Lizeng Gao

Intrinsic peroxidase-like activity of ferromagnetic nanoparticles

Nanoparticles containing magnetic materials, such as magnetite (Fe3O4), are particularly useful for imaging and separation techniques. As these nanoparticles are generally considered to be biologically and chemically inert, they are typically coated with metal catalysts, antibodies or enzymes to increase their functionality as separation agents. Here, we report that magnetite nanoparticles in fact possess an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools. Based on this finding, we have developed a novel immunoassay in which antibody-modified magnetite nanoparticles provide three functions: capture, separation and detection. The stability, ease of production and versatility of these nanoparticles makes them a powerful tool for a wide range of potential applications in medicine, biotechnology and environmental chemistry.

Carbon nanotube delivery of the GFP gene into mammalian cells.

Exogenous-gene expression and manipulation in mammalian cells has become a mainstay of biomedical research. Consequently, improving methods for efficient gene transfer to a broad range of cell types is of great interest and remains a high priority. Several classes of transfection methods have been developed, which include traditional cationic moleculemediated agents, such as Lipofectamine 20000 and FuGENE 6, viral-vector systems, and the “gene gun” approach. With the rapid development of nanobiotechnology, a variety of new materials, such as gold nanoparticles, silica nanoparticles, polymers, nanogels, and dendrimers have been investigated as biocompatible transporters. Recently, carbon nanotube—a well-studied nanomaterial— have been investigated for their ability to interact with and affect living systems. For instance, carbon nanotubes have been found to enhance DNA amplification in PCR and affect the growth pattern of neurons. Pantarotto et al. have reported the internalization of fluorescein isothiocynate (FITC) labeled nanotubes and nanotube delivery of the gene that encodes b-galactosidase into cells, with no apparent toxic effects. Kam et al. have studied the mechanism of protein-conjugated carbon nanotube uptake into cells via the endocytic pathway. Here we present our finding that amino-functionalized multiwalled carbon nanotubes (NH2-MWCNTs) are able to interact with plasmid DNA and deliver the green fluorescence protein (GFP) gene into cultured human cells. Our data strongly suggest that carbon nanotubes can be considered as a new carrier for the delivery of biomolecules, such as DNA, proteins, and peptides into mammalian cells. Therefore, this novel system might have potential applications in biology and therapy, including vaccine and gene delivery. In order to increase their biocompatibility, we introduced amino-, carboxyl-, hydroxyl-, and alkyl groups onto the surface of MWCNTs. COOH-MWCNTs were first prepared by nitric / sulfuric acid oxidation, and then NH2and CH3CH2CH2-groups were added. Finally, we obtained four types of MWCNTs with different chemical groups on their surface. Functionalized MWCNTs were observed under an electron microscope and were found to be 60–70 nm in diameter and 1–2 mm in length. Although we did not find a significant difference in size between the NH2-MWCNTs and NH2-MWCNT–DNAs, the latter appeared to have the tendency to aggregate (Figure 1B). In order to test the DNA-binding ability of amino-, carboxyl-, hydroxyl-, and alkyl-group-modified MWCNTs, we incubated them with pEGFPN1-plasmid DNA, and MWCNT–DNA mixtures were analyzed by agarose-gel electrophoresis. The results show that only NH2-MWCNT bound to DNA (Figure 2); since the NH2-MWCNT–DNA complex was too big to run into the

Fabrication of HAp-ZrO2 (3Y) nano-composite by SPS.

Nano-HAp-ZrO(2) powders and HAp-TZP(3Y) composites have been fabricated in this work. The results show that nano-HAp-ZrO(2) powders with homogeneous distribution could be synthesized by two-step precipitation method. HAp-TZP(3Y) composites with small grain size could be sintered at relatively low temperatures and very short dwelling time by using SPS technology. No reaction between HAp and ZrO(2) was found, which could be attributed to the very short sintering time of SPS.

Anti-CD146 monoclonal antibody AA98 inhibits angiogenesis via suppression of nuclear factor-κB activation

Our previous study showed that an anti-CD146 monoclonal antibody (mAb), AA98, which was raised against the vascular endothelial cells stimulated by a conditioned medium from hepatocarcinoma SMMC 7721 cells (SMMC 7721-CM), inhibited cell migration, angiogenesis, and tumor growth. However, the underlying mechanism was not elucidated. The objective of this study was to understand the mechanism by which mAb AA98 inhibits the endothelial cell migration and angiogenesis that is induced by SMMC 7721-CM. Using confocal imaging and biochemical studies, we found that SMMC 7721-CM induced nuclear factor κB (NF-κB) activation through the upstream p38 mitogen-activated protein kinase pathway, leading to the up-regulation of matrix metalloproteinase 9 and intercellular adhesion molecule 1 expression. Interestingly, all these activities stimulated by SMMC 7721-CM could be effectively inhibited by mAb AA98 in a dose- and time-dependent manner. Our data showed that the engagement of mAb AA98 with membrane protein CD146 inhibited p38 mitogen-activated protein kinase phosphorylation, suppressed NF-κB activation, and down-regulated matrix metalloproteinase 9 and intercellular adhesion molecule 1 expression, suggesting that the suppression of NF-κB is a critical point for the inhibitory function of mAb AA98 on endothelial cell migration, angiogenesis, and tumor metastasis. These results will provide clues for a better understanding of the mechanisms underlying tumor angiogenesis as well as antiangiogenesis therapy. [Mol Cancer Ther 2006;5(11):2872–8]

Label-free colorimetric detection of gelatinases on nanoporous silicon photonic films.

We report the development of a sensor platform for detection of gelatinases based on porous silicon photonic films. The sensor is made by spin-coating gelatin, a substrate protein to gelatinases, onto the porous silicon, which forms a thin, uniform, and smooth gel layer where samples can be directly spotted. The digestion products of gelatin by the active gelatinase present in the sample are able to enter the pores and induce color changes that can be detected by the naked eye. Using this sensor, we have demonstrated the detection of matrix metalloproteinase-2 (MMP-2)-an important gelatinase closely associated with tumor aggressiveness and metastatic potential-with concentrations varying from 0.1 to 1000 ng/mL in samples with volumes as small as 1 microL. The detection limit of this method, in terms of the minimum quantity of active MMP-2 in the sample that can be detected, is 2 orders of magnitude lower than what has been reported for zymography.

Functionalized tetrapod-like ZnO nanostructures for plasmid DNA purification, polymerase chain reaction and delivery

Functionalized tetrapodal ZnO nanostructures are tested in plasmid DNA experiments (1) as a solid-phase adsorbent for plasmid DNA purification, (2) as improving reagents in a polymerase chain reaction (PCR) and (3) as novel carriers for gene delivery. The amino-modification, the tetrapod-like shape of the nanostructure and its high biocompatibility all contribute to measurements showing promise for applications. A sol-gel method is used for silica coating and amino-modification. Plasmid DNA is purified through reversible conjugations of amino-modified ZnO tetrapods with DNA. Also, as additional reagents, functionalized tetrapods are shown to improve the amount of PCR product. For transfection, ZnO tetrapods provide some protection against deoxyribonuclease cleavage of plasmid DNA and deliver plasmid DNA into cells with little cytotoxicity.

Nanozymes: an emerging field bridging nanotechnology and biology

Key Laboratory of Protein and Peptide Pharmaceuticals, CAS-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, School of Medicine, Yangzhou University, Yangzhou 225001, China

Iron Oxide Nanozyme: A Multifunctional Enzyme Mimetic for Biomedical Applications

Iron oxide nanoparticles have been widely used in many important fields due to their excellent nanoscale physical properties, such as magnetism/superparamagnetism. They are usually assumed to be biologically inert in biomedical applications. However, iron oxide nanoparticles were recently found to also possess intrinsic enzyme-like activities, and are now regarded as novel enzyme mimetics. A special term, “Nanozyme”, has thus been coined to highlight the intrinsic enzymatic properties of such nanomaterials. Since then, iron oxide nanoparticles have been used as nanozymes to facilitate biomedical applications. In this review, we will introduce the enzymatic features of iron oxide nanozyme (IONzyme), and summarize its novel applications in biomedicine.

Human ferritin for tumor detection and therapy.

Ferritin, a major iron storage protein found in most living organisms, is composed of a 24-subunit protein cage with a hollow interior cavity. Serum ferritin serves as a critical marker to detect total body iron status. However, recent research reveals a number of novel functions of ferritin besides iron storage; for example, a ferritin receptor, transferrin receptor 1 (TfR1), has been identified and serum ferritin levels are found to be elevated in tumors. A particular new finding is that magnetoferritin nanoparticles, biomimetically synthesized using H-chain ferritin to form a 24-subunit cage with an iron oxide core, possess intrinsic dual functionality, the protein shell specifically targeting tumors and the iron oxide core catalyzing peroxidase substrates to produce a color reaction allowing visualization of tumor tissues. Here we attempt to summarize current research on ferritin, particularly newly identified functions related to tumors, in order to address current challenges and highlight future directions.

Binding Force Dynamics of Streptococcus mutans–glucosyltransferase B to Candida albicans

Candida albicans cells are often detected with Streptococcus mutans in plaque biofilms from children affected with early childhood caries. The coadhesion between these 2 organisms appears to be largely mediated by the S. mutans–derived exoenzyme glucosyltransferase B (GtfB); GtfB readily binds to C. albicans cells in an active form, producing glucans locally that provide enhanced binding sites for S. mutans. However, knowledge is limited about the mechanisms by which the bacterial exoenzyme binds to and functions on the fungal surface to promote this unique cross-kingdom interaction. In this study, we use atomic force microscopy to understand the strength and binding dynamics modulating GtfB–C. albicans adhesive interactions in situ. Single-molecule force spectroscopy with GtfB-functionalized atomic force microscopy tips demonstrated that the enzyme binds with remarkable strength to the C. albicans cell surface (~2 nN) and showed a low dissociation rate, suggesting a highly stable bond. Strikingly, the binding strength of GtfB to the C. albicans surface was ~2.5-fold higher and the binding stability, ~20 times higher, as compared with the enzyme adhesion to S. mutans. Furthermore, adhesion force maps showed an intriguing pattern of GtfB binding. GtfB adhered heterogeneously on the surface of C. albicans, showing a higher frequency of adhesion failure but large sections of remarkably strong binding forces, suggesting the presence of GtfB binding domains unevenly distributed on the fungal surface. In contrast, GtfB bound uniformly across the S. mutans cell surface with less adhesion failure and a narrower range of binding forces (vs. the C. albicans surface). The data provide the first insights into the mechanisms underlying the adhesive and mechanical properties governing GtfB interactions with C. albicans. The strong and highly stable GtfB binding to C. albicans could explain, at least in part, why this bacterially derived exoenzyme effectively modulates this virulent cross-kingdom interaction.