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US-12616658-B2 - Method for preparation of liposomes

US12616658B2US 12616658 B2US12616658 B2US 12616658B2US-12616658-B2

Abstract

Disclosed are methods of preparing liposomes without size exclusion steps resulting in small sized liposomes exhibiting narrow size distribution and compositions of same. The methods utilize liposome formation from lyso-PS, PS, and PC as the only phospholipids.

Inventors

  • Sathy V. Balu-Iyer
  • Nhan Hanh NGUYEN
  • Dominique WEEKS
  • Vincent CHAK
  • Milagros Riquelme Gonzalez

Assignees

  • THE RESEARCH FOUNDATION FOR THE STATE UNIVERSITY OF NEW YORK

Dates

Publication Date
20260505
Application Date
20210701

Claims (15)

  1. 1 . A method of preparing liposomes having an average diameter of less than 300 nm, wherein at least 95% of the particles have a diameter of 200-275 nm, comprising: mixing a plurality of lipids comprising phosphatidylcholine (PC) and lysophosphatidylserine (lyso-PS), wherein the ratio of PC to lyso-PS is from 90:10 to 60:40, incubating the plurality of lipids for 30 seconds to 5 minutes, and vortexing the mixture, wherein the liposomes formed have an average diameter of less than 300 nm and 95% of the particles have a diameter of 200-275 nm, and wherein the method does not comprise an extrusion step.
  2. 2 . The method of claim 1 , wherein the ratio of PC to lyso-PS is from 85:15 to 70:30.
  3. 3 . The method of claim 1 , wherein the PC is present as dimyristoyl-sn-glycero-3 phosphatidylcholine (DMPC).
  4. 4 . The method of claim 1 , wherein PC and lyso-PS are the only phospholipids present in the bilayer of the liposomes.
  5. 5 . The method of claim 1 , wherein the lyso-PS is 18:1.
  6. 6 . The method of claim 1 , wherein the lyso-PS is L-lyso-PS.
  7. 7 . The method of claim 1 , wherein the vortexing is done from 30 seconds to 3 minutes.
  8. 8 . The method of claim 1 , further comprising trigger loading a protein into the liposome.
  9. 9 . The method of claim 8 , wherein the protein is loaded into a liposomal bilayer.
  10. 10 . The method of claim 8 , wherein the conformation of the protein is altered by heating the protein to about 37° C.
  11. 11 . The method of claim 10 , wherein the protein is chosen from AAV proteins, collagens, aquaporin 4, cas proteins, insulin, Factor VIII, Adalimumab, and alpha glucosidase (GAA).
  12. 12 . The method of claim 1 , wherein the mixing comprises contacting the plurality of lipids with a buffered aqueous solution.
  13. 13 . The method of claim 12 , wherein prior to the mixing, each lipid of the plurality of lipids are thin lipid films.
  14. 14 . The method of claim 13 , wherein the thin lipid films are formed from allowing a solution to dry, wherein the solution comprises an organic solvent and one or more lipids.
  15. 15 . The method of claim 14 , wherein the organic solvent is chosen from ethanol, methanol, and isopropyl ether.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. provisional patent application No. 63/047,068, filed on Jul. 1, 2020, the disclosure of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under contract no. HL070227 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE DISCLOSURE A liposome is a closed structure made of a lipid bilayer. The bilayer encloses an aqueous phase (inner water phase). Current methods of preparation of liposomes generally start with an emulsification step where an oil phase containing lipids dissolved in an organic solvent is mixed with a water phase and stirred. The organic solvent is then evaporated leaving an aqueous solution containing liposomes. The liposomes prepared by such a process, however, are a heterogeneous size population, and generally large (1 micron and above). To reduce the size where smaller liposomes are desired, filtration or extrusion steps are carried out. However, filtration and extrusion can cause lipid loss affecting the loading efficiency of the liposomes. Moreover, the extrusion step poses challenges during manufacturing process and scaling up is not easily achieved. SUMMARY OF THE DISCLOSURE The present disclosure provides compositions and methods for preparing liposomes without size exclusion steps, but nevertheless generating small-sized liposomes exhibiting narrow size distribution that is comparable to methods using size exclusion steps. The elimination of size exclusion steps is achieved by using lyso-PS. Avoiding a size-exclusion step results in reduced lipid loss and is advantageous for the manufacturing process. BRIEF DESCRIPTION OF THE FIGURES AND TABLES FIG. 1 shows exposure of PS as a function of lipid concentration. FIG. 2 shows a schematic representation of liposomes composed of PC alone, PC containing double chain PS and PC containing lyso-PS. DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure provides methods for preparing liposomal compositions that are suitable for delivery of protein/peptides such that induction of immune tolerance is achieved. The method of preparation of liposomes uses materials such that no size exclusion step is required during the entire process and yet small-sized, uniform distribution liposome population can be obtained. Elimination of a size exclusion step is advantageous in the manufacturing process for preparation of liposomal compositions. Throughout this application, the use of the singular form encompasses the plural form and vice versa. For example, “a”, or “an” also includes a plurality of the referenced items, unless otherwise indicated. Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. By “lipidic structures” is meant liposomes and other structures such as, for example, micellar structures, liposomes, cochleates, molecular assemblies, and the like. The term “lyso” when used herein in conjunction with a phospholipid means that the glycerol part of the molecule has only one acyl chain instead of two. For example, lyso-PS has only one acyl chain whereas PS has two acyl chains. “Liposomes” may be referred to herein as lipidic nanoparticles, or nanoparticles. The liposomes may comprise PC and PS, where some or all PS is in the form of lyso-PS. The liposomes may contain PS, lyso-PS, and PC as the only phospholipids. The liposomes may contain lyso-PS and PC as the only phospholipids. The PS or lyso-PS may be in a range of from 10% to 30% of the total phospholipids in the bilayer with the remaining phospholipids being mainly PC or only PC. For example, the lyso-PS can be from 10 to 50%, or 15 to 50%, or 15 to 30%, with the remaining phospholipids being PC. The liposomes may have a ratio of PC:lyso-PS as 90:10, 80:20, 70:30, 60:40, or 50:50 molar ratios. Only the PS (some or all) is in the form of lyso-PS, while all of PC has two acyl chains. In an embodiment, phosphatidylethanolamine (PE) may be added. PE may be added at the expense of PC up to 20 mol %. In various examples, the liposomes do not contain any PG. The phospholipids for preparing the liposomes can be obtained from any available source such as plant or animal. The phospholipids are commercially available or can be synthesized by known methods. For example, PS can be obtained from porcine brain PS or plant-based soy (e.g., soya bean) PS. Lyso-PS is also available commercially. PC may have from 14 to 20 carbons (and all integer number of carbons and ranges therebetween (e.g., 14, 15, 16, 17, 18, 19, or 20)). In various examples, the PC is not be lyso-PC. The acyl chain for lyso-PS may have from 16 to 20 carbons (e.g., 16, 17, 18, 19, or 20). It should have at least one double bond.