The method developed for the synthesis of polysaccharide cryogels by photochemical crosslinking in the frozen state requires the use of water as a freezable solvent [17]. The presence of organic solvents such as ethanol, methanol, etc. might hinder both the regular cryo-structuring process and crosslinking reaction [18]. Therefore, the first step of this study involved solubilization of the hydrophobic CBD in aqueous media with the aid of block copolymer micelles. PEO-b-PPO-b-PEO triblock copolymers are commercially available products (Pluronics, Poloxamers, etc.), well known for their high capacity to solubilize hydrophobic drugs [19]. In particular, PEO₁₀₁-b-PPO₅₆-b-PEO₁₀₁ (popular under trade names Poloxamer 407, Pluronic F127, etc.) is available as a pharmaceutical grade product and used for solubilizing active substances in pharmaceutical formulations [20]. For that reason, PEO₁₀₁-b-PPO₅₆-b-PEO₁₀₁ (denoted further in the text as F127 for simplicity) was selected to prepare a micellar solution of CBD in water by applying the solvent evaporation technique. First, water was added to an ethanol solution of CBD and block copolymer, followed by evaporation of the organic solvent under reduced pressure. The final concentrations of polymer and CBD were 30 and 10 g·L⁻¹, respectively, corresponding to 25% drug loading. DLS measurements of CBD-loaded micelles were conducted to determine the micellar size and size distribution (Figure 1). The intensity-weighted plot revealed the formation of small micelles having a hydrodynamic diameter (D_h) of 21 nm and relatively narrow dispersity. These results are similar to the characteristics obtained for pure F127 micelles (Figure 1, inset). There is an extra, less intensive peak (D_h = 173 nm) in the CBD-containing sample, suggesting the existence of larger aggregates in the colloid solution. However, one can assume that the micelles are the dominant population in the sample as the second peak is not visible in the volume-weighted plot.

Next, the micellar solution was mixed with an aqueous solution of HEC, crosslinking agent BAAm (or PEGDA) and photoinitiator H₂O₂ (Figure 2A). The mixture was homogenized, frozen and kept at temperature −20 °C for 2 h, and then irradiated with UV-light for 2 min. It has been previously shown that such reaction procedure affords good quality nanocomposite cryogels of high GF yield [10]. The mechanism of formation of nanocomposite super-macroporous cryogel as follows: The cooling of the aqueous mixture at the reported conditions caused the formation of ice crystals (solid phase) surrounded by a liquid microphase (LM). It is known that the solutes, i.e., polymer, initiator, crosslinking agent and micelles are concentrated in the LM, while the crystals are considered a pure form of water [21]. Consequently, the polymer network was formed in the LM by recombination of active (macro) radicals, generated from HEC and crosslinker molecules by the UV-irradiation. In turn, the solid crystals templated the cryogel pores. The thawing of frozen samples afforded spongy materials comprising interconnected pores, filled with free water which can be squeezed by compression. In principle, a polymer network can be obtained by crosslinking of HEC only; however, the involvement of bifunctional molecules (crosslinking agent) into the crosslinking process has been shown to improve the reaction efficacy and mechanical strength of cryogels [22]. In this work, two crosslinking agents, BAAm and PEGDA, were used at the same content (30 wt.% to HEC). The gels obtained with BAAm exhibited slightly higher GF yield than those with PEGDA (Table 1). This is probably due to the existence of more reactive end groups per unit mass, as BAAm has a lower molar mass than PEGDA. The freeze-dried cryogels, containing CBD were a little yellowish unlike the white pure HEC cryogel (Figure 2B). It was found that the presence of CBD in the reaction mixture interferes to some extent the crosslinking process. However, by keeping the drug concentration up to 10% to the HEC mass, one can obtain gels of relatively high yield (80–85%). From a practical point of view the small decrease of GF yield of CBD-loaded cryogels, compared to blank ones, is negligible since the gels were compact, elastic and easy to handle. At a first glance, the swelling behavior of cryogels in water also suggested some difference in the density of polymer network obtained in the presence/absence of CBD (Table 1). Normally, the denser networks swell less in liquids. In the case of cryogels, there are some specific features making the correct interpretation of results slippery. Cryogels are highly porous heterogeneous materials and naturally the water uptake is dominated by the interconnected pores. Typically, the capillary-bound water present in the pores is about 60–70% of the total water. In some cases, this is advantageous because the diffusion of small molecules (drugs, nutrients, etc.) within the gel is facilitated. As a whole, the nanocomposite cryogels uptake a significant amount of water which, in combination with the biocompatibility of polymers, makes them suitable for drug delivery applications.

The inner morphology of nanocomposite HEC cryogels was studied using scanning electron microscopy. Cryogels were freeze-dried and fractured prior to the analyses. It was anticipated that CBD-loaded micelles are embedded into the HEC matrix due to the cryo-structuring phenomena described above. Therefore, one could expect to see nano-sized micellar structures in the cryogel walls rather than in the pores. For comparison, pure HEC cryogel was analyzed by SEM under the same conditions. Representative micrographs of pure and nanocomposite HEC cryogels are shown in Figure 3. Both materials are super-macroporous and have similar dimensions for the pores (20–30 μm) and wall thickness (1–2 μm). At a high magnification, one can clearly differentiate the smooth surface of pure HEC gel (Figure 3B) and the surface decorated with nano-sized bumps of CBD/PM/CBD containing sample (Figure 3D). Thus, the SEM study confirmed our suggestion that CBD-loaded polymeric micelles were homogeneously embedded in the cryogel walls (polymer matrix).

Nanocomposite cryogels were soft and tear-resistant upon gentle handling. Their viscoelastic characteristics, storage (G′) and loss (G″) moduli were evaluated by dynamic rheological measurements in the oscillation–frequency mode. Figure 4 compares the results from HEC/PM cryogels prepared with and without CBD. In both cases, in the 0.3–10 Hz frequency range, the storage and loss moduli were frequency-independent and G′ was more than one order of magnitude higher than G″. The G′ and G″ values of the two samples were very similar. Definitely, the nanocomposite HEC cryogels exhibited typical properties for chemically crosslinked gels and one may conclude that the incorporation of CBD at the given concentration did not alter the mechanical strength of carrier.

The advantages of the nanocomposite cryogel carrier of CBD, in comparison to pure HEC carrier, were demonstrated by the release experiments. The in vitro tests were performed in acetate buffer of pH 5.5 taking into account that the elaborated cryogels are intended for a potential dermal application and the pH of human skin is about 5.5. The evaluation of CBD release from both pure and nanocomposite HEC cryogels showed a significant difference in the profiles (Figure 5). The pure HEC cryogel carrier was characterized by an initial burst release of CBD. In fact, the carrier released approximately 50% of CBD in the first hour of the dissolution experiment and the process completed within 8 h.

In the case of nanocomposite HEC/PM cryogels (obtained either with PEGDA or with BAAm), there was no burst effect. These systems were characterized with the pronounced release of CBD for the studied period of 24 h. The observed differences in the release profiles of CDB from pure and nanocomposite cryogel systems are most likely due to the different loading procedure and localization of the active compound, respectively. Indeed, one may assume that by soaking pre-formed HEC cryogel in an ethanol solution of CBD and subsequent solvent evaporation, most of the CBD was entrapped into the gel pores. In the macroscopic pores, the fluid uptake, dissolution and diffusion of CBD are rapid. In contrast, the loading of CBD in micellar cores combined with the cryogenic procedure afforded systems with CBD embedded within the gel walls (polymer matrix). Thus, the release from micelles and diffusion of drug molecules into the polar dissolution medium was decelerated. Noteworthy, the strategy to embed a hydrophobic substance in the cryogel walls via the reported procedure could be accomplished only with a water-soluble form of CBD such as a micellar solution. Besides, the type of crosslinking agents used in the preparation of nanocomposite cryogels did not affect the release behavior of the drug.

WAXD analysis of freeze-dried samples revealed that CBD, loaded in the nanocomposite cryogel was in an amorphous state (Figure 6). To collect statistical data and to avoid any preferred orientation from the crystals, the measurement was conducted by sample rotation at 10° min⁻¹. The patterns of pure CBD and pure F127 showed many diffraction peaks and their positions represent the crystal structure of CBD and F127, respectively. In contrast, in the diffraction patterns of HEC/CBD and HEC/PM/CBD, the crystalline structure of CBD cannot be detected. This phenomenon should be ascribed to the applied preparation procedure, i.e., encapsulation of a small amount of CBD in nano-sized micelles, which was then distributed in the polymeric matrix. The specific microstructure of material obviously did not favor the crystallization of CBD as well as poly(ethylene glycol) segments from the micellar shell.

The results from WAXD measurements confirmed the amorphization of CBD when the active substance was embedded in the HEC matrix in the form of a micellar solution via cryogenic treatment. This is considered advantageous since the solubilization of amorphous CBD in physiological media is easier.

To prove that polymeric carriers do not have intrinsic cytotoxicity, the inhibition effect of blank cryogel systems on the proliferation of mouse fibroblast cells (CCL-1) was studied (Figure 7A). The non-loaded cryogels were devoid of any cytotoxicity since no suppression of the vitality of the treated cells (values in the 94–98% range) was observed. Next, a comparative study regarding the antiproliferative activity of free cannabidiol and its nanocomposite cryogel formulations was carried out. In all experiments, we used a standard cytotoxicity bioassay test, based on the enzymatic reduction of the yellow tetrazolium salt MTT to a violet MTT-formazan by the mitochondrial succinate dehydrogenase in viable cells [23] with slight modifications [24]. The in vitro antineoplastic activity was evaluated against two human tumor cell lines MJ (T-cell lymphocyte) and T-24 (urinary bladder carcinoma). The concentration-response curves are depicted in Figure 7B,C and the equieffective inhibitory concentrations (IC₅₀) derived thereof are summarized in Table 2.

Evidently, both free CBD (applied as ethanol solution) and formulated CBD exhibited concentration dependent cytotoxicity. At lower drug concentrations, the effect of free CBD is more pronounced. The IC₅₀ values of the two nanocomposite HEC/PM cryogel systems (synthesized with PEAGA and BAAm) were higher than those obtained for the free drug (with an identical magnitude of modulation) depending on cell type. However, our findings firmly indicate that the tested nanocomposite carriers represent a plausible platform for the effective topical delivery of CBD without compromising its antineoplastic effects in terms of local skin or intravesical application. The in vitro bioassay unambiguously showed that the loaded systems are able to release CBD in a sustained manner and further to retain the tumor inhibitory effects of the natural product in respective in vitro models of mycosis fungoides (CTCL; cell line MJ) and non-invasive urinary bladder cancer (cell line T-24). Those two models represent important neoplastic diseases, whose anatomical localization and biological characteristics allow regional or topical therapy.