Zheng-Mei Song

Progress in the characterization and safety evaluation of engineered inorganic nanomaterials in food

Nanotechnology has stepped into the food industry, from the farm to the table at home, in order to improve the taste and nutritional value, extend the shelf-life and monitor the food quality. In fact, as consumers we have already been in contact, via oral exposure, with a number of food products containing engineered nanomaterials (ENMs) more often than most people think. However, the fate of ENMs after entering the GI tract of the human body is not yet clearly understood. Hence, the related safety issue is raised, and attracts much attention and wide debate from the public, even including protest demonstrations against nanotechnology in food. In this review, we summarize the up-to-date information about the characterization and safety evaluation of common inorganic ENMs (with a focus on silver, titanium dioxide, silica and zinc oxide nanoparticles) in food. Based on the literature, a whole scenario of the safety issue of these ENMs in food and an outlook on the future studies are given.

Evaluation of the toxicity of food additive silica nanoparticles on gastrointestinal cells

Silica nanoparticles (NPs) have been widely used in food products as an additive; however, their toxicity and safety to the human body and the environment still remain unclear. As a food additive, silica NPs firstly enter the human gastrointestinal tract along with food, thus their gastrointestinal toxicity deserves thorough study. Herein, we evaluated the toxicity of food additive silica NPs to cells originating from the gastrointestinal tract. Four silica NP samples were introduced to human gastric epithelial cell GES‐1 and colorectal adenocarcinoma cell Caco‐2 to investigate the effect of silica sample, exposure dose and exposure period on the morphology, viability and membrane integrity of cells. The cell uptake, cellular reactive oxygen species (ROS) level, cell cycle and apoptosis were determined to reveal the toxicity mechanism. The results indicate that all four silica NPs are safe for both GES‐1 and Caco‐2 cells after 24‐h exposure at a concentration lower than 100 µg ml–1. At a higher concentration and longer exposure period, silica NPs do not induce the apoptosis/necrosis of cells, but arrest cell cycle and inhibit the cell growth. Notably, silica NPs do not pass through the Caco‐2 cell monolayer after 4‐h contact, indicating the low potential of silica NPs to cross the gastrointestinal tract in vivo. Our findings indicate that silica NPs could be used as a safe food additive, but more investigations, such as long‐term in vivo exposure, are necessary in future studies. Copyright

Biological effect of food additive titanium dioxide nanoparticles on intestine: an in vitro study

Titanium dioxide nanoparticles (TiO2 NPs) are widely found in food‐related consumer products. Understanding the effect of TiO2 NPs on the intestinal barrier and absorption is essential and vital for the safety assessment of orally administrated TiO2 NPs. In this study, the cytotoxicity and translocation of two native TiO2 NPs, and these two TiO2 NPs pretreated with the digestion simulation fluid or bovine serum albumin were investigated in undifferentiated Caco‐2 cells, differentiated Caco‐2 cells and Caco‐2 monolayer. TiO2 NPs with a concentration less than 200 µg ml–1 did not induce any toxicity in differentiated cells and Caco‐2 monolayer after 24 h exposure. However, TiO2 NPs pretreated with digestion simulation fluids at 200 µg ml–1 inhibited the growth of undifferentiated Caco‐2 cells. Undifferentiated Caco‐2 cells swallowed native TiO2 NPs easily, but not pretreated NPs, implying the protein coating on NPs impeded the cellular uptake. Compared with undifferentiated cells, differentiated ones possessed much lower uptake ability of these TiO2 NPs. Similarly, the traverse of TiO2 NPs through the Caco‐2 monolayer was also negligible. Therefore, we infer the possibility of TiO2 NPs traversing through the intestine of animal or human after oral intake is quite low. This study provides valuable information for the risk assessment of TiO2 NPs in food. Copyright

Toxicological Effects of Caco-2 Cells Following Short-Term and Long-Term Exposure to Ag Nanoparticles

Extensive utilization increases the exposure of humans to Ag nanoparticles (NPs) via the oral pathway. To comprehensively address the action of Ag NPs to the gastrointestinal systems in real situations, i.e., the long-term low-dose exposure, we evaluated and compared the toxicity of three Ag NPs (20–30 nm with different surface coatings) to the human intestine cell Caco-2 after 1-day and 21-day exposures, using various biological assays. In both the short- and long-term exposures, the variety of surface coating predominated the toxicity of Ag NPs in a descending order of citrate-coated Ag NP (Ag-CIT), bare Ag NP (Ag-B), and poly (N-vinyl-2-pyrrolidone)-coated Ag NP (Ag-PVP). The short-term exposure induced cell growth inhibition and death. The cell viability loss appeared after cells were exposed to 0.7 μg/mL Ag-CIT, 0.9 μg/mL Ag-B or >1.0 μg/mL Ag-PVP for 24 h. The short-term and higher-dose exposure also induced reactive oxygen species (ROS) generation, mitochondrial damage, cell membrane leakage, apoptosis, and inflammation (IL-8 level). The long-term exposure only inhibited the cell proliferation. After 21-day exposure to 0.4 μg/mL Ag-CIT, the cell viability dropped to less than 50%, while cells exposed to 0.5 μg/mL Ag-PVP remained normal as the control. Generally, 0.3 μg/mL is the non-toxic dose for the long-term exposure of Caco-2 cells to Ag NPs in this study. However, cells presented inflammation after exposure to Ag NPs with the non-toxic dose in the long-term exposure.

Interaction of multi-walled carbon nanotubes and zinc ions enhances cytotoxicity of zinc ions

More and more nanomaterials enter the environment along with their production, application and deposal. They may alter the biological effect of pollutants already existing in the real environment by different interactions. Therefore efforts should also be paid to investigate the combined toxicity of nanomaterials and pollutants. Herein, we studied the combined toxicity of oxidized multi-walled carbon nanotubes (O-MWCNTs) and zinc ions on cells. It is found that cytotoxicity of the combined O-MWCNTs and zinc ions elevates significantly, compared with O-MWCNTs or zinc ions alone. This result comes from the assays of cell morphology, cell viability and proliferation, cell membrane integrity, mitochondrial membrane potential and cell apoptosis. Mechanism studies indicate that O-MWCNTs absorb zinc ions and form slight aggregation. These enhance remarkably the cellular uptake of O-MWCNTs, and thus induce the death of cells by bringing in more zinc ions into cells. Our study indicates that the existence of nanomaterials could change the bioconsequence of other pollutants and emphasizes the importance of the combined toxicity research in the presence of nanomaterials.

Biological effects of agglomerated multi-walled carbon nanotubes.

The physicochemical properties of nanomaterials play crucial roles in determining their biological effects. Agglomeration of nanomaterials in various systems is a common phenomenon, however, how agglomeration affects the biological consequence of nanomaterials has not been well investigated because of its complexity. Herein, we prepared variable sized agglomerates of oxidized multi-walled carbon nanotubes (O-MWCNTs) by using Ca(2+) and studied their cellular uptake and cytotoxicity in HeLa cells. We found the altered property of O-MWCNTs agglomerates could be controlled and adjusted by the amount of Ca(2+). Agglomeration remarkably facilitated the cellular uptake of O-MWCNTs at the initial contact stage, due to the easy contact of agglomerates with cells. But agglomeration did not induce evident cytotoxicity when the concentration of O-MWCNTs was less than 150μg/mL. That was assayed by cell proliferation, membrane integrity, apoptosis and ROS generation. This study suggests us that the biological behaviors of nanomaterials could be altered by their states of agglomeration.

Chitosan-coated red fluorescent protein nanoparticle as a potential dual-functional siRNA carrier

AIMS Developing safe and efficient nano vectors is critical for the success of siRNA therapy. MATERIALS & METHODS By encapsulating red fluorescent protein (RFP) with chitosan (CS), a dual-functional siRNA delivery nano vector, RFP@CS, has been synthesized. RESULTS RFP@CS has an optimum size of 7-23 nm for siRNA delivery; and the fluorescence of RFP, protected by CS coating, provides an excellent probe to track the delivery of siRNA. RFP@CS delivers siRNA efficiently into cells and the targeted gene could be completely silenced even after 48 h. No cytotoxicity or acute toxicity in mice was observed. CONCLUSION The high transfection efficacy and safety demonstrate RFP@CS is a promising nano vector for the gene therapy.

Artificial antibody created by conformational reconstruction of the complementary-determining region on gold nanoparticles

Significance Mimicking protein-like specific interactions and functions has been a long-pursued goal in nanotechnology. The key challenge is to precisely organize nonfunctional surface groups on nanoparticles into specific 3D conformations to function in a concerted and orchestrated manner. Here, we develop a method to graft the complementary-determining regions of natural antibodies onto nanoparticles and reconstruct their “active” conformation to create nanoparticle-based artificial antibodies that recognize the corresponding antigens. Our work demonstrates that it is possible to create functions on nanoparticles by conformational engineering, namely tuning flexible surface groups into specific conformations. Our straightforward strategy could be used further to create other artificial antibodies for various applications and provides a new tool to understand the structure and folding of natural proteins. To impart biomedical functions to nanoparticles (NPs), the common approach is to conjugate functional groups onto NPs by dint of the functions of those groups per se. It is still beyond current reach to create protein-like specific interactions and functions on NPs by conformational engineering of nonfunctional groups on NPs. Here, we develop a conformational engineering method to create an NP-based artificial antibody, denoted “Goldbody,” through conformational reconstruction of the complementary-determining regions (CDRs) of natural antibodies on gold NPs (AuNPs). The seemingly insurmountable task of controlling the conformation of the CDR loops, which are flexible and nonfunctional in the free form, was accomplished unexpectedly in a simple way. Upon anchoring both terminals of the free CDR loops on AuNPs, we managed to reconstruct the “active” conformation of the CDR loops by tuning the span between the two terminals and, as a result, the original specificity was successfully reconstructed on the AuNPs. Two Goldbodies have been created by this strategy to specifically bind with hen egg white lysozyme and epidermal growth factor receptor, with apparent affinities several orders of magnitude stronger than that of the original natural antibodies. Our work demonstrates that it is possible to create protein-like functions on NPs in a protein-like way, namely by tuning flexible surface groups to the correct conformation. Given the apparent merits, including good stability, of Goldbodies, we anticipate that a category of Goldbodies could be created to target different antigens and thus used as substitutes for natural antibodies in various applications.

Intestinal injury alters tissue distribution and toxicity of ZnO nanoparticles in mice

The fast growing applications of ZnO nanoparticles (NPs) in food sector and other fields enhance the exposure possibility of human beings to ZnO NPs including via oral administration route. Although the oral toxicity of ZnO NPs has been studied, most of the research was performed on the normal animal models. Therefore, the understanding of the biological consequence of ZnO NPs on the population with diseases, especially gastrointestinal disease, is extremely limited. In this study, a mice model of inflammatory bowel disease (IBD) induced by indomethacin has been developed to comprehensively investigated the bioeffects of ZnO NPs on the specific population. The effect of the intestinal inflammation/injury on the distribution and toxicity of orally administrated ZnO NPs (nZnO, 20 nm × 100 nm and mZnO, ∼200 nm) in mice were analyzed. The results showed that there was a difference in the distribution of Zn and the essential trace elements (Fe and Cu) between the IBD mice and the normal mice. We also observed an obvious size effect. Higher hepatic Zn was detected in the IBD mice post-exposure to ZnO NPs, especially bigger ZnO NPs. In addition, the histopathological examination of main organs and biological parameters analysis showed that ZnO NPs caused slight toxicity to the liver and kidneys in the IBD mice. Our findings highlight the importance of the health status of animals on the bioeffects of nanomaterials.

Silica nanoparticle with a single His-tag for addressable functionalization, reversible assembly, and recycling

For many biomedical and catalytic applications, such as encapsulation of proteins/enzymes in nanoparticles (NPs), it is preferable to have well-dispersed small NPs that are stable in solution and behave quasi-homogeneously. However, conventional liquid phase methods for small-NP synthesis and functionalization usually face great difficulties in separation/purification and recycling. In addition, controlling the orientation of proteins inside NPs is also a crucial issue to maximize the activity of the encapsulated proteins. Herein, we report a solid phase method to solve these problems. Using His-tagged proteins as cores, well-dispersed core-shell silica NPs are facilely synthesized and functionalized in a column. The core His-tagged proteins are kept bound on the surface of the resin beads in the column during the entire process, making the separation/purification of NPs and their precursors during the multiple-step process as simple as a few-minutes procedure of draining and washing the column. Each obtained silica NP has an adjustable eccentric core-shell structure with only one His-tag sticking out of the particle. This single His-tag on the surface of each NP not only makes it easy for addressable and stoichiometric functionalization of the NP but also provides an easy way to reversibly assemble NPs into dimers or be oriented on the surface of large particles. Notably, this solid phase approach also provides a versatile means to control the orientation of proteins inside NPs, and the His-tag makes it easy to recycle those well-dispersed small NPs.