*3.3. In Vitro Drug Release Studies*

In vitro drug release studies are regularly used in the optimization process of pharmaceutical forms [91]. In this work, the cumulative drug release profiles of the preselected optimized formulations were estimated by a dialysis method using the Franz diffusion cell set-up [92] and presented in Figure 7 and Tables S1 and S2 (Supplementary Materials section). Dialysis methods are appropriate and well accepted to study drug release profiles. Two processes are involved in drug transfer from the donor to the receiver chamber: drug release from the drug reservoir and molecule diffusion through the dialysis membrane, K' (diffusion rate) and K (release rate) being their respective experimental constants. Using the fitting models, in which K > K' for first orders transports and K ≈ K' for zero order transport, we observed that K was higher than K'. Therefore, the limiting step in all cases presented is the drug release from the drug carrier [74]. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 15 of 23 last L1 samples (44, 46, and 48% at 24, 48, and 72 h, respectively) rose, suggesting that the plateau was not reached and differences may be reduced if the release were extended.

**Figure 7.** Release profiles from liposomes, transferosomes, ethosomes, and solution after 72 h (**a**) and 8 h (**b**). All results are expressed as mean ± SD (*n* = 6). \* means statistically significant differences (*p* < 0.05), n.s. means no statistically significant differences (*p* > 0.05) using two-way ANOVA followed by Bonferroni's multiple comparison test. **Figure 7.** Release profiles from liposomes, transferosomes, ethosomes, and solution after 72 h (**a**) and 8 h (**b**). All results are expressed as mean ± SD (*n* = 6). \* means statistically significant differences (*p* < 0.05), n.s. means no statistically significant differences (*p* > 0.05) using two-way ANOVA followed by Bonferroni's multiple comparison test.

*3.4. Model Fitting: Kinetic Drug Release Mechanisms*  The experimental release data were fitted to different kinetic models to better under-The B12 solution (S) was used as a control as it represents the drug diffusion profile without limitations. The rest of the lipid vesicle formulations showed a controlled release

and first order. This point is highly recommended since mathematical modeling could help to understand the further in vivo performance of the formulations [96]. Fitting parameters of all the models using the first 10 h data and 72 h data are listed in Table S1, Supplementary Materials section. The AIC was used as a comparative of the goodness of fit (also listed in Table S1, Supplementary Materials section). In general, the Korsmeyer– Peppas model presented the lowest AIC values, indicating an accurate fitting, for almost all formulations. However, in certain cases (T1d 10 h, T2c 72 h, E1 72 h and S), the first order was the best model. The Kim model is a modification of the Korsmeyer–Peppas one that considers a possible burst effect. This burst effect (represented by parameter "b") was

of drug, as shown in Figure 7b. B12 was released faster during the initial hours from all the tested formulations due to the concentration gradient established between the donor and the receiver media [93]. After 3 h, the B12 amount detected in the receptor medium was significantly higher for the solution than the liposomes, transferosomes, and ethosomes, as expected [94]. Moreover, differences between the release from liposomal and ultraflexible vesicles were also observed, being lower from the liposomes, probably due to their different rigidity. It has been reported that vesicles with considerable bilayer rigidity exhibit higher resistance to drug transport through the liposomal bilayer [95]. The long-term percentage of drug released is probably also affected by this vesicle property. The lowest percentage of drug released corresponded to the liposomes and the highest to the ethosomes, which were able to release around 100% of the encapsulated B12. Nevertheless, the final percentages of released B12 between transferosomes and ethosomes were different (Figure 7a) even though no differences were found in vesicle flexibility during the characterization stage (Figure 1a,b). Possible reasons for this are: the difference of entrapped drug amounts (higher in transferosomes than in ethosomes) that led to different gradients and the vesicle structure and components. We found similar results in B12 release from L1 and L2 formulations profiles at the initial stage (<10 h) even though the B12 entrapped amount in L1 was higher than L2 formulation (Figure 2c). Differences are observed when reaching the plateau. However, the B12 cumulative amounts found in the last L1 samples (44, 46, and 48% at 24, 48, and 72 h, respectively) rose, suggesting that the plateau was not reached and differences may be reduced if the release were extended.
