Synthesis, characterization and bioimaging of fluorescent labeled polyoxometalates.

A fluorescent labeled Wells-Dawson type POM ({P2W17O61Fluo}) was newly synthesized and characterized by a wide range of analytical methods. {P2W17O61Fluo} was functionalized with fluorescein amine through a stable amide bond, and its long time stability was verified by UV/vis spectroscopic techniques at physiologically relevant pH values. No significant impact on the cell viability or morphology of HeLa cells was observed for POM concentrations up to 100 µg mL(-1). Cellular uptake of fluorescent {P2W17O61Fluo} was monitored by confocal laser scanning microscopy. POM uptake occurs mainly after prolonged incubation times of 24 h resulting in different intracellular patterns, i.e. randomly distributed over the entire cytoplasm, or aggregated in larger clusters. This direct monitoring strategy for the interaction of POMs with cells opens up new pathways for elucidating their unknown mode of action on the way to POM-based drug development.

Chitosan-thioglycolic acid as a versatile antimicrobial agent.

As functionalized chitosans hold great potential for the development of effective and broad-spectrum antibiotics, representative chitosan derivatives were tested for antimicrobial activity in neutral media: trimethyl chitosan (TMC), carboxy-methyl chitosan (CMC), and chitosan-thioglycolic acid (TGA; medium molecular weight: MMW-TGA; low molecular weight: LMW-TGA). Colony forming assays indicated that LMW-TGA displayed superior antimicrobial activity over the other derivatives tested: a 30 min incubation killed 100% Streptococcus sobrinus (Gram-positive bacteria) and reduced colony counts by 99.99% in Neisseria subflava (Gram-negative bacteria) and 99.97% in Candida albicans (fungi). To elucidate LMW-TGA effects at the cellular level, microscopic studies were performed. Use of fluorescein isothiocyanate (FITC)-labeled chitosan derivates in confocal microscopy showed that LMW-TGA attaches to microbial cell walls, while transmission electron microscopy indicated that this derivative severely affects cell wall integrity and intracellular ultrastructure in all species tested. We therefore propose LMW-TGA as a promising and effective broad-band antimicrobial compound.

Trimethyl and carboxymethyl chitosan carriers for bio-active polymer-inorganic nanocomposites.

The carrier properties of carboxymethyl chitosan (CMC) and trimethyl chitosan (TMC) in combination with polyoxometalates (POMs) as inorganic drug prototypes are compared with respect to the influence of polymer matrix charge and structure on the emerging composites. A direct crosslinking approach with TMC and K(6)H(2)[CoW(11)TiO(40)]·13H(2)O ({CoW(11)TiO(40)}) as a representative anticancer POM affords nanocomposites with a size range of 50-90nm. The obtained POM-chitosan composites are characterized with a wide range of analytical methods, and POM encapsulation into positively charged TMC brings forward different nanocomposite morphologies and properties than CMC as a carrier material. Furthermore, uptake of fluorescein isothiocyanate (FITC) labeled POM-CMC and POM-TMC by HeLa cells was monitored, and the influence of chlorpromazine (CP) as inhibitor of the clathrin mediated pathway revealed different cellular uptake behavior of composites and pristine carriers. TMC/{CoW(11)TiO(40)} nanocomposites are taken up by HeLa cells after short incubation times around 30 min at low concentrations. The anticancer activity of pristine {CoW(11)TiO(40)} and its TMC-nanocomposites was investigated in vitro with MTT assays and compared to a reference POM.

Photodynamic mechanisms induced by a combination of hypericin and a chlorin based-photosensitizer in head and neck squamous cell carcinoma cells.

The aim of this study was to elucidate photodynamic therapy (PDT) effects mediated by hypericin and a liposomal meso-tetrahydroxyphenyl chlorin (mTHPC) derivative, with focus on their 1:1 mixture, on head and neck squamous cell carcinoma cell lines. Absorption, excitation and photobleaching were monitored using fluorescence spectrometry, showing the same spectral patterns for the mixture as measured for single photosensitizers. In the mixture mTHPC showed a prolonged photo-stability. Singlet oxygen yield for light-activated mTHPC was Φ(Δ) = 0.66, for hypericin Φ(Δ) = 0.25 and for the mixture Φ(Δ) = ~0.4. A linear increase of singlet oxygen yield for mTHPC and the mixture was found, whereas hypericin achieved saturation after 35 min. Reactive oxygen species fluorescence was only visible after hypericin and mixture-induced PDT. Cell viability was also more affected with these two treatment options under the selected conditions. Examination of death pathways showed that hypericin-mediated cell death was apoptotic, with mTHPC necrotic and the 1:1 mixture showed features of both. Changes in gene expression after PDT indicated strong up-regulation of selected heat-shock proteins. The application of photosensitizer mixtures with the features of reduced dark toxicity and combined apoptotic and necrotic cell death may be beneficial in clinical PDT. This will be the focus of our future investigations.

Targeted delivery of polyoxometalate nanocomposites.

Polyoxometalate/carboxymethyl chitosan nanocomposites with an average diameter of 130 nm are synthesized and labeled with fluorescein isothiocyanate (FITC) for a combined drug-carrier and cellular-monitoring approach. [Eu(ß(2) -SiW(11) O(39) )(2) ](13-) /CMC nanospheres as a representative example do not display cytotoxicity for POM concentrations up to 2 mg mL(-1) . Cellular uptake of fluoresecently labelled {EuSiW(11) O(39) }/FITC-CMC nanoparticles is monitored with confocal laser scanning microscopy. Nanoparticle uptake occurs after incubation times of around 1 h and no cyctotoxic effects are observed upon prolonged treatment. The preferential location of the POM/CMC nanocomposites in the perinuclear region is furthermore verified with transmission electron microscopy investigations on unlabeled nanoparticles. Therefore, this approach is a promising dual strategy for the safe cellular transfer and monitoring of bioactive POMs.

Cellular and molecular effects of the liposomal mTHPC derivative Foslipos in prostate carcinoma cells in vitro.

BACKGROUND: Meso-tetra-hydroxyphenyl-chlorine (mTHPC) is among the most powerful photosensitizers available for photodynamic therapy (PDT). However, the mechanisms leading to cell death are poorly understood. We here focused on changes at DNA and RNA levels after treatment with the liposomal mTHPC derivative Foslipos in vitro. METHODS: After determination of darktoxicity, laser conditions and uptake kinetics, PC-3 prostate carcinoma cells were subjected to PDT with Foslipos, followed by assessment of cell numbers directly (TP0) or 1h (TP1), 2h (TP2), 5h (TP5) and 24h (TP24) after illumination. Nucleic acids had been extracted for evaluation of RNA amounts and integrity as well as for estimation of abasic sites as a measure for DNA damage. Furthermore, expression changes of 84 genes related to oxidative stress were investigated by quantitative polymerase chain reaction. RESULTS: Already at TP0, the number of dead cells was significantly higher after PDT versus controls and at TP24 more than 90% of cells had been destroyed. PDT resulted in a severe damage of both RNA and DNA. Gene expression analyses revealed an impact of PDT on pathways for oxidative and metabolic stress, heat shock, proliferation and carcinogenesis, growth arrest, inflammation, DNA repair and apoptosis signaling. CONCLUSIONS: Mechanisms of Foslipos-mediated PDT comprise a combination of acute and delayed lethal effects in PC-3 cells. The latter may include death processes initiated by nucleic acid damage, activation of stress and growth arrest genes in combination with a reduced capability to adequately cope with oxidative toxicity. Our results will help to better understand molecular photodynamic effects.