*2.6. Mechanism of Enhancement Drug Release* 2.6.1. Attenuated Total Reflection FT-IR (ATR-FT-IR) of the CP Hydrogels

The ATR-FT-IR study was used to confirm the effect of the enhancers on the drug release of the CP systems. The hydrogel films were obtained from the preparation of hydrogels, and the infrared spectra were recorded using a Nicolet iS50 FT-IR spectrometer (Thermos, New York, NY, USA) within the frequency range of 500–4000 cm−<sup>1</sup> using 32 scans at a resolution of 2.

#### 2.6.2. Raman Spectroscopy

Raman spectra were also used to characterize the potential interactions among the drug, enhancers, and CP by a Raman spectrometer (Renishaw RM2000, London, England). Then, the samples were measured at 25 ◦C using a 785 nm laser source with 500 mW power.

#### 2.6.3. X-ray Diffraction (XRD)

The proportion of crystalline in different hydrogels was determined by diffracted intensity measurement using an X-ray diffractometer (SmartLab 3KW, Rigaku, Japan) of Cu Kα radiation in the 5–60◦ 2θ range with a scan rate of 10◦/min.

#### 2.6.4. Polarized Light Microscopy (PLM)

PLM measurement was conducted to confirm the results of XRD using a Nikon polarized optical microscope (edipse lv100N pol, Tokyo, Japan). The images were captured using QImaging software (Nis-Elements F) with a first-order compensator at 100× magnification.

#### 2.6.5. Differential Scanning Calorimetry (DSC)

The hydrogel films were placed in the aluminum DSC pans of a thermal analyzer (TA Q2000, TA, New Castle, Lindon, UT, USA), and then heated from 25–250 ◦C at a rate of 10 ◦C/min with 3 cycles (to eliminate the thermal history). All samples were performed under a nitrogen atmosphere (40 mL/min). The parameter glass transition temperature (Tg) was recorded at the midpoint of the transition in the curve.

#### 2.6.6. Molecular Interaction Study: Molecular Docking

Molecular docking was conducted to corroborate the results of FT-IR to calculate the intermolecular strength of the Gla (LicA)–CP and Gla (LicA)–enhancers–CP systems using Materials Studio version 8.0 (Accelrys, San Diego, CA, USA). The molecular structures of the CP, Gla, LicA, and seven kinds of enhancers were obtained from the PubChem database and subjected to geometry optimization with Forcite modules in the COMPASS II force field. Next, the mixing energy (Emix) and interaction parameters (χ) were calculated. In addition, the optimized structure of the Gla (LicA)–CP and Gla (LicA)–enhancers–CP associations were obtained.

#### 2.6.7. Molecular Dynamic Simulation

Molecular dynamic simulation was utilized to understand the drug release behaviors of different hydrogels with or without enhancers. The optimized CP, Gla (LicA), and enhancers were placed in the amorphous cell modules according to the proportions of the actual formulation, and the built systems were further optimized by For cite modules. Subsequently, NVT equilibration of 50 ps at 298 K was conducted for each system, after which NPT equilibration of 100 ps was performed at 305 K and 101.325 Kpa with a time step of 1 ps. The CED was calculated for each system. Then, snapshots of the hydrogel systems at the end of the MD were obtained.

#### *2.7. Correlation Analysis 1*

First, a linear regression analysis was conducted to investigate the relationship between the drug release amount and physicochemical parameters, including MW, log P, polarizability, and polar surface area, in different hydrogels using SPSS 20.0 software (SPSS, Chicago, IL, USA). On the other hand, the linear regression equation of the drug release amount and Emix and CED were calculated.

#### *2.8. In Vitro Skin Permeation of Drug Solution and Hydrogel*

Porcine skin (one-month-old Bama miniature pig, male, 20 kg) was supplied by Aperture Biotech Co., Ltd. (Hong Kong, China) The thickness of porcine skin was maintained at approximately 800 µm and its structural integrity was guaranteed before the experiments. The porcine skin sample was sandwiched between the donors and receptors compartment

in Franz diffusion cells with the dermal side facing downwards. Then, different drug aqueous solutions (0.3 g) and 0.3 g of the corresponding hydrogels were added to the donor receptors, and PBS/PEG400 (*v/v*, 80/20) was chosen as the medium to obtain sink conditions. In total, 1 mL of receptor vehicle was withdrawn after1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h. Others were processed similar to the in vitro release of the hydrogel. 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).

Psolution and Phydrogel represent the cumulative permeation in the solution and hydrogel, respectively.

The enhancement ratio of the drug skin permeation in the drug solution (ERpermeation) was calculated as follows:

$$\text{ER}\_{\text{permeation}} = \frac{\text{P}\_{\text{solution with enhancement}}}{\text{P}\_{\text{solution without enhancement}}} \tag{2}$$

The enhancement ratio of the drug skin permeation in hydrogel (ERcom) was calculated as follows:

$$\text{ER}\_{\text{com}} = \frac{\text{P}\_{\text{hydrogel with enhaner}}}{\text{P}\_{\text{hydrogel without enhameter}}} \tag{3}$$

βR/P was calculated to evaluate the sites of action of the enhancers [17]:

$$
\beta\_{\text{R/P}} = \frac{\text{ER}\_{\text{release}}}{\text{ER}\_{\text{permeation}}} \tag{4}
$$

The rate-limiting step of transdermal drug delivery was assessed using the following equation:

$$\mathbf{F} = \frac{\mathbf{P}\_{\text{hydrogel}}}{\mathbf{Q}\_{\text{hydrogel}}} \tag{5}$$

#### *2.9. Drug Retention*

After in vitro skin permeation, the treated skin samples were removed from the diffusion cell. Subsequently, the skin at the administration site was wiped to remove the unabsorbed drug, cut into pieces, weighed, and extracted with methanol by ultrasound for 1 h. Then, the supernatant was detected with HPLC to obtain the skin retention amount.

REsolution and REhydrogel are the cumulative retention amount of drug in the solution and hydrogel, respectively.

The enhancement ratio of the drug skin retention in solution (ERsolution retention) was calculated as follows:

$$\text{ER}\_{\text{solution iteration}} = \frac{\text{RE}\_{\text{solution with enhancement}}}{\text{RE}\_{\text{solution without enhancement}}} \tag{6}$$

The enhancement ratio of the drug skin retention in hydrogel (ERhydrogel retention) was calculated as follows:

$$\text{ER}\_{\text{hydrogel iteration}} = \frac{\text{RE}\_{\text{hydrogel with enhaner}}}{\text{RE}\_{\text{hydrogel without enhance}}} \tag{7}$$

#### *2.10. Mechanism of Enhancement Drug Permeation*

2.10.1. ATR-FT-IR Spectra of the Porcine Skin

The ATR-FT-IR study was used to investigate the effect of the enhancers on the arrangement variations of the skin lipid and protein region. The skin samples were taken from the in vitro skin permeation of drug solution, and the infrared spectra were recorded as described in Section 2.6.1.

#### 2.10.2. Confocal Laser Microscope (CLSM)

CLSM was used to visualize the LicA and Gla distribution in the skin tissue, and C6 was utilized as a substitute for Gla. Treated skin samples were processed similar to the in vitro skin permeation of drug solution with a permeation time of8 h. The samples were cut longitudinally into 6-µm-thick slices using a Cryostat microtome (Thermo HM525 NX, New York, NY, USA) after fixation. LicA and C6 were emitted at 480 and 485 nm using a confocal laser microscope (CLSM 800, ZEISS, Jena, Germany), respectively.

#### 2.10.3. Molecular Docking and Molecular Dynamic Simulation

Ceramide 2 was used as are presentative of skin lipids for molecular docking and molecular dynamic simulation due to it having the highest proportion in skin lipids [14,20]. The Emix, χ, and CED of Gla (LicA)-skin and Gla (LicA)–enhancers-skin systems and their snapshots were obtained as described.

#### *2.11. Correlation Analysis 2*

A multiple linear regression model was also used to detect the correlation between the drug retention, drug permeation amount, and physicochemical parameters of the enhancers as described before. Moreover, the relationships between the drug retention or drug permeation amount and C=O band displacement value in the FT-IR, Emix, and CED were calculated.

#### *2.12. Statistical Analysis*

All data were analyzed using SPSS 20.0 software (Chicago, IL, USA). Data were expressed as mean ± SD and subjected to one-way analysis of variance (ANOVA) or two-tailed paired Student's *t*-test. The significance level was set at *p* < 0.05.

#### **3. Results**

#### *3.1. Preparation of the CP–Gla and CP–LicA Hydrogel*

Lyophilized hydrogels were prepared to investigate the potential interaction between the drugs and CP. First, XRD (Figure 2a) and PLM analyses (Figure 2b) were used to detect the crystals in the hydrogel films, and the results demonstrated that both Gla and LicA almost completely dissolved in the CP hydrogel without the formation of obvious crystals. Moreover, LicA and Gla displayed a similar miscibility to CP. These results indicate that Gla and LicA were molecularly dispersed in the hydrogel, which laid a foundation for the hydrogen bond or the formation of other interactions [21]. The hydroxyl (−OH) and carbonyl group (C=O) of Gla and LicA, and the carboxyl (−COOH) group of CP are the functional groups that may potentially be involved in the drug–CP interaction. In the blank CP, the characteristic band at 2934.05 cm−<sup>1</sup> was assigned to –OH stretching vibration while the band at 1695.27cm−<sup>1</sup> was attributed to C=O stretching of the CP (Figure 2c). The band at 2934.05 cm−<sup>1</sup> shifted to 2933.31 and 2934.24 cm−<sup>1</sup> , and the C=O band moved to 1696.69 and 1696.22 cm−<sup>1</sup> for Gla-CP and LicA-CP, respectively, indicating weak interaction in the drug–CP systems. Furthermore, the Gla–CP system showed a stronger interaction strength than LicA-CP. The Raman spectra (Figure 2d) also confirmed the presence of the interaction due to the movement of the −OH band. The values of Emix and χ measured using molecular docking are used to estimate the strength of the intermolecular interactions. The closer Emix and χ are to 0, the greater the miscibility and the stronger the intermolecular interactions. In this case, the Gla–CP system possessed a lower Emix and χ than LicA–CP, further underscoring the stronger interaction between Gla and CP (Table 2). The optimized structures of the Gla (LicA)–CP binary associations are displayed in Figure 2e.

sociations are displayed in Figure 2e.

**Figure 2.** (**a**) X-ray powder diffractograms of different hydrogels; (**b**) PLM images of CP films; (**c**) FT-IR spectra of CP, LicA–CP, and Gla–CP; (**d**) Raman spectra of the hydrogels; (**e**) conformations of LicA-CP and Gla-CP. **Figure 2.** (**a**) X-ray powder diffractograms of different hydrogels; (**b**) PLM images of CP films; (**c**) FT-IR spectra of CP, LicA–CP, and Gla–CP; (**d**) Raman spectra of the hydrogels; (**e**) conformations of LicA-CP and Gla-CP.

sults indicate that Gla and LicA were molecularly dispersed in the hydrogel, which laid a foundation for the hydrogen bond or the formation of other interactions [21]. The hydroxyl (−OH) and carbonyl group (C = O) of Gla and LicA, and the carboxyl (−COOH) group of CP are the functional groups that may potentially be involved in the drug–CP interaction. In the blank CP, the characteristic band at 2934.05 cm−1 was assigned to –OH stretching vibration while the band at 1695.27cm−1 was attributed to C = O stretching of the CP (Figure 2c). The band at 2934.05 cm−1 shifted to 2933.31 and 2934.24 cm−1, and the C = O band moved to 1696.69 and 1696.22 cm−1 for Gla-CP and LicA-CP, respectively, indicating weak interaction in the drug–CP systems. Furthermore, the Gla–CP system showed a stronger interaction strength than LicA-CP. The Raman spectra (Figure 2d) also confirmed the presence of the interaction due to the movement of the −OH band. The values of Emix and χ measured using molecular docking are used to estimate the strength of the intermolecular interactions. The closer Emix and χ are to 0, the greater the miscibility and the stronger the intermolecular interactions. In this case, the Gla–CP system possessed a lower Emix and χ than LicA–CP, further underscoring the stronger interaction between Gla and CP (Table 2). The optimized structures of the Gla (LicA)–CP binary as-

#### *3.2. In Vitro Release of Gla–CP and LicA–CP Hydrogels*

The results (Figure 3a) showed that Gla displayed a higher release amount and release rate than LicA, indicating that the interaction strength in the drug–CP systems was not a dominating factor controlling the drug release. However, the highest release percent of Gla and LicA only reached 69.08% and 43.56%, respectively, after 48 h. Moreover, the release behaviors of Gla and LicA from CP hydrogel followed the zero-order equation, and the release equations of the release amount percent and time are listed as follows:

$$\text{R}\_{\text{hydrogel}} \text{ (LicA)} = 1.01 \times \text{t} + 0.20 \text{ (R}^2 = 0.97) \tag{8}$$

$$\text{TR}\_{\text{hydrogel}} \text{ (Gla)} = 1.61 \times \text{t} + 0.70 \text{ (R}^2 = 0.95) \tag{9}$$


**Table 2.** The Emix, χ, and CED of different Gla (LicA)–enhancers–CP systems.

**Figure 3.** (**a**) In vitro drug release profiles of hydrogel (*n* = 3); (**b**) frequency sweep (G′, G′′, and δ) of the hydrogel (*n* = 3); (**c**) LicA and Gla release percent after48 h after different enhancers were added (n = 3); (**d**) response surface plot demonstrating the effect of MW and polarizability on the ERrelease of LicA; (e) response surface plot demonstrating the effect of MW and polarizability on the ERrelease of Gla. **Figure 3.** (**a**) In vitro drug release profiles of hydrogel (*n* = 3); (**b**) frequency sweep (G0 , G00, and δ) of the hydrogel (*n* = 3); (**c**) LicA and Gla release percent after48 h after different enhancers were added (*n* = 3); (**d**) response surface plot demonstrating the effect of MW and polarizability on the ERrelease of LicA; (**e**) response surface plot demonstrating the effect of MW and polarizability on the ERrelease of Gla.

*3.3. In Vitro Release of Drug in the Presence of Enhancers*  To improve the drug release from the hydrogels, different enhancers were added to hydrogels and in vitro release experiments were performed (Figure S1c,d). We found that only PG (highest ERrelease: 1.25) and NMP significantly increased the LicA release percent while TP (highest ERrelease: 1.15), POCC, SP, and IPM all contributed to a significantly higher Gla release percent (Figure 3c and Table 3) after 48 h. Then, multivariate linear regression analysis was conducted to investigate the effect of the physicochemical parameters of the enhancers on Rhydrogel. The regression equations areexpressed as follows: Rhydrogel (LicA) = 52.87 + 0.057 × M.W − 0.74 × Polarizability (10) To demonstrate the influencing effect on the drug release, DSC study reflecting the free volume and mesh size of the hydrogels was carried out. The results (Figure S1a) demonstrated that Gla–CP showed a similar T<sup>g</sup> to LicA–CP, which is indicative of a slight effect of the mesh size on the drug release. In addition, G0 represents the rigidity of hydrogels [22,23], and G00 is a parameter used to demonstrate the friction of a molecular chain and reflect changes in the intermolecular interaction [24]. The rheological study (Figures 3b and S1b) revealed that the zero-shear viscosity, G0 , G00, and δ of Gla–CP all showed no significant difference to that of LicA–CP, indicating that the viscoelastic properties of the hydrogel also had no significant influence on the drug release.

were negatively correlated with the polarizability, and positively correlated with MW of the enhancers. Moreover, the polarizability dominated the drug release. Polarizability represents the ability of Van der Waals forces to form [25], which are the primary interaction forms in drug–enhancers–CP systems. Enhancers with higher polarizability tended to be linked with drug–CP systems, which is a sign of stronger intermolecular interactions forming in the drug–enhancers–CPternary systems, thereby decreasing the drug release percent. These results prove the interaction strength in drug–enhancers–CP

Rhydrogel (Gla) = 79.76 + 0.03 × M.W − 0.36 × Polarizability (11)

systems was an important factor determining the drug release.

#### *3.3. In Vitro Release of Drug in the Presence of Enhancers*

To improve the drug release from the hydrogels, different enhancers were added to hydrogels and in vitro release experiments were performed (Figure S1c,d). We found that only PG (highest ERrelease: 1.25) and NMP significantly increased the LicA release percent while TP (highest ERrelease: 1.15), POCC, SP, and IPM all contributed to a significantly higher Gla release percent (Figure 3c and Table 3) after 48 h. Then, multivariate linear regression analysis was conducted to investigate the effect of the physicochemical parameters of the enhancers on Rhydrogel. The regression equations areexpressed as follows:

$$\text{R}\_{\text{hydrogel}} \text{ (LicA)} = 52.87 + 0.057 \times \text{M.W} - 0.74 \times \text{Polarizability} \tag{10}$$

$$\text{R}\_{\text{hydrogel}} \text{ (Gla)} = 79.76 + 0.03 \times \text{M.W} - 0.36 \times \text{Polarizability} \tag{11}$$


**Table 3.** The enhancement efficacy parameters of enhancers in the drug release and skin penetration process.

The response surface plot (Figure 3d,e) showed that both Rhydrogel of Gla and LicA were negatively correlated with the polarizability, and positively correlated with MW of the enhancers. Moreover, the polarizability dominated the drug release. Polarizability represents the ability of Van der Waals forces to form [25], which are the primary interaction forms in drug–enhancers–CP systems. Enhancers with higher polarizability tended to be linked with drug–CP systems, which is a sign of stronger intermolecular interactions forming in the drug–enhancers–CP ternary systems, thereby decreasing the drug release percent. These results prove the interaction strength in drug–enhancers–CP systems was an important factor determining the drug release.

#### *3.4. Molecular Modeling and Correlation Analysis 1*

Then, Emix and χ of different Gla (LicA)–enhancers–CP systems were calculated (Table 2) using Materials Studio version 8.0, and their optimized ternary associations are displayed at Figure S2. For LicA–enhancers–CP, LicA–PG–CP hydrogel showed the highest χ (19.48) and Emix (11.54) while LicA–SP–CP showedthe lowest (−1.77 and −1.05). For Gla–enhancers–CP, the Gla–PG–CP system showed the worst miscibility, with χ of 8.85 and Emix of 5.24, whereas POCC showed the best miscibility with Gla–CP. Linear regression of the drug release percent and Emix was performed to clarify the effect of the interaction strength on Rhydrogel. The linear regression equation of Rhydrogel and Emix is as follows:

$$\text{R}\_{\text{hydrogel}} \text{ (LicA)} = 0.92 \times \text{E}\_{\text{mix}} + 42.10 \text{ (R}^2 = 0.57) \tag{12}$$

$$\text{R}\_{\text{hydrogel}} \text{ (Gla)} = 1.64 \times \text{E}\_{\text{mix}} + 71.75 \text{ (R}^2 = 0.82) \tag{13}$$

The equations (Figure 4a,b) showed that the better the compatibility between the enhancers and drug–CP binary systems, the lower the release amount. These findings are consistent with the above results. To further corroborate the effect of intermolecular forces on the drug release, molecular dynamics simulation was carried out to calculate the CED values to reflect the interactions among the drugs, enhancers, and CP. The results are displayed in Table 2 and snapshots of the hydrogels systems at the end of the MD are shown in Figures 4e and S3. A higher CED value means a stronger interaction [3]. Similarly, linear regression of the drug release percent and CED was also conducted, and the linear regression equations are expressed as follows:

$$\text{R}\_{\text{hydrogel}} \text{ (LicA)} = -42.21 \times \text{CED} + 144.75 \text{ (R}^2 = 0.85) \tag{14}$$

$$\text{R}\_{\text{hydrogel}} \text{ (Gla)} = -17.01 \times \text{CED} + 119.09 \text{ (R}^2 = 0.89\text{)}\tag{15}$$

**Figure 4.** (**a**) The correlation relationship between theLicA release percent and Emixof ternary systems; (**b**) linear analysis of theGla release percent and Emix of ternary systems; (**c**) the correlation relationship between the LicA release percent and CED of ternary systems; (**d**) linear analysis of theGla release percent and CED of ternary systems; (**e**) snapshots of the LicA (Gla)–enhancers–CP systems at the end of the MD (drug: ball and stick model; enhancers: CPK model). **Figure 4.** (**a**) The correlation relationship between the LicA release percent and Emix of ternary systems; (**b**) linear analysis of the Gla release percent and Emix of ternary systems; (**c**) the correlation relationship between the LicA release percent and CED of ternary systems; (**d**) linear analysis of the Gla release percent and CED of ternary systems; (**e**) snapshots of the LicA (Gla)–enhancers–CP systems at the end of the MD (drug: ball and stick model; enhancers: CPK model).

*3.5. The Release Mechanism of the Drug from the Drug–Enhancers–CP System*  To demonstrate the drug release mechanism of the ternary systems, FT-IR and PLM were conducted. LicA–CP was used as the control group, and CP showed a typical band at 2931.5 cm−1 representing the –OH group (Figure 5a,b), while the band at 1696.21 cm−<sup>1</sup> The results (Figure 4c,d) further emphasize the decreasing effect of Van der Waals forces on the drug release. The stronger the interaction strength in the compound systems, the lower the drug release percent.

belonged to the C = O band (Figure S4a and Figure 4b). Upon mixing SP or POCC with CP, the −OH bands showed a red shift to 2925.14 and 2924.66 cm−1, respectively (Figure

with CP. Moreover, only NMP and POCC induced a weakmovement of the C = O band for LicA–CP (Figure S4a). For the Gla–CP binary systems, the bands appearing at 2932.12 and 1696.6 cm−1 also represented the −OH and C = O groups, respectively. The addition of SP or POCC also led to significant movement of the−OH band (Figure 5b), which was similar to LicA–CP. In contrast, upon loading with NMP and IPM, the −OH band showed no significant difference with the control group. The bands of the C = O groups did not show significant movement except for the addition of NMP (Figure S4b). The results revealed that the –OH of CP was not the enhancement site for LicA and Gla release.

#### *3.5. The Release Mechanism of the Drug from the Drug–Enhancers–CP System*

To demonstrate the drug release mechanism of the ternary systems, FT-IR and PLM were conducted. LicA–CP was used as the control group, and CP showed a typical band at 2931.5 cm−<sup>1</sup> representing the –OH group (Figure 5a,b), while the band at 1696.21 cm−<sup>1</sup> belonged to the C=O band (Figures S4a and 4b). Upon mixing SP or POCC with CP, the <sup>−</sup>OH bands showed a red shift to 2925.14 and 2924.66 cm−<sup>1</sup> , respectively (Figure 5a), attributing to a strong interaction between SP or POCC and CP. However, the position of the −OH band did not show any significant difference when mixing CP 90 or NMP with CP. Moreover, only NMP and POCC induced a weak movement of the C=O band for LicA–CP (Figure S4a). For the Gla–CP binary systems, the bands appearing at 2932.12 and 1696.6 cm−<sup>1</sup> also represented the <sup>−</sup>OH and C=O groups, respectively. The addition of SP or POCC also led to significant movement of the−OH band (Figure 5b), which was similar to LicA–CP. In contrast, upon loading with NMP and IPM, the −OH band showed no significant difference with the control group. The bands of the C=O groups did not show significant movement except for the addition of NMP (Figure S4b). The results revealed that the –OH of CP was not the enhancement site for LicA and Gla release. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 14 of 24

**Figure 5.** (**a**) FT-IR spectra (−OH group) of LicA–enhancers–CP systems; (**b**) FT-IR spectra (−OH group) of Gla–enhancers–CP systems; (**c**) PLM images of drug–CP films after different enhancers were added. **Figure 5.** (**a**) FT-IR spectra (−OH group) of LicA–enhancers–CP systems; (**b**) FT-IR spectra (−OH group) of Gla–enhancers–CP systems; (**c**) PLM images of drug–CP films after different enhancers were added.

PLM was used to observe the re-crystallization of the drug after the addition of enhancers. Higher drug re-crystallization indicates weaker enhancers–CP interactions and a better drug release ability. After mixing with SP, CP 90, IPM, NMP, or POCC, no significant LicA crystals were detected in the drug–enhancers–CP films (Figures 5c and S4c). However, a significantly larger amount of LicA crystals were observed in the film system after PG or NMP addition. These results indicated that the enhancers, such as PG or PLM was used to observe the re-crystallization of the drug after the addition of enhancers. Higher drug re-crystallization indicates weaker enhancers–CP interactions and a better drug release ability. After mixing with SP, CP 90, IPM, NMP, or POCC, no significant LicA crystals were detected in the drug–enhancers–CP films (Figures 5c and S4c). However, a significantly larger amount of LicA crystals were observed in the film system after PG or

NMP, occupied the LicA–CP binding site, enabling LicA release from the hydrogel. For the Gla–CP system, upon adding NMP, PG, SP, or TP, a significantly larger amount of

better miscibility with the Gla–CP systems; therefore, no Gla was detected in these ter-

A comparison of the enhancement of LicA and Gla skin retention after 48 h is shown in Figure 6. The results demonstrated that the amount of Gla that accumulated in the skin within 48 h was 5.63 times higher than that of LicA (Figure 6a). Furthermore, approximately a 7.02 times increase in the amount of Gla permeating into the receptor fluids was observed when compared with LicA (Figure 6b). The addition of CP 90, POCC, SP, and IPM significantly enhanced the retention of LicAin the skin, and the enhancement effect was rank ordered as SP (ERsolution retention: 3.78) > POCC ≈ IPM >CP 90> TP> PG > NMP (Figure 6a and Table 3). CP 90, POCC, SP, and IPM also significantly facilitated LicA's permeation across the skin and POCC showed the highest ERpermeation value (Figure 6b and Table 3). However, the seven enhancers all significantly improved Gla's disposition

*3.6. In Vitro Skin Permeation and Drug Retention of Drug Solution* 

nary systems. These results are consistent with the in vitro release study.

NMP addition. These results indicated that the enhancers, such as PG or NMP, occupied the LicA–CP binding site, enabling LicA release from the hydrogel. For the Gla–CP system, upon adding NMP, PG, SP, or TP, a significantly larger amount of Gla crystals appeared in the hydrogel film. In contrast, IPM, CP 90, and POCC showed better miscibility with the Gla–CP systems; therefore, no Gla was detected in these ternary systems. These results are consistent with the in vitro release study.

#### *3.6. In Vitro Skin Permeation and Drug Retention of Drug Solution*

A comparison of the enhancement of LicA and Gla skin retention after 48 h is shown in Figure 6. The results demonstrated that the amount of Gla that accumulated in the skin within 48 h was 5.63 times higher than that of LicA (Figure 6a). Furthermore, approximately a 7.02 times increase in the amount of Gla permeating into the receptor fluids was observed when compared with LicA (Figure 6b). The addition of CP 90, POCC, SP, and IPM significantly enhanced the retention of LicAin the skin, and the enhancement effect was rank ordered as SP (ERsolution retention: 3.78) > POCC ≈ IPM > CP 90 > TP > PG > NMP (Figure 6a and Table 3). CP 90, POCC, SP, and IPM also significantly facilitated LicA's permeation across the skin and POCC showed the highest ERpermeation value (Figure 6b and Table 3). However, the seven enhancers all significantly improved Gla's disposition into the skin, and the enhancement effect followed the order of CP 90 (ERsolution retention: 2.11) >TP ≈ IPM≈ NPM >PG > POCC > SP (Figure 6a and Table 3). However, only POCC, CP 90, and NMP significantly facilitated Gla's permeation and CP 90 showed the highest ERpermeationvalue (Figure 6b and Table 3). In addition, it was observed that the permeation amount of LicA and Gla showed a positive linear relation with the LicA and Gla retention amount (R<sup>2</sup> = 0.85 and R<sup>2</sup> = 0.47), respectively, indicating that the seven enhancers all demonstrated a similar contributory effect on the drug retention and drug permeation. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 15 of 24 into the skin, and the enhancement effect followed the order of CP 90 (ERsolution retention: 2.11) >TP ≈ IPM≈ NPM >PG > POCC > SP (Figure 6a and Table 3). However, only POCC, CP 90, and NMP significantly facilitated Gla's permeation and CP 90 showed the highest ERpermeationvalue (Figure 6b and Table 3). In addition, it was observed that the permeation amount of LicA and Gla showed a positive linear relation with the LicA and Gla retention amount (R2 = 0.85 and R2 = 0.47), respectively, indicating that the seven enhancers all demonstrated a similar contributory effect on the drug retention and drug permeation.

**Figure 6.** (**a**) REsolutionof LicA and Gla after 48 h (*n* = 4); (**b**) Psolutionof LicA and Gla after 48 h (*n* = 4); (**c**) REhydrogel of LicA and Gla after 48 h (*n* = 4); (**d**) Phydrogel of LicA and Gla after 48 h (*n* = 4). **Figure 6.** (**a**) REsolution of LicA and Gla after 48 h (*n* = 4); (**b**) Psolution of LicA and Gla after 48 h (*n* = 4); (**c**) REhydrogel of LicA and Gla after 48 h (*n* = 4); (**d**) Phydrogel of LicA and Gla after 48 h (*n* = 4).

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 bandsat 2918.02 and 2850.44 cm−<sup>1</sup> represent the asymmetric VasCH2 and symmetric VsCH2 stretching vibrations of SC lipid (Figure S5a,b), and the bands at 1647.97 and 1538.07 cm−1 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−1, respectively (Figure S5a**)**. SP also caused a blueshift of the Amide I and Amide II bands to 1648.41 and 1539.55 cm−1, respectively (Figure 7a). The results indicate that POCC and SP interacted with keratin of SC and disrupted the protein structure for enhanced drug

*3.7. The Enhancement Mechanism of the LicA and Gla* 
