Anales RANF

Nannocannabinoids for brain tumor drug delivery @Real Academia Nacional de Farmacia. Spain 201 size-dependent pattern, the role played by the particle size of LNCs in the extent of targeting has concomitantly been evaluated. On the other hand, we have encapsulated CBD into the oily core of LNCs and assessed in vitro their efficacy as extended-release carriers of CBD against the U373MG cell line. The role played by the size of LNCs in drug release and cytotoxicity has also been evaluated.. 2. MATERIALS AND METHODS 2.1. Materials Labrafac ® lipophile WL 1349 (caprylic-capric acid triglycerides) and Labrafil ® M 1944 CS (6-macrogol oleic glycerides) were kindly supplied by Gattefossé. Kolliphor ® HS15 (C 18 E 15 polyethylene glycol (15) 12- hydroxystearate) and Kolliphor ® ELP (C 18Δ9 E 35 polyethylene glycol (35) ricinoleate) were gifts from BASF. Lipoid ® S75 (soybean lecithin with 70% of phosphatidylcholine) was supplied by Lipoid-Gmbh. NaCl was purchased from Panreac. De-ionized water was obtained from a MilliQ ® Purification System. The fluorescent dyes 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine 4-chlorobenzenesulfonate salt (DiD) were purchased from Invitrogen Molecular Probes. Cannabidiol (CBD) was provided by THC- Pharma. Endothelial Cell Basal Medium-2 (EBM-2) and its culture supplements were purchased from Lonza. Dulbecco’s Modified Eagle Medium (DMEM) and penicillin-streptomycin (10,000 U/mL) were provided by Gibco. Tetramethyl-rhodamine-isothiocyanate–dextran (TRITC-dextran, MW 150 kDa), type I collagen from calf skin, fibronectin from bovine plasma, Hank’s Balanced Salt Solution (HBSS), 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT), dimethyl- sulfoxide (DMSO) and sterile Nunc Lab-Tek ® chamber slides (8 wells, Permanox ® slide, 0.8 cm 2 /well) were purchased from Sigma-Aldrich. Vectashield ® mounting medium with DAPI (H-1200) was provided by Vector Laboratories. Sterile Millicell ® Hanging Cell Culture Inserts (12-well culture plates; membrane: polyethylene terephthalate membrane; pore size: 1.0 µm; membrane surface area: 1.1 cm 2 ) and Amicon® Ultra 15 mL Centrifugal Filters (MWCO: 10 kDa) were supplied by Merck Millipore. Methanol, acetonitrile and tetrahydrofuran HPLC grade were purchased from Fisher Scientific. 2.2. Cell lines The human brain endothelial hCMEC/D3 cells were seeded in collagen-coated flasks and cultured in EBM-2 medium supplemented with 2.5% foetal bovine serum (FBS), 0.025% (v/v) rhEGF, 0.025% (v/v) VEGF 0.025% IGF, 0.1% (v/v) rhFGF, 0.1% (v/v) gentamycin, 0.1% (v/v) ascorbic acid and 0.04% (v/v) hydrocortisone at 37ºC and 5% CO 2 . For all experiments, cells between passage 25 and 30 were used. The human glioblastoma U373MG cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37ºC and 5% CO 2 . For all experiments, cells between passage 15 and 25 were used. 2.3. Animals Male ICR mice (4-5 weeks old) were purchased from Envigo. The mice were housed in ventilated cages with free water and food in a 12h dark/light cycle. All in vivo experiments were approved by the Ethics Committee of the Community of Madrid (Ref. PROEX 111/14) and conducted according to Spanish and European guidelines (Directive 86/609/EEC). 2.4. Preparation and characterization of LNCs LNCs were prepared by the PIT method (43). Briefly, all excipients (namely, aqueous and oily phases along with nonionic polyethoxylated surfactants) were mixed under magnetic stirring and progressively heated over the phase inversion temperature of the system. Subsequently, the mixture was gradually cooled down until the phase inversion temperature was reached. Then, a sudden quench with cold water (5 mL) was performed to obtain the final suspension of LNCs. Different formulations of LNCs were prepared by varying the relative proportions of their excipients and the surfactant/oil affinity, i.e. changing the nature of the surfactant (between Kolliphor ® HS15 and Kolliphor ® ELP) and of the oil (between Labrafac ® lipophile WL1349 and Labrafil ® M 1944 CS). 2.4.1. Size distribution and zeta potential The average volume diameter and polydispersity index (PdI) were measured by dynamic light scattering (DLS) with a Microtrac ® Zetatrac™ Analyzer (Microtrac Inc.). The zeta potential was measured using a Zetasizer Nano ZS (Malvern Instruments). 2.4.2. Incorporation efficiency and drug content The CBD content in the CBD-decorated and CBD- loaded LNCs was determined by high performance liquid chromatography (HPLC). A mixture of methanol: acetonitrile: water (52:30:18 v/v) at a flow rate of 1.8 mL/min was used as mobile phase. The analytical column was a reversed-phase Mediterranea Sea ® C18 (5µm 15 x 0,46 cm) (Teknokroma ® ). The amount of CBD associated with LNCs in each case was determined as the difference between the total amount of CBD in suspension derived from the lysis of LNCs with tetrahydrofuran (1:5 (v/v)) and the unassociated CBD filtered with 10 kDa Amicon ® Centrifugal Filters (6000 rpm, 60 min). 2.5. In vitro evaluation of the BBB and glioma targeting ability of the CBD functionalization strategy 2.5.1. CBD decoration of LNCs The fluorescent dye DiO was encapsulated in LNCs for in vitro experiments. To this end, the fluorescent dye was dissolved in the oily core of the LNCs at a weight ratio of 15 mg of dye/g of Labrafac ® WL1349. Pre-formed fluorescently-labeled LNCs were incubated with a CBD solution (15 mg/mL) in a 3:1 (v/v) ratio. The mixture was gently stirred overnight until complete solvent