Patent Application
20060094053
This invention relates to processes for producing self-assembled arrays of lipid vesicles and uses thereof. Products made by the process are also provided.
Spatial compartmentalization is a prerequisite for the creation of living matter[1]. Without the existence of clearly defined borders[2], differentiation and diversity at the cellular level would not be possible. Most scientific disciplines that deal with dissolved molecules are concerned with the same problem of subdividing solutions in miniaturized autonomous units, either to increase the functional complexity of a system[3], reduce reagent consumption[4], monitor fast chemical kinetics[5], or even to study single-molecules[6]. We describe a method that allows the massively parallel isolation of attoliter (1 al=10−18 L) reaction volumes and their self-assembled positioning with 100-nm precision as an ordered array on a solid surface.
U.S. Pat. No. 4,282,287 and its reissue 31,712 disclose a multiple-layer product and a process of applying alternate, successive layers of avidin and biotin-containing extender material to a surface to modify its properties. But no self-assembly of vesicles on the surface or vessels which circumscribe a reaction volume to produce an array were taught or suggested.
U.S. Pat. No. 6,221,401 and U.S. Pat. No. 6,565,889 disclose a vesicle (“bilayer structure”) containing multiple reaction volumes (“containment units”). It is preferred therein that each vesicle is attached to another. But no self-assembly of the vesicle on a surface to produce an array was taught or suggested.
U.S. Pat. No. 6,270,983 also discloses a surface with avidin and biotin attached thereto. But reagents are then immobilized on the coated surface and reactions take place in solid phase. No self-assembly of vesicles on the surface or vessels which circumscribe a reaction volume to produce an array were taught or suggested.
U.S. Pat. No. 6,444,254 discloses microstamping a functionalized polymer surface with ligands. Avidin and biotin may be attached to the surface. No self-assembly of vesicles on the surface or vessels which circumscribe a reaction volume to produce an array were taught or suggested.
U.S. Pat. No. 6,444,318 discloses a self-assembling array, but no vesicle immobilized on the surface or vessels which circumscribe a reaction volume to produce the array were taught or suggested.
U.S. Pat. No. 6,855,329 discloses a patterned surface with ligands attached by a biotin-avidin-biotin linkages. But no self-assembly of vesicles on the surface or vessels which circumscribe a reaction volume to produce an array were taught or suggested.
WO 00/73798 discloses functionalized, polymer-reinforced (sterically stabilized) vesicles, but they are not self-assembled to produce an array.
US 2004/0241748 A1 discloses self-assembling arrays, but they are different from the arrays of the present invention.
US 2005/0019836 A1 and WO 02/046766 disclose vesicles which circumscribe a reaction vesicle, but they are also different from the arrays of the present invention.
The present invention is directed to an improved self-assembled array of lipid vesicles, processes for producing them, and processes for using them to address problems of the art. Other advantages and improvements are described below or would be apparent from the disclosure herein.
It is an object of the invention to self assemble an array comprising a surface (or substrate) and lipid vesicles immobilized thereon.
In one embodiment, an array may comprise (i) a surface and receptors attached thereto in a pattern and (ii) vesicles having a lipid bilayer, ligands exposed on the vesicle's exterior, and chemical reagents or proteins contained by the vesicle (i.e., encapsulated in the vesicle's aqueous interior or embedded in the lipid matrix). A vesicle may be located in one or more region(s) on the surface by specific binding between ligand exposed on the vesicle's exterior and receptor attached to the region(s). Areas, which do not have receptor attached thereto, separate regions from each other on the surface. The lipid bilayer of a vesicle is comprised of charged lipids, uncharged lipids, and hydrophilic modified lipids.
At least 104 regions, at least 105 regions, at least 106 regions, at least 107 regions, or at least 108 regions per mm2 may be formed on the surface. Each region may be separated from its nearest neighboring region by a center-to-center distance of at least 1 μm, at least 10 μm, at least 100 μm, at most 1 μm, at most 10 μm, at most 100 μm, or any combination thereof. The surface may be glass, metal conductor or metal oxide semiconductor, nonporous film or porous membrane (e.g., polyester, nylon), or polymeric material (e.g., polyacetate, polycarbonate, polystyrene).
There may be about one vesicle immobilized in each region (e.g., average number of vesicles in each region is from 0.5 to 10). For single vesicle occupancy, immobilization may be performed with a dilute concentration of vesicles. It is preferred that at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of all regions are occupied by one or more vesicles. The average diameter of the vesicles may be at least 10 nm, at least 50 nm, at least 100 nm, at least 500 nm, at least 1 μm, at most 50 nm, at most 100 nm, at most 500 nm, at most 1 μm, at most 10 μm, at most 100 μm, or any combination thereof. Small vesicles are preferred for single vesicle reactions. Vesicles may be made by electroformation, extrusion, or sonication. Vesicles of substantially uniform size are preferably made by extrusion, but they may also be selected by sizing through a membrane or gel exclusion; their use provides a substantially equal density of vesicles across each region or the entire surface.
The “pattern” may be regular or irregular with respect to a repeating arrangement of receptor-attached regions where at least one vesicle is immobilized on the surface. Examples of a regular pattern are tiling (or tessalation) of regions which cover the surface with no gaps or overlaps, and regions with rotational and/or translational symmetry which allow gaps while covering the surface. An irregular pattern is characterized by the absence of any discernible repeating arrangment of regions. Each region may be elliptical (e.g., circle) or polygonal (e.g., rectangle) on the two-dimensional surface, and separated from each other by areas where receptors are not immobilized. Thus, receptors are attached to the surface in each “region” but not in the “area” separating regions. Tiled regions may be separated from each other by areas along the region's border; symmetric regions may be separated from each other by areas which are the gaps between the regions.
Receptor and ligand may be streptavidin and biotin, respectively, or analogs thereof (e.g., monomeric avidin or streptavidin, and DSB-X biotin). Alternatively, they may be antibody binding site/hapten, complementary oligonucleotides, polyhistidine/alkaline earth metal, or electrostatic interactions between a positively-charged surface and a negatively-charged vesicle, respectively. It is preferred that the ligand-conjugated lipids be about 2% of all lipids of the vesicle (e.g., from 1 mol % to 5 mol %).
Negatively-charged (anionic) lipids of the vesicle include: cardiolipin, diacyl-glycero-phosphatidic acid, diacyl-phosphatidylglycerol, diacyl-phosphatidylinositol, and diacyl-glycero-phosphatidylserine. Positively-charged (cationic) lipids are not preferred for making the vesicle. Uncharged lipids of the vesicle include: cholesterol (in mixtures with other lipids), diacyl-phosphatidylethanolamine, and sphingomyelin. Archae lipids can be included for their chemical and mechanical stability. It is preferred that the charged lipids be about 10% of all lipids of the vesicle (e.g., from 8 mol % to 15 mol %).
The lipid bilayer may be modified with a hydrophilic polymeric chain (e.g., a PEG) or glycolipid (e.g., ganglioside GM1) to sterically stabilize the vesicle. It is preferred that the modified lipids be about 0.3% of all lipids of the vesicle (e.g., from 0.1 mol % to 1 mol %).
It is another object of the invention to provide a process for producing the array. The arrays and intermediate products made during the production process may then be subjected to further processing and/or use. It is yet another object of the invention to provide a process for using the array.
Arrays may be produced by attaching receptors to regions of the array where vesicles will be immobilized, but not to areas of the array where vesicles will not be immobilized. Vesicles are made from charged lipids, uncharged lipids, and hydrophilic modified lipids; the lipid bilayer spatially compartmentalizes the interior from the exterior of each vesicle, and ligand is exposed the exterior of vesicles. Vesicles are immobilized on the array by specific receptor-ligand binding and located at “regions” instead of “areas” of the surface.
Receptors may be patterned on the surface by contact printing (see Xia & Whitesides, Angew. Chem. Int. Ed. 37:550-575, 1998), electron beam lithography (see Stamou et al., Langmuir 20:3495-3497, 2004), or dip pen lithography using cantilevers of atomic force microscopy. Successive steps of immobilizing vesicles may be used to “activate” different regions of the surface by attaching receptors, binding ligand-bearing vesicles to receptors, and blocking unbound receptors with an excess of free ligands. Different vesicles can be localized to particular regions using a single receptor-ligand pair in many separate self-assembly steps. Alternatively, several receptor-ligand pairs (e.g., complementary oligonucleotides) may be used to “activate” different regions of the surface by attaching all of the receptors at the same time. Different vesicles can be localized to particular regions in the same self-assembly step. “Three-dimensional” patterns may be produced by building up layers through successive steps of constructing planar vesicle structures. Receptor may be attached to a region through a ligand which was previously fixed on the surface; this ligand may be the same or different from the vesicle-borne ligand.
Chemical reactions may be controlled by segregating reagents in separate vesicles, and then initiating the reaction by disrupting the lipid bilayers and/or mixing the contents of one or more vesicles. Vesicles may be released from the surface by breaking the receptor-ligand interactions (e.g., monomeric stretavidin and/or DSB-X) under mild conditions without lysing vesicles or denaturing their contents. Such elution requires a reversible receptor-ligand interaction, but covalent crosslinking may be used if reversible binding is not required. Selective release of vesicles from the surface by eluting with excess free ligand, may then be followed by chemical analysis of free vesicles and/or empty surface by an analytical technique (e.g., infrared, Raman, or mass spectroscopy). A combinatorial library comprising chemical compounds or enzymes may be immobilized in vesicles of an array, then screened for chemical reactivity or enzyme activity (e.g., hydrolysis, transferase).
Processes for using and making arrays are provided. It should be noted, however, that a claim directed to the product is not necessarily limited to a process unless the particular steps of the process are recited in the product claim.
Further aspects of the invention will be apparent to a person skilled in the art from the following description of specific embodiments and the claims, and generalizations thereto.
By using lipid-bilayer vesicles[1] as molecular shuttles[7], we transported and localized (bio)molecules encapsulated in their aqueous interior or embedded in the lipid matrix. The site-selective immobilization of intact single vesicles (SVs) was mediated by patterns of receptor molecules defined by microcontact printing (μCP)[8] on glass. One-step directed self-assembly (SA)[9] produced arrays of about 106 volume-elements per mm2 within minutes. As illustrated, this approach can additionally create random arrays of vesicles cof varied content that may serve as libraries of miniaturized (bio)chemical reaction systems.
The strategy employed to construct arrays of surface-immobilized SVs is illustrated in
The properties of vesicles can be tailored for optimum interaction with the surface by selecting an appropriate lipid composition for their bilayer. See WO 00/73798, the contents of which are incorporated by references herein.
We adjusted the lipid composition to introduce two long-range repulsive forces that prevent nonspecific interactions between the vesicles and the surface. Electrostatic repulsion was controlled by setting the ratio of charged to uncharged lipids. The presence of 10% charged lipids (e.g., 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) or POPG) also increases the bending energy[14] of the lipid membrane, which prevents vesicles from deforming and fusing upon immobilization. To establish a second barrier against intimate contact between the surface and vesicles, we employed lipids modified with a hydrophilic polymeric chain (e.g., poly(ethylene glycol) or PEG)[15]. The PEG molecules induce a force that is entropic in nature and independent of the immobilization conditions (e.g., pH, ionic strength, etc.) that would otherwise affect the electrostatic-based repulsion. To maintain the membrane in a fluid state, the main lipid constituent (e.g., 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or POPC) was chosen to have an ordered-to-fluid phase transition far below room temperature. Other lipid compositions and processes for producing vesicles are described in WO 00/73798, the contents of which are incorporated by references herein.
Balancing the contributions of the three most important interactions (i.e., receptor-ligand binding, electrostatic repulsion, and entropic force) was crucial to achieving selective deposition of intact vesicles. This may be done empirically by starting from initial conditions such as those exemplified herein.
We chose the dimensions of the vesicles and the resolution of the patterning method to be complementary to the probe volume of fluorescence-based detection techniques. Most of the vesicles used here were prepared with an average diameter of 100 nm using extrusion[16] The surface onto which the vesicles are immobilized was structured by μCP, a versatile technique to pattern surfaces with a variety of receptors[17] and a resolution of less than 100 nm[18]. In this patterning step, we directly defined both the geometry and the resolution of the subsequent process of vesicle assembly on the solid surface.
One strategy to immobilize small numbers of vesicles onto predefined regions of a surface is to reduce the number of vesicles available for binding per printed region. This is readily accomplished in the SA step by reducing the vesicle concentration. The fluorescence image in
Extension of the technique from statistical placement of small numbers of vesicles on predefined region to SV positioning required reduction of the pattern-size to the dimension of the vesicles[10]. We functionalized glass using high-resolution μCP stamps that had various features with sizes as small as 60 nm[18]. The simultaneous presence of different patterns on the surface allowed us to screen in one step the geometries and sizes that were appropriate for having a single immobilized vesicle on a printed feature. The highest occupation of printed sites by SVs, 80%, was obtained for “linelets” having dimensions of 100×400 nm2 spaced 800 nm apart (
In addition, we incubated the linelet patterns with a mixture of two vesicle populations, each tagged with a different fluorophore (
To assess the stability of the controlled environment of molecules confined inside immobilized vesicles, we investigated the permeability of the lipid bilayer. Low passive influx/efflux of ions into/out of vesicles is a prerequisite for keeping the pH or ionic composition of an SV stable, and the function of a protein or the reactivity of solutes inside an SV reliable. The pH sensitivity of CF was used to monitor the pH inside SVs, which are 0.5 μm to 1 μm in diameter, immobilized on 10 μm wide stripes. The vesicles were prepared to have a pH of about 5.5 and were immobilized in a buffer of pH 7.4. After immobilization, the pH value of the vesicles shifted to about 6.2, where it remained stable because of the established diffusion potential (
Subsequently, we addressed the interior of these tightly sealed containers and initiated a simple chemical reaction. The deprotonation of CF loaded in SVs was triggered using an ion channel-forming peptide (gramicidin A). Gramicidin A forms cation-selective channels in lipid membranes. Upon incorporation into the vesicle's lipid bilayer, the channel allows Na+ ions to diffuse down their concentration gradient, in this case into the vesicle. The sodium influx induces a pH increase (proton efflux) to maintain electroneutrality. Individual vesicles were monitored over time and how the fluorophores trapped in their interior respond to the gramicidin A stimulus was recorded (
Alternatively, the immobilized vesicle may be targeted by a vesicle in solution using a receptor-ligand interaction, and then fusion of the vesicles and mixing of their contents may be triggered through SNAREpins. SNARE-mediated fusion between vesicles will immobilize them on a surface, mix their contents, initiate a chemical reaction using any reagents that might be present, and contain any reaction product(s) within the fused vesicle. The product(s) may be released from the surface by lysing the lipid bilayer and/or breaking the receptor-ligand interaction. Only chemicals that are transformed in the reaction are considered “reagents” herein, in contrast to inert chemicals like the aqueous solution, salts, and buffer. The specificity of v- and t-SNARE interactions may be used to combinatorially vary the pairing of vesicles with different contents by faithful targeting of vesicles with complementtary SNAREs, and then fusing their lipid bilayers through SNAREpins.
Combinatorial libraries of chemical compounds or enzymes may be arrayed. A unique or a few different compound(s)/enzyme(s) may be enclosed in each vesicle, which is then immobilized. The same region on each array may be activated by attaching the receptor, at least one vesicle which contains the unique or the few different compound(s)/enzyme(s) of the combinatorial library may be immobilized on the surface through ligand on the vesicle's exterior leaflet, free receptors are blocked with an excess of ligand in solution, and the cycle is repeated on another region on each array for another unique or few different compound(s)/enzyme(s). Here, compounds/enzymes are identified by their location on the array; alternatively, each vesicle could contain an nucleic acid tag in the vesicle's aqueous interior or lipid matrix and the compound/enzyme is identified by the tag's nucleotide sequence.
Hierarchical SA is an emerging approach to the fabrication of functional nano-sized architectures[9]. Here, SA principles and receptor-ligand interactions are combined to define attoliter autonomous reaction volumes, and then to order them on a surface. Incorporation of multifunctional recognition elements, like oligonucleotides, would further increase the complexity of the assembled structures[23]. The lipid-bilayer vesicles we used as molecular vessels are arrayed at high densities (every 800 nm, about 106 per mm2). Nevertheless, each one maintains its cargo dissolved in a protective environment[24] of defined chemical composition (pH, ionic strength, etc.) and at the same time localizes its position with 100-nm precision. Such ultra-small volume libraries allow simultaneous screening[6] of (bio)chemical properties, molecular function, or confined chemical reactions over millions of samples, while scarce reagents are conserved (for a total reaction volume of a few picoliters). A natural extension of this work is the use of vesicles produced directly from cells to form arrays[25]. To array native vesicles individually, the employment of a more versatile surface modification[26] might be crucial since their properties are not so easily controlled as those of synthetic vesicles. Different enzymes may be encapsulated in the vesicle's aqueous interior or embedded in its lipid matrix, and then arrayed. Native vesicles are of primary importance as they can carry receptor proteins expressed in cell membranes and/or signal transduction machinery from the cytosol. Alternatively, functional membrane proteins (e.g., ion channels or pumps, receptors, signal transduction machinery, SNAREs, transporters) may be reconstituted in the vesicle. In array format, they may thus be used to screen binding of drug candidates (e.g., enzyme inhibitor, receptor agonist or antagonist) or the functional responses induced by such binding.
Vesicle Production
A dried lipid film was rehydrated overnight in 200 mM D-sorbitol. A vesicle cloud was then harvested (about 1 mg/ml), freeze thawed, and extruded through 100-nm filter pores (typical S.D.±40 nm). The vesicles used for the experiments in
Patterned Surface Functionalization
Poly(dimethylsiloxane) stamps for μCP were inked with 0.1 mg/ml BSA-biotin. After washing the stamps with PBS and drying them under a N2 stream, we printed BSA-biotin on a clean glass substrate that was subsequently blocked with 0.5 mg/ml BSA. The patterned substrates were then functionalized with 0.025 mg/ml streptavidin incubated for 10 min. The final streptavidin surface density was about 10% of a complete monolayer.
Calibration of pH Ratio for Carboxyfluoroscein
The carboxyfluorescein (CF) dye response (excitation ratio 488 nm/458 nm, emission bandwidth of 505 nm to 550 nm) was calibrated in solutions of different known pH values[27] (buffer: 200 mM D-sorbitol, 10 mM NaHPO4). The calibration curve (
Insertion of Gramicidin Lipid Bilayer
The stock solution of gramicidin A was 100 μM in chloroform. At the beginning of each day, an ethanol (EOH) solution of 1 μM was prepared from the stock solution. The EOH solution was finally diluted in working buffer at a ratio of 1/100 to give a final concentration of 10 nM gramicidin A[28]. The sample was mixed manually with a pipette and then equilibrated for 2 min to 3 min to assure sufficient time for insertion and dimerization of gramicidin A in the lipid bilayer[29]. Injection of the same EOH quantity did not have any effect on vesicle pH.
Sodium Influx Induced by Gramicidin
To trigger the pH increase, we used the peptide gramicidin A which has a single channel conductivity[30] of ˜40 pS and an initial current of ˜12 pA or 108 ions/sec under our conditions. The half time of the pH response upon gramicidin A-induced ion flux in vesicles has been measured before[31] but is also straightforward to approximate. Using the Nernst equation, for external and internal Na+ concentration of 0.1 M and 10−6 M, we can calculate the electrochemical equilibrium potential ENa˜0.3 V. To create this potential difference of a vesicle which is 1 μm in diameter, assuming a membrane capacitance of 1 μF/cm2, we need a net inflow of about 104 ions. So at a first approximation, the half time of Na+/pH increase is in the order of about 104 ions/108 ions sec−1=0.1 msec. In agreement to this rough calculation, the time response is reported[31] to be <1 msec. An ion channel with single channel current of ˜1 fA would have a half time about 104-fold longer (i.e., in the range of seconds).
The invention has also been described in Stamou et al. (Agnew. Chem. Int. Ed. 42, 5580-5583, 2003); the contents of which are incorporated by reference. Patents, patent applications, and other publications cited herein are also incorporated by reference in their entirety.
In stating a numerical range, it should be understood that all values within the range are also described (e.g., one to ten also includes every integer value between one and ten as well as all intermediate ranges such as two to ten, one to five, and three to eight). The term “about” may refer to the statistical uncertainty associated with a measurement or the variability in a numerical quantity which a person skilled in the art would understand does not affect operation of the invention or its patentability.
All modifications and substitutions that come within the meaning of the claims and the range of their legal equivalents are to be embraced within their scope. A claim using the transition “comprising” allows the inclusion of other elements to be within the scope of the claim; the invention is also described by such claims using the transitional phrase “consisting essentially of” (i.e., allowing the inclusion of other elements to be within the scope of the claim if they do not materially affect operation of the invention) and the transition “consisting” (i.e., allowing only the elements listed in the claim other than impurities or inconsequential activities which are ordinarily associated with the invention) instead of the “comprising” term. Any of these three transitions can be used to claim the invention.
It should be understood that an element described in this specification should not be construed as a limitation of the claimed invention unless it is explicitly recited in the claims. Thus, the granted claims are the basis for determining the scope of legal protection instead of a limitation from the specification which is read into the claims. In contradistinction, the prior art is explicitly excluded from the invention to the extent of specific embodiments that would anticipate the claimed invention or destroy novelty.
Moreover, no particular relationship between or among limitations of a claim is intended unless such relationship is explicitly recited in the claim (e.g., the arrangement of components in a product claim or order of steps in a method claim is not a limitation of the claim unless explicitly stated to be so). All possible combinations and permutations of individual elements disclosed herein are considered to be aspects of the invention. Similarly, generalizations of the invention's description are considered to be part of the invention.
From the foregoing, it would be apparent to a person of skill in this art that the invention can be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments should be considered only as illustrative, not restrictive, because the scope of the legal protection provided for the invention will be indicated by the appended claims rather than by this specification.
This application claims the benefit of provisional Appln. No. 60/624,757, filed Nov. 5, 2004; the contents of which are incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60624757 | Nov 2004 | US |
