The Influence of Silver Nanoparticles Against Toxic Effects of Philodryas olfersii Venom

Abstract

Purpose: A silver nanoparticle obtained by reducing salts with solid dispersion of curcumin (130 nm, 0.081 mg mL^-1) was used to counteract against the toxic - edematogenic, myotoxic, and neurotoxic - effects of Philodryas olfersii venom.

Methods: The edematogenic effect was evaluated by plasma extravasation in rat dorsal skin after injection of 50 µg per site of venom alone or preincubated with 1, 10, and 100 µL of AgNPs; the myotoxicity was evaluated by measuring the creatine kinase released into the organ-bath before the treatment and at the end of each experiment; and neurotoxicity was evaluated in chick biventer cervicis using the conventional myographic technique, face to the exogenous acetylcholine (ACh) and potassium chloride (KCl) added into the bath before the treatment and after each experiment. Preliminarily, a concentration-response curve of AgNPs was carried out to select the concentration to be used for neutralizing assays, which consists of neutralizing the venom-induced neuromuscular paralysis and edema by preincubating AgNPs with venom for 30 min.

Results: The P. olfersii venom-induced edema (n=6) and a complete neuromuscular blockade (n=4) that includes the total and unrecovered block of ACh and KCl contractures. AgNPs produced a concentration-dependent decrease the venom-induced edema (n=6) from 223.3% to 134.4% and to 100.5% after 10 and 100 µL AgNPs-preincubation, respectively. The preincubation of venom with AgNPs (1 µL; n=6) was able to maintain 46.5 ± 10.9% of neuromuscular response under indirect stimuli, 39.2 ± 9.7% of extrinsic nicotinic receptors functioning in absence of electrical stimulus and 28.3 ± 8.1% of responsiveness to potassium on the sarcolemmal membrane. The CK release was not affected by any experimental protocol which was like control.

Conclusion: AgNPs interact with constituents of P. olfersii venom responsible for the edema-forming activity and neuromuscular blockade, but not on the sarcolemma membrane-acting constituents. The protective effect of the studied AgNPs on avian preparation points out to molecular targets as intrinsic and extrinsic nicotinic receptors.

Keywords: Philodryas olfersii; chick biventer cervicis; neuromuscular blockade; opisthoglyphous snakes; silver nanoparticles.

Figure 1

Epilated area of the rat dorsum for random application of the different protocols (1 to 8), with n = 6 for each experimental group.

Figure 2

Images obtained from transmission electron microscopy (TEM, 60 kV, 100.000X). (A) Milli-Q water. (B) AgNPs 0.081 mg mL⁻¹ in absence of curcumin used as a reference. (C) AgNPs 0.081 mg mL⁻¹ synthetized with curcumin. The latter image denotes the successful production process of curcumin-silver nanoparticles. Bars=200 nm.

Figure 3

Extravasation of plasma in the dorsal skin of rats. The rats were anesthetized, and the dorsal skin was epilated. The animals received Evan’s blue dye (100 µL per 100 g) through the urethral bulb penile vein. Then, they were injected intradermally (fixed volume: 100 μL) of the vehicle (saline control), AgNPs (1, 10, and 100 µL) and P. olfersii venom alone (50 μg per site) or previously incubated (30 min) with 1, 10, and 100 µL of AgNPs, using a random order in balanced and standardized sites. Thirty minutes later, the diameter of the injected sites (Evan’s blue halo) was measured. Plasma leakage was expressed as a percentage of the control (% control). The columns represent the mean ± SEM (n = 6). *p <0.05 compared to the control. ^#p <0.05 compared to the poison.

Figure 4

Chick Biventer cervicis preparation. (A) Concentration-response curve of AgNPs under indirect stimuli showing a concentration-dependent effect. (B) and (C) show the contracture response to ACh and KCl, respectively, being: Krebs control (n=7) Venom (n=4) AgNPs 0.081 mg mL⁻¹ (1 µL, n=6) Preincubation (n=6). Each point represents the mean±SEM. *p<0.05 in all subsequent points, and in all concentrations compared to control. ^#p<0.05 compared among AgNPs.

Figure 5

Chick Biventer cervicis preparation. (A) The protection of AgNPs is showed in preincubation experiments. (B) Myographical register showing the irreversible neuromuscular blockade-induced by 50 µg mL⁻¹ P. olfersii venom. (C) and (D) show the contracture response to ACh and KCl, respectively, which were blockade by the venom. (E) CK activities of experimental groups. Note that the venom alone did not induce CK release in this experimental model. In preincubation assays the comparison among (A) with (C) and (D) there was a positive correlation between indirect stimulation with exogenous ACh addition, but not between exogenous KCl and CK determination. Each point represents the mean±SEM. *p<0.05 in all subsequent points and concentrations compared to control. ^#p<0.05 compared to the venom. Arrow in D is indicative of the blockade of the venom.