Betamethasone Dipropionate Nanostructured Lipid Carriers Enriched Jojoba Oil Gel for Treatment of Atopic Dermatitis

doi:10.21203/rs.3.rs-4812373/v1

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Research Article

Betamethasone Dipropionate Nanostructured Lipid Carriers Enriched Jojoba Oil Gel for Treatment of Atopic Dermatitis

https://doi.org/10.21203/rs.3.rs-4812373/v1

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Aim- To develop and evaluate topical gel containing Betamethasone Dipropionate-loaded nanostructured lipid carriers for the management of Atopic dermatitis.

Background- Atopic dermatitis is a chronic, recurrent skin inflammatory condition that affects both children and adults. Betamethasone Dipropionate, a commonly used topical corticosteroid, has limitations such as low bioavailability, inadequate penetration, and potential skin irritation.

Objective- To evaluate the nanostructured lipid carriers gel by comparison with its marketed formulation and to improve epidermal targeting and minimize the side effects associated with the conventional formulation of betamethasone dipropionate.

Method- A hot homogenization process was used to create the NLCs, and their particle size, zeta potential, entrapment efficiency, and in-vitro release were all measured. Next, the NLCs were mixed with 0.1% jojoba oil to create a gel.

Results- The optimized formulation of Betamethasone dipropionate nanostructured lipid carriers had a particle size of 164 nm, PDI 0.188, zeta potential of -11.6 mv, entrapment efficiency of 92.65%, and an in-vitro release of 90.62% after 12 hours. 0.1% jojoba oil was added to the improved recipe to increase the moisturizing effect and integrated into the gel. NLC mixed in Jojoba oil gel demonstrated much higher spreadability, occlusive action, and regulated release of 91.28% after 12 hours when compared to the marketed gel. The ex-vivo investigation on porcine ear skin demonstrated enhanced penetration and retention of Betamethasone dipropionate in skin layers. There was no sign of skin irritation, indicating that the topical application was safe.

Conclusion- The results of the cytotoxicity study (MTT assay) suggested NLC dispersion has cell proliferation potency on HaCaT cells and was non-toxic. Results indicated that betamethasone dipropionate's therapeutic efficacy might be enhanced by NLC formulation.

Betamethasone dipropionate

nanostructured lipid carriers

Jojoba oil

topical drug delivery

atopic dermatitis

The aim of Atopic dermatitis (AD) management is to rectify epidermal barrier impairments, mitigate inflammation, and forestall exacerbations. While topical therapies may confer advantages for AD, they are susceptible to drawbacks stemming from extended utilization. Conventional treatments are linked to local adverse effects and low patient compliance. Given the epidermal barrier dysfunction, long-term use may result in systemic absorption, leading to potential toxicity. (Abimbola Oninla et al., 2021).

Nanostructured lipid carriers (NLC) are lipid formulations that consist of a blend of liquid and solid lipids. Essential components of NLC formulations include solid lipids such as triglycerides, fatty acids, and waxes, as well as liquid lipids like medium-chain triglycerides, oleic acid, and isopropyl myristate.(Beloqui et al., 2016). Nanostructured lipid carriers (NLCs) are small, small molecules that interact with the stratum corneum, reducing corneocyte packing and aiding drug penetration into the skin. They create a thin lipid film, reducing water loss and boosting skin moisture, helping alleviate atopic dermatitis symptoms. NLCs enhance drug solubility, facilitate drug diffusion, and minimize drug leakage. Their unique structure allows controlled release, improved targeting precision, and drug entrapment(Li & Ge, 2012).

Topical corticosteroids (TCS) are used to treat acute atopic dermatitis flare-ups, typically twice daily, to alleviate inflammation and itching. They are recommended until significant improvement in inflammatory lesions is observed, often lasting several weeks. However, TCS can cause adverse effects like skin rashes, stretch marks, hair growth, and acne(Adcock, 2004). Betamethasone dipropionate, a lipophilic medication, is ideal for Nanostructured Lipid Carriers (NLCs) due to its anti-proliferative, immunosuppressive, and anti-inflammatory properties, making it an effective treatment for various skin inflammatory conditions(Hanna et al., 2019; Tung et al., 2019). Jojoba oil is often described as a liquid wax rather than a traditional oil or fat due to its composition, which consists of approximately 98% pure waxes primarily in the form of wax esters. It also contains small quantities of free fatty acids, alcohols, hydrocarbons, sterols, and vitamins. (“5 Final Report on the Safety Assessment of Jojoba Oil and Jojoba Wax,” 1992) Research on jojoba oil and hydrogenated jojoba oil in pharmaceutical skincare products has shown promising results in enhancing skin hydration and reducing transepidermal water loss, suggesting potential benefits for alleviating skin conditions like eczema, according to in vivo studies.(Gad et al., 2021). Numerous studies have demonstrated that jojoba oil's anti-inflammatory and emollient properties contribute to a significant enhancement in therapeutic efficacy and a reduction in skin irritation and dryness. (Shahin et al., 2011).

This study aims to develop and evaluate nanostructured lipid carriers (NLCs) containing betamethasone dipropionate and jojoba oil gel as a potential treatment for atopic dermatitis. The NLCs were prepared using hot melt emulsification and analyzed for parameters like particle size, polydispersity index, entrapment efficiency, zeta potential, and structural integrity. The gel formulations were evaluated for rheological attributes, drug release kinetics, and skin irritation potential.

2.1 Materials

Betamethasone dipropionate was gifted from Avik Pharmaceuticals Ltd, Mumbai. Gattefosse India Pvt Ltd, Mumbai gifted Compritol 888 ATO, Caproyl 90 and Precirol ATO 5. Glyceryl monostearate, Isopropyl myristate and Cetyl palmitate were obtained from Arihant Innochem Pvt Ltd, Mumbai. Tween 80, span 80, Tween 40 and Tween 60 were obtained from Mohini Organics Pvt Ltd, Mumbai. Capmul MCM, Capmul MCM, Capmul PG 8 NF, Campul PG 12, Captex 200P and Acconon AKG-6 were gifted by Abitec Corporation, Mumbai. Poloxamer 188 and Cremophor RH 40 was obtained from BASF Pvt Ltd, Mumbai. Carbopol® Ultrez 10 NF, Carbopol® 974 and Carbopol® 971 was obtained from Lubrizol life sciences. Jojoba oil was purchased from Nature’s Tattva. Potassium dihydrogen phosphate, disodium hydrogen orthophosphate, olive oil and oleic acid was obtained from S.D fines chemicals, Ltd.

2.2 Methods

2.2.1 Preformulation studies

Preliminary screening

For this research, the lipids employed included Compritol 888 ATO, Precirol ATO 5, glyceryl monostearate and Cetyl palmitate. The process involved dissolving escalating quantities of drugs in different molten solid lipids to establish the maximum solubility of the active ingredient in each lipid(Nagaich et al, 2019).

Various liquid lipids and surfactants to determine the solubility of Betamethasone dipropionate. The solubility was determined by adding Betamethasone dipropionate to the mixture, vortexed, and centrifuged for 72 hours. A UV spectrophotometer was used to examine the supernatant after it had been diluted with methanol. The study also examined the solubility of Betamethasone dipropionate in the lipids and surfactants.

Physical compatibility of solid lipid and liquid lipid

Solid lipids and liquid lipids which showed maximum solubility for betamethasone dipropionate were mixed and melted in 1:1 ratio in glass vials to determine their physical compatibility. A smear was formed on the glass slide by streaking a drop of the melted lipid and observed to determine the uniformity and phase separation of the smear which indicates the compatibility of both lipids.

Selection of ratio for solid lipid-liquid lipid

To determine the maximum solubility of Betamethasone dipropionate in various ratios of solid lipid and liquid lipid, the desired dose of drug was added in different ratios of Solid lipid: Liquid lipid (5:5, 6:4, 7:3, and 8:2, 9:1). The mixture that showed the maximum solubility of drug was used for further formulation and development.

Preliminary study for the preparation of NLCs

NLCs were produced using hot melt emulsification and sonication. The mixture of liquid and solid lipids, as well as the medication, was heated to a temperature of 10ºC over the solid lipid melting point. Simultaneously, the surfactant was dissolved in water to create the aqueous phase. The two stages were heated to the same temperature. After incorporating the aqueous phase into the lipid phase, emulsification was performed with continuous stirring at 70–80ºC for 30 minutes. The formulation procedure was then completed with homogenization utilizing probe sonication(Mahant et al., 2020).

2.2.2 Experimental design

Central Composite Design (CCD) was utilized to optimize the formulation of NLCs loaded with betamethasone dipropionate. CCD, a commonly employed response surface methodology, integrates factorial or fractional factorial designs, central, and axial points. The CCD setup was generated through Design Expert software. Response surface plots were developed to demonstrate the impact of key variables. The significance and interactions between variables were assessed using predicted plots, fit summaries, model statistics, and ANOVA. After that, the regression model was used to create contour plots and three-dimensional surfaces for a detailed examination of the interactions between independent variables(Tung et al., 2019).

2.2.3 Characterization of optimized NLC dispersions

The study used Zetasizer equipment to measure particle size and polydispersity index of Nanostructured Lipid Carriers containing Betamethasone dipropionate. The results provided insights into particle dispersion and entrapment efficiency. The structure of the NLC was investigated using a JEOL Model-JEM-2100 Transmission Electron Microscope (TEM), with negative staining using phosphotungstic acid. The NLC specimen was thinned and incubated for 10 minutes.

Fourier-transform infrared spectroscopy

The FTIR spectra of the NLC dispersion were examined and compared with a mixture of Betamethasone dipropionate, Compritol 888 ATO, Caproyl 90, and Tween 80 to confirm the encapsulation of Betamethasone dipropionate within the NLC structure.

2.2.4 Preparation of NLC-based gel

Carbopol® Ultrez 10 NF, Carbopol® 974, and Carbopol® 971 were selected for formulating the NLC gel. The chosen NLC was integrated into a gel based on Carbopol. The gel formulation was optimized by adjusting the polymer concentration, resulting in gels with 1%, 0.75%, and 0.5% gelling agent content The polymer was hydrated in purified water for 6 hours before incorporating oil and surfactant phases. The remaining water was used to prepare the NLC dispersion. Triethanolamine (TEA) was used to neutralize the Carbopol® polymer, and jojoba oil (0.1%) was added as a functional excipient. A comparison was made between Betamethasone dipropionate-NLC gel, Betamethasone dipropionate-NLC jojoba oil gel, and a commercially available gel.

2.2.5 Evaluation of NLC-based gel

Appearance

The prepared formulation was inspected for its colour, homogeneity and consistency by visual inspection

pH measurement

The pH of the formulation was determined by using a digital pH meter at constant temperature.

Spreadability

A one-centimeter diameter glass slide was layered with approximately 50 mg of gel, and subsequently topped with a second glass slide of matching size. A standard 50 g weight was set on the upper slide and left in place for 120 seconds. Following this, the spread of the gel within the 1 cm circle was measured, and the spreading coefficient was computed accordingly.

Spreadability coefficient = Area occupied after spreading the gel / Weight placed

Viscosity

The viscosity of different gel formulations was evaluated using a DVII Brookfield viscometer. The measurements were performed three times at ambient temperature, using the 94 spindle no.(Kakkar et.al. 2018).

Assay

The optimized gel, which included 10 mg of the drug, was precisely weighed and moved into a 10 ml volumetric flask. It was dissolved in a solution of Phosphate buffer 5.5 and ethanol (1:1). The drug was extracted and dissolving in buffer after 20 minutes of sonication. The solution was diluted, and drug content was determined using spectrophotometry, and the concentration was calculated using linear regression analysis.

Occlusive effect

The smaller size of NLC forms a thin film on the skin, limiting transepidermal water loss (TEWL) through the stratum corneum. This enhances skin moisture and expands inter and intra-corneocyte gaps, hence increasing permeability. The occlusive properties of Betamethasone dipropionate-NLC gel, Betamethasone dipropionate NLC-jojoba oil gel, and a commercial gel were evaluated using an in-vitro occlusion test. The water loss percentage was computed, as well as the occlusion factor(Kakkar et al., 2018).

Occlusive factor (F) = A – B / A × 100

Where A: water loss without sample and B: water loss with sample

In-vitro drug release studies

The diffusion analysis was conducted on the optimized formulations to evaluate drug release via a PVDF membrane. This experiment was carried out using a Franz diffusion cell with a 22 ml receptor compartment split by a PVDF membrane. The receptor compartment was filled with a 1:1 solution of ethanol and Phosphate Buffer 5.5. The cell was constantly agitated at 100 rpm to maintain a temperature of 37°C ± 0.5°C.One gram of the formulation was placed inside each donor compartment, which was then sealed. Samples were taken at certain intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), filtered, and UV spectrophotometry was used to determine the amount of drugs present. To maintain sink conditions consistent, new media was put to the receptor compartment after each sampling.

Ex- vivo permeation and retention studies

Permeation study

The optimized formulations underwent a diffusion study to assess drug release through a PVDF membrane. The study was conducted in a Franz diffusion cell with a 22 ml receptor compartment and a PVDF membrane. Samples were collected at specific intervals, filtered, and analyzed for drug content using UV spectrophotometry. Fresh medium was added to maintain sink conditions, and the percentage of cumulative drug release was graphed against time.

Retention study

The treated viable epidermis and dermis skin pieces were finely diced and immersed in methanol to ensure complete drug extraction. Following six hours of sonication, materials were collected, centrifuged, and filtered using a 0.2 µm filter. The concentration of betamethasone dipropionate in the supernatant was determined using a standard analytical method.

2.2.6 Determination of skin irritation potential by Hen’s egg test-chorioallantoic membrane (HET-CAM)

The HET-CAM method uses fertilized eggs' chorioallantoic membrane (CAM) to predict skin irritability. It's ideal for toxicity assessments, as the CAM reacts to harmful substances. Betamethasone dipropionate-loaded NLC formulations were tested using this method, following the ICCVAM-Recommended Test Method Protocol. Fertilized White Leghorn chicken eggs were used, and irritation effects were observed for 5 minutes(Chew & Maibach, 2006). The time taken and severity of injuries post-application of each sample were documented, and an irritation score (IS) was assigned and calculated using the following equation:

𝐼𝑆 = (301−𝑡𝐻) ∗5300 + (301−𝑡𝐿) ∗7300 + (301−𝑡𝐶) ∗9300

Where tH, tL and tC are time (in seconds) required for the occurrence of hemolysis, lysis and coagulation, respectively. The experimentation was performed in triplicate.

2.2.7 Cell viability study by MTT cytotoxicity assay

A cell viability study for atopic dermatitis was performed using the MTT assay on the HACAT - Human Skin keratinocyte cell line (Rapalli et al., 2020). This colorimetric technique evaluates cell proliferation and cytotoxicity by measuring the conversion of yellow MTT dye into insoluble formazan crystals. The assay procedure involves seeding cell suspensions, incubating, dissolving formazan crystals, and measuring absorbance at 570 nm after gentle agitation for complete dissolution. This process offers valuable information on cellular viability and the potential cytotoxic effects of substances tested. The IC50 value was calculated using the linear regression equation, Y = Mx + C.

Here, Y = 50, M and C values were derived from the viability graph.

$$\:\mathbf{\%}\:\mathbf{C}\mathbf{e}\mathbf{l}\mathbf{l}\:\mathbf{V}\mathbf{i}\mathbf{a}\mathbf{b}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{y}=\frac{\mathbf{M}\mathbf{e}\mathbf{a}\mathbf{n}\:\mathbf{A}\mathbf{b}\mathbf{s}\mathbf{o}\mathbf{r}\mathbf{b}\mathbf{a}\mathbf{n}\mathbf{c}\mathbf{e}\:\mathbf{o}\mathbf{f}\:\mathbf{S}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}-\mathbf{B}\mathbf{l}\mathbf{a}\mathbf{n}\mathbf{k}}{\mathbf{M}\mathbf{e}\mathbf{a}\mathbf{n}\:\mathbf{a}\mathbf{b}\mathbf{s}\mathbf{o}\mathbf{r}\mathbf{b}\mathbf{a}\mathbf{n}\mathbf{c}\mathbf{e}\:\mathbf{o}\mathbf{f}\:\mathbf{U}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{e}\mathbf{a}\mathbf{t}\mathbf{e}\mathbf{d}-\mathbf{B}\mathbf{l}\mathbf{a}\mathbf{n}\mathbf{k}}\mathbf{*}\:10$$

2.2.8 Stability studies of optimized nanostructured lipid carrier based gel formulation as per ICH Q1 A (R2) guidelines

Stress tests were conducted by subjecting the refined formulation to diverse thermal conditions. Each formulation was preserved in airtight glass containers under varying temperature and humidity conditions, per ICH Q1A (R2) guidelines: 5ºC ± 3ºC, 25ºC ± 2ºC/ 60% RH ± 5% RH, and 40ºC ± 2ºC/ 75% RH ± 5% RH, over 3 months. Following the stability studies, the samples were inspected for any physical alterations (like clarity or phase separation), drug concentration, and pH levels.

3.1 Preformulation studies

Preliminary screening of solid lipids, liquid lipids and surfactants

The study focuses on optimizing drug solubility within lipids for maintaining drug solubilization in non-latex lipid (NLC) formulations. Betamethasone dipropionate showed the highest solubility in Compritol 888 ATO and GMS, indicating the need for further experimentation. Caproyl 90 was chosen as the liquid lipid component due to its hydrophobic interaction with the lipid phase. Poloxamer 188 and tween 80 were chosen for initial trial batches.

Physical compatibility of solid lipid and liquid lipid

0.5 grams of both Compritol 888 ATO and Caproyl 90 were combined in glass vials and heated above the melting point of Compritol 888 ATO. A smear was then created on a glass slide. It was noted that there was no visible phase separation between Compritol 888 ATO and Caproyl 90, demonstrating their physical compatibility Similar observations were made with Glyceryl monostearate and Caproyl 90, indicating their compatibility as well.

Selection of solid lipid-liquid lipid ratio

Solubility assessments of Betamethasone dipropionate were conducted with various combinations of solid and liquid lipids. It was noted that Betamethasone dipropionate exhibited solubility exceeding 100 mg/g in glyceryl monostearate, Compritol 888 ATO, and Caproyl 90. Consequently, these specific solid and liquid lipids were utilized in the specified ratio for the formulation of nanostructured lipid carriers.

Preliminary trials for the preparation of NLCs

The study developed NLCs using a hot melt emulsification method and probe-sonication. To attain the necessary features, trial batches were generated using various formulations and parameters. The important elements influencing formulation and stabilization were identified through initial improved batches. To optimize, a central composite design was adopted. The dependent variables were particle size, polydispersity index, and entrapment efficiency, whereas the independent variables were the concentrations of liquid and solid lipids.

(Table 1).

Table 1

Central Composite Design generated by Design Expert software with measured responses
Batch No	Solid lipid %	Liquid lipid %	PS (nm)	PDI	EE (%)
1	0.7	1.3	206.4	0.315	91.62
2	0.8	1.4	248.9	0.450	89.17
3	0.8	1.2	164.4	0.188	92.65
4	0.7	1.3	206.4	0.315	91.62
5	0.6	1.2	227.3	0.322	88.5
6	0.84	1.3	212.8	0.322	90.5
7	0.55	1.3	209.3	0.383	89.89
8	0.7	1.44	229.3	0.368	91.62
9	0.7	1.3	206.4	0.315	91.62
10	0.7	1.3	206.4	0.315	91.62
11	0.7	1.15	198.1	0.269	90.52
12	0.6	1.4	195.9	0.327	92.29
13	0.7	1.3	206.4	0.315	91.62

The particle size (PS) measurements exhibited a range from 164.4 nm to 248.9 nm, while the polydispersity index (PDI) varied between 0.188 and 0.450. The entrapment efficiency (% EE) showed variability from 88.5–92.65%. Notably, a notably high % EE of 92.65% was attained in the Betamethasone dipropionate loaded NLC formulation with 0.8% solid lipid and 1.2% liquid lipid in batch F3, which demonstrated a PS of 164.4 nm and a PDI of 0.188.

Contour plots and surface response curves

Figure 1. Contour plot A) Contour plot of Particle size, B) 3D surface plot of Particle size, C) Contour plot of PDI, D) 3D surface plot of PDI, E) Contour plot of EE and F) 3D surface plot of EE

Table 2

Analysis of variance
Source		P value	F value
Analysis of variance for particle size (PS)
Model		< 0.0001	109.40		Significant
A- Solid lipid		0.5764
B- Liquid lipid		< 0.0001
AB		< 0.0001
A²		0.2168
B²		0.03162
Model summary
R-sq					0.9874
Analysis of variance for polydispersity index (PDI)
Model		< 0.0001	41.82		Significant
A- Solid lipid		0.0151
B- Liquid lipid		< 0.0001
AB		< 0.0001
A²		0.0151
B²		0.9556
Model summary
R-sq					0.9676
Analysis of variance for entrapment efficiency (EE)
Model	< 0.0001			125.71		Significant
A- Solid lipid	0.0053
B- Liquid lipid	0.0058
AB	< 0.0001
A²	< 0.0001
B²	0.0039
Model summary
R-sq						0.9890

Interpretation:

The CCD analysis revealed a statistically significant model with a linear relationship observed for particle size. Compritol ATO 888 positively influenced particle size, while an increase in Caproyl 90 concentration led to a corresponding increase in particle size. The P-value from DOE was highly significant (< 0.0001), with an r2 value of 0.9874. The graph from Design-Expert software illustrated that increasing the concentrations of Compritol ATO 888 and Caproyl 90 led to a rise in PDI. The P-value from DOE was highly significant (< 0.0001), with an r2 value of 0.9676. The graph generated from Design-Expert software indicated that both Compritol ATO 888 and Caproyl 90 positively influenced %EE. Increasing the concentration of liquid lipid resulted in enhanced drug solubility, leading to improved loading efficiency of the nanoparticles. The P-value obtained from DOE was highly significant (< 0.0001), with an r2 value of 0.9890.

Table 3

Confirmation batch by DOE
	Predicted Value	Actual value
Compritol ATO 888 (%)	0.8	-
Caproyl 90 (%)	1.2	-
Particle size (nm)	169.0	164.4
PDI	0.201	0.188
Entrapment Efficiency (%)	92.46	92.65

3.3 Characterization of optimized NLC dispersion

Morphological characterization by transmission electron microscopy

The images were analyzed using Image J software. From the results, the average diameter of the nanostructured lipid carrier was found to be 171.41.

Figure 2. Morphology of prepared NLC

Fourier transform spectroscopy

It was clear that the major peaks of Betamethasone dipropionate were not visible in the FTIR spectrum, suggesting that the drug was encapsulated within the nanostructured lipid carriers.

In vitro drug release studies

T The study showed that betamethasone dipropionate, released from a nanostructured lipid carrier dispersion, was released in a controlled manner, with 90.62% of the drug being released after 12 hours. This controlled release of the drug from the NLC ensures that therapeutic concentrations of betamethasone dipropionate are maintained at the target site for an extended period, which implies a single dosing frequency and reduced side effects associated with the drug.

Figure 3. In vitro drug release of NLC gel

3.4 Preparation and evaluation of Betamethasone dipropionate loaded NLC gel

The optimized NLC formulation was integrated into Carbopol® Ultrez 10 NF gel, with jojoba oil added for moisturization. The resulting formulations were clear and homogeneous, with pH levels aligning with skin pH. The gels had lower viscosity compared to the marketed gel, and BD-Jojoba oil NLC was more spreadable. The occlusive effect of BD-Jojoba oil NLC was higher than the gel and marketed gel, suggesting reduced water evaporation and potential skin dryness alleviation. In vitro release studies showed Betamethasone dipropionate release from both BD-NLC gel and jojoba oil was 89.32% and 91.28%, respectively, after 12 hours, compared to the marketed gel's 93.73% release in 5 hours. Ex vivo permeation studies showed enhanced permeation through skin layers over an extended period, with lower flux values indicating controlled release effects. The amount of drug retained in skin layers was higher in Betamethasone dipropionate loaded NLC jojoba gel, suggesting targeted localized efficacy with minimal systemic loss.

Table 4

Characterization of NLC gels
Parameters	BD-NLC gel	BD-NLC jojoba oil gel	Marketed gel
Appearance	Clear	Clear	Clear
Spreadability	635 gm.cm/4.5secs	675 gm.cm/4secs	550 gm.cm/6secs
Viscosity (cp)	1681 ± 0.47	1682 ± 0.82	1882 ± 0.82
Drug Content (%)	93.50 ± 0.001	94.99 ± 0.004	96.99 ± 0.008
Occlusive effect (%)	55.82 ± 1.02	58.67 ± 1.02	23.16 ± 1.10
pH	5.57 ± 0.02	5.6 ± 0.10	5.56 ± 0.01

3.5 Determination of skin irritation potential by Hen’s egg test-chorioallantoic membrane (het-cam)

The Het-Cam assay was employed to evaluate the skin irritation potential. The results indicated that the NLC dispersion, Betamethasone dipropionate NLC gel, and Betamethasone dipropionate NLC jojoba oil gel were non-irritating and non-sensitizing in nature, contrasting the marketed gel, which exhibited a slight irritation potential. This implies that encapsulating these drugs in NLC could serve as an efficient method to decrease the skin irritation associated with the marketed gel, making it a safe formulation for topical use.

Figure 4. Evaluation A) Percent Drug retained from BD-NLC jojoba oil gel, B) Percent Cell viability of pure drug, and C) Percent Cell viability of NLC dispersion

3.6 Cell viability study

In HaCaT cell lines, which comprise 95% of skin epidermal cells, the study examined the cell viability of betamethasone dipropionate solution and NLC dispersion. The keratinocyte cells exhibited minimal impact, with both the free Betamethasone dipropionate solution and the NLC dispersion showing over 50% cell viability at a high dose of 100 µg/mL. These findings indicate that the excipients in the formulations do not exert a toxic effect on keratinocyte cells.

3.7 Stability study as per ICH Q1A (R2) guidelines

The stability of the Betamethasone dipropionate NLC jojoba oil gel was assessed over a three-month period. The stability data for the formulation is detailed in Table 16. The NLC gel maintained a clear appearance and a pH within the range of 5.6 to 5.7. The drug content assay of the Betamethasone dipropionate loaded NLC-jojoba oil gel exhibited no notable changes, indicating the long-term stability of the developed product.

Betamethasone dipropionate, a potent corticosteroid, is known to have drawbacks such as limited epidermal targeting, skin atrophy, irritation, and systemic side effects with prolonged use. Therefore, a NLC-based formulation was developed for the topical delivery of Betamethasone dipropionate to mitigate these side effects. The study results demonstrated that the Betamethasone dipropionate loaded nanostructured lipid carriers jojoba oil enriched gel exhibited controlled drug release, facilitating epidermal targeting without adverse effects in comparison to that of the marketed gel.

Ex-vivo drug release studies indicated enhanced retention and permeation of Betamethasone dipropionate in the epidermal layers with the formulated gel. The inclusion of jojoba oil in the formulation improved occlusivity, potentially aiding in regulating transpirational water loss in the skin, thereby moisturizing dry atopic skin and reducing excessive flaking of epidermal cells. The absence of skin irritation in the Het-Cam assay confirmed its safety for topical use. Additionally, MTT cytotoxicity assays revealed the non-toxic nature of the NLC formulation.

Stability assessments conducted following ICH Q1 A R2 guidelines demonstrated that the formulations remained stable with no significant changes in physicochemical parameters under various storage conditions. These findings suggest that Betamethasone dipropionate NLC jojoba oil gel could offer a promising therapeutic approach for treating Atopic dermatitis.

NLC- Nanostructured lipid carriers

AD- Atopic dermatitis

BD- Betamethasone dipropionate

GMS- Glyceryl monostearate

C188- Compritol ATO 888

C90- Caproyl 90

P188- Poloxamer 188

T80- Tween 80

Acknowledgments

The authors are thankful for Betamethasone dipropionate gifted from Avik Pharmaceuticals Ltd, Mumbai. Jojoba oil was purchased from Nature’s Tattva. We acknowledge that DST-FIST during the studies.

Declaration of competing interest

No conflicts of interest are declared by the authors .

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Authors Contribution

All authors contributed to the study's conception and design. Conceptualized and guided by Prabha Singh. Material preparation, data collection and analysis were performed by Shruti Ghosalkar. Nipun Panchal wrote the draft of the manuscript. All authors reviewed and approved the final manuscript.

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Betamethasone Dipropionate Nanostructured Lipid Carriers Enriched Jojoba Oil Gel for Treatment of Atopic Dermatitis

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Abstract

Figures

1. Introduction

2. Materials and Method

2.1 Materials

2.2 Methods

2.2.1 Preformulation studies

2.2.2 Experimental design

2.2.3 Characterization of optimized NLC dispersions

2.2.4 Preparation of NLC-based gel

2.2.5 Evaluation of NLC-based gel

2.2.6 Determination of skin irritation potential by Hen’s egg test-chorioallantoic membrane (HET-CAM)

2.2.7 Cell viability study by MTT cytotoxicity assay

3. Results and Discussion

3.1 Preformulation studies

3.3 Characterization of optimized NLC dispersion

3.4 Preparation and evaluation of Betamethasone dipropionate loaded NLC gel

3.5 Determination of skin irritation potential by Hen’s egg test-chorioallantoic membrane (het-cam)

3.6 Cell viability study

3.7 Stability study as per ICH Q1A (R2) guidelines

4. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Version 1