Biomolecular Corona Research Articles

The lipid composition of few layers graphene and graphene oxide biomolecular corona

Among all the biomolecules that form the corona of nanomaterials, lipids are somehow the less studied and characterized. In the present paper, we report on the first investigation of the lipid content of the biomolecular corona formed around few layers graphene (FLG) and graphene oxide (GO) nanomaterials. Our work revealed that significantly different lipid signatures are associated with each of the two materials and that the biocorona formed in human plasma around FLG and GO is enriched or depleted in specific lipids. We also investigated if the presence of individual lipids in the corona, like sphingomyelins and triacylglycerols, might play a role in the uptake of these two materials in a blood-brain barrier cellular model.

Biomolecular Corona Associated with Nanostructures: The Potentially Disruptive Role of Raman Microscopy

AbstractWhen nanostructures and other materials are exposed to biological fluids, they are immediately covered by a layer of biological molecules, which is typically referred to as a “biomolecular corona” (BC). This represents the first component of a material that interacts with biological systems, so characterizing the composition and the dynamic evolution of BC is essential for predicting the interactions of materials and living organisms. This review provides an analysis of current BC characterization techniques, with particular attention to nanostructures involved in biomedical applications. The influence on cell–nanostructure interactions is assessed and the advantages and limitations of each technique are discussed and compared. An in‐depth analysis of Raman microscopy, a relatively unexploited tool with great potential in the characterization of BC, is then conducted. Raman microscopy can be used to analyze a vast amount of specimens without the need for staining, and can provide analysis on a spatial scale of hundreds of nanometers: it may thus represent a potentially disruptive tool for the characterization of BC, as it overcomes many of the limitations posed by current techniques.

Size Dependence Unveiling the Adsorption Interaction of High-Density Lipoprotein Particles with PEGylated Gold Nanoparticles in Biomolecular Corona Formation.

Apolipoproteins have been often found to be highly enriched in the serum protein coronas produced on various engineered nanoparticles (NPs), which is also known to greatly influence the behaviors of protein corona NPs in the biological systems. As most of the apolipoproteins in blood are associated with lipoproteins, it suggests the active involvement of lipoproteins in the formation of biomolecular coronas on NPs. However, the interactions of lipoprotein complexes with NPs in the corona formation have been rarely understood. In this study, to obtain insights into the interactions, the formation of biomolecular coronas of high-density lipoproteins (HDLs) on the PEGylated gold NPs (PEG-AuNPs) of various sizes (20-150 nm dia.) was investigated as a model system. The results of this study revealed a noticeable size dependence, which is a drastic increase in the affinity of HDL for larger NPs and thus less-curved NP surfaces. For example, only a few HDLs per NP, which correspond to 5% surface coverage, were found to constitute the hard coronas of HDLs on 20 nm PEG-AuNPs, whereas 73% surface coverage was assessed for larger 150 nm PEG-AuNPs. However, the relative affinities of HDL and apolipoprotein A-1 (APOA1) examined in competition with human serum albumin exhibited the opposite size dependences, which suggests that the adsorption of HDLs is not driven by the constituent protein, APOA1. In fact, the total strength of non-covalent intermolecular interactions between a HDL particle and a NP relies on the physical contact between the two particles, which thus depends on the varying curvatures of spherical NPs in this case. Therefore, it was concluded that it is whole HDL complex that interacts with the spherical PEG-AuNPs in the initial stage of adsorption toward biomolecular coronas, which is unveiled by the distinct size dependence observed in this study.

Internalisation and Biological Activity of Nucleic Acids Delivering Cell-Penetrating Peptide Nanoparticles Is Controlled by the Biomolecular Corona.

Nucleic acid molecules can be transferred into cells to alter gene expression and, thus, alleviate certain pathological conditions. Cell-penetrating peptides (CPPs) are vectors that can be used for transfecting nucleic acids as well as many other compounds. CPPs associate nucleic acids non-covalently, forming stable nanoparticles and providing efficient transfection of cells in vitro. However, in vivo, expected efficiency is achieved only in rare cases. One of the reasons for this discrepancy is the formation of protein corona around nanoparticles, once they are exposed to a biological environment, e.g., blood stream. In this study, we compared protein corona of CPP-nucleic acid nanoparticles formed in the presence of bovine, murine and human serum. We used Western blot and mass-spectrometry to identify the major constituents of protein corona forming around nanoparticles, showing that proteins involved in transport, haemostasis and complement system are its major components. We investigated physical features of nanoparticles and measured their biological efficiency in splice-correction assay. We showed that protein corona constituents might alter the fate of nanoparticles in vivo, e.g., by subjecting them to phagocytosis. We demonstrated that composition of protein corona of nanoparticles is species-specific that leads to dissimilar transfection efficiency and should be considered while developing delivery systems for nucleic acids.

Interaction of Differently Sized, Shaped, and Functionalized Silver and Gold Nanoparticles with Glycosylated versus Nonglycosylated Transferrin.

Exposure of nanomaterials (NMs) to biological medium results in their direct interaction with biomolecules and the formation of a dynamic biomolecular layer known as the biomolecular corona. Despite numerous published data on nano-biointeractions, the role of protein glycosylation in the formation, characteristics, and fate of such nano-biocomplexes has been almost completely neglected, although most serum proteins are glycosylated. This study aimed to systematically investigate the differences in interaction of metallic NPs with glycosylated vs nonglycosylated transferrin. To reach this aim, we compared interaction mechanisms between differently sized, shaped, and surface-functionalized silver NMs and gold NMs to commercially available human transferrin (TRF), a glycosylated protein, and to its nonglycosylated recombinant form (ngTRF). Bovine serum albumin (BSA) was also included in the study for comparative purposes. Characterization of NMs was performed using transmission electron microscopy and dynamic and electrophoretic light scattering techniques. Fluorescence quenching and circular dichroism methods were used to evaluate protein binding constants on the nanosurface and conformational changes after the protein-NM interactions, respectively. Competitive binding of TRF, ngTRF, and BSA to the surface of different NMs was evaluated by separating them after extraction from protein corona by gel electrophoresis following quantification with a commercial protein assay. The results showed that the binding strength between NMs and transferrin and the changes in the secondary protein structure largely depend not only on NM physicochemical properties but also on the protein glycosylation status. Data gained by this study highlight the relevance of protein glycosylation for all future design, development, and efficacy and safety assessment of NMs.

Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood.

Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.

Isolation and Quantification of miRNA from the Biomolecular Corona on Mesoporous Silica Nanoparticles.

To understand the factors that control the formation of the biomolecular corona, a systematic study of the adsorption of several miRNAs shown to be important in prostate cancer on amine-functionalized mesoporous silica nanoparticles (MSN-NH2) has been performed. Process parameters including miRNA type, nanoparticle concentration, incubation temperature and incubation time were investigated, as well as the potential competition for adsorption between different miRNA molecules. The influence of proteins and particle PEGylation on miRNA adsorption were also explored. We found that low particle concentrations and physiological temperature both led to increased miRNA adsorption. Adsorption of miRNA was also higher when proteins were present in the same solution; reducing or preventing protein adsorption by PEGylating the MSNs hindered adsorption. Finally, the amount of miRNA adsorbed from human serum by MSN-NH2 was compared to a commercial miRNA purification kit (TaqMan®, Life Technologies, Carlsbad, CA, USA). MSN-NH2 adsorbed six times as much miRNA as the commercial kit, demonstrating higher sensitivity to subtle up- and downregulation of circulating miRNA in the blood of patients.

Probing the Intracellular Bio-Nano Interface in Different Cell Lines with Gold Nanostars.

Gold nanostars are a versatile plasmonic nanomaterial with many applications in bioanalysis. Their interactions with animal cells of three different cell lines are studied here at the molecular and ultrastructural level at an early stage of endolysosomal processing. Using the gold nanostars themselves as substrate for surface-enhanced Raman scattering, their protein corona and the molecules in the endolysosomal environment were characterized. Localization, morphology, and size of the nanostar aggregates in the endolysosomal compartment of the cells were probed by cryo soft-X-ray nanotomography. The processing of the nanostars by macrophages of cell line J774 differed greatly from that in the fibroblast cell line 3T3 and in the epithelial cell line HCT-116, and the structure and composition of the biomolecular corona was found to resemble that of spherical gold nanoparticles in the same cells. Data obtained with gold nanostars of varied morphology indicate that the biomolecular interactions at the surface in vivo are influenced by the spike length, with increased interaction with hydrophobic groups of proteins and lipids for longer spike lengths, and independent of the cell line. The results will support optimized nanostar synthesis and delivery for sensing, imaging, and theranostics.

Inherited and acquired corona of coronavirus in the host: Inspiration from the biomolecular corona of nanoparticles

The family of coronavirus are named for their crown shape. Encoded by the genetic material inherited from the coronavirus itself, this intrinsic well-known “viral corona” is considered an “inherited corona”. After contact with mucosa or the entrance into the host, bare coronaviruses can become covered by a group of dissolved biomolecules to form one or multiple layers of biomolecules. The layers acquired from the surrounding environment are named the “acquired corona”. We highlight here the possible role of the acquired corona in the pathogenesis of coronaviruses, which will generate fresh insight into the nature of various coronavirus-host interactions.

Are Smaller Nanoparticles Always Better? Understanding the Biological Effect of Size-Dependent Silver Nanoparticle Aggregation Under Biorelevant Conditions.

PurposeSilver nanoparticles (AgNPs) are one of the most commonly investigated nanomaterials, especially due to their biomedical applications. However, their excellent cytotoxic and antimicrobial activity is often compromised in biological media due to nanoparticle aggregation. In this work, the aggregation behavior and the related biological activity of three different samples of citrate capped silver nanoparticles, with mean diameters of 10, 20, and 50 nm, respectively, were examined.MethodsFollowing nanoparticle synthesis and characterization with transmission electron microscopy, their aggregation behavior under various pH values, NaCl, glucose, and glutamine concentrations, furthermore in cell culture medium components such as Dulbecco’s Modified Eagle’s Medium and fetal bovine serum, was assessed through dynamic light scattering and ultraviolet-visible spectroscopy.ResultsThe results indicated that acidic pH and physiological electrolyte content universally induce micron-scale aggregation, which can be mediated by biomolecular corona formation. Remarkably, larger particles demonstrated higher resistance against external influences than smaller counterparts. In vitro cytotoxicity and antimicrobial assays were performed by treating cells with nanoparticulate aggregates in differing stages of aggregation.ConclusionOur results revealed a profound association between colloidal stability and toxicity of AgNPs, as extreme aggregation led to the complete loss of biological activity. The higher degree of aggregation resistance observed for larger particles had a significant impact on the in vitro toxicity, since such samples retained more of their activity against microbes and mammalian cells. These findings lead to the conclusion that aiming for the smallest possible nanoparticles might not be the best course of action, despite the general standpoint of the relevant literature.

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

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

NanoBio Interfaces: Opportunities and Challenges of Biomolecular Corona

NanoBio Interfaces: Opportunities and Challenges of Biomolecular Corona

Nanoscale characterization of the biomolecular corona by cryo-electron microscopy, cryo-electron tomography, and image simulation

The biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.

Biomolecular corona formation on CuO nanoparticles in plant xylem fluid

CuO nanoparticles selectively remove proteins from Cucurbita pepo (pumpkin) xylem to form a nanoparticle corona.

Disentangling Biomolecular Corona Interactions With Cell Receptors and Implications for Targeting of Nanomedicines.

Nanoparticles are promising tools for nanomedicine in a wide array of therapeutic and diagnostic applications. Yet, despite the advances in the biomedical applications of nanomaterials, relatively few nanomedicines made it to the clinics. The formation of the biomolecular corona on the surface of nanoparticles has been known as one of the challenges toward successful targeting of nanomedicines. This adsorbed protein layer can mask targeting moieties and creates a new biological identity that critically affects the subsequent biological interactions of nanomedicines with cells. Extensive studies have been directed toward understanding the characteristics of this layer of biomolecules and its implications for nanomedicine outcomes at cell and organism levels, yet several aspects are still poorly understood. One aspect that still requires further insights is how the biomolecular corona interacts with and is “read” by the cellular machinery. Within this context, this review is focused on the current understanding of the interactions of the biomolecular corona with cell receptors. First, we address the importance and the role of receptors in the uptake of nanoparticles. Second, we discuss the recent advances and techniques in characterizing and identifying biomolecular corona-receptor interactions. Additionally, we present how we can exploit the knowledge of corona-cell receptor interactions to discover novel receptors for targeting of nanocarriers. Finally, we conclude this review with an outlook on possible future perspectives in the field. A better understanding of the first interactions of nanomaterials with cells, and -in particular -the receptors interacting with the biomolecular corona and involved in nanoparticle uptake, will help for the successful design of nanomedicines for targeted delivery.

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

The formation of the biomolecular corona represents a crucial factor in controlling the biological interactions and trafficking of nanomaterials. In this context, the availability of key epitopes exposed on the surface of the corona, and able to engage the biological machinery, is important to define the biological fate of the material. While the full biomolecular corona composition can be investigated by conventional bottom-up proteomics, the assessment of the spatial orientation of proteins in the corona in a high-throughput fashion is still challenging. In this work, we show that labeling corona proteins with isobaric tags in their native conditions and analyzing the MS/MS spectra of tryptic peptides allow an easy and high-throughput assessment of the inner/outer orientation of the corresponding proteins in the original corona. We put our results in the context of what is currently known of the protein corona of graphene-based nanomaterials. Our conclusions are in line with previous data and were confirmed by in silico calculations.

Nanoplasmonic Paper-Based Platform for General Screening of Biomacromolecules.

Hygiene assessment in industrial and clinical environments is crucial in the prevention of health risks. Current technologies for routine cleanliness evaluation rely on the detection of specific biomolecules, thus requiring more than one test for broad-range screening. Herein, the modulation of the catalytic activity of gold nanoparticles (AuNPs) by biomacromolecules was employed to develop a nanoplasmonic platform for general hygiene screening. AuNPs were immobilized on cellulose paper by simple adsorption. When ferricyanide was dispensed onto the paper, the AuNPs catalysed the ferricyanide’s dissociation, releasing free cyanide ions that dissolved them. The AuNP dissolution produced an intense colour shift detectable with the naked eye. When biomacromolecules (e.g., proteins and polysaccharides) were present, they spontaneously attached to AuNPs, forming a biomolecular corona (biocorona), reducing their catalytic activity until complete suppression when the NPs were fully covered by molecules. The concentration-dependent decrease in the catalytic activity was here used to quantify biomacromolecules and complex samples such as milk, eggs, soy sauce and yeast extract (in 20 min), with detection limits comparable to those of standard methods, i.e., 0.25 µg mL−1 for albumin. This nano-enabled technology may be applied as a broad-range (unspecific) alert system for inexpensive cleanliness evaluation, with potential applications in sensitive sectors including productive industries and hospitals.

The Protein Corona Conundrum: Exploring the Advantages and Drawbacks of its Presence around Amphiphilic Nanoparticles.

The success of targeted drug delivery systems still requires a detailed understanding about the biological consequences of self-developed biomolecular coronas around them, since this is the surface that interacts with living cells. Herein, we report the behavior of carbohydrate-decorated amphiphilic nanoparticles in a plasma environment with regard to the formation and biological consequences of the protein corona. Naked amphiphilic nanoparticles were produced through the self-assembly of azido-PEO900-docosanoate molecules, and the coupling of N-acetylglucosamine via click chemistry enabled the fabrication of the corresponding bioactive glyco-nanostructures. Light scattering measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography-mass spectrometry, and the Pierce BCA protein assay all confirmed the presence of protein coronas around the self-assembled nanoparticles, regardless of the presence of the sugar residues, although it reduces the amount of adsorbed proteins. The protein coronas were formed mainly by human serum albumin, complement proteins, apolipoproteins, immunoglobulins, and proteins involved in the coagulation cascade (fibrinogen and prothrombin). While the presence of these protein coronas significantly reduced cellular uptake of the amphiphilic assemblies, they also notably reduced the cytotoxic and hemolytic effects that result from the contact of the nanoparticles with living cells. Accordingly, we highlight that protein coronas should not always be treated as artifacts that have to be avoided because they can also provide beneficial effects.

Analysis of the Human Plasma Proteome Using Multi-Nanoparticle Protein Corona for Detection of Alzheimer's Disease.

As the population affected by Alzheimer's disease (AD) grows, so does the need for a noninvasive and accurate diagnostic tool. Current research reveals that AD pathogenesis begins as early as decades before clinical symptoms. The unique properties of nanoparticles (NPs) may be exploited to develop noninvasive diagnostics for early detection of AD. After exposure of NPs to biological fluids, the NP surface is altered by an unbiased but selective and reproducible adsorption of biomolecules commonly referred to as the biomolecular corona or protein corona (PC). The discovery that the plasma proteome may be differentially altered during health and disease leads to the concept of disease-specific PCs. Herein, the disease-specific PCs formed around NPs in a multi-NPs platform are employed to successfully identify subtle changes in plasma protein patterns and detect AD (>92% specificity and ≈100% sensitivity). Similar discrimination power is achieved using banked plasma samples from a cohort of patients several years prior to their diagnosis with AD. With the nanoplatform's analytic ability to analyze pathological proteomic changes into a disease-specific identifier, this promising, noninvasive technology with implications for early detection and intervention could benefit not only patients with AD but other diseases as well.

Person-Specific Biomolecular Coronas Modulate Nanoparticle Interactions with Immune Cells in Human Blood.

When nanoparticles interact with human blood, a multitude of plasma components adsorb onto the surface of the nanoparticles, forming a biomolecular corona. Corona composition is known to be influenced by the chemical composition of nanoparticles. In contrast, the possible effects of variations in the human blood proteome between healthy individuals on the formation of the corona and its subsequent interactions with immune cells in blood are unknown. Herein, we prepared and examined a matrix of 11 particles (including organic and inorganic particles of three sizes and five surface chemistries) and plasma samples from 23 healthy donors to form donor-specific biomolecular coronas (personalized coronas) and investigated the impact of the personalized coronas on particle interactions with immune cells in human blood. Among the particles examined, poly(ethylene glycol) (PEG)-coated mesoporous silica (MS) particles, irrespective of particle size (800, 450, or 100 nm in diameter), displayed the widest range (up to 60-fold difference) of donor-dependent variance in immune cell association. In contrast, PEG particles (after MS core removal) of 860, 518, or 133 nm in diameter displayed consistent stealth behavior (negligible cell association), irrespective of plasma donor. For comparison, clinically relevant PEGylated doxorubicin-encapsulated liposomes (Doxil) (74 nm in diameter) showed significant variance in association with monocytes and B cells across all plasma donors studied. An in-depth proteomic analysis of each biomolecular corona studied was performed, and the results were compared against the nanoparticle-blood cell association results, with individual variance in the proteome driving differential association with specific immune cell types. We identified key immunoglobulin and complement proteins that explicitly enriched or depleted within the corona and which strongly correlated with the cell association pattern observed across the 23 donors. This study demonstrates how plasma variance in healthy individuals significantly influences the blood immune cell interactions of nanoparticles.