Keywords

Magnetic nanoparticles; Copper-organic framework; Naproxen; Drug delivery

Introduction

Naproxen is used to relieve pain from various conditions such as headaches, muscle aches, tendonitis, dental pain, and menstrual cramps. It also reduces pain, swelling, and joint stiffness caused by arthritis, bursitis, and gout attacks. This medication is known as a nonsteroidal anti-inflammatory drug (NSAID). It works by blocking the body's production of certain natural substances that cause inflammation. The structure and preparation of chemical structures used in medicinal consumption are only part of the battle against diseases. Maintaining the drug concentration in blood and other bodily fluids in suitable values and time are among the crucial issues in controlled drug release systems. Indeed, in order to maintain the concentration of drug in plasma within desirable levels, sometimes it is needed to use the medication several times a day. This, in turn, causes the drug not to have the desired effect and even bring about further side effects [1]. The first controlled drug release systems were produced in the early 1970s. Since then, such systems have been attracting a great deal of attention [2,3]. One of the vital features in drug release systems is predictable rate independent of the releasing environment. Maintaining the drug concentration levels within the desired limit, requiring direct usage of drug, and greater compliance in the patient, as well as diminished sided effects, are the advantages of controlled-release drug delivery systems [4,5]. The kinetics of drug release is of significance in the technological development of drug release in the body, which depends on various factors including heat, pH, relative humidity, enzyme, and physicochemical properties of their carriers [6,7]. Target full transfer of drug with the aims of prolonging the drug presence time, decreasing toxicity, and enhancing the drug half-life has led to significant improvements in pharmacologic treatments. This is thanks to pharmacokinetic changes in the drug in nano-based drug delivery systems [8]. Among the advantages of magnetic nanoparticles for medical applications are the size, remote controllability, and resonance reaction to field variations [9]. One of the imperative reasons that have given biomedical uses to magnetic nanoparticles is their biocompatibility. In many in vivo and in-vitro investigations, these particles have shown little toxicity. Coated nanoparticles enjoy less toxicity not only due to presence of biocompatible coating but also because of fewer attachment sites for proteins, ions, and other components [10]. When compared with heavy metal-based nanoparticles, magnetic nanoparticles have less toxicity, and even in some cases, they are regarded as nontoxic. The nanoparticles that have part of the body's physiological iron, their iron oxide content is metabolized and stored by Ferritin-transferrin and Hemosiderin protein [11].

Three-dimensional coordination polymers which have permanent pores and are known as Metal-Organic Frameworks (MOFs) are more of interest. This is because it is possible to incorporate many molecules in their networks for various applications. Selection of metal ions or secondary structural units not only can result in topological diversity, but also the pores present in walls can be programmed for various uses [12,13]. Regular porous networks which bring about accurate control of drug loading and reduction of kinetic effects, the high volume of pores for trapping of drug, high surface area which develops a high potential for drug adsorption, and functionalizing active surface using functional groups for loading and releasing drug, are among the most important uses of MOFs in drug releasing systems [14,15].

In this research, we used Fe₃O₄-Cu (BDC) for delivery of naproxen drug, one of the non-steroidal anti-inflammatory drugs and a potent cyclooxygenase enzyme inhibitor. The purpose of this study was to synthesize nanoparticles and modifying them with metal-organic framework that was studied for adsorbing naproxen in a simulated gastrointestinal medium. The particle composition size and morphology can be easily tuned to optimize the final particle properties.

Experimental

Reagent and Instrument

Tetraethyl orthosilicate (TEOS), FeCl₃.6H₂O and FeCl₂.4H₂O, and copper acetate were purchased from Merck (Germany). Ammonia aqueous (25 wt.%), and (3-Aminopropyl) trimethoxysilane (APTMS, 99%) were obtained from Sigma Aldrich (Germany). The rest of reagents were as a minimum of analytical grade. Elemental analyses (C, H, N) were determined with ECS400 Costech (Italy), IR spectra were recorded as KBr pellets on Spectrum 100 Fourier transform infrared (FT-IR) from Perkin company (USA). Field-emission scanning electron microscope (FE-SEM) images were recorded on MIRA3LMU from TESCAN Company (Czech Republic). Thermogravimetric analysis (TGA) was used to evaluate thermal stability and was carried out between 20 and 600°C under nitrogen atmosphere using a 209F1-NETZCH Company (Germany). The structure and morphology of Fe₃O₄-Cu (BDC) nanoparticles were identified by powder x-ray diffraction (PXRD) XRD 360 kV, 25 mA, 5 to 80° D5000 SIEMENS Company (Germany). The magnetization curves were measured at room temperature under a varying magnetic field from -10 000 to 10 000 Oe on a MDKF. Vibrating sample magnetometer (VSM) was from Danesh Pazhohan Company (Iran).

Preparation of Silica Nanoparticles with Magnetic Coating (Fe₃O₄@SiO₂)

For synthesizing magnetic nanoparticles by coprecipitation method, 10 mmol FeCl₃.6H₂O and 5 mmol FeCl₂.4H₂O were separately weighed and dissolved in 70 ml of distilled water and stirred with mechanical stirrer at 800 rpm. After that, 15 ml of ammonia (25%v/v) was added under stirring at 30°C gradually inside the reaction environment by a syringe until the pH of the environment reached within 11-12. In order to prevent heterogeneity of particles, the approximate injection rate was adjusted at 1 ml.min-1. The resulting black precipitate was stirred for 1 hour at room temperature. It was also exposed to 80°C for 1 h in reflux until an emulsion was prepared out of the magnetic nanoparticles (Fe₃O₄). To coat the nanoparticles with silica, Schtuber process was used [16]. For this purpose, concurrently and without separating the nanoparticles from the reaction's environment, 80 ml of ethanol and 40 ml of tetraethyl orthosilicate (TEOS) were added to a balloon. It was then allowed to be stirred by a mechanical stirrer at 40°C for one day. Following the mentioned period, the nanoparticles were separated by a permanent magnet of neodymium-iron-Bohr with a power of 1 T. It was then washed twice by distilled water and dried at 50°C centigrade for 1 day.

Functionalizing the Magnetic Nanoparticles (Fe₃O₄@SiO₂)

In order to obtain amino-functionalized Fe₃O₄ nanoparticles, 1 g of iron oxide magnetic nanoparticles modified with silica together with 358 mg of (3-aminopropyl) trimetoxysilan were stirred at 110°C for 24 h. The precipitated product was gathered by a magnet, rinsed with methanol, and dried at 50°C. Following synthesis of the functionalized magnetic bed, the solution of 1 ml bromoacetic acid in 5 ml of toluene was added dropwise to a suspension of 1 g of iron oxide magnetic nanoparticles modified by silica (Fe₃O₄@SiO₂) in 20 ml toluene. The reaction mixture was stirred for 6 h under reflux. Following termination of the reaction, the precipitate was separated by a magnet and dried at 50°C.

Preparation of the Hybrid Magnetic MOF

Fe₃O₄ @ MOF core-shell has been synthesized correctly using a versatile step-by-step strategy [17]. For this purpose, 0.05 g of the bed of the modified iron oxide magnetic nanoparticles was stirred for 15 min in 4 ml of copper acetate.2H₂O 10 mM in ethanol. Next, it was stirred in 10 mM solution of 1,4-benzene-dicarboxylic acid (BDC) in ethanol for 15 min. Between these two stages, the adsorbent was washed by ethanol. Following 60 stages, the adsorbent was washed by ethanol and dried at 60°C. The methodology of synthesizing of Fe₃O₄@SiO₂@MOF was shown in Figure 1.

Urine Extraction

In order to evaluate the performance of the synthesized polymer in the extraction of naproxen in the urine sample, the volunteer’s urine samples were taken with no consumption of the targeted drug before the study. The urine was spiked with naproxen and added to 0.02 g of the synthesized adsorbent, shaken for 15 min and centrifuged for 10 min. The product eluted by the elution solution (10% acetic acid+40% acetonitrile+50%H₂O) and the concentration of naproxen was measured by HPLC. This measurement was carried out at 1 ml/min, and wavelength of the HPLC detector was 231 nm.

Isotherm Studies

In order to investigate the capacity of the synthesized adsorbent for adsorption of naproxen, solutions with concentrations between 5 and 60 mg/L of naproxen, which contained 5 ml of optimal buffer (pH=5) were prepared. They were then shaken for 30 min and centrifuged for 10 min together with 0.02 g of Fe₃O₄-MOF adsorbent. The capacity of the synthesized polymer for adsorption of naproxen was calculated by the following formula:

q_e=(C₀-C_e) V/W

Where qe is the polymer capacity in terms of mg/g,C ₀ is the initial concentration of naproxen (mg/L), Ce is the secondary concentration (mg/L), W is the polymer weight in terms (g) and V is the solution volume (L).

In vitro Unloading of the Drug

The unloading pattern of naproxen from Fe₃O₄@SiO₂@Cu (BDC) were assessed in simulated gastric fluid with pH value of 1.2 and intestinal fluid with neutral pH of 7. The Fe₃O₄@SiO₂@Cu (BDC) structure has loaded with Naproxen drug and preserved at 37°C in beakers. Samples were obtained at arranged time interims and the drug content of each sample was ascertained using UV-Vis spectroscopy (Figure 1).

Results and Discussion

In order to prove the formation of the intended stabilized organic matrix, all stages of synthesis were taken FT-IR spectrum. In the first stage, which was related to synthesis of iron magnetic nanoparticles coated by silica, at 3422 cm^-1 a peak has been observed related to the stretching vibrations of O-H functional group. Other peaks at 1628 cm^-1 associated to stretching vibrations of out-of-plan O-H, at 1095 cm^-1 related to stretching vibrations of the Si-O functional group, and at 638 and 8035 cm^-1 concerning the vibrations of Fe-O functional groups has been observed. Next step was modification of magnetic nanoparticles with (3-aminopropyl) trimethoxysilan. Consequently, a peak at 2938 cm^-1 related to stretching vibrations of aliphatic C-H functional group, has been appeared. The next and third step was coupling of modified nanoparticles with the tri bromoacetic acid ligand and peaks at 1751 cm^-1 for the stretching vibrations of C=O functional group and at 1407 cm^-1 because of stretching vibrations of CH₂ functional group has been appeared. In the fourth and final step, which was the modification of nanoparticles with copper organic metal matrix, a peak at 1688 cm^-1 related to stretching vibrations of C=O functional group, at 1401 cm^-1 for stretching vibrations of CH₂, and 1081 cm^-1 for C-O and Si-O functional group were observed.

For metal analysis and phase detection, X-Ray Diffraction (XRD) was used. For this purpose, first the XRD pattern was obtained from the sample followed by comparing the data with well-known standards for determining the sample's phase. The diffraction peaks at 2/30º ? 5/35º, 1/43º, 8/53º, 57º,4/62º associated with crystal planes are observable in the XRD pattern, fully compatible with the known standard pattern peaks Fe₃O₄ (JCPDS card No 88-0315) and SiO₂ (JCPDS card No 76-0939) (Figure 2).

The FE-SEM results show the synthesized adsorbent particles are in agglomerate and spherical form with a size between 20 and 50 nm. Moreover, the surface of agglomerated particles is rough, with substantial pores (Figure 3).

In order to investigate the interatomic, intramolecular, and intermolecular interactions, contingent upon the exerted external changes, the final synthesized adsorbent was taken TGA thermo gravimetric analysis. As can be observed in Figure 4, the sample had lost approximately 46% of its weight, which 14% of it was for the adsorbed water, inter crystal water, and functional group attached to the surface. The derivative thermo gravimetric (DTG) results indicated that the organic part grafted on the nanoparticles was stable up until 300°C, and was destroyed at higher temperatures.

The results obtained from C, H and N elemental analysis spectrum of iron oxide nanoparticles and the synthesized polymer adsorbent reported in Table 1 suggest that the high percentage of carbon and nitrogen in the synthesized polymer adsorbent confirms proper polymer grafting onto the iron oxide nanoparticles.

Table 1: Elemental analysis results

To investigate the magnetic properties of iron oxide nanoparticles and the final metal-organic framework, VSM vibrational sample magnetometer was used. Considering the curves of nanoparticles and the synthesized polymer adsorbent, the magnetic stability changed in the final synthesized adsorbent and has shown lower magnetic stability in comparison with iron oxide nanoparticles (Figures 4 and 5 and Table 1).

Optimization of parameters in order to maximize the adsorption level by the adsorbent, the solution of Naproxen was analyzed at various pH by UV-Vis spectrophotometer. The results in Figure 5 indicated that the synthesized adsorbent could adsorb naproxen at different pH. However, the optimal pH for adsorbing naproxen is 5. Furthermore, the results obtained from the zero charge point showed that the synthesized adsorbent has a negative charge at pH higher than 5 and positive charge at pH lower than 5, while it is neutral at pH=5. Indeed, due to the acidic structure of Naproxen, at low pH, it has a molecular structure, and the adsorption enhances, whereas, at higher pH, the synthesized adsorbent is dissolved. Furthermore, the effect of contact time of the synthesized adsorbent with the naproxen solution was investigated on the level of adsorption by the adsorbent. For this purpose, the synthesized adsorbent was examined with the naproxen solution (10 mg/L) at the optimal pH (pH=5) at various times. The results indicated that the first 10 minutes, the synthesized adsorbent had the highest adsorption such that within shorter than five minutes, it adsorbs as much as 50% of naproxen. This signifies that the process has high kinetic adsorption, and there is no need for prolonged contact of the synthesized adsorbent with naproxen solution (Figures 6 and 7).

Isotherm Studies

The following equation has been used to calculate the amount of naproxen at equilibrium qe (mg g^-1) on Fe₃O₄-MOF Cu (BDC):

q_e=(C₀-C_e) V/W

C₀ and Ce (mg L^-1) are initial and equilibrium concentrations of the naproxen, V (L) is the solution volume, and W (g) is the mass amount of the Fe₃O₄-MOF, qmax is the maximum capacity of adsorbed Naproxen which is equal to the entire monolayer coating the adsorbent surface (mg g^-1).

Langmuir isotherm represents homogeneous monolayer adsorption into a mall removing the interactive effects of adsorbed molecules. Its equation is as follows:

C_e/q_e=(l/q_maxKL)+(C_e/q_max)

K_L is Langmuir constant (L mg^-1)

R_L is the main concept in Langmuir’s equation which expressed by the dimensionless separation factor

R_L=l/(l+K_LC0)

R_LR_L can have four values each referring a different state: R_L=0, irreversible adsorption; R_L=1, linear adsorption; 0<R_L<1, desirable adsorption; and R_L>1, undesirable adsorption (with R_L=0.71 indicating desirable adsorption) for naproxen sorption are listed in Table 2.

Table 2: Isotherm equation parameters determined using the linear method at 20°C

Freundlich isotherm assume a heterogeneous surface with a non-uniform distribution of adsorption heat on the surface, which is an experimental isotherm. Freundlich predicts that the concentration of material increases on the adsorbent surfaces when the concentration of material grows in the solution. The heterogeneity factor is stated by 1/n and calculated using the following empirical equation:

lnq_e=lnK_F+l/n lnC_e

where K_F is Freundlich constant, adsorption capacity per unit of concentration (mg g^-1) (L mg^-1) ^1/n and 1/n is the heterogeneity factor. If 1/n=0, the adsorption process is irreversible; if 0 < 1/n <1, the adsorption is desirable; 1/n>1, the adsorption is undesirable. The value of 1/n=0.449 shows the desirable adsorption for naproxen. Temkin isotherm takes place as a linear reduction of energy grows when a degree of completion of adsorption centers of the adsorbent increases. Its equation is as follows.

q_e=BlnA+BlnC_e

B=RT/b

In the above equations, b is the Temkin constant concerning the naproxen adsorption heat (J mol^-1), A is the Temkin constant (L g^-1), B is Temkin constant which is without dimension, R is the universal gas constant (8.314 Jmol-1K^-1), and T is absolute temperature (K). The material amount which adsorbed by each unit of the adsorbent was investigated by three isotherms including Langmuir, Freundlich, and Temkin reported in Table 2.

Method Application

In order to evaluate the performance of the synthesized polymer in the adsorption and recovery of naproxen in the urine sample, the urine sample of people who did not use Naproxen was taken. The obtained results suggest 98% recovery of the urine sample. The mean and relative standard deviations were calculated, reported in Table 3.

Table 3: Naproxen results in urine sample

Drug Delivery

Figure 8 is showing the Naproxen release through Fe₃O₄/MOF in the simulated intestine and stomach environment. The 56% of the adsorbed drug was released rapidly within 30 min in a gastric-like environment with pH value of 1.2 and 100% of the adsorbed drug was unloaded within 15 h in the intestine at a slow rate. Further, the results are congruent with the predetermined model of Higuchi, one of the most successful theories for prediction of drug release from an integrated degradable system. This model is based on some assumptions: the initial concentration of the drug in the framework is higher than solubility of the drug in it, permeation of drug takes place in only one direction, inflation and the gradation of matrix is negligible, and permeability of the drug is constant [18,19] (Figure 8).

Conclusion

This study reports high chemical stability and properties efficiency for sorption of Naproxen. In this research, by stabilizing copper metal-organic framework on to iron oxide nanoparticles, the level of adsorption of this compound was maximized and reached the highest possible adsorption, the as-synthesized MOF (Fe₃O₄-Cu (BDC)) offers several sites attached to the adsorbent body. In this way the adsorption capacity has increased significantly. The drug release study perfectly shows the fast release of drug in gastric area and total gently unloading of drug in the intestine area. Finally, the values for method application demonstrates the excellent reproducibility and repeatability parameters of the suggested technique.

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