Fig 1.
Regions of conserved (red) and variable (blue) amino acids from reviewed sequences on www.uniprot.org for PLA2 (left, 27 sequences) and 3FTXs (right, 96 sequences) from the following elapid snake venoms: Naja sputatrix, Naja mossambica, Bungarus caeruleus, Bungarus fasciatus, Naja haje, Naja melanoleuca, Naja nivea, Dendroaspis polylepsis. The core structures depicted for PLA2 and 3FTXs are from Bungarus caeruleus (PDB: 2OSN) and Dendroaspis polylepsis (PDB: 2MFA), respectively. Disulfide bonds are highlighted in yellow.
Fig 2.
(A) Method used to analyze the binding of the synthetic NPs to various venoms. *A representative venom from a species of each genus tested was further analyzed by LC-MS/MS. (B) Composition of the synthetic NPs. (C) TEM image of the NPs (scale bar = 200 nm).
Fig 3.
Nanoparticle binding to elapid snake venoms in concentrated human serum (A) Control experiments using the venom compositions of interest. (1) MW ladder. (1.1) Venom from Naja mossambica. (1.2) Venom from Bungarus caeruleus. (1.3) Venom from Bungarus fasciatus. (1.4) Venom from Naja haje. (1.5) Venom from Naja nivea. (1.6) Venom from Naja melonoleuca. (1.7) Venom from Naja sputatrix. (1.8) Venom from Dendroaspis polylepis. (B) NP selectivity results. (1) MW ladder. (2.0) Serum only control experiment. (2.1–2.8) NP selectivity results following the same venom-order described above. See S1–S10 Figs for full gel images.
Fig 4.
NP selectivity in human serum (red) and in human serum + N. nigricollis venom (blue).
In the presence of serum alone, the NP favorably associates with proteins of varying molecular weights (Serum only corona band). A distinct change in the distribution and a new dominant molecular weight band (<15 kDa) in the N. nigricollis venom-containing corona band. Proteins that comprised this NP corona were identified via in gel digestion followed by proteomic identification.
Fig 5.
Dose-response curve of inhibition of cytotoxicity in L6 muscle cells by the NP against administration of 7 μg/mL of (A) N. nigricollis venom and (B) 7 μg/mL of N. mossambica measured 14 h after exposure to the cells (n = 4–5).
Fig 6.
Inhibition of dermonecrotic activity of N. nigricollis venom by NPs.
(A) A fixed amount of venom was incubated with variable amounts of NPs to attain several ratios. Controls included venom incubated with saline solution instead of NPs. Upon incubation, aliquots of the mixtures, containing 100 μg venom, were injected intradermally in mice. Seventy-two hours after injection, the areas of necrotic lesions in the inner side of the skin were measured. Necrosis was expressed as percentage, 100% corresponding to the necrotic area (60 mm2) in mice receiving venom alone. NPs inhibited, dose-dependently, the dermonecrotizing activity of the venom. (B, C, D) Light micrographs of sections of the skin of mice 72 h after injection of 100 μg N. nigricollis venom incubated with saline solution (B), with NPs at a NP: venom (w : w) ratio of 5.0 (C), or after injection of NPs alone (D). In (B) there is ulceration, with loss of epidermis and the formation of a hyaline proteinaceous material (arrow); skin appendages are absent and there is a prominent inflammatory infiltrate. In (C) NPs inhibited the ulcerative effect, evidenced by the presence of epidermis (arrow) and skin appendages, whereas an inflammatory infiltrate is observed in the dermis. Skin injected with NPs alone (D) shows a normal histological pattern. Hematoxylin-eosin staining. Bar represents 100 μm. (E) Inhibition of dermonecrosis by N. nigricollis venom in experiments involving intradermal injection of 100 μg venom followed by the injection of NP suspension (5.5 mg/mL), in the same region of venom injection, at various time intervals. A significant reduction in the extent of dermonecrosis was observed (* p < 0.05), although the extent of inhibition decreased with time. NP alone did not induce skin damage.