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Interaction of Anionic Superparamagnetic Nanoparticles with Cells: Kinetic Analyses of Membrane Adsorption and Subsequent Internalization C. Wilhelm,† F. Gazeau,*,† J. Roger,‡ J. N. Pons,‡ and J.-C. Bacri† Laboratoire des Milieux De´ sordonne´ s et He´ te´ roge` nes, CNRS UMR 7603 and Universite´ Pierre et Marie Curie, Tour 13, Case 86, 4 place Jussieu, 75252 Paris, France, and Fe´ de´ ration de Recherche MSC, CNRS FR 2438 and Universite´ Paris, 7-Denis Diderot, Paris, France, and Laboratoire des Liquides Ioniques et Interfaces Charge´ es, Universite´ Pierre et Marie Curie, Baˆ timent F, Case 63, 4 place Jussieu, 75005 Paris, France Received March 13, 2002. In Final Form: August 8, 2002 Cell targeting with magnetic nanoparticles raises a growing interest in the fields of both cellular biology and medical imaging. We have investigated the nonspecific interactions of superparamagnetic negatively charged iron oxide nanoparticles with HeLa tumor cells and mouse RAW macrophages, qualitatively by electron microscopy and quantitatively following the particle cell uptake by two magnetic based quantification assays (magnetophoresis and electron spin resonance). The analyses of particle uptake kinetics at 4 and 37 °C led us to modelize the observed endocytosis as a two-step process: we distinguish the binding of anionic magnetic nanoparticles onto the cell surface (described as a Langmuir adsorption) from the subsequent step of cell internalization (also described as a saturable mechanism). Fits of experimental uptake kinetics result in the quantitative determination of binding parameters (adsorption rate, desorption rate, and density of binding sites) as well as internalization rate and internalization capacity. All binding parameters appear to coincide for tumor cells and macrophages, whereas their internalization capacity differs by 1 order of magnitude, reflecting the cell function specificity.
1. Introduction Attempts to use colloidal systems as drug carrier, delivery vehicle, nonviral transfection vector, imaging label, or therapeutic agent raise the recurrent question of their recognition and capture by cells. Most improvements of the particle/cell recognition lie on the association of the colloidal particle with ligands that bind specifically to targeted membrane receptors. However, the way particles, devoid of any specific recognition tag, can interact with cells is often poorly understood. So far, the detailed mechanism of their interaction with the cell membrane and their possible uptake have been studied mainly for liposomes (in relation with their lipid specificity, surface charge density, or other surface modification)1-4 and for polymer particles.5,6 Besides, the development of * Corresponding author. E-mail address:
[email protected]. Fax number: 33-(1)-44-27-38-54. † Laboratoire des Milieux De ´ sordonne´s et He´te´roge`nes, CNRS UMR 7603 and Universite´ Pierre et Marie Curie, and Fe´de´ration de Recherche MSC, CNRS FR 2438 and Universite´ Paris. ‡ Laboratoire des Liquides Ioniques et Interfaces Charge ´ es, Universite´ Pierre et Marie Curie. (1) Chenevier, P.; Veyret, B.; Roux, D.; Henry-Toulme, N. Interaction of cationic colloids at the surface of J774 cells: a kinetic analysis. Biophys J. 2000, 79 (3), 1298-309. (2) Lee, K. D.; Nir, S.; Papahadjopoulos, D. Quantitative analysis of liposome-cell interactions in vitro: rate constants of binding and endocytosis with suspension and adherent J774 cells and human monocytes. Biochemistry 1993, 32 (3), 889-99. (3) Miller, C. R.; Bondurant, B.; McLean, S. D.; McGovern, K. A.; O’Brien, D. F. Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes. Biochemistry 1998, 37 (37), 12875-83. (4) Carmona-Ribeiro, A. M.; Ortis, F.; Schumacher, R. I.; Armelin, M. C. S Interactions between cationic vesicles and cultured mammalian cells. Langmuir 1997, 13, 2215-18. (5) Jaulin, N.; Appel, M.; Passirani, C.; Barratt, G.; Labarre, D. Reduction of the uptake by a macrophagic cell line of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate). J. Drug Target 2000, 8 (3), 165-72.
bionanotechnologies based on the physical properties of nanosized nonorganic particles, such as superparamagnetic iron oxide,7 semiconductor nanocrystals (quantum rods as fluorescent labels),8,9 luminophore-doped silica nanoparticles,10 gold nanoparticles, or radioactive particles, restores the key issue of the different pathways of nanoparticle internalization by cells and especially by nonprofessional phagocytes. Among mineral nanoparticles, superparamagnetic ones are involved in an increasing number of biomedical applications from cell biology to clinical diagnosis and therapy.11 Magnetic forces are used to move cells or organelles within the cell12 in directed fashion, to hold them or target them in vivo at specific anatomical sites, and to track and separate in vitro different cell lines. From a therapeutic point of view, magnetic nanoparticles incorporated into cells behave, when submitted to a high-frequency magnetic field, as nanosources of a heat-inducing cytotoxic effect.13,14 More (6) Alyaudtin, R. N.; Reichel, A.; Lobenberg, R.; Ramge, P.; Kreuter, J.; Begley, D. J. Interaction of poly(butylcyanoacrylate) nanoparticles with the blood-brain barrier in vivo and in vitro. J. Drug Target 2001, 9 (3), 209-21. (7) Hogemann, D.; Ntziachristos, V.; Josephson, L.; Weissleder, R. High throughput magnetic resonance imaging for evaluating targeted nanoparticle probes. Bioconjugate Chem. 2002, 13 (1), 116-21. (8) Lacoste, T. D.; Michalet, X.; Pinaud, F.; Chemla, D. S.; Alivisatos, A. P.; Weiss, S. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (17), 9461-6. (9) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281 (5385), 2013-6. (10) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Conjugation of biomolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal. Chem. 2001, 73 (20), 4988-93. (11) Schu¨tt, W.; Gru¨ttner, C.; Ha¨feli, U.; Zborowski, M.; Teller, J.; Putzar, H.; Schu¨michen, C. Applications of magnetic targeting in diagnosis and therapyspossibilities and limitations: a mini-review. Hybridoma 1997, 16, 109-116. (12) Bausch, A. R.; Ziemann, F.; Boulbitch, A. A.; Jacobson, K.; Sackmann, E. Measurement of local viscoelasticity and forces in living cells by magnetic tweezers. Biophys. J. 1999, 76, 573-579.
10.1021/la0257337 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/11/2002
Anionic Superparamagnetic Nanoparticles
currently and with promising developments, magnetic nanoparticles serve as contrast agent, enhancing proton relaxation in the noninvasive diagnostic method of magnetic resonance imaging (MRI). After in vivo injection, iron oxide nanoparticles accumulate into cells of the reticuloendothelial system, but also into tumor cells.15,16 Recently, magnetic nanoparticles have been engineered to allow efficient detection of gene expression in vivo using MRI.17,18 High resolution MRI also enables us to detect magnetically labeled cells,19 providing a method, of particular interest in the evaluation of cellular therapy, to study the fate and homing of cells injected in vivo.20,21,22 Understanding and further controling the cellular uptake of magnetic nanoparticles become thereby of crucial importance for these applications.17,23 A wide variety of cell lines have been labeled with the well-known dextrancoated ultrasmall superparamagnetic iron oxide particles.16 However, the cellular uptake remains too low to enable most applications, probably because of a relatively inefficient fluid phase endocytosis pathway. In this paper, we present a qualitative and quantitative analysis of the interaction with cells of anionic magnetic nanoparticles,24 free of any dextran coating. We show that this new class of magnetic nanoparticles exhibits a high level of cellular internalization, that is mediated by a high affinity with the cell membrane and coated pits endocytosis. The present work is aimed at understanding the mechanism of cell/ nanoparticle interaction through a quantitative kinetic study. A formalism based on mass action kinetics is developed to modelize the overall uptake of particles as a two-step process: a first step of binding at the cell membrane, described as a Langmuir adsorption, followed by a subsequent internalization step. The kinetic parameters for each step as well as the binding capacity and internalization capacity are determined separately by (13) Jordan, A.; Scholz, R.; Wust, P.; Fahling, H.; Krause, J.; Wlodarczyk, W.; Sander, B.; Vogl, T.; Felix, R. Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo. Int. J. Hyperthermia 1997, 13 (6), 587-605. (14) Ito, A.; Shinkai, M.; Honda, H.; Kobayashi, T. Heat-inducible TNF-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumor-targeted therapy. Cancer Gene Ther. 2001, 8 (9), 649-54. (15) Moore, A.; Weissleder, R.; Bogdanov, A. Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages. J. Magn. Reson. Imaging 1997, 7, 1140-1145. (16) Moore, A.; Marecos, E.; Bogdanov, A., Jr.; Weissleder, R. Tumoral distribution of long-circulating dextran-coated iron oxide nanoparticles in a rodent model. Radiology 2000, 214 (2), 568-74. (17) Hogemann, D.; Josephson, L.; Weissleder, R.; Basilion, J. P. Improvement of MRI probes to allow efficient detection of gene expression. Bioconjugate Chem. 2000, 11 (6), 941-6. (18) Weissleder, R.; Moore, A.; Mahmood, U.; Bhorade, R.; Benveniste, H.; Chiocca, E. A.; Basilion, J. P. In vivo magnetic resonance imaging of transgene expression. Nat. Med. 2000, 6 (3), 351-5. (19) Dodd, S. J.; Williams, M.; Suhan, J. P.; Williams, D. S.; Koretsky, A. P.; Ho, C. Detection of single mammalian cells by high-resolution magnetic resonance imaging. Biophys. J. 1999, 76 (1 Pt 1), 103-9. (20) Bulte, J. W.; Zhang, S.; van Gelderen, P.; Herynek, V.; Jordan, E. K.; Duncan, I. D.; Frank, J. A. Neurotransplantation of magnetically labeled oligodendrocyte progenitors: magnetic resonance tracking of cell migration and myelination. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (26), 15256-61. (21) Lewin, M.; Carlesso, N.; Tung, C. H.; Tang, X. W.; Cory, D.; Scadden, D. T.; Weissleder, R. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol. 2000, 18 (4), 410-4. (22) Bulte, J. W.; Douglas, T.; Witwer, B.; Zhang, S. C.; Strable, E.; Lewis, B. K.; Zywicke, H.; Miller, B.; van Gelderen, P.; Moskowitz, B. M.; Duncan, I. D.; Frank, J. A. Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat. Biotechnol. 2001, 19 (12), 1141-7. (23) Josephson, L.; Tung, C. H.; Moore, A.; Weissleder, R. Highefficiency intracellular magnetic labeling with novel superparamagneticTat peptide conjugates. Bioconjugate Chem. 1999, 10 (2), 186-91. (24) Fauconnier, N.; Pons, J. N.; Roger, J.; Bee, A. Thiolation of maghemite nanoparticles by dimercaptosuccinic acid. J. Colloid Interface Sci. 1997, 194, 427-433.
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performing incubation at 4 °C (only binding occurs) and at 37 °C (binding plus internalization occur) and by following the cell particle load with two different quantitation magnetic assays. All parameters are obtained by the proposed model both for a macrophage cell line (RAW 264.7) and for a human ovarian tumor cell line (HeLa). 2. Materials and Methods 2.1. Chemical Synthesis and Magnetic Properties of Anionic Magnetic Nanoparticles. The anionic magnetic nanoparticles are iron oxide (maghemite γFe2O3) nanoparticles and are synthesized following the Massart’s method.25 The ionic precursor is obtained by alkalizing an aqueous mixture of iron(II) chloride and iron(III) chloride. Nanoparticles are then chelated with meso-2,3-dimercaptosuccinic acid (HOOC-CH(SH)-CH(SH)-COOH) or DMSA,24 which forms a strong complex with the surface layer of the nanoparticles and confers to the nanoparticles negative surface charges due to the carboxylate groups. The colloidal stability is thus ensured by electrostatic repulsion between charged nanoparticles.26 One finally obtains an aqueous sol of thiolated maghemite nanoparticles, stable in a large pH range (from 3 to 11), in suitable ionic strength (
