Understanding protein-nanoparticle interactions leading to protein corona formation: In vitro - in vivo correlation study.

Nanoparticles (NPs) in contact with biological fluids form a biomolecular corona through interactions with proteins, lipids, and sugars, acquiring new physicochemical properties. This work explores the interaction between selected proteins (hemoglobin and fetuin-A) that may alter NP circulation time and NPs of different surface charges (neutral, positive, and negative). The interaction with key proteins albumin and transferrin, the two of the most abundant proteins in plasma was also studied. Binding affinity was investigated using quartz crystal microbalance and fluorescence quenching, while circular dichroism assessed potential conformational changes. The data obtained from in vitro experiments were compared to in vivo protein corona data. The results indicate that electrostatic interactions primarily drive protein-NP interactions, and higher binding affinity does not necessarily translate into more significant structural changes. In vitro and single protein-NP studies provide valuable insights that can be correlated with in vivo observations, opening exciting possibilities for future protein corona studies.

Competition of opsonins and dysopsonins on the nanoparticle surface.

The biological behavior and fate of nanoparticles are dependent on their retention time in the blood circulation system. The protein corona components, especially opsonins, and dysopsonins, adsorbed on the nanoparticle surface determine their blood circulation time. The protein corona formation is a dynamic process that involves the competition between different proteins to be adsorbed on the nanoparticles. Therefore, studying how proteins compete and are oriented on the nanoparticle surface is essential. We hypothesized that the presence of opsonins (immunoglobulin (IgG)) might affect the adsorption of dysopsonins (human serum albumin (HSA)) and vice versa. Using the molecular dynamics simulations, we showed that the adsorption of HSA on the GO surface after the IgG adsorption is more probable than the opposite order of adsorption. It was also observed that the higher lateral diffusion of the HSA compared to the IgG helped the system reach a more stable configuration while the initial adsorption of the HSA limits the lateral diffusion of IgG. Therefore, replacing IgG adsorbed on the GO surface with HSA is plausible while the reverse process is less likely to occur. This study revealed that albumin might extend the blood circulation time of GO by replacing opsonins (IgG).

Identification of the Proteins Determining the Blood Circulation Time of Nanoparticles.

The therapeutic efficacy and adverse impacts of nanoparticles (NPs) are strongly dependent on their systemic circulation time. The corona proteins adsorbed on the NPs determine their plasma half-lives, and hence, it is crucial to identify the proteins shortening or extending their circulation time. In this work, the in vivo circulation time and corona composition of superparamagnetic iron oxide nanoparticles (SPIONs) with different surface charges/chemistries were analyzed over time. SPIONs with neutral and positive charges showed the longest and shortest circulation times, respectively. The most striking observation was that corona-coated NPs with similar opsonin/dysopsonin content showed different circulation times, implying these biomolecules are not the only contributing factors. Long-circulating NPs adsorb higher concentrations of osteopontin, lipoprotein lipase, coagulation factor VII, matrix Gla protein, secreted phosphoprotein 24, alpha-2-HS-glycoprotein, and apolipoprotein C-I, while short-circulating NPs adsorb higher amounts of hemoglobin. Therefore, these proteins may be considered to be determining factors governing the NP systemic circulation time.

A Comprehensive Review on Barium Titanate Nanoparticles as a Persuasive Piezoelectric Material for Biomedical Applications: Prospects and Challenges.

Stimulation of cells with electrical cues is an imperative approach to interact with biological systems and has been exploited in clinical practices over a wide range of pathological ailments. This bioelectric interface has been extensively explored with the help of piezoelectric materials, leading to remarkable advancement in the past two decades. Among other members of this fraternity, colloidal perovskite barium titanate (BaTiO3 ) has gained substantial interest due to its noteworthy properties which includes high dielectric constant and excellent ferroelectric properties along with acceptable biocompatibility. Significant progression is witnessed for BaTiO3 nanoparticles (BaTiO3 NPs) as potent candidates for biomedical applications and in wearable bioelectronics, making them a promising personal healthcare platform. The current review highlights the nanostructured piezoelectric bio interface of BaTiO3 NPs in applications comprising drug delivery, tissue engineering, bioimaging, bioelectronics, and wearable devices. Particular attention has been dedicated toward the fabrication routes of BaTiO3 NPs along with different approaches for its surface modifications. This review offers a comprehensive discussion on the utility of BaTiO3 NPs as active devices rather than passive structural unit behaving as carriers for biomolecules. The employment of BaTiO3 NPs presents new scenarios and opportunity in the vast field of nanomedicines for biomedical applications.

Characterization Challenges of Self-Assembled Polymer-SPIONs Nanoparticles: Benefits of Orthogonal Methods.

Size and zeta potential are critical physicochemical properties of nanoparticles (NPs), influencing their biological activity and safety profile. These are essential for further industrial upscale and clinical success. However, the characterization of polydisperse, non-spherical NPs is a challenge for traditional characterization techniques (ex., dynamic light scattering (DLS)). In this paper, superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyvinyl alcohol (PVAL) exhibiting different terminal groups at their surface, either hydroxyl (OH), carboxyl (COOH) or amino (NH2) end groups. Size, zeta potential and concentration were characterized by orthogonal methods, namely, batch DLS, nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), asymmetric flow field flow fractionation (AF4) coupled to multi-angle light scattering (MALS), UV-Visible and online DLS. Finally, coated SPIONs were incubated with albumin, and size changes were monitored by AF4-MALS-UV-DLS. NTA showed the biggest mean sizes, even though DLS PVAL-COOH SPION graphs presented aggregates in the micrometer range. TRPS detected more NPs in suspension than NTA. Finally, AF4-MALS-UV-DLS could successfully resolve the different sizes of the coated SPION suspensions. The results highlight the importance of combining techniques with different principles for NPs characterization. The advantages and limitations of each method are discussed here.

Titanate nanoribbon-based nanobiohybrid for potential applications in regenerative medicine.

Nanoparticles capable of mimicking natural tissues represent a major technological advancement in regenerative medicine. In this pilot study, the development of a new nanohybrid composed of titanate nanoribbons to mimic the extracellular matrix is reported. During the first phase, nanoribbons were synthesized by hydrothermal treatment. Subsequently, titanate nanoribbons were functionalized by heterobifunctional polyethylene-glycol (PEG) to graft type I collagen on their surface. Biological properties of this new nanobiohybrid such as cytotoxicity to cardiac cells and platelet aggregation ability were evaluated. The so-formed nanobiohybrid permits cellular adhesion and proliferation favoring fine cardiac tissue healing and regeneration.

In vivo protein corona on nanoparticles: does the control of all material parameters orient the biological behavior?

Nanomaterials have a huge potential in research fields from nanomedicine to medical devices. However, surface modifications of nanoparticles (NPs) and thus of their physicochemical properties failed to predict their biological behavior. This requires investigating the "missing link" at the nano-bio interface. The protein corona (PC), the set of proteins binding to the NPs surface, plays a critical role in particle recognition by the innate immune system. Still, in vitro incubation offers a limited understanding of biological interactions and fails to explain the in vivo fate. To date, several reports explained the impact of PC in vitro but its applications in the clinical field have been very limited. Furthermore, PC is often considered as a biological barrier reducing the targeting efficiency of nano vehicles. But the protein binding can actually be controlled by altering PC both in vitro and in vivo. Analyzing PC in vivo could accordingly provide a deep understanding of its biological effect and speed up the transfer to clinical applications. This review demonstrates the need for clarifications on the effect of PC in vivo and the control of its behavior by changing its physicochemical properties. It unfolds the recent in vivo developments to understand mechanisms and challenges at the nano-bio interface. Finally, it reports recent advances in the in vivo PC to overcome and control the limitations of the in vitro PC by employing PC as a boosting resource to prolong the NPs half-life, to improve their formulations and thereby to increase its use for biomedical applications.

Anti-Platelet Effect Induced by Iron Oxide Nanoparticles: Correlation with Conformational Change in Fibrinogen.

Iron oxide nanoparticles are developed for various biomedical applications, however, there is limited understanding regarding their effects and toxicity on blood components. The particles traveling in circulation inevitably interact with blood cells and plasma proteins and may interfere with hemostasis. Specifically, this study focuses on the influence of superparamagnetic iron oxide nanoparticles (SPIONs) coated with a biocompatible polymer, polyvinyl alcohol (PVA), on platelet function. Here, engineered SPIONs that are functionalized with various PVA coatings to provide these particles with different surface charges and polymer packing are described. These formulations are assessed for any interference with human platelet functions and coagulation, ex vivo. Positively charged SPIONs induce a significant change in platelet GPIIb-IIIa conformation, indicative of platelet activation at the dose of 500 µg mL-1 . Remarkably, engineered PVA(polyvinyl alcohol)-SPIONs all display a robust dose-dependent anti-platelet effect on platelet aggregation, regardless of the PVA charge and molecular weight. After assessing hypotheses involving SPION-induced steric hindrance in platelet-platelet bridging, as well as protein corona involvement in the antiplatelet effect, the study concludes that the presence of PVA-SPIONs induces fibrinogen conformational change, which correlates with the observed dose-dependent anti-platelet effect.

Taurine-Conjugated Mussel-Inspired Iron Oxide Nanoparticles with an Elongated Shape for Effective Delivery of Doxorubicin into the Tumor Cells.

Multifunctional iron oxide magnetic nanoparticles, among them nanorods, were prepared with a mussel-inspired polydopamine (pDA) surface coating agent for cancer therapeutics. Taurine, a free sulfur-containing ß amino acid, was grafted on the pDA at the iron oxide nanoparticle surface to enhance its biocompatibility and targeted delivery action. Doxorubicin (DOX), an anticancer drug, was loaded on the prepared nanovehicles with an entrapment efficiency of 70.1%. Drug release kinetics were then analyzed using UV-vis and fluorescence spectroscopies, suggesting the pH-responsive behavior of the developed nanovehicle. The developed system was then tested on PC-3 cell lines to check its cellular response. Confocal microscopy observations and (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and Annexin V-FITC assays used to evaluate cell toxicity and apoptosis reveal a dose-dependent nature of nanorods and can overcome the side effects of using free DOX with a targeted action.

Innovative Magnetic Nanoparticles for PET/MRI Bimodal Imaging.

Superparamagnetic iron oxide nanoparticles were developed as positron emission tomography (PET) and magnetic resonance imaging (MRI) bimodal imaging agents. These nanoparticles (NPs), with a specific nanoflower morphology, were first synthesized and simultaneously functionalized with 3,4-dihydroxy-l-phenylalanine (LDOPA) under continuous hydrothermal conditions. The resulting NPs exhibited a low hydrodynamic size of 90 ± 2 nm. The functional groups of LDOPA (-NH2 and -COOH) were successfully used for the grafting of molecules of interest in a second step. The nanostructures were modified by poly(ethylene glycol) (PEG) and a new macrocyclic chelator MANOTA for further 64Cu radiolabeling for PET imaging. The functionalized NPs showed promising bimodal (PET and MRI) imaging capability with high r 2 and r 2* (T 2 and T 2* relaxivities) values and good stability. They were mainly uptaken from liver and kidneys. No cytotoxicity effect was observed. These NPs appear as a good candidate for bimodal tracers in PET/MRI.

Efficient Quantification by X-ray Photoelectron Spectroscopy and Thermogravimetric Analyses of the One-Pot Grafting of Two Molecules on the Surface of Iron Oxide Nanoparticles.

In this study, Superparamagnetic iron oxide nanoparticles (SPION) were functionalized in one pot with two organic molecules. Firstly, polyethylene glycol (PEG) was mixed for 46 hours to improve steric stability and then, two hours before the end of the reaction, dimercaptosuccinic acid (DMSA) was added to provide negative charges and thiol groups for post-functionalization. Three different molecular weights of PEG were used (550, 2000 and 5000 g mol-1). The main goal of this study was to characterize and quantify accurately the surface of SPION functionalized with two organic molecules. We demonstrated the advantages of coupling thermogravimetric and X-ray photoelectron spectrometry analyses to distinguish accurately the covering of SPION's surface. Thanks to the combination of these two techniques we were able to distinguish the amount of DMSA and PEG on SPION regarding the length of the polymer. We also showed that the length of the PEG influenced the quantity of DMSA adsorbed. With the smallest PEG (550 g mol-1) the presence of DMSA is almost ten times higher than with the two other PEG used proving that long polymers prevent the adsorption of small molecules on the surface of SPION.

Elaboration of Trans-Resveratrol Derivative-Loaded Superparamagnetic Iron Oxide Nanoparticles for Glioma Treatment.

In this work, new nanohybrids based on superparamagnetic iron oxide nanoparticles (SPIONs) were elaborated and discussed for the first time as nanovectors of a derivative molecule of trans-resveratrol (RSV), a natural antioxidant molecule, which can be useful for brain disease treatment. The derivative molecule was chemically synthesized (4'-hydroxy-4-(3-aminopropoxy) trans-stilbene: HAPtS) and then grafted onto SPIONs surface using an organosilane coupling agent, which is 3-chloropropyltriethoxysilane (CPTES) and based on nucleophilic substitution reactions. The amount of HAPtS loaded onto SPIONs surface was estimated by thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) analyses at 116 µmol·g-1 SPIONs. The synthesized HAPtS molecule, as well as the associated nanohybrids, were fully characterized by transmission electron microscopy (TEM), XPS, TGA, infrared (IR) and UV-visible spectroscopies, dynamic light scattering (DLS), and zeta potential measurements. The in vitro biological assessment of the synthesized nanohybrid's efficiency was carried out on C6 glioma cells and showed that the nanovector SPIONs-CPTES-HAPtS do not affect the mitochondrial metabolism (MTT test), but damage the plasma membrane (FDA test), which could contribute to limiting the proliferation of cancerous cells (clonogenic test) at a HAPtS concentration of 50 µM. These nanoparticles have a potential cytotoxic effect that could be used to eliminate cancer cells.

Beyond Unpredictability: The Importance of Reproducibility in Understanding the Protein Corona of Nanoparticles.

While it is well established that the surface of a nanoparticle plays a pivotal role for the protein corona, the vast number of proteins present in biological media render general conclusions about affinities between nanoparticle surfaces and proteins nontrivial. Recently published articles increasingly reveal differences between systems and an ever increasing number of influencing factors for the protein corona. In contrast, the present study posits that the reported differences may, at least in part, be due to poor experimental design, which leads to biased results. The present study investigates protein adsorption onto silica nanoparticles with different chemical groups on the surface by the statistical analysis of triplicate measurements as well as control measurements. We demonstrate that 60% of the identified protein types did not have any significant affinities for the nanoparticles. Of the remaining 40%, 60% were driven by surface charges and only 40% preferentially adsorbed onto specific surface groups. Furthermore, we found that of the 20 most abundant proteins in the serum, only five bound to the nanoparticles studied here. We illustrate the importance of control replicate experiments to avoid exaggerated differences between systems and to properly quantify the differences and similarities between comparable systems.

Nanoscience-Based Strategies to Engineer Antimicrobial Surfaces.

Microbial contamination and biofilm formation of medical devices is a major issue associated with medical complications and increased costs. Consequently, there is a growing need for novel strategies and exploitation of nanoscience-based technologies to reduce the interaction of bacteria and microbes with synthetic surfaces. This article focuses on surfaces that are nanostructured, have functional coatings, and generate or release antimicrobial compounds, including "smart surfaces" producing antibiotics on demand. Key requirements for successful antimicrobial surfaces including biocompatibility, mechanical stability, durability, and efficiency are discussed and illustrated with examples of the recent literature. Various nanoscience-based technologies are described along with new concepts, their advantages, and remaining open questions. Although at an early stage of research, nanoscience-based strategies for creating antimicrobial surfaces have the advantage of acting at the molecular level, potentially making them more efficient under specific conditions. Moreover, the interface can be fine tuned and specific interactions that depend on the location of the device can be addressed. Finally, remaining important challenges are identified: improvement of the efficacy for long-term use, extension of the application range to a large spectrum of bacteria, standardized evaluation assays, and combination of passive and active approaches in a single surface to produce multifunctional surfaces.

Effectiveness of hand washing on the removal of iron oxide nanoparticles from human skin ex vivo.

In this study, the effectiveness of washing with soap and water in removing nanoparticles from exposed skin was investigated. Dry, nanoscale hematite (α-Fe2O3) or maghemite (γ-Fe2O3) powder, with primary particle diameters between 20-30 nm, were applied to two samples each of fresh and frozen ex vivo human skin in two independent experiments. The permeation of nanoparticles through skin, and the removal of nanoparticles after washing with soap and water were investigated. Bare iron oxide nanoparticles remained primarily on the surface of the skin, without penetrating beyond the stratum corneum. Skin exposed to iron oxide nanoparticles for 1 and 20 hr resulted in removal of 85% and 90%, respectively, of the original dose after washing. In the event of dermal exposure to chemicals, removal is essential to avoid potential local irritation or permeation across skin. Although manufactured at an industrial scale and used extensively in laboratory experiments, limited data are available on the removal of engineered nanoparticles after skin contact. Our finding raises questions about the potential consequences of nanoparticles remaining on the skin and whether alternative washing methods should be proposed. Further studies on skin decontamination beyond use of soap and water are needed to improve the understanding of the potential health consequences of dermal exposure to nanoparticles.

Modification of the surface of superparamagnetic iron oxide nanoparticles to enable their safe application in humans.

Combined individually tailored methods for diagnosis and therapy (theragnostics) could be beneficial in destructive diseases, such as rheumatoid arthritis. Nanoparticles are promising candidates for theragnostics due to their excellent biocompatibility. Nanoparticle modifications, such as improved surface coating, are in development to meet various requirements, although safety concerns mean that modified nanoparticles require further review before their use in medical applications is permitted. We have previously demonstrated that iron oxide nanoparticles with amino-polyvinyl alcohol (a-PVA) adsorbed on their surfaces have the unwanted effect of increasing human immune cell cytokine secretion. We hypothesized that this immune response was caused by free-floating PVA. The aim of the present study was to prevent unwanted immune reactions by further surface modification of the a-PVA nanoparticles. After cross-linking of PVA to nanoparticles to produce PVA-grafted nanoparticles, and reduction of their zeta potential, the effects on cell viability and cytokine secretion were analyzed. PVA-grafted nanoparticles still stimulated elevated cytokine secretion from human immune cells; however, this was inhibited after reduction of the zeta potential. In conclusion, covalent cross-linking of PVA to nanoparticles and adjustment of the surface charge rendered them nontoxic to immune cells, nonimmunogenic, and potentially suitable for use as theragnostic agents.

Effects of PVA-coated nanoparticles on human T helper cell activity.

Superparamagnetic iron oxide nanoparticles (SPION) are used as high-sensitive enhancer for magnetic resonance imaging, where they represent a promising tool for early diagnosis of destructive diseases such as rheumatoid arthritis (RA). Since we could demonstrate that professional phagocytes are activated by amino-polyvinyl-alcohol-coated-SPION (a-PVA-SPION), the study here focuses on the influence of a-PVA-SPION on human T cells activity. Therefore, primary human CD4+ T cells from RA patients and healthy subjects were treated with varying doses of a-PVA-SPION for 20h or 72h. T cells were then analyzed for apoptosis, cellular energy, expression of the activation marker CD25 and cell proliferation. Although, we observed that T cells from RA patients are more susceptible to low-dose a-PVA-SPION-induced apoptosis than T cells from healthy subjects, in both groups a-PVA-SPION do not activate CD4+ T cells per se and do not influence mitogen-mediated T cells activation with regard to CD25 expression and cell proliferation. Nevertheless, our results demonstrate that CD4+ T cells from RA patients and healthy subjects differ in their response to mitogen stimulation and oxygen availability. We conclude from our data, that a-PVA-SPION do neither activate nor significantly influence mitogen-stimulated CD4+ T cells activation and have negligible influence on T cells apoptosis.

Influence of Surface Charge and Polymer Coating on Internalization and Biodistribution of Polyethylene Glycol-Modified Iron Oxide Nanoparticles.

The aim of this study was to investigate the influence of the surface charge and coating of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on their in vitro and in vivo behaviors. Neutral and negatively-charged PEG-based SPIONs were synthesized and compared to Resovist, a carboxydextran-based SPION currently used in clinics. Their cytotoxicity, cell internalization, and potential as contrast agents for magnetic resonance imaging were assessed. Neutral pegylated SPIONs were internalized less readily by the reticuloendothelial system and showed a lower uptake by the liver, compared to negatively-charged SPIONs (with carboxydextran and PEG). These results suggested that the charge of functionalized SPIONs was more relevant for their biological interactions than the nature of their coating.

Significance of surface charge and shell material of superparamagnetic iron oxide nanoparticle (SPION) based core/shell nanoparticles on the composition of the protein corona.

As nanoparticles (NPs) are increasingly used in many applications their safety and efficient applications in nanomedicine have become concerns. Protein coronas on nanomaterials' surfaces can influence how the cell "recognizes" nanoparticles, as well as the in vitro and in vivo NPs' behaviors. The SuperParamagnetic Iron Oxide Nanoparticle (SPION) is one of the most prominent agents because of its superparamagnetic properties, which is useful for separation applications. To mimic surface properties of different types of NPs, a core-shell SPION library was prepared by coating with different surfaces: polyvinyl alcohol polymer (PVA) (positive, neutral and negative), SiO2 (positive and negative), titanium dioxide and metal gold. The SPIONs with different surfaces were incubated at a fixed serum : nanoparticle surface ratio, magnetically trapped and washed. The tightly bound proteins were quantified and identified. The surface charge has a great impact on protein adsorption, especially on PVA and silica where proteins preferred binding to the neutral and positively charged surfaces. The importance of surface material on protein adsorption was also revealed by preferential binding on TiO2 and gold coated SPION, even negatively charged. There is no correlation between the protein net charge and the nanoparticle surface charge on protein binding, nor direct correlation between the serum proteins' concentration and the proteins detected in the coronas.