Formulation And Evaluation of Solid Lipid Nanoparticles of Poorly Water-Soluble Drug Glimepiride by Hot Homogenization Technique

Abstract

One of the third-generation sulfonylureas used to treat type 2 diabetes is glimepiride. Due to subtherapeutic plasma drug levels, glimepiride's poor water solubility and delayed rate of dissolution might cause unpredictable clinical responses or therapeutic failure in some circumstances. Solubility, dissolution rate, and biological performance of the medication were to be improved as a result of this investigation. Nanoparticle technology is extremely helpful for increasing a substance's solubility, permeability, and bioavailability. The SLNs of glimepiride were attempted to be prepared in the current study using Glyceryl behenate as the solid lipid, Poloxamer, and Tween 80 as the surfactant and co-surfactant, respectively. Successful incorporation of glimepiride into solid lipid nanoparticles exhibited improved solubility and dissolution. This is a result of the drug particle size being reduced to the nano level by high shear homogenization and lyophilization. Solid lipid nanoparticle stability is improved by lyophilization, and lyophilizates have good re-dispersibility after being sonicated for two minutes. Tween 80 and Poloxamer, when utilized as cosurfactants, have excellent wetting and surfactant properties that contribute to the drug's increased solubility. Based on the in-vitro dissolution of all four formulations—SLN1, SLN2, SLN3, SLN4, SLN5, and SLN6—in three distinct media—distilled water, 0.1 N HCl, and phosphate buffer pH 6.8—formulation optimization was carried out. FT-IR, DSC, XRD, SEM, TEM, Particle size, and zeta potential tests were performed on the improved formulation SLN5. The characterization study's findings were compiled and assessed for solubility improvement or bioavailability.

Keywords

Introduction

Characterization of solid lipid nanoparticles of Glimepiride

Solubility study of solid lipid nanoparticles

The solubility of solid lipid nanoparticles was determined by taking an excess amount of SLNs and adding them to 10 ml of solvent, in Teflon-facing screw-capped vials. The supernatant liquid were collected and filtered through 0.2μ membrane filter and analysed by UV visible spectrophotometer at wavelength 234 nm for glimepiride. [11,12]

In Vitro Dissolution Study of Glimepiride solid lipid nanoparticles

In vitro dissolution studies Glimepiride solid lipid nanoparticles were carried out by USP type XXIV rotating basket type dissolution apparatus. The dissolution carried out in three different media deionized water, 0.1 N HCl, Phosphate Buffer 6.8 each of 900ml. SLNs were placed in each vessel and the medium was allowed to maintain at 100 rpm at 37⁰ ± 0.5⁰ c. Samples of 5ml were sink condition. The absorbance of sample was measured by UV double beam spectrophotometer at λmax 234 nm and cumulative percentage drug release were calculated. [13]

Particle size and zeta potential [15]

The mean diameter (z-average diameter) and size distribution were measured by photon correlation spectroscopy and the zeta potential was measured in capillary cells with path lengths of 10 mm, using the Nano ZS Zetasizer. Measurements were performed in deionized water obtained by a MilliQ system. [17,18]

Entrapment Efficiency

The entrapment efficiency of SLN calculated by measuring the concentration of free drug in the dispersion medium. The concentration of entrapped drug determined by substracting the concentration of free unentrapped drug from amount of initial compound used. The entrapment efficiency was calculated by the following equation: [20]

Entrapment efficiency = WInitial drug – Wfinal drugWInitial drug

 × 100

Scanning electron microscopy

For scanning electron microscopy (SEM), dried solid lipid nanoparticles loaded with model drugs were fixed on a stub using double-sided adhesive tape and then made electrically conductive by coating with a thin layer of gold for 30 seconds using JEOL fine coat. [21]

Transmission electron microscopy:

TEM study was performed to confirm the size and shape of drug dispersed in lipid. Before the examination the samples were diluted then dropped onto a carbon coated copper grids and dried in air before examination. [22]

Differential scanning calorimetry:

  Thermal characteristics of drug Glimepiride (GLP) and optimized batch of solid lipid nanoparticles (SLN5) were studied by using a differential scanning calorimeter. [24,28]

X-ray diffractometry

Powder X-ray diffractometric (PXRD) pattern of pure drug Glimepiride (GLP) and GLP loaded SLN were obtained by employing X-ray diffractometer (3000, Seifert); Ni-filtered Cu-K radiation, voltage of 40 kV, and current of 30mA radiation scattered in the crystalline regions were used and measured with a vertical goniometer. [29]

Preparation of SLN dispersion

Solid lipid nanoparticles of optimized batch F5 equivalent to weight of 50mg of drug was taken to form dosage form. Then the SLNs was then added in distilled water and homogenized at a pressure of 500 bar for 15 min 3 cycles in a high pressure homogenizer (Homogenius, Panda). The volume of obtained SLN dispersion was made upto 50ml with water and stored in well closed container. [31,33]

EVALUATION PARAMETERS

PH measurement

The pH value of the formulation was measured by immersing the electrode into the dispersion using calibrated pH meter (Systronics MK VI, Mumbai). [34]

Viscosity

The viscosity of the formulation was determined by using Brookefield Viscometer. All the experiments were performed at 25⁰C and in triplicates. [35]

Drug content

About 1 ml of SLN dispersion was diluted using solvent methanol and drug content was determined spectrophotometrically at 234 nm. [36]

In vitro diffusion study

Drug release from the formulations was studied in vitro using dialysis membrane (Himedia, India; molecular cut-off point 3500 D). Membrane was soaked in double-distilled water for 12 h before mounting in a diffusion cell. SLN dispersion (1 mL) free from any unentrapped drug was placed in dialysis tube, which was suspended in a beaker containing 250 mL PBS (pH 6.8). The contents of the beaker were stirred using a magnetic stirrer at 37±2⁰C. Samples were withdrawn periodically and replaced with the same volume of fresh media to keep the volume in the receptor compartment constant. The samples were analysed for drug content using UV visible spectrophotometry. [37]

RESULTS AND DISCUSSION

UV Spectrophotometric analysis

The ultraviolet spectrum of drug is obtained by scanning from 200 to 400 nm. The absorption maximum (λ max) of Glimepiride (GLP) was found to be 234 nm in distilled water, methanol, 0.1 N HCl and Phosphate buffer pH 6.8. Hence all the further analysis was carried out at 234 nm. [38,39]

Preparation of standard curve of Glimepiride in distilled water

Accurately weighed 10 mg of Glimepiride and transferred to 100ml volumetric flask. This was dissolved in water and volume made upto 100ml. this solution was treated as the stock solution and contains 100 µg/ml of Glimepiride solution. Further dilutions made with distilled water to obtain the concentration of 25, 38, 50, 63, 75 µg/ml. Absorbance of these solutions were measured at 234 nm against blank solution i.e. distilled water. [40]

Fig 1. UV spectrum of Glimepiride in water

Table 2: Absorbance of different concentrations of glimepiride in distilled water

Sr. No.	Concentration (µg/ml)	Absorbance
1	0	0
2	25	0.2126
3	38	0.3201
4	50	0.4223
5	63	0.5220
6	75	0.6306

 

Fig 2: Standard calibration curve of Glimepiride in distilled water

Solubility determination

Solubility Studies were performed to check the solubility enhancing property of solid lipid nanoparticles. Solubility study of drug was carried out in in different conditions and media at 37⁰c the result are shown in table 3. [43]

Table 3: Solubility of glimepiride in different media

Sr. No.	Solvents/ Media	Solubility in mg/ml
1	Distilled water	0.0336
2	Methanol	0.391
3	0.1 N HCl	0.156
4	Phosphate buffer pH 6.8	0.147

Table 4: Enhanced solubility of SLN in water (In fold)

Formulation batch	Solubility mg/ml	Enhanced solubility (In fold)
SLN1	0.163	4.66
SLN2	0.248	5.81
SLN3	0.386	11.48
SLN4	0.334	9.94
SLN5	0.453	13.48
SLN6	0.262	7.79

    FT-IR studies

The FT-IR studies are done to check the changes are done of drug with polymer. FT-IR studies of pure Glimepiride drug (GLP), physical mixtures GLP and Glyceryl beheneate (GB), GLP and Poloxamer (PLX) and Optimized formulation batch (SLN5) were performed.     

study were shown in Table no. 5

 

Figure 3: FT-IR spectrum of Pure Glimepiride Drug

Figure 4: FT-IR spectrum of Glimepiride + Glyceryl behenate

Figure 5: FT-IR Spectrum of Glimepiride + Poloxamer

Figure 6: FT-IR Spectrum of Optimized formulation SLN5

Figure 7: In vitro dissolution of SLNs in Water

Figure 8: In vitro dissolution of SLNs in 0.1 N HCl

Figure 9: In vitro dissolution of SLNs in Phosphate buffer pH 6.8

Particle size and zeta potential

Particle size of optimized formulation of solid lipid nanoparticles was analyzed by Photon correlation spectroscopy using a Nano ZS zetasizer (Malvern) equipped with at 25⁰ c. Average particle size distribution, Polydispersity index and zeta potential of the optimized formulation summarized in Table 9. From the data it is concluded that average particle size was found to be 669.1 nm (1-1000 nm) with zeta potential -0.711 and polydispersity index 0.601. Hence, it can be concluded that, SLNs are converted into nano form, this may be the reason for enhanced solubility of drug. [45]

Figure 10: Particle size distribution of SLN5.

Figure 11: Zeta potential of SLN5.

Entrapment Efficiency

          The percentage of entrapped Glimepiride in different SLN formulation with different drug, lipid, and surfactant and stabilizer concentration was determined spectrophotometrically. The results are given in the following Table 6. The drug entrapment efficiency ranged from 79.4 – 87.4 %. Highest entrapment 87.4% was found in SLN5 and lowest 77.5 in SLN1 formulation. Hence increase in drug loading increased the percentage of drug entrapped and decrease in drug loading causes decrease in entrapment efficiency. [47]

Table 6: % Entrapment efficiency in SLNs

Sr. No.	Formulation	% Entrapment efficiency
1	SLN1	77.5%
2	SLN2	89.1%
3	SLN3	84.8%
4	SLN4	81.4%
5	SLN4	87.4%
6	SLN4	80.4%

DSC studies

DSC curve of pure Glimepiride shows sharp endothermic peak at 214.35⁰ C.  DSC thermogram of SLN shows two endothermic peaks at 165.03⁰ c and 72.08⁰ C which is not same as like pure Glimepiride (GLP). The peak at 165.03⁰ c is of drug in the formulation and peak at 72.08⁰ c is of lipid in the formulation. [46]

Figure 12: DSC thermogram of pure drug Glimepiride (GLP).

Figure 13: DSC thermogram of SLN5.

XRD Studies

Powder X-Ray Diffraction (XRD) of GLP and SLN are shown in figure 14, 15. The pure GLP exhibited intense crystalline peak between 10⁰ and 30⁰. Characteristic diffraction peaks at 13.71⁰, 17.22⁰, 21.29⁰, 23.15⁰ were observed with intense peak at 21.29⁰ indicating the crystalline nature of GLP. [34]

Fig. 14: XRD graph of pure drug

Fig. 15: XRD graph of SLN

Scanning Electron Microscopy studies

 The SEM studies are generally performed to the study surface morphology of the drug particles. The morphological characteristic of drug and SLN is shown in following figures. [22,25]

Figure 16: SEM images of (A): Pure drug (GLP), (B): SLN

Figure 17: TEM images of (A): pure drug (GLP), (B): SLN

The morphology and particle size of GLP & SLN was investigated by TEM. TEM observation used to further confirm the particle size of Glimepiride nanoparticles embedded with the lipid. Very small nanoparticle are easily seen here in figure (A) and (B).  A comparison of crystalline size mentioned through X-ray line profile fit with the particle size observed from the TEM. Particle size of pure drug was found to be 200 to 500 nm and particle size of SLN which was reduced to nano scale which is clearly shown in the figure 17. TEM suggests that the SLN have mixture of single & polycrystalline structures. [42,43]

POST FORMULATION EVALUATION

PH of the formulation

The pH value of the formulation was determined by Digital pH meter by immersing the electrode into the SLN dispersion, pH was found to be 4.35.

Viscosity

he viscosity of the formulation measured by Brookfield’s viscometer and it was found to be 16 cPs.

Drug content

Drug content of our new formulated system was found to be 95.84%. This parameter is necessary for the dosage of antidiabetic formulation.

In vitro drug diffusion study [16]

In vitro drug release of the formulation was carried out by diffusion study by dialysis bag method in phosphate buffer pH 6.8. The results of in vitro drug diffusion study were shown in following table 11.

Fig. 18: In-vitro diffusion of SLN dispersion

From this study, %CDR at 24 hr was found to be 95.36. As compared to dissolution study drug release by diffusion study found to be low, this may be due to the particle size of the dispersion above 500 nm. [45]

SUMMARY

In this study we have formulated four different formulations of solid lipid nanoparticles differing in lipid concentrations by homogenization of lipid phase and aqueous surfactant solution followed by lyophilization. Glyceryl behenate used as lipid acts carrier for entrapped drug in controlled manner which causes extended release. Poloxamer used as surfactant and Tween 80 as cosurfactant possess good surfactant and wetting property which help in increasing the solubility of drug. Optimization of formulation was carried out on the basis of in vitro dissolution of all four formulations SLN1, SLN2, SLN3, SLN4, SLN5, SLN6 in three different media distilled water, 0.1 N HCl and Phosphate buffer pH 6.8. The optimized formulation SLN5 was subjected to FT-IR, DSC, XRD, SEM, TEM, Particle size and zeta potential. The result of the characterization study were collected and evaluated for solubility enhancement or bioavailability.

CONCLUSIONS

From this study, it is concluded that hot homogenization technique is effective and economical method to develop solid lipid nanoparticles. Glimepiride was successfully incorporated in solid lipid nanoparticles and revealed enhancement of solubility and dissolution. This is due to reduction of drug particle size to nano level due to High shear homogenization followed by lyophilization. Lyophilization is useful for stability of solid lipid nanoparticles and lyophilizates possess good re-dispersibility upon sonication for 2 min. SEM and TEM images given the information regarding size, shape and surface morphology of SLN. Particle size was found to be below 1000 nm and entrapment efficiency shown that drug is successfully entrapped in the lipid matrix. The characterization studies such as FT-IR, DSC, and XRD confirmed the compatibility between the drug and excipients. All the characterization studies confirm the formation of solid lipid nanoparticles. The solubility and in vitro dissolution study confirmed the application of solid lipid nanoparticles in enhancement of solubility and extended release of drug.

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