In Situ Investigation on the Protein Corona Formation of Quantum Dots by Using Fluorescence Resonance Energy Transfer

A fundamental understanding of nanoparticle–protein corona and its interactions with biological systems is essential for future application of engineered nanomaterials. In this work, fluorescence resonance energy transfer (FRET) is employed for studying the protein adsorption behavior of nanoparticles. The adsorption of human serum albumin (HSA) onto the surface of InP@ZnS quantum dots (QDs) with different chirality (d ‐ and l ‐penicillamine) shows strong discernible differences in the binding behaviors including affinity and adsorption orientation that are obtained upon quantitative analysis of FRET data. Circular dichroism spectroscopy further confirms the differences in the conformational changes of HSA upon interaction with d ‐ and l ‐chiral QD surfaces. Consequently, the formed protein corona on chiral surfaces may affect their following biological interactions, such as possible protein exchange with serum proteins plasma as well as cellular interactions. These results vividly illustrate the potential of the FRET method as a simple yet versatile platform for quantitatively investigating biological interactions of nanoparticles.     

Formation of a Monolayer Protein Corona around Polystyrene Nanoparticles and Implications for Nanoparticle Agglomeration

Nanoparticle (NP) interactions with cells and organisms are mediated by a biomolecular adsorption layer, the so‐called “protein corona.” An in‐depth understanding of the corona is a prerequisite to successful and safe application of NPs in biology and medicine. In this work, earlier in situ investigations on small NPs are extended to large polystyrene (PS) NPs of up to 100 nm diameter, using human transferrin (Tf) and human serum albumin (HSA) as model proteins. Direct NP sizing experiments reveal a reversibly bound monolayer protein shell (under saturating conditions) on hydrophilic, carboxyl‐functionalized (PS‐COOH) NPs, as was earlier observed for much smaller NPs. In contrast, protein binding on hydrophobic, sulfated (PS‐OSO₃H) NPs in solvent of low ionic strength is completely irreversible; nevertheless, the thickness of the observed protein corona again corresponds to a protein monolayer. Under conditions of reduced charge repulsion (higher ionic strength), the NPs are colloidally unstable and form large clusters below a certain protein–NP stoichiometric ratio, indicating that the adsorbed proteins induce NP agglomeration. This comprehensive characterization of the persistent protein corona on PS‐OSO₃H NPs by nanoparticle sizing and quantitative fluorescence microscopy/nanoscopy reveals mechanistic aspects of molecular interactions occurring during exposure of NPs to biofluids.     

The Human In Vivo Biomolecule Corona onto PEGylated Liposomes: A Proof‐of‐Concept Clinical Study

The self‐assembled layered adsorption of proteins onto nanoparticle (NP) surfaces, once in contact with biological fluids, is termed the “protein corona” and it is gradually seen as a determinant factor for the overall biological behavior of NPs. Here, the previously unreported in vivo protein corona formed in human systemic circulation is described. The human‐derived protein corona formed onto PEGylated doxorubicin‐encapsulated liposomes (Caelyx) is thoroughly characterized following the recovery of liposomes from the blood circulation of ovarian carcinoma patients. In agreement with previous investigations in mice, the in vivo corona is found to be molecularly richer in comparison to its counterpart ex vivo corona. The intravenously infused liposomes are able to scavenge the blood pool and surface‐capture low‐molecular‐weight, low‐abundance plasma proteins that cannot be detected by conventional plasma proteomic analysis. This study describes the previously elusive or postulated formation of protein corona around nanoparticles in vivo in humans and illustrates that it can potentially be used as a novel tool to analyze the blood circulation proteome.     

Super‐Resolution Microscopy Unveils Dynamic Heterogeneities in Nanoparticle Protein Corona

The adsorption of serum proteins, leading to the formation of a biomolecular corona, is a key determinant of the biological identity of nanoparticles in vivo. Therefore, gaining knowledge on the formation, composition, and temporal evolution of the corona is of utmost importance for the development of nanoparticle‐based therapies. Here, it is shown that the use of super‐resolution optical microscopy enables the imaging of the protein corona on mesoporous silica nanoparticles with single protein sensitivity. Particle‐by‐particle quantification reveals a significant heterogeneity in protein absorption under native conditions. Moreover, the diversity of the corona evolves over time depending on the surface chemistry and degradability of the particles. This paper investigates the consequences of protein adsorption for specific cell targeting by antibody‐functionalized nanoparticles providing a detailed understanding of corona‐activity relations. The methodology is widely applicable to a variety of nanostructures and complements the existing ensemble approaches for protein corona study.     

The Impact of Protein Corona Formation on the Macrophage Cellular Uptake and Biodistribution of Spherical Nucleic Acids

The effect of serum protein adsorption on the biological fate of Spherical Nucleic Acids (SNAs) is investigated. Through a proteomic analysis, it is shown that G‐quadruplexes templated on the surface of a gold nanoparticle in the form of SNAs mediate the formation of a protein corona that is rich in complement proteins relative to SNAs composed of poly‐thymine (poly‐T) DNA. Cellular uptake studies show that complement receptors on macrophage cells recognize the SNA protein corona, facilitating their internalization, and causing G‐rich SNAs to accumulate in the liver and spleen more than poly‐T SNAs in vivo. These results support the conclusion that nucleic acid sequence and architecture can mediate nanoparticle–biomolecule interactions and alter their cellular uptake and biodistribution properties and illustrate that nucleic acid sequence is an important parameter in the design of SNA therapeutics.     

The Influence of Nanoparticle Shape on Protein Corona Formation

Nanoparticles have become an important utility in many areas of medical treatment such as targeted drug and treatment delivery as well as imaging and diagnostics. These advances require a complete understanding of nanoparticles' fate once placed in the body. Upon exposure to blood, proteins adsorb onto the nanoparticles surface and form a protein corona, which determines the particles' biological fate. This study reports on the protein corona formation from blood serum and plasma on spherical and rod‐shaped nanoparticles. These two types of mesoporous silica nanoparticles have identical chemistry, porosity, surface potential, and size in the y‐dimension, one being a sphere and the other a rod shape. The results show a significantly larger amount of protein attaching from both plasma and serum on the rod‐like particles compared to the spheres. Interrogation of the protein corona by liquid chromatography–mass spectrometry reveals shape‐dependent differences in the adsorption of immunoglobulins and albumin proteins from both plasma and serum. This study points to the need for taking nanoparticle shape into consideration because it can have a significant impact on the fate and therapeutic potential of nanoparticles when placed in the body.     

Polymeric Nanoparticles with Neglectable Protein Corona

The current understanding of nanoparticle–protein interactions indicates that they rapidly adsorb proteins upon introduction into a living organism. The formed protein corona determines thereafter identity and fate of nanoparticles in the body. The present study evaluates the protein affinity of three core‐crosslinked polymeric nanoparticles with long circulation times, differing in the hydrophilic polymer material forming the particle surface, namely poly(N‐2‐hydroxypropylmethacrylamide) (pHPMA), polysarcosine (pSar), and poly(ethylene glycol) (PEG). This includes the nanotherapeutic CPC634, which is currently in clinical phase II evaluation. To investigate possible protein corona formation, the nanoparticles are incubated in human blood plasma and separated by asymmetrical flow field‐flow fractionation (AF4). Notably, light scattering shows no detectable differences in particle size or polydispersity upon incubation with plasma for all nanoparticles, while in gel electrophoresis, minor amounts of proteins can be detected in the particle fraction. Label‐free quantitative proteomics is additionally applied to analyze and quantify the composition of the proteins. It proves that some proteins are enriched, but their concentration is significantly less than one protein per particle. Thus, most of the nanoparticles are not associated with any proteins. Therefore, this work underlines that polymeric nanoparticles can be synthesized, for which a protein corona formation does not take place.     

Probing Adsorption Behaviors of BSA onto Chiral Surfaces of Nanoparticles

Chiral properties of nanoscale materials are of importance as they dominate interactions with proteins in physiological environments; however, they have rarely been investigated. In this study, a systematic investigation is conducted for the adsorption behaviors of bovine serum albumin (BSA) onto the chiral surfaces of gold nanoparticles (AuNPs), involving multiple techniques and molecular dynamic (MD) simulation. The adsorption of BSA onto both L‐ and D‐chiral surfaces of AuNPs shows discernible differences involving thermodynamics, adsorption orientation, exposed charges, and affinity. As a powerful supplement, MD simulation provides a molecular‐level understanding of protein adsorption onto nanochiral surfaces. Salt bridge interaction is proposed as a major driving force at protein–nanochiral interface interaction. The spatial distribution features of functional groups (? COO^?, ? NH₃⁺, and ? CH₃) of chiral molecules on the nanosurface play a key role in the formation and location of salt bridges, which determine the BSA adsorption orientation and binding strength to chiral surfaces. Sequentially, BSA corona coated on nanochiral surfaces affects their uptake by cells. The results enhance the understanding of protein corona, which are important for biological effects of nanochirality in living organisms.     

Effects of Nanoparticle Electrostatics and Protein–Protein Interactions on Corona Formation: Conformation and Hydrodynamics

All‐atom molecular dynamics simulations of plasma proteins (human serum albumin, fibrinogen, immunoglobulin gamma‐1 chain‐C, complement C3, and apolipoprotein A‐I) adsorbed onto 10 nm sized cationic, anionic, and neutral polystyrene (PS) particles in water are performed. In simulations of a single protein with a PS particle, proteins eventually bind to all PS particles, regardless of particle charge, in agreement with experiments showing the binding between anionic proteins and particles, which is further confirmed by calculating the binding free energies from umbrella sampling simulations. Simulations of mixtures of multiple proteins and a PS particle show the formation of the protein layer on the surface via the adsorption competition between proteins, which influences the binding affinity and structure of adsorbed proteins. In particular, diffusivities are much higher for proteins bound to the particle surface or to the boundary of the protein layer than for those bound to both the particle surface and other proteins, indicating the dependence of protein mobility on their positions in the layer. These findings help to explain in detail experimental observations regarding the replacement of plasma proteins at the early stage of corona formation and the difference in the binding strength of proteins in inner and outer protein‐layers.     

Quantum Dot–Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins

Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Förster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (≈6.5 kDa) histidine‐tagged albumin‐binding domain‐derived affinity proteins (ADAPTs) can efficiently self‐assemble to zwitterionic ligand–coated quantum dots (QDs). These ADAPT–QD conjugates are significantly smaller than QD‐conjugates based on IgG, Fab', or single‐domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum‐containing samples using time‐gated Tb‐to‐QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 × 10^?12m (≈8 ng mL^?1) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.     

Polyvalent Display and Packing of Peptides and Proteins on Semiconductor Quantum Dots: Predicted Versus Experimental Results

Quantum dots (QDs) are loaded with a series of peptides and proteins of increasing size, including a <20 residue peptide, myoglobin, mCherry, and maltose binding protein, which together cover a range of masses from <2.2 to ≈44 kDa. Conjugation to the surface of dihydrolipoic acid‐functionalized QDs is facilitated by polyhistidine metal affinity coordination. Increasing ratios of dye‐labeled peptides and proteins are self‐assembled to the QDs and then the bioconjugates are separated and analyzed using agarose gel electrophoresis. Fluorescent visualization of both conjugated and unbound species allows determination of an experimentally derived maximum loading number. Molecular modeling utilizing crystallographic coordinates or space‐filling structures of the peptides and proteins also allow the predicted maximum loadings to the QDs to be estimated. Comparison of the two sets of results provides insight into the nature of the QD surface and reflects the important role played by the nanoparticle's hydrophilic solubilizing surface ligands. It is found that for the larger protein molecules steric hindrance is the major packing constraint. In contrast, for the smaller peptides, the number of available QD binding sites is the principal determinant. These results can contribute towards an overall understanding of how to engineer designer bioconjugates for both QDs and other nanoparticle materials.     

Protein Corona Mediated Uptake and Cytotoxicity of Silver Nanoparticles in Mouse Embryonic Fibroblast

Medical applications of nanoparticles (NPs) require understanding of their interactions with living systems in order to control their physiological response, such as cellular uptake and cytotoxicity. When NPs are exposed to biological fluids, the adsorption of extracellular proteins on the surface of NPs, creating the so‐called protein corona, can critically affect their interactions with cells. Here, the effect of surface coating of silver nanoparticles (AgNPs) on the adsorption of serum proteins (SPs) and its consequence on cellular uptake and cytotoxicity in mouse embryonic fibroblasts are shown. In particular, citrate‐capped AgNPs are internalized by cells and show a time‐ and dose‐dependent toxicity, while the passivation of the NP surface with an oligo(ethylene glycol) (OEG)‐alkanethiol drastically reduces their uptake and cytotoxicity. The exposure to growth media containing SPs reveals that citrate‐capped AgNPs are promptly coated and stabilized by proteins, while the AgNPs resulting from capping with the OEG‐alkanethiol are more resistant to adsorption of proteins onto their surface. Using NIH‐3T3 cultured in serum‐free, the key role of the adsorption of SPs onto surface of NPs is shown as only AgNPs with a preformed protein corona can be internalized by the cells and, consequently, carry out their inherent cytotoxic activity.     

Nanoparticles Interacting with Proteins and Cells: A Systematic Study of Protein Surface Charge Effects

Despite intense research on biological and biomedical applications of nanoparticles, our understanding of their basic interactions with the biological environment is still incomplete. Systematic variation of the physicochemical properties of the nanoparticles is widely seen as a promising strategy to obtain further insights. In view of the key role of the protein adsorption layer forming on nanoparticles in contact with biofluids, we systematically varied the surface charge of proteins adsorbing onto nanoparticles by chemical modification so as to examine the effect of Coulomb forces in modulating nano‐bio interactions. We chose human serum albumin (HSA) as a model protein and ultra‐small, negatively charged fluorescent gold nanoclusters (AuNCs) as model nanoparticles. By using fluorescence and CD spectroscopies, we measured binding affinities and structural changes upon binding of the HSA variants. The strengths of the protein‐nanoparticle interactions were found to change substantially upon modifying the surface charge of HSA. Furthermore, by using inductively coupled plasma optical emission spectroscopy, confocal fluorescence microscopy, scanning transmission electron microscopy and cell viability assays, we observed that cellular interactions of the AuNCs, including their adherence to cell membranes, uptake efficiency and cytotoxicity, depended markedly on the different surface charges of the HSA variants adsorbed onto the nanoparticles. These results illustrate vividly that the cellular responses to nanoparticle exposure depend on the specific properties of the proteins that adsorb onto nanoparticles from biofluids.     

Hardening of the nanoparticle-protein corona in metal (Au, Ag) and oxide (Fe3O4, CoO, and CeO2) nanoparticles

The surface modifications of metal and metal oxide nanoparticles with sizes ranging from 7 to 20 nm dispersed in commonly used cell culture medium supplemented with serum are investigated. All the tested nanoparticles adsorb proteins onto their surface, thereby forming a protein corona through a dynamic process evolving towards an irreversible coating (hard protein corona). Despite the fact that the studied nanomaterials have similar characteristics of hydrophobicity and surface charge, different temporal patterns of the protein corona formation are observed that can be considered a fingerprint for nanoparticle identification. Some of the biological and toxicological implications of the formation of the nanoparticle-protein corona are studied using the human monocytic cell line THP-1 exposed to cobalt oxide nanoparticles. Results show that production of reactive oxygen species is decreased if the nanoparticles are preincubated for 48 h with serum.     

CdTe nanoparticles display tropism to core histones and histone-rich cell organelles

The disclosure of the mechanisms of nanoparticle interaction with specific intracellular targets represents one of the key tasks in nanobiology. Unmodified luminescent semiconductor nanoparticles, or quantum dots (QDs), are capable of a strikingly rapid accumulation in the nuclei and nucleoli of living human cells, driven by processes of yet unknown nature. Here, it is hypothesized that such a strong tropism of QDs could be mediated by charge-related properties of the macromolecules presented in the nuclear compartments. As the complex microenvironment encountered by the QDs in the nuclei and nucleoli of live cells is primarily presented by proteins and other biopolymers, such as DNA and RNA, the model of human phagocytic cell line THP1, nuclear lysates, purified protein, and nucleic acid solutions is utilized to investigate the interactions of the QDs with these most abundant classes of intranuclear macromolecules. Using a combination of advanced technological approaches, including live cell confocal microscopy, fluorescent lifetime imaging (FLIM), spectroscopic methods, and zeta potential measurements, it is demonstrated that unmodified CdTe QDs preferentially bind to the positively charged core histone proteins as opposed to the DNA or RNA, resulting in a dramatic shift off the absorption band, and a red shift and decrease in the pholuminescence (PL) intensity of the QDs. FLIM imaging of the QDs demonstrates an increased formation of QD/protein aggregates in the presence of core histones, with a resulting significant reduction in the PL lifetime. FLIM technology for the first time reveals that the localization of negatively charged QDs to their ultimate nuclear and nucleolar destinations dramatically affects the QDs' photoluminescence lifetimes, and offers thereby a sensitive readout for physical interactions between QDs and their intracellular macromolecular targets. These findings strongly suggest that charge-mediated QD/histone interactions could provide the basis for QD nuclear localization downstream of intracellular transport mechanisms.     

Insights into Stabilization of a Viral Protein Cage in Templating Complex Nanoarchitectures: Roles of Disulfide Bonds

As a typical protein nanostructure, virus‐based nanoparticle (VNP) of simian virus 40 (SV40), which is composed of pentamers of the major capsid protein of SV40 (VP1), has been successfully employed in guiding the assembly of different nanoparticles (NPs) into predesigned nanostructures with considerable stability. However, the stabilization mechanism of SV40 VNP remains unclear. Here, the importance of inter‐pentamer disulfide bonds between cysteines in the stabilization of quantum dot (QD)‐containing VNPs (VNP‐QDs) is comprehensively investigated by constructing a series of VP1 mutants of cysteine to serine. Although the presence of a QD core can greatly enhance the assembly and stability of SV40 VNPs, disulfide bonds are vital to stability of VNP‐QDs. Cysteine at position 9 (C9) and C104 contribute most of the disulfide bonds and play essential roles in determining the stability of SV40 VNPs as templates to guide assembly of complex nanoarchitectures. These results provide insightful clues to understanding the robustness of SV40 VNPs in organizing suprastructures of inorganic NPs. It is expected that these findings will help guide the future design and construction of protein‐based functional nanostructures.     

Highly Efficient Inverted Structural Quantum Dot Solar Cells

Highly efficient PbS colloidal quantum dot (QD) solar cells based on an inverted structure have been missing for a long time. The bottlenecks are the construction of an effective p–n heterojunction at the illumination side with smooth band alignment and the absence of serious interface carrier recombination. Here, solution‐processed nickel oxide (NiO) as the p‐type layer and lead sulfide (PbS) QDs with iodide ligand as the n‐type layer are explored to build a p–n heterojunction at the illumination side. The large depletion region in the QD layer at the illumination side leads to high photocurrent. Interface carrier recombination at the interface is effectively prohibited by inserting a layer of slightly doped p‐type QDs with 1,2‐ethanedithiol as ligands, leading to improved voltage of the device. Based on this graded device structure design, the efficiency of inverted structural heterojunction PbS QD solar cells is improved to 9.7%, one time higher than the highest efficiency achieved before.     

接枝层厚度对聚(N-异丙基丙烯酰胺)改性表面与蛋白质相互作用的影响

采用表面引发原子转移自由基聚合(ATRP)的方法制备了聚(N-异丙基丙烯酰胺)(PNIPAAm)改性表面,通过改变聚合时间进而调控表面接枝层厚度.水接触角和蛋白质吸附测试发现PNIPAAm改性表面的浸润性和对蛋白质吸附都具有一定的温度响应性,且随着PNIPAAm接枝层厚度的增加,该温度响应性有一定程度的增强.同时,纤维蛋白原和溶菌酶的吸附测试表明PNIPAAm改性表面对尺寸不同的蛋白质具有不同的温度响应性.     

微波等离子体在不锈钢上制备抵抗蛋白质吸附表面

本文利用微波低温等离子体表面改性技术,对医用316L型不锈钢进行改性以增强其表面抵抗蛋白质粘附的能力,提高生物相容性。经过X射线光电子能谱和衰减全反射傅立叶变换红外光谱的分析和表征,发现沉积的涂层为类PEG结构,表面主要聚集大量—CH2—CH2—O键,并且氧原子和碳原子在金属和有机物界面层之间形成共价键结合。血浆蛋白吸附试验显示,与改性前相比,等离子体沉积在不锈钢表面的类PEG涂层可以有效抵抗蛋白质粘附。     

Quantum dot size dependent J-V characteristics in heterojunction ZnO/PbS quantum dot solar cells

The current-voltage (J-V) characteristics of ZnO/PbS quantum dot (QD) solar cells show a QD size-dependent behavior resulting from a Schottky junction that forms at the back metal electrode opposing the desirable diode formed between the ZnO and PbS QD layers. We study a QD size-dependent roll-over effect that refers to the saturation of photocurrent in forward bias and crossover effect which occurs when the light and dark J-V curves intersect. We model the J-V characteristics with a main diode formed between the n-type ZnO nanocrystal (NC) layer and p-type PbS QD layer in series with a leaky Schottky-diode formed between PbS QD layer and metal contact. We show how the characteristics of the two diodes depend on QD size, metal work function, and PbS QD layer thickness, and we discuss how the presence of the back diode complicates finding an optimal layer thickness. Finally, we present Kelvin probe measurements to determine the Fermi level of the QD layers and discuss band alignment, Fermi-level pinning, and the V(oc) within these devices.