Corona Complexes Research Articles

Machine Learning-Aided 3D Dynamic SERS Strategy for Physiological Mapping: Biotoxicity of Environmentally Dimensional Aged Nanoplastics and Corresponding Protein Corona Complexes.

Nanoplastics (NPs) are emerging pollutants that undergo inevitable aging in the environment, raising concerns about human exposure and health hazards. Research on the cytotoxicity of various polymer types of NPs, aged nanoplastics (aNPs), and their interactions with proteins (aNPs-protein corona) is still nascent. Traditional cytotoxicity detection methods often rely on end point assays with restricted temporal resolution and analysis of single or multiple biomarkers. Here, we propose a novel approach integrating the 3D dynamic SERS strategy (DSS) with machine learning to rapidly analyze the cell fate and death modes induced by NPs, aNPs, and aNPs-protein corona complexes at the molecular level. PS, PVC, PMMA, and PC products from the water environment were used to prepare the corresponding NPs, and the impact of UV irradiation on their physicochemical properties was examined. DSS systematically maps the molecular changes in the cellular secretome caused by these NPs. Machine learning effectively extracts information from complex spectra, differentiating between biological samples. Results show prolonged UV exposure increases cell sensitivity to ferroptosis and cytotoxicity in various aNPs, while the protein corona on aNPs significantly mitigates toxicity associated with surface oxygen-containing functional groups, resulting in a reduced similarity to ferroptosis signatures. 3D DSS with machine learning technique analyzes the overall metabolite profile at the molecular level rather than individual biomarkers. This study is the first attempt to compare the biotoxicity of diverse polymer NPs, aNPs, and aNPs-protein coronas at cellular and molecular levels in human hepatocytes, enhancing our understanding of the complex biological impacts of NPs.

Protocols for isolation and characterization of nanoparticle biomolecular corona complexes.

Engineered nanoparticles (NPs) pose a broad spectrum of interesting properties that make them useful for many applications. However, continuous exposure to NPs requires the need to deeply understand the outcomes when these NPs interact with different biological environments. After exposure within (to) these environments, the pristine surfaces of NPs strongly interact with the molecules from the surrounding medium, including metabolites, lipids, glycan, and proteins, forming the so-called protein corona (PC). It is well established that the NP-PC strongly influences the biological fate of various NPs types, including cellular uptake, toxicity, and biodistribution. Thus, for a proper assessment of potential hazards associated with engineered NPs, it is mandatory to study and evaluate the PC that forms around NPs. Herein, we describe protocols in detail for the isolation and characterization of NP-PC complexes and cover the following aspects: 1) isolation protocols for different nanomaterials in a range of exposing media, including magnetic isolation methods for superparamagnetic NPs, 2) NP physico-chemical characterization using advanced and standard techniques available in regular laboratories, and 3) NP- PC characterization of the protein and glycan components.

Driving Role of Zinc Oxide Nanoparticles with Different Sizes and Hydrophobicity in Metabolic Response and Eco-Corona Formation in Sprouts (Vigna radiata).

Zinc oxide nanoparticles (ZnO NPs) cause biotoxicity and pose a potential ecological threat; however, their effects on plant metabolism and eco-corona evolution between NPs and organisms remain unclear. This study clarified the molecular mechanisms underlying physiological and metabolic responses induced by three different ZnO NPs with different sizes and hydrophobicity in sprouts (Vigna radiata) and explored the critical regulation of eco-corona formation in root-nano systems. Results indicated that smaller-sized ZnO inhibited root elongation by up to 37.14% and triggered oxidative burst and apoptosis. Metabolomics confirmed that physiological maintenance after n-ZnO exposure was mainly attributed to the effective stabilization of nitrogen fixation and defense systems by biotransformation of the flavonoid pathway. Larger-sized or hydrophobic group-modified ZnO exhibited low toxicity in sprouts, with 0.89-fold upregulation of citrate in central carbon metabolism. This contributed to providing energy for resistance to NP stress through amino acid and carbon/nitrogen metabolism, accompanied by changes in membrane properties. Notably, smaller-sized and hydrophobic NPs intensely stimulated the release of root metabolites, forming corona complexes with exudates. The hydrogen-bonded wrapping mechanism in protein secondary structure and hydrophobic interactions of heterogeneous functional groups drove eco-corona formation, along with the corona evolution intensity of n-ZnO > s-ZnO > b-ZnO based on higher (α-helix + 3-turn helix)/β-sheet ratios. This study provides crucial insight into metabolic and eco-corona evolution in bionano fates.

Cellular uptake and cytotoxicity of PEGylated MXene nanomaterials mediated by protein corona

A stringent analysis of the biocompatibility of MXene is a necessary condition for assessing the biological risk of MXene. Owing to high surface free energy, MXene is capable of adsorbing a large amount of blood proteins to form MXene-protein corona complexes, however, a comprehensive understanding of the relationship between MXene and cellular physiological systems remains limited. Therefore, we investigated the cellular uptake and cytotoxicity effect of MXene Ti3C2Tx and PEGylation Ti3C2Tx mediated by human serum protein corona in THP-1 cells. It was found that PEGylation can alter the interaction between Ti3C2Tx and serum proteins, inducing a significant transformation in the fingerprint of the protein corona. Following protein corona formation, both Ti3C2Tx and PEGylated Ti3C2Tx predominantly accumulated at lysosomal sites within THP-1 cells. Further analysis revealed that clathrin-mediated endocytosis was the primary mechanism of Ti3C2Tx internalization by THP-1 cells. There was no significant effect on cell viability. However, we found that Ti3C2Tx plays a dual role as both a stimulus and scavenger of ROS within THP-1 cells, influenced by its PEGylation and the formation of a protein corona. This study provides important insights for biocompatibility evaluation and rational design of nanoproducts based on Ti3C2Tx in the future.

Formation of extracellular polymeric substances corona on TiO2 nanoparticles: Roles of crystalline phase and exposed facets

Nanoparticles (NPs) in the environment can interact with macromolecules in the surrounding environment to form eco-corona on their surfaces, which in turn affects the environmental fate and toxicity of nanoparticles. Wastewater treatment plants containing large amounts of microbial extracellular polymeric substances (EPS) are an important source of NPs into the environment, where the formation of EPS coronas on NPs is critical. However, it remains unclear how the crystalline phase and exposed facets, which are intrinsic properties of NPs, affect the formation of EPS coronas on NPs. This study investigated the formation of EPS corona on three TiO2 NPs (representing the most widely used engineered NPs) with different crystalline phases and exposed facets. The protein type and abundance in EPS coronas on TiO2 NPs varied depending on the crystalline phase and exposed facets. Anatase with {101} facets and {001} facets preferred to adsorb proteins with lower molecular weights and higher H-bonding relevant amino acids, respectively, while EPS corona on rutile with {110} facets had proteins with higher hydrophobicity. In addition, the selective adsorption of proteins was primarily determined by steric hindrance, hydrogen bonding, and hydrophobic interaction between TiO2 NPs and proteins, which were affected by changes in aggregation state, surface hydroxyl density, and hydrophobicity of TiO2 NPs induced by crystalline phase and exposed facets. Moreover, crystalline phase and exposed facets-induced EPS corona changes altered the aggregation state and oxidation potential of TiO2–EPS corona complexes. These findings emphasize the important role of crystalline phase and exposed facets in the environmental behavior of nanoparticles and may provide insights into the safe design of nanoparticles.

Protein Corona Formation on Lipid Nanoparticles Negatively Affects the NLRP3 Inflammasome Activation.

The interaction between lipid nanoparticles (LNPs) and serum proteins, giving rise to a unique identification in the form of the protein corona, has been shown to be associated with novel recognition by cell receptors. The presence of the corona enveloping the nanoparticle strongly affects the interplay with immune cells. The immune responses mediated by protein corona can affect nanoparticle toxicity and targeting capabilities. But the intracellular signaling of LNPs after corona formation resulting in the change of nanoparticles' ability to provoke immune responses remains unclear. Therefore, a more systematic and delineated approach must be considered to present the correlation between corona complexes and the shift in nanoparticle immunogenicity. Here, we studied and reported the inhibiting effect of the absorbed proteins on the LNPs on the NLRP3 inflammasome activation, a key intracellular protein complex that modulates several inflammatory responses. Ionizable lipid as a component of LNP was observed to play an important role in modulating the activation of NLRP3 inflammasome in serum-free conditions. However, in the presence of serum proteins, the corona layer on LNPs caused a significant reduction in the inflammasome activation. Reduction in the lysosomal rupture after treatment with corona-LNPs significantly reduced inflammasome activation. Furthermore, a strong reduction of cellular uptake in macrophages after the corona formation was observed. On inspecting the uptake mechanisms in macrophages using transport inhibitors, lipid formulation was found to play a critical role in determining the endocytic pathways for the LNPs in macrophages. This study highlights the need to critically analyze the protein interactions with nanomaterials and their concomitant adaptability with immune cells to evaluate nano-bio surfaces and successfully design nanomaterials for biological applications.

Interaction of polystyrene nanoplastics and hemoglobin is determined by both particle curvature and available surface area

Understanding nanoplastic (NP, or nanoparticle in general) toxicity requires establishing the causal relationships between the physical properties of the nanoparticles and their biological impact. We use spectroscopic, zeta-potential, and dynamic light scattering (DLS) techniques to investigate the formation, structure, and catalytic properties of hemoglobin corona complexes with polystyrene NPs (0–10 mg/mL) of various diameters (20, 50, 100, 500, and 5000 nm). Resonance light scattering, zeta-potential analysis, and DLS demonstrated that hemoglobin corona complexes formed different forms of aggregates with NPs in terms of diameter. Medium-sized (100 nm) NPs induced the most significant conformational alterations in the protein corona compared to smaller and larger ones, which was revealed by spectroscopic assays. However, the catalase-like activity of hemoglobin was promoted in the presence of 100 nm NPs by as high as 35.2 %. NP curvature and surface area are antagonistic factors that govern the conformation of proteins together. This also suggests that 100 nm NPs are more likely to disrupt protein-dependent physiological processes at a given mass concentration than small or large NPs.

In depth characterisation of the biomolecular coronas of polymer coated inorganic nanoparticles with differential centrifugal sedimentation

Advances in nanofabrication methods have enabled the tailoring of new strategies towards the controlled production of nanoparticles with attractive applications in healthcare. In many cases, their characterisation remains a big challenge, particularly for small-sized functional nanoparticles of 5 nm diameter or smaller, where current particle sizing techniques struggle to provide the required sensitivity and accuracy. There is a clear need for the development of new reliable characterisation approaches for the physico-chemical characterisation of nanoparticles with significant accuracy, particularly for the analysis of the particles in the presence of complex biological fluids. Herein, we show that the Differential Centrifugal Sedimentation can be utilised as a high-precision tool for the reliable characterisation of functional nanoparticles of different materials. We report a method to correlate the sedimentation shift with the polymer and biomolecule adsorption on the nanoparticle surface, validating the developed core–shell model. We also highlight its limit when measuring nanoparticles of smaller size and the need to use several complementary methods when characterising nanoparticle corona complexes.

ZnO, Ag and ZnO-Ag nanoparticles exhibit differential modes of toxic and oxidative action in hemocytes of mussel Mytilus galloprovincialis

The present study investigates the cytotoxic and oxidative effects of custom-made nanoparticles (NPs) on hemocytes of Mytilus galloprovincialis, utilizing hemolymph serum (HS) as exposure medium. Specifically, hemocyte lysosomal membrane destabilization (in terms of neutral red retention time assay/NRRT), superoxide anion (O2−), nitric oxide (NO, in terms of nitrites) and lipid peroxidation content (in terms of malondialdehyde/MDA equivalents) were determined in cells treated for 1 h with different concentrations (0.1–50 μg mL−1) of ZnO NPs, Ag NPs and ZnO-Ag NPs, as well as AgNO3 and/or ZnCl2 (bulk ions, respectively). According to the results, Ag NPs were more cytotoxic than ZnO-Ag NPs and/or ZnO NPs, while NRRT values observed in AgNO3 treated cells were lower than those of ZnCl2. Furthermore, high levels of both O2− and MDA were detected in cells treated with Ag NPs, ZnO-Ag NPs, and AgNO3 at concentrations lower than 5 μg mL−1, while high NO generation was observed only in cells treated with 5–25 μg mL−1 of ZnO NPs or ZnCl2. Despite the absence of data, regarding the formation of NP-serum protein corona complexes that could mediate NP surface energy and uptake efficiency, the current study firstly revealed that ZnO NPs, probably via their surface charge, particle agglomeration, and NP Zn+ release could promote an immune-related generation of O2− and NO via the respiratory burst stimulation, a process that is questioned in the case of Ag NPs and/or ZnO-Ag NPs. Moreover, ZnO-Ag NP interaction with biological membranes and their oxidative mode of action seemed to be regulated by the release and the antagonistic/synergistic response of its ionic counterparts (ZnO+ and Ag+), but further studies are needed to elucidate the oxidative mode of action of NP metal ions in complex NP mixtures.

Structure of soft and hard protein corona around polystyrene nanoplastics-Particle size and protein types.

A major challenge in understanding nanoplastic toxicity (or nanoparticles in general) lies in establishing the causal relationships between its physical properties and biological impact. This difficulty can be attributed to surface alterations that follow the formation of a biological complex around the nanoplastic, as exemplified by protein coronae. The protein corona is known to be responsible for the biological response elicited, although its own structure and attributes remain unknown. We approach this knowledge gap by independently studying the structure of soft and hard coronae using neutron scattering techniques. We investigated the formation and the structure of corona proteins (human serum albumin and lysozyme) and the resulting protein corona complexes with polystyrene nanoplastics of different sizes (20 and 200 nm) and charges. Soft corona complexes (regardless of protein type) adopted a structure where the nanoplastics were surrounded by a loose protein layer (∼2-3 protein molecules thick). Hard corona complexes formed fractal-like aggregates, and the morphology of which is known to be harmful to cellular membranes. In most cases, hard-corona coated nanoplastics also formed fractal-like aggregates in solution. Nanoplastic size affected the structures of both the protein corona and the intrinsic protein: more significant conformational change was observed in the hard corona proteins around smaller nanoparticles compared to larger ones, as the self-association forces holding the nanoplastic/protein complex together were stronger. This also implies that protein-dependent biochemical processes are more likely to be disrupted by smaller polystyrene nanoplastics, rather than larger ones.

Immunological Responses Induced by Blood Protein Coronas on Two-Dimensional MoS2 Nanosheets.

Two-dimensional (2D) nanosheets (NSs) have a large surface area, high surface free energy, and ultrathin structure, which enable them to more easily penetrate biological membranes and promote adsorption of drugs and proteins. NSs are capable of adsorbing a large amount of blood proteins to form NSs-protein corona complexes; however, their inflammatory effects are still unknown. Therefore, we investigated the pro-inflammatory effect of 2D model nanosheet structures, molybdenum disulfide (MoS2), and the MoS2 NSs-protein complexes with four abundant proteins in human blood, i.e., human serum albumin (HSA), transferrin (Tf), fibrinogen (Fg), and immunoglobulin G (IgG). The interactions between the NSs and the proteins were analyzed by quantifying protein adsorption, determining binding affinity, and correlating structural changes in the protein corona with the uptake of NSs by macrophages and the subsequent inflammatory response. Although all of the NSs-protein complexes induced inflammation, IgG-coated and Fg-coated NSs triggered much stronger inflammatory effects by producing and releasing more cytokines. Among the four proteins, IgG possessed the highest proportion of β-sheets and led to fewer secondary structure changes on the MoS2 nanosheets. This can facilitate uptake and produce a stronger pro-inflammatory response in macrophages due to the recognition of an NSs-IgG complex by Fc gamma receptors and the subsequent activation of the NF-κB pathways. Our results demonstrate that the blood protein components contribute to the inflammatory effects of nanosheets and provide important insights for the nanosafety evaluation and the rational design of nanomedicines in the future.

A lysozyme corona complex for the controlled pharmacokinetic release of probucol from mesoporous silica particles.

Mesoporous silica particles (MSPs) enhance the release kinetics of poorly soluble compound probucol (PB) under the influence of a pore-blocking protein corona, prepared with lysozyme protein adsorption. In vivo oral administration experiments show a prolongation in the time to reach maximum systemic concentration and half-life of PB released from the lysozyme-MSP complex in comparison to the MSP alone. Specific hard protein corona complexes can act as functional diffusion barriers for the controlled release of drugs from MSP based formulations.

Isolation methods for particle protein corona complexes from protein-rich matrices.

Background: Nanoparticles become rapidly encased by a protein layer when they are in contact with biological fluids. This protein shell is called a corona. The composition of the corona has a strong influence on the surface properties of the nanoparticles. It can affect their cellular interactions, uptake and signaling properties. For this reason, protein coronae are investigated frequently as an important part of particle characterization. Main body of the abstract: The protein corona can be analyzed by different methods, which have their individual advantages and challenges. The separation techniques to isolate corona-bound particles from the surrounding matrices include centrifugation, magnetism and chromatographic methods. Different organic matrices, such as blood, blood serum, plasma or different complex protein mixtures, are used and the approaches vary in parameters such as time, concentration and temperature. Depending on the investigated particle type, the choice of separation method can be crucial for the subsequent results. In addition, it is important to include suitable controls to avoid misinterpretation and false-positive or false-negative results, thus allowing the achievement of a valuable protein corona analysis result. Conclusion: Protein corona studies are an important part of particle characterization in biological matrices. This review gives a comparative overview about separation techniques, experimental parameters and challenges which occur during the investigation of the protein coronae of different particle types.

Adsorption of Plasma Proteins on Single-Walled Carbon Nanotubes Reduced Cytotoxicity and Modulated Neutrophil Activation.

Proteins in the bloodstream bind to carbon nanotubes (CNTs) through noncovalent interactions to form a protein corona, thereby effectively influencing the biological properties and blood biocompatibility of the CNTs. Here, we investigated the binding of common plasma proteins (i.e., human immunoglobulin G (IgG), human serum albumin (HSA), and fibrinogen (FG)) to carboxylated single-walled CNTs (SWCNTs), and evaluated the effects of these different protein coronas on cytotoxicity to endothelial cells and immune response to neutrophils in the bloodstream. Measurements of adsorption parameters revealed tight binding of proteins to SWCNTs, and the SWCNTs adsorption capacities followed the order FG > HSA > IgG. In addition, the basic residues (Arg, Lys, His) were found to play an important role in the formation of protein-SWCNTs corona complexes and determine their adsorption capacity. Consistent with the higher protein adsorption capacity, FG more significantly reduced the cytotoxicity of CNTs to human umbilical vein endothelial cells than the other two proteins. However, only treatment of SWCNTs with IgG resulted in the enhancement of CNT-induced myeloperoxidase (MPO) release (i.e., neutrophil activation) in neutrophils, while MPO-dependent degradation of CNTs induced less cytotoxicity than initial nanomaterials. Consistent with these effects of protein coronas, the presence of serum attenuated the cytotoxicity of CNTs and CNTs could induce neutrophil activation in human blood plasma. Our study demonstrates the ability of adsorbed plasma proteins to influence cytotoxicity and neutrophil response caused by CNTs in the bloodstream.

Serendipitous Synthesis Found in the Nuances of Homoleptic Zinc Complex Formation.

A series of aminophenolate and aminonaphtholate homoleptic zinc complexes were obtained using a simple and unique synthetic strategy. A rigorous analysis of the byproduct supported modifications of the main course of the bis-chelation reaction. Controlled alcoholysis was followed by alternation and controlled anaerobic hydrolysis of ethyl-zinc aminophenolate or aminonaphtholate complexes. This new and intriguing reaction yielded a new class of zinc corona complexes. All the synthesized complexes were fully characterized in the solid state and in solution using X-ray and spectroscopic methods as well as density functional theory calculations.

Revealing the immune perturbation of black phosphorus nanomaterials to macrophages by understanding the protein corona

The increasing number of biological applications for black phosphorus (BP) nanomaterials has precipitated considerable concern about their interactions with physiological systems. Here we demonstrate the adsorption of plasma protein onto BP nanomaterials and the subsequent immune perturbation effect on macrophages. Using liquid chromatography tandem mass spectrometry, 75.8% of the proteins bound to BP quantum dots were immune relevant proteins, while that percentage for BP nanosheet–corona complexes is 69.9%. In particular, the protein corona dramatically reshapes BP nanomaterial–corona complexes, influenced cellular uptake, activated the NF-κB pathway and even increased cytokine secretion by 2–4-fold. BP nanomaterials induce immunotoxicity and immune perturbation in macrophages in the presence of a plasma corona. These findings offer important insights into the development of safe and effective BP nanomaterial-based therapies.

Effective lock-in strategy for proteomic analysis of corona complexes bound to amino-free ligands of gold nanoparticles

For specific applications, gold nanoparticles (GNPs) are commonly functionalized with various biological ligands, including amino-free ligands such as amino acids, peptides, proteins, and nucleic acids. Upon entering a biological fluid, the protein corona that forms around GNPs can conceal the targeting ligands and sterically hinder the functional properties. The protein corona is routinely prepared by standard centrifugation or sucrose cushion centrifugation. However, such methodologies are not applicable to the exclusive analysis of a ligand-binding protein corona. In this study, we first proposed a lock-in strategy based on a combination of rapid crosslinking and stringent washing. Cysteine was used as a model of amino-free ligands and attached to GNPs. After corona formation in the human plasma, GNP cysteine and corona proteins were quickly fixed by 5 s of crosslinking with 7.5% formaldehyde. After stringent washing using SDS buffer with sonication, the cysteine-bound proteins were effectively separated from unbound proteins. Qualitative and quantitative analyses using a mass spectrometry-based proteomics approach indicated that the protein composition of the cysteine-binding corona from the new method was significantly different from the composition of the whole corona from the two conventional methods. Furthermore, network and formaldehyde-linked site analyses of cysteine-binding proteins provided useful information toward a better knowledge of the behavior of protein-ligand and protein-protein interactions. Collectively, our new strategy has the capability to particularly characterize the protein composition of a cysteine-binding corona. The presented methodology in principal provides a generic way to analyze a nanoparticle corona bound to amino-free ligands and has the potential to decipher corona-masked ligand functions.

Interactions Between Plant Viral Nanoparticles (VNPs) and Blood Plasma Proteins, and Their Impact on theVNP In Vivo Fates.

Plant viral nanoparticles (VNPs) are currently being developed as novel vessels for delivery of diagnostic and therapeutic cargos to sites of disease. With a rapid increase in the number of VNP variants and their potential applications in nanomedicine, the properties they acquire in the bloodstream need to be investigated. Biomolecules present in plasma are known to adsorb onto the surface of nanomaterials (including VNPs), forming a biointerface called the protein corona, which is capable of reprogramming the properties of VNPs. Here we describe a few general methods to isolate and study the VNP-protein corona complexes, in order to evaluate the impact of protein corona on molecular recognition of VNPs by target cells, and clearance by phagocytes. We outline procedures for in vivo screening of VNP fates in a mouse model, which may be useful for evaluation of efficacy and biocompatibility of different VNP based formulations.

Probing the modulated formation of gold nanoparticles-beta-lactoglobulin corona complexes and their applications.

Understanding the interactions between proteins and nanoparticles (NPs) along with the underlying structural and dynamic information is of utmost importance to exploit nanotechnology for biomedical applications. Upon adsorption onto a NP surface, proteins form a well-organized layer, termed the corona, that dictates the identity of the NP-protein complex and governs its biological pathways. Given its high biological relevance, in-depth molecular investigations and applications of NPs-protein corona complexes are still scarce, especially since different proteins form unique corona patterns, making identification of the biomolecular motifs at the interface critical. In this work, we provide molecular insights and structural characterizations of the bio-nano interface of a popular food-based protein, namely bovine beta-lactoglobulin (β-LG), with gold nanoparticles (AuNPs) and report on our investigations of the formation of corona complexes by combined molecular simulations and complementary experiments. Two major binding sites in β-LG were identified as being driven by citrate-mediated electrostatic interactions, while the associated binding kinetics and conformational changes in the secondary structures were also characterized. More importantly, the superior stability of the corona led us to further explore its biomedical applications, such as in the smartphone-based point-of-care biosensing of Escherichia coli (E. coli) and in the computed tomography (CT) of the gastrointestinal (GI) tract through oral administration to probe GI tolerance and functions. Considering their biocompatibility, edible nature, and efficient excretion through defecation, AuNPs-β-LG corona complexes have shown promising perspectives for future in vitro and in vivo clinical settings.

Monitoring of the Enzymatic Degradation of Protein Corona and Evaluating the Accompanying Cytotoxicity of Nanoparticles.

Established nanobio interactions face the challenge that the formation of nanoparticle-protein corona complexes shields the inherent properties of the nanoparticles and alters the manner of the interactions between nanoparticles and biological systems. Therefore, many studies have focused on protein corona-mediated nanoparticle binding, internalization, and intracellular transportation. However, there are a few studies to pay attention to if the corona encounters degradation after internalization and how the degradation of the protein corona affects cytotoxicity. To fill this gap, we prepared three types of off/on complexes based on gold nanoparticles (Au NPs) and dye-labeled serum proteins and studied the extracellular and intracellular proteolytic processes of protein coronas as well as their accompanying effects on cytotoxicity through multiple evaluation mechanisms, including cell viability, adenosine triphosphate (ATP) content, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). The proteolytic process was confirmed by recovery of the fluorescence of the dye-labeled protein molecules that was initially quenched by Au NPs. Our results indicate that the degradation rate of protein corona is dependent on the type of the protein based on systematical evaluation of the extracellular and intracellular degradation processes of the protein coronas formed by human serum albumin (HSA), γ-globulin (HGG), and serum fibrinogen (HSF). Degradation is the fastest for HSA corona and the slowest for HSF corona. Notably, we also find that the Au NP-HSA corona complex induces lower cell viability, slower ATP production, lower MMP, and higher ROS levels. The cytotoxicity of the nanoparticle-protein corona complex may be associated with the protein corona degradation process. All of these results will enrich the database of cytotoxicity induced by nanomaterial-protein corona complexes.