A SERS/fluorescence dual-mode immuno-nanoprobe for investigating two anti-diabetic drugs on EGFR expressions.

A surface-enhanced Raman scattering (SERS)/fluorescence dual-mode nanoprobe was proposed to assess anti-diabetic drug actions from the expression level of the epidermal growth factor receptor (EGFR), which is a significant biomarker of breast cancers. The nanoprobe has a raspberry shape, prepared by coating a dye-doped silica nanosphere with a mass of SERS tags, which gives high gains in fluorescence imaging and SERS measurement. The in situ detection of EGFR on the cell membrane surfaces after drug actions was achieved by using this nanoprobe, and the detection results agree with the enzyme-linked immunosorbent assay (ELISA) kit. Our study suggests that rosiglitazone hydrochloride (RH) may be a potential drug for diabetic patients with breast cancer, while the anti-cancer effect of metformin hydrochloride (MH) is debatable since MH slightly promotes the EGFR expression of MCF-7 cells in this study. This sensing platform endows more feasibility for highly sensitive and accurate feedback of pesticide effects at the membrane protein level.

Electrostimulus-triggered reactive oxygen species level in organelles revealed by organelle-targeting SERS nanoprobes.

Electrostimulation (ES) is an important therapeutic method for diseases caused by abnormal intracellular electrical activity. Also, it can induce apoptosis of cells, which is a potential tumor treatment method. At present, there are no relevant studies on changes in intracellular reactive oxygen species (ROS) levels produced in the process of ES, or on the effects of simultaneous implementation of conventional antioxidant inhibitor drugs and ES therapy. To reveal these, two organelle-targeting core-shell plasmonic probes were designed for measuring ROS produced during ES. The probes were delivered into target organelles (nucleus and mitochondrion) before the cells were electrically stimulated for different periods of time. Surface-enhanced Raman scattering (SERS) signals were detected in situ, and the sensing mechanism for the quantitative analysis of ROS is based on the signal reduction of SERS caused by the ROS-etching effect on the silver shell. The detection results revealed that ES could trigger ROS generation in cells, and the ROS levels localized around organelles were assessed by SERS. This study has great potential for exploring abnormal organelle microenvironments via organelle-targeting probes combined with SERS technology.

Ratiometric pH-responsive SERS strategy for glioma boundary determination.

Glioma is one of the most common intracranial malignant tumors worldwide. Since the glioma is invasive and lacks a clear boundary with normal brain tissue, the neurosurgeon can only determine the extent of surgical resection based on empirical experience. Thus, accurately demarcating its boundaries has become a major challenge for surgeons. Owing to the high glycolysis metabolism of glioma cells, the acidification of the extracellular fluid has become an indicator of glioma evolution. Herein, a ratiometric pH-responsive surface-enhanced Raman scattering (SERS) strategy was developed for the rapid identification of glioma boundaries. A sensing chip composed of silver nanoparticles self-assembled film was fabricated, followed by the self-assembly of a pH-responsive SERS reporter, 4-mercaptopyridine (4-MPY). The characteristic SERS peak ratios of 4-MPY change regularly under different pH conditions. The boundary of glioma invasion was determined by measuring the pH of waterdrops infiltrated by interstitial fluids. The technology enables accurate, non-invasive, and rapid determination of local pH, thereby maximizing the removal of tumor tissue while minimizing damage to normal tissue. This technique is more rapid and simple than intraoperative pathological detection and can be possibly used for intraoperative navigation.

Thermoplasmonic Regulation of the Mitochondrial Metabolic State for Promoting Directed Differentiation of Dental Pulp Stem Cells.

Regulating stem cell differentiation in a controllable way is significant for regeneration of tissues. Herein, we report a simple and highly efficient method for accelerating the stem cell differentiation of dental pulp stem cells (DPSCs) based on the synergy of the electromagnetic field and the photothermal (thermoplasmonic) effect of plasmonic nanoparticles. By simple laser irradiation at 50 mW/cm2 (10 min per day, totally for 5 days), the thermoplasmonic effect of Au nanoparticles (AuNPs) can effectively regulate mitochondrial metabolism to induce the increase of mitochondrial membrane potential and further drive energy increase during the DPSC differentiation process. The proposed method can specifically regulate DPSCs' cell differentiation toward odontoblasts, with the differentiation time reduced to only 5 days. Simultaneously, the molecular profiling change of mitochondria within DPSCs during the cell differentiation process is revealed by in situ surface-enhanced Raman spectroscopy. It clearly demonstrates that the expression of hydroxyproline and glutamate gradually increases with prolonging of the differentiation days. The developed method is simple, robust, and rapid for stem cell differentiation of DPSCs, which would be beneficial to tissue engineering and regenerative medicine.

Electrostimulus-associated PD-L1 expression on cell membrane revealed by immune SERS nanoprobes.

Programmed cell death ligand 1 (PD-L1) is considered a major immune checkpoint protein that mediates antitumor immune suppression and response. Effectively regulating PD-L1 expression and dynamic monitoring has become a significant challenge in immunotherapy. Herein, we adopted smart surface-enhanced Raman scattering (SERS) nanoprobes to discriminate and monitor the dynamic expression of PD-L1 under external electrostimulation (ES). The PD-L1 expression levels in three cell lines (MCF-7 cells, HeLa cells, and H8 cells) were assessed before and after ES. The results reveal that ES could effectively and rapidly mediate a transformation in the PD-L1 content (or activity) on the cell membrane. Moreover, the molecular profiles of the cell membrane before and after ES were revealed by using the label-free SERS method with the help of immune plasmonic nanoparticles. The cell membrane protein information presented identifiable conformation changes after ES, showing a significant inhibitory effect on the bridge of PD-L1 and its antibody. This study indicates that ES is superior to chemical drugs due to lesser side effects because ES-based regulation does not depend on intracellular signalling pathways. This strategy is versatile and robust for discriminating and monitoring PD-L1 on cell membranes, thus providing potential clinical application value to PD-L1-mediated systems. This study also offers a practical way to assess the molecular profiles of cell membrane proteins in the presence of an external stimulus, which may be applicable to many membrane protein-related studies.

Smart Tumor Microenvironment-Responsive Nano-Prodrug for Disulfiram Toxification In Situ and the Exploration of Lethal Mechanisms in Cells.

Disulfiram (DSF) is a clinical antialcoholism drug that has been confirmed to show anticancer bioactivity after chelating with Cu2+. Therefore, how to co-deliver DSF and Cu2+ to tumor tissues and generate a smart response to the tumor microenvironment (TME) are the focus of repurposing DSF for the effective treatment of cancer. Herein, we fabricated facilely a smart nanosystem by coating tannic acid (TA) and Cu2+ network on DSF, denoted as DSF@TA-Cu, which responses well to TME and forms CuET complex in situ. In such a way, besides the chemotherapy effect of CuET, the anticancer efficacy of the resulting nano-prodrug can further be augmented by a continuous Fenton-like reaction. We then tested the cytotoxicity DSF@TA-Cu with normal and cancerous cell lines. Finally, by constructing mitochondria-targeted nanoprobes, we monitored the changes in mitochondrial metabolism and explored the lethal mechanisms in A549 cells. We found that DSF@TA-Cu showed higher toxicity to cancerous cells. By analyzing the fluorescence images and surface-enhanced Raman scattering (SERS) spectra of mitochondria, we found that the DNA damage and the decrease in mitochondrial membrane potential (MMP) were closely related to the generation and accumulation of reactive oxygen species (ROS). Although activated related pathways try to counteract the effects of elevation of ROS, excessive ROS inevitably leads to apoptosis of cancer cells.

Metformin hydrochloride action on cell membrane N-cadherin expression and cell nucleus revealed by SERS nanoprobes.

The anti-tumor effects of metformin hydrochloride (MH), an initial pharmacologic agent for type 2 diabetes, were reexamined by surface-enhanced Raman scattering (SERS) spectroscopy. A SERS immuno-tag fabricated by decorating silver nanoparticles (AgNPs) with a specific antibody was employed to trace the dynamic expression of the tumor metastasis-related N-cadherin. With the MH action, the N-cadherin expression on the cell membranes decreases, proving that MH has a pharmacological effect on prohibiting cancer cell metastasis. Another AgNP-based nucleus targeting nanoprobe was adopted to culture with the MH acted cells, which can help the label-free SERS collection of the cell nuclei to explore the MH influences on intranuclear genes and proteins. By analyzing the intranuclear SERS spectra, the find is that MH has impacts on the transcription and translation of genes, thus regulates the expression of tumor metastasis-related proteins (N-cadherins). This study presents a proof-of-concept for MH as a potential drug for diabetes patients associated with tumors. The developed plasmonic immune analytical platform can be extended to assess other substances of the cell membrane and applicable for the SERS-based screening of membrane receptor-related drugs at the cellular level.

Tumor Microenvironment-Activated Degradable Multifunctional Nanoreactor for Synergistic Cancer Therapy and Glucose SERS Feedback.

Integration of disease diagnosis and therapy in vivo by nanotechnology is a challenge in the design of multifunctional nanocarriers. Herein, we report an intelligent and degradable nanoreactor, an assembly of the 4-mercaptobenzonitrile-decorated silver nanoparticles (AgNPs@MBN) and the glucose oxidase (GOx)-loaded metal-organic-framework (ZIF-8@GOx), which can be activated by tumor microenvironment to start the catalytic cascade-enhanced chemo-starvation synergistic therapy and simultaneous self-sense of cellular glucose level. Under the mild acidic microenvironment of tumor, the nanoreactor will collapse to release GOx that triggers a catalytic cascade reaction in vivo, depleting glucose, etching AgNPs@MBN, and producing toxic H2O2, Ag+, and Zn2+ ions, all of which work together to inhibit tumor growth. The AgNPs@MBN as SERS nanoprobe reads out glucose concentration noninvasively in tumor to achieve instant feedback of therapeutic progression. This work proposes a promising example of using enzyme-encapsulated biomineralized MOFs as an effective anticarcinogen for clinical applications.

Molecular Profiling of Dental Pulp Stem Cells during Cell Differentiation by Surface Enhanced Raman Spectroscopy.

Dental pulp stem cells (DPSCs) are considered one of the key cells in tooth regeneration engineering. Understanding molecular biological information on DPSCs during differentiation is of great significance for the construction of tissue-engineered teeth. In this study, we investigated the differentiation process of DPSCs stimulated by drugs and gained molecular insights in the process. By using label-free and noninvasive surface enhanced Raman spectroscopy (SERS) to monitor molecular change profiling in the cell nucleus of single DPSCs during the differentiation process, we found that two pivotal differentiation biomarkers, alkaline phosphatase (ALP) and dentin sialophosphoprotein (DSPP), were overexpressed during the process. Continuous and intermittent monitoring of SERS spectra from the nuclear region indicated that the expression of proteins and related amino acids of tryptophan were markedly increased until peak period of differentiation (on day 14). Meanwhile corresponding transformation of DNA/RNA backbone vibrational modes was also observed during the differentiation process, indicating the occurrence of replication or transcription of DNA. The method provides a useful tool for the molecular biology studies of DPSCs differentiation, and the finding will broaden our understanding of DPSCs differentiation.

Male asymptomatic hyperuricemia patients display a lower number of NKG2D+ NK cells before and after a low-purine diet.

BACKGROUND: Aberrant activation of the immune system has been reported in asymptomatic hyperuricemia (HUA) patients. However, very few studies have elucidated the role of natural killer (NK) cells in this disease. METHODS: In this study, we evaluated the relationship between NK cells and HUA in 16 control subjects and 20 patients, who were all on a low-purine diet. We analyzed the number of circulating NK cells, its subsets, interferon-γ, and CD107 NK cells, by flow cytometry, before and after 4 and 24 weeks of diet control. We also assessed the potential association of the NK cells with clinical measures. RESULTS: The patients consistently had a lower number of NKG2D NK cells before and after low-purine diet, even the serum uric acid (SUA) levels <7 mg/dL after diet control. Moreover, a lower number of NK cells and a higher number of CD107a NK cells were observed on recruitment. Low-purine diet was benefit on the improvement of the SUA levels, body mass index (BMI), and the number and functions of NK cells. Furthermore, the number of CD3CD56 NK cells and NKG2D NK cells negatively correlated with the BMI before and after diet control. CONCLUSION: The consistent lower number of NKG2D NK cells and correlated with BMI before and after low-purine diet may be involved in the occurrence and development of HUA.

Decreased percentage of NKG2D+NK cells in patients with incident onset of Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) is characterized by absolute insulin deficiency owing to autoimmune destruction of the pancreatic ß cells. A significant decrease in natural killer (NK) cells in peripheral blood has been observed in patients with untreated T1DM. In the present study, we aimed to explore the role of NK cells and their subsets in young T1DM patients. A total of 30 children and adolescents with untreated T1DM and 27 healthy controls (HC) were recruited in this study. Flow cytometry analysis indicated that the percentage of peripheral blood CD3-CD56+ NK cells and NK cells subsets (CD56bright, CD56dim and CD56neg), were significantly decreased in the T1DM patients compared to healthy controls. In addition, the percentage of inducible CD107a+ and IFN-γ-secreting NK cells was significantly decreased compared to HC. Interestingly, the percentage of NKG2D+ NK cells negatively correlated with the level of serum TCHOL and TG in T1DM patients. Our data indicate that decreased number and impaired function of NK cells may have a role in the pathogenesis of T1DM.

Tanshinone IIA sensitizes oral squamous cell carcinoma to radiation due to an enhanced autophagy.

PURPOSE: Tanshinone IIA (TanIIA), which is derived from the roots of Salvia miltiorrhiza (Danshen), has multiple pharmacological activities. However, the radiosensitivezing activity of TanIIA in oral cancer cells has not been studied extensively. The purpose of this study is to investigate the radiosensitizing effects of TanIIA in human oral squamous cell carcinoma SCC090 and examined the underlying mechanisms. METHODS: Clonogenic assay was used to investigate the radiosensitizing effect of TanIIA on SCC090. And then, apoptosis and reactive oxygen species (ROS) induced by the combination of TanIIA with radiation were analyzed by Flow cytometry. Finally, autophagy was detected by monodansylcadervarine (MDC) staining. RESULTS: TanIIA could significantly sensitize SCC090 to radiation. Meanwhile, an increase ROS generation after exposed to the combination treatment was found. In addition, the protein levels of Beclin 1, Atg5 and LC3-II, three important proteins involved in autophagy were increased in cells. CONCLUSIONS: TanIIA exerted a strong radiosensitizing effect on SCC090 comparing with the simple drug or single radiation treatment, which was due to an enhanced ROS generation and autophagy.

A successfully treated case of hypopituitarism complicated with hyperosmolar hyperglycaemic state and rhabdomyolysis.

INTRODUCTION: This study aimed to report a rare case of hypopituitarism complicated with hyperosmolar hyperglycaemic state and rhabdomyolysis. CASE PRESENTATION: Hypopituitarism is a clinical syndrome in which there is a deficiency in hormone production by the pituitary gland. It often leads to hypoglycaemia, but in this case the patient was complicated with hyperosmolar hyperglycaemic state. The patient received prompt medical treatment, which effectively prevented the occurrence of possible acute kidney failure and other complications. CONCLUSION: This is a complicated and rare case. Our report provides some indications for the timely diagnosis and the standardised treatments for a patient who has hypopituitarism complicated with hyperosmolar hyperglycaemic state and rhabdomyolysis.

NKG2A expression and impaired function of NK cells in patients with new onset of Graves' disease.

BACKGROUND: Graves' disease (GD) is an organ-specific autoimmune disease. A significant decrease of the distribution of NK cells in the peripheral blood in children and adolescents with untreated GD has been observed. However, the role of NK and its subsets in adults with GD remains unclear. METHODS: A total of 28 adult patients with new onset of GD and 23 healthy controls (HC) were recruited. The number of activated inhibitory NK cells in peripheral blood of individual subjects was determined by flow cytometry. RESULTS: The number of CD3(-)CD56(+) and CD3(-)CD16(+)NK cells in peripheral blood was significantly decreased in the GD patients than the HC. Compared to the HCs, decreased number of NKG2D(+), NKG2C(+), NKp30(+) and NKG2A(+) NK cells and increased number of KIR3DL1(+) NK cells were detected in the GD patients. Moreover, the number of inducible CD107a(+) and IFN-γ-secreting NK cells in GD patients significantly decreased than those in HC. Interestingly, the number of NKG2A(+)NK cells was negatively correlated with the level of serum TRAb in GD patients. CONCLUSION: Our data indicate that decreased number and impaired function of NK cells may contribute to the pathogenesis of GD.

High frequency of activated natural killer and natural killer T-cells in patients with new onset of type 2 diabetes mellitus.

Chronic low-grade inflammation is crucial for the development of insulin resistance and type 2 diabetes mellitus (T2DM), and immunocompetent cells, such as T-cells, B-cells, mast cells and macrophages, regulate the pathogenesis of T2DM. However, little is known about the role of natural killer (NK) and natural killer T (NKT) cells in the pathogenic process of T2DM. A total of 16 patients with new onset T2DM and nine healthy subjects were recruited, and the frequency of peripheral blood activated and inhibitory NK and NKT cells in individual subjects was determined by flow cytometry. The frequency of spontaneous and inducible interferon gamma (IFN-γ) and CD107a(+) NK cells was further examined, and the potential association of the frequency of NK cells with clinical measures was analyzed. While there was no significant difference in the frequency of peripheral blood NK and NKT cells between patients and controls, the frequency of NKG2D(+) NK and NKT cells in patients was significantly higher than those in the controls (P = 0.011). In contrast, the frequency of NKG2A(+) and KIR2DL3(+) inhibitory NK and NKT cells in patients was significantly lower than those in the controls (P = 0.002, P < 0.0001, respectively). Furthermore, the frequencies of NKG2D(+) NK cells were correlated significantly with the values of body mass index in patients. Moreover, the frequencies of spontaneous and inducible CD107a(+), but not IFN-γ-secreting, NK cells in patients were significantly higher than those in the controls (P < 0.004, P < 0.0001). Our data indicated that a higher frequency of activated NK cells may participate in the obesity-related chronic inflammation involved in the pathogenesis of T2DM.