Animals

Male Swiss albino mice (18–20 g) used in this study was obtained from the animal house of Jamia Hamdard (Hamdard University), New Delhi. Guidelines of the Institutional Animal Ethical Committee has been followed for the care and handling of animals. All mice were housed five/cages and fed standard laboratory diet and water ad libitum with 12-h dark/light cycles at constant temperature of 25 ± 2 °C. ‘Principles of laboratory animal care’ (NIH publication No. 85-23, revised in 1985) as well as Indian laws on ‘Protection of Animals’ have been followed for all the animal experiments under the provision of authorized investigators. Experiments involving the use of mice were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of the Jamia Hamdard (Deemed University), New Delhi (permission #173/GO/Re/S/2000/CPCSEA). Experiments involving animals meet the International Guiding Principles for Biomedical Research Involving Animals (CIOMS).

Preparation of nanoparticles

The GLAD technique was carried out to deposit TiO 2 (High purity) (99.99%, MTI, USA) NPs of 5 nm range inside the electron beam chamber (Hind High Vacuum Co. (p) Ltd., 15F6) on p-type Silicon Si <100> substrate (∼30 ohm-cm) at a base pressure of ~2 × 10−5 mbar19. The substrates were rotated azimuthally with constant speed of 460 rpm at an orientation of 85° with respect to the perpendicular line between the metal source and the planar substrate holder20. A low deposition rate of 1.2 Å/s was kept constant, which was monitored by a quartz crystal. Then the grown TiO 2 -NPs were ultrasonicated in electron grade acetone to make a liquid solution. The surface morphology of randomly placed TiO 2 -NPs over n-type Si substrate of probably 5 nm was observed using an Atomic Force Microscope (BrukerInnova).

Preparation of venoms

Lyophilized snake venom from Daboia russelii (Viper), and Naja kaouthia (Cobra) were obtained from Calcutta Snake Park (Kolkata, India) and was preserved in desiccators at 4 °C in an amber-coloured glass vial until further use. The snake venom was dissolved in 0.9% saline and centrifuged at 2000 rpm for 10 min. The supernatant was used as venom and kept at 4 °C and used within three month. The venom concentration was expressed in terms of dry weight (mg/ml, stock venom solution)21.

Venom inhibiting activity

To determine the venom inhibiting activity of the nanoparticles, following pharmacological experiments were performed:

Inhibition of venom lethal effect

Snake venom toxicity assessment was done by intravenous (i.v) injection of venom prepared in 0.2 mL physiological saline at different concentration in male albino mice weighing18–20 g22,23. Assessment of in vitro antagonism was done by mixing different concentrations of venom (2.2–22 µg) with a fixed amount of TiO 2 -NPs, the mixture incubated at 37 °C for 1 hour, and then centrifuged at 2000 rpm for 10 minutes. The supernatant was injected intravenously (i.v) into male albino mice, six mice per dose. The median lethal dose (MLD 50 ) was calculated 24 hours after injection of the venom-TiO 2 -NPs mixture. Lethal toxicity was also assessed by subcutaneous (s.c) injection of various doses of venom. The neutralizing potency of TiO 2 -NPs was assessed by injection (s.c) of venom (45–225 µg) into groups of six mice followed by immediate injection of fixed dose of TiO 2 -NPs (5 ng) intravenously24.

Inhibition of venom haemorrhagic activity

The minimum haemorrhagic dose (MHD) of venom which when administered into mice causes development of haemorrhagic lesion of 10 mm diameter within 24 hours23. This lesion was measured and the estimation of neutralization of the haemorrhagic activity was done by mixing a fixed amount of TiO 2 -NPs (2 ng) with different amounts of venom (5–25 µg). The mixture of TiO 2 -NPs- venom was incubated at 37 °C for 1 hour, then spin at 2000 rpm for 10 min and finally 0.1 ml of supernatant taken were injected intradermally (i.d)25. After 24 hours haemorrhagic lesion was estimated. To assess the anti-haemorrhagic activity of venom in vivo, various amount of venom (5–15 µg) were injected (i.d) followed by the TiO 2 -NPs (5 ng, intravenously) and the haemorrhagic lesion measured after 24 hours.

Inhibition of venom necrotic activity

To assess the in vitro anti-necrosis effect of TiO 2 -NPs, various concentration of venom (5–25 µg) were incubated with fixed amount of TiO 2 -NPs and administered intradermally (i.d) into mice. The necrotic lesion was estimated after 48 hours. In in vivo study, the venom (i.d) (5–15 µg) was injected followed by injection (i.v) of TiO 2 -NPs and observed after 48 hours.

Inhibition of venom defibrinogenating activity

Defibrinogenating activity of venoms or toxins is expressed as the minimum defibrinogenating dose (MDD). MDD of DRV is defined as the minimum amount of venom which when injected (intravenously) into mice causes incoagulable blood 1 hour later. Neutralization of this activity was estimated by mixing different amount of venom (2.5–12.5 µg) with fixed amounts of TiO 2 -NPs, incubating at 37 °C for 1 hour and centrifugation. The supernatant was injected (i.v) into albino mice (18–20 g) as described above (in vitro). For in vivo studies, the MDD of venom was injected (i.v) followed by the TiO 2 -NPs (i.v) and the nature of the blood observed after 1 hour26.

Inhibition of venom PLA 2 effect

For carrying out the PLA 2 inhibition activity, 2 ml of egg yolk suspension, 0.2 ml of test material (venom, TiO 2 -NPs and Venom + TiO 2 -NPs) were mixed in the different test tube and kept for 1 hour incubation at 37 °C. The test tube containing test materials were kept on a water bath and the time required for coagulation was recorded. A blank was run with normal saline in place of test material. One unit enzyme activity was defined as the amount of venom, which increased the coagulation time of the egg yolk control by one minute27. Estimation of neutralization of the enzyme activity, was done when fixed amount of TiO 2 -NPs (2 ng) were mixed with different amount of viper venom (2–10 μg) and the mixture was incubated for 1 hour at 37 °C. Centrifuged at 2000 rpm for 10 minutes, supernatant was tested in a total of 0.2 ml for the enzyme neutralization activity.

Inhibition of venom-induced mouse paw oedema

The minimum oedematic dose (MOD) of venom/carrageenan is defined as the least amount of venom/carrageenan which when injected into male albino mice produced inflammation (oedema) in the paw. Non-fasted male albino mice (18–20 g) were treated with different doses of venom, carrageenan and TiO 2 -NPs. To assess the anti-inflammatory activity of TiO 2 -NPs various amount of venom/carrageenan (in 0.01 ml) were injected (intraplantar) followed by the intraperitoneal (i.p) injection of TiO 2 -NPs (250 ng/kg) and anti-inflammatory activity was measured. For control, equal amounts of saline were injected (intraplantar). The oedematogenic response was evaluated by the use of a screw gauge at given time intervals. Oedema was reported as the percentage difference between the values obtained from the injected paw with the carrageenan, venom, venom + TiO 2 -NPs, carrageenan + TiO 2 -NPs and saline, as described by Trebien and Calixto24,28.

Inhibition of Sterile inflammatory Markers