U.S. patent application number 10/414038 was filed with the patent office on 2004-01-08 for biologically functionalized porous microspheres.
Invention is credited to Buranda, Tione, Goparaju, Venkata Rama Rao, Huang, Jinman, Ista, Linnea, Lopez, Gabriel P., Sklar, Larry A..
Application Number | 20040005352 10/414038 |
Document ID | / |
Family ID | 43501909 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005352 |
Kind Code |
A1 |
Lopez, Gabriel P. ; et
al. |
January 8, 2004 |
Biologically functionalized porous microspheres
Abstract
The present invention provides functionalized mesoporous
microspheres comprising a microsphere including surface mesopores;
a lipid bilayer covering at least a portion of the microsphere; and
enclosed mesoporous spaces comprising the surface mesopores and
respective portions of the lipid bilayer.
Inventors: |
Lopez, Gabriel P.;
(Albuquerque, NM) ; Buranda, Tione; (Albuquerque,
NM) ; Goparaju, Venkata Rama Rao; (Albuquerque,
NM) ; Huang, Jinman; (Albuquerque, NM) ; Ista,
Linnea; (Albuquerque, NM) ; Sklar, Larry A.;
(Albuquerque, NM) |
Correspondence
Address: |
JAGTIANI + GUTTAG
10363-A DEMOCRACY LANE
FAIRFAX
VA
22030
US
|
Family ID: |
43501909 |
Appl. No.: |
10/414038 |
Filed: |
April 16, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60373201 |
Apr 16, 2002 |
|
|
|
60410830 |
Sep 16, 2002 |
|
|
|
Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 9/1617 20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 009/127 |
Goverment Interests
[0002] The United States Government has rights in this invention
pursuant to: Grant Nos. EEC-0210835 and MCB-9907611 from the
National Science Foundation; Grant No. F49620-01-1-0168, from the
Air Force Office of Scientific Research; Grant No. GM60799/EB00264
from the National Institutes of Health; and Grant No. 00-01-205-5
from the U.S. Department of Energy through the U.S./Mexico
Materials Corridor Initiative. The government may have certain
rights in this invention.
Claims
What is claimed is:
1. A product comprising: a microsphere including surface mesopores;
a lipid bilayer covering at least a portion of said microsphere;
and enclosed mesoporous spaces comprising said surface mesopores
and respective portions of said lipid bilayer.
2. The product of claim 1, wherein said enclosed mesoporous spaces
contain a dispersion.
3. The product of claim 2, wherein said enclosed mesoporous spaces
contain a solution.
4. The product of claim 2, wherein said enclosed mesoporous spaces
contain a colloidal suspension.
5. The product of claim 1, wherein said microsphere has internal
mesopores within said microsphere in communication with at least
one of said surface mesopores.
6. The product of claim 1, wherein said surface mesopores are 10-20
nm in diameter.
7. The product of claim 1, wherein said surface mesopores are 2 nm
in diameter.
8. The product of claim 1, wherein said surface mesopores are 5 nm
in diameter.
9. The product of claim 1, wherein said surface mesopores are 10 nm
in diameter.
10. The product of claim 1, wherein all of said mesopores are the
same size.
11. The product of claim 1, wherein said mesopores are divided into
a plurality of regions and within each region, said mesopores are
the same size.
12. The product of claim 1, wherein said microsphere has a diameter
of 0.5 to 20 .mu.m
13. The product of claim 1, wherein said microsphere has a diameter
of about 15 .mu.m.
14. The product of claim 1, wherein at least one of said enclosed
mesoporous spaces contains an indicator.
15. The product of claim 1, wherein at least one of said enclosed
mesoporous spaces contains a biomolecule.
16. The product of claim 1, wherein at least one of said enclosed
mesoporous spaces contains an organelle.
17. The product of claim 1, wherein at least one of said enclosed
mesoporous spaces contains a whole cell.
18. The product of claim 1, wherein at least one of said enclosed
mesoporous spaces contains a magnetic material.
19. The product of claim 1, wherein at least one of said enclosed
mesoporous spaces contains a charged material.
20. The product of claim 1, wherein said lipid bilayer includes at
least one integral protein therein.
21. The product of claim 1, wherein said lipid bilayer includes at
least one peripheral protein thereon.
22. A product comprising: at least one external phase comprising an
external dispersant; and a plurality of functionalized microspheres
dispersed in said external dispersant, each of said functionalized
microspheres comprising: a microsphere body including surface
mesopores; a lipid bilayer covering at least a portion of said
microsphere; and enclosed mesoporous spaces comprising said surface
mesophores and respective portions of said lipid bilayer.
23. The product of claim 22, wherein said enclosed mesoporous
spaces include an internal dispersant.
24. The product of claim 23, wherein said internal dispersant is
different from said external dispersant.
25. The product of claim 23, wherein said enclosed mesoporous
spaces contain a solution.
26. The product of claim 23, wherein said enclosed mesoporous
spaces contain a colloidal suspension.
27. The product of claim 22, wherein said at least one external
phase comprises an external dispersion including said external
dispersant.
28. The product of claim 22, wherein said microsphere has internal
mesopores within said microsphere in communication with at least
one of said surface mesopores.
29. The product of claim 22, wherein said surface mesopores are
10-20 nm in diameter.
30. The product of claim 22, wherein said surface mesopores are 2
nm in diameter.
31. The product of claim 22, wherein said surface mesopores are 5
nm in diameter.
32. The product of claim 22, wherein said surface mesopores are 10
nm in diameter.
33. The product of claim 22, wherein all of said mesopores are the
same size.
34. The product of claim 22, wherein said mesopores are divided
into a plurality of regions and within each region, said mesopores
are the same size.
35. The product of claim 22, wherein each of said microspheres has
a diameter of 0.5 to 20 .mu.m.
36. The product of claim 22, wherein each of said microspheres has
a diameter of about 15 .mu.m.
37. The product of claim 22, wherein at least one of said enclosed
mesoporous spaces contains an indicator.
38. The product of claim 22, wherein at least one of said enclosed
mesoporous spaces contains a biomolecule.
39. The product of claim 22, wherein at least one of said enclosed
mesoporous spaces contains an organelle.
40. The product of claim 22, wherein at least one of said enclosed
mesoporous spaces contains a whole cell.
41. The product of claim 22, wherein at least one of said enclosed
mesoporous spaces contains a magnetic material.
42. The product of claim 22, wherein at least one of said enclosed
mesoporous spaces contains a charged material.
43. A method comprising the steps of: transferring a substance from
at least one external phase comprising a dispersant to an enclosed
mesoporous space of a functionalized microsphere; and reacting said
substance with at least one internal phase in said enclosed
mesoporous space, wherein said enclosed mesoporous space comprises
a surface mesopore of said microsphere body of said functionalized
microsphere and a portion of a lipid bilayer covering said
microsphere body.
44. The method of claim 43, wherein said enclosed mesoporous space
includes an internal dispersant.
45. The method of claim 44, wherein said internal dispersant is
different from said external dispersant.
46. The method of claim 44, wherein said enclosed mesoporous spaces
contain a solution.
47. The method of claim 44, wherein said enclosed mesoporous space
contains a colloidal suspension.
48. The method of claim 43, wherein said at least one external
phase comprises an external dispersion including said external
dispersant.
49. The method of claim 43, wherein said microsphere has internal
mesopores within said microsphere in communication with at least
one of said surface mesopores.
50. The method of claim 43, wherein said surface mesopore is 10-20
nm in diameter.
51. The method of claim 43, wherein said surface mesopore is 2 nm
in diameter.
52. The method of claim 43, wherein said surface mesopore is 5 nm
in diameter.
53. The method of claim 43, wherein said surface mesopore is 10 nm
in diameter.
54. The method of claim 43, wherein all of said mesopores are the
same size.
55. The method of claim 43, wherein said mesopores are divided into
a plurality of regions and within each region, said mesopores are
the same size.
56. The method of claim 43, wherein said microsphere has a diameter
of 0.5 to 20 .mu.m
57. The method of claim 43, wherein said microsphere has a diameter
of about 15 .mu.m.
58. The method of claim 43, wherein said enclosed mesoporous space
contains an indicator.
59. The method of claim 43, wherein said enclosed mesoporous space
contains a biomolecule.
60. The method of claim 43, wherein said enclosed mesoporous space
contains an organelle.
61. The method of claim 43, wherein said enclosed mesoporous space
contains a whole cell.
62. The method of claim 43, wherein said enclosed mesoporous space
contains a magnetic material.
63. The method of claim 43, wherein said enclosed mesoporous space
contains a charged material.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application makes reference to and claims the priority
the following co-pending U.S. Patent Applications. The first
priority application is U.S. Provisional Application No. 60/373,201
entitled "Bioparticle Constructs, Applications and Methods," filed
Apr. 16, 2002. The second priority application is U.S. Provisional
Application No. 60/410,830, entitled "Monodisperse Mesoporous
Silica Microspheres Formed by Evaporation-Induced Self-Assembly of
Surfactant Templates in Aerosols" filed Sep. 16, 2002. The entire
disclosure and contents of the above applications are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to microspheres.
[0005] 2. Description of the Prior Art
[0006] In the field of biotechnology there continues to be a need
for improved methods of drug discovery and controlled drug
delivery. There also continues to be a need for systems that allow
for precise and controlled study of interactions between membrane
bound receptors and specific cytosolic components. In addition,
there continues to exist a need for systems that allow the study of
bioenergetics at the cellular level. There also exists a need for
better artificial cells than are currently available as sources of
hormonal replacement in such areas as diabetes and liver failure or
as carriers for gene therapy agents. Additionally, there continues
to exist a need, in treating viruses such as HIV, influenza, and
SARS for improved viral decoys.
SUMMARY OF THE INVENTION
[0007] It is therefore an object of the present invention to
provide a functionalized mesoporous microspheres that may be used
in improved methods of drug discovery and controlled drug
delivery.
[0008] It is a further object to provide functionalized mesoporous
microspheres that may be used to allow precise and controlled study
of interactions between membrane bound receptors and specific
cytosolic components.
[0009] It is yet another object to provide functionalized
mesoporous microspheres that allow the study of bioenergetics at
the cellular level.
[0010] It is yet another object to provide functionalized
mesoporous microspheres that may be used to make improved
artificial cells than are currently available.
[0011] It is yet another object to provide functionalized
mesoporous microspheres that may be used in making improved viral
decoys.
[0012] According to a first broad aspect of the present invention,
there is provided a product comprising: a microsphere including
surface mesopores; a lipid bilayer covering at least a portion of
the microsphere; and enclosed mesoporous spaces comprising the
surface mesopores and respective portions of the lipid bilayer.
[0013] According to a second broad aspect of the invention, there
is provided a product comprising: at least one external phase
comprising an external dispersant; and a plurality of
functionalized microspheres dispersed in the external dispersant,
each of the functionalized microspheres comprising: a microsphere
body including surface mesopores; a lipid bilayer covering at least
a portion of the microsphere; and enclosed mesoporous spaces
comprising the surface mesophores and respective portions of the
lipid bilayer.
[0014] According to a third broad aspect of the invention, there is
provided a method comprising the steps of: transferring a substance
from at least one external phase comprising a dispersant to an
enclosed mesoporous space of a functionalized microsphere; and
reacting the substance with at least one internal phase in the
enclosed mesoporous space, wherein the enclosed mesoporous space
comprises surface mesopore of the a microsphere body of the
functionalized microsphere and a portion of a lipid bilayer
covering the microsphere body.
[0015] Other objects and features of the present invention will be
apparent from the following detailed description of the preferred
embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention will be described in conjunction with the
accompanying drawings, in which:
[0017] FIG. 1 is a cross-sectional view in simplified form of a
biologically functionalized porous microsphere of one preferred
embodiment of the present invention;
[0018] FIG. 2 is a cross-sectional view in simplified form of a
biologically functionalized porous microsphere of another preferred
embodiment of the present invention;
[0019] FIG. 3 is an illustration in simplified form of a porous
microsphere of one preferred embodiment of the present
invention;
[0020] FIG. 4 is a series of micrographs of a Z-series (1 mm
slices, overlap .about.0.5 mm) of rhodamine trapped within a
membrane bound mesoporous microsphere of the present invention;
[0021] FIG. 5 is a series of micrographs of fluorescence recovery
after photobleaching rhodamine encapsulated in a lipid coated
porous microsphere. Total recovery time was 7 minutes;
[0022] FIG. 6 is a graph illustrating .alpha.-hemolysin mediated
influx of Mg2+ into membrane-enclosed microspheres;
[0023] FIG. 7 is a graph illustrating pH sensitivity of fluoroscein
as measured by emission intensity at 518 (excitation
wavelength=480);
[0024] FIG. 8 is a graph illustrating FITC response to illumination
of BR in the membrane. %=weight percent of BR incorporated into an
Egg-PC liposome;
[0025] FIG. 9 is a graph illustrating the uptake and release of
FITC from PNIPAAM modified microspheres;
[0026] FIG. 10 is a graph illustrating flow cytometric detection of
.alpha.-hemolysin using a magnesium indicator dye trapped in a
lipid coated mesoporous microsphere of the present invention;
[0027] FIG. 11 illustrates in schematic form a fluidic sample
handling system for high throughput screening using flow cytometry
and the functionalized mesoporous microspheres of the present
invention;
[0028] FIG. 12A is a confocal fluorescence microscopic section of a
porous microbead that has been loaded with rhodamine and
encapsulated in an encapsulated space of a functionalized
mesoporous microsphere of the present invention;
[0029] FIG. 12B is a confocal fluorescence microscopic section of
the functionalized mesoporous microsphere of FIG. 12A after
photobleaching;
[0030] FIG. 12C is a confocal image of the functionalized
mesoporous microsphere of FIGS. 12A and 12B after recovery, about 7
minutes later;
[0031] FIG. 13 is a set of optical micrographs (top) and flow
cytometry fluorescence histograms (bottom) for mesoporous
microspheres of the present invention and nonporous commercial
silica particles coated with fluorescent phospholipid bilayers;
and
[0032] FIG. 14 schematically depicts the structure of
functionalized mesoporous microsphere of the present invention and
presents preliminary data gathered from flow cytometry using such
microspheres.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0033] It is advantageous to define several terms before describing
the invention. It should be appreciated that the following
definitions are used throughout this application.
Definitions
[0034] Where the definition of terms departs from the commonly used
meaning of the term, applicant intends to utilize the definitions
provided below, unless specifically indicated.
[0035] For the purposes of the present invention, the term
"microsphere" refers to a sphere or bead having a diameter of 10
nm-1 mm. Preferably the microspheres have a diameter of 0.5 to 20
.mu.m, and more preferably have a diameter of about 15 .mu.m.
Although generally microspheres are roughly spherical, a
microsphere of the present invention may have any shape compatible
with being used in a column of beads or in a flow cytometer.
[0036] For the purposes of the present invention, the term
"mesopore" refers to any pore on or in a microsphere having a
diameter of 2 nm-50 nm in at least one direction. Preferably the
mesopores of the present invention are 10-20 nm in diameter and
some preferred pore sizes are 2 nm, 5 nm and 10 nm.
[0037] For the purposes of the present invention, the term
"mesoporous microsphere" refers to a microsphere or microsphere
having plurality of mesopores thereon and/or therein.
[0038] For the purposes of the present invention, the term
"functionalized microsphere" refers to a microsphere at least
partially covered by a lipid bilayer.
[0039] For the purposes of the present invention, the term
"diameter" refers to the distance from one side to an opposite side
of an object, such as a pore, microsphere, etc. in any direction
through the middle of the object. The maximum diameter of an object
is the longest diameter for that object.
[0040] For the purposes of the present invention, the term
"dispersion" refers broadly to any mixture in which one phase
comprising one or more substances is dispersed in a second phase
comprising one or more substances. Examples of dispersions include
solutions, colloidal solutions, suspensions, etc.
[0041] For the purpose of the present invention, the term
"dispersant" refers to a phase of one or more substances in which
another phase of one or more substances is dispersed. Examples of
dispersants include a solvent of a solution, a liquid in which
colloidal particles are suspended, etc.
[0042] For the purposes of the present invention, the term
"external" refers to phases that are external to a functionalized
microsphere of the present invention. For example, the terms
external dispersion, external dispersant, etc. refer to a
dispersion, dispersant, etc. in which functionalized microspheres
of the present invention are dispersed.
[0043] For the purposes of the present invention, the term
"internal" refers to phases that are internal functionalized
microsphere of the present invention. For example, the terms
internal dispersion, internal dispersant, etc. refer to a
dispersion, dispersant, etc. present in the enclosed mesopores,
channels, etc. of the functionalized microspheres of the present
invention are dispersed.
[0044] For the purposes of the present invention, the term
"integral membrane macromolecule" refers to macromolecules whose
structure are at least partially imbedded in a lipid bilayer of the
present invention.
[0045] For the purposes of the present invention, the term
"bioactive macromolecule" refers broadly to any biological active
macromolecule. Examples of bioactive macromolecules include
biomolecules, organelles, assemblies of biomolecules or organelles,
etc.
[0046] For the purposes of the present invention, the term
"biomolecule" refers broadly to the conventional meaning of the
term biomolecule. Examples of biomolecules include: nucleic acids
(DNA, RNA), plasmids, yeast artificial chromosomes (YACs), other
artificial chromosomes, enzymes, hormones, pharmaceuticals,
antibodies, etc.
[0047] For the purposes of the present invention, the term
"organelle" refers broadly to the conventional meaning of the term
organelle and includes any an assembly of biomolecules.
Description
[0048] This present invention provides membrane-enclosed mesoporous
silica microspheres that incorporate biological or biochemical
functionality, either by inclusion within a mesoporous microsphere
matrix, in or on a supported, tethered or untethered lipid bilayer
on the surface The microspheres used are similar to those described
in the prior art and are of a size 0.5-50 .mu.m, see Rama Rao, et
al., "Monodisperse Mesoporous Silica Microspheres Formed by
Evaporation-Induced Self-Assembly of Surfactant Templates in
Aerosol," Advanced Materials, 2002 (submitted), the entire contents
and disclosure of which is hereby incorporated by reference. A key
feature of this disclosure is the incorporation of bioactive
species onto the surface of the microspheres using lipid bilayers,
into the microsphere matrix itself and/or in communication between
two functionalized components (i.e. membrane and microsphere
matrix).
[0049] Unlike previous commercial microspheres, the preferred
mesoporous microspheres of the present invention contain well
ordered pores, with extremely high surface areas, (up to 1000
m.sup.2/g). In addition, the pore size, shape, spacing and density
may be changed to incorporate a variety of biological species, from
ions up to bacterial cells.
[0050] FIG. 1 is an illustration in simplified form of a
biologically functionalized porous microsphere 100 of one preferred
embodiment of the present invention. Microsphere 100 includes a
microsphere body 102 having mesopores 104 that are enclosed by a
lipid bilayer 106 to form enclosed mesoporous spaces 108. Lipid
bilayer 106 includes two lipid layers 112 and 114. Lipid bilayer
112 is made of lipids 116 having polar heads 118 and non-polar
tails 120. Lipid bilayer 114 is made of lipids 122 having polar
heads 124 and non-polar tails 126. Lipid bilayer 106 also includes
integral proteins 136, 138 and 140 and a peripheral protein 142.
Some enclosed mesoporous spaces 108 are just filled with a liquid,
as shown by arrow 152, while other spaces 108 include solid
particles 154, 156 and 158.
[0051] FIG. 2 is an illustration in simplified form of a
biologically functionalized porous microsphere 200 of one preferred
embodiment of the present invention. Microsphere 200 includes a
microsphere body 202 having mesopores 204 that are enclosed by a
lipid bilayer 206 to form enclosed mesoporous spaces 208. Lipid
bilayer 206 includes two lipid layers 212 and 214. Lipid bilayer
212 is made of lipids 216 having polar heads 218 and non-polar
tails 220. Lipid bilayer 214 is made of lipids 222 having polar
heads 224 and non-polar tails 226. Lipid bilayer 206 also includes
integral proteins 236, 238 and 240 and a peripheral protein 242.
Some enclosed mesoporous spaces 208 are just filled with a liquid,
as shown by arrow 252, while other spaces 208 include solid
particles 254, 256 and 258. Some of mesopores 204 include channels
262 that extend into microsphere 202.
[0052] Although, for simplicity, the solid particles in the
enclosed mesoporous spaces of FIGS. 1 and 2 are shown as individual
particles, in the present invention, the particles present in the
enclosed mesoporous spaces may be collections of particles.
[0053] FIG. 3 is an illustration in simplified form of a porous
microsphere 302 of one preferred embodiment of the present
invention having mesopores 312, 314, 316 clustered into groups 322,
324 and 326 respectively. Mesopores 312 are all the same size,
mesopores 314 are all the same size and mesophores are all the same
size. Mesopores 312 are smaller than mesopores 314 and mesopores
314 are smaller than mesopores 316.
[0054] For simplicity, only three different groups of mesopores are
shown in FIG. 3. However, the present invention envisages that
there could be many different groups of mesopores with each group
having mesopores of the same size. Also, for simplicity, the groups
of mesophores occupy only a small portion of the microsphere in
FIG. 3. However, the mesopores may cover almost the entire
microsphere.
[0055] Non-hollow monodisperse mesoporous silica microspheres that
may be used as mesoporous microspheres for the present invention
may be synthesized by evaporation-driven surfactant templating in
microdroplets produced by a vibrating orifice aerosol generator
(VOAG). The pore size, mesoscopic ordering, and monodisperse
particle size of the microspheres may be controlled by the
experimental conditions, precursor chemistry, and VOAG
parameters.
[0056] In one preferred embodiment, the present invention
encompasses mesoporous microspheres functionalized with lipid
bilayers or modified bilayers supported or tethered on the surface
of the microsphere. The bilayer itself may provide the
functionality, or it may further modified by inclusion of other
biomolecules, particularly proteins, protein complexes,
lipo-protein complexes, glycoproteins, or lipoglycoproteins.
Various modifications of the lipid bilayer of the present invention
are described below.
[0057] The lipid bilayer of the present invention may include a
variety of different types of integral membrane macromolecules,
such as the integral proteins depicted in FIGS. 1 and 2. Examples
of useful integral membrane proteins include: receptors,
intracellular proteins, ion channels, ion pumps, transport
proteins, electron transport chains, etc.
[0058] With respect to the use of surface receptors in the lipid
bilayer of the present invention, signal transduction via cell
surface receptors is a fundamental process in cell biology,
controlling cellular growth, movement and olfactory function. The
largest family of receptors accomplishes transmembrane signalling
by coupling to heterotrimeric guanine nucleotide (G) binding
proteins. Such receptors include olfactory receptors. The receptors
are identified by seven transmembrane .alpha. helical 1 domains,
with three connecting loops on each inner and outer face of the
membrane, and are therefore referred to as seven-loop transmembrane
receptors (7TMRs). Some general characteristics of the ligand
binding regions of the receptors have been defined. While some
small hormones bind directly to the transmembrane domains, large
ligands often bind to the extracellular loops and are brought into
contact with the transmembrane domains.
[0059] With respect to the use of intracellular proteins in the
lipid bilayer of the present invention, several of the inner
connecting loops and the carboxyl terminal tail couple the receptor
to intracellular proteins such as the G proteins. The G proteins
are a family of .alpha..beta..gamma. heterotrimeric molecules. The
.alpha. subunit contains nucleotide (GTP, cAMP) binding sites. Both
.alpha. and .beta..gamma. subunits have the capacity to interact
with catalytic enzymes or "effectors" required for cell activation.
There are several classes of .alpha., .beta., and .gamma. subunits
which provide specificity for the coupling of the receptors, G
proteins, and effectors. The intracellular loops contain
phosphorylation sites for receptor kinases that yield desensitized
receptors. There is a family of "arrestin" molecules which bind to
desensitized receptors and may prevent the receptor from being
engaged in further signal transduction, see Lefkowitz, R. J., "G
protein coupled receptors III. New Roles for receptor kinases and
beta-arrestins in receptor signaling and desentitization," Journal
of Biological Chemistry, 273:18677-18680, (1998), the entire
contents and disclosure of which is hereby incorporated by
reference. The specificity of interactions is often defined in
cellular expression systems because there have not been up to this
point convenient (i.e. sensitive, real-time) assay systems for
examining molecular assemblies between signal initiation and
transduction components.
[0060] With respect to the use of ion channels in the lipid bilayer
of the present invention, ion channels allow for the influx of ions
down their chemical gradients (i.e. do not require in the input of
cellular energy to function.) These channels are present in both
prokaryotic and eukaryotic cells and perform a variety of functions
within the cell, such as osmoregulation, signal transduction,
hormonal regulation and neurological response. These channels are
often found in association with intracellular signaling
proteins.
[0061] With respect to the use of ion pumps in the lipid bilayer of
the present invention, in contrast to ion channels, these molecules
pump ions against a gradient, requiring energetic input (i.e.
hydrolysis of ATP). These most generally function as osmoregulators
in cells.
[0062] With respect to the use of transport proteins in the lipid
bilayer of the present invention, transport proteins are proteins
that transport (as opposed to gating) ions and hydrophilic across
biological membranes. Transport proteins are important in providing
nutrition, metal ions, cellular regulation and sometimes energy to
the cell. An example of such a transport protein is
bacteriorhodopsin. This 7 TMR protein is active in extremely
halophilic archaebacteria and uses light to drive a proton pump,
creating an excess of protons inside the cellular membrane. The
proton gradient in turn fuels a proton-dependent ATP synthase which
provides chemical energy to the bacteria.
[0063] With respect to the use of electron transport chains in the
lipid bilayer of the present invention, electron transport chains
couple oxidative processes from cellular metabolism (or in the case
of photosynthesis, light energy) to the generation of chemical
energy (i.e. ATP).
[0064] With respect to the use of Intracellular transport and
signaling molecules associated with in the lipid bilayer of the
present invention, intracellular transport and signaling molecules
associated with the integral membrane macromolecules described
above, in 7 TMR receptors, several of the inner connecting loops
and the carboxyl terminal tail couple the receptor to intracellular
proteins such as the G proteins. The G proteins are a family of
.alpha..beta..gamma. heterotrimeric molecules. The .alpha. subunit
contains nucleotide (GTP, cAMP) binding sites. Both .alpha. and
.beta..gamma. subunits have the capacity to interact with catalytic
enzymes or "effectors" required for cell activation. There are
several classes of .alpha., .beta., and .gamma. subunits which
provide specificity for the coupling of the receptors, G proteins,
and effectors. The intracellular loops contain phosphorylation
sites for receptor kinases that yield desensitized receptors. There
is a family of "arrestin" molecules which bind to desensitized
receptors and may prevent the receptor from being engaged in
further signal transduction. The specificity of interactions is
often defined in cellular expression systems because there have not
been up to this point convenient (i.e. sensitive, real-time) assay
systems for examining molecular assemblies between signal
initiation and transduction components.
[0065] The lipid bilayer of the present invention may include
therein receptor-signal complexes Complete signaling complexes
composed of macromolecules such as described above and perhaps
intracellular targets, such as organelles or artificial
chromosomes.
[0066] The encapsulated spaces of the present invention may contain
a variety of different types of molecules including: biomolecules,
indicators, actuators, organelles, whole cells, magnetic materials,
etc.
[0067] The lipid bilayer of the present invention may include lipid
bilayer-associated cellular machinery. Such machinery includes
molecules and molecular assemblies involved in energy transduction
such as bacteriorhodpsin, proton dependent ATPase, photosynthetic
reaction centers, and bacterial flagellar motors.
[0068] Macromolecules that may be contained in the encapsulated
spaces of the microspheres of the present invention include
delivery systems for gene therapy, genetic discovery, direct
treatment of enzyme disorders, directed antibody, etc.
[0069] Biomolecules that may be contained in the encapsulated
spaces of the microspheres of the present invention include such
biomolecules as nucleic acids (DNA, RNA), plasmids, yeast
artificial chromosomes (YACs), other artificial chromosomes,
enzymes, hormones, pharmaceuticals, antibodies, etc.
[0070] Whole organellar components such as mitochondria and
chloroplasts, lysozomes, and nuclei or whole cells, either
prokaryotic or eukaryotic may be contained in the encapsulated
spaces of the microspheres of the present invention.
[0071] A functionalized mesoporous microsphere of the present
invention including assemblies of various materials within one or
more encapsulated spaces may form an artificial cell. Artificial
cells are currently being used in a wide variety of therapeutic
applications. These range from the use of encapsulated cultured
cells, see Pohorille and Deamer, "Artificial cells: prospects for
biotechnology," Trend Biotechnol., 20:123-128, (2002), the entire
contents and disclosure of which is hereby incorporated by
reference or bacteria to replace or augment damaged metabolic
pathways such as those which occur in diabetes or liver damage, to
encapsulated ferromagnetic particles to increase blood circulation
in those with artificial hearts, see Mitamura, et al.,
"Ferromagnetic artificial cells for artificial circulation," ASAIO
Journal., 42:M402-M406, (1996), the entire contents and disclosure
of which is hereby incorporated by reference to antigen displaying
particles that heighten or replace immune response, Than, et al.,
"Activation of antigen-specific T-cells by artificial cell
constructs having immobilized multimeric peptide class I complexes
and recombinant B7-Fc proteins," J. Immunol., (2001), the entire
contents and disclosure of which is hereby incorporated by
reference. Artificial cells are also the devices used to deliver
modified genetic material for use in gene replacement therapy.
[0072] Indicators contained in the encapsulated spaces of the
present invention may be any type of indicator used in the
biological sciences such as pH indicators, compound formation
indicators, complex formation indicators. The indicators may be
color indicators, fluorescent indicators, etc.
[0073] The encapsulated spaces of the present invention may include
whole cells. The encapsulated spaces may include magnetic materials
to allow the mesoporous microspheres to be magnetically
manipulated. The encapsulated spaces of the present invention may
also included charged materials to allow the mesoporous
microspheres to be electrically manipulated, such as by
electrophoresis.
[0074] The encapsulated spaces of the preset invention may include
actuator made of polymers that swell due to changes in pH, ion
concentration, etc., in the encapsulated space.
[0075] The materials in the encapsulated spaces of the mesoporous
microspheres of the present invention may be present by themselves
or may be part of a solution or suspension. When the functionalized
mesoporous microspheres of the present invention are present in an
external liquid, solution, or suspension, the liquid, solution or
suspension in which the materials in the encapsulated materials are
dispersed may be the same as or different from the external liquid,
solution or suspension.
[0076] Assemblies of the functionalized mesoporous microspheres of
the present invention may be for use in sensing devices. Of
particular interest in this category is the use of microspheres
with energy transfer systems (e.g. bacteriorhodopsin) to fuel
energy-requiring sensing processes (e.g. producing ATP or GTP for
use in G-protein coupled sensing reactions) or possibly to create
proton gradients necessary for function of incorporated bacterial
flagella motors, thus rendering the sensing microsphere mobile.
[0077] It is also anticipated that access to the internal of the
microsphere may be controlled by means of an environmentally
responsive or smart polymer, such as poly
(N-isopropylacrylamide).
[0078] The functionalized mesoporous microspheres of the present
invention may be used in a variety of applications. For example,
the functionalized mesoporous microspheres may be used in drug
discovery. Such microspheres may be used in flow cytometry methods
and used for high-throughput drug screening, as described in the
examples below. It is anticipated that the present invention will
have particular application within this technology.
[0079] The functionalized mesoporous microspheres of the present
invention may be used controlled drug delivery. The combination of
inclusion of smart (i.e. environmentally responsive) polymers,
already being investigated for use in drug delivery, included
within the pore with target-specific receptors or ligands displayed
on an encapsulating lipid bilayer may allow for targeted,
controlled drug release. Especially important may be the
possibility of receptor triggered activation of the smart polymer
(e.g. through increases in ionic strength within the particle) to
release the drug once it reaches its target.
[0080] The functionalized mesoporous microspheres of the present
invention may be used in proteomics. This microspheres of the
present invention allow for precise and controlled study of
interactions between membrane bound receptors and specific
cytosolic components (enzymes, organelles, nuclei) incorporated
into the microsphere matrix.
[0081] The functionalized mesoporous microspheres of the present
invention may be used in bioenergetics. Because many energy
generating biological processes depend upon ionic gradients between
membrane partitioned areas, this invention provides a unique
opportunity to exploit these processes for biotechnology. In the
examples below the use of the functionalized mesoporous
microspheres of the present invention with bacteriorhodpsin, which
generates proton gradients in response to light is described. A
system such as this could be used to fuel molecular energy (i.e.
ATP) generation, or provide a proton gradient needed to fuel
bacterial flagellar motors also located within the membrane.
[0082] The functionalized mesoporous microspheres of the present
invention may be used to provide artificial cells. Such constructs
have the potential for use as artificial cells, which are currently
being investigated as sources of hormonal replacement in such areas
as diabetes and liver failure or as carriers for gene therapy
agents.
[0083] The functionalized mesoporous microspheres of the present
invention may be used in creating viral decoys. By displaying
targets for viral interaction on the surface of the functionalized
mesoporous microspheres, invading viruses may be tricked into
attaching and fusing with artificial constructs. Since the
functionalized mesoporous microspheres possess no nuclear material
with which to propagate the viruses, they would be sequestered from
attacking cellular targets. This shows promise for those viral
diseases for which vaccines have not been developed, either due to
potential disruption of the immune response (as in HIV) or to
frequently changing viral surface epitopes (e.g. influenza).
[0084] The present invention will now be described by way of
example.
EXAMPLE I
[0085] A dye molecule, rhodamine, was encapsulated inside a
functionalized porous microsphere of the present invention, and
that the dye was accessible to all internal areas of the
microsphere as illustrated by FIG. 4. FIG. 4 is a series of
micrographs of a Z-series (1 mm slices, overlap .about.0.5 mm) of
rhodamine trapped within a membrane bound mesoporous microsphere of
the present invention.
[0086] After photobleaching of a small area, rapid recovery of
rhodamine fluorescence, as seen in FIG. 5, shows that flow within
the microsphere is possible. FIG. 5 is a series of micrographs of
fluorescence recovery after photobleaching rhodamine encapsulated
in a lipid coated porous microsphere. Total recovery time was 7
minutes.
[0087] Then the effect of perturbation of the ionic porosity of the
surrounding membrane on a contained reactive molecule was examined.
A Mg.sup.2+ sensitive dye Mag-fluo-4 (Molecular probes) was
introduced into the microspheres and the microspheres were
surrounded with a membrane. The microspheres were then incubated in
various concentrations of the bacterial toxin .alpha.-hemolysin in
magnesium-containing buffer. The resultant change in fluorescence
intensity is shown in FIG. 6. FIG. 6 depicts the .alpha.-hemolysin
mediated influx of Mg.sup.2+ into membrane-enclosed microspheres.
The Mg.sup.2+ indicator dye, Mag-Fluo-4 (0.2 mM in Tris buffer pH
7.4) was incorporated into microspheres and the microspheres
encapsulated in a lipid bilayer. The microspheres were then placed
in buffer solution containing 3 mM MgCl.sub.2 and varying
concentrations of .alpha.-hemolysin. The increase in Mag-Fluo-4
fluorescence was monitored by flow cytometry MCF (mean channel
fluorescence). The graph of FIG. 6 shows the pH sensitivity of
fluoroscein as measured by emission intensity at 518 nm (excitation
wavelength=480 nm).
[0088] The increase in fluorescence intensity corresponds to the
formation of ion pores by the incorporation of .alpha.-hemolysin
into the membrane, allowing for penetration of Mg.sup.2+ into the
microspheres to interact with the Mag-fluo-4.
[0089] The next investigation was to determine whether a integral
membrane protein, bacteriorhodopsin (BR) retained its activity and
effect a change on the contents of the microporous microsphere. The
natural activity of bacteriorhodpsin is to increase the proton
concentration (i.e. pH) upon illumination with the appropriate
wavelength of light (ca 510 nm). For this experiment, pH sensitive
fluorescent dye, fluoroscein, was incorporated into the
microspheres, as shown in FIG. 7. FIG. 7 illustrates the FITC
response to illumination of BR in the membrane, where %=weight
percent of BR incorporated into an Egg-PC liposome. Gd represents
the effect of 3 .mu.M Gd(NO.sub.3).sub.3, an inhibitor of BR.
[0090] The microspheres were then enveloped with liposomes
containing 0.1-5% reconstituted bacteriorhodpsin-containing "purple
membrane" from Halobacterium salinarum, in egg phosphatidyl
choline. Initial measurements were made regarding fluorscein
emission and then the BR-containing microspheres exposed to white
light for several minutes with FITC intensity being measure over
time using a flow cytometer, after several minutes of illumination,
FITC intensity decreased, indicating a rise in the proton
concentration within the microsphere, as shown by FIG. 8.
Furthermore, the decrease in pH was more pronounced as more BR was
incorporated into the membrane. Taken together, these results
indicate that BR is active within the microsphere and that the
contents of the microsphere are affected by activity within the
surrounding membrane.
[0091] In order to demonstrate that the contents of the microsphere
itself need not be limited to small species such as ions, and that
the activity of these species is also retainedlarger molecules were
incorporated into the microsphere-membrane system. The
environmentally responsive polymer poly-(N-isopropylacrylamide)
(PNIPAAM) was used as the basis of the experiments. PNIPAAM
exhibits a temperature dependent phase transition of 32.degree. C.
Below this temperature, the polymer is fully hydrated and fully
swollen; when the temperature rises above the transition
temperature, the polymer collapses. The pores of microspheres were
modified with surface grafted-PNIPAAM. In this situation, when the
polymer is swollen, the size of the pores should be reduced,
whereas in the collapsed state of the polymer, the pores should be
open. The uptake and release of FITC this system was examined, as
shown in FIG. 9. FIG. 9 is a graph illustrating the uptake and
release of FITC from PNIPAAM modified microspheres.
[0092] In these experiments, the FITC was incubated with
PNIPAAM-modified microspheres either at 40.degree. C. (pores open)
or at room temperature (ca 22.degree. C.; pores closed). The
microspheres were then incubated at 25.degree. C. to retain any
trapped molecules and analyzed by flow cytometry. The microspheres
were then washed in 40.degree. C. buffer in an attempt to release
trapped molecules and the mean channel fluorescence measured again.
While the results indicate that the pores were not completely
blocked (as evidenced by uptake of labeled molecules at 25.degree.
C.), there was uptake and release indicating that the incorporated
PNIPAAM was functional. Similar experiments have been attempted
with FITC-labeled insulin (MW .about.7000) and dextran (data not
shown). Preliminary results indicate that the molecules are taken
up readily into the microspheres, but unlike FITC, remain trapped.
The fact that larger molecules were taken up indicates that large,
biologically significant molecules may be incorporated into the
pores of the microspheres.
EXAMPLE II
[0093] Mesoporous microspheres useful in the product of the present
invention were made in the following ways:
[0094] Two precursor solution formulations were used: one based on
an acidic silica sol and one based on an aqueous TEOS solution. For
the acidic silica sol formulation, the solutions were synthesized
by addition of CTAB:
CH.sub.3(CH.sub.2).sub.15N(CH.sub.3).sub.3Br.sup.- (Aldrich) or
Brij-58: CH.sub.3(CH.sub.2).sub.15--(OCH.sub.2CH.sub.2).sub.20-- OH
(Aldrich) to an acidic silica sol, as reported by Lu et al.,
Nature, 1999, 398, 223. Two different silica precursor sols were
employed: a pre-hydrolyzed acidic TEOS-based sol, and an acidic
aqueous TEOS solution.
[0095] In a typical preparation, tetraethylorthosilicate (TEOS)
(Aldrich), ethanol, deionized water (conductivity less than 18.2
M.OMEGA. cm) and dilute HCl (mole ratios 1:3.8:1:0.0005) were
refluxed at 60.degree. C. for 90 min to provide the stock sol.
Then, 10 mL of stock sol was diluted with ethanol, followed by
addition of water, dilute HCI, and aqueous surfactant solution (1.5
g of surfactant dissolved in 20 mL of water) to provide final
overall TEOS/ethanol/H.sub.2O/HCI/surfactant molar ratio of
1:27:55:0.0053:0.19 and 1:22:55:0.0053:0.06 for CTAB and Brij-58
sols, respectively. For the aqueous TEOS-CTAB precursor solution,
CTAB was mixed with water (5 wt.-% CTAB) and stirred to obtain a
clear solution. To this solution TEOS and 1 N HCl were added to
give a solution with final molar ratios of TEOS/H.sub.2O/CTAB/HCI
is 1:63.28:0.15:0.0227. This sol was stirred for about 10 min
before beginning a powder synthesis run.
[0096] Monodisperse droplets were generated by means of a VOAG (TSI
Model 3450). In the VOAG, the aerosol solution was forced through a
small orifice (10 .mu.m or 20 .mu.m) by a syringe pump, with
syringe velocities of approximately 2.times.10.sup.-4 cm s.sup.-1
(.about.1.4.times.10.sup.-- 3 cm.sup.3s-1) and 8.times.10.sup.-4 cm
s.sup.-1 (.about.4.7.times.10.sup.- -3 cm.sup.-3 s.sup.-1) for the
10 .mu.m and 20 .mu.m orifices, respectively. This delivery rate
was adjusted to provide a stable operating pressure of 340-420 kPa.
The liquid stream was broken up into uniform droplets by the
vibrating orifice. The frequency range employed was 40-200 kHz,
with the final setting adjusted to eliminate satellite droplets.
The droplets were then injected axially along the center of a
turbulent air jet to disperse the droplets and to prevent
coagulation. Following the mixing of the dispersed droplets with a
much larger volume of filtered dry air, the droplet-laden gas
stream flowed through a 2.5 cm diameter quartz tube into a
three-zone furnace (0.9 m heated length) maintained at 500.degree.
C. (acidic silica sol runs) or 420.degree. C. (TEOS solution runs).
This provided a mean residence time of approximately 0.3 seconds in
the heated zone. The particles were collected on a filter
maintained at approximately 80.degree. C. by a heating tape.
Collected particles were calcined in air at 400-450.degree. C. for
4 hours (acidic silica sol runs) or at 500.degree. C. for 12 hours
(TEOS solution runs) to remove the surfactant template. The typical
powder production rate using the VOAG was 0.35 g SiO.sub.2
h.sup.-1.
[0097] The particles were characterized by scanning electron
microscope (Hitachi S-800) and X-ray diffraction (Siemens D5000, Cu
K.alpha. radiation, 2.=1.5418 .ANG.) techniques. Surface area and
pore size distribution studies were carried out by nitrogen
adsorption/desorption at 77 K using a Micromeritics ASAP 2000
porosimeter. For cross-sectional TEM (JEOL 2010, 200 KV), particles
were embedded in an epoxy and then cross-sectioned using a Sorvall
MT-5000 Ultra Microtome machine.
[0098] Particle size was controlled by varying the orifice diameter
in the VOAG and the concentration of the precursor solution.
Scanning electron micrograph (SEM) images of spherical porous
silica particles indicated that the particles are spherical,
monodisperse in size, and possess a smooth surface morphology. The
average particle size obtained using the TEOS-based sol formulation
with CTAB surfactant and a 20 .mu.m orifice was 9.9.+-.0.5 .mu.m
(mean and standard deviation of over 100 particles measured from
SEM images). The pH of the TEOS-based sol formulation minimized the
siloxane condensation rate, thereby facilitating silica surfactant
self-assembly during aerosol processing. Using the same sol
formulation with Brij-58 surfactant, a 10 .mu.m orifice resulted in
an average particle size of 6.0 .mu.m.+-.0.3 .mu.m.
[0099] Powder X-ray diffraction (XRD) of the particles revealed
only a single peak, indicating periodic short range structural
order with d-spacings of 30.4 and 45.5 .ANG. for particles produced
with CTAB and Brij-58, respectively. The absence of higher order
Bragg peaks indicates that these particles lack the high degree of
long-range structural order that is commonly seen for commercially
prepared material. Transmission electron microscopy (TEM) images
obtained from cross sections of ultra-microtomed particle slices
revealed that the particles produced from both CTAB and Brij-58
were not hollow. Particles produced with CTAB displayed a uniform
mesostructure, but with no apparent long-range ordering. Particles
produced using Brij-58 displayed a highly uniform periodic pore
structure in some regions of the particles; however, most of the
particle cross-section showed no apparent order. Electron
diffraction of the Brij-58 particles indicated a mesostructural
d-spacing of 45 .ANG., which corroborated the XRD results. Nitrogen
adsorption-desorption isotherms showed typical features for
mesoporous materials. The average (hydraulic) pore diameter d.sub.p
determined from nitrogen adsorption data (calculated as
d.sub.p-4V.sub.p/S.sub.p where V.sub.p is total pore volume and
S.sub.p, is the BET surface area) were 22.3 and 28.2 for CTAB and
Brij-58 templated powders, respectively. The corresponding BET
surface areas of the particles were 600 and 516 m.sup.2/gl for CTAB
and Brij-58, respectively. These specific surface areas are much
lower than observed for CTAB in fully ordered submicrometer
particles, but are consistent with previous results obtained using
Brij-56 surfactant.
[0100] Particles produced using the aqueous TEOS precursor solution
with CTAB surfactant displayed much better ordering than those made
with the TEOS-based sol. TEM images of an ultra-microtomed section
of one these particles at low magnification, indicated that
particles were not hollow, while higher magnification revealed that
pores were present in well-ordered domains across the entire cross
section of the particles. In some regions, the TEM images appeared
consistent with the hexagonally packed tubular pores bundles that
have been observed with the same solution and surfactant in
submicrometer particles. In some areas near the surface, pores
apparently are oriented parallel to the surface, which is
consistent with what has been normally observed in submicrometer
particles. However, in other regions the pore channels are
apparently aligned perpendicular to the surface. This provided
clear evidence of perpendicular pore alignment at the particle
surface. This is of interest because alignment of tubular pore
channels normal to the surface is optimal for unimpeded access to
the pore internals.
[0101] In summary, monodisperse spherical porous silica particles
were prepared with diameters in the 5 to 10 .mu.m range based on
evaporation-driven self-assembly of surfactants in droplets
produced from a VOAG. The method should be applicable to particles
as small as 1-2.mu.m and as large as 50 .mu.m by varying VOAG
orifice size and solution concentrations. This approach can have
several significant advantages over the traditional solution-based
self-assembly. The aerosol process is a continuous, scaleable
process in which the entire particle synthesis process occurs on a
time scale of several seconds or less. Highly spherical
non-aggregated particles are consistently produced under
appropriate conditions. Finally, any additives, dopants or
additional components that may be made into an aerosol from a
solution or dispersion are inevitably incorporated into each
particle. These features make the method attractive for producing
microspheres to be used in for example, sensor applications, where
environmentally sensitive fluorescent dyes could be incorporated
into particles. Similarly, porous internals could serve as
reservoirs for pharmaceutical agents in controlled release schemes.
Mesoporous silica microspheres in the size range produced here are
also highly promising as supports for biomolecules and
biomembranes, which is an interesting new strategy for developing
molecular affinity surfaces, biosensor devices and high-throughput
screening devices. A number of other possible applications can be
envisioned, such as optical materials, catalyst supports,
biocompatible microreactors and molecular separations media. The
convenient control of particle size and monodispersity demonstrated
this example are important complements to the control of internal
mesostructure and pore size provided by surfactant templating.
[0102] The functionalized mesoporous microspheres of the present
invention may also be used with fluorescence techniques.
Fluorescence techniques, while not having the spatial resolution of
electron microscopy, enable characterization of important dynamic
processes, both within the particles and on particle surfaces. The
fluorescence measurements that may be made may be separated into
four classes that illustrate their spatial resolution. The first
class, with lowest spatial resolution, is traditional fluorescence
spectroscopy of particle suspensions and their supernatants. These
simple measurements (e.g. centrifugation assays) will be useful in
quantifying and optimizing diffusive release or uptake by porous
mesoporous microspheres and are thus useful in controlled delivery
applications. The second class is flow cytometry measurements, in
which one obtains fluorescence information from individual
particles. Monodisperse mesoporous microspheres formed by acid
catalyzed evaporation induced self-assembly (AA-EISA using a VOAG
droplet generator are compatible with flow cytometric measurements.
Several unique tools for kinetic and high throughput analysis,
described in the examples below, when coupled with the versatile
functionalized mesoporous microspheres of the present invention
will allow an unprecedented incorporation of biochemical function
into flow cytometry methods as described below. The third class is
laser microscopy of individual particles and includes confocal
scanning laser microscopy and spectroscopic imaging.
[0103] Another class of fluorescence characterization is single
molecule measurements, which have the potential to offer the
highest level of spatial resolution. Confocal laser microscopy is
especially useful for quantifying transient processes involving
diffusion of ensembles of fluorophores within, on the surface, into
or out of the porous particles.
[0104] The mesoporous microspheres of the present invention
versatile supports for functional lipid bilayer membranes that
envelop the microspheres and biochemical reactants and reporter
groups within the encapsulated spaces of the microspheres.
[0105] The functionalized mesoporous microspheres have particular
applicability to high throughput pharmaceutical screening
technologies based on flow cytometry and affinity microcolumns. The
functionalize mesoporous microspheres of the present invention also
provide a convenient new platform for membrane biophysical studies
that are generally applicable to study of a wide range of cell
membrane functions.
EXAMPLE III
[0106] The functionalized mesoporous microspheres of the present
invention may be used for the low cost, small volume, high
throughput screening of cell and bead-based molecular target assays
at rates approaching 100,000 assays per day and for the
investigation of active transport systems such as the dopamine
transporter (DAT) and the effect of toxins such as cocaine.
[0107] The dopamine transporter (DAT), which is a major target for
a number of pharmacologically active drugs and environmental and
synthetic toxins, mediates uptake of dopamine (DA) into neurons.
Understanding the function of the DAT allow for the implementation
of high throughput screening methods for pharmacotherapeutics and
antitoxin agents, for the development of biosensors for chemical
warfare agents, and for the development of methodologies for active
sequestration of toxins in the protection of service personnel.
Further, altered DAT expression in a variety of diseases (e.g.
Parkinson's disease, substance abuse, Alzheimer's disease,
Tourette's syndrome, attention-deficit hyperactivity disorder
(ADHD), Lesch-Nyhan disease and normal aging,) suggests that the
DAT plays a critical role in normal and abnormal DA
neurotransmission. Since the cloning of DAT, much information has
been obtained regarding the structure of DAT and function and
structural variations have been linked to a number of
psychopathologies.
[0108] DAT may be investigated using the following methods.
[0109] Maintenance of the HEK-293 Cells. Suitable human embryonic
kidney (HEK) cells stablely transfected with human DAT have been
made by Dr. Zhicheng Lin at NIDA. Typical maintenance and expansion
of these cells will be performed in accordance with Dr. Lin's
protocol. Cells will be passed no more than 20 times before a new
stock is thawed and grown for use. If it is found that the amount
of DAT that can be purified from these cells is limiting, hDAT cDNA
made by Dr. Lin may be used in production of an over-expression
cell system. Additionally, Dr. Lin has developed a number of point
mutations in the DAT sequence and these may be used for study as
well.
[0110] Preparation of Solubilized DAT. The procedure for DAT
solubilization is modified from established protocols. DAT
expressing HEK cells are homogenized, centrifuged and resuspended
in solubilization buffer containing detergent (e.g.
.beta.-octyl-glucoside, CHAPS or digitonin), and asolectin. The
detergent-membrane suspension is kept on ice for 30 min before
centrifugation. Supernatant is purified by affinity chromatography
using cocaine, a DAT ligand. The receptors are eluted from the
column with 10 mM dopamine in wash buffer. Transporter containing
fractions are pooled, the pH adjusted and the samples applied to a
DEAE-Sepharose column. Transporters are eluted from the column by
the addition of an elution buffer and the pooled transporter
containing fractions are dialyzed twice. Crosslinking prior to
solubilization will be evaluated using methodsdescribed in Hastrup
H, Karlin A, Javitch J A, "Symmetrical Dimer of the Human Dopamine
Transporter Revealed by Cross-Linking Cys-306 at the Extracellular
End of the Sixth Transmembrane Segment," Proceedings Of The
National Academy Of Sciences Of The United States Of America,
98:10055-10060, (2001), the entire contents and disclosure of which
are hereby incorporated by reference. Should the above techniques
prove unsatisfactory for solubilization, a method using a
biotinylated DAT expression/purification protocol similar to that
described in Julien M, Kajiji S, Kaback R H, Gros P, "Simple
Purification of Highly Active Biotinylated PGlycoprotein:
Enantiomer-Specific Modulation of Drug-Stimulated ATPase Activity,"
BIOCHEMISTRY, 39:75-85, 2000, the entire contents and disclosure of
which are hereby incorporated by reference, will be used.
[0111] Reconstitution into Lipid Vesicles. Purified dopamine
transporters are reconstituted into various combinations of
purified lipids, sphingomyelin, and cholesterol for later
incorporation into supported lipid bilayers. Several different
combinations and ratios of membrane components will be evaluated
for efficiency and preservation of transporter function. Detergent
is removed by gel filtration using a Sephadex G50-80 column or by
the use of non-polar styrene beads. Transporters are eluted in the
void volume by a reconstitution buffer and dialyzed against
3.times.4 L of reconstitution buffer for 20 hours at 4.degree. C.
Proteoliposomes are then frozen at -80.degree. C. until needed.
[0112] Immunoprecipitation of DAT Containing Vesicles. In order to
enrich our suspensions, proteoliposomes containing transporter will
be separated using immunological techniques. Solubilized samples
are immunoprecipitated with antibody 16, directed against amino
acids 42-59 of the deduced DAT primary sequence. This antibody has
been shown by immunoprecipitation, immunoblot, and
immunohistochemistry to be highly specific for DAT. Samples are
incubated with protein Sepharose CL4B. Immune complexes are washed
twice with the Tris-Triton buffer, and samples are eluted with
SDS-polyacrylamide gel electrophoresis loading buffer. Samples are
electrophoresed on 7% polyacrylamide gels followed by
autoradiography. Positive controls for immunoprecipitations are
provided by parallel precipitation of [.sup.125I]DEEP photoaffinity
labeled dopamine transporters, as described in Vaughan R A, Agoston
G E, Lever J R, Newman A H, "Differential Binding of Tropane-Based
Photoaffinity Ligands on the Dopamine Transporter," JOURNAL OF
NEUROSCIENCE, 19: 630-636, 1999, the entire contents and disclosure
of which are hereby incorporated by reference. For peptide blocking
experiments, diluted antiserum is preincubated with either peptide
16 or peptide 18 (amino acids 580-608) prior to addition of the
sample.
[0113] Dopamine Uptake. To assure the reconstituted transporters
are functional, dopamine uptake in the proteoliposomes will be
performed as described with minor modification in Lee SH, Cho H K,
Son H, Lee Y S, "Substrate Transport and Cocaine Binding of Human
Dopamine Transporter is Reduced by Substitution of Carboxyl Tail
with that of Bovine Dopamine Transporter," NEUROREPORT, 8:
2591-2594, 1997, and Frank J. S. Lee, Zdenek B. Pristupa, Brian J.
Ciliax, Allan I. Levey and Hyman B. Niznik, "The Dopamine
Transporter Carboxyl-Terminal Tail Truncation/Substitution Mutants
Selectively Confer High Affinity Dopamine Uptake while Attenuating
Recognition of the Ligand Bindingdomain," Jouranl Of Pharmacology
And Experimental Therapeutics 271: 20885-20894, 1996, the entire
contents and disclosure of which are hereby incorporated by
reference. Aliquots of proteoliposomes in assay buffer containing
sodium are incubated for 3 min at 34.degree. C. Uptake solution
will contain [.sup.3H]DA at 1 nM except for saturation analyses
where [.sup.3H]DA will be increased to 10 nM and unlabeled dopamine
varied from 10 nM to 1 .mu.M. Nonspecific uptake is defined with
100 .mu.M cocaine. Uptake assays are carried out for 3 min at
34.degree. C. Proteoliposomes are placed on disposable Dowex 50W
resin columns and washed with 2 column volumes. Flow through is
collected and counted by liquid scintillation spectrometry.
Specific dopamine uptake in control preparations should average
1.18 pmol/min/mg protein, and data will be analyzed by EBDA and
LIGAND computer software. Human DAT protein is anticipated to have
a K.sub.m of 2100 nM and a V.sub.max of 9.0 pmol/10.sup.5
cells/min. To determine specificity of the transporters
[.sup.3H]alanine uptake will be examined using the same conditions
as for dopamine transport, except that [.sup.3H]alanine at a
concentration of 10 nM will be used.
[0114] Incorporation of DAT into Lipid Bilayers on Porous
Microbeads. DAT will be incorporated into lipid bilayers on porous
microbeads generated by procedures described in above (q.v.).
Dopamine transport will be indicated by the detection of imported
Na.sup.+ ions using the sodium sensitive dye SBFI. A similar method
with magnesium-sensitive dyes for the detection of
.alpha.-hemolysin, a bacterial transmembrane toxin, using flow
cytometry has been performed. FIG. 10 illustrates Flow cytometric
detection of .alpha.-hemolysin using a magnesium indicator dye
trapped in a lipid coated porous microbead In this procedure,
monodisperse porous beads are placed in Mag-Fluo-4 dye (a
fluorescent magnesium indicator) in a Tris-buffered solution,
sonicated and incubated overnight. Proteoliposomes are added to the
beads and the whole shaken for 5 min, followed by incubation at
room temperature for 30 minutes. Liposomes envelope the
dye-containing beads spontaneously. Unbound phospholipid and
unsequestered dye are removed by repeated centrifugation and
resuspension in buffer. Varying concentrations of .alpha.-hemolysin
are mixed with 3 mM MgCl.sub.2, and added to the bead suspension,
and incubated at 37.degree. C. Aliquots of the beads are removed at
defined time intervals and fluorescence intensity of the beads,
indicating the influx of Mg.sup.+2 ions due to the incorporation of
.alpha.-hemolysin, a porin, into the liposome is monitored using
flow cytometry. In this project similar assemblies for the
detection of active transport of DAT will be developed and will be
demonstrated as important models in the high throughput screening
of ligands, drugs, and antagonists for neurotransmitter
transporters that will be extendable to a host of other important
transporter types.
[0115] High Throughput Screening with Flow Cytometry. Initially,
porous beads containing the DAT transporter along with necessary
associated lipid and rafts will be evaluated using a high
throughput flow cytometry "plug flow" that is capable of 10 end
point assays per min, 4 on-line mixing experiments per min, and a
concentration gradient for secondary screening of a compound in
about 2 min. Later a second generation instrument (HyperCyt.TM.)
which approaches a rate of 100 samples/minute with 1% particle
carryover from well to well will be used. As it is not necessary
that the vessel containing the sample be pressurized, it has been
possible to interface the flow cytometer with devices such as a
High Throughput Pharmacology System for rapid evaluation of cell
responses to pharmacological receptor-ligand interaction. This
approach uses air bubbles to separate samples, with low carryover.
The samples are introduced by an autosampler and peristaltic pump
into a tubing line that directly connects to the flow cytometer and
is described in U.S. patent application Ser. No. 09/501,643, the
entire contents and disclosure of which is hereby incorporated by
reference. This approach uses a standard multi-well plate as well
as 60 well Terasaki plates with 10 .mu.L wells. On-line
microfluidic mixing for submicroliter samples and sampling rates of
up to 20 samples per minute has been developed. Two micromixing
approaches that are compatible with commercial autosamplers, flow
cytometry and other detection schemes that require mixing of
components that have been introduced into laminar flow have been
identified. The mixing is driven by a magnetic microstirrer
contained within the sample line. For example, the mixing may be
assessed using SBFI fluorescence of both cell sodium responses and
bead-based fluorescence. The fluorescent indicator of sodium, SBFI
comes in permeant and impermeant forms (Molecular Probes) and is
similar to the calcium intracellular indicators in use and
calibration. Porous beads that contain SBFI and that are enveloped
with phospholipid bilayers containing DAT can be used in the
development of high throughput assays for optimization of
transporter containing molecular assemblies, ruggedization of
transporter systems, and study and optimization of agonist or
antagonist function. In previous work, the number of receptor sites
occupied by a fluorescent ligand to the receptor has been related
to the calcium response in cells transfected with the formyl
peptide receptor. Similarly, the number of transporter sites
occupied by a fluorescent ligand (e.g. agonist or antagonist) may
be related to the sodium response due to active transport in cells
transfected to express DAT. The cell-based assays will be
especially useful in screening of DAT mutants and directed
evolution of transporter proteins that respond specifically to
drugs or chemical warfare agents. Such exercises will be useful in
the development of biosensors and active molecular sequestration
devices. FIG. 11 illustrates in schematic form a fluidic sample
handling system, of the type described above, for high throughput
screening using flow cytometry and the functionalized mesoporous
microspheres of the present invention.
EXAMPLE IV
[0116] A functionalized mesoporous microsphere of the present
invention was loaded with rhodamine and encapsulated in an
encapsulated space of a functionalized mesoporous microsphere of
the present invention. The functionalized mesoporous microsphere
was then photobleached. The microsphere was approximately 10 .mu.m
diameter. FIG. 12A is a confocal laser scanning micrograph of the
functionalized mesoporous microsphere before photobleaching. FIG.
12B is a confocal image of the functionalized mesoporous
microsphere after photobleaching. FIG. 12C is a confocal image of
the functionalized mesoporous microsphere after recovery, about 7
minutes later. In one region of the particle section has been
photobleached and then allowed to recover its fluorescence through
diffusion of the dye. This preliminary experiment establishes that
the particle is uniformly fluorescent and that the dye can readily
diffuse through it, suggesting that the mesoporous structure is
well connected. Fluorescence recovery after photobleaching (FRAP)
may be used to determine the diffusion characteristics (e.g.,
diffusion constants, diffusion asymmetry) of various fluorescent
probes within the mesoporous particles to gain understanding of the
pore transport characteristics (pore accessibility, conductivity,
connectivity, and tortuousity). Likewise FRAP of the labeled
lipids, or of labeled proteins, that are either incorporated or
appended to the lipid bilayer membranes, can be used to
characterize the fluidity of the membrane and its functional
components. A key issue to be examined is the role of the porous
architecture in optimizing the function of cytoplasmic domains of
transmembrane proteins. Further fluorescence characterization of
interactions between protein and mesoporous microsphere components
will include fluorescence energy transfer (FRET) and polarization
anisotropy measurements. Thus, detailed characterization of
dynamical processes both within the mesoporous microspheres and on
the surface of the mesoporous microspheres will enable the rational
design of particle interior nano-architectures for particular
applications.
EXAMPLE V
[0117] Functionalized mesoporous microspheres of the present
invention that were monodisperse in diameter in the supramicron
range (approximating the size of mammalian cells) were made by
AA-ESIA using a vibrating orifice aerosol generater (VOAG). These
microparticles had several interesting characteristics including:
(1) their accessible relatively large pores that may be used to
accommodate functional biomolecules and probe molecules (e.g.,
fluorophores); (2) their function as supports for well-organized
lipid bilayer membranes; and (3) their compatibility with flow
cytometric measurements and our recently developed techniques for
construction of mesoporous microsphere-packed microbioanalytical
microsystems. FIG. 4 presents a 2-color confocal fluorescence
micrograph of a rhodamine filled microbead encapsulated in a
fluorescein-labeled lipid bilayer. FIG. 6 compares images and
fluorescence histograms for these particles with those of
commercial, nonporous silica flow cytometry beads. The data in show
that (1) it is possible to form monodisperse mesoporous
microspheres of comparable size and dispersity to those sold
commercially for flow cytometry experiments, and (2) the simple
protocols established for formation of unilamellar phospholipid
bilayers on glass beads can be extended to the mesoporous
microsphere. The latter fact is born out by the flow cytometry data
that indicates quite comparable levels of fluorescence on the two
bead types. Further evidence that the lipid bilayers on the porous
beads are defect free and completely envelop the mesoporous
microspheres is provided by the ability of the mesoporous
microspheres to block ionic transport (vide infra). Flow cytometry
is an ideal analytical tool for studying these particles for
several reasons: (1) flow cytometry provides data from each
mesoporous microsphere thus making data gathering extremely
efficient and statistical analysis straightforward; (2) flow
cytometry provides data not only on the fluorescence
characteristics, but also size and morphology of the particles
through measurement of light scattering from each particle; (3)
flow cytometry is compatible with particle sorting so that, for a
particular particle design, particles with desired scattering and
fluorescence characteristics can be sorted and collected; and (4)
flow cytometry is compatible with high throughput reaction and
interrogation of different bead types as required, for example, for
drug screening and proteomic applications.
EXAMPLE VI
[0118] An important step toward the realization of hybrid
biosynthetic bead systems that incorporate cellular machinery as
cell mimics is the ability to incorporate functional transmembrane
proteins (TMPs) into the supported lipid bilayer. In experiments,
several proteins (including ICAM-1 and bacteriorhodopsin) were
readily incorporated into bead supported bilayers through protocols
established for nonporous beads. The functionality of such proteins
has also been established in preliminary experiments.
Bacteriorhodopsin was used as a model TMP in the experiments
described below for several reasons. Bacteriorhodopsin is
well-studied and bacteriorhodopsin's structure and proton pumping
mechanisms are well established. Furthermore, bacteriorhodopsin may
be used as a molecular machine that may be used to establish
voltage gradients across the mesoporous microsphere supported lipid
bilayer membranes, as well as being technologically important in
its own right In addition, through the availability of pH sensitive
dyes (e.g., fluorescein, SNAFL-2) it has been possible to establish
a simple assay for its activity based on flow cytometric
measurement of fluorophore loaded beads that are coated with
bacteriorhodopsin-containing phospholipid bilayers. FIG. 14
schematically depicts the structure of such a bead and presents
preliminary data gathered using flow cytometry of modified porous
beads. The initial pH is 7.2 These data clearly demonstrate the
integrity of the lipid bilayer and the photo-induced proton pumping
of the bacteriorhodopsin immobilized on the mesoporous
microspheres. This result is significant because bacteriorhodopsin
can be considered a well-characterized model for the important
class of transmembrane proteins that have structural homology
including seven transmembrane .alpha. helical domains, with three
connecting loops on each inner and outer face of the membrane.
Seven-loop transmembrane receptors (7TMRs) are one of the most
important class of proteins in the area of pharmaceutical
development. This important class of proteins is involved in a
myriad of physiological functions including intercellular
signalling, active transport, and sensing.
EXAMPLE VII
[0119] A biochemical "tool kit" was developed based on a G protein
coupled 7TMR, the formyl peptide receptor that includes the
receptor itself and several intercellular components (G protein
subunits and arrestin) that are important in intracellular
signalling following ligand binding to the 7TMR and that are
important as drug targets. Several of the inner connecting loops
and the carboxyl terminal tail couple the receptor to intracellular
proteins such as the G proteins. The G proteins are a family of
.alpha..beta..gamma. heterotrimeric molecules. The .alpha. subunit
contains nucleotide binding sites. Both .alpha. and .beta..gamma.
subunits have the capacity to interact with catalytic enzymes or
"effectors" required for cell activation. There are several classes
of the .alpha., .beta., and .gamma. subunits which provide
specificity for the coupling of the receptors, G proteins, and
effectors. The intracellular loops contain phosphorylation sites
for receptor kinases that yield desensitized receptors. There is a
family of "arrestin" molecules which bind to desensitized receptors
and may prevent the receptor from being engaged in further signal
transduction. The specificity of interactions is often defined in
cellular expression systems because there have not been up to this
point convenient (i.e., sensitive, real-time) assay systems for
examining molecular assemblies between signal initiation and
transduction components. The availability of a biomimetic molecular
assembly for analysis of potential drug compounds with various
parts of the G protein coupled receptor systems would have the
potential to greatly enhance high throughput drug discovery and
mechanistic investigation of intracellular signaling pathways.
[0120] Lipid bilayers are reconstituted that incorporate 7TMRs and
their associated intracellular protein couples on well-defined
porous particles prepared by AA-EISA. Formyl peptide receptor is
reconstituted as a model 7TMR for other G-protein coupled
transmembrane proteins and their associated intracellular machinery
into cell mimics. Our experience with production and purification
of these proteins, their labeling, assembly and immobilization will
be crucial to the success of these studies. Successful
immobilization of this receptor, and its associated G proteins
constitutes a "proof-of-principle" demonstration that will
establish the feasibility of bead-based display of a variety of
7TMRs, including those of interest in intracellular signalling and
response to toxins. In this work, concentration of precious, dilute
proteins into porous beads is enhanced by the incorporation of
stimuli-responsive polymers such as poly(N-isopropyl acrylamide)
(PNIPAAM) into the porous network of the particles. PNIPAAM
exhibits an inverse solubility transition at a critical temperature
of .about.33.degree. C.; below this temperature the polymer is
soluble in water; above it, it is insoluble (hydrophobic). Upon
exposure of the PNIPAAM modified beads (above 33.degree. C.) to
dilute solutions of intracellular proteins (e.g., G-proteins,
arrestin), the proteins will be adsorbed into the particle
interior, where they can be entrapped by lipid bilayer membranes.
Upon cooling of the particles to room temperature, where the
PNIPAAM is hydrated, the cytoplasmic proteins will be liberated
from their hydrophobic traps and will be free to assemble with each
other and with their transmembrane receptor couples. Electrokinetic
trapping may also be used to concentrate cytoplasmic proteins into
beads. Reconstituted cell-signaling pathways will thus be amenable
to incorporation into mesoporous microsphere based platforms that
will be ideal for high throughput drug screening via flow cytometry
(see below). In these flow cytometric assays, and in the
characterization of the cell mimetic reconstitution process,
fluorescence spectroscopy and imaging will be an invaluable
tool.
EXAMPLE VIII
[0121] Another fluorescence sensing platform that is used with the
biomimetic mesoporous microspheres is porous affinity biosensors
based on fluorescent bead-based microcolumns as a highly efficient
nanofluidic detection platform. These sensing systems have a number
of unique traits including the possibility for rapid, economical
analysis of very small samples without the need for reagent
addition and mixing. The new beads to be developed in this work
will have immediate application in microfluidic sensors based on
this technology and will greatly expand the nanoscopic
functionality of these systems.
EXAMPLE IX
[0122] High Throughput Screening with Flow Cytometry and
Nanofluidic Systems. In flow cytometry, the particles in a sample
flow single file through a focused laser beam at rates up to
thousands per second. The fluorescence measurements are highly
sensitive, with commercial instruments having detection limits of
several hundred fluorophores per particle. With the use of
appropriate standards and calibration protocols, the measurements
can be made quantitative. Recently, a computer controlled sample
mixing and delivery system has been coupled to a flow cytometer for
continuous kinetic analysis with subsecond resolution as
illustrated in FIG. 11. With delivery times under one half second
and the ability to perform complex, multi-step mixing protocols,
rapid mix flow cytometry has made sensitive and quantitative real
time measurements of molecular interactions a reality. Previously
dense biotinylated lipid coated beads were used to display a
variety of peptide ligands through streptavidin coupling. The use
of fluorescent peptides and nonfluorescent streptavidin allowed us
to determine the degree of specificity in ligand binding on these
beads. These methods may be extended to mesoporous particles.
Initially, porous beads containing the transmembrane proteins
(bacteriorhodopsin, formyl peptide receptor) along with necessary
associated lipid will be evaluated using a high throughput flow
cytometry "plug flow" that is capable of 10 end point assays per
min, 4 on-line mixing experiments per min, and a concentration
gradient for secondary screening of a compound in about 2 min.
Later a second generation instrument (HyperCyt.TM.) which
approaches a rate of 100 samples/minute with 1% particle carryover
from well to well will be used. As it is not necessary that the
vessel containing the sample be pressurized, it has been possible
to interface the flow cytometer with devices such as a High
Throughput Pharmacology System for rapid evaluation of
pharmacological receptor-ligand interaction. This approach uses air
bubbles to separate samples, with low carryover. The samples are
introduced by an autosampler and peristaltic pump into a tubing
line that directly connects to the flow cytometer. This approach
uses a standard multi-well plate as well as 60 well Terasaki plates
with 10 .mu.L wells. On-line microfluidic mixing for submicroliter
samples and sampling rates of up to 20 samples per minute have been
developed. Two micromixing approaches have been identified that are
compatible with commercial autosamplers, flow cytometry and other
detection schemes that require mixing of components that have been
introduced into laminar flow. The mixing is driven by a magnetic
microstirrer contained within the sample line. Porous beads that
contain pH sensitive dyes (e.g., SNAFL-2) and that are enveloped
with phospholipid bilayers containing bacteriorhodopsin can be used
in the development of high throughput assays for optimization of
7TMR containing molecular assemblies, ruggedization of functional
biomimetic assemblies, and study and optimization of agonist or
antagonist function. It has been possible to relate the number of
receptor sites occupied by a fluorescent ligand to the receptor to
the calcium response in cells transfected with the formyl peptide
receptor. It is similarly possible to relate the number of
transporter sites occupied by a fluorescent ligand (e.g., agonist
or antagonist) to the fluorescent response due to its affect on
formyl peptide receptor function. Functionality assays will be
based on fluorescence resonance energy transfer between labeled
proteins that participate in the G-protein associated signaling
process.
[0123] All documents, patents, journal articles and other materials
cited in the present application are hereby incorporated by
reference.
[0124] Although the present invention has been fully described in
conjunction with the preferred embodiment thereof with reference to
the accompanying drawings, it is to be understood that various
changes and modifications may be apparent to those skilled in the
art. Such changes and modifications are to be understood as
included within the scope of the present invention as defined by
the appended claims, unless they depart therefrom.
* * * * *