Materials
Mixture of THC and CBD was obtained from the Industrial and Medical Cannabis Research Institute (IMCRI), Iran. Tween 80 and Span 80 were purchased from Merck Chemicals (Germany). The water was deionized by Human Corporation Filter (Korea). DMEM (Dulbecco’s Modified Eagle Medium), trypsin/EDTA, F12, penicillin and streptomycin were purchased from Gibco (USA).
Preparation of NED
The NED was prepared using the titration method. A mixture of THC and CBD (1:1) at a final concentration of 300 ng/mL was infused to hemp seed oil (1.00 w/w%). The mixture was then filtered to obtain a clear oily phase. Span 80 (7.82% w/w) was added to the oil phase. The mixture was stirred at 4000 rpm for 10 min. Then, 27.18% w/w Tween 80 and 10% ethanol were added to the mixture in the same conditions. Subsequently, deionized water was added dropwise to the container.
Construction of pseudo-ternary phase diagram
Surfactants and co-surfactant (Smix) were combined in constant weight ratios of 1:1, 3.5:1, and 7:1. Samples that remained transparent with no sign of phase separation were considered as stable in the pseudo-ternary phase diagram.
Accelerated stability studies
Different accelerated stability tests, including centrifugation (5000 rpm for 30 min), heating-cooling cycles (4 °C and 40 °C for 6 times) and freeze and thaw cycles (−21 °C and room temperature for 3 times) were employed to estimate the stability of the prepared samples. The formulations that showed no evidence of instability (i.e. phase separation) were considered as having appropriate stability.
Characterization of the optimized NED
Scatterscope I (K-ONE NANO, Korea) was used to examine the mean particle size of the NEDs. The prepared NEDs were diluted 15 times with deionized water before particle sizing. The data obtained were the particles’ hydrodynamic diameter, (i.e. d50) Also, SPAN was calculated using Eq. (1).
$${\text{SPAN }}={\text{ }}({{\text{d}}_{90}}-{{\text{d}}_{10}})/{{\text{d}}_{50}}$$
(1)
The smaller the SPAN is, the more mono-dispersed preparation has been obtained. Transmission electron microscopy (TEM, LEO 906, Zeiss, Germany) was used to investigate the morphology and size of NED. The NED had been stained on a copper grid which was covered with carbon (200 mesh) and placed with 1% of phosphoric tungstic acid at room temperature for five minutes, then, checked by TEM.
The viscosity of NED was determined without dilution with an MCR300 rheometer device (Anton paar, Austria) with concentric cylinders at 25 °C.
Cytotoxicity assay
To determine the cytotoxicity, C6 cell lines (rat glioma cell line) were cultured in DMEM-F12 medium having FBS (10%) and Penicillin/Streptomycin (1%). To evaluate the cytocompatibility, C6 cells were counted and 5000 cells were seeded to each well of 96-well plates and incubated (37 °C, 24 h). Subsequently, 100 µL of the NEDs were added to each well. After 24 h, 10 µL of Alamar Blue solution was added to each well and incubated for 4 h at 37 °C. Cell viability was determined by recording the absorbance at 570 and 630 nm using an ELISA microplate reader (BioTek, USA).
Animal studies
All animal experiments followed the guidelines of the Research Ethics Committee and were approved by the Animal Welfare Ethics Committee of North Khorasan University of Medical Sciences (IR.NKUMS.REC.1400.1122). The Wistar rats (male, 10 weeks old, weight ∼ 200 g) were purchased from Royan Institute (Iran). Animals were anesthetized by IP injection of ketamine (80 mL/kg/h) and xylazine (10 mL/kg/h) before the animal studies (including cell implantation and magnetic resonance imaging (MRI)). For euthanasia, according to AVMA protocol, the animals were placed inside a CO2 chamber and CO2 was gradually introduced. To prevent distress, the CO2 flow rate was set at 20–30% of the chamber volume per minute. Heartbeat and respiration of the animals were then checked.
Hemocompatibility
To examine the potential in vivo toxicity, male rats’ blood was collected from each treatment group. Treatment groups included control (normal saline), bulk (free form of mixture of THC and CBD, 21 µg per injection), carrier (NE without THC and CBD, 0.7 mL), and NED (NE containing the mixture of THC and CBD drugs, 0.7 mL containing 21 µg of the mixture of THC and CBD per injection). 1 mL of the whole blood was drawn and placed in vacutainers containing EDTA, then, centrifuged (1500–2000 g, room temp, 10–15 min) to separate plasma (top layer), RBCs (red blood cells, bottom layer) and white blood cells. The following hemocompatibility factors were then checked: RBCs, hemoglobin, hematocrit, mean corpuscular volume of RBCs (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red cell distribution width (RDW) [17]. For determination of coagulation factors, blood was drawn in a sodium-citrate vacutainer, then centrifuged (3000 rpm, 8 °C, 20 min) to obtain platelet-poor plasma (PPP). Prothrombin time (PT) and partial thromboplastin time (PTT) were also evaluated in all groups.
In vivo efficacy studies
Before tumor implantation, rats were carefully positioned in a stereotactic machine. After scalp preparation, a borehole was drilled into the skull to inject 5 µL cell suspension containing 800,000 C6 cells at pH 7.4 unilaterally into the right striatum with a Hamilton glass syringe. The coordinates of injection were 1.5 mm lateral from the bregma, 2.5 mm deep from the skull surface and 2 mm posterior. After 15 min of injection, the needle was gently withdrawn, the hole was covered and the incision was sutured. The animals were monitored daily for 14 days post-implantation of the C6 tumor cells.
MR imaging was done in the National Brain Mapping Laboratory (Tehran, Iran) using a 3-T MRI (Slew rate: 200 mT/m/ms, maximum amplitude: 45 mT/m, Siemens Healthcare, Germany). MRI was used to determine the presence, expansion, and tumor size that had been implanted in the rat brain. T2-weighted MR images were then acquired at different time points. Tumor volume was calculated by drawing areas of interest in each slice at specified time.
Rats with cerebral glioblastoma were divided into 4 treatment groups (5 rats in each group) and treated twice a week via the tail vein. Rat deaths were documented and for each group, the Kaplan-Meier survival curve (generated using Graphpad Prism software) were plotted.
Statistical analyses
The mean and standard deviation were used for all numerical data. Differences between the two-groups and multiple-groups analyses were compared using Student’s t-test and one-way ANOVA tests, respectively. For survival analysis, the log-rank test was used. Statistics were considered significant if the p-value was less than 0.05.
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