Formulation And Characterization of Glibenclamide Nanocrystal for Enhance Solubility and Bioavability in Type 2 Diabetes Mellitus

Glibenclamide is a second-generation sulfonylurea widely prescribed for the management of Type 2 Diabetes Mellitus; however, its clinical performance is limited due to poor aqueous solubility and low oral bioavailability. The drug belongs to BCS Class II category where dissolution is the rate-limiting step for absorption, resulting in variable therapeutic response from conventional dosage forms. Therefore, enhancement of solubility and dissolution rate is essential to achieve consistent glycemic control and improved patient compliance. Nanocrystal technology has emerged as an effective strategy to overcome solubility related limitations of poorly water-soluble drugs. Reduction of particle size to nanometer range increases surface area and saturation solubility, leading to rapid dissolution as explained by the Noyes–Whitney equation. Stabilizers such as PVP K30, HPMC, and Tween 80 are commonly used to prevent aggregation and to maintain physical stability of nanocrystals. In recent years, Design of Experiment (DOE) has gained importance in pharmaceutical formulation for systematic optimization, as it allows evaluation of the effect of multiple formulation variables with minimum experimental runs. In the present study, glibenclamide nanocrystals were formulated and optimized using DOE approach to obtain a stable formulation with enhanced solubility, dissolution rate, and bioavailability for effective treatment of Type 2 Diabetes Mellitus.

Aim and Objectives

The present study was aimed at formulating and characterizing glibenclamide nanocrystals to enhance its aqueous solubility and oral bioavailability for effective management of Type 2 Diabetes Mellitus. The work focused on preparation of nanocrystals using suitable stabilizers and optimization of formulation variables through Design of Experiment (DOE) approach in order to obtain minimum particle size and maximum drug release. The study also intended to evaluate the developed formulations for particle size, polydispersity index, drug content, and in-vitro dissolution, followed by kinetic modelling to understand the mechanism of drug release. Based on these evaluations, an optimized formulation was to be selected to demonstrate the potential of nanocrystal technology in improving therapeutic performance of glibenclamide.

2.    MATERIALS AND METHOD

Materials

Glibenclamide (GLB) was received as a gift sample from Pioneer Company for Pharmaceutical Industries, PVP K15 was purchased from Otto Chemie Pvt. Ltd., Germany, while Poloxamer 188 was obtained from the Chemdyes Corporation, Rajkot. HPMC E5 and HPMC E15 were procured from Baoji, China. Tween 20 and methanol were purchased from Loba Chemie (Chemicals) Pvt. Ltd., India.

Pre-Formulation Studies

Before formulation development, pre-formulation tests were conducted to assess glibenclamide physicochemical characteristics and make sure it was compatible with the chosen excipients.

Solubility Studies

Glibenclamide solubility was assessed in a number of solvents, including methanol, distilled water, and phosphate buffer (pH 6.8). Each solvent was filled with an excess of the medication and shaken constantly for a full day at room temperature. A UV-visible spectrophotometer was used to analyze the drug content after the samples were filtered and appropriately diluted. The purpose of these investigations was to find appropriate dissolving media and solvents for formulation and assessment.(8)

Determination of λmax

A UV–visible spectrophotometer was used to scan a standard stock solution of glibenclamide in methanol between 200 and 400 nm in order to identify the maximum wavelength of absorption (λmax), which was utilized for additional analytical investigations.

Calibration Curve of Glibenclamide

The stock solution was used to create a number of standard glibenclamide solutions in phosphate buffer (pH 6.8). A UV–visible spectrophotometer was used to test each solution's absorbance at the specified λmax. To demonstrate linearity and facilitate quantitative glibenclamide estimate in later research, a calibration curve between concentration and absorbance was plotted.(9)

Drug–Excipient Compatibility Study

Fourier Transform Infrared (FTIR) spectroscopy was used in drug–excipient compatibility studies to assess any potential interactions between glibenclamide and the chosen excipients (PVP K30, HPMC E15, PVP K15, Poloxamer 188, and Tween 80). Pure glibenclamide and physical mixes' FTIR spectra were recorded, and any notable shifts or disappearances of distinctive peaks were compared.(10)

Formulation and Optimization of Nanocrystals by Central Composite Design  

Glibenclamide nanocrystal formulation was optimized using Central Composite design and Design-Expert software (version 12). Zeta potential (Y1), percent drug entrapment (Y2), and drug content (Y3) were chosen as dependent variables, whereas concentrations of polymers, such as Polyvinylpyrrolidone K15 (X1), Hydroxypropyl methylcellulose (X2), and stabilizer, were chosen as independent factors.5 and 6

Table 1: Factors and Levels for the Optimization of Nanocrystals

Table 2: Response Variable with Unit

The response variables, percent drug entrapment (Y1) and drug content (Y2), were combined following the production of nine batches. The impact of the independent variables on the dependent variables was ascertained by applying the 3D response surface methodology to the data.

Figure 1 :Drug content and drug entrapment efficiency of glibenclamide nanocrystal formulation (F1-F9)

Effect of X1 and X2 on Y1                                              Effect of X1 and X2 on Y2 

The Effect of X1 and X2 on Y1 Show by figure           The Effect of X1 and X2 on Y2 Show by figure

Figure 2: Effect of X1 and X2 on Y1        Figure 3: Effect of X1 and X2 on Y2

Preparation of GLB Nanocrystal

The antisolvent precipitation method was used to create glibenclamide nanocrystals. The organic phase was created by dissolving a precisely measured amount of glibenclamide in an appropriate proportion of methanol. The necessary concentrations of stabilizers (PVP K30, PVP K15, or HPMC E15) and surfactants (Poloxamer 188 or Tween 80) were dissolved in distilled water while being continuously stirred to create the aqueous antisolvent phase. Next, using a syringe and continuous magnetic stirring at 1200 rpm for two hours, the organic drug solution was introduced dropwise into the aqueous phase. The precipitation of glibenclamide nanocrystals was shown by the instantaneous creation of turbidity. To guarantee full solvent diffusion and stabilization of the nanocrystals, the resultant nanosuspension was further agitated. Following preparation, the nanosuspension underwent additional characterization and assessment.

Figure 4: Formulations of Nanocrystal (F1-F9)

Table 3: Formulation of Nanocrystal (F1-F9)

Characterization and Evaluation of Glibenclamide Nanocrystals

To evaluate the generated glibenclamide nanocrystals' particle size, stability, solid-state characteristics, and dissolving behavior, they underwent thorough physicochemical characterization.

Particle Size

Using a Malvern Zetasizer and the dynamic light scattering technique, the mean particle size of the nanosuspension was calculated. To prevent numerous scattering effects, the samples were appropriately diluted with distilled water prior to analysis. The average particle size was measured in nanometers and all measurements were done at room temperature.(11)

Zeta Potential

To assess the formulation's surface charge and physical stability, the nanocrystals' zeta potential was evaluated. Greater electrostatic repulsion between particles and improved stability of the nanosuspension are indicated by higher absolute values of zeta potential.(12)

Scanning Electron Microscopy (SEM)

Scanning electron microscopy was used to analyze the glibenclamide nanocrystals' surface morphology and form. Samples were placed on aluminium stubs, covered with a thin layer of gold, and examined at the appropriate magnifications.(13)

X-Ray Diffraction (XRD) Analysis

The crystalline nature of glibenclamide and potential modifications to its solid-state characteristics following nanocrystal formation were examined by X-ray diffraction investigations. An X-ray diffractometer with Cu-Kα radiation (λ = 1.5406 Å) was used to record the diffraction patterns of pure glibenclamide and optimized nanocrystals throughout a 2θ range of 5°–50°. Changes in peak intensity and crystallinity were compared between the patterns.(14)

Drug Entrapment Efficiency

Centrifuging a measured amount of nanosuspension to extract the free drug from the supernatant allowed for the measurement of the drug entrapment efficiency. UV-visible spectrophotometry was used to analyze the supernatant. A high entrapment efficiency suggests that glibenclamide was successfully included into the nanocrystal formulation.(3)

Drug Content

A precisely measured volume of the nanosuspension was diluted with methanol, filtered, and examined at the predefined glibenclamide λmax using a UV-visible spectrophotometer. The standard calibration curve was used to determine the drug content.(11)

In-Vitro Drug Release Study

USP Type II (paddle) dissolution equipment was used for the in-vitro drug release investigation. The dissolution medium was phosphate buffer pH 6.8, which was kept at 37 ± 0.5 °C with a paddle speed of 50 rpm. Samples were removed at prearranged intervals, filtered, and subjected to UV-visible spectrophotometry analysis. It was determined what the cumulative proportion of medication release was.(15)

In-Vitro Drug Release Kinetic Modeling

To assess the mechanism of drug release, the optimized glibenclamide nanocrystal formulation (F8) in vitro drug release data were fitted into various kinetic models. The Higuchi, Korsmeyer-Peppas, zero-order, and first-order models were used to analyze the cumulative percentage drug release.(16)

3.    RESULT AND DISCUSSION

3.1       Preformulating studies of Glibenclamide

3.1.1    Organoleptic evaluation

Since the drug's texture, condition, and other characteristics matched those specified in IP, it is considered pure. The sensory characteristics also aided in identifying the material and its behavior, according to IP. It demonstrates how glibenclamide properties and those of the reported drug were identical. Table 1 presents the organoleptic properties of the drug glibenclamide.

Table 4: Organoleptic Evaluation of Glibenclamide Drug

3.1.2    Solubility

The solubility of glibenclamide has been studied in a range of solvents, such as water, ethanol, methanol, and alcohol. Methanol was the main solubility. It shows that glibenclamide was soluble in organic solvents like methanol and alcohol. Glibenclamide was shown to be insoluble in water. Table 2 shows the solubility of glibenclamide.

Table 5: Solubility of Glibenclamide Drug in Different Solvent

3.1.3    Melting Point

Glibenclamide has a melting point of 169–1730 C, according to the Indian Pharmacopoeia. Glibenclamide was discovered to have a melting point of about 1700C. As a result, it uses the IP standard to show the purity of the sample. The melting point of the drug glibenclamide is shown in Table 10.

Table 6: Melting Point of Glibenclamide Drug

3.1.4    Determination of λmax of Glibenclamide Drug in 0.1N NAOH

The wavelength of maximum absorbance (λmax) of glibenclamide in 0.1 N NAOH was found to be 227 nm. The glibenclamide calibration curve in 0.01 N NAOH is shown in Figure 1. The concentration and absorbance of glibenclamide were found to be linear.

Figure 1: Determination of λmax of Glibenclamide

Preparation of Standard Calibration Curve in Methanol as per IP Method:

The absorbances of the standard solutions in methanol in the range of 2–16 µg/mL were measured at 242 nm. The standard calibration curve, which was produced by plotting absorbance (λmax) against concentration, is shown in Table 16 and Figure 22. Linearity was examined using regression analysis.

Table 7: Calibration Curve of Glibenclamide drug in Methanol

Figure 5:  Calibration Curve of Glibenclamide in Methanol

The linear reversion analysis was done on Absorbance data points. The results are as follow for standard curve

Slope = 0.045

The intercept = 0.029

The correlation coefficient (r2) = 0.985

The glibenclamide calibration curve in methanol at 242 nm was examined using a UV-visible spectrophotometer. The correlation coefficient (r2) was found to be 0.985 after the calibration curve was plotted and the slope, intercept, and correlation coefficient were calculated.

3.1.5    FTIR of Glibenclamide

Glibenclamide distinctive functional groups were identified and its chemical integrity was verified using Fourier Transform Infrared (FT-IR) spectroscopy. The KBr pellet method was used to record the spectra at a resolution of 4 cm?¹ in the 400–4000 cm?¹ scanning range.

Glibenclamide FT-IR spectrum revealed notable absorption bands at 3405 cm?¹ (O–H stretching), 3063 cm?¹ (N–H stretching), and 2943 cm?¹ (C–H stretching). Sharp peaks around 3670 and 3450 cm?¹ suggested oxygen-containing functional groups like phenolic or alcoholic moieties, while a broad band in the 3650–3250 cm?¹ range indicated the existence of hydroxyl groups.

These distinctive peaks verified that glibenclamide had urea, Sulphonamide, carbonyl, and aryl functional groups, verifying the drug's purity and structural integrity.

Figure 6: FTIR Spectrum of Glibenclamide Drug     Figure 7: FTIR Spectrum of Drug +HPMC E15

Figure 8: FTIR spectrum of drug + PVPK30          Figure 9: FTIR spectrum of drug + PVPK15

Figure 10: FTIR spectrum of drug + Poloxamer 188

3.2       Evaluation of Glibenclamide Nanocrystal

3.2.1    Particle size

A Nanotrac Wave II particle size analyzer was used to measure the glibenclamide nanocrystals' particle size at 25–26 °C. To avoid aggregation, the nanosuspension was appropriately diluted with deionized water and sonicated for one to two minutes before analysis. Triplicate measurements were made, and the mean ± SD was used to express the results. Size distribution was assessed using the polydispersity index (PDI) and Z-average particle size. With a narrow PDI and the smallest particle size, the optimized formulation (F8) demonstrated a homogenous particle size distribution.

Figure 11: Particle Size Distribution of Optimized Glibenclamide Nanocrystal Formulation

3.2.2    Zeta potential

After glibenclamide nanocrystals were appropriately diluted with deionized water, their zeta potential was assessed using a Nanotrac Wave II analyzer. With a zeta potential of –14.3 mV, the improved formulation (F8) demonstrated enough electrostatic repulsion between particles. The glibenclamide nanocrystal system's good physical stability and decreased aggregation were shown by this negative surface charge.

Figure 12: Zeta Potential of Optimized Glibenclamide Nanocrystal Formulation (F8)

3.2.3    Scanning Electron Microscopy

Using scanning electron microscopy, glibenclamide nanocrystals' surface morphology was investigated. The SEM pictures showed that the nanocrystals had a rough surface, an uneven shape, and a uniform distribution with little to no aggregation. The successful creation of glibenclamide nanocrystals was validated by the smaller particle size and distinct nature of the nanocrystals.

Figure 13: SEM Image Of Optimized Glibenclamide Nanocrystal Formulation (F8)

3.2.4    XRD (X- ray diffraction)

X-ray diffraction investigations were used to examine the crystalline nature of glibenclamide nanocrystals. The crystalline structure of pure glibenclamide was confirmed by its strong and crisp diffraction peaks. Reduced crystallinity as a result of nanosizing and the presence of stabilizer (PVP K30) was seen in the optimized nanocrystal formulation (F8), as evidenced by a decrease in peak intensity and mild peak broadening. It is anticipated that this decrease in crystallinity will increase glibenclamide solubility and rate of dissolution.

Figure 14: XRD spectra of (A) GLB, (B) GNCs and (C) GLB-GNCs

3.2.5    Drug entrapment efficacy

The entrapment efficiencies of glibenclamide nanocrystals (F1–F9) ranged from 65.42 ± 0.42% to 95.68 ± 0.38%. The enhanced formulation F8, which had the highest entrapment efficiency, indicated effective drug loading. The higher EE may be caused by the stabilizing impact of PVP K30 and Tween 80, which reduced drug leakage and enhanced drug–polymer contact.

Table 8: Drug Content and Drug Entrapment of Glibenclamide Nanocrystal

Figure 15 : Standard Calibration Curve      Figure 16: Drug entrapment efficiency of glibenclamide nanocrystal

3.2.6 Drug content

Glibenclamide nanocrystal formulations (F1–F9) varied in their drug concentration from 82.15 ± 0.42% to 91.48 ± 0.22%. The maximum drug content was found in the optimized formulation F8, suggesting that the medication was evenly distributed throughout the nanocrystal system. Good repeatability and successful medication integration are confirmed by the little variation between formulations.

Figure 17: Comparative drug content of different glibenclamide nanocrystal formulations

3.2.7    In – vitro Percentage Drug Release

When compared to a traditional formulation, the glibenclamide nanocrystals' in-vitro drug release profile demonstrated a quick and improved dissolution pattern. Improved dissolving behavior was demonstrated by the optimized formulation F8, which showed the highest cumulative drug release within 60 minutes of all batches. The greater release could be explained by the nanocrystals' larger surface area and smaller particle size, which allowed the medication to dissolve more quickly in phosphate buffer pH 6.8.

Figure 18: In - vitro Drug Release Study of Glibenclamide Nanocrystal

3.2.8    Kinetic Release Profiles

Fig  19: Zero Order Release Kinetic Plot of F3           Fig 20: First - Order Release Kinetic Plot of F3

Fig 21: Higuchi Model Plot of F3Figure        22: Korsmeyer - Peppas Release Kinetic Plot of F3

Table 9:Release Kinetic Model Fitting Data of Optimized Formulation F3

Kinetic Model Formulation F8

Fig 24: Zero - Order Release Kinetic Plot of F8    Fig 23: First - Order Release Kinetic Plot of F8

Fig 25: Higuchi Model Plot of F8     Fig 26: Korsmeyer-Peppas Release Kinetic Plot of F8