Ephemeral and/or coloured nuonic hydrogen atoms

The unexpected and, in conventional terms, yet unexplained experimental results, obtained in systematic measurements of muon transfer from muonic hydrogen to sulphur and oxygen of sulphur dioxide, seem to violate the principle of reproducibility of muon transfer data. With the hypothesis of ephemeral muonic hydrogen atoms, the number of different hydrogen atoms can be reduced from four to two. This hypothesis does, however, not help to interpret the transfer data to helium, neon and argon, where the muonic hydrogen atoms seem to wear colours.     

Multiparametric microsensor chips for screening applications

The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.     

Encapsulation of porphyrins and chlorins in biodegradable nanoparticles: the effect of dye lipophilicity on the extravasation and the photothrombic activity. A comparative study

In the present work, we performed a preclinical inter-comparison study using several photosensitizers with the goal of optimizing photodynamic therapy (PDT) for the treatment of choroidal neovascularization (CNV) associated with age-related macular degeneration. The tested molecules were the porphyrins meso-tetraphenylporphyrin (TPP) and meso-tetra-(4-carboxyphenyl)-porphyrin (TCPP), and the chlorins pheophorbide-a (Pheo-a) and chlorin e(6) (Ce(6)). Each of these molecules was entrapped in biodegradable nanoparticles (NP) based on poly(d,l-lactic acid). The influence of the degree of lipophilicity on the incorporation efficiency of the drug in the NPs, and on the dye leakage from blood vessels as well as on the photothrombic efficiency was investigated using the chick chorioallantoic membrane (CAM) as in vivo model. NP characterization showed that the dye was more effectively entrapped in the polymeric matrix when its degree of lipophilicity increased. While less lipophilic compounds (TCPP, Ce(6)) extravasate rather easily, the more lipophilic dyes (TPP, Pheo-a) tend to remain inside the blood vessels. After injection of a drug dose of 1 mg/kg body weight and a drug-light application interval of 1 min, irradiation with light doses ranging from 5 to 20 J/cm(2) led to the highest photothrombic efficiency when using the NPs loaded with the most lipophilic molecule (TPP). The latter induced vascular damage, which was significantly higher than that observed with the other molecules tested. Thus, in addition to minimal leakage from blood vessels, the TPP in NP formulation exhibited photothrombic efficiency similar to Visudyne which was also tested in the CAM model.     

Rapid determination of 5-hydroxytryptamine in whole blood by liquid chromatography with fluorimetric detection.

A rapid and simple reversed-phase (using muBondapak C18 as the stationary phase) liquid chromatographic method with fluorimetric detection is described for the quantitation of 5-hydroxytryptamine in whole blood. The rapidity and simplicity of the method are explained by the absence of a pretreatment. 5-Fluoro-dl-tryptophan was used as internal standard. The mobile phase was 0.01 M phosphate buffer (pH 4.5) with 0.0025 M 1-heptanesulfonic acid and 20% methanol. The detection wavelength were 302 nm for excitation and 340 nm for emission. Analysis time was 10 min with retention times for 5-hydroxytryptamine of 9 min and for 5-fluoro-dl-tryptophan of 7 min. This method is proposed for biological exploration of psychiatric disorders involving 5-hydroxytryptamine and would be useful for tryptophan.     

Photoinduced electron transfer at liquid|liquid interfaces: dynamics of the heterogeneous photoreduction of quinones by self-assembled porphyrin ion pairs

The initial stages of the heterogeneous photoreduction of quinone species by self-assembled porphyrin ion pairs at the water|1,2-dichloroethane (DCE) interface have been studied by ultrafast time-resolved spectroscopy and dynamic photoelectrochemical measurements. Photoexcitation of the water-soluble ion pair formed by zinc meso-tetrakis(p-sulfonatophenyl)porphyrin (ZnTPPS(4)(-)) and zinc meso-tetrakis(N-methylpyridyl)porphyrin (ZnTMPyP(4+)) leads to a charge-separated state of the form ZnTPPS(3)(-)-ZnTMPyP(3+) within 40 ps. This charge-separated state is involved in the heterogeneous electron injection to acceptors in the organic phase in the microsecond time scale. The heterogeneous electron transfer manifests itself as photocurrent responses under potentiostatic conditions. In the case of electron acceptors such as 1,4-benzoquinone (BQ), 2,6-dichloro-1,4-benzoquinone (DCBQ), and tetrachloro-1,4-benzoquinone (TCBQ), the photocurrent responses exhibit a strong decay due to back electron transfer to the oxidized porphyrin ion pair. Interfacial protonation of the radical semiquinone also contributes to the photocurrent relaxation in the millisecond time scale. The photocurrent responses are modeled by a series of linear elementary steps, allowing estimations of the flux of heterogeneous electron injection to the acceptor species. The rate of electron transfer was studied as a function of the thermodynamic driving force, confirming that the activation energy is controlled by the solvent reorganization energy. This analysis also suggests that the effective redox potential of BQ at the liquid|liquid boundary is shifted by 0.6 V toward positive potentials with respect to the value in bulk DCE. The change of the redox potential of BQ is associated with the formation of hydrogen bonds at the liquid|liquid boundary. The relevance of this approach toward modeling the initial processes in natural photosynthetic reaction centers is briefly discussed.     

Speciation of metal complexes with biomolecules by reversed-phase HPLC with ion-spray and inductively coupled plasma mass spectrometric detection

Complementarity of ion-spray MS and ICP-MS as detection techniques in reversed-phase HPLC for the characterization of metals complexed by biomacromolecules is discussed by the example of the speciation of metallothionein-bound cadmium. The commercially purified rabbit liver MT-2 isoform is eluted from a microbore C₈ column with a gradient of up to 50% methanol in acetate buffer (pH 6.0) to give one major and three minor peaks detected at 254 nm. The preparation is further characterized by using an ICP mass spectrometer interfaced with HPLC via a direct injection nebulizer which allows for the specific detection of cadmium down to the 10 ng mL^–1 level. On-line detection by mass spectrometry with an ion-spray (pneumatically-assisted electrospray) ion source further allows the determination of the molecular masses of the eluted compounds. Received: 30 July 1997 / Revised: 7 November 1997 /Accepted: 11 November 1997     

Photodynamic actinometry using microspheres: concept, development and responsivity

Photodynamic therapy (PDT) relies on three main ingredients, oxygen, light and photoactivating compounds, although the PDT response is definitively contingent on the site and level of reactive oxygen species (ROS) generation. This study describes the development of a novel, fluorescent-based actinometer microsphere system as a means of discerning spatially resolved dosimetry of total fluence and ROS production. Providing a high resolution, localized, in situ measurement of fluence and ROS generation is critical for developing in vivo PDT protocols. Alginate-poly-L-lysine-alginate microspheres were produced using ionotropic gelation of sodium alginate droplets, ranging from 80 to 200 microm in diameter, incorporating two dyes, ADS680WS (ADS) and Rhodophyta-phycoerythrin (RPE), attached to the spheres' inside and outside layers, respectively. To test the responsivity and dynamic range of RPE for ROS detection, the production of ROS was initiated either chemically using increasing concentrations of potassium perchromate or photochemically using aluminum tetrasulphonated phthalocyanine. The generation of singlet oxygen was confirmed by phosphorescence at 1270 nm. The resulting photodegradation and decrease in fluorescence of RPE was found to correlate with increased perchromate or PDT treatment fluence, respectively. This effect was independent of pH (6.5-8) and could be inhibited using sodium azide. RPE was not susceptible to photobleaching with light alone (670 nm; 150 Jcm(-2)). ADS, which absorbs light between 600 and 750 nm, showed a direct correlation between radiant exposure (670 nm; 0-100 Jcm(-2)) and diminished fluorescence. Photobleaching was independent of irradiance (10-40 mW cm(-2)). We propose that actinometer microspheres may provide a means for obtaining high spatial resolution information regarding delivered PDT dose within model systems during investigational PDT development and dosimetric information for clinical extracorporeal PDT as in the case of ex vivo bone marrow purging.