Browsing by Subject "microfluidics"
Now showing items 1-6 of 6
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(2017)Cytochrome P450 (CYP) enzymes are important catalysers in the first phase of drug metabolism. Roughly two thirds of drugs are oxidized via CYP enzymes, which enable the further modification of drugs, and their excretion. In this thesis, human liver microsomes containing the main hepatic CYP enzymes were immobilized on thiol-ene based micropillar arrays and their stability was evaluated using a CYP2C9 isoenzyme specific luminescent substrate, Luciferin-H. The aim of the study was to develop microfluidic immobilized enzyme reactors (IMERs) for studying enzyme kinetics and drug-drug interactions. For this purpose, the instability issues associated with previously reported CYP-IMERs were carefully addressed. The CYP immobilization protocol used was based on a protocol previously developed in the context of other research projects and relied on biotinylation of human liver microsomes (HLM) with help of fusogenic liposomes. The biotinylated HLMs were then attached to the streptavidin-modified thiol-ene surfaces. The CYP activity was determined by utilizing microfluidics under continuous flow conditions (typically 5 μL/min) in the presence of NADPH. The luminescent metabolite formed by the CYP2C9 enzymes was quantified with a commercial well-plate reader from fractions collected at the microreactor outlet. Half-life was used to compare the differences between enzyme stabilities reached via different immobilization conditions. The effects of flow rate and reaction temperature on the stability of the CYP-IMERs was evaluated together with addition of antioxidative agents and reactive oxygen species (ROS) scavengers. Different functionalization steps as well as storage time and conditions were studied. With Luciferin-H as the model substrate of CYP2C9, the CYP-IMERs showed higher activity and stability at room temperature than at +37 °C. The peak activity could be increased via optimization of the immobilization protocol, though long-term storage diminished the peak activity. The activity of the IMERs typically attenuated within 1-2 hours with little or no improvement achieved via optimization of the immobilization or operation conditions. Only upon addition of the ROS scavengers, the peak activity and stability of the CYP-IMERs could be slightly improved. After functionalization, the IMERs maintained their activity until the time of use when stored in +4 °C for up to 2 weeks, but re-use of IMERs was not possible.
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(2013)Polymer microspheres hold great potential as oral drug delivery system for therapeutic proteins. Microspheres prepared with biocompatible and biodegredable polymers have been extensively studied, since the oral delivery of therapeutic proteins is challenging due to the conditions in the GI-tract. The aims of this research were to apply microfluidics on polymeric microsphere preparation process, to determine what kind of formulations are suitable for this technology, to establish a controlled preparation process that produces advanced particles and to create a template for oral protein drug delivery. With microfluidic fabrication it is possible to gain control over the process and content of each droplet. However, finding suitable formulations for microfluidics is demanding. In this study, biphasic flow was employed to successfully produce double (W/O/W) emulsion droplets with ultra thin shells. Once the process and formulation variables were optimized constant droplet production was achieved. Flow rates used were 500 µl/h in the inner and in the middle phase and 2500 µl/h in the outer phase, respectively. Two formulations were selected for further characterization: 5 % poly(vinyl alcohol) in water in the outer phase, 3 % polycaprolactone in ethyl acetate in the middle phase and either 10 % or 20 % poly(vinyl alcohol) and polyethylenglycol (1:4) in water in the inner phase. All the particles were found to be intact and contain the inner phase, as verified by confocal microscopy. Further, the particles were monodisperse and non-porous, as observed by scanning electron microscopy. Particle size was found to be around 20-40 µm, variation in the particle size within one batch was small and the particles were stable up to 4 weeks. The encapsulation efficiency of the particles was remarkable; as high as 85 % loading of the model compound, bovine serum albumin. Particles released 30 % of their content within 48 hours. In conlusion, developing functional formulations for micfoluidic technology was possible, the microparticles encapsulated the model protein extremely well and all in all microfluidic technology had a lot of potential for droplet manufacturing for pharmaceutical applications.
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(2020)Throughout the history, there has been a wide selection of drugs developed for therapy of cardiovascular diseases (CVD). Despite a broad spectrum of different therapeutic strategies to deaccelerate and try to reverse the progression of cardiovascular diseases has been achieved, only a modest amelioration of the health of the CVD patients was achieved, as the mortality remains high by being the cause of nearly one in every three deaths yearly, myocardial infarction being involved in majority of these cases. Novel solutions are being studied to overcome this problem, one of them being nanoparticles, which may provide potential solution by carrying drugs to the desired location. Microfluidics technique may further improve the properties of nanoparticles, being a platform that allows the production of homogenous and repeatable batches that are non-dependent by the operator using it. In this thesis, it is described how microfluidics-based preparation of spermine-functionalised acetalated dextran nanoparticles co-loaded with a trisubstituted isoxazole and curcumin perform in physicochemical and in vitro experiments, in order to evaluate their potential in the application of ischemic myocardial injury therapy.
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Mikrofluidististen paperipohjaisten analyysilaitteistojen kehittäminen farmaseuttiseen analytiikkaan (2012)The aim of this research was to evaluate the use of microfluidic paper-based devices (µPAD) in drug analysis. Micro total analysis systems (µTAS) channels are in the range of a few micrometers and are capable of performing all steps of a chemical analysis. The advantages of miniaturization are lower sample consumption and faster analysis time. µTASs are usually fabricated of glass, silicon or polymers and their fabrication requires cleanroom facilities and specific equipment. Paper offers an inexpensive and versatile substrate for µTASs. Paper wicks liquids and no external pumps are required. µPADs advantages over µTAS are its ease of use and inexpensive and simple fabrication. µPADs are fabricated by patterning hydrophobic barriers in hydrophilic paper. There are several fabrication methods for µPADs such as photolithography, cutting and methods based on the application of wax (etching, wax printing, wax dipping). In this research wax printing was selected as the fabrication method because it's simple, rapid and inexpensive. Wax was printed using Xerox Phaser 8560DN solid ink printer. After printing the wax was melted through the paper by heating the paper at 150 °C for 120 seconds on a hotplate. Thus the wax creates a hydrophobic barrier on the hydrophilic paper which channels the liquids flow. Owing to papers anisotropic nature the wax also spreads horizontally in the paper when heated, thus reducing the wax patterns resolution and making the pattern coarse. Wax printing is an inexpensive and simple fabrication method suitable for fabricating µPADs. Also liquids flow velocity and methods for controlling the flow rate were studied. By knowing the flow velocity, one can assure that the analytes and reagents reach the reaction site. Controlling the flow velocity enables the use of multiphase reactions or the use of multiple simultaneous reactions on the µPAD. The liquid flow velocity can be controlled by changing the hydrophilic channels width, reducing the average pore size by melting a layer of wax inside the hydrophilic channel or by changing the surface tension or viscosity of the liquid used. Colorimetric assays are the most commonly used detection methods in µPADs, but also electrochemical sensing and detection methods based on fluorescence are used. In this study direct and indirect fluorescence detection methods were studied. In the detection method based on direct fluorescence, fluorescein and coumarine derivates were studied. In indirect fluorescence amino acids fluorescamine conjugates, which were created in the paper, were studied. Level of the analytes detected in direct fluorescence detection was 10-13 mol in the range of visible light and 10-12 mol in the range of UV-light. Level of the amino acids fluorescamine conjugates detected in indirect fluorescence detection was 10-9 mol. According to our results the fluorescence based detection methods used in this study are suitable for drug analysis on µPADs.
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(2015)The properties of liquid and gas flows in microscale systems differ from those in macroscale; microfluidics is a field of science in which these properties are investigated and utilized for the development of microscale systems. Acoustofluidics is a branch of microfluidics focusing on the movement (acoustophoresis) or localization (acoustic trapping) of particles in microchannels using ultrasound. In this work, the suitability of a new miniaturized method for the screening of cell-drug interactions was investigated. In the method, the cells were acoustically trapped within a glass capillary, enabling liquid movement (generated with a syringe pump) in the capillary while the trapped cell cluster remains stationary. In this manner, the trapping of cells, their incubation with a drug solution, rinsing, and the elution could be done using the same capillary. The sample preparation was done using a miniaturized solid phase extraction technique (integrated selective enrichment target, ISET), and the analysis was done with matrix assisted laser desorption/ionization mass spectrometry (MALDI MS). The drug compounds investigated were selective serotonin reuptake inhibitors (SSRI). The research was conducted in five phases. In the first phase, a suitable solid phase extraction method for the drug compounds was investigated. In the second phase, the performance of the acoustic trap was investigated by acoustically trapping polystyrene beads and Coulter counting them. In the third phase, the method was modelled by conducting drug binding studies using cation exchange beads instead of cells. In the fourth phase, the drug binding studies were conducted by investigating the binding of drug compounds to human platelets and yeast cells. Platelets were chosen due to the expression of serotonin transporter, the molecular target of SSRI drugs, on their cell membranes. Also a cell membrane preparation containing serotonin transporter was used for the binding studies. In addition, memory effects occurring in the method were investigated. In the fifth phase, comparative drug binding studies without acoustic trapping were conducted. The suitability of the method for the screening of cell-drug interactions could not be thoroughly substantiated, but further research and method development are required. The reason for this was the inadequate sensitivity of the method, because of which large drug concentrations had to be used. This lead to the increased occurrence of memory effects.
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(2023)Inhibition of the cytochrome P450 enzymes is one of the most significant factors causing drug-drug-interactions, and thus one of the most important objects of study at preclinical drug development. CYP-inhibition can be either reversible or irreversible. Although different inhibition mechanisms are well known, their evaluation in vitro is still challenging. Thus, the development of more accurate and efficient in vitro methods is important and as a continuous target of interest. Immobilized enzyme microreactors (IMER) have presumably several advantages over traditional in vitro methods and have been presented as a promising tool for drug metabolism studies in vitro. The purpose of this work was to evaluate the suitability of a novel flow-through based immobilized enzyme microreactor in determining the CYP enzyme kinetic parameters. The developed immobilization protocol is based on attaching biotinylated human liver microsomes to a thiolene-based microreactor coated with Streptavidin. To validate the developed method, the activity of the CYP2C9 enzyme was assessed using the recommended model reaction by authorities, that is 4-hydroxylation of diclofenac. The enzyme kinetic parameters i.e., enzyme affinity (Km) and activity (Vmax), determined with the developed IMER were comparable to the values previously published in the literature and determined in static in vitro conditions. In addition, the inhibition of CYP2C9 enzyme by four model inhibitors (fluconazole, nicardipine, sulfaphenazole and miconazole), was examined by determining the IC50 (half-maximal inhibitory constant) values for each compound and by monitoring the reversibility of the CYP2C9 enzyme for 90 minutes after the inhibitor was removed from the feed solution. The IC50 values determined with the developed method for all inhibitors were well in line with previous publications, showing fluconazole (IC50 22 µM) to be the weakest inhibitor of CYP2C9 enzyme and the other examined inhibitors caused more potent inhibition (IC50 for sulfaphenazole 1.3 µM; IC50 for miconazole 1.3 µM; IC50 for nicardipine 0.67-1.1 µM). The reversibility of the CYP2C9 enzyme was examined by removing the inhibitor from the feed solution and monitoring the recovery of the enzyme activity via diclofenac 4-hydroxylation. Based on the results obtained with developed IMER, the inhibition of fluconazole and sulfaphenazole was reversible and thus well in line with previous studies. In contrast, on account of data obtained with IMER, inhibition by miconazole and nicardipine was not reversible, although these compounds have previously been reported to be reversible CYP2C9 inhibitors in vitro, which may be due to the strong aggregation tendency of these compounds. The study shows that the developed flow-through based IMER is well suited for studying inhibition of CYP enzymes However, to utilize the developed technology in CYP enzyme inhibition research, it’s applicability in determining enzyme inhibition should still be evaluated with more comprehensively with several CYP isoenzymes.
Now showing items 1-6 of 6