Cannabidiol Loaded Nanoemulsion with Improved Bioaccessibility and Anti-Inflammatory Activity

Abstract

Oral delivery of cannabidiol (CBD) is challenging because of its poor water solubility and low bioavailability, which restrict its therapeutic potential. In this study, we used a CBD-loaded oil-in-water nanoemulsion prepared from octenyl succinate anhydride (OSA)-modified starch to address these challenges. The CBD-encapsulated nanoemulsion (CBD-NE) exhibited a uniform particle size of 39.18 ± 0.15 nm, high encapsulation efficiency of 99.80 ± 0.13%, and maintained colloidal stability during storage for 28 days. Simulated gastrointestinal digestion demonstrated that the bioaccessibility of CBD in CBD-NE was higher than that in unformulated CBD. Furthermore, in vitro experiments using RAW264.7 macrophages showed that CBD-NE significantly (p < 0.05) suppressed lipopolysaccharide-induced secretion of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α. Collectively, the improved bioaccessibility and potent anti-inflammatory efficacy of CBD-NE highlight its potential for therapeutic and nutraceutical applications.

1. Introduction

Cannabidiol (CBD; Figure ), a major nonpsychoactive constituent of Cannabis sativa L., has attracted increasing interest due to its diverse pharmacological effects, such as antiepileptic, neuroprotective, anti-inflammatory, and antioxidant properties. − The recent FDA approval of a CBD-based oral formulation for refractory epilepsy underscores its increasing therapeutic relevance. Despite its therapeutic potential, the practical application of CBD–particularly via the oral route–remains challenging. This limitation primarily stems from CBD’s intrinsically poor water solubility, reported as 0.023–0.1 μg/mL and its high lipophilicity (log P ≈ 6.3). , As a consequence, CBD exhibits poor dissolution in gastrointestinal (GI) fluids, leading to low oral bioavailability across species. Reported values include 6.5% in humans after a 3000 mg oral solution dose, 2.8% in rats following a single 10 mg/kg oral gavage, and 13–19% in dogs after a 180 mg oral dose. These issues not only hinder effective drug delivery but also limit the translation of its pharmacological benefits to clinical practice.

1.

Molecular structure of CBD.

To date, a variety of formulation approaches have been explored to improve the aqueous solubility and oral absorption of CBD. For instance, nanoliposome-based carriers have been used to enhance the solubility, stability, and modulated GI release of CBD. Nevertheless, liposome formations involve complex processing and often encounter stability and scalability challenges during large-scale production. Similarly, a self-emulsifying drug delivery system has been introduced to enhance oral bioavailability of CBD, although issues related to excipient selection, formulation complexity, and the risk of drug precipitation upon dilution in the GI tract remain problematic. Additionally, an oil-in-water emulsion with synthetic surfactants such as Tween 80 has been employed to improve the stability and bioaccessibility of CBD. However, the use of synthetic surfactants may pose potential risks related to long-term safety and regulatory compliance, particularly in pharmaceutical formulations. These limitations highlight the need for alternative delivery approaches to improve the stability, biocompatibility, and practical utility of CBD.

Selection of a suitable stabilizer for emulsion systems plays a critical role in determining both physical stability and biocompatibility. Octenyl succinic anhydride-modified starch (OSA-S), a food-grade biodegradable polysaccharide, has emerged as an efficient emulsifier for stabilizing oil-in-water emulsions. OSA-S offers the dual benefits of high emulsification efficiency and safety, making it suitable as formulation of delivery systems for pharmaceutical applications. Moreover, recent studies have demonstrated that OSA-S-based emulsions can significantly enhance the dispersibility and GI stability of hydrophobic compounds. − Based on the current evidence, no previous studies have systematically explored the use of OSA-S-based emulsions for CBD delivery.

In this study, we formulated a nanoemulsion system stabilized by OSA-S to improve the oral bioaccessibility of CBD. The physicochemical properties, encapsulation efficiency (EE), loading efficiency (LE), and storage stability of the formulation were characterized. Furthermore, the in vitro bioaccessibility following simulated GI digestion and anti-inflammatory activity in a macrophage cell model were evaluated. This study offers insights into the potential therapeutic applications of CBD by overcoming its inherent bioavailability.

2. Materials and Methods

2.1. Formulation of CBD-Encapsulated Nanoemulsion (CBD-NE)

The preparation of CBD-NE followed a previously established protocol with slight adjustments. To formulate the oil phase, 5 mg of CBD (Purity: 99.2%; #APICBDISO15, Averix Bio, NC) was dissolved in 5 g of sunflower oil. The aqueous phase was obtained by dispersing 5 g of OSA-S (Purity Gum 1773, Ingredion Korea Inc., Incheon, Korea) in 45 mL of distilled water. The two phases were combined and the resulting mixture was emulsified (500 W, 20 kHz) using a CP-505 ultrasonic device (Cole-Parmer, Vernon Hills, IL). Ultrasonication was performed at 30% amplitude for three cycles, each lasting 60 s. A blank nanoemulsion system (blank-NE) without CBD was prepared using the same method. The final concentration of CBD in the CBD-NE was calculated to be approximately 318 μM. CBD-NE and blank-NE were independently produced at least three times to verify the experimental consistency. To ensure microbial stability during the 28-day storage period, the emulsions were supplemented with 0.02% (w/v) sodium azide (S2002, Sigma-Aldrich, St. Louis, MO). However, sodium azide was not included in the samples used for cell viability or anti-inflammatory assays.

2.2. EE and LE of CBD-NE

The EE of CBD-NE was evaluated by measuring the concentration of unencapsulated CBD in the emulsion. To separate the unencapsulated free CBD, the emulsion solution was centrifuged at 15,000g for 30 min to acquire the supernatant. The collected supernatant was then combined with ethanol in a 1:1 volume ratio, vortexed for 2 min, and filtered through a 0.45 μm PTFE membrane filter. The resulting filtrate was analyzed using high-performance liquid chromatography (HPLC).

The CBD content in the filtrate was determined using HPLC equipped with a G1329A autosampler, G1311A pump, G1322A degasser, G1316A column oven, and G1315D diode array detector (Agilent Technologies, Santa Clara, CA). Chromatographic separation was carried out on a Zorbax Eclipse XDB-C18 column (250 mm × 4.6 mm i.d., 5 μm; Agilent Technologies), with the column temperature set at 30 °C. The mobile phase was composed of 0.1% formic acid in water (A) and pure acetonitrile (B), and the following gradient elution program was applied: 0–3 min, 5% B; 3–20 min, 5–100% B; 20–25 min, 100%; 25–26 min, 100–5%, 26–30 min, 5% B. The analysis was performed at a flow rate of 1.0 mL/min, with an injection volume of 20 μL, and UV detection was set at 280 nm. ChemStation software (Agilent Technologies) was used for data acquisition.

CBD concentrations in the samples were quantified using a standard curve generated from authentic CBD solutions (0.196, 0.393, 0.786, 7.86, 15.7, 31.4, and 157 ng/μL; Figure S2). The HPLC method was validated using key parameters involving the linear calibration equation (y = 31.316x + 8.3826), correlation coefficient (R ², 1), and detection and quantification limits of 0.01 and 0.04 ng/μL, respectively (Table S1). The EE (%) of the CBD-NE was calculated using the following formula

$$\mathrm{E}\mathrm{E}(\%)=\left(1-\frac{\mathrm{f}\mathrm{ree\; CBD}}{\mathrm{t}\mathrm{otal\; CBD}}\right)\times 100$$

LE (%) was defined as the ratio of encapsulated CBD to the total weight of the nanoemulsion and was calculated using the corresponding formula.

$$\mathrm{LE}(\%)=\frac{\mathrm{w}\mathrm{eight\; of\; CBD\; in\; nanoemulsion}}{\mathrm{t}\mathrm{otal\; weight\; of\; nanoemulsion}}\times 100$$

All measurements were conducted using three independently prepared batches of nanoemulsions (n = 3).

2.3. Morphology Observation

Emulsion droplet morphology was visualized with an Olympus CKX53SF microscope (Tokyo, Japan) connected to an MD500 digital camera, following the method described by Lee et al. Freshly prepared emulsion (15 μL) was loaded onto a 76 mm × 26 mm glass slide (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany), overlaid with an 18 mm × 18 mm coverslip (Paul Marienfeld GmbH & Co. KG), and examined at 40× magnification using an objective lens (numerical aperture = 0.55). The images were acquired using AmScope imaging software.

2.4. Particle Size and ζ-Potential

The droplet size, polydispersity index (PDI), and ζ-potential of CBD-NE and blank-NE were analyzed at 25 °C using dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Prior to the analysis, the emulsions were diluted 10-fold in distilled water to minimize interference from light scattering. Data acquisition was performed using the Zetasizer software. All measurements were conducted on three independently prepared batches of CBD-NE and blank-NE (n = 3).

2.5. Emulsion Stability Index Value

The ESI was evaluated following the method reported by Han et al. (2024) Briefly, 40 μL of freshly prepared emulsion was diluted with 4 mL of 0.1% (w/v) sodium dodecyl sulfate solution (SDS500, LPS Solution, Daejeon, Korea). The absorbance at 500 nm was recorded immediately (Abs₀) and again after 10 min (Abs₁₀). ESI values were obtained using the following equation

$$\mathrm{E}\mathrm{SI}(\min )=\frac{{\mathrm{Ab}\mathrm{s}}_0}{{\mathrm{Ab}\mathrm{s}}_0-{\mathrm{Abs}}_{10}}\times 10$$

All measurements were performed using three independently prepared batches of nanoemulsions (n = 3).

2.6. Creaming Index (CI)

CI serves as an indicator of the physical stability. To determine the CI, 7 mL of fresh emulsion sample was kept at room temperature for 28 days. The CI values were calculated by dividing the height of the cream layer (H _c) by the total height of the emulsion (H _s), and measurements were taken on days 0, 1, 3, 5, 7, 14, 21, and 28.

$$\mathrm{CI}(\%)=\frac{H_{\mathrm{C}}}{H_{\mathrm{S}}}\times 100$$

The CI values were obtained from three separately prepared nanoemulsion batches (n = 3) to ensure reproducibility.

2.7. Turbidity Measurement

The turbidity decline over time is commonly used as a measure of emulsion cloud stability. The turbidity reduction observed under accelerated (diluted) conditions is suggestive of decreased emulsion stability. The turbidity loss rate of the emulsion was assessed by monitoring the absorbance at 500 nm using a spectrophotometer (SpectraMax M3; Molecular Devices, Sunnyvale, CA). Before measurement, freshly prepared emulsions were mixed with distilled water to obtain an absorbance close to 1.0. Over a 28-day period, the diluted samples were maintained at room temperature, and turbidity measurements were performed on days 0, 1, 3, 5, 7, 14, 21, and 28. The reduction in turbidity over time was analyzed using a first-order kinetic equation. The rate constant (k-value) was obtained from the slope of the fitted linear plot, as shown in the following formula

$$\ln T_t=\ln T_0-\mathit{kt}$$

where, T ₀ and T _t denote the turbidity values at the initial time point (0) and a specific time (t), respectively. All measurements were conducted using three independently prepared batches of nanoemulsions (n = 3).

2.8. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy

The emulsions were lyophilized using an FDU-2110 vacuum freeze-dryer (Tokyo, Japan) to obtain dry powders. The lyophilized powder was subjected to ATR-FTIR analysis using an IdentifyIR system (Smiths Detection, London, UK). FTIR spectra were recorded in the ATR mode over the range of 700–3900 cm^–1 with a scan number of 256.

2.9. Differential Scanning Calorimetry (DSC)

To assess the thermal properties of CBD-NE, the lyophilized samples were analyzed based on a previously reported method. Briefly, 5 mg of each sample was sealed in an aluminum TZero DSC pan (#DSC84010 and #DSC84011, DSC Consumables, Austin, MN) and heated from 30 to 130 °C at a heating rate of 10 °C per minute. The measurements were conducted using a TA Instrument Q200 DSC (DE) fitted with a refrigerated cooling unit. Thermal transitions were identified, and their corresponding peak temperatures and enthalpy values were extracted using Universal Analysis 2000 software.

2.10. RAW264.7 Cell Maintenance and Viability Assessment

Mouse macrophage RAW264.7 cells (cell line T1B-71) were purchased from American Type Culture Collection (ATCC; Manassas, VA). RAW264.7 cells were maintained in 10% fetal bovine serum (FBS; BIOWEST, Nuaillé, France) and Dulbecco’s modified Eagle’s medium (DMEM; WelGene, Daegu, Korea) with 100 U/mL penicillin-streptomycin (Invitrogen, Carlsbad, CA), and incubated at 37 °C in a humidified environment containing 5% CO₂. To assess the cytotoxic effects, a WST-1 assay was performed using the EZ-Cytox cell viability kit (DAEIL Lab, Seoul, Korea). RAW264.7 cells were seeded into a 96-well plate (#30096, SPL, Pocheon, Korea) (2 × 10⁴ cells/well) and incubated for 24 h to allow cell stabilization. After incubation, the cells were treated for 18 h with either CBD or CBD-NE dissolved in DMEM containing 0.1% dimethyl sulfoxide (DMSO; D8418, Sigma-Aldrich) in the presence or absence of LPS (200 ng/mL; #00–4976–93, Invitrogen). Final CBD concentrations were 1.99, 3.98, 7.95, 15.9, and 31.8 μM. A distinct group of cells treated with 0.1% DMSO in DMEM served as the control. After 18 h of treatment, the medium was removed and 10% WST-1 reagent dissolved in DMEM was added to each well. The cells were then further incubated at 37 °C in a 5% CO₂ environment for 3 h. Subsequently, absorbance was measured at 450 nm using a microplate reader. Cell viability (%) was calculated relative to that of the control group. All experiments were conducted using three independently prepared emulsion samples (n = 3).

2.11. Measurements of IL-6 and TNF-α

The levels of IL-6 and TNF-α were quantified using a previously reported method. Briefly, RAW264.7 cells were introduced into a 48-well plate (#30048, SPL) (0.5 × 10⁵ cells/well) and incubated for 24 h. After incubation, the cells were treated with CBD or CBD-NE and dissolved in 0.1% DMSO/DMEM to final CBD levels of 1.99, 3.98, 7.95, 15.9, and 31.8 μM in the presence of LPS (200 ng/mL) for 18 h. The control group was treated with 0.1% DMSO in DMEM. Quantification of IL-6 (#558301) and TNF-α (#558299) was carried out using mouse CBA ELISA kits (BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. All measurements were conducted using three independently prepared cell samples treated with nanoemulsions (n = 3).

2.12. In Vitro Simulated Digestion Experiment

An in vitro digestion test was conducted according to the INFOGEST procedure with minor modifications. The composition and preparation of the simulated digestion fluids–simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF)–are summarized in Table S2. The digestion procedure was carried out in three sequential stages: salivary, gastric, and intestinal stages. (1) Salivary stage: 500 μL of CBD, CBD-NE, or blank-NE (final CBD level: 318 μM) was combined with 400 μL of 1.25 × SSF. To achieve a final α-amylase activity of 75U/mL, 50 μL of α-amylase solution (A1031, Sigma-Aldrich) was added. An additional 50 μL of distilled water was then added to adjust the mixture to a final 1× SSF concentration. The mixture was incubated at 37 °C for 2 min. The final volume for this stage was 1 mL. (2) Gastric stage: The 1 mL salivary digesta was combined with 800 μL of 1.25 × SGF. To obtain a final pepsin activity of 2000 U/mL, 50 μL of pepsin solution (KK38.1, Carl Roth, Karlsruhe, Germany) was added. The pH was adjusted to 3 using 1 N hydrochloride, and distilled water was added to dilute the solution to a final concentration of 1 × SGF. The sample was incubated at 37 °C for 2 h. Pepsin activity was stopped by adjusting the pH to 7 using 1 N sodium hydroxide. This resulted in a final volume of 2 mL. (3) Intestinal stage: The 2 mL gastric digesta was combined with 850 μL of 1.25× SIF. To reach final concentrations of 100 U/mL pancreatin (P7545, Sigma-Aldrich) and 10 mM bile (B8631, Sigma-Aldrich), 500 μL of pancreatin solution and 250 μL of bile solution were introduced. The pH was maintained at 7, and distilled water was added to adjust the mixture to a final concentration of 1 × SIF. The samples were then incubated at 37 °C for 2 h. The reaction was stopped by adding AEBSF/Pefabloc SC (#11429868001, Roche, Mannheim, Germany) to reach a final concentration of 5 mM. This resulted in a total volume of 4 mL.

Following digestion, the bioaccessibility of CBD was assessed by quantifying the amount incorporated into the micelle fraction. The intestinal digesta were centrifuged (14,000g, 4 °C, 30 min), and the micelle-containing middle layer was collected. A 2 mL aliquot of this layer was mixed with 2 mL of an ethanol/chloroform solution (2:3, v/v). The organic layer was filtered using a 0.45 μm membrane filter and analyzed by HPLC to quantify CBD. The chromatographic conditions were as described in Section . Bioaccessibility was calculated using the following equation

$$\mathrm{b}\mathrm{ioaccessibility\; of\; CBD}(\%)=\frac{C2}{C1}\times 100$$

where, C1 and C2 are the CBD concentrations in the initial nanoemulsion and micelle phases, respectively. All digestion experiments were independently performed in triplicate to ensure reproducibility (n = 3).

2.13. Statistical Analysis

All experimental procedures were conducted in triplicate or more to ensure the reliability and reproducibility of the results. Statistical analyses were carried out using GraphPad Prism software (San Diego, CA). Results are expressed as mean ± standard deviation (SD). For statistical evaluation, Student’s t-test was used for comparisons between two groups, whereas one-way analysis of variance (ANOVA) followed by Tukey-Kramer’s post hoc test was used for comparisons involving multiple groups. A p-value less than 0.05 was considered to indicate statistical significance.

3. Results and Discussion

3.1. EE and DL of CBD-NE

The EE of CBD within CBD-NE was measured as 99.80 ± 0.13% using HPLC-UV analysis (Table ). Blank-NE was subjected to identical HPLC settings, with no detectable peak at 22.7 min, indicating no interference from the formulation matrix. This high EE suggests that CBD was nearly entirely entrapped in the emulsion system. This result indicated that the formulation efficiently stabilized and retained CBD, probably because of the compatibility between the intrinsic hydrophobic nature of CBD and the oil phase. Similar EE values have been reported previously. For example, a chrysin-encapsulated emulsion exhibited an EE of 99%, which was associated with improved stability and functional properties. Moreover, the LE of CBD within CBD-NE was determined to be 0.01 ± 0.00% (Table ) by dividing the amount of entrapped CBD by the overall emulsion mass. This value is in line with the LE reported for other hydrophobic compounds, such as curcumin, which showed an LE of 0.0033%.

1. Physicochemical Profiles of CBD-NE and Blank-NE,

	zeta average (nm)	PDI	ζ-potential (mV)	EE (%)	LE (%)	ESI (min)	CI (%)	k value
CBD-NE	39.18 ± 0.15a	0.26 ± 0.01	–3.31 ± 0.24a	99.80 ± 0.13	0.01 ± 0.00	4438 ± 679	2.62 ± 0.34	–0.0007
Blank-NE	57.32 ± 0.43b	0.24 ± 0.01	–5.81 ± 0.71b			2816 ± 173	2.86 ± 0.00	–0.0009

^aValues are expressed as mean ± standard deviation (n = 3).

^bDifferent lowercase letters indicate significant differences (p < 0.05) according to the Student’s t-test.

3.2. Physicochemical Characterization of CBD-NE

Both the CBD-NE and blank-NE exhibited a milky white appearance (Figure S1) and a dispersed spherical morphology under optical microscopy (Figure ). The droplet size and ζ-potential of the emulsions were analyzed by DLS to further characterize their physicochemical properties (Table and Figure S3). CBD-NE and blank-NE exhibited average particle sizes of 39.18 ± 0.15 and 57.32 ± 0.43 nm, respectively, consistent with the nanoscale range of typical nanoemulsions. A similar trend was reported by Hady et al. (2022) who showed that the encapsulation of pomegranate peel extract led to a decline in particle size from 304 to 259 nm in an emulsion system. The PDI values of both CBD-NE and blank-NE were below 0.3, indicating uniform droplet size distribution and good dispersion stability. To further evaluate the colloidal stability, the ζ-potential of the nanoemulsions was observed. The ζ-potential of CBD-NE was measured as −3.31 ± 0.24 mV, while that of blank-NE was relatively more negative at −5.81 ± 0.71 mV. The negative surface charge observed in blank-NE is likely due to the presence of carboxylic groups (−COO−) in the OSA-S. The elevated ζ-potential of CBD-NE aligns with earlier reports showing that encapsulation of bioactive compounds such as chrysin or curcumin can modify surface charge, likely through interactions with the carrier matrix. A similar trend was observed in a previous study, where the ζ-potential of blank emulsion (−8.5 mV) increased to −0.7 mV upon chrysin loading, suggesting that the incorporation of a hydrophobic compound can reduce surface charge while maintaining dispersion stability.

2.

Morphological characteristics of freshly prepared emulsions observed under optical microscopy at 40× magnification: (A) CBD-NE; (B) blank-NE.

3.3. ATR-FTIR Characterization of CBD-NE

To investigate how the incorporation of CBD influenced the molecular interactions within the emulsion system, ATR-FTIR analysis was performed (Figure ). The FTIR bands for OSA-S at 3436, 1149, and 995 cm^–1 correspond to the stretching vibrations of O–H, C–O, and C–O–C respectively. − In blank-NE, these bands shifted toward 3334 cm^–1 (O–H), 1151 cm^–1 (C–O), and 1017 cm^–1 (C–O–C), likely because of the anchoring of the OSA residue of the starch molecules at the oil–water interface. Upon CBD encapsulation, although no change in the C–O banding signals (1151 cm^–1) was observed, the O–H and C–O–C bands in the CBD-NE appeared at 3339 and 996 cm^–1, respectively. These spectral changes suggest that CBD incorporation influenced the molecular interactions between the oil and OSA-S within the emulsion system. Such alteration between oil and emulsifier interactions due to the addition of CBD in the oil phase may contribute to a disparity between the particle size of emulsion blank-NE (57.32 ± 0.43 nm) and CBD-NE (39.18 ± 0.15 nm).

3.

ATR-FTIR spectra of CBD, CBD-NE, Blank-NE, and OSA-S (A), and their normalized and expanded spectra (B).

3.4. Assessment of Storage Stability of CBD-NE

The physical stabilities of the CBD-NE are summarized in Table . Short-term physical stability was evaluated using ESI, with values of 4438 min for CBD-NE and 2816 min for blank-NE. To evaluate the long-term physical stability, changes in CI and turbidity were monitored over a 28-day storage period at room temperature. Throughout the storage period, the CI of both the CBD-NE and blank-NE remained below 3%, suggesting good physical stability. A CI below 5% is generally considered indicative of a stable emulsion. Likewise, the turbidity of both nanoemulsions remained stable, with the absorbance at 500 nm close to 1.0 during the storage period (28 days), as indicated by k-value of −0.0007 for CBD-NE and −0.0009 for blank-NE. These results suggest that the CBD-NE retained uniform particle dispersion and colloidal stability, highlighting its potential for long-term storage and practical applications.

3.5. Thermal Behavior of CBD-NE

The thermal properties of CBD in the nanoemulsion system were assessed by DSC (Figure ). Pure CBD exhibited a sharp endothermic peak at 68 °C, with an enthalpy of 51 J/g, corresponding to its melting point and crystalline structure. In contrast, no characteristic peak of CBD was observed in the thermogram of CBD-NE. This suggests that the encapsulation process may have disrupted the crystallinity of CBD, resulting in an amorphous state. Similarly, a study on CBD-loaded nanoparticles reported the disappearance of the endothermic peak of pure CBD, indicating a complete loss of crystallinity and transformation to an amorphous form due to biopolymer interactions. Comparable findings have also been reported in other nanoparticle systems, where drug crystallinity was lost during the formation using polymers or stabilizers. , This transition to an amorphous state often correlates with enhanced solubility and bioavailability, which is considered advantageous in pharmaceutical formulations.

4.

DSC curves of CBD, CBD-NE, and blank-NE.

3.6. Inhibitory Effects of CBD-NE on LPS-Stimulated IL-6 and TNF-α in RAW264.7 Macrophages

Numerous studies have demonstrated the anti-inflammatory effects of CBD. − Among the various inflammatory mediators, IL-6 and TNF-α are widely recognized as key indicators of the inflammatory response. To assess the anti-inflammatory effect of CBD-NE, its effects on LPS-induced production of IL-6 and TNF-α were evaluated in RAW264.7 macrophages, a widely used in vitro model for inflammation studies. , Before the assessments, the cytotoxicity of CBD-NE and blank-NE was evaluated using the WST-1 assay. As shown in Figure A, treatment with raw CBD or CBD-NE at final concentrations of 1.99, 3.98, 7.95, and 15.9 μM for 24 h had no impact on cell viability. However, a notable decrease in viability was observed at 31.8 μM of raw CBD. Similarly, exposure to LPS (200 ng/mL) under identical conditions did not induce cytotoxic effects (Figure B). These results indicated that the selected concentrations (1.99–15.9 μM) were suitable for further assessment of the anti-inflammatory effects of CBD-NE in RAW264.7 cells. As shown in Figure C,D, LPS treatment considerably (p < 0.001) elevated the levels of IL-6 (1446 pg/mL) and TNF-α (1086 pg/mL), compared to the control (IL-6: 772 pg/mL; TNF-α: 94 pg/mL). Notably, CBD-NE considerably suppressed LPS-stimulated IL-6 and TNF-α concentrations at all tested concentrations (1.99–15.9 μM, p < 0.001). In comparison, blank-NE showed a negligible influence on cytokine suppression, verifying that the observed bioactivity of CBD-NE is attributable to CBD loading (Figure S4). These findings suggested that nanoemulsion encapsulation enhanced the anti-inflammatory activity of CBD.

5.

Effects of CBD-NE on cell viability and inflammatory responses in RAW264.7 cells. (A and B) Cell viability was evaluated after 24 h exposure to either CBD alone or CBD-NE at concentrations of 1.99, 3.98, 7.95, and 15.9, and 31.8 μM under LPS-free (A) or LPS-treated (B, 200 ng/mL) conditions using the WST-1 assay. Values are expressed as mean ± SD (n = 3). Statistical analysis was conducted using the Tukey-Kramer multiple comparison test; *, p < 0.05 vs control. (C and D) Suppressive effects of CBD-NE on LPS-induced IL-6 and TNF-α levels in RAW264.7 macrophages following 24 h LPS treatment (200 ng/mL). ELISA kits were employed to quantify IL-6 and TNF-α concentrations. Values are expressed as mean ± SD (n = 3). Statistical analysis was conducted using the Tukey-Kramer multiple comparison test; ^###, p < 0.001 vs control; *, p < 0.05 vs LPS-only group; **, p < 0.01 vs LPS-only group; ***, p < 0.001 vs LPS-only group.

3.7. Enhanced Bioaccessibility of CBD-NE after Digestion

Bioaccessibility refers to the proportion of a compound that is released from its matrix during GI digestion, making it available for absorption. To assess this property, in vitro digestion models are widely used to evaluate the release and absorption potential of bioactive compounds in GI systems. In the present study, we evaluated the digestibility of raw CBD and CBD-NE using a static INFOGEST model, maintaining consistent ratio of enzymes, salts, and bile acids throughout the procedure. The extent of CBD transfer into the micellar phase after digestion was measured using HPLC-UV to determine its bioaccessibility. As shown in Figure , a larger CBD peak was observed in the micellar phase of CBD-NE after digestion than that in raw CBD. Quantitative analysis (Table ) revealed that the bioaccessibility of CBD-NE was 22.77 ± 2.66%, which was considerably higher (p < 0.01) than that of raw CBD (1.03 ± 0.09%). This improvement is likely attributable to the ability of the OSA-S-stabilized nanoemulsion system to promote CBD solubilization and facilitate its transfer into micelles. These observations are in line with previous reports demonstrating that emulsion systems enhance the bioaccessibility of lipophilic compounds. , CBD is presumed to be absorbed primarily via passive diffusion across the intestinal epithelium, as suggested in previous studies. , However, future studies including in vitro Caco-2 cell permeability and in vivo pharmacokinetic analyses, are required to determine whether enhanced bioaccessibility leads to improved absorption. Collectively, the increased bioaccessibility of CBD-NE underscores the utility of the nanoemulsion platform for addressing the inherent barriers of CBD, thereby supporting its potential use in pharmaceutical formulations and therapeutic applications.

6.

HPLC-UV chromatograms for CBD determination before and after in vitro INFOGEST digestion. (A) CBD before digestion, (B) digested CBD, (C) digested CBD-encapsulated nanoemulsion (CBD-NE), and (D) digested blank nanoemulsion (blank-NE). Arrows indicate the CBD peak at approximately 22.7 min.

2. Bioaccessibility of CBD in Raw CBD and CBD-NE after Simulated Digestion,

	bioaccessibility (%)
CBD	1.03 ± 0.09a
CBD-NE	22.77 ± 2.66b

^aValues are expressed as mean ± standard deviation (n = 3).

^bDifferent lowercase letters indicate significant differences (p < 0.01) according to the Student’s t-test.

In this study, OSA-S was used as a stabilizer to prepare a nanoemulsion system to improve the oral delivery of CBD. Our findings suggest that this food-grade biocompatible emulsifier can be effectively used to address the solubility and bioaccessibility challenges associated with CBD. The emulsion exhibited physical stability, as reflected by its low CI, minimal turbidity loss, and high EE. Additionally, the improved incorporation of CBD into the micellar phase during digestion indicates that the OSA-S-based nanoemulsion may enhance bioaccessibility under GI conditions. The simplicity of the emulsification process further supports its potential industrial application in oral delivery systems for hydrophobic compounds. Collectively, these findings highlight the potential of the OSA-S-based nanoemulsion as a practical and scalable delivery system for improving the bioaccessibility of poorly soluble compounds, such as CBD. This system may also be applicable to other hydrophobic cannabinoids with similar challenges in oral delivery, such as tetrahydrocannabinol, cannabinol, and β-caryophyllene. However, further in vivo studies are required to confirm whether this improved bioaccessibility translates into greater absorption and therapeutic benefits.

4. Conclusion

In summary, this study demonstrated that an OSA-S-stabilized nanoemulsion system is highly effective in enhancing the oral bioaccessibility and anti-inflammatory activity of CBD. The formulated CBD-NE exhibited colloidal stability over 28 days of storage and achieved a high EE, supporting its suitability as a delivery vehicle. Notably, the bioaccessibility of CBD in CBD-NE increased substantially after simulated GI digestion, and CBD-NE reduced the secretion of pro-inflammatory cytokines in RAW264.7 cells. These findings suggest that OSA-S-based nanoemulsions may address the solubility and bioavailability limitations of CBD and provide a promising strategy for improving its oral delivery. Although future studies, including in vivo evaluations, are warranted, a straightforward and scalable fabrication method will further enhance the practical value of this approach for future pharmaceutical applications.

Glossary

Abbreviations

ATR-FTIR

attenuated total reflection Fourier transform infrared

Blank-NE

blank nanoemulsion system

CBD

cannabidiol

CBD-NE

cannabidiol-encapsulated nanoemulsion

CI

creaming index

DMEM

Dulbecco’s modified Eagle’s medium

DLS

dynamic light scattering

DMSO

dimethyl sulfoxide

DSC

differential scanning calorimetry

EE

encapsulation efficiency

ESI

emulsion stability index

FBS

fetal bovine serum

GI

gastrointestinal

HPLC

high-performance liquid chromatography

IL-6

interleukin-6

LE

loading efficiency

OSA-S

octenyl succinic anhydride-modified starch

PDI

polydispersity index

SD

standard deviation

SGF

simulated gastric fluid

SIF

simulated intestinal fluid

SSF

simulated salivary fluid

TNF-α

tumor necrosis factor-α

All data supporting the findings of this study are available within the manuscript and the Supporting Information files. Additional details are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04608.

• Figure S1. Visual appearance of CBD-NE and blank-NE; Figure S2. HPLC-UV analysis for CBD determination in the emulsion; Figure S3. DLS analysis of particle size distribution and zeta-potential in the emulsion; Figure S4. Effects of blank-NE on LPS-stimulated IL-6 and TNF-α levels in RAW264.7 cells; Table S1. HPLC-UV validation parameters for CBD determination in the emulsion; Table S2. Preparation and composition of in vitro digestion fluids (PDF)

Y.L.: Conceptualization; Investigation; Methodology; Formal analysis; and Writing-original draft preparation; S.H.K.: Writing-Review and editing; and Funding acquisition; H.-Y.S.: Investigation; and Methodology; E.-B.B.: Writing-Review and editing; and Supervision.

The authors declare no competing financial interest.