Magnetic nanoparticles: applications and cellular uptake

Transcription

1 Magnetic nanoparticles: applications and cellular uptake Susanne Kirsch

2 Overview superparamagnetic particles in biomedical and biotechnological applications drug/gene delivery hyperthermia magnetic resonance imaging cellular uptake mechanism: endocytosis pathways endocytosis of magnetic nanoparticles experiments with L929 fibroblasts and MG63 osteoblasts temperature colchicine literature

3 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.

4 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.

5 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 halflife life for redirecting to the desired target, the particle surface has to be labeled with ligands that specifically bind to receptors

6 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 (Sigmaaldrich)

7 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 ( C) instead of heating entire tumor regions to approximately 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)

8 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 c 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 MissouriColumbia Wang et al.

9 What do all these applications have in common? Uptake of MNPs by the cells Internalization

10 Cellular uptake mechanism: Endocytosis pathways Huth et al.

11 Endocytosis pathways macropinocytosis nonspecific uptake of extracellular molecules invagination of cell membrane to form first a pocket and second a vesicle clathrindependent specific uptake of extracellular molecules clathrin initiates the formation of a vesicle by forming a crystalline coat on the inner surface of the cell s membrane clathrinindependent (caveolaemediated) specific uptake of extracellular molecules flaskshape pits in the membrane that resemble the shape of a cave Mariana Ruiz Villarreal

12 Clathrindependent endocytosis major route for endocytosis in most mammalian cells occurs at specialized sites (coated pits), which cover 0.52% 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 clathrincoated vesicle. (a) A typical clathrincoated vesicle comprises a membranebounded 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.]

13 Clathrindependent 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 clathrincoated pits on the plasma membrane together with an adaptor complex called AP2 that links clathrin with transmembrane receptors, concluding in the formation of mature clathrincoated vesicles. ( SEMimage. Inside of the plasma membrane of a skin cell. It shows many clathrin coated pits and vesicles forming on the inner surface of the plasma membrane (John Heuser, J. Cell Biol. 84:560583, 1980)

14 Clathrindependent endocytosis AP2 large protein complex composed of four subunits (α,( β2, μ2, δ2) α: : involved in targeting AP2 2 to the plasma membrane β2: interaction with clathrin Epsin interacting protein binding to αsubunit of AP2 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.

15 Clathrindependent endocytosis Macromolecules bind to specific receptors on the cell surface a) Induction of membrane curvature Epsin recruits clathrin and AP2 complexes to the endocytic sites AP2 mediates the assembly of a clathrin cage b) Coated pit formation Epsin can link membrane curvature with coated pit formation invagination of coated pits c) Invagination deeply invaginated pits (~0,3 µm) pinch off from the membrane in a dynamindependent dependent manner d) Construction and fission fission to clathrincoated coated vesicles (may also be facilitated by actin filaments) e) Uncoating uncoating ATPase disintegrates clathrinshell into monomers transport of shell molecules to the cell membrane Mousavi et al.

16 Clathrindependent 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. 39:257272; 1979)

17 Clathrindependent 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 receptorldl complex will disassociate. The receptor will be recycled back to the plasma membrane m 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.

18 Clathrindependent endocytosis 1 min (formation of clathrincoated vesicle) 515 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.

19 Caveolaemediated endocytosis caveolae are small (50100 nm) invaginations of the plasma membrane flaskshaped shaped structure components, appearance and function are celltype dependent directly involved in internalization of membrane components, extracellular ligands,, bacterial toxins and nonenveloped viruses rich in proteins and lipids (cholesterol) caveolin plays a role in caveolae formation and maintenance Caveolaeformation (Amsterdam University:

20 Caveolaemediated endocytosis Caveolin integral membrane proteins (hairpin loop structure) 3 subtypes in mammalia (caveolin1, caveolin2, caveolin3) forms oligomers and associates with cholesterol and sphingolipids Dynamin similar to its role in coated pit fission Cholesterol

21 Caveolaemediated endocytosis Caveolaemediated endocytosis is a triggered event that involves complex signaling. 1. After binding to the membrane, virus particle are mobile until trapped in caveolae, which are linked to the actin skeleton 2. SV40 particles trigger a signal transduction cascade that leads to local protein tyrosine phosphorylation and depolymerization of the actin skeleton 3. Actin monomers are recruited to the virus loaded caveolae and an actin patch is formed 4. Dynamin is recruited and a burst of actin polymerization occurs on the actin patch 5. Virusloaded vesicles are released from the membrane and can move into the cytosol 6. After internalization, the cortical actin cytoskeleton returns to its normal pattern Caveolaemediated endocytosis (Pelkmans( L. et al., Traffic 2003; 3; ) 320)

22 Caveolaemediated endocytosis 0,25 µm Fibroblast membrane with caveolae (Rothberg et al. Cell, 1992 (68); )

23 Caveolaemediated endocytosis after internalization, caveolaederived 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 MedinaKauwe, L.K., Adv Drug Deliv Rev. 2007; 59(8): min (formation of caveolar vesicles) 1015 min (transport from cell surface to caveosome)

24 Clathrin and caveolaemediated endocytosis

25 Endocytosis of MNPs cellular uptake is energydependent and proceeds by endocytosis clathrindependent as well as caveolaemediated endocytotic uptake is involved internalization is sizedependent MNPs with a diameter <200 nm involved clathrincoated coated pits larger MNPs enter cells via caveolaemediated endocytosis the magnetic field itself does not alter the uptake mechanism (accelerated sedimentation on the cell surface)

26 Experiments L929fibroblasts with and without MNPs (SiMAGCyanuric 500nm; 24 h after MNPaddition)

27 Experiments questions: is the cellular uptakemechanism (L929 fibroblasts and MG63 osteoblasts) energydependent? internalization occurs via endocytosis? which endocytosis pathway is involved?

28 Experiments methods: examination of energydependency active transport is temperaturedependent (enzymes and ATP!) examination of endocytosis pathway blocking of intracellular transport (microtubules)

29 Examination of energydependency 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 energydependent. energydependent processes require ATP Incubation of cells with MNPs at different temperatures: 37 C C (normal culture conditions) 15 C C (slowdown of cellular metabolism) 7 C C (inhibition of cellular metabolism)

30 Examination of energydependency

31 Examination of energydependency L929 fibroblasts after 24 h incubation with SiMAGCyanuric 500 nm particles at 37 C, 15 C and 7 C MG63 osteoblasts after 24 h incubation with SiMAGCyanuric 500 nm particles at 37 C and 15 C

32 Examination of energydependency MG63 L929 MG63 L929 MG63 L C h fluidmag ARA 250 nm 7 C 37 C 24h 15 C SiMAG Cyanuric 250 nm SiMAG Cyanuric 500 nm C MNPuptake with different temperatures and incubation times.

33 Examination of energydependency good MNPuptake and internalization at 37 C no MNPuptake and internalization at 15 C C and 7 C 7 MNPuptake and internalization is temperaturedependent!! passive transport mechanisms can be excluded uptake mechanism is energydependent (receptor mediated) endocytosis!!

34 Examination of endocytotic pathway blocking of intracellular microtubule mediated transport exclusively in clathrindependent endocytosis transport from early to late endosomes is phdependent (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

35 Examination of endocytotic pathway

36 Examination of endocytotic pathway Cell count of vital L929 fibroblasts after colchicine treatment µg/ml 0,1 µg/ml 0,5 µg/ml 1 µg/ml 2,5 µg/ml 5 µg/ml cell count/ml time [h]

37 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

38 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

39 Examination of endocytotic pathway 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 clathrindependent endocytosis is unlikely MNPuptake and internalization is probably caveolaemediated!!

40 Literature Guptar, A.K. et al. Biomaterials. 2005, 26, No. 18; Dobson, J. Gene Therapy. 2006, 13, No. 4; Ito, A. et al. J. Biosci. Bioeng. 2005, Vol. 100, No. 1; Mornet,, S. et al. Prog. Solid State Chem. 2006, 34; Osaka, T. et al. Anal. Bioanal.. Chem. 2006, 384; Lu, A.H. et al. Angew. Chem. Int. Ed. 2007, 46; Barbé,, C. et al. Adv. Mater. 2004, 16, No. 21; Bertorelle, F. et al. Langmuir. 2006, 22; Kim, J.S. et al. J. Vet. Sci. 2006, 7(4); Lu, C.W. et al. Nano Lett , Vol. 7, No. 1; Won, J. et al. Science. 2005, 309; Gupta, A.K. et al. J Mater. Sci. Mater. Med. 2004, 15(4); Huth, S. et al. J Gene Med 2004, 6; Rejman, J. et al. Biochem J. 2004, 377; Pelkmans,, L. et al. Traffic 2003, 3; Bananis,, E. et al. J Cell Biol. 2000, 151; MedinaKauwe Kauwe,, L.K. Adv Drug Deliv Rev. 2007, 59(8); Rothberg et al. Cell, 1992 (68); M. M. Perry, A. B. Gilbert, J. Cell Sci. 1979, 39; ; 272; Mousavi,, S. A. et al. Biochem. J. 2004, 377; 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 , 11;

41 Thank you for your attention!!