An engineering characterisation of shaken bioreactors: flow, mixing and suspension dynamics

The thesis describes an experimental investigation of the flow, mixing and suspension dynamics in cylindrical orbitally shaken bioreactors (OSRs). Amongst the plethora of bioreactor types and geometries available for cell culture, the OSR is ubiquitous in bioprocess research and development. Offering a well defined liquid-gas interface, high throughput potential and experimental flexibility, it is the vessel of choice in early bioprocess research, either as microtiter-plates, Erlenmeyer flasks or other geometries. Despite recent advances in the field, an accurate and exhaustive engineering characterisation of OSRs from this point of view is lacking. In the present study a mixing time estimation methodology is developed and employed to assess the effect of operational parameters on the mixing of cylindrical OSRs. Particle Image Velocimetry measurements are carried out to evaluate the effect of vessel geometry modifications on the flow. Laser Induced Fluorescence is used to produce accurate description of the micro-mixing. Solid suspension studies are also undertaken to assess potential strategies to improve microcarrier culture in OSRs. Accurate determination of the mixing time in OSRs is essential for the optimisation of mixing processes and minimization of concentration gradients that can be deleterious to cell cultures. The Dual Indicator System for Mixing Time (DISMT) is employed, together with a purposely built image processing code to objectively measure mixing times in cylindrical and Erlenmeyer flask bioreactors. Relevant data acquisition aspects to optimise the accuracy of DISMT measurements are discussed in detail, with direct comparison of different mixing time measurement methodologies, including DISMT, pH probe and iodine thiosulfate decolourisation results obtained in two types of stirred reactors. The DISMT is employed to determine mixing characteristics of OSRs at different flow conditions and develop an effective feeding strategy, by evaluating the effect of the position of the feed at different radial locations in the vessel. At low Fr the flow presents a toroidal vortex below the free surface, which controls mass transport process across the entire vessel and defines two distinct regions in and outside of the vortex exhibiting different mixing rate. At higher Fr the axial flow enhances the mixing of the fluid located next to the wall as the mean flow transition to axial flow coincides with a regime flow transition and onset of turbulence fluctuations. By controlling the locations of eed addition, the flow characteristics can be exploited to enhance initial distribution of the added liquid and decrease the time required to reach homogeneity. The mixing number is highly dependent on the position of the feeding pipe. Insertion close to the vessel walls, and in the periphery of the toroidal vortex, where local shear stresses and deformation rates are highest, were found to significantly enhance mixing. In order to provide an effective scaling methodology, the results obtained in OSRs are compared with data previously reported in the literature for both cylindrical reactors and Erlenmeyer flasks. The employment of a critical Froude number shows promise for the establishment of a scaling law for mixing time across various types and sizes of shaken bioreactors. The flow dynamics in cylindrical shaken bioreactors of different conical bottom geometries (inward facing) is investigated by means of phase-resolved Particle Image Velocimetry. The cylindrical bioreactor with a conical bottom geometry is selected to assess its potential application in three-dimensional cultures, and improve solid suspension in shaken systems. The effects of conical shaped bottoms of different heights on the fluid flow are evaluated for different operating conditions with water being the working fluid. The results provide evidence that the presence of the conical bottom affects the transition from laminar to turbulent flow, increases the vorticity and generates shear stresses at well defined locations. The increased kinetic energy content measured with PIV in cylindrical OSRs with a conical bottom is found to effectively enhance solid suspension in microcarrier or embryod body cell culture. The dynamics of solid suspension is studied using commercially available Cytodex-3, stained with trypan blue for improved visual contrast and image acquisition. The presence of the conical bottom improves solid suspension by requiring lower agitation rates for the microcarriers to lift from the bottom completely. The critical Froude, which determines the flow type controlling the bioreactor, can also be used to scale the suspension of microcarriers in OSRs. Full characterisation of macro- and micro- mixing scales in OSRs for highly viscous is obtained by DIMST and pLIF, respectively. This data also provides an effective visualisation of the flow structures controlling the bioreactor transport processes. The mixing characteristics of high viscosity fluids in OSRs are investigated by means of DISMT and pLIF, for the macro- and microscales of mixing, respectively. Fluids of viscosities 2-14 times that of water, exhibit flow characteristics different to those observed at ν=10−6 m^2s^−1. A toroidal vortex, similar to that observed in water, is present at low Fr. Small increments of agitation rate provide transition to other flow structures, never reported in the literature. The pLIF measurements allow to characterise the small scale features of the flow not observable from phase averaged PIV, and visualise well defined elongation and striation dynamics for different regimes. Although extensively used, OSRs are yet to be fully characterised. Further research is required on the hydrodynamic phenomena dominating orbitally shaking vessels, to enable the development of scale-up platform to simplify and speed progress from cell line development to industrial production of bio-products. The development of the scaleup platform must be made considering the advantages and requirements of single-use technology, to provide the industry with robust and reproducible scale-up model

Protein Precipitation for the Purification of Therapeutic Protein

This thesis documents the application of precipitant scouting and analytical tools for the development of a precipitation process for the purification of therapeutic proteins in biopharmaceuticals. Precipitation has the potential to bypass the bottlenecks in productivity experienced with packed bed chromatographic separations, offering a fast, robust first purification step with volume reduction at low cost. In order to evaluate a large number of precipitation candidates, microscale investigations aided by automated liquid handling robotics were chosen, which conferred precision, speed, and complex experimental design with microliter material requirements and in-line analytics. A precipitation screening methodology was successfully built onto a Tecan liquid handling platform including product recovery and techniques for measuring soluble protein, liquid volumes, and recovered protein precipitate. The protocol was supplemented with a custombuild Excel VBA driven Tecan control tool. This accelerated progress by freeing up the potential of the liquid handling arm, which was curtailed by system software. These techniques were then used to characterise effective precipitants for the purification of monoclonal antibodies. Optimal conditions were identified on pure protein models, which were the bridged to process relevant cell culture fluid. Process performance, notably recovery yield and product quality were the investigated on the precipitant conditions brought forward. Process integration aspects were explored, by linking precipitation with an anion exchange chromatography step. This led to discussion on future work and process scale up considerations

Establishment of High Cell Density Fed-Batch Microbial Cultures at the Microwell Scale

The rate limiting steps of biopharmaceutical process development are clone evaluation and process optimisation. To improve the efficiency of this step, miniature bioreactors are increasingly being used as a tool for high throughput experimentation. At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes; however, many commercially available miniature bioreactors do not have an inbuilt feeding capacity. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. The overall aim of this project was to develop a scale-down fermentation platform suitable for the study and optimisation of high cell density cultures. The first objective of this work was to evaluate options for fed-batch cultures in a commercially available 24-well shaken microbioreactor. To achieve this, two feeding strategies were evaluated using an E. coli strain expressing a domain antibody: in situ feeding by the enzymatic release of glucose from polymeric starch, and direct feeding using a bespoke feed delivery system. In situ feeding was investigated as it is a simple option that does not require a physical method of feed delivery; cellular productivity was enhanced in comparison to batch cultures, however the glucose release was insufficient to sustain high cell density cultures representative of laboratory and pilot scale processes. To enable direct and continuous feed delivery to the microbioreactor a bespoke 3D-printed feeding system was developed that can operate at flow rates of 20μL h-1 and above, and enables up to twelve fed-batch cultures to be run in parallel. E. coli fermentations were performed on complex medium containing glycerol with direct feeding of a 23% w/v glycerol solution initiated at around 18 hours. The second objective of this project was to establish an industrially relevant feeding strategy in the microbioreactor, comparable to a laboratory scale fed-batch process. To this end, the direct feeding strategy was refined in terms of cell growth and product expression; the feed rate and concentration were modified, the DO set point was increased, and a pre-feeding hold period was implemented to allow for consumption of the inhibitory by-products generated in the batch phase. It was found that direct feeding enhanced biomass production by ~70% and product expression by ~2.4 fold in comparison to non-fed cultures. The third objective of this work was to demonstrate the applicability of the new feeding system as a tool for process optimisation experiments. The effect of IPTG concentration and post-induction temperature on product expression was performed using the both the microbioreactor feeding system and the 1L laboratory scale process. The data trends were consistent between scales; product expression was enhanced at a higher post-induction temperature, and IPTG concentration did not affect product expression over the concentration range tested. This demonstrates that the microbioreactor, is predicative of the 1L laboratory scale process terms of sensitivity to change in process conditions The fourth objective of this work was to characterise the microbioreactor in terms of oxygen transfer capability and fluid mixing. To achieve this aim, the volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) of the microbioreactor were determined. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume. The oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency (500-800rpm) and gas flow rate (0.1-20 mL min-1) over the range 3-101h-1; this is at the lower end of the range for conventional stirred tank reactors. It was demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.5-15s. The final objective this project was to demonstrate suitability of the microbioreactor as a scale-down model of an industrial fermentation process. 50L pilot scale, 1L laboratory scale, and 4mL microbioreactor fed-batch fermentations were performed under optimum conditions. The 4mL microbioreactor fed-batch process was shown to better predict the 50L pilot-scale process than the 1L laboratory-scale process based on cell growth, product expression and product quality. This could be explained by mixing and oxygen mass transfer phenomena. At 1L scale, oxygen mass transfer and fluid mixing are most efficient, meaning cell growth and productivity were the highest of the three processes. It appears that the limitations in oxygen mass transfer in the microbioreactor and fluid mixing in the 50L scale vessel, results in a comparable cellular environment, and therefore cell growth, productivity and product quality. In summary, this work has demonstrated the ability to conduct high cell density, fed-batch microbial cultures in parallel, using a shaken miniature bioreactor system. A bespoke, 3D-printed feed delivery system was developed allowing for twelve industrially-relevant microbial fed-batch cultures to be run in parallel. The microbioreactor fed-batch cultures were shown to be predictive of, a 50L pilot scale process in terms of cell growth, productivity and product quality

Vat 3D printable materials and post-3D printing procedures for the development of engineered devices for the biomedical field

L'abstract è presente nell'allegato / the abstract is in the attachmen

Morphogen Gradient Creation and Bio-modified Surfaces for Rebuilding a Stem Cell Microenvironment

Recreation of a microenvironment where stem cells maintain pluripotency or initiate differentiation is crucial especially when they are used for therapeutic needs. In this thesis, to generate more in vivo like environments, the application of soluble factors such as morphogen gradients was realized by microfluidic system and cadherin-anchored artificial substrates were generated for the cellular contact to the microenvironment by atomic force microscope based nanografting

Nanoparticle mediated toxicity and antimicrobial action

Nanomaterials are either inorganic or organic nanosized particles which have many industrial and biological applications such as in cosmetics, environmental remediation, electronics, biosensing and imaging and in drug delivery. Some have toxic effect upon release to the environment causing death of microorganisms and others are biodegradable and nontoxic to the living beings. In this work, two types of nanoparticles were investigated: inorganic titania nanoparticles which have been shown to have toxic effects and organic Carbopol Aqua SF1 microgel particles which were shown to be nontoxic and biodegradable organic nanoparticles for drug delivery.Chapter one explores the current literature relating to nanoparticles. The types, chemical and physical properties, methods of synthesis, characterization and functionalization are discussed along with general applications and the toxicity of titania nanoparticles. The role of nanomaterials as drug delivery systems and their design in terms of stability, swelling studies, encapsulation, drug loading and release, response to stimuli and targeting is also discussed. Finally the use of microfluidics for screening nanoparticle activity is discussed including microfabrications of chips cell trapping methods and microfluidic cell based assay methods.Chapter two is the experimental chapter describing the chemicals and instrumentation used. It also includes the methods used for the synthesis of titania nanoparticles and effects of pH on the zeta potential measurement. In addition to that, methods for the testing of the cytotoxic effects of uncoated and coated titania nanoparticles are described. Finally the methods employed for studying the optimization of the encapsulation of berberine and chlorhexidine into Carbopol Aqua SF1 are also included.Chapter three describe the synthesis of titania nanoparticles (TiO2NPs) and their characterization, including crystallite size, particle size distribution, surface area measurement and zeta potential. It was found that as the temperature increased from 100oC to 800oC, the crystallite size and particle size increased while the surface area decreased. At 100oC, the crystallite size, particle size, surface area and zeta potential of the titania nanoparticles were 5 nm, 25±20 nm, 163 m2 g-1 and +40±9 mV, respectively with anatase as the dominant phase. However, the phase changed to rutile at the annealing temperature of 800oC with the crystallite size, particle size, surface area and zeta potential becoming 142 nm, 145±60 nm, 7.5 m2 g-1 and -26±8 mVrespectively. In addition to that, the zeta potential of the titania nanoparticles at 25 nm size was affected by changing the pH of the suspension, at low pH, the zeta potential was +40 mV giving high stability and fully dispersed particles while the nanoparticles flocculated in the basic medium with a zeta potential of -25 mV, with the isoelectric point of titania nanoparticles being pH 6.7. Changing the pH of the solution for titania nanoparticles caused an irreversible process as it was not possibility to convert the aggregated titania nanoparticles from microscale to nanoscale.Chapter four describes the investigation into the the nanotoxicity of the titania nanoparticles (TiO2NPs) at various hydrodynamic diameters and crystallite size on C. reinhardtii microalgae and S. cerevisiae (yeast) upon illumination with UV and visible light. The cell viability was assessed for a range of nanoparticle concentrations and incubation times. It was found that uncoated TiO2NPs affect the C. reinhardtii cell viability at a much lower particle concentrations than for yeast. It was also observed that the TiO2NPs toxicity increased upon illumination with UV light compared to dark conditions due to the oxidative stress of the reactive oxygen species produced. It was also found that TiO2NPs nanotoxicity increased upon illumination with visible light which indicated that the nanoparticles might also interfere with the microalgae photosynthetic system leading to decreased chlorophyll content upon exposure to TiO2NPs. The results showed that the larger the hydrodynamic diameter of the TiO2NPs the lower their nanotoxicity, with anatase TiO2NPs generally being more toxic than rutile TiO2NPs. A range of polyelectrolyte-coated TiO2NPs were also prepared using the layer by-layer method and their nanotoxicity on yeast and microalgae was studied. It was found that the toxicity of the coated TiO2NPs alternates with their surface charge. TiO2NPs coated with cationic polyeletrolyte as an outer layer exhibited much higher nanotoxicity than the ones with an outer layer of anionic polyelectrolyte. TEM images of sectioned microalgae and yeast cells exposed to different polyelectrolyte-coated TiO2NPs confirmed the formation of a significant build-up of nanoparticles on the cell surface for bare and cationic polyelectrolyte-coated TiO2NPs. The effect came from the increased adhesion of cationic nanoparticles to the cell walls. Significantly, coating the TiO2NPs with anionic polyelectrolyte as an outer layer led to a reduced adhesion and much lower nanotoxicity due to electrostatic repulsion with the cell walls.Chapter five describes the development and characterisation of berberine-loaded and chlorhexidine-loaded polyacrylic acid based microgels. The procedure for loading the Carbopol microgels with both berberine and chlorhexidine was developed using a swelling-deswelling cycle dependent on pH. The result of this protocol was a colloidal suspension of collapsed microgel particles loaded with fixed percentage of the antimicrobial agents, berberine and chlorhexidine, respectively. The initial microgel particle concentration, as well as the initial concentrations of berberine and chlorhexidine, were optimized to allow for maximum encapsulation efficiency of the loaded reagent in the microgel while maintaining the colloidal stability of the Carbopol microgel suspension. It was determined that 0.15 wt% berberine and 0.1% chlorhexidine could be successfully incubated with 0.1 wt% Carbopol microgel while the pH was varied from 8 to 5.5 with a measurable increase of the collapsed microgel due to electrostatic conjugation of these cationic antimicrobial agents with the carboxylic groups of the microgel.While for berberine, only 10% encapsulation efficiency was achieved, for chlorhexidine over 90% encapsulation efficiency was obtained without significant impact on the colloidal stability of the microgel. The zeta potential of the loaded microgels remained negative in the range of -35 mV - -40 mV with very moderate increase of the collapsed (and loaded) microgel particle size. The release of berberine and chlorhexidine from these microgel materials was studied and sustained release from the formulations was demonstrated upon dilution over the period of up to 6 hours. The berberine- and chlorhexidine-loaded microgel particles were then further coated with cationic polyelectrolytes, PAH and PDAC. This carried out to increase the adhesion of these antimicrobial particles to the cell membranes. These studies showed a reversal of the zeta-potential of the PDAC coated microgels after their loading with berberine and chlorhexidine, respectively.In chapter six, the antimicrobial activity of both berberine and chlorhexidine loaded Carbopol microgel was studied upon incubation with algae, yeast and E.coli. It was noticed that an increase in the antimicrobial activity of berberine and chlorhexidine Carbopol microgel occurred after 6 hours incubation time for algae and after 24 hours for E.coli while there was no pronounced antimicrobial action for yeast in comparison with the antimicrobial activity of free berberine or chlorhexidine. This was due to the repulsion forces between the anionic microgel and the anionic cell membrane which did not allow the encapsulated berberine or chlorhexidine to be released and diffuse into the cytoplasm causing cell death. In addition to that, the fully anionic charged Carbopol microgel did not allow berberine and chlorhexidine to be released easily at pH 5.5 while the percentage of release increased with pH up to 7.5.The antimicrobial activity of cationic PDAC coated berberine and chlorhexidine loaded-Carbopol microgel was also studied for algae, yeast and E.coli. It was found that cationic PDAC on its own had an acute toxic effect on algae, yeast and E.coli while the toxicity of cationic PDAC reduced upon coating Carbopol microgel with cationic PDAC. Algae and E.coli stayed viable up to 0.0045 wt. % and 0.009 wt. % of PDAC coated Carbopol microgel, respectively. Yeast was resistance to the PDAC coated carbopol for a wide range of concentrations of PDAC coated Carbopol microgel up to 0.018 wt. %. This was due to the different thicknesses of the cell membrane. The Carbopol microgels with encapsulated berberine and chlorhexidine were then coated with cationic PDAC to form PDAC coated particles. The PDAC coated berberine or chlorhexidine loaded carbopol microgel were then incubated with each of algae, yeast, and E.coli. The coating appeared to increase the antimicrobial actions against algae, yeast and E.coli for short incubation times. The increase in the antimicrobial activity was attributed to the electrostatic interaction between the cationic PDAC coated berberine or chlorhexidine loaded carbopol microgel and the anionic cell membrane allowing diffusion of berberine or chlorhexidine easily through cell membrane causing cell death. TEM images showed aggregation of these cationic PDAC coated berberine or chlorhexidine loaded carbopol microgel on the surface of the cell membrane.Chapter seven describes the development of new microfluidics device for cell trapping to achieve a microscreening cell based assay. The idea involved trapping the cells in a micro chamber and then passing over suspensions of the nanomaterials and monitoring the effect. Three design of microfluidic chips were studied with different designs, channel dimensions (depth and width) cell trapping techniques and materials. Initially chemical adhesion was investigated to adhere cells into micro chamber of the microfluidics device using poly-l-lysine bu the cells detached from the surface of microchip because of shear stress forces. Synthesized magnetic yeast cells which were then investigated for trapping other cells into the micro chamber of the chip but the back pressures were too high when liquids were flowed through the system. Magnetic glass beads were then studied to trap the cells, these were synthesized by coating anionic glass beads with cationic and anionic polyelectrolytes such as PAH and PSS, however, they had a low magnetic response towards the magnet. Synthesized magnetic beads were then synthesized using a PDMS based ferrofluid but again a low magnetic response was obtained towards the magnet. Synthesis by flow focusing microfluidics was the tried to generate mono dispersed magnetic beads where SDS with water was the continuous phase and a styrene based ferrofluid was the dispersed phase but the magnetic beads were unstable and they need to be optimized. The continuous phase was then changed to use Hitenol BC20 a polymerisable surfactant, to form hydrophobic magnetic beads and and a mixing serpentine was added to the chip design to give the generated magnetic beads time to be stablilise. Despite these changes the magnetic beads stayed unstable and therefore an emulsification method was used to fabricate poly dispersed magnetic beads which produced 20μm to 50μm magnetic beads. These beads were successfully utilized for trapping cells into the micro chamber of the chip device. A new microfluidic device was designed with suitable channel dimensions which allowed the magnetic beads to move freely inside the micro chamber of the device. These beads were placed into the micro chamber could be controlled using the neodymium magnet easily move the beads

Porous Silicon-Based On-Demand Nanohybrids for Biomedical Applications

Considerable efforts have been made to fabricate nano-sized drug delivery systems (DDS) with unique and advanced features in comparison to conventional DDS. Yet, challenges still lay ahead requesting for more controllable, even on-demand drug release profiles from the DDS. Moreover, the emerging concept of personalized treatment further urges the combining of therapy and imaging regimes into a single nanocarrier. Among all the nanomaterials studied so far, porous silicon (PSi) draws increasing interest for constructing DDS due to its good biocompatibility, non-immunogenicity, large pore size/surface area and easily changeable surface properties. Herein, the aim of this thesis was to explore PSi-based DDS for multiple biomedical applications, which were designed and synthesized with specific on-demand features. Moreover, simultaneous incorporation of imaging modalities and drugs enables real-time visualization of drug release and/or cellular/tissue level disease condition, which are expected to be beneficial for personalized treatment regime. First, the potential of PSi nanoparticles for hydrophilic drug loading and on-demand release were evaluated by adapting a dynamic non-covalent bonding method. Different ligands were synthesized and applied for modifying the PSi, and the hydrophilic anti-cancer drug doxorubicin (DOX) was sequentially loaded into the fabricated DDS for pH-responsive release profiles. Meanwhile, the fluorescence spectrum of DOX can be dynamically shifted or quenched, depending on the loading and releasing process, thus facilitating the in situ visualization of the drug releasing process. For hydrophobic drugs, a physical encapsulation method was applied to seal the pores of the PSi by a polymeric matrix. Microfluidic-assisted nanoprecipitation method was applied to synthesize batches of nanohybrids with identical PSi-core/polymer-shell structures, and the release behavior was feasibly tailored by the degradation behavior of the outer polymeric matrix. The first trial was set to fabricate a core/shell nanohybrid, with PSi and gold nanoparticles co-encapsulated in a pH-responsive polymer to simultaneously deliver hydrophobic drug and increase the computed tomography signal for acute liver failure theranostics. The newly established single-step co-encapsulation of different particles endowed a system with multi-functionalities, and the polymeric shell precisely tailored the drug release behavior in a pH-dependent manner. Similarly, an acid/oxidation dual-responsive polymer was designed and further applied in encapsulating atorvastatin-loaded PSi nanoparticles. The meticulously designed system not only obtained a dynamic drug release behavior, but also showed an orchestrated cascade that facilitated bio-mimetic diabetic wound healing. To better elucidate the biocompatibility of PSi for DDS fabrication, the biological effects and immunogenicity of different PSi nanoparticles were evaluated at pre-existing lesion sites, which provided insights for further applications of PSi in DDS fabrication. In conclusion, multiple PSi-based nanohybrids with different on-demand responses were fabricated and applied as DDSs for different diseases. The newly developed nanosystems tailored drug release and obtained multiple modalities, ranging from real-time bio-imaging to bio-mimetic/bio-response alteration, as such, represent promising platforms for future therapy regimes.Nanokokoisilla lääkeainekuljettimilla on ainutlaatuisia ja kehittyneempiä ominaisuuksia perinteisiin lääkeaineen kuljetusmenetelmiin verrattuna. Niiden kehittämiseksi on tehty paljon, siitä huolimatta niillä on edessä vieläkin useita haasteita, jotka vaativat täsmällistä ja erityisen hyvin säädeltyä lääkeaineenvapautumista. Lisäksi kiinnostus potilaslähtöiseen hoitoon vaatii terapian ja kuvantamismahdollisuuksien yhdistämistä yksittäiseen lääkeaineen nanokantajaan. Yksi kiinnostavimmista tähän mennessä tutkituista materiaaleista on huokoinen pii (PSi), mikä johtuu sen bioyhteensopivuudesta, vähäisestä immunogeenisyydestä, suuresta huokosten koosta ja pinta-alasta sekä helposti muunnettavista pinnan ominaisuuksista. Tämän väitöstyön tavoite oli tutkia huokoiseen piihin pohjautuvien räätälöityjen lääkeainekuljettimien soveltuvuutta erilaisiin biolääketieteellisiin sovelluksiin. Lääkeaineiden ja kuvantamisen mahdollistavien aineiden kuormaaminen samoihin nanohiukkasiin mahdollistaa tosiaikaisen lääkeaineenvapautumisen kuvantamisen sekä taudinkuvan määrittämisen solu- ja/tai kudostasolla, joiden molempien voi odottaa olevan hyödyllisiä potilaslähtöiseen terapiaan. Ensimmäiseksi tutkittiin PSi-nanohiukkasten soveltuvuutta hydrofiilisten lääkeaineiden kantajina. Hallittua lääkeaineenvapautumista tutkittiin soveltamalla dynaamista ei-kovalenttista sidosmenetelmää. Erilaisia ligandeja syntetisoitiin ja niitä kiinnitettiin PSi-nanohiukkasten pintaan. Tämän jälkeen hiukkasiin kuormattiin syöpälääke doksorubisiinia, jonka pH-vasteellinen vapautuminen määritettiin. Doksorubisiinin fluoresenssispektri voidaan dynaamisesti siirtää tai sammuttaa eri kuormaamis- ja vapautumismenetelmillä, mikä mahdollistaa lääkeaineenvapautumis-prosessin in situ-kuvantamisen. Hydrofobisille lääkeaineille sovellettiin mikrofluidiikkaan perustuvaa kapselointia, jossa lääke ensin kuormattiin PSi:n huokosiin, jotka sitten suljettiin polymeeri-matriisilla. Lääkeaineenvapautuminen määräytyi polymeerin hajoamisnopeudesta. Ensimmäisessä kokeessa valmistettiin kuori/ydin-nanohybridejä, joissa PSi, kultananohiukkaset sekä hydrofobinen lääkeaine kapseloitiin pH-vasteelliseen polymeeriin. Näin voitiin akuutin maksan vajaatoiminnan yhteydessä säädellä lääkeaineenvapautumista pH:n avulla sekä samanaikaisesti tomografiakuvantaa. Yksivaiheisella kerakapseloinnilla, jossa yhdistetään erilaisia partikkeleita, saatiin aikaan monitoiminnallinen kuljetin, jonka polymeerikuori kykenee täsmällisesti säätelemään lääkeaineenvapautumista eri pH-olosuhteissa. Seuraavassa kokeessa pH- ja hapettumis-vasteellisella polymeerillä kapseloitiin atorvastatiinilla kuormattuja PSi-nanohiukkasia. Nämä huolellisesti suunnitellut nanohiukkaset kykenivät aktiivisesti muuttamaan lääkeaineen vapautumista mutta myös vaikuttivat aktiivisesti, muistuttaen luonnollista prosessia, diabeteksesta johtuvan haavan paranemiseen. PSi-hiukkasten biologinen yhteensopivuus lääkeaineenkuljettimeksi varmistettiin tutkimalla erilaisten PSi-nanohiukkasten biologisia vaikutuksia ja immunogeenisyyttä leesio (haavauma) kohdissa. Näin saatiin lisätietoa jatkokehittelyyn. Väitöstyössä kehitettiin useita piihiukkasiin pohjautuvia nanoyhdistelmiä lääkeainekuljetukseen erilaisten tautien hoitamiseksi. Perinteisiin lääkeaineenkuljetus-menetelmiin verrattuna uusien nanosysteemien etuja ovat tarpeenmukainen lääkeaineenvapautuminen ja monikäyttöisyys – tosiaikaisesta kuvantamisesta luonnollista prosessia muistuttavaan tai biologista vastetta muuttavaan vaikutukseen. Sellaisena ne edustavat lupaavaa menetelmää tulevaisuuden hoidoill