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The protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines-with a particular focus on liposomes-when using the traditional isolation methods that separate according to size and density., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

ObjectiveApolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia.Approach and resultsAdeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike wild-type apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others.ConclusionsSubstitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.

Flow cytometry (FCM) is a popular method used in characterisation of extracellular vesicles (EVs). Circulating EVs are often identified by FCM by exploiting the lipid nature of EVs by staining with Annexin V (Anx5) or lactadherin against the membrane phospholipid phosphatidylserine (PS) and evaluating the specificity of the labels by detergent lysis of EVs. Here, we investigate whether PS labelling and detergent lysis approaches are confounded by lipoproteins, another family of lipid‐based nanoparticles found in blood, in both frozen and fresh blood plasma. We demonstrated that Anx5 and lactadherin in addition to EVs stained ApoB‐containing lipoproteins, identified by the use of fluorophore‐labelled polyclonal ApoB‐antibody, and that Anx5 had a significantly larger tendency for labelling lipoprotein‐bound PS than lactadherin. Furthermore, detergent lysis resulted in a decrease in both EV and lipoprotein events and especially lipoproteins positive for either Anx5 or lactadherin. Taken together, our findings pose concerns to the use of lipid‐based strategies in identifying EVs by FCM and support the use of transmembrane proteins such as tetraspannins to distinguish EVs from lipoproteins. [ABSTRACT FROM AUTHOR]

For the past two decades, biomimetic high-density lipoproteins (b-HDL) have been used for various drug delivery applications. The b-HDL mimic the endogenous HDL, and therefore possess many attractive features for drug delivery, including high biocompatibility, biodegradability, and ability to transport and deliver their cargo (e.g. drugs and/or imaging agents) to specific cells and tissues that are recognized by HDL. The b-HDL designs reported in the literature often differ in size, shape, composition, and type of incorporated cargo. However, there exists only limited insight into how the b-HDL design dictates their biodistribution. To fill this gap, we conducted a comprehensive systematic literature search of biodistribution studies using various designs of apolipoprotein A-I (apoA-I)-based b-HDL (i.e. b-HDL with apoA-I, apoA-I mutants, or apoA-I mimicking peptides). We carefully screened 679 papers (search hits) for b-HDL biodistribution studies in mice, and ended up with 24 relevant biodistribution profiles that we compared according to b-HDL design. We show similarities between b-HDL biodistribution studies irrespectively of the b-HDL design, whereas the biodistribution of the b-HDL components (lipids and scaffold) differ significantly. The b-HDL lipids primarily accumulate in liver, while the b-HDL scaffold primarily accumulates in the kidney. Furthermore, both b-HDL lipids and scaffold accumulate well in the tumor tissue in tumor-bearing mice. Finally, we present essential considerations and strategies for b-HDL labeling, and discuss how the b-HDL biodistribution can be tuned through particle design and administration route. Our meta-analysis and discussions provide a detailed overview of the fate of b-HDL in mice that is highly relevant when applying b-HDL for drug delivery or in vivo imaging applications. Unlabelled Image • Biomimetic HDL (b-HDL) used for drug delivery vary in size, shape and composition • The biodistribution of the b-HDL variants is to a large extent similar • The b-HDL lipids accumulate primarily in the liver (and tumor) • The b-HDL scaffold accumulates primarily in the kidney and liver (and tumor) • The b-HDL biodistribution is tunable via particle design and administration route [ABSTRACT FROM AUTHOR]

The protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines—with a particular focus on liposomes—when using the traditional isolation methods that separate according to size and density. [ABSTRACT FROM AUTHOR]

Post-staining of extracellular vesicles (EVs) with lipid-anchored fluorophores (LAFs) such as PKH67 is a widely used strategy for studying EVs but it is associated with several pitfalls. The pitfalls discussed in this commentary are related to LAF labelling of non-EV species due to (1) lipoprotein contamination in EV samples, (2) desorption of the LAF reporters from vesicles into proteins and lipoproteins in blood and serum, and (3) the capability of the amphiphilic LAF compounds to form EV-like particles. Awareness of these challenges and developing solutions to overcome these are important to ensure that we make relevant interpretations when using LAFs to track EVs. [ABSTRACT FROM AUTHOR]