Development and Bioavailability Assessment of Simvastatin Nanoparticle Formulation

 

Suvarna G. Bhokare*, Rajendra P. Marathe

Yash Institute of Pharmacy, South City, Waluj, Aurangabad, Maharashtra, India.

Dr. Babasahed Ambedekar Marathawada University Aurangabad

 

ABSTRACT:

Aim: The aim of the present study is to prepare and evaluate nanoparticles containing Simvastatin using chitosan as the polymer. Methods: The Simvastatin loaded nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions. Nanoparticles of different core: coat ratio were formulated and evaluated for process yield, loading efficiency, particle size, zeta potential, in vitro drug release, kinetic studies and stability studies. Results: The prepared nanoparticles were white, free flowing and spherical in shape. The infrared spectra showed stable character of Simvastatin in the drug-loaded nanoparticles and revealed the absence of drug polymer interactions. The chitosan nanoparticles have a particle diameter ranging approximately 132.1±5.60 to 774.8±2.60nm and a zeta potential 11.93 to 43.23mV. The formulation with the initial Simvastatin concentration of 0.5 mg/ml provided the highest loading capacity. The in vitro release behavior from all the drug loaded batches were found to follow zero order and provided sustained release over a period of 10 h. No appreciable difference was observed in the extent of degradation of product during 90 days in which nanoparticles were stored at various temperatures. Conclusion: The best-fit release kinetics was achieved with zero order mechanism. The release of Simvastatin was influenced by the drug to polymer ratio and particle size. These results indicate that Simvastatin nanoparticles could be effective in sustaining drug release for a prolonged period.

 

 

 

INTRODUCTION:

Simvastatin (SV) is a cholesterol-lowering agent that's derived synthetically from a fermentation product of Aspergillus terreus and widely accustomed treat hypercholesterolemia.¹ When given orally, SV (a lactone) is quickly hydrolyzed in vivo to the corresponding β, δ-dihydroxy acid form, a potent competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) the enzyme that catalyzes the conversion of HMG-CoA to mevalonate, which is an early and rate-limiting step within the biosynthesis of cholesterol.² However, it's a short half-life and is practically insoluble in water.

 

It is also generally considered that compounds with poor water solubility will show dissolution rate-limited absorption in vivo and hence poor absorption, distribution, and site-specific delivery.^3,4 Conventional drug delivery system has been characterized by immediate release and repeated dosing of the drug which could result in the danger of dose fluctuation.⁵ the main objectives of designing nanoparticles as a drug delivery system are very important particle size, surface properties and to deliver pharmacologically active agents at right place, at the rational rate and dose.^6,7 Therefore it's important to introduce effective methods to boost the solubility and dissolution rate of drug, substantially resulting in its improved oral bioavailability. Sustained release formulation, nanoparticles, are reported to resolve these problems because of the alteration of its tissue distribution, improving the drug efficacy, reducing the drug toxicity, and prolonging the half-lives in blood.^8,9 Chitosan (CS) may be a natural cationic polysaccharide obtained by the N-deacetylation of chi-tin, a product found within the shells of crustaceans the first amine groups provide special properties and characteristic that make CS very useful in pharmaceutical applications.^10,11 the formed nanoparticles are biocompatible, biodegradable, non-toxic and capable to sustain the release of encapsulated materials more efficiently than either alginate or chitosan alone.¹² This necessitated the development of novel chitosan nanoparticles as novel drug delivery system for Simvastatin order to provide pH dependent, sustained drug release and increase oral bioavailability.

 

MATERIALS AND METHODS:

Materials:

Simvastatin was procured as gift sample from Aurobindo Pharma ltd. Hyderabad. Sodium tripolyphosphate was purchased from sigma-Aldrich, Mumbai; Chitosan (high viscosity) was purchased from Central Institute of Fisheries Cochin. All other reagents used were of analytical grade.

 

Experimental Methods:

Preparation of Chitosan Nanoparticles:

Chitosan nanoparticles containing Simvastatin were prepared by ionotropic gelation method. Chitosan was dissolved in 1% acetic acid solutions at various concentrations to obtain (0.1%, 0.2% and 0.3% i.e. 35 mg, 65mg and 90mg) and adjusted the pH 5-6 with 0.1N sodium hydroxide solution, while STPP was dissolved in deionized water at various concentrations to obtain 0.1%, 0.15% and 0.20% while stirring at 750rpm. Simvastatin 50 mg was dissolved in ethanol/water mixture (1:1) (1%tween 80) to obtain clear solution. Simvastatin solution was added dropwise during probe sonication with syringe needle size 0.45mm to 40ml chitosan solution. Repeat the sonication cycles for 1 h. The 20ml of STTP solution was added dropwise 0.75 ml/min. under stirring (1000rpm) at ambient temperature. The formulation was stirred for 30 minutes so as to remove ethanol content. All the formulation was sonicated at fixed time for 30 minutes. All experiments were performed in triplicates. Nanoparticles were collected by centrifugation at 9000rpm for a period of 1 h and supernant was analyzed using UV-Visible spectrophotometerically to determine encapsulation efficiency. Pellet was redissolved for sonicated for 15 min. The sample was freeze dried at -40⁰C and lyophilized to get dry powder using 2% Mannitol as cryoprotectant.^13-15

 

Freeze Drying of Nanoparticles:

Briefly, by taking 5ml of nanoparticles dispersion was filled in 10ml glass vials, covered with special stoppers for lyophilization and placed in a freeze dryer (Southern scientific lab Instrument, India) After freeze drying all sample vials were stored at 2-8°C.

 

Experimental Design:

The formulations were fabricated according to a 3² full factorial design, allowing the simultaneous evaluation of two formulation variables and their interaction.

 

Table 1: Experimental design and Parameters for 3² Full Factorial Design Batches

 

Table 2: Translation of coded levels to actual quantities

 

Evaluation of Simvastatin chitosan nanoparticles:

Determination of particle size and Polydispersity index:

The size distribution and polydispersity index (PDI) of the formulations was measured by Dynamic Light Scattering Particle Size Analyzer (Nanoplus3, Micromeretics, USA). The average diameter and a measure of the distribution width (polydispersity) were determined from the particle size distribution data. Polydispersity index varies from 0.0 to 1.0. The closer to 0 the PDI value, the more homogenous are the particles. The usual range of PDI values: 0-0.05 (monodisperse standard).

 

X-ray diffraction (XRD) analysis:

XRD patterns were obtained at room temperature using a very high-resolution Cu-K_α radiation diffraction system (Bruker D8 Advance) operating at a voltage of 40 kV and current of 30 mA. NPs were analyzed in the 2θ angle range of 0–80°.0.05-0.08 (nearly-monodisperse), 0.08-0.7 (midrange polydispersity),>0.7 (very polydisperse).¹⁶

 

Fourier transforms infrared spectroscopy (FTIR) analysis:

Infrared spectroscopy was carried out to determine the chemical composition of the prepared nanoparticles using FTIR (Nicolet, USA) operating in the wave number range of 400–4000 cm^-1 at the absorption mode.

 

Scanning electron microscopy (SEM):

The prepared microspheres were coated with a thin layer of gold by sputtering (Hitachi High E-1010, Japan) and then the microstructure were observed in a scanning electron microscope (SEM; Hitachi High S-4800, Japan) that operated at an acceleration voltage of 20 kV.¹⁶

Determinations of drug content:

A quantity of drug loaded nanoparticles equivalent to 1 mg was added to 10ml phosphate buffer pH 6.8 (1:10) and stirred continuously for 2 hr and then the final colloidal suspensions were ultracentrifuged at 10000rpm 1-2 hour. The supernatant was analyzed for drug content by measuring the absorbance at 238nm using UV spectrophotometer.¹⁷

 

Entrapment efficiency:

The encapsulation efficiency of nanoparticles was determined by the separation of drug-loaded Nanoparticles from the aqueous medium containing non-associated Simvastatin by ultracentrifugation at 12,000 rpm at 4°C for 1hr. The amount of Simvastatin loaded into the nanoparticles was calculated as the difference between the total amount used to prepare the nanoparticles and the amount that was found in the supernatant. The amount of free Simvastatin in the supernatant was measured by UV Spectrophotometer.¹⁹ Entrapment efficiency was then calculated as follows:

Entrapment efficiency was calculated by Eq.1

 

                Total amount of drug-non bound drug

 % EE= –––––––––––––––––––––––––––––––– X 100 ………….Eq 1

                    Total amount of drug added

 

Percentage yield:

Fixed volumes of Simvastatin nanosuspension were centrifuged at 9000 rpm for 30 min at 15°C. The obtained sediment was dried and weighed.^{18, 20} the percentage yield was calculated by Eq.2

 

                               Weight of nanoparticles obtained

Percentage yield = ––––––––––––––––––––––––––––– X 100 ….Eq..2

                                  Weight of drugs and excipient

 

Zeta potential:

The zeta potential value of optimized Simvastatin loaded chitosan nanoparticle formulation was measured with the Zetasizer. To determine the zeta potential, optimized formulation was diluted with double-distilled water and placed in an electrophoretic cell.²¹

 

In vitro drug release:

The release of Simvastatin from nanoparticles was evaluated using USP type II paddle apparatus over 10 hr, dialysis membrane was loaded with nanoparticle formulation containing 10mg equivalent of drug, which was suspended initially for 2 hrs in 900ml of 0.1 N HCl buffer of pH 1.2 and then in pH 6.8 phosphate buffer upto 10hr maintained at 37±0.5°C and 50rpm. At regular intervals aliquots of 1ml of the sample were withdrawn and replaced with the same volume of the respected fresh phosphate buffer solution. The amount of released drug was assessed by UV-1700 analysis at 238nm (Shimadzu UV-1700, Japan) after dilution.

 

Stability studies:

Optimized formulation was chosen to perform short term stability studies. Samples were stored in glass vials for 3 months at 5±3°C in freeze and at 30±2°C/65±5% RH. After 30, 60 and 90 days samples were observed for particle size, % entrapment efficiency and drug release were carried out for optimized formulation at every one month interval.²²

 

RESULTS AND DISCUSSION:

Particle size and Size distribution:

The mean particle size for formulations SF1 to SF9 varied in range of 132.1±3.05 to 774±3.60 (Table 3). It was observed that mean particle size increases with the increase in the polymer concentration upto a level. Further increase in the polymer concentration above the concentration range mentioned resulted in the aggregation of the particles. The mean polydispersity index values for the Simvastatin loaded chitosan alginate nanoparticle formulations SF1 to SF9 are in the range of 0.270 - 0.628 as shown in (Table 3).

 

Figure 1: Particle size analysis of formulation SF6 batch

 

 

Table 3: Average particle size, PDI, %Yield, drug content and % EE of nanoparticles

* Indicates average ± SD (n=3)

Powder X- ray diffraction (PXRD) studies:

The results of PDI can be simultaneously checked with particles size analysis. A monodisperse sample indicates PI value nearer to 0. However, PDI < 1 indicates polydisperse samples. Therefore, PI measurement was essential to confirm the size distribution of the particles.^24,25

 

XRD pattern of the Simvastatin and selected nanoparticle formulation are shown in figure. 2. The nanoparticle prepared with chitosan was characterized by less intensity of the diffraction peak when compared to that of Simvastatin. Simvastatin the characteristic peaks at 9.3°, 10.98°, 14.84°, 15.42^o, 16.39^o, 17.11^o, 17.62^o, 18.64^o, 19.16^o, 22.43^o, 26.14^o, 28.08^o, 31.68^o 2θ while diffractogram of Simvastatin NPs showed the characteristic peaks at 9.6°, 10.77°, 13.72°, 14.74°, 17.32°, 18.86°, 20.49°, 21.41°, 23.57°, 28.39°, 29.62°, 33.74^o, 36.19^o, 38.75^o, 44.08^o 2θ. This clearly indicates the reduction in the crystallanity of the Simvastatin loaded chitosan nanoparticles.

 

Compatibility Studies:

Overlay FTIR spectra of Simvastatin, chitosan, physical mixture and NPs batch are shown in figure 3, From FTIR studies it can be seen that the fundamental peaks of Simvastatin are retained. From FTIR indicated that there was no chemical interaction between Simvastatin and chitosan used in the formulation hence, can be used in the formulation of nanoparticles.

 

Figure 2: XRD pattern of pure drugs and NPs Simvastatin (SF6)

 

 

Figure 3: Overlay FTIR spectra of pure drug, chitosan, physical mixture (1:1) and optimized NPs batch

 

Scanning electron microscopy study:

The exhibited from the SEM of SIM pure drug consisted of a mixture of large crystals, indicating its crystalline nature. However, the prepared Simvastatin-loaded CS NP’s of batch SF6 had a spherical shape with a relatively uniform size of about 330.4nm in diameter and no drug crystals were present which was shown in SEM of pure Simvastatin. The SEM of pure Simvastatin (A), SF6 batches were nearly spherical in shape depicted in figure 4.

 

A                                                        B

Figure 4: Scanning electron microscopy of Simvastatin (A) andoptimized batch of SF6 (B)

 

 

Table 4: % Drug entrapment and zeta potential of Simvastatin nanoparticles batches

* Indicates average ± SD (n=3)

 

Figure 5: Zeta potential of optimized formulation (SF6)

 

Percent Drug content study:

Drug content varies in the range of 65.06±0.10 to 82.26±0.35 and was determined using the UV spectroscopic analysis at 238nm.

 

Entrapment efficiency:

Encapsulation efficiency of the nanoparticles was found to vary between 87.3±0.035 to 97.5±0.036 suggested that at intermediate concentration of chitosan and STTP the encapsulation of drug was maximum.¹⁹ Formulation SF6 (97.5±0.036) show maximum entrapment efficiency Based on entrapment efficiency and drug content formulation SF6 was taken as optimized formulation.¹⁷

 

Percentage yield:

Percentage yield was found to be 42.7±0.60% to 56.4 ±0.15% for formulation SF1 to SF9 (Table 3). Percentage practical yield depends on the concentration of polymer added, as the concentration of polymer increases there is increases in the % yield. Maximum yield obtained is 56.4± 0.15% for formulation SF6.

 

Zeta potential:

Zeta potential is an important parameter to analyze the long-term stability of the nanoparticles. It refers to the surface charge of the particles. Zeta potential of simvastatin nanoparticles is of significance tools on stability in suspension through the electrostatic repulsion between the particles.

 

Zeta potential of the optimized formulation SF6 was found to be 39.25±0.015mV (Figure 5). The zeta potentials of about 39.25mV indicate good stability of formulation. This might be attributed to surfactant which decreases the electrostatic repulsion between the particles and sterically stabilizes the nanoparticles by forming a coat around their surface.¹⁸

 

In vitro drug release:

In vitro drug release studies were carried out using USP Type II dissolution apparatus (EDT.08LX, 1292092, Elestro Lab, India), at rotation speed of 50 rpm. The cumulative percentage drug release of SV in Phosphate buffer pH 6.8 medium of F1 to F9 batches were shown in Table 5 and figure 6 respectively. Cumulative percentage drug released for SF1, SF2, SF3, SF4, SF5, SF6, SF7, SF8 and SF9 after 10 h were found to be 82.33%, 66.57%, 84.43%, 98.60%, 89.00%, 93.24%, 86.12%, 79.74% and 78.58% respectively. It was apparent that in vitro release of Simvastatin showed a very rapid initial burst, and then followed by a very slow drug release.

 

Table 5: Correlation coefficients according to different kinetic equations

 

 

Figure 6: Comparative in vitro drug release profile of SF1 to SF9 batches

 

Table 6: Effect on particle size, % Entrapment efficiency and % drug release during stability studies.

 

Kinetic studies:

The release data of optimized NPs formulation was fitted into various kinetic models such as zero-order, first-order, Higuchi, Korsmeyer–Peppas and Hixson Crowell equations in order to determine the release mechanism and regression coefficients (R2).²⁵ From chitosan NPs fitted best to Higuchi, which can be confirmed by comparing the values for the regression coefficient (R2) of the zero order, first order, Higuchi, Korsmeyer–Peppas and Hixson Crowell which are 0.995, 0.995, 0.9739, 0.967, 0.978, 0.908, 0.970, 0.997, 0.994 (calculated from mean values) respectively. Table 5.The value of ‘n’ (0.5 < n < 1), the diffusion exponent of zero order kinetic followed by Hixson Crowell equation indicated that the release of Simvastatin from CS NPs is anomalous i.e. contributed by combination of dissolution and diffusion.

 

Stability studies:

Stability studies were carried out on the optimized formulation SF6 as per ICH guidelines for 90 days. By comparing this data with initial data it was observed that there was a slight decrease in the percentage entrapment efficiency and increase in particles size due to degradation of polymer and aggregation of particles. (Table 4) There was not much change in the drug release. Formulation stored at (5±3°C) showed better stability as compared to the formulation stored at 30±2°C/65±5% RH. Simvastatin-chitosan nanoparticles can be successfully prepared by ionotropic technique. In vitro release study showed that chitosan nanoparticles showed pH dependent and sustained release of drug for a prolong period of time.²⁶

 

CONCLUSION:

This paper report, the possibility to entrap hydrophobic Simvastatin within CS-STPP nanoparticles using a modified ionotropic gelation technique, strong electrostatic interactions exist in the nanoparticles Chitosan nanoparticles herald a novel controlled drug delivery, which offer several potential benefits. Chitosan nanoparticles had shown an excellent capacity for the association of Simvastatin. The present study was aim to develop Simvastatin loaded chitosan Nanoparticles. Chitosan concentrations and drug/polymer ratio in the nanoparticles influence the physiochemical characteristics such as zeta potential polydispersity index, and average nanosize diameter or percentage encapsulation efficiency of Simvastatin. Average Nanosize diameter, Polydispersity index, zeta potential, percentage encapsulation efficiency, stability study was found to be good for optimum formulation (SF6) (0.1%, 0.2% and 0.3%) The concentration of polymer and cross-linking agent are the important factors in the development of Simvastatin nanoparticles. The in vitro release data of the optimized formulation was compared with different kinetic models to select the best fitting model. Good correlation coefficients (R2 ≥ 0.99) for Simvastatin NPs tablet could be obtained. The drug release follows zero order release kinetics and followed by Hixson Crowell mechanism. Thus resulting in improved therapeutic outcome, thereby minimizing the dose-dependent adverse effects and maximizing the patient’s compliance.

 

ACKNOWLEDGEMENT:

The authors would sincerely like to thank Aurobindo Pharma Ltd. Hyderabad for providing gift samples of Simvastatin and Central Institute of Fisheries Cochin, Kerala for providing chitosan. We also grateful to R.C. and H.R Patel College of Pharmacy Shirpur Dhule, Dr.BAM University, Aurangabad and Jalgao University’s for providing the laboratory facilities.

 

ABBREVIATIONS:

SEM: Scanning electron microscopy; XRD: X-Ray Diffraction; CS: Chitosan; PDI: Polydispersity index, SIM: Simvastatin, NPs: Nanoparticles.

 

CONFLICT OF INTEREST:

Nil.

 

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Accepted on 18.05.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1615-1621.