*2.3. Optimized Composition*

The optimized formula obtained from BBD for formulating CVC-loaded niosomes, drug (9.76 mg), surfactant (131.02 mg), and cholesterol (4.62 mg). Optimized formulation was further characterized for various parameters. The chosen formulation for the optimized niosomes was formulation code F4, which contains the drug (mg) (A) 10, surfactant (mg) (B) 120, and cholesterol (mg) (C) 5.

### *2.4. Characterization of CVC-Ns*

For measurements of the vesicle size, Polydispersity Index (PDI), and zeta potential (ZP), the optimal formulation of CVC-Ns was carefully developed based on the characteristics of obtaining the ideal vesicle size and PDI with zeta potential. For the optimized niosomal formulation, the vesicle size distribution (PDI) was 0.265 ± 0.11, and the mean vesicle size was 180.23 ± 1.21 nm. Figure 3A,B, respectively, show the size and zeta potential curve of the optimized formulation. The optimized zeta potential was found to be −31.70 ± 1.11 mV; the zeta potential magnitude gives stability potential of the formulation. All particles in suspension with a large negative or positive zeta potential will resist each other and thus have a low tendency to aggregate [49].

## Entrapment Efficiency and Drug Loading

The % entrapment efficiency and % drug loading of optimized CVC-Ns was found to be 90.61 ± 2.14% and 60.40 ± 0.99%.

was further characterized for various parameters. The chosen formulation for the optimized niosomes was formulation code F4, which contains the drug (mg) (A) 10, surfactant

For measurements of the vesicle size, Polydispersity Index (PDI), and zeta potential (ZP), the optimal formulation of CVC-Ns was carefully developed based on the characteristics of obtaining the ideal vesicle size and PDI with zeta potential. For the optimized niosomal formulation, the vesicle size distribution (PDI) was 0.265 ± 0.11, and the mean vesicle size was 180.23 ± 1.21 nm. Figure 3A,B, respectively, show the size and zeta potential curve of the optimized formulation. The optimized zeta potential was found to be −31.70 ± 1.11 mV; the zeta potential magnitude gives stability potential of the formulation. All particles in suspension with a large negative or positive zeta potential will resist each

(mg) (B) 120, and cholesterol (mg) (C) 5.

other and thus have a low tendency to aggregate [49].

*2.4. Characterization of CVC-Ns* 

**Figure 3.** (**A**). Vesicle size and distribution of CVC-loaded niosomes. (**B**). Zeta potential of CVCloaded niosomes. **Figure 3.** (**A**). Vesicle size and distribution of CVC-loaded niosomes. (**B**). Zeta potential of CVCloaded niosomes.

### *2.5. Morphology of CVC-Ns*

The optimized CVC-Ns formulation's TEM picture displays the produced vesicles to be well-defined sealed structures with uniform size distribution and spherical morphologies (Figure 4). The spherical vesicles indicate that CVC is entrapped by niosomes. These vesicles are spherical in shape and homogeneous. The other spots represent the small amount of CVC entrapment in the niosomes [50].

### *2.6. In Vitro Release Study*

The CVC-loaded optimized niosomal formulation represents a release of CVC through the dialysis bag of 70.24 ± 1.21%, compared to the in vitro CVC release of CVC-suspension, which was found to be 32.87 ± 1.03% (Figure 5A). More than 10–15% of the drug was released in the early two hours, followed by a sustained release for the next 24 h. Initially, the fast release was caused by the rapid release of CVC from the niosome surface, but later, a slower release phase was discovered. The delayed phase in drug release was controlled

by diffusion through swollen niosomal bilayers [51]. The results of the in vitro drug release experiment were analysed by applying several mathematical kinetic models to the data. It was discovered that the value of the correlation coefficient (R<sup>2</sup> ) for the release of CVC from optimized CVC-loaded niosomes for the Higuchi matrix model had the greatest value (R<sup>2</sup> = 0.9933) (Figure 5B). First-order (R<sup>2</sup> = 0.9878) and zero-order (R<sup>2</sup> = 0.9268) models were found to have the lowest R<sup>2</sup> values, as shown in Table 3 and Figure 5C,D. Consequently, after obtaining the highest value of the correlation coefficient, the optimized CVC-Ns indicated Higuchi's model as the best-fit model. The Korsmeyer–Peppas model was used to fit data to analyses of the release mechanism of CVC from optimized CVC-Ns (Figure 5E). The R<sup>2</sup> value was found to be 0.9927, and the n value was found to be 0.45 < n < 0.89, indicating that the release mechanism of CVC from optimized CVC-N follows non-Fickian diffusion [52]. Entrapment Efficiency and Drug Loading The % entrapment efficiency and % drug loading of optimized CVC-Ns was found to be 90.61 ± 2.14% and 60.40 ± 0.99%. *2.5. Morphology of CVC-Ns*  The optimized CVC-Ns formulation's TEM picture displays the produced vesicles to be well-defined sealed structures with uniform size distribution and spherical morphologies (Figure 4). The spherical vesicles indicate that CVC is entrapped by niosomes. These vesicles are spherical in shape and homogeneous. The other spots represent the small amount of CVC entrapment in the niosomes [50].

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**Figure 4.** Surface morphology of optimized niosomes. **Figure 4.** Surface morphology of optimized niosomes.

#### *2.6. In Vitro Release Study 2.7. Analysis of the Gel and Texture of the Optimized CVC-N Gel*

The CVC-loaded optimized niosomal formulation represents a release of CVC through the dialysis bag of 70.24 ± 1.21%, compared to the in vitro CVC release of CVCsuspension, which was found to be 32.87 ± 1.03% (Figure 5A). More than 10–15% of the drug was released in the early two hours, followed by a sustained release for the next 24 h. Initially, the fast release was caused by the rapid release of CVC from the niosome surface, but later, a slower release phase was discovered. The delayed phase in drug release was controlled by diffusion through swollen niosomal bilayers [51]. The results of the in The pH for the CVC-N gel formulation was 6.01, which is quite close to the skin's pH [52]. According to data on texture analysis, as shown in Figure 6, the CVC-N gel has the following properties: firmness of 239.09 g, consistency of 1587.00 gm sec, cohesiveness of −89.17 gm, and work of cohesion of −645.47 gm sec, as shown in Table 4 [53]. Peak or maximum force is used to measure firmness; the higher the number, the thicker the sample's consistency. The sample's stickiness or cohesiveness is determined by measuring the greatest negative force. The stiffer the sample is, the more negative the number [54].

### vitro drug release experiment were analysed by applying several mathematical kinetic models to the data. It was discovered that the value of the correlation coefficient (R2) for *2.8. Confocal Laser Scanning Microscopy*

the release of CVC from optimized CVC-loaded niosomes for the Higuchi matrix model had the greatest value (R2 = 0.9933) (Figure 5B). First-order (R2 = 0.9878) and zero-order (R2 = 0.9268) models were found to have the lowest R2 values, as shown in Table 3 and Figure The stratum corneum thickness of rat or human skin is reported to be 18 and 17 µm, respectively, and the active site for most of the therapeutic complications associated with the skin lies below the stratum corneum; thus, a drug moiety must penetrate at least

5C,D. Consequently, after obtaining the highest value of the correlation coefficient, the

20–200 µm across the skin to exert therapeutic effect [55]. The results showed that niosomal gel loaded with rhodamine B penetrated a deeper layer to a depth of 25.0 µm (Figure 7A). Rhodamine B hydroethanolic solution only exhibited infiltration up to 5.0 µm, suggesting that the dye was only present in the top layers of the rat's skin (Figure 7B). In contrast, because the fluorescence intensity is greater in the centre of the skin, it is probable that the formulation was maintained in the lower epidermal area of the rat skin. Many skin conditions that affect this lower epidermal skin region require the formulation loaded with rhodamine B to remain in the skin. Thus, it could be said that the created niosomal gel effectively carried rhodamine B dye deeper into the rat's skin layers.


**Table 4.** Physiochemical characterization of CVC-based niosomal gel (CVCNG).

**Figure 5.** *Cont*.

**Figure 5.** (**A**) Comparison of the optimized CVC-Ns formulation's in vitro release profile with that of CVC suspension. The investigation was performed in triplicate, and the data are depicted as mean ± SD. (**B**) Higuchi release kinetics of CVC-niosome formulation. (**C**) Release kinetics for a first order and (**D**) release kinetics for zero order of CVC-loaded niosomes. (**E**) Korsmeyer–Peppas model for predicting the release of the drug. **Figure 5.** (**A**) Comparison of the optimized CVC-Ns formulation's in vitro release profile with that of CVC suspension. The investigation was performed in triplicate, and the data are depicted as mean ± SD. (**B**) Higuchi release kinetics of CVC-niosome formulation. (**C**) Release kinetics for a first order and (**D**) release kinetics for zero order of CVC-loaded niosomes. (**E**) Korsmeyer–Peppas model for predicting the release of the drug.

The pH for the CVC-N gel formulation was 6.01, which is quite close to the skin's pH

−89.17 gm, and work of cohesion of −645.47 gm sec, as shown in Table 4 [53]. Peak or maximum force is used to measure firmness; the higher the number, the thicker the sample's consistency. The sample's stickiness or cohesiveness is determined by measuring the greatest negative force. The stiffer the sample is, the more negative the number [54].

*2.7. Analysis of the Gel and Texture of the Optimized CVC-N Gel* 

**Figure 6.** Texture analysis of optimized CVC-N gel.

**Figure 5.** (**A**) Comparison of the optimized CVC-Ns formulation's in vitro release profile with that of CVC suspension. The investigation was performed in triplicate, and the data are depicted as mean ± SD. (**B**) Higuchi release kinetics of CVC-niosome formulation. (**C**) Release kinetics for a first order and (**D**) release kinetics for zero order of CVC-loaded niosomes. (**E**) Korsmeyer–Peppas model for

The pH for the CVC-N gel formulation was 6.01, which is quite close to the skin's pH [52]. According to data on texture analysis, as shown in Figure 6, the CVC-N gel has the following properties: firmness of 239.09 g, consistency of 1587.00 gm sec, cohesiveness of −89.17 gm, and work of cohesion of −645.47 gm sec, as shown in Table 4 [53]. Peak or maximum force is used to measure firmness; the higher the number, the thicker the sample's consistency. The sample's stickiness or cohesiveness is determined by measuring the greatest negative force. The stiffer the sample is, the more negative the number [54].

**Figure 6. Figure 6.** Texture analysis of optimized CVC-N gel. Texture analysis of optimized CVC-N gel. effectively carried rhodamine B dye deeper into the rat's skin layers.

predicting the release of the drug.

*2.7. Analysis of the Gel and Texture of the Optimized CVC-N Gel* 

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**Table 4.** Physiochemical characterization of CVC-based niosomal gel (CVCNG).

**Homogeneity Appearance Washability Separation of Phase Odour**  Homogeneous Translucent Yes No odourless

**Figure 7.** Confocal laser scanning microscopy; (**A**) niosome-formulation-loaded rhodamine B dye; (**B**) hydroalcoholic solution of rhodamine B dye. **Figure 7.** Confocal laser scanning microscopy; (**A**) niosome-formulation-loaded rhodamine B dye; (**B**) hydroalcoholic solution of rhodamine B dye.

### *2.9. Dermatokinetic Studies 2.9. Dermatokinetic Studies*

As a result of the application of CVC-CF gel and CVC-N gel at a predefined time interval, the CVC concentration in the dermis and epidermis of the rat's skin is shown in Figure 8. Table 5 displays the values of the dermatokinetic parameters. The rat skin that had been exposed to CVC-CF gel displayed CSkin max values of 179.04 ± 0.96 µg/cm2 in the epidermis and 160.13 ± 0.64 µg/cm2 in the dermis. A CSkin max value of 283.54 ± 1.01 µg/cm2 was found in the epidermis, and 262.64 ± 1.12 µg/cm2 was found in the dermis of a rat As a result of the application of CVC-CF gel and CVC-N gel at a predefined time interval, the CVC concentration in the dermis and epidermis of the rat's skin is shown in Figure 8. Table 5 displays the values of the dermatokinetic parameters. The rat skin that had been exposed to CVC-CF gel displayed CSkin max values of 179.04 <sup>±</sup> 0.96 <sup>µ</sup>g/cm<sup>2</sup> in the epidermis and 160.13 <sup>±</sup> 0.64 <sup>µ</sup>g/cm<sup>2</sup> in the dermis. A CSkin max value of 283.54 <sup>±</sup> 1.01 <sup>µ</sup>g/cm<sup>2</sup> was found in the epidermis, and 262.64 <sup>±</sup> 1.12 <sup>µ</sup>g/cm<sup>2</sup> was found in the dermis of a rat whose skin had been treated with CVCN gel. AUC0-t values of 677.47 ± 0.28 and 572.23 <sup>±</sup> 0.31 <sup>µ</sup>g/cm<sup>2</sup> h, respectively, were seen in the epidermis and dermis of a rat whose skin had been treated with CVC-CF gel. AUC0-t values in the epidermis and dermis of rat skin treated with CVC-N gel, in contrast, were 1135.5 <sup>±</sup> 0.64 and 1158.7 <sup>±</sup> 1.08 <sup>µ</sup>g/cm<sup>2</sup> h, respectively. When rat skin was treated with CVC-N gel in contrast to the CVC-CF gel

formulation, a greater percentage of CVC was maintained in both rat skin layers. The epidermis and dermis both showed greater CSkin max and AUC0-t values, which demonstrate that the CVC-N gel improved drug absorption in both the epidermal and dermis layers because of its nanosized vesicles' ease of penetration into the skin's lipid layers, enhancing its therapeutic efficacy in the treatment of inflammatory diseases [56]. and dermis both showed greater CSkin max and AUC0-t values, which demonstrate that the CVC-N gel improved drug absorption in both the epidermal and dermis layers because of its nanosized vesicles' ease of penetration into the skin's lipid layers, enhancing its therapeutic efficacy in the treatment of inflammatory diseases [56].

whose skin had been treated with CVCN gel. AUC0-t values of 677.47 ± 0.28 and 572.23 ± 0.31 µg/cm2 h, respectively, were seen in the epidermis and dermis of a rat whose skin had been treated with CVC-CF gel. AUC0-t values in the epidermis and dermis of rat skin treated with CVC-N gel, in contrast, were 1135.5 ± 0.64 and 1158.7 ± 1.08 µg/cm2 h, respectively. When rat skin was treated with CVC-N gel in contrast to the CVC-CF gel formulation, a greater percentage of CVC was maintained in both rat skin layers. The epidermis

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**Figure 8.** CVC concentration on the (**A**) epidermis and (**B**) dermis of excised rat skin following ap-**Figure 8.** CVC concentration on the (**A**) epidermis and (**B**) dermis of excised rat skin following application to the skin of CVC-N gel and CVC-CF gel.

**Table 5.** Dermatokinetic parameters (mean ± SD) of CVC-N gel and CVC-CF gel.


plication to the skin of CVC-N gel and CVC-CF gel.

### *2.10. Ferric-Reducing Antioxidant Power (FRAP)*

The analysis indicated that CVC has a significant amount of antioxidant capacity and that the agent is effective at scavenging free radicals, and preventing the oxidation of antioxidant activity was found to be 60.14 ± 1.11% in free CVC, 88.41 ± 2.32% in an ascorbic acid solution, and 71.24 ± 3.31% in a CVC-N-optimized formulation. As the results indicate, CVC-Ns have been shown to have a significant antioxidant effect.

### *2.11. Stability Studies*

The data of experiments into short-term accelerated stability were evaluated for six months. Physical appearance, shape, vesicle size, PDI, zeta potential, colour appearance, phase separation, clarity, homogeneity, pH, and drug content were detected (Tables 6 and 7). This study thus validates the notion that niosomes are durable over long periods of storage.

**Table 6.** Evaluation of the CVC-loaded optimized niosome (CVC-N) formulation's short-term accelerated stability.


++ good, +++ excellent.

**Table 7.** Short-term accelerated stability evaluation of CVC-loaded optimized niosomal gel formulation.


\* satisfactory, \*\* good, \*\*\* excellent.

### **3. Conclusions**

Niosomes were effectively developed using thin film hydration. Using BBD, the niosomal formulation was optimized by taking independent and dependent factors. The optimized formulation achieved the highest amount of CVC entrapping, suggesting that it was the most ideal among all formulations, i.e., batch code F4. The optimized CVC-loaded niosomes had reduced PDI values and were in the colloidal size range, which indicated that the formulations were homogeneous. The TEM images demonstrated the spherical shapes of the vesicles, providing evidence of an entrapped CVC. According to in vitro drug release studies, the amount of CVC released from CVC-loaded niosomes was two times higher than it was from CVC suspension solution, demonstrating improved drug release from the niosome formulation due to nanosized vesicles. A CLSM study on rat skin has provided insight into the penetration of rhodamine-loaded B hydroalcoholic and rhodamine-loaded niosome formulations, and the results showed that rhodamine-B-loaded niosomal formulations penetrated the skin much more effectively than hydroalcoholic solutions of the rhodamine B dye, indicating that niosomes have improved in vivo prospects for anti-inflammatory treatment. Compared to CVC-CFG, dermatokinetic studies show that a higher concentration of CVC-NG reaches the epidermis and dermis (Cskin max and AUC0-t) due to the nanosized vesicles providing a means to cross the stratum corneum to have a profound effect. The antioxidant studies revealed that the niosome formulation has a greater potential for antioxidants than the pure drug, showing a capability to reduce the level of free radicals and reactive oxygen stress and hence to reduce inflammation. Moreover, the results of this experiment confirmed that the developed, optimized niosome formulation is an effective means of delivering CVC drugs topically and can therefore be used to treat anti-inflammatories more successfully. However, the actual skin penetration of carvacrol may vary depending on the formulation and the distinctive characteristics of the skin. While carvacrol is usually regarded as safe, the safety profile of carvacrol-containing niosomes for topical distribution has not been fully established, and additional research may be required to determine their possible toxicity and side effects and to validate the in vitro and skin permeation results by using appropriate animal models in preclinical studies.

### **4. Materials and Methods**

### *4.1. Materials*

Carvacrol oil, polyethylene glycol 400 (PEG), triethanolamine rhodamine-123, Ascorbic acid and potassium ferricyanide were procured from Sigma-Aldrich (St. Louis, MI, USA). Cholesterol and tween 80 were provided by S D Fine Chemicals Ltd. (Mumbai, India). Methanol and chloroform were provided by Merck Mumbai, India. B.S. Goodrich in Pleveland generously provided carbopol-934 as a gift sample. Other agents used in the experiment such as disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride of analytical grade were provided by S D Fine Chemicals Limited, Mumbai, India.

### *4.2. Method*

### 4.2.1. Preparation of CVC-Loaded Niosomes (CVC-Ns)

Niosomes loaded with CVC (CVC-Ns) were made using the thin-layer hydration method. Briefly, the drug (CVC), cholesterol, and surfactant were dissolved in a roundbottomed flask using a chloroform-to-methanol ratio of 2:1. The organic phase was then evaporated in a rotary evaporator (120 rpm, 60 ◦C, 1 h) to generate a thin layer. Phosphatebuffered saline solution (PBS pH—6.8, 10 mL) was then used to rehydrate the desiccated thin film at room temperature (30 ◦C) for one hour (200 rpm). CVC-loaded niosomes were then sonicated for 2 min by a probe sonicator. The samples were preserved in a refrigerator at 4 ◦C for further investigation [57].
