2.1. Materials
CBD was sourced from PM Separations in Queensland, Australia, and had a purity of ≥98%. Glyceryl distearate (Precirol® ATO 5) was obtained from Gattefosse in Lyon, France. Hydroxyethyl cellulose NF was provided by Medisca (NY, USA). Sigma-Aldrich in New South Wales, Australia provided polyethylene glycol 400, Tween 80, and liquid oil capric/caprylic triglycerides. Deionised water with a resistivity of 18.2 MΩ at 25 °C was used to prepare the formulations and all chemicals were of the highest commercial grade available.
2.2 HPLC method for quantification of CBD
HPLC (Shimadzu Corporation, Kyoto, Japan) equipped with a degasser (DGU-20A3), an autosampler (SIL-20A HT), a pump (LC-20ADXR), and a photodiode array detector (PDA) (SPD-M20A) was utilized to analyse CBD. A Luna 5 µm C8(2) 100 Å column (250 × 4.6 mm) was used. The mobile phase consisted of acetonitrile and water (80:20 v/v). The flow rate and injection volume were 1.0 mL•min -1 and 10 μL. The peak was detected at 7.9 minutes with the help of a PDA detector using a wavelength of 210 nm.
2.3 NLCs preparation and optimization
Solid lipids including Gelucire 48/16, Precirol® ATO 5, Stearic acid, Compritol® ATO 888, Dynasan® 116, and Dynasan® 118 were considered for their suitability to prepare NLCs of CBD. Precirol® ATO 5 was selected due to its relatively lower melting point (54°C) and effectiveness to produce the best cannabinoid-loaded lipid nanoparticles43. Furthermore, GDS has been shown to effectively mask the taste of bitter drugs44. Similarly, caprylic/capric oil was selected as a liquid oil due to better stability of CBD in medium-chain triglyceride. Calvi et al. demonstrated the absence of any lipid oxidation products when CBD was dissolved in medium-chain triglycerides (MCT) illustrating MCT oil matrices were less prone to oxidative degradation compared to hemp seed oil or olive oil 45. Tween 80 was used as a surfactant due to its lower irritation to the cell membrane, low toxicity, widespread use in the pharmaceutical field and success in preparing NLCs21 46.
The NLCs were prepared by hot emulsification-ultrasonication method47. Briefly, lipid phase (GDS and Caprylic/Capric oil 70:30 %w/w) was heated to 70˚C (5°C above the melting point of GDS). The aqueous phase was simultaneously prepared by mixing the surfactant (Tween 80) with de-ionised water and heating to the same temperature as the oily phase. Subsequently, the aqueous phase was poured into the lipid phase under continuous shaking and the mixture was exposed to ultrasonication (60 % amplitude, 20s on-off) (QSonica Q500, CT, USA) to form the NLCs (Fig.3). The mixture was stored in a refrigerator (4 °C) prior to the preparation of the buccal film. To produce lyophilized NLCs, the blend was cooled in a freezer at -80°C for 1h and then subjected to lyophilization using a freeze dryer (Lyph-Lock® 6, Labconco, Kansas, USA) for 48 hours at a pressure of 0.06 mbar and temperature of -45°C. This process was used to produce a blank dispersion (without CBD) and a dispersion of NLC containing CBD at a concentration of 2% (w/w). Table 1 provides details of the composition of these dispersions.
Table 1. Placebo and CBD-loaded NLCs composition
Design of Experiments (DoE) was utilized to screen and optimize the concentration of different ingredients and processing parameters. The Three-factor Box-Behnken Design was selected for the optimisation of the formulation and analysis of the effect of independent factors on dependent factors, using the Design Expert software version 13. The Box-Behnken design was preferred due to its ability to analyse quadratic response surfaces and polynomial models with the minimum possible number of runs48. The studied independent variables were the total lipid concentration (% w/v TL), surfactant concentration (v/v %), and ultrasonication time (min) at three levels (-1, 0, +1). The dependent variables analysed were particle size (Y1) and polydispersity index (Y2) (Table 2). The ratio of solid to liquid lipid (oil) was kept constant at 70:30 throughout the study. Seventeen blank NLC formulations were prepared, and the optimised formulation was utilized to prepare CBD-loaded NLCs. The significance of the effects, lack of fit, and their interactions were evaluated using a significance level of 95% (α = 0.05)41.
Table 2. Variables selected for the preparation of CBD-NLCs
The generated quadratic model for the design expert generated 17 runs is shown below.
Y= F0 + F1X1 + F2X2 + F3X3 + F12X1X2 + F13X1X3 + F23X2X3 + F11X12 + F22X22+ F33X32
In the multiple regression equation, Y represents the dependent variable, d0 is the intercept, and d1 to d33 represent the regression coefficients calculated from the observed responses of the independent variables X1 to X3 at coded levels. X1 represents the solid-to-liquid lipid ratio, X2 represents surfactant concentration, and X3 represents ultrasonication time.
2.4 In vitro characterisation of prepared CBD-loaded NLCs
2.4.1 Zeta potential, particle size, and polydispersity index
DLS was employed to determine the average polydispersity index (PDI, particle size, and zeta potential of the samples, using a zetasizer (Malvern Instruments, UK) at a temperature of 25°C. A 100-fold dilution of all samples was prepared using deionized water and then injected into a disposable. The zeta potential was measured for both the optimized formulation and CBD-loaded NLC. All measurements were carried out in triplicate (n = 3)49.
2.4.2 Drug loading (DL%) and Entrapment efficiency (EE %)
The technique used for determining the entrapment efficiency (EE) and drug loading (DL) was based on ultrafiltration/centrifugation30. To achieve this, CBD-NLC was introduced into AmiconⓇ (50-KD cut-off) ultrafiltration devices and centrifuged at 3400 rpm for 30 minutes. The NLCs held on the filter were washed three times to eliminate any free drug, and the HPLC method described above (Section 2.2) was used to determine the quantity of CBD in the filtered pool (free drug). Total amount of CBD was determined by first dissolving an aliquot of the NLCs in simulated salivary fluid and analysing using HPLC. Equations 1 and 2 were used to calculate the EE (%) and DL (%) respectively.
2.4.3 Desirability and optimization
The optimization of CBD-loaded NLCs involved the utilization of numerical optimization and the desirability function approach. The main aim was to obtain NLCs with the smallest possible particle size and PDI. To determine the optimal values for the independent variables, the desirability function method was employed. This approach entailed evaluating the desirability index for each response variable and then combining all response variables into a single desirability function that ranged from 0 to 1, indicating the ideal values of the independent parameters50.
2.5 Feed preparation and 3D printing of CBD-NLCs film
Among the evaluated mucoadhesive polymers, HEC-based formulation resulted in a good film, upon visual inspection and was used for preparing CBD-NLCs loaded buccal film using 3D printing. Briefly, the gel was prepared by dissolving 8% of HEC (H) and 2.4% PEG (Mw~ 400) in water. First, PEG was dissolved in water heated to 60 °C. The separately prepared CBD-NLC was added to the heated solution bit by bit under continuous stirring. Finally, HEC was added to the formulation and stirred until a uniform solution was formed (Fig. 4b).
A square film (20 x 20 mm², thickness=1mm) was designed using Autodesk Inventor® Professional 2021 software. The resulting designs were saved in. stl format and converted into G-code files, which were readable by the 3D printer software. PAM (Bio X, Cellink, Gothenburg, Sweden) was used to manufacture the film. Approximately 2 mL of the formulation was loaded into the printer cartridge using a 5 mL syringe. Printing was carried out at a nozzle speed of 2 mm/s and a pressure of 90 kPa using a 25 G bioprinter nozzle. The films were subsequently dried for 48 h at room temperature, protected from light (Figure 4a, b).
2.6 Characterization of the optimised CBD-NLCs and 3D-printed CBD film
2.6.1. Physical appearance
Smoothness and homogeneity were assessed for the printed films, followed by the characterization of physicochemical properties and release kinetics. The thickness and weight of the films were determined after drying them at room temperature for 48 hours using a digital micrometer and weighing balance, as outlined by Bala et al51.
2.5.2. Mechanical characteristics
A texture analyzer (Stable Micro Systems, Godalming Surrey, UK) was used to evaluate the elongation at break and tensile strength (TS). The films were pulled apart, at the loading length of 200 mm, until breakage occurs by moving the upper clamp at a rate of 1 mm/s. The lower clamp remains stationary. The measurement was done in triplicate.
2.5.3 Fourier transform infrared spectroscopy (FTIR)
FTIR-attenuated total reflectance spectra of the CBD, GDS, lipid mix (GDS +Caprylic oil), HEC, physical mixture (CBD, GDS, lipid mix and HEC), blank and CBD loaded film were obtained using FTIR spectrometer (Bruker, Massachusetts, USA). The spectra were recorded at room temperature in a range of 4000 to 450 cm−1 in transmittance mode using 4 scans per analysis at a resolution of 4.0 cm−1. A small portion of the films or powder was placed on ATR diamond crystal followed by application of force with the use of the clamp to ensure adequate contact of the sample with the crystal.
2.5.4 Differential scanning calorimetry (DSC)
DSC measurements of CBD, GDS, lipid mix (GDS +Caprylic oil), HEC, physical mixture (CBD, GDS, lipid mix and HEC), blank and CBD loaded film were taken in Discovery DSC 2920 (TA Instruments (New Castle, USA) calibrated with an indium standard. Samples weighing 4.0 ± 0.5 mg were put in aluminum pans followed by recording of thermal profiles by heating the samples from 25 - 250°C at a rate of 10 °C/min while continuously flowing nitrogen gas.
2.5.5. Scanning electron microscopy (SEM)
The morphology of the films and pure drug were evaluated using a Zeiss Merlin Field-Emission Dispersive X-Ray Spectroscopy (Jena, Germany) operating at an accelerating voltage range of 2 - 5 KV, after sputter-coating with platinum.
2.5.6 Film thickness and dry weight
Thickness of the film was determined by measuring five locations (four corners and one center) using a digital micrometer (ID-S1012, Mitutoyo, Japan) as described by Bala et al52. Dry weight of the film was determined by randomly cutting four pieces (0.64 cm2) and weighing them using a digital balance.
2.5.7 Surface pH
The surface pH of each film (n=3) was measured by adding a drop of MilliQ water to the surface and measuring with a pH meter (Orion Star A121, Thermo Scientific, USA)51.
2.5.8 Folding endurance
The folding endurance was assessed by continually folding each film at the same spot until breakage and recording the total number of folds.
2.5.9 Drug loading
To determine the drug loading, films (20*20 mm2) were placed in a Falcon tube containing a hydro-alcoholic solution (10 mL, 50:50 v/v) maintained at 37°C for 1 hour. The solution was then centrifuged at 3,000 rpm for 5 min, filtered, and analysed using HPLC.
2.5.10 In vitro release experiments
The method used to determine the in vitro release of CBD from the buccal film was similar to the one reported by our research group earlier53. The film was placed in a Falcon tube with 10 mL of simulated salivary fluid (SSF) and kept in a shaking water bath (Julabo SW22, Germany) at 37 ± 0.5 °C while being stirred at 50 rpm. At fixed time intervals of 10, 20, 30, 45, 60, 90, 120, 180, 240 and 360 min, 1 mL aliquots of the sample were withdrawn and an equal volume of fresh SSF was replaced. HPLC was used to analyse the drug content in the withdrawn samples.