Inhibition of the Naja naja venom toxicity by polymeric nanoparticles loaded with Leucas aspera methanolic extract

Abstract

Background: Snakebite is a neglected tropical disease that affects millions of people worldwide. Developing effective treatments can make a significant contribution to global health efforts and public health initiatives. To reduce mortality due to snakebite, there is an immediate need to explore novel and effective treatment methodologies. In that context, nanoparticle-based drug delivery is gaining a lot of attention. Hydrophilic nanoparticles are suitable for the delivery of therapeutic peptides, proteins, and antigens.

Methods: The present investigation is aimed at evaluating the anti-ophidian potential of the methanolic extract of the ethno-medicinal herb Leucas aspera (Willd.) loaded within chitosan nanoparticles (CNP-LA), against the Indian cobra (Naja naja) venom enzymes. For this purpose, nanoparticles were prepared using the ionic gelation method to enhance the efficacy of the extract. The physicochemical and structural features of nanoparticles were investigated using dynamic light scattering (DLS), Fourier-transform Infrared (FTIR), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD) techniques.

Results: It was found that CNP-LA has an average size of 260 nm with a polydispersity index of 0.132 (PDI) and zeta potential of 34.7 mV, with an encapsulation efficiency of 92.46%. The in vitro release study was performed at pH 5.0 and 7.4. Furthermore, in vitro studies indicated that CNP-LA inhibited the phospholipase A2, hemolytic, and caseinolytic activities of Naja naja venom with the percentage inhibition of 92.5%, 83.9%, and 94.5%, respectively.

Conclusion: This is the first report on the application of herbal methanolic extract loaded within chitosan nanoparticles for neutralizing snake venom enzymes with increased efficiency.

Keywords: Leucas aspera (Willd.); Naja naja; chitosan; controlled release; nanoparticle.

FIGURE 1

UV-visible spectrum (200–800 nm) of chitosan nanoparticles (CNP), L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA), and L. aspera methanolic extract alone (LA).

FIGURE 2

Fourier-transform infrared (FTIR) spectrum of (A) methanolic L. aspera extract (LA), (B) chitosan nanoparticles (CNPs), and (C) L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA).

FIGURE 3

Field emission scanning electron microscopy (FESEM) images with EDX of (A) chitosan nanoparticles and (B) L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA).

FIGURE 4

X-ray diffractogram of (A) chitosan nanoparticles and (B) L. aspera methanolic extract loaded within chitosan nanoparticles. The inset is the expanded view of the diffractogram in the given 2θ range.

FIGURE 5

In vitro release profiles of the L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA) plotted as a function of percentage release over the time at pH values 7.4 (circles) and 5.0 (rectangles). The values are represented as mean ± SD (n = 3).

FIGURE 6

Effect of CNP and CNP-LA on N. naja venom phospholipase A₂ (PLA₂) was evaluated by pre-incubating with 50 µg of N. naja venom with different concentrations of chitosan nanoparticles (CNP) and the L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA) at (A) 0.5 TPP, (B) 1 TPP, and (C) 2 TPP at various ratios for 1 h at 37°C. Values represent the mean ± SD of replicates (p < 0.001). In the figure, 2T represents the free nanoparticle, whereas 1P, 2P, 5P, and 10P represent the nanoparticles loaded within the plant extract.

FIGURE 7

Effect of CNP and CNP-LA on N. naja venom hemolytic inhibition was evaluated by pre-incubating with 50 µg of N. naja venom with different concentrations of chitosan nanoparticles (CNP) and the L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA) at (A) 0.5 TPP, (B) 1 TPP, and (C) 2 TPP at various ratios for 1 h at 37°C. Values represent the mean ± SD of replicates (p < 0.001). In the figure, 2T represents the free nanoparticle, whereas 1P, 2P, 5P, and 10P represent the nanoparticles loaded within the plant extract.

FIGURE 8

Effect of CNP and CNP-LA on N. naja venom caseinolytic inhibition was evaluated by pre-incubating with 50 µg of N. naja venom with different concentrations of chitosan nanoparticles (CNP) and the L. aspera methanolic extract loaded within chitosan nanoparticles (CNP-LA) at (A) 0.5 TPP, (B) 1 TPP, and (C) 2 TPP at different ratios for 1 h at 37°C. Values represent the mean ± SD of replicates (p < 0.01). In the figure, 2T represents the free nanoparticle, whereas 1P, 2P, 5P, and 10P represent the nanoparticles loaded within the plant extract.