Synthesis of copaiba (Copaifera officinalis) oil nanoemulsion and the potential against Zika virus: An in vitro study

Abstract

Zika virus (ZIKV) has spread all over the world since its major outbreak in 2015. This infection has been recognized as a major global health issue due to the neurological complications related to ZIKV infection, such as Guillain-Barré Syndrome and Zika virus Congenital Syndrome. Currently, there are no vaccines or specific treatments for ZIKV infection, which makes the development of specific therapies for its treatment very important. Several studies have been developed to analyze the potential of compounds against ZIKV, with the aim of finding new promising treatments. Herein, we evaluate the ability of a copaiba (Copaifera officinalis) oil nanoemulsion (CNE) to inhibit ZIKV. First, the highest non-cytotoxic concentration of 180 μg/mL was chosen since this concentration maintains 80% cell viability up to 96h after treatment with CNE in VERO cells resulted from MTT assay. The intracellular uptake assay was performed, and confirmed the internalization of the nanoemulsion in cells at all times analyzed. VERO cells were infected with ZIKV and simultaneously treated with CNE and the nanoformulation without oil (ENE) at the highest non-toxic concentration. The results evaluated by plaque assay revealed a viral inhibition of 80% for CNE and 70% for ENE. A dose-dependence assay revealed that the CNE treatment demonstrated a dose-dependent response in the viral RNA levels, whereas all ENE tested concentrations exhibited a similar degree of reduction. Taken together, our results suggest CNE as a promising nano-sized platform to be further studied for antiviral treatments.

Fig 1

Colloidal stability parameters of copaiba oil nanoemulsion (CNE) and nanoformulation without oil (ENE) over time of storage (1, 7, 15, 30, and 60 days) at 4°C according to hydrodynamic diameter (A), polydispersity index (B), and zeta potential (C). Macroscopic aspect of CNE and ENE (D). (Data available: S1 and S2 Figs).

Fig 2. Representative ultramicrographs of copaiba oil nanoemulsion (CNE), acquired by transmission (A) and scanning (B) electron microscopy.

Red arrows indicate CNE nanodroplets.

Fig 3. Cell viability of VERO E6 cells treated with nanoformulation without oil (ENE), copaiba oil nanoemulsion (CNE), and free copaiba oil (FCO), for 24, 48, and 96 hours in concentrations of 5.62; 11.25; 22.5; 45; 90; 180; and 360 (μg/mL).

(A) Treatment with ENE. (B) Treatment with CNE. (C) Treatment with free copaiba oil. C: control cells. Representative of statistical significant difference between concentrations of treatments and the control (*: p<0.05; **: p<0.01; ***: p<0.001). (Data available: S3–S5 Figs).

Fig 4. Fluorescent microscopy images of cellular uptake of copaiba oil nanoemulsion—CNE (180 μg/mL) in VERO E6 after different periods of incubation.

The pictures were taken (A) 1 hour (B) 2 hours, (C) 3 hours, and (D) 48 hours after nanoemulsion incubation. Scale bar: 50 μm.

Fig 5. VERO E6 cells incubated with nanoformulations presented viral activity inhibition.

(A) Representative scheme of the viral inhibition assay and the dose-dependence assay. First (I), representing the test performed with the copaiba oil nanoemulsion (CNE), and, second (II), representing the test performed with the nanoformulation without oil (ENE). (B) Relative foci number (%) resulting from the viral inhibition assay in VERO E6 cells infected with ZIKV. These assays were performed in two independent events. Both treatments showed statistical difference in relation to control cells (**: p <0.01 and ***: p <0.001). (C) ZIKV RNA resulting from the dose dependence of CNE and ENE in VERO E6 cells infected with ZIKV. (Data available: S6 and S7 Figs).