynamic therapy research. Since the efficacy of the treatment is somewhat limited by the hydrophilic nature of 5-aminolaevulinic acid, chemical modifications such as esterification with aliphatic alcohols have been made to induce higher porphyrin production. In an attempt to improve delivery of 5-aminolaevulinic acid to tissue, we have investigated the use of dendritic derivatives capable of bearing several drug molecules. The aim of this work was to evaluate in vivo and in vitro the efficacy of the first generation dendron, aminomethane tris-methyl 5-aminolaevulinic acid (containing three 5-aminolaevulinic acid residues) in terms of porphyrin synthesis. In LM3 cells, the dendron induced similar porphyrin levels compared to equimolar concentrations of 5-aminolaevulinic acid. Although the dendron is taken up with comparable efficiency to 5-aminolaevulinic acid, we found that there is only partial intracellular liberation of 5-aminolaevulinic acid residues. Both systemic and topical administration of the dendron to tumour-bearing mice induced higher porphyrin levels than the widely investigated hexyl ester derivative in most tissues studied, although it was not possible to surpass the levels induced by 5-aminolaevulinic acid. In conclusion, aminomethane tris-methyl 5-aminolaevulinic acid is capable of being taken up by cells efficiently, and liberating the active residues, although in vivo it was not possible to improve upon the efficacy of 5-aminolevulinic acid. Studies of accessibility and regulation of the esterases are needed to improve the design of these dendritic derivatives.     

Precise ligand engineering of Ir(III)-based photosensitizer with aggregation-induced emission for image-guided photodynamic therapy

Photodynamic therapy (PDT), which relies on the production of reactive oxygen species (ROS) induced by a photosensitizer to kill cancer cells, has become a non-invasive approach to combat cancer. However, the conventional aggregation-caused quenching effect, as well as the low ROS generation ability of photosensitizers, restrict their biological applications. In this work, a new Ir(III) complex with a dendritic ligand has been strategically designed and synthesized by ingenious modification of the ancillary ligand of a reported Ir(III) complex ( Ir-1 ). The extended π-conjugation and multiple aromatic donor moieties endow the resulting complex Ir-2 with obvious aggregation-induced emission (AIE) activity and bathochromic emission. In in vitro experiments, importantly, Ir-2 nanoparticles exhibit the excellent photoinduced ROS generation capabilities of O₂^•− and ¹O₂, as well as excellent biocompatibility and the lipid droplets (LDs) targeting feature. This study would provide useful guidance to design efficient Ir(III)-based photosensitizers used in biological applications in the future.     

Synthesis and in vitro testing of a pyropheophorbide-a-fullerene hexakis adduct immunoconjugate for photodynamic therapy

The employment of carriers to enhance drug selectivity is one of the strategies to increase the efficacy and reduce the side effects of antitumor therapy. The concept of a modular carrier system (MCS) was developed to construct a complex drug having a high efficacy and selectivity. An MCS employs diverse units or modules: beside the therapeutic unit, an addressing unit (e.g., an antibody) serves to direct the drug to its target, and a multiplying unit has the role of increasing the number of biological active moieties the system can carry. In this paper, we report on the synthesis of a modular carrier system in which the role of multiplying unit is given to a [5:1]fullerene hexakis adduct. This fullerene hexaadduct has five malonate spacers which can bind two therapeutic units (the photosensitizer pyropheophorbide-a) each, for a total of ten, and a longer malonate spacer which serves for the conjugation to the addressing unit, the monoclonal antibody rituximab. Confocal microscopy studies using Epstein-Barr virus-transformed B-lymphocytes and Jurkat cells showed that the antibody conjugate conserves the affinity for its receptor (CD20) and its selectivity toward CD20 positive B-lymphocytes. On the contrary, the antibody-free complex did not show any bounding or intracellular uptake.     

Synthesis and investigation of glycosylated mono- and diarylporphyrins for photodynamic therapy.

The synthesis of a diaryl substituted porphyrin bearing a galactosyl and a cholesteryloxy substituent and of a galactosyl substituted monoaryl porphyrin is described. The spectroscopic and aggregation properties of both compounds were investigated. The galactosyl substituted monoaryl porphyrin (12) was efficiently incorporated into liposomes and lipoproteins whereas the diaryl porphyrin showed no interaction with these lipids. Furthermore the binding constants of compound 12 to HDL, LDL, VLDL, and PE and DMPC liposomes were estimated.     

Synthesis and photodynamic activity of novel asymmetrically substituted fluorinated phthalocyanines

A series of asymmetrically substituted dodecafluorinated phthalocyanines has been synthesized via the Kobayashi ring expansion reaction of the corresponding dodecafluorinated boron subphthalocyanine with differently substituted 1,3-diiminoisoindolines. The mild reaction conditions employed during this ring expansion reaction gave rise exclusively to 3:1 asymmetrically substituted dodecafluorinated phthalocyanines. Metal insertion into the metal-free phthalocyanines was accomplished by heating at 40 degrees C in N,N-dimethylformamide in the presence of zinc bromide. The resulting zinc dodecafluorophthalocyanines were formulated as Cremophor EL oil-water emulsions and evaluated as photosensitizers in vitro against EMT-6 mouse mammary tumor cells. As compared to the previously studied zinc hexadecafluorophthalocyanine, these new asymmetrical zinc dodecafluorophthalocyanines exhibited improved photodynamic activity.     

Synthesis, photobiological activity and photoreactivity of methyl-thieno-8-azacoumarins, novel bioisosters of psoralen

4,4'-dimethyl-thieno-8-azacoumarin (6) and 4,4',5'-trimethyl-thieno-8-azacoumarin (8) were synthesised. Their photobiological activity was tested on human tumour cell lines. Interestingly, for 6, a photocytotoxic ability higher in HL-60, comparable in HeLa cells, with respect to that of the well-known drug 8-methoxypsoralen (8-MOP), was demonstrated. The covalent photoaddition to DNA occurs by means of the molecular mechanism already demonstrated for furocoumarins. However, it is noteworthy that no skin phototoxicity appears.