Intracranially injectable multi-siRNA nanomedicine for the inhibition of glioma stem cells.

BACKGROUND: Nanoparticle siRNA-conjugates are promising clinical therapeutics as indicated by recent US-FDA approval. In glioma stem cells (GSC), multiple stemness associated genes were found aberrant. We report intracranially injectable, multi-gene-targeted siRNA nanoparticle gel (NPG) for the combinatorial silencing of 3 aberrant genes, thus inhibiting the tumorogenic potential of GSCs. METHODS: NPG loaded with siRNAs targeted against FAK, NOTCH-1, and SOX-2 were prepared by the self-assembly of siRNAs with protamine-hyaluronic acid combination. Electron microscopy, DLS, and agarose gel electrophoresis were used for the physicochemical characterization. Cell transfection and gene-silencing efficiency were studied using human mesenchymal stem cells and rat C6 glioma-derived GSCs. Neurosphere inhibition was tested in vitro using GSCs derived from C6 cell line and glioma patient samples. Patient-derived xenograft model and orthotopic rat glioma model were used to test the effect of NPG on in vivo tumorigenicity. RESULTS: The siRNA nanoparticles with an average size ~ 250 nm and ~ 95% loading efficiency showed cellular uptake in ~95.5% GSCs. Simultaneous gene silencing of FAK, NOTCH-1, and SOX-2 led to the inhibition of neurosphere formation by GSCs, whereas normal stem cells remained unaffected and retained neuronal differentiation capability. GBM PDX models manifested significant impairment in the tumorigenic potential of NPG treated GSCs. Intracranial injection of NPG inhibited tumor growth in orthotopic rat brain tumor model. CONCLUSION: Intracranially injectable n-siRNA NPG targeted to multiple stem-cell signaling impairs glioma initiation capabilities of GSCs and inhibited tumor growth in vivo.

Brain-Tumor-Regenerating 3D Scaffold-Based Primary Xenograft Models for Glioma Stem Cell Targeted Drug Screening.

Glioma stem cells (GSC) present a critical therapeutic challenge for glioblastoma multiforme (GBM). Drug screening against GSC demands development of novel in vitro and in vivo platforms that can mimic brain microenvironment and support GSC maintenance and tumorigenesis. Here, we report, a 3-dimensionel (3D) biomimetic macro-porous scaffold developed by incorporating hyaluronic acid, porcine brain extra cellular matrix (ECM) and growth factors that facilitates regeneration of GBM from primary GSCs, ex vivo and in vivo. After characterizing with human and rat GBM cell lines and neurospheres, human GSCs expressing Notch1, Sox-2, Nestin, and CD133 biomarkers were isolated from GBM patients, cultured in the 3D scaffold, and implanted subcutaneously in nude mice to develop patient derived xenograft (PDX) models. Aggressive growth pattern of PDX with formation of intratumoral vascularization was monitored by magnetic resonance imaging (MRI). Histopathological and phenotypial features of the original tumors were retained in the PDX models. We used this regenerated GBM platform to screen novel siRNA nanotherapeutics targeting Notch, Sox-2, FAK signaling for its ability to inhibit the tumorigenic potential of GSCs. Current clinical drug, Temozolomide and an anticancer phytochemical, nanocurcumin, were used as controls. The siRNA nanoparticles showed excellent efficacy in inhibiting tumorigenesis by GSCs in vivo. Our study suggests that the brain-ECM mimicking scaffold can regenerate primary gliomas from GSCs in vitro and in vivo, and the same can be used as an effective platform for screening drugs against glioma stem cells.

Protection against Whole Body γ-Irradiation Induced Oxidative Stress and Clastogenic Damage in Mice by Ginger Essential Oil.

Radioprotective effects of ginger essential oil (GEO) on mortality, body weight alteration, hematological parameters, antioxidant status and chromosomal damage were studied in irradiated mice. Regression analysis of survival data in mice exposed to radiation yielded LD50/30 as 7.12 and 10.14 Gy for control (irradiation alone) and experimental (GEO-treated irradiated) mice, respectively, with a dose reduction factor (DRF) of 1.42. In mice exposed to whole-body gamma-irradiation (6 Gy), GEO pre-treatment at 100 and 500 mg/kg b.wt (orally) significantly ameliorated decreased hematological and immunological parameters. Radiation induced reduction in intestinal tissue antioxidant enzyme levels such as superoxide dismutase, catalase, glutathione peroxidase and glutathione was also reversed following administration of GEO. Tissue architecture of small intestine which was damaged following irradiation was improved upon administration of GEO. Anticlastogenic effects of GEO were studied by micronuclei assay, chromosomal aberration and alkaline gel electrophoresis assay. GEO significantly decreased the formation of micronuclei, increased the P/N ratio, inhibited the formation of chromosomal aberrations and protected agaisnt cellular DNA damage in bone marrow cells as revealed by comet assay. These results are supportive of use of GEO as a potential radioprotective compound.

Enhanced bioavailability and safety of curcumagalactomannosides as a dietary ingredient.

In spite of the various bioavailable formulations of curcumin for pharma and dietary supplement applications, food grade formulations suitable as a dietary ingredient, and capable of providing significant levels of plasma curcumin, are limited. The present contribution describes the safety and oral bioavailability of a novel water soluble formulation of curcumin, curcumagalactomannosides (CGM), when used as a dietary ingredient in selected food items. CGM was prepared using a food grade hydrocolloid (galactomannans) derived from the kitchen spice fenugreek (Trigonella foenum graccum), without using any synthetic excipients. The safety of the formulation was assessed through acute and subchronic toxicity studies on Wistar rats and genotoxicity studies. The efficacy of CGM as a bioavailable dietary ingredient was assessed by successfully preparing various food items and by measuring the post-blood plasma curcumin levels at various time intervals after the consumption of food items. High performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) and electrospray ionization tandem mass spectrometer (ESI-MS/MS) was employed for the quantification of plasma curcuminoids. It was observed that CGM is safe and suitable for further development and clinical studies, with a no observable adverse effect level (NOAEL) up to 2.0 g kg⁻¹ per day b.wt. CGM was found to offer seven to ten times higher bioavailability of curcumin in humans, when incorporated into various food/beverage items at 100 mg CGM per serving size, as compared to the standard unformulated curcumin.

Gastroprotective activity of essential oils from turmeric and ginger.

BACKGROUND: Turmeric (Curcuma longa) and ginger (Zingiber officianale) are widely used in Asian countries as traditional medicine and food ingredients. In the present study, we have evaluated the gastroprotective activity of turmeric essential oil (TEO) and ginger essential oil (GEO) in rats. METHODS: Turmeric and ginger were evaluated for their antiulcer activity against ethanol-induced ulcers in male Wistar rats at different doses: 100, 500 and 1000 mg/kg body weight. Ethanol was used to induce gastric ulcer in Wistar rats. Parameters such as ulcer index, histopathology and levels of antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase and glutathione (GSH) levels were measured to assess the degree of protection produced by the essential oils. RESULTS: TEO and GEO inhibited ulcer by 84.7% and 85.1%, respectively, as seen from the ulcer index. Reduced antioxidant enzymes such as GPx, SOD, catalase and GSH produced by alcohol administration were significantly (p<0.001) increased by simultaneous administration of TEO and GEO. Histopathological examination showed that ethanol-induced lesions such as necrosis, erosion and hemorrhage of the stomach wall were significantly reduced after oral administration of essential oils. CONCLUSIONS: RESULTS suggest that TEO and GEO could reduce the gastric ulcer in rat stomach as seen from the ulcer index and histopathology of the stomach. Moreover, oxidative stress produced by ethanol was found to be significantly reduced by TEO and GEO.

Chemopreventive activity of turmeric essential oil and possible mechanisms of action.

This study aimed to evaluate the antimutagenic and anticarcinogenic activity of turmeric essential oil as well as to establish biochemical mechanisms of action. Antimutagenicity testing was accomplished using strains and known mutagens with and without microsomal activation. Anticarcinogenic activity was assessed by topical application of 7, 12 - dimethylbenz[a]anthracene (DMBA) as initiator and 1% croton oil as promoter for the induction of skin papillomas in mice. Inhibition of p450 enzymes by TEO was studied using various resorufins and aminopyrene as substrate. Turmeric essential oil (TEO) showed significant antimutagenic activity (p<0.001) against direct acting mutagens such as sodium azide (NaN3), 4-nitro-O-phenylenediamine (NPD) and N-methyl- N-nitro N'nitrosoguanine (MNNG). TEO was found to have significant antimutagenic effect (>90%) against mutagen needing metabolic activation such as 2-acetamidoflourene (2-AAF). The study also revealed that TEO significantly inhibited (p<0.001) the mutagenicity induced by tobacco extract to Salmonella TA 102 strain. DMBA and croton oil induced papilloma development in mice was found to be delayed and prevented significantly by TEO application. Moreover TEO significantly (P<0.001) inhibited isoforms of cytochrome p450 (CYP1A1, CYP1A2, CYP2B1/2, CYP2A, CYP2B and CYP3A) enzymes in vitro, which are involved in the activation of carcinogens. Results indicated that TEO is antimutagenic and anticarcinogenic and inhibition of enzymes (p450) involved in the activation of carcinogen is one of its mechanisms of action.

Antimutagenic potential and modulation of carcinogen-metabolizing enzymes by ginger essential oil.

Essential oil extracted from ginger (GEO) was evaluated for its mutagenicity to Salmonella typhimurium TA 98, TA 100, TA 102, and TA 1535 strains with and without microsomal activation. GEO was found to be non-mutagenic up to a concentration of 3 mg/plate. It was also assessed for antimutagenic potential against direct acting mutagens such as sodium azide, 4-nitro-o-phenylenediamine, N-methyl-N'-nitro-N-nitrosoguanidine, tobacco extract, and 2-acetamidoflourene, which needs microsomal activation. GEO significantly inhibited (p < 0.001) the mutagenicity induced by these agents in a concentration-dependent manner. The effect of GEO to modulate the action of phase I carcinogen-metabolizing enzymes was investigated by studying its effect on various isoforms of microsomal cytochrome P450 enzymes. Significant inhibition of CYP1A1, CYP1A2, and CYP2B1/2, aniline hydroxylase (an indicator of CYP2E1 activity), and aminopyrine-N-demethylase (indicator of CYP1A, 2A, 2B, 2D, and 3A activity) was shown by GEO both in vitro and in vivo. GEO gave an IC50 value of 30, 57.5, and 40 µg for CYP1A1, CYP1A2, and CYP2B1/2, respectively, 55 µg for aniline hydroxylase, and 37.5 µg for aminopyrene-N-demethylase. GEO also significantly increased the levels of phase II carcinogen-metabolizing enzymes uridine 5'-diphospho-glucuronyl transferase and glutathione-S-transferase in vivo indicating the use of GEO as an antimutagen and as a potential chemopreventive agent.

Antioxidant, anti-inflammatory and antinociceptive activities of essential oil from ginger.

Chemical compositions of ginger oil as well as its antioxidant, anti-inflammatory and antinociceptive potential were evaluated in the present study. The main constituents as detected by GC/MS analysis was alpha-zingiberene which constituted 31% of the total area, ar-curcumene (15.4%) and a -sesquiphellandrene (14.02%). Ginger oil scavenged superoxide, DPPH, hydroxyl radicals and inhibited tissue lipid peroxidation in vitro. Intraperitoneal administration of ginger oil was found to inhibit phorbol-12-myristate-13-acetate induced superoxide radicals elicited by macrophages. Oral administration of ginger oil for one month, significantly increased superoxide dismutase, glutathione and glutathione reductase enzymes level (P < 0.001) in blood of mice and glutathione-S-transferase, glutathione peroxidase and superoxide dismutase enzymes in liver. Ginger oil produced significant reduction in acute inflammation produced by carrageenan and dextran and formalin induced chronic inflammation (P < 0.001). It also exhibited significant reduction in acetic acid induced writhing movements (P < 0.001). The present report revealed that ginger oil possesses antioxidant activity as well as significant anti-inflammatory and antinociceptive property.

Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L).

The present study investigated the acute, subchronic and genotoxicity of turmeric essential oil (TEO) from Curcuma longa L. Acute administration of TEO was done as single dose up to 5 g of TEO per kg body weight and subchronic toxicity study for thirteen weeks was done by daily oral administration of TEO at doses 0.1, 0.25 and 0.5 g/kg b.wt. in Wistar rats. There were no mortality, adverse clinical signs or changes in body weight; water and food consumption during acute as well as subchronic toxicity studies. Indicators of hepatic function such as aspartate aminotransferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) were unchanged in treated animals compared to untreated animals. Oral administration of TEO for 13 weeks did not alter total cholesterol, triglycerides, markers of renal function, serum electrolyte parameters and histopathology of tissues. TEO did not produce any mutagenicity to Salmonella typhimurium TA-98, TA-100, TA-102 and TA-1535 with or without metabolic activation. Administration of TEO to rats (1 g/kg b.wt.) for 14 days did not produce any chromosome aberration or micronuclei in rat bone marrow cells and did not produce any DNA damage as seen by comet assay confirming the non toxicity of TEO.

An evaluation of antioxidant, anti-inflammatory, and antinociceptive activities of essential oil from Curcuma longa. L.

OBJECTIVES: This study was aimed to evaluate the chemical composition, antioxidant potential in vitro and in vivo, anti-inflammatory, and antinociceptive activity of turmeric oil. MATERIALS AND METHODS: Chemical analysis of turmeric oil was done by gas chromatography/mass spectrometry. Antioxidant activities in vitro was done by six different methods and in vivo antioxidant activity was determined by measuring superoxide generation from macrophages treated with phorbol-12-myristate-13-acetate (PMA) as well as determining antioxidant level after feeding the oil orally for one month. Anti-inflammatory activity was studied in mice using carrageenan, dextran, and formalin. Antinociceptive activity was evaluated by using acetic acid-induced writhing movement in mice. RESULTS: The main constituent of essential oil of turmeric was found to be ar-turmerone (61.79%), curlone (12.48%), and ar-curcumene (6.11%). Turmeric oil was found to have in vitro antioxidant activity and IC(50) for scavenging superoxides, hydroxyl radicals, and lipid peroxidation were 135 µg/ml, 200 µg/ml, and 400 µg/ml, respectively. The ferric-reducing activity for 50 µg of turmeric essential oil was found to be 5 mM. Intraperitoneal administration of oil was found to inhibit PMA-induced superoxide radicals elicited by macrophages. Oral administration of turmeric oil for one month to mice significantly increased superoxide dismutase, glutathione, and glutathione reductase enzyme levels in blood and glutathione-S-transferase and superoxide dismutase enzymes in liver. Turmeric oil showed significant reduction in paw thickness in carrageenan, dextran-induced acute inflammation, and formalin-induced chronic inflammation. The drug produced significant antinociceptive activity (P < 0.001) at all doses studied. CONCLUSIONS: These results demonstrated that turmeric oil has potential health benefits as it can scavenge the free radicals and produce significant anti-inflammatory and antinociceptive activities.

A preliminary 13-week oral toxicity study of ginger oil in male and female Wistar rats.

Zingiber officinale Roscoe, ginger, is a major spice extensively used in traditional medicine. The toxicity profile of ginger oil was studied by subchronic oral administration for 13 weeks at doses of 100, 250, and 500 mg/kg per day to 6 groups of Wistar rats (5/sex per dose). Separate groups of rats (5/sex per group) received either paraffin oil (vehicle) or were untreated and served as comparative control groups. There was no mortality and no decrease in body weight or food consumption as well as selective organ weights during the study period. Administration of ginger oil to rats did not produce any treatment-related changes in hematological parameters, hepatic, renal functions, serum electrolytes, or in histopathology of selected organs. The major component of ginger oil was found to be zingiberene (31.08%), and initial studies indicated the presence of zingiberene in the serum after oral dosing. These results confirmed that ginger oil is not toxic to male and female rats following subchronic oral administrations of up to 500 mg/kg per day (no observed adverse effect level [NOAEL]).