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KR-20260066058-A - pH-inducible structure-converting lipid nanovectors, semi-synthetic extracellular vesicles, methods for preparing the same, and uses thereof

KR20260066058AKR 20260066058 AKR20260066058 AKR 20260066058AKR-20260066058-A

Abstract

The present disclosure provides a pH-induced structure-converting non-lamellar lipid nanovector (LNV) comprising (a) at least one ionizable cationic lipid; (b) at least one phospholipid having a critical packing parameter (CPP) value greater than 1; and (c) at least one nonionic surfactant having a CPP value less than 1 with a molar concentration of 20% to 50%, a method for preparing said LNV, a semisynthetic extracellular vesicle (ssEV) produced from the fusion of the LNV and an extracellular vesicle at a pH greater than 6 and about 10 or less, a kit, and a use of the ssEV as a pharmaceutical or diagnostic agent.

Inventors

  • 르루, 장-크리스토프
  • 몬타나리, 엘리타
  • 바더, 요하네스

Assignees

  • 에테하 쭈리히

Dates

Publication Date
20260512
Application Date
20240903
Priority Date
20230904

Claims (15)

  1. As a pH-induced structure-converting non-lamellar lipid nanovector (LNV), (a) at least one ionizable cationic lipid; (b) at least one phospholipid having a critical packing parameter (CPP) value greater than 1; and (c) At least one nonionic surfactant having a molar concentration of 20% to 50% and a CPP value of less than 1 LNV including
  2. In paragraph 1, (a) At least one ionizable cationic lipid has an apparent acid dissociation constant (pKa) of 5 to 7.5 before being incorporated into the LNV; (b) At least one phospholipid is a phosphatidylethanolamine (PE)-based lipid; (c) At least one surfactant is a polyethylene glycol ester or polysorbate of a fatty acid; or (d) LNV, which is a combination of at least two of (a) to (c).
  3. In paragraph 1 or 2, (a') At least one ionizable cationic lipid is D-Lin-MC3-DMA(MC3); (b') At least one phospholipid is 2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE); (c') At least one nonionic surfactant is a polyethylene glycol ester of a fatty acid, such as polyethylene glycol 12-hydroxystearate, preferably Kolliphor®HS15(KLP); or LNV, which is a combination of at least two of (d') (a') to (c').
  4. An LNV according to any one of claims 1 to 3, wherein the LNV is loaded with a drug or diagnostic agent, preferably the drug or diagnostic agent is a nucleic acid molecule.
  5. A composition comprising an LNV defined in any one of claims 1 to 4, and at least one pharmaceutically acceptable excipient.
  6. A method for producing an LNV defined in any one of claims 1 to 3, comprising the step of mixing (a) an organic phase comprising at least one ionizable cationic lipid, at least one phospholipid, and at least one surfactant in an organic solvent; and (b) an aqueous medium.
  7. In claim 6, (i) the organic phase further comprises a lipid-soluble drug or diagnostic agent; and (ii) the aqueous phase further comprises a water-soluble drug or diagnostic agent, preferably wherein the water-soluble drug or diagnostic agent is a nucleic acid molecule A method that is at least one of the following.
  8. In claim 7, (ii) the aqueous phase further comprises a water-soluble drug or diagnostic agent, and the pH of the aqueous medium is about 4 to about 6, method.
  9. A method according to any one of claims 6 through 8, wherein the mixing is performed using a microfluidic device.
  10. A method for producing semi-synthetic extracellular vesicles (ssEVs), comprising the step of mixing an extracellular vesicle (EV) with an LNV defined in any one of claims 1 to 4 or a composition defined in claim 5 at a pH greater than 6 and about 10 or less, thereby producing ssEVs.
  11. Semi-synthetic extracellular vesicles (ssEVs) manufactured by the method defined in paragraph 10.
  12. Essentially, a semisynthetic extracellular vesicle (ssEV) comprising all of the following components in part or completely mixed: (a) LNV defined in any one of paragraphs 1 through 4; and (b) Extracellular vesicle (EV).
  13. A composition comprising ssEV as defined in claim 11 or 12, and at least one pharmaceutically acceptable excipient.
  14. (A) (a) a composition defined in any one of claims 1 to 4 or in claim 5; and (b) (i) a solution for hydrating (a); (ii) a composition comprising an extracellular vesicle (EV) or an EV and at least one pharmaceutically acceptable excipient; (iii) instructions for using at least one of (a) and (i) and (ii); or (iv) a combination of at least two of (i) to (iii); or (B) (a') an ssEV defined in claim 11 or 12, or a composition defined in claim 13; and (b') (i') a solution for hydrating (a'); (ii') instructions for using (a') and (i'); or (iii') a kit comprising a combination of (i') and (ii').
  15. Use of the ssEV defined in paragraph 11 or 12 or the composition defined in paragraph 13 as a pharmaceutical or diagnostic agent.

Description

pH-inducible structure-converting lipid nanovectors, semi-synthetic extracellular vesicles, methods for preparing the same, and uses thereof The present disclosure relates to pH-inducible structure-converting lipid nanovectors, semi-synthetic extracellular vesicles, methods for preparing the same, and uses. More specifically, the present disclosure relates to lipid nanovectors having the ability to spontaneously fuse with extracellular vesicles. Extracellular vesicles (EVs) are cell-derived vesicle structures that serve as important intercellular communicators for transporting macromolecules, such as messenger ribonucleic acid (mRNA) or proteins, through the extracellular space (O'Brien et al., 2020). Encapsulating therapeutic nucleic acids, such as short interfering RNA (siRNA) and mRNA, in large quantities within EVs remains a challenging task, and versatile approaches to achieve this are still lacking (De Jong et al., 2019). Active loading strategies generally involve disturbing the vesicle bilayer to allow for drug encapsulation (De Jong et al., 2019). These approaches are particularly relevant for loading relatively high molecular weight drugs into EVs. For example, electroporation is widely used for nucleic acid-based therapeutics (Alvarez-Erviti et al., 2011). However, it is not fully known how destructive this method is and whether biological cargo is lost during the loading process. Similarly, sonication has been used as a method to load biological materials into EVs by temporarily destabilizing the membrane (Lamichhane et al., 2016). A simpler passive method is needed to load macromolecules (e.g., therapeutic macromolecules) into an EV without impairing EV function. This specification refers to numerous documents, the contents of which are incorporated herein by reference in their entirety. The present disclosure provides lipid nanostructures (i.e., lipid nanovectors (LNVs)) capable of spontaneously hybridizing/fusion with EVs. This passive process does not require additional external stimuli (i.e., sonication, electroporation, specific buffer components) and occurs within minutes under very mild conditions (e.g., physiological conditions of pH 7.4 and 37°C). LNVs promote a fusion stalk within EVs to generate semi-synthetic extracellular vesicle (ssEV) particles without impairing the biological activity of EVs. Various molecular and/or macromolecular therapeutic agents can be loaded onto these versatile particles. The present disclosure also provides a method for manufacturing LNV and ssEV. Although not limited thereto, it is assumed that the unique fusogenic property of the LNVs of the present disclosure is based on their non-lamellar lyotropic liquid crystal (LLC)-based structures (e.g., sponge-based structures). More specifically, the following items are provided in accordance with the present disclosure: Item 1. As a pH-inducible structure-converting non-lamellar lipid nanovector (LNV), (a) at least one ionizable cationic lipid; (b) at least one phospholipid having a critical packing parameter (CPP) value greater than 1; and (c) LNV comprising at least one nonionic surfactant having a CPP value of less than 1 and a molar concentration of 20% to 50%. Item 2. Regarding the LNV of Item 1, (a) At least one ionizable cationic lipid has an apparent acid dissociation constant (pKa) of 5 to 7.5 before being incorporated into the LNV; (b) At least one phospholipid is a phosphatidylethanolamine (PE)-based lipid; (c) At least one surfactant is a polyethylene glycol ester or polysorbate of a fatty acid; or (d) LNV, which is a combination of at least two of (a) to (c). Item 3. In the case of the LNV of Item 1 or 2, (a') At least one ionizable cationic lipid is D-Lin-MC3-DMA(MC3); (b') At least one phospholipid is 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (c') At least one nonionic surfactant is a polyethylene glycol ester of a fatty acid, such as polyethylene glycol 12-hydroxystearate, preferably Kolliphor® HS15(KLP); or LNV, which is a combination of at least two of (d') (a') to (c'). Item 4. An LNV loaded with a drug or diagnostic agent in any one of Items 1 to 3, wherein the drug or diagnostic agent is preferably a nucleic acid molecule. Item 5. A composition comprising an LNV defined in any one of Items 1 to 4, and at least one pharmaceutically acceptable excipient. Item 6. A method for preparing an LNV defined in any one of Items 1 to 3, comprising the step of mixing (a) an organic phase comprising at least one ionizable cationic lipid, at least one phospholipid, and at least one surfactant in an organic solvent; and (b) an aqueous medium. Item 7. The method of Item 6, wherein (i) the organic phase further comprises a lipid-soluble drug or diagnostic agent; and (ii) the aqueous phase further comprises a water-soluble drug or diagnostic agent, preferably wherein the water-soluble drug or diagnostic agent is at least one of a nucleic acid molecule. Item 8. The method of Item 7, wherein (ii) th