CROSS-REFERENCE TO RELATED APPLICATIONS
GOVERNMENT INTEREST STATEMENT
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
 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.
1. Field of the Invention
This invention relates to microspheres.
2. Description of the Prior Art
- SUMMARY OF THE INVENTION
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.
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.
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.
It is yet another object to provide functionalized mesoporous microspheres that allow the study of bioenergetics at the cellular level.
It is yet another object to provide functionalized mesoporous microspheres that may be used to make improved artificial cells than are currently available.
It is yet another object to provide functionalized mesoporous microspheres that may be used in making improved viral decoys.
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.
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.
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.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects and features of the present invention will be apparent from the following detailed description of the preferred embodiment.
The invention will be described in conjunction with the accompanying drawings, in which:
FIG. 1 is a cross-sectional view in simplified form of a biologically functionalized porous microsphere of one preferred embodiment of the present invention;
FIG. 2 is a cross-sectional view in simplified form of a biologically functionalized porous microsphere of another preferred embodiment of the present invention;
FIG. 3 is an illustration in simplified form of a porous microsphere of one preferred embodiment of the present invention;
FIG. 4 is a series of micrographs of a Z-series (1 mm slices, overlap ˜0.5 mm) of rhodamine trapped within a membrane bound mesoporous microsphere of the present invention;
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;
FIG. 6 is a graph illustrating α-hemolysin mediated influx of Mg2+ into membrane-enclosed microspheres;
FIG. 7 is a graph illustrating pH sensitivity of fluoroscein as measured by emission intensity at 518 (excitation wavelength=480);
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;
FIG. 9 is a graph illustrating the uptake and release of FITC from PNIPAAM modified microspheres;
FIG. 10 is a graph illustrating flow cytometric detection of α-hemolysin using a magnesium indicator dye trapped in a lipid coated mesoporous microsphere of the present invention;
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;
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;
FIG. 12B is a confocal fluorescence microscopic section of the functionalized mesoporous microsphere of FIG. 12A after photobleaching;
FIG. 12C is a confocal image of the functionalized mesoporous microsphere of FIGS. 12A and 12B after recovery, about 7 minutes later;
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
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
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.
It is advantageous to define several terms before describing the invention. It should be appreciated that the following definitions are used throughout this application.
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.
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 μm, and more preferably have a diameter of about 15 μm. 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.
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.
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.
For the purposes of the present invention, the term “functionalized microsphere” refers to a microsphere at least partially covered by a lipid bilayer.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 μm, 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).
Unlike previous commercial microspheres, the preferred mesoporous microspheres of the present invention contain well ordered pores, with extremely high surface areas, (up to 1000 m2/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.
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.
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.
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.
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.
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.
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.
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.
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.
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 α 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.
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 αβγ heterotrimeric molecules. The α subunit contains nucleotide (GTP, cAMP) binding sites. Both α and βγ subunits have the capacity to interact with catalytic enzymes or “effectors” required for cell activation. There are several classes of α, β, and γ 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.
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.
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.
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.
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).
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 αβγ heterotrimeric molecules. The α subunit contains nucleotide (GTP, cAMP) binding sites. Both α and βγ subunits have the capacity to interact with catalytic enzymes or “effectors” required for cell activation. There are several classes of α, β, and γ 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 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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).
- EXAMPLE I
The present invention will now be described by way of example.
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 ˜0.5 mm) of rhodamine trapped within a membrane bound mesoporous microsphere of the present invention.
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.
Then the effect of perturbation of the ionic porosity of the surrounding membrane on a contained reactive molecule was examined. A Mg2+ 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 α-hemolysin in magnesium-containing buffer. The resultant change in fluorescence intensity is shown in FIG. 6. FIG. 6 depicts the α-hemolysin mediated influx of Mg2+ into membrane-enclosed microspheres. The Mg2+ 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 MgCl2 and varying concentrations of α-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).
The increase in fluorescence intensity corresponds to the formation of ion pores by the incorporation of α-hemolysin into the membrane, allowing for penetration of Mg2+ into the microspheres to interact with the Mag-fluo-4.
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 μM Gd(NO3)3, an inhibitor of BR.
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.
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° 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.
- EXAMPLE II
In these experiments, the FITC was incubated with PNIPAAM-modified microspheres either at 40° C. (pores open) or at room temperature (ca 22° C.; pores closed). The microspheres were then incubated at 25° C. to retain any trapped molecules and analyzed by flow cytometry. The microspheres were then washed in 40° 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° C.), there was uptake and release indicating that the incorporated PNIPAAM was functional. Similar experiments have been attempted with FITC-labeled insulin (MW ˜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.
Mesoporous microspheres useful in the product of the present invention were made in the following ways:
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: CH3(CH2)15N(CH3)3Br− (Aldrich) or Brij-58: CH3(CH2)15—(OCH2CH2)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.
In a typical preparation, tetraethylorthosilicate (TEOS) (Aldrich), ethanol, deionized water (conductivity less than 18.2 MΩ cm) and dilute HCl (mole ratios 1:3.8:1:0.0005) were refluxed at 60° 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/H2O/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/H2O/CTAB/HCI is 1:63.28:0.15:0.0227. This sol was stirred for about 10 min before beginning a powder synthesis run.
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 μm or 20 μm) by a syringe pump, with syringe velocities of approximately 2×10 −4 cm s−1 (˜1.4×10−3 cm3s−1) and 8×10−4 cm s−1 (˜4.7×10−3 cm−3 s−1) for the 10 μm and 20 μm 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° C. (acidic silica sol runs) or 420° 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° C. by a heating tape. Collected particles were calcined in air at 400-450° C. for 4 hours (acidic silica sol runs) or at 500° C. for 12 hours (TEOS solution runs) to remove the surfactant template. The typical powder production rate using the VOAG was 0.35 g SiO2 h−1.
The particles were characterized by scanning electron microscope (Hitachi S-800) and X-ray diffraction (Siemens D5000, Cu Kα radiation, 2.=1.5418 Å) 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.
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 μm orifice was 9.9±0.5 μm (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 μm orifice resulted in an average particle size of 6.0 μm±0.3 μm.
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 Å 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 Å, which corroborated the XRD results. Nitrogen adsorption-desorption isotherms showed typical features for mesoporous materials. The average (hydraulic) pore diameter dp determined from nitrogen adsorption data (calculated as dp−4Vp/Sp where Vp is total pore volume and Sp, 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 m2/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.
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.
In summary, monodisperse spherical porous silica particles were prepared with diameters in the 5 to 10 μm 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 1-2μm and as large as 50 μm 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.
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.
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.
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.
- EXAMPLE III
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.
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.
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.
DAT may be investigated using the following methods.
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.
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. β-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.
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 3×4 L of reconstitution buffer for 20 hours at 4° C. Proteoliposomes are then frozen at −80° C. until needed.
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 [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.
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° C. Uptake solution will contain [3H]DA at 1 nM except for saturation analyses where [3H]DA will be increased to 10 nM and unlabeled dopamine varied from 10 nM to 1 μM. Nonspecific uptake is defined with 100 μM cocaine. Uptake assays are carried out for 3 min at 34° 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 Km of 2100 nM and a Vmax of 9.0 pmol/105 cells/min. To determine specificity of the transporters [3H]alanine uptake will be examined using the same conditions as for dopamine transport, except that [3H]alanine at a concentration of 10 nM will be used.
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+ ions using the sodium sensitive dye SBFI. A similar method with magnesium-sensitive dyes for the detection of α-hemolysin, a bacterial transmembrane toxin, using flow cytometry has been performed. FIG. 10 illustrates Flow cytometric detection of α-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 α-hemolysin are mixed with 3 mM MgCl2, and added to the bead suspension, and incubated at 37° C. Aliquots of the beads are removed at defined time intervals and fluorescence intensity of the beads, indicating the influx of Mg+2 ions due to the incorporation of α-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.
- EXAMPLE IV
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™) 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 μL 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 V
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 μm 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 VI
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 VII
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 α 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.
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 αβγ heterotrimeric molecules. The α subunit contains nucleotide binding sites. Both α and βγ subunits have the capacity to interact with catalytic enzymes or “effectors” required for cell activation. There are several classes of the α, β, and γ 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.
- EXAMPLE VIII
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 ˜33° 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° 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 IX
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.
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™) 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 μL 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.
All documents, patents, journal articles and other materials cited in the present application are hereby incorporated by reference.
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.