Genipin crosslinked curcumin loaded chitosan/montmorillonite K-10 (MMT) nanoparticles for controlled drug delivery applications.

Here, we have reported the influence of MMT and genipin in releasing curcumin from the Genipin crosslinked Chitosan/MMT nanoparticles, prepared by ionic gelation method. The nanoparticles were characterised using Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffractometry (XRD), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). Zeta potential and average diameter of the nanoparticles were found in the range 32-47 mV and 430-560 nm. Swelling and release of curcumin from the nanoparticles increased with the decrease in pH of the medium, MMT, and genipin content. Curcumin released from the nanoparticles reduced the viability of MCF-7 and Hep G2 cells as compared to untreated cells. The nanoparticles increased the level of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase level in human PBMCs and decreased the level of Lipid peroxidation suggesting an enhanced protection against cellular damage. Lower pH and higher MMT concentration in the nanoparticles improved the mucoadhesive properties.

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc.

Exosomes secreted under hypoxia enhance invasiveness and stemness of prostate cancer cells by targeting adherens junction molecules.

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-ß2, TNF1α, IL6, TSG101, Akt, ILK1, and ß-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.

Carboxymethyl starch-chitosan-coated iron oxide magnetic nanoparticles for controlled delivery of isoniazid.

CONTEXT: The coating material of magnetic nanoparticles plays a great role in drug delivery application. The coatings not only increase the stability of the nanoparticles but also improve the drug release pattern, biocompatibility and mucoadhesivity. OBJECTIVE: Montmorillonite (MMT) containing magnetic iron oxide nanoparticles coated with polyelectrolyte complex (PEC) of carboxymethyl starch-chitosan were prepared for controlled release applications. METHOD: The PEC-coated nanoparticles were characterised by Fourier Transmission Infra-red spectroscopy and X-ray diffraction, scanning electron microscope, transmission electron microscope, and dynamic light scattering. Cytotoxicity study was performed by MTT assay analysis. Mucoadhesivity test was performed by using in vitro wash off and ex vivo method. RESULT: The coating of PEC showed good stability, biocompatibility and mucoadhesivity of the iron oxide magnetic nanoparticles. MMT addition enhanced the swelling, drug loading and release and also the cytotoxicity and mucoadhesivity of the nanoparticles. CONCLUSION: This study revealed that the MMT incorporated PEC of CMS-CS can be effectively used for coating of iron oxide nanoparticles.

SNAI1 is critical for the aggressiveness of prostate cancer cells with low E-cadherin.

BACKGROUND: A better molecular understanding of prostate carcinogenesis is warranted to devise novel targeted preventive and therapeutic strategies against prostate cancer (PCA), a major cause of mortality among men. Here, we examined the role of two epithelial-to-mesenchymal transition (EMT) regulators, the adherens junction protein E-cadherin and its transcriptional repressor SNAI1, in regulating the aggressiveness of PCA cells. METHODS: The growth rate of human prostate carcinoma PC3 cells with stable knock-down of E-cadherin (ShEC-PC3) and respective control cells (Sh-PC3) was compared in MTT and clonogenic assays in cell culture and in nude mouse xenograft model in vivo. Stemness of ShEC-PC3 and Sh-PC3 cells was analyzed in prostasphere assay. Western blotting and immunohistochemistry (IHC) were used to study protein expression changes following E-cadherin and SNAI1 knock-down. Small interfering RNA (siRNA) technique was employed to knock- down SNAI1 protein expression in ShEC-PC3 cells. RESULTS: ShEC-PC3 cells exerted higher proliferation rate both in cell culture and in athymic nude mice compared to Sh-PC3 cells. ShEC-PC3 cells also formed larger and a significantly higher number of prostaspheres suggesting an increase in the stem cell-like population with E-cadherin knock-down. Also, ShEC-PC3 prostaspheres disintegration, in the presence of serum and attachment, generated a bigger mass of proliferating cells as compared to Sh-PC3 prostaspheres. Immunoblotting/IHC analyses showed that E-cadherin knock-down increases the expression of regulators/biomarkers for stemness (CD44, cleaved Notch1 and Egr-1) and EMT (Vimentin, pSrc-tyr416, Integrin ß3, ß-catenin, and NF-κB) in cell culture and xenograft tissues. The expression of several bone metastasis related molecules namely CXCR4, uPA, RANKL and RunX2 was also increased in ShEC-PC3 cells. Importantly, we observed a remarkable increase in SNAI1 expression in cytoplasmic and nuclear fractions, prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, SNAI1 knock-down by specific siRNA strongly inhibited the prostasphere formation, clonogenicity and invasiveness, and decreased the level of pSrc-tyr416, total Src and CD44 in ShEC-PC3 cells. Characterization of RWPE-1, WPE1-NA22, WPE1-NB14 and DU-145 cells further confirmed that low E-cadherin is associated with higher SNAI1 expression and prostasphere formation. CONCLUSIONS: Together, these results suggest that E-cadherin loss promotes SNAI1 expression that controls the aggressiveness of PCA cells.

Physical and biophysical assessment of highly fluorescent, magnetic quantum dots of a wurtzite-phase manganese selenide system.

Combining fluorescence and magnetic features in a non-iron based, select type of quantum dots (QDs) can have immense value in cellular imaging, tagging and other nano-bio interface applications, including targeted drug delivery. Herein, we report on the colloidal synthesis and physical and biophysical assessment of wurtzite-type manganese selenide (MnSe) QDs in cell culture media. Aiming to provide a suitable colloidal system of biological relevance, different concentrations of reactants and ligands (e.g., thioglycolic acid, TGA) have been considered. The average size of the QDs is ∼7 nm, which exhibited a quantum yield of ∼75% as compared to rhodamine 6 G dye(®). As revealed from time-resolved photoluminescence (TR-PL) response, the near band edge emission followed a bi-exponential decay feature with characteristic times of ∼0.64 ns and 3.04 ns. At room temperature, the QDs were found to exhibit paramagnetic features with coercivity and remanence impelled by TGA concentrations. With BSA as a dispersing agent, the QDs showed an improved optical stability in Dulbecco's Modified Eagle Media(®) (DMEM) and Minimum Essential Media(®) (MEM), as compared to the Roswell Park Memorial Institute(®) (RPMI-1640) media. Finally, the cell viability of lymphocytes was found to be strongly influenced by the concentration of MnSe QDs, and had a safe limit upto 0.5 µM. With BSA inclusion in cell media, the cellular uptake of MnSe QDs was observed to be more prominent, as revealed from fluorescence imaging. The fabrication of water soluble, nontoxic MnSe QDs would open up an alternative strategy in nanobiotechnology, while preserving their luminescent and magnetic properties intact.

Oxidative stress mediated hepatotoxicity induced by ZNP and modulatory role of fruit extract on male Wistar rat.

Zinc oxide nanoparticles (ZNP) are being used in various fields viz cosmetics industry as UV protectants, in the food packaging industry due to their anti-bacterial properties, in agriculture as micronutrients, etc. Increased applications of ZNPs in our day to day life, leading to the contamination of the surrounding environment posing a direct or indirect health risk. Various reports suggest that fruits and vegetables are a rich source of phytochemicals having antioxidant properties which help in neutralizing ROS generated on metal toxicity of the body. The present study focuses to study the ameliorative effect of apple (Pyrus malus) extract (E) on ZNP induced toxicity. Therefore, animals were grouped, six in each, exposed to various doses of ZNP (50 and 250 mg/kg), ZNP (50 and 250 mg/kg)+E. The studied parameters was: food intake, water intake, antioxidants assay, zinc accumulation, and histological alterations and was compared to control. Investigation revealed that ZNP induces toxicity as revealed by the alteration in the studied parameter, whereas those exposed to ZNP along with Pyrus malus fruit extract try to reduce the toxicity induced by nanoparticles but at low doses only. This ameliorative effect of fruit extract might be due to the presence of antioxidants scavenging the free radicals generated by ZNPs suggesting that antioxidant-rich fruit may have a protective role and have the potential to reduce the nanoparticles mediated oxidative stress.

Author Correction: Transcript expression profiling in two contrasting cultivars and molecular cloning of a SKP-1 like gene, a component of SCF-ubiquitin proteasome system from mungbean Vigna radiate L.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Assessment of intermittent exposure of zinc oxide nanoparticle (ZNP)-mediated toxicity and biochemical alterations in the splenocytes of male Wistar rat.

Nanoparticles are being used extensively and found in applications to various fields ranging from agriculture to electronic devices, diagnosis to drug delivery, and cosmetics to food packaging. Increasing usage of engineered nanomaterials (ENM) raises potential concern for human health as well as to the environment. The present study aims to explore the effects of intermittent intraperitoneal exposure of ZNP on the spleen of male Wistar rat. Animals were divided into three groups, control and ZNP-treated groups (50 mg/kg and 250 mg/kg body weight), six in each group. Experimental animals were treated with different doses of ZNP once a week for 4 weeks, whereas control groups received water. After 28 days of exposure, animals were sacrificed, spleen tissue was excised, and various parameters such as hematological, genotoxicity, antioxidants, and histopathological were studied for changes in spleen if any. Results showed that ZNP exposure manages to induce alteration in various studied hematological parameters like neutrophils, platelets, and eosinophils which are found to increase significantly after the last treatment compared with the first treatment of ZNP. However, hemoglobin content, PCV, and MCV decrease with increasing dose of ZNP significantly in last treatment, when compared with the first treatment. DNA damage was observed in rats treated with a high dose of ZNPs compared with that in the control when analyzed through comet assay. Flow cytometric study was performed for better understanding of the underlying mechanism of the ZNP-mediated toxicity. From the present investigation, an increase in ROS production, a decrease in MMP, and increased apoptosis were exhibited. Further, altered antioxidant level (SOD, CAT, LDH, CYT P450, and CYT b5 r) has been observed in the studied splenic tissue, also histopathological changes observed in the rats exposed with high doses of ZNP. Therefore, ZNP may have the potential to induce a toxic effect even when exposed intermittently.

Transcript expression profiling in two contrasting cultivars and molecular cloning of a SKP-1 like gene, a component of SCF-ubiquitin proteasome system from mungbean Vigna radiate L.

Protein degradation and turnover under various environmental stresses is basically regulated by ubiquitin-proteasome system (UPS), of which SKP1 is a very essential component. Isolation and cloning of an identified potential stress responsive candidate gene SKP1, was successfully done for the first time to fathom the role of SKP1 in drought tolerance at genetic level in drought tolerant mungbean cultivar Pratap, which was screened after a detailed physio-biochemical screening amongst seven popular mungbean cultivars. The cloned gene SKP1 (accession number KX881912) is 550 bp in length, encodes 114 amino acids. It shows high sequence homology with SKP1 from Zea mays (NP_001148633). The protein expression of isolated SKP1 was confirmed by GUS fused expression using a Histochemical assay under control as well as under drought stress. Further, up-regulation in relative expression level of SKP1 in different plant parts under drought stress confirmed its utility as a potential drought responsive candidate gene certainly demanding extensive genetic research for further incorporation in breeding programs. Moreover, the structure of VrSKP1 (Vigna radiata SKP1) has been modelled, validated and an Essential Dynamics (ED) was done on the Molecular Dynamics (MD) simulation trajectories for filtering large-scale concerted motions. Free-energy calculations on the ED revealed a complex free-energy landscape (FEL) implying the conformational diversity of the modelled VrSPK1 protein.

Exosomal microRNA profiling to identify hypoxia-related biomarkers in prostate cancer.

Hypoxia and expression of hypoxia-related biomarkers are associated with disease progression and treatment failure in prostate cancer (PCa). We have reported that exosomes (nanovesicles of 30-150 nm in diameter) secreted by human PCa cells under hypoxia promote invasiveness and stemness in naïve PCa cells. Here, we identified the unique microRNAs (miRNAs) loaded in exosomes secreted by PCa cells under hypoxia. Using TaqMan® array microRNA cards, we analyzed the miRNA profile in exosomes secreted by human PCa LNCaP cells under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions. We identified 292 miRNAs loaded in both ExoHypoxic and ExoNormoxic. The top 11 miRNAs with significantly higher level in ExoHypoxic compared to ExoNormoxic were miR-517a, miR-204, miR-885, miR-143, miR-335, miR-127, miR-542, miR-433, miR-451, miR-92a and miR-181a; and top nine miRNA with significantly lower expression level in ExoHypoxic compared to ExoNormoxic were miR-521, miR-27a, miR-324, miR-579, miR-502, miR-222, miR-135b, miR-146a and miR-491. Importantly, the two differentially expressed miRNAs miR-885 (increased expression) and miR-521 (decreased expression) showed similar expression pattern in exosomes isolated from the serum of PCa patients compared to healthy individuals. Additionally, miR-204 and miR-222 displayed correlated expression patterns in prostate tumors (Pearson R = 0.66, p < 0.0001) by The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) genomic dataset analysis. Overall, the present study identified unique miRNAs with differential expression in exosomes secreted from hypoxic PCa cells and suggests their potential usefulness as a biomarker of hypoxia in PCa patients.

Evaluation of folic acid tagged aminated starch/ZnO coated iron oxide nanoparticles as targeted curcumin delivery system.

The role of 'superparamagnetic iron oxide nanoparticles' has gained considerable interest in various biomedical applications. However extensive researches are still going on to modify the nanoparticle size, surface properties etc. in order to get full benefit of the material. In this study, iron oxide nanoparticles have been modified with folic acid tagged aminated starch/ZnO coating to formulate a targeted drug delivery system. The nanoparticles were characterized by Fourier Transform Infra-red Spectroscopy, X-ray Diffraction, Scanning Electron Microscopy and Transmission Electron Microscopy. Both the swelling and in vitro drug release was performed in both acidic and alkaline pH. The cytotoxicity of the drug loaded nanoparticles was analysed by MTT assay in human lymphocytes, HepG2 and MCF7 cell lines. The cell uptake efficiencies and Reactive Oxygen Species generation with the drug loaded nanoparticles was estimated in HepG2 cell lines. The uptake capacity of fluorescein isothiocyanate tagged nanoparticles was assessed by fluorescence microscope.

Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles.

Aminated starch coated iron oxide magnetic nanoparticles loaded with curcumin were synthesized via coprecipitation technique. The nanoparticles were crosslinked by using three different crosslinkers: glutaraldehyde, genipin and citric acid and the effect of crosslinking on different properties of the nanoparticles was evaluated. Characterisation of the nanoparticles was done with FTIR (Fourier Transform Infrared spectroscopy) and XRD (X-Ray Diffraction). Magnetic property study using VSM (Vibrating Sample Magnetometer) showed their superparamagnetic nature. Morphology of the nanoparticles was studied by SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy). Zeta potential values showed that crosslinking imparted stability to the system. Crosslinking also enhanced drug loading and encapsulation efficiency of the system. Swelling and in vitro studies of the nanoparticles showed that the release of drug was dependent on time, crosslinker nature, crosslinker concentration and pH of the medium. The aminated starch coated nanoparticles also showed good mucoadhesive character. The cell viability assessment by MTT study revealed their compatibility with human lymphocytes cells and their considerable cell growth inhibiting properties with MCF7 and HepG2 cells. The nanoparticles showed good internalization in HepG2 cells along with considerable ROS formation.

Alcohol and Tobacco Increases Risk of High Risk HPV Infection in Head and Neck Cancer Patients: Study from North-East Region of India.

BACKGROUND: Human papilloma virus (HPV) associated Head and Neck Cancers (HNCs) have generated significant amount of research interest in recent times. Due to high incidence of HNCs and lack of sufficient data on high-risk HPV (hr-HPV) infection from North -East region of India, this study was conceived to investigate hr-HPV infection, its types and its association with life style habits such as tobacco, alcohol consumption etc. METHODS: A total of one hundred and six primary HNC tumor biopsy specimens were collected. These samples were analyzed for hr-HPV DNA (13 HPV types) using hybrid capture 2 (HC2) assay and genotyping was done by E6 nested multiplex PCR (NMPCR). RESULTS: The presence of hr-HPV was confirmed in 31.13% (n = 33) and 24.52% (n = 26) of the HNC patients by nested multiplex PCR (NMPCR) and HC2 assay respectively. Among hr-HPV positive cases, out of thirteen hr- HPV types analyzed, only two prevalent genotypes, HPV-16 (81.81%) followed by HPV-18 (18.18%) were found. Significant association was observed between hr-HPV infection with alcohol consumption (p <0.001) and tobacco chewing (p = 0.02) in HNC cases. Compared to HPV-18 infection the HPV-16 was found to be significantly associated with tobacco chewing (p = 0.02) habit. CONCLUSIONS: Our study demonstrated that tobacco chewing and alcohol consumption may act as risk factors for hr-HPV infection in HNCs from the North-East region of India. This was the first study from North-East India which also assessed the clinical applicability of HC2 assay in HNC patient specimens. We suggest that alcohol, tobacco and hr- HPV infection act synergistically or complement each other in the process of HNC development and progression in the present study population.

Hypoxia induces triglycerides accumulation in prostate cancer cells and extracellular vesicles supporting growth and invasiveness following reoxygenation.

Hypoxia is an independent prognostic indicator of poor outcome in several malignancies. However, precise mechanism through which hypoxia promotes disease aggressiveness is still unclear. Here, we report that under hypoxia (1% O2), human prostate cancer (PCA) cells, and extracellular vesicles (EVs) released by these cells, are significantly enriched in triglycerides due to the activation of lipogenesis-related enzymes and signaling molecules. This is likely a survival response to hypoxic stress as accumulated lipids could support growth following reoxygenation. Consistent with this, significantly higher proliferation was observed in hypoxic PCA cells following reoxygenation associated with rapid use of accumulated lipids. Importantly, lipid utilization inhibition by CPT1 inhibitor etomoxir and shRNA-mediated CPT1-knockdown significantly compromised hypoxic PCA cell proliferation following reoxygenation. Furthermore, COX2 inhibitor celecoxib strongly reduced growth and invasiveness following hypoxic PCA cells reoxygenation, and inhibited invasiveness induced by hypoxic PCA EVs. This establishes a role for COX2 enzymatic products in the enhanced PCA growth and invasiveness. Importantly, concentration and loading of EVs secreted by PCA cells were significantly compromised under delipidized serum condition and by lipogenesis inhibitors (fatostatin and silibinin). Overall, present study highlights the biological significance of lipid accumulation in hypoxic PCA cells and its therapeutic relevance in PCA.

Flower extract of Nyctanthes arbor-tristis modulates glutathione level in hydrogen peroxide treated lymphocytes.

BACKGROUND: Nyctanthes arbor-tristis Linn (Oleaceae) is a well-known traditional medicinal plant used throughout the India as an herbal remedy for treating various infectious and non-infectious diseases. OBJECTIVE: To evaluate the antioxidative activity of hydro-alcoholic extract of flower in the lymphocytes exposed to oxidative stress induced by H(2)O(2) . MATERIALS AND METHODS: Isolated lymphocytes were treated in vitro with extract or extract+H(2)O(2,) and the level of reduced glutathione (GSH) as well as the activity of glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) were measured. RESULTS: Treatment of lymphocyte with flower extract (50, 100, and 200 µg/ ml) significantly increased the level of GSH and decreased the activity of GST. The LDH activity measured in the cell-free medium decreased significantly. Pre-treatment of lymphocyte with flower extract protects the lymphocyte from the H(2)O(2) induced oxidative stress by significantly increasing the levels of GSH as compared to the cells treated only with H(2)O(2). Pre-treatment also reduced the activity of LDH significantly as compared to the cells treated only with H(2)O(2). The LDH activity in cell-free medium is associated with membrane damage, the decreased levels of LDH activity reflects the reduced level of membrane damage due to H(2)O(2). CONCLUSION: The present findings suggest the protective role of the hydro-alcoholic extracts of the flower of Nyctanthes arbor-tristis against membrane damage induced by H(2)O(2). The results also suggest that the extract might be rich in phytochemicals with antioxidant/radical scavenging potentials, which might find application in antioxidant therapy.