*3.7. The Enhancement Mechanism of the LicA and Gla* 3.7.1. ATR-FT-IR of the Skin

ATR–FT-IR was conducted to elucidate the effects of the enhancers on the lipid and keratin arrangement of the porcine skin, and to further characterize drug–enhancers–skin interactions. The characteristic infrared absorption bands at 2918.02 and 2850.44 cm−<sup>1</sup> represent the asymmetric VasCH<sup>2</sup> and symmetric VsCH<sup>2</sup> stretching vibrations of SC lipid (Figure S5a,b), and the bands at 1647.97 and 1538.07 cm−<sup>1</sup> correspond to Amide I and Amide II of keratin (Figure 7a,b). In the LicA–skin control group, when POCC was added, the VasCH2, VsCH2, and Amide II moved to 2920.05, 2851.60, and 1538.65 cm−<sup>1</sup> , respectively (Figure S5a). SP also caused a blueshift of the Amide I and Amide II bands to 1648.41 and 1539.55 cm−<sup>1</sup> , respectively (Figure 7a). The results indicate that POCC and SP interacted with keratin of SC and disrupted the protein structure for enhanced drug permeation and retention. However, PG and NMP did not induce any changes in the lipids and keratin bands, suggesting an insignificant enhancement of LicA retention. When Gla–skin was considered as an entirety, the seven enhancers all changed the Amide I and Amide II bands to a higher position (blueshift) to different degrees (Figure 7b), indicating that the enhancers promoted Gla deposition by changing the secondary structures of the proteins. However, no linear correlation between the drug retention amount and the Amide I and Amide II bands' displacement values was observed. These results prove that the C=O group of porcine skin was the main enhancement site for LicA and Gla permeation. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 16 of 24 permeation and retention. However, PG and NMP did not induce any changes in the lipids and keratin bands, suggesting an insignificant enhancement of LicA retention. When Gla–skin was considered as an entirety, the seven enhancers all changed the Amide I and Amide II bands to a higher position (blueshift) to different degrees (Figure 7b), indicating that the enhancers promoted Gla deposition by changing the secondary structures of the proteins. However, no linear correlation between the drug retention amount and the Amide I and Amide II bands' displacement values was observed. These results prove that the C = O group of porcine skin was the main enhancement site for LicA and Gla permeation.

**Figure 7.** (**a**) FT-IR spectra (C = O group) of LicA–enhancers–skin systems; (**b**) FT-IR spectra (C = O group) of Gla–enhancers–skin systems; (**c**) CLSM images of the penetration depth and fluorescence intensity LicA and C6 in porcine skin treated withenhancers (Bar = 100 μm, the red arrows and white arrows represent the SC and hair follicles of the skin, respectively). **Figure 7.** (**a**) FT-IR spectra (C=O group) of LicA–enhancers–skin systems; (**b**) FT-IR spectra (C=O group) of Gla–enhancers–skin systems; (**c**) CLSM images of the penetration depth and fluorescence intensity LicA and C6 in porcine skin treated with enhancers (Bar = 100 µm, the red arrows and white arrows represent the SC and hair follicles of the skin, respectively).

The results of CLSM are shown in Figures 7c and S5c. Coumarin 6 was used as a

LicA fluorescence was distributed in the epidermis and dermis when compared with the parent LicA. However, PG, TP, and NMP did not facilitate drug permeation into deeper skin layers. For Gla, the seven enhancers all improved the drug retention amount and drug fluorescence intensity, and CP 90 and TP had the most significant improvementef-

3.7.2. CLSM

#### 3.7.2. CLSM

The results of CLSM are shown in Figures 7c and S5c. Coumarin 6 was used as a probe as a substitute for Gla in this study. It was observed that LicA permeated to a deeper skin layer in the presence of CP 90, POCC, SP, and IPM, and significantly stronger LicA fluorescence was distributed in the epidermis and dermis when compared with the parent LicA. However, PG, TP, and NMP did not facilitate drug permeation into deeper skin layers. For Gla, the seven enhancers all improved the drug retention amount and drug fluorescence intensity, and CP 90 and TP had the most significant improvement effect. The results are in accordance with the in vitro skin permeation and retention study. Interestingly, we found that hair follicles were the main permeation routes for LicA and Gla, and most fluorescence was located in the hair follicles.

#### 3.7.3. Molecular Modeling and Correlation Analysis 2

Gla showed a higher Emix (12.24) with skin than that of LicA (9.96), indicating that LicA showed better miscibility with skin than that of Gla. However, CED of Gla–skin was similar to LicA–skin. Then, the Emix, χ and CED values of different drug–enhancers–skin ternary systems were calculated as before (Table 4).The optimized ternary associations are displayed in Figures 8d, S6 and S7. After enhancers were added, they could occupy the site of drug–skin interaction and link with the skin, leading to better compatibility between the enhancers and skin. Thus, a lower Emix value is indicative of better enhancers–skin interaction and higher drug permeation [17].

**Table 4.** Molecular docking and molecular dynamics (MD) simulation results of Gla (LicA)–enhancers– skin systems.


Next, multivariate linear regression analysis was conducted to confirm the correlation between the Psolution, REsolution, and physicochemical parameters of enhancers and the regression equations are expressed as follows:

<sup>P</sup>solution (LicA) = 1.90 + 7.59 <sup>×</sup> log P (R<sup>2</sup> = 0.88) (16)

$$\text{RE}\_{\text{solution}}\text{ (LicA)} = -2.26 + 0.49 \times \log \text{P (R}^2 = 0.89\text{)}\tag{17}$$

$$P\_{\text{solution}}\text{ (Gla)} = -1.57 + 3.84 \times \text{Polarizability} - 9.12 \times \log P \tag{18}$$

$$\text{RE}\_{\text{solution}} \text{ (Gla)} = 34.90 - 0.34 \times \text{M.W} + 3.85 \times \text{Polarizability} \tag{19}$$

**Figure 8.** (**a**) Correlation analysis between LicAREsolutionand CED of ternary systems; (**b**) correlation analysis between LicAREsolutionand Emixof ternary systems; (**c**) the correlation relationship between GlaREsolutionand CED of ternary systems; (**d**) snapshots of LicA(Gla)–enhancers–skin systems at the end of the MD (drug: ball and stick model; enhancers: CPK model). **Figure 8.** (**a**) Correlation analysis between LicAREsolution and CED of ternary systems; (**b**) correlation analysis between LicAREsolution and Emix of ternary systems; (**c**) the correlation relationship between GlaREsolution and CED of ternary systems; (**d**) snapshots of LicA(Gla)–enhancers–skin systems at the end of the MD (drug: ball and stick model; enhancers: CPK model).

The results (Figure 8a,b**)** indicated that both the retention of LicA and Gla increased as the CED or Emixvalue decreased, revealing the inhibitory effect of the intermolecular force on the drug retention. Taken together, for LicA–skin, enhancers with a higher log P showed better miscibility with skin, which resulted in increasedLicA retention inthe skin. For the Gla–skin binary system (Figure 8c), enhancers with a higher polarizability tended to occupy the site of the skin, thereby facilitating greater Gla deposition onthe skin.

The results (Figure 9a,b) showed that both REsolution and Psolution of LicA were positively correlated with log P of the enhancers. The response surface plots (Figure 9c,d) showed that REsolution and Psolution of Gla increased as the polarizability increased. A linear regression of the drug retention amount and Emix or CED was also carried out to explain the interaction force on drug retention, respectively, and the linear regression equations are expressed as follows:

$$\text{RE}\_{\text{solution}} \text{ (LicA)} = -60.59 \times \text{CED} + 102.21 \text{ (R}^2 = 0.85) \tag{20}$$

$$\text{RE}\_{\text{solution}} \text{ (LicA)} = -0.50 \times \text{E}\_{\text{mix}} + 28.19 \text{ (R}^2 = 0.82) \tag{21}$$

$$\text{RE}\_{\text{solution}} \text{ (Gla)} = -108.57 \times \text{CED} + 202.96 \text{ (R}^2 = 0.79) \tag{22}$$

**Figure 9.** (**a**) The correlation relationship between LicAREsolutionand log P of enhancers; (**b**) linear analysis ofLicAPsolutionand log P of enhancers; (**c**) response surface plot demonstrating the effect of MW and polarizability on REsolution of Gla; (**d**) response surface plot demonstrating the effect of log P and polarizability on Psolutionof Gla. **Figure 9.** (**a**) The correlation relationship between LicAREsolution and log P of enhancers; (**b**) linear analysis of LicAPsolution and log P of enhancers; (**c**) response surface plot demonstrating the effect of MW and polarizability on REsolution of Gla; (**d**) response surface plot demonstrating the effect of log P and polarizability on Psolution of Gla.

*3.8. In Vitro Skin Permeation and Drug Retention of Drug Hydrogel*  For the hydrogel system, only PG and TP could facilitate a significantly higher amount of LicA accumulation in the skin, and PG possessed the highest ERhydrogel retention value. The seven enhancers all improved Gla retention in the CP systems, and the ERhydrogel retention value was ranked as SP>POCC> IPM> CP>NMP>TP>PG (Figure 6c and Table 3). Moreover, a significantly higher amount of LicA was detected in the diffusion cells The results (Figure 8a,b) indicated that both the retention of LicA and Gla increased as the CED or Emix value decreased, revealing the inhibitory effect of the intermolecular force on the drug retention. Taken together, for LicA–skin, enhancers with a higher log P showed better miscibility with skin, which resulted in increased LicA retention in the skin. For the Gla–skin binary system (Figure 8c), enhancers with a higher polarizability tended to occupy the site of the skin, thereby facilitating greater Gla deposition on the skin.

#### from hydrogel after the intervention of TP and POCC. Furthermore, only CP 90 and POCC could significantly disrupt the skin barrier for Gla penetration from the hydrogel *3.8. In Vitro Skin Permeation and Drug Retention of Drug Hydrogel*

(Figure 6d). βR/P values >1 indicate that the enhancers mainly facilitated the drug release process while βR/P values <1 indicate that the enhancement action site was mainly skin [17]. The results (Table 3) showed that the βR/P values of PG were 2.28 and 1.63 for the LicA–CP and Gla–CP systems, respectively, which proves that the site of action of the enhancement was mainly the CP matrix for PG. For LicA, the βR/P values of POCC, SP, IPM, and CP were all less than 0.5, demonstrating that skin was the main site of action of the enhancement. **4. Discussion**  Although we observed an ascending trend for the utilization of enhancers in whitening products for anti-pigmentation, the interaction of whitening compounds, enhancers, and CP or skin in the drug release or permeation process has been neglected, which has resulted in the unreasonable utilization of enhancers and unscientific design of cos-For the hydrogel system, only PG and TP could facilitate a significantly higher amount of LicA accumulation in the skin, and PG possessed the highest ERhydrogel retention value. The seven enhancers all improved Gla retention in the CP systems, and the ERhydrogel retention value was ranked as SP > POCC > IPM > CP > NMP > TP > PG (Figure 6c and Table 3). Moreover, a significantly higher amount of LicA was detected in the diffusion cells from hydrogel after the intervention of TP and POCC. Furthermore, only CP 90 and POCC could significantly disrupt the skin barrier for Gla penetration from the hydrogel (Figure 6d). βR/P values >1 indicate that the enhancers mainly facilitated the drug release process while βR/P values <1 indicate that the enhancement action site was mainly skin [17]. The results (Table 3) showed that the βR/P values of PG were 2.28 and 1.63 for the LicA–CP and Gla–CP systems, respectively, which proves that the site of action of the enhancement was mainly the CP matrix for PG. For LicA, the βR/P values of POCC, SP, IPM, and CP were all less than 0.5, demonstrating that skin was the main site of action of the enhancement.

#### metic formulations. This study systematically demonstrated the quantitative enhance-**4. Discussion**

ment efficacy of the release and permeation of Gla and LicA by enhancers with different physicochemical parameters, providing a comprehensive understanding of the interaction of drugs, enhancers, and polymer or skin. More importantly, we provided strategies Although we observed an ascending trend for the utilization of enhancers in whitening products for anti-pigmentation, the interaction of whitening compounds, enhancers, and CP or skin in the drug release or permeation process has been neglected, which has resulted in

the unreasonable utilization of enhancers and unscientific design of cosmetic formulations. This study systematically demonstrated the quantitative enhancement efficacy of the release and permeation of Gla and LicA by enhancers with different physicochemical parameters, providing a comprehensive understanding of the interaction of drugs, enhancers, and polymer or skin. More importantly, we provided strategies for reasonable selection of enhancers in hydrogel formulations to obtain high drug release and permeation.

Both LicA and Gla showed an anti-melanogenic effect in our previous studies; however, their poor solubility and high partition coefficient affected their formulation design and storage stability. In this work, to ensure the complete dispersion of drug in the CP system, 5% Gla and LicA (*w/w*) were added, respectively, and the XRD and PLM study confirmed this. FT-IR and Raman studies together indicated weak Van der Waals forces interactions present in the Gla–CP and LicA–CP systems. Moreover, the interaction of Gla–CP was significantly higher than that of the LicA–CP binary system due to the better compatibility between the drug and CP. Correspondingly, LicA–CP was expected to possess a higher release percent than that of the Gla–CP due to its easier escape from the hydrogel network. However, the result was contrary to this. Previous studies concluded that the intermolecular force, viscoelasticity, and mesh size of the drug-loaded hydrogels jointly influenced the drug release from hydrogel [4]. The mesh size can be tested by thermal analysis, which was performed to reflect the molecular mobility of the hydrogel and is described by Tg. A lower T<sup>g</sup> is indicative of good molecular flexibility and a larger mesh size [26]. Next, the study confirmed that the G0 , G", and mesh size of the hydrogel did not significantly contribute to the drug release percent supported by rheological studies and DSC. In fact, drug release from hydrogel is due to the two processes of hydrogel relaxation and drug diffusion while intermolecular force, viscoelasticity, and the mesh size mainly influence the hydrogel relaxation process [27–29]. When diffusion dominates drug release, the drug release percent is primarily inhibited by the drug solubility in receptor fluids. In this work, it was found that Gla had a significantly higher solubility in PEG 400/PBS (20/80, *v/v*) (Table 1), thereby facilitating a higher Gla release percent.

To obtain a higher drug release percent, hydrophilic and hydrophobic enhancers were added. Moreover, the proportion of enhancers was chosen to be 10% to acquire an apparent release and penetration enhancement effect, and to obtain a stronger drug–enhancers–CP or drug–enhancers–skin interaction. Both the LicA and Gla release amount promoted an increase in the MW of the enhancers, which is attributed to the enhanced mesh size induced by the increasing MW. This conclusion is supported by the results showing that molecules with high MW can form a larger pore size in the hydrogel network, leading to a higher drug release amount [3,30]. More importantly, we found that enhancers with high polarizability (α) had higher ERrelease for both LicA and Gla. The higher polarizability suggests that the drug was more easily polarized by polar molecules, which indicates stronger Van der Waals forces [16]. As a result, enhancers, which had higher polarizability, could link with the drug–CP binary systems to form drug–enhancers–CP ternary systems, or occupied drug–CP sites to form enhancers–CP binary systems. The detailed explanation is discussed below. To further confirm these results, we calculated the Emix and χ values of different drug–enhancers–CP systems and CED of the built amorphous cell systems. Interestingly, th eLicA and Gla release percent showed a good positive correlation with Emix and a negative relationship with CED. Thus, the key intermolecular interaction (Van der Waals forces) was theoretically consistent with the results of the correlation analysis, underscoring the dominant role of interaction forces in hindering drug release.

Next, FT-IR was used to demonstrate the enhancement site of the different enhancers and the enhancement efficacy can be reflected by the displacement degree of –OH and C=O groups. In this work, dry CP rather than aqueous CP was utilized to measure the whole system's energy. Previous studies revealed that the C=O group show a redshift in CP systems from a dry to a hydrated state, leading to a discrepancy in the interaction between drugs and CP in the two systems. The presence of water reduces the ionic interaction force and increases the H-bonding between the drug and polymers [31,32]. In this work, Van der Waals forces were the dominating forces that controlled the drug release; therefore, dry CP is suitable as an alternative to hydrated CP to measure the interaction in different hydrogel systems. For LicA–CP and Gla–CP systems, POCC and SP generated stronger Van der Waals forces with CP by linking with the –OH group, which inhibited the drug release. However, the addition of CP 90 or NMP did not cause movement of the –OH group. These results proved that –OH of CP was not the enhancement site of drug release, which was different from the –OH enhancement site for loxoprofen release from the PSA matrix in a previous study [17]. The re-crystallization of the drug after the addition of enhancers was also necessary to assess the enhancement mechanism and higher drug re-crystallization indicates a better drug release ability. It was seen that a significantly larger amount of LicA crystals were re-crystallized in the film after the addition of PG or NMP while CP 90 and TP contributed to Gla re-crystallization. This result was consistent with the in vitro release study. It further proved that the drug–CP interaction was destroyed by the enhancers and enhancers–CP interactions occurred.

From the FP/Q value (Table 3), the skin barrier is still the dominating rate-limiting step for Gla and LicA permeation. The amount of Gla retention was significantly higher than LicA retention due to the higher log P of LicA, resulting in better miscibility with skin, which hindered LicA molecules' penetration into the skin. This result was consistent with previous reports. Usually, permeation enhancers improve the permeation of drug into the skin by disordering the arrangement of lipids and improving the lipid flexibility of SC [11,33]. Moreover, this process is dependent on the physicochemical properties of the enhancers rather than the amount of enhancers [34]. In this work, more LicA and Gla molecules were expected to accumulate in the skin to exert a remarkable anti-melanogenic effect. The seven enhancers chosen could all improve the LicA permeation and retention proportionally. For LicA, enhancers with high log P showed the highest ERpermeation and ERsolution retention, whereas enhancers with high polarizability facilitated a higher amount of Gla retention. Enhancers with high log P, such as POCC, weakened the LicA–skin interaction and then decreased the interaction forces of the whole LicA–enhancers–skin ternary systems, thereby improving the diffusion of LicA into the skin. Thus, a good linear relation was observed between Qretention (LicA) and CED or Emix after different enhancers were used. However, Gla–skin interaction, which was weaker than LicA–skin, was easier to be destroyed by enhancers with higher polarizability. Enhancers with higher polarizability could also occupy the Gla–skin binding sites to decrease the intermolecular force of the whole system. Taken together, the addition of enhancers disrupted the drug– skin interaction and then reduced the interaction forces of the whole systems, resulting in an improvement of the drug retention or permeation.

FT-IR is an analytical tool used to measure the disorder degree of proteins and lipids in the skin. Porcine skin was used for in vitro skin penetration studies because it possesses a similar epidermal thickness, lipid composition, low frequency impedance, and more importantly, permeability with human skin. Therefore, the VasCH2, VsCH<sup>2</sup> and Amide I, Amide II displacement values were not as significant as rat skin [35,36]. A slight movement of the CH<sup>2</sup> or amide groups results in disarrangement of lipids or distortion of the protein structure. POCC and SP enhanced LicA retention by interacting with the C=O group of the ceramide, thereby a blueshift of the Amide I and Amide II bands was observed. Similarly, C=O was also the main enhancement site for Gla retention by the seven enhancers, indicating enhancers decreased the barrier's resistance by distorting the protein structure for enhanced LicA and Gla retention. Since Gla showed weaker interaction forces with skin than that of LicA, the seven enhancers all occupied the Gla–skin binding sites and improved Gla accumulation in the skin. CLSM is another tool that was used to confirm the effect of the enhancers on LicA and Gla retention. Higher fluorescence intensity and deeper skin location were indicative of a stronger penetration enhancement effect. This result was also consistent with the in vitro permeation study. Interestingly, the enhancers mainly facilitated drug retention via hair follicle pathways, which was mainly attributed to large hair follicles and a high number of hair follicles in porcine skin [37].

CP hydrogel is a complicated system including CP, water, and drug. Thus, the drug permeation and retention behaviors of hydrogel are not simply a combination of drug release and skin penetration. For LicA, the seven enhancers all improved LicA accumulation in the skin from the solution; however, PG showed the highest ERhydrogel retention and ERrelease for LicA, indicating that both the drug release and skin permeation processes limited LicA's permeability. Interestingly, the seven enhancers all showed a significant enhancement effect on Gla retention from hydrogel, which was different from the enhanced effect of Gla retention by CP, POCC, and PG in solution. These results further indicate that the main rate-limiting of Gla's penetration is its skin permeability.

#### **5. Conclusions**

In this work, a systematic approach was established to evaluate the enhanced release and retention of whitening agents from CP hydrogel in the presence of enhancers based on interactions among the drug, enhancers, and CP or skin. ERrelease, ERpermeation, ERcom, ERsolution retention, and ERhydrogel retention were utilized to evaluate the quantitative enhanced effect, and βR/P was calculated to evaluate the enhancement action sites of the enhancers. We found that the release and retention enhancement effect were closely related to the structures of the enhancers. Gla–CP hydrogel showed higher drug release and retention ability than LicA–CP, which was attributed to the higher solubility in medium and better miscibility with skin of Gla than that of LicA. Enhancers with higher MW and lower polarizability showed a higher ERrelease for both LicA and Gla, whereas enhancers with higher log P and polarizability displayed a higher ERsolution retention for LicA and Gla. More importantly, Van der Waals forces among the drug, enhancers and CP showed a negative correlation with the drug release percent, and intermolecular interaction between the drug, enhancers, and skin also showed a linear decreasing effect on drug retention. Additionally, the C=O group of the ceramide was the enhancement site for drug permeation by the enhancers. Consequently, TP and PG, and the seven enhancers showed a higher ERhydrogel retention for LicA–CP and Gla–CP respectively. Taken together, the conclusions provide a strategy for reasonable utilization of enhancers and formulation optimization in whitening topical hydrogels.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics14020262/s1, Methods S1: The HPLC methods of LicA and Gla, Figure S1: (a) DSC curves of different hydrogels; (b) Flow characterization of the hydrogels (n = 3); (c) In vitro drug release profiles of LicA-CP hydrogel when enhancers were added (n = 3); (d) In vitro drug release profiles of LicA-CP hydrogel when enhancers were added (n = 3), Figure S2: Conformation of LicA-enhancers-CP and Gla-enhancers-CP ternary systems, Figure S3: The snapshots of LicA (Gla)-enhancers-CP systems at the end of the MD. (Drug: Ball and stick model; Enhancers: CPK model), Figure S4: (a) FT-IR spectra (C=O group) of LicA-enhancers-CP systems; (b) FT-IR spectra (C=O group) of Gla-enhancers-CP systems; (c) PLM images of drug-CP films after different enhancers were added, Figure S5: (a) FT-IR spectra (CH<sup>2</sup> - group) of LicA-enhancers-skin systems; (b) FT-IR spectra (CH2-group) of Gla-enhancers-skin systems; (c) CLSM images of penetration depth and fluorescence intensity LicA and C6 in porcine skin treated by enhancers (bar = 100 µm), Figure S6: Conformations of LicA-enhancers-skin and Gla-enhancers-skin ternary systems, Figure S7: The snapshots of LicA (Gla)-enhancers-skin systems at the end of the MD. (Drug: Ball and stick model; Enhancers: CPK model).

**Author Contributions:** Conceptualization, Z.W. and Q.L.; methodology, Z.Z.; software, Y.H.; validation, Y.W. (Yufan Wu), Q.Z. and Z.Z.; formal analysis, Y.W. (Yuan Wang); investigation, Z.W. and Y.X.; resources, Q.L.; data curation, Y.X. and C.J.; writing—original draft preparation, Z.W. and Y.X.; writing—review and editing, C.J. and C.S.; visualization, L.L. and H.Z.; supervision, Q.L.; project administration, H.Z. and Q.L.; funding acquisition, Q.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China, grant number 82074023, 81874346.

**Institutional Review Board Statement:** All animal experiments were performed in accordance with the "Guiding Principles in the Care and Use of Animals" (China), and approved by the Ethics Committee of Southern Medical University (L2019036, date of approval: 13 April 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to send their gratitude to the National Natural Science Foundation of China.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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