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Controlled release of ofloxacin from chitosan–montmorillonite hydrogel
Applied Clay Science 50 (2010) 112–117
Contents lists available at ScienceDirect
Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l a y
Controlled release of ofloxacin from chitosan–montmorillonite hydrogel Shuibo Hua a,b, Huixia Yang a,b, Wenbo Wang a,b, Aiqin Wang a,⁎ a b
Center for Eco-material and Green Chemistry, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 11 August 2009 Received in revised form 7 July 2010 Accepted 12 July 2010 Available online 23 July 2010 Keywords: Chitosan Montmorillonite Nanocomposite Drug release Ofloxacin
a b s t r a c t A series of ofloxacin/montmorillonite/chitosan (OFL/MMT/CTS) hydrogels were prepared by solution intercalation and ionic crosslinking with sodium tripolyphosphate (TPP). The structure and drug loading of the beads were characterized by Fourier transform infrared spectroscopy (FTIR). The formation of intercalated nanocomposites was confirmed by X-ray diffraction (XRD). The swelling and degradation of the beads were influenced by the pH of test medium and the MMT content. Compared to pure CTS beads, the incorporation of MMT enhanced the drug entrapment, improved the swelling behavior and reduced the drug release. The CTS beads without MMT dissolved after a 3 h immersion in pH 1.2 solution. No notable disintegration was visually observed for MMT-containing beads even after a 10 h immersion. The observations suggested that the electrostatic interaction between CTS and MMT enhanced the stability of the beads and showed good potential for the use as drug carriers for sustained release. © 2010 Published by Elsevier B.V.
1. Introduction Drug delivery systems recently received great interests because they realized the effective and targeted delivery of drug and minimized the side effects resulting from the traditional drug-dosage form in the pharmaceutical field. A large number of drug delivery systems have been conceived and developed (Ding et al., 2002; Li et al., 2004; Ginebra et al., 2006; Malmsten, 2006; Tomonori and Hiroshi, 2008; Taa et al., 2009). Clay mineral composites as known drug delivery systems showed unique hybrid properties superior to the components and the capability to incorporate various drug substances (Byrne and Deasy, 2005; Aguzzi et al., 2007; Lin et al., 2007). MMT exhibits enhanced gel strength, mucoadhesive capability to cross the gastrointestinal (GI) barrier and adsorb bacterial and metabolic toxins such as steroidal metabolites (Herrera et al., 2000). MMT is a common ingredient as both the excipient and active substance in pharmaceutical products (Takahashi et al., 2005). Cationic drugs can be intercalated into MMT by ion exchange (Zheng et al., 2007; Jung et al., 2008; Pongjanyakul et al., 2009). The regulation of the amount and rate of drug release is difficult when the drug is intercalated by ion exchange. Chitosan (CTS) is an amine-bearing, linear polysaccharide derived from the N-deacetylation and depolymerization of chitin (i.e., poly-Nacetyl-glucosamine) (Agnihotri et al., 2004). As a unique cationic polysaccharide, CTS shows gel and film forming properties and found
⁎ Corresponding author. Tel.: + 86 931 4968118; fax: + 86 931 8277088. E-mail address: [email protected] (A. Wang). 0169-1317/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.clay.2010.07.012
potential in the pharmaceutical industry as a drug delivery system (Thanou et al., 2000; Dang and Leong, 2006). CTS can form nanocomposites with MMT, and there are several reports about the drug release behavior from the CTS/MMT nanocomposite films, scaffolds and nanoparticles (Wang et al., 2008; Depan et al., 2009). From these points of view, the combination of MMT with CTS beads as a drug carrier is attractive. In our study, ofloxacin (Fig. 1), the fluoroquinolone antibiotic, commonly used for treatment of prophylaxis of a variety of bacterial diseases, was tested as the cationic model drug in in vitro experiments. OFL/MMT nanocomposites were prepared by the solution intercalation technique. Without filtering out the free drug, OFL/MMT intercalated CTS beads were obtained through ionic crosslinking and their morphology and structure were characterized by scanning electron microscopy (SEM), FTIR and XRD. Finally, swelling and in vitro drug release were studied in simulated gastric fluids (SGF, pH 1.2) and simulated intestinal fluids (SIF, pH 7.4). 2. Materials and methods 2.1. Materials Chitosan (CTS, deacetylation degree, 81%; mass average molar mass, 9 × 105) from shrimp shell was purchased from Yuhuan Ocean Biochemical Co. (Taizhou, China). Ofloxacin (OFL) was obtained from Kunshan Double-crane Pharmaceutical Co., Ltd, China. High purity montmorillonite (MMT, the reference substance for scientific research; the content of montmorillonite is 99% determined by XRD technology) was purchased from Zhejiang Sanding Technology Co., Ltd (Shaoxing, China), and the contents of SiO2 and Al2O3 are
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aqueous solution containing 4.0 g ammonium acetate and 7.0 g sodium perchlorate were adjusted to pH 2.2 with phosphoric acid. The drug content was determined by comparing with the standard curve of OFL which was achieved from OFL solutions in PBS 7.4 with concentration from 0.002 to 0.01 g/L. The drug entrapment efficiency (EE) is expressed as follows: EE = ðPractical drug loading = Theoretical drug loadingÞ × 100% ð1Þ Fig. 1. Structure of OFL.
2.4. Swelling studies 59.48 mass% and 21.93 mass%, respectively. Tripolyphosphate (TPP) was purchased from Sinopharm Chemical Reagent Co., Ltd, China. Simulated gastric fluid (SGF, pH 1.2) containing 21.25 mL HCl, 11.18 g KCl in 3000 mL distilled water and simulated intestinal fluid (SIF, phosphate buffer solutions, PBS, pH 7.4), containing 20.4 g K2HPO4 and 4.8 g NaOH in 3000 mL distilled water were prepared as described in US Pharmacopoeia 30. All other chemicals were of analytical grade and used as received. 2.2. Preparation of OFL/MMT/CTS hydrogel beads The hydrogel beads were prepared according to an improved technique using TPP as the gelling counterion (Sezer and Akbuga, 1995). A series of the samples with different amounts of MMT were prepared according to the following procedure. 0.2 g MMT was dispersed in 20 mL of 2 mass% acetic acid solution, then 0.20 g OFL were added into the dispersion, followed by a continuous stirring at room temperature for 1 h. CTS (0.60 g) was dissolved in the OFL/MMT mixture to reach the final concentration of 3% (mass/v) and stirred for 0.5 h. The beads were formed by dropping the bubble-free solution or dispersion through a disposable syringe onto a gently agitated TPP solution (50 mL, 5 mass%; pH 5.0, adjusted with standard 1 mol/L HCl solution). After 0.5 h, the obtained beads were filtrated with a 100mesh stainless screen and rinsed with distilled water for three times. Finally, the gel-like beads were air-dried for 24 h, followed by drying at 70 °C for 6 h. Similar procedures were used to prepare OFL-free placebo beads. The beads corresponded to 0, 0.10, 0.20, 0.30, 0.40 and 0.50 g MMT were denoted as M0, M1, M2, M3, M4 and M5. The M0 and M4 beads without adding OFL were termed as M0C and M4C (Table 1). 2.3. Entrapment efficiency The amount of OFL entrapped in the beads was calculated by measuring the content of OFL in the gelling medium. The floating small MMT particles were removed through a 0.45 μm membrane filter. The clear superficial solution was analyzed by HPLC (WatersTM 600 Pump, 2998 Photodiode Array Detector) using the C18 column. The mixture of acetonitrile and buffer solutions (25:75, v/v) was used as the mobile phase. OFL was detected at the wavelength of 294 nm. The buffer solution was prepared according to USP 30: 1300 mL of an Table 1 Feed composition and drug content of the samples. Code
CTS (g)
OFL (g)
MMT (g)
OFL content (mass%)
M0 M1 M2 M3 M4 M5 M0C M4C
0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6
0.2 0.2 0.2 0.2 0.2 0.2 0 0
0 0.1 0.2 0.3 0.4 0.5 0 0.4
7.81 10.00 10.92 12.02 12.74 12.83 – –
Swelling studies and in vitro degradation of the beads were carried out in two aqueous media: 0.1 mol/L HCl solution (pH 1.2) as simulated gastric fluid and phosphate buffer solutions (pH 7.4) as simulated intestinal fluid. Beads (100 mg) were placed at 37 °C in the dissolution test apparatus (ZRS-8G, Tianjing University Wireless factory, China) containing 500 mL of the medium at 50 rpm. At regular intervals, the beads were reweighed after carefully wiping off excess liquid with a tissue paper. The mass change of the wet beads Qw and of the dry beads Q d with respect to time was determined as follows: Q w = ðWw –W0 Þ = W0
ð2Þ
Q d = ðWd –W0 Þ = W0
ð3Þ
where Ww and Wd are the wet and dry mass of the beads at time t, respectively; Wo is the initial mass of beads. 2.5. In vitro drug release The in vitro drug release tests were carried out using the USP 30 NO.2 dissolution test apparatus fixed with six rotating paddles. The release medium is a simulated intestinal fluid (pH 7.4) or simulated gastric fluid (pH 1.2) (500 mL, 37.5 ± 0.5 °C), and the speed of rotation was 50 ± 1 rpm. 50 mg samples were placed into each of the six cups, and 5 mL solution was collected from the release medium at regular intervals. After each sample collection, the equal amount of fresh release medium at the same temperature was added. The amount of drug released was monitored by a UV–vis spectrophotometer (SPECORD 200, Analytik Jera AG) at 294 nm based on the established UV standard absorbance curve for OFL. In the concentration range (2.5 ~ 12.5 × 10−5 mol/L) investigated, the UV absorbance obeyed the Beer's law. 2.6. Characterization FTIR spectra were recorded on a FTIR spectrophotometer (Thermo Nicolet, NEXUS, TM) in the range of 4000–400 cm−1 using KBr pellets. The surface morphology was observed by scanning electron microscopy (JSM-5600LV, JEOL) after coating the samples with gold film using an acceleration voltage of 20 kV. Powder XRD analyses were performed using a diffractometer with Cu anode (PAN analytical X'pert PRO), running at 40 kV and 30 mA, scanning from 3° to 40° at 3°/min. 2.7. Statistical analysis Each experiment was carried out in triplicate and the results were averaged. The influence of MMT content on the entrapment and the release of the drug from beads were statistically analyzed by one-way ANOVA. The data were considered to be significantly different at p b 0.05.
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electrostatic interaction between OFL and MMT. The FTIR results confirmed that CTS was crosslinked with TPP in the beads and OFL was encapsulated in the beads or stabilized in the interlayer space of MMT.
3. Results and discussion 3.1. Morphology of the beads When the dispersion of CTS in acetic acid was dropped into TPP solution, gelled spheres formed instantaneously due to the electrostatic interaction between positively charged CTS and negatively charged TPP. Generally, the wet beads were spherical with a diameter of about 5 mm and possessed a smooth surface. After air drying, the diameter of test beads decreased to about 2 mm but still kept the spherical shape. For any batches of nanocomposite beads with various amounts of MMT, there was no obvious variation of the bead size. The large size of wet beads suggested high swelling and water retention ability. The surface morphology showed severe wrinkles, which was caused by partial collapsing of the polymer network during drying (Fig. 2). Compared to placebo M0C beads, little particles appeared on the surface of the OFL-loaded beads (M0) due to the leakage of OFL crystals from the network space of beads. However, the OFL crystals disappeared with increasing MMT content, suggesting that the loading of OFL was remarkably improved due to the incorporation of MMT. 3.2. FTIR analysis The intense characteristic bands of CTS at 1651 and 1602 cm−1 (Fig. 3) were ascribed to the absorptions of amide I and amide II (Papadimitriou et al., 2008), which shifted to 1640 cm− 1 and 1557 cm− 1 after the formation of CTS beads with TPP addition. This information indicated that the amide formed ionic bonds. This interaction reduced the solubility of CTS and contributed to the separation of CTS from the solution in the form of beads. The absorption band of MMT at 3623 cm− 1 (stretching vibration of the interlayer –OH groups) almost disappeared and the band at 1714 cm− 1 (characteristic absorption of the C = O group of OFL) was weakened in the spectra of the beads (M4). This result indicated
3.3. XRD analysis The reflection of CTS at around 20° (Fig. 4) was characteristic of the hydrated crystalline structure (Liu et al., 2008), but its intensity was largely weakened after forming the beads. Crystalline CTS was not observed. OFL showed characteristic intense reflections at 6° and 10.9° due to the presence of OFL crystals. These reflections disappeared in the pattern of M4, which indicated that OFL was dispersed at the molecular level in the polymer matrix or intercalated for organic-nanoclay hybrid systems (Zheng et al., 2007; Depan et al., 2009) and no crystals were found in the drug-loaded matrices. The XRD pattern of MMT showed the basal reflection at about 6.68° with a basal spacing of 1.32 nm. The basal spacing increased from 1.32 nm to 2.14 nm after OFL addition indicating the intercalation of OFL. However, due to its coiled or helicoidal structures, CTS was only adsorbed on the surface of MMT or led to the very less intercalation to MMT as the basal spacing only increased from 1.32 nm to 1.36 nm (M4C). This result was in agreement with a previous study (Günister et al., 2007). 3.4. Entrapment efficiency The EE of the beads increased with the amount of MMT added (Fig. 5). The entrapment of OFL in beads without MMT (M0) was 31.2, whereas the corresponding entrapment in the MMT-containing beads varied from 45.3 to 83.5. A comparison of EE with the different MMT contents was evaluated by ANOVA and the F value was 906.41 (df = 17, p = 0). Thus, the incorporation of MMT in the beads very effectively improved the entrapment of OFL.
Fig. 2. SEM images of (a) M0C, (b) M0, (c) M2, and (d) M4.
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Fig. 3. FTIR spectra of CTS, OFL, MMT, M0C and M4 beads.
In order to simulate the possible effect of pH on drug release rate, a swelling study was conducted in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) at physiological temperature of 37 °C (Fig. 6). The swelling ratio at pH 7.4 was very small (about 30%). At pH 1.2, with increasing the MMT content, the initial swelling ratio decreased (b1.2 h), but the swelling ratio within 10 h increased significantly (p b 0.05). The mass of M0 beads increased by 4.68 fold
for the initial 1 h, and then was sharply reduced until the beads were disintegrated for the subsequent 1 h immersion. However, the mass of M2 beads increased by 6 fold for the first 4 h, and then reduced to 4.3 times of their initial values for the subsequent 6 h immersion. For swelling of M4 beads, the mass of M4 beads increased by 12 fold within 10 h. A greater extent of crosslinking of the beads restricted the mobility of the polymer chains and thus limited the swelling. The swelling was mainly influenced by ionic interactions between CTS chains and MMT interlayers, which depended on the crosslinking density. The incorporation of MMT strengthened the network of prepared beads. Liu et al. (2008) studied the drug release from CTS–MMT hydrogels by electrostimulation, and considered that exfoliated MMT layers can act as crosslinkers between CTS and MMT (Liu et al., 2008). In our study, some MMT layers could also act as crosslinker between the CTS chains. However, in contrast to decreased swelling by increasing MMT content as reported by Liu et al. (2008), a greater swelling at pH 1.2 for 10 h incubation was obtained, probably because the crosslinking between CTS and TPP was broken. Meanwhile, electrostatic repulsion occurred between protonated amine groups on CTS causing higher water uptake as in the simulated gastric fluid. However, no such repulsion was effective in the simulated intestinal fluid, which caused only a small swelling at pH 7.4. If the crosslinking density became too small, interactions were no longer strong enough to avoid disintegration and the beads decomposed completely within 3 h (M0).
Fig. 4. XRD patterns of OFL, MMT, CTS, M0C, M4 and M4C beads.
Fig. 5. Entrapment efficiency of OFL at different MMT contents of the beads.
During the reaction with TPP, it is likely that a part of the drug would leak into the solution. The addition of MMT obviously decreased the leakage of OFL (Fig. 2). This significant improvement can be best ascribed to the following facts. MMT with the large specific area adsorbs OFL not only at the external surfaces but also in the interlayer space. On the other hand, the addition of MMT increased the viscosity of the dispersions and retarded the diffusion of OFL (Fig. 2). Sriamornsak et al. previously studied the rheological properties, size of the aggregates, and the zeta potential of CTS–MAS (magnesium aluminum silicate) dispersions (Sriamornsak et al., 2007). They considered that the electrostatic interactions between the surface charges of MAS and the positively charged amino groups (–NH+ 3 ) of CTS may affect the zeta potential and the flow behavior of the dispersion.
3.5. Swelling studies
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Fig. 6. Mass change of wet beads (M0, M2 and M4) at pH 7.4 and 1.2. Fig. 8. Effect of MMT content on the release profile at pH 7.4.
The dry masses of M0, M2 and M4 beads at pH 7.4 decreased rapidly within the first hour due to the leakage of the loaded beads. No notable disintegration was visually observed, and the spherical shape of the beads was retained even after 10 h. However, the dry mass of M0, M2 and M4 beads at pH 1.2 decreased strongly and disintegration was clearly observed. Formation of more stable gels and slower disintegration due to the addition of MMT was also reported with diclofenac/ magnesium aluminum silicate/Ca-alginate beads (Puttipipatkhachorn et al., 2005) and films of chitosan-g-lactic acid and MMT (Depan et al., 2009). From these results, a possible model for the nanocomposite beads is shown in Fig. 7. The physical crosslinking between CTS and MMT layers formed the network structure of the beads accompanied by the ionic crosslinking between CTS and TPP and thus enhanced the stability of the beads. 3.6. In vitro release studies In vitro release of OFL from the beads with various MMT contents at pH 7.4 and 1.2 are shown in Figs. 8 and 9. The drug released at pH 1.2 was relatively faster than at pH 7.4. The noticeably higher release rate of OFL at pH 1.2 can be attributed to the higher solubility of OFL in acidic medium (Cui et al., 2008) and the degradation behavior at pH 1.2 (Figs. 6 and 10). The drug release was correlated to the swelling and degradation of the beads. The entrapped OFL in M0 bead was released almost completely within 2 h because of the almost complete
Fig. 7. Schematic illustration of interactions between CTS, OFL and MMT.
degradation of the beads in SGF. The swelling of M4 beads in SGF, especially for the initial 4 h, caused about 66% of entrapped OFL to migrate out. For the subsequent 6 h, the bead swelling was minimal and only about 13% of entrapped OFL was further released. At pH 1.2, the cumulative amount of OFL released for the test beads without MMT addition (M0) was about 90%. In contrast, at any time of incubation, the level of release was higher in the order of M0, M1, M2, M3, M4 and M5, which clearly indicated that increased amount of MMT reduced the extent of drug release (p b 0.05). After 10 h, none of the MMT-containing samples (M1 to M5) completely released the encapsulated drug. Also, prolonged time did not markedly increase the release (Nunes et al., 2007). This fact was more significant in the release profile of M5, where the release was below 35% and 70% in SIF and SGF within 10 h. Based on the above analyses, the addition of a certain amount of MMT not only improved the drug EE, but also provided a slower and continuous drug release. Thus, the incorporation of MMT particles into biopolymers may be useful for the sustained delivery formations of drugs and other bioactive molecules. 4. Conclusions CTS/OFL/MMT hydrogel beads were prepared and characterized by SEM, FTIR and XRD techniques. The beads showed improved drug loading and controlled release. These properties were highly dependent on the MMT content. The disintegration of the pure CTS beads at pH 1.2 was overcome by introducing MMT, which made this system more useful in targeting at low pH. The drug release rate of the
Fig. 9. Effect of MMT content on the release profile at pH 1.2.
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Fig. 10. Mass change of dry beads (M0, M2 and M4) at pH 7.4 and 1.2.
beads was also influenced by pH of the medium. The cumulative drug release of OFL from the nanocomposite beads at pH 7.4 was slower than that at pH 1.2, which decreased with increasing MMT content. A sustained release system can be prepared by incorporating MMT into the nanocomposite beads. Acknowledgement The authors would like to thank the Western Action Project of CAS (No. KGCX2-YW-501) and the Special Research Fund of Scholarship of the Dean of CAS” for financial support of this research. References Agnihotri, S.A., Mallikarjuna, N.N., Aminabhavi, T.M., 2004. Recent advances on chitosanbasedmicro-and nanoparticles in drugdelivery.J. Control.Release 100,5–28. Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Appl. Clay Sci. 36, 22–36. Byrne, R.S., Deasy, P.B., 2005. Use of porous aluminosilicate pellets for drug delivery. J. Microencapsul. 22, 423–437. Cui, Y., Zhang, Y., Tang, X., 2008. In vitro and in vivo evaluation of ofloxacin sustained release pellets. Int. J. Pharm. 360, 47–52. Dang, J.M., Leong, K.W., 2006. Natural polymers for gene delivery and tissue engineering. Adv. Drug Deliv. Rev. 58, 487–499. Depan, D., Kumar, A.P., Singh, R.P., 2009. Cell proliferation and controlled drug release studies of nanohybrids based on chitosan-g-lactic acid and montmorillonite. Acta Biomater. 5, 93–100.
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Contents lists available at ScienceDirect
Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l a y
Controlled release of ofloxacin from chitosan–montmorillonite hydrogel Shuibo Hua a,b, Huixia Yang a,b, Wenbo Wang a,b, Aiqin Wang a,⁎ a b
Center for Eco-material and Green Chemistry, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 11 August 2009 Received in revised form 7 July 2010 Accepted 12 July 2010 Available online 23 July 2010 Keywords: Chitosan Montmorillonite Nanocomposite Drug release Ofloxacin
a b s t r a c t A series of ofloxacin/montmorillonite/chitosan (OFL/MMT/CTS) hydrogels were prepared by solution intercalation and ionic crosslinking with sodium tripolyphosphate (TPP). The structure and drug loading of the beads were characterized by Fourier transform infrared spectroscopy (FTIR). The formation of intercalated nanocomposites was confirmed by X-ray diffraction (XRD). The swelling and degradation of the beads were influenced by the pH of test medium and the MMT content. Compared to pure CTS beads, the incorporation of MMT enhanced the drug entrapment, improved the swelling behavior and reduced the drug release. The CTS beads without MMT dissolved after a 3 h immersion in pH 1.2 solution. No notable disintegration was visually observed for MMT-containing beads even after a 10 h immersion. The observations suggested that the electrostatic interaction between CTS and MMT enhanced the stability of the beads and showed good potential for the use as drug carriers for sustained release. © 2010 Published by Elsevier B.V.
1. Introduction Drug delivery systems recently received great interests because they realized the effective and targeted delivery of drug and minimized the side effects resulting from the traditional drug-dosage form in the pharmaceutical field. A large number of drug delivery systems have been conceived and developed (Ding et al., 2002; Li et al., 2004; Ginebra et al., 2006; Malmsten, 2006; Tomonori and Hiroshi, 2008; Taa et al., 2009). Clay mineral composites as known drug delivery systems showed unique hybrid properties superior to the components and the capability to incorporate various drug substances (Byrne and Deasy, 2005; Aguzzi et al., 2007; Lin et al., 2007). MMT exhibits enhanced gel strength, mucoadhesive capability to cross the gastrointestinal (GI) barrier and adsorb bacterial and metabolic toxins such as steroidal metabolites (Herrera et al., 2000). MMT is a common ingredient as both the excipient and active substance in pharmaceutical products (Takahashi et al., 2005). Cationic drugs can be intercalated into MMT by ion exchange (Zheng et al., 2007; Jung et al., 2008; Pongjanyakul et al., 2009). The regulation of the amount and rate of drug release is difficult when the drug is intercalated by ion exchange. Chitosan (CTS) is an amine-bearing, linear polysaccharide derived from the N-deacetylation and depolymerization of chitin (i.e., poly-Nacetyl-glucosamine) (Agnihotri et al., 2004). As a unique cationic polysaccharide, CTS shows gel and film forming properties and found
⁎ Corresponding author. Tel.: + 86 931 4968118; fax: + 86 931 8277088. E-mail address: [email protected] (A. Wang). 0169-1317/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.clay.2010.07.012
potential in the pharmaceutical industry as a drug delivery system (Thanou et al., 2000; Dang and Leong, 2006). CTS can form nanocomposites with MMT, and there are several reports about the drug release behavior from the CTS/MMT nanocomposite films, scaffolds and nanoparticles (Wang et al., 2008; Depan et al., 2009). From these points of view, the combination of MMT with CTS beads as a drug carrier is attractive. In our study, ofloxacin (Fig. 1), the fluoroquinolone antibiotic, commonly used for treatment of prophylaxis of a variety of bacterial diseases, was tested as the cationic model drug in in vitro experiments. OFL/MMT nanocomposites were prepared by the solution intercalation technique. Without filtering out the free drug, OFL/MMT intercalated CTS beads were obtained through ionic crosslinking and their morphology and structure were characterized by scanning electron microscopy (SEM), FTIR and XRD. Finally, swelling and in vitro drug release were studied in simulated gastric fluids (SGF, pH 1.2) and simulated intestinal fluids (SIF, pH 7.4). 2. Materials and methods 2.1. Materials Chitosan (CTS, deacetylation degree, 81%; mass average molar mass, 9 × 105) from shrimp shell was purchased from Yuhuan Ocean Biochemical Co. (Taizhou, China). Ofloxacin (OFL) was obtained from Kunshan Double-crane Pharmaceutical Co., Ltd, China. High purity montmorillonite (MMT, the reference substance for scientific research; the content of montmorillonite is 99% determined by XRD technology) was purchased from Zhejiang Sanding Technology Co., Ltd (Shaoxing, China), and the contents of SiO2 and Al2O3 are
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aqueous solution containing 4.0 g ammonium acetate and 7.0 g sodium perchlorate were adjusted to pH 2.2 with phosphoric acid. The drug content was determined by comparing with the standard curve of OFL which was achieved from OFL solutions in PBS 7.4 with concentration from 0.002 to 0.01 g/L. The drug entrapment efficiency (EE) is expressed as follows: EE = ðPractical drug loading = Theoretical drug loadingÞ × 100% ð1Þ Fig. 1. Structure of OFL.
2.4. Swelling studies 59.48 mass% and 21.93 mass%, respectively. Tripolyphosphate (TPP) was purchased from Sinopharm Chemical Reagent Co., Ltd, China. Simulated gastric fluid (SGF, pH 1.2) containing 21.25 mL HCl, 11.18 g KCl in 3000 mL distilled water and simulated intestinal fluid (SIF, phosphate buffer solutions, PBS, pH 7.4), containing 20.4 g K2HPO4 and 4.8 g NaOH in 3000 mL distilled water were prepared as described in US Pharmacopoeia 30. All other chemicals were of analytical grade and used as received. 2.2. Preparation of OFL/MMT/CTS hydrogel beads The hydrogel beads were prepared according to an improved technique using TPP as the gelling counterion (Sezer and Akbuga, 1995). A series of the samples with different amounts of MMT were prepared according to the following procedure. 0.2 g MMT was dispersed in 20 mL of 2 mass% acetic acid solution, then 0.20 g OFL were added into the dispersion, followed by a continuous stirring at room temperature for 1 h. CTS (0.60 g) was dissolved in the OFL/MMT mixture to reach the final concentration of 3% (mass/v) and stirred for 0.5 h. The beads were formed by dropping the bubble-free solution or dispersion through a disposable syringe onto a gently agitated TPP solution (50 mL, 5 mass%; pH 5.0, adjusted with standard 1 mol/L HCl solution). After 0.5 h, the obtained beads were filtrated with a 100mesh stainless screen and rinsed with distilled water for three times. Finally, the gel-like beads were air-dried for 24 h, followed by drying at 70 °C for 6 h. Similar procedures were used to prepare OFL-free placebo beads. The beads corresponded to 0, 0.10, 0.20, 0.30, 0.40 and 0.50 g MMT were denoted as M0, M1, M2, M3, M4 and M5. The M0 and M4 beads without adding OFL were termed as M0C and M4C (Table 1). 2.3. Entrapment efficiency The amount of OFL entrapped in the beads was calculated by measuring the content of OFL in the gelling medium. The floating small MMT particles were removed through a 0.45 μm membrane filter. The clear superficial solution was analyzed by HPLC (WatersTM 600 Pump, 2998 Photodiode Array Detector) using the C18 column. The mixture of acetonitrile and buffer solutions (25:75, v/v) was used as the mobile phase. OFL was detected at the wavelength of 294 nm. The buffer solution was prepared according to USP 30: 1300 mL of an Table 1 Feed composition and drug content of the samples. Code
CTS (g)
OFL (g)
MMT (g)
OFL content (mass%)
M0 M1 M2 M3 M4 M5 M0C M4C
0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6
0.2 0.2 0.2 0.2 0.2 0.2 0 0
0 0.1 0.2 0.3 0.4 0.5 0 0.4
7.81 10.00 10.92 12.02 12.74 12.83 – –
Swelling studies and in vitro degradation of the beads were carried out in two aqueous media: 0.1 mol/L HCl solution (pH 1.2) as simulated gastric fluid and phosphate buffer solutions (pH 7.4) as simulated intestinal fluid. Beads (100 mg) were placed at 37 °C in the dissolution test apparatus (ZRS-8G, Tianjing University Wireless factory, China) containing 500 mL of the medium at 50 rpm. At regular intervals, the beads were reweighed after carefully wiping off excess liquid with a tissue paper. The mass change of the wet beads Qw and of the dry beads Q d with respect to time was determined as follows: Q w = ðWw –W0 Þ = W0
ð2Þ
Q d = ðWd –W0 Þ = W0
ð3Þ
where Ww and Wd are the wet and dry mass of the beads at time t, respectively; Wo is the initial mass of beads. 2.5. In vitro drug release The in vitro drug release tests were carried out using the USP 30 NO.2 dissolution test apparatus fixed with six rotating paddles. The release medium is a simulated intestinal fluid (pH 7.4) or simulated gastric fluid (pH 1.2) (500 mL, 37.5 ± 0.5 °C), and the speed of rotation was 50 ± 1 rpm. 50 mg samples were placed into each of the six cups, and 5 mL solution was collected from the release medium at regular intervals. After each sample collection, the equal amount of fresh release medium at the same temperature was added. The amount of drug released was monitored by a UV–vis spectrophotometer (SPECORD 200, Analytik Jera AG) at 294 nm based on the established UV standard absorbance curve for OFL. In the concentration range (2.5 ~ 12.5 × 10−5 mol/L) investigated, the UV absorbance obeyed the Beer's law. 2.6. Characterization FTIR spectra were recorded on a FTIR spectrophotometer (Thermo Nicolet, NEXUS, TM) in the range of 4000–400 cm−1 using KBr pellets. The surface morphology was observed by scanning electron microscopy (JSM-5600LV, JEOL) after coating the samples with gold film using an acceleration voltage of 20 kV. Powder XRD analyses were performed using a diffractometer with Cu anode (PAN analytical X'pert PRO), running at 40 kV and 30 mA, scanning from 3° to 40° at 3°/min. 2.7. Statistical analysis Each experiment was carried out in triplicate and the results were averaged. The influence of MMT content on the entrapment and the release of the drug from beads were statistically analyzed by one-way ANOVA. The data were considered to be significantly different at p b 0.05.
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electrostatic interaction between OFL and MMT. The FTIR results confirmed that CTS was crosslinked with TPP in the beads and OFL was encapsulated in the beads or stabilized in the interlayer space of MMT.
3. Results and discussion 3.1. Morphology of the beads When the dispersion of CTS in acetic acid was dropped into TPP solution, gelled spheres formed instantaneously due to the electrostatic interaction between positively charged CTS and negatively charged TPP. Generally, the wet beads were spherical with a diameter of about 5 mm and possessed a smooth surface. After air drying, the diameter of test beads decreased to about 2 mm but still kept the spherical shape. For any batches of nanocomposite beads with various amounts of MMT, there was no obvious variation of the bead size. The large size of wet beads suggested high swelling and water retention ability. The surface morphology showed severe wrinkles, which was caused by partial collapsing of the polymer network during drying (Fig. 2). Compared to placebo M0C beads, little particles appeared on the surface of the OFL-loaded beads (M0) due to the leakage of OFL crystals from the network space of beads. However, the OFL crystals disappeared with increasing MMT content, suggesting that the loading of OFL was remarkably improved due to the incorporation of MMT. 3.2. FTIR analysis The intense characteristic bands of CTS at 1651 and 1602 cm−1 (Fig. 3) were ascribed to the absorptions of amide I and amide II (Papadimitriou et al., 2008), which shifted to 1640 cm− 1 and 1557 cm− 1 after the formation of CTS beads with TPP addition. This information indicated that the amide formed ionic bonds. This interaction reduced the solubility of CTS and contributed to the separation of CTS from the solution in the form of beads. The absorption band of MMT at 3623 cm− 1 (stretching vibration of the interlayer –OH groups) almost disappeared and the band at 1714 cm− 1 (characteristic absorption of the C = O group of OFL) was weakened in the spectra of the beads (M4). This result indicated
3.3. XRD analysis The reflection of CTS at around 20° (Fig. 4) was characteristic of the hydrated crystalline structure (Liu et al., 2008), but its intensity was largely weakened after forming the beads. Crystalline CTS was not observed. OFL showed characteristic intense reflections at 6° and 10.9° due to the presence of OFL crystals. These reflections disappeared in the pattern of M4, which indicated that OFL was dispersed at the molecular level in the polymer matrix or intercalated for organic-nanoclay hybrid systems (Zheng et al., 2007; Depan et al., 2009) and no crystals were found in the drug-loaded matrices. The XRD pattern of MMT showed the basal reflection at about 6.68° with a basal spacing of 1.32 nm. The basal spacing increased from 1.32 nm to 2.14 nm after OFL addition indicating the intercalation of OFL. However, due to its coiled or helicoidal structures, CTS was only adsorbed on the surface of MMT or led to the very less intercalation to MMT as the basal spacing only increased from 1.32 nm to 1.36 nm (M4C). This result was in agreement with a previous study (Günister et al., 2007). 3.4. Entrapment efficiency The EE of the beads increased with the amount of MMT added (Fig. 5). The entrapment of OFL in beads without MMT (M0) was 31.2, whereas the corresponding entrapment in the MMT-containing beads varied from 45.3 to 83.5. A comparison of EE with the different MMT contents was evaluated by ANOVA and the F value was 906.41 (df = 17, p = 0). Thus, the incorporation of MMT in the beads very effectively improved the entrapment of OFL.
Fig. 2. SEM images of (a) M0C, (b) M0, (c) M2, and (d) M4.
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Fig. 3. FTIR spectra of CTS, OFL, MMT, M0C and M4 beads.
In order to simulate the possible effect of pH on drug release rate, a swelling study was conducted in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) at physiological temperature of 37 °C (Fig. 6). The swelling ratio at pH 7.4 was very small (about 30%). At pH 1.2, with increasing the MMT content, the initial swelling ratio decreased (b1.2 h), but the swelling ratio within 10 h increased significantly (p b 0.05). The mass of M0 beads increased by 4.68 fold
for the initial 1 h, and then was sharply reduced until the beads were disintegrated for the subsequent 1 h immersion. However, the mass of M2 beads increased by 6 fold for the first 4 h, and then reduced to 4.3 times of their initial values for the subsequent 6 h immersion. For swelling of M4 beads, the mass of M4 beads increased by 12 fold within 10 h. A greater extent of crosslinking of the beads restricted the mobility of the polymer chains and thus limited the swelling. The swelling was mainly influenced by ionic interactions between CTS chains and MMT interlayers, which depended on the crosslinking density. The incorporation of MMT strengthened the network of prepared beads. Liu et al. (2008) studied the drug release from CTS–MMT hydrogels by electrostimulation, and considered that exfoliated MMT layers can act as crosslinkers between CTS and MMT (Liu et al., 2008). In our study, some MMT layers could also act as crosslinker between the CTS chains. However, in contrast to decreased swelling by increasing MMT content as reported by Liu et al. (2008), a greater swelling at pH 1.2 for 10 h incubation was obtained, probably because the crosslinking between CTS and TPP was broken. Meanwhile, electrostatic repulsion occurred between protonated amine groups on CTS causing higher water uptake as in the simulated gastric fluid. However, no such repulsion was effective in the simulated intestinal fluid, which caused only a small swelling at pH 7.4. If the crosslinking density became too small, interactions were no longer strong enough to avoid disintegration and the beads decomposed completely within 3 h (M0).
Fig. 4. XRD patterns of OFL, MMT, CTS, M0C, M4 and M4C beads.
Fig. 5. Entrapment efficiency of OFL at different MMT contents of the beads.
During the reaction with TPP, it is likely that a part of the drug would leak into the solution. The addition of MMT obviously decreased the leakage of OFL (Fig. 2). This significant improvement can be best ascribed to the following facts. MMT with the large specific area adsorbs OFL not only at the external surfaces but also in the interlayer space. On the other hand, the addition of MMT increased the viscosity of the dispersions and retarded the diffusion of OFL (Fig. 2). Sriamornsak et al. previously studied the rheological properties, size of the aggregates, and the zeta potential of CTS–MAS (magnesium aluminum silicate) dispersions (Sriamornsak et al., 2007). They considered that the electrostatic interactions between the surface charges of MAS and the positively charged amino groups (–NH+ 3 ) of CTS may affect the zeta potential and the flow behavior of the dispersion.
3.5. Swelling studies
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Fig. 6. Mass change of wet beads (M0, M2 and M4) at pH 7.4 and 1.2. Fig. 8. Effect of MMT content on the release profile at pH 7.4.
The dry masses of M0, M2 and M4 beads at pH 7.4 decreased rapidly within the first hour due to the leakage of the loaded beads. No notable disintegration was visually observed, and the spherical shape of the beads was retained even after 10 h. However, the dry mass of M0, M2 and M4 beads at pH 1.2 decreased strongly and disintegration was clearly observed. Formation of more stable gels and slower disintegration due to the addition of MMT was also reported with diclofenac/ magnesium aluminum silicate/Ca-alginate beads (Puttipipatkhachorn et al., 2005) and films of chitosan-g-lactic acid and MMT (Depan et al., 2009). From these results, a possible model for the nanocomposite beads is shown in Fig. 7. The physical crosslinking between CTS and MMT layers formed the network structure of the beads accompanied by the ionic crosslinking between CTS and TPP and thus enhanced the stability of the beads. 3.6. In vitro release studies In vitro release of OFL from the beads with various MMT contents at pH 7.4 and 1.2 are shown in Figs. 8 and 9. The drug released at pH 1.2 was relatively faster than at pH 7.4. The noticeably higher release rate of OFL at pH 1.2 can be attributed to the higher solubility of OFL in acidic medium (Cui et al., 2008) and the degradation behavior at pH 1.2 (Figs. 6 and 10). The drug release was correlated to the swelling and degradation of the beads. The entrapped OFL in M0 bead was released almost completely within 2 h because of the almost complete
Fig. 7. Schematic illustration of interactions between CTS, OFL and MMT.
degradation of the beads in SGF. The swelling of M4 beads in SGF, especially for the initial 4 h, caused about 66% of entrapped OFL to migrate out. For the subsequent 6 h, the bead swelling was minimal and only about 13% of entrapped OFL was further released. At pH 1.2, the cumulative amount of OFL released for the test beads without MMT addition (M0) was about 90%. In contrast, at any time of incubation, the level of release was higher in the order of M0, M1, M2, M3, M4 and M5, which clearly indicated that increased amount of MMT reduced the extent of drug release (p b 0.05). After 10 h, none of the MMT-containing samples (M1 to M5) completely released the encapsulated drug. Also, prolonged time did not markedly increase the release (Nunes et al., 2007). This fact was more significant in the release profile of M5, where the release was below 35% and 70% in SIF and SGF within 10 h. Based on the above analyses, the addition of a certain amount of MMT not only improved the drug EE, but also provided a slower and continuous drug release. Thus, the incorporation of MMT particles into biopolymers may be useful for the sustained delivery formations of drugs and other bioactive molecules. 4. Conclusions CTS/OFL/MMT hydrogel beads were prepared and characterized by SEM, FTIR and XRD techniques. The beads showed improved drug loading and controlled release. These properties were highly dependent on the MMT content. The disintegration of the pure CTS beads at pH 1.2 was overcome by introducing MMT, which made this system more useful in targeting at low pH. The drug release rate of the
Fig. 9. Effect of MMT content on the release profile at pH 1.2.
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Fig. 10. Mass change of dry beads (M0, M2 and M4) at pH 7.4 and 1.2.
beads was also influenced by pH of the medium. The cumulative drug release of OFL from the nanocomposite beads at pH 7.4 was slower than that at pH 1.2, which decreased with increasing MMT content. A sustained release system can be prepared by incorporating MMT into the nanocomposite beads. Acknowledgement The authors would like to thank the Western Action Project of CAS (No. KGCX2-YW-501) and the Special Research Fund of Scholarship of the Dean of CAS” for financial support of this research. References Agnihotri, S.A., Mallikarjuna, N.N., Aminabhavi, T.M., 2004. Recent advances on chitosanbasedmicro-and nanoparticles in drugdelivery.J. Control.Release 100,5–28. Aguzzi, C., Cerezo, P., Viseras, C., Caramella, C., 2007. Use of clays as drug delivery systems: possibilities and limitations. Appl. Clay Sci. 36, 22–36. Byrne, R.S., Deasy, P.B., 2005. Use of porous aluminosilicate pellets for drug delivery. J. Microencapsul. 22, 423–437. Cui, Y., Zhang, Y., Tang, X., 2008. In vitro and in vivo evaluation of ofloxacin sustained release pellets. Int. J. Pharm. 360, 47–52. Dang, J.M., Leong, K.W., 2006. Natural polymers for gene delivery and tissue engineering. Adv. Drug Deliv. Rev. 58, 487–499. Depan, D., Kumar, A.P., Singh, R.P., 2009. Cell proliferation and controlled drug release studies of nanohybrids based on chitosan-g-lactic acid and montmorillonite. Acta Biomater. 5, 93–100.
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