Transferrin coated nanoparticles: study of the bionano interface in human plasma

It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.     

Apatinib-loaded nanoparticles inhibit tumor growth and angiogenesis in a model of melanoma

Anti-angiogenic drugs are an effective therapeutic method for the treatment of melanomas. Apatinib is a small-molecule tyrosine kinase inhibitor, which has potent inhibitory activity on tumor angiogenesis. Due to the low water solubility and stability of Apatinib, we aimed to design and develop poly (lactic-co-glycolic acid) (PLGA) and Poloxamer 407 nanoparticles to encapsulate Apatinib (Apa/p NPs) to improve the efficacy of application in melanoma treatment. The size and morphology of the nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro proliferation assays were used to assess the capacity of Apa/p NPs to suppress the growth of B16 cells. Furthermore, we constructed melanoma models using C57BL/6 mice, and preliminary evaluation of the effect and mechanism of Apa/p NPs on tumor inhibition was performed in vivo. The results showed that the size of Apa/p NPs averaged 136 ± 0.27 nm and the nanoparticles were evenly dispersed. Moreover, Apa/p NPs significantly inhibited the growth of B16 cells and melanoma tumors, compared with the naked drug treatment and control groups. The protein levels of VEGFR-2, phosphorylated (p)-VEGFR-2 and p-ERK1/2 in tumor tissues were inhibited by Apa/p NP treatment, as detected by Western blot. The results of this study suggested that Apa/p NPs could inhibit the growth of melanoma tumors by inhibiting the phosphorylation and expression of VEGFR-2 and downstream ERK1/2, providing a theoretical basis for the clinical application of Apatinib in the treatment of melanoma.     

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO₂ NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO₂ NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.     

In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria

In situ cell separation and immobilization of bacterial cells for biodesulfurization were developed by using superparamagnetic Fe₃O₄ nanoparticles (NPs). The Fe₃O₄ NPs were synthesized by coprecipitation followed by modification with ammonium oleate. The surface-modified NPs were monodispersed and the particle size was about 13 nm with 50.8 emu/g saturation magnetization. After adding the magnetic fluids to the culture broth, Rhodococcus erythropolis LSSE8-1 cells were immobilized by adsorption and then separated with an externally magnetic field. The maximum amount of cell mass adsorbed was about 530 g dry cell weight/g particles to LSSE8-1 cells. Analysis showed that the nanoparticles were strongly absorbed to the surface and coated the cells. Compared to free cells, the coated cells not only had the same desulfurizing activity but could also be easily separated from fermentation broth by magnetic force. Based on the adsorption isotherms and Zeta potential analysis, it was believed that oleate-modified Fe₃O₄ NPs adsorbed bacterial cells mainly because of the nano-size effect and hydrophobic interaction.     

Preparation of CoS nanoparticles-cisplatin bio-conjugates and investigation of their effects on SH-SY5Y neuroblastoma cell line

Neuroblastoma is one of the most widely seen under the age of 15 tumors that occur in the adrenal medulla and sympathetic ganglia. Cisplatin, an antineoplastic drug, is a Platinum-based compound and is known to inhibit the proliferation of neuroblastoma cells. Effective applications of nanoparticles in biomedical areas such as biomolecular, antimicrobial detection and diagnosis, tissue engineering, theranostics, biomarking, drug delivery, and anti-cancer have been investigated in many studies. This study aims to prepare the bioconjugates of CoS (cobalt sulfide) nanoparticles (NPs) with cisplatin combination groups and to evaluate their effects on the neuroblastoma cell line. Nanoparticle synthesis was done using the green synthesis technique using Punica granatum plant extract. The size and shape of CoS NPs were characterized by SEM, FT-IR, and XRD. Zeta potential was confirmed by the DLS study. For this purpose, the SH-SY5Y neuroblastoma cell line was cultured in a suitable cell culture medium. Cisplatin 5 µg and different concentrations (Cisplatin + CoS NPs bioconjugates (5, 10, 25, 50, 75 μg) doses were applied to SH-SY5Y neuroblastoma cell lines for 24 h. TAC, TOS and MTT tests were performed 24 h after the application. According to the MTT test results, cisplatin and CoS NP combinations reduced the proliferation of neuroblastoma cells by 78 to 57% compared to the cisplatin control. From the findings obtained; the most effective Bio-conjugate group was Cisplatin 5 μg/mL + CoS 75 μg/mL.     

Effect of the surface charge density of nanoparticles on their translocation across pulmonary surfactant monolayer: a molecular dynamics simulation

Interaction between nanoparticles (NPs) and pulmonary surfactant monolayer plays a very significant role in nanoparticle-based pulmonary drug delivery system. Previous researches have indicated that different properties of nanoparticles can affect their translocation across pulmonary surfactant monolayer. Here we performed coarse-grained molecular dynamics simulation aimed at nanoparticles’ surface charge density effect on their penetration behaviours. Several hydrophilic nanoparticles with different surface charge densities were modelled in the simulations. The results show that NPs’ surface charge density affects their translocation capability: the higher the surface charge densities of NPs are, the worse their translocation capability is. It will cause the structural changes of pulmonary surfactant monolayer, and inhibit the normal phase transition of the monolayer during the compression process. Besides, charged NPs can be adsorbed on the surface of the monolayer after translocation as a stable state, and the adsorption capability of NPs increases generally with the increase of surface charge densities. Our simulation results suggest that the study of nanoparticle-based pulmonary drug delivery system should consider the nanoparticles’ surface charge density effect in order to avoid biological toxicity and improve efficacy.     

The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure

This report extensively explores the benefits of including chitosan into poly-ε-caprolactone (PCL) nanoparticles (NPs) to obtain an improved protein/antigen delivery system. Blend NPs (PCL/chitosan NPs) showed improved protein adsorption efficacy (84%) in low shear stress and aqueous environment, suggesting that a synergistic effect between PCL hydrophobic nature and the positive charges of chitosan present at the particle surface was responsible for protein interaction. Additionally, thermal analysis suggested the blend NPs were more stable than the isolated polymers and cytotoxicity assays in a primary cell culture revealed chitosan inclusion in PCL NPs reduced the toxicity of the delivery system. A quantitative 6-month stability study showed that the inclusion of chitosan in PCL NPs did not induce a change in adsorbed ovalbumin (OVA) secondary structure characterized by the increase in the unordered conformation (random coil), as it was observed for OVA adsorbed to chitosan NPs. Additionally, the slight conformational changes occurred, are not expected to compromise ovalbumin secondary structure and activity, during a 6-month storage even at high temperatures (45°C). In simulated biological fluids, PCL/chitosan NPs showed an advantageous release profile for oral delivery. Overall, the combination of PCL and chitosan characteristics provide PCL/chitosan NPs valuable features particularly important to the development of vaccines for developing countries, where it is difficult to ensure cold chain transportation and non-parenteral formulations would be preferred.     

Toward a molecular understanding of nanoparticle–protein interactions

Wherever nanoparticles (NPs) come in contact with a living organism, physical and chemical interactions take place between the surfaces of the NPs and biomatter, in particular proteins. When NP are exposed to biological fluids, an adsorption layer of proteins, a “protein corona” forms around the NPs. Consequently, living systems interact with the protein-coated NP rather than with a bare NP. To anticipate biological responses to NPs, we thus require comprehensive knowledge of the interactions at the bio–nano interface. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP–protein interactions. In this brief review, we present the latest findings regarding the composition of the protein corona as it forms on NPs in the blood stream. We also discuss molecular aspects of this adsorption layer and its time evolution. The current state of knowledge is summarized, and issues that still need to be addressed to further advance our understanding of NP–protein interactions are identified.     

Synthesis of tumor-targeted folate conjugated fluorescent magnetic albumin nanoparticles for enhanced intracellular dual-modal imaging into human brain tumor cells

Superparamagnetic iron oxide nanoparticles (SPIO NPs), utilized as carriers are attractive materials widely applied in biomedical fields, but target-specific SPIO NPs with lower toxicity and excellent biocompatibility are still lacking for intracellular visualization in human brain tumor diagnosis and therapy. Herein, bovine serum albumin (BSA) coated superparamagnetic iron oxide, i.e. γ-Fe₂O₃ nanoparticles (BSA-SPIO NPs), are synthesized. Tumor-specific ligand folic acid (FA) is then conjugated onto BSA-SPIO NPs to fabricate tumor-targeted NPs, FA-BSA-SPIO NPs as a contrast agent for MRI imaging. The FA-BSA-SPIO NPs are also labeled with fluorescein isothiocyanate (FITC) for intracellular visualization after cellular uptake and internalization by glioma U251 cells. The biological effects of the FA-BSA-SPIO NPs are investigated in human brain tumor U251 cells in detail. These results show that the prepared FA-BSA-SPIO NPs display undetectable cytotoxicity, excellent biocompatibility, and potent cellular uptake. Moreover, the study shows that the made FA-BSA-SPIO NPs are effectively internalized for MRI imaging and intracellular visualization after FITC labeling in the targeted U251 cells. Therefore, the present study demonstrates that the fabricated FITC-FA-BSA-SPIO NPs hold promising perspectives by providing a dual-modal imaging as non-toxic and target-specific vehicles in human brain tumor treatment in future.     

Core-shell Au@Co-Fe hybrid nanoparticles as peroxidase mimetic nanozyme for antibacterial application

Nanozymes have been developed as alternative for enzymes to overcome practical limitations of enzymes in industry and medicine. Infectious diseases are becoming severe threat to public health. Hence, peroxidase nanozyme for combating bacteria have been designed. Core-Shell Au@Co-Fe hybrid nanoparticles (Au@Co-Fe NPs) were synthesized. The structure of Au@Co-Fe NPs was characterized by UV–vis and FT-IR spectroscopic methods. The size, zeta potential and spherical morphology of Au@Co-Fe NPs were determined by DLS, TEM and AFM techniques. Au@Co-Fe NPs has been evaluated as peroxidase mimic nanozyme. The peroxidase mimetic activity of gold nanoparticles, Co (II) and Fe (III) were measured and compared with that obtained for native HRP. The enzymatic measured activity was 50% of native horse radish peroxidase. Additionally, Au@Co-Fe NPs was evaluated as antibacterial agent against four selected standard pathogenic bacteria as Escherichia coli, Pseudomonas aeruginosa (as gram negative) and Staphylococcus aureus, and Bacillus cereus (as gram positive).     

Identifying Metal Nanoparticle Size Effect on Sensing Common Human Plasma Protein by Counting the Sensitivity of Optical Absorption Spectra Damping

Colloidal nanoparticles (NPs) interact with biological fluids such as human plasma to form a protein coating (corona) on the surface of NPs (NP-protein complex). However, the impact of size and type of NPs on binding of the hard corona to the surface of NPs as well as damping of their optical spectra has not been systematically explored. To elucidate the interaction between biological environment (human plasma) and NPs, a photophysical measurement was conducted to quantify the interaction of two different types of NPs (gold (Au) and silver (Ag)) with common human plasma proteins. The colloidal AuNPs and AgNPs were electrostatically stabilized and varied in diameter from 10 to 80 nm in the presence of common human plasma. The sizes of the NPs were determined using transmission electron microscopy (TEM). Optical absorption spectra were obtained for the complexes. Dynamic light scattering (DLS) measurement and zeta potential were used to characterize the sizes, hydrodynamic diameters, and surface charges of the protein-NPs complexes. Protein separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate and identify the protein bands. The absorption of proteins to the NPs was found to be strongly dependent on the size and type of NPs. The distance between surface of NPs by absorbed protein bound to the NPs gradually increased with size of NPs, particularly for AgNPs with primary diameter of < 50 nm. The chi-square test proved that AgNPs are a good candidate in sensing the protein complex in human plasma compared with AuNPs mainly for the AgNPs with diameter sized 50 nm.

     

Application of a fluorescent biosensor based‐on magneto‐γ‐Fe2O3–methyldopa nanoparticles for adsorption of human serum albumin

Understanding and controlling the interaction between the polymer methyldopa (2‐amino‐3‐(3,4‐dihydroxyphenyl)‐2‐methyl‐propanoic acid) (PMDP)–γ‐Fe₂O₃ nanoparticles and biological fluids is important if the potential of nanoparticles (NPs) in biomedicine is to be realized. Physicochemical studies on the interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of the NP surface, interactions between human serum albumin (HSA) and PMDP–γ‐Fe₂O₃ NPs were investigated. Here, the adsorption of HSA onto small (10–30 nm diameter) PMDP–γ‐Fe₂O₃ NPs was quantitatively analyzed using spectroscopic methods. The fluorescence quenching data were checked for the inner‐filter effect, the main confounding factor in the observed quenching. The binding constants, K_a, were calculated at different temperatures, using a nonlinear fit to the experimental data, and the thermodynamic parameters ?H, ?S and ?G were given. The obtained thermodynamic signature suggests that hydrophobic interactions at least are present. This result indicates that the structure of the protein turns from a structureless denatured state at pH 3 into an ordered biologically active native state on addition of PMDP–γ‐Fe₂O₃ NPs. Copyright © 2015 John Wiley & Sons, Ltd.     

Targeted Anticancer Drug Delivery Using Chitosan,Carbon Quantum Dots,and Aptamers to Deliver Ganoderic Acid and 5-Fluorouracil

Breast cancer is a malignancy that affects mostly females and is among the most lethal types of cancer. The ligand-functionalized nanoparticles used in the nano-drug delivery system offer enormous potential for cancer treatments. This work devised a promising approach to increase drug loading efficacy and produce sustained release of 5-fluorouracil (5-FU) and Ganoderic acid (GA) as model drugs for breast cancer. Chitosan, aptamer, and carbon quantum dot (CS/Apt/COQ) hydrogels were initially synthesized as a pH-sensitive and biocompatible delivery system. Then, CS/Apt/COQ NPs loaded with 5-FU-GA were made using the W/O/W emulsification method. FT-IR, XRD, DLS, zeta potentiometer, and SEM were used to analyze NP's chemical structure, particle size, and shape. Cell viability was measured using MTT assays in vitro using the MCF-7 cell lines. Real-time PCR measured cell apoptotic gene expression. XRD and FT-IR investigations validated nanocarrier production and revealed their crystalline structure and molecular interactions. DLS showed that nanocarriers include NPs with an average size of 250.6 nm and PDI of 0.057. SEM showed their spherical form, and zeta potential studies showed an average surface charge of +37.8 mV. pH 5.4 had a highly effective and prolonged drug release profile, releasing virtually all 5-FU and GA in 48 h. Entrapment efficiency percentages for 5-FU and GA were 84.7±5.2 and 80.2 %±2.3, respectively. The 5-FU-GA-CS-CQD-Apt group induced the highest cell death, with just 57.9 % of the MCF-7 cells surviving following treatment. 5-FU and GA in CS-CQD-Apt enhanced apoptotic induction by flow cytometry. 5-FU-GA-CS-CQD-Apt also elevated Caspase 9 and downregulated Bcl2. Accordingly, the produced NPs may serve as pH-sensitive nano vehicles for the controlled release of 5-FU and GA in treating breast cancer.     

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

PhaP介导EGFR靶向多肽修饰的肿瘤靶向PHBHHx纳米药物递送载体的构建

聚羟基脂肪酸(PHA)颗粒表面结合蛋白Pha P具有与疏水性高分子材料表面紧密结合的能力,本研究将EGFR靶向多肽(ETP)与PhaP进行融合表达,构建了ETP-PhaP融合蛋白表达的重组工程菌Escherichia coli BL21(DE3)(pPI-ETP-P)。经对工程菌株的诱导表达及ETP-PhaP融合蛋白的纯化后,通过PhaP蛋白介导能够有效地将ETP-PhaP融合蛋白修饰于3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)纳米微球表面,构建成为具有EGFR靶向作用的药物递送载体。分别检测宫颈癌细胞系SiHa(EGFR高表达)和CaSKi(EGFR低表达)对ETP-PhaP修饰的PHBHHx纳米药物载体和未经修饰的纳米药物载体的吞噬情况。结果显示,纯化的ETP-PhaP融合蛋白能够很好地吸附于PHBHHx颗粒的表面,经ETP-PhaP融合蛋白修饰的PHBHHx纳米药物载体对EGFR高表达的宫颈癌Si Ha细胞的靶向效果强于EGFR低表达的CaSKi细胞系。这一结果表明了PhaP介导的PHBHHx纳米微球表面EGFR靶向多肽修饰具有简便、高效的优势,为疏水性纳米药物载体表面功能多肽修饰提供了一种新策略。     

Assessing protein oxidation by inorganic nanoparticles with enzyme‐linked immunosorbent assay (ELISA)

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme‐linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn₂O₃, and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS‐dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Biotechnol. Bioeng. 2013; 110: 694–701. © 2012 Wiley Periodicals, Inc.     

Probing the interaction of silver nanoparticles with tau protein and neuroblastoma cell line as nervous system models

Interestingly pharmaceutical sciences are using nanoparticles (NPs) to design and develop nanomaterials-based drugs. However, up to recently, it has not been well realized that NPs themselves may impose risks to the biological systems. In this study, the interaction of silver nanoparticles (AgNPs) with tau protein and SH-SY5Y neuroblastoma cell line, as potential nervous system models, was examined with a range of techniques including intrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and acridine orange/ethidium bromide (AO/EB) dual staining method. Fluorescence study showed that AgNPs with a diameter of around 10–20 nm spontaneously form a static complex with tau protein via hydrogen bonds and van der Waals interactions. CD experiment revealed that AgNPs did not change the random coil structure of tau protein. Moreover, AgNPs showed to induce SH-SY5Y neuroblastoma cell mortality through fragmentation of DNA which is a key feature of apoptosis. In conclusion, AgNPs may induce slight changes on the tau protein structure. Also, the concentration of AgNPs is the main factor which influences their cytotoxicity. Since, all adverse effects of NPs are not well detected, so probably additional more specific testing would be needed.     

Effects of surface charge and ligand type of Au nanoparticles on green fluorescent protein-expressing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effects of the surface charge and ligand type of three types of Au nanoparticles (NPs), namely anionic polyethylene glycol (PEG)-Au NPs, anionic citrate (Cit)-Au NPs, and cationic branched polyethylenimine (bPEI)-Au NPs, on green fluorescent protein (GFP)-expressing Escherichia coli were evaluated through the combined analysis of optical density (OD) and fluorescence intensity (FI). OD and FI can provide information about cell growth and metabolism of bacteria, respectively. The results demonstrated that PEG- and Cit-Au NPs had no major effects on the OD and FI of GFP-expressing bacteria. However, it was found that Cit-Au NPs may slightly influence cell metabolism at higher concentrations, although it is necessary to perform further in-depth study to clarify this issue. Cationic bPEI-Au NPs showed significant effects on cell density and metabolism of E. coli, with an especially strong effect on metabolism. The combined analysis of OD and FI may be useful for monitoring the effects of a wide range of nanomaterials on microorganisms.     

Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass,surface charge,and nanoparticle–cell interaction

The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood–brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS–PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30 kDa and 100 kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30 kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30 min-incubation.     

Toxicity evaluation of manufactured CeO2 nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells

Background

Engineered nanomaterials may release nanosized residues, by degradation, throughout their life cycle. These residues may be a threat for living organisms. They may be ingested by humans through food and water. Although the toxicity of pristine CeO₂ nanoparticles (NPs) has been documented, there is a lack of studies on manufactured nanoparticles, which are often surface modified. Here, we investigated the potential adverse effects of CeO₂ Nanobyk 3810™ NPs, used in wood care, and their residues, altered by light or acid.

Results

Human intestinal Caco-2 cells were exposed to residues degraded by daylight or in a medium simulating gastric acidity. Size and zeta potential were determined by dynamic light scattering. The surface structure and redox state of cerium were analyzed by transmission electronic microscopy (TEM) and X-ray absorption spectroscopy, respectively. Viability tests were performed in Caco-2 cells exposed to NPs. Cell morphology was imaged with scanning electronic microscopy. Gene expression profiles obtained from cells exposed to NPs before and after their alteration were compared, to highlight differences in cellular functions.No change in the cerium redox state was observed for altered NPs. All CeO₂ NPs suspended in the culture medium became microsized. Cytotoxicity tests showed no toxicity after Caco-2 cell exposure to these various NPs up to 170 μg/mL (24 h and 72 h). Nevertheless, a more-sensitive whole-gene-expression study, based on a pathway-driven analysis, highlighted a modification of metabolic activity, especially mitochondrial function, by altered Nanobyk 3810™. The down-regulation of key genes of this pathway was validated by qRT-PCR. Conversely, Nanobyk 3810™ coated with ammonium citrate did not display any adverse effect at the same concentration.

Conclusion

The degraded nanoparticles were more toxic than their coated counterparts. Desorption of the outside layer was the most likely cause of this discrepancy in toxicity. It can be assumed that the safe design of engineered nanoparticles could include robust protective layers conferring on them greater resistance to alteration during their life cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-700) contains supplementary material, which is available to authorized users.