**4. Discussion**

The structures revealed by SEM in the films after they were treated with an acid (Figure 3) allow for conclusion that the formation of the modified films is based on a phase transformation in a gelatin-CAP mixture at a preparation stage. Due to the fact that pH of the utilized type B gelatin solution was around 4.5, one can expect that CAP should constitute a separate phase as this polymer is insoluble at low pH. The CAP phase can be considered continuous; due to the high temperature at the stage of mixture preparation, which is well above the glass transition temperature (Tg) of CAP, the particles can appear in a rubbery state and coagulate easily. Therefore, the two separate phases: gelatin gel and CAP phase, are physically mixed, forming a bi-continuous network with discreet separate microdomains. The structures revealed with SEM sugges<sup>t</sup> that the phase separation proceeds with a spinodal decomposition mechanism, which is spontaneously initiated, and kinetically limited by increase in the viscosity of gelatin during the gelling process when the temperature drops at the casting stage. Similar "kinetic arrestation" of the phase separation process in the gelatin-containing mixtures was described by Lorén et al. [10] and by Tromp et al. [11].

To better explain the phase separation in the discussed systems, an additional experiment was performed. A premix of GA composition was placed in a glass vial and slowly heated to reach 80 ◦C. After 5 min at 80◦C the temperature was lowered stepwise by 10 ◦C each 5 min. The appearance of turbidity indicated the phase separation process. After reaching 40 ◦C the sample was heated again to 80 ◦C and kept at that temperature for 48 h. The second heating revealed that the temperature-dependent phase separation is reversible (the sample became transparent again). The results are presented in Figure 11.

**Figure 11.** The visible phase-separation on lowering the temperature of the sample, and suspected spinodal decomposition process on storage at 80 ◦C for a prolonged time.

However, the storage at 80 ◦C for a prolonged time caused irreversible phase separation, with a fibrillar/sponge-like appearance of the precipitated phase within the liquid continuous phase. Additionally, the alteration of color of the sample and the fact that the gel was partially liquid at room temperature indicated gelatin degradation. The overall results of this experiment confirm that the phase separation process proceeds at high temperature and can be stopped by immobilization of the growing CAP structure in a gelatin gel when the temperature drops below approx. 50 ◦C. This supports the thesis that the separate phase of CAP acts like a "reinforcement" for the gelatin network, decreasing the rate of penetration of the acidic medium into the water-soluble phase, and explains the structure integrity of the films in acidic media. Furthermore, it appears that the temperature/time balance in the preparation procedure allows to exploit the natural imbalance between phases for the favor of functionality of the films.

The mechanism behind formation and acid-resistance of both binary (GA) and ternary (GAC) polymer systems appears the same. However, in comparison to the GA films, a more significant hindering of disintegration and dissolution can be observed in case of GAC films [5]. This can be explained by a possible interaction between gelatin and carrageenan, which is suspected to be a polyelectrolyte complex formation. This non-covalent interaction has already been reported and is widely described [6,12–16]. Even though carrageenan is present in the film in a small amount, it still can significantly impact the viscosity of the gel phase. Therefore, by increasing the density of the polymer network, it leads to formation of a gel di ffusion layer more viscous than the gelatin alone. That causes higher swelling degree and slower dissolution rate of soluble ingredients present in the films, as it was previously described [5]. It appears also possible that the carrageenan in the film-forming mixture accumulates on the CAP-gelatin interface, supporting the CAP sca ffold during the immersion in an acid. However, we believe that the more irregular appearance of the CAP sca ffolds visible in Figure 4 are actually CAP coated with undissolved gelatin-carrageenan complex. This hypothesis appears to be supported by the results of Raman microscopy (see Figures 6 and 7), in which after immersion in acid, the structure shows a pattern in Raman spectra with the same features as observed in the spectra of the films before the acid treatment (both CAP and GAC), what suggests that during the immersion the gelatin was not fully dissolved and it is still present on the surface of the residual CAP sca ffold structure.

The CLSM study (Figure 4) appears to correspond well with the SEM results. Additionally, it was discovered that there are clear di fferences in the density of the solid material left in structure inside the films and on its surface. Due to the fact that the soluble fraction of the film composition is supposedly gelatin-based gel and plasticizer, the mechanism of erosion of the film when placed in an acid is likely based on the di ffusion of the medium through the gel layer. The erosion can be additionally limited by the presence of the insoluble CAP phase, which acts also as a sca ffold. Therefore, it can be suspected that the penetration of the acidic medium into the membrane is delayed and can depend on both density of the CAP sca ffold and viscosity of the gelatin-based phase.

The higher barrier properties of a ternary system (GAC) than the binary one (GA) also was demonstrated by the oxygen permeability test. However, in the literature the results of oxygen permeability can be found only for very thin gelatin films obtained from dilute gelatin solutions [17], where the values of oxygen permeability can be around 350–600 cm<sup>3</sup>/(m<sup>2</sup> × 24 h × 0.1 MPa). In the present study the permeability was measured for films with thickness around 650 μm, at which the measured values were between 3.5 and 7.5 cm<sup>3</sup>/(m<sup>2</sup> × 24 h × 0.1 MPa). Although, after addition of CAP, the oxygen permeability increased slightly, the di fferences between formulations were still very low and one can conclude that there is a lack of significant influence of the film ingredients on gas barrier properties.

During the formation of modified GA or GAC films, the CAP spherical particles (average size of 0.43 μm) are being incorporated in the gel structure. QCM-D is a surface sensitive technique which can be applied to analyze the interaction of the particles of CAP with gelatin. Quantitative values can be obtained with well-defined model systems. Saurebrey and Johannsman models [18] are often used to calculate the surface excess after adsorption. In our particular case, those models will not give reliable approximation because of the large size of the CAP particles. However, a qualitative information on interaction between CAP particles and gelatin films can be obtained. The large decrease in frequency of the vibration as soon as the CAP particles were introduced into the flow cell reflects the adsorption of CAP particles on the gelatin films. Due to the fact that the measurements were performed at 25 ◦C, a coalescence of the CAP is rather negligible, therefore such interaction should be based purely on surface charge of the particles. Although the test could not be performed at high temperature (80 ◦C), the confirmed high a ffinity of these two materials at 25 ◦C may be also relevant at higher temperature. We assume that such type of interaction can potentially stabilize the CAP inside the gel matrix and allow formation of a network structure during the preparation of the film-forming mass.

Preparation of capsules on a lab scale with the proposed steel mold was a simple process, allowing for application of a liquid fill, and for obtaining visually sealed capsules. However, the results of the disintegration tests show large variability because of the significant tendency of capsules to disrupt at the sealing area. On the other hand, the results prove that formation of the capsules using GAC composition is generally possible, and the capsules can be filled with liquid oil, PEG or melted fatty alcohol. In addition, no case in which a capsule disintegrated in acid at other region than the sealing was observed. This confirms that the films being in contact with a filling, still retain their structural integrity when submersed in acid.

For the purpose of investigation, whether the filling formulation has an impact on acid-resistance of the capsule shell, the capsules were filled with three types of substances: PEG, MCT oil and cetearyl alcohol. The results show that the capsules filled with solid fatty alcohol show lower resistance of the sealing to disintegration in acid. On the other hand, there is no noticeable di fference between the capsules filled with MCT oil or PEG. Overall, the mechanism of disintegration of the capsules appears to be related more to the capsule formation process, than to the filling composition. It was observed that the lab-manufactured capsules are prone to leakages on the sealing zone, especially when more intensive mechanical stress was involved, as in the tablet disintegration apparatus. We believe that the imperfect capsule sealing can be corrected when encapsulation process involves the conventional soft capsule manufacturing machines.

The imperfections in the sealing region did not allow to further test the capsules in the drug release test. This is why this study was performed in a vertical di ffusion cell placed in a paddle dissolution apparatus. The test was performed to investigate whether the films display barrier properties against di ffusion of diclofenac sodium at acidic pH. In addition, it was important that the films allow to release the API after switching the pH to neutral (6.8). In one of our previous articles, the barrier properties of the films towards radio-labeled water were described [5], and preliminary data on the di ffusion-hindering by the modified gelatin-CAP compositions was obtained. The present investigation confirms appropriate barrier properties of GAC film, because no di ffusion of diclofenac during 2 h in 0.1 M HCl was observed. However, the reproducibility of the diclofenac release at pH 6.8 is not very high. The release was initiated at di fferent time points, what results from the mechanism of film rupture—not dissolving totally in a specified time, but forming a breach. Since at lower stirring rates the release of diclofenac did not occur or was accidental, the proposed model requires higher stirring rates, which shows the significance of the mechanical factor in the dissolution of the GAC film in the pH 6.8 bu ffer.

Although the performed experiment with diclofenac as a model drug demonstrates lack of the drug di ffusion through the modified gelatin film immersed for 2 h in an acid, di ffusion of an acid through the membrane was not measured in the course of this stage of the research. Impermeability of the new capsule-forming material to the acid is a condition for using it in the capsules filled with an acid-labile drugs.
