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JP-7856800-B2 - Method for synthesizing apolipoprotein-containing hybrid nanoparticles

JP7856800B2JP 7856800 B2JP7856800 B2JP 7856800B2JP-7856800-B2

Inventors

  • ジョン，ミ ヨン
  • リー，ウ スン
  • キム，セ ヨン
  • キム，ヨン テ

Assignees

  • メプスジェン カンパニー リミテッド

Dates

Publication Date
20260511
Application Date
20230613
Priority Date
20220613

Claims (10)

  1. In a vortex micro-vortex device having a first inlet, a second inlet, and an outlet, the steps include injecting phospholipids into the first inlet and injecting polymers into the second inlet, The steps include mixing phospholipids and polymers using swirling microvortices, In a vortex microvortex device, the steps include injecting hybrid nanoparticles into the first injection port and injecting protein into the second injection port, The process includes a step in which proteins are added to hybrid nanoparticles by a swirling microvortex , The phospholipids are DPPC and DSPE-PEG, and the polymer is PLGA (poly(lactic acid/glycolic acid copolymer)), characterized in that A method for producing protein-containing hybrid nanoparticles.
  2. A method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the Reynolds number in the vortex micro-vortex device is 50 to 300.
  3. The method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the protein is an apolipoprotein or a polymer having amphoteric properties.
  4. The method for producing protein-containing hybrid nanoparticles according to claim 1, characterized in that the apolipoprotein is at least one selected from the group consisting of apolipoproteins A1, A2, E2, E3, J, and M.
  5. A method for producing protein-containing hybrid nanoparticles according to claim 3, characterized in that the synthetic weight ratio of the hybrid nanoparticles to the apolipoprotein is 20:1 to 0.5:1.
  6. A method for producing protein-containing hybrid nanoparticles according to claim 3, characterized in that the synthetic weight ratio of the hybrid nanoparticles to the apolipoprotein is 20:1 to 2:1.
  7. A method for producing protein-containing hybrid nanoparticles according to claim 1, further comprising the step of recovering protein-containing nanoparticles from the aforementioned outlet.
  8. A method for producing protein-containing hybrid nanoparticles according to claim 1 , characterized in that the molecular weight of PEG in the DSPE-PEG is 2000 to 5000.
  9. A method for producing protein-containing hybrid nanoparticles according to claim 1 , characterized in that the synthetic blending weight ratio of DPPC and DSPE-PEG is 2.3:1 to 1:1.
  10. A method for producing protein-containing hybrid nanoparticles according to claim 1 , characterized in that the hybrid nanoparticles are polymer-lipid hybrid nanoparticles (PHNPs).

Description

This invention relates to a method for synthesizing apolipoprotein-containing hybrid nanoparticles. Specifically, this invention relates to a method for synthesizing hybrid nanoparticles containing various functional proteins, including apolipoproteins, using a vortex micro-vortex flow apparatus. The manufacturing method of this invention allows for the production of small, uniform, and stable nanoparticles with diverse physiological activities. Nanoparticles refer to particles with a diameter of micrometers or less. Due to their small size, they can move freely within the body and can be imparted various physical properties depending on their constituent materials, making them a promising next-generation drug carrier. Drug delivery nanoparticles are composed of various components, such as phospholipids and polymers, as needed, and possess different properties depending on their composition. Nanoparticles made of inorganic materials are primarily used for diagnostic purposes, while polymer nanoparticles are mainly used as drug carriers to regulate drug release rates and in vivo circulation time. Phospholipid nanoparticles are amphiphilic, easily self-assembled, and possess various molecular sieves; therefore, various types of phospholipid nanoparticles are being studied as drug carriers. In recent years, hybrid nanoparticles, which combine the advantages of such nanoparticles, have been studied. However, conventional nanoparticle manufacturing methods mainly consist of unstandardized multi-step processes such as nanoprecipitation and emulsion-based solvent evaporation. Therefore, mass production of hybrid nanoparticles with uniform particle size using conventional methods presents many difficulties. In particular, when macromolecules such as functional proteins are included in nanoparticles, their synthesis is complex, making it difficult to synthesize them in a single step. Manufacturing involves many processes, leading to a loss of uniformity and stability, and posing many challenges in mass production. The inventors of this invention have researched a synthesis method that allows functional proteins to be incorporated into polymer-lipid hybrid nanoparticles (PHNPs) using PLGA (poly(lactic-co-glycolic acid)), a polymer that is biodegradable and biocompatible and can protect drugs from degradation, and have completed the present invention. Toth et al. , Robust manufacturing of lipid-polymer nanoparticles through feedback control of parallelized swirling microvortices. 2017, Lab. Chip. , 17.16: 2805-2813.Manon, R. , et al. Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer, Scientific reports, 12:9483 (2022)Bekard, I. , et al. The Effects of Shear Flow on Protein Structure and Function. Biopolymers, 95, 11, 733-745 (2011) This is a schematic diagram showing the synthesis process of hybrid nanoparticles (PHNP-E3/A1) containing both ApoE3 and ApoA1, and the morphology of PHNP-E3/A1.This graph compares the particle size of hybrid nanoparticles (PHNP-2000) at various synthetic weight ratios of DSPE-PEG2000 to DPPC.Figure 2 shows TEM images of hybrid nanoparticles (PHNP-2000) at various synthetic weight ratios of DSPE-PEG2000 to DPPC.This graph compares the particle size of hybrid nanoparticles (PHNPs) at different PEG molecular weights for DSPE-PEG.Figure 4 shows TEM images of hybrid nanoparticles (PHNPs) at various PEG molecular weights in DSPE-PEG.Figure 4 shows a graph illustrating the stability of each hybrid nanoparticle (PHNP) synthesized in the diagram, measured on the day of synthesis and three days after synthesis.This is a schematic diagram showing the synthesis process of ApoE3-containing hybrid nanoparticles (PHNP-E3) using a vortex micro-vortex flow apparatus, and the morphology of PHNP-E3.This graph compares the particle sizes of PHNP-PEG2000 and PHNP-E3.This is a TEM image of PHNP-E3.This graph compares the particle sizes of PHNP-E3 synthesized under various Reynolds number conditions.This graph shows the yield of ApoE3 contained in PHNP-E3.This is a schematic diagram showing the synthesis process of ApoA1-containing hybrid nanoparticles (PHNP-A1) and the morphology of the hybrid nanoparticles (PHNP-A1).This graph compares the particle sizes of PHNP-PEG2000 and PHNP-A1.This is a TEM image of PHNP-A1.This is a fluorescence image of rhodamine-labeled hybrid nanoparticles (PHNP-PEG2000) delivered into HAEC cells.This is a fluorescence image of rhodamine-labeled hybrid nanoparticles (PHNP-A1) delivered into HAEC cells.Figures 15 and 16 are graphs comparing the quantified fluorescence intensity of rhodamine.This graph compares the particle size and polydispersity index (PDI) of synthesized PHNP-A1 (SMR) and PHNP-A1 (BT) with and without a vortex micro-vortex device.This graph compares the yields of ApoA1 produced by synthesizing PHNP-A1 (SMR) and PHNP-A1 (BT) at various synthesis weight ratios of PHNP to ApoA1, and then using ELISA.This graph compares the yields of ApoA1 produced