Cannabidiol/β-Cyclodextrin Inclusion Complex-Loaded Poly(Vinyl Alcohol) Semi-solid Hydrogels for Potential Use in Wound Management

Semi-solid poly(vinyl alcohol) (PVA)-borax hydrogels containing a cannabidiol (CBD)/β-cyclodextrin (β-CD) inclusion complex were prepared and aimed for being used as wound management materials. The inclusion complex was prepared by the precipitation method and loaded within PVA semi-solid hydrogels which had various contents of CBD/β-CD inclusion complex (i.e., 0.5%, 1.0%, and 1.5% w/w). The obtained inclusion complexes and their corresponding hydrogels were characterized for their physicochemical properties and mechanical properties. The potential use as wound management of the obtained hydrogels in terms of their release profile, antioxidant activities, antibacterial capabilities, cytotoxicity, and anti-inflammatory efficacy. The CBD/β-CD inclusion complex was successfully prepared, as confirmed by FT-IR, 1H-NMR, XRD, and SEM. The complexation efficiency was 84.73 ± 0.64% and the loading capacity was 32.70 ± 0.32%. The water solubility of CBD was significantly increased to 0.336, which was increased by 16-fold, as compared with that of raw CBD. The cumulatively released amount of CBD from the hydrogels increased with higher amounts of inclusion complex. The prepared hydrogels provided an antibacterial capability with S. aureus and MRSA, while no antibacterial capability was observed for E. coli. However, adding CBD to the hydrogel was beneficial for improving the antioxidant properties of the obtained hydrogels. All the hydrogels loaded with the CBD/β-CD inclusion complex at concentrations below 1.25 mg/mL for the extraction medium were non-cytotoxic towards RAW 264.7 cells and also provided the ability to suppress nitric oxide (NO) production by more than 75% compared with the LPS treated group. These findings suggest a new application for semi-solid PVA-borax hydrogels containing the CBD/β-CD inclusion complex in biomedical applications.


Introduction
Research into Cannabis sativa L. has received increasing attention in recent years due to its active chemical compounds produced by secondary metabolism such as cannabinoids, flavonoids, terpenoids, and alkaloids [1][2][3]. According to a number of pre-clinical studies, cannabinoids may be helpful in the treatment of patients with skin wounds, both chronic and acute [4]. Cannabidiol (CBD as shown in Fig. 1a) is the second most abundant chemical in the cannabinoid family with non-psychoactive properties, and it has been extensively studied from a pharmaceutical perspective [4][5][6][7][8]. Various studies have shown its pharmacological activities, including as an antioxidant, antibacterial, anti-inflammatory, anti-proliferative, and neuroprotective properties [3,[9][10][11][12]. Nevertheless, CBD's hydrophobic nature and degradation under processing or storage conditions limit its bioavailability and therapeutic uses [3,13]. To overcome these drawbacks, CBD can be encapsulated in other materials to enhance its solubility and retain its bioactivity, in addition to controlling its release profile.
Various approaches have been utilized to encapsulate the active compound in other materials [14]. Molecular inclusion in β-cyclodextrin (β-CD as shown in Fig. 1b), a cyclic oligosaccharide with a hydrophobic internal cavity and a hydrophilic external surface, is a versatile and effective method for encapsulation due to it being able to host a wide variety of hydrophobic guest molecules with molecular weights between 200 and 800 g/mol in its cavity [15,16]. β-CD and also its derivatives are often used to enhance the water solubility and chemical stability of numerous active natural compounds [15,[17][18][19][20][21][22][23][24][25]. Additionally, β-CD provides advantages over other materials in terms of its ease of production and reasonable price [26]. Many studies have attempted to encapsulate CBD in the cavity of β-CD and its derivatives using a variety of techniques, including physical mixing [20,21], suspension [20], saturated aqueous solution [17], and co-precipitation [19,21]. Co-precipitation is the most widely used technique, in which guest molecules dissolved in a solvent (e.g., ethanol) are added to the β-CD solution and stirred for several hours to achieve complexation and crystallization of the complexed molecules [27]. This method has been reported to encapsulate various natural active entities, such as cannabidiol [19][20][21], curcumin [22], catechin [23], and numerous other essential oils [15,[24][25][26][27][28].
Hydrogels are physically or chemically crosslinked macromolecular networks of hydrophilic polymers and are extremely versatile materials used in a wide variety of pharmaceutical and biomedical applications due to their ability to absorb large amounts of water or other biological fluids [29]. In wound dressing applications, hydrogels are wellknown for promoting the wound healing processes by providing a moist environment and absorbing wound exudates [30]. They are biocompatible with the human body since their characteristics resemble natural tissue and provide high sensitivity to physiological environments [30,31]. Moreover, they do not adhere to the wound, which reduces pain and makes them more comfortable dressings for patients [32][33][34]. Over the past two decades, hydrogels with great biocompatibility and mechanical strength have attracted more attention for use as wound dressings [30,31]. Among the various types of hydrogels, poly(vinyl alcohol) (PVA) hydrogel is considered to be a promising candidate as a wound dressing material due to its remarkable biomedical properties [35][36][37][38][39]. There are numerous ways to crosslink PVA to form chemically crosslinked hydrogels and physically crosslinked hydrogels [30,38,39]. Even though chemically crosslinked hydrogels have strong elasticity and sufficient cohesive qualities, one of their drawbacks is that they cannot conform to irregularly shaped surfaces, which causes them to only partially adhere to the wound bed. To enhance the flow ability of hydrogels for use in wound management, physically crosslinked hydrogels that form temporary networks are prepared. Sodium tetraborate decahydrate (Na 2 B 4 O 7 ⋅10H 2 O), also known as borax, has been used as a physical crosslinking agent for PVA [40][41][42]. The addition of borax solution to PVA solution can significantly enhance the viscoelasticity of the obtained PVA hydrogel via a temporary ionic crosslink reaction [43]. Despite numerous reports focusing on the use of chemical crosslink agents to form PVA hydrogels as wound dressing materials [39], few studies have investigated the potential use of physical crosslink agent to form semi-solid PVA hydrogels for these purposes. In the present study, we attempted to develop semi-solid PVA-borax hydrogels loaded with a CBD/β-CD inclusion complex to serve as wound management materials. The CBD/β-CD inclusion complex was prepared using the co-precipitation method. To explore the physical and chemical properties of the obtained inclusion complex, we evaluated its physicochemical properties, complexation efficiency, loading capacity and water solubility. Furthermore, semi-solid PVA-borax hydrogels containing various amounts of the CBD/β-CD inclusion complex were developed. We hypothesized that the obtained semi-solid hydrogels can exhibit beneficial for wound healing. To verify these hypotheses, the potential use of the obtained semisolid hydrogels was evaluated in terms of their release characteristics, antibacterial capability, antioxidant activities, cytotoxicity and anti-inflammatory. A pristine semi-solid PVA-borax hydrogel was used as the internal control.

Preparation of Cannabidiol/β-Cyclodextrin Inclusion Complex
The solid complex of CBD/β-CD was prepared by the coprecipitation method, similar to that previously published [20] with a slight modification. Briefly, an ethanolic CBD solution (20 mg/mL) was prepared and gradually added to an aqueous of β-CD solution (1.7% w/v) over a 2 h-period while stirring simultaneously. The resulting precipitant was centrifuged and then the supernatant was removed. The precipitant was then freeze-dried and stored in an airtight container. The mole ratio of CBD to β-CD (CBD:β-CD) was fixed at 1:1.

Physicochemical Characterization The CBD/β-CD Inclusion Complex
The chemical structures of the CBD, β-CD and CBD/β-CD inclusion complexes were investigated using a Nicolet FT-IR spectrophotometer (Nicolet Instruments, USA), and the 1 H-NMR spectra was obtained on a Bruker Advanced III HD spectrometer at 500 MHz (Oxford Instruments, United Kingdom). Tetramethylsilane (TMS) was used as a reference. CBD was dissolved in DMSO-d6, whereas β-CD and CBD/β-CD inclusion complexes were dissolved in D 2 O and filtered prior to conducting all NMR investigations. The crystalline structure of the CBD, β-CD and their corresponding CBD/β-CD inclusion complex was verified by an X-ray Power Diffractometer (XRD; Bruker AXS, United Kingdom). All the samples were scanned from 2θ = 5° to 60°. The morphological appearance of CBD, β-CD, and their corresponding CBD/β-CD inclusion complex was observed using a LEO 1450 VP Scanning Electron Microscopy (SEM; Carl Zeiss, Germany).

Determination of the Complexation Efficiency, Loading Capacity and Water Solubility of the CBD/ β-CD Inclusion Complex
To determine the CBD content in the complex, 5 mg of the CBD/β-CD inclusion complex was dissolved in 20 ml methanol in glass tubes and sonicated for 1 minute in order to extract CBD. The amount of CBD in methanol was quantified using Lambda 35 UV-vis spectrophotometer (PerkinElmer, USA) at a wavelength of 273 nm and its concentration was calculated using the calibration curve. For each measurement, the baseline was established using blank methanol as a reference. The complexation efficiency (CE) and loading capacity (LC) was calculated by the following equations: To determine water solubility, a surplus of each sample (equivalent to 10 mg raw CBD) was suspended in a conical flask containing 20 mL of deionized water and left for 24 h at 37 °C while being stirred magnetically at a speed of 100 rpm. After a 5-minute centrifugation at 4000 rpm, the suspension was examined.

Preparation of Semi-solid PVA-Borax Hydrogels Loaded with the CBD/β-CD Inclusion Complex
The cannabidiol incorporated semi-solid PVA hydrogels were prepared according to the modified method previously described [35] with a slight modification. Briefly, 2% w/v of borax powder was dissolved in various amount of CBD/β-CD complex aqueous suspensions with continuous stirring at 25 °C for 30 minutes. Subsequently, 5% w/v of PVA powder was slowly sprinkled onto the surface of the aqueous solutions and stirred for 30 minutes at room temperature. After a complete swelling of the PVA powder, the solutions were heated to 90 °C and agitated for 2 h. The PVA powder started to dissolve as the temperature increased, and the mixture eventually grew homogenous and viscose. Following the thorough dissolution of PVA, homogenous solutions containing a well-dispersed CBD/β-CD combination were produced. The solutions were slowly cooled to room temperature to form the hydrogels. The amount of CBD/β-CD inclusion complex in the aqueous suspensions was varied to be 0.5, 1.0, and 1.5% w/w. Table 1 shows the sample codenames and their compositions. The obtained hydrogels were kept at room temperature in a sealed container for 48 h to reach equilibrium before the application of any tests.

Water Content of the Semi-solid PVA-Borax Hydrogels Loaded with the CBD/β-CD Inclusion Complex
Each sample was dried in a vacuum oven at 50 °C until obtaining a constant weight. The water content (Wc) of the hydrogels was calculated from the following equation: where Wi is the initial weight of each hydrogel and Wd is the weight of sufficiently dried hydrogel. Each study was conducted in triplicate and reported as the mean ± SD.

Texture Properties and Adhesiveness
Textural properties and adhesiveness of the hydrogels were evaluated using a texture analyzer (TA XTplus) in texture profile analysis (TPA) mode and the adhesive mode, respectively. In the TPA mode, a tubular probe was crushed twice into each sample, with a delay of 15 s in between compressions, to a depth of 5 mm. By employing texture profile analysis to create force-time graphs, the hardness and compressibility were determined. The highest force of the first positive curve on the force-time plots was used to determine the hardness. The area under the first positive curve on the force-time charts was used to calculate compressibility. In the adhesive mode, the pig skin was firmly attached to the upper part of the instrument. The probe was then lowered to the sample's surface loaded in a plastic petri dish filled with 8 g of each sample and held for 30 s at 0.02 N. After which time, the probe was then moved upwards at 5 mm/s. The force maxima of the force-time plots created by separating the pig skin from the sample surface were used to define the adhesiveness. Each study was conducted in triplicate and reported as the mean ± SD.

In Vitro CBD/β-CD Release Study
The precise amount of CBD in the hydrogels were determined prior to studying the release characteristics of CBD. Briefly, 200 mg of each sample was dissolved in a phosphate buffer saline solution (PBS; pH 7.4) and sonicated for 30 minutes to extract the CBD. The obtained solution was then measured using a UV-visible spectrophotometer at a wavelength of 273 nm. The actual contents of CBD were then back-calculated from the obtained data against a predetermined CBD calibration curve. The release characteristics of CBD from each sample were investigated by the transdermal diffusion method using the vertical franz diffusion cell. The release study was evaluated across a polycarbonate membrane cell culture insert with an 8 µm pore size to allow for unhindered diffusion of CBD. A total of 200 mg of each hydrogel were added to the cell culture insert (Corning, USA). Each sample was then placed in a chamber containing a phosphate buffer saline solution (PBS; pH 7.4) and incubated at 37 °C under continuous stirring. After the specified time, 1 ml of the released medium was withdrawn and then 1 ml of fresh PBS was refilled. The sample solution was determined by UV-vis spectrophotometer at a wavelength of 273 nm. The obtained data was calculated to determine the amount of CBD released from the samples. Each study was conducted in triplicate and reported as the mean ± SD.

Total Phenolic Content and Antioxidant Capability
The total phenolic content of the semi-solid PVA-borax hydrogels loaded various amounts of the CBD/β-CD inclusion complex was determined based on the Folin-Ciocalteau test [44]. Briefly, all the hydrogels were immersed in water for 3 h and the solution was then reacted with 2 N Folin and Ciocalteu's phenol reagent solution before being immediately vortexed. After being vortexed, 7%w/v Na 2 CO 3 solution was added to the mixture. The mixture was then shaken and left to incubate for 2 h and absorbance was measured using a microplate reader at 765 nm. Gallic acid was used as standard reagent, and the total phenolic content was expressed as mg gallic acid equivalent per gram of the sample (GAE/g extract). Each study was conducted in triplicate and reported as the mean ± SD. The antioxidant activities of the semi-solid PVA-borax hydrogels loaded with the CBD/β-CD inclusion complex were determined using 2,2′-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical cation scavenging assay, and Ferric reducing antioxidant power (FRAP).

DPPH Radical Scavenging Assay
The DPPH radical scavenging activity of the semi-solid PVA-borax hydrogels loaded with various amounts of the CBD/β-CD inclusion complex was determined according to the method described by Pang et al. [45]. In this, 0.3 mM of DPPH was added to the reaction mixture containing various hydrogel samples in methanol. The mixture was kept in the dark for 30 minutes. The absorbance of the reaction mixture was then measured using a microplate reader at a wavelength of 517 nm. The scavenging activity (%) was calculated as follows: where A blank is the absorbance values of the blank solution (0.3 mM DPPH solution) and A sample is the sample solution. Each study was conducted in triplicate and reported as the mean ± SD.

ABTS Radical Cation Scavenging Assay
The ABTS radical cation scavenging activity of the semisolid PVA-borax hydrogels loaded with various amounts of the CBD/β-CD inclusion complex was determined according to the procedure described by Chuysinuan et al. [46]. The ABTS radical cation solution was obtained from reacting 2.6 mM potassium persulfate in water with 7.4 mM ABTS in methanol for 12 h in the dark. The stock solution was diluted with methanol to obtain an absorbance of 0.7 ± at 734 nm with a microplate reader. Then the released solution of various samples was reacted with ABTS solution and incubated for 2 h. The ABTS radical scavenging capacity was calculated using the following formula: where A blank is the absorbance of the ABTS radical without the antioxidant materials and A sample is the absorbance of the ABTS radical in the presence of the antioxidant materials after 2 h. Each study was conducted in triplicate and reported as the mean ± SD.

Ferric Reducing Antioxidant Power (FRAP)
The antioxidant capacity of the semi-solid PVA-borax hydrogels loaded various amounts of the CBD/β-CD inclusion complex was estimated spectrophotometrically following the procedure of Yang et al. [47]. The Ferric reducing antioxidant power (FRAP) reagent was prepared by mixing an acetate buffer, 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ) solution TPTZ in HCl and FeCl 3 at a 10:1:1 proportion. The freshly prepared working FRAP reagent was pipetted using a micropipette and mixed with the solution. An intense blue color complex formed when the ferric tripyridyl triazine (Fe 3+ TPTZ) complex was reduced to a ferrous (Fe 2+ ) form and recorded at 593 nm against a reagent blank (FRAP reagent). The calibration curve was prepared by plotting the absorbance at 593 nm versus known concentrations of FeSO 4 , while Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the positive control. The reducing power was expressed as mg of Trolox equivalent per gram of sample (mg TE/g extract). Each study was conducted in triplicate and reported as the mean ± SD.

Antibacterial Activities
The antibacterial capabilities of the semi-solid PVA-borax hydrogels loaded various amounts of the CBD/β-CD inclusion complex were investigated according to the disc diffusion method against gram-positive Staphylococcus aureus ATCC 25923 (S. aureus), gram-negative Escherichia coli ATCC 25922 (E. coli), and Methicillin-resistant Staphylococcus aureus (MRSA) as model bacteria. First, each model bacteria was cultured in a tryptone soy broth (TSB) at 37 °C for 18 h and then diluted with normal saline solution (NSS; 0.85% NaCl) to prepare a 10 8 CFU/ml inoculum. The bacterial inoculum were then swabbed onto agar plates and each hydrogel sample was subsequently placed on the top of the bacterial plates. The zone of inhibition of the hydrogel sample was measured after 24 h of incubation at 37°C. Each study was conducted in triplicate and reported as the mean ± SD.

Cytotoxicity Evaluation
To evaluate the potential biomedical applications of the semi-solid PVA-borax hydrogels loaded with various amounts of the CBD/β-CD inclusion complex, their biocompatibility in terms of indirect cytotoxicity towards RAW 264.7 cells (mouse macrophage cell line, ATCC TIB-71, Rockville, MD, USA) was evaluated in vitro compared to the corresponding pristine PVA-borax hydrogels and tissueculture polystyrene (TCPS) by MTT colorimetric assay in accordance with ISO10993-5 standard test protocol [48]. Briefly, RAW 264.7 cells were seeded in 96-well tissue culture polystyrene plates (TCPS) with 30,000 cells/100 µL/ well in phenol red-free medium (Gibco, Thermo Fisher Scientific, USA) enriched with 10% v/v fetal bovine serum and 1% penicillin/streptomycin. The cells were incubated at 37 °C for 24 h under CO 2 (5%) atmosphere. Next, the hydrogels were cut into a circular shape with 2.5 cm in diameters and sterilized by UV irradiation for 30 min and then immersed in phenol red-free DMEM cell culture medium for 24 h to produce sample extract (i.e., 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/mL). After that, the sample extract was replaced to each well and further incubated for 24 h. A modified MTT assay was employed to determine viability of the treated cells [49]. Briefly, 25 µL of the prepared MTT solution was added to the well and incubated in CO 2 incubator for 4 h at 37 °C. Then, lysis solution (20% SDS in 10 mM HCl) (100 µL) was added to each well in order to lyse and solubilize the formazan crystals formed by mitochondrial respiratory cycle. The plate was placed in the dark at room temperature for 2 days. Eventually, the absorbance was read at 550 nm and subtracted with absorbance at 650 nm. Each study was conducted in triplicate and reported as the mean ± SD. The percentage of cell viability was calculated using untreated control cells by the following formula:

Anti-inflammatory Assay
After cytotoxicity test, the anti-inflammatory activity of the CBD/β-CD loaded hydrogels towards RAW 264.7 cells was assessed by measuring nitric oxide (NO) released as nitrite via a Griess assay. Briefly, RAW 264.7 cells were seeded at a density of 30,000 cells/well into 96-well TCPS plates in the phenol red-free medium supplemented with 10% v/v fetal bovine serum and 1% penicillin/streptomycin. Three dilutions of the sample extract (0.315, 0.625, and 1.25 mg/mL) were added to each well and further incubated for 3 h. Subsequently, the culture medium was activated by lipopolysaccharide (LPS, 10 µg/mL) for 24 h in order to stimulate NO production. After that, the level of NO was determined using Griess reagent (Promega, USA). Thereafter, 50 µL of the collected medium was mixed with 100 µL of Griess reagent and incubated for 20 min in the dark at room temperature. The concentration of nitrite was determined at 540 nm using a microplate reader and calculated using a sodium nitrite standard curve (0-100 µM). Cells treated with vermelhotin acted as the positive control and cells without any treatment acted as the negative control.

Statistical Analysis
Means and standard errors of the means (n = 3) are used to present the data. The means of the various data sets were compared using a one-way analysis of variance (one-way ANOVA) and Turkey's post hoc test in SPSS (SPSS, IBM, Armonk, NY, USA), with statistical significance accepted at a 0.05 confidence level.

Results and Discussion
Wound management is the main part of the wound therapy. The creation of promising materials for wound care to fulfill crucial dressing needs has been substantially accelerated by recent developments in hydrogel-based wound dressings [30]. Although there are currently several hydrogel-based dressings available, new solutions for wound care treatment are urgently needed to meet rising demand. Due to its capacity to remove tissue debris by flowing into the wound bed, semi-solid hydrogel wound dressing in particular could be a novel option for wound treatment. Furthermore, because known pathogens are becoming increasingly resistant to (6) % Cell viability = absorbance of treated cells absorbance of treated cells × 100 the therapeutic treatments now in use, plant-derived active chemicals may offer a new choice for wound healing.
In this work, we attempted to prepare physically crosslinked PVA borax hydrogels to generate transient networks improves the flow ability of hydrogels into the irregularly-shaped of wound bed for use in wound treatment. The semi-solid PVA hydrogels loaded various amounts of the CBD/β-CD inclusion complex were prepared based on ionic crosslinking method as shown in Scheme 1. This is attributed to the formation of temporary crosslinks between tetrahydroxyborate anion and hydroxyl functional groups of PVA in its repeating unit to form didiol complexation [43]. These characteristics of hydrogels and prospective applications are examined in the next section.

FT-IR Analysis
FT-IR spectroscopy was investigated to prove the alteration of characteristic peak of many groups in a molecule. As a result, it is frequently used to research how hosts and guests interact in inclusion complexes [50]. If CBD forms the inclusion complexes with β-CD, the characteristic peaks of CBD probably change. Figure 2  For the CBD/β-CD inclusion complex, all the peaks belonging to β-CD were observed while the characteristic peaks of CBD disappeared, indicating that some interaction occurred between CBD and the β-CD molecules. These results further proved that CBD/β-CD inclusion complex was successfully formed.

H-NMR Analysis
To reveal the characteristics of the inclusion complexes, the 1 H-NMR spectra of CBD, β-CD and their inclusion complex were analyzed, as illustrated in Fig. 3. Corresponding with the FT-IR results, the 1 H-NMR spectrum of CBD dissolved in DMSO-d6 also showed relatively distinct characteristic peaks at 0.  The chemical environment of the protons inside the cavity will be altered if a guest molecule is encapsulated in the cavity of β-CD. The chemical shifts of the protons will then be altered, while those of the protons outside the cavity will remain unchanged [21]. Following encapsulation, the chemical shifts of the protons belonging to the guest molecule will also alter [51]. The proton peaks of the CBD generally are not clearly visible when D 2 O was utilized as a solvent because of its weak water solubility. However, the 1 H-NMR spectrum of the CBD/β-CD inclusion complex dissolved in D 2 O revealed distinctive peaks that were the sum of the CBD and β-CD spectra. However, the insignificant alteration in chemical shifts might be due to the weak non-covalent forces between CBD and β-CD [51].

XRD Analysis
The crystalline nature of the complexation between guest molecules and β-CD could be examined by X-ray diffraction (XRD) analysis. The XRD pattern will not be a straightforward superimposition of the crystalline of the host and guest molecules because the crystalline character of the host and guest molecules is destroyed once they form inclusion complex [52,53]. As evidenced in Fig. 4, the diffraction patterns of CBD exhibited distinct peaks at 2θ angles around 9.7, 11.7, 13.1, 15.1, 16.6, 17.4, 18.8, 21.6, 22.2, and 22.7 showed a high degree of crystallinity of CBD. Identically, β-CD displayed many diffraction peaks at 2θ angles around 9.0, 10.7, 12.5, 15.4, 18.9, 20.7, 22.7, 27.0, 34.9, and 45.2 which indicate the crystalline form. This pattern corresponds with the cage-type packing in the molecular organization of β-CD [54]. However, the spectra of the inclusion complexes were noticeably different from those of CBD and β-CD. The

Morphology of CBD/β-CD Inclusion Complex
SEM is employed as a supplementary method to verify the morphology before and after inclusion complex. Figure 5a shows that CBD mainly appeared as an irregularly shape of various sizes, while β-CD (Fig. 5b) displayed a three-dimensional block structure with an even shape. For the inclusion complex, the original morphology of both components disappeared as shown in Fig. 5c. These results were in line with the findings of FT-IR, 1 H-NMR and XRD, indicating that the inclusion complexes were produced successfully.

Encapsulation Efficiency, Loading Capacity and Water Solubility
The complexation efficiency (%CE) and loading capacity (%LC) are two important parameters, which are frequently employed to estimate the amount of guest molecule entrapped into the cavity of CDs [55]. %CE is the percentage of the drug that is successfully entrapped into the β-CD, whereas %LC is the percentage of the drug in the complexation. In this work, %CE and %LC were quantified by UV-visible analysis at a wavelength of 273 nm and calculated using Equations (1) and (2), respectively. As shown in Table 2, %CE and %LC of the obtained CBD/β-CD inclusion complex were 84.73 ± 0.64% (n = 3) and 32.70 ± 0.32% (n = 3), respectively. These results confirm that CBD encapsulated in β-CD were successfully prepared and can be used as a healing drug in the hydrogel matrices.
The water solubility of CBD/β-CD inclusion complex was assessed by preparing their saturated aqueous solutions. As compared with that of raw CBD (0.021 µg/ml), the water solubility of CBD was significantly increased to 0.336 µg/ ml which was promoted by 16-fold. As mentioned above, β-CD has a hydrophilic external surface and a hydrophobic internal cavity that can bind to CBD molecules via intermolecular hydrogen bonds and van der waals force. Therefore, utilizing the water-soluble β-CD as a medicinal carrier could improve CBD's solubility.

FT-IR Analysis
FT-IR spectroscopy was studied to characterize the chemical structure and intermolecular interaction of the as-prepared hydrogels. As illustrated in Fig. 6, PVA_0 showed a broad peak around 3400-3200 cm −1 (vibration of O-H groups and hydrogen bonds), 2937 cm −1 (asymmetric stretching of CH 2 ), 2908 cm −1 (symmetric stretching of CH 2 ), and 1650 cm −1 (due to water absorption). Additionally, important peaks related to B-O-C asymmetric stretching vibration appeared at 1417 and 1332 cm −1 [56]. This observation indicates that borate ions ( BO 3− 3 ), obtained from borax dissociation in the water, acted as inter and intra crosslinking agents by means of tetrahedral complexes and hydrogen bonding with diol units of PVA chains to form temporary physical crosslinks. For the hydrogels loaded with the CBD/β-CD inclusion complexes (PVA_0.5, PVA_1.0, and PVA_1.5), the FT-IR spectra displayed characteristic absorption identical to the spectrum of PVA_0, except the peak at 1650 cm −1 which shifted to the lower wavenumber attributed to overlap deformation vibration of the absorbed water molecules presented in the PVA hydrogel and CBD/β-CD inclusion complex.

Water Content
The water content of the as-prepared hydrogels is shown in Table 3. The water content of all hydrogels ranged between 94.11 ± 0.64% to 95.64 ± 0.28%. The amount of water content in the samples gradually decreased with increasing amounts of the inclusion complex. This may be the result of the high interaction between hydroxyl groups of β-CD and PVA, resulting in decreased water interaction of the hydrogels.

Mechanical Properties
For wound management application, the semi-solid hydrogels must possess the required mechanical characteristics to withstand the physiological stress brought on by the movement of skin. These hydrogels should also firmly adhere to the skin during the healing process and be simple to apply and remove without causing damage or leaving behind residue [57,58]. Thus, the hardness, compressibility, and adhesiveness of the semi-solid PVA-borax hydrogels loaded CBD/β-CD inclusion complex must be evaluated and the results are shown in Fig. 7. The hardness and compressibility of the obtaines hydrogels were significantly lower than those of the pure semi-solid PVA hydrogels. These findings may be the result of the steric hindrance of CBD molecules, which reduces the ability of PVA and tetrahydroxyborate anion to form a complex. However, the adhesiveness values of the obtained hydrogels were not significantly different.   Fig. 7 Mechanical properties of the semi-solid PVA-borax hydrogels loaded CBD/β-CD inclusion complex with various contents. *p < 0.05 compared with pure PVA hydrogels

In Vitro CBD/β-CD Release from the Semi-solid PVA-Borax Hydrogels
The actual contents of the CBD/β-CD inclusion complex in the obtained hydrogels were determined before investigating the CBD release characteristics from the hydrogels. The results show that the actual contents of CBD loaded PVA hydrogels were 0.0507 ± 0.0088 g, 0.1072 ± 0.0546 g, and 0.1284 ± 0.0404 g for PVA_0.5, PVA_1.0, and PVA_1.5, respectively. The CBD release characteristics from the as-prepared hydrogels were investigated by the transdermal diffusion method using the vertical franz diffusion cell over a period of 72 h in a phosphate buffer saline solution (PBS; pH 7.4) and incubated at 37 °C under continuous stirring. As shown in Fig. 8, the cumulative release profiles of CBD from all the hydrogels were presented as the percentage of the weight of CBD released divided by the actual weight of CBD. For PVA_0.5 and PVA_1.0, the cumulatively released content of CBD from these hydrogels gradually increased with increased dissolution time and then reached a plateau value. However, the cumulative released from PVA_1.5 increased rapidly with increased dissolution time, continued to increase more gradually afterward, and then reached a plateau value. Normally, an initial faster and a large content of drug release were found at higher loading doses of the drug. This can be explained by the fact that the higher drug concentration in the hydrogels produced a significant concentration gradient, which in turn raised the driving force for diffusion and increased the amount of drug released [59]. The maximum contents of CBD released from PVA_0.5, PVA_1.0, and PVA_1.5 were 67.14 ± 5.96%, 79.74 ± 4.82%, and 92.09 ± 6.86%, respectively. Additionally, the cumulatively released contents of CBD from PVA_1.5 was greater than those from both PVA_0.5 and PVA_1.0 due to the higher loading of the CBD/β-CD complex, which caused a cluster of active molecules to collect on the hydrogels' surfaces and made it easier for the CBD molecules to diffuse out of the hydrogels.

Total Phenolic Content and Antioxidant Activity
Phenolic compounds are the important plant constituents with redox characteristics that are in charge of their antioxidant activity [60]. The hydroxyl groups in plant extracts are accountable for facilitating free radical scavenging. Normally, The Folin-Ciocalteu reagent was typically used to calculate the phenolic content of each extract. Table 4 presents the total phenolic contents of all the semi-solid hydrogels. The total phenolic contents increased as the amount of CBD loaded increased. PVA_0.5, PVA_1.0, and PVA_1.5 had the average total phenolic content of 0.54 ± 0.02, 0.65 ± 0.05, and 0.73 ± 0.01 mg GAE/g extract, respectively.
An antioxidant is a molecule which is capable of inhibiting the oxidation of other molecules. They are believed to speed up wound healing by reducing wound oxidative stress [61]. Additionally, they play a crucial role as mediators in controlling possible damage to biological components like DNA, protein, lipids, and body tissue when reactive species are present [62]. For CBD, the antioxidant activity came from its pentyl-substituted bisphenol aromatic group linked to an alkyl-substituted cyclohexene terpene ring system. Commonly accepted assays, such as DPPH, FRAP, and ABTS, are used to evaluate the antioxidant activity of plant extracts [63]. The antioxidant capacity analysis results   [64,65]. Therefore, the free radicals show several resonance structures in which the unpaired electrons are mainly distributed on alkyl and hydroxyl groups, as well as on the benzene ring [65]. Subsequently, adding CBD to the hydrogel is beneficial for improving the antioxidant properties of wound management applications.

Antibacterial Activities
Antibacterial wound dressings have become increasingly popular in recent years because bacteria are the main issue affecting wound treatment [66]. The threat of infection, contamination, or colonization must be addressed by using wound dressings which offer broad-spectrum antibacterial capabilities [67]. CBD has some remarkably useful antimicrobial activity against a wide range of gram-positive bacteria and a small subset of gram-negative bacteria [68]. In this work, the disc diffusion method, or clear zone, was used to evaluate against gram-negative Escherichia coli (E. coli), gram-positive Staphylococus aureus (S. aureus), and Methicillin-resistant Staphylococcus aureus (MRSA). The resulting inhibition zone of bacteria is illustrated in Table 5.
The as-prepared hydrogels are only able to prevent S. aureus and MRSA, while no zone of inhibition was observed for E.
coli. The largest diameter of the zone of inhibition with S. aureus and MRSA observed for PVA_1.5 was 0.7 ± 0.3 mm and 1.0 ± 0.3 mm, respectively. A greater inhibition against gram-positive bacteria compared to gram-negative strains of bacteria was observed, which is likely the result of variations in the cell wall structure of these microorganisms. According to Blaskovich et al. research, the presence of the outer membrane and lipopolysaccharide is a factor in ineffectiveness of CBD against gram-negative bacteria [68].

Cytotoxicity Evaluation
Cytotoxicity is a basic property of materials used in biomedical applications [69]. Thus, all samples were investigated to explore the potential use as wound management materials in terms of indirect cytotoxicity towards RAW 264.7 cells via MTT reduction assay. Cytotoxicity of CBD in the culture medium was also been evaluated according to the ISO 10993-5-2009 standard. According to the ISO standards, the viability of cells exposed to the material is higher than 70% is considered as non-cytotoxic materials [70]. Figure 9 shows the viability of the cells obtained from the MTT assay after the cells were cultured with various concentrations of extraction media which extracted from pristine semi-solid hydrogel (PVA_0) and their corresponding hydrogels loaded CBD/β-CD inclusion complex (PVA_0.5, PVA_1.0 and PVA_1.5), as compared to that obtained after the cells had been cultured with phenol red-free DMEM cell culture medium. Evidently, the viability of RAW 264.7 cells with all the extraction media concentrations from PVA_0 was in the range of 93-98%. This indicates that PVA_0 does not release some cytotoxic substances into the culture media, implying the biocompatibility of pristine materials. For any  Fig. 9 Indirect cytotoxicity of semi-solid PVA-borax hydrogels loaded with various amounts of CBD/β-CD inclusion complex cultured with RAW 264.7 cells. *p < 0.05 compared with phenol red-free DMEM cell culture medium at any given extraction ratio given concentration of the extraction media, the viability of the cultured cells extremely decreased in line with increased concentrations. The viability of the cultured cells with all concentrations of extraction media from PVA_0.5, PVA_1.0, and PVA_1.5 were in the range of 84-98%, 7-103%, and 6-104%, respectively. Additionally, the cell viability was greater than 70% in all cases of the hydrogels loaded with the CBD/β-CD complex when the extraction medium concentration of 0.3125-1.25 mg/mL. However, the cell viability was lower than 70% for PVA_1.0 and PVA_1.5 when the extraction medium concentration exceeded 5 and 2.5 mg/mL, respectively. This result confirms toxicity in these conditions. According to the previous study, CBD-induced cytotoxicity increased in a dose-and time-dependent manner [71]. Meanwhile, all the cases of hydrogels loaded with the CBD/β-CD complex at concentrations below 1.25 mg/mL for the extraction medium had cell viabilities in excess of 70%, indicating that these conditions were non-cytotoxic and are therefore have potential in wound dressing applications.

Anti-inflammatory Activity
In the early phase of wound healing, inflammatory cells are drawn to the wounded site in order to clean the region of bacteria and debris and to promote the development of granulation tissue and neovascularization [66]. However, the continued presence of inflammatory cells causes the creation of tissue-degrading proteinases to be stimulated, which can obstruct the healing process. Therefore, anti-inflammatory components in wound dressings have the ability to reduce this process and create a suitable environment for wound healing [72]. It is generally known that polyphenolic chemicals have anti-inflammatory effects [73,74]. Therefore, the anti-inflammatory properties of the hydrogels loaded CBD/ β-CD inclusion complex were assessed in vitro against RAW 264.7 cells in terms of their ability to suppress nitric oxide (NO) production. NO is a proinflammatory mediator that triggers inflammation under unusual conditions, making it a promising marker for the treatment of inflammatory diseases [75]. Lipopolysaccharide (LPS), an external stimulation for the overproduction of NO, has the ability to activate macrophages. As a result of the incredibly short physiological half-life of NO, it is quickly converted to nitrite, which can then be quantified using the Griess assay [76]. Figure 10 shows the anti-inflammatory activity of the cells after treating with various concentrations of extraction media which extracted from the obtained hydrogels. Evidently, all the extraction media concentrations from PVA_0 were no significant difference when compared to the LPS treatment group, indicating that there were no antiinflammatory activity of pristine materials. However, for any given concentration of the extraction media from PVA_0.5, PVA_1.0, and PVA_1.5, the LPS stimulated NO production was decreased in line with increased extract medium concentrations. Especially for the extraction media of all formulas at the concentration of 1.25 mg/mL, the NO production could be significantly reduced to less than 25% of the LPS treated group, indicating the efficacy of the CBD released from the obtained hydrogels to improve anti-inflammatory effects. Jean-Gilles et al. also showed that CBD can indirectly enhance anti-inflammatory effects by reducing the generation of pro-inflammatory cytokines, preventing T cell proliferation, and reducing immune cell migration and adhesion [77].

Conclusion
In this work, the researchers successfully prepared semisolid PVA-borax hydrogels containing the CBD/β-CD inclusion complex. The inclusion complex was prepared via the co-precipitation method. The FT-IR, 1 H-NMR, XRD and SEM results indicate the successful formation of the CBD/ β-CD inclusion complex. The complexation efficiency was 84.73 ± 0.64%, the loading capacity was 32.70 ± 0.32% and the water solubility of CBD was significantly increased by 16-fold, as compared with that of raw CBD. The obtained inclusion complex was incorporated into the semi-solid hydrogels with various contents. The hydrogels were evaluated for their potential for use as wound management materials in terms of their release characteristics, antibacterial capability, antioxidant activities, and cytotoxicity. The cumulatively released amounts of CBD from the hydrogels increased with higher amounts of the inclusion complex. The prepared hydrogels provided an antibacterial capability Fig. 10 Anti-inflammatory activity of RAW 264.7 cells after treating with various concentrations of extraction media which extracted from the CBD/β-CD inclusion complex loaded hydrogels, vermelhotin alone (positive control), and cells without any treatment (negative control). *p < 0.05 compared with LPS treated group with S. aureus and MRSA, while no antimicrobial capability was observed for E. coli. However, the addition of CBD to the hydrogel would be beneficial for improving the antioxidant properties of the obtained hydrogels. All the hydrogels loaded with the CBD/β-CD inclusion complex at concentrations below 1.25 mg/mL for the extraction medium were non-cytotoxic towards RAW 264.7 cells and also provided a significantly reduction of NO (more than 75%) compared with the LPS treated group. These results indicated the potential use of these hydrogels for wound management applications.