Extracellular Vesicles for Intracellular Drug Delivery

Extracellular vesicles (EVs) are nanoparticles released from cells, which play an important role in cell-cell communication. EVs cause phenotypic changes in recipient cells through the transfer of biomolecules (lipids, proteins, nucleic acids). However, the mechanism behind this cargo transfer is poorly understood. In this thesis, using a combination of light and electron microscopy, and a novel molecular probe-assay, we show that EVs are taken up by cells via endocytosis, and release their cargo via fusion with endosomes. In addition, we apply EV-mediated cargo transfer for the treatment of neurodegenerative diseases, in particular Huntington’s disease (HD), i.e., a protein aggregation disease. EVs loaded with the chaperone protein DNAJB6 are shown to reduce protein aggregation in cellular and in vivo models of HD. Proteomic analysis of the DNAJB6-EVs confirmed that they contain the DNAJB6 chaperone, but also other chaperones. Next, as EVs show intrinsic organotrophic properties, we explored EVs isolated from brain-trophic neural stem cells. Indeed, these EVs efficiently crossed the blood-brain barrier (BBB) in an in vitro BBB model, and carried encapsulated cargo along with them. This property could be attributed to their interactions with heparan sulfate proteoglycans at the BBB. Summarized, this thesis describes the development of EVs as vehicle for intracellular drug delivery, providing insight into EV transport across the BBB and mechanistic detail on the process of intracellular cargo delivery, while aiming at delivery of molecular chaperones as a therapeutic intervention for neurodegenerative diseases

Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-theart molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody- expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/ fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol

DNAJB6b-enriched small extracellular vesicles decrease polyglutamine aggregation in in vitro and in vivo models of Huntington disease

Huntington disease (HD) is a devastating neurodegenerative disorder characterized by aggregation of huntingtin (HTT) protein containing expanded polyglutamine (polyQ) tracts. DNAJB6, a member of the DNAJ chaperone family, was reported to efficiently inhibit polyQ aggregation in vitro, in cell models, and in vivo in flies, xenopus, and mice. For the delivery of exogenous DNAJB6 to the brain, the DNAJB6 needs to be protected against (enzymatic) degradation and show good penetration into brain tissue. Here, we tested the potential of small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) for delivery of DNAJB6 as anti-amyloidogenic cargo. Administration of sEVs isolated from DNAJB6-overexpressing cells to cells expressing expanded polyQ tracts suppressed HTT aggregation. Furthermore, intrathecal injection of DNAJB6-enriched sEVs into R6/2 transgenic HD mice significantly reduced mutant HTT aggregation in the brain. Taken together, our data suggest that sEV-mediated molecular chaperone delivery may hold potential to delay disease onset in HD

Heparan sulfate proteoglycan-mediated dynamin-dependent transport of neural stem cell exosomes in an in vitro blood-brain barrier model

Drug delivery to the brain is greatly hampered by the presence of the blood-brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood-brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, that is, exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB

Erratum: Breaking free: endocytosis and endosomal escape of extracellular vesicles

The authors want to make the following corrections to this paper[1]. In the section “Intracellular trafficking of EVs”, the citations after the sentence “Using another cell-free assay, Morandi et al. provided interesting insights into the process of endosomal fusion in EV cargo release”and “Murphy et al. tackled this question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in terms of uptake and cargo (specifically gRNA) delivery” were missed. Correctly modify as follows: Using another cell-free assay, Morandi et al. provided interesting insights into the process of endosomal fusion in EV cargo release[22,68,87]. Murphy et al. tackled this question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in terms of uptake and cargo (specifically gRNA) delivery[135]. Corresponding subsequent citation numbers also need to be adjusted: reference 140 is changed to reference 135, reference 135 is changed to reference 136, reference 136 is changed to reference 137, reference 137 is changed to reference 138, reference 138 is changed to reference 139, and reference 139 is changed to reference 140. We apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated

Preparation of chaperone-loaded neural stem cell-derived extracellular vesicles to reduce protein aggregation in Huntington’s disease cellular models

Summary: Here, we present a protocol using genetic engineering techniques to prepare small extracellular vesicles (sEVs) enriched in the chaperone protein DNAJB6. We describe steps to prepare cell lines overexpressing DNAJB6, followed by the isolation and characterization of sEVs from cell conditioned media. Further, we describe assays to examine effects of DNAJB6-loaded sEVs on protein aggregation in Huntington’s disease cellular models. The protocol can be readily repurposed to study protein aggregation in other neurodegenerative disorders or extended to other therapeutic proteins.For complete details on the use and execution of this protocol, please refer to Joshi et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Designing a sky-scanning Fabry–Perot interferometer system with a large size integrating sphere

Integrating spheres can be manufactured at sizes ranging from 1 mm to 3 m in diameter. It has been agreed widely by manufacturers that fitting a large-sized integrating sphere as per the customer requirement is a complex process. Thus, these spheres due to its large beam divergence from the exit port, have never been deployed for the calibration of aeronomy applications. In this article a optical system is described to integrate a large-sized integrating sphere with the Fabry–Perot etalon and thus devise a set-up to generate the concentric ring pattern and record it on the detector. The experimental set-up was verified by optical simulation using ZEMAX (OpticStudio) so as to validate the system’s performance in the laboratory. The important parameter finesse is calculated and its accuracy is found to be 80%

Research on impact of financial technologies on commercial banks profitability ratios

Tyrime siekiama įvertinti finansinių technologijų įtaką komercinio banko pelningumo rodikliams. Straipsnyje analizuojama mokslinė literatūra, aprašomi naudojami tyrimo metodai, atliekama panelinių duomenų regresinė analizė (XLSTAT programa) siekiant įvertinti įtaką. Atliekant tyrimą analizuojama Lietuvoje veikiančių komercinių bankų naudojamos finansinės technologijos (mokėjimo kortelės, bankomatai, mokėjimo kortelių skaitytuvai, elektroninė bankininkystė) ir turto grąžos pelningumo rodiklis (ROA) bei nuosavo kapitalo grąžos rodiklis (ROE) laikotarpyje nuo 2014 m. iki 2018 m. Atlikus tyrimą atmetamos iškeltos hipotezės, kad finansinės technologijos daro įtaką komercinių bankų pelningumo rodikliams, kadangi atlikto tyrimo rezultatai neparodė reikšmingos finansinių technologijų įtakos komercinių bankų pelningumo rodikliams. Tyrimo rezultatai gali įspėti komercinius bankus dėl investicijų atsiperkamumo diegiant finansines technologijas. Tačiau svarbu atkreipti dėmesį į apribojimus, kadangi šio tyrimo rezultatai remiasi tik Lietuvoje veikiančių komercinių bankų duomenimis, o dėl mažos duomenų imties rezultatai gali būti netikslūs.The aim of this study is to evaluate impact of financial technologies on commercial banks profitability. The research analyzes scientific literature, distinguishes the research methods used, and performs linear regression analysis to evaluate the impact. The research analyzes financial technologies used by commercial banks operating in Lithuania (payment cards, ATMs, EFTPOS, electronic banking) and return on assets (ROA) and return on equity (ROE) ratios. The study rejects the hypotheses that financial technologies have an impact on commercial banks “profitability ratios, as the results of the research did not show significant impact of financial technologies on commercial banks” profitability ratios. The results of the study may alert commercial banks to the return on investment of financial technology. However, it is important to note the limitations as the results of this study are based only on commercial banks operating in Lithuania and the small sample size may lead to inaccurate results