U.S. patent application number 13/265916 was filed with the patent office on 2012-03-29 for methods and devices for capturing circulating tumor cells.
This patent application is currently assigned to THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOI. Invention is credited to David Eddington, Seungpyo Hong, Cari Launiere, Ja Hye Myung.
Application Number | 20120077246 13/265916 |
Document ID | / |
Family ID | 43011770 |
Filed Date | 2012-03-29 |
United States Patent
Application |
20120077246 |
Kind Code |
A1 |
Hong; Seungpyo ; et
al. |
March 29, 2012 |
Methods and Devices for Capturing Circulating Tumor Cells
Abstract
A method of capturing a Circulating Tumor Cell (CTC) from a
sample includes introducing a sample into a microfluidic device
having a cell capture surface and a flow modification surface under
conditions that allow a CTC to bind to a cell rolling-inducing
agent and a capturing agent disposed on the cell capture surface.
The flow modification surface induces a rotational flow within the
sample as it flows through the microfluidic device.
Inventors: |
Hong; Seungpyo; (Naperville,
IL) ; Eddington; David; (Wheaton, IL) ; Myung;
Ja Hye; (Daejeon, KR) ; Launiere; Cari;
(Chicago, IL) |
Assignee: |
THE BOARD OF TRUSTEES OF THE
UNIVERSITY OF ILLINOI
Urbana
IL
|
Family ID: |
43011770 |
Appl. No.: |
13/265916 |
Filed: |
April 23, 2010 |
PCT Filed: |
April 23, 2010 |
PCT NO: |
PCT/US10/32266 |
371 Date: |
October 24, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61172454 |
Apr 24, 2009 |
|
|
|
61174602 |
May 1, 2009 |
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Current U.S.
Class: |
435/174 ;
422/502 |
Current CPC
Class: |
G01N 33/574
20130101 |
Class at
Publication: |
435/174 ;
422/502 |
International
Class: |
C12N 11/00 20060101
C12N011/00; B01L 3/00 20060101 B01L003/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. CBET-0931472 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1. A method of capturing a Circulating Tumor Cell (CTC) from a
sample comprising the step of introducing said sample into a
microfluidic device under conditions that allow a CTC to bind to a
cell rolling-inducing agent and a capturing agent, the device
inducing a rotational flow with the sample, the device comprising
an immobilized cell rolling-inducing agent and an immobilized
capturing agent.
2. The method of claim 1 further comprising applying a shear stress
between 0.05 and 10 dyn/cm.sup.2 on the sample introduced into the
device.
3. The method of claim 2 wherein the shear stress is between 0.1
and 2.0 dyn/cm.sup.2.
4. The method of claim 3 wherein the shear stress is about 0.16
dyn/cm.sup.2.
5. The method of claim 1 wherein the cell rolling-inducing agent is
a selectin or a CTC binding fragment of a selectin.
6. The method of claim 5 wherein the selectin is selected from the
group consisting of E-selectin, P-selectin, and L-selectin.
7. The method of claim 1 wherein the capturing agent specifically
binds a moiety on a CTC cell surface, the capturing agent selected
from the group consisting of an antibody, an antibody fragment, an
engineered antibody, folic acid, transferrin, a peptide, and an
aptamer.
8. The method of claim 7 wherein the antibody is anti-EpCAM.
9. The method of claim 7 wherein the peptide is an RGD peptide.
10-22. (canceled)
23. A microfluidic device for capturing a Circulating Tumor Cell
(CTC) from a sample, comprising: a channel comprising a cell
capture surface and a flow modification surface, wherein the cell
capture surface comprises a cell rolling-inducing agent and a
capturing agent immobilized on the cell capture surface, and the
flow modification surface comprises one or more ridges extending
into the channel and arranged to induce a rotational flow in a
sample flowing through the channel.
24. The device of claim 23, wherein the cell rolling-inducing agent
is a selectin or a CTC binding fragment of a selectin.
25. The device of claim 24, wherein the selectin is selected from
the group consisting of E-selectin, P-selectin, and L-selectin.
26. The device of claim 23, wherein the capturing agent is adapted
to specifically bind to a moiety on a CTC cell surface, and the
capture agent is selected from the group consisting of an antibody,
an antibody fragment, an engineered antibody, folic acid,
transferrin, a peptide, and an aptamer.
27. The device of claim 26, wherein the antibody is anti-EpCAM.
28. The device of claim 26 wherein the peptide is an RGD
peptide.
29. The device of claim 23, wherein the capturing agent is
immobilized on the cell capture surface by direct attachment of the
capturing agent to the cell capture surface.
30-56. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/172,454, filed Apr. 25, 2009, and U.S.
Provisional Patent Application No. 61/174,602, filed May 1, 2009,
the disclosures of which are incorporated herein by reference in
their entirety.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The invention relates to a method of capturing Circulating
Tumor Cells from a sample, and a microfluidic device for performing
the method.
[0005] 2. Brief Description of Related Technology
[0006] Cancer remains one of the world's most devastating diseases,
with more than 10 million new cases every year. Although recent
advances in diagnostic and therapeutic methods to treat primary
tumors have resulted in a decrease in mortality of cancer for the
past two years, metastasis of cancer still poses a great challenge
as patients often relapse. Disseminated and Circulating tumor cells
(DTCs and CTCs, respectively) are known to induce secondary tumor
formation at distant sites from primary tumors, known as
metastasis. Two major theories describing cancer metastasis, the
seed and soil hypothesis and the mechanical trapping theory, are
available and the extravasation process for each are similar,
consisting of three sequential steps. The metastasis mechanism is
known to be initiated by cell rolling--the naturally occurring
process utilized to recruit leukocytes to sites of inflammation. In
the second step, the cells firmly attach to the endothelial cells.
In the third step, the cells transmigrate through the endothelium
(diapedesis), resulting in secondary tumor formation.
[0007] Research efforts on diagnosis and prognosis of metastatic
cancer have concentrated on the detection of DTCs in bone marrow
(BM) and CTCs in blood. Detection of DTCs requires aspiration of
BM--a process that is invasive, time-consuming, and often painful
for the patients, precluding repeated samplings that are necessary
for prognosis studies along with therapeutic treatments.
Consequently, effective detection of CTCs in peripheral blood of
cancer patients holds a promise as an alternative due to its
minimal invasiveness and easy samplings (i.e. blood drawing).
However, the clinical usage of CTCs has not yet been implemented
for routine clinical practice. In fact, the clinical significance
of CTCs in patient blood is less clear than that for DTCs in BM.
Unlike DTCs in BM that are relatively easy to enrich using
Ficoll-based assays or the OncoQuick approach, and other
immunomagnetic enrichment procedures, CTCs are extremely rare
(estimated to be in the range of one tumor cell in the background
of 10.sup.6-10.sup.9 normal blood cells), presenting a tremendous
challenge for efficient, clinically significant detection of
CTCs.
[0008] Thus, there exists in the art a need for devices and methods
to efficiently isolate circulating tumor cells with enhanced
sensitivity and specificity to aid in diagnosis and prognosis of
cancer.
SUMMARY OF THE INVENTION
[0009] In one aspect of the disclosure, there is provided a method
of capturing a circulating tumor cell (CTC) in a sample comprising
the step of introducing said sample into a microfluidic device
under conditions that allow a CTC to bind to a cell
rolling-inducing agent and a capturing agent, the device inducing a
rotational flow with the sample, the device comprising an
immobilized cell rolling-inducing agent and an immobilized
capturing agent.
[0010] In one aspect, the method further comprises applying a shear
stress between 0.05 dyn/cm.sup.2 and 10 dyn/cm.sup.2 on the sample
introduced into the device.
[0011] In another aspect of the method, the shear stress is between
0.1 dyn/cm.sup.2 and 2 dyn/cm.sup.2.
[0012] In yet another aspect of the method, the shear stress is
about 0.16 dyn/cm.sup.2.
[0013] In another aspect of the method, the cell rolling-inducing
agent is a selectin or a CTC binding fragment of a selectin. In one
aspect, the selectin is selected from the group consisting of
E-selectin, P-selectin, and L-selectin.
[0014] In another aspect of the method, the capturing agent
specifically binds a moiety on a CTC cell surface, the capturing
agent selected from the group consisting of an antibody, an
antibody fragment, an engineered antibody, folic acid, transferrin,
a peptide, and an aptamer. In one aspect, the antibody is
anti-EpCAM. In another aspect, the peptide is an RGD peptide.
[0015] In one aspect of the method, the capturing agent is
immobilized via attachment to a surface of the microfluidic device.
In another aspect, the capturing agent is immobilized via
attachment to a linker and the linker is attached to a surface of
the device. In one aspect, the linker is a polymeric nanolinker. In
another aspect, the polymeric nanolinker comprises a modified
poly(amidoamine) dendrimer covalently attached to polyethylene
glycol. In another aspect, the modified poly(amidoamine) dendrimer
is selected from the group consisting of a generation 3, a
generation 4, a generation 5, a generation 6, a generation 7, a
generation 8, and a generation 9 modified poly(amidoamine)
dendrimer.
[0016] In another aspect of the method, the polymeric nanolinker
comprises polyester-n-carboxylate-1-alkyne dendron covalently
attached to polyethylene glycol, wherein n is 8, 16, 32, 64, or
128.
[0017] In another aspect of the method, the sample comprises
blood.
[0018] In another aspect of the method, the immobilized cell
rolling-inducing agent and the immobilized capturing agent are
arranged in a substantially uniform manner.
[0019] In yet another aspect of the method, the immobilized cell
rolling-inducing agent and the immobilized capturing agent are
arranged in a pattern.
[0020] In still another aspect of the method, the cell-rolling
inducing agent is covalently attached to a surface of the
microfluidic device.
[0021] In another aspect of the method, the cell-rolling inducing
agent is covalently attached to the surface via a chemical moiety
selected from the group consisting of an epoxy group, a carboxyl
group, a thiol group, an alkyne group, an azide group, a maleimide
group, a hydroxyl group, an amine group, an aldehyde group, and a
combination thereof.
[0022] In yet another aspect of the method, the cell-rolling
inducing agent is immobilized to a surface of the microfluidic
device via a linker. In one aspect, the linker is selected from the
group consisting of a dendrimer, a dendron, a dextran, polyethylene
glycol, poly(L-lysine), poly(L-glutamic acid), polyvinyl alcohol,
polyethylenimine, poly(lactic acid), poly(glycolic acid), and a
combination thereof.
[0023] Also provided herein is a microfluidic device for capturing
a circulating tumor cell (CTC) from a sample, comprising a channel
comprising a cell capture surface and a flow modification surface,
the cell capture surface comprising a cell rolling-inducing agent
and a capturing agent immobilized on the cell capture surface, and
the flow modification surface comprising one or more ridges
extending into the channel and arranged to induce a rotational flow
in a sample flowing through the channel.
[0024] In one aspect of the device, the cell rolling-inducing agent
is a selectin or a CTC binding fragment of a selectin. In another
aspect of the device, the selectin is selected from the group
consisting of E-selectin, P-selectin, and L-selectin.
[0025] In another aspect of the device, wherein the capturing agent
is adapted to specifically bind to a moiety on a CTC cell surface,
and in various aspects, the capture agent is selected from the
group consisting of an antibody, an antibody fragment, an
engineered antibody, folic acid, transferrin, a peptide, and an
aptamer. In one aspect, the antibody is anti-EpCAM. In another
aspect, the peptide is an RGD peptide.
[0026] In another aspect of the device, the capturing agent is
immobilized on the cell capture surface by direct attachment of the
capturing agent to the cell capture surface.
[0027] In yet another aspect of the device, the capturing agent is
immobilized on the cell capture surface by attachment to a linker
directly attached to the cell capture surface. In one aspect, the
linker is a polymeric nanolinker. In another aspect, the polymeric
nanolinker comprises a modified poly(amidoamine) dendrimer
covalently attached to polyethylene glycol. In an another aspect,
the modified poly(amidoamine) dendrimer is selected from the group
consisting of a generation 3, a generation 4, a generation 5, a
generation 6, a generation 7, a generation 8, and a generation 9
modified poly(amidoamine) dendrimer. In another aspect, the
polymeric nanolinker comprises polyester-n-carboxylate-1-alkyne
dendron covalently attached to polyethylene glycol, wherein n is 8,
16, 32, 64, or 128.
[0028] In another aspect of the device, the cell rolling-inducing
agent and the capturing agent are arranged in a substantially
uniform manner.
[0029] In another aspect of the device, the cell capture surface
comprises a pattern of first and second regions, the first region
comprising the cell rolling-inducing agent, and the second region
comprising the capture agent. In one aspect, the first region
further comprises the capture agent.
[0030] In another aspect of the device, the first and second
regions are arranged in an alternating pattern.
[0031] In another aspect of the device, the cell-rolling inducing
agent is covalently attached to the cell capture surface. In
another aspect, the covalent attachment is through a chemical
moiety selected from the group consisting of an epoxy group, a
carboxyl group, a thiol group, an alkyne group, an azide group, a
maleimide group, a hydroxyl group, an amine group, an aldehyde
group, and combinations thereof.
[0032] In another aspect of the device, the cell-rolling inducing
agent is immobilized to a surface of the microfluidic device via a
linker. In another aspect, the linker is selected from the group
consisting of a dextran, a dendrimer, polyethylene glycol,
poly(L-lysine), poly(L-glutamic acid), polyvinyl alcohol,
polyethylenimine, poly(lactic acid), poly(glycolic acid), and a
combination thereof.
[0033] In another aspect of the device, the one or more ridges are
angled obliquely relative to the channel.
[0034] In still another aspect of the device, the one or more
ridges are at a 45 degree angle relative to the channel.
[0035] In yet another aspect of the device, the one or more ridges
have a linear shape.
[0036] In another aspect of the device, the one or more ridges have
a herringbone shape.
[0037] In another aspect of the device, the one or more ridges are
arranged in a pattern. In another aspect, the pattern comprises
first pattern regions and second pattern regions disposed between
adjacent first pattern regions, wherein the first pattern regions
comprise the one or more ridges and the second pattern regions are
devoid of ridges. In another aspect, the pattern comprises first
and second pattern regions, the first and second pattern regions
each comprising one or more ridges, and the ridges in the first
pattern region are oriented, sized, and/or shaped differently than
the ridges in the second pattern region.
[0038] In another aspect of the device, the pattern further
comprises a third pattern region disposed between adjacent first
and second pattern regions, the third pattern region being devoid
of ridges.
[0039] In another aspect of the device, the ridges have a height of
about 50 .mu.m to about 300 .mu.m.
[0040] In another aspect of the device, the ridges have a width of
about 50 .mu.m to about 300 .mu.m.
[0041] In another aspect of the device, the ridges have a spacing
of about 50 .mu.m to about 500 .mu.m.
[0042] In yet another aspect of the device, the flow modification
surface is disposed opposite the cell capture surface.
[0043] In another aspect of the device, the channel has a height of
about 50 .mu.m to about 600 .mu.m.
[0044] In another aspect of the device, the channel has a width of
about 200 .mu.m to about 2000 .mu.m.
[0045] Other features and advantages of the present disclosure will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples, while indicating preferred embodiments of the
disclosure, are given by way of illustration only, because various
changes and modifications within the spirit and scope of the
disclosure will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a cross-sectional view of a microfluidic device
having a cell capture surface and a flow modification surface in
accordance with an embodiment of the disclosure.
[0047] FIG. 2 is a schematic representation of a flow modification
surface having a herringbone structure of ridges in accordance with
an embodiment of the disclosure.
[0048] FIG. 3 is a schematic illustration of a method of making a
cell capture surface in accordance with an embodiment of the
disclosure.
[0049] FIG. 4 is schematic diagram of CTC capturing on a cell
capture surface using iterative cell rolling (E-selectin: red) and
multivalent stationary adhesion (anti-EpCAM: green) in accordance
with an embodiment of the disclosure. The inset diagram represents
immobilized anti-EpCAM through flexible polymer nanolinkers
(dendrimers and PEG: blue) by which the multivalent effect can be
achieved through locally concentrated anti-EpCAM. The flow
direction appears to be linear for simple illustration but actual
flow in the chip will be rotated by the flow modification surface
(not shown) disposed in the channel.
[0050] FIG. 5 illustrates a synthetic scheme for G5 PAMAM
dendrimer-based nanodevices with AF488 and different numbers of FA
molecules. The number average molecular weights and PDIs were
determined by GPC. All numbers of functional attachment were
calculated from GPC results. The total number of end groups (110)
was determined by potentiometric titration.
[0051] FIG. 6 illustrates a) a comparison of effect of the number
of FA per dendrimer molecules between model study using SPR (blue)
and in vitro study using FACS (red). The nanodevice with 2.6 FA
shows a lower degree of cellular binding and association constant
(K.sub.a) than the rest of the nanodevices. FACS data were obtained
after incubation with dendritic nanodevices with FAR
over-expressing KB cells at 37.degree. C. for 1 hr and were
averaged from 12 different samples at each condition. Those of
association constants were averaged values from at least three runs
of the SPR measurements for each point. The association constant
(K.sub.A=1/K.sub.D) is plotted in this case as it provides the best
visual comparison to the FACS data. b) Association rate constant
(k.sub.a) (M.sup.-1s.sup.-1) and dissociation rate constant k.sub.d
(s.sup.-1) of dendrimers with varying numbers of folic acid as
measured by SPR. The k.sub.a value increases linearly with the
number of folic acids per dendrimer whereas the k.sub.d value
decays exponentially with the increasing number of folic acid
ligands.
[0052] FIG. 7 is a schematic of covalent immobilization of
P-selectin on PEG functionalized surfaces using various
chemistries.
[0053] FIG. 8 are a) and b) SPR sensorgrams of density controlled
P-selectin immobilization. As shown in a) and b), by changing the
ration between OEG-COOH (OEG-NH.sub.2) and OEG-OH, the amount of
P-selectin immobilized is controlled by wavelength changes up to 20
nm (corresponds to .about.300 ng/cm.sub.2 of immobilized protein)
and is proportional of the content of bifunctional OEGs. c) Effect
of P-selectin orientation in antibody binding response on the
unoriented P-selectin (using OEG-COOH, FIG. 7a) and oriented
P-selectin (using OEG-biotin, FIG. 7b). For this binding curve, two
chips with the same amount of immobilized P-selectin were used.
[0054] FIG. 9 are time-course images of HL-60 cells (a) and b)) and
MCF-7 cells (c) and d)) on the surfaces coated with E-selectin.
Both cell lines exhibit the rolling behavior in an E-selectin
specific manner. The rolling velocities of HL-60 and MCF-7 cells
were 2.12.+-.0.15 and 4.24.+-.0.31 .mu.m/sec at a shear stress of
0.32 dyn/cm2, respectively.
[0055] FIG. 10A is a graph illustrating the rolling velocity of
HL-60 and MCF-7 cells on E-selectin coated surfaces at four
different flow rates (50, 200, 400, and 800 .mu.l/min).
[0056] FIG. 10B is a graph illustrating the rolling velocity of
HL-60 and MCF-7 cells on E-selectin coated on surfaces at four
different shear stresses (0.08, 0.32, 0.64, and 1.28 dyn/cm.sup.2),
which correspond to the flow rates of FIG. 6A.
[0057] FIG. 11 are time-course images of MCF-7 cells on anti-EpCAM
coated surfaces under shear stress of 0.32 dyn/cm.sup.2. a) and b):
time interval is 5 seconds. c) and d): time interval is 60 seconds.
Note that MCF-7 cells were stationary adhered on anti-EpCAM coated
region but very slowly moving on the surface (>3 .mu.m/min).
[0058] FIG. 12 are images of HL-60 and DsRED-transfected MCF-7
cells (red cells) on a) P-selectin, b) E-selectin, c) anti-EpCAM,
and d) patterned E-selectin/anti-EpCAM coated surfaces, under shear
stress of 0.32 dyn/cm.sup.2. The patterned surface with E-selectin
and anti-EpCAM (d) achieved efficient isolation of
DsRED-transfected MCF-7 (a CTC model) cells from the mixture with
HL-60 (a leukocyte model), on the anti-EpCAM coated region.
[0059] FIG. 13 are graphs illustrating the a) number of captured
cells and b) capturing efficiencies of the surfaces immobilized
with the mixtures of anti-EpCAM and E-selectin at various
composition ratios (c) and d)). The number of DsRED-MCF-7 cells on
each surface was counted and the capturing efficiency was
calculated based on the total number of the MCF-7 cells injected
into the flow chamber. The flow experiments were performed at the
shear stress of 0.16 dyn/cm2. The average capturing efficiency of
the surfaces with mixture of E-selectin and anti-EpCAM were
generally higher than those with anti-EpCAM only. With an increase
of added concentrations of E-selectin, the capturing efficiency of
the surfaces was further enhanced. Error bars: standard error.
[0060] FIG. 14 is a fluorescent image of a micropatterned
fluorescently labeled albumin formed by plasma ablation.
[0061] FIG. 15 is a schematic illustration of immobilization of
anti-EpCAM via polymeric nanolinkers. Anti-EpCAM will be conjugated
with G7 PAMAM dendrimer, followed by covalent immobilization on
PEGylated surface. The local concentration of anti-EpCAM will be
substantially increased by dendrimer that is flexible enough to be
deformed to allow a maximal number of binding events to occur
simultaneously in a small area.
[0062] FIG. 16 is a schematic illustration of conjugation between
polyester-64-carboxylate-1-alkyne dendron (dendron) and anti-EpCAM
and immobilization of the conjugate to the surface through a PEG
linker. Anti-EpCAM will be first conjugated with G6 polyester
dendron, followed by covalent immobilization on PEGylated surface.
The terminal carboxylic acid groups will provide reactive sites for
anti-EpCAM and the alkyne group in the core will allow controlled
immobilization on the PEGylated surface through click chemistry
(i.e. only one PEG chain will be conjugated to the dendron
conjugate). Note that G3 dendron is used in the figure for better
illustration but G6 dendron that has 64 carboxylate groups will be
actually employed.
[0063] FIG. 17 is a graph illustrating the ability of anti-EpCAM-
and E-selectin-functionalized microfluidic devices to capture
circulating tumor cells. The mixer device induces a rotational flow
through the channel while the control device does not. The mixer
device with both anti-EpCAM- and E-selectin-functionalized surfaces
captures circulating tumor cells significantly more efficiently
than the mixer device with only anti-EpCAM or the corresponding
control devices.
DETAILED DESCRIPTION OF THE INVENTION
[0064] It must be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise.
[0065] In one aspect, a device for capturing circulating tumor
cells (CTCs) from a sample includes a channel that includes a cell
capture surface 12 and a flow modification surface 20. The cell
capture surface 12 includes a cell rolling-inducing agent and a
capturing agent 18. The flow modification surface 20 includes one
or more structures arranged to induce a rotational flow in the a
sample flowing through the channel. Referring to FIG. 4, a method
for capturing CTCs from a sample, in one aspect, includes
introducing the sample into the device 10 under conditions that
allow a CTC to bind to the cell-rolling-inducing agent and a
capturing agent 18. The flow modification surface 20 induces a
rotational flow in the sample, which may allow for enhanced contact
of the cells with the cell capture surface 12, and, thus, for more
efficient CTC capture.
[0066] The methods of the invention provide for high throughput
separation of biological samples in a physiological range of flow
rates from about 200 to 500 .mu.L/min. In some embodiments, a shear
stress of between 0.05 dyn/cm.sup.2 and 10 dyn/cm.sup.2 is applied
to the sample introduced into the microfluidic device 10. In some
embodiments, a shear stress of between 0.1 dyn/cm.sup.2 and 2
dyn/cm.sup.2 is applied to the sample introduced into the
microfluidic device 10. In some embodiments, the shear stress is
about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about
0.30, about 0.35, about 0.40, about 0.45, about 0.50, about 0.55,
about 0.60, about 0.65, about 0.70, about 0.75, about 0.80, about
0.85, about 0.90, about 0.95, about 1.0, about 1.1, about 1.2,
about 1.3, about 1.4, about, 1.5, about 1.6, about 1.7, about 1.8,
about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4,
about, 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0,
about 3.1, about 3.2, about 3.3, about 3.4, about, 3.5, about 3.6,
about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2,
about 4.3, about 4.4, about, 4.5, about 4.6, about 4.7, about 4.8,
about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4,
about, 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0,
about 6.1, about 6.2, about 6.3, about 6.4, about, 6.5, about 6.6,
about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2,
about 7.3, about 7.4, about, 7.5, about 7.6, about 7.7, about 7.8,
about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4,
about, 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0,
about 9.1, about 9.2, about 9.3, about 9.4, about, 9.5, about 9.6,
about 9.7, about 9.8, about 9.9, about 10.0 dyn/cm.sup.2. In some
embodiments, the shear stress is about 0.16 dyn/cm.sup.2.
I. Microfluidic Device
[0067] Referring to FIG. 1, the device 10 includes a channel having
a cell capture surface 12 and a flow modification surface 20. The
cell capture surface 12 can be disposed opposite the flow
modification surface 20. For example, the cell capture surface 12
can be disposed on the bottom surface of the channel and the flow
modification surface 20 can be disposed on the top surface of the
channel, opposite the cell capture surface 12. Alternatively, the
flow modification can be disposed adjacent to the cell capture
surface 12. In yet another embodiment, the cell capture surface 12
and the flow modification surface 20 can be incorporated into a
single surface. The channel can be, for example, a closed channel
having four walls. The cell capture surface 12 and/or the flow
modification surface 20 can be disposed on multiple walls of the
channel.
[0068] The channel can have any suitable cross-sectional shape. For
example, the channel can be rectangular, triangular, circular, or
elliptical. The dimension of the microfluidic device 10 can be
optimized to maximum fluid rotation while minimizing fluid
resistance using the following equation:
R = 12 .mu. L wh 3 ##EQU00001##
where .mu. is the kinematic viscosity, L is the channel length, w
is the channel width, and h is the channel height.
[0069] For example, the channel can have a height of about 50 .mu.m
to about 600 .mu.m, about 100 .mu.m to about 500 .mu.m, about 200
.mu.m to about 400 .mu.m. Other suitable heights include, for
example, about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,
550, or 600 .mu.m. The channel can have a width of about 200 .mu.m
to about 2000 .mu.m, about 400 .mu.m to about 1500 .mu.m, about 500
.mu.m to about 1000 .mu.m, or about 600 .mu.m to about 800 .mu.m.
Other suitable widths include, for example, about 200, 300, 400,
500, 600, 700, 800, 900, 1000, 1100, 1200, 1500, 1600, 1700, 1800,
1900, or 2000 .mu.m. The channel can have a length of about 200
.mu.m to about 5000 .mu.m, about 400 .mu.m to about 4000 .mu.m,
about 600 .mu.m to about 2000 .mu.m, or about 800 .mu.m to about
1000 .mu.m. Other suitable lengths include, for example, about 200,
300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,
3500, 4000, 4500, or 5000 .mu.m.
[0070] The cell capture surface 12 includes a cell-rolling inducing
agent 16 and a capturing agent 18 attached to a substrate 14. The
substrate 14 can be, for example, glass, plastics (or
polymer-coated), hydrogels, matrigel, or extracellular matrix
(ECM)-coated substrates. The cell-rolling inducing agent 16 and the
capturing agent 18 can be immobilized on the substrate 14 either
directly or indirectly, using, for example a linker. The
cell-rolling inducing agent 16 and the capturing agent 18 can be
arranged uniformly across the cell capture surface 12. For example,
as illustrated in FIG. 1, the cell capture surface 12 can include
alternating regions having the cell-rolling inducing agent 16 and
the capture agent. The alternating regions can have substantially
the same widths or the widths can vary among the regions. The
regions including the cell rolling-inducing agent and the capturing
agent 18 can be arranged, for example, as parallel to or at angles
relative to the direction of the flow through the channel. For
example, the regions can be arranged tangentially to the direction
of the flow through the channel.
[0071] Flow modification surfaces are well known in the art. Any
known flow modification surface 20 can be used. For example, the
flow modification surface 20 can include one or more ridges 22,
extending from the surface into the channel. The ridges 22 are
shaped, sized, and oriented so as to induce a rotational flow in a
sample flowing through the channel. The cell capture surface 12 and
the flow modification surface 20 can be included on a single
surface of the device, for example, by coating the flow
modification surface with the cell-rolling inducing agent 16 and
the capture agent 18. For example, the ridges 22 can be coated with
the cell-rolling inducing agent 16 and the capture agent 18. All or
portions of the ridges 22 can be coated. For example, the side
walls of the ridges 22 can be coated with the cell-rolling inducing
agent 16 and the capture agent 18. The induction of rotational flow
in the sample can enhance cell capture efficiency. Cells having low
diffusivity will have a tendency to remain the region of the
channel at which they enter. For example, hematologic cells have an
inherently low diffusivity due to their large diameter. This
detrimentally affects the cell capture process when the cells enter
the channel distant from the cell capture surface 12. For example,
if a blood cell enters the microfluidic channel near the top, it
will likely remain near the top as it travels several centimeters
along a microchannel, limiting interaction of the cells with
biofunctionalized substrates located at the bottom of the channel.
The induction of a rotational flow in the sample will force the
cells towards the cell capture surface 12, thereby enhancing the
contact between the cells and the cell capture surface 12.
[0072] The ridges 22 can have any suitable cross sectional shape,
such as, for example, rectangular, circular, elliptical, or
triangular. The ridges 22 can have a thickness t of about 10 .mu.m
to about 300 .mu.m, about 50 .mu.m to about 300 .mu.m, about 100
.mu.m to about 250 .mu.m, or about 150 .mu.m to about 200 .mu.m.
Other suitable thicknesses t include about 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175,
200, 225, 250, 275, or 300 .mu.m. The ridges 22 can have a width w
of about 50 .mu.m to about 300 .mu.m, about 100 .mu.m to about 250
.mu.m, or about 150 .mu.m to about 200 .mu.m. Other suitable widths
w include about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or
300 .mu.m. The distance between adjacent ridges 22 can be about 50
.mu.m to about 500 .mu.m, about 100 .mu.m to about 400 .mu.m, or
about 200 .mu.m to about 300 .mu.m. Other suitable distances
include about 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500
.mu.m. The distance between adjacent ridges 22 can be substantially
uniform across the flow modification surface 20 or can vary.
[0073] The ridges 22 can be substantially linear, extending, for
example, in a direction perpendicular to the flow. As shown in FIG.
2, the ridges 22 can have a herringbone structure. The ridges 22
can be angled relative to the direction of flow F. For example, the
ridges 22 can be angled perpendicularly or obliquely relative to
the direction of flow F. In one embodiment, the ridges 22 are at a
45.degree. angle relative to the direction of flow F. Other
suitable angles include about 45.degree., 50.degree., 55.degree.,
60.degree., 65.degree., 70.degree., 75.degree., 80.degree.,
85.degree., 90.degree., 100.degree., 110.degree., 120.degree.,
130.degree., 140.degree., 150.degree., 160.degree. and 170.degree..
The one or more ridges 22 can be angled uniformly. Alternatively,
the angle of the ridges 22 can vary across the channel to induce
different rotational properties to a sample flowing through the
channel.
[0074] The one or more ridges 22 can be arranged in a pattern. For
example, the flow modification surface 20 can include first regions
having the one or more ridges 22 and second regions that are devoid
of ridges 22. Alternatively, the second regions can include ridges
22 that are oriented, sized, and/or shaped differently than the
ridges 22 of the first regions. The flow modification surface 20
can further include third regions that are completely devoid of
ridges 22. Any suitable number of regions having differently sized,
oriented, and/or shaped ridges 22 can be included on the flow
modification surface 20. The first and second regions can
alternate, for example, uniformly across the flow modification
surface 20.
[0075] The microfluidic device 10 can be fabricated by preparing
the cell capture surface 12 with regions having the cell
rolling-inducing agent and regions having the capturing agent 18. A
microfluidic channel having a flow modification surface 20 can then
be attached to the cell capture surface 12.
[0076] The cell capture surface 12 can be formed by patterning the
cell rolling-inducing agent and the capturing agent 18 on a
substrate 14, for example, a glass slide. Any known method of
forming regions of a cell rolling-inducing agent and a capturing
agent 18 can be used to form the cell capture surface 12. Referring
to FIG. 3, the cell rolling-inducing agent and the capturing agent
18 can be patterned, for example, using a polymer stencil, for
example, a PDMS stencil. The stencil can be formed as is known in
the art, for example, using photolithography. A photoresist can be
coated on a wafer and selectively exposed using a photomask. The
photoresist is then developed, resulting in unexposed portions of
the photoresist being removed, thereby forming a negative mold for
the stencil. A polymer, such as PDMS, can then be poured onto the
negative mold and cured, thereby resulting in the stencil. The
stencil includes one or more structures protruding from a first
surface. The size, orientation, and shape of the protruding
structures are substantially the same as the size, orientation, and
shape of the desired regions of the cell rolling inducing agent and
the capturing agent 18. When placed on a substrate 14, the
protruding structures function to mask portions of the substrate
14. The cell rolling-inducing agent or the capturing agent 18 can
be attached to the unmasked portions of the substrate 14. The
stencil can then be removed and the exposed portions of the
substrate 14 can be filled with the cell rolling-inducing agent or
the capturing agent 18, thereby forming the cell capture surface
12.
[0077] The cell rolling-inducing agent and the capturing agent 18
can be attached to the substrate 14 using, for example,
physisorption or plasma ablation. For example, the agents can be
attached using microfluidic adsorption in which the desired agent
is placed in a soluble media and injected through a microfluidic
channel placed onto the substrate 14. The solution is allowed to
adsorb to the surface over several hours. This technique is
advantageous when the desired agents are sensitive to or damaged by
heat.
[0078] Methods are well known in the art for preparing surfaces
with different densities and patterns of suitable groups for
covalent bonding (e.g., see Rusmini et al., 8 Biomacromolecules
1775-89 (June 2007) and Leckband et al., 37 Biotechnology and
Bioengineering 227-237 (1991), the entire contents of both of which
are incorporated herein by reference). In some embodiments, the
density of a capturing agent 18 and/or a cell rolling-inducing
agent ranges from about 10 ng/cm.sup.2 to about 600 ng/cm.sup.2. In
some embodiments, the density of a capturing agent 18 and/or a cell
rolling-inducing agent is greater than about 30 ng/cm.sup.2. For
example, in some embodiments, the density of a capturing agent 18
and/or a cell rolling-inducing agent ranges from about 30
ng/cm.sup.2 to about 360 ng/cm.sup.2. In some embodiments, the
density of a capturing agent 18 and/or a cell rolling-inducing
agent ranges from about 50 ng/cm.sup.2 to about 300 ng/cm.sup.2. In
some embodiments, the density of a capturing agent 18 and/or a cell
rolling-inducing agent ranges from about 100 ng/cm.sup.2 to about
200 ng/cm.sup.2.
[0079] The channel and the flow modification surface 20 can be
formed as is known in the art. See Stroock et al., 295 Science
647-51, the disclosure of which is incorporated herein by reference
in its entirety. The flow modification surface 20 can be formed
using soft-lithography. For example, the flow modification surface
20 can be formed using a photoresist, such as a dual height SU-8
photoresist mold. The mold is prepared by first spinning and
patterning the microfluidic channel. Before developing the channel
pattern, a second photoresist layer is spun onto the mold to
generate a pattern for the ridges 22 of the flow modification
surface 20. Alignment markers may be added to facilitate proper
orientation of the second photoresist layer. The mold is then
exposed, hard baked, and developed, thereby resulting in a mold
that contains a channel with structures that will cast the ridges
22 of the flow modification surface 20. The resulting mold is then
coated with a polymer, for example, PDMS, to form the channel and
the flow modification surface 20.
II. Cell Rolling
[0080] In some aspects of the disclosure, the microfluidic device
10 comprises an immobilized cell rolling-inducing agent. The
formation of transient ligand-receptor interactions occurs commonly
between cells flowing in the blood and the vascular endothelium;
this physiological process is known as cell rolling. Cell rolling
is known to play a key role in biologically important processes
such as recruitment of leukocytes to sites of inflammation, homing
of hematopoietic progenitor cells after intravenous injection, and
CTC-induced metastasis. This behavior is typically mediated by
dynamic interactions between selectins (e.g., E-, P-, L-selectins)
on the vascular endothelial cell surface and membrane proteins
including P-selectin glycoprotein ligand-1 (PSGL-1). A person of
skill in the art will recognize that any molecule capable of
inducing CTCs to undergo cell rolling can be used to practice the
disclosed methods and prepare the disclosed devices.
[0081] In some embodiments of the disclosure, the cell
rolling-inducing agent is a selectin. In another embodiment, the
selectin is endothelial (E)-selectin. In yet another embodiment,
the selectin is P-selectin. In another embodiment, the selectin is
L-selectin. Moreover, fragments of selectins which retain the
ability to bind CTCs are specifically envisioned to be within the
scope of the disclosure.
[0082] E-selectin (CD62E) is particularly noteworthy in disease by
virtue of its expression on activated endothelium and on bone-skin
microvascular linings and for its role in cell rolling, cell
signaling, and chemotaxis. Many studies point to the key role
played by E-selectin in being involved in the adhesion and homing
of various types of cancer cells such as prostate, breast, and
colon carcinoma cells. E-selectin is synthesized de novo by
endothelial cells in response to inflammatory cytokines, such as
interleukin-1.beta. (IL-1.beta.) and tumor necrosis factor-.alpha.
(TNF-.alpha.). Thus, cell separation based on the cell rolling
behavior is being exploited as it mimics physiological processes
and eliminates labeling and label removal steps that are necessary
for other immune-labeling detection methods. However, given that a
large class of cells, including leukocytes, platelets, neutrophils,
mesenchymal and hematopoietic stem cells, and metastatic cancer
cells, exhibits rolling on selectins, rolling-based detection for
specific cell types from cell mixtures or whole blood has
limitations to achieve sufficient specificity, which has hindered
translation of the technology to a clinically significant device
10. The methods and devices of the disclosure overcome this
limitation by coupling the cell rolling-inducing agent with an
immobilized capturing agent 18. Without intending to be bound by
any particular theory, it is believed that the cell
rolling-inducing agent causes a circulating tumor cell (CTC) to
exhibit the "rolling" behavior (described above) on a surface of
the microfluidic device 10. The rolling CTC then contacts the
immobilized capturing agent 18, and is thereby captured (i.e.
immobilized) by the device 10.
[0083] Any covalent chemistry may be used to immobilize cell
rolling-inducing agents to a surface of the microfluidic device 10.
In some embodiments, cell rolling-inducing agents are attached to a
surface through one or more chemical moieties. In general, the bond
between the chemical moiety and the surface is covalent. Without
limitation, in some embodiments, the chemical moiety comprises an
epoxy group, a carboxyl group, a thiol group, an alkyne group, an
azide group, a maleimide group, a vinyl group, a hydroxyl group, an
amine group, an aldehyde group, and combinations thereof.
[0084] In some embodiments, the cell-rolling inducing agent 16 is
immobilized to a surface of the microfluidic device 10 via a
linker. In some embodiments, the linker is selected from the group
consisting of a dendrimer, a dendron, a dextran, polyethylene
glycol, poly(L-lysine), poly(L-glutamic acid), polyvinyl alcohol,
polyethylenimine, poly(lactic acid), poly(glycolic acid), and
combinations thereof.
III. Cell Capture
[0085] A. Capturing Agent
[0086] In some aspects of the disclosure, the microfluidic device
10 comprises an immobilized capturing agent 18. A person of skill
in the art will appreciate that any molecule capable of selectively
binding to circulating tumor cells will be useful as a capturing
agent 18. Specific examples of such molecules exhibiting selective
binding to circulating tumor cells include antibodies (or antibody
fragments), folic acid, transferrin, certain peptides, and
aptamers. Examples of antibodies include, but are not limited to,
Trastuzumab (Herceptin), Bevacizumab (Avastin), anti-CD33 antibody
(Mylotarg), anti-CD20 antibodies (Zevalin and Bexxar), and their
fragments and engineered forms (e.g. diabody, avimer, etc.).
Examples of peptides include, but are not limited to, RGD and
NGR
[0087] Epithelial cell adhesion molecule (EpCAM) is frequently
overexpressed by a variety of carcinomas such as lung, colorectal,
breast, prostate, head and neck, and hepatic origin, but is absent
from hematologic cells. Thus, to allow specific binding (i.e.
"capturing") of CTCs while avoiding binding of non-CTC cells, in
some embodiments, the capturing agent 18 is an anti-EpCAM antibody.
Anti-EpCAM antibody is commercially available from several sources
including, for example, R&D Systems, Abcam, and Millipore.
Alternatively, anti-EpCAM antibodies useful for practicing the
methods of the disclosure or generating the devices of the
disclosure can be generated by any method known in the art.
[0088] As used herein, the terms "antibody" and "immunoglobulin"
are understood to mean (i) an intact antibody (for example, a
monoclonal antibody or polyclonal antibody), (ii) antigen binding
portions thereof, including, for example, an Fab fragment, an Fab'
fragment, an (Fab').sub.2 fragment, an Fv fragment, a single chain
antibody binding site, an sFv, (iii) bi-specific antibodies and
antigen binding portions thereof, and (iv) multi-specific
antibodies and antigen binding portions thereof.
[0089] As used herein, the terms "bind specifically," "specifically
bind" and "specific binding" are understood to mean that the
antibody has a selective binding affinity for a particular antigen
of at least about 10.sup.6 M.sup.-1, more preferably, at least
about 10.sup.7 M.sup.-1, more preferably at least about 10.sup.8
M.sup.-1, and most preferably at least about 10.sup.10 M.sup.-1.
Appropriate controls can be used to distinguish between "specific"
and "non-specific" binding.
[0090] In some embodiments, the capturing agent 18 is transferrin.
Transferrin is an iron binding transport protein, which can bind
two atoms of ferric iron in association with the binding of an
anion, for example, bicarbonate. Transferrin is responsible for the
transport of iron from sites of absorption and heme degradation to
those of storage and utilization. The transferrin receptor (TfR) is
known to be overexpressed in a broad range of cancers, making
transferrin useful as a capturing agent 18.
[0091] In some embodiments, the capturing agent 18 is an RGD
peptide, a cRGD peptide, RGD mimetics, peptides or proteins
containing the RGD sequence, structural or functional equivalents
thereof, or combinations thereof. The RGD or RGD mimetics described
herein include any peptides or peptide mimetics resulting from the
modification of the cyclic Arg-Gly-Asp peptide. The modification
can be on the pendant groups and/or on the backbone of the peptide.
Peptide synthesis, including the synthesis of peptide mimetics, is
well documented and can be readily achieved via, for example,
combinatorial chemistry.
[0092] In some embodiments, the capturing agent 18 is folic acid.
Folic acid is known to bind to a tumor-associated antigen known as
the folate receptor (FR), making folic acid useful as a capturing
agent 18.
[0093] B. Multivalent Effect
[0094] Multivalent interactions--the simultaneous binding event of
multiple ligands to multiple receptors in biological systems--have
been extensively investigated to promote targeting of specific cell
types. These activities are also central to a number of
pathological processes, including the attachment of viral,
parasitic, mycoplasmal, and bacterial pathogens. Studies with
biological multivalent inhibitors have yielded quantitative
measurements of binding avidities, with increases on the order of 1
to 9 orders of magnitude.
[0095] In some aspects of the disclosure, the multivalent effect is
accomplished by immobilizing the capturing agent 18 on the
substrate 14 of the cell capture surface 12 via attachment to a
linker, which is directly attached to the substrate 14 of the cell
capture surface 12. In some embodiments, the linker is a polymeric
nanolinker. In some embodiments, the polymeric nanolinker is a
modified poly(amidoamine) (PAMAM) dendrimer.
[0096] The nanolinker may be a dendritic polymer. Any of the known
dendritic architectures may be used, including, for example,
dendrimers, tecto-dendrimers, regular dendrons, dendrigrafts, and
hyperbranched polymers. Dendritic star-branched polymers having a
plurality of arms emanating from a nucleus may also be used.
Accordingly, as used herein, dendritic polymers are polymers with
densely branched structures having a large number of terminal
reactive groups. A dendritic polymer includes several layers or
generations of repeating units, usually referred to as branch
cells, which all contain one or more branch points. Dendritic
polymers, including dendrimers and hyperbranched polymers, are
prepared by reaction of monomeric units having two or more reactive
groups, or a combination of monomeric units in which at least one
of the monomeric units has at least three reactive groups. The
dendrimers which can be used include those comprised of a plurality
of dendrons that emanate from a common core which can be a single
atom or a group of atoms. Each dendron generally consists of
terminal surface groups, interior branch junctures having branching
functionalities greater than or equal to two, and divalent
connectors that covalently connect neighboring branching
junctures.
[0097] Methods of preparing and characterizing dendrimers,
dendrons, hyperbranched polymers, star-branched polymers, dense
star-branched polymers and hypercomb-branched polymers are all well
known in the art and thoroughly described in the literature.
Dendrons are regular-branched polymeric molecules and their
structures can be precisely controlled at the molecular level and
they have unique properties. They are wedge-shaped and comprise a
focal point from which the branches originate. Different dendrons
may have different numbers of branches extending from each branch
and different numbers of layers. In some embodiments of the
disclosure, the polymeric nanolinker comprises
polyester-n-carboxylate-1-alkyne dendron covalently attached to
polyethylene glycol, wherein n is 8, 16, 32, 64, or 128.
[0098] Specific examples of dendritic polymers that may be used
include poly(amidoamine) (PAMAM) dendrimers, dendrigrafts and
hyperbranched polymers; poly(benzylether) dendrimers, dendrigrafts
and hyperbranched polymers; polyester dendrimers and hyperbranched
polymers; poly(propyleneimine) (PPI) dendrimers, dendrigrafts and
hyperbranched polymers; organo silicon-containing dendrimers,
dendrigrafts and hyperbranched polymers, polystyrene arborescent
polymers.
[0099] PAMAM dendrimers have been reported to be an excellent
mediator for facilitated multivalent effect because the geometry of
the dendrimer preorganizes the ligands into a small region of space
as compared to what is obtained if one conjugates the ligands to a
similar molecular weight linear polymer. Thus, one has "prepaid"
the entropy penalty for localizing the ligands. Second, the
dendrimer structure allows all ligands to address the cell surface.
This is not necessarily the case for a similar molecular weight
hyperbranched polymer in which tangled or cross-linked chains may
prevent the needed ligand orientation. PAMAM dendrimers are quite
flexible and easily deform from the spherical shape adopted in
isotropic media to a disc-like structure upon interaction with a
surface. This combination of preorganization, polymer backbone
topology, and easy deformability, makes the PAMAM dendrimer an
effective material for achieving multivalent binding to cell
surfaces. Furthermore, the multivalent effect can significantly
increase specificity and sensitivity of detection of target
proteins or cells. By immobilizing PAMAM dendrimers conjugated with
cancer cell specific markers such as anti-EpCAM, specificity and
sensitivity of the surface is substantially increased by the
multivalent effect.
[0100] In some embodiments, the PAMAM dendrimer is covalently
attached to polyethylene glycol.
[0101] In some embodiments, the PAMAM dendrimer is selected from
the group consisting of a generation 3 PAMAM dendrimer, a
generation 4 PAMAM dendrimer, a generation 5 PAMAM dendrimer, a
generation 6 PAMAM dendrimer, a generation 7 PAMAM dendrimer, a
generation 8 PAMAM dendrimer, and a generation 9 PAMAM
dendrimer.
EXAMPLES
[0102] The following examples are provided for illustration and are
not in any way to limit the scope of the invention.
Example 1
Multivalent Effect
[0103] The recent development of nanotechnology has demonstrated
many breakthroughs in a range of biomedical
applications--particularly for cancer treatment. For a new design
of effective targeted drug delivery/imaging vectors based on
nanotechnology, multivalent effects are desirable as they
dramatically enhance active targeting efficacy. The similar
enhancement can be also achieved in specific capturing when the
delivery vectors are immobilized on the surfaces.
[0104] Preparation of PAMAM dendrimer-based nanodevice: the PAMAM
dendrimer-based folate receptor (FAR) targeting nanodevices were
synthesized as summarized in FIG. 3. Briefly, G5 PAMAM dendrimers
were partially acetylated (70 of the 110 total primary amines),
resulting in G5-Ac70. The remaining 40 primary amine groups were
used for reaction to further functionalize the dendrimers. Note
that a G5 PAMAM dendrimer molecule has approximately 110 primary
amine termini according to the previous titration measurement. To
fluorescently label the dendrimers, AlexaFluor.RTM. 488 (AF488,
Molecular Probes) dissolved in DMSO was added to the dendrimer/H 2
0 solution at a molar ratio of 5:1 (AF488:dendrimer) in the
presence of 1 M NaHCO 3 and the reaction mixture was stirred at RT
for 48 hr. The resulting mixture of the dendrimer conjugate
(G5-Ac.sub.70-AF488) was then dialyzed in water for 2 days and
lyophilized for 2 days, followed by 10 cycles of ultrafiltration
with PBS (with Ca.sup.2+ and Mg.sup.2+) and water using a 10,000
molecular weight cut-off membrane at 21.degree. C., 5000 rpm for 30
min each. G5-Ac 70-AF488 conjugate in H.sub.2O was then reacted
with FA preactivated by
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide/HCI (EDC) in
DMF/DMSO at different molar ratios (3:1, 6:1, 9:1, 12:1, 15:1) of
FA to G5-Ac.sub.70-AF488. The same purification process was carried
out as described in the AF488 conjugation. Lastly, full acetylation
of the remaining primary amine group was completed, yielding our
final products G5-Ac-AF488-FA.sub.0, G5-Ac-AF488-FA.sub.2.6,
G5-Ac-AF488-FA.sub.4.7, G5-Ac-AF488-FA.sub.7.2,
G5-Ac-AF488-FA.sub.11.5, and G5-Ac-AF488-FA.sub.13.7. Since all the
nanodevices were conjugated with the same number of AF488,
differences in fluorescence intensities from the nanodevices in
later FACS data (FIG. 4a-red squares) can be regarded as a result
of differences in nanodevice binding and/or uptake by KB cells. The
nanodevices become more polydisperse and ultimately give a bimodal
distribution as the number of attached FA increases. In the case of
G5-Ac-AF488-FA.sub.13.7, the polydispersity index
(PDI=M.sub.w/M.sub.n) is 12.41 which is significantly greater than
PDIs of previously reported dendrimer conjugates.
[0105] Quantitative analysis of multivalent effect mediated by
PAMAM dendrimers: The material properties required for maximal
multivalent effects include: 1) flexibility for conformational
deformation to increase interacting surface area at a low cost of
entropy and 2) localized reactive groups for targeting agents to
utilize receptor clustering effect for a maximal number of
simultaneous binding events in the given area. A series of
experiments to quantitatively measure the multivalent targeting has
been conducted using PAMAM dendrimers that satisfy the two
pre-requisite properties, yielding a substantial enhancement in
binding avidity as high as .about.170,000 fold compared to the
monovalent binding counterpart. To study the interaction of
FA-conjugated G5 PAMAM-based nanodevices (G5-Ac-AF488-FAx: x=2.6,
4.7, 7.2, 11.5, or 13.7) with folate binding protein (FBP), the
surface plasmon resonance (SPR) technique using BIAcore X
(Pharmacia Biosensor AB, Uppsala, Sweden) was employed. FBP was
immobilized on the sensor chip surface (channel 2) of a
carboxylated dextran-coated gold film (CM 5 sensor chip) by amine
coupling as described. The dendritic nanodevices (30 .mu.l) were
injected at concentrations of 500 nM, 1 mM, and 2 mM at a flow rate
of 10 .mu.l/min, allowing the nanodevices to flow in both channels
(channel 1 for reference and channel 2 with FBP) for 3 min. The
final SPR sensorgrams were obtained from the signals from channel 2
subtracted by those from channel 1. Binding parameters of free FA
with FBP were evaluated by the same condition but at different
concentrations (1 and 2 mM used for free FA). The binding curves
were fit using the 1:1 Langmuir binding model in BIAevaluation
software. Associations and dissociations were fit separately since
there was turbulence in the curves between association and
dissociation phases in the process of subtracting signals from the
reference channel. Dissociation constants (K.sub.D) for each
dendrimer were obtained by averaging at least three different sets
of results which had .chi..sup.2 values lower than 3.0. All runs
were independently analyzed for errors associated with mass
transport by exporting the data files to Excel and plotting dR/dt
versus R following the analysis described by Glaser.
[0106] To compare the SPR results to in vitro cell level data, the
KB cell line (ATCC, Manassas, Va.) was employed and grown
continuously as a monolayer at 37.degree. C. and 5% CO.sub.2 in
RPMI 1640 medium (Mediatech, Herndon, Va.) supplemented with
penicillin (100 units/ml), streptomycin (100 mg/ml), and 10%
heat-inactivated fetal bovine calf serum (FBS) before use. KB cells
were also cultured in RPMI 1640 medium without folic acid
(Mediatech) for at least 4 days before experiments, resulting in
the folic acid receptor overexpressing KB (FAR.sup.+ KB) cell line.
For the FACS measurements, the FAR-KB cells were seeded on a
24-well plate for tissue culture at a concentration of
2.times.10.sup.5 cells/well and at 37.degree. C., 5% CO2 for 24 hr.
The cells were then incubated with the series of the prepared
nanodevices at 37.degree. C. for 1 hr. After removal of
supernatants, cells were trypsinized and collected into FACS tubes,
followed by centrifugation at 1500 rpm for 5 min to obtain cell
pellets. The pellets were washed with PBS (Ca.sup.2+, Mg.sup.2+)
twice using a repetitive centrifugation and resuspension process
and then finally resuspended in PBS with 0.1% bovine serum albumin.
The FACS sample preparation was performed on ice to inhibit
cellular reactions such as further uptake. Fluorescence signal
intensities from the samples were measured using a Coulter EPICS/XL
MCL Beckman-Coulter flow cytometer, and data were analyzed using
Expo32 software (Beckman-Coulter, Miami, Fla.).
[0107] As shown in FIG. 4a, an optimum number of targeting
molecules (folic acid (FA)) appeared to be .about.5 where the
dendrimers showed an exponential increase in binding avidity and
sustained their monodispersed properties. Note that conjugation of
more than 10 FA molecules caused substantial deterioration of
homogeneity of the materials. Based on this optimized design
criteria, engineered dendritic anti-cancer nanodevices utilizing
the multivalency have exhibited great efficacy in targeting and
killing cancer cells both in vitro and in vivo without apparent
harmful side effects. This study supports the idea that
nanoparticle based drug delivery systems can be significantly
improved in targeting efficacy if the optimization process is
conducted to maximize the multivalent effect without compromising
the material's properties.
[0108] The significance of these results regarding the multivalent
effect is three-fold: 1) the ability of PAMAM dendrimer-based
scaffolds to afford a functional multivalent effector system is
demonstrated 2) the in vivo effect is demonstrated to arise from
the substantial enhancement of K.sub.D, not an increased rate of
endocytosis and 3) the on-rate, k.sub.a, increases linearly with
the number of targeting agents and shows no cooperativity whereas
the off-rate, k.sub.d, decreases exponentially with the number of
targeting agents (4b).
[0109] Although the experiments on the multivalent effect are
focused on the targeted drug delivery, the results of enhanced
binding avidity as high as 170,000 fold clearly indicates that this
naturally occurring effect can substantially enhance device
sensitivity that is highly desirable for detection and isolation of
extremely rare cells such as CTCs. Furthermore, the multivalent
effect (exponential increase in binding avidity) appeared to be
primarily due to the exponential decrease in dissociation rate
constants (FIG. 4b), indicating that capturing efficiency of the
multivalent capturing device will be enhanced as the CTCs will
likely remain adhered on the surface even at high flow rates
(>200 .mu.L/min).
Example 2
Controlled Immobilization of P-Selectin
[0110] Covalent immobilization of biologically active species has a
number of advantages such as controlling the density, conformation,
and enhanced stability of the species. Although covalent
immobilization procedures for peptides and enzymes have been
extensively studied for decades, covalent immobilization of large
molecular weight biomolecules such as selectins present significant
challenges due to the increase of binding to non-specific sites and
due to the requirement for mild processing conditions to prevent
protein inactivation. Given that preparation of devices proposed in
this work requires a high level of control over the selectin
presentation on surfaces, it is desirable to control density and
conformation of selectin, and to introduce controlled
co-immobilization capacity for secondary molecules that facilitates
selective separation of target CTCs. We have developed covalent
immobilization chemistries along with a set of appropriate
analytical tool in order to achieve stable and tunable adhesive
properties of surfaces with minimal batch-to-batch variations.
[0111] Enhanced controllability of surfaces by covalent
immobilization: Previously, we have shown the surfaces with
covalently immobilized P-selectin present enhanced functional
stability by approximately 10 times, as compared to those with
physisorbed P-selectin. The surface stability was assessed using
cell-mimicking microspheres as well as live neutrophils. We also
developed immobilization chemistries (as shown FIG. 5) on gold
coated, non-foulding PEGylated surfaces to employ the flow-based
SPR technique that quantitatively analyze the surface functions,
offering: 1) easy surface functionalization using thiol chemistries
due to the presence of a gold layer and 2) quantitative and real
time monitoring of binding events without any modification of
analytes. Thus, the SPR technique is useful particularly for
determining controllability of density and orientation of
P-selectin. The chemistries described in FIG. 5 were developed to
achieve non-fouling surfaces and to provide reactive sites for
subsequent P-selectin immobilization using oligo(ethylene
glycol)-alkanethiols (Prochimia, Poland) on gold coated SPR chips.
Briefly, the gold chip surface was cleaned before the subsequent
formation of mixed self assembled monolayers (SAMs) by washing with
absolute ethanol and drying by nitrogen blowing. SAMs were formed
by soaking gold coated substrates in a solution containing a 100
.mu.M total OEG-alkanethiol concentration in ethanol at RT
overnight. Following mixtures of different OEG-alkanethiols were
used at the indicated molar ratios: OEG-COOH (or --NH 2):OEG-OH
(1:39, 1:9, 3:7, 5:5) and OEG-biotin:OEG-OH (1:9). All the SAMs
were then rinsed extensively with water and ethanol, followed by
drying in a stream of nitrogen. All the buffers and solutions were
degassed under vacuum for 30 min before applied into the SPR
system. P-selectin was immobilized onto the surface of the mixed
SAMs as follows. The chemistry used for the mixed SAMs of
OEG-COOH/OEG-OH (FIG. 5a), 10 mM phosphate buffer (PB) was first
flowed into a chip at a flow rate of 50 .mu.t/min for 5 min. A 1:1
(v/v) mixture of EDC at 76.68 mg/mL and NHS at 11.51 mg/mL was
injected to activate carboxyl groups on the SAMs for 10 min. After
flowing for 5 min, P-selectin at a concentration of 20 .mu.g/mL in
PB was injected and flowed to be immobilized for 7 min. The chip
surface was then washed with PB for 5 min, followed by ethanolamine
(100 mM in PB) to inactivate remaining active ester groups and to
remove loosely bound P-selectin from the surface. For P-selectin
immobilization on the mixed SAM of OEG-biotin/OEG-OH (FIG. 5b),
P-selectin was first biotinylated using maleimide-PEO.sub.2-biotin
(Pierce) before the SPR measurement as shown in FIG. 5. A solution
of P-selectin at 50 .mu.l of 1 mg/ml P-selectin in PBS was mixed
with 50 molar excess maleimide-PEG 2-biotin solution at 4.degree.
C. overnight. The reaction mixture was purified by 4 cycles of
ultrafiltration using a 10K molecular weight cut-off membrane. Each
cycle was performed at 14,000.times.g for 30 min. The mixed SAM of
OEG-biotin/OEG-OH was mounted on the SPR and 10 .mu.g/mL
streptavidin in PBS was flowed for 10 min to create binding sites
for the biotinylated P-selectin. P-selectin was then immobilized
under the same condition used for other mixed SAM surfaces via
strong biotin/avidin binding. For the mixed SAM of
OEG-NH.sub.2:OEG-OH (FIG. 5c), the surface was first immersed in a
solution of sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce,
Rockford, Ill.) at room temperature for 1 hr to convert the amine
groups to maleimide groups that specifically binds to a cysteine
residue in P-selectin. The chip was mounted on the SPR sensor and
PB and PBS were sequentially flowed into the channels. P-selectin
was immobilized under the same conditions.
[0112] FIGS. 6a and b show that the amount of P-selectin was
successfully controlled by varying mixture ratios of the SAM
components. The amount of immobilized P-selectin was proportional
to the amount of --COOH (or --NH.sub.2) containing SAM component in
a linear fashion. This results also suggest that the OEG based SAMs
reduce non-specific adsorption of P-selectin because only the
OEG-COOH or --SH groups on SMCC provides reactive sites for
--NH.sub.2 or --SH residues in P-selectin, respectively.
Orientation effect of P-selectin was also examined by comparing the
two different chemistries in FIG. 5a (random conformation) and FIG.
5b (oriented conformation). Because a P-selectin molecule has many
amine groups that can react with --COOH groups on the surface,
conformation of P-selectin ought to be random. In contrast,
P-selectin is known to possess only one cysteine as its 766.sup.th
amino acid (P-selectin used in this study is composed of 1-771
amino acids of its natural form) on the other side of active
binding sites at N terminal. The pre-biotinylation step involves a
chemical reaction between the thiol group on P-selectin and the
maleimide group on the linker and thus there should be only one
biotin per P-selectin molecule, resulting in oriented conformation
of P-selectin immobilized through following biotin-streptavidin
binding. For channels prepared using the both chemistries,
comparable amounts of P-selectin were first immobilized (-12 nm in
wavelength shift), followed by flowing 20 .mu.g/mL P-selectin
antibody (eBioscience) at a flow rate of 20 .mu.L/min. As a result,
the channels with oriented P-selectin exhibit a significantly
greater binding response than that from the channels with randomly
immobilized P-selectin (FIG. 6c), indicating that orientation of
P-selectin was controlled by utilizing thioether chemistry.
Example 3
Rolling Assays of a Tumor Cell Line as a CTC Model
[0113] Patterning of E-selectin on a glass substrate: Recombinant
human E-selectin chimera (R&D systems, Minneapolis, Minn.) was
patterned on an epoxy functionalized glass surface
(SuperEpoxy.RTM., ArrayIt Inc, Sunnyvale, Calif.) using a silicone
gasket to block a part of the glass substrate during E-selectin
immobilization, resulting in the clear interface between E-selectin
coated and uncoated regions as shown in FIG. 9 (the yellow dotted
lines). An 1.5 cm.times.6 cm silicone gasket was placed on a
SuperEpoxy.RTM. glass slide, along with a small piece (0.3
cm.times.1 cm) of silicone in the center of the slide. The larger
gasket was filled with PBS to rinse the surface, followed by
incubation with 5 .mu.g/mL E-selectin at RT overnight. The slide
was then rinsed with PBS, the small piece was removed, and the
entire surface was blocked with 1% BSA solution.
[0114] Cell rolling of tumor cells on E-selectin: The rolling
response of tumor cells on E-selectin coated surfaces was assessed
using a commercially available rectangular parallel-plate flow
chamber (Glycotech, Gaithersburg, Md.). A breast cancer cell line
MCF-7 (ATCC, Manassas, Va.) was employed as a CTC model. The
rolling behavior of the MCF-7 cells was compared with that of HL-60
cells, a human myeloid cell line that expresses high levels of
sialyl Lewisx and exhibits rolling on selectins mediated primarily
by PSGL-1. With no expression of PSGL-1, MCF-7 cells express CD24
that interacts with selectins and that has been known to be a
marker for a subpopulation of MCF-7 cells with higher potential to
cause metastasis than CD24-MCF-7 cells. For the flow experiments,
the flow chamber with a gasket with thickness of 250 .mu.m and
length of 6 cm was placed on the E-selectin coated surface. HL-60
and MCF-7 cells at a 2-8.times.105 cells/mL concentration were
perfused into the chamber at shear stresses of 0.08 and 0.32
dyn/cm2 using a syringe pump (New Era Pump Systems, Inc., Wantagh,
N.Y.). During each experiment, flow was interrupted for 1 min,
followed by image recording for 2 min at 1 fps image capturing. The
average velocities were obtained by averaging rolling velocities of
at least 40 cells.
[0115] FIG. 9 shows video frames of HL-60 and MCF-7 cells on an
E-selectin coated surface at t=0 and 10 sec. The both HL-60 and
MCF-7 cells exhibited the typical rolling behavior that was
specific to E-selectin-conjugated region. No cell adhesion was
observed on the regions that were not functionalized with
E-selectin. MCF-7 cells exhibited faster rolling velocity (4.23
.mu.m/s) as compared to HL-60 cells (2.12 .mu.m/s). Note that the
free flow on the wall should be 80 .mu.m/s at 0.32 dyn/cm2,
indicating that the speed of rolling cells was significantly
reduced, as compared to non-interacting cells. Furthermore, it was
observed that not all MCF-7 cells showed rolling. This is likely
due to the CD24- subpopulation, indicating that this technology can
also be used as a separation tool for the two subpopulations (CD24+
and CD24-) of the cells without modification steps, such as cell
labeling.
[0116] The rolling velocities of HL-60 and MCF-7 cells were
measured at 4 different flow rates and corresponding shear stresses
as plotted in FIGS. 10A and 10B, respectively. Note that the
rolling velocity of MCF-7 cells was significantly increased with an
increase of the flow rate (shear stress) whereas the rolling
response of HL-60 cells was not as dependent upon the flow rate
change.
Example 4
Tumor Cell Specific Capturing Using Anti-EpCAM
[0117] Surface preparation and the flow chamber experiments: The
anti-EpCAM coated surfaces were prepared using a similar protocol
that is described above. Instead of E-selectin, 5 .mu.g/mL of
anti-EpCAM was incubated on the Epoxy group functionalized glass
slides (SuperEpoxy.RTM., ArrayIt Inc, Sunnyvale, Calif.).
[0118] As shown in FIG. 11, MCF-7 cells were captured on the
anti-EpCAM coated region. HL-60 cells (used as a leukocyte model)
did not interact with the surface functionalized with anti-EpCAM
(data not shown).
Example 5
Enhanced Separation of Cancer Cells Using Combination of Anti-EpCAM
and E-Selectin
[0119] To demonstrate the enhancement of separation efficiency of
tumor cells through mimicking the naturally occurring process of
cell rolling and multivalent effects, mixture of the two cell lines
under flow was observed on the various surfaces functionalized by
P-selectin, E-selectin, anti-EpCAM, and E-selectin/anti-EpCAM
combination (FIG. 12). For easier recognition from the cell
mixture, MCF-7 cells were transfected using HIV-1-based lentiviral
vector and Discosoma sp. Red fluorescent protein
(DsRED)-transfected MCF-7 (DsRED-MCF-7) cells were isolated from
non-transfected cells prior to the flow chamber experiments.
P-selectin (FIG. 12a) induced rolling of HL-60 but no interaction
with DsRED-MCF-7 cells was observed. As expected from FIG. 5,
E-selectin, on the other hand, caused both cell types to roll as
presented in FIG. 12b. The anti-EpCAM coated surface induced
stationary adhesion of DsRED-MCF-7 cells exclusively (FIG. 12c).
The combination of E-selectin and anti-EpCAM provided separation of
pure MCF-7 population from HL-60 and MCF-7 mixture on the
anti-EpCAM immobilized region (the right-hand side) as shown in
FIG. 12d. This result indicates that iterative rolling and
stationary binding using combination of E-selectin and anti-EpCAM
may enhance the separation capability of the surface, as compared
to the surfaces functionalized with one of the two proteins
alone.
Example 6
Enhanced Capturing Efficiency of Anti-EpCAM-Functionalized Surfaces
with Addition of E-Selectin
[0120] The enhanced separation efficiency observed in FIG. 12 was
further supported by a quantitative analysis of capturing
efficiency of the various surfaces. The surfaces were prepared as
follows: the mixtures of anti-EpCAM and E-selectin at different
composition ratios were prepared using the protocols described
earlier. After placing a home-made gasket made of
polydimethylsiloxane (PDMS) (one panel: 10 mm (L).times.25 mm (W))
on the epoxy-functionalized glass substrate, 300 .mu.l of mixtures
were added and incubated for 4 hrs at RT. DsRED-MCF-7 cells were
used again to easily distinguish the cells of interest from cell
debris. Number of captured cells was counted using a microscope
(Olympus 1.times.70 inverted microscope) at each cycle, which was
composed of forward flow (from left to right) for 2.5 min, and
backward flow (from right to left) for 2.5 min at a flow rate of
100 .mu.l/min that is correspondent to a shear stress of 0.16
dyn/cm.sup.2. As the fixed number of DsRED-MCF-7 cells were
perfused into the flow chamber, the number of the captured cells
could be translated into the capturing efficiency (%).
[0121] FIGS. 13a and 13b demonstrate enhanced capturing efficiency
with the surface immobilized with mixture (anti
EpCAM:E-selectin=1:1) as compared to the surface with anti EpCAM
only. Furthermore, as shown in FIGS. 13c and 13d, the average
number of captured cells and average capturing efficiency of the
surfaces with the two proteins was enhanced as the concentration of
E-selectin was increased. Taken together, the combination of
E-selectin, which induces rolling of various cell types, and
anti-EpCAM, which recognizes/captures tumor cells, greatly enhances
the capturing of the cells, most likely because E-selectin-induced
tumor cell rolling maximizes the chance of the tumor cells
interacting with anti-EpCAM on the surface, resulting in the
enhanced capturing efficiency. The isolation efficiency of the
surface is further enhanced by incorporating microfluidic channels
that induce rotation of flow, which is described below.
Example 7
Generating Defined Microdomains
[0122] Plasma Ablation: A simple 100 .mu.m wide line of Albumin was
micropatterned onto a glass substrate using plasma ablation.
Briefly, fluorescently labeled albumin was adsorbed onto a
coverglass substrate at a pH of 7.4 for two hours. After this, a
PDMS etch mask was placed onto the dried surface with PDMS covering
regions 100 .mu.m wide, spaced every 1000 .mu.m. After exposure to
an oxygen plasma at 100 W for 2 minutes, the mask was removed and
the substrate was imaged as shown in FIG. 14. This technique can be
extended to micropatterning domains of selectins and anti-EpCAM by
first patterning the lines of anti-EpCAM similar to how the albumin
was patterned, followed by backfilling with E-selectin to adsorb to
the remaining surfaces. Additionally, plasma ablation will work to
pattern covalently immobilized ligands in a similar matter as the
oxygen plasma is very reactive and will oxidize and effectively
burn up any organic molecule in contact with the plasma. For work
in this project, either physisorption or plasma ablation is used,
however it is important to note both require stencils and are
similar techniques.
[0123] Microfluidic Adsorption: Micropatterns of EpCAM and
E-Selectin can also be generated by using a microfluidic device 10
that can be placed onto a glass substrate. The desired surface
molecules placed in soluble media and are injected through the
microfluidic channels. Then the injection is stopped and the
solution is allowed to adsorb to the surface over several hours.
After this, the microfluidic network is peeled off and the desired
micropattern is achieved. This technique is advantageous when the
desired molecules are damaged by the heat generated during plasma
ablation.
Example 8
Immobilization of Anti-EpCAM Through Polymeric Nanolinkers
[0124] The surface is functionalized with anti-EpCAM coated domains
via a polymeric nanolinker. The nanolinkers are composed of PAMAM
dendrimers and PEG, dendron and PEG, and other linear polymers. An
example of the immobilization process of anti-EpCAM is outlined in
FIG. 15. For an enhanced multivalent effect, G7 PAMAM dendrimers
(Sigma-Aldrich, St. Louis, Mo.) were partially acetylated and
conjugated with multiple number of anti-EpCAM molecules, which
increases the local concentration of anti-EpCAM that facilitates
the multivalent binding between the surface and cells. The
resulting dendrimer-anti-EpCAM conjugates were immobilized on the
PEGylated surface through amide bond formation. A variety of
parameters can be tested such as PEG chain length, size of
dendrimer, number of conjugated anti-EpCAM, and degree of
acetylation, in order to determine an optimal condition for various
purposes.
[0125] In addition to the use of a PAMAM dendrimer as a component
of the nanolinker, polyester-64-carboxylate-1-alkyne dendron
(dendron) can also be used. Dendron has an advantage as it can use
click chemistry for immobilization on the PEGylated surface and use
amine (or carboxyl) based chemistries for conjugation with
anti-EpCAM (and other biomarkers specific to cancer).
Example 9
Fabrication of a Biomimetic Microfluidic Chip
[0126] The microfluidic device 10 was fabricated in two steps.
First, the cell capture surface 12 was prepared forming
microdomains of the cell rolling-inducing agent and the capturing
agent 18 on a glass substrate 14. Anti-EpCAM-dendrimer conjugates
for CTC binding and was used by the capturing agent 18. The
Anti-EpCAM was surrounded by E-Selectin, which was used as the cell
rolling-inducing agent. Following this, a PDMS based microfluidic
channel have a flow modification disposed on the ceiling was
attached to the micropatterned substrate.
[0127] The capturing agent 18 was fabricated using a PDMS stencil.
The PDMS stencil was formed using previously described and are well
established procedures. Briefly, the microchannel layout was
designed in AutoCAD and printed onto a high resolution (5080 dpi)
transparency. This transparency was used as a photomask to
selectively crosslink a photoresist which was spin coated onto a
silicon wafer at a desired thickness (the spin velocity and time
dictate the thickness). Following exposure, the unexposed,
uncrosslinked photoresist was washed away, resulting in a negative
mold of the desired device 10 structure. Next, PDMS is poured on to
the negative mold and cured. Following curing, the PDMS stencil is
peeled from the mold master and ready for use. As shown in FIG. 3,
the stencil contains a micropatterned surface with features
protruding from the surface, such that when the features contact
the substrate 14, regions of the substrate 14 covered by the PDMS
features will be masked.
[0128] The PDMS stencil was applied to a glass substrate 14 to mask
regions of the substrate 14. The capturing agent 18, Anti-EpCAM,
was applied to the unmasked regions of the substrate 14. The PDMS
stencil was then removed and the substrate 14 was backfilled with
the cell rolling-inducing agent E-Selectin, thereby attaching the
E-Selectin to the exposed portions of the substrate 14.
[0129] Flow rotation in the microfluidic device 10 is induced by
integrating a flow modification surface 20 into the microfluidic
channel. The flow modification surface 20 included a herringbone
structure. Standard soft lithographic techniques like those used to
fabricating the stencil for micropatterning the
dendrimer-anti-EpCAM were used to form the herringbone structure
flow modification surface 20. A dual height SU-8 photoresist was
used to form the mold for the flow modification surface 20. This
dual height SU8 mold was prepared by first spinning and patterning
the microfluidic channel. Before the pattern was developed a second
photoresist layer was spun onto the surface to generate a pattern
for the herringbone shaped ridges 22 the flow modification surface
20. Alignment marks were added to the designs to facilitate proper
orientation of the second photoresist layer. Following selective
exposure and a second hard bake the entire wafer was developed and
the resulting mold contained both the microchannel and structures
for casting the ridges 22 of the flow modification surface 20. PDMS
is then cast onto the mold to form a microchannel having a flow
modification surface 20. The resulting microfluidic channel was
then attached to the cell capture surface 12.
[0130] The dimensions of the microchannel and the flow modification
surface 20 can be optimized to maximize fluid rotation while
minimizing the fluidic resistance which is governed by the
following equation:
R = 12 .mu. L wh 3 ##EQU00002##
[0131] Where .mu. is the kinematic viscosity, L is the channel
length, w is the channel width, and h is the channel height. In
addition to the height and width of the microchannel, the
dimensions of the herringbone ridges 22 can be optimized. Previous
studies showed an optimal dimension of the herringbone ridges 22 to
be a height h1 of 85 .mu.m, a width w of 200 .mu.m, a .theta. of 60
degrees, placement of the ridges 2/3 from one side. The ridges 22
have a thickness t of about 15 .mu.m. Increasing rotations can
increase the contact of cells to the cell capture surface 12.
Example 10
Enhanced CTC Capture Efficiency is Provided by Microfluidic Devices
that Induce Rotational Flow
[0132] Polydimethylsiloxane and glass microfluidic devices were
fabricated using soft-lithography. The glass bottom was coated with
either adsorbed anti-EpCAM or a mixture of adsorbed anti-EpCAM and
E-selectin, creating a cell capture surface 12. Control devices
consisted of ten rectangular channels 45 mm long, 730 .mu.m wide
and 120 .mu.m high. Additionally, microfluidic devices had 120
.mu.m height slanted ridged ceiling structures as the flow
modification surface 20. The flow modification surface 20 included
360 .mu.m wide grooves and 110 .mu.m wide ridges 22 aligned at 45
degree angles to the channel wall. These ceiling structures induce
downward flows which carry cells downward toward the capture
surface. These microfluidic devices, designed to isolate carcinoma
cells from blood samples, were tested with a suspension of
MDA-MB-468 breast adenocarcinoma cells in buffer solution. Cell
suspensions were injected into the device 10 at a constant flow
rate using a syringe pump with flow rates that corresponded to a
shear stress of 0.5 dyn/cm.sup.2 on the control channel floor. Five
minutes after flow into the device 10 was started, a 865
.mu.m.times.660 .mu.m section of the device 10 was imaged at one
frame per second for one minute. The total number of captured
stationary cells and total cells in bulk flow were counted. To
estimate capture efficiency, the total numbers of captured cells
were divided by the number of cells in bulk flow entering the
device 10 over one minute. The cell capture was then normalized to
the maximum capture efficiency. The mean data for a total of three
trials is shown in FIG. 17. The results indicate that the
microfluidic device 10 having the flow modification surface 20 had
significantly improved capture efficiency over the control device
10, and that the protein mixture devices had more cell capture than
the devices with only anti-EpCAM immobilized on the capture
surface.
* * * * *