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The generation of bio-targetable photosensitizers is of utmost importance to the emerging field of photodynamic therapy and antimicrobial (photo-)therapy. A synthetic strategy is presented in which chelating dipyrrin moieties are used to enhance the known photoactivity of iridium(III) metal complexes. Formed complexes can thus be functionalized in a facile manner with a range of targeting groups at their chemically active reaction sites. Dipyrrins with N- and O-substituents afforded (dipy)iridium(III) complexes via complexation with the respective Cp*-iridium(III) and ppy-iridium(III) precursors (dipy=dipyrrinato, Cp*=pentamethyl-η5 -cyclopentadienyl, ppy=2-phenylpyridyl). Similarly, electron-deficient [IrIII (dipy)(ppy)2 ] complexes could be used for post-functionalization, forming alkenyl, alkynyl and glyco-appended iridium(III) complexes. The phototoxic activity of these complexes has been assessed in cellular and bacterial assays with and without light; the [IrIII (Cl)(Cp*)(dipy)] complexes and the glyco-substituted iridium(III) complexes showing particular promise as photomedicine candidates. Representative crystal structures of the complexes are also presented.

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A synthetic strategy to BODIPY dyes is presented giving access to a range of new compounds relevant in the context of antimicrobial photodynamic therapy (aPDT). BODIPYs with the 8-(4-fluoro-3-nitrophenyl) and the 8-pentafluorophenyl substituents were used for the synthesis of new mono- and dibrominated BODIPYs. The para-fluorine atoms in these electron-withdrawing groups facilitate functional modification via nucleophilic aromatic substitution (SNAr) with a number of amines and thio-carbohydrates. Subsequently, the antibacterial phototoxic activity of these BODIPYs has been assessed in bacterial assays against the Gram-positive germ S. aureus and also against the Gram-negative germ P. aeruginosa. The bacterial assays allowed to identify substitution patterns which ensured antibacterial activity not only in phosphate-buffered saline (PBS) but also in the presence of serum, hereby more realistically modelling the complex biological environment that is present in clinical applications.

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A post-functionalization route to tris(dipyrrinato) metal complexes is presented giving access to a range of new complexes relevant in the context of medicinal inorganic chemistry. A pentafluorophenyl group in the meso-position of the dipyrrin ligand serves as an anchor for the connection with alcohols and thiocarbohydrates. The photochemotherapeutic activity of the complexes has been assessed in cellular assays with tumor cell lines and against the Gram-positive bacterium S. aureus. Finally, it is shown that this post-functionalization is also applicable to other dipyrrinato metal complexes.

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An adaptable approach toward cleavable nanoparticle carrier systems for photodynamic therapy (PDT) is presented, comprising a biocompatible carrier loaded with multiple photosensitizer (PS) molecules related to the clinically employed PS Temoporfin, two linkers cleavable under different triggers and glyco-targeting with mannose. A synthetic pathway to stimuli responsive hyperbranched polyglycerol (hPG) porphyrin conjugates via the copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC) or the strain-promoted alkyne-azide cycloaddition (SPAAC) has been developed. The PS 10,15,20-tris(3-hydroxyphenyl)-5-(2,3,4,5,6-pentafluorophenyl)porphyrin was functionalized with disulfide containing cystamine and acid-labile benzacetal linkers. Conjugates with reductively and pH labile linkers were thus obtained. Cleavage of the active PS agents from the polymer carrier is shown in several different release studies. The uptake of the conjugates into the cells is demonstrated via confocal laser scanning microscopy (CLSM) and flow cytometry. Finally, the antitumor and antibacterial phototoxicity of selected conjugates has been assessed in four different tumor cell lines and in cultures of the bacterium Staphylococcus aureus. The conjugates exhibited phototoxicity in several tumor cell lines in which conjugates with reductively cleavable linkers were more efficient compared to conjugates with acid-cleavable linkers. For S. aureus, strong phototoxicity was observed for a combination of the reductively cleavable and the pH labile linker and likewise for the cleavable conjugate with mannose targeting groups. The results thus suggest that the conjugates have potential for antitumor as well as antibacterial PDT.

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The antibacterial photodynamic activity of hyperbranched polyglycerol (hPG) loaded with zinc porphyrin photosensitizers and mannose units was investigated. hPG, with a MW of 19.5 kDa, was functionalized with about 15 molecules of the photosensitizer {5,10,15-tris(3-hydroxyphenyl)-20-[4-(prop-2-yn-1-ylamino)tetrafluorophenyl]porphyrinato}-zinc(II) by using copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC). These nanoparticle conjugates were functionalized systematically with increasing loadings of mannose in the range of approximately 20 to 110 groups. With higher mannose loadings (ca. 58-110 groups) the water-insoluble zinc porphyrin photosensitizer could thus be transferred into a water-soluble form. Targeting of the conjugates was proven in binding studies to the mannose-specific lectin concanavalin A (Con A) by using surface plasmon resonance (SPR). The antibacterial phototoxicity of the conjugates on Staphylococcus aureus (as a typical Gram-positive germ) was investigated in phosphate-buffered saline (PBS). It was shown that conjugates with approximately 70-110 mannose units exhibit significant antibacterial activity, whereas conjugates with approximately 20-60 units did not induce bacterial killing at all. These results give an insight into the multivalency effect in combination with photodynamic therapy (PDT). On addition of serum to the bacterial cultures, a quenching of this antibacterial phototoxicity was observed. In fluorescence studies with the conjugates in the presence of increasing bovine serum albumin (BSA) concentrations, protein-conjugate associations could be identified as a plausible cause for this quenching.

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Photodynamic inactivation (PDI) of bacteria is a promising approach for combating the increasing emergence of antibiotic resistance in pathogenic bacteria. To further improve the PDI efficiency on bacteria, a bacteria-targeting liposomal formulation was investigated. A generation II photosensitizer (temoporfin) was incorporated into liposomes, followed by conjugation with a specific lectin (wheat germ agglutinin, WGA) on the liposomal surface. WGA was successfully coupled to temoporfin-loaded liposomes using an activated phospholipid containing N-hydroxylsuccinimide residue. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were selected to evaluate the WGA modified liposomes in terms of bacteria targeted delivery and in vitro PDI test. Fluorescence microscopy revealed that temoporfin was delivered to both kinds of bacteria, while flow cytometry demonstrated that WGA- modified liposomes delivered more temoporfin to bacteria compared to nonmodified liposomes. Consequently, the WGA- modified liposomes eradicated all MRSA and significantly enhanced the PDI of P. aeruginosa. In conclusion, the WGA- modified liposomes are a promising formulation for bacteria targeted delivery of temoporfin and for improving the PDI efficiency of temoporfin on both Gram-positive and Gram-negative bacterial cells.

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Photodynamic antimicrobial chemotherapy (PACT) and antimicrobial peptides (AMPs) are two promising strategies to combat the increasing prevalence of antibiotic-resistant bacteria. To take advantage of these two strategies, we integrated a novel antimicrobial peptide (WLBU2) and a potent generation II photosensitizer (temoporfin) into liposomes by preparing WLBU2-modified liposomes, aiming at bacteria targeted delivery of temoporfin for PACT. WLBU2 was successfully coupled to temoporfin-loaded liposomes using a functional phospholipid. The delivery of temoporfin to bacteria was confirmed by fluorescence microscopy and flow cytometry, thus demonstrating that more temoporfin was delivered to bacteria by WLBU2-modified liposomes than by unmodified liposomes. Consequently, the WLBU2-modified liposomes eradicated all methicillin-resistant Staphylococcus aureus (MRSA) and induced a 3.3 log(10) reduction of Pseudomonas aeruginosa in the in vitro photodynamic inactivation test. These findings demonstrate that the use of AMP-modified liposomes is promising for bacteria-targeted delivery of photosensitizers and for improving the PACT efficiency against both gram-positive and gram-negative bacteria in the local infections.

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BACKGROUND AND OBJECTIVES: Enterococcus faecalis is frequently found in persistent endodontic infections. In this context, the antimicrobial photodynamic therapy (aPDT) could become a modern alternative to existing antibacterial treatment approaches. The aim of this study was to investigate the effect of aPDT on E. faecalis using the photosensitizer (PS) 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) enriched in liposomes. MATERIALS AND METHODS: Enterococcus faecalis was cultivated in Schaedler submerged culture for 24 hours, then isolated and adjusted in PBS to 10(8) cells/ml. The bacterial suspension was pipetted into a black microtitration plate and incubated for 15 minutes in the dark with mTHPC in various concentrations (10, 30, and 50 µM). The photosensitized suspensions were subjected to laser light (652 nm) at a light fluence of 100 J cm(-2) (test group A). In addition, the suspension sensitized with 50 µM mTHPC was irradiated with 25, 50, and 75 J cm(-2) (test group B). The following controls were used: non-irradiated bacterial suspension in the absence of mTHPC (C); irradiated bacterial suspension in the absence of mTHPC (D); non-irradiated bacterial suspension incubated with mTHPC (E). Dilution series (10(0)-10(-6)) were made of all groups and applied on Schaedler agar. After anerobic cultivation (4 days), the colony-forming units (CFU/ml) were determined. RESULTS: Enterococcus faecalis was suppressed completely after incubation with 50 µM mTHPC and illumination with 100 J cm(-2). Photodynamic treatment with 10 and 30 µM mTHPC caused reduction in CFU by 5.8 and 6.7 log-units. The application of an energy fluence <100 J cm(-2) resulted in a decline of antibacterial efficiency. Irradiation of the non-photosensitized solution showed no suppressing impact. Incubation of the PS without additional irradiation caused a maximal reduction in CFU by 1.5 log-units. CONCLUSION: The results show that aPDT using the PS mTHPC incorporated in liposomes could be a new approach to adjuvant treatment of endodontic infections with E. faecalis.

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In the case of cutaneous malignant or non-malignant diseases, topical photodynamic therapy (PDT) with a temoporfin (mTHPC)-containing formulation would be advantageous. Unfortunately, mTHPC is a highly hydrophobic drug with low percutaneous absorption and novel mTHPC-loaded invasomes for enhanced skin delivery were developed. The purpose of this study was to investigate photodynamic efficacy of mTHPC-loaded invasomes in vitro in two cell lines, i.e. the human colorectal tumour cell line HT29 and the epidermoid tumour cell line A431. Invasomes are vesicles containing besides phospholipids a mixture of terpenes or only one terpene and ethanol. Dark toxicity, phototoxicity and intracellular localization of mTHPC were studied. Laser scanning microscopy indicated perinuclear localization of mTHPC. Results revealed that mTHPC-invasomes and mTHPC-ethanolic solution used at a 2µM mTHPC-concentration and photoirradiation at 20J/cm(2) were able to reduce survival of HT29 cells and especially of A431 cells, being more sensitive to PDT. In contrast to HT29 cells, where there was not a significant difference between cytotoxicity of mTHPC-ethanolic solution and mTHPC-invasomes, in A431 cells mTHPC-invasomes were more cytotoxic. Survival of about 16% of A431 cells treated with mTHPC-invasomes is very promising, since it demonstrates invasomes' potential to be used in topical PDT of cutaneous malignant diseases.

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The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to kill antibiotic-resistant bacteria uses light in combination with a photosensitizer to induce a phototoxic reaction. Concentrations of 1, 10 and 100microM of tetrahydroporphyrin-tetratosylat (THPTS) and different incubation times (30, 90 and 180min) were used to measure photodynamic efficiency against two Gram-positive strains of S.aureus (MSSA and MRSA), and two Gram-negative strains of E.coli and P.aeruginosa. We found that phototoxicity of the drug is independent of the antibiotic resistance pattern when incubated in PBS for the investigated strains. Also, an incubation with 100microM THPTS followed by illumination, yielded a 6lg (> or =99.999%) decrease in the viable numbers of all bacteria strains tested, indicating that the THPTS drug has a high degree of photodynamic inactivation. We then modulated incubation time, photosensitizer concentration and monitored the effect of serum on the THPTS activity. In doing so, we established the conditions to obtain the strongest bactericidal effect. Our results suggest that this new and highly pure synthetic compound should improve the efficiency of photodynamic therapy against multiresistant bacteria and has a significant potential for clinical applications in the treatment of nosocomial infections.

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In order to increase topical delivery of temoporfin (mTHPC), a highly hydrophobic photosensitizer with low percutaneous penetration, neutral, anionic and cationic flexible liposomes (i.e. flexosomes) were prepared and investigated for their penetration enhancing ability. The in vitro skin penetration study was performed using human abdominal skin mounted in Franz diffusion cells. Besides the effect of surface charge of flexosomes on skin penetration of mTHPC, also its effect on physical properties (particle size, polydispersity index, lamellarity) and physicochemical stability of vesicles was investigated. Photon-correlation spectroscopy revealed that vesicles had after preparation a small particle size and low polydispersity index, while cryo-electron microscopy confirmed that these vesicles were mostly unilamellar and of a spherical shape. Regarding stability, contrasting to anionic flexosomes showing lack of long-term stability, neutral and cationic flexosomes were stable during 9 months storage at 4 degrees C. As to the penetration enhancing ability, cationic flexosomes possessed the highest, i.e. they delivered the highest mTHPC-amount to stratum corneum and deeper skin layers compared to conventional liposomes, neutral and anionic flexosomes. In conclusion, mTHPC-loaded cationic flexosomes could be a promising tool for delivering mTHPC to the skin, which would be beneficial for the photodynamic therapy of cutaneous malignant or non-malignant diseases.

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Temoporfin (mTHPC) is a potent second-generation photosensitizer. The primary object of this study was to develop a topical mTHPC-loaded liposomal hydrogel able to deliver mTHPC into the stratum corneum (SC) and deeper skin layers. This study was conducted (1) to determine the effect of carbomer concentration, used as a gelling agent, and the effect of phosphatidylcholine (PC) content of lecithin, used for the liposome preparation, on viscoelastic properties and viscosity of liposomal gels and (2) to determine the relationship between rheological properties of gels and the skin penetration of mTHPC. Liposomal hydrogels revealed plastic flow behaviour. The increase of carbomer concentration induced a domination of elastic over viscous behaviour of gels. There was an inverse relationship between the elasticity of gels and mTHPC-penetration. Viscosity also increased with the increment of carbomer concentration, reducing the mTHPC-penetration. Liposomal gels containing lecithin of smaller PC-content (i.e. smaller purity) exhibited a more elastic solid behaviour than gels containing lecithin with high PC-content, and showed smaller mTHPC-penetration. The gel containing 0.75%, w/w, carbomer and lecithin with high PC-content was considered to be the optimal formulation, since it delivered high amounts of mTHPC to the SC and deeper skin layers, and it possessed desirable rheological properties.

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Both the phytopathogenic fungus Ramularia collo-cygni and the hyperparasite R. uredinicola biosynthesize a number of red and yellow anthraquinone derivatives called rubellins. The new compounds uredinorubellins I and II, which were isolated from R. uredinicola, contribute to understanding the biosynthesis pathway that leads from simple anthraquinones to the rubellins. In addition, we isolated for the first time such simple compounds as chrysophanol and helminthsporin from both Ramularia species. A blue compound isolated from the mycelium of R. collo-cygni was revealed to be a unique 9,4-anthracenedione derivative. Structure elucidation by (1)H and (13)C NMR of the new but unstable compound caeruleoramularin was possible only by feeding the fungus different labeled (13)C acetates. The photodynamic activity of the uredinorubellins was comparable to rubellin D, whereas chrysophanol and caeruleoramularin did not display such activity.

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Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is one of the most potent photodynamically active substances in clinical use. Treatment protocols for Temoporfin-mediated photodynamic therapy often rely on drug-light intervals of several days in order for the photosensitizer to accumulate within the target tissue, though tumor selectivity is limited. Here, the mTHPC localization was studied at 2-8 h following systemic administration of a liposomal Temoporfin formulation (0.15 mg kg(-1) b.w.) in HT29 human colon adenocarcinoma in NMRI nu/nu mice. Photosensitizer distribution within tumor and internal organs was investigated by means of high performance liquid chromatography following chemical extraction, as well as in situ fluorescence imaging and point-monitoring fluorescence spectroscopy. For tumor tissue, the Temoporfin concentrations at 4 h (0.16+/-0.024 ng mg(-1)) and 8 h (0.18+/-0.064 ng mg(-1)) were significantly higher than at 2 h (0.08+/-0.026 ng mg(-1)). The average tumor-to-muscle and the tumor-to-skin selectivity were 6.6 and 2, respectively, and did not vary significantly with time after photosensitizer injection. In plasma, the Temoporfin concentration was low (0.07+/-0.07 ng mg(-1)) and showed no significant variation with time. Our results indicate a rapid biodistribution and clearance from the bloodstream. Within the same type of organ, data from both fluorescence methods generally exhibited a significant correlation with the extraction results.

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