In this paper, micronization with the SAS technique has been carried out to obtain corticosteroid inclusion complexes, which have shown benefits in many fields of application. In particular, the active ingredients selected, i.e., DEX and PRED, have been micronized in the presence of a hydrophilic carrier to increase the relative rate of dissolution in water and, consequently, the bioavailability of the same within the human body. Therefore, the SAS micronization has been carried out by varying different operating conditions to identify the most suitable ones. During the tests, the effect of pressure (P), the active substance/carrier ratio, and the total concentration (Ctot) of solutes (active substance and β-cyclodextrin) in the solvent used (DMSO) are evaluated. The performed experiments are summarized in
Table 1, in which the morphology obtained, the average diameter of the particles (MD), and the standard deviation (SD) of the particle size distributions on a volumetric basis are indicated. In addition, the operating conditions investigated are shown, namely, the pressure, the total concentration of solutes in the injected solution (Ctot), and the active compound/carrier ratio.
3.4 Micronization of PRED/β-CD Complexes
PRED /β-CD system has been studied by evaluating the effect of pressure, the drug/polymer molar ratio, and concentration on morphology and particle size.
3.4.1 Effect of Pressure
The effect of the pressure was evaluated by fixing the PRED/β-cyclodextrin ratio at 1/2 mol/mol and the total solute concentration at 200 mg/mL; however, the pressure varied from 90 bar to 120 bar. For the initial test, a pressure of 90 bar was used in the chamber. Analysis through FE-SEM microscopy of the resulting samples confirmed the production of both microparticles and large crystals, as visible in
Figure 6a,b.
The average diameter is 0.435 μm and 2.414 μm, respectively, for the two tests.
As can be observed from
Figure 6a,b, the increase in operating pressure leads to the formation of well-defined micrometric particles. Indeed, at 120 bar (
Figure 7b), the complete disappearance of the crystalline form is observed; this can be explained by observing the thermodynamics of crystal formation at that specific pressure and temperature. Specifically, at 90 bar, the crystallization time is faster than the solvent removal time for this mixture, leading to the formation of large crystals and small particles precipitated onto them.
Overall, looking at
Figure 6c, it can be observed that the increase in pressure leads to particles with a larger diameter but, simultaneously, the disappearance of the crystalline form.
3.4.2 Effect of Molar Ratio PRED:β-CD
Another investigated parameter is the active principles/carrier molar ratio; therefore, two different values will be used: 1/1 mol/mol and 1/2 mol/mol. The parameters set for each test are a pressure equal to 120 bar, a temperature of 40 ºC, and a concentration of 200 mg/mL.
The first test was conducted with an active substance/carrier molar ratio of 1/1 mol/mol. From
Figure 7, a high degree of coalescing microparticles and the presence of crystals can be observed. Therefore, the particle size distribution was not evaluated.
The second test, carried out with a 1/2 mol/mol PRED/β-cyclodextrin molar ratio, as shown in the previous section, has led to the attainment of only microparticles, some of which coalescing (
Figure 6b).
Therefore, after the tests are carried out, it is possible to observe that the variation of the active principle/carrier molar ratio leads to a significant alteration of the obtained morphology. Passing from the test at 1/1 mol/mol to the test at 1/2 mol/mol, the crystals disappear at the end of the process. Therefore, it can be concluded that the greater amount of active substance present in the test 1/1 involves a shift of the MCP (mixture critical point) towards higher pressure values, and working at the same operating conditions, the operating point fell within the miscibility gap.
3.4.3 Effect of Total Concentration
The parameter investigated in this section is the effect of the total concentration; in particular, two different values were examined: 20 mg/mL and 200 mg/mL.
Therefore, the parameters left unchanged for testing are the molar ratio of 1/1 mol/mol (1/3 w/w), the pressure of 120 bar, and the temperature of 40 ºC.
The first test was conducted with a 20 mg/mL concentration value. FESEM images in
Figure 8 show the formation of coalescing microparticles. Due to coalescence, plotting the distribution of particle sizes was impossible.
The second test was then conducted at 200 mg/ml. In this case, as mentioned in the previous section, it is noted that no well-distinguished spherical particles were obtained.
Figure 7 shows an evident irregularity in the shape and a high degree of coalescence beyond the presence of crystals.
Therefore, the variation in the concentration of solutes inside the solvent leads to a remarkable alteration of the obtained morphology that can undoubtedly be explained by observing the thermodynamics of the considered system.
3.6 Characterization
Fourier transform spectroscopic analyses (FT-IR) were carried out to obtain information on the formation of inclusion complexes and to verify if there are interactions between the functional group of active ingredients and the polymer carrier.
Figure 9a,b show and compare the FT-IR spectra of unprocessed active ingredients, the pure carrier, physical mixtures, and powders obtained by the SAS process. In particular, the FT-IR spectrum of β-CD displays a broad absorption band between 3571 cm
-1 and 3220 cm
-1 corresponding to the -OH group stretching; a band at about 2928 cm
-1 and 1158 cm
-1, which corresponds to the stretching of the bond -CH
2 and -C-C and a peak at 1029 cm
-1 attributable to the bending of the -O-H group [
30].
The spectrum of DEX alone (
Figure 9a) shows two bands at about 3522 cm
-1 and 3381 cm
-1 that correspond to the stretching of the -OH and -OH ... H (hydrogen bond), respectively; a peak at 2935 cm
-1 corresponding to the stretching of the bond -C-H; three additional peaks at about 1702 cm
-1, 1658 cm
-1 and 1620 cm
-1 for stretching the bond -C=O; a broad band at 1261 cm
-1 due to stretching of the -C-F bond [
31,
32].
With regards to the spectrum of pure PRED (
Figure 9b), it is possible to observe a band in the range 3200-3500 cm
-1 characteristic of the -OH group, a peak at about 2947 cm
-1 due to the -C-H group, and three very intense peaks at 1708 cm
-1, 1654 cm
-1 and 1614 cm
-1 due to the -C=O carboxylic groups [
31].
Looking at
Figure 9a, it is clear that the characteristic bands of the polymer predominate in the spectra of processed powders and the physical mixture. In addition, the vertical line in black highlights the characteristic peak of DEX in the two processed SAS samples and the physical mixture (at 1620 cm
-1), while the vertical lines in pink show the characteristic peaks of the drug (at 1702 cm
-1 and 1658 cm
-1) which are present on the test spectrum 1/2 mol/mol and not on the test spectrum 1/1 mol/mol. This indicates that using an active ingredient/carrier molar ratio equal to 1/1 increases the complexation degree.
From
Figure 9b, where PRED spectra are shown, it can be observed that the physical mixture and processed powders have a more significant predominance of the characteristic bands of the polymer, demonstrating that the guest molecule (prednisolone) is embedded in the host molecule cavity (β-CD). Hence, the polymer hides its characteristic bands.
In addition, the characteristic peaks of the drug detectable in SAS powders and the physical mixture (at 2947 cm
-1 and 1654 cm
-1) are indicated by vertical lines in pink, while through vertical lines in black, the characteristic peaks of the drug at 1708 cm
-1 and 1614 cm
-1 absent in SAS powder with active principle/carrier ratio 1/2 mol/mol molar (this is since from the test 1/1 mol/mol, as shown in the FESEM images in
Figure 7, were obtained both crystal and microparticles).
In the spectra, a lower intensity of the peaks characteristic of the drug can also be noted, indicating that prednisolone formed partial inclusion complexes with β-CD (that is, only a tiny part of prednisolone is outside the cavity).
Once the presence of the active substance inside the samples produced by the SAS technique has been verified, using UV-vis spectroscopy, the dissolution profiles of the pure active principle and the microparticles obtained by the SAS technique are compared, using PBS (a saline phosphate buffer) at pH 7.4 and a temperature of 37ºC as the dissolving medium. The objective was also to assess the effect of carrier presence on the dissolution of the active substance.
Figure 10 compares the release profiles obtained for both active principles.
Figure 10a shows the release kinetics of pure DEX and those obtained from particles produced by the SAS technique. From
Figure 10a, it can be observed that SAS microparticles can make the drug release about 3 times faster than pure DEX. The unprocessed DEX has release times in an aqueous environment of about 22 hours, while all samples release for much better times of about 8 hours. Apart from the sample obtained using a molar ratio of 1/2 mol/mol and a total concentration of 200 mg/mL, the release time of this sample was approximately 14 hours. This could be explained by observing the sample's morphology obtained; the latter is characterized by large drug crystals and agglomerated expanded particles. Then, from the comparison of kinetics, shown in
Figure 10a, it is possible to observe that the release time tends to decrease with the decrease of the molar ratio of the active substance/vector. In particular, the sample with the lowest release time is the one with a molar ratio of 1/1 and a concentration of 20 mg/mL.
Finally, the dissolution time of pure PRED is about 18 hours. In this case, it can be observed that sample processing with the SAS technique can effectively accelerate the release of the active substance for up to 4 hours. But even this sample obtained using a molar ratio of 1/2, a total concentration of 200 mg/mL, and a pressure of 90 bar (red line) has longer release times, comparable to pure PRED. This phenomenon can be explained by looking at the sample’s morphology shown in
Figure 6a, where the formation of large crystals of drug covered by polymer microparticles can be observed. In addition, the kinetic comparison in
Figure 10b shows that the release time decreases significantly for samples produced with a molar ratio of 1/1 active substance/vector. In particular, the most efficient release profile is obtained in the case of the sample with a 1:1 molar ratio and a total concentration of 20 mg/ml (pink line); the drug is released in about 2 hours.
Therefore, the best release profile for both active ingredients is obtained from the sample produced using an active ingredient/polymer molar ratio of 1/1, total concentration of 20 mg/mL, pressure of 120 bar, and temperature of 40 ºC.
These results could indicate the effective complexation of the active substance in the β-cyclodextrin cavity using a 1/1 molar ratio.