Browsing by Subject "surface plasmon resonance"
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(2020)The solubility of a poorly water-soluble drug can be improved by converting the crystalline drug into an amorphous form. However, the amorphous form is metastable due to the higher energy state and recrystallization may occur during storage and dissolution. The amorphous form can be stabilized by forming an amorphous solid dispersion (ASD), where the drug molecules are dispersed to the solid medium, e.g. hydrophilic polymer. One preparation method for amorphous solid dispersions is spray drying, where a solution containing a drug and polymer is converted into small droplets in a drying chamber, in which the solvent evaporates in a hot gas stream and solid particles are formed. The aim of this study was to investigate whether an ASD of a poorly water-soluble drug can be prepared by spray drying using 20:80 (V/V) ethanol-water mixture as a solvent in a feed solution. Indomethacin (γ-polymorph) was used as a model drug and polyvinylpyrrolidone vinyl acetate (PVPVA) as a polymer. The aim was to find a suitable formulation where the drug is in the amorphous form after spray drying and remains in the amorphous form during storage. The ratios of the drug to polymer in the spray-dried formulations were 1:4, 1:6, 1:8, 1:10, 1:12 and 1:16. The study also examined whether a change in one process parameter, pump feed rate, affects the amorphous nature and stability of the resulting spray-dried solid dispersions. Two different pump feed rates, a higher 30% and a lower 15%, were used in the study. X-ray powder diffraction (XRPD) was used to characterize the solid-state of the spray-dried formulations. XRPD measurements were performed immediately after spray drying and on selected time points during storage. Formulations 1:10 at 30% feed rate, 1:12 at both feed rates and 1:16 at 30% feed rate were amorphous after spray drying. In 1:12 (30%) and 1:16 (30%) formulations indomethacin remained in amorphous form over the study periods (22 and 56 days, respectively). In other formulations, indomethacin was found to be in crystalline α-form immediately after spray drying or recrystallization to the α-form occurred during storage. The interaction between indomethacin and PVPVA was studied by surface plasmon resonance spectroscopy (SPR). The aim of the SPR measurements was to understand the interaction between these substances in the feed solution used in spray drying. PVPVA solutions of various concentrations (1%, 0.5%, 0.1% and 0.01%) were injected to the surface of the gold sensor coated with crystalline γ-indomethacin, and the changes in the SPR signal responses were monitored during the interaction. The same measurements were also performed on a pure gold sensor without indomethacin. An interaction between indomethacin and PVPVA can be observed, and based on the measurements, a polymer layer with a thickness of about 1 nm was formed on the surface of the indomethacin sensor regardless of the concentration of the polymer solution. Thus, even a small amount of polymer in solution is sufficient to cover the indomethacin crystals. This may also occur in the feed solution during spray drying, but further studies with SPR are still needed, especially with amorphous indomethacin. This study showed that an ASD of indomethacin and PVPVA can be successfully prepared by spray drying using an aqueous feed solution. Spray-dried 1:12 and 1:16 formulations at a higher pump feed rate were found to be stable enough for further studies. If the spray-dried material is further formulated into a pharmaceutical product, indomethacin must remain in amorphous form throughout the shelf-life of the product to maintain the improved solubility.
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(2018)Nanolääkkeiden pinnalle elimistössä muodostuva biomolekyylikerros eli proteiinikorona vaikuttaa muun muassa jakautumiseen, toksisuuteen ja soluvuorovaikutuksiin. Koronan ominaisuuksien tuntemus jakautumisen eri vaiheissa on siten edellytys tehokkaampien ja turvallisempien nanolääkkeiden kehittämiselle, mutta kehitystyötä on hidastanut soveltuvien menetelmien puute. Turvallisuuden ja tehon ennakoinnin osalta on korostettu leimavapaiden in vitro -menetelmien tarvetta. Tutkielmassa kehitettiin multiparametriseen pintaplasmoniresonanssilaitteistoon ja laskennalliseen mallinnukseen perustuva menetelmä liposomien koronan tiheyden ja paksuuden määrittämiseen. Toisin kuin koronan tutkimiseen yleisesti käytetyt menetelmät, valoon perustuva kajoamaton ja leimavapaa menetelmä ei vaikuta koronan rakenteeseen. Näin voidaan tutkia myös löyhemmin sitoutuneista proteiineista muodostunutta pintakerrosta, mikä vastaa keskeisimpään kirjallisuuskatsauksessa todettuun menetelmäpuutteeseen. Menetelmää sovellettiin neljän biosensorille immobilisoidun liposomiformulaation pinnalle ihmisen seerumissa muodostuvan koronan tutkimiseen. Sen avulla oli mahdollista määrittää ensimmäistä kertaa tiiviin ja löyhän koronan tiheys ja paksuus laimentamattomassa seerumissa. Tulokset tukevat käsitystä ns. erotteluhypoteesin kuvaamasta erillisestä löyhästä proteiinikerroksesta ja avaavat uusia mahdollisuuksia sen biologisen merkityksen arviointiin. Lisäksi voitiin määrittää ensi kerran opsoniinimolekyylien sitoutumiskinetiikka liposomien pinnalle, minkä avulla voidaan arvioida nanolääkkeiden taipumusta poistua verenkierrosta ja aktivoida sisäsyntyinen immuunipuolustus. Menetelmä soveltuu siten liposomien koostumuksen ja pinta-arkkitehtuurin optimointiin prekliinisessä lääkekehitysvaiheessa.
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(2021)Nowadays, targetability studies usually require sample modifications and quite often, examination requires the use of directed light in harmful wavelengths. The surface plasmon resonance (SPR) technique does not need either of those actions. With SPR technology, the targetability of biomolecules can be studied in real-time and without any additional labels. The SPR response is received by measuring the change in surface plasmon resonance conditions due to refractive index changes caused by material interactions in the vicinity of a metal sensor surface. In the present study, the targetability of neonatal Fc receptor (FcRn) was studied by SPR. FcRn-mediated targetability studies were performed against protein A and human colorectal adenocarcinoma (Caco-2) immobilized on SPR sensors. The aim of the study was to confirm the FcRn targetability with bare Fc-fragment and Fc-fragment modified nanoparticles (NPs) designed for oral drug delivery. The NPs consisted of a core porous silicon (PSi) particle, entrapped into a lignin capsule, and finally functionalized with the FcRn-targeting ligand. Results confirmed the binding efficacy of bare Fc-fragment with protein A at pH 6.5, which was the critical pH value for preserving the lignin capsule around the PSi NPs. The cell-based SPR response was significantly higher for FcRn-targeted NPs when compared with non-functionalized NPs. According to these results, FcRn-mediated transcytosis emerges with great potential for oral drug delivery via Fc-functionalized NPs.
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(2013)There is a strong need for new in vitro methods in early drug development that predict in vivo conditions more reliably. One of the prerequisites for successful drug therapy is sufficient permeability. A drug needs to be transported through a cell membrane before it can have a pharmacological effect. Therefore, the drug-cell interactions are studied in the early stage of the drug development process. The literature review of this work covers the traditional in vitro and in silico methods of predicting the permeability of drugs across the intestinal membrane. The widely applied methods are reviewed briefly and the predictability of the methods is evaluated. Moreover, the surface plasmon resonance (SPR) technique is introduced. The principle of SPR and its applications for predicting intestinal permeability using lipid membranes resembling the intestinal membrane and for studying drug-cell interactions are discussed. The advantage of the SPR technique is that it is an optical method which allows real-time monitoring under a constant flow without labeling agents. The aim of the experimental part of this work was to evaluate the suitability of the SPR technique for cell-based studies to monitor drug-cell interactions in native cellular environments. Previously, the SPR technique has been almost merely used in routine biomolecular interaction analysis. Recently, the SPR technique has also been applied to cellbased assays but in those studies the reason for the SPR signal responses is generally poorly discussed. The objective of the experimental study was to evaluate and optimize different cell culturing approaches for living cell sensing for SPR, i.e. cells immobilized on the roof of the PDMS molded flow channel in the SPR instrument and cells immobilized directly on the SPR sensor surface. ARPE-19 cells were immobilized on the PDMS substrates but the challenge of imaging cell monolayers on PDMS molded SPR flow channels suggested that immobilizing the cells directly on the SPR sensor surface would be a more straightforward procedure. Hence, ARPE-19 and MDCKII cell culturing protocols were optimized for successful immobilization of confluent cell monolayers directly on the SPR sensor surface. However, ARPE-19 cells showed poor resistance against shear stress in the flow channel; whereas MDCKII cells showed much better resistance against shear stress in the flow channel. Therefore, only MDCKII cells immobilized on the SPR sensor surfaces were used for drug-cell interaction studies. After three days of culture MDCKII cells were exposed to test compounds in separate SPR measurements. The used test compounds were propranolol, D-mannitol, D-glucose and HSPC:Chol liposomes. During the SPR measurements, the changes in the SPR peak minimum angular position and SPR peak minimum intensity were recorded in real-time, and these were further used for analysis after the measurements. The results showed that clear differences in both SPR signals between propranolol and D-mannitol were observed when the cells were exposed to the test compounds. Propranolol diffuses effectively by the transcellular pathway into cells whereas D-mannitol uses the paracellular pathway. This indicates that the introduced SPR approach may be a potential in vitro method in order to provide real-time information on the permeability of drugs and possibly on cell uptake mechanisms of nanoparticles for a better mechanistic understanding of drug-cell interactions on a cellular level.
Now showing items 1-4 of 4