*3.5. Tape-Stripping Drug Penetration Studies*

The stratum corneum layers depth in porcine skin was determined by infrared densitometry using a standardized procedure [66]. The total thickness obtained was around

20 µm, according with the standard values between 17–28 µm, previously reported by Jacobi et al. [99]. Tape-stripping studies were performed using the most promising formulations according to the afore-presented data: L1, L2, T1d, T2d, and E1. As incubation times, 2 h, 4 h, and 6 h were selected, covering the longest time a topical formulation would be maintained onto the skin. Besides, longer incubation times lead to overhydration of the skin and removal of the whole stratum corneum in a single strip [83]. The B12 solution 0.5% *w/v* (S) was used as a reference since it represents the free diffusion, and no enhancing absorption effects are expected from the vehicle. B12 has been delivered through the skin using chemical and physical methods by other authors. Recently, Ramöller et al. achieved an effective delivery of B12 to plasma in a rat model. Rapidly dissolving microneedle allowed the permeation of 60% of dose after 2 h post-insertion, achieving 0.4 μg/mL plasma levels [28]. Yang et al. studied in vitro the effects of chemical enhancers and physical methods in topical administration of B12. Chemical enhancers (ethanol, oleic acid, and propylene glycol) allowed an effective permeability of B12 in comparison with passive diffusion. The permeability enhancement of-

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 17 of 23

Figure 8 shows the progressive B12 penetration in the different layers of the stratum corneum at different incubation periods. In general, the amounts of B12 detected in the deeper layers of the skin increased as a function of the incubation time, as expected. As shown, B12 solution presented the highest amount of B12 extracted in the first strip regardless of the incubation time. Two reasons can explain this fact: immediate availability of B12 on the skin leading to a quick distribution over the first stratum corneum layer, where it is retained; and a deficient cleaning of the skin surface. Concerning the latter, many authors discard this strip because the inaccuracy of the data is typically high and considered as an experimental artifact [100]. However, the rest of B12 from the solution did not penetrate the stratum corneum nor the deeper skin layers, and only low quantifiable concentrations were detected up to the third strip, which corresponds to 6 µm depth, after 6 h. These findings confirm the inability of B12 to diffuse through intact skin. The ethosome samples presented the lowest quantifiable amounts among all the vesicles tested, probably due to the low B12 load and the gradual release. Samples could have easily been under the detection/quantification limits. fered by iontophoresis was around 2-fold in comparison with a combination of all enhancers (50% ethanol, 10% oleic acid, and 40% propylene glycol) [27]. Although these methods have proven to effectively delivery B12, they present several inconveniences in B12 delivery for atopic dermatitis. For example, they were not able to localize their effects in the epidermis and dermis leading to an absorption of B12 to systemic circulation, which is unnecessary for psoriatic and atopic dermatitis patients. Additionally, local skin reactions and poor effectiveness in combination with hydrophilic molecules has been reported for chemical enhancers [102]. Moreover, ultraflexible lipid vesicles are technically easier to produce and a low cost in comparison to microneedles and iontophoresis devices [102]. From the results presented here, it can be concluded that the developed formulations are able to efficiently deliver B12 to the deeper skin layers after 6 h, as desired. We also demonstrate that B12 does not diffuse through the stratum corneum if formulated in water media. Further studies should be performed to formulate these vesicles in adequate dosage forms for an effective topical application of B12 that can be transferred to the clinic.

**Figure 8.** *Cont.*

**Figure 8.** Penetration profiles of B12 delivered from liposomes, transferosomes, ethosomes, and solution after 2 h (**a**), 4 h (**b**), and 6 h (**c**). All results are expressed as mean ± SD (*n* = 3). **Figure 8.** Penetration profiles of B12 delivered from liposomes, transferosomes, ethosomes, and solution after 2 h (**a**), 4 h (**b**), and 6 h (**c**). All results are expressed as mean ± SD (*n* = 3).

**4. Conclusions**  Enhanced penetration of B12 through the skin is possible using the lipid vesicle formulations (L1, T2d, and T1d) designed in this work, which opens the possibility to improve the clinical results obtained in previous studies and to investigate its utility for topical treatment of atopic dermatitis and psoriasis. Several particle properties such as size, stability, and purification method were revealed as key parameters to achieve a suitable and efficient production of lipid vesicles. Skin permeability studies should be further investigated to elucidate if these nanosystems are able not only to increase the penetration in the skin but also to allow the passage of B12 to the systemic circulation, thus broadening the possible applications of the systems for the treatment of other pathologies related with The best penetration results were obtained using liposomes and transferosomes. L2 carries less B12 amounts than the other prototypes, consequently showing a considerably lower B12 penetration amounts, only until approximately 15 µm depth (Figure 8c). Nevertheless, L1, T1d, and T2d vesicles allowed the B12 to reach the dermis (>25 µm). L1 and T2d contained similar B12 doses, but after 4 and 6 h of incubation, the transferosomes showed higher permeation rates up to the deepest layers (Figure 8b,c). This means that the stratum corneum is saturated in B12 at that time point, and a diffusion gradient to the deeper layers starts. T1d showed the highest permeation profiles. Our penetration results suggest that transferosome enhancing effect was much higher than the one of liposomes and are in agreement with Abd et al. [101], who compared the penetration of a hydrophilic drug (caffeine) delivered from liposomes, transferosomes, and solution.

B12 deficiencies. **Supplementary Materials:** The following are available online at www.mdpi.com/xxx/s1, Figure S1: Release parameters (10 and 72 h) for release kinetic models: Higuchi, Korsmeyer–Peppas, Kim, Peppas–Sahlin, zero order, and first order; Figure S2: *R*2 values of Korsmeyer–Peppas and first order models for the B12 vesicles release data (10 and 72 h). B12 has been delivered through the skin using chemical and physical methods by other authors. Recently, Ramöller et al. achieved an effective delivery of B12 to plasma in a rat model. Rapidly dissolving microneedle allowed the permeation of 60% of dose after 2 h post-insertion, achieving 0.4 µg/mL plasma levels [28]. Yang et al. studied in vitro the effects of chemical enhancers and physical methods in topical administration of B12. Chemical enhancers (ethanol, oleic acid, and propylene glycol) allowed an effective permeability of B12 in comparison with passive diffusion. The permeability enhancement

offered by iontophoresis was around 2-fold in comparison with a combination of all enhancers (50% ethanol, 10% oleic acid, and 40% propylene glycol) [27]. Although these methods have proven to effectively delivery B12, they present several inconveniences in B12 delivery for atopic dermatitis. For example, they were not able to localize their effects in the epidermis and dermis leading to an absorption of B12 to systemic circulation, which is unnecessary for psoriatic and atopic dermatitis patients. Additionally, local skin reactions and poor effectiveness in combination with hydrophilic molecules has been reported for chemical enhancers [102]. Moreover, ultraflexible lipid vesicles are technically easier to produce and a low cost in comparison to microneedles and iontophoresis devices [102].

From the results presented here, it can be concluded that the developed formulations are able to efficiently deliver B12 to the deeper skin layers after 6 h, as desired. We also demonstrate that B12 does not diffuse through the stratum corneum if formulated in water media. Further studies should be performed to formulate these vesicles in adequate dosage forms for an effective topical application of B12 that can be transferred to the clinic.
