Cinnamyl-Modified Polyglycidol/Poly(ε-Caprolactone) Block Copolymer Nanocarriers for Enhanced Encapsulation and Prolonged Release of Cannabidiol

2.2. Synthesis of Polyglycidol-Block-Poly(Propylene Oxide)-Block-Poly(α-Cinnamyl-ɛ-Caprolactone-co-ɛ-Caprolactone)-Block-Poly(Propylene Oxide)-Block-Polyglycidol Block Copolymer

The polymer precursor N₃-[(PCyCL)₄–co-(PCL)₄₀]-N₃ (0.076 g, 0.0154 mmol, 1 eq) and CuBr (0.0220 g, 0.154 mmol, 10 eq) were added to a 50 mL round-bottom flask under an argon atmosphere. Dry THF (3 mL) was added via a syringe, and the solution was purged with argon and stirred vigorously for 20 min. Monoalkyne-terminated PEEGE₅₀–b-PPO₄-C≡CH (0.247 g, 0.03087 mmol, 2 eq) was dissolved in dry THF (2 mL) and added to the solution along with PMDETA (0.0396 g, 0.228 mmol, 20 eq). The click coupling reaction was carried out at 30 °C for 24 h. The reaction mixture was cooled to RT, diluted with THF (30 mL), and filtered through a column filled with neutral alumina to remove copper complexes. The excess THF was evaporated; the crude product was dissolved in methanol (10 mL) and dialyzed against a methanol/water mixture (10:1 v/v, membrane, MWCO 8 kDa) for 72 h. The methanol was removed using a rotary vacuum evaporator, and the copolymer was recovered by freeze-drying. Yield: 0.258 g (80%); M_n = 15,000 g·mol⁻¹, M_w/M_n = 1.3.

PEEGE blocks were derivatized into PG ones by treatment with AlCl₃·6H₂O, as described elsewhere [54,55]. PEEGE₅₀–b-(PO₄)-b-[(PCyCL)₄–co-(PCL)₄₀]-b-(PO₄)-b-PEEGE₅₀] (0.100 g, 1 eq) was dissolved in methanol (8.9 mL, 3200 eq) at 40 °C, and then AlCl₃·6H₂O (0.020 g, 10 eq) was added under stirring. The hydrolysis was conducted at the same temperature for 48 h until the complete disappearance of the methine proton signal at 4.75 ppm in the ¹H-NMR spectrum. The reaction mixture was filtered through Hyflo Super Cel diatomaceous earth, and then methanol was evaporated under reduced pressure.

2.3. Preparation of Polymeric Micelles

A copolymer solution (5 mg of copolymer in 5 mL of methanol) was added dropwise to purified water (5 mL) at room temperature under vigorous stirring (1000 rpm). After 30 min, the organic solvent was evaporated under reduced pressure at 37 °C to obtain a slightly opalescent, colorless micellar dispersion with a concentration of 1 mg·mL⁻¹.

2.4. Loading of CBD

The micelles of the novel (PG₅₀-b-PPO₄-b-[P(CyCL)₄-co-(CL)₄₀]-b-PPO₄-b-PG₅₀) and referent (PG₄₅-b-PCL₃₅-b-PG₄₅) copolymers were loaded with CBD according to two protocols, schematically depicted in Scheme 1. The encapsulation efficiency (EE) was calculated from the following equation:

Protocol A: The selected copolymer (5 mg) and CBD (0.5 mg) were dissolved in methanol (5 mL) at a copolymer/CBD-mass ratio of 10:1. After that, the organic solution was added dropwise to purified water (5 mL) at room temperature under vigorous stirring (1000 rpm). The resulting mixture was stirred additionally for 30 min at the same temperature. Finally, the organic solvent was evaporated by a rotary vacuum evaporator at 37 °C, yielding a slightly opalescent, colorless aqueous micellar dispersion. The dispersion was filtered (Nylon, 0.22 μm) and the filter was rinsed with ethanol. The collected filter fraction was quantified by spectrophotometric measurements (λ = 274 nm) to determine the mass of free, unentrapped CBD.

Protocol B: The selected copolymer (5 mg) was dissolved in methanol (5 mL). After that, the organic solution was added dropwise to purified water (5 mL) at room temperature under vigorous stirring (1000 rpm). The resulting mixture was stirred additionally for 30 min at the same temperature. Next, the organic solvent was evaporated by a rotary vacuum evaporator at 37 °C. The CBD (0.5 mg) (copolymer/CBD-mass ratio 10:1) dissolved in 40 µL methanol was added to the as-prepared micellar dispersion. The resulting mixture was stirred additionally for 30 min at the same temperature. Finally, the traces of organic solvent were evaporated under argon, yielding a slightly opalescent, colorless aqueous micellar dispersion. The dispersion was filtered (Nylon, 0.22 μm) and the filter was rinsed with ethanol. The collected filter fraction was quantified by spectrophotometric measurements (λ = 274 nm) to determine the mass of free, unentrapped CBD.

2.5. Proton Nuclear Magnetic Resonance (¹H-NMR)

¹H-NMR measurements were conducted on a Bruker Avance II spectrometer operating at 600 MHz using CDCl₃, DMSO-d_6, or CD₃OD at 25 °C.

2.6. Size Exclusion Chromatography (SEC)

Analyses were performed on Shimadzu Nexera HPLC chromatograph, equipped with a degasser, a pump, an auto-sampler, a RI detector, and three columns: 10 µm PL gel mixed-B, 5 µm PL gel 500 Å, and 50 Å. THF was used as the eluent at a flow rate of 1.0 mL·min⁻¹ and a temperature of 40 °C. The sample concentration was 1 mg·mL⁻¹ and GPC was calibrated with polystyrene standards.

2.7. Transmission Electron Microscopy (TEM)

Micrographs were obtained using an HRTEM JEOL JEM-2100 transmission electron microscope operating at 200 kV. The samples were prepared by depositing a drop of the dispersions onto a carbon grid and the subsequent evaporation of the solvent under a vacuum. To visualize the hydrophilic corona with high contrast, a uranyl acetate staining protocol prior to sample preparation was utilized [56].

2.8. Atomic Force Microscopy (AFM)

The images were obtained using a Bruker Dimension Icon Instrument operating at a 1.00 Hz scan rate under ambient conditions. Moreover, 2 μL of the copolymer dispersions was placed onto a freshly cleaned glass substrate (1 cm²) and spin-casted at 2000 rpm for a minute. AFM measurements were performed in ScanAsyst (Peak Force Tapping) mode.

2.9. Dynamic and Electrophoretic Light Scattering

The preliminary assessment of the particle size and size distribution was carried out by dynamic light scattering (DLS) measurements on a NanoBrook 90 Plus PALS instrument (Brookhaven Instruments Corporation), equipped with a 35 mW red diode laser (λ = 660 nm) at a scattering angle of 90 °. The measurements were taken at 25 °C and 37 °C applying a dust cut-off of 30 micellar dispersions at concentrations of 1 mg·mL⁻¹ and fixed volumes of 1.7 mL.

The apparent hydrodynamic radii (R_h⁹⁰) were determined according to the Stokes–Einstein equation:

where k is the Boltzmann constant, η is the solvent viscosity at temperature T in Kelvin and D₉₀ is the diffusion coefficient measured at an angle of 90°. Each measurement was performed in triplicate.

The electrophoretic light scattering measurements were carried out on the same instrument at a scattering angle of 15° and 25 °C and 37 °C. The principle of phase analysis light scattering (PALS) was applied for the measurements of electrophoretic mobility. The ζ potentials were calculated using the Smoluchowski equation:

where η is the solvent viscosity, υ is the electrophoretic mobility, and ε is the dielectric constant of the solvent.

2.10. Multiangle Dynamic and Static Light Scattering

Dynamic light scattering measurements were performed on a Brookhaven BI-200 goniometer with vertically polarized incident light at a wavelength λ = 633 nm supplied by a He–Ne laser operating at 35 mW and equipped with a Brookhaven BI-9000 AT digital autocorrelator. The scattered light was measured for dilute aqueous dispersions of the empty and CBD-loaded micellar dispersions in the concentration range 0.417–1.0 mg·mL⁻¹ at 25 °C. Measurements were made at angles θ in the range of 50–130°. The autocorrelation functions were analyzed using the constrained regularized algorithm CONTIN [57] to obtain the distributions of the relaxation rates (Γ). The latter provided distributions of the apparent diffusion coefficient (D = Γ/q²), where q is the magnitude of the scattering vector given by q = (4πn/λ)sin(θ/2), n is the refractive index of the medium. The mean hydrodynamic radius was obtained by the Stokes–Einstein equation:

where k is the Boltzmann constant, η is the solvent viscosity at temperature T in Kelvin, and D₀ is the diffusion coefficient at infinite dilution.

Static light scattering (SLS) measurements were carried out in the interval of angles from 40 to 140° at 25 °C using the same light scattering set. The SLS data were analyzed using the Zimm plot software provided by Brookhaven Instruments. Information on the weight-average molar mass, M_w, the radius of gyration, R_g, and the second virial coefficient, A₂, was obtained from the dependence of the quantity (Kc/R_θ) on the concentration (c) and scattering angle (θ). Here, K is the optical constant given by K = 4π²n₀²(dn/dc)²/N_Aλ⁴, where n₀ is the refractive index of the solvent, N_A is Avogadro’s constant, λ is the laser wavelength, and R_θ is the Rayleigh ratio at angle θ. dn/dc is the refractive index increment measured in water in separate experiments on an Orange GPC19 DNDC refractometer. The dn/dc values of the investigated systems were in the 0.131–0.133 g·mL⁻¹ range. The DLS and SLS measurements were performed at 25 and 37 °C.

2.11. Spectrophotometric Determination of the Critical Micelle Concentration (CMC)

Furthermore, 20 μL of a 0.4 mM solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) in methanol was added to 2 mL micellar dispersions with increasing concentrations in the range 9.765 × 10⁻⁴–1.0 mg·mL⁻¹. The samples were incubated in the dark for 24 h at room temperature. UV-vis absorption spectra of DPH in the wavelength interval λ = 300–500 nm at room temperature were recorded on a Beckman Coulter DU 800 UV-vis spectrometer. The intensities of the absorption peak at 356 nm were plotted against the polymer concentration. The CMC value was determined as the break in the absorbance intensity versus the concentration curve.

2.12. Drug-Release Study

The release of CBD was evaluated as a function of time using the dialysis method [58]. In short, 2 mL of tested formulations was inserted into a dialysis bag (MWCO 12,000–14,000, Sigma-Aldrich, Steinheim, Germany) and placed in a 50 mL dissolution medium of phosphate-buffered saline pH 7.4 and 10% ethanol to retain the solubility of the released CBD. The acceptor medium was in constant motion (200 rpm) and the circulating water jacket (Huber, Germany) maintained the temperature at 37 ± 0.5 °C during the study. At predetermined time points, 1 mL samples from the released medium were withdrawn and analyzed by UV–vis spectroscopy at 274 nm using a calibration curve with linearity in the concentration range of 0.0025 to 10 µg·mL⁻¹ (correlation coefficient R² = 0.995). The aliquots were replaced with equal volumes of fresh medium. The drug-release studies were carried out threefold.

2.13. Cytotoxicity Assessment

The cytotoxicity of the copolymers and the micelles prepared thereof was evaluated by standard MTT-dye reduction assay. The assay is based on the aptitude of the mitochondrial succinate dehydrogenase of viable cells to metabolize the yellow tetrazolium MTT dye to a violet formazan. The experiment was performed as described by Mosman [59] with small modifications [60]. Cells in the exponential phase were seeded in 96-well microplates (100 μL/well) at a density of 1 × 10⁵ cells·mL⁻¹ and incubated at 37 °C for 24 h. Afterward, they were exposed to various concentrations of empty micelles or free or micellar CBD for a period of 72 h. For each test group, a set of at least eight wells were used. After the exposure time, to each well aliquots of 10 μL of MTT solution (10 mg·mL⁻¹ in PBS) was added. Next, the microplates were incubated for 4 h at 37 °C and the MTT-formazan crystals formed were dissolved by the addition of 100 μL/well of 5% formic acid solution in 2-propanol. The MTT-formazan absorption was recorded using a Beckman–Coulter DTX800 multimode microplate reader at 580 nm. Thereafter the cell survival fractions were calculated as a percentage of the untreated control. In addition, IC₅₀ values were derived from the concentration-response curves.

3.2. Preparation of Blank and CBD-Loaded Polymeric Micelles

The critical micelle concentration of the modified copolymer was determined by dye solubilization. The method employs specific photophysical properties of the hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene: its UV absorbance in water is minimal, whereas it increases substantially in a hydrophobic environment so that the appearance of a characteristic maximum at 356 nm and its sharp increase upon increasing copolymer concentration indicates an abrupt change in the properties of the system— the formation of hydrophobic domains (presumably cores of the micelles). DPH solubilization has frequently been employed for the determination of the CMC of conventional surfactants and amphiphilic polymers [48,61,62,63]. The CMC value was determined from the break of the absorbance intensity vs. copolymer concentration curve as shown in Figure 2.

The resulting value (0.12 mg·mL⁻¹) was slightly larger than that of the reference copolymer PG₄₅–b-PCL₃₅–b-PG₄₅ (0.10 mg·mL⁻¹) [48] and probably reflected the effects of the longer hydrophilic polyglycidol blocks.

At concentrations ca. one order of magnitude higher than the CMC, the copolymer micelles were loaded with CBD by applying different protocols, depicted in Scheme 1—loading during micelle formation (protocol A) and loading in preformed micelles (protocol B). Based on previous studies [51,53] and preliminary concentration-dependent measurements, the drug/copolymer-mass ratio of 1:10 at a copolymer concentration of 1 mg·mL⁻¹ was determined to be the most effective and optimal, ensuring the highest encapsulation efficiency and drug loading. Preliminary screening of the size and size distributions, performed by DLS at a single angle (90°), revealed monomodal size distributions and apparent hydrodynamic radii, R_h⁹⁰, in the 50–60 nm range (Table 2). All particles exhibited low ζ potential (very slightly positive or negative, practically neutral), which is in line with the non-ionic nature of the two copolymers and the drug (Table 2). The simultaneous formation and loading of the micelles (protocol A) yielded higher encapsulation efficiency than protocol B, that is, the loading of the drug into preformed micelles (Table 2). The aggregates of the novel copolymer invariably exhibited higher encapsulation efficiency than those of the referent copolymer (Table 2), which apparently reflected the introduction of cinnamyl-bearing units intended to increase the loading efficiency. Although exhibiting low ζ potential, the micelles of the two copolymers (blank or CBD-loaded) are characterized by enhanced colloidal stability for at least three months. The enhanced colloidal stability is provided by the hydrophilic corona built of polyglycidol chains.

Multiangle dynamic and static light scattering was performed to fully characterize the empty micelles of the novel copolymer and the micelles of the novel copolymer loaded by protocol A. In the investigated concentration range (0.417–1.0 mg·mL⁻¹), which is slightly above the CMC, the relaxation time distributions were predominantly monomodal, indicating the existence of only one population of particles. Additional modes of low amplitude (mainly fast modes of amplitude below 4%) were occasionally and unsystematically observed at certain angles. A representative time distribution is shown in Figure 3a. More relaxation time distributions and converted therefrom particle size distributions are presented in Figure S4 in the ESI. The diffusion coefficients were determined from the angular dependence of the relaxation rate (inversely proportional to relaxation time), measured at different scattering angles and then plotted against concentration to obtain the diffusion coefficient at zero concentration, D₀, as shown in Figure 3b and c. D₀ was used to calculate the hydrodynamic radii, R_h, of the empty and loaded micelles. The R_h values of the empty and CBD-loaded micelles are summarized in Table 3.

Static light scattering was performed to determine the weight-average molar mass (M_w), radius of gyration (R_g), and second virial coefficient (A₂) of the empty and loaded micelles. The static parameters were evaluated by the Zimm plot method. Zimm diagrams of the empty and loaded micelles are presented in Figure 4, whereas the derived parameters are collected in Table 3.

Evident from the results in Table 3 is that the CBD-loaded micelles are slightly smaller in size than the empty micelles, whereas the molar masses are comparable within the standard deviation of the method. These findings implied the formation of more compact and dense particles and revealed the effect of the hydrophobic drug molecules as nucleation sites on which copolymer self-assembly occurred, bringing about the enhancement of particle density. A realistic assessment of the different compactness levels of the empty and loaded particles is given by the particle density, ρ (Table 3), calculated from the molar mass and hydrodynamic volume data while assuming the spherical morphology of the particles (see ESI for the calculation of the particle density). Apparently, the ρ value of the CBD-loaded micelles was higher than that of the empty micelles. In addition, R_g/R_h attained values close to unity, which was compatible with the structure of both the empty and loaded micelles—relatively dense and compact particles with “hairy” surfaces [64,65]. A₂ values were very small in magnitude (of the order of 10⁻⁶ mL·mol/g²), which is in accordance with the high molar mass of the micelles, and negative. The negative values of A₂ normally indicate unfavorable particle–solvent interactions. As the A₂ values are very small, we may speculate here that there are weak attractive interactions between the micelles, previously observed for self-assembled structures of polyglycidol-based copolymers [48,66,67].

Another parameter that can be extracted from the light scattering data is the loading capacity expressed as the number of CBD molecules loaded in one micelle of the novel and referent copolymer. The light scattering characterization data of the referent copolymer micelles loaded with CBD as well as the calculation of loading capacity are presented in Figure S5 and Table S1 in the ESI. Evident from the values of this quantity was that the micelles of the novel copolymer bore about 32% more CBD molecules than those of the referent copolymer, which undoubtedly revealed the effect of the copolymer design, namely, the introduction of cinnamyl-bearing units in the PCL block of the copolymer. Measurements were also performed at the physiological temperature of 37 °C. As many of the constituent elements of the copolymer micelles did not exhibit- sensitivity to temperature variations in this temperature range, the characterization parameters were practically the same as those at 25 °C and, therefore, not presented.

Furthermore, data for the enhanced encapsulation efficiency and loading capacity of the modified block copolymer micelles were in excellent agreement with the calculated Flory–Huggins solubilization parameters (χ_sp) for the two core-forming blocks (Table S2 in the ESI). χ_sp for the modified block P(CyCL-co-CL) was 0.0039 vs. 0.1195 for PCL, indicating the significantly greater affinity of the former to CBD [68,69,70].

TEM and AFM were performed to resolve the morphology of the empty and CBD-loaded micelles (Figure 5 and Figure 6). The objects were well-separated with dimensions in a dry state that was consistent with the results from the dynamic light scattering. The spherical morphology is dominant; some irregularities in the sphericity were occasionally observed and could be attributed to dehydration-induced artifacts. We suggested that the increased contrast at the periphery of the micelles was due to a selective interaction between PG corona and the staining agent—uranyl acetate. The loading of the micelles with CBD did not affect their morphology.

Research equipment of Distributed Research Infrastructure INFRAMAT, part of the Bulgarian National Roadmap for Research Infrastructures, supported by the Bulgarian Ministry of Education and Science, was used in this investigation. D.M. acknowledges the support by the European Union-Next Generation EU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, Project BG-RRP-2.004-0004-C01. Georgi Momekov from the Department of Pharmacology, Pharmacotherapy and Toxicology at Faculty of Pharmacy, Medical University-Sofia is greatly acknowledged for the in vitro pharmacological evaluation of the developed nanosystems.