Anales RANF

Juan Aparicio Blanco, Ignacio A. Romero, Jean P Benoit, Ana I. Torres Suárez @Real Academia Nacional de Farmacia. Spain 202 evaporation. 2.5.2. Uptake experiments Flow cytometry was used to quantitatively evaluate the BBB and glioma targeting ability in vitro . hCMEC/D3 and U373MG cells were separately seeded into 6-well plates. After cells had been confluent for 48 hours, the culture medium was replaced by fluorescently-labeled LNCs at an equivalent dye concentration of 1.65 µg DiO/mL of medium. After 24 hours incubation, cells were rinsed, trypsinized and finally resuspended in 0.3 mL of HBSS. The fluorescence intensity of cells treated with fluorescent-LNCs was analyzed with a flow cytometer (FACScalibur, BD Biosciences). Cells treated with blank LNCs served as control. Confocal microscopy was used to qualitatively illustrate the BBB and glioma targeting ability in vitro . hCMEC/D3 and U373MG cells were separately seeded into chamber slides. After cells had been confluent for 48 hours, the culture medium was replaced by fluorescently- labeled LNCs at an equivalent dye concentration of 1.65 µg DiO/mL of medium. After 24 hours incubation, cells were rinsed and mounted with Vectashield ® with DAPI mounting medium. The cells were then observed with a Leica SP5 microscope (405 nm for DAPI, 488 nm for DiO). Cells treated with blank LNCs served as control. 3D imaging reconstruction was made with IMARIS software. 2.5.3. BBB permeability experiments hCMEC/D3 cells were seeded into collagen- and fibronectin-coated hanging cell culture inserts at confluence and incubated for 72 hours in complete EBM- 2. The monolayer integrity was assessed by determining the permeability coefficient across the hCMEC/D3 monolayer of TRITC-dextran both in the presence and the absence of LNCs. Briefly, a TRITC-dextran solution (2 mg/mL) was added in the apical chamber and at 2, 4, 6, 8, 12 and 24 hours, 200 µL from the basolateral compartment were sampled and replaced with fresh medium. At 24 hours, the apical compartment was likewise sampled (100 µL). Similarly, in a separate experiment, fluorescently- labeled LNCs at an equivalent dye concentration of 1.65 µg DiO/mL were added in the apical chamber to determine the permeability coefficient of the different formulations across the hCMEC/D3 monolayer. The concentration of TRITC-dextran and DiO were determined using a microplate reader (FLUOstar Omega, BMG Labtech; λ exc dextran: 544 nm, λ em dextran: 590 nm, λ exc DiO: 485 nm, λ em DiO: 520 nm). These concentrations were used to calculate the permeability coefficients using the equations from (44). 2.6. In vivo evaluation of the BBB targeting ability of CBD-decorated LNCs For biodistribution studies in healthy mice, DiO was replaced by the fluorescent dye DiD. Mice (n=4-5 per group) were injected via the tail vein with 150 µL of DiD- fluorescently-labeled LNCs. Ninety minutes after administration, mice were sacrificed, and the brain, liver, spleen, kidneys, lungs, heart and blood were collected and homogenized in ethanol for dye extraction. The concentration of DiD was measured using a microplate reader (Varioskan Flash, Thermo Scientific, excitation wavelength: 644 nm, emission wavelength: 665 nm). Results were expressed as percentage of the injected dose per gram of organ. 2.7. In vitro efficacy of CBD-loaded LNCs against U373MG cells 2.7.1. CBD loading into the LNCs core CBD was encapsulated in the oily core of LNCs for in vitro efficacy experiments by dissolving it at a concentration of 15 % CBD/ Labrafac ® WL1349 (w/w). Then, the remaining excipients were added and progressively heated and cooled down around the phase inversion temperature as indicated above. 2.7.2. Cytotoxicity experiments U373MG cells were seeded into 96-well plates. After 48 hours of incubation, cells were treated with LNCs (200 µL) for 48 and 96 hours. Then, the in vitro cytotoxicity of CBD-loaded LNCs was determined using an MTT assay. For each formulation of CBD-loaded LNCs, U373MG cells treated with their blank counterparts served as control. Cell viability was expressed as a percentage relative to that of control. The half-maximal inhibitory concentration (IC 50 ) was calculated in each case. 2.8. Statistical analysis The data are expressed as mean ± SD of at least three different experiments. Unpaired Student’s t test was used for two-group comparisons. One-way ANOVA followed by post-hoc Tukey test were used for multiple-group analysis. Statistical significance was fixed as *: p<0.05, **: p<0.01, ***: p<0.001. All the data were analyzed using the GraphPad Prism 7 software. 3. RESULTS AND DISCUSSION 3.1. Determination of the parameters that control the size distribution of LNCs prepared by the PIT method The parameters that control the properties of nanocarriers can be classified into formulation or preparation variables. For low-energy methods (as it is the case of the PIT method), the formulation variables, and particularly the relative proportion of excipients, are the key parameters, as these methods do not rely either on physical energy input or on shear forces. Since Morales et al (45) showed that water only acts as a dilution medium for the dispersed phase, we hypothesized that surfactant and oil should be considered the formulation-driving parameters. In this respect, particle size is expected to be reduced with increasing amounts of surfactant due to the decrease in interfacial tension and to grow with increasing amounts of oil, as it constitutes the liquid core of the nanocapsules. As a result, the oily phase/surfactant mass ratio seems to be a suitable variable for prediction of the particle size of LNCs prepared by the PIT method. Effectively, as shown in Figure 1 and Table 2, we have