Huai-Hong Cai

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells.

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA–Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA–Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA–Se NPs in MCF-7 cells. The results indicated that the 70-nm FA–Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA–Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca2+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA–Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA–Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA–Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.

Gold nanoprobes-based resonance Rayleigh scattering assay platform: Sensitive cytosensing of breast cancer cells and facile monitoring of folate receptor expression

A rapid, facile assay for sensitive cytosensing of breast cancer cells should help to guide potential medical evaluation for breast cancer. Here, we report development of novel resonance Rayleigh scattering (RRS) cytosensor for cell recognitions and folate (FA) receptor expression analyses on living cells. Using FA-conjugated gold nanoparticles (FA-AuNPs) as nanoprobes, the constructed nanoprobes-assembled recognition interface could increase the binding capacity for cell recognition, amplify Au-aggregates-enhanced RRS signal, and then enhance the sensitivity for membrane antibody assay. FA-AuNPs-based RRS measurements enabled a distinct 34-times-enhancement in RRS intensities after incubation with human breast cancer cells, compared with normal cells. Receptor-targeted cytosensor was used to quantitatively detect human breast cancer MCF-7, liver cancer HepG2 and normal cells, which expressing different amount of FA receptor, respectively. The detection limit for MCF-7 cells was 12 cells/mL with good selectivity and reproducibility. Furthermore, the proposed cytosensor allowed for dynamic evaluation of FA receptor expression on different living cells after dihydroartemisinin stimulus. This assay platform shows the good potential for clinical diagnostics and antibody-targeted drug screening.

Synthesis and synergetic effects of chrysin-organogermanium (IV) complex as potential anti-oxidant.

Organogermanium(IV) (Ge) is considered to play an important role in the anti-oxidative activities of some Chinese medicines. Here, a new chrysin-organogermanium (Chry-Ge) complex was synthesized and investigated for its potential biological activities. The radicals-sensitive Ge-O bond was introduced to Chry-Ge complex to enhance bioactivities of organic Ge or Chry. Results showed that Chry-Ge complex possessed great anti-oxidative activities, showing stronger hydroxyl scavenging effects than their corresponding ligands. We also demonstrated Chry-Ge complex inhibited ROS-dependent oxidative damage in cells. Moreover, the morphological and biophysical recoveries in oxidation-damaged cells induced by Chry-Ge complex were characterized by atomic force microscopy. All these results collectively suggested that Chry-Ge complex has synergetic effect for radicals scavenging and could be served as promising pharmacologically active agent against anti-oxidative treatment.

Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells

Graphical abstract Figure. No Caption available. ABSTRACT As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM‐SMFS) was used for real‐time and in‐situ detection of EGF‐EGFR interactions in living esophageal cancer KYSE‐150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE‐150 cells. AFM‐SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE‐150 cells by decreasing the unbinding force and binding probability for EGF‐EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM‐SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF‐EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF‐EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF‐EGFR interactions in ROS dependent mechanism, but also highlighted AFM‐SMFS as a powerful technique for pharmacodynamic studies by detecting ligand‐receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs.

Hydroxyl radical scavenging mechanism of human erythrocytes by quercetin–germanium (IV) complex

Quercetin is a popular flavonoid in plant foods, herbs, and dietary supplement. Germanium, a kind of trace elements, can enhance the body immunity. This study investigated the hydroxyl-radical-scavenging mechanism of the quercertin-germanium (IV) (Qu-Ge) complex to human erythrocytes, especially the effects on ultrastructure and mechanical properties of cell membrane, plasma membrane potential and intracellular free Ca(2+) concentration. Results showed that QuGe(2), a kind of the Qu-Ge complex, could reduce the oxidative damage of erythrocytes, change the cell-surface morphology, and partly recover the disruption of plasma membrane potential and intracellular free Ca(2+) level. Atomic force microscopy (AFM) was used to characterize the changes of the cell morphology, cell-membrane ultrastructure and biophysical properties at nanoscalar level. QuGe(2) has triggered the antioxidative factor to inhibit cellular damage. These results can improve the understanding of hydroxyl-radical-scavenging mechanism of human erythrocytes induced by the Qu-Ge complex, which can be potentially developed as a new antioxidant for treatment of oxidative damage.

Qualitative and Quantitative Analysis of ROS-Mediated Oridonin-Induced Oesophageal Cancer KYSE-150 Cell Apoptosis by Atomic Force Microscopy.

High levels of intracellular reactive oxygen species (ROS) in cells is recognized as one of the major causes of cancer cell apoptosis and has been developed into a promising therapeutic strategy for cancer therapy. However, whether apoptosis associated biophysical properties of cancer cells are related to intracellular ROS functions is still unclear. Here, for the first time, we determined the changes of biophysical properties associated with the ROS-mediated oesophageal cancer KYSE-150 cell apoptosis using high resolution atomic force microscopy (AFM). Oridonin was proved to induce ROS-mediated KYSE-150 cell apoptosis in a dose dependent manner, which could be reversed by N-acetylcysteine (NAC) pretreatment. Based on AFM imaging, the morphological damage and ultrastructural changes of KYSE-150 cells were found to be closely associated with ROS-mediated oridonin-induced KYSE-150 cell apoptosis. The changes of cell stiffness determined by AFM force measurement also demonstrated ROS-dependent changes in oridonin induced KYSE-150 cell apoptosis. Our findings not only provided new insights into the anticancer effects of oridonin, but also highlighted the use of AFM as a qualitative and quantitative nanotool to detect ROS-mediated cancer cell apoptosis based on cell biophysical properties, providing novel information of the roles of ROS in cancer cell apoptosis at nanoscale.

Construction of An Electrochemical Cytosensor Based on Polyaniline Nanofiber/Gold Nanoparticle Interface and Application to Detection of Cancer Cells

Abstract Polyaniline nanofibers (PANI-NF) were first synthesized with diameters of approximately 60–80 nm. PANI-NF/gold nanoparticle (PANI-NF/AuNP) nanocomposite was then fabricated through a simple electrostatic reaction between the positive charges on the surface of PANI-NF and the negatively charged AuNPs. Thereafter, a novel electrochemical cytosensor was designed by the self-assembly of folate on PANI-NF/AuNP nanocomposite film. An electrochemical impedance method was used to detect cancer cells based on the specific interaction between the folate receptor on cancer cell membranes and folate immobilized on PANI-NF/AuNP surface. Experimental results showed that the PANI-NF/AuNP cytosensor specifically recognized cancer cells, such as HeLa cells, which was reflected by the change in electrode impedance after attachment of the cancer cells. The linear detection range was from 1.0 × 104 to 6.4 × 106 cells mL−1, and the detection limit was 2000 cells mL−1. This cytosensor has several advantages, such as easy construction, short response time, high sensitivity, easy regeneration, good stability, and freedom of additional labeling, which can be thus used for a highly sensitive detection of cancer cells.

GE11 peptide conjugated selenium nanoparticles for EGFR targeted oridonin delivery to achieve enhanced anticancer efficacy by inhibiting EGFR-mediated PI3K/AKT and Ras/Raf/MEK/ERK pathways

Abstract Selenium nanoparticles (Se NPs) have attracted increasing interest in recent decades because of their anticancer, immunoregulation, and drug carrier functions. In this study, GE11 peptide-conjugated Se NPs (GE11-Se NPs), a nanosystem targeting EGFR over-expressed cancer cells, were synthesized for oridonin delivery to achieve enhanced anticancer efficacy. Oridonin loaded and GE11 peptide conjugated Se NPs (GE11-Ori-Se NPs) were found to show enhanced cellular uptake in cancer cells, which resulted in enhanced cancer inhibition against cancer cells and reduced toxicity against normal cells. After accumulation into the lysosomes of cancer cells and increase of oridonin release under acid condition, GE11-Ori-Se NPs were further transported into cytoplasm after the damage of lysosomal membrane integrity. GE11-Ori-Se NPs were found to induce cancer cell apoptosis by inducting reactive oxygen species (ROS) production, activating mitochondria-dependent pathway, inhibiting EGFR-mediated PI3K/AKT and inhibiting Ras/Raf/MEK/ERK pathways. GE11-Se NPs were also found to show active targeting effects against the tumor tissue in esophageal cancer bearing mice. And in nude mice xenograft model, GE11-Ori-Se NPs significantly inhibited the tumor growth via inhibition of tumor angiogenesis by reducing the angiogenesis-marker CD31 and activation of the immune system by enhancing IL-2 and TNF-α production. The selenium contents in mice were found to accumulate into liver, tumor, and kidney, but showed no significant toxicity against liver and kidney. This cancer-targeted design of Se NPs provides a new strategy for synergistic treating of cancer with higher efficacy and reduced side effects, introducing GE11-Ori-Se NPs as a candidate for further evaluation as a chemotherapeutic agent for EGFR over-expressed esophageal cancers.

Atomic force microscopy based investigations of anti-inflammatory effects in lipopolysaccharide-stimulated macrophages.

A new method based on atomic force microscopy (AFM) was developed to investigate the anti-inflammatory effects of drugs on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The LPS-stimulated RAW264.7 macrophage cell line is a widely used in vitro cell model for the screening of anti-inflammatory drugs or the study of anti-inflammatory mechanisms. In this work, the inhibitory effects of dexamethasone and quercetin on LPS–CD14 receptor binding in RAW264.7 macrophages was probed by LPS-functionalized tips for the first time. Both dexamethasone and quercetin were found to inhibit LPS-induced NO production, iNOS expression, IκBα phosphorylation, and IKKα/β phosphorylation in RAW264.7 macrophages. The morphology and ultrastructure of RAW264.7 macrophages were determined by AFM, which indicated that dexamethasone and quercetin could inhibit LPS-induced cell surface particle size and roughness increase in RAW264.7 macrophages. The binding of LPS and its receptor in RAW264.7 macrophages was determined by LPS-functionalized AFM tips, which demonstrated that the binding force and binding probability between LPS and CD14 receptor on the surface of RAW264.7 macrophages were also inhibited by dexamethasone or quercetin treatment. The obtained results imply that AFM, which is very useful for the investigation of potential targets for anti-inflammatory drugs on native macrophages and the enhancement of our understanding of the anti-inflammatory effects of drugs, is expected to be developed into a promising tool for the study of anti-inflammatory drugs.

Efficient electrochemical detection of cancer cells on in situ surface-functionalized MoS2 nanosheets

Surface engineering is crucial to improve the biocompatibility and sensing response of two-dimensional (2D) nanomaterials. For nanostructured MoS2 biosensors, post functionalization via cumbersome procedures unfortunately leads to inevitable structural damage and thus reduced functionalities. Herein, in situ surface functionalization by the reactant thiourea (TU) was employed to one-step fabricate TU-capped MoS2 (TU-MoS2) nanosheets. The amino-group terminated surface of TU-MoS2 favours immobilization of the GE11 peptide that can specifically recognize cancer cells. The resulting sensor shows high sensitivity and selectivity in detecting cancer cells, relying on the varied expression of the epidermal growth factor receptor (EGFR) on cell membranes. In the case of human liver cancer cells, it is featured by a wide linear range (50–106 cells mL−1) and a low detection limit (50 cells mL−1) in electrochemical impedance spectroscopy, as the variation of charge-transfer resistance is plotted against cell concentration. Furthermore, it exhibits good efficiency in monitoring the dynamic variation of EGFR expression on living cells in response to drug treatment, which is promising for clinical diagnosis and drug screening in miniaturization. By elucidating an efficient biosensing platform on the basis of surface engineered MoS2 nanosheets, this work sheds some light on the development of biosensing technology and relevant materials.