1. INTRODUCTION
Globally, diabetes mellitus is a serious public health concern. The International Diabetes Federation estimates that 536.6 million people between the ages of 20 and 79 years, or 10.5% of the world’s adult population, had diabetes in 2021. By 2045, this figure is expected to increase by 46% to 783.2 million [1]. One of the hallmarks of diabetes mellitus is hyperglycemia, which is usually caused by decreased insulin production and/or insulin resistance [2]. About 90% to 95% of diabetes cases are type 2 diabetes mellitus (T2DM), which is influenced by a number of risk factors, including sedentary lifestyles, obesity, urbanization, population aging, and longer life expectancies. The worldwide epidemiological shift from infectious to chronic diseases is also correlated with these causes [3].
The standard treatment for type 1 diabetes consists of lifelong insulin replacement therapy. In contrast, individuals with T2DM are typically prescribed various oral hypoglycemic agents, such as metformin, sulfonylureas, thiazolidinediones, meglitinides, incretin-based therapies, sodium-glucose cotransporter-2 (SGLT-2) inhibitors, and amylin analogues. In instances of insufficient glycemic control with monotherapy, the use of a combination of antidiabetic agents is frequently advised [4]. Dipeptidyl peptidase-4 (DPP-4) inhibitors, commonly known as gliptins, are extensively utilized in the management of T2DM. Vildagliptin, a selective DPP-4 inhibitor, inhibits the degradation of incretin hormones (GLP-1 and GIP), which increases insulin secretion and reduces glucagon levels. This mechanism reduces blood glucose levels, presenting a low risk of hypoglycemia and no weight gain, thus serving as a tolerable and effective choice for glycemic management in T2DM patients [5].
Vildagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, belongs to the Biopharmaceutical Classification System (BCS) Class III, characterized by high solubility and low permeability. It has a short elimination half-life of 1–3 hours, necessitating frequent dosing to maintain therapeutic plasma concentrations. Chemically designated as (S)-1-[N-(3-Hydroxy-1-adamantyl) glycyl] [pyrrolidine-2-carbonitrile], vildagliptin is a white or nearly white crystalline powder (molecular weight 303.4) with a melting point of 148°C–152°C. It is highly soluble in DMF, methanol, 0.1 M HCl, and 0.1 M NaOH, moderately soluble in ethanol and acetonitrile, and exhibits pKa values of 14.71, 9.03, and 7.6, along with a logP value of 0.9, indicating moderate lipophilicity [6]. The development of controlled-release drug delivery systems is necessary to overcome these limitations, as they extend therapeutic action, enhance patient adherence, and reduce dosing frequency. Controlled-release oral formulations increasingly utilize diverse polymers and biocompatible carriers to modulate drug release profiles [7]. Approximately 84% of the leading 50 pharmaceutical products worldwide are delivered orally, frequently employing generally recognized as safe (GRAS) excipients to improve bioavailability and reduce off-target effects [8]. Advanced drug delivery technologies, including microemulsions, microspheres, niosomes, self-emulsifying systems, and liposomes, have been investigated to enhance oral absorption and therapeutic efficacy [9,10]. Liposomes are phospholipid-based vesicles that encapsulate active pharmaceutical ingredients, providing protection against enzymatic degradation and enabling controlled drug release. The inclusion of components like cholesterol, glycolipids, surfactants, and membrane proteins improves stability, bioavailability, and targeting efficiency [11,12]. Liposomes have emerged as a promising platform for the delivery of various therapeutics due to their advantageous properties, including reduced toxicity, sustained release, and adaptability [13,14].
The incorporation of herbal therapies alongside synthetic pharmaceutical agents is increasingly recognized, driven by cultural preferences and the potential for synergistic effects. Trigonella foenumgraecum, or fenugreek, is a traditional medicinal plant noted for its antidiabetic properties, especially in South Asia and North Africa. Fenugreek exhibits antihyperglycemic activity in multiple experimental models, including glucose-loaded normal animals and diabetic models [15,16].
The present investigation seeks to develop and optimize fenugreek integrated vildagliptin liposomes to enhance antidiabetic efficacy by mitigating GLP-1 and GIP degradation through DPP-4 inhibition. Specifically, the study aims to employ a factorial design approach for formulation optimization, undertake comprehensive physicochemical characterization, delineate in vitro release kinetics, and evaluate in vivo antidiabetic performance in alloxan-induced diabetic rats to establish a sustained glycemic control platform.
2. MATERIALS AND METHODS
2.1 Materials
Vildagliptin (Eskayef Bangladesh Limited), cholesterol and lecithin (Alfa Aesar, UK), diethyl ether, methanol, chitosan, dicetyl phosphate (DCP), and PLGA (Merck, Germany) were used. All other reagents were of analytical grade.
2.2 Preparation of vildagliptin-loaded liposomes by ether injection method (EIM)
According to Tables 1 and 2, lecithin and cholesterol were dissolved in 10 ml diethyl ether, followed by the addition of vildagliptin, chitosan, PLGA, and DCP. The mixture was injected at a rate of 1 ml/min into 20 ml of pH 1.2 acetate buffer under constant stirring at 50°C–55°C. The temperature gradient between the organic and aqueous phases facilitated rapid ether vaporization, leading to spontaneous vesiculation and liposome formation. All liposomal formulations were stored at 25°C ± 2?°C to ensure stability and reproducibility.
Table 1. Independent variables and their levels in experimental design.
| Factors | Independent variables | |||
|---|---|---|---|---|
| Levels | ||||
| Unit | Low (−1) | High (+1) | ||
| X1: Lecithin (mg) | 100 | 200 | ||
| X2: Cholesterol (mg) | 75 | 150 | ||
| X3: Chitosan (mg)/PLGA | 100 | 250 | ||
| Dependent variables | Goals | |||
| Y1 | Drug entrapment efficiency (%) | Maximize | ||
| Y2 | Cumulative drug release (%) | Minimize | ||
Table 2. Design layout of experiments as per user-defined factorial design.
| Formulation Code | Vildagliptin (mg) | Fenugreek (mg) | Coded variable | Actual coded variable | Response | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| X1 | X2 | X3 | X1 | X2 | X3 | Y1 | Y2 | |||
| (mg) | (mg) | (mg) | % | % | ||||||
| CHF-1 | 50 | 100 | −1 | −1 | +1 | 100 | 75 | 250 | 87.99 ± 0.84 | 85.23 |
| CHFE-2 | 50 | 100 | −1 | +1 | +1 | 100 | 150 | 250 | 90.63 ± 2.28 | 83.67 |
| PLF-3 | 50 | 100 | −1 | −1 | +1 | 100 | 75 | 250 | 91.87 ± 1.12 | 72.23 |
| PLFE-4 | 50 | 100 | +1 | −1 | +1 | 200 | 75 | 250 | 92.52 ± 0.71 | 70.98 |
2.3 Characterization of formulated liposomes
2.3.1 Drug-excipient compatibility study by FTIR spectroscopy
Studies on drug-excipient compatibility were conducted to assess potential interactions between the drug and the polymer utilized to generate the liposomes. FTIR spectroscopy was utilized to verify that vildagliptin and the surfactants were compatible.
2.3.2 Drug entrapment efficiency (DEE)
Drug entrapment efficiency represents the ratio of the actual drug content to the theoretical drug loading in the formulation. Approximately 20 mg of the prepared microspheres were finely powdered using a mortar and pestle and dissolved in 20 ml of freshly prepared buffer solution (pH 1.2). The mixture was continuously agitated for three hours to ensure complete dissolution, followed by filtration. The drug content in the filtrate was then quantified using ultraviolet (UV) spectroscopy.
The calculation was performed by referencing the unentrapped free drug. The following formula has been used for the calculation of DEE.
2.3.3 Determination of particle size and PDI
The particle size and polydispersity index (PDI) of the formulated vildagliptin loaded liposomes were determined using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK). A small aliquot of the liposome suspension was diluted appropriately with double-distilled water to avoid multiple scattering effects and placed in a quartz cuvette. The measurements were performed at 25°C, maintaining a fixed scattering angle (typically 90°). The average hydrodynamic diameter (z-average) and PDI were recorded. The PDI value indicates the uniformity of particle size distribution, with values closer to 0 representing more uniform systems.
2.3.4 Determination of zeta potential
The zeta potential was determined using the zeta master apparatus (Malvern Instruments, Malvern, UK) by measuring the electrophoretic mobility of the particles. Zeta potential, measured in millivolts, refers to the ionic potential at the interface between this unit and the surrounding medium.
2.3.5 Analysis of morphological features by scanning electron microscopy (SEM)
An optical microscope was used to determine the vesicle size of the selected liposomal dispersion. On the other hand, the vesicle size, shape, and surface properties of the chosen formula were examined using the scanning electron microscope.
2.3.6 Kinetic analysis of dissolution
The Higuchi, first, and zero-order equations were utilized to examine the release data and investigate the process of drug release from the liposomes [17]. The following formula is used: log (Mt/mf??) = log K + log t
Where Mt represents the amount of drug released after infinite time, and k denotes the release rate constant, which encompasses the structural and geometric characteristics influencing the mechanism of drug release.
2.3.8 Successive fractional dissolution time
The following equations were employed to calculate T25%, T50% (mean dissolution time), and T80% from dissolution data to characterize the drug release rate across different experimental conditions: T25% = (0.25/k) (1), T50%= (0.5/k) (1), and T80%= (0.8/k) (1). The mean dissolution time (MDT) was calculated using the following equation:
MDT= (n/n+1). K
2.3.9 Optimization using the desirability function
In the current work, the numerical optimization approach offered by the design-expert program was used to simultaneously optimize all three answers using a desired function. For the preparation of vildagliptin-loaded liposomes, a two-factor three-level user-defined design was adopted in accordance with Tables 1 and 2.
In this design, three factors were evaluated each at two levels, and experimental trials were performed using all possible eight combinations. The amount of lecithin (X1), cholesterol (X2), and chitosan/Poly(lactic-co-glycolic acid) (PLGA) (X3) was selected as three independent variables, whereas physicochemical properties of the prepared liposomes as entrapment efficiency (Y1) and cumulative drug release(Y2) were selected as dependent variables.
2.3.10 Permeability study using Franz diffusion cell
In this step, each formulation, prepared using the emulsification solvent diffusion method, was provided with 10 mg as a dry powder to the donor compartment over the dialysis membrane to eliminate the sodium azide preserving agent. Samples were analyzed using Franz diffusion cell at 32°C [18].
2.3.10.1 In vitro release study using cellulose dialysis membrane
An in vitro release study was done using a dialysis membrane in the USP II paddle method. Dialysis membrane was cut into 9 cm in length and soaked in 500 ml distilled water at room temperature for 30 minutes to remove the sodium azide preserving agent. The release study was checked for eight hours.
2.3.10.2 Ex vivo release study using chicken intestinal sac
To study ex vivo release, acetic buffer (pH 1.2) was used as the dissolution medium. Dissolution studies were conducted in a dissolution apparatus using the USP II paddle method. An ex vivo release study was carried out by using a chicken intestinal sac. The sac was dipped into the receptor compartment containing the dissolution medium, 900 ml of phosphate buffer was stirred continuously at 100 rpm, and the temperature was maintained at 37°C. A 10 ml of the sample was withdrawn at predetermined intervals from each basket, filtered, and the media was replenished with fresh medium. The permeability study was checked for 8 hours.
2.3.11 IVIVC-based plasma profile prediction
Plasma drug concentrations were predicted from in vitro dissolution data using a validated Level A IVIVC approach. The fraction of drug released in vitro was converted to the fraction absorbed in vivo using the Wagner–Nelson method for a one-compartment model. The absorbed fraction was then used to calculate plasma concentration at each time point using the equation:
where C(t) is the plasma concentration, Fabs(t) is the fraction absorbed, D is the administered dose (50?mg), Vd is the volume of distribution, and ke? is the elimination rate constant. Plasma concentrations are expressed in µg/ml.
2.3.12 Statistical analysis
Statistical analysis was performed using the analysis of variance (ANOVA) design in Design Expert® software (version 7.1.6, Stat-Ease Inc., MN). Eight experimental runs were conducted for the software trial, with three parameters selected at two levels each (low and high). Design specialists generated regression parameters and graphical representations for each response, ensuring statistical significance (p < 0.05).
2.4 Stability studies
The stability studies of the optimized liposomal formulation were performed at different temperatures, and the effect of the formulations was noted. The liposomal dispersions (6 mg vildagliptin/1ml) were kept in the air light containers and stored at refrigeration temperature (2oC–8oC) and at room temperature (30oC) for 21 days. During this storage, 1 ml sample from each formulation was withdrawn at a 7-day interval (7,14, and 21 days). The stability of the formulation was analyzed by measuring drug entrapment efficiency.
2.5 In vivo study
2.5.1 Animals used in the experiment
Initially, male albino rats weighing between 100 and 120 grams and six to eight weeks old were obtained from the animal house at Jahangirnagar University. The rats were confined in an enclosure that was maintained at a constant 22°C temperature and 40%–60% humidity, with a 12-hour day-night cycle. During the investigative trial, rats were provided with pellets (Rat Feed, Bangladesh) and unrestricted access to distilled water.
2.5.2 Experimental diabetes induction
In order to induce diabetes, rats were administered a single intraperitoneal injection of alloxan monohydrate, which had been recently synthesized and dissolved in purified water, at a dosage of 130 mg/kg body weight (Table 3). Following an overnight fast, the rats’ blood glucose levels were assessed 48 hours after they were administered alloxan. Rats with blood glucose levels between 200 and 300 mg/dl were considered to have diabetes [19].
Table 3. Experimental protocol of mouse model.
| Group | No. of Rats | Description |
|---|---|---|
| Group I (Negative Control) | 6 | Normal rats receiving daily saline treatment served as the negative control. |
| Group II (Positive Control) | 6 | Animals receiving a single intraperitoneal dose of alloxan monohydrate (130 mg/kg) to induce diabetes served as the positive control. |
| Group III (Disease control + MP) | 6 | Diabetic rats were administered pure vildagliptin at a dosage of 2.5 mg/kg body weight. |
| Group IV (Disease control + PLFE-4) | 6 | Diabetic rats were administered vildagliptin-containing liposomes at a dose of 2.5 mg/kg body weight. |
2.5.3 Long-term treatment protocol for HbA1c assessment
For the evaluation of long-term glycemic control, diabetic rats were randomly divided into treatment groups following confirmation of hyperglycemia. Vildagliptin and liposome-loaded DPP-4 inhibitor formulations were administered orally at a dose of 2.5 mg/kg body weight once daily for a period of three months using a calibrated oral gavage.
Blood glucose levels were measured daily at predetermined time intervals following each dose to monitor short-term postprandial glucose variations as well as overall glycemic trends throughout the treatment period. Glycosylated hemoglobin (HbA1c) levels were assessed using the diagnostic reagent kits described earlier, allowing evaluation of glycemic control across the entire three-month treatment duration.
This experimental design ensured that the sustained-release profile observed after each dose was consistently maintained over the entire treatment period, providing clinically relevant evidence of long-term glycemic control.
2.5.4 Collection of blood from the rats
Following the experimental course of the drug, the rats were slaughtered by cervical dislocation after undergoing moderate chloroform anesthesia. After the victim was beheaded, blood was drawn, and the serum was extracted using centrifugation at 2000 rpm for 20 minutes.
2.5.5 Blood glucose concentration estimation
Following the induction of diabetes, both vildagliptin, administered as a pure drug, and liposomes loaded with a DPP-4 inhibitor were suspended in distilled water and orally administered using a syringe. Subsequently, blood samples were collected at different time intervals from the tail vein for the measurement of blood glucose levels using an electronic glucometer.
2.5.6 Estimation of glycosylated hemoglobin (HbA1c)
HbA1c was assayed using a diagnostic reagent kit manufactured by Crescent Diagnostic Ltd, Atlas Medical Diagnostics Ltd, and Stanbio Diagnostic Ltd. Each supernatant was poured directly into separate cuvettes for absorbance measurements. Absorbance (Agly) of standard, unknown versus water was read at 415 nm. Total hemoglobin was assayed by pipetting 5 ml deionized water into tubes labeled Standard (S), Unknown (U), then pipetting 20 μl of hemolysate into the appropriately labeled tube and mixing well. Absorbance (Atot) of standard and unknown versus water was taken at 415 nm within 60 minutes [20]. Calculation was done using the following method:
Glycohemoglobin (%) = (Runknown/ Rstd) X Concentration of glycohemoglobin standard [Here, Concentration of glycohemoglobin standard (%) = 10]
3. RESULTS AND DISCUSSION
3.1 Drug-excipient compatibility study
In order to examine the interaction between the drugs and polymers, FTIR analysis was done as shown in Figure 1. The liposomal formulations of chitosan, chitosan + fenugreek, PLGA, and PLGA + fenugreek-loaded with vildagliptin were analyzed, as illustrated in Figure 1. In the FTIR spectrum of the pure drug, there were characteristic amide peaks at 3,400.50, 2,117.84, and 1,643.35 per cm, urea carbonyl stretching (urea N-H stretching) vibrations at 1,489.05 and 1,359.82 per cm, and SO2 stretching vibrations at 1,358 and 1,222.87 per cm.
 | Figure 1. FTIR spectra of a) vildagliptin+chitosan b) vildagliptin+chitosan + fenugreek c) vildagliptin+ PLGA and d) vildagliptin+ PLGA +fenugreek, respectively. [Click here to view] |
The FTIR spectra of the liposomal formulations containing vildagliptin revealed comparable important drug characteristics peaks that remained consistent within the formulation, thereby confirming that the polymer and pure drug did not interact substantially in the functional group [21,22]. The principal functional group peaks for the formulation and the pure drug are within the range of previously reported values, which means that there are no drug polymer interactions [23].
3.2 Drug entrapment efficiency (%DEE)
The %DEE of the formulated liposomes was evaluated, and the results are represented in Table 4. The %DEEs of various formulations, including chitosan, chitosan + fenugreek, PLGA, and PLGA + fenugreek, were prepared by the ether injection method (EIM), ranging from 87.99% to 92.52%. The PLGA ratio can control the release percentage of the encapsulated drug, achieving a very small particle size [24]. The effectiveness of drug entrapment was enhanced by PLGA and fenugreek at their maximum concentrations. By increasing the fluidity of the liposomes, PLGA reduces the amount of drug that leaks out. Moreover, adding more polymers to formulations improved the efficiency of drug entrapment. This is probably because the outer lipid bilayer layer was coated with PLGA and fenugreek oil, increasing the amount of vildagliptin trapped between the layers, which led to a reduction in drug leakage [25].
Table 4. Drug entrapment efficiency, particle size, PDI, and zeta potential of different formulations.
| Formulation code | % Drug entrapment efficiency | Mean particle size (nm) | PDI | Zeta potential (mV) |
|---|---|---|---|---|
| CHF-1 | 87.99 ± 0.84 | 253.153 ± 6.71 | 0.821 ± 0.05 | −20.45 ± 0.33 |
| CHFE-2 | 90.63 ± 2.28 | 116.297 ± 10.04 | 0.765 ± 0.02 | −30.33±0.21 |
| PLF-3 | 91.87 ± 1.12 | 104.153 ± 8.25 | 0.434 ± 0.03 | −29.5 ± 0.16 |
| PLFE-4 | 92.52 ± 0.71 | 93.513 ± 5.33 | 0.236 ± 0.03 | −35.5 ± 0.17 |
CH = Chitosan, PL = PLGA, F = Formulation and FE = Fenugreek.
3.3 Analysis of particle size, PDI, and zeta potential
Particle size analysis was performed on different formulations, yielding mean particle sizes of 104.153 ± 0.07 nm and 93.513 ± 0.03 nm, respectively.
PDI is a depiction of the distribution of the size of populations within a given sample. The numerical value of PDI ranges from 0.0 (for a perfectly uniform sample concerning the particle size) to 1.0 (for a highly polydisperse sample with multiple particle size populations). Values of PDI were observed ranging from 0.236 to .0821.
The zeta potential of the liposomal formulation was found to be stable. The zeta potential value of formulation CHF-1, CHFE-2, PLF-3, and PLFE-4 was −30.33 mV, −20.45 mV, −29.5 mV and −35.5 mV, respectively.
The results of particle size analysis in Table 4 showed that the combination of PLGA and fenugreek produced smaller particles measuring 93.513 ± 5.33 nanometers. In drug delivery applications using lipid-based carriers, such as liposome formulations, a PDI of 0.3 and below is considered acceptable and indicates a homogenous population of phospholipid vesicles [26].
The electrostatic repulsive interactions between individual particles, liposomal vesicles with a large positive or negative zeta potential, imply strong physical stability of liposomal suspensions [27]. It is commonly accepted that a zeta potential value larger than −30 mV to +30 mV has enough repulsive force to achieve improved physical stability. Zeta potential of all formulations found to be −20.45 to −35.5 mV. The findings demonstrated that the produced liposomes have a satisfactory level of physical stability, and liposomal vesicle coalescence is not possible. The result also demonstrated that the same independent factors that increased droplet charge also decreased droplet size [28,29].
3.4 Particle surface morphology
Scanning electron microscopy was utilized to examine the surface properties of liposomes. Figure 2a illustrates that pure vildagliptin displays an irregular crystalline morphology characterized by rough surfaces and sharp-edged structures, signifying its native crystalline state. Conversely, Figure 2b exhibited notable morphological changes subsequent to formulation. The PLGA-based fenugreek-loaded vildagliptin liposomes exhibited a smoother, fibrous, and amorphous surface structure, indicating effective drug encapsulation within the polymeric matrix. The observed reduction in crystallinity and the emergence of a more uniform texture in the formulated liposome suggest potential enhancements in drug solubility, stability, and controlled release properties [30].
 | Figure 2. SEM microscopy of a) vildagliptin and b) PLGA-based fenugreek-loaded vildagliptin liposome, respectively. [Click here to view] |
3.5 Effects of polymers on vildagliptin release from liposomes
This study examines the influence of polymer combinations on drug release from liposomes, specifically by formulating vildagliptin liposomes with varying concentrations of polymeric materials. Lecithin, cholesterol, and chitosan were utilized in the formulation of CHF-1. After 8 hours, 87.99% of the drug was released from the liposomes. Chitosan and fenugreek were utilized in the formulation of CHFE-2, in contrast to lecithin and cholesterol. After eight hours of degradation, 90.63 percent of the drug was released from the liposomes. Formulation PLF-3 was developed by combining PLGA, lecithin, and cholesterol, resulting in a drug release of 91.87%. After 8 hours, 92.52% of the drug was released from the liposomes composed of PLGA, fenugreek, lecithin, and cholesterol, resulting in the formulation PLFE-4, as illustrated in Figure 3.
 | Figure 3. Comparative dissolution profile of prepared liposomes. [Click here to view] |
3.6 Kinetic profile of the prepared liposomes
To determine the mechanism of drug release, controlled-release vildagliptin liposomes were subjected to analysis using various mathematical models. Table 5 displays the computed “n” values and the correlation coefficients (R2). The polymer concentration affects the values of n [31,32].
Table 5. The release rate constants (n) and correlation coefficient (R2) values of different formulations of vildagliptin–fenugreek liposomes using various polymers via the ether injection technique.
| Formulation code | Zero-order model | First-order model | Higuchi model | Korsmeyer-Peppas model | Best-fitted model | Drug release mechanism | |
|---|---|---|---|---|---|---|---|
| R2 | R2 | R2 | R2 | n | |||
| CHF-1 | 0.899 | 0.965 | 0.988 | 0.969 | 0.463 | Higuchi | Non-Fickian (Anomalous) Diffusion |
| CHFE-2 | 0.928 | 0.964 | 0.990 | 0.980 | 0.522 | Higuchi | Non-Fickian (Anomalous) Diffusion |
| PLF-3 | 0.932 | 0.986 | 0.994 | 0.986 | 0.531 | Higuchi | Non-Fickian (Anomalous) Diffusion |
| PLFE-4 | 0.992 | 0.986 | 0.937 | 0.972 | 0.864 | Zero order | Non-Fickian (Anomalous) Diffusion |
| MP-1 | 0.836 | 0.996 | 0.975 | 0.978 | 0.409 | First order | Fickian diffusion |
The optimal strategy for enhancing therapeutic efficacy and minimizing adverse effects of the drug is to achieve zero-order drug release [33,34]. In zero-order release, the drug concentration-time profile remains flat as the combination effects of the fenugreek and vildagliptin, along with other excipients, for the best fitted formulation PLFE-4 showed a constant rate, and the drug release mechanism was non-Fickian in comparison with other formulations.
3.7 Successive fractional dissolution time
Figure 4 shows the dissolution profiles of vildagliptin-loaded liposomal formulations. PLFE-4 exhibited the longest T25%, T50%, T80%, and MDT values, with a notably extended T80% of 13.194 hours and MDT of 3.796 hours, indicating superior sustained release. These results suggest that the PLGA-based fenugreek formulation (PLFE-4) provides enhanced prolonged drug release, likely due to its polymeric matrix.
 | Figure 4. Successive fractional dissolution profile of vildagliptin-loaded liposomes by EIM. [Click here to view] |
3.8 Optimization using the desirability function
The adequacy and significance of the developed models for all four formulations (CHF-1, CHFE-2, PLF-3, and PLFE-4) were evaluated using analysis of variance (ANOVA) for two response variables (Y1 and Y2). The statistical results are presented in Tables 6 and Table 7. For all formulations, the model F-values were considerably high, and the corresponding p-values were less than 0.05, indicating that the models were statistically significant for both Y1 and Y2. Specifically, the highest F-values were observed in CHFE-2 for Y1 (177.07) and Y2 (98.65), suggesting an excellent fit and strong influence of independent variables on the response variables. Even the lowest F-values, as seen in PLF-3 (Y1 = 52.00; Y2 = 27.50), were still well within the range of statistical significance, affirming the robustness of the models. Furthermore, the lack-of-fit F-tests yielded p-values greater than 0.1 in all cases, indicating that the lack of fit was not statistically significant [35,36]. This confirms the adequacy of the models in fitting the experimental data without unexplained systematic variation.
Table 6. Results of ANOVA and lack of fit tests for the responses (Y1 and Y2).
| Formulation codes | Response | F value | Probability > F | Comment | |
|---|---|---|---|---|---|
| CHF-1 | Y1 | Model | 50.81 | 0.0001a | Significant |
| Lack of Fit | 0.77 | 0.68b | Not significant | ||
| Y2 | Model | 29.32 | 0.0001a | Significant | |
| Lack of Fit | 1.52 | 0.64b | Not significant | ||
| CHFE-2 | Y1 | Model | 177.07 | <0.0001a | Significant |
| Lack of Fit | 0.35 | 0.52b | Not significant | ||
| Y2 | Model | 98.65 | 0.0001a | Significant | |
| Lack of Fit | 0.17 | 0.56b | Not significant | ||
| Model | 52.00 | 0.0001a | Significant | ||
| PLF-3 | Y1 | Lack of Fit | 0.68 | 0.59b | Not significant |
| Model | 27.50 | 0.0025a | Significant | ||
| Y2 | Lack of Fit | 1.02 | 0.44b | Not significant | |
| Model | 89.00 | <0.0001a | Significant | ||
| PLFE-4 | Y1 | Lack of Fit | 0.43 | 0.63b | Not significant |
| Model | 65.00 | <0.0001a | Significant | ||
| Y2 | Lack of Fit | 0.29 | 0.73b | Not significant | |
aSignificance probability values (Probability > F) less than 0.05 imply that the model is significant; bNon-significant lack of fit (p value > 0.1) proves the adequacy of the model.
Table 7. Determination of optimized formulation using desirability function.
| Formulation Codes | X1 (mg) | X2 (mg) | X3 (mg) | Y1 (%) | Y2 (%) | Desirability |
|---|---|---|---|---|---|---|
| CHF-1 | 100 | 75 | 250 | 87.99 ± 0.84 | 85.23 | 0.825 |
| CHFE-2 | 100 | 150 | 250 | 90.63 ± 2.28 | 83.67 | 0.895 |
| PLF-3 | 200 | 150 | 250 | 91.87 ± 1.12 | 72.23 | 0.912 |
| PLFE-4 | 200 | 75 | 250 | 92.52 ± 0.71 | 70.98 | 0.998 |
3.9 Permeability study of vildagliptin-loaded liposomes
3.9.1 In vitro permeability study using cellulose membrane
A comparative study is shown in Figure 5a, for pure drug, marketed products, and liposome formulation with natural product, fenugreek. After 8 hours, the liposome formulations containing vildagliptin and fenugreek demonstrated greater diffusion compared to both pure drugs and commercially available products.
 | Figure 5. a) Comparative permeability study of vildagliptin-loaded liposomes using cellulose membrane b) Comparative permeability study of vildagliptin-loaded liposomes using chicken intestinal sac. [Click here to view] |
Figure 5a compares drug release profiles of pure vildagliptin, a marketed formulation, and PLFE-4 over 8 hours. The pure drug showed rapid release, while the marketed product had a slower, sustained profile. PLFE-4 exhibited the most controlled release, with significantly lower drug permeation, indicating effective sustained delivery through liposomal encapsulation.
3.9.2 Ex vivo permeability study using chicken intestinal sac
Ex vivo release kinetics were carried out for 8 hours using the USP II paddle method as described earlier. Drug release from the liposomes was studied with a zero-order kinetic model. Vildagliptin-loaded liposomes and liposomes containing fenugreek exhibited increased diffusion compared to both pure drugs and commercial products. Increased permeability is indicated by the increased diffusion, which results from the polymeric behavior of the liposomes [37]. The comparison also showed that the controlled drug release property of these liposomes was significantly better than that of pure vildagliptin.
Figure 5b shows ex vivo permeation of vildagliptin across intestinal tissue. The unformulated drug showed rapid, high permeation, while the marketed formulation had a slower but substantial release. PLFE-4 exhibited the most controlled and sustained permeation, with significantly lower cumulative release, indicating its potential to enhance bioavailability and prolong therapeutic effect by regulating intestinal absorption.
The sustained-release formulations of vildagliptin depicted a fine-tuned reduction in blood glucose level till 8 hours. From the stated result, it can be said that PLFE-4 could have some antihyperglycemic effect. Moreover, a comparative study showed a greater diffusion of our formulated liposomes with fenugreek compared to others after eight hours. Further studies with higher dose levels may be used to evaluate the actual impact [38].
3.10 IVIVC-based plasma profile prediction
Model-based plasma concentration–time profiles were generated using a Level A IVIVC approach with ex vivo release data and published vildagliptin disposition parameters [39]. The predicted profiles for PLFE-4 showed reduced Cmax, delayed Tmax, and comparable AUC versus the immediate-release references in Figure 6.
 | Figure 6. Predicted plasma concentration - time profile (IVIVC-based). [Click here to view] |
3.11 Stability studies
To obtain stability data for different liposomal formulations, they were kept under different conditions for several days, which are given in Figure 7 as graphical representations.
 | Figure 7. Diagram of stability studies of PLFE-4 (DEE= Drug entrapment efficiency, RFT= Refrigeration temperature, RMT= Room temperature, ET= Elevated temperature, CH= Chitosan, PL= PLGA, F =Formulation, and FE = Fenugreek [Click here to view] |
The stability study of PLFE-4, as shown in Figure 7, demonstrates that drug entrapment efficiency (DEE%) is highest at refrigeration temperature (4°C ± 2°C) and slightly declines when stored at room temperature (30°C ± 2°C) and elevated temperature (40°C ± 2°C). However, the reduction in DEE% across these conditions is relatively minor, indicating good overall stability. Interestingly, a slight recovery or stabilization in DEE% is observed from room to elevated temperatures. These results suggest that PLFE-4 maintains its stability well across different storage conditions, with cooler temperatures being more favorable for optimal drug entrapment.
3.12 In vivo animal model test
3.12.1 Investigation of antihyperglycemic effects on albino rats
Table 8 illustrates average blood glucose levels over 8 hours in different experimental groups. The negative control remained stable with the lowest glucose levels, while the disease control group showed consistently high levels, confirming hyperglycemia. The placebo liposome control group produced only a slight reduction in glucose levels; however, this change was statistically non-significant (p > 0.05), indicating no therapeutic benefit. Treatment with the marketed product and optimized formulation [PLFE-4] significantly reduced glucose levels (*p < 0.05, **p < 0.01), indicating their antihyperglycemic potential. Pure drug administration led to a brief glucose drop, followed by a return to baseline, highlighting poor sustained release. In contrast, polymer-based formulations ensured prolonged glucose control over 8 hours. Fenugreek alone treatments produced no significant changes in blood glucose compared with the disease control (p > 0.05), indicating that the glycemic benefit was specific to the vildagliptin–fenugreek formulation.
Table 8. Average blood glucose levels over 8 hours across different groups.
| Time (Hours) | Negative control (mg/dl) | Disease control (mg/dl) | Market product (mg/dl) | PLFE-4 (mg/dl) | Fenugreek only (mg/dl) | Placebo-liposome control (mg/dl) |
|---|---|---|---|---|---|---|
| 2 | 72 ± 2.1 | 315 ± 4.3 | 170 ± 5.2 | 200 ± 6.1 | 290 ± 3.4 | 308 ± 4.6 |
| 4 | 71 ± 3.2 | 310 ± 5.5 | 250 ± 4.1 | 155 ± 5.7 | 280 ± 4.3 | 302 ± 5.2 |
| 6 | 75 ± 2.8 | 312 ± 6.1 | 310 ± 3.7 | 100 ± 4.5 | 295 ± 6.0 | 305 ± 5.1 |
| 8 | 70 ± 3.5 | 315 ± 5.4 | 312 ± 4.6 | 75 ± 3.2 | 300 ± 4.8 | 309 ± 3.9 |
The negative control maintained the lowest levels, while the disease control showed persistently high HbA1c, indicating chronic hyperglycemia. The use of the marketed product and PLFE-4 resulted in a significant (**p < 0.01) reduction in HbA1c by the third month, demonstrating their ability to help maintain stable blood sugar levels by inhibiting the breakdown of GLP-1 and GIP, as well as the DPP-4 inhibitor combined with Trigonella foenum-graecum liposomes, as mentioned in Figure 8.
 | Figure 8. Comparative glycosylated hemoglobin (HbA1c) assay results in a mouse model using the prepared liposome PLFE-4. The average reduction in HbA1c level (mg/dl) for the negative control, disease control, marketed product, and optimized formulation [PLFE-4] (* denotes statistically significant changes (p < 0.05) at 95% confidence level, and * * denotes highly significant changes (p < 0.01) at 95% CL). [Click here to view] |
4. CONCLUSION
In conclusion, the development of vildagliptin-loaded liposomes containing fenugreek (Trigonella foenumgraecum) represents notable progress in overcoming the pharmacokinetic limitations of traditional solid dosage forms. The modified liposomal system, specifically the PLGA-based PLFE-4, demonstrated higher drug entrapment efficiency, sustained drug release, improved permeability, and extended therapeutic effectiveness through strategic formulation using a full factorial design and the ether injection method. Comprehensive physicochemical and in vivo tests confirmed the formulation’s ability to significantly improve glycemic control, as shown by consistent reductions in blood glucose levels and positive HbA1c results in diabetic rats, by preventing GLP-1 and GIP degradation of DPP-4 inhibitors (vildagliptin) and fenugreek-loaded liposomes. These findings collectively highlight the potential of this innovative liposomal delivery system as a more effective and biocompatible option for the long-term management of T2DM.
5. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
6. FINANCIAL SUPPORT
There is no funding to report.
7. ETHICS STATEMENT
The study protocol was approved by the Research Ethic Committee of the Department of Pharmacy, University of Asia Pacific, Dhaka, Bangladesh and followed the laboratory animal guidelines for ethical review of animal welfare (Approval no.: UAP/REC/2022/112).
8. DATA AVAILABILITY
All the data is available with the authors and shall be provided upon request.
9. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
10. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
11. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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