The pH of developed DE gel was found to be 6.2, which is close to skin i.e., 4.5&#;6.4 as per literature. As pH of the formulation is neutral or closer to the pH of the skin, the formulation can be considered as safe or may not cause any skin irritation on application.
The micro-mechanical properties of gels can be assessed by viscometric analysis. The flow properties of DE-gel were recorded by using cone and plate viscometer and the values of parameters were computed using Herschel-Bulkley model. The value of flow index (n&#;=&#;0.252), obtained from the slope (Fig. 6A), is less than 1 which indicates DE-gel formulation observed a pseudoplastic (shear-thinning) behavior, thus, reflecting decreased formulation viscosity with increase in shear rate (Fig. 6B). The appearance of pseudoplastic behaviour can be attributed to the underlying colloidal network structure, which exhibits deformation and adjustment in response to the direction of flow. The flow behaviour observed in this context might be associated with the existence of microstructures within a three-dimensional lattice network. The consistency index K (Pa.sn) and yield value were found to be 109.18 Pa and 120.55 respectively, which could be due to gel structure rigidity within the hydrogel which required more force to initiate its flow. However, the intrinsic viscosity of DE-gel was found to be 106.7 Pa.s. Attaining a specific phase and viscosity is a crucial prerequisite in the formulation of DE-gel to facilitate their convenient transportation and storage at an optimal temperature.
Figure 6(A) Graph of the Herschel-Bulkey model showing the relationship between the natural logarithm of shear stress and the natural logarithm of shear rate; (B) Plot of shear rate vs viscosity and shear stress for the DE-gel formulation; (C) The texture analysis of the developed DE-gel formulation.
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Figure 6C and Table 1 both represent the textural curve along with the various values of textural parameters such as firmness, consistency, cohesiveness, and index of viscosity that were acquired from texture analysis of the developed DE-gel formulation. The visual illustration of the DE-gel formulation shows better gel strength, suggesting greater capability to hold the gel at the topical site for a longer period of time, exhibiting smooth extrudability, and ensuring that the gel is easy to spread.
Table 1 Shows the data for the various parameters obtained from the texture analysis of the developed DE-gel formulation.Full size table
The final DE-gel was finally studied for desired attributes with regard to permeation of ETO across skin vis-à-vis the conventional gel of equivalent strength. The data presented in Fig. 7 demonstrates the permeation profiles of two formulations. It is evident that the developed DE-gel formulation exhibiting 31.33% drug permeation across of the rodent skin, exhibited superior transport properties compared to the conventional gel formulation (10.34% ETO permeation). This study provides evidence for the better transport properties of the newly developed carrier-based formulation in comparison to the conventional formulation. The enhanced penetration of ETO from the DE-gel formulation can be attributed to the phospholipid's compatibility with the skin, which is in turn attributable to the effective drug movement properties of the vesicular carriers. Similar findings with phospholipids have been frequently reported and these make the phospholipids are one of the important constituents of the topical formulations68. Figure 7 Inset depicts the results of drug deposition in skin reveal that the DE-gel formulation exhibits 648.68 µg/cm2 (i.e., 33.13%) and conventional gel exhibits 304.08 µg/cm2 (i.e., 14.71%). DE-gel formulation is 2.25 times significantly higher drug deposition than that of conventional gel (p&#;<&#;0.01). This suggested DE-gel effectively makes the drug more readily available within the different dermal layers, also depositing them within target sites by means of closely integration of phospholipids to the lipids of the skin.
Figure 7Ex vivo permeation profile of DE-gel and conventional gel formulations. Each bar indicates&#;±&#;SD (n&#;=&#;3). Inset figure depicting bar diagram for the amount of drug deposited by the skin for DE-gel and conventional formulations. Each cross bar indicates&#;±&#;SD. (n&#;=&#;3).
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The CLSM technique was used to determine the extent and pattern of DE formulation penetration into the skin layers. The efficacy of a coumarin-6 dye loaded-DE formulation was assessed via topical administration on rat skin for a duration of 6 h. The Fig. 8A depicts the skin treated with coumarin-6 dye alone and Fig. 8B depicts transdermal penetration of coumarin-6 dye loaded-DE formulation across the skin barrier. The confocal images demonstrated that the carrier containing the dye was evenly dispersed throughout the stratum corneum, epidermis, and dermis, exhibiting a significantly higher fluorescent intensity. The findings are consistent with prior literature indicating that the utilization of a flexible vesicular system is significant in enhancing drug solubility and optimizing the distribution of the drug molecule into skin tissue69.
Figure 8(A) CLSM of coumarin-6 dye alone; (B) coumarin-6 dye loaded-DE formulation in the deeper layer of the skin; (C) ETO concentration time profile in epidermis and dermis of Wistar rats after single application of Conventional gel formulation; (D) ETO concentration time profile in epidermis and dermis of Wistar rats after single application of DE-gel formulation. Each cross bar indicates&#;±&#;SD (n&#;=&#;4).
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Dermatokinetic investigations were conducted to assess the dermal pharmacokinetics therapeutic efficacy of the newly formulated DE-gel in comparison to the conventional gel in distinct skin compartments, specifically the epidermis and dermis. Figure 8C and D demonstrate a difference in the permeation of ETO concentration between the epidermal and dermal skin layers following a single application of conventional gel and DE-gel. This observation indicates that the drug concentration over time followed the principles of a one-compartment open body model (1CBM) through the use of dermatokinetic modelling. The administration of DE-gel loaded with ETO exhibited a statistically significant increase (p&#;<&#;0.05) in the transdermal delivery when compared to the conventional gel.
Table 2 displays the values of the following parameters like AUC0&#;12 h (µg/ cm2), \({C}_{max}^{Skin}\)(µg/cm2), \({T}_{max}^{Skin}\)(h) and Kp (h&#;1). It is evocative from the results that the duration of stay of the drug was strongly augmented by the developed DE-gel formulation in deep skin layers and \({T}_{max}^{Skin}\)(h) decreased markedly. The lower Tmax by developed formulation vis-à-vis control signifies better penetration and assures faster onset of action. Apart from this \({C}_{max}^{Skin}\) in both the layers i.e., dermal and epidermal and AUC in dermis increased significantly. Therefore, the data ratified that the DE-gel formulation has prospective outcome in regard to enhanced delivery of ETO across the skin in comparison to the conventional formulation.
Table 2 Various dermatokinetic parameters (Mean&#;±&#;SD) of ETO topical formulations in epidermis and dermis.Full size table
The cell viability assay results using MTT dye are depicted in Fig. 9A. HaCaT cells were used to study the cytotoxic possibility of the formulations. The % viability of untreated cells which served as the control was considered to be 100%. The developed formulation i.e., DE and comparative blank DE without ETO did not show any toxicity till 100 µg/mL after 48 h treatment with almost 91.99% and 93.88% viability. In contrast, the pure drug, namely ETO, demonstrated notable toxicity, resulting in less than 21.5% viability in comparison to the control. The findings confirmed that the formulations developed in-house exhibit no cytotoxic effects on HaCaT cells. Although there was no statistically significant variance in the percentage of cell viability comparing the control group and the created formulations, there was a statistically significant variance between the control group and the pure medication (p&#;<&#;0.05). The observed phenomenon may be attributed to the biocompatible properties inherent in phospholipids.
Figure 9(A) Cell viability assay results after 48 h of treatment of pure drug ETO, ETO-DE, blank DE. The results are expressed as % cell viability, taking the viability of control as 100%. Each cross bar indicates&#;±&#;SD (n&#;=&#;4;****p&#;<&#;0.; ns-non significant); (B) Images depict HaCaT cells subjected to treatment with coumarin-6 alone at 40&#;×; and (C) Coumarin-6 loaded DE at 40&#;×&#;objective, respectively, as observed through fluorescent microscopy.
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The findings of the investigations on cellular uptake portray the internalization of Coumarin-6 loaded DE formulation by HaCaT cells. As demonstrated in Fig. 9C, the cellular uptake assay of Coumarin-6 DE revealed the proficient internalization of the formulation within the cytosol of HaCaT cells within a time frame of 3 h. The test formulations were observed to induce a distinct green fluorescence in the nuclei of cells, as evidenced by the visualization of coumarin-6. Similarly, coumarin-6 dye labelling alone demonstrated the efficient assimilation of the developed formulations by keratinocytes, as illustrated by the green fluorescence in Fig. 9B.
Results of stability testing of the developed formulations indicated all the formulations were stable. As discerned from the results given in Fig. 10, the ETO loaded DE-gel formulation is stable at all the studied storage conditions for 6 months. The effect of temperature on the drug migration from one phase to other was not substantial and was suited for topical products.
Figure 10Figure depicting the % drug assay of DE-gel at different storage conditions and time intervals.
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Effect of temperature on the drug content was found to be insignificant at high temperature conditions (40 °C&#;±&#;2 °C/75%&#;±&#;5% RH) which can be ascribed to the bilayer packing alteration of vesicles at high temperatures. Similarly, the DE-gel formulation stored at controlled room temperature (25 °C&#;±&#;2 °C/60%&#;±&#;5% RH) conditions also showed better stability for the studied period.
The observations for a period of 6 months were recorded for various physical parameters are enlisted in Table 3. The DE formulation showed macroscopic stability on the studied parameters for 6 months at 25 °C&#;±&#;2 °C/60%&#;±&#;5% RH and 40 °C&#;±&#;2 °C/75%&#;±&#;5% RH storage conditions. The DE-gel formulation was devoid of notable discoloration and change in odour. The gel consistency also remained good with absence of drug crystals and phase separation. The particle size alteration was below 13%, indicating acceptable variation. However, the present study is limited in scope w.r.t. the changes in the lamellae and viscosity.
Table 3 Physical stability assessment studies on DE gel formulation.Full size table
The developed DE-gel formulation was evaluated for any irritating effect on the skin. The erythemal grading (ranging from 0 to 4) were recorded for 7 days. Absence of erythema on skin was observed in case of DE-gel formulation, whereas moderate to severe erythema (light red) scores were observed in case of conventional product as shown in Fig. 11 and the scoring is tabulated in Table 4.
Figure 11Shows the animals and histology at the end of seven days (A) untreated animal (B) conventional gel formulation (C) DE-gel formulation.
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Table 4 Mean erythemal scores observed for group, (A) untreated, (B) conventional gel formulation, and (C) DE-gel formulation for 7 days.Full size table
In conformity with the histopathology, the skin section of various animal groups treated with DE-gel formulation and conventional gel formulation were stained with eosin-hematoxylin and evaluated for the histological changes occurred during the period of exposure. Figure 11A showed the photograph of untreated skin, which was normal. Figure 11B showed epidermal thickening and inflammation in the dermis layer on the skin treated with conventional gel. Therefore, the microscopic examination directed that the viable formulation had compromised the normal healthy skin. Furthermore, the skin section treated with DE-gel formulation was found to be healthy with no inflammation in the dermis tissue. It revealed DE-gel did not damage the normal healthy skin Fig. 11C. The study's findings demonstrated the safety and effectiveness of biocompatible phospholipid in the DE-gel system. These positive outcomes can be due to phospholipids interaction with skin components and their ability to establish a skin-depot. The study reported here align with prior literature, which suggests that lipid-based formulations are both safer and more compatible with the skin55,56.
Figure 12A showed DE-gel formulation exhibited remarkably advanced anti-inflammatory activity versus conventional formulation. The % swelling of treated ear was reduced by 2.99 times (DE-gel formulation) and 1.33 times (conventional gel formulation) with respect to untreated ear. Thus, the efficacy of the formulated DE-gel was significantly 2.2-folds higher than that of conventional (p&#;<&#;0.01). The outcomes of the animal study conducted exposed the edge of the vesicular delivery systems as compared to the conventional systems. This accredited to their better interaction with the skin and skin-depot forming potential. As per the histopathological studies, there was division into three groups viz. disease control causing swelling, epidermis stretching and detachment of epidermis from dermis as seen in Fig. 12B. The histopathology of untreated ear as shown in Fig. 12C was normal whereas Fig. 12E displayed DE-gel group with healing of ear with intact epidermal and dermal layers and no edema formation. In contrast, Fig. 12D was the group treated with conventional gel formulation reporting incomplete recovery which was manifested from histopathology where disordered articular cartilage with greater number of inflammatory cells was experienced.
Figure 12(A) Comparison of present ear swelling after application of conventional gel and DE-gel formulations. Each cross bar indicates&#;±&#;SD (n&#;=&#;4;***p&#;=&#;0.001; ****p&#;<&#;0.); (B) Histopathology of disease control ear; (C) histopathology of normal ear; (D) histopathology of conventional gel formulation treated ear; (E) histopathology of DE-gel formulation treated ear.
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To understand the anti-arthritic activity in diseased rats after topical application of DE-gel formulation, the most vital parameter that is % arthritis swelling reduction was calculated. A significant rise in swelling was measured for CFA rats which didn&#;t receive any treatment. In the conventional gel, a slight reduction in % swelling was detected (i.e., 27.27%), however in the DE-gel formulation, a significant decrease in swelling was noted (i.e., 3.89%), as depicted in Fig. 13A. Thus, efficacy of the DE-gel formulation was sevenfold higher than that of group treated with conventional gel formulation (p&#;<&#;0.01). This shows the superior activity of ETO loaded DE-gel over conventional gel in arthritis and indicated better penetration of drug to the site of action.
Figure 13(A) Comparison of % arthritis swelling after application of DE-gel formulation and conventional gel formulations. Each cross bar indicates&#;±&#;SD (n&#;=&#;4); (B) Histopathology of normal paw joint; (C) Disease control paw joint; (D) conventional gel formulation treated paw joint; (E) DE-gel formulation treated paw joint.
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The histological examination of paw joint and paw skin of animals suffering from arthritis treated with different ETO formulations was executed to evaluate the level of inflammation and morphological behaviour in the internal structure of the paw joint and paw skin.
Figure 13C represents paw joint of CFA (untreated) induced arthritic rat experienced accretion of synovial fluid, lymphocytes in synovium, tissues granulation along with formation of pannus adding up to the high level of inflammation extended into joint synovium recorded as compared to the normal joint (Fig. 13B). Figure 13D presents a little lower inflammation with the joint space surrounded by inflammatory cells with moderate pannus formation and accretion of synovial fluid in the case of paw joints treated with conventional gel formulation. Almost no signs of inflammation were observed in the paw joint treated with DE-gel formulation attaining improved joint bone health to its normal structure as seen in Fig. 13E.
Similar results were recorded for the infected paw skin in Fig. 14 further divided into 4 groups i.e., control, untreated, conventional gel, DE-gel treated rat. Figure 14B showed acute inflammation, augmented thickness of the layers, disrupted layers with separation and hyperkeratosis in the untreated rat paw skin. The normal paw skin was observed to be intact having the natural anatomy of skin (Fig. 14A). In comparison to the rat paw skin treated with conventional gel which showed moderate inflammation with slightly higher thickness of the skin layers, edema and hyperkeratosis in Fig. 14C vis-à-vis negligible level of inflammation was detected in case of DE-gel treated rat skin as shown in Fig. 14D.
Figure 14(A) Histopathology of normal paw; (B) Disease control paw; (C) conventional gel formulation treated paw; (D) DE-gel formulation treated paw.
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The severity degree for treated groups is described as a score of inflammation and arthritis from 0 to&#;+&#;&#;+&#;&#;+&#;&#;+&#;(0 indicates normal &&#;+&#;&#;+&#;&#;+&#;&#;+&#;indicates severe). The severity of inflammations for paw skin was observed in the following order: CFA (untreated&#;>&#;conventional gel formulation&#;>&#;DE-gel formulation&#;=&#;control (normal rat).
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