ABSTRACT
The formation of a protein corona gives nanomedicines a distinct biological identity, profoundly influencing their fate in the body. Nonspecific nanoparticle-protein interactions are typically highly heterogeneous, which can lead to unique biological behaviors and in vivo fates for individual nanoparticles that remain underexplored. To address this, we have established an in situ approach that allows quantitative examination of nanoparticle-protein adsorption at the individual nanoparticle level. This method integrates dual fluorescence quantification techniques, wherein the nanoparticles are first individually analyzed via nanoflow cytometry to detect fluorescent signals from adsorbed proteins. The obtained fluorescence intensity is then translated into protein quantities through calibration with microplate reader quantification. Consequently, this approach enables analysis of interparticle heterogeneity of nano-protein interactions, as well as in situ monitoring of protein adsorption kinetics and nanoparticle aggregation status in blood serum, preconditioning for a comprehensive understanding of nano-bio interactions, and predicting in vivo fate of nanomedicines.
Subject(s)
Blood Proteins , Nanoparticles , Adsorption , Nanoparticles/chemistry , Blood Proteins/chemistry , Blood Proteins/analysis , Humans , Protein Corona/chemistry , Fluorescence , KineticsABSTRACT
Introducing engineered nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of a selective and reproducible protein corona at the particle-protein interface, driven by the relationship between protein-NP affinity and protein abundance. This enables scalable systems that leverage protein-nano interactions to overcome current limitations of deep plasma proteomics in large cohorts. Here the importance of the protein to NP-surface ratio (P/NP) is demonstrated and protein corona formation dynamics are modeled, which determine the competition between proteins for binding. Tuning the P/NP ratio significantly modulates the protein corona composition, enhancing depth and precision of a fully automated NP-based deep proteomic workflow (Proteograph). By increasing the binding competition on engineered NPs, 1.2-1.7× more proteins with 1% false discovery rate are identified on the surface of each NP, and up to 3× more proteins compared to a standard plasma proteomics workflow. Moreover, the data suggest P/NP plays a significant role in determining the in vivo fate of nanomaterials in biomedical applications. Together, the study showcases the importance of P/NP as a key design element for biomaterials and nanomedicine in vivo and as a powerful tuning strategy for accurate, large-scale NP-based deep proteomic studies.
Subject(s)
Nanoparticles , Protein Corona , Protein Corona/chemistry , Proteome , Proteomics , Nanoparticles/chemistry , NanomedicineABSTRACT
A new theoretical framework that enables the use of differential dynamic microscopy (DDM) in fluorescence imaging mode to quantify in situ protein adsorption onto nanoparticles (NP) while simultaneously monitoring for NP aggregation is proposed. This methodology is used to elucidate the thermodynamic and kinetic properties of the protein corona (PC) in vitro and in vivo. The results show that protein adsorption triggers particle aggregation over a wide concentration range and that the formed aggregate structures can be quantified using the proposed methodology. Protein affinity for polystyrene (PS) NPs is observed to be dependent on particle concentration. For complex protein mixtures, this methodology identifies that the PC composition changes with the dilution of serum proteins, demonstrating a Vroman effect never quantitatively assessed in situ on NPs. Finally, DDM allows monitoring of the evolution of the PC in vivo. This results show that the PC composition evolves significantly over time in zebrafish larvae, confirming the inherently dynamic nature of the PC. The performance of the developed methodology allows to obtain quantitative insights into nano-bio interactions in a vast array of physiologically relevant conditions that will serve to further improve the design of nanomedicine.
Subject(s)
Nanoparticles , Protein Corona , Animals , Blood Proteins , Nanoparticles/chemistry , Polystyrenes/chemistry , Protein Corona/chemistry , ZebrafishABSTRACT
As drugs/drug carriers, upon encountering physiological fluids, nanoparticles adsorb biological molecules almost immediately to form a biocorona, which is often simply called a corona. Once the corona is formed, it dictates the subsequent fate of the drug nanoparticle as a therapeutic agent. Protein adsorption on micron-size or even bigger particles was originally described by the Vroman effect. It has served as a useful framework to understand the corona formation. Proteins that are irreversibly adsorbed on nanoparticles form what is called a hard corona. Beyond that is the exchangeable population of proteins, which constitute the dynamic structure called a soft corona. More than the abundance, the affinity of the proteins toward the nanoparticles decides which ones end up in the corona. For example, the more common serum albumin, which is deposited initially, is displaced by fibrinogen, which has a higher affinity for gold nanoparticles. The curvature of the particle is a crucial parameter with bigger particles generally able to bind a more diverse population of proteins from the physiological milieu. The earlier perception of the corona formation being a challenge for drug targeting, etc. has been turned into an opportunity by engineering corona to manipulate properties like circulating half-lives, capacity to evade the immune system, and targeting or even overcoming the blood-brain barrier. The most commonly used techniques for particle characterization, including dynamic light scattering (DLS), differential sedimentation centrifugation, transmission electron microscopy (TEM), and SDS-PAGE, have been adopted to study corona formation in the past. Many newer tools, for example, a combination of capillary electrophoresis with mass spectrometry, are being used to study the corona composition. The comparison of interlaboratory results is a problem because of the lack of standard protocols. This has hindered the ability to obtain more precise information about the corona composition. That, in turn, affects our prospects to use nanoparticles as drugs/drug carriers. This overview is an attempt to assess our understanding of corona formation critically and to outline the complexities involved in gaining precise information. The discussion is largely focused on findings of the last year or so.
Subject(s)
Drug Carriers/chemistry , Drug Carriers/metabolism , Gold/chemistry , Magnetic Iron Oxide Nanoparticles/chemistry , Protein Corona/chemistry , Protein Corona/metabolism , Adsorption , Animals , Betaine/analogs & derivatives , Betaine/chemistry , Humans , Ligands , Liposomes/chemistry , Liposomes/metabolism , Particle Size , Polyethylene Glycols/chemistry , Serum Albumin, Human/chemistry , Serum Albumin, Human/metabolismABSTRACT
The interaction of inorganic nanoparticles and many biological fluids often withstands the formation of a Protein Corona enveloping the nanoparticle. This Protein Corona provides the biological identity to the nanoparticle that the immune system will detect. The formation of this Protein Corona depends not only on the composition of the nanoparticle, its size, shape, surface state and exposure time, but also on the type of media, nanoparticle to protein ratio and the presence of ions and other molecular species that interfere in the interaction between proteins and nanoparticles. This has important implications on immune safety, biocompatibility and the use of nanoparticles in medicine.
Subject(s)
Biocompatible Materials/metabolism , Immune System , Nanoparticles/metabolism , Protein Corona/metabolism , Animals , Biocompatible Materials/chemistry , Humans , Nanomedicine , Nanoparticles/chemistry , Particle Size , Protein Corona/chemistryABSTRACT
In this study, we propose a new polymer substrate that is able to covalently couple intended proteins and reduce nonspecific protein fouling. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-ran-N-methacryloyl-(L)-tyrosinemethylester (MAT)] [P(MPC/MAT)] was synthesized by free-radical polymerization. The photooxidation of the MAT unit in the copolymer was observed under ultraviolet (UV) light at 254 nm. P(MPC/MAT) was spin-coated on silicon (Si) and gold substrates. Without UV irradiation of the polymer-coated surface, P(MPC/MAT) physisorbed on the substrates, and the thickness of the polymer layer was less than 10 nm, regardless of the polymer concentration in the coating solution. In contrast, when the polymer-coated surface was irradiated with UV light, the thickness of the polymer layer could be controlled by changing the polymer concentration of the coating solution. Competitive protein adsorption on P(MPC/MAT) was studied. Bovine serum albumin was first contacted with the surface and later challenged with bovine fibrinogen. On bare gold and Si substrates, a large amount of albumin was adsorbed, and the competitive adsorption of albumin and fibronectin was observed. In contrast, the non-UV-irradiated P(MPC/MAT) surface effectively reduced protein adsorption. Interestingly, on the UV-irradiated P(MPC/MAT) surface, the primary protein preferably bonded, and significantly less secondary protein was adsorbed compared to primary protein. Cell adhesion was also tested on the substrate to clarify the effects of proteins existing on the substrates. On the bare Si surface, many adherent cells were observed, regardless of the protein pretreatment. On the non-UV-irradiated P(MPC/MAT) surface, cell adhesion was effectively reduced along with protein adsorption. Cell adhesion on the UV-irradiated P(MPC/MAT) surface depended strongly on the type of protein that was initially in contact with the surface. We concluded that the desired proteins could be immobilized on the photo-activated P(MPC/MAT) surface while preserving their function. Moreover, competitive protein exchange and multilayer adsorption hardly occurred on the surface.
Subject(s)
Fibronectins , Phosphorylcholine , Serum Albumin, Bovine , Adsorption , Animals , Cattle , Fibronectins/chemistry , Mice , Photoelectron Spectroscopy , Serum Albumin, Bovine/chemistry , Surface Plasmon Resonance , Surface Properties , Ultraviolet RaysABSTRACT
Interaction of metal or oxide nanoparticles (NPs) with biological soft matter is one of the central phenomena in basic and applied biology-oriented nanoscience. Often, this interaction includes adsorption of suspended proteins on the NP surface, resulting in the formation of the protein corona around NPs. Structurally, the corona contains a "hard" monolayer shell directly contacting a NP and a more distant weakly associated "soft" shell. Chemically, the corona is typically composed of a mixture of distinct proteins. The corresponding experimental and theoretical studies have already clarified many aspects of the corona formation. The process is, however, complex, and its understanding is still incomplete. Herein, we present a kinetic mean-field model of the formation of the "hard" corona with emphasis on the role of (i) protein-diffusion limitations and (ii) interplay between competitive adsorption of distinct proteins and irreversible reconfiguration of their native structure. The former factor is demonstrated to be significant only in the very beginning of the corona formation. The latter factor is predicted to be more important. It may determine the composition of the corona on the time scales comparable or longer than a few hours.
Subject(s)
Models, Biological , Nanoparticles , Protein Corona , KineticsABSTRACT
Heavy metal ions, i.e., Cu(2+), are harmful to the environment and our health. In order to detect them, and circumvent or alleviate the weaknesses of existing detecting technologies, we contrive a unique Surface Plasmon Resonance (SPR) biosensor combined with competitive adsorption of proteins, termed the Vroman effect. This approach adopts native proteins (albumin) as bio-receptors that interact with Cu(2+) to be denatured. Denaturation disrupts the conformation of albumin so that it weakens its affinity to adsorb on the sensing surface. Through the competitive adsorption between the denatured albumins and the native ones, the displacement occurs adjacent to the sensing surface, and this process is real-time monitored by SPR, a surface-sensitive label-free biosensor. The affinities of native albumin is significantly higher than that of denatured albumin, demonstrated by measured KD of native and denatured albumin to gold surafce, 5.8±0.2×10(-5) M and 5.4±0.1×10(-4) M, respectively. Using our biosensor, Cu(2+) with concentration down to 0.1mg/L is detected in PBS, tap water, deionized water, and bottled water. The SPR biosensor is characterized for 5 different heavy metal ions, Cu(2+), Fe(3+), Mn(2+), Pb(2+), and Hg(2+), most common heavy metal ions found in tap water. At the maximum contaminant level (MCL) suggested by the United States Environmental Protection Agency (EPA), the SPR biosensor produces 13.5±0.4, 1.5±0.4, 0, 0, and 0 mDeg, respectively, suggesting the biosensor may be used to detect Cu(2+) in tap water samples.