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ZnO-DOX@ZIF-8 Core-Shell Nanoparticles for pH-Responsive Drug Delivery Cunchuan Zheng, Yang Wang, Soo Zeng Fiona Phua, Wei Qi Lim, and Yanli Zhao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00435 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017

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ACS Biomaterials Science & Engineering

ZnO-DOX@ZIF-8 Core-Shell Nanoparticles for pH-Responsive Drug Delivery Cunchuan Zheng,†,‡ Yang Wang,‡ Soo Zeng Fiona Phua,‡ Wei Qi Lim,‡ Yanli Zhao*‡,§

†

College of Chemistry and Chemical Engineering, Southwest Petroleum University, No. 8

Xindu Road, Chengdu 610500, P. R. China ‡

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 §

School of Materials Science and Engineering, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798 *E-mail: [email protected]

Abstract: Developing multifunctional hybrid nanosystems for controlled drug delivery is a challenging task. In this work, we prepared hierarchical core-shell nanoparticles (ZnO-DOX@ZIF-8) composed of mesoporous ZnO core and microporous ZIF-8 shell, in which the core serves as the drug storage reservoir for the loading of anticancer drug doxorubicin (DOX) and the shell could be used to prevent premature release of loaded drug at physiological environment. The mesoporous ZnO nanoparticles were firstly prepared, followed by DOX drug loading. Such ZnO nanoparticles were then employed as the zinc source to react with 2-methylimidazole for the formation of ZnO-DOX@ZIF-8 core-shell nanoparticles. The core-shell nanoparticles exhibit good dispersibility and stability as well as pH-responsive drug release property. While only up to 20% of loaded DOX was released in

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the buffer of pH 7.4, over 80% of DOX was released in the buffer of pH 5.5 owing to the decomposition of the ZIF-8 shell as well as the dissolution of the ZnO core under acidic conditions. The confocal microscopy studies show that the core-shell nanoparticles could be efficiently internalized by cancer cells, and the loaded DOX in the nanoparticles could be successfully released under acidic intracellular environment. The in vitro cytotoxicity measurements demonstrate that the core-shell nanoparticles free of drug exhibit a significant cytotoxicity when the concentration was above 25 µg/mL on account of the production of reactive oxygen species. The reactive oxygen species are only generated in acidic condition, which could combine with DOX for a synergistic cancer treatment with satisfactory therapeutic efficacy. On the other hand, the nanoparticles were stable and non-toxic in physiological environment. Thus, the ZnO-DOX@ZIF-8 core-shell nanoparticles are a promising pH-responsive drug delivery system for the cancer therapy. Keywords: core-shell nanoparticles, drug delivery, mesoporous ZnO, microporous ZIF-8, pH-responsiveness

Multifunctional nanoparticles as drug delivery systems have attracted great attention on account of their good biosafety, easy functionalization, and capability of intracellular uptake.1-4 In these drug carriers, mesoporous nanoparticles are of intense interests owing to their high drug encapsulation efficiency and controllable drug release.5-7 Examples of such mesoporous nanoparticles include mesoporous silica,8,9 Fe3O4,10 TiO2,11 ZnO,12 MoO3-x,13,14 metal-organic

frameworks

(MOFs),6,15,16

and

carbon

nanomaterials.17

Particularly,

mesoporous silica nanoparticles and their composites with regular channels have been widely

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employed in the drug delivery, while their biodegradability in physiological conditions poses a potential safety concern. Mesoporous Fe3O4 and TiO2 nanoparticles are also restricted by the similar problem as mesoporous silica for further biological applications. Meanwhile, mesoporous ZnO nanoparticles have emerged as a new kind of nanosystem for the drug delivery on account of their pH-responsive property. The mesoporous ZnO nanoparticles are stable under near-neutral condition and physiological environment, while it could be dissociated to zinc ions at acidic environment below the pH value of 6.5.18-23 Therefore, when drug-loaded ZnO nanoparticles are internalized by cancer cells, under acidic conditions in cancer cells, especially in the lysosome and endosome of cancer cells, ZnO nanoparticles could be dissociated to release the encapsulated drugs for the cancer therapy. Owing to the pH-responsive property, ZnO nanoparticles as drug carriers have been developed rapidly in the past few years. For example, Xiong et al24 prepared a ZnO@polymer composite as the drug carrier, which had a high drug loading capacity of over 20 wt%. Furthermore, more than 90% of doxorubicin (DOX) loaded into the ZnO@polymer composite was released in buffer solution (pH 5.0) due to the decomposition of the ZnO@polymer composite in acidic conditions. Zhu and co-workers25 firstly prepared ZnO quantum dots and then used them as the gatekeeper on mesoporous silica to control the drug release. ZnO quantum dots were stable under physiological environment, but rapidly dissolved in acidic condition of cancer cells. In addition, stable ZnO nanoparticles in physiological environment exhibited low cytotoxicity, whilst dissolving ZnO nanoparticles into zinc ions above a certain concentration in cancer cells resulted in marked cytotoxicity. Zinc ions could generate reactive oxygen species (ROS) to kill cancer cells, presenting combinational therapy along

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with released drug.26

Scheme 1. Schematic illustration for the construction of ZnO-DOX@ZIF-8 and its pH-responsive drug release mechanism for cancer therapy.

On the other hand, zeolitic imidazolate frameworks (ZIFs) have been used in gas storage and separation as well as drug carriers for biological applications on account of its high thermal and hydrothermal stability.27-31 ZIF-8 is a kind of ZIF prepared from zinc ion and 2-methylimidazolate, exhibiting low toxicity under certain experimental conditions.32,33 Furthermore, ZIF-8 is stable at nearly neutral aqueous solution, and could be decomposed in acidic conditions. Thus, it could be employed to construct pH-sensitive drug delivery systems. In addition, there are a plenty of pore cavities in ZIF-8 with the diameter of 11.6 Å, to be used

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for the drug storage. However, the diameter of the pore opening on ZIF-8 is only 3.4 Å, which is smaller than most anticancer drugs.34,35 Therefore, such small pore opening might prevent premature drug release in physiological conditions, and loaded drugs in the pore cavity are only released in cancer cells after the decomposition of nanoparticles under acidic conditions.36,37 Interestingly, ZnO could be used as the zinc source and template to prepare ZIF-8 for the formation of hierarchical ZnO/ZIF-8 composites.38-42 Integrating mesoporous ZnO nanoparticles with ZIF-8 by taking full advantage of their properties43-47 may lead to a new generation of pH-responsive drug delivery systems. Inspired by the composition and unique pore structure of ZIF-8, we firstly prepared mesoporous ZnO nanoparticles as drug carriers, and then employed the obtained ZnO nanoparticles as the zinc source to synthesize ZnO@ZIF-8 core-shell nanoparticles (Scheme 1). 2-Methylimidazolate ligand was used to coordinate with the generated zinc ions to form the ZIF-8 shells. In this process, mesoporous ZnO nanoparticles serve as not only the drug carriers, but also the zinc source to prepare the ZIF-8 shell. When DOX is loaded in the mesoporous ZnO core (ZnO-DOX@ZIF-8), the microporous ZIF-8 shell with the pore opening of only 3.4 Å could prevent the drug release at physiological environment. After internalizing by cancer cells, the ZIF-8 shell is decomposed at acidic conditions and the loaded drugs could be released from the mesoporous ZnO core for the cancer therapy. The core of ZnO nanoparticles could also be degraded at acidic environment. Even the pore volume of the mesoporous ZnO nanoparticles is generally lower than that of mesoporous silica, they showed higher drug loading efficiency. The drug release of DOX-loaded ZnO@ZIF-8 was investigated at both physiological environment and acidic

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condition. Furthermore, cellular uptake and intracellular drug release of DOX-loaded ZnO@ZIF-8 were confirmed with confocal laser scanning microscopy (CLSM). Finally, the cytotoxicity

of

the

drug

delivery

system

was

studied

by

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The schematic illustration for the preparation of ZnO-DOX@ZIF-8 nanoparticles with encapsulated drug molecules is presented in Scheme 1. Detailed synthesis and characterization data could be found in the Supporting Information. Initially, highly dispersed ZnO nanoparticles were prepared with zinc acetate hexahydrate in triethylene glycol via a solvothermal method at the temperature of 160 oC, which could serve as drug carriers on account of the mesoporous property and dissociation in acidic solution. Subsequently, the mesoporous ZnO nanoparticles were redispersed in aqueous solution to load the DOX anticancer drug at nearly neutral environment. Functional groups of DOX, such as hydroxyl, carbonyl and amino groups, could form weak coordination interactions with zinc species of the mesoporous ZnO nanoparticles. Therefore, the DOX molecule could diffuse into the nanoparticles through mesoporous pores and adsorb onto the internal surface through coordination interactions, leading to higher drug loading amount as compared with conventional mesoporous silica. After the drug loading, 2-methylimidazole was dropwise added into the ZnO aqueous dispersion, and the pH value of the solution increased sharply. When the pH value of the solution exceeded twelve, the ZnO nanoparticles were partially dissolved to zinc ions. At the same time, the dissolved zinc ions were coordinated with 2-methylimidazole to form a ZIF-8 shell on the surface of mesoporous ZnO nanoparticles, resulting in hierarchical core-shell

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nanoparticles. The final nanocomposites consist of mesoporous ZnO core loaded with DOX surrounded by microporous ZIF-8 shell that prevents the drug premature release under physiological environment as the diameter of the pore opening in the ZIF-8 shell is smaller than the size of the DOX molecule. During the process of the nanocarrier fabrication, the mesoporous ZnO core serves as not only the drug carriers, but also the zinc source to prepare the ZIF-8 shell. The added 2-methylimidazole causes partial dissolution of ZnO nanoparticles, and subsequently coordinates with the dissolved zinc ions to form the ZIF-8 shell. Therefore, no additional zinc precursors were required in the reaction process. Since excess amount of 2-methylimidazole was added, the dissolved zinc ions were linked immediately by 2-methylimidazole to form the ZIF-8 shell on the ZnO core.

Figure 1. SEM images of (a) mesoporous ZnO and (b) ZnO-DOX@ZIF-8 nanoparticles. TEM images of (c) mesoporous ZnO and (d) ZnO-DOX@ZIF-8 nanoparticles. (e) Zeta-potential values of ZnO, ZnO@ZIF-8 and ZnO-DOX@ZIF-8 nanoparticles. (f) Size distributions of ZnO and ZnO-DOX@ZIF-8 nanoparticles determined by DLS.

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As shown in Figure 1a, well-dispersed mesoporous ZnO nanoparticles were observed by scanning electron microscopy (SEM), which were comprised of plenty of small nanocrystals with the diameter about 20 nm. When the ZnO nanoparticles were encapsulated within the ZIF-8 shell, the surface became smoother than that of the ZnO nanoparticles (Figure 1b). Transmission electron microscopy (TEM) images of the ZnO and ZnO-DOX@ZIF-8 nanoparticles were displayed in Figure 1c,d. An obvious core-shell structure can be observed in the TEM image (Figure 1d) of ZnO-DOX@ZIF-8. Furthermore, the zeta potential (Figure 1e) was changed from positive to negative charge after coating ZIF-8 on ZnO nanoparticles, indicating the variation of the surface property. Energy-dispersive X-ray (EDX) elemental mapping analysis (Figure S1) also indicates that the ZIF-8 shell was coated onto ZnO and DOX was loaded successfully. Dynamic light scattering (DLS) measurements showed that the obtained ZnO nanoparticles have a mean diameter of 240 nm (Figure 1f). The particle size increased from 240 to 270 nm after the formation of ZnO-DOX@ZIF-8 nanoparticles, meaning that the thickness of the shell was approximate 15 nm. All these properties confirmed that the ZnO-DOX@ZIF-8 core-shell nanoparticles were prepared successfully. The sharp diffraction peaks in powder X-ray diffraction (XRD) patterns (Figure 2a) indicate that the ZnO, ZIF-8, and ZnO-DOX@ZIF-8 nanoparticles are of high crystallinity. The presence of characteristic peaks of both ZnO (30 < 2θ < 60) and ZIF-8 (5 < 2θ < 30) in the powder XRD pattern of ZnO-DOX@ZIF-8 nanoparticles indicates the existence of both crystal forms in the ZnO-DOX@ZIF-8 nanoparticles. As shown in Figure 2b, the nitrogen adsorption/desorption analysis of ZnO nanoparticles presents a typical type IV curve

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accompanied with a hysteresis loop, indicating that the ZnO nanoparticles are mesoporous with the surface area of 34.8 m2/g. Its pore size distribution (Figure 2c) was determined via Barrett-Joyner-Halenda (BJH) method and the average pore size was about 3 nm, also proving its mesoporous property. With the presence of microporous ZIF-8 shell, the surface area of ZnO-DOX@ZIF-8 nanoparticles increased dramatically from 34.8 to 637.4 m2/g (Figure 2b), and the pore volume also increased from 0.06 to 0.98 cm3/g (Figure 2d,e).

Figure 2. (a) Powder XRD patterns of ZnO, ZIF-8 and ZnO-DOX@ZIF-8 nanoparticles. (b) N2 adsorption/desorption isotherms of ZnO and ZnO-DOX@ZIF-8 nanoparticles. Corresponding BJH pore size distributions of (c) ZnO and (d) ZnO-DOX@ZIF-8 nanoparticles. (e) BJH pore size distribution of ZnO-DOX@ZIF-8 nanoparticles at the range from 2 to 4 nm.

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Both the ZnO core and ZIF-8 shell are stable under physiological conditions and could be decomposed in acidic condition. Thus, the loaded drug is prevented under physiological environment in normal organs, minimizing premature drug release with unspecific cytotoxicity. On the other hand, when the core-shell nanoparticles are endocytosed into cancer cells, the ZIF-8 shell could be dissolved due to the acidic intracellular environment of cancer cells, and the loaded DOX could be released for the cancer therapy.

Figure 3. pH responsive release of DOX from the ZnO-DOX@ZIF-8 nanoparticles at 37 °C.

Thus, we studied the DOX release of ZnO-DOX@ZIF-8 in different pH values to evaluate its potential as a drug delivery system for cancer therapy. The ZnO-DOX@ZIF-8 nanoparticles were dissolved in a nitric acid solution (1 mol/L), and then measured with the fluorescence spectrometer under the fluorescence emission of 560 nm and the excitation wavelength of 480 nm. The drug loading amount of the ZnO-DOX@ZIF-8 nanoparticles was 11.2%, which was obviously higher than conventional mesoporous silica (about 5%) even the 10


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surface area and pore volume of the ZnO-DOX@ZIF-8 nanoparticles are lower than that of the mesoporous silica.8,9,48,49 Figure 3 shows the pH-responsive cumulative release profiles of ZnO-DOX@ZIF-8 nanoparticles. There was only about 20% DOX release from ZnO-DOX@ZIF-8 nanoparticles even after 25 h at the pH value of 7.4 at 37 °C, which was presumably attributed to the drug adsorbed on the surface of the ZIF-8 shell of through coordination interactions. While the loaded DOX was released quickly at lower pH of 5.5, and more than 70% of the loaded DOX was released in 5 h, exhibiting superior pH-responsive release capability.

Figure 4. CLSM images of HeLa cancer cells incubated with ZnO-DOX@ZIF-8 nanoparticles for different time periods. For each panel, the images from left to right are 11



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bright field images, DOX fluorescence in cells (red), and overlay of the two images.

The release mechanism of the ZnO-DOX@ZIF-8 core-shell nanoparticles is different from that of other conventional drug delivery systems such as mesoporous silica50 and MOFs.6,16,51,52 For the drug release of mesoporous silica and MOFs, drugs desorb from the surface of the pore and diffuse out of the delivery systems, while the carriers normally remain intact and non-degradable. In our case, the drug release from ZnO-DOX@ZIF-8 nanoparticles at acidic condition is associated with the dissociation of the ZIF-8 shell and ZnO core. As such, the ZnO-DOX@ZIF-8 nanoparticles could be stable with minimal drug release under physiological environment, whereas almost all loaded drugs are released in acidic condition after their internalization in acidic tumor microenvironment. Thus, ZnO-DOX@ZIF-8 nanoparticles are a promising pH-responsive drug delivery system for the cancer therapy. According to the conclusion discussed above, the ZnO-DOX@ZIF-8 nanoparticles are unstable in acidic condition, and the drugs loaded in the nanoparticles could be released with the dissociation of the ZIF-8 shell. It is well known that intracellular pH of some cancer cells is acidic, especially in lysosomes and endosomes. Thus, the dissolution of ZnO-DOX@ZIF-8 nanoparticles in cancer cells would aid in the drug release. On the other hand, since the nanoparticles are stable under nearly neutral environment, an obvious drug release would not occur in normal cells having nearly neutral environment. The cellular uptake of the ZnO-DOX@ZIF-8 nanoparticles and its DOX release inside cancer cells were investigated with CLSM under different incubation times (Figure 4). After treating with ZnO-DOX@ZIF-8 nanoparticles for two hours, the fluorescence of

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DOX from the cancer cells were observed, indicating that the nanoparticles could be endocytosed by cancer cells and the loaded DOX was released from the core-shell nanoparticles successfully. According to pH-responsive release profile, the drug release from the core-shell nanoparticles was rapid in acidic condition, and more than 60% of DOX was released after one hour owing to the thin thickness of the ZIF-8 shell (about 15 nm). It could be obviously observed that the fluorescence of DOX was present mainly in the cytoplasm and accumulated around the cell nuclei after two hours. Only weak red fluorescence was observed in the perinucleus region of cells after 2 hours of incubation. The reason may be that the uptake of ZnO-DOX@ZIF-8 nanoparticles is relatively slow, and the released DOX did not yet diffuse into the cell nuclei after 2 hours. After the incubation of 4 h, stronger DOX fluorescence was observed in the cell nucleus region, which was attributed to the sustained internalization of the ZnO-DOX@ZIF-8 nanoparticles and acid-induced DOX release in the HeLa cells. The CLSM study indicates that the ZnO-DOX@ZIF-8 nanoparticles could be internalized by cancer cells and the loaded drugs could be released successfully. The cytotoxicity of drug-free ZnO@ZIF-8 was evaluated by MTT assay at different concentrations using HeLa cells. As shown in Figure 5, the ZnO@ZIF-8 nanoparticles exhibited obvious cytotoxicity, especially at concentrations above 25 µg/mL. The cell viability was only about 35% when the concentration of the ZnO@ZIF-8 nanoparticles was 25 µg/mL, and almost all the cancer cells were dead at the concentration of 50 µg/mL. The cytotoxicity of ZnO@ZIF-8 was probably owing to the generation of ROS by dissolved zinc ions in cancer cells.25,26 However, it is worth to mention that both of the ZnO nanoparticles and ZIF-8 were stable under physiological environment and nontoxic in normal cells. The

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ZnO@ZIF-8 nanoparticles were cytotoxic only in acidic cancer microenvironment. This cytotoxicity could work together with anticancer drugs for combinational therapy of cancer.

Figure 5. In vitro anticancer activity of HeLa cells after the incubation with different concentrations of free DOX, ZnO@ZIF-8 and ZnO-DOX@ZIF-8 for 24 h. The cell viability of HeLa cells without any treatment was regarded as 100%.

The free DOX and ZnO-DOX@ZIF-8 nanoparticles were also investigated for the cancer therapy. As compared with free DOX, ZnO-DOX@ZIF-8 nanoparticles showed excellent therapeutic efficacy for HeLa cells upon the increase of the dose. After the treatment with free DOX at a concentration of 0.4 µg/mL for 24 h, the cell viability fell from the baseline level to 68%. The values were lower (about 45%) after the cells were treated with free DOX at a concentration of 1.6 µg/mL. After the treatment with ZnO-DOX@ZIF-8 nanoparticles, the cell viability decreased to about 50% at the concentration of 3.125 µg/mL and was even lower 14


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than 20% when the concentration was up to 12.5 µg/mL. The toxicity of ZnO-DOX@ZIF-8 was significantly higher than that of ZnO@ZIF-8 as well as equal amount of free DOX, which was caused by synergistic toxic effect of released DOX and decomposed ZnO@ZIF-8 from ZnO-DOX@ZIF-8 under acidic tumor microenvironment. In summary, core-shell ZnO-DOX@ZIF-8 nanoparticles have been developed as a pH-responsive nanosystem for the drug delivery in the cancer therapy. The mesoporous ZnO acts as the drug carrier and the microporous ZIF-8 shell could prevent the drug release under physiological environment. Since the mesoporous ZnO core and microporous ZIF-8 shell could be dissociated in acidic conditions, more than 80% of the drugs in the nanoparticles could be released at the pH value of 5.5, exhibiting good pH-responsive drug release property. Thus, the drug-loaded core-shell nanoparticles could be efficiently endocytosed by HeLa cancer cells, and the loaded DOX could be released under acidic intracellular environment, exhibiting a satisfactory therapeutic efficacy to HeLa cells. When compared with other core-shell drug delivery systems such as Fe3O4@SiO2 and CuS@SiO2, the present core-shell nanoparticles show specific advantages. Firstly, the ZnO@ZIF-8 core-shell nanoparticles could be prepared easily in a mild condition. The ZIF-8 shell was prepared using the ZnO core as the zinc resource, and no other zinc ions need to add during the synthesis, thus affording ZIF-8 coating onto the surface of ZnO easily to form the core-shell structure. Secondly, the ZnO@ZIF-8 core-shell nanoparticles present low toxicity for normal cells and high toxicity in cancer cells under acidic conditions, which could kill cancer cells selectively. Thirdly, both of the ZnO core and the ZIF-8 shell are stable under nearly neutral microenvironment, but could dissolve under acidic conditions, endowing the

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system with pH responsive property and selective release capability in cancer cells. These results conclude that the ZnO-DOX@ZIF-8 core-shell nanoparticles are a promising pH-responsive drug delivery system in the cancer therapy.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions All authors have given contributions to the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by the SingHealth-NTU Research Collaborative Grant (SHS-NTU/009/2016). Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Materials, synthesis and characterization details, drug release procedures, cellular uptake study, in vitro cytotoxicity study, SEM image, and EDX elemental mapping analysis.

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Table of Contents Graphic

ZnO-DOX@ZIF-8 Core-Shell Nanoparticles for pH-Responsive Drug Delivery Cunchuan Zheng,†,‡ Yang Wang,‡ Soo Zeng Fiona Phua,‡ Wei Qi Lim,‡ Yanli Zhao*‡,§

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ACS Biomaterials Science & Engineering - ACS Publications

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