In-vitro in-vivo correlation (IVIVC) in nanomedicine: Is protein corona the missing link?

One of the unmet challenges in nanotechnology is to understand and establish the relationship between physicochemical properties of nanoparticles (NPs) and its biological interactions (bio-nano interactions). However, we are still far from assessing the biofate of NPs in a clear and unquestionable manner. Recent developments in the area of bio-nano interface and the understanding of protein corona (PC) has brought new insight in predicting biological interactions of NPs. PC refers to the spontaneous formation of an adsorbed layer of biomolecules on the surface of NPs in a biological environment. PC formation involves the spatiotemporal interplay of an intricate network of biological, environmental and particle characteristics. NPs with its PC can be viewed as a biological entity, which interacts with cells and barriers in a biological system. Recent studies on the bio-nano interface have revealed biological signatures that participate in cellular and physiological bioprocesses and control the biofate and toxicity of NPs. The ability of in-vitro derived parameters to forecast in-vivo consequences by developing a mathematical model forms the basis of in-vitro in-vivo correlation (IVIVC). Understanding the effect of bio-nano interactions on the biological consequences of NPs at the cellular and physiological level can have a direct impact on the translation of future nanomedicines and can lead to the ultimate goal of developing a mathematical IVIVC model. The review summarizes the emerging paradigms in the field of bio-nano-interface which clearly suggests an urgent need to revisit existing protocols in nanotechnology for defining the physicochemical correlates of bio-nano interactions.

Assessment of stress in effect to pyrethroid insecticides, λ-cyhalothrin and cypermethrin, in a freshwater fish, Channa punctatus (BLOCH).

The present study was planned to see the changes in the levels of different biochemical stress markers such as the level of lipid peroxidation and the specific activities of lactate dehydrogenase (LDH), acid and alkaline phosphatases in different organs such as brain, liver, kidney, gills and muscle of a freshwater muddy fish, Channa punctatus in effect to pyrethroid insecticides, cypermethrin and λ-cyhalothrin treated for 96 h. The results showed significant increase in the levels of lipid peroxidation as well as the activities of LDH, acid and alkaline phosphatases in a dose dependent manner. The remarkable increase in the levels of these stress biomarkers indicates strong stress inducing potential of these insecticides in fishes. The importance of the current study lies in indicating the potential risk of muddy freshwater fishes due to strong soil binding property of pyrethroids along with their slow metabolism in fishes as compared to that of mammals.

Non-ionic surfactant vesicles mediated transcutaneous immunization against hepatitis B.

Transcutaneous immunization (TI) has many practical merits compared to parenteral routes of administration. In the present study, non ionic surfactant vesicular carrier, i.e. niosomes, was evaluated for topical delivery of vaccines using hepatitis B surface protein as an antigen and cholera toxin B as an adjuvant. Niosomes were characterized for size, shape, entrapment efficiency and in process antigen stability. In vitro permeation and skin deposition studies of antigen were performed using human cadaver skin. Skin penetration efficiency of niosomes was assessed by confocal laser scanning microscopy. The immune stimulating activity of these vesicles was studied by measuring the serum IgG titer, isotype ratio IgG2a/IgG1 and mucosal immune responses following transcutaneous immunization in Balb/c mice and results were compared with the alum adsorbed HBsAg given intramuscularly and topically administered plain HBsAg solution. The result shows that optimal niosomal formulation could entrap 58.11 ± 0.71 of antigen with vesicle size range of 2.83 ± 0.29 µm. Serum IgG titers after three consecutive topical administrations were significantly better than single administration of hepatitis antigen with niosomal system, suggesting an effective stimulation of serum immune response; higher IgG1/IgG2a ratio revealed CTB mixed niosomes elicit both Th1 and Th2 responses. This study suggests that topical immunization with cholera toxin B is potential adjuvant for cutaneous immune responses when coadministered with the HBsAg encapsulated niosomes. Results also suggest that the investigated niosomes systems can be effective as topical delivery of vaccines.

Evaluation of ISCOM vaccines for mucosal immunization against hepatitis B.

Immune stimulating complexes (ISCOMs) incorporating recombinant hepatitis B surface antigen (HBsAg) was prepared for induction of humoral, cellular and mucosal immunity by intranasal administration. Prepared ISCOMs were characterized for its size, shape, incorporation efficiency, zeta potential, and antigen integrity. Designed ISCOMs possessed negative zeta potential (-21.7 mV) and an average size of 44.1 nm with antigen incorporation efficiency approximately 39 %. Serum anti-HBsAg IgG titer after three high nasal doses of ISCOMs was comparable with titer recorded after alum-HBsAg administered subcutaneously. Similarly, modest but higher cellular response (cytokines level in spleen homogenates) and significantly higher secretory sIgA response in mucosal secretions was observed (P < 0.001) in case of HBsAg ISCOM vaccines. Whereas, alum-HBsAg vaccine did not elicit considerable cellular or mucosal response. Thus, ISCOMs produced humoral, mucosal, and cellular immune responses upon nasal administration although high and multidose administrations were required to elicit potent immune responses. These data demonstrate potential of ISCOMs in their use as a carrier adjuvant for nasal subunit vaccines against hepatitis B.

Evaluation of ISCOMs for immunization against hepatitis B.

Immune stimulating complexes (ISCOMs) incorporating recombinant hepatitis B surface antigen (rHBsAg) were prepared for induction of humoral and cellular immunity by subcutaneous administration. Prepared ISCOMs were characterized for their size, shape, incorporation efficiency, zeta potential, antigen integrity, antigen conformation and immunogenicity by biophysical and immunological techniques including transmission electron microscopy (TEM), Dynamic light scattering (DLS), SDS-PAGE, fluorescence spectroscopy, in vitro potency test and in vivo humoral and cellular immune stimulatory efficacy in Balb/c mice. Prepared ISCOM particles show characteristic cage like morphology with average size of 44 approximately nm, polydispersity index 0.1, negative zeta potential (-21.7 mV) and antigen association efficiency approximately 39%. Tryptophan emission fluorescence and in vitro potency assay data suggest that association of rHBsAg with ISCOMs results in local electrostatic interactions, motional restriction of tryptophan residues of the protein resulting in reduction of anti-rHBsAg monoclonal antibodies binding affinity. Immunization with rHBsAg ISCOMs resulted in upregulation of specific cellular (IFN-gamma and IL-2) as well as IgG response (IgG2a isotype biased) humoral response in Balb/c mice. Immune responses were significantly higher than those produced by of alum-adsorbed antigen (alum-rHBsAg) after (one booster) (p < 0.001). These data demonstrate that although the conformation of rHBsAg after incorporation into ISCOMs was moderately altered but due to strong adjuvant ability, rHBsAg ISCOMs were highly immunogenic as compared to marketed rHBsAg formulations by subcutaneous route of administration.

Effect of Asparagus racemosus rhizome (Shatavari) on mammary gland and genital organs of pregnant rat.

Asparagus racemosus (AR) Willd (family Liliaceae) is commonly known as Shatavari. The alcoholic extract of its rhizome was administered orally to adult pregnant female albino rats at a dose of 30 mg/100 g body weight, daily for 15 days (days 1-15 of gestation). The macroscopic findings revealed a prominence of the mammary glands, a dilated vaginal opening and a transversely situated uterine horn in the treated group of animals. The weight of the uterine horns of the treated group was found to be significantly higher (p < 0.001) but the length was shorter (p > 0.01). Microscopic examination of the treated group showed proliferation in the lumen of the duct of mammary gland. It was obliterated due to hypertrophy of ductal and glandular cells. Hyperplasia of the glandular and muscular tissue and hypertrophy of the glandular cells were observed in the genital organs. The parenchyma of the genital organs showed abundant glycogen granules with dilated blood vessels and thickening of the epithelial lining. The oviduct in the treated group showed hypertrophied muscular wall, whereas the ovary revealed no effect of the drug. The results suggest an oestrogenic effect of Shatavari on the female mammary gland and genital organs.

Anti-inflammatory properties of BHUx, a polyherbal formulation to prevent atherosclerosis.

BHUx is a polyherbal formulation consisting of water-soluble fractions of five medicinal plants (Commiphora mukul, Terminalia arjuna, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica). The present study was undertaken to evaluate its antioxidant and antiinflammatory effects. BHUx, standardized by HPLC fingerprinting and filtered through 0.2 microm filter paper, was employed for different studies under in vivo and in vitro conditions. Under in vivo conditions, BHUx significantly reduced inflammation in the carrageenan-induced rat paw oedema model of inflammation, suggesting its anti-inflammatory properties. In order to test the mechanism of action of BHUx, further in vitro studies were undertaken on cumene-hydroperoxide-induced lipid peroxidation (CHP) in liver homogenate, LPS-induced NO production in peritoneal macrophages and on key enzymes of arachidonic acid cascade, involved in the mediation of inflammation. Under the conditions, BHUx showed concentration-dependent inhibition of CHP-induced lipid peroxidation in liver homogenate, suggesting its antioxidant properties. Similarly the potent anti-inflammatory effects of BHUx are evident by (a) preferential inhibition of COX-2 (IC50 for COX-2 = 80 microg/ml and IC50 for COX-1 = 169 microg/ml), (b) low ratios in the IC50 values of COX-2/COX-1 (0.47), (c) decreased production of NO in LPS-induced peritoneal macrophages and (d) inhibition of 5-LOX (IC50 = 795 microg/ml). BHUx also showed a preference for inhibiting 15-lipoxygenase (IC50 = 44 microg/ml), a key enzyme implicated in LDL oxidation. These studies suggest that BHUx is acting mainly at three levels, i.e., as a potent natural antioxidant, by reduction of key inflammatory mediators of arachidonic acid cascade and by preventing 15-LOX-mediated LDL oxidations, to prevent atherosclerosis.

Semecarpus anacardium L, nuts inhibit lipopolysaccharide induced NO production in rat macrophages along with its hypolipidemic property.

Traditionally S. anacardium is used for rejuvenation, rheumatoid arthritis, fever and neurological disorders. In the present study it was observed that a fraction of S. anacacrdium at dose of 1 mg/100 g body wt, significantly reduced serum cholesterol from 378.87 mg/dl in the rats fed with atherogenic diet (AD) to 197.99 mg/dl (45-52%) in the rats fed with AD diet and increased serum HDL-cholesterol (33-37%). The same fraction also inhibited LPS induced NO production in the culture activated rat peritoneal macrophages in the dose dependent manner with IC50 value at 50 ng/ml of the culture medium. The drug in the above doses was completely safe and non-toxic, (no change in the enzymes), to liver and kidney functions.

Toxicology and free radicals scavenging property of Tamra bhasma.

Free radicals are implicated in various chronic diseases. There has always been a search for new antioxidants. In this paper we have investigated Tamra bhasma, a metallic ayurvedic preparation. It is a time-tested medicine in Ayurveda and is in clinical use for various ailments specifically the free radical mediated diseases. Our results show that Tamra bhasma inhibits lipid peroxidation (LPO), prevents the rate of aerial oxidation of reduced glutathione (GSH) content and induces the activity of superoxide dismutase (SOD) in rat liver homogenate in the bi-phasic manner. The drug was orally given for 7, 15 and 30 days in different doses. Best protective response was found at the dose of 0.5mg/100g body weight in albino rats, although it showed some histopathological changes at the dose of 20mg/100g body weight. The results suggest that this Ayurvedic preparation is not merely a source of copper metal, but it is a strong anti-oxidant with no detectable adverse effect in lower doses of therapeutic range.

Does higher cardiovascular response to ECT predict early antidepressant effect?

BACKGROUND: Effects of diencephalic seizure generalization during ECT, e.g., cardiovascular response, may be relevant in indexing its therapeutic potency. A trend for greater rate pressure product (RPP=heart rate x systolic blood pressure) response to modified ECT in responders than in nonresponders is reported. Atropine used in modified ECT is known to increase RPP. This study examined if cardiovascular response during ECT with or without atropine predicts antidepressant effect. METHODS: Twenty nine consenting, major depressive disorder patients received ECTs. Atropine premedication was randomly withheld in the second or third ECT session. RPP was recorded during ECT. Severity of depression was measured at twice weekly intervals. RESULTS: Fifteen patients remitted at the end of 2 weeks. These early remitters had significantly higher poststimulus RPP than the rest in the ECT session without atropine but not so in the session with atropine. Cumulative poststimulus RPP predicted the early antidepressant response. Corresponding motor or EEG seizure durations were not associated with antidepressant effect. LIMITATIONS: Most patients continued to receive antidepressants. ECT stimulus laterality was not controlled. The study focussed on only short term antidepressant effects. CONCLUSIONS: RPP response to ECT recorded under no-atropine condition may reflect its physiological effects relevant to therapeusis and may have the potential to index seizure adequacy.

FSH induced stimulation of catalase activity in goat granulosa cells in vitro.

Reactive oxygen species scavenging enzymes like catalase play diverse role in mammals. The presence of catalase in mammalian ovary is now well established. In the present investigation, changes in catalase activity in granulosa cells isolated from follicles at various stages of differentiation in response to FSH were studied. The follicles were dissected out from goat ovaries and classified as small (<3mm), medium (3-6mm) or large (>6mm). Granulosa cells were isolated from categorized follicles. Results showed that there was a three-fold increase in catalase activity in granulosa cells from large follicles as compared to small and medium follicles. The catalase activity was stimulated significantly when granulosa cells were treated with FSH in vitro. The minimum effective dose that could stimulate catalase activity and estradiol secretion in case of granulosa cells from small and medium sized follicles was 100 ng/ml; for larger follicles, this value was 200 ng/ml. Concomitant to the increase in catalase activity, the estradiol secretion was significantly enhanced when cultured goat granulosa cells were treated with FSH. It was concluded that enzyme catalase may have a functional role in goat ovarian follicular development under endocrine regulation.

Effect of epidermal growth factor on steroidogenesis by caprine granulosa cells in culture: interaction with FSH.

Besides gonadotrophins various peptide growth factors have been implicated in the ovarian folliculogenesis. In this study, the effect of epidermal growth factor (EGF) (0, 0.1, 1.0, 10ng/ml culture medium) on steroidogenesis by caprine granulosa cells at various stages of maturation was investigated using serum free culture medium. Caprine granulosa cells were obtained from ovarian follicles and classified into three classes: small (<3mm), medium (3-6mm) and large (>6mm in diameter). EGF (10ng/m culture medium) alone reduced estradiol secretion in granulosa cell from small, medium and large follicles by 62, 48 and 29%, respectively, as compared with control. This inhibition was 50, 36 and 21%, respectively, when EGF (10ng/ml culture medium) was applied in combination with FSH (100ng/ml culture medium). EGF alone stimulated the secretion of progesterone in granulosa cells from all the three categories of follicles only at highest dose tested (10ng/ml culture medium). FSH acted synergistically with EGF in stimulating progesterone secretion by cultured granulosa cells. EGF in combination with FSH (100ng/ml culture medium) significantly (P<0.05) stimulated progesterone secretion by cultured granulosa cells from all three categories of follicles even at the lowest dose (0.1ng/ml culture medium) tested. In conclusion, EGF significantly influences the steroidogenesis by caprine granulosa cells in vitro and may play important role in the follicular growth and maturation.

Activity of glutathione related enzymes and ovarian steroid hormones in different sizes of follicles from goat and sheep ovary of different reproductive stages.

The investigations on enzymes related to glutathione like glutathione-S-transferase (GST) and glutathione peroxidase (GSH-Px) have been carried out mostly in human and rat ovaries, however the studies on these enzymes in ruminants are relatively absent. In the present study the changes in the activity of these enzymes, in different sizes of follicles from goat and sheep ovaries of different reproductive stages, were investigated. The results demonstrated that the activity of the enzyme GST increased with the increase in size of the follicles from small to large follicles of follicular phase ovary and from small to medium follicles of luteal phase ovary in both the species, thereafter it decreased in large follicles of luteal phase ovary. There was increasing pattern in the activity of GSH-Px in the follicular phase follicles and a decreasing pattern in the luteal phase follicles from both the species. Thus the changes in the activity of glutathione related enzymes namely GST and GSH-Px in different size follicles from both the species during different reproductive phases are evident from the results. It is reasonable, therefore, to assume that these enzymes may have functional role in the steroid hormone metabolism in ruminant ovary as reported in human ovary.

Biochemical and hormonal characterization of follicles from follicular and luteal phase ovaries of goat and sheep.

Follicles from goat and sheep ovaries were characterized for their biochemical and hormonal parameters to investigate the effect of developmental stage of follicles on ovarian steroidogenesis. The follicles were isolated mechanically from follicular and luteal phase ovaries and divided in 6 morphologically different groups (small, medium and large follicular and small, medium and large luteal). Follicles were characterized for their contents of protein, DNA, estradiol-17 beta and progesterone and the activity of 3 beta-hydroxysteroid dehydrogenase. There was a progressive increase in the contents of all these biomolecules and activity of the enzyme as size of follicles increased in both the follicular and luteal phase ovaries. Follicles from follicular phase ovaries exhibited higher estradiol-17 beta content than those shown by luteal phase follicles. The reverse pattern was obtained for progesterone content. The results provide the basic data on biochemical and hormonal entities at different stages of follicular development in small ruminants which may be useful for in vitro studies on regulation of follicular development and steroidogenesis.

Effect of insulin-like growth factor-1 on steroidogenesis in cultured carp ovarian follicles: interactions with estradiol.

The biological action of insulin like growth factor-1 (IGF-1) on follicular steroidogenesis during follicular development in common carp was examined. Studies were carried out by culturing small (1-2 mm diam.) and large (> 2 mm diam.) follicles. IGF-1 (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48 hr culture of small follicles. Progesterone accumulation by large follicles was also unaffected by IGF-1 over the same period, although aromatase activity was stimulated in a dose dependent manner (8-fold increase over basal levels with a maximum stimulatory dose of 30 ng IGF-1/ml). In contrast, small and large follicles responded to IGF-1 in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone and 6 days for aromatase). Concurrent treatment of small follicles with estradiol (10(-7) M) enhanced the action of IGF-1 on both indices of steroidogenesis and advanced the time at which IGF-1 stimulated activity was first detectable. The effect of estradiol on follicular IGF-1 responsiveness were independent of cell number. In summary, these results demonstrate varied actions of IGF-1 carp ovarian follicular steroidogenesis in vitro. The results indicate that carp follicles acquire responsiveness to IGF-1 in terms of aromatase activity during follicular development in vivo and that estradiol can induce the response in vitro. The results also suggest that estrogen and progesterone biosynthesis by cultured carp ovarian follicles is differentially regulated by IGF-1. Together, these results provide new insights into the biological actions of IGF-1 in fish ovary.

Naltrexone in primary hyperphagic obesity wity hypochondriacal disorder - a clinical study.

Six well investigated patients of primary hyperphagic obesity with hypochondrical disorder were sequentially treated with psychoeducational methods alone and psychoeducational methods with naltrexone hydrochloride 50 mg daily orally for six weeks each. RMANOVA revealed no statically significant (p>0.05) decrease in body mass index suggesting that psychoeducational methods with naltrexone were as ineffective in reducing obesity as psychoeducational methods alone. The limitations of the study and implications for future research are discussed.

Changes in superoxide dismutase activity and estradiol-17 beta content in follicles of different sizes from ruminants.

Subcellular distribution of oxygen free radical scavenging enzymes has recently been demonstrated in goat ovary. In the present study the follicles of the follicular phase were isolated mechanically from goat and sheep ovaries and grouped as small (< 3 mm), medium (3-6 mm) and large (> 6 mm) follicles. The specific (units/mg protein) and total (units/g tissue) activity of superoxide dismutase (SOD) was estimated in homogenate of different sizes of follicles. The small follicles of both the species contained highest specific and total activity (6.99 +/- 1.49, 107.9 +/- 3.1 in sheep and 3.57 +/- 0.09, 76.5 +/- 4.0 in goat, respectively), whereas large follicles showed lowest specific and total activity (32.02 +/- 0.25, 80.4 +/- 5.5 in sheep and 1.71 +/- 0.06, 63.20 +/- 2.20 in goat, respectively). The estradiol-17 beta was also estimated and expressed as ng/follicle. The results show that estradiol-17 beta content was highest in large follicle (8.00 +/- 0.46 in sheep and 4.5 +/- 0.73 in goat). The content of estradiol-17 beta was increased progessively as the size of the follicles increased. From these findings, an inverse relationship was observed between SOD activity and estradiol-17 beta content in the follicles of different sizes from ruminants which may have functional role in follicle development and steroidogenesis.

Changes in catalase activity and hydrogen peroxide level in rat ovary during estrous cycle and induction of catalase in rat ovary by estradiol-17 beta.

Catalase activity in the whole ovary homogenate and hydrogen peroxide level in the differentially centrifuged fractions of the ovary homogenate during each stage of estrous cycle were measured. The highest catalase activity was observed in the metestrous which declined in the estrous and proestrous and was lowest in the diestrous. An inverse relationship was found between catalase activity and hydrogen peroxide production. Treatment of immature (28-29 days old) female rats with estradiol-17 beta (5 micrograms in 0.2 ml oil/animal/day for consecutive 3 days, s.c.) increased the ovarian catalase activity. The findings indicate that the free radical-scavanger system may have functional role in the ovary.

Glutathione-S-transferase in rat ovary: its changes during estrous cycle and increase in its activity by estradiol-17 beta.

Glutathione-S-transferases (GSTs) are drug metabolizing and detoxification enzymes, involved in the intracellular transport and metabolism of steroid hormones. This study indicated that the enzyme was heterogeneously distributed and changed during estrous cycle. The enzyme was found to be predominantly located in the cytosolic fraction while considerable activity was also observed in the mitochondrial and microsomal fractions. The activity of GST was measured during different stages of estrous cycle, viz proestrous, estrous, metestrous and diestrous. The change in its activity was observed during different stages of estrous cycle. Estrous stage was observed to have the highest GST activity. Immature (28-29 days old) rats treated with estradiol-17 beta (5 micrograms in 0.2 ml oil/animal/day, and 10 micrograms in 0.2 ml oil/animal/day, for 3 days), ovarian GST activity seemed to increase significantly (P < 0.01). This increase in the activity and the heterogeneous distribution of GST indicates the functional role of this enzyme in the ovary under endocrine regulation.