Browsing by discipline "Biopharmaceutics"
Now showing items 1-12 of 12
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(2017)In vitro liver cell models are important systems to study for example hepatotoxicity, which is one of the major causes for safety-related failures of drug candidates. 2D cell culture-based tests for compound screening are standard procedures in drug discovery, but reliable data for in vivo studies is hard to obtain because cells in a monolayer are in unnatural microenvironment. In turn, cells in 3D culture systems have more natural interactions with other cells and extracellular matrix, and their responses to drugs resemble more in vivo responses. In drug discovery and development, automation of the cell culture processes and compound screening saves time and costs, and improves the consistency and sterility of the procedures. As 3D cell culture systems are becoming more compatible with automation, they are also more promising to be used in drug discovery and development. The aim of the study was to develop and optimize automated processes for preparing 3D cell cultures into 96-well plates. HepG2, a human liver cancer cell line, cultures in nanofibrillar cellulose were prepared into well plates manually or by using automated liquid handling system. To our knowledge, this was the first time that automated processes for cell seeding into NFC were used to prepare 3D cell cultures. Cell seeding steps that could be automated were identified and optimized based on visual analysis of the wells and viability of the cells after seeding. After optimization, manual and automated processes were compared by studying cell viability, morphology and functionality. Alamar blue assay, Live/Dead assay and fluorescence-activated cell sorting were used to study cell viability, and F-actin staining, differential interference contrast microscopy and light microscopy were used to investigate cell morphology. Cell functionality was analyzed by studying albumin secretion. Cells seeded by using automation secreted normal amounts of liver-specific albumin. Cells maintained viability, morphology and functionality for four days after seeding although the results of viability varied. Alamar blue assays showed decreased development of viability although viability of manually seeded cells increased, but in other experiments the results from cultures seeded manually or by using automation were more similar. For example, lower viscosity of nanofibrillar cellulose and longer waiting time of cells at room temperature before automated processes are possible explanations for differences, as well as the natural variability in cell studies. In the future, automated high-throughput screening of compounds could be performed in 3D cell cultures prepared by using automation. That would save time and costs, and increase the correlation between in vitro and in vivo studies.
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(2020)Monoclonal antibodies (mAbs) are widely used in the treatment of several diseases such as cancer and autoimmune diseases. Due to their high prices and growing consumption, therapeutic mAbs have become potential targets of falsification. This generates a demand for quick and efficient analytical procedures for identifying and characterizing mAbs in a case of suspected falsification. The structure of therapeutic mAbs consists of human or murine IgG framework, where unique complementarity determining regions (CDRs) are engineered with different recombinant techniques. Given the complex nature of the mAbs, they must be identified using multiple complementary analytical methods. Ten full-sized therapeutic mAbs, Fab-fragment abciximab and CTLA4-Fc-fusion protein belatacept were studied in order to find analytical methods for efficient characterization and identification. All studied antibodies were characterized by their charge and molecular weight by isoelectric focusing (IEF) in polyacrylamide gels, native and reduced SDS-PAGE, and size exclusion chromatography (SEC). Six mAbs, abciximab and belatacept were digested with trypsin, and the cleaved peptides were further analysed by RPLC-MS. In addition, quantification methods including SEC peak area measurements and Bradford protein assay were performed for all antibodies. As expected, SDS-PAGE of non-reduced and reduced mAbs gave little distinction between the mAbs. Both methods were however shown to be useful in the identification of the mAb nature, as they confirmed the presence of heavy chains, light chains, and disulfide bonds. IEF showed potential in mAb identification, as clear, partly distinguished patterns of charge variants were obtained. However, some improvements to the pH gradient are needed to enable better separation and pI estimation of basic variants. Determination of molecular size with SEC was found to be difficult, as there seemed to be no consistency between the calculated molecular weights based on measured elution times, and the theoretical molecular weights. Nevertheless, SEC brings added value in mAb quantification and detection of protein aggregation and fragmentation. Finally, RPLC-MS analysis of tryptic peptides resulted in mAb identification, with the measured sequence coverage of 87-97 %. Identification process may be enhanced by focusing on the known CDR-peptides prior the constant frame peptides. Given the structural similarity of therapeutic mAbs, identification of an unknown mAb requires combination of multiple analytical methods. If available, the use of reference mAb product obtained from a reliable source is recommended, as the identification may be based on comparative analyses using simpler analytical steps, e.g. IEF, SDS-PAGE and SEC. If no reference product is available, identification of the mAb requires peptide mapping and determination of the CRD sequences by RPLC-MS analysis. Further research is needed to find a suitable set of analytical methods for identification of all therapeutic mAbs.
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(2019)Background: The World Health Organization (WHO) outlined in their report published in 2014 that antimicrobial resistance (AMR) is a real public health threat worldwide and the actions against it should be taken. Otherwise, the post-antibiotic era where common community-acquired infections can lead to death, could hypothetically become true. The discovery and development of novel antibiotics (ATBs) against Gram-negative bacteria (GNB)-related infections is difficult due to a dual defence mechanism: the extra protection barrier called the outer membrane and efflux pumps which GNB utilize to protect themselves against external noxious compounds. Efflux pumps are expressed at the basal level in GNB, such as E. coli, but when exposed to sub-inhibitory concentrations of ATBs and the intrinsic extruding capacity is exceeded, GNB start overexpressing these “so-called” multi-drug resistance (MDR) efflux pumps. The most abundant and studied MDR efflux pump in E. coli is a tripartite protein complex AcrAB-TolC which traverse through the bacterial cell envelope and is capable of extruding a broad range of structurally unrelated compounds, thus leading to cross-resistance against several classes of ATBs. It has been suggested that antibacterial activity of existing ATBs could be restored again by inhibiting increased efflux activity through efflux pump inhibitors (EPIs). Objectives: Define the optimal assay conditions and a positive control (EPI) to be used in high throughput screening (HTS) of novel EPIs. The assay consists of one E. coli strain of clinical relevance with high intrinsic efflux activity, one ATB and one EPI, both of them at specific concentrations defined during this study. Methods: The intrinsic efflux activities of seven E. coli strains were studied by Hoechst 33342 (H33342) accumulation assay, both in the absence and presence of five commercially available EPIs. The same assay was used in the dose-response studies in which an optimal concentration of EPIs was identified for further to be utilized in the checkerboard assays. The minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to Clinical and Laboratory Standards Institute. The synergistic effects of ATB and EPI in terms of decreasing the intrinsic MIC value of the ATB were determined in the checkerboard assays partially performed by the Biomek i7 Automated Workstation. The data was analysed by using Microsoft Excel and IBM SPSS Statistics, version 25. Results and discussion: E. coli ATCC 25922 had statistically significantly the highest efflux activity of all wild-type pathogenic and non-pathogenic E. coli strains. However, when H33342 accumulation assay was carried out in conjunction with EPIs, E. coli BAA1161 (uropathogenic strain) had the highest median increase in the intracellular level of H33342. Mefloquine showed to be the most potent of all EPIs at the tested concentrations. However, mefloquine increased the intracellular H33342 accumulation even in efflux-deficient E. coli JW5503 (ΔtolC), thus possible additional modes of action or inhibitory activity towards other efflux pumps might exist. Dose-response studies carried out in ΔtolC E. coli JW5503 suggested that CCCP at 1.25 g/ml and mefloquine at 0.5 g/ml were the optimal concentrations. However, for mefloquine, when tested at 0.5 g/ml, the intracellular level of H33342 was not increased in six remaining E. coli strains. Therefore higher concentrations up to ½ MIC were tested in the checkerboard assays. In the antibacterial susceptibility testing, E. coli BAA1161 was the only strain showing resistance to tetracycline and piperacillin, resulting in MIC ratios (MIC wild-type/MIC mutant) of 512 to 2048. Piperacillin and ofloxacin, which showed a MIC ratio of 4 in two E. coli strains, were chosen to the checkerboard assays in which mefloquine reduced the intrinsic MIC of piperacillin by 16-fold and CCCP by 32-fold in E. coli BAA1161. Conclusions: E. coli BAA1161 was chosen to be used as a model strain in HTS due to the highest median increase in intracellular H33342 accumulation and also for being the only strain with resistance towards the ATBs tested. Mefloquine (16 g/ml) was the EPI of choice for the positive control in HTS because the synergistic effects observed between piperacillin and mefloquine were most probably explained by efflux pump inhibition and not by antibacterial activity of mefloquine itself. Piperacillin (256 g/ml) was selected to be used as an ATB in HTS because it was the only ATB which was potentiated by the tested EPIs.
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(2020)The human immune system can provide a powerful tool in developing therapies against various cancers. Even though the idea of an immune system actively searching for and disposing of potential mutated tumor cells is over a century old, only recent developments in various fields such as mass spectrometry, immuno-checkpoint blockade strategies and in silico modelling have enabled the realization of the full potential of recruiting immune system to fight cancer and the possibilities of personalized therapies. These therapeutic methods, including but not limited to oncolytic virus therapies, T-cell therapies and cancer vaccines, are based on the body’s ability to recognize mutated antigen peptides presented on the cell surface by MCH-receptors (also known as HLA-receptors in humans) and the disposal of the malignant cells by cytotoxic T-cells. Thus, the capability to map the individual HLA-presented peptidome and differentiate the immunogenic peptides is a foundation for this plethora of therapies and is in focus of ongoing research. This master thesis is a part of a project aiming to set up immunoaffinity-purification/MS based method in order to analyse the ligandome and determine T-cell recognized cancer associated antigens from tumor cells. Objectives of the work: 1. Characterizing tumor cell lines. 2. Immunological assay set up. 3. Collecting cell culture material for the ligandome affinity purification. 4. In silico prediction if the immunogenicity of selected peptides and assessing their source proteins. Methods used: 1. Cell culture. 2. FACS-analysis. 3. MTS-viability assay. 4. Immunological assays (ELISA, ELISPOT). 5. Immunological bioinformatics analysis tools (IEDB) and database search (UniPROT). Results: 1. Flow cytometric analysis provided essential information of the cell line HLA-1 expression. Additional information of PD-L1 expression can be used to evaluate cell line’s immune-evasion abilities. Preliminary MTS assay is used to determine linear range and optimal time frame for the PBMC/cancer cell co-culture killing assay. 2. Interferon γ cytokine secretion was determined by ELISPOT to assess PBMC response against known antigens in a preliminary experiment to approximate usable range for the following antigen specific PBMC assays. ELISA is used to confirm the presence of HLA-I receptors in the ligandome affinity purification eluates and to estimate the efficacy of purification. 3. Feasibility of in silico methods in the prediction of immunogenic peptides was explored. The experiments provided information that can be applied to the further development of the immune ligandome discovery project. In silico methods were successfully used to characterize previously identified HLA-restricted peptides and one previously identified immunogenic T-cell epitope. Even if the data acquired in silico can be considered only nominally verified at this stage, the results are encouraging.
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(2020)Pharmacogenomic test are laboratory tests that are performed in order to find out what kind of a variable response to a specific drug is most likely for a patient, or in order to determine the background of a deviating adverse drug reaction. Individual drug responses can be caused by a variation in a gene that codes for a protein that is involved in the pharmacokinetics or the pharmacodynamic response of the drug. These genes are called pharmacogenes. Pharmacogenomic tests are most commonly DNA tests that look for the most frequent variations in the pharmacogenes or variations that are associated with variable drug response even if located in the non-coding region of the DNA. Pharmacogenomics is a rapidly emerging branch in scientific research, and the number of known pharmacogenes and available pharmacogenomic tests is constantly growing. Pharmacogenomic tests can be helpful in avoiding ineffective medication, and decrease the probability of severe adverse drug reactions. Several international specialist consortiums, such as Clinical Pharmacogenetics Implementation Consortium (CPIC) and Dutch Pharmacogenetics Working Group (DPWG), have published guidelines regarding dosing of spesific drugs or drug classes when a patient has a certain variation in one of known pharmacogenes. Due tothe amount of knowledge constantly increasing, use of pharmacogenomic tests is believed to expand in the healthcare. The aim of this study was to examine the use of pharmacogenomic test in Finnish healthcare during years 2016 and 2017. A questionnaire was send to Finnish laboratories by email. Purpose of the questionnaire was to collect information of the amount of available and performed pharmacogenomic tests and respondents opinions regarding the future of pharmacogenomic tests. Questionnaire was carried out in co-operation with Ministry of Social Affairs and Health and Division of Pharmaceutical Biosciences in University of Helsinki, using Webropol survey tool. The survey was renewed the following year, without major alterations to its content. Unfortunately, due to the somewhat low response rate of the survey in both 2016 and 2017, the results of the survey cannot be considered to sufficiently represent the collective views of the target group. However, from the giver replies we can rather reliably conclude that pharmacogenomic tests were not used in high volume during years 2016 and 2017. The laboratories reported mainly very small amounts of performed tests. The most abundantly reported test was TPMT gene test, regardless of the year. In spite of the low test volume, respondents were, however, widely unanimous that the use of pharmacogenomic test and their significance in healthcare are very likely to increase in the near future.
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(2018)Cancer afflicts an ever-growing number of people globally each year. In part due to a complex pathophysiology where much is still unknown, the need for new cancer treatments has been persistent, fuelled further by the issue of treatment resistance. An emerging field holding much promise in oncology is immunotherapy, a subgroup of which is oncolytic virus treatments. These treatments utilize the inherent or acquired ability of certain viruses to selectively replicate in tumor cells to fight cancer. One of these viruses is the adenovirus. With these viruses it is possible to modulate the immune response e.g. through the expression of certain genes. The thesis focuses on genetically arming an oncolytic adenovirus in an effort to enhance treatment efficacy. The transgene of choice is the CD40 ligand (CD40L), a costimulatory molecule capable of aiding in the development of systemic antitumor immunity. Adenoviruses have previously been designed expressing the CD40L, however, a novel aspect was introduced with the design and incorporation of a soluble a form of the protein. The main aim of the study was the construction of four functional oncolytic adenoviruses, encoded with either the human or mouse variants of the two CD40L proteins (full-length and soluble). Successful completion required protocols for the cloning, bacterial colony screening, and primary virus production to be established. Insertion of the CD40L transgenes into the E3-gp19k region of the chosen Ad5Δ24 backbone was first attempted with the traditional approach of homologous recombination. The method that ultimately proved successful was a one-step Gibson Assembly® reaction. Screening the bacterial colonies with colony polymerase chain reaction, the potential CD40L positive clones underwent restriction analysis to affirm the presence of the transgene in the viral genome, as well as the retainment of critical elements. Two out of three recombined plasmids carrying the full-length CD40L proceeded to transfection and virus propagation in A549 cells, after which the presence of the adenovirus and CD40L expression was confirmed with immunostaining. Finally, a protocol was successfully established by the construction of one of the intended four viruses. The protocol entails all the main steps from cloning until primary virus production, additionally offering the option of applying it to the genetic arming of the Ad5Δ24 with other transgenes of interest. In terms of future perspectives for the project, following construction of the remaining viruses, the intentions are to validate transgene expression and functionality for all constructs, as well as compare the immunogenicity between the full-length and soluble CD40L. In the event of promising results, the project will hopefully proceed to in vivo studies.
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(2018)Liposomes are nanosized drug delivery vesicles composed of phospholipid membranes. They present an attractive drug delivery system due to their bioavailability and flexibility. Liposomes can be prepared by different techniques. They can carry both hydrophobic and hydrophilic molecules and their surface can be modified with targeting molecules. Coating the liposome surface with the PEG derivative makes their pharmacokinetics easier to predict. There are several liposome-based medicinal products already on the market. Triggering of drug delivery systems by different external or internal stimuli allows precise control of drug release. Light-triggered drug release is an attractive alternative due to the easy control and regulation of the stimulus. The problem with light-triggered therapy has previously been the need to use high-energy ultraviolet light that penetrates badly to the tissues and is not safe. In TTA-UC process the low-energy red or green light is converted to high-energy blue light. In this process photosensitive molecules are excited by visible light and after that the energy is transferred from sensitizer to annihilator molecules. Collision of two annihilators leads to the excitation of the other molecule while the other returns back to its general energy state. The excitation breaks up with fluorescence. In this process the highly permeable and safe red light is converted to blue light which has enough energy to induce drug release. The aim of this work was to optimize liposomal preparation method and prepare a pegylated and stabile liposome formulation for TTA-UC process. Hydrophobic light sensitive molecules were loaded into the phospholipid membrane as much as possible. One of the problems in this work was to find proper methods to measure the concentrations of these molecules. The lipid composition for formulation was chosen after thermostability studies. As a quality control, the size, capability to load calcein and phase transition temperature of liposomes were measured. The quality control of light sensitive molecules was operated too. In this work, the formulation for TTA-UC was prepared. In further studies TTA-UC process happened with sufficient efficacy. The formulation was pegylated and stable in physiological conditions and the concentrations of the molecules were high enough. This was the very first time to get TTA-UC to happen in this kind of liposome formulation that may be useful as a drug carrier. Long-term stability studies and further optimization of TTA-UC method are needed in the future. Some drug release studies are important to arrange in the future, too.
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(2019)Background: Antibiotics have been an important factor in the dramatic decrease of infectious disease mortality in the 20th century. Bacteria are, however, very quick to respond to the changes in their environment because of their short life cycle. Thus, the development of bacterial antibiotic resistance is a natural consequence of the enormous worldwide antibiotic use. The current situation is that the antibiotic resistance develops faster than novel antibiotics are found and developed. The three main resistance strategies of Gram-negative bacteria are: modification of the antibiotic target, enzymatic inactivation of the antibiotic and reduce of the intracellular antibiotic concentration by changing the function of the outer membrane. To decrease the intracellular antibiotic concentration bacteria use efflux pumps. RND efflux pumps are the most important family of efflux pumps regarding antibiotic resistance. They typically function as a part of a tripartite structure which allows the efflux of antibiotics to the extracellular space. Multiple inhibitors have been developed against RND efflux pumps but none has reached the clinical stage of drug development. Objectives: Development and testing of a 384-well plate method for screening efflux pump inhibitors for E. coli (BAA1161) efflux pumps. Methods: Verifying that the absorbance measurement is a sensitive enough method for measuring the bacterial (BAA1161) growth in 384-well plate format. The antibiotic chosen to be used in the screening method was piperacillin and the positive control efflux pump inhibitor was mefloquine. Determining the minimum growth inhibiting concentrations (MICs) of piperacillin and mefloquine in 96- and 384-well plate formats. Verification of the synergistic growth inhibitory effect of piperacillin and mefloquine with the checkerboard method in 96- and 384-well plate formats. Determining the positional effect in the 384-well plate. Determining the highest DMSO concentration without effect on the growth of BAA1161. Screening of 126 natural compounds in 384-well plates to test the developed method. Screening was done in quadruplicates based on the growth inhibitory effect of the natural compounds when combined with piperacillin. Dose-response assay was conducted in combination with and without piperacillin with the compounds that showed growth inhibiting effect during screening. Results and discussion: Absorbance measurement was sensitive enough method for measuring the BAA1161 growth in the 384-well plate. MIC value of mefloquine was 32 μg/ml in both plate formats. Piperacillin’s MIC was 1024 μg/ml in the 96-well plate, but on the 384-well plate there was variation in the MIC. Piperacillin and mefloquine showed synergistic effect on BAA1161 growth inhibition in the checkerboard assays. Positional effect could not be determined, because of the variation in the BAA1161 growth inhibition effect of piperacillin. This randomly occurring phenomenon were piperacillin inhibited BAA1161 growth completely or almost completely with sub-MIC concentration was encountered in all the subsequent experiments in the 384-well plate format. One possible reason for this phenomenon, occuring in the 384-well plate format, could be piperacillin heteroresistance of BAA1161 strain. In the test screen, four compounds, which all included gallic acid ester, showed promising activity. These compounds were: epigallocatechin gallate, hamamelitannin, isopropyl gallate and octyl gallate. In the dose-response assay, hamamelitannin’s and octyl gallate’s effect was synergistic with piperacillin. Conclusions: The developed method can be used to screen novel efflux pump inhibitors. However, to increase the reliability of the method, further optimization is required to eliminate the variability in the effect of piperacillin. When plate format of a method is changed, factors which could affect the functionality of the method in the new format should be carefully assessed. Based on the test screed, gallic acid esters are interesting compounds which combined effects with antibiotics should be studied in the future experiments.
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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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(2019)Background and objectives: Cells secrete extracellular vesicles (EV) and it has been found that cells communicate via EVs. EVs are liposome-like vesicles. Membrane is consisting of a lipid bilayer and hydrophilic moiety is inside the vesicle. It has been found that EVs carry e.g. nucleic acids, lipids and proteins. The aim of this master thesis was to determine whether EVs can transport non-coding RNA (siRNA) into the central nervous system through the blood-brain barrier. In the literature review, investigated methods which has been used to load siRNA into the EVs and how EVs are transported through the blood-brain barrier. The aim of the experimental part was to produce and isolate EVs and to load FAM-labeled dsDNA and siRNA into EVs by physical methods such as sonication and electroporation. Fluorescence measurements were taken to demonstrate FAM-labeled DNA loading into EVs and the functionality of the siRNA-loaded EVs was measured by measuring the expression level of the gapdh gene. Methods: Extracellular vesicles were produced in ARPE-19 and PC-3 cells. EVs were isolated from the cell culture medium by two-step differential centrifugation (DC) and further purified by gradient centrifugation (GC) by using the OptiPrep™-reagent. OptiPrep™-reagent was purified by Amicon 10kDa filtration tubes. The average particle size and size distribution of the isolated EVs were determined by NTA analysis, protein concentration was measured by colorimetric BCA method and EVs were characterized by Western blot method using HSP70 and CD9 antibodies. EVs were loaded with 21 bp length FAM-labeled dsDNA or siRNA by sonication or electroporation. Free nucleic acid and OptiPrep™-reagent were purified from EVs by the size-exclusion chromatography with Sephacryl (S-300) column. Loading efficient of the EVs were studied by measuring the fluorescence (ex 485 nm, em 520 nm) and qPCR method was used to demonstrate the functionality of the siRNA loaded EVs. In qPCR, the expression level of the gapdh gene was measured in dividing ARPE-19 cells. Results: DC and GC purified ARPE-19 and PC-3 EVs had an average particle size of about 140 nm and were successfully characterized by Western blot method. PC-3 EVs were produced in the bioreactor and the yields were enough for loading experiments. ARPE-19 cells produced only small amounts of EVs in culture flasks. The size-exclusion chromatography was a good method to purification free nucleic acids from EVs. The sonication method did not cause EVs to be degradation under the conditions used. Based on fluorescence measurement, FAM-labeled dsDNA could not be loaded into EVs. The functionality of siRNA-loaded EVs could not be demonstrated in ARPE-19 cell experiments. After electroporation large number of EVs were lost and this method of loading siRNA into EVs did not proved to be suitable. Conclusions: ARPE-19 EVs must be produced in the bioreactor to produce enough EVs for loading experiments. The EV purification protocol should be further optimized since the recovery-% of EVs were low after several purification steps. The size-exclusion chromatography is suitable for the purification of the free siRNA from EVs, but the chromatography method needs further optimization and miniaturization. Loaded EVs should be produced by aseptically or alternatively sterilized prior to ARPE-19 cell assay. Physical loading method, such as sonication, can be scaled to larger scale. Sonication method should be optimized e.g. by experimenting with higher temperatures and longer sonication times. The probe sonicator should be tested instead of the water bath sonicator. According to the literature review, the use of extracellular vesicles as carriers for biomolecule delivery into the central nervous system seems to be promising.
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(2018)Liposomes are nano-sized vesicles in which the aqueous phase is surrounded by lipid-derived bilayer. They are excellent drug vehicles for example in ocular drug delivery because they can, among other things, increase the bioavailability and stability of the drug molecules and reduce their toxicity. Liposomes are known to be safe to use, because they degrade within a certain period of time and they are biocompatible with the cells and tissues of the body. Owing to its structure, the surface of liposomes can also be easily modified and functionalized. Light-activated ICG liposomes allow drug release in a controlled manner at a given time and specific site. Their function is based on a small molecule called indocyanine green (ICG) which, after being exposed to laser light, absorbs light energy and thereby locally elevates the temperature of the lipid bilayer. As a result, the drug inside is released into the surroundings. The blood circulation time of liposomes has often been prolonged by coating the liposomes with polyethylene glycol (PEG). Although PEG is generally regarded as a safe and biocompatible polymer, it has been found to increase immunological reactions and PEG-specific antibodies upon repeated dosing. Conversely, hyaluronic acid (HA), is an endogenous polysaccharide, which is present in abundance for instance in vitreous. Thus, it could serve as a stealth coating material which extends the otherwise short half-life of liposomes. One of the main objectives of this thesis was to find out whether HA could be used to coat liposomes instead of PEG. In order to prepare HA-coated liposomes, one of the lipid bilayer phospholipids, DSPE, had to be first conjugated with HA. For the conjugation, potential synthesis protocols were sought from the literature. Ultimately two different reductive amination-based protocols were tested. Consequently, the protocol in which the conjugation was achieved via the aldehyde group of HA, proved to be working. Thereafter, HA-coated liposomes were prepared by thin film hydration from the newly synthesised conjugate as well as DPPC, DSPC and 18:0 Lyso PC. Calcein was encapsulated in the liposomes. HA-covered liposomes were then compared with uncoated and PEGylated liposomes by examining their phase transition temperatures, ICG absorbances, sizes, polydispersities, and both light and heat-induced drug releases. The aforementioned tests were also conducted when the effects of the HA and ICG doubling were examined and the possibility to manufacture HA liposomes with small size was assessed. HA-liposomes showed similar results as PEG-coated liposomes. In addition, successful extrusion of HA-liposomes through a 30 nm membrane was also demonstrated in the results. Doubling of HA did not significantly affect the results. In contrast, increasing the molar amount of ICG by double caused spontaneous calcein leakage even before any heat or light exposure. Based on these findings, HA could work as a coating material instead of PEG, yet further studies are required for ensuring this conclusion. The other key objective was to evaluate the stability of four different formulations, named as AL, AL18, AL16 and AL14, in storage and biological conditions. Based on the differences in the formulation phospholipid composition, the assumption was that AL would be the most stable of the group and that the stability would decrease so that AL18 and AL16 would be the next most stable and eventually AL14 would be the least stable formulation. As in the previous study, the liposomes were prepared by thin film hydration with calcein being encapsulated inside the liposomes. In the storage stability test, liposomes were stored in HEPES buffer at either 4 °C or at room temperature for one month. In the test conducted in physiological conditions, the liposomes were added either to porcine vitreous or fetal bovine serum (FBS) and the samples were incubated at 37 ºC for five days. Regardless of the experiment, phase transition temperatures as well as light and heat-induced drug releases were initially measured. As the test progressed, calcein release, ICG absorbance, size, and polydispersity were measured at each time point. The initial measurements confirmed the hypothesis about the stability differences of tested formulations. In the storage stability test, all formulations, except AL14, appeared to be stable throughout the study and no apparent differences between the formulations or temperatures were observed. On the other hand, the stability of liposomes stored in biological matrices varied so that the liposomes were more stable in vitreous than in FBS and the stability decreased in both media as expected.
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(2018)Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.
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