Magnetic-Graphene-Oxide-Incorporated Alginate Hydrogel Beads as a Potential Controlled Drug Delivery System

Abstract

The aim of this study was to synthesize alginate hydrogel beads using ionotropic gelation containing pH-sensitive magnetic reduced graphene oxide (MGO). MGO was prepared using a hydrothermal method and surrounded by alginate beads. FTIR, XRD, FESEM, TEM, VSM and TGA showed that the synthesized beads have a quasi-spherical structure, exhibit superparamagnetic behavior, and are thermally stable up to 350 °C. The model drug, quercetin, was loaded into these particles with an efficiency of 25.8%. These particles showed a pH-dependent release. HFF-2 and Caco-2 cells were used to investigate cytotoxicity. At a concentration of 140 μg/mL, more than 80% viability was observed in HFF-2 cells and anticancer effects were observed on Caco-2 cells with a decrease in viability of less than 50% at a concentration of 200 μg/mL. The obtained cell culture results indicate that the hydrogel beads are biocompatible and act as a drug delivery system.

1. Introduction

Controlled oral drug delivery systems are one of the new exciting methods in drug delivery systems. This method has advantages such as reducing the number of doses, reducing side effects, toxicity, and optimal drug control [1,2]. Recently, biodegradable, biocompatible, and non-toxic hydrogels have been studied in several types of research on oral targeted drug delivery [3].

Hydrogels are three-dimensional networks of natural or synthetic polymers. Hydrogels have many applications in several forms [4]. The porosity and physical structure of hydrogels play an essential role in controlling their properties, which are affected by temperature, electric and magnetic fields, light, etc. [5]. The high capacity of water absorption in hydrogels, generally 70–99%, makes hydrogels similar to the body’s natural environment [4]. In pharmaceutical formulation, polymer-based hydrogels play an essential role in biomedical applications. Hydrogels have good biocompatibility, hydrophilicity, and biodegradable properties, but drug release and swelling are not controlled. Depending on the manufacturing method, hydrogels can be beads, films, or matrices [6]. The beads have a regular size and shape, a large surface, and a high ability to load different drugs and biological materials [7]. Hydrogel beads made of polysaccharides such as alginate, chitosan, and cellulose have been used as carriers in drug delivery [8]. One of the most popular anionic polysaccharides is alginate.

Sodium alginate (SA) is an anionic linear polysaccharide. That is obtained from brown algae and consists of (L)-mannuronic acid and (D)-guluronic acid units [4]. The distribution of these units affects properties such as the ability to create a gel, the degree of polymerization, etc. This polymer is considered one of the most popular natural polymers for biomedical applications [9]. SA has versatile properties such as biocompatibility, biodegradability, and a high ability for drug delivery [10,11]. SA is chelated in the vicinity of polyvalent cations such as Ca²⁺, Sr²⁺, Ba²⁺, and Fe²⁺, building the three-dimensional structure of bead hydrogels [12].

Hydrogel nanocomposites are composites in which nanoparticles are entrapped in a three-dimensional hydrogel network and have both advantages [13]. One of the nanoparticles is a magnetic nanoparticle, which makes the hydrogel respond to a magnetic field. Several types of research have been conducted in drug delivery with magnetic hydrogels; for example, Rafi et al used SA-magnetic pH-sensitive hydrogels to deliver naproxen to the colon [14].

Graphene, as a two-dimensional material, has been investigated since 2008 as a nanocarrier for various drugs [15]. Graphene is highly interesting in biomedical studies due to its high drug loading [16]. Its use is still doubtful due to the toxicity caused by synthesis or surface modification. To overcome this drawback, using toxic reducing agents such as hydrazine to convert graphene oxide to reduced graphene oxide can be replaced by methods such as hydrothermal [17].

Magnetic nanoparticles have many applications due to their magnetic properties, easy surface modification, and biocompatibility [18]. Magnetite is of interest in MRI imaging and targeted drug delivery [19], which can overcome the low solubility of drugs and rapid drug clearance in the body, and deliver the drug to the appropriate tissue.

In this study, we prepared superparamagnetic alginate hydrogel-based beads for quercetin delivery as a drug model (Figure 1). The physicochemical properties of the synthesized hydrogel beads were studied by several methods, such as FTIR, X-ray, FESEM, TEM, etc.

Figure 1.

The synthesized uniform magnetic hydrogel beads containing quercetin (aqueous (A), dry (B) hydrogel) and their magnetic properties (C).

2. Results and Discussion

Synthesis of alginate beads was carried out by cross-linking the polymer network with Ca²⁺ ions. The increase in these two substances together creates hydrogel beads in the early moments.

Magnetic beads are commonly employed in water-based magneto-driven applications [20]. Consequently, it holds immense significance to explore the stability of alginate beads when immersed in aqueous solutions with varying pH levels over a specified duration. For this purpose, investigations were carried out on alginate beads in water solutions at pH 2.0 ± 0.2, 7.0 ± 0.2, and 12.0 ± 0.2 up to 21 h. It is worth noting that alginate beads typically undergo distinct swelling and shrinking responses in acidic and basic pH solutions. Alginate polymer comprises carboxylate groups (–COO–) that transform into carboxylic acid (–COOH) under acidic conditions. This transformation leads to interactions with other hydroxyl (–OH) and –COOH groups through intermolecular hydrogen bonding. Consequently, the beads contract over time. In contrast, in pH values exceeding 7, especially under basic conditions, the carboxylate groups remain as negatively charged COO^– anions. These negative charges induce electrostatic repulsion forces between these groups, resulting in bead expansion and the absorption of additional water molecules. This expansion continues until equilibrium is achieved [21].

2.1. Characterization of the Hydrogel Beads

FT-IR spectroscopy was employed to confirm the chemical composition of magnetic hydrogel beads and to evaluate the successful loading of quercetin (Figure 2).

Spectrum (a) corresponds to the unloaded magnetic hydrogel beads. The symmetric and asymmetric stretching vibration of carboxylate groups (-COO⁻) are shown at 1429 cm⁻¹ and 1573 cm⁻¹, respectively. A broad band at 3424 cm⁻¹ is attributed to O-H stretching vibrations associated with hydroxyl groups and absorbed water molecules [22].

The FT-IR spectrum of pure quercetin (spectrum b), a polyphenolic flavonoid compound, exhibits a broad and intense band around 3400 cm⁻¹ corresponding to the O–H stretching vibrations of phenolic hydroxyl groups. The characteristic stretching vibration of the carbonyl (C=O) group appears at approximately 1660 cm⁻¹. In addition, the aromatic C=C stretching vibrations observed at 1608 cm⁻¹ and 1518 cm⁻¹ are associated with the benzene rings present in the quercetin structure [23].

In the spectrum of quercetin-loaded magnetic alginate beads (spectrum c), the characteristic absorption bands of quercetin are not distinctly visible due to overlap with the broad O–H stretching band and the carboxylate vibrations of the alginate matrix [24,25]. However, the absence of new bands or significant peak shifts suggests that quercetin is physically incorporated into the alginate matrix rather than chemically bonded. This behavior is consistent with non-covalent interactions such as hydrogen bonding and π–π interactions with the MGO component.

Figure 2.

FT-IR spectrum of magnetic hydrogel beads (a), quercetin (b) and magnetic-hydrogel-beads–quercetin (c).

Energy-dispersive X-ray spectroscopy (see Figure 3) confirmed the elemental makeup of MGO-alginate beads. The spectra confirmed the presence of carbon, oxygen, iron, and calcium. The detection of iron is attributed to the Fe₃O₄ nanoparticles, while calcium originates from Ca²⁺ ions used during ionotropic cross-linking of the alginate matrix. Trace amounts of sodium were also detected, which can be ascribed to residual sodium-containing reagents used during the MGO synthesis process.

Figure 3.

EDX spectrum of MGO-alginate beads.

2.2. XRD Study

The synthesized MGO filler was characterized using X-ray powder diffraction (XRD) and the details are given below. The powder XRD patterns of the synthesized rGO (a) and MGO (b) confirmed the phase formation and crystallinity of the fillers as shown in Figure 4. The XRD data confirmed the formation of rGO (JCPDS card number: 41-1487) by the reduction of GO by hydrothermal method [26]. After reduction, the (002) peak was observed at 2θ = 25.4°, which is related to the good arrangement of the interlayer spacing of graphene. The XRD pattern of MGO (b) showed all the peaks related to Fe₃O₄ (JCPDS card number 19-0629). The peaks at 2θ values of 30.0°, 35.5°, 43.0°, 53.6°, 57.2°, and 63° corresponded to (220), (311), (400), (422), (511) and (440) crystal planes of magnetic Fe₃O₄, respectively. The Fe₃O₄ nanoparticles are uniformly deposited on the surface of rGO sheets and prevent the restacking of graphene sheets. This causes the regular and crystalline structure of rGO sheets to be destroyed and their characteristic peak does not appear [19]. The XRD pattern of MGO-alginate beads (c) shows only three diffraction peaks at 2θ = 35.5, 57.2 and 43.0° without any other prominent sharp diffraction peaks (4c), indicating the semi-crystalline nature of the studied biopolymer [27], and these findings are consistent with previous studies [28].

Figure 4.

XRD patterns of (a) rGO, (b) MGO, and (c) MGO-alginate beads.

2.3. Morphological Study

To study the morphology of the designed nanocomposites and beads, FESEM and TEM imaging were carried out (Figure 5). The FESEM image in Figure 5a shows the formation of alginate hydrogel beads with a relatively uniform morphology. At higher magnification (Figure 5b), the bead surface exhibits a porous structure, which is consistent with previous reports on ionotropically gelled alginate hydrogel systems [29]. Such surface porosity facilitates the penetration of aqueous media into the hydrogel network and contributes to bead swelling behavior.

TEM images acquired at two different magnifications from representative regions of the sample (Figure 5c,d) show the presence of graphene oxide–Fe₃O₄ nanocomposites embedded within the alginate matrix. The Fe₃O₄ nanoparticles appear as clustered domains distributed on the surface of graphene oxide sheets, indicating the retention of the nanocomposite structure after encapsulation.

Figure 5.

FESEM (a,b) and TEM (c,d) of MGO-alginate beads with different magnification obtained from different regions of the sample.

The initial weight loss observed below approximately 220 °C can be attributed to the removal of physically and chemically adsorbed water molecules (Figure 6). The major weight loss occurring in the temperature range of 300–500 °C is mainly associated with the thermal degradation of alginate chains and hydroxyl-containing functional groups [30]. According to previously reported TGA studies on Fe₃O₄/GO nanocomposites, the decomposition of graphene-based components typically occurs between 450 and 550 °C [31]. The residual mass remaining above ~600–700 °C is therefore assigned to the thermally stable iron oxide phase. Based on these observations and the literature comparisons, the high-temperature residue in the present composite is attributed to the Fe₃O₄/GO component.

Figure 6.

TGA-DSC thermogram of magnetic hydrogel beads.

2.4. Magnetic Properties

Figure 7 shows the hysteresis loops of the magnetic hydrogel beads investigated by VSM technique between ±10 kOe at 298 K. The saturation magnetization of graphene oxide-Fe₃O₄ nanocomposite and magnetic hydrogel beads were obtained to be about 63 and 28.1 emu/g, respectively. As shown in the figure, both samples exhibit negligible coercivity and remanence, indicating superparamagnetic behavior. The obtained magnetization of the magnetic hydrogel beads is sufficient for magnetic manipulation and separation in biological environments [32]. The saturation magnetization in this study is comparable to or higher than many reported magnetic alginate systems. For example, Mahdvinia et al. reported a value of 3.4 emu/g for magnetic κ-carrageenan beads [33], and synthesized magnetic halloysite nanotubes (MHNTs) by Polat et al. that have a saturation magnetization of 22.7, and the addition of alginate coating reduces the saturation magnetization to 8.17 [34]. The decrease in magnetization from 63 to 28.1 emu/g in this study is due to the non-magnetic alginate content (~55 wt%) and is considered sufficient for magnetic separation and potential targeted drug delivery applications.

Figure 7.

VSM graph of graphene oxide-Fe₃O₄ nanocomposite (A) and magnetic hydrogel beads (B).

2.5. Drug Loading and Release Study

For practical applications, the drug loading capacity of the beads or carriers is a critical factor, as higher drug loading makes the carriers more cost-effective [35]. The drug loading of the hydrogel was 25.8 ± 1.3%. Figure 8a,b show the drug release profile of magnetic hydrogel beads in pH 1.2 and 7.4 buffer solutions at 37 ± 1 °C. The results show that both the rate and percentage of quercetin release from magnetic hydrogel beads were higher at pH 7.4 compared to pH 1.2. These findings are consistent with the swelling rate of hydrogel beads, which were higher in neutral to basic pH environments than in acidic environments [36]. At pH values above the pKa of alginate (≈3.5), alginate carboxyl groups (-COOH) deprotonate to produce negatively charged carboxylate ions (-COO⁻), inducing electrostatic repulsion in the polymer network, matrix expansion, and higher uptake of water. Conversely, under pH 1.2 (<pKa), the protonated -COOH form prevails and gives rise to a compact, hydrogen-bonded structure with reduced porosity and swelling. Such pH-dependent structural transformation directly facilitates improved drug diffusion and release in simulated intestinal fluid, thus making alginate-based carriers promising candidates for oral and site-specific delivery applications [37,38]. It should be noted that under pH 7.4 conditions, the alginate beads gradually cracked and partially disintegrated; therefore, the release profile represents drug release from bead fragments and polymer remnants rather than from fully intact spheres.

It should be noted that quercetin is known to exhibit limited chemical stability under physiological pH conditions. Therefore, the release experiments conducted at pH 7.4 reflect not only the release behavior of quercetin from the hydrogel matrix but also its intrinsic pH-dependent instability. Similar observations regarding quercetin degradation at neutral and alkaline pH values have been reported in previous studies [39,40]. In this study, the released quercetin was quantified immediately after sampling to minimize degradation-related errors. Cadena-Volandia et al. [23] reported alginate microparticles containing quercetin with a loading of 1.43% w/w and a high encapsulation efficiency of 96.21%. Drug release was carried out by inhomogeneous swelling and erosion due to particle surface defects. In contrast, in our study, a significantly higher loading (25.8%) was achieved, which was attributed to the high surface area and π-π interactions provided by the embedded magnetic graphene oxide (MGO) nanoparticles. In another report, Liu et al. [41] fabricated alginate–inulin–chitosan microspheres with encapsulation efficiencies of 53.2% and 80.3% of quercetin. It showed delayed release in the colon (within 3 h) and complete metabolism by the gut microbiota within 24 h. Our beads show comparable efficacy but pH-dependent release (83% at pH 7.4 vs. 45% at pH 2.1). The release is likely enhanced by the presence of superparamagnetic Fe₃O₄ nanoparticles in MGO, which may contribute to enhanced drug release at neutral pH. These comparisons highlight the advantages of incorporating MGO in improving loading capacity and intestinal targeted release for oral drug delivery applications.

Figure 8.

Release profile of quercetin in (a) pH 7.4 and (b) pH 1.2 at 37 °C.

2.6. Hemolysis Assay

Hemocompatibility of magnetic hydrogel beads was evaluated through the standard hemolysis assay on human RBCs. As shown in Figure 9, no significant membrane damage was observed in concentrations below 62.5 µg/mL after 48 h incubation, whereas hemolysis percentages were below 5% for all the tested components (quercetin, MGO, and beads). Such good blood compatibility can be attributed to the biocompatibility character of the alginate matrix and the protective effect of the hydrogel coating that minimizes the potential interactions between graphene oxide and RBC membranes.

Figure 9.

Effect of components on RBC membrane integrity.

2.7. Cell Cytotoxicity

Safety and toxicity characteristics are critical parameters for nanocomposites intended for biomedical applications. Figure 10 shows the cytotoxicity of magnetic hydrogel beads in vitro, where cell viability was evaluated using MTT assay on HFF-2 cell lines. Different concentrations of magnetic hydrogel beads were incubated in cells for 24 and 48 h. As shown in Figure 10, the cell viability remained relatively stable even after 48 h, indicating the biocompatibility of the synthesized magnetic hydrogel beads. These findings show that the synthesized magnetic hydrogel beads are promising as a potential drug carrier for anticancer applications. Figure 10 shows the in vitro anticancer results of quercetin, magnetic hydrogel beads and quercetin-loaded magnetic hydrogel beads on human colorectal epithelial adenocarcinoma (Caco-2) cells over 48 h with different concentrations. The MTT results showed that the magnetic hydrogel beads exhibited significant toxicity, leading to a decrease in cell viability below 50% at a concentration of 200 μg/mL, thus suggesting a concentration-dependent anticancer potential in vitro. This effect showed much more lethality with the quercetin loading. These data show that the cytotoxicity is directly related to the concentration of magnetic hydrogel beads. Beyond its cytotoxic effects, quercetin has also been reported to actively interact with intestinal epithelial cells through regulated biological pathways.

Figure 10.

(a) MTT assay of magnetic hydrogel beads on HFF-2 cell line after incubation for 24, and 48 h. The values represented as mean ± SD (n = 3), (b) MTT assay of magnetic hydrogel beads (H), quercetin-loaded magnetic hydrogel beads (H-Q) and free quercetin (Q) on Caco-2 cell line after incubation for 48 h. The values represented as mean ± SD (n = 3).

Previous studies have reported that quercetin exhibits dose-dependent cytotoxicity against Caco-2 cells, mainly through apoptosis induction and cell cycle arrest, while showing lower toxicity toward normal fibroblast cells [42,43]. The observed reduction in Caco-2 cell viability in the present study is consistent with these reports, although the sustained release behavior of the hydrogel system may moderate the immediate cytotoxic effect compared to free quercetin.

Previous studies have demonstrated that quercetin interacts effectively with Caco-2 intestinal epithelial cells and exhibits concentration-dependent biological activity. For instance, transport studies across Caco-2 monolayers have shown that quercetin can cross the intestinal epithelial barrier, although its permeability is limited by its physicochemical properties and cellular metabolism. In addition, quercetin has been reported to modulate epithelial barrier function in Caco-2 cells by increasing the expression of tight junction proteins such as claudin-4, indicating its active involvement in cellular regulatory pathways rather than nonspecific toxicity [39].

In line with these reports, the observed response of Caco-2 cells to the quercetin-loaded magnetic hydrogel beads in the present study suggests that the released quercetin retains its biological activity. The moderated cytotoxic effect observed compared to free quercetin may be attributed to the sustained release behavior of the carrier system, which is consistent with previously reported quercetin Caco-2 interactions.

3. Conclusions

MGO-alginate hydrogel beads were prepared using a green ionotropic gelation technique. The beads were also superparamagnetic at 28.1 emu/g, and the beads are temperature-resistant up to 350 °C. An EDX analysis indicated that they consist of carbon (45.2 wt%), oxygen (40.8 wt%), iron (10.5 wt%), and calcium (3.5 wt%), forming successful cross-linking between MGO and alginate. They could load 25.8% quercetin and showed a pH-dependent release profile, releasing 83% at pH 7.4 in 200 min, which makes them suitable for drug targeting. Their biocompatibility was excellent, with greater than 80% viability of HFF-2 at 140 µg/mL, while their anticancer activity reduced Caco-2 cell viability to below 50% at 200 µg/mL. The beads were also stable for a pH range of 2.0 to 12.0 and had retained high magnetic properties, demonstrating their potential to act as drug delivery system. Additional research must investigate their in vivo properties, scaling, and potential for exposure of other drug candidates to the clinic.

4. Materials and Methods

4.1. Materials

Graphite, SA and calcium chloride, magnesium chloride, iron (III) chloride hexahydrate (FeCl₃.6H₂O), iron (II) chloride tetrahydrate (FeCl₂.4H₂O), potassium permanganate (KMnO₄), hydrochloric acid, sodium acetate, ethylene glycol, hydrogen peroxide, and sulfuric acid were bought from Merck (Darmstadt, Germany). Quercetin (MW = 302.2 and Tween 80 (Polysorbate 80, (C₆₄H₁₂₄O₂₆)) were purchased from Sigma Aldrich (St. Louis, MO, USA). Ammonium hydroxide (NH₄OH, 25% (v/v)) and ethanol were obtained from Dr. Mojallali Co. (Teheran, Iran). All materials were used without further purification.

4.2. Methods

4.2.1. Graphene Oxide Preparation

Graphene oxide was synthesized by Hammer’s method. 200 mg of graphite powder was added to 24 mL of sulfuric acid and stirred in an ice bath for one hour. Then 1.2 g of KMnO₄ was added to the medium and stirred again for 15 min. The mixture was stirred at room temperature for another 24 h. Then the temperature reached 100 °C. In the meantime, we added deionized water (15 mL) and allowed it to sit for two hours to remove excess KMnO₄. 15 mL of deionized water/H₂O₂ mixture (2:1) was added slowly to the reaction medium. The obtained graphene oxide was separated with the help of a centrifuge and washed with 5% hydrochloric acid. Finally, it was dried in an oven at 60 °C and stored for further use.

4.2.2. Graphene Oxide-Fe₃O₄ Nanocomposite Preparation

Graphene oxide (0.3 g) was dispersed into 30 mL of ethylene glycol by sonication. 125 mg of FeCl₃.6H₂O and 1.32 g of sodium acetate were added and stirred for 15 min. Then the mixture was transferred to a hydrothermal bomb and placed in an oven at 180 °C for 8 h. Finally, precipitation was dried at 60 °C [44].

4.2.3. Magnetic Hydrogel Beads Preparation

First step: 1.5 g of sodium alginate was dissolved in 50 mL distilled water. A total of 3 mg of quercetin was dissolved in the minimum ethanol and added to the previous solution. Second step: step 4.2.2 nanocomposite was dispersed in 20 mL of distilled water by sonication. Then it was added to the first step solution under stirring. The third step: The prepared colloidal solution was added drop by drop by syringe to the 0.3 M calcium chloride solution under vigorous stirring. As soon as the composition of alginate increases, hydrogel beads are formed. After 5 min of mixing, hydrogels were separated by an external magnet and dried at room temperature (Scheme 1).

Scheme 1.

Hydrogel beads preparation.

4.2.4. Characterization Methods

Magnetic hydrogel bead structures were characterized by Fourier transform infrared spectroscopy (FTIR) (Bruker, Tensor 27, New York, NY, USA). The crystallinity of the prepared magnetic hydrogel beads were analyzed with X-ray diffraction analysis (XRD) (Philips PW1730, North Castle, NY, USA). The magnetic measurements of the hydrogels and the thermal stability were carried out with a vibrating sample magnetometer (VSM) (Kashan Desert Magnet Company LBKFB, Kashan, Iran) and thermal gravimetric analysis (TGA) (TAQ600, New Castle, DE, USA), respectively. The morphology by transmission electron microscopy (TEM) (TEM Philips EM 208S, Beaverton, OR, USA) at an accelerating voltage of 100 kV and field emission scanning electron microscopy (FESEM) (ZEISS Sigma 300, Cambridge, MA, USA) was characterized.

4.2.5. Drug Loading Determination

Drug loading of quercetin in the hydrogel beads was determined by an extraction method followed by UV–Vis spectrophotometric analysis. Briefly, 10 mg of dried beads were crushed and dispersed in 10 mL of ethanol and sonicated for 30 min at room temperature to ensure complete extraction of the loaded drug. The suspension was then analyzed using a UV–Vis spectrophotometer at 335 nm. The concentration of quercetin was calculated from a previously established calibration curve (R² = 0.999).

Drug loading (%) was calculated using the following equation:

Drug loading (%) = (weight of quercetin loaded in the beads/weight of dried beads) × 100

All experiments were performed in quadruplicate (n = 4), and the results are reported as mean ± standard deviation.

4.2.6. In Vitro Drug Release

Quercetin release from hydrogel beads was studied at pH of physiological (7.4) and acidic (5.5 and 2.1). The hydrogel beads were placed in 2 mL phosphate-buffered saline into the dialysis bag (Mw cutoff: 12 KDa) and immersed in 18 mL phosphate-buffered saline with 0.5% (w/w) tween 80. Sampling was done at predetermined times and replaced by the same volume of fresh buffer. The amount of release was measured at the wavelength of 335 nm by UV-Vis spectroscopy.

4.2.7. Hemolysis Assay

Red blood cells (RBC) were washed with medium (centrifugation at 2000× g for 5 min) and red blood cells were resuspended in the same medium at a final hematocrit of 5%. The suspension was transferred in triplicate to 1.5 mL sterile microfuge tubes. Two-fold serial dilutions of quercetin and magnetic hydrogel beads were tested starting at 400 µg ml⁻¹. Medium supplemented with 1% Triton X-100 was used as a positive control and 0.5% DMSO as a negative control. Microtubes were incubated at 37 °C for 2 h. The microtube was then centrifuged at 2000× g for 5 min and 100 μL of supernatant was transferred to a microtiter plate. Absorbance of free hemoglobin was measured at 550 nm. Percent hemolysis was calculated using a 1% Triton X-100 positive control for hemolysis of human RBCs taken as 100%. The hemolysis assay was performed in accordance with the ethical guidelines of Zanjan University of Medical Sciences and approved by the Institutional Ethics Committee (Approval No. IR.ZUMS.REC.1401.094).

4.2.8. Cytotoxicity Assay

Methyl thiazolyl tetrazolium (MTT, Sigma–Aldrich, St. Louis, MO, USA) assay was used to determine the in vitro cytotoxicity. Human colorectal adenocarcinoma cells (Caco-2) were used as a cancer cell line to test the anticancer potential of the samples, while human foreskin fibroblast cells (HFF-2) were used as a normal cell line to test the cytocompatibility of the samples.

Caco-2 and HFF-2 cells were seeded onto a 96-well plate with 1 × 10⁴ cells per well, then incubated in 5% CO₂ at 37 °C for 24 h. At that point, the Caco-2 cells were incubated with 100 µL fresh medium containing different concentrations (0–140 µgml⁻¹) of quercetin, rGO/Fe₃O₄, and magnetic-hydrogel-beads–quercetin for 24 h.

Abbreviations

The following abbreviations are used in this manuscript:

MGO	Magnetic graphene oxide
RBC	Red blood cells

Author Contributions

Conceptualization, S.S., A.K. (Akram Khanmohammadi); methodology, S.S.; software, A.K. (Akram Khanmohammadi); validation, A.K. (Abolfazl Kordloo), M.R.H.; formal analysis, N.F., T.M.; investigation, N.F.; data curation, S.S.; writing—original draft preparation, S.S.; writing—review and editing, S.S.; visualization, Z.K., A.K. (Akram Khanmohammadi); supervision, S.S.; project administration, S.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original findings presented in this work are fully included in the article. Further details may be obtained by contacting the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.