Cellulolytic bacteria from soils in harsh environments

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60A degrees C) in liquid media (trypic soy broth-TSB and minimum salt medium-MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254-46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254-9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.

Enhancement of laser induced Au nanoparticle formation by femtosecond pulse shaping

We report the control of Au nanoparticle (NP) formation by using shaped 30 fs pulses, in a solution containing HAuCl4 and chitosan. By using a sinusoidal spectral phase, a periodic train of pulses is generated. When the period of the pulse train matches certain Raman resonances of chitosan, the reducing agent of the process, an enhancement of the Au NP formation is observed. Theoretical quantum chemical calculations indicate that the outer groups of the chitosan are mostly influenced by low Raman frequencies, which is in reasonably agreement with the experimental data and indicates an enhancement in the Au NP formation as the pulse train period increases (low frequency).

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Investigations of cooperative interactions in template induced crystallization processes and kinetic studies of nucleation and growth by small-angle neutron scattering

Zusammenfassung Um zu einem besseren Verständnis des Prozesses der Biomineralisation zu gelangen, muss das Zusammenwirken der verschiedenen Typen biologischer Makromoleküle, die am Keimbildungs- und Wachstumsprozess der Minerale beteiligt sind, berücksichtigt werden. In dieser Arbeit wird ein neues Modellsystem eingeführt, das aus einem SAM (self-assembled monolayer) mit verschiedenen Funktionalitäten und unterschiedlichen, gelösten Makromolekülen besteht. Es konnte gezeigt werden, dass die Kristallisation von Vaterit (CaCO3) sowie Strontianit (SrCO3) Nanodrähten der Präsenz von Polyacrylat in Kooperation mit einer COOH-funktionalisierten SAM-Oberfläche zugeschrieben werden kann. Die Kombination bestehend aus einer polaren SAM-Oberfläche und Polyacrylat fungiert als Grenzfläche für die Struktur dirigierende Kristallisation von Nanodraht-Kristallen. Weiter konnte gezeigt werden, dass die Phasenselektion von CaCO3 durch die kooperative Wechselwirkung zwischen einer SAM-Oberfläche und einem daran adsorbierten hb-Polyglycerol kontrolliert wird. Auch die Funktionalität einer SAM-Oberfläche in Gegenwart von Carboxymethyl-cellulose übt einen entscheidenden Einfluss auf die Phasenselektion des entstehenden Produktes aus. In der vorliegenden Arbeit wurden Untersuchungen an CaCO3 zur homogenen Keimbildung, zur Nukleation in Gegenwart eines Proteins sowie auf Kolloiden, die als Template fungieren, mittels Kleinwinkel-Neutronenstreuung durchgeführt. Die homogene Kristallisation in wässriger Lösung stellte sich als ein mehrstufiger Prozess heraus. In Gegenwart des Eiweißproteins Ovalbumin konnten drei Phasen identifiziert werden, darunter eine anfänglich vorhandene amorphe sowie zwei kristalline Phasen.

Synthese und radioaktive Markierung potentieller PET- bzw. SPECT-Liganden zur Quantifizierung und Visualisierung der pankreatischen beta-Zell-Masse

Abstract Due to the ongoing efforts in transplanting b-cell mass there is also a great medical interest in specific b-cell imaging agents to quantify the acceptance of transplanted islets in humans in vivo. Additionally, in the context of type 1 diabetes mellitus the chronic and progressive loss of b-cells caused by autoimmune destruction has led to concerted efforts to prevent further loss of b-cells by autoantigen-specific immunotherapy of pre-diabetic patients. nateglinide and glibenclamide are SUR1 ligands used to stimulate insulin secretion in type 2 diabetic patients. They bind to a class of molecules known as the ATP-sensitive potassium channels, located on the insulin producing b-cells of the islets of Langerhans and are therefore excellent candidates as b-cell specific tracers. To obtain a precursor for a direct labelling of nateglinide with [18F]fluoride, the aromatic system of the phenylalanine structure element was derivatised to obtain a phenolic OH-group in 4-position which is capable of further derivatisation. The formed phenylether N-(trans-4-isopropylcyclohexanecarbonyl)-O-(2-hydroxyethyl)-D-tyrosin benzylester was tried to be tosylated according to several literature procedures but none of them was applicable. The catalytic influence of ytterbium(III)triflate in the reaction of toluenesulfonic acid anhydride and the alcohol was investigated. It was found that Yb(III) facilitates the tosylation of the alcohol under non-basic conditions and was extended to the tosylation of a great variety of different alcohols to prove its applicability in general. The radioactive labelling of N-(trans-4-isopropyl-cyclohexanecarbonyl)-O-(2-[18F]fluoroethyl)-D-tyrosine with [18F]F-/ Kryptofix® 222/ K2CO3-system was achieved in radiochemical yields (RCY) of 10 % after deprotection with Pd/ C and H2. In addition to the direct labelling approach, a labelling procedure applying 2[18F]fluoroethyltosylate and N-(trans-4-isopropyl-cyclohexanecarbonyl)-D-tyrosin was performed in 40 % RCY. Unfortunately the determination of the KD value of N-(trans-4-isopropylcyclohexanecarbonyl)-O-(2-fluoroethyl)-D-tyrosine revealed a significant decrease in affinity compared to original nateglinide. The in vivo evaluation of some 18F-labelled glibenclamide derivatives in humans and animals revealed that longer measuring times are warranted because a high liver uptake spoiles the data acquisition and the activity washout proceeds very slowly. Therefore glibenclamide was labelled with a radioisotope with a longer half life such as 99mTc (t1/2 = 6 h) to lengthen the possible time frame for image acquisition. The synthesis of a 99mTc labelled hydrophilic glibenclamide derivative was performed. It is hoped that gliben-clamide is internalised into the b-cell and there binds to the 95 % of intracellular SUR-1 receptors with eventual metablolisation and thus trapping in the cell. The KD-value of the corresponding Re-compound was determined to be 0.5 nM and the insulin secretion properties were similar to those of original glibenclamide. The labelling precursor N-{4-[N,N-bis-(carboxy-methyl)-aminoethyl)-5-chlorobenzene-carboxamido]-ethyl}-benzene-sulfonyl-N'-cyclohexyl urea tris sodium salt was reacted with [99mTc(I)(OH2)3(CO)3] Cl to yield the final N-{4-[99mTc(I)-tricarbonyl-N,N-bis-(carboxymethyl)-aminoethyl)-5-chloro-benzene-carboxamidoethyl]-benzene-sulfonyl}-N'-cyclo-hexyl-urea sodium salt in 70% RCY.

Assembly and characterization of supramolecular architectures for biosensor applications

The research has included the efforts in designing, assembling and structurally and functionally characterizing supramolecular biofunctional architectures for optical biosensing applications. In the first part of the study, a class of interfaces based on the biotin-NeutrAvidin binding matrix for the quantitative control of enzyme surface coverage and activity was developed. Genetically modified ß-lactamase was chosen as a model enzyme and attached to five different types of NeutrAvidin-functionalized chip surfaces through a biotinylated spacer. All matrices are suitable for achieving a controlled enzyme surface density. Data obtained by SPR are in excellent agreement with those derived from optical waveguide measurements. Among the various protein-binding strategies investigated in this study, it was found that stiffness and order between alkanethiol-based SAMs and PEGylated surfaces are very important. Matrix D based on a Nb2O5 coating showed a satisfactory regeneration possibility. The surface-immobilized enzymes were found to be stable and sufficiently active enough for a catalytic activity assay. Many factors, such as the steric crowding effect of surface-attached enzymes, the electrostatic interaction between the negatively charged substrate (Nitrocefin) and the polycationic PLL-g-PEG/PEG-Biotin polymer, mass transport effect, and enzyme orientation, are shown to influence the kinetic parameters of catalytic analysis. Furthermore, a home-built Surface Plasmon Resonance Spectrometer of SPR and a commercial miniature Fiber Optic Absorbance Spectrometer (FOAS), served as a combination set-up for affinity and catalytic biosensor, respectively. The parallel measurements offer the opportunity of on-line activity detection of surface attached enzymes. The immobilized enzyme does not have to be in contact with the catalytic biosensor. The SPR chip can easily be cleaned and used for recycling. Additionally, with regard to the application of FOAS, the integrated SPR technique allows for the quantitative control of the surface density of the enzyme, which is highly relevant for the enzymatic activity. Finally, the miniaturized portable FOAS devices can easily be combined as an add-on device with many other in situ interfacial detection techniques, such as optical waveguide lightmode spectroscopy (OWLS), the quartz crystal microbalance (QCM) measurements, or impedance spectroscopy (IS). Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) allows for an absolute determination of intrinsic rate constants describing the true parameters that control interfacial hybridization. Thus it also allows for a study of the difference of the surface coupling influences between OMCVD gold particles and planar metal films presented in the second part. The multilayer growth process was found to proceed similarly to the way it occurs on planar metal substrates. In contrast to planar bulk metal surfaces, metal colloids exhibit a narrow UV-vis absorption band. This absorption band is observed if the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as the localized surface plasmon resonance (LSPR). LSPR excitation results in extremely large molar extinction coefficients, which are due to a combination of both absorption and scattering. When considering metal-enhanced fluorescence we expect the absorption to cause quenching and the scattering to cause enhancement. Our further study will focus on the developing of a detection platform with larger gold particles, which will display a dominant scattering component and enhance the fluorescence signal. Furthermore, the results of sequence-specific detection of DNA hybridization based on OMCVD gold particles provide an excellent application potential for this kind of cheap, simple, and mild preparation protocol applied in this gold fabrication method. In the final chapter, SPFS was used for the in-depth characterizations of the conformational changes of commercial carboxymethyl dextran (CMD) substrate induced by pH and ionic strength variations were studied using surface plasmon resonance spectroscopy. The pH response of CMD is due to the changes in the electrostatics of the system between its protonated and deprotonated forms, while the ionic strength response is attributed from the charge screening effect of the cations that shield the charge of the carboxyl groups and prevent an efficient electrostatic repulsion. Additional studies were performed using SPFS with the aim of fluorophore labeling the carboxymethyl groups. CMD matrices showed typical pH and ionic strength responses, such as high pH and low ionic strength swelling. Furthermore, the effects of the surface charge and the crosslink density of the CMD matrix on the extent of stimuli responses were investigated. The swelling/collapse ratio decreased with decreasing surface concentration of the carboxyl groups and increasing crosslink density. The study of the CMD responses to external and internal variables will provide valuable background information for practical applications.

Enteric coated mucoadhesive micropellets in rotary agglomeration process for wet spheronization

In this work a generally applicable method for the preparation of mucoadhesive micropellets of 250 to 600µm diameter is presented using rotor processing without the use of electrolytes. The mucoadhesive micropellets were developed to combine the advantages of mucoadhesion and microparticles. It was possible to produce mucoadhesive micropellets based on different mucoadhesive polymers Na-CMC, Na-alginate and chitosan. These micropellets are characterized by a lower friability (6 to 17%) when compared to industrial produced cellulose pellets (Cellets®) (41.5%). They show great tapped density and can be manufactured at high yields. The most influencing variables of the process are the water content at the of the end spraying period, determined by the liquid binder amount, the spraying rate, the inlet air temperature, the airflow and the humidity of the inlet air and the addition of the liquid binder, determined by the spraying rate, the rotor speed and the type of rotor disc. In a subsequent step a fluidized bed coating process was developed. It was possible to manifest a stable process in the Hüttlin Mycrolab® in contrast to the Mini-Glatt® apparatus. To reach enteric resistance, a 70% coating for Na-CMC micropellets, an 85% for chitosan micropellets and a 140% for Na-alginate micropellets, based on the amount of the starting micropellets, was necessary. Comparative dissolution experiments of the mucoadhesive micropellets were performed using the paddle apparatus with and without a sieve inlay, the basket apparatus, the reciprocating cylinder and flow-through cell. The paddle apparatus and the modified flow-through cell method turned out to be successful methods for the dissolution of mucoadhesive micropellets. All dissolution profiles showed an initial burst release followed by a slow release due to diffusion control. Depending on the method, the dissolution profiles changed from immediate release to slow release. The dissolution rate in the paddle apparatus was mainly influenced by the agitation rate whereas the flow-through cell pattern was mainly influenced by the particle size. Also, the logP and the HLB values of different emulsifiers were correlated to transfer HLB values of excipients into logP values and logP values of API´s into HLB values. These experiments did not show promising results. Finally, it was shown that manufacture of mucoadhesive micropellets is successful resulting in product being characterized by enteric resistency combined with high yields and convincing morphology.

Enantioselective Reactions Promoted by Organocatalytic Species From The Natural Chiral Pool

During the course of my Ph.D. in the laboratories directed by Prof. Alfredo Ricci at the Department of Organic Chemistry “A. Mangini” of the University of Bologna, I was involved in the study and the application of a number of organocatalytic systems, all coming from the natural chiral pool. The first part of this thesis will be devoted to new homogeneous organocatalytic reactions promoted by Cinchona alkaloid-based organocatalysts. Quinine based catalysts were found to be a very effective catalyst for Diels-Alder reactions involving 3-vinylindoles. Excellent results in terms of yields and enantioselectivities were achieved, outlining also a remarkable organocatalytic operational mode mimicking enzymatic catalysis. The same reaction with 2-vinylindoles showed a completely different behaviour resulting in an unusual resolution-type process. The asymmetric formal [3+2] cycloaddition with in situ generated N-carbamoyl nitrones using Cinchona-derived quaternary ammonium salts as versatile catalysts under phase transfer conditions, outlines another application in organocatalysis of this class of alkaloids. During the seven months stage in the Prof. Helma Wennemers’ group at the Department of Chemistry of the University of Basel (Switzerland) I have been involved in organocatalysis promoted by oligopeptides. My contribution regarded the 1,4-addition reaction of aldehydes to nitroolefins. In the work performed at the Department of Organic Chemistry “A. Mangini” of the University of Bologna, in collaboration with the ‘Institut Charles Gerhardt-Montpellier, of Montpellier (France) the possibility of performing for the first time heterogeneous organocatalysis by using a natural polysaccharide biopolymer as the source of chirality was disclosed. With chitosan, derived from deacetylation of chitin, a highly enantioselective heterogeneous organocatalytic aldol reaction could be performed. The use of an eco-friendly medium such as water, the recyclability of the catalytic specie and the renewable nature of the polysaccharide are assets of this new approach in organocatalysis and open interesting perspectives for the use of biopolymers.

New biosensor applications of surface plasmon and hydrogel optical waveguide spectroscopy

Rapid and sensitive detection of chemical and biological analytes becomes increasingly important in areas such as medical diagnostics, food control and environmental monitoring. Optical biosensors based on surface plasmon resonance (SPR) and optical waveguide spectroscopy have been extensively pushed forward in these fields. In this study, we combine SPR, surface plasmon-enhanced fluorescence spectroscopy (SPFS) and optical waveguide spectroscopy with hydrogel thin film for highly sensitive detection of molecular analytes.rnrnA novel biosensor based on SPFS which was advanced through the excitation of long range surface plasmons (LRSPs) is reported in this study. LRSPs are special surface plasmon waves propagating along thin metal films with orders of magnitude higher electromagnetic field intensity and lower damping than conventional SPs. Therefore, their excitation on the sensor surface provides further increased fluorescence signal. An inhibition immunoassay based on LRSP-enhanced fluorescence spectroscopy (LRSP-FS) was developed for the detection of aflatoxin M1 (AFM1) in milk. The biosensor allowed for the detection of AFM1 in milk at concentrations as low as 0.6 pg mL-1, which is about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.rnrnIn addition, LRSPs probe the medium adjacent to the metallic surface with more extended evanescent field than regular SPs. Therefore, three-dimensional binding matrices with up to micrometer thickness have been proposed for the immobilization of biomolecular recognition elements with large surface density that allows to exploit the whole evanescent field of LRSP. A photocrosslinkable carboxymethyl dextran (PCDM) hydrogel thin film is used as a binding matrix, and it is applied for the detection of free prostate specific antigen (f-PSA) based on the LRSP-FS and sandwich immunoassay. We show that this approach allows for the detection of f-PSA at low femto-molar range, which is approximately four orders of magnitude lower than that for direct detection of f-PSA based on the monitoring of binding-induced refractive index changes.rnrnHowever, a three dimensional hydrogel binding matrix with micrometer thickness can also serve as an optical waveguide. Based on the measurement of binding-induced refractive index changes, a hydrogel optical waveguide spectroscopy (HOWS) is reported for a label-free biosensor. This biosensor is implemented by using a SPR optical setup in which a carboxylated poly(N-isoproprylacrylamide) (PNIPAAm) hydrogel film is attached on a metallic surface and modified by protein catcher molecules. Compared to regular SPR biosensor with thiol self-assembled monolayer (SAM), HOWS provides an order of magnitude improved resolution in the refractive index measurements and enlarged binding capacity owing to its low damping and large swelling ratio, respectively. A model immunoassay experiment revealed that HOWS allowed detection of IgG molecules with a 10 pM limit of detection (LOD) that was five-fold lower than that achieved for SPR with thiol SAM. For the high capacity hydrogel matrix, the affinity binding was mass transport limited.rnrnThe mass transport of target molecules to the sensor surface can play as critical a role as the chemical reaction itself. In order to overcome the diffusion-limited mass transfer, magnetic iron oxide nanoparticles were employed. The magnetic nanoparticles (MNPs) can serve both as labels providing enhancement of the refractive index changes, and “vehicles” for rapidly delivering the analytes from sample solution to an SPR sensor surface with a gradient magnetic field. A model sandwich assay for the detection of β human chorionic gonadotropin (βhCG) has been utilized on a gold sensor surface with metallic diffraction grating structure supporting the excitation of SPs. Various detection formats including a) direct detection, b) sandwich assay, c) MNPs immunoassay without and d) with applied magnetic field were compared. The results show that the highly-sensitive MNPs immunoassay improves the LOD on the detection of βhCG by a factor of 5 orders of magnitude with respect to the direct detection.rn

Strategie formulative per la veicolazione nasale di farmaci

Microparticelle a base di complessi polielettrolitici di Chitosano/Pectina per il rilascio nasale di Tacrina cloridrato. Lo scopo di questo studio è stata la ricerca di nuove formulazioni solide per la somministrazione nasale di Tacrina cloridrato allo scopo di ridurre l’eccessivo effetto di primo passaggio epatico ed aumentarne la biodisponibilità a livello del Sistema Nervoso Centrale. La Tacrina è stata incapsulata in microparticelle mucoadesive a base di complessi elettrolitici di chitosano e pectina. Le microparticelle sono state preparate mediante due diversi approcci tecnologici (spray-drying e spray-drying/liofilizzazione) e analizzate in termini di caratteristiche dimensionali, morfologiche e chimico-fisiche. Nanoparticelle di Chitosano reticolate con Sodio Cromoglicato per il trattamento della rinite allergica. Il Sodio Cromoglicato è uno dei farmaci utilizzati per il trattamento della rinite allergica. Come noto, la clearance mucociliare provoca una rapida rimozione dei farmaci in soluzione dalla cavità nasale, aumentando così il numero di somministrazioni giornaliere e, di conseguenza, riducendo la compliance del paziente. Per ovviare a tale problema, si è pensato di includere il sodio cromoglicato in nanoparticelle di chitosano, un polimero capace di aderire alla mucosa nasale, prolungare il contatto della formulazione con il sito di applicazione e ridurre il numero di somministrazioni giornaliere. Le nanoparticelle ottenute sono state caratterizzate in termini di dimensioni, resa, efficienza di incapsulazione e caricamento del farmaco, potenziale zeta e caratteristiche mucoadesive. Analisi quantitativa di Budesonide amorfa tramite calorimetria a scansione differenziale. È stato sviluppato un nuovo metodo quantitativo allo stato solido basato sulla Calorimetria a Scansione Differenziale (DSC) in grado di quantificare in modo selettivo e accurato la quantità di Budesonide amorfa presente in una miscela solida. Durante lo sviluppo del metodo sono stati affrontati problemi relativi alla convalida di metodi analitici su campioni solidi quali la miscelazione di polveri solide per la preparazione di miscele standard e il calcolo della precisione.

Einfluss von Hilfsstoffen auf die Bioverfügbarkeit von Substanzen der BCS Klasse III

In dieser Arbeit wurde der Effekt verschiedener Hilfsstoffe auf die Permeabilität von Substanzen der BCS Klasse III untersucht. Drei pharmazeutische Hilfsstoffe wurden hinsichtlich der Möglichkeit ihres Einsatzes als Permeationsverbesserer in Arzneistoffformulierungen untersucht. Außerdem wurde die Beteiligung von Gallensalzen an der Nahrungsmittel-Interaktion von Trospium untersucht.rnEs wurden Komplexe aus Trospium und λ-Carrageen hergestellt. Eine verbesserte Permeation, die höchstwahrscheinlich durch Mukoadhäsion zustande kam, war im Ussing-Kammer-Modell sehr gut reproduzierbar. In vivo war der Effekt nur bei einigen Tieren zu sehen und es kam zu hohen Standardabweichungen.rnTrospium bildet Ionenpaare mit Gallensalzen, welche zu einer besseren Permeabilität des Wirkstoffes führten. In Gegenwart von Nahrungsfetten blieb dieser Effekt aus. Eine Beteiligung der Interaktion von Trospium und Gallensalzen am Food-Effekt kann auf Basis dieser Ergebnisse als wahrscheinlich gelten.rnIm Caco-2-Modell konnte bereits eine Verbesserung der Permeabilität von Trospium durch Zusatz von Eudragit E gezeigt werden. Nun konnte gezeigt werden, dass durch den Hilfsstoff auch in vivo in Ratten eine verbesserte Permeation erreicht werden kann.rnDie Permeationsverbesserung von Aciclovir durch Zusatz von Chitosan-HCl sollte untersucht werden. Im Caco-2-Modell kam es zu einer signifikanten Permeationsverbesserung. Im Ussing-Kammer-Modell wurde die Permeation nicht verbessert. In Loop-Studien konnte nur bei hohen Hilfsstoff-Konzentrationen eine Tendenz zur Permeationsverbesserung erkannt werden.rn

Nanoparticle preparation process using novel microjet reactor technology for enhancing dissolution rates of poorly water soluble drugs

In this study a novel method MicroJet reactor technology was developed to enable the custom preparation of nanoparticles. rnDanazol/HPMCP HP50 and Gliclazide/Eudragit S100 nanoparticles were used as model systems for the investigation of effects of process parameters and microjet reactor setup on the nanoparticle properties during the microjet reactor construction. rnFollowing the feasibility study of the microjet reactor system, three different nanoparticle formulations were prepared using fenofibrate as model drug. Fenofibrate nanoparticles stabilized with poloxamer 407 (FN), fenofibrate nanoparticles in hydroxypropyl methyl cellulose phthalate (HPMCP) matrix (FHN) and fenofibrate nanoparticles in HPMCP and chitosan matrix (FHCN) were prepared under controlled precipitation using MicroJet reactor technology. Particle sizes of all the nanoparticle formulations were adjusted to 200-250 nm. rnThe changes in the experimental parameters altered the system thermodynamics resulting in the production of nanoparticles between 20-1000 nm (PDI<0.2) with high drug loading efficiencies (96.5% in 20:1 polymer:drug ratio).rnDrug releases from all nanoparticle formulations were fast and complete after 15 minutes both in FaSSIF and FeSSIF medium whereas in mucodhesiveness tests, only FHCN formulation was found to be mucoadhesive. Results of the Caco-2 studies revealed that % dose absorbed values were significantly higher (p<0.01) for FHCN in both cases where FaSSIF and FeSSIF were used as transport buffer.rn

Hydrodynamics and solid dosage form disintegration/dissolution : immediate release tablets and novel in situ polyelectrolyte gastroretentive drug delivery systems

Solid oral dosage form disintegration in the human stomach is a highly complex process dependent on physicochemical properties of the stomach contents as well as on physical variables such as hydrodynamics and mechanical stress. Understanding the role of hydrodynamics and forces in disintegration of oral solid dosage forms can help to improve in vitro disintegration testing and the predictive power of the in vitro test. The aim of this work was to obtain a deep understanding of the influence of changing hydrodynamic conditions on solid oral dosage form performance. Therefore, the hydrodynamic conditions and forces present in the compendial PhEur/USP disintegration test device were characterized using a computational fluid dynamics (CFD) approach. Furthermore, a modified device was developed and the hydrodynamic conditions present were simulated using CFD. This modified device was applied in two case studies comprising immediate release (IR) tablets and gastroretentive drug delivery systems (GRDDS). Due to the description of movement provided in the PhEur, the movement velocity of the basket-rack assembly follows a sinusoidal profile. Therefore, hydrodynamic conditions are changing continually throughout the movement cycle. CFD simulations revealed that the dosage form is exposed to a wide range of fluid velocities and shear forces during the test. The hydrodynamic conditions in the compendial device are highly variable and cannot be controlled. A new, modified disintegration test device based on computerized numerical control (CNC) technique was developed. The modified device can be moved in all three dimensions and radial movement is also possible. Simple and complex moving profiles can be developed and the influence of the hydrodynamic conditions on oral solid dosage form performance can be evaluated. Furthermore, a modified basket was designed that allows two-sided fluid flow. CFD simulations of the hydrodynamics and forces in the modified device revealed significant differences in the fluid flow field and forces when compared to the compendial device. Due to the CNC technique moving velocity and direction are arbitrary and hydrodynamics become controllable. The modified disintegration test device was utilized to examine the influence of moving velocity on disintegration times of IR tablets. Insights into the influence of moving speed, medium viscosity and basket design on disintegration times were obtained. An exponential relationship between moving velocity of the modified basket and disintegration times was established in simulated gastric fluid. The same relationship was found between the disintegration times and the CFD predicted average shear stress on the tablet surface. Furthermore, a GRDDS was developed based on the approach of an in situ polyelectrolyte complex (PEC). Different complexes composed of different grades of chitosan and carrageenan and different ratios of those were investigated for their swelling behavior, mechanical stability, and in vitro drug release. With an optimized formulation the influence of changing hydrodynamic conditions on the swelling behavior and the drug release profile was demonstrated using the modified disintegration test device. Both, swelling behavior and drug release, were largely dependent on the hydrodynamic conditions. Concluding, it has been shown within this thesis that the application of the modified disintegration test device allows for detailed insights into the influence of hydrodynamic conditions on solid oral dosage form disintegration and dissolution. By the application of appropriate test conditions, the predictive power of in vitro disintegration testing can be improved using the modified disintegration test device. Furthermore, CFD has proven a powerful tool to examine the hydrodynamics and forces in the compendial as well as in the modified disintegration test device. rn

Studien zur Totalsynthese von Monilicin und strukturverwandten Naturstoffen

Ziel dieser Arbeit war die Totalsynthese von Monilicin. Seine Chlor- und Brom-Derivate wurden aus Monilinia fructicola isoliert und zeigten fungizide Wirkung. Die Schlüsselschritte der Synthese sind der Aufbau des ε-Lakton, die Einführung der exozyklischen Carboxymethyl-Gruppe und der Einbau der Doppelbindung in das Lakton. Es wurden drei Synthesestrategien verfolgt, wobei die Bildung des Laktons über eine Veresterung erfolgen sollte.rnÜber enantioselektive Syntheseschritte sollten die reinen Enantiomere erhalten werden. Ausgehend vom Orcinol erfolgte auf allen Syntheserouten zuerst der Aufbau des 5-Hydroxy-7-methylchromon-Grundgerüstes, und anschließend dessen Funktionalisierung in den Positionen 2 und 3. Der Ringschluss zum ε-Lakton gelang über eine Steglich-Veresterung. Syntheseweg A lieferte nach der Oxidation der primären exozyklischen Alkoholgruppe und anschließender Methylierung das Dihydromonilicin. Auf dem Syntheseweg B gelang die Einführung der späteren exozyklischen Carboxymethyl-Gruppe vor der Laktonisierung. Aus der Dicarbonsäure konnte zum ersten Mal auch der Naturstoff Oxalicumon C totalsynthetisch dargestellt und seine absolute Konfiguration aufgeklärt werden. Nach selektiver Hydrolyse konnte aus Oxalicumon C ebenfalls das Dihydromonilicin synthetisiert werden. Die Darstellung von Monilicin durch Einführung der Doppelbindung in das Dihydromonilicin oder bereits vor der Laktonisierung (Syntheseweg C) konnte nicht erreicht werden. Einige der Chromon-Derivate zeigten fungizide und zytotoxische Aktivitäten. rn

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as (111)In and (68)Ga.