Cannabidiol-Loaded Solid Lipid Nanoparticles Ameliorate the Inhibition of Proinflammatory Cytokines and Free Radicals in an In Vitro Inflammation-Induced Cell Model

2.2. Validation of the Optimized Conditions in Formulating CBD-SLNs

In the development of our CBD-SLN, we determined the optimal formulation conditions to be GMS at 1.60 g, polysorbate 80 at 0.62 g, and CBD at 20 mg. This composition was optimized through systematic testing to enhance SLN characteristics such as particle size, DL, and EE, as detailed in Table 3. The use of methanolic CBD involved using a minimal amount of methanol to dissolve the CBD during the initial formulation stages. This choice was due to methanol’s ability to quickly evaporate at the high temperatures used for lipid melting, effectively leaving no residual solvent in the final product. To confirm the accuracy and precision of the RSM, the CBD-SLNs were prepared under the optimal conditions determined for the quantities of GMS (A), polysorbate 80 (B), and CBD concentration (C), as specified in Table 3. Subsequently, the observed values for size (Y₁), PDI (Y₂), EE (Y₃), and DL (Y₄) obtained from the experiment were compared with the predicted responses generated by the software. The analysis of the predicted values against the observed values for each response showed a high level of agreement within the 95% confidence interval (Table 3). Additionally, the computed error was found to be <10% (Equation (5)), further indicating the excellent accuracy and precision of the RSM models used in predicting the outcomes of the experiment.

2.3. Characterization of the CBD-SLNs

The optimal formulation demonstrated a size of 123.40 ± 2.00 nm and a PDI of 0.2099 ± 1.00, suggesting a monodisperse system with a single peak observed in DLS analysis (Figure 2A). The International Standard Organization (ISO) guidelines suggest that formulations with a PDI exceeding 0.7 are prone to aggregation [43]. For lipid-based drug delivery, a PDI of 0.3 or less is considered appropriate [34]. Furthermore, the zeta potential values of investigated CBD-SLNs have been found to range between −33.13 and −28.23 mV depending on the specific composition. Under optimal conditions, the SLN formulation exhibited a zeta potential of −31.25 ± 0.21 mV (Figure 2B). Maintaining a surface charge of at least ±30 mV is crucial for stable colloidal dispersions through electrostatic repulsion between particles [44]. These CBD-SLNs were spherical, as depicted by transmission electron microscopy (TEM) (Figure 2C,D). The EE and DL of nanoparticles represent their capacity to efficiently incorporate and retain a substantial amount of the drug within their structure. In the case of CBD-SLNs, it exhibited a high EE of 95.16 ± 0.14 and DL of 2.36 ± 0.05.

The X-ray diffractometer (XRD) patterns of free CBD, lipid, blank SLNs, and CBD-SLNs are depicted in Figure 2E. The comparison between CBD and CBD-SLNs diffraction indicates a notable distinction. The analysis of pure CBD displayed a sequence of sharp diffraction peaks between 2θ values of 5° and 30°, consistent with various research findings that show its crystalline properties [45,46]. Importantly, these characteristic peaks were not observed in the XRD pattern of the CBD-SLNs. This suggests that the CBD was effectively dissolved within the lipid matrix of the SLNs and stabilized in an amorphous state. Figure 2F displays the FTIR spectra of pure CBD, CBD-SLNs, blank SLNs, and GMS, aiding in identifying the stability and interactions of CBD-SLNs. Fourier-transform infrared spectroscopy (FTIR) is a well-established method employed in pharmaceutical formulations to analyze interactions between drugs and excipients or detect chemical incompatibilities. When a drug and an excipient interact or incompatibility arises, discernible changes manifest in the infrared spectra. These alterations encompass shifts in peak positions, variations in intensity, or the emergence/disappearance of specific peaks. The comprehensive analysis of these spectral changes is instrumental in identifying potential issues that may contribute to the drug’s instability or diminished efficacy within the formulation [47,48]. The FT-IR spectrum of CBD exhibits distinctive molecular vibrations within specific regions: 3405–3518 cm⁻¹ for O–H (aromatic) stretching, 3000 cm⁻¹ for C–H stretching (phenyl), 2923 cm⁻¹ for methyl/methylene groups, 1581 cm⁻¹ for C=C stretching (phenyl ring), and 1214 cm⁻¹ for C–O stretching vibrations [49]. Notably, some bands in CBD-SLNs and CBD overlap with lipid/GMS bands, such as the 2914 cm⁻¹ peak representing –CH2 and –CH3 groups’ stretching vibration and the 1195–1219 cm⁻¹ bending vibration of C–O. [50,51]. Pure CBD’s distinct peaks at 3000–3518 cm⁻¹ are absent in GMS and blank SLNs, indicating their absence. Moreover, the peaks attributed to CBD in the CBD-SLNs exhibit slight shifts: from 3518 to 3517 cm⁻¹, 3405 to 3402 cm⁻¹, and 2923 to 2915 cm⁻¹. It is important to note that the absence of new peaks and the lack of significant changes in the characteristic CBD peaks further demonstrate the excellent compatibility of CBD with the lipidic matrix and the successful incorporation of the bioactive compounds into the NP structures, consistent with findings reported by Verdanega et al. [52] and Matarazzo et al. [53] in their CBD-loaded lipid NPs.

2.4. In Vitro Release Study

In assessing the release profile of CBD from the SLNs, it was crucial to consider the molecular weight of CBD relative to the MWCO of the dialysis membrane used in our study. CBD, with a molecular weight of approximately 314.47 g/mol, required a dialysis membrane capable of effectively distinguishing between the encapsulated compound and the free drug released from the SLNs. Therefore, we employed a dialysis membrane with an MWCO of 3.5 kDa (equivalent to 3500 g/mol), allowing unencapsulated CBD to freely pass through while retaining the larger SLN particles within the dialysis bag. This methodological choice enabled a precise evaluation of CBD release dynamics from the NPs, aligning with standard protocols for NPs drug release studies [54,55]. The CBD release study from the SLNs was conducted to evaluate release behavior and kinetics in the presence of proteins. In the presence of human serum albumin (HSA), free CBD leads to a notably higher cumulative release of 86.08% after 12 h and 96.00 after 24 h (Figure 3A). In contrast, CBD-SLNs exhibit a distinct release pattern, characterized by a sustained release profile. The cumulative CBD release percentages for CBD-SLNs were markedly lower, at 63.01% and 75.83% after 12 and 24 h, respectively. Moreover, it can be noted that the free CBD exhibited higher release rates than CBD-SLNs over 24 h, considering that CBD has low water solubility. This difference can be attributed to the specific composition of the release medium, which contained a surfactant and a 70:30 mixture of PBS and ethanol. These components significantly enhanced the solubility of free CBD, promoting its rapid dissolution and diffusion in the surrounding medium. Conversely, CBD-SLNs are expected to provide controlled and sustained release characteristics. CBD encapsulated within the SLNs must diffuse out of the lipid matrix, leading to a slower release profile than freely dissolved CBD. The lipid matrix of the SLNs acts as a barrier that governs the rate of CBD release into the release medium, resulting in a more controlled and prolonged release pattern. To further substantiate these findings, De Gaetano et al. [56] and Hassan et al. [57] have reported similar observations regarding the controlled release behavior of non-polar compounds from SLNs. These studies reinforce our understanding of how formulation strategies can influence the release characteristics of compounds like CBD, underscoring the importance of customized delivery systems in enhancing CBD’s therapeutic effectiveness. In this study, HSA significantly influences CBD release profiles. Without HSA, CBD suspension reached 96.08% at 12 h and 98.48% at 24 h (Figure 3B). In contrast, CBD-SLNs had lower cumulative release: 75.95% at 12 h and 83.04% at 24 h. This suggests that, in the absence of HSA, higher levels of CBD can be released from both samples under investigation.

The cumulative release percentages vary due to intricate interactions among CBD, the lipid carrier, and HSA, impacting the drug release mechanism [58]. HSA, a prominent plasma protein, is known for its capacity to bind and transport various substances, including drugs [59]. CBD binds to HSA, forming the CBD-HSA complex, which affects the solubility and diffusion of CBD [60,61]. Typically, HSA binding restricts drug diffusion, diminishing the release of CBD from delivery systems such as suspensions or SLNs [62]. Furthermore, the presence of HSA on the surface of CBD-SLNs can enhance protein adsorption, thereby forming a barrier that impacts the release of CBD from these nanoparticles. Additionally, the adsorption of HSA onto the dialysis membrane can alter the drug’s permeability and create resistance to diffusion. To avoid the interaction of the drug with HSA inside the dialysis bag, which might block the penetration of the released CBD through the membrane due to CBD’s high affinity for HSA, we purposely did not include HSA inside the dialysis bag in this study. This approach was deliberately chosen, considering that HSA, with an MW of 66.5 kDa, binds to CBD, forming the CBD-HSA complex, which cannot traverse the dialysis membrane with an MWCO of 3.5 kDa. Studies have shown that buffers containing additives such as surfactants, salts, or chaotropic agents can significantly reduce protein adsorption on dialysis membranes [63,64]. Notably, we integrated surfactant poloxamers into the release media in our experiment, thereby minimizing the potential adsorption of HSA onto the dialysis membrane.

To elucidate CBD release patterns from the SLNs, data were fitted into different empirical models, including zero-order, first-order, Higuchi, Korsmeyer–Peppas, and Hixson–Crowell models, using the DDsolver add-in program in Microsoft Excel 2010 (Tables S6 and S7) [65]. The optimal model was chosen based on a high R² value close to 1, a high model selection criterion (MSC), and a low Akaike information criterion (AIC) [66]. The Korsmeyer–Peppas model best fit most samples, except for free CBD without HSA, where the first-order mechanism was optimal. Release mechanisms were determined by diffusional index (n) values: n < 0.45 indicates quasi-Fickian diffusion, n = 0.45 signifies Fickian release, 0.45 < n < 0.89 suggests non-Fickian/anomalous release, n = 0.89 represents case II/zero-order release, and n > 0.89 indicates super case II release (Table 4) [67]. All samples fitting the Korsmeyer–Peppas model exhibited quasi-Fickian diffusion, consistent with prior studies on nanoparticle formulations, particularly lipid-based nanoparticles [68].

Our in vitro release study acknowledges the differences between laboratory conditions and the physiological environment, posing challenges to directly applying our findings in a clinical context [69]. While our results demonstrate a promising sustained release profile of CBD from SLNs, it is important to note that our study focused on oral administration. The sustained release observed in our SLN formulation offers significant therapeutic potential for systemic conditions, such as chronic inflammatory diseases, where continuous drug delivery can enhance treatment efficacy by maintaining consistent drug levels over time. This sustained release mechanism is particularly advantageous for conditions requiring prolonged therapeutic intervention, providing a more controlled and effective delivery of CBD through oral administration. Factors such as metabolic processes, elimination pathways, and interactions with biological matrices can profoundly influence the pharmacokinetics and efficacy of the drug [70]. Therefore, to validate our initial in vitro findings and evaluate the clinical relevance of CBD-SLNs, comprehensive in vivo studies are essential. These investigations should involve examining the pharmacokinetics, distribution, and therapeutic efficacy of CBD-SLNs in vivo. Bridging the gap between in vitro and in vivo data is crucial for the clinical translation of our research, emphasizing the need for in vivo experiments to confirm the potential of CBD-SLNs as a sustained-release drug delivery system for conditions that benefit from ongoing CBD therapy.

2.5. Stability of CBD-SLNs

Stability tests were focused on the aqueous dispersion of SLNs, chosen for its relevance to intended oral applications. The colloidal stability of nanoparticles in biologically relevant media was assessed by examining the size (Figure 4A) and PDI (Figure 4B) before conducting cell assays. The culture medium used in these experiments contained various biomolecules, including proteins like serum albumin and globulins, along with amino acids and ionic salts, all of which can significantly affect nanoparticle behavior [71,72]. CBD-SLN samples showed consistent size and PDI in phosphate buffer solution (PBS) and Dulbecco’s modified eagle medium (DMEM), but changes occurred with 10% fetal bovine serum (FBS), aligning with the release study results. Serum affects CBD-SLN release and characteristics via protein interactions. To mitigate this, serum-free media were employed in future experiments, improving nanoparticle control in biological applications.

The long-term physical stability of CBD-SLNs was also assessed over 6 months to check for particle aggregation or drug leakage during storage. CBD-SLNs stored at 4 °C for up to 4 months showed no significant changes in size (Figure 4C), zeta potential (Figure 4D), EE (Figure 4E), or drug content (Figure 4F). In the 5th month, size increased, indicating aggregation, with a higher PDI. EE and drug content dropped by 9% and 7%, possibly due to drug diffusion from the lipid matrix. CBD-SLNs stored at room temperature (RT) had significant changes earlier. By the 4th month, size increased, surface charge decreased, and there was a 14% EE reduction. Temperature fluctuations and humidity during storage likely caused physical instability, potentially leading to drug expulsion and reduced EE. Additionally, chemical degradation of the drug or lipid components over time could impact overall drug content and EE.

Overall, storing CBD-SLNs at 4 °C is favorable for stability, underlining the importance of proper storage conditions to maintain formulation characteristics [73]. These insights are valuable for CBD-SLN development and storage in pharmaceutical or food applications.

3.2. Extraction, Isolation, and Purification of CBD

The inflorescence of Cannabis sativa L. subsp. Sativa was obtained from Chiang Rai Province, Thailand, and authenticated by one of the authors (W.T.). The hemp inflorescence was dried at 50 °C and pulverized into powder before extraction. CBD extraction was performed using a customized in-house method to maximize yield and purity. The CBD powder was derived from hemp plants through a cold ethanol extraction process. Initially, 1 kg of mature hemp plants was harvested and dried to minimize moisture content. Subsequently, the dried hemp material was finely ground to augment the surface area, facilitating enhanced solvent penetration. The finely ground plant material was then introduced into an in-house extractor, and cold ethanol (maintained between −35 and −40 °C) was added to the extraction machine in a 1:1 ratio (weight of dried plant to the volume of ethanol). Ethanol at temperatures ranging from −30 °C to −40 °C was utilized for extraction. Specifically, ethanol at a significantly lower temperature range was selected due to its efficacy in solubilizing CBD while minimizing the extraction of unwanted nonpolar compounds, such as chlorophylls and waxes, which exhibit less solubility at lower temperatures [90]. Notably, waxes can impede CBD crystallization during recrystallization, potentially leading to the observed low final yield after purification. The mixture was stirred at 50 rpm for 30 min, allowing the cold ethanol to dissolve cannabinoids and other desired components. The extraction process was conducted while maintaining the temperature between −35 and −40 °C. The liquid extract was concentrated using a rotary evaporator to remove the ethanol. The concentrated oil and ethanol underwent freezing to eliminate unwanted waxes and lipids, which solidify during freezing. The resultant mixture was filtered to eliminate solidified impurities. The winterized oil underwent further concentration to yield a more refined oil. This refined oil was heated to convert any remaining CBDA to CBD. The decarboxylated CBD oil was subjected to fractional distillation for purification, resulting in more concentrated CBD distillates. CBD crude powder was isolated from CBD distillates through crystallization in pentane. The crude powder underwent recrystallization to achieve high-purity CBD, resulting in a purity greater than 99.5% (40.5 g). The yield was about 4% from dried hemp, IR (KBr): υ_max 3405–3518 (O–H (aromatic)), 3000 (C–H (alkene)), 2923 (C–H (alkane)), 1581 (C=C (phenyl)), 1214 (C–O) cm⁻¹ (Supplementary Figure S1); ¹H NMR (CDCl₃, 500 MHz): δ 0.86 (3H, t, J = 6.8 Hz), 1.29 (4H, m), 1.54 (2H, q, J = 7.6 Hz), 1.64 (3H, s), 1.79 (3H, s), 1.83 (2H, m), 2.09 (1H, m), 2.24 (1H, m), 2.40 (1H, m), 2.43 (2H, t, J = 7.4 Hz), 3.84 (1H, dm, J = 11.8 Hz), 4.54 (cis, 1H, m), 4.64 (trans, 1H, m), 4.82 (1H, s(br)), 5.55 (1H, s), 5.97 (1H, s(br)), 6.16 (1H, s(br)), 6.25 (1H, s(br)) (Figure S2); ¹³C NMR (CDCl₃, 125 MHz): δ 13.99, 20.39, 22.50, 23.64, 28.35, 30.35, 30.60, 31.46, 35.44, 37.13, 46.14, 107.96, 109.68, 110.82, 113.73, 124.105, 139.99, 142.99, 149.25, 153.89, 155.99 (Figure S3); and HRMS (m/z): 337.21309 (C₂₁H₃₀NaO₂) [M + Na⁺] (Figure S4).

3.3. Preparation of CBD-Loaded SLNs

The formulation of our CBD-SLNs was meticulously developed through initial experiments aimed at identifying the optimal lipid matrix. This process was underpinned by an extensive review of the literature focusing on lipid-based NPs for drug delivery, emphasizing their physicochemical characteristics, biocompatibility, and performance in encapsulating hydrophobic compounds like CBD [91,92]. GMS is widely utilized in lipid nanoparticle development, notably in SLNs, owing to its favorable properties and safety profile. Numerous studies have highlighted GMS’s efficacy in enhancing drug solubility and achieving controlled drug release of hydrophobic compounds [93,94,95]. The choice of GMS as the lipid component for our SLN formulation was determined by assessing the solubility of CBD. GMS was considered a suitable candidate due to the absence of drug crystals; the visual inspection of CBD solubility in melted lipids confirmed GMS’s ability to dissolve CBD completely [96,97,98]. The CBD-SLNs were prepared using low-energy hot homogenization and ultrasonication methods described by Kaisit et al., with some modifications [97]. In brief, food-grade GMS lipid, considered GRAS (generally recognized as safe), was melted with 3 mL ethanol at 80 °C. Simultaneously, polysorbate 80 was mixed with 80 mL of water at the same temperature and mixed at 1000 rpm for 10 min. Methanolic CBD (1 mL) was added to the melted lipid and stirred for 3 min. The lipid phase was then added to the aqueous phase, resulting in emulsion formation, and was continuously mixed for 30 min. Hot water was added to maintain the volume at 100 mL due to evaporation during mixing. A bath sonicator (Powersonic™ P230D, Crest Ultrasonics, NJ, USA) was used for 30 min to reduce emulsion size further. The colloidal dispersion was cooled at room temperature (RT) and left overnight to ensure complete lipid crystallization.

3.4. Experimental Design for the Optimization of CBD-SLNs

BBD is a valuable statistical tool with a three-factor, three-level design. In this study, critical parameters for CBD-SLN formulation were lipid/GMS (A), surfactant/polysorbate 80 (B), and CBD amount (C), chosen based on initial experiments. A three-level BBD with three replicated center points for each factor was used to optimize the formulation and assess its impact on particle size, PDI, % EE, and % DL (Table 1). Design-Expert^® version 23.1 (Stat-Ease^® Inc., Minneapolis, MN, USA) generated 15 formulation conditions (Table 2). ANOVA validated the statistical significance of polynomial equations. Three-dimensional response surface and contour plots from the same software identified the optimal formulation. Predicted values were compared to experimental results to assess optimization accuracy. The experimental design process involved utilizing a BBD to generate a comprehensive set of experimental conditions encompassing varying levels of GMS, polysorbate 80, and methanolic CBD. This allowed for systematically exploring formulation combinations on specific SLN characteristics such as size, PDI, EE, and DL. Subsequently, the responses obtained from these experiments, including size, PDI, EE, and DL, were subjected to regression analysis and model fitting followed by graphical interpretation to discern individual factors’ impact and interactions on the desired SLN properties. By analyzing response surface plots and contour maps derived from the BBD, optimal formulation conditions were identified that concurrently optimized the targeted SLN characteristics essential for effective CBD delivery. Finally, these optimized conditions were rigorously validated through experimental verification to ensure the reproducibility and robustness of the formulated CBD-SLNs, consolidating the reliability of the study outcomes.

3.5. Physicochemical Characterization of CBD-Loaded SLNs

The aqueous dispersion of CBD-SLN was diluted directly in normal saline for size and PDI measurements using dynamic light scattering (DLS, Nano-ZS, Malvern PANalytical Ltd., Malvern, UK). The diluted SLN dispersion was gently stirred to ensure uniform mixing before the measurements were conducted. No ultrasonication was used during the dilution process to minimize potential disruption of the SLN structure. At the same time, the zeta potential was determined by analyzing its electrophoretic mobility using the same equipment. The morphology of the CBD-SLNs was observed through TEM (JEM-1400, JEOL Ltd., Tokyo, Japan).

The DL (Equation (6)) and EE (Equation (7)) of the CBD-SLNs were determined by measuring the dry weight of the separated NPs and the absorbance of the supernatant, respectively. The CBD-SLNs were ultracentrifuged at 4 °C at 105,000× g for 1 h. The absorbance was then measured at 210 nm (Agilent Cary 60, Agilent Technology, Santa Clara, CA, USA), and the amount of free CBD in the supernatant was calculated against the standard curve. The DL and EE of the CBD-SLNs were calculated using the following equations:

where Wt is the initial amount of CBD added to the formulation, Ws is the amount of free CBD in the supernatant, and Wnp is the weight of the CBD-SLNs after lyophilization. The lyophilization process was performed after removing the supernatant and collecting the SLN at the bottom. The NPs were frozen for 5 min at −80 °C to solidify the water content rapidly and then subjected to freeze-drying under vacuum using the FreeZone Freeze Dry System (Labconco Corp., Kansas City, MO, USA) at −55 °C for 24 h and weighed accordingly.

IR spectra and XRD patterns of CBD-SLNs, blank SLNs, and their components were analyzed to assess CBD encapsulation and crystallinity post-preparation. FTIR spectra ranged from 4000 to 400 cm⁻¹ (Spectrum one™, PerkinElmer Inc., Waltham, MA, USA). XRD patterns were obtained with a wide-angle XRD (PANalytical X’Pert Pro model, PANalytical, Kassel-Waldau, Germany) scanning at 0.05°/min from 3 to 70° at room temperature.

3.6. In Vitro Release Study

The release of CBD from an SLN formulation was investigated in vitro using the dialysis bag method designed for precision and reproducibility. In the release test, CBD-loaded SLNs were enclosed in a dialysis bag (MWCO: 3.5 kDa) to isolate them from the release medium. The release medium was a blend of phosphate-buffered saline (PBS), ethanol (70:30), and 3% Poloxamer (pH 7.4), selected to ensure CBD solubility while mimicking physiological conditions to some extent. A total of 5 mL of the dispersion (CBD suspension or aqueous dispersion of CBD-SLNs) was sealed in a dialysis bag and placed in 200 mL of pH 7.4 release medium at 37 ± 0.5 °C with continuous stirring at 50 rpm to maintain sink conditions throughout the experiment. This setup facilitated the diffusion of released CBD without saturating the medium. Thus, solubility studies were conducted prior to the release tests to confirm that CBD’s solubility surpassed its concentration in the release medium throughout the experiment, ensuring sink conditions. With a solubility exceeding 30 μg/mL, this step was crucial for accurately simulating drug release from the SLNs, preventing saturation, and facilitating the diffusion process effectively. To further study the interaction of SLN with proteins, Human Serum Albumin (HSA) was introduced to the external medium, considering its stability in 30% ethanol [99,100]. Additionally, 1 mL aliquots were collected at set intervals and replaced with fresh release medium. CBD concentration was measured using spectrophotometry to calculate % cumulative CBD released using the formula below (Equation (8)):

where CR (%) is cumulative CBD released, V_e is collection volume (mL), V_o is the volume of the medium (mL), Cn is the concentration of the time point (mg/mL), and m is total CBD (mg) in the dialysis bag. Finally, the release profiles of the samples were subjected to mathematical modeling using several well-established equations, including the zero-order, first-order, Korsmeyer–Peppas, Hixon–Crowell, and Higuchi equations [101].

3.7. Storage and Colloidal Studies

The storage stability of the CBD-SLNs was investigated over six months by conducting regular evaluations of the nanoparticle’s size, total drug content, zeta potential, and EE. The formulations were stored in light-resistant glass vials at two different temperatures: 4 °C and room temperature [102]. In our investigation into the stability of SLN formulations, we opted to store the formulations as aqueous dispersions within light-resistant glass vials. This decision was predicated on preserving the formulations in a state that mirrors their intended application closely, ensuring that any findings related to stability accurately reflect their performance in practical scenarios. The choice of light-resistant vials was strategic, intended to mitigate any potential degradation of the encapsulated CBD caused by light exposure, thereby maintaining the formulation’s integrity and therapeutic efficacy over time.

The colloidal stability of SLNs was assessed in various biologically relevant media, including PBS, PBS with 10% FBS, serum-free DMEM, and DMEM with 10% FBS. SLN dilutions (1:200) were prepared in each medium, and size and PDI were measured as previously described. Samples were then incubated at 37 °C for varying durations before analysis [103].

3.8. In Vitro Inflammation Study

We thank the Pharmaceutical Research Instrument Center and the Metallurgy and Materials Science Research Institute at Chulalongkorn University for their research facilities and Inspector and Engineering Co., Ltd. for their generosity. Special thanks to Chawanphat Muangnoi (Mahidol University), Waraluck Chaichompoo (Chulalongkorn University), and Wisut Wichitnithad (Pharma Nueva Co., Ltd.) for their valuable contributions. We also acknowledge the Ratchadaphiseksomphot Endowment Fund for supporting Khent Primo Alcantara’s Postdoctoral Fellowship.