Nanoparticle Corona Research Articles

Mimicking the transit of nanoparticles through the body: when the path determines properties at the destination

To test if the transit of a nanoparticle across different organs in the body affects its properties at the final destination, we devised an in vitro system that mimics the passage of nanoparticles from the blood to the lung to the brain and, occasionally, back to the blood. Each of the three media provided a unique corona to the nanoparticles, comprising mainly secreted, blood-specific proteins in the plasma and membrane proteins in the lung and the brain. The hard corona changed during the transit of the nanoparticle between different organs. Cellular uptake, antibacterial activity, and drug release kinetics were analyzed to assess the difference in the biological response to nanoparticles depending on the transit route. Drug release assays most consistently demonstrated the effects of the history of the nanoparticle passage across different media on its present properties. Both the uptake analysis and antibacterial assays showed that the key proteins for these two processes in the brain replace their analogues from the lung, but are ineffective in replacing the blood plasma content from the nanoparticle surface. Notwithstanding that some routes can erase the history of the passage of the nanoparticle across different organs, the route traversed by it mostly affects the properties it displays at the destination. A nanoparticle sent to a circular journey through an array of media can have different properties upon its return to the point of origin. History is an essential determinant of the properties of nanoparticles in biological milieus, the memory of which is inscribed in the nanoparticle corona.

Pay Attention to Biological Nanoparticles when Studying the Protein Corona on Nanomedicines.

The protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines-with a particular focus on liposomes-when using the traditional isolation methods that separate according to size and density.

Pay Attention to Biological Nanoparticles when Studying the Protein Corona on Nanomedicines

AbstractThe protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines—with a particular focus on liposomes—when using the traditional isolation methods that separate according to size and density.

Targeting Tumorigenicity of Breast Cancer Stem Cells Using SAHA/Wnt-b Catenin Antagonist Loaded Onto Protein Corona of Gold Nanoparticles.

BackgroundAmong various theories for the origin of cancer, the “stemness phenotype model” suggests a dynamic feature for tumor cells in which non-cancer stem cells (non-CSCs) can inter-convert to CSCs. Differentiation with histone-deacetylase inhibitor, vorinostat (SAHA), can induce stem cells to differentiate as well as enforces non-CSCs to reprogram to CSCs. To avoid this undesirable effect, one can block the Wnt-βcatenin pathway. Thus, a dual delivery system of SAHA and a Wnt-βcatenin blocker will be beneficial in the induction of differentiation of CSCs. Protein corona (PC) formation in nanoparticle has a biologic milieu, and despite all problematic properties, it can be employed as a medium for dual loading of the drugs.Materials and MethodsWe prepared sphere gold nanoparticles (GNPs) with human plasma protein corona loaded with SAHA as differentiating agent and PKF118-310 (PKF) as a Wnt-βcatenin antagonist. The MCF7 breast cancer stem cells were treated with NPs and the viability and differentiation were evaluated by Western blotting and sphere formation assay.ResultsWe found that both drugs loaded onto corona-capped GNPs had significant cytotoxicity in comparison to bare GNP-corona. Data demonstrated an increase in stem cell population and upregulation of mesenchymal marker, Snail by SAHA-loaded GNPs treatment; however, the combination of PKF loaded GNPs along with SAHA-loaded GNPs resulted in a reduction of stem cell populations and Snail marker. We have shown that in MCF7 and its CSCs simultaneous treatment with SAHA and PKF118-310 induced differentiation and inhibition of Snail induction.ConclusionOur study reveals the PC-coated GNPs as a biocompatible career for both hydrophilic (PKF) and hydrophobic (SAHA) agents which can decrease breast cancer stem cell populations along with reduced stemness state regression.

Synergistic Effects of Physicochemical Parameters on Bio-Fabrication of Mint Silver Nanoparticles: Structural Evaluation and Action Against HCT116 Colon Cancer Cells.

BackgroundPhysicochemical parameters such as temperature, pH, the concentration of the AgNO3 and ratio of reactants act synergistically to influence the reaction kinetics, molecular mechanics, enzymatic catalysis and protein conformations that aid to affect the size, shape and biochemical corona of nanoparticles. The present study was performed to investigate the influence of reaction parameters on the bio-fabrication of silver nanoparticles (AgNPs) by using Mentha arvensis and to determine their potential to control the proliferation of colon cancer cells'.MethodsPlant-mediated method was used for the bio-fabrication and stabilization of AgNPs. Reaction parameters were arranged, and surface plasmon resonance (SPR) bands of AgNPs were collected by using a UV-Visible spectrophotometer. NPs were characterized structurally and optically by using SEM, AFM, EDX and DLS techniques. AgNPs and plant aqueous extract were tested against HCT116 colon cancer cells by using SRB assay, Annexin V assay and cell cycle analysis.ResultsSpectrophotometric comparison of various reaction conditions manifested that 5 mM of AgNO3, 60 °C in an acidic pH and a mixing ratio of 1:9 of plant extract and AgNO3, respectively, are the optimized conditions for AgNP synthesis. Structural evaluation by SEM, AFM and particle size analysis confirmed that the NPs are <100 nm and are anisotropic, spherical, triangular and moderately dispersed in the colloidal mixture. SRB assay expressed biomass-stabilized AgNPs as effective cytotoxic particles against HCT116 colon cancer cells, and the IC50 was measured at 1.7 µg/mL. Annexin V apoptosis assay further confirmed that the AgNPs induce apoptosis in a dose-dependent manner. Experimental evidence manifested that the AgNPs arrest cell cycle and expressed entrapment of a greater number of cells in the Sub-G1 phase, further verifying the anticancer abilities of AgNPs.ConclusionThese findings explain the synergistic effects of physicochemical parameters to optimize the phytosynthesis of biocompatible AgNPs to overcome the limitations of conventional chemotherapeutic treatments of colon cancer cells.

On‐Chip Electrical Monitoring of Real‐Time “Soft” and “Hard” Protein Corona Formation on Carbon Nanoparticles

AbstractWhen nanoparticles encounter a biological fluid, proteins, lipids, and other biomolecules adsorb on the nanoscale surface consequently leading to the evolution of a protein shell or “corona.” The corona formed is dynamic in nature and depends on the “synthetic identity” of the NPs, ultimately affecting their biological response. In this paper, an integrated microfluidic platform coupled with an electrical resistance measurement setup is developed to monitor and investigate the real‐time formation of a biomolecular corona of carbon nanoparticles. “Soft” and “hard” corona formation stages are effectively discriminated based on their nanoscale surface chemistries when combined with a time‐frequency tool known as wavelet transform and machine‐learning techniques. Additionally, the corona and its composition are studied using different techniques such as dynamic light scattering, nanoparticle tracking analysis, zeta potential, excitation–emission profiles, 1D sodium dodecyl polyacrylamide gel electrophoresis and subsequently, liquid chromatography‐mass spectrometry analysis. The dynamic setup can eventually be used as a valuable tool for screening of any nanoparticles formulations with distinct surface chemistries for the purpose of reduced protein adsorption/weaker protein corona formation and consequently enhance the success of targeted drug delivery.

Recent Advances in Understanding the Protein Corona of Nanoparticles and in the Formulation of "Stealthy" Nanomaterials.

In the last decades, the staggering progress in nanotechnology brought around a wide and heterogeneous range of nanoparticle-based platforms for the diagnosis and treatment of many diseases. Most of these systems are designed to be administered intravenously. This administration route allows the nanoparticles (NPs) to widely distribute in the body and reach deep organs without invasive techniques. When these nanovectors encounter the biological environment of systemic circulation, a dynamic interplay occurs between the circulating proteins and the NPs, themselves. The set of proteins that bind to the NP surface is referred to as the protein corona (PC). PC has a critical role in making the particles easily recognized by the innate immune system, causing their quick clearance by phagocytic cells located in organs such as the lungs, liver, and spleen. For the same reason, PC defines the immunogenicity of NPs by priming the immune response to them and, ultimately, their immunological toxicity. Furthermore, the protein corona can cause the physical destabilization and agglomeration of particles. These problems induced to consider the PC only as a biological barrier to overcome in order to achieve efficient NP-based targeting. This review will discuss the latest advances in the characterization of PC, development of stealthy NP formulations, as well as the manipulation and employment of PC as an alternative resource for prolonging NP half-life, as well as its use in diagnostic applications.

Nanoparticle corona artefacts derived from specimen preparation of particle suspensions

Progress in the implementation of nanoparticles for therapeutic applications will accelerate with an improved understanding of the interface between nanoparticle surfaces and the media they are dispersed in. We examine this interface by analytical scanning transmission electron microscopy and show that incorrect specimen preparation or analysis can induce an artefactual, nanoscale, calcium phosphate-rich, amorphous coating on nanoparticles dispersed in cell culture media. We report that this ionic coating can be induced on five different types of nanoparticles (Au, BaTiO3, ZnO, TiO2 and Fe2O3) when specimen preparation causes a significant rise in pH above physiological levels. Such a pH change reduces ionic solubility in the suspending media to permit precipitation of calcium phosphate. Finally, we demonstrate that there is no indication of a calcium-phosphorus-rich coating on BaTiO3 nanoparticles suspended in culture media when prepared without alteration of the pH of the suspending media and imaged by cryo-STEM. Therefore we recommend that future reports utilising nanoparticles dispersed in cell culture media monitor and report the pH of suspensions during sample preparation.

The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles.

Multivalent nanoparticle binding to cells can be of picomolar avidity making such interactions almost as intense as those seen with antibodies. However, reducing nanoparticle design exclusively to avidity optimization by the choice of ligand and its surface density does not sufficiently account for controlling and understanding cell-particle interactions. Cell uptake, for example, is of paramount significance for a plethora of biomedical applications and does not exclusively depend on the intensity of multivalency. In this study, it is shown that the mobility of ligands tethered to particle surfaces has a substantial impact on particle fate upon binding. Nanoparticles carrying angiotensin-II tethered to highly mobile 5 kDa long poly(ethylene glycol) (PEG) chains separated by ligand-free 2 kDa short PEG chains show a superior accumulation in angiotensin-II receptor type 1 positive cells. In contrast, when ligand mobility is constrained by densely packing the nanoparticle surface with 5 kDa PEG chains only, cell uptake decreases by 50%. Remarkably, irrespective of ligand mobility and density both particle types have similar EC50 values in the 1-3 × 10-9 m range. These findings demonstrate that ligand mobility on the nanoparticle corona is an indispensable attribute to be considered in particle design to achieve optimal cell uptake via multivalent interactions.

Metabolomic analyses of the bio-corona formed on TiO2 nanoparticles incubated with plant leaf tissues

BackgroundThe surface of a nanoparticle adsorbs molecules from its surroundings with a specific affinity determined by the chemical and physical properties of the nanomaterial. When a nanoparticle is exposed to a biological system, the adsorbed molecules form a dynamic and specific surface layer called a bio-corona. The present study aimed to identify the metabolites that form the bio-corona around anatase TiO2 nanoparticles incubated with leaves of the model plant Arabidopsis thaliana.ResultsWe used an untargeted metabolomics approach and compared the metabolites isolated from wild-type plants with plants deficient in a class of polyphenolic compounds called flavonoids.ConclusionsThese analyses showed that TiO2 nanoparticle coronas are enriched for flavonoids and lipids and that these metabolite classes compete with each other for binding the nanoparticle surface.

Tuning liposome composition to modulate corona formation in human serum and cellular uptake

Nano-sized objects such as liposomes are modified by adsorption of biomolecules in biological fluids. The resulting corona critically changes nanoparticle behavior at cellular level. A better control of corona composition could allow to modulate uptake by cells. Within this context, in this work, liposomes of different charge were prepared by mixing negatively charged and zwitterionic lipids to different ratios. The series obtained was used as a model system with tailored surface properties to modulate corona composition and determine the effects on liposome interactions with cells. Uptake efficiency and uptake kinetics of the different liposomes were determined by flow cytometry and fluorescence imaging. Particular care was taken in optimizing the methods to isolate the corona forming in human serum to prevent liposome agglomeration and to exclude residual free proteins, which could confuse the results. Thanks to the optimized methods, mass spectrometry of replicate corona isolations showed excellent reproducibility and this allowed semi-quantitative analysis to determine for each formulation the most abundant proteins in the corona. The results showed that by changing the fraction of zwitterionic and charged lipids in the bilayer, the amount and identity of the most abundant proteins adsorbed from serum differed. Interestingly, the formulations also showed very different uptake kinetics. Similar approaches can be used to tune lipid composition in a systematic way in order to obtain formulations with the desired corona and cell uptake behavior. Statement of significanceLiposomes and other nano-sized objects when introduced in biological fluids are known to adsorb biomolecules forming the so-called nanoparticle corona. This layer strongly affects the subsequent interactions of liposomes with cells. Here, by tuning lipid composition in a systematic way, a series of liposomes with tailored surface properties has been prepared to modulate the corona forming in human serum. Liposomes with very different cellular uptake kinetics have been obtained and their corona was identified in order to determine the most enriched proteins on the different formulations. By combining corona composition and uptake kinetics candidate corona proteins associated with reduced or increased uptake by cells can be identified and the liposome formulation can be tuned to obtain the desired uptake behavior.

A method to measure the denatured proteins in the corona of nanoparticles based on the specific adsorption of Hsp90ab1.

The protein corona influences and determines the biological function of nanoparticles (NPs) in vivo. Analysis and understanding of the activities of proteins in coronas are crucial for nanobiology and nanomedicine research. Misfolded proteins in the corona of NPs theoretically exist, and a protein denaturation-related cellular response might occur in this process as well as in related diseases. The exact evaluation of protein denaturation in the corona is valuable to assess the bioactivities of NPs. Here, we found that the level of adsorbed heat shock protein 90 kDa α class B member 1 (Hsp90ab1) by the denatured protein in iron-cobalt-nickel alloy NPs (FeCoNi NPs) and iron oxide NPs (Fe3O4 NPs) was correlated with circular dichroism (CD) analysis and 1-anilinonaphthalene-8-sulfonate (ANS) analysis. The content of Hsp90ab1 in the corona could be easily analysed by western blotting (WB). Further analysis suggested that the method could precisely show the time-dependent protein denaturation on Fe3O4 NPs, as well as the influence of the size and the surface modification. More importantly, this method could be applied to other proteins, like lysozyme, other than albumin. Based on the results and the correlation analysis, incubation and detection of Hsp90ab1 in the NP-corona complex can be used as a new and feasible method to evaluate protein denaturation induced by NPs.

The biomolecule corona of lipid nanoparticles contains circulating cell-free DNA.

The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely investigated over the last decade. Characterisation of the protein composition of the 'biomolecule corona' has dominated research efforts, whereas other classes of biomolecules, such as nucleic acids, have received no interest. Scarce, speculative statements exist in the literature about the presence of nucleic acids in the biomolecule corona, with no previous studies attempting to describe the contribution of genomic content to the blood-derived NP corona. Herein, we provide the first experimental evidence of the interaction of circulating cell-free DNA (cfDNA) with lipid-based NPs upon their incubation with human plasma samples, obtained from healthy volunteers and ovarian carcinoma patients. Our results also demonstrate an increased amount of detectable cfDNA in patients with cancer. Proteomic analysis of the same biomolecule coronas revealed the presence of histone proteins, suggesting an indirect, nucleosome-mediated NP-cfDNA interaction. The finding of cfDNA as part of the NP corona, offers a previously unreported new scope regarding the chemical composition of the 'biomolecule corona' and opens up new possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids.

Revealing the pulmonary surfactant corona on silica nanoparticles by cryo-transmission electron microscopy.

When inhaled, nanoparticles (NPs) deposit in alveoli and transit through the pulmonary surfactant (PS), a biofluid made of proteins and phospholipid vesicles. They form a corona reflecting the PS–nanomaterial interaction. Since the corona determines directly the NPs' biological fate, the question of its nature and structure is central. Here, we report on the corona architecture formed after incubation of positive or negative silica particles with Curosurf®, a biomimetic pulmonary surfactant of porcine origin. Using optical, electron and cryo-electron microscopy (cryo-TEM), we determine the pulmonary surfactant corona structure at different scales of observation. Contrary to common belief, the PS corona is not only constituted by phospholipid bilayers surrounding NPs but also by multiple hybrid structures derived from NP–vesicle interaction. Statistical analysis of cryo-TEM images provides interesting highlights about the nature of the corona depending on the particle charge. The influence of Curosurf® pre- or post-treatment is also investigated and demonstrates the need for protocol standardization.

Relating the composition and interface interactions in the hard corona of gold nanoparticles to the induced response mechanisms in living cells.

Understanding the formation of the intracellular protein corona of nanoparticles is essential for a wide range of bio- and nanomedical applications. The innermost layer of the protein corona, the hard corona, directly interacts with the nanoparticle surface, and by shielding the surface, it has a deterministic effect on the intracellular processing of the nanoparticle. Here, we combine a direct qualitative analysis of the hard corona composition of gold nanoparticles with a detailed structural characterization of the molecules in their interaction with the nanoparticle surface and relate both to the effects they have on the ultrastructure of living cells and the processing of the gold nanoparticles. Cells from the cell lines HCT-116 and A549 were incubated with 30 nm citrate-stabilized gold nanoparticles and with their aggregates in different culture media. The combined results of mass spectrometry based proteomics, cryo soft X-ray nanotomography and surface-enhanced Raman scattering experiments together revealed different uptake mechanisms in the two cell lines and distinct levels of induced cellular stress when incubation conditions were varied. The data indicate that the different incubation conditions lead to changes in the nanoparticle processing via different protein-nanoparticle interfacial interactions. Specifically, they suggest that the protein-nanoparticle surface interactions depend mainly on the surface properties of the gold nanoparticles, that is, the ζ-potential and the resulting changes in the hydrophilicity of the nanoparticle surface, and are largely independent of the cell line, the uptake mechanism and intracellular processing, or the extent of the induced cellular stress.

Influence of the physicochemical features of TiO2 nanoparticles on the formation of a protein corona and impact on cytotoxicity.

Due to their unique properties TiO2 nanoparticles are widely used. The adverse effects they may elicit are usually studied in relation to their physicochemical features. However, a factor is often neglected: the influence of the protein corona formed around nanoparticles upon contact with biological media. Indeed, although it is acknowledged that it can strongly influence nanoparticle toxicity, it is not systematically considered. The aim of this study was to characterize the formation of the protein corona of TiO2 nanoparticles as a function of the main nanoparticle properties and investigate potential relationship with the cytotoxicity nanoparticles induce in vitro in human lung cells. To that purpose, five TiO2 nanoparticles differing in size, shape, agglomeration state and surface charge were incubated in cell culture media (DMEM or RPMI supplemented with 10% fetal bovine serum) and the amount and profile of adsorbed proteins on each type of nanoparticle were compared to their toxicological profile. While nanoparticle size and surface charge were found to be determinant factors for protein corona formation, no clear impact of the shape and agglomeration state was observed. Furthermore, no clear relationship was evidenced between the protein corona of the nanoparticles and the adverse effect they elicited.

Effect of a protein corona on the fibrinogen induced cellular oxidative stress of gold nanoparticles.

The protein corona of nanoparticles is becoming a tool to understand the relation between intrinsic physicochemical properties and extrinsic biological behaviour. A diverse set of characterisation techniques such as transmission electron microscopy, mass spectrometry, dynamic light scattering, zeta-potential measurements and surface enhanced Raman spectroscopy are used to determine the composition and physical properties of the soft and hard corona formed around spherical gold nanoparticles. Advanced characterisation via small angle X-ray scattering and cryo-transmission electron microscopy suggests the presence of a thin hard corona of a few nm on 50 nm gold nanoparticles. The protein corona does not cause changes in cell viability, but inhibits the generation of reactive oxygen species in microglia cells. When a pre-incubated layer of fibrinogen, a protein with high affinity for the gold surface, is present around the nanoparticles before a protein corona is formed in bovine serum, the cellular uptake is significantly increased with an inhibition of ROS. The selective sequential pre-formation of protein complexes prior to incubation in cells is demonstrated as a viable method to alter the biological behaviour of nanoparticles.

Proteomic analysis of intracellular protein corona of nanoparticles elucidates nano-trafficking network and nano-bio interactions.

The merits of nanomedicines are significantly impacted by the surrounding biological environment. Similar to the protein corona generated on the surface of nanoparticles in the circulation system, the intracellular protein corona (IPC) might be formed on nanoparticles when transported inside the cells. However, little is known currently about the formation of IPC and its possible biological influence.Methods: Caco-2 cells, a classical epithelial cell line, were cultured in Transwell plates to form a monolayer. Gold nanoparticles (AuNPs) were prepared as the model nanomedicine due to their excellent stability. Here we focused on identifying IPC formed on the surface of AuNPs during cell transport. The nanoparticles in the basolateral side of the Caco-2 monolayer were collected and analyzed by multiple techniques to verify IPC formation. High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics was utilized to analyze the composition of IPC proteins. In particular, we established a dual-filtration strategy to exclude various interference in IPC identification. Based on the subcellular localization of specific IPC proteins, we elicited the nano-trafficking network of AuNPs. The transport pathways of AuNPs identified by proteomic analysis were also verified by various conventional technologies. Finally, we explored the influence of IPC on the uptake and stress response of endothelium.Results: The existence of IPC was demonstrated on the surface of AuNPs, in which 227 proteins were identified. Among them, 40 proteins were finally ascertained as the specific IPC proteins. The subcellular location analysis indicated that these “specific” IPC proteins could back-track the transport pathways of nanoparticles in the epithelial cell monolayer. According to the subcellular distribution of IPC proteins and co-localization, we discovered a new pathway of nanoparticles from endosomes to secretory vesicles which was dominant during the transcytosis. After employing conventional imageology and pharmacology strategies to verify the result of proteomic analysis, we mapped a comprehensive intracellular transport network. Our study also revealed the merits of IPC analysis, which could readily elucidate the molecular mechanisms of transcytosis. Besides, the IPC proteins increased the uptake and stress response of endothelium, which was likely mediated by extracellular matrix and mitochondrion-related IPC proteins.Conclusion: The comprehensive proteomic analysis of IPC enabled tracing of transport pathways in epithelial cells as well as revealing the biological impact of nanoparticles on endothelium.

Validation of a field deployable reactor for in situ formation of NOM-engineered nanoparticle corona

A realistic exposure of n-TiO2 nanoparticles to river water by using a dialysis bag as a passive reactor: DOM of the river water diffuses inside while n-TiO2 nanoparticles remain inside.

Corona of Thorns: The Surface Chemistry-Mediated Protein Corona Perturbs the Recognition and Immune Response of Macrophages.

The significance of protein coronas on the biological fates of nanoparticles has been widely recognized. Therefore, the alterations on biological effects caused by protein coronas need systemic study and interpretation to design novel safe and efficient nanomedicines. In the present study, we present a comprehensive quantitative analysis of the protein coronas on gold nanorods modifiedwith various surface ligands of different chemical compositions and charges. The design of surface ligands is of utmost importance for the functionalization of nanoparticles, and further, the ligand-induced biological identity determines the fate of nanoparticles in the human body. We found that the surface chemistry influences the composition of the protein corona more profoundly than surface charge. Since the first and most important challenge for administrated nanomedicines is navigating the interaction with macrophages, we further investigated how the surface chemistry-induced specific protein corona affects the phagocytosis and immune responses of macrophages exposed to the corona-nanoparticle complexes. Our results reveal that the protein corona alters the internalization pathways of goldnanorods by macrophages via the interactions of the predominant coronal proteins with specific receptors on the cell membrane. The cytokine secretion profile of macrophages is also highly dependent on the adsorption pattern of the protein corona. The more abundant proteins involved in immune responses, such as acute phase, complement, and tissue leakage proteins, present in the acquired nanoparticle corona, the more macrophage interleukin-1β (IL-1β) released is stimulated. The ligand-protein corona composition-immune response coefficient analysis may serve next-generation nanomedicines with high efficiency and good safety for better clinical translation.