Barbara Pucelik

Design of Pluronic-Based Formulation for Enhanced Redaporfin-Photodynamic Therapy against Pigmented Melanoma

The therapeutic outcome of photodynamic therapy (PDT) with redaporfin (a fluorinated sulfonamide bacteriochlorin, F2BMet or LUZ11) was improved using Pluronic-based (P123, F127) formulations. Neither redaporfin encapsulated in Pluronic nor micelles alone exhibited cytotoxicity in a broad concentration range. Comprehensive in vitro studies against B16F10 melanoma cells showed that redaporfin-P123 micelles enhanced cellular uptake and increased oxidative stress compared with redaporfin-F127 or photosensitizer alone after short incubation times. ROS-sensitive fluorescent probes showed that the increased oxidative stress is due, at least in part, to a more efficient formation of hydroxyl radicals, and causes strong light-dose dependent apoptosis and necrosis. Tissue distribution and pharmacokinetic studies in tumor-bearing mice show that the Pluronic P123 formulation of redaporfin increases its bioavailability as well as the tumor-to-muscle and tumor-to-skin ratios, in comparison with Cremophor EL and Pluronic F127 formulations. Redaporfin in P123 was most successful in the PDT of C57BL/6J mice bearing subcutaneously implanted B16F10 melanoma tumors. Vascular-targeted PDT combining 1.5 mg kg(-1) redaporfin in P123 with a light dose of 74 J cm(-2) led to 100% complete cures (i.e., no tumor regrowth over one year post-treatment). This remarkable result reveals that modification of redaporfin with Pluronic block copolymers overcomes the resistance of melanoma cells to PDT possibly via increased tumor selectivity and enhanced ROS generation.

New hybrid materials based on halogenated metalloporphyrins for enhanced visible light photocatalysis

Photophysical and photochemical studies on 5,10,15,20-tetrakis(2,6-difluoro-5-N-methylsulfamylophenyl)porphyrin (F2PMet) and its cobalt(III) and zinc(II) complexes, including spectroscopic characteristics, photostability and photocatalytic activity, were carried out. The hybrid materials resulting from adsorption of these tetrapyrroles at the surface of titanium dioxide were prepared and examined in terms of their morphological, optical and functional properties applying absorption spectroscopy, scanning electron microscopy (SEM), photoelectrochemistry and photocatalytic tests. Our studies revealed that MF2PMet@TiO2 photocatalysts can be considered as the hybrid organic/inorganic photoactive materials enabling photodegradation of a synthetic opioid such as tramadol hydrochloride (TRML) and a model pollutant, 4-chlorophenol, in aqueous solution under visible light irradiation (λ > 400 nm). To elucidate mechanisms of photochemical processes, the photocatalytic activity of investigated metalloporphyrins was compared in homo- and heterogeneous systems. The results indicate that impregnation of TiO2 (P25) with functionalized porphyrins can improve its photoactivity. ZnF2PMet@TiO2 exhibited a superior photocatalytic performance towards TRML degradation. The role of singlet oxygen and hydroxyl radicals in photodegradation processes has been elucidated both for MF2PMet and MF2PMet@TiO2 systems.

Towards tuning PDT relevant photosensitizer properties: comparative study for the free and Zn2+ coordinated meso-tetrakis[2,6-difluoro-5-(N-methylsulfamylo)phenyl]porphyrin

The spectroscopic, photochemical, and biological studies of 5,10,15,20-tetrakis[2,6-difluoro-5(N-methylsulfamylo)phenyl]porphyrinate Zn(II) (ZnF2PMet) were carried out including absorption and fluorescence spectra, fluorescence quantum yields, triplet absorption spectra, triplet lifetimes, singlet oxygen quantum yield, and reactive oxygen species (ROS) detection under biological conditions and compared with its free-base analog (F2PMet). Zinc coordination into the porphyrin ring results in decrease of hydrophobicity and in higher cellular uptake. F2PMet localized specifically in endoplasmic reticulum and mitochondria while the ZnF2PMet is more diffused all over the cell, bonded to membrane proteins, as assessed by fluorescence microscopy. Zn-porphyrin exhibits greater singlet oxygen quantum yield than its free-base analog. Studies with fluorescent probes confirm that the ZnF2PMet produces mostly singlet oxygen, whereas F2PMet generates more hydroxyl radicals as the ROS. F2PMet is a more effective photosensitizer in vitro than its zinc complex, thus, the final photodynamic effect depends more on the nature of ROS than on the higher cellular uptake.

Properties of halogenated and sulfonated porphyrins relevant for the selection of photosensitizers in anticancer and antimicrobial therapies

The impact of substituents on the photochemical and biological properties of tetraphenylporphyrin-based photosensitizers for photodynamic therapy of cancer (PDT) as well as photodynamic inactivation of microorganisms (PDI) was examined. Spectroscopic and physicochemical properties were related with therapeutic efficacy in PDT of cancer and PDI of microbial cells in vitro. Less polar halogenated, sulfonamide porphyrins were most readily taken up by cells compared to hydrophilic and anionic porphyrins. The uptake and PDT of a hydrophilic porphyrin was significantly enhanced with incorporation in polymeric micelles (Pluronic L121). Photodynamic inactivation studies were performed against Gram-positive (S. aureus, E. faecalis), Gram-negative bacteria (E. coli, P. aeruginosa, S. marcescens) and fungal yeast (C. albicans). We observed a 6 logs reduction of S. aureus after irradiation (10 J/cm2) in the presence of 20 μM of hydrophilic porphyrin, but this was not improved with incorporation in Pluronic L121. A 2–3 logs reduction was obtained for E. coli using similar doses, and a decrease of 3–4 logs was achieved for C. albicans. Rational substitution of tetraphenylporphyrins improves their photodynamic properties and informs on strategies to obtain photosensitizers for efficient PDT and PDI. However, the design of the photosensitizers must be accompanied by the development of tailored drug formulations.

Photodynamic therapy as an alternative to antibiotic therapy for the treatment of infected leg ulcers

Infected leg ulcers are painful, debilitating and reduce a patients quality of life, therefore they are becoming a significant clinical and socioeconomic problem. Increasing resistance to antibiotics, is one of the most urgent challenge to medicine worldwide and requires searching for new, innovative and more efficient medical strategies. One of the opportunities for the cure of leg ulcers is photodynamic inactivation (PDI), which has been widely used in the treatment of various bacterial, fungal and viral infections. PDI encompasses three independently non-toxic elements: a photosensitizer (PS), light of an appropriate wavelength, and molecular oxygen that lead to generation of reactive oxygen species (ROS) responsible for inactivation of microorganisms, including those present in the form of biofilm in chronic wounds. PDI, due to its multiple mechanism of action, low invasiveness and lack of significant side effects, offers an interesting potential alternative for combating the microbial resistance in the infected leg ulcers. It also significantly decreases the area of leg ulcers, or may even heal them completely and thus remarkably improves a patients health.

Translating phototherapeutic indices from in vitro to in vivo photodynamic therapy with bacteriochlorins: TRANSLATING PHOTOTHERAPEUTIC INDICES

To compare hydrophilic and lipophilic bacteriochlorin photosensitizers in the photodynamic therapy of cancer, and relate their properties and in vitro phototoxicities to the efficacy of in vivo PDT treatments.

Palette of polarity-tunable halogenated bacteriochlorins for efficient photodynamic therapy in cellular and animal models

We have previously reported the development of a telodendrimer, comprised of dendritic cholic acids and a linear polyethylene glycol (PEG), that can selfassemble and encapsulate hydrophobic chemotherapeutic drugs to form 25 to 50nm micellar-based nanoparticles. Using a highly efficient onebead-one-compound (OBOC) combinatorial technology, we have discovered several peptidic ligands that target cancer cell surface receptorswith high affinity and specificity. Such ligands can be easily conjugated to the telodendrimers via click chemistry, such that the ligands are displayed on the surface of the nanoparticles for efficient in vivo tumor targeting and intracellular drug delivery. Recently, we have developed a hybrid telodendrimer comprised of PEG, cholic acid and pyropheophorbibe. Like our standard telodendrimers, such hybrid telodendrimer can also self-assemble and efficiently encapsulate hydrophobic drugs to from 25 to 30nm nanoparticles. Such nanoporphyrin has proven to be extremely potent photosensitizer for in vivo phototherapy in both transgenic and xenograft tumor models. In addition to efficiently generate reactive oxygen species when illuminated with light, heat can also generated, thus allowing simultaneous photodynamic and photothermal therapy. In addition, we have discovered that low dose doxorubicin and/or heat shock protein inhibitor can be efficiently loaded into nanoporphyrin and greatly enhance the phototherapeutic effects. Clinical applications of nanoporphyrins include phototherapy of superficial tumors such as oral cancer and bladder cancer, and intraoperative phototherapy. Furthermore, nanoporphyrin can also be loaded with Gd(III) for MRI and Cu-64 for PET, making it a highly versatile theranostic agent.