System for Preparing Vacuum Medium Liposomes and Method of Using the Same

Abstract

A liposome preparation system is provided. Aspects of the system include a liposome collection container and a membrane holder. In an embodiment, the liposome collection container has an open first end, a closed bottom, a wall, and an opening in the wall. The system can be operably connected to a vacuum source, such that a vacuum force can be applied through the opening, thereby pulling liposomes through the porous membrane and generating a population of liposomes. Also provided are a method of manufacturing liposomes, as well as an apparatus and a kit for carrying out the method.

Description

Technical Field

[0001] A liposome is a spherical vesicle having one or more lipid bilayers.

Background Art

[0002] Liposomes containing a single lipid bilayer may be referred to as unilamellar liposomes, while liposomes containing multiple lipid bilayers may be referred to as multilamellar vesicles. Liposomes can be prepared using different methods, which can depend on multiple factors such as the lipid composition of the lipid bilayer of the liposome, the type of medium in which the lipid vesicles are dispersed, the desired size of the liposome, the desired polydispersity of the liposome, the robustness and batch-to-batch reproducibility of the manufacturing method, and other factors such as the intended use of the resulting liposomes. For example, liposomes can contain substances such as drugs and can be used to deliver the substances to a target area in a patient. Thus, the methods used to produce such liposomes can also depend on the physicochemical properties of the substances entrapped in the liposomes, the concentration of the entrapped substances, or additional processes involved during the application / delivery of the liposomes to the patient.

Summary of the Invention

Problems to be Solved by the Invention

[0003] After a suspension of liposomes such as a suspension of large multilamellar vesicles is produced, it may be desirable to produce liposomes having sizes within a certain size range. One common technique for sizing liposomes is extrusion, but by that process, large multilamellar vesicles may be disrupted and downsized by being extruded through a polycarbonate membrane having a defined pore size. Currently, in the adopted extrusion protocol, phospholipids are first suspended in buffered saline to obtain large multilamellar vesicles (LMV). Then, the vesicles are repeatedly extruded through a polycarbonate membrane having a defined pore size and are shuttled by a specially modified airtight syringe attached to a membrane support chamber.

Means for Solving the Problems

[0004] The inventors have understood that by employing a vacuum device, the membrane extrusion procedure can be substantially improved, accelerated, and simplified. Instead of special glass airtight syringes and metal membrane support chambers, the method of embodiments of the present invention employs plastic parts including a membrane holding unit, a collection tube, and a vacuum tube, and the membrane holding unit and the collection tube can be disposable. By eliminating the need for glass and metal liposome manufacturing extrusion devices, support filters, and polycarbonate membranes, and eliminating the time for assembling and disassembling such devices, the vacuum device of embodiments of the present invention saves time and money. The switch from a special extrusion device to a disposable unit eliminates the possibility of post-work cleaning and cross-contamination between liposome samples.

[0005] A system for manufacturing a population of liposomes is provided. Aspects of the system include a liposome collection container and a membrane holder. In an embodiment, the liposome collection container has an open first end, a closed bottom, a wall, and an opening in the wall. The system can be operably connected to a vacuum source, such that a vacuum force can be applied through the opening, thereby pulling liposomes through a porous membrane and generating a population of liposomes. A method for manufacturing liposomes, as well as an apparatus and a kit for practicing the method, are also provided.

Brief Description of the Drawings

[0006]

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DETAILED DESCRIPTION OF THE INVENTION

[0007] A system for producing a population of liposomes is provided. Aspects of the system include a liposome recovery container and a membrane holder. In an embodiment, the liposome recovery container has an open first end, a closed bottom, a wall, and an opening in the wall. The system can be operably connected to a vacuum source such that a vacuum force can be applied through the opening, thereby pulling liposomes through a porous membrane and generating a population of liposomes. A method of manufacturing liposomes, as well as an apparatus and a kit for practicing the method, are also provided.

[0008] Before describing the present invention in more detail, it is to be understood that the invention is not limited to the specific embodiments described, and thus can naturally vary. Since the scope of the present invention is limited only by the appended claims, it is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0009] When a range of values is provided, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, and any other stated or intervening values in the stated range are understood to be included in the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are included in the present invention subject to any limits specifically excluded in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention.

[0010] A range is presented herein and the term “about” is prefixed to a number. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as numbers that are close to or substantially the same as the number that the term precedes. In determining whether a number is close to or substantially equal to a specifically recited number, the unrecited numbers that are close to or approximate the specifically recited number may be numbers that result in substantial equivalence for the specifically recited number in the context in which it is presented.

[0011] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar to or equivalent to those described herein can also be used in the practice or testing of the present invention, but representative exemplary methods and materials are described herein.

[0012] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and incorporated by reference herein to describe and explain the manner and / or materials in which the publications are cited in relation. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the present invention has no right to antedate such publication by virtue of prior invention. Further, the provided publication dates may differ from the actual publication dates which may need to be independently confirmed.

[0013] Note that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional element. Thus, this description is intended to serve as a basis for using exclusive terms such as "solely", "only", etc. in connection with the recitation of claim elements, or for using "negative" limitations.

[0014] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with those of any of several other embodiments without departing from the scope or spirit of the invention. Any recited method can be performed in the order of recited events or in any other order that is logically possible.

[0015] The apparatus and method are described, or are to be described, with functional descriptions for grammatical fluidity, but unless explicitly formulated under 35 U.S.C. § 112, the claims should not be construed as necessarily limited in any way by the construction of "means" or "step" limitations, and the full scope of the meaning and equivalents provided by the claims under the doctrine of equivalents should be given. When the claims are explicitly formulated based on 35 U.S.C. § 112, it should be explicitly understood that full statutory equivalents under 35 U.S.C. § 112 are granted.

[0016] Method for manufacturing liposomes A method for manufacturing a population of liposomes is provided. For example, the method can include providing a liposome extrusion system that includes a liposome collection vessel and a membrane holder. The liposome collection vessel has an open first end and a closed bottom separated by walls that collectively define an interior. The collection vessel also has an opening in the wall. The membrane holder has an open first end and a porous membrane at a second end that defines a liposome suspension holding volume, i.e., the interior of the collection vessel, separated by walls that collectively define the interior. The membrane holder is arranged such that the porous membrane is positioned inside the liposome collection vessel.

[0017] The method can further include a step of introducing a liposome suspension into a liposome suspension holding volume portion. Thus, the liposome suspension can contact the upper side of a porous membrane, for example. Thereafter, liposomes are drawn into the interior of the recovery container through the porous membrane, and a vacuum force can be applied through an opening in the wall of the liposome recovery container so as to generate a population of liposomes. In other words, the vacuum force can be transmitted from the opening in the wall of the recovery container into the interior of the recovery container, and the porous membrane is disposed inside the recovery container. In this way, the vacuum force can be applied through the pores of the porous membrane, whereby the liposomes can be drawn out through the pores of the porous membrane. When the liposomes cross the membrane, the liposomes pass through the pores in the porous membrane to generate a population of liposomes.

[0018] In some cases, providing the system includes disposing a second end of the membrane holder inside the liposome recovery container. Thus, the porous membrane at the second end of the membrane holder is also disposed inside the liposome recovery container. Accordingly, the liposome suspension holding volume portion of the membrane holder is in fluid communication with the interior of the liposome recovery container via the porous membrane. "Fluid communication" means that a fluid (for example, a gas or a liquid including a suspension of liposomes) can flow from one region to another region. For example, in the liposome extrusion system disclosed herein, the fluid can flow from the liposome suspension holding volume portion of the membrane holder through the porous membrane into the interior of the liposome recovery container.

[0019] In some cases, the opening in the wall of the liposome collection container is located above the porous membrane. Thus, when the liquid of the liposome suspension is drawn through the porous membrane by the vacuum force, the amount of liquid exiting the liposome collection container through the opening can be minimized or eliminated. In other words, by disposing the opening above the membrane, it is easily suppressed that the liquid is accidentally sucked into the opening, thereby reducing the liquid loss to the outside of the container. In some examples, the opening in the wall is oriented perpendicular to the porous membrane. For example, the porous membrane can be oriented horizontally, while the opening can be disposed on the vertical or substantially vertical wall of the collection container. In some examples, the opening is oriented within 30° from the vertical, such as within 15°, or within 5°.

[0020] In some examples, the membrane holder is oriented such that the porous membrane is located directly below the open first end. In other words, the membrane holder is oriented such that there is a vertical line that intersects both the open first end and the porous membrane.

[0021] Furthermore, in some embodiments, the volume of the liposome collection container below the porous membrane is larger than the liposome suspension holding volume portion. Thus, even if the liposome suspension holding volume portion is completely filled with the liposome suspension, the amount of liquid will not be sufficient to rise to the level of the opening and overflow from the liposome collection container. In some cases, the volume of the liposome collection container below the porous membrane can exceed the liposome suspension holding volume portion by 5% or more, such as 20% or more, or 50% or more. Such a relative volume can, for example, reduce the possibility of liquid overflowing into the opening when the user handles the system.

[0022] In certain embodiments, the suspension of liposomes can be introduced into a liposome extrusion system such that the suspension of liposomes is disposed on one side of the membrane. For example, the suspension of liposomes can be contained in a liposome suspension holding volume disposed on one side of the membrane. During the production of a population of liposomes, the suspension of liposomes can traverse from one side of the membrane, through the porous membrane, to the other side of the membrane. For example, the suspension of liposomes can first be contained in a liposome suspension holding volume on a first side of the membrane and then, during the production of the population of liposomes, can traverse the porous membrane such that it is contained within the interior of a liposome collection vessel on the other side of the membrane.

[0023] In certain aspects, the suspension of liposomes is introduced into the liposome extrusion system using any convenient liquid handling technique. For example, the amount of the suspension of liposomes, although not limited, can be added to the liposome extrusion system using any convenient liquid handling device, such as a syringe, needle, pipette, aspirator, among other liquid handling devices.

[0024] In some embodiments, one or both of the membrane holder and the liposome collection vessel can have a circular cross-section. In some examples, both such elements have a circular cross-section. Further, the membrane holder and the liposome collection vessel can be arranged such that the circular cross-section of the membrane holder is coaxial with the circular cross-section of the liposome collection vessel. In other words, each circular cross-section can have a center of the circle, and the axis is positioned perpendicular to the cross-section and intersects the center of the circle.

[0025] In some cases, the system further includes a sealing element that seals the membrane holder to the liposome collection vessel. The seal between such elements can be liquid-tight, gas-tight, or both. As used herein, "liquid-tight" means that liquid does not substantially leak through the connection between the elements, while "gas-tight" means that gas does not substantially leak through the connection between the elements. In some embodiments, the sealing element is a gasket, such as an O-ring, that seals the membrane holder to the liposome collection vessel.

[0026] By applying a vacuum force to the suspension of liposomes within the liposome suspension holding volume portion, the liposomes can be drawn through the porous membrane. In some cases, the vacuum force is generated by a vacuum source such as a vacuum ejector, rotary vane pump, diaphragm pump, liquid ring pump, reciprocating compressor, scroll compressor, and lobe pump. For example, a vacuum ejector uses the Venturi effect to generate a vacuum due to the flow of a fluid, and a vacuum ejector may also be referred to as a suction pump. The vacuum force depends on the pressure difference between the vacuum source and the atmosphere surrounding the device. Atmospheric pressure depends on several factors including altitude, temperature, and weather. The average atmospheric pressure at sea level is about 101 kPa, but it can vary to higher or lower values such as up to about 105 kPa at a specific location on Earth during certain meteorological events. In some cases, the atmospheric pressure during the method is about 101 kPa, and thus the vacuum force has a magnitude less than about 101 kPa. In some cases, the magnitude of the vacuum force can be in the range of 0.1 kPa to 105 kPa, such as 0.1 kPa to 101 kPa, 0.1 kPa to 100 kPa, 10 kPa to 100 kPa, 25 kPa to 100 kPa, 50 kPa to 95 kPa, or 70 kPa to 90 kPa. The vacuum can also be described by the pressure within the vacuum source. In some cases, the vacuum source pressure is in the range of 100 kPa to 0.1 kPa, and can be referred to herein as rough vacuum or low vacuum. For example, the vacuum source pressure can be in the range of 95 kPa to 0.5 kPa, such as 90 kPa to 1 kPa, 80 kPa to 2 kPa, 70 kPa to 3 kPa, 60 kPa to 4 kPa, or 50 kPa to 5 kPa. In some embodiments, the vacuum source pressure is in the range of 100 Pa to 0.1 Pa, and is referred to herein as fine vacuum or medium vacuum. In other cases, the vacuum source pressure is in the range of 0.1 Pa to 10 -5 Pa, and is referred to herein as high vacuum.

[0027] In some examples, the system used to implement the method further includes a vacuum adapter, and the vacuum force can be transmitted from a vacuum source to the vacuum adapter and then to an opening in the wall of the liposome collection container. In some cases, the system is configured such that there is a fluid communication between the vacuum source, the vacuum adapter, and the interior of the liposome collection container through the opening of the liposome collection container. The vacuum adapter can have a first opening operably coupled to the vacuum source and a second opening operably coupled to the opening of the liposome collection container, together with the wall of the vacuum adapter that collectively defines the interior of the vacuum adapter.

[0028] In some embodiments, at least a portion of the liposome collection container is disposed inside the vacuum adapter. The vacuum adapter can have a circular cross-section. For example, the circular cross-section of the vacuum adapter is coaxial with the circular cross-section of the liposome collection container. The system can also include a sealing element that seals the vacuum adapter to the collection container. For example, the sealing element can be a gasket, such as an O-ring.

[0029] In some cases, the introduction and application steps are repeated one or more additional times to draw the liposomes through the membrane. In such cases, the membrane holder can be separated from the liposome collection container, for example, by lifting the membrane holder vertically. Then, the liposomes can be removed from the liposome collection container, for example, by a user-operated or robot-operated pipette, and then dispensed into the liposome suspension holding volume of the membrane holder. Then, the elements can be arranged such that the porous membrane is located inside the liposome collection container and the vacuum force can be applied again. The introduction and application steps can be repeated one or more times, for example, two or more times, three or more times, four or more times, six or more times, eight or more times, or ten or more times.

[0030] In some embodiments, the method further includes transferring a population of liposomes from a liposome collection vessel to a receiving vessel. For example, the membrane holder can be separated from the liposome collection vessel by lifting the membrane holder vertically. Thereafter, it is withdrawn from the liposome collection vessel, for example, by a user-operated or robotic-operated pipette and then dispensed into the receiving vessel.

[0031] Liposome In some examples, the population of liposomes generated by the method is a population of liposomes of defined size. By "defined size" is meant that the sizes of the various liposomes in the population are known for the method by which the liposomes are produced, specifically that the range of liposome sizes in the population is known. In some cases, the liposomes have substantially the same average size. For example, in the case of spherical liposomes, the population of liposomes can have substantially the same average diameter. By "average" is meant the arithmetic mean. Values that are substantially identical are within 50% or less of each other, such as 45% or less, or 40% or less, or 35% or less, or 30% or less, or 25% or less, or 20% or less, or 15% or less, or 10% or less, or 5% or less, or 3% or less, or 1% or less, or 0.5% or less, and include different values. In some cases, values that are substantially identical are within 10% or less of each other and include different values. In some cases, values that are substantially identical are within 5% or less of each other and include different values. In some cases, values that are substantially identical are within 3% or less of each other and include different values. In some cases, values that are substantially identical are within 1% or less of each other and include different values. In some cases, values that are substantially identical are within 0.5% or less of each other and include different values. The average size of the liposomes can vary by 50% or less, such as 45% or less, or 40% or less, or 35% or less, or 30% or less, or 25% or less, or 20% or less, or 15% or less, or 10% or less, or 5% or less, or 3% or less, or 1% or less, or 0.5% or less, in some examples. In some cases, the average size of the liposomes varies by 10% or less. In some cases, the average size of the liposomes varies by 5% or less. In some cases, the average size of the liposomes varies by 3% or less. In some cases, the average size of the liposomes varies by 1% or less. In some cases, the average size of the liposomes varies by 0.5% or less.

[0032] In certain embodiments, a population of liposomes can be described by the polydispersity of the liposomes. "Dispersity" or "polydispersity" is a measure of the heterogeneity of the sizes of particles in a mixture. In the context of liposomes, the polydispersity can range from 0 to 1, where a polydispersity of 0 indicates a monodisperse population of liposomes (e.g., liposomes having the same average size), and a polydispersity of 1 indicates a heterogeneous mixture of liposomes. In some cases, the size (and thus the polydispersity) of the liposomes can be determined by dynamic light scattering (DLS).

[0033] In certain embodiments, the methods of the present disclosure are sufficient to generate a population of liposomes from a suspension of liposomes (e.g., an aqueous suspension of liposomes). In some cases, the starting suspension of liposomes contains a population of liposomes having heterogeneous sizes. Thus, the methods of the present disclosure include starting from a suspension of liposomes (e.g., a population of liposomes having heterogeneous sizes) and generating a population of liposomes from the starting suspension of liposomes. The population of liposomes in the starting suspension of liposomes may also be referred to herein as the initial population of liposomes, i.e., to distinguish such liposomes from the population of liposomes generated by pulling them through a porous membrane.

[0034] In some embodiments, the method includes generating a population of liposomes from a suspension of heterogeneous liposomes, and the average size of the resulting population of liposomes is different by 50% or less, such as 45% or less, or 40% or less, or 35% or less, or 30% or less, or 25% or less, or 20% or less, or 15% or less, or 10% or less, or 5% or less, or 3% or less, or 1% or less, or 0.5% or less. In some cases, the method includes generating a population of liposomes from a suspension of heterogeneous liposomes, and the average size of the resulting population of liposomes is different by 10% or less. In some cases, the method includes generating a population of liposomes from a suspension of heterogeneous liposomes, and the average size of the resulting population of liposomes is different by 5% or less. In some embodiments, the method includes generating a population of liposomes from a suspension of heterogeneous liposomes, and the average size of the resulting population of liposomes is different by 3% or less. In some cases, the method includes generating a population of liposomes from a suspension of heterogeneous liposomes, and the average size of the resulting population of liposomes is different by 1% or less. In some cases, the method includes generating a population of liposomes from a suspension of heterogeneous liposomes, and the average size of the resulting population of liposomes is different by 0.5% or less. In still other examples, the average size of the heterogeneous liposome members of the population can be different by 50% or more, such as 75% or more, and 100% or more. For example, the average size of the heterogeneous liposome members in the starting suspension of liposomes can be different by 50% or more, such as 75% or more, and 100% or more.

[0035] In some examples, the starting suspension of liposomes has a higher polydispersity compared to the resulting population of liposomes. Thus, the methods of the present disclosure are useful for producing a population of liposomes having a polydispersity less than that of the starting suspension of liposomes.

[0036] In some cases, the polydispersity of the generated population of liposomes is 0.9 or less, for example 0.8 or less, or 0.7 or less, or 0.5 or less, or 0.4 or less, or 0.3 or less, or 0.2 or less, or 0.1 or less, or 0.05 or less, or 0.01 or less. For example, the polydispersity of the generated population of liposomes can be 0.5 or less. In some cases, the polydispersity of the generated population of liposomes can be 0.4 or less. In some cases, the polydispersity of the generated population of liposomes can be 0.3 or less. In some cases, the polydispersity of the generated population of liposomes can be 0.2 or less. In some cases, the polydispersity of the generated population of liposomes can be 0.1 or less. In some cases, the polydispersity of the generated population of liposomes can be 0.05 or less. In some cases, the polydispersity of the generated population of liposomes can be 0.01 or less. In certain examples, the polydispersity of the generated population of liposomes is in the range of 0.01 - 0.5, for example 0.01 - 0.4, or 0.01 - 0.3, or 0.01 - 0.2, or 0.01 - 0.1. In other embodiments, the polydispersity of the generated population of liposomes is in the range of 0.01 - 0.5, for example 0.05 - 0.5, or 0.1 - 0.5, or 0.1 - 0.4, or 0.1 - 0.3. In other embodiments, the polydispersity of the generated population of liposomes is in the range of 0.01 - 0.5, for example 0.05 - 0.5, or 0.1 - 0.5, or 0.2 - 0.5, or 0.2 - 0.4.

[0037] In some cases, the polydispersity of the starting suspension of liposomes is 0.5 or more, for example 0.6 or more, or 0.7 or more, or 0.8 or more, or 0.9 or more. In some cases, the polydispersity of the starting suspension of liposomes is 1. For example, the polydispersity of the starting suspension of liposomes can be in the range of 0.5 - 1, for example 0.6 - 1, or 0.7 - 1, or 0.8 - 1, or 0.9 - 1.

[0038] In some examples, the starting suspension of liposomes comprises liposomes having a size larger than the population of liposomes produced. In some examples, the starting suspension of liposomes comprises liposomes having an average size (e.g., average diameter) of 500 nm or more, such as 600 nm or more, or 700 nm or more, or 800 nm or more, or 900 nm or more, or 1000 nm or more, or 1250 nm or more, or 1500 nm or more, or 1750 nm or more, or 2000 nm or more, or 2250 nm or more, or 2500 nm or more, or 2750 nm or more, or 3000 nm or more, and in some examples, the size is 5000 nm or less, such as 4000 nm or less, and 3000 nm or less. For example, the starting suspension of liposomes can comprise large multilamellar vesicles (LMVs), such as multilamellar vesicles having an average size in the range of 200 nm or more, such as 200 nm to 3000 nm. In some examples, the starting suspension of liposomes can comprise large unilamellar vesicles (LUVs), such as unilamellar vesicles having an average size of 100 nm or more, such as in the range of 100 nm to 1000 nm.

[0039] In some cases, embodiments of the method can include the step of generating a starting suspension of liposomes. As noted above, the suspension of liposomes can be heterogeneous with respect to the size of the liposomes in the liposome suspension. A suspension of heterogeneous liposomes can be produced using any convenient method for manufacturing liposomes, such as, but not limited to, the solvent dispersion method (e.g., the Bangham method including dissolution of lipids in an organic solvent followed by removal of the organic solvent, such as by evaporation of the organic solvent), the detergent removal process (e.g., formation of detergent-lipid micelles followed by removal of the detergent to form liposomes), the injection process (e.g., lipids are dissolved in an organic solvent and the resulting lipid solution is injected into an aqueous medium), the microfluidic process (e.g., when a stream of lipids dissolved in an organic solvent passes between two aqueous streams within a microfluidic channel), the mechanical dispersion process, the sonication process, combinations thereof, and the like.

[0040] Liposomes useful in embodiments of the present disclosure are composed of lipids. In certain embodiments, the lipid is amphiphilic. An amphiphilic lipid may include a hydrophilic group and one or more lipophilic groups covalently bonded to the hydrophilic group. In some cases, the hydrophilic group is a charged group such as an anionic group or a cationic group. In some examples, the hydrophilic group is a non-charged polar group. In some embodiments, the hydrophilic group includes a charged group and a polar group. Examples of hydrophilic groups include, but are not limited to, phosphate, phosphocholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositol, ethylphosphosphorylcholine, polyethylene glycol, polyglycerol, sphingosine, phosphoshingosine, tri-nitrilotriacetic acid, melamine, glucosamine, trimethylamine, spermine, spermidine, and conjugated carboxylates, sulfates, borates, sulfonates, sulfates, carbohydrates, amino acids, etc. In some cases, the hydrophilic group includes phosphocholine.

[0041] In certain embodiments, the lipophilic group includes an aliphatic chain such as a saturated or unsaturated, straight or branched, substituted or unsubstituted aliphatic chain. For example, the lipophilic group may include an aliphatic chain having 2 to 40 carbon atoms in length, and may be saturated or unsaturated, straight or branched, substituted or unsubstituted. For example, the lipophilic group may include a saturated or unsaturated, straight or branched, substituted or unsubstituted hydrocarbon chain having 2 to 40 carbon atoms, such as 4 to 30 carbon atoms, or 4 to 25 carbon atoms, or 6 to 24 carbon atoms, or 10 to 20 carbon atoms. In certain cases, the lipophilic group includes a saturated or unsaturated, straight or branched hydrocarbon chain having 18 carbon atoms. In certain cases, the lipophilic group includes a saturated or unsaturated, straight or branched hydrocarbon chain having 16 carbon atoms. In embodiments where the lipid includes more than one lipophilic group, the lipophilic groups may be the same or, in other cases, different. The liposome may be composed of the same type of lipid or a combination of two or more different types of lipids.

[0042] Embodiments of liposomes can include liposomes having a detectable label. In some cases, the detectable label stably associates with a support. "Stably associates" means that a moiety binds or otherwise associates with another moiety or structure under standard conditions. The binding can include, but is not limited to, covalent and non-covalent bonds such as ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, etc. In certain embodiments, the detectable label covalently binds to the liposome. For example, as described above, the lipid comprising the liposome can include a hydrophilic group, which in some cases can include an activating functional group that provides a covalent bond to a detectable label. Any convenient activating functional group useful in chemical synthesis can be used to covalently bond a detectable label to a hydrophilic group of the lipid, such as, but not limited to, amine, carboxyl, amide, hydroxy, azide, maleimide, bromoacetyl, 2-pyridyldithiol, haloalkyl, alkene, or propargyl, etc.

[0043] Liposomes according to embodiments of the present disclosure can include a payload associated with the liposome. As used herein, "payload" refers to a component included within the structure of a liposome that is present within the bilayer of the lipid particle or attached to the surface of the liposome (e.g., by covalent or non-covalent attachment). Thus, the payload can include components encapsulated by the liposome (e.g., pharmaceutically active agents, nutritional supplements, cosmetic agents, imaging agents, radiopharmaceuticals, magnetic resonance imaging contrast agents, etc.). In certain embodiments, the encapsulated payload can be present in solution or as a crystal, as a powder, or as a combination thereof. For example, in embodiments where it is desired to provide liposomes having an encapsulated payload (e.g., an encapsulated drug, therapeutic agent, contrast agent, etc.), such an agent can be included in the aqueous phase inside the liposome. Alternatively, in embodiments where the agent is hydrophobic and thus poorly soluble in water, the hydrophobic agent can be included within a portion of the lipid bilayer.

[0044] The method of the present disclosure is useful for producing a population of liposomes as described above, for example, a population of liposomes of a defined size. In some embodiments, the population of liposomes has an average size that is less than the average size of the starting suspension of liposomes. In some cases, the population of liposomes produced has an average size (e.g., average diameter) of 1000 nm or less, such as 900 nm or less, or 800 nm or less, or 700 nm or less, or 600 nm or less, or 500 nm or less, or 400 nm or less, or 300 nm or less, or 250 nm or less, or 200 nm or less, or 150 nm or less, or 100 nm or less, or 75 nm or less, or 50 nm or less, or 25 nm or less, or 20 nm or less, or 15 nm or less, or 10 nm or less, or 5 nm or less, or 1 nm or less, and in some examples, the average size is 1 nm or more, such as 5 nm or more. In certain examples, the population of liposomes produced has an average size of 1000 nm or less. In certain examples, the population of liposomes produced has an average size of 800 nm or less. In certain examples, the population of liposomes produced has an average size of 500 nm or less. In certain examples, the population of liposomes produced has an average size of 400 nm or less. In certain examples, the population of liposomes produced has an average size of 300 nm or less. In certain examples, the population of liposomes produced has an average size of 250 nm or less. In certain examples, the population of liposomes produced has an average size of 200 nm or less. In certain examples, the population of liposomes produced has an average size of 100 nm or less. In certain examples, the population of liposomes produced has an average size of 50 nm or less. For example, the population of liposomes produced can include small unilamellar vesicles (SUVs), such as unilamellar vesicles having an average size in the range of 100 nm or less, such as 10 nm to 100 nm.

[0045] In certain examples, the starting suspension of liposomes has an average size that is larger than the pores of the membrane. In some examples, pulling larger-sized liposomes through smaller-sized pores in the membrane resizes the liposomes to an average size that is approximately the same as the size of the pores of the membrane.

[0046] Additional aspects In certain embodiments, the method also includes introducing a suspension of liposomes into a liposome extrusion system and then mixing the contents of the liposome extrusion system. The mixing can be carried out using any convenient protocol. For example, mixing can be carried out using a stirrer. The stirrer can be any convenient stirrer sufficient to mix the liquid in the liquid container, which includes, among other stirring protocols, vortexers, sonicators, shakers (e.g., manual, mechanical, or electric shakers), rockers, rocking plates, magnetic stirrers, static mixers, rotators, blenders, mixers, tumblers, orbital shakers, but is not limited thereto.

[0047] In some cases, the method also includes assaying the population of liposomes generated. Assaying the population of liposomes can be carried out using any suitable assay device. For example, the assay can be an assay for determining the average size of the population of liposomes, the polydispersity of the population of liposomes, or a combination thereof. In some cases, the assay can be carried out by dynamic light scattering (DLS). In some cases, the assay device can be a flow cytometer. In these embodiments, assaying includes flow cytometrically analyzing the population of liposomes. In certain embodiments, the liposomes include a fluorescent label, and thus a particular embodiment of assaying includes contacting the population of liposomes with electromagnetic radiation (e.g., light), e.g., electromagnetic radiation having a wavelength corresponding to the excitation maximum of the fluorescent label of the liposomes. Assaying can further include detecting the light emitted from the excited fluorescent label. For example, the method can include detecting the emitted light from the excited fluorescent label at one or more wavelengths corresponding to the emission maximum of the fluorescent label. In certain embodiments, the population of liposomes can be used in a method for calibrating a flow cytometer, e.g., the population of liposomes can be used as a calibration standard for a flow cytometer.

[0048] In certain embodiments, the fluorescent label comprises one or more detectable moieties or markers that are detectable based on, for example, fluorescence emission maxima, fluorescence polarization, fluorescence lifetime, light scattering, mass, molecular weight, or combinations thereof. In certain embodiments, the fluorescent label comprises a fluorophore (i.e., a fluorescent label, a fluorescent dye, etc.). Fluorophores of interest can include, but are not limited to, dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.). A number of dyes (e.g., small molecule dyes) are commercially available from a variety of sources such as, for example, Molecular Probes (Eugene, OR) and Exciton (Dayton, OH). For example, fluorophores of dyes include 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine, and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; brilliant yellow; coumarin, and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyanine, and derivatives such as cyanosin, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7; 4',6-diamidin-2-phenylindole (DAPI); 5',5”-dibromopyrogallol-sulfonphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriaminepentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4'-Dimethylaminophenylazo)benzoic acid (DABCYL); 4-Dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); Eosin, and derivatives such as eosin and eosin isothiocyanate; Erythrosin, and derivatives such as erythrosin B and erythrosin isothiocyanate; Ethidium; Fluorescein, and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein and QFITC (XRITC); Fluorescamine; IR144; IR1446; Green fluorescent protein (GFP); Reef coral fluorescent protein (RCFP); Lissamine (trademark); Lissamine rhodamine, Lucifer yellow; Malachite green isothiocyanate; 4-Methylumbelliferone; Orthocresolphthalein; Nitrotyrosine; Pararosaniline; Nile red; Oregon green; Phenol red; B-Phycoerythrin (PE); o-Phthalaldehyde; Pyrene, and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrenebutyrate; Reactive red 4 (Cibacron (trademark) brilliant red 3B-A); Rhodamine, and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulphonyl chloride derivative of sulforhodamine 101 (Texas red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC); Riboflavin; Rosolic acid and terbium chelate derivatives; Xanthene; Carotenoid-protein complexes, for example, peridinin chlorophyll protein (PerCP); Allophycocyanin (APC);Or it may be a combination of these.;

[0049] Flow cytometry systems and methods suitable for analyzing samples that can be employed in the method of the present invention include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt1):17-28; Linden et al., Semin Throm Hemost. 2004 Oct; 30(5):502-11; Alison et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference.In certain examples, flow cytometry systems of interest include BD Biosciences FACSCanto(TM) and FACSCantoII(TM) flow cytometers, BD Biosciences FACSVantage(TM), BD Biosciences FACSort (TM), BD Biosciences FACSCount(TM), BD Biosciences FACScan(TM), and BD Biosciences FACSCalibur(TM) systems, BD Biosciences Influx(TM) cell sorters, BD Biosciences Accuri(TM) C6 flow cytometers, BD Biosciences LSRFortessa(TM) flow cytometers, BD Biosciences LSRFortessa(TM) X-20 flow cytometers, BD Biosciences FACSVerse(TM) flow cytometers, BD Biosciences FACSAria(TM) III and BD FACSAria(TM) Fusion flow cytometers, BD Biosciences FACSJazz(TM) flow cytometers, and the like. In certain embodiments, the system of the subject is a flow cytometry system such as those described in U.S. Patent Nos. 3,960,449; 4,347,935; 4,667,830; 5,245,318; 5,464,581; 5,483,469; 5,602,039; 5,643,796; 5,700,692; 6,372,506 and 6,809,804, the disclosures of which are incorporated herein by reference in their entirety.

[0050] Although not limiting, other analytical methods may also be used, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry systems. For example, assaying may involve the use of an analytical separation device such as a liquid chromatograph (LC) including a high performance liquid chromatograph (HPLC), a micro or nano liquid chromatograph or an ultra high pressure liquid chromatograph (UHPLC) device, capillary electrophoresis (CE), or a capillary electrophoresis chromatograph (CEC) device. A mass spectrometer (MS) system may be used to assay the dye composition. Examples of mass spectrometers may include, but are not limited to, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron impact (El), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI), or inductively coupled plasma (ICP) ionization, or any combination thereof. Similarly, any of a variety of different mass spectrometers may be employed, including time of flight (TOF), Fourier transform ion cyclotron resonance (FTICR), ion trap, quadrupole or double focusing magnetic electric sector mass analyzers, or any hybrid thereof.

[0051] In certain embodiments, the method also includes storing the liposomes in, for example, a receiving vessel, or another container, for a period of time. The liposomes may be stored for a period of time before and / or after manufacturing a population of liposomes. In some examples, the liposomes are stored for a period of time such as 1 hour or more, or 4 hours or more, or 6 hours or more, or 12 hours or more, or 18 hours or more, or 24 hours or more, or 48 hours or more, or 72 hours or more, or 4 days or more, or 5 days or more, or 6 days or more, or 1 week or more.

[0052] Method embodiments may further include transporting liposomes to a remote location. A "remote location" is a location other than where the liposomes are manufactured. For example, the remote location can be another location within the same city (e.g., an office, a research institute, etc.), another location within a different city, another location within a different state, another location within a different country, and so on. Thus, if one item is shown as being "away" from another item, what this means is that the two items can be separated within the same room, or at least in different rooms or different buildings, and can be at least 1 mile, 10 miles, or 100 miles or more apart.

[0053] In some cases, a population of liposomes can be produced without using a centrifuge to apply centrifugal force to the liposomes. Such a centrifuge can generate a large force on the liposomes. Thus, in some cases, the maximum acceleration force experienced by the liposomes is 10 m / s 2 as follows, and the acceleration due to gravity on the surface of the earth is approximately 9.8 m / s 2 is. Centrifuging the liposomes can generate a centrifugal acceleration force significantly exceeding 10 m / s 2 .

[0054] As described above, the method includes applying a vacuum force to the liposomes. In some cases, the atmospheric pressure is about 101 kPa during the method, and thus, the vacuum force has a magnitude of less than about 101 kPa. In some embodiments, the method is performed without using a pump to generate a pressure higher than the atmospheric pressure, i.e., without using a pressure such as to extrude the liposomes through a porous membrane. Thus, in some cases, the maximum pressure difference that the liposome suspension undergoes during the method is 110 kPa or less, such as 105 kPa or less, 100 kPa or less, or 90 kPa or less. Further, in some cases, the membrane holder and the liposome recovery container contact each other without a fastener for fastening the two elements together. Thus, in some cases, the membrane holder and the liposome recovery container can be separated without removing or disabling the fastener. In some cases, the only force substantially resisting the separation of such elements is gravity. In some embodiments, separating the membrane holder and the liposome recovery container can be performed with a force of less than 20 N, such as less than 10 N, less than 5 N, or less than 2.5 N, in the absence of a vacuum force.

[0055] Device Aspects of the present disclosure include a liposome extrusion device. The liposome extrusion device of the present disclosure is useful for the production of populations of liposomes.

[0056] The liposome extrusion device includes a liposome recovery container, a membrane holder, and a vacuum source. As will be described below, the liposome extrusion system includes a liposome recovery container and a membrane holder. Thus, the liposome extrusion device can also be referred to as comprising a liposome extrusion system and a vacuum source.

[0057] When implementing the method described herein, the membrane holder is arranged such that the porous membrane is positioned inside the liposome recovery vessel, whereby a vacuum force can draw liposomes through the porous membrane into the interior of the recovery vessel. Further, a vacuum source is operably connected to an opening in the wall of the liposome recovery vessel. Thus, the vacuum source can transmit a vacuum force through the opening in the wall of the recovery vessel, through the interior of the recovery vessel, through the porous membrane, to the liposome suspension. In some embodiments, the apparatus further includes a vacuum adapter operably connected to an opening in the wall of the liposome recovery vessel. The vacuum force is transmitted from the vacuum source to the opening in the wall of the liposome recovery vessel during the method, whereby liposomes can be drawn through the porous membrane.

[0058] Membrane holder The membrane holder can be said to have a longitudinal axis and one or more transverse axes perpendicular to the longitudinal axis. For example, the porous membrane can form a plane, and the longitudinal axis can be perpendicular to the plane. Thus, in some cases, the membrane holder is arranged such that the porous membrane is horizontal and the longitudinal axis is vertical, and when a vacuum force is applied, liposomes are moved downward through the porous membrane. In such an orientation, the open first end is located at the top of the membrane holder, while the second end at the membrane holder is located at the bottom. Additionally, the membrane holder has a transverse axis perpendicular to the longitudinal axis, for example, the transverse axis is horizontal and parallel to the porous membrane.

[0059] Thus, in some cases, the membrane holder has a first dimension (i.e., the transverse axis) in the range of 0.5 cm to 20 cm, for example 0.75 cm to 15 cm, or 1 cm to 10 cm, parallel to the porous membrane. In some examples, the membrane holder has a second dimension (i.e., the longitudinal axis) in the range of 1 cm to 25 cm, for example 1.5 cm to 20 or 2 cm to 15 cm, perpendicular to the porous membrane. In some examples, the first dimension, i.e., the transverse axis, is a diameter. In some examples, such dimensions are outer dimensions, i.e., dimensions of one or more outer surfaces of the membrane holder.

[0060] The size of the membrane holder, and thus the volume of the liposome suspension it holds, can in some cases range from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or from 0.1 ml to 800 ml, or from 0.1 ml to 700 ml, or from 0.1 ml to 600 ml, or from 0.1 ml to 500 ml, or from 0.1 ml to 400 ml, or from 0.1 ml to 300 ml, or from 0.1 ml to 200 ml, or from 0.1 ml to 100 ml, or from 0.1 ml to 50 ml, or from 0.1 ml to 25 ml, or from 0.1 ml to 10 ml, or from 0.1 ml to 5 ml, or from 0.1 ml to 2 ml, or from 0.1 ml to 1.5 ml, or from 0.1 ml to 1 ml, or from 0.1 ml to 0.5 ml. In certain examples, the membrane holder is configured to have a liposome suspension holding volume in the range of 1 ml to 750 ml, such as 5 ml to 500 ml, or 10 ml to 200 ml. The liposome suspension holding volume portion can also be referred to as the interior of the membrane holder.

[0061] The open first end of the membrane holder exposes the interior of the liquid container to the ambient environment, for example, such that the contents of the liquid container are at the same atmospheric pressure as the ambient environment. For example, the open first end can be used to allow access to the interior of the liquid container, such as for introducing a suspension of liposomes into the liquid container or removing a suspension of liposomes from the liquid container.

[0062] Embodiments of the membrane holder can be compatible with liquids and / or liposomes or other components that can come into contact with the membrane holder. Examples of suitable membrane holder materials for a liposome extrusion device include, but are not limited to, plastics such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK). In some examples, the membrane holder comprises a polymeric material, such as a plastic material. Exemplary polymeric materials include polycarbonate, polyester, nylon, cellulose, cellulose acetate, polyethylene terephthalate, and the like. In some cases, the membrane holder does not comprise a metallic material. In some embodiments, the membrane holder does not comprise a glass material.

[0063] Porous membrane In certain embodiments, the membrane pores pass through the membrane in a non-meandering path. For example, the porous membrane can include pores having a longitudinal axis that is substantially perpendicular to the surface of the membrane. In some cases, the porous membrane can include pores having a longitudinal axis at an angle of less than 90° with respect to the surface of the membrane. In a particular example, the porous membrane does not include a web-like or matrix structure in which the network of pores is interconnected to form a meandering path through the membrane. In other words, the porous membrane can include discrete pores that pass through the membrane without intersecting other pores in the membrane.

[0064] In certain embodiments, the porous membrane is composed of one or more layers of membrane material. For example, the membrane can be composed of a single layer of membrane material. In other embodiments, the membrane is composed of two or more layers of membrane material. For example, the membrane can include two layers of membrane material, such as three or more layers, or four or more layers, or five or more layers, or six or more layers, or seven or more layers, or eight or more layers, or nine or more layers, or ten or more layers of membrane material. In some cases, the membrane includes two layers of membrane material. In some cases, the membrane includes three layers of membrane material. In embodiments including two or more layers of membrane material, the membrane materials of each layer can be the same or different. In certain embodiments, the pore size of each of two or more layers of membrane material is the same. In other embodiments, the pore sizes of at least two of two or more layers of membrane material are different.

[0065] The membrane can be composed of any suitable membrane material. In some cases, the membrane material is compatible with the liquid and / or liposomes that contact the membrane. For example, the membrane material can be a liquid-compatible membrane material such as a hydrophilic membrane material. In some cases, the liposomes can be in an aqueous liquid, and in these cases, the membrane material can be compatible with the aqueous medium. "Compatible" means that the membrane material is substantially inert (e.g., does not significantly react or decompose) in the presence of the liquid and / or liposomes or other components that contact the membrane. Examples of suitable membrane materials include polymer materials such as, but not limited to, polymers such as polycarbonate, polyester, nylon, cellulose, cellulose acetate, polyethylene terephthalate, etc. In some examples, the membrane material is polycarbonate. In some examples, the membrane material is polyester.

[0066] Liposome recovery container The liposome recovery container that can be used in this method includes an open first end and a closed bottom (i.e., a closed second end) separated by walls that collectively define the interior. The recovery container further includes an opening in the wall to the interior.

[0067] The liposome recovery container can be said to have a longitudinal axis and one or more transverse axes perpendicular to the longitudinal axis. For example, the longitudinal axis can be perpendicular to the open first end. Thus, in some cases, the recovery container is arranged such that the longitudinal axis is vertical and the transverse axis is horizontal. For example, the open first end is at the top of the recovery container. In some embodiments, the opening in the wall of the recovery container is arranged on the side wall of the recovery container. Thus, in some cases, the opening is perpendicular to the transverse axis.

[0068] Thus, in some cases, the recovery container has a first dimension (e.g., the transverse axis) in the range of 1.5 cm to 21 cm, for example, 1.75 cm to 16 cm, or 1.5 cm to 10.5 cm. In some cases, the first dimension is the diameter. In some examples, the recovery container has a second dimension (e.g., the longitudinal axis) in the range of 1 cm to 26 cm, for example 1.5 cm to 25 or 2 cm to 21 cm. In some examples, such dimensions are the outer dimensions, i.e., the dimensions of one or more outer surfaces of the recovery container.

[0069] The size of the liquid container may depend on the amount of liquid held in the liquid container. For example, the liquid container may be configured to hold an amount (e.g., the amount of liquid) in the range of 0.1 ml to 1000 ml, such as 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 2 ml, or 0.1 ml to 1.5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml. In certain examples, the liquid container is configured to hold an amount in the range of 0.1 ml to 5 ml, such as 0.5 ml, or 1 ml, or 1.5 ml, or 2 ml, etc. In other examples, the liquid container is configured to hold an amount in the range of 0.1 ml to 10000 ml, such as 1 ml to 750 ml, 10 ml to 550 ml, 50 ml to 250 ml, or 20 ml to 150 ml.

[0070] Embodiments of the liposome recovery container can be compatible with liquids and / or liposomes, or other components that can contact the membrane holder. Examples of suitable recovery container materials for the liposome extrusion device include, but are not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK), etc. In some examples, the recovery container comprises a polymeric material such as, for example, a plastic material. Exemplary polymeric materials include polycarbonate, polyester, nylon, cellulose, cellulose acetate, polyethylene terephthalate, etc. In some cases, the recovery container does not comprise a metallic material. In some embodiments, the recovery container does not comprise a glass material.

[0071] Vacuum adapter The vacuum adapter can be configured to transmit a vacuum force from a vacuum source to an opening in the wall of the liposome recovery container. For example, the vacuum adapter can have a first opening (i.e., a vacuum intake opening) and a second opening (i.e., a vacuum output opening) separated by a wall that collectively defines the interior of the vacuum adapter. In some cases, the vacuum source can be operably connected to the first vacuum intake opening, i.e., such that the vacuum source can transmit a vacuum force into the interior of the vacuum adapter. The first vacuum intake opening is in fluid communication with the vacuum source, thereby allowing the vacuum force to be transmitted. In some cases, another element such as a hose is used to connect the vacuum source to the first vacuum intake opening. Further, the second vacuum output opening can be operably connected to an opening in the wall of the liposome recovery container. For example, at least a portion of the liposome recovery container can be inserted into the interior of the vacuum adapter through the second vacuum output opening. Thus, when a vacuum force is generated inside the vacuum adapter, such a vacuum force also acts on the opening of the liposome recovery container. This vacuum force is then transmitted through the interior of the liposome recovery container, through the porous membrane, to the liposome suspension, and the liposome suspension is drawn out through the porous membrane. Thus, there is fluid communication from the vacuum source, through the vacuum adapter, to the opening in the wall of the liposome recovery container, thereby allowing the transmission of the vacuum force.

[0072] The vacuum adapter can be said to have a longitudinal axis and one or more transverse axes perpendicular to the longitudinal axis. For example, the longitudinal axis can be perpendicular to the second opening. Thus, in some cases, the vacuum adapter is arranged such that the longitudinal axis is vertical and the transverse axis is horizontal, and for example, the first opening is at the top of the vacuum adapter. Thus, when the recovery container is at least partially disposed within the vacuum adapter, both elements are vertically oriented.

[0073] Thus, in some cases, the vacuum adapter has a first dimension (e.g., a lateral axis) in the range of 2 cm to 21.5 cm, such as 2 cm to 16.5 cm or 2 to 11 cm. In some cases, the first dimension is a diameter. In some examples, the collection container has a second dimension (e.g., a longitudinal axis) in the range of 1.5 cm to 26.5 cm, such as 2 cm to 25.5 cm or 2.5 cm to 21.5 cm. In some examples, such dimensions are outer dimensions, i.e., the dimensions of one or more outer surfaces of the collection container. In embodiments where the collection container is at least partially inserted and disposed inside the vacuum adapter, the vacuum adapter can have an inner dimension larger than the outer dimension of the collection container, thereby enabling insertion.

[0074] Examples of suitable vacuum adapter materials for the liposome extrusion device include, but are not limited to, plastics such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK), etc. In some examples, the vacuum adapter comprises a polymeric material such as a plastic material. Exemplary polymeric materials include polycarbonate, polyester, nylon, cellulose, cellulose acetate, polyethylene terephthalate, etc. In some cases, the vacuum adapter does not comprise a metallic material. In some embodiments, the vacuum adapter does not comprise a glass material.

[0075] System The system of the present disclosure includes a liposome collection container and a membrane holder as described herein. The liposome collection container has an open first end and a closed bottom separated by walls that collectively define an interior. The collection container further includes an opening in the interior wall. The membrane holder includes an open first end and a porous membrane disposed at the second end, the first end and the second end being separated by a wall therebetween that collectively defines a liposome suspension volume.

[0076] As described above, the apparatus includes a liposome collection container, a membrane holder, and a vacuum source. Thus, the apparatus can also be referred to as including a system and a vacuum source.

[0077] The membrane holder is configured to be arranged such that the porous membrane can be positioned inside the liposome collection container. For example, the outer dimension (e.g., outer diameter) of the membrane holder at the second end can be made smaller than the inner dimension (e.g., inner diameter) of the liposome collection container at its open first end. Thus, the second end having the porous membrane can be moved inside the collection container without the need to break or deform any of the elements.

[0078] In some cases, the membrane holder is arranged such that the porous membrane is positioned inside the liposome collection container. In other cases, the membrane holder is arranged such that the porous membrane is positioned outside the inside of the liposome collection container.

[0079] In some embodiments, the system further includes, for example, a vacuum adapter as described above. In some cases, the vacuum adapter is operably coupled to the collection container, and in other cases, the vacuum adapter is not coupled to the collection container.

[0080] The system can also include one or more sealing elements, such as gaskets, that can be used to seal the elements to each other. For example, the sealing element can be configured to seal the membrane holder to the collection container, and another sealing element can be configured to seal the collection container to the vacuum adapter.

[0081] Kit Aspects of the present disclosure also include a liposome extrusion system configured to hold a system and a kit including the packaging. The packaging can be a sealed packaging, such as a water-resistant and / or water-vapor resistant container optionally under airtight and / or vacuum sealing. In certain examples, the packaging is a sterile packaging configured to maintain the system enclosed within the packaging in a sterile environment. "Sterile" means substantially free of microorganisms (such as fungi, bacteria, viruses, spore forms, etc.).

[0082] The kit can further include a liquid. For example, the kit can include a buffer such as a sample buffer, a wash buffer, an assay buffer. In some cases, the kit can include a liquid suitable for suspending liposomes. The kit can further include additional reagents such as, but not limited to, detectable labels (such as fluorescent labels, colorimetric labels, chemiluminescent labels, multicolor reagents, avidin-streptavidin related detection reagents, radiolabels, gold particles, magnetic labels, etc.).

[0083] In certain embodiments, the kit may also include a calibration standard. For example, the kit may include a set of labeled beads, such as a set of reference fluorescently labeled beads. The calibration standard can be useful for determining the accuracy of the assay device and for ensuring consistency between subsequent assays. For example, the calibration standard can be useful for determining the accuracy of a flow cytometer. In some cases, the calibration standard includes labeled beads, such as fluorescently labeled beads. The fluorescently labeled beads can typically be reference fluorescently labeled beads used as a calibration standard. Examples of reference fluorescently labeled beads include, but are not limited to, fluorescently labeled microparticles or nanoparticles. In some cases, the fluorescently labeled beads are configured to remain suspended in the assay mixture and not substantially sediment or aggregate. In some embodiments, the fluorescently labeled beads include, but are not limited to, fluorescently labeled polystyrene beads, fluorescein beads, rhodamine beads, and other beads tagged with fluorescent dyes. Further examples of fluorescently labeled beads are described in U.S. Patent Nos. 6,350,619, 7,738,094, and 8,248,597, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0084] In addition to the above components, the subject kit may further include instructions for practicing the subject method. These instructions can be present in the subject kit in a variety of forms, one or more of which can be present within the kit. One form in which these instructions can be present is as information printed on a suitable medium or substrate, e.g., one or more sheets of paper on which the information is printed, the packaging of the kit, a package insert, etc. Another form is a computer-readable medium, e.g., a CD, DVD, Blu-Ray, computer-readable memory (e.g., flash memory) in which the information is recorded or stored. Yet another form that can be present is a website address that can be used via the Internet to access the information at a destination site. Any convenient form of the instructions can be present in the kit.

[0085] Utility The present methods, systems, and apparatuses find use in applications where a population of liposomes of defined size may be desired, for example, for research or laboratory testing or for the manufacture of therapeutic compositions. In some embodiments, the subject methods, apparatuses, and systems facilitate accurate analysis of analytes (e.g., cells) obtained from biological samples (e.g., organs, tissues, tissue fragments, fluids). In certain instances, the subject methods, apparatuses, and systems find use in testing the accuracy of devices used for the analysis of such analytes for research or laboratory testing. For example, the subject methods, apparatuses, and systems find use in testing the accuracy of flow cytometers. In some cases, a population of liposomes produced using the methods, apparatuses, and systems of the present disclosure is used as a calibration standard for devices such as flow cytometers. Thus, the subject methods, apparatuses, and systems find use in the efficient preparation of a population of liposomes from a suspension of heterogeneous liposomes.

[0086] For example, the present method enables the production of a population of liposomes with relatively common and inexpensive laboratory equipment. For example, some liposome extrusion devices involve using a centrifuge or a high-pressure pump to force liposomes through an extrusion membrane. However, the present method includes the vacuum source and system described herein. Since the vacuum source can be more common and less expensive compared to centrifuges and high-pressure pumps, the present method enables such advantages over other methods of producing a population of liposomes.

[0087] In addition, centrifuges and pumps can generate fairly high forces on components, thereby requiring more durable and expensive materials. However, this vacuum force can have a relatively low magnitude, allowing the use of less expensive materials and thus, in some cases, enabling the use of disposable items. Further, by using disposable items, there is no need to clean the item between different uses. For example, an extrusion membrane within a pump system may require disassembly and cleaning between two different samples, but the used membrane holder can simply be discarded and a new second membrane holder can be used for the second sample. Also, in some cases, the membrane holder can be simply placed on top of the collection vessel without the need for fasteners to hold the elements together. Thus, the system can be quickly and easily disassembled after use to collect liposomes.

[0088] The subject methods, devices, and systems find use in the preparation of populations of liposomes for therapeutic applications, such as liposomes containing substances such as drugs, proteins, fluorescent compounds, etc., which can be used to deliver the substances to a target region in a subject. For example, the subject methods, devices, and systems find use in the preparation of populations of liposomes for drug delivery, cell therapy and in vivo applications, as well as for analytical applications such as for immunoassays (fid).

Example

[0089] The following examples are set forth to provide a complete disclosure and description to those skilled in the art of how to make and use the invention and are not intended to limit the scope that the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s), etc.

[0090] General Procedure The dry lipid mixture was produced by lyophilization or by drying under a stream of inert gas, followed by removal of water under vacuum.

[0091] The dry lipid was then hydrated with an aqueous solution for 30 - 60 minutes. For lipids having a high phase transition temperature, the aqueous solution was preheated before hydration. In some cases, the hydrated lipid suspension was subjected to freeze / thaw cycles to increase the capture efficiency of water-soluble compounds.

[0092] Next, the membrane holder was placed into the liposome recovery container, thereby fabricating the system. FIG. 1A shows the membrane holder 10, which has an open first end 11 (at the top) and a porous membrane 12 disposed at the second end 13, and these are separated by a wall 14 therebetween that collectively defines a liposome suspension holding volume 15. FIG. 1A also shows an O-ring 30 disposed under a flange 16 at the first end of the membrane holder. FIG. 1B shows a liposome recovery container 20 having an open first end 21 (at the top) and a closed bottom 22 separated by a wall 23 that collectively defines an interior 24. The wall also has an opening 25 and a top flange 26, and an O-ring 40 is disposed below the flange. FIG. 1C shows how the membrane holder can be disposed inside the liposome recovery container, and one of the O-rings serves to seal the two flanges, thereby helping to form a seal.

[0093] Next, the system was placed into a vacuum adapter so that a vacuum force could be transmitted through an opening in the wall of the liposome recovery container. FIG. 2 shows the placement of the system inside a vacuum adapter 50, and a second O-ring 40 serves to form a seal between the liposome recovery container and the vacuum adapter. The vacuum adapter 50 includes an open first end 51 (at the top), an opening 52 at a second end 53 (at the bottom), and a wall 54 therebetween that collectively defines an interior 55. A flange 56 is also shown that can seal the flange 26 of the recovery container via the O-ring 40. In addition, an opening in the wall of the liposome recovery container connects the interior of the liposome recovery container and the interior of the vacuum adapter. The vacuum adapter 50 in FIG. 2 has an open end 52 at the bottom end 53 of the adapter, shown to enable connection to a vacuum source. Thus, the vacuum source can exert a vacuum force through the vacuum adapter and through the opening in the wall of the liposome recovery container onto the porous membrane.

[0094] A sample was loaded into the liposome suspension holding volume inside the membrane holder. Next, a vacuum force was applied, thereby pulling liposomes through the porous membrane into the interior and generating a population of liposomes.

[0095] In some cases, the extrusion procedure was repeated. In such cases, the vacuum force was removed and the membrane holder was taken out of the liposome collection container. The aqueous fluid containing the liposomes was removed, the membrane holder was repositioned to its original position, and the liposomes contained in the fluid were placed inside the membrane holder. The vacuum force was applied again, thereby pulling the liposomes out through the porous membrane again.

[0096] Example 1 Using the general procedure described above, LMV consisting of 42 mol% PMPC, 14% DOPC, 13% DOPE, 30 mol% cholesterol, and 1 mol% DHPE-F was extruded 5 times in a system having three polycarbonate membranes with a pore size of 400 nm under a vacuum of 88 kPa. Dynamic light scattering (DLS) showed that the generated liposomes had a size of 336.7 + / - 11.7 nm, as shown in Figure 3.

[0097] Example 2 Using the general procedure described above, 42 mol% PMPC, 14% DOPC, 13% DOPE, 30 mol% cholesterol, and 1 mol% DHPE-F were extruded 10 times in a system having three polycarbonate membranes with a pore size of 800 nm under a vacuum of 88 kPa. The liposomes were then extruded an additional 5 times in a system having one polycarbonate membrane with a pore size of 200 nm under a vacuum of 88 kPa. The size of the liposomes was determined by DLS and was 195.8 + / - 13.7 nm, as shown in Figure 4.

[0098] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses. 1. A method for producing a population of liposomes, the method comprising: (a) a liposome collection container comprising an open first end and a closed bottom separated by walls that collectively define an interior, and further comprising an opening in the wall to the interior, and A membrane holder comprising an open first end and a porous membrane disposed at a second end, the first and second ends being separated by a wall therebetween that collectively defines a liposome suspension holding volume, the membrane holder being arranged such that the porous membrane is positioned inside a liposome recovery container To provide a liposome extrusion system comprising (b) introducing a liposome suspension into the liposome suspension holding volume (c) drawing liposomes inwardly through the porous membrane and applying a vacuum force through an opening in a manner sufficient to create a population of liposomes and including 2. The method according to clause 1, wherein providing includes disposing the second end of the membrane holder inside the liposome recovery container 3. The method according to any one of clauses 1 to 2, wherein an opening in the wall of the liposome recovery container is located above the porous membrane 4. The method according to any one of clauses 1 to 3, wherein an opening in the wall of the liposome recovery container is oriented perpendicular to the porous membrane 5. The method according to any one of clauses 1 to 4, wherein the membrane holder is oriented such that the porous membrane is located directly below the open first end 6. The method according to any one of clauses 1 to 5, wherein the volume of the liposome recovery container below the porous membrane is larger than the liposome suspension holding volume 7. The method according to any one of clauses 1 to 6, wherein the membrane holder has a circular cross-section that is coaxial with the circular cross-section of the liposome recovery container 8. The method according to any one of clauses 1 to 7, further comprising a gasket for sealing the membrane holder to the liposome recovery container 9. The method according to any one of clauses 1 to 8, further comprising a vacuum adapter, wherein a vacuum force can be transmitted from a vacuum source to the vacuum adapter and then to an opening in the wall of the liposome recovery container 10. The method according to clause 9, wherein at least a portion of the liposome recovery container is disposed inside the vacuum adapter 11. The vacuum adapter is the method according to any one of clauses 9 to 10, having a circular cross-section coaxial with the circular cross-section of the liposome recovery container. 12. The method according to any one of clauses 9 to 11, further comprising a gasket for sealing the vacuum adapter to the liposome recovery container. 13. The liposome suspension has an average diameter of 1000 nm or more, and is the method according to any one of clauses 1 to 12. 14. The porous membrane has a pore diameter of 1000 nm or less, and is the method according to any one of clauses 1 to 13. 15. The porous membrane has a pore diameter in the range of 1 nm to 500 nm, and is the method according to clause 14. 16. The liposome population has an average diameter in the range of 75% to 125% of the pore diameter of the porous membrane, and is the method according to any one of clauses 1 to 15. 17. The membrane holder has a first dimension parallel to the porous membrane in the range of 1 cm to 10 cm and a second dimension perpendicular to the porous membrane in the range of 1.5 cm to 20 cm, and is the method according to any one of clauses 1 to 16. 18. The first dimension of the membrane holder is the diameter, and is the method according to clause 17. 19. The liposome suspension holding volume is in the range of 5 ml to 500 ml, and is the method according to any one of clauses 1 to 18. 20. The liposome suspension holding volume is in the range of 10 ml to 100 ml, and is the method according to clause 19. 21. The liposome recovery container has a first dimension in the range of 1.5 cm to 10.5 cm and a second dimension in the range of 2 cm to 21 cm, and is the method according to any one of clauses 1 to 20. 22. The first dimension of the liposome recovery container is the diameter, and is the method according to clause 21. 23. The interior of the liposome recovery container has a volume in the range of 10 ml to 550 ml, and is the method according to any one of clauses 1 to 22. 24. The interior of the liposome recovery container has a volume in the range of 20 ml to 150 ml, and is the method according to clause 23. 25. The vacuum adapter has a first dimension in the range of 2 cm to 11 cm and a second dimension in the range of 3 cm to 22 cm, and the method according to any one of clauses 9 to 24. 26. The vacuum adapter has a volume in the range of 20 ml to 600 ml, and the method according to any one of clauses 9 to 25. 27. One or more of the membrane holder, the liposome collection container, and the vacuum adapter comprise a polymer material, and the method according to any one of clauses 1 to 26. 28. One or more of the membrane holder, the liposome collection container, and the vacuum adapter do not comprise a metallic material, and the method according to any one of clauses 1 to 27. 29. One or more of the membrane holder, the liposome collection container, and the vacuum adapter do not comprise a glass material, and the method according to any one of clauses 1 to 28. 30. The porous membrane comprises a polycarbonate material, and the method according to any one of clauses 1 to 29. 31. The system does not comprise a fastener for fastening the membrane holder to the liposome collection container, and the method according to any one of clauses 1 to 30. 32. In the absence of a vacuum force, the membrane holder can be separated from the liposome collection container with a force of 10 N or less, and the method according to any one of clauses 1 to 31. 33. The vacuum force has a magnitude in the range of 0.1 kPa to 105 kPa, and the method according to any one of clauses 1 to 32. 34. The magnitude of the vacuum force is in the range of 50 kPa to 95 kPa, and the method according to clause 33. 35. The maximum pressure difference received by the liposome suspension in the method is 105 kPa or less, and the method according to any one of clauses 1 to 34. 36. The maximum acceleration force received by the liposome suspension in the method is 10 m / s 2 or less, and the method according to any one of clauses 1 to 35. 37. The method further includes transferring a population of liposomes from the liposome collection container to the receiving container, and the method according to any one of clauses 1 to 36. 38. A liposome extrusion device for manufacturing a population of liposomes, comprising a liposome collection container having an open first end and a closed bottom separated by a wall that collectively defines an interior, and further comprising an opening in the wall into the interior; a membrane holder comprising an open first end and a porous membrane disposed at a second end, the first and second ends being separated by a wall therebetween that collectively defines a liposome suspension holding volume, the membrane holder being arranged such that the porous membrane is positioned inside the liposome collection container; and a vacuum source operably connected to an opening in the wall of the liposome collection container. A liposome extrusion device comprising the above. 39. The device according to clause 38, wherein the opening in the wall of the liposome collection container is located above the porous membrane. 40. The device according to any one of clauses 38 to 39, wherein the opening in the wall of the liposome collection container is oriented perpendicular to the porous membrane. 41. The device according to any one of clauses 38 to 40, wherein the membrane holder is oriented such that the porous membrane is positioned directly below the open first end. 42. The device according to any one of clauses 38 to 41, wherein the volume of the liposome collection container below the porous membrane is larger than the liposome suspension holding volume. 43. The device according to any one of clauses 38 to 42, wherein the membrane holder has a circular cross-section that is coaxial with the circular cross-section of the liposome collection container. 44. The device according to any one of clauses 38 to 43, further comprising a gasket that seals the membrane holder to the liposome collection container. 45. The device according to any one of clauses 38 to 44, further comprising a vacuum adapter, wherein the vacuum force can be transmitted from the vacuum source to the vacuum adapter and then to the opening in the wall of the liposome collection container. 46. The device according to clause 45, wherein at least a part of the liposome collection container is disposed inside the vacuum adapter. 47. The vacuum adapter is the device according to any one of clauses 45 to 46, having a circular cross-section that is coaxial with the circular cross-section of the liposome recovery container. 48. The device according to any one of clauses 45 to 47, further comprising a gasket for sealing the vacuum adapter to the liposome recovery container. 49. The device according to any one of clauses 38 to 48, further comprising a suspension of liposomes located in the liposome suspension holding volume portion. 50. The device according to clause 49, wherein the suspension of liposomes has an average diameter of 1000 nm or more. 51. The device according to any one of clauses 49 to 50, wherein the porous membrane has a pore diameter of 1000 nm or less. 52. The device according to clause 51, wherein the porous membrane has a pore diameter in the range of 1 nm to 500 nm. 53. The device according to any one of clauses 49 to 52, wherein the population of liposomes has an average diameter in the range of 75% to 125% of the pore diameter of the porous membrane. 54. The device according to any one of clauses 38 to 53, wherein the membrane holder has a first dimension parallel to the porous membrane in the range of 1 cm to 10 cm and a second dimension perpendicular to the porous membrane in the range of 1.5 cm to 20 cm. 55. The device according to any one of clauses 38 to 54, wherein the first dimension of the membrane holder is the diameter. 56. The device according to any one of clauses 38 to 55, wherein the liposome suspension holding volume is in the range of 5 ml to 500 ml. 57. The device according to clause 56, wherein the liposome suspension holding volume is in the range of 10 ml to 100 ml. 58. The device according to any one of clauses 38 to 57, wherein the liposome recovery container has a first dimension in the range of 1.5 cm to 10.5 cm and a second dimension in the range of 2 cm to 21 cm. 59. The device according to clause 58, wherein the first dimension of the liposome recovery container is the diameter. 60. The device according to any one of clauses 38 to 59, wherein the interior of the liposome recovery container has a volume in the range of 10 ml to 550 ml. 61. The device according to any one of clauses 38 to 60, wherein the interior of the liposome collection container has a volume in the range of 20 ml to 150 ml. 62. The device according to any one of clauses 45 to 61, wherein the vacuum adapter has a first dimension in the range of 2 cm to 11 cm and a second dimension in the range of 3 cm to 22 cm. 63. The device according to any one of clauses 45 to 62, wherein the vacuum adapter has a volume in the range of 20 ml to 600 ml. 64. The device according to any one of clauses 38 to 63, wherein one or more of the membrane holder, the liposome collection container, and the vacuum adapter comprise a polymer material. 65. The device according to any one of clauses 38 to 64, wherein one or more of the membrane holder, the liposome collection container, and the vacuum adapter do not comprise a metallic material. 66. The device according to any one of clauses 38 to 65, wherein one or more of the membrane holder, the liposome collection container, and the vacuum adapter do not comprise a glass material. 67. The device according to any one of clauses 38 to 66, wherein the porous membrane comprises a polycarbonate material. 68. The device according to any one of clauses 38 to 67, wherein the device does not comprise a fastening tool for fastening the membrane holder to the liposome collection container. 69. The device according to any one of clauses 38 to 68, wherein the membrane holder can be separated from the liposome collection container with a force of 10 N or less in the absence of a vacuum force. 70. A system for manufacturing a population of liposomes, a liposome collection container comprising an open first end and a closed bottom separated by walls that collectively define an interior, and further comprising an opening in the wall to the interior, a membrane holder comprising an open first end and a porous membrane disposed at a second end, the first and second ends being separated by a wall therebetween that collectively defines a liposome suspension holding volume, and the membrane holder being configured such that the porous membrane is positioned inside the liposome collection container, comprising a system. 71. The system according to clause 70, further comprising a gasket configured to seal the membrane holder to the liposome recovery container. 72. The system according to any one of clauses 70 to 71, further comprising a vacuum adapter configured to transmit a vacuum force from a vacuum source to the vacuum adapter and then to an opening in the wall of the liposome recovery container. 73. The system according to clause 72, further comprising a gasket configured to seal the vacuum adapter to the liposome recovery container. 74. The system according to any one of clauses 70 to 73, wherein the membrane holder has a first dimension in the range of 1 cm to 10 cm parallel to the porous membrane and a second dimension in the range of 1.5 cm to 20 cm perpendicular to the porous membrane. 75. The system according to clause 74, wherein the first dimension of the membrane holder is a diameter. 76. The system according to any one of clauses 70 to 75, wherein the liposome suspension holding volume is in the range of 5 ml to 500 ml. 77. The system according to clause 76, wherein the liposome suspension holding volume is in the range of 10 ml to 100 ml. 78. The system according to any one of clauses 70 to 77, wherein the liposome recovery container has a first dimension in the range of 1.5 cm to 10.5 cm and a second dimension in the range of 2 cm to 21 cm. 79. The system according to clause 78, wherein the first dimension of the liposome recovery container is a diameter. 80. The system according to any one of clauses 70 to 79, wherein the interior of the liposome recovery container has a volume in the range of 10 ml to 550 ml. 81. The system according to any one of clauses 70 to 80, wherein the interior of the liposome recovery container has a volume in the range of 20 ml to 150 ml. 82. The system according to any one of clauses 72 to 81, wherein the vacuum adapter has a first dimension in the range of 2 cm to 11 cm and a second dimension in the range of 3 cm to 22 cm. 83. The system according to clause 82, wherein the vacuum adapter has a volume in the range of 20 ml to 600 ml. 84. The system according to any one of clauses 72 to 83, wherein one or more of the membrane holder, the liposome recovery container, and the vacuum adapter comprise a polymer material. 85. The system according to any one of clauses 72 to 84, wherein one or more of the membrane holder, the liposome recovery container, and the vacuum adapter do not comprise a metallic material. 86. The system according to any one of clauses 72 to 85, wherein one or more of the membrane holder, the liposome recovery container, and the vacuum adapter do not comprise a glass material. 87. The system according to any one of clauses 72 to 86, wherein the porous membrane comprises a polycarbonate material. 88. A kit for manufacturing a population of liposomes, comprising: the system according to any one of clauses 70 to 87; packaging configured to hold the system; and a kit.

[0099] In at least some of the foregoing embodiments, one or more of the elements used in the embodiments can be used interchangeably in another embodiment, provided that such substitution is not technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0100] In general, those skilled in the art will understand that the terms used in this specification, particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be "open" terms (e.g., the term "comprising" should be construed as "including but not limited to", the term "having" should be construed as "having at least", the term "including" should be construed as "including but not limited to", etc.). If a specific number of recited elements in a claim is intended, such intent will be expressly recited in the claim, and those skilled in the art will further understand that such intent does not exist if there is no such recitation. For example, for purposes of illustration, the following appended claims may include the use of introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to limit any particular claim that recites an element introduced by the indefinite article "a" or "an" to embodiments that contain only one such recited element, even if the claim includes such introductory phrases as "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce claim recitations. Further, even if a specific number of recited elements in a claim is expressly recited, those skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., a bare recitation of "two recited elements" without other modifiers means at least two recited elements, or two or more recited elements).Furthermore, in those examples where conventional expressions similar to "at least one of A, B, and C, etc." are used, generally, such a structure is intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, and C" would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those examples where conventional expressions similar to "at least one of A, B, or C, etc." are used, generally, such a structure is intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, or C" would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It should be further understood by one of ordinary skill in the art that, wherever in the specification, claims, or drawings, substantially any alternative word and / or phrase presenting two or more alternative terms is to be understood as contemplating the possibility of including one of the terms, any of the terms, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B".

[0101] In addition, when features or aspects of the present disclosure are described with respect to a Markush group, one of ordinary skill in the art will recognize that the present disclosure thereby describes it with respect to any individual member or subgroup of the members of the Markush group.

[0102] As will be understood by those skilled in the art, all ranges disclosed herein include any and all sub-ranges and combinations of sub-ranges for any and all purposes, such as for the purpose of providing the recited description. Any recited range can be readily recognized as fully describing and enabling the same range as would be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily decomposed into, for example, a lower third, a middle third, and an upper third. Also, as will be understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", etc., refer to a range that includes the recited number and can then be decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having from 1 to 3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having from 1 to 5 articles refers to a group having 1, 2, 3, 4, or 5 articles, and so on.

[0103] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but it will be readily apparent to those skilled in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims in light of the teachings of this invention.

[0104] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated by those skilled in the art that, although not explicitly described or shown herein, various configurations that embody the principles of the present invention and fall within its spirit and scope can be devised. Further, all of the examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Moreover, all descriptions of the principles, aspects, and embodiments of the present invention herein, as well as their specific examples, are intended to encompass both their structural and functional equivalents. Further, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any development element that performs the same function regardless of structure. Additionally, nothing disclosed herein is intended to be dedicated to the public, whether or not such disclosure is explicitly recited in the claims.

[0105] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is explicitly defined to apply to a claim limitation only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim, and 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) does not apply when such exact phrase is not used in the claim limitation.

[0106] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to the filing date of U.S. Provisional Patent Application No. 63 / 340,091, filed May 10, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

Claims

1. A method for producing a population of liposomes, comprising: (a) a liposome recovery container having an open first end and a closed bottom separated by a wall that collectively defines an interior, further comprising an opening in the wall into the interior, and a membrane holder having an open first end and a porous membrane disposed at a second end, the first and second ends being separated by a wall therebetween that collectively defines a liposome suspension holding volume, the membrane holder being arranged such that the porous membrane is located inside the interior of the liposome recovery container providing a liposome extrusion system comprising; (b) introducing a liposome suspension into the liposome suspension holding volume; (c) drawing liposomes through the porous membrane into the interior and applying a vacuum force through the opening in a manner sufficient to produce a population of liposomes comprising.

2. The method of claim 1, wherein said providing comprises disposing the second end of the membrane holder inside the interior of the liposome recovery container.

3. The method according to any one of claims 1 to 2, wherein the opening in the wall of the liposome recovery container is located above the porous membrane.

4. The method according to any one of claims 1 to 3, wherein the opening in the wall of the liposome recovery container is oriented perpendicular to the porous membrane.

5. The method according to any one of claims 1 to 4, wherein the membrane holder is oriented such that the porous membrane is located directly below the open first end.

6. The method according to any one of claims 1 to 5, wherein the volume of the liposome recovery container below the porous membrane is larger than the liposome suspension holding volume.

7. The method according to any one of claims 1 to 6, wherein the membrane holder has a circular cross-section that is coaxial with the circular cross-section of the liposome recovery container.

8. The method according to any one of claims 1 to 7, further comprising a gasket for sealing the membrane holder to the liposome recovery container.

9. The method according to any one of claims 1 to 8, further comprising a vacuum adapter, wherein a vacuum force can be transmitted from a vacuum source to the vacuum adapter and then to the opening in the wall of the liposome recovery container.

10. The method according to any one of claims 1 to 9, wherein the porous membrane has a pore size of 1000 nm or less.

11. The method according to any one of claims 1 to 10, further comprising transferring the population of liposomes from the liposome recovery container to the receiving container.

12. A liposome extrusion device for producing a population of liposomes, A liposome recovery container having an open first end and a closed bottom separated by a wall that collectively defines an interior, and further comprising an opening in the wall into the interior; A membrane holder having an open first end and a porous membrane disposed at a second end, the first and second ends being separated by a wall therebetween that collectively defines a liposome suspension holding volume, the membrane holder being arranged such that the porous membrane is located inside the liposome recovery container; A vacuum source operably connected to the opening in the wall of the liposome recovery container A liposome extrusion device comprising.

13. The device according to claim 12, wherein the opening in the wall of the liposome recovery container is located above the porous membrane.

14. A system for producing a population of liposomes, A liposome recovery container having an open first end and a closed bottom separated by a wall that collectively defines an interior, and further comprising an opening in the wall into the interior; A membrane holder having an open first end and a porous membrane disposed at a second end, the first and second ends being separated by a wall therebetween that collectively defines a liposome suspension holding volume, the membrane holder being configured such that the porous membrane is located inside the liposome recovery container; A system comprising.

15. A kit for producing a population of liposomes, The system according to claim 14, Packaging configured to hold the system A kit comprising.

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