The collected small EVs were stored at ?20 C for further analysis

The collected small EVs were stored at ?20 C for further analysis. The urinary small EVs were further purified using qEV exosome isolation size-exclusion chromatography (SEC) columns (Izon Science, Oxford, UK) according to the manufacturers instructions, and the eluted fractions were analysed for exosome size and concentration using nanoparticle tracking analysis (NTA) as described below. 4.3.2. obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells. tachyzoites (Physique 5). Open in a separate window Physique 5 Combination of parasite contamination with urinary exosome uptake in RPE1 cells. (A) through interactions with reactive oxygen species (ROS) [25]. The tendency of nanoparticles to accumulate in specific tissues supports the idea of using nanoparticles to target parasite cysts in the host tissue. Such particles were synthesized in combination with extracts of natural plants (date palm and nabka leaves) and were tested and compared to alternative sulfadiazine (oral: 100 mg/kg) drug therapy [26], with the outcome showing that this treated nanoparticles prevented the occurrence of hepatotoxicity more effectively than did standard treatments. In another study, two different drugs against ocular toxoplasmosis were successfully loaded into polystyrene latex particles during polymerization, generating coreCshell nanoparticles [27]. However, the drug-loaded particles were no more effective than the unloaded particles. Orally administered triclosan-loaded liposomes have also been investigated in terms of their efficacy against a virulent strain of [28]: Both the drug alone and drug-loaded liposomes reduced parasite burden and infectivity of tachyzoites harvested from the peritoneal fluid of infected treatment groups, and the tachyzoites caused disintegration of plasma and nuclear membranes and vacuolization in the cytoplasm. Altogether, ideal carriers should be non-toxic, non-immunogenic, well-tolerated, and effective. To date, these criteria are best met by small EVs, since nanoparticles still raise concerns about cytotoxicity, low drug-loading capacity, and insufficient delivery. Small EVs can GLPG0634 also be successfully loaded with drugs such as anticancer therapeutics as described in [29], where small EVs obtained from a macrophage cell Rabbit Polyclonal to EDG4 line are loaded with paclitaxel and doxorubicin by mixing and subsequent electroporation or sonication. To date, the only in vivo study on small EVs for use against toxoplasmosis involved the development of a vaccine against congenital toxoplasmosis in mice, with the small EVs obtained from a splenic dendritic cell line pulsed with antigens [30]. These data are expected to provide a reliable basis for further studies dealing with drug loading and with assessing the anti-parasitic action on infected ARPE-19 cells. Interestingly, our results showed that internalized small EVs are localized in close vicinity to the parasites in the ARPE-19 cells, thus making them a promising tool for targeted drug delivery. One of the major concerns in drug delivery is whether the drugs can be released GLPG0634 sufficiently, i.e., in therapeutic amounts. The regular fate of the small EVs after their uptake by energy-dependent endocytic internalization is usually fusion with lysosomes. To promote drug delivery, premature lysosomal degradation of small EVs must be prevented. This prevention may be achieved by modifying the small EVs with unsaturated dioleoyl phosphatidylethanolamine (DOPE), which triggers the fusion of the liposomal membrane with GLPG0634 the endosomal membrane, releasing the drug into the cytoplasm. In [31], this combination of a nanoparticle with pH-sensitive DOPE was described and indicated that this DNA was released into the cytoplasm during gene transfection. Prior to therapeutic applications, more work will obviously be needed to shed more light on the current black box; that is, researchers GLPG0634 must investigate uptake mechanisms, the intracellular fate and the release of cargo from drug-loaded small EVs. In one study, the intracellular fate of labelled small EVs was tracked by live imaging using lysosomal markers (LysoTracker) and ER trackers as described in [32], and it was shown that the small EVs joined the cells via filopodia, were sorted in endocytic vesicles and targeted to the ER before reaching the lysosome. Within 16 h post-labelling, we did not observe substantial colocalization with lysotracker or LAMP-1 (not shown). A time course (high resolution microscopy) using lysosomal markers and ER trackers will shed more light around the time-course of exosome uptake events such as early endosomal localization (EEA-1), late endosomes (CD71 as a marker), and lysosomal degradation (LAMP1). The lack.