Biomimetic Interfaces for Surface Sensitive Drug Discovery Techniques

For the last few decades, the expences of pharmaceutical development and drug discovery have been constantly increasing whereas the amount of new pharmaceutical products reaching the market has been diminishing. The drug discovery methods today rely heavily on different screening technologies in the early discovery phase. High-throughput screening is usually the dominant approach along with different computational methods, but these methods lack the ability to monitor the interactions between drugs and cells in real-time. The ability to measure drug-cell interactions and cell responses during drug stimulation in real-time could provide complementary kinetic information to traditional methods already used in drug discovery. This time-resolved information should help to build a better mechanistic understanding of the effect of drug formulation design on the drug release actions, the drug delivery process and the efficacy of the drug, especially when it comes to new biological drugs and nanoparticle formulations. This dissertation addresses challenges in developing functional surfaces and analysis methods based on the surface plasmon resonance technique for pharmaceutical research purposes. The research in this thesis spans from traditional drug-protein interaction studies and preparation of cell model surfaces to interaction studies with living cells. An approach where proteins were immobilized in a hydrogel was used for studying the interaction kinetics between protein kinase C ε and both an activating and an inhibiting single-chain antibody. The affinities determined for the interactions were able to predict the level of activation or inhibition in subsequent cell culture assays. This thesis also presents two types of new analysis methods, i.e. label-enhanced and multi-wavelength surface plasmon resonance (SPR) methods were developed in order to improve the sensitivity of bioassays and accuracy for characterizing ultra-thin films, respectively. The label-enhanced SPR method was shown to improve assay sensitivity up to 100-fold, whereas the multi-wavelength SPR analysis provided the means to characterize organic layers in the range from a few nanometers to hundreds of nanometers, i.e. layer thicknesses of relevance to biological membranes and hydrogels. New surface coating chemistries based on dextran and thiol-PEG were also developed in this thesis in order to enable the preparation of robust biomimetic membranes by vesicle spreading or adsorption. The dextran-based and PEG-based coatings promoted supported lipid bilayer and adsorbed vesicle layer formation, respectively. The new analysis approaches developed in this thesis were further utilized in order to characterize the optical properties of the formed lipid layers on the dextran- and PEG-based coatings. Finally, a new analytical approach for signal processing of the real-time and label-free SPR measurements performed together with living cells is introduced which provides the mean to differentiate between para- and transcellular cell absorption routes of drug molecules. This dissertation contributes to the pharmaceutical research field by introducing new measuring tools, improved in vitro biomimetic models and new approaches for processing of the signal from label-free measurements in order to provide relevant real-time and complementary information to traditional drug development and discovery tools. This will hopefully benefit the pharmaceutical research field and possibly enable a more efficient development of new pharmaceuticals and therapies in the future.Uusien lääkkeiden kehitys on yleisesti ottaen hidasta, ja lääkekehityksen vaatima rahallinen panostus on viime aikoina vain kasvanut entisestään. Tämän lisäksi myös uusien markkinoille saapuvien lääkevalmisteiden määrä on vuosia jatkuvasti laskenut. Vaikka lääkekehitysmenetelmät ovat kehittyneet viime vuosina merkittävästi varsinkin laskennallisten menetelmien osalta, esikliinisen vaiheen tutkimusten ennustavuus lääkevalmisteen käyttäytymisestä elävässä eläimessä tai ihmisessä ei ole vielä toivottavalla tasolla. Paremmilla aikaisen vaiheen tutkimusmenetelmillä voitaisiin nopeuttaa ja vähentää lääkekehityksen kustannuksia, ja mahdollisesti myös kehittää tehokkaampia ja turvallisempia lääkeaineita. Tämän väitöstutkimuksen tärkeimpänä tavoitteena on kehittää uusia tutkimusmenetelmiä perustuen biologiaa matkiviin pinnoitteisiin ja näiden käyttämiseen lääkeaineiden fysikaalisten ja kemiallisten ominaisuuksien määrittämiseen. Väitöksessä käytetyt tutkimusmenetelmät perustuvat mittausmenetelmiin, joissa ei käytetä leimaus- tai merkkiaineita (ns. leimavapaa menetelmä) ja joiden avulla voidaan mitata nanometri kokoluokan pintarakenteita ja vuorovaikutuksia erilaisten aineiden välillä. Väitöstutkimuksen kuluessa on kehitetty mittateknologiaan uusia laskentatyökaluja, mittausten herkkyyttä parantavia fysikaalis-kemiallisia menetelmiä, elävien solujen käyttöä mittauksissa sekä kemiallisia menetelmiä muokata pintoja soveltuvaksi mittauksiin. Erityisesti elävien solujen pintarakennetta matkivien pinnoitteiden kehitys on ollut tärkeässä roolissa väitöstutkimuksessa. Väitöstutkimuksessa kehitettyjen menetelmillä voidaan tulevaisuudessa täydentää lääkekehityksessä käytettyjen menetelmien työkalupakkia . Tutkimuksen menetelmät voivat siis tulevaisuudessa auttaa kehittämään tehokkaampia lääkehoitoja, nykyistä nopeammin ja nykyistä pienemmillä kustannuksilla

Label-Enhanced Surface Plasmon Resonance: A New Concept for Improved Performance in Optical Biosensor Analysis

Peer reviewe

Elucidating the Signal Responses of Multi-Parametric Surface Plasmon Resonance Living Cell Sensing: A Comparison between Optical Modeling and Drug-MDCKII Cell Interaction Measurements

Peer reviewe

Spin coated chitin films for biosensors and its analysis are dependent on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.Peer reviewe

Kinetics of PKC epsilon Activating and Inhibiting Llama Single Chain Antibodies and Their Effect on PKC epsilon Translocation in HeLa Cells

Peer reviewe

Challenges in land use and transport planning integration in Helsinki metropolitan region—a historical-institutional perspective

Funding Information: The unevenness of resources in metropolitan transport and land use planning also follows from the above differences in their institutionalization. The transport system planning by the HSL is funded by its member municipalities and the state for its institutionally designated duties, while there are no extra funds reserved for metropolitan land use planning. While the HSYK is a light cooperative organ, there is no actual land use planning agency at the metropolitan level. Instead, the municipal land use planners assign the time that is left to metropolitan level land use planning concerns after having to handle their municipal planning duties, institutionally assigned to them. What follows is that the actions of the municipal land use planners are much more closely observed by the municipal decision makers, and the conflict between the metropolitan and municipal land use interests is evident in their everyday planning work. While a metropolitan body to take charge of land use planning at that level has been missing, a logical consequence was that HSL, with its organizational prowess and resources, assumed the coordinating role in the MAL 2019 planning process. This unavoidably also led to an integrated land use and transport planning context where the transport planning approach is more influential and decisive in the MAL 2019 planning process. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Land use and transport integration has been considered a must-have approach in achieving sustainable urban development. However, successful applications of the concept have been few, as institutional reforms to support land use and transport integration have lagged behind. Accordingly, this article argues that understanding difficulties in land use and transport integration requires an analysis of the long-term evolution of formal and informal institutional frameworks in planning practices. For this purpose, this article presents a case study of land use and transport planning in Finland’s Helsinki Metropolitan Region, which combines interview research on planners’ perceptions with a document analysis of the historical trajectories of the region’s plans, policy documents and related institutional and organizational changes. The historical-institutional approach of the article draws on discursive institutionalism as a novel analytical approach for studying how land use and transport integration is institutionally conditioned.Peer reviewe

Supplement 1: Surface plasmon resonance for characterization of large-area atomic-layer graphene film

Supplemental-document Originally published in Optica on 20 February 2016 (optica-3-2-151

Effects of charge ratios of xylan-poly(allylamine hydrochloride) complexes on their adsorption onto different surfaces

The adsorption behavior of polyelectrolyte complexes (PECs) of poly(allylamine hydrochloride) (PAH) and 4-O-methylglucuronoxylan (Xyl) onto silica surface, cellulose nanofibrils (CNFs) model surfaces, and unbleached softwood kraft fibers was studied. Different charge ratios and ionic strengths of the liquid medium (q−/q+: 0.3, 0.5 and 0.8 in 1 mM NaCl, and q−/q+: 0.2, 0.3 and 0.5 in 10 mM NaCl), at pH 7.5 were considered. First, the complexes obtained were characterized by measuring the charge density, the particle size and the zeta potential. Then, the adsorption of PAH alone and PECs on silica and CNF model surfaces were studied by quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR). The QCM-D test indicated that: (a) adsorbed cationic layers of PECs were soft in 10 mM NaCl, (b) higher adsorption was observed at higher ionic strength and (c) the highest adsorption of complex and coupled water was found at a charge ratio of 0.3. The SPR results analyzed together with the corresponding QCM-D results revealed that a significant portion of the adsorbed layers corresponded to coupled water on the cationic PEC structures. Morphology and structures of the adsorbed PEC layers studied using atomic force microscopy showed that the majority of the PECs were spherical, while some bigger aggregates were also found. Finally, the retention of different PECs on an unbleached softwood pulp was determined and the maximum retention was obtained when the PEC charge ratio was 0.5 in a 10 mM NaCl solution.Fil: Galván, María Verónica. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología Celulósica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Peresin, María Soledad. No especifíca;Fil: Mocchiutti, Paulina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología Celulósica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Granqvist, Niko. No especifíca;Fil: Zanuttini, Miguel Angel Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología Celulósica; ArgentinaFil: Tammelin, Tekla. No especifíca

Effects of charge ratios of xylan-poly(allylamine hydrochloride) complexes on their adsorption onto different surfaces

The adsorption behavior of polyelectrolyte complexes (PECs) of poly(allylamine hydrochloride) (PAH) and 4-O-methylglucuronoxylan (Xyl) onto silica surface, cellulose nanofibrils (CNFs) model surfaces, and unbleached softwood kraft fibers was studied. Different charge ratios and ionic strengths of the liquid medium (q - /q +: 0.3, 0.5 and 0.8 in 1 mM NaCl, and q - /q +: 0.2, 0.3 and 0.5 in 10 mM NaCl), at pH 7.5 were considered. First, the complexes obtained were characterized by measuring the charge density, the particle size and the zeta potential. Then, the adsorption of PAH alone and PECs on silica and CNF model surfaces were studied by quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR). The QCM-D test indicated that: (a) adsorbed cationic layers of PECs were soft in 10 mM NaCl, (b) higher adsorption was observed at higher ionic strength and (c) the highest adsorption of complex and coupled water was found at a charge ratio of 0.3. The SPR results analyzed together with the corresponding QCM-D results revealed that a significant portion of the adsorbed layers corresponded to coupled water on the cationic PEC structures. Morphology and structures of the adsorbed PEC layers studied using atomic force microscopy showed that the majority of the PECs were spherical, while some bigger aggregates were also found. Finally, the retention of different PECs on an unbleached softwood pulp was determined and the maximum retention was obtained when the PEC charge ratio was 0.5 in a 10 mM NaCl solution

Kretschmann configuration and key parameters obtained from the full SPR angular spectra.

<p>A) A simplified chart of the Kretschmann configuration enabling plasmon excitations and SPR measurements. The intensity of the reflected light from a monochromatic light source is measured as a function of incident light angle (θ). The light passes from a high refractive index medium (glass, ε₀) to a low refractive index medium (air or liquid, ε₁+ε_bulk). In between, the light is reflected from an interface containing a metal with a high density of free electrons and an optimal thickness for plasmon excitation (gold 50 nm, ε₂) to a photodetector. The surface plasmons on the metal surface are excited at a certain incident light angle (θ) and the evanescent field created by the plasmon extends to the adjacent low refractive index medium (ε₁) where samples are introduced to the system. B) A schematic full SPR angular spectrum showing the positions of the TIR region, the main SPR peak angular position and the main SPR peak minimum intensity.</p