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Magnetic nanoparticles: applications and cellular uptake Susanne Kirsch 20.02.2008 Overview - superparamagnetic particles in biomedical and biotechnological applications • • • - cellular uptake mechanism: endocytosis • • - pathways endocytosis of magnetic nanoparticles experiments with L929 fibroblasts and MG63 osteoblasts • • - drug/gene delivery hyperthermia magnetic resonance imaging temperature colchicine literature Superparamagnetic particles in biomedical and biotechnological applications Biomedical applications based on the controlled interactions between living cells and biologically activated magnetic nanoparticles. (PennWell) Magnetic particles ranging from nanometer to micrometer scale are being widely used in biomedical and biotechnological applications. Superparamagnetic particles in biomedical and biotechnological applications Particles used for biomedical and biotechnological applications are "superparamagnetic", meaning that they are attracted to a magnetic field but retain no residual magnetism after the field is removed. Therefore, suspended superparamagnetic particles tagged to the biomaterial of interest can be removed from a matrix using a magnetic field, but they do not agglomerate (i.e., they stay suspended) after removal of the field. Superparamagnetic particles in biomedical and biotechnological applications - small enough for administration (intravenous, oral, inhalation, etc.) → method to reach any target organ or tissue • • - - must reside in vivo long enough to reach its target avoid immunological reactions, toxicity, rapid excretion and captation by undesired tissues the smaller, the more neutral and the more hydrophilic the particle surface, the longer is its plasma half-life for redirecting to the desired target, the particle surface has to be labeled with ligands that specifically bind to receptors Drug/gene delivery - - inject magnetic particles to which drug molecules are attached, guide these to a chosen site under the localized magnetic field gradients, hold them there and remove them after therapy controlled drug delivery implies the ability to control the distribution of therapeutic agents both in space and time • increases the efficiency of the drug by maintaining the drug concentration within the optimum range and below the toxicity threshold Schematic representation of reservoir diffusion controlled drug delivery device, monolithic (matrix) diffusion controlled drug delivery device and biodegradable (bioerodible) drug delivery device (Sigma-aldrich) Hyperthermia - - efficient tool in cancer therapy based on the principle that under the influence of an alternating magnetic field, a magnetic particle can generate heat by hysteresis loss concept of intracellular hyperthermia (55-60°C) instead of heating entire tumor regions to approximately 42.5-44.0°C • - deactivation of normal cellular processes → thermal ablation (necrosis) superparamagnetic iron oxide (SPIO) particles have to be at least 10 nm in diameter for increasing the therapeutic effect: • • ability to encapsulate therapeutic drugs or genes surface can be chemically modified in order to enable targeting to a specific tissue (tumor specific antibodies) Magnetic resonance imaging (MRI) - primarily used in medical imaging to visualize the structure and function of the body superparamagnetic iron oxide particles (SPIO) as contrast agent • • • • increase the ability to distinguish between differences in soft tissues are composed of biodegradable iron, which is biocompatible and can be recycled by cells using biochemical pathways for iron metabolism surface coating allows chemical linkage of functional groups and ligands disadvantage: large size and fast clearance rate by phagocytic cells University of Missouri-Columbia Wang et al. What do all these applications have in common? Uptake of MNPs by the cells → Internalization Cellular uptake mechanism: Endocytosis pathways Huth et al. Endocytosis pathways macropinocytosis clathrin-dependent clathrin-independent (caveolae-mediated) non-specific uptake of extracellular molecules specific uptake of extracellular molecules specific uptake of extracellular molecules invagination of cell membrane to form first a pocket and second a vesicle clathrin initiates the formation of a vesicle by forming a crystalline coat on the inner surface of the cell‘s membrane flask-shape pits in the membrane that resemble the shape of a cave Mariana Ruiz Villarreal Clathrin-dependent endocytosis - major route for endocytosis in most mammalian cells occurs at specialized sites (coated pits), which cover 0.5-2% of the cell surface mediated by the molecule clathrin: assists in the formation of a coated pit on the inner surface of the plasma membrane of the cell Structure of a clathrin-coated vesicle. (a) A typical clathrin-coated vesicle comprises a membrane-bounded vesicle (tan) about 40 nm in diameter surrounded by a fibrous network of 12 pentagons and 8 hexagons. The fibrous coat is constructed of 36 clathrin triskelions, one of which is shown here in red. (b) Detail of a clathrin triskelion. Each of the three clathrin heavy chains has a specific bent structure. A clathrin light chain is attached to each heavy chain near the center. [Part (a) see B. M. F. Pearse, 1987, EMBO J. 6:2507; part (b) see B. Pishvaee and G. Payne, 1998, Cell 95:443.] Clathrin-dependent endocytosis - - formation of coated pits appears to start at specific assembly sites on the plasma membrane → coated pit zones it is unknown how clathrin assembles into a closed lattice Clathrin and cargo molecules are assembled into clathrin-coated pits on the plasma membrane together with an adaptor complex called AP-2 that links clathrin with transmembrane receptors, concluding in the formation of mature clathrin-coated SEM-image. Inside of the plasma membrane of a skin vesicles. (www.bookworm.org) cell. It shows many clathrin coated pits and vesicles forming on the inner surface of the plasma membrane (John Heuser, J. Cell Biol. 84:560-583, 1980) Clathrin-dependent endocytosis - AP-2 • large protein complex composed of four subunits (α, β2, μ2, δ2) - - Epsin • interacting protein - - α: involved in targeting AP-2 to the plasma membrane β2: interaction with clathrin binding to α-subunit of AP-2 interaction with clathrin → promotes assembly Dynamin • • GTPase activity (can bind and hydrolyze GTP) involved in the scission of newly formed vesicles from the membrane of one cellular compartment Mousavi et al. Clathrin-dependent endocytosis Macromolecules bind to specific receptors on the cell surface Induction of membrane curvature a) • • Epsin recruits clathrin and AP-2 complexes to the endocytic sites AP-2 mediates the assembly of a clathrin cage Coated pit formation b) • • Epsin can link membrane curvature with coated pit formation invagination of coated pits Invagination c) • deeply invaginated pits (~0,3 µm) pinch off from the membrane in a dynamin-dependent manner Construction and fission d) • fission to clathrin-coated vesicles (may also be facilitated by actin filaments) Uncoating e) • • uncoating ATPase disintegrates clathrin-shell into monomers transport of shell molecules to the cell membrane Mousavi et al. Clathrin-dependent endocytosis Electron micrographs showing the sequence of events in the formation of a clathrin coated vesicle at the surface of the plasma membrane. (M. M. Perry, A. B. Gilbert, J. Cell Sci. 39:257-272; 1979) Clathrin-dependent endocytosis The internalization of cholesterol from the extracellular fluid. The coated vesicle will lose its coat of clathrin proteins prior to fusion with an early endosome. In the endosome, the receptor-LDL complex will disassociate. The receptor will be recycled back to the plasma membrane in a recycling endosome; whereas, the LDL particle will be transported to the lysosme, and then, degraded by hydrolytic enzymes, releasing free cholesterol that can be used by the cell. Clathrin-dependent endocytosis 1 min (formation of clathrin-coated vesicle) 5-15 min (transport from cell surface to late endosome) The endosomal pathway. The early endosome is transported via microtubules from cell periphery towards nucleus. Macromolecules will be transported into the late endosome, which fuses with vesicles from the trans face of the Golgi complex that are filled with precursor lysosomal hydrolases. In the acidic pH of the endosome, the lysosomal hydrolases are activated and the late endosome matures into an active lysosome. Alternatively, the endosome can fuse with a preexisting mature lysosome. In the lysosome, the endocytosed material is degraded. Caveolae-mediated endocytosis - caveolae are small (50-100 nm) invaginations of the plasma membrane flask-shaped structure components, appearance and function are cell-type dependent directly involved in internalization of membrane components, extracellular ligands, bacterial toxins and non-enveloped viruses rich in proteins and lipids (cholesterol) • caveolin plays a role in caveolae formation and maintenance Caveolae-formation (Amsterdam University: www.science.uva.nl) Caveolae-mediated endocytosis - Caveolin • • • - integral membrane proteins (hairpin loop structure) 3 subtypes in mammalia (caveolin-1, caveolin-2, caveolin-3) forms oligomers and associates with cholesterol and sphingolipids Dynamin • similar to its role in coated pit fission Cholesterol Caveolae-mediated endocytosis Caveolae-mediated endocytosis is a triggered event that involves complex signaling. 1. 2. 3. 4. 5. 6. After binding to the membrane, virus particle are mobile until trapped in caveolae, which are linked to the actin skeleton SV40 particles trigger a signal transduction cascade that leads to local protein tyrosine phosphorylation and depolymerization of the actin skeleton Actin monomers are recruited to the virusloaded caveolae and an actin patch is formed Dynamin is recruited and a burst of actin polymerization occurs on the actin patch Virus-loaded vesicles are released from the membrane and can move into the cytosol After internalization, the cortical actin cytoskeleton returns to its normal pattern CaveolaeCaveolae-mediated endocytosis (Pelkmans (Pelkmans L. et al., al., Traffic 2003; 3; 311311-320) Caveolae-mediated endocytosis 0,25 µm Fibroblast membrane with caveolae (Rothberg et al. Cell, 1992 (68); 673-682) Caveolae-mediated endocytosis - - - - after internalization, caveolae-derived vesicles travel to caveosomes, which are distinct from endosomes in content and pH in caveosomes, ligands or membrane consistuents could reside, be sorted to the Golgi complex, or to the endoplasmic reticulum (ER) whether ligands or consistuents can cycle from caveosomes directly back to the plasma membrane has not yet been studied an exchange between caveosome and endosome is not possible 1-5 min (formation of caveolar vesicles) 10-15 min (transport from cell surface to caveosome) Medina-Kauwe, L.K., Adv Drug Deliv Rev. 2007; 59(8): 798-809 Clathrin- and caveolae-mediated endocytosis Endocytosis of MNPs - - - cellular uptake is energy-dependent and proceeds by endocytosis clathrin-dependent as well as caveolae-mediated endocytotic uptake is involved internalization is size-dependent • • - MNPs with a diameter <200 nm involved clathrin-coated pits larger MNPs enter cells via caveolae-mediated endocytosis the magnetic field itself does not alter the uptake mechanism (accelerated sedimentation on the cell surface) Experiments L929L929-fibroblasts with and without MNPs (SiMAG(SiMAG-Cyanuric 500nm; 24 h after MNPMNP-addition) Experiments - questions: • is the cellular uptake-mechanism (L929 fibroblasts and MG63 osteoblasts) energy-dependent? • internalization occurs via endocytosis? • which endocytosis pathway is involved? Experiments - methods: • examination of energy-dependency - • active transport is temperature-dependent (enzymes and ATP!) examination of endocytosis pathway - blocking of intracellular transport (microtubules) Examination of energy-dependency - Passive transport (diffusion, osmosis) means moving biochemicals and other atomic or molecular substances across membranes. Unlike active transport, this process does not involve chemical energy. In endocytosis many proteins and enzymes are involved which are temperature- and energy-dependent. energy-dependent processes require ATP - Incubation of cells with MNPs at different temperatures: - - • • • 37°C (normal culture conditions) 15°C (slowdown of cellular metabolism) 7°C (inhibition of cellular metabolism) Examination of energy-dependency Examination of energy-dependency L929 fibroblasts after 24 h incubation with SiMAG-Cyanuric 500 nm particles at 37°C, 15°C and 7°C MG63 osteoblasts after 24 h incubation with SiMAG-Cyanuric 500 nm particles at 37°C and 15°C Examination of energy-dependency fluidMAG ARA 250 nm 6h 24h 37°C 7°C 37°C 15°C 7°C MG63 - - - - - L929 - - - - - SiMAG Cyanuric 250 nm MG63 - - + - - L929 ++ - +++ - - SiMAG Cyanuric 500 nm MG63 + - ++ - - L929 +++ - ++++ - - MNP-uptake with different temperatures and incubation times. Examination of energy-dependency - good MNP-uptake and internalization at 37°C no MNP-uptake and internalization at 15°C and 7°C ¾ MNP-uptake and internalization is temperature-dependent!! ¾ passive transport mechanisms can be excluded ¾ uptake mechanism is energy-dependent (receptormediated) endocytosis!! - Examination of endocytotic pathway - blocking of intracellular microtubulemediated transport • • • - exclusively in clathrin-dependent endocytosis transport from early to late endosomes is pH-dependent (endosomes have acidic pH, whereas caveosomes are neutral) blocking of microtubules with colchicine • • highly poisonous mitosis inhibitor inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules Examination of endocytotic pathway Examination of endocytotic pathway Cell count of vital L929 fibroblasts after colchicine treatment 1000000 0 µg/ml 0,1 µg/ml 0,5 µg/ml 900000 1 µg/ml 2,5 µg/ml 800000 5 µg/ml cell count/ml 700000 600000 500000 400000 300000 200000 100000 0 0 10 20 30 40 time [h] 50 60 70 80 Examination of endocytotic pathway - colchicine: 0.5 µg/ml MNPs: SiMAG Cyanuric 500 nm (35 µg/ml) L929 L929: without MNPs and colchicine L929: without MNPs and with colchicine L929: with MNPs and without colchicine L929: with MNPs and colchicine Examination of endocytotic pathway - colchicine: 0.5 µg/ml MNPs: SiMAG Cyanuric 500 nm (35 µg/ml) MG63 MG63: without MNPs and colchicine MG63: without MNPs and with colchicine MG63: with MNPs and without colchicine MG63: with MNPs and colchicine Examination of endocytotic pathway - ¾ ¾ ¾ colchicine has as expected no effect on MNP uptake colchicine has little or no effect on intracellular particle transport microtubule transport seems not to be involved clathrin-dependent endocytosis is unlikely MNP-uptake and internalization is probably caveolae-mediated!! Literature Guptar, A.K. et al. Biomaterials. 2005, 26, No. 18; 3995-4021 Dobson, J. Gene Therapy. 2006, 13, No. 4; 283-287 Ito, A. et al. J. Biosci. Bioeng. 2005, Vol. 100, No. 1; 1-11 Mornet, S. et al. Prog. Solid State Chem. 2006, 34; 237-247 Osaka, T. et al. Anal. Bioanal. Chem. 2006, 384; 593-600 Lu, A.-H. et al. Angew. Chem. Int. Ed. 2007, 46; 1222-1244 Barbé, C. et al. Adv. Mater. 2004, 16, No. 21; 1959-1966 Bertorelle, F. et al. Langmuir. 2006, 22; 5385-5391 Kim, J.-S. et al. J. Vet. Sci. 2006, 7(4); 321-326 Lu, C.-W. et al. Nano Lett. 2007, Vol. 7, No. 1; 149-154 Won, J. et al. Science. 2005, 309; 121-125 Gupta, A.K. et al. J Mater. Sci. Mater. Med. 2004, 15(4); 493-496 Huth, S. et al. J Gene Med 2004, 6; 923-936 Rejman, J. et al. Biochem J. 2004, 377; 159-169 Pelkmans, L. et al. Traffic 2003, 3; 311-320 Bananis, E. et al. J Cell Biol. 2000, 151; 179-186 Medina-Kauwe, L.K. Adv Drug Deliv Rev. 2007, 59(8); 798-809 Rothberg et al. Cell, 1992 (68); 673-682 M. M. Perry, A. B. Gilbert, J. Cell Sci. 1979, 39; 257-272; Mousavi, S. A. et al. Biochem. J. 2004, 377; 1-16 B. M. F. Pearse, 1987, EMBO J. 6; 2507 B. Pishvaee and G. Payne, 1998, Cell 95; 443 Dissertation of Ulrich Stephan Huth, 2005, Heidelberg Wang, Y. X. J. et al. Eur. Radiol. 2001, 11; 2319-2331 Thank you for your attention!!