Methods of creating substance with different freezing points by encapsulation

Abstract

To provide compositions and methods for manufacturing biomaterials that form flowable and injectable cold slurries.SOLUTION: The present disclosure provides a composition containing a plurality of liposomes where the encapsulated internal liposomal media and external liposomal media have different freezing points.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 878,108, filed July 24, 2019, the entire contents of which are incorporated herein by reference.

[0002] Technical Field The present disclosure relates generally to compositions and methods for producing biomaterials that form flowable, injectable cold slurries. More specifically, the present disclosure relates to compositions containing multiple liposomes, wherein the encapsulated intraliposomal medium and the extraliposomal medium have different freezing points. [Background technology]

[0003] background Cold slurries (e.g., ice slurries) are known in the art as compositions consisting of sterile water ice particles, varying amounts of excipients or additives (e.g., freezing-point depressants), and optionally one or more active pharmaceutical ingredients, as described in U.S. Patent Application No. 15 / 505,042 ("'042" application, Publication No. US2017 / 0274011), the entire contents of which are incorporated herein by reference. The cold slurries can be delivered, preferably via injection, to the tissues of a subject (preferably a human patient) for selective or non-selective cryotherapy and / or cryolysis for preventive, therapeutic, or cosmetic purposes. The injectable cold slurries can be used to treat a variety of disorders requiring the inhibition of neurotransmission. For example, U.S. Patent Application No. 15 / 505,039 (the "'039" application, Publication No. US2017 / 0274078), the entire contents of which are incorporated herein by reference, discloses the use of a slurry to induce reversible degeneration of nerves (through Wallerian degeneration) by causing lipid crystallization in the nerve myelin sheath. The '039 application also discloses the use of an injectable cold slurry to treat a variety of other disorders requiring somatic or autonomic nerve inhibition, including motor spasms, hypertension, hyperhidrosis, and urinary incontinence.

[0004] A method for preparing a cold slurry is shown in U.S. Patent Application No. 16 / 080,092 (the "'092" application, Publication No. US2019 / 0053939). However, the '092 application requires the care site to manufacture the cold slurry by installing a medical ice slurry manufacturing system. This technique also requires the care site to take steps to maintain the sterility of the cold slurry during manufacturing and before administration.

[0005] A need exists for compositions and methods that allow for the easy transport, storage, and preparation of cold, flowable, injectable slurries at clinical sites without compromising the sterility of the slurry during preparation, without requiring manufacturing facilities to be available at the site, and without compromising the sterility of biomaterials at the site. The present disclosure addresses this need by providing improved compositions and methods that reduce the time required to provide patients with therapeutic substances (e.g., injectable slurries) that are easily transported and stored. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. US2017 / 0274011 [Patent Document 2] U.S. Patent Application Publication No. US2017 / 0274078 [Patent Document 3] U.S. Patent Application Publication No. US2019 / 0053939 Summary of the Invention

[0007] overview The present disclosure provides a composition comprising a multiplicity of liposomes that separate an internal medium from an external medium, the internal medium and the external medium having different freezing points (i.e., the temperature at which the medium freezes). The present disclosure further provides a composition that, due to the different freezing points of the composition, can be converted into an injectable slurry at the point of care by placing it in a standard freezer.

[0008] In one aspect, disclosed herein is a composition comprising water, at least one liposome, and at least one excipient, wherein the liposome is configured to encapsulate a first volume of the composition, the excipient is configured to be restricted to a second volume of the composition outside the liposome and configured to be isolated from the encapsulated first volume, and wherein the first freezing point of the first encapsulated volume is higher than the second freezing point of the second volume.

[0009] In some embodiments, the liposome is composed of a lipid selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg sphingomyelin (DPSM), dipalmitoylphosphatidylcholine (DPPC), dicetyl phosphate (DCP), L-α-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and combinations thereof. In some embodiments, the lipid is L-α-phosphatidylcholine (PC).

[0010] In some embodiments, the excipient is selected from the group consisting of salts, ions, lactated Ringer's solution, sugars, biocompatible surfactants, polyols, and combinations thereof. In some embodiments, the excipient is a polyol. In some embodiments, the polyol is polyethylene glycol 1000 (PEG1000).

[0011] In some embodiments, the composition further comprises a second excipient in both the first and second volumes, hi some embodiments, the second excipient is saline or phosphate buffered saline (PBS).

[0012] In some embodiments, the encapsulated first volume is about 20% to about 50% of the total volume of the composition. In some embodiments, the encapsulated first volume is about 30% to about 40% of the total volume of the composition. In some embodiments, the encapsulated first volume is about 40% to about 50% of the total volume of the composition. In some embodiments, the encapsulated first volume is about 35% to about 40% of the total volume of the composition. In some embodiments, the encapsulated first volume is about 40% to about 45% of the total volume of the composition. In some embodiments, the encapsulated first volume is about 38% of the total volume of the composition. In some embodiments, the encapsulated first volume is about 43% of the total volume of the composition.

[0013] In some embodiments, the first freezing point of the encapsulated first volume is about −2° C. to about 0° C. In some embodiments, the second freezing point of the second volume is about −20° C. to about −10° C. In some embodiments, the average freezing point of the total volume of the composition comprising the first volume, the second volume, and the liposomes is about −10° C. to about −5° C.

[0014] In some embodiments, the encapsulated first volume is configured to form a plurality of ice particles when the composition is cooled to a predetermined temperature. In some embodiments, the ice particles comprise about 30% to about 50% by weight of the total weight of the composition. In some embodiments, the predetermined temperature is about -20°C to about -5°C. In some embodiments, the predetermined temperature is about -20°C. In some embodiments, the predetermined temperature is about -5°C.

[0015] In another aspect, disclosed herein is a method of preparing a composition for administration to a patient in a clinical setting, the method comprising: preparing a composition having a plurality of liposomes, wherein an aqueous medium fills an intraliposomal volume and an extraliposomal volume; adding at least one excipient to the extraliposomal volume, wherein the at least one excipient lowers a first freezing point of the extraliposomal volume below a second freezing point of the intraliposomal volume; and cooling the composition to a predetermined temperature such that a cold slurry having a plurality of ice particles within the intraliposomal volume is formed.

[0016] In some embodiments, the liposome is composed of a lipid selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg sphingomyelin (DPSM), dipalmitoylphosphatidylcholine (DPPC), dicetyl phosphate (DCP), L-α-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and combinations thereof. In some embodiments, the lipid is L-α-phosphatidylcholine (PC).

[0017] In some embodiments, the excipient is selected from the group consisting of salts, ions, lactated Ringer's solution, sugars, biocompatible surfactants, polyols, and combinations thereof. In some embodiments, the excipient is a polyol. In some embodiments, the polyol is polyethylene glycol 1000 (PEG1000).

[0018] In some embodiments, the aqueous medium is composed of water, saline, or phosphate buffered saline (PBS).

[0019] In some embodiments, the intraliposomal volume is about 20% to about 50% of the total volume of the composition. In some embodiments, the intraliposomal volume is about 30% to about 40% of the total volume of the composition. In some embodiments, the intraliposomal volume is about 40% to about 50% of the total volume of the composition. In some embodiments, the intraliposomal volume is about 35% to about 40% of the total volume of the composition. In some embodiments, the intraliposomal volume is about 40% to about 45% of the total volume of the composition. In some embodiments, the intraliposomal volume is about 38% of the total volume of the composition. In some embodiments, the intraliposomal volume is about 43%.

[0020] In some embodiments, the first freezing point of the extraliposomal volume is about −20° C. to about −10° C. In some embodiments, the second freezing point of the intraliposomal volume is about −2° C. to about 0° C. In some embodiments, the average freezing point of the total volume of the composition comprising the first volume, the second volume, and the liposomes is about −10° C. to about −5° C.

[0021] In some embodiments, the ice particles comprise about 30% to about 50% by weight of the biomaterial.

[0022] In some embodiments, the predetermined temperature is about -20° C. to -5° C. In some embodiments, the predetermined temperature is about -20° C. In some embodiments, the predetermined temperature is about -5° C.

[0023] In some embodiments, the bilayer composition of the liposome is selected from the group consisting of unilamellar vesicles, multilamellar vesicles, oligolamellar vesicles, multivesicular vesicles, and combinations thereof.

[0024] In some embodiments, the liposomes have an average diameter of about 0.1 μm to about 2 μm.

[0025] In another aspect, disclosed herein is a method for producing a flowable, injectable encapsulated ice solution, the method comprising: providing a plurality of biodegradable liposomes configured to form vesicles selected from the group consisting of multilamellar vesicles, oligolamellar vesicles, multivesicular vesicles, giant unilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles, or combinations thereof; encapsulating water within at least two of the plurality of liposomes to generate liposomes filled with a first volume comprising water; adding an excipient to a second volume outside the liposomes isolated from the first volume, wherein the excipient alters the freezing point of the second volume relative to the first volume; freezing the plurality of filled liposomes to generate a plurality of ice particles within the filled liposomes; and controlling the average diameter of each of the plurality of ice particles to a predetermined size.

[0026] In some embodiments, the first volume is about 20% to about 50% of the total volume of the composition. In some embodiments, the first volume is about 20% to about 50% of the total volume of the composition. In some embodiments, the first volume is about 30% to about 40% of the total volume of the composition. In some embodiments, the first volume is about 40% to about 50% of the total volume of the composition. In some embodiments, the first volume is about 35% to about 40% of the total volume of the composition. In some embodiments, the first volume is about 40% to about 45% of the total volume of the composition. In some embodiments, the first volume is about 38% of the total volume of the composition. In some embodiments, the first volume is about 43% of the total volume of the composition.

[0027] In some embodiments, the excipient is PEG1000. [Brief explanation of the drawings]

[0028] The following figures illustrate exemplary aspects of the present disclosure.

[0029] [Figure 1] FIG. 1 is a schematic diagram of a composition containing liposomes having different freezing points for the intraliposomal and extraliposomal media. [Figure 2] FIG. 2 shows the freezing point depression graph for water and a solution containing 47% PEG1000 by volume (v / v). [Figure 3] FIG. 3 is a graph of the solid-liquid phase transition of a cold slurry with a crystallization set point of −6.5° C. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description The present disclosure relates to compositions and methods for preparing injectable biomaterials (e.g., sterile cold slurries). The biomaterials preferably contain suspended substances (e.g., liposomes) that separate internal and external media having different freezing points. Due to the different freezing points of the internal and external media, the liposomes preferably encapsulate the internal media, which freezes at a predetermined temperature (e.g., 0°C), while the external media remains liquid at that temperature. In a preferred embodiment, the biomaterials form a flowable, injectable slurry containing a plurality of ice particles. The ice particles are preferably held within a plurality of liposomes and maintained in a state separated from the solution by a liposome barrier. The internal media is preferably pure water, and the external media is preferably a solution containing water and inactive excipient materials. In another embodiment, the slurry further comprises a known active pharmaceutical compound.

[0031] The present disclosure relates to flowable, injectable ice slurries containing ice particles with a precise particle size distribution, which are biodegradable, biocompatible, and can be stored for long periods (e.g., two years or more). In some embodiments, pure water or saline is encapsulated in a biocompatible, biodegradable material (e.g., liposomes), allowing the aqueous phase solution to be isolated from the solid phase material when the composition is placed in a standard freezer (e.g., a freezer set at approximately -20°C) to create flowable, injectable encapsulated ice particles. Different freezing points are created by adding a freezing-point depressant to the extraliposomal medium. Water encapsulation allows for control of the size and shape of the ice particles when the biomaterial is exposed to freezing temperatures. An example of a material that can be used to encapsulate water / ice is liposomes. Liposomes are widely used in medicine to deliver active molecules and drugs to target tissues. However, the present disclosure relates to the use of liposomes to encapsulate ice particles to create cold slurry compositions as therapeutic biomaterials.

[0032] In some embodiments, the biomaterial is a cold slurry (e.g., an ice slurry) that can be delivered directly to the tissue of a human patient or subject via injection for prophylactic, therapeutic, or cosmetic purposes. The injectable slurry can be used for selective or non-selective cryotherapy or cryolipolysis.

[0033] In some embodiments, liposomes are used to create solutions or mixtures with different freezing points for the purpose of creating flowable, cold slurries. Referring to Figure 1, a schematic diagram of a biomaterial shows an orthographic view of a liposome with an internal medium composed of water (or saline) with a freezing point of 0°C, and an external medium in which the liposomes are suspended, composed of water (or saline) and at least one excipient (e.g., polyethylene glycol 1000, "PEG1000") with a freezing point below 0°C. In some embodiments, the freezing point of the internal liposome medium is below about -2°C, between about -2°C and about 0°C, between about 0°C and about 2°C, or above about 2°C. In some embodiments, the freezing point of the external liposome medium is below about -15°C, between about -15°C and about -10°C, between about -10°C and about -5°C, or above about -5°C. If the internal and external media have different freezing points, when the biomaterial is subjected to cooling at a specific temperature, ice particles will form in the intraliposomal media while the extraliposomal media remains aqueous, creating a flowable, injectable, cooled slurry composition. In some embodiments, the biomaterial can also have the ice particles partially melt before administering the biomaterial to a patient to create an injectable, flowable slurry.

[0034] Liposomes are spherical vesicles that can be made from non-toxic lipids / phospholipids. As shown in Figure 1, phospholipids have a hydrophilic head group 13 and two long hydrophobic tails 14, which give them the ability to self-form bilayer vesicles when suspended in water. In some embodiments, liposomes are synthesized from commonly used lipids / phospholipids known in the art, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg sphingomyelin (DPSM), dipalmitoylphosphatidyl (DPPC), dicetyl phosphate (DCP), L-α-phosphatidylcholine (e.g., egg yolk PC or soybean PC), phosphatidylethanolamine (e.g., egg yolk PE or soybean PE), phosphatidylserine (PS), and phosphatidylglycerol (PG), or any combination thereof. Various phospholipids can be selected to create liposomes with the desired level of fluidity and permeability. In preferred embodiments, the liposome composition contains cholesterol to improve bilayer stability and reduce lipid aggregation. Liposomes can further be synthesized with (or coated with) polymers, such as poly(lactic-co-glycolic acid) (PLGA) or polyethylene glycol (PEG), to improve stability. In some embodiments, the liposomes further contain one or more surfactants (e.g., sodium cholate). In preferred embodiments, the liposomes are composed entirely of biodegradable and non-immunogenic components.

[0035] In some embodiments, liposomes can be composed of one or more phospholipid bilayers. In some embodiments, liposomes are unilamellar vesicles, i.e., vesicles composed of a single lipid bilayer, including giant unilamellar vesicles (GUVs; >1 μm (vesicle diameter)), large unilamellar vesicles (LUVs; >0.1 μm), and small unilamellar vesicles (SUVs; <0.1 μm) (e.g., as depicted in FIG. 1 ). In some embodiments, liposomes are multilamellar / oligolamellar vesicles (MLVs / OLVs) composed of multiple lipid bilayers organized into concentric phospholipid spheres. In some embodiments, liposomes are multivesicular vesicles (MVVs) composed of multiple non-concentric vesicles encapsulated within a single bilayer. In some embodiments, the charge of the phospholipids in the liposomes is neutral, anionic, or cationic. The lipid composition, lipid chain length and saturation, size, preparation method, and vesicle charge can all be adjusted to alter liposome properties. In some embodiments, liposomes are synthesized with short, unsaturated phospholipid chains that allow the outer aqueous phase of the bilayer to be separated from the inner solid ice without freezing or deformation of the liposome.

[0036] Any method known in the art can be used to prepare the liposomes disclosed herein. For example, liposomes according to the present disclosure can be produced according to the method disclosed in Dua JS, et al., Liposome: methods of preparation and applications, 3 Int. J. Pharm. Stud. Res. 14-20 (Apr. 2012), the entire contents of which are incorporated herein by reference. Such methods include mechanical dispersion methods (e.g., lipid film hydration, sonication, lyophilization, freeze-thawing, French pressure cell, microemulsification), solvent dispersion methods (e.g., ethanol injection, reverse phase evaporation, double emulsion), and detergent removal methods (e.g., dialysis, dilution, column chromatography). In some embodiments, the sonication of mechanical dispersion methods is used to produce small liposome vesicles of specific diameters, as further disclosed herein. Liposomes are produced in a medium that allows encapsulation (i.e., intraliposomal medium) of pure water, saline, or phosphate-buffered saline (PBS). These liposomes can be lyophilized and then rehydrated in another medium (i.e., the extraliposomal medium). In some embodiments, the extraliposomal medium is composed of a solvent (e.g., pure water, saline, or phosphate-buffered saline) and at least one excipient (e.g., PEG1000) that can function as a freezing point depressant (see Figure 1).

[0037] The present disclosure also relates to the production of a flowable, injectable encapsulated ice solution that can be produced at a central facility, delivered to a treatment site at room temperature (e.g., about 19°C), and immediately converted to an ice slurry at the treatment site by simply lowering the temperature of the biomaterial using a standard freezer. This eliminates the need for the treatment site to manufacture the biomaterial or to worry about maintaining the sterility of the biomaterial. The aqueous biomaterial containing liposomes can be placed in a standard freezer at a clinical treatment site set to temperatures below about -25°C, between about -25°C and about -20°C, between about -20°C and about -15°C, between about -15°C and about -10°C, between about -10°C and about -5°C, between about -5°C and about 0°C, and warmer than about 0°C. In some embodiments, the biomaterial is placed in the freezer for a predetermined time to allow the temperature of the biomaterial to drop to a desired level to form a cold slurry having a predetermined ratio of ice particles.

[0038] In some embodiments, the final liposome composition (including the internal and external media and lipids) is subjected to a sterilization process and maintained sterile from the manufacturing site to the point of administration when it is loaded into a transport container (e.g., a bag or syringe). In some embodiments, the intraliposomal and / or extraliposomal media are sterilized during liposome preparation and maintained sterile throughout the manufacturing, transport, and storage process. In some embodiments, the liposome composition is sterilized at the treatment site using any sterilization method known in the art (e.g., using heat, radiation, high pressure, etc.). In some embodiments, the liposome composition is sterilized in the container (e.g., a bag or syringe).

[0039] In some embodiments, biomaterials are transformed into cold slurry through flash freezing.In such embodiments, ice particles are created inside liposomes by pressure changes.When pure water freezes, it expands.Starting with encapsulated water of a certain shape or size, the temperature is lowered below 0°C under high pressure, and the water cannot freeze until the pressure is released and the water can expand, thus causing the instant freezing of the volume inside the liposome.Flash freezing does not require a thermal gradient.

[0040] The disclosed liposome technology allows for the creation of liposomes of consistent sizes for various applications. In some embodiments, vigorous sonication is used during liposome preparation to restrict the size of phospholipids to ensure their injectability. Size can also be controlled by creating a minimum lamellar size that is energetically favorable and prevents diffusion from the intraliposomal volume. The free energy barrier of such minimum-sized liposomes can trap water under conditions of higher osmotic pressure outside the liposome vesicle. The disclosed method achieves cold slurry solutions with highly precise particle sizes over a wide range of diameters, from about 0.02 μm to about 100 μm. In preferred embodiments, the average diameter of the liposomes in the composition is less than about 0.1 μm, from about 0.1 μm to about 0.5 μm, from about 0.5 μm to about 1 μm, from about 1 μm to about 1.5 μm, from about 1.5 μm to about 2 μm, or greater than about 2 μm. In some embodiments, the average diameter of the liposomes in the composition is from about 0.2 μm to about 0.4 μm, or from about 1.1 μm to about 1.3 μm.

[0041] Liposome size distribution is measured using standard techniques known in the art, such as electron microscopy, dynamic light scattering (DLS), atomic force microscopy (AFM), size exclusion chromatography (SEC), etc. In some embodiments, the size of the ice particles can be controlled to achieve flowability through containers of various sizes (e.g., needle gauge sizes from about 7 to about 43) as described in the '042 application, which is incorporated herein by reference in its entirety. In some embodiments, the average diameter is measured by dynamic light scattering (DLS).

[0042] In some embodiments, the average diameter is the mean diameter.

[0043] In some embodiments, one or more excipients are included in the slurry. An excipient is any substance that is not itself a therapeutic agent and is used as a diluent, adjuvant, and / or vehicle for delivering a therapeutic agent to a subject or patient, and / or is added to a composition to improve its handling, stability, or storage. To create a biomaterial with different freezing points between the intraliposomal and extraliposomal media, one or more freezing-point depressants can be added to the extraliposomal solution as an excipient to lower the freezing point of the extraliposomal solution (e.g., below about 0°C). After preparing and suspending the liposomes in an aqueous medium, the excipients are added to the external medium. The lowering of the freezing point of the extraliposomal medium allows the final slurry mixture to contain an effective proportion of ice particles while maintaining fluidity and remaining injectable. Suitable freezing point depressants include salts (e.g., sodium chloride, Betadex sulfobutylether sodium), ions, lactated Ringer's solution, sugars (e.g., glucose, sorbitol, mannitol, hetastarch, sucrose, (2-hydroxypropyl)-β-cyclodextrin, or combinations thereof), biocompatible surfactants such as glycerol (also known as glycerin or glycerine), other polyols (e.g., polyvinyl alcohol, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 1000, propylene glycol), other sugar alcohols, or urea, etc. Other exemplary freezing point depressants are disclosed in the '042 application, which is incorporated herein by reference in its entirety.

[0044] Preferably, the freezing point depressant added to the extraliposomal medium is polyethylene glycol 1000 (PEG1000). PEG1000 is particularly suitable for the present disclosure due to its large molecular weight / size (i.e., approximately 1000 kDa), which prevents it from crossing the lipid membrane of the liposome and entering the intraliposomal medium and disrupting the freezing point differential generated across the liposome. Other suitable freezing point depressants include any excipient that depresses the freezing point of the extraliposomal medium without crossing the membrane or disrupting the freezing point differential.

[0045] The concentration of the freezing point depressant can determine the proportion of ice particles in the slurry and its flowability and injectability. In some embodiments, the freezing point depressant (e.g., PEG 1000) comprises about 10% v / v to about 70% v / v of the extraliposomal medium. In some embodiments, the freezing point depressant comprises less than about 30% v / v, about 30% v / v to about 40% v / v, about 40% to about 50% v / v, about 50% to about 60% v / v, or more than about 60% v / v of the extraliposomal medium.

[0046] Referring to Figure 2, a freezing point depression graph is shown for pure water T1 and a mixture of water and 47% v / v PEG1000 T2. In this graph, all materials were placed in a freezer with a constant temperature of -20°C. Temperatures were measured using thermometers placed within each material / slurry. This graph shows that the mixture of water and PEG1000 may have a freezing point different from that of pure water, meaning that the solution can be cooled below 0°C and only partially crystallized. The graph shows that cooling causes pure water T1 to crystallize at its equilibrium freezing point of 0°C. This is indicated by the period in which pure water T1 is maintained at a temperature of approximately 0°C from approximately 1.3 hours to approximately 4.4 hours, beginning shortly after pure water T1 passes its supercooling point of approximately -6°C. The presence of an equilibrium window of crystallization (i.e., the "horizontal" portion of pure water T1 in Figure 2) is typical for pure solvents. In the case of 47% PEG1000 solution T2, upon cooling, the solution begins to crystallize after just under one hour with an initial freezing point of about −6.5° C., and as this crystallization continues, the temperature of the solution further decreases to about −19° C. after about 2.5 hours. Initial crystallization occurs shortly after 47% PEG1000 solution T2 passes the supercooling point of about −15° C., as shown after just under one hour. In some embodiments, the presence of a lower temperature window of crystallization in 47% PEG1000 solution T2 is typical of a solution (i.e., an impure mixture).

[0047] In some embodiments, the final product administered via injection to a human patient or subject (e.g., a non-patient human or non-human animal) is a cold slurry composed of sterile water ice particles, liposome-forming lipids, and varying amounts of excipients or additives, such as freezing point depressants (e.g., PEG1000).

[0048] In some embodiments, the ice particles are generally restricted to the intraliposomal medium, hi some embodiments, the total volume of the intraliposomal medium is either completely or partially crystallized.

[0049] In some embodiments, the proportion of ice particles in the total volume of the cold slurry composition comprises less than about 10% by weight of the slurry, between about 10% and about 20% by weight, between about 20% and about 30% by weight, between about 30% and about 40% by weight, between about 40% and about 60% by weight, greater than about 60% by weight, etc.

[0050] In some embodiments, the size of the ice particles is controlled to achieve flowability through containers of various sizes (e.g., needle gauge sizes from about 7 to about 43) as described in the '042 application and incorporated herein by reference. In some embodiments, the biomaterial is first cooled to a specific temperature (as disclosed hereinabove) and further subjected to thawing to achieve a desired ratio of ice particles.

[0051] The proportion of ice particles in the slurry composition can be controlled, in part, through the encapsulation volume in the liposome composition. The higher the encapsulation volume, the higher the final proportion of ice particles when the composition is placed in the freezer. The encapsulation volume is the ratio of what is located within the intraliposomal medium to the total volume of the composition (e.g., a 40% encapsulation volume means that 40% of the composition comprises the intraliposomal medium and 60% of the composition comprises lipids, excipients, and extraliposomal medium). In some embodiments, the encapsulation volume of the biomaterial is less than about 20%, between about 20% and about 30%, between about 30% and about 40%, between about 40% and about 50%, or greater than about 50%. In some embodiments, the encapsulation volume is about 38%. In alternative embodiments, the encapsulation volume is about 43%. In some embodiments, the desired encapsulation volume is achieved using multiple filtrations of the composition, concentrating the liposomes while reducing the volume of the extraliposomal medium. The encapsulated volume can be estimated during the preparation of the biomaterial using methods known in the art, including the method described in Oku, N, et al., "A simple procedure for the determination of the trapped volume of liposomes," 691 Biochim. Biophys. Acta 332-340 (1982), the entire contents of which are incorporated herein by reference. Briefly, Oku reported the preparation of liposomes in a solution containing the fluorescent dye calcein. Once liposomes are formed, cobalt cations are added to the external medium, which acts to quench the fluorescence of calcein only in the external medium; therefore, the encapsulated volume is the proportion of fluorescence remaining after quenching. Other standard methods known in the art for determining encapsulated volume can also be used in the present disclosure.

[0052] Referring to Figure 3, two different slurry compositions (batches) are characterized with respect to their temperature profiles. The temperature traces show two separately produced slurry batches with the same composition: 47% v / v PEG1000 in the extraliposomal medium and 38% liposome encapsulation volume, with a measured freezing point of -6.5°C. The two slurry batches were placed on a copper plate heated to 40°C with thermocouple wires measuring the temperature change of the slurry and copper plate over time. The plotted data show the temperature change over time of two different slurry batches, both of which were cooled to -18°C in a freezer just before being placed on the heated copper plate. The temperature was measured at the implant location inside the copper plate (trace A) for each slurry. C and B C ) and the middle of the copper plate exposed outside the plate (Trace A M and B M When the slurry batch was first placed on the copper plate, the thermocouple wire embedded inside the plate (trace A) C and B C ) is the initial temperature measurement of the heated plate (trace A) C and B C Both are approximately 38°C at time 0), and then reach equilibrium at a lower temperature due to the cooling effect of the placed slurry (trace A). C In about 5 minutes, the temperature reached 19°C, and trace B C (The temperature reached 24°C in approximately 6 minutes for the thermocouple wire located in the middle of the plate.) On the other hand, when the slurry is first placed on the copper plate, the exposed wire immediately comes into contact with the thermocouple wire. Therefore, at this middle position, the crystallized slurry comes into contact with the wire, resulting in a negative temperature reading at first (trace A at time 0). M at -15°C, trace B M (-17°C at 200°C) and then equilibrates at warmer temperatures as the slurry melts on the heated plate (trace A). M Trace B: 16°C in approximately 12 minutes M(The temperature reached 19°C in approximately 8 minutes.) The thermocouple wire exposed to the slurry (trace A) M and B M ) can be used to detect the phase transition where the crystallization slurry begins to melt. This graph shows that both slurry compositions reach a similar point (trace A). M and B M The graph shows that the phase transition is reached in approximately 5 minutes for both batches. The graph also shows that the two slurry batches reach equilibrium in a similar time frame and at similar temperatures (as measured by the two thermocouple wire positions) ranging from about 17°C to about 24°C, depending on the thermocouple position (middle / bottom). Figure 3 therefore demonstrates that batch-to-batch consistency exists between slurries with the same composition.

[0053] Equivalence and Scope In the claims, articles such as "a," "an," and "the" can mean one or more, unless indicated otherwise or clear from the context. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise associated with a product or process, unless indicated otherwise or clear from the context. The present disclosure includes embodiments in which only one member of the group is present in, employed in, or otherwise associated with a product or process. The disclosure also includes embodiments in which two or more or all of the group members are present in, employed in, or otherwise associated with a product or process.

[0054] Furthermore, the present disclosure encompasses all derivations, combinations, and permutations in which one or more limitations, elements, clauses, and statements from one or more of the enumerated claims are introduced into another claim. For example, any claim that recites another claim can be modified to include one or more limitations found in any other claim that recites the same base claim. Where elements are presented as lists, such as a Markush group list, each subgroup of the elements is also disclosed, and any element can be removed from the group. In general, when the present disclosure or aspects of the disclosure are referred to as comprising certain elements and / or features, it should be understood that certain embodiments of the present disclosure or aspects of the disclosure consist of, or consist essentially of, such elements and / or features. For brevity, these embodiments have not been specifically set forth in that language herein. It should also be noted that the terms "comprising," "including," and "containing" are intended to be open-ended and permit the inclusion of additional elements or steps. Where ranges are presented, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed in ranges may assume in different aspects of this disclosure any specific value or subrange within the stated range, down to one-tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise.

[0055] This application references various issued patents, published patent applications, literature articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification will control. In addition, any particular aspect of the present disclosure that falls within the prior art may be expressly excluded from any one or more claims. Because such aspects are deemed known to those skilled in the art, they may be excluded even if such exclusion is not expressly set forth herein. Any particular aspect of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0056] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the inventive embodiments described herein is not intended to be limited to the above description, but is instead set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to the description can be made without departing from the spirit or scope of the present disclosure, as defined in the appended claims.

Claims

1. water, at least one liposome, and At least one excipient A composition comprising: the liposome is configured to encapsulate a first volume of the composition; the excipient is configured to be confined to a second volume of the composition outside the liposome and configured to be sequestered from the encapsulated first volume; a first freezing point of the encapsulated first volume is higher than a second freezing point of the encapsulated second volume; composition.

2. 2. The composition of claim 1, wherein the liposome is composed of a lipid selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg sphingomyelin (DPSM), dipalmitoylphosphatidylcholine (DPPC), dicetyl phosphate (DCP), L-α-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and combinations thereof.

3. 3. The composition of claim 2, wherein the lipid is L-α-phosphatidylcholine (PC).

4. 10. The composition of any one of the preceding claims, wherein the excipient is selected from the group consisting of salts, ions, lactated Ringer's solution, sugars, biocompatible surfactants, polyols, and combinations thereof.

5. 10. The composition of claim 1, wherein the excipient is a polyol.

6. 6. The composition of claim 5, wherein the polyol is polyethylene glycol 1000 (PEG1000).

7. 10. The composition of any one of the preceding claims, further comprising a second excipient in both the first and second volumes.

8. 8. The composition of claim 7, wherein the second excipient is saline or phosphate buffered saline (PBS).

9. 10. The composition of claim 1, wherein the encapsulated first volume is about 20% to 50% of the total volume of the composition.

10. 10. The composition of claim 9, wherein the encapsulated first volume is about 38% of the total volume of the composition.

11. 10. The composition of claim 9, wherein the encapsulated first volume is about 43% of the total volume of the composition.

12. 2. The composition of claim 1, wherein the first freezing point of the encapsulated first volume is from about -2°C to about 0°C.

13. 2. The composition of claim 1, wherein the second freezing point of the second volume is from about -20°C to about -10°C.

14. 10. The composition of claim 1, wherein the total volume of the composition, including the first volume, the second volume, and the liposomes, has an average freezing point of about -10°C to about -5°C.

15. 10. The composition of claim 1, wherein the encapsulated first volume is configured to form a plurality of ice particles when the composition is cooled to a predetermined temperature.

16. 16. The composition of claim 15, wherein the ice particles comprise from about 30% to about 50% by weight of the total weight of the composition.

17. 17. The composition of claim 15 or 16, wherein the predetermined temperature is about -20°C to -5°C.

18. 1. A method for preparing a composition for administration to a patient in a clinical setting, comprising: preparing a composition having a plurality of liposomes, wherein an aqueous medium fills the intraliposomal volume and the extraliposomal volume; adding at least one excipient to the extraliposomal volume, wherein the at least one excipient reduces a first freezing point of the extraliposomal volume below a second freezing point of the intraliposomal volume; and cooling the composition to a predetermined temperature so as to form a cold slurry having a plurality of ice particles within the intraliposomal volume. A method comprising:

19. 19. The method of claim 18, wherein the liposome is composed of a lipid selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg sphingomyelin (DPSM), dipalmitoylphosphatidylcholine (DPPC), dicetyl phosphate (DCP), L-α-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and combinations thereof.

20. 20. The method of claim 19, wherein the lipid is L-α-phosphatidylcholine (PC).

21. 21. The method of any one of claims 18 to 20, wherein the excipient is selected from the group consisting of salts, ions, lactated Ringer's solution, sugars, biocompatible surfactants, polyols, and combinations thereof.

22. 22. The method of any one of claims 18 to 21, wherein the excipient is a polyol.

23. 23. The method of claim 22, wherein the polyol is polyethylene glycol 1000 (PEG1000).

24. 24. The method of any one of claims 18 to 23, wherein the aqueous medium is composed of water, saline, or phosphate buffered saline (PBS).

25. 25. The method of any one of claims 18 to 24, wherein the intraliposomal volume is about 20% to 50% of the total volume of the composition.

26. 26. The method of claim 25, wherein the intraliposomal volume is about 38% of the total volume of the composition.

27. 26. The method of claim 25, wherein the intraliposomal volume is about 43% of the total volume of the composition.

28. 28. The method of any one of claims 18 to 27, wherein the first freezing point of the extraliposomal volume is from about -20°C to about -10°C.

29. 29. The method of any one of claims 18 to 28, wherein the second freezing point of the intraliposomal volume is from about -2°C to about 0°C.

30. 30. The method of any one of claims 18 to 29, wherein the average freezing point of the total volume of the composition comprising the first volume, the second volume, and the liposomes is from about -10°C to about -5°C.

31. 31. The method of any one of claims 18 to 30, wherein the ice particles comprise about 30% to about 50% by weight of the biomaterial.

32. 32. The method of any one of claims 18 to 31, wherein the predetermined temperature is about -20°C to -5°C.

33. 33. The method of any one of claims 18 to 32, wherein the bilayer composition of the liposome is selected from the group consisting of unilamellar vesicles, multilamellar vesicles, oligolamellar vesicles, multivesicular vesicles, and combinations thereof.

34. 34. The method of any one of claims 18 to 33, wherein the liposomes have an average diameter of about 0.1 μm to about 2 μm.

35. 1. A method for producing a flowable, injectable encapsulated ice solution, comprising: providing a plurality of biodegradable liposomes configured to form vesicles selected from the group consisting of multilamellar vesicles, oligolamellar vesicles, multivesicular vesicles, giant unilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles, or combinations thereof; encapsulating water within at least two of the plurality of liposomes to form liposomes filled with a first volume containing water; adding an excipient to a second extraliposomal volume isolated from the first volume, the excipient altering the freezing point of the second volume relative to the first volume; freezing the plurality of filled liposomes to generate a plurality of ice particles within the filled liposomes; and controlling the average diameter of each of the plurality of ice particles to a predetermined size; A method comprising:

36. 36. The method of claim 35, wherein the first volume is about 20% to 50% of the total volume of the composition.

37. 37. The method of claim 36, wherein the first volume is about 38% of the total volume of the composition.

38. 37. The method of claim 36, wherein the first volume is about 43% of the total volume of the composition.

39. The method of any one of claims 35 to 38, wherein the excipient is PEG1000.

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