Exploring the potential of GO-based composite hydrogels and their swelling property for controlled drug delivery

. Swelling studies are essential for hydrogels with potential applications in biomedical areas, as the materials will be exposed to biological fluids. This study obtained composite hydrogels by physically cross-linked carboxymethyl cellulose (CMC) with GO. CMC is known to be non-toxic, non-allergenic, and possesses good biodegradability. To produce GO, a ‘greener’ modified Hummers’ method was first emplo yed by removing the use of sodium nitrate in the oxidation process to avoid the generation of toxic NOx gases. Iron (III) chloride was then used as a cross-linker in composite preparation. The responses of GO-CMC hydrogel networks to various solvents and temperatures were studied by measuring their swelling property. The solvents included water, salt solution, ethanol, hexane, and phosphate buffer solutions with various pH (pH 2.1, 5.0, and 7.4). The effect of temperature on swelling was studied at temperatures of 25, 35, and 45 o C. Results showed that the presence of GO within CMC matrixes altered the structures and properties whilst enhancing the swelling property compared to its native CMC hydrogel, at studied temperatures. It was also observed that the swelling property of GO-CMC composite hydrogels depended significantly on the pH of the environment, a great attribute for drug carriers with pH-sensitive behavior..


Introduction
Graphene oxide (GO) is a single atomic layer sheet created from graphite via oxidation and exfoliation, frequently using potassium permanganate in concentrated sulfuric acid [1,2].It can also be derived from graphene through the introduction of covalent C-O bonds [3].GO has been studied for decades, as early as 1859 by a British chemist B.C. Brodie.Since then, many studies have been reported on the development of GO formation as summarized by Dreyer et al. [1].These include the Brodie method, the Staudenmaier method, and the most well-known to date Hummers' method which combines potassium permanganate and sulfuric acid.
In Hummers' method, many carbon atoms are transformed into C-O bonds, as hydroxyl and epoxy groups, and C=O bonds as carbonyl and carboxyl groups [4].During oxidation, the sodium nitrate (NaNO3) acts as a reaction catalyst.The mechanism involves three steps as reported by Dimiev and Tour [3] and Gupta et al. [5].Graphite is first transformed into sulfuric acid-graphite intercalation compound (H2SO4-GIC) before being transformed into pristine graphite oxide (PGO).Following the transformation, the PGO is converted to GO by water [3,5].However, to obtain relatively uniform GO products, a reproducible synthesis method is required.
Studies have been conducted on GO's oxygen-containing groups and structure.In principle, the hydroxyl and epoxy groups are located on the basal planes, while the carboxyl and carbonyl groups are located at the edges of GO sheets [6,7].The distribution of these groups varies depending on the used method and is associated with the GO oxidation degree.However, the information on this matter is still limited.This information is essential to better understand the formation mechanism of the surface groups of GO [6].Another issue worth considering is the presence of amorphous and berthollide compounds on the GO structure [1].
Oxygen-containing functional groups on GO provide good hydrophilicity, dispersity, and biocompatibility.Therefore, GO has been used for wide applications such as polymer composites, energy-related materials, sensors, and biomedical applications [1].GO sheets have large surface areas on both sides which is useful for physical adsorption through π-π stacking [8].Studies have also reported that GO has low toxicity, a mandatory attribute for biomedical applications.Combining these with the anionic-exchange properties, GO is a great candidate for cationic drug carriers in drug delivery systems [9].
Biopolymers present good biocompatibility, but their mechanical properties are often poor [10].Combination of GO with biopolymers such as CMC [9,10], chitosan and its derivatives [8,11,12], alginate [10,13], and gelatin [14] has been of great interest because it may improve solubility and bioavailability of GO, tailor temperature-and pH-responsive function, and mechanical properties.However, the characteristics of the resultant materials varied depending on the methods employed.
Among various biopolymers, chitosan (CS) and its derivatives such as carboxymethyl chitosan have been the most widely studied to prepare functional hydrogels for biomedical applications due to their biocompatibility, low-cost, and non-toxic [8,12].CS is insoluble in water, organic solvents, and aqueous bases, while it is soluble after stirring in acids such as acetic, nitric, hydrochloric, perchloric, and phosphoric acids.The incorporation of GO into CS matrix is thus challenging.Compared to CS, carboxymethyl chitosan may be an option since it has better water solubility and can be applied in wider biomedical systems [8].Due to the ease of the formation process and uniformity of product size and shape, gel beads CS hydrogels have been greatly considered as drug carriers with controlled-release system [15].
CMC is a biopolymer with multiple carboxyl groups.It can form hydrogels in the presence of metal ions by simple coordination between metal ions and the polymer carboxyl groups [9].CMC is known to be non-toxic, non-allergenic, and possesses good biodegradability [10].CMC may be composited with GO making a hydrogel nanocomposite bead by physically cross-linked CMC with iron(III) chloride hexahydrate (FeCl3•6H2O).Results showed that the nanocomposite beads can perform complete release of drugs in the acidic media (pH 6.8).Therefore, CMC can be used as a polymer matrix that protects GO and drugs passing through the gastrointestinal tract with controlled-release properties [9].
Swelling studies on hydrogels for biomedical applications are important, as the hydrogels will be exposed to biological fluids.The swelling ability of hydrogels depends on composition and environment.Some hydrogels are temperature-dependent, while others are pH-dependent.Wan et al. [16] stated that swelling behavior can be used to predict the drugrelease behavior of a hydrophilic matrix.An inverse relationship between the drug release rate and matrix swelling rate of hydroxypropyl methylcellulose (HPMC) materials was reported.However, inverse swelling trends were observed in the other materials.More studies are thus needed to give a better understanding of the swelling behavior of composite hydrogels before studying their application as drug carriers.
The objectives of this study were therefore (1) to synthesize GO-CMC composite hydrogels using a modest and eco-friendly approach, and (2) to investigate the swelling behavior of the resultant composite hydrogels in various solvents and temperatures.GO was firstly prepared by 'greener' Hummers' method with minor modifications.To identify the presence of certain functional groups on GO and its derived composite hydrogels, FT-IR analyses were performed.Thermal stability of GO-CMC composite hydrogels was also investigated.

Materials
All chemicals used in this work, such as graphite powder (<20 μm), potassium permanganate, hydrogen peroxide, sodium carboxymethyl cellulose (of medium viscosity), and iron(III) chloride, were of analytical grade and purchased from Sigma-Aldrich (St. Louis, Missouri, USA).Sulfuric acid (98%) was purchased from Scharlau (Sentmenat, Spain).These chemicals were used without further purification.Potassium bromide for FT-IR analysis was also purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Preparation of GO
GO was prepared from graphite powder with oxidation and exfoliation processes according to modified Hummers' method described by Singh et al. [17].A concentrated H2SO4 and 1 to 4 ratio of graphite powder and KMnO4 were used in the experiments.The mixture was stirred for 24 h at room temperature.Fifty milliliters of deionized water (DI) were then added to the mixture.To stop the reaction, hydrogen peroxide solution (30%) was added dropwise until the mixture color turned yellowish-brown (foaming gold) -Fig.1a.After repetition of centrifugation to remove the unreacted graphite oxide, a brown-colored GO paste (Fig. 1b) was obtained.The paste was washed thoroughly with DI water to neutralize its pH and then freeze-dried for 24 h before storage.A freshly prepared GO dispersion was obtained by dispersing the freeze-dried GO in DI water followed by ultrasonic exfoliation in an ultrasonic bath.

Preparation of GO-CMC composite hydrogels
GO-CMC composite hydrogels were prepared using the following steps.A certain amount of CMC was dissolved in the as-prepared GO dispersion.The solutions were added dropwisely using a syringe into 0.2 M FeCl3 solution as the cross-linking agent.The formed beads were then allowed to crosslink with Fe 3+ in FeCl3 solution.After being left for 20 minutes, the beads were filtered and washed with DI water several times to remove unreacted FeCl3 and neutralize the pH of the beads.Finally, the beads were oven-dried for 24 h at 40•C.For comparison, CMC hydrogel beads were also prepared similarly without the use of GO dispersion.ImageJ software was employed to analyse the size of the beads, before and after drying, taken from digital pictures.

Characterizations
Several characterizations were carried out on the prepared GO and GO-CMC composite hydrogels.These include FT-IR and TGA analyses.FT-IR spectra of all samples were recorded in the scanning range of 4000-400 cm -1 using a Bio-Rad FTS-3500 (Bio-Rad Laboratories Inc., USA).The KBr method was applied to all FT-IR analyses.The thermal stability of the prepared materials was analyzed using Diamond TG/DTA (Perkin Elmer, USA) in a highly purified nitrogen atmosphere and a programming temperature range from 30 to 750°C with a heating rate of 10°C/min.Samples were weighed at approximately 3 to 4 mg and placed in a platinum crucible pan for every analysis.

Swelling behavior analysis
To study the effect of different environments on GO-CMC composite hydrogels, the swelling behavior of the prepared composites was studied in various solvents and at various temperatures.The equilibrium swelling (ES) of the prepared composites was determined in phosphate buffer solutions, DI water, and sodium chloride solution.The water equilibrium swelling of the prepared composites was studied at temperatures of 25, 35, and 45 o C.
In general, the prepared composite hydrogels were immersed in the designated solutions at a certain temperature until equilibrium.The ES of composite hydrogels was then determined according to Equation (1) [9].
where W1 is the initial weight of dried hydrogels, and W2 is the weight of the corresponding hydrogels after being immersed in a medium for 48 h.

Preparation and characterization of GO and GO-CMC composite hydrogels
In the preparation of GO from graphite powder, washing, and centrifugation steps were very crucial.These steps dictated the pH of the obtained GO paste and affected the functional groups on GO, as shown in the FT-IR spectra of Fig. 2. It was confirmed that graphite did not have any oxygen-functional groups, whilst GOs had these groups.These oxygenfunctional groups were created due to the harsh oxidation treatment of graphite during preparation.The presence of oxygen-functional groups on GO provided properties that potential for diverse applications [1].The crosslinking process of GO dispersion and CMC was successfully carried out physically using Fe 3+ in solution.Using ImageJ software, the obtained beads of GO-CMC composite hydrogels (Fig. 3) were known to have average diameters of about 0.250 + 0.01 cm and 0.085 + 0.01 cm, before and after being dried respectively.Due to drying, the amount of water being removed from the beads was approximately 94%.The incorporation of CMC into the GO solution with Fe 3+ ion as the crosslinking agent has changed the structure and content of functional groups on GO-CMC composite hydrogels compared to the parent materials, i.e.GO and CMC.The alteration was clearly visible in Figure 4 and proved the successful formation of GO-CMC composite hydrogels.Peaks of 3420 and 1608 cm -1 were detected, which were attributed to the characteristics of CMC, namely hydroxyl stretching and carboxylate bending modes, respectively.Moreover, the adsorption bands at 2921 and 1134 cm -1 related to C-H stretching and bending modes, respectively were also observed [9,18].In general, hydrogels are porous materials with high absorption capacity of liquid molecules such as water within its matrix.TGA analysis is often used to reveal the water content and thermal stability of hydrogels.The TGA thermograms of the resultant composite hydrogels exhibited a multistage degradation curve (Fig. 5), which agreed with Santoso et al. [19].The thermograms first began to degrade at about 100 o C, attributed to water evaporation.The second degradation started at about 200 o C and lasted until about 300 o C when the thermal decomposition of hydrogel components was completed.

Swelling studies of GO-CMC composite hydrogels
Water equilibrium (WE) swellings of GO-CMC and CMC at different temperatures (25 to 45 o C) were shown in Fig. 6.WE swelling of GO-CMC at 35 o C showed the highest values even though the difference was not significant to that of 25 o C and 45 o C. Given the potential exposure to biological fluids (typically at 35-37oC), swelling studies on hydrogels must be conducted before application in the biomedical field.Jalalvandi et al. [20] mentioned that the extent of swelling depends on factors such as polymer nature, rigidity of its chains, crosslinking degree, and temperature and pH conditions.Rasoulzadeh and Namazi [9] explained that with increasing pH, the electrostatic repulsion increased.The carboxyl groups on the CMC/GO chain were converted into negatively charged carboxylate ions.Thus, water penetrated, and the swelling rate increased.Results shown in Fig. 7 agreed with this statement.GO-CMC hydrogel beads had lower swelling ratio in DI water and salt solution (within the range of about 2%-10%), compared to that on phosphate buffer solution with pH 6.8 and 7.4 (above 350% or 10 folds higher).

Conclusion
GO and GO-CMC composite hydrogels were prepared using a simpler and "greener" approach, a modified Hummers' method without using sodium nitrate catalyst.The resultant composite hydrogels showed promising application as materials with temperature-and pHsensitive behaviors.The presence of GO within CMC matrixes altered the structures and properties whilst also enhancing the swelling behavior compared to its native CMC beads, especially at a temperature of 35 o C.Moreover, the swelling behavior of GO-CMC composite hydrogels were significantly influenced by the pH of the environment.Further studies shall thus be carried out to explore the potential application of the obtained composite hydrogels as drug carriers.
The authors would like to thank LPPM Widya Mandala Surabaya Catholic University for funding this research.

Fig. 7 .
Fig. 7. Swelling of GO-CMC composite hydrogel in various mediums at a temperature of 35 o C.