Liposomes have the ability to carry and deliver both hydrophilic and hydrophobic drugs and to protect them when injected into the circulatory system. They thus provide an attractive vehicle for drug delivery. However, problems of rapid clearance and inability to target liposomes to specific cells and tissues remain unresolved. Rapid clearance has been attributed to adsorption of opsonins, and one approach to reduce such adsorption is to create sterically stabilized liposomes by modifying the surface with polyethylene glycol (PEG) or dextran. To deliver their drug "payload" liposomes must interact with the membranes of target cells. Interactions with cellular components of the vascular walls have been observed for various sulfated polysaccharides such as heparin and functionalised dextrans. Based on the above considerations, the purpose of this work was to investigate the ability of various polymeric modifiers on liposomes to reduce protein adsorption and promote incorporation into target cells. Liposomes of composition PC/PEI cholesterol (70/10/20 mol %) were surface modified with PEG, dextran, heparin, and functionalised dextran. Protein adsorption was studied from solutions of IgG in buffer and from plasma. Adsorption from buffer was measured by radio labelling methods. For the plasma work, a total protein assay was used to determine the amount of protein adsorbed to the liposome surface, while gel electrophoresis and immunoblotting methods were used to examine the profiles of protein binding. Liposome incorporation into vascular smooth muscle and endothelial cells was evaluated using fluorescent labelling and radio labelling techniques. The IgG adsorption studies showed reduced adsorption on all polymer-modified liposomes. Plasma adsorption data showed that adsorbed protein layer compositions on the different liposome types were similar, but different from that of the plasma itself, showing that the plasma was fractionated on the liposome surfaces. Cell interaction studies showed that liposomes modified with dextran and sulfated dextran were incorporated into both cell types. The unmodified, PEG-and heparin-modified liposomes were not incorporated to any significant extent. / Thesis / Master of Engineering (ME)

Yes / A set of water-swollen core−shell particles was synthesized by emulsion polymerization of a 1,3-dioxolane functional monomer in water. After removal of the 1,3- dioxolane group, the particles’ shells were shown to swell in aqueous media. Upon hydrolysis, the particles increased in size from around 70 to 100−130 nm. A bicinchoninic acid assay and ζ-potential measurements were used to investigate the adsorption of lysozyme, albumin, or fibrinogen. Each of the core−shell particles adsorbed significantly less protein than the noncoated core (polystyrene) particles. Differences were observed as both the amount of difunctional, cross-linking monomer and the amount of shell monomer in the feed were changed. The core−shell particles were shown to be resistant to protein adsorption, and the degree to which the three proteins adsorbed was dependent on the formulation of the shell. / EPSRC and MRC

Es ist schon seit längerer Zeit bekannt, dass nach Kontakt des Biomaterials mit der biologischen Umgebung bei Implantation oder extrakorporaler Wechselwirkung zunächst Proteine aus dem umgebenden Milieu adsorbiert werden, wobei die Oberflächeneigenschaften des Materials die Zusammensetzung der Proteinschicht und die Konformation der darin enthaltenden Proteine determinieren. Die nachfolgende Wechselwirkung von Zellen mit dem Material wird deshalb i.d.R. von der Adsorbatschicht vermittelt. Der Einfluss der Oberflächen auf die Zusammensetzung und Konformation der Proteine und die nachfolgende Wechselwirkung mit Zellen ist von besonderem Interesse, da einerseits eine Aussage über die Anwendbarkeit ermöglicht wird, andererseits Erkenntnisse über diese Zusammenhänge für die Entwicklung neuer Materialien mit verbesserter Biokompatibilität genutzt werden können. In der vorliegenden Habilitationsschrift wurde deshalb der Einfluss der Zusammensetzung von Polymeren bzw. von deren Oberflächeneigenschaften auf die Adsorption von Proteinen, den Aktivitätszustand der plasmatischen Gerinnung und die Adhäsion von Zellen untersucht. Dabei wurden auch Möglichkeiten zur Beeinflussung dieser Vorgänge über eine Veränderung der Volumenzusammensetzung oder durch Oberflächenmodifikationen von Biomaterialien vorgestellt. Erkenntnisse aus diesen Arbeiten konnten für die Entwicklung von Membranen für Biohybrid-Organe genutzt werden. / The implantation of biomaterials or the contact of blood with extracorporal devices leads to the rapid adsorption of proteins from the surrounding biological fluids. The surface properties of materials determine the composition of the adsorption layer and the conformation of adsorbed proteins. Hence, the subsequent interaction of cells with biomaterials is dependent on the adsorption layer of proteins. The detailed knowledge on the role of surface properties in protein adsorption and cellular interactions is a useful means to learn about the biomedical applicability of materials and to develop novel materials with improved biocompatibility. The thesis describes the influence of polymer composition and surface properties on protein adsorption, the activation of blood clotting and adhesion of cells. The thesis presents options to modify the reactions of the biological system by the modification of bulk or surface composition of polymers. Results of these studies have been used to develop polymer membranes for biohybrid organs.

Protein adsorption at the solid/liquid interface is of wide ranging importance in many different areas of science such as biomaterial design, the fate of nanoparticles and in the food industry. As a result, many studies have been undertaken with varying foci but there still remains a lack of agreement between many working in this field and fundamental questions regarding the adsorption of proteins at the solid/liquid interface. Neutron reflectivity is a powerful technique for probing the properties of adsorbed layers at interfaces due to its high structural resolution and the possibility of using isotopic substitution to distinguish between components of a mixture. In this work, neutron reflectivity has been used as the primary technique for the investigation of proteins adsorbed sequentially or from a binary mixture. Initially, the adsorption of four proteins (carbonic anhydrase II, lysozyme, human serum albumin and maltose binding protein) onto a clean silica surface was investigated which revealed the importance of electrostatic interactions and entropic contributions to the driving forces for adsorption. Most of the adsorbed layers were described by a 2-layer model with a thinner, denser layer adjacent to the surface and a thick, diffuse layer extending into the bulk solution. The presence of impurities is also shown to have a significant impact on the adsorption of HSA. A study of the HSA/myristic acid system shows that the presence of small amphiphiles can inhibit HSA adsorption and also remove a pre-adsorbed layer. A comparison was made between the protonated and deuterated forms of two proteins, HSA & MBP, showing the deuterated proteins to have a higher affinity for the surface with adsorption occurring in a 3:1 ratio when from a 1:1 mixture. Likewise, d-MBP displaced h-MBP more readily than vice versa in an investigation into the effect of incubation time on the properties of the protein layer. The extent of desorption into protein free buffer is not affected by incubation time but the extent to which d-MBP was displaced by h-MBP showed a clear trend of decreased exchange with increasing incubation time indicating an active exchange process was occurring. This was also observed to a lesser extent for the sequential adsorption of binary protein systems, HSA & LYS and HSA & MBP. When investigating binary protein mixtures the higher propensity for deuterated proteins to adsorb is observed. LYS dominates when adsorbed from a mixture with h-HSA but from a d-HSA & LYS mix both proteins were adsorbed. The marked difference between the adsorption characteristics of perdeuterated proteins and their protonated counterparts provides a good case study for testing the neutron reflectivity technique when investigating systems with more than one component. This thesis assesses the limitations of the methodology of contrast variation for investigating mixtures as well as using different solvent contrasts. A comparison of neutron reflectivity and dual polarisation interferometry (DPI) shows that the two techniques are similar and any small differences can be attributed to the small change in surface chemistry. This comparison also highlights the advantages of DPI; high throughput of samples and detailed information but the restriction to using a 1-layer model limits its use.

At the intersection of materials chemistry and biology, biomaterials have been successfully employed in an array of medical applications. From diagnostic tools to targeted drug delivery, the modular physical and chemical properties of these materials provide numerous applications. For example, porous nanoparticles have been widely integrated as vehicles to carry chemotherapeutics to localized tumor sites. By encapsulating these cytotoxic compounds within a porous framework, the commonly associated adverse side effects of conventional chemotherapeutics, such as Doxorubicin, have been greatly reduced. One such material, mesoporous silica, has received widespread attention due to its excellent biocompatibility, high surface area to mass ratio, tunable pore diameters and volumes, and robust surface chemistry. However, recent studies have demonstrated that exposing silica nanoparticles, and other synthetic materials, to biological milieu envelops the particles in layers of proteins and biomolecules. The resulting protein coat, known as the "protein corona", has been shown to have profound effects on bioavailability, cellular targeting, and cytotoxicity. Thus, in order to develop safe and effective particle-based therapies, it is of utmost importance to establish a more thorough understanding of this process. To examine how changes in surface chemistry influence protein adsorption, monodisperse, spherical mesoporous silica nanoparticles, ca. 50 nm, were modified with a variety of surface functionalizations, -NH2, -COOH, and -PEG. Exposing these materials to biological fluid revealed drastically different protein fingerprints, suggesting a strong correlation between the surface chemistry and the identity and composition of the protein corona. Quantification of the protein corona, i.e. mg protein/mg particles, was then achieved by performing thermogravimetric analysis. These values, in concert with spectral counts obtained by shotgun proteomics, illustrates a method for quantifying individual proteins present in the corona. Spherical, silica particles of varying diameters, 70-900 nm, were then synthesized to investigate how particle diameter may affect the biomolecular identity of the protein corona. Applying the previously described methods, it was found that mesoporous particles exhibit a higher affinity for low-molecular weight proteins compared to dense silica particles of similar diameters. Finally, stochastic optical reconstruction microscopy (STORM) was used to map protein adsorption/diffusion throughout as-prepared (pore diameter ~ 30 Å) .and large pore (pore diameter > 60 Å) mesoporous silica particles. By collecting three-dimensional data on the protein-adsorbed materials, a sphere-fitting algorithm could be applied to determine the center and radius of the host particle. This calculation demonstrated that the depth by which specific proteins diffused into the porous framework was a function of both the protein's molecular weight as well as the pore diameter.

This work deals with the development of a new label-free detection technique called Pulsed Streaming Potential (PSP). Its novelty relies on the adaptation of a classical electrokinetic phenomenon (streaming potential) into a tool which can evaluate molecular interplay in label-free fashion. Implementation of PSP to microfluidic platforms allowed the label-free sensing of binding events to plastic (modified and unmodified) surfaces. It was demonstrated the use of real time PSP in plastic microfluidic platforms for determination of kinetic parameters of the interaction of proteins and plastic surfaces. Moreover, initial change of PSP after adsorption of proteins showed to be proportional to the bulk concentration of proteins and it was used for quantification of Lysozyme in the nanomolar range. Several approaches were studied to manipulate the surface of microfluidic channels in order to improve selectivity of PSP through reduction of non-specific adsorption. These approaches included the fabrication of composite surface of polyacrilic acid (PAA) and polyethylene glycol acrylate (PEGA) on cyclic olefin copolymer microchannels, as well as adsorption of nanospheres on COC-PEGA channels.

Esta tese apresenta o estudo do efeito do solvente acetato de etila e acetona no comportamento em solução dos polímeros acetato butirato celulose (CAB) e acetato butirato carboximetil celulose (CMCAB) e nas características dos seus filmes finos obtidos pela técnica de revestimento rotacional ou por adsorção. As medidas de viscosidade e espalhamento de raio-X a baixo ângulo (SAXS) mostraram que o acetato de etila é um melhor solvente para CAB e CMCAB do que a acetona. A caracterização dos filmes foi feita através de medidas de elipsometria, microscopia de força atômica (AFM), espectrocospia vibracional por geração de soma de freqüências (SFG) e medidas de ângulo de contato. Os filmes de CMCAB obtidos por revestimento rotacional são mais espessos quando preparado em acetona do que em acetato de etila. Imagens de AFM mostraram que os filmes de CMCAB oriundos de soluções em acetato de etila são mais homogêneos e lisos do que aqueles preparados a partir de acetona. Medidas de SFG comprovaram a forte afinidade da acetona com SiO2/Si, mostrando que esse solvente cria uma nova camada para os filmes de CAB e CMCAB. Os valores de energia superficial calculados para CAB e CMCAB foram semelhantes ~ (49,0 ± 0,5) mJ/m², sendo a contribuição da componente dispersiva maior que a da componente polar. A adsorção das proteínas lisozima, albumina do soro bovino (BSA), concanavalina A e jacalina foram mais pronunciadas sobre os filmes de CMCAB do que sobre CAB. Indicando que a presença do grupo carboximetil (CM) contribui significativamente no processo de adsorção das biomoléculas. O efeito da rugosidade dos filmes de CAB e CMCAB sobre o processo de adsorção das proteínas foi estudado. No caso do CMCAB, a adsorção das proteínas foi mais pronunciada sobre o filme rugoso do que sobre o filme mais liso. Entretanto, para os filmes de CAB a rugosidade não teve um efeito significativo na adsorção das proteínas / The effect of ethyl acetate and acetone on the solution behavior of cellulose acetate butyrate (CAB) and carboxymehtylcellulose acetate butyrate (CMCAB) and on the characteristics of films obtained either by spin coating or adsorption was investigated. Viscosity and small angle X-ray scattering (SAXS) measurements showed that ethyl acetate is a better solvent than acetone for CAB e CMCAB. Films were characterized by means of ellipsometry, atomic force microscopy (AFM), sum frequency generation (SFG) and contact angle measurements. Spin-coated films of CMCAB from ethyl acetate solutions were thicker than those deposited from acetone solutions. AFM images revealed that CMCAB spin coated films from ethyl acetate solutions were homogeneous and flat. However, films obtained from solutions in acetone were very rough. SFG spectra showed that acetone binds strongly to SiO2/Si wafers, creating a new surface for CAB and CMCAB films. Surface energy values determined for spin-coated CAB and CMCAB were similar ~ (49,0 ± 0,5) mJ/m² with the dispersive component larger than the polar component. The adsorption of lysozyme, bovine serum albumin (BSA), concanavalin A and jacalin was more pronounced onto CMCAB films than that onto CAB films. Indicating that carboxymethyl group favored the adsorption process. The influence of surface roughness of CAB and CMCAB on protein adsorption has been investigated. In the case of CMCAB, protein adsorption was morepronounced onto rough films than that onto flat films. However, the roughness of CAB films exerted no significant influence on proteins adsorption

<p>Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle.</p><p>Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles.</p><p><i>Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.</i></p>

Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle. Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles. Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.

Calcium phosphates are the most frequently used ceramics for bone regeneration due to their biocompatibility and favorable resorption properties. Their performance can however be improved if they are associated to growth factors. In order to control the release of growth factors, we have inted to synthesize calcium phosphates with controlled mesoporosity. This thesis represents the first work that combines mesoporous calcium phosphates with the growth factors TGF and VEGF. To obtain hydroxyapatite with controlled mesoporosity, we propose new synthesis pathways: the hydroxyapatite is synthesized inside the porosity of silica or carbon templates by infiltration of aqueous precursor solutions. The template is eliminated by chemical etching with NaOH (silica template) or by selective oxidation (carbon template). Six ceramics have been chosen for the analysis of their protein adsorption and release properties. First, the experimental protocol is defined using the model proteins BSA and Cytochrom C. Then, the growth factors TGF and VEGF have been used. By this study, we were able to determine which samples were the most efficient in terms of protein adsorption and release.