Hongying Jia

Targeted surface-functionalized gold nanoclusters for mitochondrial imaging

Due to mitochondria involved in both apoptotic and necrotic cell death, labeling and imaging mitochondria has attracted considerable interest. However, conventional organic dyes used for mitochondrial imaging are limited because of their poor photostability. Considering that gold nanoclusters (AuNCs) possess some advantages over considerable interest, such as excellent photostability and strong fluorescence emission, we herein prepared a mitochondria-targeted fluorescent probe, AuNCs@CS-TPP, based on a covalent link between triphenylphosphonium (TPP) cations and chitosan-coated AuNCs (AuNCs@CS). The as-prepared AuNCs@CS-TPP exhibited a bluish fluorescence emission at 440 nm with a quantum yield of 8.5%. Meanwhile, the fluorescence intensity of AuNCs@CS-TPP labeled HeLa cells did not show apparent decrease after 8 min irradiation. Cytotoxicity assay showed that AuNCs@CS-TPP did not display any appreciable cytotoxicity on cells even at a concentration of 60 μg mL(-1). In addition, the result of fluorescence co-localization imaging in vitro indicated that AuNCs@CS-TPP could selectively accumulate into mitochondria of HeLa cells and HepG2 cells. These findings demonstrated that AuNCs@CS-TPP possessed superior photostability, low cytotoxicity, high sensitivity and target-specificity to mitochondria, allowing labeling and imaging of the mitochondria in living cells.

Protecting effect of phosphorylation on oxidative damage of D1 protein by down-regulating the production of superoxide anion in photosystem II membranes under high light

The physiological significance of photosystem II (PSII) core protein phosphorylation has been suggested to facilitate the migration of oxidative damaged D1 and D2 proteins, but meanwhile the phosphorylation seems to be associated with the suppression of reactive oxygen species (ROS) production, and it also relates to the degradation of PSII reaction center proteins. To more clearly elucidate the possible protecting effect of the phosphorylation on oxidative damage of D1 protein, the degradation of oxidized D1 protein and the production of superoxide anion in the non-phosphorylated and phosphorylated PSII membranes were comparatively detected using the Western blotting and electron spin resonance spin-trapping technique, respectively. Obviously, all of three ROS components, including superoxide anion, hydrogen peroxide and hydroxyl radical are responsible for the degradation of oxidized D1 protein, and the protection of the D1 protein degradation by phosphorylation is accompanied by the inhibition of superoxide anion production. Furthermore, the inhibiting effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a competitor to QB, on superoxide anion production and its protecting effect on D1 protein degradation are even more obvious than those of phosphorylation. Both DCMU effects are independent of whether PSII membranes are phosphorylated or not, which reasonably implies that the herbicide DCMU and D1 protein phosphorylation probably share the same target site in D1 protein of PSII. So, altogether it can be concluded that the phosphorylation of D1 protein reduces the oxidative damage of D1 protein by decreasing the production of superoxide anion in PSII membranes under high light.

New photostable naphthalimide-based fluorescent probe for mitochondrial imaging and tracking.

Monitoring mitochondria morphological changes temporally and spatially exhibits significant importance for diagnosing, preventing and treating various diseases related to mitochondrial dysfunction. However, the application of commercially available mitochondria trackers is limited due to their poor photostability. To overcome these disadvantages, we designed and synthesized a mitochondria-localized fluorescent probe by conjugating 1,8-naphthalimide with triphenylphosphonium (i.e. NPA-TPP). The structure and characteristic of NPA-TPP was characterized by UV-vis, fluorescence spectroscopy, (1)HNMR, (13)CNMR, FTIR, MS, etc. The photostability and cell imaging were performed on the laser scanning confocal microscopy. Moreover, the cytotoxicity of NPA-TPP on cells was evaluated using (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The results showed that NPA-TPP not only has high sensitivity and specificity to mitochondria, but also exhibits super-high photostability, negligible cytotoxicity and good water solubility. In short, NPA-TPP indicates great potential for targeting mitochondria and enables a real-time and long-term tracking mitochondrial dynamics changes.

Mechanism and Cellular Kinetic Studies of the Enhancement of Antioxidant Activity by Using Surface-Functionalized Gold Nanoparticles

The enhanced antioxidant activity of surface-functionalized gold nanoparticles (AuNPs) synthesized by self-assembly has attracted great attention, but little is known about the mechanism behind the enhanced activity. To address this challenge, the antioxidant activity of Au@PEG3SA (i.e., surface-functionalization of spherical AuNPs with the antioxidant salvianic acid A) was used as an example to illustrate the mechanism of the enhanced activity. Evaluation of the antioxidant activity was performed in a radical-scavenging reaction between Au@PEG3SA and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. As expected, the rate constant for the reaction of Au@PEG3SA with DPPH was about nine times greater than that for the salvianic acid A monomer. A comparative analysis of the spectral characteristics of Au@PEG3SA and the salvianic acid A monomer further imply that the enhancement of the antioxidative reaction kinetics may be ascribed to the variation in the transition state for the DPPH-radical scavenging reaction through π-π stacking interactions between and among adjacent groups on the surface of Au@PEG3SA. On the other hand, the kinetic enhancement of Au@PEG3SA on reactive-oxygen-species (ROS) scavenging can be observed in living cells and in vivo, which possibly provides new insight for the bioapplication of self-assembly of surface-functionalized AuNPs.

Polyamidoamine dendrimer conjugated chitosan nanoparticles for the delivery of methotrexate

Encapsulating anticancer drugs to synthetic polymer is a promising approach to improve the efficiency and reduce the side effects of anticancer drugs. In this study, novel chitosan derivatives with polyamidoamine moieties (CS-PAMAM) were synthesized and characterized by morphology, particle size, and zeta potential. Then the anticancer drug-methotrexate-encapsulated CS-PAMAM was prepared by hydrophobic-hydrophilic interactions. The drug release assay showed that the amount of the methotrexate release from CS-PAMAM was pH depended. Meanwhile, the cell viability assay illustrated that CS-PAMAM was suitable for the drug delivery because of its low cytotoxicity on cells. Moreover, our results showed that the CS-PAMAM could significantly improve the cytotoxicity of free methotrexate on A549 cells. These results demonstrate that CS-PAMAM may provide a suitable platform for the water-insoluble drug delivery.

Detection of nitric oxide in macrophage cells for the assessment of the cytotoxicity of gold nanoparticles

In this paper, an electrochemical method using a nitric oxide sensor was employed for quantitative evaluation of NO released from AuNPs-treated macrophage cells. Our results indicate that the AuNPs initiate NO release from macrophage cells and the amount of NO released is positively correlated with concentration of AuNPs. Meanwhile, total nitrite/nitrate concentrations in the AuNPs-treated macrophage cells have been determined via the Griess reaction and we demonstrate that the variation of the nitrite/nitrate concentrations is in accordance with that measured by the electrochemical method. In contrast to the citrate-coated gold nanoparticles (CT-AuNPs), when AuNPs were protected by thiolated poly ethylene glycol (PEG), the NO-releasing in macrophage upon the addition of AuNPs was relieved, implying that the PEG-coated AuNPs having less cytotoxicity and oxidative stress potential is probably due to inhibition of NO production. In conclusion, this work has demonstrated an effective sensing platform for the evaluation of the cytotoxicity of AuNPs by detecting the extracellular NO released from macrophage cells.

Hydroxyethylated graphene oxide as potential carriers for methotrexate delivery

In this study, we presented a simple approach to prepare hydroxyethylated graphene oxide (HE-GO) with high water solubility and physiological stability. The successful synthesis of HE-GO was confirmed by UV–Vis spectroscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The loading of anticancer drug methotrexate (MTX) onto this nanocarrier (MTX/HE-GO) was investigated. The results of in vitro drug release experiment showed that the rate of MTX release from MTX/HE-GO was pH dependent. Moreover, cell viability assay demonstrated that HE-GO loaded with MTX exhibits higher anticancer activity against human lung adenocarcinoma epithelial cell line than non-vehicle MTX.

Highly sensitive free radical detection by nitrone-functionalized gold nanoparticles

The detection of free radicals and related species has attracted significant attention in recent years because of their critical roles in physiological and pathological processes. Among the methods for the detection of free radicals, electron spin resonance (ESR) coupled with the use of the spin trapping technique has been an effective approach for characterization and quantification of these species due to its high specificity. However, its application in biological systems, especially in in vivo systems, has been greatly limited partially due to the low reaction rate between the currently available spin traps with biological radicals. To overcome this drawback, we herein report the first example of nitrone functionalized gold nanoparticles (Au@EMPO) as highly efficient spin traps in which the thiolated EMPO (2-(ethoxycarbonyl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide) derivative was self-assembled on gold nanoparticles. Kinetic studies showed that Au@EMPO has a 137-fold higher reaction rate constant with ˙OH than PBN (N-tert-butyl-α-phenylnitrone). Owing to the high rate of trapping ˙OH by Au@EMPO as well as the high stability of the resulting spin adduct (t½ ∼ 56 min), Au@EMPO affords 124-fold higher sensitivity for ˙OH than EMPO. Thus, this new nanospin trap shows great potential in trapping the important radicals such as ˙OH in various biological systems and provides a novel strategy to design spin traps with much improved properties.

An effective strategy for the synthesis of biocompatible gold nanoparticles using danshensu antioxidant: prevention of cytotoxicity via attenuation of free radical formation

Abstract To suppress the cytotoxicity of gold nanoparticles (AuNPs), danshensu, a naturally occurring polyphenol antioxidant isolated from Chinese herb, was used to provide a fundamental protection layer for AuNPs, to alleviate oxidative stress and as a reducing agent to react with chloroauric acid. Besides danshensu, gum arabic was chosen as an auxiliary stabilising agent to improve the stability of AuNPs against aggregation. As expected, the prepared GA–DS–AuNPs (gum arabic–danshensu–gold nanoparticle) was remarkably stable in various buffer solutions. More interestingly, the GA–DS–AuNPs not only did not show any appreciable cytotoxicity, but also could alleviate the oxidative damage induced by AuNPs. Meanwhile, the ROS/RNS scavenging activities of GA–DS–AuNPs was evaluated by electron spin resonance spectroscopy (ESR), potentiometric nitric oxide (NO) sensor and cell confocal imaging. The results suggest that GA–DS–AuNPs might have effectively reduced the AuNPs-induced cytotoxicity and oxidative stress by downregulation of ROS/NOS production. The GA–DS–AuNPs may provide potential opportunities for the application in nanomedicine and nanobiology.

The protective effects of Trolox-loaded chitosan nanoparticles against hypoxia-mediated cell apoptosis

Antioxidants have potentials to treat hypoxia-mediated oxidative stress related diseases. However, their therapeutic efficacy is restricted due to its poor cellular uptake efficiency and poor cell membrane permeability. To resolve these issues, we prepare the hydroxyethylated chitosan nanoparticles as drug carriers for the delivery of 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox), which was considered as a model compound. The experiment on cellular uptake and subcellular localization of Trolox-loaded chitosan nanoparticles (Trolox-CSNPs) indicate that Trolox-CSNPs enter the cells via the caveolae-mediated endocytosis pathway and traffic with endosomes. Furthermore, compared with Trolox, Trolox-CSNPs exert a higher protective effect against the hypoxia-mediated oxidative stress. Molecular basis of apoptosis study reveals that Trolox-CSNPs can directly block the mitochondria dependent apoptotic pathway through up-regulation of Bcl-2 expression and inhibiting the activation of Bax, Caspase-3 expression. In conclusion, the hydroxyethylated chitosan is a promising drug nanocarrier to deliver antioxidants for the treatment of hypoxia-mediated disease. From the clinical editor: Antioxidants are potentially beneficial in oxidative stress-related diseases, although cellular uptake of most antioxidants is suboptimal. In this study, hydroxyethylated chitosan nanoparticles are demonstrated as promising drug carriers in a Trolox-model system.