*3.2. Stability Studies*

Physical and chemical long-term stability was monitored by measuring the vesicle size, PDI, and EE% every week for 2 months and the drug content for 3 months. Figure 5a shows that no significant differences in size as a function of storage time at 4 ◦C, except for the liposomal formulations after 4 weeks of storage. This fact has been previously reported by other authors [86,87]. On the other hand, intra-type significant changes in PDI were not observed, pointing out a homogeneous evolution in size of vesicles populations (Figure 5b).

Significant changes in drug content related to the initial amount were detected in all prototypes between weeks 7 and 8 (Figure 6). Recent works have also reported a drug leakage and an effective content loss, especially for liposomes containing hydrophilic drugs, thus supporting our observations [87,88]. Considering these findings, B12 lipid vesicles seemed to exhibit short- and medium-term stability (at least 1 month for all the parameters checked), which makes them suitable for clinical applications. However, to increase the stability for longer-term use, liposome lyophilization could be investigated in the future for these B12 lipid vesicles, since it has been shown as an excellent method for ensuring liposome long-term stability [89,90].

**Figure 3.** Short-term stability data: (**a**) L1 transmission data; (**b**) L1 backscattering data; (**c**) T1d transmission data; (**d**) T1d backscattering data; (**e**) E1 transmission data; (**f**) E1 backscattering data. **Figure 3.** Short-term stability data: (**a**) L1 transmission data; (**b**) L1 backscattering data; (**c**) T1d transmission data; (**d**) T1d backscattering data; (**e**) E1 transmission data; (**f**) E1 backscattering data.

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 13 of 23

**Figure 4.** Short-term stability data; L1 TSI (blue), E1 TSI (yellow), and T1d TSI (red). **Figure 4.** Short-term stability data; L1 TSI (blue), E1 TSI (yellow), and T1d TSI (red). (Figure 5b).

**Figure 5.** (**a**) Vesicle size over 2 months of storage (4 °C); (**b**) PDI over 2 months of storage (4 °C). All results are expressed as mean ± SD (*n* = 3). \* means statistically significant differences when compared to the initial values (*p* < 0.05) using oneway ANOVA followed by Tukey's multiple comparison test. **Figure 5.** (**a**) Vesicle size over 2 months of storage (4 ◦C); (**b**) PDI over 2 months of storage (4 ◦C). All results are expressed as mean ± SD (*n* = 3). \* means statistically significant differences when compared to the initial values (*p* < 0.05) using one-way ANOVA followed by Tukey's multiple comparison test. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 14 of 23

the future for these B12 lipid vesicles, since it has been shown as an excellent method for ensuring liposome long-term stability [89,90]. **Figure 6.** Remaining percentage of B12 amounts during 2 months of storage (4 °C). All results are expressed as mean ± SD (*n* = 3). \* means statistically significant differences when compared to initial values (*p* < 0.05) using one-way ANOVA followed by Tukey's multiple comparison test. **Figure 6.** Remaining percentage of B12 amounts during 2 months of storage (4 ◦C). All results are expressed as mean ± SD (*n* = 3). \* means statistically significant differences when compared to initial values (*p* < 0.05) using one-way ANOVA followed by Tukey's multiple comparison test.

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

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 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

Significant changes in drug content related to the initial amount were detected in all

in all cases presented is the drug release from the drug carrier [74].
