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Cell surface molecules such as receptors play an important role to regulate many essential cellular processes, including cell adhesion, tissue development, cellular communication, inflammation, tumor metastasis, and microbial infection. Specially, these events often involve multimolecular interactions occurring on a nanometer scale, and how to image the distribution and organization of cell surface molecules are becoming increasingly required in Cell and Molecular biology Sciences. By combing atomic force microscopy (AFM), near-field scanning microscopy (NSOM) and quantum dots (QD) labeling, a novel AFM and NSOM/QD-based dual-color nanoscale imaging system is constructed to directly visualize the distribution and organization of these molecules on cell-membrane surface. And this will supply a powerful tool for direct molecular visualization at the single-cell level.

At the prosperous fields of nanoscience and biology, the nanoscale analysis of cells and cell membrane using atomic force microscopy (AFM) is an exciting and rapidly developing research area. Over the past decade, there has been tremendous progress in the use of AFM to observe living cells and membrane proteins at high resolution. Remarkable advances have been made in applying AFM-based force spectroscopy techniques to characterize surface biomechanical properties, to map receptor sites on cell surface, and to measure cellular interactions at the single-cell or single-molecule level. Moreover, recent developments in AFM combining advanced optical microscopy, such as confocal microscopy and near-field scanning optical microscopy, opened up new avenues for cell imaging at nanoscale resolution.

Identification of the nanoscale changes that take place in cell membrane (CM) morphology or cell biomechanical properties (CBPs) in disease states or in response to drug treatment enable for a better understanding of the effects of the drugs on disease pathogenesis and recovery. CM proteins and CBPs have a crucial role in the regulation of many physiological and pathological processes. Direct assessment of the CM and CBPs is therefore useful not only for a better appreciation of cell structure but also for a better understanding of cell pathophysiology. Atomic force microscopy (AFM) is a technique that can be employed to assess CM structure and CBPs at the nanometer scale. In the first part of this chapter, we describe the principles of AFM and appraise its value in the assessment of CM morphology and CBPs. In the second part, we review examples of disease- or drug-dependent changes in CM morphology and CBPs that have been elucidated using AFM.

Summary Although the importance of B7/CD28 co-stimulation has been widely studied, little is known about their nano-spatial localization and their corresponding cells' biophysical and biomechanical properties. Here, we investigated the morphological, biophysical, and biomechanical properties of T cells and dendritic cells (DCs) by atomic force microscopy (AFM) and force curves. The nano-spatial distribution of CD28 and CD86 antigen on T cells and DCs was detected by CD86 or CD28 antibody-functionalized AFM tip. Single-molecule force spectroscopy (SMFS)-based force volumes and quantum dots (QDs)-based fluorescence imaging demonstrated that the co-stimulatory molecules were not randomly distributed over the cells' surface, but more than 80% of CD28 and CD86 molecules appeared to be expressed as 100–200 nm nanoclusters and polarize dominantly in the peak of the cell membrane fluctuations. AFM imaging and quantitative analysis showed that the roughness of mature DCs (mDCs) was higher than that of immature DCs (iDCs). The adhesion force distribution of iDCs and mDCs was heterogeneous while the elasticity distribution was homogeneous locally. In addition, mDCs had a fourfold increase of Young's modulus of iDCs, indicating the contribution of the actin cytoskeleton to the elastic properties of the cells. Taken together, the nano-cluster distribution of CD28 and CD86, the rough mDCs surface, the higher adhesion force and elasticity of mDCs may facilitate to the occurrence of B7/CD28 co-stimulation signals and the formation of immune synapse. These nanoscale findings provide new insights into the antigen-presenting function of DCs, the T cell activation and ultimate immune response. SCANNING 9999:1–11, 2015. © 2015 Wiley Periodicals, Inc.

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.

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.

Water-soluble germanium-doped cadmium sulfide quantum dots (Ge:CdS q-dots) were successfully synthesized by a facile one-pot method, and used as fluorescent probes for labeling live MCF-7 cells. The optical properties of the q-dots and several key parameters of the synthesis conditions were studied, and the mechanism of these parameters was also discussed. After doping with germanium, the PL intensity increased 50%, while improving the photostability and fluorescence quantum yield. Furthermore, the toxicity of CdS quantum dot decreased after doping with germanium due to its low toxicity, which greatly improved the CdS quantum dots biocompatibility. Then the doped quantum dots modified with folic acid (FA) to use as fluorescent probes, which expanded the application of semiconductor quantum dots in the biomedical field.

Summary Colorectal cancer, a kind of malignant cancer, has more than 1 million new patients and results in 0.5 million deaths every year globally based on the estimation of Globocan in 2008. One of the most important issues against colon cancer is tumor metastasis. Anti-angiogenesis, a form of targeted therapy uses drugs or other substances to prevent the new blood vessel formation, which is critical for tumor metastasis. In our previous studies, we have demonstrated a simple method to synthesize Chry–Ge complex through the reaction between chrysin and triphenylgermanium bromide. In this work, we investigated the mechanism of Chry–Ge induced Colo205 cell apoptosis. We found that Chry–Ge could induce apoptosis in Colo205 cells in mitochondrial-dependent pathway, cause the reorganization of cytoskeleton and induce the damage of nucleus in Colo205 cells. Besides, Chry–Ge was also found to induce membrane ultrastructural changes in Colo205 cells by AFM. Further, we found that Chry–Ge can inhibit tube formation of human umbilical vascular endothelial cell in vitro. Chry–Ge was also tested in vivo in the chicken chorioallantoic membrane (CAM) assay and found to inhibit bFGF-treated CAMs development. These results suggested that Chry–Ge could induce Colo205 cell apoptosis by mitochondrial pathway and anti-angiogenesis, highlighting the use of organic germanium agents for the treatment of colorectal cancer. SCANNING 37:246–257, 2015. © 2015 Wiley Periodicals, Inc.

Quartz crystal microbalance with dissipation (QCM-D) monitoring was used for real-time and label-free detection of changes and folate receptor (FR) expression levels on living MCF-7 cells for evaluating the anticancer activity of resveratrol. Here, the mechanical changes of cellular responses to resveratrol were tracked by a poly(l-lysine) (PLL) modified QCM-D sensor, and the inhibition effect of resveratrol on FR expression levels on MCF-7 cells was monitored by chitosan-folic acid (CS-FA) composite membrane functionalized Au substrate for the first time. Changes in morphology and the cellular state of MCF-7 cells stimulated by resveratrol at different concentrations were detected by inverted fluorescence microscopy and flow cytometry. Atomic force microscopy confirmed that resveratrol influenced the cellular mechanical properties. The results indicated that the MCF-7 cells lose their original elasticity and increase their stiffness induced by resveratrol. It was further observed by confocal fluorescence imaging that resveratrol reduced the FR expression levels on the living cells surface. This study established a typical model of the QCM-D biosensor to evaluate the protein biomarker expression levels on the cells surface. QCM-D, which was used to investigate potential targets for an antitumor drug on living cells and realize a better understanding of the drug action mechanism, was expected to be developed into a promising tool for the screening of drugs.

Type 1 diabetes is an insulin-dependent metabolic disorder always associated with ketoacidosis and a high morbidity rate in teenagers. The in situ single molecule detection of insulin receptors on healthy and diseased erythrocytes is helpful to understand the pathomechanism of type 1 diabetes ketoacidosis (T1-DKA), which would also benefit the diagnosis and treatment of T1-DKA. Here, we demonstrated, for the first time, the single molecule interaction between insulin and insulin receptor on erythrocytes from a healthy volunteer and a T1-DKA patient using high sensitivity atomic force microscopy (AFM) in PBS solution. The single molecule force results demonstrated the decreased binding force and binding probability between insulin and insulin receptor on T1-DKA erythrocytes, implying the deficit of insulin receptor functions in T1-DKA. The binding kinetic parameters calculated from dynamic force spectroscopy indicated that the insulin-insulin receptor complexes on T1-DKA erythrocytes were less stable than those from healthy volunteer. Using high resolution AFM imaging, a decreased roughness was found both in intact T1-DKA erythrocytes and in the purified membrane of T1-DKA erythrocytes, and an increased stiffness was also found in T1-DKA erythrocytes. Moreover, AFM, which was used to investigate the single molecule interactions between insulin-insulin receptor, cell surface ultrastructure and stiffness in healthy and diseased erythrocytes, was expected to develop into a potential nanotool for pathomechanism studies of clinical samples at the nanoscale.

Nanoparticle aggregates induced by synergistic effect of electrostatic interaction and hydrogen-bonding recognition between melamine and 3-mercaptopriopionic acid (MA), which conjugated on the surface of gold nanoparticles (AuNPs) are used for detection of melamine. MA molecules are conjugated on AuNP surfaces to form MA-modified AuNPs (MA-AuNPs), acting as nanoprobes in the detection of melamine. Since such nanoparticle aggregates-mediated signal amplification can be measured by absorption spectroscopy, the method enables sensitive and real-time detection of melamine at the detection limit as low as 0.4 μg/ml, and the linear detection ranging from 0.6 μg/ml to 42 μg/ml. The color change can be readily seen by the naked eye at 30 μg/ml melamine without the aid of any advanced instruments. This method has been successfully applied to detect melamine in infant formula with good reproducibility, and it has the potential of being used in food safety detection.

Poor bioavailability and non-specificity of chemotherapeutic agents are major challenges in breast cancer treatment. Antibodies and small molecules that block cell signaling pathways have shown promise in the clinic, but their application is also limited by the high costs and treatment dosages required. Novel therapies that aim to rapidly and specifically target malignant cells with long-lasting impact in the tumor microenvironment may ultimately improve clinical outcome in cancer patients. Here, we demonstrate that epidermal growth factor receptor (EGFR)-targeting GE11 peptides conjugated with PEGylated polylactic-co-glycolic acid (PLGA) nanoparticles can be used to effectively deliver an anti-cancer agent, curcumin, into EGFR-expressing MCF-7 cells in vitro and in vivo. Treatment of breast cancer cells and tumor-bearing mice with these curcumin-loaded nanoparticles gave rise to reduced phosphoinositide 3-kinase signaling, decreased cancer cell viability, attenuated drug clearance from the circulation, and suppressed tumor burden compared with free curcumin or non-EGFR targeting nanoparticles. The targeted nanoscale drug delivery system we describe here may provide a new strategy for the design of targeted cancer therapy vectors. Our study provides evidence that the efficacy of pharmacologic anti-cancer agents can be enhanced through their delivery in the form of modified nanoparticles that effectively target specific malignant cell types.

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.

Summary Apigenin is a flavonoid, which has been proved to possess effective anti-cancer bioactivities against variety of cell lines. However, little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, human breast cancer line (MCF-7) was selected to be as a cell model to investigate the effects of apigenin on cell growth, proliferation, apoptosis, cellular morphology, etc. MTT assay showed that the growth inhibition induced by apigenin was in a dose-dependent manner when treated with different concentrations of apigenin while had little cytotoxic effects on human normal cells (MCF-10A). Fluorescence-based flow cytometry was used to detect cellular apoptosis and ROS production. The results showed that 80 µM apigenin could effectively induce apoptosis and overproduction of ROS in MCF-7 cells. Here, atomic force microscopy (AFM) was utilized to detect the shapes and membrane structures of MCF-7 cells at cellular or subcellular level. The results showed that the control MCF-7 cells presented typical elongated-spindle shapes with abundant pseudopodia, while after treated with apigenin, the cells shrunk and became round, the pseudopodia diminished. Moreover, the images of ultrastructure indicated that the cell membrane was composed of nanoparticles of 49 nm, but with the treated concentrations of apigenin increasing, the sizes of membrane particles significantly increased to 400 nm. These results can improve our understanding of apigenin, which can be potentially developed as a new agent for treatment of cancers. SCANNING 36:622–631, 2014. © 2014 Wiley Periodicals, Inc.

The aim of this study was to isolate and characterise porcine spermatogonial stem cells (PSSCs). The putative porcine germline stem cells from testis were isolated successfully by an improving way of enrichment with lymphocyte separation medium (LSM). Results from RT-PCR analyses showed that PSSCs were positive for OCT4, SOX2, NANOG, PGP9.5, c-MYC, KEL4 and PRDM-14 which are multipotent stem cell markers. At the protein level, the results of immunofluorescence analyses showed that PSSCs were positive for OCT4, PGP9.5, SOX2 and CD29. We successfully differentiated these PSSCs into adipocytes and muscle cells and then defined their characteristics, including morphology, surface stem cell markers, and mechanical properties. But the experiment of teratoma formation was negative. The results indicated the PSSCs could be multipotent. Atomic force microscopy was used to characterise the morphological and mechanical properties of undifferentiated PSSCs, as well as the differentiated adipocytes and muscle cells, which could be potentially useful for distinguishing PSSCs from differentiated cells.

The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In this review, we attempt to summarize the characteristics of these advanced techniques for use in the in situ single molecule imaging of cell membranes. We believe that this work will help to promote the technological and methodological developments of super-resolution techniques for the single molecule imaging of cell membranes and help researchers better understand which technique is most suitable for their future exploring of membrane biomolecules; ultimately promoting further developments in cell biology, immunology and medicine.

A label-free and aggregation-based gold nanorods (AuNRs) probe has been developed for the detection of Pb2+ in aqueous solution, based on the fact that Pb2+ ions induce assembly mediated signal enhancement of cysteine-functionalized AuNRs. Cysteine (Cys) molecules are conjugated on AuNR surfaces to form cysteine-modified AuNRs (Cys-AuNRs), acting as nanoprobes in the detection of Pb2+. Transmission electron microscopy (TEM) and UV–vis absorption spectroscopy data reveal the formation of controlled side-by-side assembly of the AuNRs in the presence of Pb2+. The formation of aggregation of AuNRs significantly enhances detection signals, leading to dramatic decrease in the longitudinal surface plasmon resonance (SPR) absorption. The experiment conditions, including AuNRs aspect ratio, reaction time, pH value and salt concentration, are optimized. The Cys-AuNRs probe is highly sensitive (LOD = 0.1 nM) and selective toward Pb2+ ions, with a liner detection range from 0.1 nM to 1.0 nM. This system only becomes less sensitive when other metal ion is present at a very high concentration (i.e., >0.5 μM). The cost-effective nanoprobes allow rapid and simple determination of the concentration of Pb2+ ions in city tap water samples, with results showing its practicality for the detection of lead in real samples.

A sensitive and facile electrochemical biosensor has been developed for monitoring the protein nitration damage affected by the nitro free radicals(NO 2). The NO 2 radicals is generated from hemin-catalyzed oxidation of nitrite (NO−2) in the presence of hydrogen peroxide (H2O2). In this work, nanocomposite films of graphene-gold nanoparticles (EG-AuNPs) were modified on the glassy carbon electrode by electropolymerization. Bovine serum albumin (BSA) was then further assembled on EG-AuNPs film through Au–S bond. The damage of BSA molecule was caused by the NO 2 radicals which was generated from the NaNO2/hemin/H2O2 nitration reagent. The differential pulse voltammetry was used to detect the damage of BSA molecule. Fluorescence spectra and circular dichroism spectrum further confirmed the nitration damage of BSA. Moreover, the lowest concentration at which the BSA damage was detected is 28.9 µM NO−2 or H2O2, and the volume ratio of NO−2 and H2O2 was 1:1 in the hemin/NO−2/H2O2 nitration reagent. The results demonstrated that the proposed electrochemical method can be used to detect protein damage affected by nitration reagent. The developed electrochemical biosensor is envisioned to have promising applications in protein damage studies.

The early stages of angiogenesis can be divided into three steps: endothelial cell proliferation, migration, and tube formation. Vascular endothelial growth factor (VEGF) is considered the most important proangiogenic factor; in particular, VEGF165plays a critical role in angiogenesis. Here, we evaluated whether gold nanoparticles (AuNPs) could inhibit the VEGF165-induced human umbilical vein endothelial cell (HUVEC) migration and tube formation. AuNPs and VEGF165were coincubated overnight at 4°C, after which the effects on cell migration and tube formation were assessed. Cell migration was assessed using a modified wound-healing assay and a transwell chamber assay; tube formation was assessed using a capillary-like tube formation assay and a chick chorioallantoic membrane (CAM) assay. We additionally detected the cell surface morphology and ultrastructure using atomic force microscopy (AFM). Furthermore, Akt phosphorylation downstream of VEGFR-2/PI3K in HUVECs was determined in a Western blot analysis. Our study demonstrated that AuNPs significantly inhibited VEGF165-induced HUVEC migration and tube formation by affecting the cell surface ultrastructure, cytoskeleton and might have inhibited angiogenesis via the Akt pathway.

Selenium nanoparticles (Se NPs) have been served as promising materials for biomedical applications, especially for cancer treatment. The anti-cancer effects of Se NPs against cancer cells have been widely studied in recent years, but whether Se NPs can induce the changes of cell membrane bio-mechanical properties in cancer cells still remain unexplored. In this Letter, we prepared Se NPs for investigating the intracellular localization of Se NPs in MCF-7 cells and determined the effects of Se NPs on apoptosis and necrosis in MCF-7 cells. Especially, we reported for the first time about the effects of Se NPs on the bio-mechanical properties of cancer cells and found that Se NPs could remarkably decrease the adhesion force and Young's modulus of MCF-7 cells. To further understand the potential mechanisms about how Se NPs affect the bio-mechanical properties of MCF-7 cells, we also investigated the expression of CD44 molecules, the structure and the amounts of F-actin. The results indicated that the decreased adhesion force between AFM tip and cell membrane was partially due to the changes of membrane molecules induced by Se NPs, such as the down-regulation of trans-membrane CD44 molecules. Additionally, the decrease of Young's modulus of MCF-7 cells was due to the dis-organization and down-regulation of F-actin induced by Se NPs. These results collectively suggested that cell membrane was of vital importance in Se NPs induced toxicity in cancer cells, which could be served as a potential target for cancer treatment by Se NPs.

Wnt/β-catenin signaling (WNT) has widespread roles during stem cell differentiation. Whether WNT suppresses or promotes insulin-producing cell (IPC) differentiation and function is still not known. In this study, we investigated the role of WNT signaling during human adipose-derived stem cell (hADSC) differentiation into IPCs. Western blot analysis revealed that several key components of WNT were dynamically regulated in a 12-day IPC differentiation assay. Specifically, protein levels of Wnt1, β-catenin, and GSK3β steadily increased from day 0 to day 9 and rapidly decreased by day 12 of differentiation. Similarly, endonuclear β-catenin levels peaked at day 9 and then, fell to pre-differentiation levels. The expression of two WNT pathway targets, TCF-1 and cyclin D1, closely followed the same pattern of regulation, confirming that WNT signaling was transiently activated during IPC differentiation. Interestingly, the inhibition of WNT signaling did not block IPC differentiation; instead, it resulted in the upregulation of IPC-specific markers, including PDX-1, insulin, IRS-1, and IRS-2. Notably, another IPC marker, glucokinase, remained downregulated since it is a direct target of WNT signaling. Next, we examined the effect of maintaining active WNT signaling from day 9 to day 12 of IPC differentiation. Differentiating cells were treated with Wnt1 on day 9, when WNT signaling is typically turned off, and subjected to gene expression analysis on day 12. Remarkably, Wnt1 treatment resulted in reduced expression of IPC-specific markers. Taken together, these data indicate that WNT may not be necessary for IPC differentiation but may be involved in IPC maturation.

Chrysin (5,7-dihydroxylflavone, Chry) is a natural product extracted from plants, honey, and propolis. In this work, a novel chrysin–organogermanium(IV) complex (Chry–Ge) with enhanced anticancer activities was synthesized, and its potential anticancer effects against cancer cells were measured using various methods. MTT results showed that Chry–Ge had significant inhibition effects on the proliferation of MCF-7, HepG2 and Colo205 human cancer cell lines in a dose-dependent manner while had little cytotoxic effects on MCF-10A human normal cells (MCF-10A cells) with the same treatment of Chry–Ge. These results suggested that Chry–Ge possessed enhanced anticancer effects and high selectivity between cancer cells and normal cells. The immuno-staining results showed that the nuclei of MCF-7 cells represented a total fragmented morphology and a disorganized cytoskeletal network in MCF-7 cells after Chry–Ge treatment. Besides, atomic force microscopy (AFM) was applied to detect the changes of ultrastructural and biomechanical properties of MCF-7 cellular membrane induced by Chry–Ge. The AFM data indicated that Chry–Ge treatment directly caused the decrease of cell rigidity and adhesion force of MCF-7 cells, suggesting that membrane toxicity might be one of the targets for Chry–Ge in MCF-7 cells. Moreover, the fluorescence-based flow cytometric analysis demonstrated that Chry–Ge could induce apoptosis in MCF-7 cells in ROS-dependent mitochondrial pathway. All results collectively showed that Chry–Ge could be as a promising anticancer drug for cancer therapy.

A simple method for preparing water-dispersable multifunctional germanium nanoparticles (GeNPs) in chitosan (CS) colloid was reported, which met the growing interest of green chemistry. The monodisperse CS–GeNPs were synthesized through the formation of Ge–N bond with a mean particle size of 60 nm and were characterized by UV–visible absorption spectroscopy, FTIR, HR-TEM and XRD measurements. The size and dispersibility of CS–GeNPs were controlled through tuning the initial dosage of CS. To test its potential in biological applications, CS–GeNPs were functionally loaded with doxorubicin (Dox) drug and conjugated with folic acid (FA), the resulting Dox-loaded FA–CS–GeNPs were capable of effectively killing MCF-7 human cancer cells. Fluorescent images showed that Dox-loaded FA–CS–GeNPs targeted cancer cells through FA receptors-mediated pathway. Furthermore, GeNPs had high efficiency of photothermal conversion. This study showed a new type of multifunctional Ge nanoparticles to achieve synergetic effect between drug-targeted delivery and photothermal therapy.

Transplantation of functional insulin-producing cells (IPCs) provides a novel mode for insulin replacement, but is often accompanied by many undesirable side effects. Our previous studies suggested that IPCs could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. To obtain a better method through which to acquire more similar IPCs, we compared the difference between IPCs of the GLP-1 group and IPCs of the non-GLP-1 group in the morphological features in cellular level and physiological function. The levels of insulin secretion were measured by ELISA. The insulin and glucagon-like peptide-1 (GLP-1) mRNA gene expression was determined by real-time quantitative PCR. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy (LCSM). Intracellular Ca(2+) levels and Glucagon-like peptide-1 receptor (GLP-1R) levels were determined by flow cytometer (FCM). We found that IPCs of the GLP-1 group had bigger membrane particle size and average roughness (Ra ) than IPCs of the non-GLP-1 group but still smaller than normal human pancreatic beta cells. The physiology function of IPCs of the GLP-1 group were much closer to normal human pancreatic beta cells than IPCs of the non-GLP-1 group. GLP-1 could improve the similarity of IPCs from human adipose tissue-derived mesenchymal stem cells and pancreatic beta cells in cellular ultrastructure and function.

A novel poly dopamine-hyaluronic acid (PDA-HA) electrochemical sensor was successfully constructed based on electrostatic interactions. Electrochemical impedance method was used to detect H2O2 based on the degradation processes between hyaluronic acid and hydrogen peroxide. The results showed that the sensor could be successfully applied to the detection of H2O2 in 0.01 M PBS (pH 7.0) medium after reaction for 30–40 min. The linear range was 1.0 × 10−5 −4.0 × 10−4 M and the detection limit was 2.0 × 10−6 M. The electrochemical sensor showed advantages of high sensitivity, low cost, satisfactory stability and reproducibility.

Activation of lymphatic cells is associated with changes in morphology, ultrastructure and adhesion force. We have investigated the activation efficiency of Staphylococcus aureus (SAC) on B-cell chronic lymphatic leukaemia (B-CLL) cells using atomic force microscopy (AFM), and found changes in the above properties. Cell viability and proliferation were measured using Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA). AFM clearly showed that the volume and nuclear-cytoplasm ratio of cells increased significantly with activated time. It also showed that pseudopodia and immunological synapses began to appear at 24 h. In the activation process, nano-structures of the cell surface became aggregated, and adhesion increased. In conclusion, the results indicate a close relationship between membrane reconstruction and multiplication process of B-CLL cells.

Integrin-mediated human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix plays a fundamental role in tumor-induced angiogenesis. Celastrol, a traditional Chinese medicine plant, has possessed anticancer and suppressed angiogenesis activities. Here, the mechanism underling the antiangiogenesis capacity of celastrol was investigated by exploring the effect of celastrol on β1(CD29) integrin-mediated cell adhesion and migration. Flow cytometry results showed that the HUVECs highly expressed CD29 and cell adhesion assay indicated that celastrol specifically inhibited the adhesion of HUVECs to fibronectin (FN) without affecting nonspecific adhesion to poly-L-lysine (PLL). After cell FN adhesion being inhibited, the cell surface nanoscale structure and adhesion force were detected by atomic force microscope (AFM). High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with celastrol. The membrane average roughness (Ra) and the major forces were decreased from 31.34 ± 4.56 nm, 519.60 ± 82.86 pN of 0 μg/ml celastrol to 18.47 ± 6.53 nm, 417.79 ± 53.35 pN of 4.0 μg/ml celastrol, 10.54 ± 2.85 nm, 258.95 ± 38.98 pN of 8.0 μg/ml celastrol, respectively. Accompanying with the decrease of adhesion force, the actin cytoskeleton in the cells was obviously disturbed by the celastrol. All of these changes influenced the migration of HUVECs from the wound-healing migration assay. Taken together, our results suggest that celastrol can be as an inhibitor of HUVEC adhesion to FN. This work provides a novel approach to inhibition of tumor angiogenesis and tumor growth.

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.

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. © 2016 Wiley Periodicals, Inc.

Summary Due to its extensive antitumor activity, curcumin has been focused on by more researchers. But, its antiproliferative mechanisms are still unknown. Here we studied the antiproliferative activity of curcumin in human liver cancer HepG2 cells. In order to analyze the cytotoxic activity and anticancer mechanisms of curcumin, we carried out cytotoxicity tests using 3-[4,5-dimethyl-2-thiazolyl]-2,5 diphenyltetrazolium bromide (MTT) assay. The HepG2 cell cycle distribution and the expression of tubulin were detected by flow cytometry. Alterations in morphological and cytoskeletal properties of HepG2 cells were investigated using atomic force microscopy (AFM). Simultaneously, the effects of curcumin on the growth and proliferation of HepG2 cells were also assayed by MTT method. Cells were incubated with different doses of curcumin (0–80 μmol/l) for 24 h, the cell viability decreased from 91.10 ± 3.2% to 10.84 ± 4.0%, and the 50% inhibiting concentration (IC50) was 23.15 ± 0.37 μmol/l. Moreover, flow cytometry quantitatively detected that curcumin treatment resulted in a dose-dependent accumulation of HepG2 cells in G2/M phase with concomitant losses from G0/G1 phase, so curcumin caused cell-cycle arrest at G2/M phase. Furthermore, we discovered that curcumin was able to upregulate the expression of tubulin in HepG2 cells. In addition, AFM analysis including cell-membrane structure and cytoskeleton networks is helpful to explain the relationship between the changes of cells and external pharmacologic stimulation. SCANNING 35: 253-260, 2013. © 2012 Wiley Periodicals, Inc.

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, Ca(2)+ 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.

Summary: The integrity and adhesion properties ofendothelium playvital roles during atherosclerosis. Itis well known that oxidized low-density lipoprotein(Ox-LDL)influencesmanyphysiologicalactivitiesormechanical properties of endothelial cells. However,the effects of Ox-LDL on the integrity and nonspe-cific adhesion properties of endothelial cells are stillunclear. In this study, using the topographical imag-ing and force measurement functions of atomic forcemicroscopy(AFM),wefoundthatOx-LDLcantran-sientlyweakentheintegrityofendotheliumbyimpair-ingcellspreadingofendothelialcellsanddecreasetheattachment of irrelevant blood cells to endotheliumby impairing the nonspecific adhesion property ofendothelial cells. The AFM-based data provide im-portant information for understanding the effects ofOx-LDLonendothelialcellsorduringatherogenesis.SCANNING 00: 1–8, 2012. C 2012 Wiley Periodi-cals, Inc. Key words: atherosclerosis, oxidized low-densitylipoprotein (Ox-LDL), human umbilical veinendothelial cells (HUVECs), atomic forcemicroscopy (AFM), cell spreading, adhesive force

We successfully differentiated human adipose tissue-derived mesenchymal stem cells (haMSCs) into insulin-producing cells (IPCs) in vitro and did not use any insulin which might be absorbed by cells during in vitro culture. Expression of insulin gene was massively increased by 28,000-fold at day 12 compared with haMSCs (P

A novel polyaniline nanofiber (PANI-NF)-based electrochemical sensor was constructed for specific detection of nitrite and sensitive monitoring of ascorbic acid scavenging nitrite. The electrochemical behaviors of PANI-NF were studied by cyclic voltammetry and amperometry in the presence of nitrite. Results showed that nitrite reduction was enhanced by the nanostructure of PANI-NF film. Under low potential difference and acid medium, PANI-NF catalyzed the conversion of nitrite ion to nitric oxide, and then realized the reduction of nitrite ion. Under optimal experimental conditions, PANI-NF-based sensor demonstrated a fast response time of 5 s and a detection limit of 0.05 μM nitrite (signal-to-noise ratio, S/N = 3), a linear range from 0.2 μM to 35 mM. This electrochemical sensor has been successfully applied to sensitively monitor ascorbic acid scavenging nitrite, and it has the potential of being used in nitrite detection.

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.

In this study, functionalized gold nanoparticles, in combination with fluorescence resonance energy transfer (FRET), was utilized to visually detect glutathione (GSH) in aqueous samples and then demonstrated its applicability by estimating GSH level in living cells. We first modified the Au NPs with rhodamine B (RB), ON state of FRET, using the electrostatic interaction. In the presence of GSH, the competitive reaction between RB-functionalized nanoprobes (RB-Au NPs) and GSH caused the release of RB molecules from Au NP surface, OFF state of FRET, which was monitored simply by the naked eye. Fluorescence spectra and UV–vis absorption spectra were employed to investigate the spectroscopic characteristics of nanoprobes. Furthermore, the assay conditions, such as nanoprobe concentration, reaction time and the influence of interferent, were investigated. The visible color change of RB-Au NPs solution from dark blue to red in the presence of GSH was observed when the GSH concentration exceeded 10 μM. This one-step naked eye detection depicted here successfully applied for the sensing of human hepatoma (HepG2) intracellular GSH level, showing the potential use in clinical nanodiagnosis. This method allows the rapid colorimetric detection of intracellular GSH without any preliminary treatment and specific instruments.

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.

B-lymphocyte activation plays an important role in humoral immune system, and its process has been studied well in vivo and in vitro. However, the ultrastructure and adhesion property changes remain unclear. In this study, changes in the morphology and mechanical properties of human peripheral blood B lymphocytes were first studied by atomic force microscopy (AFM). B lymphocytes were treated with the mitogen, pokeweed mitogen (PWM), and Staphylococcus aureus Cowan strain I (SAC) for 24 hr. After B lymphocyte is stimulated by the mitogen, the cell height, diameter, and volume are changed in different degree. The ultrastructure of the B lymphocytes membrane obviously displayed proteins gathering, corresponding with larger changes of average roughness and mean height of particles on cell membrane. Meanwhile, we detected the adhesion force of B lymphocytes after being stimulated by PWM and SAC. We found that the treated cells had a higher adhesion force of 304.16 ± 60.30 pN (PWM) and 249.63 ± 58.03 pN (SAC) than that of control group (104.28 ± 21.77 pN). Therefore, our results could provide new information to further understand the B-lymphocyte activation process and their structure-function analyses. SCANNING 34: 60–67, 2012. © 2011 Wiley Periodicals, Inc.

Alterporriol L, a new bianthraquinone derivative, was isolated from a marine fungus Alternaria sp. ZJ9-6B. The cytotoxic activity and anticancer mechanisms of alterporriol L towards breast cancer cells lines were detected using MTT assay, immunofluorescence, and flow cytometry. Simultaneously, the changes in morphological properties of cells were detected before and after treatment with alterporriol L by atomic force microscope (AFM) at a nanometer scale. MTT assay showed that alterporriol L could effectively inhibit the growth and proliferation, and there was a dose-dependent manner of cell death. Moreover, the alterporriol L could induce cancer cell apoptosis or necrosis. Furthermore, the reactive oxygen species, mitochondrial membrane potential, and cytosolic free calcium level were changed after treatment with alterporriol L, suggesting that alterporriol L played vital roles in breast cancer cells through destroying the mitochondrial. And all these alterations are in accord with changes of morphology detected by AFM, which suggested that the AFM is a useful tool to detect the morphological changes of the cancer cells.

Gene transfer has been proven to be a promising approach for treatment of several diseases. The cytotoxicity of transfection reagents is one of the key factors for clinical applications. The cytotoxicity of liposome has been extensively studied. However, its effects on the adhesion and spreading of transformed cells are still unclear. In this study, the cytotoxic effects of liposome on cell viability and mitochondrial membrane potential of HEK293 cells were first evaluated. Then, an atomic force microscope (AFM) was recruited to investigate the effects of liposome on the adhesion and spreading of HEK293 cells. AFM data indicated that liposome induced a significant decrease in number of cellular pseudopodia and cell-surface particles, in cell-surface roughness, and in average adhesion force of cell membranes. The AFM data implied that liposome impaired the adhesion and spreading of HEK293 cells.

The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 μM curcumin-treated) and 50-120 pN (20 μM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

Hematoporphyrin monomethyl ether (HMME) has been effectively used to treat solid tumors of some types. However, its application in nasopharyngeal carcinoma has not been studied yet. In this paper, the detailed sonodynamic effects of HMME-SDT (sonodynamic therapy) on CNE-2 cells including cell growth inhibition, apoptosis induction, and membrane toxicity were investigated. It was found that HMME alone had less cytotoxicity whereas HMME-SDT could suppress the cell proliferation in a dose-dependent manner as detected by MTT assay. The annexin V-based flow cytometric data indicated that upon SDT, different concentrations of HMME induce distinct types of cell death, apoptosis by low concentration (60 µg/ml) of HMME and necrosis by higher concentration (120 µg/ml). The immunofluorescence of cytoskeleton and nuclei morphology showed that upon HMME-SDT, the cells became rounding and the cytoskeletal network disappeared, and, the nuclei represented a total fragmented morphology of nuclear bodies. These alternations showed the apoptosis induction by HMME-SDT. Further AFM study showed that the cell membrane structure and cytoskeleton networks were destroyed, and, the Young's modulus, tip-cell-surface adhesion force decreased to 0.22 ± 0.11 Mpa, 35.4 ± 12.8 pN of cells with 120 µg/ml HMME-SDT from 0.48 ± 0.21 Mpa, 69.6 ± 22.3 pN of native cells, respectively. These membrane changes caused the collapse of mitochondrial transmembrane potential and disturbance of intracellular calcium homeostasis, which was consistent with the results detected by flow cytometry. Therefore, membrane toxicity and cytoskeleton disrupture induced by HMME-SDT maybe important factors to induce cell apoptosis, and, the disturbance of mitochondrial transmembrane potential and calcium channels might be the apoptosis mechanisms.

The pathological changes of erythrocytes were detected at the nanometer scale, which was important for revealing the onset of diseases, early diagnosis, and effective therapies. Diseases may disturb the morphology and function of erythrocytes at molecular scale. There were dramatic surface deformations in topography of erythrocytes from a patient with elliptocytosis complicating idiopathic thrombocytopenic purpura (ITP). The overall shape and surface membrane of the healthy, pre- and post-therapeutic erythrocytes have been studied by high-resolution atomic force microscopy imaging. The results showed that we can detect healthy and pathological erythrocytes by the morphologic parameters of the length, width, ratio of length to width, peak, valley, valley-to-peak, surface fluctuation, and standard deviations of the erythrocytes. Therefore, the morphologic information of erythrocytes is very important indictor for diagnosing the healthy and disease, as well as evaluating therapeutic effect.

In this paper, nano-hydroxyapatite/chitosan (n-HA/CS) composites with high compressive strength were prepared by the urease-catalyzed method (UCM) under mild conditions. The samples were characterized by infrared spectroscopy (IR), X-ray diffraction (XRD), laser diffraction particle size analyzer (LDPSA) and transmission electron microscopy (TEM). The results showed that the shape, size and crystallinity index of nano-HA crystals in composites were similar to that of nature bone. The n-HA/CS samples prepared by UCM had smaller particle size compared with those prepared by traditional co-precipitation technique. Moreover, mechanical testing showed that the maximum compressive strength of the composite materials was 130.3±9.3 MPa, which was close to that of cortical bone. UCM has great potential in fabricating non-sintering and drug-loaded bioceramics with improved mechanical features in bone tissue engineering.

CD44 is the principle cell surface receptor for the extracellular matrix. The altered expression or dysfunction of CD44 proteins contributes to numerous pathological processes. Therefore, it is very necessary to detect the distribution and density of CD44 proteins on cell surface. In this paper, the unbinding force between the tip of an atomic force microscope modified with anti-human CD44 antibody (a kind of CD44 pathway ligation proteins, currently used to induce the apoptosis of some types of tumors) and B16 (human melanoma cell line) cells was measured. The results indicated that the distribution of CD44 was nonuniform and represented clusters on B16 cell surface. And, the data of kinetics of CD44 antibody-antigen binding experiments indicated that the CD44 signal pathway in B16 cells could be blocked by anti-CD44 monoclonal antibody. This methodology can be extended to the evaluation and screening of molecular targeted drugs for pharmacological use.