Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the efficient and cost effective delivery of shRNA to cancer cells.

In this study, shRNA against VEGFA was packaged in bacterial minicells and surface of minicells was modified with folic acid. Analysis of cellular internalization revealed that folic acid conjugated minicells internalized through receptor mediated endocytosis in folate and PSMA receptor positive KB and LNCaP cells, respectively. In contrast, A549 (folate receptor negative) cells showed minute internalization. In vitro delivery of FAminicellsVEGFA significantly reduced the expression of VEGFA mRNA in KB and LNCaP cells whereas expression of VEGFA remained unaltered in A549 cells. FAminicellsVEGFA significantly reduced tumor volume in mice bearing KB and LNCaP xenograft. On contrary, gradual increase in the tumor volume was recorded in A549 xenograft. FAminicellsVEGFA significantly silenced the VEGFA mRNA in KB and LNCaP xenograft. Expression of VEGFA remained same in FAminicellsVEGFA delivered A549 xenograft. In vivo biodistribution study showed that majority of FAminicellsVEGFA were localized in the tumor followed by intravenous administration.

Minicell-Based Targeted Delivery of shRNA to Cancer Cells: An Experimental Protocol.

Bacterial minicell has emerged as a novel targeted delivery system for RNAi-based therapeutics. In this chapter, we have described the detailed protocol for the preparation of minicell-based targeted delivery system for shRNA. Initially, minicell-producing parent bacterial cells were transformed with plasmid vector containing shRNA. Subsequently, shRNA-packaged minicells were purified from parent bacterial cells. Purified minicells were characterized by fluorescence microscopy and transmission electron microscopy. In the next step, targeting ligand was conjugated on the minicell surface for the active targeting of cancer cell surface receptors. Eventually, target-specific delivery of minicells was explored in vitro in selected cancer cell line and in vivo in mice bearing tumor xenograft.

Antithrombocytopenic activity of carpaine and alkaloidal extract of Carica papaya Linn. leaves in busulfan induced thrombocytopenic Wistar rats.

ETHNOPHARMACOLOGICAL RELAVANCE: The decoction of Carica papaya Linn. leaves is used in folklore medicine in certain parts of Malaysia and Indonesia for the treatment of different types of thrombocytopenia associated with diseases and drugs. There are several scientific studies carried out on humans and animal models to confirm the efficacy of decoction of papaya leave for the treatment of disease induced and drug induced thrombocytopenia, however very little is known about the bio-active compounds responsible for the observed activity. The aim of present study was to identify the active phytochemical component of Carica papaya Linn. leaves decoction responsible for anti-thrombocytopenic activity in busulfan-induced thrombocytopenic rats. MATERIALS AND METHODS: Antithrombocytopenic activity was assessed on busulfan induced thrombocytopenic Wistar rats. The antithrombocytopenic activity of different bio-guided fractions was evaluated by monitoring blood platelet count. Bioactive compound carpaine was isolated and purified by chromatographic methods and confirmed by spectroscopic methods (LC-MS and 1D/2D-1H/13C NMR) and the structure was confirmed by single crystal X-ray diffraction. Quantification of carpaine was carried out by LC-MS/MS equipped with XTerra(®) MS C18 column and ESI-MS detector using 90:10 CH3CN:CH3COONH4 (6mM) under isocratic conditions and detected with multiple reaction monitoring (MRM) in positive ion mode. RESULTS: Two different phytochemical groups were isolated from decoction of Carica papaya leaves: phenolics, and alkaloids. Out of these, only alkaloid fraction showed good biological activity. Carpaine was isolated from the alkaloid fraction and exhibited potent activity in sustaining platelet counts upto 555.50±85.17×10(9)/L with no acute toxicity. CONCLUSIONS: This study scientifically validates the popular usage of decoction of Carica papaya leaves and it also proves that alkaloids particularly carpaine present in the leaves to be responsible for the antithrombocytopenic activity.

Anti-inflammatory, Anti-estrogenic, and Anti-implantation Activity of Bergia suffruticosa (Delile) Fenzl.

BACKGROUND: Bergia suffruticosa (Delile) Fenzl (Syn. Bergia odorata Edgew) (Elatinaceae family) is used traditionally to repair bones and is applied as a poultice on sores. It is also used for stomach troubles and as an antidote to scorpion stings. So far, very little scientific work has been reported to validate its ethnomedical uses in the alleviation of pain, bone repair, etc. OBJECTIVE: This study was designed to explore the anti-inflammatory and anti-implantation potential of n-hexane extract of B. suffruticosa whole plant in mice along with identification of its chemical constituents. MATERIALS AND METHODS: n-Hexane extract of B. suffruticosa whole plant was screened for acute and chronic anti-inflammatory activity followed by an anti-estrogenic activity. Eventually, n-hexane extract was tested for anti-implantation activity by exploiting markers of uterine receptivity, lipid peroxidation, and superoxide enzyme activity. The extract was administered orally at a dose of 100 mg/kg body weight in each study. RESULTS: Thin layer chromatography fingerprint profile of n-hexane extract revealed the presence of lupeol and ß-sitosterol. The n-hexane extract reduced the edema by 80% in acute inflammation, whereas it reduced edema to 75% on the 5(th) day in chronic inflammation. The n-hexane extract reduced elevated malonaldehyde level from 6 to 2.5 nmol/g × 10(-5) and increased superoxide dismutase enzyme activity from 0 to 350 units/g in treated animals on the 5(th) day of pregnancy. Moreover, extract decreased uterine weight from 0.33 to 0.2 g in estradiol treated animals. CONCLUSION: These results indicate that n-hexane extract of B. suffruticosa is having potent anti-inflammatory, anti-estrogenic, and anti-implantation activity. This is the first report of all the pharmacological activities of B. suffruticosa mentioned above. SUMMARY: TLC fingerprint profile of n-hexane extract of Bergia suffruticosa whole plant revealed the presence of lupeol and ß-sitosteroln-Hexane extract showed in vivo anti-inflammatory activity in both acute and chronic model of inflammation in ratsn-Hexane extract possess significant anti-estrogenic activityn-Hexane extract altered the levels superoxide anion radical and superoxide dismutase enzyme activity during the blastocyst implantationAnti-implantation activity of n-hexane extract is attributed to its anti-inflammatory and anti-estrogenic potential. Abbreviations used: TLC: Thin layer chromatography; LPO: Lipid peroxidation; SOD: Superoxide dismutase; B. suffruticosa: Bergia suffruticosa; TNF-α: Tumor necrosis factor-α; NO: Nitric oxide; IL-1: Interleukin-1; LIF: Leukemia inhibitory factor; CSF-1: Colony-stimulating factor; COX: Cyclooxygenase; SDS: Sodium dodecyl sulfate; IAEC: Animal House Ethics Committee; CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; HBSS: Hank's balanced salt solution; MDA: Malonaldehyde; and TBA: Thiobarbituric acid.

Antiestrogenic and Anti-Inflammatory Potential of n-Hexane Fraction of Vitex negundo Linn Leaf Extract: A Probable Mechanism for Blastocyst Implantation Failure in Mus musculus.

The anti-implantation potential of different fractions of Vitex negundo Linn leaf extract was evaluated in female Swiss Albino mice. Animals from different groups were dosed orally either with 0.2% agar (vehicle) or with fractions of V. negundo leaf extract (n-hexane, chloroform, n-butanol, and remnant fractions) at 10:00 a.m., from day 1 to day 6 of pregnancy. The pregnant females from each group were sacrificed on different days of pregnancy (n = 6), and uterus was excised and used for estimation of lipid peroxidation and assay of superoxide dismutase activity as a marker for blastocyst implantation. Animals treated with n-hexane fraction showed altered level of superoxide anion radical and superoxide dismutase activity as compared to control animals. The probable mechanism by which this extract exhibits inhibition of blastocyst implantation is through the anti-inflammatory and antiestrogenic potential.

An improved and versatile immunosuppression protocol for the development of tumor xenograft in mice.

BACKGROUND: The objective of the present study was to evaluate the efficacy of a simple, versatile and cost-effective immunosuppression protocol, using cyclosporine, ketoconazole and cyclophosphamide drug regimen to develop human tumor xenograft in mice. MATERIALS AND METHODS: Cyclosporine, ketoconazole and cyclophosphamide drug regimen was administered to C57BL/6 mice to induce immunosuppression. Five million A549, LNCaP and KB cells were injected subcutaneously in the immunocompromised mice for the development of tumor xenograft. Tumor volume was calculated every week. Histopathology of tumor tissue was analyzed. RESULTS: Prolong immunosuppression was achieved by this combination treatment. The average tumor volume was found to be greater than 600 mm(3). Histopathology of tumor tissue revealed the presence of large and irregular nucleus and scanty cytoplasm, which are characteristic of malignant cells. CONCLUSION: A versatile immunosuppression protocol was developed which was validated for xenograft development using three different cell lines, with a 100% take rate and no mortality.

Purification and Characterization of Haloalkaline, Organic Solvent Stable Xylanase from Newly Isolated Halophilic Bacterium-OKH.

A novel, alkali-tolerant halophilic bacterium-OKH with an ability to produce extracellular halophilic, alkali-tolerant, organic solvent stable, and moderately thermostable xylanase was isolated from salt salterns of Mithapur region, Gujarat, India. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. Maximum xylanase production was achieved at pH 9.0 and 37°C temperature in the medium containing 15% NaCl and 1% (w/v) corn cobs. Sugarcane bagasse and wheat straw also induce xylanase production when used as carbon source. The enzyme was active over a range of 0-25% sodium chloride examined in culture broth. The optimum xylanase activity was observed at 5% sodium chloride. Xylanase was purified with 25.81%-fold purification and 17.1% yield. Kinetic properties such as Km and Vmax were 4.2 mg/mL and 0.31 µmol/min/mL, respectively. The enzyme was stable at pH 6.0 and 50°C with 60% activity after 8 hours of incubation. Enzyme activity was enhanced by Ca(2+), Mn(2+), and Mg(2+) but strongly inhibited by heavy metals such as Hg(2+), Fe(3+), Ni(2+), and Zn(2+). Xylanase was found to be stable in organic solvents like glutaraldehyde and isopropanol. The purified enzyme hydrolysed lignocellulosic substrates. Xylanase, purified from the halophilic bacterium-OKH, has potential biotechnological applications.

Evaluation of GABAergic Transmission Modulation as a Novel Functional Target for Management of Multiple Sclerosis: Exploring Inhibitory Effect of GABA on Glutamate-Mediated Excitotoxicity.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) where the communication ability of nerve cells in the brain and spinal cord with each other gets impaired. Some current findings suggest the role of glutamate excitotoxicity in the development and progression of MS. An excess release of glutamate leads to the activation of ionotropic and metabotropic receptors, thus resulting in accumulation of toxic cytoplasmic Ca(2+) and cell death. However, it has been observed that gamma-aminobutyric acid-A (GABAA) receptors located in the nerve terminals activate presynaptic Ca(2+)/calmodulin-dependent signaling to inhibit depolarization-evoked Ca(2+) influx and glutamate release from isolated nerve terminals, which suggest a potential implication of GABAA receptor in management of MS. With this proof of concept, we tried to explore the potential of selective GABAA receptor agonists or positive allosteric modulators (diazepam and phenobarbitone sodium) and GABAA level enhancer (sodium valproate) for management of MS by screening them for their activity in experimental autoimmune encephalomyelitis (EAE) model in rats and cuprizone-induced demyelination model in mice. In this study, sodium valproate was found to show the best activity in the animal models whereas phenobarbitone sodium showed moderate activity. However, diazepam was found to be ineffective.

A combination approach for rapid and high yielding purification of bacterial minicells.

A method for bacterial minicell purification was developed by combining antibiotic (ceftriaxone) lysis and filtration. This method is fast, cost effective and facilitates high yield of purified minicells, with no parent strain contamination as confirmed by fluorescent microscopy, average particle size and polydispersity index.