MATERIALS AND METHODS

Hydrogenated palm oil (Softisan 154 or S154) was a gift from Condea. Hydrogenated soybean lecithin (lipoid S100-3, containing 90% phosphatidylcholine, including 12-16% palmitic acid, 83-88% stearic acid, oleic acid and isomers and linoleic acid) was a gift from lipoid. Thimerosal, mercury ((o-carboxyphenyl)thio) ethyl sodium salt and sorbitol, (2S,3R,4R,5R)-hexane-1,2,3,4,5,6-hexol, were purchased from Sigma. Oleyl alcohol (octadecenol or cis-9-octadecen-1-ol) was purchased from Fluka. Ethanol and nitazoxanide, [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl] ethanoate were obtained from sigma-aldrich. Double-distilled water was used in this experiment.

Preparation of nitazoxanide-loaded SLNs: Nitazoxanide-loaded SLN was prepared using the high-pressure homogenization technique¹¹. A mixture of S154 and lipoid S100 at a ratio of 70:30 was ground in a ceramic crucible. The mixture was then heated to 65-70°C while being stirred with a PTFE coated magnet until a clear-yellowish Lipid Matrix (LM) solution was obtained. A solution containing 1 mL oleyl alcohol, 0.005 g thimerosal, 4.75 g sorbitol and 89.25 mL bidistilled water (all w/w) at the same temperature was added to 5 g of LM. A pre-emulsion of SLN was obtained using the homogenizer (Ultra Turrax, Ika, Staufen, Germany) at 13,000 rpm for 20 min and high-pressure homogenizer (EmulsiFlex-C50 CSA10, Avestin, Ottawa, Canada) at 1000 bar, 20 cycles and 60°C. The lipophilic drug Nitazoxanide was dissolved in ethanol and mixed with SLN pre-emulsion. This mixture was then incubated overnight at 50-60°C, stirred periodically with a PTFE coated magnet at 500 rpm and finally exposed to air to solidify. Three different types of NTX-loaded SLNs were prepared, which were of the following composition: 1 mg NTX incorporated in 5 mg SLN (NTX-SLN^a), 1 mg NTX incorporated in 10 mg SLN (NTX-SLN^b) and 1 mg NTX incorporated in 20 mg SLN (NTX-SLN^c).

Characterization of nitazoxanide-loaded SLN: The mean particle sizes (i.e., diameter±standard deviation) and size distribution (polydispersity index or PI) of SLNs and nitazoxanide-loaded SLNs were determined using a high-performance particle sizer (Mal 1033452, Malvern Instruments, UK). Measurements were performed at 25°C in triplicate. Before measurement, each sample was dispersed in filtered bidistilled water by a sonic water bath. The zeta potential (i.e., electrophoretical movement) of the SLN and nitazoxanide-loaded SLN was then measured by an analyzer (Mal 1033452, Malvern Instruments, UK) in triplicate.

Differential scanning calorimetry: The melting points of the bulk lipid and SLN formulation were measured using Differential Scanning Calorimetry (DSC). The DSC analysis was performed using the Mettler Toledo DSC 823e (Switzerland) and thermograms were recorded in the 20-120°C temperature range with a heating rate of 5°C min^–1. An empty aluminum sample pan was used as a reference.

Fourier transforms infrared spectroscopy: Fourier transform infrared (FTIR) spectroscopy (Perkin Elmer, Spectrum 65, England) was used to characterize bonding characteristics of the NTX, SLN and NTX-SLN.

Entrapment efficiency and drug loading: A series of nitazoxanide concentrations ranging from 5000-80000 ppm was used to obtain a calibration curve (Y = 0.0783X-0.0011) with linear regression (r = 0.999). To determine Entrapment Efficiency (EE) and Drug Loading (DL), nitazoxanide-loaded SLNs were dispersed in ethanol. The dispersion was centrifuged at 20,000 g for 60 min to remove SLNs. The amount of drug in the supernatant that was not incorporated into the SLNs was analyzed by UV-Vis spectrophotometer (Cecil aquarius 900, UK). Absorbance of the drug was determined at 420 nm¹². The EE and DL% were obtained¹³ using the following Eq. 1 and 2:

(1)

(2)

Nitazoxanide release from nanoparticles in human plasma: nitazoxanide release profile from SLN was assayed in vitro. Five similar batches of nitazoxanide-loaded SLNa containing 30 μg of nitazoxanide in 2 mL of human plasma were prepared. The experiment was conducted in room temperature with mechanical stirring. At predetermined intervals, each sample was centrifuged at 20,000 g for 60 min and the SLN removed. Plasma proteins in the supernatant were precipitated at a 1:1 ratio with trichloroacetic acid and centrifuged at 1500 g for 15 min. The amount of free drug in the supernatant that was neither incorporated into the SLNs nor linked to albumin was estimated by UV-Vis spectrophotometer (Cecil aquarius 900, UK). Free drug at concentrations ranging from 5000-80000 ppm in methanol (50%) was used to obtain the calibration curve.

Drug release at pH 7.4: Drug release in pH = 7.4 was assayed at 5 prefixed time intervals. For this purpose, 5 batches of nitazoxanide-loaded SLN^a containing 30 μg of Nitazoxanide in 2 mL of phosphate buffer saline with pH = 7.4 were prepared and kept at room temperature under mechanical stirring. At suitable time intervals, samples were centrifuged at 20,000 g for 60 min at 4°C, to remove the SLNs and analyzed by UV-Vis spectrophotometer (Cecil aquarius 900, UK). Each experiment was carried out in triplicate.

Statistical analysis: The data obtained were subjected to statistical analysis. The differences in means among the groups were expressed as Mean±Standard Deviation. All the data were subjected to one-way analysis of variance (ANOVA) followed by post hoc multiple comparison and Duncan test after verification of the normal distribution of the data. The Statistical Package for the Social Sciences (SPSS)¹⁴ version 16.0 was used to perform all statistical tests and p-value less than 0.05 were considered significant.

RESULTS

The authors used high-pressure homogenization to prepare SLN because the matrix lipid composed of palm oil (i.e., a triglyceride mixture of natural, hydrogenated and unbranched fatty-acid chains) was suitable for the incorporation of lipophilic drugs¹⁰, such as nitazoxanide. Soy lecithin was the most useful surfactant in SLN dispersions. The SLN and nitazoxanide-loaded SLN were characterized in vitro for particle size, particle-size distribution and zeta potential (Table 1).

Table 1:	Particle sizes, particle size distribution, specific surface area and zeta potential of NTX-loaded SLN formulations with different amounts of nitazoxanide
NTX-SLNa, NTX-SLNb and NTX-SLNc represent 1 mg NTX incorporated in 5, 10 and 20 mg SLN, respectively

Fig. 1:	DSC thermogram and melting point of bulk lipid, Solid Lipid nanoparticle (SLN), nitazoxanide (NTX) and NTX-loaded SLN

The diameters of NTX-loaded SLN dispersions were between 228.50±9 and 448±16.4 with acceptable particle size distribution (PI). The average sizes of the NTX-loaded SLN were larger than the free SLN. However, in the formulations, the particle size of NTX-loaded SLN was increased with concentration of the drug, although generally the size of the particles showed observable increase in following order: SLN^c>SLN^b>SLN^a. According to the Table 1 the surfaces of nitazoxanide-loaded SLN carried a negative charge.

The DSC thermogram and melting point of SLN, NTX and NTX-loaded SLN are shown in Fig. 1. The melting point of the drug-free SLN and free Nitazoxanide were 71.54 and 201.51°C, respectively. Incorporating Nitazoxanide into the SLN reduced its melting point to 60.28°C.

The FTIR spectra for drug-free SLN, NTX and NTX-loaded SLN are shown in Fig. 2. The specific peaks of SLN were in the following profile: at 3366, 3291.5, 2846.9 and 2918.6 cm^–1 (relate to the stretching of O-H and C-H bonds) the peaks at 1470 and 1728.1 cm^–1 (relate to the stretching of C=O and C=C bonds) and at 1079 cm^–1 single bonds of C-C and C-O were appeared. As it is shown in Fig. 2, the specific peaks of NTX were 3358.73 cm^–1 (stretching of N-H bond), 3087,94 cm^-1 (stretching of C-H and O-H bonds), 1772.86 and 1662 cm^–1 (stretching of C=O, C=C and C=N bonds and bending N-H bond) and 1473.35 and 1364.72 cm^–1 (stretching and bending of C=O and C-N). The curve is presented in Fig. 2 also revealed a shift from 3358,73 cm^–1 (related to NTX) to 3354.6 cm^–1 (related to NTX-SLN) and wider spectra peak at 3354.6 cm^–1.

In the present study the three NTX-loaded SLNs (NTX-SLN^a, NTX-SLN^b and NTX-SLN^c) were prepared and analyzed for Entrapment Efficiency (EE%) and drug loading (DL%). The results of EE (%), (NTX-SLN^a 91.81±1.5, NTX-SLN^b 74.54±1.06, NTX-SLN^c 60.45±2.15) and DL (%), (NTX-SLN^a 18.26±1.9, NTX-SLN^b 7.45±1.1, NTX-SLN^c 3.02±1.6) from fresh samples are as in Fig. 3 and 4, respectively.

The release profile of Nitazoxanide-loaded SLN^a in human plasma is shown in Fig. 5. The release rate remained low for the first 2 h, only increasing by near 1% for that period. Immediately after 2 h there was a sudden burst of drug release approaching 12.57% by 4 h’ post-drug incorporation. After 4 h the release rate remained low by the last hour, increasing by 14.5% for that period. Figure 5 shows slowly release profile of NTX from NTX-SLN^a in PBS solution (pH = 7.4). The NTX-loaded SLN^a showed a 4.7% release during 240 min and increasingly 8.32% release after 480 min.

DISCUSSION

Particle size is an important characteristic for pharmaceutical applications because it significantly affects in vitro and in vivo studies¹⁵.

Fig. 2:	FTIR spectra of SLN, NTX and NTX-SLN

Fig. 3:	Drug loading of nitazoxanide-loaded solid lipid nanoparticles. NTX-SLNa, NTX-SLNb and NTX-SLNc represent 1 mg NTX incorporated in 5, 10 and 20 mg SLN, respectively. Means with superscripts a-c are significantly different (p<0.05), n = 3

The result from this study is in agreement with that from another study¹⁶ using Ubidecarenone (Q₁₀) as a drug model, which showed increase in the size of the carrier nanoparticle, tripalmitin SLN. This may be assigned to the fact that drug is generally incorporated within the oily core and on the particle surface of the SLN. With NTX incorporation into SLN, the increase in size of particles suggests that the loaded NTX is either adsorbed to particle surface or entangled in aliphatic chains of triglycerides.

Fig. 4:	Entrapment efficiency of nitazoxanide-loaded solid lipid nanoparticles. NTX-SLNa, NTX-SLNb and NTX-SLNc represent 1 mg NTX incorporated in 5, 10 and 20 mg SLN, respectively. Means with superscripts a-c are significantly different (p<0.05), n = 3

According to Memisoglu-Bilensoy et al.¹⁷ when the size of drug-loaded nanoparticulate system is decreased with respect to blank nanoparticles, the drug generally encapsulated in large amounts within the oily core of the nanoparticulate carrier and when the size increased, the drug probably is accommodating onto the particle surface. When nitazoxanide was incorporated into SLNs, the increase in particle size suggested that loaded nitazoxanide was either adsorbed onto the particle surface or entangled in the aliphatic chains of triglycerides.

Fig. 5:	Phosphate buffer (pH = 7.4) and human plasma concentration profiles of NTX-loaded SLN. Each point represents the Means±standard deviation, (n = 3)

Drugs incorporated nanoparticles have many useful effects such as increased bioavailability¹⁸. Incorporation of the poorly soluble lipophilic drugs such as NTX in particles of nanosize will improve its bioavailability.

Zeta potential is also an important factor when evaluating the stability of colloidal systems¹⁹. In the presence of the drug, it is probable some of the negative charges were neutralized by the complex formation. The positive charge also might be raised by the nitazoxanide amino group and by nitazoxanide localization on the surface of SLNs¹³. Therefore, in our study it seems that NTX is encapsulated in the oily core and some of the drug could also be located on the surface of the SLN.

With regard to the melting point and crystallization behavior of SLNs, incorporating nitazoxanide reduced the melting point of SLN from 71.54-60.28°C. Nitazoxanide thus is probably in an amorphous state and not crystalline. A substance with a less ordered crystal or amorphous state requires less energy to overcome lattice forces and perfect crystalline substances require high melting enthalpy²⁰. The shape of the drug-free SLN and nitazoxanide DSC thermogram were a sharp trough, whereas, those of the nitazoxanide-loaded SLNs was broader indicating dispersion¹¹ and the amorphous state may contribute to drug loading capacity²⁰. This study showed that the nitazoxanide was entrapped into the SLN during preparation and decrease in melting temperature could be attributed to the small particle size²¹. Since the thermal behavior of SLN is affected by thermodynamic factors, which depend on quality of the interaction between the components, the presence of guest molecules like NTX would alter the melting point¹⁵. According to Westesen et al.⁹ and Radtke et al.²², in drug-free and drug-loaded SLNs the less-ordered crystals and amorphous state will favor high-drug loading. Lipoid S100-3 used in this study is a purified soybean lecithin with >94% phosphatidylcholine (linolic acid, lineolic acid, oleic acid, palmitic acid and stearic acid) content²³. Therefore, it is expected that the drug becomes incorporated into the crevices or in-between the fatty acid chains¹⁵.

The results obtained from FTIR spectrum confirmed the DSC experiment. The peak at 3354.6 cm^–1 related to NTX-loaded SLN was wider than corresponded peak in SLN is attributed to the participation of NTX in hydrogen bonding and -NH group interactions²⁴. A shift from 3,358.73 cm^–1 of NTX (N-H stretching) to lower peak at 3,354.6 cm^–1 can be related to coordination binding between SLN and NTX through amide N-H bond²⁵. This result proves successful loading of NTX in SLN and also indicates some interactions between SLN and NTX.

The EE of NTX-loaded SLNs was quite high (i.e., 91.81%). The palm oil used to prepare the SLN dispersion produced the highest entrapment efficiency. Triglycerides with various fatty acids offer relatively better drug solubilization²⁶. Wong et al.²⁷ used doxorubicin, verapamil HCl, propranolol HCl and quinidine sulfate in SLNs stabilized with tween 80 and showed that increasing drug concentration led to a significant increase (p<0.05) in DL and significant decrease (p<0.05) in EE. These effects were a result of reduced SLN dispersions and high solubility of the drug in high lipid concentrations. Other researchers showed a positive correlation between particle size and drug loading^28,29.

High entrapment efficiency is one of the important benefits of using SLNs as carriers^30,31. The factors that affected loading capacity of drug in nanoparticles are solubility of drug in melted lipid, miscibility of drug and lipid melt, chemical and physical structure of solid lipid matrix and polymorphic state of lipid material. Therefore, high solubility of the drug in the lipid matrix led to adequate loading capacity⁹. The chemical structure and crystalline state of the lipid has an important role in expulsion or incorporation of the drug into the carrier system²⁰. A complete lattice is formed by high crystalline state of the lipid would lead to drug expulsion. Therefore, lattice imperfection in the lipid structure of the SLN may provide drugs accommodation. The result of our study showed that as the percentage of SLN increased entrapment efficiency was decreased (ranging from 91.81-60.45%). This may be due to drug expulsion through SLN modification as lipid and surfactant increased.

Among the factors that support fast drug release from SLNs are a large surface area, small molecular size, low matrix viscosity and short diffusion distance of the drug³². In the authors’ study, however, the release of the drug from SLN, which contains a lipid matrix, was retarded. Characterization of NTX-loaded SLNs suggested that NTX was either encapsulated within the matrix and membrane of palm oil or entrapped in aliphatic chains. This situation could explain the burst release of the drug from SLNs after 2 h. Considering that SLN is solid at room temperature and that the incorporated drug is released relatively slowly, SLNs have potential as a sustained-release drug carrier. According to Fig. 5 release of NTX from NTX-SLNa in PBS (3%) is considerably lower than plasma (4.27%) within 2 h. It has been suggested this typical kinetics of drug release is due to enzymatic reactions that are involved in degradation of SLN causing drug solubilization in plasma^13-33. In the present study the profile of drug release in pH = 7.4 may be due to low solubility of NTX in PBS solution and the burst drug release in human plasma is more likely a consequence of rapid surface desorption drug molecules from a high specific surface area provided by particles at nanoscale.

CONCLUSION

This study presented a study on the optimization of parameters for preparation of nitazoxanide (NTX) incorporation into SLN. Of the three types of NTX-loaded SLN studied, the NTX:SLN ratio of 1:5 showed the highest drug loading and entrapment efficiency. Characterization of NTX-loaded SLN indicated that the drug is either encapsulated within the matrix and membrane of palm oil or entrapped in aliphatic chains. In conclusion, SLN can serve as a good drug carrier for sustained-released of NTX.

ACKNOWLEDGMENTS

We would like to thank Hamadan University of Medical Sciences for financial support (grant number; 930126252) and facilities.