Development and Characterization of Dissolving Microneedles for the Buccal Delivery of Cannabidiol (CBD)

TL;DR

This study developed dissolving microneedles for delivering cannabidiol (CBD) through the mouth, offering a more effective and patient-friendly alternative to traditional oral methods.

Contribution

The study introduces a novel method using 3D-printed molds to customize microneedle geometry for buccal CBD delivery.

Findings

Needles were successfully inserted into buccal tissue and released CBD in a controlled manner.

The approach bypasses first-pass metabolism, potentially improving CBD bioavailability.

Histological evaluation confirmed the safety and integrity of the mucosa after microneedle application.

Abstract

This study aimed to develop dissolving microneedles (MNs) for the buccal delivery of cannabidiol (CBD). CBD is a non-psychotomimetic phytocannabinoid with anti-inflammatory and anxiolytic properties. The MN arrays were produced using micromolding, which has the ability of scalability. However, this approach lacks the ability to customize needle geometry; thus, additive manufacturing was implemented in the study. Digital Light Processing (DLP) printing is a promising way to produce molds with customized MN architecture. In the present study, molds were fabricated from 3D-printed MN arrays to prepare dissolving MNs for buccal administration. Polymeric needles based on Eudragit L100-55 and Eudragit RSPO were produced from reverse molds and they were evaluated regarding their physiochemical and mechanical properties, followed by in vitro and ex vivo studies using porcine buccal mucosa. Visualization studies were conducted using confocal scanning laser microscopy, whereas the membrane integrity of the porcine mucosa upon application of the MNs was assessed by histological evaluation. Our results suggest that the needles can be effectively inserted into the buccal tissue and release the active pharmaceutical ingredient (API) in a controlled manner. This approach offers a patient-friendly alternative to oral CBD delivery, bypassing first-pass metabolism.

Figures

Microscopic images of (A) L100-55 MNs and (B) RSPO MNs. SEM micrographs of (C) L100-55 MNs and (D) RSPO MNs. CLSM images of (E) L100-55 MNs and (F) RSPO MNs.

(A) CLSM images of buccal tissue after L100-55 MN application; (Ai–Aiii) show cross-sections and top views. (B) CLSM images after RSPO MN application; (Bi–Biii) show similar views. (C–E) Histological images of control, L100-55, and RSPO MN-treated tissues.

Full text

1. Introduction

Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid derived from Cannabis sativa, known for its many therapeutic abilities including in the treatment of epilepsy, chronic pain, anxiety, or even some neurodegenerative conditions [1]. However, these therapeutic effects are limited due to its poor solubility and low bioavailability when administered per os [2]. Even though the oral route is the most convenient for patients, the bioavailability of CBD ranges from 9 to 13%, as it has a significant first-pass metabolism and it is prone to oxidation starting in the intestine [3]. To address these problems, oromucosal delivery of CBD has been proposed using sprays or gels [3,4]. In this way, first-pass metabolism is avoided and bioavailability is increased. The buccal mucosa provides a competent route of administration and is has been suggested that it may serve as a reservoir for CBD. Nonetheless, issues regarding the washout of the API through the saliva limit the development of buccal formulations containing CBD [5]. Therefore, it is crucial to design drug delivery systems that can improve mucosal adhesion, retention, and penetration, while reducing discomfort.

Oromucosal applications of CBD have been proposed for both topical and systemic indications. The oral cavity has many endocannabinoid receptors offering therapeutic potential for CBD in addressing oral illness. The cannabis plant has been used to treat a wide range of conditions, including oral and dental problems such as mouth ulcers, oral mucositis, microbial infections of the oral cavity, periodontal abscesses, inflammatory oral diseases, and tooth pain [4]. To increase the bioavailability and stability of cannabis extracts, d’Angelo et al. developed a nanoemulsion for buccal application [6]. Sativex^®^ is the first cannabis-derived buccal spray approved by the European Medicine Agency (EMA) for the treatment of neuropathic pain associated with multiple sclerosis [7]. However, this spray has a high concentration of ethanol, limiting its use as it can cause irritation and discomfort in the oral cavity [8]. Therefore, in the present study, buccal microneedles were designed for the buccal delivery of CBD.

Microneedle (MN) technology can address these issues and improve the absorption of CBD though the oral cavity. MNs are a non-invasive drug delivery system used to deliver drugs though the skin [9], the buccal tissue [10], the cornea [11], and the intestine [12]. There are many types of MNs, namely solid, coated, hollow, and dissolving. Dissolving MNs are designed to dissolve completely upon insertion, releasing the API directly into the targeted tissue without requiring device removal [13]. Thus, they are considered an optimal option for buccal applications.

In this study, dissolving MNs were fabricated using Eudragit L100-55 (L100-55) and Eudragit RSPO (RSPO). CBD was incorporated in the produced arrays and methylcellulose (MC) was used to create a backing layer to enhance the structural integrity of the MNs and to enable the unidirectional release of CBD by preventing its ingestion [14]. The developed formulations were characterized in terms of morphology, mechanical behavior, drug release, tissue permeation, and histological compatibility. This approach seeks to provide a possible and effective platform for CBD delivery via the buccal route, addressing current formulation and bioavailability challenges associated with CBD therapy.

2. Materials and Methods

2.1. Materials

Eudragit^®^ L100-55 (L100-55) and Eudragit^®^ RSPO (RSPO) were obtained from Evonik (Essen, Germany). Ethanol was purchased from Honeywell International Inc. (Charlotte, NC, USA). Cannabidiol (CBD) in crystalline form was kindly donated from Enecta Nature’s Nectars (Bologna, Italy). All other reagents were of analytical grade.

2.2. Methods

2.2.1. Fabrication of Polymeric MNs Using Micromolding

Buccal MNs were prepared using a mold casting technique combined with vacuum and centrifugation cycles (2000 rpm, 10 min) to ensure structural uniformity and reproducibility. Polymeric solutions of L100-55 or RSPO (30% w/v) in ethanol were prepared and CBD was dissolved completely into them at a concentration of 5% w/v. This concentration was selected based on preliminary experiments to ensure the formation of MNs with structural integrity. Water-soluble polymers like PVP and PVA were also tested; however, CBD is a highly hydrophobic API with very low solubility in water (12.6 mg/L, logP 6.3) [15]. Thus, Eudragit-based polymers were employed to ensure the solubility of all the components in ethanol. Silicone molds were produced from 3D-printed MNs. The MNs were designed in AutoCAD 2021 (Autodesk Inc., San Francisco, CA, USA) and printed using a DLP printer (PartPro ×100, XYZ, Taiwan). The needles were designed to be in pyramid shape with 0.5 mm base and 1 mm height. A biocompatible resin (Dental SG, Formlabs, Somerville, MA, USA) was used for the production of the master templates. The biocompatible resin was selected to ensure that no toxic materials would be incorporated into the produced formulations. The master templates were printed directly onto the platform to ensure smoother surface and dimensional accuracy, with a layer height of 0.05 mm, and after printing, the MN templates were washed with isopropyl alcohol (IPA) to remove any residual resin, for 15 min [16]. Following this procedure, the printed templates were cured for 1 h to ensure full polymerization. After printing and curing, silicone was casted onto them and allowed to set overnight, yielding in flexible molds ready for MN fabrication. To enhance mechanical strength and prevent premature MN degradation due to humidity, a methylcellulose (7.5% w/v in water) backing layer was integrated onto the needles, followed by vacuum and centrifugation cycles.

The entire assembly was then dried at room temperature for 24 h to allow solvent evaporation and full structural stabilization. Once dried, the MNs were carefully demolded and stored in desiccators until further use.

2.2.2. Physiochemical Characterization

Physiochemical characterization was conducted using Infrared Spectroscopy and thermal analysis to examine the interactions between the polymers and the API.

2.2.3. Fourier-Transform Infrared (FTIR) Analysis

FTIR spectroscopy was employed to characterize all individual components as well as the prepared MN formulations to identify possible interactions between the API and carriers. This technique was used to ensure compatibility and to detect possible changes in functional groups that might indicate interactions or chemical modifications. Spectral analysis was carried out using an IR Prestige-21 FTIR spectrophotometer (Shimadzu, Kyoto, Japan). For this study, FTIR spectra were acquired within 500–4500 cm^−1^, at a resolution of 4 cm^−1^, providing detailed information on the molecular vibrations and structural characteristics of the samples.

2.2.4. Thermal Analysis

Thermal analysis of the MNs and their individual components was conducted using differential scanning calorimetry (DSC). The analysis was performed using a DSC 204 F1 Phoenix instrument (NETZSCH, Selb, Germany). Approximately 5 mg of each sample, was weighed and sealed in standard aluminum pans. Samples were heated from 25 °C to 150 °C at a constant rate of 10 °C/min under a continuous flow of dry nitrogen gas at 70 mL/min to ensure an inert atmosphere. Thermal events such as endothermic and exothermic transitions were recorded to identify potential polymorphic changes, assess polymer-drug interactions, and determine the physical state of CBD within the polymer matrix.

2.2.5. Morphological Characterization

The morphology of the MNs was first examined using an optical microscope (Celestron MicroDirect 1080p HD Hand-held Digital Microscope, Celestron, Torrance, CA, USA) to provide an initial qualitative assessment of their structural integrity. ImageJ 1x software (NIH, Bethesda, MD, USA) was used to analyze key dimensional parameters including base width, height, and needle sharpness. Further morphological assessment was carried out using scanning electron microscopy (SEM) with a Zeiss SUPRA 35VP (Zeiss, Oberkochen, Germany), to obtain detailed surface microstructure and high-resolution topographical information.

2.2.6. Mechanical Properties

The mechanical strength of the microneedles was evaluated using a universal testing machine M500-50AT (Testometric Company, Rochdale, UK). For compression testing, MN arrays L100-55 and RSPO were fixed to a metal rod and pressed against a metal plate at a rate of 0.5 mm/min until structural failure occurred. Insertion testing was performed on excised porcine buccal tissue. The patches were mounted on a cylindrical probe and inserted at 0.5 mm/min under a force of 40 N for 30 s. Post-insertion inspection of the tissue was followed to ensure effective penetration. The mechanical evaluations were conducted under quasi-static conditions to isolate material strength and fracture thresholds relevant to manual buccal application. The method was adopted from literature with slight modifications [17]. All the tests were performed in triplicate.

2.2.7. Visualization of the MNs Using Confocal Laser Scanning Microscopy (CLSM)

Confocal laser scanning microscopy (CLSM) was employed to investigate the diffusion and the uniformity of the API loaded into the MNs. The Nile red-loaded arrays were placed on glass slides and examined under the CLSM (Zeiss LSM 780 CLSM, Oberkochen, Germany). Z-stack images were captured by scanning each array from needle tip to base. Image acquisition was performed using ZEN lite 2011 software. Porcine buccal tissue was also examined under CLSM after MN application. Z-stack scanning was conducted to acquire sequential optical sections through the depth of each needle, allowing for full 3D visualization of the structure and dye penetration.

2.2.8. In Vitro Release of CBD

The MN patches were immersed in 50 mL PBS containing 3% w/v Tween 20. The system was maintained at 37 °C and at predetermined time points 1 mL was withdrawn and analyzed using high performance liquid chromatography (HPLC). Prior to analysis, drug loading was performed to ensure the amount of API loaded onto the arrays. The loading was calculated after dissolving the MNs in 100 mL ethanol and analyzing them using HPLC.

2.2.9. Quantification of CBD

The quantification of CBD was performed using HPLC. The HPLC system consisted of a UV–Vis detector (SPD-10A VP), a binary pump (LC-10 AD VP), and an autosampler (SIL-20A HT), all from Shimadzu (Kyoto, Japan). Chromatographic separation was achieved using a reversed-phase C18 column (3 μm particle size). The mobile phase was composed of acetonitrile:water (80:20, v/v), degassed for 15 min to prevent baseline instability and false peaks caused by dissolved gases. The system was operated at a flow rate of 1.0 mL/min and the detection wavelength was set at 230 nm. All samples and standards were analyzed in triplicate.

2.2.10. Ex Vivo Permeation Study

Tissue samples were mounted between the donor and receptor chambers of Franz diffusion cells (PermeGear, Hellertown, PA, USA). The receptor compartment, with filled with PBS (pH 7.4) and the donor with Simulated Saliva (SS, pH 6.8). The produced MNs were manually inserted into the mucosa. The system was maintained at 37 °C with gentle stirring to simulate physiological conditions. Sampling was conducted at predetermined time points and the amount of CBD permeated was analyzed using HPLC. At the end of the experiment, tissue samples were transferred to Falcon tubes containing 10 mL of ethanol to extract residual CBD. The samples were sonicated for 1 h and analyzed by HPLC.

2.2.11. Histological Evaluation

Following the permeation study, tissue samples were removed and fixed in 4% paraformaldehyde. After that, the specimens were embedded in paraffin wax. Sections of 10 μm thickness were cut using a microtome (Microm, LabX, Midland, ON, Canada) and stained using hematoxylin and eosin (H&E). After staining, the sections were observed under an optical microscope (Olympus EX41, Olympus, Tokyo, Japan).

2.2.12. Data Analysis

Statistical analysis was conducted using Origin software v. 7.5 (OriginLab, Northampton, MA, USA). All results are presented as mean ± SD.

3. Results

3.1. Physiochemical Characterization

FTIR was employed to investigate the individual components and the produced formulations. Figure 1A presents the characteristic peaks of the polymers, the API, and the MNs. CBD exhibited a characteristic band at 1600 cm^−1^ (indicated by the red arrow), attributed to C=C stretching vibrations [18]. However, these characteristic CBD peaks were not present in the MN spectra, suggesting that the drug was molecularly dispersed within the polymer matrix and not simply attached to surface.

DSC results are presented in Figure 1B. Pure crystalline CBD exhibited a sharp endothermic peak at approximately 70 °C (indicated by the red arrow), corresponding to its melting point. However, this melting peak was absent from the formulations, indicating a homogeneous dispersion within the polymer matrix and implying its amorphization [19]. These findings support the compatibility between CBD and both polymers and further confirm that the fabrication process resulted in stable systems with optimized dispersion of the API.

3.2. Mechanical Properties

Mechanical integrity is critical factor for a successful buccal delivery as the needles need to withstand the mechanical stress during application. Figure 1C,D present the compression and insertion test conducted on porcine buccal tissue, respectively. The compression test confirmed that the needles can withstand forces higher than 3 N per needle, rendering them appropriate for on-body applications [20].

The insertion test revealed that L100-55 MNs had slightly higher displacement due to their sharper tips, which contributed to a successful penetration. Figure 1E shows the perforation sites of the needles onto the buccal tissue, suggesting the needles can bypass the mucosa and effectively deliver the API into the tissue.

3.3. Morphological Characterization

3.3.1. Optical Microscopy

Optical microscopy analysis was conducted to evaluate the needles’ architecture. Images were taken to measure their length and base and tip diameters, as presented in Figure 2A,B. L100-55 MNs were 0.67 ± 0.05 mm in length, 0.53 ± 0.05 mm in base diameter, and 0.05 ± 0.02 mm in tip diameter. RSPO MNs were 0.54 ± 0.06 mm, 0.48 ± 0.06 mm, and 0.06 ± 0.02 mm, respectively. L100-55 exhibited lower viscosity and better rheological behavior as a water-soluble polymer. These properties facilitate mold filling and allow the formation of taller and sharper MNs. On the other hand, RSPO formed more viscous mixtures with higher viscosity that hindered mold filling, particularly at the needle tip regions, resulting in shorter MNs.

3.3.2. Scanning Electron Microscopy (SEM)

Figure 2C,D depict the SEM micrographs of the produced MNs, offering detailed insights into their morphology and structural integrity. Needle structures closely matched the geometry of the silicone molds, while their surface revealed layered features. These features are attributed to the replication of the surface texture from the 3D-printed MN masters used in mold fabrication. These findings suggest that the fabrication technique was successful as the produced MNs were identical to the master arrays used as templates.

3.3.3. Confocal Laser Scanning Microscopy (CLSM)

CLSM studies were performed to further investigate the structural integrity and API distribution to the MN arrays. Nile red, a lipophilic fluorescent dye was incorporated in L100-55 and RSPO MNs for visualization. Figure 2E,F illustrates the Z-stack images taken from each type of MN. Both MNs showed homogeneous dye distribution, with clearly defined layers and shapes, confirming consistent drug incorporation and proper molding.

Microscopic images of (A) L100-55 MNs and (B) RSPO MNs. SEM micrographs of (C) L100-55 MNs and (D) RSPO MNs. CLSM images of (E) L100-55 MNs and (F) RSPO MNs.

3.4. Drug Loading

Quantitative analysis using HPLC was performed to calculate the total amount of CBD incorporated into the produced MN arrays. The total drug content per L100-55 and RSPO MN array was 9.49 ± 1.22 mg and 11.92 ± 1.88 mg, respectively. The high drug content achieved in the present study is close to the therapeutic doses of the FDA-approved CBD products (e.g., Sativex^®^) [5].

3.5. In Vitro Release Study

The in vitro release study was conducted to evaluate the dissolution and drug release profiles of the API from the produced MNs (Figure 3A). As evident from Figure 3B, L100-55 MNs exhibited a controlled release, reaching up to 70% release of CBD within 240 min. This behavior is due to the pH-sensitive nature of the polymer, which dissolves at pH > 5.5, matching the PBS (pH 7.4) medium used. Conversely, RSPO MNs showed a total release of 5% at the same time, suggesting that the release of CBD is governed only by the swelling ration of RSPO resulting in poor release of the API at the time scale of 250 min.

The release data were fitted to the Korsmeyer–Peppas kinetic model (R2 = 0.9816 and 0.9418 for L100-55 and RSPO MNs, respectively). In both cases n > 0.5 suggesting that the API from both formulations has a non-Fickian diffusion.

3.6. Ex Vivo Permeation Study and CLSM

The ex vivo permeability study was conducted to evaluate the mucosal delivery of CBD through porcine buccal tissue following the application of MNs. The study was conducted in Franz diffusion cells, as evident from Figure 3C. Permeation of CBD after 4 h monitoring is shown in Figure 3D. L100-55 and RSPO MNs presented a similar permeation profile for the API. Although L100-55 MNs demonstrated better in vitro performance, their rapid drug release may result in local saturation of the tissue interface, potentially limiting further transportation of the API. Conversely, the slower, prolonged release of RSPO allows a steady and continuous flux of CBD across the buccal tissue, promoting sustained absorption and potentially improved tissue concentration over time [21]. Additionally, this was further confirmed after tissue extraction. The extraction of CBD after RSPO MN application was up to 2-fold higher than that of L100-55 MNs (395 ± 26 μg compared to 216 ± 13 μg, respectively) (t-test, p < 0.01)

MNs loaded with Nile red, a model lipophilic dye, were applied onto the mucosa, and 4 h after application the buccal tissues were removed and scanned with CLSM. Figure 4A,B depict the Z-stack images representing the diffusion of the dye into the tissue. L100-55 MNs increased the penetration depth of the dye, suggesting that the transportation of the API can be increased after MN application. On the other hand, RSPO MNs did not completely dissolve in the tissue sample and some fragments were visible after scanning. These fragments may serve as drug reservoirs, supporting a controlled release of CBD, leading to improved patient compliance through reduced dosing frequency.

3.7. Histological Evaluation

A histological study was conducted to evaluate the safety of the produced MNs. Hematoxylin and eosin (H&E) staining was performed on tissue sections with and without MN application (Figure 4C–E). The MN-treated samples did not present any tissue damage. Therefore, clearly, the MNs can bypass the first layer of the oral epithelium to deliver the API in the deeper layers of the tissue, increasing its absorption though the buccal route (Figure 4D). Figure 4C,D present a well-developed epithelium, suggesting that the after MN penetration the tissue holds its integrity [22]. These results indicate that the produced formulations could increase CBD transportation though buccal administration without damaging the tissue.

(A) CLSM images of buccal tissue after L100-55 MN application; (Ai–Aiii) show cross-sections and top views. (B) CLSM images after RSPO MN application; (Bi–Biii) show similar views. (C–E) Histological images of control, L100-55, and RSPO MN-treated tissues.

4. Discussion

MN technology has emerged as a versatile platform with wide range of applications. PVA-based MNs were fabricated to increase the absorption of catechin, a natural polyphenolic compound found in green tea. Catechin has very poor oral bioavailability, which makes a suitable candidate for MN-based delivery. PVA was combined with CMC and sugars, leading to different release profiles for catechin [23]. Therefore, in the present study, a natural compound, CBD, was incorporated in different Eudragit-based polymers for MN fabrication, with different release characteristics. Oral delivery of CBD was widely explored using different fabrication methods. Electrospinning was employed to produce fibers loaded with CBD using polyvinyl(pyrrolidone) (PVP) and Eudragit L-100 [24]. Due to low absorption of CBD when administered per os, different administration roads were explored. Adhesive cannabinoid-loaded transdermal patches were developed using extracts of Cannabis sativa and Eudragit^®^ E100 along with different permeation enhancers to increase the transdermal absorption of the actives [25]. In the present study, Eudragit L100-55 and RSPO were implemented for the fabrication of MNs for the buccal delivery of CBD.

Physiochemical characterization (Figure 1A,B) revealed the successful incorporation of the API inside the polymer matrix. Due to poor water solubility of CBD, amorphization has been suggested for CBD using Kollidon^®^ VA 64, Parteck^®^ MXP, and Eudragit^®^ EPO. EPO was considered more suitable to produce amorphous solid dispersion with CBD, indicating that the use to Eudragit-based polymers could enhance the solubility and bioavailability of CBD [26].

The produced MNs were evaluated regarding their mechanical properties and their ability to penetrate the buccal mucosa (Figure 1C,D). The results showed that both MN formulations are able bypass the first layer of the buccal mucosa without breaking or creating fragments, suggesting that they are suitable for buccal application. Eudragit E100 and Eudragit RS100 have been employed in the development of MN patches for the transdermal delivery of resveratrol [27]. Mechanical evaluation showed that the needles have sufficient mechanical strength to penetrate the skin, confirming that Eudragit polymers can produce appropriate MN arrays for on-body applications.

The morphological characteristics of the MNs were further evaluated using optical microscopy and SEM (Figure 2A–D). The MN have a characteristic layer-like surface, confirming that the methods used (3D printing and micromolding) can produce identical drug delivery devices based on 3D-printed master templates. The combination of additive manufacturing and molding is a promising way to manufacture MNs as 3D printing provides full customization regarding needle number and geometry, while molding provides the ability for scale-up manufacturing. To visualize the distribution of Nile red and, consequently, the distribution of CBD, CLSM imaging was performed (Figure 2E,F). CLSM is often used to examine the distribution of the actives onto the surface of the needles [28]. Both MNs had homogeneous dye distribution confirming the complete incorporation of the API into the MNs.

Due to their chemical compatibility, hydrophilic compounds integrate more uniformly into the hydrophilic MN matrix, promoting consistent device performance, whereas hydrophobic compounds tend to exhibit heterogeneous distribution, frequently resulting in low delivery efficiency. Dissolving MNs are the most extensively investigated platform for the delivery of hydrophobic compounds, owing to their comparatively high drug-loading capacity and formulation versatility. To address the challenges associated with hydrophobicity and improve drug solubilization for MN incorporation, a range of formulation strategies has been developed, including the use of co-solvency approaches. Thus, polymeric solutions in ethanol were selected to produce these buccal MN arrays. Aggregation was avoided as CBD is highly soluble in ethanol and the concentration remained below saturation [29,30].

Drug-loading and release studies were conducted to assess the release profile of the API after buccal administration. The MNs were able to incorporate high amounts of CBD similar to those of Sativex^®^, indicating that these formulations are very promising as drug delivery systems for CBD in the buccal tissue. L100-55 MNs exhibited a controlled release, reaching up to 70% release of CBD within 240 min. Conversely, RSPO MNs showed a total release of 5% at the same time (Figure 3B). These variations between the two polymers are due to their different physiochemical characteristics. L100-55 is a pH-sensitive polymer, which dissolves at pH > 5.5, matching the PBS (pH 7.4) medium used for the study. Thus, the release of CBD was more rapid. RSPO is a polymer mainly used for sustained release formulations [31], suggesting that CBD will be released in a slower manner. The release data were fitted to Korsmeyer–Peppas model suggesting that the diffusion of the API is non-Fickian (n > 0.5). These findings suggest that CBD has an anomalous transport and its diffusion is followed by erosion of the matrix [32].

The ex vivo permeability study (Figure 3D) presented a similar permeation behavior for the two MNs. Although L100-55 MNs exhibited superior in vitro performance, their rapid drug release may cause local saturation at the mucosa interface, potentially restricting further transport of the API. In contrast, the slower and more prolonged release profile of RSPO supports a consistent and continuous flux of CBD across the buccal tissue, facilitating sustained absorption and potentially higher tissue concentrations over time [21]. Tissue extraction could also confirm that, as the extraction of CBD after RSPO MN application was up to 2-fold higher than that of L100-55 MN.

Four hours after application of Nile red-loaded MNs, the porcine buccal tissues were visualized using CLSM to further examine the diffusion of the dye into the tissue specimens (Figure 4A,B). CLSM is commonly used to assess the penetration behavior of fluorescent dyes and is regarded as a semi-quantitative evaluation method [33]. In the present study only one time point was assessed. L100-55 MNs enhanced the dye’s penetration depth, indicating that API transport can be increased following MN application. In contrast, RSPO MNs did not fully dissolve within the tissue sample, with some fragments still visible after scanning. These residual fragments may act as drug reservoirs, enabling controlled CBD release and potentially improving patient compliance by reducing dosing frequency. The buccal site is commonly utilized for sustained-release formulations for both systemic and topical treatment. Buccal films were developed for the controlled delivery of omeprazole [34] while extended-release patches were fabricated to treat xerostomia [35].

The data provided in this study regarding the in vitro and ex vivo behavior of the produced MNs are important steps towards the clinical use of MNs. Emerging clinical data demonstrated that MNs could be better drug delivery systems than spray formulations. Dissolving MN patches proved to have a better immune response against influenza compared to intranasal vaccination [36]. MNs are promising drug delivery systems and have potential for clinical use. Other findings suggest that in vitro and ex vivo studies are an important step prior to clinical studies, as they provide important insight and prediction of their in vivo application [37].

Histological evaluation of the treated buccal tissues was carried out to ensure the MNs will not permanently damage the mucosa (Figure 4C–E). The images show no permanent damage after MN application, indicating that buccal MNs are safe and can be used for drug delivery. Other studies also evaluated the histological responses of buccal MNs showing that there is no significant or permanent damage after application [38]. Moreover, Li et al. examined the biosafety of MNs in the oral cavity by recording the recovery process of the mucosa after MN application [39]. Further studies regarding the inflammatory response of the buccal tissue are required to ensure MNs’ safety for clinical uses.

5. Conclusions

This study demonstrated the successful development of MN systems using two polymers, L100-55 and RSPO, for the buccal delivery of CBD. Additive manufacturing and micromolding techniques were combined for a successful fabrication. Microscopic analysis revealed well-defined needle geometry and surface characteristics, while physicochemical characterization confirmed the molecular dispersion of the API in the polymer matrix. Mechanical strength and insertion tests confirmed the ability of the MNs to penetrate the buccal tissue effectively, while the in vitro and ex vivo studies showed the potential of increased CBD bioavailability through the buccal route. In conclusion, the developed drug delivery systems are promising for buccal applications and further investigation is required to confirm their efficacy and safety.