Browsing by study line "Biofarmasia"

Chronic wounds are a worldwide health problem that produce a lot of costs for society and can have a substantial impact on patients’ quality of life. Human adipose stem cells (hASCs) have been studied as a treatment option for chronic wounds as they can induce wound healing in many ways. Extracellular vesicles (EVs) produced by hASCs are a great solution to acquire the benefits of hASCs while avoiding their problems such as possible mutagenicity. HASC-EVs have been found to induce wound healing by for example enhancing angiogenesis and fibroblast proliferation. HASCs can be grown in 2D where the cells attach to the bottom of the cell culture vessel or in 3D where the cells attach to each other and create a spheroid. 2D cell culturing is easy and inexpensive but 3D cultured cells resemble in vivo –like conditions more. Because of these in vivo -like features, hASCs grown in 3D might produce EVs that resemble the properties of host cells in natural environment more than 2D. The aim of this thesis was to compare 2D culture, matrix-based nanofibrillar cellulose (NFC) hydrogel culture, and matrix-free suspension culture in ultra-low attachment (ULA) wells as growing platforms for hASCs and as continuous EV production methods. During culturing, the conditioned media was collected after which, the EVs were isolated, and the EV concentration and size range was measured with nanoparticle tracking analysis (NTA). After culturing, the metabolic activity of hASCs was measured and the cells were collected for immunocytochemistry (ICC) assay, western blot (WB) assay, and for quantitative PCR (qPCR) to examine the stemness and differentiation of hASCs grown in different cell cultures. The hypothesis of this thesis was that the NFC cell culture would produce the best EV yield and the best EVs for therapeutic use. Based on the acquired results, this hypothesis could not be supported. When visually inspecting the cells, all three cell cultures were viable but the metabolic activity of hASCs in NFC hydrogel was low compared to 2D and suspension cultures. Also, the EV, protein and RNA yield were lower in NFC. ICC, western blotting, and qPCR results were inadequate to make a straightforward implication of what cell culturing condition is the best for EV production and they would need repetition and optimization. Looking at the overall results, 2D cell culturing produced the best EV and RNA yield, had the highest metabolic activity and was least laborious cell culturing method which makes it a good option for continuous EV production. Suspension culture on the other hand resembles in vivo -like environment which could possibly produce better EVs for therapeutic use. The metabolomic assays on the EVs would be interesting to perform in the future to examine if the in vivo –like features affect the quality of EVs.

During the past few decades, the explosion of discovery in cancer and immunological research has led to the increased understanding of the interactions between the immune system and tumors. These developments have provided vital information about the immune system’s role in cancer development. It is evidenced that the immunity system is capable to distinguish tumor cells from normal tissue by recognizing tumor antigens that are exclusively expressed on tumor cells or are presented in greater amounts on tumor cells than normal cells. Consequently, the immune cells start to attack tumors for protecting the host. The possibility to use the immune system as a weapon against cancer cells leaded to the promising innovation – cancer immunotherapy – which aims to activate the body’s own immune system and its components to mount antitumor immune responses for eliminating cancer cells. The antitumor efficacy and high safety profile of several immunotherapeutic strategies have already been demonstrated thereby resulting in their integration into clinical practice. However, most patients have not benefited from cancer immunotherapy as a single treatment. In this regard, new innovative methods are clearly needed to overcome the obstacles hindering the clinical success of this field. Therapeutic cancer vaccines are emerging as attractive immunotherapies currently being evaluated in both pre-clinical and clinical studies. The purpose of cancer vaccines is to eradicate tumor cells by eliciting antitumor CD8+ T cell responses against the injected tumor antigens. Due to the ability to specifically kill tumor cells and simultaneously trigger immune responses against tumor antigens via direct oncolysis and by encoding transferred tumor antigens, oncolytic viruses are of significant interest for being used as in situ cancer vaccines. Despite these unique properties, several factors such as tumor immunosuppression and immune tolerance to targeted tumor antigens resembling antigens of normal tissues hamper the use of oncolytic vaccines in clinic. Instead of focusing only on CD8+ T cells, it has been suggested that giving more attention to CD4+ T helper cells, which are required for priming and expansion of CD8+ T cell responses, could be the key to improve the efficacy of cancer vaccines. Researchers have also demonstrated that an ongoing antigen-specific CD4+ T cell response can lead to the bystander activation of surrounding T cells with unrelated antigen specificities. Based on this theory, the hypothesis of this study was to employ the pre-existing immunological CD4+ memory against infectious pathogens in generating bystander CD8+ immunity against solid tumors. In this study, mice transplanted with poorly immunogenic B16-OVA tumors were pre-immunized with the chosen vaccine to induce immunological CD4+ memory against an infectious pathogen. Tumors were then treated with already developed cancer vaccine, which was peptide-coated conditionally replicating adenovirus (PeptiCRAd) complex. PeptiCRAd was constructed by electrostatically coating adenovirus with both pathogen-derived and tumor-derived peptide. The intratumorally injected double-coated PeptiCRAd complex was assumed to activate peptide-specific T cells and thus, result in anti-pathogen CD4+ T cell recall responses and the following bystander activation of antitumor CD8+ T cells, which can then mount an effective immune response to destroy cancer cells. The efficacy of this treatment was observed in pre-immunized mice by measuring the growth of injected tumors. The experiment was repeated identically with non-immunized naïve mice to see the difference in the results. The immunological background of this treatment approach was investigated by analyzing mouse tissue samples with standard immunological techniques including ELISA, IFN-γ ELISPOT and flow cytometry. This study showed that long-term immunological memory against the pathogen was successfully accomplished and the strongest inhibition of tumor growth in pre-immunized mice was achieved with double-coated PeptiCRAd, whereas the antitumor efficacy was not seen in naïve mice. Additionally, a new ex vivo method to detect pathogen-specific CD4+ T cells from spleen was developed and the stimulation of cell-mediated immunity by this treatment was supported by finding the highest levels of pathogen-specific CD4+ Th1 cells from mice treated with double-coated PeptiCRAd. Some encouraging results concerning the beneficial immune cell composition of tumors and tumor draining lymph nodes were also obtained from other performed experiments. Though further immunological analyses are required for understanding the precise mechanisms of action behind the treatment, the increased immunogenicity and antitumor efficacy of double-coated PeptiCRAd can still be considered as a consequence of the bystander effect, which can possibly be utilized for developing improved strategies to win the fight against cancer.

P-glycoprotein is an efflux transporter of the ABC family. It is expressed mainly in tissues that have a role in limiting the absorption and distribution of xenobiotics in the body or their elimination. P-glycoprotein is known to have an important role for example in the blood-brain barrier and in protecting the fetus from xenobiotics in the mother’s blood stream. Genetic polymorphisms in transporter proteins can cause individual differences in the pharmacokinetics of drug substances, which can lead to differences in drug efficacy or side effects. In the ABCB1 gene, which codes for p-glycoprotein, several polymorphisms have been discovered. The frequencies of these polymorphisms vary in different ethnic populations. Previous studies have shown that the effects of these polymorphisms are often substrate-dependent. Since there are several confounding factors usually present in clinical association studies, in vitro studies are needed to clarify the effects of individual polymorphisms. Polymorphisms can be studied in vitro by making intentional mutations to the gene sequence and expressing the variant gene in a suitable cell line. In this study four variant p-glycoprotein genes (c.781A>G, c.1199G>T, c.2005C>T and c.3421T>A) were created by site-directed mutagenesis, and expressed in HEK293 cells using a baculovirus recombinant protein expression method. The effects of the polymorphisms were studied by determining the expression level and the transport acitivity of the variant proteins compared to the wild-type. Western blot was used to determine the expression level and a calcein accumulation assay in HEK293 cells was used to compare the transport activities. Also a membrane vesicle transport assay with n-methyl quinidine was set up and optimized, but the variants were not yet studied with this method during this study. In this study no statistically significant differences were found in the transport activities of any of the four variants compared to the wild-type p-glycoprotein. Also the differences in protein expression level between wild-type and variant proteins were small. However, because of the previously reported substrate dependency of polymorphism effects, it would be beneficial to study the variants with at least one other substrate and one other assay method, and thus the membrane vesicle transport assay would be useful to further compare the transport activities of variant proteins to the wild-type p-glycoprotein.

Physiologically based pharmacokinetic modelling (PBPK) can be used to predict pharmacokinetic behaviour of new drug molecules in human. PBPK model represents the body anatomically and physiologically with compartments connected to each other and combines those to drug specific parameters. PBPK modelling can be used to predict the absorption, disposition, and time-concentration profiles of drug molecules. The purpose of the study was to build a PBPK model for new drug molecule under research (compound A) and predict pharmacokinetics in human, to support the selection of dosing interval, formulation, and sampling time points for the first clinical trial. In this work it is described the building of the model in the ”bottom-up”-approach using in vitro parameters in GastroPlusTM-software. The modelling was done also for preclinical species (mouse, rat, dog) comparing the simulations to the observed in vivo data, which gave the confidence to the methods used in the modelling also for human. The model was first built for systemic kinetics and thereafter it was used for predicting pharmacokinetics after oral dosing. Parameters of systemic kinetics were compared also to the predictions from allometric scaling. Based on the preclinical species the most predictive method for the volume of distribution of compound A was the method by Lukacova, which predicted the volume of distribution to be moderate in human (1.7 l/kg). From the in vitro-to-in vivo -extrapolation methods the most predictive method to predict the clearance was the method by Poulin, which predicted low clearance in human (8.1-14.3 l/h). Empirical scaling factors based on the preclinical data were not needed, as the models predicted well the observed in vivo data. Allometric methods predicted the systemic kinetic parameters to be in the similar range. Advanced compartmental absorption transit -model (ACAT) integrated to GastroPlusTM-software predicted the absorption after oral dosing well in the preclinical species (predicted/observed ratio 0.8-1.3 for systemic exposure) despite the low solubility of the compound A. The model predicted the absorption in human to be sensitive to particle size and absorption rate to be clearly affected by the particle size. The feeding status was also predicted to affect on the absorption with larger particle sizes. The gut metabolism was not predicted to limit the oral exposure notably, whereas moderate bioavailability was predicted to be achievable. Compound A could be given in a capsule if the target particle size distribution could be achieved. The built PBPK-model can be used in the future to predict the first clinical doses by comparing the predicted plasma concentrations to in vitro pharmacodynamic parameters and to the plasma concentrations needed for efficacy in the pharmacodynamic models. The model can also be used to predict the drug-drug interactions.

Native nanofibrillated cellulose is wood-derived, animal-free biocompatible biomaterial which has proved the suitability of nanoscale cellulose fiber based hydrogels for 3D cell culturing and wound healing applications. The problem of freeze-drying nanofibrillated cellulose hydrogel (NFCh) has been the aggregation of the hydrophilic fibrils of the NFC during freeze-drying, which leads deformed freeze-dried cake and unsuccessful reconstitution of the sample. Molecular Dynamic (MD) simulations have been earlier applied in formulation design of NFCh for freeze-drying successfully by screening excipients based on their attraction to the surface of NFCh. The weakness of MD simulations is it can only model the fresh formulation system intend to freeze-dry, but not the actual freeze-drying process and the effect of it and the excipients to the material. To evaluate the protecting properties of excipients and therefore the accuracy of the MD simulations detailed information about changes in the physical state and molecular orientation of the formulation before and after freeze-drying is needed. Non-invasive and label-free Raman spectroscopy can be used to determine vibrational modes of molecules to investigate changes in molecular orientation of the material. The aim of this study was to investigate the possible molecular changes induced by freeze-drying of NFCh-based formulations utilizing Raman spectroscopy and evaluate the connection of the results to MD simulations. NFCh with different excipients was freeze-dried and physicochemical properties, rheology and Raman signal were measured before and after freeze-drying and compared to the literature of MD simulations. The principal component analysis (PCA) was done to the Raman spectra and differences evaluated. The spectra of all formulations differed before and after freeze-drying, and more detailed analysis was done to two most potential 0.8% NFCh based formulations, lactose 300 mM and lactose 250 mM + glycine 50 mM. They had great attraction to NFCh in MD simulations and very similar rheological properties before and after freeze-drying and reconstitution. The spectra of different state of both formulations different on areas between 400 - 500 cm-1 and 850 - 900 cm-1 based on PCA analysis contributing the mutarotation of lactose during freeze-drying and reconstitution. Freeze-drying and the absence of water molecules in NFCh formulation favor different ratios of β and α anomers than the fresh hydrated state which could be detected utilizing Raman spectroscopy. Therefore, Raman spectroscopy was confirmed to be a sensitive option to assess subtle changes in molecular orientation in fresh, freeze-dried, and reconstituted NFCh-based formulations, resulting in a detail knowledge of the molecular behavior of excipients which could be applied in MD simulations and design of better freeze-drying formulations in future.

Silmätipat ovat silmälääkkeiden yleisin annostelumuoto, mutta silmään imeytyvän lääkeaineen osuus jää pieneksi. Esimerkiksi glaukoomaa sairastaa maailmanlaajuisesti noin 80 miljoonaa potilasta, mutta glaukoomalääkkeiden hyötyosuus etukammiossa on vain 1–7 %. Päällimmäiset syyt heikkoon hyötyosuuteen ovat lääkeaineen nopea poistuminen silmän pinnalta mm. systeemiverenkiertoon sekä sarveiskalvon heikko läpäisevyys. Tästä syystä lääkeaineen permeaatio sarveiskalvon läpi on ollut tutkimuksen kiinnostuksen kohteena. Lääkeaine voi imeytyä silmän pinnalta etukammioon sarveiskalvon läpi passiivisella diffuusiolla ja aktiivisesti transporttereiden välityksellä. On vielä pitkälti epäselvää, että mitä transporttereita löytyy aktiivisena ihmisen sarveiskalvosta ja että kuinka paljon eri lääkeaineet hyödyntävät transporttereita imeytyessään. Tämän tutkimuksen tarkoituksena oli kartoittaa transportterien aktiivisuutta ihmisen sarveiskalvon epiteelisolulinjassa (HCE) sekä kanin eristetyssä sarveiskalvossa. Tutkimuksessa käytettiin radioleimattuja lääkeaineita, joita käytetään silmälääkkeenä (kliinisesti tai tutkimusvaiheessa) ja joiden tiedetään olevan eräiden transporttereiden substraatteja ja/tai inhibiittoreita. HCE-soluilla tehtiin in vitro aika-lineaarisuus- ja inhibitiosolunottokokeita ja kanin eristetyllä sarveiskalvolla permeabiliteettikokeita ex vivo. Tulosten mukaan kaikilla lääkeaineilla näyttäisi olevan aktiivista kuljetusta HCE-soluissa, mutta aktiivisen kuljetuksen osuus ja että mitkä transportterit ovat vastuussa kuljetuksesta, on epäselvää. Kiinnostava tulos oli, että inhibiittoreista MK-571 inhiboi metotreksaatin solunottoa HCE-soluissa sekä permeabiliteettia kanin sarveiskalvon läpi apikaali-basolateraalisuunnassa. Tulokset viittaavat influksitransportterien inhibitioon, mutta tarkempia johtopäätöksiä on hankala tehdä. Silmän ja sarveiskalvon transportteritutkimus on vielä alkutekijöissä ja lisää tutkimustietoa aiheesta tarvitaan.

Extracellular vesicles (EVs) are a very heterogeneous group of cell originated nanoparticles that act as mediators of intercellular communication. Accurate characterization of EVs is essential to enable their wider use and development as possible biomarkers, drug carriers, and vaccines. There is no validated reference material with EV-like properties currently available. A validated reference material would improve the reliability and reproducibility of EV studies. Nanoerythrosomes (NanoE) have been studied as a possible option for biological reference material. We aimed to further characterize and compare properties of NanoEs and erythrocyte-derived EVs (EryEV) and assess their stability concerning concentration and size distribution at most commonly applied storage temperatures, +4°C, -20°C, and -80°C for 12 weeks. Characterization was done using nanoparticle tracking analysis and flow cytometry. In addition, we studied the surface protein expression including CD235a, CD47, and CD41 of NanoEs and EryEV and conducted a preliminary cellular uptake test using PC-3 cells, CFSE-labeled NanoE, and EryEV particles. For both, NanoE and EryEV samples, 20°C was the worst storage condition. NanoEs stay stable at +4°C for a month and at -80°C, there were some drops in concentration during the 12 weeks of the experiment. EryEVs stay stable at +4°C and -80°C for 12 weeks. Both NanoE and EryEV particles seemed to be taken into the PC-3 cells, but due to problems with autofluorescence we conclude that confirming studies with different labeling protocols or another method need to be conducted. Both NanoEs and EryEVs samples had a significant number of CD47-positive particles.

Plasmidit ovat geneettisiä elementtejä, joita voidaan käyttää esimerkiksi geeninsiirtovektoreina. Transposonit ovat DNA-fragmentteja, joilla on kyky siirtyä genomissa paikasta toiseen. Tutkimuksessa käytettävä transposoni on piggyBac, joka on eristetty tupsumetalliyökkösen (Trichoplusia ni) soluista. Transpositiossa piggyBac tunnistaa ITR-osat (käännetty terminaalinen toistojakso) siirtäen osien välissä olevan DNA:n. Tutkimuksen tavoitteena oli tuottaa kaksi erilaista plasmidia. pAc5.1-piggyBac-plasmidiin sisällytettiin piggyBac ja pMT-In-EGFP-PB-ITR-plasmidiin ITR-osat sekä niiden väliin hygromysiiniresistenssigeeni sekä EGFP-geeni. BTI-Tn-5B1-4-solujen DNA:sta eristettiin piggyBac sekä ITR-osat ja ne siirrettiin plasmidiin pTOPO-piggyBac-R. Tästä plasmidista irrotettiin erilleen piggyBac ja ITR-osat, joista välivaiheiden kautta rakennettiin lopulliset plasmidit. Plasmidit rakennettiin pitkälti pilkkomalla DNA-fragmentteja restriktioentsyymeillä ja yhdistämällä niitä ligaatiolla. Plasmideja tuotettiin suurempia määriä siirtämällä niitä transformaation avulla E.Coli-soluihin lämpöshokkimenetelmällä ja eristämällä tämän jälkeen saadut plasmidit. Tuotettujen plasmidien onnistuminen varmistettiin pilkkomalla ne restriktioentsyymeillä ja tutkimalla DNA-fragmenttien kokoa agaroosigeelielektroforeesilla. Plasmidinäytteet myös sekvensoitiin osittain. Banaanikärpäsen (Drosophila melanogaster) S2-solut transfektoitiin kehitetyillä plasmideilla ja solukonsentraatioita sekä elinkelpoisuutta mitattiin 8 päivän ajan transfektion jälkeen. Tavoitteena oli hyödyntää EGFP-geeniä fluoresenssimittauksiin. Solunäytteisiin lisättiin kokeen aikana hygromysiini, jotta voitiin selvittää, olivatko viljellyt solut saavuttaneet hygromysiiniresistenssin. Tutkimuksen tuloksena plasmidit saatiin kehitettyä, mutta solukokeiden tulokset jäivät epäselviksi. Solunäytteissä ilmeni kasvatuksen aikana kontaminaatioita. Lisäksi EGFP-osia ei voitu luotettavasti mitata käytössä olleella laitteella. Transfektio tulee siis toistaa transposonisysteemin toiminnan tutkimiseksi. Lisäkokeilla voidaan selvittää tarkemmin kehitetyn transposonisysteemin mahdollisuuksia sekä toiminnan yksityiskohtia.