Browsing by Subject "extracellular vesicles"

Even though cancer treatment modalities have improved during last decades, there is still lack of specific, efficient and curative treatments especially in case of advanced and metastatic cancers. One relatively new approach is to use oncolytic adenoviruses, which selectively infect and kill cancerous cells leaving healthy cells unharmed. These viruses have shown to be effective especially when administered intratumorally and in combination with chemotherapeutics. However this approach has multiple challenges like rapid clearance by antibody neutralization in systemic administration. Another challenge is the cell entry of oncolytic adenovirus, which is mainly mediated by the Coxsackie-Adenovirus receptor and this receptor is downregulated in various cancer cells. Rapid clearance and reduced cell entry thus lead to decreased amount of oncolytic adenovirus in target cells and decreased efficacy. In order to overcome these limitations, this study explored the possibility to use cancer cell derived extracellular vesicles (EVs) as drug delivery system for oncolytic adenovirus. Since oncolytic adenoviruses have shown to be effective especially in combination with chemotherapeutics, the ability of EVs to deliver both oncolytic adenoviruses and chemotherapeutic drug paclitaxel was studied. The aims of this study were to i) study whether oncolytic adenoviruses could be encapsulated inside EVs (EV-virus complex) and load this complex with paclitaxel (EV-virus-PTX complex), ii) discover whether the surface charge or size distribution of EV-virus and EV-virus-PTX complexes differs from the control EVs and iii) study the infectivity/efficacy of EV-virus and EV-virus-PTX complex in comparison to noncapsulated adenovirus in vitro. Since this is a novel approach, the literature review focused on the characteristics, advantages and challenges of oncolytic adenoviruses and EVs. In order to determine whether cancerous cell are able to encapsulate oncolytic adenoviruses inside EVs, A549 lung cancer and PC-3 prostate cancer cells were infected with oncolytic adenovirus and the formed EVs were isolated form conditioned media using differential centrifugation. Paclitaxel was loaded into these EV-virus complexes with incubation. EV-virus complexes were imaged using transmission electron microscopy (TEM) (i). The characteristics of these EV-virus and EV-virus-paclitaxel complexes were studied by determining the surface charge by electrophoretic light scattering and the size distribution by nanoparticle tracking analysis (ii). In order to determine the infectivity/efficacy of these complexes in autologous use, three in vitro level assays were performed (cell viability, immunocytochemistry and transduction assay) (iii). In addition confocal microscopy was used to observe the localization of EV-virus complexes inside the cell. These studies pointed out that both cell lines were able to encapsulate oncolytic adenovirus inside EVs, which was observed by TEM. The size distribution of these EV-virus and EV-virus-PTX complexes may support this observation and the size was in range 50-500 nm. In addition the determined surface charge was shown to be similar in EV-virus and EV-virus-PTX- complexes when compared to control EVs derived from noninfected cells - however more specific assays in order to characterize the surface properties of EV-virus complexes are needed. As a main finding, these EV-virus and EV-virus-PTX complexes were shown to significantly increase the efficacy of oncolytic adenovirus in comparison to free oncolytic adenovirus, paclitaxel and paclitaxel+virus combination in all three in vitro assays. In addition localization of the EV-virus complex was seen with confocal microscopy imaging. These results indicate that EVs may enhance the delivery of oncolytic adenovirus into cancerous cells. Using EVs as a drug delivery system for both oncolytic adenovirus and chemotherapeutic drug paclitaxel was shown to increase the efficacy of oncolytic adenovirus in comparison to free virus. This characteristic could potentially enhance the targeting ability to cancerous cells and thus lead to decreased amount of side-effects of healthy tissues especially in case of chemotherapeutics. These promising results of this novel approach are however preliminary due to relatively low number of repetitions (n~3) and more research is needed especially in order to characterize, purify and concentrate the EV-virus complexes.

Cells release different types of phospholipid bilayer-limited vesicles into the extracellular space. These are commonly referred to as extracellular vesicles (EVs). Exosomes (EXOs), ca 50-100 nm in diameter and microvesicles (MVs), ca 100-1000 nm in diameter, having different intracellular origin, are the two main subpopulations of EVs. EVs have been demonstrated to carry a range of proteins and nucleic acids subsequently delivered to recipient cells, making them attractive as drug delivery vehicles. Several mechanisms for the cellular uptake of EVs have been established. When a nanoparticle is introduced into blood plasma, plasma proteins are adsorbed to its surface, forming a protein corona. The formation of the corona is a dynamic process, governed by individual protein concentrations as well as their respective affinities for the surface. Proteins of the corona interact with surrounding cells, thus being able to influence the cellular uptake of the nanoparticle. In the current study, the uptake of PC-3-derived EVs into PC-3 cells was investigated. Moreover, the impact of a human blood plasma-derived protein corona on said uptake was assessed. EVs were isolated from collected PC-3 cell culture medium using differential centrifugation. Experiments were performed separately for MVs (20000xg EV-fraction) and EXOs (110000xg EVfraction). SDS-PAGE analysis revealed adsorption of plasma proteins to EVs, following their exposure to plasma. Prior to uptake experiments DiO-labelled EVs were either incubated or not incubated in plasma. Plasma incubation lasted overnight. PC-3 cells were then treated with either of the two EV-preparations. Following incubation, EV uptake was assessed using confocal microscopy by determining the percentage of positive fluorescent cells in cell cultures. Pre-study plasma incubation resulted in a reduced or unchanged uptake of MVs and in a reduced uptake of EXOs, when compared to their native counterparts. In conclusion, the plasma-derived protein corona was shown not to improve EV uptake. It is worth noting that the current study limits itself to the use of PC-3-derived EVs and PC-3 cells as recipient cells in uptake experiments.

Background and objectives: Cells secrete extracellular vesicles (EV) and it has been found that cells communicate via EVs. EVs are liposome-like vesicles. Membrane is consisting of a lipid bilayer and hydrophilic moiety is inside the vesicle. It has been found that EVs carry e.g. nucleic acids, lipids and proteins. The aim of this master thesis was to determine whether EVs can transport non-coding RNA (siRNA) into the central nervous system through the blood-brain barrier. In the literature review, investigated methods which has been used to load siRNA into the EVs and how EVs are transported through the blood-brain barrier. The aim of the experimental part was to produce and isolate EVs and to load FAM-labeled dsDNA and siRNA into EVs by physical methods such as sonication and electroporation. Fluorescence measurements were taken to demonstrate FAM-labeled DNA loading into EVs and the functionality of the siRNA-loaded EVs was measured by measuring the expression level of the gapdh gene. Methods: Extracellular vesicles were produced in ARPE-19 and PC-3 cells. EVs were isolated from the cell culture medium by two-step differential centrifugation (DC) and further purified by gradient centrifugation (GC) by using the OptiPrep™-reagent. OptiPrep™-reagent was purified by Amicon 10kDa filtration tubes. The average particle size and size distribution of the isolated EVs were determined by NTA analysis, protein concentration was measured by colorimetric BCA method and EVs were characterized by Western blot method using HSP70 and CD9 antibodies. EVs were loaded with 21 bp length FAM-labeled dsDNA or siRNA by sonication or electroporation. Free nucleic acid and OptiPrep™-reagent were purified from EVs by the size-exclusion chromatography with Sephacryl (S-300) column. Loading efficient of the EVs were studied by measuring the fluorescence (ex 485 nm, em 520 nm) and qPCR method was used to demonstrate the functionality of the siRNA loaded EVs. In qPCR, the expression level of the gapdh gene was measured in dividing ARPE-19 cells. Results: DC and GC purified ARPE-19 and PC-3 EVs had an average particle size of about 140 nm and were successfully characterized by Western blot method. PC-3 EVs were produced in the bioreactor and the yields were enough for loading experiments. ARPE-19 cells produced only small amounts of EVs in culture flasks. The size-exclusion chromatography was a good method to purification free nucleic acids from EVs. The sonication method did not cause EVs to be degradation under the conditions used. Based on fluorescence measurement, FAM-labeled dsDNA could not be loaded into EVs. The functionality of siRNA-loaded EVs could not be demonstrated in ARPE-19 cell experiments. After electroporation large number of EVs were lost and this method of loading siRNA into EVs did not proved to be suitable. Conclusions: ARPE-19 EVs must be produced in the bioreactor to produce enough EVs for loading experiments. The EV purification protocol should be further optimized since the recovery-% of EVs were low after several purification steps. The size-exclusion chromatography is suitable for the purification of the free siRNA from EVs, but the chromatography method needs further optimization and miniaturization. Loaded EVs should be produced by aseptically or alternatively sterilized prior to ARPE-19 cell assay. Physical loading method, such as sonication, can be scaled to larger scale. Sonication method should be optimized e.g. by experimenting with higher temperatures and longer sonication times. The probe sonicator should be tested instead of the water bath sonicator. According to the literature review, the use of extracellular vesicles as carriers for biomolecule delivery into the central nervous system seems to be promising.