Preparation of graphene oxide–chitosan nanocapsules and their applications as carriers for drug delivery

Abstract

In this paper, graphene oxide (GO)–chitosan (Cs) nanocapsules [(GO/Cs)_x, x represents the number of GO/Cs bilayers] were prepared via layer-by-layer self-assembly method. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) images show that the (GO/Cs)_x nanocapsules have a hollow structure with a uniform diameter of 570 nm. The (GO/Cs)₅ nanocapsules were crosslinked with genipin. FESEM and TEM images indicated that the crosslinking process can improve the morphology and stability of the (GO/Cs)₅ nanocapsules in aqueous solution. Furthermore, the drug loading and release behaviors of ibuprofen using (GO/Cs)₅ and G(GO/Cs)₅ nanocapsules as carriers were investigated. The results show the G(GO/Cs)₅ nanocapsules exhibit a higher drug loading ratio and longer release time than the (GO/Cs)₅ nanocapsules.

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1. Introduction

At present, polyelectrolyte capsules have attracted wide interest for a variety of applications such as microreactors,¹ drug delivery^2,3 and optical biosensors.⁴ Several methods such as simple coacervation,⁵ complex coacervation,⁶ and emulsion polymerization,⁷ have been employed to assemble capsules. Among these methods, layer-by-layer (LbL) technology has attracted great attention by sequential deposition of oppositely charged materials onto substrates and/or templates. Since the pioneering work of Decher⁸ in 1997, the LbL method has been widely used to encapsulate various materials (for example, dyes,^9,10 cells,¹¹ aptamers¹² and trypsin¹³). The frequently-used shell materials of LbL microcapsules are synthetic polyelectrolytes such as polystyrene sulfonate (PSS) and polyallylamine hydrochloride (PAH).¹⁴

Graphene oxide (GO), a precursor of chemically converted graphene, has plenty of oxygen atoms on its basal plane and edge in the form of oxygen-containing groups such as epoxy, carboxyl and hydroxyl.¹⁵ These negative oxygen-containing groups can bind positively charged compound through electrostatic attraction. This unique molecular structure provides a promising lab bench for synthesizing, manipulating, and characterizing low-dimensional materials.¹⁶ GO is now very much attractive due to its unique properties such as good dispersion in water, excellent biocompatibility and prolonged blood circulation as compared to other carbon based nanomaterials.¹⁷ And its potential applications include electrochemical devices, energy storage, catalysis, adsorption of enzyme, cell imaging, drug delivery, biosensors¹⁸ and antibacterial paper.¹⁹

Chitosan (Cs), a low cost natural biomaterial, has aroused general interest because of its good biocompatibility, biodegradability and multiple functional groups. It has been applied in the different fields such as water treatment,²⁰ separation membrane,²¹ food package, tissue engineering and drug delivery.²²

With comparation to the traditional method of using two oppositely charged polymers in LbL assembly method, the GO–polymer capsules prepared by LbL assembly method have been reported rarely and we believe that GO in the capsules can yield unique properties, such as to prevent the breakage or deformation of capsules during the process of drug uptake and/or release. It should find a wide range of application in the fabrication of GO–polymer capsules via LbL assembly technology through its oxygen-containing function groups.

Continuous advances have been made for GO or graphene based capsules to improve the application in recent years. Zhang et al.²³ found the graphene microcapsules on the zeolite surface show slow-release characteristics as drug carrier. Kurapati et al.²⁴ fabricated the composite LbL capsules of GO–PAH using CaCO₃ particles as template. Hong et al.²⁵ used positively charged reduced GO (rGO–NH₃⁺) and negatively charged reduced GO (rGO–COO⁻) as shell materials for the layer-by-layer assembly onto polystyrene (PS) colloids. Chen et al.²⁶ identified the strong electrostatic interaction between GO and Cs in hydrogel system. With addition of GO, the chitosan-based composites display a significantly improvement in tensile strength and Young's modulus.²⁷ Moreover, the addition of GO into Cs matrix also improves the resistance to degradation in vitro.²⁸ Li et al.²⁸ identified the synergistic effect of hydrogen bonding and electrostatic interaction between GO and Cs. Fan et al.⁹ prepared nanoadsorbent by fabricating β-cyclodextrin–chitosan modified Fe₃O₄ onto GO nanosheets. Zuo et al.²⁹ prepared GO–Cs composites films. Singh et al.³⁰ employed GO–Cs composites for the development of DNA-based electrochemical biosensor for diagnosis of typhoid. The reported GO-based capsules still have many drawbacks, such as the irregular morphology and different sizes.

However, to date, nobody has fabricated GO–Cs nanocapsules. Here, in order to solve the aforementioned problem, we prepared (GO/Cs)_x nanocapsule by assembling GO sheets and Cs chains on SiO₂–NH₂ cores via LbL technique and examined the loading and release profiles of the (GO/Cs)₅ nanocapsule carriers using ibuprofen as model drug.

2. Experimental

2.1 Materials

Chitosan (Cs, 90% degree of deacetylation) and 3-aminopropyltriethoxysilane (APTES) were purchased from Aladdin Chemical Co. Ltd. Tetrathoxysilane (TEOS), graphite powder, ethanol, hydrogen peroxide (H₂O₂, 30.0%) and hydrofluoric acid (HF) were purchased from Sinopharm Chemical Reagent Co. Ltd. Genipin was purchased from Zhixin Bio-technology Co. Ltd (Jiangxi, China). Methylbenzene, sulphuric acid (H₂SO₄, 98.0%), potassium permanganate (KMnO₄, 99.9%), hydrochloric acid (HCl, 37.0%) and acetic acid (99.9%) were purchased from Lingfeng Chemical Reagent Co. Ltd (Shanghai, China). Sodium nitrate (NaNO₃) was purchased from Xilong Chemical Co. Ltd (Guangdong, China). Ibuprofen was purchased from Bright Chemical Co. Ltd (Wuhan, China). All reagents were of analytical grade without further purification.

2.2 Preparation of SiO₂–NH₂ nanospheres

Silica nanospheres were prepared by a modified Stöber method.³¹ The APTES-methylbenzene solution was obtained by mixing 8 mL APTES with 50 mL methylbenzene. The as-prepared SiO₂ (0.5 g) was dispersed into the before-mentioned APTES-methylbenzene solution by stirring for 30 min to get a homogeneous suspension. Then, the homogeneous suspension was kept at 60 °C for 24 h under stirring to ensure complete reaction of the silanization. The SiO₂–NH₂ nanospheres were dried at 60 °C overnight after filtered and washed twice with methylbenzene, ethanol and deionized water, respectively.

2.3 Preparation of GO

GO was prepared by a modified Hummers method.³² Briefly, 1 g graphite, and 46 mL H₂SO₄ were added in a 500 mL round bottom flask under agitation. Subsequently, 2.5 g NaNO₃ and 6 g KMnO₄ was successively added into the mixture under ice bath. Then the suspension was transferred to a water bath at 35 °C and stirred for 1 h. Subsequently, 92 mL deionized water was added gradually. Then, the temperature of this system was increased to 98 °C and the reaction was maintained at 98 °C for 15 min. Successively, 150 mL deionized water and 5 mL H₂O₂ solution (30%) were added dropwise into the mixture solution until the color changed from brown to bright yellow. The graphite oxide was collected from the mixture solution by filtration. The graphite oxide was washed by 5 wt% aqueous hydrochloric acid solution to remove the metal ions. The washing procedure was repeated with deionized water until the leaching solution was neutral. The graphene oxide (GO) was obtained by ultrasonication of the graphite oxide in deionized water for 30 min. Solid GO powders were obtained after filtration and freeze drying. To prepare the GO dispersion solution, 100 mg GO powders were dispersed in 50 mL deionized water and sonicated at room temperature for 10 min.

2.4 Preparation of (GO/Cs)_x nanocapsules

The suspension solution of SiO₂–NH₂ nanospheres (0.4 g) in 20 mL deionized water was added dropwise in 50 mL GO solution (2 mg mL⁻¹). The GO/SiO₂–NH₂ suspension solution was kept to be stirred for another 4 h to ensure to good dispersion of the solid materials. The suspension solution was centrifuged at 9000 rpm for 10 min and washed with deionized water. There must be at least two centrifugation-washing cycles so that the unadsorbed GO could be removed completely. Then, the SiO₂–NH₂@GO nanospheres dispersion solution (0.02 g mL⁻¹) was added into 50 mL chitosan solution (10 mg mL⁻¹). The GO layer and the near outside chitosan layer was defined as a GO/Cs layer. The SiO₂–NH₂@(GO/Cs)_x were formed after repeated this procedure, where the x represents the number the GO/Cs layer. The SiO₂–NH₂ cores of the SiO₂–NH₂@(GO/Cs)_x nanospheres were dissolved by 1 wt% HF solution at room temperature overnight (about 12 h), and then the hollow (GO/Cs)_x nanocapsules were obtained.

Genipin was used to crosslink the (GO/Cs)₅ nanocapsules. One milliliter genipin (5 mg mL⁻¹) was added into 20 mL SiO₂–(GO/Cs)₅ nanospheres dispersion solution (0.02 g mL⁻¹), stirred at 37 °C for 12 h. The SiO₂–NH₂ cores of the SiO₂–NH₂@G(GO/Cs)₅ nanospheres were dissolved by 1 wt% HF solution at room temperature overnight (about 12 h). All the preparation procedure of the (GO/Cs)₅ and G(GO/Cs)₅ nanocapsules were depicted in Fig. 1.

Fig. 1 Schematic of formation process of (GO/Cs)_x and G(GO/Cs)_x nanocapsules via LbL technology.

2.5 (GO/Cs)_x and G(GO/Cs)₅ as drug carrier

2.5.1 Drug uptake and release. One hundred milligrams (GO/Cs)_x or G(GO/Cs)₅ nanocapsules were dispersed in 10 mL IBU/hexane solution (30 mg mL⁻¹) under water bath chader (180 rpm, 37 °C) for given time. The solutions were covered with polyethylene film to prevent the evaporation of hexane. After centrifugation (7500 rpm, 15 min), 1 mL supernatant liquid was taken using plastic transfer pipette, and diluted 100 times with hexane. The remaining concentration of IBU after centrifugation was obtained through UV-vis spectrometry. The absorbance–concentration relation for IBU absorbance in hexane and IBU concentration in hexane was examined.

Then, 100 mg IBU loaded nanocapsules were dispersed in 100 mL simulated body fluid (SBF) solution stored a plastic bottle, and the plastic bottles were immersed into water bath chader (180 rpm, 37 °C) for given time. The 5 mL suspension solution was withdrawn using plastic transfer pipette, followed with centrifugation (9000 rpm, 15 min), and another 5 mL fresh SBF solution was added into the plastic bottle to keep the same volume of the solution. The release IBU concentration was determined by measuring UV-vis spectrometry.

2.5.2 Equations of drug uptake and drug release. Drug loading ratio was calculated as the following:

DLC = (C₀V₀ − C₁V₁)/m₀

where DLC is the drug loading ratio, C₀ and V₀ are the mass concentration and volume of the IBU initial solution, respectively; C₁ and V₁ are the mass concentration and volume of the supernatant IBU solution, respectively; and m₀ is the mass of drug carrier.

Encapsulation efficiency was calculated as the following:

DEC = (C₀V₀ − C₁V₁)/m₁

where DEC is drug encapsulation efficiency, C₀ and V₀ are the mass concentration and volume of the IBU initial solution, respectively; C₁ and V₁ are the mass concentration and volume of the supernatant IBU solution, respectively; and m₁ is the initial mass of IBU added.

Drug release ratio was calculated as the following:

DR_i = (C₁V₁ + C₂V₁ + ⋯ + C_iV₁)/(C₀V₀ − C₁V₁)

where DR is cumulative drug release ratio, C_i denotes the mass concentration of supernatant IBU liquid after centrifugation, V₁ denotes the volume taken out from solution, i denotes different time (i > 0), C₀ represents initial IBU mass concentration and V₀ represents initial volume.

2.6 Characterization of the nanocapsules

Field emission scanning electron microscopy (FESEM) images of the SiO₂–NH₂, SiO₂–NH₂@(GO/Cs)_x and SiO₂–NH₂@G(GO/Cs)₅ nanospheres were recorded with a Hitachi S-4800 scanning electron microscope at an acceleration voltage of 5.0 kV. The transmission electron microscopy (TEM) images were taken on a JEOL JEM-2010 transmission electron microscope at an accretion voltage of 200 kV. Fourier Transform Infrared Spectroscopy (FTIR) spectra were taken on a Nicolet-Nexus 670 FTIR spectrometer in the range from 500 to 4000 cm⁻¹ with a resolution of 4 cm⁻¹ using KBr method. The thermal gravimetric analysis (TGA) was carried out on Netzsch DSC204 differential scanning calorimetry from room temperature to 1000 °C under air atmosphere to confirm the relative amount of GO and Cs absorbed on the SiO₂–NH₂ nanospheres with a heating rate of 20 °C min⁻¹. UV spectra were taken on a Gold spectrumlab-54 UV-vis spectrometer in the range from 200 to 500 nm.

3. Results and discussion

3.1 Preparation of SiO₂–NH₂ nanospheres and GO sheets

Fig. 2A shows TEM image of the as-prepared SiO₂ nanospheres. The SiO₂ nanospheres prepared by the modified Stöber method have a diameter of around 480 nm and have good monodispersity. Negatively charged GO nanosheets, whose surface have a lot of oxygen-containing groups, cannot be immobilized on the negatively charged SiO₂ nanospheres. The positive amide groups should be grafted on the surface of the as-prepared SiO₂ nanospheres to form SiO₂–NH₂ nanospheres by soaking the as-prepared SiO₂ nanospheres into APTES-methylbenzene solution at 60 °C for 24 h. Then, GO nanosheets can be deposited on positively charged SiO₂–NH₂ nanospheres by electrostatic interaction.³³ Fig. 2B shows TEM image of the SiO₂–NH₂ nanospheres. The diameter and morphology of the SiO₂ nanospheres were not affected after modification with amide groups. Fig. 2C shows FTIR spectra of the as-prepared SiO₂ and SiO₂–NH₂ nanospheres. The intensity of the peak at 950 cm⁻¹, which belongs to stretching vibration of Si–OH, decreases obviously after modification. Moreover, the curve a in Fig. 5A is TGA curve of the SiO₂–NH₂ nanospheres. The weight loss in 400–600 °C further verifies that the SiO₂ nanospheres are modified by organic APTES. These evidences indicate that amino groups were successfully grafted on the as-prepared SiO₂ nanospheres.

Fig. 2 TEM images of the as-prepared SiO₂ (A) and SiO₂–NH₂ (B) nanospheres, FTIR spectra of the as-prepared SiO₂ (a) and SiO₂–NH₂ (b) nanospheres (C), and TEM (D) and AFM (E) images of GO nanosheet. The inset is digital image of the aqueous GO dispersion in a glass bottle.

The GO nanosheet is several hundred nanometers in size as revealed by TEM image (Fig. 2D). The thickness of GO nanosheet determined from the height profile of the AFM image (Fig. 2E) is about 1.3 nm, which is consistent with the previous result.³⁴ This indicates that our GO has single-layer structure. In FTIR spectrum (curve b in Fig. 5B), the bands at 1718 and 1060 cm⁻¹, respectively, are ascribed to the COOH and C–O groups from GO nanosheets; the bands at 1170 and 1230 cm⁻¹ are indicator of C–OH peak; the bands at 1389 and 1731 cm⁻¹ are ascribed to the –OH and C=O from the –COOH group, respectively. In view of the FTIR and TEM results, GO with oxygen-containing groups such as –OH and –COOH was successfully synthesized by the modified Hummer's method in our work.

3.2 Preparation of (GO/Cs)_x and G(GO/Cs)_x nanocapsules

The negative GO and positive Cs were used as wall materials of the (GO/Cs)_x nanocapsules, respectively. The Cs chain has –OH, –NH₂ and O=C–NH₂ groups.³⁵ These functional groups can form hydrogen bond and/or electrostatic interaction with the –COOH and –OH groups of GO nanosheets.³⁶ These interactions lead to the adsorption of Cs chains on the GO surface.

Fig. 3 shows FESEM and TEM images of the SiO₂–NH₂ nanospheres after dispersed in GO and Cs solutions alternately for several times. The SiO₂–NH₂ nanospheres are covered by sheet-like materials after soaked in GO and Cs solution alternately. For SiO₂–NH₂@(GO/Cs)₁ nanospheres prepared by soaking the SiO₂–NH₂ nanospheres into GO solution and Cs solution alternately for only one time, thin and little shell materials are adsorbed on the surface of the SiO₂–NH₂ nanospheres, and the GO/Cs coating cannot encapsulate the nanospheres completely (Fig. 3A). For SiO₂–NH₂@(GO/Cs)₃ nanospheres prepared by soaking the SiO₂–NH₂ nanospheres into GO and Cs solutions alternately for three times, the irregular structure on the SiO₂–NH₂ nanospheres are obviously observed, the GO/Cs coating encapsulates the nanospheres completely although the thickness of the outer shell (GO/Cs coating) is not uniform (Fig. 3B). For SiO₂–NH₂@(GO/Cs)₅ nanospheres prepared by soaking the SiO₂–NH₂ nanospheres into GO and Cs solutions alternately for five times, the thickness of outer shell (GO/Cs coating) becomes uniform (Fig. 3C). It is worth noting that the sheet-like structures (solid arrow shown in Fig. 3C1) appear on the surface of all the SiO₂–NH₂@(GO/Cs)_x nanospheres. Moreover, the diameters of the SiO₂–NH₂@(GO/Cs)_x nanospheres are about 480 nm, similar to the ones of the unencapsulated SiO₂–NH₂ nanospheres.

Fig. 3 FESEM images of SiO₂–(GO/Cs)₁ (A1), SiO₂–(GO/Cs)₃ (B1) and SiO₂–(GO/Cs)₅ (C1); TEM images of SiO₂–(GO/Cs)₁ (A2); SiO₂–(GO/Cs)₃ (B2); SiO₂–(GO/Cs)₅ (C2).

The uniform SiO₂ nanospheres were used as sacrificial templates for fabricating the capsules because SiO₂ nanospheres can be dissolved in the HF solution according to the following chemical reaction:

SiO₂ + 4HF = SiF₄ + 2H₂O

In order to obtain (GO/Cs)_x nanocapsules, the SiO₂–NH₂@(GO/Cs)_x nanospheres were soaked into 1 wt% HF solution overnight (about 12 h). After removal the SiO₂–NH₂ cores, the suspension solution became a little transparent solution. Then the nanocapsule solutions were diluted and observed by TEM. The TEM images are shown in Fig. 4. The hollow structures are broken in the (GO/Cs)₁ and (GO/Cs)₃ samples (Fig. 4A and B) because of their thin wall and swelling of Cs in aqueous solution. That is, the perfect nanocapsules were rarely prepared by soaking the templates into GO and Cs solutions, alternately, for no more than three times. Fortunately, the integrated nanocapsules were obtained in the (GO/Cs)₅ sample (Fig. 4C), although their morphology is not completely spherical.

Fig. 4 TEM images of the nanospheres and nanocapsules: SiO₂–NH₂@(GO/Cs)₁ (A1), (GO/Cs)₁ (A2) and their overview image (GO/Cs)₁ (A3); SiO₂–NH₂@(GO/Cs)₃ (B1), (GO/Cs)₃ (B2) and their overview image (GO/Cs)₃ (B3); SiO₂–NH₂@(GO/Cs)₅ (C1), (GO/Cs)₅ (C2) and their overview image (GO/Cs)₅ (C3); SiO₂–NH₂@G(GO/Cs)₅ (D1), G(GO/Cs)₅ (D2) and their overview image G(GO/Cs)₅ (D3).

Genipin was employed to crosslink the weak nanocapsules, because the crosslinked chitosan chains can tighten the wall, reduce the swelling ratio and slow down the degradation rate. As expected, the crosslinked SiO₂–NH₂@(GO/Cs)₅ nanospheres (Fig. 4D1) have a good spherical shape, compared with the uncrosslinked ones (Fig. 4C1). The genipin crosslinked (GO/Cs)₅ nanocapsules [G(GO/Cs)₅] have good integrated spherical morphology under TEM in the dry state (Fig. 4D2 and D3), which implies that the crosslinked nanocapsules have strong structure and can keep its spherical morphology in aqueous solution for long time. Hence, genipin increases the stability of the (GO/Cs)_x nanocapsules and avoids the collapse of the capsules stored in aqueous solution. Moreover, the morphological integration of G(GO/Cs)₅ nanocapsules has been improved obviously (Fig. 4D3). It is worthy to note that the G(GO/Cs)₅ nanocapsules with a diameter of 570 nm are bigger than the (GO/Cs)₅ nanocapsules.

FTIR spectra (Fig. 5B) were employed to particularly identify the interaction between GO and Cs. In the spectrum of Cs (curve a in Fig. 5B), there are two characteristic bands at 1650 and 1595 cm⁻¹, which correspond to the C=O stretching vibration of amide I and the N–H bending of –NH₂, respectively.²⁸ In the spectrum of GO (curve b in Fig. 5B), the bands at 1080, 1423, and 1634 cm⁻¹ correspond to C–O–C stretching vibrations, C–OH stretching, and the C–C stretching, respectively, while the bands at 1730 and 3280 cm⁻¹ correspond to C–O stretching vibration of –COOH groups and O–H stretching vibration, respectively.³⁷ In the spectrum of SiO₂–NH₂@G(GO/Cs)₅, the bands at 1730, 1596 and 1029 cm⁻¹ disappears. In consideration of the low magnitude of GO in SiO₂–NH₂@G(GO/Cs)₅, the possible reason accounting for this phenomena is the intensive and broad peak of Si–O–Si (at 1100 cm⁻¹) and –OH (at 1630 cm⁻¹). After removal of the SiO₂–NH₂ core, the spectrum of G(GO/Cs)₅ nanocapsules (curve c in Fig. 5B) presents a combination of characteristics similar to those of the Cs and GO. Not only the characteristic absorption peaks of SiO₂ at 808, 950 and 1100 cm⁻¹ disappear, but also the characteristic absorption peak of GO at 1718 cm⁻¹ and characteristic absorption peak of Cs at 1596 cm⁻¹ appears. The absence of absorption peaks of SiO₂ in G(GO/Cs)₅ nanocapsules means that silica templates were completely removed after soaked in 1 wt% HF solution for overnight.

Fig. 5 (A) TGA curves for SiO₂–NH₂ (a), SiO₂–NH₂@GO (b), SiO₂–NH₂@(GO/Cs)₁ (c), SiO₂–NH₂@(GO/Cs)₅ (d), and G(GO/Cs)₅ (e); (B) FTIR spectra of Cs (a), GO (b), G(GO/Cs)₅ (c) and SiO₂–NH₂@G(GO/Cs)₅ (d).

Comparing the TGA curves between SiO₂–NH₂ and SiO₂–NH₂@GO (Fig. 5A), we can clearly see weight loss of GO nanosheets is at 200–300 °C. There are three weight loss steps in TGA curves (Fig. 5A). The first weight loss at around 100 °C is attributed to water evaporation. The second one at around 200 °C corresponds to the decomposition of GO.³⁸ The third weight loss at around 300 °C corresponds to the decomposition of Cs.³⁹ After calcinations, the remnant of 72 wt% of the core–shell nanospheres indicates that the SiO₂ core is the main constitution of the SiO₂–NH₂@(GO/Cs)₅ nanospheres. So the amount of GO and Cs assembled on the surface of the SiO₂–NH₂ templates is about 20 wt%. The G(GO/Cs)₅ nanocapsules were completely burnt out at 600 °C. This indicates that the SiO₂ cores were totally removed in G(GO/Cs)₅ nanocapsules after soaked in 1 wt% HF solution for overnight.

3.3 IBU uptake and release

To verify the feasibility of G(GO/Cs)₅ and (GO/Cs)₅ nanocapsules as drug carrier, IBU was selected as model drug. We compare the drug loading and release profiles between G(GO/Cs)₅ and (GO/Cs)₅. The drug loading results (Fig. 6A) shows that the loading ratio of IBU on SiO₂–NH₂@GO, SiO₂–NH₂@(GO/Cs)₅, SiO₂–NH₂@G(GO/Cs)₅ and (GO/Cs)₅ and G(GO/Cs)₅ are 28%, 65%, 56%, 77% and 78%, respectively. Encapsulation of the SiO₂–NH₂ cores can improve the drug loading obviously. Moreover, more drugs can be loaded in (GO/Cs)₅ and G(GO/Cs)₅ nanocapsules because of hollow structure.

Fig. 6 Drug loading (A) and release profiles (B and C) of IBU using SiO₂–NH₂@GO, SiO₂–NH₂@ (GO/Cs)₅, SiO₂–NH₂@G(GO/Cs)₅, (GO/Cs)₅ and G(GO/Cs)₅ as drug carrier (DEC: encapsulation efficiency, DLC: drug loading).

Fig. 6B and C show the curve of drug release profile of the SiO₂–NH₂@GO, SiO₂–NH₂@(GO/Cs)₅, SiO₂–NH₂@G(GO/Cs)₅, (GO/Cs)₅ and G(GO/Cs)₅ carriers. At the beginning of 1 h, drug release of the SiO₂–NH₂@GO nanospheres reaches to about 57%, while those in the other four curves (SiO₂–NH₂@(GO/Cs)₅, SiO₂–NH₂@G(GO/Cs)₅, (GO/Cs)₅ and G(GO/Cs)₅) reaches about 51%, 48%, 36% and 37%, respectively (Fig. 6C). The initial fast-release may be associated with two factors:⁴⁰ (1) the drug concentration needs to reach a concentration equilibrium; (2) the very outside layer of drug molecules interact more weekly with the composite surface and is easier to be dissolved into SBF. After 10 h, the drug release of SiO₂–NH₂@GO nanospheres reaches release balance and almost 100% drugs have been released. At the same time, the drug release ratios of the SiO₂–NH₂@G(GO/Cs)₅ nanospheres, SiO₂–NH₂@G(GO/Cs)₅ nanospheres, (GO/Cs)₅ nanocapsules and G(GO/Cs)₅ nanocapsules are more less than 100%. The drug release rate of G(GO/Cs)₅ nanocapsules is the slowest and the drug release time of G(GO/Cs)₅ is 100 h when all drugs were released. GO and Cs on the SiO₂–NH₂ nanospheres (encapsulation) can slow the release of IBU drug. The GO nanosheets in Cs matrix can lower the diffusion of IBU from the capsule wall. Moreover, crosslinking by genipin can prevent swelling of Cs matrix in SBF solution and decrease permeation of IBU through capsule wall during drug release in SBF solution. These two factors leads to longer release time of IBU in G(GO/Cs)₅ nanocapsules. That is, G(GO/Cs)₅ nanocapsules have a relative high drug loading ratio and longer drug release time than (GO/Cs)₅ nanocapsules.

Our (GO/Cs)_x and G(GO/Cs)₅ nanocapsules/nanospheres have potential application as drug carriers in human body. However, their biocompatibility and toxicity need to be examined in biological environment and/or in vivo. Fortunately, the raw materials (GO and Cs) used in our work are biocompatible and nontoxic.^41,42 Although, the biological properties of the nanocapsules/nanospheres will be investigated in the future study.

4. Conclusions

In conclusion, negatively charged GO was successfully employed to prepared (GO/Cs)_x and G(GO/Cs)₅ nanocapsules by LBL assembly of different bilayers of GO and Cs on the SiO₂–NH₂ cores. The SiO₂–NH₂ cores were decomposed in HF solutions at pH < 1. G(GO/Cs)_x nanocapsules with size around 570 nm. The drug IBU was loaded into (GO/Cs)₅ and G(GO/Cs)₅ nanocapsules. We found the use of crosslinking agent (genipin) can slightly increase drug loading ratio and help to improve release time. The loading ratio of IBU and the release time of G(GO/Cs)₅ nanocapsules are 78% and about 100 h, respectively. The G(GO/Cs)₅ nanocapsules have potential to apply as carriers in nanomedcine.

Acknowledgements

This work was supported by Nature Science Foundation of China (No. 50802042), Nature Science Foundation of Jiangsu province (No. BK2011076) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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