Toxicological assessment of photoactivated tetra-cationic porphyrin molecules under white light exposure in a Caenorhabditis elegans model.

Photodynamic therapy (PDT) utilizes the potential of photosensitizing substances to absorb light energy and produce reactive oxygen species. Tetra-cationic porphyrins, which have organic or coordination compounds attached to their periphery, are heterocyclic derivatives with well-described antimicrobial and antitumoral properties. This is due to their ability to produce reactive oxygen species and their photobiological properties in solution. Consequently, these molecules are promising candidates as new and more effective photosensitizers with biomedical, environmental, and other biomedical applications. Prior to human exposure, it is essential to establish the toxicological profile of these molecules using in vivo models. In this study, we used Caenorhabditis elegans, a small free-living nematode, as a model for assessing toxic effects and predicting toxicity in preclinical research. We evaluated the toxic effects of porphyrins (neutral and tetra-cationic) on nematodes under dark/light conditions. Our findings demonstrate that tetra-methylated porphyrins (3TMeP and 4TMeP) at a concentration of 3.3 µg/mL (1.36 and 0.93 µM) exhibit high toxicity (as evidenced by reduced survival, development, and locomotion) under dark conditions. Moreover, photoactivated tetra-methylated porphyrins induce higher ROS levels compared to neutral (3TPyP and 4TPyP), tetra-palladated (3PdTPyP and 4PdTPyP), and tetra-platinated (3PtTPyP and 4PtTPyP) porphyrins, which may be responsible for the observed toxic effects.

Computational Assessment of a Dual-Action Ru(II)-Based Complex: Photosensitizer in Photodynamic Therapy and Intercalating Agent for Inducing DNA Damage.

A combined quantum-mechanical and classical molecular dynamics study of a recent Ru(II) complex with potential dual anticancer action is reported here. The main basis for the multiple action relies on the merocyanine ligand, whose electronic structure allows the drug to be able to absorb within the therapeutic window and in turn efficiently generate 1O2 for photodynamic therapy application and to intercalate within two nucleobases couples establishing reversible electrostatic interactions with DNA. TDDFT outcomes, which include the absorption spectrum, triplet states energy, and spin-orbit matrix elements, evidence that the photosensitizing activity is ensured by an MLCT state at around 660 nm, involving the merocyanine-based ligand, and by an efficient ISC from such state to triplet states with different characters. On the other hand, the MD exploration of all the possible intercalation sites within the dodecamer B-DNA evidences the ability of the complex to establish several electrostatic interactions with the nucleobases, thus potentially inducing DNA damage, though the simulation of the absorption spectra for models extracted by each MD trajectory shows that the photosensitizing properties of the complex remain unaltered. The computational results support that the anti-tumor effect may be related to multiple mechanisms of action.

An oxygen-economical nano-photosensitizer with a high photodynamic therapeutic outcome via simultaneous reduction of the cellular respiration and oxygen depletion of PDT.

The development of photodynamic nanomedicines that can alleviate intratumoral oxygen deficiency during photodynamic therapy (PDT) is of great significance for improving the therapeutic outcome of solid tumors characterized by severe hypoxia. Massive oxygen consumption due to vigorous cellular respiration, i.e., mitochondrial-associated oxidative phosphorylation (OXPHOS), is another major cause of severe tumor hypoxia in addition to insufficient oxygen supply. Moreover, oxygen depletion during PDT further exacerbates the shortage of intratumoral oxygen. In this work, we engineered a novel oxygen-economical nano-photosensitizer via co-encapsulation of an OXPHOS inhibitor (ATO) and a newly developed type-I photosensitizer (IPS) into a polymeric micelle of PEG-b-PCL. By controlling the length of hydrophobic PCL segments, we successfully optimized the micelle size to around 30 nm for enhanced tumor penetration. The orchestration of the two functional components, ATO and IPS, can simultaneously hinder the two major tumor oxygen-consuming pathways, where ATO targets mitochondrial complex III to inhibit cellular respiration, while IPS generates ROS through a low oxygen-consuming type-I photochemical pathway, enabling remarkable PDT efficacies in both hypoxic cells and a 4T1 tumor-bearing BALB/c mouse model. This work sheds new light on the construction of nano-photosensitizers to rejuvenate PDT against hypoxic solid tumors.

Broaden sources and reduce expenditure: Tumor-specific transformable oxidative stress nanoamplifier enabling economized photodynamic therapy for reinforced oxidation therapy.

Cancer cells immersed in inherent oxidative stress are more vulnerable to exogenous oxidative damages than normal cells. Reactive oxygen species (ROS)-mediated oxidation therapy preferentially aggravating tumor oxidative stress to disrupt redox homeostasis, has emerged as an effective and specific anticancer treatment. Herein, following an ingenious strategy of "broaden sources and reduce expenditure", we designed a versatile tumor-specific oxidative stress nanoamplifier enabling economized photodynamic therapy (PDT), to achieve synergistic oxidative stress explosion for superior oxidation therapy. Methods: Cinnamaldehyde (CA) as a therapeutic ROS generator was first conjugated to hyaluronic acid (HA) through acid-labile hydrazone bond to synthesize tailored amphiphilic HA@CA conjugates, which could surprisingly self-assemble into uniform nanofibers in aqueous media. Photosensitizer protoporphyrin (PpIX) was efficiently encapsulated into HA@CA nanofibers and transformed HA@CA nanofibers to final spherical HA@CAP. Results: With beneficial pH-responsiveness and morphology transformation, improved bioavailability and selective tumor accumulation, HA@CAP combining ROS-based dual chemo/photodynamic treatment modalities could induce cytotoxic ROS generation in a two-pronged approach to amplify tumor oxidative stress, termed "broaden sources". Moreover, utilizing CA-induced H2O2 production and cascaded Fenton reaction in mitochondria to consume intracellular overloaded Fe(II), HA@CAP could skillfully block endogenic heme biosynthesis pathway on site to restrain undesired elimination of PpIX for economized PDT, termed "reduce expenditure". Both in vitro and in vivo results demonstrated the superior antitumor performance of HA@CAP. Conclusion: This study offered an inspiring strategy of "broaden sources and reduce expenditure" to specifically boost tumor oxidative stress for reinforced oxidation therapy.

Lipid monolayer as a simple model membrane for comparative assessment of the photodynamic therapy photosensitizer efficiency via macroscopic measurements.

Cellular membrane is one of the main targets of photodynamic therapy. Its high complexity has led to the study of the efficiency of photosensitizers on artificial lipid systems mimicking membranes. However, the preliminary analysis of this efficiency remains limited due to difficulty of the model construction and/or implementation of the required measurement techniques. Hereby, we propose a quite simple way for the rapid comparative assessment of novel photosensitizers in terms of membrane photodegradation, based on simple and fast measurements, such as wetting angle and surface plasmon resonance spectroscopy. As a proof of concept, we applied this methodology to two bacteriopurpurinimide derivatives. We have shown in particular that such complementary techniques can be employed not only for the multiparametric monitoring of the kinetics of the photodegradation, but also for the comparison of the damaging efficiency of the photosensitizers in the lipid structures as well.

Validation of combined Monte Carlo and photokinetic simulations for the outcome correlation analysis of benzoporphyrin derivative-mediated photodynamic therapy on mice.

We compare previously reported benzoporphyrin derivative (BPD)-mediated photodynamic therapy (PDT) results for reactive singlet oxygen concentration (also called singlet oxygen dose) on mice with simulations using a computational device, Dosie™, that calculates light transport and photokinetics for PDT in near real-time. The two sets of results are consistent and validate the use of the device in PDT treatment planning to predict BPD-mediated PDT outcomes in mice animal studies based on singlet oxygen dose, which showed a much better correlation with the cure index than the conventional light dose.

DANPY (dimethylaminonaphthylpyridinium): an economical and biocompatible fluorophore.

Dyes with nonlinear optical (NLO) properties enable new imaging techniques and photonic systems. We have developed a dye (DANPY-1) for photonics applications in biological substrates such as nucleic acids; however, the design specification also enables it to be used for visualizing biomolecules. It is a prototype dye demonstrating a water-soluble, NLO-active fluorophore with high photostability, a large Stokes shift, and a favorable toxicity profile. A practical and scalable synthetic route to DANPY salts has been optimized featuring: (1) convergent Pd-catalyzed Suzuki coupling with pyridine 4-boronic acid, (2) site-selective pyridyl N-methylation, and (3) direct recovery of crystalline intermediates without chromatography. We characterize the optical properties, biocompatibility, and biological staining behavior of DANPY-1. In addition to stability and solubility across a range of polar media, the DANPY-1 chromophore shows a first hyperpolarizability similar to common NLO dyes such as Disperse Red 1 and DAST, a large two-photon absorption cross section for its size, substantial affinity to nucleic acids in vitro, an ability to stain a variety of cellular components, and strong sensitivity of its fluorescence properties to its dielectric environment.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Ultralow-Power Near Infrared Lamp Light Operable Targeted Organic Nanoparticle Photodynamic Therapy.

Tissue penetration depth is a major challenge in practical photodynamic therapy (PDT). A biocompatible and highly effective near infrared (NIR)-light-absorbing carbazole-substituted BODIPY (Car-BDP) molecule is reported as a class of imaging-guidable deep-tissue activatable photosensitizers for PDT. Car-BDP possesses an intense, broad NIR absorption band (600-800 nm) with a remarkably high singlet oxygen quantum yield (ΦΔ = 67%). After being encapsulated with biodegradable PLA-PEG-FA polymers, Car-BDP can form uniform and small organic nanoparticles that are water-soluble and tumor-targetable. Rather than using laser light, such nanoparticles offer an unprecedented deep-tissue, tumor targeting photodynamic therapeutic effect by using an exceptionally low-power-density and cost-effective lamp light (12 mW cm-2). In addition, these nanoparticles can be simultaneously traced in vivo due to their excellent NIR fluorescence. This study signals a major step forward in photodynamic therapy by developing a new class of NIR-absorbing biocompatible organic nanoparticles for effective targeting and treatment of deep-tissue tumors. This work also provides a potential new platform for precise tumor-targeting theranostics and novel opportunities for future affordable clinical cancer treatment.

Nanoparticle-aided external beam radiotherapy leveraging the Cerenkov effect.

This study investigates the feasibility of exploiting the Cerenkov radiation (CR) present during external beam radiotherapy (EBRT) for significant therapeutic gain, using titanium dioxide (titania) nanoparticles (NPs) delivered via newly designed radiotherapy biomaterials. Using Monte Carlo radiation transport simulations, we calculated the total CR yield inside a tumor volume during EBRT compared to that of the radionuclides. We also considered a novel approach for intratumoral titania delivery using radiotherapy biomaterials (e.g. fiducials) loaded with NPs. The intratumoral distribution/diffusion of titania released from the fiducials was calculated. To confirm the CR induced enhancement in EBRT experimentally, we used 6MV radiation to irradiate human lung cancer cells with or without titania NPs and performed clonogenic assays. For a radiotherapy biomaterial loaded with 20µg/g of 2-nm titania NPs, at least 1µg/g could be delivered throughout a tumor sub-volume of 2-cm diameter after 14days. This concentration level could inflict substantial damage to cancer cells during EBRT. The Monte Carlo results showed the CR yield by 6MV radiation was higher than by the radionuclides of interest and hence greater damage might be obtained during EBRT. In vitro study showed significant enhancement with 6MV radiation and titania NPs. These preliminary findings demonstrate a potential new approach that can be used to take advantage of the CR present during megavoltage EBRT to boost damage to cancer cells. The results provide significant impetus for further experimental studies towards the development of nanoparticle-aided EBRT powered by the Cerenkov effect.

Tumor reactive ringlet oxygen approach for Monte Carlo modeling of photodynamic therapy dosimetry.

Photodynamic therapy (PDT) is an emergent technique used for the treatment of several diseases. It requires the interaction of three components: a photosensitizer, a light source and tissue oxygen. Knowledge of the biophysical aspects of PDT is important for improving dosimetry protocols and treatment planning. In this paper we propose a model to simulate the spatial and temporal distribution of ground state oxygen ((3)O2), cumulative singlet excited state oxygen ((1)O2)rx and photosensitizer, in this case protoporphyrin IX (PpIX) in an ALA mediated PDT treatment. The results are analyzed in order to improve the treatment dosimetry. We compute the light fluence in the tissue using Monte Carlo simulations running in a GPU system. The concentration of (3)O2, ((1)O2)rx and the photosensitizer are calculated using this light fluence and a set of differential equations describing the photochemical reactions involved in PDT. In the model the initial photosensitizer concentration depends on tissue depth and type, moreover we consider blood vessel damage and its effect in the ground state oxygen concentration in the tissue. We introduce the tumor reactive single oxygen (TRSO) as a new dosimetry metric. It represents the amount of singlet oxygen per tumor volume that reacts, during the treatment, with the molecules in the tumor. This quantity integrates the effect of the light irradiance, the optical properties of the tumor and the normal tissue, the oxygen consumption and supply, and the photosensitizer biodistribution on the skin.

Nanocarriers for photodynamic therapy-rational formulation design and medium-scale manufacture.

The development and manufacture of novel nanocarriers for drug delivery has proved challenging with regards to scale-up and pharmaceutical quality. Polymeric nanocarriers composed of poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) were prepared and the photosensitizer meso-tetrakis(3-hydroxyphenyl) chlorin (mTHPC) was effectively encapsulated. Furthermore, the interplay of various process and formulation parameters and their impact on the most important product specifications were investigated by using a factorial design and a central composite design in a microfluidic manufacturing process. These nanoparticles for intravenous administration with a size of 97 ± 0.13 nm, narrow size distribution, and an encapsulation efficiency of more than 80% were produced at high throughput. In vitro stability and in vitro drug release testing were applied for quality control purposes. Finally, the toxicity of the photosensitizer was tested in vitro. The cytotoxicity was successfully reduced while the efficacy of the formulation was maintained. First observations using in vivo imaging suggest effective distribution of the nanocarrier system after injection into rodents. Thus, further in vivo testing of the beneficial effects of nanoencapsulation into the matrix system and its formulation will be considered for the delivery of mTHPC to tumor tissues during photodynamic therapy.

Assessment of the action spectrum for photooxidation in full fat bovine milk.

The action spectrum for photooxidation in full fat bovine milk was measured. Samples of milk with air or argon in headspace were exposed to narrow wavelength bands of light in the range 400-700 nm. Photooxidation in terms of off-flavors was measured by a sensory panel, volatile compounds by headspace solid phase micro extraction (SPME-GC-MS), and photobleaching of photosensitizers in milk (riboflavin, protoporphyrin IX and a chlorophyllic compound) by front face fluorescence spectroscopy. The action spectrum deviated significantly from the absorption spectrum of milk. Significant oxidation was induced by wavelengths around 400 nm and 500-650 nm in milk with air in headspace. Argon in headspace gave significant oxidation also at 700 nm. It is suggested that protoporphyrin IX and chlorophyll are responsible for oxidation induced by wavelengths >500 nm, and that also riboflavin is contributing from 400 to 500 nm.

The European Medicines Agency approval of 5-aminolaevulinic acid (Ameluz) for the treatment of actinic keratosis of mild to moderate intensity on the face and scalp: summary of the scientific assessment of the Committee for Medicinal Products for Human Use.

The European Commission has recently issued a marketing authorisation valid throughout the European Union for 5-aminolaevulinic acid (Ameluz). The decision was based on the favorable opinion of the CHMP recommending a marketing authorization for 5-aminolaevulinic acid for treatment of actinic keratosis of mild to moderate intensity on the face and scalp. The active substance is a sensitizer used in photodynamic/radiation therapy (ATC code L01XD04). The gel should cover the lesions and approximately 5 mm of the surrounding area with a film of about 1 mm thickness. The entire treatment area should be illuminated with a red light source, either with a narrow spectrum around 630 nm and a light dose of approximately 37 J/cm(2) or a broader and continuous spectrum in the range between 570 and 670 nm with a light dose between 75 and 200 J/cm(2). One session of photodynamic therapy should be administered for single or multiple lesions. Non- or partially responding lesions should be retreated in a second session 3 months after the first treatment. 5-aminolaevulinic acid is metabolized to protoporphyrin IX, a photoactive compound which accumulates intracellularly in the treated actinic keratosis lesions. Protoporphyrin IX is activated by illumination with red light of a suitable wavelength and energy. In the presence of oxygen, reactive oxygen species are formed which causes damage of cellular components and eventually destroys the target cells. The benefit with 5-aminolaevulinic acid is its ability to improve the complete response rate of actinic keratosis lesions. The most common side effects are reactions at the site of application. The objective of this article is to summarize the scientific review of the application. The detailed scientific assessment report and product information, including the summary of product characteristics (SmPC), are available on the EMA website (www.ema.europa.eu).

Effect of 1O2 quencher depletion on the efficiency of photodynamic therapy.

Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) fluorescence and photodynamic oxygen consumption were monitored during AlPcS4-photodynamic therapy (PDT) of Mat LyLu cells in suspension. These measurements were used to calculate the PDT efficiency, which is defined as the oxygen consumption rate divided by the sensitizer concentration. As a function of the intracellular oxygen concentration consumed by PDT, the normalized PDT efficiency fell off more quickly at lower photosensitizer concentrations. The changes in PDT efficiency were compared to models of PDT in which the photosensitizer (PS) and singlet oxygen quencher (A) were either free to diffuse or were fixed. The model in which PS and A are free to diffuse did not agree with the experimental data because this model predicts that the reduction in PDT efficiency is independent of [PS]. A Monte Carlo model was written to simulate PDT when both PS and A are stationary. This model was found to describe the experimental data when the initial intracellular [A] = 90 mM and when the initial and final (i.e. after all A has been depleted) singlet oxygen lifetimes were 0.4 and 1.2 µs respectively.

Role of intercalation and redox potential in DNA photosensitization by ruthenium(II) polypyridyl complexes: assessment using DNA repair protein tests.

Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids.

Printing ink compounds in foods: UK survey results.

Three hundred and fifty foodstuffs packaged in printed paper/board were purchased from UK retail outlets. Solvent extracts of all foods and associated quality assurance samples were analysed by gas chromatography-mass spectrometry (GC-MS) to determine the presence and concentrations of 20 printing ink compounds: benzophenone, 4-methylbenzophenone, 2-methylbenzophenone, 3-methylbenzophenone, 4-hydroxybenzophenone, 2-hydroxybenzophenone, 4-phenylbenzophenone, methyl-2-benzoylbenzoate, 1-hydroxycyclohexyl phenyl ketone, 2-isopropylthioxanthone, 4-isopropylthioxanthone, 2,4-diethyl-9H-thioxanthen-9-one, 2,2-dimethoxy-2-phenylacetophenone, 2-methyl-4'-(methylthio)-2-morpholinopropiophenone, 4-(4-methylphenylthio)benzophenone, ethyl-4-dimethylaminobenzoate, 2-ethylhexyl-4-(dimethylamino)benzoate, N-ethyl-p-toluene-sulphonamide, triphenyl phosphate and di-(2-ethylhexyl) fumarate. The presence of one or more of the compounds benzophenone, 4-phenylbenzophenone, methyl-2-benzoylbenzoate, 1-hydroxycyclohexyl phenyl ketone, 2,2-dimethoxy-2-phenylacetophenone, 4-(4-methylphenylthio)benzophenone, ethyl-4-dimethylaminobenzoate, 2-ethylhexyl-4-dimethylaminobenzoate and triphenyl phosphate was confirmed in some food samples. Analysis of the associated packaging material was also carried out to confirm whether or not it was likely that the occurrence of these compounds in the foods was due to migration from the printed paper/board packaging. With the exception of triphenyl phosphate, detected in one foodstuff, all the packaging material contained the substance(s) found in the food.

Continuous flow photocatalysis enhanced using an aluminum mirror: rapid and selective synthesis of 2'-deoxy and 2',3'-dideoxynucleosides.

A unique photochemical flow reactor featuring quartz tubing, an aluminum mirror and temperature control has been developed for the photo-induced electron-transfer deoxygenation reaction to produce 2'-deoxy and 2',3'-dideoxynucleosides. The continuous flow format significantly increased the efficiency and selectivity of the reaction.

Syntheses of furo[3,4-c]coumarins and related furyl coumarin derivatives via intramolecular Wittig reactions.

A new and general strategy for highly functional furo[3,4-c]coumarins and related furyl coumarin derivatives has been developed, which is based on an extraordinarily facile intramolecular Wittig reaction, starting from α,ß-unsaturated ketones, tributylphosphine, and acyl chlorides. The phosphorus ylides were proposed to be the key intermediates for constructing the crucial furan ring, leading to a wide variety of substituted furyl coumarins in one step.