Nanoparticulate delivery of LHRH analogue for the treatment of prostate cancer.

Goserelin acetate (Gos) is a luteinizing hormone-releasing hormone agonist, used in treatment of prostate cancer in which desired concentration of Gos in blood is maintained for longer duration. The aim of this study is to improve the efficacy of Gos targeted at the site of action and eliminate the need for frequent administration. Gos-encapsulated nanoparticles were fabricated by double emulsification process. The physicochemical traits of the nanoparticles including morphology, particle size, zeta-potential, entrapment efficiency, and in-vitro release profile were studied. The in-vitro cytotoxicity of the blank nanoparticles and Gos-loaded nanoparticles were also evaluated on LNCaP cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Blank methoxy PEG-poly(ε-caprolactone) (mPEG-PCL) nanoparticles exhibited low cytotoxicity, which increased with increase in concentration of Gos-loaded nanoparticles. Serum Gos and testosterone levels were analyzed after subcutaneous administration in Wistar rats. In-vivo study showed that a sustained serum level of Gos successfully suppressed the plasma testosterone concentration to castration level. So, it can be concluded that mPEG-PCL nanoparticles might prove to be useful for site specific and sustain protein delivery.

"Prevention of structural perturbation and aggregation of hepatitis B surface antigen: screening of various additives".

The instability of protein and antigen(s) during encapsulation in biodegradable polymers by water-in-oil-in-water (w/o/w) encapsulation is well established. The aim of present study is to screen various additives to prevent the inactivation and loss of immunogenicity of HBsAg upon its exposure to the water/CH(2)Cl(2) (methylene chloride) interface by simulating the formulation steps involved in the preparation of microspheres. The secondary structure of HBsAg, recovered under different conditions after primary emulsification, was investigated by FTIR spectroscopy and Circular Dichorism. Subsequently, PLGA microspheres were formulated and characterized for their size, shape, incorporation efficiency, antigen integrity, and immunogenicity. The immunogenicity and the HBsAg recovery under different conditions were tested in BALB/c mice. Inulin and trehalose were found to be better stabilizing agents to prevent the aggregation, the structural perturbations and immunogenicity of HBsAg. This study substantiated that inulin could overcome the aggregation and denaturing effects of the water/CH(2)Cl(2) interface upon HBsAg during emulsification step and upon encapsulation.

Antioxidant and cytotoxic potential of a new thienyl derivative from Tagetes erecta roots.

CONTEXT: The search for newer compounds against pathogenic species continues unabated due to drug resistance. Traditionally, Tagetes erecta Linn. (Compositae) has been used for the treatment of various parasitic and microbial diseases. OBJECTIVE: To evaluate the antioxidant activity of the ethanol extract of Tagetes erecta roots and its cytotoxicity against prostate and HeLa cancer cell lines followed by activity-guided isolation. MATERIALS AND METHODS: The antioxidant screening was carried out using diphenylpicrylhydrazyl (DPPH) radical scavenging assay with serial concentrations ranging from 2 to 100 µg/mL, and cytotoxicity was evaluated against prostate (PC-3) and HeLa cell lines using microculture tetrazolium test (MTT) assay with concentrations ranging from 500 to 1.89 µg/mL. Isolation of the ethanol extract was carried out using column chromatography whereby 21 isolates were obtained (T1-T21), and the most active isolate was subjected for characterization using ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), and mass spectroscopic techniques. RESULTS: The ethanol extract scavenged DPPH free radicals thereby exhibiting antioxidant activity with an IC50 of 35.9 µg/mL. In addition, the extract conferred noticeable cytotoxicity against the HeLa (LD50 of 164.28 µg/mL) and PC-3 cell lines (LD50 of 407.3 µg/mL). Among all the isolates, T3 showed antioxidant activity with IC50 of 11.56 µg/mL and cytotoxicity with LD50 of 12.5 µg/mL against HeLa and 30.25 µg/mL against PC-3 cell lines and was characterized as 2-ethynyl-5-(thiophen-2-yl) thiophene. DISCUSSION: The new thienyl compound (T3) exhibited profound antioxidant activity and cytotoxicity at relatively lower concentrations than the extract. CONCLUSION: The observations provide support for the ethnobotanical use of the plant.

Targeted novel surface-modified nanoparticles for interferon delivery for the treatment of hepatitis B.

The purpose of the present work was to develop hepatitis B surface antigen (HBsAg) surface-adsorbed cationic poly (d,l-lactic-co-glycolic acid) PLGA nanoparticles for interferon alpha (IFNα) delivery targeted to hepatocytes. Cationic PLGA nanoparticles loaded with IFNα were prepared using the double emulsification technique. Delipidated HBsAg was passively adsorbed on the surface of nanoparticles by using the simple dipping and drying method. Surface morphology and size distribution of nanoparticles were analyzed by scanning electron microscopy and dynamic light-scattering method, respectively. The biodistribution behavior of plain and HBsAg-coated (99m)Tc-tagged PLGA nanoparticles was also examined followed by intravenous injection. The results revealed that ∼75% of the radioactivity was recovered in the liver after 4 h of injection that was nearly 3-fold greater in magnitude than the plain PLGA nanoparticles. These data demonstrated that the novel formulation of nanoparticles has potential application in hepatic-targeted drug delivery.

Development characterizations and evaluation of Poly(-ε-caprolactone)-based microspheres for hepatitis B surface antigen delivery.

The major disadvantage of several currently available vaccines is the need for repeated administrations. The aim of the study was to develop long-acting microspheres based on poly(-ε-caprolactone) (PCL) for delivery of recombinant hepatitis B surface antigen (rHBsAg). PCL microspheres were prepared for induction of humoral and cellular immunity by intramuscular administration. Microspheres were characterized for their size, shape, incorporation efficiency, zeta potential, antigen integrity, antigen conformation and immunogenicity. DSC (Differential Scanning Calorimetry) studies revealed that better encapsulation efficiency between high and low mol wt polymer. The Circular Dichorism spectroscopy (CD) of antigen, released from PCL microspheres revealed that the secondary structure of antigen was unperturbed. Antigen integrity was evaluated by SDS-PAGE. Immunization with HBsAg PCL microspheres resulted in upregulation of specific cellular (IFN-γ and IL-2) as well as IgG response in BALB/c mice. Immune responses were found significantly higher than the conventional alum adjuvant following a single intramuscular immunization. These results highlight the enhanced efficiency of these PCL microspheres as an adjuvant and their prospective use in the prevention of hepatitis B.

Goserelin loaded nanoparticles inhibit growth and induce apoptosis in human prostate cancer cell lines.

Goserelin acetate (Gos) is a synthetic analogue of luteinizing hormone-releasing hormone (LHRH) used in treatment of prostate cancer. The objective of this study is to investigate the in vitro cytotoxic effect of Goserelin loaded nanoparticles on both androgen dependent and androgen independent cell lines. Goserelin causes cell death of prostate cancer cell lines by inducing apoptosis. Treatment of Goserelin loaded nanoparticles inhibited the proliferation of LNCaP and DU145 in the dose-dependent manner, however did not affect the cell proliferation in PC-3 cell lines. Blank nanoparticles exhibited negligible cytotoxicity on cell lines. In addition, immunocytochemical studies indicated that Gos induced apoptosis in prostate cancer cells. The presence and characteristics of LHRH receptors and their messenger ribonucleic acid (mRNA) expression in human prostate cancer cell line LNCaP were investigated by polymerase chain reaction. Changes in nuclear morphology and DNA fragmentation associated with Gos induced apoptosis were clearly seen in both LNCaP and DU145 cell lines by DNA studies. The PCR product of the expected size of 319 bp for human LHRH receptors was obtained in cell line sample. Goserelin loaded nanoparticles are the potential tool for site specific delivery for prostate cancer treatment.

Influence of fungal elicitation on glycyrrhizin production in transformed cell cultures of Abrus precatorius Linn.

BACKGROUND: Glycyrrhizin, obtained from Abrus precatorius (Indian liquorice), is a phytoconstituent of importance for pharmaceutical and food industries. MATERIALS AND METHODS: High producing and fast growing cell lines of A. precatorius were developed by transformation with Agrobacterium tumefaciens for glycyrrhizin production. Its maximum transformation efficiency of 85% was obtained by infecting leaves with A. tumefaciens MTCC-431 supplemented with 50 µM acetosyringone. Thorough culture growth kinetics with sugar consumption profiles was established. RESULTS: A twofold increase in glycyrrhizin productivity was obtained in transformed A. precatorius cell suspension cultures over the untransformed cultures. The fungal elicitors prepared from Aspergillus niger and Rhizopus stolonifer were tested at different concentrations to enhance glycyrrhizin production in transformed cell suspension cultures of A. precatorius. Maximum enhancement of 4.9- and 3.8-fold in glycyrrhizin contents, were obtained with A. niger (7.5% v/v) and R. stolonifer (5.0% v/v), respectively, on the 5th day after elicitor treatment. CONCLUSION: This study indicates the prospective of the amalgamation of elicitation methodology with transformed cell cultures for the large-scale production of glycyrrhizin.

Development and characterization of chitosan coated poly-(ɛ-caprolactone) nanoparticulate system for effective immunization against influenza.

In this study surface coated poly-(ɛ-caprolactone) (PCL) nanoparticles with chitosan (CS) were developed as a carrier system for nasal immunization using recombinant Influenza A virus (A/California/07/2009) H1N1 hemagglutinin (HA) protein, for the induction of humoral, cellular and mucosal immunity. CS coated PCL (CS-PCL) nanoparticles were characterized in vitro for their percent yield, size, shape, entrapment efficiency, loading capacity and zeta potential. The in vitro release and antigen integrity were also evaluated. Particles were prepared by an emulsion-diffusion-solvent evaporation method. The coated cationic nanoparticles of average size 125.64±6.51 nm with a narrow size distribution (pdi: 0.185±0.032) and a positive surface charge (+22.88 mV) were obtained. HA antigen was efficiently entrapped in CS-PCL nanoparticles (entrapment efficiency 74.84±4.51%, loading capacity 14±2% (w/w)). The molecular weight and antigenicity of the entrapped HA was maintained as shown by polyacrylamide gel electrophoresis and Western blotting, respectively. In vitro release study of antigen showed that about 66.47% of entrapped antigen was released within 63 days. The immune-stimulating activity was studied by measuring hemagglutination inhibition (HAI) titer, IgG, IgG1 and IgG2a titer, secretory IgA level in nasal and lung lavage (mucosal secretions) following nasal administration of modified CS-PCL nanoparticles in Balb/c mice and compared with soluble HA antigen administered intramuscular (IM) and with PCL (uncoated) nanoparticles administered intranasal (IN). The numbers of IFN-γ or IL-4 secreting cells in spleen homogenates were also measured 21 day after third immunization. Single IN or IM immunization with antigen-loaded CS-PCL nanoparticles resulted in strong HAI and total IgG responses. These responses were higher than those achieved after booster IM administration of the subunit antigen, whereas the IgG1/IgG2a profile did not change substantially. The IN administered antigen-CS-PCL nanoparticles induced higher immune responses compared to the other IN antigen formulations, and these responses were enhanced by IN booster vaccinations. Moreover, IM administered soluble HA antigen did not elicit s-IgA in mucosal secretions as it was induced and measured in the case of nasal administration of CS-PCL nanoparticles. In contrast to IM administered antigen CS-PCL nanoparticles induced a balanced Th1 and Th2 response. CS-PCL nanoparticles (cationic nanoparticles) thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. These findings demonstrate high potential of CS-PCL nanoparticles for their use as a carrier adjuvant for nasal administered influenza antigens.