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LDPE composite for copper-containing intrauterine contraceptive devices
Acta Biomaterialia 8 (2012) 897–903
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
A porous Cu/LDPE composite for copper-containing intrauterine contraceptive devices Weiwei Zhang, Xianping Xia ⇑, Cheng Qi, Changsheng Xie, Shuizhou Cai State Key Laboratory of Material Processing and Die & Mould Technology, Department of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 27 May 2011 Received in revised form 7 August 2011 Accepted 20 September 2011 Available online 24 September 2011 Keywords: Porous Cu/LDPE composite DTBHQ Ethyl acetate Injection molding and particulate leaching Cupric ion release
a b s t r a c t To improve the rates of both cupric ion release and the utilization of copper in non-porous copper/lowdensity polyethylene (Cu/LDPE) composite, a porous Cu/LDPE composite is proposed and developed in the present work. Here 2,5-di-tert-butylhydroquinone was chosen as the porogen, ethyl acetate was chosen as the solvent for extraction, and the porous Cu/LDPE composite was obtained by using injection molding and the particulate leaching method. After any residual ethyl acetate remaining inside the porous Cu/LDPE composite had been removed by vacuum drying, the composite was characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, gas chromatography– mass spectrometry and absorption measurement. For comparison, a non-porous Cu/LDPE composite was also characterized in the same way. The results show that the porous structure was successfully introduced into the polymeric base of the non-porous Cu/LDPE composite, and the porous Cu/LDPE composite is a simple hybrid of copper particles and porous LDPE. The results also show that the introduction of a porous structure can improve the cupric ion release rate of the non-porous Cu/LDPE composite with a certain content of copper particles, indicating that the utilization rate of copper can be improved either the introduction of a porous structure, and that the porous Cu/LDPE composite is another promising material for copper-containing intrauterine devices. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Copper-containing intrauterine devices (Cu-IUDs) are contraceptive devices containing copper on a plastic frame or a thread (frameless) that are inserted through the cervix and placed within the uterine cavity. They are one of the most commonly used forms of fertility control worldwide, and are cheap, safe, reliable, convenient, reversible and culturally acceptable [1]. However, side effects caused by existing Cu-IUDs, such as pain and bleeding, have not yet been overcome [2–5]. It is believed that at least some of these side effects may be related to the burst release of cupric ions within the first few months after insertion of the Cu-IUD [6,7]. The cytotoxic and genotoxic effects of cupric ion released from metallic copper have been investigated in Chinese hamster ovary cells [8,9], and the results show that a reduction in mitochondrial activity can be observed when the cupric ion concentration exceeds 7.42 lg ml 1; in addition, a decrease in cell viability of up to 90% was found at a cupric ion concentration of 10.85 lg ml 1; finally, copper-induced DNA damage is detected when the cupric ion concentration is in the range of 5.67–7.42 lg ml 1. Thus, the optimal
⇑ Corresponding author. Tel.: +86 27 87556544; fax: +86 27 87543778. E-mail address: [email protected] (X. Xia).
cupric ion concentration released from Cu-IUDs in uterine solution should be less than 5.50 lg ml 1. To eliminate these deficiencies of the existing Cu-IUDs, a novel system for the controlled release of cupric ions, the non-porous copper/low-density polyethylene (Cu/LDPE) composite, has been developed for Cu-IUDs [10,11]. Our previous investigations [12– 14] have verified that a Cu/LDPE composite and its devices can lessen the side effects, such as pain and bleeding, that are caused by the existing Cu-IUDs remarkably, and have the same excellent contraceptive efficacy as the existing Cu-IUDs. However, our recent study [15] shows that the cupric ion release rate of such a non-porous Cu/LDPE composite IUD, with 25 wt.% content of copper particles, in 20 ml of simulated uterine solution (SUS) was equivalent to only 2.50 lg ml 1 in uterine fluid (no more than 0.8 ml [16]) during the early stage of the stable release, and its predicted lifespan is only about 5 years. This indicates that the lifespan of composite IUDs is relatively short, which means that the effective utilization of copper particles in composite IUDs will only be about 30 wt.% even if close to 100% of the copper particles can be converted into cupric ions in SUS [17]. Fortunately, non-porous Cu/LDPE composite devices are only one kind of drug delivery system, and a number of investigations on drug delivery systems [18–20] have shown that the introduction of pores into the drug carrier can remarkably improve its drug
1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.09.024
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release rates. Therefore, in the present study we have attempted to improve the cupric ion release rate of novel Cu/LDPE composite IUDs by introducing a porous structure into the matrix of the device. Several methods can be used for the preparation of porous composites, such as extrusion assisted by supercritical carbon dioxide [21], co-extrusion and gas foaming [22], injection molding and particulate leaching [23], gas foaming [24], freeze-drying [25] and phase separation [26]. Here, we used the injection molding and particulate leaching technique to prepare a porous Cu/LDPE composite. 2,5-Di-tertbutylhydroquinone (DTBHQ) was chosen as the porogen due to its extraordinary antioxidation property, its non-toxicity [27–30] and its excellent solubility in solvents such as ethyl acetate. Ethyl acetate was chosen as the solvent for extraction. Most importantly, DTBHQ and ethyl acetate do not react with copper particles or with the LDPE. After samples of the porous Cu/LDPE composite had been obtained, their structures were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), and their residual DTBHQ and ethyl acetate were analyzed by gas chromatography–mass spectrometry (GC–MS). Finally, the cupric ion release rate of the porous Cu/LDPE composite samples was measured by absorption measurement. For comparison, a non-porous Cu/LDPE composite was also characterized in the same way. 2. Materials and methods 2.1. Materials LDPE, with a melt index of 1.8–3.2 g/10 min at 463 K/2.16 kg and with a softening temperature of 108–126 °C, was purchased as pellets from Qilu Petrochemical Corporation of China. Before use, the LDPE pellets were ground into microsize powders with an average size of 250 lm. Copper microparticles, with an average size of about 2 lm and a purity of 99.95%, was purchased from Shanghai Longxin (China). DTBHQ, with a melting point of 212–218 °C and purity of 99.9%, was purchased from Guangzhou Taijun (China). The average DTBHQ particle size was about 35 lm. 2.2. Preparation of DTBHQ/Cu/LDPE composite Two types of samples were used in this study: one consisting of disks with a diameter of 15 mm and a thickness of 3 mm, the other c-shaped composite specimens as described previously [31]. Briefly, all the c-shaped composite specimens have a pair of symmetrical transverse arms with total length of 30 mm and a longitudinal stem with total height of 32 mm, both the transverse arms and the longitudinal stem have a same diameter of 2.0 mm, in addition, the diameter of the hemispheroidal end of each transverse arm is 2.5 mm and that of the longitudinal stem is 2.0 mm. Specimens of DTBHQ/Cu/LDPE composite, with 20 wt.% of DTBHQ and 25 wt.% of copper microparticles, were prepared by injection molding using an injection molding extruder (SA600/100, Ningbo Haitian, China). All the samples were prepared by using the same process: mixing the LDPE powders, copper microparticles and DTBHQ powders in a tumble mixer for 15 min, heating the extruder from hopper to die to 145, 220, 220 and 165 °C for 30 min, putting the homogeneous mixtures into the hopper of the injection molding extruder and then injecting, to produce samples of DTBHQ/Cu/LDPE composite. For comparison, samples of non-porous Cu/LDPE composite with the same content of copper particles were prepared using the same process.
2.3. Preparation of porous Cu/LDPE composite In this experiment, ethyl acetate was selected as the solvent for extraction due to the excellent solubility of DTBHQ in it. To ensure that experimental samples are immersed completely in the ethyl acetate throughout the entire experiment, a modification was made to the base of the Soxhlet extractor by the addition of an overflow pipe connecting the extractor directly to the top of the siphon. In this way, even if the siphon is broken, the ethyl acetate can be maintained at a level sufficient to keep the experimental samples completely immersed. In addition, the excess ethyl acetate extracted from the experimental samples will return to the distillation flask through the overflow pipe, thereby ensuring that the concentrations of DTBHQ in the ethyl acetate in the extractor decrease as the extraction time increases. After the samples of DTBHQ/Cu/LDPE composite has been kept in the extractor, filled with fresh ethyl acetate (distilled from the distillation flask at 90 °C), for at least for 14 h, all the samples were removed and put into a vacuum drying oven (DZF-6020, Shanghai Suopu, China) to remove any residue of ethyl acetate from the composite. The vacuum drying was performed at a temperature of 40 °C at least for 1 day. Thus the porous Cu/LDPE composite was obtained. 2.4. Characterization The structures of the non-porous Cu/LDPE composite, DTBHQ/ Cu/LDPE composite and porous Cu/LDPE composite were characterized by XRD, SEM and FTIR. The DTBHQ and ethyl acetate remaining in the porous Cu/LDPE composite were characterized by GC–MS. All the specimens used here were the disks of the various composites. The cupric ion release rates of the porous Cu/LDPE composite and the Cu/LDPE composite without a porous structure were measured by absorption measurement. 2.4.1. XRD analysis XRD was used to evaluate the phases of non-porous Cu/LDPE composite, DTBHQ/Cu/LDPE composite and porous Cu/LDPE composite. All samples were measured with an X’Pert PRO X-ray diffractometer (PANalytical B.V.) using Cu Ka radiation (40 kV, 40 mA, k = 1.54060 Å). The experiments were performed in a range of 2h from 5 to 80°, with a scan rate of 5°min 1. For comparison, XRD patterns of the microparticles, the pure LDPE and the pure DTBHQ were also characterized by the same equipment using the same process. 2.4.2. FTIR analysis FTIR was applied to evaluate the change of functional group of the non-porous Cu/LDPE composite, the DTBHQ/Cu/LDPE composite and the porous Cu/LDPE composite. The FTIR transmission spectra were obtained in the range of 500–4000 cm 1 using a VERTEX 70 FTIR spectrophotometer (Bruker, Germany). 2.4.3. SEM analysis SEM was employed to evaluate microstructures of the non-porous Cu/LDPE composite, the DTBHQ/Cu/LDPE composite and the porous Cu/LDPE composite. The SEM images were obtained by using a Quanta 200 scanning electron microscope (FEI, Holland) with the acceleration voltage of 10 kV. The specimens for microstructure observation were obtained by cooling the samples in liquid nitrogen for about 5 min, then breaking them into pieces. All the specimens were gilded before being observed. 2.4.4. GC–MS analysis GC–MS was applied to evaluate the residual ethyl acetate and residual DTBHQ in the porous Cu/LDPE composite. The parameters
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W. Zhang et al. / Acta Biomaterialia 8 (2012) 897–903 Table 1 GC–MS operating conditions. Column
19091S-433: 30 m 250 lm 0.25 lm
Carrier gas: flow rate Oven temperature Injector system Injector temperature MS quad temperature MS source temperature Mass range m/z
Helium: 1 ml min–1 50 °C for 2 min, then 20 °C min split mode, Split Ratio:50:1 250 °C 150 °C 250 °C 30–550
1
to 300 °C for 10 min
Table 2 The composition of the SUS. Composition (g l
1
)
NaCl
KCl
CaCl2
NaHCO3
Glucose
NaH2PO42H2O
Serum albumin
4.97
0.224
0.167
0.25
0.50
0.072
0.50
of the GC–MS analysis (7890A/5975, Agilent, USA) are presented in Table 1. For the analysis, a standard sample solution, composed of 10 mg of DTBHQ, 10 ll of ethyl acetate and 10 ml of ethanol, was used to obtain the standard GC–MS spectra of ethyl acetate and DTBHQ, respectively. The porous Cu/LDPE composite, which weighed 1.0095 g, was cut into pieces and immersed in ethanol for 2 days, then removed and the immersion solution was analyzed by GC–MS. 2.5. Cupric ion release from the experimental samples Five prepared c-shaped samples of porous Cu/LDPE composite were taken at random, and each of them was put into a conical flask with 20 ml of SUS. The composition (g l 1) of the SUS [11,17,32] is given in Table 2. Experiments were performed at a constant temperature of 37.0 ± 0.5 °C. Twenty-four hours later, the cupric ion concentration in the SUS was measured using a UV-2102PC ultraviolet spectrophotometer (Unico (Shanghai), China). To avoid retarding the cupric ion release, the SUS was replaced by a freshly prepared batch once a week, and the cupric ion concentration in the SUS was measured again after 24 h of incubation. The samples of Cu/LDPE composite without the introduction of a porous structure were treated using the same method as the porous Cu/LDPE composite, and their cupric ion release rates were also investigated using the same equipment and process. 3. Results and discussion 3.1. Structures of the porous Cu/LDPE composite Structures of the non-porous Cu/LDPE composite, the DTBHQ/ Cu/LDPE composite and the porous Cu/LDPE composite were characterized by XRD, SEM and FTIR. All three kinds of composites contained the same content of copper microparticles, i.e. 25 wt.%. The XRD patterns of copper microparticles, pure LDPE, nonporous Cu/LDPE composite, porous Cu/LDPE composite, DTBHQ/ Cu/LDPE composite and pure DTBHQ are illustrated in Fig. 1. It can be seen that there are three diffraction peaks, located at 43.33, 50.45 and 74.09o, in the XRD pattern of copper (Fig. 1(a)); these are denoted as peaks 1. Two diffraction peaks, located at 21.48 and 23.85o, are found in the XRD pattern of pure LDPE (Fig. 1(b)), and are denoted as peaks 2; three diffraction peaks, located at 9.79, 18.39 and 19.63o, are found in the XRD pattern of pure DTBHQ (Fig. 1(c)), and are denoted as peaks 3. Only two kinds of peaks (peaks 1 and peaks 2) can be found in the XRD patterns of the non-porous Cu/LDPE composite (Fig. 1(d)) and the porous Cu/LDPE composite (Fig. 1(e)), while all the three kinds of peaks (peaks 1,
Fig. 1. XRD patterns for: (a) the copper microparticles, (b) the pure LDPE, (c) the pure DTBHQ, (d) the non-porous Cu/LDPE composite, (e) the porous Cu/LDPE composite and (f) the DTBHQ/Cu/LDPE composite, respectively.
peaks 2 and peaks 3) can be found in the XRD pattern of the DTBHQ/Cu/LDPE composite (Fig. 1(f)). These results indicate that no new phases have been found in the non-porous Cu/LDPE composite, the porous Cu/LDPE composite and the DTBHQ/Cu/LDPE composite. That is to say, either the non-porous Cu/LDPE composite or the porous Cu/LDPE composite is a simple hybrid of the copper microparticles and the pure LDPE, and the DTBHQ/Cu/LDPE composite is also a simple hybrid of the copper microparticles, the pure DTBHQ and the pure LDPE. These results also indicate that the porogen DTBHQ can be removed completely from the DTBHQ/Cu/LDPE
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Fig. 2. FTIR spectra for: (a) the pure LDPE, (b) the non-porous Cu/LDPE composite, (c) the pure DTBHQ, (d) the DTBHQ/Cu/LDPE composite and (e) the porous Cu/LDPE composite, respectively.
composite at the particulate leaching stage of the injection molding/ particulate leaching process. The FTIR spectra of pure LDPE, non-porous Cu/LDPE composite, pure DTBHQ, DTBHQ/Cu/LDPE composite and porous Cu/LDPE composite are shown in Fig. 2. Four absorption peaks can be found in the FTIR spectrum of pure LDPE (Fig. 2(a)), the peaks around 2916, 2848, 1465 and 720 cm 1 corresponding to the asymmetric stretching vibration of methylene, the symmetric stretching vibration of methylene, the asymmetric deformation vibration of methylene and the inner rock vibration of methylene, respectively. These four absorption peaks are the typical absorption peaks of the pure LDPE [33,34]. After injection molding, the non-porous Cu/LDPE composite (Fig. 2(b)) has the same FTIR spectrum as that of the raw LDPE, indicating that no interactions between the copper and the LDPE have occurred during the injection molding process. From the FTIR spectrum of pure DTBHQ (Fig. 2(c)), absorption peaks around 3406 and 1208 cm 1 can be seen; these correspond, respectively to the stretching vibration and deformation vibration of phenol. The absorption peaks around 1655, 1524 and 1470 cm 1 indicate the existence of the benzene ring; the peaks around 1117– 852 cm 1 correspond to the H out-of-plane deformation in aromatic rings [35]; the absorption peak at 789 cm 1 is associated with p-substitution [36]; the absorption peaks around 1395 and 1360 cm 1 indicate the existence of tertiary butyls; and those around 2953 and 2869 cm 1 are associated, respectively with the asymmetric and symmetric stretching vibrations of methyl. All these absorption peaks are typical absorption peaks of pure DTBHQ. The only peaks found in the FTIR spectrum of the DTBHQ/Cu/LDPE composite (Fig. 2(d)) are the characteristic peaks of the LDPE and DTBHQ, which appeared at 2916, 2849, 1466, 720 cm 1 and 3421, 1645, 1524, 1383, 1360, 1268, 1171, 1019, 790 cm 1, respectively; no other new absorption peaks were
found. This indicates that no interactions between the copper, the DTBHQ and the LDPE have occurred during the process of injection molding, i.e. the structures of LDPE and DTBHQ have not been affected. From the FTIR spectrum of the porous Cu/LDPE composite (Fig. 2(e)), it can be seen that, whereas the characteristic peaks of the LDPE are present, all the characteristic peaks of DTBHQ have disappeared, indicating that the DTBHQ has been removed completely after extraction for 14 h. In short, these FTIR spectra results show that no interactions have occurred between the copper microparticles, the pure LDPE and the pure DTBHQ. The SEM images of copper microparticles, pure DTBHQ, nonporous Cu/LDPE composite, DTBHQ/Cu/LDPE composite and porous Cu/LDPE composite are given in Fig. 3. From the SEM image of the copper microparticles (Fig. 3(a)), it can be seen that their average particle size is about 4 lm, and they show some degree of agglomeration. The SEM image of DTBHQ (Fig. 3(b)) shows that it is a kind of hexagonal flake, with an average particle size of about 35 lm. From the SEM images of the non-porous Cu/LDPE composite (Fig. 3(c) and (d)), it can be seen that the surface is smooth with no obvious pores (Fig. 3(c)), and that the copper microparticles are dispersed in the LDPE matrix and show some degree of agglomeration (Fig. 3(d)). From the SEM images of the DTBHQ/Cu/LDPE composite (Fig. 3(e) and (f)), the surface is also seen to be smooth with no obvious pores (Fig. 3e). The cross-sectional appearance of the DTBHQ/Cu/LDPE composite is very different from that of the non-porous Cu/LDPE composite due to the introduction of DTBHQ, as shown in Fig. 3(f). It can also be seen that the appearance of DTBHQ in the DTBHQ/Cu/LDPE composite is very different from that of the original DTBHQ, owing to the melting point of DTBHQ being lower than the injection temperature of the DTBHQ/Cu/LDPE composite and recrystallization of DTBHQ has taken place during the process of injection molding. The SEM images of porous Cu/ LDPE composite are shown in Fig. 3(g) and (h), where it can be seen that the surface and cross-sectional appearance are very different from those of the non-porous Cu/LDPE composite and the DTBHQ/Cu/LDPE composite. After the DTBHQ/Cu/LDPE composite has been leached by ethyl acetate for 14 h, the porous Cu/LDPE composite, with a highly porous surface and well-interconnected pores inside the surface, is obtained, as shown in Fig. 3(g) and (h). The good interconnection of pores is due to the melting point of DTBHQ being lower than the injection temperature of the DTBHQ/Cu/LDPE composite; thus the molten DTBHQ can be distributed uniformly and continuously in the DTBHQ/Cu/LDPE composite. From Fig. 3(g), it can be seen that the size of the pore in the porous Cu/LDPE composite is about 1–2 lm. This could be because of the size of the DTBHQ particles that had recrystallized after molting during the process of injection molding. As we described in the section on the preparation of the DTBHQ/Cu/LDPE composite, the temperatures of the extruder from hopper to die were 145, 220, 220 and 165 °C, whereas the melting point of DTBHQ is 212–218 °C. When the mixture of DTBHQ, copper particles and LDPE was put into the hopper, the DTBHQ would melt and remain in the liquid state in the middle of the extruder. The DTBHQ would then recrystallize in the die of the extruder, where the temperature was only 165 °C. In this process, the size of the DTBHQ changes to about 1–2 lm in the composite with 20 wt.% of DTBHQ. When the recrystallized DTBHQ is removed, pores form that are the size of the recrystallized DTBHQ, i.e. 1–2 lm in the experimental sample. 3.2. Residual ethyl acetate and residual DTBHQ in the porous Cu/LDPE composite During the preparation of the porous Cu/LDPE composite, DTBHQ was chosen as the porogen and ethyl acetate was selected as the solvent for extraction due to the excellent solubility of
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Fig. 3. SEM images for: (a) copper microparticles, (b) pure DTBHQ, (c, d) non-porous Cu/LDPE composite, (e, f) DTBHQ/Cu/LDPE composite and (g, h) porous Cu/LDPE composite, respectively.
DTBHQ in it. However, ethyl acetate is a slightly toxic organic solvent [37] and is thus not allowed to remain in porous Cu/LDPE composites that are used for Cu-IUDs. Therefore, the residual ethyl acetate in the porous Cu/LDPE composite needs to be measured by GC–MS. The amount of residual DTBHQ in porous Cu/LDPE composite must also be ascertained in the same way. The GC–MS spectra of the standard sample solution and the porous Cu/LDPE composite is shown in Fig. 4. From the chromatogram of the standard sample solution, which is composed of 10 mg of DTBHQ, 10 ll of ethyl acetate and 10 ml of ethanol, and is shown in Fig. 4(a), it can be seen that only two obvious peaks, at 1.877 and 11.045 min, can be found. According to the mass spectra of these peaks, which are presented in Fig. 4(b) and (d), the peak eluting at 1.877 min can be attributed to ethyl acetate and that eluting at 11.045 min can be attributed to DTBHQ. The chromatogram of the porous Cu/LDPE composite is shown in Fig. 4(c). It can be seen
that ethyl acetate and DTBHQ were not observed in the sample of the porous Cu/LDPE composite. That is to say, DTBHQ has been removed completely after extraction for 14 h and ethyl acetate has been removed completely after vacuum drying for 1 day, respectively. 3.3. The cupric ion release rate of the porous Cu/LDPE composite The curves of cupric ion release rate vs. immersion time of the porous Cu/LDPE composite and the non-porous Cu/LDPE composite with the same content (25 wt.%) of copper particles are presented in Fig. 5. It can be seen that the cupric ion release rate of the porous Cu/LDPE composite is much higher than that of the composite without the porous structure. For the non-porous Cu/LDPE composite, the route for SUS to enter and the route for cupric ions to diffuse out are mainly attributed to the immanent pores of the
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Fig. 4. GS/MS spectra: (a) GS for standard sample solution contain both DTBHQ and ethyl acetate, (b) MS for spectrum A, (c) GS for the immersion solution of porous Cu/LDPE composite and (d) MS for spectrum B, respectively.
rate of cupric ions by the porous Cu/LDPE composite that is much higher than that of the Cu/LDPE composite without the introduction of the porous structure. 4. Conclusion A porous Cu/LDPE composite can be successfully prepared using the injection molding and particulate leaching method, and it is a simple hybrid of copper microparticles and porous LDPE. One of the results of the introduction of a porous structure is the remarkable improvement in the cupric ion release rate of this novel CuIUD material, indicating that the utilization rate of copper particles can be improved and the lifespan of Cu-IUDs prepared with this kind of material can be prolonged. Acknowledgements
Fig. 5. Curves of the cupric ion release rate of composites in 20 ml of SUS for (a) the porous Cu/LDPE composite and (b) the non-porous Cu/LDPE dense composite.
polymer matrix and the pores acquired from the corrosion of copper particles [11]. For the porous Cu/LDPE composite, not only the pores referred above but also those introduced by the injection molding and particulate leaching method can provide routes for the entry of SUS and the diffusion of the cupric ions. The more SUS that enters into the composite, the greater the chances that copper particles will meet with the SUS and the greater the amounts of cupric ions that will be produced, resulting in a release
The authors gratefully acknowledge the financial supported by the National Natural Science Foundation of China (Grant No. 50271029 and 50803023), the National ‘‘the eleven-fifth’’ Project of Ministry of Science and Technology (Grant No. 2006BAI03B01) and the Self-determined and Innovative Research Funds of HUST (Grant No. M2009048). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2 and 5, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2011.09.024.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
A porous Cu/LDPE composite for copper-containing intrauterine contraceptive devices Weiwei Zhang, Xianping Xia ⇑, Cheng Qi, Changsheng Xie, Shuizhou Cai State Key Laboratory of Material Processing and Die & Mould Technology, Department of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 27 May 2011 Received in revised form 7 August 2011 Accepted 20 September 2011 Available online 24 September 2011 Keywords: Porous Cu/LDPE composite DTBHQ Ethyl acetate Injection molding and particulate leaching Cupric ion release
a b s t r a c t To improve the rates of both cupric ion release and the utilization of copper in non-porous copper/lowdensity polyethylene (Cu/LDPE) composite, a porous Cu/LDPE composite is proposed and developed in the present work. Here 2,5-di-tert-butylhydroquinone was chosen as the porogen, ethyl acetate was chosen as the solvent for extraction, and the porous Cu/LDPE composite was obtained by using injection molding and the particulate leaching method. After any residual ethyl acetate remaining inside the porous Cu/LDPE composite had been removed by vacuum drying, the composite was characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, gas chromatography– mass spectrometry and absorption measurement. For comparison, a non-porous Cu/LDPE composite was also characterized in the same way. The results show that the porous structure was successfully introduced into the polymeric base of the non-porous Cu/LDPE composite, and the porous Cu/LDPE composite is a simple hybrid of copper particles and porous LDPE. The results also show that the introduction of a porous structure can improve the cupric ion release rate of the non-porous Cu/LDPE composite with a certain content of copper particles, indicating that the utilization rate of copper can be improved either the introduction of a porous structure, and that the porous Cu/LDPE composite is another promising material for copper-containing intrauterine devices. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Copper-containing intrauterine devices (Cu-IUDs) are contraceptive devices containing copper on a plastic frame or a thread (frameless) that are inserted through the cervix and placed within the uterine cavity. They are one of the most commonly used forms of fertility control worldwide, and are cheap, safe, reliable, convenient, reversible and culturally acceptable [1]. However, side effects caused by existing Cu-IUDs, such as pain and bleeding, have not yet been overcome [2–5]. It is believed that at least some of these side effects may be related to the burst release of cupric ions within the first few months after insertion of the Cu-IUD [6,7]. The cytotoxic and genotoxic effects of cupric ion released from metallic copper have been investigated in Chinese hamster ovary cells [8,9], and the results show that a reduction in mitochondrial activity can be observed when the cupric ion concentration exceeds 7.42 lg ml 1; in addition, a decrease in cell viability of up to 90% was found at a cupric ion concentration of 10.85 lg ml 1; finally, copper-induced DNA damage is detected when the cupric ion concentration is in the range of 5.67–7.42 lg ml 1. Thus, the optimal
⇑ Corresponding author. Tel.: +86 27 87556544; fax: +86 27 87543778. E-mail address: [email protected] (X. Xia).
cupric ion concentration released from Cu-IUDs in uterine solution should be less than 5.50 lg ml 1. To eliminate these deficiencies of the existing Cu-IUDs, a novel system for the controlled release of cupric ions, the non-porous copper/low-density polyethylene (Cu/LDPE) composite, has been developed for Cu-IUDs [10,11]. Our previous investigations [12– 14] have verified that a Cu/LDPE composite and its devices can lessen the side effects, such as pain and bleeding, that are caused by the existing Cu-IUDs remarkably, and have the same excellent contraceptive efficacy as the existing Cu-IUDs. However, our recent study [15] shows that the cupric ion release rate of such a non-porous Cu/LDPE composite IUD, with 25 wt.% content of copper particles, in 20 ml of simulated uterine solution (SUS) was equivalent to only 2.50 lg ml 1 in uterine fluid (no more than 0.8 ml [16]) during the early stage of the stable release, and its predicted lifespan is only about 5 years. This indicates that the lifespan of composite IUDs is relatively short, which means that the effective utilization of copper particles in composite IUDs will only be about 30 wt.% even if close to 100% of the copper particles can be converted into cupric ions in SUS [17]. Fortunately, non-porous Cu/LDPE composite devices are only one kind of drug delivery system, and a number of investigations on drug delivery systems [18–20] have shown that the introduction of pores into the drug carrier can remarkably improve its drug
1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.09.024
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release rates. Therefore, in the present study we have attempted to improve the cupric ion release rate of novel Cu/LDPE composite IUDs by introducing a porous structure into the matrix of the device. Several methods can be used for the preparation of porous composites, such as extrusion assisted by supercritical carbon dioxide [21], co-extrusion and gas foaming [22], injection molding and particulate leaching [23], gas foaming [24], freeze-drying [25] and phase separation [26]. Here, we used the injection molding and particulate leaching technique to prepare a porous Cu/LDPE composite. 2,5-Di-tertbutylhydroquinone (DTBHQ) was chosen as the porogen due to its extraordinary antioxidation property, its non-toxicity [27–30] and its excellent solubility in solvents such as ethyl acetate. Ethyl acetate was chosen as the solvent for extraction. Most importantly, DTBHQ and ethyl acetate do not react with copper particles or with the LDPE. After samples of the porous Cu/LDPE composite had been obtained, their structures were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), and their residual DTBHQ and ethyl acetate were analyzed by gas chromatography–mass spectrometry (GC–MS). Finally, the cupric ion release rate of the porous Cu/LDPE composite samples was measured by absorption measurement. For comparison, a non-porous Cu/LDPE composite was also characterized in the same way. 2. Materials and methods 2.1. Materials LDPE, with a melt index of 1.8–3.2 g/10 min at 463 K/2.16 kg and with a softening temperature of 108–126 °C, was purchased as pellets from Qilu Petrochemical Corporation of China. Before use, the LDPE pellets were ground into microsize powders with an average size of 250 lm. Copper microparticles, with an average size of about 2 lm and a purity of 99.95%, was purchased from Shanghai Longxin (China). DTBHQ, with a melting point of 212–218 °C and purity of 99.9%, was purchased from Guangzhou Taijun (China). The average DTBHQ particle size was about 35 lm. 2.2. Preparation of DTBHQ/Cu/LDPE composite Two types of samples were used in this study: one consisting of disks with a diameter of 15 mm and a thickness of 3 mm, the other c-shaped composite specimens as described previously [31]. Briefly, all the c-shaped composite specimens have a pair of symmetrical transverse arms with total length of 30 mm and a longitudinal stem with total height of 32 mm, both the transverse arms and the longitudinal stem have a same diameter of 2.0 mm, in addition, the diameter of the hemispheroidal end of each transverse arm is 2.5 mm and that of the longitudinal stem is 2.0 mm. Specimens of DTBHQ/Cu/LDPE composite, with 20 wt.% of DTBHQ and 25 wt.% of copper microparticles, were prepared by injection molding using an injection molding extruder (SA600/100, Ningbo Haitian, China). All the samples were prepared by using the same process: mixing the LDPE powders, copper microparticles and DTBHQ powders in a tumble mixer for 15 min, heating the extruder from hopper to die to 145, 220, 220 and 165 °C for 30 min, putting the homogeneous mixtures into the hopper of the injection molding extruder and then injecting, to produce samples of DTBHQ/Cu/LDPE composite. For comparison, samples of non-porous Cu/LDPE composite with the same content of copper particles were prepared using the same process.
2.3. Preparation of porous Cu/LDPE composite In this experiment, ethyl acetate was selected as the solvent for extraction due to the excellent solubility of DTBHQ in it. To ensure that experimental samples are immersed completely in the ethyl acetate throughout the entire experiment, a modification was made to the base of the Soxhlet extractor by the addition of an overflow pipe connecting the extractor directly to the top of the siphon. In this way, even if the siphon is broken, the ethyl acetate can be maintained at a level sufficient to keep the experimental samples completely immersed. In addition, the excess ethyl acetate extracted from the experimental samples will return to the distillation flask through the overflow pipe, thereby ensuring that the concentrations of DTBHQ in the ethyl acetate in the extractor decrease as the extraction time increases. After the samples of DTBHQ/Cu/LDPE composite has been kept in the extractor, filled with fresh ethyl acetate (distilled from the distillation flask at 90 °C), for at least for 14 h, all the samples were removed and put into a vacuum drying oven (DZF-6020, Shanghai Suopu, China) to remove any residue of ethyl acetate from the composite. The vacuum drying was performed at a temperature of 40 °C at least for 1 day. Thus the porous Cu/LDPE composite was obtained. 2.4. Characterization The structures of the non-porous Cu/LDPE composite, DTBHQ/ Cu/LDPE composite and porous Cu/LDPE composite were characterized by XRD, SEM and FTIR. The DTBHQ and ethyl acetate remaining in the porous Cu/LDPE composite were characterized by GC–MS. All the specimens used here were the disks of the various composites. The cupric ion release rates of the porous Cu/LDPE composite and the Cu/LDPE composite without a porous structure were measured by absorption measurement. 2.4.1. XRD analysis XRD was used to evaluate the phases of non-porous Cu/LDPE composite, DTBHQ/Cu/LDPE composite and porous Cu/LDPE composite. All samples were measured with an X’Pert PRO X-ray diffractometer (PANalytical B.V.) using Cu Ka radiation (40 kV, 40 mA, k = 1.54060 Å). The experiments were performed in a range of 2h from 5 to 80°, with a scan rate of 5°min 1. For comparison, XRD patterns of the microparticles, the pure LDPE and the pure DTBHQ were also characterized by the same equipment using the same process. 2.4.2. FTIR analysis FTIR was applied to evaluate the change of functional group of the non-porous Cu/LDPE composite, the DTBHQ/Cu/LDPE composite and the porous Cu/LDPE composite. The FTIR transmission spectra were obtained in the range of 500–4000 cm 1 using a VERTEX 70 FTIR spectrophotometer (Bruker, Germany). 2.4.3. SEM analysis SEM was employed to evaluate microstructures of the non-porous Cu/LDPE composite, the DTBHQ/Cu/LDPE composite and the porous Cu/LDPE composite. The SEM images were obtained by using a Quanta 200 scanning electron microscope (FEI, Holland) with the acceleration voltage of 10 kV. The specimens for microstructure observation were obtained by cooling the samples in liquid nitrogen for about 5 min, then breaking them into pieces. All the specimens were gilded before being observed. 2.4.4. GC–MS analysis GC–MS was applied to evaluate the residual ethyl acetate and residual DTBHQ in the porous Cu/LDPE composite. The parameters
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W. Zhang et al. / Acta Biomaterialia 8 (2012) 897–903 Table 1 GC–MS operating conditions. Column
19091S-433: 30 m 250 lm 0.25 lm
Carrier gas: flow rate Oven temperature Injector system Injector temperature MS quad temperature MS source temperature Mass range m/z
Helium: 1 ml min–1 50 °C for 2 min, then 20 °C min split mode, Split Ratio:50:1 250 °C 150 °C 250 °C 30–550
1
to 300 °C for 10 min
Table 2 The composition of the SUS. Composition (g l
1
)
NaCl
KCl
CaCl2
NaHCO3
Glucose
NaH2PO42H2O
Serum albumin
4.97
0.224
0.167
0.25
0.50
0.072
0.50
of the GC–MS analysis (7890A/5975, Agilent, USA) are presented in Table 1. For the analysis, a standard sample solution, composed of 10 mg of DTBHQ, 10 ll of ethyl acetate and 10 ml of ethanol, was used to obtain the standard GC–MS spectra of ethyl acetate and DTBHQ, respectively. The porous Cu/LDPE composite, which weighed 1.0095 g, was cut into pieces and immersed in ethanol for 2 days, then removed and the immersion solution was analyzed by GC–MS. 2.5. Cupric ion release from the experimental samples Five prepared c-shaped samples of porous Cu/LDPE composite were taken at random, and each of them was put into a conical flask with 20 ml of SUS. The composition (g l 1) of the SUS [11,17,32] is given in Table 2. Experiments were performed at a constant temperature of 37.0 ± 0.5 °C. Twenty-four hours later, the cupric ion concentration in the SUS was measured using a UV-2102PC ultraviolet spectrophotometer (Unico (Shanghai), China). To avoid retarding the cupric ion release, the SUS was replaced by a freshly prepared batch once a week, and the cupric ion concentration in the SUS was measured again after 24 h of incubation. The samples of Cu/LDPE composite without the introduction of a porous structure were treated using the same method as the porous Cu/LDPE composite, and their cupric ion release rates were also investigated using the same equipment and process. 3. Results and discussion 3.1. Structures of the porous Cu/LDPE composite Structures of the non-porous Cu/LDPE composite, the DTBHQ/ Cu/LDPE composite and the porous Cu/LDPE composite were characterized by XRD, SEM and FTIR. All three kinds of composites contained the same content of copper microparticles, i.e. 25 wt.%. The XRD patterns of copper microparticles, pure LDPE, nonporous Cu/LDPE composite, porous Cu/LDPE composite, DTBHQ/ Cu/LDPE composite and pure DTBHQ are illustrated in Fig. 1. It can be seen that there are three diffraction peaks, located at 43.33, 50.45 and 74.09o, in the XRD pattern of copper (Fig. 1(a)); these are denoted as peaks 1. Two diffraction peaks, located at 21.48 and 23.85o, are found in the XRD pattern of pure LDPE (Fig. 1(b)), and are denoted as peaks 2; three diffraction peaks, located at 9.79, 18.39 and 19.63o, are found in the XRD pattern of pure DTBHQ (Fig. 1(c)), and are denoted as peaks 3. Only two kinds of peaks (peaks 1 and peaks 2) can be found in the XRD patterns of the non-porous Cu/LDPE composite (Fig. 1(d)) and the porous Cu/LDPE composite (Fig. 1(e)), while all the three kinds of peaks (peaks 1,
Fig. 1. XRD patterns for: (a) the copper microparticles, (b) the pure LDPE, (c) the pure DTBHQ, (d) the non-porous Cu/LDPE composite, (e) the porous Cu/LDPE composite and (f) the DTBHQ/Cu/LDPE composite, respectively.
peaks 2 and peaks 3) can be found in the XRD pattern of the DTBHQ/Cu/LDPE composite (Fig. 1(f)). These results indicate that no new phases have been found in the non-porous Cu/LDPE composite, the porous Cu/LDPE composite and the DTBHQ/Cu/LDPE composite. That is to say, either the non-porous Cu/LDPE composite or the porous Cu/LDPE composite is a simple hybrid of the copper microparticles and the pure LDPE, and the DTBHQ/Cu/LDPE composite is also a simple hybrid of the copper microparticles, the pure DTBHQ and the pure LDPE. These results also indicate that the porogen DTBHQ can be removed completely from the DTBHQ/Cu/LDPE
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Fig. 2. FTIR spectra for: (a) the pure LDPE, (b) the non-porous Cu/LDPE composite, (c) the pure DTBHQ, (d) the DTBHQ/Cu/LDPE composite and (e) the porous Cu/LDPE composite, respectively.
composite at the particulate leaching stage of the injection molding/ particulate leaching process. The FTIR spectra of pure LDPE, non-porous Cu/LDPE composite, pure DTBHQ, DTBHQ/Cu/LDPE composite and porous Cu/LDPE composite are shown in Fig. 2. Four absorption peaks can be found in the FTIR spectrum of pure LDPE (Fig. 2(a)), the peaks around 2916, 2848, 1465 and 720 cm 1 corresponding to the asymmetric stretching vibration of methylene, the symmetric stretching vibration of methylene, the asymmetric deformation vibration of methylene and the inner rock vibration of methylene, respectively. These four absorption peaks are the typical absorption peaks of the pure LDPE [33,34]. After injection molding, the non-porous Cu/LDPE composite (Fig. 2(b)) has the same FTIR spectrum as that of the raw LDPE, indicating that no interactions between the copper and the LDPE have occurred during the injection molding process. From the FTIR spectrum of pure DTBHQ (Fig. 2(c)), absorption peaks around 3406 and 1208 cm 1 can be seen; these correspond, respectively to the stretching vibration and deformation vibration of phenol. The absorption peaks around 1655, 1524 and 1470 cm 1 indicate the existence of the benzene ring; the peaks around 1117– 852 cm 1 correspond to the H out-of-plane deformation in aromatic rings [35]; the absorption peak at 789 cm 1 is associated with p-substitution [36]; the absorption peaks around 1395 and 1360 cm 1 indicate the existence of tertiary butyls; and those around 2953 and 2869 cm 1 are associated, respectively with the asymmetric and symmetric stretching vibrations of methyl. All these absorption peaks are typical absorption peaks of pure DTBHQ. The only peaks found in the FTIR spectrum of the DTBHQ/Cu/LDPE composite (Fig. 2(d)) are the characteristic peaks of the LDPE and DTBHQ, which appeared at 2916, 2849, 1466, 720 cm 1 and 3421, 1645, 1524, 1383, 1360, 1268, 1171, 1019, 790 cm 1, respectively; no other new absorption peaks were
found. This indicates that no interactions between the copper, the DTBHQ and the LDPE have occurred during the process of injection molding, i.e. the structures of LDPE and DTBHQ have not been affected. From the FTIR spectrum of the porous Cu/LDPE composite (Fig. 2(e)), it can be seen that, whereas the characteristic peaks of the LDPE are present, all the characteristic peaks of DTBHQ have disappeared, indicating that the DTBHQ has been removed completely after extraction for 14 h. In short, these FTIR spectra results show that no interactions have occurred between the copper microparticles, the pure LDPE and the pure DTBHQ. The SEM images of copper microparticles, pure DTBHQ, nonporous Cu/LDPE composite, DTBHQ/Cu/LDPE composite and porous Cu/LDPE composite are given in Fig. 3. From the SEM image of the copper microparticles (Fig. 3(a)), it can be seen that their average particle size is about 4 lm, and they show some degree of agglomeration. The SEM image of DTBHQ (Fig. 3(b)) shows that it is a kind of hexagonal flake, with an average particle size of about 35 lm. From the SEM images of the non-porous Cu/LDPE composite (Fig. 3(c) and (d)), it can be seen that the surface is smooth with no obvious pores (Fig. 3(c)), and that the copper microparticles are dispersed in the LDPE matrix and show some degree of agglomeration (Fig. 3(d)). From the SEM images of the DTBHQ/Cu/LDPE composite (Fig. 3(e) and (f)), the surface is also seen to be smooth with no obvious pores (Fig. 3e). The cross-sectional appearance of the DTBHQ/Cu/LDPE composite is very different from that of the non-porous Cu/LDPE composite due to the introduction of DTBHQ, as shown in Fig. 3(f). It can also be seen that the appearance of DTBHQ in the DTBHQ/Cu/LDPE composite is very different from that of the original DTBHQ, owing to the melting point of DTBHQ being lower than the injection temperature of the DTBHQ/Cu/LDPE composite and recrystallization of DTBHQ has taken place during the process of injection molding. The SEM images of porous Cu/ LDPE composite are shown in Fig. 3(g) and (h), where it can be seen that the surface and cross-sectional appearance are very different from those of the non-porous Cu/LDPE composite and the DTBHQ/Cu/LDPE composite. After the DTBHQ/Cu/LDPE composite has been leached by ethyl acetate for 14 h, the porous Cu/LDPE composite, with a highly porous surface and well-interconnected pores inside the surface, is obtained, as shown in Fig. 3(g) and (h). The good interconnection of pores is due to the melting point of DTBHQ being lower than the injection temperature of the DTBHQ/Cu/LDPE composite; thus the molten DTBHQ can be distributed uniformly and continuously in the DTBHQ/Cu/LDPE composite. From Fig. 3(g), it can be seen that the size of the pore in the porous Cu/LDPE composite is about 1–2 lm. This could be because of the size of the DTBHQ particles that had recrystallized after molting during the process of injection molding. As we described in the section on the preparation of the DTBHQ/Cu/LDPE composite, the temperatures of the extruder from hopper to die were 145, 220, 220 and 165 °C, whereas the melting point of DTBHQ is 212–218 °C. When the mixture of DTBHQ, copper particles and LDPE was put into the hopper, the DTBHQ would melt and remain in the liquid state in the middle of the extruder. The DTBHQ would then recrystallize in the die of the extruder, where the temperature was only 165 °C. In this process, the size of the DTBHQ changes to about 1–2 lm in the composite with 20 wt.% of DTBHQ. When the recrystallized DTBHQ is removed, pores form that are the size of the recrystallized DTBHQ, i.e. 1–2 lm in the experimental sample. 3.2. Residual ethyl acetate and residual DTBHQ in the porous Cu/LDPE composite During the preparation of the porous Cu/LDPE composite, DTBHQ was chosen as the porogen and ethyl acetate was selected as the solvent for extraction due to the excellent solubility of
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Fig. 3. SEM images for: (a) copper microparticles, (b) pure DTBHQ, (c, d) non-porous Cu/LDPE composite, (e, f) DTBHQ/Cu/LDPE composite and (g, h) porous Cu/LDPE composite, respectively.
DTBHQ in it. However, ethyl acetate is a slightly toxic organic solvent [37] and is thus not allowed to remain in porous Cu/LDPE composites that are used for Cu-IUDs. Therefore, the residual ethyl acetate in the porous Cu/LDPE composite needs to be measured by GC–MS. The amount of residual DTBHQ in porous Cu/LDPE composite must also be ascertained in the same way. The GC–MS spectra of the standard sample solution and the porous Cu/LDPE composite is shown in Fig. 4. From the chromatogram of the standard sample solution, which is composed of 10 mg of DTBHQ, 10 ll of ethyl acetate and 10 ml of ethanol, and is shown in Fig. 4(a), it can be seen that only two obvious peaks, at 1.877 and 11.045 min, can be found. According to the mass spectra of these peaks, which are presented in Fig. 4(b) and (d), the peak eluting at 1.877 min can be attributed to ethyl acetate and that eluting at 11.045 min can be attributed to DTBHQ. The chromatogram of the porous Cu/LDPE composite is shown in Fig. 4(c). It can be seen
that ethyl acetate and DTBHQ were not observed in the sample of the porous Cu/LDPE composite. That is to say, DTBHQ has been removed completely after extraction for 14 h and ethyl acetate has been removed completely after vacuum drying for 1 day, respectively. 3.3. The cupric ion release rate of the porous Cu/LDPE composite The curves of cupric ion release rate vs. immersion time of the porous Cu/LDPE composite and the non-porous Cu/LDPE composite with the same content (25 wt.%) of copper particles are presented in Fig. 5. It can be seen that the cupric ion release rate of the porous Cu/LDPE composite is much higher than that of the composite without the porous structure. For the non-porous Cu/LDPE composite, the route for SUS to enter and the route for cupric ions to diffuse out are mainly attributed to the immanent pores of the
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Fig. 4. GS/MS spectra: (a) GS for standard sample solution contain both DTBHQ and ethyl acetate, (b) MS for spectrum A, (c) GS for the immersion solution of porous Cu/LDPE composite and (d) MS for spectrum B, respectively.
rate of cupric ions by the porous Cu/LDPE composite that is much higher than that of the Cu/LDPE composite without the introduction of the porous structure. 4. Conclusion A porous Cu/LDPE composite can be successfully prepared using the injection molding and particulate leaching method, and it is a simple hybrid of copper microparticles and porous LDPE. One of the results of the introduction of a porous structure is the remarkable improvement in the cupric ion release rate of this novel CuIUD material, indicating that the utilization rate of copper particles can be improved and the lifespan of Cu-IUDs prepared with this kind of material can be prolonged. Acknowledgements
Fig. 5. Curves of the cupric ion release rate of composites in 20 ml of SUS for (a) the porous Cu/LDPE composite and (b) the non-porous Cu/LDPE dense composite.
polymer matrix and the pores acquired from the corrosion of copper particles [11]. For the porous Cu/LDPE composite, not only the pores referred above but also those introduced by the injection molding and particulate leaching method can provide routes for the entry of SUS and the diffusion of the cupric ions. The more SUS that enters into the composite, the greater the chances that copper particles will meet with the SUS and the greater the amounts of cupric ions that will be produced, resulting in a release
The authors gratefully acknowledge the financial supported by the National Natural Science Foundation of China (Grant No. 50271029 and 50803023), the National ‘‘the eleven-fifth’’ Project of Ministry of Science and Technology (Grant No. 2006BAI03B01) and the Self-determined and Innovative Research Funds of HUST (Grant No. M2009048). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2 and 5, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2011.09.024.
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