Browsing by Subject "liposomes"
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(2016)Extracellular vesicles are cell-derived vesicles which consist of two lipid layers. Extracellular vesicles involve in intercellular communication, maintaining of homeostase and development of pathophysiological states in human body. Extracellular vesicles are promising biomarkers and drug carriers in future. The aim of this study was to develop a method based on time resolved fluorescence microscopy and autologous extracellular vesicles labelled with environmentally sensitive fluorescent probes for studying the distribution of mitose-inhibitor paclitaxel in prostate cancer cells (PC-3) carried by extracellular vesicles. The efficacy of paclitaxel loaded extracellular vesicles was compared to synthetic liposomes. The two subpopulations of extracellular vesicles, exosome -and microvesicle-enriched, were isolated from the PC-3 cell media by differential ultracentrifugation. The size distribution and particle concentration of extracellular vesicles was determined by nanoparticle tracking analysis. DSPC-Cholesterol liposomes were prepared by reverse-phase evaporation method and the size distribution of the liposomes was determined by dynamic laser diffraction and nanoparticle tracking analysis. Paclitaxel was loaded into the liposomes in hydration phase and into the extracellular vesicles by incubating vesicles and paclitaxel. Unbound paclitaxel was removed from samples by ultracentrifugation. The the dose-dependent sytotoxicity of paclitaxel loaded extracellular vesicles and liposomes was evaluated with Alamar Blue viability assay. The release and distribution of paclitaxel from extracellular vesicles in living PC-3 cells was investigated by confocal microscopy and time-resolved fluorescence microscopy. The exosomes had approximately 50 nm smaller diameter than microvesicles and exosome particle concentrations were significantly higher compared to microvesicles. According to viability assays conducted with wide range of concentrations, paclitaxel loaded in microvesicles were slightly more effective than paclitaxel loaded in exosomes. The time-resolved fluorescence microscopy was useful method for investigating the release and distribution of extracellular vesicle bound paclitaxel, since we succesfully detected changes in Paclitaxel-OregonGreen fluorescence lifetime in different phases of the drug delivery process. With confocal microscopy we detected that paclitaxel loaded extracellular vesicles were already uptaken inside the cells after two hours of incubation and after few hours, paclitaxel was detected in microtubules of PC-3 cells and killed PC-3 cells. Extracellular vesicles may improve the accumulation of paclitaxel into tumor cells thus preventing the side-effects of paclitaxel. Nevertheless, PC-3 cell derived extracellular vesicles have ability to increase the PC-3 cell viability, which limits their potential use as drug carrier due to safety issues. In addition, extracellular vesicles characterization and isolation methods lack standardization and the isolation of exosomes and microvesicles is impossible due to this fact. Extracellular vesicles involvement in physiological and pathophysiological states should be investigated throughoutly and their safety as drug carriers should be examined both in animal and human.
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(2022)Liposomes are biocompatible spherical nanosized vesicles consisting of hydrophobic phospholipid bilayer encasing an aqueous core. They can be utilized as drug carriers by either encapsulating molecules inside the core or embedding them in the bilayer accordingly to achieve numerous advantages such as prevention of rapid clearance and reduction of adverse effects as systemic exposure is reduced. Despite the marked efforts in designing the liposomes to improve therapeutic outcomes, only limited drug concentrations are achieved at the target sites such as in solid tumors. Stimuli-responsive liposomes could be applied as potential delivery systems to achieve spatiotemporally controlled drug delivery, i.e., the drug release could be pinpointed and restrained to the target site. In this thesis, the objective was to study the light-activated indocyanine green (ICG) liposomes as nanocarriers for peptide-based anti-tumor agents. The physicochemical characteristics, stability and functionality of the prepared liposomes were determined alongside optimizing the formulation as needed and utilizing different model peptides as encapsulated compounds. Additionally, the peptide stability during near-infrared (NIR) light illumination and the effects of the anti-angiogenic model peptides in vitro were investigated. The stability of the liposomes was assessed by monitoring the size of the liposomes, intactness of ICG, and passive leakage of the peptides over time, and by determining the phase transition temperatures of the different formulations. The liposomes remained adequately stable in different relevant conditions, and the observed phase transition temperatures did not indicate the lipid bilayer becoming permeable in physiological temperatures. However, the rate of passive leakage was rather high in all formulations, although with stiffer lipid bilayer in the “rigid” formulation, the unintended release was able to be decreased slightly in comparison to the other formulations. On the other hand, light-triggered release upon illuminating the liposomes remained considerably low in all formulations. The intactness of peptides seemed to not be impacted by the illumination. Also, no cytotoxic effects were observed after exposing human umbilical vein endothelial cells (HUVEC) to the peptides. The final “rigid” formulation showed the best functionality out of those included in the studies. It remains to be investigated whether the formulation could be improved further for optimal functionality and stability, and to what degree do the properties of the cargo molecule affect the performance of the liposomes.
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(2020)Liposomes are nano-sized vesicles, that are composed of a phospholipid bilayer structure. They can be utilized as drug carriers, in which case the drug is incorporated either to their hydrophilic internal cavity, or into their hydrophobic bilayer structure. For anticancer drugs, liposomal formulations have exhibited their capability in reducing adverse effects of anticancer drugs. This is achieved mainly by the enhanced permeability and retention (EPR) effect, in which liposomes accumulate into tumour tissue. However, the conventional liposomes release their drug content passively, and a proportion of drug is distributed to off-target tissues. Therefore, there is a demand to develop liposomes from which the content can be released in a controlled manner, by an external stimulus. The objectives of this master’s thesis project were to determine the potential of light-activated paclitaxel (PTX) liposomes for the treatment of lung cancer, and to optimize a dynamic cell culture system, QuasiVivo® (QV), to study the off-target effects of light-activated PTX liposomes. The hypothesis was that the induction of the light-activated PTX liposomes would increase the efficiency of paclitaxel treatment. For QV experiments, it was expected that the presence of flow would improve the viability of the cells. The encapsulation efficiency of PTX into the liposomes and the effect of the PTX incorporation into the phase transition temperature of the liposomes were determined. The stability of liposomes was determined by monitoring the liposomal size and light sensitizer absorbance during a storage period. The cells of lung cancer cell line A549 were cultured inside QV system, and their viability was monitored with two commercial cell viability assays. Incorporation of PTX decreased the phase transition temperature, but the liposomes remained stable in the studied conditions. The PTX liposome treatments with and without light activation resulted in the similar efficacy as free PTX treatment did. A549 cells failed to display superior viability inside the QV compared to static conditions. Cells cultured under lower flow rate portrayed modestly higher viability. The light-activated PTX liposomes did not improve the efficacy of PTX treatment. Neither of the flow rates were optimal for A549 cells, as the variation between experiments was high. The EPR effect is the main reason for the improved effects of liposomal anticancer drugs, therefore, it is likely that in vivo experiments would elicit the differences between the efficacy of the liposomal and free PTX. The non-existent effects of light activation on the viability are likely caused by the low total concentration of the light sensitizer in the treatment solution.
Now showing items 1-3 of 3