Method of providing liposome solution for forming microvesicles

Abstract

The present invention relates to a method of providing a liposome solution composed of a phospholipid blend for forming microvesicles. The invention also relates to the liposome solution for forming the microvesicles obtained by the method, and the microvesicles formed by the liposome solution. Furthermore, the present invention relates to the use of a liposome solution or microvesicles as a drug delivery enhancer or as a drug delivery vehicle for a drug to a patient.

Description

[0001] Description

[0002] The present invention relates to a method for providing a liposome solution for forming microvesicles, which consists of a phospholipid blend. The present invention also relates to a liposome solution for forming microvesicles obtained by this method, and microvesicles composed of this liposome solution. In addition, the present invention relates to the use of the liposome solution or microvesicles as a drug delivery enhancer or as a drug delivery carrier to a patient for a drug.

[0003] Microvesicles (also known in the art as nanodroplets / microdroplets, microcapsules, microbubbles) are heterogeneous membrane-bound objects that have a core encapsulated by an outer membrane, such as a fluid (i.e., a gas or a liquid), and the outer membrane is preferably a lipid bilayer membrane in the case of lipid-stabilized droplets, or more preferably a lipid monolayer membrane in the case of lipid-stabilized gaseous microbubbles. Microvesicles are typically produced from a liposome solution containing phospholipids for generating microvesicles in a dedicated system. Such microvesicles can be used as drug delivery enhancers, for example, by co-administering intravenously with a parenteral drug in the circulatory (blood) system to treat a patient, or for parenteral administration of a drug or treatment to a patient, for example, by intravenously administering drug-loaded microvesicles in the patient's circulatory (blood) system. For effective drug delivery, it is important that the microvesicles have a predetermined size and / or size distribution, where the distribution is preferably as narrow as possible.

[0004] A liposome solution composed of a phospholipid blend is typically produced by dissolving the lipids in a solvent and then reducing the volume by freeze-drying or distillation. Such a method for preparing a liposome solution for forming microvesicles utilizes highly toxic carcinogenic solvents in a wide-ranging multi-step method, as described, for example, in Segers et al. (Langmuir 2017, 33(39)). The production is typically carried out according to the thin-film hydration method using toxic organic solvents, where the lipids are mixed and dissolved in a mixture of chloroform and methanol, which is carried out at ambient pressure and a temperature of 60 °C or higher. Then the pressure is reduced to evaporate the solvent. Then the resulting lipid film is dried overnight in a vacuum oven at 100 mbar pressure, and the next day, the lipids are rehydrated again at 60 °C in a mixture of TRIS buffer water, propylene glycol (PG), and glycerin / glycerol. After rehydration, the lipid dispersion is immediately homogenized by sonication using, for example, a tip sonicator, and the dispersion is cooled to room temperature.

[0005] Current methods for generating microvesicles use toxic carcinogenic solvents. A drawback of this process is the possible presence of toxic solvents in the final product. Considering microvesicles formed from liposome solutions and their subsequent drug delivery applications, post-treatment to remove trace organic solvents is required, as well as additional clinical testing to demonstrate the non-toxicity of the product. Additionally, sonication can introduce undesirable iron particles into the liposome solution, which are shed from the sonicator. Further, a high concentration of glycerol (typically 5 vol%) results in foam formation due to gas dissolution, for example, during stabilization after microbubble formation by microfluidic flow focusing. Additionally, glycerol increases the volumetric viscosity, which can have a negative effect on the microfluidic flow focusing process. Glycerol is used for rehydration of lipids in the mixture, however, it results in an increase in the viscosity of the liposome solution and a reduction in high-quality microbubbles generated by microfluidic flow focusing. A gas mixture and mixing device are required to reduce foam formation during microbubble stabilization after microbubble formation, resulting in a long stabilization time (>1 hour). Current methods are suitable for small laboratory-scale generation, but scaling up to production-scale quantities typically presents problems, including maintaining the uniformity of the liposome solution (and resulting microvesicles) during the solvent removal step due to the different solubilities of the lipids, and minimizing the solvent volume in view of the reduced toxicity or improved environmental footprint of the production process.

[0006] Finally, elevated temperatures (>60 °C) are required to obtain a liposome solution in which the phospholipids are dissolved and for microvesicle formation, followed by immediate cooling to reduce microvesicle coalescence during high-flow microfluidic formation, which is less sustainable and difficult to scale up due to increased energy costs. Currently, hydration of phospholipids at lower temperatures results in a turbid suspension with relatively large and non-uniform particle size distribution (0.1 μm to 10 μm). Such a suspension cannot be filtered at lower (e.g., ambient) temperatures when the suspension solution temperature is below the phase transition temperature of the lipid. The lipid will accumulate in the filter, resulting in restricted flow and ultimately clogging the filter.

[0007] In view of the above, there is a need in the art for simplified, large-scale, more environmentally friendly, and more energy-efficient methods for preparing liposome solutions for forming microvesicles. Additionally, there is a need in the art for more environmentally friendly liposome solutions for forming microvesicles.

[0008] Among other purposes, an object of the present invention is to address the above needs in the art. Among other purposes, the objects of the present invention are achieved by the present invention as outlined in the appended claims.

[0009] Specifically, among other purposes, the above purpose is achieved by the present invention according to the first aspect by providing a method for forming a liposome solution for microvesicles, wherein the liposome solution is composed of a phospholipid blend of at least two phospholipids having different phase transition temperatures (Tm), and the method comprises the following steps:

[0010] a) Dissolve a first phospholipid having the lowest Tm in a preheated organic non-aqueous solvent at a temperature lower than the Tm of the first phospholipid to provide a first liposome solution.

[0011] b) Add a second phospholipid to the first liposome solution to obtain a liposome dispersion, or

[0012] Add the second phospholipid to a preheated organic aqueous solvent at a temperature lower than the Tm of the second phospholipid to obtain a second liposome dispersion.

[0013] c) Keep the liposome dispersion at a temperature lower than the Tm of the first phospholipid for 30 minutes to 2 hours, preferably 45 minutes to 90 minutes, more preferably 60 minutes to 75 minutes, preferably with stirring, to obtain a further liposome solution in which the phospholipids are dissolved, preferably with stirring, or

[0014] Keep the second liposome solution at a temperature lower than the Tm of the second phospholipid for 30 minutes to 2 hours, preferably 45 minutes to 90 minutes, more preferably 60 minutes to 75 minutes, to obtain a second liposome solution in which the phospholipids are dissolved, preferably with stirring.

[0015] d) Add a preheated organic aqueous solvent at a temperature lower than the Tm of the first phospholipid to the further liposome solution, preferably with stirring, or

[0016] Add the second liposome solution to the first liposome solution, preferably with stirring.

[0017] To obtain a pre-final liposome solution with a ratio of organic non-aqueous solvent to organic aqueous solvent as follows: 5 vol% to 25 vol% non-aqueous: 75 vol% to 95 vol% aqueous, preferably 7.5 vol% to 20 vol% non-aqueous: 80 vol% to 92.5 vol% aqueous, even more preferably 8 vol% to 15 vol% non-aqueous: 85 vol% to 92 vol% aqueous, most preferably about 10 vol% non-aqueous: 90 vol% aqueous.

[0018] e) Keep the pre-final liposome solution at a temperature lower than the Tm of the first phospholipid for 2 to 12 hours, preferably 4 to 10 hours, more preferably 6 to 8 hours, preferably with stirring, to obtain a final liposome solution.

[0019] The method of the present invention provides a liposome solution composed of a homogeneous lipid blend. Experiments have shown that, compared with known methods for preparing liposome solutions, the method of the present invention for preparing such solutions provides a more uniform liposome distribution, resulting in a more uniform and less coalesced population of microbubbles. Coalescence of microbubbles leads to a polydisperse population of microbubbles. To maintain a monodisperse population of microbubbles, coalescence should be avoided. The method does not contain toxic carcinogenic organic solvents, thus eliminating the need for further purification steps, operates at relatively low temperatures, and is a simple process that is easily scalable without the need for excessive heating or cooling to obtain the liposome solution.

[0020] Liposomes are mainly composed of phospholipids, which are different types of compounds consisting of a hydrophilic head group covalently linked to a pair of hydrophobic fatty acids. When added to an aqueous solution, phospholipid molecules spontaneously form a bilayer because the phospholipids are driven to orient their head groups towards the water and protect their fatty acid tails from water through hydrophobic interactions. Phospholipids can undergo a phase transition under suitable environmental conditions. The phase transition temperature (Tm) of a phospholipid is the temperature required to induce a change in the physical state of the lipid from an ordered gel phase to a disordered liquid crystal phase. The main driving force for most phase transitions is temperature. Temperatures above the lipid Tm will cause the lipid to transition to the liquid phase, while colder temperatures will result in a transition to a solid-like phase. However, due to different structural characteristics, the Tm can vary between lipids and strongly depends on environmental conditions (such as solvent and mixture conditions). Considering known methods for generating liposome solutions, the outstanding feature of the method of the present invention is that the lipids are dissolved in an organic solvent at a relatively low temperature (more specifically, below the Tm of the lipid). Compared with known methods, an increased volume of non-aqueous organic solvent (preferably 7.5 vol% or greater) is preheated to a temperature below the Tm of the respective phospholipid.

[0021] The range of the non-aqueous solvent is preferably greater than or equal to 7.5 vol% based on the total volume of the organic solvent in the liposome solution to obtain an optimally dissolved lipid solution at the claimed temperature. The mixture of the organic non-aqueous solvent and the organic aqueous solvent of the liposome solution contains 5 vol% to 25 vol% of the organic non-aqueous solvent based on the total volume of the solution. Adding the organic solvent to the first or second phospholipid results in the lipid being preferably completely dissolved in the organic solvent. However, since the temperature is below the Tm of the lipid, time (preferably with stirring / gentle mixing) is required to completely dissolve the lipid. After adding the lipid to the organic solvent, at least 90 mol% of the lipid is dissolved within the disclosed time, preferably at least 92 mol%, more preferably at least 95 mol%, even more preferably at least 99 mol%, and most preferably 100 mol% is dissolved, providing a homogeneous lipid blend.

[0022] According to a preferred embodiment, the present invention relates to a method, wherein the method further comprises step f) flowing and extruding or filtering the final liposome solution at a temperature below the Tm of the first phospholipid, preferably at 15 to 40 °C, more preferably at room temperature of 18 to 25 °C, so as to provide a final liposome solution for forming microvesicles. The flowing extrusion or filtration of the liposome solution can be carried out using a sterilizing filter, preferably through a 0.2 μm polycarbonate / cellulose acetate membrane. This extrusion or filtration step improves the liposome solution to provide more uniform and monodisperse microvesicles. The method of the present invention provides microvesicles prepared at room temperature and quickly stabilized (<30 minutes), and the product can be quickly used after formation. In contrast to known methods for forming microvesicles in the art, the present invention can generate microvesicles by microfluidic flow focusing using the liposome solution of the present invention with an increased volume of non-aqueous organic solvent. The extruded final liposome solution can be stored, for example, at 4 °C for an extended period of time.

[0023] According to another preferred embodiment, the present invention relates to a method, wherein the temperature of the preheated organic non-aqueous or aqueous solvent is at most 20%, more preferably at most 10%, more preferably at most 5%, and most preferably at most 1% lower than the respective Tm values of the first or the second phospholipid. The advantage of the method of the present invention is that the lipids are uniformly mixed and dissolved at a low temperature (i.e., below the Tm of the phospholipids used in the lipid blend). The closer the temperature of the preheated solvent is to the Tm value of the phospholipid, the more effectively the process of dissolving the lipids in the solvent is achieved. However, when the temperature is reduced too much, a uniform lipid blend can no longer be achieved. The method of the present invention uses an increased volume of non-aqueous organic solvent (preferably 7.5 vol% or more), providing a phospholipid blend optimized for large-scale production at low temperature.

[0024] According to another preferred embodiment, the present invention relates to a method, wherein the preheated organic non-aqueous solvent with a temperature below the Tm of the first phospholipid in step a and the preheated organic aqueous solvent with a temperature below the Tm of the first phospholipid in step d are 20 °C to 55 °C, preferably 25 °C to 45 °C, more preferably 35 °C to 39 °C, and most preferably 38 °C.

[0025] According to another preferred embodiment, the present invention relates to a method, wherein the preheated organic aqueous solvent with a temperature below the Tm of the second phospholipid in step b is 40 °C to 60 °C, preferably 45 °C to 55 °C, more preferably 48 °C to 50 °C, and most preferably 50 °C.

[0026] According to a preferred embodiment, the present invention relates to a method in which the addition of the preheated organic aqueous solvent or the second liposome solution in step d is carried out in two to five separate steps, preferably three to four separate steps, wherein the added volume is equally divided among these steps. The addition in step d is preferably carried out in a multi-step manner to reduce the possibility of precipitation of the lipid moiety or even prevent precipitation of the lipid moiety and to obtain a liposome solution composed of a homogeneous lipid blend.

[0027] According to another preferred embodiment, the present invention relates to a method in which the final liposome solution contains 60 mol% to 95 mol%, more preferably 70 mol% to 90 mol%, even more preferably 75 mol% to 87 mol%, and most preferably 80 mol% to 85 mol% of the first phospholipid. It is difficult to obtain monodisperse microvesicles by microfluidic flow focusing when the first phospholipid in the liposome solution is outside these mol% ranges.

[0028] According to a preferred embodiment, the present invention relates to a method in which the final liposome solution contains 5 mol% to 40 mol%, more preferably 10 mol% to 30 mol%, even more preferably 13 mol% to 25 mol%, and most preferably 15 mol% to 20 mol% of the second phospholipid. It is difficult to obtain monodisperse microvesicles by microfluidic flow focusing when the second phospholipid in the liposome solution is outside these mol% ranges.

[0029] According to another preferred embodiment, the present invention relates to a method wherein the first phospholipid is selected from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), egg phosphatidylcholine (EPC), and hydrogenated soybean phosphatidylcholine (HSPC), preferably DSPC, more preferably DPPC. DSPC and DPPC are mainly used in current ultrasound contrast agents and have a good safety profile in patients. DPPC is more preferred due to its low Tm value and smaller liposome size.

[0030] According to a preferred embodiment, the present invention relates to a method wherein the second phospholipid is a phospholipid polyethylene glycol (PEG) conjugate, preferably selected from N-(carbonyl-methoxypolyethylene glycol (750 to 10,000))-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG(750 to 10,000)), N-(carbonyl-methoxypolyethylene glycol (1000 to 5000))-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-mPEG(1000 to 5000)), preferably DPPE-mPEG2k, more preferably DSPE-mPEG5000, most preferably DPPE-mPEG5000. The molecular weight of the PEG chain in the phospholipid can vary from about 750 daltons to about 10,000 daltons.

[0031] According to a preferred embodiment, the present invention relates to a method in which phospholipids, more specifically the sum of a first phospholipid and a second phospholipid, are present in the final liposome solution at a concentration of 1 to 25 mg / ml, preferably 5 to 20 mg / ml, more preferably 10 to 15 mg / ml. The phospholipid concentration within the claimed scope facilitates microbubble formation by microfluidic flow focusing. Due to such a high concentration of lipid vesicles in the liposome solution, the coalescence of microbubbles is greatly reduced or even absent. A high concentration of lipid vesicles is required to prevent coalescence. Unexpectedly, by using the liposome solution of the present invention prepared at low temperature, monodisperse microbubbles can be formed by microfluidic flow focusing.

[0032] According to another preferred embodiment, the present invention relates to a method in which the non-aqueous organic solvent is one or more selected from the following: propylene glycol, ethylene glycol, preferably propylene glycol.

[0033] According to another preferred embodiment, the present invention relates to a method in which the aqueous organic solvent is one or more selected from the following: phosphate buffer saline (PBS), buffered aqueous solution, saline solution, preferably PBS.

[0034] According to a preferred embodiment, the present invention relates to a method in which the method or the liposome solution does not contain glycerol or diphenylphosphoryl azide (DPPA) and toxic carcinogenic organic solvents such as chloroform. Advantageously, the liposome solution does not contain DPPA. Phospholipid compositions prepared by prior art methods are difficult to sterilize by filtration or extrude at low temperature. In addition, in view of the use of liposome solutions or microvesicles as drug delivery enhancers or as drug-loaded microvesicles and subsequent use in patients, it is considered that DPPA may contribute to an undesired immune response caused by microvesicles.

[0035] According to a second aspect, the present invention relates to a liposome solution for forming microvesicles obtained by the method as described herein, the liposome solution being composed of a lipid blend, the lipid blend being composed of a first phospholipid and a second phospholipid having different phase transition temperatures (Tm) dissolved in a mixture of an organic non-aqueous solvent and an organic aqueous solvent, and wherein the ratio of the organic non-aqueous solvent to the organic aqueous solvent in the liposome solution is from 5% to 25% by volume non-aqueous: 75% to 95% by volume aqueous, preferably from 7.5% to 20% by volume non-aqueous: 80% to 92.5% by volume aqueous, even more preferably from 8% to 15% by volume non-aqueous: 85% to 92% by volume aqueous, and most preferably about 10% by volume non-aqueous: 90% by volume aqueous. Considering the known methods for producing liposome solutions, the outstanding feature of the method of the present invention is that the lipids are dissolved in the organic solvent at a relatively low temperature (more specifically below the Tm of the lipids). Compared with the known methods, an increased volume of the non-aqueous organic solvent (preferably 7.5% by volume or more) is preheated to a temperature below the Tm of the corresponding phospholipid, thereby providing a relatively high concentration of solvent in the lipid solution. The range of the non-aqueous solvent is preferably greater than or equal to 7.5% by volume based on the total volume of the organic solvents in the liposome solution to obtain an optimal dissolved lipid solution at the claimed temperature.

[0036] According to a preferred embodiment, the present invention relates to a liposome solution, wherein the first phospholipid is selected from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), egg phosphatidylcholine (EPC) and hydrogenated soy phosphatidylcholine (HSPC), preferably DSPC, more preferably DPPC, and wherein the second phospholipid is selected from N-(carbonyl-methoxypolyethylene glycol (750 to 10,000))-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG(750 to 10,000)), N-(carbonyl-methoxypolyethylene glycol (1,000 to 5,000))-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-mPEG(1,000 to 5,000)), preferably DPPE-mPEG2k, more preferably DSPE-mPEG5000, most preferably DPPE-mPEG5000.

[0037] According to another preferred embodiment, the present invention relates to a liposome solution, wherein the phospholipids, more specifically the sum of the first phospholipid and the second phospholipid, are present in the final liposome solution at a concentration of 1 to 25 mg / ml, preferably 5 to 20 mg / ml, more preferably 10 to 15 mg / ml.

[0038] According to a preferred embodiment, the present invention relates to a liposome solution, wherein the non-aqueous organic solvent is one or more selected from the following: propylene glycol, ethylene glycol, preferably propylene glycol.

[0039] According to yet another preferred embodiment, the present invention relates to a liposome solution, wherein the aqueous organic solvent is one or more selected from the following: phosphate buffered saline (PBS), buffered aqueous solution, saline solution, preferably PBS.

[0040] According to a preferred embodiment, the present invention relates to a liposome solution, wherein the liposome solution does not contain glycerol, diphenylphosphoryl azide (DPPA) and toxic carcinogenic organic solvents such as chloroform.

[0041] According to another aspect, the present invention relates to microvesicles composed of the liposome solution disclosed herein, wherein the microvesicles are monodisperse, with a diameter of the particle size of 1 to 8 μm, preferably 2 to 6 μm, more preferably 3 to 5 μm. The monodisperse microvesicles disclosed herein are preferably microbubbles of approximately similar size, i.e., having a uniform size distribution, with a geometric standard deviation (GSD) ≤ 1.1. The liposome solution of the present invention enables the formation of foam-free monodisperse lipid-coated microbubbles with a reduced tendency to coalesce at low temperatures. The median diameter of the monodisperse microbubbles disclosed herein is less than 10 μm, preferably 2 to 6 μm, more preferably 3 to 5 μm, and the geometric standard deviation GSD is preferably less than or equal to 1.1. Microbubbles with a larger diameter (>10 μm) are not desirable because they will be filtered out of the blood circulation by the lungs after intravenous injection. Microbubbles that are too small (<1 μm) will be inefficient because they are acoustically over-damped and will not resonate in an ultrasound field.

[0042] According to a preferred embodiment, the present invention relates to such microvesicles, wherein the microvesicles are drug delivery enhancers or drug-loaded microvesicles for delivering drugs to a patient, preferably for parenteral drug delivery.

[0043] According to another aspect, the present invention relates to the use of the liposome solution or microvesicles disclosed herein for delivering drugs to a patient, preferably by parenteral administration, more preferably by intravenous co-administration.

[0044] The present invention will be further described in detail in the following examples and drawings, wherein:

[0045] Figure 1 : shows the size distribution of monodisperse microbubbles (formulation B) obtained after microfluidic flow focusing using a liposome composition obtained by the method according to the present invention, compared to microbubbles (formulation A) obtained by a method known in the art.

[0046] Figure 2: Shows the size distribution of monodisperse microbubbles obtained after microfluidic flow focusing in the case of liposome compositions L1 and L2, measured using a Beckman Coulter Counter Multisizer 4e. Both lipid formulations were prepared according to the method of the present invention and consist of a primary lipid (DSPC) and a secondary pegylated lipid (DPPE-mPEG5k) in a molar percentage ratio of 80 / 20. The total lipid concentration was 15 mg / ml. The ratio of the organic non-aqueous solvent to the organic aqueous solvent for L1 was 5 vol% propylene glycol (PG) and 95 vol% phosphate buffered saline (PBS). The ratio of the organic non-aqueous solvent to the organic aqueous solvent for L2 was 7.5 vol% propylene glycol (PG) and 92.5 vol% phosphate buffered saline (PBS). The L1 formulation showed multiple peaks (N1, N2) due to coalescence, while L2 showed a single sharp peak (N1).

[0047] Figure 3 : Shows the relative intensity-based (%) size distribution of lipid vesicles of L2 and L3 formulations (total lipid concentration of 15 mg / ml) by dynamic light scattering (DLS) using a Zetasizer NanoS. DLS measurements showed that compared to lipid formulation L3, smaller liposomes <100 nm were predominantly present in lipid formulation L2, and vice versa, with L3 formulation mainly containing liposomes >100 nm.

[0048] Figure 4 : Shows the optical transmission (%) at 700 nm of the three lipid formulations L1, L2, and L3 of Example 3 using a Jenway spectrophotometer 630. All formulations had the same total lipid concentration of 15 mg / mL. The higher the transmission reading, the smaller the lipid vesicles in the lipid formulation. L2 showed a higher optical transmission reading than lipid formulations L1 and L3, indicating different size distributions of lipid vesicles in the three lipid solutions. Compared to lipid formulations L1 and L3, L2 consisted of the smallest-sized lipid vesicles. Since the total lipid concentration of all three lipid formulations was the same, this indicates a higher concentration of lipid vesicles in lipid formulation L2 compared to lipid formulations L1 and L3, which is beneficial for preventing microbubble coalescence. Example

[0049] Example 1 - Preparation of Liposomal Formulations for Large-Scale Microvesicle Formation

[0050] Liposome solutions for subsequent microvesicle formation were provided according to the method of the present invention by methods A and B.

[0051] Method A

[0052] Preheat propylene glycol (10% by volume) at 38 °C. Then, add the first lipid DPPC (80 mol%) with the lowest Tm, and gently stir for about 20 minutes until the lipid is dissolved. Then add the second lipid DPPE-mPEG5k (20 mol%) to the same vial containing the dissolved first lipid. The total lipid concentration is 15 mg / mL. Maintain the lipid solution in a water bath at 38 °C and gently stir for about 1 hour. Next, slowly add the preheated PBS solution at 38 °C with stirring to obtain a solution containing 10% / 90% v / v propylene glycol / PBS lipid. Keep the liposome solution at 38 °C with stirring for about 6 hours. Next, flow-extrude the liposome solution through a 0.2 μm polycarbonate / cellulose acetate membrane at 38 °C to obtain a liposome solution capable of forming monodisperse microvesicles by microfluidic flow focusing. The resulting liposome solution can then be stored, preferably at 4 °C.

[0053] Method B

[0054] Prepare the first stock solution by dissolving the first lipid DPPC (80 mol%) in preheated propylene glycol (10% by volume) as described in Method A. Prepare the second stock solution separately with the second lipid DPPE-mPEG5k (20 mol%) and add it to the preheated PBS at 50 °C to obtain a ratio of about 10% by volume / 90% by volume with the organic solvent when the first and second stock solutions are subsequently combined. Keep the second stock solution at 50 °C with gentle stirring. Keep the two stock solutions at 38 °C and 50 °C respectively and gently stir, preferably for about 1 hour. Then, slowly add the second stock solution to the first stock solution, preferably in three to four steps gradually to reduce or even prevent partial precipitation of the lipid to obtain a liposome solution. Keep the liposome solution at 38 °C with stirring for about 6 hours to obtain a clear and homogeneous solution in which the liposomes are completely dissolved. Next, flow-extrude the liposome solution through a 0.2 μm polycarbonate / cellulose acetate membrane at 38 °C to obtain a liposome solution capable of enabling the formation of monodisperse microvesicles by microfluidic flow focusing. The resulting liposome solution can then be stored, preferably at 4 °C.

[0055] Example 2 - Monodisperse Microbubble Formation by Microfluidic Flow Focusing

[0056] Two-phase microfluidic flow focusing is a technique using a microfluidic chip, in which the dispersed phase is focused by the continuous phase through a narrow orifice, where the dispersed phase undergoes capillary instability and pinchoff to release monodisperse particles. In the case of using a gas as the dispersed phase (e.g., C3F8) and a liquid as the continuous phase (e.g., liposome solution), the particles consist of gas-filled monodisperse microbubbles stabilized by a lipid monolayer. Here, a microfluidic flow focusing device with a channel geometry as described by Segers et al. (Soft Matter 2018, 14, 9550 - 9561) is used. Both the gas pressure and the liquid flow rate are controlled by a pressure regulator (i.e., pressure-based liquid supply flow control). In this way, the size and concentration of the microbubbles can be controlled by the ratio between the gas and liquid pressures.

[0057] Monodisperse microbubbles are formed using pure C3F8 as the gas (i.e., no gas mixture is used) and two liposome formulations (liposome formulation A and B) at a constant 25 °C (controlled temperature on the chip). Liposome formulation A is obtained according to the prior art method, where formulation A is prepared at 72 °C using an 80 / 20 mol% DPPC / DPPE-mPEG5k and a 5 vol% / 95 vol% propylene glycol / PBS ratio. Briefly, a hydrated phospholipid solvent mixture is prepared by dissolving DPPC and DPPE-mPEG5k in propylene glycol at 72 °C to form a dissolved phospholipid solvent mixture. Next, an aqueous phosphate buffer is added and mixed with the dissolved phospholipid solvent mixture to obtain the hydrated phospholipid solvent mixture. Liposome formulation B is obtained according to method A as described above (i.e., according to the method of the present invention, i.e., prepared at 38 °C using an 80 / 20 mol% DPPC / DPPE-mPEG5k and a 10 vol% / 90 vol% propylene glycol / PBS ratio).

[0058] The gas pressure for microbubble formation is 1750 mbar; the gas pressure for controlling the liquid flow rate is 2015 mbar for formulation A and 2050 mbar for formulation B. The outlet of the flow focusing device is connected to a 24G needle through a PEEK tube for collecting microbubbles in a sealed glass vial; the glass vial is pre-filled with 1 mL of 0.9% NaCl and purged with C3F8 gas so that the headspace is filled with C3F8. A second needle is inserted through the closed rubber stopper and used as an exhaust port by positioning it near the bottom of the glass vial, and the glass vial is placed upside down during microbubble collection. Microbubbles are collected for 5 to 7 minutes. After microbubble formation is complete, the needles are removed and the vial is left to stand for 1 hour to allow the microbubbles to stabilize in the vial. After 1 hour, the size distribution is measured using a Coulter Counter Multisizer 4e.Figure 1 Shows the microbubble size distribution as measured with a Coulter Counter Multisizer 4e. It shows the size distribution of monodisperse microbubbles obtained with formulation A- (blue) and formulation B- (red). In Table 1, the size distribution statistics of the monodisperse microbubble samples obtained with formulations A and B are listed.

[0059] Table 1. Microbubble (MB) size distribution statistics (GSD = geometric standard deviation).

[0060]

[0061] From Figure 1 and the above results in Table 1, it can be concluded that monodisperse microbubbles formed by microfluidic flow focusing can be obtained by using 100% C3F8 as the gas (without gas mixture) and a liposome solution prepared according to the method of the present invention.

[0062] The method of the present invention provides a desired small microbubble diameter of 3 to 5 μm, and most of the generated microbubbles are within this size distribution range. Compared with the microbubbles obtained with formulation A, the microbubbles obtained with formulation B are at least comparable and even slightly improved. No microbubble coalescence and foam formation leading to a polydisperse size distribution were observed in the Coulter Counter measurement, and formulation B is slightly improved compared with formulation A.

[0063] Example 3 - Characteristics of Lipid Formulations

[0064] Three lipid formulations (L1 to L3) were prepared, which consisted of a major lipid (DSPC) and a minor PEGylated lipid (DPPE-mPEG5000) in a molar percentage ratio of 80 / 20;

[0065] - L1 was prepared according to method A of Example 1 at a temperature of about 52 °C, with a ratio of organic non-aqueous solvent to organic aqueous solvent of 5 vol% propylene glycol (PG) and 95 vol% phosphate buffered saline (PBS).

[0066] - L2 was prepared according to method A of Example 1 at a temperature of about 52 °C, with a ratio of organic non-aqueous solvent to organic aqueous solvent of 7.5 vol% propylene glycol (PG) and 92.5 vol% phosphate buffered saline (PBS).

[0067] - L3 was prepared according to the method disclosed in WO2022139582A1 at a temperature of about 72 °C, with a ratio of organic non-aqueous solvent to organic aqueous solvent of 5 vol% propylene glycol (PG) and 95 vol% phosphate buffered saline (PBS).

[0068] For all lipid formulations L1, L2, and L3, the same total lipid concentration of 15 mg / ml was used.

[0069] As in Example 2, a microfluidic flow focusing device was used to form monodisperse microbubbles using the prepared liposome formulations at a constant 40 °C (controlled temperature on the chip). For lipid formulations L1 and L2, the microbubble size distribution was measured using a Beckman Coulter Counter Multisizer 4e according to the manufacturer's instructions, see Table 2. Compared to the microbubbles formed using the L1 formulation (the second peak in the size distribution), the microbubbles formed using the L2 formulation showed reduced microbubble coalescence. In fact, no coalescence was observed during microbubble formation when using L2. In contrast, significant coalescence was observed during microbubble formation when using L1 ( Figure 2 ), resulting in multiple peaks.

[0070] The coalescence factor (CF) of the microbubbles formed using the L1 formulation was calculated according to the following equation:

[0071] CF = (2*N2) / (N1 + 2*N2)*100 (1)

[0072] where N2 is the number of microbubbles in the second peak of the size distribution, and N1 is the number of microbubbles in the first peak of the size distribution. The number of microbubbles in the second peak (N2) is multiplied by 2 because the microbubbles that are part of the second peak are initially formed from two microbubbles that are part of the first peak. If no coalescence occurs, then (N1 + 2*N2) is equal to the total number of microbubbles in the main peak.

[0073] Table 2. Microbubble Coulter Counter statistics (microbubble (MB) number concentration and median diameter) and derived coalescence factor when using different lipid formulations.

[0074]

[0075] It was also observed that the lipid solution L2 prepared using 7.5 vol% propylene glycol during preparation was easier to filter compared to L1 when using 5 vol% propylene glycol during lipid formulation preparation. This is most likely due to the better solubility of the phospholipids in L2.

[0076] Next, the size distribution of the lipid vesicles of L2 and L3 was measured in more detail by using a Zetasizer NanoS for dynamic light scattering (DLS) measurements. DLS measurements provide information on the average particle size as well as the particle size distribution. Figure 3is an intensity-based size distribution obtained by DLS measurement using a Zetasizer NanoS. This type of measurement determines the size of individual particles based on the amount of scattered light. It does not give direct information about the absolute number of particles. However, since larger particles scatter more light than smaller particles, the peak of larger particles usually dominates in the intensity-based size distribution. In cases where the peak of smaller-sized particles dominates, it indicates the presence of a large number of small particles in the solution.

[0077] DLS measurements showed that smaller liposomes <100 nm were predominantly present in lipid formulation L2, and in lipid formulation L3, and vice versa for formulation L3, predominantly present were liposomes >100 nm ( Figure 3 ). It is known in the art that for a fixed total lipid concentration, an increase in liposome size increases the likelihood of coalescence, which may be due to a decrease in liposome concentration and a decrease in relative viscosity. It is shown here that when microfluidic flow focusing is used to form microbubbles, a high concentration of small liposomes (<100 nm) is advantageous in reducing or even preventing microbubble coalescence. A high concentration of small liposomes, i.e., below 100 nm, is favorable for preventing microbubble coalescence. Additionally, since a lower temperature is required to provide formulation L2 compared to formulation L3, less energy is consumed during the preparation of lipid formulation L2 compared to L3.

[0078] Finally, optical transmission was measured using a Jenway spectrophotometer 630, which measures the amount of light (700 nm) transmitted through the lipid solution compared to a reference measurement. The reference measurement was performed on phosphate buffered saline (PBS), which is the main component of the lipid solution. The optical transmission reading of the reference sample was set to 100%. The closer the optical transmission reading is to 100%, the more comparable the transparency of the sample is to the reference measurement. An optical transmission reading below 100% indicates that the loaded sample absorbs / scatters some of the incident light. Also in this case, smaller particles scatter less, resulting in a higher optical transmission reading, and vice versa, larger particles scatter more, resulting in a lower optical transmission reading. Although the three lipid formulations L1, L2, and L3 have the same total lipid concentration of 15 mg / mL, lipid formulation L2 showed a higher optical transmission reading than lipid formulations L1 and L3 ( Figure 4 ). This means that the sizes of the lipid vesicles in the three lipid solutions are different. Additionally, this means that due to the higher transmission reading, smaller lipid vesicles are present in lipid formulation L2 compared to lipid formulations L1 and L3, which, as mentioned earlier, is favorable for preventing microbubble coalescence.

Claims

1. A method for providing a liposome solution for forming microvesicles, wherein the liposome solution is composed of a phospholipid blend of at least two phospholipids having different phase transition temperatures (Tm), the method comprising the following steps: a) Dissolving a first phospholipid having the lowest Tm in a preheated organic non-aqueous solvent at a temperature lower than the Tm of the first phospholipid to provide a first liposome solution, b) Adding a second phospholipid to the first liposome solution to obtain a liposome dispersion, or Adding the second phospholipid to a preheated organic aqueous solvent at a temperature lower than the Tm of the second phospholipid to obtain a second liposome dispersion, c) Maintaining the liposome dispersion at a temperature lower than the Tm of the first phospholipid for 30 minutes to 2 hours, preferably 45 minutes to 90 minutes, more preferably 60 minutes to 75 minutes, to obtain a further liposome solution in which the phospholipids are dissolved, preferably with stirring, or Maintaining the second liposome dispersion at a temperature lower than the Tm of the second phospholipid for 30 minutes to 2 hours, preferably 45 minutes to 90 minutes, more preferably 60 minutes to 75 minutes, to obtain a second liposome solution in which the phospholipids are dissolved, preferably with stirring, d) Adding a preheated organic aqueous solvent at a temperature lower than the Tm of the first phospholipid to the further liposome solution, preferably with stirring, or Adding the second liposome solution to the first liposome solution, preferably with stirring, To obtain a pre-final liposome solution with a ratio of organic non-aqueous solvent to organic aqueous solvent as follows: 5 vol% to 25 vol% non-aqueous: 75 vol% to 95 vol% aqueous, preferably 7.5 vol% to 20 vol% non-aqueous: 80 vol% to 92.5 vol% aqueous, even more preferably 8 vol% to 15 vol% non-aqueous: 85 vol% to 92 vol% aqueous, most preferably approximately 10 vol% non-aqueous: 90 vol% aqueous, e) Maintaining the pre-final liposome solution at a temperature lower than the Tm of the first phospholipid for 2 to 12 hours, preferably 4 to 10 hours, more preferably 6 to 8 hours, preferably with stirring, to obtain a final liposome solution.

2. The method according to claim 1, wherein the method further comprises step f) flowing and extruding or filtering the final liposome solution at a temperature lower than the Tm of the first phospholipid, thereby providing a final liposome solution for forming microvesicles, preferably at 15°C to 40°C, more preferably at room temperature of 18°C to 25°C.

3. The method according to claim 1 or 2, wherein the temperature of the preheated organic non-aqueous solvent or preheated organic aqueous solvent is at most 20% lower, more preferably at most 10% lower, more preferably at most 5% lower, most preferably at most 1% lower than the respective Tm values of the first phospholipid or the second phospholipid.

4. The method according to any one of claims 1 to 3, wherein the preheated organic non-aqueous solvent having a temperature lower than the Tm of the first phospholipid in step a and the preheated organic aqueous solvent having a temperature lower than the Tm of the first phospholipid in step d are at 20°C to 55°C, preferably 25°C to 45°C, more preferably 35°C to 39°C, and most preferably 38°C.

5. The method according to any one of claims 1 to 4, wherein the preheated organic aqueous solvent having a temperature lower than the Tm of the second phospholipid in step b is at 40°C to 60°C, preferably 45°C to 55°C, more preferably 48°C to 50°C, and most preferably 50°C.

6. The method according to any one of claims 1 to 5, wherein the addition of the preheated organic aqueous solvent or the second liposome solution in step d is carried out in two to five separate steps, preferably in three to four separate steps, and the volumes added are equally divided among these steps.

7. The method according to any one of claims 1 to 6, wherein the final liposome solution contains 60 mol% to 95 mol% of the first phospholipid, more preferably 70 mol% to 90 mol%, even more preferably 75 mol% to 87 mol%, and most preferably 80 mol% to 85 mol%.

8. The method according to any one of claims 1 to 7, wherein the final liposome solution contains 5 mol% to 40 mol% of the second phospholipid, more preferably 10 mol% to 30 mol%, even more preferably 13 mol% to 25 mol%, and most preferably 15 mol% to 20 mol%.

9. The method according to any one of claims 1 to 8, wherein the first phospholipid is selected from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), egg phosphatidylcholine (EPC), and hydrogenated soy phosphatidylcholine (HSPC), preferably DPPC or DSPC.

10. The method according to any one of claims 1 to 9, wherein the second phospholipid is a phospholipid polyethylene glycol (PEG) conjugate, preferably selected from N-(carbonyl-methoxypolyethylene glycol (750 to 10,000))-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG(750 to 10,000)), N-(carbonyl-methoxypolyethylene glycol (1,000 to 5,000))-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-mPEG(1,000 to 5,000)), preferably DPPE-mPEG2000, more preferably DSPE-mPEG5000, most preferably DPPE-mPEG5000.

11. The method according to any one of claims 1 to 10, wherein the phospholipid, more specifically the sum of the first phospholipid and the second phospholipid, is present in the final liposome solution at a concentration of 1 to 25 mg / ml, preferably 5 to 20 mg / ml, more preferably 10 to 15 mg / ml in the final liposome solution.

12. The method according to any one of claims 1 to 11, wherein the non-aqueous organic solvent is one or more selected from the following: propylene glycol, ethylene glycol, preferably propylene glycol.

13. The method according to any one of claims 1 to 12, wherein the aqueous organic solvent is one or more selected from the following: phosphate buffered saline (PBS), buffered aqueous solution, saline solution, preferably PBS.

14. The method according to any one of claims 1 to 13, wherein the method or the liposome solution does not contain glycerol or diphenylphosphoryl azide (DPPA) and toxic carcinogenic organic solvents such as chloroform.

15. A liposome solution obtained by the method according to any one of claims 1 to 14, which is used for forming microvesicles, the liposome solution being composed of a lipid blend, the lipid blend being composed of a first phospholipid and a second phospholipid having different phase transition temperatures (Tm) dissolved in a mixture of an organic non-aqueous solvent and an organic aqueous solvent, and wherein the ratio of the organic non-aqueous solvent to the organic aqueous solvent in the liposome solution is 7.5 vol% to 20 vol% non-aqueous: 80 vol% to 92.5 vol% aqueous, preferably 8 vol% to 15 vol% non-aqueous: 85 vol% to 92 vol% aqueous, more preferably about 10 vol% non-aqueous: 90 vol% aqueous.

16. The liposome solution according to claim 15, wherein the first phospholipid is selected from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), egg phosphatidylcholine (EPC), and hydrogenated soy phosphatidylcholine (HSPC), preferably DSPC, more preferably DPPC, and wherein the second phospholipid is selected from N-(carbonyl-methoxypolyethylene glycol (750 to 10,000))-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-mPEG (750 to 10,000)), N-(carbonyl-methoxypolyethylene glycol (1000 to 5000))-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-mPEG (1000 to 5000)), preferably DPPE-mPEG2000, more preferably DSPE-mPEG5k, most preferably DPPE-mPEG5000.

17. The liposome solution according to claim 15 or 16, wherein the sum of the first phospholipid and the second phospholipid is present in the final liposome solution at a concentration of 1 to 25 mg / ml, preferably 5 to 20 mg / ml, more preferably 10 to 15 mg / ml.

18. The liposome solution according to any one of claims 15 to 17, wherein the non-aqueous organic solvent is one or more selected from the following: propylene glycol, ethylene glycol, preferably propylene glycol.

19. The liposome solution according to any one of claims 15 to 18, wherein the aqueous organic solvent is one or more selected from the following: phosphate buffered saline (PBS), buffered aqueous solution, saline solution, preferably PBS.

20. The liposome solution according to any one of claims 15 to 19, wherein the liposome solution does not contain glycerol, diphenylphosphoryl azide (DPPA), and carcinogenic organic solvents such as chloroform.

21. A microvesicle composed of the liposome solution according to any one of claims 15 to 20, wherein the microvesicle is monodisperse and has a particle size of 1 to 8 μm in diameter, preferably 2 to 6 μm, more preferably 3 to 5 μm.

22. The microvesicles according to claim 21, wherein the microvesicles are drug delivery enhancers or drug-loaded microvesicles for delivering a drug to a patient, preferably for parenteral drug delivery.

23. Use of a liposome solution or microvesicles according to any one of claims 15 to 22 for delivering a drug to a patient, preferably by parenteral administration, more preferably by intravenous administration.

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