Download Magnetic nanoparticles: applications and cellular uptake

Magnetic nanoparticles:
applications and cellular uptake
Susanne Kirsch
20.02.2008
Overview
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superparamagnetic particles in biomedical and
biotechnological applications
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cellular uptake mechanism: endocytosis
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pathways
endocytosis of magnetic nanoparticles
experiments with L929 fibroblasts and MG63 osteoblasts
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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
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small enough for administration (intravenous, oral,
inhalation, etc.) → method to reach any target organ or
tissue
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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
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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
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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
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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
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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:
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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)
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primarily used in medical imaging to visualize the structure and function
of the body
superparamagnetic iron oxide particles (SPIO) as contrast agent
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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
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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
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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
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AP-2
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large protein complex composed of four subunits (α, β2, μ2, δ2)
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Epsin
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interacting protein
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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
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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)
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Epsin recruits clathrin and AP-2
complexes to the endocytic sites
AP-2 mediates the assembly of a
clathrin cage
Coated pit formation
b)
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Epsin can link membrane curvature
with coated pit formation
invagination of coated pits
Invagination
c)
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deeply invaginated pits (~0,3 µm)
pinch off from the membrane in a
dynamin-dependent manner
Construction and fission
d)
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fission to clathrin-coated vesicles
(may also be facilitated by actin
filaments)
Uncoating
e)
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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
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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)
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caveolin plays a role in caveolae
formation and maintenance
Caveolae-formation (Amsterdam University: www.science.uva.nl)
Caveolae-mediated endocytosis
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Caveolin
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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
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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
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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
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cellular uptake is energy-dependent and proceeds by
endocytosis
clathrin-dependent as well as caveolae-mediated
endocytotic uptake is involved
internalization is size-dependent
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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
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questions:
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is the cellular uptake-mechanism (L929 fibroblasts and
MG63 osteoblasts) energy-dependent?
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internalization occurs via endocytosis?
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which endocytosis pathway is involved?
Experiments
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methods:
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examination of energy-dependency
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active transport is temperature-dependent (enzymes and ATP!)
examination of endocytosis pathway
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blocking of intracellular transport (microtubules)
Examination of energy-dependency
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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
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Incubation of cells with MNPs at different temperatures:
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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
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L929
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SiMAG Cyanuric 250 nm
MG63
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+
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L929
++
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+++
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SiMAG Cyanuric 500 nm
MG63
+
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++
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L929
+++
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++++
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MNP-uptake with different temperatures and incubation times.
Examination of energy-dependency
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good MNP-uptake and internalization at 37°C
no MNP-uptake and internalization at 15°C and 7°C
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MNP-uptake and internalization is temperature-dependent!!
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passive transport mechanisms can be excluded
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uptake mechanism is energy-dependent (receptormediated) endocytosis!!
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Examination of endocytotic pathway
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blocking of intracellular microtubulemediated transport
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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
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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
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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
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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
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¾
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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
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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
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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!!