No description available.

As one of the natural polyphenols, resveratrol possesses hydroxyl substituted trans-stilbene structure and exerts impact on health by inhibiting multiple human enzymes, such as cyclooxygenase, F1 ATPase, and tyrosinase. Resveratrol has to be bound by human serum albumin (HSA) to keep a high concentration in serum, since its solubility is low in water. To improve water solubility and bioavailability, two resveratrol aliphatic acids and their esters have been designed and synthesized. The solubilities of the resveratrol and its derivatives have been measured using a standard procedure. The two aliphatic acids showed better solubilities in pure water and phosphate buffer (pH 7). The binding affinities of resveratrol derivatives for HSA were also measured, and the drug-protein interaction mechanism was investigated using fluorescence, UV-vis, and NMR spectroscopies. Interestingly, resveratrol hexanoic acid (5) was found to be a much better ligand (Ka = (6.70 ± 0.10) × 106 M-1) for HSA than resveratrol (Ka = (1.64 ± 0.07) × 105 M-1), and there was 41-fold improvement for the binding affinity. It was the first time that the increase of fluorescence of resveratrol moiety was observed during the binding to HSA, suggesting that 5 should be bound tightly by HSA. The UV-vis absorption spectroscopy revealed a maximum absorption shift from 318 to 311 nm with decreasing intensity by 20% upon complexation, suggesting that the π-π conjugation of the stilbene structure was impaired during the binding. Although HSA was reported to have only one binding site for resveratrol, the Job's and molar ratio plots suggested that HSA should bind two molecules of 5. NMR study suggested that phenyl group (B ring) in the center of the molecule of 5 should be involved in the π-π stacking interactions with HSA aromatic amino acid residues. Molecular geometry calculation of 5 with Spartan software showed that the stilbene structure had two conformers, orthogonal and planar ones. The former (E = -1.432 KJ/mol) was more stable than the latter (E = -0.128 KJ/mol), suggesting that the former should be the conformer of 5 in the complexation with HSA.

Zinc is an essential metal ion for the activity of multiple enzymes and transcription factors. Among many other transporting proteins human serum albumin (HSA) is the main carrier of Zn(II) in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids. Hence, HSA controls the availability and distribution of those molecules throughout the body. Previous studies have established that the existence of one zinc site with high affinity (MBS-A) that is modulated by the presence of fatty acids. Therefore, the fatty acid concentration in the blood influences zinc distribution which may result in a significant effect on both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc ion, here, we attempted to investigate zinc interactions and coordination with HSA and the effect of different fatty acids on the protein structure, stability and on Zn(II) binding. By NMR titration, we examine the Zn(II) binding to HSA and the spectra show distinct movements of some resonances showing a conformational change has occurred as a result of Zn(II) binding. Isothermal calorimetry titrations study was performed to evaluate zinc binding affinity to HSA in the absence and presence of fatty acids. Free HSA results indicates the existence of one high affinity site and multiple low affinity sites. Upon the binding of fatty acids to HSA, three distinct behaviors of Zn(II) affinity was observed ranging from no effect to moderate to significant depending on the FAs. By the use of circular dichroism, we investigate secondary and tertiary structure of HSA in the presence and absence of FAs and Zn(II). We found albumin is predominately α-helical and the overall conformation of the protein remains unchanged even after interacting with FAs and Zn(II) with some exception. The structural stability of HSA was evaluated by obtaining the denaturation temperature in the presence and absence of fatty acid and we found the thermal denaturation of HSA increases with the increase of amount of fatty acids.

The purpose of this study was to determine if post-operative serum albumin and percentage of weight loss improved in patients who received formalized pre-operative nutrition counseling. Nutrition intervention was measured quantitatively. A retrospective review of records was conducted on 77 RYGB patients (68 female subjects and 9 male subjects), ages 21-64, during January 2001 through January 2006. The results indicated that patients who received pre-operative nutrition intervention had better clinical outcomes of serum albumin than those with no nutrition intervention from a registered dietitian. However, outcomes regarding percentage of weight loss varied. Both pre-operatively and at the 3 month post-operative visit the weight of subjects who received nutrition intervention seemed to be increasing by the 6 month post-operative visit the subjects with no nutrition intervention had lost more weight.

No description available.

<p>Aggregation of the<strong> </strong>Alzheimer’s Aβ peptide in the brain and blood plasma is controlled by endogenous Aβ binding proteins. The structural basis for the interaction between the Aβ peptide and the Aβ binding proteins is critical not only to understand how Aβ amyloids are controlled in vivo, but also to guide the design of novel Aβ-self association inhibitors. However, the current knowledge of the structures of the Aβ/Aβ binding protein complexes is still sparse. This thesis focuses mainly on the interaction of the Aβ peptide with Human Serum Albumin (HSA). It is known that HSA binds ~90% of the Aβ in human plasma and prevents the Aβ self-association into amyloid fibrils. However, the mechanism of Aβ self-association inhibition by albumin was not understood prior to our work. We have shown that albumin preferentially binds toxic Aβ oligomers and fibrils inhibiting their growth into larger Aβ assemblies through a “monomer competitor” mechanism. Using a combination of NMR, domain deletion mutants, dynamic light scattering and ultrafiltration we have investigated the stoichiomery and affinity of the Aβ oligomer: HSA complexes. Our results indicate that all three domains of HSA bind Aβ oligomers and fibrils with an affinity in the 1-100 nM range. Such binding site degeneracy explains how albumin minimizes competition by other ligands such as fatty acids and drugs. Moreover we have used the soluble and NMR suitable domain 3 of albumin to dissect further the determinants of the Aβ oligomer binding to albumin at subdomain and peptide resolution. We show that both subdomains of the HSA domain 3 (<em>i.e</em>. 3A and 3B) bind the Aβ oligomers. In addition, we identified a peptide sequence within subdomain 3B that displays significant potency in the inhibition of Aβ self-association.</p> / Doctor of Philosophy (PhD)

The emergence of microneedle (MN) technologies offers a route for a pain free, straightforward and efficient way of transdermal drug delivery, but technological barriers still exist which pose significant challenges for manufacture of MN systems with high volume outputs at low cost. The main aim of this research was to develop new ways for MN manufacture primarily using micro-injection moulding processes with high performance engineering thermoplastics. During the moulding process these polymeric melts will be subjected to extreme stress and temperature gradients and detailed material characterisation combined with in-line monitoring is desirable to optimise the moulding parameters and will help in achieving sharp microneedles with acceptable quality. Hence high shear rheology of these selected materials was performed at wall shear rates carried out in excess of 107 s-1 over a range of temperatures to predict the flow behaviour of polymer melts at such high shear strain rates. This information was fed into injection moulding simulation software tools (Moldflow) to assist the MN production process design. The optimal design was then used to produce a full 3D solid model of the injection mould and mould insert. Furthermore various design of experiments were conducted considering input parameters such as injection pressure, injection speed, melt temperature, filling time and mould cavity temperature. Response variables including product quality and data acquired from the cavity pressure and temperature transducers were used to optimise the manufacturing process. The moulded MNs were geometrically assessed using a range of characterisation techniques such as atomic force microscopy, confocal microscopy and scanning electron microscopy. An attempt to make hollow MNs was performed and encountered many challenges like partial cavity filling and part ejection during processing. Studies were carried out to understand the problem and identified the major problem was in tool design and improvements to the moulding tool design were recommended. Plasma treatment and mechanical abrasion were employed to increase the surface energy of the moulded polymer surfaces with the aim of enhancing protein adsorption. Sample surface structures before and after treatment were studied using AFM and surface energies have been obtained using contact angle measurement and calculated using Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness resulting in better adsorption and release of BSA. To assist design-optimisation and to assess performance, a greater understanding of MN penetration behaviour is required. Contact stiffness, failure strength and creep behaviour were measured during compression tests of MN against a steel surface, and in-vitro penetration of MNs into porcine skin. The MN penetration process into porcine skin was imaged using optical coherence tomography. Finally, a finite element model of skin was established to understand the effect of tip geometry on penetration. The output of findings from this research will provide proof of concept level development and understanding of mechanisms of MN penetration and failure, facilitating design improvements for micro-injection moulded polymeric MNs.

Yes / Plasma technology has been widely used to increase the surface energy of the polymer surfaces for many industrial applications; in particular to increase in wettability. The present work was carried out to investigate how surface modification using plasma treatment modifies the surface energy of micro-injection moulded microneedles and its influence on drug delivery. Microneedles of polyether ether ketone and polycarbonate and have been manufactured using micro-injection moulding and samples from each production batch have been subsequently subjected to a range of plasma treatment. These samples were coated with bovine serum albumin to study the protein adsorption on these treated polymer surfaces. Sample surfaces structures, before and after treatment, were studied using atomic force microscope and surface energies have been obtained using contact angle measurement and calculated using the Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness of the microneedles resulting in better adsorption and release of BSA.

Yes / The role of human serum albumin (HSA) in the transport of molecules predicates its involvement in the determination of drug distribution and metabolism. Optimization of ADME properties are analogous to HSA binding thus this is imperative to the drug discovery process. Currently, various in silico predictive tools exist to complement the drug discovery process, however, the prediction of possible ligand-binding sites on HSA has posed several challenges. Herein, we present a strong and deeper-than-surface case for the prediction of HSA-ligand binding sites using multi-cavity molecular descriptors by exploiting all experimentally available and crystallized HSA-bound drugs. Unlike previously proposed models found in literature, we established an in-depth correlation between the physicochemical properties of available crystallized HSA-bound drugs and different HSA binding site characteristics to precisely predict the binding sites of investigational molecules. Molecular descriptors such as the number of hydrogen bond donors (nHD), number of heteroatoms (nHet), topological polar surface area (TPSA), molecular weight (MW), and distribution coefficient (LogD) were correlated against HSA binding site characteristics, including hydrophobicity, hydrophilicity, enclosure, exposure, contact, site volume, and donor/acceptor ratio. Molecular descriptors nHD, TPSA, LogD, nHet, and MW were found to possess the most inherent capacities providing baseline information for the prediction of serum albumin binding site. We believe that these associations may form the bedrock for establishing a solid correlation between the physicochemical properties and Albumin binding site architecture. Information presented in this report would serve as critical in provisions of rational drug designing as well as drug delivery, bioavailability, and pharmacokinetics.

Ziel der vorliegenden Arbeit ist die photophysikalische Untersuchung Photosensibilisator-beladener Nanopartikel als Transportsysteme für aktives und passives Tumor-Targeting. Zu diesem Zweck wurden sowohl stationäre, als auch zeitaufgelöste spektroskopische Methoden angewandt. Der erste Teil beschäftigt sich mit der photophysikalischen Charakterisierung von Pheo-HSA-Nanopartikeln. Mittels stationärer und zeitaufgelöster Messungen konnte gezeigt werden, dass die Wechselwirkungen zwischen Phäophorbid a und den HSA-Nanopartikeln sehr stark ist. Diese Wechselwirkungen bewirken eine geringe Singulettsauerstoffquantenausbeute (0,07) in D2O verglichen mit dem von Phäophorbid a in Ethanol (0,52). Im Gegensatz dazu konnte nach der Inkubation in Jurkat- und HT-29-Zellen eine intrazelluläre Singulettsauerstoffgenerierung der Pheo-HSA-NPs nachgewiesen werden. Im zweiten Teil wurden mit den Photosensibilisatoren mTHPP and mTHPC beladene HSA- und PLGA-Nanopartikel untersucht. Es konnte gezeigt werden, dass die Photosensibilisator-Beladungsrate die photophysikalischen Eigenschaften der HSA- und PLGA-Nanopartikel stark beeinflusst. Für die HSA-Nanopartikel dominieren bei geringen Beladungsraten die Wechselwirkungen zwischen HSA und den Photosensibilisatormolekülen. Mit steigender Beladung spielen Wechselwirkungen zwischen den Photosensibilisatormolekülen eine zunehmende Rolle. Diese Wechselwirkungen verringern bei hoher Beladung der HSA-Nanopartikel die Generierung von Singulettsauerstoff. Auch für die PLGA-Nanopartikel konnte mit zunehmender Beladung ein verstärktes Singulettsauerstoffquenching nachgewiesen werden. Im dritten Teil dieser Arbeit wurden, für aktives Targeting von Tumorzellen, Oberflächenmodifizierte PLGA- und HSA-Nanopartikel untersucht. Die intrazellulären Singulettsauerstoffmessungen weisen auf eine erleichterte Aufnahme in Tumorzellen von Antikörper- und PEG-modifizierten HSA-Nanopartikeln in vitro hin. / The main goal of this PhD thesis is the photophysical investigation of biodegradable photosensitizer-nanoparticle carrier systems achieving passive and active tumour targeting strategies. For this purpose both steady state and time-resolved spectroscopic methods accompanied by data analysis were utilized. This work contains three main parts: First the photophysical properties of Pheo-HSA nanoparticles were compared to free pheophorbide a. Steady-state and time-resolved fluorescence experiments have already proved that the interaction between pheophorbide a and HSA nanoparticles is strong. This interaction leads to low singlet oxygen quantum yield (0.07) in D2O compared to free Pheo (0.52) in ethanol. But when incubated in Jurkat and HT-29 cell lines, Pheo-HSA nanoparticles have been proved to generate singlet oxygen inside cells. In the second part the well-known photosensitizers mTHPP and mTHPC were loaded to HSA- and PLGA- nanoparticles. It was found that the loading ratio determines the photophysical properties of both photosensitizer-loaded HSA and PLGA nanoparticles. For HSA nanoparticles, photosensitizer-nanoparticle interaction is the preferential mechanism in low loading ratio sample. But in high loading ratio sample, photosensitizer-photosensitizer interaction becomes the determining interaction. This interaction prevents singlet oxygen generation from high loading sample. For PLGA nanoparticles, high drug loading ratio also leads to a strong singlet oxygen quenching. At high drug loading ratio PLGA nanoparticles, some photosensitizer molecules may be localized deeply inside PLGA matrices and far away from surface. In the third part of this work, active tumour targeting behaviour achieved by surface modification of HSA and PLGA nanoparticles has been tested. Intracellular singlet oxygen measurement reveals that HSA nanoparticles, both with antibody and PEG surface modification have an enhanced targeting of tumour cells in vitro.