Formulation and evaluation of folic acid-loaded binary ethosomes for enhanced skin permeation

Formulation and evaluation of folic acid-loaded binary ethosomes for enhanced skin permeation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Formulation and evaluation of folic acid-loaded binary ethosomes for enhanced skin permeation Shraddha Singh Raghav, Bhavna Kumar, Poorvi Varshney, Abdul Ahad, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8382960/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diabetes-related foot ulcers are a serious health concern and can have detrimental impacts on both physical and mental health. The objective of present study was to prepare folic acid-loaded binary ethosomes. The cold technique was used to prepare the folic acid-loaded binary ethosomes formulation, and evaluated for vesicles size, PDI, zeta potential, FTIR, entrapment efficiency, drug loading. Further the optimized binary ethosomes formulation was converted to gel formulation and assessed for in-vitro drug release, ex-vivo skin penetration, and formulation stability. The optimized binary ethosomes formulation F6 exhibited a vesicles size of 401.8 ± 1.2 nm, with a PDI of 0.32 and a zeta potential of − 29.67 ± 0.5 mV. The entrapment efficiency was found to be 82.24 ± 1.2%. The comparative in vitro drug release profile of the binary ethosomes formulation gel showed a significantly higher release (76.21%) compared to the control gel (39.32%) at 1440 minutes. In ex vivo permeation studies, the flux of the binary ethosomes -loaded gel was 4.98 µg/cm²/h at 2160 minutes. Organoleptic evaluation revealed that the formulated gel was yellowish in color, odorless, and had a pH of 5.5. Rheological studies confirmed that the formulation followed a non-Newtonian shear-thinning behavior, which is favorable for topical application. Overall, optimized F6 was identified as the best formulation based on its physicochemical properties, drug release performance, and rheological characteristics. Hence, the developed folic acid loaded binary ethosomes gel showed potential for improving folic acid efficacy in topical administration. Diabetic foot ulcer Folic acid Binary ethosomes Gel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Diabetes-related foot ulcers are a serious health concern for those who have the disease since they can have detrimental impacts on both physical and mental health. Recurrence, amputation, and death risks are also linked to diabetes-related foot ulcers. Effective prevention and management techniques are required in diabetes patients to lower morbidity and mortality. Approximately 20% of diabetics have both foot ulcers and neuropathy, compared to an estimated 80% who have foot ulcers and 50% who have neuropathy. Most cutaneous ulcers in diabetic foot problems are caused by mechanical or physical trauma. Neutrophils, fibroblasts, and leukocyte levels are reduced in diabetic individuals, though inflammatory cells have lower efficiency at the ulcer site. The risk of foot issues is further increased by additional macrovascular disease risk factors like hypertension, hyperlipidemia, and smoking. The main contributor to the onset of diabetic foot ulcers is inadequate glucose management. Its primary role is to promote the formation of new cells in the body, which includes DNA synthesis, repair, and methylation. Folic acid is a water-soluble B vitamin that can be found naturally in a range of foods including legumes, vegetables, liver, and milk [ 1 , 2 ]. It participates in a variety of metabolic activities important for cell division, including purine and pyrimidine production, DNA/RNA biosynthesis, and amino acid metabolism [ 1 , 3 ]. Because mammals cannot synthesize folic acid, they must obtain it from their diet. Even though folic acid is widely available in foods, folic acid deficiency is nevertheless common due to a variety of factors such as a poor diet [ 4 , 5 ] disease-related malabsorption, medication-related depletion [ 6 ], or vitamin B12 deficiency [ 7 ]. Folic acid deficiency has been associated with several health problems, such as anemia [ 8 , 9 ], cancer [ 10 ], cardiovascular diseases [ 11 ], neural tube defects in newborns, neuropsychiatric dysfunction [ 12 ], depression [ 13 ], inflammatory diseases [ 14 , 15 ], and eye diseases [ 16 ]. Folic acid considerably reduced levels of lipid peroxidation, protein nitro tyrosination, and glutathione depletion in wounds, as vitamins play a vital role in the prevention and treatment of diabetic foot ulcers and wounds, among other things. Folic acid supplementation's role is to heal damaged wounds by decreasing oxidative stress. Vitamins are the ideal sources since they are inexpensive, widely available, cure disease at its base, are toxic-free, and have no or minimum side effects. Folic acid has antioxidant, anticancer, cardiovascular, and neuroprotective effects. Folic acid's antioxidant activity is mediated through multiple mechanisms, including a decrease in plasma homocysteine concentrations, which may increase total antioxidant capacity and reduce reactive oxygen species formation. Thus its inbuilt activity of antioxidant its plays an important role in inflammatory, inhibits microbial growth, and healing wounds, or/and promotes the synthesis of collagen fibers to increase supply of vital to the affected area or wound site that enhanced the wound healing process by expanding the feasibility of collagen fibrils, increased the collagen strands strength, increased the circulation or avoidance of cell harm, or promotes the synthesis of DNA [ 16 ]. Folic acid, a low soluble and low permeability drug, was chosen as the model of biopharmaceutical classification system class IV. It may also have dietary effects, poor absorption, and significant variation in inter- and intra-patient dose response [ 17 ]. Folic acid has poor solubility in water or any other commonly used media, and is poorly absorbed by the body. Vitamins degrade when exposed to the outside environment, which is one of the reasons why developing pharmaceutical dose forms are difficult. To address this issue, various ways have been considered, including the development of suspensions, solubilization in organic solvents, the use of drug salts, the development of prodrugs, and the development of innovative drug delivery systems. In addition, insoluble solid drug forms pose a significant problem in terms of their limited bioavailability in common media such as water [ 17 , 18 ]. As liposomes are unstable and localize predominantly to the reticuloendothelial system, mainly the liver and spleen, complex drug carriers such as liposomes have limited utility. As conventional solvents and solubility enhancers are toxic and irritating when injected into humans, they are mainly used to treat acute, life-threatening diseases where medical experts are continually available to administer palliative treatments to counteract the negative effects of the solvents. Solvents/enhancers that are water miscible also have the disadvantage of causing medicine to precipitate rapidly when exposed to water. When a medication candidate's water solubility is insufficient to allow solution formulations, solubility enhancers are frequently used to boost solubility. A solubility enhancer can increase solubility several orders of magnitude. Solubility enhancers such as propylene glycol, polyethylene glycols, ethanol, and sorbitol are commonly used [ 17 , 19 ]. Adding a co-solvent to the solvent can lower its dielectric constant, thereby boosting the solubility of hydrophobic molecules. With solubility enhancers, drug precipitation can occur when dilutions of solvents are made, as well as tissue injury and pain during injection. They are more difficult to administer and prepare because of their low dissolving rate, poor bioavailability, and poor penetration [ 17 ]. Thus, novel dosage formulations are necessary to overcome the solubility and permeability difficulties of such therapeutic candidates. The compositions of ethosomes that are hydro/alcoholic/phospholipid systems with a high alcohol concentration. A system that increases skin penetration and has the ability to produce ethosomes, which are "soft" vesicles made of phospholipid when water and ethanol (alcohol) are present. Ethosomes offer numerous significant benefits, such as boosting therapeutic efficacy, enhancing patient comfort and compliance, and reducing overall treatment expenses. Ethosomes are non-invasive drug delivery systems that enable drugs to penetrate deep layers of skin before entering the bloodstream. The improved distribution of bioactive substances through cellular and epidermal membranes via an ethosomes carrier poses a number of challenges as well as opportunities for further research and the creation of innovative, better treatments[ 20 ]. As a result, developing folic acid-loaded ethosomes may be a viable option for improving solubility and permeability in diabetic foot ulcers. As a result, the invention is about developing novel dosage forms as ethosomes formulations can resolve the issues of solubility and permeability, providing a new avenue for treatment approaches for ulcers that are easier to apply in the form of topical gels and have fewer side effects when compared to other routes of administration. Furthermore, shorter half-lives have a superior safety profile than other routes of administration. 2. Materials and methods 2.1 Materials Folic acid was bought from Dhamtec Pharma and Consultants in Mumbai, India. Soya lecithin, ethanol, propylene glycol, and cholesterol were purchased from Sigma-Aldrich Chemicals (Germany). All other compounds were classified using analytical grades. 2.2 Preparation method of folic acid loaded binary ethosomes Folic acid binary ethosomes were developed by cold method [ 21 ], where 300–400 mg of lipids, 10–30% v/v of ethanol, 5mg of Folic acid (the drug), propylene glycol (PG)- 5% and percentage of water was primarily taken. Further, the former solution was kept on a magnetic stirrer (Remi Equipment, Mumbai) and distilled water was added with continuous stirred (700 rpm) [ 22 ]. The solution formed was retained at 4°C in refrigerator, and sonication was performed for 3 cycles with probe sonicator for 5 min (Table 1 ) [ 23 , 24 ]. Table 1 Composition of folic acid loaded binary ethosomes formulations. Formulation code Drug (mg) Soya lecithin (mg) Ethanol (%) Propylene glycol (%) F1 5 300 10 5 F2 5 400 10 5 F3 5 300 15 5 F4 5 400 15 5 F5 5 300 20 5 F6 5 400 20 5 F7 5 300 30 5 F8 5 400 30 5 F9 5 300 25 5 F10 5 400 25 5 2.3 Characterization of prepared folic acid loaded binary ethosomes 2.3.1 Vesicles size and PDI Vesicle size distribution and PDI of formulated binary ethosomes were done by Malvern zeta sizer 2000 MU (Malvern instrument UK) [ 25 , 26 ]. 2.3.2 Zeta potential The zeta potential of the preparation may analyze using a Malvern zeta sizer 2000MU (Malvern instrument UK). A value of 25 mV, regardless of its polarity, may serve as the threshold that distinguishes surfaces with low charge from those with high charge. The importance of zeta potential lies in its capacity to be correlated with the stability of colloidal dispersions [ 25 , 26 ]. 2.3.3 Transmission electron microscopy (TEM ) The shape and surface morphology of formulated binary ethosomes was examined using TEM (TEM-Tecknai G2, FEI, 200 kV, Tokyo, Japan) [ 27 ]. 2.3.4 FTIR Spectroscopy The functional groups of formulated binary ethosomes were examined using an FTIR spectrometer (IRAffinity-1S, Shimadzu, Kyoto, Japan) with KBr pellets and FTIR wave numbers ranging from 4000 cm -1 to 450 cm -1 [ 27 ]. 2.3.5 Entrapment efficiency % The produced binary ethosomes ability to entrap drug was determined using centrifugation method. A cold centrifuge was used to spin the centrifugation tubes at a speed of 15000 RPM for 30 minutes at 4°C after adding 1 ml of a drug-loaded binary ethosomes suspension. The unentrapped drug concentration was determined spectrophotometrically at 283 nm. The amount of free drug in the supernatant represents the total amount of untrapped drug. The entrapment efficiency was then calculated using the given formula (1) and expressed as a percentage of drugs trapped [68]. \(\:\text{E}\text{E}\text{%}\:=\frac{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{d}\text{r}\text{u}\text{g}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}-\text{F}\text{r}\text{e}\text{e}\:\text{d}\text{r}\text{u}\text{g}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{d}\text{r}\text{u}\text{g}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}}\times\:100\) ………Eq. (1 ) 2.4 Preparation of folic acid loaded binary ethosomes gel Gels were prepared using carbopol (grade 934k in 0.5% and 1% w/v concentrations) [ 25 , 26 ]. Carbopol 934k was precisely weighed, soaked in the least amount of water, and continuously stirred at 400 rpm until homogenous gel bases with a smooth texture and no lumps were formed. After being weighed, the optimized binary ethosomes loaded with 5 mg of folic acid were added to the swollen polymer. It was mixed using a homogenizer to form homogenous gels [ 27 ]. A sufficient quantity of TEA (triethanolamine) was added drop by drop to neutralize pH 6.8 [ 28 ]. A gel with 200 mg of pure medication was also prepared using the same procedures. Table 2 lists the components and amounts needed to form a folic acid-loaded binary ethosomes gel. Table 2 Composition of folic acid control gel and folic acid loaded binary ethosomes gel. Formulations code Polymer name (%) Weight of the polymer (w/v) (%) Ethosomes loaded into the gel base Purified water F1 Carbopol 934k 1 Ethosomes suspension equivalent to 5 mg Upto 100 ml F2 Carbopol 934k 0.5 Ethosomes suspension equivalent to 5 mg Upto 100 ml F3 Carbopol 934k 1 5 mg free Upto 100 ml F4 Carbopol 934k 0.5 5 mg free Upto 100 ml 2.6 Characterization of prepared folic acid loaded binary ethosomes gel 2.6.1 Physical Evaluation The organoleptic properties were evaluated to assess the folic acid-loaded ethosomes gel. 2.6.2 Determination of pH A pH meter was used to measure pH of the folic acid loaded ethosomes gel. Three duplicate readings of the data were taken, and the average value was determined pH meter (Labtronics, India) [ 29 ] . 2.6.3 Spreadability Spreadability of gel was assessed by glass slide method where 0.5 g of gel was placed on the lower glass slide in 1 cm marked circle on it and using the weight of 500 g, the upper slide is placed on the lower glass slide for 5 min and measured the gel diameter by its spreadability [ 30 ]. S = \(\:\frac{\mathbf{M}\times\:\mathbf{L}}{\mathbf{T}}\) ……………….Eq. (2 ) Where, S = Spreadability; M = Weight tied on the upper plate; L = Length (cm) of glass plate; T = Time taken (second) 2.6.4 Viscosity Viscosity of formulated gel was assessed by viscometer (Anton Paar MCR 72 rheometers, India). The viscosity of gel was evaluated by temperature shear sweep method at temperature ranging from 25°C to 60°C and the shear rate ranging from 20–100 rpm. 2.6.5 In-vitro drug release study The folic acid-loaded ethosomes gel was placed into a pre-soaked dialysis membrane (molecular weight cut-off: 12–14 kDa), securely sealed, and suspended in phosphate buffer (pH 7.4) maintained at 37 ± 0.5°C. Samples were withdrawn at predetermined intervals and immediately replaced with an equal volume of fresh buffer to maintain sink conditions. Using a dialysis bag (diameter: 2.5 cm), the release of folic acid from the formulation was assessed at time intervals of 5, 15, 30, 45, 60, 75, 90, 120, 180, 360, 720, 1080, and 1440 minutes. The absorbance of each sample was measured using a UV–Visible spectrophotometer (Shimadzu 1800, Japan) at 283 nm. The Higuchi, Korsmeyer–Peppas, zero-order, and first-order release models were among the kinetic models to which the cumulative release data were fitted [ 28 ]. 2.6.7 Ex-vivo permeation study of folic acid loaded binary ethosomes gel Ex-vivo drug permeability was measured using Franz's diffusion cell method and a semipermeable membrane made from freshly excised goat abdomen skin. For subsequent use, the skin was first washed with physiological salt solution, followed by distilled water, and then soaked in phosphate-buffered saline (pH 7.4) [ 31 ]. The skin was clamped with Franz's diffusion cell containing 6.5 ml of phosphate-buffered saline and 1.7 cm 2 of effective permeation area. In Franz's diffusion cell, the gel was carefully weighed and added to the donor compartment while being continually stirred with a magnetic stirrer (Expo India Ltd., Mumbai, India) at 37.5°C. To maintain the sink condition, an equal volume of blank receptor fluid was injected into the receptor compartment at predetermined intervals (0, 5, 15, 30, 45, 60, 120, 180, 360, 720, 1080, 1440 and 2160 minutes) and the drug concentration was determined using UV spectroscopy. As a result of the triplicate experiment, the apparent permeation coefficient, flux, and permeation amount were determined using the equation below (3) [ 31 ]. \(\:Apparent\:permeation\:coefficient=\:\frac{Flux}{Area\:of\:permeation\:\times\:\:Amount\:of\:folic\:acid\:in\:formulation}\) …………. Eq. (3) 2.7 Stability study of folic acid loaded binary ethosomes gel The formulated gel was assessed for statistical analysis as per the ICH guidelines. The prepared folic acid loaded binary ethosomes gel was kept in the dark for 90 days at 4 ºC. Analyze the size of the vesicles, PDI, and zeta potential were performed to determine the stability of the formulated binary ethosomes gel [ 32 ]. 3. Result and discussion 3.1 Result of folic acid loaded binary ethosomes 3.1.1 Vesicles size, PDI and zeta potential Binary ethosomes were measured using a Malvern zeta sizer 2000MU to determine their size, PDI, and zeta potential. The size of vesicles influences drug disposition, diffusion, release factors, and skin absorption. The size and amount of drug loaded into vesicles are influenced by a variety of factors, including chemical structure, the type and quantity of drug and lipid used, and experimental procedures [ 33 , 34 ]. As indicated in Fig. 1 , the vesicles size of optimized binary ethosomes (F6) was 401.8 ± 1.2 nm. A PDI value in the range of 0.1–0.7 demonstrates the presence of uniform size vesicles in the formulation, indicating homogeneous dispersion and a narrow size distribution. [ 35 ]. The PDI of the prepared gel was determined to be within this range, i.e., 0.3, indicating great vesicles size distribution homogeneity. As indicated in Fig. 2 , the zeta potential of optimized binary ethosomes (F6) was − 29.67 ± 0.5 mV. The negative charge on the zeta potential implies electrostatic repulsion between the charged ions, which is a crucial element in preventing vesicles aggregation. Thus, the Zeta potential reflects the formulation's electrostatic stability; the greater the negative charge, the greater the stability [ 36 ]. 3.1.2% Entrapment efficiency The entrapment efficiency (%) of the optimized formulation (F6) were determined to be 82.24%. The high entrapment efficiency in formulation is attributed to the presence of a membrane composed of ethanol or propylene glycol, as well as an increased number of C-H bonds between the hydrophilic tail structures and the lipophilic nature of the folic acid. The hydrophobic structure of the membrane allows for sustained drug release. Such binary ethosomes, like other vesicles that are considered stable vesicles with higher drug permeability release, operate as drug depots [ 37 – 39 ]. 3.1.3 FTIR analysis The peaks of Folic acid (A) are displayed in Fig. 3 . The C-O stretching is at 1072.44 cm-1, the CH2 bending is at 1401 cm-1, the C = C stretching is at 1683 cm-1, and the C-H stretching is at 1944 cm-1 and 3036 cm-1. Figure 3 (C)showed soya lecithin peaks are shown at 1039 cm -1 indicates the C-O stretching, 1308 cm -1 showed CH 2 bending, 1633 cm- 1 showed C = C stretching and 1965 cm -1 & 2974 cm -1 showed the C-H bending, while the FTIR Peaks of folic acid loaded binary ethosomes (Fig. 3 (B)) are shown at 1043 cm- 1 revealed C-O stretching, 1452 cm-1 due to CH 2 bending, 1726 cm -1 showed C = O stretching, 2062 cm- 1 and 2848 cm -1 indicates the C = H stretching. The FTIR Spectrum of folic acid loaded binary ethosomes demonstrated that there was no peak formation occurs in the formulation in comparative to the added ingredients. Thus, we can conclude that there was no chemical interaction shown in the formulation with the active ingredients. 3.1.4 TEM analysis The shape of nanovesicles were investigated using transmission electron microscopy (TEM) by placing the nanovesicles on a 300-mesh coated carbon film and swirling for 10 minutes until the correct air dried. The sample was then mixed with a 2% w/v phosphotungstic acid solution and dried at room temperature before the spectra was examined [ 40 , 41 ]. The results of the TEM analysis are displayed in Fig. 4 . The combination of 10–30% v/v ethanol and 5% propylene glycol resulted in spherical shaped, nonporous smooth surface and multilamellar binary ethosomes with increased and high entrapped drug concentration. 3.2 Evaluation of folic acid loaded binary ethosomes gel 3.2.1 Physical Evaluation The formulated gel exhibited a yellowish appearance and was odorless. 3.2.3 pH measurement The pH of the formulated gel was measured to be 5.5, indicating suitability for skin application and minimal risk of irritation. 3.2.3 Spreadability The spreadability of the gel was measured using the glass slide method. Both the initial and final diameters before and after spreading were recorded as 44 mm. 3.2.4 Viscosity of gel Viscosity is important in both stability and applicability. As shown in Fig. 5 , the viscosity of the formed folic acid loaded binary ethosomes gel reduces with increasing shear rate, indicating pseudoplastic behavior. The viscosity reduces due to the colloidal network structure in the direction of shear rate. The polymer chains disarranged to fit in the flow of the stream with increased shear stress. This direction reduced the material's internal resistance, allowing for a larger shear rate with each successive shearing force. This resulted in a decrease in apparent viscosity. The curve of the formulation graph demonstrates non-Newtonian essence. The rheological characteristics listed above were excellent for the formulations designed for topical application. 3.2.5 In-vitro drug release analysis The release profile of folic acid loaded binary ethosomes gel and folic acid was compared in-vitro on the dialysis bag at pH 7.4 (phosphate buffer), as shown in Fig. 6 . The folic acid loaded binary ethosomes gel demonstrated high drug release kinetics, with 76.21% at 1440 minutes compared to 39.32% at 1440 minutes for the folic acid loaded gel. As a result of the nano formulation, binary ethosomes loaded gel demonstrated a greater in-vitro drug release profile. As shown in Table 3 , kinetic modeling indicates that the Korsmeyer-Peppas model has a higher R 2 value than the other models. The R 2 value of the folic acid-loaded binary ethosomes gel was 0.9843, and the release exponent (n) of the Korsmeyer-Peppas model was less than 0.45 [ 27 , 42 ]. Consequently, during drug release, the produced folic acid-loaded binary ethosomes gel followed Fick's law of diffusion. It was strongly concluded that the formulated folic acid loaded binary ethosomes gel can be used for the topical delivery of the local action in the condition of diabetic foot ulcer because of the adhesive and hydrophilic nature of the gelling agent, i.e., propylene glycol, as well as their other physical properties and the effective permeation of binary ethosomes loaded gel due to Fickian based diffusion [ 43 – 45 ]. Table 3 Data of release kinetics profile of folic acid loaded binary ethosomes gel. Formulation Zero order First order Higuchi Korsemeyer- Peppas Folic acid loaded binary ethosomes R 2 K O R 2 K O R 2 K O R 2 K O N 0.7600 0.047 0.8767 0.001 0.9827 1.541 0.9843 1.974 0.45 3.2.6 Ex-vivo permeation study Ex-vivo permeation analysis was performed on goat abdomen skin at pH 7.4 to compare the data of folic acid and folic acid-loaded binary ethosomes gel, as shown in Fig. 7 . Results showed that the folic acid loaded binary ethosomes gel showed high retention of drug on the skin and the flux value in comparison with the folic acid loaded binary ethosomes gel at same time of duration i.e., 2160 minutes. Hence, folic acid loaded binary ethosomes gel should be indicated for topical use in the treatment of foot ulcer because of high retention of drug due to binary ethosomes formulation of the drug in aspect of folic acid formulation [ 46 ]. 3.3 Stability studies Lipid vesicular formulation, such as folic acid-loaded binary ethosomes gel, frequently undergo changes such as fusion or dissertation while in storage. The drug leaking from the vesicles affects VS, PDI, and ZP while decreasing the EE% [ 47 , 48 ]. To improve the physical and chemical stability of binary ethosomes gel, it was recommended that it be stored in a refrigerator (4°C). Table 4 shows the VS, PDI, ZP, and %EE of freshly prepared folic acid-loaded binary ethosomes gel after three months of refrigeration (4°C). These results showed that the folic acid loaded binary ethosomes gel was stable because it did not significantly differ from those obtained before storage. When kept at 4°C, binary ethosomes gel (which contains PG as another alcohol) was good stability [ 45 ]. Therefore, it was proposed that PG increases viscosity and antihydrolysis to improve binary ethosomes gel stability. Table 4 Effect of storage on properties of selected formulation (F6). Parameter Fresh prepared F6 F6 after 3 months of storage at 4 °C Vesicles size (nm) 401.8 ± 1.2 420 ± 1.70 PDI 0.32 ± 0.82 0.36 ± 1.14 ZP -29.67 ± 0.5 -29.23 ± 1.4 %EE 82.24 ± 1.2% 71.10 ± 1.20 4. Conclusion The present study successfully developed and characterized a folic acid-loaded binary ethosomes gel using propylene glycol as a permeation enhancer for potential use in diabetic foot ulcer treatment. The optimized formulation (F6) demonstrated good stability and nanoscale uniformity with a vesicle size of 401.8 ± 1.2 nm, a PDI of 0.32, and a zeta potential of − 29.67 ± 0.5 mV. Entrapment efficiency was notably high at 82.24 ± 1.2%, with a drug loading of 44.16%, suggesting efficient incorporation of folic acid into the vesicles. In-vitro release studies demonstrated that the binary ethosomes gel achieved a cumulative drug release of 76.21% at 1440 minutes, nearly double that of the conventional gel (39.32%). Ex vivo permeation through goat skin revealed a flux value of 4.98 µg/cm²/h at 2160 minutes, confirming enhanced skin penetration and retention. Stability studies indicated minimal changes in vesicle size, PDI, zeta potential, and entrapment efficiency over 3 months at 4°C, validating good storage stability. Overall, the incorporation of propylene glycol significantly improved the solubility, permeability, and retention of folic acid in the binary ethosomes gel, making it a promising topical delivery system for diabetic foot ulcer management with potential to enhance therapeutic efficacy and patient compliance. Declarations Conflicts of Interest: “The authors declare no conflict of interest”. Author Contribution “Conceptualization, BK, and SSR; Supervision, BK; Formal analysis, SSR; Methodology, BK, SSR and PV; Writing—original draft, SSR and BK; Writing—review and editing, SSR, PV, BK, AA and IK; Software: SSR, AA and IK. All authors have reviewed and agreed to the published version of the manuscript”. Acknowledgement All the authors extend their appreciation to the Ongoing Research Funding Program, (ORF-2026-959), King Saud University, Riyadh, Saudi Arabia, for supporting this project.” Data Availability Statement: “Data are contained within the article”. References Iyer R, Tomar SK Folate: a functional food constituent 2009; 74: R114–R122 Lyon P, Strippoli V, Fang B, Cimmino L (2020) B vitamins and one-carbon metabolism: implications in human health and disease. 12:2867 Farias N, Ho N, Butler S, Delaney L, Morrison J, Shahrzad S et al (2015) The effects of folic acid on global DNA methylation and colonosphere formation in colon cancer cell lines. 26:818–826 Hildebrand LA, Dumas B, Milrod CJ, Hudspeth JC Folate deficiency in an urban safety net population 2021; 134: 1265–1269 Iimura Y, Kurokawa T, Kanemoto Y, Yazawa K, Tsurita G, Ahiko Y et al Severe thrombocytopenia induced by chemotherapy after total gastrectomy: a report of three cases 2022; 60: 36 Arcot J, Shrestha A (2005) Folate: methods Anal 16:253–266 Fishman SM, Christian P, West KP Jr The role of vitamins in the prevention and control of anaemia 2000; 3: 125–150 Fleming CR. Modern nutrition in health and disease., Mautice E, Shils JA, Olson, Shike M (1994) 1885 pp. $ 99.50 (2 volumes) Lea & Febiger, Malvern, Pennsylvania, ISBN 0-8121-1485-X 1994 Erdman JW Jr, Macdonald IA, Zeisel SH (2012) Present knowledge in nutrition. Wiley Duthie SJ Folic acid deficiency and cancer: mechanisms of DNA instability 1999; 55: 578–592 Wald DS, Law M, Morris JK (2002) Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. 325:1202 Shea TB, Lyons-Weiler J, Rogers E, Homocysteine (2002) folate deprivation Alzheimer neuropathology 4:261–267 Falade J, Onaolapo AY, Onaolapo OJ The role of folate-supplementation in depression: a narrative review 2021; 10: 115–122 Suzuki H, Kunisawa J Vitamin-mediated immune regulation in the development of inflammatory diseases 2015; 15: 212–215 Jones P, Lucock M, Scarlett CJ, Veysey M, Beckett EL Folate and inflammation–links between folate and features of inflammatory conditions 2019; 18: 100104 Sijilmassi O Folic acid deficiency and vision: a review 2019; 257: 1573–1580 Radhakrishnan R Solubility enhancer and use thereof 2015 Bhalani DV, Nutan B, Kumar A, Singh Chandel AK (2022) Bioavailab enhancement techniques poorly aqueous soluble drugs Ther 10:2055 Suksaeree J, Simchareon W, Pichayakorn W (2021) Effect of glycols permeation enhancer on the release and permeation of meloxicam-natural rubber film through pig skin. 66:102874 Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M (2000) Ethosomes—novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. 65:403–418 Fathalla D, Youssef EMK, Soliman GM Liposomal and ethosomal gels for the topical delivery of anthralin: preparation, comparative evaluation and clinical assessment in psoriatic patients 2020; 12: 446 Castro NR, Cristal dos Santos CP, de Campos VEB, Cardoso V, Vermelho AB, dos Santos EP et al (2021) Development of hybrid vesicular nanosystems composed of lipids and chitosan for octyl methoxycinnamate encapsulation. ; 608: 125476 Rakesh R, Anoop KR Formulation and optimization of nano-sized ethosomes for enhanced transdermal delivery of cromolyn sodium 2012; 4: 333–340 Shumilov M, Bercovich R, Duchi S, Ainbinder D, Touitou E (2010) Ibuprofen transdermal ethosomal gel: characterization and efficiency in animal models. 6:569–576 Campos EVR, Proença PLF, da Costa TG, de Lima R, Fraceto LF, de Araujo DR (2022) Using chitosan-coated polymeric nanoparticles‐thermosensitive hydrogels in association with limonene as skin drug delivery strategy 2022; : 9165443 Khan ZU, Razzaq A, Khan A, Rehman NU, Khan H, Khan T et al Physicochemical characterizations and pharmacokinetic evaluation of pentazocine solid lipid nanoparticles against inflammatory pain model 2022; 14: 409 Ijaz M, Akhtar N (2020) Fatty acids based α-Tocopherol loaded nanostructured lipid carrier gel: In vitro and in vivo evaluation for moisturizing and anti‐aging effects. ; 19: 3067–76 Akhtar N, Akhtar N Development of stable tocopherol succinate-loaded ethosomes to enhance transdermal permeation: in vitro and in vivo characterizations 2022; 21: 4942–4955 Sharif A, Akhtar N Fabrication of niosomes containing N-acetyl glucosamine: In-vitro and ex-vivo characterizations 2020 Pereira TA, Guerreiro CM, Maruno M, Ferrari M, Rocha-Filho PA (2016) Exotic vegetable oils for cosmetic o/w nanoemulsions: In. vivo evaluation 21:248 Khalil RM, Abdelbary GA, Basha M, Awad GEA, El-Hashemy HA (2017) Design and evaluation of proniosomes as a carrier for ocular delivery of lomefloxacin HCl. 27:118–129 Pokharana M, Vaishnav R, Goyal A, Shrivastava A (2018) Stab Test guidelines Pharm Prod 8:169–175 Iyer SS, Barr WH, Karnes HT (2006) Profiling in vitro drug release from subcutaneous implants: a review of current status and potential implications on drug product development. 27:157–170 Nair R, Arun Kumar KS, Vishnu Priya K, Sevukarajan M Recent advances in solid lipid nanoparticle based drug delivery systems 2011; 3: 368–384 Thang L, Hanh N, Duong D Study on cause-effect relations and optimization of exemestane-loaded nanostructured lipid carriers 2017; 9: 68–74 Sargam Y, Wang K, Tsyrenova A, Liu F, Jiang S (2021) Effects of anionic and nonionic surfactants on the dispersion and stability of nanoSiO2 in aqueous and cement pore solutions. 144:106417 Basiri L, Rajabzadeh G, Bostan A (2017) Physicochemical properties and release behavior of Span 60/Tween 60 niosomes as vehicle for α-Tocopherol delivery. 84:471–478 Junyaprasert VB, Singhsa P, Suksiriworapong J, Chantasart D Physicochemical properties and skin permeation of Span 60/Tween 60 niosomes of ellagic acid 2012; 423: 303–311 Fu X, Shi Y, Wang H, Zhao X, Sun Q, Huang Y et al (2019) Ethosomal gel for improving transdermal delivery of thymosin β-4 : 9275–9284 Pyrz WD, Buttrey DJ (2008) Particle size determination using TEM: a discussion of image acquisition and analysis for the novice microscopist. 24:11350–11360 Chourasia MK, Kang L, Chan SY Nanosized ethosomes bearing ketoprofen for improved transdermal delivery 2011; 1: 60–67 Abd El-Alim SH, Kassem AA, Basha M (2014) Proniosomes as a novel drug carrier system for buccal delivery of benzocaine. 24:452–458 Shirsand SB, Para MS, Nagendrakumar D, Kanani KM, Keerthy D (2012) Formulation and evaluation of Ketoconazole niosomal gel drug delivery system. 2:201 Budhiraja A, Dhingra G Development and characterization of a novel antiacne niosomal gel of rosmarinic acid 2015; 22: 723–730 Abdulbaqi IM, Darwis Y, Khan NAK, Assi RA, Khan AA Ethosomal nanocarriers: the impact of constituents and formulation techniques on ethosomal properties, in vivo studies, and clinical trials 2016: 2279–2304 Arora D, Nanda S (2019) Quality by design driven development of resveratrol loaded ethosomal hydrogel for improved dermatological benefits via enhanced skin permeation and retention. 567:118448 Paliwal S, Tilak A, Sharma J, Dave V, Sharma S, Yadav R et al (2019) Flurbiprofen loaded ethosomes-transdermal delivery of anti-inflammatory effect in rat model. 18:1–15 Verma P, Pathak K (2012) Nanosized ethanolic vesicles loaded with econazole nitrate for the treatment of deep fungal infections through topical gel formulation. 8:489–496 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8382960","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":576198536,"identity":"2a56c0c8-f974-4514-a95c-a533843b8c4d","order_by":0,"name":"Shraddha Singh Raghav","email":"","orcid":"","institution":"Dehradun Institute of Technology University","correspondingAuthor":false,"prefix":"","firstName":"Shraddha","middleName":"Singh","lastName":"Raghav","suffix":""},{"id":576198537,"identity":"73f0010c-f44a-47ed-8de4-e789ba3d5caa","order_by":1,"name":"Bhavna Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACAwkogx9EJBSQokWyAaTFgBQtBgfAJBFazKWbn0kX1NyRMz6/OvHDAwMGeX6xA/i1WM45ZiY949gzY7MbbzdLAB1mOHN2AgGH3UgwNuZhO5y47cbZDSAtCQa3CWpJ/2zM8+9w4uYZZzf/IFJLjuFj3rbDiRv4e7cRZ4vljJzCx7x9h40lbvBus0gwkCDsF3OJ9A2Heb4dluPvP7v55o8KG3l+aQJaEEACrFKCgCoUwH+AFNWjYBSMglEwkgAAZY5Fleg+cqoAAAAASUVORK5CYII=","orcid":"","institution":"Dehradun Institute of Technology University","correspondingAuthor":true,"prefix":"","firstName":"Bhavna","middleName":"","lastName":"Kumar","suffix":""},{"id":576198538,"identity":"b73a7c03-e1cd-465f-96cb-2d273a21c2bf","order_by":2,"name":"Poorvi Varshney","email":"","orcid":"","institution":"Dehradun Institute of Technology University","correspondingAuthor":false,"prefix":"","firstName":"Poorvi","middleName":"","lastName":"Varshney","suffix":""},{"id":576198539,"identity":"712f9b65-db36-4bfa-b2eb-42302c8c69c8","order_by":3,"name":"Abdul Ahad","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Ahad","suffix":""},{"id":576198540,"identity":"77edc952-a5f8-4447-a3e9-619848eab9f2","order_by":4,"name":"Islauddin Khan","email":"","orcid":"","institution":"University of Illinois","correspondingAuthor":false,"prefix":"","firstName":"Islauddin","middleName":"","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2025-12-17 07:53:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8382960/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8382960/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100562375,"identity":"f8d11990-3184-48e9-83b6-77c181d3ac7d","added_by":"auto","created_at":"2026-01-19 08:44:54","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":704871,"visible":true,"origin":"","legend":"","description":"","filename":"FinalManuscipt2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/7c30191bd762b311d7ca975c.docx"},{"id":100595113,"identity":"ab15f464-6dd4-407f-9ef5-731fb6f822ed","added_by":"auto","created_at":"2026-01-19 13:47:27","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6816,"visible":true,"origin":"","legend":"","description":"","filename":"aad61c7012aa45f6ae65ca696bd805c2.json","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/ccba5b49b468273d5546aad9.json"},{"id":100562353,"identity":"1425bded-65b5-4db2-897c-3424169fe566","added_by":"auto","created_at":"2026-01-19 08:44:50","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104896,"visible":true,"origin":"","legend":"","description":"","filename":"aad61c7012aa45f6ae65ca696bd805c21enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/505a374b6b30b753c81483e7.xml"},{"id":100594871,"identity":"f8ce5cb6-effa-41d3-86da-6175d3421de3","added_by":"auto","created_at":"2026-01-19 13:45:53","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58586,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/bd2ab1c10552016757a9cc29.png"},{"id":100562399,"identity":"1a3fc64a-46c1-4f8f-8855-34e18d2273cd","added_by":"auto","created_at":"2026-01-19 08:44:55","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":54297,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/a427b80cf90b3f42ca8b5858.png"},{"id":100595188,"identity":"b25e2afc-7118-4447-9ffa-3ce33a870ad1","added_by":"auto","created_at":"2026-01-19 13:47:48","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":43699,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/75b17ef6d3d44c7c4ca0909c.png"},{"id":100595346,"identity":"12ad94d0-0a3e-4bba-a9c4-66bc102e09a5","added_by":"auto","created_at":"2026-01-19 13:48:16","extension":"emf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":556244,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.emf","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/93913b7626903e1d64e3764b.emf"},{"id":100562423,"identity":"f4c5751c-59af-4cea-9584-5196018dc357","added_by":"auto","created_at":"2026-01-19 08:44:56","extension":"emf","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":820296,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.emf","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/fe7b09a098fe2d613fe11d31.emf"},{"id":100562380,"identity":"9c4de5a7-95d8-4eaa-8a02-26ef7ee36299","added_by":"auto","created_at":"2026-01-19 08:44:54","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44203,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/6746f0b12fd449947dc03aff.png"},{"id":100562420,"identity":"ac52ab9e-145b-4cda-8a35-3aefe5d8b5bc","added_by":"auto","created_at":"2026-01-19 08:44:55","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":44114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/7f7e0bc7dbf989d315b9f771.png"},{"id":100562409,"identity":"3ec3d59a-691a-4d06-958f-ab6681d18271","added_by":"auto","created_at":"2026-01-19 08:44:55","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8674,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/47bbafe35e5f405df5ebe780.png"},{"id":100562450,"identity":"84e21173-7727-4e55-9653-bb4d56f2c315","added_by":"auto","created_at":"2026-01-19 08:44:58","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7203,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/0a30e1ee0a142f03061630cc.png"},{"id":100562359,"identity":"0312018e-ab28-4c56-8cf1-3b32abf0198a","added_by":"auto","created_at":"2026-01-19 08:44:52","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11697,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/5ea32ae04c80d70b554a2940.png"},{"id":100562369,"identity":"49535036-007a-4d2c-97ca-21031f2c9947","added_by":"auto","created_at":"2026-01-19 08:44:53","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49961,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/d6d8b7b4de03148b140b853d.png"},{"id":100595500,"identity":"19e35dd4-82cc-41ee-a78e-538d494deee1","added_by":"auto","created_at":"2026-01-19 13:48:37","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15379,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/ecd8958d7f38da9413d9648d.png"},{"id":100562429,"identity":"06374d26-cf4b-482a-a401-e9213949e005","added_by":"auto","created_at":"2026-01-19 08:44:56","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11988,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/cf464b167295d6e0b2c1581e.png"},{"id":100594865,"identity":"5fc71e6e-2c6a-469a-a870-932f43d34b3c","added_by":"auto","created_at":"2026-01-19 13:45:48","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11948,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/dfd4a8b69ed4de91939a6c22.png"},{"id":100562370,"identity":"93ccce9b-7456-4faa-939b-2e3db21d4fae","added_by":"auto","created_at":"2026-01-19 08:44:54","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105703,"visible":true,"origin":"","legend":"","description":"","filename":"aad61c7012aa45f6ae65ca696bd805c21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/f7e4dbf97c53045a0068e85e.xml"},{"id":100594860,"identity":"ce6bdbbf-6960-4c61-b8ed-5c3ab3ead84c","added_by":"auto","created_at":"2026-01-19 13:45:46","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115290,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/c57a5ca17ba95bf79bdc222e.html"},{"id":100562449,"identity":"0028cfed-952e-4491-9114-6609cebb6d4c","added_by":"auto","created_at":"2026-01-19 08:44:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48948,"visible":true,"origin":"","legend":"\u003cp\u003eVesicles size analysis of optimized folic acid loaded binary ethosomes formulation (F6).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/2ab12b74b49a5e1a1c7c4f9e.png"},{"id":100562447,"identity":"0096bff4-d2ce-42e8-bda3-6edd3c4a9315","added_by":"auto","created_at":"2026-01-19 08:44:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51262,"visible":true,"origin":"","legend":"\u003cp\u003eZeta Potential analysis of optimized folic acid loaded binary ethosomes formulation (F6).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/e944319b5571d1c92582ee90.png"},{"id":100594870,"identity":"dcc8253c-4464-46d4-ba56-a7130cc39bf1","added_by":"auto","created_at":"2026-01-19 13:45:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44741,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR Analysis of A (Folic Acid), B (Folic Acid binary ethosomes) and C (Soyalecithin).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/d190320a4fe08d0a07cf027b.png"},{"id":100562378,"identity":"2db439a8-d891-4b90-84d5-4b6ab5611192","added_by":"auto","created_at":"2026-01-19 08:44:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222044,"visible":true,"origin":"","legend":"\u003cp\u003eTEM Analysis of optimized folic acid loaded binary ethosomes formulation (F6).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/65285ce9f471a601d28dc1b3.png"},{"id":100562356,"identity":"cad65ea0-2a85-45b8-aff0-bfed049a5d36","added_by":"auto","created_at":"2026-01-19 08:44:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74431,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity Graph of formulated folic acid loaded binary ethosomes gel.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/136fe692b9c94f000d88f8fa.png"},{"id":100594831,"identity":"55953da5-b955-47dd-b2a2-4ae6d690c798","added_by":"auto","created_at":"2026-01-19 13:45:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn-Vitro\u003c/em\u003e comparative drug release analysis between folic acid and folic acid loaded binary ethosomes gel.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/c10d11d612a66fefc1f9907d.png"},{"id":100562301,"identity":"31cf312b-ecf8-499c-be7e-d3d024722266","added_by":"auto","created_at":"2026-01-19 08:44:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":47881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEx-vivo\u003c/em\u003e comparative drug permeation analysis between folic acid and folic acid loaded binary ethosomes gel.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/1633d320c145bb3d47cd7e44.png"},{"id":103715590,"identity":"7290383b-003a-4ad2-b149-c5b0342e2179","added_by":"auto","created_at":"2026-03-02 05:40:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1730945,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8382960/v1/f276502d-dc3b-4fd5-a9f0-f7ccf98d1079.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Formulation and evaluation of folic acid-loaded binary ethosomes for enhanced skin permeation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetes-related foot ulcers are a serious health concern for those who have the disease since they can have detrimental impacts on both physical and mental health. Recurrence, amputation, and death risks are also linked to diabetes-related foot ulcers. Effective prevention and management techniques are required in diabetes patients to lower morbidity and mortality. Approximately 20% of diabetics have both foot ulcers and neuropathy, compared to an estimated 80% who have foot ulcers and 50% who have neuropathy. Most cutaneous ulcers in diabetic foot problems are caused by mechanical or physical trauma. Neutrophils, fibroblasts, and leukocyte levels are reduced in diabetic individuals, though inflammatory cells have lower efficiency at the ulcer site. The risk of foot issues is further increased by additional macrovascular disease risk factors like hypertension, hyperlipidemia, and smoking. The main contributor to the onset of diabetic foot ulcers is inadequate glucose management.\u003c/p\u003e \u003cp\u003eIts primary role is to promote the formation of new cells in the body, which includes DNA synthesis, repair, and methylation. Folic acid is a water-soluble B vitamin that can be found naturally in a range of foods including legumes, vegetables, liver, and milk [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It participates in a variety of metabolic activities important for cell division, including purine and pyrimidine production, DNA/RNA biosynthesis, and amino acid metabolism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Because mammals cannot synthesize folic acid, they must obtain it from their diet. Even though folic acid is widely available in foods, folic acid deficiency is nevertheless common due to a variety of factors such as a poor diet [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] disease-related malabsorption, medication-related depletion [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], or vitamin B12 deficiency [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Folic acid deficiency has been associated with several health problems, such as anemia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], cardiovascular diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], neural tube defects in newborns, neuropsychiatric dysfunction [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], depression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], inflammatory diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and eye diseases [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFolic acid considerably reduced levels of lipid peroxidation, protein nitro tyrosination, and glutathione depletion in wounds, as vitamins play a vital role in the prevention and treatment of diabetic foot ulcers and wounds, among other things. Folic acid supplementation's role is to heal damaged wounds by decreasing oxidative stress. Vitamins are the ideal sources since they are inexpensive, widely available, cure disease at its base, are toxic-free, and have no or minimum side effects. Folic acid has antioxidant, anticancer, cardiovascular, and neuroprotective effects. Folic acid's antioxidant activity is mediated through multiple mechanisms, including a decrease in plasma homocysteine concentrations, which may increase total antioxidant capacity and reduce reactive oxygen species formation. Thus its inbuilt activity of antioxidant its plays an important role in inflammatory, inhibits microbial growth, and healing wounds, or/and promotes the synthesis of collagen fibers to increase supply of vital to the affected area or wound site that enhanced the wound healing process by expanding the feasibility of collagen fibrils, increased the collagen strands strength, increased the circulation or avoidance of cell harm, or promotes the synthesis of DNA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Folic acid, a low soluble and low permeability drug, was chosen as the model of biopharmaceutical classification system class IV. It may also have dietary effects, poor absorption, and significant variation in inter- and intra-patient dose response [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFolic acid has poor solubility in water or any other commonly used media, and is poorly absorbed by the body. Vitamins degrade when exposed to the outside environment, which is one of the reasons why developing pharmaceutical dose forms are difficult. To address this issue, various ways have been considered, including the development of suspensions, solubilization in organic solvents, the use of drug salts, the development of prodrugs, and the development of innovative drug delivery systems. In addition, insoluble solid drug forms pose a significant problem in terms of their limited bioavailability in common media such as water [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As liposomes are unstable and localize predominantly to the reticuloendothelial system, mainly the liver and spleen, complex drug carriers such as liposomes have limited utility. As conventional solvents and solubility enhancers are toxic and irritating when injected into humans, they are mainly used to treat acute, life-threatening diseases where medical experts are continually available to administer palliative treatments to counteract the negative effects of the solvents. Solvents/enhancers that are water miscible also have the disadvantage of causing medicine to precipitate rapidly when exposed to water. When a medication candidate's water solubility is insufficient to allow solution formulations, solubility enhancers are frequently used to boost solubility. A solubility enhancer can increase solubility several orders of magnitude. Solubility enhancers such as propylene glycol, polyethylene glycols, ethanol, and sorbitol are commonly used [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Adding a co-solvent to the solvent can lower its dielectric constant, thereby boosting the solubility of hydrophobic molecules. With solubility enhancers, drug precipitation can occur when dilutions of solvents are made, as well as tissue injury and pain during injection. They are more difficult to administer and prepare because of their low dissolving rate, poor bioavailability, and poor penetration [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, novel dosage formulations are necessary to overcome the solubility and permeability difficulties of such therapeutic candidates. The compositions of ethosomes that are hydro/alcoholic/phospholipid systems with a high alcohol concentration. A system that increases skin penetration and has the ability to produce ethosomes, which are \"soft\" vesicles made of phospholipid when water and ethanol (alcohol) are present.\u003c/p\u003e \u003cp\u003eEthosomes offer numerous significant benefits, such as boosting therapeutic efficacy, enhancing patient comfort and compliance, and reducing overall treatment expenses. Ethosomes are non-invasive drug delivery systems that enable drugs to penetrate deep layers of skin before entering the bloodstream. The improved distribution of bioactive substances through cellular and epidermal membranes via an ethosomes carrier poses a number of challenges as well as opportunities for further research and the creation of innovative, better treatments[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a result, developing folic acid-loaded ethosomes may be a viable option for improving solubility and permeability in diabetic foot ulcers. As a result, the invention is about developing novel dosage forms as ethosomes formulations can resolve the issues of solubility and permeability, providing a new avenue for treatment approaches for ulcers that are easier to apply in the form of topical gels and have fewer side effects when compared to other routes of administration. Furthermore, shorter half-lives have a superior safety profile than other routes of administration.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eFolic acid was bought from Dhamtec Pharma and Consultants in Mumbai, India. Soya lecithin, ethanol, propylene glycol, and cholesterol were purchased from Sigma-Aldrich Chemicals (Germany). All other compounds were classified using analytical grades.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation method of folic acid loaded binary ethosomes\u003c/h2\u003e \u003cp\u003eFolic acid binary ethosomes were developed by cold method [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], where 300\u0026ndash;400 mg of lipids, 10\u0026ndash;30% v/v of ethanol, 5mg of Folic acid (the drug), propylene glycol (PG)- 5% and percentage of water was primarily taken. Further, the former solution was kept on a magnetic stirrer (Remi Equipment, Mumbai) and distilled water was added with continuous stirred (700 rpm) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The solution formed was retained at 4\u0026deg;C in refrigerator, and sonication was performed for 3 cycles with probe sonicator for 5 min (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of folic acid loaded binary ethosomes formulations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDrug (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSoya lecithin\u003c/p\u003e \u003cp\u003e(mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthanol (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePropylene glycol (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of prepared folic acid loaded binary ethosomes\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Vesicles size and PDI\u003c/h2\u003e \u003cp\u003eVesicle size distribution and PDI of formulated binary ethosomes were done by Malvern zeta sizer 2000 MU (Malvern instrument UK) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Zeta potential\u003c/h2\u003e \u003cp\u003eThe zeta potential of the preparation may analyze using a Malvern zeta sizer 2000MU (Malvern instrument UK). A value of 25 mV, regardless of its polarity, may serve as the threshold that distinguishes surfaces with low charge from those with high charge. The importance of zeta potential lies in its capacity to be correlated with the stability of colloidal dispersions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.3.3 Transmission electron microscopy (TEM\u003c/b\u003e)\u003c/h2\u003e \u003cp\u003eThe shape and surface morphology of formulated binary ethosomes was examined using TEM (TEM-Tecknai G2, FEI, 200 kV, Tokyo, Japan) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 FTIR Spectroscopy\u003c/h2\u003e \u003cp\u003eThe functional groups of formulated binary ethosomes were examined using an FTIR spectrometer (IRAffinity-1S, Shimadzu, Kyoto, Japan) with KBr pellets and FTIR wave numbers ranging from 4000 cm \u003csup\u003e-1\u003c/sup\u003e to 450 cm\u003csup\u003e-1\u003c/sup\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 Entrapment efficiency %\u003c/h2\u003e \u003cp\u003eThe produced binary ethosomes ability to entrap drug was determined using centrifugation method. A cold centrifuge was used to spin the centrifugation tubes at a speed of 15000 RPM for 30 minutes at 4\u0026deg;C after adding 1 ml of a drug-loaded binary ethosomes suspension. The unentrapped drug concentration was determined spectrophotometrically at 283 nm. The amount of free drug in the supernatant represents the total amount of untrapped drug. The entrapment efficiency was then calculated using the given formula (1) and expressed as a percentage of drugs trapped [68].\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{E}\\text{E}\\text{%}\\:=\\frac{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{d}\\text{r}\\text{u}\\text{g}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}-\\text{F}\\text{r}\\text{e}\\text{e}\\:\\text{d}\\text{r}\\text{u}\\text{g}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{d}\\text{r}\\text{u}\\text{g}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}}\\times\\:100\\)\u003c/span\u003e \u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;Eq.\u0026nbsp;(1\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of folic acid loaded binary ethosomes gel\u003c/h2\u003e \u003cp\u003eGels were prepared using carbopol (grade 934k in 0.5% and 1% w/v concentrations) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Carbopol 934k was precisely weighed, soaked in the least amount of water, and continuously stirred at 400 rpm until homogenous gel bases with a smooth texture and no lumps were formed. After being weighed, the optimized binary ethosomes loaded with 5 mg of folic acid were added to the swollen polymer. It was mixed using a homogenizer to form homogenous gels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A sufficient quantity of TEA (triethanolamine) was added drop by drop to neutralize pH 6.8 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A gel with 200 mg of pure medication was also prepared using the same procedures. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e lists the components and amounts needed to form a folic acid-loaded binary ethosomes gel.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition of folic acid control gel and folic acid loaded binary ethosomes gel.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulations code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolymer name (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeight of the polymer (w/v) (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthosomes loaded into the gel base\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePurified water\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbopol 934k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthosomes suspension equivalent to 5\u0026thinsp;mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUpto 100 ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbopol 934k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEthosomes suspension equivalent to 5\u0026thinsp;mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUpto 100 ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbopol 934k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u0026thinsp;mg free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUpto 100 ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbopol 934k\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 mg free\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUpto 100 ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Characterization of prepared folic acid loaded binary ethosomes gel\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Physical Evaluation\u003c/h2\u003e \u003cp\u003eThe organoleptic properties were evaluated to assess the folic acid-loaded ethosomes gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Determination of pH\u003c/h2\u003e \u003cp\u003eA pH meter was used to measure pH of the folic acid loaded ethosomes gel. Three duplicate readings of the data were taken, and the average value was determined pH meter (Labtronics, India) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Spreadability\u003c/h2\u003e \u003cp\u003eSpreadability of gel was assessed by glass slide method where 0.5 g of gel was placed on the lower glass slide in 1 cm marked circle on it and using the weight of 500 g, the upper slide is placed on the lower glass slide for 5 min and measured the gel diameter by its spreadability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eS =\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\mathbf{M}\\times\\:\\mathbf{L}}{\\mathbf{T}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.Eq.\u0026nbsp;(2\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhere, S\u0026thinsp;=\u0026thinsp;Spreadability; M\u0026thinsp;=\u0026thinsp;Weight tied on the upper plate; L\u0026thinsp;=\u0026thinsp;Length (cm) of glass plate; T\u0026thinsp;=\u0026thinsp;Time taken (second)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4 Viscosity\u003c/h2\u003e \u003cp\u003eViscosity of formulated gel was assessed by viscometer (Anton Paar MCR 72 rheometers, India). The viscosity of gel was evaluated by temperature shear sweep method at temperature ranging from 25\u0026deg;C to 60\u0026deg;C and the shear rate ranging from 20\u0026ndash;100 rpm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.6.5 \u003cem\u003eIn-vitro\u003c/em\u003e drug release study\u003c/h2\u003e \u003cp\u003eThe folic acid-loaded ethosomes gel was placed into a pre-soaked dialysis membrane (molecular weight cut-off: 12\u0026ndash;14 kDa), securely sealed, and suspended in phosphate buffer (pH 7.4) maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. Samples were withdrawn at predetermined intervals and immediately replaced with an equal volume of fresh buffer to maintain sink conditions. Using a dialysis bag (diameter: 2.5 cm), the release of folic acid from the formulation was assessed at time intervals of 5, 15, 30, 45, 60, 75, 90, 120, 180, 360, 720, 1080, and 1440 minutes. The absorbance of each sample was measured using a UV\u0026ndash;Visible spectrophotometer (Shimadzu 1800, Japan) at 283 nm. The Higuchi, Korsmeyer\u0026ndash;Peppas, zero-order, and first-order release models were among the kinetic models to which the cumulative release data were fitted [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.6.7 \u003cem\u003eEx-vivo\u003c/em\u003e permeation study of folic acid loaded binary ethosomes gel\u003c/h2\u003e \u003cp\u003eEx-vivo drug permeability was measured using Franz's diffusion cell method and a semipermeable membrane made from freshly excised goat abdomen skin. For subsequent use, the skin was first washed with physiological salt solution, followed by distilled water, and then soaked in phosphate-buffered saline (pH 7.4) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The skin was clamped with Franz's diffusion cell containing 6.5 ml of phosphate-buffered saline and 1.7 cm\u003csup\u003e2\u003c/sup\u003e of effective permeation area. In Franz's diffusion cell, the gel was carefully weighed and added to the donor compartment while being continually stirred with a magnetic stirrer (Expo India Ltd., Mumbai, India) at 37.5\u0026deg;C. To maintain the sink condition, an equal volume of blank receptor fluid was injected into the receptor compartment at predetermined intervals (0, 5, 15, 30, 45, 60, 120, 180, 360, 720, 1080, 1440 and 2160 minutes) and the drug concentration was determined using UV spectroscopy. As a result of the triplicate experiment, the apparent permeation coefficient, flux, and permeation amount were determined using the equation below (3) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:Apparent\\:permeation\\:coefficient=\\:\\frac{Flux}{Area\\:of\\:permeation\\:\\times\\:\\:Amount\\:of\\:folic\\:acid\\:in\\:formulation}\\)\u003c/span\u003e \u003c/span\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;. \u003cb\u003eEq.\u0026nbsp;(3)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Stability study of folic acid loaded binary ethosomes gel\u003c/h2\u003e \u003cp\u003eThe formulated gel was assessed for statistical analysis as per the ICH guidelines. The prepared folic acid loaded binary ethosomes gel was kept in the dark for 90 days at 4 \u0026ordm;C. Analyze the size of the vesicles, PDI, and zeta potential were performed to determine the stability of the formulated binary ethosomes gel [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Result of folic acid loaded binary ethosomes\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Vesicles size, PDI and zeta potential\u003c/h2\u003e \u003cp\u003eBinary ethosomes were measured using a Malvern zeta sizer 2000MU to determine their size, PDI, and zeta potential. The size of vesicles influences drug disposition, diffusion, release factors, and skin absorption. The size and amount of drug loaded into vesicles are influenced by a variety of factors, including chemical structure, the type and quantity of drug and lipid used, and experimental procedures [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the vesicles size of optimized binary ethosomes (F6) was 401.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 nm. A PDI value in the range of 0.1\u0026ndash;0.7 demonstrates the presence of uniform size vesicles in the formulation, indicating homogeneous dispersion and a narrow size distribution.\u003c/p\u003e \u003cp\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The PDI of the prepared gel was determined to be within this range, i.e., 0.3, indicating great vesicles size distribution homogeneity. As indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the zeta potential of optimized binary ethosomes (F6) was \u0026minus;\u0026thinsp;29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mV. The negative charge on the zeta potential implies electrostatic repulsion between the charged ions, which is a crucial element in preventing vesicles aggregation. Thus, the Zeta potential reflects the formulation's electrostatic stability; the greater the negative charge, the greater the stability [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2% Entrapment efficiency\u003c/h2\u003e \u003cp\u003eThe entrapment efficiency (%) of the optimized formulation (F6) were determined to be 82.24%. The high entrapment efficiency in formulation is attributed to the presence of a membrane composed of ethanol or propylene glycol, as well as an increased number of C-H bonds between the hydrophilic tail structures and the lipophilic nature of the folic acid. The hydrophobic structure of the membrane allows for sustained drug release. Such binary ethosomes, like other vesicles that are considered stable vesicles with higher drug permeability release, operate as drug depots [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.1.3 FTIR analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe peaks of Folic acid (A) are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The C-O stretching is at 1072.44 cm-1, the CH2 bending is at 1401 cm-1, the C\u0026thinsp;=\u0026thinsp;C stretching is at 1683 cm-1, and the C-H stretching is at 1944 cm-1 and 3036 cm-1. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(C)showed soya lecithin peaks are shown at 1039 cm\u003csup\u003e-1\u003c/sup\u003e indicates the C-O stretching, 1308 cm\u003csup\u003e-1\u003c/sup\u003e showed CH\u003csub\u003e2\u003c/sub\u003e bending, 1633 cm-\u003csup\u003e1\u003c/sup\u003e showed C\u0026thinsp;=\u0026thinsp;C stretching and 1965 cm\u003csup\u003e-1\u003c/sup\u003e \u0026amp; 2974 cm\u003csup\u003e-1\u003c/sup\u003e showed the C-H bending, while the FTIR Peaks of folic acid loaded binary ethosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(B)) are shown at 1043 cm-\u003csup\u003e1\u003c/sup\u003erevealed C-O stretching, 1452 cm-1 due to CH\u003csub\u003e2\u003c/sub\u003e bending, 1726 cm\u003csup\u003e-1\u003c/sup\u003e showed C\u0026thinsp;=\u0026thinsp;O stretching, 2062 cm-\u003csup\u003e1\u003c/sup\u003e and 2848 cm \u003csup\u003e-1\u003c/sup\u003e indicates the C\u0026thinsp;=\u0026thinsp;H stretching. The FTIR Spectrum of folic acid loaded binary ethosomes demonstrated that there was no peak formation occurs in the formulation in comparative to the added ingredients. Thus, we can conclude that there was no chemical interaction shown in the formulation with the active ingredients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 TEM analysis\u003c/h2\u003e \u003cp\u003eThe shape of nanovesicles were investigated using transmission electron microscopy (TEM) by placing the nanovesicles on a 300-mesh coated carbon film and swirling for 10 minutes until the correct air dried. The sample was then mixed with a 2% w/v phosphotungstic acid solution and dried at room temperature before the spectra was examined [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The results of the TEM analysis are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The combination of 10\u0026ndash;30% v/v ethanol and 5% propylene glycol resulted in spherical shaped, nonporous smooth surface and multilamellar binary ethosomes with increased and high entrapped drug concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Evaluation of folic acid loaded binary ethosomes gel\u003c/h2\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Physical Evaluation\u003c/h2\u003e \u003cp\u003eThe formulated gel exhibited a yellowish appearance and was odorless.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 pH measurement\u003c/h2\u003e \u003cp\u003eThe pH of the formulated gel was measured to be 5.5, indicating suitability for skin application and minimal risk of irritation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Spreadability\u003c/h2\u003e \u003cp\u003eThe spreadability of the gel was measured using the glass slide method. Both the initial and final diameters before and after spreading were recorded as 44 mm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4 Viscosity of gel\u003c/h2\u003e \u003cp\u003eViscosity is important in both stability and applicability. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the viscosity of the formed folic acid loaded binary ethosomes gel reduces with increasing shear rate, indicating pseudoplastic behavior. The viscosity reduces due to the colloidal network structure in the direction of shear rate. The polymer chains disarranged to fit in the flow of the stream with increased shear stress. This direction reduced the material's internal resistance, allowing for a larger shear rate with each successive shearing force. This resulted in a decrease in apparent viscosity. The curve of the formulation graph demonstrates non-Newtonian essence. The rheological characteristics listed above were excellent for the formulations designed for topical application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.2.5 \u003cem\u003eIn-vitro\u003c/em\u003e drug release analysis\u003c/h2\u003e \u003cp\u003eThe release profile of folic acid loaded binary ethosomes gel and folic acid was compared in-vitro on the dialysis bag at pH 7.4 (phosphate buffer), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The folic acid loaded binary ethosomes gel demonstrated high drug release kinetics, with 76.21% at 1440 minutes compared to 39.32% at 1440 minutes for the folic acid loaded gel. As a result of the nano formulation, binary ethosomes loaded gel demonstrated a greater \u003cem\u003ein-vitro\u003c/em\u003e drug release profile. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, kinetic modeling indicates that the Korsmeyer-Peppas model has a higher R\u003csup\u003e2\u003c/sup\u003e value than the other models. The R\u003csup\u003e2\u003c/sup\u003e value of the folic acid-loaded binary ethosomes gel was 0.9843, and the release exponent (n) of the Korsmeyer-Peppas model was less than 0.45 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Consequently, during drug release, the produced folic acid-loaded binary ethosomes gel followed Fick's law of diffusion. It was strongly concluded that the formulated folic acid loaded binary ethosomes gel can be used for the topical delivery of the local action in the condition of diabetic foot ulcer because of the adhesive and hydrophilic nature of the gelling agent, i.e., propylene glycol, as well as their other physical properties and the effective permeation of binary ethosomes loaded gel due to Fickian based diffusion [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eData of release kinetics profile of folic acid loaded binary ethosomes gel.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eZero order\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eFirst order\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eHiguchi\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eKorsemeyer- Peppas\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eFolic acid loaded binary ethosomes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003csub\u003eO\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003csub\u003eO\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK\u003csub\u003eO\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eK\u003csub\u003eO\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.9843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.2.6 \u003cem\u003eEx-vivo\u003c/em\u003e permeation study\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEx-vivo\u003c/em\u003e permeation analysis was performed on goat abdomen skin at pH 7.4 to compare the data of folic acid and folic acid-loaded binary ethosomes gel, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Results showed that the folic acid loaded binary ethosomes gel showed high retention of drug on the skin and the flux value in comparison with the folic acid loaded binary ethosomes gel at same time of duration i.e., 2160 minutes. Hence, folic acid loaded binary ethosomes gel should be indicated for topical use in the treatment of foot ulcer because of high retention of drug due to binary ethosomes formulation of the drug in aspect of folic acid formulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Stability studies\u003c/h2\u003e \u003cp\u003eLipid vesicular formulation, such as folic acid-loaded binary ethosomes gel, frequently undergo changes such as fusion or dissertation while in storage. The drug leaking from the vesicles affects VS, PDI, and ZP while decreasing the EE% [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. To improve the physical and chemical stability of binary ethosomes gel, it was recommended that it be stored in a refrigerator (4\u0026deg;C). Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the VS, PDI, ZP, and %EE of freshly prepared folic acid-loaded binary ethosomes gel after three months of refrigeration (4\u0026deg;C). These results showed that the folic acid loaded binary ethosomes gel was stable because it did not significantly differ from those obtained before storage. When kept at 4\u0026deg;C, binary ethosomes gel (which contains PG as another alcohol) was good stability [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Therefore, it was proposed that PG increases viscosity and antihydrolysis to improve binary ethosomes gel stability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of storage on properties of selected formulation (F6).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFresh prepared F6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF6 after 3 months of storage at 4\u0026thinsp;\u0026deg;C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVesicles size (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e401.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e420\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-29.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u0026thinsp;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e%EE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e82.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e71.10 \u0026plusmn;\u0026nbsp;1.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe present study successfully developed and characterized a folic acid-loaded binary ethosomes gel using propylene glycol as a permeation enhancer for potential use in diabetic foot ulcer treatment. The optimized formulation (F6) demonstrated good stability and nanoscale uniformity with a vesicle size of 401.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 nm, a PDI of 0.32, and a zeta potential of \u0026minus;\u0026thinsp;29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mV. Entrapment efficiency was notably high at 82.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%, with a drug loading of 44.16%, suggesting efficient incorporation of folic acid into the vesicles. \u003cem\u003eIn-vitro\u003c/em\u003e release studies demonstrated that the binary ethosomes gel achieved a cumulative drug release of 76.21% at 1440 minutes, nearly double that of the conventional gel (39.32%). Ex vivo permeation through goat skin revealed a flux value of 4.98 \u0026micro;g/cm\u0026sup2;/h at 2160 minutes, confirming enhanced skin penetration and retention. Stability studies indicated minimal changes in vesicle size, PDI, zeta potential, and entrapment efficiency over 3 months at 4\u0026deg;C, validating good storage stability. Overall, the incorporation of propylene glycol significantly improved the solubility, permeability, and retention of folic acid in the binary ethosomes gel, making it a promising topical delivery system for diabetic foot ulcer management with potential to enhance therapeutic efficacy and patient compliance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003e\u0026ldquo;The authors declare no conflict of interest\u0026rdquo;.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e\u0026ldquo;Conceptualization, BK, and SSR; Supervision, BK; Formal analysis, SSR; Methodology, BK, SSR and PV; Writing\u0026mdash;original draft, SSR and BK; Writing\u0026mdash;review and editing, SSR, PV, BK, AA and IK; Software: SSR, AA and IK. All authors have reviewed and agreed to the published version of the manuscript\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAll the authors extend their appreciation to the Ongoing Research Funding Program, (ORF-2026-959), King Saud University, Riyadh, Saudi Arabia, for supporting this project.\u0026rdquo;\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003e\u0026ldquo;Data are contained within the article\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIyer R, Tomar SK Folate: a functional food constituent 2009; 74: R114\u0026ndash;R122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLyon P, Strippoli V, Fang B, Cimmino L (2020) B vitamins and one-carbon metabolism: implications in human health and disease. 12:2867\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarias N, Ho N, Butler S, Delaney L, Morrison J, Shahrzad S et al (2015) The effects of folic acid on global DNA methylation and colonosphere formation in colon cancer cell lines. 26:818\u0026ndash;826\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHildebrand LA, Dumas B, Milrod CJ, Hudspeth JC Folate deficiency in an urban safety net population 2021; 134: 1265\u0026ndash;1269\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIimura Y, Kurokawa T, Kanemoto Y, Yazawa K, Tsurita G, Ahiko Y et al Severe thrombocytopenia induced by chemotherapy after total gastrectomy: a report of three cases 2022; 60: 36\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArcot J, Shrestha A (2005) Folate: methods Anal 16:253\u0026ndash;266\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFishman SM, Christian P, West KP Jr The role of vitamins in the prevention and control of anaemia 2000; 3: 125\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFleming CR. Modern nutrition in health and disease., Mautice E, Shils JA, Olson, Shike M (1994) 1885 pp. \u003cspan\u003e$\u003c/span\u003e99.50 (2 volumes) Lea \u0026amp; Febiger, Malvern, Pennsylvania, ISBN 0-8121-1485-X 1994\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErdman JW Jr, Macdonald IA, Zeisel SH (2012) Present knowledge in nutrition. Wiley\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuthie SJ Folic acid deficiency and cancer: mechanisms of DNA instability 1999; 55: 578\u0026ndash;592\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWald DS, Law M, Morris JK (2002) Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. 325:1202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShea TB, Lyons-Weiler J, Rogers E, Homocysteine (2002) folate deprivation Alzheimer neuropathology 4:261\u0026ndash;267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalade J, Onaolapo AY, Onaolapo OJ The role of folate-supplementation in depression: a narrative review 2021; 10: 115\u0026ndash;122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki H, Kunisawa J Vitamin-mediated immune regulation in the development of inflammatory diseases 2015; 15: 212\u0026ndash;215\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones P, Lucock M, Scarlett CJ, Veysey M, Beckett EL Folate and inflammation\u0026ndash;links between folate and features of inflammatory conditions 2019; 18: 100104\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSijilmassi O Folic acid deficiency and vision: a review 2019; 257: 1573\u0026ndash;1580\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadhakrishnan R Solubility enhancer and use thereof 2015\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhalani DV, Nutan B, Kumar A, Singh Chandel AK (2022) Bioavailab enhancement techniques poorly aqueous soluble drugs Ther 10:2055\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuksaeree J, Simchareon W, Pichayakorn W (2021) Effect of glycols permeation enhancer on the release and permeation of meloxicam-natural rubber film through pig skin. 66:102874\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTouitou E, Dayan N, Bergelson L, Godin B, Eliaz M (2000) Ethosomes\u0026mdash;novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. 65:403\u0026ndash;418\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFathalla D, Youssef EMK, Soliman GM Liposomal and ethosomal gels for the topical delivery of anthralin: preparation, comparative evaluation and clinical assessment in psoriatic patients 2020; 12: 446\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastro NR, Cristal dos Santos CP, de Campos VEB, Cardoso V, Vermelho AB, dos Santos EP et al (2021) Development of hybrid vesicular nanosystems composed of lipids and chitosan for octyl methoxycinnamate encapsulation. ; 608: 125476\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRakesh R, Anoop KR Formulation and optimization of nano-sized ethosomes for enhanced transdermal delivery of cromolyn sodium 2012; 4: 333\u0026ndash;340\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShumilov M, Bercovich R, Duchi S, Ainbinder D, Touitou E (2010) Ibuprofen transdermal ethosomal gel: characterization and efficiency in animal models. 6:569\u0026ndash;576\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampos EVR, Proen\u0026ccedil;a PLF, da Costa TG, de Lima R, Fraceto LF, de Araujo DR (2022) Using chitosan-coated polymeric nanoparticles‐thermosensitive hydrogels in association with limonene as skin drug delivery strategy 2022; : 9165443\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan ZU, Razzaq A, Khan A, Rehman NU, Khan H, Khan T et al Physicochemical characterizations and pharmacokinetic evaluation of pentazocine solid lipid nanoparticles against inflammatory pain model 2022; 14: 409\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIjaz M, Akhtar N (2020) Fatty acids based α-Tocopherol loaded nanostructured lipid carrier gel: In vitro and in vivo evaluation for moisturizing and anti‐aging effects. ; 19: 3067\u0026ndash;76\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkhtar N, Akhtar N Development of stable tocopherol succinate-loaded ethosomes to enhance transdermal permeation: in vitro and in vivo characterizations 2022; 21: 4942\u0026ndash;4955\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharif A, Akhtar N Fabrication of niosomes containing N-acetyl glucosamine: In-vitro and ex-vivo characterizations 2020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePereira TA, Guerreiro CM, Maruno M, Ferrari M, Rocha-Filho PA (2016) Exotic vegetable oils for cosmetic o/w nanoemulsions: In. vivo evaluation 21:248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil RM, Abdelbary GA, Basha M, Awad GEA, El-Hashemy HA (2017) Design and evaluation of proniosomes as a carrier for ocular delivery of lomefloxacin HCl. 27:118\u0026ndash;129\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePokharana M, Vaishnav R, Goyal A, Shrivastava A (2018) Stab Test guidelines Pharm Prod 8:169\u0026ndash;175\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIyer SS, Barr WH, Karnes HT (2006) Profiling in vitro drug release from subcutaneous implants: a review of current status and potential implications on drug product development. 27:157\u0026ndash;170\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNair R, Arun Kumar KS, Vishnu Priya K, Sevukarajan M Recent advances in solid lipid nanoparticle based drug delivery systems 2011; 3: 368\u0026ndash;384\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThang L, Hanh N, Duong D Study on cause-effect relations and optimization of exemestane-loaded nanostructured lipid carriers 2017; 9: 68\u0026ndash;74\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSargam Y, Wang K, Tsyrenova A, Liu F, Jiang S (2021) Effects of anionic and nonionic surfactants on the dispersion and stability of nanoSiO2 in aqueous and cement pore solutions. 144:106417\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasiri L, Rajabzadeh G, Bostan A (2017) Physicochemical properties and release behavior of Span 60/Tween 60 niosomes as vehicle for α-Tocopherol delivery. 84:471\u0026ndash;478\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJunyaprasert VB, Singhsa P, Suksiriworapong J, Chantasart D Physicochemical properties and skin permeation of Span 60/Tween 60 niosomes of ellagic acid 2012; 423: 303\u0026ndash;311\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu X, Shi Y, Wang H, Zhao X, Sun Q, Huang Y et al (2019) Ethosomal gel for improving transdermal delivery of thymosin β-4 : 9275\u0026ndash;9284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePyrz WD, Buttrey DJ (2008) Particle size determination using TEM: a discussion of image acquisition and analysis for the novice microscopist. 24:11350\u0026ndash;11360\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChourasia MK, Kang L, Chan SY Nanosized ethosomes bearing ketoprofen for improved transdermal delivery 2011; 1: 60\u0026ndash;67\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbd El-Alim SH, Kassem AA, Basha M (2014) Proniosomes as a novel drug carrier system for buccal delivery of benzocaine. 24:452\u0026ndash;458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShirsand SB, Para MS, Nagendrakumar D, Kanani KM, Keerthy D (2012) Formulation and evaluation of Ketoconazole niosomal gel drug delivery system. 2:201\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBudhiraja A, Dhingra G Development and characterization of a novel antiacne niosomal gel of rosmarinic acid 2015; 22: 723\u0026ndash;730\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdulbaqi IM, Darwis Y, Khan NAK, Assi RA, Khan AA Ethosomal nanocarriers: the impact of constituents and formulation techniques on ethosomal properties, in vivo studies, and clinical trials 2016: 2279\u0026ndash;2304\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArora D, Nanda S (2019) Quality by design driven development of resveratrol loaded ethosomal hydrogel for improved dermatological benefits via enhanced skin permeation and retention. 567:118448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaliwal S, Tilak A, Sharma J, Dave V, Sharma S, Yadav R et al (2019) Flurbiprofen loaded ethosomes-transdermal delivery of anti-inflammatory effect in rat model. 18:1\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma P, Pathak K (2012) Nanosized ethanolic vesicles loaded with econazole nitrate for the treatment of deep fungal infections through topical gel formulation. 8:489\u0026ndash;496\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic foot ulcer, Folic acid, Binary ethosomes, Gel","lastPublishedDoi":"10.21203/rs.3.rs-8382960/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8382960/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetes-related foot ulcers are a serious health concern and can have detrimental impacts on both physical and mental health. The objective of present study was to prepare folic acid-loaded binary ethosomes. The cold technique was used to prepare the folic acid-loaded binary ethosomes formulation, and evaluated for vesicles size, PDI, zeta potential, FTIR, entrapment efficiency, drug loading. Further the optimized binary ethosomes formulation was converted to gel formulation and assessed for in-vitro drug release, ex-vivo skin penetration, and formulation stability. The optimized binary ethosomes formulation F6 exhibited a vesicles size of 401.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 nm, with a PDI of 0.32 and a zeta potential of \u0026minus;\u0026thinsp;29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mV. The entrapment efficiency was found to be 82.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%. The comparative \u003cem\u003ein vitro\u003c/em\u003e drug release profile of the binary ethosomes formulation gel showed a significantly higher release (76.21%) compared to the control gel (39.32%) at 1440 minutes. In \u003cem\u003eex vivo\u003c/em\u003e permeation studies, the flux of the binary ethosomes -loaded gel was 4.98 \u0026micro;g/cm\u0026sup2;/h at 2160 minutes. Organoleptic evaluation revealed that the formulated gel was yellowish in color, odorless, and had a pH of 5.5. Rheological studies confirmed that the formulation followed a non-Newtonian shear-thinning behavior, which is favorable for topical application. Overall, optimized F6 was identified as the best formulation based on its physicochemical properties, drug release performance, and rheological characteristics. Hence, the developed folic acid loaded binary ethosomes gel showed potential for improving folic acid efficacy in topical administration.\u003c/p\u003e","manuscriptTitle":"Formulation and evaluation of folic acid-loaded binary ethosomes for enhanced skin permeation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 08:36:29","doi":"10.21203/rs.3.rs-8382960/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a6c5267-094c-4d3c-9272-e7bf71d0b315","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T05:40:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 08:36:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8382960","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8382960","identity":"rs-8382960","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}