Introduction

In modern medicine, it is crucial to find ways to maximize the absorption of drugs by the skin. Transdermal drug administration has emerged as a promising field of study, rivaling that of oral medication1. An increasingly popular method of administering drugs systemically, transdermal administration involves applying a small but significant dose of the medicine directly to the skin2. Because biopharmaceuticals comprise a constantly expanding share of novel treatments and are often administered intravenously, there is a pressing need for more effective methods of drug administration3. The creation of new medical treatments with current pharmaceuticals has been made possible thanks to the discovery of new medicinal agents and the corresponding innovation in drug delivery technology. Transdermal patches have evolved into a well-established technique over the past two decades, offering the prospect of novel chemical delivery in a noninvasive, practical manner via the skin4.

Compared to more traditional administration methods, the transdermal route may be preferable because it reduces the likelihood of unwanted side effects, allows for the use of drugs with a shorter half-life, enhances physiological and pharmacological response, eliminates fluctuations in drug levels, and is more convenient for the patient5. However, the limited rate of penetration into the outermost layer of skin is a key issue in transdermal medication administration6.

Psoriasis is an inflammatory disease caused by the immune system, namely, T cells and inflammatory cytokines7. Psoriatic lesions are characterized by rapid keratinocyte proliferation and maturation that prevents cells from fully differentiating. Researchers have observed that vitamin D analogs can bring keratinocyte hyperproliferation back to normal8,9,10. The function of vitamin D analogs as immune system regulators has come into focus in recent years. Vitamin D, which acts as a hormone in the body, regulates calcium and phosphorus levels in the blood11. Calcium and phosphorus metabolism, cell development and differentiation, and immunological function are all governed by vitamin D3’s action on the vitamin D receptor (VDR)12. Vitamin D3 suppresses cytotoxic T-cell and natural killer cell activity in addition to reducing IL-2 and IL-6 production and interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA transcription13,14,15. Vitamin D3 regulates epidermal development and suppresses the keratinocyte proliferation in culture16. Vitamin D responsive genes have a VDR that attaches to them and turns on their transcription17,18. Calcitriol has been proven in experiments to modulate the immune system in a way that is unique to monocytes, macrophages, T cells and dendritic cells19. The role of vitamin D3 as an immune-modulator, especially in autoimmune disorders has been evidenced in many studies. Reports suggest high doses of vitamin D3 up to 50,000 IU daily can be safely used in autoimmune conditions20.

The ethosomes is a slightly modified version of the conventional drug carrier liposome. Phospholipids, a high concentration of alcohol, and water make up the lipid vesicles known as ethosomes. In the previous ethosomes researcher used the synthetic polymers along with phospholipids with 40 °C, but we have used room temperature with phospholipids and significant drug loading was achieved. Ethosomes can range in size from a few nano to microns21. Ethosomes have a much greater transdermal flux and may easily pass through many layers of skin22. The medicine would be protected from immune reaction and other removal mechanisms, the release rate could be controlled over a prolonged period of time, and the drug’s concentration could be maintained for longer periods of time if ethosomes vesicles were used23. Ethanol, water and phospholipids (phosphatidyl choline) make up the bulk of ethosomal carriers. It was discovered that ethosomes may pass through the skin and improve the transport of different compounds to the dermal layers or the bloodstream24.

We have developed an ethosomal gel for transdermal administration of vitamin D3. Water cannot dissolve vitamin D3 since it is a lipophilic molecule. Vitamin D3, despite its high lipophilicity, may permeate the skin at therapeutic levels with the help of appropriate penetration enhancers25,26. It is possible that this is not always the case with lipophilic substances27. The epidermis, dermis, and hypodermis are the three primary layers of the skin’s distinctive structure. The stratum corneum (SC) is the uppermost layer of the epidermis. This dead layer has no metabolic activity; hence, diffusion across it is the limiting step. Percutaneous absorption is mostly mediated by intercellular lipids in the SC28. After the administration of the formulation to the skin, the drug substance must be firstly absorbed. To increase the absorption of drugs into the deeper layer of skin, different approaches are considered on the basis of increasing permeability and water solubility, which are rate-limiting steps for absorption. To examine how well vitamin D3 can pass across a synthetic artificial membrane, we employed Strat-M. The Strat-M artificial membrane is designed to look and feel like human skin. It may stand in for real skin during permeation tests on humans and animals29. To treat psoriasis, we initially developed a gel for topical use by transforming a vitamin D3-loaded ethosomal solution.

The purpose of this study is to create vitamin D3-loaded ethosomal based gels and assess these by utilizing a variety of instruments in light of the previously provided information. For the first time, we have created ethosomes gel for psoriasis. We don’t use any of the chemicals that cause negative effects even after prolonged use were used by us. Additionally, a diffusion investigation was conducted to compare the resulting vitamin D3-loaded ethosomal-based gel systems with the physical combination in order to assess any potential improvements in these activities. Still there are no any commercial products available for topical delivery so we used vitamin D3 raw material suspension to compare with our formulation. To ensure the stability and effectiveness of our medication for the treatment of psoriasis, we carried out characterizations.

Materials and methods

Chemicals

Soya lecithin, propylene glycol (PG), ethanol (99%), cholesterol, carbopol 934, triethanol amine (TEA), and PEG 6000 were purchased from Daejung Chemicals & Metals in Siheung-si, South Korea. For scientific purposes, 99% pure vitamin D3 was provided by Saffron Pharmaceutical of Faisalabad, Pakistan. Distilled water was utilized in this study. To examine vitamin D3 absorbance in the skin, we used a synthetic membrane (Strat-M), which was purchased from Merck Millipore Burlington, MA, USA.

Synthesis of vitamin D3 loaded ethosomes

For vitamin D3 drug preparation, a cold process was used with a little bit of modification. This approach was proposed by Touitou et al. for preparing ethosomes. Phospholipids (1–8 mL), ethanol (20 mL), propylene glycol (10 mL), cholesterol (0.01 g), and water (100 mL make-up) were used in the experiment and are shown in Table 1. Ethosomes size and loading was observed by using Phospholipids concentration (1–8 mL) and on this basis we have selected the optimized formulation.

At room temperature, a mixture of soya lecithin, propylene glycol, and cholesterol was stirred (1500 rpm) for 30 min. In phase two, 50 mg of vitamin D3 was dissolved in ethanol using a stirrer (1500 rpm) at room temperature in a glass container. The contents of the first beaker were slowly added to the phase two mixture in the center of the covered vessel, where they were agitated at 1000 rpm for 15 min. The resulting combination was sonicated with a probe sonicator (Hoverlabs)(Model No-HV-PRO-650) for 15 min. Sonication or extrusion can be used to reduce the vesicle size of an ethosomal formulation to an optimal level. The finished product is then refrigerated. Figure 1 depicts the process of spontaneous ethosomes formation, while Table 1 displays the concentration of the formulation used.

Preparation of vitamin D3 Ethosomes Gel

Carbopol gel (1, 1.2 and 1.5% w/w) was used to integrate the most effective ethosomal vesicle suspension (ED3-7). On the basis of maximal drug entrapment, we used the ED3-7 formulation. For 30 min, distilled water was heated to 100 °C while the correct quantity of carbopol 934 powders was gently added with continuous stirring (1500 rpm). Then, we gradually added triethanolamine (TEA) and phosphate buffer to adjust the pH (5.5). The gel base was then mixed with 100 mL of the vitamin D3 ethosomes formulation ED3-7. During the formulation process, q.s. Water was added together with the other components and mixed well with constant stirring (1500 rpm). Table 2 shows the results of a similar procedure employing 1%, 1.2%, and 1.5% Carbopol to create a gel containing vitamin D3 ethosomes. The final pH level was controlled at 5.5.

Table 1 Composition of ethosomal formulations.
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Table 2 Composition of the gel formulation.
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Determination of the drug entrapment efficiency of ethosomes

We tested the efficiency of drug entrapment using the solvent mixture (ethanol and freshly prepared pH 7.4 phosphate buffer USP, 30:70) (United States pharmacopeia). Sonication for 5 min using the same solvent was used to extract vitamin D3 from the ED3-7 suspension. After sonication, the supernatant was collected and dissolved in the mobile phase (phosphate buffer and acetonitrile 30:70) (pH 5.0). HPLC was set to a maximum wavelength of 265 nm to test the solution containing vitamin D3 into ethosomes30,31. The entrapment effectiveness of the prepared ethosomes for vitamin D3 was calculated using Eq. (1)32.

$${\text{Entrapment}}\;{\text{efficiency~}}\;\% =\frac{{~{\text{drug}}\;~{\text{contents}}\;~{\text{in}}~\;{\text{ethomoes}}~}}{{{\text{drug}}\;~{\text{content}}\;~{\text{within}}\;~{\text{solution}}}} \times 100$$
(1)

Measurement of particle size, potential, and Polydispersity Index

The zeta potential and sizer of ethosomal vesicles were measured using a computerized inspection system called the Malvern Nano-ZetaSizer (Nano-ZS, Malvern Instruments, Worcestershire, UK). This parameter is crucial because it affects both vesicular properties such as stability and skin-vesicle interactions. Based on considerations of the zeta sizer and drug entrapment potential, we chose an optimum formulation. ED3-7 was the most effective optimized formulation when measuring the zeta sizer, potential, and polydispersity index.

Scanning electron microscopy of ethosomes

Scanning electron microscopy (SEM) was used to analyze the vitamin D3-loaded ethosomal suspension for surface morphology (roundness, smoothness, and ethosomal aggregate formation). For SEM imaging, we chose the formulation that worked best for entrapping the drugs. For SEM, the most optimized formulation was ED3-7.

Fourier transform infrared spectroscopy of ethosomes

It is a strategy for understanding how the structural groups of interest are related to the groups of pure components. Vitamin D3, soya lecithin, propylene glycol, ethanol, and ethosomal formulations were tested to determine the functional groups and interactions. The value range of 4000 to 500 cm− 1 for spectrum scans was achieved using a Bruker FTIR (Tensor 27 Series-Bruker Corporation Germany) instrument and attenuated-total-reflection (ATR) technology33,34,35.

TGA analysis of Ethosomes

Thermogravimetric analyzers from the Q5000 series (West Sussex, United Kingdom) were used for the thermal analysis. Vitamin D3, lipid (soya lecithin), propylene glycol (PG), and ethosomes were subjected to thermogravimetric analysis (TGA). Therefore, TGA was used to monitor any mass change or reduction of the sample. A synthetic atmosphere, or air with 22% oxygen and 78% nitrogen, was used for the TGA, and the testing temperature ranged from 25 to 300 °C36.

Differential scanning calorimetry of ethosomes

The glass transition temperature (Tg) was measured using Differential Scanning Calorimetry (DSC) on a formulation of vitamin D3, soy lecithin, and ethosomes. A TA USA Q2000 (DCS) was utilized. Under 20 mL/min of nitrogen gas purging, we weighed and packed 100 mg of each sample into an aluminum pan for analysis. The heating rate was set at 20 °C/min, and the temperature range was 25–300 °C37,38.

Release study of ethosomes gel formulation

The dissolution of the drug was studied using USP Dissolution Apparatus II at a pH of 5.5, which is the skin pH. The ethosomes suspension was put into dialysis membrane pouches, hung in 900 mL of the dissolution liquid (buffer pH 5.5), and constantly spun at 50 rpm to keep the drug concentration constant within the dissolving medium. A pure vitamin D3 water suspension underwent the same treatment. The temperature of the dissolving media was adjusted to 37 °C. According to the plan, we took samples at 1, 2, 3, 4, 6, 8, 10, and 12 h. New material was continuously substituted for the measured volume. Vitamin D3 release at 265 nm was measured by high-performance liquid chromatography39. The following formula was used to determine the percentage release (2):

$$\% ~\;{\text{Release}}=\frac{{{\text{Absorbance}}~\;{\text{of}}\;~{\text{the}}~\;{\text{sample}}~\;{\text{solution~}}}}{{{\text{Absorbance}}\;~{\text{of}}\;~{\text{the}}\;{\text{~standard~}}\;{\text{solution}}}} \times 100$$
(2)

Rheological study of ethosomal gel

Viscosity, the resistance of a fluid to flow, is measured in centipoise and is one of the variables studied in rheology. Using spindle No. 7 and a Brookfield viscometer Model DV2T we determined the room-temperature viscosity of ethosomal gel at several rotational speeds. Using a rheometer, we measured the concentration-dependent viscosity (1%, 1.2%, 1.5%).

Spreadability and pH of ethosomal gel

Spreadability (g cm/s) is expressed in terms of the time taken in seconds by two slides to slip off from the ethosomal gel placed between them under a certain load. The standardized weight tied on the upper plate was 20 g, and the length of the glass slide was 6 cm. The less time taken for the separation of the two slides, the better the spreadability. The spreadability was calculated by using the following equation:

$${\text{S}}={\text{M}}/{\text{T}}$$

where S is the spreadability in g/s, M is the mass in grams and T is the time in seconds.

For pH, one gram of ethosomal gel (pH 7.0–7.5) was dissolved in thirty milliliters of distilled water. The ethosomal gel pH was tested using a digital pH meter by placing the meter’s probe in the sample.

Physical stability and microbial assessment of ethosomes

The prepared ethosomal gel was kept in accelerated stability studies (40 ± 2 °C/75 ± 5% RH) for 6 months. A physicochemical analysis including description, odor, pH, viscosity, and microbial assessments was performed in the initial, 3rd, and 6th months.

Hemolytic investigations of ethosomes

We collected human blood (5 mL) and centrifuged it (3000 rpm for 5 min) to separate red blood cells (RBCs). The RBCs were washed with 4% phosphate buffer. Then, the ethosomal suspension was mixed at different concentrations, and the mixture was placed in blood tubes containing ethylenediaminetetraacetic (EDTA) acid and incubated at 37 ± 1 °C for 4 h and 12 h. This mixture was then centrifuged at 1500 rpm for 5 min in a tube containing EDTA. After removing the supernatant, the absorbance of the supernatant was measured at 541 nm. In this study, Triton X-100 was used as a positive control, while phosphate-buffered saline was used as a negative control. Hemolysis in red blood cells was measured using Eq. (3)40. All experiments were conducted in compliance with the World Medical Association’s Declaration of Helsinki and were authorized by the Rashid Latif College of Pharmacy’s (RLCP) Ethical Review Board (IRB No. RLCP/EP/142/2023).

$$\% ~{\text{Hemolysis}}=\frac{{{\text{ABS}}\;~{\text{sample}} - {\text{ABS}}~\;{\text{blank}}}}{{{\text{ABS}}~\;{\text{positive}} - {\text{ABS}}\;~{\text{blank}}}} \times 100\%$$
(3)

In vitro permeability study of ethosomal gel

In this we have checked the drug diffusion through the skin and we have used artificial skin for this. With an effective permeation area of 4.25 cm2 and a receptor cell capacity of 25 mL, a transdermal Franz diffusion sampling device was used to study ethosome skin penetration ex vivo. The receiver vehicle (phosphate buffer saline, pH 5.5) was kept at 37 °C and swirled at 50 rpm with a magnetic stirrer. To determine how well vitamin D3-loaded ethosomal gel can pass across the synthetic artificial membrane Strat-M (molecular weight from 180) we conducted penetrance tests with ethsomsal gel equivalent ro 1.25 mg vitamin D3 and compared with vitamin D3 raw material suspension equivalent to 1.25 mg of vitamin D3. The Strat-M artificial membrane is designed to look and feel like human skin. The stratum corneum side of the artificial skin was placed toward the donor compartment and attached to a receptor compartment. The donor compartment received one gram of weighed ethosomes. Over the course of 12 h, several samples were taken from the receptor compartment via the sampling port and evaluated for drug content. After each sample was taken, an equivalent volume of new buffer was added to the receptor phase.

Skin irritation test of ethosomes

To achieve this goal, rabbits where taken from Rashid Lateef khan University animal House under ethical approval (RLCP/EP/142/2023). Notably, male rabbits were used, and 24 h before the mixture was applied, the rabbit’s abdominal skin was shaved. Rabbits hair were removed and pasted 0.1 g of a gel (ED3-7) which was containing 1.25 mg of vitamin D3 dispersed evenly over an area of 4.25 cm2 of their skin. After 24 h, the skin’s surface was examined for any outward signs of alteration, such as erythema (redness). No erythema = 0, light erythema (barely noticeable; light pink) = 1, moderate erythema (dark pink) = 2 and severe erythema (extreme redness) = 3.

Analytical statistics

One-way analysis of variance and Tukey’s test were among the statistical procedures run in Graph-Pad Prism v.5 (https://www.graphpad.com/support/prism-5-updates). The mean and standard deviation were used to symbolize the data (SD). A P value of 0.05 was used as the cutoff for statistical significance41.

Results and discussion

Preparation and evaluation of vitamin D3-Loaded ethosomes

Effect of the vitamin D3/soya lecithin ratio on vitamin D3 entrapment efficiency

Ethosomes loaded with vitamin D3 were prepared using the cold technique, with varying concentrations of soya lecithin (Table 3). This is one of the simplest methods to prepare ethosomes on a lab scale and can be transferred to production scale up42. Due to the low hydrolysis rate in the medium, soya lecithin was chosen as the primary lipid component in this investigation.

Vitamin D3/soya lecithin ratios were varied from 1 mL to 8 mL, whereas the cholesterol/propylene glycol ratio was held constant during ethosome preparation. The drug entrapment efficiency was increased from 77.22 0.5% to 96.21 0.3% when the soya lecithin quantity was raised from 1 mL to 8 mL (P 0.05) Table 3. The entrapment effectiveness was decreased, and the suspension’s color was altered when the lipid content was more than 7 mL. Vitamin D3 is hydrophobic (practically insoluble in water) (soluble in lipids) and may be responsible for its easy absorption into the lipid bilayers of ethosomes, leading to the high encapsulation efficiency observed during formulation. We have repeated the experiment with scale up technology and found the trapping efficiency of vitamin D3 which was not affected. Drug loading was also measured and mentioned in Table 3 and found significant in ED3-7 formulation.

Table 3 Drug entrapment efficiency (%) of formulations.
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Determination of drug entrapment efficiency

To make a suspension of vitamin D3 ethosomes, the optimal formulation was taken and checked with HPLC30. Each formulation, ED3-1 through ED3-8, was tested using the HPLC analytical technique and Table 3 displays the findings about drug entrapment. Drug entrapment was shown to be significant in the ED3-7 formulation, in which we used 7 mL of soya lecithin. We have performed HPLC to determine the entrapment efficiency. Both ethosomes and concentrated solution was tested by HPLC and found results. We found that in the formulation which was rich with soya lecithin has maximum drug entrapment. ED3-7 formulation was found best for entrapment but after some concentration of lecithin drug entrapment was reduces.

Measurement of particle size and Polydispersity Index

A Malvern Nano-ZetaSizer (Nano-ZS, Malvern Instruments-UK) with a He-Ne laser at 633 nm and an avalanche photodiode detector was used for these studies. Measurements were taken in triplicate at room temperature, and the optimized vitamin D3 ethosomal formulation (ED3-7) was diluted ethanol. The average particle size of ethosmes varied, and Fig. 2 showed two peaks in size distributions. Peak one is on 148 and peak 2 is 657 nm and this same formulation showed high entrapment efficiency (96.25%± 0.3%). There is a crucial impact of particle size on drug absorption through the skin. The ethosome formulation had a PDI value of 0.770 ± 0.12 along with negatively charge − 14 ± 3. Particle size and polydispersity index (PDI) are critical factors for particle characterization because of their impact on safety, stability, effectiveness, and in vivo performance43. Values of the particle size dispersion index (PDI) for ethosomes below 0.8 suggest a very monodispersed formulation44. Ethosomes are considered to be homogeneous with phospholipid vesicles when their PDI is near 0.345. Ethosomal vesicles have a particle size much smaller than conventional liposomes and the presence of high ethanol contents makes their size independent of phospholipid concentration and due this its penetration into skin is very high46.

SEM

The ethosome suspension (ED3-7) underwent SEM examination to reveal its morphological features. Figure 3A also depicts the form and structure of pure vitamin D3 crystals. The SEM data (Fig. 3B) show that the formulations have a nearly spherical shape. There was no clear delineation between the wall and the vesicular outer membrane. The wall or the vesicular outer membrane was not definite. In most of the ethosome preparations, scientists used two types of lipids, and we used only one lipid phase. No leakage was observed in SEM images of our ethosomes, which indicated a more stable product. The ethosome wall and the vesicular outer membrane could not be clearly distinguished in the SEM pictures47. This finding raises the possibility of a well-integrated wall and outer membrane, which could point to a more uniform and continuous lipid bilayer.

FTIR

Figure 4 displays the FTIR spectra of vitamin D3, soya lecithin, propylene glycol, and ethosomes. A scanning range of 4000 to 500 cm− 1 was used to analyze all of the samples. Bands at 2972 and 2881 cm− 1 in the vitamin D3 spectrum are related to C–H stretching, while the band at 3213 cm− 1 is due to O–H hydrogen bonds. However, the band at 890 cm− 1 might be due to C=CH2 vibrations. There were two additional bands linked with the angular deformation of geminal dimethyl at 1650.23 and 1277.23 cm− 148,49.

In this FTIR analysis, the N(CH3)3 group bond in pure soya lecithin was detected at 969 cm− 1, the P–O bond at 821 cm− 1, and the P = O bond at 1231–1232 cm− 1. These chemical linkages indicated that soya lecithin’s molecular structure showed some typical standard bonds as the bond on phosphatidylcholine. Additionally, a signal-attributable peak due to C-H stretching of CH3 was detected at 2867.20 cm− 1.

The FTIR spectrum of propylene glycol is shown in Fig. 4. The C–H stretching of CH3 is represented by bands at 2859 and 2970 cm− 1. The region at approximately 1089 cm− 1 is also significant for propylene glycol, and ether is stretched at this point.

Figure 4 shows the FTIR spectra of vitamin D3 ethosomes. The presence of vitamin D3 in the ethosomal formulation is evidenced by the presence of distinctive absorption bands in the spectra at 1267.22 cm− 1 and 1670.23 cm− 1. The C–H stretching of CH3 was detected as a peak at 2907.12 cm− 1 in the spectra of pure soy lecithin. The FTIR spectrum of a vitamin D3-loaded ethosome shows that propylene glycol and soya lecithin peaks are still present with a slight shift. The peak intensities decreased, indicating that vitamin D3 was taken up by the ethosomes. This suggested a possibility of a slight interaction between vitamin D3 and the ethosomal contents, which is nonsignificant.

Thermogravimetric analysis (TGA)

Vitamin D3, soya lecithin, propylene glycol, and ethosomes were subjected to a thermal examination, the results of which are shown in Fig. 5. Vitamin D3 thermogravimetric analysis (TGA) is shown in Fig. 5 to have a melting point of 81.57 °C, corresponding to a 30% mass loss of the whole sample. Vitamin D3 degradation causes a 70% mass loss between 163.66 °C and 180.97 °C50.

Thermogravimetric analysis (TGA) of a lipid (soya lecithin) reveals a small loss of mass (5%). Water evaporation could account for a small amount of the loss. Only at temperatures over 90.97 °C did a second weight loss (30%) occur. This indicates that the soya lecithin sample has excellent resistance to high temperatures. Figure 5 shows, however, that at 260.41 °C, a massive weight loss of approximately 50% was observed. The results of De Alvarenga and colleagues are very comparable to these51. At 110 °C, propylene glycol lost 30% of its weight, and at 120 °C, it showed a loss of 70%.

The TGA of ethosomes is shown in Fig. 5 and includes the aforementioned components. This demonstrates that at high temperatures, mass was lost in three distinct phases. A 20% weight loss arises below 78.64 °C, which was due to vitamin D3 and indicated successful incorporation of vitamin D3 into ethsomes. Within a temperature range of 183.52 °C, a second mass loss of 40% was observed. At 250.55 °C, a drastic reduction of almost 80% was observed, indicating a total breakdown of the formulation. The results show that the vitamin D3 contained within the designed ethosomes is more stable but that it is ruptured at high temperatures due to the presence of other chemicals, including cholesterol and alcohol.

DSC Analysis

Vitamin D3, soya lecithin, propylene glycol, and ethosomes were subjected to differential scanning calorimetry (DSC) using a TA USA Q-2000 differential calorimeter. A temperature range of 30 °C to 300 °C was set, with a heating rate of 20 °C/min. The reactions of vitamin D3, soya lecithin, propylene glycol, and ethosomes to thermal stress are shown in Fig. 6. Figure 6 shows a DSC curve of vitamin D3, which has a prominent endothermic peak at 80 °C, which is due to its melting point. The vitamin D3 melting point has been proven to be within this range by a number of articles. Vitamin D3 degrades at a temperature of 180 °C, creating a second large endothermic peak.

Two endothermic peaks, one at 40.9 °C and another at 60.3%, were observed in differential scanning calorimetry (DSC) of lipids (soya lecithin). The melting of lipids and the consequent dehydration and removal of other volatile components account for the exothermic peak observed at 140.08 °C.

Figure 6 shows an endothermic peak for propylene glycol at 140.22 °C, indicating polymer breakdown.

Multiple peaks at various temperatures were observed when DSC was run on exosomes containing vitamin D3. DSC analysis of exosomes revealed a total of five peaks. We also found two endothermic peaks at 40.28 °C and 53.22 °C in soya lecithin. These thermographs represent the presence of soya lecithin, which is an integral ingredient of ethosomes. The existence of vitamin D3 and its melting point, evidenced by a third strong peak at 78.34 °C, were confirmed. At 134.28 °C, we observed another peak, which was identical to the peak seen in propylene glycol. Due to mixing of different polymers melting temperature of vitamin D3 change towards high because PG and lecithin are more stable then vitamin D3 pure. A broad and irregular endothermic peak was also observed at 178.88 °C, which was attributed to the breaking of bonding among the polymeric and lipid networks52.

Drug release study of ethosomal gel formulation

The release profile of vitamin D3 from the ethosomes was measured by conducting dissolution at pH 5.5 for 12 h at varied intervals. The absorbance of the samples was measured at a fixed wavelength (265 nm maximum) using a high-performance liquid chromatograph (HPLC) (Shimadzu, Germany). We checked the release profile of the best formulation, ED3-7, at a pH of 5.5. Figure 7 shows the medication release characteristics of ethosomes ED3-7 and a raw vitamin D3 suspension. Vitamin D3 was encapsulated into ethosomes, and its release was shown to be both rapid and sustained over the course of 12 h. No sufficient release pattern was observed in the raw vitamin D3 suspension during the course of the 12-hour investigation. In the current release study, we observed that vitamin D3 release from ethosomes was 30.43%  ± 3 in the 1st hour, which indicated burst release, and it is best to give a loading dose of vitamin D3 on the skin. After this time, the 12 h of controlled release continued, and the final hour produced 94.42% ± 3 of the desired amount of vitamin D3. In contrast, the limited solubility of raw vitamin D3 resulted in a total 2 releases of just 21.72% ± 0.56. Diffusion studies using the same formulations and observing the same trend for ethosomes were also performed; however, only a minor quantity of vitamin D3 from the raw vitamin D3 suspension was able to diffuse over the membrane.

The release of ethosomes ED3-7 showed a controlled release phase after an initial burst release, which did not precisely match zero-order kinetics. The entire release profile appears to be more complex, even though the early phase may meet zero-order kinetics. The sustained release phase of the ethosomal gel may exhibit first-order kinetics (R2 0.9592), in which the rate of release falls as the ethosomes’ vitamin D3 concentration gradually drops.

Table 4 The fitted equations of ethosomes gel (ED3-7).
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Rheological properties of ethosomal gels

As shown in Fig. 8, the constant shear flow curves of ethosomal gels are a function of concentration. When subjected to shear rates ranging from 0 to 1000 s − 1, the viscosities of ethosomal gels of varied concentrations exhibited the characteristics of shear-thinning pseudoplastic fluids. Table 4 shows that different carobopol % of the gel samples were evaluated for spreadability and found to be suitable. The spreadability behavior values of all ethosomal gel concentrations decreased continuously, and the steady shear viscosity of all % gels increased with concentration from 1 to 1.5%, showing the high dependence of viscosity on concentration (Table 5).

Table 5 Evaluation of ethosomal gel viscosity, spreadability, and pH.
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Spreadability and pH of ethosomal gels

The gel was examined and determined to be homogeneous and devoid of any air molecules. All % were determined to have pH values between 5.6 and 5.8. Spreadability testing showed that gels made from formulated ethosomes will be readily dispersed with a minimum shearing force. Ethosome gel containing vitamin D3 was shown to have a spreadability of 2.2 to 1.8 cm/5 min. We also measured the viscosity of the ethosomal gel and found from 26,200 ± 623 cPs to 29,100 ± 184 cPs. It was concluded that the % of polymeric content directly affected its viscosity.

Physical stability and microbial assessment

We tested the vitamin D3 ethosomal gel for stability and total aerobic microbial count (TAMC). This product is for skin use because it is mandatory to test the microbial load in the formulation, and it must be according to pharmacopeia. The vitamin D3 ethosomal gel was tested for stability by keeping it in a stability chamber for six months at 40 °C. Vitamin D3 ethosomal gel order, description, pH, and viscosity were all measured on day one, three months, and six months. The results showed that the vitamin D3 ethosomal gel had a pH of 5.5 to 5.4, the optimal range for skin care products. With a viscosity of 89–82 cpi at 25 °C, this formulation is also skin-friendly. Its homogeneity and clarity were evaluated and found to be adequate. When manufactured at a larger scale, vitamin D3 ethosomal gel must meet al.l of the standards set forth by USP 4353. The microbiological parameters of the vitamin D3 ethosomal gel were also checked. TAMCs, TYMCs, Staphylococcus, and Pseudomonas were examined in vitamin D3 ethosomal gel for up to six months. To prepare the plates, Sabouraud dextrose agar and nutrient agar were used, and the method was described in the publication by Ballal et al.54. The plates were incubated in Memmert incubators for 72 h with TAMCs and seven days with TYMCs at different temperatures (32 °C ± 2 and 22 °C ± 2).parens results shown in Table 6 are quite satisfactory until the 6th month.

Table 6 Stability results until 6 months with 40 °C
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Hemolysis assay

The ethosomal hemolysis assay was carried out because it is a quick and reliable method for determining whether a substance is compatible with human blood35,41,55,56. The hemolysis assay evaluates the erythrolysis and hemoglobin dissociation that occurs when blood is in contact with a suspension of ethosomes. Ethosome suspension weights in mg (50, 100, 150, 200, and 400 mg/mL) (0.05 mg/μL, 0.1 mg/μL, 0.15 mg/μL, 0.2 mg/μL and 0.4 mg/μL) were immersed in blood for 2 h and 12 h. The ethosome suspension was in mg, and blood was taken in mL for the final concentration. A spectrophotometer was then used to determine the blood compatibility. The negative control was PBS (not displayed), while the positive control was Triton-X (97.73% lysis). The results of a 2-hour blood compatibility test using dosages of 50 mg (2.83%), 100 mg (4.35%), 150 mg (5.35%), 200 mg (6.92%), and 400 mg (10.34%) were all within limits. After 12 h of the same treatment, blood compatibility data were collected for doses of 50 mg (4.16%), 100 mg (5.35%), 150 mg (8.68%), 200 mg (11.58%), and 400 mg (14.91%) (Fig. 9). All lysis percentages were less than 15%, and for 50 mg and 100 mg doses, they were less than 6%. Our findings suggest that ethosomes are not harmful to blood and can be used. This might be due to the high biocompatibility of vitamin D3 and the other components with blood because we have used all ingredients that are biocompatible according to FDA guidelines. Our findings imply that ethosomes have long-lasting hemostatic potential for use due to their adaptable mechanical, physical, and biological properties and can be used in the long term on skin.

In vitro permeability

The primary objective of the current study was to test the in vitro permeability of the ED3-7 formulation through a silica membrane at a pH of 5.5. In this study, we employed dry, clean receptor cells that were filled with an oxygen-free buffer. At 37 °C, the mixture was stirred continuously for 30 min with a magnetic stirrer. Para-film was used to seal the holes and prevent water loss through evaporation. A constant speed of 50 rpm was used to agitate the receptor compartment. Vitamin D3 permeation was evaluated utilizing a synthetic artificial membrane called Strat-M to quantify drug diffusion. The Strat-M artificial membrane is designed to look and feel like human skin. To conduct UV analysis at 295 nm, a 0.5 mL sample was drawn from the receptor cell with a glass syringe. At 1, 2, 3, 4, 6, 8, 10, and 12 h, 0.5 mL samples of the receptor phase were removed and replaced with a new buffer solution in the cell. This process was repeated six times over the course of 12 h. The results, depicted in Fig. 10, showed that at pH 5.5, the ethosomal formulation diffused twice as much as the pure vitamin D3 solution in a short period of time. The results demonstrated a much higher permeability of ethosomes than that of pure vitamin D3. Analysis of the withdrawn solution showed that 22.5%  3 of vitamin D3 was diffused into the membrane during the first hour (Fig. 9). Over the course of 12 h, 95.34% ± 3 vitamin D3 was penetrated through the membrane. These findings demonstrated the connection between dissolution and penetration, and they were extremely similar to the dissolution release profile. As seen in Fig. 3A (SEM), the high particle size of vitamin D3 significantly slowed its penetration when compared to pure vitamin D3. The improvement is due to the smaller particle sizes achieved during the precipitation process of producing ED3-7. Ethosomes are vesicles made of lipids that have been enhanced with ethanol. Water, ethanol, and phospholipids are usually present. Compared to conventional liposomes, ethosomes have a higher ethanol concentration, which makes them more flexible and improves their capacity to pass through the skin barrier. Ethanol disrupts the lipid bilayer of the stratum corneum (the outermost layer of the skin), increasing skin permeability. This disruption facilitates the delivery of the drug through the skin barrier.

Skin irritation test

The in vivo rabbit test has become the standard against which innovative methods of assessing skin irritation are judged. For this regulatory objective, there is currently no viable alternative to animal testing. We applied 0.1 g of a suspension of ED3-7 ethosomes to rabbit skin after shaving. After 24 h, the skin’s surface was examined for any outward signs of alteration, such as erythema (redness). The results revealed that there was no skin irritation in rabbit skin after 24 h. Analysis of the results led to the conclusion that ED3-7 ethosomes may be considered relatively safe and nontoxic. Figure 11 shows the skin picture after 24 h (A- before applying, B- after applying). Before conducting experiment on animal we have got approval from committee and certifying that such experiments were performed in accordance with all national or local guidelines and regulations.

Conclusion

In this pilot research we have synthesized ethosomes loaded with vitamin D3 and transformed them into a gel which is easily applicable to the pharmaceutical industry. This is first time we have developed gel for topical use against psoriasis disease and it can be easily scalable for pharmaceutical industries. Features including particle size and images, encapsulation, release rate, penetration study, compatibility, and stability are frequently seen in successful formulations. The toxicity was evaluated with hemolysis assay and with skin irritation test. Vitamin D3 ethosomes were shown to have much better membrane penetration in ex vivo permeability testing than raw vitamin D3 suspensions. Since micron-sized particles aid in quick absorption via the skin, this is an important finding. We used FTIR and thermal analyses to investigate the molecular interactions between lipids and vitamin D3. These findings suggest that ethosomes containing vitamin D3 will be useful in overcoming psoriasis.

Fig. 1
figure 1

Synthesis of ethosomal suspensions and gels (solution A contain PG, Cholesterol and phospholipids) (solution B contain ethanol and vitamin D3) and (solution C contain both mixture of solution A and B).

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Fig. 2
figure 2

Size distribution index image of vitamin D3 ethosomal suspensions.

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Fig. 3
figure 3

SEM image of raw material (vitamin D3 powder) (A) and vitamin D3 ethosomes (B).

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Fig. 4
figure 4

FTIR spectra of vitamin D3, lipid (soy lecithin), PG (propyleneglycol) and ethosomes.

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Fig. 5
figure 5

TGA of vitamin D3, lipid (soy lecithin), PG (propylene glycol) and ethosomes.

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Fig. 6
figure 6

DSC of vitamin D3, lipid (soy lecithin), PG (propylene glycol) and ethosomes.

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Fig. 7
figure 7

Percent drug release of ethosomes gel (ED3-7) at pH 5.5.

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Fig. 8
figure 8

Shear flow curves of ethosomal gels with 1%, 1.2% and 1.5% Carbopol.

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Fig. 9
figure 9

Hemolysis graph of ethosomes after 2 h and 12 h.

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Fig. 10
figure 10

Permeation study using an artificial membrane at pH 5.5.

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Fig. 11
figure 11

Photograph of the skin of a rabbit before applying the formulation (A) and 24 h after applying the test formulation (B).

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