Molecular Sciences I.
Krisztina Balázs-Németh 1,2, Zoltán Varga 3,4, Dorina Lenzinger 1, Edit Buzás 1,2,5
1 Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest
2 ELKH-SE Translational Extracellular Vesicle Research Group, Budapest
3 Research Centre for Natural Sciences, Institute of Materials and Environmental Chemistry, Budapest
4 Department of Biophysics and Radiation Biology, Semmelweis University, Budapest
5 Hungarian Centre of Excellence for Molecular Medicine, Semmelweis University Extracellular Vesicle Research Group, Budapest
Extracellular vesicles (EVs) are mainly spherical particles bounded by a phospholipid bilayer. In their lumen, they transport cargo molecules in a protected way. Our group has shown earlier that in the blood circulation, plasma proteins attach to the surface of the nascent EVs, forming a protein corona.
Our aim was to study the effect of ApoB-rich protein corona on the physicochemical properties and biodistribution of EVs.
Small EVs (sEVs) were separated by differential centrifugation from serum-free conditioned media of HEK293T-palmGFP cells. The sEVs were characterized by particle number, protein, lipid content and GFP expression. sEVs were labeled with 99mTc-duramycin and incubated with human serum albumin (HSA) or HSA-ApoB mixture. One hour after intravenous injection to adult female BalbC mice, the distribution of sEVs was examined by SPECT/CT. The effect of the corona-forming proteins on the number and size of sEVs was analysed by nanoparticle tracking analysis and flow cytometry. The stability of sEVs was determined by TritonX-100 and phospholipaseA2 (PLA2) lysis. The uptake of nascent and protein-coated sEVs was examined in THP-1 monocyte culture.
After in vitro coating of the vesicles, ApoB protein was successfully detected on the surface of sEVs. The sEVs accumulated mainly in the liver and spleen. The HSA-ApoB mixture significantly reduced sEV accumulation in the bladder and spleen. The particle number and the size of sEVs were significantly increased in the presence of ApoB. The TritonX-100 lysis of the sEVs was reduced by HSA or HSA-ApoB mixture. PLA2 decreased the number of nascent sEVs by 30%, while increased the number of protein-coated sEVs by 30%.
Our results suggest that the preformed protein corona may affect the physical properties and the in vivo behavior of sEVs. When using EVs for therapeutic purposes, it is important to keep in mind that we need to focus not only on the composition of EVs, but also on the protein-corona surrounding our EVs.
The project was funded by the following grants: 2019-2.1.7-ERA-NET-2021-00015, STIA-KFI-2022, RRF-2.3.1-21-2022-00003, TKP2021-EGA-23 and ELKH-SE Extracellular Vesicle Research Group. Krisztina Balázs-Németh was supported by the ÚNKP-22-4-I-SE-13 new national excellence program of the ministry for culture and innovation from the source of the national research, development and innovation fund.