2.1. Materials
The PLGA polymer with M.wt (7000–17000 Dalton) 50:50 RESOMER® RG502H was purchased from Sigma-Aldrich. The curcumin 95% was purchased from LOBA CHEMIE in India. The polyvinyl alcohol (PVA), dichloromethane, methanol, zinc acetate, formaldehyde, Dulbecco's Modified Eagle's medium (DMEM), Fetal Bovine Serum (FBS), Bovine Serum Albumin (BSA), and potassium hydroxide were purchased from Merck Millipolids Co., Ltd.
2.2. Methods
2.2.1. Preparation of stoichiometric and non-stoichiometric Cur-PLGA
Cur-PLGA was prepared using the solid-in-oil-in-water (s/o/w) solvent evaporation technique as previously described [11]. In a beaker, one gram of PLGA was dissolved in dichloromethane until it formed a uniform PLGA solution. Then the beaker was filled with 0.023 gm of curcumin. Because this is the exact stoichiometric amount, we referred to this product as stoichiometric Cur-PLGA. A second beaker was prepared in the same manner as the previous one, but this time 0.039 grams of curcumin was added to the PLGA solution resulting in a light yellow fluorescence solution. In the second beaker, an excess of curcumin was evenly distributed throughout the solution for few seconds concurrently with the light yellow fluorescence color formation and then was encapsulated by PLGA self-assembly behavior, forming a dark yellow solution with no fluorescence, which we named this product as a non-stoichiometric Cur-PLGA. Both the stoichiometric and non-stoichiometric Cur-PLGA were sonicated for 2 min in a probe sonicator (VCX-1500, Sonics) in order to generate the s/o primary emulsion. Then the stoichiometric and non-stoichiometric Cur-PLGA s/o primary emulsion were added in-separate drops through filter paper into two separate flasks containing 160 mL of PVA solution (1% w/v) and sonicated for 3 min to generate the final s/o/w emulsion. The organic solvent was removed through rotary vacuum evaporation at 50 oC, while the free PLGA/PVA polymers were eliminated by centrifugation at 3000 rpm using a Hermle Z32 (German centrifuge) for 10 min. Finally, solutions were lyophilized to be stored as solids at 4 oC for further use.
2.2.2. Preparation of Cur-ZnO nano composite with different percentages of curcumin
The ZnO nanoparticles were first prepared following Thanasi et al.[12]. In brief, a solution of potassium hydroxide in methanol (2% W/V) was added to a solution of zinc acetate dehydrate (2% W/V) as a dropwise addition on a magnetic hot plate stirrer at 60oC for 2 h, forming a precipitate, which was separated, washed twice with methanol and dried at 60oC. Loading of curcumin on ZnO-Nps was done according to Al-Ajmi et al. [13] with some modifications. Three beakers were prepared in which the previously prepared ZnO-Nps were added to 50 ml of ethanol. In each beaker, 0.95, 0.9, and 0.80 gm .Then curcumin powder were added to them with a percent of 0.05, 0.1, and 0.2, respectively. All beakers were sonicated for 30 minutes to trap curcumin on ZnO, and then the solutions were centrifuged at 10,000 rpm for 10 minutes to remove solvent and unreacted materials before drying at 60oC overnight. In final, three preparations of Cur-ZnO were obtained with curcumin percentages of 5%, 10% and 20%. We refer to them as X = 5, X = 10 and X = 20, respectively.
2.3 Characterizations techniques
2.3.1. X-Ray Diffraction (XRD)
Empyrean Malverpanalytical and Netherland X-ray diffraction were used to document the XRD data. The device worked with a 30 mA current, a functioning voltage of 40 kV (power of 1200 W), and a scanning speed of 2/min (step size = 0.04 degree and step time = 1.0s) in the 10-80 scanning range (2 Theta scale) at room temperature. The crystallite size was calculated from the XRD peaks using Scherrer's equation.
D = 0.94λ/β cos θ (Eq. 1).
Where D is the average crystallite size, λ is the radiation wavelength, β is the corrected full width at half the maximum of the diffraction peak, and θ is the Bragg’s diffraction angle.
2.3.2. Fourier-transform infrared (FTIR)
The functional groups were determined by FTIR spectra that were obtained using a Bruker (Vertex 70 RAM II IR (Bruker, Germany) spectrometer. Samples were ground and mixed with potassium bromide (KBr) to make pellets. It was compressed under high pressure to prepare pellets of 10.0 mm and 1–2 mm thick. The pellets were scanned over a range of 4000 to 500 cm−1. The spectral of free Curcumin and free PLGA and free ZnO in addition to the five prepared formulas were recorded in the transmission mode. The resolution used in the scans was 1 cm−1, and the spectra were averaged over three scans.
2.3. 3. Morphological and size analysis
The size determination of all five prepared curcumin nanoparticles was determined using a JEOL JEM-2100 high resolution transmission electron microscope at an accelerating voltage of 200 KV. All samples were diluted by water, and some samples were treated with phosphotungstic acid (PTA) to improve the resolution.
2.4. Antiviral Study
2.4.1. Vero-E6 cells with SARS-CoV-2 infection
To culture Vero-E6 cells, Dulbecco's Modified Eagle's medium (DMEM) was used. The cells were supplemented with 10% Fetal Bovine Serum (FBS) (Invitrogen) and 1% Penicillin/Streptomycin mixture (pen/strep) and then incubated at 37 °C in a humidified incubator with 5% CO2. The hCoV-19/Egypt/NRC-3/2020 SARS-CoV-2 "NRC-03-nhCoV" virus was proliferated in Vero-E6 cells. Simply, confluent VERO-E6 cell monolayers were infected with NRC-03-nhCoV in infection medium (DMEM with 1% pen/strep, 0.2% Bovine Serum Albumin (BSA), and 1 µg/ml TPCK-treated trypsin) at a multiplicity of infection (MOI) of 0.1 for 2 h. Following virus adsorption, the infection medium containing the inoculum of the virus was discarded and replaced with new infection media. The infected VERO-E6 monolayers were incubated for 4 days post-infection at 37°C in a humidified incubator with 5% CO2. The cell supernatants were collected and centrifuged at 2500 rpm for at least 5 min to remove cellular remains. The clear supernatant containing propagated viruses was then liquated and preserved at -80°C in a freezer. An aliquot of the saved NRC-03-nhCoV virus was subjected to virus titration using a plaque titration assay.
2.4.2. Cytotoxicity assay
2.4.2.1 Cytotoxic Concentration CC50
To assess the cytotoxic concentration CC50 of the prepared products as described before (citations ), stock solutions were prepared by dissolving the mentioned formulae in 10% DMSO "in 1X DMEM" and serially diluting them with 1X DMEM to prepare the working concentrations. The CC50 of each formula was examined in Vero-E6 cells by using a crystal violet assay in which 100 µl of the VERO-E6 cell suspension was dispersed into 96-well plates (3*105 cells/mL). Incubation for the seeded plates then occurred at 37°C in a 5% humidified CO2 incubator for 24 h. Then cell monolayers were co-incubated with a combination of different concentrations of each drug in triplicates at 37°C in a 5% humidified CO2 incubator. After three days, the media supernatants were thrown away and the cell monolayers were washed once with 1X PBS and fixed with 10% formaldehyde for 1 h at room temperature (RT). The plates were later dried and stained at RT with 0.1% crystal violet for 20 min on a bench rocker. Again, the monolayers were then washed, dried, and the crystal violet dye in each well was then treated with 200 µL methanol for 20 min on a bench rocker at RT. Eventually, the absorbance was measured at λmax 570 nm using the Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The cytotoxicity concentrations of the mentioned formulae to control were compared using nonlinear regression analysis. The cytotoxicity concentrations of the mentioned formulae to control were compared using nonlinear regression analysis.
2.4.2.2. Inhibitory concentration 50 (IC50)
The IC50 values were determined as previously described by crystal violet assay (citations ). Briefly, after washing the 24 h cultured VERO-E6 monolayers in the 96-well tissue culture plates once with 1x PBS. The NRC-03-nhCoV "virus was co-incubated with serial diluted concentrations of the mentioned formulae for 1 h at 37 °C. The Vero-E6 cells were classified as follows: the uninfected cells were represented as control, the infected cells which remain untreated represent the virus control, and the infected cells were treated with mentioned formulae. All cells were kept in a 5% CO2 incubator for 72 h at 37 °C. The fixation of the cell monolayers took place by the addition of 100 μL of 10% formaldehyde for 20 min, and the staining process occurred by the addition of 0.1% crystal violet "in distilled water" for 15 min at RT. Then 100 μL of absolute methanol was added per well to dissolve the crystal violet dye. The optical density of the color was measured at λmax 570 nm using the Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC50 values were calculated using nonlinear regression analysis through plotting log inhibitor versus normalized response.