U.S. patent application number 14/563268 was filed with the patent office on 2015-07-16 for plasma membrane isolation.
This patent application is currently assigned to KATHOLIEKE UNIVERSITEIT LEUVEN, K.U. LEUVEN R&D. The applicant listed for this patent is Wim Annaert, Gustaaf Borghs, Liesbet Lagae, Deepak Balaji Thimiri Govinda Raj. Invention is credited to Wim Annaert, Gustaaf Borghs, Liesbet Lagae, Deepak Balaji Thimiri Govinda Raj.
Application Number | 20150197742 14/563268 |
Document ID | / |
Family ID | 43248879 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150197742 |
Kind Code |
A1 |
Thimiri Govinda Raj; Deepak Balaji
; et al. |
July 16, 2015 |
Plasma Membrane Isolation
Abstract
The present invention relates to a population of monodisperse
magnetic nanoparticles with a diameter between 1 and 100 nm which
are coated with a layer with hydrophilic end groups. Herein the
layer with hydrophilic end groups comprises an inner layer of
monosaturated and/or monounsaturated fatty acids bound to said
nanoparticles and bound to said fatty acids, an outer layer of a
phospholipid conjugated to a monomethoxy polyethyleneglycol (PEG)
comprising a hydrophilic end group, or comprises a covalently bound
hydrophilic layer bound to said nanoparticles.
Inventors: |
Thimiri Govinda Raj; Deepak
Balaji; (Leuven, BE) ; Lagae; Liesbet;
(Leuven, BE) ; Annaert; Wim; (Kontich, BE)
; Borghs; Gustaaf; (Leuven, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Thimiri Govinda Raj; Deepak Balaji
Lagae; Liesbet
Annaert; Wim
Borghs; Gustaaf |
Leuven
Leuven
Kontich
Leuven |
|
BE
BE
BE
BE |
|
|
Assignee: |
KATHOLIEKE UNIVERSITEIT LEUVEN,
K.U. LEUVEN R&D
Leuven
BE
IMEC
Leuven
BE
|
Family ID: |
43248879 |
Appl. No.: |
14/563268 |
Filed: |
December 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13111428 |
May 19, 2011 |
8936935 |
|
|
14563268 |
|
|
|
|
Current U.S.
Class: |
435/173.7 ;
252/62.54; 427/127; 435/180 |
Current CPC
Class: |
G01N 33/5076 20130101;
H01F 1/01 20130101; C12N 11/08 20130101; B82Y 5/00 20130101; Y10S
977/962 20130101; Y10S 977/703 20130101; Y10S 977/702 20130101;
Y10S 977/713 20130101; Y10S 977/779 20130101; Y10S 977/773
20130101; Y10S 977/783 20130101; C12N 13/00 20130101; G01N 33/6842
20130101 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12N 11/08 20060101 C12N011/08; H01F 1/01 20060101
H01F001/01 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2010 |
EP |
10163644 |
Claims
1. A population of monodisperse positively charged magnetic
nanoparticles which have a zeta potential in the range of 10-30 mV
at pH 7 and a diameter between 1 and 100 nm which are coated with a
layer with hydrophilic end groups, wherein said coating has an
inner layer of monosaturated and/or monounsaturated fatty acids
bound to said nanoparticles and bound to said fatty acids and an
outer layer of a phospholipid conjugated to a monomethoxy
polyethyleneglycol (PEG).
2. The population according to claim 1, wherein said nanoparticles
with an inner layer of fatty acids bound and an outer layer of a
phospholipid do not comprise a peptide moiety.
3. The population according to claim 1, wherein said hydrophilic
end group is a phosphonate, an amine, a C.sub.1-C.sub.20 alkane, a
C.sub.1-C.sub.20 alkene, a C.sub.1-C.sub.20 alkyene, an azido, an
epoxy, an NH.sub.2, a COOH, unsubstituted or substituted PEG, PDP,
CHO or SH.
4. The population according to claim 1, wherein the phospholipids
in the outer layer are
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol-
)-2000](DSPE-PEG-COOH),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Amine (polyethylene
glycol)-2000] (DSPE-PEG-Amine),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000](DSPE-PEG), DSPE-Folate, DSPE-PEG(2000) Maleimide or
DSPE-PEG(2000) Carboxyfluroscein.
5. The population according to claim 1, wherein the covalently
bound hydrophilic layer is silane, dimercaptosuccinic acid (DMSA)
or ammonium chloride.
6. A method of preparing a monodisperse population of magnetic
nanoparticles with a diameter between 1 and 100 nm which are coated
with a layer with hydrophilic end groups, comprising a) providing a
magnetic material, b) applying a layer of monounsaturated and/or
monosaturated fatty acid to said material by thermal decomposition
under conditions to provide magnetic nanoparticles with a diameter
between 1 and 100 nm, c) precipitating said fatty acid coated
magnetic nanoparticles with an alcohol, d) discarding from the
nanoparticles of step c) the population of aggregated nanoparticles
and collecting the population of monodisperse nanoparticles, e)
applying a layer of lipids comprising a hydrophilic end group to
the monodisperse nanoparticles of step d). f) selecting from the
nanoparticles obtained in step e) the population of monodisperse
nanoparticles in the presence of solvent, or instead of e) and f),
g) replacing the fatty acid coating with a hydrophilic layer in the
presence of a nonaqueous solvent (chloroform), and h) selecting
from the nanoparticles obtained in step g) the population of
monodisperse nanoparticles in the presence of said non aqueous
solvent.
7. The method of claim 6, wherein in g), said layer with
hydrophilic end group is DMSA, Silane, Tetramethylammonium
hydroxide (TMAOH) or ammonium chloride.
8. A monodisperse population of magnetic nanoparticles obtained by
the method according to claim 6.
9. An isolated complex of a nanoparticle according to claim 1 with
the plasma membrane or with a plasma membrane derived
organelle.
10. A method for isolating a plasma membrane of a cell, a fraction
thereof, or a plasma membrane derived organelle, comprising 1.
providing a population of intact and suspended cells at a
temperature where endocytic uptake by a cell is inhibited, 2.
contacting said intact cells with magnetic nanoparticles of claim
1, thereby allowing the binding of magnetic nanoparticles to and
into the cell plasma membrane, 3. removing unbound magnetic
nanoparticles, 4. disrupting the cells, 5. removing cellular
organelles, 6. isolating from the disrupted cells by magnetic
attraction the plasma membranes with magnetic nanoparticles.
11. A preparation of a plasma membrane, wherein at least 60% of the
proteins in said preparation are integral membrane proteins or
proteins associated therewith.
12. The population according to claim or 2, wherein said
hydrophilic end group is a phosphonate, an amine, a
C.sub.1-C.sub.20 alkane, a C.sub.1-C.sub.20 alkene, a
C.sub.1-C.sub.20 alkyene, azido, epoxy, NH.sub.2, COOH,
unsubstituted or substituted PEG, PDP, CHO or SH.
13. The population according to claim 2, wherein the phospholipids
in the outer layer is
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene
glycol)-2000](DSPE-PEG-COOH),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Amine (polyethylene
glycol)-2000] (DSPE-PEG-Amine),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000](DSPE-PEG), DSPE-Folate, DSPE-PEG(2000) Maleimide or
DSPE-PEG(2000) Carboxyfluroscein.
14. An isolated complex of a nanoparticle according to claim 8 with
a plasma membrane or with a plasma membrane derived organelle.
15. A method for isolating a plasma membrane of a cell, a fraction
thereof, or a plasma membrane derived organelle, comprising: a)
providing a population of intact and suspended cells at a
temperature where endocytic uptake by a cell is inhibited, b)
contacting said intact cells with magnetic nanoparticles of claim
8, thereby allowing the binding of magnetic nanoparticles to and
into the cell plasma membrane, c) removing unbound magnetic
nanoparticles, d) disrupting the cells, e) removing cellular
organelles, f) isolating from the disrupted cells by magnetic
attraction the plasma membranes with magnetic nanoparticles.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 13/111,428 filed on May 19, 2011, which claims
priority to European Patent Application No. 10163644 filed on May
21, 2010, the disclosures of which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the manufacture and use of
coated magnetic nanoparticles. More particularly the present
invention relates to the use of these nanoparticles for the
purification of plasma membranes and endosomes from cells. The
present invention further relates to the analysis of the repertoire
of proteins, lipids and carbohydrates which are present in the
isolated plasma membranes and endosomes.
BACKGROUND OF THE INVENTION
[0003] Advances in protein separation technologies and innovations
in MS (mass spectrometry) have greatly increased whole genome
approaches in biology. However the explosion of information in the
fields of genomics and proteomics has not been matched by a
corresponding advancement of knowledge in the field of organellar
proteomics, lipids and glycans, which is largely due to the
structural complexity and the lack of powerful tools for their
analysis. Hence, it has become more apparent that whole cells and
tissues are not currently amenable to satisfactory whole "Omics"
analysis. This is due to complexity and extreme dynamic range of
protein expression in a whole cell (for example less abundant
proteins are masked by those expressed at higher levels).
[0004] However, whole "Omics" analysis of subcellular compartments
is hampered by difficulties inherent in purifying organelles as
disclosed in Dreger (2003) Mass Spectrom. Rev. 22(1), 27-56.
Particularly the analysis or proteins, lipids and carbohydrates in
the plasma membrane and endosomal/lysosomal compartment system
(EE/Lys) poses major hurdles as it is most dynamic in nature with
strongly overlapping buoyant densities making it impossible to
physically separate closely related populations. In addition,
effective isolation and protein purification from subcellular
compartments is the most crucial step for a whole genome analysis
where only minute quantities are available. However, even the best
optimized conventional purification methods such as density
gradient centrifugation and colloidal silica based plasma membrane
fractionation often lead to only partially purified compartments
[Arjunan (2009) Cell Biochem. Biophys. 53(3), 135-143.].
[0005] Magnetic particles with targeting groups, such as antibodies
have been used to isolate particular proteins or cells. Lipid
coated magnetic particles have been used to deliver substances
intracellularly. The use of such magnetic particles for the
isolation of distinct cell membranes, such as e.g. plasma membrane
and endosomes/lysosomes is unexplored. The present inventors
disclosed the advantageous properties of a magnetic particle that
would remain associated with a plasma membrane but are silent on
the composition of beads that would have such properties [Nanotech
Montreux meeting 17-19 Oct. 2008].
[0006] Other attempts to isolate plasma membrane derived endosomes
are described in e.g. Riviere et al. (2007) Eu. Phys J. E. soft
matter 22, 1-10 wherein magnetic particles are used which are not
homogenous in size, resulting in an inefficient isolation process
with low yield and purity and contamination with plasma
membranes.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention relates to a population
of monodisperse magnetic nanoparticles with a diameter between 1
and 100 nm which are coated with a layer with hydrophilic end
groups. This layer with hydrophilic end groups comprises
a) an inner layer of monosaturated and/or monounsaturated fatty
acids bound to the nanoparticles and bound to the fatty acids, an
outer layer of a phospholipid conjugated to a monomethoxy
polyethyleneglycol (PEG) comprising a hydrophilic end group, or
comprises b) a covalently bound hydrophilic layer bound to the
nanoparticles.
[0008] In particular embodiments the population according to claim
1 a), does not carry a peptide moiety such as membrane targeting
peptides.
[0009] The nanoparticles can partially labelled with a detectable
marker on the coating.
[0010] In other particular embodiment the hydrophilic end group is
selected from the group consisting of phosphonate, amine,
C.sub.1-C.sub.20 alkane, C.sub.1-C.sub.20 alkene, C.sub.1-C.sub.20
alkyene, azido, epoxy, NH.sub.2, COOH, unsubstituted or substituted
PEG, PDP, CHO and SH.
[0011] In other particular embodiments, the phospholipids in the
outer layer are selected from the group consisting of
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene
glycol)-2000](DSPE-PEG-COOH),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Amine (polyethylene
glycol)-2000] (DSPE-PEG-Amine),
[0012]
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000](DSPE-PEG), DSPE-Folate, DSPE-PEG(2000) Maleimide and
DSPE-PEG(2000) Carboxyfluroscein.
[0013] In particular embodiments, the covalently bound hydrophilic
layer is selected from the group of consisting of silane,
dimercaptosuccinic acid (DMSA) and ammonium chloride. Optionally
the silane is substituted with trimethoxy silyl, methoxyl silyl,
ethoxy siliyl or silanol or the end group of the silane is
substituted with a group selected from phosphonate, amine, thiol,
C.sub.1-C.sub.20 alkyl, C.sub.1-C.sub.20 alkene, C.sub.1-C.sub.20
alkyne, azido and epoxy.
[0014] In other particular embodiments, the covalently bound
hydrophilic layer which is bound to the nanoparticles is further
modified with an endocytic pathway specific molecule wherein the
pathways is selected from the group consisting of the
clathrin-dependent, caveolae-dependent, ARF6-specific,
clathrin-independent and caveolae-independent pathway.
[0015] The endocytic pathway specific molecule is selected from a
peptide or protein (e.g. a receptor or receptor ligand, an
antibody), a carbohydrate, a biotin, a virus and optionally further
comprising a chromophoric molecule, for example a fluorescent
label.
[0016] These endocytic pathway specific molecules can be conjugated
to the nanoparticles via e.g. an amide, a disulfide or an ester
bond.
[0017] Another aspect of the present of the invention is a method
of preparing a monodisperse population of magnetic nanoparticles
with a diameter between 1 and 100 nm which are coated with a layer
with hydrophilic end groups, comprising the steps of:
a) providing a magnetic material, b) applying a layer of
monounsaturated and/or monosaturated fatty acid to the material by
thermal decomposition under conditions to provide magnetic
nanoparticles with a diameter between 1 and 100 nm, c)
precipitating the fatty acid coated magnetic nanoparticles with an
alcohol, d) discarding from the nanoparticles of step c) the
population of aggregated nanoparticles and collecting the
population of monodisperse nanoparticles, e) applying a layer of
lipids comprising a hydrophilic end group to the monodisperse
nanoparticles of step d). f) selecting from the nanoparticles
obtained in step e) the population of monodisperse nanoparticles in
the presence of solvent, or instead of step e) and f), performing
the step of g) replacing the fatty acid coating with a hydrophilic
layer in the presence of a nonaqueous solvent (chloroform), h)
selecting from the nanoparticles obtained in step g) the population
of monodisperse nanoparticles in the presence of the non aqueous
solvent.
[0018] Herein in step g, the layer with hydrophilic end group can
be selected from the group consisting of DMSA, Silane,
Tetramethylammonium hydroxide (TMAOH) and ammonium chloride.
[0019] In step g) the non aqueous solvent can be selected from the
group consisting of organic-like alcohols, hydrocarbons and benzene
derivatives. in step g) the non aqueous solvent can be selected
from the group consisting of toluene, cyclohexane, methanol,
ethanol, mixtures of ethanol and toluene, chloroform,
dimethylsulfoxide (DMSO) and dimethylformamide (DMF).
[0020] A further aspect of the invention relates to a monodisperse
population of magnetic nanoparticles obtainable by the method
described above.
[0021] A further aspect of the invention relates to an isolated
complex of a nanoparticle with the plasma membrane or with a plasma
membrane derived organelle such as an endosome.
[0022] A further aspect of the invention relates to the use of a
population of nanoparticles as described or prepared above for the
isolation of cell plasma membranes or for the isolation of
endosomes.
[0023] Yet a further aspect of the invention relates to a method
for isolating a plasma membrane of a cell, a fraction thereof, or a
plasma membrane derived organelle, comprising the step of: [0024]
a) providing a population of intact and suspended cells at a
temperature where endocytic uptake by a cell is inhibited, [0025]
b) contacting the intact cells with magnetic nanoparticles as
described or prepared above, thereby allowing the binding of
magnetic nanoparticles to and into the cell plasma membrane, [0026]
c) removing unbound magnetic nanoparticles, [0027] d) disrupting
the cells, [0028] e) removing cellular organelles, [0029] f)
isolating from the disrupted cells by magnetic attraction the
plasma membranes with magnetic nanoparticles.
[0030] An optional further step is the isolation of caveolae by
treating the plasma membrane with mechanic shearing and detergents
followed by a gradient separation.
[0031] A further optional step is the of isolating GPI anchored
protein domains wherein plasma membranes or caveolae are treated
with detergents and subjected to gradient separation.
[0032] In particular embodiments, in step e) the cell organelles
are removed under conditions of 0.1 to 2 M of salt concentrations
and/or a pH between 10 and 12.
[0033] A further aspect of the invention to plasma membranes
obtained by the method described above, for the analysis of
biological molecules comprised in the cell membranes. Typically
these biological molecules are selected from proteins,
carbohydrates and lipids. For example these proteins are an
enzymatically active complex of gamma secretase.
[0034] A further aspect of the invention relates to a preparation
of a plasma membrane, characterised in that at least 60% of the
proteins in the preparation are integral membrane proteins or
proteins associated therewith.
[0035] A further aspect of the invention relates to a method for
isolating endosomes of a cell, comprising the step of: [0036] a)
providing a population of intact cells at a temperature whereby
endocytic mechanisms take place, [0037] b) contacting the intact
cells for a period between 1 and 30 minutes at 37.degree. C. with
the nanoparticles described above, thereby allowing the uptake of
nanoparticles into the endosomes.
[0038] This method further comprises the step of [0039] c) removing
the unbound magnetic nanoparticles which have not been taken up by
the cells, [0040] d) disrupting the cells and [0041] e) isolating
by magnetic attraction endosomes with magnetic nanoparticles bound
thereto from the disrupted cells.
[0042] Optionally the method further comprises after step b) the
step of: [0043] maintaining the population of cells for a further
period of between 1 and 180 minutes at a temperature whereby
endocytic uptake by a cell takes place.
[0044] A further aspect of the invention relates to the use of
endosomes obtained by the method described above, for the analysis
of a population of biological molecules comprised in the endosomes,
such as proteins, carbohydrates or lipids. A particular example
hereof is an enzymatically active complex of gamma secretase.
[0045] Isolation of subcellular organelles present an attractive
target for whole proteomics, lipidomics and glycomics, as their
proteins, lipids and glycans complexity is reduced and lower
abundant ones that are specifically enriched on subcellular
organelles compared to whole cell lysates could be identified. In
addition subcellular approach is also advantageous in that
identified proteins are linked to functional units. For novel
proteins, the connection to an organelle can provide the first
clues as to the protein functionality. Moreover, a global analysis
on the organelle provides insights and understanding in the
functional roles of the organelles.
[0046] The present invention discloses Magnetic NanoParticle
(MNP)s-based methodologies for the isolation of plasma membrane and
endosomes from control and disease-related cell lines and allows to
analyze the proteome, glycome and lipidome content of such plasma
membrane and endosomes. The invention allows to perform a
comparative analysis supported by bioinformatics, and allows the
identification of aberrant protein expression patterns from which
potentially causal gene products and/or novel biomarkers may be
identified.
[0047] The present invention relates to the development and
characterization of biocompatible MNPs.
[0048] The present invention further allows to define parameters
for cellular uptake and isolation of plasma membrane and endosomal
compartments.
[0049] The present invention further provides an optimization of
MNPs based plasma membrane isolation method for proteomics,
glycomics and lipidomics.
[0050] Particular embodiments relate to the analysis of proteome,
glycome and lipidome content of diseased cell lines or models of
diseased cell lines, such as a Presenilins (PSEN)-deficient mouse
embryonic fibroblast cell lines.
[0051] The invention relates to a method for synthesis of
monodisperse and surface functionalized lipid coated MNPs for
plasma membrane isolation. Herein an alcohol etching is performed
and the lipid composition is adjusted for specific cell type plasma
membrane isolation.
[0052] The invention relates to a method for synthesis of
monodisperse and surface functionalized DMSA/Silane/TMAOH coated
MNPs for endosomal isolation. Herein the surface composition is
adjusted for specific endosomal compartmental isolation. Optionally
an appropriate pathway-specific and application-specific
bioconjugate is selected for endosomal specific targeting and
isolation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1: DLS Size measurement in Relative number (%) [0054]
a) SPMNPs in Hexane, b) DMSA-SPMNPs in H.sub.2O, c)
Amino-Lipid-SPMNPs in H.sub.2O, 1d) COOH-Lipid-SPMNPs in H.sub.2O,
1e) PEG-Lipid-SPMNPs in H.sub.2O, 1f) TMAOH-SPMNPs in H.sub.2O,
1(g) COO-TMACl-SPMNPs in H.sub.2O, 1(h) SPMNPs in Medium DMEM/F12.
These measurements illustrate the monodisperse character of the
nanoparticles. The most abundant fraction is presented as 100%.
1(i) UV-Vis measurement of SPMNPs in medium at 1000 nm for 4 hours
indicates the stability of the nanoparticles in medium, 1(j) Trypan
blue based cell viability test in HeLa Cells for 2 hours SPMNPs
incubation, indicates that there is no toxic effect of any of the
surface coatings. 1(k) shows exemplary illustrations of the various
coated particles referenced in FIGS. 1(a)-1(j).
[0055] FIG. 2(A) Magnetically Tagged HeLa Cells (% labelled cells
of total cell amount). After 2 hours of SPMNPs incubation in medium
at 37.degree. C. These data show that the hydrophobic nature of the
nanoparticle coating has an effect on the labelling of specific
cell types.
[0056] FIG. 2B Sequential incubation of SPMNPs (100 .mu.g/ml) in
HeLa Cells for increasing time period 15, 30, and 60 minutes.
[0057] FIG. 3: Nanoparticle Synthesis and Characterization.
Transmission Electron Microscope images of Fe.sub.3O.sub.4
nanoparticles coated with oleic acid (3A) and coated with lipids
(3B). Dynamic Light Scattering (DLS) graphs of Fe.sub.3O.sub.4
nanoparticles coated with oleic acid (3C) and coated with amino
end-group lipids (3D), illustrating the monodisperse nature of the
nanoparticle population. The most abundant fraction is presented as
100%. FIG. 3E: Magnetic properties measurement by alternating
gradient field magnetometer (AGFM)--Oleic Acid coated (Dark line)
and Lipid coated Fe.sub.3O.sub.4 nanoparticles (dotted line) where
X-axis is magnetic field (KOe) and Y-axis is magnetization (emu/g
nanoparticles). The data illustrate the superparamagnetic
properties of the nanoparticles, FIG. 3F: zeta potential
measurement on amino end-group lipid coated Fe.sub.3O.sub.4
nanoparticles for different pH range (2-11) shows that the
nanoparticles are nanoparticle are positively charged and suitable
for the plasma membrane isolation.
[0058] FIG. 4: Plasma membrane Omics Analysis. A) Step flow diagram
for SPMNPs based plasma membrane isolation and different omics
analysis (left side). B) Schematic representation of SPMNPs-Cell
interaction and magnetic plasma membrane isolation (right
side).
[0059] FIG. 5: SPMNPs--localize to the cell surface of MEFs cells.
FIGS. 5A-C: confocal laser scanning microscopy images of MEFs Wt
incubated with fluorescent modified SPMNPs for Pulse period of 15
minutes at 37.degree. C. The data show that the SPMNPs co-localize
with a validated membrane marker.
[0060] FIGS. 5D-F: Transmission electron microscopy images at
different resolution of MEFs wt cell membranes incubated SPMNPs for
a pulse period of 15 minutes at 4.degree. C.
[0061] FIG. 5G: Western blot analysis of various proteins with
appropriate primary and secondary antibodies. Protein sample was
resolved in a 4-12% SDS-Page gradient gel in each lane. Lane 1-3
represents Post Nuclear Supernatant (PNS), fraction which is
unbound to the membrane (UB) and fraction which is bound to the
membrane (B).
[0062] List of marker proteins and corresponding organelle marker
used: Na.sup.+K.sup.+ATPase--Plasma Membrane, Lamin A--Nucleus,
GM130--Golgi Apparatus, GADPH-Cytosol, RER1p, BIP &
RBI--Endoplasmic Reticulum, Actin--Cytoskeleton, GM130 for Golgi
compartments, Rab 7--Late Endosomes, HSP60--Mitochondria,
P58--Intermediate Compartments. FIG. 5H: Western blot protein
signal intensity quantification--Y-axis represent--total percentage
retainment of protein in Bound fraction with respect to PNS and
X-axis represents organelle marker proteins.
[0063] FIGS. 5I and J: Gamma-Secretase Cell free activity Assay on
PNS and Plasma Membrane fraction and Intensity quantification of
total AICD level and enrichment with respect to PNS.
[0064] FIG. 5K: glycoprotein specific Ponceau staining of PNS, UB
and B fraction of MEFs Wildtype and MEFs PSDKO.
[0065] FIG. 6: N-Glycoproteomics analysis on MEFs Wt plasma
membrane fraction. Identification of the glycoproteins in the PM
shows that about 75% of the identified proteins are plasma membrane
derived.
[0066] FIG. 7A: Blue-Native Gel based gamma-secretase complex
isolation: FIG. 7B: Levels of gamma-secretase components (NCT,
APh-1a, PEN-2, PSEN1-NTF, PSEN1-CTF) at the plasma membrane
relative to PNS fractions, showing a significant enrichment of the
gamma secretase components in the PM; FIG. 7C: percentage of full
gamma-secretase complexes at the plasma membrane relative to PNS
and % enrichment at the plasma membrane versus PNS.
[0067] FIGS. 8A and 8B: DMSA-SPMNPs based Endosomal Compartmental
isolation: a) Confocal laser scanning microscopy images of HeLa
incubated with fluorescent modified DMSA-SPMNPs for Pulse period of
15 minutes (A) & Chase 30 minutes (B) at 37.degree. C. and
Pulse period of 30 minutes at 37.degree. C.
[0068] FIG. 8C: Western blot protein signal intensity
quantification--Y-axis represent--Relative enrichment versus PNS
and X-axis represent Chase time incubation for 30, 60 and 120
minutes. List of marker proteins and corresponding organelle marker
used: Na.sup.+K.sup.+ATPase--Plasma Membrane, EEA1--Early Endosomes
and RAB7--Late Endosomes.
[0069] FIG. 9 shows a schematic overview of the manufacture of
magnetic nanoparticles in accordance with certain embodiments of
the invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0070] "Nanopartide" (abbreviated as NP) in the context of the
present invention refers to spherical particles with a diameter
between 1 to 100 nm.
[0071] "Magnetic" material in the context of the present invention
refers to magnetic (M), paramagnetic (PM) and superparamagnetic
material (SPM).
[0072] "Monodisperse" in the context of the present invention
related to nanoparticles which are homogeneous in nanoparticle
shape and size. In the context of the present invention this refers
to a population of nanoparticles wherein 90-95% of the
nanoparticles fall within the defined size range.
[0073] "Plasma membrane" or "cell membrane" or "plasmalemma" refers
to the lipid bilayer which surrounds the protoplasm of a cell. This
membrane is not to be confused with the cell wall which covers
plant and yeast cells.
[0074] "gamma secretase" refers to a protein complex comprising at
least the proteins presenilin (PSEN1-NTF and PSEN1-CTF or PSEN2-NTF
and PSEN2-CTF), nicastrin (NCT), APH-1 (anterior pharynx-defective
1) 1a (or APH-1 b or APH-1c), and PEN-2 (presenilin enhancer
2).
[0075] A first aspect of the invention relates the modification of
magnetic material. The magnetic material which is as such
hydrophilic, is modified in order to obtain a hydrophilic outer
layer which allows the nanoparticles to interact with and bind to
the plasma membrane of a cell.
[0076] The present invention discloses two types of nanoparticles
which have these advantageous properties.
[0077] The nanoparticles can be either magnetic paramagnetic or
superparamagnetic. In a particular embodiment the nanoparticles are
superparamagnetic.
[0078] The magnetic material can be any magnetic material known to
the skilled person such as iron oxide, cobalt oxide, manganese
oxide, nickel oxide zinc oxide or a combination of any of these
materials.
[0079] A first type of nanoparticles relates to a population of
monodisperse magnetic nanoparticles with a diameter between 1 and
100 nm which are coated with a hydrophobic layer with hydrophilic
end groups. These nanoparticles contain an inner layer of
monosaturated/mono-unsaturated/a combination of fatty acids bound
to the nanoparticles. These mono-unsaturated fatty acids are
selected from the group of myristoleic acid, palmitoleic acid,
sapienic acid, oleic acid and erucic acid. The saturated fatty
acids are selected from the group of lauric acid, myristic acid,
palmitic acid, stearic acid, and arachidic acid.
[0080] Bound thereto is an outer layer of lipids comprising a
hydrophilic end group. Suitable lipids in the context of the
present invention comprise
Distearoyl-sn-glycero-3-phosphoethanolamine or other phospholipids
conjugated to monomethoxy polyethyleneglycol (PEG).
[0081] These lipids are further substituted with a hydrophilic
group such as NH.sub.2, COOH, unsubstituted or substituted PEG
(polyethylene glycol), PDP (-(2-pyridyldithio)propionate), CHO
(aldehyde) or SH (thiol).
[0082] Particular embodiments of substituted liquids comprise
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol-
)-2000](DSPE-PEG-COOH),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Amine(polyethylene
glycol)-2000] (DSPE-PEG-Amine),
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethylene glycol)-2000](DSPE-PEG), DSPE-Folate, DSPE-PEG(2000)
Maleimide or DSPE-PEG(2000) Carboxyfluoroscein, and DSPE-PEG(2000)
Biotin.
[0083] A second type of nanoparticles relates to a population of
monodisperse magnetic nanoparticles with a diameter between 1 and
100 nm which have covalently bound thereto a hydrophilic layer.
[0084] Typically the same types of nanoparticles carrying a layer
of fatty acids bound are used for the preparation. However instead
of adding lipids to this layer, the layer is replaced by a
hydrophilic layer. Suitable hydrophilic compounds for this layer
are Silane, DMSA, and ammonium chloride.
[0085] The physicochemical properties of the nanoparticles
determine the fusion with the plasma membrane of a cell.
[0086] One property is the size of the nanoparticles, which is
between 1 and 100 nm. In preferred embodiments the monodisperse
population of nanoparticles has a diameter around 10 nm (between 5
and 15 nm or between 8 and 12 nm). In embodiments where
nanoparticles are targeted and internalised via phagocytosis
nanoparticles with a diameter between 60 and 100 nm are
envisaged.
[0087] The other properties of the nanoparticles are the
hydrophilic and charge nature of the nanoparticles which can be
measured by determining the zeta potential of the nanoparticles. In
order to achieve a maximal uptake by a plasma membrane the zeta
potential of the nanoparticles is measured and the composition of
the coating is accordingly measured.
[0088] Zeta potential has been used to detect the cellular
interaction with charged nanoparticles and in general negatively
charged nanoparticle will decrease the surface Zeta potential and
positively charged nanoparticle will increase the surface Zeta
potential. These interaction effects can be observed with the
changes in the zeta potential values are relative to cell surface
charge and nanoparticle surface charge. Firstly, the binding of the
nanoparticles to the plasma membrane will change the zeta potential
value of the cells. During endocytosis, cells take up external
materials by invaginating a small portion of the cell surface
plasma membrane to form a new intracellular vesicle around the
substance to transport inside the cells. Since the cell membrane is
overall negatively charged, the loss of negatively charged cell
membrane during vesicular transport and charged nanoparticles
loaded inside the vesicles will cause the zeta potential values to
become to less negative. Hence in order to use the nanoparticle for
cell surface plasma membrane isolation or for endosomal
compartmental isolation, nanoparticle's surface composition should
be optimized depending on the zeta potential of the cell surface
and culture medium. This allows the skilled person to design and
select for any type of cell the appropriate type of nanoparticles
which can fuse with the plasma membrane or which can be
internalised in the endosomes. For example if the cell type has a
zeta potential of -31.16 mV at pH7, positively charged
nanoparticles (NH.sub.2-lipid end group nanoparticles) which has a
zeta potential in the range of 10-30 mV at pH 7.
[0089] The nanoparticles and methods of the present invention allow
to isolate plasma membranes of any eukaryotic cell type, including
fungi, yeasts, plants, and animals such as mammals. The properties
of the plasma membrane are highly similar, allowing to application
of the technology to any eukaryotic cell, when the eventual cell
wall covering the plasma membrane is removed or disrupted.
[0090] The size and physicochemical nature of the nanoparticles
allow the fusion with the plasma membrane and/or the uptake by the
endosomes without the additional presence of a targeting element
such as an antibody, ligand, receptor, for a plasma membrane
specific protein.
[0091] However for the targeting of specific subpopulations of
endosomes, nanoparticles which have a covalently bound hydrophilic
layer bound to the nanoparticles can be further modified with
antibodies, ligands of receptors, receptors, carbohydrates and
other compounds which provide specific targeting to a subpopulation
of endosomes.
[0092] Optionally magnetic nanoparticles can be further labelled
with a detectable marker, such as radioactive magnetic metal or
radioactive or isotope labelling of the layer around the magnetic
material. Alternatively, a part of the layer is functionalised with
a fluorescent, or other chromophoric group.
[0093] The present invention demonstrates that the efficiency of
the labelling of plasma membranes and/or endosomes is influenced by
the size distribution of the nanoparticles and the efficiency of
coating the nanoparticles with the fatty acid layer. These
advantageous properties are obtained by manufacturing of the
nanoparticles using the methods as disclosed in the present
invention.
[0094] In the methods of the present invention magnetic
nanoparticles are manufactured by a thermal decomposition method as
published in Sun and Zeng (2002) J. Am. Chem. Soc. 124(28),
8204-8205 or in US20040134565. This method has the advantage that
nanoparticles with a controlled size are obtained. Briefly a
mixture of a metal acetylacetone,
1,2-RCH(OH)CH.sub.2OH+RCOOH+RNH.sub.2 is heated at prolonged time.
Herein the metal can be any of Fe, Co, Mn, NI, Zn or a combination
thereof and R is C.sub.12 to C.sub.24. The reaction temperature and
time can be adjusted to obtain a population of magnetic
nanoparticles of desired diameter. Although different methods have
been used to generate magnetic nanoparticles for biological imaging
and cell and/or organelle targeting, the importance of the
homogeneity of the nanoparticle size population has been
underestimated for the targeting of nanoparticle to plasma
membranes. Only nanoparticles with a well defined size will fuse
with the membrane. Larger nanoparticle, will not interact with the
membrane or will bind aspecifically and temporarily to the
membrane, as has been documented for e.g. cationic silica beads.
Smaller nanoparticles will be taken up by the cell at an unwanted
rate or via an unwanted mechanism. Such nanoparticles will end up
in organelles or in the cytoplasm. Upon disruption of cell, the
cellular material which will be purified with magnetic
nanoparticles will consequently be contaminated with intracellular
material. Until present the use of magnetic nanoparticles which are
prepared with a thermal decomposition method for the binding to
plasma membranes has been unexplored.
[0095] In the prior art methods, the use of an organic coating,
such as oleic acid, to prevent agglomeration is described in
US20040134565. However the nanoparticles described therein are used
in the production of larger structures wherein the nanoparticles
are compacted or compressed.
[0096] For the purpose of the present invention it has been found
that the method of isolating the nanoparticles from the reaction
mixture has a significant effect on further applications wherein
the nanoparticles are used for binding to plasma membranes.
[0097] It has been found that due to the organic coating, such as
oleic acid, the coated nanoparticles can be precipitated with an
alcohol such as methanol, ethanol, propanol, butanol or mixtures
thereof, whereby the unreacted magnetic material remains in
solution. On the other hand it was found that under conditions
whereby e.g. more than 75% (V/V) of ethanol is used, a part, or
all, of the organic coating is removed from the magnetic
nanoparticles.
[0098] Under the appropriate conditions, the precipitation step as
explained above generated magnetic nanoparticles which have a
homogeneous coating. This has the advantage that the
physicochemical character of the coating and the modifications
thereof will be equally of an unexpected homogeneity.
[0099] In summary, the homogenous size and homogeneous coating of
nanoparticles which are prepared as explained above provides a
population of nanoparticles which will also behave more
homogeneously and predictable when these nanoparticles are
contacted with cells.
[0100] The coated nanoparticles as obtained by the thermal
decomposition methods are further modified by two alternative
methods. In a first method the coating is further modified. In a
second method the coating is replaced.
[0101] In the method wherein the coating is modified the coated
nanoparticles are mixed with one or more phospholipids in the
presence of an organic solvent such as chloroform to obtain
block-copolymer micelles following a procedure as published by
Dubertret (2002) Science 298(5599), 1759-1762. Prior to the ligand
addition step the nanoparticles are precipitated with an alcohol
(less than 75% v/v).
[0102] In the method wherein the coating is replaced a ligand
exchange method is used. Briefly. nanoparticles were dispersed in
an organic solvent and then DMSA, Silane, or TMAOH is added
dissolved in an alcohol or solvent such as chloroform or DMSO
following a procedure as published by Lee et al (2207) Nat Med
13(1), 95-99; Salgueirino-Maceira et al. (2004) Langmuir 20(16)
6946-6950 and Song et al. (2005) J. Am. Chem. Soc. 127(28),
9992-9993. For effective ligand exchange method, complete surface
etching is performed using 75% v/v) alcohol precipitation step.
[0103] Whereas other methods such as chemical co-precipitation also
lead to nanoparticles which are functionalised with the same
functional groups, these methods inherently lead to a less
homogeneous population in size and coating and make the less
suitable for the specific targeting to plasma membrane.
[0104] The magnetic nanoparticles of the present invention make it
possible to isolate plasma membranes and endosomes with a yield and
purity which is superior over prior methods. In addition methods as
described in the present invention require less steps than prior
art methods, and result in plasma membranes and/or endosomes
wherein the proteins or protein complexes have an activity which is
significantly higher, or results even in isolated proteins which
previously could not be isolated without loss of activity.
[0105] Accordingly one aspect of the present invention relates to a
method for the isolation of a plasma membrane of a cell, a fraction
thereof, or a plasma membrane derived organelle, comprising the
following steps: [0106] A population of intact and suspended cells
is provided at a temperature where endocytic uptake by a cell is
inhibited, e.g. below 25.degree. C., below 18.degree. C., below
12.degree. C., between 2 and 6.degree. C. (such as at about
4.degree. C.). [0107] The intact cells are contacted with magnetic
nanoparticles as described above. The physicochemical parameters
(size of the nanoparticle and hydrophobic nature or the coating)
allow the binding of these magnetic nanoparticles to and into the
plasma membrane. The defined size distribution and low temperature
allows targeting the plasma membrane with only a negligible uptake
into other cell organelles. [0108] Unbound magnetic nanoparticles
are removed by one or more washing steps, in a buffer which
preserves the integrity of the cells. [0109] Thereafter the cells
are disrupted, by e.g. osmotic shock or mechanical sheering
(typically using a ball-bearing cell cracker) and organelles such
as nuclei are removed by centrifugation. [0110] The fraction of the
disrupted cells which comprises magnetic nanoparticles is isolated
using magnetic purification methods, typically with a magnetic
force in the range of 0.1-0.5 Tesla magnetic. In particular
embodiments the fraction of the disrupted cells which comprises
magnetic nanoparticles is bound on a column of ferromagnetic beads
in the presence of a magnetic field, allowing to further wash to
bound fraction or to perform additional manipulations on the bound
fraction.
[0111] Alternatively or in addition, contaminating cell organelles
are washed out under high salt condition (between 0.1 and 2 M salt)
and/or in the presence of alkaline buffers with a pH between 10 and
12.
[0112] The methods as described above allow the isolation of plasma
membranes which have a significant higher degree of purity than
prior preparations, as illustrated in the accompanying experimental
data.
[0113] In addition to the above described general method, fractions
of plasma membranes can be equally isolated.
[0114] For example, isolated plasma membranes can be further
treated by mechanical shearing and the addition of detergents to
release cholesterol-rich microdomains or caveolae from the plasma
membrane. The caveolae are separated from the plasma membrane by a
gradient separation.
[0115] Another aspect of the present invention relates to the
isolation of endosomes from a cell population. These methods
comprise the following steps: [0116] A population of intact and
viable cells is provided and maintained a temperature where
endocytic mechanisms take place, generally above 25.degree. C.,
typically at about 37.degree. C. [0117] These cells are then
contacted for a predetermined period (typically between 1 and 30
minutes with magnetic nanoparticles, which are taken up by the
cells into the endosomes. (this step is also described as the
"pulse") [0118] Optionally, the above "pulse" step is followed by a
so-called "chase" step whereby the unbound magnetic nanoparticles
are removed, and whereafter the cells are further incubated at
temperature where endocytic mechanisms take place for a period
which may range from about 1 to 180 min. During this chase step,
magnetic nanoparticles will be further sorted from the plasma
membrane into the endosomes.
[0119] Hereafter the cells are also disrupted whereafter the
endosomal fraction is removed from the lysate by magnetic
attraction.
[0120] The methods of the present invention provide a number of
unprecedented advantages as indicated in table 1. Due to their
small size (<100 nm) the magnetic nanoparticle, do not interfere
with mass spectrometry methods and behave as a salt, and this in
contrast with silica beads. The absence of detergents in the
isolation method for plasma membranes preserves the structure and
function of proteins, carbohydrates and lipids.
TABLE-US-00001 TABLE 1 comparison of various plasma membrane
isolation techniques Magnetic Antibody nanoparticles Density
conjugated of present Properties Silica Bead Gradient Microbeads
invention Cell type Universal Universal Antibody Universal specific
specific Yield Low yield Low Yield Yield depends High Yield
affinity Process One-Step Multiple step Multiple steps One-step
Purity Low Purity Low purity High Purity High resolution Proteomics
good good good Good Lipidomics None none None Good Glycomics None
None None Good Enzyme none None none Good Activity
Examples
Example 1
Synthesis of Superparamagnetic Fe.sub.3O.sub.4 Nanoparticles
(SPMNPs)
[0121] Fe.sub.3O.sub.4 nanoparticles were synthesized using thermal
decomposition method as reported by Sun (cited before). In a
typical synthesis for 8 nm Fe.sub.3O.sub.4 nanoparticles, Iron
(III) acetylacetonate (2 mmol), 1,2-hexadecanediol (10 mmol), oleic
acid (6 mmol), oleyl amine (6 mmol) and benzyl ether (20 ml) were
mixed and magnetically stirring the under N.sub.2 flow conditions.
The mixture was heated to 200.degree. C. for 2 hours and then
refluxed at 300.degree. C. for 1 hour. The black colored mixture
was cooled to room temperature by removing the heat source. Further
black material (SPMNPs with oleic acid layer as shell coating) was
precipitated by adding ethanol at appropriate percentage depending
on the further functionalization step and was magnetically
separated using a rare earth magnet. Finally, SPMNPs were dispersed
in hexane and centrifuged step (5000 rpm, 10 minutes) was performed
to remove aggregates.
[0122] These Fe.sub.3O.sub.4 nanoparticles were coated with DSPE
(2000) phospholipids by adopting the ligand addition procedure
described for water soluble quantum dots (Dubertret cited
before).
[0123] In a typical experiment, 5 mg of SPMNPs were dissolved in 1
ml of chloroform. DSPE (2000)-PEG-NH.sub.2 (10 mg) was added to the
solution and vortexed for 4 hours followed by the removal of
chloroform by evaporation. The residual solid was dried by N.sub.2
flow for 5 minutes, and 1 ml of deionized water was added
immediately. After 5 minutes of vigorous stirring, a uniform
transparent water soluble SPMNPs aqueous solution was formed.
Further centrifugation for 10 minutes at 5000 rpm was performed in
order to remove the aggregates. The supernatant was further
purified by running through Miltenyi MACS LS column in the presence
of magnetic field. Finally the bound fraction (lipid coated SPMNPs)
were resuspended in 1 ml of PBS solution. Further the SPMNPs
concentration and size were determined using TGA and DLS
respectively.
[0124] To increase the cationic property of SPMNPs and for enhanced
electrostatic interaction with cells, the stoichiometric ratio
between SPMNPs and DSPE phospholipids can be adjusted.
[0125] For example: For MEFs wild-type, 1:1 ratio of SPMNPs (5 mg)
and DSPE phospholipids (5 mg) were used for functionalization.
While for MEFs PSDKO, 1:2 ratio of SPMNPs (5 mg) and DSPE
phospholipids (10 mg) were used for lipid functionalization. The
ideal stoichiometric ratio for maximum plasma membrane sheet
isolation can be determined by setting pilot experiments wherein
different ratios are tested. To generate fluorescently labeled
SPMNPs, 1:4 compositions (CF (fluorescent label) versus NH.sub.2
substituted phospholipids) were used in the functionalization
step.
[0126] DMSA (dimercaptosuccinic acid), TMAOH (Tetramethylammonium
hydroxide), COOH-TMACl ((3-carboxylpropyl)trimethylammonium
chloride) coated SPMNPs were synthesized by ligand exchange
methodology as cited above in [Lee (2007) Salgueirino-Maceira, et
al. (2004) and Song et al. (2005). After the reaction, SPMNPs were
dissolved in water, magnetic purified and then adjusted the pH of
the solution to be 7. (FIG. 1)
[0127] Several techniques were applied to the SPMNPs as a quality
control.
Transmission EM.
[0128] SPMNP suspensions were adhered onto a carbon-coated copper
grid, dried and imaged on a 300 kV Philips CM30 instrument equipped
with a field emission gun electron source.
Thermal Gravimetric Analysis (TGA).
[0129] SPMNP concentration measurements were performed on a TA
instruments Q5000 IR under N.sub.2 atmosphere.
Dynamic Light Scattering (DLS) and Zeta Potential Measurement.
[0130] The hydrodynamic diameters and zeta potential measurement of
SPMNPs were measured using Zetasizer Nano-ZS DLS system (Malvern
Instruments Ltd., England) and reported as number average using DTS
application software
Magnetic Characterization of SPMNPs.
[0131] Magnetization measurements were made using a standard
alternating gradient field magnetometer (AGFM Model 2900, Princeton
Instruments NJ).
Stability in SPMNPs.
[0132] Stability of SPMNPs in medium/H.sub.2O was performed using
UV-vis spectroscopy conducted on a Shimadzu UV-1601 PC
spectrophotometer and recorded between 300 and 1000 nm with a 0.5
nm resolution.
Example 2
Development and Functionalization of SPMNPs for Subcellular
Compartmental Isolation
[0133] SPMNPs are inorganic nanocrystals characterized by
superparamagnetic and size controlled physical properties which can
be fine-tuned depending on the application of interest. In
particular for subcellular isolation, nanoparticle-cell surface
interaction is the critical step and mainly governed by three major
physiochemical properties such as size, shape and surface coating
[Nel et al., (2009) Nat Mater 8, 543-557; Verma & Stellacci
(2010), Small. 6(1), 12-21]. Hence, the effect of surface coating
dependent selection of SPMNPs for subcellular localization and
magnetic isolation was investigated. Herein SPMNPs with a 10 nm
diameter have been synthesized by the thermal decomposition method
generating monocrystalline Fe.sub.3O.sub.4 with narrow size
distribution and high magnetization value (.about.60 emu/g).
However these SPMNPs were hydrophobic due to oleic acid coating and
were further functionalized using two alternative
methodologies:
a) Ligand exchange: DMSA/TMAOH/COO-TriMACl, or b) Ligand addition:
DSPE-Lipids. Functionalized SPMNPs were characterized for their
physical properties showing retention of superparamagnetism and
slight increase in size.
Example 3
SPMNPs--Cell Interaction and Magnetic Cell Isolation
[0134] HeLa and MEFs (wild-type) were grown to 70% confluence in 4
plates with a diameter of 10 cm dish plates. Initially cells were
washed three times with PBS (37.degree. C.) and incubated with
SPMNPs in DMEM/F12 medium for various time periods and at
increasing nanoparticle concentration. After incubation, cells were
trypsinized and harvested by centrifugation at 1000 rpm for 10
minutes. Further cell viability analysis was performed using trypan
blue staining and magnetic cell isolation using SuperMACSII
magnetic separation system.
[0135] MEFs (WT, PSENDKO, PSEN1r) cells grown to confluence in 8
plates with a diameter of 10 cm, were initially incubated in ice
cold DMEM for 30 minutes at 4.degree. C., washed three times with
ice cold phosphate buffered saline (PBS) and then incubated with
SPMNPs in PBS (2 mg/ml) for 20 minutes at 4.degree. C. with slow
horizontal shaking. After the incubation, cells were harvested in
PBS, centrifuged (1000 rpm, 10 minutes) and homogenized in
homogenizing buffer HB (250 mM sucrose, 10 mM Hepes and 1 mM EDTA
pH 7.4 supplemented with protease inhibitors) using a ball-bearing
cell cracker (20 passages, clearance 10 .mu.m, Isobiotec, Germany).
After low-speed centrifugation (200 g, 10 minutes), the post
nuclear supernatant (PNS) were loaded on to equilibrated LS column
in presence of SuperMACSII magnetic system (Miltenyi Biotec),
extensively washed sequentially with ice-cold HB, high salt 1M KCl
and high pH 0.1M Na.sub.2CO.sub.3 respectively, and the purified
plasma membrane fraction was eluted by removal from the magnet. The
plasma membrane fraction was enriched by ultracentrifugation
(55,000 rpm, 1 hour) and resuspended in 200 .mu.l HB buffer for
further analysis. For western blot and total protein analysis,
samples concentration were determined using a Bradford assay
(Bio-Rad) and protein separation were run in NUPAGE Novex pre-cast
4-12% gradient Bis-Tris gel (Invitrogen). Further processing was
performed using ECL detection protocol (Western Lightning,
PerkinElmer).
[0136] Of the different types of nanoparticles DMSA and
Lipid-SPMNPs showed medium stability and hence were used for
cellular (HeLa and MEFs) viability and uptake. No detectable cell
death was detected with trypan blue staining after 2 hours of
SPMNPs incubation. A surface coating-dependent variation in
cellular uptake of SPMNPs was observed with the following trend in
magnetic cell isolation
(NH.sub.2-lipid>DMSA>PEG-lipid>COOH-lipid-SPMNPs, whereby
NH.sub.2-lipid coated SPMNPs have the highest cellular uptake).
[0137] Further experiments on surface coating based selection were
performed with NH.sub.2-lipid/DMSA coated SPMNPs for Pulse-Chase
labeling and subcellular compartmental isolation without the use of
any biomolecule (i.e. targeting moieties such as antibodies,
peptides, ligand, etc).
[0138] Using DMSA/lipid-SPMNPs, the Pulse-Chase methodology for
magnetic endosome isolation was optimized based on concentration,
chase time and by studying the level of subcellular compartment
proteins in the magnetic fraction (MF) using western blot (WB)
analysis.
[0139] Depending on the chase period, MFs from DMSA-SPMNPs were
enriched either in early endosomal marker EEA1 (15-30 minutes), or
the late endosomal marker Rab7 (60 minutes). However MF from
lipid-SPMNPs (NH.sub.2 endgroup) showed enrichment in the plasma
membrane marker Na.sup.+K.sup.+ATPase for all Pulse-Chase
conditions. Based on these results, NH.sub.2-lipid-SPMNPs and
DMSA-SPMNPs were selected for plasma membrane and endosomal
isolation respectively.
Example 4
Isolation of Endosomal Fraction
[0140] 90% confluent HeLa cells grown on 10-cm dishes were
incubated for 15 minutes at 37.degree. C. with SPMNPs [DMSA] in
medium (200 .mu.g/ml). Then they were washed in PBS and chased at
37.degree. C. for various time periods. Cells were washed and
harvested in PBS, centrifuged and homogenized in homogenizing
buffer HB using a ball-bearing cell cracker. After low-speed
centrifugation (400 g, 10 minutes), the Post Nuclear Supernatant
(PNS) were loaded in SuperMACSII magnetic system, washed with
ice-cold HB and the concentrated magnetic fraction was resuspended
in HB buffer. The magnetic fraction (MF) was pelleted by
ultracentrifugation (55,000 rpm for 1 hour) and resuspended in 200
.mu.l HB buffer for further western blot analysis.
Example 5
Analysis of Plasma Membrane Proteomics Glycoproteomics and
Lipids
Lipid Extraction and ESI-MS/MS Based Analysis.
[0141] A qualitative and quantitative Proteomics, Glycomics and
Lipidomics analysis was performed between the wild type, PSENDKO
[Presenilin 1 and 2 double knock out] and PSEN1r [Presenilin
rescued with human PSEN1] MEF.
[0142] Peter et al. (2007). J Biomol Tech. 18(5), 287-297 described
a magnetic carrier based methods for plasma membrane isolation.
These magnetic purification procedures require additional
detergent/acid wash steps to release proteins from the magnetic
beads for suitable MS analysis.
[0143] The nanoparticles as used in the present invention have a
very high surface to volume ratio and small size. These
nanoparticles do not interfere with MS analysis and provide a clear
enrichment for plasma membrane (protein, lipids and glycans)
compared to the total fraction. Accordingly the present invention
allows to perform a novel detergent-free plasma membrane isolation
method which is compatible with MS analysis compared to existing
antibody/detergent based magnetic purification methods.
Proteomics
[0144] For separation of N-terminal peptides by COFRADIC, cell
membrane fraction were lysed, disulfide bonds were reduced and
alkylated prior to acetylation of N-termini with trideutero-acetyl
N-Hydroxy-Succinimide. Samples were digested with trypsin, V8
protease or chymotrypsin overnight at 25.degree. C. or 37.degree.
C., respectively. Purified dried peptides were reconstituted in
0.1% trifluoroacetic acid (TFA) and separated on an Ultimate 3000
LC system. During the primary run, 16 fractions of 4 minutes each
were collected and dried under vacuum. N-termini of internal
peptides were derivatized with TNBS. Afterwards, fractions were
applied to a secondary RP-HPLC run with identical chromatographic
conditions. Fractions were collected in the same time intervals as
before, dried under vacuum and prepared for LC-MS/MS analysis. The
quantitative differential aspect of the procedure was also
performed by reacting peptides with propionylC.sup.13-sulfo NHS
(heavy) and propionylC.sup.12-sulfo NHS (light). Two such
experiments were performed comparing wild-type and PS-/- MEFS with
heavy and light labels switched for the repeat experiments.
[0145] Proteomics analysis of plasma membrane fractions showed
clear enrichments for integral membrane proteins (70%) compared to
the PNS fraction. Fragmented peptide spectra were identified using
Mascot search algorithm and identified approximately 2000 unique
proteins in the plasma membrane fraction. Similar trends were
observed with respect to PSENDKO and PSEN1r samples. This degree of
purity is substantially higher compared to existing technologies
such as cell surface biotinylation, density gradient centrifugation
and antibody based magnetic purification. Furthermore and using
Gene Ontology (GO) based database analysis, 150 unique proteins
were observed which are present only in the wildtype plasma
membrane and 300 unique proteins which are present only in the
PSENDKO plasma membrane fraction. Significantly more cell migration
related proteins are represented in the wildtype plasma membrane
proteome (Wildtype--6%, PSENDKO--1%), while adhesion related
proteins are more present in the PSENDKO plasma membrane fraction
(Wildtype--6%, PSENDKO--8%). These results were confirmed using
cell migration assays and confocal analysis on the indicated cell
lines.
[0146] ICAT labeling based differential proteomics observed the
absence of certain proteins in PSDKO and a, increase of certain
proteins in the wildtype MEFs (about 80 proteins).
Lipidomics
[0147] Cell membrane was isolated using lipid coated SPMNPs as
stated previously. To prepare lipid extracts for ESI-MS/MS
analysis, the cell membrane fraction and Post Nuclear Supernatant
(PNS) were mixed with 0.9 ml of 1N HCl:Methanol 1:8 (v/v).
CHCl.sub.3 (0.8 ml) and 200 .mu.g/ml of the anti-oxidant
2,6-di-tert-butyl-4-methylphenol (Sigma) were added. After addition
of the lipid standards, the organic fractions were collected by
centrifugation at 200 g for 5 minutes. Samples were evaporated and
reconstituted in CH.sub.3OH:CHCl.sub.3:NH.sub.4OH (90:10:1.25,
v/v/v) and the lipids were analyzed by electrospray ionization
tandem mass spectrometry (ESI-MS/MS) on a hybrid quadrupole linear
ion trap mass spectrometer (4000 QTRAP system; Applied Biosystems,
Foster City, Calif.) equipped with a robotic nanoflow/ion source
(Advion Biosciences). The system was operated in the MRM mode for
quantification of individual species. Data were expressed as fold
change relative to the control samples (wild-type) and were
presented as heatmaps using the Heatmap Builder software (Clifton
Watt, Stanford University, USA).
Cholesterol and Total Spingomyleinase Activity Determination
[0148] Cholesterol levels were determined using the Amplex-Red
cholesterol assay (Molecular Probes). Similarly Spingomyleinase
activity was measured using the Amplex-Red Sphingomyelinase Assay
Kit (Molecular Probes). Total SM concentrations were determined
enzymatically using a modified assay from the Amplex-Red
Sphingomyelinase Assay Kit (Molecular Probes). Briefly, for SM
determination, membrane fraction was adjusted to a protein
concentration of 0.15 mg/ml using a bicinchoninic acid assay. A 100
.mu.l sample was added to a 100 .mu.l assay solution, which
contains 100 .mu.M Amplex Red reagent, 2 U ml-1 HRP, 0.2 U ml-1
choline oxidase, 8 U ml-1 of alkaline phosphatase, 1 mU nSMase and
0.1 M Tris-HCl, 10 mM MgCl.sub.2, pH 7.4. After preincubation for 1
h at 37.degree. C. under light exclusion conditions, fluorescence
was measured for 30 minutes using excitation at 530.+-.2.5 nm and
fluorescence detection at 590.+-.2.5 nm. The slope, which has to be
0, was calculated to scrutinize the completeness of the reaction.
The values were corrected from the background signal that was
determined by samples treated in the same way as described above
but which did not contain any SMs.
[0149] A lipid profiling on total and plasma membrane fractions in
WT and PSENDKO MEFs was performed for the following lipids:
Cholesterol (Chol), Phosphatidylcholine (PC), Phosphatidylserine
(PS), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI) and
Sphingomylein (SM) lipids using ESI/MS analysis. We observed clear
enrichments at the plasma membrane and deciphered the hallmarks of
plasma membrane lipid composition including an increase in SM (8%
increase) and decrease in PI (4% decrease) with respect to the PNS.
Furthermore we performed quantitative and qualitative comparisons
of plasma membrane lipid level vs. total between WT and PSENDKO
plasma membrane fractions. In these analyses, we observed
significant increase in SM and Chol levels in PSENDKO PNS fractions
compared to the wildtype levels as published in Grimm (2005) Nat.
Cell Biol. 7(11), 1118-1123. In addition, we also identified a
reverse trend, i.e. decrease in Chol level, at the plasma membrane
of PSENDKO MEFs. This was next confirmed using filipin staining and
confocal analysis demonstrating clear intracellular enrichments for
Chol while less staining was observed at the cell surface. With
respect to the other phoshopholipids PC, PS and PI, there was no
significant difference between the wildtype and PSENDKO in terms of
lipid levels. At the same time, we identified variations between
the lipid species in the PC and SM families. For example SM species
(16:1, 18:0 & 18:1) were significantly higher in PSENDKO PNS
fraction compared to wildtype, while there was no significant
variation in the plasma membrane fraction. In addition we observed
increased levels of unsaturated PC species at plasma membrane and
similar trends for PI, PS, and PE with respect to PNS and plasma
membrane for PSDKO vs. wildtype. Currently we are comparing the
lipid profiles with the one of the PSEN1 rescued MEFs (PSEN1r) to
validate our analysis. With respect to cholesterol levels, no
significant difference between the wildtype and PSEN1r (PM, PNS)
was observed thereby showing recovery in cholesterol trafficking.
Further differential lipid profiling will be performed in order to
generate complete lipidomics on PSEN1 r and also to further
scrutinize our methodology.
Glycomics
[0150] N-termini of internal peptides fractions were pooled as
stated previously. Prior to an identical secondary RP-HPLC
separation, each pooled fraction was redissolved in 85 .mu.l of a
freshly prepared 50 mM ammonium bicarbonate buffer (pH 7.8), and
0.8 units of peptide N-glycosidase (PNGase F) from Chryseobacterium
(Flavobacterium) meningsepticum (proteomics grade, g95%,
Sigma-Aldrich) was added. Hydrolysis of N-linked sugar chains from
peptide backbone asparagines was allowed for 20 minutes at
30.degree. C. The reaction was stopped by adding 10 .mu.l of 50%
acetic acid. Each PNGase F treated pool of primary fractions was
loaded onto the same RP-HPLC column and the same solvent gradient
was applied as during the primary separation. The altered peptides
were collected in secondary fractions, dried under vacuum and
prepared for LC-MS/MS analysis.
[0151] Based on the identified sites and using GO databases, we
could identify the proteins and locate known N-glycosylation sites
(For example: Nicastrin potential N-glycosylation site at 44.sup.th
amino acid was identified). Based on the MS and database analysis,
235 glycosylated proteins were identified in which 65 are known and
100 are potential sites, respectively. Next, we demonstrate that
the SPMNPs-based plasma membrane extraction can be interfaced with
downstream Fluorophore Assisted Carbohydrate Electrophoresis as
published in Laroy et al. (2006) Nat Protoc. 1(1), 397-405,
resulting in glycan profiling. Using this approach, we observed
significantly higher N-glycan sialylation levels in PSENDKO
compared to wildtype plasma membrane fractions. This difference
could not be clearly observed when analyzing PNS, likely due to the
high content of immature N-glycans in the spectra. Further
structural analysis confirms that structures are typical bi- and
tri-antennary. We also confirmed the absence glycans originating
from any serum related protein which in general is a limiting
factor for cell surface glycomics. Currently we are studying
whether there is any recovery in N-glycan sialylation level in the
PSEN1 r plasma membrane fraction similar to the WT.
Example 6
Isolation of Active Protein Complexes from Cell Membrane
[0152] Cell membrane was isolated using lipid coated SPMNPs as
stated previously. To prepare microsomal membranes from the cell
membrane, pelleted magnetic fraction was resuspended in PIPES
buffer (20 mM PIPES pH7, 140 mM KCl, 0.25M Sucrose, 5 mM EGTA,
Protease inhibitor) containing 1% CHAPS (sigma-Aldrich) and
solubilized for 1 hour at 4.degree. C. Following sequential
ultracentrifugation for 30 minutes and 15 minutes (100,000 g
4.degree. C.) the cleared supernatant was collected. Similarly,
microsomal membranes were isolated from the PNS and further
obtained supernatants (from PNS & Bound) were used for cell
free enzyme activity studies.
Example 7
Development of a Cell Free Gamma-Secretase Assay
[0153] PM and PNS fractions were extracted in CHAPS and mixed with
recombinant APP-C99-FLAG affinity isolated from transiently
transfected Aph1.sup.-/-/- MEFs as described in Spasic et al.
(2007) J Cell Biol 176(5), 629-640. Newly produced APP
intracellular domain (AICD) was separated on 10% precasted gels
(NuPAGE) in MES buffer and analyzed for Western blotting.
[0154] The formation and levels of .gamma.-secretase complexes at
the PM, protein complexes were extracted from microsomal membranes
from PNS and plasma membrane with 0.5% dodecylmaltoside (DDM) and
were run on a Native gel.
[0155] The novel technology of the present invention was compared
it with established methods like cell surface biotinylation (CSB)
to study the levels of gamma-secretase components like Nicastrin
(Nct), Presenilin 1 & 2 (PS1&2), Anterior pharynx
defective-1 (APH1a) and Presenilin enhancer-2 (PEN2) at the cell
surface. All methods (including confocal analysis of isolated
plasma membrane sheets) confirmed the higher abundancies of PS1,
NCT, PEN2 and APH1a at the cell surface while much lower levels
(about 1-2%) for PSEN2 were measured. Next we quantified the levels
of intact gamma-secretase complexes as well as activity in i SPMNP
isolated PMs using blue native electrophoresis (complexes) as well
as a cell free gamma-secretase assay. These data directly establish
that most if not all components exist in active gamma-secretase
complexes at the PM. Moreover, we also observed a significant
enrichment in terms of quantity and quality of gamma-secretase
complexes when comparing plasma membrane fractions with PNS. In
summary the present inventions provides an SPMNPs based plasma
membrane method, which is detergent-free and does not rely on IP
(Immune precipitations) for the isolation of pure and biological
active PMs including active gamma-secretase complexes. These data
are part of a larger project and paper studying the distinct
distribution of PSEN1 and PSNE2-containing complexes in the
endocytic pathway and cell surface.
Example 8
Validation of Results Based on Fluorescent Labeled SPMNPs
[0156] Transmission EM. After magnetic labeling, cells were washed
twice with PBS-/- and sequentially fixed in double strength
fixative for 30 minutes. Fixed cells were coated with 1% gelatin,
scraped and the cell pellet repeatedly washed. Next, cell pellets
were fixed in 2% osmium (1 hr), rinsed with dH.sub.2O, and
subsequently dehydrated using an ethanol series (50-100%) and
embedded in Epon. Ultrathin sections of 70 nm were examined on a
JEOL120CX TE microscope.
[0157] Confocal Laser scanning microscopy. Following incubation
with fluorescently labeled SPMNPs, cells were washed in PBS-/-,
fixed with 4% paraformaldehyde, and mounted in Moviol. Fluorescence
was captured on confocal microscope (Radiance 2100, Zeiss)
connected to an upright Nikon E800 microscope and Image processing
was done using Lasersharp 2000 (Zeiss) and Photoshop (Adobe,
CA).
[0158] Fluorescent modified lipid-SPMNPs were developed to confirm
the WB results on the Pulse-Chase method using confocal analysis.
We observed lipid-SPMNPs predominately localized at the plasma
membrane even for prolonged chase period. Furthermore, by using TEM
analysis on MEFs incubated with SPMNPs at 4.degree. C. for 20
minutes, we could observe SPMNPs localized exclusively at the cell
surface. Based on the fluorescence and TEM analysis results, we
designed a detergent- and conjugation-free SPMNP based approach to
isolate plasma membrane fractions with a high purity and yield
(Method section). We validated the method by studying the level of
enrichment and purity of our plasma membrane fractions by WB
analysis of an extensive number of compartment specific marker
proteins. Our isolated plasma membrane fraction is of high purity
and could be further enriched by combining high salt/high pH washes
resulting in less than 2%--ER/Golgi contaminations. Similar quality
of plasma membrane fractions was isolated from other cell lines
like wildtype, PSEN deficient and rescued (PSEN1r) mouse embryonic
fibroblasts (MEF).
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