RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial

No. 61/926,179, filed January 10, 2014, entitled "Systems and Methods Involving Interactions Such As Protein or Virus Interactions," incorporated herein by reference.

FIELD

The present invention generally relates to systems and methods involving interactions such as protein or virus interactions.

BACKGROUND

One of the biggest threats to humanity nowadays is the virus. Placed at the top end on the rate of evolution scale, viruses are fast evolving and continuously generating new and deadly diseases every year. Unfortunately, to date, once a subject is infected by a virus, very little can be done to help, as there are not many drugs that can eliminate the virus threat effectively from the subject.

The virus is, in principle, a small and very basic machine incapable of decision or control over its own path or fate. Floating arbitrarily in large quantities, some viruses coincidently meet a cell, which can serve as a host cell, penetrate it and hijack its bio- machinery for replication. The virus is merely an envelope containing a nucleic acid (genetic) cargo, yet it is effective in infecting cells and replicating at high rates, thus disrupting normal function of the host organism.

SUMMARY

The present invention generally relates to systems and methods involving interactions such as protein or virus interactions, as well as other interactions. For example, in some cases, the interactions may be of a lipid with a target (e.g., a labeled target). The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, the present invention is generally directed to an article. In one set of embodiments, the article comprises a microfluidic device comprising a plurality of compartments, at least some of which comprise a surface film comprising at least about 75 wt lipid. In another set of embodiments, the article comprises a microfluidic device comprising a plurality of compartments, at least some of which comprise a surface film comprising lipid, wherein the film, when exposed to water, produces liposomes from the lipid.

The present invention, in another aspect, is generally directed to a method. In one set of embodiments, the method includes an act of forming, in a plurality of compartments within a microfluidic device, a surface film comprising at least about 75 wt lipid. In another set of embodiments, the method includes an act of forming, in a plurality of compartments within a microfluidic device, a surface film comprising lipid. In some cases, the film, when exposed to water, produces liposomes from the lipid.

The method, in yet another set of embodiments, is generally directed to providing a microfluidic device comprising a plurality of compartments, at least some of which comprise a surface film comprising lipid, producing liposomes in at least some of the compartments from the lipid, exposing at least some of the liposomes to a target, and determining an amount of the target associated with the liposomes within the

compartments. In another set of embodiments, the method includes acts of producing liposomes in a plurality of compartments contained within a microfluidic device, exposing at least some of the liposomes to a target, and determining an amount of the target associated with the liposomes within the compartments.

In one set of embodiments, the method includes an act of administering, to a subject suspected of being infected with a virus, a liposome that the virus preferentially associates with, relative to cells within the subject. In another set of embodiments, the method includes an act of administering a liposome to a subject suspected of being infected with a virus, wherein the virus associates with the liposome such that the virus inserts nucleic acid into the liposome.

In another aspect, the present invention encompasses methods of making one or more of the embodiments described herein. In still another aspect, the present invention encompasses methods of using one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

Figs. 1A-1C illustrate a microfluidic system in accordance with one embodiment of the invention, at various resolutions;

Figs. 2A-2D illustrate determination of the association of cholera toxin with certain lipids, in another embodiment of the invention;

Figs. 3A-3B illustrate the introduction of a dye in one embodiment of the invention;

Fig. 4 illustrates association of the envelope protein of the Dengue virus to various lipids, in another embodiment of the invention;

Figs. 5A-5C illustrate the binding of certain proteins to various lipids, in yet another embodiment of the invention;

Fig. 6 illustrates viruses that associate with liposomes as "decoys" of cells, in still another embodiment of the invention;

Figs. 7A-7D illustrates an example of image processing;

Figs. 8A-8C illustrate a microfluidic system in accordance with certain embodiments of the invention;

Figs. 9A-9B illustrate Cholera Toxin subunit b binding, in accordance with another embodiment of the invention;

Figs. 1 OA- IOC illustrate affinity of Trans-Membrane Domains from human proteins to a liposome library, in yet another embodiment of the invention;

Figs. 11A-11D illustrate measurement of fluorescence, in certain embodiments of the invention; Figs. 12 and 13 show a table of various lipid species, in certain embodiments;

Fig. 14 shows detailed information used in Figs. 12 and 13;

Figs. 15A-15B show affinity tables of certain viruses, in accordance with another embodiment of the invention; and

Figs. 16A-16D illustrate liposome decoys in certain embodiments of the invention.

DETAILED DESCRIPTION

The present invention generally relates to systems and methods involving interactions such as protein or virus interactions. In one aspect, the present invention is generally directed to systems and methods of determining interactions or associations between lipids or liposomes, and targets such as proteins or viruses. In some cases, microfluidic systems or other systems may be used to determine such interactions. For example, in some embodiments, a plurality of compartments containing various lipids in a microfluidic system may be exposed to a target, and interactions or associations between the target and the lipids in the compartments may be determined. In another aspect, the invention is generally directed to liposome decoys that can be administered to a subject. The liposomes may exhibit certain preferential interactions with infectious agents such as proteins or viruses within the subject, which may be used to inhibit the infectious agents.

One aspect of the present invention is generally directed to systems and methods for determining interactions between lipids or liposomes, and targets such as proteins or viruses. In some cases, microfluidic or other systems may be used to determine such interactions in parallel. For example, referring to Fig. 1A, microfluidic system 10 comprises a plurality of compartments 15, arranged in any suitable order, e.g., in a regular array as depicted here. The compartments may be arranged, in some instances, such that the compartments share a common inlet 11 and a common outlet 12. In other embodiments, however, more than one inlet and/or more than one outlet may be used. In some cases, the compartments may be connected with suitable channels such that the path distance (the distance the fluid travels) between the common inlet and the common outlet is substantially the same for some or all of the compartments.

Fig. IB is an expanded view of Fig. 1A, showing a single, representative compartment 15, including an inlet channel 21 and an outlet channel 22 to the compartment. The meandering pathway of the inlet channel, in this embodiment, is used to ensure that the path distance between the common inlet and the common outlet for each of the compartments is substantially the same, as noted above. Also shown in Fig. IB are a plurality of posts 30. The posts are optional, but may be used, for example, to direct or control fluid flow within compartment 15, and/or to reduce the formation of air bubbles within the compartment.

Some or all of the compartments may contain lipids and or liposomes. In some cases, the compartments may contain lipids that may be formed into liposomes upon exposure to a suitable environment, e.g., an aqueous fluid. The lipids and/or liposomes within the compartments may be the same or different. For instance, each of the compartments may contain the same lipids and/or liposome composition, and/or there may be differences between some or all of the compartments, e.g., with respect to types of lipids and/or liposomes, and/or concentrations of lipids and/or liposomes. For example, in certain cases, some of the compartments may contain the same lipids and/or liposome composition, e.g., to provide for replications or redundancies. In one set of embodiments, the lipid may be formed as a film on a surface of the channel, and upon the addition of water (or another suitable fluid), the lipids may be induced to form liposomes 35, as is shown in Fig. 1C, which is an expanded view of Fig. IB.

In one set of embodiments, a target is introduced to some or all of the

compartments. More than one target may be used in some cases. The target molecule may be any molecule that can associate with the lipids and/or liposomes in some fashion, e.g., by fusing or binding to the lipids and/or liposomes. Examples of targets include, but are not limited to, proteins, viruses, and/or other molecules as discussed below. The target may be a molecule or may, in some cases, comprise a plurality of molecules (for example, as in a virus). In some cases, the targets may be labeled, e.g., with a dye or a fluorescent compound. Targets may be introduced to the compartments before, during, and/or after introduction or formation of liposomes within the compartments.

After exposure of the target molecule with the lipids and/or liposomes within the compartments, the amount of binding or other association of the target with the lipids and/or liposomes may be determined. In some cases, a label may be used to facilitate such determination. For example, in one set of embodiments, the amount of absorbance or fluorescence of the compartments may be determined to determine the amount of binding or other association for each compartment, which may not necessarily be the same for each compartment (e.g., if different lipid and/or liposome compositions are used, and/or if different targets are used). Image processing techniques may be used in some cases. An example may be seen in Fig. 7, where fluorescence (Fig. 7A) and light (Fig. 7B) images can be processed to identify posts (Fig. 7C) in an image, which can be ignored, and liposomes (Fig. 7D).

In addition, in some cases, a dye may also be introduced to the compartments. The dye may be used to determine the flow of fluid within the compartment, and/or to determine the amount of liposomes or lipid within the compartments (for example, if the dye is hydrophilic, and/or if the dye is one that does not readily bind or associate with the liposomes). This may be used, for example, to determine what portion of the

compartment contains liposomes, and in some cases, to normalize the results when comparing different compartments.

The above discussion is a non-limiting example of one embodiment of the present invention that can be used to determine interactions between lipids and/or liposomes, and suitable targets. However, other embodiments are also possible. For example, in certain aspects, the present invention is generally directed to determining interactions between lipids or liposomes, and targets such as proteins or viruses using a microfluidic system comprising a plurality of compartments. Those of ordinary skill in the art will be familiar with microfluidic systems and methods of forming microfluidic systems, e.g., from materials such as glass or PDMS.

The compartments themselves may be of any suitable volume or dimension, and may be formed of or defined by any suitable material. The compartment may have any suitable shape, and in certain cases, the compartment may be or from part of a microfluidic channel. In some cases, the compartment has a largest dimension, or a side dimension (e.g., length or width, which may be independently chosen), of no more than about 10 mm, no more than about 5 mm, no more than about 3 mm, no more than about 1 mm, no more than about 500 micrometers, no more than about 300 micrometers, no more than about 100 micrometers, no more than about 50 micrometers, no more than about 30 micrometers, no more than about 10 micrometers, or no more than about 5 micrometers, etc. For example, in one set of embodiments, the compartments may have an average dimension of between 10 mm x 10 mm x 100 nm and 1.2 mm x 1.2 mm x 10 nm.

In certain embodiments, the compartments may have an average volume of at least about 10 nl, at least about 30 nl, at least about 50 nl, at least about 100 nl, at least about 300 nl, at least about 500 nl, at least about 1 microliter, at least about 3 microliters, at least about 5 microliters, at least about 10 microliters, etc. In some cases, the compartments may have an average volume of no more than about 10 microliters, no more than about 5 microliters, no more than about 3 microliters, no more than about 1 microliter, no more than about 500 nl, no more than about 300 nl, no more than about 100 nl, no more than about 50 nl, no more than about 30 nl, or no more than about 10 nl. Combinations of any of these are also possible in certain embodiments; for example, the compartment may have an average volume of at least about 50 nl but no more than about 500 nl.

In one set of embodiments, a compartment may have an average planar surface area of at least about 1,000 micrometers 2 , at least about 3,000 micrometers 2 , at least about 5,000 micrometers 2 , at least about 10,000 micrometers 2 , at least about 30,000 micrometers 2 , at least about 50,000 micrometers 2 , at least about 100,000 micrometers 2 , at least about 300,000 micrometers 2 , at least about 500,000 micrometers 2 , at least about 1 mm 2 , at least about 3 mm 2 , at least about 5 mm 2 , at least about 10 mm 2 , at least about 30 mm 2 , at least about 50 mm 2 , at least about 100 mm 2 , etc. The planar surface area may be taken as the area of the surface of one face of the walls forming the compartment (for example, the bottom face of the compartment). It should be understood that this is used in a relative sense. For example, in some embodiments as discussed herein, a solvent containing a lipid is introduced into a compartment and the solvent removed (e.g., through evaporation) to form a film on one surface of the compartment. Upon removal of the solvent, the film may be coated on one wall of the compartment. However, the wall coated with the film may not necessarily always be the bottom, for example, if the microfluidic device is put into a different orientation afterwards.

Any number of compartments may be present. For example, the device may contain 1, 2, 3, 4, 5, 7, or 10 or more compartments. In some cases, there may be at least 20, at least 30, at least 50, at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 compartments. If more than one compartment is present, the compartments may each independently have the same or different volumes or dimensions, including any of the volumes or dimensions given herein. For example, each of the compartments may have the same volume and/or the same surface area, and/or some or all of the compartments may have different volumes and/or surface areas (e.g., planar surface areas).

In addition, in some cases, the compartments may contain one or more posts. The posts may have any shape (e.g., circular, triangular, diamond- shaped, square, rectangular, semicircular, polygonal, irregular, etc.), and may extend partially or completely from one surface of the compartment to an opposed surface of the

compartment. In some cases, the posts may be used to direct the flow of fluids, e.g., liquids or gases, through the compartment. For example, the posts may be arranged to channel liquids through the compartment, to prevent or reduce the ability of gases (e.g., air) to become trapped within the compartment upon introduction of a liquid, to ensure or facilitate mixing within the compartment, or the like.

The compartments may also contain more than one inlet and/or more than one outlet, e.g., for introducing or removing fluids. In some cases, e.g., as shown in Fig. 1, each compartment includes only one inlet and one outlet. However, in other cases, there may be 2, 3, 4, or more inlets and/or 2, 3, 4, or more outlets. In some cases, the inlets of some or all of the compartments may be in fluid communication with a common inlet, and/or the outlets of some or all of the compartments may be in fluid communication with a common outlet.

In addition, in certain cases, the path distance from the common inlet to the common outlet, through a compartment, may be substantially the same for each of the compartments in fluid communication with the common inlet and the common outlet, or at least the path distances through each of the compartments may each be between about 80% and about 120%, between about 90% and about 110%, or between about 95% and about 105% of the average path distance. The path distance may be taken as the minimal distance that fluid has to travel to go from the common inlet to the common outlet (i.e., as opposed to the actual distance between the common inlet and the common outlet). For instance, the path between the common inlet and the common outlet is not necessarily linear, but there may be bends, serpentine or meandering sections, etc. in the fluidic pathway to direct the flow of fluid, e.g., to various compartments or the outlet, etc.

In some embodiments, at least some of the channels within the article are microfluidic channels. "Microfluidic," as used herein, refers to a device, article, or system including at least one fluid channel having a cross- sectional dimension of less than about 1 mm. The "cross-sectional dimension" of the channel is measured perpendicular to the direction of net fluid flow within the channel. Thus, for example, some or all of the fluid channels in an article can have a maximum cross- sectional dimension less than about 2 mm, and in certain cases, less than about 1 mm. In one set of embodiments, all fluid channels in an article are microfluidic and/or have a largest cross sectional dimension of no more than about 2 mm or about 1 mm. In certain embodiments, the fluid channels may be formed in part by a single component (e.g. an etched substrate or molded unit). Of course, larger channels, tubes, chambers, reservoirs, etc. can be used to store fluids and/or deliver fluids to various elements or systems in other embodiments of the invention. In one set of embodiments, the maximum cross- sectional dimension of the channels in an article is less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 25 micrometers, less than about 20 micrometers, less than about 15 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 100 nm, or less than about 50 nm.

A channel can have any cross- sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross- section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlets and/or outlets or openings. An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid. The fluid within the channel may partially or completely fill the channel. In some cases where an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).

The channel may be of any size, for example, having a largest dimension perpendicular to net fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. In some cases, the dimensions of the channel are chosen such that fluid is able to freely flow through the article or substrate. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel may be used.

The channel may also have any suitable cross-sectional aspect ratio. The "cross- sectional aspect ratio" is, for the cross-sectional shape of a channel, the largest possible ratio (large to small) of two measurements made orthogonal to each other on the cross- sectional shape. For example, the channel may have a cross-sectional aspect ratio of less than about 2: 1, less than about 1.5: 1, or in some cases about 1: 1 (e.g., for a circular or a square cross- sectional shape). In other embodiments, the cross- sectional aspect ratio may be relatively large. For example, the cross- sectional aspect ratio may be at least about 2: 1, at least about 3: 1, at least about 4: 1, at least about 5: 1, at least about 6: 1, at least about 7: 1, at least about 8: 1, at least about 10: 1, at least about 12: 1, at least about 15: 1, or at least about 20: 1.

In some aspects, one or more lipids may be introduced into a compartment. In some cases, the lipids may be introduced using a solvent or other liquid. The solvent or other liquid may be removed, e.g., through heating or evaporation, thereby resulting in deposition of the lipid within the compartment, e.g., as a film on one or more surfaces within the compartment. However, it should be understood that solvents are not required. Other methods may be used to introduce lipids to the compartments. For example, pure lipid may be introduced into the compartment, or a reaction that produces the lipid may be used within the compartment. In addition, if more than one

compartment is present, the same or different lipids may be used within the

compartments. In some cases, each compartment may contain a different lipid composition (e.g., having different lipids, and/or having the same lipids but at different concentrations and/or amounts, etc.), although in other cases, some of the compartments may contain the same lipid compositions and/or concentrations.

Any suitable solvent or other liquid able to carry or transport lipids to the compartment may be used. In some cases, the liquid is an organic solvent, or the liquid is one that is not miscible in water, e.g., the liquid may be able to form a separate phase when exposed to water under ambient conditions. In some cases, the liquid may be one that is relatively volatile. For example, in some cases, a liquid may be used to introduce a lipid to a compartment, then the liquid may be removed through evaporation.

However, in other cases, the liquid is not necessarily volatile, and/or other techniques may be used to remove the liquid, e.g., via chemical reaction, heating, or the like. In addition, if more than one compartment is present, the same or different solvents or other liquids may independently be used to introduce lipids into the compartments. The liquids may be added to the compartments in any suitable order, e.g., sequentially and/or simultaneously. Examples of suitable organic or non-polar liquids include, but are not limited to, chloroform, methylene chloride, carbon tetrachloride, acetone,

tetrahydrofuran, ethyl acetate, acetonitrile, pentane, benzene, toluene, diethyl ether, 1,4- dioxane, or the like.

In one set of embodiments, the lipids are added to the compartments prior to sealing of the microfluidic device. For example, the lipids may be added directly to the compartments instead of being introduced via an inlet. In this fashion, for instance, different lipids may be added to different compartments, even if the compartments are in fluidic communication with a common inlet and/or a common outlet. In one

embodiment, for example, the microfluidic device comprises a top and a bottom, which are joined together to form the device; the lipids may be added to some or all of the compartments prior to the joining of the top and bottom to finish the microfluidic device.

Any of a wide variety of lipids may be introduced into the compartments.

Examples of lipids include, but are not limited to, phosphatidylcholine (e.g.,

hydrogenated phosphatidylcholine), phosphatidylethanolamine, 1,2-dipalmitoyl-sn- glycerol, lysophosphatidylcholine, sphingomyelin, ganglioside extract, a cerebroside, ceramide, N-palmitoylglycine, dioleoylglycerol pyrophosphate,

phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine,

phosphatidylinositol, cardiolipin, phosphatidic acid, cholesterol, stigmasterol, ergosterol, hopanoid, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, etc. The lipids may be natural or synthetic, and the lipids may be saturated or unsaturated. The lipids may include sterols, sphingolipids, glycerols, or the like. Many such lipids are commercially available.

In some cases, the lipids are contained within the compartments as a film or coating on a surface of the compartment. The film may be evenly or unevenly distributed on the surface, and may be pure lipid or contain other components as well. For example, the surface film may comprise at least about 50 wt lipid, at least about 60 wt lipid, at least about 70 wt lipid, at least about 75 wt lipid, at least 80 wt lipid, at least about 85 wt lipid, at least about 90 wt lipid, or at least about 95 wt lipid.

In some cases, the lipids within the compartment may be induced to form liposomes upon the addition of water or another suitable aqueous fluid, for example, ethanol, a saline solution, PBS, or the like. The aqueous fluid may be introduced into the compartments individually or in parallel. In some cases, the aqueous fluid may be introduced into the compartments through a common inlet of the microfluidic device. Without wishing to be bound by any theory, it is believed that upon exposure of lipids to the aqueous fluid, the lipids may from liposomes or other vesicles (e.g., micelles) within the compartments. The liposomes may be single- walled and/or multilamellar. In some embodiments, additional techniques, such as heat or sonication, may be applied to facilitate liposome production. For instance, in some cases, the compartment may also be heated to a temperature above the T _m of the lipids, thereby facilitating formation of liposomes. Heating may be performed, for instance, by applying heat to the

compartment, by heating the water or other aqueous fluid entering the compartment, etc.

In addition, other methods of introducing liposomes into the compartments may be used, instead of or in addition to the techniques described above. For instance, in another set of embodiments, liposomes may be prepared separately, then added to the compartments, directly and/or through inlets to the compartments. Although the above discussion is generally directed to lipids and liposomes, it should be understood that this is by way of example only, and other species may be present within the compartments, instead of or in addition to lipids and/or liposomes. In some cases, such species may be used for testing or other purposes such as is discussed in more detail herein. Examples include, but are not limited to, nucleic acids or proteins.

The compartments containing lipids and/or liposomes (or other suitable species) may be exposed to one or more targets, e.g., to determine association of the target to the lipids and/or liposomes. The association may include direct bonding, adsorption, absorption, reaction, non-specific or surface binding, or other associations. In some case, after suitable exposure, the amount and/or nature of the associations may be determined for the compartments. The targets may be introduced into the compartments before, during, and/or after formation of lipids and/or liposomes, depending on the application.

Examples of suitable targets include, but are not limited to, viruses, proteins, drugs or other small molecules (e.g., having a molecular weight of less than about 2 kDa or less than about 1 kDa), pesticides, microbicides, antibiotics, or the like. In addition, in some cases, organisms such as mold, bacteria, etc. may be used as targets.

The association between the target and the lipids and/or liposomes (if any) may be determined using any suitable technique. For example, in one set of embodiments, the target may be labeled, and the label may be determined to determine the amount of binding or other association. For example, the label may include a fluorescent entity, a radioactive entity, an absorbent entity, or the like. The label may be covalently bound to the target or incorporated into the target, in some cases. Any suitable method may be used to determine the label, and such determination may be quantitative and/or qualitative. For example, a fluorescent entity may be determined by exciting the entity and determined its emissions, an absorbent entity may be determined by determining the amount of light it absorbs, a radioactive entity may be determined by determining the amount of radioactivity within each compartment, etc.

Examples of fluorescent entities include, but are not limited to, fluorescein, rhodamine, cynanine, indocarbocyanine, Oregon green, Texas Red, Nile red, Nile blue, cresyl violet, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, etc. Fluorescent proteins may also be used in some cases, such as GFP (green fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein), etc. Examples of radioactive entities include, but are not limited to, ³ H, ¹⁴ C, ²² Na, ³⁶ C1, ³⁵ S, ³³ P, ³² P, ¹²⁵ I, etc.

In addition, in some cases, a dye may also be introduced to the compartments. The dye may be used to determine the flow of fluid within the compartment, and/or to determine the amount of liposomes or lipid within the compartments (for example, if the dye is hydrophilic and/or if the dye is one that does not readily bind or associate with the liposomes). This may be used, for example, to determine what portion of the

compartment contains liposomes, and in some cases, to normalize the results when comparing different compartments. An example can be seen in Figs. 3 A and 3B, where the dye does not enter the liposomes or solid posts found within a compartment (same views shown, with and without dye present; the liposomes can be seen as relatively dark circular regions near the square posts). Examples of dyes include, but are not limited to, crystal violet, trypan blue, cresyl blue, acridine orange, bismark brown, carmine, cresyl violet, Coomassie blue, DAPI, methylene blue, methyl green, malachite green, rhodamine, or the like.

In addition, a variety of image processing techniques may be used, e.g., for analyzing the data and determining binding or association of the target (or the label) with lipids and/or liposomes within the compartments. For example, in some cases, extraneous information (e.g., the presence of posts or walls within the image) may be corrected or deleted. In addition, in some cases, differences between compartments, e.g., due to lighting, useable volume within a compartment, different labels, etc. may be normalized using techniques such as image processing techniques.

Various aspects of the present invention may find use in a variety of different applications. As a non-limiting example, in one set of embodiments, a plurality of compartments may contain a variety of different lipids, and a virus may be introduced into the compartments to determine the association or affinity of the virus to each of the lipids contained within the compartments, for example, producing an "affinity map" relating the affinity or association of the virus to each of the lipids within the

compartments.

One non-limiting example is shown by reference to Fig. 2, where cholera toxin

(Figs. 2A and 2B) is used as a target and lipids such as GM1

(monosialotetrahexosylganglioside) and GM2 (a monosialic ganglioside having a slightly different structure) are present within various compartments. The cholera toxin, in native form, is an oligomeric complex made up of six protein subunits: a single copy of the A subunit (part A, enzymatic), and five copies of the B subunit (part B, receptor binding), denoted as AB5. Subunit B binds while subunit A activates the G protein which activates adenylate cyclase. See Figs. 2A and 2B. However, testing using the cholera toxin can also performed using subunits of the cholera toxin (e.g., using only subunit B). As one example, GM1 can be introduced into certain compartments but not introduced into other compartments, such that the loading of the compartments encodes specific patterns as is shown in Fig. 2C with lighter colors representing compartments containing GM1 and darker colors representing compartments that do not contain GM1. In addition, Fig. 2D shows the binding of cholera toxin with a liposome library as another example, showing that the cholera toxin binds specifically only to liposomes containing gangliosides, which are marked with white squares within Fig. 2D.

As another non-limiting example, the target may be a virus such as the virus that causes Dengue fever, which enters cells via endocytosis. The endocytosed virus is then contained within an endosome within the cell, which becomes a late-endosome and then a lysosome, accompanied by a decrease in pH (e.g., from about 6-6.5 to about 4-5). This may induce the virus to fuse to the lipid bilayer walls of the lysosome, thereby allowing the virus to inject its cargo (nucleic acid) into the cell, thereby infecting the cell. In this example, the virus can be introduced to a plurality of compartments containing various lipids at various temperatures and/or pHs, which can be used to determine which lipids are recognized by the virus. As is shown in Fig. 4, the envelope protein of the virus may associate with lipids such as BMP (bis(monoacylglycero)phosphate), DGPP

(diacylglycerol pyrophosphate), PS (phosphatidylserine), or PA (phosphatidic acid).

As yet another example, proteins that associate with membranes may be used as targets. For example, such proteins may be exposed to a plurality of compartments containing a variety of different lipids. In some cases, information about the structure and/or affinity of such proteins may be determined by determining which lipids associate with the proteins. For instance, proteins that bind to particular membranes (e.g., under certain conditions) may be determined. As a non-limiting example, portions of three proteins (pdl, p24, and p23) were exposed to compartments containing various lipids. The portions used were the transmembrane domains of the proteins, which interacts with cell membranes. The portions were additionally linked to a GFP protein as a fluorescent label. The transmembrane domains of these proteins that were studied were as follows: pdl ( gggsgggGQFQTLVVGWGGLLGSLVLLV VLAVI ) ( _SE Q J _{D n0:} 1), _p 24

( gggsgggsDNTNS VLWSFFEALVLVAMTLGQIY ) (SEQ ID NO: 2), and p23

( gggsQggsESTNTRVLYFSi SMFCLiGLAT QVF) (SEQ ID NO: 3). The associations of each protein to the lipids are shown in Figs. 5A-5C. p24 (Fig. 5A) and p23 (Fig. 5B) were observed to show relatively specific binding to certain lipids, while pdl (Fig. 5C) was relatively less specific and showed greater association with a variety of different lipids.

In another set of embodiments, proteins that are used as targets may contain hydrophobic domains, and such proteins may be modified to determine portions of the protein that can bind to or otherwise associate with membranes. In some cases, proteins that are used as targets may be difficult to synthesize within a suitable lipid membrane or other structure. In some cases, proteins may be synthesized under cell-free conditions and exposed to lipids and/or liposomes within compartments to determine how such proteins interact with lipids.

Other targets besides proteins and viruses may also be studied, in still other embodiments of the invention. For example, in one set of embodiments, one or more drug candidates or small molecules (or other candidate species) may be analyzed to determine lipid binding. For example, certain lipids may be present in certain cells, and candidate species that associate with such lipids may be determined. In some cases, as discussed above, some species may exhibit preferential specificity to particular lipids, and such species can be determined in accordance with certain embodiments. Such species may be, for example, candidates that exhibit preferential targeting to certain desired cells (e.g., cancer cells). As another non-limiting example, fungi typically contain ergosterol instead of cholesterol in their cell walls, and candidate species that preferentially associate with ergosterol relative to cholesterol may be identified, e.g., as potential fungicides.

As another set of embodiments, molds or other potentially infective species may be analyzed by exposure to compartments containing lipids to determine a pattern of associations. In some cases, certain molds may preferentially associate with certain lipids; thus, the pattern of associations may be used as a "fingerprint" to identify the mold or other potentially infective species. Other species may also be determined or identified using a similar approach, including infective species such as bacteria, spores, etc., or chemical species such as dust, protein, prions, toxins, drugs, etc. The species may be, for example, airborne, waterborne, blood-borne, etc. Thus, for example, an affinity map may be used for prognosis in some embodiments, i.e. to identify harmful entities such as toxins, molds, viruses, other infective species, etc. in a sample.

In still another set of embodiments, protein that associate with certain lipids may be useful for identifying suitable protein/lipid associations, e.g., for structural or crystallographic purposes. For instance, a protein/lipid complex (e.g., of proteins such as membrane proteins that are relatively hydrophobic or hard to crystallize in isolation) may be easier to crystallization upon association with a suitable lipid. Thus, in some cases, suitable protein/lipid pairings (or other target/lipid pairings) may be determined.

In addition, in some cases, the protein may be a denaturated or a mis-folded protein. For example, certain types of proteins (e.g., prions or amyloids) may bind preferentially to certain lipids, relative to the normal protein. Thus, by determining binding of a protein to various lipids, the status of the protein (e.g., whether it is correctly folded or not) may be determined. Such a test may be useful, for example, for diagnosing certain types of diseases involving misfolded proteins, e.g., Alzheimer's disease, Parkinsons disease, certain prion diseases such as transmissible spongiform encephalopathy, or the like.

In yet another set of embodiments, lipids and/or liposomes that a virus can associate with may be determined. As noted above, some viruses may associate with specific lipids and/or liposomes, which may be similar to a target cell of the virus. In some cases, such lipids and/or liposomes may be determined as discussed herein, and such lipids and/or liposomes may represent suitable targets for the virus. In some cases, for example, a virus may recognize a liposome as a target cell, and upon association of the virus with the liposome, the virus may be induced to deliver or inject its nucleic acid into the liposome, e.g., thereby "infecting" the liposome.

In some aspects, such liposomes may be delivered or administered to a subject. Liposomes within the subject may be decoy "targets" of the virus, which may be useful, for example, to reduce the virus load of a subject infected with the virus. For example, a virus circulating within the subject may be induced to associate with and "infect" (e.g., fuse) with a liposome rather than a real cell of the subject, thereby reducing the severity or loading of the virus infection within the subject. The subject may be human, or a non- human subject, such as a non-human primate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla, etc.), a dog, a cat, a rat, a pig, a bird, etc. Thus, for example, as is shown in Fig. 6, one or more viruses 80 may associate with a liposome 85 in order to "infect" the liposome.

In addition, in some embodiments, the liposome may contain other components that can interact with the virus. For example, the liposome may contain nucleases (e.g., DNA or RNA nucleases) which can degrade the nucleic acid inserted by the virus, and/or proteinases to inhibit viral enzymes from the virus. In some cases, the liposome may contain adjuvants or other components which can be used to boost the immune response of the subject, e.g., upon "infection" of the liposome with the virus. In some cases, the virus, after association with the liposome, may be in a weakened state, which may allow the immune system to recognize the virus or more readily produce an appropriate immune response.

The virus may be any virus, e.g., that is capable of infecting a subject. The subject may have the virus, or be at risk of becoming infected by the virus. The virus may be, for example, Dengue virus, common cold virus, influenza virus, chicken pox virus, or a virus that causes diseases such as Ebola virus disease, AIDS (i.e., the HIV virus), avian influenza, and SARS. Other non-limiting examples of viruses include adenovirus, Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus, type 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus, human bocavirus, Parvovirus Bl, human astro virus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coltivirus, or Banna virus. In one embodiment, the virus is not HIV. In another embodiment, the virus is not influence virus. In yet another embodiment, the virus is not one or more of the viruses given above. Thus, another aspect of the invention provides methods of administering any composition discussed herein, such as a liposome, to a subject. When administered, the compositions of the invention are applied in a therapeutically effective, pharmaceutically acceptable amount as a pharmaceutically acceptable formulation. Any of the

compositions (e.g., comprising liposomes) may be administered to the subject in a therapeutically effective dose. When administered to a subject, effective amounts will depend on the particular condition being treated and the desired outcome. A

therapeutically effective dose may be determined by those of ordinary skill in the art, for instance, employing factors such as those described herein and using no more than routine experimentation.

In certain embodiments of the invention, the administration of such compositions may be designed so as to result in sequential exposures to the composition over a certain time period, for example, hours, days, weeks, months, or years. This may be

accomplished, for example, by repeated administrations of a composition by one or more of the methods described herein, or by a sustained or controlled release delivery system in which the composition is delivered over a prolonged period without repeated administrations. Administration of the composition using such a delivery system may be, for example, by a transdermal patch. Maintaining a substantially constant concentration of the composition may be preferred in some cases.

The compositions may additionally comprise one or more adjunct ingredients, for instance, pharmaceutical drugs. For example, the compositions may include adjuvants such as salts, buffering agents, diluents, excipients, chelating agents, fillers, drying agents, antioxidants, antimicrobials, preservatives, binding agents, bulking agents, silicas, solubilizers, or stabilizers. Non-limiting examples include species such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch, gelatin or acacia; lubricating agents such as magnesium stearate, stearic acid, or talc; time-delay materials such as glycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone; dispersing or wetting agents such as lecithin or other naturally-occurring phosphatides; thickening agents such as cetyl alcohol or beeswax; buffering agents such as acetic acid and salts thereof, citric acid and salts thereof, boric acid and salts thereof, or phosphoric acid and salts thereof; or preservatives such as benzalkonium chloride, chlorobutanol, parabens, or thimerosal. Suitable concentrations can be determined by those of ordinary skill in the art, using no more than routine experimentation. Those of ordinary skill in the art will know of other suitable formulation ingredients, or will be able to ascertain such, using routine experimentation.

Preparations can include sterile aqueous or nonaqueous solutions, suspensions and emulsions, which can be isotonic with the blood of the subject in certain

embodiments. Examples of nonaqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oil such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, organic esters such as ethyl oleate, or fixed oils including synthetic mono or di-glycerides. Aqueous solvents include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like. Those of skill in the art can readily determine the various parameters for preparing and formulating the compositions of the invention without resort to undue experimentation.

Any medically acceptable method may be used to administer the composition to the subject. The administration may be localized (i.e., to a particular region,

physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition may be administered orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally through parenteral injection or implantation, via surgical administration, or any other method of

administration. Examples of parenteral modalities that can be used with the invention include intravenous, intradermal, subcutaneous, intracavity, intramuscular,

intraperitoneal, epidural, or intrathecal.

Other delivery systems which can be include time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing convenience to the subject. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones and/or combinations of these; nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based- systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. Specific examples include, but are not limited to, erosional systems in which the composition is contained in a form within a matrix (for example, as described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152), or diffusional systems in which an active component controls the release rate (for example, as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686). The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some embodiments, the system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation containing the composition. In addition, a pump-based hardware delivery system may be used to deliver one or more embodiments of the invention.

In another aspect, the present invention is directed to a kit including one or more of the compositions discussed herein. A kit may include a package or an assembly including one or more of the compositions of the invention, and/or other compositions associated with the invention, for example, as described herein. Each of the

compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form (e.g., a dried powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit.

A kit of the invention may, in some cases, include instructions in any form that are provided in connection with the compositions of the invention in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with the compositions of the invention. For instance, the instructions may include instructions for the use, modification, mixing, diluting, preserving, administering, assembly, storage, packaging, and/or preparation of the composition and/or other compositions associated with the kit. In some cases, the instructions may also include instructions for the delivery and/or administration of the compositions, for example, for a particular use, e.g., to a sample and/or a subject. The instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic

communications (including Internet or web-based communications), provided in any manner.

The following documents are incorporated herein by reference in their entirety for all purposes: U.S. Provisional Patent Application Serial No. 61/926,179, filed January 10, 2014, entitled "Systems and Methods Involving Interactions Such As Protein or Virus Interactions"; U.S. Provisional Application Serial Number 61/980,541, filed April 16, 2014, entitled "Systems and Methods for Producing Droplet Emulsions with Relatively Thin Shells"; International Patent Publication Number WO 2004/091763, filed April 9, 2004, entitled "Formation and Control of Fluidic Species," by Link et al. ; International Patent Publication Number WO 2004/002627, filed June 3, 2003, entitled "Method and Apparatus for Fluid Dispersion," by Stone et al. ; International Patent Publication Number WO 2006/096571, filed March 3, 2006, entitled "Method and Apparatus for Forming Multiple Emulsions," by Weitz et al.; International Patent Publication Number WO 2005/021151, filed August 27, 2004, entitled "Electronic Control of Fluidic Species," by Link et al. ; International Patent Publication Number WO 2008/121342, filed March 28, 2008, entitled "Emulsions and Techniques for Formation," by Chu et al; International Patent Publication Number WO 2010/104604, filed March 12, 2010, entitled "Method for the Controlled Creation of Emulsions, Including Multiple Emulsions," by Weitz et al.; International Patent Publication Number WO 2011/028760, filed September 1, 2010, entitled "Multiple Emulsions Created Using Junctions," by Weitz et al.; International Patent Publication Number WO 2011/028764, filed September 1, 2010, entitled "Multiple Emulsions Created Using Jetting and Other Techniques," by Weitz et al.; International Patent Publication Number WO 2009/148598, filed June 4, 2009, entitled "Polymersomes, Phospholipids, and Other Species Associated with Droplets," by Shum, et al. ; International Patent Publication Number WO 2011/116154, filed March 16, 2011, entitled "Melt Emulsification," by Shum, et al. ; International Patent Publication Number WO 2009/148598, filed June 4, 2009, entitled

"Polymersomes, Colloidosomes, Liposomes, and other Species Associated with Fluidic Droplets," by Shum, et ah; International Patent Publication Number WO 2012/162296, filed May 22, 2012, entitled "Control of Emulsions, Including Multiple Emulsions," by Rotem, et al. ; International Patent Publication Number WO 2013/006661, filed July 5, 2012, entitled "Multiple Emulsions and Techniques for the Formation of Multiple Emulsions," by Kim, et al. ; and International Patent Publication Number WO

2013/032709, filed August 15, 2012, entitled "Systems and Methods for Shell

Encapsulation," by Weitz, et al.

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1

Lipid rafts are important to functionality by acting as templates that choose which proteins will be incorporated. The lipid composition in a membrane may control the protein population on it. This example demonstrates a method to study the specificity of a protein to lipid rafts and determine out of hundreds lipid compositions which are the protein's "favorite" rafts. As examples, this example shows the lipids a Dengue virus requires for fusion to the membrane, and the lipid selectivity of transmembrane domains.

This example studies the binding of fluorescently labeled proteins (from the

Dengue virus) to a library of membrane lipids. The library has more than 200 different vesicles, made of 70 lipid species. The vesicles (or liposomes) were grown inside microfluidic compartments upon exposure to water. After expose of the vesicles to the fluorescently labeled proteins, the compartments were analyzed using confocal microscopy to determine the association of the proteins with the different vesicles.

It was found that the Dengue virus fuses with the late endosome membrane. Dengue virus requires certain lipids for fusion. For example, phosphatidylserine exists in the plasma membrane and probably also in the endosome. However, it may be located on the inner leaflet, i.e. on the other side of the membrane, as the virus approaches from the outer leaflet side, and thus phosphatidylserine may not interact with the Dengue virus during the initial fusion process. Fig. 4 shows an affinity matrix of the Dengue virus to various lipids, with lighter shadings showing more binding and darker shadings showing less binding to various lipids. This matrix shows that the Dengue virus exhibits relatively specific binding to certain lipids but not others.

In addition, most membrane proteins are immiscible in water. Thus, in order to study the transmembrane domains of the proteins, in vitro protein synthesis was used in another set of experiments. The transmembrane domains were linked to GFP and were expressed inside the microfluidic compartments, adjacent to the lipid vesicles. All of the non-binding peptides were then aggregated and flushed out of the microfluidic device.

These results serve as an additional proof to the lipid domains hypothesis and indicate that there is a high specificity in protein-lipid interactions. Specificity can be determined also by the hydrophobic core of the membrane, as observed in transmembrane domains.

EXAMPLE 2

This example is generally directed to determining the specificity of a protein's trans-membrane domain to lipid compositions by combinatorial screening of liposomes, in accordance with certain embodiments of the invention.

Membranes differ significantly by their lipid and protein contents, according to the cell type and organelles they encompass. The variety of lipids plays a vital role in protein transport and function. The protein content in the membrane is often tightly related with the lipid types composing it, indicating that proteins selectively bind to specific lipid types. Determining the specificity of proteins to lipids is essential to the basic understanding of membrane function. It may also be an important step for designing drugs that will target specific cell membranes or tissues according to their lipid contents. In addition, defining the lipids with which the protein is in contact with may be fundamental for structural studies of the membrane proteins, and may serve as better substrate for crystallization. Lipid-protein interactions have been, therefore, increasingly studied over the years, and liposomes have become the preferred in-vitro substrates, often used to measure the binding of fluorescently labeled proteins to lipid membranes. These studies, however, were limited to soluble proteins and not feasible for the grand majority of membrane proteins, due to their exposed hydrophobic moiety that leads to aggregation of the proteins. Therefore, most integral membrane proteins, which are the significant portion of the human genome encoded membrane proteins, cannot be studied. This example shows combinatorial screening of Trans-Membrane Domains (TMDs) - lipid interactions using a liposome library and identify their specificity. In order to reproduce and compare the composition conditions at the binding site of the protein, a liposome library was created, made of the most abundant lipids in cells or highlighted in recent studies, where each liposome was made of at least three different lipid types. 108 different liposome compositions were grown and measured in parallel on a microfluidic chip, where TMDs linked to Green Fluorescence Protein (GFP) were synthesized in a cell-free system, at a close proximity with the liposomes. Affinity studies of the hydrophobic moiety of human proteins to the liposome library revealed high specificity to lipid composition, and showed that the specific interaction of a protein to lipids depends not only on the presence of a single lipid, but on a combination of a few. The data shown here, contributes to knowledge of how specific are the protein-lipid interactions, and further support our understanding of the rich lipid content that exists in biological membranes.

Liposome Library. Lipid compositions in cell membranes have shown high variability depending on cell organelle and type. However, most of the data sources are derived from the average total composition of the membrane and of cell population. To date, knowledge of the lipid compositions located at the binding site for each protein is limited. Therefore, when choosing the lipid mixtures in the liposome library, liposomes were not created with the total lipid composition as found to exist in cell membranes. Instead, a list of the most abundant lipids and lipids at the focus of interest in recent studies was gathered. A single alpha helix domain passing through the membrane has a periphery of at least 4-5 lipids, indicating that TMD specificity to membrane may depend on the presence of a combination of lipid types. Therefore, a liposome library was created made of mixtures of the lipid species; dividing the lipids into saturated and non- saturated fats, and subdividing the saturated lipids into glycerol and sphingo groups according to their backbone connecting the alkyl chains, all possible mixtures were made among the three groups. On top of these groups, each liposome mixture contained cholesterol, which is abundant in all vertebrate membranes, and the unsaturated 1- palmitoyl-2-oleoyl-5'w-glycero-3-phosphocholine (POPC) was included in all saturated mixtures for enabling spontaneously swelling into liposomes upon hydration. See Fig. 12. 120 lipid mixtures were made, dissolved in chloroform, and stored in glass vials at - 20 °C. It is believed to be the largest library reported to date. See Fig. 13.

The number of lipid molecules inside each well of the microfluidic device was substantially higher than the number of introduced proteins. Hence, an addition of even a merely 0.1% molar ratio of a lipid, as performed by many previous experimental studies on lipid-protein interaction, may serve as enough binding sites for proteins, and could lead to "false positive" signals. Therefore, in the method shown here, any additions to the membrane such as polymers, charged lipids, PEGylated (polyethylene glycol) lipids or fluorescently labeled lipids, was completely avoided to prevent any possible protein labeling of sites on the membrane, other than the lipids in question.

Microfluidic Device. Polydimethylsiloxane (PDMS) microfluidic devices were fabricated by standard soft lithographic methods. Using 3D printed parts, a capillary brush was used to collect a few microliters from 12 lipid compositions at a time and seeded on the PDMS microfluidic device. The process was repeated 9 times in order to seed all 108 wells. Most solvent evaporated within seconds and the PDMS device was bonded shut, and kept under vacuum until experiment. Fig. 8. Prior to experiment, the microarray device was filled with de-gassed water/PBS and placed in a 65 °C oven for 60 minutes for catalyzing liposome swelling. The liposome swelling was uncontrolled and lead to multi-lamellar membranes. This in fact was a virtue, since attempts to repeat the experiment on uni-lamellar liposomes were proven difficult because of their fragility and tendency to collapse during protein expression. In contrast, the multi-lamellar liposomes were robust and as the binding occurred on the outer membrane, the additional membrane layers were unlikely to play a role or lead to different results. The microfluidic device allowed parallel labeling measurements at 108 different wells with a total volume of about 40 microliters, each well contained specific lipid mixture.

Measurement. Using an automated stage, XYZ scans of all wells in the device were performed by resonance confocal microscopy. Fluorescence from each well was collected three times: 1. Control— prior to protein binding to liposomes. 2.

Fluorescently labeled proteins were introduced to the liposomes in all wells. Each well was then flushed with water or PBS 40 times its volume to exclude unbound proteins or aggregates. 3. Dyed water was flushed throughout the device to verify the flow through all wells, and, if necessary, compensate by normalizing the fluorescence data from wells with partial flow. The use of the dye is also beneficial for detecting low light scattering unilamellar liposomes that were difficult to detect by transmitted light microscopy at low magnification. Fig. 11.

Data Analysis. The lipid swelling occurred spontaneously, uncontrolled and the resulting liposomes varied greatly in size and structure, depending on the lipids composing the liposome. Often, there was a big variation in the number of liposomes in each well, which may cause a rather big error in the binding measurement, as the binding is estimated by fluorescence per unit area. In addition, the PDMS surface may have also served as a binding site and contributed to non-specific fluorescence signal. In order to minimize these effects, the data was collected using a confocal microscope which provided Z- stacks images. Fluorescence originated from all surfaces of PDMS was cleared out, by the use of a homemade image processing.

Cholera toxin subunit B is one of the few proteins that were well characterized and its selectivity to lipids was previously studied. Upon purification of the protein from cell cultures, gangliosides (GM1) residues were found in addition to the toxin, which indicated strong interactions with the lipid. As a proof of concept of the liposome method, 0.1 micromolar of fluorescently labeled cholera toxin on the liposome library was run. The affinity matrix obtained from this measurement indeed showed fluorescent labeling of all the liposomes containing GM1, and more importantly had negligible false positive labeling any of the liposomes which do not contain GM1. The variability of fluorescence was calculated and normalized to the amount of GM1 in the liposomes as the error scale of the system, even though variability in the lipid ratio alone have shown to affect the cholera toxin labeling.

Trans-Membrane Domains. Unlike the cholera toxin, binding studies of TMDs could not be performed by flowing a solution of the TMDs into the microfluidic chip, since the hydrophobic TMDs instantly aggregated. Instead, in order to study the labeling of a single TMD with the lipid membrane in an aqueous environment, a synthetic protein of TMD fused to a green fluorescent protein (GFP) was constructed. The GFP-TMD fusion protein is expressed by a reconstituted cell-free protein synthesis system, which will be referred to as in-vitro Transcription and Translation (IVTT). See, e.g., U.S. Pat. Apl. Ser. No. 62/054,263, filed September 23, 2014, entitled "Two-Hybrid Systems and Methods in Droplets and Other Compartments," incorporated herein by reference in its entirety. The microfluidic device was filled with the IVTT reagents along with DNA expressing GFP-TMD, and heated to 37 °C for 2.5 hours. Under these conditions, GFP- TMD proteins expressed at the vicinity of a liposome may embed into the lipid membrane, or aggregate into micelles. All non-bound aggregates of GFP-TMDs were washed off prior to confocal scans.

Specificity studies, unlike affinity studies, utilize non-labeled molecules to compete with the molecule of interest, over the binding sites. Such a competitor should cancel fluorescence labeling of non-specific sites, to which the molecule may have some affinity but no more than another similar molecule. In these examples, the competitor purpose was to eliminate binding of the GFP-TMD to less favorable lipids, for which SNAP-TMDs with different sequences as competitors were used. SNAP is a non- fluorescent protein with a similar size as the GFP protein, hence estimated to be expressed at similar levels as the GFP-TMD. The competitor addition was performed by including SNAP-TMDs DNA's into the IVTT reagents. To increase competition, the ratio between the SNAP-TMD and GFP-TMD DNAs was 2: 1. The competitors that were used for each experiment are detailed in the affinity table (Fig. 13). Interesting to note, for some lipid composition, e.g. SM:Chol at 70:30%, several TMDs show high affinity even though these were used as each other competitors, suggesting that the number of binding sites may be substantially higher than proteins, leaving enough sites for all TMDs to bind.

In contrast to the common concept that a hydrophobic moiety of a protein would always tend to go to the hydrophobic membrane regardless of its lipid contents, the hydrophobic TMDs studied here were found to be highly specific, with the exception of the PD1 protein, where 40% of all liposomes in library had labeling intensity of over 25% and 70% of the library had intensity above 10%.

In a recent study the TMD of CD40 in Murine T cells was replaced with TMD of CD45, and as a result the mutated CD40/45 protein did not form clusters in cell membranes upon stimulation with its ligand, as its wild-type CD40 has. It was suggested that the CD40 TMD was selective to certain lipid domains, the so called lipid rafts, which are defined to be mainly composed of cholesterol and lipid of sphingolipids family. Comparison of the affinity matrices of CD40 and CD45 show indeed that though the two share a few of the lipid compositions, generally each TMD has a distinct specificity. The affinity matrices also teach us that for both TMDs all the lipid compositions that had above 20% labeling level, included some type of sphingolipid, suggesting that each have preferential to different lipid rafts (Fig. 13).

A recent study comparing the TMDs interactions with Sphingomyelin (SM) showed that P24 had higher labeling to 18 carbon long SM when compared to other chain lengths of SM. Moreover, P23 did not have a strong affinity to any of the SM lipids. The SM used in these studies was a combination of several chain lengths among which is SM-18. These measurements correspond well with the study, showing significantly lower labeling of P23 to any of the SM mixtures, as compared to the P24. The data also revealed that the P24 labeling of the SM mixtures strongly depended on the additional lipid in the composition, with highest labeling to SM combinations with PC, DAG, Ceramide and Cerebroside. Both P23 and P24 showed high affinities to other mixtures as well. The fluorescence from the highest affinity composition for the P24 was 7 times higher than of the P23 (Fig. 13).

The liposomes labeling by the GFP-TMDs as detected here was believed likely to be a result of two separate events: the entry of the peptide to the membrane, where the transformation from solution into the hydrophobic membrane may energetically favor certain lipid combinations, and once embedded in the membrane the TMD may diffuse freely and find its preferable lipid domain that would prolong its lifetime in the membrane. It may be speculated that TMDs embed in all membranes and are at equilibrium with the bulk and after the long wash with PBS all micelles were drained and the wells were left only with proteins which were strongly associated with their membranes. However, when expressing the TMD-GFP proteins outside of the microfluidic device the protein aggregated, and formed micelles. Upon introduction of the micelles to the liposome library, a negligible amount of fluorescence was detected (data not shown). Therefore, a more likely explanation is that the selectivity observed here is a result of lipid preference for entry, rather than equilibrium with micelles in the bulk.

In contrast to intuition that a small hydrophilic headgroup would perform as a lower barrier for a peptide embedment, comparison between the binding levels of CenChol, both of which have hydroxyl headgroup, and SM:Chol where SM has a larger phosphocholine headgroup, shows higher labeling to the later, as seen in Fig. 13. Unlike cells, the methodology introduced here which uses the bacterial IVTT does not include aids for membrane entry such as chaperones that escort the unfolded polypeptide, or translocons, which assist in translocation of the peptide through the membrane. The data presented here were the result of spontaneous embedding of the polypeptide. It should also be considered that the membranes studied here are symmetrical, meaning the lipid composition on both leaflets of the membrane is identical, whereas cell membranes are asymmetric. Nevertheless, the P23/24 specificity to SMI 8 in vitro study that was performed with TMDs on was also supported by experiments on cells, suggesting that the TMD binding to liposomes concept is relevant for studying cellular systems, despite of the above discrepancies.

Discussion. It has been established that lipids are not spread homogeneously in the cell membrane. Instead they segregate into domains, which were hypothesized to be functional by interacting differently with specific proteins according to lipid

composition, i.e. act as a protein sorting machinery and physically positions proteins in the bilayer. The terms "lipid domain" or "lipid rafts" have a wide variety of definitions. It should be understood that the liposomes in these studies were not an attempt to imitate any domain, but rather the structural and chemical composition requirements for the affinity of a protein at the scale of its binding site. Phase separations of most lipid mixtures introduced here were never studied. Therefore, it is unknown if the detected binding is to a homogenous phase or one of the separated phases.

Several mechanisms have been proposed, which partially explain the selective protein interactions with lipid membranes. Most common are: Lipid phases— order and disorder domains determine lipid fluidity of the domain, membrane curvature and membrane thickness, and indeed, these mechanisms were demonstrated to have an effect on protein transport in cells. The data shown here naturally demonstrate that structural and chemical complementarities should also be considered and suggest that there could be a much larger variety of selective, hence functional domains, as all five TMDs studied here showed affinity to many compositions. The actual lipid compositions of domains in many cell membranes are currently unknown, and it may be that the TMDs studied here would have a stronger affinity to domains which are not in the liposome library, yet do exist in cells. Nevertheless, these data provide experimental support to the hypothesis of lipid function by specificity to proteins and exemplifies further the importance of lipid variability. It is believed that the methodology introduced here, is not only important for our basic understanding of membrane function, but may also serve as a tool for designing cell targeting drugs, antibiotics and diagnosis.

Out of 29375 human genes, 7299 were predicted to contain transmembrane alpha-helix and 3461 of those are of a single alpha-helix TMDs, and their lipid environment is currently unknown. Approximately 60% of today's drugs target membrane proteins, yet surprisingly, there are limited tools to study these proteins. It is believed that a liposome library can play a vital role to understand the protein's required lipid environment, valuable for understanding its transport and for simulation studies, which are currently being performed without any previous knowledge of the actual lipids the proteins are in contact with. It may also serve as a tool for choosing the right lipid compositions for protein crystallization for structure and function studies. Similar to the "draggable genome" concept which include all genes that are believed to interact with drags, it is believed that the methodology discussed here will lead to "draggable lipids" where a list of peptides will be found to interact with certain lipid mixtures, hence able to differentiate between membranes, and target specific organelles, cells or species.

The microfluidic device may have several inlets all of which lead through separate channels into all wells evenly, with an identical channel lengths and widths to all wells. Drainage of each well leads directly to the outlet of the device and does not pass through another well, to avoid lipid contamination between wells. The thickness of inlet channels was 15 micrometers, the wells were 50 micrometers, and the outlets were 75 micrometers, for preventing upstream flow from drainage to wells. Each well had PDMS rectangular poles to increase the surface area for liposome swelling, act as physical barrier to prevent liposomes from flushing out, and to prevent persistent air bubbles from staying within the wells upon hydration. Air bubbles trapped in the wells were caused due to the lipids tendency to create air-water interface, and may cause interference with the flow of material in/out of the well.

Imaging: Using a lOx dry objective with a numerical aperture of 0.3 on a confocal microscope (Leica), bright field and fluorescence images were acquired, simultaneously, using multi-track mode. For this, an argon (488 nm) laser was used as an excitation source with a pinhole size of 100 micrometers and fluorescence emission was collected by the PMT detectors through bandpass filters between 525 nm and 567 nm. All scans were performed at room temperature.

Image processing. Whenever possible, the surface area of the liposomes was estimated by the image analysis software, or using the negative image of the dye, which can reveal liposomes unrecognized by the transmittance image. Each experiment was reproduced at least 3 times and after image processing the binding error from average was up to 40%. Nevertheless, it is believed that the method, in its current format, is sufficient for finding a "hit" of a suitable membrane composition out of a large library, which can then be studied further more accurately by conventional means. Control scans of the liposome library, performed prior to introduction of fluorescently labeled proteins, were subtracted from the scans performed with the proteins, and then normalized to the amount flow that went through each well, as measured by flowing in fluorescent dyed water at the end of each experiment as exemplified in Fig. 11.

Materials: For the complete list of lipid products see Fig. 13. Cholera toxin subunit B (recombinant), Alexa Fluor® 488 conjugate, Life Technologies. The reconstituted cell-free system (IVTT) was the PURExpress system (New England Biolabs). Water was dyed using sulforhodamine B (Sigma) or fluorescein (Sigma) at O.lg/L.

Fig. 7 shows confocal microscope images of (Fig. 7A) fluorescence, (Fig. 7B) transmittance. Scans undergo image analysis for identifying (Fig. 7C) PDMS poles, Fluorescence and (Fig. 7D) liposomes features.

Fig. 8. Microfluidic device. Fig. 8A shows a preparation scheme of PDMS microfluidic device: 1. PDMS device with channels is plasma treated using a hand held corona treater BD20-C A. 2. 3D printed capillary brush collects solvents from 12 glass vials simultaneously and seeds the lipid mixtures on PDMS device. The process is repeated 9 times for seeding all 108 wells. Fig. 8B shows schematics of a device containing 108 liposome farms. Enhancements are of a single well and (Fig. 8C) a confocal microscope transmitted beam where liposomes swelling off from the rectangular PDMS walls can be seen.

Fig. 9 shows Cholera Toxin subunit b binding to liposome library. Fig. 9A shows the structure of the cholera toxin subunit b pentamer and the structure of the Ganglioside lipid (GM1). Fig. 9B shows a labeling matrix to the liposome library. Bar height represents the fluorescence per unit area found on liposomes features. Liposomes which contain the GM1 lipid are marked with an aurora. Lipid composition along with relative binding for each bar can be found in the tables.

Fig. 10 shows affinity of Trans-Membrane Domains from human proteins to the liposome library. Fig. 10A shows a binding matrix of GFP-TMDs to the liposome library. Each of the matrices is normalized relatively to its highest intensity. The difference between the matrices shows the unique specificity each TMD possess. Fig. 10B are sequences of the TMD peptides that were introduced to the liposomes. The sequences, from top to bottom in Fig. 10B, correspond to SEQ ID NOs: 4, 5, 6, 7, and 8. Fig. IOC is a scheme of a GFP-TMD embedded in a lipid bilayer. Peptide was drawn as passing through the membrane as would a trans-membrane protein. The actual orientation of the embedded peptide in the liposomal membrane is unknown.

Fig. 11. As a final step of experiment, after measurement of fluorescence from all microfluidic wells, the microfluidic chip is filled with a fluorescent dye. This was performed for checking how much fluorophore each well was exposed to. Fig. 11A. Addition of the dye can at times be used to identify liposomes which are difficult to observe by the transmitted light image. Fig. 1 IB. Mapping the dye also can explain localization of fluorescence and provide the actual concentration of flow the liposomes were exposed to. The total fluorescence measured from the liposomes can then be normalized to the actual concentration of particles it was exposed to. Lipid compositions in a and b are different and were each taken from different wells. Fig. 11C shows the total dye that was measured from each well at two location scans as illustrated in Fig. 1 ID that also correlates to positions a-b. As can be seen, there are variations in flow to each well, due to clogging that was usually caused by high liposome concentrations at inlet channels.

Fig. 12 shows a table of the lipid species that compose the liposome library. There are 67 different lipid species in total. Lipid ratios of all liposomes in the library are detailed in Fig. 13. Product details of each lipid are available in Fig. 14.

In Fig. 13, each of the matrices is normalized relatively to its highest intensity. A comparison of the highest labeling positions between all TMDs showed a ratio of:

11:4:3:7: 1 for PD1:CD40:CD45:P24:P23, respectively. However, the IVTT is highly sensitive to minor changes in its environment and likely has different yields from one experiment to another.

Fig. 14 shows detailed information of all lipid products and their acronyms as appear in Figs. 12 and 13.

EXAMPLE 3

This example illustrates the affinity for various lipids influenza proteins (HA1 and HA2) which fuse with the host membrane, and VSV-Ebola virus, produced using techniques similar to the ones discussed above (Fig. 15 A), and the envelope protein of Dengue virus serotype 2 (Fig. 15B).

Each of these has a different affinity table; however, it can be seen that all bind well to most compositions that contain negatively charged lipids such as: PI, PA, PS, DGPP and BMP. These also show some binding to the neutral PE, N-palmitoid and also to sugar-containing lipids: cerebrosides and gangliosides.

EXAMPLE 4

Viruses pose a major threat and there is an increasing demand for anti-viral drugs. This example shows an approach for designing anti- viral drugs, where instead of attempting to inhibit the virus's life cycle process, "traps" or "decoys" are created for diverting the virus from encountering the host cell. These membrane based traps were screened out of a liposome library by binding measurements of the virions to specific lipid compositions. These examples show dengue virus using an infectivity assay.

Humans are locked in an evolutionary struggle with viruses, and as population density rises the risk of new pandemics is ever increasing. While vaccines have proven effective in preventing infection and the spread of stable viruses, their long development times means they are far less effective against rapidly mutating viruses— the very class of viruses which pose the greatest infections threat. In addition, vaccines have little therapeutic value to patients already infected with a virus and can instead be treated with an anti- viral drug. Such anti-viral drugs are designed to either interrupt viral synthesis or disrupt its penetration into the host cell. Both approaches are successful at times, and are usually based on either genetic sequences or protein structure for effective inhibition. However, side effects and toxicity are not uncommon, especially when attempting to tamper with viral replication as it occurs inside the host cell, because it utilizes much of the host's endogenous cellular machinery. It may be for this reason that, to date, only a limited number of effective anti- viral agents have been developed.

Enveloped viruses enter cells by fusing with a target membrane. This example shows an approach for the development of anti-viral drugs where viruses are induced to fuse with a "decoy" membrane before encountering the host cell. Cell membranes differ significantly in their lipid contents, according to cell type and the organelle they encompass. The wide variety of these lipids plays a vital role in protein transport and function, since each membrane associated protein tends to bind selectively to a limited combination of lipid types. Similarly, the interaction of the virus with the host membrane is also lipid dependent. Therefore, for every virus type, a suitable lipid composition with which the virus has a high affinity to can be determined.

This examples uses a microfluidic setup that enables high-throughput screening over a large library of lipid membranes to identify the lipid selectivity profile of any target protein. The library is composed of over 200 liposomes. Using this methodology, the lipid selectivity of dengue virions, vesicular stomatitis virions pseudotyped with the Ebolavirus glycoprotein, and recombinant influenza virus hemaglutinnin protein were profiled. In each case, distinct lipid compositions recognized with high affinity and selectivity were identified. Notably, the high affinity was detected even without the presence of a receptor protein and at pH 7.4. This suggests that the virus would bind to liposomes with similar composition, in e.g. blood and pulmonary environments with identical pH levels. It es believed that upon contact with a decoy liposome, composed of its target lipid composition, the virus will no longer pose a threat to the cells.

Viral fusion is a key aspect of viral adaptation for cell entry. In fact, viral fusion with the lipid membrane of the host cell is shared by all enveloped viruses, whether the fusion is with the plasma membrane or the endosome membrane (after endocytosis). Any modification of the viral fusion process with its lipid membrane target would be devastating to its infectious potential, therefore, anti- viral agents that exploit the fusion are more likely to last.

To test the hypothesis, liposomes of defined composition can inhibit the infectivity of dengue virus in cell culture were studied. Cells were incubated with the virus along with liposome decoys. Twenty-four hours later, corresponding to a single cycle of infection, the yield of infectious particles was quantified as a measure of the viral infectivity. Liposome decoys (#52, 83, 89, 95) were found to lower infectivity as predicted by microfluidics experiments, whereas negative control liposomes ("neg lip") caused no significant reduction in infectivity. See Fig. 16. For two compositions, #89 and #95, cell infectivity was completely diminished (Fig. 16A). Cells infected along with non-decoy liposomes ("neg lip") showed similar levels as cells infected with a virus alone ("no lip"). Cell viability was unaffected by the presence of the liposomes.

Liposomes #3 and 29 did not seem to have lower the infectivity at these levels, however when repeated with liposome #3, cell infectivity was significantly reduced at higher concentrations. The liposomes were biocompatible and are not recognized by the immune system, especially if coated with a polyethylene glycol (PEG) layer. PEGylated liposomes were also tested and shown to be effective as decoys (Fig. 16B).

In addition, as the concentration of Liposome #3 (composed of 47% BMP, 38% SM and 15% Cholesterol) is increased, the infectivity goes down accordingly, i.e. the higher the dosage the better the infectivity inhibition. See Fig. 16C. In addition, when the liposomes were exposed to cells infected with another type of virus, the VSV virus, it was found that the liposomes had little effect on cell infectivity. This shows that the composition of liposomes was specific to the type of virus. See Fig. 16D. Thus, when repeating the infectivity assay on a different type of virus (VSV) using the same controls and decoy, it was found that in for the VSV case, none of the liposomes had any significant effect and neither showed significant reduction in infectivity.

These liposomes can therefore, be considered as nano-medicines which can be used as anti-viral drugs, effectively lowering the number of active viruses and aiding the immune system. While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.

Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.