Inhibition of snake venom induced sterile inflammation and PLA2 activity by Titanium dioxide Nanoparticles in experimental animals

Sterile inflammation (SI) is an essential process in response to snakebite and injury. The venom induced pathophysiological response to sterile inflammation results into many harmful and deleterious effects that ultimately leads to death. The available treatment for snakebite is antiserum which does not provide enough protection against venom-induced pathophysiological changes like haemorrhage, necrosis, nephrotoxicity and often develop hypersensitive reactions. In order to overcome these hindrances, scientists around the globe are searching for an alternative therapy to provide better treatment to the snake envenomation patients. In the present study TiO2 (Titanium dioxide)-NPs (Nanoparticles) has been assessed for antisnake venom activity and its potential to be used as an antidote. In this study, the synthesis of TiO2-NPs arrays has been demonstrated on p-type Silicon Si < 100 > substrate (∼30 ohm-cm) and the surface topography has been detected by Field-emission scanning electron microscopy (FESEM). The TiO2-NPs successfully neutralized the Daboia russelii venom (DRV) and Naja kaouthia venom (NKV)-induced lethal activity. Viper venom induced haemorrhagic, coagulant and anticoagulant activities were effectively neutralized both in in-vitro and in vivo studies. The cobra and viper venoms-induced sterile inflammatory molecules (IL-6, HMGB1, HSP70, HSP90, S100B and vWF) were effectively neutralised by the TiO2-NPs in experimental animals.

of Titanium (Ti), Oxygen (O 2 ) and Si (Substrate) in the sample. Raman analysis confirms the crystalline nature of TiO 2 -NPs with Anatase and Rutile modes.
Present day lifestyle increases the risk of several deadly diseases including cardiovascular disorder, diabetes, cancer, etc. In these cases early detection and treatment can decrease the mortality rates. Apart from these lifestyle related diseases, some clinical conditions such as snake envenomation also pose a major public health challenge across the world. About 5.4 million annual snakebite cases are reported globally and more than 100,000 people die each year as a result of snakebites of which more than 50,000 deaths occur in India alone 9 . Majority of deaths reported are particularly from rural and agriculturally active areas. WHO has also declared snakebite as a 'neglected tropical disease' and is now a global health priority [9][10][11][12] . Antiserum is the only choice to treat snakebite patients, which again is not always effective against venom-induced haemorrhage, necrosis, nephrotoxicity and often develop hypersensitive reaction [13][14][15][16][17] . Antiserum development in animal is time consuming and needs cold chain management. Therefore, there is a need to develop suitable therapeutic agent for the treatment of snakebite. Now-a-days use of nanoparticles are quite popular and frequently used as a drug-delivery vehicles 2,18 . TiO 2 is one such promising material because of its wide spectrum properties that varies with different techniques used for its synthesis 3 and has been reported for various applications in the field of biomedical research [4][5][6] .
Sterile inflammation is an essential process in response to snakebite and injury. The pathophysiological response to sterile inflammation results into many harmful and deleterious effects that ultimately lead to death. The harmful effects of snake envenomation are haemorrhage, necrosis, renal failure, cardiotoxicity, neurotoxicity, myotoxicity and hematoxicity. These deleterious effects could be due to the increased formation of sterile molecules: HMGB1, HSPs, S100B and IL6. HMGB1 which has recently been drawn much attention because of its binding ability to cell signalling receptors like RAGE, TLR2, TLR4 and TLR9 receptors. These receptors have major role in the initiation of inflammatory response and they also participate in development and progression of haemorrhage, necrosis, and cell death. Similar pathophysiological changes were also observed in case of snake envenomation. Therefore, in this paper we have shown the expression of the sterile inflammatory marker molecules developed in response to snakebite in experimental animals and its neutralization by TiO 2 .

Materials chemicals.
All chemical used in this study were of analytical grade. The solvents used were prepared in distilled water.Anti-Hsp70 antibody, Anti-Hsp90 antibody, Anti-IL-6 antibody, Anti-Von Willebrand Factor antibody, Anti-S100 beta antibody, Anti-HMGB1 antibody-ChIP Grade were purchased from Abcam (Cambridge, MA, USA); and O-Phenylenediamine dihydrochloride and bovine serum albumin were purchased from Sigma Aldrich (St. Louis, MO, USA). MaxiSorp flat-bottom 96-well plates were purchased from Nunc GmbH & Co, Langenselbold, Germany.

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 mbar 19 . 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 holder 20 . 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 g 22,23 . 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) intravenously 24 .
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 hours 23 . 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 hour 26 .
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 minute 27 . 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. 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 Calixto 24,28 . www.nature.com/scientificreports www.nature.com/scientificreports/ Inhibition of Sterile inflammatory Markers. Different pro-inflammatory markers such as heat shock proteins (HSP70 and HSP90), high mobility group box 1 (HMGB1), interleukin 6 (IL-6), von Willbrand factor(vWF), and S100 calcium-binding protein B (S100B) was done by ELISA as described by Engvall et al. 29 . Briefly, 50 μg of proteins were used with bicarbonate coating buffer to coat the wells in 96-well plates and incubated over night at 4 °C. Nonspecific binding was blocked with 5% BSA and then the samples were washed with PBST (0.05% Tween-20) and incubated for 3 hours with respective primary antibodies (against Hsp70; Hsp90; HMGB1); IL-6; vWF; S100b (1:1000). Next, the wells were washed and incubated with HRP conjugated secondary antibodies for 1 hour, and then washed with PBST and incubated with OPD substrate solution. The reaction was terminated by adding H 2 SO 4 (1 N) and absorbance was measured at 450 nm. Using similar methods, a standard ELISA curve for absorbance versus specific antigen concentration was constructed using respective recombinant proteins.

Results
topographic image of tio 2 -nps. In case of GLAD, the TiO 2 -NPs are formed due to the atomic shadowing effect on the arbitrarily deposited seeds. The diffusivity of the landed atoms on the substrate depends on the rate of evaporation. A lower evaporation rate of the material will enable the landed atoms to diffuse for longer distances before they get impinged by next arriving atoms. This can lead to an increase in the capture radius of the deposited atoms and hence the atoms nucleation area increases. Figure 1A shows a top view of grown TiO 2 -NPs

Venom-NPs incubated 37 °C, 60 min then added to egg yolk suspension
TiO 2 (2 ng) 10 5 Table 4. Inhibition of Phospholipase A 2 activity of Viper venom by TiO 2 -NPs. Results are expressed as mean of ten observations. 1 unit = one unit of enzyme activity of DRV was defined as the amount of venom which increased the coagulation time of egg yolk by one minute.  Fig. 1B. Figure 1C projects the particle counts histogram of FEG: SEM image 1 A, the grown particles were in the range of 1-14 nm, where maximum number of NPs had a count of 5-6 nm. The Raman spectra processing of TiO 2 samples shows that the grown nanoparticles are having two Anatase and Rutile modes 30 (Fig. 1D).

Raman analysis. Raman spectroscopy is a flexible and most authenticated technique used for ubiquitous
sectors. It is a very prominent tool for nano-biomedical studies and also helpful in the analysis of food and pharmaceutical non-materials. It has a great impact on drug analysis due to its simplicity in minimal sample handling and the significant differences in scattering strength between packaging materials tablet excipients, and active drug components 31 . Figure 1D shows the dependence of the Raman scattering intensity on the incident laser wavelength (laser excitation 532 nm). We have noticed that our prepared TiO 2 -NPs have crystalline nature and also have some characteristic peaks.
properties of snake venoms and tio 2 -nps. LD  Lethal activity. In in-vitro study, viper and cobra venoms (1-10 LD 50 ) were incubated with TiO 2 -NPs (2 ng) and gave protection against venom-induced lethality. In in vivo study, cobra venom (1-5 LD 50 ) was injected (s.c) into male albino mice followed by TiO 2 -NPs (5 ng/mouse, i.v). Viper and Cobra venom-induced lethality was significantly antagonized by TiO 2 -NPs as compared with the control animal (venom only) ( Table 1). The ED 50 of the TiO 2 -NPs was observed as 2 ng in vitro and 4 ng in in vivo respectively against viper venom (Table 1).
Haemorrhagic and necrotic activity. In in-vitro study, Viper venom (20 µg) incubated with TiO 2 (2 ng) and injected 0.1 ml (i.d) into mice. It showed protection against venom induced haemorrhagic activity ( Table 2). In in-vivo study, venom (10 µg) injected (i.d) into mice followed by the injection (i.v) of TiO 2 -NPs (5 ng) gave protection against venom-induced haemorrhagic activity. The degree of protection in in-vivo was less than that of in-vitro studies.
Defibrinogenation activity. The TiO 2 -NPs effectively antagonized the viper venom-induced defibrinogenating activity. In in-vitro study, the TiO 2 -NPs (2 ng) gave protection up to 2 MDD (5 µg) against venom-induced defibrinogenation. In in-vivo study, venom-induced defibrinogenation was antagonised by TiO 2 -NPs. The fold of protection was always higher in in vitro studies ( Table 3).
Neutralisation of Venom Phospholipase A 2 (PLA 2 ) activity. Egg yolk coagulation method was performed to assess the PLA 2 activity of viper venom. 1 unit of venom activity which increased the coagulation time by 1 minute was found to be 2 μg (control 0.9% saline, coagulation time was found to be 45 ± 1.16 seconds). The TiO 2 -NPs were tested for Phospholipase A 2 activity by incubating with different amount of venom (10 μg). The venom PLA 2 was effectively neutralized by the TiO 2 -NPs (Table 4).
Mouse paw oedema. The assessment of anti-inflammatory activity of the TiO 2 -NPs was done by mouse paw oedema. Mouse paw oedema was induced by venom, attained its peak at 1 hour of observation. The TiO 2 -NPs at a dose of 5 ng/mouse (i.p) was found to produce significant inhibition of venom-induced inflammation. Inhibition of inflammation induced by TiO 2 -NPs was maximum (51.3 ± 2.5%) at 2 hours of observation, as compared with   www.nature.com/scientificreports www.nature.com/scientificreports/ aspirin (42.8 ± 1.2%) and indomethacin (40.3 ± 1.3%). Carrageenan (300 µg in 0.01 ml) injection produced significant inflammation in mouse paws. Pre-treatment with TiO 2 -NPs (5 mg/kg, i.p) before carrageenan injection produced a significant reduction in oedema (Table 5).

Discussion
Injury and death due to snake bite is major health hazards in tropical and subtropical countries. In India more than 250 species of snakes are found, out of which 50 are venomous. Cobra and viper are the common snakes found throughout India [32][33][34] . The majority of death occurs due to the bites of these snakes. Snake envenomation is characterized by severe damage caused by the toxic action of venom components. The venom contains multiple components that induce haemorrhage, necrosis, inflammation, nephrotoxicity, cardiotoxicity, hematoxicity and often leads to death 35,36 . Viper and cobra venom contains major parts of Phospholipase A 2 (PLA 2 ) and metalloproteinase. Viper venom is a rich source of PLA 2 enzyme that leads to many pathophysiological disturbances in www.nature.com/scientificreports www.nature.com/scientificreports/ the victims 34 . These components directly damage the micro-vessels, with consequent increase in haemorrhage and oedema 34 . The inflammatory effect induced by viper envenomation is due to the presence of PLA 2 enzyme in venom [34][35][36][37] . The inflammatory effects induced by PLA 2 in response to cobra and viper venoms are primarily related to the action of this enzyme on the membrane phospholipids and the release of eicosanoid precursors 25,[38][39][40][41][42] . In present work, we characterized the oedematogenic, haemorrhagic, lethal and anti-inflammatory effects induced by the viper venom and its neutralization by TiO 2 -NPs (Fig. 4). Nanomedicine is a rapidly evolving field that provide enhanced diagnostic imaging and treatment of diseases known as theranostic. On account of improved health care, theranostic approaches are employed to make drug delivery more efficient and target specific. Theranostic nanomedicine can also be used for different purposes like non-invasive assessment of the pharmacokinetics, bio-distribution and the target site localization of conjugated or entrapped active agents 43,44 . Therefore TiO 2 -NPs is considered as a suitable target for medicine delivery as well as for detection of target cells due to its high fluorescence property under the excitation of normal white light in in vitro condition.
In this study, we have shown that TiO 2 -NPs are effective agent that may be used as a treatment in snake venom induced pathophysiological changes. TiO 2 -NP arrays have been fabricated on p-type Si substrate using a cost effective (GLAD, PVD) technique. The surface topography revealed that the average size of TiO 2 -NPs is in nano-metric range. The element mapping of FESEM image indicates the presence of Titanium (Ti) and Oxygen (O) in the sample. The stability of TiO 2 -NPs synthethised by glancing angle deposition technique was calculated by its zeta potential. The continuous SAED analysis of TiO 2 -NPs reveals that particles are having crystalline behaviour with fine stability. So far, we have seen that TiO 2 -NPs effectively neutralize the viper and cobra venom-induced lethal activities both in in vitro and in vivo studies. TiO 2 -NPs also effectively neutralized the viper venom induced haemorrhagic activity in experimental animals. The fold of venom induced lethal and haemorrhagic activity was always found to be higher in in vitro than in vivo studies. The TiO 2 -NPs were also found to be more effective in viper venom induced pathophysiological changes than the cobra venom. The exact mechanism of action is still unclear. Among the several, one of the consequences of snake envenomation is increased DAMP molecules. Here, we have shown for the first time that the sterile inflammatory markers like IL-6, HSP, vWF, S100B and HMGB1 were increased by the administration of both cobra and viper venoms in male albino mice. The venoms may act through the stimulation of arachidonic acid pathway that leads to the generation of ROS and cytokines which leads to haemorrhage and tissue injury 41,45,46 . Tissue injury caused due to snake envenomation could be because of different mechanism. One of the mechanisms is inflammatory response which entails the generation of Hsp70, Hsp90, HMGB1, IL6, vWF and S100b. They are all generated through the arachidonic acid pathway. The metabolism of arachidonic acid by lipo-oxygenase or cyclo-oxygenase causes the release of pro-inflammatory marker proteins. Presence of PLA 2 in viper venom helps in the conversion of the phospholipids to arachidonic acid which causes release of cytokines and leukotrienes 25,47,48 . The TiO 2 -NPs inhibits the production of sterile inflammatory proteins in venom-induced experimental rodents. The fold of protection is higher in case of viper venom than the cobra venom may be because of the difference in PLA 2 concentration. PLA 2 is www.nature.com/scientificreports www.nature.com/scientificreports/ higher in viper venom than the cobra venom. Further study may provide new biological probes in the treatments of snakebite. TiO 2 -NPs have shown better results in in vitro than in in vivo experiments. Our present investigation showed that the TiO 2 -NPs is effective against venom-induced pathophysiological conditions and could be taken as a potential antidote. The present work may be helpful to treat victims of snakebite especially in rural parts of India where availability of antiserum is limited. We are engage to explore the mechanism of venom inhibition by TiO 2 -NPs as a potential therapeutic agent against snake envenomation 49,50 .