Systems for vacuum mediated liposome preparation and methods of use thereof

Abstract

The invention provides a liposome preparation system. 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 is operably connectable to a vacuum source such that a vacuum force can be applied through the opening to draw liposomes through the porous membrane and produce a population of liposomes. The invention also provides methods of producing liposomes, as well as devices and kits for performing the methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to the filing date of U.S. Provisional Patent Application Serial No. 63 / 340,091, filed on May 10, 2022; the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Liposome is a spherical vesicle with one or more lipid bilayers. Liposomes comprising a single lipid bilayer can be referred to as unilamellar liposomes, and liposomes comprising multiple lipid bilayers can be referred to as multilamellar vesicles. Liposomes can be prepared using different methods, which can depend on factors such as the lipid composition of the lipid bilayer of the liposome, the type of medium in which the lipid vesicle is dispersed, the desired size of the liposome, the desired polydispersity of the liposome, the robustness of the production method and the reproducibility between batches and other factors, such as the intended use of the resulting liposome. For example, liposomes can include substances such as drugs, and can be used to deliver the substance to the target area of ​​the patient. Therefore, the method for producing such liposomes can also depend on the physicochemical characteristics of the substance to be embedded in the liposome, the concentration of the embedded substance, or other processes involved in applying the liposome to / delivering the patient.

[0004] After a liposome suspension (such as a suspension of large multilamellar vesicles) has been produced, it may be desirable to produce liposomes with a size within a certain size range. A common technique for determining liposome size is extrusion (a process by which large multilamellar vesicles can be destroyed and reduced by extrusion through a polycarbonate membrane with a defined pore size). In the currently employed extrusion scheme, first, phospholipids are suspended in a buffered saline solution to obtain large multilamellar vesicles (LMVs). The vesicles are then repeatedly extruded through a polycarbonate membrane with a defined pore size, extruded back and forth by a specially modified gastight syringe attached to the membrane support chamber. Summary of the invention

[0005] The inventor has recognized that by using a vacuum device, the film extrusion process can be significantly improved, accelerated and simplified. The method of the embodiment of the present invention is not to use a special glass airtight syringe and a metal film supporting chamber, but to use a plastic component comprising a film holding unit, a collecting tube and a vacuum tube, wherein the film holding unit and the collecting tube can be disposable. By not using glass and metal liposomes to make extrusion devices, supporting filters and polycarbonate membranes, and eliminating the time of assembling and disassembling such devices, the vacuum device of the embodiment of the present invention saves time and money. Converting from a special extrusion device to a disposable device eliminates the possibility of cross contamination between post-work cleaning and liposome samples.

[0006] The present invention provides a system for producing a population of liposomes. 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 so that: a vacuum force can be applied through the opening to draw the liposomes through the porous membrane and produce a population of liposomes. Also provided are methods of producing liposomes, and apparatus and kits for performing the methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A A membrane holder and an O-ring are shown according to an embodiment of the present invention.

[0008] Figure 1B A liposome collection container and an O-ring according to an embodiment of the present invention are shown.

[0009] Figure 1C The placement of a membrane holder into a liposome collection container is shown, wherein an O-ring seals the flange of the membrane holder to the flange of the liposome collection container, according to an embodiment of the present invention.

[0010] Figure 2 The placement of a membrane holder and a liposome collection container into a vacuum adapter according to an embodiment of the present invention is shown.

[0011] Figure 3 Shown is the particle size distribution determined by dynamic light scattering (DLS) analysis of liposomes drawn through a membrane at 400 nm.

[0012] Figure 4 Shown is the particle size distribution determined by dynamic light scattering (DLS) analysis of liposomes drawn through a membrane at 200 nm. DETAILED DESCRIPTION

[0013] The present invention provides a system for producing a population of liposomes. 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 so that: a vacuum force can be applied through the opening to draw the liposomes through the porous membrane and produce a population of liposomes. Also provided are methods of producing liposomes, and apparatus and kits for performing the methods.

[0014] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as these embodiments may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be restrictive, as the scope of the present invention will only be limited by the appended claims.

[0015] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of the range (up to one tenth of the lower limit unit, unless the context clearly indicates otherwise) and any other stated value or intermediate value within the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits within the stated ranges. Where the stated range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0016] Certain ranges of values ​​given herein are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number to which it is preceded, as well as numbers that are close to or approximately the number to which the term is preceded. In determining whether a number is close to or approximately a specifically enumerated number, a close or approximately unenumerated number may be a number that, in the context in which it is presented, provides a substantial equivalence to the specifically enumerated number.

[0017] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials will now be described.

[0018] All publications and patents cited in this specification are incorporated herein by reference, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in conjunction with the cited publications. The citation of any publication is for disclosures prior to the filing date, and should not be construed as an admission that the present invention cannot obtain an earlier filing date than these publications because of prior inventions. In addition, the publication date provided may be different from the actual publication date, which may need to be independently confirmed.

[0019] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0020] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that may be readily separated or combined with the features of any other embodiments without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0021] Although the apparatus and method have been or will be described for the sake of grammatical fluency and functional explanation, it should be clearly understood that unless expressly recited pursuant to 35 U.S.C. §11, the claims should not be interpreted as necessarily being limited in any way to the "apparatus" or "step" limitations, but should be consistent with the full scope of the meaning and equivalents provided by the definition of the claims under the doctrine of equivalents, and in the event that the claims are expressly recited pursuant to 35 U.S.C. §112, they will be given full legal equivalents pursuant to 35 U.S.C. §112.

[0022] Method for producing liposomes

[0023] The present invention provides a method for producing a liposome population. For example, the method may include: providing a liposome extrusion system, the liposome extrusion system including a liposome collection container and a membrane holder. The liposome collection container includes: an open first end separated by a wall and a closed bottom, the first end, the bottom and the wall together define an internal space. The collection container also has an opening located in the wall. The membrane holder has an open first end separated by a wall and a porous membrane located at the second end, the first end, the second end and the wall together define a liposome suspension holding volume, i.e., the internal space of the collection container. The membrane holder is positioned so that: the porous membrane is located in the internal space of the liposome collection container.

[0024] The method may further comprise the step of introducing a liposome suspension into a liposome suspension holding volume. In this way, the liposome suspension may contact a porous membrane (e.g., the top side of the porous membrane). Thereafter, a vacuum force may be applied through an opening in the wall of the liposome collection container so that: the liposomes are sucked into the interior space of the collection container through the porous membrane and a liposome population is produced. In other words, the vacuum force may be transferred from the opening in the wall of the collection container to the interior space of the collection container, wherein the porous membrane is located in the interior space of the collection container. In this way, the vacuum force may be applied through the pores of the porous membrane, thereby sucking the liposomes through the pores of the porous membrane. When the liposomes pass through the membrane, the liposomes pass through the pores in the porous membrane to produce a liposome population.

[0025] In some cases, providing a system includes: placing the second end of the membrane holder into the interior space of the liposome collection container. In this way, the porous membrane at the second end of the membrane holder is also positioned in the interior space of the liposome collection container. Therefore, the liposome suspension holding volume of the membrane holder is fluidly connected to the interior space of the liposome collection container through the porous membrane. "Fluid communication" means that a fluid (e.g., a gas or liquid, which includes a suspension of liposomes) can flow from one area to another. For example, in the liposome extrusion system disclosed herein, the fluid can flow from the liposome suspension holding volume of the membrane holder through the porous membrane to the interior space of the liposome collection container.

[0026] In some cases, the opening in the wall of the liposome collection container is located above the porous membrane. In this way, when the liquid of the liposome suspension is sucked through the porous membrane under the action of the vacuum force, the amount of liquid leaving the liposome collection container through the opening can be minimized or eliminated. In other words, by positioning the opening above the membrane, it is possible to inhibit the liquid from being accidentally sucked into the opening easily, thereby reducing the loss of liquid to the outside of the container. In some cases, the opening in the wall is perpendicular to the porous membrane orientation. For example, the porous membrane can be horizontally oriented, and the opening can be positioned in the vertical or nearly vertical wall of the collection container. In some cases, the opening is oriented within 30 ° of the vertical direction or less, such as 15 ° or less, or 5 ° or less.

[0027] In some cases, the membrane holder is oriented so that the porous membrane is located directly below the first end of the opening. In other words, the membrane holder is oriented so that there is a vertical line that intersects both the first end of the opening and the porous membrane.

[0028] In addition, in some embodiments, the volume of the liposome collection container below the porous membrane is greater than the liposome suspension holding volume. Therefore, even if the liposome suspension holding volume is completely filled with the liposome suspension liquid, the liquid volume will not be enough 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 by 5% or more, such as 20% or more, or 50% or more. Such relative volume can reduce the chance that liquid will overflow into the opening (for example when the user carries the system).

[0029] In certain embodiments, a suspension of liposomes can be introduced into a liposome extrusion system such that: the suspension of liposomes is positioned on one side of the membrane. For example, the suspension of liposomes can be contained in a liposome suspension holding volume positioned on one side of the membrane. During the generation of a liposome population, the suspension of liposomes can pass through the porous membrane from one side of the membrane and reach the other side of the membrane. For example, the suspension of liposomes can initially be contained in a liposome suspension holding volume positioned on a first side of the membrane, and then during the generation of the liposome population, can pass through the porous membrane and be contained in the interior space of a liposome collection container positioned on the other side of the membrane.

[0030] In certain aspects, the suspension of liposomes is introduced into the liposome extrusion system using any convenient liquid handling technique. For example, a volume of the suspension of liposomes can be added to the liposome extrusion system using any convenient liquid handling device, such as, but not limited to, a syringe, a needle, a pipette, an aspirator, or the like.

[0031] In some embodiments, one or both of the membrane holder and the liposome collection container may have a circular cross-section. In some cases, both elements have a circular cross-section. In addition, the membrane holder and the liposome collection container may be positioned so that: the circular cross-section of the membrane holder is coaxial with the circular cross-section of the liposome collection container. In other words, each circular cross-section may have a center of circle, wherein the axis is positioned perpendicular to the cross-section and intersects with the center of circle.

[0032] In some cases, the system also includes a sealing element that seals the film holder to the liposome collection container. The seal between these elements can be liquid-tight, air-tight or both. As used herein, "liquid-tight" refers to that liquid will not leak through the connection between the elements, and "air-tight" refers to that gas will not leak through the connection between the elements. In certain embodiments, the sealing element is a gasket, such as an O-ring, which seals the film holder to the liposome collection container.

[0033] Liposomes can be sucked through a porous membrane by applying vacuum force to the suspension of liposomes in the liposome suspension holding volume. In some cases, vacuum force is produced by a vacuum source, such as a vacuum ejector, a rotary vane pump, a diaphragm pump, a liquid ring pump, a reciprocating compressor, a scroll compressor and a cam pump. For example, a vacuum ejector utilizes the Venturi effect to produce a vacuum by fluid flow, wherein the vacuum ejector is sometimes referred to as an aspirator. Vacuum force depends on the pressure difference between the vacuum source and the atmosphere around 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 also become a higher or lower value, such as during some weather events, the air pressure at some locations on the earth is up to about 105 kPa. In some cases, the atmospheric pressure during the method is about 101 kPa, so the size of the vacuum force is less than about 101 kPa. In some cases, the vacuum force may range from 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. Vacuum may also be described by the pressure within the vacuum source. In some cases, the vacuum source pressure range is 100 kPa to 0.1 kPa, which may be referred to herein as a rough vacuum or a low vacuum. For example, the vacuum source pressure range may be 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 range is 100 Pa to 0.1 Pa, which may be referred to herein as a fine vacuum or a medium vacuum. In other cases, the vacuum source pressure range is 0.1 Pa to 10 -5 Pa is referred to as high vacuum in this article.

[0034] In some cases, the system for performing the method further comprises 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. In some cases, the system is configured such that: fluid communication is achieved between the vacuum source, the vacuum adapter, and the interior space of the liposome collection container through the opening of the liposome collection container. The vacuum adapter can have a first opening and a second opening and a wall of the vacuum adapter, the first opening being operably coupled to the vacuum source, the second opening being operably coupled to the opening of the liposome collection container, and they together define the interior space of the vacuum adapter.

[0035] In some embodiments, at least a portion of the liposome collection container is located within the interior space of the vacuum adapter. The vacuum adapter can have a circular cross-section, for example, wherein 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.

[0036] In some cases, repeat introducing and applying step, so that suction liposomes pass through film again once or more times.In such case, for example, by vertically lifting film holder, film holder can be separated from liposome collection container.Afterwards, for example, by user operation or robot-operated pipette, liposomes can be extracted from liposome collection container, and then be assigned to the liposome suspension holding volume of film holder.Then element can be positioned so that: porous membrane is located in the inner space of liposome collection container, and vacuum force can be applied again.Introducing and applying step can be repeated 1 time or more times, for example 2 times or more times, 3 times or more times, 4 times or more times, 6 times or more times, 8 times or more times or 10 times or more times.

[0037] In some embodiments, the method further comprises: transferring the liposome population from the liposome collection container to the receiving container. For example, the membrane holder can be separated from the liposome collection container, for example, by vertically lifting the membrane holder. The liposomes are then extracted from the liposome collection container, for example, by a user-operated or robot-operated pipette, and then dispensed into the receiving container.

[0038] Liposomes

[0039] In some instances, the liposome population produced by the method is a liposome population of a defined size. "Defined size" means that due to the manner in which the liposomes are prepared, the size of each liposome in the population is known, and in particular, 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 liposome population may have substantially the same average diameter. "Average" means the arithmetic mean. Substantially identical values ​​include values ​​that differ from each other 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, substantially identical values ​​include values ​​that differ from each other by 10% or less. In some cases, substantially identical values ​​include values ​​that differ from each other by 5% or less. In some cases, substantially the same values ​​include values ​​that differ by 3% or less from each other. In some cases, substantially the same values ​​include values ​​that differ by 1% or less from each other. In some cases, substantially the same values ​​include values ​​that differ by 0.5% or less from each other. In some instances, the average size of the liposomes may differ 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 average size of the liposomes differs by 10% or less. In some cases, the average size of the liposomes differs by 5% or less. In some cases, the average size of the liposomes differs by 3% or less. In some cases, the average size of the liposomes differs by 1% or less. In some cases, the average size of the liposomes differs by 0.5% or less.

[0040] 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 particle size in a mixture. In the context of liposomes, polydispersity can range from 0 to 1, where a polydispersity of 0 represents a monodisperse population of liposomes (e.g., liposomes of the same average size), and where a polydispersity of 1 represents a heterogeneous mixture of liposomes. In some cases, the size (and therefore the polydispersity) of the liposomes can be determined by dynamic light scattering (DLS).

[0041] 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 includes a population of liposomes having non-uniform sizes. Therefore, the methods of the present disclosure include: starting with a suspension of liposomes (e.g., a population of liposomes having non-uniform 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 an initial population of liposomes, i.e., to distinguish such liposomes from a population of liposomes generated by aspiration of a porous membrane.

[0042] In some embodiments, the method comprises generating a population of liposomes from a suspension of heterogeneous liposomes, wherein the average size of the resulting population of liposomes varies 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 comprises generating a population of liposomes from a suspension of heterogeneous liposomes, wherein the average size of the resulting population of liposomes varies by 10% or less. In some cases, the method comprises generating a population of liposomes from a suspension of heterogeneous liposomes, wherein the average size of the resulting population of liposomes varies by 5% or less. In some cases, the method comprises generating a population of liposomes from a suspension of heterogeneous liposomes, wherein the average size of the resulting population of liposomes varies by 3% or less. In some cases, the method comprises generating a population of liposomes from a suspension of heterogeneous liposomes, wherein the average size of the resulting population of liposomes differs by 1% or less. In some cases, the method comprises generating a population of liposomes from a suspension of heterogeneous liposomes, wherein the average size of the resulting population of liposomes differs by 0.5% or less. In yet other examples, the average size of different liposome members of the population may differ by 50% or more, such as 75% or more, including 100% or more. For example, the average size of different liposome members in the starting suspension of liposomes may differ by 50% or more, such as 75% or more, including 100% or more.

[0043] In some instances, the starting suspension of liposomes has a higher polydispersity than the population of liposomes produced. Thus, the methods of the present disclosure can be used to produce a population of liposomes having a polydispersity less than the polydispersity of the starting suspension of liposomes.

[0044] In some cases, the polydispersity of the liposome population produced is 0.9 or less, such as 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 liposome population produced can be 0.5 or less. In some cases, the polydispersity of the liposome population produced can be 0.4 or less. In some cases, the polydispersity of the liposome population produced can be 0.3 or less. In some cases, the polydispersity of the liposome population produced can be 0.2 or less. In some cases, the polydispersity of the liposome population produced can be 0.1 or less. In some cases, the polydispersity of the liposome population produced can be 0.05 or less. In some cases, the polydispersity of the liposome population produced can be 0.01 or less. In certain instances, the polydispersity of the liposome population produced is in the range of 0.01 to 0.5, e.g., 0.01 to 0.4, or 0.01 to 0.3, or 0.01 to 0.2, or 0.01 to 0.1. In other embodiments, the polydispersity of the liposome population produced is in the range of 0.01 to 0.5, e.g., 0.05 to 0.5, or 0.1 to 0.5, or 0.1 to 0.4, or 0.1 to 0.3. In other embodiments, the polydispersity of the liposome population produced is in the range of 0.01 to 0.5, e.g., 0.05 to 0.5, or 0.1 to 0.5, or 0.2 to 0.5, or 0.2 to 0.4.

[0045] In some cases, the polydispersity of the starting suspension of liposomes is 0.5 or greater, e.g., 0.6 or greater, or 0.7 or greater, or 0.8 or greater, or 0.9 or greater. In some cases, the polydispersity of the starting suspension of liposomes is 1. For example, the polydispersity of the starting suspension of liposomes can range from 0.5 to 1, e.g., 0.6 to 1, or 0.7 to 1, or 0.8 to 1, or 0.9 to 1.

[0046] In some examples, the starting suspension of liposomes includes liposomes having a size greater than the population of liposomes produced. In some examples, the starting suspension of liposomes includes liposomes having an average size (e.g., average diameter) of 500 nm or greater (e.g., 600 nm or greater, or 700 nm or greater, or 800 nm or greater, or 900 nm or greater, or 1000 nm or greater, or 1250 nm or greater, or 1500 nm or greater, or 1750 nm or greater, or 2000 nm or greater, or 2250 nm or greater, or 2500 nm or greater, or 2750 nm or greater, or 3000 nm or greater), wherein, in some examples, the size is 5000 nm or less, such as 4000 nm or less, including 3000 nm or less. For example, the starting suspension of liposomes may include large multilamellar vesicles (LMVs), such as multilamellar vesicles having an average size of 200 nm or more (e.g., in the range of 200 nm to 3,000 nm). In some examples, the starting suspension of liposomes may include large unilamellar vesicles (LUVs), such as unilamellar vesicles having an average size of 100 nm or more (e.g., in the range of 100 nm to 1000 nm).

[0047] In some cases, embodiments of the method may include: the step of generating a starting suspension of liposomes. As described above, with respect to the size of the liposomes in the suspension of liposomes, the suspension of liposomes may be inhomogeneous. The suspension of inhomogeneous liposomes may be generated using any convenient method for producing liposomes, such as, but not limited to, solvent dispersion (e.g., Bangham method, which includes dissolving lipids in an organic solvent, then removing the organic solvent, such as by evaporating the organic solvent), detergent removal (e.g., wherein detergent-lipid micelles are formed, then detergent is removed to form liposomes), injection (e.g., wherein lipids are dissolved in an organic solvent and the resulting lipid solution is injected into an aqueous medium), microfluidic method (e.g., wherein a lipid stream dissolved in an organic solvent is passed between two water streams in a microfluidic channel), mechanical dispersion, ultrasonic treatment, and combinations thereof, etc.

[0048] The liposomes that can be used in the embodiments of the present disclosure are composed of lipids. In certain embodiments, the lipids are amphiphilic. Amphiphilic lipids 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 instances, the hydrophilic group is an uncharged polar group. In certain embodiments, the hydrophilic group includes a charged group and a polar group. Examples of hydrophilic groups include, but are not limited to: phosphate, phosphorylcholine, phosphoglycerol, phosphoethanolamine, phosphoserine, phosphoinositide, ethylphosphocholine, polyethylene glycol, polyglycerol, sphingosine, phosphoinosine, trinitrotriacetic acid, melamine, aminoglucose, trimethylamine, spermine, spermidine and conjugated carboxylates, sulfates, boric acid, sulfonates, sulfates, carbohydrates, amino acids, etc. In some cases, the hydrophilic group includes phosphorylcholine.

[0049] In certain embodiments, the lipophilic group includes a fatty chain, such as a saturated or unsaturated, straight or branched, substituted or unsubstituted fatty chain. For example, the lipophilic group may include a fatty chain of 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 with 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 some cases, the lipophilic group includes a saturated or unsaturated, straight or branched hydrocarbon chain with 18 carbon atoms. In some cases, the lipophilic group includes a saturated or unsaturated, straight or branched hydrocarbon chain with 16 carbon atoms. In the embodiment where lipid includes more than one lipophilic group, the lipophilic group may be identical, or may be different in other cases. Liposomes can be composed of the same type of lipid, or of a combination of two or more different types of lipids.

[0050] Embodiments of the liposomes may include: liposomes with detectable labels. In some cases, the detectable labels are stably associated with the support. "Stably associated" means: under standard conditions, a certain part is bonded to another part or structure, or otherwise associated with another part or structure. The bond may include covalent bonds and non-covalent interactions, such as but not limited to ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces) and dipole-dipole interactions, etc. In certain embodiments, the detectable label is covalently bonded to the liposome. For example, as described above, the lipids comprising the liposomes may include: a hydrophilic group, which may include in some cases: an activated functional group provided for covalent connection with the detectable label. Any convenient activated functional group useful in chemical synthesis may be used to covalently bond the detectable label to the hydrophilic group of the lipid, such as but not limited to amine, carboxyl, amide, hydroxyl, azide, maleimide, bromoacetyl, 2-pyridyldithiol, haloalkyl, olefin or propargyl, etc.

[0051] Liposomes according to embodiments of the present disclosure may include a payload associated with the liposome. As used herein, "payload" refers to a component contained in a liposome structure, present in a bilayer of lipid particles, or attached to a liposome surface (e.g., by covalent bonds or non-covalent interactions). Therefore, the payload may include a component (e.g., a pharmaceutically active agent, a nutritional agent, a cosmeceutical, an imaging agent, a radiopharmaceutical, and a nuclear magnetic resonance contrast agent, etc.) encapsulated by the liposome. In certain embodiments, the encapsulated payload is in a solution, or may be present as a crystal, a powder, or a combination thereof. For example, in embodiments of liposomes having an encapsulated payload (e.g., an encapsulated agent, a therapeutic agent, or an imaging agent, etc.), such an agent may be contained in an aqueous phase within the liposome. Alternatively, in embodiments where the agent is hydrophobic and therefore less soluble in water, a hydrophobic agent may be contained within a portion of the lipid bilayer.

[0052] The method of the present disclosure can be used to produce a liposome population as described above, such as a liposome population with a determined size. In some embodiments, the average size of the liposome population is less than the average size of the starting suspension of the liposome. In some cases, the liposome population produced has an average size (e.g., average diameter) of 1000nm or less (e.g., 900nm or less, or 800nm ​​or less, or 700nm or less, or 600nm or less, or 500nm or less, or 400nm or less, or 300nm or less, or 250nm or less, or 200nm or less, or 150nm or less, or 100nm or less, or 75nm or less, or 50nm or less, or 25nm or less, or 20nm or less, or 15nm or less, or 10nm or less, or 5nm or less, or 1nm or less), wherein, in some instances, the average size is 1nm or greater, such as 5nm or greater. In some instances, the liposome population produced has an average size of 1000nm or less. In some instances, the liposome population produced has an average size of 800 nm or less. In some instances, the liposome population produced has an average size of 500 nm or less. In some instances, the liposome population produced has an average size of 400 nm or less. In some instances, the liposome population produced has an average size of 300 nm or less. In some instances, the liposome population produced has an average size of 250 nm or less. In some instances, the liposome population produced has an average size of 200 nm or less. In some instances, the liposome population produced has an average size of 100 nm or less. In some instances, the liposome population produced has an average size of 50 nm or less. For example, the liposome population produced can include small unilamellar vesicles (SUVs), such as unilamellar vesicles having an average size of 100 nm or less (e.g., in the range of 10 nm to 100 nm).

[0053] In some instances, the average size of the starting suspension of liposomes is larger than the pores of the membrane. In some instances, pumping larger sized liposomes through smaller sized pores in the membrane adjusts the size of the liposomes to an average size that is approximately the same as the size of the pores of the membrane.

[0054] Other aspects

[0055] In certain embodiments, the method further comprises: mixing the contents of the liposome extrusion device after the liposome suspension is introduced into the liposome extrusion device. Any convenient scheme can be used for mixing. For example, a stirrer can be used for mixing. The stirrer can be any convenient stirrer that is enough to mix the liquid in the liquid container, including but not limited to a vortexer, sonicator, oscillator (e.g., manual, mechanical or electric oscillator), rocker, oscillating plate, magnetic stirrer, static mixer, rotator, blender, mixer, drum, orbital oscillator and other stirring schemes.

[0056] In some cases, the method further comprises: measuring the liposome population produced. The measurement of the liposome population can be performed using any suitable measurement device. For example, the measurement can be a measurement for determining the average size of the liposome population, the polydispersity of the liposome population, or a combination thereof. In some cases, the measurement can be performed by dynamic light scattering (DLS). In some cases, the measurement device can be a flow cytometer. In these embodiments, the measurement comprises: analyzing the liposome population flow cytometry. In certain embodiments, the liposomes include a fluorescent marker, and therefore certain embodiments of the measurement comprise: contacting the liposome population with electromagnetic radiation (e.g., light), such as a wavelength of electromagnetic radiation corresponding to an excitation maximum of the fluorescent marker of the liposome. The measurement can also comprise: detecting emission light from the excited fluorescent marker. For example, the method can comprise: detecting emission light from the excited fluorescent marker at one or more wavelengths corresponding to the emission maximum of the fluorescent marker. In certain embodiments, the liposome population can be used in a method for calibrating a flow cytometer, for example, the liposome population can be used as a calibration standard for the flow cytometer.

[0057] In certain embodiments, fluorescent labels include: one or more detectable parts or markers, which are detectable based on, for example, fluorescence emission maximum, fluorescence polarization, fluorescence lifetime, light scattering, mass, molecular weight, or a combination thereof. In certain embodiments, fluorescent labels include fluorophores (i.e., fluorescent markers, fluorescent dyes, etc.). The fluorophores of interest may include, but are not limited to, dyes suitable for analytical applications (e.g., flow cytometry, imaging, etc.). A large number of dyes (e.g., non-polymeric dyes) can be commercially available from various sources, such as Molecular Probes (Eugene, Oregon) and Exciton (Denton, Ohio).For example, the fluorophore of the dye can be: 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; o-aminobenzamide; Brilliant Yellow; coumarin and derivatives, such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151); cyanines and derivatives, such as tetrachlorotetrabromofluorescein (cyanosine), Cy3, Cy3.5, Cy5, Cy5.5 and Cy7; 4',6-diaminidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 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; erythrosine and derivatives, such as erythrosine B and erythrosine isothiocyanate; ethidium bromide (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), chlorotriazinylfluorescein (fluorescein chlorotriazinyl), naphthofluorescein and QFITC ​​(XRITC); fluorescamine; IR144; IR1446; green fluorescent protein (GFP); reef coral fluorescent protein (RCFP); lissamine. TM; Lissamine rhodamine, fluorescent yellow; malachite green isothiocyanate; 4-methylumbelliferone; o-cresolphthalein; nitrotyrosine; para-carmine; Nile Red; Oregon Green; phenol red; B-phycoerythrin (PE); o-phthalaldehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron TM 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, the sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rhodamine and terbium chelate derivatives; xanthene; carotenoid-protein complexes, such as peridinin-chlorophyll protein (PerCP); allophycocyanin (APC); or combinations thereof.

[0058] Suitable flow cytometry systems and methods for analyzing samples that can be used in the methods 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 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 disclosure of which is incorporated herein by reference. In certain embodiments, flow cytometry systems of interest include: BD Biosciences FACSCantoTM and FACSCanto II TM Flow cytometer, BD Biosciences FACSVantage TM BD Biosciences FACSort TM BD Biosciences FACSCount TM BD Biosciences FACScan TM , and BD Biosciences FACSCalibur TM System, BD Biosciences Influx TM Cell sorter, BD Biosciences Accuri TM C6 flow cytometer; BD Biosciences LSR Fortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSAria TM III and BD FACSAria TM Fusion Flow Cytometer or BD Biosciences FACSJazz TM Flow cytometers, etc. In certain embodiments, the subject system is a flow cytometry system, such as those described in U.S. Pat. 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 entireties.

[0059] Other analytical methods can also be used, such as but not limited to liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry systems.For example, measuring can include using analytical separation device, such as liquid chromatograph (LC) (comprising high performance liquid chromatograph (HPLC), micron or nano liquid chromatograph or ultra-high pressure liquid chromatograph (UHPLC) device), capillary electrophoresis (CE) or capillary electrophoresis chromatograph (CEC) device.Mass spectrometer (MS) system can also be used for measuring dye composition.The example of mass spectrometer can include but not limited to electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron bombardment (EI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI) or inductively coupled plasma (ICP) ionization or such as any combination thereof.Similarly, any one of various mass analyzers can be adopted, comprising time-of-flight (TOF), Fourier transform ion cyclotron resonance (FTICR), ion trap, quadrupole or double focusing magnetoelectric sector mass analyzer or its any mixing.

[0060] In certain embodiments, the method further comprises: storing the liposomes for a period of time, for example in a receiving container or in another container. The liposomes can be stored for a period of time before and / or after the liposome population is generated. In some instances, the liposomes are stored for a period of time, such as 1 hour or longer, or 4 hours or longer, or 6 hours or longer, or 12 hours or longer, or 18 hours or longer, or 24 hours or longer, or 48 hours or longer, or 72 hours or longer, or 4 days or longer, or 5 days or longer, or 6 days or longer, or 1 week or longer.

[0061] Embodiments of the method may further include transporting the liposomes to a remote location. A "remote location" is a location other than the location where the liposomes are produced. For example, the remote location may be another location in the same city (e.g., an office, a laboratory, etc.), another location in a different city, another location in a different state, another location in a different country, etc. Thus, when one project is indicated as being "remote" from another project, this means that the two projects may be in the same room but separate, or at least in different rooms or different buildings, and may be at least one mile, ten miles, or a hundred miles or more apart.

[0062] 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 large forces on the liposomes. Thus, in some cases, the maximum acceleration experienced by the liposomes is 10 m / s. 2 or less, where the gravitational acceleration on the Earth's surface is about 9.8 m / s 2 Centrifugation of liposomes can generate speeds far exceeding 10 m / s 2 The centrifugal acceleration.

[0063] As mentioned above, the method comprises: applying vacuum force to liposome. In some cases, atmospheric pressure is about 101kPa during the method, so the size of vacuum force is less than about 101kPa. In certain embodiments, the method is performed without using a pump to generate a pressure higher than atmospheric pressure, that is, without using the pressure that pushes the liposome through the porous membrane. Therefore, in some cases, the maximum pressure difference experienced by the suspension of liposomes during the method is 110kPa or less, such as 105kPa or less, 100kPa or less, or 90kPa or less. In addition, in some cases, the film retainer and the liposome collection container contact each other without using the fasteners that fasten the two elements together. Therefore, in some cases, the film retainer and the liposome collection container can be separated without removing or deactivating the fasteners. In some cases, the only force that substantially prevents these elements from separating is gravity. In certain embodiments, in the absence of vacuum force, the film retainer and the liposome collection container can be separated with a force less than 20N (such as less than 10N, less than 5N or less than 2.5N).

[0064] Device

[0065] Aspects of the present disclosure include a liposome extrusion device.The liposome extrusion device of the present disclosure can be used to produce a population of liposomes.

[0066] The liposome extrusion device comprises a liposome collection container, a film retainer and a vacuum source. As described below, the liposome extrusion system comprises a liposome collection container and a film retainer. Therefore, the liposome extrusion device can also be referred to as comprising a liposome extrusion system and a vacuum source.

[0067] When performing the methods described herein, the membrane holder is positioned so that: the porous membrane is located in the interior space of the liposome collection container, thereby allowing the vacuum force to draw the liposomes through the porous membrane and into the interior space of the collection container. In addition, the vacuum source is operably connected to the opening in the wall of the liposome collection container. Therefore, the vacuum source is able to transmit the vacuum force through the opening in the wall of the collection container, through the interior space of the collection container, through the porous membrane, and into the suspension of liposomes. In some embodiments, the device also includes: a vacuum adapter, which is operably connected to the opening in the wall of the liposome collection container. During the method, the vacuum force can be transmitted from the vacuum source to the opening in the wall of the liposome collection container, thereby drawing the liposomes through the porous membrane.

[0068] Membrane holder

[0069] The membrane holder can be referred to as having 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. Therefore, in some cases, the membrane holder is positioned so that: the porous membrane is horizontal and the longitudinal axis is vertical, wherein the application of vacuum force will move the liposomes downward through the porous membrane. Under such orientation, the first end of the opening is positioned toward the top of the membrane holder, and the second end with the membrane holder is positioned toward the bottom. In addition, the membrane holder has a transverse axis perpendicular to the longitudinal axis, for example, wherein the transverse axis is horizontal and parallel to the porous membrane.

[0070] Therefore, in some cases, the film retainer has: a first dimension (i.e., transverse axis), which is parallel to the porous membrane and has a scope of 0.5 cm to 20 cm, such as 0.75 cm to 15 cm or 1 cm to 10 cm. In some cases, the film retainer has: a second dimension (i.e., longitudinal axis), which is perpendicular to the porous membrane and has a scope of 1 cm to 25 cm, such as 1.5 cm to 20 cm or 2 cm to 15 cm. In some cases, the first dimension (i.e., transverse axis) is a diameter. In some cases, these dimensions are external dimensions, i.e., the dimensions of one or more outer surfaces of the film retainer.

[0071] In some cases, the size of the membrane holder and therefore the liposome suspension holding volume can range from 0.1 ml to 1000 ml, for example 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 some cases, the membrane holder is configured to have a liposome suspension holding volume ranging from 1 ml to 750 ml, such as 5 ml to 500 ml or 10 ml to 200 ml. The liposome suspension volume may also be referred to as the interior space of the membrane holder.

[0072] The open first end of the membrane holder exposes the interior space of the liquid container to the surrounding environment, for example, so that the contents of the liquid container are at the same atmospheric pressure as the surrounding environment. For example, the first open end can be used to allow access to the interior space of the liquid container, for example, for introducing a suspension of liposomes into the liquid container or removing a suspension of liposomes from the liquid container.

[0073] The embodiment of film retainer can be compatible with liquid and / or liposome or other compositions that may contact with film retainer.The example of suitable film retainer material for liposome extrusion device includes but is not limited to plastics, such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK) etc. In some cases, film retainer comprises polymer material, for example plastic material.Exemplary polymer material comprises polycarbonate, polyester, nylon, cellulose, cellulose acetate, polyethylene terephthalate etc. In some cases, film retainer does not comprise metal material.In certain embodiments, film retainer does not comprise glass material.

[0074] porous membrane

[0075] In some embodiments, the membrane pores pass through the membrane in a non-tortuous path. For example, the porous membrane may include: holes having a longitudinal axis substantially perpendicular to the membrane surface. In some cases, the porous membrane may include: holes having a longitudinal axis at an angle of less than 90° relative to the membrane surface. In some instances, the porous membrane does not include: a mesh or matrix structure in which a network of pores is interconnected to form a tortuous path through the membrane. In other words, the porous membrane may include: different pores that pass through the membrane without intersecting other pores in the membrane.

[0076] 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 monolayer of membrane material. In other embodiments, the membrane is composed of two or more layers of membrane material. For example, the membrane can include 2 layers of membrane material, such as 3 or more layers, or 4 or more layers, or 5 or more layers, or 6 or more layers, or 7 or more layers, or 8 or more layers, or 9 or more layers, or 10 or more layers of membrane material. In some cases, the membrane includes 2 layers of membrane material. In some cases, the membrane includes 3 layers of membrane material. In the embodiment comprising two or more layers of membrane material, the membrane material of each layer can be the same, or can be different. In certain embodiments, the pore size of each of the two or more layers of membrane material is the same. In other embodiments, the pore size of at least two of the two or more layers of membrane material is different.

[0077] The film can be made up of any suitable membrane material. In some cases, the membrane material is compatible with the liquid and / or liposome in contact with the film. For example, the membrane material can be a liquid-compatible membrane material, such as a hydrophilic membrane material. In some cases, the liposome can be in an aqueous liquid, and in these cases, the membrane material can be compatible with an aqueous medium. "Compatible" refers to: the membrane material is substantially inert (for example, not significantly reacting or degrading with it) in the presence of the liquid and / or liposome or other components in contact with the film. The example of suitable membrane material includes polymeric materials, such as polymers, such as but not limited to polycarbonate, polyester, nylon, cellulose, cellulose acetate and polyethylene terephthalate etc. In some instances, the membrane material is polycarbonate. In some instances, the membrane material is polyester.

[0078] Liposome collection container

[0079] The liposome collection container that can be used for the present method comprises: an open first end separated by a wall and a closed bottom (i.e., a closed second end), wherein the wall, the first end, and the second end together define an interior space. The collection container also comprises: an opening in the wall leading to the interior space.

[0080] A liposome collection container can be referred to as having a longitudinal axis and one or more transverse axes perpendicular to the longitudinal axis. For example, the longitudinal axis can be perpendicular to the first end of the opening. Thus, in some cases, the collection container is positioned so that: the longitudinal axis is vertical, and the transverse axis is horizontal, for example, wherein the first end of the opening is located at the top of the collection container. In some embodiments, the opening in the wall of the collection container is located on the side wall of the collection container. Thus, in some cases, the opening is perpendicular to the transverse axis.

[0081] Thus, in some cases, the collection container has: a first dimension (e.g., a transverse axis) ranging from 1.5 cm to 21 cm, such as 1.75 cm to 16 cm or 1.5 cm to 10.5 cm. In some cases, the first dimension is a diameter. In some cases, the collection container has: a second dimension (e.g., a longitudinal axis) ranging from 1 cm to 26 cm, such as 1.5 cm to 25 cm or 2 cm to 21 cm. In some cases, these dimensions are external dimensions, i.e., the dimensions of one or more outer surfaces of the collection container.

[0082] The size of the liquid container can depend on the volume of the liquid contained in the liquid container. For example, the liquid container can be configured to contain a volume (e.g., liquid volume) ranging from 0.1 ml to 1000 ml (e.g., 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 some cases, the liquid container is configured to hold a volume ranging from 0.1 ml to 5 ml, such as 0.5 ml, 1 ml, 1.5 ml, or 2 ml. In other cases, the liquid container is configured to hold a volume ranging from 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.

[0083] The embodiment of liposome collection container can be compatible with liquid and / or liposome or other composition that may contact with film holder.The example of suitable collection container material for liposome extrusion device includes but is not limited to plastics, such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkane (PFA), polyethylene terephthalate (PET), polyethylene (PE) and polyetheretherketone (PEEK) etc. In some cases, collection container comprises polymer material, for example plastic material.Exemplary polymer material comprises polycarbonate, polyester, nylon, cellulose, cellulose acetate, polyethylene terephthalate etc. In some cases, collection container does not comprise metal material.In certain embodiments, collection container does not comprise glass material.

[0084] Vacuum adapter

[0085] The vacuum adapter can be configured to transfer the vacuum force from the vacuum source to the opening in the wall of the liposome collection container. For example, the vacuum adapter can have: a first opening (i.e., a vacuum suction opening) and a second opening (i.e., a vacuum output opening) separated by a wall, and the first opening, the second opening and the wall together define the internal space of the vacuum adapter. In some cases, the vacuum source is operably connected to the first vacuum suction opening, that is, so that: the vacuum force can be transferred from the vacuum source to the internal space of the vacuum adapter. The first vacuum suction opening is in fluid communication with the vacuum source, thereby allowing the vacuum force to be transferred. In some cases, another element such as a hose is used to connect the vacuum source to the first vacuum suction opening. In addition, the second vacuum output opening can be operably connected to the opening in the wall of the liposome collection container. For example, at least a portion of the liposome collection container can be inserted into the internal space of the vacuum adapter through the second vacuum output opening. Therefore, when a vacuum force is generated in the internal space of the vacuum adapter, the vacuum force is also applied to the opening of the liposome collection container. Then, the vacuum force is transferred to the liposome suspension through the internal space of the liposome collection container and the porous membrane, causing the liposome suspension to be sucked through the porous membrane. In this way, there is fluid communication from the vacuum source through the vacuum adapter to the opening in the wall of the liposome collection container, allowing the vacuum force to be transmitted.

[0086] The vacuum adapter can be referred to as having 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 positioned such that: the longitudinal axis is vertical and the transverse axis is horizontal, such as where the first opening is located at the top of the vacuum adapter. Thus, if the collection container is at least partially placed in the vacuum adapter, both elements are oriented vertically.

[0087] Thus, in some cases, the vacuum adapter has: a first dimension (e.g., a transverse axis) that ranges from 2 cm to 21.5 cm, such as 2 cm to 16.5 cm, or 2 cm to 11 cm. In some cases, the first dimension is a diameter. In some cases, the collection container has: a second dimension (e.g., a longitudinal axis) that ranges from 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 cases, these dimensions are external dimensions, i.e., dimensions of one or more outer surfaces of the collection container. In embodiments where the collection container is at least partially inserted into and located within the interior space of the vacuum adapter, the interior dimensions of the vacuum adapter can be larger than the exterior dimensions of the collection container to allow insertion.

[0088] Examples of suitable vacuum adapter materials for liposome extrusion devices include, but are not limited to, plastics, such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), and polyetheretherketone (PEEK), etc. In some cases, the vacuum adapter comprises a polymer material, such as a plastic material. Exemplary polymer materials include polycarbonate, polyester, nylon, cellulose, cellulose acetate, and polyethylene terephthalate, etc. In some cases, the vacuum adapter does not include a metal material. In some embodiments, the vacuum adapter does not include a glass material.

[0089] system

[0090] 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 separated by a wall and a closed bottom, the first end, the bottom and the wall together defining an interior space. The collection container also includes an opening in the wall of the interior space. The membrane holder includes: an open first end and a porous membrane at a second end, the first end and the porous membrane are separated by a wall located therebetween, the first end, the second end and the wall together defining a liposome suspension volume.

[0091] As described above, a device includes a liposome collection container, a membrane holder and a vacuum source. Therefore, a device can also be said to include a system and a vacuum source.

[0092] The membrane holder is configured to be positioned so that the porous membrane can be located in the interior space of the liposome collection container. For example, the outer dimension (e.g., outer diameter) of the membrane holder at the second end can be smaller than the inner dimension (e.g., inner diameter) of the liposome collection container at its open first end. Therefore, the second end with the porous membrane can be moved into the interior space of the collection container without destroying or deforming any element.

[0093] In some cases, the membrane holder is positioned so that the porous membrane is located in the interior space of the liposome collection container. In other cases, the membrane holder is positioned so that the porous membrane is located outside the interior space of the liposome collection container.

[0094] In some embodiments, the system further comprises: a vacuum adapter, such as described above. In some cases, the vacuum adapter is operably coupled to the collection container, while in other cases, the vacuum adapter is not coupled to the collection container.

[0095] The system may also include: one or more sealing elements, such as gaskets, which can be used to seal the elements to each other. For example, a sealing element can be configured to seal the film holder to the collection container, and another sealing element can be configured to seal the collection container to the vacuum adapter.

[0096] Kits

[0097] Aspects of the present disclosure also include kits, which include: the subject liposome extrusion system and a package configured to hold the system. The package can be: a sealed package, such as a waterproof and / or water vapor-proof container, which is optionally airtight and / or vacuum-sealed. In some instances, the package is: a sterile package, which is configured to maintain the device enclosed in the package in a sterile environment. "Sterile" means: substantially free of microorganisms (such as fungi, bacteria, viruses, spore forms, etc.).

[0098] The kit may also include a liquid. For example, the kit may include a buffer, such as a sample buffer, a wash buffer, and an assay buffer. In some cases, the kit may include a liquid suitable for a suspension of liposomes. The kit may also include additional reagents, such as, but not limited to, detectable labels (e.g., fluorescent labels, colorimetric labels, chemiluminescent labels, multicolor reagents, avidin-streptavidin related detection reagents, radioactive labels, gold particles, magnetic labels, etc.), etc.

[0099] In certain embodiments, the kit may also include calibration standards. For example, the kit may include a set of labeled beads, such as a set of fluorescently labeled standard beads. Calibration standards can be used to determine the accuracy of the assay device and to ensure consistency between subsequent assays. For example, calibration standards can be used to determine the accuracy of a flow cytometer. In some cases, calibration standards include labeled beads, such as fluorescently labeled beads. Fluorescently labeled beads can be fluorescently labeled standard beads that are commonly used as calibration standards. Examples of fluorescently labeled standard beads include, but are not limited to, fluorescently labeled microparticles or nanoparticles. In some cases, fluorescently labeled beads are configured so that they remain suspended in the assay mixture and are substantially non-precipitated or aggregated. In some embodiments, fluorescently labeled beads include, but are not limited to, fluorescently labeled polystyrene beads, fluorescein beads, rhodamine beads, and other beads labeled with fluorescent dyes. Other examples of fluorescently labeled beads are described in U.S. Patent Nos. 6,350,619; 7,738,094; and 8,248,597, the disclosure of each of which is incorporated herein by reference in its entirety.

[0100] In addition to the above-mentioned components, the theme kit can also include instructions for practicing the theme method. These instructions can be present in the theme kit in various forms, one or more of which can be present in the kit. A form in which these instructions can exist is as information printed on suitable medium or substrate (for example, one or more sheets of paper on which information is printed), information printed in the packaging of the kit, information printed in a package insert, etc. Another form will be a computer-readable medium, such as CD, DVD, Blu-Ray, computer-readable memory (for example, flash memory) etc., which have recorded or stored information thereon. Another form that can exist is the website address that can obtain the information of deleted websites via the Internet. Any convenient form of instructions can be present in the kit.

[0101] Practicality

[0102] The methods, systems and devices of the present invention can be used for applications where a liposome population with a determined size may be required, such as for research or laboratory testing, or for the production of therapeutic compositions. In some embodiments, the subject methods, devices and systems contribute to the accurate analysis of analytes (e.g., cells) obtained from biological samples (e.g., organs, tissues, tissue fragments, fluids). In some instances, the subject methods, devices and systems can be used to test the accuracy of devices for analyzing such analytes for research or laboratory testing. For example, the subject methods, devices and systems can be used to test the accuracy of flow cytometers. In some cases, the liposome populations produced using the methods, devices and systems disclosed herein are used as calibration standards for devices (e.g., flow cytometers). Therefore, the subject methods, devices and systems can be used to effectively prepare liposome populations from a suspension of inhomogeneous liposomes.

[0103] For example, the present method allows the use of relatively common and inexpensive laboratory equipment to produce liposome populations. For example, some liposome extrusion devices involve the use of a centrifuge or a high-pressure pump to force the liposomes through an extrusion membrane. However, the present method involves a vacuum source and the system described herein. Since vacuum sources may be more common and less expensive than centrifuges and high-pressure pumps, the present method has these advantages over other methods of making liposome populations.

[0104] In addition, centrifuges and pumps can generate considerable forces on the components, thus requiring more durable and more expensive materials. However, current vacuum forces can be of relatively low magnitude, thereby allowing the use of cheaper materials, so disposable items can be used in some cases. In addition, the use of disposable items does not require the items to be cleaned between uses. For example, although the extruded membrane in a pump system may need to be disassembled and cleaned between two different samples, the used membrane holder can be simply discarded and a new second membrane holder can be used for the second sample. In addition, in some cases, the membrane holder can be simply placed on top of the collection container without the use of fasteners to hold the components together. Therefore, the system can be quickly and easily disassembled after being used to collect liposomes.

[0105] The subject methods, devices and systems can be used to prepare liposome populations for therapeutic applications, such as liposomes containing a substance (e.g., a drug, a protein, a fluorescent compound, etc.), which can be used to deliver the substance to a target area of ​​a subject. For example, the subject methods, devices and systems can be used to prepare liposome populations for drug delivery, cell therapy and in vivo applications, as well as analytical applications (e.g., for immunoassays).

[0106] Example

[0107] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the present disclosure, and the examples are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all experiments 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 considered. Unless otherwise indicated, parts are parts 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, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; etc.

[0108] General Routines

[0109] The dry lipid mixture is produced by drying under an inert gas flow or lyophilization followed by drying under vacuum.

[0110] The dried lipids are then hydrated with an aqueous solution for 30-60 minutes. For lipids with higher phase transition temperatures, the aqueous solution is preheated prior to hydration. In some cases, the hydrated lipid suspension undergoes freeze / thaw cycles to improve the encapsulation efficiency of water-soluble compounds.

[0111] Next, the membrane holder was placed into the liposome collection container, thereby generating the system. Figure 1A A membrane holder 10 is shown having an open first end 11 (at the top) and a porous membrane 12 at a second end 13 (at the bottom), the first end and the second end being separated by a wall 14 therebetween, the first end 11, the second end 13 and the wall 14 together defining a liposome suspension holding volume 15. Figure 1A Also shown is an O-ring 30 which is positioned beneath the flange 16 at the first end of the membrane holder. Figure 1B A liposome collection container 20 is shown having an open first end 21 (at the top) and a closed bottom 22, the first end 21 and the bottom 22 being separated by a wall 23, the first end 21, the bottom 22 and the wall 23 together defining an interior space 24. The wall also has an opening 25 and a flange 26 at the top, with an O-ring 40 positioned below the flange. Figure 1C It shows how the membrane holder is placed into the interior space of the liposome collection container, with one of the O-rings helping to seal the two flanges, thereby helping to form a seal.

[0112] The system was then placed into a vacuum adapter so that the vacuum force could be transmitted through the opening in the wall of the liposome collection container. Figure 2 The system is shown placed into a vacuum adapter 50, wherein a second O-ring 40 helps to form a seal between the liposome collection 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, the first end 51, the second end 53 and the wall 54 together defining an interior space 55. A flange 56 is also shown, which can be sealed to the flange 26 of the collection container by the O-ring 40. In addition, the opening in the wall of the liposome collection container connects the interior space of the liposome collection container and the interior space of the vacuum adapter. Figure 2 The vacuum adapter 50 is shown with an open end 52 at the bottom end 53 of the adapter, allowing connection to a vacuum source. Thus, the vacuum source can apply a vacuum force to the porous membrane through the vacuum adapter, the opening in the wall of the liposome collection container.

[0113] The sample is loaded into the liposome suspension holding volume of the membrane holder. A vacuum force is then applied, thereby drawing the liposomes through the porous membrane into the interior space and generating a liposome population.

[0114] In some cases, the extrusion routine is repeated. In these cases, the vacuum force is removed and the membrane holder is removed from the liposome collection container. The aqueous fluid containing the liposomes is removed, the membrane holder is returned to its original position, and the liposomes containing the fluid are placed in the membrane holder. The vacuum force is applied again, thereby sucking the liposomes through the porous membrane again.

[0115] Example 1

[0116] Using the general procedure described above, LMVs consisting of 42 mol% PMPC, 14% DOPS, 13% DOPE, 30 mol% cholesterol, and 1 mol% DHPE-F were extruded five times in a system with three polycarbonate membranes with a pore size of 400 nm under 88 kPa vacuum. Dynamic light scattering (DLS) showed that the size of the produced liposomes was 336.7 + / - 11.7 nm, as shown in Figure 2. Figure 3 shown.

[0117] Example 2

[0118] Using the general routine described above, 42 mol% PMPC, 14% DOPS, 13% DOPE, 30 mol% cholesterol, and 1 mol% DHPE-F were extruded 10 times under 88 kPa vacuum in a system of 3 polycarbonate membranes with a pore size of 800 nm. The liposomes were then extruded 5 more times under 88 kPa vacuum in a system of 1 polycarbonate membrane with a pore size of 200 nm. The size of the liposomes was 195.8 + / - 13.7 nm, as determined by DLS and shown in Figure 4 In.

[0119] Notwithstanding the appended claims, the present disclosure is defined by the following terms:

[0120] 1. A method for producing a liposome population, the method comprising:

[0121] (a) providing a liposome extrusion system, which comprises:

[0122] A liposome collection container comprising: an open first end separated by a wall and a closed bottom, wherein the first end, the bottom and the wall together define an interior space, and the liposome collection container further comprises an opening in the wall leading to the interior space;

[0123] A membrane holder comprising: an open first end and a porous membrane at a second end, the first end and the second end being separated by a wall therebetween, the first end, the second end and the wall together defining a liposome suspension holding volume, wherein the membrane holder is positioned such that the porous membrane is located at the liposome collection

[0124] In the interior space of the container;

[0125] (b) introducing a liposome suspension into the liposome holding volume; and

[0126] (c) applying a vacuum force through the opening in a manner sufficient to draw liposomes through the porous membrane into the interior space and produce a population of liposomes.

[0127] 2. The method according to clause 1, wherein providing a liposome extrusion system comprises placing the second end of the membrane holder into the inner space of the liposome collection container.

[0128] 3. The method according to any one of clauses 1-2, wherein the opening in the wall of the liposome collection container is located above the porous membrane.

[0129] 4. The method according to any one of clauses 1 to 3, wherein the openings in the wall of the liposome collection container are oriented perpendicular to the porous membrane.

[0130] 5. The method according to any of clauses 1 to 4, wherein the membrane holder is oriented so that the porous membrane is located directly below the open first end.

[0131] 6. The method according to any one of clauses 1 to 5, wherein the volume of the liposome collection container below the porous membrane is larger than the liposome suspension holding volume.

[0132] 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 collection container.

[0133] 8. The method according to any one of clauses 1-7, further comprising: a gasket sealing the membrane holder to the liposome collection container.

[0134] 9. The method according to any one of clauses 1-8, further comprising a vacuum adapter, wherein a 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.

[0135] 10. The method according to clause 9, wherein at least a portion of the liposome collection container is located in the interior space of the vacuum adapter.

[0136] 11. The method according to any one of clauses 9-10, wherein the vacuum adapter has: a circular cross-section that is coaxial with the circular cross-section of the liposome collection container.

[0137] 12. The method according to any one of clauses 9-11, further comprising: a gasket sealing the vacuum adapter to the liposome collection container.

[0138] 13. The method according to any one of clauses 1 to 12, wherein the suspension of liposomes has an average diameter of 1000 nm or more.

[0139] 14. The method according to any one of clauses 1 to 13, wherein the porous membrane has a pore size of 1000 nm or less.

[0140] 15. The method according to clause 14, wherein the porous membrane has a pore size ranging from 1 nm to 500 nm.

[0141] 16. The method according to any one of clauses 1 to 15, wherein the average diameter of the liposome population ranges from 75% to 125% of the pore size of the porous membrane.

[0142] 17. A method according to any one of clauses 1 to 16, 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.

[0143] 18. The method of clause 17, wherein the first dimension of the membrane holder is a diameter.

[0144] 19. The method according to any one of clauses 1 to 18, wherein the liposome suspension is maintained in a volume ranging from 5 ml to 500 ml.

[0145] 20. The method according to clause 19, wherein the liposome suspension is maintained in a volume ranging from 10 ml to 100 ml.

[0146] 21. The method according to any one of clauses 1 to 20, wherein the liposome collection container has a first dimension ranging from 1.5 cm to 10.5 cm and a second dimension ranging from 2 cm to 21 cm.

[0147] 22. The method of clause 21, wherein the first dimension of the liposome collection container is a diameter.

[0148] 23. The method according to any one of clauses 1 to 22, wherein the internal space of the liposome collection container has a volume ranging from 10 ml to 550 ml.

[0149] 24. The method according to clause 23, wherein the internal space of the liposome collection container has a volume ranging from 20 ml to 150 ml.

[0150] 25. The method of any of clauses 9-24, 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.

[0151] 26. A method according to any of clauses 9-25, wherein the vacuum adapter has a volume in the range of 20ml to 600ml.

[0152] 27. The method according to any one of clauses 1 to 26, wherein one or more of the membrane holder, the liposome collection container and the vacuum adapter comprises a polymer material.

[0153] 28. The method according to any one of clauses 1-27, wherein one or more of the membrane holder, the liposome collection container and the vacuum adapter does not contain metal materials.

[0154] 29. The method according to any one of clauses 1-28, wherein one or more of the membrane holder, the liposome collection container and the vacuum adapter does not comprise glass material.

[0155] 30. The method according to any one of clauses 1 to 29, wherein the porous membrane comprises a polycarbonate material.

[0156] 31. The method of any one of clauses 1-30, wherein the system does not comprise: a fastener for fastening the membrane holder to the liposome collection container.

[0157] 32. The method according to any one of clauses 1 to 31, 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.

[0158] 33. The method of any one of clauses 1-32, wherein the vacuum force has a magnitude ranging from 0.1 kPa to 105 kPa.

[0159] 34. The method of clause 33, wherein the vacuum force is in the range of 50 kPa to 95 kPa.

[0160] 35. A method according to any one of clauses 1 to 34, wherein the maximum pressure differential to which the suspension of liposomes is subjected during the method is 105 kPa or less.

[0161] 36. A method according to any one of clauses 1 to 35, wherein the maximum acceleration force to which the suspension of liposomes is subjected during the method is 10 m / s 2 or smaller.

[0162] 37. The method according to any one of clauses 1-36, further comprising: transferring the liposome population from the liposome collection container to a receiving container.

[0163] 38. A liposome extrusion apparatus for producing a liposome population, the apparatus comprising:

[0164] A liposome collection container comprising: an open first end separated by a wall and a closed bottom, wherein the first end, the bottom and the wall together define an interior space, and the liposome collection container further comprises: an opening in the wall leading to the interior space;

[0165] a membrane holder comprising: an open first end and a porous membrane at a second end, the first end and the second end being separated by a wall therebetween, the first end, the second end and the wall together defining a liposome suspension holding volume, wherein the membrane holder is positioned such that the porous membrane is located in the interior space of the liposome collection container; and

[0166] A vacuum source is operably connected to the opening in the wall of the liposome collection container.

[0167] 39. The device according to clause 38, wherein the opening in the wall of the liposome collection container is located above the porous membrane.

[0168] 40. The device according to any of clauses 38-39, wherein the openings in the wall of the liposome collection container are oriented perpendicular to the porous membrane.

[0169] 41. The device of any of clauses 38-40, wherein the membrane holder is oriented so that the porous membrane is located directly below the open first end.

[0170] 42. The device according to any one of clauses 38-41, wherein the volume of the liposome collection container below the porous membrane is larger than the liposome suspension holding volume.

[0171] 43. The device according to any one of clauses 38-42, wherein the membrane holder has: a circular cross-section that is coaxial with the circular cross-section of the liposome collection container.

[0172] 44. The device according to any one of clauses 38-43, further comprising: a gasket that seals the membrane holder to the liposome collection container.

[0173] 45. The device according to any of clauses 38-44, further comprising a vacuum adapter, wherein a 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.

[0174] 46. ​​The device of clause 45, wherein at least a portion of the liposome collection container is located within an interior space of the vacuum adapter.

[0175] 47. The device according to any of clauses 45-46, wherein the vacuum adapter has: a circular cross-section that is coaxial with the circular cross-section of the liposome collection container.

[0176] 48. The device according to any of clauses 45-47, further comprising: a gasket that seals the vacuum adapter to the liposome collection container.

[0177] 49. The device according to any of clauses 38-48, further comprising: a suspension of liposomes located in the liposome suspension holding volume.

[0178] 50. The device of clause 49, wherein the suspension of liposomes has an average diameter of 1000 nm or greater.

[0179] 51. The device according to any of clauses 49-50, wherein the porous membrane has a pore size of 1000 nm or less.

[0180] 52. The device according to clause 51, wherein the porous membrane has a pore size ranging from 1 nm to 500 nm.

[0181] 53. The device according to any of clauses 49-52, wherein the average diameter of the liposome population ranges from 75% to 125% of the pore size of the porous membrane.

[0182] 54. The device according to any of clauses 38-53, 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.

[0183] 55. The device of any of clauses 38-54, wherein the first dimension of the membrane holder is a diameter.

[0184] 56. A device according to any one of clauses 38-55, wherein the liposome suspension holding volume ranges from 5 ml to 500 ml.

[0185] 57. The device according to clause 56, wherein the liposome suspension holding volume ranges from 10 ml to 100 ml.

[0186] 58. The device according to any of clauses 38-57, wherein the liposome collection container has a first dimension ranging from 1.5 cm to 10.5 cm and a second dimension ranging from 2 cm to 21 cm.

[0187] 59. The device of clause 58, wherein the first dimension of the liposome collection container is a diameter.

[0188] 60. The device according to any of clauses 38-59, wherein the inner space of the liposome collection container has a volume ranging from 10 ml to 550 ml.

[0189] 61. The device according to any of clauses 38-60, wherein the inner space of the liposome collection container has a volume ranging from 20 ml to 150 ml.

[0190] 62. The device of any of clauses 45-61, wherein the vacuum adapter has a first dimension ranging from 2 cm to 11 cm and a second dimension ranging from 3 cm to 22 cm.

[0191] 63. Apparatus according to any of clauses 45-62, wherein the vacuum adapter has a volume range of 20ml to 600ml.

[0192] 64. The device according to any of clauses 38-63, wherein one or more of the membrane holder, the liposome collection container and the vacuum adapter comprises a polymer material.

[0193] 65. The device according to any of clauses 38-64, wherein one or more of the membrane holder, the liposome collection container and the vacuum adapter does not contain metal material.

[0194] 66. The device according to any one of clauses 38-65, wherein one or more of the membrane holder, the liposome collection container and the vacuum adapter does not comprise glass material.

[0195] 67. A device according to any of clauses 38 to 66, wherein the porous membrane comprises a polycarbonate material.

[0196] 68. The device according to any one of clauses 38-67, wherein the device does not comprise: a fastener to fasten the membrane holder to the liposome collection container.

[0197] 69. The device according to any of clauses 38-68, 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.

[0198] 70. A system for producing a population of liposomes, comprising:

[0199] A liposome collection container comprising: an open first end separated by a wall and a closed bottom, wherein the first end, the bottom and the wall together define an interior space, and the liposome collection container further comprises: an opening in the wall leading to the interior space;

[0200] A membrane holder comprising: an open first end and a porous membrane at a second end, the first end and the second end being separated by a wall therebetween, the first end, the second end and the wall together defining a liposome suspension holding volume, wherein the membrane holder is configured to be positioned so that the porous membrane is located in the interior space of the liposome collection container.

[0201] 71. The system of clause 70, further comprising: a gasket configured to seal the membrane holder to the liposome collection container.

[0202] 72. The system of any of clauses 70-71, further comprising: a vacuum adapter configured to enable transfer of a vacuum force from the vacuum source to the vacuum adapter and then to an opening in a wall of the liposome collection container.

[0203] 73. The system of clause 72, further comprising: a gasket configured to seal the vacuum adapter to the liposome collection container.

[0204] 74. A system according to any of clauses 70-73, 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.

[0205] 75. A system according to clause 74, wherein the first dimension of the membrane holder is a diameter.

[0206] 76. A system according to any one of clauses 70-75, wherein the liposome suspension holding volume ranges from 5 ml to 500 ml.

[0207] 77. A system according to clause 76, wherein the liposome suspension holding volume ranges from 10 ml to 100 ml.

[0208] 78. The system of any of clauses 70-77, wherein the liposome collection container has a first dimension ranging from 1.5 cm to 10.5 cm and a second dimension ranging from 2 cm to 21 cm.

[0209] 79. The system of clause 78, wherein the first dimension of the liposome collection container is a diameter.

[0210] 80. The system according to any of clauses 70-79, wherein the internal space of the liposome collection container has a volume ranging from 10 ml to 550 ml.

[0211] 81. The system according to any of clauses 70-80, wherein the internal space of the liposome collection container has a volume ranging from 20 ml to 150 ml.

[0212] 82. The system of any of clauses 72 to 81, wherein the vacuum adapter has a first size ranging from 2 cm to 11 cm and a second size ranging from 3 cm to 22 cm.

[0213] 83. A system according to clause 82, wherein the vacuum adapter has a volume ranging from 20 ml to 600 ml.

[0214] 84. The system of any one of clauses 72-83, wherein one or more of the membrane holder, the liposome collection container, and the vacuum adapter comprises a polymer material.

[0215] 85. The system of any one of clauses 72-84, wherein one or more of the membrane holder, the liposome collection container, and the vacuum adapter does not comprise metal material.

[0216] 86. The system of any one of clauses 72-85, wherein one or more of the membrane holder, the liposome collection container, and the vacuum adapter does not comprise glass material.

[0217] 87. The system of any of clauses 72-86, wherein the porous membrane comprises a polycarbonate material.

[0218] 88. A kit for producing a population of liposomes, comprising:

[0219] A system according to any of clauses 70-87; and

[0220] A package configured to house the system.

[0221] In at least some of the previously described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed object. All of these modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0222] Those skilled in the art will understand that, in general, the terms used herein, especially the terms used in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "comprising" should be interpreted as "including but not limited to", etc.). Those skilled in the art will also understand that if a specific number of introduced claim statements is intended to be introduced, such an intent will be explicitly stated in the claim, and in the absence of such a statement, no such intent is present. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of these phrases should not be interpreted as implying that any particular claim containing such introduced claim statements by introducing claim statements without quantifiers will be limited to embodiments containing only one such statement, even when the same claim includes the introductory phrases "one / kind or more / kind" or "at least one / kind" and no quantifiers (e.g., should be interpreted as meaning "at least one / kind" or "one / kind or more / kind"); the same applies to the use of definite articles used to introduce introduced claim statements. In addition, even if a specific number of introduced claim statements is explicitly cited, those skilled in the art will recognize that such statements should be interpreted to mean at least the stated number (e.g., "two statements" without other modifiers means at least two statements, or two or more statements). Moreover, where a convention similar to "at least one of A, B, and C, etc." is used, generally, such construction is intended to be in the sense that those skilled in the art understand the convention (e.g., "a system having at least one of A, B, and C" would include but is not limited to a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where a convention similar to "at least one of A, B, or C, etc." is used, generally such construction is intended to be in the sense that one skilled in the art understands the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One skilled in the art would also understand that virtually any separate word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, one of the terms, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0223] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0224] As will be understood by those skilled in the art, for any and all purposes, such as in providing a written description, all ranges disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to fully describe and be able to divide the same range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily divided into the lower third, the middle third, and the upper third, etc. It will also be understood by those skilled in the art that all languages ​​(such as "at most", "at least", "greater than", "less than", etc.) include the numbers listed, and refer to the ranges that can be subsequently divided into sub-ranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1 to 3 items refers to a group with 1, 2 or 3 items. For example, a group with 1 to 5 items refers to a group with 1, 2, 3, 4 or 5 items, etc.

[0225] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art that certain changes and modifications may be made in light of the teachings of this invention without departing from the spirit or scope of the appended claims.

[0226] Therefore, the foregoing merely illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various arrangements that, although not explicitly described or indicated herein, embody the principles of the present invention and are included in its spirit and scope. Moreover, all embodiments and conditional language stated herein are primarily intended to help the reader understand the principles of the present invention and the concepts contributed by the inventor to promote the art, and should be interpreted as not being limited to these specifically stated embodiments and conditions. Moreover, all statements stating the principles, aspects and embodiments of the present invention and their specific examples herein are intended to cover their structural and functional equivalents. In addition, it is intended that such equivalents include: currently known equivalents and equivalents developed in the future, i.e., any element developed that performs the same function, regardless of the structure. Moreover, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is explicitly stated in the claims.

[0227] Therefore, 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 expressly defined as follows: Only when the exact phrase "means" or the exact phrase "step" is cited at the beginning of such limitation in the claim, the limitation in the claim is invoked; if such exact phrase is not used in the limitation of the claim, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is not invoked.

Claims

1. A method for producing a liposome population, the method comprising: (a) providing a liposome extrusion system, the liposome extrusion system comprising: A liposome collection container, the liposome collection container comprising: an open first end separated by a wall and a closed bottom, the first end, the bottom and the wall together defining an interior space, the liposome collection container further comprising: an opening in the wall leading to the interior space; a membrane holder, the membrane holder comprising: an open first end and a porous membrane at a second end, the first end and the second end being separated by a wall therebetween, the first end, the second end and the wall together defining a liposome suspension holding volume, wherein the membrane holder is positioned such that the porous membrane is located in the interior space of the liposome collection container; (b) introducing a liposome suspension into the liposome holding volume; and (c) applying a vacuum force through the opening in a manner sufficient to draw the liposomes through the porous membrane into the interior space and produce a population of liposomes.

2. The method according to claim 1, wherein: The providing of the liposome extrusion system comprises: placing the second end of the membrane holder into the inner space of the liposome collection container.

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

4. The method according to any one of claims 1 to 3, wherein: The openings in the wall of the liposome collection container are 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 collection 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 collection container.

8. The method of any one of claims 1-7, further comprising a gasket sealing the membrane holder to the liposome collection 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 the vacuum source to the vacuum adapter and then to the opening in the wall of the liposome collection container.

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

11. The method according to any one of claims 1 to 10, further comprising: The liposome population is transferred from the liposome collection vessel to a receiving vessel.

12. A liposome extrusion device for producing a liposome population, the device comprising: a liposome collection container, the liposome collection container comprising: an open first end separated by a wall and a closed bottom, the wall, the first end and the bottom together defining an interior space, the liposome collection container further comprising an opening in the wall leading to the interior space; a membrane holder comprising: an open first end and a porous membrane at a second end, the first end and the second end being separated by a wall therebetween, the first end, the second end and the wall together defining a liposome suspension holding volume, wherein the membrane holder is positioned such that the porous membrane is located in the interior space of the liposome collection container; and A vacuum source is operably connected to the opening in the wall of the liposome collection container.

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

14. A system for producing a liposome population, comprising: a liposome collection container, the liposome collection container comprising: an open first end separated by a wall and a closed bottom, the wall, the first end and the bottom together defining an interior space, the liposome collection container further comprising an opening in the wall leading to the interior space; A membrane holder, the membrane holder comprising: an open first end and a porous membrane located at a second end, the first end and the second end being separated by a wall therebetween, the first end, the second end and the wall together defining a liposome suspension holding volume, wherein the membrane holder is configured to be positioned so that the porous membrane is located in the interior space of the liposome collection container.

15. A kit for producing a liposome population, comprising: The system according to claim 14; as well as A package configured to house the system.

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