Faculty of Pharmacy

 

Recent Submissions

  • Zhang, Feng (Helsingin yliopisto, 2021)
    By the virtue of the divers physicochemical properties, nanomaterials have emerged as a powerful platform to improve the pharmacokinetic properties of drug molecules. Furthermore, in view that the created or constructed materials own the same size level of biomacromolecules, and can be endowed with various biochemical functions, nanotechnology has been treated as the most promising technology to develop smart therapeutics with flexible articifal controllability to innovate the current medicine. Generally, the anchor or the breakthrough point of these technologies depends on the nanomaterials-based drug delivery systems (DDS), which has been in tremendous development for more than thirty years. However, clinical transition of DDS are facing great challenges related to the insufficient targeting accumulation in the cells/tissues. The in vivo delivery of nanotherapeutics is a multi-stage process and needs to conquer multiple biological barriers. In this case, conventional DDS is not competent enough to cope with the various biological barriers. Thus, the transformable design of DDS with tunable surface properties in responsive to stimuli-signals at different barriers are in urgent need. In this thesis, the focus was on constructing DDS with different stimulus and functions to achieve the task-oriented NPs’ transformation, including surface charge inversion, sequential antifouling surface, in situ size modulation and multi-stage signal interaction. Firstly, it was fabricated a PSi DDS with receptor-mediated surface charge inversion. The negatively charged surface can convert into positive charged surface in response to cancer micro- environment, driven by the AS1411-nucliolin interaction. Secondly, it was modified the biotin- PEI nanoparticles with acrylates-ortho-nitrobenzyl-PEG5000, which further acted as the primary antifouling surface to prevent the formation of protein corona and avoid off-targeting effect. After UV-irradiation, the PEG surface can be cleaved to generate carboxyl group on the biotin- PEI surface, forming a secondary zwitterionic anti-fouling surface. This dual-antifouling modification can efficiently avoid protein adsorption on the NPs’ surface in human serum. Moreover, the secondary zwitterionic surface can guarantee the effective exposure of active targeting segments for improving cell uptake. Simultaneously, the reduced size facilitates deep tissue penetration of the NPs. Thirdly, it was constrcuted a photo-driven size tunable DDS, which can increase the size after tumor accumulation in situ to prolong the tumor retention time and also to improve cancer cell uptake. Finally, it was developed a multistage signal interactive system on NPs through integrating the Self-peptide and YIGSR peptide into a chimeric form, with a hierarchical signaling interface involving “don’t eat me” and “eat me” signals. This biochemical transceiver can act as both signal receiver for amantadine to achieve NP transformation and signal conversion, as well as the signal source with different signals by reversible self-mimicking. Throughout chemical and biological testing, it was demonstrated these designed DDS have efficient signal-induced transformation behavior and enhanced controllability of NP-cell interactions for improving the cancer therapeutic efficacy. Overall, this dissertation provides a new insight of targeting drug delivery and nano-tools to facilitate clinical transition of nanomedicines.
  • Zanjanizadeh Ezazi, Nazanin (Helsingin yliopisto, 2021)
    Biomaterials science denotes a multidisciplinary science combining materials engineering, biomedical engineering, and biology. Tissue engineering is defined as an engineering system field using biomaterials to regenerate, restore tissue, or improve its function. This thesis focuses on the fabrication of scaffolds and patches for bone and heart regeneration using a conductive polypyrrole (PPy) polymer. PPy is widely used in different tissue engineering applications due to its biocompatibility, high electrical properties and conductivity, ease to synthesize, and environmental stability, such as in water and air. First, the role of this polymer in attracting protein and osteoblast cells was investigated. The bone scaffold’s mechanical properties using PPy were analyzed. The results showed no difference between conductive and non-conductive bone scaffolds, presenting almost the same young modulus between the two systems. In addition, more proteins were adsorbed on the surface of the conductive polymers. The same results were obtained in conductive cardiac patches, which showed higher blood coverage on the conductive 2D patch using PPy compared to the non-conductive one. In addition, cellulose was used to 3D print and fabricate a porous conductive cardiac patch. The in vitro investigations showed that cell attachment and proliferation on the conductive bone scaffold and cardiac patches were higher. Biomineralization was induced in simulated body fluid on the bone scaffold’s conductive surface. PPy was also combined with bacterial cellulose (BC) to make conductive hydrogel for the treatment of myocardial infarction. The positive chains of PPy improved the loading of negative charged drug-loaded nanoparticles (NPs). All four systems were successfully developed, fabricated, and loaded with different drugs with different methods to make multifunctional bioengineered systems for drug delivery applications. The results showed that PPy controlled the release of the vancomycin, an antibiotic, and the GATA4-targeted compound 3i-1000, inside the pores of 3D bone scaffold and 3D printed cardiac patch, respectively, which suitable for long-term therapy. Furthermore, bone scaffold, 2D cardiac patch, and conductive BC were combined with microparticles of silica, NPs of porous silicon, and acetylated dextran, respectively, presenting the high potential of in situ drug delivery system enabling and dual delivery of drugs. Overall, this thesis demonstrated that PPy-based biocomposites are successfully developed showing high biocompatibility and high functionality within hard scaffolds and soft patches, and thus, they are very promising platforms for future bone and heart tissue regeneration applications in vivo studies.
  • Snirvi, Jasmi (Helsingin yliopisto, 2021)
    Since wound healing represents an increasing burden and a clinical challenge for healthcare professionals, there is a continuous need for more efficient therapy methods. Although current methods include wound dressings, skin grafts, and more recently skin substitutes that often offer a working therapy method, lack of transplantation material and time-consuming preparation time have limited their use. The aim of this thesis was to study the interactions between human adipose-derived mesenchymal stromal cells (hASCs) and wood-derived nanofibrillar cellulose (NFC) dressings and further to evaluate the function of NFC dressing as a cell delivery vehicle in vitro and a wound-healing process in vivo. Additionally, the biocompatibility of two different NFC hydrogel types was evaluated in vitro and their effects on wound healing in vivo. First, the interactions between hASCs and three differentially manufactured NFC dressings (Types 1, 3, and 4) were studied by analyzing cell characteristics. Furthermore, the immunological properties of hASCs were evaluated using enzymelinked immunosorbent and monocyte migration assays. Second, Type 4 NFC dressing was studied clinically by comparing it with a commercial wound dressing when treating skin graft donor site wounds. Third, the biocompatibility of native and anionic NFC hydrogels was evaluated in vitro by analyzing hASCs’ viability and morphology and in vivo using a splinted full-thickness mouse wound healing model. Three different NFC dressings demonstrated different effects on hASCs. Based on our studies, Type 3 NFC dressing offered a functional cell culture platform and potential cell delivery vehicle for hASCs in a cell density-dependent manner without affecting hASCs’ gene or protein expression levels. Further studies suggested that immunological properties of hASCs were also maintained regarding secretion of vascular endothelial growth factor and transforming growth factor-b1 as well as their positive effect on monocyte migration capacity when cultured on top of Type 3 NFC dressing. In contrast, Type 1 and Type 4 NFC dressing did not support the cell culture of hASCs. However, when studied clinically, Type 4 NFC dressing performed comparably with a commercial wound dressing. No statistically significant differences were observed regarding wound healing time determined as the detachment day of the dressing or in percentage of skin epithelialization. Moreover, no difference was observed between Type 4 NFC dressing and commercial wound dressing or between Type 4 NFC dressing and healthy skin regarding skin elasticity in terms of viscoelasticity, elastic modulus, and transepidermal water loss six months postoperatively. Importantly, Type 4 NFC dressing also presented a trend toward less pain experienced by the patients during treatment. Similar to NFC dressings, both native and anionic NFC hydrogels were biocompatible in vitro and supported hASC spheroid formation and high cell viabilities during three-dimensional cell culturing. In vivo, neither NFC hydrogel showed statistically significant differences in wound healing compared with untreated control wounds, and presented normal inflammatory response, granulation-tissue maturation, wound closure rate, re-epithelialization, and vascularization with no signs of fibrosis. To summarize, NFC dressing offers a functional cell culture platform for hASCs and potential therapy material for patient wound treatment. Similarly, native and anionic NFC hydrogels appear to be promising materials for wound treatment. This thesis describes the potential biomaterial NFC for wound healing treatment that is easy to prepare, modify, and use in different forms. NFC dressing is applicable both as a cell scaffold and for patient wound treatment, while native and anionic NFC hydrogels appear to be promising materials for wound treatment. This thesis further describes cell-biomaterial interactions between hASCs and NFC that show relevance for future cell therapies. These findings offer insight into different wound therapy effects of NFC-based materials as well as their possible effects on hASCs, which could improve (and have already) the design of new wound healing therapy methods.
  • Oliveira Martins, João Pedro (Helsingin yliopisto, 2021)
    Managing a chronic disease throughout the lifetime is an enormous challenge, and diabetes mellitus (DM) is no exception. For the time being, DM cannot be cured but only managed, if and when appropriately diagnosed. However, the vast majority of the currently available glucose-lowering interventions provide diabetic patients with only invasive and distressing treatment opportunities, and an undeniably poor compliance to the therapy. Therefore, it is of utmost importance to develop alternative treatment modalities. Success in the oral delivery of anti-diabetic proteins and peptides could represent a paradigm shift in the management of DM and improve the quality of life of millions of patients. Over the years, nanoparticles (NPs) emerged with great potential to carry and deliver drugs in a precisely tuned and controllable manner. Moreover, nanocarrier systems aimed at oral drug delivery have demonstrated the ability to overcome a variety of biochemical, physical, and mechanical barriers that are intrinsic to the physiological functioning of gastrointestinal tract (GIT). Yet, drug absorption at the intestinal microenvironment is still severely mitigated due to the presence of the tightly organized and restrictive cellular barrier of the intestinal epithelium. The exploration of the mechanisms by which molecules presented in the lumen can be naturally transported into the blood circulation has recently drawn attention to the neonatal Fc receptor (FcRn). FcRn is a membrane receptor responsible for maintaining the homeostasis of immunoglobulin G (IgG) and albumin, via mechanisms of recycling and transcytosis. Hence, NPs functionalized with FcRn-targeting ligands could hijack the FcRn transport pathway to promote the transport of drugs across the intestinal cell wall. Therefore, the main aim of this dissertation was to design and develop different FcRn-targeted NPs for efficient oral anti-diabetic drug delivery. Porous silicon (PSi) NPs were used as core drug carriers due to the unprecedented advantages shown as drug nanocarriers. Recombinant human insulin and GLP-1 were loaded into the PSi NPs as model anti-diabetic peptides. Drug-loaded PSi NPs were further encapsulated into different pH-responsive polymers (hypromellose acetate succinate and lignin), via different preparation techniques (emulsification-evaporation, microfluidics and desolvation). The surface of the NPs was functionalized with either albumin or the Fc portion of IgG, to investigate the underexplored potential of the FcRn in increasing the intestinal absorption of orally administered drug-loaded NPs. Overall, the developed NP formulations showed a small size and narrow size distribution. Microscopy images showed the successful encapsulation of the NPs into the pH-sensitive polymeric matrices. Moreover, these pH-responsive matrices remained intact in acidic conditions, dissolving only at specific pH conditions, thereby enabling the controlled release of the drugs. When functionalized with FcRn-targeting ligands, the NPs presented high cytocompatibility and increased levels of interaction with intestinal cells, in which the FcRn expression was also confirmed. Also importantly, the targeted NPs showed augmented drug permeability across in vitro intestinal models. Hence, this dissertation provides new insights on the design and development of FcRn-targeted NPs, which emerge as a toolbox to explore the potential of the FcRn transcytotic capacity for oral anti-diabetic drug delivery.
  • Tarvainen, Ilari (Helsingin yliopisto, 2021)
    Cancer can be regarded as a group of diseases involving the uncontrolled division of cells and it is a leading cause of death worldwide. Abnormal function of protein kinases underlies pathogenesis of various cancers, making them one of the most important primary drug targets in search of novel cancer therapies. Protein kinase C (PKC) is a serine/threonine kinase; the activation of which could be beneficial in treating some forms of cancer. We have previously discovered and synthesized a series of isophthalic acid derivatives (HMIs) that act as partial agonists of PKC without inducing down-regulation of PKC. Some of them have previously shown potent anti-proliferative activity in cultured human cervical cancer cells and prostate cancer cell lines. The first aim of this thesis project was to characterise the effects of isophthalate HMI-1a3 on colorectal cancer (CRC) cells and to unravel the non-PKC mediators of HMI-1a3 induced effects. HMI-1a3 exhibited cytotoxic, antiproliferative and apoptosis inducing effects in CRC cell lines. Cytotoxic effects were not PKC-dependent, but instead, the kinase profile together with commercially available pharmacological tools revealed that protein kinase A (PKA) plays a key role in the HMI-1a3 induced decrease in CRC cell viability. The second aim of this thesis was to further develop and characterise novel PKC activators. We characterised the structure-activity relationships (SAR) of novel pyrimidine and pyridine analogues of HMIs and prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) targeted 4β-phorbol ester derivatives. The results demonstrated that pyrimidine analogues of HMIs showed no affinity to PKCα, whereas pyridine derivatives demonstrated similar binding and PKC-dependent ERK1/2 phosphorylation as HMIs. The 4β-phorbol ester derivatives bind to PKCα and decrease the viability of prostate cancer cell lines apart from the PSA or PSMA expression. In addition, 4β-phorbol ester derivatives downregulated PKCα, PKCδ and PSMA. In conclusion, PKA was recognised as a novel target of HMI-1a3 and the antiproliferative and cytotoxic effects of HMI-1a3 on CRC cells reinforce its potential as a lead for anti-cancer drug discovery. Furthermore, these studies confirm the importance of developing PKC targeted compounds that do not induce PKC downregulation. Pyridine analogues were also identified as a novel scaffold for future assessment and development of HMIs.
  • Bogacheva, Mariia (Helsingin yliopisto, 2021)
    Investigation of the hepatotoxicity of therapeutic compounds in vitro before clinical studies requires a reliable human liver cell model. Due to the disadvantages of using primary cell lines, hepatocellular carcinoma cell lines, and animal models, stem cell-derived liver models represent a promising tool for drug testing. Existing stem cell-derived liver models have immature characteristics, causing a demand for deeper investigation and improvements in hepatic differentiation procedures. Hepatocytes, the most abundant cell population in the liver, are responsible for drug metabolism. This feature makes them the most desirable liver cell population for drug testing. Pluripotent stem cell (PSC) differentiation into hepatocytes occurs in a few stages, namely, definitive endoderm (DE), hepatic progenitors (hepatoblasts), fetal hepatocytes, and adult hepatocytes. Each stage is controlled by defined external biochemical signals and physical environmental features provided by the surrounding extracellular matrix (ECM). Successful hepatic differentiation in vitro depends on the ability of the chosen conditions to mimic natural development. The right approach should focus on the assessment of the cellular state at every step of the differentiation. This thesis aimed to develop an effective method for human PSC differentiation into functional hepatocyte-like cells (HLCs) in two-dimensional (2D) and three-dimensional (3D) environments via spatially and temporally controlled physical and biochemical cues. First, we compared six previously developed differentiation medium compositions for their effectiveness in PSC differentiation into DE. We conducted differentiation for human induced PSCs (hiPSCs) and human embryonic stem cells (hESCs). We assessed the dynamic effectiveness of the differentiation using different combinations of growth factors (activin A and Wnt-3a) and/or small molecules (sodium butyrate salt and IDE1). We demonstrated activin A to be an efficient and sufficient agent for DE induction, while sodium butyrate and Wnt-3a were dispensable and IDE1 was inefficient for this purpose. Second, we applied the protocol for generating DE cells in 2D culture to the differentiation of hiPSCs into DE in 3D. We investigated the influence of the formed spheroid size and the presence of scaffold on spheroid morphological characteristics as well as the success of DE formation. We demonstrated a reverse correlation between spheroid size and the efficacy of DE formation. A scaffold-based 3D environment can be beneficial for spheroid culture. However, it can negatively affect cell aggregation if the scaffold material possesses strong cell affinity. Moreover, the hydrogel-based scaffold can also impede mass transport and therefore slow down cell differentiation. Third, we investigated the effect of the thyroid hormone triiodothyronine (T3) in the hepatic differentiation of hiPSCs-derived hepatic cells. We found that T3 increased the expression of thyroid hormone responsive protein THRSP (or SPOT14) and decreased the expression of alpha-1-fetoprotein (AFP). To further investigate the role of AFP in thyroid hormone - mediated hepatic differentiation, we developed an effective protocol based on CRISPR/Cas9-mediated genome editing to create an AFP knockout (AFP-KO) cell line. The improved genome editing approach includes a staggered transfection and the use of a Cas-9-linked selection marker. By comparing wild type and AFP-KO cell lines, we found that AFP does not determine the action of T3 in hepatocyte differentiation. This study suggests an effective method for hPSCs differentiation into definitive endoderm cells which is necessary for further liver cell formation. It demonstrates the importance of the culture environment and control of spheroid size for spheroid formation and hiPSCs differentiation in 3D culture conditions. It shows that the thyroid hormone affects the expression of particular fetal and liver maturation genes, thereby participating in hepatic differentiation of hiPSCs. Furthermore, finally, this study suggests an improved methodology for RNA-based CRISPR-Cas9-mediated genome editing in hPSCs for the study of gene functions during the differentiation of hPSCs in vitro. Taken together, these findings are important for the further development of hPSC-derived liver cell models.
  • Kallio, Sonja (Helsingin yliopisto, 2021)
    The burden that medication-related problems and risks cause to health systems has globally increased because populations are ageing. This burden has forced communities to find new strategies and tools to prevent, solve, and reduce these problems and risks. This study originates from the Finnish Medicines Agency’s (Fimea) programme to promote rational medicines use among older adults in Finland that was run during 2012-2015. The programme aimed to learn from existing collaborative medicines optimisation practices in different social and healthcare contexts to build up guidelines for best practices. The field-work findings of the programme raised the need for more extensive research on community pharmacists’ involvement in medication risk management. Therefore, this study focused on exploring community pharmacists’ contribution to the prospective medication risk management for older adults. The specific objectives of the studies I-IV were: I) To explore challenges and potential solutions for interprofessional collaboration in the optimisation of medicines use in older adults. II) To identify Medication Review Interventions (MRIs) for older adults that involve community pharmacists and the evidence of the outcomes of these interventions. III) To investigate community pharmacists’ contributions to medication risk management in Finland and clarify what risk management actions are taken as part of routine dispensing. IV) To identify gaps in community pharmacists’ competence and their need for continuing education in medication risk management in routine dispensing in Finland. Study I was based on interviews with Fimea’s network participants (group discussions, pair and individual interviews, n=15) in the formation phase of the interprofessional network in 2012. The study explored the challenges and potential solutions experienced by existing healthcare teams in managing medications for older adults at: 1) the individual and team level (micro level), 2) organisational level (meso level), and 3) structural level (macro level). Network theory was the theoretical framework applied to the study. Study II was a systematic review. Cinahl, MEDLINE (Ovid), Scopus, International Pharmaceutical Abstracts, and Cochrane Library were searched for articles published between January 2000 and February 2016. Articles that involve community pharmacists in medication reviews for outpatients aged 65 and older were included. Community pharmacists’ contribution to MRIs and evidence of economic, clinical, and humanistic outcomes of the interventions were summarised. Studies III-IV based on the same national cross-sectional online survey targeted to all community pharmacies in Finland (n=576) in October 2015. One pharmacist from each pharmacy was recommended to report on behalf of their outlet. Reason’s Human Error theory with a systems approach served as the theoretical framework of the studies. Study I indicated that challenges in interprofessional collaboration, problems with patient record systems, the organisation of work and lack of resources were present at all the levels contributing to patients’ medication problems. The study participants generally experienced that no one truly takes comprehensive responsibility for patient care. They suggested multiple potential solutions to improve interprofessional collaboration, sharing the tasks and responsibilities, using pharmaceutical expertise, and developing tools as the most commonly mentioned. In Study II, 16 articles were found that described 12 MRIs. Six were compliance and concordance reviews, four were clinical medication reviews, and two were prescription reviews according to a previously developed typology by Clyne et al. (2008). The community pharmacists’ contributions to MRIs varied from sending the dispensing history to other healthcare providers to comprehensive involvement in medication management. The most commonly assessed outcomes of the interventions were medication changes leading to a reduction in actual or potential medication-related problems (n=12) and improved adherence (n=5). In Studies III and IV, responses were received from 169 pharmacies (response rate 29%). Study III demonstrated that when dispensing, pharmacists were oriented to solve poor adherence and technical problems in prescriptions. In contrast, responsibility for therapeutic risks was transferred to the patient to resolve them with the physician. The pharmacies rarely had local agreements with other healthcare providers to solve medication-related risks. In study IV, pharmacists reported having good competence for confirming prescribed dosages and identifying drug-drug interactions. The major gaps were related to applied clinical and geriatric pharmacotherapy, reviewing medications, and using medication risk management databases. The competence gaps regarding the use of the risk management tools concerned: 1) skills to use the tools and 2) to interpret and apply patient-specific risk information into practice. Optimising medication use of older adults requires new systemic solutions within and between different system levels. The main challenges could be solved by clarifying responsibilities, enhancing communication and applying practices that involve pharmacists and the use of information technology. In routine dispensing in community pharmacies, the solving of medication-related risks is still minor and concentrates principally on technical issues related to prescriptions. More attention needs to be paid to identifying and solving potential therapeutic risks in medications, especially in older adults. The study indicates an urgent need to invest in improving patient care-oriented competencies in applied geriatric pharmacotherapy. Better participation of community pharmacists in medication risk management requires stronger integration and an explicit mandate to solve therapeutic risks.
  • Gilbert-Girard, Shella (Helsingin yliopisto, 2021)
    Increasing antibacterial resistance is one of the most important challenges of modern medicine and new antibiotics are greatly needed to combat multi-drug resistant pathogens. Moreover, bacteria are often found in the form of sessile multicellular communities called biofilms, which are known for their exceptional tolerance to antibiotic treatment, and account for the majority of chronic microbial infections. This thesis covers the screening of chemical libraries against the pathogen Staphylococcus aureus to identify compounds with antibacterial and anti-biofilm activities, the characterization of selected active compounds and the structural optimization of a lead compound. A screening platform was first optimized and was used to test over 3000 chemicals, from natural compounds to FDA-approved drugs. From the active antibacterial compounds identified in the screening, six were selected for follow-up studies (honokiol, tschimganidin, ferutinin, oridonin, deoxyshikonin and fingolimod). The capacity of the compounds to kill bacterial cells and to prevent biofilm formation was assessed against various bacterial species, with fingolimod displaying the highest potency. The six selected hits were also tested against pre-formed biofilms and ferutinin proved highly effective. The follow-up studies also included the evaluation of resistance development, cytotoxicity, quorum sensing inhibition and activity in a co-culture of bacteria and human cells. Based on the results, fingolimod, a drug approved for the treatment of multiple sclerosis, was selected for structural optimization. Some of the synthesized derivatives displayed a more potent antibacterial and anti-biofilm activity than fingolimod against one or more bacterial species. Overall, this work covered the results of a complete screening approach and novel information was gathered on the antibacterial and anti-biofilm activities of six compounds. Particularly, ferutinin was found to be an interesting anti-biofilm compound whereas fingolimod was demonstrated to be a potent antibacterial candidate with a novel quorum sensing inhibitory activity.
  • Hiltunen, Anna Katariina (Helsingin yliopisto, 2021)
    Biofilm, a major lifestyle of bacteria, refers to bacterial communities surrounded by a self-produced protective slimy matrix. Due to this matrix and the reduced metabolic activity of bacteria within them, biofilms usually withstand the eradication of conventional antibiotics, leading to recalcitrant and recurrent infections. Therefore, there is an immense need for discovering novel truly effective anti-biofilm treatment strategies that do not promote the development of antibiotic resistance. Biofilms colonizing permanently inserted indwelling medical devices are particularly challenging as their eradication always requires some kind of invasive surgery. Even though a wide variety of biomaterials are used in medical devices, the thorough understanding of biofilm characteristics or formation on such materials is still limited. In this thesis, a multidimensional orthogonal approach was taken to characterize biofilm dynamics on different biomaterials (borosilicate glass, plexiglass, hydroxyapatite, titanium, and polystyrene) at different maturation points (18, 42, and 66 h) (study I). This study covered (i) biomaterial surface properties (and the correlation with material susceptibility to biofilm formation), (ii) biofilm matrix structures (via proteosurfaceomics and polysaccharide/protein content determinations), and (iii) biofilm functionality (antimicrobial tolerance studies). Staphylococcus aureus, which is a major pathogen in medical device-associated infections, was chosen as a model organism for this study. The matrix-associated polysaccharide content was observed to play an important role in the initial stages of biofilm formation as its amount decreased towards the end of the observation period. In turn, the matrix-associated protein content seemed to increase over time. Interestingly, the classical surface proteins that have been deemed as the most attractive targets in antibiotic development, demonstrated high biomaterial-dependent variability in their amounts. In turn, the presence of non-classical surface proteins “moonlighting proteins”, forming the major portion of the core proteosurfaceome, did not appear to be strongly dependent on the material. It is known that biofilm inhabitants reutilize cytoplasmic proteins as moonlighting constituents in the extracellular space, offering enhanced bacterial attachment, virulence, and antibiotic tolerance. Therefore, inhibiting moonlighting activity would offer a novel target for anti-biofilm agent/material development. Finally, biofilms formed on hydroxyapatite were in many cases more susceptible to antibiotics than titanium-associated biofilms. Also, according to our findings, the biofilm age did not always correlate with the increased antibiotic tolerance. To the best of our knowledge, study I is among the pioneer investigations shedding light onto the matrix-associated proteosurfaceomes of S. aureus biofilms developed on different biomaterials and at diverse biofilm formation points. The study offers further mechanistic insights into biofilm formation and the findings may facilitate the development of new anti-biofilm compounds. After focusing on the more fundamental aspects of biofilm formation on different materials, the next goal was to study whether one of these biomaterials, hydroxyapatite, could be protected from biofilm colonization with a novel non-antibiotic combination product. This was studied in two in vitro biofilm infection models: for prosthetic joint infections (PJIs) (modelled using S. aureus and Staphylococcus epidermidis; study II) and periodontitis (using Aggregatibacter actinomycetemcomitans; study III). Both diseases cause significant individual burden and would benefit from an approach with both biofilm-eradicating and bone-preserving capabilities. The rise of antibiotic resistance is a top threat to public health, and thus innovative treatment options reducing the use of conventional antibiotics are desired. Based on this, bioactive glass S53P4 (BAG), a bone-constructing material with a completely divergent (i.e. physical-based) bactericidal mechanism, was chosen as a focus here. Additionally, it has been previously reported that the bone construction ability of other bioactive glasses can be enhanced by combining them with anti-osteoporotic drugs, i.e. bisphosphonates (BPs). However, how the addition of BPs (alendronate, clodronate, etidronate, risedronate, and zoledronate) influences the anti-biofilm effect of BAG has not been previously studied, and this was the main research question in studies II and III. Etidronate-BAG and risedronate-BAG were found to be the most promising combinations from the perspective of both studies (II and III), while clodronate-BAG was not effective against any of the strains. Risedronate was the only BP that had an intrinsic anti-biofilm effect against all the tested strains. Moreover, an enhanced anti-biofilm effect was systematically observed in study III, probably due to the longer treatment period (48 h) compared to the one used in study II (24 h). Finally, a possible mechanistic perspective to the anti-biofilm effect of BP-BAG combinations was undertaken. The observed anti-biofilm effects could not be rationalized alone with lowered pH values. Increased osmotic pressure or another yet-unknown mechanism seems to contribute to the observed anti-biofilm effect. In conclusion, the results of studies II and III further support the use of the most effective BP-BAG combinations in protecting biomaterials from biofilm infections in PJI and periodontal applications.  
  • Kilpeläinen, Tommi (Helsingin yliopisto, 2021)
    Most neurodegenerative diseases (NDD) are characterized by accumulation of toxic protein species leading to the loss of movement and cognitive disorders. In the two most common NDD Parkinson´s disease (PD) and Alzheimer´s disease (AD), there are almost 60 million patients worldwide. In the U.S, there were one million PD patients and an estimated economic burden of 51.9 billion dollars in 2017. As there is no disease-modifying therapy available, NDD lead to loss of life quality and eventually to untimely death. Current drug therapies for PD can only relieve symptoms of the disease by increasing dopaminergic activity in the brain but they do not address the underlying mechanisms of the disease such as clearance of detrimental protein aggregates. In previous studies, prolyl oligopeptidase (PREP) has been shown to negatively regulate autophagy and enhance the aggregation of alpha-synuclein (αSyn). Thus, PREP inhibitors have been shown to reduce aggregation and increase clearance of αSyn aggregates via enhanced autophagy. These mechanisms are mediated by protein-protein -interactions (PPI) of PREP with other proteins. Several small molecular inhibitors of PREP have been developed to target memory disorders, cancer, and NDD. However, none of these PREP inhibitors are in therapeutical use. The aim of this study was to investigate the effect of these inhibitors in two neurodegeneration-related functions of PREP: autophagy and αSyn dimerization. A further aim was to synthesize novel PREP ligands targeted to modulate these PPI-mediated functions of PREP. In the first part of this study, a structurally diverse set of 12 potent PREP inhibitors were tested in in vitro assays of autophagy and αSyn dimerization. The study revealed that the least potent inhibitor with IC50-value of 1010 nM had the most prominent effect on reduction of αSyn dimers and enhancing autophagy, while some of the highly potent inhibitors with IC50-value ranging from 0.32 to 1.2 nM did not have an effect on autophagy or αSyn dimerization. In the second part of the study, novel 4-phenylbutanoyl-α-aminoacyl-2(S)-tetrazolyl-pyrrolidines were designed and synthesized to target αSyn dimerization. Synthesized compounds had IC50-values ranging from 12 nM to 200 000 nM, but they all reduced αSyn dimerization in a cellular assay. Furthermore, molecular docking studies showed that these novel compounds have another putative binding mode to PREP compared to typical PREP inhibitors. These findings combined with the findings from the first part of the study suggest that the PREP mediated reducing effect on αSyn dimerization and enhancing effect on autophagy is not dependent on the magnitude of PREP inhibition but more likely due to conformational stabilization in PREP caused by ligand binding. Another speculative explanation is that PREP has another binding site, which modulates the conformation of PREP and thereby the PPI mediated functions of PREP. In the third part of the study, a human αSyn transgenic mouse, carrying A30P and A53T point mutations in αSyn was characterized and evaluated as a model for early onset PD. The model displayed behavioral alternations at an early age with differences in dopaminergic neurotransmission in the nigrostriatal pathway and accumulation of oligomeric αSyn leading to decreased tyrosine hydroxylase in the striatum and substantia nigra. Furthermore, we evaluated the effect of seven days treatment by the novel PREP inhibitor, HUP-55, in this model. Results in this study showed that HUP-55 was able to decrease the level of oligomeric αSyn in the striatum and substantia nigra. In summary, this study revealed and highlighted the disconnected structure-activity relationships between inhibition of the proteolytic activity and the PPI mediated functions of PREP. Moreover, this finding led to the development of a novel PREP ligand that was successfully tested in a PD mouse model.  
  • Siiskonen, Satu Johanna (Helsingin yliopisto, 2021)
    This doctoral dissertation summarises current knowledge on risk factors for the most common types of skin cancer and describes some of the important methodological challenges related to research on skin cancer aetiology. The three original studies each assess a different type of risk factor for cutaneous squamous cell carcinoma (SCC) or cutaneous malignant melanoma (CM) using large data sources in the USA and/or the Netherlands. Study I was one of the first published genome-wide association studies on SCC. The most significant finding was the association between a single nucleotide polymorphism (SNP) rs8063761 in the DEF8 gene and SCC, which has since been replicated by other research groups. The role of the DEF8 gene in skin carcinogenesis is yet to be determined. A single finding from a genome-wide association study with a modest effect size has no clinical value in screening individuals at high risk for SCC. It can, however, be useful when constructing polygenic risk scores to predict an individual’s genetic predisposition to SCC development. Study II was a large cohort study with time-dependent variables to investigate the association between alcohol intake and SCC. Alcohol consumption was associated with SCC and SCC in situ in a dose-dependent manner in men and women. The results of this study also suggest an association between white wine consumption and SCC, when the analyses were adjusted for the total alcohol intake. However, residual confounding by other factors, such as time spent in the sun, cannot be ruled out. Alcohol use is a modifiable risk factor for many cancers and other diseases, and its use should be minimised. Study III reports an association between the use of phototoxic quinolone antibiotics and propionic acid derivative analgesics, and CM. Even short-term use may be relevant, and the risk estimates were different for men and women. It cannot, however, be excluded that the detected associations are partly due to bias or confounding by unmeasured factors. Although the relationship between phototoxic reactions and development of CM is still a topic of debate, phototoxic reactions themselves can cause significant morbidity. Healthcare professionals should therefore advise patients to avoid sun exposure while taking phototoxic drugs. Identifying and assessing risk factors for skin cancer is methodologically challenging due to lacking knowledge on relevant exposure time periods and often incomplete or absent information on exposure to UV radiation and other known strong predictors of skin cancer, such as skin phenotype and the use of immunosuppressants. The data source used in etiologic skin cancer research should, ideally, include information on the known risk factors and validated skin cancer diagnosis and allow for careful examination of relevant exposure time periods.  
  • Štukelj, Jernej (Helsingin yliopisto, 2021)
    The two parameters used to characterize the effect of a drug in the body are potency and selectivity. Despite their importance, the biopharmaceutical classification system (BCS) and the developability classification system (DCS) are constructed based on permeability and solubility. Furthermore, solubility is not only a molecular property as it is highly affected by the solid-state of a compound. Thus, obtaining a complete understanding of the solid-state impact on the solubility of a drug candidate early in the drug development process is crucial for well-informed decision-making. The overall aim of this thesis was to detect, investigate and quantify the impact of different solid-state forms on the solubility of pharmaceutical compounds using the image-based single-particle analysis (SPA) method. First, the apparent solubility of polymorphs in different solvents was measured. It was demonstrated that the solubility ratio between the two polymorphs is determined by the difference in Gibbs free energy between the polymorphs and it is not affected by changes in pH, ionic-strength and surfactant concentration in dissolution media. Next, the amorphous solubility of a particulate quench-cooled amorphous sample was measured directly. The results correlated well with the two orthogonal methods – standardized supersaturation and precipitation method, and theoretical estimation based on thermal analysis. Moreover, the direct approach of measuring amorphous solubility has proven superior when dealing with highly hygroscopic and fast crystallizing samples. Furthermore, the newly established direct approach of measuring amorphous solubility was then, concurrently with X-ray powder diffraction, used to track two amorphous samples stored at different conditions over an extended period of time. Differences in the crystallization process and its impact on solubility were observed. Machine-vision-detected solid-state changes were demonstrated to provide additional information for the interpretation of the measured pH-solubility profiles of pharmaceutical salts. Based on pH-solubility profiles, values such as pKa, pHmax and Ksp were easily obtained. Moreover, the potential effect of the microenvironmental pH shift was evaluated in the context of solubility measurements. Finally, the solubility of extremely poorly soluble drug – itraconazole – and its co-crystals was measured across a wide pH range and also in biorelevant buffers. A considerable improvement in solubility was detected upon co-crystal formation. Moreover, the solubility of itraconazole in biorelevant buffers was in good agreement with the results in literature obtained previously by the conventional shake-flask method. In summary, all of the above was achieved with less than 100 μg of a sample per measurement. Moreover, non-specificity and the demonstrated broad applicability make the SPA method highly suitable for use in the early stages of the drug discovery and development. The possibility of acquiring a broad level of physicochemical characterization using a single analytical method could significantly accelerate and improve data acquisition leading to enhanced understating of solid-state impact on solubility. Furthermore, with the data quality comparable to the current ‘gold standard’ technique the SPA method could thread the path towards more accurate in silico solubility predictions. Thus, eventually leading to more informed decision-making processes and, finally, optimized and more affordable drug products.
  • Karhu, Tuuli (Helsingin yliopisto, 2021)
    Heart failure is a common costly disease that remains one of the leading causes of mortality in the world. Current treatments benefit heart failure patients by relieving the symptoms and improving the quality of life; however, they do not provide a cure for the disease. The adult human heart has a very limited capacity to regenerate cardiomyocytes. Therefore, following cardiac injury, the cell loss results in the formation of a fibrotic scar and consequent remodelling process, which includes thickening and stiffening of the ventricular wall. Eventually, this leads to impaired cardiac function. The fundamental goal of heart failure therapy is to restore the cardiac function and to prevent the pathological remodelling process. The transcription factor GATA4 plays an important role in cell survival, myocardial regeneration, and cardiomyocyte hypertrophy, and is therefore considered as an attractive drug target for cardiac repair. On the other hand, several studies have suggested that protein kinase C (PKC) plays a role in cardiac fibrosis. However, the results are conflicting and the role of PKC specifically in cardiac fibroblasts is unclear. Early drug discovery projects have traditionally used immortalized cell lines and primary cells from experimental animals to assess the effects of novel compounds. The translatability of the results from these models to human, however, is limited. Today, the emergence of the human induced pluripotent stem cell (hiPSC) technology allows for the production of human-based cell models, which more closely resemble the physiological cell types. The aim of this thesis project was to characterise pharmacological and toxicological effects of small molecules targeted to GATA4 or PKC, to compare different cell models in in vitro compound testing, and to establish a chronic cardiotoxicity model using human-based cardiomyocytes. In the study, nine different cell models were utilised, including hiPSCs, cardiomyocytes derived from hiPSCs, cardiomyocytes isolated from neonatal rats, and cardiac fibroblasts isolated from adult mice. The effects of the small molecules on cell viability, proliferation, and morphology were investigated using colorimetric cytotoxicity assays, immunocytochemistry, and high-content analysis (HCA). The effects on gene expression, protein levels, and cellular kinase activity were studied using the quantitative polymerase chain reaction (qPCR), western blotting, and AlphaLISA® technology, respectively. The toxicity screening of GATA4-targeted compounds revealed a characteristic molecular structure that is predominantly responsible for the toxic outcome in hiPSCs, which proved to be the most sensitive screening tool to identify toxicity. The results also demonstrate that long-term low-dose exposure of hiPSC-derived cardiomyocytes can be used as an in vitro model of delayed doxorubicin-induced cardiotoxicity and provide evidence that targeting GATA4 by small molecules may counteract doxorubicin-induced cardiotoxicity. Finally, the results reveal distinct PKC-dependent regulation of cardiac fibroblast transdifferentiation and proliferation and suggest that fibroblast-targeted PKC activation may be a promising strategy to inhibit cardiac fibrosis. Overall, the results highlight the importance of selecting an appropriate cell type and experimental model for compound testing and support further development of both GATA4-targeted compounds and PKC activators.
  • Tanner, Timo (Helsingin yliopisto, 2021)
    Developing new pharmaceuticals is costly and time-consuming. New methods are always in demand for various stages of product development. Investing in the early phases of development can save a significant amount of resources in the long term. Tablet is still the most commonly used pharmaceutical dosage form. Tablets are often produced by powder compression. Powder particles fragment and deform under pressure, allowing new bonds to form between them. Modern machines can produce over one million tablets per hour. The mechanical properties of the powders have a remarkable impact on compact formation. For example, excessive elasticity in a powder mixture can lead to weak or defected tablets being produced. Therefore, the mechanical properties need to be studied. Devices known as tableting simulators have been designed to aid in developing adequate tablet formulations. These machines are useful, but they can still be quite expensive and large. The results obtained by these machines are not always universally applicable, and further interpretation is often required. In this thesis, a novel gravitation-based high-velocity compaction (G-HVC) method was developed to study the compressibility and tabletability of powders in a cost-efficient and straightforward manner. The method is based on a freely falling steel bar, which compresses the powder sample inside a custom-made die. The movement of the bar and the deformation wave of the system base were monitored by high-accuracy displacement sensors. Displacement graphs could then be derived further. All data obtained by the method was ultimately only based on the displacement data. First, microcrystalline cellulose (MCC) and starch samples were compressed to demonstrate the functionality of the method. MCC was shown to be more compressible and less elastic than starch. Apparent differences in the relative volume decrease and the compression behaviour of these two materials could be seen. Next, various materials were studied more comprehensively. Two different setups with varying pressure were in use. Lactose grades and glucose showed effective fragmentation and reached true density with both setups. MCC grades were clearly pressure-dependent and showed slower gradual deformation, indicating plastic behaviour. Compression pressure was not high enough to effectively fragment calcium phosphate. Starch showed most elasticity of all the samples. In summary, all examined materials could be successfully categorized in terms of their mechanical properties. Finally, the practical relevance of the method was shown by creating a model between the compaction energy values determined by G-HVC method and the tensile strength of tablets produced with a tableting machine. Three different formulations consisting of MCC, calcium phosphate, theophylline and HPMC were granulated utilizing a fluid bed system. There was a good correlation between compaction energy and tensile strength. In summary, the G-HVC method was proven to be a reliable and cost-efficient tool in the examination of the mechanical properties of powders. The method was also capable of producing practically relevant results. The method fits well in modern pharmaceutical research where material-sparing, straightforward and reliable methods are in demand.
  • Hassan, Ghada (Helsingin yliopisto, 2021)
    Antibiotic resistance is a current threat to modern medicine. Not only is it challenging to treat but it also adds considerable costs to the healthcare systems. At the current rate of rising drug resistance, approximately 10 million people will die annually by 2050. The World Health Organization (WHO) has listed the six most threatening pathogens for which new antibiotics and approaches are urgently needed. One of the fastest evolving and most notorious Gram-positive bacteria in the list is Staphylococcus aureus. In addition to developing resistance to a vast number of antibiotics, this bacterium can attach to surfaces and form biofilms. This lifestyle allows bacteria to protect themselves from the immune system and impairs treatment. Thus, new innovative approaches and antibacterial agents are needed to fight bacterial resistance. As microbial adhesion is the first step of biofilm formation, the first aim of this study was to develop novel antibacterial surfaces to hamper the accumulation of antibiotic-resistant microbes. Cellulose is the most abundant natural polymer with various appealing characteristics. It is renewable, biocompatible, biodegradable, and possesses excellent mechanical properties. Cellulose nanofibers are prepared by mechanical disintegration of cellulose. Thin films of cellulose nanofiber can be prepared, and their surface can be modified to provide cellulose with new properties. Terpenoids such as abietic acid and dehydroabietic acid originating from conifer resin possess antibacterial and antifungal activities. We have previously shown that the amino acid-bearing derivatives of dehydroabietic acid possess antimicrobial properties. Herein, I report the design and the synthesis of new wide-spectrum contact-active non-leaching antibacterial cellulose nanofiber films by coupling the films with dehydroabietylamine, dehydroabietic acid and their derivatives. Different techniques and measurements were used to study the new biomaterials including contact angle (CA), streaming current measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), oxygen transmission rate (OTR), water vapor transmission rate (WVTR), scanning electron microscope (SEM), tensile strength, and Young’s modulus. Our unique design rendered four anionic surfaces highly active against the Gram-positive S. aureus, including the drug resistant methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative Escherichia coli. The proposed modes of action and the fact that the compounds are based on a new chemical class account for a low potential to spread resistance. Our most active material 87 was tested against bacterial colonization in both biofilm and artificial dermis models. The bacterial colonization was efficiently prevented in both models. Material 87 proved to be biocompatible as it nurtured fibroblast growth at its surface without causing significant hemolysis. Our originally designed surfaces represent a new class of renewable biomaterials suitable for biomedical applications. The second aim of this thesis was to develop a new class of pyrimidine derivatives against S. aureus biofilms. From our novel set of pyrimidines, compounds 89, 99e, and 100e displayed potent activities. They inhibited biofilm formation and were active against pre-formed biofilms of S. aureus ATCC 25923 and Newman strains with IC50 values ranging between 11.6 to 62.0 μM. The compounds were also effective against planktonic cells with minimum inhibitory concentration (MIC) values lower than 60 μM. Only marginal cytotoxicity was revealed against human Hep2 cells at concentrations comparable to their pre-exposure IC50 values. Overall, this study resulted in the synthesis of four new antibacterial biomaterials and 26 new pyrimidine derivatives. This thesis offers novel approaches to target one of the most dangerous antibiotic-resistant bacteria S. aureus and efficiently limit its proliferation.
  • Kiiski, Iiro (Helsingin yliopisto, 2021)
    Drug metabolism is an enzyme-catalyzed process that has major implications for a drug’s safety and efficacy. Consequently, evaluating the metabolic properties of a new drug candidate is of paramount importance already in the preclinical phase of drug development. Although the available in vitro techniques for drug metabolism research have improved over recent years, the state-of-the-art methodology relies on enzyme assays conducted in static conditions, with limited spatiotemporal control of assay variables. Conducting metabolic assays under flow-through conditions could pave the way for more in-depth understanding of the mechanistic basis underlying drug-enzyme and drug-drug interactions. This, however, requires that drug-metabolizing enzymes be immobilized onto a solid support material without compromising protein folding or enzyme function. Although a wealth of different enzyme immobilization strategies are currently available, most of them are not amenable to immobilization of the microsomal, drug-metabolizing enzymes. The aim of this thesis was to establish immobilized enzyme microreactor (IMER) platforms for studying drug metabolism under flow conditions, thereby improving the in vitro-in vivo prediction of the metabolic fate of new drug candidates. The use of microfluidics and microfabrication technology facilitated the straightforward implementation of flow-through assays and furthermore allows their multiplexing (parallelism) and integration with other operational units on a single platform, minimizing both reagent consumption and dead volumes. In the first sub-project (publication I), a novel strategy for the immobilization of the membrane-bound enzymes cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) was designed and implemented on microreactors fabricated from off-stoichiometric thiol-enes (OSTE). The strategy was based on biotinylation of human liver microsomes via biotin-tagged fusogenic liposomes, and utilized the tunable surface chemistry of OSTEs to allow easy functionalization of the microreactor surface. The IMER platform was preliminarily characterized to ascertain enzyme stability and preservation of key enzyme kinetic parameters, with an emphasis on CYP-mediated (phase I) oxidoreductive reactions. In the second sub-project (publication II), the feasibility of the IMER platform for studying the kinetics of UGT-mediated drug conjugation (phase II) was assessed, with an emphasis on the mechanistic basis for the pronounced underestimation of in vivo clearance kinetics by the currently available static in vitro techniques. In particular, the effect of membrane disruption and fatty-acid inhibition on UGT-kinetics in vitro was studied in detail. The kinetics of zidovudine glucuronidation (the model reaction used in this study) under flow-through conditions was shown to be in good agreement with that obtained using microsomal incubations in static conditions without the need for added pore-forming agents such as alamethicin. In the third sub-project (publication III), the technology was further developed by incorporating a highly overlooked dimension in the assay design: the impact of oxygen partial pressure on the metabolic fate of drug candidates. This was achieved by exploiting the unique material-induced oxygen scavenging property of thiol-ene polymers. The developed chip design allowed the rapid and simple control of oxygen concentration in enzymatic assays, which is difficult to achieve with conventional static assay methods. Overall, the developed methodology was shown to retain enzyme activity and native enzyme kinetic parameters of both CYP and UGT enzymes, an unconditional prerequisite for drug metabolism assays. With the immobilization method utilizing membrane biotinylation via liposome fusion, common problems (such as diffusion-limited kinetics and enzyme inactivation) associated with conventional immobilization approaches were circumvented. Furthermore, the universal applicability of the immobilization method for all membrane-bound enzymes was preliminarily demonstrated with recombinant CYP enzymes in sub-project/publication I. The methodology developed here also enabled mechanistic studies focused on the alamethicin-induced membrane disruption (commonly used in UGT assays to overcome mass transfer limitations) and the proposed inhibitory effects of fatty acids, which may shed light on the foundations behind the poor in vivo correlation associated with UGT reactions in vitro. The material-induced oxygen scavenging facilitated by OSTE polymers, together with microfluidic actuation, was shown to be an easily controllable approach for adjusting the oxygen partial pressure on demand. The advantage of this approach is that, unlike in typical approaches, complex chip designs or pressurized compartments are not needed. Beyond the demonstrated feasibility of the IMER platform for studying the impact of NADPH-cytochrome P450 oxidoreductase (POR)-mediated metabolism, the theoretical framework established here for OSTE-enabled oxygen control in microfluidic assays is likely to find many applications, particularly in organ-on-a-chip research. In conclusion, the microreactor platform developed in this thesis offers an enabling toolbox for conducting in vitro drug metabolism assays under flow conditions that circumvents many of the key shortcomings of current state-of-the-art (static) in vitro enzyme assay methodology, as well as those of previously reported IMER platforms. At the time of publication, the methodology developed herein has already been utilized in several follow-up studies and has shown utility in diverse applications beyond this work.
  • Renko, Juho-Matti (Helsingin yliopisto, 2021)
    Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of nigrostriatal dopamine neurons and propagating Lewy body pathology. Dopamine depletion in the striatum gives rise to the cardinal motor symptoms of PD. Current PD medications are based on replenishing striatal dopamine and provide symptomatic relief to the motor deficits. However, troublesome adverse effects and diminished efficacy complicate their long-term use. There is a great unmet medical need for a therapy that could slow or halt the progression of the disease. Neurotrophic factors (NTFs) are secreted proteins that promote neuronal growth, differentiation and survival. They are able to prevent the progression of neurodegeneration and restore aberrant neuronal function in a variety of preclinical models of PD. Nonetheless, outcomes from clinical trials have been disappointing. The purpose of this work was to characterize the effects of cerebral dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF) and novel small molecule receptor tyrosine kinase RET agonists (BT13 and BT44) on nigrostriatal dopamine system and support their preclinical development as potential neurotrophic therapies of PD. To further clarify the functional effects of glial cell line-derived neurotrophic factor (GDNF), CDNF and MANF in the normal rat brain, microdialysis measurements were performed after a bolus injection of NTFs into the striatum. We saw augmented stimulus-evoked dopamine release and elevated dopamine turnover in the striatum of MANF-injected rats. GDNF injection increased in vivo tyrosine hydroxylase (TH) and catechol-O-methyltransferase activity and decreased monoamine oxidase A activity. These data are relevant when considering exogenously administered NTFs as a potential therapeutic approach for PD, since they have to be compatible with the existing dopaminergic medications of the patients. We also investigated the distribution properties of 125I-labeled and unlabeled CDNF after a nigral injection to intact rats. CDNF readily diffused into the brain areas surrounding the injection site and colocalized with TH-immunoreactive neurons in the substantia nigra. We did not detect active transportation of CDNF to distal brain areas. This characterization provides valuable insights into the selection of optimal delivery site and protocol for CDNF therapy. Our in vitro assays showed that RET agonists BT13 and BT44 were able to induce RET phosphorylation and activate downstream pro-survival signaling cascades Akt and ERK. They also supported the survival of cultured midbrain dopamine neurons from wild-type, but not from RET knockout, mice. The functional effects of BT13 and BT44 were evaluated in a unilateral 6-hydroxydopamine rat model of PD, where both compounds alleviated amphetamine-induced turning behavior. BT44 also showed potential to restore striatal TH-immunoreactive fibers. As blood-brain barrier penetrating compounds, BT13 and BT44 serve as promising leads that can be further developed into a disease-modifying therapy for PD.
  • Suutari, Teemu (Helsingin yliopisto, 2020)
    Cell-based assays have become an integral part of pharmaceutical research and drug development. They offer researchers not only a means to investigate basic cellular functions and different disease states, but also a way to study drug action in cells. Nowadays, during the long journey that new drug candidates take from an early discovery to a final product, all drug candidates undergo a preclinical test phase where their properties are evaluated using various cell-based assays. While perhaps the most common and important goal for these cell-based assays is to ensure that the investigated molecules are not harmful to cells, cell-based assays are also used to investigate the biological activity, mechanisms of action, and possible interactions, such as interaction with other drugs. With the traditionally used cell-based assays it is a common practice to alter the investigated pharmaceuticals or cells, or both, to produce a measurable signal, such as radioactivity, chemiluminescence, or fluorescence. However, there are well-known issues with this strategy, not the least that by subjecting the pharmaceuticals and/or cells to these modifications, an artificial system is created where the cells or pharmaceuticals under investigation are no longer in their original state. This can have a profound impact on the properties of the pharmaceuticals or behavior of the cells, thereby resulting in erroneous readouts. Because of this, different label-free cell-based assays are an attractive and logical alternative. Label-free technologies have been applied in the field of drug research and discovery since the 1980s. During the turn of the last millennium an increasing number of studies have implemented these methods to measure cellular activity, such as receptor activation. Nowadays, some of the commercial label-free devices are designed specifically with cell-based assays in mind. Of course, these technologies are not without their own challenges. The most prominent of these stems from the inherit property of these technologies: they are not sensitive to just a single predefined cellular event or response, but instead they measure cell activity as a whole. While well-controlled experimental design can mitigate the contribution of non-relevant cell responses to the measured label-free signals, advanced signal analysis methods have made, and can be expected to continue to make, label-free cell-based assays even more powerful tools for cell studies and help researchers to better understand the effect of different cellular activities on the label-free signals. Amongst the different label-free techniques, optical methods have been widely adopted for cell-based assays. While resonant waveguide grating (RWG) has been mainly utilized in the investigation of membrane receptors, namely G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), surface plasmon resonance (SPR) has been applied in the study of a variety of cellular processes, such as cell adhesion, detachment, spreading, contraction, cytoskeleton rearrangement, cell-to-cell contacts, monolayer formation and cell death. Surprisingly, studies on GPCRs with SPR are relatively low in number, even though they should be of high interest as around 35% of all current drugs mediate their effect via these receptors. Although SPR has already been shown to be a powerful tool for studying various cellular processes, its wider use in life sciences is hampered by the challenge in interpreting the contribution of the cell responses of interest and other non-relevant responses interfering with the measured signals. A variety of NPs are used as drug carriers, especially for biological drugs, in order to protect them from degradation and guide them to diseased cells or other desired location within the human body, while EVs are naturally produced nanosized structures that function as message carriers between cells. Because of the inherent property of EVs to penetrate cellular barriers, there is a growing interest in using them as “natural” and personalized drug carriers. This thesis investigates the use of SPR in the context of cellular uptake studies of nanoparticles (NPs) and extracellular vesicles (EVs), and GPCR-mediated signaling. Various approaches to analyze real-time label-free SPR signals are implemented in order to improve the correlation between the measured SPR responses and cell activities. The results show, for the first time, how SPR can be used to measure cellular uptake of NPs and EVs. However, because NP and EV uptake lead to ambiguous SPR signals, different signal analysis strategies are investigated and implemented to provide meaningful SPR responses. Three strategies proved to be effective: (1) varying the measurement temperature allowed to investigate possible cell entry mechanisms of different NPs in HeLa cells; (2) correcting the SPR angle response by removing the contribution of total internal reflection (TIR) angle shift allowed the quantification of the uptake efficacy of functionalized NPs into HT-29 colorectal adenocarcinoma cells; (3) comparing the responses measured with two different wavelengths corrected for the large response variation observed across different experiments for EV uptake in PC-3 prostate cancer cells. In addition, multiple label-free SPR parameters from the full SPR curves were analyzed after stimulating different GPCR subtypes, signaling primarily via Gs-, Gq- or Gi-coupled pathways. It was revealed that by combining two key label-free parameters, unique label-free signal profiles for each pathway could be generated, which makes it possible to recognize the pathway coupling of the receptor subtypes from the label-free responses alone, which has not been possible before. Collectively, these results demonstrate how SPR can be effectively utilized in cell-based assays to investigate NP and EV uptake and cell signaling. Cell-based SPR provides high information content, and with the help of the developed and used analysis methods, the otherwise hard-to-interpret label-free signals provide meaningful information on these cell responses.
  • Kortesoja, Maarit (Helsingin yliopisto, 2020)
    Chlamydia pneumoniae is an obligate intracellular human pathogen whose primary site of infection is the respiratory tract. In addition to respiratory tract infections, such as sinusitis and pharyngitis, C. pneumoniae has been related to several chronic inflammatory diseases of which atherosclerosis is the most widely studied. In order to contribute to the pathogenesis e.g. in atheromatous arteries, C. pneumoniae must disseminate from lungs to other tissues. This translocation occurs via peripheral blood mononuclear cells (PBMCs), mainly monocytes and macrophages. The presence of C. pneumoniae has been reported to induce inflammatory molecule production and alter redox balance in these cells, while also promoting their foam cell formation, migration and adherence. The ability of C. pneumoniae to persist inside the PBMCs, and thus become refractory to conventional antibiotics complicates the management of the associated underlying infection in chronic inflammatory diseases. Most C. pneumoniae susceptibility studies are currently based on the use of permissive cell lines, such as epithelial and endothelial cell lines in which the bacteria are actively replicating, which leaves the persistent infections unnoticed. Therefore, in addition to new active compounds against C. pneumoniae, new methods to study their effects against the persistent chlamydial infection are urgently needed. In this study, dibenzocyclooctadiene lignans, new antichlamydial compounds originating from berries of medicinal plant, Schisandra chinensis, were profiled regarding different aspects of C. pneumoniae-PBMC interactions. Human THP-1, a monocyte and macrophage cell line, and a murine macrophage cell line RAW264.7 were used in the studies to examine the effects of the schisandrin lignans on C. pneumoniae infection and their influence on host cell responses. Oxidative stress is the basic pathological mechanism underlying a spectrum of chronic inflammatory diseases, and it largely contributes to the consequences of C. pneumoniae infection as well. In this work, C. pneumoniae was found to induce proinflammatory cytokine, interleukin (IL)-12 secretion in the human monocytic cell line. It also increased the intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels in macrophages, and it had an impact on the concentration of glutathione (GSH), the major small-molecule antioxidant, in macrophages. Gene expression analysis of murine macrophages revealed that C. pneumoniae suppressed the peroxisome proliferator-activated receptor γ (PPARγ) transcription in the cells, which influences lipid metabolism and inflammatory responses in these cells. Schisandrin lignans had an impact on C. pneumoniae-induced alterations in these cell models. Schisandrin B and schisandrin C reduced the elevated interleukin (IL) -12 cytokine levels, as well as the LPS induced IL-6 and IL-12 levels. Schisandrin lignans also affected cellular oxidative balance by elevating the basal ROS levels while simultaneously reducing the ROS or NO levels induced by infection or LPS. The redox balance alteration was also shown within the GSH levels, which were reduced by the lignans in THP-1 monocytes and macrophages but elevated in RAW264.7 cells. Schisandrin B additionally upregulated the transcription of genes involved in GSH synthesis, GCL and GGT-1. Schisandrin B and schisandrin C also reduced the C. pneumoniae-induced macrophage foam cell formation and altered the related expression of PPARγ and ABCA1 genes. In this work, a new platform for studying the C. pneumoniae transfer between lung epithelial cells and phagocytes, is also introduced. The platform can be used in lead compound profiling studies against C. pneumoniae infection. Mitogen-activated protein kinase (MAPK) inhibitors were found to inhibit the transfer of the infection, serving as reference compounds in future profiling studies. This work provides new information about the C. pneumoniae infection in monocyte-macrophage cell models, while also offering valuable insight on the antichlamydial lead compounds, dibenzocyclooctadiene lignans. These lignans were shown to suppress the C. pneumoniae induced pathological changes in the host cells. With newly identified antichlamydial activities and novel study methods, we can explore more impactful approaches to overcoming C. pneumoniae-induced chronic inflammatory diseases.
  • Kari, Otto (Helsingin yliopisto, 2020)
    Nanoparticles administered into the body are rapidly covered by biomolecules and assume a biological identity that mediates their biological interactions. Methodological constraints have limited our ability to study the formation of this “protein corona”. To increase our understanding of its formation and evolution in different biological environments, and hence improve the safety and efficacy of nanomedicines, there is a need for non-invasive and label-free methods that better mimic the conditions inside the human body. This thesis describes the development of a novel label-free workflow to determine the structure and composition of the hard and soft corona on liposomes. Multi-parametric surface plasmon resonance is adapted to probe corona structure without interference, including the soft corona of loosely bound proteins. Microfluidics is used to elute the corona subsections for proteomics analysis of their compositions by nanoliquid chromatography tandem mass spectrometry. The in vivo relevance of the method is improved by the use of undiluted biological fluids under dynamic flow conditions. The workflow was applied to the preclinical development of pH-responsive and light- activated liposomes intended for tumour targeting and ocular drug delivery. A novel hyaluronic acid (HA)-coated liposome was also synthesized. The results indicate that thin and sparse monolayer hard coronas form on the liposomes in plasma and vitreous. It is covered by a soft corona of monolayer thickness in plasma, but in vitreous, the soft corona is less defined. Formulation-dependent differences in corona structure and relative protein composition were observed in plasma, and the liposomes clustered based on their surface charge. In vitreous, corona composition was independent of formulation properties. In both environments, there is a high overlap in hard and soft corona compositions, and the most abundant proteins are predominantly hydrophilic and negatively charged irrespective of liposome properties. A common set of surfactant and immune system associated proteins adsorbs in both plasma and vitreous, possibly in response to the high surface free energy of lipid bilayers, labelling them “non-self” lipid particles. However, liposomal lipids induced the enrichment of stealth-mediating proteins in the plasma hard coronas regardless of pegylation. These also enriched in the soft coronas of most other formulations, suggesting that the soft corona is part of the biological identity and modulated by liposome surface properties. Protein-specific, rather than formulation- specific factors, are drivers of protein adsorption in the vitreous, possibly due to the molecular crowding activity of structural HA. Although the HA-coated liposome bound more proteins in vitreous, which may interact with its structural meshwork, corona formation did not significantly influence the vitreal mobility of HA or PEG-coated anionic liposomes. The HA-coating yielded improved plasma stability of the light-activated liposomes with otherwise comparable properties, making it a promising coating for both intravenous and ocular drug delivery applications. The workflow addresses significant methodological gaps in the research field by providing information on truly complementary hard and soft corona compositions together with their in situ structural properties. This is achieved with undiluted biofluids and under dynamic conditions without the use of interfering labels. The first description of vitreal corona formation on drug delivery systems contributes to our understanding of ocular pharmacokinetics. The method was also applied to determine the kinetics of opsonin binding directly on liposomes for the first time. The convenient and easily reproducible workflow is well-suited for the preclinical development of liposomes, and it can be combined with other omics methodologies and adapted to accelerate the preclinical development of different types of nanomedicines.

View more