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Li, Y., Feng, Y., Jing, J., and Yang, F. (2019). "Cellulose/guar gum hydrogel microspheres as a magnetic anticancer drug carrier," BioRes. 14(2), 3615-3629.

Abstract

A novel magnetic anticancer drug carrier based on cellulose, guar gum, and Fe3O4 hydrogel microspheres was synthesized by chemical crosslinking. These microspheres were crosslinked with epoxy chloropropane and loaded with 5-fluorouracil (5-fu). The effect of the ratio of cellulose to guar gum on bead size, drug loading, and in vitro release behaviors were investigated. The influence of the magnetic content on drug loading and in vitro release behaviors were also evaluated. The magnetic hydrogel microspheres were characterized via an optical microscope, Fourier transform infrared spectroscopy, swelling behavior analysis, vibrating sample magnetometer, and ultraviolet absorption spectroscopy. The results showed that as the ratio of cellulose to guar gum increased from 3:1 to 5:1, the particle size increased from 395 to 459 um. Moreover, the drug loading capacity, encapsulation efficiency, and in vitro release behavior were influenced by the ratio of cellulose/guar gum and Fe3O4 content. Finally, the Fe3O4 particle had an adsorption effect on the drug, thereby reducing the maximum cumulative release.


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Cellulose/Guar Gum Hydrogel Microspheres as a Magnetic Anticancer Drug Carrier

Yanli Li,a,b Yucheng Feng,a,b Jun Jing,a and Fei Yang *,a,b

A novel magnetic anticancer drug carrier based on cellulose, guar gum, and Fe3O4 hydrogel microspheres was synthesized by chemical crosslinking. These microspheres were crosslinked with epoxy chloropropane and loaded with 5-fluorouracil (5-fu). The effect of the ratio of cellulose to guar gum on bead size, drug loading, and in vitro release behaviors were investigated. The influence of the magnetic content on drug loading and in vitro release behaviors were also evaluated. The magnetic hydrogel microspheres were characterized via an optical microscope, Fourier transform infrared spectroscopy, swelling behavior analysis, vibrating sample magnetometer, and ultraviolet absorption spectroscopy. The results showed that as the ratio of cellulose to guar gum increased from 3:1 to 5:1, the particle size increased from 395 to 459 um. Moreover, the drug loading capacity, encapsulation efficiency, and in vitro release behavior were influenced by the ratio of cellulose/guar gum and Fe3O4 content. Finally, the Fe3O4 particle had an adsorption effect on the drug, thereby reducing the maximum cumulative release.

Keywords: Cellulose; Guar gum; Fe3O4 nanoparticle; Hydrogel microsphere; Drug controlled release

Contact information: a: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China; b: United Lab of Plant Resources Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China;

* Corresponding author: yangfei@scut.edu.cn

INTRODUCTION

Currently, cancer is one of the most life-threatening diseases worldwide, especially in China where cancer incidence and mortality has continued to increase and has become a major public health problem (Chen et al. 2016). The treatment for cancer includes surgery, radiation, and chemotherapy. Chemotherapy is one of the most useful treatments for cancer. However, the improvement in quality of life and increasing safe awareness assure that high quality and safe drug therapies are a priority for policymakers and multidisciplinary scientists. Controlled drug delivery technology has had a more dominant role in recent years due to its advantages compared to the conventional medicine system, including improved efficiency and reduced dosage (Uhrich et al. 1999; Babu et al. 2007). Drug carriers have an important role in the drug release system and are a main factor in affecting drug efficacy (Uekama and Otagiri 1987). Polymeric hydrogels are cross-linked hydrophilic materials that have a three-dimensional network structure and can absorb large quantities of water without dissolving (O’Connor and Gehrke 2015; Liu et al. 2009). In recent years, much attention has been focused on hydrogel microspheres for the delivery of water-solution drugs due to their outstanding characteristics such as excellent water-absorption, water-retention, and slow-release (Korsmeyer et al. 1983; Liu et al. 2006a; Karaaslan et al. 2010).

Biopolymeric resources are promising feedstocks to produce sustainable products (Nurunnabi et al. 2015; Aravamudhan et al. 2016; Shaghaleh et al. 2018). Cellulose is the most abundant natural polymer on Earth (Bledzki and Gassan 1999; Fan et al. 2017). Because cellulose has an abundant number of hydroxyl groups, it can be used to prepare hydrogels easily with fascinating structures and properties (Chang and Zhang 2011; Xu et al. 2018). For example, previous work exploited carboxymethyl cellulose (CMC) beads via a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently bonded to the CMC beads (Yakup 2015). Cellulose (or its derivatives) blended with other polymers is an extremely attractive, inexpensive, and advantageous method to obtain new structural materials (Bajpai et al. 2008), such as chitosan (Nan and Bai 2005), starch (Faroongsarng and Sukonrat 2008), and polyvinyl alcohol (Salmawi 2007; Chang et al. 2010). Guar gum is one of the most abundant natural polysaccharides in the world, consisting of a 1,4-β-D-mannose backbone and 1,6-α-D-galactose side chain, with a ratio of galactose/mannose of 1:2 (Sinha and Kumria 2001; Li et al. 2008). It has been suggested as a vehicle for oral controlled release purposes and for colon targeting due to its biodegradability and biocompatibility (Huang et al. 2007). However, to the authors’ knowledge, there have been few reports on the investigation of cellulose blended with guar gum microspheres.

Recently, magnetic nanoparticles have gained attention because of their unique features (Rani et al. 2010; Fan et al. 2011). They can be used in medical applications, such as drug targeting for drug control release and targeted release, especially for cancer therapy (Liu et al. 2006b). This innovative strategy for targeted drug delivery consists of coupling the drug to magnetic nanoparticles that can be guided to the target by means of external magnetic fields (Barbucci et al. 2012). In the present paper, the objective is to prepare a magnetic hydrogel microsphere, consisting of cellulose, guar gum, and Fe3O4, while the microspheres were located with 5-fluorouracil (5-fu). The effect of the ratio of cellulose to guar gum on bead size and drug loading as well as the in vitro release behaviors were investigated. The influence of magnetic content on drug loading and in vitro release behaviors were also studied. The magnetic hydrogel microspheres were characterized using an optical microscope, Fourier transform infrared spectroscopy (FTIR), and swelling behavior, vibrating sample magnetometer (VSM) to investigate the shape synthesis. Additionally, ultraviolet (UV) absorption spectroscopy was performed to evaluate the drug loading and release.

EXPERIMENTAL

Materials

Jute pulp was supplied by China Tobacco Mauduit (Guangdong, China). Guar gum was bought from Wuhan Shengruiyuan Biotechnology Co., Ltd. (Wuhan, China), Liquid paraffin, epoxy chloropropane, sodium hydroxide, and urea were supplied by Guangzhou Congyuan Instrument Corporation (Guangzhou, China). Iron vitriol and anhydrous ferric chloride were bought from Tianjin Damao Chemical Reagent Factory (Guangzhou, China) and 5-fluorouracil (5-fu) was supplied by Aladdin Reagent Co., Ltd. (Shanghai, China). All of the chemical reagents were of analytical grade without further purification.

Methods

Preparation of cellulose/guar gum hydrogel microspheres

Jute pulp was dissolved in a NaOH/urea aqueous solution at a low temperature to form a cellulose solution with a concentration of 2 wt% as previously described (Qin et al. 2013). Guar gum was dissolved in water by stirring overnight to form a guar gum solution with a concentration of 1 wt%. The gel beads were produced in a 250-mL round-bottom flask equipped with a stirrer. Liquid paraffin (100 mL) in a continuous phase was added to provide a reaction environment and Tween 80 was added as a 0.1 mL/mL mixed solution as dispersants. Then, the mixture of cellulose and guar gum was poured into the continuous phase while it was being stirred at a speed of 800 r/min. When the mixed solution was dispersed into droplets in liquid paraffin, epoxy chloropropane was added as a 0.1 mL/mL mixed solution. The solution was kept at 30 ℃ for 30 min and then heated to 60 ℃. Five hours later, the reaction was stopped, and the gel beads were obtained. The microspheres were then frozen and freeze-dried in a lyophilizer (FD5-2.5; Gold SIM, Los Angeles, USA) (-58℃, 0.22 mbar) for 48 h before further analysis.

Preparation of cellulose/guar gum/Fe3O4 hydrogel microspheres

The Fe3O4 magnetic fluid was prepared via chemical coprecipitation of iron vitriol and anhydrous ferric chloride solution (0.25 mol/L) with ammonia as the precipitant (Deng et al. 2003). To the resultant suspension, the Fe3O4 magnetic fluid was washed by deionized water until pH = 7 and was then stored in 50 mL of alcohol. The obtained magnetic nanoparticles were added to a cellulose/guar gum mixture during the process of the polymerization reaction. The magnetic hydrogel microspheres were treated with Fe3O4 magnetic fluid with different volumes of 0.2 mL, 0.4 mL, and 0.6 mL and were coded as M1, M2, and M3, respectively. The samples that were loaded with Fe3Owere calcined at 600 C for 5 h to obtain iron oxide. Then, the content of Fe3O4 loaded into the hydrogel was calculated from the Fe2O3.

Morphology of hydrogel microspheres

An optical microscope (SZX12; Olympus, Tokyo, Japan) was used to observe the size of the gel beads. The bead size was the average size determined by measuring 200 samples of beads (O’Connor and Gehrke 2015).

Fourier transform infrared spectroscopy

The FTIR spectral measurements were performed using a Nicolet FTIR spectrometer (Nicolet Nexus 670; Nicolet Instrument Technologies, Inc., Madison, WI, USA) to confirm the presence of cross-linking in the cellulose-guar gum gel beads. The blended particles were finely ground with KBr to prepare the pellets, and the spectra were scanned between 600 and 3700 cm-1.

Swelling behavior analysis

Swelling experiments were conducted in distilled water at 25 ℃. The weighed mass of dry microspheres were dipped in the distilled water. After preset time intervals, the sample was blotted with filter paper to remove excess water from the hydrogel surface and the sample was weighed. The swelling ratio (S) was calculated according to,

(1)

where W(g) and W(g) are the weights of microsphere at any time (min) and the initial weight of the dry gels, respectively.

Magnetic properties analysis

The magnetic properties of the microspheres were measured with a vibrating sample magnetometer (VSM, Lake Shore, Columbus, OH, USA) at 25 ℃, and the hysteresis loop was obtained in a magnetic field that was varied from 1.0 to +1.0 T.

Drug loaded into hydrogel microspheres

A sample of 20 mg of 5-fu was dissolved into the mixture of cellulose and guar gum (mixture of cellulose, guar gum, and Fe3O4 magnetic fluid) during the heat-initiated free radical polymerization process. The syntheses of composite microsphere and drug loaded process were presented in Fig. 1. After polymerization, drug-loaded hydrogels were frozen and then freeze-dried in a lyophilizer (-58 ℃, 0.22 mbar) for 48 h. The drug-loading content (Q) and encapsulation efficiency (EE) were determined using a UV-spectrophotometer (UV-2600, Shimadzu, Suzhou, China) at 265 nm.

where Wt is the total amount (g) of 5-fu employed and Wa is Wt minus the amount (g) of unloaded 5-fu and W is the weight (g) of microsphere.

In vitro release

A sample of 20 mg of drug-loaded cellulose/guar gum (cellulose/guar gum/Fe3O4) hydrogel microspheres were dispersed in 20 mL of artificial simulation intestinal fluid, and then was stirred at 100 rpm at 37 ℃. The drug release behavior from the microspheres was studied in the intestinal fluid. At regular intervals of time, 2 mL of release media was removed and replaced by fresh release media. The concentration of 5-fu released was determined using a UV-spectrophotometer (UV-2600; Shimadzu, Suzhou, China) at 265 nm.

Fig. 1. Preparation of the drug-loaded cellulose/guar gum/Fe3O4 hydrogel nanosphere

RESULTS AND DISCUSSION

Microscopic Study

The microspheres prepared by different methods had different particle sizes, as shown in Table 1 and Fig. 2.

Table 1. Results of Particle Size of Different Ratios of Cellulose to Guar Gum

Fig. 2. The size distribution of gel beads. (a) C-1, (b) CG-1, (c) CG-2, (d) CG-3

Results indicated that gel bead diameter was influenced by the guar gum content of the microsphere. It is known that the particle size of the pristine cellulose was higher than those of the cellulose/guar gum composite microspheres. Table 1 also shows that as the ratio of cellulose to guar gum increased from 3:1 to 5:1, the average size increased from 359 to 459 um; this change can be seen by size distribution of gel beads (Fig. 2). The size distributions generally could be fitted to the Gaussian distribution. With an increase of guar gum content, the size of beads moves toward to lower size. As the amount of guar gum in the microsphere increased, the interfacial viscosity of the polymer droplets in the emulsion also increased. Because the number of free sites available for cross-linking was more with the increasing amount of guar gum, the size of the microspheres also decreased. The optical microscope micrographs of C-1 are shown in Fig. 3.

Fig. 3. Optical microscope micrographs of C-1

FTIR Spectroscopy Analysis

IR spectra of composite hydrogel are depicted in Fig. 4.

Fig. 4. FTIR spectra of gel beads: (a): CG-1 (Cellulose/guar gum = 5:1), (b): CG-3 (Cellulose/guar gum = 3:1), (c): C-1 (pure cellulose) and (d) DC-1 (Drug loaded C-1 )

There were strong absorption peaks at about 3384 to 3367 cm-1 in Fig. 4 (a, b and c), attributed to the stretching of -O-H from gel beads. In addition, the peak at 1053 cm-1 was assigned to the C-O-C, which suggests the cross-linking reaction of cellulose or guar gum with epoxy chloropropane. As Fig. 4 shows, the vibration peak of -O-H was stronger and exhibited a shift towards a slightly lower wavenumber direction with increased guar gum content relative to cellulose. This suggested that cellulose/guar gum was well mixed and led to noticeable changes in the interaction between the molecules. In Fig. 4 (d), a significant new peak appears at 1732 cm-1 (C=O stretching) of carbonyl group of 5-Fu; this result indicated that the 5-fu were loaded with C-1.

Swelling Behavior Analysis

The swelling kinetics curves of samples in distilled water at 25 °C were displayed in Fig. 5. As we can see, compared with CG composited hydrogel, pristine cellulose hydrogel has much lower swelling ratio. With an increase of guar gum content, the equilibrium swelling ratios of the hydrogels increases from 14.0 to 20.1 wt%. The more guar gum content, the more available free volume of the expanded polymer matrix and availability of group such as -OH able to form hydrogen bonds with water. As a consequence, compared with the same hydrogels with lower guar gum content, high guar gum content hydrogel showed a higher swelling ratio.

Fig. 5. Swelling kinetics curves of C-1, CG-1, CG-2, and CG-3 in distilled water at 25 °C

Magnetic Properties

Figure 6 shows the images of M1, M2, and M3 in a magnetic field. As can be seen, the samples could align in the magnetic field. Also, the magnetic response of hydrogel microsphere was more sensitive with the increasing of Fe3O4 content. This revealed that the magnetic hydrogel microsphere could move directionally by introducing a magnetic field. Therefore, the goal of site-specific drug delivery of the magnetic beads loaded drug can be achieved by using an external magnetic field.

The magnetization of M1, M2, and M3 as a function of the magnetic field at 298 K is shown in Fig. 6. As can be seen, their magnetization increased with an increase of the magnetic field. This clearly shows that they exhibited an extremely small hysteresis loop and low coercivity, which demonstrated the superparamagnetism of the particles. The result indicated that the magnetic composite microspheres possessed good magnetic property and thus, showed promise in biomedical applications.