Drug release material hosted by natural montmorillonite with proper modification

Applied Clay Science 148 (2017) 123–130

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Applied Clay Science journal homepage: www.elsevier.com/locate/clay

Research paper

Drug release material hosted by natural montmorillonite with proper modification

MARK

Limei Wua,⁎, Guocheng Lvb,⁎, Meng Liub, Danyu Wangb a

School of Materials Science and Engineering, Shenyang Jianzhu University, Shenyang 110168, China Beijing Key Laboratory of Materials Utilization of Nonmetallic Minerals and Solid Wastes, National Laboratory of Mineral Materials, School of Materials Science and Technology, China University of Geosciences, Beijing 100083, China

b

A R T I C L E I N F O

A B S T R A C T

Keywords: Montmorillonite Ciprofloxacin Drug release Layer charge

In developing new generations of coatings for drug and drug controlled release, there is a need for self-assembled materials that provide controlled sequential release of multiple therapeutics, while provide a tunable approach to time dependence and the potential for sequential or staged release. Herein, we demonstrated the ability to develop a self-assembled, in ciprofloxacin intercalated montmorillonite (CIP-Mt), the release rate and amount of interlayer ciprofloxacin (CIP) could be controlled by modifying the layer charge of montmorillonite (Mt). Compared with common sustained release materials, this composite had better effect on antibacterial and disinfection. The CIP-Mt system effectively blocked diffusion-based release, leading to approximately 50% reduction in bolus doses and 10-fold increase in the release timescale. Mt was a non-toxic and non-polluting sustained release material, could be applied to drug release research, and the release rate and time of its interlayer CIP can be controlled by modifying layer charge.

1. Introduction Nowadays, microorganism threat on human health and environmental safety has become a serious public concern (Carey et al., 2011; Touré et al., 2014). Pathogenic bacteria, fungi and viruses, etc., are responsible for the transmission of most of the serious diseases (Ghosh et al., 2012; Harimawan et al., 2013). It is thus quite essential to conduct studies with aspects of sterilization, prevention, disinfection, etc., to control microbial pollution. Antibacterial agents offer a solution to this microbial problem (Sahiner and Yasar, 2013). However, the overuse and misuse of antibiotics has led to the emergence of antibioticresistant bacteria, compromising the effectiveness of antimicrobial therapy because the infectious organisms are becoming resistant to most antibiotics (Pruneau et al., 2011; Marti and Balc-azar, 2013). Since a high local concentration of antibiotics is provided through sustained release, bacteria are killed before they grow into biofilm (Diaz-Rodriguez et al., 2011; Deacon et al., 2015). In fact, the emergence and spread of antibiotic resistant bacteria has been classified by the World Health Organization (WHO) as one of the three biggest threats to public health in the 21st century. These antibiotics have good efficacy, while excessive intake would cause some side effects (Li and Chauhan, 2006), and especially for longtime excessive intake would decline antibiotic effect and human

⁎

Corresponding authors. E-mail addresses: [email protected] (L. Wu), [email protected] (G. Lv).

http://dx.doi.org/10.1016/j.clay.2017.07.034 Received 2 March 2017; Received in revised form 26 July 2017; Accepted 27 July 2017 Available online 30 August 2017 0169-1317/ © 2017 Published by Elsevier B.V.

immunity. For example, a rapid rise of drug concentration after intake is considered to cause poisoning, allergies and other symptoms (Schwartz and Calvert, 1990; Akahane et al., 1993; Lomaestro, 2000). A short half-life drug leads to rapid loss of metabolism, reducing the duration and concentration of the antibiotics in the human body and weakening the efficacy. The preparation of sustained release of antibiotic composite is significant for improving the efficacy. The sustained release of drug can be realized by a combination of active ingredient and molecule host, where a slow released drug agent with strong efficacy can be obtained by controlling release rate of drug by diffusion and penetration (Cheow et al., 2010; Ochs et al., 2010; Mayol et al., 2014). Sustained release of drug not only prolongs the action, thus reducing the frequency of drug administration, but also attempts to maintain drug levels within the therapeutic window to avoid potentially hazardous reflections in drug concentration. In recent years, clays, such as Mt, intercalated by drug molecules have attracted much interest from researchers due to their novel physical and chemical properties (Lin et al., 2002; Park et al., 2008; Joshi et al., 2009, 2010). Moreover, it has also been reported that intercalation of some drugs onto Mt can overcome the problem of oral administration in clinical application (Lin et al., 2002; Donga and Feng, 2005). As one of the smectite groups, Mt is composed of silica tetrahedral sheets layered between alumina octahedral sheets. The imperfection of the crystal lattice and the

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2.3. Materials characterization

isomorphous substitution induces a net negative charge that leads to the adsorption of alkaline earth metal ions in the interlayer spaces. Such imperfection is responsible for the activity and exchange reactions with organic compounds. Mt is a natural mineral exhibiting good adjustability among common inorganic layered materials, as well as non-toxic, nonirritating and without side effect. Thus, Mt can be applied to drug release research, and the release rate and time of its interlayer CIP can also be controlled by modifying layer charge. In the research, a ciprofloxacin-montmorillonite (CIP-Mt) composite was prepared as a sustained release drug system, and the sustained ability can be controlled by adjustment of layer charge. The improved sustained ability indicates potential applications in retard drugs. When CIP enters Mt interlayer space, the releases of CIP is affected by the distribution of CIP in the interlayer spaces and its interaction with the Mt layers. Changes in charge density of Mt (AMt) lead to variations in electric strength in the interlayer space region. This affects amount of intercalated CIP to Mt and electrostatic attraction between CIP and Mt layers. After adjustment of layer charge, release time of CIP-AMt is 700 min longer than that of CIP-Mt, and release amount is also stable and controllable, whose characteristics are applicable to practical retard drug system.

The equilibrium CIP concentrations were analyzed by a UV–Vis spectrophotometer (Model T6 New Century 1650, made by General Instrument, Inc. LLT, Beijing China) at the wavelength of 275 nm, corresponding to its maximal absorbance. Calibrations were made following standards of 10, 20, 30, 40, 50, and 60 mg/L with a regression coefficient of 0.9998. The amount of CIP adsorbed was calculated as the difference between the initial and final concentrations. Powder XRD analyses were performed on a Rigaku D/max-III a diffractometer (Tokyo, Japan) with a Ni-filtered CuKα radiation at 30 kV and 20 mA. Orientated samples were scanned from 3° to 10° or 3° to 70° with a scanning step of 0.01°. Powder samples were packed in horizontally held trays. The changes in the XRD reflection positions reflected the intercalation of the dye into layered silicates. The Bragg equation was applied to calculate the basal spacing of Mt layers. The interlayer space sizes of intercalated hybrids were deduced from XRD reflection positions of (001) of the hybrids. Elemental compositions of Mt were determined by X-Ray Fluorescence spectrometry (XRF) to confirm the dissolution of Si after acid treatment. It was carried out by using a portable XRF spectrometer (Oxford Instruments) with a molybdenum anode, at 25 kV and 0.1 mA. A Si-PIN detector from AMPTEK was employed and characterized by an energy resolution of about 200 eV at 5.9 keV. Thermogravimetric analyses were carried out on TGA Q-500 (TA Instruments, New Castle, USA) from room temperature to 800 °C with a heating rate of 10 °C/min under a nitrogen flow of 60 mL/min. TG curves were used to determine the percentage of mass loss. Differential scanning calorimetry (DSC) was performed using a differential scanning calorimeter (TA Instruments Q100) fitted with a cooling system using liquid nitrogen. It was calibrated with an indium standard. Samples of 6 mg Mt were accurately weighed into aluminum pans, sealed and then heated from 30 to 800 °C at 10 °C/min under a nitrogen flow of 60 mL/ min. Molecular simulation was performed under the module ‘CASTEP’ of Materials Studio 6.1 software to investigate the sorption sites of CIP on Mt. The primitive unit cell of Mt was optimized with the generalized gradient approximation (GGA) for the exchange-correlation potential (PW91) that was appropriate for the relatively weak interactions present in the models studied. The resulting primitive unit cell was characterized by the parameters a = 15.540 Å, b = 17.940 Å, c = 12.56 Å, and α = γ = 90°, β = 99°. Based on the primitive unit cell, a series of (3 × 2 × 1) supercells were built with the spacing of layers set to15.24 and 15.04 Å, respectively. The number of cycles was 3 and the steps of one cycle were 106, a representative part of the interface devoid of any arbitrary boundary effects.

2. Experiment and methods 2.1. Materials The montmorillonite used was SWy-2 obtained from the Clay Mineral Repositories in Purdue University (West Lafayette, IN) without further purification. It had a chemical formula of (Ca0.12 Na0.32 K0.05)[Al3.01 Fe (III)0.41 Mg0.54][Si7.98 Al0.02]O20(OH)4, a CEC of 85 ± 3 mmolc/100 g (Borden and Giese, 2001), a layer charge of 0.32 eq/mol per (Si,Al)4O10 (Chipera and Bish, 2001), an external surface area (ESA) of 23 m2/g, respectively (Mermut and Lagaly, 2001), and a mean particle size of 3.2 μm with a d25 to d75 in the range of 3–10 μm. Ciprofloxacin (CIP) was used as received without further purification. CIP hydrochloride (purity > 99.6%) was purchased from Beijing Solarbio life sciences Co., Ltd., (China). Its pKa1 and pKa2 values were 6.1 and 8.7, respectively (Chang et al., 2016). 2.2. Experiment Natural Mt was treated by HCl at concentrations of 0, 0.05, 0.1, 0.5, and 1.0 mol/L to adjust its structure and charge density (AMt). After the acid treatments, the Mt was washed with two portions of distilled water to remove the residual Cl. The samples (denoted as AMt) were dried naturally before interaction of CIP into Mt. The initial CIP concentrations varied from 5 to 10,000 mg/L for the intercalation isotherm study. The mass of Mt was 0.5 g and the volume of solution was 10 mL for all studies, except for the kinetic study (for which 50 mL of solution was used). The solid and solution were combined in each 50 mL centrifuge tube and shaken for 4 h at 150 rpm at room temperature for all studies, except for the kinetic study. The mixtures were centrifuged at 10000 rpm for 20 min, then dried at 60 °C and ground before characterizations. A mixture, composed of 0.5 g and 50 mL NaCl (the concentration was 10 mol/L), was shaken at 150 rpm for 5–1440 min at room temperature. P. aeruginosa, a strain isolated from the institute of microbiology Chinese academy of sciences, was preserved in our laboratory, and was identified to have excellent algicidal activity on M. aeruginosa. Briefly a nutrient agar (pH 7.0–7.2) was prepared by dissolving 10 g peptone, 3 g beef extract, 5 g NaCl, 20 g agar in 1 L distilled water. After sterilization in an autoclave (100 kPa, 120 °C, 20 min), the nutrient agar was casted on Petri dishes and cooled to the room temperature. Then, 100 μl diluted degrading medium containing microorganisms was coated on the nutrient agar surface. After being incubated at 37 °C for 24 h, the number of microorganism colonies, representing the living microorganisms, was counted.

3. Results and discussions 3.1. The modification of Mt and preparation of CIP-Mt The structure modification of Mt and the preparation process of CIPMt are shown in Fig. 1. Over the course of fabrication, Mt was treated by HCl firstly at concentrations of 0.05, 0.1, 0.5 and 1.0 M (mol/L) to adjust its structure. The acid treatment would result in Mt with different charge densities. However, the crystal structure of Mt after acid treatment was intact as observed by XRD analyses (Fig. 1a). Meanwhile, the intensity of the reflection at 27.8° reduced gradually until disappeared, and the main phase was feldspar. The small changes in d001 value could be attributed to the changes in layer charge (Wu et al., 2016). This study was done to mimic the behavior of Mt with CIP. In contrast to many drug projects that have used completely purified and modified Mt, the experiment was done on pure and modified Mt as it has CIP for cation exchange processes (Majzik and Tombacz, 2007). Initial concentration of CIP solution had a profound influence on the amount of Mt intercalation. The removal of CIP by Mt and AMt 124

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(a)

d=14.52

1.0 mol/L

d=14.67

0.05 mol/L

Intensity

Intensity

0.5 mol/L 0.1 mol/L

d=15.72 d=16.28

Raw Mt

10 20 30 40 50 60 70 80 90 100

2 Theta (° )

(b)

d=15.24

3

4

5 6 7 8 2 Theta (° )

9 10

Fig. 1. Schematic illustration for the modification of Mt and preparation of CIP-AMt. X-ray diffraction patterns spectra of Mt, acid treated Mt (a), and CIP-AMt (b). (d/Å).

favorable substitution of CIP for the inorganic cations in the Mt interlayer space. However, as charge density increased further, the strong interaction between the inorganic cations, the amount of CIP intercalation and d001 decreased accordingly.

increased as the concentration of CIP solution increasing. At CIP concentration of 2000 mg/L, the removal of CIP by Mt was 296 mg/g, while at CIP concentration of 5000 mg/L, the removal amount peaked at 310 mg/g (Fig. 2a). The removal amount of Mt was obvious higher than AMt. 0.05 M–AMt and 0.1 M–AMt, with relative lower acid modification degree, had similar removal amount to Mt, and reached a plateau at 290 mg/g when CIP concentration was 5000 mg/L. However, for 0.5 M–AMT and 1.0 M–AMt modified by higher acid concentration, when the CIP concentration was 10,000 mg/L, the highest removal amount reached 265 mg/g and 177 mg/g, respectively. It is considered that, with increasing acid modification, the removal amount of CIP by Mt reduced while the initial concentration of CIP increased. It was because CIP existing as a cation in acid solution, the removal of which by Mt was mainly by ways of cation exchange (intercalation). It could be inferred that the mechanism of CIP intercalation was mainly via cation exchange by electrostatic interactions rather than forming micelles (Cohen et al., 1984; Chattopadhyay and Traina, 1999; Lv et al., 2014). The above results revealed that the addition in acid modification (i.e. increase in Mt sheet charge) led to decrease in intercalation amount of CIP to Mt, as well as increase in intercalation difficulty. The d001 value of raw Mt was 12.04 Å, and this value increased to 15.24 Å after it was in contact with CIP at an initial concentration of 2000 mg/L (Fig. 1b). In response, the amount of CIP intercalated increased and the corresponding d001 value of AMt became larger. These results indicated a higher charge density would result in a more

3.2. The characterization of Mt with different layer charge and mechanism analysis of CIP-Mt The XRF results of the AMt showed Si, Al, and Fe as the major components, with the content of SiO2 as high as 67.47% (Table 1). Most importantly, with increasing the HCl concentration, the SiO2 content decreased systematically due to progressive dissolution of Si from the tetrahedral sites (Table 1). Then, the layer charges became progressively more negative, resulting charge densities of −0.45, −0.61, − 0.70, −0.83, and −0.91 charge per half (Si, Al)4O10(OH)4 after acid treatment with initial HCl concentrations of 0, 0.05, 0.1, 0.5, and 1.0 M, respectively (Table 2). Corresponding to these treatments, the CEC values, measured by ammonium exchange method, were 0.80, 1.08, 0.84, 0.72, and 0.48 mmol/g. The structural formula, calculated by the XRF data, indicated the progressive increase in the amount of interlayer cations Na+ and Ca2 + per (Si, Al)4O10(OH)4 formula. Thus, the Si content played a vital role in the layer charge and CEC of the Mt (Christidis and Eberl, 2003). Studied the drug release from Mt interlayer space, and considered that exfoliated Mt layers could act as cross linkers between CIP and Mt 125

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Remove quantity (mg/g)

350

(a)

0 mol/L 0.05 mol/L 0.1 mol/L 0.5 mol/L 1.0 mol/L

300 250 200 150 100 50 0 0

2000

4000

6000

8000

10000

Concentration (mg/L) 1.1

(b)

140

0 mol/L 0.05 mol/L 0.1 mol/L 0.5 mol/L 1.0 mol/L

(c)

1.0 0.9

120

Release rate

Release quantity (mg/g)

160

100 80 60 40

0 0

200

400

600

800

0.7 0.6 0.5 0.4 0.3

0 mol/L 0.05 mol/L 0.1 mol/L 0.5 mol/L 1.0 mol/L

20

0.8

0.2 0.1 0.0

1000 1200 1400 1600

100

150

200

Time (min)

250

300

600

Time (min)

Fig. 2. CIP intercalated on acid treated Mt with different CIP concentration (a); Acid treated Mt released CIP with different reaction time (b), and release rate of Mt (c).

(Liu et al., 2008). Computer simulation was used to calculate interaction between Mt layers and interlayer CIP in different CIP-AMt. For Mt modified by acid with various concentrations, Si in silicon-oxygen tetrahedron and Al in aluminum-oxygen octahedron were dissolved out. In analysis of interlayer arrangement of CIP in Mt, Si was used to represent position of Mt sheet and N (positively charged groups in CIP) to represent CIP position, and then simulation results were shown in Fig. 3a–f. Mt sheet (Si) located in position of 5.5–14.8 Å, and interlayer CIP in Mt and AMt (0 to 1.0 M acid modified) in 21–30 Å, 19–26 Å, 18–25 Å, 17–27 Å and 15–20 Å, respectively. It can be observed that, with increasing acid concentration (i.e. increased layer charge of Mt),

Table 2 Property of six kind of acid treated Mt. HCl(mol/L)

0

0.05

0.1

0.5

1.0

Layer charge CEC(mmol/g)

0.45 0.80

0.61 1.08

0.70 0.84

0.83 0.69

0.92 0.48

distance of CIP to silicon-oxygen tetrahedron in Mt gradually reduced. It proves more layer charge of Mt sheet and more interaction force to interlayer CIP caused better efficacy. In addition, arrangement of CIP and position relation of positively

Table 1 Chemical composition of Mt and their chemical formula. Chemical composition (%)

0 mol/L

0.05 mol/L

0.1 mol/L

0.5 mol/L

1.0 mol/L

SiO2 Al2O3 Fe2O3 MgO CaO Na2O Crystal structure formula

67.42 17.34 6.34 5.11 2.43 1.35 (Na0.15Ca0.15) (Al1.23Fe0.28Mg0.45)[Si4O10] (OH)2

62.19 18.79 7.48 4.26 3.87 0.83 (Na0.11Ca0.25) (Al1.14Fe0.34Mg0.38) [(Si3.78Al0.22)O10](OH)2

62.1 19.11 7.46 4.29 4.64 0.67 (Na0.1Ca0.30) (Al1.04Fe0.34Mg0.40) [(Si3.7Al0.3)O10](OH)2

61.1 16.81 8.68 5.78 5.71 0.63 (Na0.09Ca0.37) (Al1.04Fe0.34Mg0.53) [(Si3.7Al0.3)O10](OH)2

60.09 16.11 9.68 6.54 6.35 0.51 (Na0.08Ca0.42) (Al0.88Fe0.45Mg0.62) [Si3.7Al0.3O10](OH)2

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(a)

Relation concentration

0

10

20

16 14 12 10 8 6 4 2 0

30

(c)

0

10

20

16 14 12 10 8 6 4 2 0

30

(e)

0

10

20

16 14 12 10 8 6 4 2 0

Si

30

40

50

60

N-0.05 mol/L

40

50

50

10

20

16 14 12 10 8 6 4 2 0

60

60

Distance (Angstrom)

30

(d)

0

10

20

16 14 12 10 8 6 4 2 0

N-0.5 mol/L

40

(b)

0

Relation concentration

16 14 12 10 8 6 4 2 0

30

(f)

0

10

20

30

Fig. 3. Molecular dynamic simulation of intercalation of CIP into Mt. Location of Si in Mt sheet (a) and the N in the CIP molecules (b–f) from the Mt layers.

N-0 mol/L

40

50

60

N-0.1 mol/L

40

50

60

N-1.0 mol/L

40

50

60

Distance (Angstrom)

were not consistent over time. The results confirmed cation exchange mechanism for CIP adsorption and desorption on charged mineral surfaces (Wu et al., 2013). The effect of incorporation of Mt layers could be significantly observed as reduced release rate at initial stage of immersion. Initially, the specimen was solvated, which facilitated the lateral diffusion of drugs (Takahashi and Yamaguchi, 1999). The release rate was moderate and sustained over the time. Clay minerals, which are layered silicates, have large surface area and can accommodate the various substances in interlayer space to form nanohybrids because of their organized structures (Depan et al., 2009). Release rate of Mt was higher than AMt and reached the release balance at 120 min, maximum release amount was 130 mg/g (Fig. 2b). The maximum of 0.05 M–AMt was obtained at 300 min, where release rate obviously slowed down after 180 min and almost finished at 300 min. For this AMt sample, the release balance was at the range of 180–300 min and notably longer than Mt. Still, time for maximum release amount elongated as increase the layer charge of AMt. Release amount of 1.0 M acid modified Mt balanced at 900 min and the release rate slowed down at 600 min. They indicated that the release balancing time was 600–900 min, and was 700 min longer than that of Mt. The above results indicated that AMt had a great influence on extending the

charged groups and silicone hexagonal ring in Mt can also be known from molecule dynamics simulation. Plan view of position of CIP and Mt layers are shown in Fig. 4. Positively charged groups (− N+) in CIP were above silicone hexagonal ring, which was over the Mg replacing Al site in aluminum oxygen octahedron. It illustrated that the position had strong electrostatic force, for the intercalated CIP mainly arranged by electrostatic force. In 1.0 M–AMt, acid treatment dissolved all Si in silicon-oxygen tetrahedron. Positive groups in CIP finally located above the site where Si dissolved out, meaning stronger electrostatic force than Mg replacing Al site before. In conclusion, after CIP intercalating into different Mt interlayer space, increasing acid concentration could enhance the electrostatic force of Mt sheet and CIP, and increase the release energy of CIP which could extend release time. 3.3. The release of CIP-Mt with different layer charges and characterization of intercalation and release of CIP-Mt To investigate the interaction force between interlayer cation and Mt sheet with different charges, the release amount of CIP-Mt in NaCl over time has been studied. Acid modified Mt with 175 mg/g CIP intercalated samples were used. The release processes of Mt and AMt

(a)

(b)

Fig. 4. Molecular dynamic simulation of intercalation of CIP into Mt. Location of the CIP and [SiO4], 0 mol/L (a) and 1.0 mol/L (b). For all species, C = gray, N = blue, H = white, O = red, Si = yellow, Al = pink, Mg = green, F = light blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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arrangements and amount of intercalated CIP molecules (Wu et al., 2010). Comparing XRD patterns of two kinds of Mt before and after releasing, d001value decrement of Mt was more than that of AMt, indicating release amount of CIP decreased after acid modifying, and stronger interaction force of Mt sheet to interlayer CIP rendering less detachment. In temperature range of 50–200 °C, the mass loss of Mt was due to the removal of the adsorbed surface and interlayer water (Li and Jiang, 2009). In this temperature range, the mass losses of Raw Mt, CIP-Mt, Release-CIP-Mt, 1.0 M CIP-AMt, and Release-1.0 M CIP-AMt were 10.8%, 2.3%, 4.2%, 2.6% and 3.3% respectively (Fig. 6a). Clearly, CIPMt had the least surface absorbed and interlayer water, while Raw Mt was the highest. The higher the degree of organic modification, the less surface absorbed and interlayer water. In temperature range of 200–450 °C, the raw Mt showed no mass loss. The mass losses of CIPMt, Release-CIP-Mt, 1.0 M CIP-AMt, and Release-1.0 M CIP-AMt were 6.2%, 3.4%, 4% and 3.6% respectively, due to the decomposition of CIP. For all five Mts, a mass loss of 5% was detected in the range from 450 to 800 °C attributed to the dehydroxylation of the structural OHgroups. The losses of intercalated Mt were 16%, 14.8%, 12.4% and 11.2% respectively. The different mass losses indicated different interaction forces. The differences of layer charge content for different Mts showed the differences in the ability of interlayer CIP for different decomposition amount. The interaction force made significant influence on the potential barrier associated to the cation hopping process (Belarbi et al., 2007). In other words, amount of layer charge affects the diffusion of the interlayer cations and force of structure layers. The DTA curves show a similar trend in the range of 50–150 °C (Fig. 6b). The endothermic valley was due to removal of interlayer and surface adsorbed water. The second peak of raw Mt appeared in the range from 600 to 700 °C due to the dissociation of the structural hydroxyl group. In contrast, the CIP-Mt, Release-CIP-Mt, 1.0 M CIP-AMt, and Release-1.0 M CIP-AMt were one strong and two weak endothermic peaks in the range from 200 to 800 °C. The peak at 200–250 °C was linked to the decomposition of CIP in the interlayer of Mt, while the peaks at 250–400 °C and 550–750 °C corresponded to the decomposition of the benzene ring in the interlayer of Mt. For intercalated Mt, the decomposition occurred at two different temperatures because the distribution and the binding force of the alkyl chain and the benzene ring in the interlayer of Mt were different (He et al., 2005; Shanmugharaj et al., 2006; Piscitelli et al., 2010). The temperature of the endothermic valley indicated the force between CIP and Mt layers, which are in the order for the five Mts: Release-1.0 M CIP-AMt, 1.0 M CIP-AMt, Release-CIP-Mt, CIP-Mt, Raw Mt. The less mass loss temperature meant stronger electrostatic attraction for the interlayer cation. Therefore, the amount of layer charge played an important role in facilitating the cation exchange of Mt.

releasing time of Mt to CIP, and also reducing release time (maximum 130 mg/g of Mt and 104 mg/g of 1.0 M acid modified Mt, reduced by 26 mg/g). For CIP intercalated Mt, concentration of modified acid affected the release amount, demonstrating different interaction force of Mt with various layer charges to CIP. The interaction between silicate sheets and guest molecules could be Coulombic interactions, van der Waals force, and H-bonding (Takahashi and Yamaguchi, 1991). In addition, the inclusion of nanolayers like Mt into biopolymers may be used for the sustained delivery of drug and other bioactive molecules (Griffith and Naughton, 2002). Differentiating the release amount of various acid modified Mt in Fig. 2b to get release rate at different times are illustrated in Fig. 2c (only 90–600 min was shown). At 90 min, rate of Mt outrun AMt, with increasing modified acid concentration, the rate of AMt went down. At 120 min, release rate of Mt decreased while rate of 0.05 M–AMt reached a peak. Then at 180 min, rate of 0.1 M–AMt was the highest. After 600 min, when all release rates of Mt were nearly zero, while 1.0 M–AMt still released. Above results demonstrated different acid modified Mt had diverse impact on CIP release rate, meaning adjustment of Mt sheet charge considerably extends release time. Directed synthesis of Mt is possible to obtain samples with various drug-release profiles in simulated bacteria environment (Golubeva et al., 2015). This expansion was readily measured by XRD. Modified by the CIP, the interlayer spacing of the Mt was expanded. The basal and interlayer spacing observed for the different minerals are shown in Fig. 5. The differences between the different CIP-Mts were caused by different incorporation and arrangement of the intercalation (Wu et al., 2016). This result was in agreement with a previous study (Günister et al., 2007). The degree of CIP intercalation could be reflected by d001 value of CIP-Mt calculated from the XRD patterns. Initial d001 value of Mt (without intercalation) was 12.04 Å, and this value increased to 15.24 Å when initial concentration of CIP solution was 2000 mg/L. After releasing for 300 min, d001 value of CIP-Mt decreased to 13.14 Å, indicating reduction of CIP in Mt interlayer space, and then reducing the interlayer spacing. According to intercalation amount in different concentrations in Fig. 5, 1.0 M–Mt intercalated at 5000 mg/L of CIP solution, the interlayer spacing was extended to 15.04 Å, indicating a successful intercalation, which intercalation amount and interlayer spacing are similar to Mt. However, after 300 min of releasing, interlayer spacing of Mt decreased to 13.14 Å and that of AMt decreased to 13.75 Å, which shrink of AMt (1.30 Å) was less than shrink of Mt (2.10 Å). The different basal spacing of CIP-adsorbed Mt under acidic, neutral, and alkaline conditions may reflect different interlayer

13.75 R-1.0mol/L 15.05

3.4. The influence of Mt with different layer charges on released CIP affecting bacteria reproduction

Intensity

1.0mol/L The influence of the charge density of Mt in the CIP-Mt on the physical properties and drug-release behavior was the main purpose of this study. At drug release study, CIP showed initial release effect for 24 h and then continuously released for 72 h. Their antibacterial activity was assayed by the inhibitory zone method (Meng et al., 2009). As Mt with different layer charge affected release rate of CIP, the reproduction of pseudomonas aeruginosa around CIP-Mt pills was observed to detect disinfection of CIP-Mt. To simulate the intra-abdominal environment and situation, we observed the antibacterial behavior for 24 h at particular point, such as third to fourth day, rather than a period of cumulative release time, such as 0 to 7th day (Zhang et al., 2015). CIP-Mt pills were numbered as No.1 (blank), No.2 (0 M), No.3 (0.05 M), No.4 (0.1 M), No.5 (0.5 M) and No.6 (1.0 M). In the first 24 h of antibacterial test, all samples with drugs presented big inhibition zone and high antibacterial efficacy (Fig. 7a). Bacteria in No.1 reproduced

13.14 R-0 mol/L

15.24

0 mol/L 12.01 Raw Mt

3

4

5

6

7

8

9

10

2 Theta (°) Fig. 5. X-ray diffraction patterns of Mt intercalated with CIP and released with CIP (d/Å). a = 5.18 Å, b = 8.97 Å.

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102 100 98 96 94 92 90 88 86 84 82 80 78 76 74

(a)

0.0000

(b)

2.3

2.6

3.3 4.2

6.2

-0.0002

4 3.6 3.4

10.8

4.8 11.2

Mt 0 mol/L R-0 mol/L 1.0 mol/L R-1.0 mol/L 100

200

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L. Wu et al.

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-0.0012

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Temperature (°C) Fig. 6. TG (a) and DTA (b) curves of CIP-Mt.

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Fig. 7. Pseudomonas inhibition zone of CIP-Mt on agar plates after incubating specified time.

4. Conclusion

evenly, covering nearly whole Petridish. While in No.2 Petridish, bacteria did not reproduce in a pill centered circle, showing some CIP released from pill to preclude bacteria production. And other pills were also surrounded by circles in different sizes, where the area of circles decreased as the concentration of modifying acid increased, and indicating decrease in CIP release amount. At the third day (Fig. 7b), reduced circle size in No.2 illustrated decreased release amount of CIP, rendering thriving bacteria reproduction outstrips released antibiotics and approached to the pill. Differently, disinfection of other pills gradually increased. Circle area of No.3 grew due to release amount increase, so did circle area in other Petridishes, indicating longer release time of acid modified Mt. At the fifth day (Fig. 7c), circle in No.2 almost disappeared and circle in other dishes also diminished, however, circle area in No.6 Petri dish slightly enlarged and kept good efficacy till the fifth day. Cultivating to the seventh day (Fig. 7d), all pills lost their disinfection and bacteria occupied whole Petri dishes. The above results demonstrated the more modifying acid, the longer disinfection time maintaining, for instance disinfection time of No.6 was 4 days longer than that of No.2. Clay minerals played a very crucial role in modulating drug delivery (Joshi et al., 2009). This indicated that the sustained drug release behavior ensured its sustained long-term antibacterial activity. As short half-life of CIP (2 − 3h), efficacy would not take action to human body without sustained release treatment. Still, frequent intake of drugs would cause great side effect conversely and also develop resistance of bacteria. This sustained release material effectively elongates release time of antibiotic like CIP, and keep long time of efficacy and the half-life. These targeted implantations of loading drugs could extremely decrease the possible side effects.

In this study, a CIP-Mt composite drug release system is prepared, whose sustained ability has been greatly improved, exhibiting slower release rate and longer release time. The CIP-Mt system effectively blocked diffusion-based release, leading to approximately 50% reduction in bolus doses and 10-fold increase in the release timescale. The sustained ability is enhanced by layer charge adjustment, and the improvement shows the potential of application in drug delivery to elongate half-life of drugs. When CIP intercalated to Mt interlayer, the releases of CIP is affected by the distribution of CIP in the interlayer spaces and its interaction with the Mt layers. Changes in charge density of Mt lead to variations in electric strength in the interlayer. The CIP-Mt extends release time, and release amount is also stable and controllable, whose characteristics are applicable to practical retard drug system. Acknowledgments This research was jointly funded by Scientific Research Project of Shenyang Jianzhu University (2017044) and National Natural Science Foundation of China (51604248). References Akahane, K., Kato, M., Takayama, S., 1993. Involvement of inhibitory and excitatory neurotransmitters in levofloxacin- and ciprofloxacin-induced convulsions in mice. Antimicrob. Agents Chemother. 37, 1764–1770. Belarbi, H., Haouzi, A., Douillard, J.M., Giuntini, J.C., Henn, F., 2007. Hydration of a

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