Extracellular Vesicle Delivery of Neferine for the Attenuation of Neurodegenerative Disease Proteins and Motor Deficit in an Alzheimer’s Disease Mouse Model

Abstract

Exosomes are nano-extracellular vesicles with diameters ranging from 30 to 150 nm, which are secreted by the cell. With their role in drug cargo loading, exosomes have been applied to carry compounds across the blood–brain barrier in order to target the central nervous system (CNS). In this study, high-purity exosomes isolated by the ultra-high-speed separation method were applied as the natural compound carrier, with the loading efficiency confirmed by UHPLC-MS analysis. Through the optimization of various cargo loading methods using exosomes, this study compared the efficiency of different ways for the separation of exosomes and the exosome encapsulation of natural compounds with increasing molecular weights via extensive in vitro and in vivo efficacy studies. In a pharmacokinetic study, our data suggested that the efficiency of compound’s loading into exosomes is positively correlated to its molecular weight. However, with a molecular weight of greater than 1109 Da, the exosome-encapsulated natural compounds were not able to pass through the blood–brain barrier (BBB). In vitro cellular models confirmed that three of the selected exosome-encapsulated natural compounds—baicalin, hederagenin and neferine—could reduce the level of neurodegenerative disease mutant proteins—including huntingtin 74 (HTT74), P301L tau and A53T α-synuclein (A53T α-syn)—more effectively than the compounds alone. With the traditional pharmacological role of the herbal plant Nelumbo nucifera in mitigating anxiety, exosome-encapsulated-neferine was, for the first time, reported to improve the motor deficits of APP/PS1 (amyloid precursor protein/ presenilin1) double transgenic mice, and to reduce the level of β-amyloid (Aβ) in the brain when compared with the same concentration of neferine alone. With the current trend in advocating medicine–food homology and green healthcare, this study has provided a rationale from in vitro to in vivo for the encapsulation of natural compounds using exosomes for the targeting of BBB permeability and neurodegenerative diseases in the future.

Extracellular vesicles in the study of Alzheimer's and Parkinson's diseases: Methodologies applied from cells to biofluids

“…However, comparatively to TEI the size of EVs extracted by UC were closer to the expected exosomal size range (Tang et al, 2022).…”

“…On the other hand, comparing EVs preparations from N2a cells, using UC and TEI, revealed that the concentration of EVs with exosome‐like characteristics was superior using the PP method. However, comparatively to TEI the size of EVs extracted by UC were closer to the expected exosomal size range (Tang et al, 2022). Furthermore, the enrichment of preparations in exosomes has also been achieved using immuno‐affinity with an exosomal marker (e.g.…”

“…Regarding the isolation methods, UC is by far the most used in EVs with exosome‐like characteristics secreted from cellular models (Asai et al, 2015; Bai et al, 2021; Beretta et al, 2020; Bodart‐Santos et al, 2019; Candelario et al, 2020; Chen et al, 2020; Delenclos et al, 2017; Ding et al, 2022; Dou et al, 2021; Eitan et al, 2016; Emmanouilidou et al, 2010; Grey et al, 2015; Gustafsson et al, 2018; Harischandra et al, 2018; Harischandra et al, 2019; Imanbekova et al, 2021; Izadpanah et al, 2020; Koinuma et al, 2021; Lauritzen et al, 2019; Li et al, 2020; Li, Li, et al, 2022; Marzano et al, 2019; Meng et al, 2020; Micci et al, 2019; Nikitidou et al, 2017; Park et al, 2020; Santa‐Maria et al, 2012; Tang et al, 2022; Vrancx et al, 2021; Wang et al, 2012; Wei et al, 2020; Willén et al, 2017; Yang et al, 2020; Yuyama et al, 2019; Zarrin et al, 2021; Zhang, Xu, et al, 2020), whereas dgUC was employed in EVs isolation derived from brain tissue in nearly all studies presented (Asai et al, 2015; Cheng et al, 2020; Clayton et al, 2021; Cohn et al, 2021; Ding et al, 2022; Hurwitz et al, 2018a; Karampetsou et al, 2020; Lauritzen et al, 2019; Muraoka et al, 2021; Pérez‐González et al, 2012; Pérez‐González et al, 2020; Polanco et al, 2016; Polanco et al, 2021; Ruan et al, 2021; You et al, 2022; Yuyama et al, 2019).…”

“…Nevertheless, PP‐based isolation kits as ExoQ (Aulston et al, 2019; Ding et al, 2018; Guo et al, 2020; Park et al, 2020; Wang et al, 2012) and TEI (Lee et al, 2022; Liu et al, 2022; Marzano et al, 2019; Ozansoy et al, 2020; Peng et al, 2022; Tang et al, 2022) are also being employed in some studies, as well as UF kit Exo:pure (Zhdanova et al, 2021). To note, the employment of different methods for EVs isolation from diverse cell lines render in different results.…”

“…(Ding et al, 2022;Grey et al, 2015;Koinuma et al, 2021;Tang et al, 2022;Willén et al, 2017), MC65(Imanbekova et al, 2021), H4(Eitan et al, 2016), SL1SL2(Delenclos et al, 2017) and MN9D(Harischandra et al, 2018;Harischandra et al, 2019). In all studies undifferentiated cells were used.…”