Browsing by Subject "biofarmasia"

Sort by: Order: Results:

Now showing items 1-10 of 10
  • Wikström, Jonna (Helsingin yliopisto, 2013)
    Cells have multiple functions in the body, including maintenance of the tissue structure and physiological homeostasis. The cells express and secrete proteins and other factors that exert actions in other cells. These principles form the underlying basis for cell therapy and cell transplantations. Transplanted cells can be used to regenerate tissue structures and homeostasis or they can be used as platform for secretion of therapeutic molecules. Biomaterials can be used to augment the cell growth, differentiation and viability in cell therapy. In addition, the biomaterial matrix may help the surgical placement of the cells into the target site. Importantly, the biomaterial may protect the cell from the immunological and inflammatory reactions after transplantation. The immunological protection of the transplanted therapeutic cells is based to selectively permeable artificial membrane. The membrane prevents the passage of high-molecular weight substances such as large antibodies and cytotoxic immune cells, but permits the passage of smaller molecules, like the secreted therapeutic molecules, nutrients, waste products and oxygen. Lately the interest in cell encapsulation and biomaterial cell interactions has increased due to the emerging techniques of cellular engineering and stem cell differentiation. Storage of microencapsulated cells in freeze-dried form would improve the logistics of the cell therapies (e.g. shipment to the hospitals for reconstitution and use). Otherwise, the microencapsulated cells should be kept viable in continuous culture conditions. The goal of this work was to evaluate alginate based microencapsulation of retinal pigment epithelial cell line (ARPE-19) for cell therapy. Cell viability was evaluated with stably expressed secreted alkaline phosphatase (SEAP), live/dead imaging and oxygen consumption. An empirical kinetic model was built based on FITC-dextran release and protein secretion to describe, release and potential accumulation of therapeutic proteins in the cell microcapsules. Primary animal experiments were done to evaluate the protein release and functionality in the cell microcapsules. Alginate based cell microcapsules were frozen and freeze-dried in order to evaluate the possibility for cell microcapsule preservation in dry powder form. In conclusion, ARPE-19 is a potential cell line for long-term cell therapy based on the expression of transgenes. ARPE-19 cells remain vital in the alginate microcapsules, and they are able to express stably transfected transgene over long periods (at least 20 months). The best cell viability was obtained with alginate microcapsules with calcium and barium cross-linking. This method results in adequate pore sizes that allowed secretion of SEAP. The same microcapsules showed biocompatibility after intraperitoneal administration in preliminary animal experiments. Empirical kinetic simulation model was able to predict the possibility of accumulation inside the alginate microcapsules and demonstrated that the accumulation potential depends on the microcapsule structure. Lyophilization of the cell microcapsules showed that the cells were able to retain some viability during freeze-drying and reconstitution when lyoprotectants were used.
  • Vellonen, Kati-Sisko (2010)
    Drug discovery and development from its very onset up to market approval is a long process lasting 10-15 years. New research tools are needed to accelerate and rationalize this process. Ocular drug research still relies heavily on animal testing with rabbits and other rodents. Computational methods and cell culture models are promising tools for early pharmacokinetic studies and may partly replace the animals in pharmacokinetic and toxicological studies. Computational methods are initially based on experimental data, but thereafter their application is straightforward and they can be used to reduce, partly replace and refine further experimental studies. Similarly, cell culture models may enable absorption and toxicity testing of drug candidates with continuously growing cells of human origin, and thereby reduce the need for animal experiments. The cornea is the main route of ocular drug absorption after topical administration, and the corneal epithelium is the most important barrier to drug permeation. Membrane transporter proteins play an important role in the general pharmacokinetics and toxicology. However, their role in ocular pharmacokinetics is still poorly understood. Based on literature analysis many ocular drugs seem to be substrates of transporters, but the expression of these proteins in the eye is largely unknown. The goal of this work was to develop and evaluate cellular and computational tools for ocular pharmacokinetics and toxicology, and to characterise the active drug transporters in the corneal epithelium. The expression of monocarboxylate transporters and ATP-binding cassette (ABC) class efflux proteins was studied in the corneal epithelium and human corneal epithelial (HCE) cell model. Human corneal epithelium expressed monocarboxylate transporters 1 and 4 (MCT1 and MCT4), efflux transporters multidrug resistance-associated protein 1 and 5 (MRP1 and MRP5), and breast cancer resistance protein (BCRP). Cultured human corneal epithelial cells over-expressed several ABC class efflux proteins and MCT1 and MCT4. The functionality of efflux and monocarboxylate transport was demonstrated in HCE cells and in the rabbit cornea ex vivo. The MTT test is a widely used cytotoxicity test in cell research. It was demonstrated that substrates and inhibitors of ABC class efflux proteins may interfere with the MTT test, presumably by inhibiting dye efflux from the cells. This may lead to an underestimation of toxicity in the MTT test. Quantitative structure property relationship (QSPR) models are commonly used in early drug discovery to predict ADME properties of novel compounds. Multivariate analysis was used to develop QSPR models for in silico prediction of the corneal permeability. Two factors, the distribution coefficient (logD7.4 /logD8.0) and hydrogen binding potential, were shown to be the parameters that determine the transcorneal permeability of a compound. These models were able to predict intracameral steady state drug concentrations in rabbit eyes. In conclusion, the new in silico QSPR model can make reliable predictions for passive drug permeability in the cornea, while the HCE model seems to over-express some membrane transporters as compared to the normal human corneal epithelium. Even if these investigated methods have some restrictions they are still very useful tools for drug discovery purposes.
  • Malinen, Melina (Helsingin yliopisto, 2014)
    New organotypic liver cell cultures are needed to predict the metabolism, excretion, and safety of chemical compounds. Liver cell models are particularly important since the liver largely regulates the ultimate fate of compounds in the body. Approximately 70% of the drugs administered to the body are metabolized or excreted by the liver. Animal models, cell cultures, and cell-free assays are the most common liver models. However, animal models and animal cells do not represent humans due to the interspecies differences in drug metabolizing enzymes and transporters. Instead, the most common cell-free methods, microsomes, are appropriate for drug metabolism studies, but the lack of drug transporters and transcription machinery prevents the complete evaluation of compounds. Primary human hepatocytes are capable of both drug metabolism and drug transport, and are, therefore, considered the gold standard to assess metabolism and toxicity of compounds in vitro. Primary hepatocytes, however, suffer limited availability, high functional variability, and difficulty with maintaining differentiated phenotypes and functions in cell cultures. Therefore, continuous human liver cell lines, such as HepG2 and HepaRG, have been widely used to evaluate drugs and chemicals even though they have defects in their biotransformation functions. The advantages of cell lines are their good availability, easy maintenance, and inducible drug metabolism. Generally, these cells are cultured in a two-dimensional (2D) manner that deviates from the physiological morphology and functions of the hepatocytes. The flattened 2D phenotype leads to reduced polarization and loss of important signaling pathways; this is likely to be a major reason for the failure in the prediction of drug metabolism, pharmacokinetics, and hepatotoxicity. It is believed that for more predictive in vitro models, the liver cells should be maintained in a three-dimensional (3D) microenvironment that allows reconstruction of polarization, and cell-cell and cell-extracellular matrix (ECM) contacts. The 3D cell cultures have been generated by different methods, such as cultures in matrices, scaffolds, bioreactors, and microfluidic platforms. Biomaterial hydrogels have demonstrated great potential for 2D liver cell culturing, but their potential to generate functional 3D liver cell cultures is largely unknown. The main goal of this thesis was to establish improved 3D liver cell cultures with biomaterial hydrogels. Particular attention was focused on the effects of 3D hydrogels on drug metabolism and excretion, cytoarchitecture, and cellular differentiation of HepG2 and HepaRG cell lines. As a starting point, we studied the suitability of wood-derived nanofibrillar cellulose (NFC) hydrogel as a cell culture matrix. NFC hydrogel has not been studied in cell culture before; however, as a novel, defined, animal-free, and abundantly available material, it evoked interest for testing. Herein, the wood-derived NFC was proven to own rheological and structural characters that allow 3D cell culture. Moreover, the NFC was compatible with the HepG2 and HepaRG cells, allowing for the formation of 3D multicellular aggregates with increased apicobasal polarity. When compared to commercial hydrogels, the NFC supported the albumin secretion, an indicator of hepatocellular synthetic function, from HepG2 and HepaRG cells as well or even better. These results demonstrate the potential of wood- derived NFC to function as an ECM analogue, and present the first HepaRG aggregate cultures. Next, the effect of the RAD16-I peptide hydrogel on the HepG2 cell line was investigated in more detail. Immunofluorescence staining and vectorial transport showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of canalicular efflux transporters, multidrug resistance-associated protein 2 (MRP2), and multidrug resistance protein 1 (MDR1), in the spherical HepG2 cell aggregates. The study clearly demonstrated that the peptide hydrogel increases the apicobasal polarity and appearance of bile canaliculi structures in HepG2 cell cultures. The plasticity of HepaRG liver cells was exploited to investigate the impact of 3D NFC and hyaluronan-gelatin (HG) hydrogel cultures on the phenotype of both undifferentiated HepaRG cells (early liver progenitors) and differentiated HepaRG cells (hepatocyte-like cells together with cholangiocyte-like cells). Based on the expression and activity of hepatic markers, drug metabolizing enzymes, and drug transporters, the 3D NFC and HG hydrogels promoted the differentiation of HepaRG liver progenitor cells when compared to the standard 2D technique. Instead, the 3D hydrogel cultures could not really improve the properties of differentiated HepaRG cells. In conclusion, these findings reveal the capability of the NFC, RAD16-I peptide, and HG hydrogels to improve the properties of HepG2 and HepaRG human liver cells. The new spheroid cultures of HepG2 and HepaRG cells may represent added value for pharmacokinetic and toxicity predictions, showing a liver-like cytoarchitecture and demonstrating applicability for drug metabolism and transport studies. Overall, the results deepen our knowledge of the 3D liver cell cultures.
  • Kartal-Hodzic, Alma (Helsingin yliopisto, 2012)
    Tobacco smoke is a major risk factor for the development of cancers in the upper parts of gastrointestinal tract. It has been estimated that the risk of oral cancer among smokers is 7 10 times higher than for never-smokers. Acetaldehyde is formed during the tobacco smoking burning process and may be one of the most toxic compounds in tobacco smoke. According to the International Agency for Research on Cancer (IARC), in 2004 acetaldehyde was classified as a possible carcinogen in humans (Group 2B). In 2009, acetaldehyde was classified in Group 1, as a carcinogen to humans. A non-essential-amino acid such as L-cysteine, is able to bind acetaldehyde and form 2-methylthiziolidine-4-carboxylic acid (MTCA). The general aim of this study was to through formulation studies to explore the ability of L-cysteine to eliminate carcinogenic acetaldehyde present in saliva. In addition, the aim was to develop user-friendly L-cysteine containing chewing gum to reduce acetaldehyde formed during tobacco smoking. The main variables were the chemical form of L-cysteine used (L-cysteine or L-cysteine hydrochloride) and the chewing gum preparation method (traditional and novel direct compression method). Furthermore, the aim was to obtain more information on the optimal formulation properties, using approaches such as stability studies and possible interactions between cysteine and used excipients. Caco-2 cell lines were used to access the ability of L-cysteine and MTCA to absorb from the gastrointestinal tract. A computational model was developed to analyse the effects of different physiological factors and effect of formulation parameters on tobacco smoke acetaldehyde. The combined results of these studies suggested that tobacco smoke carcinogenic acetaldehyde can be successfully eliminated with prepared L-cysteine chewing gums. Compared to the traditional manufacturing process the directly compressed gum formulation can offer an alternative method to traditional chewing gum production. Due to the slower dissolution rate, better compatibility with excipients, and better stability under higher temperature and humidity, L-cysteine as a free base is a better candidate for chewing gum formulation than cysteine hydrochloride. The Caco-2 permeability studies indicate no significant risk of the locally administered L-cysteine being absorbed before binding to acetaldehyde. Permeability results also indicated that MTCA is not absorbed locally from the gastrointestinal tract, which reduces the risk of systemic effects. An MTT assay, a widely used cytotoxicity test, demonstrated that neither L-cysteine nor MTCA was toxic to the Caco-2 cells. A computational model that was developed was able to show how sensitive acetaldehyde is to changes in the amount of L-cysteine as well as in saliva excretion rates. The model can be used as a tool for the prediction of drug amount and the local effect in the mouth of water-soluble compounds, such as L-cysteine. In conclusion, elimination of acetaldehyde, not only carcinogen, but also agent which possibly increases the addictive potential of tobacco, might help in the fight against smoking and make smoking cessation programs more efficient. L-cysteine, a non-essential amino acid, is able to prevent the harmful effects of acetaldehyde by binding acetaldehyde and forming MTCA. It should be kept in mind that acetaldehyde elimination does not make smoking completely harmless and tobacco smoke contains other carcinogens and addictives. The best way to protect from tobacco induced diseases is to refrain from smoking. However, besides the fact that most smokers want to quit but most attempts fail and since tobacco smoke contains many carcinogenic compounds, in the future, developed computational models can offer a new view in eliminating or reducing not only one toxic compound from tobacco smoke but also many other compounds using only one formulation containing various active compounds.
  • Hirvinen, Mari (Helsingin yliopisto, 2016)
    Cancer is the leading cause of death worldwide creating a need for novel cancer treatments that are more efficient but also safer and more specific. Oncolytic viruses (OVs) have shown a solid safety profile in clinical trials. OVs are nowadays considered immunotherapies because of to their ability to stimulate the host immune system to fight against cancer. Promising efficacy has been seen in some trials, however, efficacy is often seen only in a small group of patients. The purpose of the thesis was to improve the efficacy of OV therapies by boosting the immunogenicity of the viruses, and to optimize the therapeutic efficacy by selecting favorable patient populations and by developing a method to tailor the drug individually for each patient. In the first study, an oncolytic adenovirus (OAd) was modified to express human tumor necrosis factor alpha (hTNFα), a potent immunomodulatory cytokine. The TNFα-virus showed effective tumor cell killing associated with signs of immunogenic cell death and enhanced recruitment of immune cells to the infection site. We also saw potential for combining the TNFα-virus therapy with radiation. In another study the immunogenicity of an oncolytic vaccinia virus was enhanced by modifying it to express DNA-dependent activator of interferon-regulatory factors (DAI), a potent inducer of innate immune responses during virus infection. We showed that the DAI-virus induces expression of genes involved in immune responses, and treatments with the virus showed improved cancer-killing efficacy and immunogenicity in murine and human melanoma models, suggesting applicability also in vaccine design. Response rates after virotherapies vary between patients, and there is a lack of markers that would help predict the patient cohorts who would benefit from the therapy. We screened over 200 cancer patients treated with OAds for two Fc gamma receptor (FcγR) polymorphisms to determine if these polymorphisms would affect the responsiveness to the treatments. We observed a certain FcγR genotype combination (FcγRIIIa-VV + FcγRIIa-HR) to be predictive of poor overall survival after OAd treatments. To tailor the OV therapy for enhanced specificity, we developed a novel platform (PeptiCRAd) to coat a virus with tumor-specific antigens (peptides) for improved induction of cancer-specific immunity. Efficacy and immunogenic potency of the PeptiCRAd were shown in several in vivo models. Our results suggest that administration of tumor-specific peptides on the surface of OVs increases the anti-tumor efficacy compared to treatments with viruses or peptides alone. This platform has potential to be used as a carrier and adjuvant for patient-specific peptides to trigger anti-tumor immunity in a personalized manner.
  • 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.
  • Marvola, Janne (Helsingin yliopisto, 2008)
    The per oral route of administration is the most convenient and commonly used means of drug administration. However, many drugs are ineffectively absorbed per orally, or the dosing frequency is inconveniently short. Controlled-release (CR) technologies offer means to optimize the resulting plasma concentration-time profiles of such drugs. Transit of dosage forms in the gastro-intestinal (GI) tract is one of the major factors that determine their overall in vivo performance. Gamma scintigraphy is one of the most appropriate means of studying the fates of drug formulations in the human GI tract. For complex dosage forms imaging methods involving use of stable (non-radioactive) markers during preparation of the products are preferable. Samarium oxide can be incorporated in a formulation during normal manufacture and later it can be activated in a thermal neutron flux of a nuclear reactor to 153Sm2O3. A number of scintigraphic studies have been published during the last two decades, but only a few studies about neutron activation in relation to oral administration. In per oral drug delivery, the rate and extent of drug absorption is determined by the drug, the formulation and the properties of the gastrointestinal tract. Modelling helps to reveal the relative importance of different factors and to predict the biopharmaceutical impact of formulation changes. So far, the published computational models have included only stomach and small intestine, but not colon, even though major part of drug release from the CR formulations takes place in the colon. Pharmacokinetic simulation models can be designed based on parameters in relation to the transit of the formulation and the physiological environment in the gut obtained from imaging studies. In this study, neutron activation-based scintigraphic methods were developed and evaluated for various CR formulations in the human GI tract, from the oesophagus to the colon. The developed methods were successfully utilized in imaging the transit of dosage forms and in verifying drug release for a maximum of 24 h after administration. A total of 48 healthy volunteers were imaged in five clinical studies. The in vivo transit characteristics of capsule formulations were studied in the oesophagus. Muco adhesion of chitosan granule formulations was studied in the stomach. Sites and rates of drug release and disintegration of different CR capsules and tablets were investigated in the small intestine and the colon. In one imaging study, colon targeted drug delivery using a CR capsule formulation was also verified in vivo. Results of these studies revealed new information in relation to the fates of the studied dosage forms in the GI tract and provided a basis for planning subsequent in vitro and in silico studies for further development of these formulations. The developed imaging methods and equipment used were proven to be valuable tools in CR per oral drug delivery studies The fate of one CR caffeine tablet formulation was studied further by means of computational pharmacokinetic simulation modelling by designing an extended compartmental model that takes into account drug release, transit and absorption in the small intestine and the colon. Three colon segment compartments were added to the existing seven-compartment small intestinal transit and absorption model. The new model revealed that the inter-individual differences in the kinetics of the tablet are due to differences in drug metabolism, rather than in the dosage form transit.
  • Saari, Heikki (Helsingin yliopisto, 2020)
    Drug delivery aims to optimize the systemic distribution of administered drugs to reach their target tissue in an effective and specific manner. This approach is arising also in the field of cancer therapy, since traditional small molecule chemotherapeutics can cause severe side effects and more sophisticated biomolecular therapeutics cannot always reach the target cells on their own. For these purposes, therapeutics can be packed into carriers that improve their pharmacokinetics by providing a suitable, protected environment for the cargo to travel in the body, armed with targeting molecules that guide them to the site of interest. Synthetic drug carriers include liposomes, porous silica and different polymer particles that can be modified to contain necessary surface structures for targeted delivery and improved biocompatibility. While they have shown much promise in research settings, their success in cancer treatment has been limited to only a handful of commercially available formulations, some of which have presented liver toxicity as a result of continuous administration. Additionally, targeting cancer cells is difficult because cancer is a very individual disease, and heterogeneity exists even within tumors, so finding any universal cancer-specific markers to target is extremely challenging. Nature has adopted a similar approach for delivering molecules containing biologically functional cargo from cell to cell. These naturally occurring carriers are called extracellular vesicles (EVs), in contrast to intracellular vesicles that mediate cargo trafficking inside of the cells. EVs are 50 – 1000 nm in diameter, consisting of a lipid bilayer with embedded membrane proteins that encloses water-soluble biological cargo within. Their exact composition and cargo vary from cell to cell and depending on the condition of the cells that produce them. Additionally, cells have multiple pathways for EV secretion and production. For these reasons EVs comprise a very heterogeneous population of vesicles in composition and they also have multiple biological functions that have not been completely clarified yet. EVs have been found in practically all body fluids and organisms that have been studied, taking part in the normal and pathological functions of the organism. For example, procoagulant EVs are found in blood and saliva that participate in hemostasis, while EVs secreted by cancer cells promote the survival, growth and metastasis of the tumor. They are able to affect cells via membrane interactions and by delivering functional cargo into their recipient cells. It has been shown that EVs are even able to target cells selectively by binding to specific cellular receptors, enabling targeted cargo delivery. Given their natural cargo delivery properties, EVs present remarkable potential for drug delivery applications. As cancer cells also use EVs to communicate with each other, their EVs reflect the same heterogeneity that exists within the cells themselves, and may provide useful insights for cancer-targeted drug delivery. However, the internalization mechanisms of drug-carrying EVs and especially the fate of the drugs they carry is not well understood. In this thesis, the use of EVs was assessed for the delivery of chemotherapeutic drug paclitaxel and an oncolytic adenovirus that represent small molecular and biological chemotherapeutics, with cancer cell-derived EVs as the model carrier. The studies presented here focus on the preparation, characterization, intracellular tracking and effectiveness of these EVs for cancer therapy. The obtained results provide novel methods and knowledge for the future development of EV-based therapeutics for the treatment of cancer. First, EVs were loaded with paclitaxel by incubation, which enhanced the cytotoxic effect of the drug, changing its internalization from passive diffusion to endocytosis. A novel approach using fluorescence lifetime microscopy was introduced for tracking the release of paclitaxel from the EVs inside of the cells, identifying distinct patterns of subcellular drug release in individual cells and its intracellular kinetics. This method was able to show details about the drug release mediated by EVs that could not be observed with conventional fluorescence microscopy. Additionally, the relationship between EVs and adenoviruses was explored, revealing a previously undocumented pathway of spreading adenoviral infection via EVs. This EV-mediated infective delivery of the viral genome occurred separately from the classical pathway of adenoviral life cycle, and produced infective particles resembling EVs more than virions. EVs can in theory enhance the pharmacokinetics of oncolytic viruses by hiding them from the immune system and providing them alternative pathways for cell targeting and internalization, however it was found that infective EVs do react with adenovirus neutralizing antibodies. Taken together, these results suggest that EVs can act as versatile carriers of therapeutic cargo for cancer treatment, ranging from small molecule chemotherapeutics to oncolytic viruses, though they require further development. New methods are reported constantly for preparing and studying therapeutic EVs with new, innovative approaches that will help in the future treatments of cancer and other diseases.
  • Rojalin, Tatu (Helsingin yliopisto, 2016)
    Biophotonics is an emerging area of scientific research that uses light of photons to probe biological specimens, such as tissues, cells and molecules. The field of biophotonics is broad and considerably multidisciplinary. Therefore the prerequisite for understanding biophotonics is the capability to integrate the fundamental knowledge of the physics of light with perspectives of engineering of devices and instruments used to generate, modify, and manipulate light. Also, the fundamentals of biology and medicine are essential particularly comprehension of the biochemical and cellular phenomena that occur in living systems, and how such phenomena can be scaled up to concern the physiology of organisms, for example humans. Biological pathways and processes differ in the healthy and diseased state, and that is why it is essential to develop understanding of pathophysiology and various states of disease such as cancer, neurodegenerative disease or infectious states. Consequently, solid insights into the functions of medical treatments, including biopharmaceutics, are needed. Raman and surface plasmon resonance (SPR) are both light-based technologies enabling label-free measurements with high sensitivity. The primary aim of this Thesis was to tackle the emerging hardships encountered in the fundamental biopharmaceutical research, clinical settings, or in the pharmaceutical industry by introducing applications and data analysis methods based on the cutting edge Raman and SPR technologies. These techniques represent the biophotonic cornerstones as tools for biopharmaceutical applications throughout this Thesis. The scope of this Thesis was essentially broad. First, small drug molecules were investigated with state-of-the-art time-gated Raman technology, showing that the interfering photoluminescent backgrounds can be effectively suppressed thus improving the acquired Raman data significantly. Additionally, EVs were studied with laser tweezers Raman spectroscopy (LTRS) as larger scale analytes and representatives of a highly interesting topic in the current nanomedical field. When Raman data from several different types of single EVs was examined using sophisticated data analysis, distinct subpopulations were observed, and the differences could be related to the biochemical compositions of the vesicle membranes. For the first time, the study showed the importance of measuring single EVs instead of a pool of vesicles. Multi-parametric SPR (MP-SPR) technology was harnessed to develop applications and data analysis methods for small and larger scale analytes. Hence, a new small drug molecule, spin-labeled fluorene (SLF), was investigated in the context of Alzheimer s disease (AD) particularly its potential to interfere with the detrimental amyloid peptide aggregation processes. The developed bio-functional in vitro platform in combination with rigorous data analysis and computational simulations demonstrated the capabilities of SLF when it was employed in various biomimetic aggregation schemes. Moreover, liposomes were examined with the MP-SPR as larger scale nanomedical particles for the purposes of safe and effective nanocarrier development. The administration of a liposomal nanocarrier into the blood circulation was mimicked in the designed bionanophotonic in vitro schemes. Undiluted serum was made to interact with immobilized model liposomes in dynamic flow conditions. The findings revealed that the variation in surface chemistries of the liposomes plays a role when serum essentially immune system components are interacting with the liposomes. In particular, distinct soft and hard protein coronas were observed and characterized during the interactions. Collectively, the results and findings in this Thesis underline the broad potential of biophotonics for biopharmaceutical applications. The technical improvements in instrumentation, and creativity in the application and data analysis development make the future of biophotonics bright.
  • 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.