Materials
Chondroitin 4-sulfate sodium salt from a bovine trachea, low molecular weight chitosan (75–85% DDA and 50–190 kDa), and chloroauric acid 99.99% (HAuCl4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Roche (Mannheim, Germany). Doxorubicin HCl (DOX) was obtained from Actoverco pharmaceutical company (Tehran, Iran). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), antibiotic solutions, and phosphate-buffered saline (PBS) were obtained from Gibco (NY, USA). All other materials were purchased from Merck (Darmstadt, Germany) and were analytical reagent grade. Chitosan was further purified by the precipitation procedure [44].
Cell lines and culture
Briefly, MDA-MB-468 cells were cultured in DMEM supplemented with 10% FBS, 100 IU mL−1 penicillin, 100 IU mL−1 streptomycin at 37 °C in a humidified incubator with 5% CO2 levels. Then, the growth patterns and morphology of cells were regularly examined using an inverted microscope [45]. After 48 h incubation, the cells were harvested by trypsinization to perform cell passaging and backup cultures. Furthermore, βTC3 cells in the presence of 2 mM l-glutamine were cultured similarly in a proper culture medium [45, 46]. Human fibroblast as the normal cell was isolated by the procedure described in our previous work [45, 47]. All the cell culture ingredients were supplied from Auto-cell Co. (Warsaw, Poland).
Synthesis and preparation of NPs
Green synthesis of GNPs capped with CHS (CHS-GNPs)
20 ml of HAuCl4 (0.39 mg mL−1) with 10 mL of CHS (5 mg mL−1) were reacted (in a 2:1 ratio v/v %) while the pH of the solution was maintained at 7.5 by NaOH solution (0.1 N) and HCl solution (0.1 N). The solution was stirred at 60 °C for 70 min until a deep-red color appeared. After three times centrifuging (8000 rpm, 30 min), the reaction was performed in a stirring aqueous environment at room temperature without the need for UV light, autoclave, microwave, and laser irradiation (CHS-GNPs) [34, 48].
Conjugation of DOX and CHS-GNPs
The conjugation of DOX onto CHS-GNPs was performed by mixing 2 mg of CHS-GNPs with 20 µL DOX (1% w/v) in distilled water with a ratio of 1:10 w/w. The product was shaken at 37 °C and 100 rpm for 30 min. It is assumed that positively charged DOX can be complexed with negatively charged CHS-coated GNPs through electrostatic interaction. Eventually, the solution was centrifuged at 14,000 rpm for 20 min. The yielded pellet was dispersed in 300 μL of distilled water to determine the percentage of CHS-GNPs loaded with DOX.
Modification of DOX-CHS-GNPs by CS
For this purpose, 3 µL of CS (2 w/v %) was added dropwise to 300 µL of DOX-CHS-GNPs solution (in a ratio of 1:100 v/v %). Then, the mixture was shaken for 3 min, incubated at room temperature for 30 min, and centrifuged at 14,000 rpm for 20 min. The DOX loading and supernatant concentrations were determined. Different DOX concentrations (3.125, 6.25, 12.5, 25, 60, 80, and 100 μgmL−1) in distilled water were prepared to evaluate the amount of drug-loaded on the surface of synthesized NPs. The DOX concentration in the supernatant and the precipitate was determined using UV–Vis spectroscopy at 480 nm. A step-by-step schematic procedure for preparing DOX-CHS-GNPs-CS by the green synthesis method was presented in Additional file 1: Fig. S1.
Physicochemical properties of NPs
UV–Vis spectroscopy Cary 100 (Varian) is a reliable technique to evaluate the formation, and crystal growth of NPs. [48]. To this end, the solution of DOX-CHS-GNPs-CS with Dox concentration 20 μg mL−1 was diluted by distilled water in a 2:1 ratio v/v % at pH 7 and prepared using UV–vis spectrophotometry.
To verify the DOX-CHS-GNPs-CS synthesis, FT-IR spectra were recorded by Prestige-21 Shimadzu Spectrometer (Kyoto, Japan). The suitable amount of samples, including CHS, CHS-GNPs, DOX, DOX-CHS-GNPs, CS, and DOX-CHS-GNPs-CS, was compressed into KBr, and samples were then tested in the range of 400–4000 cm−1 with a resolution of 4 cm−1. Furthermore, particle size, zetapotential and polydispersity index of CHS-GNPs, DOX-CHS-GNPs, and DOX-CHS-GNPs-CS were evaluated using DLS following distilled water in a 1:9 ratio v/v % at pH 7.
Transmission electron microscopy (TEM) was used to identify the morphology and particle size of CHS-GNPs and DOX-CHS-GNPs by using LEO 906 E microscope (Carl Zeiss, Germany) with an accelerating voltage of 100 kV. Before analysis, GNPs were deposited on carbon-copper grids. In addition, the surface morphology of DOX-CHS-GNPs-CS was investigated through TEM. Finally, GNPs represent consistent stability between − 30 and + 30 mV of their surface potential values because ambient ionic liquids can produce electrostatic forces to increase GNPs stability which was quite evident from higher zeta potential value versus aqueous extract value [49, 50].
X-ray diffraction (XRD) analysis
To confirm the crystalline nature of the synthesized CHS-GNPs, XRD analysis was carried out (Xpert pro, Panalytical, Holland), which was operated at a voltage of 40 kV and a current of 30 mA with cu k−1 radiation.
In vitro cytotoxicity assay
The MTT assay was utilized to evaluate the cytotoxicity of free DOX and NPs-DOX on MDA-MB-468, βTC-3, and HFb cell lines, as described previously [46, 51]. To evaluate cell viability, 1 × 104 cells were cultured in a 24-well plate with the proper culture medium for 24 h. Then, 20 µL of treatment solutions of DOX, DOX-CHS-GNPs, and DOX-CHS-GNPs-CS, were added at different concentrations into each well for another 48 h. Finally, the cell viability was evaluated by analyzing optical densities (ODs) obtained from the SpectraMax 190 absorbance microplate reader (Bio Tek Instruments, USA) at 570 nm.
In vitro release study of DOX
In order to evaluate the release of DOX at the surface of DOX-CHS-GNPs and DOX-CHS-GNPs-CS, cellulose acetate dialysis membranes (Spectra, MWCO 12 kDa) in phosphorus buffer solution (PBS) at pH 7.4 were used [46, 52]. DOX-CHS-GNPs and DOX-CHS-GNPs-CS were located inside the membrane separately and were dialyzed against 7 mL PBS for continuous stirring at 100 rpm. Every hour, to read the absorbance of DOX, a total solution of 7 mL of PBS dialysis solution was replaced completely with 7 mL of fresh PBS. The amount of released DOX was analyzed by determining the absorbance at 481 nm, and the release of DOX was calculated according to the standard equation of the released drug. The released DOX amounts from DOX-CHS-GNPs and DOX-CHS-GNPs-CS were measured in the different time intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6, 18, 24, 27, 30, 42, and 45 h). Finally, the results were repeated three times for each sample, and the calibration curve of DOX in the incubation medium was calculated.
CAM assay
Chick chorioallantoic membrane (CAM) assay was used to evaluate the effect of different therapeutic groups on reducing angiogenesis and tumor growth. In summary, fertilized hen’s eggs were incubated at 37 °C and 60% humidity. After 10 days, an incision (square dimension: 0.5 cm2) was made on the eggshell under sterile conditions to access the CAM layer. Subsequently, 0.5–1 × 106 cells (10 µl of the cell suspension) treated with DOX-CHS-GNPs-CS, DOX-CHS-GNPs, free DOX, and CS-CHS-GNPs were injected onto the CAM surface. The incised area was then covered, and the eggs were reincubated for 7 days. Finally, the shell was opened, and CAM was extracted to assess tumor weight, size, and angiogenesis, and images were taken.