Materials Science and Engineering C 29 (2009) 1161–1166

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Materials Science and Engineering C 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 / m s e c

Antibacterial activity of ciprofloxacin-loaded zein microsphere films Jian-Xi Fu a,b,1, Hua-Jie Wang c,1, Yan-Qing Zhou b, Jin-Ye Wang a,c,⁎ a b c

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China Henan Normal University, 46 East Construction Road, Xinxiang, Henan 453007, China College of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China

a r t i c l e

i n f o

Article history: Received 7 May 2008 Received in revised form 16 August 2008 Accepted 15 September 2008 Available online 30 September 2008 Keywords: Antibacterial Ciprofloxacin Zein Microsphere Sustained release

a b s t r a c t Our aim was to produce an antibiotic-emitting coating composed of zein microspheres for the prevention of bacterial infection on implanted devices. Ciprofloxacin-loaded zein microspheres were prepared using a phase separation procedure, with particle sizes between 0.5 and 2 µm. Drug encapsulation and drug loading varied with the amount of both zein and ciprofloxacin, and the highest encapsulation efficiency was 8.27% (2 mg/ml ciprofloxacin and 20 mg/ml zein; n = 3). A ciprofloxacin-loaded zein microsphere film (CF-MS film) was generated via solvent evaporation. Continuous drug release from a trypsin-degraded microsphere film was observed for up to 28 days. The liberation of ciprofloxacin from the trypsin-degraded film and the biodegradation of the microsphere film were highly correlated. Proliferation assay of the growth of human umbilical vein endothelial cells (HUVECs) by the MTT method showed that the microsphere film had no toxicity when compared with cells grown on Corning culture plates alone and plates with a zein film alone. Quantification of bacteria adhesion showed that adhesion on the microsphere film is significantly suppressed. In addition, according to the results of bacterial growth tests, ciprofloxacin-loaded microsphere films maintained antibacterial activity for more than 6 days. In contrast, a control medium containing a zein film allowed constant bacterial growth. These results indicate that CF-MS films might be useful as antibacterial films on implanted devices. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Bacterial infection on implanted devices is a significant clinical problem and is associated with bacterial adhesion, bacterial proliferation, and biofilm formation [1]. The most common reason for infections is the presence of such bacterial strains as Staphylococcus aureus, Escherichia coli, Enterococcus spp., and Pseudomonas aeruginosa [2,3]. Traditional treatments for bacterial infection include systemic and local administration of antibiotics. Because systemic antibiotic treatment can cause several side effects (sensitivity, resistant strains, superinfections), the local administration of antibiotics has received considerable attention [4]. One method for local administration involves coating or impregnating biomaterials with antibiotics. However, there are important limitations to this technique, such as the fact that antibacterial activity cannot be sustained for a long period due to physical adsorption of the antibiotic onto the biomaterial [5,6]. Recently, researchers have attempted to resolve this problem using controlled delivery systems for a number of antimicrobial drugs [7–11]. Treatment of bacterial infection with localized drug delivery systems is ⁎ Corresponding author. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China. Tel.: +86 2154925330; fax: +86 2164166128. E-mail address: [email protected] (J.-Y. Wang). 1 The authors contributed equally to this work. 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.09.031

becoming more prevalent, with a number of products now commercially available [12,13]. So far, local release of antibiotics has only been clinically applied with nondegradable carrier materials such as polymethyl methacrylate (PMMA), which have the disadvantage of requiring a second surgical procedure for their removal. To overcome these disadvantages, biodegradable polymers have been examined as carrier materials for antibiotics [1]. Ciprofloxacin (CF) is a third-generation fluoroquinolone with a broad spectrum of antibacterial activity, and it has been effectively used to treat a variety of bacterial infections including symptomatic urinary tract infections, gastrointestinal infections, and skin and bone infections. It has been reported that ciprofloxacin is more effective against both Gram-negative and Gram-positive pathogens in vitro and in vivo than many other antibiotics [14,15]. Ciprofloxacin has been shown to have a superior ability to penetrate most tissues compared to other antibiotics, and ciprofloxacin can reach organs including the livers, lungs, and lymph nodes [16]. However, ciprofloxacin does not preferentially accumulate in these tissues and may therefore not reach sustained therapeutic levels at these sites unless higher drug doses are used [17]. Furthermore, the frequent use of ciprofloxacin has been associated with arthritic damage in children and possible stimulation of the nervous system [18]. One potential method to solve these problems is to use microspheres as a sustained delivery system.

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Zein is the major storage protein of corn and comprises 40–50% of the protein in corn. It was first identified in 1897, based on its solubility in aqueous alcohol solutions (60–95%) [19]. We have used zein to prepare films composed of microspheres with diameters of 100–2500 nm with good biocompatibility [20]. Zein microspheres have been used to deliver insulin, but these microspheres are quite heterogeneous in size [21]. In previous work, we successfully produced ivermectin-loaded and heparin-loaded zein microspheres [22,23]. The major aim of the present study was to develop a biodegradable ciprofloxacin–zein microsphere film with controlled release characteristics and to evaluate its antibacterial properties.

2.5. In vitro release of ciprofloxacin from microspheres

2. Materials and methods

2.6. In vitro release and degradation studies of ciprofloxacin-loaded zein microsphere film

Ciprofloxacin microsphere powder was washed with water to remove unencapsulated drug, then introduced into dialysis bags and dialyzed against 30 ml PBS (pH 7.4) at 37 °C. A release protocol was adopted as follows: 3 ml of supernatant was taken from the system while adding the same volume of PBS to keep the total volume constant at 30 ml. The supernatant was diluted five times with PBS (pH 7.4) and the absorbance at 322 nm was recorded. The cumulative amount released was integrated from each measurement, with concentrations determined from a calibration curve.

2.1. Materials Biochemically pure zein was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), ciprofloxacin was purchased from Jiachen Chemical Industries, Ltd. (Shanghai, China), endothelial cell growth supplement (ECGS) was from Upstate Biotechnologies Inc. (Lake Placid, NY, USA), and other reagents were reagent grade. Strains of E. coli and S. aureus were obtained from Henan Normal University. All strains were maintained as freeze-dried cultures in our laboratory and reconstituted by growth on nutrient agar plates. 2.2. Preparation of ciprofloxacin-loaded zein microspheres and film Ciprofloxacin-loaded zein microspheres (CF-MS) were obtained using a phase separation procedure. Typically, 300 mg zein and 60 mg ciprofloxacin were dissolved in 10 ml of 60% ethanol, then 5 ml of MilliQ water (PALL, USA) was added immediately to form a microsphere suspended solution [22]. The above solution was poured onto a glass disc surface and volatilized at 30 °C to prepare a ciprofloxacin-loaded zein microsphere film (CF-MS film). The film was washed with water for 10 min before the next use. The lyophilized microsphere suspended solution was used for encapsulation efficiency determination. 2.3. Morphology analysis A scanning electron microscope (JSM-6700F, JEOL, Japan) was used to observe the morphology of microspheres and microsphere films, which were mounted onto brass stubs, vacuum-dried at room temperature and sputter-coated with gold prior to examination. Surface morphology was also determined as roughness using an atomic force microscopy (AFM, Nanoscope IIIa, Digital Instruments, CA, USA) at ambient temperature in non-contact mode with a conical silicon high resonance frequency probe. The roughness parameter for the surface, Ra, which is the centerline average or the distance between the highest and the lowest point of the surface irregularities, was calculated by Nanoscope III software (Version 5.30r3sr3). 2.4. Ciprofloxacin loading and encapsulation efficiency Quantification of ciprofloxacin was analyzed using an ultraviolet– visible spectrophotometer at 322 nm (HITACHI, U-3010, Japan), at which there was no adsorption for zein. The standard curve of ciprofloxacin could be represented as: A=0.0357C+0.0458 in PBS, and A=0.0332C+ 0.0147 in 66.7% ethanol (5 µg/ml–30 µg/ml, r2 =0.9999). The encapsulation efficiency of ciprofloxacin was determined by the following method. Lyophilized microsphere powder was washed 3× with 1 ml water and vacuum-dried. Microspheres were then dissolved in aqueous ethanol and analyzed spectrometrically. Ciprofloxacin encapsulation efficiency and ciprofloxacin loading were determined by the equations: encapsulation efficiency (% w/w)=(amount of ciprofloxacin in microspheres/ciprofloxacin initially added)×100 and ciprofloxacin loading (% w/w)= (amount of ciprofloxacin in microspheres/amount of microspheres)×100.

Release and degradation studies were conducted in trypsin solution (100 nfu/ml in PBS, pH 7.4) and collagenase solution (100 units/ml in PBS, pH 7.4), respectively. Ciprofloxacin-loaded zein microsphere film was immersed in the trypsin solution and incubated at 37 °C. At prescribed time intervals, samples were removed from the releasing medium and placed in fresh releasing solution. Ciprofloxacin content was measured spectrophotometrically at 322 nm. The degradation rate of microsphere film was analyzed using an ultraviolet–visible spectrophotometer at 215 nm and 225 nm. Degraded zein content was determined according to the absorption difference between 215 nm and 225 nm [22]. The percentage of ciprofloxacin cumulative release (% w/w) and the zein degradation rate were examined as a function of incubation time. Each experiment was performed in four replicates. 2.7. In vitro cytotoxicity evaluations Human umbilical vein endothelial cells (HUVECs) were harvested from fresh umbilical cords [24] and maintained in a humidified atmosphere of 5% CO2/air at 37 °C. Third passage HUVECs were detached using trypsin (0.25%)/EDTA (0.02%), resuspended in RPMI 1640 medium (20% fetal calf serum, 30 µg/ml endothelial cell growth supplement (ECGS), 100 units/ml penicillin, 100 µg/ml streptomycin), and seeded at 1.2 × 104 cells/ml into 96-well Corning culture microplates with ciprofloxacin-loaded microsphere films or zein films. Other groups included a zein film with 20 µg/ml ciprofloxacin group and a Corning culture microplate control group. The medium was changed after 2 days. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were performed to detect viable cells after 1, 2, and 3 days of culture. 2.8. Quantification of bacterial adhesion in vitro Testing for bacterial adhesion was performed in vitro using the CFU plate counting method, which is the most basic method for bacterial enumeration [25]. The bacteria used in this study were E. coli and S. aureus. Before testing, bacteria were grown in Luria–Bertani (LB) medium, and E. coli and S. aureus cultures were diluted such that the bacterial concentrations were 1.24 × 106 cfu/ml and 2.18 × 106 cfu/ml, respectively. Glass discs, zein films, and microsphere films were sterilized with UV irradiation for 1 h before experiments. Glass discs, ciprofloxacin-loaded microsphere films, and zein films were placed in the diluted bacterial suspension and cultured at 37 °C for 24 h. Samples from the three groups were removed from the cultures and rinsed 3× in PBS. Adhered bacteria were detached from samples by ultrasonic cleaning, and viably detached bacteria were counted using the CFU plate counting method. 2.9. Tests of sustained antibacterial activity of microsphere films in vitro Ciprofloxacin-loaded microsphere films were immersed in PBS (pH 7.4) and incubated at 37 °C. Samples were removed at prescribed

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intervals (1–6 days), washed with PBS (pH 7.4), and the ciprofloxacin released from the microsphere films was measured at each time point. The zein films was tested as the control. The antibacterial activity of ciprofloxacin was tested using E. coli and S. aureus. Before testing, inocula of each bacterium were grown in LB medium. Inocula of E. coli were used at 1.25 × 108 and 1.25 × 106 cfu/ml and inocula of S. aureus were used at 2.16 × 108 and 2.16 × 106 cfu/ml. Inocula of each bacterial strain were mixed with LB medium containing sample films. After a 24 h incubation period at 37 °C, samples from each experiment were tested for bacterial growth by measuring the optical density at 550 nm. 3. Results and discussion 3.1. Characterization of ciprofloxacin-loaded zein microspheres and films In this study, we used the phase separation method to prepare ciprofloxacin-loaded zein microspheres. Scanning electron micrographs of microspheres and a microsphere film are shown in Fig. 1. Microspheres were spherical in shape and had nearly smooth surfaces. Fig. 1A also shows many ciprofloxacin crystals in cross-sections of microspheres, and microspheres had diameters between 0.5 and 2 µm. Fig. 1B shows that microsphere films are composed of microspheres, but no ciprofloxacin crystals were visible. This could be due to the ciprofloxacin penetrating deep into the film during evaporation. 3.2. Ciprofloxacin loading and encapsulation efficiency Drug loading levels for ciprofloxacin-loaded zein microspheres, expressed as absolute drug loading and encapsulation efficiency, are

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Table 1 Ciprofloxacin loading and encapsulation efficiency in zein microspheres (n = 3). Initial concentrations of ciprofloxacin and zein

Encapsulation efficiency (% w/w)a

Ciprofloxacin loading (% w/w)b

2 3 4 5 4 4 4 3 2

8.29 ± 0.3 7.89 ± 0.39 7.06 ± 0.22 6.27 ± 0.27 6.09 ± 0.5 5.24 ± 0.17 4.97 ± 0.09 7.02 ± 0.63 5.74 ± 0.05

0.87 ± 0.05 1.18 ± 0.1 1.54 ± 0.06 1.64 ± 0.03 1.47 ± 0.01 1.74 ± 0.02 2.41 ± 0.18 1.68 ± 0.11 1.32 ± 0.05

mg/ml ciprofloxacin, 20 mg/ml zein mg/ml ciprofloxacin, 20 mg/ml zein mg/ml ciprofloxacin, 20 mg/ml zein mg/ml ciprofloxacin, 20 mg/ml zein mg/ml ciprofloxacin, 16 mg/ml zein mg/ml ciprofloxacin, 12 mg/ml zein mg/ml ciprofloxacin, 8 mg/ml zein mg/ml ciprofloxacin, 12 mg/ml zein mg/ml ciprofloxacin, 8 mg/ml zein

a Encapsulation efficiency (% w/w) = amount of ciprofloxacin in microspheres / ciprofloxacin initially added. b Ciprofloxacin loading (% w/w) = amount of ciprofloxacin in microspheres / amount of microspheres.

presented in Table 1. Absolute drug loading levels between 8.7 and 24 µg drug/mg microspheres and encapsulation efficiencies between 4.97% and 8.29% were measured. Drug loading and encapsulation efficiency differentially depended on the ratio of ciprofloxacin to zein: encapsulation efficiency increased while loading decreased with a decreasing ratio of ciprofloxacin to zein. For ciprofloxacin, encapsulation efficiency did not change significantly when the drug concentration was increased from 2 mg/ml to 5 mg/ml, the drug loading increased significantly from 8.7 to 16.4 µg drug/mg microspheres. This increase in loading is likely to be due simply to the increased overall amount of drug. As shown in Table 1, the optimum condition to get higher ciprofloxacin loading and encapsulation efficiency was: 4 mg/ml ciprofloxacin and 20 mg/ml zein. We can find that the average range for ciprofloxacin loading and encapsulation efficiency is above 1.5 and 7%, respectively in this condition. 3.3. Release kinetics of ciprofloxacin from microspheres To understand the characteristics of drug release from ciprofloxacinloaded microspheres, in vitro release experiments were carried out. The profile of drug release from microspheres is illustrated in Fig. 2. The drug release rate of ciprofloxacin-loaded microspheres fitted best to first-order release kinetics, with a correlation coefficient of 0.97 (ln(1−Mt /M∞) = −0.216t, where Mt is the accumulative amount of drug released at t, M∞ is the total drug concentration, t is the time). The release profile consisted of an initial rapid phase (nearly 70% of the drug was released after 4 h) followed by a sustained release period for 2 days, with nearly 100% of the ciprofloxacin eventually being released. The result indicated that there was no significantly sustained efficacy in ciprofloxacin-loaded microsphere suspension system.

Fig. 1. Scanning electron micrographs of ciprofloxacin-loaded zein microspheres (upper) and a microsphere film (lower).

Fig. 2. In vitro release profile of ciprofloxacin from zein microspheres in PBS (pH 7.4, 37 °C, n = 3).

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Fig. 5. Proliferation of HUVECs on Corning plate controls (●), zein films (○), zein films treated with ciprofloxacin (△, 20 µg/ml), and ciprofloxacin-loaded zein microsphere films (□) after 3 days in culture.

are so small that they may also lead to a fast initial release because of the high ratio of surface area to volume. Then a slow release period was followed in which the remaining drug captured in the microsphere structure was released in a controlled fashion. 3.4. In vitro release and degradation of ciprofloxacin-loaded zein microsphere films

Fig. 3. Influence of enzyme degradation on ciprofloxacin release from ciprofloxacin-loaded zein microsphere films at pH 7.4 and 37 °C. (A) In vitro release curve of microsphere film (○, PBS), trypsin-degraded microsphere film (□, 100 nfu/ml trypsin) and collagenasedegraded microsphere film (△, 100 units/ml collagenase); (B) in vitro degradation of microsphere films with enzyme (□, 100 nfu/ml trypsin; △, 100 units/ml collagenase).

There are several parameters corresponding to the drug release profile of microspheres, such as the rate of water uptake, drug dissolution/diffusion rate and degradation rate [26,27]. Some studies have demonstrated that the release of low molecular drugs such as progesterone or phenothiazines from polycaprolactone-based microparticles was rapid [28], this phenomenon is attributed to the molecular dispersion of the drugs in the polymer. Because ciprofloxacin is a water-soluble low molecular drug, fast release rate of ciprofloxacin from microspheres was observed in the initial phase. On the other hand, microspheres with an average diameter of about 1 µm

We next investigated the release and degradation characteristics of CF-MS films (4 mg/ml ciprofloxacin and 20 mg/ml zein) using trypsin and collagenase. Fig. 3A shows the release of ciprofloxacin from CF-MS films with or without enzyme treatments. A burst effect was also observed in all groups even the films have been washed before analysis. Washing treatment could only remove the unencapsulated drug on the film surface, which could be considered as the noneffective drug when the film is implanted and contacted with body fluids such as blood. In the burst stage, over 50% of ciprofloxacin was released within 12 h, which might be due to the unencapsulated drug near/under the surface and that adsorbed on the surface of the microsphere films. After the initial drug release, significant differences were observed in the subsequent “slower” phase: the release rate of microsphere film in collagenase solution was a little higher than in PBS, and the rate in trypsin solution was the highest. After 28 days, drug release from the microsphere films in PBS and in collagenase reached 67% and 75%, respectively. While for microsphere film in

Fig. 4. SEM images of the zein films after incubation at 37 °C for 20 days with and without enzyme. (A) microsphere film in PBS; (B) collagenase-degraded microsphere film (100 units/ml collagenase); (C) trypsin-degraded microsphere film (100 nfu/ml trypsin).

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on zein film, ciprofloxacin-loaded zein microsphere film, and zein film treated with ciprofloxacin (20 µg/ml) was observed compared with HUVECs on Corning culture microplate, but cells on zein film treated with ciprofloxacin and microsphere film showed no significant difference from the zein film control group. A potential advantage of microsphere films as drug delivery systems is the ability to control ciprofloxacin concentrations to avoid side effects resulting from excessively high drug concentrations [18]. These results show that microsphere films are not cytotoxic, and on the contrary, have good biocompatibility and promote cell growth. This suggests that when the drug is released from the microsphere film, cells around the film can proliferate at the same time. 3.6. Bacterial adhesion

Fig. 6. Number of bacteria adhered on glass controls, zein films and ciprofloxacin-loaded microsphere films after incubation for 24 h (n = 4). ⁎Compared with glass discs, p b 0.05; # compared with zein films, p b 0.01. The inocula used were: E. coli, 1.5× 106 cfu/ml; S. aureus, 2.4 × 106 cfu/ml.

trypsin solution, drug release eventually reached 100%. Fig. 3B shows the degradation prosperities of microsphere films as a function of time. The degradation rate of trypsin-degraded microsphere film was significantly higher than for collagenase-degraded microsphere film. The film was degraded nearly 80% in trypsin solution, whereas only 7% of the film was degraded in collagenase solution after 28 days. The correlation between the release and degradation rate is noteworthy: the correlation coefficient was 0.965. SEM observation of the films further confirmed the release mechanism in the subsequent “slower” phase. When there was no digestive enzyme existed, the film surface was smooth and the drug entrapped into microspheres could not continue to be released by diffusion into the medium even after 20 days (Fig. 4A). So its release kinetics of drug–zein microspheres was characterized as no erosion incomplete release. While a significant erosion of the film incubated with trypsin was observed at 20 days (Fig. 4C). Its release kinetics showed almost a zero-order behavior after an initial burst period, with 100% of the drug being eventually released. These results suggest that the faster release rate corresponds to a faster degradation of the microsphere film. Because zein is highly degradable in vivo [29], we speculate that complete drug release from zein microsphere films might occur in vivo.

In this experiment, the bacteriostatic activity of microsphere films was tested in the presence of E. coli and S. aureus strains. Fig. 6 demonstrates that the adhesion of both strains on microsphere film was significantly suppressed by ciprofloxacin (p b 0.01). The adhered concentration of bacteria on the surface of CF-MS film is only about 1% of that on zein film. However, more bacteria adhered to zein film than to glass disc. Bacterial infection begins with bacterial adhesion, followed by colonization. Therefore, it is important to inhibit the initial adhesion of bacteria on implanted device surfaces [31]. According to the results of the bacterial adhesion test (see above), relatively more bacteria adhered to the zein film, which was consistent with our previous results of the cell culture (NIH3T3 and HL-7702). The zein film is beneficial for two kinds of cells for adhesion, comparing to the culture plate [20]. Another possible reason may be the rough surface of the zein microsphere film (Ra = 104.4 ± 43.9 nm, n = 10), which could support more bacterial adhesion than the smooth surface such as glass (Ra = 1.3 ± 0.4 nm, n = 10). However, the number of bacteria adhered to the microsphere film was significantly reduced by ciprofloxacin release. Recently, the use of antibacterial-coated (or -impregnated) medical devices has become widespread within the health care environment. Owing to unsatisfactory cell adhesion and the susceptibility of the implants to bacterial infections, titanium-based implant materials have specific complications associated with their application [32]. According to the results of this study, CF-MS film was highly effective in reducing the adherence of Gram-positive and Gramnegative strains and thus has the potential to prevent these complications. 3.7. Sustained antibacterial tests

3.5. In vitro cytotoxicity evaluations To evaluate the cytotoxicity of microsphere films, HUVEC proliferation was measured using the MTT method [30]. Fig. 5 shows the viability of HUVECs detected by the MTT assay as a function of culture time. An obvious enhancement of mitochondrial activity from HUVECs

We observed sustained bactericidal activity of the microsphere film against tested bacterial strains. As Table 2 shows, the tested bacteria (at each infecting dose) did not grow in LB medium exposed to the CF-MS film. When cultured in media exposed to films for 1 to 6 days, only very low E. coli growth was observed when infecting

Table 2 Optical density measurements of E. coli and S. aureus incubated in LB medium at 37 °C after 24 h in the presence of ciprofloxacin-loaded zein microsphere films that had released for different times in PBS (pH 7.4) (n = 4). Strain

Infecting dose (cfu/ml)

OD at 550 nm measured Release time (days) 1

E. coli S. aureus

1.25 × 108 1.25 × 106 2.16 × 108 2.16 × 106

2

4

6

M

C

M

C

M

C

M

C

0.06 ± 0.05 0 0 0

1.03 ± 0.20 0.57 ± 0.11 0.88 ± 0.08 0.68 ± 0.15

0.07 ± 0.02 0 0 0

1.15 ± 0.28 0.68 ± 0.16 0.71 ± 0.08 0.96 ± 0.07

0.06 ± 0.02 0 0 0

1.03 ± 0.19 0.77 ± 0.08 0.80 ± 0.12 0.77 ± 0.20

0.06 ± 0.04 0 0 0

1.16 ± 0.22 0.70 ± 0.11 0.78 ± 0.16 0.58 ± 0.04

M: CF-MS film; C: zein film control. Note: Prior to measurements, S. aureus incubated in LB medium in the presence of zein films after a 24 h cultivation period were diluted 1:2 (v/v) in LB medium to obtain concentrations in the measure range. Other groups were analyzed directly without dilution.

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doses of 1.25 × 108 cfu/ml were used. In the control sample, robust bacterial growth was observed (p b 0.01). Because ciprofloxacin was continuously released from the microsphere film, antibacterial activity continued for at least 6 days. This property has the potential to protect the biomaterial (as well as surrounding liquids) from bacterial infections and prevent biofilm formation on the film surface [33]. This time period seems to be sufficient to avoid biofilm formation and to reduce the frequency of infections after implantation. According to the results of the release and degradation studies (see above), ciprofloxacin was released quickly when the microsphere film was being gradually hydrolyzed by trypsin, so we assume that any antibacterial activity will be enhanced by the activity of the patient's own proteases in vivo. 4. Conclusion To decrease the frequency of bacterial infection on implanted devices, it is essential to eradicate the pathogens from the implantation site, both immediately and several days after implantation. In this study, we prepared ciprofloxacin-loaded zein microsphere film. As zein is a hydrophobic material, the solubility of drug might be the most important issue in encapsulation of the drug. Besides, drug loading and encapsulation efficiency depend on the ratio of drug to zein: encapsulation efficiency increased while loading decreased with a decreasing ratio of ciprofloxacin to zein. Although most amount of the drug could be removed in the burst release phase, sufficient amount of the drug to avoid the bacterial adhesion could still be achieved with our drug–zein microsphere film for at least 6 days. This microsphere film has the potential to be used as a drug delivery system with clinical applications for the prevention or treatment of biomaterial-related infections. Further tests and clinical investigations should be carried out to improve the functional properties and to enhance the applicability of the system. Acknowledgements This study was supported by the National Program on Key Basic Research Projects of China (973 Program, 2005CB724306) and the National Natural Science Foundation of China (30870635). References [1] A. Montali, Injury 37 (Suppl.) (2006) 81. [2] G. Metan, P. Zarakolu, B. Ca̧kira, G. Hascelik, O. Uzun, International Journal of Antimicrobial Agents 26 (2005) 254.

[3] P. Nordmann, T. Naas, N. Fortineau, L. Poirel, Current Opinion in Microbiology 10 (2007) 436. [4] M. Diefenbeck, T. Mückley, G.O. Hofmann, Injury 37 (suppl.) (2006) 95. [5] J.K. Baveja, M.D.P. Willcox, E.B.H. Hume, N. Kumar, R. Odell, L.A. Poole-Warren, Biomaterials 20 (2004) 5003. [6] J.A. Berry, J.F. Biedlingmaier, P.J. Whelan, Otolaryngology—Head and Neck Surgery 123 (2000) 246. [7] G. Ginalska, D. Kowalczuk, M. Osińska, International Journal of Pharmaceutics 288 (2005) 131. [8] N. Blanchemain, S. Haulon, B. Martel, M. Traisnel, M. Morcellet, H.F. Hildebrand, European Journal of Vascular and Endovascular Surgery 29 (2005) 628. [9] S. Radin, P. Ducheyne, Biomaterials 28 (2007) 1721. [10] H.-I. Chang, Y. Perrie, A.G.A. Coombes, Journal of Controlled Release 110 (2006) 4141. [11] L. Pasquardini, L. Lunelli, L. Vanzetti, M. Anderle, C. Pederzolli, Colloids and Surfaces B: Biointerfaces 62 (2008) 265. [12] H.M. Kelly, P.B. Deasy, E. Ziaka, N. Claffey, International Journal of Pharmaceutics 274 (2004) 167. [13] L. Montanaro, D. Campoccia, C.R. Arciola, Biomaterials 28 (2006) 5155. [14] D.A. Talan, K.G. Naber, J. Palou, D. Elkharrat, International Journal of Antimicrobial Agents 23 (Suppl.) (2004) 54. [15] K. Dillen, J. Vandervoort, G. Van den Mooter, A. Ludwig, International Journal of Pharmaceutics 314 (2006) 72. [16] M.S. Hanan, E.M. Riad, N.A. El-Khouly, Deutsche Tierarztlliche Wochenschr 107 (2000) 151. [17] J.P. Wong, H. Yang, K.L. Blasetti, G. Schnell, J. Conley, L.N. Schofield, Journal of Controlled Release 92 (2003) 265. [18] Z. Mao, L. Ma, C. Gao, J. Shen, Journal of Controlled Release 104 (2005) 193. [19] R. Shukla, M. Cheryan, Industrial Crops and Products 13 (2001) 171. [20] J. Dong, Q.S. Sun, J.-Y. Wang, Biomaterial 25 (2004) 4691. [21] H. Bernstein, E. Morrel, E. Mathiowitz, K. Schwaller, T.R. Beck, US Patent (1997) No. 5679377. [22] X.M. Liu, Q.S. Sun, H.J. Wang, L. Zhang, J.Y. Wang, Biomaterial 26 (2005) 109. [23] H.J. Wang, Z.X. Lin, X.M. Liu, S.Y. Sheng, J.Y. Wang, Journal of Controlled Release 105 (2005) 120. [24] E.A. Jaffe, R.L. Nachman, C.G. Becker, Journal of Clinical Investigation 52 (1973) 2745. [25] J.X. Li, J. Wang, L.R. Shen, Z.J. Xu, P. Li, G.J. Wan, N. Huang, Surface and Coatings Technology 201 (2007) 8155. [26] F. Oneda, M.I. Ré, Powder Technology 130 (2003) 377. [27] O. Franssen, R.J.H. Stenekes, W.E. Hennink, Journal of Controlled Release 59 (1999) 219. [28] R.-K. Chang, J.C. Price, C.W. Whitworth, Drug Development and Industrial Pharmacy 13 (1987) 249. [29] H.J. Wang, S.J. Gong, Z.X. Lin, J.X. Fu, S.T. Xue, J.C. Huang, J.Y. Wang, Biomaterial 28 (2007) 3952. [30] P. Da Silva Melo, N. Durán, M. Haun, Toxicology Letters 116 (2000) 237. [31] Y.L. Jeyachandran, S. Venkatachalam, B. Karunagaran, Sa.K. Narayandass, D. Mangalaraj, C.Y. Bao, C.L. Zhang, Material Science and Engineering C 27 (2007) 35. [32] P.-H. Chua, K.-G. Neoh, E.-T. Kang, W. Wang, Biomaterial 29 (2008) 1412. [33] G. Ginalska, A. Uryniak, M. Osińska, T. Urbanik-Sypniewska, Polski Przeglad Chirurgiczny 76 (2004) 1063.