Funtionalization of magnetic nanoparticles for bio applications

FUNCTIONALIZATION OF MAGNETIC NANOPARTICLES FOR BIO-APPLICATIONS WUANG SHY CHYI (B. Eng (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to my supervisors, Professors K. G. Neoh, Daniel Pack, E.-T. Kang and Deborah Leckband for their continued guidance, invaluable suggestions and profound discussion throughout this work. Without their enthusiasm and help, this project would not be possible. The knowledge gained under their supervision and the research experiences pave the way for my lifelong study. I would also like to thank the other members of my committee for their help and time, as well as the research staff and laboratory officers, both in the National University of Singapore and the University of Illinois at Urbana-Champaign. Finally I thank my family, colleagues and numerous friends for their love, support and encouragement. ii TABLE OF CONTENTS ACKNOWLEDGEMENT ii TABLE OF CONTENTS iii SUMMARY v LIST OF TABLES vi LIST OF FIGURES vii NOMENCLATURE x CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW 2.1 Magnetic nanoparticles 2.2 Magnetic drug delivery 2.3 Cancer targeting 12 2.4 Magnetic Fluid Hyperthermia 14 2.5 Magnetic nanoparticles in imaging 15 2.6 Biocompatible polymers 18 CHAPTER 3: HEPARINIZED MAGNETITE NANOPARTICLES 3.1 Introduction 20 3.2 Methods and materials 21 3.3 Results and discussion 28 3.4 Chapter Conclusion 43 CHAPTER 4: DOXORUBICIN-ATTACHED MAGNETITE NANOPARTICLES 4.1 Introduction 44 4.2 Methods and materials 45 4.3 Results and discussion 51 iii 4.4 Chapter Conclusion 63 CHAPTER 5: ANTIBODY-ATTACHED MAGNETITE NANOPARTICLES 5.1 Introduction 65 5.2 Methods and materials 66 5.3 Results and discussion 70 5.4 Chapter Conclusion 80 CHAPTER 6: POLYPYRROLE-MAGNETITE NANOSPHERES 6.1 Introduction 81 6.2 Methods and materials 82 6.3 Results and discussion 91 6.4 Chapter Conclusion 124 CHAPTER 7: SUMMARY CONCLUSION 126 CHAPTER 8: RECOMMENDATIONS FOR FUTURE WORK 129 REFERENCES 132 LIST OF PUBLICATIONS 149 LIST OF CONFERENCES 150 iv SUMMARY Magnetite nanoparticles were modified to render them suitable for bio-applications, namely drug delivery and hyperthermia, using two different approaches. The first approach is to graft polymers onto the nanoparticles using surface-initiated atom transfer radical polymerization, followed by chemical linking of biomolecules onto the grafted polymers. The monomers used include N-isopropylacrylamide, Nvinylformamide and methacrylic acid while the immobilized biomolecules include heparin, folic acid, doxorubicin and anti-HER2/neu antibodies. It was found that the heparinized nanoparticles could reduce macrophage uptake, and at the same time inhibit plasma clotting. The doxorubicin-bearing nanoparticles were able to release a greater amount of the drug under acidic conditions as opposed to physiological pH, and could potentially serve as drug depots. Particles that were functionalized with anti-HER2/neu antibodies showed a preferential binding to cancer cells and may be useful for imaging purposes. The second approach was to encapsulate these magnetite nanoparticles into polypyrrole nanospheres via emulsion polymerization for potential use as hyperthermia causing agents. The nanospheres were then functionalized with folic acid or herceptin to impart onto them cancer cell-targeting properties. These functionalized nanospheres target cancer cells in vitro and possess good magnetization which is useful for magnetic fluid hyperthermia. v LIST OF TABLES Table 3.1 Dispersion of as-synthesized and functionalized magnetite in various solvents at 25ºC. Table 3.2 Comparison of PRT obtained in the absence and presence of magnetic nanoparticles. Table 6.1 Characteristics of the PPY nanospheres (Scale bar = 200nm). Table 6.2 Properties of NS(PVA). Table 6.3 Properties of NS(HA) and NS(HA)-HER2. Table 6.4 Amount of iron associated with SK-Br-3 and MDA-MB-231 cells after incubation with NS(HA) and NS(HA)-HER2. vi LIST OF FIGURES Figure 3.1 Schematic representation of the process for preparing MNP-NP-He. Figure 3.2 XPS C 1s and S 2p core-level spectra of as-synthesized MNP (a, c), magnetite-Cl (b, d) and N 1s core-level spectra of magnetite-Cl (e) and MNP-NP (f). Figure 3.3 XPS C 1s and S 2p core-level spectra of MNP-NP (a, c) and MNP-NPHe (b, d). Figure 3.4 FTIR spectra of MNP (a), magnetite-Cl (b), MNP-NP(c) and MNPNP-He (d) Figure 3.5 Room temperature magnetization curves of as-synthesized and functionalized magnetite nanoparticles as a function of applied magnetic field: MNP (a), MNP-NP (b) and MNP-NP-He (c). Figure 3.6 Optical microscopy images of macrophages cultured with control cells (a, b), MNP (c, d), MNP-NP (e, f) and MNP-NP-He (g, h) after and 24 h respectively. Figure scale bar = 50μm, inset scale bar = 25μm. Figure 3.7 Total uptake of as-synthesized and functionalized nanoparticles by macrophages after 2, and 24 h. Figure 3.8 Cytotoxicity of as-synthesized and functionalized magnetite nanoparticles, as measured by the viability of macrophages grown in media containing 0.2 mg/ml of these nanoparticles relative to the nontoxic control. T represents the results obtained with the toxic control. Results are represented as mean ± standard deviation. Figure 4.1 Schematic for synthesis of doxorubicin-conjugated particles. Figure 4.2 XPS C 1s core-level spectra of MNP-P(MAA)-NHNH2 (a), MNPP(MAA)-NH-N=Dox (b) and doxorubicin hydrochloride (c). Figure 4.3 Magnetization profiles of MNP-P(MAA)-NH-N=Dox in the solid state (a), and as dispersed in 1% agarose (b). Figure 4.4 In vitro doxorubicin release from MNP-P(MAA)-NH-N=Dox at 37°C or 42°C in various pHs as indicated. . Figure 4.5 Figure 4.6 magnetite In vitro doxorubicin release from MNP-P(MAA)-NH-N=Dox at 37°C. Arrow indicates point of pH change from 7.4 to 5.5 or 6.6. In vitro doxorubicin release from MNP-P(MAA)-NH-N=Dox. Arrow indicates point of temperature change from 37°C to 42°C and pH change from 7.4 to 5.5 or 6.6. vii Figure 4.7 Figure 4.7: Viabilities of MDA-MB-231 cells (relative to non-toxic controls) incubated with medium containing free doxorubicin (Dox) or MNP-P(MAA)-NH-N=Dox. “*”, “#”, “**” and “##” denote statistical differences (P < 0.05) between the similarly marked samples. Figure 4.8 Optical microscopy images of prussian blue staining of MDA-MB-231 cells cultured with MNP-P(MAA)-NH-N=Dox in pH 5.5 (a) and pH 7.4 (b). Scale bar = 25μm. Figure 5.1 Schematic for synthesis of MNP-NVAM-PEG-Ab. Figure 5.2 FTIR spectra of MNP-NVAM (a), MNP-NVAM-PEG (b) and MNPNVAM-PEG-Ab (c) Figure 5.3 Optical microscopy images of SK-Br-3 cells after a h incubation with MNP-NVAM-PEG-Ab (a) and co-treatment with MNP-NVAM-PEGAb and free antibody (b). Scale bar = 25 µm. Figure 5.4 Prussian blue staining of the liver (a), bladder (b), heart (c), kidney (d), spleen (e), lung (f), tumors (g-i) and the site of injection, tail (j). Scale bar = 50 µm. Figure 5.5 Prussian blue staining of the liver (a), bladder (b), heart (c), kidney (d), spleen (e), lung (f), tumors (g-i) and the site of injection, tail (j). Scale bar = 50 µm. Figure 5.6 MMOCT spectra of negative control (a), and phantom with an equivalent particle concentration of 23 µg Fe/ml (b). Scale bar = 250 μm. Figure 6.1 Schematic representation of the preparation route NS(PVA)-FA. Figure 6.2 (a) FTIR spectra of Fe3O4, PPY nanospheres and NS(PVA) (b) XRD spectrum of NS(PVA). Figure 6.3 Room temperature magnetization curves of NS(PVA) as a function of applied magnetic field. Figure 6.4 FESEM and TEM images of NS(PVA) with (a, b) %, (c, d) 23.5 %, (e, f) 28.0 % and (g, h) 38.8 % Fe3O4 content respectively. Figure 6.5 XPS C 1s core-level and wide scan spectra of (a, b) NS(PVA) and (c, d) NS(PVA)-FA. Viabilities of 3T3 fibroblasts incubated with medium containing 0.2 mg/ml of NS(PVA) with the indicated Fe3O4 content. FA-(28.0%) denoted NS(PVA)-FA with 28.0% of Fe3O4. “*” denotes statistical differences (P < 0.05) compared to the control experiment. Figure 6.6 viii Figure 6.7 Figure 6.8 Viabilities of MCF cells incubated with medium containing 0.2 mg/ml of nanospheres containing 28.0 % of Fe3O4. “*” denotes statistical differences (P < 0.05) compared to the control experiment. Optical microscopy images of MCF-7 cells cultured with: (a) no nanospheres (control) (b) NS(PVA) and (c) NS(PVA)-FA after 24 h. Figure scale bar = 50μm. Figure 6.9 Schematic for preparation of NS(HA) and subsequent functionalization with herceptin. Figure 6.10 Uptake of the nanospheres by HCC1954 cells: (a) Optical microscopy images of cells after 24 h incubation with (i) NS(HA), and (ii) NS(HA)-HER1. Figure scale bar = 50 μm. (b) Amount of iron in cells after h and 24 h incubation with NS(HA) and NS(HA)-HER1 determined using ICP. Figure 6.11 Schematic representation of the preparation of NS(HA)-HER2. Figure 6.12 XPS C 1s core-level spectra of (a) NS(NH2), (b) NS(HA)-HER2 and (c) herceptin. Figure 6.13 Amount of iron associated with SK-Br-3 cells after 2, and 24 h incubation with NS(HA), NS(HA)-HER2 and NS(HA)-HER2 with free herceptin. Three sets of duplicates were done for each data point. The iron association of NS(HA)-HER2 is significantly higher (P < 0.01) than those for NS(HA) and NS(HA)-HER2 with free herceptin at all time points. Figure 6.14 Transmission electron micrographs of cells cultured with (a) NS(HA)HER2 (b) NS(HA)-HER2 with pre- and co-treatment of 200 µg/ml free herceptin, for 4h. Figure 6.15 Cytotoxicities of NS(HA)-HER2 and NS(HA) with various concentrations of herceptin (HER), as measured by the viabilities of SK-Br-3 cells grown in media containing 0.2 mg/ml of these nanospheres relative to the non-toxic control. Results are represented as mean ± standard deviation. “*” denotes statistical differences (P < 0.05) compared to the control experiment. Figure 6.16 Plot of viability of cells versus iron uptake by breast cancer cells. Tested cell lines include SK-Br-3 (■), MDA-MB-231 (♦) and MCF-7 (▲). Figure 6.17 Magnetization curves of NS(HA)-HER2 in different environments (a) NS(HA)-HER2 solid, (b) NS(HA)-HER2 dispersed in culture medium with 1% agarose and (c) endocytosed NS(HA)-HER2 dispersed in culture medium with 1% agarose. ix NOMENCLATURE ATRP Atom-transfer radical polymerization Bpy 2-2’-bipyridyl CPA 3-chloropropionic acid CT Computed tomography CTCS 2-(4-chlorosulfonylphenyl) ethyltrichlorosilane DMF Dimethyl formamide DMSO Dimethyl sulphoxide EDC 1-ethyl-3-(3-dimethylamino)-propyl carbodiimide EGFR Human epidermal growth factor receptor FMOC-NH-PEG-SCM Fluorenylmethoxycarbonyl-poly(ethylene glycol)-succinimidyl carboxymethyl FTIR Fourier-transform infrared HA Hyaluronic acid HER2 Human epidermal growth factor receptor ICP Inductively coupled plasma spectroscopy LCST Lower critical solution temperature mAb Whole monoclonal antibodies MFH Magnetic fluid hyperthermia MMOCT Magnetomotive OCT MNP Magnetite nanoparticles MNP-NP poly(NIPAAM)-grafted MNP MNP-NP-He Magnetite-poly(NIPAAM)-Heparin MNP-P(MAA) P(MAA)-grafted MNP x References nanoparticles as drug carriers, European Biophysics J. 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Szoka, Acid-triggered transformation of diortho ester phosphocholine liposome, J. Am. Chem. Soc. 1281 (2006) 60-61. [227] D.J. Javier, N. Nitin, M. Levy, A. Ellington, R. Richards-Kortum, Aptamertargeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging, Bioconjug. Chem. 196 (2008) 1309-1312. - 148 - Publications LIST OF PUBLICATIONS S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “HER-2mediated Endocytosis of Magnetic Nanospheres and the Implications in Cell Targeting and Particle Magnetization” Biomaterials (2008) 29, 2270-2279. S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Synthesis and Functionalization of Polypyrrole-Fe3O4 Nanoparticles for Applications in Biomedicine” J. Mater. Chem. (2007) 17, 3354-3362. S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Polypyrrole Nanospheres with Magnetic and Cell-Targeting Capabilities.” Macromol. Rapid Comm. (2007) 28, 816-821. (Cover article) S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Heparinized Magnetic Nanoparticles: In-Vitro Assessment for Biomedical Applications” Adv. Funct. Mater. (2006) 16, 1723–1730. F. J. Xu, S. C. Wuang, B. Y. Zong, E.-T. Kang, K. G. Neoh. “Immobilization of Functional Oxide Nanoparticles on Silicon Surfaces via Si-C Bonded Polymer Brushes.” J. Nanosci. Nanotechnol. (2006) 6, 1458-1463. - 149 - Conferences LIST OF CONFERENCES S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Targeting Breast Cancer Cells via HER-2-mediated Endocytosis” Poster Presentation, Controlled Release Society Annual Meeting (2008), New York, New York S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Surface Functionalization of Magnetic Nanospheres for Cancer Cell Targeting” Poster Presentation, SBE's 3rd International Conference on Bioengineering and Nanotechnology (2007) Singapore S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Surface Functionalization of Magnetic Nanospheres for Cancer Cell Targeting” Poster Presentation, CNST Nanotechnology Workshop (2007) Urbana-Champaign, Illinois S. C. Wuang, F. X. Hu, K. G. Neoh and E.-T. Kang “Surface Functionalization of Magnetic Nanoparticles via Surface-Inititated Atom-Transfer Radical Polymerization (ATRP)” Poster Presentation, OLS-NUSNNI Workshop on Nanobiotechnology and Nanomedicine (2006) Singapore S. C. Wuang, K. G. Neoh, E.-T. Kang, D. W. Pack, and D. E. Leckband. “Functionalization of Magnetic Nanoparticles with Biomolecules” Oral Presentation, Particles 2006 Conference, (2006) Orlando, Florida S. C. Wuang, F. X. Hu, K. G. Neoh and E.-T. Kang “Surface Functionalization of Magnetic Nanoparticles via Surface-Inititated Atom-Transfer Radical Polymerization (ATRP)” Poster Presentation, Particles 2006 Conference, (2006) Orlando, Florida F. X. Hu, S. C. Wuang, K. G. Neoh and E.-T. Kang. “Surface Functionalized Magnetic Nanoparticles for Biomedical Applications” Poster Presentation, 2nd MRS-S Conference on Advanced Materials (2006) Singapore - 150 - [...]... saturation of ferromagnetic iron oxide [15] This behavior allows the tracking of such particles in a magnetic field gradient without losing the advantage of a stable colloidal suspension Nanotechnology has allowed for the production, characterization and functionalization of magnetic nanoparticles for specialized clinical applications Extensive research has been done on use of magnetic nanoparticles for bioapplications... are of great interest for researchers from a wide range of disciplines, including catalysis [8, 9], data storage [10], environmental remediation [11, 12] and more recently in biotechnology/biomedicine [13] Some of the more specific biomedical applications of magnetic nanoparticles include their use as magnetic contrast agents in magnetic resonance imaging (MRI), hyperthermia agents, where the magnetic. .. layer was first formed on the surface of the magnetite nanoparticles via ATRP followed by the immobilization of heparin onto the poly(NIPAAM) shell The poly(NIPAAM) layer also allows for the dispersion of the nanoparticles in formamide, the medium used for heparin immobilization Recent trends have shown that poly(NIPAAM) can be used for a number of potential biomedical applications such as for drug release... superparamagnetic nanoparticles very attractive for a broad range of biomedical applications because the risk of forming agglomerates is -4- Chapter 2 negligible at room temperature Superparamagnetic iron oxide (SPIO) nanoparticles are small synthetic γ-Fe2O3 or Fe3O4 particles with a core size of ~10 nm and an organic or inorganic coating Superparamagnetic magnetization is, compared to normal paramagnetic... with these biological entities Through manipulation by an external magnetic field, magnetic nanoparticles are potentially very useful in the transport and immobilization of magnetically tagged biological cargoes, and also in transferring energy from the exciting field The focus of this research project is to functionalize or modify magnetic nanoparticles to render them suitable for biomedical applications. .. application of a high frequency oscillating magnetic field, and magnetic drug delivery In most biomedical applications, magnetic nanoparticles perform best when the size of the nanoparticles is around 10–20 nm [14] Each nanoparticle then becomes a single magnetic domain and shows superparamagnetic behavior when the temperature is above the Curie temperature Superparamagnetism is a phenomenon by which magnetic. .. solution phases present during the synthesis Fe3O4 and CoFe2O4 nanoparticles can be prepared in very uniform sizes of about 9 and 12 nm, respectively Hydrothermal synthesis is a relatively little explored method for the synthesis of magnetic nanoparticles, although it allows the synthesis of high- -7- Chapter 2 quality nanoparticles To date, magnetic nanoparticles prepared from co-precipitation and thermal... imaging [19] and gene delivery [20, 21] applications The main advantages of magnetic nanoparticles in biomedicine are that they can be (i) visualized by magnetic resonance imaging due to their ability to change the T1 or T2 relaxation times of the surrounding tissues; (ii) manipulated by means of a magnetic field (i.e in magnetic drug delivery); and (iii) heated in a magnetic field to trigger drug release... years, magnetic nanoparticles have been proposed for use in a number of biomedical applications such as drug delivery, hyperthermia and chemotherapy and as radiotherapy enhancement agents because of their special physical properties [1-3] Magnetic nanoparticles have controllable sizes, smaller than cells and comparable to proteins and other biological entities, and hence they can be modified for interaction... temperature [32]; drug -8- Chapter 2 release can also be magnetically triggered from the drug-conjugated magnetic nanoparticles In biomedicine, one major hurdle that underlies the use of nanoparticle therapy is the problem of getting the particles to a particular site in the body [1] A potential benefit of using magnetic nanoparticles is the use of localized magnetic field gradients to attract the particles . FUNCTIONALIZATION OF MAGNETIC NANOPARTICLES FOR BIO-APPLICATIONS WUANG SHY CHYI (B. Eng (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL. application of a high frequency oscillating magnetic field, and magnetic drug delivery. In most biomedical applications, magnetic nanoparticles perform best when the size of the nanoparticles. use of magnetic nanoparticles for bio- applications in recent years. For instance, dendrimer modified magnetic nanoparticles have been synthesized to improve protein binding [16]. Iron oxide nanoparticles