| Abstract [eng] |
The main aim of this master thesis project was to investigate microglial exosome potential as chemotherapy delivery vehicle. To accomplish this goal, the following objectives have been set: 1) to isolate exosomes from human microglial cells and characterise them based on their size, morphology and specific markers; 2) to track exosome uptake into human glioblastoma cells; 3) to evaluate the viability of glioblastoma cells after treatment with natural microglial exosomes 4) to load chemotherapy agent doxorubicin hydrochloride into microglial exosomes by using electroporation method and determine drug loading efficiency; 5) to evaluate doxorubicin hydrochloride loaded microglial exosome effectiveness in eliminating human glioblastoma cells. Exosomes were isolated using precipitation technique with polyethylene glycol and centrifugation. In order to find the optimal conditions for exosome isolation, microglial cells were grown in serum depleted growth media in 37oC and 39oC temperatures. The concentration of isolated exosomes was determined by measuring total exosomal protein concentration by using spectrophotometric Bradford assay. The size of exosomes was measured using dynamic light scattering technique, their morphology was examined using transmission electron microscopy and the concentration of exosomal marker CD9 was assessed using the ELISA method. Exosome uptake into glioblastoma cells was monitored by labelling exosomes with RNA dye conjugated with Alexa Fluor 555 and visualised using a fluorescence microscope. Doxorubicin hydrochloride was loaded into exosomes using the electroporation method and the amount of encapsulated drug was determined by measuring the intrinsic fluorescence of doxorubicin. To evaluate the effect of doxorubicin hydrochloride loaded microglial exosomes and natural microglial exosomes on the viability of human glioblastoma cells, the cells were incubated with drug-loaded and empty exosomes as well as free doxorubicin hydrochloride with appropriate concentrations. The cell viability was measured after 48 and 72 hour incubation using spectrophotometric analysis and fluorescence microscopy. The fluorescence image analysis was performed using ImageJ software. Statistical data analysis was performed by SigmaPlot 14.0 software. The experiments revealed that microglial cells excrete more exosomes when they are grown in serum depleted media in 37oC temperature. The size range of isolated exosomes was 60-90 nm and their morphology corresponded to a typical exosome (round shape and enclosed with plasma membrane). ELISA CD9 assay further confirmed that isolated particles indeed were exosomes. From exosome uptake studies, it was revealed that exosomes can be internalised by glioblastoma cells in 2 hours and remained inside the cells for 24 hours. Glioblastoma cell viability assay, where the cells were treated with natural microglial exosomes, revealed that after 72 hour treatment the viability was significantly reduced compared to untreated control. Doxorubicin hydrochloride was loaded into exosomes via electroporation. The encapsulated drug concentration was 342 ng/ml and loading efficiency was 0.34%. After 72 hour incubation with doxorubicin hydrochloride loaded exosomes, a statistically significant reduction in glioblastoma cell viability was observed as compared to untreated control, cells treated with natural microglial exosomes and free doxorubicin hydrochloride. |
