1. INTRODUCTION:

Diabetes mellitus (DM) is a metabolic disease which affects the quality of life of numerous people around the world. More than 1.5 million deaths have been reported every year by the world Health Organization (WHO)¹. There are several forms of diabetes and diabetes Type 2 is the most common. This is caused by some effective factors for instance: stress, low physical activity, unhealthy food habits, obesity, genetics, age, and inflammation^2,3,4. Type 2 diabetes is a lifestyle related disease which is controllable by healthy lifestyle like exercising and well diet. A combination of treatment strategies can help you manage the condition to live a healthy life and prevent complications. In most cases people have to use anti-diabetes drugs or insulin.

Metformin hydrochloride is used widely for treating diabetes type 2 for more than 120 million people over the world^5,6. Two main effects of Metformin have reported as 1- reduction of produced glucose in liver, 2- strong effect on insulin in order to decrease of blood glucose level^7,8,9. Increasing the treatment effects of drugs without any side effects on healthy organs is the main advantage of drug delivery system. Drug delivery system(DDS) has been researched by Paoul and his co-workers for the first time¹⁰. An ideal DDS should act for sustainable and purposeful carrying and releasing the drug in the target organ. Nanoparticles(NPs) with some advantages including hydrophobic surface and high surface area could be the well candidate as neutral, nontoxic and fast carrier for drug delivery approaches. Fe₃O₄ magnetic nanoparticles have special properties such as highly dispersible in solutions, easy to modify and isolation by magnetic field¹¹. In the recent years, the researchers have been focused on surface modification of magnetic nanoparticles in order to use for various purpose^12-17. There are several reports about using of nanoparticles in purpose of DDS^18-20. Accordingly, in the present work iron oxide magnetite nanoparticles(MNPs) were prepared using a simple coprecipitation technique and they were coated for more stability and biocompatibility with silica/agarose. The prepared MNPs were then impregnated with Fe³⁺ ions to obtain the Fe₃O₄/SiO₂/Agarose/Fe³⁺ nano carrier. In every stage of the synthesis, the nanoparticles were characterized by Fourier transform infrared spectroscopy(FT-IR), dynamic light scattering(DLS), field emission scanning electron microscopy (FESEM) and energy dispersive x-ray spectroscopy(EDX). Finally, the modified MNPs were used for adsorption of metformine on the nano carrier.

2. EXPERIMENTAL:

2.1. Instrumental:

A double beam UV-vis spectrophotometer (Shimadzu PC-1650, Japan) with a quartz cell of 1 cm optical length was used to measure absorbance of analyte. A FT-IR spectrometer (Shimadzu 8400, Japan) was used for measuring the FT-IR spectrum of samples. In every stage, the morphology of synthesized and modified MNPs was investigated by field emission scanning electron microscopy (FESEM, MIRA3 TESCAN, Czech Republic) equipped with an oxford energy-dispersive X-ray spectrometer (EDS) system, under acceleration voltage of 20 kv. Dynamic light scattering (DLS) and Zeta potential were carried out by (Zetasizer Malvern ZEN3600, UK). A pH meter model (Microprocessor P211, Hanna) was used for solution pH adjustment.

2.2. Reagents:

All solutions were prepared using ultra-pure water (18.2 MΩ). The glassware was kept overnight in a 5% (v/v) nitric acid solution and subsequently washed with deionized water. All reagents were used of analytical grade. Hydrochloric acid (VWR international, USA), TEOS (Tetraethyl orthosilicate, Acrose, USA) and Agarose (Merk, Darmstadt, Germany) with high purity were available and used without further purification. Working solutions of Metformin (1000 mg L−1) were prepared daily by diluting of appropriate amount of stock solution. The pH of solutions was adjusted by using Phosphate-buffered saline and Ammonium acetate.

2.3. Synthesized of Fe₃O₄ magnetic nanoparticles:

Iron magnetic nanoparticles (Fe₃O₄-MNPs) were prepared by co-precipitation of Fe²⁺ and Fe³⁺ ions with molar ratio of 1:2^21,22. In this method, 0.324 g of FeCl₃ and 0.198 g of FeCl₂.4H₂O were diluted to 50 ml with deionized water and sonicated for 15 min in order to de-oxygenation. Then, while the solution vigorously stirred under the nitrogen atmosphere and at 80 ^oC, 5 mL NH₄OH was added dropwise into the solution until the orange-red solution becomes a black suspension. The reaction was stopped and the obtained suspension was cooled at room temperature. The product was left onto the magnet for settled of MNPs. Then the supernatant was discarded and the nanoparticles were washed with distilled water (twice time) until neutrality.

2.4. Preparation of modified: Fe₃O₄/SiO₂/Agarose/Fe³⁺:

To prepare modified MNPs, 5mL of synthesized magnetic nanoparticles (Fe₃O₄) was suspended in a mixture of 80 mL ethanol, 16mL deionized water and 2 mL Ammonium and sonicated for 15min (at 25^oC). Then, while the solution was stirring, 1.0mL TEOS was added dropwise for about 17 h. Silica coated MNPs were separated from solution with a magnet and were washed with ethanol (three times)²³. After that, 50mL Ammonium acetate (0.1mol L^-1) was added and the mixture was sonicated for 15min at 25^oC. then, 10mL Agarose (0.05%) and 0.324g Fe³⁺ were added and stirred for 11h (at pH=7) (fig.1). The resulted mixture was separated with a strong magnet and immersed in ethanol 20% at 4^oC.

Fig. 1: Schematic of synthesis and modification of Fe₃O₄/SiO₂/ Agarose/Fe³⁺ MNPs.

2.5. General procedure:

For the adsorption of Metformin, 5mL of adsorbent was added into 5mL aliquot of solution containing 5mg L^-1 Metformin. The pH was adjusted at desired pH value of 9 with the aid of Phosphate-buffered saline and Ammonium acetate. After adjusting of pH, the mixture was stirred for 10 min. After that, the adsorbent was separated with a strong magnet and the supernatant solution was introduced for subsequent UV-Vis determination (234nm).

2.6. Adsorption of Metformin studies:

Quantification of Metformin concentrations was performed based on the calibration curve achieved by plotting absorbance vs. Metformin concentration. In order to determine the adsorption capacity, the concentration of Metformin was determined before and after adsorption by adsorbent. To determine the adsorption capacity, following equation is used:

q = ((C_o−C_t). v) / M (1)

Where Co (mg L^-1) and C_t (mg L^-1) are the concentrations of Metformin before and after adsorption, respectively. q (mg g^-1) is adsorption capacity, v (mL) is the volume of aqueous solution and M (mg) is the mass of adsorbent.

3. RESULTS AND DISCUSSION:

3.1. Characterization of adsorbent:

The FT-IR spectrums of the modified magnetic nanoparticles are shown in Fig. 2. As can be seen, the peaks at 574 cm⁻¹, 3400 and 3805 cm⁻¹ are related to strength vibration of Fe-O (which is the main peak for Fe₃O₄) and symmetric/asymmetric strength vibration of –OH, respectively (fig. 2a). figure 2b Showed the peak of Si-O-Si in 1091 cm^-1. Moreover, all peaks of Agarose were coverd with other functional groups (fig. 2c).

Fig. 2: FT-IR spectra: (a) naked Fe₃O₄, (b) Fe₃O₄/SiO₂ (c) Fe₃O₄/SiO₂/Agarose/Fe³⁺ MNPs.

The SEM images of the Fe₃O₄/SiO₂ and Fe₃O₄/SiO₂/ Agarose/Fe³⁺ magnetic nanoparticles are shown in figure 3. By consideration of these figures it can be concluded that synthesized and modified particles are in size of nanometer (about 16.28 nm). Moreover, as can be seen, the surface morphology of these nanoparticles is relatively smooth and homogeneous.

Fig. 3: FESEM images of magnetic nanoparticles: (a) Fe₃O₄/SiO₂, (b) Fe₃O₄/SiO₂/Agarose/Fe³⁺ MNPs.

Additionally, the Energy-dispersive X-ray Spectrometry (EDS) analysis was carried out to study the elemental composition of the Fe₃O₄ and modified Fe₃O₄ magnetic nanoparticles figure 4 (EDX graphes). As is showed, Fe, Si, O and C are the main elements in synthesized nanoparticles. The Silicon atom revealed that Fe₃O₄is modified with TEOS. In addition, the obvious increase of Carbon atom was observed in elemental analysis, which confirmed modification of Fe₃O₄/SiO₂ with Agarose (Table 1).

Table 1. EDS analysis of the adsorbent.

Elemet

Fe₃O₄/SiO₂ W (%)

Fe₃O₄/SiO₂/Agarose/Fe³⁺ W (%)

14.56

64.99

13.71

6.74

18.83

72.62

17.03

10.35

Notes: W (%): weight percent; (a) Fe₃O₄/SiO₂,

(b) Fe₃O₄/SiO₂/Agarose/Fe³⁺

Dynamic light scattering (DLS) was carried out in order to determination of size of magnetic nanoparticles and investigation the ability of these MNPs to form aggregates in solvent (fig 5). As can be seen, the average diameter of Fe₃O₄/SiO₂/Agarose/Fe³⁺ particles is 248.5 nm. Also, surface charge of MNPs was investigated -41.6 based on Zeta potential analysis.

3.2. Effect of Ph:

The pH of the solution is one of the most important parameters in the adsorption of Metformin on the surface of MNPs. It is well known that adsorption might have different behavior in different pH values. For this purpose, pH of the solution was investigated in the range of 1.6-9. As can be seen in figure 6, adsorption of Metformin was gradually increased by the increase in the pH. And Metformin had the highest adsorption in the pH=9. It might be because that this point (pH=9) is the pH_pzc (pH point zero charge) which MNPs has the neutral surface charge. So, pH=9 was selected as the optimum point for pH of the adsorption of Metformin.

Fig. 6: Effect of pH in the adsorption of Metformin. Metformin concentration (mg L^-1): 20, contact time (min): 10, adsorbent amount (mL): 5, Temperature (^oC): 20.

3.3. Effect of contact time:

In the proposed method, effect of contact time in the adsorption of Metformin on the surface of MNPs was investigated in the range of 5-35min. Based on the results, the adsorption efficiency was decreased slightly and remained constant over the 10-30min experiments time. In the beginning, there was the highest level of adsorption for Metformin (fig. 7). It could be concluded that modified MNPs has dramatic adsorption for Metformin.

Fig. 7: Effect of contact time in the adsorption of Metformin. Metformin concentration (mg L^-1): 20, pH: 9, adsorbent amount (mL): 5, Temperature (^oC): 20.

3.4. Effect of temperature:

In order to investigate the effect of temperature on the adsorption recovery, various temperatures (^oC) were studied. Based on the figure 8, the adsorption of Metformin is declined slowly by increasing the temperature. Therefore, 20^oC was selected as the best condition for adsorption of Metformin on the MNPs.

Fig. 8: Effect of Temperature in the adsorption of Metformin. Metformin concentration (mg L^-1): 20, pH: 9, adsorbent amount (mL): 5, contact time (min): 10.

3.5. Determination of adsorbent capacity:

The adsorbent capacity of modified MNPs was studied in optimum conditions. For this purpose, modified MNPs was used for adsorption of 10-2000 (mg L^-1) Metformin. After adsorption process, the retained Metformin was determined by using UV-VIS. As is clear, the absorbent has not reached saturation. Moreover, the results showed that, the maximum adsorbent capacity was 111.20 mg g^-1 (fig 9).

Fig. 9: Effect of concentration of Metformin in adsorption capacity. pH: 9, adsorbent amount (mL): 5, Temperature (^oC): 20, contact time (min): 10.

3.6. Drug release investigation:

Release of Metformin was investigated in optimal conditions obtained from previous sections (Metformin: 20mg L^-1, adsorbent: 5mL, Temperature: 20^oC). Additionally, the pH was adjusted in pH=1.6 (the pH of stomach internal medium). As the chart showed (fig 10), the release of drug rose over the time.

Fig. 10: Release of Metformin. pH: 1.6, adsorbent amount (mL): 5, Temperature (^oC): 20, Metformin concentration (mg L^-1): 20.

CONCLUSION:

The proposed method was presented as a suitable and successful drug delivery system for Metformin. In this method, a twice modification step of magnetic nanoparticles with silicon, Agarose and Iron were applied as adsorbent. The Fe₃O₄/SiO₂/Agarose/Fe³⁺ MNPs were synthesized successfully and investigated with SEM-EDX and FT-IR in each step. The short adsorption time, high efficiency, simplicity and huge surface to volume ratio were some of synthesized MNPs.

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Received on 29.11.2023 Modified on 17.04.2024

Asian J. Pharm. Res. 2024; 14(3):221-226.

DOI: 10.52711/2231-5691.2024.00035