Metformin HCl-loaded transethosomal gel; development, characterization, and antidiabetic potential evaluation in the diabetes-induced rat model

Abstract

Herein we designed, optimized, and characterized the Metformin Hydrochloride Transethosomes (MTF-TES) and incorporate them into Chitosan gel to develop Metformin Hydrochloride loaded Transethosomal gel (MTF-TES gel) that provides a sustained release, improved transdermal flux and improved antidiabetic response of MTF. Design Expert® software (Ver. 12, Stat-Ease, USA) was applied for the statistical optimization of MTF-TES. The formulation with Mean Particle Size Distribution (MPSD) of 165.4 ± 2.3 nm, Zeta Potential (ZP) of −21.2 ± 1.9 mV, Polydispersity Index (PDI) of 0.169 ± 0.033, and MTF percent Entrapment Efficiency (%EE) of 89.76 ± 4.12 was considered to be optimized. To check the chemical incompatibility among the MTF and other formulation components, Fourier Transform Infrared (FTIR) spectroscopy was performed and demonstrated with no chemical interaction. Surface morphology, uniformity, and segregation were evaluated through Transmission Electron Microscopy (TEM). It was revealed that the nanoparticles were spherical and round in form with intact borders. The fabricated MTF-TES has shown sustained release followed by a more pronounced effect in MTF-TES gel as compared to the plain MTF solution (MTFS) at a pH of 7.4. The MTF-TES has shown enhanced permeation followed by MTF-TES gel as compared to the MTFS at a pH of 7.4. In vivo antidiabetic assay was performed and results have shown improved antidiabetic potential of the MTF-TES gel, in contrast to MTF-gel. Conclusively, MTF-TES is a promising anti-diabetic candidate for transdermal drug delivery that can provide sustained MTF release and enhanced antidiabetic effect.

Keywords:

• Transethosomes
• antidiabetic effect
• nanoparticle
• metformin hydrochloride transethosomes
• statistical optimization

1. Introduction

Diabetes is a chronic metabolic illness distinguished by hyperglycemia having heterogeneous etiopathology which includes insulin resistance, lack of its secretion, or both (Patiño-Herrera et al., 2019). It is one of the major causes of mortality having several concomitant complications such as hypertension, varied lipid metabolism, and cardiovascular diseases (Torabian et al., 2022). Type 2 diabetes mellitus (T2DM) is characterized by irregular glucose metabolism caused by decreasing pancreatic β cells working with the classical indications of polyuria, polydipsia, and polyphagia (Udokang, 2012; San Tang, 2019). It is now a universal health problem accounting for almost 463 million people worldwide and spending about 11% (∼612.2 billion USD) of the global healthcare disbursement as reported by the International Diabetes Federation (IDF) and by 2045 the number of diseased patients is expected to escalate to 700 million people, updated last by 2019 (Kumar et al., 2017; Luo et al., 2022).

Myriad oral pharmacotherapies for the management of T2DM are available including Biguanides (Luft et al., 1978), meglitinides (Black et al., 2007), sulfonylureas (Del Prato & Pulizzi, 2006), dipeptidyl peptidase IV inhibitors (Demuth et al., 2005), α-glucosidase inhibitors (Van De Laar et al., 2005) and thiazolidinediones (Soccio et al., 2014), however, their unwanted side effects such as nausea, diarrhea, weight gain, hypoglycemia, and heart failure contribute significantly to their limited use (Cetin et al., 2013). Among them, Metformin (MTF) from the category of biguanides has been widely prescribed as a foundation drug for all patients with initially diagnosed T2DM (Momoh et al., 2013). MTF inhibits hepatic gluconeogenesis, suppresses glucose absorption by the intestine, and ameliorates glucose consumption by muscle cells (Migdadi et al., 2018). It belongs to the class III of the biopharmaceutical classification system (BCS) and is presented with high solubility and low permeability (Kim et al., 2022). Despite being widely used, MTF when administered orally is associated with a plethora of side effects and problems that reduce its therapeutic effectiveness entailing poor and partial gastrointestinal (GIT) absorption (40–60%) (Kenechukwu et al., 2022) and relatively rapid pre-systemic clearance which results in limited bioavailability and a short elimination half-life of nearly 6.2 h (Cesur et al., 2022). These pharmacokinetic characteristics of MTF culminate into frequent dosing, high dose, elevated incidence of dose-related adverse effects, and curtailed patient adherence (Kamboj & Verma, 2018). The GIT-related adverse effects of MTF may include diarrheal stomach pain, anorexia, vomiting, and nausea. It is also related to several other undesirable side effects such as lactic acidosis having symptoms like muscle pain, tremors, severe drowsiness, dizziness, fatigue, difficulty in breathing, and irregular heartbeat (Sankhyan & Pawar, 2013; Saini et al., 2016).

Transdermal drug delivery system (TDDS) promises significant advantages over the oral route as it is non-invasive, can circumvent the first pass effect and alleviates the GIT side effects (Kumar & Utreja, 2020). However, the transdermal bioavailability of therapeutics is constrained by the stratum corneum (SC) which exhibits the most resilient impediment to drug absorption through the skin (Ahad et al., 2018). Drug nanoencapsulation is growing as a potential substitute that provides deeper skin penetration, controlled release, and site-specific delivery of drugs (Rostamkalaei et al., 2019). Transethosomes (TES) are new-generation ultra-deformable lipid-based nanovesicles containing lipids, an edge activator, and ethanol. Contrary to the traditional liposomes, which are reluctant to penetrate the deeper layers of skin and stay restricted to the SC these ultra-deformable vesicles could penetrate deep and perform their work (Ahmed et al., 2021). They are characterized by elevated thermodynamic stability and loading capacity having properties of both ethosomes and transfersomes. TES have an edge over other drug delivery systems as they have ethanol in them (Costanzo et al., 2021). Ethanol improves drug penetration from the minute holes created in the SC as a result of lipid bilayer membrane fluidization, thus decreasing the strength of the lipid bilayer. They become ultra-deformable and very elastic due to the presence of an edge activator and its ability to destabilize the phospholipid bilayer (Song et al., 2012). On topical application, these nanocarriers not only save individuals from intrusive parental therapy but also prevent the undesirable side effects produced by the parenteral or oral route. Therefore, it is anticipated that these drug-laden vesicles will have a positive effect on therapeutic potential (Majumder et al., 2019).

Gels are believed to be potential delivery systems for retaining nanoparticles or medications at the intended application site. Owing to their mucoadhesive nature, they also lengthen the drug residence time. Retaining the TES at the desirable site for an extended time duration is challenging by their low viscosity (Rabia et al., 2020). Therefore, metformin-loaded transethosomes (MTF-TES) were encapsulated into chitosan gel for effective skin distribution and to achieve optimal conditions for transdermal drug delivery. Nowadays, there is a significant amount of interest in the utilization of chitosan in medical and pharmaceutical applications. Its intriguing inherent qualities are undoubtedly the key causes of this growing attention (Berger et al., 2004). Chitosan gel was used to incorporate MTF-TES into the gel to fabricate the MTF-TES laden gel (MTF-TES gel) as it is biodegradable, biocompatible, nontoxic, and possesses enhanced stability (Verma et al., 2020).

The present research work, nonetheless, aimed at the fabrication of MTF-TES and loading them into chitosan gel that would provide sustained release, ameliorates the incidences of unwanted side effects, and reduces the necessity of repeated dosing. By using Box Behnken design^®, the formulation has been optimized systematically and after that, the characterizations were carried out. Moreover, the antidiabetic model was developed in which fasting blood glucose and body weight of the rats treated with MTF-TES were determined and were compared with the rats treated with free MTF which confirmed its enhanced antidiabetic activity.

2. Materials and methods

2.1. Reagents and chemicals

Metformin HCl was purchased from Sigma Aldrich (Darmstadt, Germany). A phospholipid Phospholipon 90 G was taken as a gift of research from Lipoid GmBH, Switzerland. Tween 80^® as an Edge activator and LMW chitosan was taken from Saint-Priest, France. Sigma Aldrich also provides our lab Ethanol, Methanol, isopropyl alcohol, and Chloroform. Streptozotocin, a diabetes inducer, was purchased from Sigma Aldrich (Germany).

2.2. Equipment and apparatus

Equipment including weight balance (Make: OHAUS, USA, Model: PA 214 C), Transmission electron microscopy (Make: Philips, Japan, Model: CM10), Magnetic stirrer with Hot plate, FTIR (Model: United States Perkin-Elmer), Rotary Evaporator (Preparation: Germany, Heidolph GmBH, Model: Hei Vap Core) and Vortex mixer were used. Similarly, Franz-diffusion cell (Manufacturer: United States Permegear), Microtiter plate reader (Make: BioTek, United States), Centrifuge (Manufacturer: Germany HERMLE Labortechnik, Model: Z 216 MK), Zeta Sizer (Model: Nano ZS, UK), UV spectrophotometer (Manufacturer: United States), pH meter (Manufacturer: Unites States, Bante Instruments, Model: PHS 25 GW), and lyophilizer Manufacturer: Germany GmBH Martin Christ, Model: Alpha 1–2 LD Plus) were also employed in this study.

2.3. Animals

BALB/c mice approximately 30–35 g were purchased from NIH, Islamabad for use in the ex vivo penetration experiment through the skin. For in vivo antidiabetic assay, 6–8 weeks old, 20 Sprague Dawley rats were purchased from Riphah International University, Islamabad weighing 250 ± 30 g. The antidiabetic study was done after the approval of Quaid-i-Azam University ethical committee (BEC-FBS-QAU2022-385) as per the Guide for the Care and Use of Laboratory Animals (8th Edition) and ARRIVE (Version 2) guidelines. All the animals were housed and acclimated in the Quaid-i-Azam University animal house at 25.0 ± 2 °C with a 12 h/12 h light-dark cycle. Moreover, all the animals were fed with filtered tap water and standard feed.

2.4. Fabrication of MTF-TES

MTF-TES were fabricated by the thin film hydration method as reported earlier (Nayak et al., 2020). Generally, a thin organic layer was prepared by dissolving the Phospholipon 90 G (PL 90 G) in methanol. This blend was then placed in a flask in the rotary evaporator that functioned under vacuum; the temperature was kept at 60 °C and rotated at a speed of 100 rpm to make a layer of the thin film at the bottom of the flask. Erstwhile, tween 80^® and MTF in 30% v/v hydroethanolic solution were used to prepare the hydration phase, and it was afterward used to hydrate the already formed thin film and again put on the rotary evaporator for 1 h at the previously stated temperature. Furthermore, to reduce the size of the prepared TES, extrusion was done from filters (3 times from 0.45 µm and 2 times from 0.22 µm filters) as illustrated in Figure 1 (Khalid et al., 2022). Various concentrations of hydroethanolic solution were formulated in pre-formulation studies and 30% were found to be the appropriate one. A hydroethanolic solution of 30% v/v was prepared by adding 3 ml ethanol in 7 ml distilled water which was used as an aqueous phase (Kumar et al., 2016).

Figure 1. Thin-film hydration method for the fabrication of MTF-TES.

2.5. Optimization of MTF-TES

Preformulation studies were performed to determine the upper and lower limits of independent variables and the blank formulations were also prepared. To statistically optimize the formulation, Box-Behnken design using Design Expert^® software (Ver. 12) was applied and 14 runs were given by it. All the formulations were fabricated and the effect on the dependent variable (%EE, PDI, ZP, and P.S) by variation of independent factors (PL 90 G, Tween 80^®, and Ethanol) was evaluated. All the factors; independent as well as dependent are stated in Table 1 (Moolakkadath et al., 2018).

Table 1. Levels of independent and desired outcome of dependent factor employed in Box-Behnken design.

Independent factor	Highest level	Median level	Low level
	(+1)	(0)	(−1)
X1 = PL 90 G Conc. (mg)	95	80	65
X2 = Tween 80® Conc. (mg)	35	20	5
X3 = Ethanol Conc. (%v/v)	30	20	10
Dependent factor	Desired outcome
A1 = MPSD (nm)	Minimum
A2 = PDI	Minimum
A3 = Zeta Potential (mV)	Maximum
A4 = %EE	Maximum

2.6. Characterization of fabricated MTF-TES

The characterization of optimized MTF-TES was done in terms of myriad factors including mean particle size distribution (MPSD), polydispersity index (PDI), and zeta potential (Z.P), and was determined using Malvern Zeta Sizer (Model: ZS90, Worcestershire, UK) which was placed in 25 ± 0.5 °C temperature-maintained environment. The study was performed in triplicate and the samples were formulated by diluting 10 µl MTF-TES into 990 µl deionized water. Standard error as a Mean ± S.D. was also depicted in the results (Dar et al., 2018).

2.7. Percent entrapment efficiency

The indirect method was used for the determination of entrapment efficiency. The formulated MTF-TES were centrifuged for 3 h. The temperature of −4 °C and the speed of 15,000 rpm were maintained to isolate the free dug. After centrifugation, samples were taken and the supernatant was separated and analyzed under a UV spectrophotometer (Model: HALO DB 20, Make: Dynamica, UK) at the wavelength of 234 nm. The following formula was used to check the EE: $$\mathrm{\%}EE=\frac{D_1}{D_2}\times 100$$

Where, D₁ is the amount of entrapped MTF inside the nano-vesicle and D₂ is the amount of cumulative drug added (Dar et al., 2018).

2.8. TEM analysis

Surface morphology, uniformity, and segregation were evaluated using TEM as it is imperative to characterize this for nanosized particles. Before analysis, the sample was treated negatively with phosphotungstic acid and then the analysis was done at a voltage of 100 kV (Rizvi et al., 2019). Furthermore, the ImageJ software was employed to analyze the particle size from the TEM micrograph.

2.9. FTIR analysis

To check any incompatibility or chemical interaction between the formulation and its components FTIR was used (Bibi et al., 2022). For this, the optimized TES, PL 90 G, drug, and physical mixture of drug and PL 90 G was consigned to FTIR scanning as per the protocols, previously stated (El-Gizawy et al., 2020).

2.10. Fabrication of MTF-TES gel

The formulated MTF-TES were encapsulated within a hydrogel made of chitosan to be applied to the skin efficiently. Chitosan gel with different concentrations were prepared (ranging from 1.5 to 4%). Among them 2% chitosan gel was found to be the appropriate one, with respect to its optimum viscosity (Hurler et al., 2012). For the preparation of chitosan gel, 2% w/v of the low molecular weight (LMW) chitosan was added in 1% acetic acid solution and the formulation was then added with continuous stirring to fabricate the MTF-TES gel. To prepare a 1% acetic acid solution, 1 ml acetic acid was added to 99 ml distilled water (Ternullo et al., 2019).

2.11. Characterization of MTF-TES gel

The pH, clarity, homogeneity, and rheological properties of the MTF-TES gel were evaluated. A digital meter was used to determine the pH of MTF-TES GEL. Briefly, 30 ml distilled water was poured over 1 g of chitosan gel, was thoroughly dissolved, followed by assessing the pH in triplicate (Salim et al., 2020). Brookfield Viscometer (Model: DV3T, Middleborough, MA) was used to measure the flow behavior of the MTF-TES gel. To assess the impact of stress or strain, the chitosan gel viscosity was evaluated at various speeds (0.5, 1, 2, 3.5, 4, 5.4, 6, 9.5, 14, 22, 36, 58, 64, and 100 rpm) with the torque of (4.6, 9.1, 13.4, 17.9, 20.0, 22.6, 24.5 27.4, 35.3, 37.1 49.7, 59.3, 81.3, 85.9 and 94.3%) and flow patterns at 25 ± 2 °C. A stress-associated dependent or independent viscosity pattern was observed (Jamshaid et al., 2022). Visual examination was used to assess the consistency and homogeneity of the MTF-TES gel. The gel was examined to see whether or not there were any bunches or agglomerates, and any clumps or agglomerates were documented. To assess the transparency of the MTF-TES gel, visual observation was used (Aggarwal et al., 2012; Amin et al., 2013). To determine the Spreadability, the petri dish method was employed. With this technique, a circle of 1 cm² was drawn upon the petri dish and the MTF-TES gel of 0.5 g was then placed on that circle. The petri dish was covered with a glass lid with a weight of 500 g placed over it. The precise area across which the gel was placed was finally calculated (Ahad et al., 2016). The diameter increment has been measured using the following equation. $$S_i=d_2\times \frac{\pi }{4}$$

Whereas, S_i is the spread area in mm² and symbol d denotes the spread diameter of the gel (Bachhav & Patravale, 2009). The formulated MTF-TES gel was put in a beaker with methanol and left there for 48 h while being stirred. After the specified time duration, all the collected formulations were evaluated, and using a UV spectrophotometer, (Patil et al., 2015) percent drug content was calculated using the following formula; $$\mathrm{\%}\mathrm{ }\mathit{Drug}\mathrm{ }\mathit{content}=\frac{\mathit{Amount}\mathrm{ }of\mathrm{ }\mathit{drug}\mathrm{ }\mathit{detected}}{\mathit{Total}\mathrm{ }\mathit{theoratial}\mathrm{ }\mathit{amount}\mathrm{ }of\mathrm{ }\mathit{drug}\mathrm{ }in\mathrm{ }\mathit{gel}}\times 100$$

2.12. In vitro drug release

To determine the release pattern of MTF, the dialysis bag method was used. For this, the release of MTF from MTF-TES and MTF-TES gel was determined and their comparison with metformin HCl solution (MTFS) was done. MTFS, MTF-TES, and MTF-TES gel were all placed in individual dialysis bags, having the same quantity of each formulation carrying 3 mg of the drug, and then placed in beakers containing 50 ml of phosphate buffer solution (PBS). Then, the beakers were set in a shaking water bath and the temperature of the water shaker bath was maintained at 37 ± 0.5 °C and the speed at 100 rpm. At pH of 5.5 and 7.4, the in vitro drug release study was conducted. Before starting the study, the dialysis bag was soaked for 30 min in the phosphate buffer solution (PBS) to make sure it was completely moist (Nahar & Jain, 2009). Samples of 1 ml were removed at a predetermined time duration (0.5, 1, 2, 4, 6, 8, 12, and 24 h), and to sustain sink conditions promptly, the same quantity of PBS was added. The speed of the shaker was set at 100 rpm. Utilizing a UV visible spectrophotometer (Model: HALO DB 20, Make: Dynamica, UK), the concentration of the MTF in the samples was taken at the wavelength of 234 nm. Graphs were plotted between the time and % cumulative release of MTF. By running the data through various mathematical models using the DD Solver software, including first order, zero order, Higuchi Hixson-Crowell, and Korsmeyer-Pappas release models, it was possible to calculate the release kinetics of MTF from MTFS, MTF-TES and MTF-TES gel (Chaubey & Mishra, 2014; Yuan et al., 2022).

2.13. Ex vivo skin permeation study

2.13.1. Skin preparation for the study

Healthy BALB/c mice were euthanized for this experiment utilizing 4% isoflurane inhalation cylinders. This was followed by cardiac ex-plantation of deeply anesthetize animal as per protocols employed by previously published study (Batool et al., 2021). The skin of sacrificed mice was taken for the conduction of an ex vivo skin permeation study. Shaving was done from the abdomen to remove hair and the shaved skin was separated. The subcutaneous layer was carefully peeled, and any fat tissues that were still adhering were cleaned by using a cotton bud immersed in isopropyl alcohol. PBS was used to wash the skin, and the skin was then maintained at −20 °C until further process.

2.13.2. Permeation study

A Franz diffusion cell apparatus (consisting of two compartments) was used to perform the ex vivo skin permeation study. The major goal of this experiment was to check the permeation potential of MTF from various test samples. Among the donor chamber and the acceptor compartments, which contained the MTF-TES and the PBS, correspondingly, the removed BALB/c mice skin was placed. In individual cells, the identical configuration was built for MTFS-TES, MTF-TES gel, and plain MTF-loaded gel (MTF-gel). Samples of 1 ml were obtained at specific times (0.25, 0.5 1, 2, 4, 6, 8, 12, and 24 h), and the concentration of MTF was determined by measuring absorbance at the wavelength of 234 using a UV visible spectrophotometer. The cumulative quantity of MTF that permeated through the skin as a function of time was displayed (Zhu et al., 2008; Van Staden et al., 2020).

2.14. In vivo antidiabetic assay on Sprague Dawley rats

The antidiabetic potential of MTF-TES was determined by evaluating its blood sugar-lowering response in twenty STZ-induced diabetic Sprague Dawley rats. The fasting glucose level and body weight of all the rats were determined using a life care glucometer (Make: Lifecare GmBH) and weight machine (Sharma et al., 2010). All the animals were injected with 50 mg/kg/animal sterile solution of STZ in sodium citrate buffer (pH = 4.5), intraperitoneally followed by administration of 5% glucose solution using oral gavage. After 72 h of STZ injection, the fasting glucose level of all the animals was evaluated by pricking the ear vein, and the animals having blood glucose above 200 mg/dl were recruited and randomly divided into 4 experimental groups. Although, initially 20 rats were for the induction of disease, yet some of them didn’t showed the required blood glucose level and thus they were not included in the study. Thus, each group contained 4 Sprague-Dawley rats for this study. Group-1 was the negative control group treated with plain distilled water (placebo). Group-2 was the positive control group and was administered with plain metformin (100 mg/kg/d). However, group-3 was treated with a topical application of MTF-TES gel daily (containing 75 mg/kg/d of MTF per animal). Group-4 was treated with plain TES gel. The treatment was continued for 28 d. On days 7th, 14th, 21st, and 28th, the fasting blood glucose and its effect on body weight during the study course were evaluated (Lari et al., 2021). The study protocol is also tabulated in Table 2.

Table 2. In-vivo anti-diabetic study treatment and dosing.

Group name	Treatment	Dosing
Group-1	Placebo	–
Group-2	Oral MTF	100 mg/kg/d
Group-3	MTF-TES gel	75 mg/kg/d
Group-4	Plain TES gel	–

2.15. Statistical analysis

All the data presented in this research study are presented as Mean ± S.D. with each experiment performed in triplicate. The formulation was optimized using the Design Expert^® (Ver. 12). The TEM micrograph was analyzed by using ImageJ software. Furthermore, the statistical analysis including multiple comparison Tukey’s test, was performed using GraphPad Prism (Ver. 9.0.0) (Chen et al., 2017; Opatha et al., 2020).

3. Results and discussion

3.1. Fabrication and optimization of MTF-TES

As it was mentioned in the previous section, the Box-Behnken model was applied for the MTF-TES optimization. For this purpose, the Design-Expert^® (Ver. 12) software was used. The MTF-TES were prepared by utilizing a thin-film hydration process. The results of all 14 experimental runs (generated by Box-Behnken design) are stated in Table 3. In addition, the model characteristic for Box-Behnken design including adequate precision, and adjusted and predicted R² values are also mentioned in Table 4. The impact of the independent variable on the individual response factor (dependent factor) is discussed in the next sub-sections.

Table 3. Formulation chart of MTF-TES obtained by Box-Behnken design and the determined response factors.

Formulations	Independent factors			Response factors
	PL90G (mg)	Tween 80^® (mg)	Ethanol (%v/v)	MPSD (nm)	Zeta Potential (mV)	PDI	EE (%)
TE1	95	20	30	240.5 ± 6.7	−15.9 ± 1.4	0.232 ± 0.012	93.29 ± 1.67
TE2	80	20	20	165.4 ± 3.3	−14.5 ± 2.2	0.169 ± 0.045	83.73 ± 4.97
TE3	80	35	10	199.2 ± 4.1	−10.2 ± 1.7	0.334 ± 0.100	75.66 ± 5.85
TE4	80	35	30	154.8 ± 2.6	−9.84 ± 1.5	0.066 ± 0.011	82.28 ± 5.43
TE5	95	5	20	290.3 ± 5.1	−27.6 ± 3.8	0.398 ± 0.067	94.73 ± 2.81
TE6	95	20	10	269.5 ± 5.5	−14.9 ± 2.4	0.526 ± 0.104	85.66 ± 4.77
TE7	95	20	20	165.4 ± 2.3	−21.6 ± 1.9	0.169 ± 0.033	89.76 ± 4.12
TE8	65	35	20	105.7 ± 1.3	−7.85 ± 0.8	0.091 ± 0.015	62.32 ± 3.59
TE9	65	20	30	92.5 ± 1.0	−13.1 ± 1.4	0.033 ± 0.004	66.67 ± 2.72
TE10	80	5	10	208.2 ± 3.7	−26.2 ± 3.2	0.184 ± 0.027	73.41 ± 4.95
TE11	65	20	10	136.8 ± 2.6	−13.6 ± 2.0	0.102 ± 0.019	48.91 ± 5.16
TE12	80	20	20	165.4 ± 4.2	−14.5 ± 1.4	0.169 ± 0.024	83.76 ± 4.43
TE13	80	5	30	169.1 ± 4.8	−21.2 ± 1.7	0.233 ± 0.087	85.66 ± 2.96
TE14	65	5	20	141.2 ± 2.5	−15.9 ± 2.1	0.164 ± 0.076	84.11 ± 4.71

Table 4. Summary of the Box-Behnken Design for the optimization of MTF-TES.

Dependent/Response factors	R^2	Predicted R^2	Adjusted R^2	Significant factors	Adequate precision	Mean optimized parameters
A1: MPSD	0.9161	0.8373	0.8933	X1, X2, X3	18.56	165.4
A2: PDI	0.7372	0.4551	0.6655	X1, X3	10.16	0.169
A3: Zeta Potential	0.9052	0.8061	0.8793	X1, X2	18.16	−21.6
A4: Entrapment efficiency (%)	0.7097	0.4382	0.6306	X1, X2, X3	8.82	89.76

3.1.1. Impact of independent factors on MPSD

The MTF-TES vesicle’s MPSD is regarded as being one of the very important characteristics of a transdermal drug delivery system. The MPSD of the MTF-TES was substantially influenced by all the independent factors including, PL 90 G, tween 80^®, and the concentration of ethanol with a p-value of <.0001, <.0048, and <.0225, respectively. As it can be observed in Figure 2(A,B), a noticeable increase in particle size was accompanied by a decrease in concentration of tween 80^® and an increase in PL 90 G amount. As it is depicted in the graphs, by increasing the concentration of PL 90 G, MPSD increased. This could be because of more bilayer formation ultimately leading to the formation of multilamellar vesicles occurs (Chen et al., 2017). Moreover, by increasing the concentration of tween 80^® particle size was decreased. This behavior could most probably be because of the adsorption of surfactant on the surface which may prevent the growth of TES (Jamshaid et al., 2022). Moreover, it reduces the surface free energy resulting in the reduction in the agglomeration and hence decreases the particle size. As far as the effect of ethanol concentration is concerned, by increasing the concentration of ethanol, the MPSD of MTF-TES was decreased, because it also acts as a co-surfactant and reduces the interfacial tension and hence decreases the particle size (Opatha et al., 2020; Akram et al., 2022). In conclusion, there is a direct relation between MPSD with the concentration of PL 90 G, and an opposite was observed with an augmented concentration of ethanol and tween 80^®.

Figure 2. 3D optimization graphs showing the effects of independent variables over the dependent variables.

3.1.2. Impact of independent factors on PDI

It is noteworthy that the PDI of nanoparticles measures their degree of uniformity. The stability of nanoparticles is interconnected with their PDI. A higher PDI (>0.5) is the indicator of non-uniformity which leads to the formation of unstable formulation. There is a marked effect of the concentration of independent factors including PL 90 G (p-value = .0007) and ethanol (p-value = .0196) on PDI. As shown in Figure 2(B,C), a remarkable increase in PDI was linked with an increase in PL 90 G amounts. By increasing the concentration of PL 90 G, PDI increases because there is the formation of more non-uniform size, multilamellar, and dispersed vesicles. Owing to the effect on steric stabilization, ethanol reduces the MPSD and PDI of TES (Albash et al., 2019). Conclusively, there is an inverse relationship between the concentrations of ethanol and a direct relationship between PDI and PL 90 G.

3.1.3. Impact of independent factors on ZP

Determining the ZP of MTF-TES is a crucial factor to evaluate its stability owing to the fact that it determines the charge carried by the TES because as the repulsive charges of the formulation increase, its stability increases. All the independent variables; such as PL 90 G, tween 80^® except the concentration of ethanol having the p-value of .0027 and <.0001, respectively, had a significant impact on the ZP of the MTF-TES. As shown in Figure 2(E,F), a considerable increase in ZP was associated with a decrease in the concentration of tween 80^® and an increase in the concentration of PL 90 G. By increasing the concentration of PL 90 G, ZP increases because the higher lipid content carries a more negative charge (owing to the presence of a negatively charged phosphate group). Furthermore, because of the particles and skin cells’ electrostatic interaction, the drug permeates the skin more readily (Khalid et al., 2022). As far as tween 80^® concentration is concerned, by increasing the concentration of tween 80^®, ZP decreases because the surfactant provide a shielding effect. Regarding the impact of ethanol content, there is no marked effect of the concentration of ethanol on ZP. In a nutshell, ZP directly incremented with the augmentation in the concentration of PL 90 G, but with an enhanced concentration of tween 80^®, the results were the opposite i.e. ZP was reduced.

3.1.4. Impact of independent factors on %EE

The percentage of a drug that is successfully entrapped is the main factor for therapeutic effectiveness. %EE of the MTF-TES was markedly impacted by the concentration of PL 90 G, tween 80^®, and ethanol, and the p-value was found to be .0012, .0401, and .0296, respectively. As shown in Figure 3, a prominent increase in %EE was observed with a decreased tween 80^® concentration and an increase in PL 90 G amounts. By increasing the concentration of PL 90 G, the %EE was increased, which could be because of the development of large-size particles which ultimately increased the space for the entrapment of the drug. Then, by increasing the concentration of tween 80^®, particle size was decreased because the surfactant may get adsorbed on the surface and may prevent the growth of TES. Moreover, with a reduction particle size, less space may be available for the drug to be incorporated. Additionally, the surfactant may cause leakage of the drug from nanoparticles by rupturing them, hence reduced %EE may be observed (Moolakkadath et al., 2018). By increasing the concentration of ethanol, the %EE of MTF inside the MTF-TES increases because of the direct increment of MTF solubilization from 10% ethanol to 30% ethanol (Opatha et al., 2020). Overall, there is a direct correlation between %EE and PL 90 G as well as ethanol concentration, whereas tween 80® concentrations have an inverse relationship.

Figure 3. (A) MPSD and PDI of MTF-TES TE7 (optimized formulation). (B) Zeta potential of MTF-TES TE7 (optimized formulation). (C) TEM micrograph of MTF-TES TE7 (optimized formulation). (D) FTIR of MTF-TES TE7 (optimized formulation).

3.1.5. Optimized MTF-TES formulation

According to the results of Box-Behnken software, formulation TE7 was optimized with the MPSD value of 165.4 ± 2.3 nm, zeta potential of −21.6 ± 1.9 mV, and PDI of 0.169 ± 0.033, these results are displayed in Figure 3(A,B), and 89.76 of %EE as depicted in Table 3, because the response factors were in our desired limits. Furthermore, the summary of the Box-Behnken design is also presented in Table 4.

3.2. TEM analysis for surface morphology assessment

Surface morphology, uniformity, and segregation were evaluated through TEM analysis. The fabricated MTF-TES TEM micrograph is depicted in Figure 3(B). It revealed that the nanoparticles were spherical with round morphology and intact borders. These outcomes also supported the particle size determined using Zeta Sizer as the ImageJ software has predicted the average particle size of 162.50 nm, from the TEM micrograph.

3.3. FTIR analysis confirmed the drug-excipients chemical compatibility

To check any incompatibility or chemical interaction between the MTF-TES, MTF, PL 90 G, and physical mixture, an FTIR study was performed. According to Figure 3(D), the MTF FTIR curve showed peaks at 3336 cm⁻¹ and (C = N) at 1675 cm⁻¹. Similarly, PL 90 G exhibited its peaks of (C = O) at 1739 cm⁻¹. The peaks of both MTF and PL 90 G remained intact in the final formulation as depicted in the figure and no chemical and physical interaction between the formulation and its component was observed. Moreover, all the double and triple bonds of MTF and PL 90 G were reserved in the final formulation and physical mixture. These findings demonstrated the drug-excipient stability of MTF-TES. By this, we can conclude that our preparation is stable, and the biological and physicochemical characteristics of the excipients are preserved (Chen et al., 2017).

3.3.1. Characterization of MTF-TES gel

The pH, clarity, viscosity, Spreadability, homogeneity, and drug content of the chitosan-based transethosomal gel (MTF-TES) were evaluated. Chitosan was selected for application to the skin in accordance with the standard criteria defined for gels and in light of the suitable physicochemical qualities (Usman et al., 2016). The gel was clear and uniform. An important consideration is to examine the Spreadability test’s flow pattern for chitosan gel. The appropriate Spreadability of MTF-TES gel is crucial for effective topical application and the results have shown that the Spreadability of 336.7 ± 5.8% is appropriate for the topical application of gels. The Brookfield Viscometer was used to measure the flow behavior of the MTF-TES gel. To assess the impact of stress or strain, the chitosan gel’s viscosity was evaluated at various speeds (0.5, 1, 2, 3.5, 4, 5.4, 6, 9.5, 14, 22, 36, 58, 64, and 100 rpm) and flow patterns and the results have shown that the formulation has followed non-Newtonian flow (graph is shown in Figure S1, please refer to the supplementary file). The pH meter was used to determine the MTF-TES gel pH and the results have shown a pH of 5.36 ± 0.06 which is appropriate for the topical application of gels. All the assessed parameters of MTF-TES gel are presented in Table S1 (supplementary file)

3.4. Sustain release pattern of MTF-TES and MTF-TES gel

In vitro release profile of MTF was determined by plotting % cumulative drug release versus time as reported in Figure 4(A). Release of MTF was determined from MTF-TES and MTF-TES gel and was compared with MTFS at pH 5.5 and 7.4. At pH 7.4, after 4 h, 93.67 ± 2.65% of the cumulative MTF was released from the MTFS, while only 50.94 ± 3.91% of the drug was released from MTF-TES gel followed by only 63.56 ± 2.08% drug release from MTF-TES. While, after 12 h, only 81.01 ± 2.51% of the cumulative drug was released from MTF-TES and 66.02 ± 3.23% was released from MTF-TES gel. Finally, at 24 hr time point, the MTF release was extended up to 87.43 ± 2.47% by MTF-TES, and 72.18 ± 3.21% MTF was released from MTF-TES gel. The MTF-TES has shown sustained release followed by a more pronounced effect in MTF-TES gel as compared to the MTFS because the ultra-deformable lipid-based nanoparticles exhibit a sustained drug release profile (Jamshaid et al., 2022) and by incorporating the drug into this drug delivery system the release of the drug has been delayed and by further loading them into the gel their release was further delayed because of the 3D structure of the gel which holds them for longer period of time as compared to the burst release as shown by the solution (Khan et al., 2022). At 5.5 pH, less percent cumulative drug release was occurred in contrast to the drug release observed at pH 7.4. According to the graph in Figure 4(B), 95.86 ± 3.51% cumulative MTF was released from the MTFS; owing to the acidic nature of the medium, it showed less drug release as compared to pH 7.4 (where comparable drug release was encountered within 4 h), while only 37.03 ± 4.99% of the drug was released from MTF-TES. However, from MTF-TES gel, the cumulative drug release in the first 6 hr was found to be 22.05 ± 2.40%. The release percentage of the drug was finally extended to 39.59 ± 5.91% and 28.50 ± 3.62% drug release from MTF-TES and MTF-TES gel, respectively. Additionally, the kinetic release mathematical modeling, including Higuchi, Zero-order, Hixson Crowell, Korsmeyer-Peppas, and First order, was applied using a DD-solver (MS Excel Add-Ins). The Korsmeyer-Peppas has been determined as being the most accurate (best fit) release kinetic model at 7.4 pH based on the calculated regression coefficient values. In Table 5, the R² values for each kinetic release model are listed. The ‘n’ value is crucial for the Korsmeyer-Peppas paradigm. Since the ‘n’ number for every both MTF-TFS and MTF-TFS gel was smaller than 0.45, thus it follows release patterns by non-fickian diffusion.

Figure 4. (A) Percent Cumulative MTF release from MTF soln, MTF-TES and MTF-TES gel at pH 7.4. (B) Percent cumulative MTF release from MTF soln, MTF-TES and MTF-TES gel at pH 5.5 (C) ex vivo cutaneous permeation of MTF from MTF soln, MTF-TES and MTF-TES gel.

Table 5. R² values of MTF release kinetic of various kinetic model.

Release Model	MTF-TES	MTF-TES gel
Zero order	0.135	0.6114
First order	0.2014	0.2443
Higuchi	0.8115	0.882
Hixon-Crowell	0.6314	0.7681
Korsmeyer-Peppas (n)	0.9106 (0.333)	0.9306 (0.436)

3.5. Improved ex vivo cutaneous permeation of MTF with MTF-TES and MTF-TES gel

To determine the permeation profiles of MTF under simulated conditions (at pH 7.4; showing blood pH across the skin), an ex vivo skin permeation study was conducted. An Ex vivo skin permeation experiment was used to evaluate the MTF-TES ability to pass the skin’s physical barriers. Figure 4(C) shows the cumulative cutaneous permeation of MTF from MTF gel, MTF-TES, and MTF-TES gel. As can be seen, MTF-TES has shown enhanced permeation followed by MTF-TES gel as compared to the MTFS. The preliminary cutaneous permeation (from 0.5 to 1 h) of MTF from MTFS, MTF-TES, and MTF-TES gel was nearly negligible. MTF began to penetrate inside the SC barrier after the afore-stated period. In 4 h negligible cumulative drug permeation occurred from the MTFS, however, an increased amount of drug (66.41 ± 11.41 µg/cm²) was permeated from MTF-TES gel. Moreover, a more increased amount of MTF (176.67 ± 15.29 µg/cm²) was permeated from MTF-TES. Upon extension to 24 h, only 177.78 ± 19.65 µg/cm² of MTF was permeated from the MTFS, whereas, 893.41 ± 66.62 µg/cm² of the cumulative drug was permeated from MTF-TES. Similarly, 793.94 ± 59.08 of MTF from MTF-TES gel was permeated across the skin barrier. The MTF-TES has shown enhanced permeation followed by a more pronounced effect in MTF-TES gel as compared to the MTFS because the ultra-deformable lipid-based nanoparticles exhibit a deformable nature (Song et al., 2012) which can help them in crossing the SC barrier. Another important effect is that the presence of ethanol causes the fluidization of the skin’s stratum corneum lipid molecules and improves the mobility of phospholipid chains to the deeper skin tissues (Mishra et al., 2019; Gupta et al., 2020). By incorporating the drug into an ultra-deformable system, the permeation of the drug was significantly enhanced as compared to the solution which showed poor permeation (Jamshaid et al., 2022). Similar sort of results are shown by the research studies previously carried out (Chen et al., 2017; Moolakkadath et al., 2018), in which the transethosomal nanocarriers are shown to have markedly incremented drug dermal penetration than the conventional gel systems. Following the calculation of the enhancement ratio, it was determined that the permeation of MTF from MTF-TES and MTF-TES gel was 4.38 and 3.86 times incremented, correspondingly, compared to the MTF gel. The permeation parameters including transdermal flux, enhancement ratio, and permeation coefficient are stated in Table 6.

Table 6. MTF cutaneous permeation profile from MTF-gel, MTF-TES and MTF-TES gel.

Formulation	Drug permeated in 24 h Q (μg/cm^2)	^aFlux Jmax (μg/cm^2/h)	^bTransdermal Flux Jss (μg/cm^2/h)	Enhancement ratio	Permeation coefficient
MTF-TES	893.41 ± 66.62	37.22 ± 3.50	49.44 ± 5.93	4.38	0.049 ± 0.005
MTF-TES gel	793.94 ± 59.08	33.08 ± 2.10	43.52 ± 3.26	3.86	0.043 ± 0.003
MTF-gel	177.78 ± 19.65	7.40 ± 1.7	11.28 ± 2.17	–	0.011 ± 0.002

^aJ_max is the drug flux after 24 h.

^bJ_ss is the drug flux at steady state concentration (slope).

3.6. MTF-TES gel presented with marked antidiabetic effect in STZ-induced diabetic rats

Post successful optimization and in vitro characterization of MTF-TES gel, its therapeutic efficacy in terms of lowering fasting glucose level (FBG) level was assessed and compared with oral marketed MTF. As illustrated in Figure 5, at day-0, animals of all the groups were highly hyperglycemic and the treatment was initiated. In both, MTF-TES gel-treated group and the oral MTF-treated group, the FBG started to be lowered as the treatment was initiated. After 28 d of treatment with MTF-TES gel, the FBG level lowered from 557.67 ± 28.50 mg/dl to 104.00 ± 16.70 mg/dl. It can clearly be seen from Figure 5(A,B) that results of 75 mg/kg/d of MTF-TES gel has produced better results, in terms of FBG reduction. Unlikely, as obtained with oral MTF after 28 d of treatment even with 100 mg/kg/d of oral MTF, the FBG was reduced to 171.33 ± 24.91 mg/dl, which is significantly higher as compared to the MTF-TES gel treated group. However, in the negative control group and plain TES gel-treated group, there was no significant effect encountered in FBG reduction in rats. STZ-induced diabetic rats were found to be associated with a marked reduction in body weight. The prevention in weight reduction of the animal was determined and compared for each experimental group. MTF-TES gel-treated group was presented with significantly different behavior as observed with the negative control group. After 28 d of treatment with MTF-TES gel the body weight was reduced from 263.33 ± 19.14 g to 232.01 ± 12.49 g, which was significantly high as compared to the oral MTF treated group, which showed weight reduction of 265.75 ± 21.25 g to 193.01 ± 16.51 g (Figure 5(C)). One of the most interesting findings encountered by the results of the antidiabetic assay was the efficacy of nano-formulation of MTF when compared with marketed oral MTF, even in lower doses. These results demonstrated the fact that the MTF-TES gel has markedly enhanced the transdermal permeation of MTF and produced a significantly variable lowering of FBG levels in comparison to that obtained from oral MTF. The pretext is that the nanoparticles optimize the aqueous solubility of MTF, which probably leads to enhanced retention time and slow excretion from the body (Usman et al., 2016). This causes comparable FBG lowering of oral MTF and MTF-TES gel (with low doses of MTF-TES gel).

Figure 5. (A) in vivo antidiabetic assay on Sprague Dawley rats using STZ induced diabetic Model. (B) evaluation of fasting blood glucose levels in diabetic rats during study. (C) effect on the body weight of animals throughout the antidiabetic assay.

4. Conclusion

Metformin hydrochloric acid-loaded TES were successfully fabricated with maximum entrapment efficiency and small particle size. MTF-TES were successfully loaded to Chitosan gel. In-vitro release and Ex-vivo permeation confirmed the potential of MTF-TES as a nanocarrier to deliver drug across the skin, with enhanced permeability. The results of in vivo antidiabetic assay demonstrated the significantly improved antidiabetic potential of MTF after its incorporation inside the TES system. By the assessment of all these findings, we can conclude that the MTF-TES gel system can be an optimal alternative for oral MTF tablets, however, further evaluation of pharmacokinetic profiling is required.

Author contributions

Kainat Nousheen, Humzah Jamshaid: Conception, design, investigation and drafting. Rabia Afza, Saif Ullah Khan, Maimoona Malik: Design, analysis and interpretation of the data, intellectual content evaluation. Zakir Ali, Sibgha Batool, Alam Zeb: Design approval, drafting of the paper, visualization, approval of the draft. Abid Mehmood Yousaf, Saud Alqahtani: Methodology, software, validation, revision of the manuscript critically for intellectual content. Salman Khan, Gul Majid Khan: Design, validation, resources, final approval of the version to be published. Fakhar ud Din, Ali H Almari: Conception, design, supervision, funding, final approval for submission. Moreover, all the authors agree to be accountable for all aspects of the work.

Ethical approval

The animal study was performed as per the Guide for the Care and Use of Laboratory Animals (8^th Edition) and ARRIVE (Version 2) guidelines, after the approval of Quaid-i-Azam University ethical committee via approval number (BEC-FBS-QAU-2022-385). Further information on animal use is provided in Sections 2.3, 2.13 and 2.14.

Justification for use of animals

The cellular and molecular effects of a therapeutic agent could be predicted using in vitro systems and the usefulness cannot be underemphasized as it allows high throughput rapid screening of candidate compounds. However, a proper understanding of the complex physiological response needs a whole organism that could exhibit the signs and symptoms of diseases. Thus, animal studies have been introduced as a subset of test systems employed at the preclinical stage of drug development for the prediction of clinical effectiveness in humans. This has been possible due to the ability to correlate data generated from animal studies to humans.

Rodents (mice and rats) were used in this study, as they are the most commonly used animal models due to their short lifecycle and lifespan. These offer complementary benefits of cost and time savings as they can reproduce quickly, yielding faster results. Moreover, they have been reported to have 90-95% resemblance with human anatomy. As discussed earlier (Sections 2.3, 2.13 and 2.14) standard operating procedures were adopted to perform animal studies, including the use of minimal number with marginal pain and discomfort.

Acknowledgement

The authors are thankful to the Higher Education Commission of Pakistan, National institute of Health Islamabad Pakistan and Department of Pharmacy, Quaid-i-Azam University Islamabad Pakistan for their facilitation in conducting this research.

Disclosure statement

The authors declare no potential competing interest.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [Fakhar Ud Din], upon reasonable request.

Additional information

Funding

The authors are thankful to the Higher Education Commission of Pakistan for funding this research work through its grant No: 20-14604/NRPU/R&D/HEC/2021. Moreover, the authors are grateful to the Deanship of Scientific Research at King Khalid University for funding this study through the Research Group Project, under grant number RGP2/436/44.