Formulation and Evaluation of Trimetazidine Hydrochloride Loaded Solid Lipid Nanoparticles

Introduction

Oral route is a preferable route for administration because of higher compliance. However due to poor stability, poor permeability, bioavailability is affected. The physicochemical and metabolic instability in the stomach and the liver negatively influences the drug concentration in the blood.

Due to degradation of the drug (via hepatic first pass metabolism), there is chance for increased toxicity and the desired concentration of drug may not be able to reach the site of action (target site). Hence to overcome these problems associated with the oral route, a new nanoparticulate drug delivery system was developed which can provide controlled and targeted drug delivery system.^1,2

According to NNI (National Nanotechnology Initiative), nanoparticles are defined as ‘the solid colloids in size of nanometers ranging from 10-1000 nm (generally 50-500 nm), to which drugs can be adsorbed, entrapped, encapsulated or covalently attached’. Nanoparticles can be formulated using solid lipid, natural polymer (chitosan, guar gum, gelatin, bovine serum albumin, human serum albumin, sodium alginate), synthetic polymer (poly d lactide, poly- E-caprolactane, polymethacrylate), semisynthetic polymer and protein. The materials for formulating the nanoparticles are selected according to their encapsulation capacity, drug stability, drug release pattern and their targeting capacity.³

Nano-particles possess the ability to penetrate through several anatomical barriers due to their nanometer size and they can also release the drug in a sustained manner.

Trimetazidine hydrochloride is an anti-anginal drug. Classified as a ‘fatty acid oxidation inhibitor,’ it helps prevent and alleviate angina symptoms. This drug supports the energy metabolism of heart muscle cells, safeguarding them against the impacts of reduced oxygen supply. Trimetazidine hydrochloride belongs to BCS Class I.

Trimetazidine hydrochloride is administered two times a day which causes peaks and valleys in plasma drug concentration. To avoid fluctuations of plasma drug concentration, sustained release dosage forms are preferred. To achieve constant drug levels in the plasma, in the current study, we aimed to formulate and evaluate Trimetazidine loaded solid lipid nanoparticles (SLNs). Thus developed Trimetazidine SLNs, can enhance stability and efficacy of Trimetazidine and patient compliance.⁴

Materials and Methods

Trimetazidine hydrochloride was purchased from Yarrow Chemicals. Tristearin from Sasol Germany, Glycerol mono stearate from Research-Lab Fine chem. Industries, Compritol from Gattefosse-France, Soy lecithin was purchased from Hi Media Laboratories Pvt. Ltd, and Tween 80, chloroform and methanol were purchased. All the chemicals and solvents utilized were of analytical grade.

Fourier-transform infrared spectroscopy (FT-IR)

Drug-solid lipid interactions were studied by FTIR spectroscopy. The following samples were subjected to FTIR studies. Scanning of the samples in FTIR were done in the wavelength of 400–4000 cm-1 using FTIR spectrophotometer.⁵

a. Pure drug (Trimetazidine hydrochloride)

b. Physical mixture, drug + Tristearin (1:1)

c. Physical mixture, drug + GMS (1:1)

d. Physical mixture, drug + Compritol (1:1)

Preparation of Trimetazidine hydrochloride SLN using lipids (Compritol, Tristearin and Glycerol monostearate)

SLNs were prepared by using lipids (Compritol 888, Tristearin and glycerol monostearate) and surfactants (Tween 80 and Poloxamer 188). Lipid was first melted by heating in a boiling tube and then soy lecithin and drug were added to the lipid melt, which was then heated to the temperature 5°C above the melting point of the lipid. Simultaneously, surfactant (Poloxamer 188/Tween 80) and water were taken in a test tube and heated to temperature equal to that of lipid phase. This aqueous phase was slowly added to lipid phase in small quantities by continuous homogenization. After homogenizing the mixture for 15 minutes at 20,000 rpm, it was subjected to a probe ultrasonicator and run for 15 minutes at 75% amplitude. In a similar fashion, blank nanoparticles were made without the use of trimetazidine hydrochloride.⁶

Determination of Particle size, Polydispersity index and Zeta potential

Particle size estimation

Using the principle of dynamic light scattering, the particle size was estimated with a Malvern Zetasizer. The experiments were conducted at a temperature of 25.0±0.1 ºC, with the measurement angle set at 90º to the incident beam.⁷

Polydispersity index (PDI)

PDI is a metric derived from a cumulants analysis of the intensity autocorrelation function recorded by dynamic light scattering (DLS). The polydispersity index had been determined using the same tool, the Malvern Zetasizer.⁷

Zeta potential

The zeta potential was measured using a Malvern Zetasizer. Following dilution with distilled water, the nanoparticles were transferred to a transparent, disposable zeta cell and kept at 25°C. Three zeta runs were conducted on the sample to determine its size and zeta potential.⁸

Drug content

Five mL of methanol and about 0.2 mL of drug-loaded SLNs were put to a centrifuge tube. After centrifuging the mixture for 30 minutes at 5000 rpm, it was vortexed for 10 minutes. The collected supernatant was used. The amount of drug in the supernatant was measured at 269 nm using a UV spectrophotometer for trimetazidine hydrochloride.⁹

Drug content was calculated using following formula

Entrapment efficiency of SLN (%DEE)

The sample recovery chamber was positioned on top of approximately two mL of SLN loaded with trimetazidine hydrochloride that was placed in the Centrisart device’s outer chamber. The unit was centrifuged at 5000 rpm for 20 minutes.

The aqueous phase was filtered through a membrane into the sample recovery chamber, while the SLN and the encapsulated drug remained in the outer chamber. The resulting aqueous phase was analyzed at 269 nm using a UV spectrophotometer to measure trimetazidine hydrochloride. The entrapment efficiency was then calculated using the following relationship.⁹

Percentage Drug Loading Efficiency

Loading efficiency help us to deal with SLN’s after their separation from the medium and to know their drug content.

In vitro Drug Release

Study Franz diffusion cell was used to conduct in vitro drug release investigations. Two mL of nanoparticle dispersion was employed. Drug-containing nanoparticles were inserted into the donor compartment, and 22 mL of phosphate buffer (PB) pH 6.8 maintained at 25±2 °C was included in the receiver compartment. The magnetic beads rotational speed was kept at 50 rpm. A 2 mL sample was taken out at pre-fixed intervals. The samples’ drug content was determined using a UV spectrophotometer set at 269 nm. To examine the pattern of drug release, the percentage cumulative drug release from the collected data was computed and plotted against the function of time.¹⁰

Kinetic Modelling of Drug Dissolution Profiles

The following data treatment modes were used to plot the in vitro release profile results for each formulation:

1. Zero order - Cumulative % drug released versus time.

2. First order - Log cumulative percentage drug remaining versus time.

3. Higuchi’s - Cumulative percentage drug released versus square root of time.

4. Korsmeyer & Peppas - Log cumulative percentage drug released versus log time.¹¹

Results

Drug-polymer interaction study by FT-IR spectrophotometer

An FT-IR spectroscopy study had been carried out separately to check the compatibility between the drug (Trimetazidine hydrochloride) and the lipids (TS, compritol and GMS) used for the preparation of nanoparticles. The FT-IR was performed for drug, lipids and physical mixture of trimetazidine hydrochloride and individual lipids. The spectra obtained from FT-IR spectroscopy study at wave number from 4000 to 400 cm^-1 are shown below.

Determination of ƛmax of Trimetazidine hydrochloride in PB pH 6.8 and solvent methanol

A UV spectrophotometer was used to scan a solution of trimetazidine hydrochloride between 200 and 400 nm. Results showed absorbance max at 270 nm in case of methanol and 269 nm for PB pH 6.8.

Standard graph of Trimetazidine hydrochloride in solvent methanol and PB of pH 6.8

Standard graphs of Trimetazidine hydrochloride were plotted in 20 to 500 μg /mL concentration range in potassium dihydrogen orthophosphate buffer of pH-6.8 at 269 nm using UV- spectrophotometer. Regression equation obtained was y = 0.0016x + 0.0244 and R2 = 0.9996. This equation was used for estimation of drug in case of release studies, drug content estimation and determination of entrapment efficiency.

Particle size, PDI, Zeta potential

Trimetazidine hydrochloride SLNs developed with lipids (Glyceryl monostearate, Tristearin and Compritol) using soy lecithin as stabilizer and Tween 80 as surfactant were evaluated for their zeta potential, particle size and also PDI. Blank SLN was prepared using tristearin and tween 80. Particle size of blank nanoparticles was found to be 98.1 nm, whereas drug loaded particles were found to be between 49.7 to 523.7 nm

Drug Content

Percentage drug release of various formulations obtained was fitted to various kinetic models. To evaluate the kinetics and mechanism of drug release, R² values for zero order kinetics were ranged from 0.8168 to 0.9189 and for the first order kinetics, were ranged from 0.9628 to 0.9963 and for the Higuchi model, ranged from 0.9432 to 0.9907 and Peppa’s model were ranged from 0.9513 to 0.9846. Data were fitted to first order better with higher R² values, which indicate the drug release followed was first order. Since, the “n” value derived from the plots of Korsmeyer and Peppa ranged from 0.4230 to 0.5716, this indicated that mechanism of release of all formulations F1-F9 was Quasi-Fickian and anomalous i.e. non- Fickian diffusion.

Discussion

FT-IR Compatibility

FT-IR spectra of trimetazidine hydrochloride, lipids (Tristearin, Compritol and glyceryl monostearate), physical mixture of trimetazidine hydrochloride and lipids in 1:1 ratio are shown in Figure 1. The characteristic peaks of trimetazidine hydrochloride of pure spectrum were retained in the FTIR spectra of physical mixture of trimetazidine hydrochloride with tristearin, compritol, and GMS. Therefore, there was no drug lipid interaction. As a result, SLN were prepared using these lipids.

SLNs’ size, PDI and Zeta potential

Trimetazidine hydrochloride SLNs made with lipids (Compritol, Tristearin and Glyceryl monostearate) using soy lecithin as stabilizer and Tween 80 as surfactant were evaluated for their particle size, PDI and zeta potential. Report of size and zeta potential was obtained from the zeta sizer. Blank SLN was prepared using tristearin and tween 80. The particle size of blank nanoparticles was found to be 98.1 to 104 nm, whereas drug loaded particles were found to be between 49.7 to 523.7 nm.

PDI of all formulations were good within the range of 0.170 to 0.358. The zeta potential of blank SLNs was -15.2 mV. Zeta potential for drug-loaded SLN ranged from -24.2 to -38.0 mV.

Drug Content

Drug content of prepared SLN of three different lipids was determined by extracting drug with methanol and estimating using UV spectrophotometer. The values for drug content varied from 95.85 to 99.98%. These drug content values were utilized in case of release and entrapment efficiency studies.

Entrapment Efficiency

In all formulations, the average entrapment effectiveness of trimetazidine hydrochloride solid-liquid nanoparticles was found to be between 84.42% and 93.82%. Percentage entrapment efficiency for final selected formulation F1 showed 90.59%.

Loading Efficiency

Loading efficiency of trimetazidine hydrochloride SLN prepared with tristearin as lipid decreased for formulation F1 (18.118) to F3 (6.165). This was because with the drug’s continual addition to the formulation (10 mg), the amount of lipid increased from 50 mg to 150 mg. As the lipid concentration increased, the projected loading efficiency obviously decreased. The formulation F3 also may have good loading efficiency i.e., obtained by increasing the amount of drug in the formulation. Similarly, the loading efficiency decreased in case of GMS. However, the highest loading efficiency was observed for F1.

In Vitro Release Studies 

Diffusion studies for the prepared trimetazidine hydrochloride SLNs were performed using Franz diffusion cell. Cumulative drug release for different formulations is shown in the Table 6. The percentage CDR of trimetazidine hydrochloride from different trimetazidine hydrochloride nano-particles varied from 75.43 to 93.59% depending upon the drug to lipid ratio and the kind of lipid used.

In Vitro Release Kinetics

To evaluate the kinetics and mechanism of drug release, R2 values for zero order kinetics ranged from 0.8168 to 0.9189 and for the first order, kinetics ranged from 0.9628 to 0.9963 and for the Higuchi model ranged from 0.9432 to 0.9907 and for Peppa’s model ranged from 0.9513 to 0.9846. Data were fitted to first order better with higher R² values, which indicated the drug release followed to be first order. The “n” value derived from the Korsmeyer- Peppa’s plots ranged from 0.4230 to 0.5716 indicating that mechanism of release of all formulations F1-F9 was Quasi-Fickian and anomalous i.e. non-Fickian diffusion

Conclusion

This research work attempted to develop SLNs of trimetazidine hydrochloride using compritol, tristearin and glyceryl monostearate as carrier matrices, Tween 80 as surfactant, soy lecithin as stabilizer.

FT-IR was used to investigate possible interactions between the chosen drug and lipids (GMS and TS). It was found that the selected drug and lipids did not interact with each other.

Trimetazidine hydrochloride SLNs were prepared by homogenization technique at a temperature slightly above melting point of lipids. This technique proved successful in yielding stable nanoparticles of proper range. Formulations F1 to F9 showed high entrapment efficiencies. Three distinct lipid kinds were used for developing SLNs. For optimizing the lipid and surfactant in batch F1, particle size, zeta potential, and in vitro drug release profile were considered among all the batches.

SLN’s size, PDI and zeta potential of each formulations developed were in the acceptable and suitable range. Average entrapment efficiency for most of trimetazidine hydrochloride SLNs was determined to be 90.35%, while the optimized formulations F1 showed 90.59% entrapment.

Release kinetics studies showed that drug release from the nano-particles follows quasi-Fickian diffusion and non-Fickian diffusion. The data lead to the conclusion that the trimetazidine hydrochloride lipid nanoparticulate delivery system, which was produced utilizing widely recognized and physiologically safe lipids, was able to demonstrate sustained release properties for 24 hours. As a result, they may decrease the frequency of dose, which would minimize the likelihood of side effects, enhance the drug’s absorption, and improve its effectiveness.

Conflict of interest

None