Digital microfluidics for drug metabolism research in droplet-scale

Drug metabolism is a detoxification process by which the body converts pharmaceuticals into more hydrophilic metabolites. Understanding of the drug metabolism process and metabolic profiling plays a vital role in drug development processes by ensuring the safety and efficacy of treatments. Cytochromes P450 (CYP) are a superfamily of enzymes that are primarily responsible for metabolizing the majority of clinically relevant drugs. In preclinical drug development research, there is a constant need for the identification of metabolites and their CYP isoenzyme-specific elimination route, as well as possible drug-drug interactions thereof using high speed in vitro techniques. Miniaturization of the drug metabolism assays and related processes could further improve the throughput via parallelism and integration of several analytical steps on a single platform, as well as reducing the consumption of expensive reagents substantially. Micro total analysis systems (µTAS) usually refer to microfabricated devices that integrate several analytical unit operations, such as sample preparation, extraction, separation, and analysis on a single platform. These µTAS platforms can be either continuous-flow microchannel based systems or discrete droplet systems. Digital microfluidics (DMF) is one such technology, where sample droplets are manipulated individually on an array of electrodes. In DMF, the droplets of hundreds of nanolitres to a few microliters of volume can be dispensed, split, mixed, and merged independently via programmed and automated voltage application. In this thesis, several DMF-based bioanalytical concepts were developed and their feasibility for implementing droplet-scale drug metabolism assays was evaluated. In the first sub-project, droplet-scale immobilized enzyme reactors were developed by immobilizing CYP enzymes on porous polymer monoliths affixed onto a DMF platform. Assay incubation at physiological temperature was facilitated by localized heating of the DMF platform using integrated inkjet-printed microheaters. For the on-chip detection of drug metabolites, a protocol facilitating interfacing of the DMF device with a commercial wellplate reader was developed. In the second sub-project, the developed DMF platform, featuring the CYP reactors, were interfaced with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). For in situ identification of the drug metabolites by DAPPI-MS, the chip design was optimized to be able to control the critical surface sensitive processes, such as sample precipitation and subsequent desorption/ionization directly from DMF surfaces. In addition, the feasibility of the same platform for a droplet-based liquid-liquid extraction of pharmaceuticals was demonstrated. All pharmaceuticals and metabolites analyzed could be detected with lower limits of detection in the range of a few picomoles. In the third sub-project, DMF droplet manipulation was interfaced with channel microfluidics to facilitate more versatile sample preparation such as separation of target analytes after the droplet-based enzyme reactions and prior to detection. To support the scaling up of the developed technology toward mass manufacturing, the entire device was assembled using low-cost inkjet printing and non-cleanroom polymer processing techniques. To achieve this interfacing, off-stoichiometric thiol-ene (OSTE) polymers were introduced as a new alternative dielectric material for the coating of inkjet-printed DMF electrode arrays, as well as for the integration of the microchannels with a DMF platform. In the fourth sub-project, magnetic bead based enzyme immobilization protocol was developed to facilitate screening the individual variation of CYP activities in donor-derived human liver microsomes (HLM) in droplet-scale. A CYP1A isoenzyme-specific model reaction was chosen to assess the inter-individual variation in the activities of this metabolic route in the liver microsomes collected from five individuals. The demonstrated protocol was shown to be technically feasible for biopsy-scale samples. In all, the new droplet-scale concepts developed in this thesis are first-in-their-kind examples of droplet-scale drug metabolism assays on DMF platform. The methods developed are generally qualitative or semi-quantitative and thus, in their present form, best feasible for the preliminary determination of metabolic clearance via CYP or identification of the produced metabolites of new drug candidates in vitro. Further development of the technology, particularly the enzyme immobilization process and the quantification of the produced metabolites, is needed to improve the wider applicability of the assays. It is noteworthy however that all of the fabrication processes and interfacing approaches taken in this thesis were carried out in regular, non-cleanroom laboratory conditions, which is foreseen to significantly improve the adaptability of the technology in any bioanalytical laboratories.Lääkeainemetabolia on elimistön suojamekanismi, joka muuntaa yleensä hyvin rasvaliukoiset lääkeaineet entsymaattisesti vesiliukoisemmiksi metaboliiteiksi. Lääkeainemetabolialla on siksi keskeinen rooli lääkkeenkehityksessä lääkehoidon tehon ja turvallisuuden varmistamiseksi. Valtaosa kliinisessä käytössä olevista lääkeaineista metaboloituu sytokromi P450 (CYP) –entsyymien kautta. Yksi prekliinisen lääkekehityksen tärkeimpiä tehtäviä on tunnistaa lääkeaineiden metaboliitit ja niiden CYP-isoentsyymikohtaiset eliminaatioreitit sekä ennakoida mahdollisia lääkkeiden haitallisia yhteisvaikutuksia nopeiden in vitro –tekniikoiden avulla. Lääkeainemetaboliatutkimuksessa käytettävien menetelmien miniatyrisointi mahdollistaa paralleelien ja toisiinsa integroitujen analyysiyksiköiden valmistamisen, mikä tehostaa lääkkeiden seulontaa sekä vähentäisi kalliiden reagenssien kulutusta. TAS-konsepti (engl. Micro total analysis systems) viittaa mikrovalmistusmenetelmillä tuotettuihin analyysilaitteisiin, joissa samalle alustalle on integroitu useita analyyttisiä yksikköoperaatioita kuten näytteenkäsittely, uutto, erotus ja havainnointi. µTAS-laitteet voivat olla esimerkiksi mikrokanavia sisältäviä jatkuvan virtauksen laitteita tai yksittäisten pisaroiden liikuttelun mahdollistavia laitteita. Digitaalimikrofluidistiikka (engl. digital microfluidics, DMF) on eräs tekniikka, joka mahdollistaa pisaroiden kontrolloidun käsittelyn sähköelektrodien päällä. DMF:ssa pisarat ovat tilavuudeltaan satoja nanolitroja – muutama mikrolitra ja niitä voidaan syöttää, jakaa, sekoittaa ja yhdistää yksitellen käyttäen ohjelmoitavaa ja automaattista jännitteen syöttöä. Tässä väitöskirjassa kehitettiin useita DMF:aan perustuvia bioanalyyttisia sovelluksia tavoitteena selvittää DMF-teknologian soveltuvuutta lääkeainemetaboliatutkimukseen pisaramittakaavassa. Ensimmäisessä osajulkaisussa kehitettiin pisarakokoluokan entsyymireaktori kiinnittämällä CYP-entsyymejä huokoiseen polymeerimonoliittiin, joka oli kiinnitetty DMF-alustaan. Entsyymireaktorin lämmittämiseksi fysiologiseen lämpötilaan kehitettiin mustesuihkutulostettu lämmitinelementti, joka kiinnitettiin DMF-alustaan. Metaboliittien havainnoimiseksi kehitettiin menetelmä, joka mahdollisti DMF-mikrosirun yhdistämisen kaupalliseen kuoppalevylukijaan. Toisessa osajulkaisussa DMF-pohjaisia CYP-entsyymireaktorit liitettiin massaspektrometriaan hyödyntämällä desorptio/fotoionisaatiota ilmanpaineessa (engl. desoprtion atmospheric pressure photoionization, DAPPI). Jotta lääkeainemetaboliitit voitiin tunnistaa massaspektrometrisesti suoraan DMF-alustalta, mikrosiru optimoitiin siten, että näytteen haihtumista ja desoprtio/ionisaatio-prosessia pystyttiin kontrolloimaan pinnan ominaisuuksien avulla. Kehitetyn DMF-alustan soveltuvuus osoitettiin myös lääkeaineiden pisarapohjaisessa neste-neste-uutossa. Kaikkien lääkeaineiden ja metaboliittien havaintoalarajat olivat pikomolaarisella tasolla. Kolmannessa osajulkaisussa pisaroiden käsittelyyn kehitetty DMF-alusta yhdistettiin mikrokanavarakenteisiin, mikä mahdollistaa monipuolisemman näytteenkäsittelyn kuten reaktiotuotteiden erotuksen ennen havainnointia. Tässä osajulkaisussa käytettiin edullisia, massatuotantoon skaalautuvia valmistusmenetelmiä, kuten mustesuihkutulostusta ja polymeeriprosessointia. DMF-alustan ja mikrokanavien yhdistämiseksi osajulkaisussa tutkittiin uuden, epästoikiometrisen tioleenipolymeerin soveltuvuutta DMF-teknologiaan sekä dielektrisenä kerroksena että mikrokanavien valmistusmateriaalina. Neljännessä osajulkaisussa kehitettiin magneettipartikkelipohjainen entsyymi-immobilisointimenetelmä, joka mahdollisti luovuttajakohtaisten maksamikrosomien käytön pisarakokoluokan CYP-aktiivisuusmäärityksissä. DMF-alustan soveltuvuutta yksilöllisten erojen selvittämiseen tutkittiin CYP1A-entsyymille spesifisen mallireaktion avulla viiden eri luovuttajan maksamikrosomeilla. Menetelmän todettiin teknisten ominaisuuksiensa puolesta soveltuvan CYP-aktiivisuuden määrittämiseen jopa biopsianäytteistä. Kokonaisuudessaan tässä väitöskirjassa kehitetyt pisarapohjaiset, bioanalyyttiset menetelmät ovat ensimmäisiä DMF-teknologian sovelluksia lääkeainemetaboliatutkimuksessa. Kaikki kehitetyt menetelmät ovat joko kvalitatiivisia tai semikvantatiivisia ja soveltuvat siten nykyisessä muodossaan lähinnä alustaviin lääkeaineen metaboliareittiä selvittäviin tutkimuksiin tai uusien lääkeainekandidaattien metaboliittien nopeaan tunnistamiseen in vitro. Tekniikan jatkokehitys, erityisesti entsyymien immobilisoinnin sekä metaboliittien kvantitoinnin osalta, on kuitenkin tarpeellista menetelmien laajemman käytettävyyden parantamiseksi. Huomionarvoista kuitenkin on, että kaikki tässä työssä käytetyt valmistusmenetelmät soveltuvat normaaleihin laboratorio-olosuhteisiin, myös puhdastilojen ulkopuolelle, mikä mahdollistaa kehitettyjen tekniikoiden soveltamisen missä tahansa bioanalytiikan laboratoriossa

Drug-loaded supramolecular gels prepared in a microfluidic platform: distinctive rheology and delivery through controlled far-from-equilibrium mixing

It is shown here that controlled mixing of a gelator, drug, solvent, and antisolvent in a microfluidic channel leads to faster setting gels and more robust materials with longer release profiles than the physical gels of the same composition obtained using random mixing in solution. The system is similar to a related gelator system we had studied previously, but we were unable to apply the same gelling procedure because of the instability of the colloid caused by the small structural modification (length of the alkyl chain in the bis-imidazolium head group). This situation holds true for the gels formed with varying compositions and under different conditions (gelator/drug ratio, solvent proportion, and flow rates), with the most significant differences being the improved gel rheology and slower drug release rates. Very importantly, the gels (based on a previously unexplored system) have a higher water content ratio (water/EtOH 4:1) than others in the family, making their medicinal application more attractive. The gels were characterized by a variety of microscopy techniques, X-ray diffraction and infrared spectroscopy, and rheology. Salts of the antiinflammatory drugs ibuprofen and indomethacin were successfully incorporated into the gels. The diffraction experiments indicate that these composite gels with relatively short alkyl chains in the gelator component contrast to previous systems, in that they exhibit structural order and the presence of crystalline areas of the drug molecule implying partial phase separation (even though these drug crystallites are not discernible by microscopy). Furthermore, the release study with the gel incorporating ibuprofenate showed promising results that indicate a possible drug delivery vehicle application for this and related systems

Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 mu g microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.Peer reviewe

Interfacing Digital Microfluidics with Ambient Mass Spectrometry Using SU-8 as Dielectric Layer

This work describes the interfacing of electrowetting-on-dielectric based digital microfluidic (DMF) sample preparation devices with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). The DMF droplet manipulation technique was adopted to facilitate drug distribution and metabolism assays in droplet scale, while ambient mass spectrometry (MS) was exploited for the analysis of dried samples directly on the surface of the DMF device. Although ambient MS is well-established for bio- and forensic analyses directly on surfaces, its interfacing with DMF is scarce and requires careful optimization of the surface-sensitive processes, such as sample precipitation and the subsequent desorption/ionization. These technical challenges were addressed and resolved in this study by making use of the high mechanical, thermal, and chemical stability of SU-8. In our assay design, SU-8 served as the dielectric layer for DMF as well as the substrate material for DAPPI-MS. The feasibility of SU-8 based DMF devices for DAPPI-MS was demonstrated in the analysis of selected pharmaceuticals following on-chip liquid-liquid extraction or an enzymatic dealkylation reaction. The lower limits of detection were in the range of 1-10 pmol per droplet (0.25-1.0 mu g/mL) for all pharmaceuticals tested.Peer reviewe

Digital microfluidic immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research

We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (n = 10) turnover rate was 20.3 +/- 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.Peer reviewe

A Digital-to-Channel Microfluidic Interface via Inkjet Printing of Silver and UV Curing of Thiol-Enes

Microfluidic sample manipulation is a key enabler in modern chemical biology research. Both discrete droplet-based digital microfluidic (DMF) assays and continuous flow in-channel assays are well established, each featuring unique advantages from the viewpoint of automation and parallelization. However, there are marked differences in the applicable microfabrication materials and methods, which limit the interfacing of DMF sample preparation with in-channel separation systems, such as the gold standard microchip electrophoresis. Simultaneously, there is an increasing demand for low-cost and user-friendly manufacturing techniques to foster the adaptation of microfluidic technology in routine laboratory analyses. This work demonstrates integration of DMF with in-channel separation systems using only low-cost and accessible (non-cleanroom) manufacturing techniques, i.e., inkjet printing of silver for patterning of the driving electrodes and UV curing of off-stoichiometric thiol-ene (OSTE) polymers both for dielectric coating of the electrode arrays and replica molding of the microchannel network. As a dielectric, OSTE performs similar to Parylene C (a gold standard dielectric in electrowetting), whereas its tunable surface and bulk properties facilitate straightforward bonding of the microchannel with the dielectric layer. In addition, a new chip design that facilitates efficient droplet transfer from the DMF part to the microchannel inlet solely by electrowetting is showcased.Peer reviewe

Pulmonary delivery of siRNA-loaded lipid-polymer hybrid nanoparticles: Effect of nanoparticle size

Nanomedicines based on nanoparticles rely both on the potency of the drug as well as the efficiency of the delivery system, for which particle size plays a crucial role. For the intracellular delivery of small interference RNA (siRNA), lipid-polymer nanoparticle (LPN) hybrid systems constitute a safe and highly effective class of delivery systems. In the present study, we employ a microfluidics method for the manufacturing of spherical siRNA-loaded LPNs for pulmonary delivery with distinct size distributions with average diameters of approximately 70, 110, and 220 nm. We designed an optically clear, inexpensive thiol-ene polymeric microfluidic chip prototype that is compatible with standard ‘soft-lithography’ techniques, allows for replica molding, and is resistant to harsh solvents. By using SPECT/CT in vivo imaging, we show comparable pulmonary clearance patterns of all three differently sized LPN formulations following intratracheal administration. Also, negligible accumulation in the liver was observed