Alopecia is defined as a non-cancerous autoimmune hair disorder in which extensive hair loss occurs from various body regions, especially from the scalp [1]. It is the most common dermatological complaint with a varying prevalence rate throughout the globe [2]. It has an immense psychosocial influence over the person’s mind [3]. The pathophysiological mechanism of alopecia is an enigma, but hormones, infections, medications, and hair styling practices are the various triggering factors that are also responsible for the occurrence of alopecia [4].

Treatment options for alopecia include masks, wigs, and sunscreen, administration of plasma injection rich in platelets and corticosteroids, and administration of topical prescription drugs like anti-androgens, vasodilators, etc [5, 6, 7, 8]. Among the different drugs used for alopecia treatment, minoxidil is the first USFDA-approved poorly water-soluble anti-hypertensive drug, which is known to prolong the growth phase (anagen phase) in the hair follicle with prominent anti-hair loss results [9]. It does not bind to plasma proteins and exhibits a shorter elimination half-life (4.2 hours). The oral ingestion of this drug, however, leads to different adverse effects such as hypotension, palpitations, weight gain, hypertrichosis, and itching, etc, with 80% first pass metabolism [10]. Therefore, it necessitates the formulation of a nanocarrier, which would overcome the drawbacks associated with the oral traditional formulation and improve the drug’s overall efficacy. Further, it would be advantageous if the formulated nanocarrier provided a controlled release of the drug as it would also contribute to reducing the dose and the dose-related side effects. The controlled drug delivery systems developed and reported in the literature for the treatment of alopecia include nano lipid carriers [11, 12] solid lipid nanoparticles [13, 14], polymeric nanoparticles [15, 16], Nanoemulsions [17, 18], liposomes [19, 20], transferosomes [21,22], Niosomes [23, 24], Ethosomes [25, 26], cubosomes [27], metallic nanoparticles [28, 29], Liquid Crystalline nanoparticles [30]. However, since most of the above-mentioned dosage forms are in liquid form, they limit the retention of the drug at the application site. In order to overcome this drawback in the present study, a submicron emulsion-based gel was formulated to deliver minoxidil.

Submicron emulsions are thermodynamically stable isotropic mixtures of drugs with suitable concentrations of oil, surfactants, co-surfactants, and water with a mean droplet size of 10-500 nm. This nanocarrier has gained the attention of many scientists due to its maximum solubilization capability, smaller droplet size, and maximum stability [31]. However, to overcome the problem of its low viscosity, the formulation of submicron emulsion as a topical gel is a better option. The submicron emulsion-based gel is defined as the gelling system that can be obtained after the addition of the submicron emulsion system into the gel matrix. The presence of sebum in hair follicles makes it a most promising approach for delivering minoxidil [32]. This nanocarrier enhances the permeation of active drug moiety into the skin and provides sustained release, which leads to improved patient compliance and higher therapeutic effect [33]. This study provides the preparation and characterization of submicron emulsion-based topical gel containing minoxidil for treating alopecia. The gel would not only provide targeted delivery of the drug at the site of application but, due to its gel state, would enhance the retention of the drug at the application site, which would, in turn, lead to an overall improvement in the therapeutic efficacy of the drug and in turn patient compliance.

The study was conducted at Khalsa college of Pharmacy, Amritsar and didnot utilise any living animals or humans and therefore no permission was taken from the Institutional Ethical Committee. Minoxidil was procured as a gift sample from Kwality Pharmaceutical Ltd. (Amritsar, India). Capryol 90 and Transcutol P were procured from Saint-Priest, France (Gattefosse) as a gift sample. Polysorbate 20and triethanolamine wereobtained from S.D. Fine Chem Ltd. (Tamilnadu, India). Carbopol 934 and ferric chloride were obtained from Himedia laboratories Pvt. Ltd. (Mumbai, India). All other analytical reagents & chemicals were purchased from Merck Specialities Pvt. Ltd. (Mumbai, India) and Qualikem Fine Chemicals Pvt. Ltd. (New Delhi, India).

Formulation of submicron emulsion

Solubility and Miscibility Studies

The solubility determination is the prerequisite to selecting the appropriate components for formulating a submicron emulsion. To commence with the solubility studies, an excess amount of the drug was added in Eppendorf tubes containing different oils (Clove oil, Oleic acid, Isopropyl Myristate, Capryol 90, Tea tree oil and Olive oil), surfactants (Kolliphor HS 15; Tween 40; Tween 20, Span 80, Span 20, Tween 60 and Tween 80) and co-surfactants (Transcutol P, PEG 400 and PEG 600) individually. The contents in Eppendorf tubes were mixed using a vortex mixer and then kept on a biological shaker for 3 days at 37\(\pm\)2. After this, the centrifugation of samples was done at 3000 rpm for 15 minutes, and the supernatant layer was removed, followed by filtration. It was then analyzed using a UV spectrophotometer at a max of 286 nm. The estimations were performed in triplicate.

For the miscibility studies, one ml of each selected surfactant and co-surfactant was taken in a 1:1 ratio in Eppendorf tubes and mixed using a vortex mixer at 25\(\pm\)1. The obtained mixtures were kept overnight to determine the miscibility of S\(_{\text{mix}}\) [18].

Pseudo-Ternary Phase Diagram Construction and Formulation Development

The method used for the construction of pseudo-ternary phase diagrams was the aqueous titration method in which the different S\(_{\text{mix}}\) (Surfactant: Co-surfactant) ratios (1:0, 1:1, 1:2, 2:1, 3:1, and 4:1) were prepared followed by the preparation of various oil: S\(_{\text{mix}}\) ratios. The aqueous phase was slowly added into this oil: S\(_{\text{mix}}\) ratios to produce a clear, transparent, and less viscous submicron emulsion system. The prepared emulsions were vigorously vortexed and kept for 24 hours to attain equilibrium [34]. After this, the combinations for preparing placebo submicron emulsions were selected from the constructed phase diagrams to achieve a formulation with suitable oil concentration for better solubilization of the drug with minimum S\(_{\text{mix}}\) and high water content. The same procedure was repeated to prepare submicron emulsion containing minoxidil in a dose of 1%w/v [29].

Physical Stability Studies

The minoxidil-containing submicron emulsions were checked for stability by subjecting them to different physical stability tests. The first test, i.e., the heating-cooling cycle, was performed by storing the emulsion formulations at \(4^{\circ}\) and \(40^{\circ}\). each for 48 hours. The second stability test was the centrifugation test, in which the centrifugation of prepared submicron emulsion was performed for 30 minutes at 3000 rpm to examine any physical instability. The third test, i.e., freeze-thaw cycle or accelerated stability testing, was done by storing the formulations at \(-21^{\circ}\) and \(+25^{\circ}\) for at least 2 days [35]. The stable formulations were further characterized based on different parameters.

Characterization of submicron emulsion

pH

The pH of submicron emulsions was examined using digital pH meter at room temperature as too high or too low pH could lead to unwanted side effects [35].

In Vitro Drug Release Study & Determination of Release Kinetics

For the drug release study, the dialysis membrane was first treated with 0.3% w/v sodium sulphide solution and other reagents. It was then mounted on a Franz diffusion cell between two half-cell compartments. The phosphate buffer pH 5.5 was added in the receptor compartment as release medium at \(37^{\circ}\) with continuous stirring at 75 rpm. The donor compartment consisted of one ml of 1% w/v drug-loaded submicron emulsions, and the aliquots were taken at pre-defined time periods up to 6 hours from the receptor compartment. Were the collected samples assessed using a UV spectrophotometer \(\lambda_{\max}\) 286 nm, and the results were compared with those obtained using ethanol’s drug solution (1%w/v). The experiment was repeated in triplicate [36].

The data produced from the in vitro release investigation was plotted in different release models to study the release kinetics of prepared submicron emulsions. The model for which the coefficient correlation (R2) value was near unity was considered the release model [37].

Particle Size, PDI and Droplet Charge Assessment

The size of particles and polydispersity index of submicron emulsion were examined by the dynamic light scattering method. For this, the dilution of the samples was performed 100 times with distilled water and assessed using a zeta-sizer. After this, they were directly placed into module [38]. The charge on the average globules was measured by using Malvern zeta-sizer [39].

Ex Vivo Skin Permeation Study & Data Analysis

For the evaluation of drug permeation, the excised rat skin sample was obtained from the pharmacology department, followed by the complete removal of hair and underlying fat from the body. After this, the excised skin was completely stabilized, and a Franz diffusion cell was used to conduct this study. Phosphate buffer (pH 7.4) was put into the receptor compartment, while the donor compartment consisted of 1 mL of the 1% w/v minoxidil-loaded submicron emulsion. The samples were taken at pre-defined time intervals (6-hrs study) and analyzed by U.V spectrophotometer at \(\lambda_{\max}\) 286 nm. The experiment was done in triplicate, and the comparison was done with 1% w/v minoxidil ethanolic solution [40]. The drug flux (Jss) was obtained by plotting the amount of minoxidil submicron-sized emulsion permeated in steady state conditions v/s time. The permeability coefficient (kp) was measured by dividing the flux obtained by the initial drug concentration (Co) present in the donor compartment.

Formulation of submicron emulsion based topical gel

Submicron emulsion-based topical gel was prepared by dissolving different quantities of selected gelling agent, i.e.carbopol 934, into the optimized submicron emulsion (M5) formulation with continuous stirring till the equilibrium was attained. The pH adjustment to a neutral value was done using triethanolamine. The gel formulations (M5\(^{1\%w/v}\), M5\(_{1.5\%w/v}\), M5\(_{2\%w/v}\)) were then kept overnight and further evaluated [41].

Characterization of submicron emulsion based topical gels (M51%w/v, M51.5%w/v, M52%w/v)

Physical Appearance, pH, Homogeneity & Grittiness

The prepared submicron gel formulations were checked under normal sunlight to examine their physical appearance. The pH was assessed using a digital pH meter, while the homogeneity and grittiness were measured by rubbing the gel formulations on the back of the hand [42]. Drug content

To determine the drug content,minoxidil-loaded submicron gels were mixed with methanol and shaken for 2 hours. The obtained solutions were filtered, followed by removing the supernatant layer. The dilution of the supernatant layer was done using methanol and analyzed by a UV spectrophotometer at a wavelength of 286 nm [43].

Ex Vivo Skin Permeation Studies of Topical Submicron Gel Formulations & Data Analysis

The ex vivo skin permeation study of topical submicron emulsion-based gels was done using Franz diffusion cells. The skin was properly excised, stabilized, and placed between the half-cell compartments. The different concentrations (M5\(_{1\%w/v}\), M5\(_{1.5\%w/v}\), M5\(_{2\%w/v}\)) of submicron topical gel were separately placed in the donor compartment while the phosphate buffer pH 7.4 was used as a release medium. The study was continued for 6 hours with the continuous withdrawal of the sample at predetermined time intervals followed by replacement with fresh release medium. The results were compared with those obtained from plain minoxidil gel. The experiment was performed in triplicate [44]. The flux and permeability coefficient was also calculated.

Drug Retention Studies

After the permeation studies, the remaining formulation was removed from the skin and cleaned with cotton soaked in 0.05% SLS, followed by subsequent washing using purified water. Further, the skin was weighed and chopped into small pieces. It was then dissolved in a suitable solvent and kept for sonication. The resulting solution was centrifuged and filtered. It was then analyzed using a U.V spectrophotometer at \(\lambda_{\max}\) 286 mn [32].

Histopathological Investigation

The skin samples utilized in the ex vivo permeation study were subjected to histopathological study. Skin samples were excised from rat bodies and placed in a saline solution for control. The treated and control samples were kept for storage in 10% formaldehyde solution. Afterward, the samples were chopped vertically and stained with paraffin wax. After staining, the treated and untreated samples were examined using a microscope [45].

Viscosity and in Vitro Bioadhesion Study

The viscosity of the submicron topical gels was determined by Anton Paar rheometer. The gel was put on the plate and kept for equilibration at \(25 \pm 0.1^{\circ}\) for a few minutes. The measurement was performed in triplicate [46].

For the in vitro bioadhesion study, an agar plate was first prepared. The gel samples to be studied were placed in the center of the plate and further attached to the IP disintegration assembly. At room temperature, the plates were continuously moved up and down in phosphate buffer media (pH 7.4). The residence time of the test samples on the agar plate was measured visually. The experiment was repeated in triplicate [47].

Antioxidant activity of submicron emulsion based topical gel formulation

The antioxidant activity of minoxidil loaded submicron emulsion based topical gel was determined by using two methods;

DPPH Method

Firstly, the stock solution (1mg/ml) of standard (ascorbic acid) and test formulation was prepared separately in methanol. Following this, the different serial dilutions (1-20 \(\mu\)g/ml) of standard and test formulations were also prepared separately. From these dilutions, one ml was taken and dissolved in methanolic solution of DPPH (0.004%w/v). After 30 minutes, the prepared solutions were analyzed at 515 nm against methanol as blank [48]. The formula used for measuring percent inhibition was as follows: \[\% \text{inhibition}= \left[\frac{\text{A}_{\text{Blank}}-\text{A}_{\text{Formulation}}}{\text{A}_{\text{Blank}}}\right].\]

Stability studies

The stability studies of optimized gel formulation was performed by storing the submicron emulsion based gel formulations at referigerator temperature (4\(^{\circ}\)) for the period of 3 months [49].

Statistical analysis

All the results were represented as mean \(\pm\) standard deviation. The software used for analysis of resultsproduced by various test groups was Graph pad Instat 3 (two tailed unpaired t-test). The p-values \(\leq 0.05\) were found to be significant.

Solubility studies & miscibility assessment

The determination of minoxidil solubility was done in various components and is depicted in Table 1.

Construction of Pseudoternary phase diagrams for formulation selection

Pseudoternary phase diagrams were constructed for each S\(_{\text{mix}}\) ratios (1:0, 1:1, 1:2, 2:1, 3:1 & 4:1) by aqueous titration method (Figure 1) to determine the extent of submicron emulsion region.

Figure 1: Pseudoternary Phase Diagrams Constructed for Different S\(_{\text{mix}}\) Ratios for Preparation of Submicron Emulsion; (A) Represents 1:0 S\(_{\text{mix}}\) Ratio, (B) - 1:1, (C) - 1:2, (D) - 2:1, (E) - 3:1, (F) - 4:1 S\(_{\text{mix}}\) Ratio

Formulation development and thermodynamic stability studies

The placebo submicron emulsions which were found to be stable after physical stability testing were further chosen for loading the drug in a suitable dose (1%w/v). The prepared minoxidil loaded submicron emulsion were again checked for stability and the composition of formulations which passed the stability testing is given in Table 2.

Characterization and optimization of submicron emulsion

pH

The pH of all submicron emulsion formulations were found to be in the range of 4.5 to 6.6.

In Vitro Drug Release Study & Determination of Release Kinetics

The comparision of in vitro release study of different submicron emulsion formulations with minoxidil solution is illustrated in Figure 2.

Figure 2: Comparison of in Vitro Release Profiles of Minoxidil Loaded Submicron Emulsion Formulations (M1, M5, N2, N6 & H1) with Minoxidil Solution (1%w/v) over a Period of 6 hours

Ex Vivo Skin Permeation Study and Data Analysis

The submicron emulsion (M5) was further subjected to ex vivo skin permeation study using excised rat skin obtained from the pharmaclogy department. The results obtained were compared with those obtained by using minoxidil solution (1%w/v) and are depicted in Figure 3.

Figure 3: Comparison of Ex Vivo Permeation Profile of Submicron Emulsion Formulation (M5) and Minoxidil Solution (1%w/v)

The data obtained from ex vivo skin permeation study is summarized in Table 3.

Particle Size, PDI and Zeta Potential

The graphs obtained from particle size, PDI and zeta potential determination are given in Figure 4.

Figure 4: (A) The Particle Size and PDI Value of Optimized Submicron Emulsion Formulation (M5); (B) The Zeta Potential of Optimized Submicron Emulsion Formulation (M5)

Formulation of Minoxidil loaded submicron emulsion based topical gel

The optimized minoxidil loaded submicron emulsion formulation (M5) was converted into gel formulation by using carbopol 934 as gelling agent.

Characterization and optimization of submicron emulsion based gel formulations (M5\(_{1\%w/v}\), M5\(_{1.5\%w/v}\), M5\(_{2\%w/v}\))

Physical Evaluation, pH, Drug Content, Homogeneity and Grittiness

The gel formulations were visually evaluated to examine their physical appearance. The results are shown in Table 4.

Ex Vivo Skin Permeation Study Using Excised Rat Skin & Data Analysis

The cumulative amount of minoxidil permeated from different submicron emulsion-based gel formulations (M5\(_{1\%w/v}\), M5\(_{1.5\%w/v}\), M5\(_{2\%w/v}\)) was found to be 64.28\(\pm\)0.15%, 56.29\(\pm\)0.18% and 51.86\(\pm\)0.18% respectively, which was higher in contrast to the plain gel (33.23 \(\pm\) 0.18). Figure 5 depicts the outcome of ex vivo permeation study.

Figure 5: Comparison of Ex Vivo Skin Permeation Profile of Different Minoxidil Loaded Submicron Emulsion Based Gels and Plain Minoxidil Gel

The Flux and Permeability Coefficient Values of Different Minoxidil Loaded Submicron Emulsion Based Gels and Plain Minoxidil Gel were also Measured and Reported in Table 5.

Drug Retention Study

The amount of drug retained (mg) and percent drug retained in skin layers were also measured and results are given in Figures 6(a) and Figure 6(b). The drug retention from submicron emulsion based gel formulation M5\(_{2\%w/v}\) (16.5 \(\pm\) 0.28%) was also significantly higher \((p\leq 0.05)\) as compared to the drug retention from plain gel (5 \(\pm\) 0.2%).

Figure 6: The Comparision of Amount of Drug Retained in Skin Layers (mg) (Left) and Percentage of Drug Retained in Skin Layers from Differentminoxidil Loaded Gels (Right)

Figure 6 represents the comparision of amount of drug retained in skin layers (mg) and percentage of drug retained in skin layers from differentminoxidil loaded gels.

Histopathological Study

The histopathological study of treated and saline treated skin samples to demonstrate the safety of the developed formulation was done as per the given procedure. The data collected from this study is shown in Figure 7 and 8.

Figure 7: Saline Treated Excised Rat Skin (magnification 10X) (Left); Excised Rat Skin Sample Treated with Submicron Emulsion Based Gel

Viscosity Determination & in Vitro Bioadhesion Study

The optimized submicron emulsion based gel formulation M52%w/v exhibited viscosity of 3854 \(\pm\) 0.027 mpas and bioadhesion of 104 minutes \(\pm\) 11.24. The bioadhesion time possesed by minoxidil loaded submicron emulsion based gel was significantly higher \((p\leq 0.05)\) in contrast to plain minoxidil gel (23 minutes \(\pm\) 6.73). Antioxidant activity of optimized submicron emulsion based gel

DPPH Method

The results of DPPH assay are given in Figure 8.

Figure 8: Antioxidant Activity Activity of Ascorbic Acid and Drug Loaded Submicron Emulsion (Left); Log Dose Response of Ascorbic Acid and Drug Loaded Submicron Emulsion Based Gel (Right)

Stability Study

The results of stability study are given in Table 6.

The gel formulation (M5\(_{2\%w/v}\)) after 3 months storage is given in Figure 9.

Figure 9: Optimized Minoxidil Loaded Gel Formulation after Storage Period of 3 Months at \(4^{\circ}\)

Solubility studies & miscibility assessment

The solubility determination of the drug in the oil, surfactant, and cosurfactant is considered the most important criterion in the formulation of submicron emulsion. Among all the oils, the maximum solubility of minoxidil was found in clove oil (51.83 \(\pm\) 0.52 mg/ml) (Table [t1]). Thus, clove oil was chosen to formulate submicron emulsion as it would reduce the incidence of alopecia from oxidative stress at dermal papillae cells due to its antioxidant property [50].

On screening surfactants and cosurfactants (Table [t1]), it was observed that the drug exhibited maximum solubility in Tween 20 (5.82 \(\pm\) 0.4 mg/ml) and Transcutol P (11 \(\pm\) 0.3 mg/ml), which were chosen as surfactant and co-surfactant phase respectively. The right blend of surfactants leads to a stable submicron emulsion upon dilution with water [51].

Construction of Pseudo ternary phase diagrams for formulation selection

Pseudo ternary phase diagrams were constructed for each S\(_{\text{mix}}\) ratio (1:0, 1:1, 1:2, 2:1, 3:1 & 4:1) by the aqueous titration method to determine the extent of the submicron emulsion region (Figure 1). In S\(_{\text{mix}}\) ratio 1:0, the submicron emulsion region was found to be much less in contrast to S\(_{\text{mix}}\) ratio 1:1, indicating that the surfactant alone could not solubilize the oil completely. When the co-surfactant (Transcutol P) was increased from 1:1 to 1:2, the submicron emulsion area was found to be decreased. This is due to the fact that further enhancement in the concentration of cosurfactant did not result in lower interfacial tension. In the case of 2:1 surfactant-cosurfactant ratio, the area was comparatively higher than previous ratios but lower than that found in the case of 3:1. The largest submicron emulsion area was found in the S\(_{\text{mix}}\) ratio 4:1, which might be due to the complete solubilization of oil phase, reduced interfacial tension and formation of flexible film surrounding the oil droplets of the dispersed phase. Therefore, combinations for developing placebo formulations were selected from a S\(_{\text{mix}}\) ratio of 4:1 and subjected to physical stability testing. The drug was loaded into the stable placebo submicron emulsion formulations.

Formulation development and thermodynamic stability studies

The placebo submicron emulsions which were found to be stable after physical stability testing were further chosen for loading the drug in a suitable dose (1%w/v). The prepared minoxidil loaded submicron emulsion were again checked for stability.

Characterization and optimization of submicron emulsion

pH

The pH of all submicron emulsion formulations were found to be in the range of 4.5 to 6.6, similar to the pH of skin and hair [52]. This pH value indicated that the prepared formulations were safe for topical application.

In Vitro Drug Release Study & Determination of Release Kinetics

This study measured the drug release from prepared minoxidil containing submicron emulsions and minoxidil solution (1%w/v) over a period of 6 hours ( Figure 2). The results showed that minoxidil solution (1% w/v) showed higher release (98.01\(\pm\)0.19%) compared to the prepared submicron emulsion formulations. It was due to the presence of the drug in its free form [53]. However, a sudden decrease in drug release from the minoxidil solution was observed after 2 hours.

On the other hand, submicron emulsions showed sustained and controlled release of the drug. Among the submicron emulsion formulations, M5 exhibited an in vitro release of 95.08\(\pm\) 0.36%, which was found to be comparatively higher than M1 (88.08\(\pm\) 0.36%), N2 (79.91\(\pm\) 0.26%), N6 (68.83 \(\pm\)0.29%) and H1 (67.66 \(\pm\) 0.22) respectively. Therefore, M5 was considered an optimized formulation and selected for further evaluation. Kinetic analysis of the in vitro release profile of optimized submicron emulsion (M5) was done by fitting the data obtained in various kinetic models. The correlation coefficient values obtained for zero order, first order, Higuchi model, and Peppas-Korsemeyer were found to be 0.8659, 0.9648, 0.683, and 0.6415, respectively. Since the correlation coefficient (R2) for the first order model was near 1, it was concluded that the release of minoxidil from submicron emulsion formulation (M5) followed first-order kinetics.

Ex Vivo Skin Permeation Study and Data Analysis

The data obtained from this study revealed that minoxidil loaded submicron emulsion (M5) exhibited maximum drug permeation (79.36 \(\pm\) 0.18%) in comparison with minoxidil solution (49.79 \(\pm\) 0.15%). The high drug permeation from submicron emulsion was due to smaller globule size, which resulted in direct and higher drug permeation into skin. Another reason of higher drug permeation from submicron emulsion was due to the presence of Transcutol P as co-surfactant, which is reported to be a potent permeation enhancer [54]. The flux and the permeability coefficient values were found to be remarkably higher for submicron emulsion in comparison with drug solution.

Particle Size, PDI and Zeta Potential

The average droplet size of submicron emulsion (M5) was observed to be 181.3 nm with a PDI value of 0.239 (Figure 4), which is less than 1 respectively. The polydispersity value less than 1 indicates monodisperse and homogeneous nature of the formulation. These results confirmed the ability of formulation to achieve the desired transfollicular and transcellular penetration in skin [49,12]. The formulation M5 exhibited the zeta potential of -8.80 mV.

Formulation of Minoxidil Loaded Submicron Emulsion Based Topical Gel

The optimized minoxidil loaded submicron emulsion formulation (M5) was converted into gel formulation by using different concentrations of carbopol 934 as gelling agent.

Characterization and optimization of submicron emulsion based gel formulations(M5\(_{1\%w/v}\), M5\(_{1.5\%w/v}\), M5\(_{2\%w/v}\))

Physical Evaluation, pH, Drug Content, Homogeneity and Grittiness

The gel formulations were visually evaluated to examine their physical appearance (Table [t3]) and found to be stable and homogeneous.

Ex Vivo Skin Permeation Study Using Excised Rat Skin & Data Analysis

The results of this study revealed that high drug permeation was observed in the case of submicron emulsion-based gel formulations in contrast to the plain gel, which might be due to the presence of translator P, which has a permeation-enhancing activity [53].

The drug permeation was reduced as the quantity of gelling agent enhanced from 1% to 2%. For effective topical delivery, the drug permeated from the submicron emulsion-based gel must not come in contact with blood circulation. Due to this, the formulation with the least permeation (M5\(_{2\%w/v}\))was selected as the optimized submicron emulsion-based gel formulation. The flux and permeability coefficient values of different minoxidil-loaded submicron emulsion gels and plain minoxidil gel were significantly higher than the plain drug-loaded gels due to surfactants.

Drug Retention Study

The drug retention from submicron emulsion-based gel formulation M5\(_{2\%w/v}\) was also significantly higher \((p\leq 0.05)\) as compared to the drug retention from plain gel, which was due to the submicron size and also due to the presence of transcutol P, which is an efficient penetration enhancer. The presence of sebum in the follicular compartment also results in the accumulation of the drugs in lipid environments or skin appendages, thus enhancing the retention of hydrophobic drugs in the skin appendages and follicles [55].

Histopathological Study

From the observations of te histopathological studies conducted, it was concluded that both the skin samples (Treated or untreated) showed no evidence of inflammation, indicating that the prepared formulation was not irritating to the rat skin. Thus, the minoxidil loaded submicron emulsion based gel was within the limit of skin tolerance and safe to use for topical applications[56].

Viscosity Determination & in Vitro Bioadhesion Study

The bioadhesion time possesed by minoxidil loaded submicron emulsion based gel was significantly higher \((p\leq 0.05)\) in contrast to plain minoxidil gel, leading to higher residence time and better therapeutic activity.

Antioxidant activity of optimized submicron emulsion based gel

DPPH Method

The significant antioxidant activity showed by minoxidil loaded gel was due to the presence of clove oil, which is a potent anti-oxidant [57].

Stability Study

The results of stability study revealed that there was no significant change in the value of pH, viscosity and drug content of optimized minoxidil loaded submicron emulsion based gel formulation after the storage period of 3 months.

The efficacy of the dosage form would be dependent on the storage conditions. In this regards the patients have to be counselled.

The minoxidil-loaded submicron emulsion-based topical gel was formulated. It showed a controlled drug release, optimum particle size with a PDI value of less than 1, significantly higher permeation, and drug retention with prolonged stability. It also showed a potent anti-oxidant activity, which would, in turn, result in decreased incidence of alopecia. Thus, the stabilized submicron emulsion-based topical gel of minoxidil would be a beneficial topical nanostrategy in contrast to other topical dosage formulations introduced for alopecia therapy.

The authors acknowledge the facilities provided by Khalsa College of Pharmacy, Amritsar, Punjab.

This research paper received no external funding.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

1. Norwood, O. T. (1975). Male-pattern baldness: Classification and incidence. Southern Medical Journal, 68(11), 1359-1370.
2. Hillmer, A. M., Hanneken, S., Ritzmann, S., Becker, T., Freudenberg, J., Brockschmidt, F. F., ... & N\"othen, M. M. (2005). Genetic variation in the human androgen receptor gene is the major determinant of common early-onset androgenetic alopecia. The American Journal of Human Genetics, 77(1), 140-148.
3. Farrant, P., Messenger, A. G., & McKillop, J. (2012). British Association of Dermatologists-Guidelines for the management of Alopecia. British Journal of Dermatology, 166(916), 1-3.
4. Ito, T. (2013). Recent advances in the pathogenesis of autoimmune hair loss disease alopecia areata. Clinical and Developmental Immunology, 2013, 1-6.
5. Shapiro, J. (2013). Current treatment of alopecia areata. Journal of Investigative Dermatology Symposium Proceedings, 16}, S42-S44.
6. Semalty, M., Semalty, A., Joshi, G. P., & Rawat, M. S. M. (2011). Hair growth and rejuvenation: An overview. Journal of Dermatological Treatment, 22, 123-132.
7. Alsantali, A. (2011). Alopecia areata: A new treatment plan. Clinical, Cosmetic and Investigational Dermatology, 4, 107-115.
8. Kanti, V., Messenger, A., Dobos, G., Reygagne, P., Finner, A., Blumeyer, A., Trakatelli, M., Tosti, A., Marmol, V., Piraccini, B. M., Nast, A., & Blume-Peytavi, U. (2018). Evidence-based (S3) guideline for the treatment of androgenetic alopecia in women and in men - short version. Journal of the European Academy of Dermatology and Venereology, 32(1), 11-22.
9. Santos, A. C., Silva, M. P., Guerra, C., Costla, D., Peixoto, D., Pereira, I., Pita, I., Ribeiro, A. J., & Veiga, F. (2020). Topical Minoxidil-Loaded nanotechnology Strategies for Alopecia. Cosmetics, 7(21), 1-25.
10. Silchenko, S., Nessah, N., Li, J., Huang, Y., & Hidalgo, I. J. (2019). Ex vivo dissolution absorption system (IDAS2): Use for the prediction of food viscosity effects on drug dissolution and absorption form oral solid dosage forms. Reference Module in Biomedical Sciences, 143, 105164.
11. Uprit, S., Sahu, R. K., Roy, A., & Pare, A. (2013). Preparation and characterization of minoxidil loaded nanostructured lipid carrier gel for effective treatment of alopecia. Saudi Pharmaceutical Journal, 21, 379-385.
12. Gomes, M. J., Martins, S., Ferreira, D., Segundo, M. A., & Reis, S. (2014). Lipid nanoparticles for topical and transdermal application for alopecia treatment: development, physicochemical characterization, and in vitro release and penetration studies. International Journal of Nanomedicine, 9, 1231-1242.
13. Padois, K., Centieni, C., Bertholle, V., Bardel, C., Pirot, F., & Falson, F. (2011). Solid lipid Nanoparticles versus Commercial solutions for dermal delivery of Minoxidil. International Journal of Pharmaceutics, 416, 300-304.
14. Matos, B. N., Reis, T. A., Gratieri, T., & Gelfuso, G. M. (2015). Chitosan Nanoparticles for targeting and sustaining Minoxidil sulpate delivery into hair follicles. International Journal of Biological Macromolecules, 75, 225-229.
15. Khalil, R., Hashem, F., Zaki, H., & El-Arini, S. (2014). Polymeric Nanoparticles as potential carriers for topical delivery of Colchicine: Development and in vitro characterization. International Journal of Pharmaceutical Sciences and Research, 5(5), 1746-1756.
16. Roque, L. V., Dias, I. S., Cruz, N., Rebelo, A., Roberto, A., Rijo, P., & Reis, C. P. (2017). Design of finasteride-loaded nanoparticles for potential treatment of Alopecia. Skin Pharmacology and Physiology, 30(4), 197-204.
17. Abd. E., Benson, H. A. E., Roberts, M. S., & Grice, J. E. (2018). Follicular penetration of caffeine from topically applied nanoemulsion formulations containing penetration enhancers in vitro human skin studies. Skin Pharmacology and Physiology, 31(5), 252-260.
18. Cardoso, S. A., & Baraddass, T. N. (2020). Developing formulations for drug follicular targeting: Submicron-sized emulsions loaded with minoxidil and clove oil. Journal of Drug Delivery Science and Technology, 59, 1-10.
19. Kumar, R., Singh, B., Bakshi, G., & Katare, O. P. (2007). Development of liposomal systems of finasteride for topical applications: Design, characterization, and in vitro evaluation. Pharmaceutical Development and Technology, 12(6), 591-601.
20. Jain, B., Singh, B., Katare, O. P., & Vyas, S. P. (2010). Development and characterization of minoxidil-loaded liposomal system for delivery to pilosebaceous units. Journal of Liposome Research, 20(2), 105-114.
21. Ahmed, O. A. A., & Rizg, W. Y. (2018). Finasteride nano-transferosomal gel formula for management of androgenetic alopecia: Ex vivo investigational approach. Drug Design, Development and Therapy, 12, 2259-2265.
22. Ramezani, V., Honarvar, M., Seyedabadi, M., Karimollah, A., Ranjbar, A. M., & Hashemi, M. (2018). Formulation and optimization of transferosomes containing minoxidil and caffeine. Journal of Drug Delivery Science and Technology, 44, 129-135.
23. Mali, N., Darandale, S., & Vavia, P. (2013). Niosomes as a vesicular carrier for topical administration of minoxidil: Formulation and in vitro assessment. Drug Delivery and Translational Research, 3, 587-592.
24. Khatereh, Z., Payam, K., Abbas, P., & Mehdi, R. (2017). Preparation and physicochemical characterization of topical niosomal formulation of minoxidil and tretinoin. Global Journal of Pharmaceutical Sciences, 3(2), 555-606.
25. Wilson, V., Siram, K., Rajendran, S., & Sankar, V. (2018). Development and evaluation of finasteride loaded ethosomes for targeting to the pilosebaceous units. Artif Cells Nanomed Biotechnol, 46(8), 1892-1901.
26. Pravalika, G., Chandhana, P., Chiranjitha, I., & Dhurke, R. (2020). Minoxidil ethosomes for treatment of alopecia. International Journal of Recent Scientific Research, 11(1), 37112-37117.
27. Kwon, T.K., & Kim, J.C. (2010). In-vitro skin permeation of monoolein nanoparticles containing $\beta$-cyclodextrins/minoxidil complex. International Journal of Pharmaceutical Sciences, 392, 268-273.
28. Boca, S., Berce, C., Jurj, A., Petrushev, B., Pop, L., Gafencu, G.A., Selicean, S., Moisoiu, V., Temian, D., Micu, W.T., Astilean, S., Braicu, C., Tomuleasa, C., & Berindan-Neagoe, I. (2017). Ruxolitinib-conjugated gold nanoparticles for topical administration: An alternative for treating alopecia? Medical Hypotheses, 109, 42-45.
29. Nagai, N., Iwai, Y., Sakamoto, A., Otake, H., Oaku, Y., Abe, A., & Nagahama, T. (2019). Drug delivery systems based on minoxidil nanoparticle promotes hair growth in C57BL/6 Mice. Drug Delivery, 14, 7921-7931.
30. Madheswaran, T., Baskaran, R., Thapa, R.K., Rhyu, J.Y., Choi H.Y., Kim, J.O., Yong,C.S., & Yoo, B.K. (2013). Design and in vitro evaluation of finasteride-loaded liquid crystalline nanoparticles for topical delivery. AAPS PharmSciTech, 14(1), 45-52.
31. Mundada, V., Patel, M., & Sawant, K. (2016). Submicron emulsions and their applications in oral delivery. Critical Reviews\(^{\text{TM}}\) in Therapeutic Drug Carrier Systems, 33(3), 265-308.
32. Benson, A.E., Abd, E., Roberts, M.S., & Grice, J.E. (2018). Minoxidil Skin delivery from submicron-sized emulsion formulations containing eucalyptol or oleic acid: enhanced diffusivity and follicular targeting. Pharmaceutics, 10(19), 1-12.
33. Singh, R.P. (2014). Emulgel: A recent approach for topical drug delivery system. Asian Journal of Pharmaceutical Research and Development, 2(2), 13-15.
34. Srivastava, M., Kohli, K., & Ali, M. (2016). Formulation development of novel in situ nanoemulgel of ketoprofen for the treatment of periodontitis. Drug delivery, 23(1), 154-166.
35. Sunitha, S., Jitendra, W., Sujatha, D., & Kumar, M.S. (2013). Design & Evaluation of topical gel-thickened microemulsions for topical delivery of MXD. Iranian Journal of Pharmaceutical Science, 9(4), 1-14.
36. Rai, V.K., Yadav, N.P., Sinha, P., Mishra, N., Luqman, S., & Dwivedi, H. (2014). Development of cellulosic polymer based gel of novel ternary mixture of miconazole nitrate for buccal delivery. Carbohydrate Polymers, 103, 126-133.
37. Dhawan, B., Aggarwal, G., & Harikumar, S.C. (2014). Enhanced transdermal permeability of piroxicam through novel nanoemulgels formulation. International Journal of Pharmaceutical Investigation, 4(2), 65-76.
38. Sood, S., Jain, K., & Gowthamarajan, K. (2014). Optimization of curcumin nanoemulsion for intranasal delivery using design of experiment and its toxicity assessment. Colloids and Surfaces B: Biointerfaces, 113, 330-337.
39. Choudhury, S., Dasgupta, S., Patel, D. K., Ramani, Y. R., Ghosh, S. K., & Mazumder, B. (2013). Nanoemulsion as a carrier for topical delivery of aceclofenac. In Advanced Nanomaterials and Nanotechnology: Proceedings of the 2nd International Conference on Advanced Nanomaterials and Nanotechnology, Dec 8-10, 2011, Guwahati, India (pp. 1-19). Springer Berlin Heidelberg.
40. Teichmann, A., Jacobi, U., Ossadnik, M., Richter, H., Koch, S., Sterry, W., & Lademann, J. (2005). Differential stripping: Determination of the amount of topically applied substances penetrated into the hair follicles. Journal of Investigative Dermatology, 125, 264-269.
41. Sharma, S., Sahni, J.K., & Ali, J. (2015). Effect of high-pressure homogenization on formulation of drug loaded nanoemulsion of rutin - pharmacodynamic and antioxidant studies. Drug Delivery, 22(4), 541-551.
42. Indora, N., & Kaushik, D. (2015). Design, development and evaluation of ethosomal gel of fluconazole for topical fungal infection. International Journal of Engineering Science Invention Research & Development, 1, 280-306.
43. Talele, S., Nikam, P., Ghosh, B., Deore, C., & Jaybhave, A.A. (2017). Nanogel as topical promising drug delivery for Diclofenac sodium. Indian Journal of Pharmaceutical Education and Research, 51(4), 580-587.
44. Upadhyay, D. K., Sharma, A., Kaur, N., Gupta, G. D., Narang, R. K., & Rai, V. K. (2021). Nanoemulgel for efficient topical delivery of finasteride against androgenic alopecia. Journal of Pharmaceutical Innovation, 16, 735-746.
45. Zheng, Y., Ouyang, W., Wei, Y., Shahid, F.S., Chao, S.H., & Yan, H. (2016). Effect of Carbopol 934 proportion on nanoemulsion gel for topical and transdermal drug delivery: a skin permeation study. International Journal of Nanomedicine, 2016 5971-5987.
46. Biradar, S.V., Dhumal,R.S., & Paradkar, A. (2009). Rheological investigation of self emulsification process. Journal of Pharmaceutical Science, 12(1), 17-31.
47. Zubairu, Y., Negi, L.M., Talegoankar, S., & Iqbal, Z. (2015). Design and development of novel bioadhesive niosomal formulation for the transcorneal delivery of anti-infective agent: in vitro and in vivo investigations. Asian Journal of Pharmaceutical Sciences, 10, 322-330.
48. Jufri, M., & Dhyaksa, M.L. (2021). Formulation and physical stability of nanoemulsion gel (nanoemulgel) containing Belimbing Wuluh (Averrhoa Bilimbi L.,) ethanolic extract. World Journal of Pharmaceutical and Life Sciences, 7(3), 11-19.
49. Shakeel, F., Shafiq, S., Haq, N., Alanazi, F.K., & Alsarra, I.A. (2012). Nanoemulsions as potential vehicles for transdermal and dermal delivery of hydrophobic compounds: an overview. Expert Opinion on Drug Delivery, 9(8), 953-974.
50. Gulcin, I. (2012). Antioxidant activity of eugenol: A structure-activity relationship study. Journal of Medicinal Food, 14(9), 975-985.
51. Kommuru, T.R., Gurley, B., Khan, M.A., & Reddy, I.K. (2001). Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: Formulation development and bioavailability assessment. International Journal of Pharmaceutics, 212, 233-246.
52. Pichler, J., Adriano, A.R., Cecato, P.M., De Almeida, A.M., & Gavazzoni, M.F. (2014). The shampoo pH can affect the hair: Myth or Reality. International Journal of Trichology, 6(3), 95-99.
53. Sarkar, N., Bose, S., & Banerjee, D. (2018). Effects of PCL, PEG and PLGA polymers on curcumin release from calcium phosphate matrix for in vitro and in vivo bone regeneration. Today Chemistry, 8, 110-120.
54. Baboota, S., Shakeel, F., Ahuja, A., Ali, J., & Shafiq S. (2007). Design, development and evaluation of novel submicron emulsion formulations for transdermal potential of celecoxib. Acta Pharmaceutica, 57(3), 315-332.
55. Lademann, J., Knorr, F., Richter, H., Blume-Peytavi, U., Vogt, U., Antoniou, C., Sterry, W., & Patzelt, A. (2008). Hair follicles - an efficient storage and penetration pathway for topically applied substances. Skin Pharmacology and Physiology, 21, 150-155.
56. Mao, L., Yang, J., Xu, D., Yuan, F., & Gao, Y. (2019). Effects of homogenization models and emulsifiers on the physicochemical properties of beta-carotene nanoemulsions. Journal of Dispersion Science and Technology, 31, 986-993.
57. Prie, B.E., Iosif, L., Tivig, I., Stoian, I., & Giurcaneanu, C. (2016). Oxidative stress in androgenetic alopecia. Journal of Medicine and Life, 9(1), 79-83.