Novel microfilaricidal activity of nanosilver

Abstract

Purpose: The currently available drug repertoire against lymphatic filariasis, a major health hazard in the developing world, is inadequate and is fraught with serious limitations. Thus, the development of an effective antifilarial strategy has become a global research thrust mandated by the World Health Organization. Nanoparticles of silver endowed with antibacterial potency are known to induce apoptosis in eukaryotic cells. The present study was designed to investigate the possible microfilaricidal efficacy of silver nanoparticles and to establish the validity of apoptotic rationale in antifilarial drug designing.

Methods: This report analyzed the effect of nanoparticles of silver as well as gold (size range: 10-15 nm) on the microfilariae of Brugia malayi obtained from the lavage of peritoneal cavities of infected jirds (Meriones unguiculatus). The study included a microfilarial motility assay, a trypan blue exclusion test, a poly(adenosine diphosphate-ribose) polymerase activity study, ethidium bromide/acridine orange differential staining, and transmission, as well as scanning electron microscopic evaluation of ultrastructural changes in microfilariae.

Results: The study demonstrates that nanoparticles of silver, but not of gold, elicited significant loss in microfilarial motility. Differential staining of parasites with ethidium bromide and acridine orange, poly(adenosine diphosphate-ribose) polymerase activity in microfilarial lysate, and electron microscopic findings underscored apoptotic death of parasites attributable to nanosilver. In a trypan blue exclusion test, the 50% lethal dose of nanosilver was measured to be 101.2 μM, which was higher than the recorded complete inhibitory concentration value (50.6 μM), thus supporting nanosilver as a potential drug candidate against lymphatic filariasis.

Conclusion: The present report provides the first ever conclusive proof in support of apoptosis as a novel stratagem in antifilarial drug designing and nanoscale silver as a valid lead in research on antifilarial therapeutics. The main embargo about the current drug diethylcarbamazine citrate is its empirical use without rationale. Effective microfilaricidal activity of nanosilver at relatively low concentrations as reported in this study, with evidence of the induction of apoptosis in microfilariae, projects nanosilver as a potential drug adjuvant against lymphatic filariasis. The much higher 50% lethal dose value of nanosilver compared to the complete inhibitory concentration value reported in this study argues in favor of a safe therapeutic window of this agent in its antifilarial efficacy.

Keywords: apoptosis; lymphatic filariasis; microfilaricidal agent; parasitic diseases; silver nanoparticles.

Figure 1

Characterization of silver nanoparticles. (A) Silver nanoparticles showing spherical, monodispersed particles (scale bar, 50 nm). The inset shows one single particle of silver (scale bar, 5 nm). (B) Particle size distribution showing preponderance of particles in the size range of 10–15 nm. (C) Electron diffraction pattern of nanoparticles showing various crystallographic planes. (D) Optical spectra of silver before (1) and after reduction (2). The inset shows the corresponding change in color.

Figure 2

Ethidium bromide/acridine orange differential staining of microfilarial forms for the detection of apoptosis. Untreated (A) and gold nanoparticles preincubated (D) nuclei showed green staining due to acridine orange permeation, while organisms treated with silver nanoparticles (B) and staurosporine (C) appeared orange-yellow due to ethidium bromide, suggesting loss of integrity of surface membrane of the parasite shown. Note: Data are representative of three different experiments.

Figure 3

Scanning electron micrographs of (A) untreated control parasite in Roswell Park Memorial Institute medium; (B) microfilariae treated with staurosporine (0.5 μM); (C) microfilariae treated with silver nanoparticles (50 μM), and (D) microfilariae treated with gold nanoparticles (50 μM). Note: Data are representative of three different experiments.

Figure 4

Transmission electron micrographs through sections of (A) untreated control parasite in Roswell Park Memorial Institute medium; (B) microfilariae treated with staurosporine (0.5 μM); (C) microfilariae treated with silver nanoparticles (50 μM); and (D) microfilariae treated with gold nanoparticles (50 μM). Notes: Arrows point to the vacuoles; data are representative of three different experiments. Abbreviation: V, vacuoles.