U.S. patent application number 15/730658 was filed with the patent office on 2018-02-22 for construct for promoting absorption of molecules by a cell and methods of using the contstruct.
This patent application is currently assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH (A*STAR). The applicant listed for this patent is AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH (A*STAR), NATIONAL UNIVERSITY OF SINGAPORE. Invention is credited to Seok Hong GOH, Tanu Suryadi KUSTANDI, Hong Yee LOW, Kim Kiat TEO, King Fai Evelyn YIM.
Application Number | 20180051107 15/730658 |
Document ID | / |
Family ID | 48014639 |
Filed Date | 2018-02-22 |
United States Patent
Application |
20180051107 |
Kind Code |
A1 |
YIM; King Fai Evelyn ; et
al. |
February 22, 2018 |
CONSTRUCT FOR PROMOTING ABSORPTION OF MOLECULES BY A CELL AND
METHODS OF USING THE CONTSTRUCT
Abstract
The present disclosure is directed to a construct for promoting
absorption of molecules by a cell and the application thereof in
drug and gene delivery. The present disclosure further describes
topographical modulation of endocytosis for drug and gene
delivery.
Inventors: |
YIM; King Fai Evelyn;
(Singapore, SG) ; LOW; Hong Yee; (Singapore,
SG) ; KUSTANDI; Tanu Suryadi; (Singapore, SG)
; TEO; Kim Kiat; (Singapore, SG) ; GOH; Seok
Hong; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH (A*STAR)
NATIONAL UNIVERSITY OF SINGAPORE |
Singapore
Singapore |
|
SG
SG |
|
|
Assignee: |
AGENCY FOR SCIENCE, TECHNOLOGY AND
RESEARCH (A*STAR)
Singapore
SG
NATIONAL UNIVERSITY OF SINGAPORE
Singapore
SG
|
Family ID: |
48014639 |
Appl. No.: |
15/730658 |
Filed: |
October 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13602616 |
Sep 4, 2012 |
9815921 |
|
|
15730658 |
|
|
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61529969 |
Sep 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2533/30 20130101;
C08F 112/08 20130101; C12N 2535/00 20130101; C12N 5/0068 20130101;
G03F 7/0002 20130101; Y10T 428/24355 20150115 |
International
Class: |
C08F 112/08 20060101
C08F112/08; C12N 5/00 20060101 C12N005/00; G03F 7/00 20060101
G03F007/00 |
Claims
1. A method of promoting absorption of molecules by cells, wherein
the method comprises: providing a construct comprising a plurality
of micro and/or nanoscale protrusions located at the surface of the
construct; wherein the protrusions have a size and are spaced apart
from each other at a distance that promotes absorption of molecules
by said cell; and seeding and culturing at least one cell at the
surface of the construct under conditions suitable for absorption
of molecules by the cells.
2. The method of claim 1, wherein the absorption of molecules is
via endocytosis, or pinocytosis, or phagocytosis, or wherein
absorption of the molecules uses a non-viral carrier or a viral
carrier.
3. (canceled)
4. The method of claim 1, wherein the molecules to be absorbed are
selected from the group consisting of nucleic acid, nucleic acid
vectors, siRNA, microRNA, magnetic nanoparticles, gold
nanoparticles, fluorescent nanoparticles, quantum dots, aptamers
(oligonucleic acid or peptide), peptides, growth factors,
therapeutically active substances, biomarkers, colouring agents,
and any one of the aforementioned molecules attached to a
microparticles.
5. A method of cell transfection, or drug-delivery or
high-throughput screening arrays using a construct for promoting
absorption of molecules by a cell located at the surface of the
construct; wherein the construct comprises: a plurality of micro
and/or nanoscale protrusions located at the surface of the
construct; wherein the protrusions have a size and are spaced apart
from each other at a distance that promotes absorption of molecules
by said cell.
6. The method of claim 1, wherein the construct comprises a
plurality of protrusions located at the surface of the construct;
wherein the protrusions are in the form of pillars having a
diameter of between about 200 nm to about 2 .mu.m; wherein the
pitch between the protrusion from edge to edge is between 150 to
300 nm such that the pitch between the protrusions is less than the
size of the cells to be located at the surface of the construct;
wherein the protrusions have a size and are spaced apart from each
other at a distance that promotes absorption of molecules by said
cell and wherein the absorption of molecules is facilitated by
endocytosis, or receptor mediated endocytosis, or pinocytosis, or
phagocytosis; wherein the pillars are collapsed pillars lying at
the surface of the construct; and wherein the collapsed pillars
have a length of between about 50 nm to about 5 .mu.m.
7. The method of claim 1, wherein the protrusions are arranged in
an isotropic pattern.
8. The method of claim 1, wherein the protrusions are arranged in
an anisotropic pattern.
9. The method of claim 1, wherein the protrusions are located at
the surface of the construct in a detachable form.
10. The method of claim 1, wherein the protrusions are located at
the surface of the construct in a non-detachable form.
11. The method of claim 1, wherein the protrusions are round.
12. The method of claim 1, wherein the protrusions are
polygonal.
13. The construct of claim 1, wherein the pillars are extend about
50 nm to 4 .mu.m, or about 100 nm to about 2 .mu.m above the
surface of the construct.
14. The construct of claim 1, wherein the pitch between the pillars
from edge to edge is between 200 nm to 250 nm.
15. The construct of claim 1, wherein a residual layer is arranged
between the pillars and the surface of the construct.
16. The construct of claim 1, wherein the construct is obtained via
nano-imprinting lithography.
17. The construct of claim 16, wherein the nano-imprinting
lithography is thermal nano-imprinting lithography or UV
nano-imprinting lithography.
18. The construct of claim 16, wherein the construct and/or the
protrusions are made of a polymer.
19. The construct of claim 18, wherein the polymer is a synthetic
polymer, or a rigid synthetic polymer, or a bioresorbable polymer,
or a biodegradable polymer.
20. The construct of claim 19, wherein the synthetic polymer is
selected from the group consisting of poly(methyl methacrylate)
(PMMA), polydimethylsiloxane (PDMS), polystyrene (PS) and mixtures
thereof, or wherein the biodegradable polymer is selected from the
group consisting of chitosan, poly(.epsilon.-caprolactone),
polyglycolic acid, poly(lactic acid), polyphosphoester (PPE) and
mixtures thereof, or wherein the rigid synthetic polymer is
polystyrene, optionally the polystyrene is tissue-culture grade
polystyrene (TCPS).
21. The construct of claim 18, wherein during manufacture the
polymer is mixed with molecules which are to be absorbed by the
cell to be located on the construct, optionally wherein the
molecules are attached to the surface of the protrusions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/602,616, filed Sep. 4, 2012, which claims the benefit
of U.S. Provisional Application Ser. No. 61/529,969, filed Sep. 1,
2011, each of which are incorporated herein by reference in their
entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of devices for
supporting growth and modification of biological cells.
BACKGROUND OF THE INVENTION
[0003] The topography of extracellular microenvironment can
influence cellular responses from attachment and migration to
differentiation and production of new tissue. Cells in their
natural environment interact with extracellular matrix that
contains structures in the nanometer scale. Likewise, cells
cultured on surfaces with nanotopography show alteration in their
biological properties with respect to attachment, motility and
proliferation and the like.
[0004] In particular, endocytocytic properties of cells are also
modulated in response to the topography of the extracellular
environment. Recent studies have highlighted the influence of
cell-topography interactions on the modulation of cellular
processes, including protein expression and cytoskeletal behaviors
implicated in endocytosis. Endocytosis plays a key role in
intracellular molecular, drug and gene (or nucleic acid) delivery.
However, topographical control of cell transfectability remains
largely unexplored.
[0005] Delivery of molecules, drugs and genes to a cell can be
classified into viral and non-viral vector delivery. Viral delivery
provides good transfection efficiency. However, the risk of
potential adverse immunological responses has hindered its
development in clinical settings.
[0006] Non-viral delivery has been shown to be safe as it avoids
the complication of using viral components. However, very low
transfection efficiency and non-specific delivery have limited the
practical application of non-viral delivery methods.
[0007] There is therefore a need to provide an improved delivery
technique that overcomes the disadvantages mentioned above to
enhance intracellular delivery of molecules, drugs and nucleic
acids.
SUMMARY OF THE INVENTION
[0008] According to a first aspect, there is provided a construct
for promoting absorption of molecules by a cell located at the
surface of the construct; wherein the construct comprises: [0009] a
plurality of micro and/or nanoscale protrusions located at the
surface of the construct; [0010] wherein the protrusions have a
size and are spaced apart from each other at a distance that
promotes absorption of molecules by said cell.
[0011] According to a second aspect, there is provided a method of
promoting absorption of molecules by cells, wherein the method
comprises: [0012] providing a construct as defined above; [0013]
seeding and culturing at least one cell at the surface of the
construct under conditions suitable for absorption of molecules by
the cells.
[0014] According to a third aspect, there is provided a method of
cell transfection, or drug-delivery or high-throughput screening
arrays using a construct as defined above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The invention will be better understood with reference to
the detailed description when considered in conjunction with the
non-limiting examples and the accompanying drawings, in which:
[0016] FIG. 1 shows the verification of topographical structures on
the substrate. Scanning electron microscopy of poly(methyl
methacrylate) (PMMA) nano- and micro-structures used in this study
shows (A) a 2 .mu.m diameter pillar with 2 .mu.m height (top view),
(B) a 200 nm diameter pillar with 400 nm height (top view), (C) a
250 nm grating with 250 nm height (top view), (D) 2 .mu.m PMMA
pillars with residual layer (cross sectional view), (E) 200 nm
Rhodamine-PS pillar without residual layer (cross sectional view)
and (F) 2 .mu.m Rhodamine-polystyrene collapse pillar (top view).
It can be seen from FIG. 1 that all SEM images showed high fidelity
and dimensions that were in accordance to the initial mold
used.
[0017] FIG. 2 shows magnified images of fluorescently stained
F-actin (red--bright colour) hMSC (A-D), COS7 (E-H) and MCF7 (I-L)
cultured on PMMA control, 2 .mu.m pillars, 200 nm pillars and 250
nm gratings, respectively. It can be seen from FIG. 2 that
actin-dense ring regions coincide with the underlying pillar
topography on the micron sized structures as indicated by the white
arrows while they were also faintly observed in the nanopillar
substrates. It can also be observed that F-actin in 250 nm gratings
were aligned and elongated to the grating axis (indicated by the
double ended arrows). (White bars=10 .mu.m)
[0018] FIG. 3 comprises FIGS. 3A and 3B. FIG. 3 presents the flow
cytometry analysis of FITC-dextran internalization in COS 7
fibroblastic cells cultured on various topographies using two
different FITC-dextran tracers of different molecular weights after
24 hours of incubation time. Low MW refers to 40,000 while high MW
refers to 500,000 in molecular weight. FIG. 3A shows the overall
percentage of fluorescent population of COS 7 on 2 .mu.m pillars,
200 nm pillars, 250 nm gratings and PMMA control, where 1 mg/ml of
FITC-dextran (low MW) was added. (P<0.01 *-vs control 1 mg/ml,
#-vs 200 nm gratings, .dagger.-vs from 200 nm pillars, n=3). FIG.
3B shows the overall percentage of fluorescent population of COS 7
on 2 .mu.m pillars, 200 nm pillars, 250 nm gratings and PMMA
controls, where 1 mg/ml of FITC-dextran (high MW) was added.
(P<0.01, *-vs control 1 mg/ml, #-vs 200 nm gratings, .dagger.-vs
200 nm pillars, n=3). An independent PMMA control with 2 mg/ml of
FITC-dextran was carried out as the positive control in both FIGS.
3A and 3B. It can be seen from FIG. 3 that the observed trend of
dextran intake among the topographical patterns is similar for the
different molecular weights.
[0019] FIG. 4 shows the flow cytometry analysis of FITC-dextran
internalization in MCF 7 breast cancer cells cultured on 2 .mu.m
pillars, 200 nm pillars and 250 nm gratings with PMMA control (2
mg/ml) after 24 hours of incubation time. It can be seen from FIG.
4 that no statistical difference was observed amongst the different
topographies in the breast cancer cell population.
[0020] FIG. 5 comprises FIG. 5A to 5D and shows the flow cytometry
analysis of FITC-dextran internalization in hMSC cultured on 2
.mu.m pillars, 200 nm pillars, 250 nm grating and PMMA control (1
mg/ml). PMMA control (2 mg/ml) represents the positive control for
the experiment. The percentage of fluorescent hMSC population was
analyzed at 18 hours (FIG. 5A), 6 hours (FIG. 5B), 3 hours (FIG.
5C) and 2 hours (FIG. 5D) of incubation time. It can be seen from
FIG. 5 that hMSCs that were cultured on 2 .mu.m pillars after 3
hours of incubation time showed significantly increased dextran
internalization compared to 200 nm pillars, 250 nm gratings and 1
mg/ml PMMA control. (P<0.01 *-vs control 1 mg/ml, #-vs 250 nm
gratings, .dagger.-vs 200 nm pillars, n=3).
[0021] FIG. 6 shows the flow cytometry analysis of hMSCs cultured
on 2 .mu.m pillars, 200 nm pillars, 250 nm grating and blank PMMA
control that were transfected for 3 hr with the GFP plasmid with
the aid of lipofectamine 2000. It can be seen from FIG. 6 that
there is a statistical difference in the percentage of fluorescence
between cells cultured on the 200 nm pillars compared to PMMA
control. (P<0.05, *-vs PMMA control, n=5).
[0022] FIG. 7 shows confocal z-stack fluorescent images of hMSCs
cultured on 200 nm upright pillars without the residual layer after
24 hours. Each successive image represents a 0.1 .mu.m z-step from
the baso-lateral surface to the apical surface of the cell. Cells
are stained for actin filaments using Oregon Green 488 Phalloidin
(green--circles with interrupted line), DAPI (blue--circles with
dotted line) and upright 200 nm pillars are rhodamine tagged
(red--circles with solid line). Arrows indicate internalized
pillars and scale bars represent 50 .mu.m. It can be seen from FIG.
7 that the hMSCs appear to have internalized some of the upright
pillars.
[0023] FIG. 8 shows scanning electron microscope images of COS 7
(A-B), MCF7 (C-D) and hMSCs (E-F) cultured on 200 nm upright
pillars without the residual layer after 24 hours. It can be seen
from FIG. 8 that all cell types show increased filopodia extensions
directed towards the nanopillars on these substrates and appear to
"grab" the pillars towards themselves, detaching the pillars from
the substrate as indicated by arrows seen in E, D and F. It can
also be seen that the large numbers of filopodias long extensions
were particularly noticeable in hMSCs. (Scale bars=5 .mu.m in each
image)
[0024] FIG. 9 shows fluorescent images of human mesenchymal stem
cells (hMSCs), COS7 and MCF7 cells on unpatterned (A, E and I), 2
.mu.m pillars (B, F and J), 200 nm pillars (C, G and K), 250 nm
gratings PMMA substrates (D, H and L) 24 hours after cell seeding
respectively. Cells were stained for F-actin using Alexa Fluor 546
Phalloidin (bright colour) and counterstained with DAPI (circles
with dotted line). Arrows indicate the direction of nanogratings
while white boxes indicate the region magnified for easier
visualization (shown in FIG. 2). (Bars=50 .mu.m). It can be seen
from FIG. 9 that generally cells are more spread out on unpatterned
and pillar substrates as compared to grating substrates where they
exhibit an elongated morphology.
[0025] FIG. 10 shows scanning electron microscopy images of hMSCs
on 200 nm pillars residual free substrates. It can be seen from A
that long filopodia extensions were observed in an attempt by the
cells to grab onto these structures while B shows cortical actin
was highly expressed in these cells on these substrates.
[0026] FIG. 11 shows the confocal Z-stack of COS7 (A to D), MCF7 (E
to H) and hMSC (I to L) cells cultured on PDMS substrates
containing collapsed 2 .mu.m PS pillar structures after 24 hours.
Each successive image represents a 0.3 .mu.m z step from the
baso-lateral surface to the apical surface of the cell. Cells are
stained for actin filaments using Oregon Green 488 Phalloidin
(green-dashed arrows), DAPI (blue-circles with dotted lines) and
collapsed pillar structures are rhodamine-tagged (red-circles with
solid line). Solid arrows indicate pillars that have been
internalized and bars represent 20 .mu.m. It can be seen from FIG.
11 that hMSCs appear to have internalized the larger sized 2 .mu.m
collapsed pillars.
[0027] FIG. 12 shows a schematic diagram of the fabrication process
for collapsed polystyrene (PS) structures. (i) Upright PS pillar
structures were first fabricated using nanoimprinting lithograpy;
(ii) contact initiation between the top surface of PS pillars and
PDMS slab; (iii) breaking of PS upright structures via shearing;
(iv) transferring collapsed PS pillars onto the PDMS slab during
the process of shearing. The collected PS pillars are then used for
subsequent studies.
[0028] FIG. 13 shows the summary of the various substrates used for
baso-lateral phagocytosis study. The top row shows the schematic
diagram of the cross-section of the substrate used while the bottom
row shows the representative images that were discussed relevant to
the corresponding substrates. The substrates that were used were
mainly those of 200 nm pillars with residue (A), 200 nm pillars
residual-free (B) and 2 .mu.m collapsed structures (C). Note the
residual-free topographical structures comparing A and B while in C
the topographical structures were detached from the underlying
material for easy internalization.
DEFINITIONS
[0029] The following words and terms used herein shall have the
meaning indicated:
[0030] The terms "promoting" or "promotion" indicate that uptake of
molecules is possible or is improved or enhanced as determined by
comparison to a control, wherein the control is an unpatterned
surface.
[0031] The term "endocytosis" refers to the process where
eukaryotic cells internalize segments of plasma membrane,
cell-surface receptors and other essential soluble components such
as nutrients from the extracellular fluid. Different regions of the
plasma membrane of polarized cells, such as epithelial and
endothelial cells, exhibit different biochemistry and endocytic
mechanisms. Endocytosis activity at the basal-lateral surface (side
interacting with the basement membrane) may be distinct from that
at the apical surface. The endocytic pathway, surface receptor
expression and functions may be different in the different regions
of plasma membrane and in various cell types.
[0032] Endocytosis mechanisms may include but are not limited to
receptor mediated endocytosis, pinocytosis and phagocytosis.
[0033] Examples of receptor mediated endocytosis may include but
are not limited to caveolae-mediated endocytosis and
clathrin-mediated endocytosis.
[0034] Caveolae-mediated endocytosis is defined by the involvement
of flask-shape pits in the membrane known as caveolae to uptake
extracellular molecules.
[0035] Clathrin-mediated endocytosis is defined by the involvement
of vesicles that have a coat made up of a complex of proteins that
are mainly associated with the cytosolic protein clathrin.
Clathrin-mediated endocytosis is involved in Lipofectamine 2000
aided transfection.
[0036] "Pinocytosis" refers to the non-receptor mediated process
where cells can uptake large volumes of extracellular fluids and
materials. Pinocytosis of large volumes is also known as
macropinocytosis. Macropinosomes can be >1 .mu.m in diameter.
Pinocytosis uses a mechanism which involves active formation of
plasma membrane ruffles and protrusions. Macropinocytosis is one of
the processes involved in FITC-dextran internalization.
[0037] Phagocytosis may refer to the process by which cells bind
and internalize large particulate matter (>1 .mu.m).
[0038] The term "microscale" is to be interpreted to include any
dimensions that are in the range of about 1 .mu.m to about 1000
.mu.m.
[0039] The term "nanoscale" or "submicron" is to be interpreted to
include any dimensions that are below 1 .mu.m.
[0040] The term "protrusion" is to be broadly interpreted as any
topographical formation on a surface extending away from the
surface and/or above the surface.
[0041] The terms "isotropic" and "anisotropic" refer to uniform and
non-uniform arrangements of the protrusions respectively. Uniform
arrangement may include an ordered array of protrusions or other
topographical structures. Non-uniform arrangement may include a
disordered or random array of protrusions or other topographical
structures. Uniform may also refer to the height, width, diameter,
pitch or shape of the protrusions.
[0042] The term "pillar" refers to a substantially vertical
structure extending from the surface of the construct.
[0043] The term "grating" refers to a series of parallel disposed
grooves or slit formations on the surface of a solid surface having
dimensions in the micro and/or nanoscale range.
[0044] The term "biodegradable" refers to the ability to be broken
down by biological means.
[0045] The term "tissue culture grade" refers to a substrate
suitable for culturing cells and tissues in vitro.
[0046] The term "basolateral cells" refer to cells that interact
with the basal membrane. Examples include but are not limited to
acinar cells, lacrimal acinar cells, gastrointestinal epithelial
cells, skin keratinocytes and retinal pigment epithelium.
[0047] The term "apical cells" refer to cells that are located at
the opposite pole of a biological structure relative to the basal
membrane. Apical cells generally refer to cells that interact with
a lumen of a biological structure for example, the intestine and
blood vessels. Examples of apical cells include but are not limited
to apical corneal epithelial cells and cells of the intestinal
villi.
[0048] The present application does not only enable promoting
absorption of molecules by basolateral and apical cells located or
growing on the construct of the present invention but all types of
cells. Thus, not only polarized cells, such as basolateral cells,
can be used but also cells which are not polarized. Cells which are
not polarized, upon attachment to the extracellular matrix or a
substrate in vitro, the side or the area of the cell surface
interacting with the substrate would be defined as "basal"
membrane. Therefore, the application is not limited only to polar
cells or basolateral cells. For example, fibroblasts, which are not
polarized cells, have been shown in the experiments to have
interaction with the nano-imprinted substrate; the cell surface
that interacted with the imprinted substrate would be referred to
as "basal" or "basolateral"; and the internalization through the
cell-substrate interacting surface would be referred to as
"basolateral internalization" or "basolateral endocytosis".
[0049] As used herein, the term "nucleic acid" means any single or
double-stranded RNA or DNA molecule, such as mRNA, cDNA, genomic
DNA and xeno DNA.
[0050] The term "nucleic acid vector" refers to a nucleic acid
molecule that is used to introduce foreign genetic material into a
target cell.
[0051] The term "siRNA" refers to small interfering ribonucleic
acids (RNA) or RNA analogs comprising between about 10 to 50 or 10
to 30 nucleotides (or nucleotide analogs) capable of directing or
mediating the RNA interference pathway. These molecules can vary in
length and can contain varying degrees of complementarity to their
target messenger RNA (mRNA). The term "siRNA" includes duplexes of
two separate strands, i.e. double stranded RNA, as well as single
strands that can form hairpin structures comprising of a duplex
region.
[0052] The term "miRNA" refers to micro RNA. miRNA is generally a
single stranded molecule that averages about 20 nucleic acids.
[0053] The term "nanoparticles" refers to particles comprising
nanoscale or submicron features.
[0054] The term "microparticles" refers to particles comprising
microscale features.
[0055] The term "quantum dots" refer to tiny particles of a
semiconductor material, traditionally chalcogenides (selenides or
sulfides) of metals like cadmium or zinc (CdSe or ZnS, for
example), which range from 2 to 10 nanometers in diameter.
[0056] The term "aptamer" refers to nucleic acids or peptides
having a desirable action on a target. A desirable action includes,
but is not limited to, binding of the target, catalytically
changing the target, reacting with the target in a way which
modifies/alters the target or the functional activity of the
target, covalently attaching to the target as in a suicide
inhibitor, facilitating the reaction between the target and another
molecule.
[0057] An aptamer may be a nucleic acid aptamer or a peptide
aptamer. A nucleic acid aptamer refers to a nucleic acid that binds
a target molecule through a mechanism which predominantly depends
on Watson/Crick base pairing or triple helix binding, wherein the
aptamer does not have the known physiological function of binding
the target molecule. A peptide aptamer refers to combinatorial
recognition molecules that consist of a constant scaffold protein,
typically thioredoxin (TrxA) which contains a constrained variable
peptide loop inserted at the active site.
[0058] The term "growth factor" refers to a substance that is
capable of stimulating cellular growth, proliferation or cellular
differentiation. Growth factors typically act as signaling
molecules between cells. Examples of growth factors include but are
not limited to bone morphogenic protein, fibroblast growth factor
and vascular endothelial growth factor.
[0059] The term "high-throughput screening" refers to experimental
methods that involve rapid collection of large amounts of data.
High throughput assays employ robotics, data processing and control
software, liquid handling devices, and sensitive detectors to
generate and process data.
[0060] The term "attached" as used herein refers to the binding of
a molecule onto a surface via chemical bonding.
[0061] The term "immobilized" as used herein refers to the adhesion
of a molecule onto a surface wherein the adhesion does not involve
chemical bonding.
[0062] The term "transformation" or "transformed", as used herein,
refers to the genetic alteration of a cell resulting from the
uptake, incorporation and expression of exogenous genetic material
(for example exogenous DNA).
[0063] The term "biocompatible" as used herein refers to the
property of a material that does not cause adverse biological
reactions to the human or animal body.
[0064] The terms "nanoimprinting lithography" or "NIL" refers to
nano-fabrication that allows the production of nano- to
micron-scale features with complex structures on a wide range of
materials.
[0065] The term "thermal NIL" refers to a hot embossing process
wherein the pattern is frozen-in once the material is cooled
down.
[0066] The term "UV-NIL" refers to imprinting that uses UV-curable
resin, where the resin is polymerized/cured in-situ during the
imprint process.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0067] Exemplary, non-limiting embodiments of a construct for
promoting absorption of molecules by a cell located at the surface
of the construct will now be disclosed. In one embodiment, the
construct comprises: [0068] a plurality of micro and/or nanoscale
protrusions located at the surface of the construct; [0069] wherein
the protrusions have a size and are spaced apart from each other at
a distance that promotes absorption of molecules by said cell.
[0070] In one embodiment, the absorption of molecules is
facilitated by endocytosis, or receptor mediated endocytosis, or
pinocytosis, or phagocytosis.
[0071] In one embodiment, the protrusions are arranged in an
isotropic or anisotropic pattern.
[0072] In one embodiment, the isotropic pattern may be a uniform or
ordered array of protrusions.
[0073] Also, the maximum number of protrusions, such as pillars is
only limited by the sample area. For example, if the sample area is
2 cm.times.2 cm, and the pitch of 200 nm pillar pattern is 400 nm,
the total number of pillar per row will be 2 cm/400 nm, which will
be 50000. Therefore, it could be 50000.times.50000. A typical cell
culture area can be ranged from 0.5 cm.sup.2 to 150 cm.sup.2, Thus,
it can be range up to .about.500,000.times..about.500,000.
Generally, the size and shape of the sample area of the construct
thus depends on the application and the platform on which this
construct is used. For example, the construct can be part of a
device for high-throughput screening. Such a device can comprise
multiple constructs. The multiple constructs can be either used for
locating the same or different cell types on its surface.
[0074] In a further embodiment, the protrusions may have a density
of 2 or more, or from 2 to 10, from 2 to 20, from 2 to 30, from 2
to 40, from 2 to 50, from 2 to 60, from 2 to 70, from 2 to 80, from
2 to 90 or from 2 to 100 protrusions in a defined area. Generally,
the density will depend on the pitch size and sample area size. For
example, for 1 mm.times.1 mm sample, using the 400 nm pitch as an
example, it will be 50000. Thus, it could also range from 2 to
50000.
[0075] In one embodiment, the protrusions are located at the
surface of the construct in a detachable or non-detachable form.
Located in a detachable form means that the protrusions are not
fixed to the surface. For example, in one embodiment protrusions,
such as pillars are imprinted into the surface of the construct
base material as described herein. After formation of the construct
and the protrusions, such as pillars, the protrusions are
disconnected from the surface of the construct by shearing. One
specific example of this general process is illustrated in FIG. 12.
This detaching allows for example to transfer the protrusions made
of one material to a surface made of another material.
[0076] In one embodiment, the protrusions are pillars in the form
of micropillars or nanopillars or in the form of a grating.
[0077] In one embodiment, the protrusions are cylindrical or
polygonal. The polygonal protrusions may be triangular (3-sided),
rectangular (4-sided), square (4-sided), pentagonal (5-sided),
hexagonal (6-sided), 7-sided, 8-sided, 9-sided, 10-sided, 11-sided,
12-sided, 13-sided, 14-sided or 15-sided. The polygon may be
equilateral or non-equilateral.
[0078] In one embodiment, the diameter or maximal width of the
pillars may be in the nanoscale or microscale. The diameter of the
pillars may be selected from the group consisting of between 10 nm
to 5 .mu.m, between 50 nm to 4 .mu.m, between 200 nm to 2 .mu.m,
about 200 nm, about 300 nm, about 400 nm and about 500 nm.
[0079] In one embodiment, the diameter or maximal width of the
pillars may be between about 200 nm to about 2 .mu.m.
[0080] The aspect ratio (ratio between the width and height of the
protrusion) may or may not be significant. For example, for PMMA,
because of its high modulus, the aspect ratio of the topography
does not play a significant role. However, when involving a low
modulus polymer, the aspect ratio can affect the effective elastic
modulus experience by the cells and altered its endocytosis uptake.
A suitable range depends on the polymer use as it is linked to the
fabrication limitation of the material. In some embodiments, an
aspect ratio of 1:2 (width:height) was used for the 200 nm pillars
and 1:1 was used for the 2 .mu.m pillar and 250 nm grating. Thus,
the aspect ratio can be between about 1:1 to 1:10 or between about
1:1 to 1:5 or between about 1:1 to 1:3 or it can be 1:1, 1:2, 1:3,
1:4 or 1:5.
[0081] In one embodiment, the gratings may have a width of between
about 10 nm to about 2 .mu.m, or between about 100 nm to about 1.5
.mu.m.
[0082] In one embodiment, the pillars are extending substantially
perpendicular from the surface of the construct or wherein the
pillars are collapsed pillars lying at the surface of the
construct; wherein in case the pillars are collapsed pillars they
have a length of between about 50 nm to about 5 .mu.m.
[0083] In one embodiment, the pillars of the grating are extending
between about 50 nm to 4 .mu.m, or between about 100 nm to about 2
.mu.m above the surface of the construct.
[0084] In one embodiment the protrusions may be spaced apart from
each other by between 200 nm to 12 .mu.m, or 400 to 10 .mu.m, or 5
nm to about 1 .mu.m. In other words the "pitch" is equal to the
center to center of 2 adjacent protrusions, such as pillars or
gratings.
[0085] In one embodiment, the protrusions are micropillars and the
pitch between the micropillars from an edge of one micropillar to
an edge of another micropillar may be between 1 to 10 .mu.m, or
about 9.5 .mu.m.
[0086] In one embodiment, the protrusions are nanopillars and the
pitch between the nanopillars from an edge of one nanopillar to an
edge of another nanopillar may be between 150 to 300 nm, or 200 nm
to 250 nm.
[0087] In one embodiment, the pitch between the protrusions may be
less than the size of the cells to be located at the surface of the
construct.
[0088] In one embodiment, a residual layer may be arranged between
the pillars and the surface of the construct. In one embodiment,
the residual layer may be removed from between the pillars by
reactive ion etching. The residual layer can be made of the same or
different material than the pillars and/or the surface of the
construct.
[0089] In one embodiment, the construct may be obtained via
nano-imprinting lithography (NIL).
[0090] In one embodiment, the nano-imprinting lithography may be
thermal nano-imprinting lithography or UV nano-imprinting
lithography.
[0091] In one embodiment, the construct and/or the protrusions may
be made of a polymer. In one embodiment, the polymer may be a
synthetic polymer, or a rigid synthetic polymer, or a biodegradable
polymer.
[0092] In one embodiment, the synthetic polymer may be selected
from the group consisting of poly(methyl methacrylate) (PMMA),
polydimethylsiloxane (PDMS), polystyrene (PS) and mixtures
thereof.
[0093] In one embodiment, the biodegradable polymer include, but
are not limited to of chitosan, poly(.epsilon.-caprolactone),
polyglycolic acid, poly(lactic acid), polyphosphoester (PPE),
poly(lactide-co-glycolide), poly(lactide-co-caprolactone),
poly(glycolide-co-caprolactone), polydioxanone,
polytrimethylenecarbonate, poly(glycolide-co-dioxanone),
polyamideester, polypeptide, polyorthoesters, polymaleic acid,
polyphosphazene, polyanhydride, polycebacicanhydride,
polyhydroxyalkanoate, polyhydroxybutylate, polycyanoacrylate and
mixtures thereof.
[0094] In one embodiment, a bioresorbable polymer is used. Examples
of bioresorbable polymers include, but are not limited to a mixture
of two or more bioresorbable homopolymers derived from the
polymerization of alpha-hydroxy carboxylic acids, and a mixture of
one or more bioresorbable terpolymers derived from the condensation
of a dicarboxylic acid, an alpha hydroxy carboxylic acid and an
aliphatic diol and one or more homopolymers derived from the
polymerization of alpha-hydroxy carboxylic acids, said homopolymers
and said terpolymers having an average molecular weight equal to or
greater than about 150,000 as measured by gel permeation
chromatography and wherein at least one of said homopolymers and
said terpolymers has an average molecular weight of from about
234,000 to about 320,000 as measured by gel permeation
chromatography. Other examples of bioresorbable polymers include,
but are not limited to polyhydroxyalkanoates such as
poly(hydroxybutyrate) (PHB), poly(hydroxyvalerate) (PHV) and
poly(hydroxybutyrate-co-valerate) (PHBV), polylactones, such as
polycaprolactone (PCL), poly(L-lactic acid) (PLA), poly(glycolic
acid) (PGA), poly(lactide-co-glycolide) (PLGA), polydioxanone,
polyorthoesters, polyanhydrides, poly(glycolic acid),
poly(D,L-lactic acid), poly(lactide-co-caprolactone), poly(glycolic
acid-co-trimethylene carbonate), polydioxanone, polyorthoesters,
polyphosphoesters, polyphosphoester urethanes, polyanhydrides,
poly(amino acids), polyacrylates, cyanoacrylates, poly(trimethylene
carbonate), polyurethanes, poly(iminocarbonate),
copoly(ether-esters) (e.g. PEO/PLA), polyalkylene oxalates,
polyphosphazenes and biomolecules, such as fibrin, fibrinogen,
starch, collagen, hyaluronic acid, etc., other natural polymers
such as alginate, polysaccharides such as dextran and cellulose,
etc. and mixtures thereof.
[0095] In one embodiment, the rigid synthetic polymer may be
polystyrene. In one embodiment, the polystyrene may be
tissue-culture grade polystyrene (TCPS).
[0096] For manufacturing purposes or even after manufacturing the
construct can be mounted or attached to another support surface,
such as a metal surface. As illustrated for example in FIG. 12, the
construct can be attached to a semiconductor surface, such as a
silicon surface.
[0097] In one embodiment, during manufacture of the construct, the
polymer may be mixed with molecules which are to be absorbed by the
cell to be located on the construct. In another embodiment, the
molecules may be attached to the surface of the protrusions. The
molecule may be immobilized via chemical bonding or adsorbed on the
surface of the protrusion, wherein adsorption does not involve
chemical binding.
[0098] In one embodiment, the cell comprising cell types comprising
an apical membrane or cell types comprising a basolateral
membrane.
[0099] In yet another embodiment, a method of promoting absorption
of molecules by cells is disclosed. The method comprises: [0100]
providing a construct as defined above; [0101] seeding and
culturing at least one cell at the surface of the construct under
conditions suitable for absorption of molecules by the cells.
[0102] In one embodiment, the absorption of molecules is via
endocytosis, or pinocytosis, or phagocytosis.
[0103] In another embodiment, absorption of the molecules may be
via a non-viral carrier or a viral carrier. This is advantageous as
non-viral vectors do not trigger an immune response and may be
delivered by processes including but not limited to endocytosis.
Examples of non-viral vectors include nano-particles and liposomes.
In one embodiment, the viral carrier may be a virus that is used to
deliver material into cells. Examples of suitable viral vectors
include retroviruses, lentiviruses, adenoviruses and
adeno-associated viruses.
[0104] In one embodiment, the molecules to be absorbed may be
selected from the group consisting of nucleic acid, nucleic acid
vectors, siRNA, microRNA, magnetic nanoparticles, gold
nanoparticles, fluorescent nanoparticles, quantum dots, aptamers
(oligonucleic acid or peptide), peptides, growth factors,
therapeutically active substances, biomarkers, colouring agents,
and any one of the aforementioned molecules attached to a
microparticles.
[0105] In one embodiment, a method of cell transfection, or
drug-delivery or high-throughput screening arrays using a construct
is defined above is disclosed.
[0106] The invention illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including", "containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0107] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0108] Other embodiments are within the following claims and
non-limiting examples. In addition, where features or aspects of
the invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is also
thereby described in terms of any individual member or subgroup of
members of the Markush group.
EXPERIMENTAL SECTION
[0109] Non-limiting examples of the invention and comparative
examples will be further described in greater detail by reference
to specific Examples, which should not be construed as in any way
limiting the scope of the invention.
Example 1
[0110] This example demonstrates the verification of topographical
structures on the substrate.
[0111] Material and Methods
[0112] Fabrication of Upright-Patterned Structures
[0113] Patterned substrates were fabricated using nanoimprint
lithography as previously documented. Briefly poly(methyl
methacrylate) (PMMA) (Microresist, PMMA, MW 35000 g/mol) was first
spin-coated on a clean silicon substrate to form a thin PMMA film
before a silanized silicon mold was placed on top of the
spin-coated surface and the imprinting was carried out at
150.degree. C. under a pressure of 60 bar for 10 minutes.
Subsequently, the system was cooled before demolding the silicon
master from the imprinted PMMA polymer layer. The fabricated
upright PS pillar structures can also be collapsed and collected
using a polydimethylsiloxane (PDMS) slab, hence forming an ordered
array of detachable structures. All structures used in the study to
be described in the following were fabricated using PMMA except for
collapsed pillar structures.
[0114] Fabrication of Residual Free Pillar Structures
[0115] Upright PMMA pillars were fabricated as described above with
an addition of Rhodamine B (Sigma-Aldrich, Rhodamine 110 chloride,
M.sub.W 366.8 g/mol). Subsequently, a reactive ion etching machine
(Plasmalab 80plus, Oxford) was used to remove the residual layer of
the imprinted substrate.
[0116] Verification of the Imprinted Structure by Scanning Electron
Microscope (SEM)
[0117] The fidelity of imprinted structures was verified using a
SEM (JEOL, JSM-6700F). Samples were coated for 20 seconds using a
gold coating machine (JEOL, JFC-1200) to achieve a gold film
thickness of approximately 10 nm. The structures were viewed using
an accelerating voltage of 5 kV, at a working distance of 6 mm.
[0118] Results
[0119] Scanning electron microscopy (SEM) was used to verify the
micro and nano structures formed on the substrate after
nano-imprint lithography. For the PMMA upright structures with
residual layer, 3 structures were used, 2 .mu.m pillars, 200 nm
pillars and 250 nm gratings. For the residual layer free PMMA
upright structures, 200 nm pillars were used. For the polystyrene
(PS) collapsed pillars, 2 .mu.m pillars were used. FIGS. 1A-C and
FIGS. 1D-E show the top and cross sectional SEM images of the
various structures fabricated for the experiments respectively,
while FIG. 1F shows the top view SEM image of the collapsed
detachable pillars.
[0120] These results indicate that all SEM images showed high
fidelity and dimensions that were in accordance with the initial
mold used.
Example 2
[0121] This example demonstrates fluorescence imaging of cellular
morphologies on different topographies.
[0122] Materials and Methods
[0123] Cell Culture
[0124] Bone marrow human mesenchymal stem cells (hMSCs) (CD105+,
CD166+, CD29+, CD44+, CD14-, CD34-, CD45-, Lonza, Poietics) were
cultured and expanded in serum containing Mesenchymal Stem Cell
Growth Medium (MSCGM, Lonza) according to the manufacturer's
protocol. The hMSCs used for experiments were at passages 5-7. MCF7
breast cancer cells (ATCC) were cultured in Eagle Minimum Essential
Medium (Sigma) containing 10% fetal bovine serum (Gibco,
Invitrogen), 1% Penicillin-Streptomycin (Gibco, Invitrogen) and
0.01 mg/ml bovine insulin (Sigma). COS7 fibroblasts (ATCC) were
cultured in a medium containing DMEM (Gibco, Invitrogen), 10% fetal
bovine serum and 1% Penicillin-Streptomycin (Gibco,
Invitrogen).
[0125] Topographical substrates were first sterilized under
ultra-violet light for 20 minutes, before respective cells were
seeded at 10,000 cells/cm.sup.2 in a 6-well tissue culture plate.
Collapsed PS pillars on PDMS substrates were rinsed with 70%
ethanol before being air dried and subsequently seeded with hMSCs
at 5,000 cells/cm.sup.2.
[0126] Fluorescence Imaging of Cell Morphology on Topographies
[0127] Cells cultured on different substrates were stained for
F-actin using AlexaFluor 546 Phalloidin and counterstained with 4',
6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Briefly, cells
were fixed in 4% PFA in PBS before cell permeabilization using 1%
Triton-X-100 in PBS for 15 minutes. The samples were then incubated
with 1:750 Alexa Fluor 546 Phalloidin (Molecular Probes,
Invitrogen) and 1:2500 DAPI for twenty minutes before mounting.
Samples were observed and imaged using a fluorescence microscope
(Leica epifluorescence microscope Leica DM IRB). For visualization
of dextran internalization, FITC-dextran (lysine fixable, Molecular
Probes) at 1 mg/ml was added to the culture medium before cells
were fixed for F-actin staining after 24 hours.
[0128] Results
[0129] Fluorescently labeled hMSCs, COST and MCF7 cells showed
characteristic morphologies on the various topographies after 24
hours (FIG. 2). Generally, cells were more spread out on
unpatterned and pillar substrates as compared to grating substrates
where they exhibited an elongated morphology (FIG. 9). F-actin
stress fibers showed the different distribution of the actin
cytoskeleton on these topographies. Cells that were cultured on the
2 .mu.m pillars showed intracellular actin rich rings that outlined
the top surface of the upright pillar substrates the cells were in
contact with (FIG. 2). The actin rich regions were of a higher
intensity in the micron sized pillars compared to the nano-sized
pillars while no such actin rich regions were seen on the grating
topography. Stress fibers on these cells bridged these actin rich
regions, seemingly connecting these rings together in a web like
fashion while cells that were cultured on the 250 nm gratings
substrate showed a distribution of stress fibers that were aligned
to the grating axis.
[0130] Accordingly, these results show that morphological changes
are closely linked to cellular adhesions, the focal adhesions, that
have an intertwined regulation with actin cytoskeleton.
Example 3
[0131] This example demonstrates the effect of topographical
structures on apical FITC-dextran internalization.
[0132] Materials and Methods
[0133] Internalization of FITC-Dextran
[0134] Respective cells were cultured on various patterned
substrates for 24 hours before fluorescein isothiocyanate
(FITC)-dextran molecule (Sigma Aldrich, MW 40,000) was added to all
samples at a concentration of 1 mg/ml with the exception of the
positive control where a higher concentration of 2 mg/ml was added
to cells cultured on unpatterned PMMA substrates. At the specific
time points of interest, the cells were detached for subsequent
flow cytometry analysis. For the comparison study between two
FITC-dextrans of different molecular weights, the experiment was
similarly carried out using an additional type of FITC-dextran with
higher molecular weight (Sigma Aldrich, MW 500,000).
[0135] Flow Cytometry Analysis of Internalized Dextran in Cells
[0136] For FITC-dextran internalization studies, patterned
substrates containing the cells were rinsed with phosphate buffered
saline (PBS) solution and subsequently detached from the substrate
using trypsin-EDTA for COS7 and MCF7 while Accutase (Stem Cell
Technologies) was used for hMSCs. The appropriate medium was then
added to neutralize the enzymatic detachment process before the
cells were washed, re-suspended in PBS and fixed in 0.5%
paraformaldehye (PFA). Cells were passed through a 60 .mu.m pore
size nylon filter before analyzing using a Dako flow cytometry
Analyzer (Dako Cytomation Cyan LX). Cells cultured in the absence
of FITC-dextran were used as the gating and negative controls where
a minimum of 10,000 events were recorded for each of the triplicate
samples.
[0137] Statistics
[0138] For comparisons of internalization efficiency between
different topography and control, one-way ANOVA analysis was
performed before Bonferroni's Multiple Comparison Tests were
carried out between different topographies and control, with a
p-value of at least <0.05 considered as significant. Errors bars
denote the standard deviation of at least 2 independent
experiments. In flow cytometry analysis of FITC dextran and GFP
transfection, the percentage of fluorescence population and mean
fluorescence intensity was studied.
[0139] Results
[0140] Flow cytometry analysis was carried out to investigate the
effect of topographical cues on the uptake of FITC-dextran mainly
from the apical surface of the cell membrane. The three different
cell types used in this study were COS7, MCF7 and hMSCs,
representing fibroblastic cells, cancer cells and multipotent stem
cells respectively.
[0141] Effect of Topography and FITC-Dextran Molecular Weight on
COS7 Internalization
[0142] FIG. 3 shows the fluorescent population of COS7 cells when
they were cultured on different topographies in FITC-dextran
containing medium for 24 hours. In FIG. 3A, COS 7 cells seeded on 2
.mu.m pillar (13.36.+-.0.44%), 200 nm pillar (10.99.+-.0.33%) and
250 nm grating (9.01.+-.0.45%) had a significantly larger
fluorescent population compared to the PMMA unpatterned control
(4.5%).
[0143] Comparing between the topographies, 2 .mu.m pillars were
also significantly different from 200 nm pillars and 250 nm
gratings, inducing the largest increase in FITC-dextran uptake
among the topographies tested while 250 nm gratings showed the
least increase in FITC-dextran uptake compared to an unpatterned
surface. The FITC-dextran used in this experiment was of a
relatively lower molecular weight (40,000) compared to FIG. 3B.
[0144] A similar experiment was carried out using a higher
molecular weight of FITC-dextran molecule (500,000) as shown in
FIG. 3B. Using a higher molecular weight FITC-dextran resulted in a
general decrease in fluorescent population across all substrates
except for the 2 .mu.m pillars (13.56.+-.0.33% vs. 13.36.+-.0.44%).
All other topographies showed a decrease in fluorescent population
(high MW vs. low MW), 200 nm pillars (7.20.+-.0.42% vs.
10.99.+-.0.33%), 250 nm gratings (2.18.+-.0.36% vs. 9.01.+-.0.45%).
The generic decrease in higher molecular weight FITC-dextran
uptake, however, did not affect the trends observed among the
different topographies. 2 .mu.m pillars similarly induced the
highest amount of FITC-dextran internalization while 250 nm
gratings were the least. When 200 nm pillars and 250 nm gratings
were compared to the unpatterned control substrate, the fluorescent
population was significantly lower while the 2 .mu.m pillars
remained significantly higher.
[0145] Effect of Topography on MCF7 FITC-Dextran
Internalization
[0146] Results from a repeat experiment using MCF7 are shown in
FIG. 4. MCF7 on 2 .mu.m pillars (59.99.+-.7.98%), 200 nm pillars
(58.28.+-.4.02%) and 250 nm gratings (56.78.+-.3.10%) did not show
any significant difference in the fluorescent population when
cultured on these topographies compared to the unpatterned PMMA
control (50.35.+-.1.68%).
[0147] Effect of Topography and Incubation Time on hMSC
FITC-Dextran Internalization
[0148] The results for similar experiments performed using hMSCs
are shown in FIG. 5. In addition to a 24 hour time point (data not
shown) as previously used for both COST and MCF7 cells, the
experiments were carried out at different cell incubation times in
FITC-dextran containing medium. FIG. 5 shows the results obtained
for the fluorescent population of hMSCs on different topographies
at incubation times of 18 hours (FIG. 5A), 6 hours (FIG. 5B), 3
hours (FIG. 5C) and 1 hour (FIG. 5D) respectively. Results from
FIGS. 5A and 5B showed the saturation of FITC-dextran
internalization where hMSCs cultured on the different topographies
all showed a similar absolute population fluorescence at 18 hours
(2 .mu.m pillars: 98.01.+-.1.35%, 200 nm pillars: 93.45.+-.5.66%,
250 nm gratings: 96.64.+-.3.64%). Results obtained for the 6 hour
time point was similar to the fluorescent population on 2 .mu.m
pillars at 84.43.+-.1.77%, 200 nm pillars at 94.55.+-.6.34% and 250
nm gratings at 86.73.+-.1.99%. These results demonstrated that the
uptake of FITC-dextran was saturated at these time points with the
specified FITC-dextran concentration in medium.
[0149] When the analysis was carried out after 3 hours of FITC
dextran incubation, the 2 .mu.m pillar showed a significantly
higher percentage of fluorescent population (P<0.01) as compared
to the control (1 mg/ml), 250 nm grating and 200 nm grating,
similar to earlier observations in COS7 cells. When the same
analysis was carried out at the 1 hour time point, the percentage
of fluorescent hMSCs on 2 .mu.m pillars (16.02.+-.8.38%), 200 nm
pillars (21.44.+-.9.37%) and 250 nm gratings (16.97.+-.10.88%) were
higher than the unpatterned control (15.41.+-.8.12%) although these
differences were not statistically significant.
[0150] Accordingly the enhanced internalization of FITC-dextran in
COS7 and hMSCs on 2 .mu.m pillars show that micron sized topography
is able to increase the pinocytosis rate of cells cultured on these
patterned substrates.
[0151] Macropinocytosis is initiated from actin-rich regions of the
plasma membrane called ruffles that are closely coordinated by
actin and its key regulators, the family of Rho GTPases (Rho,
Cdc42, Rac). In hMSCs and COS 7 on both the micron and nano sized
pillar substrates (FIG. 2), intracellular rich actin rings were
observed residing on the top surface of the pillars, with the
micron sized pillars showing actin rings which were larger and of a
higher fluorescence intensity. Micron sized pillars were able to
induce more actin dense regions in hMSCs and COS7 compared to the
nano pillars and gratings, changing the intracellular contractility
and upregulating RhoGTPases to enhance ruffling and thus
macropinocytosis. Indeed, results from the FITC-dextran experiment
demonstrated that 2 .mu.m sized pillars induced the strongest
enhancement in macropinocytosis rate in both COS7 and hMSC.
Similarly, lesser such actin-dense regions observed in MCF7
corresponded to an insignificant difference in FITC-dextran
internalization when the MCF7 cells were cultured on the different
topographical patterns. This can also be due to the increased
ruffling in MCF7 metastatic cells compared to the other cell lines,
masking the effect of topography on macropinocytosis since cancer
cells often exhibit increased motility.
[0152] On the other hand, the hMSCs that were cultured on different
substrate topographies exhibited different integrin subunits
profiles, suggesting different integrin trafficking rates in hMSCs
on different topographies. Cell migration requires integrins to be
redistributed from disassembling focal adhesions to new assembling
focal adhesions at the leading edges and the initial step of
integrin redistribution has been shown to involve clathrin-mediated
endocytosis of (3-1 integrins. Cell stimulation using
platelet-derived growth factor for cell migration also causes a
rapid redistribution of integrins to the dorsal circular ruffles
before being internalized through macropinocytosis.
[0153] The increased rates of macropinocytosis in the examples may
be a synchronized result of increased integrin transport on micron
sized topography. Cellular migrations are generally lower on
gratings compared to pillar substrates while the micron size pillar
substrates are more widely spaced apart compared to the dense
nano-sized pillars patterns which gives difficulty for cells to
find a suitable path for attachment and thus migration, exhibiting
slower motility in these nanopillar substrates.
[0154] Accordingly, topography like growth factors, can be a potent
stimulus for cellular migration, exhibiting differential cell
migration rates on different substrate topographies.
[0155] It is also interesting to note that hMSCs on different
topographies adopted morphology that had vastly different surface
area, with the largest being on the unpatterned control. These
results suggest that the effect of topography on macropinoctyosis
is independent of the surface area of contact with cargo
FITC-dextran. In addition, the results of employing two different
molecular weight of FITC dextran tracers (MW 40000 versus 500000)
suggests that the topographical effects on the macropinocytosis
were only partly dependent on the conformation and size of cargo,
with a decrease in the fluorescence population yet still observing
a similar trend.
[0156] Results from the time point study suggest that topographical
effects on the FITC-dextran internalization in hMSCs is the most
apparent after 3 hours of incubation in dextran-containing medium
as the dextran-internalization process seemed to be saturated after
6 and 18 hours of incubation, while 1 hour was insufficient to
distinguish a significant difference between the patterned and
unpatterned substrates. The results also indicate that
topographical effects can only be observed at earlier time points
before a steady-state of internationalization is reached.
[0157] Application of topography to increase the cellular uptake of
naked DNA or drug-containing nanoparticles have the potential to
make these macropinocytosis-dependent processes more efficient.
Based on our results, we demonstrate that micro-structures enhance
cellular internalization by the macropinocytosis pathway.
Example 4
[0158] This example demonstrates the effect of topographical
structures on non-viral GFP transfection of hMSCs.
[0159] Materials and Methods
[0160] Green Fluorescence Protein (GFP) Plasmid Amplification
[0161] Pmax FP-Green-C vector (Lonza, 4.7 kb) which expressed
maxFP-Green in mammalian cells were amplified in Escherichia coli
DH5.alpha. and purified using an AxyPrep plasmid midiprep kit
(Axygen Bioscience). GFP plasmid was obtained in elution buffer and
its concentration was measured at the absorbance wavelength of 260
nm (Nanodrop 2000, Thermo Scientific).
[0162] Transfection of Bone Marrow Human Mesenchymal Stem Cells
(hmSC) on Topographies
[0163] The transfection experiment was carried out similar to the
FITC-dextran internalization study. 24 hours after seeding hMSCs on
substrates placed in a 6 well plate, the cells were transfected
with GFP plasmid using Lipofectamine 2000 (Invitrogen) reagent
volume (.mu.l) to GFP plasmid mass (.mu.g) at a ratio of 1:2.5 in
Opti-MEM (Invitrogen). After 3 hours of incubation, the
transfection medium was replaced by fresh MSCGM medium with serum.
The hMSCs were detached for flow cytometry analysis 18 hours after
transfection, to allow time for GFP to be expressed.
[0164] Flow Cytometry Analysis of Non-Viral GFP Transfection of
hMSCs
[0165] For transfection efficiency studies, hMSCs were similarly
detached with Accutase, neutralized with MSCGM and washed with PBS.
In order to determine cell viability, the Live/Dead cells stain
(Invitrogen, Molecular Probes) was used according to the supplier's
instructions. Cells were analyzed by flow cytometry as described
above. Non-transfected cells were used as the negative control.
Cells that stained positive for both GFP and Live/Dead assay were
used as positive controls, while dead cells were used as negative
controls to set the compensation for the individual
populations.
[0166] Results
[0167] Flow cytometry analysis of Lipofectamine aided GFP
transfection of hMSCs is shown in FIG. 6. Human MSCs cultured on
unpatterned PMMA showed a mean fluorescent population of
1.8.+-.1.18%. When hMSCs were transfected while attached to various
topographical structures, an increase in the fluorescent population
was observed. The hMSCs cultured on 2 .mu.m pillars, 200 nm pillars
and 250 nm gratings show a mean value of 2.60.+-.1.8%,
5.00.+-.1.16% and 3.32.+-.1.83%, respectively (FIG. 6). Comparison
between the three different topographies indicate that 200 nm
pillars showed a 2.5 fold significant increase (p<0.05) in
fluorescent population compared to blank PMMA.
[0168] This example inventigates the effect of GFP (+) transfection
in hMSCs using both micron and nano-sized topography. Human MSCs
are known to be highly sensitive and notoriously difficult to
transfect, causing research groups to resort to electroporation for
enhanced transfection efficiency, although often at the expense of
increased cell death. Cellular uptake of lipofectamine-mediated GFP
plasmid occurs mainly by clathrin-mediated endocytosis,
distinctively different from the earlier experiments targeted at
macropinocytosis.
[0169] The amount of protein binding onto surfaces depends on the
surface energy and exposed surface area. Nanoscale topography is
able to significantly influence both the amount and the
conformation of these adsorbed proteins. Substrate topography
provides a 3-dimensional surface area for the adsorption of
proteins while nanoscale topography further enhances the surface
areas. Such differences in protein adsorption can have implications
for cells, which exhibit intrinsic ability to sense minute-scale
physical differences in the extra-cellular matrices.
[0170] However, the results suggest that surface area is unlikely
to play an important role in the direct modulation of both
macropinocytosis and clathrin-mediated pathway (FIG. 9) since
surface area of the cells are largest on the unpatterned
control.
[0171] Adherent cells respond to their surrounding microenvironment
through the modeling of focal adhesions, which are also used in
cellular motility. These focal adhesions can be modulated using
substrate topography and the size of focal adhesions are a
reflection of the state of intracellular actin-contractility.
[0172] hMSCs that were cultured on nanometer gratings showed
reduced vinculin expression compared to unpatterned control,
indicating a decreased actin cytoskeletal contractility on such
topographical surface.
[0173] Actin cytoskeletal tension plays an important role in
regulating endocytosis related proteins including integrins,
clathrin and caveolin-1. The results show that the use of
topography allows the modulation of cytoskeletal arrangement within
the cell, in turn regulating the plasma membrane tension which
changes the cell endocytosis rate.
[0174] The results also show that hMSCs on nanosized structures
have a higher affiliation to the receptor-mediated endocytosis;
caveolae-mediated encytosis and clathrin-mediated endocytosis.
[0175] Without being bound by any specific theory, we consider that
nanotopography can increase hMSCs transfection through 1)
differential protein adsorption on nanotopographies compared to
micron and unpatterned substrates, 2) modulation of focal adhesion
turnover on substrates with different patterns and sizes and 3)
different intracellular contractility on different substrates.
Example 5
[0176] This example demonstrated the baso-lateral uptake of
topographical structures in hMSCs.
[0177] Materials and Methods
[0178] Fabrication of Collapse Pillar Structures
[0179] FIG. 12 illustrates the fabrication of the polystyrene
collapse pillar structures using polydimethylsiloxane (PDMS, Dow
Corning, Sylgard 184). Upright polystyrene (PS) pillars were first
fabricated by nanoimprinting lithography. Polystyrene (PS) (Sigma
Aldrich, Mw=45,000 g/mol), and Rhodamine B (Sigma Aldrich,
Rhodamine 110 chloride, Mw=366.8 g/mol) were purchased from Sigma
Aldrich and used as received. PS films were prepared by
spin-coating of their solutions (2.5 wt % for PMMA and 23 wt % for
PS, in toluene) on silicon substrate at 2000 rpm. Rhodamine B was
added as the staining fluorescent dye in PS solutions. The
imprinting process of PS was carried out at 180.degree. C. at 60
bars for 10 minutes. To collapse the pillars, a PDMS slab was used.
PDMS was mixed at a ratio of 1:10 curing agent to elastomeric base
according to the manufacturer's protocol, degassed and cured at
80.degree. C. for 3 hours. The cured PDMS was brought into contact
with the top surface of upright PS pillars. A shearing force was
then applied parallel to the substrate to overcome the cohesive
force between the PS pillars and the imprinted layer. During the
process of shearing, collapsed PS pillar will be transferred onto
the PDMS, which will be used for the subsequent basal-lateral
uptake experiment.
[0180] Internalization of Residual Free Pillar and Collapsed
Pillar
[0181] Human MSCs that were seeded on rhodamine labeled residual
free and collapsed pillar structures were stained for F-actin using
Oregon Green 488 Phalloidin (Invitrogen Molecular Probe) and
counterstained with DAPI. Fluorescently stained hMSCs for F-actin
and DAPI were imaged using a laser scanning confocal microscope
(Olympus FluoView FV1000). Z-sections of the hMSCs on residual free
and collapsed pillar substrates were taken at 0.1 .mu.m and 0.3
.mu.m intervals respectively, for the visualization of collapsed
pillars present within hMSCs.
[0182] SEM of Cells on Pillars
[0183] Samples with hMSCs, COS7 and MCF7 seeded were fixed in 4%
PFA, washed in 0.1 M sodium cacodylate, and post-fixed in 2% OsO4
in 0.1 M Na cacodylate, pH 7.2. After post-fixation, the samples
were dehydrated in a graded ethanol series. After drying by
evaporation of hexamethyldisilazane (HMDS), the samples were
sputter-coated with gold and viewed with SEM (STEM, Quanta FEG 200,
I-IV mode) at 10-15 kV.
[0184] Results
[0185] Internalization of 200 nm Residual-Free Upright Pillars
[0186] hMSCs were cultured onto substrates containing residual-free
200 nm pillars as visualized using confocal microscopy and SEM in
FIGS. 7 and 8, respectively. FIG. 7A-F show confocal z-sections in
0.1 .mu.m successive steps of hMSCs fluorescently stained for
F-actin and nucleus. A reconstructed 3-D representation of the cell
using collected z-stacks showed these structures to be located
within the pen-nuclear region of these stem cells (data not shown).
Upright pillars without the residual layer were embedded with
rhodamine in red to differentiate these structures from cellular
organelles and intracellular structures. When the focal planes were
moved from the basal surface (maximum pillar signal intensity) to
the apical surface (pillars are off focus), intense red fluorescent
signals originating from the pillars (denoted by arrows in FIG.
7A-F) appear to remain in focus. The hMSCs appeared to have
internalized some of the upright pillars. SEM of the COS7, MCF7 and
hMSCs residing on these residual free nano-pillars showed long and
extensive filopodia projections (FIG. 8A-F). The close up images of
filopodia extensions and cortical actin in hMSCs are shown in FIG.
10. A similar experiment carried out using the collapse pillar
structures gave similar results that hMSCs appear to have
internalized the larger sized 2 .mu.m collapsed pillars (FIG.
11).
[0187] The results from the SEM images (FIG. 8) show that cells
extended long filopodia extensions to reach for these nano-sized
structures. Filopodia are thin membrane protrusions that act to
probe the extracellular environment. Also known as microspikes,
these extensions can be found at the migrating front end of cells.
The SEM observations indicate that cells were able to specifically
target their filopodia onto these nano-sized pillars, affix
themselves onto these structures and exert a pulling force onto
these structures for cellular locomotion. In the disclosed system
of structure internalization, the residual free topographical
structures act to provide both topographical cues and a medium of
possible drug delivery. The imprinted topographical cues can
therefore regulate cellular behaviors including proliferation,
spreading and even differentiation in stem cells. Upon exertion of
cellular force onto these nanotopographical cues, the imprinted
structures can be detached and internalized by the residing cells
as a drug delivery system.
[0188] These results show an increased cortical actin activity in
the cells residing on these particles. Cortical actin plays
important roles in clathrin-mediated endocytotic pathways. Earlier
studies employing 1 .mu.m cylindrical PRINT particles were shown to
be internalizing mainly via clathrin-mediated endocytotic pathways
as well as macropinocytosis. However, due to the proximity of the
structures to the baso-lateral surface of the residing cells, entry
from the apical surface will be little or not significant.
Therefore, the internalization of residual free pillars is unlikely
to be similar to the previous mentioned study. Nonetheless, the
results show that multiple internalized particles were similarly
observed. As smaller sized structures were used in this study, it
is possible for these particles to gain entry through other
pathways.
[0189] Internalization of 2 .mu.m Collapsed Pillars.
[0190] Adherent cells can undergo endocytosis at both the apical as
well as the baso-lateral surface. Accordingly, confocal imaging of
fluorescently stained COS7, MCF7 and hMSCs cultured on rhodamine
embedded 2 .mu.m pillars after 24 hours incubation was performed
(FIG. 11). Each successive image shows 0.3 .mu.m step increase in
the z-directions from the maximal red intensity indicating the
focal plane of the collapsed pillars to the apical surface of the
cells. F-actin was stained using Oregon Green 488 Phalloidin while
the nuclei were counterstained with DAPI.
[0191] Confocal imaging of COS7 and MCF7 cells (FIG. 11E-H) on
these collapsed structures did not show any form of cellular
internalization although a large number of pillars appear to reside
in the baso-lateral surface of the MCF7 cells, as indicated by the
red fluorescence intensity. This suggests the attempted endocytosis
of these structures may be hindered due to the size.
[0192] On the other hand, the hMSCs cultured on these collapsed
structures appeared to internalize some of these collapsed
structures. It can also be observed that most of the internalized
particles reside within the pen-nuclear region of these stem cells
as indicated by the white arrows in FIG. 11L. Pillars indicated by
red intensity appeared to remain in focus when the focal plane was
adjusted towards the apical surface of the cells where surrounding
pillars that were not internalized became out of focus (FIG.
11I-K).
[0193] This study employing 2 .mu.m collapsed structures allows
cells to undergo particle internalization by exerting less pulling
force to "self-detach" the collapsed topographical structures. The
results indicate that cells were able to internalize larger sized
collapsed structures compared to residual free nanostructures as
the adhesion forces between the structures and the underlying
substrates were much reduced. The preferential internalization by
hMSCs might also be due to the larger intracellular contractility
present in hMSCs. The careful design of these substrates can thus
be used for specific trafficking in a target population within a
mixed population.
* * * * *