Photodynamic inactivation of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa in municipal wastewater by tetracationic porphyrin and violet-blue light: The impact of wastewater constituents.

There is an increasing need to discover effective methods for treating municipal wastewater and addressing the threat of multidrug-resistant (MDR) strains of bacteria spreading into the environment and drinking water. Photodynamic inactivation (PDI) that combines a photosensitiser and light in the presence of oxygen to generate singlet oxygen and other reactive species, which in turn react with a range of biomolecules, including the oxidation of bacterial genetic material, may be a way to stop the spread of antibiotic-resistant genes. The effect of 5,10,15,20-(pyridinium-3-yl)porphyrin tetrachloride (TMPyP3) without light, and after activation with violet-blue light (VBL) (394 nm; 20 mW/cm2), on MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and K. pneumoniae OXA-48 in tap water and municipal wastewater was investigated. High toxicity (~2 µM) of TMPyP3 was shown in the dark on both strains of K. pneumoniae in tap water, while on P. aeruginosa toxicity in the dark was low (50 µM) and the PDI effect was significant (1.562 µM). However, in wastewater, the toxicity of TMPyP3 without photoactivation was much lower (12.5-100 µM), and the PDI effect was significant for all three bacterial strains, already after 10 min of irradiation with VBL (1.562-6.25 µM). In the same concentrations, or even lower, an anti-adhesion effect was shown, suggesting the possibility of application in biofilm control. By studying the kinetics of photoinactivation, it was found that with 1,562 µM of TMPyP3 it is possible to achieve the complete destruction of all three bacteria after 60 min of irradiation with VBL. This study confirmed the importance of studying the impact of water constituents on the properties and PDI effect of the applied photosensitiser, as well as checking the sensitivity of targeted bacteria to light of a certain wavelength, in conditions as close as possible to those in the intended application, to adjust all parameters and perfect the method.

Combination of Two Photosensitisers in Anticancer, Antimicrobial and Upconversion Photodynamic Therapy.

Photodynamic therapy (PDT) is a special form of phototherapy in which oxygen is needed, in addition to light and a drug called a photosensitiser (PS), to create cytotoxic species that can destroy cancer cells and various pathogens. PDT is often used in combination with other antitumor and antimicrobial therapies to sensitise cells to other agents, minimise the risk of resistance and improve overall outcomes. Furthermore, the aim of combining two photosensitising agents in PDT is to overcome the shortcomings of the monotherapeutic approach and the limitations of individual agents, as well as to achieve synergistic or additive effects, which allows the administration of PSs in lower concentrations, consequently reducing dark toxicity and preventing skin photosensitivity. The most common strategies in anticancer PDT use two PSs to combine the targeting of different organelles and cell-death mechanisms and, in addition to cancer cells, simultaneously target tumour vasculature and induce immune responses. The use of PDT with upconversion nanoparticles is a promising approach to the treatment of deep tissues and the goal of using two PSs is to improve drug loading and singlet oxygen production. In antimicrobial PDT, two PSs are often combined to generate various reactive oxygen species through both Type I and Type II processes.

Photodynamic Inhibition of Herpes Simplex Virus 1 Infection by Tricationic Amphiphilic Porphyrin with a Long Alkyl Chain.

Photodynamic therapy (PDT) is broadly used to treat different tumors, and it is a rapidly developing approach to inactivating or inhibiting the replication of fungi, bacteria, and viruses. Herpes simplex virus 1 (HSV-1) is an important human pathogen and a frequently used model to study the effects of PDT on enveloped viruses. Although many photosensitizers (PSs) have been tested for their antiviral properties, analyses are usually limited to assessing the reduction in viral yield, and thus the molecular mechanisms of photodynamic inactivation (PDI) remain poorly understood. In this study, we investigated the antiviral properties of TMPyP3-C17H35, a tricationic amphiphilic porphyrin-based PS with a long alkyl chain. We show that light-activated TMPyP3-C17H35 can efficiently block virus replication at certain nM concentrations without exerting obvious cytotoxicity. Moreover, we show that the levels of viral proteins (immediate-early, early, and late genes) were greatly reduced in cells treated with subtoxic concentrations of TMPyP3-C17H35, resulting in markedly decreased viral replication. Interestingly, we observed a strong inhibitory effect of TMPyP3-C17H35 on the virus yield only when cells were treated before or shortly after infection. In addition to the antiviral activity of the internalized compound, we show that the compound dramatically reduces the infectivity of free virus in the supernatant. Overall, our results demonstrate that activated TMPyP3-C17H35 effectively inhibits HSV-1 replication and that it can be further developed as a potential novel treatment and used as a model to study photodynamic antimicrobial chemotherapy.

Targeting Microenvironment of Melanoma and Head and Neck Cancers in Photodynamic Therapy.

BACKGROUND: Photodynamic therapy (PDT), in comparison to other skin cancers, is still far less effective for melanoma, due to the strong absorbance and the role of melanin in cytoprotection. The tumour microenvironment (TME) has a significant role in tumour progression, and the hypoxic TME is one of the main reasons for melanoma progression to metastasis and its resistance to PDT. Hypoxia is also a feature of solid tumours in the head and neck region that indicates negative prognosis. OBJECTIVE: The aim of this study was to individuate and describe systematically the main strategies in targeting the TME, especially hypoxia, in PDT against melanoma and head and neck cancers (HNC), and assess the current success in their application. METHODS: PubMed was used for searching, in MEDLINE and other databases, for the most recent publications on PDT against melanoma and HNC in combination with the TME targeting and hypoxia. RESULTS: In PDT for melanoma and HNC, it is very important to control hypoxia levels, and amongst the different approaches, oxygen self-supply systems are often applied. Vascular targeting is promising, but to improve it, optimal drug-light interval, and formulation to increase the accumulation of the photosensitiser in the tumour vasculature, have to be established. On the other side, the use of angiogenesis inhibitors, such as those interfering with VEGF signalling, is somewhat less successful than expected and needs to be further investigated. CONCLUSION: The combination of PDT with immunotherapy by using multifunctional nanoparticles continues to develop and seems to be the most promising for achieving a complete and lasting antitumour effect.

Photodynamic Inactivation of Legionella pneumophila Biofilm Formation by Cationic Tetra- and Tripyridylporphyrins in Waters of Different Hardness.

The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs' stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages.

Cationic Porphyrins as Effective Agents in Photodynamic Inactivation of Opportunistic Plumbing Pathogen Legionella pneumophila.

Legionella pneumophila is an environmental bacterium, an opportunistic premise plumbing pathogen that causes the Legionnaires' disease. L. pneumophila presents a serious health hazard in building water systems, due to its high resistance to standard water disinfection methods. Our aim was to study the use of photodynamic inactivation (PDI) against Legionella. We investigated and compared the photobactericidal potential of five cationic dyes. We tested toluidine blue (TBO) and methylene blue (MB), and three 3-N-methylpyridylporphyrins, one tetra-cationic and two tri-cationic, one with a short (CH3) and the other with a long (C17H35) alkyl chain, against L. pneumophila in tap water and after irradiation with violet light. All tested dyes demonstrated a certain dark toxicity against L. pneumophila; porphyrins with lower minimal effective concentration (MEC) values than TBO and MB. Nanomolar MEC values, significantly lower than with TBO and MB, were obtained with all three porphyrins in PDI experiments, with amphiphilic porphyrin demonstrating the highest PDI activity. All tested dyes showed increasing PDI with longer irradiation (0-108 J/cm2), especially the two hydrophilic porphyrins. All three porphyrins caused significant changes in cell membrane permeability after irradiation and L. pneumophila, co-cultivated with Acanthamoeba castellanii after treatment with all three porphyrins and irradiation, did not recover in amoeba. We believe our results indicate the considerable potential of cationic porphyrins as effective anti-Legionella agents.

Marine Natural Products with High Anticancer Activities.

This review covers recent literature from 2012-2019 concerning 170 marine natural products and their semisynthetic analogues with strong anticancer biological activities. Reports that shed light on cellular and molecular mechanisms and biological functions of these compounds, thus advancing the understanding in cancer biology are also included. Biosynthetic studies and total syntheses, which have provided access to derivatives and have contributed to the proper structure or stereochemistry elucidation or revision are mentioned. The natural compounds isolated from marine organisms are divided into nine groups, namely: alkaloids, sterols and steroids, glycosides, terpenes and terpenoids, macrolides, polypeptides, quinones, phenols and polyphenols, and miscellaneous products. An emphasis is placed on several drugs originating from marine natural products that have already been marketed or are currently in clinical trials.

Porphyrins and related macrocycles: Combining photosensitization with radio- or optical-imaging for next generation theranostic agents.

This review summarises recent research into combining the photosensitizing properties of porphyrins with imaging techniques such as PET and NIR fluorescence for so called "theranostic" applications, which combine biomedical imaging and therapeutic potential into a single administered substance. The photophysical mechanisms of both the therapeutic and diagnostic properties of porphyrins are discussed, as well as key characteristics that are required in order to deliver the most effective treatment.

In Vitro Photodynamic Activity of N-Methylated and N-Oxidised Tripyridyl Porphyrins with Long Alkyl Chains and Their Inhibitory Activity in Sphingolipid Metabolism.

A series of N-methylated and N-oxidised tripyridyl porphyrins were synthesised, characterised, and their PDT activity was studied with six cell lines. All the tested porphyrins with a long alkyl chain, except one, were more efficient for PDT than an N-methylated hydrophilic porphyrin and N-oxidised porphyrin without the long alkyl chain. Generally, N-methylated tripyridyl porphyrins were more active than those N-oxidised, but IC50 values for phototoxicity of two N-oxides, named TOPyP3-C17 H33 O and TOPyP3-C17 H35 , were still in the nanomolar concentration range for most of the tested cell lines. However, TOPyP3-C17 H35 did not show phototoxicity on human foreskin fibroblast cells. Two methylated amphiphilic porphyrins, named TMPyP3-C17 H33 and TMPyP4-C17 H35, showed significant dark toxicity, whereas none of the oxidopyridyl porphyrins were toxic without light activation. The selected photosensitisers were shown to be apoptosis inducers, and had inhibitory effects on the clonogenic growth of HCT116 and HeLa cells. All three N-methylated amphiphilic porphyrins significantly reduced the migratory potential of HCT116 cells. Porphyrins TMPyP3-C17 H35 and TOPyP3-C17 H35 reduced the activity of acid ceramidase, whereas TOPyP3-C17 H33 O had a significant inhibitory effect on sphingosine kinase 1 activity in HeLa cells. Compounds with this dual activity were shown to be the most promising photosensitisers, with potential to treat invasive cancers.

Porphyrin-based cationic amphiphilic photosensitisers as potential anticancer, antimicrobial and immunosuppressive agents.

Photodynamic therapy (PDT) combines a photosensitiser, light and molecular oxygen to induce oxidative stress that can be used to kill pathogens, cancer cells and other highly proliferative cells. There is a growing number of clinically approved photosensitisers and applications of PDT, whose main advantages include the possibility of selective targeting, localised action and stimulation of the immune responses. Further improvements and broader use of PDT could be accomplished by designing new photosensitisers with increased selectivity and bioavailability. Porphyrin-based photosensitisers with amphiphilic properties, bearing one or more positive charges, are an effective tool in PDT against cancers, microbial infections and, most recently, autoimmune skin disorders. The aim of the review is to present some of the recent examples of the applications and research that employ this specific group of photosensitisers. Furthermore, we will highlight the link between their structural characteristics and PDT efficiency, which will be helpful as guidelines for rational design and evaluation of new PSs.

Synthesis, characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate.

Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by (1)H NMR, UV-vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2mWcm(-2)) of red light (643nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC50 values. The obtained IC50 values for 5 were 1.4µM at 15min irradiation time and 0.7µM when the time of irradiation was 30min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications.

An analysis of the pollution problem in Slavonski Brod (eastern Croatia).

H2S, PM2.5, O3, NO2, SO2 and meteorological parameters such as temperature, relative humidity, precipitation, wind speed and wind direction were measured simultaneously in an eastern Croatian town called Slavonski Brod during the season winter/spring 2010. Emissions from the nearby cross-border (Bosnia and Herzegovina) oil refinery were identified as sources of temporary elevated concentrations of H2S. The maximum daily averages of PM2.5 concentrations during the winter period were as high as 240 microg m(-3) which is a value 10 times greater than the threshold prescribed by the World Health Organization. It is considered that the heating season, dense traffic, intense industrial activities and temperature inversion during stable weather conditions are prevailing contributors to higher winter concentrations of PM2.5. The results of the principal component analysis technique (PCA) have shown that lower air temperature, lower wind speed and higher relative humidity play a significant role in the winter pollution episodes. From a public health point of view, implementation of measures aimed at reducing the levels of H2S and PM2.5 should be considered.

Mono- and tri-cationic porphyrin-monoclonal antibody conjugates: photodynamic activity and mechanism of action.

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.

Development and characterization of novel photosensitizer : scFv conjugates for use in photodynamic therapy of cancer.

Photodynamic therapy (PDT) is becoming an evermore useful tool in oncology but is frequently limited by side-effects caused by a lack of targeting of the photosensitizer. This problem can often be circumvented by the conjugation of photosensitizers to tumour-specific monoclonal antibodies. An alternative is the use of single chain (sc) Fv fragments which, whilst retaining the same binding specificity, are more efficient at penetrating tumour masses because of their smaller size; and are more effectively cleared from the circulation because of the lack of an Fc domain. Here we describe the conjugation of two isothiocyanato porphyrins to colorectal tumour-specific scFv, derived from an antibody phage display library. The conjugation procedure was successfully optimized and the resulting immunoconjugates showed no loss of cell binding. In vitro assays against colorectal cell lines showed these conjugates had a selective photocytotoxic effect on cells. Annexin V and propidium iodide staining of treated cells confirmed cell death was mediated principally via an apoptotic pathway. This work suggests that scFv : porphyrin conjugates prepared using isothiocyanato porphyrins show promise for use as targeted PDT agents.

Isothiocyanato boron dipyrromethenes--the first BODIPY analogues of fluorescein isothiocyanate.

Two boron complexes of 5-phenyldipyrromethenes bearing isothiocyanate groups on the phenyl ring have been synthesized for the first time. The utility of these new fluorescence probes for labeling biologically relevant proteins is demonstrated on two monoclonal antibodies that bind to antigens overexpressed on cancer cells. Spectral comparison of the two structures reveals significant photophysical differences, including bathochromically shifted excitation and emission bands, increased molar absorptivity and a large increase in fluorescence quantum yield of approximately 10 times. Differences in photophysical parameters are linked to hindered rotation of the phenyl ring in one of the probes.

Synthesis and in vitro investigation of cationic 5,15-diphenyl porphyrin-monoclonal antibody conjugates as targeted photodynamic sensitisers.

The synthesis of three cationic 5,15-diphenyl porphyrins, bearing an isothiocyanate group for conjugation to proteins is described. The potential of these compounds as targeted photosensitisers for use in photodynamic therapy (PDT) is demonstrated by their efficient conjugation to monoclonal antibodies and the ability of the bioconjugates to bind and photodynamically inactivate cancer cells that express the corresponding antigen.