*3.5. In Vitro Drug Release Study*

In vitro drug release studies are needed for predicting the reproducibility of the rate and duration of drug release. The results from the in vitro drug release studies show that the release from the formulated patches increases with an increase in the concentration of the hydrophilic polymer (HPMC). The formulations from F1 to F5 showed higher release, while formulations from F6 to F9 showed lower release over a time period of 24 h in Figure 3. Hence, from the drug release profile of all formulations, F5 showed the best controlled-release profile of 82.71%. This may be attributed to the presence of the hydrophobic polymer (EC) and hydrophilic polymer (HPMC) in the ratio of 1:5. It was

noted that the hydrophilic polymer released the drug at a faster rate than the hydrophobic polymer. The F5 formulation is an optimized formulation. The diffusion mechanism is responsible for drug release for transdermal drug delivery systems. This mechanism involves drug transport from the polymer matrix into the respective medium based on the concentration gradient. The variation in the concentration gradient leads to drug release, as well as a greater distance for diffusion. This could be the most probable reason for the comparatively slower rate of drug diffusion when the distance for diffusion increases.

**Figure 3.** Release profile of MTX from F1 to F9, data were expressed as mean ± SD, *n* = 3.

### *3.6. Drug Release Kinetics*

All the data obtained from the formulated patches were fitted to the Korsmeyer– Peppas model for the confirmation of the exact drug release behavior showed in Table 5. In our present study, the F5 formulation showed the best fit with the Korsmeyer–Peppas equation (R<sup>2</sup> = 0.974), showing an anomalous or non-Fickian diffusion mechanism of drug release (*n* = 0.50131). There was complete and controlled drug release of methotrexate found over a period of 24 h. The optimized formulation for the current study was F5. Thus, F5 releases the drug at the predefined rate for a prolonged period of time into the systemic circulation, leading to minimal dose frequency and adverse effects.

**Table 5.** Drug release kinetics (F1–F9), Data were expressed as mean ± SD, *n* = 3.


#### *3.7. Ex Vivo Drug Permeation Study*

The ex vivo permeation result of methotrexate-loaded patches having EC and HPMC at different concentrations showed in Figure 4. The F5 formulation exhibited a maximum percent cumulative amount of drug permeation (34.68%), producing a significant difference (*p* < 0.05) compared to the F1 formulation (25.11%) in 24 h. The F1 formulation produced 9.14 μg/h/cm2, which is less than the required flux of 20.11 μg/h/cm2. An increase in HPMC concentration increases flux values; thus, the F5 formulation, containing a greater amount of HPMC, produced about 1.5-fold greater flux compared to the target flux. Similarly, the F9 formulation exhibited a cumulative drug permeation of 21.68% compared to the F1 formulation, which exhibited a cumulative drug permeation of 25.11%. The flux value of F9 was found to be 9.23 μg/h/cm2.

**Figure 4.** Percent cumulative amount of MTX permeated (F1–F9), Data were expressed as mean ± SD, *n* = 3. Significant compared to formalin (*p* < 0.05).
