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Distinguishing Protein Corona from Nanoparticle Aggregate Formation in Complex Biological Media Using X-ray Photon Correlation Spectroscopy
Abstract
In biological systems, nanoparticles interact with biomolecules, which may undergo protein corona formation that can result in noncontrolled aggregation. Therefore, comprehending the behavior and evolution of nanoparticles in the presence of biological fluids is paramount in nanomedicine. However, traditional lab-based colloid methods characterize diluted suspensions in low-complexity media, which hinders in-depth studies in complex biological environments. Here, we apply X-ray photon correlation spectroscopy (XPCS) to investigate silica nanoparticles (SiO 2) in various environments, ranging from low to high complex biological media. Interestingly, SiO 2 revealed Brownian motion behavior, irrespective of the complexity of the chosen media. Moreover, the SiO 2 surface and media composition were tailored to underline the differences between a corona-free system from protein corona and aggregates formation. Our results highlighted XPCS potential for real-time nanoparticle analysis in biological media, surpassing the limitations of conventional techniques and offering deeper insights into colloidal behavior in complex environments.
... After incubation with S-protein, the fetuin-AuNPs-S-protein structure formed, and this can lead to decrease in absorbance spectra (Figure 3c) due to corona formation. [32][33][34][35] Moreover, as can be seen from Figure 3, whereas the absorbance of colloidal AuNPs was determined as approximately 1.0 (Figure 3a), this absorbance value increased approximately to 1.3 after it was functionalized with fetuin ( Figure 3b). Meanwhile, the absorbance value showed almost no change when phosphate buffer was added to the medium instead of S-protein ( Figure 3c). ...
... Protein corona and aggregation are two distinct phenomena that can occur when proteins interact with nanoparticles, but they differ in terms of the processes involved and the effects they have on the systems. [33][34][35][36] The protein corona refers to the layer of proteins that adsorb onto the surface of nanoparticles when they are introduced into a biological environment (such as blood, interstitial fluid, or other bodily fluids). This process happens due to the interaction between the nanoparticle surface and the proteins in the surrounding environment. ...
... The protein corona has been formed as nanoparticles interact with proteins from the surrounding fluid, typically driven by electrostatic forces, van der Waals forces, hydrophobic interactions, or other weak molecular forces. [33][34][35][36] Aggregation refers to the process where individual nanoparticles or proteins stick together (aggregate) to form clusters or larger structures. This can occur due to various factors such as changes in environmental conditions (e.g., pH, ionic strength), protein-protein interactions, or the properties of the nanoparticles themselves. ...
This work aims to investigate the possible role of sialic acids as host cell receptors for SARS‐CoV‐2 infection and hence to develop a practical spectroscopic protocol that relies on the interaction between fetuin and spike protein (S‐protein) for SARS‐CoV‐2 detection. For that purpose, firstly, synthesized gold nanoparticles (AuNPs) were functionalized with fetuin. Then, fetuin functionalized AuNPs were incubated with S‐protein and the difference in absorbance value of AuNPs at 530 nm was measured spectroscopically. The interaction between fetuin and S‐protein was characterized by using fourier transform infrared spectroscopy and atomic force microscopy methods whereas sole AuNP structure was investigated by using transmission electron microscopy technique. The experimental parameters such as fetuin amount (0, 5, 10, and 20 µg/mL) and incubation duration (0, 10, 20, 30, and 40 min) were optimized and analytical characteristics of developed biosensors were investigated. The detection limit value was calculated as 2.40 ng/mL, the linear range of detection was between 4.98 and 95.2 ng/mL, and the relative standard deviation value was 0.72% for 9.95 ng/mL S‐protein. Finally, the developed biosensor was applied to real samples that were taken from SARS‐CoV‐2 non‐infected and infected individuals.
... Another interesting scenario is when colloids are suspended in biological milieux (Semeraro et al., 2018a;Otto et al., 2024;Silva et al., 2024), where subtle non-equilibrium effects may be elicited. Out-of-equilibrium dynamics are prevalent in biology; for example, active transport in cellular media involves anomalous diffusion via Lé vy flight or fractional Brownian motion (Nolte, 2024). ...
The high brilliance of fourth-generation synchrotron sources coupled with advanced X-ray detectors enables a wide range of dynamic studies of colloids and other soft-matter systems. In particular, the higher fraction of coherent flux provided by these new sources is a major boost for X-ray photon correlation spectroscopy (XPCS). As a result, not only can equilibrium dynamics be accessed but also relatively fast out-of-equilibrium processes can be investigated by XPCS. This article briefly recalls the statistical properties of coherent scattering and then demonstrates a case study of non-equilibrium fluctuations in a driven colloidal system. A simple example is the resuspension of colloids by vigorous shaking, where the inhomogeneous flow generates local variations in number density of particles leading to strong velocity fluctuations. The Brownian motion of the particles homogenizes the suspension with time and the system gradually returns to pure diffusive dynamics. On the other hand, in a uniformly sheared suspension of particles, such concentration gradients do not form and upon cessation of shear the return to Brownian dynamics is rapid. These transient non-equilibrium effects can inadvertently influence micrometre-range particle size measurement by means of dynamic scattering methods.
Billions of people are exposed to fine particulate matter (PM2.5) levels above the USEPA’s annual standard of 9 μg/m³. Common emission sources are anthropogenic, producing complex aerosolized toxins. Ultrafine particulate matter (UFPM) and industrial nanoparticles (NPs) have major detrimental effects on the brain, but the USA does not measure UFPM on a routine basis. This review focuses on the development and progression of common neurodegenerative diseases, as diagnosed through neuropathology, among young residents in Metropolitan Mexico City (MMC). MMC is one of the most polluted megacities in the world, with a population of 22 million residents, many of whom are unaware of the brain effects caused by their polluted atmosphere. Fatal neurodegenerative diseases (such as Alzheimer’s and Parkinson’s) that begin in childhood in populations living in air polluted environments are preventable. We conclude that UFPM/NPs are capable of disrupting neural homeostasis and give rise to relentless neurodegenerative processes throughout the entire life of the highly exposed population in MMC. The paradigm of reaching old age to have neurodegeneration is no longer supported. Neurodegenerative changes start early in pediatric ages and are irreversible. It is time to invest in preventive medicine.
Polymer‐coated nanoparticles are widely studied in the context of nanomedicine and it is therefore of utmost importance to understand not only how their structure but also how their colloidal dynamics are affected by physiologically relevant conditions. A characteristic feature of the cytosol of cells is the very high concentration of proteins among other matrix components, often termed macromolecular crowding. Here, the structure and colloidal dynamics of poly(ethylene glycol) (PEG)‐coated gold nanoparticles in the presence of bovine serum albumin (BSA) concentrations ranging from 0 to 265 mg/mL are studied with X‐ray photon correlation spectroscopy. For protein–nanoparticle mixtures with high BSA concentrations, comparable to intracellular levels, a significant deviation of the apparent viscosity from expectations for pure BSA solutions is found. The findings strongly indicate that the nanoscopic viscous properties of the dense protein solutions are significantly affected by the nanoparticles. At these high concentrations, the colloidal stability of the samples depends on the molecular weight of the coating PEG–ligand, whereas at lower concentrations no differences are observed.
Prion diseases are characterized by prion protein (PrP) transmissible aggregation and neurodegeneration, which has been linked to oxidative stress. The physiological function of PrP seems related to sequestering of redox-active Cu ²⁺ , and Cu ²⁺ dyshomeostasis is observed in prion disease brain. It is unclear whether Cu ²⁺ contributes to PrP aggregation, recently shown to be mediated by PrP condensation. This study indicates that Cu ²⁺ promotes PrP condensation in live cells at the cell surface and in vitro through copartitioning. Molecularly, Cu ²⁺ inhibited PrP β-structure and hydrophobic residues exposure. Oxidation, induced by H 2 O 2 , triggered liquid-to-solid transition of PrP:Cu ²⁺ condensates and promoted amyloid-like PrP aggregation. In cells, overexpression of PrP C initially protected against Cu ²⁺ cytotoxicity but led to PrP C aggregation upon extended copper exposure. Our data suggest that PrP condensates function as a buffer for copper that prevents copper toxicity but can transition into PrP aggregation at prolonged oxidative stress.
Recently, fourth-generation synchrotron sources with several orders of magnitude higher brightness and higher degree of coherence compared with third-generation sources have come into operation. These new X-ray sources offer exciting opportunities for the investigation of soft matter and biological specimens by small-angle X-ray scattering (SAXS) and related scattering methods. The improved beam properties together with the advanced pixel array detectors readily enhance the angular resolution of SAXS and ultra-small-angle X-ray scattering in the pinhole collimation. The high degree of coherence is a major boost for the X-ray photon correlation spectroscopy (XPCS) technique, enabling the equilibrium dynamics to be probed over broader time and length scales. This article presents some representative examples illustrating the performance of SAXS and XPCS with the Extremely Brilliant Source at the European Synchrotron Radiation Facility. The rapid onset of radiation damage is a significant challenge with the vast majority of samples, and appropriate protocols need to be adopted for circumventing this problem.
The protein corona forms spontaneously on nanoparticle surfaces when nanomaterials are introduced into any biological system/fluid. Reliable characterization of the protein corona is, therefore, a vital step in the development of safe and efficient diagnostic and therapeutic nanomedicine products. 2134 published manuscripts on the protein corona are reviewed and a down‐selection of 470 papers spanning 2000–2021, comprising 1702 nanoparticle (NP) systems is analyzed. This analysis reveals: i) most corona studies have been conducted on metal and metal oxide nanoparticles; ii) despite their overwhelming presence in clinical practice, lipid‐based NPs are underrepresented in protein corona research, iii) studies use new methods to improve reliability and reproducibility in protein corona research; iv) studies use more specific protein sources toward personalized medicine; and v) careful characterization of nanoparticles after corona formation is imperative to minimize the role of aggregation and protein contamination on corona outcomes. As nanoparticles used in biomedicine become increasingly prevalent and biochemically complex, the field of protein corona research will need to focus on developing analytical approaches and characterization techniques appropriate for each unique nanoparticle formulation. Achieving such characterization of the nano‐bio interface of nanobiotechnologies will enable more seamless development and safe implementation of nanoparticles in medicine.
Diffusion of proteins on length scales of their size is crucial for understanding the machinery of living cells. X-ray photon correlation spectroscopy (XPCS) is currently the only way to access long-time collective diffusion on these length scales, but radiation damage so far limits the use in biological systems. We apply a new approach to use XPCS to measure cage relaxation in crowded α-crystallin solutions. This allows us to correct for radiation effects, obtain missing information on long time diffusion, and support the fundamental analogy between protein and colloid dynamical arrest.
How soft corona, the protein corona’s outer layer, contributes to biological identity of nanomaterials is largely because capturing protein composition of the soft corona in situ remains challenging. We herein develop an in situ Fishing method that can monitor the dynamic formation of protein corona on ultra-small chiral Cu 2 S nanoparticles (NPs) allowing us to directly separate and identify the corona protein composition. Our method detects spatiotemporal processes in the evolution of hard and soft coronas on chiral NPs, revealing subtle differences in NP − protein interactions even within several minutes. This study highlights the importance of in situ and dynamic analysis of soft/hard corona, provides insights into the role of soft corona in mediating biological responses of NPs, and offers a universal strategy to characterize soft corona to guide the rational design of biomedical nanomaterials.
X‐ray photon correlation spectroscopy (XPCS), a synchrotron source‐based technique to measure sample dynamics, is used to determine hydrodynamic diameters of gold nanoparticles (Au NPs) of different sizes in biological environments. In situ determined hydrodynamic diameters are benchmarked with values obtained by dynamic light scattering. The technique is then applied to analyze the behavior of the Au NPs in a biological environment. First, a concentration‐dependent agglomeration in the presence of NaCl is determined. Second, concentration‐dependent increase in hydrodynamic diameter of the Au NPs upon the presence of proteins is determined. As X‐rays in the used energy range are barely scattered by biological matter, dynamics of the Au NPs can be also detected in situ in complex biological environments, such as blood. These measurements demonstrate the possibility of XPCS for in situ analytics of nanoparticles (NPs) in biological environments where similar detection techniques based on visible light would severely suffer from scattering, absorption, and reflection effects.
Protein corona formation and nanoparticles' aggregation have been heavily discussed over the last years since the lack of fine-mapping of these two combined effects has hindered the targeted delivery evolution and the personalized nanomedicine development. We present a multi-technique approach that combines Dynamic Light and Small-Angle X-ray Scattering techniques with cryo-Transmission Electron Microscopy in a given fashion that efficiently distinguishes protein corona from aggregates formation. This methodology was tested using ~ 25-nm model silica nanoparticles incubated with either model proteins or biologically relevant proteomes (such as fetal bovine serum and human plasma) in buffers of low and high ionic strengths to precisely tune particle-to-protein interactions. In this work, we were able to differentiate protein corona, small aggregates formation, and massive aggregation, as well as obtain fractal information of the aggregates reliably and straightforwardly. The strategy presented here can be expanded to other particle-to-protein mixtures and might be employed as a quality control platform for samples that undergo biological tests.
Nanomaterials offer promising solutions as drug delivery systems and imaging agents in response to the demand for better therapeutics and diagnostics. However, the limited understanding of the interaction between nanoparticles and biological entities is currently hampering the development of new systems and their applications in clinical settings. Proteins and lipids in biological fluids are known to complex with nanoparticles to form a “biomolecular corona”. This has been shown to affect particles' morphology and behavior in biological systems and their interactions with cells. Hence, understanding how nanomaterials' physicochemical properties affect the formation and composition of this biocorona is a crucial step. This work evaluates existing literature on how morphology (size and shape), and surface chemistry (charge and hydrophobicity) of nanoparticles influence the formation of protein corona. The latest evidence suggest that although surface charge promotes the interaction with proteins and lipids, surface chemistry plays a leading role in determining the affinity of the nanoparticle for biomolecules and, ultimately, the composition of the corona. More recently the study of additional nanoparticles' properties like shape and surface chirality have demonstrated a significant effect on protein corona architecture, providing new tools to tailor biomolecular corona formation.
This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies
Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
This review shows the most common and promising strategies to generate colloidally stable silica nanoparticles (NPs) in simulated biological fluids and sheds light on the latest advances in producing degradable silica-based structures. Silica NPs can be synthesized in a wide variety of morphologies, porosity levels, and sizes. This versatility makes silica NPs one of the most promising nano-platforms for imaging and disease treatment. Nonetheless, biological barriers can decrease the success of translating them for therapeutic applications since the media composition can induce their colloidal stability loss. It can, consequently, lead to the NPs aggregation and affect their degradation profile. The interplay between NPs aggregation and degradation has been scarcely explored in the literature when biological fluids are seriously taken into account. Herein we discuss the theory behind the colloidal stability of silica NPs, the processes leading to their aggregation, and some strategies to overcome this issue (mainly focused on NPs surface functionalization). Furthermore, we addressed the main issues that affect the degradability of NPs in biological fluids, and explored some strategies, such as chemical surface modification, which are able to tune these degradation-driven profiles. Thus, the understanding of the silica NPs behavior in body fluids is essential for the approval of nanomedicines and, therefore, more investigations concerning the dynamics, thermodynamics, biological response, and structural parameters of silica-based NPs are of utmost importance.
Nanoparticles are under investigation as diagnostic and therapeutic agents for joint diseases, such as osteoarthritis. However, there is incomplete understanding of nanoparticle diffusion in synovial fluid, the fluid inside the joint, which consists of a mixture of the polyelectrolyte hyaluronic acid, proteins, and other components. Here, we show that rotational and translational diffusion of polymer-coated nanoparticles in quiescent synovial fluid and in hyaluronic acid solutions is well described by the Stokes-Einstein relationship, albeit with an effective medium viscosity that is much smaller than the macroscopic low shear viscosity of the fluid. This effective medium viscosity is well described by an equation for the viscosity of dilute polymer chains, where the additional viscous dissipation arises because of the presence of the polymer segments. These results shed light on the diffusive behavior of polymer-coated inorganic nanoparticles in complex and crowded biological environments, such as in the joint.
While the interplay between liquid-liquid phase separation (LLPS) and glass formation in biological systems is highly relevant for their structure formation and thus function, the exact underlying mechanisms are not well known. The kinetic arrest originates from the slowdown at the molecular level, but how this propagates to the dynamics of microscopic phase domains is not clear. Since with diffusion, viscoelasticity, and hydrodynamics, distinctly different mechanisms are at play, the dynamics needs to be monitored on the relevant time and length scales and compared to theories of phase separation. Using x-ray photon correlation spectroscopy, we determine the LLPS dynamics of a model protein solution upon low temperature quenches and find distinctly different dynamical regimes. We observe that the early stage LLPS is driven by the curvature of the free energy and speeds up upon increasing quench depth. In contrast, the late stage dynamics slows down with increasing quench depth, fingerprinting a nearby glass transition. The dynamics observed shows a ballistic type of motion, implying that viscoelasticity plays an important role during LLPS. We explore possible explanations based on the Cahn-Hilliard theory with nontrivial mobility parameters and find that these can only partially explain our findings.
An experimental setup to measure X-ray photon correlation spectroscopy during continuous sample translation is presented and its effectiveness as a means to avoid sample damage in dynamics studies of protein diffusion is evaluated. X-ray damage from focused coherent synchrotron radiation remains below tolerable levels as long as the sample is translated through the beam sufficiently quickly. Here it is shown that it is possible to separate sample dynamics from the effects associated with the transit of the sample through the beam. By varying the sample translation rate, the damage threshold level, Dthresh = 1.8 kGy, for when beam damage begins to modify the dynamics under the conditions used, is also determined. Signal-to-noise ratios, Rsn ≥ 20, are obtained down to the shortest delay times of 20 µs. The applicability of this method of data collection to the next generation of multi-bend achromat synchrotron sources is discussed and it is shown that sub-microsecond dynamics should be obtainable on protein samples.
The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identify a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins are revealed as modulators of nanoparticle-cell association mainly through their dynamic nature. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.
In this perspective article we highlight research opportunities and challenges in probing structural dynamics of molecular systems using X-ray Photon Correlation Spectroscopy (XPCS). The development of new X-ray sources such as 4th generation storage rings and X-ray free-electron lasers (XFELs) provides promising new insights into molecular motion. Employing XPCS at these sources allows to capture a very broad range of timescales and lengthscales, spanning from femtoseconds to minutes and atomic scales to the mesoscale. Here, we discuss the scientific questions that can be addressed with these novel tools for two prominent examples: the dynamics of proteins in biomolecular condensates and the dynamics of supercooled water. Finally, we provide practical tips for designing and estimating feasibility of XPCS experiments as well as on detecting and mitigating radiation damage.
Nanomaterials have high potential as powerful tools for nanomedicine in a wide range of most promising applications as highly specific devices for diagnosis and therapy. Yet, despite enormous research activities in the design and synthesis of nanomaterials for biomedicine, only a small number of those have made their way to clinical use. The unavoidable formation of a biomolecular adsorption layer, the ‘protein corona’ or ‘biomolecular corona’ around nanoparticles (NPs) has been recognized as a major roadblock on the way toward the efficient design of nanomedicines. It masks the generic NP properties and creates a new ‘biological identity’ that largely controls the interactions with the biological environment. Therefore, for successful design of nanomedical devices, researchers must anticipate formation of this protein adlayer and its ensuing effects. In this review, we summarize our current knowledge in the field and focus on three topics that appear to be important for furthering progress in our ability to predict in-vivo responses to NP incorporation from in-vitro studies. First, we address fundamental physicochemical issues of protein corona formation as revealed by recent in-vitro studies, with a focus on the underlying mechanistic details. Second, we illustrate with recent examples how our present, still incomplete understanding can already be exploited to control protein corona formation in the organism, including important processes involving the immune system. Third, recent advances in the transition from in-vitro to in-vivo studies of protein adsorption will be summarized, which is obviously a key step in NP development for nanomedicine. We conclude this review with an outlook on possible future developments in the field. Keywords: Nanomaterials, Protein adsorption, Protein corona, Mechanisms, Nanomedicine
Nanotechnology is a multidisciplinary science covering matters involving the nanoscale level that is being developed for a great variety of applications. Nanomedicine is one of these attractive and challenging uses focused on the employment of nanomaterials in medical applications such as drug delivery. However, handling these nanometric systems require defining specific parameters to establish the possible advantages and disadvantages in specific applications. This review presents the fundamental factors of nanoparticles and its microenvironment that must be considered to make an appropriate design for medical applications, mainly: (i) Interactions between nanoparticles and their biological environment, (ii) the interaction mechanisms, (iii) and the physicochemical properties of nanoparticles. On the other hand, the repercussions of the control, alter and modify these parameters in the biomedical applications. Additionally, we briefly report the implications of nanoparticles in nanomedicine and precision medicine, and provide perspectives in immunotherapy, which is opening novel applications as immune-oncology.
Nano-sized objects, such as nanoparticles and other drug carriers used in nanomedicine, once in contact with biological environments are modified by adsorption of biomolecules on their surface. The presence of this corona strongly affects the following interactions at cell and organism levels. It has been shown that corona proteins can be recognized by cell receptors. However, it is not known whether the composition of this acquired layer can also affect the mechanisms nanoparticles use to enter cells. This is of particular importance when considering that the same nanoparticles can form different coronas for instance In Vitro when exposed to cells in different serum amounts, or In Vivo depending on the exposure or administration route. Thus, in this work, different coronas were formed on 50 nm silica by exposing them to different serum concentrations. The uptake efficiency in HeLa cells was compared, and the uptake mechanisms were characterized using transport inhibitors and RNA interference. The results showed that the nanoparticles were internalized by cells via different mechanisms when different coronas were formed, and only for one corona condition uptake was mediated by the LDL receptor. This suggested that corona of different composition can be recognized differently by cell receptors, and this in turn leads to internalization via different mechanisms. Similar studies were performed using other cells, including A549 cells and primary HUVEC, and different nanoparticles, namely 100 nm liposomes and 200 nm silica. Overall, the results confirmed that the corona composition can affect the mechanism of nanoparticle uptake by cells.
This study explores the possibility of measuring the dynamics of proteins in solution using X-ray photon correlation spectroscopy (XPCS) at nearly diffraction-limited storage rings (DLSRs). We calculate the signal-to-noise ratio (SNR) of XPCS experiments from a concentrated lysozyme solution at the length scale of the hydrodynamic radius of the protein molecule. We take into account limitations given by the critical X-ray dose and find expressions for the SNR as a function of beam size, sample-to-detector distance and photon energy. Specifically, we show that the combined increase in coherent flux and coherence lengths at the DLSR PETRA IV yields an increase in SNR of more than one order of magnitude. The resulting SNR values indicate that XPCS experiments of biological macromolecules on nanometre length scales will become feasible with the advent of a new generation of synchrotron sources. Our findings provide valuable input for the design and construction of future XPCS beamlines at DLSRs.
An experimental study of the influence of multiple-scattering events on data acquired by small-angle X-ray scattering (SAXS) and X-ray photon correlation spectroscopy (XPCS) techniques in the ultra-small-angle range is presented. Both techniques rely on the single-scattering assumption and any deviation may lead to erroneous interpretation of structural and dynamical results. Typical effects of multiple scattering on measured static and dynamic scattering from model colloidal suspensions are presented, and it is demonstrated that the same information content as in a single-scattering experiment can be extracted after applying appropriate corrections. The perturbations induced by multiple scattering on resolution, photon statistics and speckle visibility are evaluated and the required optimal experimental conditions are discussed. In many cases, multiple scattering might be tolerable to a certain extent, especially if any shortcomings are outweighed by beneficial effects from counting statistics, acquisition time and signal-to-background ratio. The presented study may help to optimize future experiments and serve as a guide for data evaluation for this type of system.
In vitro incubation of nanomaterials with plasma offer insights on biological interactions, but cannot fully explain the in vivo fate of nanomaterials. Here, we use a library of polymer nanoparticles to show how physicochemical characteristics influence blood circulation and early distribution. For particles with different diameters, surface hydrophilicity appears to mediate early clearance. Densities above a critical value of approximately 20 poly(ethylene glycol) chains (MW 5 kDa) per 100 nm² prolong circulation times, irrespective of size. In knockout mice, clearance mechanisms are identified for nanoparticles with low and high steric protection. Studies in animals deficient in the C3 protein showed that complement activation could not explain differences in the clearance of nanoparticles. In nanoparticles with low poly(ethylene glycol) coverage, adsorption of apolipoproteins can prolong circulation times. In parallel, the low-density-lipoprotein receptor plays a predominant role in the clearance of nanoparticles, irrespective of poly(ethylene glycol) density. These results further our understanding of nanopharmacology.
The use of nanocarriers in biology and medicine is complicated by the current need to understand how nanoparticles interact in complex biological surroundings. When nanocarriers come into contact with serum, proteins immediately adsorb onto their surface, forming a protein corona which defines their biological identity. Although the composition of the protein corona has been widely determined by proteomics, its morphology still remains unclear. In this study we show for the first time the morphology of the protein corona using transmission electron microscopy. We are able to demonstrate that the protein corona is not, as commonly supposed, a dense, layered shell coating the nanoparticle, but an undefined, loose network of proteins. Additionally, we are now able to visualize and discriminate between the soft and hard corona using centrifugation-based separation techniques together with proteomic characterization. The protein composition of the ~15 nm hard corona strongly depends on the surface chemistry of the respective nanomaterial, thus further affecting cellular uptake and intracellular trafficking. Large diameter protein corona resulting from pre-incubation with soft corona or Apo-A1 inhibits cellular uptake, confirming the stealth-effect mechanism. In summary, the knowledge on protein corona formation, composition and morphology is essential to design therapeutic effective nanoparticle systems.
In this study, based on hydrolysis and condensation via Stöber process of sol-gel method, synthesis of mono-dispersed silica nanoparticles was carried out with hydrophilic solvent. This operation was expected to be a more simplified process than that with organic solvent. Based on the sol-gel method, which involves simply controlling the particle size, the particle size of the synthesized silica specimens were ranged from 30 to 300 nm by controlling the composition of tetraethylorthosilicate (TEOS), DI water and ammonia solution, and by varying the stirring speeds while maintaining a fixed amount of ethanol. Increasing the content of DI water and decreasing the content of ammonia caused the particle size to decrease, while controlling the stirring speed at a high level of RPMs enabled a decrease of the particle size. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were utilized to investigate the success factors for synthesizing process; Field emission scanning electron microscopy (FE-SEM) was used to study the effects of the size and morphology of the synthesized particles. To analyze the dispersion properties, zeta potential and particle size distribution (PSD) analyses were utilized.
Surface modifications of highly monodisperse citrate-stabilized gold nanoparticles (Au NPs) with sizes ranging from 3.5 to 150 nm after their exposure to cell culture media supplemented with serum have been studied and characterized by the combined use of UV-Vis spectroscopy, Dynamic Light Scattering and Zeta Potential measurements. In all the tested particles, a dynamic process of protein adsorption has been observed, evolving towards the formation of an irreversible hard protein coating. Interestingly, the thickness and density of this protein coating is strongly dependent on the NP size, being possible to identify different transition regimes as the size of the NP increases: (i) NP-protein complexes (or incomplete coating), (ii) the formation of a single dense protein corona layer and (iii) the formation of a multilayer corona. Besides, the different temporal patterns in the formation of the protein coating is faster for small particles than for the larger ones, further revealing that size plays a significant role in the kinetics of this process. Since the biological identity of the NPs is ultimately determined by the protein corona and different NP–biological interactions take place at different timescales, obtained results have a relevant impact in biological and toxicological studies.
Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurf(TM) and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurf(TM) promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate ∼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.
In recent years, X-ray photon correlation spectroscopy (XPCS) has emerged as one of the key probes of slow nanoscale fluctuations, applicable to a wide range of condensed matter and materials systems. This article briefly reviews the basic principles of XPCS as well as some of its recent applications, and discusses some novel approaches to XPCS analysis. It concludes with a discussion of the future impact of diffraction-limited storage rings on new types of XPCS experiments, pushing the temporal resolution to nanosecond and possibly even picosecond time scales.
The formation and composition of the protein corona on silica (SiO2) nanoparticles (NP) with different surface chemistries was evaluated over time. Native SiO2, amine (-NH2) and carboxy (-COO(-)) modified NP were examined following incubation in mammalian growth media containing fetal bovine serum (FBS) for 1, 4, 24 and 48 hours. The protein corona transition from its early dynamic state to the later more stable corona was evaluated using mass spectrometry. The NP diameter was 22.4 ± 2.2 nm measured by scanning transmission electron microscopy (STEM). Changes in hydrodynamic diameter and agglomeration kinetics were studied using dynamic light scattering (DLS). The initial surface chemistry of the NP played an important role in the development and final composition of the protein corona, impacting agglomeration kinetics and NP toxicity. Particle toxicity, indicated by changes in membrane integrity and mitochondrial activity, was measured by lactate dehydrogenase (LDH) release and tetrazolium reduction (MTT), respectively, in mouse alveolar macrophages (RAW264.7) and mouse lung epithelial cells (C10). SiO2-COO(-) NP had a slower agglomeration rate, formed smaller aggregates, and exhibited lower cytotoxicity compared to SiO2 and SiO2-NH2. Composition of the protein corona for each of the three NP was unique, indicating a strong dependence of corona development on NP surface chemistry. This work underscores the need to understand all aspects of NP toxicity, particularly the influence of agglomeration on effective dose and particle size. Furthermore, the interplay between materials and local biological environment is emphasized and highlights the need to conduct toxicity profiling under physiologically relevant conditions that provide an appropriate estimation of material modifications that occur during exposure in natural environments.
The determination of the intrinsic viscosity by single-point determinations, like that based on the Solomon–Ciuta equation,
have been proposed as efficient alternatives to dilution procedures. With a basis on theoretical analysis and computer simulation,
we evaluate the systematic and global bias of the Solomon–Ciuta result and show how it depends on the strength of the concentration
dependence of the solution viscosity (represented by the intrinsic viscosity and the Huggins constant) and the concentration
of the single measured solution. We propose that an estimated Huggins constant can be employed in a corrected Solomon–Ciuta
procedure, which may yield results for the intrinsic viscosity that are even more accurate than those from the Huggins extrapolation.
This gives support and utility to the use of the single-point procedure when the intrinsic viscosity has to be determined
for a unique concentration. We also pinpoint specific circumstances where the dilution–extrapolation procedures should be
preferred.
Nanomaterials hold promise as multifunctional diagnostic and therapeutic agents. However, the effective application of nanomaterials is hampered by limited understanding and control over their interactions with complex biological systems. When a nanomaterial enters a physiological environment, it rapidly adsorbs proteins forming what is known as the protein 'corona'. The protein corona alters the size and interfacial composition of a nanomaterial, giving it a biological identity that is distinct from its synthetic identity. The biological identity determines the physiological response including signalling, kinetics, transport, accumulation, and toxicity. The structure and composition of the protein corona depends on the synthetic identity of the nanomaterial (size, shape, and composition), the nature of the physiological environment (blood, interstitial fluid, cell cytoplasm, etc.), and the duration of exposure. In this critical review, we discuss the formation of the protein corona, its structure and composition, and its influence on the physiological response. We also present an 'adsorbome' of 125 plasma proteins that are known to associate with nanomaterials. We further describe how the protein corona is related to the synthetic identity of a nanomaterial, and highlight efforts to control protein-nanomaterial interactions. We conclude by discussing gaps in the understanding of protein-nanomaterial interactions along with strategies to fill them (167 references).
Hydrated proteins undergo a transition in the deeply supercooled regime, which is attributed to rapid changes in hydration water and protein structural dynamics. Here, we investigate the nanoscale stress-relaxation in hydrated lysozyme proteins stimulated and probed by X-ray Photon Correlation Spectroscopy (XPCS). This approach allows us to access the nanoscale dynamics in the deeply supercooled regime (T = 180 K), which is typically not accessible through equilibrium methods. The observed stimulated dynamic response is attributed to collective stress-relaxation as the system transitions from a jammed granular state to an elastically driven regime. The relaxation time constants exhibit Arrhenius temperature dependence upon cooling with a minimum in the Kohlrausch-Williams-Watts exponent at T = 227 K. The observed minimum is attributed to an increase in dynamical heterogeneity, which coincides with enhanced fluctuations observed in the two-time correlation functions and a maximum in the dynamic susceptibility quantified by the normalized variance χT. The amplification of fluctuations is consistent with previous studies of hydrated proteins, which indicate the key role of density and enthalpy fluctuations in hydration water. Our study provides new insights into X-ray stimulated stress-relaxation and the underlying mechanisms behind spatiotemporal fluctuations in biological granular materials.
The protein corona spontaneously develops and evolves on the surface of nanoscale materials when they are exposed to biological environments, altering their physiochemical properties and affecting their subsequent interactions with biosystems. In this Review, we provide an overview of the current state of protein corona research in nanomedicine. We next discuss remaining challenges in the research methodology and characterization of the protein corona that slow the development of nanoparticle therapeutics and diagnostics, and we address how artificial intelligence can advance protein corona research as a complement to experimental research efforts. We then review emerging opportunities provided by the protein corona to address major issues in healthcare and environmental sciences. This Review details how mechanistic insights into nanoparticle protein corona formation can broadly address unmet clinical and environmental needs, as well as enhance the safety and efficacy of nanobiotechnology products.
Silica nanoparticles present an enormous potential as controlled drug delivery systems with high selectivity towards diseased cells. This application is directly related to the phenomenon of protein corona, characterized by the spontaneous adsorption of proteins on the nanoparticle surface, which is not fully understood. Here, we report an investigation on the influence of pH, ionic strength and temperature on the thermodynamics of interaction of bovine serum albumin protein (BSA) with non-functionalized silica nanoparticles (SiO2NPs). Complementary, we also investigated the ability of polyethylene glycol (PEG) and zwitterionic sulfobetaine (SBS) surface-modified nanoparticles to prevent the adsorption of BSA (protein negatively charged at physiological pH) and lysozyme (protein positively charged at physiological pH). We showed that BSA interaction with SiO2NPs is enthalpically governed. On the other hand, functionalization of silica nanoparticles with PEG and SBS completely prevented BSA adsorption. However, these functionalized nanoparticles presented a negative zeta potential and were not able to suppress lysozyme anchoring due to strong nanoparticle-protein electrostatic attraction. Due to the similarity of BSA with Human Serum Albumin, this investigation bears a resemblance to processes involved in the phenomenon of protein corona in human blood, producing information that is relevant for the future biomedical use of functionalized nanoparticles.
The polycation/polyanion based coacervate platform for therapeutic protein delivery finds difficulty in their colloidal stability under physiological environment. To overcome this issue, PEGylation could be a versatile strategy to enhance colloidal stability. Herein, a poly(ethylene arginyl aspartate diglyceride) (PEAD) polycation was synthesized, and a series of methoxy polyethylene glycols (mPEG) including mPEG350, mPEG750 and mPEG2000 were attached with PEAD polycation to obtain mPEG350-PEAD, mPEG750-PEAD and mPEG2000-PEAD respectively. The PEAD and mPEGylated PEADs were complexed with heparin (HEP) to fabricate coacervate (Coa) and coacersome (mP_Coa) particles respectively. The colloidal stability of Coa and mP_Coa coacersomes has been investigated by dynamic light scattering (DLS) and microscopic techniques. The vascular endothelial growth factor 165 (VEGF-165) was encapsulated in the Coa or mP_Coa particles and administered to human umbilical vein endothelial cells (HUVECs) to induce a tubular network formation in vitro. All the polycations are highly biocompatible and exhibit more than 94 % of VEGF-165 loading efficiency. An effect of mPEG chain length on colloidal stability of mP_Coa and in vitro tubular formation ability of HUVECs has been investigated.
The dynamics of concentrated suspensions of the eye-lens protein alpha crystallin have been measured using x-ray photon correlation spectroscopy. Measurements were made at wave vectors corresponding to the first peak in the hard-sphere structure factor and volume fractions close to the critical volume fraction for the glass transition. Langevin dynamics simulations were also performed in parallel to the experiments. The intermediate scattering function f(q,τ) could be fit using a stretched exponential decay for both experiments and numerical simulations. The measured relaxation times show good agreement with simulations for polydisperse hard-sphere colloids.
Aim:
To study freeze-drying of silica nanoparticles (SiO2NPs) in order to find suitable conditions to produce lyophilized powders with no aggregation after resuspension and storage.
Methods:
SiO2NPs were synthesized using a Stöber-based procedure, and characterized by scanning electron microscopy, dynamic light scattering and nitrogen adsorption/desorption isotherms. SiO2NPs hydrodynamic diameters were compared prior and after freeze-drying in the presence/absence of carbohydrate protectants.
Results:
Glucose was found to be the most suitable protectant against the detrimental effects of lyophilization. The minimum concentration of carbohydrate required to effectively protect SiO2NPs from aggregation during freeze-drying is influenced by the nanoparticle's size and texture. Negligible aggregation was observed during storage.
Conclusion:
Carbohydrates can be used during SiO2NPs freeze-drying process to obtain redispersable solids that maintain original sizes without residual aggregation.
REFLECTED light from a coherent light source such as a laser shows a graininess known as speckle. In general, a speckle pattern is produced whenever randomly distributed regions of a material introduce different phase shifts into the scattering of coherent incident light. If the arrangement of the regions evolves with time, the speckle pattern will also change. Observation of the intensity fluctuations at a single point in the speckle pattern provides a direct measure of the time correlation function of the inhomogeneity. This leads to a technique1,2, alternatively called light-beating spectroscopy, dynamic light scattering or intensity fluctuation spectroscopy, which has been widely used with visible light to study processes such as critical fluctuations near phase transitions in fluids and the diffusion of particles in liquids. But it is not possible to study processes involving length scales less than about 200 nm, or those in opaque materials, using visible light. If intensity fluctuation spectroscopy could be carried out using coherent X-rays, however, one could probe the dynamics of processes involving atomic length scales in a wide range of materials. Here we show that by using a high-brilliance X-ray source it should be possible to perform this type of measurement using radiation of wavelength ~0.15nm. Specifically, we have observed a speckle pattern in the diffraction of coherent X-rays from randomly arranged antiphase domains in a single crystal of the binary alloy Cu3Au.
X Ray Photon Correlation Spectroscopy, XPCS, is a novel technique developed for the study of slow dynamics in condensed matter. The principle of this technique is based on the time variations of the speckle pattern originated by the scattering of coherent light from a material with spatial inhomogeneities. Although laser photon correlation spectroscopy was long established, XPCS has only been possible with the advent of new synchrotron radiation X-ray sources that can provide sufficient coherent flux. The results from these techniques and future perspectives in the field of polymer science are discussed here.
Zwitterionic polymers and poly(ethylene glycol) (PEG) have been reported as promising nonfouling materials and strong surface hydration has been proposed as a significant contributor to the nonfouling mechanism. Better understanding of the similarity and difference between these two types of materials in terms of hydration and protein interaction will benefit the design of new and effective nonfouling materials. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied for in situ and real time assessment of the surface hydration of the sulfobetaine methacrylate (SBMA) and oligo(ethylene glycol) methacrylate (OEGMA) polymer brushes, denoted as pSBMA and pOEGMA, in contact with proteins. Whereas a majority of strongly hydrogen-bonded water was observed at both pSBMA and pOEGMA surfaces, upon contact with proteins, the surface hydration of pSBMA remained unaffected, but the water ordering at the pOEGMA surface was disturbed. The effects of free sulfobetaine, free PEG chains with two different molecular weights, and PEG coated gold nanoparticles on the surface hydration of proteins were investigated. The results indicated that free sulfobetaine could strengthen the protein hydration layer, but free PEG chains greatly disrupt the protein hydration layer and likely directly interact with the protein molecules. In contrast to free PEG, the PEG chains anchored on the nanoparticles behave similarly to the pOEGMA surface and could induce strong hydrogen-bonding of the water molecules at the protein surfaces.
Nanoparticles possess unique properties beyond that of classical materials, and while these properties can be used for designing a dedicated functionality, they may also pose a problem to living organisms, to human health and the environment. The specific primary routes by which nanoparticles may interact with the human body include inhalation, injection, ingestion and application to the skin. Independent of the entry route, the particles inevitably encounter a complex physiological fluid populated with e.g. proteins, vitamins, lipids and salts/ions. Different consequences of such an encounter may include formation of a surface-bound protein layer, particle dissolution or aggregation, which are expected to have a crucial impact on cellular interaction. Understanding cellular responses to nanoparticle interactions starts with understanding particle behavior in physiological fluids. Nanoparticles are now available in practically any size, shape and functionalization, to promote distinct optical, magnetic, and physico-chemical properties, making the prediction of their behavior, in physiological fluids, not a trivial task. Characterization has therefore become of paramount importance. In this review, we give an overview about the diversity of physiological fluids as well as present an inventory of the most relevant experimental techniques used to study plasmonic nanoparticles.
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A protein corona is formed at the surface of nanoparticles in the presence of biological fluids, masking the surface properties of the particle and complicating the relationship between chemical functionality and biological effects. We present here a series of zwitterionic NPs of variable hydrophobicity that do not form coronas at moderate levels of serum protein and do not form hard coronas at physiological serum concentrations. These particles provide platforms to evaluate nanobiological behavior such as cell uptake and hemolysis dictated directly by chemical motifs at the nanoparticle surface.
It is well known that nanomaterials properties and applications are dependent on size, shape and morphology of these structures. Among nanomaterials, silver nanoparticles (AgNPs) have attracted attention since they present considerably versatile properties, such as variable surface area to volume ratio, which is very useful for many biomedical and technological applications. Within this scenario, small nanoparticle aggregates can have their properties reduced due to the increased size and alterations in their shape/morphology. In this work, silver nanoparticles aggregation was studied through the chemical reduction of silver nitrate in presence of sodium borohydride (reducing agent) and sodium citrate (capping agent). By changing the amount of reducing agent along the reaction, unaggregated and partially aggregated samples were obtained and characterized by UV-Vis, zeta-potential and SAXS techniques. pH was measured in every step of the reaction in order to correlate these results with the ones obtained from structural techniques. The addition of the reducing agent first causes the reduction of Ag+ to silver nanoparticles. For higher concentrations of sodium borohydrate, the average AgNPs size is increased and NPs aggregation is observed. It was found that zeta potential and pH values have a strong influence on AgNPs formation, since reducing agent addition can induce the partial removal of citrate weakly associated on the AgNPs surface and increase the ionic strength of the solution, promoting partial aggregation of the particles. This aggregation state was duly identified by coupling SAXS, zeta potential and pH measurements. In addition, SAXS technique showed that the aggregates formed along the process are elongated-like particles due to the exponential decay evidenced through SAXS curves.
The study of protein corona formation on nanoparticles (NPs) represents an actual main issue in colloidal, biomedical and toxicological sciences. However, little is known about the influence of polymer shells on the formation and time evolution of protein corona onto functionalized NPs. Therefore, silica-poly(ethylene glycol) core-shell nanohybrids (SNPs@PEG) with different polymer molecular weights (MW) were synthesized and exhaustively characterized. Bovine serum albumin (BSA) at different concentrations (0.1-6wt%) was used as model protein to study protein corona formation and time evolution. For pristine SNPs and SNPs@PEG (MW=350g/mol), zeta potential at different incubation times show a dynamical evolution of the nanoparticle-protein corona. Oppositely, for SNPs@PEG with MW≥2000g/mol a significant suppression of corona formation and time evolution was observed. Furthermore, AFM investigations suggest a different orientation (side-chain or perpendicular) and penetration depth of BSA toward PEGylated surfaces depending on the polymer length which may explain differences in protein corona evolution.
The search for understanding the interactions of nanosized materials with living organisms is leading to the rapid development of key applications, including improved drug delivery by targeting nanoparticles, and resolution of the potential threat of nanotechnological devices to organisms and the environment. Unless they are specifically designed to avoid it, nanoparticles in contact with biological fluids are rapidly covered by a selected group of biomolecules to form a corona that interacts with biological systems. Here we review the basic concept of the nanoparticle corona and its structure and composition, and highlight how the properties of the corona may be linked to its biological impacts. We conclude with a critical assessment of the key problems that need to be resolved in the near future.
A system of chemical reactions has been developed which permits the controlled growth of spherical silica particles of uniform size by means of hydrolysis of alkyl silicates and subsequent condensation of silicic acid in alcoholic solutions. Ammonia is used as a morphological catalyst. Particle sizes obtained in suspension range from less than 0.05 μ to 2 μ in diameter.
We extend the work of Stober, Fink and Bohn (J. Colloid Interfac Sci. 26 (1968) 62) to establish the ranges of reagent concentrations which result in the precipitation of monodisperse silica particles from ethanol solutions containing ammonia, water, and tetraethyl orthosilica. A correlation is presented which can be used to predict final particle sizes over concentrations of 0.1-0.5 M TEOS, 0.5–17.0 M H2O and 0.5-3 M NH3. The maximum particle size achievable for these conditions at 25°C is ⋍ 800 nm and near the maximum, monodispersity is difficult to maintain. The effects of reaction temperature are studied and particle size distributions are analyzed. A seeded growth technique for preparing larger particles and/or increasing solids mass fraction up to a theoretical limit of ⋍ 24 wt% of silica is described.
Nanoparticles are an essential component in the emerging field of nanomedical imaging and therapy. When deployed in vivo, these materials are typically protected from the immune system by polyethylene glycol (PEG). A wide variety of strategies to coat and characterize nanoparticles with PEG has established important trends on PEG size, shape, density, loading level, molecular weight, charge and purification. Strategies to incorporate targeting ligands are also prevalent. This article presents a background to investigators new to stealth nanoparticles, and suggests some key considerations needed prior to designing a nanoparticle PEGylation protocol and characterizing the performance features of the product.
It is now known that nanoparticles, when exposed to biological fluid, become coated with proteins and other biomolecules to form a 'protein corona'. Recent systematic studies have identified various proteins that can make up this corona, but these nanoparticle-protein interactions are still poorly understood, and quantitative studies to characterize them are few in number. Here, we have quantitatively analysed the adsorption of human serum albumin onto small (10-20 nm in diameter) polymer-coated FePt and CdSe/ZnS nanoparticles by using fluorescence correlation spectroscopy. The protein corona forms a monolayer with a thickness of 3.3 nm. Proteins bind to the negatively charged nanoparticles with micromolar affinity, and time-resolved fluorescence quenching experiments show that they reside on the particle for approximately 100 s. These new findings deepen our quantitative understanding of the protein corona, which is of utmost importance in the safe application of nanoscale objects in living organisms.