Key Transdermal Patch Using Cannabidiol-Loaded Nanocarriers with Better Pharmacokinetics in vivo

Introduction

Cannabidiol (CBD) is a promising therapeutic drug with low addictive potential and a favorable safety profile.¹ Studies have shown that CBD does not exhibit the psychoactive effects commonly associated with tetrahydrocannabinol (THC), making it a safer option for therapeutic use. Its non-addictive nature makes it an appealing choice for patients seeking effective treatments without the risk of dependency. Clinical studies have indicated that CBD holds promise for potential applications in treating various chronic diseases and neurological conditions, with possible positive effects for managing conditions such as epilepsy,^2,3 Alzheimer’s disease,⁴ Parkinson’s disease,^5,6 sleep disorders,^7,8 depression and psychotic disorders,⁹ anxiety,⁹ schizophrenia,¹⁰ and post-traumatic stress disorder.¹¹ However, CBD has faced certain challenges affecting its administration and efficacy. One challenge has concerned its hydrophobic nature, with poor water solubility posing difficulties in formulating CBD into aqueous solutions, limiting its options for oral delivery.^12,13 Another disadvantage has been the low oral bioavailability of CBD. CBD undergoes extensive first-pass metabolism in the liver when taken orally, significantly reducing its bioavailability.^14,15 Hence, only a fraction of the orally administered CBD reaches the systemic circulation in an active form, reducing its therapeutic effectiveness. Despite these drawbacks, ongoing research has aimed to develop innovative strategies to improve CBD’s solubility and oral bioavailability, including nano formulations and other delivery systems. These advancements may enhance the effectiveness of CBD as a therapeutic drug and expand its applications in various medical conditions.

In the pharmaceutical industry, oral intake remains the most preferred method of drug administration due to its advantages, such as the availability of various dosage forms, painless administration, the feasibility of solid formulations, self-administration, and patient compliance.^16–18 However, oral drug delivery also has disadvantages, including drug stability in the gastrointestinal tract, first-pass metabolism, poor water solubility, and limited absorption through physiological barriers.^19,20 To address these drawbacks, transdermal drug delivery has emerged as a promising alternative. A transdermal drug delivery system (TDDS) utilizes the skin to administer therapeutic agents.^21,22 These agents permeate the skin, are absorbed into the blood vessels, and are distributed throughout the body.^23–25 TDDS offer non-invasiveness, minimal pain, first-pass metabolism bypassing, ease of administration, independence from medical personnel, and potential applicability to a wide range of hydrophilic and hydrophobic drugs.^26–30 However, one limitation of transdermal drug delivery is the difficulty of drug penetration through the human skin, which is hard and resistant to infiltration.³¹ The human skin comprises three main layers: the epidermis, dermis, and hypodermis. The low water concentration gradient from 75% in the viable epidermis to only 10–30% in the stratum corneum contributes to the lower permeability of molecules and drugs across the stratum corneum.³²

In recent years, nanoparticle drug delivery systems in TDDS have shown significant potential for targeted drug delivery, increased drug stability, prolonged drug action, and improved bioavailability.^33–38 Several nanocarrier formulations have been developed to enhance transdermal drug delivery, including liposomes, ethosomes, transfersomes, polymeric micelles, self-nanoemulsifying drug delivery system (SNEDDS), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLCs), and polymeric nanoparticles.^39–44 Despite the growing popularity of TDDS, there have been limited developments in formulations specifically for CBD. Nanocarriers formulation provides a promising platform for enhancing the delivery of CBD. Indeed, encapsulating CBD within nanocarriers could overcome its limited solubility in water, improve stability, and potentially prolong its release. These nanocarriers could be designed to facilitate the penetration of CBD through the skin barrier, allowing for efficient absorption into systemic circulation. Thus, integrating this nanocarriers-based CBD formulation into a transdermal patch offers a synergistic approach to optimizing CBD delivery. The patch provides a convenient, non-invasive method for delivering CBD, while the nanocarriers formulation enhances its bioavailability by facilitating its permeation through the skin. This combined approach holds great potential for the treatment using of CBD.

Therefore, this study aimed to harness the potential of CBD by combining it with a TDDS. This innovative approach sought to develop a transdermal patch dosage form with micellar vesicularnanocarriers to enhance the bioavailability of CBD, leading to improved therapeutic outcomes. We aimed to address the challenges associated with CBD’s hydrophobicity and low oral bioavailability by utilizing micellar vesicular nanocarriers technology combined with formulation materials skin-penetrating properties. Micellar vesicular nanocarriers demonstrated promise for enhancing the delivery of CBD. Thus, encapsulating CBD within nanocarriers could overcome its limited solubility in water, improve stability, and potentially prolong its release. These nanocarriers could be designed to facilitate the penetration of CBD through the skin barrier, allowing for efficient absorption into systemic circulation.

Materials and Methods

Materials included Strat-M membrane (Strat-M Membrane, Transdermal Diffusion Test Model, 25 mm, Merck, Darmstadt, Germany), d-limonene 97% (Sigma-Aldrich, USA), Tween 80 (Sigma-Aldrich, USA), cannabidiol (SCI Pharmtech, Inc., Taiwan), 2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt solution (AMPS, Sigma-Aldrich, USA), N, N-methylene-bis-acrylamide (NMBA, Sigma-Aldrich, USA), hydroxyethyl cellulose (HEC, Sigma-Aldrich, USA), glycerol (Sigma-Aldrich, USA), diethylene glycol monoethyl ether (DEGME, Sigma-Aldrich, USA), and 2-hydroxy-2-methylpropiophenone (UVC-1173, TCI, Japan). All chemicals were of analytical grade and used without further purification.

Preparation and Characterization of Transdermal Nanocarriers

Transdermal nanocarriers were produced using a nano emulsion method of Tween 80 as the surfactant phase combined with limonene as the oil phase in different wt% (Table S1). After mixing the surfactant and oil phases at 50 °C 1000 rpm, we added D.D water inside and sonication with the condition of 600 W in ultrasonicator (Sonicator 3000, Misonix, Inc., New York, USA) for 30 min. The total weight of the solution was 1 g. After water addition, changes in sample appearance were visually monitored to determine if a precipitate, turbidity, or homogeneous solution was constituted.⁴⁵ The transdermal nanocarriers product are referred to as TD-NCs. The physical characterization of the developed TD-NCs used the dynamic light scattering (DLS) technique carried out by Malvern ZetaSizer Nano ZS (Malvern Instruments; Malvern, UK), and data were exported in Malvern Zetasizer Software v7.02.

Development and Characterization of CBD Loaded Nanocarriers

The method of preparing CBD loaded TD-NCs (CTD) was similar to preparing TD-NCs. First, 200 mg CBD was dissolved individually in 100 mg and 200 mg limonene and sonicated with the condition of 600 W in ultrasonicator (Sonicator 3000, Misonix, Inc., New York, USA) for 30 min. After achieving two groups of transparent solution, 400 mg and 100 mg of Tween 80 were added individually at 50 °C 1000 rpm. Next, 500 mg and 700 mg of D.D water were added individually inside and sonicated with the condition of 600 W in ultrasonicator (Sonicator 3000, Misonix, Inc., New York, USA) for 30 min. The total weight of the solution was 1.2 g. Finally, CTD-41 was made from 200 mg CBD, 100 mg limonene, 400 mg Tween 80, and 500 mg D.D water, while CTD-12 was made from 200 mg CBD, 200 mg limonene, 100 mg Tween 80, and 700 mg D.D water. Samples were stored at 4 °C for further use. The physical characterization of CTD used the DLS technique by Malvern ZetaSizer Nano ZS (Malvern Instruments; Malvern, UK), and data were exported in Malvern Zetasizer Software v7.02. The morphology of the CTD was observed using high-resolution transmission electron microscopy (HR-TEM; Tecnai G2 20 STWIN). For the sample preparation of TEM imaging, the CTD were diluted 10-fold with 2%wt phosphotungstic acid. After mixing, 20 ul solution was dropped onto a 200 mesh formvar/carbon copper mesh (TED PELLA INC, USA) and dried using an oven at 37 °C before taking on HR-TEM.

The encapsulation efficiency (EE%) was measured and determined using HPLC and calculated using the Equation 1:

(1)

Where W_total means the initial weight of CBD added into the system and W_free means the weight of CBD did not in NPs. The drug loading (DL%) were measured with freeze-dried method and determined using HPLC and calculated using the Equation 2:

(2)

Where W_{CBD in NPs} was the weight of CBD in formulation after being freeze-dried, and W_NPs was the dry weight of the formulation after being freeze-dried.

The HPLC analysis method was modified from previous studies.^46–48 Next, the appropriate standard of CBD was dissolved in MeOH diluted with MeOH to obtain solutions of known concentration, nine points in a concentration range between 0.98 and 250 μg/mL (ie, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125.00, and 250.00 μg/mL). The standard solutions were stored away from light at −20 °C. The analysis was performed on an Agilent 1260 Infinity II LC System, consisting of a quaternary pump, a vials autosampler, a multicolumn thermostat column compartment, and a diode array detector (DAD). A ReproSil 100 C18 column (4.6 × 250 mm, 5 μm, Dr. Maisch GmbH, Germany) was used with the flow rate was set at 1 mL/min for 20 min and a mobile phase composed of 15% 0.5%-acetic acid and 85% MeOH. The column temperature was set at 30° C. The sample injection volume was 20 μL. The UV/DAD acquisitions were carried out in the 190–300 nm range, and chromatograms were acquired at 220 nm. Each group was prepared in triplicate, while the sample was previously filtered with a 0.22 μm PTFE filter.

Evaluation the Skin Permeation Ability of Nanocarriers

The skin permeation ability used Strat-M™ membrane with a transdermal diffusion system (DHC-6T, LOGAN Inc, USA).^1,49 Thus, 10 mg CBD and CTD containing the same weight CBD were added on the top of the Strat-M™ membrane. The diffusion experiment was initiated by charging the receiver compartment containing 6%wt Tween 80 in PBS for all groups. Besides, the free CBD group was using PBS to dispersed in donor receiver compartment. Three independent release experiments were conducted for each sample group, collecting full receiver solution and recharging fresh solutions at each time point: 0.5, 1, 2, 4, 6, 8, 12, and 24 hr. The collecting solutions were stored away from light at a temperature of 4 °C and filtered with a 0.22 μm PTFE filter before analysis in HPLC. At the final time point, the membrane was recollected and cut into pieces, adding 10 mL MeOH with sonication for 1 hr to elute the CBD inside the membrane for analysis. The HPLC analysis protocol for CBD was the same with we mentioned before in this study. Skin permeation ability data were plotted as the cumulative amount of drug collected in the receiver compartment as a function of time. Additionally, using the cumulative data and the equations, we calculated the flux (Equation 3), permeation ratio (Equation 4), release ratio (Equation 5), area under the curve (AUC, Equation 6), and relative formulation availability (FA, Equation 7) of each group:

(3)

where C_CBD was the cumulative CBD content at each time point during 24 hr, t_number was the time point during 24 hr, Area_M was the diffusion area between the membrane and donor compartment, M_CBD was the CBD content in Strat-M™ membrane, and AUC_0–24.was the area under curve during 24 hr.

Loaded CTD-12 into AMPS Gel Formulation

CTD-12 loaded AMPS gel formulation used a mixing and UV-curing method. Thus, 0.5 g NMBA was dissolved in 25 g AMPS solution and gently stirred. Next, 9.5 g glycerol was mixed and dispersed with 0.5 g HEC at 50 °C, followed by adding 9 g D.D water inside. Finally, the two solutions were mixed at room temperature to acquire the AMPS pre-mixture. The pre-mixture was stored away from light at a temperature of 4 °C for further use. In order to find the optimal ratio of CTD-12 loaded AMPS gel, we tried different combination parameters of AMPS pre-mixture with or without CTD-12 by fixing the photo initiator UVC-1173 (0.5 wt%) and co-solvent DEGME (4.5 wt%) at 5wt% in total formulation. Subsequently, the AMPS mixed with X wt% CTD-12 are referred to as CTD-12-X%. The optimal ratio is based on water content, contact angle, and gel curing appearance. The following uses the optimized formulation as a production example to illustrate the preparation steps. Subsequently, 5 g CTD-12 was mixed with 4.5 g AMPS pre-mixture at 800 rpm with 0.45 g DEGME added inside. Next, 0.05 g UVC-1173 was added inside the solution and stirred at 300 rpm while avoiding light and bubbles. The mixed solution was evenly coated on a glass slip and UV-curing (OPAS XLite 500, Taiwan) the sample for 45 sec. Next, the water content and contact angle of the sample were measured using a moisture analyzer (Sartorius MA37, Goettingen, Germany) and a contact angle meter (First Ten Angstroms 1000B, Newark, the USA). Subsequently, the optimized mixed solution was evenly coated on a melt-blown cloth with a manual coating rod at a thickness of 700 um. Finally, UV-curing (OPAS XLite 500, Taiwan) was applied to the sample at 45 sec and cut into 4 cm² (2*2 cm). The preparation patch was stored in a dry, light-proof location for future use. The patch was cut into pieces, adding 10 mL MeOH with sonication for 1 hr to elute the CBD inside the patch for analysis. The HPLC analysis protocol for CBD was the same with we mentioned before in this study. Subsequently, the terms prototype CTD-12 loaded AMPS gel patch are referred to as PCTD-12. Moreover, the free CBD AMPS patch were prepared and analyzed in the same protocol by using 1 mL MeOH dissolve CBD and mixing with D.D water to adjust the same wt% to PCTD-12.

Evaluation the Skin Permeation Ability of PCTD-12

The evaluation experiment of skin permeation ability for transdermal patch was the same protocol with we mentioned before in this study. Briefly, Free CBD patch and PCTD-12 were added on the top of the Strat-M™ membrane. The diffusion experiment was initiated by charging the receiver compartment containing 6%wt Tween 80 in PBS for all groups. Three independent release experiments were conducted for each sample group, collecting full receiver solution and recharging fresh solutions at each time point: 0.5, 1, 2, 4, 6, 8, 12, and 24 hr. The collecting solutions were stored away from light at a temperature of 4 °C and filtered with a 0.22 μm PTFE filter before analysis in HPLC. At the final time point, the membrane was recollected and cut into pieces, adding 10 mL MeOH with sonication for 1 hr to elute the CBD inside the membrane for analysis. The HPLC analysis protocol for CBD was the same with we mentioned before in this study. Skin permeation ability data were plotted as the cumulative amount of drug collected in the receiver compartment as a function of time. Additionally, using the cumulative data and the equations, we calculated the flux (Equation 3), permeation ratio (Equation 4), release ratio (Equation 5), AUC (Equation 6), and FA (Equation 7) of each group.

In vivo Pharmacokinetic Evaluation

The animal study was conducted according to the guidelines of the Animal Care Committee of the National Chung Hsing University and approved by the Committee (IACUC No. 111–127) and performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Specific-pathogen-free male Wistar rats aged 6 weeks were purchased from BioLASCO (licensed by Charles River, Taiwan) per the Animal Ethics Committee of IACUC registration IACUC No. 111–127. For the pharmacokinetic study, seven rats were used: four for the oral route (Oral 10 mg CBD) and 3 for PCTD-12. During the experiment, each animal was housed in a standard laboratory cage and raised separately under the following conditions: controlled temperature, 25 °C ± 2 °C; humidity, 30–70%; light/dark cycle, 6 a.m. lights on/6 p.m. lights off; and access to water and standard diet, ad libitum. The animals were acclimatized to the animal facility for 1 week. On the day before the experiments were conducted, the abdomen was shaved, with the shaved area as the constant section of the entire abdomen 2 cm below the forelimbs and 1 cm above the hind limbs.

A PCTD-12 patch was applied to the abdomen and fixed with an elastic bandage. At the time points of 0.5, 1, 2, 4, 8, 12, and 24 hr, 0.2 mL of blood was collected in 1 mL micro blood collection tubes for subsequent serum extraction analysis. Following blood collection for 24 hr, all rats were sacrificed under gas anesthesia. The blood was centrifuged at 6000 rpm and 4 °C for 2 min immediately after collection, and the serum was extracted and stored at –80 °C until used for CBD extraction.

The CBD extraction step was modified from the reference.^50–52 First, 20 µL 10M KOH was added to 100 µL serum and heated at 70 °C for 30 min, subsequently adding 5 µL formic acid left standing for 5 min. Next, 625 µL of ACN was added, mixed well, and ultrasonicated for 30 min, then centrifuged at 5000 G, 4 °C for 5 minutes. After completion, the supernatant was extracted and stored in a –20 °C refrigerator. After removing the supernatant, the underlying pellet was added to 625 µL of ACN and extracted twice, resulting in 1875 µL of extracted samples at each time point. The standard curve was prepared with the CBD stock solution, a concentration of 1 mg/mL (1000 ppm) in ACN, and diluted to the concentration range of 5000–9.76 ppb. The plasma standard curve was prepared with the following protocol: adding 100 µL of blank serum in a micro tube, then adding 500 µL of CBD standard solution prepared in ACN in a range of 5000–9.76 ppb. After mixing for 1 min, the preparation was sonicated for 30 min and centrifuged at 4 °C, 5000 G for 5 min. Subsequently, the supernatant was extracted and stored in a –20 °C refrigerator. After removing the supernatant, the underlying pellet was added to 500 µL of ACN and extracted twice, resulting in 1500 µL of extracted plasma standard samples of different concentrations.

The CBD concentration in plasma was analyzed using LC-MS/MS from the extracted samples. LC-MS/MS analyses were modified from the reference and performed on the Liquid Chromatograph-Tandem Mass Spectrometer TSQ Altis (Thermo Scientific, CA, United States).^52–58 Samples were analyzed on an XBridgeTM C18 column (2.1 × 100 mm; 3.5 μm particle size; Waters, Ireland). The mobile phase was composed of (A) 0.2% formic acid and (B) acetonitrile using the following gradient program: 0.0–0.2 min, 25% (B); 0.2–4.0 min, linear gradient from 25 to 100% (B); 4.0–4.2 min, 100% (B); 4.2–4.3 min, linear gradient from 100 to 25% (B); and 4.3–10.0 min, 25% (B). The flow rate was 0.2 mL/min, and the column temperature was 25 °C. The injection volume was 5 μL, and the injector needle was washed with methanol while the autosampler was maintained at room temperature.

The chromatographic conditions were optimized by analyzing the standard solutions and extracts of whole blood spiked with the target analytes. Analytic software was used for instrument control, data acquisition, and qualitative and quantitative data analyses. Detection and quantitation of all serum-extracted samples used a single reaction monitoring mode (SRM) due to the high selectivity and sensitivity. Optimized instrument settings were as follows: ionization mode, negative; sheath gas, 35 arb; aux gas, 7 arb; sweep gas, 0 arb; ion spray voltage, 2500 V; ion transfer tube temperature, 325 °C; vaporizer temperature, 275 °C. Quantification was performed using the transition m/z 313.4–245.1 (CE = 23 V, 100 msec) and the second transition 313.4–179.1 (CE = 20 V, 100 msec) for CBD with a retention time of 5.8 min. Pharmacokinetics data were plotted as the plasma concentration of CBD extracted from the serum as a function of time. Additionally, the data in the equations were used to calculate each group’s area under the curve of PK (AUC_{PK 0–24}, Equation 8) and relative bioavailability (BA, Equation 9):

(8)

where P_CBD was the plasma concentration of CBD extracted from serum at each time point during 24 hr, t_number was the time point during 24 hr, and Dose_group was the group dosage.

Results and Discussion

Various ratios of the oil phase and surfactant were tested to develop TD-NCs. The best composition for loading with CBD was selected based on the appearance of the solution, size, and PDI of TD-NCs. Limonene, a hydrocarbon lipophilic terpene, was chosen as the oil phase due to its high compatibility with CBD, solubility properties, and enhanced penetration abilities.^59–61 Tween 80, a nonionic surfactant known to enhance skin penetration,^62,63 was selected as the surfactant. The ability of these materials to enhance skin penetration arose from their interaction with and incorporation into stratum corneum components, which led to the disruption of the skin barrier function and an increase in skin interface permeability.⁶⁴ The ternary phase diagram in Figure 1 shows a combination of limonene, Tween 80, and D.D water used to determine the homogeneous range. The mixing conditions and steps to reach equilibrium were consistent for all mixtures. After equilibration, the mixtures were visually assessed for transparency, covering the entire phase region. In the diagram, the red area represents the milky range, the light gray area represents the precipitate region, and the dark gray area represents the turbidity region.

Figure 1 Ternary phase diagram. Light grey area represents the precipitate region, while the dark grey area represents the turbidity. Red area represents the milky region. Tween 80 = surfactant, Limonene = oil phase.

Moreover, the appearance of TD samples is shown in Figure S1. The selection of TD-NCs was based on the presence of homogeneously dispersed, hydrodynamic size, and PDI data. Table S2 overviews the appearance and DLS results of TD-NCs. Homogeneous appearance was observed in the range of 1:1.25 to 1:4 of Tween 80 to limonene weight percent (T:L). However, the size, PDI and Zeta potential of the solution did not exhibit a consistent pattern in response to changes in the T:L composition. One of the selected TD-NCs (TD-41) consisted of 40% Tween 80, 10% limonene, and 50% D.D water. Although TD-41 had higher PDI and higher zeta potential than others, it had the smallest hydrodynamic size among all the groups due to the high ratio of Tween 80. Another selected TD-NP (TD-12) was chosen based on the best PDI, the most negative zeta potential and smaller hydrodynamic size among all the groups. It consisted of 10% Tween 80, 20% limonene, and 70% D.D water. The developed TD-NCs were loaded with 16.66% CBD (total weight of CBD in solution / total weight of solution). CBD was dissolved in limonene and mixed, followed by sonication with Tween 80 and D.D water. Adding CBD decreased the PDI for both selected TD-NCs formulations, indicating increased uniformity of TD-NCs due to CBD’s high compatibility with limonene. The higher DL% in CTD-12 was attributed to its higher limonene composition ratio, offering good solubility for CBD.

Figure 2A and B showed the TEM images of both CTD. As shown in Figure 2A, CTD-41 exhibited a morphology resembling a micellar system, attributed to its higher emulsifier ratio, consistent with previous report.⁶¹ Interestingly, CTD-12 displayed a uniformly spherical shape, suggesting a self-assembled system (Figure 2B), similar with studies by Zhu et al and Lu et al^65,66 This may be attributed to the hydrophobic properties of limonene and CBD, leading to a stronger cohesive effect when emulsified with Tween 80, resulting in a more spherical appearance (Figure 2C). Additionally, from the TEM images of CTD, it can be inferred that the particle aggregation ability of CTD-41 and CTD-12 may vary. Factors influencing this phenomenon stem from the surfactant’s capability within the formulation. Surfactants can potentially lower interfacial tension, providing space and electrostatic repulsion to prevent particle agglomeration and stabilize the emulsion.^67,68

Figure 2 Morphology of different nano emulsion transdermal nanoparticle by TEM. (A) CTD-41. (B) CTD-12. (C) Schematic of the structural difference between CTD-41 and CTD-12. Arrows indicate the direction of change in formulation ratio.

The above experiments demonstrated that due to the differences in T:L ratios, CTD-12 and CTD-41 had different morphologies and sizes. Therefore, the potential application of CTD as a transdermal formulation was further explored. The transdermal ability of CTD through artificial skin membranes was quantified using a transdermal diffusion system with a Strat-M^TM membrane, which has been widely used in transdermal diffusion testing.^69–72 The receiver channel was filled with a 6% wt% Tween 80 solution in PBS, which provided suitable sink conditions during the assays and was compatible with the skin membranes. The total amount of CBD in all groups was fixed at 10 mg, and the CBD concentration in the receiver channel was measured at different time points.

Figure 3A shows the 24-hr cumulative in vitro permeation CBD curves for the free CBD, 6% Tween 80, and CTD. The flux and permeation ratio of the drug permeated through the Strat-M^TM membrane at the end of 24 hr for all groups are provided in (Figure 3B and C), respectively. Table 1 presents the numerical results of skin penetration from 0–24 hr for all groups. In the initial period of the skin permeation test, the Tween 80 group exhibited faster penetration and higher flux than the other groups within 2 hr, which was attributed to its chemical properties.^62–64 However, a significant increase in total CBD content was observed after changing the formulation into transdermal nanoparticles (CTD). These phenomena occurred due to the presence of Tween 80 and Limonene, which reduced the skin’s barrier function by dissolving the lipid bilayer of the stratum corneum and enhanced the penetration ability of CBD into the skin interface.^61,62,64,73 CTD-41 began to exceed the free CBD curve at 4 hr and exceeded the 6% Tween 80 curve at 6 hr, while CTD-12 exceeded the free CBD curve at 1 hr and the 6% Tween 80 curve at 6 hr. Moreover, the AUC_0-24hr of CTD-41 and CTD-12 was higher than the AUC_0-24hr of the free CBD and the 6% Tween 80 groups. Surprisingly, the relative flux enhancement factors (FA) of CTD-41 and CTD-12 compared to the free CBD group were 2.43 ± 0.04-fold and 3.68 ± 0.17-fold, respectively, indicating their significant potential for future applications. Furthermore, CTD-12 performed better than CTD-41 in cumulative CBD content, flux and permeation ratio from 0–24 hr. In Table S3, we compared previous research on various CBD nano formulations and examined patent results related to CBD’s transdermal capabilities. Whether it was the use of CBD with penetration enhancers alone or in a gel combined with CBD and penetration enhancers, CTD-12 consistently demonstrated superior performance compared to these patents.^74–77 When compared to other nano formulation systems, such as transfersome, NLC and so on, CTD-12 still indicated higher performance.^78–82 Therefore, CTD-12 was chosen as a promising transdermal formulation for further prospective application use in transdermal drug patches.

Table 1 Numerical Results of 0–24 hr Skin Permeation for Free CBD, 6% Tween 80, CTD-41 and CTD-12

Figure 3 (A) Cumulative CBD content (B) Flux (C) Permeation ratio of Free CBD, 6% Tween 80, CTD-41 and CTD-12 during 0–24 hr at skin permeation experiment.

AMPS, a monomer used in superabsorbent hydrogels, proved suitable for establishing hydrogel interconnections and was chosen for use in this research.^83–85 The focus here was on maximizing the content of CBD, representing the optimal mixing ratio of CTD-12 and AMPS. In addition to drug content, criteria for judging the selection included gel appearance, UV curing integrity, contact angle, and water content. Figure S2 displayed the curing appearance, water content, and contact angle of different ratios of AMPS hydrogel mixed with CTD-12. As the water ratio in the formulation increased, the contact angle decreased, water content increased, and the AMPS hydrogel maintained a transparent appearance. However, when changing the distilled water into CTD-12, the appearance of the CTD-12-AMPS hydrogel became milky and not transparent. Most importantly, as the CTD-12 ratio increased to 60%, the gel would not cure completely. This indicated that CTD-12-50% was the best mixing ratio, composed of 50% CTD-12, 45% AMPS pre-mixture, 4.5% co-solvent, and 0.5% photo initiator. Subsequently, to cure the CTD-12-AMPS hydrogel into a specific shape, melt-blown cloth was chosen as the base material to support the shape. The prototype CBD-loaded transdermal nanocarrier drug patch was referred to as PCTD-12.

The above experiments demonstrated the optimal mixing ratio of CTD-12 and AMPS hydrogel and finally produced the prototype transdermal nanocarrier drug patch PCTD-12. Therefore, the potential of PCTD-12 as a transdermal drug patch was further explored. The transdermal ability of PCTD-12 through artificial skin membranes was quantified using the same method as we mention in this study before. (Figure 4A) shows the 24-hr cumulative in vitro permeation CBD curves for the free CBD patch, and PCTD-12. The flux and permeation ratio of the CBD permeated through the Strat-M^TM membrane at the end of 24 hr for the free CBD patch and PCTD-12 are provided in (Figure 4B) and (Figure 4C), respectively. Table 2 presents the numerical results of skin penetration from 0–24 hr for the free CBD patch and PCTD-12. In the initial period of skin permeation testing, PCTD-12 exhibited faster permeation and higher flux than the free CBD patch within 4 hr, and it still retained a certain penetration capacity after 4 hours. Within 24 hr permeation testing, PCTD-12 demonstrated the better performance than free CBD patch, which is due to the ability of the CTD-12 nanocarrier. These results indicated the potential of PCTD-12 as a transdermal nanocarrier drug patch for CBD.

Table 2 Numerical Results of 0–24 hr Skin Permeation for Free CBD Patch and PCTD-12

Figure 4 (A) Cumulative CBD content (B) Flux (C) Permeation ratio of Free CBD patch and PCTD-12 during 0–24 hr at skin permeation experiment.

PCTD-12 was used in a preliminary pharmacokinetic study in rats to demonstrate the potential of the developed transdermal nanocarrier drug patch for future applications. The mean plasma concentration-time profiles of oral administration CBD at a dose of 10 mg per rat and PCTD-12 in one patch per rat are presented in Figure 5. Additionally, the numerical results of the 0–24 hr plasma concentration curve for oral and PCTD-12 administrations are summarized in Table 3. The pharmacokinetic parameters in rats exhibited several differences among the oral and PCTD-12 groups. In contrast to PCAS-12 groups, the Oral group showed lower C_max, cumulative CBD concentration, and AUC values. Notably, PCTD-12 exhibited significant potential in the transdermal route, with a relative bioavailability (based on oral administration) of 9.47 ± 0.88-fold, higher than the oral group. The parameters and plots obtained in this study were similar to more recently reported findings.^57,58,86 However, few pharmacokinetic studies have been reported for CBD in rats, particularly through the IV or Oral route.^{57,58,86–89} Notably, PCTD-12 increased the plasma concentration of CBD during the 0–4 hr period. After 4 hr, PCTD-12 maintained plasma concentrations above 100 ng/mL for up to 24 hr. This sustained release phenomenon was due to the transdermal nanocarrier (CTD-12) in the patch, allowing for penetration through the skin. Tables S4 compare research and patent results regarding CBD transdermal ability and pharmacokinetics.^50,90–92

Table 3 Numerical Results of 0–24 Hr Plasma Concentration Curve of Oral and PCTD-12

Figure 5 Plasma concentration curve of oral and PCTD-12 for 0–24 hours.

In general, larger nanoparticles (size > 200 nm) encounter difficulty in penetrating the skin barrier and delivering the carried drug into the bloodstream, with the primary challenge lying in the ineffective permeation through the stratum corneum barrier.^93,94 Moreover, in non-nano carrier systems, surfactants cause a greater lipid disorientation effect in the stratum corneum and result in higher levels of cutaneous absorption compared to terpenes and solvents.⁹⁵ Increasing the concentration ratio of the surfactant in the nano carrier composition may not always lead to enhanced permeability of the carrier; conversely, it could impede the release of the drug or carrier due to interfacial interactions.^96–98 Additionally, the soft and hard segments in surfactants influences the micellar system, contingent upon the ratio of surfactants in the formulation, and has been demonstrated to affect the structural properties of the carrier, enhancing micellar packing, tuning mechanical properties, and improving interactions between oil and water interfaces.^99–102 In this study, CTD-12 chose materials such as Tween 80 (surfactants) and limonene (terpenes) as the main structural components of the nano carrier, which also served as penetration enhancers, facilitating carrier disruption of sebum, permeation through the stratum corneum, or across skin tissue layers. It is speculated that through the carrier design of CTD-12, the self-assembly characteristics enable it to maintain the shape and function of the nano carrier when penetrating the skin barrier, achieving better skin penetration through the tensile properties brought by the soft material and the lipid disturbance caused by the characteristics of the carrier material. The skin penetration data of CTD-41 (micelle system-like, containing more surfactant Tween 80) and CTD-12 (self-assembly system-like, containing less surfactant Tween 80) in this study further confirm this speculation. Once these nano-carriers breach the stratum corneum barrier and enter the epidermis and dermis tissues, the drug within the carrier can diffuse or be absorbed through various pathways, including intercellular, intracellular, and through follicular routes, ensuring effective drug delivery.^64,73,93,103 Furthermore, the endothelial cells of blood vessels and lymphatic vessels in the skin tissues served as pathways for the diffusion of drugs from the carrier into the bloodstream. Despite the size of nano-carriers hindering direct penetration into the vessels, CTD-12 still achieve enhanced drug bioavailability through optimized interactions, improved skin permeability, and effective drug delivery pathways.

Microneedle systems are widely believed to facilitate complete drug penetration through the stratum corneum, achieving optimal transdermal absorption.^104,105 In this study, it demonstrates that CTD-12 exhibits superior performance compared to previous CBD microneedle patents.⁷⁷ The reasons for this extend beyond differences in skin source and solution selection compared to patented technology. Notably, the patent under discussion relies on a conventional microneedle drug delivery system without incorporating nanocarrier technology. Instead, it directly employs CBD prodrug and CBD with microneedle patch technology. Traditional microneedle formulations have drawbacks, including the need for tailored design to match specific drug molecules, limited drug loading capacity, potential for skin allergies, and issues such as pore blockage or drug deposition at the application site.^106,107 Despite efforts to enhance microneedle systems and incorporate nanocarrier technology for next-generation transdermal drug delivery patches, the high production costs remain a significant challenge.^108–111 This study introduces the CTD-12 transdermal carrier formulation, tailored for CBD properties, as a cost-effective and easily scalable solution with excellent performance. Ultimately, both CTD-12 as a transdermal formulation and PCTD-12 as a drug patch demonstrate significant potential for future applications.

Conclusion

This study highlighted the promising attributes of CTD-12, a transdermal nanoparticle formulation, including small particle size, high encapsulation efficiency, and a favorable drug-loaded ratio for CBD. While demonstrating potential in drug patch systems, exploration of diverse patch formulations, such as oil gels or hydrogel systems, in tandem with CTD-12 was not undertaken in this study. Future research should focus on comprehensive bioavailability studies and diverse formulation combinations to advance CTD-12 development in TDDS. Overall, CTD-12 presented a promising transdermal delivery option for CBD, potentially improving treatment outcomes for various chronic diseases where CBD could be applied.