Asymmetric liposomes for drug delivery and other applications

Abstract

Some aspects of the present disclosure are generally related to systems including asymmetric liposomes, for example, for drug delivery. In some embodiments, the asymmetric liposomes facilitate the encapsulation of therapeutic agents. In some embodiments, the system may include a plurality of asymmetric liposome and a plurality of cells, e.g., within a subject. According to some embodiments, cells may uptake the asymmetric liposomes at an enhanced rate when compared to conventionally used symmetric liposomes, e.g., while also comparatively being less cytotoxic. In some embodiments, the enhanced rate of uptake of the asymmetric liposomes by cells improve delivery of a therapeutic agent encapsulated within the asymmetric liposomes to the cells. Still other aspects are generally directed to methods of making and using the liposomes, kits containing the liposomes, or the like.

Description

[0001] ASYMMETRIC LIPOSOMES FOR DRUG DELIVERY AND OTHER APPLICATIONS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 552,502, filed February 12, 2024, entitled “Asymmetric Liposomes for Drug Delivery and Other Applications,” by Weitz, et al., and of U.S. Provisional Patent Application Serial No. 63 / 572,453, filed April 1, 2024, entitled “Asymmetric Liposomes for Drug Delivery and Other Applications,” by Weitz, et al., each of which is incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] Systems containing asymmetric liposomes for drug delivery and other applications are generally described.

[0006] BACKGROUND

[0007] Delivery of therapeutic agents to cells, e.g., within a subject, involves various challenges. A conventional method of delivery is direct administration of the therapeutic agent to the cells, for example, by having a subject ingest a tablet containing the therapeutic agent. However, therapeutic agents often are not sufficiently water soluble, may degrade, and / or may not be uptaken by the cells at a sufficient rate for therapeutic effects in the cells to be observed. Accordingly, improved systems and methods are needed.

[0008] SUMMARY

[0009] Systems containing asymmetric liposomes for drug delivery and 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] Some aspects are related to systems. In some embodiments, a system comprises a subject having cancer; and a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer, contained within the subject’s blood. In some embodiments, a system comprises a solution containing a plurality of cancer cells; and a plurality of asymmetric liposomes in the solution, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating cancer. In some such embodiments, the solution is blood or cell media. In some embodiments, a system comprises a subject having a disease; and a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease, contained within the subject’s blood. In some embodiments, a system comprises a solution containing a plurality of disease cells; and a plurality of asymmetric liposomes in the solution, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the disease.

[0011] Some aspects are related to methods. In some embodiments, a method of making liposomes comprises extruding a water-in-oil emulsion stabilized by a first lipid through a membrane having an average pore size of less than or equal to 200 microns; and forming an asymmetric liposome by passing the extruded emulsion through a monolayer comprising a second lipid different from the first lipid. In some embodiments, a method of making liposomes comprises providing an emulsion having an average droplet size of less than or equal to 100 microns, the emulsion comprising droplets stabilized by a first lipid; and forming asymmetric liposomes by passing the droplets through a second lipid different from the first lipid. In some embodiments, a method of making liposomes comprises providing an emulsion having an average droplet size of less than or equal to 100 microns by extruding a first lipid through a membrane having an average pore size of less than or equal to 200 microns into an aqueous medium, the emulsion comprising droplets stabilized by a first lipid; and forming asymmetric liposomes by passing the droplets through a second lipid different from the first lipid.

[0012] In some embodiments, a method of treating a subject having cancer comprises administering a plurality of asymmetric liposomes to the subject having cancer, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the cancer. In some embodiments, a method of treating a subject having a disease comprises administering a plurality of asymmetric liposomes to the subject having a disease, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the disease.

[0013] In some embodiments, a method comprises exposing a plurality of cancer cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer. In some embodiments, a method comprises exposing, in vitro, a plurality of cancer cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer. In some embodiments, a method comprises exposing, in vivo, a plurality of cancer cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer. In some embodiments, a method comprises exposing a plurality of disease cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease. In some embodiments, a method comprises exposing, in vitro, a plurality of disease cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease. In some embodiments, a method comprises exposing, in vivo, a plurality of disease cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease. In some embodiments, use of a composition to treat a subject having cancer is generally described, the composition comprising a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer. In some embodiments, use of a composition to treat a subject having a disease is generally described, the composition comprising a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease.

[0014] 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.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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: FIG. 1A is a schematic illustration of a system including a plurality of asymmetric liposomes and a plurality of cells, according to some embodiments;

[0017] FIG. IB is a schematic diagram of an asymmetric liposome, according to some embodiments;

[0018] FIG. 2A is a schematic representation of the process used to form small asymmetric vesicles, according to some embodiments;

[0019] FIG. 2B is a plot of intensity percent as a function of hydrodynamic diameter as revealed via dynamic light scattering of small unilamellar asymmetric vesicles, according to some embodiments;

[0020] FIG. 2C is a plot of dithionite quenching assay of asymmetric vesicles with NBD-labeled lipids in the outer leaflet, according to some embodiments;

[0021] FIGS. 2D-2Q show plots of liposome yield or fluorescence intensity from liposomes as a function of lipid concentration and / or electrolyte composition, according to some embodiments;

[0022] FIG. 3A is a schematic representation of the pendant drop method used to measure the interfacial tension between different water and lipids in mineral oil, according to some embodiments;

[0023] FIG. 3B is a plot of interfacial tension between different water / mineral oil formulations in presence of DODMA, POPC, POPS or in the absence of lipids, according to some embodiments;

[0024] FIG. 3C is a schematic representation of the extraction of Rhodamine 6G from the aqueous to the oil phase due to inverted micelle formation of lipid molecules that enclose rhodamine 6G molecules, according to some embodiments;

[0025] FIG. 3D is a plot showing rhodamine 6G absorbance (Xex = 500 nm) in the aqueous solution over time partitioning experiment, according to some embodiments;

[0026] FIG. 3E is a plot showing lipid concentration correlating to the vesicle concentration formed with DODMA, POPC and POPS (inner-outer leaflet) , according to some embodiments;

[0027] FIG. 3F is an image of an experimental setup for lipid partitioning, according to some embodiments; FIG. 4A shows confocal images of HEK293 cells after incubation with liposomes for 24 h (labeled with Rhodamine B, Acx = 561 nm, n>20, Mean= +SD) with a scale bars of 10 microns, according to some embodiments;

[0028] FIG. 4B is a plot showing liposome uptake of cells analyzed from confocal images, according to some embodiments;

[0029] FIG. 4C is a plot showing liposome uptake of cells analyzed with flow cytometry (n> 15300, Mean= + / - SD), according to some embodiments;

[0030] FIGS. 4D-4E show cell uptake data for symmetric liposomes, according to some embodiments;

[0031] FIG. 4F shows the GFP fluorescence intensity measured from cells expressing GFP as a function of the liposome membrane compositions, according to some embodiments;

[0032] FIG. 5A is a schematic representation of the liposome uptake and mRNA delivery to HEK293 cells, according to some embodiments;

[0033] FIG. 5B shows confocal overlay images of HEK293 cells transfected with GFP (Xex = 488 nm) with scale bars of 20 microns, according to some embodiments;

[0034] FIG. 5C is a plot of mRNA transfection efficiency measured as ratio of transfected cells, according to some embodiments;

[0035] FIG. 5D is a plot of cell cytotoxicity of asymmetric vesicle, according to some embodiments;

[0036] FIGS. 5E-5G show additional plots of confocal overlays, mRNA transfection efficiency, and cell cytotoxicity as a function of liposome lipid composition, according to some embodiments; and

[0037] FIGS. 5H-5J, 6A-6C, and 7A-7B show various plots of liposome properties as a function of experimental parameters, according to some embodiments;

[0038] FIGS. 7C-7G show images of liposomes being uptaken by cells and plots detailing the uptake rates, transfection efficiency, and cytotoxicity of the vesicles various, according to some embodiments;

[0039] FIGS. 8A-8B show images and a plot detailing the cellular uptake of various proteins encapsulated within vesicles, according to some embodiments; and

[0040] FIGS. 9A-9D show images and a plot detailing the hybrid lipid-polymer vesicles, according to some embodiments. DETAILED DESCRIPTION

[0041] Some aspects of the present disclosure are generally related to systems including asymmetric liposomes, for example, for drug delivery. In some embodiments, the asymmetric liposomes facilitate the encapsulation of therapeutic agents, for example, when the therapeutic agent is hydrophobic. In some embodiments, the system may include a plurality of asymmetric liposomes and a plurality of cells, e.g., within a subject. According to some embodiments, cells may uptake the asymmetric liposomes at an enhanced rate when compared to conventionally used symmetric liposomes, e.g., while the asymmetric liposomes are also comparatively being less cytotoxic than symmetric liposomes. In some embodiments, the enhanced rate of uptake of the asymmetric liposomes by cells improve delivery of a therapeutic agent encapsulated within the asymmetric liposomes to the cells. Still other aspects are generally directed to methods of making and using the liposomes, kits containing the liposomes, or the like.

[0042] Delivery of therapeutic agents to cells, e.g., within a subject, involves various challenges. A conventional method of delivery is direct administration of the therapeutic agent to the cells, for example, by having a subject ingest a tablet containing the therapeutic agent. However, therapeutic agents often are not sufficiently water soluble and / or may not be uptaken by the cells at a sufficient rate for therapeutic effects in the cells to be observed. Accordingly, some aspects of the present disclosure are generally related to systems including asymmetric liposomes, e.g., for the enhanced delivery of therapeutic agents to a plurality of cells.

[0043] Cells exposed to liposomes may uptake the liposomes to facilitate the uptake of a therapeutic agent contained therein, in some embodiments. For example, cells of a subject may be exposed to a liposome containing a therapeutic agent, where the liposome may be endocytosed by the cell to deliver the therapeutic agent to the interior of the cell. In these conventional cases, symmetric liposomes are used due to the ease of fabrication. However, the inventors have recognized that using asymmetric liposomes for the delivery of therapeutic agents to cells (e.g., in vitro or in vivo) may advantageously increase the rate of uptake of the liposomes and / or decrease the cytotoxicity of the liposomes, when compared to symmetric liposome counterparts. FIG. 1A shows an example system 100 containing asymmetric liposomes. In this schematic diagram, the system 100 includes a plurality of asymmetric liposomes 110 present in a solution with a plurality of cells 150. As described elsewhere herein in more detail, the solution containing the cells and asymmetric liposomes may vary depending on the setting, e.g., an in vitro or in vivo setting. FIG. IB is a schematic diagram showing a single asymmetric liposome 110. The asymmetric liposome 110 includes a lipid membrane (e.g., a lipid bilayer in this case) having an exterior surface 120 comprising a first lipid 125 and an interior surface 130 comprising a second lipid 135. The lipid membrane defines an interior volume 140 containing a solution therein. In some embodiments, the solution within the interior volume 140 may contain a therapeutic agent. For instance, a system may comprise a solution containing a plurality of cancer cells and a plurality of asymmetric liposomes in the solution, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating cancer or other diseases.

[0044] Note that while the systems and methods described herein are generally described in the context of asymmetric liposomes, it should be understood that this is done for the sake of simplicity. Those of ordinary skill in the art will understand the systems and methods described herein may further be used with asymmetric vesicles, polymersomes, asymmetric droplets, etc. that may include a lipid membrane defining an interior volume that may contain a therapeutic agent, where the lipid membrane may have an interior surface comprising a first lipid and an exterior surface comprises a second lipid. It also will be understood that other asymmetric liposomes (e.g., and / or vesicles, droplets, etc.) are also possible. For instance, in some embodiments, the asymmetric liposome may include one or more (e.g., two or more) polymers, e.g., in place of one or both of the two lipids comprising the interior and exterior surfaces.

[0045] The system may include an asymmetric liposome, according to some embodiments. An asymmetric liposome is a liposome having a membrane defining an interior volume where an interior surface of the membrane and an exterior surface of the membrane are different, in some embodiments. For example, the membrane of the asymmetric liposome may be a lipid bilayer as shown in FIG. IB, where the exterior surface 120 of the liposome 110 may include a first lipid 125 and the interior surface 130 may include a second lipid 135 where the first lipid 125 and second lipid 135 are different. While described in the context of a first and second lipid, it will be understood that the depiction shown in FIG. IB may similarly represent asymmetric liposomes comprising a lipid and a polymer and / or asymmetric liposomes comprises a first polymer and second polymer.

[0046] While illustrated as a simple lipid bilayer comprising only a first and second lipid in FIG. IB, it should be understood that asymmetric liposomes may include various other lipids and / or molecules in one or both of the layers. For instance, in some embodiments, both the interior and the exterior layer may include a first and second lipid, but the first and second lipids may be present in the different layers in different ratios (e.g., less than or equal to 1:9 in the interior layer and greater than or equal to 1:1 in the exterior layer), thus making the liposome asymmetric. In addition, in some embodiments, some of the asymmetric liposomes may be multilamellar liposomes, e.g., having more than one layer of lipids and / or molecules. In some embodiments, one or more (or all) of the layers of the multilamellar liposome may be asymmetric.

[0047] While described above and elsewhere herein as a single asymmetric liposome, it is contemplated that there may be a plurality of asymmetric liposomes, in certain embodiments. The plurality of asymmetric liposomes may comprise any number of liposomes, in accordance with some embodiments, and optionally in solution. In some cases, the asymmetric liposomes may be present in a solution 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 micro mole / 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 asymmetric liposomes 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. According to some embodiments, an asymmetric liposome of the system may have any of a variety of shapes. In some embodiments, the shape of the asymmetric liposome may be substantially spherical at equilibrium when no net forces are present. In other embodiments, the asymmetric liposome may be shaped irregularly, e.g., elongated, tubulated, and / or stretched, for example, in the presence of a force or pressure applied anisotropically to the liposome.

[0048] The system may include a plurality of asymmetric liposomes having any of a variety of sizes, according to some embodiments. In some embodiments, the average maximum dimension of a plurality of asymmetric liposomes may be chosen by the method by which the liposomes are made, e.g., as described in more detail elsewhere herein. In some embodiments, the average maximum dimension of the plurality of asymmetric liposomes may be determined by performing dynamic light scattering measurements of a solution containing the plurality of asymmetric liposomes. In some embodiments, the plurality of asymmetric liposomes may have an average maximum dimension of greater than or equal to 50 nm, greater than or equal to 75 nm, greater than or equal to 100 nm, greater than or equal to 200 nm, greater than or equal to 300 nm, greater than or equal to 500 nm, greater than or equal to 750 nm, greater than or equal to 1 micron, greater than or equal to 1.5 microns, greater than or equal to 2 microns, greater than or equal to 2.5 microns, greater than or equal to 3 microns, greater than or equal to 5 microns, greater than or equal to 10 microns, greater than or equal to 25 microns, greater than or equal to 50 microns, or greater than or equal to 75 microns. In some embodiments, the plurality of asymmetric liposomes may have an average maximum dimension of less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 25 microns, less than or equal to 10 microns, less than or equal to 5 microns, less than or equal to 3 microns, less than or equal to 2.5 microns, less than or equal to 2 microns, less than or equal to 1.5 microns, less than or equal to 1 micron, less than or equal to 750 nm, less than or equal to 500 nm, less than or equal to 300 nm, less than or equal to 200 nm, less than or equal to 100 nm, or less than or equal to 75 nm. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 50 nm and less than or equal to 100 microns). Other ranges are also possible. According to some embodiments, a plurality of asymmetric liposomes may have a relatively uniform size distribution. For example, a maximum dimension of an asymmetric liposome may not vary much from an average maximum dimension of the plurality of liposomes. In some embodiments, the maximum dimension of an asymmetric liposome may vary by no more than 100%, no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the average maximum dimension of the plurality of liposomes. In some embodiments, the uniformity of the size of the plurality of asymmetric liposomes may be achieved due to the method of making the liposomes, e.g., by including an extrusion step through a membrane as described in more detail elsewhere herein.

[0049] An asymmetric liposome may comprise any of a variety of components, including one or more lipids (e.g., at least two lipids) forming a membrane of the liposome, a liquid within the membrane, and / or one or more therapeutic agents contained within the liquid in the membrane. In some embodiments, the asymmetric liposome comprises one or more polymers (e.g., at least two polymers) for forming the membrane of the liposome. In some embodiments, combinations of lipids and polymers may be used to form the membrane of the liposome.

[0050] A membrane of an asymmetric liposome of a system may include 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, 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 liposome may be precisely known, in accordance with some embodiments. Example lipids suitable for forming asymmetric liposomes, in some embodiments, include l-palmitoyl-2-oleoyl- sn-glycero-3-phospho-L-serine (POPS), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), l,2-dioleyloxy-3-dimethylaminopropane (DODMA), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (LissRhod-PE), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-l,3-benzoxadiazol-4-yl) (NBD-PE), and / or 1 -palmitoyl-2- { 6- [(7 -nitro-2- 1 ,3-benzoxadiazol-4-yl)amino]hexanoyl } -sn- glycero-3-phosphocholine (NBD-PC). Other lipids are also possible.

[0051] In some embodiments, an interior surface of an asymmetric liposome may comprise a first lipid and an exterior surface of an asymmetric liposome may comprise a second lipid, different from the first lipid. In some such embodiments, the first lipid may comprise POPC and the second lipid comprises POPS. Other combinations of the lipids as the first and second lipid are also possible, including selections from the above-recited lipids as well as others.

[0052] A membrane of an asymmetric liposome of a system may include any of a variety of suitable polymers, in accordance with some embodiments. For example, the polymers may be amphiphilic and / or block copolymers. Other types of polymers are also possible. In some embodiments, when the interior surface and / or the exterior surface of the asymmetric liposome comprises a polymer, the polymer may be any of a variety of suitable sizes. For instance, in some embodiments, the polymer has a number average molecular weight of greater than or equal to 300 Da, greater than or equal to 500 Da, greater than or equal to 800 Da, greater than or equal to 1 kDa, greater than or equal to 3 kDa, greater than or equal to 3 kDa, greater than or equal to 5 kDa and / or less than or equal to 10 kDa, less than or equal to 20 kDa, less than or equal to 30 kDa, or less than or equal to 50 kDa. Other ranges are also possible.

[0053] In some embodiments, the polymer comprises one or more of polylactide (PLA), polyethylene glycol (PEG), polycaprolactone (PCL), polyethylene oxide (PEO), polybutadiene (PBut), polydimethylsiloxane (PDMS), poly(2-methyl-2-2oxa-zoline) (PMOXA), polyisobutylene (PIB), and polyacrylic acid (PAAc). Other polymers are also possible. In some embodiments, copolymers and / or grafter polymers may be used. For instance, in some embodiments, the membrane comprises one or more of polylactide- block-poly (ethylene glycol) copolymer (PLA-b-PEG), poly (ethylene oxide)-block- poly(s-caprolactone) copolymer (PEO-b-PCL), poly(ethylene glycol)-block-poly(lactide- co-glycolide) copolymer (PEG-b-PLGA), poly(butadiene)-block-poly(ethylene oxide) copolymer (PBut-b-PEO), poly(dimethylsiloxane)-graft-PEO copolymer (PDMS-g- PEO), PDMS-block-poly(2-methyl-2-2oxa-zoline) copolymer (PDMS-b-PMOXA), poly-isobutylene-block-PEO copolymer (PIB-b-PEO), (poly(acrylic acid)-graft-PEO copolymer (PAAc-g-PEO), and / or PMOXA-b-PDMS-b-PMOXA. Other copolymers and grafted polymers are possible. Additionally, not all of the polymers present in the membrane of the liposome may be precisely known, in accordance with some embodiments. As noted elsewhere herein, in some embodiments, the membrane may include one or more polymers and one or more other molecules (e.g., one or more lipids, etc.).

[0054] In some embodiments, an interior surface of an asymmetric liposome may comprise a first polymer and an exterior surface of an asymmetric liposome may comprise a second polymer, different from the first polymer. In some embodiments, the membrane of the asymmetric liposome may comprise a single type of polymer, e.g., an asymmetric polymer, such that an interior surface of the liposome is different from the exterior surface of the liposome. In some embodiments, an interior surface of an asymmetric liposome may comprise a lipid and an exterior surface of an asymmetric liposome may comprise a polymer. In some embodiments, an interior surface of an asymmetric liposome may comprise a polymer and an exterior surface of an asymmetric liposome may comprise a lipid.

[0055] An asymmetric liposome, in some embodiments, includes a membrane comprising one or more lipids and defining an interior volume. In some embodiments, an asymmetric liposome includes a membrane comprising one or more polymers and defining an interior volume. In some embodiments, an inner liquid may be present within the interior volume. The inner liquid, in some embodiments, may be hydrophilic, for example, and aqueous solution. In other embodiments, the inner liquid may be hydrophobic. According to some embodiments, the inner liquid of an asymmetric liposome may solubilize a therapeutic agent.

[0056] Forming asymmetric polymers using polymers may provide a number of advantages, in some embodiments. For example, in some embodiments, polymers are lower cost than lipids, and thus asymmetric liposomes formed therefrom are less expensive than lipid counterparts. In some embodiments, polymers have simple and / or well-known synthetic methods, and thus are easily modified. In some such embodiments, easy modification of constituent polymers (e.g., modifying length, molecular weight, functional groups, charge, etc.) may facilitate tailoring of the properties of resulting asymmetric liposomes (e.g., charge, stiffness). Tailoring the properties of the asymmetric liposomes may advantageously enhance cellular uptake of the liposomes and / or provide other desirable properties (e.g., the ability to evade immune cells, etc.). In some embodiments, while lipid liposomes may facilitate uptake and delivery of certain molecules (e.g., mRNA, Cas9), the delivery of large proteins may be challenging due to complex charge profiles of the protein. Advantageously, in some embodiments, asymmetric liposomes having membranes comprising one or more polymers may improve uptake of such proteins and subsequent delivery to cells.

[0057] According to some embodiments, the system includes a plurality of asymmetric liposomes encapsulating a therapeutic agent. In some embodiments, the therapeutic agent may be suitable for treating a disease or condition. For example, in some embodiments, the therapeutic agent may be suitable for treating cancer. In some embodiments, the therapeutic agent may be suitable for administering to a subject having cancer and in need of the therapeutic agent thereof. In some embodiments, a therapeutic agent may be configured to stimulate an immune response in a subject, e.g., after delivery of the therapeutic agent to the subject. In some such embodiments, the therapeutic agent may be a vaccine, e.g., comprising mRNA. In some embodiments, the therapeutic agent may be experimental, and may only be suitable for in vitro studies as described elsewhere herein. Other diseases or conditions may also be treated in other embodiments, e.g., instead of (or in addition to) cancer. Accordingly, it should be understood that cancer is described in one embodiment for ease of understanding, but that in other embodiments, other diseases or conditions may also be treated.

[0058] Any of a variety of therapeutic agents may be contained within an asymmetric liposome of the system, in some embodiments. In accordance with some embodiments, given the size of the therapeutic agent is suitable to be contained within the asymmetric liposome. In some embodiments, the therapeutic agent 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, siRNA, etc.), DNA, peptides (e.g., having a length of less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, etc.), proteins, polymers, vesicles, nanoparticles, etc. In some embodiments, the therapeutic agents within the asymmetric liposome may consist of a single type of therapeutic agent. In other cases, the therapeutic agents within the asymmetric liposome may comprise multiple types of entities, for example, DNA and nanoparticles. In some cases, the identity of the one or more types of therapeutic agents may determine a potential function for the system. For example, a system including an asymmetric liposome containing a DNA molecule may be suitable for transfecting cells, in accordance with some embodiments. In some such cases, system may further include a cell or other organism to endocytose the asymmetric liposome such that the DNA may enter the cell and be amplified. In some embodiments, wherein the therapeutic agent comprises a small molecule (i.e., less than or equal to 1000 Da), the asymmetric liposome may be suitable for drug delivery.

[0059] Non-limiting examples of anti-cancer therapeutic agents that can be used include, but are not limited to, any one or more of 20-epi-l,25 dihydroxyvitamin D3, 4- ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizing morphogenetic protein- 1, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin, baccatin III derivatives, balanol, batimastat, benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride, bisaziridinylspermine, bisnafide, bisnafide dimesylate, bistratene A, bizelesin, bleomycin, bleomycin sulfate, BRC / ABL antagonists, breflate, brequinar sodium, bropirimine, budotitane, busulfan, buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone, camptothecin derivatives, canarypox IL-2, capecitabine, caracemide, carbetimer, carboplatin, carboxamide-amino-triazole, carboxyamidotriazole, carest M3, carmustine, cam 700, cartilage derived inhibitor, carubicin hydrochloride, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cedefingol, cetrorelix, chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam, cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, dronabinol, duazomycin, duocarmycin SA, ebselen, ecomustine, edatrexate, edelfosine, edrecolomab, eflornithine, eflomithine hydrochloride, elemene, elsamitrucin, emitefur, enloplatin, enpromate, epipropidine, epirubicin, epirubicin hydrochloride, epristeride, erbulozole, erythrocyte gene therapy vector system, esorubicin hydrochloride, estramustine, estramustine analog, estramustine phosphate sodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide, etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine, fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil, flurocitabine, forfenimex, formestane, fosquidone, fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabine hydrochloride, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride, idoxifene, idramantone, ifosfamide, ilmofosine, ilomastat, imidazoacridones, imiquimod, immuno stimulant peptides, insulin-like growth factor- 1 receptor inhibitor, interferon agonists, interferon alpha-2A, interferon alpha-2B, interferon alpha-Nl, interferon alpha-N3, interferon beta-IA, interferon gamma-IB, interferons, interleukins, iobenguane, iododoxorubicin, iproplatin, irinotecan, irinotecan hydrochloride, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, lanreotide acetate, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide acetate, leuprolide / estrogen / progesterone, leuprorelin, levamisole, liarozole, liarozole hydrochloride, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantrone hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, merbarone, mercaptopurine, meterelin, methioninase, methotrexate, methotrexate sodium, metoclopramide, metoprine, meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growth factor- saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid a / myobacterium cell wall SK, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1 -based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, mycophenolic acid, myriaporone, n-acetyldinaline, nafarelin, nagrestip, naloxone / pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, nocodazole, nogalamycin, n-substituted benzamides, 06- benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole, peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin, piritrexim, piroxantrone hydrochloride, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, propyl bis- acridone, prostaglandin J2, prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, puromycin, puromycin hydrochloride, purpurins, pyrazo furin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes, RII retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex, rubiginone Bl, ruboxyl, safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI 1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, simtrazene, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin, spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide, teniposide, teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan hydrochloride, topsentin, toremifene, toremifene citrate, totipotent stem cell factor, translation inhibitors, trestolone acetate, tretinoin, triacetyluridine, triciribine, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tropisetron, tubulozole hydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, velaresol, veramine, verdins, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin stimalamer, and zorubicin hydrochloride, as well as salts, homologs, analogs, polymorphs, derivatives, enantiomers, and / or functionally equivalent compositions thereof.

[0060] One non-limiting example of a therapeutic agent includes one or more components suitable for gene editing. In some such embodiments, the therapeutic agent includes one or more components suitable for CRISPR-Cas9 gene editing. In some embodiments, components of CRISPR-Cas9 that may be included as the therapeutic agent include a Cas9 enzyme and guide RNA (e.g., a single guide RNA, or crRNA and tracrRNA). In some embodiments, the CRISPR-Cas9 components are configured for gene editing. For instance, in some embodiments, the Cas9 enzyme and the guide RNA are configured to selectively target, cut, mutate, and / or remove a portion of a DNA sequence present within a subject receiving the asymmetric liposomes containing the therapeutic agent. In some such embodiments, a portion of the guide RNA (e.g., crRNA) may be complementary to a portion of DNA present within the subject (e.g., a portion of the DNA of the subject), and thus the guide RNA guides the Cas9 enzyme to the location of the complementary DNA where the Cas9 is configured to cut the DNA. It will be understood that the location of the DNA complementary to the guide RNA may be adjacent to a protospacer adjacent motif (PAM). According to some such embodiments, the guide RNA, in combination with a Cas9 enzyme, may be configured to cut DNA present in a subject to induce a mutation (e.g., upon repair of the cut DNA) to knockdown or knock-out a gene found at the location in the DNA.

[0061] In some embodiments, the therapeutic agent comprises multiple guide RNAs and corresponding Cas9 enzymes, where each of the guide RNAs may be at least partially complementary to different portion of DNA present within a subject. In some such embodiments, the multiple guide RNAs and Cas9 enzymes may be configured to cut DNA present within a subject in multiple locations, e.g., where each location that is cut corresponds to a location of the subject’s DNA is complementary to a corresponding guide RNA. According to some such embodiments, the multiple guide RNAs, in combination with a Cas9 enzyme, may be configured to cut DNA present in a subject at multiple locations to remove DNA and knock-out a gene. In some embodiments, where the therapeutic agent includes a Cas9 enzyme and one or more guide RNAs, the therapeutic agent may further include exogenous DNA, for example, for knock-in gene editing.

[0062] In some embodiments, where the therapeutic agent comprises a guide RNA and a Cas9 enzyme, the Cas9 enzyme may be deactivated so that it may not cut DNA but may still target a sequence at least partially complementary to the guide RNA. In some such embodiments, the Cas9 enzyme may be modified and / or configured to repress or overexpress a gene of interest (e.g., rather than cutting a gene of interest). For instance, the Cas9 enzyme may be modified with a repressive domain or an transcriptional activation domain to repress or over-express a gene, respectively.

[0063] According to some embodiments, when the therapeutic agent comprises components configured for CRISPR-Cas9 gene editing, the therapeutic agent may be suitable for treating cancer. According to some embodiments, when the therapeutic agent comprises components configured for CRISPR-Cas9 gene editing, the therapeutic agent may be suitable for treating autoimmune diseases or conditions.

[0064] The therapeutic agents may be present at any of a variety of concentrations within the asymmetric liposomes, in accordance with some embodiments, which may be selected by the composition of the solution in which the asymmetric liposomes are first formed. For example, if the asymmetric liposomes are made in a solution comprising 1 micromolar of DNA molecules (or other entities), the inner solution of the resulting asymmetric liposomes may have a concentration of DNA (or other entities) of 1 micromolar. In some embodiments, the therapeutic agents may be present within the asymmetric liposomes 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 therapeutic agents may be present within the asymmetric liposomes 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.

[0065] In some embodiments, the interior layer and the exterior layer of a plurality of asymmetric liposomes are highly asymmetric. In some embodiments, the asymmetry between the exterior and interior layers of the liposomes may be assessed using a quenching assay. For instance, in some embodiments, the exterior layer of the liposomes may be labeled with a fluorescence probe such as NBD-PC and measuring a first fluorescence intensity. Following this first fluorescence measurement, in some embodiments, a quenching assay (e.g., dithionite) may be added to the solution containing the liposomes, whereafter a second fluorescence measurement may be determined. In some embodiments, depending on the amount of decrease measure in the fluorescence intensity between the first and second measurements, the amount of the fluorescent probe present within the interior of the liposomes (e.g., separate from the quenching assay dithionite) may be determined, which may provide information regarding the asymmetry of the liposomes. In some embodiments, the average asymmetric of the plurality of liposomes is greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or greater than or equal to 99%. In some embodiments, the average asymmetric of the plurality of liposomes is less than or equal to 100%, less than or equal to 99%, less than or equal to 98%, less than or equal to 97%, less than or equal to 96%, less than or equal to 95%, less than or equal to 94%, less than or equal to 93%, less than or equal to 92%, or less than or equal to 91%. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 90% and less than or equal to 99%). Other ranges are also possible.

[0066] A plurality of asymmetric liposomes may have any of a variety of suitable average bending rigidities, according to some embodiments. In some embodiments, the average bending rigidity of the plurality of asymmetric liposomes may be measured using atomic force microscopy. According to some embodiments, an average bending rigidity of the plurality of asymmetric liposomes is greater than or equal to 2 kT, greater than or equal to 3 kT, greater than or equal to 5 kT, greater than or equal to 10 kT, greater than or equal to 20 kT, greater than or equal to 30 kT, greater than or equal to 50 kT, greater than or equal to 75 kT, greater than or equal to 100 kT, greater than or equal to 125 kT, greater than or equal to 150 kT, or greater than or equal to 175 kT. In some embodiments, an average bending rigidity of the plurality of asymmetric liposomes is less than or equal to 200 kT, less than or equal to 175 kT, less than or equal to 150 kT, less than or equal to 125 kT, less than or equal to 100 kT, less than or equal to 75 kT, less than or equal to 50 kT, less than or equal to 30 kT, less than or equal to 20 kT, less than or equal to 10 kT, less than or equal to 5 kT, or less than or equal to 3 kT. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 2 kT and less than or equal to 200 kT). Other ranges are also possible.

[0067] A plurality of asymmetric liposomes may have any of a variety of suitable average stretching modulus, according to some embodiments. In some embodiments, the average stretching modulus of the plurality of asymmetric liposomes may be measured using atomic force microscopy. In some embodiments, the average stretching modulus for the plurality of asymmetric liposomes is greater than or equal to 20 mN / m, greater than or equal to 30 mN / m, greater than or equal to 40 mN / m, greater than or equal to 50 mN / m, greater than or equal to 60 mN / m, greater than or equal to 70 mN / m, greater than or equal to 80 mN / m, greater than or equal to 90 mN / m, greater than or equal to 100 mN / m, greater than or equal to 150 mN / m, greater than or equal to 200 mN / m, greater than or equal to 250 mN / m, greater than or equal to 300 mN / m, greater than or equal to 400 mN / m, greater than or equal to 500 mN / m, greater than or equal to 600 mN / m, greater than or equal to 700 mN / m, greater than or equal to 800 mN / m, or greater than or equal to 900 mN / m. In some embodiments, the average stretching modulus for the plurality of asymmetric liposomes is less than or equal to 1000 mN / m, less than or equal to 900 mN / m, less than or equal to 800 mN / m, less than or equal to 700 mN / m, less than or equal to 600 mN / m, less than or equal to 500 mN / m, less than or equal to 400 mN / m, less than or equal to 300 mN / m, less than or equal to 250 mN / m, less than or equal to 200 mN / m, less than or equal to 150 mN / m, less than or equal to 100 mN / m, less than or equal to 90 mN / m, less than or equal to 80 mN / m, less than or equal to 70 mN / m, less than or equal to 60 mN / m, less than or equal to 50 mN / m, less than or equal to 40 mN / m, or less than or equal to 30 mN / m. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 20 mN / m and less than or equal to 1000 mN / m). Other ranges are also possible.

[0068] In some embodiments, the lipids within the membrane of the liposomes may have any of a variety of lipid diffusivities. In accordance withs some embodiments, the lipid diffusivity of the lipids within the membranes of the asymmetric liposomes may be measured by incorporating a fluorescent membrane probe into the membrane of the asymmetric liposomes and subsequently performing fluorescence recovery after photobleaching experiments. In some embodiments, the average lipid diffusivity within the membranes of the plurality of asymmetric liposomes is greater than or equal to 0.05 microns2 / second, greater than or equal to 0.1 microns2 / second, greater than or equal to 0.15 microns2 / second, greater than or equal to 0.2 microns2 / second, greater than or equal to 0.25 microns2 / second, greater than or equal to 0.3 microns2 / second, greater than or equal to 0.4 microns2 / second, greater than or equal to 0.5 microns2 / second, greater than or equal to 0.6 microns2 / second, greater than or equal to 0.7 microns2 / second, greater than or equal to 0.8 microns2 / second, greater than or equal to 0.9 microns2 / second, greater than or equal to 1 micron2 / second, greater than or equal to 1.5 microns2 / second, greater than or equal to 2 microns2 / second, greater than or equal to 2.5 microns2 / second, greater than or equal to 3 microns2 / second, greater than or equal to 4 microns2 / second, greater than or equal to 5 microns2 / second, greater than or equal to 6 microns2 / second, greater than or equal to 7 microns2 / second, greater than or equal to 8 microns2 / second, or greater than or equal to 9 microns2 / second. According to some embodiments, the average lipid diffusivity within the membranes of the plurality of asymmetric liposomes is less than or equal to 10 microns2 / second, less than or equal to 9 microns2 / second, less than or equal to 8 microns2 / second, less than or equal to 7 microns2 / second, less than or equal to 6 microns2 / second, less than or equal to 5 microns2 / second, less than or equal to 4 microns2 / second, less than or equal to 3 microns2 / second, less than or equal to 2.5 microns2 / second, less than or equal to 2 microns2 / second, less than or equal to 1.5 microns2 / second, less than or equal to 1 microns2 / second, less than or equal to 0.9 microns2 / second, less than or equal to 0.8 microns2 / second, less than or equal to 0.7 microns2 / second, less than or equal to 0.6 microns2 / second, less than or equal to 0.5 microns2 / second, less than or equal to 0.4 microns2 / second, less than or equal to 0.3 microns2 / second, less than or equal to 0.25 microns2 / second, less than or equal to 0.2 microns2 / second, less than or equal to 0.15 microns2 / second, or less than or equal to 0.1 microns2 / second. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 0.05 microns2 / s and less than or equal to 10 microns2 / second). Other ranges are also possible.

[0069] In some embodiments, the asymmetric liposomes may be formed within a solution containing one or therapeutic agents, as described elsewhere herein. In some embodiments, the plurality of asymmetric liposomes, when forming, exhibit an encapsulation efficiency towards the one or more therapeutic agents. In some embodiments, the encapsulation efficiency of the plurality of asymmetric liposomes may be determined by using a probe fluorescent molecule in place of the therapeutic agent. Thus, when forming the asymmetric liposomes in the solution containing the probe molecule, the fluorescence of a supernatant remaining after forming the asymmetric liposomes may be measured to determine the amount of the probe that was encapsulated by the liposomes. In some embodiments, the encapsulation efficiency is the fraction of the therapeutic agent (e.g., or probe in this case) encapsulated by the liposomes out of the of the initial concentration of therapeutic agent present in the solution.

[0070] In some embodiments, the plurality of asymmetric liposomes may exhibit an encapsulation efficiency of greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or greater than or equal to 99%. According to some embodiments, the plurality of asymmetric liposomes may exhibit an encapsulation efficiency of less than or equal to 100%, less than or equal to 99%, less than or equal to 98%, less than or equal to 97%, less than or equal to 96%, less than or equal to 95%, less than or equal to 94%, less than or equal to 93%, less than or equal to 92%, or less than or equal to 91%. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 90% and less than or equal to 99%). Other ranges are also possible.

[0071] A plurality of liposomes may have any of a variety of suitable average zeta potentials, according to some embodiments, which may vary based on the constituent lipids of the liposome. According to some embodiments, the average zeta potential of the asymmetric liposomes may be measured by taking a solution containing the plurality of asymmetric liposomes and using a zetasizer. In some embodiments, the average zeta potential of a plurality of asymmetric liposomes is greater than or equal to -50 mV, greater than or equal to -40 mV, greater than or equal to -30 mV, greater than or equal to -20 mV, greater than or equal to -10 mV, greater than or equal to 0 mV, greater than or equal to 10 mV, greater than or equal to 20 mV, greater than or equal to 30 mV, or greater than or equal to 40 mV. According to some embodiments, the average zeta potential of a plurality of asymmetric liposomes is less than or equal to 50 mV, less than or equal to 40 mV, less than or equal to 30 mV, less than or equal to 20 mV, less than or equal to 10 mV, less than or equal to 0 mV, less than or equal to -10 mV, less than or equal to -20 mV, less than or equal to -30 mV, or less than or equal to -40 mV. Combinations of the foregoing ranges are possible (e.g., greater than or equal to -50 mV and less than or equal to 50 mV). Other ranges are also possible.

[0072] In some embodiments, a plurality of asymmetric liposomes may be uptaken by a plurality of cells at a relatively high rate. In some embodiments, the uptake rate may be determined by introducing a plurality of asymmetric liposomes encapsulating a fluorescent probe to a plurality of cells. The portion of cells that uptake the asymmetric liposomes fluoresce. In some embodiments, 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 0.08, greater than or equal to 0.1, greater than or equal to 0.15, greater than or equal to 0.20, greater than or equal to 0.25, greater than or equal to 0.3, greater than or equal to 0.4, greater than or equal to 0.5, greater than or equal to 0.6, greater than or equal to 0.7, greater than or equal to 0.8, or greater than or equal to 0.9 of cells exposed to the plurality of asymmetric liposomes uptake the liposomes. In some embodiments, 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 0.4, 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 0.08, 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 to 0.02 of cells exposed to the plurality of asymmetric liposomes uptake the liposomes. Combinations of the foregoing range are possible (e.g., greater than or equal to 0.01 and less than or equal to 1, greater than or equal to 0.05 and less than or equal to 0.9, greater than or equal to 0.1 and less than or equal to 0.8, or the like). Other ranges are also possible. According to some such embodiments, a therapeutic agent encapsulated within an asymmetric liposome may correspondingly be uptaken by a cell at a higher rate than when encapsulated within the symmetric liposome.

[0073] Unexpectedly, according to some embodiments, asymmetric liposomes may be uptaken at a higher rate by cells than symmetric liposomes having identical compositions. Thus, such asymmetric liposomes may be surprisingly effective, for example, as drug delivery vehicles, when administered to cells, (e.g., HEK cells, immune cells, cancer cells, stem cells, or the like) or to a subject, etc.

[0074] In some embodiments, an uptake rate of asymmetric liposomes by cells 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 0.08, greater than or equal to 0.1, greater than or equal to 0.15, greater than or equal to 0.20, greater than or equal to 0.25, greater than or equal to 0.3, greater than or equal to 0.4, greater than or equal to 0.5, greater than or equal to 0.6, greater than or equal to 0.7, greater than or equal to 0.8, or greater than or equal to 0.9 greater than an uptake rate of symmetric liposomes having an identical composition. According to some embodiments, an uptake rate of asymmetric liposomes by cells 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 0.4, 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 0.10, less than or equal to 0.08, 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 to 0.02 greater than an uptake rate of symmetric liposomes having identical compositions. Combinations of the foregoing range are possible (e.g., greater than or equal to 0.01 and less than or equal to 1, greater than or equal to 0.05 and less than or equal to 0.9, greater than or equal to 0.1 and less than or equal to 0.8, or the like). Other ranges are also possible. To determine the difference in uptake rate, in some embodiments, the uptake rate of a plurality of asymmetric liposomes by a plurality of cells may be compared to the uptake rate of a plurality of symmetric liposomes by a plurality of cells.

[0075] In some cases, delivery of cargo for transfection (e.g., mRNA) via a plurality of asymmetric liposomes may lead to relatively high rates of cell transfection compared to using symmetric liposomes. In some embodiments, the transfection rate may be determined by using asymmetric liposomes encapsulating green fluorescent protein (GFP)-encoding RNA, where the uptake and transfection of the liposomes by the cells leads to the cells expressing GFP. The transfection rate, in some such embodiments, may then be determined by counting the number of cells expressing GFP out of the total number of cells (e.g., by manual counting, using software, etc.). For example, in some cases, an average transfection efficiency of a plurality of asymmetric liposomes 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 asymmetric liposomes 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.

[0076] In some embodiments, the average transfection efficiency of a plurality of asymmetric liposomes is greater than a plurality of symmetric liposomes (e.g., wherein an exterior layer of the symmetric and asymmetric liposomes is substantially the same and / or wherein an interior layer of the symmetric and asymmetric liposomes is substantially the same). In some embodiments, the average transfection efficiency of a plurality of asymmetric liposomes is greater than or equal to 0.01, greater than or equal - Zi 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

[0077] 0.15, greater than or equal to 0.2, greater than or equal to 0.25, greater than or equal to

[0078] 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 more than the average transfection efficiency of a plurality of symmetric liposomes. In some embodiments, the average transfection efficiency of a plurality of asymmetric liposomes 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 more than the average transfection efficiency of a plurality of symmetric liposomes. 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.

[0079] In some embodiments, a plurality of asymmetric liposomes may result in a low rate of cytotoxicity within a plurality of cells. The rate of cytotoxicity may be determined using a live-dead assay on a plurality of cells, e.g., following uptake of the plurality of asymmetric liposomes by the plurality of cells, according to some embodiments. In some embodiments, the rate of cell death in a plurality of cells following exposure to a plurality of asymmetric liposomes is greater than or equal to 0, greater than or equal to

[0080] 0.01, greater than or equal to 0.02, greater than or equal to 0.03, greater than or equal to

[0081] 0.04, greater than or equal to 0.05, greater than or equal to 0.06, greater than or equal to

[0082] 0.07, greater than or equal to 0.08, greater than or equal to 0.09, greater than or equal to

[0083] 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, or greater than or equal to 0.9. In some embodiments, the rate of cell death in a plurality of cells following exposure to a plurality of asymmetric liposomes 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.

[0084] In some embodiments, the rate of cell death experienced by a plurality of cells when exposed to a plurality of asymmetric liposomes is lower than when the plurality of cells is exposed to a plurality of symmetric liposomes (e.g., wherein an exterior layer of the symmetric and asymmetric liposomes is substantially the same and / or wherein an interior layer of the symmetric and asymmetric liposomes is substantially the same). In some embodiments, the cell death rate 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, or greater than or equal to 0.9 less when the cells are exposed to asymmetric liposomes than when the cells are exposed to symmetric liposomes. In some embodiments, the cell death rate 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 less when the cells are exposed to asymmetric liposomes than when the cells are exposed to symmetric liposomes. 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.

[0085] In some embodiments, the systems described herein may include a plurality of cells. Any of a variety of cell types are possible within the plurality of cells of the system, in accordance with some embodiments. Non-limiting types of cells include muscle cells, stem cells, bone cells, blood cells, nerve cells, immune cells, HEK cells, and fat cells. In some embodiments, the cells within the system may be diseased and / or in need of a treatment via a therapeutic agent, for example, cancer cells.

[0086] According to some embodiments, the cells may originate from a subject. In some embodiments, a system containing the plurality of cells and / or a plurality of asymmetric liposomes may include a solution. The solution may contain the plurality of cells and / or a plurality of asymmetric liposomes, in some embodiments. In some embodiments, the solution may be aqueous. For example, in some embodiments, the solution may be blood, a cell media, phosphate buffer solution, or the like. For instance, a system may comprise a subject having cancer (or another disease or condition) and a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer and contained within the subject’s blood. In some embodiments, the solution may be nonaqueous.

[0087] A subject, in some embodiments, refers to an individual organism such as a human or animal. In some embodiments, the subject is a mammal, e.g., a human, a domesticated animal, or a laboratory animal. In some embodiments, the subject is a human. According to some embodiments, the subject is a rodent, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a pig, or a sheep.

[0088] Some aspects of the present disclosure are generally related to methods of making the systems described herein, e.g., that contain a plurality of asymmetric liposomes. In some embodiments, the methods relate to making asymmetric liposomes. According to some embodiments, the methods comprise providing an emulsion. In some embodiments, providing an emulsion may comprise providing a water-in-oil emulsion, where the droplets of the emulsion are stabilized by a lipid (e.g., a first lipid). In some embodiments, the size of the droplets of the emulsion, as well as the identity of the lipid, are described elsewhere herein. In some embodiments, providing an emulsion may comprise providing a water-in-oil emulsion, where the droplets of the emulsion are stabilized by a polymer (e.g., a first polymer).

[0089] In some embodiments, the method may comprises extruding an emulsion. In some embodiments, extruding an emulsion comprises extruding a water-in-oil emulsion stabilized by a lipid (e.g., a first lipid) through a membrane having an average pore size of less than or equal to 200 microns. In some embodiments, extruding an emulsion comprises extruding a water-in-oil emulsion stabilized by a polymer (e.g., a first polymer) through a membrane having an average pore size of less than or equal to 200 microns. According to some embodiments, the extruding of the emulsion may result in the droplets of the emulsion having a size distribution as described elsewhere herein for a plurality of asymmetric liposomes (e.g., the average maximum dimension and / or the uniformity of the distribution). In some such embodiments, the emulsion may be extruded through a membrane, a pipette, a needle, and / or a nozzle. Other media through which the emulsion may be extruded are possible. In some embodiments, it may be advantageous to use a membrane for membrane extrusion.

[0090] In some embodiments, where an emulsion is passed through a membrane to obtain an emulsion having a size distribution as described elsewhere herein, the pores of the membrane may be similarly sized to desired average size of the droplets of the emulsion. According to some embodiments, the pore size of the membrane may be larger than the desired average size for the droplets of the emulsion. For example, in some embodiments, an average pore size of a membrane through which the droplets of the emulsion is extruded is greater than or equal to 50 nm, greater than or equal to 75 nm, greater than or equal to 100 nm, greater than or equal to 200 nm, greater than or equal to 300 nm, greater than or equal to 500 nm, greater than or equal to 750 nm, greater than or equal to 1 micron, greater than or equal to 1.5 microns, greater than or equal to 2 microns, greater than or equal to 2.5 microns, greater than or equal to 3 microns, greater than or equal to 5 microns, greater than or equal to 10 microns, greater than or equal to 25 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns greater than or equal to 125 microns greater than or equal to 150 microns greater than or equal to 175 microns. In some embodiments, an average pore size of a membrane through which the droplets of the emulsion is extruded is less than or equal to 200 microns, less than or equal to 175 microns, less than or equal to 150 microns, less than or equal to 125 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 25 microns, less than or equal to 10 microns, less than or equal to 5 microns, less than or equal to 3 microns, less than or equal to 2.5 microns, less than or equal to 2 microns, less than or equal to 1.5 microns, less than or equal to 1 micron, less than or equal to 750 nm, less than or equal to 500 nm, less than or equal to 300 nm, less than or equal to 200 nm, less than or equal to 100 nm, or less than or equal to 75 nm. Combinations of the foregoing ranges are possible (e.g., greater than or equal to 50 nm and less than or equal to 200 microns). Other ranges are also possible.

[0091] In some embodiments, the method comprises forming asymmetric liposomes. In some embodiments, where an emulsion of water-in-oil droplets stabilized by a polymer (e.g., a first polymer) is provided, the emulsion may then be used to form asymmetric liposomes. In some embodiments, wherein an emulsion of water-in-oil droplets stabilized by a lipid (e.g., a first lipid) is provided, the emulsion may then be used to form asymmetric liposomes. For example, in some embodiments, forming asymmetric liposomes comprises passing the water-in-oil droplets through a layer of a second lipid. In some such embodiments, the first and second lipid are different. In some embodiments, forming an asymmetric liposome comprises passing the extruded emulsion through a monolayer comprising a second lipid different from the first lipid. Passing the extruded emulsion through a layer of a second lipid may proceed via the application of a force, e.g., via centrifugation, a pressure differential, gravity, etc., in accordance with some embodiments. In some embodiments, the layer comprising the second lipid may be formed at an interface between an oil phase (e.g., containing the extruded emulsion) and an aqueous phase. Thus, in some embodiments, transferring the extruded emulsion to the aqueous phase may result in the forming of asymmetric lipids. Additionally, while the methods disclosed herein generally describe forming asymmetric liposomes in the context of a first and second lipid, it will be appreciated that the first lipid may be replaced with a first plurality of lipids and the second lipid may be a second plurality of lipids, as long as the compositions of the first and second plurality of lipids differ and yield asymmetric liposomes. In some embodiments, the second lipid may be a polymer (e.g., a first or second polymer) For instance, in some embodiments, water-in- oil droplets stabilized by a polymer (e.g., a first polymer) may be passed through a membrane comprising a second polymer. In still other embodiments, water-in-oil droplets stabilized by a lipid may be passed through a membrane comprising a polymer. Other combinations of polymers and lipids for the asymmetric liposomes are contemplated, as this disclosure is not so limited.

[0092] An example method of making asymmetric liposomes is shown in FIG. 2A, where a water-in-oil droplet 200 stabilized by lipid A is passed through a membrane 210 to obtain a plurality of water-in-oil droplets 220 having an average size. The solution containing the plurality of water-in-oil droplets 220 is then centrifuged to pass the plurality of water-in-oil droplets 220 through a layer of lipid B, thereby forming a plurality of asymmetric liposomes 230.

[0093] Some aspects of the present disclosure are generally directed to methods of using the systems described herein, e.g., which may include asymmetric liposomes. In some embodiments, use of a composition to treat a subject, e.g., a subject having cancer. For instance, the composition may include a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer, or another disease or condition. According to some embodiments, the method may include exposing a plurality of cells to a plurality of asymmetric liposomes. In some such embodiments, the plurality of cells may be cancer cells, and the plurality of asymmetric liposomes may encapsulate a therapeutic agent for treating the cancer. Other cells are also possible in other embodiments, e.g., disease cells. Advantageously, in some embodiments, the plurality of asymmetric liposomes may encapsulate any of a variety of therapeutic agents as described elsewhere herein, and the method may comprise exposing the plurality of asymmetric liposomes to a plurality of cells in need thereof of the therapeutic agent.

[0094] In some embodiments, exposing the plurality of cells to a plurality of asymmetric liposomes may occur in vitro. According to some such embodiments, the exposing of the cells to the asymmetric liposomes may be performed in order to determine an effectiveness of treating the type of cells with the asymmetric liposomes, e.g., and / or a therapeutic agent encapsulated therein, e.g., to treat a disease or condition. For example, cancer cells may be exposed to asymmetric liposomes containing a therapeutic agent, e.g., an experimental therapeutic agent intended for treating cancer. In some embodiments, cancer cells may be exposed to asymmetric liposomes containing a therapeutic agent suitable for treating cancer. Other types of cells may be exposed to the asymmetric liposomes in vitro, in some embodiments, and may be selected based on the therapeutic agent encapsulated by the plurality of asymmetric liposomes.

[0095] In some embodiments, a plurality of asymmetric liposomes may be exposed to cells in vivo, e.g., within a subject. Similarly, to the in vitro exposure, the plurality of asymmetric liposomes encapsulating a therapeutic agent may be exposed to a plurality of cells in need of the therapeutic agent encapsulated therein, in some embodiments. For instance, a plurality of cancer cells (or other disease cells) may be exposed to a plurality of asymmetric liposomes encapsulating a therapeutic agent suitable for treating cancer. According to some embodiments, the plurality of asymmetric liposomes and / or the therapeutic agent encapsulated therein may be biocompatible, e.g., in some cases, the liposomes and / or the therapeutic agent do not invoke an adverse reaction within the subject and / or the reaction is medically acceptable in view of the treatment provided by the therapeutic agent.

[0096] Exposing a plurality of cells to a plurality of asymmetric liposomes may proceed by any of a variety of methods, in accordance with some embodiments. For example, in vitro exposure may proceed by introducing solution containing a plurality of asymmetric liposomes to the plurality of cells. In some such embodiments, introducing the solution may proceed by using a pipette, a needle, or pouring the solution onto a substrate containing the cells and / or into a solution containing the cells. Other methods are possible, as this disclosure is not so limited. In vivo exposure, in accordance with some embodiments, may proceed by administering the plurality of asymmetric liposomes to the subject containing the plurality of cells. In some embodiments, administering the plurality of asymmetric liposomes may proceed by injecting the asymmetric liposomes into the subject, having the subject ingest the plurality of asymmetric liposomes, and / or topically administering the plurality of asymmetric liposomes to the subject. The asymmetric liposomes may also be administered by any route that results in a therapeutically effective outcome, including but not limited to intradermal, intramuscular, intranasal, and / or subcutaneous administration. Other suitable methods of administering a composition to a subject in need thereof are also possible, as this disclosure is not so limited. In some embodiments, the method includes administering a plurality of asymmetric liposomes to the subject having cancer, or another disease or condition wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the cancer, disease, or condition.

[0097] Examples of cancer that a subject may have include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS- associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen’s disease and Paget’s disease; liver cancer; lung cancer; lymphomas including Hodgkin’s disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi’s sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma, teratomas, choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms’ tumor. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer. In general, an effective amount of the one or more compositions for treating cancer will be that amount necessary to inhibit mammalian cancer cell proliferation in situ. Those of ordinary skill in the art are well- schooled in the art of evaluating effective amounts of anti-cancer agents.

[0098] In some embodiments, the method includes administering a plurality of asymmetric liposomes to the subject having an autoimmune disease or condition. In some embodiments, when administering the asymmetric liposomes to a subject having an autoimmune disease or condition, the therapeutic agent may be configured for gene editing. In some embodiments, when the therapeutic agent is configured for gene editing, the therapeutic agent may be suitable for treating the autoimmune disease or condition. For instance, following administering the asymmetric liposomes to the subject, the therapeutic agent may be configured to cut DNA of the subject within the subject, to knock-in a gene in the subject, and / or to knock-out a gene in the subject. Examples of autoimmune diseases or conditions that may be suitable for treatment by a therapeutic agent configured for gene editing include, but are not limited to, diabetes (e.g., type 1 diabetes), rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, celiac disease, vitiligo, and psoriasis. Other autoimmune diseases or conditions are also possible. In some such embodiments, the liposomes may be at least a portion of a composition that is used to treat a subject. In some embodiments, the liposomes may encapsulate a therapeutic agent suitable for treating cancer, and the liposomes may accordingly be at least a portion of a composition used to treat a subject having cancer. Additionally, as described above, various therapeutic agents may be encapsulated by asymmetric liposomes, and in some such embodiments, the liposomes may be at least a portion of the composition that is used to treat a subject in need of the therapeutic agent encapsulated therein.

[0099] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 552,502, filed February 12, 2024, entitled “Asymmetric Liposomes for Drug Delivery and Other Applications,” by Weitz, et al., and of U.S. Provisional Patent Application Serial No. 63 / 572,453, filed April 1, 2024, entitled “Asymmetric Liposomes for Drug Delivery and Other Applications,” by Weitz, et al., each of which is incorporated herein by reference in its entirety.

[0100] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

[0101] EXAMPLE 1 The following example describes the formation of asymmetric liposomes and various properties thereof, in accordance with one embodiment.

[0102] Lipid vesicles are aqueous compartments formed by amphiphilic lipid molecule bilayers. Under a balanced osmotic pressure, lipid vesicles are spherical and find widespread use as drug delivery vehicles to protect an encapsulated drug from degradation. Their lipid membrane properties determine the encapsulation efficiency, cell uptake and the release of drugs into the cell. In total, small vesicles have been successfully implemented in 14 FDA-approved liposomal products. However, lipid vesicles are generally symmetric, having the same lipid composition in each membrane leaflet, which restricts the degree of tailoring vesicle properties for drug delivery. This not only limits ongoing progress in the field but also reduces treatment efficiency and increases cost. Only a few examples show the formation of small asymmetric vesicles using the enzymatic conversion of lipids on the outer leaflet or by exchanging lipids across leaflets after vesicle formation. However, they do not investigate the impact of leaflet asymmetry for drug delivery because of the already complex formation methods. The inverted emulsion technique is a different approach to engineer asymmetric vesicles, where the leaflets are assembled independently. This approach offers more flexibility and full leaflet programmability because it does not rely on specific enzymes fueling the asymmetry.

[0103] Here, the assembly of nanoscale lipid vesicles with asymmetric leaflets using the inverted emulsion technique coupled to an extrusion step is described. Water-in-oil emulsions were extruded and centrifuged through a second lipid monolayer to form small asymmetric lipid vesicles. The leaflet asymmetry was verified of up to 99% using fluorescence quenching measurements. It was determined that leaflet asymmetry could improve lipid vesicle uptake by cells, the mRNA transfection efficiency, and reduce cell cytotoxicity. The uniqueness of asymmetric lipid vesicles broadens the parameter space for the engineering of drug delivery vehicles and leads to many potential applications, including the formation of more efficient drug carriers with reduced toxicity. Results & Discussion

[0104] Nanoscale lipid vesicles with asymmetric leaflets were developed using an inverted emulsion technique combined with an extrusion step. The process was initiated with an inverted emulsion consisting by water droplets dispersed in mineral oil, which was stabilized by lipids serving as the inner leaflet. Subsequently, water / mineral oil emulsions were extruded through a nanoscale film to generate nano-sized emulsions, the size of the emulsion was tunable by using polycarbonate membranes with different diameters, as shown in FIG. 2A. These emulsions were then centrifuged through a second lipid / water monolayer to produce small asymmetric lipid vesicles, schematized in FIG. 2A. To confirm the nanoscale structure of the asymmetric lipid vesicles, dynamic light scattering was used to measure their size. The diameter of asymmetric POPC-POPS lipid vesicles made by 200 nm membrane is 300 nm, as shown in FIG. 2B. The asymmetry property of the lipid vesicles was assessed through a quenching assay, where the outer leaflet is labeled with a NBD-PC fluorescent probe. The fluorescent intensity decreased to approximately 2% after the addition of the dithionite quenching assay, further reducing to 1% after the addition of Triton X to completely destroyed the lipid membrane. This result suggests that a 90% asymmetry could be achieved. Further, the asymmetric properties of various lipid vesicles, are shown in FIG. 2D. The vesicles’ asymmetry stability over time as well was assessed. Comparing to the results at 0 hours (circle), the quenching assay demonstrated consistent effects after 24 hours, providing evidence for the stability of the lipid vesicles’ asymmetry, as illustrated in FIG. 2C. The asymmetry of the liposomes could be influenced by changing the medium. For example, in the presence of sucrose, the asymmetry of vesicles diminished, as shown in FIG. 2E. However, the presence of ions did not influence the asymmetry, as illustrated in FIGS. 2F-2L.

[0105] Lipids are typically classified according to the charge of their headgroup into anionic, neutral, and cationic. The yield is a key property of lipid vesicles and critically depends on the choice of lipids. To comprehensively investigate how the choice of lipids influences the yield, POPS (anionic), POPC (neutral), and DODMA (cationic)were utilized to design a large variety of possible lipid vesicles. The formation method of lipid vesicles is closely related to the interfacial properties between water with lipids. So, before the lipid vesicles were fabricated, the interfacial tension of water in oil with the presence of lipids was assessed. A pendant drop method to measure the interfacial tension between the water and lipids in mineral oil was used, as schematized in FIG. 3A. The interfacial tension between water / POPS is lower than water / POPC and water / DODMA, as shown in FIG. 3B. During the formation of asymmetric lipid vesicles, it was crucial for the lipid-stabilizing emulsions to translocate from the oil phase to the water phase under centrifugation. The more emulsions that successfully entered the water phase, the higher the yield was. Therefore, a partitioning analysis was performed to examine the accumulation of POPS, POPC and DODMA lipid molecules at the Rhodamine 6G in water-oil interface. The Rhodamine 6G molecules diffused from water to oil phase and lipids molecules encapsulate the Rhodamine 6G molecules to form micelles, as schematized in FIG. 3C. As the Rhodamine 6G molecules moved from the water to the oil phase, the fluorescent concentration in the water phase decreased over time, the interfacial tension of POPS was a smaller than that of POPC and DODMA, POPS more easily formed micelles compared to POPC and DODMA. This was reflected in the decrease in fluorescent intensity between water / POPS in oil in FIG. 3D. To understand the relationship between fluorescent intensity and lipid concentration, different POPC-POPC lipid vesicles were measured via extrusion. A linear relationship between lipid concentration and fluorescent intensity was found, which helped determine the asymmetric lipid vesicles’ concentrations according to the correlation.

[0106] By employing this method, lipid vesicles with arbitrary compositions and charges in both the inner and outer leaflets could be fabricated. For example, even the inner cationic-outer anionic (POPS-DODMA) and inner anionic-outer cationic lipid vesicles could be fabricated. The yield of the lipid vesicles was calculated according to the fluorescent intensity. Compared to POPC and DODMA, POPS in outer leaflet had the highest concentration. This result was consistent with the interfacial tension and partition results, indicating that POPS, being more hydrophilic, had a greater propensity to form lipid vesicles, as illustrated in FIG. 3E. Further, it was observed that the presence of the ions would decrease the yields, as shown in FIGS. 2M-2Q. For example, the same phenomenon was observed when using MgCh with NaCl as the medium, as shown in FIGS. 2M-2N. Although the yield of lipid vesicles was relative to the lipid choice, the method make designing lipid vesicles with arbitrary composition and charges was possible.

[0107] Engineering asymmetric SUVs

[0108] Small lipid vesicles have emerged as promising carriers vehicles for a variety of nanomedicine applications. However, the lipid vesicles for drug delivery are all symmetric structures. The drug delivery potential of asymmetric lipid vesicles was explored by investigating their cell uptake by Human Embryonic Kidney (HEK) cells. The liposomes were labeled with a fluorescent lipid Lissamine Rhodamine B to evaluate the liposome uptakes. The prepared liposomes with a lipid concentration of 25 micromolar were added to HEK cells and then the liposome uptake was observed 24 hours later. There were differences of liposome uptake between POPC-POPC, POPS- POPS and POPC-POPS, as shown in FIG. 4A through confocal microscope images. The asymmetric POPC-POPS highest liposome uptake efficiency compared to symmetric POPS-POPS and POPC-POPC. To evaluate the liposomes uptake efficiency, flow cytometry was used. Both the confocal image analysis and flow cytometry results indicated that POPC-POPS liposomes demonstrated a greater cell uptake efficiency than POPC-POPC and POPS-POPC, as shown in FIGs. 4B and 4C. The uptake of liposomes with a larger stiffness was easier for the HEK cells than the uptake of ones with smaller stiffness. Soft liposomes were easily to deformed during endocytosis, which increases the contact areas between liposomes with cell membrane and larger energy for cells to uptake liposomes. It is possible that asymmetric liposomes have a larger Young’s modulus thus more stiffness. Additionally, it was determined that POPS on the outer leaflet increased the uptake compared to POPC on the out leaflet. To further support this, symmetric POPC-POPC and POPS-POPS liposomes formed via the extrusion method were added to HEK cells, and it was determined that the cellular uptake of POPS-POPS was much larger than the one of POPC-POPC (FIGS. 4D-4E). additionally, FIG. 4F shows the GFP fluorescence intensity measured as a function of lipid composition of the membranes of the liposomes.

[0109] The superior cell uptake efficiency of asymmetric lipid vesicles compared to symmetric lipid vesicles suggests they can be used as carriers for biomolecules like mRNA. To assess mRNA transfection efficiency, GFP-encoding mRNA (GFP-mRNA) was encapsulated in these liposomes. Upon cell uptake, the liposomes underwent endocytosis. Once the liposomes successfully escaped from the endosomes, the mRNA was released from the liposomes and translated into fluorescent proteins, as schematized in FIG. 5A. Upon introducing GFP mRNA-loaded POPC-POPS, POPS-POPS, and POPC-POPC liposomes to HEK cells, mRNA transfection was observed after 48 hours with a fluorescent confocal microscope. Cells that were successfully transfected with GFP-mRNA expressed the protein and displayed fluorescence, as shown in FIG. 5B. Subsequently, the mRNA transfection efficiency was calculated by determining the percentage of fluorescent cells relative to the total cell number. The transfection efficiency of POPC-POPS was 9 times higher than POPC-POPC and POPS-POPS, as shown in FIG. 5C. The HEK cells were able to uptake more POPC-POPS, leading to a higher mRNA has more chance to expressed by HEK cells, which is consistent with the result of the liposome uptake experiment. FIGS. 5E-5G show similar data to FIGS. 5B- 5D, but include the asymmetric POPS-POPC.

[0110] The cell plasma membrane is asymmetric, with various lipids in inner and outer leaflet. Conventional symmetric lipid vesicles taken up by cells cause disruption of the cell membrane integrity leading to cell apoptosis. To determine, if asymmetric lipid vesicles could reduce the damage to cell membrane, both the symmetric and asymmetric liposomes were added to HEK cells and the cell cytotoxicity was assessed using the LDH method. Although POPC-POPS exhibited the highest cell uptake efficiency, its cytotoxicity was significantly lower than that of POPS-POPS, as depicted in FIG. 5D. Without wishing to be bound by any particular theory, it is believed that this difference may arise from the fusion of cell membranes with lipid bilayers during liposome uptake. Asymmetric lipid vesicles resulted in a reduced cell cytotoxicity. Overall, the properties of asymmetric lipid vesicles (e.g., as compared to symmetric vesicles) revealed an increased cell uptake efficiency, enhanced mRNA transfection, and lower toxicity makes them efficient and unique drug delivery carriers that offer full programmability over their biochemical and biophysical properties.

[0111] Conclusion

[0112] This technique offers a systematic approach to design nano-scale asymmetric lipid vesicles with distinct lipid compositions in inner and outer leaflet. In comparison to symmetric lipid vesicles, this data indicates that asymmetric leaflet configuration enhances lipid vesicle uptake by cells, improves mRNA transfection efficiency, and reduces cell cytotoxicity. The ability to design nanoscale asymmetric lipid vesicles opens up important applications for drug delivery. The asymmetric lipid vesicles hold great potential for drug delivery with enhancing encapsulation efficiency. They also allow to encapsulate cargo beyond nucleic acids like proteins or small molecules. Their asymmetric structures allow for the utilization of arbitrary lipid compositions and screening a large library of lipid compositions. Additionally, these vesicles can mimic cell plasma membrane structures, further enhancing their compatibility and potential applications in drug delivery systems.

[0113] Experimental Section

[0114] Emulsion preparation and extrusion

[0115] To form asymmetric lipid vesicles, a mineral oil (Sigma Aldrich) solution was prepared for the inner and outer leaflets. Each solution contained a concentration of 0.0125 mg / mL of the respective lipid. The lipids used were l-palmitoyl-2-oleoyl-sn- glycero-3-phospho-L-serine (POPS), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), l,2-dioleyloxy-3-dimethylaminopropane (DODMA), l,2-dioleoyl-sn-glycero-3 phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (LissRhod-PE), 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-l,3-benzoxadiazol-4-yl) (NBD-PE). Lipids were purchased from Av anti Polar Inc., dissolved in chloroform and stored at -20 °C until use. Lipids were dried under a stream of nitrogen and resuspended in mineral oil. The lipid composition contained 99 mol% of POPS, POPC or DODMA, respectively, and 1 mol% LissRhod-PE, if not stated otherwise. To facilitate the resuspension, the mineral oil solutions were sonicated and incubated for 45 minutes. Then lipid- stabilized emulsion was formed by mixing 10 pL lx PBS (Gibco) with 500 p L mineral oil / lipid solution. The mixture was briefly vortexed to from water-in-oil droplets. Subsequently, the droplets were extruded nine times (Avanti Polar) through a polycarbonate membrane with a diameter of 100 nm (unless otherwise specified). After extrusion, the droplets were incubated for 2 hours to form a closely-packed lipid monolayer. In the meantime, 500 pL of the second mineral oil / lipid solution was layered on top of 500 pL lx PBS and also left to incubate for 2 hours. Then, 100 ul of the droplets were added on top of the second oil solution and centrifuged for 10 minutes at 10 000 g. Due to the density gradient between water and mineral oil, the lipid-stabilized emulsions were centrifuged through the lipid monolayer at the oil-water interface and dissolved in the aqueous phase. Finally, the aqueous phase containing asymmetric vesicles was pipetted out with a syringe needle (Air-Tite) and stored in LoBind tubes (Eppendorf) for up to one day. Typically, vesicles were used immediately after their formation.

[0116] Dynamic light scattering

[0117] The hydrodynamic diameter of liposomes was determined with a ZetaSizer Nano ZS instrument (Malvern Pananalytical) that utilizes a He-Ne laser with a wavelength of 632 nm and applies the phase analysis light scattering technique. Lipid vesicles were diluted 1:10 for corresponding hydrodynamic diameter measurements by placing 100 pL of dispersion in ZEN0040 disposable plastic microcuvettes. The size measurement results from DLS measurements are displayed as an intensity weighted distribution function of particle size, with determined intensity weighted Z average (mean size) and the poly dispersity index (PDI). Example DLS measurements are shown in FIG. 5H. FIG. 61 shows the average diameter of emulsion droplets as a function of the pore size of the membrane used during extrusion of the emulsion.

[0118] Quenching assay

[0119] The leaflet asymmetry was measured using fluorescence quenching experiments. For these, 1 mol% fluorescent lipid tracer molecule (NBD-PE, Avanti) was added to the outer leaflet. FIG. 5J shows example fluorescence emission from lipi membranes containing a fluorescence tracer (MemGlow). After vesicle formation, 100 pL of vesicles were placed in a well and its fluorescence measured every minute with a microplate reader. The excitation wavelength was set to 465 nm and the emission was detected at 536 nm. After three minutes, 10 pL of 200 mM sodium dithionite (Sigma Aldrich) were added to the vesicles. Dithionite quenches NBD-fluorophores and leads to a reduction of fluorescence intensity, when NBD is present in the outer leaflet. NBD on the inside of liposomes is not quenched. After 15 minutes, the quenching reaction saturated. To prove that the remaining fluorescence originates from NBD-PE in the inner leaflet of liposomes, 5 pL of Triton-X were added to disrupt the liposome stability and expose the remaining NBD to dithionite quenching.

[0120] Pendant drop

[0121] The interfacial tension between mineral oil and lx PBS was measured using the pendant drop method (Droplet Lab). The respective lipid was dissolved in mineral oil at a concentration of 0.06 mM. Since mineral oil has a lower density than water, an oil droplet was pushed into the aqueous solution using a syringe needle. The trace of the oil droplet was detected and analyzed with a Young-Laplace fit to determine the interfacial tension.

[0122] Partitioning assay

[0123] Rhodamine 6G (Sigma Aldrich) was dissolved to a concentration of 1 mM in lx PBS. To study the partitioning of lipids and micelle formation, 1 ml of this solution was placed in a cuvette and 1 ml of the respective mineral oil / lipid solution was layered on top (FIG. 3F). The cuvettes were imaged after 0, 3, 6, 12 and 24 hours. In addition, 100 pL of the aqueous solution was removed at each time point and placed in a 96-well plate. Its absorbance and fluorescence emission spectrum after excitation with 470 nm measured. Determination of vesicle concentration

[0124] The vesicle concentration was measured using 1 mol% of a rhodamine-labeled fluorescent lipid in the lipid composition. Following the centrifugation step, 100 pL of the vesicle solution was placed in a 96-well plate. The fluorescence intensity of each well was measured with a microplate reader (Tecan Spark) using an excitation wavelength of 540 nm and detecting the emission at 570 nm. The fluorescence intensity is proportional to the vesicle concentration and was used to normalize the amount of vesicles that was added to cells. Cell culture

[0125] Human embryonic kidney (HEK) cells are dispersed in 10 mL of Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10 % fetal bovine serum and 1 % pennicilin / streptomycin in a 75 mL flask. The cells are kept in an incubator at 37 °C and 5% CO2. Every 48 hours the media is exchanged by aspirating the media using a vacuum and washing the cells with 10 mL of lx phosphate buffered saline (PBS) upon which 10 mL of fresh media is added. When the cell density was around 80%, the cells were passaged. The media was removed and 2.5 mL of trypsin is added in the flask for 3 min to detach cells from the bottom of the flask. Then the flask was taken out of the incubator and 5 mL of media are added. The cells suspension was centrifuged for 5 min at 500 g. Once the cells were concentrated at the bottom of the tube, the media and trypsin were aspirated and the cells resuspended in fresh media. The cell concentration was measured before centrifugation to calculate the volume of media that needs to be added at this step to tune the cell concentration used in the uptake and transfection experiments.

[0126] Confocal microscopy

[0127] Confocal microscopy was performed on a confocal laser scanning microscope (LSM980 or LSM900) by Zeiss (Carl Zeiss AG). The pinhole was set to 1 airy unit and imaging was performed at 37 °C and 5 % CO2. Lor image acquisition a 20x air (Plan- Apochromat 20x / 0.8 M27), a 40x water immersion objective (Plan- Apochromat 40x / 1.0 DIC M27) and a 63x oil immersion (HC PL APO 63x / l,40 OIL CS2) were used. Images were analyzed and adjusted in ImageJ.

[0128] Liposome uptake and. mRNA transfection

[0129] For the cell experiments, 12 or 24 well plates were used with cell concentrations of approximately LOxlO5cells / mL. The first step of the cell experiments was to seed the cells on day 0 in the wells, then add freshly made SUVs on day 1 and observe them with confocal microscopy on days 2, 3 and 4 after having exchanged the media to remove all remaining SUVs that had not been yet taken up by the cells. Before addition of liposomes, their concentration was measured with the microplate reader. This was used to normalize the amount of vesicles added to each well. Typically, 100-200 pL of liposomes were added to each well. To measure the amount of liposome uptake per cell, the contour of 20 cells per image was determined using ImageJ. Then the mean vesicle fluorescence intensity inside each cell is measured. The mean for the 20 cells per well as the SD is then calculated for each condition. To measure the cell transfection efficiency, 2.5 ul GFP-encoding mRNA was added to the aqueous phase before the extrusion of emulsion droplets. Confocal images were taken on day 1, 2 and 3. For each well, multiple images were taken and for each image the following ratio was determined measured: Ntransfected / (Ntransfected + Nuntransfected). Multiple ratios per well were obtained to calculate the mean transfection ratio and standard deviation for each condition. Complementing the GFP fluorescence, LissRhod PE was used as fluorescent lipid. Flow cytometry

[0130] Following the liposome uptake, cells were trypsinized and concentrated to 10 x 106 ccll / mL. Flow cytometry was performed using a BD FACSymphony A3 Lite (BD Biosciences) instrument with laser wavelength 561 nm. The data was analyzed with FCS express software. Cells were gated to remove cell debris (FSC-A / SSC-A). For each condition 20000 cells were analyzed. Cell cytotoxicity assay

[0131] Cell cytotoxicity was determined using a CyQUANT LDH cytotoxicity assay (Thermo Fisher). Following the manufacturers protocol, cells were first measured the LDH activity as a function of the cell number revealing an ideal cell number of 2500 cells / well. Cells were seeded in triplicates per condition and the respective liposomes added after 24 hours. After another 24 hours, the spontaneous, maximum and liposome-induced LDH activity was measured with a plate reader for each condition. The cytotoxicity was then calculated according to: % cytotoxicity = 100 * ([Liposome-induced LDH -spontaneous LDH] / [maximum LDH -spontaneous LDH]) Statistical Analysis

[0132] All the experimental data in this example were reported as mean +SD from n experiments. The respective value for n is stated in the corresponding figure captions. All experiments were repeated at least twice. To analyze the significance of the data, a Student’ s t-test with Welch’ s correction was performed using Prism GraphPad (Version 9.1.2) and p-values correspond to ****: p <0.0001, ***: p <0.001, **: p <0.01, *: p <0.05 and ns: p >0.05.

[0133] EXAMPLE 2

[0134] The following example describes the formation of asymmetric liposomes and various properties thereof, in accordance with one embodiment.

[0135] The delivery of therapeutics to cells is crucial for the treatment and prevention of diseases. To enhance targeting and protect therapeutics from degradation, they are often encapsulated in drug delivery vehicles like lipid nanoparticles, liposomes and viral vectors. However, there is no universal vehicle for all cargo types including small molecules, nucleic acids and proteins. In this Example, a method for engineering lipid vesicles with asymmetric leaflets is presented and the ability of the vesicles to deliver mRNA and proteins to cells is demonstrated. The results show that leaflet asymmetry enhances vesicle uptake by cells and increases the transfection efficiency with mRNA up to 5-fold. Additionally, that asymmetric vesicles are shown to be able to deliver a variety of proteins. In particular, the delivery of Cas9 proteins and Cas9 / sgRNA complexes for gene-editing is demonstrated. This Example describes some of the design parameters for drug delivery vehicles, facilitating more efficient, universal drug carriers and protein delivery.

[0136] Introduction

[0137] To protect therapeutics from degradation, evade immune clearance, and facilitate their uptake by target cells, drugs are often packaged into delivery vehicles. Following the uptake by cells, these drug delivery vehicles release the drug into the cytoplasm. The drugs can vary widely in size and their biochemical properties, ranging from relatively simple small molecules to nucleic acids and even proteins such as the gene-editing complex CRISPR / Cas9. As drugs and therapeutics have become more complex, so have their delivery vehicles, due to the increasing challenges posed by the large sizes and intricate charge properties of modern drugs.

[0138] Lipid nanoparticles, which have been prominently used for the C0VID19 vaccines, are extremely successful because they can encapsulate negatively charged nucleic acids such as mRNA. The electrostatic interactions of positively charged lipids and negatively charged cargo leads to the formation of nanoparticles with high encapsulation efficiencies. However, lipid nanoparticles still face limitations in delivering neutral or positively charged therapeutics. Additionally, a change in the lipid composition affects the encapsulation efficiency and lipid nanoparticle size. This can be particularly important for the encapsulation and delivery of more complex cargos such as proteins. For example, proteins perform of a vast array of functions within organisms and, unlike mRNA or DNA plasmids, they do not require cellular expression and are immediately active upon delivery. However, proteins are often larger than nucleic acids and have an intricate charge profile, making their encapsulation into lipid nanoparticles particularly difficult. The CRISPR / Cas9 ribonucleoproteins typically lack the negative charge density needed for efficient encapsulation into lipid nanoparticles with cationic lipids, making it challenging to find or to engineer useful Cas9 proteins.

[0139] By contrast, viral vectors are an alternate type of delivery vehicle and do not rely on electrostatic interactions to form. Instead, they use natural virus capsids as compartment to host protein cargo. They are very promising for the delivery of proteins and have been used to efficiently transfect cells. However, they are difficult to produce in large-scales and are often immunogenic, leading to an elevated immune response and clearance by the liver. Moreover, their size is set by the virus capsid and cannot be easily tuned causing many viral vectors to either be too small for the efficient loading with proteins or too large to transfect cells in vivo.

[0140] Lipid vesicles are a third compartment type formed by amphiphilic lipid molecule bilayers. They can be assembled at high concentrations and in large quantities, while having complete control over their size which can range from 30 nm to 100 pm. Even though their charge can be tuned through the lipid composition, the encapsulation of therapeutics is challenging because the cargo is typically loaded after the vesicle has formed. Therefore, lipid vesicles are currently only used for small molecules that can permeate the membrane upon chemical manipulation. Lipid vesicles have been implemented in 14 US Food and Drug Administration (FDA)-approved treatments for small molecules, but the delivery of more complex cargo such as mRNA or proteins is difficult. Thus, while these delivery vehicles have important applications, none provide universal cargo delivery, such as encapsulating arbitrary macromolecular cargo and maintaining size control. A universal cargo delivery vehicle is desirable, it would facilitate the treatment of many diseases that are currently out of the scope of conventional delivery vehicles.

[0141] In this Example, lipid vesicles capable of encapsulating and transfecting cells with therapeutics, including proteins and nucleic acids, are engineered. To achieve this, nanoscale lipid vesicles with asymmetric leaflets were assembled using an inverted emulsion technique. A water-in-oil emulsion was extruded to make the vesicles sufficiently small, whereafter they are centrifuged through a second lipid monolayer to form small asymmetric lipid vesicles. Leaflet asymmetry of the vesicles of up to 93% was confirmed using fluorescence quenching experiments. Remarkably, leaflet asymmetry was found to improve lipid vesicle uptake by cells, the mRNA transfection efficiency, reduce cell cytotoxicity, and facilitate the delivery of a wide range of proteins to cells. In particular, the delivery of Cas9 proteins and Cas9 / sgRNA complexes for gene-editing is demonstrated. The uniqueness of asymmetric lipid vesicles broadens the parameter space for the engineering of drug delivery vehicles and is useful for many applications, including the formation of more efficient drug carriers with reduced toxicity while also facilitating new treatment options that require the delivery of proteins.

[0142] Results & Discussion

[0143] Nanoscale lipid vesicles with asymmetric leaflets were engineered using an inverted emulsion technique coupled to an extrusion step. An inverted emulsion consisting of water droplets dispersed in mineral oil was formed, which contains lipids as surfactants. The lipids form a monolayer that stabilizes the water-in-oil droplet and serves as the inner leaflet of the lipid vesicle. Subsequently, the water-in-mineral oil emulsions is extruded through a polycarbonate membrane with holes of a fixed diameter to generate monodisperse nanoscale emulsions. These emulsions were then centrifuged through a second lipid monolayer at a second water / oil interface to produce asymmetric lipid vesicles, schematized in FIG. 2A. This method yields asymmetric vesicles because both monolayers form independently as they are spatially separated until the centrifugation step. The diameter of asymmetric lipid vesicles can be adjusted by using polycarbonate membranes (e.g., or other types of membranes) with different pore sizes. For example, with a 200 nm membrane, the hydrodynamic vesicle diameter is about 250 nm; by using a 100 nm membrane we reduce the vesicle diameter to 150 nm, whereas a 30 nm membrane yields vesicles with a hydrodynamic diameter of 65 nm, as shown in FIG. 6A. In addition to the scattering experiments, the vesicle size was also validated with transmission electron microscopy (TEM).

[0144] An advantage of this method for forming vesicles is that it yields asymmetric vesicles. The degree of asymmetry with a dithionite quenching assay, where the outer leaflet is labeled with an NBD-PE fluorescent probe. NBD is quenched upon exposure with membrane impermeable dithionite, allowing the quantification of the amount of lipids that has exchanged to the inner leaflet. The fluorescent intensity was observed to decrease to approximately 10% after the addition of the dithionite and further reduces to 1% after the addition of Triton X, which destroys the lipid vesicles. This confirms that lipid vesicles with over 90% asymmetry in their lipid composition was formed. The asymmetric properties was confirmed to remain even after 24 h, as illustrated in FIG. 2C. To confirm that the composition and charge of lipids in both the inner and outer leaflet can be controlled, the zeta potential of lipid vesicles with different ratios of positively and negatively charged lipids was measured. By keeping the inner leaflet containing uncharged POPC lipids fixed and changing the amount of positively charged EPC lipids in the outer leaflet, it was found that the zeta potential increased proportionally to the amount of positively charged lipids up to 50 mV, as shown in FIG. 6B. This value is consistent with symmetric lipid vesicles made purely from EPC with extrusion. When the EPC lipids were replaced with negatively charged POPS lipids, a reduction of the zeta potential to-50 mV was observed, as further shown in FIG. 6B. By contrast, when the outer leaflet was uncharged and the ratio of the charged lipids in the inner leaflet was increased, the zeta potential remains constant, as shown in FIG. 6C. This shows that the zeta potential is only influenced by the lipids in the outer leaflet. The properties of lipid vesicles critically depend on the lipid composition and charge. To determine if a wide variety of asymmetric lipid vesicles could be assembled, the vesicle formation with four different charged lipid compositions was tested: uncharged POPC, negatively charged POPS, positively charged DODMA, and positively charged EPC. Strikingly, vesicles for all permutations of lipids in each leaflet were formed, including vesicles with oppositely charged lipids in each leaflet (e.g., negatively charged lipids on the interior and positively charged lipids on the exterior). However, it was observed that each lipid composition has a different yield of vesicles. The yield of lipid vesicles was measured by adding a small amount of a fluorescently labelled lipid to each leaflet. Thus, the fluorescence intensity was directly proportional to the yield of lipid vesicles. It was observed that the yield is always highest when POPS is in the outer leaflet irrespective of the inner leaflet, as shown in FIG. 7A. When the outer leaflet is composed of POPC, the yield is reduced by more than 3-fold compared to POPs, with further reductions when compared with EPC or DODMA.

[0145] During the formation of asymmetric lipid vesicles, it is crucial for the lipid- stabilizing emulsions to translocate from the oil phase to the water phase. For this reason, the effect of lipids at the water / oil interface were investigated by measuring the interfacial tension with the pendant drop method, as schematized in FIG. 3A. The interfacial tension results show that DODMA has the highest interfacial tension with 61.5+3.5 mN / m. By contrast, EPC (25 mN / m) significantly reduces the interfacial tension followed by POPC (20 mN / m) and POPS (12 mN / m), as shown in FIG. 7B. This reveals an interesting correlation between the yield of vesicles and the interfacial tension at the second water / oil interface. The lower the interfacial tension, the higher the yield of vesicles. It was also found that lipids that reduce the interfacial tension lead to a larger amount of partitioning of hydrophilic molecules from the water to the aqueous phase. The relationship between interfacial tension and lipid vesicles yield suggests that the addition of co- surfactants could increase the yield of lipid vesicles.

[0146] To investigate the potential of asymmetric lipid vesicles as drug delivery vehicles, vesicles were labelled with a fluorescent lipid and added to human embryonic kidney (HEK) cells. The concentration of the vesicles added to the cells was calibrated by comparing the yield with extruded lipid vesicles. The same lipid concentration of lipid vesicles (approximately 25 pM) was added to HEK cells and the lipid vesicle uptake after 24 hours of incubation was observed. Confocal images (FIG. 7C) indicated the cells barely take up the symmetric POPC-POPC (inner-outer leaflet) vesicles. By contrast, the uptake for the symmetric POPS-POPS is significantly higher. When the lipid vesicle uptake per cell was quantified from confocal images, a higher uptake of POPS-POPS vesicles compared to POPC-POPC vesicles was observed. This observation was confirmed using symmetric extruded lipid vesicles . Thus, the incorporation of POPS in lipid vesicles enhances the cell uptake.

[0147] To quantify a larger number of cells, flow cytometry was used to analyze the lipid vesicle uptake per cell. Unexpectedly, a 2-times higher lipid vesicle uptake for asymmetric POPC-POPS lipid vesicles was observed, as compared to symmetric POPS- POPS vesicles as shown in FIGS. 7C-7D. This reveals that not only the outer leaflet, which directly interacts with the cell membrane, dictates the lipid vesicle uptake but also the lipids in the inner leaflet. The uptake of asymmetric POPS-POPC vesicles with the inverted lipid composition is 4 times lower than the uptake of POPC-POPS. To test by which mechanism the lipid vesicles are taken up by cells, the lipid vesicle uptake for cells was investigated while inhibiting a specific endocytosis pathway. As shown in FIG. 7E, the lipid vesicle uptake decreases 3-fold when we add cytochalasin D and dynasore, two drugs that inhibit the clathrin-mediated endocytosis and macropinocytosis pathways.

[0148] To explain the difference in the uptake by cells, it is believed that the inner leaflet changes the biophysical properties of the asymmetric lipid vesicles such that they are softer than their symmetric counterparts. Soft lipid vesicles deform easily during endocytosis, increase the contact area between lipid vesicles and the cell membrane and hence decrease the energy barrier required for cells to take up lipid vesicles. To test this hypothesis, vesicles with different stiffness by changing the length of the fatty acids were prepared. When lipid vesicles containing 18:0 PS lipids were added, the cell uptake is smaller than for 16:0 PS (POPS) lipid vesicles. By contrast, the observed uptake for 15:0 PS lipid vesicles is almost two times higher than for POPS. This shows that the cell uptake increases for vesicles made from lipids with shorter fatty acids, which likely correlates with softer vesicles. These results indicate that vesicle stiffness, in addition to the choice of lipids on the outer leaflet, are important factors that drive the lipid vesicle uptake by cells. To investigate whether asymmetric lipid vesicles can also release cargo into the cytoplasm of cells through endosomal escape, vesicles were loaded with an mRNA that encodes the green fluorescent protein (GFP). After the uptake of lipid vesicles by cells, the cargo ideally releases from the lipid vesicle and escapes from the endosome leading to the translation of the mRNA into GFP as schematized in FIG. 5A. Upon encapsulation of GFP-encoding mRNA into POPC-POPS, POPS-POPS, and POPC-POPC lipid vesicles and their addition to HEK cells, the mRNA transfection was observed after 48 hours via confocal imaging.lt was observed that cells are successfully transfected with GFP as shown in FIG. 5B, indicating that a fraction of the cargo can escape the endosome. The mRNA transfection efficiency by calculating the percentage of fluorescent cells relative to the total cell number. The transfection efficiency of POPC- POPS vesicles is 9 times higher than POPC-POPC and 7 times higher than POPS-POPS, as shown in 7F. It was also observed that the intensity of the GFP signal is significantly higher for POPC-POPS compared to the other conditions, indicating that more mRNA has been released into the cytoplasm. These results show that asymmetric vesicles are taken up more than symmetric vesicles and that they are more efficient in undergoing endosomal escape.

[0149] Cell membranes are asymmetric, with a precise positioning of lipids in the inner or outer leaflet. Conventional symmetric lipid vesicles that are taken up by cells can cause a disruption of the cell membrane and lipidome leading to cell apoptosis. To investigate if asymmetric lipid vesicles that resemble the structure of cell membranes and could reduce the cell cytotoxicity, a lactate dehydrogenase (LDH) assay was employed to measure the cell viability after lipid vesicle addition. It was found that the cytotoxicity of POPC-POPS is significantly lower than that of POPS-POPS despite its 2-fold higher uptake by cells, as depicted in FIG. 7G. Thus, asymmetric lipid vesicles can result in a reduced cell cytotoxicity likely because their membrane resembles the cell membrane, which contains PS-lipids on the inner leaflet. Overall, the properties of asymmetric lipid vesicles revealing an increased cell uptake efficiency, enhanced mRNA transfection, and lower toxicity make them an efficient and unique drug delivery carrier that offers full programmability over their biochemical and biophysical properties.

[0150] The delivery of proteins is a critical strategy for disease treatment, as proteins can directly modulate cellular functions without relying on the expression processes required for mRNA or DNA. This eliminates the challenges associated with ensuring simultaneous peak levels of multiple essential components or preventing their degradation during expression. To further explore the versatility of the asymmetric lipid vesicles as delivery vehicles beyond mRNA, different fluorescently labeled proteins were encapsulated within asymmetric vesicles and added them to HEK cells. The confocal images in FIG. 8A reveal the presence of proteins in cells. These proteins have very different functions and molecular weights ranging from 60 kDa for streptavidin to 240 kDa for B-phycoeryhtrin. By comparing the signal of lipid vesicles and proteins, it was also found that a fraction of proteins has been released into the cytoplasm because the signal is not colocalized with the lipid vesicles. Protein functionality was also verified by using a Cas9 labeled with GFP; the observed GFP fluorescence confirms that the protein is functional. Strikingly, it was observed that GFP-Cas9 gradually targets the nucleus within 6 hours, due to the presence of nuclear localization signals on the protein surface. By analyzing the fluorescence intensity of GFP-Cas9 inside the cell, it was found that almost 40% of the Cas9 is localized in the nucleus, as shown in FIG. 8B. This further shows that the Cas9 protein is released from asymmetric lipid vesicles, remains functional, and can target the nucleus. To utilize Cas9 delivery, a single guide RNA (sgRNA) designed to target the TTR gene in human cells , which is responsible for the production of transthyretin, was synthesized. After incubation of sgRNA and Cas9, the Cas9 / sgRNA complex was encapsulated within asymmetric vesicles and delivered to cells. The sequencing results reveal that 7.1 % of insertions or deletions were achieved within 48 h in the HEK cell genome, proving the successful delivery of complex ribonucleoproteins and the gene-editing desired.

[0151] Conclusion

[0152] The engineering of asymmetric lipid vesicles with distinct lipid compositions in the inner and outer leaflets enables the design of new types of delivery vehicles. The method described in this Example allows for the generation of small lipid vesicles with properties that could not be assessed before. It facilitates the investigation of membrane biophysics and biochemistry with full control over the positioning of individual lipids. Compared to symmetric lipid vesicles, the results demonstrate that an asymmetric leaflet configuration enhances lipid vesicle uptake by cells, improves mRNA transfection efficiency, and facilitates effective delivery of functional proteins. Thus, the ability to design asymmetric lipid vesicles on the nanoscale has important applications for drug delivery ranging from vaccines to cancer treatment. As an example, the delivery of ribonucleoprotein complexes that successfully edit the cell genome was shown. Further modification of gene editing efficiency and in vivo testing will lead to the full potential of the method for drug delivery applications.

[0153] EXAMPLE 3

[0154] The following example describes the formation of lipid-polymer asymmetric liposomes and various properties thereof, in accordance with one embodiment.

[0155] Lipid vesicles are excellent vehicles for the delivery of small molecules due to their biocompatibility, but directly grafting chemical units onto lipid molecules to modify lipid vesicles is challenging. Polymers are a different class of macromolecules that can be modified much more easily than lipids, while providing a wider range of molecular weights, subunits, hydrophilicity, and functionalizations that could be used for the design of delivery vehicles. Moreover, polymer synthesis is also more than an order of magnitude cheaper than lipid synthesis, decreasing the expense of asymmetric vesicles containing polymers compared to vesicles made exclusively from lipids.

[0156] In this Example, a method for making asymmetric lipid-polymer vesicles with one lipid leaflet and one polymer leaflet is described. A first solution containing the polymer PEG2000-PLA3000 dissolved in mineral oil at a concentration of 5 mg / mL and a second solution containing either the POPS or DOPC lipid dissolved in mineral oil at a concentration of 0.05 mg / mL were prepared. Then 700 pL of the second solution was layered on top of 600 pL of phosphate buffered solution (PBS) and left to incubate for 2 hours.

[0157] Polymer- stabilized emulsions were formed by mixing 10 pL each of water and the first solution. The mixture was briefly shaken by hand to from water-in-oil droplets. Then, 100 pL of the emulsion of the first solution were added on top of the second solution (i.e., layered on top of the PBS) and centrifuged for 5 min at 600 g. Due to the density gradient between water and mineral oil, the polymer- stabilized emulsion (i.e., the emulsion formed from the first solution) was centrifuged through the lipid monolayer (i.e., the layer of the second solution) at the oil-water interface and dissolved in the aqueous phase. Finally, the aqueous phase containing asymmetric lipid polymer vesicles was pipetted out with a syringe needle and imaged with a confocal microscope and fluorescence microscopy (FIGS. 9A-B), the images from which were overlaid to show the colocalization of the polymer and lipid (FIG. 9C). By extruding the vesicles through a 200nm polycarbonate membrane, small lipid-polymer vesicles with sizes ranging from 100-500 nm were obtained, which was determined using dynamic light scattering of the solution containing the vesicles (FIG. 9D).

[0158] The use of lipid-polymer hybrid vesicles for drug delivery applications may facilitate a wide range of applications including increased encapsulation of entities (e.g., proteins or the like), targeted delivery of such entities, increased stability of vesicles, increased cellular uptake, increased endosomal escape, and / or provide an overall larger parameter space for tailoring the biophysical properties of drug delivery vehicles.

[0159] 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. 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.”

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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. A system, comprising: a subject having cancer; and a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer, contained within the subject’s blood.

2. A system, comprising a solution containing a plurality of cancer cells; and a plurality of asymmetric liposomes in the solution, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating cancer.

3. The system of claim 2, wherein the solution is blood.

4. The system of claim 2, wherein the solution is cell media.

5. A system, comprising: a subject having a disease; and a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease, contained within the subject’s blood.

6. A system, comprising a solution containing a plurality of disease cells; and a plurality of asymmetric liposomes in the solution, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the disease.

7. The system of any one of the preceding claims, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises l-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (POPS), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2- dioleyloxy-3-dimethylaminopropane (DODMA), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (LissRhod-PE), and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7 -nitro-2- 1 ,3-benzoxadiazol-4-yl) (NBD-PE).

8. The system of any one of the preceding claims, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a lipid bilayer comprising a first lipid and an exterior surface of the lipid bilayer comprising a second lipid, different from the first lipid.

9. The system of claim 8, wherein the first lipid comprises l-palmitoyl-2-oleoyl- glycero-3-phosphocholine (POPC) and the second lipid comprises l-palmitoyl-2-oleoyl- sn-glycero-3-phospho-L-serine (POPS).

10. The system of any one of the preceding claims, wherein the plurality of asymmetric liposomes is suitable for administration to a subject.

11. The system of any one of the preceding claims, wherein the therapeutic agent comprises a small molecule less than or equal to 1000 Da.

12. The system of any one of the preceding claims, wherein the therapeutic agent comprises mRNA.

13. The system of any one of the preceding claims, wherein the therapeutic agent comprises siRNA.

14. The system of any one of the preceding claims, wherein the therapeutic agent comprises DNA.

15. The system of any one of the preceding claims, wherein the therapeutic agent comprises a protein.

16. The system of any one of the preceding claims, wherein the therapeutic agent comprises a peptide.

17. The system of any one of the preceding claims, wherein the therapeutic agent comprises a polymer.

18. The system of any one of the preceding claims, wherein the therapeutic agent comprises a vesicle.

19. The system of any one of the preceding claims, wherein the therapeutic agent comprises a nanoparticle.

20. The system of any one of the preceding claims, wherein the therapeutic agent is configured for gene editing.

21. The system of any one of the preceding claims, wherein the therapeutic agent is configured for CRISPR-Cas9 gene editing.

22. The system of any one of the preceding claims, wherein the therapeutic agent comprises a Cas9 enzyme and a guide RNA.

23. The system of claim 22, wherein the guide RNA is a single guide RNA.

24. The system of claim 22 or 23, wherein the guide RNA is at least partially complementary to DNA of the subject.

25. The system of any one of the preceding claims, wherein the therapeutic agent comprises a Cas9 enzyme and two or more guide RNAs.

26. The system of any one of claims 22-25, wherein the therapeutic agent further comprises an exogenous DNA.

27. The system of any one of the preceding claims, wherein an average maximum dimension of the plurality of asymmetric liposomes is greater than or equal to 50 nm and less than or equal to 100 microns.

28. The system of any one of the preceding claims, wherein an average bending rigidity of the plurality of asymmetric liposomes is greater than or equal to 2 kT and less than or equal to 200 kT.

29. The system of any one of the preceding claims, wherein an average stretching modulus of the plurality of asymmetric liposomes is greater than or equal to 20 mN / m and less than or equal to 1000 mN / m.

30. The system of any one of the preceding claims, wherein an average lipid diffusivity of a lipid within the membranes of the plurality of asymmetric liposomes is greater than or equal to 0.05 microns2 / second and less than or equal to 10 microns2 / second.

31. The system of any one of the preceding claims, wherein an average zeta potential of the plurality of asymmetric liposomes is greater than or equal to -50 mV and less than or equal to 50 mV.

32. The system of any one of the preceding claims, wherein an uptake rate of the plurality of asymmetric liposomes by a plurality of cells exposed to the plurality of asymmetric liposomes is greater than or equal to 0.01 and less than or equal to 1 greater than an uptake rate of a plurality of symmetric liposomes by the plurality of cells exposed to the plurality of symmetric liposomes.

33. The system of any one of the preceding claims, wherein an inner liquid of the plurality of asymmetric liposomes comprises water.

34. Use of a composition to treat a subject having cancer, the composition comprising a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer.

35. Use of a composition to treat a subject having a disease, the composition comprising a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease.

36. A method of treating a subject having cancer, comprising: administering a plurality of asymmetric liposomes to the subject having cancer, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the cancer.

37. A method of making liposomes, comprising: extruding a water-in-oil emulsion stabilized by a first lipid through a membrane having an average pore size of less than or equal to 200 microns; and forming an asymmetric liposome by passing the extruded emulsion through a monolayer comprising a second lipid different from the first lipid.

38. A method of making liposomes, comprising: providing an emulsion having an average droplet size of less than or equal to 100 microns, the emulsion comprising droplets stabilized by a first lipid; and forming asymmetric liposomes by passing the droplets through a second lipid different from the first lipid.

39. The method of claim 38, wherein providing an emulsion comprises extruding a first lipid through a membrane having an average pore size of less than or equal to 200 microns into an aqueous medium.

40. A method, comprising: exposing a plurality of cancer cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the cancer.

41. The method of claim 40, wherein the exposing occurs in vitro.

42. A method of treating a subject having a disease, comprising: administering a plurality of asymmetric liposomes to the subject having a disease, wherein the plurality of asymmetric liposomes encapsulates a therapeutic agent for treating the disease.

43. A method, comprising: exposing a plurality of disease cells to a plurality of asymmetric liposomes encapsulating a therapeutic agent for treating the disease.

44. The method of any one claims 36-43, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises l-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (POPS), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2- dioleyloxy-3-dimethylaminopropane (DODMA), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (LissRhod-PE), and / or 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7 -nitro-2- 1 ,3-benzoxadiazol-4-yl) (NBD-PE).

45. The method of any one claims 36-44, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a lipid bilayer comprises a first lipid and an exterior surface of the lipid bilayer comprises a second lipid, different from the first lipid.

46. The method of claim 45, wherein the first lipid comprises l-palmitoyl-2-oleoyl- glycero-3-phosphocholine (POPC) and the second lipid comprises l-palmitoyl-2-oleoyl- sn-glycero-3-phospho-L-serine (POPS).

47. The method of any one of claims 36-46, wherein the plurality of asymmetric liposomes is suitable for administration to a subject.

48. The method of any one of claims 36-47, wherein the therapeutic agent comprises a small molecule less than or equal to 1000 Da.

49. The method of any one of claims 36-48, wherein the therapeutic agent comprises mRNA.

50. The method of any one of claims 36-49, wherein the therapeutic agent comprises siRNA.

51. The method of any one of claims 36-50, wherein the therapeutic agent comprises DNA.

52. The method of any one of claims 36-51, wherein the therapeutic agent comprises a protein.

53. The method of any one of claims 36-52, wherein the therapeutic agent comprises a peptide.

54. The method of any one of claims 36-53, wherein the therapeutic agent comprises a polymer.

55. The method of any one of claims 36-54, wherein the therapeutic agent comprises a vesicle.

56. The method of any one of claims 36-55, wherein the therapeutic agent comprises a nanoparticle.

57. The method of any one of claims 36-56, wherein the therapeutic agent is configured for gene editing.

58. The method of any one of claims 36-57, wherein the therapeutic agent is configured for CRISPR-Cas9 gene editing.

59. The method of any one of claims 36-58, wherein the therapeutic agent comprises a Cas9 enzyme and a guide RNA.

60. The method of claim 59, wherein the guide RNA is a single guide RNA.

61. The method of claim 59 or 60, wherein the guide RNA is at least partially complementary to DNA of the subject.

62. The method of any one of claims 36-61, wherein the therapeutic agent comprises a Cas9 enzyme and two or more guide RNAs.

63. The method of any one of claims 59-62, wherein the therapeutic agent further comprises an exogenous DNA.

64. The method of any one of claims 36-63, wherein an average maximum dimension of the plurality of asymmetric liposomes is greater than or equal to 50 nm and less than or equal to 100 microns.

65. The method of any one of claims 36-64, wherein an average bending rigidity of the plurality of asymmetric liposomes is greater than or equal to 2 kT and less than or equal to 200 kT.

66. The method of any one of claims 36-65, wherein an average stretching modulus of the plurality of asymmetric liposomes is greater than or equal to 20 mN / m and less than or equal to 1000 mN / m.

67. The method of any one of claims 36-66, wherein an average lipid diffusivity of a lipid within the membranes of the plurality of asymmetric liposomes is greater than or equal to 0.05 microns2 / second and less than or equal to 10 microns2 / second.

68. The method of any one of claims 36-67, wherein an average zeta potential of the plurality of asymmetric liposomes is greater than or equal to -50 mV and less than or equal to 50 mV.

69. The method of any one of claims 36-68, wherein an uptake rate of the plurality of asymmetric liposomes by a plurality of cells exposed to the plurality of asymmetric liposomes is greater than or equal to 0.01 and less than or equal to 1 greater than an uptake rate of a plurality of symmetric liposomes by the plurality of cells exposed to the plurality of symmetric liposomes.

70. The method of any one of claims 36-69, wherein an inner liquid of the plurality of asymmetric liposomes comprises water.

71. The method of any one of claims 36-70, further comprising encapsulating the therapeutic agent in the liposome.

72. The system of any one of claims 1-31, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a membrane comprising a first polymer and an exterior surface of the membrane comprising a second polymer.

73. The system of any one of claims 1-31, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a membrane comprising a lipid and an exterior surface of the membrane comprising polymer.

74. The system of any one of claims 1-31, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a membrane comprising a polymer and an exterior surface of the membrane comprising lipid.

75. The system of any one of claims 1-31 or 72-74, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises one or more of polylactide (PLA), polyethylene glycol (PEG), polycaprolactone (PCL), polyethylene oxide (PEG), polybutadiene (PBut), polydimethylsiloxane (PDMS), poly(2-methyl-2-2oxa-zoline) (PMOXA), polyisobutylene (PIB), polyacrylic acid (PAAc), and copolymers thereof.

76. The method of any one of claims 36-70, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a membrane comprising a first polymer and an exterior surface of the membrane comprising a second polymer.

77. The method of any one of claims 36-70 or 76, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a membrane comprising a lipid and an exterior surface of the membrane comprising polymer.

78. The method of any one of claims 36-70 or 76-77, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises an interior surface of a membrane comprising a polymer and an exterior surface of the membrane comprising lipid.

79. The method of any one of claims 36-70 or 76-78, wherein an asymmetric liposome of the plurality of asymmetric liposomes comprises one or more of polylactide (PLA), polyethylene glycol (PEG), polycaprolactone (PCL), polyethylene oxide (PEG), polybutadiene (PBut), polydimethylsiloxane (PDMS), poly(2-methyl-2-2oxa-zoline)(PMOXA), polyisobutylene (PIB), polyacrylic acid (PAAc), and copolymers thereof.

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