Solution NMR of Nanoparticles in Serum: Protein Competition Influences Binding Thermodynamics and Kinetics

Frontiers in Physiology ◽

10.3389/fphys.2021.715419 ◽

2021 ◽

Vol 12 ◽

Author(s):

Joanna Xiuzhu Xu ◽

Nicholas C. Fitzkee

Keyword(s):

Serum Proteins ◽

Drug Carrier ◽

Protein Corona ◽

Solution Nmr ◽

Binding Thermodynamics ◽

Thermodynamics And Kinetics ◽

Nanoparticle Interactions ◽

Invaluable Tool ◽

Solution Nuclear Magnetic Resonance

The spontaneous formation of a protein corona on a nanoparticle surface influences the physiological success or failure of the synthetic nanoparticle as a drug carrier or imaging agent used in vivo. A quantitative understanding of protein-nanoparticle interactions is therefore critical for the development of nanoparticle-based therapeutics. In this perspective, we briefly discuss the challenges and limitations of current approaches used for studying protein-nanoparticle binding in a realistic biological medium. Subsequently, we demonstrate that solution nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to monitor protein competitive binding in a complex serum medium in situ. Importantly, when many serum proteins are competing for a gold nanoparticle (AuNP) surface, solution NMR is able to detect differences in binding thermodynamics, and kinetics of a tagged protein. Combined with other experimental approaches, solution NMR is an invaluable tool to understand protein behavior in the nanoparticle corona.

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In vitro and in vivo interactions of selected nanoparticles with rodent serum proteins and their consequences in biokinetics

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.5.180 ◽

2014 ◽

Vol 5 ◽

pp. 1699-1711 ◽

Cited By ~ 39

Author(s):

Wolfgang G Kreyling ◽

Stefanie Fertsch-Gapp ◽

Martin Schäffler ◽

Blair D Johnston ◽

Nadine Haberl ◽

...

Keyword(s):

Protein Binding ◽

Physicochemical Properties ◽

Serum Proteins ◽

Particle Surface ◽

Protein Corona ◽

Surface Modifications ◽

Body Fluids ◽

Binding Studies

When particles incorporated within a mammalian organism come into contact with body fluids they will bind to soluble proteins or those within cellular membranes forming what is called a protein corona. This binding process is very complex and highly dynamic due to the plethora of proteins with different affinities and fractions in different body fluids and the large variation of compounds and structures of the particle surface. Interestingly, in the case of nanoparticles (NP) this protein corona is well suited to provide a guiding vehicle of translocation within body fluids and across membranes. This NP translocation may subsequently lead to accumulation in various organs and tissues and their respective cell types that are not expected to accumulate such tiny foreign bodies. Because of this unprecedented NP accumulation, potentially adverse biological responses in tissues and cells cannot be neglected a priori but require thorough investigations. Therefore, we studied the interactions and protein binding kinetics of blood serum proteins with a number of engineered NP as a function of their physicochemical properties. Here we show by in vitro incubation tests that the binding capacity of different engineered NP (polystyrene, elemental carbon) for selected serum proteins depends strongly on the NP size and the properties of engineered surface modifications. In the following attempt, we studied systematically the effect of the size (5, 15, 80 nm) of gold spheres (AuNP), surface-modified with the same ionic ligand; as well as 5 nm AuNP with five different surface modifications on the binding to serum proteins by using proteomics analyses. We found that the binding of numerous serum proteins depended strongly on the physicochemical properties of the AuNP. These in vitro results helped us substantially in the interpretation of our numerous in vivo biokinetics studies performed in rodents using the same NP. These had shown that not only the physicochemical properties determined the AuNP translocation from the organ of intake towards blood circulation and subsequent accumulation in secondary organs and tissues but also the the transport across organ membranes depended on the route of AuNP application. Our in vitro protein binding studies support the notion that the observed differences in in vivo biokinetics are mediated by the NP protein corona and its dynamical change during AuNP translocation in fluids and across membranes within the organism.

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A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

Microorganisms ◽

10.3390/microorganisms8101627 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1627

Author(s):

Tecla Ciociola ◽

Pier Paolo Zanello ◽

Tiziana D’Adda ◽

Serena Galati ◽

Stefania Conti ◽

...

Keyword(s):

Antifungal Activity ◽

Human Serum ◽

Fungicidal Activity ◽

Galleria Mellonella ◽

Serum Proteins ◽

Morphological Alterations ◽

C Terminus ◽

Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

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Probing the Intracellular Bio-Nano Interface in Different Cell Lines with Gold Nanostars

Nanomaterials ◽

10.3390/nano11051183 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1183

Author(s):

Cecilia Spedalieri ◽

Gergo Péter Szekeres ◽

Stephan Werner ◽

Peter Guttmann ◽

Janina Kneipp

Keyword(s):

Cell Line ◽

Cell Lines ◽

Early Stage ◽

Protein Corona ◽

Fibroblast Cell ◽

Ultrastructural Level ◽

Epithelial Cell Line ◽

Animal Cells ◽

Gold Nanostars

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.

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Unraveling the In Vivo Protein Corona

Cells ◽

10.3390/cells10010132 ◽

2021 ◽

Vol 10 (1) ◽

pp. 132

Author(s):

Johanna Simon ◽

Gabor Kuhn ◽

Michael Fichter ◽

Stephan Gehring ◽

Katharina Landfester ◽

...

Keyword(s):

Plasma Proteins ◽

Ex Vivo ◽

Protein Corona ◽

Blood Proteins ◽

Therapeutic Application ◽

Complex Situation ◽

Ex Vivo Approach ◽

In Vivo Administration

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed “ex vivo” as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

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EVIDENCE THAT THE L-ASPARAGINASE OF GUINEA PIG SERUM IS RESPONSIBLE FOR ITS ANTILYMPHOMA EFFECTS

Journal of Experimental Medicine ◽

10.1084/jem.118.1.99 ◽

1963 ◽

Vol 118 (1) ◽

pp. 99-120 ◽

Cited By ~ 177

Author(s):

J. D. Broome

Keyword(s):

Guinea Pig ◽

Guinea Pigs ◽

Serum Proteins ◽

Deae Cellulose ◽

Salt Precipitation ◽

Asparaginase Activity ◽

Pig Serum

A number of the properties of the L-asparaginase present in guinea pig serum have been examined and shown to be indistinguishable from those of the agent responsible for inhibiting cells of lymphoma 6C3HED in vivo. The patterns of instability of the enzyme to changes in temperature and pH were found to parallel closely those of the antilymphoma agent. L-Asparaginase activity was essentially absent from the serum of newborn guinea pigs and this failed to inhibit 6C3HED cells. On separating guinea pig serum proteins by salt precipitation, electrophoresis, and chromatography on DEAE cellulose, antilymphoma activity was found only in fractions which contained L-asparaginase.

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Detection of Protein Changes in Serum of Workers following Inhalation Exposure to Toluene Diisocyanate Vapors

Toxicology and Industrial Health ◽

10.1177/074823379200800605 ◽

1992 ◽

Vol 8 (6) ◽

pp. 407-413 ◽

Cited By ~ 5

Author(s):

Adam B. Czuppon ◽

Boleslaw Marczynski ◽

Xaver Baur

Keyword(s):

Serum Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Inhalation Exposure ◽

Staining Intensity ◽

Peak Height ◽

Toluene Diisocyanate ◽

Serum Samples ◽

Chi Square ◽

Challenge Tests

Serum samples of 10 workers undergoing occupational type inhalative challenge tests by toluene diisocyanate (TDI) were investigated by anion-exchange fast-protein-liquid-chromatography (FPLC) and polyacrylamide-gel electrophoresis (PAGE-SDS). Their serum chromatography profiles were compared to those of 20 unexposed individuals. The peak height of the first prealbumin peak in sera of workers after inhalative challenge tests was significantly different (p > 0, 01 Chi-square test) compared to that obtained before exposure and to that of unexposed subjects. In addition, qualitative changes of these peaks were also noted in sera of workers exposed to TDI. In the cases of exposed individuals, that peak was more diffuse with some shoulders and less symmetric in appearance. Similarly, PAGE-SDS of the serum proteins, followed by silver nitrate staining, revealed a different banding pattern after in vivo TDI exposure. One of the serum components at approximately 15 kD showed an increase of staining intensity after exposure (n = 10), compared to unexposed subjects or to patients before exposure. This serum fraction has not yet been identified. The results here demonstrate that it is possible to detect changes of serum proteins in TDI-exposed individuals within a relatively short analysis time. This could be useful for biological monitoring of exposure, since no method for such is yet available.

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