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Tropomyosin kinase receptor B (TrkB) has been explored as a therapeutic target for neurological and psychiatric disorders. However, the development of TrkB agonists was hindered by our poor understanding of the TrkB agonist binding location and affinity (both affect the regulation of disorder types). This motivated us to develop a combined computational and experimental approach to study TrkB binders. First, we developed a docking method to simulate the binding affinity of TrkB and binders identified by our magnetic drug screening platform from Gotu kola extracts. The Fred Docking scores from the docking computation showed strong agreement with the experimental results. Subsequently, using this screening platform, we identified a list of compounds from the NIH clinical collection library and applied the same docking studies. From the Fred Docking scores, we selected two compounds for TrkB activation tests. Interestingly, the ability of the compounds to increase dendritic arborization in hippocampal neurons matched well with the computational results. Finally, we performed a detailed binding analysis of the top candidates and compared them with the best-characterized TrkB agonist, 7,8-dyhydroxyflavon. The screening platform directly identifies TrkB binders, and the computational approach allows for the quick selection of top candidates with potential biological activities based on the docking scores. 

Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20–100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells. 

Exosomes are intrinsic cell-derived membrane vesicles in the size range of 40–100 nm, serving as great biomimetic nanocarriers for biomedical applications. These nanocarriers are known to bypass biological barriers, such as the blood–brain barrier, with great potential in treating brain diseases. Exosomes are also shown to be closely associated with cancer metastasis, making them great candidates for tumor targeting. However, the clinical translation of exosomes are facing certain critical challenges, such as reproducible production and in vivo tracking of their localization, distribution, and ultimate fate. Recently, inorganic nanoparticle-loaded exosomes have been shown great benefits in addressing these issues. In this review article, we will discuss the preparation methods of inorganic nanoparticle-loaded exosomes, and their applications in bioimaging and therapy. In addition, we will briefly discuss their potentials in exosome purification.