Jiang Pi

Folate-Chitosan Nanoparticles Loaded with Ursolic Acid Confer Anti-Breast Cancer Activities in vitro and in vivo.

Ursolic acid (UA) has proved to have broad-spectrum anti-tumor effects, but its poor water solubility and incompetent targeting property largely limit its clinical application and efficiency. Here, we synthesized a nanoparticle-based drug carrier composed of chitosan, UA and folate (FA-CS-UA-NPs) and demonstrated that FA-CS-UA-NPs could effectively diminish off-target effects and increase local drug concentrations of UA. Using MCF-7 cells as in vitro model for anti-cancer mechanistic studies, we found that FA-CS-UA-NPs could be easily internalized by cancer cells through a folate receptor-mediated endocytic pathway. FA-CS-UA-NPs entered into lysosome, destructed the permeability of lysosomal membrane, and then got released from lysosomes. Subsequently, FA-CS-UA-NPs localized into mitochondria but not nuclei. The prolonged retention of FA-CS-UA-NPs in mitochondria induced overproduction of ROS and destruction of mitochondrial membrane potential, and resulted in the irreversible apoptosis in cancer cells. In vivo experiments showed that FA-CS-UA-NPs could significantly reduce breast cancer burden in MCF-7 xenograft mouse model. These results suggested that FA-CS-UA-NPs could further be explored as an anti-cancer drug candidate and that our approach might provide a platform to develop novel anti-cancer drug delivery system.

Detection of lipopolysaccharide induced inflammatory responses in RAW264.7 macrophages using atomic force microscope

In recent years, LPS activated RAW264.7 cells are widely used as an in vitro inflammatory model for the screen of effective anti-inflammation drugs and the investigation of exact anti-inflammation mechanism of these drugs. But up to now, there are few data about the effect of LPS on the morphology, especially on the membrane ultrastructure and bio-mechanical properties of RAW264.7 macrophages. In this work, the topographical and biophysical changes of RAW264.7 macrophages upon LPS stimulation are detected by high resolution atomic force microscopy (AFM). The AFM results suggested that LPS activated RAW264.7 macrophages changed to be much bigger than control cells with some holes emerged on cell surface. The size of membrane protein clusters and the roughness of membrane significantly increased after LPS exposure. In addition, the AFM force measurement results demonstrated that LPS stimulation increased the adhesion force of RAW264.7 macrophages, and also increased the stiffness of RAW264.7 macrophages, which were attributed to the re-distribution of intracellular F-actin structures induced by LPS. These findings suggested that LPS stimulation could also induce the pathophysiological changes of RAW264.7 macrophages, which would benefit our understanding of the inflammatory processes in macrophages upon pathogen stimulation at nano-scale.

Synthesis, characterization and anticancer activity of kaempferol-zinc (II) complex

According to the previous studies, the anticancer activity of flavonoids could be enhanced when they are coordinated with transition metal ions. In this work, kaempferol-zinc(II) complex (kaempferol-Zn) was synthesized and its chemical properties were characterized by UV-VIS, FT-IR, (1)H NMR, elemental analysis, electrospray mass spectrometry (ES-MS) and fluorescence spectroscopy, which showed that the synthesized complex was coordinated with a Zn(II) ion via the 3-OH and 4-oxo groups. The anticancer effects of kaempferol-Zn and free kaempferol on human oesophageal cancer cell line (EC9706) were compared. MTT results demonstrated that the killing effect of kaempferol-Zn was two times higher than that of free kaempferol. Atomic force microscopy (AFM) showed the morphological and ultrastructural changes of cellular membrane induced by kaempferol-Zn at subcellular or nanometer level. Moreover, flow cytometric analysis indicated that kaempferol-Zn could induce apoptosis in EC9706 cells by regulating intracellular calcium ions. Collectively, all the data showed that kaempferol-Zn might be served as a kind of potential anticancer agent.

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.

Dihydromyricetin suppresses inflammatory responses in vitro and in vivo through inhibition of IKKβ activity in macrophages

Dihydromyricetin (DMY) a flavonoid derived from medicinal plant Ampelopsis grossedentata, possesses anti-oxidative and anti-inflammatory effects in vitro, however, the in vivo anti-inflammatory action of DMY remains unknown. In the current study, carrageenan-induced paw edema in rat, an acute inflammation model, and RAW264.7 macrophages activated by LPS were employed to evaluate the anti-inflammatory potency of DMY in vivo and in vitro. Results showed that DMY significantly attenuated rat paw edema induced by carrageenan. Also, DMY markedly inhibited NO secretion, iNOS, and COX-2 protein expression, as well as p65 phosphorylation via suppression of IKKβ activity and IKKα/β phosphorylation in RAW264.7 cells. And using high resolution Atomic Force Microscope (AFM), we also proved that DMY prevented morphological change and membrane alterations of RAW 264.7 macrophages caused by LPS stimulation. As activation of macrophages is one of major factors in carrageenan-induced paw edema of rats, the anti-inflammatory action of DMY is suggested to be closely associated with suppression of macrophage activation. These findings indicate that DMY is valuable of being further investigated as a candidate new agent for treating inflammatory conditions, and suggest that AFM could be a powerful nanotool for anti-inflammatory investigations. SCANNING 38:901-912, 2016.

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.

Apigenin induced apoptosis in esophageal carcinoma cells by destruction membrane structures

Apigenin has shown to have killing effects on some kinds of solid tumor cells. However, the changes in cell membrane induced by apigenin on subcellular- or nanometer-level were still unclear. In this work, human esophageal cancer cells (EC9706 and KYSE150 cells) were employed as cell model to detect the cytotoxicity of apigenin, including cell growth inhibition, apoptosis induction, membrane toxicity, etc. MTT assay showed that apigenin could remarkably inhibit the growth and proliferation in both types of cells. Annexin V/PI-based flow cytometry analysis showed that the cytotoxic effects of apigenin in KYSE150 cells were mainly through early apoptosis induction, while in EC9706 cells, necrosis, and apoptosis were both involved in cell death. The morphological and ultrastructural properties induced by apigenin were investigated at single cellular- or nanometer-level using atomic force microscopy (AFM). Additionally, lactate dehydrogenase (LDH) leakage was measured to assess the changes in membrane permeability. The results indicated that apigenin increased the membrane permeability and caused leakage of LDH, which was consistent with damages on membrane ultrastructure detected by AFM. Therefore, membrane toxicity, including membrane ultrastructure damages and enhanced membrane permeability, played vital roles in apigenin induced human esophageal cancer cell apoptosis. SCANNING 38:322-328, 2016.

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.

Investigation of quercetin-induced HepG2 cell apoptosis-associated cellular biophysical alterations by atomic force microscopy

Quercetin, a wildly distributed bioflavonoid, has been proved to possess excellent antitumor activity on hepatocellular carcinoma (HCC). In the present study, the biophysical properties of HepG2 cells were qualitatively and quantitatively determined using high resolution atomic force microscopy (AFM) to understand the anticancer effects of quercetin on HCC cells at nanoscale. The results showed that quercetin could induce severe apoptosis in HepG2 cells through arrest of cell cycle and disruption of mitochondria membrane potential. Additionally, the nuclei and F-actin structures of HepG2 cells were destroyed by quercetin treatment as well. AFM morphological data showed some typical apoptotic characterization of HepG2 cells with increased particle size and roughness in the ultrastructure of cell surface upon quercetin treatment. As an important biophysical property of cells, the membrane stiffness of HepG2 cells was further quantified by AFM force measurements, which indicated that HepG2 cells became much stiffer after quercetin treatment. These results collectively suggest that quercetin can be served as a potential therapeutic agent for HCC, which not only extends our understanding of the anticancer effects of quercetin against HCC cells into nanoscale, but also highlights the applications of AFM for the investigation of anticancer drugs.

Atomic force microscopy study of ionomycin-induced degranulation in RBL-2H3 cells

Mast cell degranulation is the typical anaphylaxis process of mast cells associated with the release of cytokines, eicosanoids and their secretory granules, which play very important roles in the allergic inflammatory response of the human body upon anaphylactogen stimulation. The calcium ionophore ionomycin is widely used as a degranulation induction agent for mast cell degranulation studies. In the present work, ionomycin-induced degranulation of RBL-2H3 basophilic leukemia cell line cells was investigated in vitro by high resolution atomic force microscopy (AFM). Ionomycin, which could increase the intracellular free Ca2+ level and β-Hexosaminidase release, was found to induce the formation of a kind of peculiar vesicles in the cytoplasm area of RBL-2H3 cells. Those vesicles induced by ionomycin would desintegrate to release a larger amount of granules surrounding RBL-2H3 cells by the controlling of F-actin. These results provide the precise morphological information of ionomycin-induced mast cell degranulation at nanoscale, which could benefit our understanding of ionomycin-induced mast cell anaphylaxis model and also validate the applicability of AFM for the detection of allergic inflammatory response in mast cells. SCANNING 38:525-534, 2016.