Saponin is Essential for the Isolation of Proteins and RNA from Biological Nanoparticles

Extracellular vesicles (EVs), biomimetics, and other biological nanoparticles (BNs) produced from human cells are gaining increasing attention in the fields of molecular diagnostics and nanomedicine for the delivery of therapeutic cargo. In particular, BNs are considered prospective delivery vehicles for different biologics, including protein and RNA therapeutics. Moreover, EVs are widely used in molecular diagnostics for early detection of disease-associated proteins and RNA. Technical approaches for measuring biologics mostly originated from the field of EVs and were later adopted for other BNs, such as extracellular vesicle-mimetic nanovesicles, membrane nanoparticles (nanoghosts), and hybrid nanoparticles, with minimal modifications. Here, we demonstrate that BNs are highly resistant to protocols that severely underestimate the protein and RNA content of BNs, and provide the relevance of these data both for general BNs characterization and practical applications of CRISPR/Cas-based therapies. We demonstrate that the addition of saponin leads to an ∼2- to 7-fold enhancement in protein isolation and an ∼2- to 242-fold improvement in RNA recovery rates and detection efficiency. Differences in the proteolipid contents of BNs, measured by Raman and surface-enhanced Raman spectroscopy, correlate with their susceptibility to saponin treatment for cargo extraction. Finally, we develop a unified protocol using saponin to efficiently isolate proteins and RNA from the BNs. These data demonstrate that previously utilized protocols underestimate BN cargo contents and offer gold standard protocols that can be broadly adopted into the field of nanobiologics, molecular diagnostics, and analytical chemistry.

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Publication Details

Journal
Analytical Chemistry
Published
2024-10-14
DOI
https://doi.org/10.1021/acs.analchem.4c04607
Primary Topic
RNA Interference and Gene Delivery
Type
article
Field-Weighted Citation Impact
0.00

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article

Saponin is Essential for the Isolation of Proteins and RNA from Biological Nanoparticles

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Analytical Chemistry
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Saponin is Essential for the Isolation of Proteins and RNA from Biological Nanoparticles

Sergey Brezgin, Zhanna V. Bochkova, Vladimir Chulanov, Marina Belikova, Olesja Eliseeva, E. Yu. Parshina, E. V. Khaydukov, Andrey A. Zamyatnin, Polina A. Demina, Dmitry Kostyushev, Anastasiya Kostyusheva, Ilya Gordeychuk, Ivan Karandashov, Vadim S. Pokrovsky, O. V. Slatinskaya, Alexander N. Lukashev, Oleg Latyshev, Anastasia S. Frolova, Д. Н. Силачев, Г. В. Максимов, Artyom Kachanov, V. I. Gegechkory, Andrey Tikhonov, Н.В. Пономарева, Ekaterina Bayurova
article en

Abstract

Extracellular vesicles (EVs), biomimetics, and other biological nanoparticles (BNs) produced from human cells are gaining increasing attention in the fields of molecular diagnostics and nanomedicine for the delivery of therapeutic cargo. In particular, BNs are considered prospective delivery vehicles for different biologics, including protein and RNA therapeutics. Moreover, EVs are widely used in molecular diagnostics for early detection of disease-associated proteins and RNA. Technical approaches for measuring biologics mostly originated from the field of EVs and were later adopted for other BNs, such as extracellular vesicle-mimetic nanovesicles, membrane nanoparticles (nanoghosts), and hybrid nanoparticles, with minimal modifications. Here, we demonstrate that BNs are highly resistant to protocols that severely underestimate the protein and RNA content of BNs, and provide the relevance of these data both for general BNs characterization and practical applications of CRISPR/Cas-based therapies. We demonstrate that the addition of saponin leads to an ∼2- to 7-fold enhancement in protein isolation and an ∼2- to 242-fold improvement in RNA recovery rates and detection efficiency. Differences in the proteolipid contents of BNs, measured by Raman and surface-enhanced Raman spectroscopy, correlate with their susceptibility to saponin treatment for cargo extraction. Finally, we develop a unified protocol using saponin to efficiently isolate proteins and RNA from the BNs. These data demonstrate that previously utilized protocols underestimate BN cargo contents and offer gold standard protocols that can be broadly adopted into the field of nanobiologics, molecular diagnostics, and analytical chemistry.

Analytical ChemistryVol. 96(43)
Peoples' Friendship University of Russia (RU), Russian Academy of Sciences (RU), Moscow State Pedagogical University (RU), Sechenov University (RU), Lomonosov Moscow State University (RU), Institute for Systems Biology (US), Faculty (United Kingdom) (GB), Sirius University of Science and Technology (RU), InSysBio (Russia) (RU), Institute of Philosophy (HU), Ministry of Health of the Russian Federation (RU), Physico-Technical Institute (RU), National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I.Kulakov of the Ministry of Healthcare of the Russian Federation (RU), Kementerian Kesehatan Republik Indonesia (ID), Bioengineering Center (RU), Chumakov Institute of Poliomyelitis and Viral Encephalitides (RU), Moscow State University (TJ), Institute Of Perinatology Obstetrics And Gynaecology (AM), Institute of Parasitology (CZ)
Russian Science Foundation, Ministry of Science and Higher Education of the Russian Federation
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