Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: COMPOSITIONS AND METHODS FOR PROMOTING LIPOSOMAL
AND CELLULAR ADHESION
[0001] The present application relates to the incorporation of
complementary, bio-orthogonal reactive functional groups into liposomes and
the use of the resulting compositions for promoting liposomal and cellular
adhesion.
BACKGROUND OF THE APPLICATION
[0002] Cells that make up tissues and organs exist and
communicate
within a complex, three-dimensional (3D) environment. The spatial orientation
and distribution of extracellular matrix (ECM) components directly influences
the manner in which cells receive, integrate, and respond to a range of input
signals.1 As such, cellular interactions with ECM molecules and/or other cells
have been extensively investigated for fundamental studies in development,
cell motility, differentiation, apoptosis, paracrine signaling, and
applications in
tissue engineering.23 There has been tremendous effort toward the design
and fabrication of 3D scaffolds that mimic ECM properties and induce tissue
formation in vitro, utilizing various biomaterials, biodegradable polymers,4
collagen,5 and hydrogels." Among the major challenges facing the use of
these technologies for tissue engineering are the abilities to force contact
between multiple cell types in 3D to control the spatial and temporal
arrangement of cellular interactions and tailor and mold the biomaterial to
recapitulate the 3D, in vivo environment under laboratory constraints. Without
the use of engineered scaffolds in culture, most cells are unable to form the
necessary higher-order 3D structure required for the anatomical mimicry of
tissue and are limited to random migration, generating two-dimensional (2D)
= monolayers, As a result, several approaches, including the use of
dielectrophoretic forces," laser-guided writing,10-12 surface manipulation,13
and a number of lithographic printing techniques14-17 have been integrated
with 30 scaffold designs to produce multi-type cellular arrays9'11'17'18 or 3D
cell
clusters or spheroids:7'5.13 In a recent study, 3D aggregates consisting of
multiple cell types were formed within a hydrogel matrix through DNA
hybridization after cell surfaces were engineered with complementary short
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oligonucleotides via a metabolic labeling approach.7 However, for some
applications, the presentation of cell-surface DNA may not be stable for
extended time periods in cell culture or in vivo.
[0003] Cell-surface engineering methodologies have primarily
been of
interest in molecular biology. As such, biosynthetic approaches have been
employed to introduce different functional groups on cell surfaces. In a
pioneering study, an unnatural derivative of N-acetyl-mannosamine, which
bears a ketone group, was converted to the corresponding sialic acid and
metabolically incorporated onto cell-surface oligosaccharides, resulting in
the
cell surface display of ketone groups.19 However, metabolic or genetic
methods may alter many of the biochemical pathways required for normal cell
= function and not all cell lines possess this metabolic machinery. Thus,
there is
a growing demand for general tools that can provide simple alternatives to the
complex genetic and biosynthetic methods. Other approaches to cell-surface
engineering have also been undertaken to incorporate a functional group into
a target biomolecule, such as an endogenous protein, utilizing a cell's
biosynthetic machinery.20,21 These strategies aim to produce a site that can
= then be covalently modified with its delivered counterpart or probe.
However,
most of these protein-based tags are large and bulky and become problematic
when interacting with the other glycans and biomolecules on the cell
suface.2423 Additionally, the perturbation of cellular physiology with
biomolecules at the cell surface may result in the interference of significant
biochemical pathways or cellular functions.24.26.
[0004] Membrane fusion processes are ubiquitous in biology and
span
multi-cellular communication, extracellular signaling, the reconstruction of
damaged organelles, and integration of cells into complex tissues and
organs.26 As a result, there has been much interest in developing model
systems to mimic biological membranes to investigate the mechanisms of
= fusion and for use in various biotechnological applications. For example,
cells
secrete and display proteins and lipids during vesicle trafficking events that
either diffuse into the ECM or become components of the cell membrane after
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fusion.27 Naturally, lipid vesicles provide an ideal platform for such studies
and
have been widely used to examine various membrane-related processes,
including fusion.28-30 In order for fusion to occur, the membranes must be
brought into close proximity, followed by bilayer destabilization.31 Fusion of
such lipid vesicles or liposomes can be initiated by using divalent cations,
polycations,32 positively charged amino acids33 and membrane-disrupting
peptides.34.35 Historically, synthetic chemical agents have also been
employed to fuse vesicle membranes36-39 through non-specific interactions.
However, recent efforts to improve selectivity and control over vesicle fusion
= 10 have been achieved through the use of small, synthetic molecular
recognition
pairs.40-41 Since vesicle fusion is a natural process and has been shown to
influence the construction of cells into multicellular organisms, much
research
has focused on using liposomes to deliver cargoes, reagents, nanomaterials,
and therapeutic agents to cells.
[0005] Noncovalent cell-surface engineering strategies via cationic graft
copolymer adsorption and a fluorescent cell labeling technique via cationic
and aromatic lipid fusion have been previously reported.42.
SUMMARY OF THE APPLICATION
[0006] The present application describes compounds,
compositions
and methods for incorporating chemoselective and bio-orthogonal
= complementary functional groups, such as ketone and oxyamine groups, into
liposomes. In one embodiment of this application, alkyl ketone and oxyamine
molecules spontaneously inserted into separate liposomes upon synthesis.
When these two types of liposomes were mixed, chemical recognition
occurred, producing stable oxime bonds under physiological conditions. The
liposomes combined in this manner reacted chemoselectively to form an
interfacial, covalent oxime linkage, resulting in liposome docking and
adhesion. Adhered liposomes either fused or formed multiadherent structures.
[0007] Accordingly, the present application includes a mixture
comprising a plurality of liposomes of type A and a plurality of liposomes of
type B, wherein the liposomes of type A comprise a reactive functional group
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that reacts with a reactive functional group comprised in the liposomes of
type
B to form a chemical interaction that results in adhesion of the liposomes of
type A and the liposomes of type B.
[0008] In an embodiment of the application, the adhesion of the
liposomes of type A and the liposomes of type B results in formation of
multiadherant liposomes, the partial fusion of liposomes of type A and the
liposomes of type B and/or the complete fusion of the liposomes of type A and
type B.
[0009] Ills an embodiment of the application that the reactive
functional
groups n the liposomes of type A and B are bio-orthogonal. In an
embodiment, the reactive functional group is comprised in an amphiphatic
= molecule wherein the reactive functional group is located in the
hydrophilic
portion of the molecule. In a further embodiment of the application, the
reactive functional group in the liposomes of type A is a ketone and the
reactive functional group in the liposomes of type B is an oxyamine.
Accordingly, in another embodiment the present application includes a
liposome comprising an amphiphatic molecule wherein the hydrophilic portion
= of the amphiphatic molecule comprises a ketone. In a further embodiment
the
present application includes a liposome comprising an amphiphatic molecule
wherein the hydrophilic portion of the amphiphatic molecule comprises an
oxyamine. In a specific embodiment, the amphiphatic molecule comprising a
ketone in the hydrophilic portion is R1C(0)R2 and the amphiphatic molecule
comprising an oxyamine in the hydrophilic portion is R3-0-NH2, wherein
= wherein R1 and R3 are independently selected from C6_30alkyl and
C6.30alkenyl
and R2 is C1..2alkyl.
[0010] In an embodiment of the application, aside from the
amphiphatic
molecule comprising a reactive functional group, the liposomes further
comprise any suitable amphipatic molecule, or mixture of molecules, that form
liposomes. In general, liposome-forming amphiphatic molecules are lipids, in
particular phospholipids. In a further embodiment, the amphiphatic molecules
are selected based on the proposed use of the liposome.
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[0011] In yet another embodiment, the liposomes further comprise
other functional molecules, such as, fluorescent molecules, dyes and/or other
indicator molecules, so that when the liposomes of type A and type B are
fused, a physical change, such as a change in fluorescence, color or smell,
5 occurs.
[0012] The present application also includes a method for promoting
adhesion of liposomes comprising contacting a plurality of liposomes of type A
with a plurality of liposomes of type B, wherein the liposomes of type A
comprise a reactive functional group that reacts with a reactive functional
group comprised in the liposomes of type B to form a chemical interaction that
results in the adhesion of the liposomes of type A and the liposomes of type
B.
[0013] The present application also describes compounds,
compositions and methods for tethering chemoselective and bio-orthogonal
complementary functional groups, such as ketone and oxyamine groups, from
cell surfaces by liposome delivery toward the goal of rewiring the cell
surface.
In one embodiment, the liposomes described above comprising ketone and
oxyamine groups were cultured with various cell types resulting in membrane
fusion and the display of ketones and oxyamines on the cell surface in a
manner such that they were available for further chemical manipulation.
Therefore the synthetic ketone and oxyamine molecules fused on the cell
membrane serve as cell-surface receptors, providing tools for the attachment
of other functional materials, biomolecules, and probes on the cell surface.
In
sum, liposome fusion to cell membranes is employed herein as a method to
deliver small chemical functional groups to tailor the cell membrane for
subsequent bio-orthogonal and chemoselective ligation reactions.
[0014] The present application therefore includes a method for
promoting the adhesion of cells comprising:
(a) contacting a first cell population with a liposome of type A under
conditions
for the fusion of the liposome of type A with the first cell population;
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(b) contacting a second cell population with a liposome of type B under
conditions for the fusion of the liposome of type B with the second cell
population; and
(c) contacting the fused first cell population with the fused second cell
population,
wherein the liposomes of type A comprise a reactive functional group that
reacts with a reactive functional group comprised in the liposomes of type B
to
form a chemical interaction that results in the adhesion of the first and
second
cell populations.
[0015] Thus, the present application includes a methodology that
combines cell-surface modification, without the use of molecular biology
techniques or biomolecules, and a simple, stable bio-orthogonal conjugation
bottom-up approach that is capable of directing tissue formation and that will
greatly benefit a range of medical applications. This platform should also
find
wide use in studying fundamental cell behavior and provide a range of new
tools for tissue engineering and biomedical applications.
[0016] Other features and advantages of the present application
will
become apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific examples
while indicating embodiments of the application are given by way of
illustration
only, since various changes and modifications within the spirit and scope of
the application will become apparent to those skilled in the art from this
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present application will now be described in greater detail
with reference to the drawings in which:
[0018] Figure 1 shows a general schematic and corresponding
lipid
components for the formation of fused and adhered liposomes based on
chemoselective oxime conjugation. (a) When mixed, ketone- and oxyamine-
tethered liposomes react chemoselectively to form an interfacial, covalent
=
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oxime linkage, resulting in liposome docking and adhesion. Docked liposomes
either fuse or form multi-adherent structures. (b) Dodecanone molecules were
incorporated into neutral, POPC at a ratio of 5:95 to form keto-LUVs (1),
while
0-dodecyloxyamine molecules were incorporated into POPC and negatively
charged, POPG at a ratio of 5:75:20 to form oxyamine-LUVs (2). These
liposomes were used for liposome-liposome fusion studies. (c) Dodecanone
molecules were incorporated into POPC and fluorescence donor, NBD-PE at
a ratio of 5:93:2 to form keto-NBD-PE LUVs (3). 0-Dodecyloxyamine
molecules were incorporated into POPC, POPG, and fluorescence acceptor,
rhod-PE at a ratio of 5:73;20:2 to form oxyamine-rhod-PE LUVs (4). These
liposomes were used for FRET studies. (d) Dodecanone molecules were
incorporated into POPC and positively charged, DOTAP at a ratio of 5:97:2 to
form ketone-presenting liposomes (5). O-Dodecyloxyamine molecules were
incorporated into POPC and DOTAP at a ratio of 5:93:2 to form oxyamine-
presenting liposomes (6). These liposomes were used for cell-liposome fusion
studies.
[0019] Figure 2 shows the characterization of the formation of fused
and adhered liposomes based on chemoselective oxime conjugation. (a)
Mass spectrometry (MS) data representing the oxime ligation of keto-LUVs to
self-assembled monolayers (SAMs) of oxyamine-terminated alkanethiol on a
gold surface is displayed. Matrix-assisted laser desorption/ionization (MALDI)
was performed after keto-LUVs were delivered to the surface, and a mass of
387 units was detected, confirming oxime conjugation. (b) Structural analyses
using transmission electron microscopy (TEM), representing the adhesion and
fusion of keto- (1) and oxyamine- (2) LUVs over time. The following images
are shown from left to right: multi-adherent liposomes that are not fused;
partially fused liposomes; and a single, large liposome after complete fusion.
The scale bars represent 60 nm. (c) Fluorescence resonance energy transfer
(FRET) analysis of liposome adhesion and fusion was monitored over 2 h.
Fluorescence emission of keto-NBD-PE/PC LUVs (3), excited at 460 nm, was
observed by scanning 475-600 nm (left-side trace). Fluorescence emission of
keto-NBD-PE/PC LUVs (3) mixed with oxyamine-rhod-PE/PC/POPG LUVs (4)
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is represented (right-side trace). A new FRET emission peak is observed at
578 nm showing mixed liposome adhesion. (d) Dynamic light scattering (DLS)
was performed upon mixing liposomes (1 and 2) to monitor vesicle size
change as a function of time. Increases in vesicle size were observed due to
aggregation, adhesion, or fusion (top trace). Liposome saturation was
reached ¨80 min after mixing. Without the presence of ketone and oxyamine
functional groups, the LUV size remains constant (bottom trace).
[0020] Figure 3 (Top) shows a schematic describing the delivery
and
subsequent fusion of fluorescent liposomes to cell surfaces with
corresponding brightfield and fluorescent images. (a) Oxy-LUVs (6, 3 mM)
were reacted with fluorescein-ketone (7, 0.15 mM, 2 h) to generate green
fluorescent liposomes. The fluorescent liposomes were then added to fbs in
= culture, resulting in the fluorescent labeling of cells after liposome
fusion to
the cell membrane. Micrographs show (b) control cells where liposomes not
containing oxyamine groups were incubated with fluorescein-ketone and
added to fbs in culture for 2 h. and (c) green fluorescently labeled cells
after
oxyamine-functionalized liposomes were incubated with fluorescein-ketone
and delivered to fbs (2 h). Figure 3 (Bottom) shows a general schematic and
= images for cell-surface tailoring using liposome fusion and
chemoselective
oxime chemistry. (d) Keto-LUVs (6, 3 mM) were added and fused with the
cells to display these groups from the cell surface (9). Addition of rhod-
oxyamine (8, 0.7 mM in H20, 2 min) resulted in chemoselective oxime
formation and red fluorescent labeling of the cells. Images display (e)
control
fbs where liposomes not displaying ketones were fused to the membrane (2 h)
and rhod-oxyamine was added and no fluorescence was observed and (f)
fluorescently labeled cells after ketone-functionalized liposomes were fused
to
fbs (2 h) and cells were -incubated with rhod-oxyamine. Scale bars for b and c
and d and e represent 50 and 30 j.im, respectively.
[0021] Figure 4 shows schematics and fluorescent micrographs of
rewired cells adhered to patterned self-assembled monolayers (SAMs) of
alkanethiolates on gold substrates. (a and b) Keto- (5) and oxyamine-LUVs (6,
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3 mM, 4 h) were cultured with separate fb populations, producing ketone- and
oxyamine-presenting fbs (9 and 10, respectively). These cells were then
seeded (-102 per mL, 2 h) to patterned, oxyamine- and aldehyde-terminated
SAMs (10 %), respectively, and allowed to adhere through stable oxime
conjugation. The unpatterned surface regions present tetra(ethylene glycol),
which resists cell and protein adsorption. The cells then grew and
proliferated
only filling out the oxyamine- and aldehyde-tethered surface regions,
respectively. (c) A fluorescent micrograph of patterned ketone-fbs (9),
adhered to an oxyamine-terminated SAM is shown. (d and e) Fluorescent
micrographs of patterned oxyamine-fbs (10), adhered to an aldehyde-
terminated SAM are demonstrated. Cells were stained with DAPI (blue,
nucleus) and phalloidin (red, actin).
[0022] Figure 5 shows cell surface molecule quantification using flow
cytometry. (a) Oxyamine-LUVs (6, 3 mM) were added to fbs in culture (4 h),
resulting in membrane fusion and subsequent display of oxyamine groups
from cell surfaces (10). Ketone-functionalized fluorescein (7, 0.15 mM 2 h)
was then reacted with the fbs, generating fluorescently labeled cells. (b)
Liposomes with varying oxyamine mol % (0 %, 1 %, 5 %, and 10 %) were
generated and cultured with separate populations of fbs. After reacting with
ketone-fluorescein, the cell populations were washed with PBS, trypsinized,
centrifuged, resuspended in RPM! media, and tested using FACS analyses.
As shown, the fluorescence intensity increased with increasing oxyamine
concentration. (c) The number of molecules present at the cell surface with
respect to oxyamine concentration was quantified using flow cytometry. A
bead with a known FITC molecule density was employed as a standard
comparison to calculate the number of oxyamines after oxy-LUVs (6) with 0 /0,
1 %, 5 %, and 10 % oxyamine was cultured with cells. As the oxyamine
concentration increased, the molecules per cell increased linearly (0 %, 128;
1 %, 1600; 5 %, 9800; and 10 %, 17400). Twenty thousand cells were
counted for each sampling.
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[0023] Figure 6 shows fluorescent, phase contrast, and scanning
electron micrographs (SEM) describing 3D spheroid formation via liposome
fusion and chemoselective cell-surface tailoring. Two fb populations were
cultured separately with ketone- (1) or oxyamine- (2) containing liposomes,
5 resulting in membrane fusion and subsequent tethering of ketones and
oxyamines from the cell surface. The oxyamine-tethered rat2 fibroblasts (12)
contained a fluorescent m-cherry nuclear label. The ketone-presenting Swiss
albino 3T3 fibroblasts (9) were not fluorescently labeled. (A) Two fibroblast
populations were cultured separately with ketone- (1) or oxyamine- (2)
10 containing liposomes. Due to the presence of a positively charged
liposome,
fusion occurred, producing ketone- (9) and oxyamine- (12) tethered cells.
Upon mixing these cell populations, clustering and tissue-like formation,
based on chemoselective oxime conjugation, occurred. (B) Control
experiments (overlay image) demonstrate no spheroid formation for cells that
did not contain either ketone or oxyamine groups. (C and D) However, when
two cell populations displaying ketone (9) and oxyamine (12) recognition
groups are mixed, interconnected spheroid assemblies form (overlay images).
(E-G) Representative SEM images of (E) control cells and (E and F) spheroid
assemblies, as described above, are displayed. For all spheroid assemblies
depicted, cell populations were mixed and cultured together for 3 h before
imaging at ¨104 cells/mL.
[0024] Figure 7 shows a general schematic and images of oxime-
mediated, 3D tissue-like structure formation with controlled
interconnectivity.
(A) Ketone- (1) and oxyamine- (2) containing liposomes were added to two
separate fb populations, resulting in membrane fusion and subsequent
presentation of the ketone (9) and oxyamine (12) groups from cell surfaces.
By culturing these cells on substrates, alternating cell population seeding
layer-by-layer, gave rise to multi-layered, tissue-like cell sheets through
stable
oxime chemistry. (B) A 3D reconstruction and (C) confocal micrograph
showing only a monolayer of cells after oxyamine-presenting cells (12) were
cultured with adhered non-functionalized cells. (E) A 3D reconstruction and
(F)
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confocal micrograph of multiple cell layers after oxyamine-presenting cells
(12)
were added to substrates presenting ketone-containing cells (9). (D and G)
Intact, 3D multi-layered cell sheets can be removed from the surface by gentle
agitation as displayed by brightfield and fluorescent images. The insets in B
and E show a z-plane cross-section that indicates the thickness of the cell
layers. Cells were stained with DAPI (nucleus) and phalloidin (actin).
[0025] Figure 8 shows
confocal images representing 2D monolayer and
3D multi-layered tissue-like structures of fbs with spatial control. (A) A
circular,
2D monolayer of fbs (control) result after ketone-functionalized fbs (9) and
fbs
(not functionalized with oxyamines) were patterned on a circular, microcontact
printed region, presenting fibronectin and allowed to grow for 5 days. (B-D)
Fbs, functionalized with ketone groups (9) were seeded onto microcontact
printed regions containing fibronectin and allowed to grow for 2 days. Fbs,
functionalized with oxyamine groups (12) were then seeded and allowed to
grow for 2-3 more days. Confocal images demonstrating 3D tissue formation
in (B) circle, (C) bar, and (D) square geometries are depicted. The
corresponding z-plane cross-sections that indicate the thickness of the cell
layers are shown as an inset; scale bars represent 30 rn. Cells were stained
with DAPI (nucleus) and phalloidin (actin).
[0026] Figure 9 shows
general schematic and brightfield images
representing oxime-mediated, 3D tissue-like structure formation with hMSC/fb
co-cultures and subsequent induced adipocyte differentiation to generate 3D
adipocyte/fb co-culture structures. (A) Ketone-tethered hMSCs (11) were
seeded onto a surface, followed by the addition of oxyamine-functionalized
fbs (12). The co-culture was allowed to grow and divide for 3 d at which
point,
adipogenic differentiation was induced with the addition of the appropriate
media. This resulted in a 3D multi-layer of adipocytes and ft. (B) A confluent
2D monolayer of ketone-presenting hMSCs is represented. (C) A brightfield
image displaying a 3D multi-layer co-culture of hMSCs (11) and oxyamine-
functionalized fbs (12) is shown. (D) Adipogenic differentiation was induced
with media resulting in 3D multi-layered adipocyte and fb co-culture
structures,
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represented by low and (E) high-resolution brightfield images (after 10 days
in
culture). Adipocytes were stained with Oil Red 0 (lipid vacuoles) and Harris
Hemotoxylin (nucleus).
DETAILED DESCRIPTION OF THE APPLICATION
I. Definitions
[0027] Unless otherwise indicated, the definitions and embodiments
described in this and other sections are intended to be applicable to all
embodiments and aspects of the application herein described for which they
are suitable as would be understood by a person skilled in the art.
[0028] As used in this application, the singular forms "a", "an" and "the"
include plural references unless the content clearly dictates otherwise. For
example, an embodiment including "a lipid" should be understood to present
certain aspects with one lipid, or two or more additional lipids.
[0029] In embodiments comprising an "additional" or "second"
component, such as an additional or second lipid, the second component as
used herein is chemically different from the other components or first
component. A "third" component is different from the other, first, and second
components, and further enumerated or "additional" components are similarly
different.
[0030] In understanding the scope of the present disclosure, the term
"comprising" and its derivatives, as used herein, are intended to be open
ended terms that specify the presence of the stated features, elements,
components, groups, integers, and/or steps, but do not exclude the presence
of other unstated features, elements, components, groups, integers and/or
steps. The foregoing also applies to words having similar meanings such as
the terms, "including", "having" and their derivatives. The term "consisting"
and its derivatives, as used herein, are intended to be closed terms that
specify the presence of the stated features, elements, components, groups,
integers, and/or steps, but exclude the presence of other unstated features,
elements, components, groups, integers and/or steps. The term "consisting
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essentially of, as used herein, is intended to specify the presence of the
stated features, elements, components, groups, integers, and/or steps as well
as those that do not materially affect the basic and novel characteristic(s)
of
features, elements, components, groups, integers, and/or steps.
[0031] The term "bio-orthogonal" as used herein refers to non-native,
non-perturbing chemical functional groups that are introduced into naturally
occurring, living systems and are modified in these living systems through
selective reactions that do not interfere with any other chemical moieties in
the natural surroundings.
[0032] The term "amphiphatic" or "amphiphilic" refers to a compound
comprising both hydrophilic (water loving) and lipophilic (fat loving)
portions.
= [0033] The term "liposomes" as used herein refers to
artificially
prepared vesicles, the surface of which is a bilayer formed from amphiphatic
molecules.
[0034] The term "reactive functional group" as used herein refers to a
group of atoms or a single atom that will react with another group of atoms or
a single atom (so called "complementary functional group") under bio-
orthogonal reaction conditions to form a chemical interaction between the two
groups or atoms.
[0035] The term "reacts with" as used herein generally means that
there is a flow of electrons or a transfer of electrostatic charge resulting
in the
formation of a chemical interation.
[0036] The term "chemical interaction" as used herein refers to
the
formation of either a covalent of ionic bond between the reactive functional
groups. The chemical interaction is one that is strong enough to promote the
adhesion of liposomes or cells.
[0037] The term "adhere" or "adhesion" as used herein means to
bring
= two or more entities, such as two or more liposomes or two or more cells,
into
close proximity to each other and to remain in contact with each other. The
adhered liposomes may remain as separate entities or, their membranes may
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destabilize and fuse together to result in the formation of a single liposome.
The adhered cells may communicate with each other and may divide and
multiply forming, for example, tissues.
[0038] The term "alkyl" as used herein means straight or
branched
chain, saturated alkyl groups. The number of carbon atoms in the chain is
defined by the C#_# prefix preceding the term. For example, the term
C6.30alkyl
means an alkyl group having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19,
20, 21, 22, 24, 25, 26, 27, 28, 29 or 30 carbon atoms.
[0039] The term "alkenyl" as used herein means straight or
branched
chain, unsaturated alkyl groups containing one or more, suitably one or three,
more suitable one or two, double bonds. The number of carbon atoms in the
chain is defined by the C#4 prefix preceding the term. For example, the term
C6_30alkyl means an alkenyl group having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms.
[0040] The term "oxyamine" as used herein refers to the functional
group "-0-NH2".
[0041] The term "ketone" refers to the functional group "-C(0)-
".
[0042] Terms of degree such as "substantially", "about" and
"approximately" as used herein mean a reasonable amount of deviation of the
modified term such that the end result is not significantly changed. These
= terms of degree should be construed as including a deviation of at least
5%
of the modified term if this deviation would not negate the meaning of the
word it modifies.
II. Liposome Adhesion
[0043] The present application includes a mixture comprising a plurality
of liposomes of type A and a plurality of liposomes of type B, wherein the
liposomes of type A comprise a reactive functional group that reacts with a
reactive functional group comprised in the liposomes of type B to form a
chemical interaction that results in the adhesion of the liposomes of type A
and the liposomes of type B.
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[0044] In an embodiment of the application, the adhesion of the
liposomes of type A and the liposomes of type B results in formation of
multiadherant liposomes, the partial fusion of liposomes of type A and the
liposomes of type B and/or the complete fusion of the liposomes of type A and
5 type B. In an further embodiment, the adhesion of the liposomes of type A
and the liposomes of type B results in the complete fusion of the liposomes of
type A and the liposomes of type B.
[0045] As would be understood by a person skilled in the art,
the
reactive functional groups in the liposomes of type A differ, but are
10 complementary to, the reactive functional groups in the liposomes of
type B.
By complementary it is meant that the reactive functional groups interact, or
react with each other, to form a chemical interaction that is strong enough to
promote the adhesion of the two types of liposomes to each other. In an
embodiment, the chemical interaction is a covalent bond or an ionic bond. In
15 another embodiment, the chemical interaction is a covalent bond.
[0046] It is an embodiment of the application that the reactive
functional
groups in the liposomes of type A and B are bio-orthogonal. Examples of
= complementary, bio-orthogonal pairs of reactive functional groups
include, but
are not limited to:
(1) ketones and oxyamines which react to form an oxime;
(2) ketones and hydrazines which react to form a hydrazone;
(3) dienes and dienophiles which react to form a six membered ring (DieIs
Alder reaction); and
= (4) azides and alkynes which react to form a triazole (Huisgen reaction).
[0047] It is an embodiment that the complementary, bio-orthogonal pair
of reactive functional groups are ketones and oxyamines which react to form
an oxime.
[0048] In an embodiment, the reactive functional group is
comprised in
an amphiphatic molecule wherein the reactive functional group is located in
the hydrophilic portion of the molecule. In an embodiment, the reactive
functional group forms the hydrophilic portion of the amphiphatic molecule
CA 02776618 2012-05-10
16
and the lipophilic portion of the amphiphatic molecule is a long hydrocarbon
chain, optionally comprising one or more double bonds.
[0049] In a further
embodiment of the application, the reactive
functional group in the liposomes of type A is a ketone and the reactive
functional group in the liposomes of type B is an oxyamine. Accordingly, in
another embodiment the present application includes a liposome comprising
an amphiphatic molecule wherein the hydrophilic portion of the amphiphatic
molecule comprises a ketone. In a further
embodiment the present
application includes a liposome comprising an amphiphatic molecule wherein
the hydrophilic portion of the amphiphatic molecule comprises an oxyamine.
In a specific embodiment, the amphiphatic molecule comprising a ketone in
the hydrophilic portion is R1C(0)R2 and the amphiphatic molecule comprising
an oxyamine in the hydrophilic portion is R3-0-NH2, wherein R1 and R3 are
independently selected from C6_30alkyl and C6_30alkenyl and R2 is C1.2alkyl.
[0050] In a further
embodiment of the application, the reactive
functional group in the liposomes of type A is a ketone and the reactive
functional group in the liposomes of type B is a hydrazine. Accordingly, in
another embodiment the present application also includes a liposome
comprising an amphiphatic molecule wherein the hydrophilic portion of the
amphiphatic molecule comprises a hydrazine. In a specific embodiment, the
amphiphatic molecule comprising a hydrazine in the hydrophilic portion is R4-
NH-NH2, wherein R4 is C6_30alkyl.
[0051] In a further
embodiment of the application, the reactive
functional group in the liposomes of type A is an azide and the reactive
functional group in the liposomes of type B is an alkyne. Accordingly, in
another embodiment the present application includes a liposome comprising
an amphiphatic molecule wherein the hydrophilic portion of the amphiphatic
molecule comprises an azide. In a further
embodiment the present
application includes a liposome comprising an amphiphatic molecule wherein
the hydrophilic portion of the amphiphatic molecule comprises an alkyne. In a
specific embodiment, the amphiphatic molecule comprising an azide in the
CA 02776618 2012-05-10
17
hydrophilic portion is R6-N3 and the amphiphatic molecule comprising an
oxyamine in the hydrophilic portion is R6-CE-CR7, wherein R6 and R6 are
independently selected from C6_30alkyl and C6_30alkenyl and R7 is H or C1-
2alkyl.
5 [0052] In a further embodiment
of the application, the reactive
functional group in the liposomes of type A is a diene and the reactive
functional group in the liposomes of type B is a dienophile. Accordingly, in
another embodiment the present application includes a liposome comprising
an amphiphatic molecule wherein the hydrophilic portion of the amphiphatic
molecule comprises a diene. In a further embodiment the present application
includes a liposome comprising an amphiphatic molecule wherein the
hydrophilic portion of the amphiphatic molecule comprises a dienophile. In an
embodiment, the hydrophobic portion of these amphiphatic molecules is C6_
30alkyl.
[0053] The present application
also include compositions comprising
one or more of the above-identified liposomes. In a further embodiment, the
composition further comprises a solvent, diluent or carrier, such as an
aqueous buffer.
[0054] In an embodiment, the
present application includes a
composition comprising the liposome comprising an amphiphatic molecule
wherein the hydrophilic portion of the amphiphatic molecule comprises a
ketone and a solvent, diluent or carrier. In a further embodiment the present
application also includes a composition comprising the liposome comprising
an amphiphatic molecule wherein the hydrophilic portion of the amphiphatic
molecule comprises an oxyamine and a solvent, diluent or carrier, such as an
aqueous buffer.
[0055] In an embodiment of the
application, aside from the amphiphatic
molecule comprising a reactive functional group, the liposomes further
comprise any suitable amphiphatic molecule, or mixture of molecules, that
form liposomes. In general, liposome-forming amphiphatic molecules are
lipids, in particular phospholipids. In a further embodiment, the liposome-
.
CA 02776618 2012-05-10
18
forming amphiphatic molecules are selected based on the proposed use of
the liposome. For example, if the liposomes are to be adhered to each other,
the liposome-forming amphiphatic molecule is any suitable neutral, positively
charged or negatively charged amphiphatic molecule or a mixture thereof. In
general, to enhance the attraction between the two entities to be adhered or
fused, the charges on each entity are opposite. Examples of suitable
liposome-forming amphiphatic molecules are diverse and the present
application is not limited to any specific type. Selection of the liposome-
forming amphiphatic molecule and methods for the formation of liposomes are
well within the skill of a person in the art.
[0056] For example, the liposomes are formed by dissolving the
amphiphatic molecule comprising a reactive functional group in an organic
solvent and thoroughly combining the resulting solution with the liposome-
forming amphiphatic molecule(s), also dissolved in an organic solvent,
followed by removal of all of the organic solvents. The dried samples are then
= reconstituted and brought to the desired concentration in an aqueous
buffer
solution, such as an aqueous buffer having a pH of about 7 to about 7.5.
Sonication and warming may be used to obtain a clear solution of large
unilamellar vesicles (LUVs).
[0057] As an example, the liposome-forming amphiphatic molecule is
selected from egg palmitoyl-oleoyl phosphatidylcholine (POPC ¨ a neutral
phospholipid), egg 1-palmitoy1-2-oleoyl-phophatidylglycerol (POPG, a
negatively charged phospholipid) and 1,2,-dioleoy1-3-trimethylammonium-
propane (DOTAP ¨ a positively charged or cationic lipid).
[0058] In an embodiment, the amount of the amphiphatic molecule
comprising a reactive functional group in the liposome is about 1 mol% to
about 10 mol%, or about 5 mol%. It is another embodiment, that the liposome
comprises about 90 mol% to about 99 mol% of a neutral lipid and, optionally,
about 1 mol% to about 5 mol% of a charged lipid.
[0059] In another embodiment of the application, the liposomes of type
A and type B further comprise fluorescent reporter molecules. In one
CA 02776618 2012-05-10
19
embodiment, the fluorescent reporter molecules are incorporated into the
liposome-forming amphiphatic molecules.
[0060] In yet another embodiment, the liposomes further
comprise
other functional molecules, such as fluorescent molecules, dyes and/or other
indicator molecules, so that when the liposomes of type A and type B are
fused, a physical change, such as a change in color, fluorscence or smell,
occurs. These functional molecules may be entrapped within the liposomes
or be incorporated into the liposome-forming amphiphatic molecules.
[0061] In a further embodiment of the application, the
liposomes of type
A and/or B further comprise biologically active agents, such as nucleic acids,
proteins, peptides, small molecule drugs, carbohydrates and the like, and
mixtures thereof, and fusion of the liposomes with the cell population results
in
the delivery of the biological agents into the cells. The biologically active
agents may be entrapped within the liposome or may be incorporated into the
liposome membrane.
[0062] In another embodiment of the application, the liposomes
of type
A and type B further comprise fluorescent reporter molecules. In one
embodiment, the fluorescent reporter molecules are incorporated into the
liposome-forming amphiphatic molecules. When present in the liposome-
forming amphiphatic molecules, it is an embodiment that these molecules are
incorporated into the liposomes in an amount of about 0.5 mol % to about 5
mol %, or about 2 mol%. As a representative example, the fluorescent
phospholipids, egg 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine-N-(7-
= nitro-2-1,3-benzoxadiazol-4-y1) (ammonium salt) (NBD-PE), and egg 1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B
sulfonyl) (ammonium salt) (Rhod-PE), are used. The incorporation of
fluorescent reporter molecules into the liposome-forming amphiphatic
molecules allows for easy monitoring of liposome adhesion and fusion. For
example, the use of NBD-PE (a fluorescence donor) in the liposomes of type
A and rhod-PE (a fluorescence acceptor) in the liposomes of type B results in
a gradual decrease in the donor emission peak and increase in the acceptor
CA 02776618 2012-05-10
emission peak upon adhesion of the liposomes of type A to the liposomes of
type B.
[0063] The present application also includes a method for promoting
adhesion of liposomes comprising contacting a plurality of liposomes of type A
5 with a plurality of liposomes of type B, wherein the liposomes of type A
comprise a reactive functional group that reacts with a reactive functional
group comprised in the liposomes of type B to form a chemical interaction that
results in the adhesion of the liposomes of type A and the liposomes of type
B.
[0064] The present application further includes kits or commercial
10 packages for performing the method of promoting the adhesion of
liposomes.
In an embodiment, the kit or package comprises, in separate containers, a
solution of a plurality of liposomes of type A and a solution of a plurality
of
liposomes of type B, wherein the liposomes of type A comprise a reactive
functional group that reacts with a reactive functional group comprised in the
15 liposomes of type B to form a chemical interaction that results in the
adhesion
of the liposomes of type A and the liposomes of type B, along with
instructions
for performing the method. In one embodiment, the kit or package further
comprises separate means for forming bubbles with the each of the plurality
of liposomes of type A and a plurality of liposomes of type B. Any means for
20 forming bubbles may be used, such as any shaped device upon which a film
of the solution comprising the liposomes of type A and the solution of the
liposomes of type B can form and the user can apply a flow of a gas, such as
air, to form bubbles. Examples of such means includes the typical bubble
blowing devices that are found in children's bubble forming toy products. In
an embodiment, the instructions include directions to form a bubble from each
of the solutions of liposomes of types A and B and to bring the bubbles into
contact with each other. In a further embodiment, each of the liposomes of
type A and type B further comprise an indicator molecule, such as a dye, and
contact of the bubbles of type A with the bubbles of type B results in a fused
bubble having a different detectable property, such as a different colour. In
an
CA 02776618 2012-05-10
21
embodiment, these kits and commercial packages are used or sold as novelty
items or toys.
III. Liposome fusion to cells
[0066] Liposome fusion to mammalian cell membranes was directed
through the use of a cationic lipid and a molecular recognition pair for
chemoselective ligation. In one embodiment, vesicles were tailored with
ketone (dodecanone) or oxyamine (0-dodecyloxyamine) molecules, a neutral
lipid, egg palmitoyl-oleoyl phosphatidylcholine (POPC), and a cationic lipid,
1,2-dioleoy1-3-trimethylammonium-propane (DOTAP). The resulting two
vesicle populations were then integrated with mammalian cells in culture.
Applications for this strategy, include, but are not limited to, small
molecule
delivery, cell-surface modification, and tissue engineering. By employing this
membrane tailoring strategy, the assembly of 3D spheroid clusters and tissue-
like structures were directed after culturing two cell populations
functionalized
with oxyamine- and ketone-containing groups. Because this method is
general, bio-orthogonal, chemically stable, and non-cytotoxic, patterned multi-
layered tissue-like structures of different geometric shapes could also be
fabricated without the use of 3D scaffolds to confine the cell populations. It
has also been shown that this method has promising use in stem cell
transplantation by co-culturing human mesenchymal stem cells (hMSCs) with
fibroblasts (fbs) and inducing adipocyte differentiation while in a 3D multi-
layered tissue-like structure.
[0066] The present application therefore includes a method for
promoting the adhesion of cells comprising:
(a) contacting a first cell population with a liposome of type A under
conditions
for the fusion of the liposome of type A with the first cell population;
(b) contacting a second cell population with a liposome of type B under
conditions for the fusion of the liposome of type B with the second cell
population; and
CA 02776618 2012-05-10
22
(c) contacting the fused first cell population with the fused second cell
population,
wherein the liposomes of type A comprise a reactive functional group that
reacts with a reactive functional group comprised in the liposomes of type B
to
= 5 form a chemical interaction that results in the adhesion of
the first and second
cell populations.
[0067] To
promote the fusion of the liposomes to cells, a mixture of
neutral, positively and/or negatively charged liposome-forming amphiphatic
molecules may be used. For example, fusion to mammalian cells types,
whose membranes comprise a negative charge, is promoted by incorporating
positively charged lipids in to the liposome. While not wishing to be limited
by
theory, the positively charged lipid enhances membrane fusion via
electrostatic destabilization. In an
embodiment, the positively charged
liposomes are incorporated in an amount of 1 mol% to about 5 mol%, or about
2 mol%. Promotion of liposome fusion to other cell types, including plants,
bacteria, viruses and the like, can be done using a similar strategy depending
on the characteristics of the cell membrane.
[0068] The
conditions for the fusion of the liposomes with the cell
populations generally involve adding an aqueous buffered solution of the
liposomes to the cells in culture and incubating the cells in the presence of
the
liposomes for example, for 6 to about 24 hours. In an embodiment the
= solution of the liposomes is added at a concentration of about 0.5 to 5
mM
and about 1 to about 10 mL of this solution is added to about 1 to about 10
mL of the cultured cells. When the cell populations are incubated with the
liposomes comprising a reactive functional group, membrane fusion occurs,
resulting in the presentation of the reactive functional groups from the cell
surfaces. These reactive functional groups are available for further reaction
so that when these cell populations are contacted together, interconnected,
3D tissue-like structures form, mediated through chemoselective reactions
between the complementary functional groups.
CA 02776618 2012-05-10
23
[0069] The contacting the fused first cell population with the
fused
second cell population can be done using any suitable means. For example,
the cell populations may be combined in solution. As a representative
example, oxyamine presenting rat2 fibroblasts were combined in solution with
ketone-presenting Swiss albino 3T3 fibroblasts and, upon mixing, these two
cell populations formed clusters and tissue-like masses. This is a significant
finding as current methods to generate these types of structures require the
support of a 3D hydrogen matrix and/or assisted assembly through an
external stimulus.
[0070] Alternatively, one of the cell populations may be grown on a
substrate and the second cell population added as a layer on top of the first
population, followed by addition of alternate layers of the first and second
population of cells. In this embodiment, larger, dense 3D tissue-like networks
are formed with geometric control. In this embodiment, the 3D-tissue like
networks are released from the substrate using, for example, agitation or
washing, accordingly, this method provides the possibility for applications in
tissue engineering and cellular transplantation.
[0071] Another alternative is to combine the two cell
populations in a
continuous fashion, for example, by flowing one stream comprising the first
population of cells into a second stream comprising the second population of
cells.
[0072] In an embodiment, at least one of the population of
cells is a
stem cell and adhesion of a second population of a specific cell type results
in
induced differentiation and proliferation of the stem cells as the second cell
type. This result holds great potential for areas of regenerative medicine and
= stem cell transplantion.
[0073] In a further embodiment of the application, the
liposomes of type
A and/or B further comprise biologically active agents, such as nucleic acids,
proteins, peptides, small molecule drugs, carbohydrates and the like, and
mixtures thereof, and fusion of the liposomes with the cell population results
in
the delivery of the biological agents into the cells. The biologically active
CA 02776618 2012-05-10
24
agents may be entrapped within the liposome or may be incorporated into the
liposome membrane.
[0074] In yet another embodiment, the liposomes of type A
and/or B
further comprise other functional molecules, such as fluorescent molecules,
dyes and/or other indicator molecules, so that when the first and second cell
populations are adhered, a physical or sensory change, such as a change in
color or fluorscence occurs. These functional molecules may be entrapped
within the liposomes or be incorporated into the liposome-forming amphiphatic
molecules.
[0075] In another embodiment of the application, the liposomes of type
A and type B further comprise fluorescent reporter molecules. In one
embodiment, the fluorescent reporter molecules are incorporated into the
liposome-forming amphiphatic molecules. When present in the liposome-
forming amphiphatic molecules, it is an embodiment that these molecules are
incorporated into the liposomes in an amount of about 0.5 mol % to about 5
mol %, or about 2 mol%. As a representative example, the fluorescent
phospholipids, egg 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine-N-(7-
. nitro-2-1,3-benzoxadiazol-4-y1) (ammonium salt) (NBD-PE), and egg
1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B
sulfonyl) (ammonium salt) (Rhod-PE), are used. The incorporation of
fluorescent reporter molecules into the liposome-forming amphiphatic
molecules allows for easy monitoring of liposome fusion and subsequent cell
adhesion.
[0076] In a further embodiment of the application, the
liposomes of type
A and/or B further comprise biologically active agents, such as nucleic acids,
proteins, peptides, small molecule drugs, carbohydrates and the like, and
mixtures thereof, and fusion of the liposomes with the cell population results
in
the delivery of the biological agents into the cells. The biologically active
agents may be entrapped within the liposome or may be incorporated into the
liposome membrane.
CA 02776618 2012-05-10
[0077] The present application also includes cell populations
whose
surfaces have been modified with reactive functional groups by fusion with the
liposomes of type A and/or B, compositions comprising these cell populations
and all uses thereof.
5 [0078] The following non-limiting examples are illustrative of the
present application:
EXAMPLES
Materials and Methods
[0079] All chemical reagents were of analytical grade and used
without
10 further purification. Lipids, egg palmitoyl-oleoyl phosphatidylcholine
(POPC),
egg 1-palmitoy1-2-oleoyl-phosphatidylglycerol (POPG), 1,2-dioleoy1-3-
trimethylammonium-propane (DOTAP), egg 1,2-diphytanoyl-sn-glycero-3-
phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-y1) (ammonium salt)
(N BD-PE), and egg 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
.
15 (lissamine rhodamine B sulfonyl) (ammonium salt) (Rhod-PE) were
purchased
from Avanti Polar Lipids (Alabaster, AL). Antibodies and fluorescent dyes
were obtained from Invitrogen (Carlsbad, CA). FITC labeled beads were
purchased from Spherotech, Inc. (Forest Lake, IL) and all other chemicals
were obtained from Sigma-Aldrich or Fisher. Swiss 3T3 albino mouse
20 fibroblasts (fbs) were obtained from the Tissue Culture Facility at the
University of North Carolina (UNC).
[0080] Transmission electron microscopy (TEM) images were
acquired
using a TF30He Polara G2 (FEI company) electron cryo microscope,
operating at 300 keV. Images were recorded using a Tietz single port model
25 415 4k x 4k CCD camera with a 15-pm pixel size. Fluorescence resonance
= energy transfer measurements (FRET) were performed using a SPEX
Fluorolog-3 Research T-format Spectrofluorometer with an excitation
wavelength of 471 nm. Dynamic light scattering was performed using a
Nikomp model 200-laser particle sizer with a 5 mW HeNe laser at an
excitation wavelength of 632.8 nm and using a Wyatt DynoPro plate reader.
CA 02776618 2012-05-10
26
Flow cytometry was performed using a Dako CyAn ADP (Beckman-Cou)ter,
=
Brea, CA), and the data was analyzed with Summit 4.3 software. Phase
contrast and fluorescent imaging was performed and processed using a Nikon
TE2000-E inverted microscope and Metamorph software, respectively.
[0081] Tetra(ethylene glycol)-terminated alkanethiol (EG4) was
synthesized as previously reported." Fluorescein-ketone (7) was synthesized
as previously reported.45 The syntheses of 0-dodceyloxyamine (A) (Scheme
1) and rhod-oxyamine (8) are described below.
0
Br H
Reagents and conditions. (i) N-hydroxyphthalimide (1.5 eq), NaHCO3 (1.5 eq),
DMF, reflux, 80 C, 12 h; 87 % and (ii) hydrazine (6 eq), dry DCM, N2, 12 h;
74%.
= Scheme 1
Example 1: 2-(dodecyloxy)isoindoline-1,3-dione (B)
[0082] As shown in Scheme 1, 1-bromododecane was added to a
solution of N-hydroxyphthalimide (1.96 g, 12.04 mmol, 1.5 eq) and sodium
bicarbonate (10.11 g, 12.04 mmol, 1.5 eq) in DMF (20 mL) at 80 C (1.93 mL,
8.02 mmol). The mixture was refluxed and stirred for 12 h. The reaction was
diluted with DCM and washed with H20 (6 x 50 mL), 1 M NaHCO3 (3 x 50 mL),
and H20 (2 x 50 mL), dried over MgSO4, and concentrated to afford a white
solid, B (2.66 g, 87 %). 1H NMR (400 MHz, CDCI3) 6 0.91 (m, 3H), 1.28 (bm,
16H), 1.47-1.49 (m, J = 9.2 Hz, 2H), 1.77-1.83 (m, J = 22.0 Hz, 2H), 4.20-4.23
(t, J = 13.6 Hz, 2H), 7.28-7.30, 7.75-7.77 (dm, J = 4.8, Hz, J = 5.6 Hz, 2H,
2H).
(ESI) (m/z) [M + 332.28.
CA 02776618 2012-05-10
27
Example 2: 0-dodecyloxyamine (A).
[0083] As shown in Scheme 1, hydrazine was slowly added to a
solution of B (2.65 g, 8.00 mmol) in dry DCM (30 mL) under inert atmosphere
(Ar) (1.53 mL, 48.00 mmol, 6 eq). Upon addition, a white precipitate
immediately formed. The mixture was stirred for 12 h. The reaction was
diluted with DCM and washed with H20 (6 x 50 mL), dried over MgSO4, and
concentrated to afford a pale yellow oil, A (1.18 g, 74 %). 1H NMR (400 MHz,
CDCI3) 6 0.88-0.91 (t, J = 13.6 Hz, 3H), 1.28 (s, 18H), 1.57-1.60 (m, J = 14.0
Hz, 2H), 3.65-3.69 (t, J = 13.2 Hz, 2H). (ESI) (m/z) [M + Hi: 201.22.
')4. 0 tr
loco NII
1. 0
.03S '03S, 01S
.0
0'
0
''),= 0 - tc-
roe.).
iiiiv
- 35
,0 H H .0 H
Cr'S cr,S:g
8
Reagents and conditions. (i) N-B0C-1,4-diaminobutane (1.5 eq), TEA (1.5 eq),
CHCI3, Nz 25 C, 8 h; 95 %, (ii) triisopropylsilane (TIPS)/H20/TFA (2.5: 2.5:
95), N2, 25 C, 3 h; 85 %, (iii) N-hydroxysuccinimide (NHS, 2 eq), N,N'-
dicyclohexylcarbodiimide (DCC, 2 eq), aminooxy acetic acid (2 eq), TEA
(excess), DMF, N2, 25 C, 4 h; 60 A, and (iv) TIPS/H20/TFA (2.5 : 2.5 : 95),
N2, 25 C, 3 h; 81 %.
Scheme 2
CA 02776618 2012-05-10
28
Example 3: (N-(4-(tert-butoxycarbonylamino)butyl)sulfamoy1)-2-(6-
(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yObenzenesulfonate (C):
[0084] As shown in
Scheme 2, to a solution of rhodamine lissamine
(0.880 g, 1.53 mmol) in chloroform (CHCI3, 30 mL) at room temperature (RI)
was added N-B0C-1,4-diaminobutane (0.431 g, 2.29 mmol, 1.5 eq) and TEA
(0.305 mL, 2.29, 1.5 eq). The mixture was stirred for 8 h and then extracted
with H20 (6 x 25 mL). The organic layers were concentrated to afford a dark
purple solid C. 1H NMR was taken in CDCI3 to confirm C (1.045 g, 95 %). TLC
conditions for entire synthesis: CHC13:Me0H (7.5:2.5). 1H NMR (400 MHz,
Me0D) 6 1.09-1.07 (t, J = 8.1 Hz, 6H), 1.36-1.33 (m, J = 12.3, 15H), 1.66-1.64
(m, J = 8.6 Hz, 4H), 3.47-3.44 (m, J = 12.1, 6H), 4.20-4.18 (q, J = 7.8 Hz,
4H),
5.66 (s, 1H), 5.77 (d, 1H), 6.01 (d, 1H), 6.34-6.30 (m, J = 16.1 Hz, 2H), 7.21
(d,
1H), 7.29 (d, 1H), 7.98 (d, 1H), 8.04 (d, 1H). (ESI) (m/z) [M + H4): 716.31.
Example 4: 5-(N-(4-
aminobutyl)sulfamoy0-2-(6-(diethylamino)-3-
(diethyliminio)-3H-xanthen-9-yl)benzenesulfonate (D):
[0085] As shown in
Scheme 2, to C (0.600 g, 0.837 mmol) was added a
solution of TFA, H20, and triisopropylsilane (TIPS) in a ratio of 95: 2.5: 2.5
(10
mL). The mixture was stirred at RT under N2 for 3 h and was then extracted
with CHCI3 and H20 (4 x 25 mL). The organic layers were dried and
concentrated to afford a purple solid, D (0.45 g, 85 %). 1H NMR (400 MHz,
Me0D) 6 1.11-1.09 (t, J = 8.7, 6H), 1.33-1.31 (m, J = 7.4 Hz, 6H), 1.70-1.67
(m, 4H, J = 12.5, 4H), 2.63-2.62 (m, J = 4.6 Hz, 2H), 3.51-3.49 (m, J = 8.7
Hz,
6H), 4.20-4.18 (q, J = 7.8 Hz, 4H), 5.64 (s, 1H), 5.71 (d, 1H; Ar-H), 6.02 (d,
1H), 6.32-6.30 (m, J = 8.3 Hz, 2H), 7.24 (d, 1H), 7.30 (d, 1H), 7.98 (d, 1H),
8.04 (d, 1H). (ESI) (m/z) [M + HI: 628.27.
Example 5: 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-
(2,2-dimethy1-4,8-dioxo-3,6-dioxa-5,9-diazatridecan-13-
yl)sulfamoyl)benzenesulfonate (E):
[0086] As shown in
Scheme 2, to a solution containing N,N'-
dicyclohexylcarbodiimide (DCC, 0.394 g, 1.91 mmol, 2 eq), N-
CA 02776618 2012-05-10
29
hydroxysuccinimide (NHS, 0.220 g, 1.91 mmol 2 eq), and aminooxy acetic
acid (0.356 g, 1.91 mmol, 2 eq) in DMF was stirred under N2 for 0.5 h. D (0.43
g, 0.684 mmol) was then added in DMF (20 mL), followed by TEA (excess).
The mixture was strirred for 4 h and then concentrated. Flash chromatography
was performed using CHC13:Me0H (8:2) to elute, E. The product was
concentrated to afford a purple solid E (0.32 g, 60 %). 1H NMR (400 MHz,
Me0D) 6 1.10-1.08 (t, J = 8.8, 6H), 1.39-1.36 (m, J = 12.3 Hz, 15H), 1.65-1.63
(m, J = 7.9, 4H), 3.08-3.06 (m, J = 8.0, 2H), 3.48-3.46 (m, J = 8.3, 6H), 4.17-
4.15 (q, J = 7.7, 4H), 4.38 (s, 2H), 5.61 (s, 1H), 5.73 (d, 1H), 6.02 (d, 1H),
6.31-6.30 (m, J = 4.4, 2H), 7.24 (d, 1H), 7.32 (d, 1H), 7.96 (d, 1H), 8.09 (d,
1H). (ESI) (m/z) [M + H]: 801.31.
Example 6: 5-(N-(4-(2-(aminooxy)acetamido)butyl)sulfamoy1)-2-(8-
(diethylamino)-3-(diethyliminio)-31-1-xanthen-9-yObenzenesulfonate
(rhod-oxyamine, 8):
[0087] As shown in Scheme 2, to E (0.30 g, 0.374 mmol) was added a
solution of TFA, H20, and triisopropylsilane (TIPS) in a ratio of 95: 2.5: 2.5
(10
mL). The mixture was stirred at RT under N2 for 3 h and was then extracted
with CHCI3 and H20 (4 x 25 mL). The organic layers were dried and
concentrated to afford a purple solid and flash chromatography was
performed using CHC13:Me0H (8:2) to elute, 8 (0.21 g, 81 %) 1H NMR (400
MHz, CDC13) 5 1.12-1.00 (t, J = 8.2, 6H), 1.42-1.40 (m, J = 7.9, 6H), 1.62-
1.60
= (m, J = 7.7, 4H), 3.07-3.05 (m, J = 8.0, 2H), 3.45-3.42 (m, J = 12.4,
6H), 4.11-
4.09 (q, J = 8.4, 4H), 4.24 (s, 2H), 5.64 (s, 1H), 5.75 (d, 1H), 6.02 (d, 1H),
6.29-6.27 (m, J = 4; 2H), 7.28 (d, 1H), 7.31 (d, 1H), 7.92 (d, 1H), 8.05 (d,
1H).
(ESI) (m/z) [M + Hi: 701.28.
Example 7: Formation of lipid vesicles. Liposome fusion studies.
= [0088] Dodecanone (55 pL, 10 mM in CHCI3 at 5 mol %)
was dissolved
with egg palmitoyl-oleoyl phosphatidylcholine (POPC) (430 pL, 10 mg/mL in
CHC13, at 95 mol %) and 0-dodecyloxyamine (60 pL, 10 mM in CHCI3 at 5
mol %) was mixed with POPC (410 pL, 10 mg/mL in CHCI3 at 75 mol %), and
egg 1-palmitoy1-2-oleoyl-phosphatidylglycerol (POPG) (92 pL, 10 mg/mL in
CA 02776618 2012-05-10
CHCI3 at 20 mol %). Both lipid sample mixtures were then concentrated under
high vacuum for 4 h. The dried lipid samples were reconstituted and brought
to a final volume of 3 mL in PBS buffer, pH 7.4. The contents of the vial were
warmed to 50 C and sonicated for 20 min, in a tip sonicator, until the
solution
5 became clear and large unilamellar vesicles (LUVs) containing ketone
(keto-
LUV, 1) or oxyamine (oxy-LUV, 2) groups were formed (see Figure 1b).
Example 8: FRET fusion studies
[0089] NBD-PE and rhod-PE were added to two separate vials at 2
mol %. The dried lipid samples were then reconstituted in 2.43 mL of PBS
10 buffer, pH 7.4. The contents of the vial were warmed to 50 C and
sonicated
for 20 min, in a tip sonicator, until the solution became clear, and LUVs
containing ketone (keto-NBD-PE LUVs, 3) or oxyamine (oxy-rhod-PE LUVs, 4)
groups were formed (see Figure 1c).
Example 9: Liposome fusion to cells
15 [0090] To generate ketone- and oxyamine-containing liposomes for cell
fusion studies, dodecanone (55 pL, 10 mM solution in CHCI3 at 5 mol %) or
0-dododecyloxyamine (60 pL, 10 mM solution in CHCI3 at 5 mol %) were
= dissolved with egg-POPC (424 pL, 10 mg/mL in CHCI3 at 93 mol %) and 1,2-
dioleoy1-3-trimethylammonium-propane (DOTAP, 10 pL, 10 mg/mL in CHCI3
20 at 2 mol %) in chloroform followed by concentration under high vacuum
for 4 h.
The dried lipid samples were then reconstituted and brought to a final volume
of 3 mL in PBS buffer, pH 7.4. The contents of the vial were warmed to 50 C
and sonicated for 20 min, in a tip sonicator, until the solution became clear,
and LUVs containing ketone (5) or oxyamine (6) groups were formed (Figure
25 1d).
Example 10: Matrix-assisted laser-desorptionlionization mass
spectrometry (MALDI-MS). Preparation of gold-coated MALDI sample
plates.
[0091] Gold-coated MALDI sample plates (123 x 81 mm) (Applied
30 Biosystems, Foster City, CA) were prepared by electron-beam deposition
31
(Thermionics Laboratory Inc, Hayward, CA) of titanium (5 nm) and then gold
(12 nm). In order to form self-assembled monolayers (SAM) of alkanethiolates
on the plates, the slides were immersed in a 1-mM solution of
aminooxyundecanethiol in Et0H for approximately 1 min, rinsed with Et0H
and dried, and then backfilled with a 1-mM solution of mercaptoundecanol in
Et0H for 1 h. Once removed from solution, the surfaces were rinsed with
Et0H and dried before use.
[0092] Keto-LUVs (1) were generated as described above and were
then delivered and allowed to react with the oxyamine-terminated MALDI
sample plate (90 min). The plates were then washed with water (3 x 3 mL)
and Et0H (2 x 3 mL) and dried before use.
[0093] MS analyses were carried out using an AB SCIEX TOF/TOFTm
5800 System (Applied Biosystems, Foster City, CA) (see Figure 2a).
Example 11: Dynamic light scattering (DLS)
[0094] Keto- (1) and oxyamine- (2) LUVs were generated as described
above and tested by DLS for monodispersity and uniformity. Light scattering
experiments were performed using a Nikomp Model 200 Laser Particle Sizer
with a 5 mW Helium-Neon Laser at an exciting wavelength of 632.8 nm.
Standard deviation determinations were made using Gaussian analysis. A
WyattTM DynoPro Dynamic Scattering Plate Reader was used to collect the
light scattering data.
Example 12: FRET analyses
[0095] Keto- (3) and oxyamine- (4) LUVs containing NBD-PE and rhod-
PE, respectively, were generated as described above and tested by FRET. All
fluorescence measurements were performed in a SPEX Fluorolog-3 Research
T-format Spectrofluorometer. NBD fluorescence was measured at 471 nm
(excitation) and 531 nm (emission), maintaining narrow excitation slits to
reduce light scattering interference. To obtain FRET measurements, the NBD
dye was excited at 471 nm, and the emission was scanned through 600 nm,
and the emission signal for rhod-PE was observed at 578 nm. Fluorescence
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was followed immediately after mixing oxy-rhod-PE LUV (4, 3 mM in PBS,
100 IA) with keto-NBD-PE LUV (3, 3 mM in PBS, 100 L) for approximately 2
h at 2 min intervals. The total lipid concentrations were adjusted to 0.2 mM,
and the two LUV populations were had a 1:1 molar ratio. A constant flow of
water was passed through the cuvette holder for temperature control. The
temperature was maintained at 25 C (see Figure 2c).
Example 13: TEM analyses
[0096] Keto- (1) and oxyamine- (2) LUVs were made as described
above (0.2 mM in PBS, pH 7.4). The two vesicle solutions (1:1) were mixed at
room temperature for 30 min. 4 pL of vesicles suspended in buffer were
applied to standard lacey carbon EM grids which were prepared according to
published methods. The specimens were blotted from behind and then
submerged into aurenyl acetate solution for staining. The hydrated specimens
were then placed into a TF30He PolaraTM G2 (FEI company) electron cryo
microscope operating at 300 keV. Images were recorded using a TietzTm
single port model 415 4k x 4k CCD camera with a 15 micron pixel size on the
chip. Pixel sizes at the specimen level were used to calculate accurate
dimensions for the specimen (see Figure 2b).
Example 14: Fibroblast (Fb) culture
[0097] Swiss 3T3 albino mouse fbs and Rat2 fbs were cultured in
Dulbecco's Modified Eagle Medium (Gibco) containing 10 % calf bovine
serum (CBS) and 1 % penicillin/streptomycin at 37 C in 5 % CO2.
[0098] Cells were seeded onto a tissue culture plate and allowed
to
grow for 48 h at 37 C in 5 % CO2 in CBS media.
Example 15: Cell-surface engineering
[0099] Two cell-surface engineering methods were employed to
fluorescently label fbs. In this first method, a solution of oxyamine vc-LUVs
(6,
3 mM) was incubated with a ketone-functionalized fluorescein (7, 0.15 mM, 1
eq, 2 h), forming fluorescently labeled liposomes. The liposomes were then
added to fbs in culture for 2 h. After fusion, the cells were washed with PBS
(3
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x 2 mL), trypsinized (1 mL, 5 min, 37 C, 5 % CO2), diluted with CBS-
containing media (-102/mL), and seeded to a glass substrate (1 x 1 cm2, 2 h).
The cells were then imaged under a fluorescence microscope with an
exposure time of 1/1200 s. In the second method, a solution of keto-LUVs (5,
200 pL, 0.6 mM) was added to fbs in culture for 2 h, resulting in membrane
fusion and subsequent display of ketones from the cell surface (9). Rhod-
.
oxyamine (8, 100 pL, 0.7 mM in H20) was then added the cells for 2 h. After
oxime formation, the fbs were washed with PBS (3 x 2 mL), trypsinized (1 mL,
5 min, 37 C, 5 % CO2), diluted with CBS-containing media (-102/mL), and
seeded to a glass substrate (1 x 1 cm2, 2 h). The cells were then imaged
under a fluorescence microscope with an exposure time of 1/1200 s (see
= Figure 3).
Example 16: Cell adhesion patterning
[00100] Self-
assembled monolayers (SAMs) presenting aldehyde or
oxyamine and tetra(ethylene glycol) (EG4) groups were patterned using
microfluidic oxidation and microfluidic lithography, respectively.34'36 Eat
has
= been shown to passivate substrates against cell and protein adsorption.36
Therefore, the ratio of EG4 and aldehyde or oxyamine groups was 90:10 to
ensure that fbs were only adhering to the patterned surface regions that
presented 10 % oxyamine or aldehyde groups, driven via oxime conjugation.
Fbs were separately cultured with keto- (5) or oxyamine- (6) LUVs as
previously described and were then seeded (-102 cells/mL, 2 h) to the
= patterned oxyamine or aldehyde surfaces, respectively. Media that 10 %
calf
bovine serum (CBS) and 1 % penicillin/streptomycin was then added, and the
substrates were incubated at 37 C in 5 % CO2 for 4 d. Cells cultured with
liposomes, not containing the key functional groups, did not attach to the
patterned surfaces. Substrates were then stained and imaged by fluorescence
microscopy. An exposure time of 400 and 1200 ms were used to image nuclei
and actin, respectively (see Figure 4).
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=
34
Example 17: Flow cytometry
[00101] Liposomes with varying oxyamine mol % (i.e., 0 %, 1 %, 5 %,
and 10 %) were generated and cultured with separate populations of fbs (6, 3
mM, 4 h), resulting in membrane fusion and subsequent display of oxyamine
groups from cell surfaces (10). Ketone-functionalized fluorescein (7, 0.15 mM
2 h) was then reacted with the fbs, generating fluorescently labeled cells.
The
control cells (i.e., not displaying oxyamine groups) were incubated with
ketone-fluorescein for 2 h each, under the same conditions. The cells were
then washed with PBS (3 x 5 mL), trypsinized (1 mL, 5 min, 37 C, 5 % CO2),
centrifuged (5 min, 1000 rpm), resuspended in RPMI (without phenol red),
centrifuged (5 min, 1000 rpm), and resuspended in RPM! (-107 cells/2 mL).
Fluorescence-assisted cell sorting analyses (FACS) of the control and fbs with
1 %, 5 %, and 10 % oxyamine were then performed (2 x 103 cells).
Fluorescence measurements were calibrated using RCP-5-30 beads (-107
beads/mL, 2 x 103 beads counted, Spherotech, Inc., Lake Forest, IL) of known
fluorescein equivalent molecule density.37 The RCP-5-30 beads contain a
mixture of several similar size particles with different fluorescence
intensities
and a blank. Every particle contains a mixture of fluorophores that allows
excitation at any wavelength from 365 to 650 nm. As a result, the RCP-30-5
beads have a two-fold purpose: (1) calibrate the different channels in the
flow
cytometer being used and (2) cross-calibrate the relative number of
fluorophores with cells or particles stained with known number of spectral
matching fluorophores, such as FITC, to estimate the number of fluorophores
on stained cells. No background is required to be subtracted because the
different fluorophores are calibrated to the different flow cytometer
channels.
The raw data obtained in Figure 5(b) was cross-calibrated to the calibration
curve that is generated with the RCP-30-5 beads to obtain the values seen in
Figure 5(c). The approximations of FITC molecules per cell in Figure 5(c)
were determined by cross calibrating the 0% (control), 1%, 5%, and 10%
oxyamine-containing liposomes to the standard curve (blank and 5
fluorophores) generated using the manufacturer's excel spreadsheet and
35
instructions (Spherotech, Inc., Lake Forest, IL). After generating a standard
calibration curve with the RCP-30-5 beads, the mean fluorescence intensities
obtained from the FITC channel, were cross calibrated with the curve using
the manufacturer's spreadsheet to produce an approximation of the number of
molecules per cell. The number of counted beads and each sample were the
same.
[00102] Fluorescent intensities based on number of cells counted
were
compared to the standard bead and control cells lacking fluorescent molecule
conjugation and approximate numbers of fluorescent compound bound to the
surface was calculated. Flow cytometry was carried out using a Dako TM CyAn
ADP (Beckman-Coulter, Brea, CA), and data was analyzed with Summit 4.3
software.
Example 18: 30 spheroid generation
[00103] Keto- (1) and oxyamine-LUVs (2) were added to two separate
fb
populations in culture for (3 mM in tris buffer, 400 ?IL added to 4 mL, 12 h),
resulting in fusion and display of ketones and oxyamines from the cell
surface.
Oxyamine-presenting Rat2 fbs (10) contained an m-cherry label (nucleus) for
enhanced visualization, while the ketone-presenting Swiss 3T3 albino mouse
fb (9) contained no fluorescent label. These two cell populations were then
trypsinized and mixed together (-204 cells/mL, 4 mL total) in serum containing
(10 % CBS, pH of 7.4) media in a 10 mL-flask and incubated at 37 C and 5 %
CO2 for 3 h. After mixing, the cells were seeded on a glass surface (-204
cells/mL, 1 mL) and visualized under a Nikon TM TE2000-E inverted
microscope or by scanning electron microscopy. Image acquisition and
processing was performed using Metamorph software. An exposure time of
75 ms was used to image all spheroids.
Example 19: Scanning electron microscopy (SEM) of 3D spheroids
[00104] Spheroids were assembled in solution (reaction for 3 h as
described above), delivered to a glass slide (-204 cells/mL, 1 mL, 0.8 x 0.8
cm2), and then fixed with 10 % formalin in PBS for 15 min. The substrate was
2554653
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36
then washed with water (15 min), and cells were then dehydrated stepwise in
30, 50, 70, 90, and 100 A ethanolic solutions for 15 min each. After critical
point drying and sputtering 2 nm of gold, the sample was ready for imaging
using a HitachiTM S-4700 field emission scanning electron microscope (Hitachi
High Technologies America, Inc., Schaumburg, Illinois).
Example 20: Human mesenchymal stem (hMSC) cell culture
[00105] hMSCs and basic, growth, and differentiation media were
obtained from Lonza (Basel, Switzerland). hMSCs were cultured in
Dulbecco's Modified Eagle Medium (Gibco) containing 10 % fetal bovine
serum (FBS) and 1 % penicillin/streptomycin at 37 C in 5 % CO2. Culturing
with induction medium as described in the Lonza protocol induced Adipogenic
differentiation.
Example 21: lmmunohistochemishy
[00106] After the growth of 3D tissue-like structures and co-
culture with
Swiss 3T3 albino mouse fb, surfaces were fixed with formaldehyde (4 % in
PBS, 30 min). Substrates were then immersed in a solution containing water
and 60 A) isopropyl alcohol (3-5 min), followed by staining with Oil Red 0 (5
min) and Harris Hemotoxylin (1 min) (6,7). Substrates were visualized by
phase contrast microscopy using a Nikon TE2000-E inverted microscope.
Image acquisition and processing was performed using Metamorph software.
An exposure time of 75 ms was used to image all HMSCs.
Example 22: Directed 3D tissue-like multi-layers
[00107] Ketone-functionalized fbs (9) were seeded (-104 cells/mL)
to
microcontact printed patterned (1 mM hexadecanethiol in Et0H, printed on
gold 5 s, backfilled with 1 mM EG4 in Et0H, 16 h) surfaces presenting
fibronectin (10 mg/mL, 2 h) for 2 h. The cells were allowed to grow for 3 d
(37
C in 5 % 002).29 Oxyamine-functionalized fbs (10) (-104 cells/mL) were then
seeded to surfaces for 2 h, followed by addition of serum-containing (10 %
CBS) media to promote cell growth. The cells were cultured for 3 more d
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37
before imaging. After generation, substrates were fixed, stained, and imaged
by confocal microscopy as described below.
Example 23: Cell staining for imaging
[00108] Cells were fixed with formaldehyde (4 % in PBS) and
permeated
(PBS containing 0.1 % Triton X-100). A fluorescent dye mixture, containing
phalloidin-TRITC (actin) and DAPI (nucleus) was then made in PBS
containing 5 % normal goat serum and 0.1 % Triton X ¨100. Cells were
incubated with the dye solution for 2 h. The substrates were then secured in
fluorescence mounting medium (Dako, Carpinteria, CA, USA), which
enhances the visualization of cells when viewed under a fluorescent
microscope on a glass cover slip. An exposure time of 400 and 1200 ms were
used to image nuclei and actin, respectively.
Example 24: Con focal microscopy
[00109] Cell clusters and tissue formation were visualized with a
Nikon
Eclipse TE2000-E inverted microscope (Nikon USA, Inc., Melville, NY). The
data were recorded using LeicaTM software and a spectral confocal
microscope (LeicaTMMicrosystems, Bannockburn, IL). An average of 84 image
scans were used to generate the 3D reconstructions with Volocity software.
Example 25: 3D Co-culture spheroid and multi-layer generation.
Spheroids
[00110] Keto- (1) and oxyamine LUVs (2) were generated as
previously
described and were added to hMSCs and fbs (3 mM in tris buffer, 400 piL
added to 4 mL, 12 h), respectively, and were cultured, resulting in fusion and
display of ketones and oxyamines from the cell surface. These two cell
populations were then trypsinized and mixed together in serum containing
(10% FBS, pH of 7.4) media in a 10 mL flask and incubated at 37 C and 5%
CO2 for 1, 2, 3, and 5 h. After mixing for the allotted time, cells were
seeded
onto a glass surface and visualized under a Nikon TE2000-E inverted
microscope under the brighffield setting (75 ms exposure time). Controls were
also performed where hMSCs displaying ketone groups were co-cultured with
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38
fbs (not displaying oxyamine groups) for each of the corresponding time
points, 1, 2, 3, and 5 h, seeded onto glass, and imaged under the brightfield
setting (75 ms). Image acquisition and processing was performed using
Metamorph software.
Example 26: Multi-layers
[00111] Keto-
(1) and oxyamine-LUVs (2) were added to hMSC and fbs
(3 mM in tris buffer, 400 added
to 4 mL, 12 h), respectively, and were
cultured, resulting in fusion and display of ketones and oxyamines from the
cell surface. hMSCs (7) displaying ketone groups were trypsinized and
= 10 cultured on glass slides (105 cells/mL) and allowed to grow
for 2 d. Fbs
presenting oxyamines (10) were then trypsinized and added (105 cells/mL) to
the hMSCs. These cells were co-cultured in media (10 % FCS) for 3, 5, and 7
d, resulting in the formation of 3D multi-layered, tissue-like structures of
hMSCs and fbs.
Example 27: Cell viability assay
[00112] Cell
viability of 3D spheroid and multi-layered tissue-like
structures was assessed using a trypan blue viability assay (Hyclone, Fisher
Sci, Pittsburgh, PA). Fb spheroid and multi-layer structures were prepared as
previously described. A solution of 0.4 % trypan blue in PBS was made and
diluted in CBS (1:1) containing the spheroids (1, 3, and 5 h after mixing, 204
cells/mL) in solution and multi-layer cell sheets (3, 5, and 7 d after a
second fb
population was added, 105 cells/mL) on a glass slide. Trypan blue was
allowed to react with the cells for 2 min, at which time spheroids and
surfaces
were imaged and false colored with blue for enhanced visualization using a
Nikon TE2000-E inverted microscope. As a control, cells were cultured for 7 d
to generate a multilayer and were then fixed as mentioned above. Trypan blue
was allowed to react for 2 min, and cells were imaged. For phase contrast and
fluorescent imaging, exposure times of 75 and 400 ms were used,
respectively.
CA 02776618 2012-05-10
39
Results and Discussion
[00113] An oxime
ligation strategy was employed herein to generate a
number of large unilamellar vesicles (LUVs) that present ketone or oxyamine
functional groups. Liposome-liposome fusion events were first initiated
through molecular recognition and subsequent oxime bond formation and the
fusion was characterized using fluorescence resonance energy transfer
(FRET), matrix-assisted laser-desorption/ionization mass spectrometry
(MADLI-MS), dynamic light scattering (DLS), and transmission electron
microscopy (TEM). Next, liposomes containing ketone and oxyamine groups
.. were cultured with 3T3 Swiss albino fibroblasts, resulting in membrane
fusion
and display of oxyamines and ketones from the cell surface for further
fluorescent probe conjugation. For the liposome-liposome fusion events
studied by MALDI-MS (Figure 2a), DLS (Figure 2d), and TEM (Figure 2b),
dodecanone and dodecyloxyamine molecules were incorporated, separately,
into neutral, egg palmitoyl-oleoyl phosphatidylcholine (POPC) at a ratio of
5:95 to form keto-LUVs (1) and oxyamine-LUVs (2), respectively (Figure la
and 1b). When observing liposome fusion via FRET analyses (Figure 2c),
dodecanone molecules were mixed with POPC and fluorescence donor, egg
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-
benzoxadiazol-4-y1) (NBD-PE) at a ratio of 5:93:2 to form keto-NBD-PE-LUVs
(3), while dodecyloxyamine molecules were incorporated into POPC,
negatively charged, egg 1-palmitoy1-2-oleoyl-phosphatidylglycerol (POPG),
and fluorescence acceptor, egg 1,2-
dipalmitoyl-sn-glycero-3-
phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhod-PE) at a ratio
of 5:73:20:2 to form oxyamine-rhod-PE-LUVs (4). Finally, liposomes that
contained dodecanone, POPC, and cationic lipid, 1,2-dioleoy1-3-
trimethylammonium-propane (DOTAP) (5:93:2, 5) and liposomes that
composed of dodecyloxyamine, POPC, and DOTAP (5:93:2, 6) were
generated to investigate liposome-cell fusion processes (Figure 1d). Cationic
lipid, DOTAP, was incorporated to induce membrane fusion due to the
electrostatic interactions with the negatively charged cell surface.36'47
= CA 02776618 2012-05-10
[00114] Fusion
methodology and DLS analyses. An embodiment of
the general fusion methodology is described in Figure la. Two liposome
populations (1 & 2, 3 & 4, or 5 & 6) were mixed, resulting in liposome
docking,
adhesion, and finally fusion due to the formation of stable, interfacial oxime
5 bonds. Depending on the application, liposomes fuse to each other, forming
larger liposomal structures (Figure 2b) or to cell surfaces and then be
further
conjugated with the corresponding oxime component. DLS was performed
upon mixing liposomes 1 and 2 over 2 h to monitor vesicle size change as a
function of time. Increases in vesicle size were observed due to aggregation,
10 adhesion, or fusion (top trace, Figure 2d). Liposome saturation was reached
-80 min after mixing. It is believed that in some cases, liposomes 1 and 2
associate with each other through oxime chemistry and initiate docking /
adhesion until enough liposomes have clustered to induce a sharp growth in
size. The ketone and oxyamine concentrations were initially varied and it was
15 found through cell-surface engineering experiments and FACS analyses
that
the higher the ketone and oxyamine concentration led to increases of these
= functional groups on the cell surface. However, increasing the
concentration
of functional groups led to faster fusion events but did not necessarily
increase the liposome size after liposome-liposome fusion. In
control
20 reactions, LUVs not presenting ketones were reacted with LUVs containing
oxyamines (1) (bottom trace, Figure 2d). Likewise, LUVs containing ketone
groups (2) were mixed with LUVs that did not display oxyamines. For both of
these control experiments, no size change was observed over time. This
result strongly supports that liposome adhesion and fusion are driven by
25 chemoselective oxime bond formation between the ketone- and oxyamine-
alkanes.
[00115] TEM.
Structural insight into the formation of different adhered
and fused liposomes was observed through TEM (Figure 2b). Vesicles of
different sizes and shapes result after 2 h of liposome mixing (keto-LUV, 1
30 and oxyamine-LUV, 2). The liposome size gradually increases with
time and
is consistent with the data collected from other sizing experiments (e.g.,
DLS).
Upon reaction, the following three structures were observed: multi-adherent
= CA 02776618 2012-05-10
41
liposomes that were not fused, partially fused liposomes, and completely
fused, large uni- and multi-lamellar liposomes (Figure 2b).
[00116] FRET. Figure 2c shows a
liposome fusion assay involving FRET
characterization. A lipid-bound FRET pair, NBD-PE (donor) and rhod-PE
(acceptor), were incorporated at 2 mol % concentration during liposome
generation to produce keto-NBD-PE LUVs (3) and oxyamine-rhod-PE LUVs
(4), respectively. Hypothetically, fusion of these vesicles should result in a
gradual decrease in the donor emission peak and an increase in acceptor
emission peak due to the close proximity of these dyes. As shown, vesicle
mixing resulted in this FRET fusion signature. Fusion was observed
immediately upon mixing 3 and 4, slowing within 2 h to a stable population,
which is similar to earlier sizing results. An emission peak was not observed
for the acceptor rhodamine dye when performing control experiments that
tested the energy transfer with an LUV that did not contain oxyamines. Similar
results were observed when LUVs that did not contain ketones or oxyamines
were mixed. This data further supports that liposome aggregation and fusion
is based on chemoselective oxime bond formation.
[00117] MALDI-MS. Oxime
conjugation, after keto-LUV (1) fusion, was
confirmed by MALDI-MS analysis. Self-assembled monolayers (SAMs) of
aminooxyundecanethiol were formed on a gold-coated, sample plate. A
solution containing keto-LUVs (1) was then allowed to fuse and react with the
surface for 90 min, followed by MALDI-MS examination. A mass of 387 units
was detected, confirming successful oxime conjugation, resulting from
liposome fusion on the surface (Figure 2a).
= 25 [00118] Cell-surface labeling. In an embodiment of the present
application, oxime chemistry is used to tailor and fluorescently label cell
surfaces via a novel liposome fusion strategy. As mentioned, cationic lipid,
DOTAP, was incorporated within keto- and oxyamine-LUVs to initiate
electrostatic destabilization and subsequent fusion to the cell membrane. As
such, the minimum DOTAP concentration required to facilitate liposome-cell
fusion was determined to be 2 % through fluorescence labeling optimization.
CA 02776618 2012-05-10
42
Keto-LUVs were generated using DOTAP and POPC concentrations that
ranged from 0.5 % to 5 % and 90 % to 94.5 %, respectively, while maintaining
a 5-% ketone concentration. These liposomes were incubated with fibroblasts
(fbs) for 4 h, conjugated with an oxyamine-tethered rhodamine (rhod-
oxyamine, 8) (0.7 mM, 2 h), and the cell fluorescence intensities were then
compared. From 2 % to 5 % DOTAP, the intensities were almost identical,
indicating that 2 % DOTAP is sufficient to initiate fusion. The liposomes for
liposome-cell fusion events were approximately -60 nm in diameter, similar to
those used for the liposome-liposome fusion characterization.
1001191 Given this optimized lipid ratio (POPC/ketone or
oxyamine/DOTAP at 93:5:2), two cell-surface engineering methods were
employed to fluorescently label fbs. Similar to the optimization experiments,
a
solution of keto-LUVs (5, 200 pL, 0.6 mM) was added to fbs in culture for 2 h,
resulting in membrane fusion and subsequent display of ketones from the cell
surface (9) (Figure 3d). Rhod-oxyamine (8, 100 pL, 0.7 mM in H20) was then
added the cells for 2 h. After oxime formation, the fbs were washed with PBS,
trypsinized, diluted with CBS-containing media (-102/mL), seeded to a glass
substrate, and imaged under a fluorescent microscope. As observed in Figure
3f, the conjugation of rhod-oxyamine with ketone-presenting fbs resulted in
the fluorescence labeling of cells. When the control fbs (i.e., no ketone
groups
present) were reacted with rhod-oxyamine (8) and then imaged, no
fluorescence was observed (Figure 3e). Demonstrating the flexibility of this
liposome-based surface labeling strategy, fb surfaces were modified to
present a ketone-functionalized fluorescein dye (7) after oxyamine-LUV-
ketone-fluorescein conjugation and subsequent membrane fusion (Figure 3a).
A solution of oxyamine-LUVs (6, 3 mM) was incubated with a ketone-
functionalized fluorescein (7, 0.15 mM, 1 eq, 2 h), generating fluorescently
labeled liposomes. The liposomes were then added to fbs in culture for 2 h.
After fusion, the cells were washed with PBS, trypsinized, diluted with CBS-
containing media (-102/mL), seeded to a glass substrate, and imaged under a
fluorescent microscope. Figure 3c presents fluorescently labeled fbs after
fusion with fluorescein-functionalized LUVs. Through fluorescent and confocal
CA 02776618 2012-05-10
43
imaging, it appears that after membrane fusion and/or endocytosis of cultured
liposomes, fluorescence is also observed in several membrane organelles.
This is an advantage of the system in that ketone or oxyamine groups are
present at the cell surface and also decorate various internal membranes. It
=
may be possible to label internal organelles with oxyamine chemistry for
future targeting studies and applications. These lipids and fluorophores are
likely packaged and trafficked to and from the cell surface and internal
compartments. However, enough functional groups are present on the cell
surface to provide handles for further oxime chemistry conjugation to tailor
cell
surfaces. When liposomes, not containing oxyamine groups were incubated
with fluorescein-ketone and added to fbs in culture for 2 h, no fluorescence
was observed (Figure 3b). Thus, control images indicated that reaction and
labeling does not occur without the proper oxime recognition pair (Figure 3b
and 3e). Furthermore, under these conditions, no changes were observed in
cell behavior upon liposome fusion to cells, which is a very important feature
for future in vivo applications. Thus, by combining liposome fusion and oxime
chemistry, the cell surface was tailored with either ketone groups or oxyamine
groups, which act as chemoselective cell-surface receptors for a range of
small molecules, ligands, biomolecules, and nanoparticles.
[00120] Cell patterning: Rewiring adhesion. The ability to pattern and
adhere cells to different materials, such as thin metal films, polymer
scaffolds,
and nanoparticles, with a simple and straightforward chemoselective and bio-
orthogonal approach would be beneficial for cell biology, tissue engineering,
and biotechnology. Thus, the liposome fusion was employed for cell-surface
engineering to modify and rewire cell surface to adhere to patterned 2D
substrates, directed through stable oxime bond conjugation. Figure 4a and 4b
illustrate the strategy to rewire cell surfaces for the goal of cell adhesion
to
self-assembled monolayers (SAMs) of alkanethiolates on gold substrates.
Employing microfluidic oxidation" and lithography," aldehyde and oxyamine
SAMs, respectively, were patterned at a ratio of 10 %. The remaining 90 % of
the surface was backfilled with tetra(ethylene glycol) alkanethiol, which is
known to pacify biomaterials against nonspecific protein adsorption and cell
CA 02776618 2012-05-10
44
adhesion.55 Meanwhile, fbs were cultured separately with keto- (5) and
oxyamine-LUVs (6, 3 mM, 4 h), resulting in membrane fusion and subsequent
display of ketones (9) and oxyamines (10) from cell surfaces. The resulting
ketone- and oxyamine-presenting fbs were then seeded (-102 cells/mL, 2 h)
to the patterned oxyamine and aldehyde substrates, respectively, and allowed
to react and form stable oxime linkages in the patterned regions. The cells
were cultured for 4 d on these substrates, growing and proliferating in the
patterned regions. The results of patterned keto-fbs on oxyamine SAMs are
shown in Figure 4c; patterned oxy-fbs on aldehyde SAMs are displayed in
Figure 4d and 4e. Furthermore, unmodified cells did not attach to the surface.
Thus, this strategy allows for a bottom-up, bio-orthogonal synthetic approach
= to rewire how cells adhere to materials and does not require metabolic or
genetic cell manipulations.
[00121] Flow cytometry. Flow cytometry was performed to further
verify
the ability of tailoring small molecules to cell surfaces through covalent
oxime
bond formation. This method also enables the quantification of ketone and
oxyamine molecules that are present at the cell surface after liposome
delivery and subsequent membrane fusion. Liposomes that incorporated
varying oxyamine concentrations of (i.e., 0, 1, 5, and 10 mol %) were
generated (6, 3 mM) cultured with separate fb populations for 4 h, resulting
in
membrane fusion and oxyamine display (10, Figure 5a). A ketone-modified
fluorescein dye (7, 0.15 mM, 2 h) was then conjugated to the cell surfaces in
each population, producing green fluorescently labeled fbs. The FACS
analyses results are demonstrated in Figure 5b. Twenty thousand cells were
counted for all samples. As shown, the fluorescence intensity increases with
increasing number of oxyamine molecules present for fluorescein conjugation.
Additionally, the control cell population that was fused with unmodified
liposomes and reacted with ketone-fluorescein (7) demonstrated the lowest
intensity. Furthermore, a bead with known FITC molecule density was
calibrated and used as a standard comparison to quantify the number of
oxyamine molecules present at the cell surface after fusion.51 Figure 5c
displays the correlation between oxyamine mol % and oxyamine molecules
CA 02776618 2012-05-10
per cell counted by FACS analyses. The calculated molecules per cell for the
control fbs and oxyamine-presenting fbs that were fused with 1 %, 5 %, and
10 A) oxyamine were approximately 128, 1300, 9800, and 17400, respectively.
A linear trend was observed; as the molecule concentration increased, the
5 fluorescence intensity and number of molecules at the cell surface
increased.
Thus, the density of molecules that decorate cell surfaces can be controlled
and quantified using this liposome fusion-based methodology for cell-surface
engineering.
[00122] 3D spheroid assembly. The ability to generate
multicellular
10 connected tissues of multiple cell types in vitro is useful for studying
the
complex interplay of cells in a range of organs in vivo and for developing
strategies for synthetic tissue transplantation. With varying successes, a
number of current strategies to generate 3D cell connections rely on forcing
mixed cell populations into complex microfabricated wells or vessels.
15 Therefore, in the present liposome fusion technology, an oxime-based
strategy was used to generate 3D spheroid assemblies of interconnected cells
using two different cell-type populations (Figure 6). The oxyamine-presenting
rat2 fbs (10) contained a nuclear m-cherry fluorescent label so that the cell
clustering to non-fluorescent ketone-tethered cells (9) could be easily
20 observed. During a 3-hour period of mixed-culturing (-104 cells/mL) in
solution,
cells formed spheroid structures due to the presence of complementary
recognition groups (Figure 6C and 60). Furthermore, when oxyamine-
presenting fbs (10) were cultured with control fbs (cells not functionalized
with
ketone groups), spheroid assembly did not occur (Figure 6B). Studies were
25 also performed to test whether spheroid size and cell composition could be
controlled. Ketone-presenting hMSCs (11) were co-cultured with oxyamine-
.
functionalized fbs (10) for 1, 2, 3, and 5 h. After 1 h, clusters comprised
only
with a few cells were observed. As the co-culturing duration was increased,
larger spheroid structures were observed. Notably, control experiments were
30 performed simultaneously to ensure that spheroid generation was being
directed through chemoselective oxime conjugation. Tissue structure
formation did not occur without the proper complementary pair displayed from
CA 02776618 2012-05-10
=
46
cell surfaces, regardless of the mixing duration (1-5 h). Thus, size and
composition of 3D cell assemblies in solution could be controlled, showing
great promise for applications in stem cell transplantation and regenerative
medicine.
[00123] Spheroid formation was also characterized by scanning electron
microscopy (SEM) (Figure 6E-G). Cells functionalized with oxyamine (10) and
ketone (9) groups were able to generate clusters when mixed in solution, as
displayed in Figure 6F and 6G. However, spheroid assemblies were not
observed when ketone-presenting fbs were reacted with non-functionalized
cells; fbs spread out on the surface, migrated, but remained alone (Figure
6E).
Notably, cells were able to form stable, interconnected 3D structures in
solution simply upon mixing two tailored cell populations. Currently, methods
to generate these structures require the support of a 3D hydrogel matrix
and/or assisted assembly through an external stimulus.5:7-9'13
[00124] 3D multi-layered tissues. In addition to forming small, 3D cell
clusters or spheroid structures in solution, this strategy may be employed to
direct larger, dense 3D tissue-like networks on a surface with geometric
control. Full substrates were used (Figure 7), as well as surfaces that were
patterned with cell adhesive and non-adhesive regions to generate multi-
layered sheets and patterned tissue structures (Figure 8), respectively.52
Ketone- (1) and oxyamine- (2) tailored liposomes were cultured with separate
fb populations, resulting in membrane fusion and subsequent presentation of
chemoselective sites for oxime conjugation from the surface (9 and 10,
respectively) (Figure 7A). Culturing these groups on a solid support (-105
cells/mL) and in a layer-by-layer deposition manner gave rise to multi-
layered,
tissue-like cell sheets, which were characterized by confocal microscopy, as
shown in Figure 7E and 7F. Fbs naturally only form a single monolayer once
they become contact-inhibited. However, fb-fb clustering has been
successfully induced though oxime-mediated, cell-surface engineering based
on liposome fusion.
CA 02776618 2012-05-10
47
100125] To ensure that oxime chemistry was aiding in the
formation of
3D tissue-like structures, several control experiments were performed. Cells
that did not present ketone or oxyamine functionality were seeded onto
separate surfaces. A second cell population presenting oxyamine (6) or
ketone (3) groups from the cell surface was added, resulting in the formation
of only a 2D monolayer of cells (Figure 4B and 4C). Similarly, two different
cell
populations that were tethered with oxyamine (10) groups were mixed
together, and only a 2D monolayer was generated after 4 d of culture. The
same results were observed after culturing two different ketone-functionalized
= 10 cell populations (9) for 4 d. These results further support
the hypothesis that
multi-layered cell interconnectivity is driven by complementary, oxime
chemistry. This strategy was also extended toward the generation of 3D multi-
layered co-cultures with hMSCs and fbs. Ketone-functionalized hMSCs (11)
were first cultured on a substrate (-105 cells/mL), and stem cells were
allowed
to spread out and grow for 2 d. Oxyamine-presenting fbs (12) were then
= added (-105 cells/mL) and co-cultured for an additional 2 d. 3D Multi-
layered
cell sheets (4 layers) were formed. The proper controls were conducted;
without the oxime pair, only a 2D monolayer of stem cells and fbs was formed.
100126] 3D tissue release and cell viability. During multi-layer
culture,
it was possible to control the release of the tissues from the surface with
gentle agitation (Figure 7D and 7G). The ability to release tissue after
surface-
supported growth in vitro shows great potential for applications in tissue
engineering and cellular transplantation. Cell viability was also tested for
3D
spheroid and multi-layered structures of fbs and hMSC/fb co-cultures using
the trypan blue assay.53 After spheroid (1, 2, 3, and 5 hours of mixing in
solution) and multi-layer (3, 5, and 7 days on a surface) formation, cells
were
incubated with trypan blue (0.4 %, 2 min). Viability was 100 % for all cells
in
the spheroid assemblies (1-5 hours) and multi-layer structure at day 3. After
5
and 7 days of multi-layer generation, cells showed an approximate viability of
91 % and 84 %, respectively. The blue intensity (fluorescence false colored
for enhanced visualization) was compared to a control cell population by
linescan analysis. The control cells were cultured for 7 days to generate 3D
CA 02776618 2012-05-10
48
multi-layers and were then fixed. Trypan blue was allowed to react for 2 min,
followed by imaging and quantification. Overall, the viability of cells in
conducting membrane fusion to generate 3D tissue-like structures in solution
and on a solid support is high. Therefore, this method may be very useful for
applications in tissue engineering and stem cell transplantation.
[00127] 3D tissue patches with geometrical control. Spatial
control
was demonstrated by generating a number of 3D multi-cellular micropatterns.
Microcontact printing52 was used to produce a variety of patterns and
geometries on a gold substrate. Employing SAM and microfabrication
technologies, hexadecanethiol (1 mM in Et0H) was printed on a gold surface.
The surface was then backfilled with EG4 (1 mM in Et0H, 16 h) to render the
remaining regions inert to nonspecific protein absorption. Fibronetin, a cell-
adhesive protein was then added (10 mg/mL in CBS, 2 h), adhering only to
the hydrophobic, patterned areas. As shown by the confocal image in Figure
8A, only a 2D, circular cell pattern arises after ketone-presenting fbs (9)
were
cultured with fbs, not functionalized with oxyamine molecules. However, when
liposome fusion occurs to display complementary ketone and oxyamine
groups from cell surfaces (9 and 10, respectively), multi-layered 3D cell
= patterns were formed (Figure 8B-D). Circular, bar, and square circular
tissue-
like structures are depicted in Figure 8B-D. The ability to generate 3D
tissues
with controlled geometry would find great use in tissue transplantation, in
which specifically tailored patches are required.
[00128] 3D stem cell co-cultures with induced adipocyte
differentiation. The general use of the present liposome fusion method was
explored to delivered ketone and oxyamine groups to different cell lines, and
it
was demonstrated that 3D spheroid and multi-layer can be generated using
co-cultures of hMSCs and fbs. The methodology was next extended toward
stem cell differentiation to determine whether 3D multi-layered co-cultures
could be induced to generate tissues of differentiated hMSCs and fbs. As
shown in Figure 9A, ketone-functionalized hMSCs (11) were first cultured on a
substrate for 3 d, producing a 2D monolayer of cells (Figure 9B). Oxyamine-
.
49
tethered fbs (12) were then co-cultured with the hMSCs, and the cells were
allowed to grow and proliferate for 2 d (Figure 9C). Adipogenic induction
media was then added, the 3D multi-layered co-culture was stained for nuclei
and lipid vacuoles, which are characteristic of adipocytes (fat cells). The
phase contrast images in Figure 9D and 9E demonstrate the successful
generation of tissue-like structures, comprising induced adipocytes and fbs.
The ability to co-culture stem cells with many other cell types and induce
differentiation shows great promise in the field of regenerative medicine and
stem cell transplantation.
[00129] While the present application has been described with reference
to what are presently considered to be the preferred examples, it is to be
understood that the application is not limited to the disclosed examples. To
the contrary, the application is intended to cover various modifications and
equivalent arrangements included within the spirit and scope of the appended
claims.
[00130] Where a term in the present application is found to be
defined
differently in a referenced document, the definition provided herein is to
serve
as the definition for the term.
2554653
CA 2776618 2018-09-24
CA 02776618 2012-05-10
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