Glycolipid functionalized liposomes or other vesicles for use as drug delivery systems or other applications

Abstract

Some aspects are generally related to articles comprising a lipid membrane (e.g., a vesicle) and a glycolipid at least partially embedded within the lipid membrane. In some cases, the glycolipid may be an agonist for a receptor and / or change at least one property of the article comprising the lipid membrane, which may make the article more suitable for associating with cells and / or being endocytosed by cells. In some cases, the lipid membranes may encapsulate entities such as small molecules, DNA and / or proteins, and thus the article may be suitable for drug delivery or other applications. Some aspects are related to articles comprising multiple glycolipids, which may impart various properties to the articles, for example, decreased zeta potentials and / or altered surface diffusion rates of lipids of the article. Still other aspects are generally directed to methods of making or using the articles, kits comprising the articles, or the like.

Description

[0001] GLYCOLIPID FUNCTIONALIZED LIPOSOMES OR OTHER VESICLES FOR USE AS DRUG DELIVERY SYSTEMS OR OTHER APPLICATIONS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 517,528, filed August 3, 2023, by Weitz, el al., incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] Articles comprising glycolipid functionalized lipid membranes, for example, for use as drug delivery systems or other applications, are generally described.

[0006] BACKGROUND

[0007] Articles comprising lipid membranes, e.g., vesicles, are often used for transporting cargo (e.g., DNA, RNA, and / or proteins) within cells and / or to cells. However, in some cases, endocytosis of such articles, and thus delivery of the cargo contained in the articles to cells, may be relatively slow. Moreover, articles comprising lipid membranes generally and uniformly associate with all types of cells, and thus targeted delivery of cargo within the articles to certain types of cells is not possible. Accordingly, improved vesicles and / or methods of making and / or using vesicles are needed.

[0008] SUMMARY

[0009] Articles comprising glycolipid functionalized lipid membranes, for example for use as drug delivery systems or other applications, are generally described. The subject matter of the present disclosure 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.

[0010] One aspect is generally directed to an article. In one set of embodiments, the article comprises a lipid membrane and / or a membrane comprising amphiphilic molecules, and a sterol covalently linked to a polysaccharide. In certain embodiments, the sterol is at least partially embedded within the lipid membrane and / or the membrane comprising amphiphilic molecules and the polysaccharide is at least partially exposed externally of the lipid membrane.

[0011] The article, in another set of embodiments, comprises a vesicle comprising a lipid membrane, and a sterol covalently linked to a polysaccharide. In certain embodiments, the sterol is at least partially embedded within the lipid membrane and the polysaccharide is at least partially exposed externally of the vesicle.

[0012] The article, in yet another set of embodiments, comprises a vesicle comprising a lipid membrane and cholesterol covalently linked to chondroitin sulfate.

[0013] According to still another set of embodiments, the article comprises a vesicle comprising a lipid membrane, and cholesterol covalently linked to chondroitin sulfate. In certain embodiments, the cholesterol is at least partially embedded within the lipid membrane and / or the chondroitin sulfate is at least partially exposed externally of the vesicle.

[0014] Yet another set of embodiments is generally directed to cholesterol covalently linked to chondroitin sulfate.

[0015] Another aspect is drawn to a method of making an article. In some embodiments, the method comprises mixing a first solution comprising a plurality of vesicles with a second solution comprising cholesterol covalently linked to chondroitin sulfate.

[0016] Still another aspect is generally directed to a method of drug delivery. In one set of embodiments, the method comprises introducing a plurality of vesicles to a population of cells. In some cases, a cholesterol covalently linked to chondroitin sulfate is embedded in a lipid membrane of at least some of the plurality of vesicles.

[0017] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure 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.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Non-limiting embodiments of the present disclosure 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 unless otherwise indicated. 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 disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures: FIGS. 1A-1B show illustrative diagrams of glycolipids and vesicles comprising glycolipids, according to some embodiments;

[0020] FIG. 2A shows fluorescence micrographs of vesicles, according to some embodiments;

[0021] FIG. 2B is a plot of the ratio of fluorescence intensity at the periphery of the vesicles in FIG. 2A to the fluorescence intensity measured in solution as a function of cholesterol-linked chondroitin sulfate, according to some embodiments;

[0022] FIGS. 2C shows electron micrographs of vesicles, according to some embodiments;

[0023] FIGS. 3A-3B show data obtained from a fluorescence recovery after photobleaching experiment, according to some embodiments;

[0024] FIGS. 4A-4D shows data obtained from a fluorescence recovery after photobleaching experiment to determine the diffusion coefficient of lipids of the vesicles, according to some embodiments;

[0025] FIGS. 5A-5B shows data obtained from a micropipette aspiration experiment, according to some embodiments;

[0026] FIGS. 6A-6Hillustrate the stretching modulus of vesicles as a function of vesicle composition, according to some embodiments;

[0027] FIGS. 7A-7D is data obtained from an experiment to measure the uptake rate and transfection efficiency of vesicles by cells, according to some embodiments;

[0028] FIGS. 8A-8B are plots of zeta potential and liposome uptake, respectively, as a function of cholesterol-linked chondroitin sulfate concentration, according to some embodiments;

[0029] FIGS. 9A-9B show plots of intensity percent of SUVs with and without Chol-CS modification against SUV size (n=3) and zeta potential of SUVs with different lipid compositions (DOTAP / DOPC 50:50mol%, green; DODMA / DOPC 50:50 mol%; DOPC 100 mol%) as a function of Chol-CS concentration (n=3), respectively, according to some embodiments;

[0030] FIGS. 10A-10C and 10E shows liposome uptake by HEK cells as a function of lipid composition (DOPC 100 mol%, DODMA / DOPC 50:50 mol%, DOTAP / DOPC 25:75 mol%, DOTAP / DOPC 50:50 mol%, DOTAP / DOPC 75:25 mol%) (n=20), confocal images of a confluent cell layer after incubation with CS-modified (FITC- labeled) SUVs (rhodamine-labeled) with a 50 micrometer scale bar, and liposome (DOTAP / DOPC 50:50mol%) uptake as a function CS concentration (n=40), respectively, according to some embodiments;

[0031] FIG. 10D shows flow cytometry data of cells after being exposed to different vesicles, according to some embodiments;

[0032] FIG. 11A shows a schematic representation of a CS-modified SUVs encapsulating Cy5-labeled GFP-encoding mRNA, which are added to HEK cells, according to some embodiments;

[0033] FIG. 11B shows the transfection efficiency as a function of CS concentration (n=7; 287-908 total cells), according to some embodiments;

[0034] FIG. 11C shows the transfection efficiency plotted against liposome uptake for different CS concentrations, according to some embodiments;

[0035] FIG. 12A shows confocal overview images of HEK cells after two days of incubation with CS-modified liposomes containing different concentrations of GFP- encoding mRNA (scale bar is 50 micrometers), according to some embodiments; and

[0036] FIGS. 12B-12C show transfection efficiency and GFP intensity as a function of mRNA concentration for different concentrations of SUVs (n=86-269 in B and n=10 in C), according to some embodiments.

[0037] DETAILED DESCRIPTION

[0038] Some aspects of the present disclosure are generally related to articles comprising a lipid membrane (e.g., a vesicle) and a glycolipid at least partially embedded within the lipid membrane. In some cases, the glycolipid may be an agonist for a receptor and / or change at least one property of the article comprising the lipid membrane, which may make the article comprising the lipid membrane more suitable for associating with cells and / or being endocytosed by cells. In some cases, the lipid membranes of the article may encapsulate entities, and thus the article may be suitable for drug delivery or other applications when the entities comprise small molecules (i.e., 1000 Da or less), DNA and / or proteins, in some embodiments. Some aspects are related to articles comprising lipid membranes comprising multiple glycolipids, which may impart various properties to the articles, for example, decreased zeta potentials and / or altered surface diffusion rates of lipids of the article. Still other aspects are generally directed to methods of making or using the articles, kits comprising the articles, or the like.

[0039] Articles comprising lipid membranes, e.g., vesicles, are often used for transporting cargo (e.g., DNA, RNA, and / or proteins) within cells and / or to cells. However, in some cases, endocytosis of such articles, and thus delivery of the cargo contained in the articles to cells, may be relatively slow. Moreover, articles comprising lipid membranes generally and uniformly associate with all types of cells, and thus targeted delivery of cargo within the articles to certain types of cells is not possible. Accordingly, some aspects of the present disclosure are generally related to articles comprising such lipid membranes for improved targeting of cells and / or articles that are endocytosed by certain cells at a relatively higher rate compared to the whole population cells.

[0040] FIG. 1A shows one such exemplary embodiment of an article 100. Here, vesicle 110 comprises a lipid membrane 120, wherein glycolipids 130 are partially embedded within the lipid membrane 120 of the vesicle 110. The glycolipids, according to some embodiments, may impart and / or change various properties of the vesicles of the article. However, it should be understood that in other embodiments, other types of articles comprising lipid membranes and / or membranes comprising amphiphilic molecules (e.g., lipid nanoparticles, surfactant- stabilized water-in-oil droplets, polymersomes, etc.) may be present, and / or different types of glycolipids may be partially or fully embedded within the lipid membrane and / or the membrane comprising amphiphilic molecules.

[0041] Still other aspects are related to methods of making and / or using the articles comprising the lipid membranes. In some cases, the lipid membrane of the article may have a substantially uniform thickness and / or a composition consisting of lipids. After forming the lipid membrane, according to some embodiments, the lipid membrane of the article may then be modified by introducing glycolipids. However, in other cases, the lipid membrane of the article may be formed such that its composition comprises lipids and / or glycolipids from the outset. Such lipid membranes of the article may contain (e.g., encapsulate) any of a variety of entities such as small molecules (i.e., less than or equal to 1000 Da), DNA, RNA, or the like, which may make the article suitable for applications such as drug delivery, transfecting cells, and / or other applications. Some aspects pertaining to such methods are described in more detail elsewhere herein. In some embodiments, the article comprises a lipid membrane and a lipid covalently linked to a polysaccharide. In some embodiments, the article comprises a lipid membrane and a sterol covalently linked to a polysaccharide. In some such cases, the sterol is at least partially embedded within the lipid membrane and the polysaccharide is at least partially exposed externally of the lipid membrane. In some such cases, the sterol is essentially completely embedded within the lipid membrane and the polysaccharide is essentially completely exposed externally of the lipid membrane. In some embodiments, the article comprises a vesicle comprising a lipid membrane and a sterol covalently linked to a polysaccharide. In some such cases, the sterol is at least partially embedded within the lipid membrane and the polysaccharide is at least partially exposed externally of the vesicle. In some such cases, the sterol is essentially completely embedded within the lipid membrane and the polysaccharide is essentially completely exposed externally of the vesicle. In some cases, the article comprises a vesicle comprising a lipid membrane and cholesterol covalently linked to chondroitin sulfate. According to some embodiments, the article comprises a vesicle comprising a lipid membrane and cholesterol covalently linked to chondroitin sulfate. In some such cases, the cholesterol is at least partially embedded within the lipid membrane and the chondroitin sulfate is at least partially exposed externally of the vesicle. In some such cases, the cholesterol is essentially completely embedded within the lipid membrane and the chondroitin is essentially completely exposed externally of the vesicle.

[0042] According to some embodiments, the article comprises a lipid membrane and / or a membrane comprising amphiphilic molecules. In some cases, the article comprising the lipid membrane and / or the membrane comprising amphiphilic molecules may be artificially made, as described elsewhere herein. The article comprising the lipid membrane and / or the membrane comprising amphiphilic molecules, according to some embodiments, may be any of a variety of types of articles comprising a lipid membrane and / or membranes comprising amphiphilic molecules. For example, the article comprising the lipid membrane and / or membranes comprising amphiphilic molecules may be a vesicle, small unilamellar vesicle (SUV), a giant unilamellar vesicle (GUV), a multilamellar vesicle, a liposome, a polymersome, a lipid nanoparticle, a surfactant- stabilized water-in-oil droplet, or the like. While some aspects are generally directed to articles comprising lipid membranes and / or membranes comprising amphiphilic molecules, for ease of presentation, much of the present disclosure describes an exemplary article comprising a lipid membrane, i.e., a vesicle. It should be understood, however, that other lipid membrane and / or membranes comprising amphiphilic molecules containing structures are also contemplated.

[0043] In some cases, articles comprising lipid membranes are described. In some embodiments, the article comprises a vesicle comprising a lipid membrane, which in some cases may be a lipid bilayer. The vesicle may be unilamellar, in some cases. The vesicle may be multilamellar, in some embodiments, such that the vesicle may comprise multiple concentric layers, such as two layers, three layers, four layers, and so forth (e.g., “onion” vesicles). For example, the vesicle may comprise a first lipid membrane within a second lipid membrane. The present disclosure generally describes unilamellar vesicles modified with glycolipids and containing an entity, in some cases, for ease of presentation. However, in some embodiments, multiple lipid membranes may be present instead of a unilamellar vesicle, e.g., where each lipid membrane may be modified with at least one glycolipid and / or each layer may contain an entity therein. In some embodiments, the entity contained within each layer may be the same or different from the entities contained within other layers. In other embodiments, only the outer most vesicle layer may comprise a glycolipid.

[0044] The vesicle may comprise any of a variety of lipids, in some embodiments. Exemplary lipids may be selected from different types of lipid classes which include, but are not limited to, phospholipids, phosphoglycerides, sterols, and sphingolipids. Other lipids are also possible. The lipid membrane of the vesicle may consist of a single type of lipid, in some cases. In some embodiments, the lipid membrane of the vesicle may comprise multiple lipids. Additionally, not all of the lipids present in the lipid membrane of the vesicle may be precisely known, in accordance with some embodiments.

[0045] The vesicle may have any of a variety of shapes, in accordance with some embodiments. In some embodiments, the vesicle is substantially spherical at equilibrium when no net forces are present. In some embodiments, the shape of the vesicle may be elongated, tubulated, and / or stretched, for example, when a force is applied anisotropically to the vesicle.

[0046] The vesicle may be any of a variety of sizes, in some embodiments. In some cases, relatively large vesicles may be used such that the interior volume of the vesicle is relatively large compared to small vesicles, which in some cases may facilitate the encapsulation of a larger number of entities when compared to relatively small vesicles, as described in more detail elsewhere herein. In some embodiments, relatively small vesicles may be used, which may lead to vesicles with higher diffusion coefficients and / or uptake coefficients, when compared to relatively larger vesicles.

[0047] While described above and elsewhere herein as a single vesicle, it is contemplated that there may be a plurality of vesicles in certain embodiments. The plurality of vesicles may comprise any number of vesicles, in accordance with some embodiments. In some cases, the vesicles may be present in a concentration of greater than or equal to 1 picomole / liter, greater than or equal to 10 picomole / liter, greater than or equal to 100 picomole / liter, greater than or equal to 1 nanomole / liter, greater than or equal to 10 nanomole / liter, greater than or equal to 100 nanomole / liter, greater than or equal to 1 micromole / liter, greater than or equal to 10 micromole / liter, greater than or equal to 100 micromole / liter, greater than or equal to 1 millimole / liter, or greater than or equal to 10 millimole / liter. In some embodiments, the vesicles may be present in a concentration of less than or equal to 100 millimole / liter, less than or equal to 10 millimole / liter, less than or equal to 1 millimole / liter, less than or equal to 100 micromole / liter, less than or equal to 10 micromole / liter, less than or equal to 1 micromole / liter, less than or equal to 100 nanomole / liter, less than or equal to 10 nanomole / liter, less than or equal to 1 nanomole / liter, less than or equal to 100 picomole / liter, or less than or equal to 10 picomole / liter. Combinations of the foregoing ranges are possible. Other ranges are also possible.

[0048] In accordance with some embodiments, the average diameter of a vesicle in a plurality of vesicles is less than or equal to 100 micrometers, less than or equal to 50 micrometers, less than or equal to 10 micrometers, less than or equal to 1 micrometer, less than or equal to 800 nanometers, less than or equal to 600 nanometers, less than or equal to 400 nanometers, less than or equal to 200 nanometers, less than or equal to 100 nanometers, less than or equal to 60 nanometers, or less than or equal to 50 nanometers. In some cases, the average diameter of a vesicle of a plurality of vesicles is greater than or equal to 30 nanometers, greater than or equal to 50 nanometers, greater than or equal to 60 nanometers, greater than or equal to 100 nanometers, greater than or equal to 200 nanometers, greater than or equal to 400 nanometers, greater than or equal to 600 nanometers, greater than or equal to 800 nanometers, greater than or equal to 1 micrometer, greater than or equal to 10 micrometers, or greater than or equal to 50 micrometers. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 30 nanometers and less than or equal to 200 nanometers, greater than or equal to 30 nanometers and less than or equal to 1 micrometer, greater than or equal to 30 nanometers and less than or equal to 100 micrometers). Other ranges are also possible. According to some embodiments, relatively small vesicles (e.g., a plurality of vesicles having an average diameter of less than or equal to 1 micron, less than or equal to 200 nanometers, and / or less than or equal to 100 nanometers) may be advantageous because they may be uptaken by cells at a higher rate than relatively large vesicles (e.g., a plurality of vesicles having an average diameter of greater than 1 micron). In some embodiments, the articles comprising vesicles may comprise a glycolipid. In some cases, the glycolipid may comprise a lipid covalently linked to a polysaccharide, e.g., a lipid- linked polysaccharide. In accordance with some embodiments, the glycolipid comprises a sterol covalently linked to a polysaccharide, e.g., a sterol-linked polysaccharide. The glycolipid, in some embodiments, may comprise cholesterol covalently linked to chondroitin sulfate, e.g., a cholesterol-linked chondroitin sulfate. In the glycolipid, the lipid and the polysaccharide may be linked in any of a variety of methods. For example, in some cases, the lipid and the polysaccharide may be covalently linked. Any of a variety of chemistries may be suitable for covalently linking the lipid to the polysaccharide. Exemplary chemistries include click chemistry, substitution reactions (e.g., NHS-chemistry), elimination reactions, or the like. Other chemistries for linking the lipid and the polysaccharide are also possible.

[0049] The lipid in the glycolipid may be any of a variety of suitable lipids, in accordance with some embodiments. Exemplary lipids may be selected from different types of lipid classes which include, but are not limited to, steroids, ceramides, sphingolipid, diglycerides, phospholipids, fatty acids, and triglycerides. For example, in some embodiments, the lipid may comprise a sterol such as cholesterol, dihydroceramides, 6-hydroxyceramids, phytoceramides, acylceramides, sphingomyelin, and / or a sphingosine. Other lipids are also possible. The lipid of the glycolipid may consist of a single type of lipid, in some cases. In some embodiments, the lipid of the glycolipid may comprise multiple lipids. For example, in some cases, a first glycolipid may comprise a sterol whereas a second glycolipid may comprise a ceramide, where both the first and second glycolipids may embed into a lipid membrane of a single vesicle. In accordance with some embodiments, and as described elsewhere herein, the lipids of the glycolipid may be configured such that, in the presence of a vesicle, it may spontaneously self-assemble into the lipid membrane of the vesicle. In some embodiments, the lipid of the glycolipid may be different from the lipid(s) of the lipid membrane of the vesicle. In other cases, the lipid of the glycolipid may be the same as at least one of the lipids of the vesicle.

[0050] The polysaccharide in the glycolipid may be any of a variety of suitable polysaccharides, in accordance with some embodiments, such as storage polysaccharides, heteropolysaccharides and / or homopolysaccharides. In some embodiments, the polysaccharide may be chondroitin sulfate, chitin, hyaluronic acid, heparin, starch (e.g., amylose), glycogen, galactogen, inulin, cellulose, pectin, and / or glycogen. Other polysaccharides are also possible.

[0051] According to some embodiments, any lipids and / or polysaccharides of the glycolipid may be functionalized. Such modifications to the lipids and / or polysaccharides include functionalizing the structure of the lipids and / or polysaccharides to comprise additional functional groups or molecular moieties (e.g., leaving groups), including, but not limited to, ethers, esters, carboxyl groups, amides, amines, alcohols, alkenes, alkynes, alkanes, or the like. Such modifications to the lipids and / or polysaccharides may facilitate the covalent bonding between the polysaccharide and / or the lipid, and / or may change the properties of the resulting glycolipid and thus the article (e.g., vesicle) comprising the glycolipid.

[0052] According to some embodiments, the article may comprise a vesicle comprising a lipid membrane and a glycolipid (e.g., a sterol-linked polysaccharide, etc.) as described elsewhere herein at least partially embedded within the vesicle. In some cases, the article comprises a vesicle comprising a lipid membrane wherein a sterol is at least partially embedded within the lipid membrane and a polysaccharide is covalently linked to the sterol and is at least partially exposed externally of the vesicle. In accordance with some such embodiments, the sterol is essentially completely embedded within the lipid membrane and / or the polysaccharide is essentially completely exposed externally of the vesicle. In some embodiments, there may be a plurality of vesicles such that at least a portion of the vesicles comprises at least one glycolipid (e.g., a sterol-linked polysaccharide, etc.). In some embodiments, at least a portion of the plurality of vesicles comprises at least one sterol-linked polysaccharide. In some embodiments, essentially each of the plurality of vesicles comprises at least one sterol-linked polysaccharide. The average number of glycolipids (e.g., a sterol-linked polysaccharide, etc.) on each vesicle in the plurality of vesicles may vary based on any of a variety of parameters, according to some embodiments, for example, the parameters used when making the vesicles, the vesicle size, the polysaccharide size, the zeta potential of the vesicle, or the like.

[0053] The average number of glycolipids on each vesicle of the plurality of vesicles may be determined by any of a variety of suitable techniques, according to some embodiments. The average number of glycolipids on each vesicle of the plurality of vesicles may be approximated by confocal microscopy, in some embodiments. Other techniques that can be used include, but are not limited to, dynamic light scattering, atomic force microscopy or mass spectrometry, e.g., to determine the polysaccharide density on the membrane. In some cases, the average number of glycolipids on each vesicle of the plurality of vesicles may be greater than or equal to 1, greater than or equal to 10, greater than or equal to 100, greater than or equal to 1,000, or greater than or equal to 10,000. In some embodiments, the average number of glycolipids on each vesicle of the plurality of vesicles may be less than or equal to 100,000, less than or equal to 10,000, less than or equal to 1,000, less than or equal to 100, or less than or equal to 10. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 1 and less than or equal to 100,000). Other ranges are also possible.

[0054] In some cases, the maximum average number of glycolipids present on each vesicle of the plurality of vesicles may depend on the size of the vesicle. For example, in some embodiments, approximately 1 glycolipid may be embedded for every 10 lipids present in the lipid membrane of the vesicle, yielding an approximate average ratio of glycolipids to lipids in the lipid membrane of each vesicle of 1:10. In some cases, the average ratio of glycolipids to lipids in the lipid membrane of each vesicle may be higher than 10 (e.g., greater than or equal to 1:10, greater than or equal to 1:5, greater than or equal to 1:2, or the like) or lower than 10 (e.g., less than or equal to 1:10, less than or equal to 1:20, less than or equal to 1:50, less than or equal to 1:100, less than or equal to 1:1,000, less than or equal to 1:10,000), for example, based on the size of the glycolipid, the size of the lipid, and / or the concentration of glycolipids in solution when forming the articles. When determining such ratios, the average number of lipids present in the lipid membrane of each vesicle may be estimated from the size of the vesicle and the identity of the lipid, in accordance with some embodiments. Accordingly, in some cases, the number of glycolipids possible on each vesicle of a plurality of vesicles is dependent on, among other factors, the size of the vesicles.

[0055] In some embodiments, the total average number glycolipids on each vesicle of the plurality of vesicles may consist of a single type of glycolipid. In other cases, the total average number glycolipids on each vesicle of the plurality of vesicles may comprise multiple types of glycolipids, e.g., at least two types of glycolipids, at least three types, at least four types, and so forth.

[0056] In some embodiments, essentially all of the polysaccharide of each glycolipid may be present external of the lipid membrane, and thus the vesicle may be considered asymmetric. In some cases, on a single vesicle, at least a portion of a first polysaccharide of a first glycolipid may be present external of the lipid membrane and at least a portion of a second polysaccharides of a second glycolipid may be present internal of the lipid membrane. In some such cases, the vesicle comprising the lipid membrane may be considered symmetric.

[0057] According to some embodiments, the vesicle may further comprise an entity within the lipid membrane. In some cases, the vesicle may comprise a lipid membrane and a glycolipid comprising a sterol covalently linked to a polysaccharide. In some such cases, the sterol is at least partially embedded within the lipid membrane, the polysaccharide is at least partially exposed externally of the vesicle, and an entity is present within the lipid membrane.

[0058] Any of a variety of entities may be contained within the vesicle, in accordance with some embodiments, given the size of the entity is suitable to be contained within the vesicle. In some embodiments, the entity may comprise one or multiple different types of species, including, but not limited to, small molecules (i.e., less than or equal to 1000 Da), RNA (e.g., mRNA), DNA, proteins, peptides, polymers, vesicles, nanoparticles, etc. In some embodiments, the entities within the vesicle may consist of a single type of entity. In other cases, the entities within the vesicle may comprise multiple types of entities, for example, DNA and nanoparticles. In some cases, the identity of the one or more type of entities may determine a potential function for the articles. For example, an article comprising a vesicle a containing a DNA molecule may be suitable for transfecting cells, in accordance with some embodiments. In some such cases, the article may be introduced to a cell or other organism and be endocytosed such that the DNA may enter the cell and be amplified (e.g., transfect). In some embodiments, wherein the entity comprises a small molecule (i.e., less than or equal to 1000 Da), the vesicle may be suitable for drug delivery.

[0059] The entities may be present at any of a variety of concentrations within the vesicles, in accordance with some embodiments, which may be selected by the composition of the solution in which the vesicles are first formed. For example, if the vesicles are made in a solution comprising 1 micromolar of DNA molecules (or other entities), the interior solution of the resulting vesicles may have a concentration of DNA (or other entities) of 1 micromolar. In some embodiments, the entities may be present within the vesicles at a concentration of greater than or equal to 1 picomole / liter, greater than or equal to 10 picomole / liter, greater than or equal to 100 picomole / liter, greater than or equal to 1 nanomole / liter, greater than or equal to 10 nanomole / liter, greater than or equal to 100 nanomole / liter, greater than or equal to 1 micromole / liter, greater than or equal to 10 micromole / liter, greater than or equal to 100 micromole / liter, greater than or equal to 1 millimole / liter, or greater than or equal to 10 millimole / liter. In some embodiments, the entities may be present within the vesicles at a concentration of less than or equal to 100 millimole / liter, less than or equal to 10 millimole / liter, less than or equal to 1 millimole / liter, less than or equal to 100 micromole / liter, less than or equal to 10 micromole / liter, less than or equal to 1 micromole / liter, less than or equal to 100 nanomole / liter, less than or equal to 10 nanomole / liter, less than or equal to 1 nanomole / liter, less than or equal to 100 picomole / liter, or less than or equal to 10 picomole / liter. Combinations of the foregoing ranges are possible. Other ranges are also possible.

[0060] An article comprising a vesicle comprising a lipid membrane and a glycolipid (e.g., a sterol-linked polysaccharide) may have different properties than a substantially identical vesicle in the absence of the glycolipid. In some cases, the presence of the glycolipid may impart such different properties. In some embodiments, the identity and / or the surface concentration of glycolipids on each vesicle may affect the properties of the vesicle. In some such cases, where there are two or more different types of glycolipids, the properties of the resulting vesicle comprising the glycolipids may change based on the identity, surface concentration, and / or relative surface concentrations of each type of glycolipid at least partially embedded in the lipid membrane of the vesicle.

[0061] For example, in some embodiments, the presence of the glycolipid may decrease the zeta potential of the vesicle compared to vesicles in the absence of the glycolipid. In some cases, the zeta potential may be measured using a zeta potential analyzer. According to some embodiments, the average zeta potential of a plurality of vesicles is less than or equal to 10 mV, less than or equal to 5 mV, less than or equal to 0 mV, or less than or equal to -5 mV. In some cases, the average zeta potential of a plurality of vesicles is greater than or equal to -10 mV, greater than or equal to -5 mV, greater than or equal to 0 mV, or greater than or equal to 5 mV. Combinations of the foregoing ranges are possible (e.g., greater than or equal to -10 mV and less than or equal to 0 mV). Other ranges are also possible.

[0062] The lipid diffusion coefficient on the surface of each vesicle may be relatively low for vesicles comprising a glycolipid (e.g., a sterol-linked polysaccharide), in accordance with some cases. In some embodiments, the lipid diffusion coefficient may be determined using fluorescence recover after photobleaching (FRAP) experiments. In some cases, the average lipid diffusion coefficient on the surface of each vesicle of a plurality of vesicles is less than or equal to 2.8 micrometers2 / second, less than or equal to 2.6 micrometers2 / second, less than or equal to 2.4 micrometers2 / second, less than or equal to 2.2 micrometers2 / second, less than or equal to 2.1 micrometers2 / second, less than or equal to 2 micrometers2 / second, or less than or equal to 1.8 micrometers2 / second. In some cases, the average lipid diffusion coefficient on the surface of each vesicle of a plurality of vesicles is greater than or equal to 1.6 micrometers2 / second, greater than or equal to 1.8 micrometers2 / second, greater than or equal to 2 micrometers2 / second, greater than or equal to 2.1 micrometers2 / second, greater than or equal to 2.2 micrometers2 / second, greater than or equal to 2.4 micrometers2 / second, or greater than or equal to 2.6 micrometers2 / second. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 2 micrometers2 / second and less than or equal to 2.6 micrometers2 / second). Other ranges are also possible. According to some embodiments, vesicles comprising a glycolipid (e.g., a sterol- linked polysaccharide) may be relatively flexible when compared to a vesicle in the absence of the glycolipid. Flexibility, in some cases, may facilitate the vesicle being deformed from an equilibrium state (e.g., substantially spherical) due to the application of a force without the vesicle permanently degrading and / or otherwise deforming the vesicle and thus releasing any entities within the vesicle. The flexibility of the vesicles may be determined, in some cases, by measuring micropipette aspiration, and may be represented by a stretching modulus of the vesicle. In some cases, the average stretching modulus of a vesicle of a plurality of vesicles is less than or equal to 250 mN / m, less than or equal to 225 mN / m, less than or equal to 200 mN / m, or less than or equal to 175 mN / m. In some embodiments, the average stretching modulus of a vesicle of a plurality of vesicles is greater than or equal to 150 mN / m, greater than or equal to 175 mN / m, greater than or equal to 200 mN / m, or greater than or equal to 225 mN / m. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 175 mN / m and less than or equal to 225 mN / m). Other ranges are also possible.

[0063] In some cases, micropipette aspiration may involve applying a suction pressure of 1-2 millibar into the glass capillary of ~7 micrometers in diameter. The low pressures required to aspirate the vesicle into the pipette indicate that the membrane is fluid-like. By increasing the suction pressure, vesicles may be deformed from their spherical shape and aspirated into the capillary. From the tongue length within the glass capillary at a given suction pressure, the membrane tension and area expansion can be determined. By plotting the membrane tension as a function of the area expansion, the stretching modulus of the membrane can be determined. The stretching modulus may be the slope of a linear fit for tensions greater than 1 mN / m and may be representative of membrane compressibility under tension.

[0064] Flexibility of the vesicle, in some embodiments, may be represented by a bending rigidity. In some embodiments, bending rigidity may be similarly determined from micropipette aspiration as to the stretching modulus by plotting the membrane tension as a function of the area expansion. However, the bending rigidity may be determined from the slope of a linear fit on a log scale from the plot for tensions smaller than 1 mN / m.

[0065] In some embodiments, a bending rigidity of a vesicle comprising a glycolipid (e.g., a sterol-linked polysaccharide) may be reduced relative to a vesicle absent the glycolipid. The average bending rigidity of a vesicle of a plurality of vesicles, in some embodiments, is less than or equal to 40 I<BT. less than or equal to 35 I<BT. less than or equal to 30 I<BT. less than or equal to 25 I<BT. less than or equal to 20 I<BT. less than or equal to 15 I<BT. or less than or equal to 10 I<BT. In some embodiments, the average bending rigidity of a vesicle of a plurality of vesicles is greater than or equal to 5 I<BT. greater than or equal to 10 I<BT. greater than or equal to 15 I<BT. greater than or equal to 20 I<BT, greater than or equal to 25 I<BT. greater than or equal to 30 I<BT. or greater than or equal to 35 I<BT. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 5 I<BT and less than or equal to 40 I<BT. greater than or equal to 10 I<BT and less than or equal to 20 I<BT . Other ranges are also possible.

[0066] Altering the physical properties of the vesicles by introducing glycolipids (e.g., sterol-linked polysaccharides), in some embodiments, may increase the rate at which cells uptake (e.g., by endocytosis) the vesicles. For example, without wishing to be bound by theory, it is believed that the lowered zeta potential (e.g., a less positive and / or more negative zeta potential) of the vesicles comprising glycolipids compared to vesicles in the absence of glycolipids may facilitate the interaction and / or association between vesicles and cells. Furthermore, in some embodiments, the identity of the polysaccharide of the glycolipid may facilitate the association of the vesicle with cells, for instance, by the polysaccharide acting as an agonist of a receptor located on cells. In some such cases, at least a portion of the polysaccharide of the glycolipid that is external of the vesicle is configured to associate with a cell, for instance, a receptor on a cell. In some embodiments, the polysaccharide may act as an agonist for a receptor, wherein the specific type of receptor is only present on a subset of cells (e.g., blood cells, muscle cells, bone cells, neural cells, fat cells, cancer cells, etc.). Accordingly, in some such embodiments, the vesicles comprising the glycolipids comprising the polysaccharides may associate more strongly with the subset of cells compared to the full population of cells, and thus the vesicles may be uptaken, in some embodiments, at a higher rate by the subset of cells relative to the whole population of cells.

[0067] In some cases, when a plurality of vesicles is introduced to a population of cells, vesicles comprising a glycolipid may be more efficiently uptaken by the cells than vesicles in the absence of a glycolipid. In some such cases, using vesicles may provide a route for introducing molecules into a cell which may not regularly be uptaken by the cell. In some embodiments, the vesicles may be uptaken by each cell at a rate of greater than or equal to 10 vesicles / day, greater than or equal to 100 vesicles / day, greater than or equal to 1,000 vesicles / day, greater than or equal to 5,000 vesicles / day. In some cases, the vesicles may be uptaken by each cell at a rate of less than or equal to 10,000 vesicles / day, less than or equal to 5,000 vesicles / day, less than or equal to 1,000 vesicles / day, or less than or equal to 100 vesicles / day. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 100 and less than or equal to 5,000 vesicles / day). Other ranges are also possible, and may be dependent on the concentration of cells and / or concentration of vesicles.

[0068] In some embodiments, because the vesicles comprising glycolipids are uptaken by cells at relatively high rates compared to vesicles in the absence of glycolipids, any cargo contained by the vesicles may be delivered to the cells at a relatively high rate, e.g., at a rate less than or equal to the uptake rate for applications such as drug delivery and / or transfecting the cells. In some cases, delivery of any cargo for transfection via vesicles comprising glycolipids may lead to relatively high rates of cell transfection compared to using vesicles in the absence of glycolipids. For example, in some cases, average transfection efficiency of a plurality of vesicles is greater than or equal to 0.01, greater than or equal to 0.02, greater than or equal to 0.03, greater than or equal to 0.04, greater than or equal to 0.05, greater than or equal to 0.06, greater than or equal to 0.07, greater than or equal to 0.08, greater than or equal to 0.09, greater than or equal to 0.1, greater than or equal to 0.15, greater than or equal to 0.2, greater than or equal to 0.25, greater than or equal to 0.3, greater than or equal to 0.35, greater than or equal to 0.4, greater than or equal to 0.45, greater than or equal to 0.5, greater than or equal to 0.55, greater than or equal to 0.6, greater than or equal to 0.65, greater than or equal to 0.7, greater than or equal to 0.75, greater than or equal to 0.8, greater than or equal to 0.85, greater than or equal to 0.9, greater than or equal to 0.95, etc. In some cases, the average transfection efficiency of a plurality of vesicles is less than or equal to 1, less than or equal to 0.9, less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, less than or equal to 0.5, less than or equal to 0.45, less than or equal to 0.4, less than or equal to 0.35, less than or equal to 0.3, less than or equal to 0.25, less than or equal to 0.2, less than or equal to 0.15, less than or equal to 0.1, less than or equal to 0.1, less than or equal to 0.09, less than or equal to 0.08, less than or equal to 0.07, less than or equal to 0.06, less than or equal to 0.05, less than or equal to 0.04, less than or equal to 0.03, or less than or equal 0.02. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 0.03 and less than or equal to 0.07, greater than or equal to 0.03 and less than or equal to 0.3, greater than or equal to 0.7 and less than or equal to 1, greater than or equal to 0.1 and less than or equal to 0.5, or the like). Other ranges are also possible.

[0069] The vesicles may be formed through any of a variety of methods, in accordance with some embodiments. For example, forming the plurality of vesicles may comprise electroforming the plurality of vesicles. In some cases, the vesicles may be formed by hydration and / or swelling of thin lipid films. In some such cases, vesicles may be disrupted and / or agitated by sonicating the solution comprising the vesicles. In some embodiments, a solution comprising lipids may be mixed (e.g., by a stir bar, a vortex machine, etc.) to spontaneously form the vesicles. Other methods for forming the vesicles are also possible.

[0070] Electroforming vesicles comprises applying an AC electric field to solution comprising the constituent lipid of the resulting lipid membrane, according to some embodiments. In some cases, the voltage of the AC electric field is greater than or equal to 1 V, greater than or equal to 1.5 V, greater than or equal to 2 V, greater than or equal to 2.5 V, greater than or equal to 3 V, greater than or equal to 3.5 V, greater than or equal to 4 V, or greater than or equal to 4.5 V. In some embodiments, the voltage is less than or equal to 5 V, less than or equal to 4.5 V, less than or equal to 4 V, less than or equal to 3.5 V, less than or equal to 3 V, less than or equal 2.5 V, less than or equal to 2 V, or less than or equal to 1.5 V. Combinations of the foregoing ranges are possible. Other ranges are also possible.

[0071] In some embodiments, the frequency of the AC electric field during electroforming is greater than or equal to 1 Hz, greater than or equal to 2 Hz, greater than or equal to 3 Hz, greater than or equal to 4 Hz, greater than or equal to 5 Hz, greater than or equal to 6 Hz, greater than or equal to 7 Hz, greater than or equal to 8 Hz, or greater than or equal to 9 Hz. In some embodiments, the frequency of the AC electric field is less than or equal to 10 Hz, less than or equal to 9 Hz, less than or equal to 8 Hz, less than or equal to 7 Hz, less than or equal to 6 Hz, less than or equal to 5 Hz, less than or equal to 4 Hz, less than or equal to 3 Hz, or less than or equal to 2 Hz. Combinations of the foregoing ranges are possible. Other ranges are also possible. The vesicles may be electroformed for any of a variety of time periods. For example, in some cases, the vesicles may be electroformed for greater than or equal to 10 min, greater than or equal to 30 min, greater than or equal to 1 hour, greater than or equal to 2 hours, greater than or equal to 3 hours, or greater than or equal to 4 hours. In some cases, the vesicles may be electroformed for less than or equal to 5 hours, less than or equal to 4 hours, less than or equal to 3 hours, less than or equal to 2 hours, less than or equal to 1 hour, or less than or equal to 30 minutes. Combinations of the foregoing ranges are possible. Other ranges are also possible.

[0072] The vesicles may then be passed through an extruder (e.g., a membrane, multiple membranes). Extruders may have any of a variety of pore sizes, in accordance with some embodiments, to obtain vesicles having average diameters corresponding to the pore size of the extruder. For example, in some cases the average pore size of the extruder may be less than or equal to 1 micrometer, less than or equal to 800 nanometers, less than or equal to 600 nanometers, less than or equal to 400 nanometers, less than or equal to 200 nanometers, less than or equal to 100 nanometers, or less than or equal to 60 nanometers. In some cases, it may be beneficial to pass the vesicles through the extruder multiple times and / or pass the vesicles through multiple extruders to obtain narrow size distributions of the vesicles and / or vesicles having average diameters in a specific range. For instance, in some embodiments, the vesicles may be passed through an extruder having an average pore size of less than or equal to 800 nm, wherein the remaining vesicles are passed through an extruder having an average pore size of less than or equal to 200 nm. In some such cases, the vesicles retained at the second extruder may have an average diameter of greater than or equal to 200 nm and less than or equal to 800 nm and the vesicles passed through the second membrane may have a diameter of less than or equal to 200 nm. Of course, obtaining vesicles having different size distributions are possible using extruders having different average pore sizes.

[0073] In addition, other techniques may be used in other embodiments. For example, in some embodiments, hydration and / or swelling may be used to generate vesicles such as multilamellar and polydisperse liposomes. These may have sizes, for example, of at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1 micrometer, at least 2 micrometers, at least 3 micrometers, at least 4 micrometers, at least 5 micrometers, etc., and / or no more than 5 micrometers, no more than 4 micrometers, no more than 3 micrometers, no more than 2 micrometers, no more than 1 micrometer, no more than 900 nm, no more than 800 nm, no more than 700 nm, no more than 600 nm, no more than 500 nm, no more than 400 nm, no more than 300 nm, no more than 200 nm, no more than 100 nm, no more than 50 nm, no more than 30 nm, no more than 20 nm, no more than 10 nm, etc. Combinations of any of these ranges are also possible in certain embodiments.

[0074] In some cases, the vesicles may be formed in the presence of the glycolipid (e.g., a sterol-linked polysaccharide, or the like) as described elsewhere herein. In some such embodiments, the lipid portion of the glycolipid may spontaneously insert itself into the lipid bilayer of the vesicle. Accordingly, in cases wherein there are multiple glycolipids on each vesicle, a first saccharide of a first glycolipid may be internal of the lipid membrane and a second saccharide of a second glycolipid may be external of the lipid membrane, therefore forming symmetric vesicles. In some cases, the vesicles are formed in a first solution, and then the first solution is mixed with a second solution comprising a glycolipid (e.g., a sterol-linked polysaccharide, or the like). In some such cases, at least a portion of the lipid of the glycolipid may spontaneously self-assemble into the lipid membrane of the vesicle, thus leaving the polysaccharide portion of the glycolipid external of the lipid membrane and forming asymmetric vesicles. In some such embodiments, the glycolipid comprises cholesterol covalently linked to chondroitin sulfate. In some cases, at least a portion of the lipid of the glycolipid may self-assemble, e.g., spontaneously, into the lipid membrane of the vesicle such that the polysaccharide portion of the glycolipid is internal to the lipid membrane and thus forming asymmetric vesicles.

[0075] In accordance with some embodiments, the degree to which a glycolipid is introduced into a membrane of a vesicle may be influenced by the concentration of glycolipid present in solution when introduced to the vesicles. For instance, a first solution containing the vesicles may be introduced to a second solution containing a glycolipid, where the concentration of the glycolipid may alter the degree to which the glycolipid is incorporated into the vesicle membranes and thus alter the properties of the vesicles. In accordance with some embodiments, the portion of the glycolipid that is not within the membrane of the vesicles (e.g., the polysaccharide portion, chondroitin sulfate) may be charged, and thus it is believed the repulsion between adjacent glycolipids within the membrane may limit the amount of glycolipids incorporated into the vesicles and the resulting properties of the modified vesicles. In accordance with some embodiments, the second solution may further include a salt comprising cations (e.g., monovalent cations such as Na+and K+, divalent cations such Mg2+and Ca2+), which may facilitate the incorporation of more glycolipids into the membrane of the vesicles relative to when the glycolipid is introduced in the absence of salts. In some embodiments, higher valency salts (e.g., Mg2+vs Na+) may advantageously facilitate further incorporation of glycolipids in the vesicle membrane. Additional glycolipid in the vesicle membranes, in some embodiments, may further alter the mechanical properties of the vesicles relative to when there is no glycolipid present in the vesicle membrane.

[0076] In some embodiments, at least a portion of the polysaccharide of the glycolipid external of the vesicle is configured to associate with a cell. For example, in some cases, at least a portion of the polysaccharide of the glycolipid is an agonist for a receptor located on a cell, and thus vesicles comprising the glycolipid may more closely associate with cells comprising the receptor for which the at least the portion of the polysaccharide of the glycolipid is an agonist. As described elsewhere herein, in some embodiments, multiple types of glycolipids may be present on the vesicles, and thus the vesicles, in some cases, may comprise agonists for multiple types of receptors and may accordingly associate with different types of cells.

[0077] Directed association between the vesicles and specific subsets of cells may facilitate higher uptake rates of the vesicles by the specific subsets of the cells. According to some such embodiments, the higher uptake rates by specific subsets of cells may facilitate the delivery of any entities within the vesicles to the specific subset of cells, such as small molecules (i.e., less than or equal to 1000 Da) and / or DNA for drug delivery and / or transfecting cells, respectively.

[0078] In some embodiments, where the vesicles are configured to be used as drug delivery articles, the vesicles may be administered to a patient by injection (e.g., intravenous, intramuscular, etc.), ingestion, topically, or any other suitable method for introducing the vesicles to a desired region of a patient. The vesicles may be present within a delivery solution, a capsule, a lotion, or the like. U.S. Provisional Patent Application Serial No. 63 / 517,528, filed August 3, 2023, by Weitz, et al., is incorporated herein by reference in its entirety.

[0079] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0080] EXAMPLE 1

[0081] The following describes an exemplary embodiment of the articles described herein.

[0082] As shown in FIG. IB, fluorescently labelled, amine-functionalized chondroitin sulfate 134 was crosslinked (e.g., covalently bound) with NHS-cholesterol 132 to form cholesterol-linked chondroitin sulfate 130. This was done by introducing 100 mg of cholesterol-PEG-NHS to 1 mL of solution containing 1 mM amine-chondroitin sulfate- FITC and 0.1 M NaHCOa, and then stirring the solution overnight (e.g., approximately 12 hours). The solution was then dialyzed against deionized water for 72 hours to obtain the cholesterol-linked chondroitin sulfate 130.

[0083] Vesicles (e.g., giant unilamellar vesicles) were then electroformed in 270 mM sucrose using a Vesicle Prep Pro device, in a manner known to those of ordinary skill in the art. For example, 30 microliters of a lipid mix (99 mol% DOPC and 1 mol% Atto633-DOPE dissolved in chloroform at a concentration of 5 mM) were spread homogeneously on the conductive site of a first indium-tin-oxide (ITO) electrode. The lipid-film-coated ITO slide was desiccated for 15 minutes to remove remaining chloroform, whereafter a rubber ring was placed on top of the lipid-coated ITO slide. The rubber ring was then was filled with approximately 270 microliters of an aqueous sucrose solution (270 mM) and formed a sealed chamber by placing a second ITO electrode on top of the rubber ring. The electrodes were then used to generate an AC field of 3 V at 5 Hz for 2 hours at 37 degrees C to form the vesicles.

[0084] A solution comprising 90 microliters of cholesterol-linked chondroitin sulfate and 270 mM sucrose was then mixed with 10 microliters of the vesicle solution. The solution was allowed to incubate for 20 minutes, wherein the cholesterol-linked chondroitin sulfate spontaneously self-assembled by hydrophobic forces into the lipid membrane of the vesicles to form the articles comprising vesicles comprising a lipid membrane wherein the cholesterol-linked chondroitin sulfate is at least partially embedded within the lipid membrane, as shown as article 100 in FIG. 1A. Due to the fluorescent label of the amine-functionalized chondroitin sulfate, the location of the cholesterol-linked chondroitin sulfate relative to the vesicles can be tracked by fluorescence microscopy. For example, FIG. 2A shows a fluorescence micrograph of vesicles in a solution comprising the fluorescently labelled cholesterol- linked chondroitin sulfate (scale bar is 50 microns). The micrograph shows the cholesterol-linked chondroitin sulfate is present outside and along the periphery of the vesicles, but not within the vesicles. FIG. 2B shows a plot of the ratio of (fluorescence intensity along the periphery of a vesicle) / (fluorescence intensity in solution) as a function of cholesterol-linked chondroitin sulfate (Chol-CS) in solution. The plot shows the ratio is higher at low concentrations of Chol-CS, indicating the Chol-CS is in higher concentrations in the periphery of the vesicles and suggest the Chol-CS preferentially partitions into the lipid membrane of the vesicle rather than stay in solution. Moreover, FIG. 2C shows negative stain electron microscopy images of vesicles in the absence and presence of the cholesterol-linked chondroitin sulfate (scale bar is 150 nm for the unmodified vesicles and 100 nm for the vesicles comprising Chol-CS). The images show the cholesterol-linked chondroitin sulfate forms a visible layer outside the lipid membrane of the vesicle. This indicates the formation of asymmetric vesicles.

[0085] Fluorescence recovery after photobleaching (FRAP) experiments were performed to determine the rate of diffusion within the lipid membranes of the vesicles. FIG. 3 A shows fluorescence micrographs of a vesicle (i) before, (ii) immediately after photobleaching, and (iii) after recovery of the fluorescence during a FRAP experiment (scale bar is 10 microns), and FIG. 3B is a plot of fluorescence intensity of the dotted circle in (ii) as a function of time throughout the experiment. FIG. 3B shows the lipids in the lipid membrane, and thus the Chol-CS, diffuse over time to recover any fluorescence that was lost during photobleaching.

[0086] A similar FRAP experiment as shown in FIG. 3 is shown in FIG. 4. FIG. 4A shows the three structures of vesicles tested. Each structure has a lipid bilayer comprising l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The second structure further contains cholesterol, whereas the third further contains Chol-CS. Exemplary micrographs of the FRAP experiment are shown in FIG. 4B (scale bar is 10 microns), which were performed to determine the diffusion coefficient of the lipids within the lipid membranes. FIG. 4C shows the average diffusion coefficient of samples tested having each of the three structures shown in FIG. 4A. Here, the diffusion coefficient of lipids within the membranes of vesicles comprising the Chol-CS is decreased relative to the diffusion coefficient of lipids within the membranes of vesicles in the absence of the Chol-CS. A similar experiment was performed where lacto syl-phosphoethanolamine (i.e., l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl (ammonium salt); lactosyl-PE) was used in place of the Chol-CS. FIG. 4D shows the diffusion coefficients of lipids within the membranes of vesicles in this case with varying amounts of lactosyl- PE remains approximately the same. Accordingly, small carbohydrates directly couples to phospholipids and in the absence of cholesterol do not change lipid diffusion in the membrane of the vesicles. This further indicates the decrease of lipid diffusion seen in FIG. 4C is related to the insertion of cholesterol into the membrane of the vesicle, and not the presence of the polysaccharide.

[0087] The stretching modulus as a function of vesicle composition was then measured using micropipette aspiration. As shown the images shown in FIG. 5A (scale bar is 5 microns), a micropipette was brought into contact with the vesicles in each case, negative pressures were applied via the micropipettes, and the stretching of the vesicle was used to determine the stretching modulus for each vesicle composition. FIG. 5B shows the average stretching moduli recorded for each composition of vesicle and shows vesicles comprising the Chol-CS have lower average stretching moduli than the vesicles tested in the absence of Chol-CS.

[0088] A second experiment to test the stretching moduli of vesicle compositions was performed in FIG. 6A, performed similarly as in FIG. 5A, wherein the tested vesicles were vesicles having a membrane comprising DOPC, vesicles comprising DOPC and cholesterol linked to polyethylene glycol (PEG), and vesicles comprising DOPC and Chol-CS (n is 19 to 22 for each vesicle type). FIG. 6A shows the average stretching moduli measured for each vesicle composition, and shows the vesicles comprising the Chol-CS have a lower stretching modulus than the DOPC-only and the DOPC-Chol-PEG vesicles. This demonstrates the lowering of the stretching modulus is associated with the polysaccharide identity, and not only the presence of a polymer tethered to the membrane.

[0089] FIGS. 6B-6D show confocal images (scale bar is 10 microns) of vesicles corresponding to those in FIG. 6A and having membranes comprising DOPC (FIG.6B), DOPC and cholesterol linked to PEG (FIG. 6C), and DOPC and Chol-CS (FIG.6D) when deflating the vesicles to an osmolarity ratio of cOut / cm = 1.5, where cout is the osmolarity outside and Cinthe osmolarity inside the vesicle. Upon deflation, all tested vesicles underwent lipid nanotube formation. Pure DOPC vesicles formed lipid nanotubes and undergo pearling toward the GUV interior and additionally exhibit strong membrane fluctuations (FIG. 6B). In both FIG. 6C and 6D, it can be seen that membrane fluctuations were suppressed when deflating the vesicles and that lipid nanotubes formed toward the vesicle exterior, which is consistent with positive spontaneous curvature generation due to asymmetric attachment of CS and PEG to the vesicles. Moreover, the amount and length of lipid nanotubes increased for Chol-CS vesicles (FIG. 6D), which indicates a softening of the membrane and a stronger curvature generation. Thes results indicate the electrostatic repulsion of Chol-CS lowers the resistance to stretching (e.g., and thus the stretching modulus), whereas uncharged polymers like Chol-PEG do not. This further indicates that the nature of the polymer that is attached to the vesicle membrane may unexpectedly affect the resultant mechanical properties.

[0090] FIG. 6E shows confocal images of a vesicle formed in a solution comprising 25 mM NaCl and a corresponding line plot of the fluorescence measured along the white dashed line of the image. FIG. 6F shows an analogous vesicle to FIG. 6E but formed in a solution comprising 25 mM MgCh, again with a corresponding line plot of the fluorescence along the dashed white line of the image. FIGS.6E and 6F show that the presence of the MgC12 resulted in membranes having a larger number of glycolipids, as evidenced by the increased fluorescence at the vesicle membrane. The importance of the increased number of glycolipids present in the membrane is shown in FIG. 6G, which plots the stretching modulus of vesicles having different compositions in the presence of different buffer conditions (i.e., phosphate buffered saline (PBS) of approximately 50 mM vs. a similar solution containing 25 mM MgCh in place of the NaCl of the PBS). Notably, the presence of 25 mM Mg2+in the buffer further lowered the stretching modulus of the vesicles, compared to just the presence of the Chol-CS. This indicates that the presence of additional glycolipid (e.g., Chol-CS) facilitated by the Mg2+in solution may further alter mechanical properties of the vesicle (e.g., the stretching modulus as measured here). FIG. 6H shows the bending rigidity of a variety of vesicles. It was observed that the presence of the Chol-CS in the membrane of the vesicles reduced the bending rigidity of the vesicles. Note that the bending rigidity did not decrease with only the addition of cholesterol to the vesicle membranes. Accordingly, the membrane -bound glycolipids change the mechanical properties of the vesicles and lead to an unexpected softening of the membrane.

[0091] The above description in this Example is generally related to giant unilaminar vesicles (GUVs) due to the relative ease of measuring properties thereof. An illustration of the uptake of small unilamellar vesicles (SUVs) by cells is shown in FIG. 7A, the rate of which was then measured as a function of vesicle composition. By tracking the fluorescence of the SUVs within a population of cells as shown in FIG. 7B, uptake rates were determined. In these experiments, the SUVs contained mRNA coding for green fluorescent protein (GFP), and thus cells exhibiting GFP were tracked by their fluorescence as shown in FIG. 7C (scale bar = 5 microns, same scale bar for images in FIG. 7B) to measure transfection efficiency. The rates for uptake and transfection efficiency (e.g., the ratio of GFP expressing cells divided by the total amount of cells) as a function of vesicle composition are plotted in FIG. 7D, which shows SUVs comprising high concentrations of Chol-CS were uptaken at the highest rate and transfected cells at the highest efficiency compared to the other vesicle compositions tested.

[0092] Finally, in FIG. 8A, the zeta potential of vesicles comprising different lipids, e.g., DOPC, l,2-dioleyloxy-3-dimethylaminopropane (DODMA), and / or l,2-dioleoyl-3- trimethylammonium-propane (DOTAP) was measured in solutions having a variable concentration of Chol-CS. The zeta potential decreases as a function of Chol-CS in solution because the cholesterol of the Chol-CS spontaneously embeds in the lipid membrane of the vesicles, giving a lower (e.g., less positive and / or more negative) zeta potential.

[0093] It is known in the art that, generally, increasing zeta potentials (e.g., more positive) of vesicles is related to increased uptake of said vesicles by cells, and thus uptake of the vesicles was expected to decrease given the decreasing zeta potentials. However, as shown in FIG. 8B (arbitrary units), the uptake rate of each vesicle composition remained approximately constant and / or increased as the concentration of Chol-CS in solution increased (e.g., as the zeta potential of the vesicles decreased). Without wishing to be bound by any particular theory, it is believed that the trends observed in FIGS. 8A-B are related to the identity of the Chol-CS and it associating with the cells in which it is being uptaken.

[0094] EXAMPLE 2

[0095] This Example describes the functionalization of lipid vesicles with chondroitin sulfate conjugated to a sterol to improve mRNA delivery to cells.

[0096] The efficacy of drug delivery depends on the biophysical and biochemical properties of drug delivery vehicles, which protect drugs from degradation and facilitate cell-specific targeting. Lipid vesicles are often employed as drug delivery vehicles because their surface properties can be tuned. However, many typical membrane modifications impede the cellular uptake of vesicles or cause an unwanted immunogenic response.

[0097] In this Example, the outer leaflet of lipid vesicles is modified with the biocompatible polysaccharide chondroitin sulfate and test their potential for drug delivery. Unexpectedly, the modified vesicles show an increased uptake by cells indicating a specific interaction between polysaccharides and cell membranes. Upon encapsulating GFP-encoding mRNA, chondroitin sulfate-modified vesicles were observed to increase the transfection efficiency up to 3-fold in human embryonic kidney cells. The transfection efficiency can be tuned via the chondroitin sulfate and mRNA concentration.

[0098] This Example introduces a versatile approach to enhance the uptake and drug release of lipid vesicles by harnessing polysaccharide-mediated interactions with cell membranes. These specific interactions show great potential to achieve the effective and targeted delivery of therapeutics.

[0099] Introduction

[0100] The delivery of therapeutics is an important way to treat and prevent disease. To protect therapeutics from degradation, they are often encapsulated into drug delivery vehicles. The surface of drug delivery vehicles can also be modified to facilitate cellspecific targeting. This is crucial to deliver therapeutics to the right cell type and to prevent side effects, both of which are significant challenges in modern medicine. Typical methods of tuning surface properties of drug delivery vehicles such as lipid vesicles or lipid nanoparticles is to modify their membrane with antibodies, other cell- targeting proteins, or synthetic polymers like polyethylene glycol. Such modifications may target certain cells, but often impede the cellular uptake of vesicles and / or cause an unwanted immunogenic response.

[0101] Polysaccharides are an alternative class of macromolecules that can be used to functionalize lipid vesicles. Polysaccharides are structurally stable, biocompatible and non-immunogenic polymers with low toxicity. They are also important constituents of cell membranes, where they guide cell-cell adhesion and cell signaling. Moreover, their diverse chemical structures and functional groups offer a versatile platform for fine- tuning the interactions between drug carriers and cells. However, the potential of polysaccharides to alter the biophysical properties of lipid membranes was generally unrecognized, with only a few examples use the electrostatic binding of the polysaccharide chitosan to lipid membranes to engineer the biophysical and biochemical properties of lipid vesicles for drug delivery. These electrostatic interactions did not leverage covalent linking to a lipid (e.g., sterol) to facilitate coupling of a polysaccharide to the lipid vesicle (e.g., in the form of a glycolipid), and were thus not as robust or facile to integrate into the lipid membranes as in this Example. Accordingly, other polysaccharides have generally not been explored for modifying properties of lipid membranes.

[0102] In this Example, lipid vesicles are functionalized with the naturally occurring polysaccharide chondroitin sulfate, which is found in connective tissues. The vesicles performance as drug delivery vehicles is additionally tested. The chondroitin sulfate is anchored to the outside of the membrane by coupling it to cholesterol. It is found that chondroitin sulfate modification lowers the electrical potential such as the zeta potential of lipid vesicles but, remarkably, still increases their uptake by human embryonic kidney cells two-fold compared to unmodified vesicles. It is also shown that vesicles modified with chondroitin sulfate increase the transfection of cells when encapsulating an mRNA into the vesicles. By increasing the mRNA concentration, transfection efficiencies of up to 30% were achieved. Thus, this work shows that the polysaccharide functionalization of lipid membranes can increase the performance of lipid-based drug delivery vehicles, opening new avenues for the treatment of many diseases that require an effective cell uptake and potentially the targeting of specific cell types.

[0103] Results & Discussion Small unilamellar lipid vesicles (SUVs) were formed by extruding multilamellar vesicles through a polycarbonate membrane with a diameter of 100 nm. Dynamic light scattering revealed that the SUVs were monodisperse in size having a mean hydrodynamic diameter of 138 nm and a polydispersity index of 0.12. To modify SUVs with the polysaccharide chondroitin sulfate, chondroitin sulfate (CS) was coupled to cholesterol (e.g., covalently bound). The cholesterol-modified chondroitin sulfate (Chol- CS) was incorporated into the outer leaflet of SUVs due to hydrophobic interactions, as schematically illustrated in FIGS. IB and 4A.

[0104] After functionalizing SUVs with Chol-CS, the hydrodynamic diameter of SUVs increased by 20 nm as shown in FIG. 9A. This confirmed confirms the Chol-CS was incorporated into the outer leaflet. To investigate the effect of Chol-CS modification on the electrical surface properties of lipid vesicles, the zeta potential of SUVs after functionalization with Chol-CS was measured. SUVs containing positively charged DOTAP lipids were prepared and the zeta potential was measured as a function of the Chol-CS concentration. Without the Chol-CS modification, the zeta potential was 18 + 1 mV. With increasing CS concentrations, the zeta potential decreased because the CS of the Chol-CS is negatively charged and neutralizes the positive charge of DOTAP lipids. Initially, a slight increase in the zeta potential was observed after adding the Chol-CS, which contrasts with the standard electrokinetic model but can result from specific ion binding or the presence of layers of polymeric brushes. Further Chol-CS addition decreased the overall zeta potential as expected. For Chol-CS concentrations above 5 pM, the zeta potential saturates at -6 mV as seen in FIG. 9B due to a balance of a fully saturated leaflet layer by Chol-CS and charge screening by the presence of counterions. The same trend was observed for a lipid composition comprising DODMA, which has a lower initial positive zeta potential than DOTAP in the absence of CS. The DODMA vesicles modified with Chol-CS saturated to the same zeta potential in the presence of Chol-CS. Even for initially uncharged DOPC lipids (e.g., in the absence of Chol-CS), the zeta potential saturated at the same value. These results indicate that this zeta potential is solely due to the presence of CS and complete outer leaflet saturation of the vesicle membranes.

[0105] To test the effect of lipid charge on the vesicle uptake by cells, SUVs with different lipid compositions were prepared and incubated with cells. After one day of incubation, vesicle uptake was analyzed with confocal microscopy. As expected, due to the negatively charged cell membrane, the vesicle uptake by HEK cells increased with increasing zeta potential. The uptake in the presence of the positively charged DOTAP lipids was 2.4-fold higher than for uncharged DOPC lipids, as shown in FIG. 10A. Negatively charged SUVs containing DOPG lipids were not taken up.

[0106] To test the impact of chondroitin sulfate on the vesicle uptake, the experiments were repeated with chondroitin sulfate-modified vesicles. The confocal images in FIG. 10B show the fluorescence intensities of Chol-CS and the SUVs were colocalized, indicating that the cells up took CS-modified SUVs and that the. The colocalization of CS and SUVs proves the successful functionalization of lipid vesicles. The uptake of CS- modified SUVs despite their decrease in the zeta potential indicates that CS is not interacting electrostatically with the cell membrane. It is believed that the CS is likely interacting with specific lectin or lectin-like receptors to facilitate vesicle endocytosis. This interaction with the cell membrane of CS-modified SUVs compared to unmodified SUVs leads is what is believed to lead to different amounts of vesicle uptake. When quantifying the amount of vesicle uptake, CS-modified vesicles were observed to be taken up 2.7 times more than unmodified vesicles, with the CS-modified vesicle uptake being dependent on the CS concentration (FIG. 10C). Increased uptake of CS-modified SUVs into cells was further confirmed with flow cytometry (FIG. 10D), showing the fluorescence intensity associated with the SUVs per cell having membranes with Chol- CS was higher than that of cells having unmodified membranes or membranes modified with Chol-PEG. The results show increased uptake of modified vesicles by HEK cells. To show the method is generalizable, FIG. 10E further plots vesicle uptake by mouse embryonic fibroblasts (MEF cells) as a function of vesicle type. This data shows CS- modified vesicles where CS is present in higher concentrations were uptaken by the MEF cells at higher rates than unmodified vesicles, similarly to the HEK cells. (FIG. 10C).

[0107] For successful drug delivery, the vesicles need to be taken up by cells and then need to release their cargo into the cytoplasm of the cell. To test if CS-modified SUVs can deliver cargo to cells, an mRNA was encapsulated into the vesicles. An mRNA that can be translated into a fluorescent protein GFP was chosen as a readout for successful delivery. To independently track the localization of the mRNA, an mRNA that itself is fluorescently labeled with Cy5 was used, as sketched in FIG. 11 A. After incubation of mRNA-containing CS-modified SUVs with HEK cells, internalization of the vesicles by cells was observed. The confocal images in FIGS. 7B-7C show two representative cells that took up SUVs, while illustrating that the mRNA is colocalized with SUVs. This indicates successful encapsulation of mRNA. In addition, it was observed that some cells expressed GFP, proving that a fraction of the mRNA has been released into the cytoplasm. To analyze the degree of successful delivery, the transfection efficiency was quantified, which is the ratio of GFP expressing cells of the whole cell population. The transfection efficiency of cells was measured after three days to reach a steady state in the output value. The transfection efficiency was observed to be 4-fold higher when lipid vesicles are modified with CS compared to unmodified vesicles, as shown in FIG. 1 IB. By plotting the transfection efficiency as a function of vesicle uptake, it was observed that an increased vesicle uptake leads to an increased transfection efficiency (FIG. 11C). The more lipid vesicles a cell internalizes, the higher the probability of endosomal escape and mRNA release. Importantly, the mRNA release shows that the CS modification does not prevent endosomal escape of the vesicle.

[0108] While CS-modified SUVs led to an increase of the transfection efficiency compared to unmodified SUVs, the overall percentage of transfected cells was approximately 5%. Different concentrations of mRNA were encapsulated into SUVs to test the effect thereof on the number of transfected cells. The confocal overview images in FIG. 12A show that the number of cells which express GFP increased with an increasing mRNA concentration encapsulated by the vesicles. By analyzing the transfection efficiency, it was observed that there was an almost linear dependence of the transfection efficiency as a function of mRNA concentration. As shown in FIG. 12B, the transfection efficiency reached up to 30%. Unexpectedly, an increase in the SUV concentration did not further increase the transfection efficiency. This indicates that the cell uptake of SUVs saturates at lipid concentrations of approximately 10 pM. In accordance with the increasing transfection efficiency, a higher GFP intensity for higher mRNA concentrations was also measured (FIG. 12C). This proves that more mRNA was released into the cytosol of cells.

[0109] Perspective

[0110] The approach described in the Example to functionalize the membrane of vesicles is easy to implement (e.g., only requiring a single mixing step after vesicle formation) and is a versatile tool for drug delivery, allowing the use of any membrane-based delivery vehicle including lipid vesicles, lipid nanoparticles, lipid-polymer hybrid vesicles, and amphiphilic polymer shells. The approach can be extended to other polysaccharides linked to lipids (e.g., sterols, cholesterol) because the approach only requires the polysaccharide contains a functional group capable of being coupled to the lipid. This includes commonly available thiol-, amine-, carboxyl-, and / or azide modifications.

[0111] In this Example, it is shown that a polysaccharide modification can improve the performance of lipid vesicles as drug delivery vehicles. Therefore, this approach may be used to enhance the uptake and delivery of commercially available therapeutics to cells.

[0112] By designing polysaccharide-modified lipid vesicles that match specific cell receptors of different cell types, the presented method may be used to target specific cell types and organs. Moreover, polysaccharides are biocompatible and natural components of any cell; thus, it is generally believed that they have fewer side effects than other polymers, making them more than suitable for drug delivery in vivo. In some embodiments, this facilitates the treatment of many diseases where targeting is essential, including cancer, neurological disorders, and genetic disorders.

[0113] 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, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0114] 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.”

[0115] 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. 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 unless clearly indicated to the contrary. 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 without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0116] 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.

[0117] 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.

[0118] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0119] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0120] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” 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.

Claims

CLAIMSWhat is claimed is:

1. An article, comprising: a lipid membrane and / or a membrane comprising amphiphilic molecules; and a sterol covalently linked to a polysaccharide, wherein the sterol is at least partially embedded within the lipid membrane and / or the membrane comprising amphiphilic molecules and the polysaccharide is at least partially exposed externally of the lipid membrane.

2. An article as in claim 1, wherein the sterol is essentially completely embedded within the lipid membrane.

3. An article as in any one of claims 1 or 2, wherein the polysaccharide is essentially completely exposed externally of the lipid membrane and / or a membrane comprising amphiphilic molecules.

4. An article as in any one of claims 1-3, wherein the polysaccharide is an agonist for a receptor located on a cell.

5. An article as in any one of claims 1-4, further comprising small molecules within the lipid membrane and / or a membrane comprising amphiphilic molecules.

6. An article as in any one of claims 1-5, further comprising RNA within the lipid membrane and / or a membrane comprising amphiphilic molecules.

7. An article as in any one of claims 1-6, further comprising DNA within the lipid membrane and / or a membrane comprising amphiphilic molecules.

8. An article as in any one of claims 1-7, further comprising proteins within the lipid membrane and / or a membrane comprising amphiphilic molecules.

9. An article as in any one of claims 1-8, further comprising nanoparticles within the lipid membrane and / or a membrane comprising amphiphilic molecules.

10. An article as in any one of claims 1-9, wherein the lipid membrane and / or a membrane comprising amphiphilic molecules is substantially spherical.

11. An article as in any one of claims 1-10, further comprising a second polysaccharide covalently linked to a second sterol that is embedded within the lipid membrane.

12. An article as in any one of claims 1-11, wherein the second polysaccharide and the first polysaccharide are not identical.

13. An article, comprising: a vesicle comprising a lipid membrane; and a sterol covalently linked to a polysaccharide, wherein the sterol is at least partially embedded within the lipid membrane and the polysaccharide is at least partially exposed externally of the vesicle.

14. An article as in claim 13, wherein the sterol is essentially completely embedded within the lipid membrane.

15. An article as in any one of claims 13 or 14, wherein the polysaccharide is essentially completely exposed externally of the vesicle.

16. An article as in any one of claims 13-15, wherein the vesicle is a liposome.

17. An article as in any one of claims 13-16, wherein the vesicle is asymmetric.

18. An article as in any one of claims 13-17, wherein the polysaccharide is an agonist for a receptor located on a cell.

19. An article as in any one of claims 13-18, further comprising small molecules within the vesicle.

20. An article as in any one of claims 13-19, further comprising RNA within the vesicle.

21. An article as in any one of claims 13-20, further comprising DNA within the vesicle.

22. An article as in any one of claims 13-21, further comprising proteins within the vesicle.

23. An article as in any one of claims 13-22, further comprising nanoparticles within the vesicle.

24. An article as in any one of claims 13-23, wherein the vesicle is substantially spherical.

25. An article as in any one of claims 13-24, further comprising a second polysaccharide covalently linked to a second sterol that is embedded within the lipid membrane.

26. An article as in claim 25, wherein the second polysaccharide and the first polysaccharide are not identical.

27. A plurality of articles as in any one of claims 13-26, comprising a plurality of vesicles.

28. An article as in claim 27, wherein an average zeta potential of the plurality of vesicles is less than or equal to -5 mV.

29. An article as in any one of claims 27 or 28, wherein an average diameter of the plurality of vesicles is less than or equal to 200 nanometers.

30. An article as in any one of claims 27-29, wherein an average diameter of the plurality of vesicles is less than or equal to 60 nanometers.

31. An article as in any one of claims 27-30, wherein an average stretching modulus of the plurality of vesicles is less than or equal to 200 mN / m.

32. An article as in any one of claims 27-31, wherein an average transfection efficiency of the plurality of vesicles is greater than or equal to 0.05.

33. An article as in any one of claims 27-32, wherein an average lipid diffusion coefficient of the plurality of vesicles less than or equal to 2.1 micrometers2 / second.

34. An article, comprising: a vesicle comprising a lipid membrane and cholesterol covalently linked to chondroitin sulfate.

35. An article as in claim 34, further comprising a second polysaccharide covalently linked to a second sterol that is embedded within the lipid membrane.

36. An article as in claim 35, wherein the second polysaccharide and the chondroitin sulfate are not identical.

37. An article as in any one of claims 35 or 36, wherein the chondroitin sulfate and / or the second polysaccharide is an agonist for a receptor located on a cell.

38. An article as in any one of claims 34-37, further comprising small molecules within the vesicle.

39. An article as in any one of claims 34-38, further comprising RNA within the vesicle.

40. An article as in any one of claims 34-39, further comprising DNA within the vesicle.

41. An article as in any one of claims 34-40, further comprising proteins within the vesicle.

42. An article as in any one of claims 34-41, further comprising nanoparticles within the vesicle.

43. An article, comprising: a vesicle comprising a lipid membrane; and cholesterol covalently linked to chondroitin sulfate, wherein the cholesterol is at least partially embedded within the lipid membrane and the chondroitin sulfate is at least partially exposed externally of the vesicle.

44. An article as in claim 43, further comprising a second polysaccharide covalently linked to a second sterol that is embedded within the lipid membrane.

45. An article as in claim 44, wherein the second polysaccharide and the chondroitin sulfate are not identical.

46. An article as in any one of claims 44 or 45, wherein the chondroitin sulfate and / or the second polysaccharide is an agonist for a receptor located on a cell.

47. An article as in any one of claims 43-46, further comprising small molecules within the vesicle.

48. An article as in any one of claims 43-47, further comprising RNA within the vesicle.

49. An article as in any one of claims 43-48, further comprising DNA within the vesicle.

50. An article as in any one of claims 43-49, further comprising proteins within the vesicle.

51. An article as in any one of claims 43-50, further comprising nanoparticles within the vesicle.

52. An article, comprising: cholesterol covalently linked to chondroitin sulfate.

53. A article as in claim 52, wherein the article is embedded within a lipid membrane.

54. A article as in claim 53, wherein a vesicle comprises the lipid membrane.

55. A article as in any one of claims 53 or 54, further comprising a second polysaccharide covalently linked to a second sterol that is embedded within the lipid membrane.

56. An article as in claim 55, wherein the second polysaccharide and the chondroitin sulfate are not identical.

57. A method of making an article, comprising: mixing a first solution comprising a plurality of vesicles with a second solution comprising cholesterol covalently linked to chondroitin sulfate.

58. The method as in claim 57, further comprising forming the plurality of vesicles.

59. The method as in claim 58, wherein forming the plurality of vesicles comprises hydrating a thin lipid film.

60. The method as in any one of claims 58 or 59, wherein forming the plurality of vesicles comprises applying an AC electric field to a solution comprising a lipid.

61. The method as in claim 60, wherein a voltage of the AC electric field greater than or equal to 2 V.

62. The method as in any one of claims 60 or 61, wherein a frequency of the AC electric field greater than or equal to 1 Hz.

63. The method as in any one of claims 60-62, wherein the AC electric field is applied to the solution for greater than or equal to 10 minutes.

64. The method as in any one of claims 57-63, further comprising passing the plurality of vesicles through an extruder.

65. A method of drug delivery, comprising: introducing a plurality of vesicles to a population of cells, wherein a cholesterol covalently linked to chondroitin sulfate is embedded in a lipid membrane of at least some of the plurality of vesicles.

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