Materials
Poloxamer 407 (Lutrol F127) was from BASF AG (D-Ludwigshafen), cholesterol, sucrose, tween 80 and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) were from Sigma-Aldrich (D-Steinheim), egg phosphatidyl choline (EPC) was obtained from Lipoid GmbH (D- Ludwigshafen), monoolein (GMOrphic-801) from Eastman Chemical Company (Kingsport, TN), methanol was from Carl Roth GmbH (D-Karlsruhe), ethanol and chloroform all from VWR International (D-Darmstadt) , and Hepes and sodium chloride were from AppliChem GmbH (D-Darmstadt). The cell lines were purchased from ATCC, USA. All cell culture materials were purchased from Lonza Bioscience (Morristown, USA). Purified water was prepared by filtration and deionization/reverse osmosis (Milli RX 20, Millipore, D-Schwalbach)
Plant collection
Ficus carica L. fam. Moraceae was collected from Wadi Fatima, Makkah, KSA and identified by Dr. Hany Gouda, Department of Pharmacognosy, Faculty of Pharmacy, Najran University. A voucher specimen (UQU-2019-1) of the plant is available in the herbarium, Department of Pharmacognosy, Faculty of Pharmacy, Umm al-Qura University. Dried plant leaves were powdered and extracted with petroleum ether. The obtained extract was evaporated and then subjected to separation using preparative HSCCC.
Isolation and spectroscopic analysis of bergapten
In order to determine the most suitable two-phase solvent system for bergapten its partition coefficient had to be calculated first in different solvent systems as previously described [23] using the formula: K = Aupper/Alower where Aupper is the absorbance of bergapten in the upper phase and Alower its absorbance in the lower phase of the solvent system determined by calculation of the area under the peak obtained for bergapten by HPLC analysis. Separation of bergapten using preparative HSCCC was carried out according to the method previously reported in literature. After successful purification of the compound it was subjected to MS and NMR analysis and its identity was confirmed by comparison of the obtained data with literature [22, 23].
Cubosomes preparation
Cubosomes were prepared using 4.4% monoolein and 0.6% poloxamer 407. Poloxamer was mixed with the molten monoolein containing bergapten followed by the dropwise addition of this mixture to water under stirring for 1 day at room temperature. As reported before, these preparation procedures resulted in the formation of cubic gel which was converted to cubosomes by homogenization [20]. Consequently, this cubic gel was subjected to a homogenization process for 15 minutes at 350 bar and 40°C followed by autoclaving these homogenized dispersions at 121°C for 15 minutes [19, 20, 24].
For quantitative cellular uptake study, Nile red was used as a florescent lipid soluble dye as it was reported that the incorporation of this dye into cubosomes did not alter particle size, zeta potential and drug loading of cubosomes [5]. Florescent cubosomes were prepared by dissolving Nile red (0.15 mg/ml) in molten monoolein and completing the procedures as previously described.
Preparation of acceptor multilamellar vesicles (MLV)
Multilamellar vesicles (MLV) were utilized as acceptor particles to measure the transfer of bergapten from cubosomes instead of measuring the release with the normal conventional methods which showed many drawbacks as reported before [17, 25-30]. Egg phosphatidylcholine (EPC) and cholesterol were dissolved in chloroform with molar ratio 4:1 (EPC: cholesterol) [31].
After evaporating chloroform under vacuum (Büchi Rotavapor R-114, D-Essen), the remaining thin lipid film was subjected to nitrogen to ensure complete removal of chloroform. 1 ml of warm 300 mM sucrose solution was added to the lipid film and the mixture was transferred to Eppendorf tube where it was subjected to centrifugation at 5000 rpm for 10 minutes to separate MLV in the supernatant from the excess sucrose solution below.
The separated MLV were washed two times with 0.5 ml HBS pH 7.4 and finally stored in HBS at refrigerator temperature.
Particle size and zeta potential measurements
Particle sizes of blank dispersions, dispersions containing bergapten and florescent dispersions before and after autoclaving were measured by photon correlation spectroscopy (PCS) (Malvern Zetasizer Nano UK-Worcestershire) after diluting the samples with filtered demineralized water. Polydispersity index (PDI) were determined to evaluate the homogeneity of the different dispersions. To confirm the homogeneity of these nanoparticles, particle sizes of the different monoolein dispersions and the acceptor MLV were measured using a laser diffractometer with PIDS technology (polarization intensity differential scattering), Coulter LS 230 Particle Sizer (Beckman Coulter, D-Krefeld,). The mean volume distribution of 8 measurements was calculated using the evaluation model Mie theory.
Zeta potential is an important feature for carriers or nanoparticles containing an anticancer drug. Thus, the zeta potential of all dispersions was measured using the same Malvern instrument after diluting the samples with 10 mM tris buffer pH 7.4.
Small angle X-ray diffraction
The existence of the cubic structure and the type of the cubic phase were determined from small angle X-ray measurements of the monoolein dispersions before and after the autoclaving process. Briefly, these measurements were carried out for 1-2 h with a SWAX camera based on a Kratky collimator system (Hecus M. Braun, Optical Systems GmbH, A-Graz). From the peaks spacing ratios and the lattice parameter (a = d√2), the type of the cubic structure was determined.
Entrapment efficiency
Ultrafiltration centrifugation technique was used to separate the excess drug from cubosomes and consequently measuring the entrapment efficiency. 1 ml of cubosomes was diluted to 10 ml with deionized water followed by centrifugation for 15 min at 5000 rpm. The amount of bergapten which might be adsorbed on the membrane was determined after filtering a known concentration of bergapten solution through the membrane and measuring the drug concentration in the filtrate.
The following equation was applied to calculate the entrapment efficiency.
The amount of free bergapten is represented by the amount of bergapten in the filtrate added to the amount adsorbed by the membrane.
In-vitro release from cubosomes
MLV were used as acceptor particles to measure the transfer of bergapten instead of measuring the release by usual conventional methods. To perform the transfer experiments with two lipid molar ratios 1:25 and 1:100 (cubosomes: MLV), different amounts of cubosomes containing bergapten were added to 600 µl MLV in Eppendorf tubes and the volume was completed to 1 ml with HBS. The tubes were incubated at 37 °C in a shaking water bath for different time intervals. At each time interval, samples were taken and centrifuged for 15 min at 5000 rpm to separate the donor cubosomes in the supernatant as a creamy layer from the acceptor MLV as precipitant pellets. The amount of bergapten remained in the cubosomes was calculated after dissolving the separated cubosomes in methanol and measuring the UV absorbance at 221 nm. On the other hand, MLV pellets were washed twice with 250 µl HBS followed by centrifugation. After washing and centrifugation the MLV pellets were collected, dissolved in methanol and the UV absorbance was measured at 221 nm. Furthermore, the two washes were combined and dissolved in methanol followed by measuring the UV absorbance at 221 nm.
The percentage of bergapten recovery was calculated by adding both the percentage retained and transferred.
Transfer kinetics
Analysis of bergapten transfer was carried out using Microcal Origin 6.0 software where the best fitted equation for the transfer curves was:
Aacc is the percentage of bergapten transferred to the acceptor MLV at time t, Afinal is the final percentage transferred (the plateau), A is a pre-exponential coefficient and k is the transfer rate constant.
In-vitro cytotoxicity
Cytotoxicity of cubosomes containing bergapten was studied on colorectal cells (C-26) using MTT assay. These cells were cultured in RPMI 1640 medium which contained 10% fetal bovine serum (FBS) in addition to streptomycin (100 µg/ml), penicillin (100 units/ml) and 0.25 µg/ml amphotericin B at 37ºC and 5% CO2. The cells were seeded (3000 cells/well) in a 96-well plate at 37ºC and 5% CO2 for 24 hours. These cells were treated with blank cubosomes, cubosomes containing bergapten and free bergapten solution for 48 and 72 hours. After removing the old medium, MTT solution was added and incubated with the cells for 4 hours to form formazan crystals. The percentage cell viability was calculated after adding dimethyl sulfoxide (DMSO) to each well and measuring the UV absorbance at 570 nm. Finally, IC50 (concentration of bergapten inhibits cell growth by 50%) was determined from the percentage viability (y-axis) and log concentrations (x-axis).
Cellular uptake
C-26 cells were seeded in 24-well plate at a density of 105 cells/well followed by incubating them with the florescent cubosomes for different time periods. Afterwards, cells were washed several times with cold PBS in order to remove excess cubosomes. The percentage of the florescent cubosomes that have been taken by the cells was calculated using the flow cytometer (Epics XL MCL, Beckman Coulter Inc., US-Fullerton) [32] after diluting the samples with PBS in a measurement tube. The count rate was 250 events/second and after 10,000 events the measurements were stopped. The detection was carried out at photomultiplier tube number 4 (FL4) with a detection wavelength range of 665-685 nm. Between each measurement, cleaning of the device was performed to avoid contamination of particles of preceding samples.
Antitumor activity
The animal studies protocols were approved from the ethics committee at the Faculty of Pharmacy, Umm Al Qura University with approval number (UQU-Pharmacy, 1200). The animal experiments were carried out according to EU Directive 2010/63/EU for animal experiments. In the shaved right flank of male mice with average weight 18-20 g and age 5 weeks, C-26 cells were injected. After 1 week, the mice were distributed randomly into 4 groups (5 mice/group). Different amounts of blank cubosomes, cubosomes containing bergapten, free bergapten solution and 5% dextrose were injected into the mice tail through intravenous route followed by measuring the tumor size using a digital caliper [33, 34]. After 22 days the mice were scarified, and the tumor weight was measured. Mice lost more than 15% of their body weight and those with a tumor volume greater than 1000 mm3 were excluded from the experiments.
Statistical analysis
Statistical analysis was performed using Student’s t-test. The differences between the results were significant when the p values were smaller than 0.05.