Caractérisation des mécanismes moléculaires liés à p53 régulant l’expression du gène P2RY6.

Le récepteur P2Y[indice inférieur 6] est un récepteur couplé à une protéine G responsable de l’activation de nombreuses voies de signalisation. Dans l’épithélium du côlon, il participe au maintien de l’équilibre hydrique, mais il a été montré que le récepteur P2Y[indice inférieur 6] participait à l’aggravation des symptômes inflammatoires dans la maladie de Crohn ou dans la colite ulcéreuse. Les maladies inflammatoires de l’intestin sont des facteurs pouvant mener au cancer colorectal. En effet, il existe deux types de cancers colorectaux : le cancer sporadique et le cancer associé à la colite qui se différencient notamment par la séquence d’apparition de mutations génétiques. Par exemple, le gène TP53 est muté de façon tardive dans le cancer colorectal sporadique et muté de façon précoce dans le cancer associé à l’inflammation. Puisque le récepteur P2Y[indice inférieur 6] est impliqué dans la création d’un environnement pro-inflammatoire, nous nous sommes intéressés au rôle de p53 sur l’expression du gène P2RY[indice inférieur 6] et avons formulé l’hypothèse suivante : la présence de TP53 mutant va réguler de façon différentielle l’expression du gène P2RY[indice inférieur 6] dans le cancer colorectal. L’objectif général des travaux est le suivant : caractériser les mécanismes moléculaires liés à TP53 régulant l’expression du gène P2RY[indice inférieur 6] dans le cancer colorectal. Les objectifs spécifiques pour ce projet de recherche sont donc : (1) déterminer et caractériser les régions promotrices du gène P2RY[indice inférieur 6] dans les cellules épithéliales intestinales cancéreuses et (2) étudier l’effet de la protéine p53 de type sauvage ou mutée sur l’expression du récepteur P2Y[indice inférieur 6]. Le gène P2RY[indice inférieur 6] code pour 8 variants d’ARN messagers. Les variants 1, 2, 3, 5, 6, 7 et 8 codent pour l’isoforme 1 du récepteur P2Y[indice inférieur 6], forme connue du récepteur. Le variant 9 code pour l’isoforme 2, non caractérisée. Nos travaux ont permis de mettre en évidence l’existence de quatre régions promotrices potentielles du gène P2RY[indice inférieur 6] et la présence du variant 9, codant pour l’isoforme 2 du récepteur P2Y[indice inférieur 6] dans la lignée cellulaire Caco-2. Nous avons également montré que les formes normale et mutée de p53 régulent de façon différentielle l’expression du récepteur P2Y[indice inférieur 6]. Enfin, le rôle de l’isoforme 2 reste à étudier, mais les tests effectués suggèrent qu’elle est activable par l’UDP.

Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21waf1/cip1

Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.

Hepatocyte growth factor stimulates invasion across reconstituted basement membranes by a new human small intestinal cell line

Hepatocyte growth factor/scatter factor (HGF/SF) is a protein growth factor whose pleiotropic effects on epithelial cells include the stimulation of motility, mitosis and tubulogenesis. These responses are mediated by the cell surface tyrosine kinase receptor c-met. Because both the cytokine and receptor are found in the gastrointestinal tract, we have studied the effects of HGF/SF on transformed gut epithelial cells which express c-met. Here we describe the response of a new transformed human jejunal epithelioid cell line (HIE-7) to HGF/SF. Morphologically HIE-7 cells are immature. Their epithelial lineage was confirmed by reactivity with the epithelial specific antibodies AE1/AE3, Cam 5.2, Ber-EP4 and anti-EMA and is consistent with their expression of c-met mRNA and protein. In addition, electron microscopic analysis revealed the presence of primitive junctions and rudimentary microvilli, but features of polarization were absent. When grown on reconstituted basement membranes, HIE-7 cells formed closely associated multicellular cord-like structures adjacent to acellular spaces. However, the cells did not mature structurally, form lumen-like structures or express disaccharidase mRNA, even in the presence of recombinant HGF (rHGF). On the other hand, rHGF induced HIE-7 cells to scatter and stimulated their rapid migration in a modified wound assay. To determine whether the motogenic effect caused by rHGF is associated with HIE-7 cell invasiveness across reconstituted basement membranes, a Boyden chamber chemoinvasion assay was performed. rHGF stimulated a 10-fold increase in the number of HIE-7 cells that crossed the basement membrane barrier, while only stimulating a small increase in chemotaxis across a collagen IV matrix, suggesting that the cytokine activates matrix penetration by these cells. rHGF also stimulated the invasion of basement membranes by an undifferentiated rat intestinal cell line (IEC-6) and by two human colon cancer cell lines which are poorly differentiated (DLD-1 and SW 948). In contrast, two moderately well differentiated colon cancer cell lines (Caco-2 and HT-29) did not manifest an invasive response when exposed to rHGF. These results suggest that HGF/SF may play a significant role in the invasive behavior of anaplastic and poorly differentiated gut epithelial tumors.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

The role of probiotics in Helicobacter pylori infection

Helicobacter pylori (H. pylori) infection is a major cause of chronic gastritis and peptic ulcer disease, and it is also designated as a class-I carcinogen for stomach cancer. The role of probiotics in the treatment of gastrointestinal infections is increasingly documented as an alternative or complement to antibiotics, with the potential to decrease the use of antibiotics or reduce their adverse effects. These studies were conducted to investigate the role of probiotics in the treatment of H. pylori infection. Various aspects included: an investigation of the effects of a probiotic combination consisting of Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 or B. lactis Bb12 as a supplementation to H. pylori eradication therapy, with special reference to tolerability, effectiveness, and microbiota alterations following the treatment; discovering the role of probiotics in vivo with H. pylori infected and uninfected patients, as well as with an in vitro model of H. pylori infection. The probiotic combination therapy was able to reduce significantly the total symptom score, which takes into account both the frequency and the severity of the adverse effects, during the eradication treatment. The supplementation did not improve the success of the eradication treatment significantly, though some difference was seen in the eradication percentages (91% vs. 79%). The quantities of predominant bacterial groups were altered significantly following the triple treatment. Probiotics slightly counteracted the effects of anti-H. pylori treatment, monitored as significantly less alterations in the total numbers of aerobes and lactobacilli/enterococci group bacteria. After probiotic intervention, L. rhamnosus GG adhered to a minority of the patients upper gastrointestinal mucosa, but all of the probiotics survived well through the gastrointestinal tract transit with and without antimicrobial treatment. Probiotic intervention decreased gastrin-17 levels in H. pylori infected patients and appeared to decrease the 13C-urea breath test values. In in vitro Caco-2 cell line experiments, probiotics inhibited H. pylori adhesion to intestinal epithelial cells. Both L. rhamnosus strains, P. freudenreichii ssp. shermanii JS and the combination inhibited the H. pylori-induced acute cell leakage. Simultaneously, both L.rhamnosus strains and the combination transiently improved the epithelial barrier function. The pro-inflammatory effects prevailed when the probiotics were used in combination. According to this series of studies, probiotic combination could have some potential in reducing adverse effects induced by H. pylori eradication treatment and beneficial effects on H. pylori infected subjects.

Mass spectrometry and n-in-one analytics in early drug discovery: Combinatorial chemistry libraries, lipophilicity and absorption screening

This thesis describes current and past n-in-one methods and presents three early experimental studies using mass spectrometry and the triple quadrupole instrument on the application of n-in-one in drug discovery. N-in-one strategy pools and mix samples in drug discovery prior to measurement or analysis. This allows the most promising compounds to be rapidly identified and then analysed. Nowadays properties of drugs are characterised earlier and in parallel with pharmacological efficacy. Studies presented here use in vitro methods as caco-2 cells and immobilized artificial membrane chromatography for drug absorption and lipophilicity measurements. The high sensitivity and selectivity of liquid chromatography mass spectrometry are especially important for new analytical methods using n-in-one. In the first study, the fragmentation patterns of ten nitrophenoxy benzoate compounds, serial homology, were characterised and the presence of the compounds was determined in a combinatorial library. The influence of one or two nitro substituents and the alkyl chain length of methyl to pentyl on collision-induced fragmentation was studied, and interesting structurefragmentation relationships were detected. Two nitro group compounds increased fragmentation compared to one nitro group, whereas less fragmentation was noted in molecules with a longer alkyl chain. The most abundant product ions were nitrophenoxy ions, which were also tested in the precursor ion screening of the combinatorial library. In the second study, the immobilized artificial membrane chromatographic method was transferred from ultraviolet detection to mass spectrometric analysis and a new method was developed. Mass spectra were scanned and the chromatographic retention of compounds was analysed using extract ion chromatograms. When changing detectors and buffers and including n-in-one in the method, the results showed good correlation. Finally, the results demonstrated that mass spectrometric detection with gradient elution can provide a rapid and convenient n-in-one method for ranking the lipophilic properties of several structurally diverse compounds simultaneously. In the final study, a new method was developed for caco-2 samples. Compounds were separated by liquid chromatography and quantified by selected reaction monitoring using mass spectrometry. This method was used for caco-2 samples, where absorption of ten chemically and physiologically different compounds was screened using both single and nin- one approaches. These three studies used mass spectrometry for compound identification, method transfer and quantitation in the area of mixture analysis. Different mass spectrometric scanning modes for the triple quadrupole instrument were used in each method. Early drug discovery with n-in-one is area where mass spectrometric analysis, its possibilities and proper use, is especially important.

Mesoporous silica- and silicon-based materials as carriers for poorly water soluble drugs

New chemical entities with unfavorable water solubility properties are continuously emerging in drug discovery. Without pharmaceutical manipulations inefficient concentrations of these drugs in the systemic circulation are probable. Typically, in order to be absorbed from the gastrointestinal tract, the drug has to be dissolved. Several methods have been developed to improve the dissolution of poorly soluble drugs. In this study, the applicability of different types of mesoporous (pore diameters between 2 and 50 nm) silicon- and silica-based materials as pharmaceutical carriers for poorly water soluble drugs was evaluated. Thermally oxidized and carbonized mesoporous silicon materials, ordered mesoporous silicas MCM-41 and SBA-15, and non-treated mesoporous silicon and silica gel were assessed in the experiments. The characteristic properties of these materials are the narrow pore diameters and the large surface areas up to over 900 m²/g. Loading of poorly water soluble drugs into these pores restricts their crystallization, and thus, improves drug dissolution from the materials as compared to the bulk drug molecules. In addition, the wide surface area provides possibilities for interactions between the loaded substance and the carrier particle, allowing the stabilization of the system. Ibuprofen, indomethacin and furosemide were selected as poorly soluble model drugs in this study. Their solubilities are strongly pH-dependent and the poorest (< 100 µg/ml) at low pH values. The pharmaceutical performance of the studied materials was evaluated by several methods. In this work, drug loading was performed successfully using rotavapor and fluid bed equipment in a larger scale and in a more efficient manner than with the commonly used immersion methods. It was shown that several carrier particle properties, in particular the pore diameter, affect the loading efficiency (typically ~25-40 w-%) and the release rate of the drug from the mesoporous carriers. A wide pore diameter provided easier loading and faster release of the drug. The ordering and length of the pores also affected the efficiency of the drug diffusion. However, these properties can also compensate the effects of each other. The surface treatment of porous silicon was important in stabilizing the system, as the non-treated mesoporous silicon was easily oxidized at room temperature. Different surface chemical treatments changed the hydrophilicity of the porous silicon materials and also the potential interactions between the loaded drug and the particle, which further affected the drug release properties. In all of the studies, it was demonstrated that loading into mesoporous silicon and silica materials improved the dissolution of the poorly soluble drugs as compared to the corresponding bulk compounds (e.g. after 30 min ~2-7 times more drug was dissolved depending on the materials). The release profile of the loaded substances remained similar also after 3 months of storage at 30°C/56% RH. The thermally carbonized mesoporous silicon did not compromise the Caco-2 monolayer integrity in the permeation studies and improved drug permeability was observed. The loaded mesoporous silica materials were also successfully compressed into tablets without compromising their characteristic structural and drug releasing properties. The results of this research indicated that mesoporous silicon/silica-based materials are promising materials to improve the dissolution of poorly water soluble drugs. Their feasibility in pharmaceutical laboratory scale processes was also confirmed in this thesis.

Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by New Cationic Gemini Tocopheryl Lipids

Nonviral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We present here six new tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient coliposomal formulations derived from each of these geminis and a helper lipid, dioleoylphosphatidylethanolamine (DOPE), were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized coliposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz., HEK 293T, HeLa, and Caco-2, significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and in the presence of serum (FBS). Notably, the knockdown activity of coliposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR, and Western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin, significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.

A delocalizable cationic headgroup together with an oligo-oxyethylene spacer in gemini cationic lipids improves their biological activity as vectors of plasmid DNA

Lipoplex nano-aggregates constituted of plasmid DNA (pDNA) pEGFP-C3 and mixed cationic liposomes, consisting of several percentages of a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl imidazolium) oxyethylene series, referred to as (C(16)Im)(2)(C2O)(n), with oxyethylene spacers (n = 1, 2 or 3) between the imidazolium cationic groups and the DOPE zwitterionic helper lipid, have been characterized by various biophysical and biological approaches carried out at several GCL compositions (alpha), and either the mass or the effective charge ratio of the lipoplex. The electrochemical study by zeta-potential confirms that the three GCLs yield a 10% lower effective charge than the nominal one, while compacted pDNA yields only a 25% effective negative charge. The SAXS study reveals, irrespective of the spacer length (n) and effective charge ratio (rho(eff)), the presence of two lamellar structures, i.e., one (L-alpha,L-main) in the whole GCL composition and another (L-alpha,L-DOPE,L-rich) with higher periodicity values that coexists with the previous one at low GCL composition (alpha = 0.2). The cryo-TEM analysis shows two types of multilamellar structures consisting of cationic lipidic bilayers with pDNA sandwiched between them: a cluster-type (C-type) at low alpha = 0.2 and a fingerprint-type (FP-type) at alpha >= 0.5, both with similar interlamellar spacing (d) in agreement with the L-alpha,L-main structure determined by SAXS. Transfection efficacies (TEs) of each lipid mixture were determined in four different cell lines (HEK293T, HeLa, Caco-2 and A549) at several alpha and rho(eff) values in the absence and presence of serum (FBS). The optimized formulations (alpha = 0.2 and rho(eff) = 2.0) substantially transfect cells much better than a commercial transfection reagent, Lipofectamine 2000 and previously studied efficient lipoplexes containing other cationic head groups or spacers both in the absence and presence of serum. The activity of optimized formulations may be attributed to the combination of several factors, such as: (a) the fusogenic character of DOPE which results in higher fluidity of the lipoplexes at alpha = 0.2, (b) the coexistence of two lamellar structures at alpha = 0.2 that synergizes the TE of these lipid vectors, and mainly (c) the higher biocompatibility of the GCLs reported in this work due to the presence of two imidazolium cationic groups together with an oligo-oxyethylene spacer. The length of the spacer in the GCL seems to have less impact, although (C(16)Im)(2)(C2O)(n)/DOPEpDNA lipoplexes with n = 1 and 3 show higher gene transfection than n = 2. All the optimum formulations reported herein are all highly efficient with negligible levels of toxicity, and thus, may be considered as very promising gene vectors for in vivo applications.

Efficacious redox-responsive gene delivery in serum by ferrocenylated monomeric and dimeric cationic cholesterols

Herein, we present the design and synthesis of new redox-active monomeric and dimeric (gemini) cationic lipids based on ferrocenylated cholesterol derivatives for gene delivery. The cationic cholesterols are shown to be transfection efficient after being formulated with the neutral helper lipid DOPE in the presence of serum (FBS). The redox activity of the resulting co-liposomes and their lipoplexes could be regulated using the alkanyl ferrocene moiety attached to the ammonium head groups of the cationic cholesterols. Atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements were performed to characterize the co-liposomal aggregates and their complexes with pDNA. The transfection efficiency of lipoplexes could be tuned by changing the oxidation state of the ferrocene moiety. The gene transfection capability was assayed in terms of green fluorescence protein (GFP) expression using pEGFP-C3 plasmid DNA in three cell lines of different origins, namely Caco-2, HEK293T and HeLa, in the presence of serum. The vesicles possessing ferrocene in the reduced state induced an efficient transfection, even better than a commercial reagent Lipofectamine 2000 (Lipo 2000) as evidenced by flow cytometry and fluorescence microscopy. All the co-liposomes containing the oxidized ferrocene displayed diminished levels of gene expression. Gene transfection events from the oxidized co-liposomes were further potentiated by introducing ascorbic acid (AA) as a reducing agent during lipoplex incubation with cells, leading to the resumption of transfection activity. Assessment of transfection capability of both reduced and oxidized co-liposomes was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Overall, we demonstrate here controlled gene transfection activities using redox-driven, transfection efficient cationic monomeric and dimeric cholesterol lipids. Such systems could be used in gene delivery applications where transfection needs to be performed spatially or temporally.

Photonic monitoring of chitosan nanostructured alginate microcapsules for drug release

By using a novel microfluidic set-up for drug screening applications, this study examines delivery of a novel risedronate based drug formulation for treatment of osteoporosis that was developed to overcome the usual shortcomings of risedronate, such as its low bioavailability and adverse gastric effects. Risedronate nanoparticles were prepared using muco-adhesive polymers such as chitosan as matrix for improving the intestinal cellular absorption of risedronate and also using a gastric-resistant polymer such as sodium alginate for reducing the gastric inflammation of risedronate. The in-vitro characteristics of the alginate encapsulated chitosan nanoparticles are investigated, including their stability, muco-adhesiveness, and Caco-2 cell permeability. Fluorescent markers are tagged with the polymers and their morphology within the microcapsules is imaged at various stages of drug release.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.