The inherent physicochemical properties of engineered nanomaterials (ENMs) are known to control the sorption of proteins, but knowledge on how the release of ENMs to the environment prior to protein exposure affects this reaction is limited. In this study, time-resolved, in situ infrared spectroscopy was used to investigate the sorption of a model protein, bovine serum albumin (BSA), onto two different types of titanium dioxide (TiO 2 ) ENMs (catalytic-grade P90 and food-grade E171) in the presence and absence of a simple dissolved organic carbon molecule, oxalate. Infrared spectroscopy results showed that oxalate adsorbed to P90 through chemisorption interactions, but it adsorbed to E171 through physisorption interactions due to the presence of inherent surface-bound phosphates. Secondary structure and two-dimensional correlation spectroscopy analyses showed that BSA interacted with and unfolded on the surface of P90, but not E171, presumably due to the repulsive forces from the negatively charged phosphates on E171. When oxalate was pre-adsorbed to either P90 or E171, the unfolding of BSA occurred, but along different pathways. This suggests both the “outer” surface chemistry ( e.g. , oxalate layers) and the mechanism by which this layer is bound to the ENM play a significant role in the adsorption of proteins. Collectively, the results indicate the exposure of ENMs to natural and engineered environments prior to biological uptake affects the resulting protein corona formation, and thus the transport and bioactivity of ENMs.
more »
« less
Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer–dimer mixture in situ
Protein adsorption on surfaces greatly impacts many applications such as biomedical materials, anti-biofouling coatings, bio-separation membranes, biosensors, antibody protein drugs etc. For example, protein drug adsorption on the widely used lubricant silicone oil surface may induce protein aggregation and thus affect the protein drug efficacy. It is therefore important to investigate the molecular behavior of proteins at the silicone oil/solution interface. Such an interfacial study is challenging because the targeted interface is buried. By using sum frequency generation vibrational spectroscopy (SFG) with Hamiltonian local mode approximation method analysis, we studied protein adsorption at the silicone oil/protein solution interface in situ in real time, using bovine serum albumin (BSA) as a model. The results showed that the interface was mainly covered by BSA dimers. The deduced BSA dimer orientation on the silicone oil surface from the SFG study can be explained by the surface distribution of certain amino acids. To confirm the BSA dimer adsorption, we treated adsorbed BSA dimer molecules with dithiothreitol (DTT) to dissociate these dimers. SFG studies on adsorbed BSA after the DTT treatment indicated that the silicone oil surface is covered by BSA dimers and BSA monomers in an approximate 6 : 4 ratio. That is to say, about 25% of the adsorbed BSA dimers were converted to monomers after the DTT treatment. Extensive research has been reported in the literature to determine adsorbed protein dimer formation using ex situ experiments, e.g. , by washing off the adsorbed proteins from the surface then analyzing the washed-off proteins, which may induce substantial errors in the washing process. Dimerization is a crucial initial step for protein aggregation. This research developed a new methodology to investigate protein aggregation at a solid/liquid (or liquid/liquid) interface in situ in real time using BSA dimer as an example, which will greatly impact many research fields and applications involving interfacial biological molecules.
more »
« less
- Award ID(s):
- 1904380
- NSF-PAR ID:
- 10332736
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 13
- Issue:
- 4
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 975 to 984
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Protein structures at solid/liquid interfaces mediate interfacial protein functions, which are important for many applications. It is difficult to probe interfacial protein structures at buried solid/liquid interfaces in situ at the molecular level. Here, a systematic methodology to determine protein molecular structures (orientation and conformation) at buried solid/liquid interfaces in situ was successfully developed with a combined approach using a nonlinear optical spectroscopic technique – sum frequency generation (SFG) vibrational spectroscopy, isotope labeling, spectra calculation, and computer simulation. With this approach, molecular structures of protein GB1 and its mutant (with two amino acids mutated) were investigated at the polymer/solution interface. Markedly different orientations and similar (but not identical) conformations of the wild-type protein GB1 and its mutant at the interface were detected, due to the varied molecular interfacial interactions. This systematic strategy is general and can be widely used to elucidate protein structures at buried interfaces in situ .more » « less
-
Nanoparticle (NP)-based therapeutics have ushered in a new era in translational medicine. However, despite the clinical success of NP technology, it is not well-understood how NPs fundamentally change in biological environments. When introduced into physiological fluids, NPs are coated by proteins, forming a protein corona (PC). The PC has the potential to endow NPs with a new identity and alter their bioactivity, stability, and destination. Additionally, the conformation of proteins is sensitive to their physical and chemical surroundings. Therefore, biological factors and protein–NP-interactions can induce changes in the conformation and orientation of proteins in vivo . Since the function of a protein is closely connected to its folded structure, slight differences in the surrounding environment as well as the surface characteristics of the NP materials may cause proteins to lose or gain a function. As a result, this can alter the downstream functionality of the NPs. This review introduces the main biological factors affecting the conformation of proteins associated with the PC. Then, four types of NPs with extensive utility in biomedical applications are described in greater detail, focusing on the conformation and orientation of adsorbed proteins. This is followed by a discussion on the instances in which the conformation of adsorbed proteins can be leveraged for therapeutic purposes, such as controlling protein conformation in assembled matrices in tissue, as well as controlling the PC conformation for modulating immune responses. The review concludes with a perspective on the remaining challenges and unexplored areas at the interface of PC and NP research.more » « less
-
null (Ed.)Gold nanoparticles (AuNPs) are now being used in such areas as diagnostics, drug delivery, and biological sensing. In these applications, AuNPs are frequently exposed to biological fluids. These fluids contain many different proteins, any of which may interfere with the intended function of the nanoparticle. In this work, we examine the thermodynamic consequences of proteinnanoparticle binding using a combined spectroscopic and calorimetric approach. We monitored binding using UV-Vis spectroscopy, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Six proteins were studied based on their differing chemical properties, and both 15 nm and 30 nm citrate-coated AuNPs were investigated. We interpreted the UV-Vis data using two different models: the commonly-used Langmuir isotherm model and a more complex mass transport model. Both models can be used to determine Kd values for the 30 nm AuNP data; however, the mass transport model is more appropriate for 15 nm AuNPs. This is because, when fitting the Langmuir model, it is commonly assumed that most proteins are not surface-associated, and this assumption fails for 15 nm AuNPs. The DSC thermograms show two transitions for a globular protein adsorbed to a 15 nm AuNP: one high-temperature transition that is similar to global protein unfolding (68 C), and one low-temperature transition that may correspond to unfolding at the surface (56 C). Conversely, ITC experiments show no net heat of adsorption for GB3, even at high protein/AuNP concentrations. Together, the spectroscopic and calorimetric data suggest a complex, multi-step process for protein-nanoparticle adsorption. Moreover, for the proteins studied, both AuNP curvature and protein chemistry contribute to protein adsorption, with proteins generally binding more weakly to the larger nanoparticles. In the future, this work may lead to principles for improving the design of AuNPbased therapeutics and sensors.more » « less
-
Particles in biopharmaceutical products present high risks due to their detrimental impacts on product quality and safety. Identification and quantification of particles in drug products are important to understand particle formation mechanisms, which can help develop control strategies for particle formation during the formulation development and manufacturing process. However, existing analytical techniques such as microflow imaging and light obscuration measurement lack the sensitivity and resolution to detect particles with sizes smaller than 2 μm. More importantly, these techniques are not able to provide chemical information to determine particle composition. In this work, we overcome these challenges by applying the stimulated Raman scattering (SRS) microscopy technique to monitor the C−H Raman stretching modes of the proteinaceous particles and silicone oil droplets formed in the prefilled syringe barrel. By comparing the relative signal intensity and spectral features of each component, most particles can be classified as protein−silicone oil aggregates. We further show that morphological features are poor indicators of particle composition. Our method has the capability to quantify aggregation in protein therapeutics with chemical and spatial information in a label-free manner, potentially allowing high throughput screening or investigation of aggregation mechanisms.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d1sc04300e
