- Email: [email protected]
Heparin-coated superparamagnetic iron oxide nanoparticles as highly effective MRI contrast agent for cell labeling
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Abstracts / Journal of Controlled Release 152 (2011) e192–e269
Acknowledgements This work was supported by the research fund of Hanyang University (HY-2009-N).
Fig. 2. Immunohistochemistry of intrahepatically xenotransplanted islets in the liver of mice. Platelet marker, anti-CD41a; Neutrophil marker, anti-CD45, Insulin, anti-insulin.
References [1] D.Y. Lee, K. Yang, S. Lee, S.Y. Chae, K.W. Kim, M.K. Lee, D.J. Han, Y. Byun, Optimization of monomethoxy-polyethylene glycol grafting on the pancreatic islet capsulesJ. Biomed. Mater. Res. 62 (2002) 372–377. [2] D.Y. Lee, J.H. Nam, Y. Byun, Functional and histological evaluation of transplanted pancreatic islets immunoprotected by PEGylation and cyclosporine for 1 year, Biomaterials 28 (2007) 1957–1966.
doi:10.1016/j.jconrel.2011.09.016
Heparin-coated superparamagnetic iron oxide nanoparticles as highly effective MRI contrast agent for cell labeling Min Jin Jung, Young Eun Ha, Dong Yun Lee Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea E-mail address: [email protected] (M.J. Jung). Abstract summary The purpose of this study was to provide highly efficient MR T2 contrast agent based on heparin-coated superparamagnetic iron oxide nanoparticles (SPIO). Two types of biocompatible magnetic agent probes were developed using unfractionated (UFH) and lowmolecular-weight-heparin (LMWH), UFH-SPIO and LMWH-SPIO, respectively. The newly developed SPIOs with high signal intensity, stability, biocompatibility and magnetization can be efficiently internalized into Ins-1 cells, without changing viability and function. Keywords: Superparamagnetic iron oxide nanoparticles (SPIO), Cell uptake, MRI
Introduction SPIO nanoparticles are frequently used material for various applications in biotechnology and biomedicine. The nanoparticles can be used as MRI agents in diagnosis and therapy [1]. Currently, one of the most important and rapidly growing fields is the use of paramagnetic iron oxide particles as contrast agent for MRI [2]. A commercially available T2 contrast agent, dextran-SPIO (Feridex®) was synthesized by an alkaline coprecipitation process of Fe2O3 and Fe3O4 precursors. Feridex® has been widely applied to the cell labeling. However, Feridex® requires long incubation times, generally over 24 h, to be efficiently internalized into cells. Recent studies indicated that the high-temperature process was favorable for the synthesis of SPIO with better monodispersity, crystallinity, a higher magnetic moment, and a tighter surface bond of surfactant than other methods [3]. In order to develop a more sensitive imaging agent, we optimized the preparation process of unfractionated and low molecular weight heparin (UFH-SPIO and LMWH-SPIO)-coated SPIO. These contrast agent probes enhanced proton magnetization relaxation rates and thus decreased relaxation times at their accumulation sites. It was demonstrated that pharmacokinetics, biodistribution, uptake efficiency, metabolism and biocompatibility of these SPIOs were highly improved in comparison with Feridex®. Experimental methods Labeling Ins-1 cell with two types of heparin-SPIO. Ins-1 cells were cultured in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C under 5% CO2. For labeling, cells were incubated with complete media containing various concentrations of UFH-SPIO and LMWH-SPIO (equivalent to 0, 3, 6,15, 24, 30 and 45 μg/mL Fe concentrations) for 2 h. Dextran-SPIO was also added to Ins-1 cells at the same Fe concentration for 2 h. Cell viability assay. Ins-1 cells, pancreatic islet beta-cell line, were seeded on 6-well culture plates at a density of 1 × 107 cells/well. Cells were incubated with complete media containing various concentrations of UFH-SPIO, LMWH-SPIO, and Dextran-SPIO (equivalent to 0, 3, 6, 9, 15, 24 and 30 μg/mL Fe concentrations) for 2 h. Results and discussion The biocompatibility of heparin-SPIO was evaluated in Ins-1 cells by measuring cell viability. As shown in Fig. 1(a (1 day)), no statistical difference between the two types of heparin-SPIO was observed below Fe concentrations of 45 μg/mL. The long-term proliferation ability of Ins-1 cells was further examined to confirm whether both heparin-SPIO affect cell proliferation (Fig. 1(b (2 days)) and (c (4 days))). In the test conditions, two types of heparin-SPIO-labeled cells showed higher proliferative ability than dextran-labeled cells did (Fig. 1(d)), suggesting that the newly developed two types of heparin-SPIO can be applied for long-term MR monitoring with minimal toxicity. Ins-1 cell labeling efficiencies of both heparin-SPIOs were also demonstrated in in vitro MRI on T2-weighted images taken by using a clinical 3.0 T MR scanner (Fig. 2). SPIO-based particles generate a negative signal contrast (T2 relaxation contrast) in MR imaging. No obvious signal was observed in unlabeled cells (white color). However, the superparamagnetic signal intensities of cells labeled with UFH-SPIO, LMWH-SPIO and dextran-SPIO were Fe-concentration-dependent. In particular, two types of heparin-SPIO-labeled cells demonstrated more negative contrast enhancement than dextranlabeled cells. The cellular uptake of those heparin-SPIOs was highly efficient compared with dextran-SPIO when the cells were incubated at the same iron oxide concentrations. These results lead us to conclude that heparin-SPIOs can be developed as excellent MRI or cell tracking probe because Ins-1-cells were more efficiently labeled by simple incubation with UFH-SPIO.
Abstracts / Journal of Controlled Release 152 (2011) e192–e269
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doi:10.1016/j.jconrel.2011.09.017
The surface modification of nano-sized porous membrane with catechol conjugated poly(n-isopropyl acrylamide) for thermal responsive diffusion Jee Seon Kim, Taek Gyung Kim, Tae Gwan Park Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Republic of Korea E-mail address: [email protected] (J.S. Kim).
Fig. 1. The effects of two types of heparin-SPIO on Ins-1 cell viability ((a), (b) and (c)) and proliferation (d). CCK-8 assay showed that the cell viability was not affected by two types of heparin-SPIO or dextran-SPIO at iron concentrations below 45 μg/mL ((a), (b) and (c)). Two types of heparin-SPIO-labeled cells (45 μg/mL for 2 h) showed similar proliferation ability compared with unlabeled cells and higher proliferative ability than dextran-labeled cells (d).
Fig. 2. T2-weighted MR image of heparin-SPIOs and dextran-SPIO. The results showed that the signal intensity of cells-labeled with two types of heparin-SPIO and dextranSPIO was Fe-concentration-dependent. (A) Unlabeled Ins-1 cells without SPIO internalization, (B) Dextran-SPIO internalized Ins-1 cells, (C) LMWH-SPIO internalized Ins-1 cells, (D) UFH-SPIO internalized Ins-1 cells.
Conclusion We developed highly efficient and long-term MR T2 contrast agents based on heparin-SPIO nanoparticles. Heparin-SPIO labeling showed better cell viability and proliferation than dextran-SPIO, suggesting excellent biocompatibility. In addition, our study shows that Ins-1-cells can be efficiently labeled by simple incubation with heparin-SPIO and detected with a clinical 3.0 T MR scanner. In conclusion, both UFH- and LMWH-SPIO will be promising cell tracking probes for cell labeling. Acknowledgments This study was supported by the Converging Research Center Program (Grant No. 20090081879) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea. References [1] A. Bjornerud, L. Johansson, The utility of superparamagnetic contrast agents in MRI: theoretical consideration and applications in the cardiovascular system, NMR Biomed. 17 (2004) 465–477. [2] J.H. Tai, et al., Imaging islets labeled with magnetic nanoparticles at 1.5 Tesla, Diabetes 55 (2006) 2931–2938. [3] F. Hu, L. Wei, Z. Zhou, Y. Ran, Z. Li, M. Gao, Preparation of biocompatible magnetite nanocrystals for in vivo magnetic resonance detection of cancer, Adv. Mater. 18 (2006) 2553–2556.
Abstract summary In this study, we prepared PNIPAam of MW 12,000 by RAFT polymerization with narrow dispersity (PDI 1.09). The end functional group of the synthesized PNIPAam was conjugated to catechol and fluorescence dye, respectively. Not only the surface but also the inside of the pores of anodized aluminum oxide membranes could be coated with PNIPAam, which was verified with confocal microscopy. Additionally, thermal responsiveness was demonstrated by static oil contact angle and permeability tests. Keywords: poly(N-isopropylacrylamide), catechol, RAFT, thermal responsive polymer Introduction Nano structured materials are diversely used due to the advantages of large surface area and high aspect ratio. Therefore, the interest for functionalized nano structured materials has increased. In addition, the surface modification of the nano architecture with environmental responsive polymers leads to smart materials. Stimuli-responsive smart polymers are intriguing materials owing to the wide applications such as controlled release of drugs. One of the representative examples is poly(N-isopropylacrylamide) (PNIPAam) which shows a thermal transition from coil to globule depending on the lower critical solution temperature (LCST). The coil to globule transition may induce an on/off gating effect as well as turbidity changes. Most of the methods to functionalize materials, however, are limited to using small molecules such as surface initiated polymerization, plasma graft pore-filling or chemical vapor deposition. It is inconvenient to introduce the adhesive functional group to the bulk polymer and difficult to make the material to penetrate the pore evenly. Conventional acrylic polymerization occurs by radical polymerization which cannot introduce the functional group to the polymer. Thus, the design of an adhesive anchored linear polymer is essential. Catechol, a side chain of an unusual amino acid of 3, 4-dihydroxy-lphenylalanine (DOPA) found extensively in mussel-adhesive proteins, plays a role as a surface-independent anchor molecule [1]. Being grafted with catechol, PNIPAam acts as a thermal responsive and surface adhesive polymer. To realize this concept, radical addition-fragmentation chain transfer polymerization (RAFT) was adopted which facilitates the introduction of the end functional group into the polymer and the production of highly monodisperse polymer [2]. Here, we suggest a versatile method to modify the nanostructure of membranes with a polymer, which is made by RAFT polymerization and which contains a catechol moiety. Experimental methods RAFT polymerization of N-isopropylacrylamide. A mixture of NIPAam monomer (3.93 g, 34.7 mmol), CTA (88.13 mg, 0.32 mmol) and AIBN (12.93 mg, 0.08 mmol) ([monomer]/[CTA] = 110/1, [CTA]/ [initiator] = 4/1)was dissolved in 1,4-dioxane (16 mL). The solution was degassed in 10 min under nitrogen. RAFT polymerization took
Abstracts / Journal of Controlled Release 152 (2011) e192–e269
Acknowledgements This work was supported by the research fund of Hanyang University (HY-2009-N).
Fig. 2. Immunohistochemistry of intrahepatically xenotransplanted islets in the liver of mice. Platelet marker, anti-CD41a; Neutrophil marker, anti-CD45, Insulin, anti-insulin.
References [1] D.Y. Lee, K. Yang, S. Lee, S.Y. Chae, K.W. Kim, M.K. Lee, D.J. Han, Y. Byun, Optimization of monomethoxy-polyethylene glycol grafting on the pancreatic islet capsulesJ. Biomed. Mater. Res. 62 (2002) 372–377. [2] D.Y. Lee, J.H. Nam, Y. Byun, Functional and histological evaluation of transplanted pancreatic islets immunoprotected by PEGylation and cyclosporine for 1 year, Biomaterials 28 (2007) 1957–1966.
doi:10.1016/j.jconrel.2011.09.016
Heparin-coated superparamagnetic iron oxide nanoparticles as highly effective MRI contrast agent for cell labeling Min Jin Jung, Young Eun Ha, Dong Yun Lee Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul 133-791, Republic of Korea E-mail address: [email protected] (M.J. Jung). Abstract summary The purpose of this study was to provide highly efficient MR T2 contrast agent based on heparin-coated superparamagnetic iron oxide nanoparticles (SPIO). Two types of biocompatible magnetic agent probes were developed using unfractionated (UFH) and lowmolecular-weight-heparin (LMWH), UFH-SPIO and LMWH-SPIO, respectively. The newly developed SPIOs with high signal intensity, stability, biocompatibility and magnetization can be efficiently internalized into Ins-1 cells, without changing viability and function. Keywords: Superparamagnetic iron oxide nanoparticles (SPIO), Cell uptake, MRI
Introduction SPIO nanoparticles are frequently used material for various applications in biotechnology and biomedicine. The nanoparticles can be used as MRI agents in diagnosis and therapy [1]. Currently, one of the most important and rapidly growing fields is the use of paramagnetic iron oxide particles as contrast agent for MRI [2]. A commercially available T2 contrast agent, dextran-SPIO (Feridex®) was synthesized by an alkaline coprecipitation process of Fe2O3 and Fe3O4 precursors. Feridex® has been widely applied to the cell labeling. However, Feridex® requires long incubation times, generally over 24 h, to be efficiently internalized into cells. Recent studies indicated that the high-temperature process was favorable for the synthesis of SPIO with better monodispersity, crystallinity, a higher magnetic moment, and a tighter surface bond of surfactant than other methods [3]. In order to develop a more sensitive imaging agent, we optimized the preparation process of unfractionated and low molecular weight heparin (UFH-SPIO and LMWH-SPIO)-coated SPIO. These contrast agent probes enhanced proton magnetization relaxation rates and thus decreased relaxation times at their accumulation sites. It was demonstrated that pharmacokinetics, biodistribution, uptake efficiency, metabolism and biocompatibility of these SPIOs were highly improved in comparison with Feridex®. Experimental methods Labeling Ins-1 cell with two types of heparin-SPIO. Ins-1 cells were cultured in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C under 5% CO2. For labeling, cells were incubated with complete media containing various concentrations of UFH-SPIO and LMWH-SPIO (equivalent to 0, 3, 6,15, 24, 30 and 45 μg/mL Fe concentrations) for 2 h. Dextran-SPIO was also added to Ins-1 cells at the same Fe concentration for 2 h. Cell viability assay. Ins-1 cells, pancreatic islet beta-cell line, were seeded on 6-well culture plates at a density of 1 × 107 cells/well. Cells were incubated with complete media containing various concentrations of UFH-SPIO, LMWH-SPIO, and Dextran-SPIO (equivalent to 0, 3, 6, 9, 15, 24 and 30 μg/mL Fe concentrations) for 2 h. Results and discussion The biocompatibility of heparin-SPIO was evaluated in Ins-1 cells by measuring cell viability. As shown in Fig. 1(a (1 day)), no statistical difference between the two types of heparin-SPIO was observed below Fe concentrations of 45 μg/mL. The long-term proliferation ability of Ins-1 cells was further examined to confirm whether both heparin-SPIO affect cell proliferation (Fig. 1(b (2 days)) and (c (4 days))). In the test conditions, two types of heparin-SPIO-labeled cells showed higher proliferative ability than dextran-labeled cells did (Fig. 1(d)), suggesting that the newly developed two types of heparin-SPIO can be applied for long-term MR monitoring with minimal toxicity. Ins-1 cell labeling efficiencies of both heparin-SPIOs were also demonstrated in in vitro MRI on T2-weighted images taken by using a clinical 3.0 T MR scanner (Fig. 2). SPIO-based particles generate a negative signal contrast (T2 relaxation contrast) in MR imaging. No obvious signal was observed in unlabeled cells (white color). However, the superparamagnetic signal intensities of cells labeled with UFH-SPIO, LMWH-SPIO and dextran-SPIO were Fe-concentration-dependent. In particular, two types of heparin-SPIO-labeled cells demonstrated more negative contrast enhancement than dextranlabeled cells. The cellular uptake of those heparin-SPIOs was highly efficient compared with dextran-SPIO when the cells were incubated at the same iron oxide concentrations. These results lead us to conclude that heparin-SPIOs can be developed as excellent MRI or cell tracking probe because Ins-1-cells were more efficiently labeled by simple incubation with UFH-SPIO.
Abstracts / Journal of Controlled Release 152 (2011) e192–e269
e215
doi:10.1016/j.jconrel.2011.09.017
The surface modification of nano-sized porous membrane with catechol conjugated poly(n-isopropyl acrylamide) for thermal responsive diffusion Jee Seon Kim, Taek Gyung Kim, Tae Gwan Park Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Republic of Korea E-mail address: [email protected] (J.S. Kim).
Fig. 1. The effects of two types of heparin-SPIO on Ins-1 cell viability ((a), (b) and (c)) and proliferation (d). CCK-8 assay showed that the cell viability was not affected by two types of heparin-SPIO or dextran-SPIO at iron concentrations below 45 μg/mL ((a), (b) and (c)). Two types of heparin-SPIO-labeled cells (45 μg/mL for 2 h) showed similar proliferation ability compared with unlabeled cells and higher proliferative ability than dextran-labeled cells (d).
Fig. 2. T2-weighted MR image of heparin-SPIOs and dextran-SPIO. The results showed that the signal intensity of cells-labeled with two types of heparin-SPIO and dextranSPIO was Fe-concentration-dependent. (A) Unlabeled Ins-1 cells without SPIO internalization, (B) Dextran-SPIO internalized Ins-1 cells, (C) LMWH-SPIO internalized Ins-1 cells, (D) UFH-SPIO internalized Ins-1 cells.
Conclusion We developed highly efficient and long-term MR T2 contrast agents based on heparin-SPIO nanoparticles. Heparin-SPIO labeling showed better cell viability and proliferation than dextran-SPIO, suggesting excellent biocompatibility. In addition, our study shows that Ins-1-cells can be efficiently labeled by simple incubation with heparin-SPIO and detected with a clinical 3.0 T MR scanner. In conclusion, both UFH- and LMWH-SPIO will be promising cell tracking probes for cell labeling. Acknowledgments This study was supported by the Converging Research Center Program (Grant No. 20090081879) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, Republic of Korea. References [1] A. Bjornerud, L. Johansson, The utility of superparamagnetic contrast agents in MRI: theoretical consideration and applications in the cardiovascular system, NMR Biomed. 17 (2004) 465–477. [2] J.H. Tai, et al., Imaging islets labeled with magnetic nanoparticles at 1.5 Tesla, Diabetes 55 (2006) 2931–2938. [3] F. Hu, L. Wei, Z. Zhou, Y. Ran, Z. Li, M. Gao, Preparation of biocompatible magnetite nanocrystals for in vivo magnetic resonance detection of cancer, Adv. Mater. 18 (2006) 2553–2556.
Abstract summary In this study, we prepared PNIPAam of MW 12,000 by RAFT polymerization with narrow dispersity (PDI 1.09). The end functional group of the synthesized PNIPAam was conjugated to catechol and fluorescence dye, respectively. Not only the surface but also the inside of the pores of anodized aluminum oxide membranes could be coated with PNIPAam, which was verified with confocal microscopy. Additionally, thermal responsiveness was demonstrated by static oil contact angle and permeability tests. Keywords: poly(N-isopropylacrylamide), catechol, RAFT, thermal responsive polymer Introduction Nano structured materials are diversely used due to the advantages of large surface area and high aspect ratio. Therefore, the interest for functionalized nano structured materials has increased. In addition, the surface modification of the nano architecture with environmental responsive polymers leads to smart materials. Stimuli-responsive smart polymers are intriguing materials owing to the wide applications such as controlled release of drugs. One of the representative examples is poly(N-isopropylacrylamide) (PNIPAam) which shows a thermal transition from coil to globule depending on the lower critical solution temperature (LCST). The coil to globule transition may induce an on/off gating effect as well as turbidity changes. Most of the methods to functionalize materials, however, are limited to using small molecules such as surface initiated polymerization, plasma graft pore-filling or chemical vapor deposition. It is inconvenient to introduce the adhesive functional group to the bulk polymer and difficult to make the material to penetrate the pore evenly. Conventional acrylic polymerization occurs by radical polymerization which cannot introduce the functional group to the polymer. Thus, the design of an adhesive anchored linear polymer is essential. Catechol, a side chain of an unusual amino acid of 3, 4-dihydroxy-lphenylalanine (DOPA) found extensively in mussel-adhesive proteins, plays a role as a surface-independent anchor molecule [1]. Being grafted with catechol, PNIPAam acts as a thermal responsive and surface adhesive polymer. To realize this concept, radical addition-fragmentation chain transfer polymerization (RAFT) was adopted which facilitates the introduction of the end functional group into the polymer and the production of highly monodisperse polymer [2]. Here, we suggest a versatile method to modify the nanostructure of membranes with a polymer, which is made by RAFT polymerization and which contains a catechol moiety. Experimental methods RAFT polymerization of N-isopropylacrylamide. A mixture of NIPAam monomer (3.93 g, 34.7 mmol), CTA (88.13 mg, 0.32 mmol) and AIBN (12.93 mg, 0.08 mmol) ([monomer]/[CTA] = 110/1, [CTA]/ [initiator] = 4/1)was dissolved in 1,4-dioxane (16 mL). The solution was degassed in 10 min under nitrogen. RAFT polymerization took