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BACKGROUND: It has been found that baicalin have anti-inflammatory effects since it reduces the elevated levels of pro-inflammatory cytokines. Meanwhile, it has also been shown that baicalin brings positive effects against rheumatoid arthritis (RA). However, little is observed on its beneficial effects on adjuvant arthritis. OBJECTIVE: To consider the anti-inflammatory influence of baicalin on adjuvant arthritis rats and its related autophagy mechanism. METHODS: In this research, there are six groups of rats, each has 10 rats in it. These groups are normal group (normal saline), model group (normal saline), dexamethasone group (0.125 mg/kg dexamethasone), low-dose baicalin group (50 mg/kg baicalin), medium-dose baicalin group (100 mg/kg baicalin) and high-dose baicalin group (200 mg/kg baicalin). The degrees of adjuvant-induced swelling in rats’ feet were measured every 4 days and the arthritis scores were calculated every 7 days. The inflamed joint tissues were taken after rats were sacrificed. The rat’ joints showed pathological changes, which were observed by HE staining. The relative expression levels of inflammatory factors IL-6, IL-1, IL-17, TNF-α, COX2, and COX1 in the rats’ snovial tissues were detected by RT-PCR. As for the expression levels of autophagy markers Beclin1, Atg5, Atg7, Atg12, microtubule-associated protein-light chain3-II (LC3-II), Bcl-2, and Bax in the synovial tissue, they were discoverd by Western blot. RESULTS: Baicalin could significantly inhibit the inflammatory response of adjuvant arthritis rats. CONCLUSIONS: RT-PCR studies showed that the different doses of baicalin could inhibit the expression of TNF-a, IL-6, IL-1, IL-17, COX2 and COX1 in the synovial tissue (P< 0.05 or P< 0.01). Western blot studies showed that the different doses of baicalin could reduce the expression of Atg5, Atg7, Atg12, LC3-II, Beclin1 and Bcl-2 proteins, and increase the expression of Bax proteins in the synovial tissue.

The synergistic manipulation of autophagy blocking with tumor targeting and penetration effects to enhance cancer cell killing during photothermal therapy (PTT) remains a substantial challenge. Herein, we fabricated a biomimetic nanoplatform by precisely coating homologous prostate cancer cell membranes (CMs) onto the surface of mesoporous polydopamine nanoparticles (mPDA NPs) encapsulating the autophagy inhibitor chloroquine (CQ) for synergistically manipulating PTT and autophagy for anticancer treatment. The resulting biomimetic mPDA@CMs NPs-CQ system could escape macrophage phagocytosis, overcome the vascular barrier, and home in the homologous prostate tumor xenograft with high tumor targeting and penetrating efficiency. The mPDA NPs core endowed the mPDA@CMs NPs-CQ with good photothermal capability to mediate PTT killing of prostate cancer cells, while NIR-triggered CQ release from the nanosystem further arrested PTT-induced protective autophagy of cancer cells, thus weakening the resistance of prostate cancer cells to PTT. This combined PTT killing and autophagy blocking anticancer strategy could induce significant autophagosome accumulation, ROS generation, mitochondrial damage, endoplasmic reticulum stress, and apoptotic signal transduction, which finally results in synergistic prostate tumor ablation in vivo. This prostate cancer biomimetic nanosystem with synergistically enhanced anticancer efficiency achieved by manipulating PTT killing and autophagy blocking is expected to serve as a more effective anticancer strategy against prostate cancer. Statement of significance Autophagy is considered as one of the most efficient rescuer and reinforcement mechanisms of cancer cells against photothermal therapy (PTT)-induced cancer cell eradication. How to synergistically manipulate autophagy blocking with significant tumor targeting and penetration to enhance PTT-mediated cancer cell killing remains a substantial challenge. Herein, we fabricated a biomimetic nanoplatform by precisely coating homologous cancer cell membranes onto the surface of mesoporous polydopamine nanoparticles with encapsulation of the autophagy inhibitor chloroquine for synergistic antitumor treatment with high tumor targeting and penetrating efficiency both in vitro and in vivo. This biomimetic nanosystem with synergistically enhanced anticancer efficiency by manipulating PTT killing and autophagy blocking is expected to serve as a more effective anticancer strategy.

Gambogic acid (GA), a kind of polyprenylated xanthone derived from Garcinia hanburyi tree, has showed spectrum anticancer effects both in vitro and in vivo with low toxicity. However, up to now, there is little information about the effects of GA on esophageal cancer. In this study, we aim to test the anticancer effects of GA on esophageal cancer EC9706 cells. We established a nanoscale imaging method based on AFM to evaluate the reactive oxygen species- (ROS-) mediated anticancer effects of GA on esophageal cancer regarding the morphological and ultrastructural changes of esophageal cancer cells. The obtained results demonstrated that GA could inhibit cell proliferation, induce apoptosis, induce cell cycle arrest, and induce mitochondria membrane potential disruption in a ROS-dependent way. And using AFM imaging, we also found that GA could induce the damage of cellular morphology and increase of membrane height distribution and membrane roughness in EC9706 cells, which could be reversed by the removal of GA-induced excessive intracellular ROS. Our results not only demonstrated the anticancer effects of GA on EC9706 cells in ROS-dependent mechanism but also strongly suggested AFM as a powerful tool for the detection of ROS-mediated cancer cell apoptosis on the basis of imaging.

Disease-modifying antirheumatic drugs (DMARDs) are the first line medications to treat rheumatoid arthritis (RA), a chronic and systemic autoimmune disease affecting multiple joints. Sinomenine (SIN) is thought a natural DMARD (nDMARD) and effectively utilized to treat RA in clinic for several decades in China. Here we reported that it is not methotrexate (MTX), a representative drug of DMARDs, but SIN protected joints from destruction to alleviate the symptoms of the mice with arthritis, indicating that the underlying mechanism of SIN is different from MTX to treat arthritis. Due to the dominate role of synovium fibroblasts in the joint destruction of arthritis, we applied synovium fibroblasts derived from RA patients (RASFs) to investigate the anti-arthritic effect and explore the underlying mechanism of SIN. We found that SIN significantly inhibited the secretion of IL-6 and IL-33 and ROS production in RASFs to mediate protective effect on bone destruction to mediate anti-arthritis effect. Underlying mechanistic study showed that SIN induced phosphorylation of p62 at Ser349 and Thr269/Ser272 to activate Keap1-Nrf2 signaling in RASFs. In line with the results, we then observed that the anti-arthritic effect of SIN was significantly attenuated in Nrf2 deficient (Nrf2-/-) mice. Notably, we found that p62 expression and phosphorylation at Thr269/Ser272 remarkably reduced, while p62 phosphorylation at Ser351 was up-regulated in Nrf2 deficient mice compared to its wild littermates, indicating that Nrf2 probably negative regulates p62 phosphorylation at Ser351. Collectively, our findings demonstrate that SIN phosphorylated p62 at Ser351 (corresponding to human Ser349) to degrade Keap1 expression and accumulate Nrf2 expression, increased p62 expression and phosphorylation at Thr269/Ser272 to activate p62-Keap1-Nrf2 axis, and finally exerted anti-arthritic effect. The current study not only clarified the anti-arthritic characteristics of SIN but also provided the clue to elucidate the correlation of p62 phosphorylation sites and Nrf2 signaling activation.

How to synergistically manipulate autophagy blocking with significant tumor targeting and penetration for the reinforcement of cancer cell killing during photo-thermal therapy (PTT) remains a substantial challenge. Herein, we fabricated a biomimetic nano-platform by exactly coating homologous cancer cell membranes (CMs) onto the surface of the assembly mesoporous polydopamine nanoparticles (mPDA NPs) with autophagy inhibitor chloroquine (CQ) encapsulating, for synergistically anticancer treatment manipulating PTT and autophagy. The resulting biomimetic mPDA@CMs NPs-CQ system could escape from the macrophage phagocytosis, overcome the vascular barrier, and home in the homologous tumor with high tumor targeting and penetrating efficiency. mPDA NPs core endow the mPDA@CMs NPs-CQ with excellent photo-thermal capability to mediate PTT killing of cancer cells, while NIR-triggered CQ release from mPDA@CMs NPs-CQ could further arrest PTT-induced protective autophagy of cancer cells, thus weakening the resistance of cancer cells to PTT. This PTT killing and autophagy blocking combined anticancer strategy could synergistically induce autophagosome accumulation, ROS generation, mitochondria damage, endoplasmic reticulum stress and apoptotic signal transduction, which leads to synergistic tumor cell inhibition both the in vitro and in vivo. This biomimetic nano-system with synergistically enhanced anticancer efficiency by manipulating PTT killing and autophagy blocking is expected to serve as more effective anticancer strategy.

How to actively target tumor sites manipulating the controllable release of the encapsulated anticancer drugs and photosensitizers for synergistic anticancer therapy remains a big challenge. In this study, a cancer cell-targeted, near-infrared (NIR) light-triggered and anticancer drug loaded liposome system (LPs) was developed for synergistic cancer therapy. Photosensitizer indocyanine green (ICG) and chemotherapy drug Curcumin (CUR) were coencapsulated into the liposomes, followed by the surface conjugation of GE11 peptide for epidermal growth factor receptor (EGFR) targeting on the cancer cell surface. Strictly controlled by NIR light, GE11 peptide modified and CUR/ICG-loaded LPs (GE11-CUR/ICG-LPs) could introduce hyperthermia in EGFR overexpressed A549 cancer cells for photothermal therapy, which could also trigger the increased release of CUR for enhanced cancer cell inhibition. GE11-CUR/ICG-LPs synergized photochemotherapy could induce reactive oxygen species (ROS) generation and cytoskeleton disruption to activate stronger apoptotic signaling events than the photothermal therapy or chemotherapy alone by regulating Bax/Bcl-2 and PI3K/AKT pathways. This EGFR-targeted drug-delivery nanosystem with NIR sensitivity may potentially serve in more effective anticancer therapeutics with reduced off-target effects.

Disease-modifying antirheumatic drugs (DMARDs) are the first line medications to treat rheumatoid arthritis (RA), a chronic and systemic autoimmune disease affecting multiple joints. Sinomenine (SIN) is thought a natural DMARD (nDMARD) and effectively utilized to treat RA in clinic for several decades in China. Here we reported that it is not methotrexate (MTX), a representative drug of DMARDs, but SIN protected joints from destruction to alleviate the symptoms of the mice with arthritis, indicating that the underlying mechanism of SIN is different from MTX to treat arthritis. Due to the dominate role of synovium fibroblasts in the joint destruction of arthritis, we applied synovium fibroblasts derived from RA patients (RASFs) to investigate the anti-arthritic effect and explore the underlying mechanism of SIN. We found that SIN significantly inhibited the secretion of IL-6 and IL-33 and ROS production in RASFs to mediate protective effect on bone destruction to mediate anti-arthritis effect. Underlying mechanistic study showed that SIN induced phosphorylation of p62 at Ser349 and Thr269/Ser272 to activate Keap1-Nrf2 signaling in RASFs. In line with the results, we then observed that the anti-arthritic effect of SIN was significantly attenuated in Nrf2 deficient (Nrf2

How to enable protein degradation pathways including the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) to enhance the efficacy of anticancer treatments remains a substantial challenge. Cobalt oxide nanoparticles (Co3O4 NPs) have attracted interest in recent years for their potential use as a synergistic anticancer treatment, although their therapeutic mechanisms of action are still poorly understood. Here, we describe the synergistic use of Co3O4 NPs as an autophagy inhibitor, chemosensitizer and photosensitizer, which manipulate protein degradation pathways (ALP and UPS) and photothermal therapy for enhanced anticancer treatments both in vitro and in vivo. We show that Co3O4 NPs can induce autolysosome accumulation and lysosomal functions damage by inhibiting lysosomal proteolytic activity and reducing intracellular ATP levels. Notably, Co3O4 NPs can be combined with the proteasome inhibitor, Carfilzomib (Cfz), to promote the accumulation of autophagic substrates, protein ubiquitination, and endoplasmic reticulum stress, and in doing so, inhibit cancer progression. By taking advantage of their photothermal conversion efficiency, Co3O4 NPs can also serve as photothermal sensitizer, which synergistically enhances the anticancer efficacy of Cfz both in vitro and in vivo. In summary, we provide evidence of a nanomaterial-synergized, photothermal anticancer strategy that synergistically targets cancer cell survival pathways and may eventually serve to enhance the anticancer efficacy of established cancer therapeutics.

Accumulating evidences suggest that amyloid β (Aβ)-peptide plays a key role in pathogenesis of Alzheimer's disease (AD) through aggregation and deposition into plaques in neuronal cells. Membrane components such as cholesterol and gangliosides not only enhance the production of amyloidogenic Aβ fragments, but also appear to strengthen Aβ-membrane interaction. Ginsenoside Rb1 (GRb1) is a major active component of Panax, which is widely used to improve learning and memory. In the present study, whether ginsenoside Rb1 could protect pheochromocytoma cells (PC12 cells) from Aβ

How to effectively enable both protein degradation pathways, which include autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS), with photothermal therapy synergistically for enhanced anticancer treatment in vivo remains a big challenge. Cobalt oxide nanoparticles (Co3O4 NPs) have attracted great interests for their biological application, including potential use for anticancer treatment although their exact mechanisms are still poorly understood. Here, we firstly report on the synergistic use of Co3O4 NPs as autophagy inhibitor, chemosensitizer and photosensitizer to manipulate protein degradation pathways (ALP and UPS) and photothermal therapy for enhanced anticancer treatments both in vitro and in vivo . Co3O4 NPs are found to block autophagic flux to induce autophagosome accumulation and lysosomal functions damage by inhibiting the lysosomal proteolytic activity and reducing the intracellular ATP level. Notably, Co3O4 NPs can further sensitize proteasome inhibitor Carfilzomib (Cfz) to promote autophagic substrates and ubiquitinated protein accumulation with increased endoplasmic reticulum stress for enhanced cancer inhibition. More importantly, taking advantages of the excellent photothermal conversion efficiency, Co3O4 NPs can further serve as photothermal sensitizer for photothermal therapy, which synergistically enhanced the anticancer efficacy of Cfz both in vitro and in vivo . This novel nanomaterial-synergized anticancer strategy synergistically manipulating autophagy, ubiquitin-proteasome system and photothermal therapy may potentially serve in more effective therapeutics against cancer.

Oridonin, an active diterpenoid isolated from Rabdosia rubescens, has been reported for its antitumor activity on several cancers. However, its effect on human esophageal cancer remains unclear. In this study, we demonstrated that oridonin could inhibit the growth of human esophageal cancer cells both in vitro and in vivo. Oridonin not only suppressed the proliferation, but also induced cell cycle arrest and mitochondrial-mediated apoptosis in KYSE-30, KYSE-150, and EC9706 cells with dose-dependent manner. Further mechanism studies revealed that oridonin led cell cycle arrest in esophageal cancer cells via downregulating cell cycle-related proteins, such as cyclin B1 and CDK2, while upregulating p53 and p21. Oridonin also increased proapoptotic protein Bax and reduced antiapoptotic protein Bcl-2, as well as the increased expression of cleaved caspase-3, -8, and -9. In addition, oridonin treatment could significantly inhibit the PI3K/Akt/mTOR and Ras/Raf signaling pathway. In vivo results further demonstrated that oridonin treatment markedly inhibited tumor growth in the esophageal cancer xenograft mice model. Taken together, these results suggest that oridonin may be a potential anticancer agent for the treatment of esophageal cancer.

Background Chemotherapy is a widely used treatment for cancer. However, the development of acquired multidrug resistance (MDR) is a serious issue. Emerging evidence has shown that the extracellular vesicles (EVs) mediate MDR, but the underlying mechanism remains unclear, especially the effects of chemotherapeutic agents on this process. Methods Extracellular vesicles isolation was performed by differential centrifugation. The recipient cells that acquired ATP-binding cassette sub-family B member 1 (ABCB1) proteins were sorted out from co-cultures according to a stringent multi-parameter gating strategy by fluorescence-activated cell sorting (FACS). The transfer rate of ABCB1 was measured by flow cytometry. The xenograft tumor models in mice were established to evaluate the transfer of ABCB1 in vivo. Gene expression was detected by real-time PCR and Western blotting. Results Herein, we show that a transient exposure to chemotherapeutic agents can strikingly increase Rab8B-mediated release of extracellular vesicles (EVs) containing ABCB1 from drug-resistant cells, and accelerate these EVs to circulate back onto plasma membrane of sensitive tumor cells via the down-regulation of Rab5. Therefore, intercellular ABCB1 transfer is significantly enhanced; sensitive recipient cells acquire a rapid but unsustainable resistance to evade the cytotoxicity of chemotherapeutic agents. More fascinatingly, in the xenograft tumor models, chemotherapeutical drugs also locally or distantly increase the transfer of ABCB1 molecules. Furthermore, some Non-small-cell lung carcinoma (NSCLC) patients who are undergoing primary chemotherapy have a rapid increase of ABCB1 protein in their monocytes, and this is obviously associated with poor chemotherapeutic efficacy. Conclusions Chemotherapeutic agents stimulate the secretion and recycling of ABCB1-enriched EVs through the dysregulation of Rab8B and Rab5, leading to a significant increase of ABCB1 intercellular transfer, thus assisting sensitive cancer cells to develop an urgent resistant phenotype. Our findings provide a new molecular mechanism of how chemotherapeutic drugs assist sensitive cancer cells in acquiring an urgent resistance.

Atomic force microscopy (AFM) is appropriately applied to the examination of hard surfaces and soft samples with extremely high resolution and ultrasensitive force, which cannot be obtained by other imaging techniques, including optical and electron microscopy. In the current study, AFM was employed to evaluate the anti-arthritic effect of licochalcone A (LCA), a flavonoid isolated from the root of Chinese medicinal herb Glycyrrhiza inflate, on rheumatoid arthritis synovial fibroblasts (RASFs) at the nanoscale for the first time. The morphology, ultrastructure and stiffness of RASFs was modified by LCA as determined by AFM, suggesting that LCA most likely exerts an anti-arthritic effect based on the key role of RASFs in the progression of RA. Further studies showed that the inhibitory effect of LCA on IκBα phosphorylation and degradation as well as on p65 nuclear translocation and phosphorylation contributed to altering the morphology, ultrastructure and stiffness of the RASF membrane. Interestingly, IKKβ phosphorylation was not detectable in RASFs, indicating that LCA altered the morphology, ultrastructure and stiffness of the RASF membrane by inhibiting NF-κB activation independent of IKKβ phosphorylation. Antigen-induced arthritis (AIA) was established in Sprague Dawley (SD) rats to validate the anti-arthritic effect of LCA, and LCA significantly decreased both the arthritis scores and paw swelling in the AIA rats, suggesting that LCA inhibits the progression and development of arthritis in vivo. Collectively, AFM provides evidence at the nanoscale to predict the anti-arthritic effect of drugs on RASFs, and LCA should be further investigated as a candidate agent for the treatment of arthritis.

Cobalt is essential to the metabolism of all animals due to its key role in cobalamin, also known as vitamin B12, the primary biological reservoir of cobalt as an ultra-trace element. Current cancer treatment strategies, including chemotherapy and radiotherapy, have been seriously restricted by their side effects and low efficiency for a long time, which urges us to develop new technologies for more effective and much safer anticancer therapies. Novel nanotechnologies, based on different kinds of functional nanomaterials, have been proved to act as effective and promising strategies for anticancer treatment. Based on the important biological roles of cobalt, cobalt oxide nanoparticles (NPs) have been widely developed for their attractive biomedical applications, especially their potential for anticancer treatments due to their selective inhibition of cancer cells. Thus, more and more attention has been attracted to the preparation, characterization and anticancer investigation of cobalt oxide nanoparticles in recent years, which is expected to introduce novel anticancer treatment strategies. In this review, we summarize the synthesis methods of cobalt oxide nanoparticles to discuss the advantages and restrictions for their preparation. Moreover, we emphatically discuss the anticancer functions of cobalt oxide nanoparticles as well as their underlying mechanisms to promote the development of cobalt oxide nanoparticles for anticancer treatments, which might finally benefit the current anticancer therapeutics based on functional cobalt oxide nanoparticles.

Development of rapid and, facile assays for quantification and evaluation of intracellular reactive oxygen species (ROS) activity in cancerous cells is of great importance to clinical diagnosis and therapeutic monitoring. Here, a straightforward, sensitive and quantitative assay for cytosensing and biodetecting of ROS from normal and cancerous cells were designed by taking advantage of targeted nanoprobes and size-sensitive Resonance Rayleigh scattering (RRS) technique for the first time. Using glutathione (GSH)-modified gold nanoparticles as nanoprobes, the constructed ROS-activated GSH dimerization, GSH disulfides-triggered interparticles couplings and nanoprobe-aggregation-assisted RRS signal amplification are used for improving selectivity and sensitivity. Fenton reaction-based RRS measurements enabled a sensitively signal enhancement in detection after introduction of FeCl2, and the detection limit of H2O2 was calculated to be 51.0 nM. GSH-targeted RRS cytosensor was further applied in quantitatively detecting human breast cancer HepG2 cell and normal cells, by recognizing changes in GSH dimerization from different cell types. The detection limit for HepG2 cells was 25 cells/mL and the intracellular ROS amount of 0.05–0.20 μM in 100–1000 cells/mL could be calculated with good selectivity and reproducibility. This assay demonstrates its potential in clinical diagnostic and therapeutic monitoring.

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.

Acute myocardial infarction (AMI) initiation and progression follow complex molecular and structural changes in the nanoarchitecture of platelets. However, it remains poorly understood how the transformation from health to AMI alters the ultrastructural and biomechanical properties of platelets within the platelet activation microenvironment. Here, we show using an atomic force microscope (AFM) that platelet samples, including living human platelets from the healthy and AMI patient, activated platelets from collagen-stimulated model, show distinct ultrastructural imaging and stiffness profiles. Correlative morphology obtained on AMI platelets and collagen-activated platelets display distinct pseudopodia structure and nanoclusters on membrane. In contrast to normal platelets, AMI platelets have a stiffer distribution resulting from complicated pathogenesis, with a prominent high-stiffness peak representative of platelet activation using AFM-based force spectroscopy. Similar findings are seen in specific stages of platelet activation in collagen-stimulated model. Further evidence obtained from different force measurement region with activated platelets shows that platelet migration is correlated to the more elasticity of pseudopodia while high stiffness at the center region. Overall, ultrastructural and nanomechanical profiling by AFM provides quantitative indicators in the clinical diagnostics of AMI with mechanobiological significance.

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.

Full-color emissive CDs (F-CDs) are synthesized by a green method, which avoids toxic reagents, harsh reaction conditions and multistep processes. The water-soluble, 6 ± 1 nm uniform-sized F-CDs exhibit tunable multicolor emission in gradient colors from blue to red. The emission wavelength and intensity of the fluorescence and the wavelength of the absorption peak can be tuned by controlling the concentration of the F-CD aqueous dispersions. The remarkable low cytotoxicity, good photostability and successful application in imaging of the adhesion of erythrocytes to endothelial cells demonstrate the great applicability of the F-CDs as multiplexed bioimaging reagents. The present research not only offers a facile, green, one-pot method for the preparation of F-CDs, but also presents a novel, experimentally convenient research mode for studying cellular functions and provides useful enlightenment for enlarging the application fields of CDs.

Pathogenesis hallmarks for tuberculosis (TB) are the Mycobacterium tuberculosis (Mtb) escape from phagolysosomal destruction and limited drug delivery into infected cells. Several nanomaterials can be entrapped in lysosomes, but the development of functional nanomaterials to promote phagolysosomal Mtb clearance remains a big challenge. Here, we report on the bactericidal effects of selenium nanoparticles (Se NPs) against Mtb and further introduce a novel nanomaterial-assisted anti-TB strategy manipulating Ison@Man-Se NPs for synergistic drug-induced and phagolysosomal destruction of Mtb. Ison@Man-Se NPs preferentially entered macrophages and accumulated in lysosomes releasing Isoniazid. Surprisingly, Ison@Man-Se/Man-Se NPs further promoted the fusion of Mtb into lysosomes for synergistic lysosomal and Isoniazid destruction of Mtb. Concurrently, Ison@Man-Se/Man-Se NPs also induced autophagy sequestration of Mtb, evolving into lysosome-associated autophagosomal Mtb degradation linked to ROS-mitochondrial and PI3K/Akt/mTOR signaling pathways. This novel nanomaterial-assisted anti-TB strategy manipulating antimicrobial immunity and Mtb clearance may potentially serve in more effective therapeutics against TB and drug-resistant TB.

Additional file 4: Table S1. Primer sequences for PCR. Table S2. The efficient targeting seqences for specific genes are shown.

Accumulating evidences suggest that amyloid β (Aβ)-peptide plays a key role in pathogenesis of Alzheimer's disease (AD) through aggregation and deposition into plaques in neuronal cells. Membrane components such as cholesterol and gangliosides not only enhance the production of amyloidogenic Aβ fragments, but also appear to strengthen Aβ–membrane interaction. Ginsenoside Rb1 (GRb1) is a major active component of Panax, which is widely used to improve learning and memory. In the present study, whether ginsenoside Rb1 could protect pheochromocytoma cells (PC12 cells) from Aβ25-35-induced cytotoxicity including inhibiting cell growth, inducing apoptosis, producing reactive oxygen species (ROS), destroying the cytoskeleton and bringing about membrane toxicity was investigated. Our results indicated that ginsenoside Rb1 could serve as an agonist of peroxisom proliferator-activated receptor-γ (PPARγ) and reduce the level of cholesterol in AD model cells. Reduction of the Aβ25-35-induced cytotoxicity by lowering cholesterol was evidenced by reduction of ROS production, lipid peroxidation, and protection of cytoskeleton and membrane surface rigidity. Most importantly, the viability of PC12 cells increased from 50.42 ± 5.51% for the AD group to 102.72 ± 4.34% for the 50 μM ginsenoside Rb1 group with cholesterol reduction. Our results suggested that ginsenoside Rb1 might function as an effective candidate to promote reverse cholesterol transport and lower ROS production, therefore providing a new insight into prevention and treatment of AD.

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 Polysaccharide compounds (PCs), which composed of different kinds of polysaccharides always isolated from different kinds of traditional Chinese medicine, are now attracting more and more attentions due to their strong immunomodulatory activities beyond the corresponding one-component polysaccharides. In this study, we demonstrated for the first time that PCs-1 and PCs-2 had strong immunomodulatory effects on macrophages both in in vitro and in vivo models by atomic force microscopy (AFM). By high resolution AFM imaging, PCs-1 and PCs-2 were found to inhibit LPS induced cell surface particle size and roughness increase in RAW264.7 macrophages, demonstrating the anti-inflammatory effects of PCs-1 and PCs-2 on macrophages. PCs-1 and PCs-2 were also proved to increase the particle size and roughness of resting RAW264.7 macrophages, which suggested that PCs could activate resting RAW264.7 macrophages. And additionally, PCs-1 and PCs-2 were also found to reverse the surface particle size and roughness decrease of peritoneal macrophages isolated from cyclophosphamide induced immunosuppressive mice, suggesting the activation effects of PCs-1 and PCs-2 on immunosuppressive macrophages. These results further enhanced our understanding of macrophage activations by direct imaging of cell surface ultrastructure and also highlighted AFM as a novel nanotool for macrophage detections. And most importantly, these results also indicated the outstanding immunomodulatory effects of PCs on macrophages, which therefore suggested that PCs could be served as a kind of novel immunomodulatory agents that would benefit human health. SCANNING 9999:1–10, 2016. © 2016 Wiley Periodicals, Inc.

Kaempferol has been identified as a potential cancer therapeutic agent by an increasing amount of evidences. However, the changes in the topography of cell membrane induced by kaempferol at subcellular- or nanometer-level were still unclear. In this work, the topographical changes of cytomembrane in human cervical cancer cell (SiHa) induced by kaempferol, as well as the role of kaempferol in apoptosis induction and its possible mechanisms, were investigated. At the macro level, MTT assays showed that kaempferol inhibited the proliferation of SiHa cells in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that kaempferol could induce SiHa cell apoptosis, mitochondrial membrane potential disruption, and intracellular free calcium elevation. At the micro level, fluorescence imaging by laser scanning confocal microscopy (LSCM) indicated that kaempferol could also destroy the networks of microtubules. Using high resolution atomic force microscopy (AFM), we determined the precise changes of cellular membrane induced by kaempferol at subcellular or nanometer level. The spindle-shaped SiHa cells shrank after kaempferol treatment, with significantly increased cell surface roughness. These data showed structural characterizations of cellular topography in kaempferol-induced SiHa cell apoptosis and might provide novel integrated information from macro to nano level to assess the impact of kaempferol on cancer cells, which might be important for the understanding of the anti-cancer mechanisms of drugs. SCANNING 38:644-653, 2016. © 2016 Wiley Periodicals, Inc.

Angiogenesis provides necessary nutrients and oxygen for tumor growth and metastasis; thus, every stage of angiogenesis process is the potential target for cancer therapies. Ursolic acid (UA) is reported to decrease tumor burden through anti-angiogenesis pathway, but its poor water solubility greatly limits its efficiency and clinical application. Here, a simple method for preparing UA-loaded chitosan nanoparticles (CH-UA-NPs) with anti-angiogenesis and anti-tumor activity was demonstrated. In vitro, CH-UA-NPs could significantly inhibit the proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs). After uptake by HUVECs, CH-UA-NPs were mainly localized in lysosomes and mitochondria, but not nuclei. CH-UA-NPs induced the destruction of lysosome membrane integrity, collapse of mitochondrial membrane potential, and reorganization of cell cytoskeleton. All these changes led to the apoptosis or necrosis in HUVECs. In vivo, CH-UA-NPs could inhibit the angiogenesis in chicken chorioallantoic membrane (CAM) model and H22 xenograft model. Notably, comparing with free UA, such synthesized CH-UA-NPs could save about tenfold of UA doses, implying that this could significantly decrease the side effects induced by high doses of UA in biological organism. Our data showed that CH-UA-NPs and this nanoparticle-based drug delivery system could be as a potential drug candidate for anti-angiogenesis treatment.

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. © 2016 Wiley Periodicals, Inc.

The article describes a quartz crystal microbalance (QCM) biosensor for the determination of nucleic acids via a DNA-templated assembly of silver nanoclusters (AgNCs) which represents a novel way for efficient signal amplification. A QCM was modified with probe DNA to specifically capture target DNA. Then, DNA-templated AgNCs were assembled to enhance the sensitivity of the QCM sensor via Ag(I) ions attached to the DNA skeleton, this followed by hydroquinone-induced reductive formation of the AgNCs. TEM and AFM were used to further confirm the formation of DNA-templated AgNCs. The results showed that frequency response of QCM sensor is up to 87 times larger when using this mode of amplification. A linear relationship was obtained between the frequency response and DNA concentration over the 0.6 to 130 nM range, with a 0.1 nM detection limit. In our perception, this scheme for improved sensitivity provides a straightforward and widely applicable tool for sensing DNA.

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.

Oridonin has been found to be a potential anti-angiogenesis agent. However, its functional targets and the underlying mechanisms are still vague. In vitro studies we found that oridonin not only inhibited VEGF-induced cell proliferation, migration and tube formation but also caused G2/M phase arrest and triggered cellular apoptosis in HUVECs. In mechanistic studies revealed that oridonin exhibited the anti-angiogenic potency, at least in part, through the down-regulation of VEGFR2-mediated FAK/MMPs, mTOR/PI3K/Akt and ERK/p38 signaling pathways which led to reduced invasion, migration, and tube formation in HUVECs. Our results could provide evidence that oridonin exerts strong anti-angiogenesis activities via specifically targeting VEGFR2 and its signaling pathway.

As a high-resolution imaging technique, AFM has been found to be a novel tool for cell topography and its quantitative imaging. This chapter is focused on the introduction of AFM cell topography and its quantitative imaging, which includes the basic principle of AFM imaging, basic operation modes of AFM imaging, AFM imaging of biological sample, critical tips for AFM cell topography and its quantitative imaging, applications of AFM cell topography and its quantitative imaging, and perspective. We believe that this work will help to promote the technological and methodological developments of AFM cell topography and its quantitative imaging, promoting further application of AFM in cell biology, immunology, and medicine.

Graphene oxides (GO) is a promising building material to fabricate desired drug delivery system due to its excellent physicochemical properties. In this study, an innovative nano-drug (Ori@GE11-GO) was constructed based on GE11 peptide functionalized GO for targeted delivery of oridonin to realize the specific recognition of tumour cells and enhance anticancer efficiency. GE11 surface modification onto GO significantly increased the cellular uptake of GO in EGFR overexpressed oesophageal cancer cells (KYSE-30 and EC109 cells) than that of normal cells, indicating the EGFR targeting effects of Ori@GE11-GO. The internalized Ori@GE11-GO could accumulate into lysosomes and significantly inhibit the viability of cancer cells. Moreover, Ori@GE11-GO could effectively induce KYSE-30 and EC109 cells cycle arrest, apoptosis, mitochondrial membrane potential (△Ψm) disruption through the activation of apoptotic signalling pathways and the inhibition of EGFR/Ras/Raf/MEK/ERK signalling pathway, showing potential use of Ori@GE11-GO for cancer treatment. Taken together, this study demonstrates a good strategy for the construction of bio-functionalized GO drug delivery nanosystem to improve the cancer targeting efficiency of anticancer medicines.

The authors describe a dual-mode (colorimetric-fluorometric) nanoprobe for H2O2 that was fabricated by covering molybdenum disulfide nanosheets (MoS2 NS) with ortho-phenylenediamine (OPD). The probe (OPD-MoS2 NS) was applied to the optical determination of H2O2, to the quantitation of cell numbers, and to the detection of intracellular concentrations of H2O2. Oxidation by H2O2 leads to a colored and fluorescent product (oxidized OPD) with absorption/excitation/fluorescence peaks at 450/450/557 nm. The nanoprobe can detect H2O2 in down to 500 nM concentrations, and HeLa cells at levels of 100 cells mL−1. The detection limit for intracellular H2O2 is in the 5.5 to 12.6 μM concentration range when the method is applied to cells at levels of 102–106 cells mL−1. Due to its good biocompatibility and easy cell uptake, the nanoprobe also permits sensitive fluorometric imaging of intracellular H2O2. It can also comparatively discriminate the change of intracellular oxidation state in living cancerous and normal cells.

Tuberculosis (TB), caused by M.tuberculosis (Mtb), has become a top killer among infectious diseases. Enhancing the ability of anti-TB drugs to kill intracellular Mtb in host cells remains a big challenge. Here, an innovative nano-system was developed to increase drug delivery and Mtb-killing efficacy in Mtb-infected macrophages. We employed mannose surface decoration to develop mannosylated and PEGylated graphene oxide (GO-PEG-MAN). Such nano-platform exhibited increased uptake by macrophages via mannose receptor-mediated endocytosis in vitro. Interestingly, drug-loaded GO-PEG-MAN was preferentially up-taken by mannose receptor-expressing mucosal CD14+ macrophages isolated from Mtb-infected rhesus macaques than drug-loaded GO-PEG. Consistently, the drug concentration was also significantly higher in macrophages than that in T and B cells expressing no or low mannose receptor, implicating a useful macrophage/mannose receptor-targeted drug-delivery system relevant to the in vivo settings. Concurrently, rifampicin-loaded GO-PEG-MAN (Rif@GO-PEG-MAN) significantly increased rifampicin uptake, inducing long-lasting higher concentration of rifampicin in macrophages. Such innovative Rif@GO-PEG-MAN could readily get into the lysosomes of the Mtb host cells, where rifampicin underwent an accelerated release in acidic lysosomic condition, leading to explosive rifampicin release after cell entry for more effective killing of intracellular Mtb. Most importantly, Rif@GO-PEG-MAN-enhanced intracellular rifampicin delivery and pharmacokinetics significantly increased the efficacy of rifampicin-driven killing of intracellular BCG and Mtb bacilli in infected macrophages both in vitro and ex vivo. Such innovative nanocarrier approach may potentially enhance anti-TB drug efficacy and reduce drug side effects.

The authors describe a dual-mode (colorimetric-fluorometric) nanoprobe for H

Matrine, as a natural alkaloid isolated from the traditional herb medicine sophora flavescens, has been proved to possess excellent biological activities, including anticancer effects. Now, this research aims to assess the anticancer activities and the mechanism of matrine against esophageal cancer cells, we investigated the proliferative inhibition, apoptosis induction, as well as the underlying mechanism of matrine on esophageal cancer KYSE-150 cells. It was found that matrine could suppress KYSE-150 cell proliferation and significantly mediate cell apoptosis in a dose-dependent relation by increasing intracellular reactive oxygen species level and triggering mitochondrial membrane potential disruption. More precise mechanism studies demonstrated that matrine could up-regulate the expression of Bax proteins and down-regulate the expression of Bcl-2 proteins, as well as the activation about caspase-3, 8 and 9 in KYSE-150 cells. The morphological analysis of KYSE-150 cells exhibited that matrine could destroy the F-actin and nuclei structures and induce morphological damage with increased surface height distribution and roughness of cell membrane. These results not only demonstrated the potential anticancer activity mechanism of matrine at nanoscale, but also provide preliminary guidance for the treatment of esophageal cancer using matrine.

Atomic force microscopy (AFM) has recently made great progress in mapping local mechanical properties of a single cell including living cell. These mechanical properties can provide not only cellular nano- and micro-structure, but also physiological and pathological information of cells. Here, the recent applications of AFM for the study of mechanical characteristics of normal cells and disease cells are reviewed.