1. Introduction
Cannabidiol (CBD) is a nonpsychoactive phytocannabinoid naturally found in
Cannabis sativa L. During the past decade, Cannabis extracts and CBD in particular have received attention in food, pharmaceutical, and cosmetic applications due to their numerous benefits. Several skin benefits have been reported for Cannabis extracts, including anti-inflammatory, antioxidant, antiaging, and cytoprotective properties without psychoactive effects [
1,
2]. CBD has been used to treat numerous skin conditions, including urticaria, persistent psoriasis, acne, and epidermolysis bullosa [
3,
4,
5]. As an antioxidant, CBD regulates the redox states of cells by reducing the formation of reactive oxygen species (ROS) and raising the level and activity of both nonenzymatic and enzymatic endogenous antioxidants at the transcriptional level. Additionally, CBD directly protects against functioning antioxidants [
6,
7]. Recently, Giacoma et al., revealed that hemp water extract containing CBD, cannabidiolic acid, and rutin protected human fibroblasts and keratinocytes from cytotoxicity and apoptosis induced by oxidative stress. In addition, hemp water extracts reduced hydrogen peroxide-induced L-dopa turnover, prostaglandin-E2 production, and the ratio of kynurenine/tryptophane in isolated rat skins, indicating anti-inflammatory and antioxidant effects [
6]. Furthermore, CBD treatment of fibroblasts and keratinocytes from psoriatic patients intensified some changes (phospholipid content and membrane charge) caused by radiation and prevented structural and functional changes in healthy skin membranes during phototherapy [
2].
CBD is a highly lipophilic molecule with a log P of 6.3 and a molecular weight of 314.46 g/mol [
8,
9]. Therefore, CBD tends to accumulate in the upper skin layer and permeates poorly into deeper skin layers due to its high lipophilicity [
8]. Junaid et al. reported the effects of solvent, drug concentration, chemical enhancer, and essential oil on the percutaneous absorption of CBD [
9]. The total amounts of CBD absorbed (CBD in the skin and receptor medium) from 5% (242.41 ± 12.17 µg/cm
2) and 10% (232.79 ± 20.82 µg/cm
2) CBD solutions after 24 h were comparable but significantly higher than that of a 1% CBD solution (23.02 ± 4.74 µg/cm
2). In addition, the enhancement of skin penetration after 4 h was observed when using oleic acid (5%
w/
w) as a chemical enhancer but was not found when using Transcutol
® P (40%
w/
w) and isopropyl myristate (10%
w/
w) [
9]. Moreover, 5.0%
w/
w peppermint oil or eucalyptus oil did not enhance the skin absorption of CBD. In addition, Stinchcomb et al. found that ethanol concentrations between 30 and 33% significantly increased the in vitro human skin transdermal flux of Delta-8-tetrahydrocannabinol and CBD due to an increase in the CBD solubility [
10]. CBD can be degraded by light, temperature, and oxidation [
11,
12]. Therefore, the development of innovative formulations is essential for improving CBD stability and permeability.
Several drug delivery systems have been used to deliver and enhance the stabilities of drugs, such as liposomes, niosomes, ethosomes, polymeric nanoparticles, and lipid nanoparticles [
13,
14,
15,
16,
17,
18]. Lipid nanoparticles, including solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs), are among the nanoencapsulation materials used to protect and enhance the stabilities of labile drugs [
19,
20]. They can be used to encapsulate lipophilic, hydrophilic, and amphiphilic drugs. However, particularly high encapsulation efficiencies were reported for lipophilic drugs. The lipid matrices of SLNs are composed of a solid lipid, whereas those of NLCs are prepared from a combination of a solid lipid and oil(s). NLCs have been proven to overcome some drawbacks of SLNs prepared from solid lipids alone, for example, low drug payload, unexpected dynamics of polymorphic transitions, and drug expulsion during storage. The incorporation of oil into the lipid matrix leads to imperfections in the crystals of the lipid matrix, which provides more space for drug accommodation. Previous researchers found that varying the solid lipid, oils, and surfactants altered the characteristics of the lipid nanoparticles [
21,
22].
Recently, positively charged NLCs containing CBD were prepared by hot microemulsion using stearic acid (1.25%) and OA (0.75%) as the lipid matrix and stabilized with cetylpyridium chloride (0.05%) and Span 20 (0.25%) [
23]. An encapsulation efficiency (E.E.) and a drug loading of 99.99% and 18.75%, respectively, were reported. The in vitro release studies showed a biphasic release pattern with a burst release (50% of CBD released in 5 min), followed by slow and sustained release. The cumulative percentages of CBD released from CBD-NLC dispersion and CBD-NLC gel were comparable. The burst release of CBD from NLCs was explained by the enrichment of CBD on the surface layers of the NLCs. However, the physicochemical properties of NLCs also depend on the compositions, such as emulsifier and oil types [
19,
20,
21,
22]. Therefore, it was of our interest to investigate the effect of emulsifier (TG450 and P188) and different oils (Transcutol P, MCT, and OA) on the properties and performance of CBD-loaded NLCs.
In this study, we aimed to develop NLCs for topical/dermal applications. Cetyl palmitate (CP) was used as a solid lipid for the CBD-NLCs. To study the effects of formulation compositions on the performance of CBD-NLCs, Tego® Care 450 (TG450) or poloxamer 188 (P188) was used as a surfactant, and three different oils were used to prepare CBD-NLCs, including Transcutol® P, medium chain triglycerides (MCT), and oleic acid (OA). The physicochemical properties of CBD-NLCs were evaluated and compared in terms of particle sizes, size distributions, zeta potentials, crystallinities, encapsulation efficiencies (E.E.), and polymorphism. The stabilities of the CBD extract in the NLCs were evaluated after exposure to simulated sunlight. Furthermore, the in vitro skin performance of CBD was evaluated for release and skin permeation by using Strat-M® as a skin model. Cell cytotoxicity was determined in keratinocytes and fibroblasts, while the anti-inflammatory study was evaluated with lipopolysaccharide (LPS)-induced RAW264.7 cell macrophages.
2. Materials and Methods
2.1. Materials
Cetyl palmitate (CP) was obtained from SABO S.p.A. (Levate (BG), Italy). Labrafac lipophile WL1340 (medium chain triglycerides, MCT) and Transcutol® P (diethylene glycol monoethyl ether) were gifts from Gattafossé (Cedex, France). Tego® Care 450 (TG450, polyglyceryl-3 methylglucose distearate) was obtained from Evonik Industries AG (Essen, Germany). Poloxamer 188 (P188) was provided by BASF (Ludwigshafen, Germany). Tween 20 and oleic acid (OA) were obtained from Croda (Singapore). CBD extract (87% CBD purity) was purchased from Greenleaf (Bangkok, Thailand). Lipopolysaccharide from Escherichia coli O55:B5 was obtained from Sigma-Aldrich (Missouri, USA). Sodium chloride was acquired from Carlo Erba Reagenti (Cornaredo MI, Italy). Phosphoric acid and potassium dihydrogen orthophosphate were obtained from Fisher Scientific (Loughborough, UK). Methanol was of HPLC grade.
2.2. Preparation of CBD-Loaded NLCs
CP was used as a solid lipid matrix, and TG450 or P188 was used as surfactants. The addition of Transcutol
® P, MCT, or OA as an oil into a solid matrix was performed to prepare the CBD-NLCs. All lipid nanoparticles were produced by the hot high-pressure homogenization technique (HPH) [
24]. In the preparation, the lipid phase containing a solid lipid, oil, and CBD extract (1.0%
w/
w) was melted at between 75 °C and 80 °C. Then, the hot aqueous surfactant solution (TG450 or P188) at 80–85 °C was added to the melted lipid phase under stirring by an Ultra-Turrax
® T25 (IKA, Staufen, Germany) at 8000 rpm for 1 min. Subsequently, the pre-emulsion was then placed in the high-pressure homogenizer (APV Gaulin, Lübeck, Germany) for three cycles at 500 bar. The hot nanoemulsions were subsequently cooled to room temperature under ambient conditions, generating lipid nanoparticles. Finally, Unigerm G2 was added to the CBD-NLC dispersions to preserve formulations from microorganisms. The compositions of all prepared formulations are shown in
Table 1.
2.3. Particle Sizes and Size Distributions
The mean particle size and the particle size distribution (polydispersity index, PDI) were determined by photon correlation spectroscopy (PCS) using a Zetasizer NanoZS (Malvern Instruments, Worcestershire, UK). The sample was diluted with sterile water for injection to obtain a suitable scattering intensity. The mean particle size and PDI were obtained by averaging the values from three measurements at an angle of 173° in a 10 mm diameter cell at 25 °C.
2.4. Zeta Potential
The zeta potential (ZP), also known as the electrokinetic potential, is defined as the value of the electrical potential at the shear plane of a particle and indicates the physical stability of a colloidal system. The ZP was determined with a Malvern Zetasizer NanoZS by averaging the results of three measurements performed at 25 °C. The ZP values were calculated using the Helmholtz–Smoluchowski equation. To reduce the fluctuations in water conductivity between days, all samples were diluted with distilled water and adjusted the conductivity to 50 S/cm with a 0.9% sodium chloride solution before the measurement.
2.5. Particle Morphology
The particle morphologies of CBD-NLCs were examined with a transmission electron microscope (TEM) (Hitachi model HT-7700, Japan). One drop of CBD-NLCs was deposited on a copper grid after being diluted with filtered water and negatively stained with 1% uranyl acetate. The test samples were air-dried at room temperature for one hour. The CBD-NLC grids were stored in a desiccator until they were used.
2.6. Drug Encapsulation Efficiency (E.E.)
To determine the amount of CBD entrapped in NLCs, the ultrafiltration technique was used to separate CBD in lipid nanoparticles and free CBD in the filtrate. Briefly, approximately 1 g of lipid nanoparticles was added onto the filter membrane of the ultracentrifuge tube with a molecular weight cutoff of 50 kDa (Amicon
® Ultra15 centrifugal filter, Merck KGaA, Darmstadt, Germany). Then, the separation of free CBD and CBD entrapped in lipid nanoparticles was performed by centrifugation at 4000 rpm for 60 min with a Centrifuge Model 5430 (Eppendorf, Germany). Afterward, the free CBD in the filtrate was analyzed by high-performance liquid chromatography (HPLC) using a Shimadzu LC-20AD system (Shimadzu, Japan). The stationary phase was Platisil ODS (5 µm, 250 mm × 4.6 mm, Dikma, CA, USA). The mobile phase consisted of methanol and water (85:15
v/
v). The flow rate of mobile phase was 1.2 mL/min. The wavelength for detection was set at 220 nm. The injection volume of the standard and test samples was 20 µL. The E.E. of CBD in NLCs was calculated with Equation (1).
2.7. Thermal Analysis
The thermal behaviors of CP, CBD extract, and CBD-NLCs were analyzed with a Mettler DSC1 apparatus (Mettler Toledo, Switzerland). CBD-NLCs samples weighing approximately 10–20 mg (related to 1–2 mg of lipid content) were placed into a 40 µL aluminum pan and covered with an aluminum pan lid. The sample was heated from 20 °C to 85 °C and then cooled to 20 °C with a heating/cooling rate of 5 °C/min and a nitrogen flushing rate of 80 mL/min. An empty aluminum pan covered with an aluminum pan lid was employed as a reference. The melting temperatures (T
m), melting enthalpies (∆H), and onset temperatures (T
onset) were analyzed using STAR
e Software version 16.10. The degree of lipid crystallinity was calculated as the percentage of CI (%CI) according to Equation (2):
2.8. X-ray Diffraction (XRD)
The polymorphism forms of the lipid matrix were also assessed by utilizing a wide-angle X-ray diffractometer (Miniflex 600, Rigaku, The Woodlands, TX, USA) with a copper anode (Cu-Kα radiation, 40 kV, 15 mA, λ = 1.54056 A°) and a goniometer as a detector. The test samples (i.e., bulk CP, CBD extract, and CBD-NLCs) were set up on a quartz sample holder. The CBD-NLCs were mixed with locust bean gum to form a paste before the measurements. The experiments were performed with a scan rate of 0.02°/min within the diffraction angle range.
2.9. Photostabilities of CBD-NLCs
The stabilities of CBD entrapped in NLCs were evaluated by exposure to simulated direct sunlight. The samples were kept in clear glass vials. The photostabilities were determined using a Q-Sun Xe-1 Xenon Test Chamber connected to a cooling system (Q-Lab Corporation, Westlake, OH, USA). The Daylight-BB optical filter was used to generate outdoor sunlight. The temperature was controlled at 30–35 °C. The CBD remaining in the NLCs was analyzed after exposure to 100, 300, 500, 700, and 1000 kJ/m2 of simulated sunlight. To determine the amount of CBD remaining in NLCs, the CBD-NLC dispersion after exposure to simulated light was added to a volumetric flask, then methanol was added and sonicated for 15 min to extract CBD from NLCs. After cooling down to room temperature, the solution was filtered through a 0.22 μm cellulose acetate syringe membrane filter (Filtrex, Milano, Italy) and analyzed by HPLC as previously described. One percent CBD extract in absolute ethanol was used as a reference for comparison.
2.10. In Vitro Release Study
In vitro release studies of CBD-NLCs were performed using static Franz diffusion cells (7 mL, surface area 0.60 cm2, DHC-6T, Logan Instruments Corporation, New Jersey, NJ, USA). A polycarbonate membrane filter with pore sizes of 0.05 µm (SterliTech, Washington, DC, USA) was mounted between the donor and receptor compartments. The temperature was controlled at 32 ± 0.3 °C. The receptor medium was a 2% Tween 20 solution (pH~5.0–5.5), and it was continuously stirred with a magnetic stirrer at 500 rpm. The 300 µL test samples were added to the donor compartment. At each time point (2, 4, 6, 8, 10, 12, and 24 h), 500 µL of the receptor medium was collected and replaced with fresh receptor medium. As previously stated, the quantity of CBD in the receptor phase was determined using HPLC. The experiments were performed in triplicate for each formulation.
To evaluate the release kinetics of the CBD-NLCs, the cumulative amount of CBD per area (µg/cm
2) in the receptor phase was plotted as a function of time and fitted to kinetic models, including zero-order kinetics, first-order kinetics, Higuchi’s kinetics, and Korsmeyer–Peppas kinetics [
25], as described in Equations (3)–(6):
where M
t and M
0 are the amounts of drug released at time t and at time = 0, respectively, k is the release rate constant, M
t/M
α is the fraction of drug released, and n is the diffusional release exponent, which is indicative of the release mechanism.
2.11. In Vitro Skin Permeation Study
In vitro skin permeation studies of CBD-NLCs were conducted with static Franz diffusion cells (7 mL, surface area 0.60 cm
2, DHC-6T, Logan Instruments Corporation, Somerset, NJ, USA) with a Strat-M
® membrane (EMD Millipore, Burlington, MA, USA) mounted between the donor and receptor compartments, with the shiny side of the Strat-M
® membrane in contact with the donor compartment [
26]. The temperature was controlled at 37 ± 0.3 °C. The receptor compartment was filled with 2% Tween 20 in a phosphate buffer (pH~7.4) and stirred with a magnetic stirrer at 500 rpm. The membrane was equilibrated at 37 ± 0.3 °C for 30 min before the test samples (300 µL) were added to the donor compartment. At predefined intervals (2, 4, 6, 8, 10, 12, and 24 h), 500 µL of the receptor medium was withdrawn and replaced with new receptor medium. The amount of CBD in the receptor phase was measured using HPLC. The experiments for each formulation were performed in triplicate. The cumulative amount of CBD permeated through Strat-M
® membrane from each formulation was compared.
2.12. Cytotoxicity in Skin Cells
To evaluate the cytotoxicities of the CBD extract and CBD-NLCs, the test samples were assessed using mitochondrial reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] or MTS (CellTiter 96® AQueous One Solution Reagent, Promega, Fitchburg, WI, USA) in the human foreskin fibroblast cell line (HDF) (ATCC® PCS-201-012) and the human immortalized keratinocyte cell line (HaCaT) (CLS Cell Lines Service GmbH, Germany).
2.12.1. Cell Preparation
First, the cells were cultured in a 75 cm3 tissue culture flask using culture media containing 10% FBS and 90% DMEM with 1 mM sodium pyruvate, 100 U/mL penicillin G, and 100 g/mL streptomycin. The culture media was replaced every 48 h until the cells were at approximately 80% confluence in a humidified incubator (5% CO2 at 37 °C). Then, a 0.05% trypsin/EDTA solution was used to trypsinize the cells. Subsequently, approximately 10,000 fibroblast cells or 8000 keratinocyte cells were seeded in each well of a flat transparent 96-well plate at a volume of 100 µL per well.
2.12.2. MTS Cell Proliferation Assay
The cytotoxicities were determined with the MTS assay, as previously described by Wang et al. [
27] and Riebeling et al. [
28] with some modifications. After seeding for 24 h, the well-plates were treated with different concentrations of samples. The cells treated with the culture medium were used as a control (untreated). After 24 h of incubation, the supernatant was removed, and the cells gently rinsed with PBS (0.01 mM, pH 7.4) twice. Then, 120 µL of culture medium containing MTS solution at a ratio of 5:1 (
v/
v) was added and incubated (5% CO
2 at 37 °C) for 3 h. After 3 h of incubation, the formation of soluble formazan was evaluated by the colorimetric method at 490 nm using a microplate reader (CLARIOstar Plus, BMG Labtech, Ortenberg, Germany). Finally, cell viabilities after the different treatments were calculated by comparing the treated cells to the untreated cells as in Equation (7):
where A and B are the absorbance of treated and untreated samples, respectively.
2.13. Anti-Inflammatory Study
2.13.1. Cell Culture Preparation
The anti-inflammatory activities of the CBD extract and CBD-NLCs were evaluated in RAW264.7 cell macrophages (ATCC TIB-71TM) by determining the in vitro production of interleukin-6 (IL-6). RAW 264.7 cell macrophages were cultured in DMEM supplemented with 10% FBS, 1% NEAAs, and 1% penicillin/streptomycin (P/S). Cells were incubated at 37 °C in 5% CO2 until 80% confluence was observed.
2.13.2. Cell Viability
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine cell viability. In brief, RAW264.7 cell macrophages were seeded in a 96-well, flat-bottom cell culture plate at a concentration of 1 × 104 cell/well in 180 µL of cell culture medium, and the cells were incubated overnight at 37 °C under 5% CO2. Afterward, the cells were treated with the test samples in triplicate at different concentrations for 24 h. Then, the culture medium was removed and replaced with 100 µL of MTT solution. After 30 min of incubation, the MTT solution was removed by aspiration. The insoluble formazan in RAW 264.7 cell macrophages was extracted using 180 µL of DMSO, and the optical density (OD) was detected at 570 nm with a microplate reader. The cell viability was computed according to Equation (7).
2.13.3. Measurement of IL-6 Production
CBD extract and CBD-NLCs with concentrations showing cell viabilities of higher than 80% were incubated with RAW264.7 cell macrophages as previously described by Teeranachaideekul et al. [
29] with some modifications. The cells were plated in a 24-well plate at a concentration of 1 × 10
5 cells/well and incubated at 37 °C under 5% CO
2 for 24 h. After that, the culture medium was removed and replaced with fresh culture medium containing test samples for 2 h. Then, 1 µg/mL lipopolysaccharide (LPS) was added to each well for 24 h of incubation at 37 °C under 5% CO
2. RAW264.7 cell macrophages treated with and without LPS were used as positive and negative controls, respectively. Diclofenac sodium at the concentration of 1000 μM was used as a standard anti-inflammatory agent [
29]. The concentration of IL-6 in culture media was determined using ELISA MAX
TM Standard Set Mouse IL-6 (Biolegend, California, USA) according to the manufacturer’s instructions in a 96-well plate.
2.14. Statistical Analysis
All data are expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS version 18. One-way ANOVA was applied to determine significant differences among samples. The p-value for a significant difference was set at less than or equal to 0.05 (p < 0.05).