WO2017155293A2 - Procédé de production d'une structure complexe métal-liposome à l'aide d'une technique de programmation, et structure complexe métal-liposome ainsi produite - Google Patents

Procédé de production d'une structure complexe métal-liposome à l'aide d'une technique de programmation, et structure complexe métal-liposome ainsi produite Download PDF

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WO2017155293A2
WO2017155293A2 PCT/KR2017/002485 KR2017002485W WO2017155293A2 WO 2017155293 A2 WO2017155293 A2 WO 2017155293A2 KR 2017002485 W KR2017002485 W KR 2017002485W WO 2017155293 A2 WO2017155293 A2 WO 2017155293A2
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metal
liposome
complex structure
reducing agent
programmed
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Korean (ko)
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WO2017155293A3 (fr
Inventor
강태욱
이진호
신용희
황금래
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Sogang University Research Foundation
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Sogang University Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/02—Inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials

Definitions

  • the present specification relates to a method for preparing a metal-liposomal composite structure using a programming technique and a metal-liposomal composite structure prepared accordingly.
  • the metal nanostructures have the effect of amplifying the electromagnetic field around the metal nanostructures by interacting with light of a specific wavelength incident from the outside and condensing them.
  • the electromagnetic amplification effect is applied in various fields such as catalyst, molecular detection, solar energy concentrator, and especially in the biomedical field, the development of in vivo imaging, monitoring, and treatment techniques using these metal nanostructures is in many ways. It is done.
  • the preparation method may further include an organic molecule in a material supported in the liposome.
  • organic molecules may be further included in the material transferred from the external environment.
  • the programmed liposome forming step forms a programmed liposome preloaded with a reducing agent or metal precursor, and then separates the programmed liposomes so that one or more of their size or shape is controlled.
  • the programmed liposome forming step includes forming liposomes from a lipid membrane in a solution containing a reducing agent or metal precursor to form a programmed liposome having a reducing agent or metal precursor supported therein; And separating and filtering the programmed liposomes from the solution comprising the programmed liposomes to control one or more of the size or shape of the programmed liposomes.
  • the size of the liposomes may be 10 ⁇ m or less, such as 30 nm to 100 nm, or greater than 100 nm and 10 ⁇ m or less.
  • the liposome form may comprise spherical, rod-shaped, bundled.
  • the metal precursor may be a precursor of metals consisting of, for example, Ag, Au, Cu, Pt, Al, Fe, Co, Ni, Ru, Rh and Pd.
  • the reducing agent may be an organic reducing agent or an inorganic reducing agent, such as sodium citrate, hydroxyamine, ascorbic acid, sodium borohydride, and the like. can do.
  • a metal-liposomal complex structure wherein the liposome forms an outer skeleton and metal particles and a reducing agent are present therein.
  • 2a to 2d are transmission electron microscope pictures of liposomes carrying a reducing agent or a metal precursor in one embodiment of the present invention.
  • FIG. 4 is a graph showing the stability of the metal (gold) -liposomal complex structure prepared in a complex environment similar to in vivo in one embodiment of the present invention.
  • Figures 5a to 5d shows the transfer efficiency of the prepared metal (gold) -liposomal complex structure to cells in one embodiment of the present invention, the amount of the structure delivered into the cell (Fig. 5a) by measuring the scattering intensity Is a qualitatively showing image (FIG. 5B) and a quantitative graph (FIG. 5D) compared to the case of the conventional gold nanoparticles (FIG. 5D).
  • FIG. 6 shows an application technique of detecting and imaging intracellular molecules by applying the prepared metal (gold) -liposomal complex structure to cells in one embodiment of the present invention.
  • Figures 6a and 6b is a result of delivering the produced gold-liposomal complex structure into the cell (Fig. 6a) and to obtain the Raman signal of specific molecules present in the cell (Fig. 6b).
  • 6C and 6D show the results of imaging the cells based on the Raman signal of the obtained intracellular molecule (FIG. 6C) (FIG. 6D).
  • the present inventors provide an innovative approach to fabricating metal-liposomal composite structures using programming techniques.
  • the yield of the metal-liposomal complex structure is higher than that of the conventional method.
  • the metal may be stably present inside the liposome rather than between the lipid membrane of the liposome or the outer wall of the liposome, thereby preventing damage to the complex, and the size and shape of the metal-liposome complex may be uniformly obtained.
  • FIG. 1 is a schematic view showing a manufacturing process of a metal-liposomal complex structure in one embodiment of the present invention.
  • a lipid bilayer is formed on a glass substrate in a solution containing a reducing agent or a metal precursor, an organic molecule, and a liposome is formed through hydration of the lipid membrane. do.
  • a reducing agent or metal precursor and organic molecules may be supported in the liposome membrane during liposome formation (referred to as programming).
  • the lipids that form liposomes include neutral, negatively charged, and positively charged.
  • the number of carbons forming the skeleton may include up to 10-30.
  • the reducing agent can use an organic reducing agent or an inorganic reducing agent.
  • the inorganic reducing agent it is preferable to use an organic reducing agent because of its high reducing power, but there is a possibility of deformation of organic molecules.
  • the reducing agent may include sodium citrate, hydroxyamine, ascorbic acid, sodium borohydride.
  • the organic molecule may include one or more organic molecules selected from the group consisting of DNA, RNA, fluorescent material, Raman molecule, nucleic acid, protein.
  • the liposomes obtained as above may be, for example, in size within the range of 10 ⁇ m or less, or 30 nm to 100 nm or more than 100 nm and 10 ⁇ m or less. When used in vivo, those of 100 nm or less are suitable. On the other hand, when aiming to use in vitro, a micro-sized one can be used.
  • the size of the metal particles in liposomes can be, for example, 20-100 nm when used in vivo, and can be micro size when used in vitro.
  • the metal precursor or the reducing agent is further introduced into the solution containing the programmed liposome in which the reducing agent or the metal precursor and the organic molecules are carried therein.
  • a metal precursor or a reducing agent is present in the programmed liposome external environment, and thus the metal precursor or reducing agent introduced is transferred into the programmed liposome through a diffusion process.
  • the metal precursor or reducing agent diffused into the programmed liposomes causes a reduction reaction through interaction with a reducing agent or metal precursor previously supported inside the liposome. Through this reduction reaction, metal particles are synthesized.
  • the synthesis reaction of the metal particles may be selectively performed only in the liposome, and as a result, the metal-liposome complex structure in which the liposome forms the outer skeleton of the metal nanoparticles at the same time as the synthesis of the metal nanoparticles can be prepared.
  • the metal-liposomal composite structure thus obtained has the following singularities structurally as it is manufactured by the above-described manufacturing method. That is, in the obtained metal-liposomal complex structure, liposomes form an outer skeleton and metal particles and a reducing agent are present therein. According to the conventional manufacturing method, since no reducing agent is used, the reducing agent cannot be present inside the liposome. On the other hand, according to exemplary embodiments of the present invention, there is a structural singularity in which the reductant used to reduce the metal precursor and the metal precursor simultaneously with the metal particles inside the liposome forming the outer skeleton.
  • the conventional method of the embodiments of the present invention that is, not a method of binding liposomes to the surface of the metal nanoparticles prepared in advance, but a programming technique, that is, using a liposome loaded with a reducing agent or a metal precursor to synthesize the metal particles therein While the liposomes also form an external skeleton, the metal-liposomal complex structure can be controlled to a constant size and shape, and a metal-liposomal complex structure having a constant size and shape can be produced with a significantly higher yield than the conventional method. have.
  • the metal-liposomal complex structure according to the embodiments of the present invention as the metal nanoparticles are prepared, and also the liposomes form the external skeleton of the metal particles and the reducing agent, such as in vivo in which a high concentration of electrolyte or a myriad of biomolecules are present. It can provide high stability and improved intracellular delivery efficiency in complex environments.
  • the metal-liposomal complex structure can be widely used in biomedical fields such as real-time monitoring at the cellular and molecular level, imaging and customized therapy, and other catalysts and energy fields using molecular detection and photothermal effects. have.
  • lipids eg 1,2-disteraoyl-sn-glycero-3-phosphocholine (DSPC)
  • chloroform is completely evaporated using a rotary evaporator.
  • a lipid thin film was formed.
  • a reducing agent eg 300 mM trisodium citrate dehydrate
  • the lipid is dissolved while maintaining the temperature at 55-60 ° C. (critical temperature of the phospholipid), and then the reducing agent is subjected to sonication and through an extruder with filters of various sizes (30 nm, 50 nm, 100 nm, and 200 nm). Supported liposomes of constant size were prepared.
  • pellet pellet
  • liposome loaded with a reducing agent was selectively secured by repeated washing with distilled water and centrifugation.
  • FIGS. 2a to 2d are transmission electron microscope pictures of liposomes carrying a reducing agent in one embodiment of the present invention.
  • Figures 2a, 2b, 2c, 2d shows liposomes having a constant size of 30, 50, 100, 200 nm, respectively.
  • the pH was adjusted to adjust the ionization state of the metal precursor.
  • a metal precursor eg, HAuCl 4 3H 2 O, tetrachloroauric acid trihydrate
  • the neutralized metal precursor existing only at a specific pH does not destroy the phospholipid membrane and passes through the liposome. The diffusion at this time takes place in mm seconds.
  • the metal precursor may be more effectively delivered into the liposome by the osmotic effect by the difference between the salt concentration inside the liposome and the external salt concentration.
  • 3A to 3D are transmission electron micrographs of metal-liposomal composite nanostructures formed by selectively synthesizing metal nanoparticles only in liposomes in one embodiment of the present invention.
  • Figures 3a, 3b, 3c, 3d shows a metal-liposomal complex structure having a constant size of 30, 50, 100, 200 nm, respectively.
  • the metal precursor (or reducing agent in the case of pre-supporting the metal precursor) delivered into the liposome selectively reacts with the reducing agent (or metal precursor) supported only inside the liposome. Based on the liposome having a size of 100 nm, the metal precursor delivered into the liposome forms metal particles within the liposome without damaging the phospholipid membrane after 2 hours. After 12 hours of reaction, the inside of the liposome is completely filled with metal particles, thereby forming a metal-liposomal complex structure in which the liposome naturally forms an external skeleton.
  • 4A and 4B are graphs showing the stability of the metal (gold) -liposomal complex structure fabricated in a complex environment similar to in vivo in one embodiment of the present invention.
  • Figure 4a compares the stability of the previously reported spherical gold nanoparticles (GNP) and rod-shaped gold nanoparticles (GNR) and the gold-liposomal composite structure (GLN) prepared in an embodiment of the present invention under various solution conditions As a result, it can be seen that the gold-liposomal composite structure prepared in the embodiment of the present invention shows high stability in various environments compared to the previously reported gold nanoparticles.
  • 5a to 5d show the transfer efficiency of the prepared metal (gold) -liposomal complex structure to cells in one embodiment of the present invention.
  • Figure 5a is an image of the cell (U87-MG cell; control) itself
  • Figure 5c is a qualitative image showing the amount of the structure transferred into the cell in the case of conventional gold nanoparticles (GNP)
  • Figure 5b Is a qualitative image showing the amount of the structure delivered into the cell in the case of a gold-liposomal complex structure (GLN).
  • Figure 5d is a graph showing the amount of the structure delivered to the cell quantitatively measuring the scattering intensity, respectively.
  • the metal-liposomal complex structure shows higher intracellular delivery efficiency .
  • FIG. 6 shows an application technique of detecting and imaging intracellular molecules by applying the prepared metal (gold) -liposomal complex structure to cells in one embodiment of the present invention.
  • Figures 6a and 6b is a result of delivering the prepared gold-liposomal complex structure (GLN) into the cell (Fig. 6a) and to obtain the Raman signal of specific molecules present in the cell (Fig. 6b).
  • 6b also shows Raman signals obtained using conventional spherical gold nanoparticles (GNS).
  • GLS conventional spherical gold nanoparticles
  • 6C and 6D show results of imaging cells based on Raman signals of intracellular molecules (FIG. 6C) obtained in the case of the gold-liposomal complex structure (GLN) (FIG. 6D).
  • the liposome forming the external skeleton serves as a protective film, so that a high concentration of electrolyte or a large number of biomolecules exist in vivo. It has high stability even in complex environment such as In addition, through this it is possible to smoothly perform the original function by maintaining a constant size and shape.
  • Such metal-liposomal complex structures can be utilized in various fields such as biomedical and catalysts, and energy.

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Abstract

La présente invention concerne une structure complexe métal-liposome dans laquelle un liposome forme le squelette externe d'une particule métallique et qui peut être produite de la manière suivante: un liposome dans lequel un agent réducteur ou un précurseur métallique est transporté à l'avance est préparé, le précurseur métallique ou l'agent réducteur est fourni à l'extérieur du liposome pour être diffusé dans le liposome, et une particule métallique est synthétisée sélectivement à l'intérieur du liposome. Par conséquent, une structure complexe métal-liposome ayant une taille uniforme et une forme uniforme peut être produite à un rendement élevé.
PCT/KR2017/002485 2016-03-08 2017-03-08 Procédé de production d'une structure complexe métal-liposome à l'aide d'une technique de programmation, et structure complexe métal-liposome ainsi produite Ceased WO2017155293A2 (fr)

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KR10-2016-0027791 2016-03-08
KR1020160027791A KR101852153B1 (ko) 2016-03-08 2016-03-08 프로그래밍기법을 이용한 금속-리포좀 복합 구조체 제조 방법 및 이에 따라 제조된 금속-리포좀 복합 구조체

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WO2017155293A3 WO2017155293A3 (fr) 2018-08-02

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US5560960A (en) * 1994-11-04 1996-10-01 The United States Of America As Represented By The Secretary Of The Navy Polymerized phospholipid membrane mediated synthesis of metal nanoparticles
DE19721601A1 (de) * 1997-05-23 1998-11-26 Hoechst Ag Polybetain-stabilisierte, Palladium-haltige Nanopartikel, ein Verfahren zu ihrer Herstellung sowie daraus hergestellte Katalysatoren zur Gewinnung von Vinylacetat
AU2003224981A1 (en) * 2002-04-25 2003-11-10 General Electric Company Preparation of nanosized copper (i) compounds
JP5429710B2 (ja) * 2009-03-30 2014-02-26 独立行政法人放射線医学総合研究所 治療薬剤の標的部位への集積及び放出を追跡可能な治療薬剤含有リポソームおよびその製造方法
KR101174470B1 (ko) * 2010-04-08 2012-08-16 한국세라믹기술원 자성 실리카 리포좀 나노입자 및 이의 제조방법

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KR101852153B1 (ko) 2018-04-25
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