Development and Characterization of the Novel Cannabidiol Loaded Polycaprolactone Nanoparticles to Boost the Bioavailability in Medicinal Oral Supplements

doi:10.21203/rs.3.rs-3974993/v1

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Development and Characterization of the Novel Cannabidiol Loaded Polycaprolactone Nanoparticles to Boost the Bioavailability in Medicinal Oral Supplements

https://doi.org/10.21203/rs.3.rs-3974993/v1

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The potential therapeutic applications of cannabidiol (CBD) extend to various health conditions such as multiple sclerosis, Parkinson's disease, cancer, nausea, insomnia, and anxiety. However, the practical use of CBD for medicinal purposes is hindered by its high lipophilicity, posing challenges in dosage and bioavailability. To address this, sustained release of CBD was assured by encapsulating CBD in biodegradable and biocompatible polymer polycaprolactone (PCL). Ultrasonication and oil in water emulsion was employed and 5, 10, 15, and 30 mg CBD were used for encapsulation into PCL nanoparticles, namely CBD-PCL NPs-1, NPs-2, NPs-3, NPs-4, respectively. The study evaluated morphological and physicochemical characteristics through FE-SEM, STEM, zeta size, HPLC, FT-IR and DSC analyses. The blank NPs-0 and CBD loaded NPs-1-4, exhibited mean particle diameters in between 207.5 nm and 227.7 nm with charges ranging − 23.37 and − 9.40 mV. Release kinetics of CBD-NPs were assessed and found to fit Zero-order kinetic model at pH 1.2 and Higuchi kinetic model at 7.4. The internalization of NPs into fibroblast cells and the impact of CBD on cell proliferation were also communicated. The findings suggest the potential application of innovative NPs as carriers for sustained release of CBD to be used in oral supplement formulations.

Cannabidiol

oral supplement

polycaprolactone

sustained release

nanoparticles

Based on research conducted by the World Health Organization (WHO), approximately 70% of the global population utilizes plant-based products within "traditional medicine" for the management of various conditions and the prevention of illnesses. Despite an estimated 70,000 species of medicinal plants, the WHO asserts that only about 21,000 species are deemed appropriate for inclusion as medicinal ingredients [1]. Hemp (Cannabis sativa L.), an adaptable annual plant species containing cannabinoids such as tetrahydrocannabinol (THC) and cannabidiol (CBD) as active components, has been employed for medicinal purposes since ancient times. THC induces psychoactive effects, altering mental states, while CBD demonstrates therapeutic properties [2]. CBD has recently gained substantial attention in the food and health sectors. Hemp is honored by a rich global history, with the earliest recorded medical use documented around 2800 BCE in China by Emperor Shen Nung in his pharmacopoeia "Shen Nung Pen Ts'ao Ching" [3]. CBD has been utilized to address various health issues, including anxiety, depression, insomnia, epilepsy, skin wounds, pain, and inflammation. Despite its benefits, CBD comes with drawbacks such as low water solubility, high lipophilicity, and limited oral bioavailability [4].

The surface properties and sizes of nanoparticles modifiable which make them attractive as medication carriers. There are many different kinds of nanoparticles some of which are liposomes, micelles, metal nanoparticles, dendrimers, nanospheres, and nanocapsules [5]. The goal of several studies and authorized nanotechnological products is to expand on the benefits of nanoparticles in medicine. Targeted drug release and transportation is greatly aided by nanoparticles. According to Wicki et al. (2015), nanoparticles have tunable surface and physicochemical characteristics especially for a target, which enhances their therapeutic qualities by modulating the pharmacokinetic and pharmacodynamic aspects of biologically active ingredient [6]. In recent times, researchers have shown significant interest in polymeric nanoparticles (NPs) and nanocapsules due to their ability to protect drug molecules until they reach the intended site, customizable drug release, and versatile properties. When developing polymeric carrier systems, natural polymers are preferred for their biodegradable and biocompatible features, while synthetic polymers offer advantages such as controlled chemical structures [7]. Polycaprolactone (PCL), an aliphatic polyester polymer, stands out as a suitable material for creating polymeric NPs for drug delivery. This is attributed to its capacity to encapsulate numerous drug molecules, non-toxicity, biodegradability, and other favorable characteristics [7]. PCL holds FDA approval as a biocompatible polymer that enhances drug bioavailability, facilitating targeted delivery and sustained release without requiring removal from the body after application [8].

This study aimed to develop novel CBD-loaded PCL NPs, emphasizing the application of nanotechnological methods to overcome CBD's hydrophobic properties, thereby enhancing its effectiveness and bioavailability. The primary objective was to contribute to the creation of a new product suitable as a safe food supplement for preventive medicine. The process began with the production of blank (placebo) PCL NPs (PCL NPs-0) with optimized physicochemical characteristics. Subsequently, CBD-loaded PCL NPs were prepared. Dynamic light scattering (DLS) was employed to analyze particle size and charge. Structural analysis and surface morphology were examined using Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (FE-SEM and STEM), respectively. Differential scanning calorimetry (DSC) analysis determined the polymer-CBD compatibility. High-performance liquid chromatography (HPLC) was used to analyze the encapsulated CBD in NPs, and Nil red-encapsulated PCL-NPs demonstrated NPs permeability into fibroblast cells. Furthermore, the impact of CBD on cell proliferation was assessed through cell viability experiments with L929 healthy fibroblast cells and MCF-7 breast cancer cells after 24, 48, and 72 hours of CBD incubation.

2.1. Chemicals and Materials

Cannabidiol (≥ 98%) was purchased from Cayman, USA. Poly-ε-Caprolactone (Mw: 14000, Mw: 45000, and Mw: 80000), Polyvinyl alcohol (Mw: 30000–70000), Acetonitrile (HPLC grade), Tween®80, Sodium Chloride, Nile Red Fluorescent dye, and Gentamicin were purchased from Sigma-Aldrich, Germany. Methanol (chromatography grade), dichloromethane (for analysis), Phenolphthalein, Glacial Acetic Acid and Ethanol (chromatography grade) were purchased from Merck, Germany. Phosphate Buffered Saline (Invitrogen), Hydrochloric Acid (Isolab), RPMI 1640 (L-Glutamine, Lonza), DMEM (1 g/L Glucose without L-Glutamine, Lonza), and Fetal Bovine Serum (Heat-Inactivated European Grade, Biological Industries) were purchased, and all chemicals and reagents were used as received. Ultra-pure water from a Millipore UV Water Purification system from Merck was used for experiments and washing procedures.

2.2. Method

2.2.1. Preparation of PCL NPs

Polycaprolactone (PCL) with varying molecular weights (14000, 45000, and 80000 g/mol) were utilized through solvent evaporation technique to obtain PCL NPs of optimal size. Manufacturing parameters are given in Table 1. Initially, 150 mg of the polymer was dissolved in 5 mL of dichloromethane at room temperature, resulting in an oil phase (O). For the aqueous phase (W), a polyvinyl alcohol (PVA) solution (0.5%) was prepared by stirring for 45 min at 60°C. Then, the oil phase was added to 50 mL of the PVA solution, and the mixture was homogenized using an Ultra-Turrax (IKA® T25 D Ultra-Turrax) at 25000 rpm. The ultrasonic homogenizer (Bandelin GM 3200, 200 W, 20 kHz, KE 76 prop) was used for 5 min at 72% amplitude to obtain NPs as the second experiment group [9]. The resulting solution was continuously stirred at 600 rpm using a magnetic stirrer at room temperature for 3 h to allow the evaporation of dichloromethane and the solidification of NPs. The NPs were recovered by centrifugation at 9000 rpm for 15 min. To remove PVA from the NPs, pellet was washed three times with deionized water, discarding the supernatant. Finally, to prevent polymer hydrolysis during storage, the NPs were frozen at -80°C for 24 h and then lyophilized at -110°C.

Table 1

The parameters to manufacture placebo PCL NPs
PCL Mw (g/mol)	Homogenizer	PCL (mg)	Rpm/ %Amp	PVA (w/v)	Duration (min)
14000	Ultra-turrax	150	25000	%0.5	5
45000	Ultra-turrax	150	25000	%0.5	5
80000	Ultra-turrax	150	25000	%0.5	5
14000	Ultrasonic	150	72%	%0.5	5
14000^a	Ultrasonic	150	72%	%0.5	5
45000	Ultrasonic	150	72%	%0.5	5
80000	Ultrasonic	150	72%	%0.5	5

^a) Filtered through a 0.22 µm filter.

2.2.2. Particle size, polydispersity index, and zeta potential measurements

The particle size, polydispersity index (PDI), and zeta potential of the NPs were determined using a zetasizer (Nanoplus-3, Micromeritics, USA). The average particle size was obtained as the mean of three consecutive measurements, each comprising 50 readings. After subjecting each sample to 10 min of ultrasonic bath treatment (ISOLAB Laborgeräte GmbH, 180 W), it was diluted in a 20:1 ratio with deionized water in a quartz cell to prevent multiple scattering and subsequently placed in the instrument for analysis. The average particle size was determined for each sample. To ensure isotonic stability, 1 mM NaCl was added prior to zeta potential analysis [10]. The particle size and zeta potential values were measured in triplicate for each sample, and the average values were calculated. The polydispersity index (PDI) was calculated using the equipment software of the Nanoplus 3 instrument for dynamic light scattering (DLS) measurements.

2.2.3. CBD encapsulation

As the parameters for the optimum size and PDI were determined for placebo PCL NPs at the previous step, the CBD encapsulated NPs were manufactured by following the same optimum steps that of placebo NP manufacturing. To prepare the oil phase (O), the active ingredient (5, 10, 15, 30 mg CBD) and 150 mg of the polymer (PCL, 14000 g/mol) were dissolved in 5 mL of dichloromethane at room temperature. The oil phase was added in to 50 mL of PVA solution which is aqueous phase (W), and ingredients were homogenized at 72% amplitude for 5 min. The resulting oil-in-water (O/W) emulsion was continuously stirred at 600 rpm using a magnetic stirrer at room temperature for 3 h to allow for the evaporation of the organic solvent and the solidification of NPs. The NPs were recovered by centrifugation at 9000 rpm for 15 min. To remove PVA from the NPs, pellet washing was performed three times using deionized water, followed by discarding the supernatant. Finally, the CBD-PCL NPs were frozen at -80°C for 24 h and then lyophilized at -110°C. The particle size, polydispersity index (PDI), and zeta potential of the CBD-PCL NPs were determined using a zetasizer (Nanoplus-3, Micromeritics, USA) as described in the section 2.2.2.

The maximum loading capacity of NPs was evaluated by encapsulation with different initial active ingredient-to-polymer ratios (PCL NPs-0, PCL NPs-1, PCL NPs-2, PCL NPs-3, and PCL NPs-4 for 0, 5, 10, 15, 30 mg CBD per 150 mg PCL, respectively). The encapsulation efficiency of CBD in PCL NPs was calculated using an HPLC (Shimadzu Prominence LC-20 AD, Japan). The analysis was performed using an SPD M20A UV-VIS DAD detector and a reverse-phase C18 column (5 µm, 150 x 4.6 mm) from Restek, at a wavelength of 205 nm. The loading capacity (LC) and encapsulation efficiency (EE) of CBD-loaded PCL NPs was calculated using Eq. 1 and Eq. 2, respectively. To determine the amount of encapsulated CBD, 15 mg of NPs was dissolved in 1 mL of DCM and then diluted in mobile phase. The solution was filtered through a 0.45 µm syringe filter and analyzed. A mixture of acetonitrile:water (85:15 v/v) was used as the mobile phase, delivered at a flow rate of 1 mL/min [11]. A calibration curve was generated using a cannabidiol standard purchased from Cayman and ranging from 0.1 to 10 µg/mL (ppm) (r² = 0.9997). The total analysis time was 10 min with a retention time of 4.35 min. All measurements were performed three times.

$$LC \left(\text{\%}\right)=\frac{\text{A}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{l}\text{o}\text{a}\text{d}\text{e}\text{d} \text{C}\text{B}\text{D}}{\text{A}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{C}\text{B}\text{D} \text{l}\text{o}\text{a}\text{d}\text{e}\text{d} \text{P}\text{C}\text{L} \text{N}\text{P}\text{s}} x 100$$

$$EE \left(\text{\%}\right)=\frac{\text{A}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{l}\text{o}\text{a}\text{d}\text{e}\text{d} \text{C}\text{B}\text{D} \left(\text{m}\text{g}\right)}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} \text{C}\text{B}\text{D} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)} x 100$$

2.2.4. Reaction efficiency

After the lyophilization process, the weight of the dried CBD-loaded PCL NPs was measured using a precision balance, and the reaction yield (%RY) was calculated according to Eq. 3.

$$\text{\%} \text{R}E=\frac{\text{M}\text{a}\text{n}\text{u}\text{f}\text{a}\text{c}\text{t}\text{u}\text{r}\text{e}\text{d} \text{n}\text{a}\text{n}\text{o}\text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \left(\text{m}\text{g}\right)}{Initial added CBD \left(mg\right)+ PCL amount \left(mg\right)} x 100$$

2.2.5. Morphology of NPs

The surface and shape of the NPs were examined using a field emission scanning electron microscope (FE-SEM, ZEISS GeminiSEM 500). Double-sided carbon tape was placed on a flat aluminum plate, and the NPs were dried on this plate at room temperature. Subsequently, the samples were coated with an average of 4 nm iridium under an argon atmosphere. The samples were observed using a FE-SEM under an extra high voltage of 2 kV. Furthermore, the morphological characterization of the placebo and CBD loaded PCL NPs was performed by using the STEM (scanning transmission electron microscopy) mode of a FE-SEM device. The placebo and CBD-loaded NPs were diluted with pure water at a ratio of 1:20 and subjected to sonication in an ultrasonic ice bath for 3 min prior to analysis. A drop of placebo and CBD-PCL NPs was placed on 200 mesh copper grids. The analyses were performed with an acceleration voltage of 30 kV.

2.2.6. Fourier transform infrared spectroscopy (FTIR) analysis

The interactions between the active ingredient and the polymer in the synthesized NPs were determined using FTIR spectroscopy (Shimadzu IRTracer-100, Japan). The interactions between the active ingredient and the polymer were detected through the shifts and changes in the IR spectra of functional groups [12]. In order to confirm the interaction between CBD and PCL and the occurrence of encapsulation, the analyses were performed on lyophilized CBD-loaded PCL NPs, placebo PCL NPs, raw PCL, and CBD. Spectra in the range of 350 cm^-1 to 4500 cm^-1 were scanned 16 times.

2.2.7. CBD-PCL solubility and compatibility

To evaluate the physical state and interaction between the active substance and polymer, 15 mg of pure CBD, pure PCL, and PCL-CBD physical mixtures (at ratios of 5:1, 10:1, 15:1, and 30:1, (w/w) respectively) were analyzed using differential scanning calorimeter (Setaram - Labsys Evo, France). The samples were heated at a rate of 10°C/min from 25 to 300°C under a nitrogen flow of 20 mL/min for analysis [13].

2.2.8. In vitro release studies

In vitro release studies were conducted at pH 1.2 and pH 7.4 to simulate the stomach and intestine pH values, respectively. Lyophilized CBD loaded-PCL NPs (15 mg) were suspended in 30 mL of buffer solutions including Tween^®80 (0.1 w/v) and continuously shaken at 35 strokes/min (Unimax 1010, Shaking incubator, Heidolph Inc., Germany) at 37°C. The supernatant was collected (28 mL) and filtered through a 0.45 µm filter at certain time intervals (hourly for the first 8 h, and once every 24 h for 5 days), 28 mL of filtered, and analyzed for CBD content by UV/Vis spectrophotometer (Multiskan SkyHigh Microplate UV/Vis Spectrophotometer, Thermo Fisher, USA) and HPLC for pH 1.2 and 7.4, respectively. The volume was replenished with 28 mL of buffer.

2.2.9. Cellular uptake of NPs

NPs containing Nile Red fluorescent dye were prepared using the same method by dissolving the dye in the oil phase at a concentration of 2 mg/mL. Encapsulation of fluorescent dye in PCL NPs were performed as described in section 2.2.3, by loading 10 mg of the dye into 150 mg of PCL polymer. Due to its hydrophobic nature [14]. Nile Red was efficiently encapsulated in the PCL NPs. After incubation with L929 rat fibroblast cells at 37°C for 2 h, the cells were washed with PBS and imaged at 40X and 60X magnification at using an inverted fluorescence microscope (BAB-TAM-F, Turkey). Separation of fluorescence channels was achieved using the Chroma Technology filter set AT-TRICT-REDSHFT 540/25x, 620/60m, AT565DC.

2.2.10. In vitro cell proliferation assay

In order to determine the effects of CBD on the proliferation of L292 normal fibroblast and MCF-7 breast cancer cells, the cells were initially cultured in complete medium containing 10% fetal bovine serum (FBS) and antibiotics (DMEM and RPMI 1640, respectively). CBD was distributed in the wells at 9 different concentrations ranging from 100 µM to 0.39 µM, while wells designated as cell control and medium control were supplemented with CBD-free medium. Actively proliferating cells were seeded in a 96-well cell culture plate at a density of 5000 cells per well in 50 µL of medium, excluding the wells designated as medium controls. The plates were then incubated in a CO₂ incubator at 37°C for 24, 48, and 72 h. Following incubation, XTT cytotoxicity solution (Sartorius) containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which measures mitochondrial activity of viable cells, was added to each well and incubated for 4 h. Absorbance in the wells was measured at 450 nm using an ELISA plate reader to calculate the percentage of viable cells. The effect of CBD on cell viability was calculated as a percentage of the control cells’ proliferation, where the proliferation of CBD-free control cells corresponded to 100% viability. Results and standard deviations were calculated from at least eight wells for each time point, and a t-test was applied (p < 0.05). The effects of CBD on L929 and MCF-7 cells, as well as the IC₅₀ (the concentration that inhibits 50% of the cell viability), were calculated based on the percentage of cell proliferation.

3.1. Physicochemical Characterization of PCL NPs

The average particle size, polydispersity index (PDI), and zeta potential measurements were conducted for PCL placebo NPs produced by different manufacturing techniques. Dynamic Light Scattering (DLS) and Zeta Potential measurements were performed for each sample, and therefore PCL NPs-0 are summarized in Table 2.

The smallest diameter of NPs was achieved with 14000 g/mol PCL using the ultrasonic homogenizer after filtration through a 0.22 µm filter, yielding a particle size of 202.4 nm. The nanoparticle sizes exhibited no substantial change after filtration; therefore, the filtration step was eliminated from the manufacturing process. The ideal nanoparticle size for oral administration falls within the range of 100 nm to 800 nm, with particles smaller than 100 nm being suitable for parenteral administration. Conversely, larger particles exceeding 1000 nm may pose a risk of phagocytosis [15]. Consequently, it was concluded that nanoparticles obtained through both Ultra-Turrax and ultrasonic homogenizer methods were suitable for oral use.

The PDI represents the particle size distribution, and the PDI values of particles generated by ultrasonication were found to be less than 0.20. For the polymer-based NPs, PDI values below 0.2 are considered to be favorable [16]. According to the PDI results, NPs produced with ultrasonic homogenization demonstrate good uniformity and homogeneity in terms of particle size and distribution. However, in the case of processes conducted using Ultra-Turrax, PDI values were higher than 0.20, thus not deemed suitable in terms of uniformity and homogeneity.

Javaid et al. reported a zeta potential of -16.8 mV for placebo PCL NPs and − 11.1 mV for sefotaxime-loaded PCL NPs [17]. Mahmoudi et al. declared that placebo PCL NPs had zeta potential − 29.3 mV and memantine-loaded PCL NPs had − 26.5 mV zeta potential [18]. Diyanat et al. found that placebo PCL NPs had − 25.8 mV and pretilachlor-loaded PCL NPs had − 23.0 mV zeta potentials [19]. Based on these results, it can be inferred that as the hydrophobicity of the active substance to be encapsulated in PCL increases, the absolute zeta potential value decreases.

Based on the evaluation of the results, considering both size and stability, as well as standardization aspects, the most suitable production condition was achieved using 14000 g/mol PCL by ultrasonication at a 72% amplitude. Collectively, CBD encapsulation studies were proceeded with these conditions.

Table 2

The size, PDI and zeta potential for PCL NPs-0 (n = 3)
PCL (g/mol)	Homogenizer	rpm/ %Amp	Mean diameter	PDI	Zeta Potential (mV)
14000	Ultra-turrax	25000	327.9 ± 6.51	0.259 ± 0.027	-14.98
45000	Ultra-turrax	25000	338.1 ± 4.46	0.271 ± 0.038	-17.17
80000	Ultra-turrax	25000	392.4 ± 13.79	0.250 ± 0.011	-15.72
14000	Ultrasonic	72%	207.5 ± 0.45	0.114 ± 0.044	-23.37
14000^a	Ultrasonic	72%	202.4 ± 6.36	0.110 ± 0.018	-20.16
45000	Ultrasonic	72%	235.6 ± 2.29	0.107 ± 0.020	-21.12
80000	Ultrasonic	72%	264.6 ± 6.85	0.108 ± 0.020	-24.11

^a) Filtered through a 0.22 µm filter.

3.1.1. Particle size, polydispersity index, and zeta potential of NPs

Average particle size, PDI and zeta potential for CBD-PCL NPs loaded with different amounts of CBD are presented in Table 3. For the CBD-loaded PCL NPs (5 mg, 10 mg, 15 mg, and 30 mg CBD), the zeta potential values were determined as -10.21, -9.62, -9.93, and − 9.40 mV, respectively. These values may indicate the presence of CBD partially on the particle surface despite being trapped within the polymer matrix, suggesting partial adsorption of CBD on the PCL shell and its incorporation with PCL. Consequently, it can be suggested that CBD increases the zeta potential in a positive direction by enhancing the surface charge of PCL NPs. Zeta potential values between 0 and ± 5 mV indicate a tendency of particles to aggregate or come together, leading to rapid particle aggregation. Particles between ± 5 and ± 20 mV are minimally stable, while particles between ± 20 and 40 mV are moderately stable [20]. Zeta potential values higher than ± 30 mV can increase stability but may also lead to toxicity [15]. NPs produced by the ultrasonic homogenizer exhibit moderate stability, while CBD-PCL NPs demonstrate minimal stability. Collectively, both placebo and CBD-PCL NPs exhibited zeta potential values below the risk threshold for toxicity.

Table 3

Mean diameter, PDI, and zeta potential for CBD-loaded PCL NPs, (n = 3)^a
NPs	Mean diameter (nm)	PDI	Zeta Potential (mV)
PCL NPs-1	227.2 ± 2.35	0.100 ± 0.004	-10.21
PCL NPs-2	225.5 ± 0.53	0.133 ± 0.029	-9.62
PCL NPs-3	227.7 ± 1.55	0.094 ± 0.014	-9.93
PCL NPs-4	216.9 ± 6.81	0.126 ± 0.031	-9.40

^a) Ultrasonic homogenizer was employed with 72% amplitude.

3.1.2. FTIR analysis

FT-IR analysis was conducted to investigate the molecular interaction between the active ingredient and the polymer. The characteristic bands of pure PCL and pure CBD are presented in Fig. 1, while the FT-IR spectra of pure PCL, pure CBD, placebo PCL NPs (PCL NPs-0), and CBD-loaded PCL NPs are revealed in Fig. 2.

Characteristic bands of CBD were identified at 3518 cm^-1, 3404 cm^-1, 1628 cm^-1, and 1585 cm^-1 [21]. In the FT-IR spectrum of CBD, the prominent bands in the range of 3404 cm^-1 to 3518 cm^-1 corresponded to O-H stretching vibrations, while the bands at 3074 cm^-1 represented C-H stretching (phenyl), the band at 2924 cm^-1 indicated methyl and methylene groups, the band at 1585 cm^-1 represented C = C stretching in the phenyl ring, and C-O stretching vibrations were observed at 1213 cm^-1 [22].

Characteristic bands of PCL were observed at 2866 cm^-1 for symmetric aliphatic stretching and at 2943 cm^-1 for asymmetric aliphatic stretching. The strong characteristic carbonyl (C = O) group was observed at the stretching band of 1720 cm^-1. The C-O and C-C stretching vibration bands of PCL were observed at 1292 cm^-1. Additionally, the symmetric C-O-C vibration band was observed at 1168 cm^-1 and the asymmetric C-O-C vibration band at 1238 cm^-1 [8].

The FT-IR spectrum of PCL NPs-4 exhibited characteristic bands at 1585 cm^-1 and 1628 cm^-1 corresponding to the chemical structure of CBD. The stretching vibration caused by the OH group was present in all CBD-loaded PCL NPs at bands 2860 cm^-1 and 2945 cm^-1. The band at 1442 cm^-1, attributed to the deformation of methylene or methoxy groups (C-H deformation), cannot be considered as a representative band for CBD [23]. The presence of CBD within PCL can be emphasized by comparing the PCL bands with the presence of bands belonging to aliphatic groups (= C-H) at 1585 cm^-1 and 1628 cm^-1. These two specific bands have also been observed in previous study of Andriotis et al. in their CBD/cyclodextrin formulation and cannabidiol orodispersible tablets formulated by Vlad et al. [23, 24]. In this work, the peaks of CBD were observed in PCL NPs-4 sample more profoundly than in PCL NPs-1, 2, 3. This may be due to the molecular dispersion of low CBD dose within the PCL polymer matrix which was also previously observed by Nawaz et al in their studies with PCL encapsulated acriflavine [10]. To conclude, FTIR spectra demonstrates CBD and PCL exhibited no chemical incompatibility in NPs.

3.2. Morphology of NPs

The morphological characteristics of PCL NPs-0 and PCL NPs-4 were examined using FE-SEM. The STEM images of placebo PCL NPs-0 and NPs-4 are presented in Fig. 3 furthermore, the FE-SEM images are displayed in Fig. 4.

Uniform spherical NPs were observed by STEM, while near-spherical, irregular shaped NPs were observed by FE-SEM imaging, as observed in previous studies [18]. Partial agglomeration was observed in FE-SEM imaging, which can be attributed to the surface tension of water that affects the NPs during the drying process [25]. Additionally, the bridges observed between NPs in the FE-SEM images are formed due to the adhesive nature of the PVA used in the formulation. Removing PVA completely, even after particle washing, is challenging due to its inherently sticky nature [26]. FE-SEM images confirmed the formation of solid NPs with a purely polymeric structure. Overall, the morphologies of STEM images of NPs in liquid medium and the FE-SEM images of dried particles confirmed the manufacture of spherical and near spherical PCL encapsulated CBD NPs with O/W emulsion technique using ultrasonication.

3.3. Reaction Efficiency, Loading Capacity and Encapsulation Efficiency

The reaction efficiencies for placebo NPs and CBD-loaded PCL NPs were calculated to be 79.75% and 75.26%, 71.71%, 73.71%, and 70.91%, respectively, using Eq. 2 (Table 4). These results indicate that the reaction efficiency of placebo NPs is higher than that of CBD-PCL NPs. This means that the increased mass of hydrophobic bioactive ingredient in the reaction pot, decreases the number of manufactured NPs. Although the reaction experienced a 9% decrease in efficiency, this is still acceptable in terms of the function of the final product. The loading capacity of nanoparticles increased as the loaded ingredient amount increased. For instance, for the NP-4 formulation the loading capacity was about 22% with the starting 30mg CBD amount. This means that the 22% of NP-4 is composed of CBD by weight. The NPs seem to have capacity to be loaded by more CBD than used in this work.

The values of %EE for PCL NPs containing different amounts of CBD are given in Table 4. These results indicate a high loading of CBD into the PCL polymer matrix. The increase in the initial agent-to-polymer ratio resulted in an increase in loading, leading to encapsulation efficiencies ranging from 74.39–94.75%. This can be attributed to the high lipophilic nature of cannabidiol, which tends to be entrapped within the polymer matrix rather than dissolving in the external aqueous phase. This situation was also experienced by Pandey et al. that is, increased amount of betamethasone valerate per polymer amount resulted in increased encapsulation efficiency [27].

Table 4

Reaction efficiencies (n = 1) and encapsulation efficiencies of NPs (n = 3)
NPs	% Loading Capacity	% Reaction Efficiency	%Encapsulation Efficiency
PCL NPs-0	-	79.75	-
PCL NPs-1	3.23 ± 0.03	75.26	74.39 ± 1.44
PCL NPs-2	6.87 ± 0.05	71.71	82.06 ± 0.19
PCL NPs-3	10.97 ± 0.08	73.71	88.74 ± 0.95
PCL NPs-4	22.05 ± 0.06	70.91	94.75 ± 0.61

3.4. CBD-PCL Solubility and Compatibility

Pure CBD, pure PCL, and PCL-CBD physical mixtures were evaluated in terms of their form and degradation profiles. The DSC thermograms of pure CBD, pure PCL, and PCL-CBD physical mixtures are demonstrated in Fig. 5, and the results are presented in Table 5. Consistent with previous studies, a sharp endothermic peak was observed at 69.82°C (corresponding to the melting point) for pure CBD, with an enthalpy of 77.45 J/g [26, 28]. Similarly, a sharp endothermic peak was observed at 68.81°C (corresponding to the melting point) for pure PCL, with an enthalpy of 77.45 J/g [11, 29]. This sharp peak is in accordance with the crystalline nature of CBD. The thermograms of PCL-CBD mixtures exhibited endothermic behavior corresponding to the melting endotherms of PCL polymer. For the CBD/ PCL ratios of 5/150, 10/150, 15/150, and 30/150 (mg/mg), the thermograms exhibited endothermic peaks at 68.93°C (enthalpy of 89.34 J/g), 68.81°C (enthalpy of 87.76 J/g), 68.71°C (enthalpy of 85.21 J/g), and 68.14°C (enthalpy of 82.46 J/g), respectively. In this work according to the thermal studies CBD powder, PCL powder and PCL-CBD mixtures exhibited a simple eutectic phase diagram. Accordingly, it may be concluded that physical mixtures of PCL and CBD have similar melting temperatures which may be implied that two components do not chemically interact with each other during melting.

Table 5

DSC analysis results of pure CBD, pure PCL and PCL-CBD physical mixtures
Materials	T(°C)	Enthalpy (J/g)
Pure PCL	68.81	77.45
Pure CBD	69.82	77.45
5/150	68.93	89.34
10/150	68.81	87.76
15/150	68.71	85.21
30/150	68.14	82.46

3.5. In Vitro Release of CBD from PCL-NPs

The release kinetics of nanoparticles offer crucial insights into their capacity to influence drug release. Therefore, it stands as a significant parameter to be taken into account when evaluating the safety, effectiveness and quality of nanoparticles. In vitro drug release studies were conducted in buffer solutions at two different conditions, pH 1.2 and pH 7.4, to simulate gastric and intestinal conditions, respectively. The studies were carried out at 37°C for 8 h at pH 1.2 and for 96 h at pH 7.4. The aim was to develop a sustained dosage form suitable for the use of CBD in dietary supplements, due to the fact that the dietary supplement may remain in the stomach for a maximum of 8 h, in vitro release kinetics measurements were conducted at gastric pH 1.2 for 8 h to observe any stationary phase in drug release profile [30]. The release profile did not demonstrate the stationary release phase until 8 h release measurements.

The cumulative CBD release percentage at pH 1.2 was found to be significantly lower when compared to the CBD release at pH 7.4 from CBD-loaded PCL NPs, in 8 h. This indicates that the CBD-PCL NPs are pH-responsive nanocarriers for hydrophobic drugs as also found by Saqib et al. with their Amphotericin B loaded PCL NPs [31]. The slow and sustained release observed in 96 h may be attributed to the diffusion of CBD within the hydrophobic core of the PCL matrix [11, 32]. and is inherent to the hydrophobic nature of PCL. Crystalline and hydrophobic polymers generally exhibit slower degradation rates. Additionally, this slow release indicates that a small amount of CBD is adsorbed on the surface of PCL NPs. Snehalatha et al. reported that the degradation rate from PCL nanoparticle matrix is redated due to its hydrophobic nature and it was also reported that release of hydrophobic ingredients is also slower from hydrophobic matrices [33]. Wang et al. manufactured CBD encapsulated zein-whey protein composite nanoparticles. They observed about 20% CBD release at gastric fluid in 2h and then 90% of CBD was released at intestinal fluid after two additional hours [34]. Here the results imply that CBD/PCL nanoparticles allowed a limited CBD release when compared to the aforementioned study. As CBD being hydrophobic in nature, it exhibits limited release from the hydrophobic PCL nanoparticle matrix. The CBD/PCL combination exhibited the sustained release profile in this study.

A near-complete release of was observed for PCL NPs-4 (94.28%) at pH 7.4 at the end of 96 h. This limitation may be due to the usage of lower molecular weight of the polymer used in this study. It was previously reported that release from higher molecular weight polymers can be extended for months due to the slow degradation of the polymers in the release medium [35]. The release profile of CBD-loaded PCL NPs was fitted to zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell kinetic models to evaluate the release kinetics best fitting to the CBD loaded NPs. Zero-order kinetic model refers to drug delivery systems that release a constant amount of active substance at the same time intervals, regardless of the concentration of the active substance [36]. On the other hand, the first-order kinetic model describes drug delivery systems where the amount of released active substance depends on the concentration of the active substance [37]. The Higuchi kinetic model explains the release of the active substance from homogeneous or heterogeneous matrix systems, while the Hixson-Crowell kinetic model illustrates the release of the active substance associated with changes in particle surface area [36, 38]. The Korsmeyer-Peppas kinetic model represents the release process where the diffusion and degradation of the polymeric structure are involved [39].

The correlation coefficients for the kinetic models of this work are presented in Table 6 and Table 7. The kinetic models with the highest correlation coefficients were depicted for the zero-order kinetic model at pH 1.2 and for the Higuchi kinetic model at pH 7.4.

In oral drug administration, the gastric retention time of the drug varies between 5 min and 5 h, depending on the individual's digestive state [40]. In the fasting state, this time can extend up to 8 h [30]. According to the results, the highest correlation coefficient for CBD-loaded NPs was observed in the Zero-Order Release Kinetic model. This model demonstrates that the NPs released the same amount of CBD at the same time intervals independent of its concentration [36]. This may indicate the CBD's partial presence on the particle surface.

Release kinetics of CBD from PCL NPs at pH 7.4 was found to fit to the Higuchi kinetic model. This model suggests that drug release from a homogeneous matrix system is controlled by the square root of time and occurs through Fickian diffusion [38]. Higuchi kinetics model presents a decrease in the amount of drug released over time. This is due to the initial release of the drug from the surface area, which forms the shortest diffusion path. However, as the dissolution process of the drug progresses, the surface area exposed to the dissolution medium decreases. After the depletion of the drug on the surface, the next layer of the film starts releasing the drug, leading to an increase in the length of the diffusion path. Additionally, the Higuchi model takes into account the relaxation of polymer chains that affect the drug release mechanism, in addition to Fickian diffusion, thereby demonstrating its impact on the drug release mechanism [41]. Furthermore, the results obtained at intestinal medium are consistent with other studies in the literature [41–43]. For instance, Kolluru et al. declared that hydrophobic Docetaxel encapsulated PCL NP system fitted to the Higuchi model at pH 7.4 [42]. Wadhwa et al. proposed that the molecular weight of the PCL polymer is one of the important factors determining the release rate from PCL nanoparticles [44].

Table 6

Correlation coefficients of the release kinetic models at pH 1.2
NP	Zero Order	First Order	Korsmeyer-Peppas	Higuchi	Hixson-Crowell
PCL NPs-1	0.9878	0.9846	0.9883	0.8739	0.9859
PCL NPs-2	0.9813	0.9760	0.9839	0.8509	0.9781
PCL NPs-3	0.9843	0.9790	0.9804	0.8601	0.9811
PCL NPs-4	0.9956	0.9957	0.8222	0.8739	0.9959
Average	0.9873	0.9838	0.9687	0.8647	0.9853

Table 7

Correlation coefficients of the release kinetic models at pH 7.4
NP	Zero Order	First Order	Korsmeyer-Peppas	Higuchi	Hixson-Crowell
PCL NPs-1	0.9619	0.9908	0.9541	0.9883	0.9868
PCL NPs-2	0.9476	0.9882	0.9035	0.922	0.9838
PCL NPs-3	0.9659	0.9783	0.9330	0.9907	0.9909
PCL NPs-4	0.9414	0.9621	0.8569	0.9883	0.9852
Average	0.9542	0.9799	0.9119	0.9899	0.9867

3.6. Internalization of PCL NPs into the Cells

Internalization of Nile Red encapsulated PCL NPs into L929 fibroblast cells was visualized under a fluorescent microscope (Fig. 7). It is apparently seen that PCL NPs were internalized into the cells and the dye was carried into the cytoplasm. This shows the PCL NPs encapsulation system may also be used as a sustained drug delivery system to carry bioactive agents into the cells. It was also previously reported that internalization of PCL NPs into the cells may be caused by random interaction of the cell membrane and polymer [42].

3.7. In vitro cell proliferation studies

An in vitro cell proliferation assay was conducted to determine the effects of the active compound CBD on L929 normal fibroblast and MCF-7 breast cancer cells. According to the test results, MCF-7 cells, which are used as a model for breast cancer, exhibited a resistant proliferation profile compared to L929 cells in response to CBD. The IC₅₀ values calculated at different time intervals are listed in Table 8.

Table 8

The effect of CBD active compound on cell proliferation
Time (h)	IC₅₀ (µM)
	Cell line
	L929	MCF-7
24	0.79 ± 0.08	19.88 ± 0.87
48	1.12 ± 0.05	18.73 ± 0.75
72	6.17 ± 0.12	18.17 ± 0.82

The antiproliferative effect of CBD on L929 cells decreased as the duration of cell exposure increased. The cytotoxic effect of CBD on normal fibroblast cells decreased at 24, 48, and 72 h, respectively (Table 8). This could be attributed to the decrease in CBD concentration per cell as the cell number increased. The antiproliferative effect of CBD on MCF-7 breast cancer cells, although less than that on L929 cells, did not show a significant difference over time. CBD had a 3-fold greater antiproliferative effect on normal fibroblast cells after 72 h compared to its effect on breast cancer cells. This may be due to the drug resistance of cancer cells to various active substances. According to the results of our Molecular Docking study recently published, the LD₅₀ value of CBD when administered intravenously in rats was 799 mg per kg and was found to be safe in oral administration [45]. In their study, Mato et al. reported that the application of 10 µM CBD for 24 h resulted in a 30% antiproliferative effect on oligodendrocyte nerve cells [46]. The results here indicate that CBD has a reducing effect on the proliferation of both normal and cancer cells (Fig. 8, Fig. 9). In order to evaluate the effect of CBD in cancer treatment, nano formulations may be developed to target cancerous tissues in order to avoid damaging normal cells. In the design of the NP formulation targeting cancer cells, specific modifications targeting cancer tissues will be required. These modifications should enable the anticancer CBD-NP formulation to reach the cancer tissue without damaging normal tissues.

The high lipophilic properties of CBD make its use and dosing challenging and limit its pharmaceutical applications. The results of this study indicate that novel CBD-PCL NPs manufactured by O/W emulsion technique have potential to provide solution to the aforementioned challenge and are suitable as prolonged CBD release system having therapeutic advantages over free CBD oil consumption. Overall findings suggest the potential application of innovative NPs as carriers for sustained release of CBD to be used in medicinal oral supplement formulations. The promising results opened the window for the demonstration of the oral dosage form of CBD NPs in oral soft gelatin capsules. The oral soft gelatin formulation encapsulating the CBD-NPs in hemp oil is at the optimization and quality control phase in collaboration with pharmaceutical company Zade Vital, Konya.

The novel CBD-PCL NPs manufactured in this study have many of the featured properties of nanoparticles such as; tunable particle size, pH responsiveness, CBD-PCL compatibility and permeability in to the cells. Here the results shed the light for manufacturing the nanoparticles to increase the bioavailability of CBD in digestion system by in vitro analyses. Furthermore, CBD encapsulated PCL NPs may be tested on three-dimensional disease tissue models, and then in vivo preclinical studies may be carried out as the subsequent step. These studies will ease to elucidate the factors such as in vivo absorption rate, metabolism, safety and effectiveness of CBD released from NPs. In addition, biological activity may be enhanced by targeting the marker proteins of specific diseases by functionalizing the NPs with different ligands such as lipids, proteins or carbohydrates. The storage time and storage conditions of CBD encapsulated PCL and its soft gel formulation will be the potential research to be conducted.

Conflicts of Interest

The authors declare no conflict of interest.

Funding

This project was funded by Necmettin Erbakan University Scientific Research Projects (BAP) Commission with grant number 211451004. TUBITAK 2244 University Industry Collaboration Project (119C100) is also acknowledged.

Author Contribution

Conceptualization, E.F.E. and M.D.K.; methodology, M.D.K. and K.Ç.; software, E.F.E.; formal analysis, E.F.E.; investigation, E.F.E.; resources, M.D.K.; data curation, E.F.E. and K.Ç. writing—original draft preparation, E.F.E and M.D.K.; writing—review and editing, M.D.K.; visualization, K.Ç.; supervision, M.D.K.; project administration, M.D.K.; funding acquisition, M.D.K. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We gratefully acknowledge Zade & Zade Vital İbn-i Sina R&D Center for their support.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Development and Characterization of the Novel Cannabidiol Loaded Polycaprolactone Nanoparticles to Boost the Bioavailability in Medicinal Oral Supplements

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Chemicals and Materials

2.2. Method

2.2.1. Preparation of PCL NPs

2.2.2. Particle size, polydispersity index, and zeta potential measurements

2.2.3. CBD encapsulation

2.2.4. Reaction efficiency

2.2.5. Morphology of NPs

2.2.6. Fourier transform infrared spectroscopy (FTIR) analysis

2.2.7. CBD-PCL solubility and compatibility

2.2.8. In vitro release studies

2.2.9. Cellular uptake of NPs

2.2.10. In vitro cell proliferation assay

3. Results and Discussion

3.1. Physicochemical Characterization of PCL NPs

3.1.1. Particle size, polydispersity index, and zeta potential of NPs

3.1.2. FTIR analysis

3.2. Morphology of NPs

3.3. Reaction Efficiency, Loading Capacity and Encapsulation Efficiency

3.4. CBD-PCL Solubility and Compatibility

3.5. In Vitro Release of CBD from PCL-NPs

3.6. Internalization of PCL NPs into the Cells

3.7. In vitro cell proliferation studies

4. Conclusions

Declarations

Conflicts of Interest

Funding

Author Contribution

Acknowledgments

Data Availability Statement

References

Additional Declarations

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