WO2003057191A1 - Encapsulation efficace dans des liposomes - Google Patents

Encapsulation efficace dans des liposomes Download PDF

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Publication number
WO2003057191A1
WO2003057191A1 PCT/US2002/041846 US0241846W WO03057191A1 WO 2003057191 A1 WO2003057191 A1 WO 2003057191A1 US 0241846 W US0241846 W US 0241846W WO 03057191 A1 WO03057191 A1 WO 03057191A1
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gel
liquid containing
aqueous medium
gel particles
weight
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PCT/US2002/041846
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English (en)
Inventor
Xingong Li
Shangguan Tong
Alla Polozova
Paul R. Meers
Walter R. Perkins
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Elan Pharmaceuticals, Inc.
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Priority to AU2002367321A priority Critical patent/AU2002367321A1/en
Publication of WO2003057191A1 publication Critical patent/WO2003057191A1/fr

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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—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes

Definitions

  • This invention concerns liposomes, methods of preparing liposomes, especially liposomes containing a biologically active substance encapsulated therein, and methods of using the liposomes containing the biologically active substance.
  • the methods of preparing the liposomes of the present invention have the advantages of being simple and able to generate primarily small liposomes of relatively homogeneous particle size with a high entrapment efficiency.
  • Liposomes are lipid vesicles having at least one aqueous phase completely enclosed by at least one lipid bilayer membrane. Liposomes can be unilamellar or multilamellar. Unilamellar liposomes are liposomes having a single lipid bilayer membrane. Multilamellar liposomes have more than one lipid bilayer membrane with each lipid bilayer membrane separated from the adjacent lipid bilayer membrane by an aqueous layer. The cross sectional view of multilamellar vesicles is often characterized by an onion-like structure.
  • Liposomes are known to be useful in drug delivery, so many studies have been conducted on the methods of liposome preparation. Descriptions of these methods can be found in numerous reviews (e.g. , Szoka et al. , "Liposomes: Preparation and Characterization", in Liposomes: From Physical Structure to Therapeutic Applications , edited by Knight, pp. 51-82, 1981; Deamer et al., “Liposome Preparation: Methods and Mechanisms", in Liposomes ', edited by Ostro, pp. 27-51, 1987; Perkins, “Applications of Liposomes with High Captured Volume", in Liposomes Rational Design, edited by Janoff, pp. 219-259, 1999).
  • SUVs Small unilamellar vesicles
  • Huang, Biochemistry 8:346-352, 1969 A phospholipid was dissolved in an organic solvent to form a solution, which was dried under nitrogen to remove the solvent.
  • An aqueous phase was added to produce a suspension of vesicles.
  • the suspension was sonicated until a clear liquid was obtained, which contained a dispersion of SUVs.
  • Other methods for the preparation of liposomes were discovered in the
  • Liposomes produced by the solvent-infusion method were mostly unilamellar.
  • the organic solvent was then removed from the mixture in a two-step procedure, in which the mixture was evaporated at 200-400 mm Hg until the emulsion became a gel, which was then evaporated at 700 mm Hg to remove all the solvent allowing the micelles to coalesce to form a homogeneous dispersion of mainly unilamellar vesicles known as reverse-phase evaporation vesicles (hereinafter referred to as REVs) (e.g. , see Papahaduopoulos, U.S. Patent No. 4,235,871).
  • REVs reverse-phase evaporation vesicles
  • Minchey et al. (U.S. Patent No. 5,415,867) described a modification of the method of Fountain et al.
  • a phospholipid, a water-miscible organic solvent, an aqueous phase and a biologically active agent were mixed to form a cloudy mixture.
  • the solvents in the mixture were evaporated, but not to substantial dryness, under a stream of air in a warm water bath at 37°C until the mixture formed a monophase, i.e., a clear liquid.
  • the mixture became opaque and gelatinous, in which the gel state indicated that the mixture was hydrated.
  • the purging was continued for 5 minutes to further remove the organic solvent.
  • Figure 1 shows, under a light microscope (magnification 400X), NAPE/DOPC (in a 70/30 molar ratio, with a volume ratio of aqueous phase: ethanol of 2: 1) liposomes prepared according to the method of the present invention before (top panel) and after (bottom panel) extrusion through a membrane filter having a 0.4 ⁇ m pore size.
  • Figure 2 depicts the appearance of NAPE/DOPC (70/30) liposomes prepared according to the method of the present invention under freeze-fracture electron microscopy.
  • Figure 5 shows the results of fractionation of NAPE/DOPC liposomes prepared according to the method of the present invention in a 5-20% sucrose gradient.
  • the lipids were homogeneously distributed with no phase separation.
  • the liposomes in the peak fractions had entrapment of 2.1 +/- 0.2 ⁇ l/ ⁇ mol of lipids.
  • the open squares, labeled "p/pc" represented the phosphate to choline weight ratios, as determined by the respective assays, of the fractions separated by the sucrose gradient.
  • Figure 11 shows the transferrin mediated binding of N-C12-DOPE/DOPC (70/30) liposomes prepared by the gel-hydration method of the present invention using ethanol as the water-miscible organic solvent (see Example 13). The binding experiment was conducted in the presence of 10% FBS.
  • Figure 14 shows the encapsulation efficiencies, for dextran fluorophores, of NAPE/DOPC (70/30) liposomes prepared using the gel hydration method of the present invention or using a process for making stable plurilamellar vesicles (SPLV).
  • the NAPE/DOPC liposomes prepared according to the gel-hydration method of the present invention had a much higher encapsulation efficiency than the NAPE/DOPC liposomes prepared using the SPLV process.
  • the methods of preparing liposomes of the present invention involve hydration of a mixture of at least one liposome-forming lipid and a water-miscible organic solvent in the form of a gel or a liquid containing gel particles.
  • the lipid is typically dissolved in the water-miscible organic solvent, preferably at a high concentration.
  • the mixture is mixed with, typically a small amount of, an aqueous medium to form the gel or the liquid containing gel particles. Hydration of the gel leads to formation of liposomes without any additional manipulation, such as evaporation or sonication, normally required in prior art methods.
  • the gel or gel particles may go through a wax stage before forming liposomes, but no additional manipulation, such as evaporation or sonication, is required other than hydration of the gel or the gel particles in the liquid followed by hydration of a waxy substance formed from the hydration of the gel or gel particles.
  • the gel or gel particles go through the wax stage upon hydration before liposome formation.
  • step (B) (i) mixing the mixture of step (I)(B) with aqueous medium Y and the biologically active substance to form a clear gel or a liquid containing clear gel particles;
  • step (III) of the general gel hydration method of the present invention or step (III) of the method of preparing the liposomes containing the at least one biologically active substance encapsulated therein means that the liposomes are formed without requiring any additional procedure or manipulation, such as evaporation or sonication, other than going through an optional intermediate stage of formation of a waxy substance if certain liposome-forming lipids are used.
  • the aqueous solution can be added to the mixture of the at least one liposome-forming lipid and the water-miscible organic solvent at an appropriate volume ratio to create a gel or a liquid containing gel particles.
  • the aqueous medium Y, aqueous medium Zl and/or aqueous medium Z2 is preferably an aqueous buffer.
  • the "liposome-forming lipid” is any lipid that is capable of forming liposomes.
  • the “liposome-forming lipid” is a lipid that can form lipid bilayers.
  • the liposome-forming lipid include phospholipids, glycolipids and sphingolipids.
  • the phospholipids that are liposome-forming include phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and N-acyl phospatidylethanolamine.
  • the liposome-forming phospholipid examples include phospholipids selected from the group consisting of dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-oleoyl- 2-palmitoy l-sn-glycero-3 -phosphocholine , 1 , 2-dioleoy l-sn-glycero-3- [phospho-rac- (1-glycerol)] , 1 ,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] , 1 ,2- distearoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] , 1 ,2-dimy
  • the at least one charged lipid can be added in step (I) to form the mixture, or added in step (II) in which the gel or the liquid containing gel particles is formed.
  • the at least one charged lipid is the same or different from the at least one liposome-forming lipid.
  • the "charged lipid” is a lipid having a net negative or positive charge in the molecule. Examples of the charged lipid include N-acyl phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol (i.e., cardiolipin) and phosphatidic acid.
  • the liposomes prepared by any of the methods of the present invention preferably comprises at least one fusogenic lipid.
  • the mixture of step (I) in the methods of the present invention preferably comprises the at least one fusogenic lipid.
  • the at least one liposome-forming lipid is also a fusogenic lipid.
  • the at least one liposome-forming lipid is a N-acyl phosphatidylethanolamine
  • the N-acyl phosphatidylethanolamine is liposome- forming and also increases the fusogenicity of the liposomes (see Meers et al, U.S. Patent No. 6,120,797, the disclosure of which is herein incorporated by reference).
  • N-acyl phosphatidylethanolamine that can be used include 1,2- dioleoyl-sn-glycero-N-decanoyl-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero- N-dodecanoy 1-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-N-tetradecanoyl-3- phosphoethanolamine , 1 ,2-dipalmitoy l-sn-glycero-N-decanoyl-3 - phosphoethanolamine, 1 ,2-dipalmitoyl-sn-glycero-N-dodecanoyl-3- phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-N-tetradecanoyl-3- phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-N-tetradecanoyl-3
  • the mixture of step (I) of the method of the present invention can further comprise a sterol.
  • the sterol is cholesterol.
  • the water-miscible organic solvent is an organic solvent that, when mixed with water, forms a homogeneous liquid, i.e., with one phase.
  • the water-miscible organic solvent can be selected from the group consisting of acetaldehyde, acetone, acetonitrile, allyl alcohol, allylamine, 2-amino-l-butanol, 1-aminoethanol, 2-aminoethanol, 2-amino-2-ethyl-l,3- propanediol, 2-amino-2-methyl-l-propanol, 3-aminopentane, N-(3- aminopropyl)morpholine, benzylamine, bis(2-ethoxy ethyl) ether, bis(2- hydroxyethyl) ether, bis(2-hydropropyl) ether, bis(2-methoxy ethyl) ether, 2- bromoethanol, meso-2,3-butanedi
  • Acetonitrile, C r C 3 alcohols and acetone are preferred examples of the water- miscible organic solvent.
  • the C r C 3 alcohols are preferably methanol, ethanol, 1- propanol, 2-propanol, ethylene glycol and propylene glycol, and more preferably ethanol, 1-propanol or 2-propanol, with ethanol being the most preferred.
  • an organic solvent such as ethanol or acetone, of relatively low toxicity can be used.
  • the liposomes prepared according to the method of the present invention would not be expected to pose any significant toxicity threat even when the liposomes contain a residual amount of the water-miscible organic solvent.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) can also be from about 5% to about 80%, about 10% to about 80%, about 15% to about 80%, about 20% to about 80% , about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80% , about 70% to about 80%, about 10% to about 70%, about 20% to about 60% , or about 30% to about 50% by weight of the gel or the liquid containing gel particles.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) ranges from about 60% to about 90%, or is about 45%, by weight of gel or the liquid containing gel particles.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) can range from about 1 % by weight of the gel or the liquid containing gel particles to the solubility limit of the at least one liposome-forming lipid in the water-miscible organic solvent.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) can have a lower limit of about 5%, about 10% , about 15%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70% by weight of the gel or the liquid containing gel particles, and an upper limit of about 90% by weight of the gel or the liquid containing gel particles.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) can have a lower limit of about 5% , about 10% , about 15%, about 20% , about 30%, about 40% , about 50% , about 60% or about 70% by weight of the gel or the liquid containing gel particles, and an upper limit of about 85% by weight of the gel or the liquid containing gel particles.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) can also be from about 5% to about 80%, about 10% to about 80%, about 15% to about 80% , about 20% to about 80% , about 30% to about 80%, about 40% to about 80% , about 50% to about 80% , about 60% to about 80%, about 70% to about 80% , about 10% to about 70%, about 20% to about 60%, or about 30% to about 50% by weight of the gel or the liquid containing gel particles.
  • the amount of the at least one liposome-forming lipid in the gel or the liquid containing gel particles of step (II) is about 45% by weight of gel or the liquid containing gel particles.
  • aqueous medium Zl is preferably mixed with the gel or the liquid containing gel particles in increments.
  • Mixing in increments has the advantage of yielding a higher entrapment efficiency compared with mixing the entire amount of the aqeuous medium Zl with the gel or the liquid containing gel particles in one step.
  • the size of the increment can be up to about 100% , up to about 90% , up to about 80% , up to about 70% , up to about 60% , up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 5% , up to about 2%, up to about 1 %, up to about 0.5%, up to about 0.1 % , up to about 0.05 % or up to about 0.01 % of the weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with any aqueous medium Zl .
  • the size of the increment can also be from about 0.001 % to about 10%, from about 0.001 % to about 5% , from about 0.001 % to about 1 % or from about 0.001 % to about 0.1 % of the weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with any aqueous medium Zl.
  • Figures 7 and 16-18 show the phase diagrams of several lipid(s)/water- miscible organic solvent/aqueous medium systems used in the gel hydration method of the present invention, wherein the lipid(s) were NAPE/DOPC (70/30), pure POPC, POPC/POPG (95:5) and POPC/POPG (9: 1).
  • the ternary phase diagrams are used to show the general relationship between the fluid zone, gel zone and liposome zone for the particular lipid(s)/water-miscible organic solvent/aqueous medium systems used in the method.
  • Liposomes are useful as delivery vehicles of encapsulated substances.
  • the method of the present invention can be used to encapsulate at least one biologically active substance in liposomes.
  • the liposomes containing the at least one biologically active substance encapsulated therein prepared by the method of the present invention have the advantages of a high entrapment efficiency and a relatively homogeneous particle size. Due to the simplicity of the procedures, the method of preparing the liposomes of the present invention allows relatively rapid production of the liposomes at a low cost.
  • the method of the present invention has the additional advantage of being easily controlled and modified, e.g., by selecting a batch or continuous operation, to fit the special requirements of different formulations.
  • liposomes are especially useful as delivery vehicles for hydrophobic pharmaceutical agents because the liposomes contain a significant amount of lipids with which the hydrophobic pharmaceutical agents can associate.
  • the at least one pharmaceutical agent to be encapsulated in the liposomes prepared by the method of the present invention can be hydrophobic.
  • bioactive lipids are especially suited for encapsulation in the liposomes prepared by the method of the present invention.
  • the DNA that can be encapsulated in the liposomes prepared according to the present invention includes a plasmid DNA.
  • the plasmid DNA can be of up to 20 kb, up to 15 kb, up to 10 kb, from about 0.5 kb to about 20 kb, from about 1 kb to about 15 kb, from about 2 kb to about 10 kb or from about 3 kb to about 7 kb in size.
  • Liposomes of the present invention containing the plasmid DNA are useful in gene therapy, transfection of eukaryotic cells and transformation of prokaryotic cells. It was discovered that the liposomes prepared by the method of the present invention containing a plasmid DNA encapsulated therein have a high transfection efficiency.
  • the liposomes of the present invention having at least one biologically active substance encapsulated therein can be administered to a subject in need of the biologically active substance via an oral or parenteral route (e.g., intravenous, intramuscular, intraperitoneal and intrathecal routes) for therapeutic or diagnostic purposes.
  • the dose of the liposomes to be administered is dependent on the biologically active substance involved, and can be adjusted by a person skilled in the art based on the health of the subject and the medical condition to be treated or diagnosed. For diagnostic purposes, some the liposomes of the present invention can be used in vitro.
  • a method of preventing or treating a health disorder in a subject in need of the treatment or prevention comprises administering the liposomes containing a biologically active substance encapsulated therein as prepared by one of the above methods in the subject, wherein the biologically active substance is a pharmaceutical agent or a nucleic acid.
  • the liposomes containing the biologically active subtance encapsulated therein prepared by the method of the present invention can further comprise a targeting agent to facilitate the delivery of the biologically active substance to a proper target in a biological system.
  • a targeting agent include antibodies, a molecule containing biotin, a molecule containing streptavidin, or a molecule containing a folate or transferrin molecule.
  • DOPC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • POPG l-palmitoyl-2-oleoy l-sn-g
  • N-C12-DOPE and 14.2 mg of DOPC were co-dissolved in 100 ⁇ l ethanol.
  • a volume of 100-200 ⁇ l of an aqueous solution containing a biological active substance was injected into the lipid ethanol solution under intense mixing.
  • 1.8 ml of a hydration buffer 300 mM sucrose, 10 mM Tris, 1 mM NaCl, pH 7.0 was slowly added to the sample to form a suspension of liposomes.
  • any unencapsulated material was removed by washing (one wash consisted of (1) sedimenting the liposomes in an aqueous phase, (2) replacing the supernatant with fresh aqueous phase, and (3) resuspending the pellet) the liposomes three times via 10,000 g centrifugation. If the biologically active substance to be encapsulated was a EGFP plasmid
  • the gel was hydrated by adding an aqueous buffer (10 mM Tris, 1 mM sodium chloride, 300 mM sucrose, pH 7.0) under intense mixing. The gel turned cloudy and finally collapsed after additional aqueous solution was added. The so formed liposome suspension was washed (a wash was (1) sedimenting the liposomes in an aqueous phase, (2) replacing the supernatant with a fresh aqueous phase, and (3) resuspending the pellet) by centrifugation to remove any free plasmid DNA.
  • an aqueous buffer (10 mM Tris, 1 mM sodium chloride, 300 mM sucrose, pH 7.0) under intense mixing.
  • the gel turned cloudy and finally collapsed after additional aqueous solution was added.
  • the so formed liposome suspension was washed (a wash was (1) sedimenting the liposomes in an aqueous phase, (2) replacing the supernatant with a fresh aqueous phase, and (3) resuspending the pellet)
  • NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of DOPC and 400 ⁇ g of EGFP plasmid DNA.
  • Light micrographs (Olympus BH-2, New York/New Jersey Scientific) of these liposomes before and after five passes of extrusion tlirough a membrane filter with 400 nm pore size were taken at a magnification of 400X (see Figure 1, top and bottom panels).
  • NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of
  • the liposomes had DNA: lipid ratios of about 1-2 ⁇ g/ ⁇ mole ( Figure 4), as determined by a phosphate assay and
  • Picogreen assay (Shangguan et al., Gene Therapy, 769-783, 2000), respectively.
  • the plasmid DNA was protected against DNase I digestion as described in Shangguan et al.
  • a 5-20% continuous sucrose gradient was obtained by mixing a 10 mM Tris buffer, pH 7, containing 140 mM NaCl, and the same buffer, containing 20% sucrose instead of NaCl.
  • the liposomes were loaded on top of the gradient and centrifuged for 17 hours at 35,000 rpm. The centrifugation yielded a single band of liposomes centered at approximately 10% sucrose.
  • the contents of the centrifuge tubes were fractionated starting from the bottom.
  • the concentrations of the total phosholipids and DOPC were determined using phosphate and choline assays. In all fractions examined, the phosphate to choline ratios were nearly the same: 3 ⁇ 0.2 (see Figure 5), which indicates compositional homogeneity of mixed lipid liposomes.
  • NAPE/DOPC lipid mixtures (70:30, molar ratio) were suspended in 34-77 mg of a 5 mM HEPES buffer (pH 7.5) to reach lipid concentrations of 25%, 33%, 43 % , and 60% (wt/wt). Ethanol was added incrementally to the lipid suspensions at increments of 15-30 mg under intense mixing. The total weight of added ethanol was recorded each time when the mixtures underwent a phase change. A ternary lipids - ethanol - aqueous phase diagram was constructed by connecting the critical points at which the mixture underwent any phase change (Figure 6).
  • a volume of 85.7 ⁇ l of a EGFP plasmid DNA stock solution (3.5mg EGFP plasmid DNA/ml) was added to each of 0-97% (wt/wt) ethanol solutions.
  • the ethanol solution contained 200 mM NaCl. 90° light scattering of the EGFP plasmid DNA at 875 nm in different ethanol solutions was presented in Figure 7. This experiment was conducted to determine the effect of ethanol on the plasmid DNA.
  • the 200 mM NaCl solution was used to mimic the ionic strength in the gel containing N-C12-DOPE.
  • Transfection solutions (0.1 ml/well for 96 well plates) were prepared by dilution of appropriate liposome samples to approximately 2 mM total lipid (for equal lipid transfection) into medium with 0.5% FBS. The plates were aspirated to remove medium and washed once with Dulbecco's phosphate buffered saline (PBS) followed by aspiration. After an addition of 1 mM CaCl 2 and 0.4 mM MgCl 2 , the transfection solution was then added to the wells and incubated at 37 °C for 3 hours. After incubation, the wells were aspirated and a medium containing 10% heat inactivated FBS was added to each well.
  • PBS Dulbecco's phosphate buffered saline
  • the N-C12-DOPE/DOPC (70:30) liposomes containing PGL-3 plasmid were made by the gel hydration method as set forth in Example 1. Transfections without transferrin were performed as described in example 10, except that in one of the transfection assays, 10% FBS instead of 0.5% FBS was used.
  • the liposome samples were first mixed with equal volumes of a 2 mg/ml poly-lysin transferrin conjugate at a concentration of 20 mM for 10 minutes, and then this mixture was diluted 10 times with Hank's balanced salt solution (HBSS) without Ca 2+ /Mg 2+ containing 10% FBS before being applied to the cells.
  • HBSS Hank's balanced salt solution
  • the level of luciferase expression was determined by the Bright-glow luciferase assay (Clontech).
  • the N-C12-DOPE/DOPC (70:30) liposomes had transfection activity at physiological concentrations of Ca 2+ -Mg 2+ , i.e. , about 1.2 mM Ca 2+ and 0.8 mM Mg 2+ ( Figure 10).
  • the N-C12-DOPE/DOPC (70:30) liposomes containing fluorescent lipid probe Dil at a 0.1 % (wt%) concentration were prepared by the ethanol gel hydration method as set forth in Example 1.
  • the liposomes were incubated with OVCAR-3 cells in the presence of 10% FBS and various concentrations of transferrin as described in Example 11. After a 3 hour incubation at 37°C, the cells were washed three times with PBS and dissolved in 1 % C12E8. Cell associated Dil fluorescence was measured at an emission wavelength of 620 nm, with an excitation wavelength of 560 nm. Binding of the liposome sample showed a small increase with increasing transferrin concentration (Figure 11).
  • the liposomes containing a EGFP plasmid DNA and the following lipids or lipid mixtures including 100% DOPC, DOPC/N-C12-DOPE (8:2 molar ratio), DOPC/N-C12-DOPE (6:4 molar ratio), DOPC/N-C12-DOPE (4:6 molar ratio), DOPC/N-C12-DOPE (2:8 molar ratio), and 100% N-C12-DOPE, were made by the ethanol gel hydration method as set forth in Example 1. The transfection assay was performed as described in Example 10.
  • N-C12-DOPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7mg of N-C12-DOPE, 14.2 mg of DOPC and 100 ⁇ l of one of the following dextran stock solutions (5 mg/ml): tetramethyl rhodamine (MW 70,000), tetramethyl rhodamine (MW 2,000,000) or fluorescein (MW 70,000, lysine fixable).
  • NAPE/DOPC liposomes 70:30, molar ratio
  • SPLV method 1.13 ml of N-C12-DOPE/DOPC lipid mixtures(60 mM total lipid, 70:30 molar ratio) in chloroform were mixed with 100 ⁇ l of one of the following dextran stock solutions (5 mg/ml): tetramethyl rhodamine (MW 70,000), tetramethyl rhodamine (MW 2,000,000) or fluorescein (MW 70,000, lysine fixable). The mixture was sonicated briefly to form an emulsion.
  • the fluorescence intensity was measured using 560/20 and 620/40 nm bandpass excitation and emission filters, respectively.
  • a lamellarity assay based on NBD-PE reduction by dithionite was used as described by Mclntyre and Sleight (1991). All measurements of liposomes' captured volumes and lamellarity in Examples 18 through 25 were conducted according to the procedures described in this example.
  • Example 19 An amount of 46 mg of POPC was mixed with 4 mg of POPG and dissolved in 50 mg of anhydrous EtOH.
  • the fluid solution became a viscous gel after addition of 20 ⁇ l of the buffer.
  • the gel became a turbid liposome-like suspension after addition of a total of 70 ⁇ l of the buffer.
  • the captured volume of the resultant liposomes was 7.1 ⁇ l/ ⁇ mol, as estimated using SR101 fluorescence. Their average diameter was 550nm, as measured by dynamic light scattering ( Figure 15).
  • Example 20 (POPC-POPG 95:5) An amount of 47.5 mg of POPC was mixed with 2.5 mg of POPG and dissolved in 75 mg of anhydrous EtOH. An amount of 0.12 mg of NBD-PE was added to serve as a lamellarity probe. A 100 mM Tris buffer, containing 5 ⁇ M sulforhodamine 101 (SRI 01) as aqueous volume marker, was added to the lipid solution in 20 ⁇ l aliquots upon rigorous vortexing. The fluid solution became a viscous gel after addition of a total of 40 ⁇ l of the buffer. The gel became a turbid liposome-like suspension after addition of totally 120 ⁇ l of the buffer.
  • SRI 01 sulforhodamine 101
  • Amounts of 90 mg of DMPC, 13 mg of cholesterol and 2.5 mg of PA were dissolved in 115.5 mg of anhydrous EtOH.
  • a viscous gel was obtained.
  • the gel was hydrated by 2 ml of the same buffer, but containing no ⁇ -amyloid peptide, and the sample was dialyzed against 1 L of the same buffer to remove ethanol and non-entrapped peptide.
  • the resultant liposomes captured 40% of the peptide added in the first step.
  • Amounts of 78 mg of DSPC and 25 mg of cholesterol were dissolved in 103 mg of anhydrous EtOH.
  • the sample and the titration buffer were maintained at 55 °C throughout the mixing process.
  • the fluid solution became a viscous gel after an addition of 40 ⁇ l of the buffer.
  • the gel became a turbid liposome-like suspension after an addition of totally 70 ⁇ l of the buffer.
  • the liposomes were cooled to room temperature upon vortexing, and dialyzed against 1 L of a 100 mM Tris buffer, pH 7, buffer overnight to remove ethanol and nonentrapped material.
  • the captured volume of the resultant liposomes was 1.2 uL/umol, as estimated using SR 101 fluorescence ( Figure 15)
  • Example 24 (DSPC-DSPG-cholesterol 5:1:4) Amounts of 20.3 mg of DSPC, 3.3 mg of DSPG and 6.3 mg of cholesterol were dissolved in 30 mg of anhydrous EtOH at 60 °C. A 100 mM Tris buffer, pH 7, containing 5 ⁇ M sulforhodamine 101, was added to the lipid solution in 5 ⁇ l aliquots upon rigorous vortexing. The sample and the titration buffer were maintained at 60 °C throughout the mixing process. The fluid solution became a viscous gel after an addition of 5 ⁇ l of the buffer. The gel became a turbid liposome-like suspension after adding a total of 20 ⁇ l of the buffer.
  • Amounts of 42.8 mg of DSPC, 21.7 mg of DSPG and 21 mg of cholesterol were dissolved in 132 mg of anhydrous EtOH at 60 °C.
  • An amount of 0.17 mg of NBD-PE was added to serve as a lamellarity probe.
  • the sample and the titration buffer were maintained at 60 °C throughout the mixing process.
  • the fluid solution became a viscous gel after an addition of 40 ⁇ l of the buffer.
  • the gel became a turbid liposome-like suspension after an addition of totally 130 ⁇ l of the buffer.
  • the ternary phase diagram of a lipid/water-miscible organic solvent/aqueous medium system was produced, wherein the lipid was POPC, the water-miscible organic solvent was ethanol and the aqueous medium was a 100 mM Tris buffer. Varying amounts (30-80 mg) of POPC were dissolved in anhydrous ethanol to form a lipid solution at concentrations of 10%, 20%, 30% , 50%, 60% and 70% (wt/wt). The 100 mM Tris buffer was added to the lipid solution in 10 ⁇ l increments upon vigorous vortexing. Appearance of the samples after each titration step was recorded.
  • the lipid solution first turned into a gel, which turned into a liposome suspension upon further incremental addition of the 100 mM Tris buffer.
  • a ternary phase diagram was constructed mapping the locations of liquid, gel and liposomal states of the samples based on their visual appearance. The boundary between the solution zone and the gel zone was as indicated by the open circles and dotted line in the ternary phase diagram of Figure 16. Additional amounts of the 100 mM Tris buffer were added to the gel with mixing to form liposomes. The boundary between the gel zone and the liposome zone was as indicated by the open circles and lines in the ternary phase diagram of Figure 16. In six different preparations (represented by six different symbols: stars, triangles, pentagons, inverted triangles, circles and squares), the 100 mM Tris buffer was added in 10 ⁇ l increments as represented by the individual symbols in Figure 16.
  • the ternary phase diagram of a lipid/water-miscible organic solvent/ aqueous medium system was produced, wherein the lipids were POPC and DOPG in a 95:5 molar ratio, the water-miscible organic solvent was ethanol and the aqueous medium was a 100 mM Tris buffer. Varying amounts (30-80 mg) of the POPC: DOPG mixture (95:5) were dissolved in anhydrous ethanol to form a lipid solution at concentrations of 10 % , 20 % , 30 % , 50 % , 60 % and 70 % (wt/wt) . The 100 mM Tris buffer was added to the lipid solution in 10 ⁇ l increments upon vigorous vortexing.
  • the 100 mM Tris buffer was added to the lipid solution in 10 ⁇ l increments upon vigorous vortexing. Appearance of the samples after each titration step was recorded. With the incremental addition of the 100 mM Tris buffer, the lipid solution first turned into a gel, which turned into a liposome suspension upon further incremental addition of the 100 mM Tris buffer.
  • a ternary phase diagram was constructed mapping the locations of liquid, gel and liposomal states of the samples based on their visual appearance. The boundary between the solution zone and the gel zone was as indicated by the dashed line in the ternary phase diagram of Figure 18. Additional amounts of the 100 mM Tris buffer were added to the gel with mixing to form liposomes.
  • POPG and 44 mg of anhydrous ethanol were prepared in separate sample tubes (POPC/POPG 9: l(mol/mol), lipid/ethanol 1: 1 (wt/wt)).
  • One mixture was rapidly mixed 23 mg of 100 mM Tris buffer (pH 7), containing 5 mM of SR101 (as an aqueous volume marker) (sample 1), another one was mixed with 69 mg of the same buffer (sample 2).
  • SR101 as an aqueous volume marker
  • the samples were dialyzed against 2000X excess of 100 mM Tris buffer to remove ethanol and non-encapsulated SR101. Encapsulation efficiency of resultant liposomes was evaluated by measuring fluorescence intensity of aqueous volume marker SR101.
  • the entrapment efficiency of the liposomes in sample 1, prepared from the gel with low content of aqueous phase was only 10%, while the entrapment efficiency of the liposomes in sample 2, prepared from the gel containing more aqueous phase, was 67% .

Abstract

La présente invention concerne une méthode de préparation de liposomes contenant au moins une substance biologiquement active encapsulée dans ces derniers. La méthode consiste: (I) (A) (a) à dissoudre au moins un lipide formant liposome et la ou les substances biologiquement actives dans un solvant organique miscible avec l'eau pour former un mélange; (b) (i) à dissoudre au moins un lipide formant liposome dans un solvant miscible avec l'eau pour former une solution organique; (ii) à dissoudre la ou les substances biologiquement actives dans un milieu aqueux X pour former une solution aqueuse; et (iii) à mélanger la solution organique et la solution aqueuse pour former un mélange; ou (c) à mélanger au moins un lipide formant liposome, la ou les substances biologiquement actives et le solvant organique miscible avec l'eau pour former un mélange; ou (B) à mélanger au moins un lipide formant liposome et un solvant organique miscible avec l'eau pour former un mélange; (II) (A) à mélanger le mélange de l'étape (I)(A) avec un milieu aqueux Y et facultativement la ou les substances biologiquement actives pour former un gel ou un liquide contenant des particules de gel; ou (B) à mélanger le mélange de l'étape (I) (B) avec la ou les substances biologiquement actives et le milieu aqueux Y pour former un gel ou un liquide contenant des particules de gel; et ensuite (III) (A) à mélanger le gel ou le liquide contenant des particules de gel avec un milieu aqueux Z1 pour former directement les liposomes contenant la ou les substances biologiquement actives encapsulées dans les liposomes; ou (B) (i) à mélanger le gel ou le liquide contenant des particules de gel avec un milieu aqueux Z1 pour former une substance cireuse; et (ii) à mélanger la substance cireuse avec un milieu aqueux Z2 pour former directement les liposomes contenant la ou les substances biologiquement actives encapsulées dans les liposomes; lesdits milieux aqueux X, Y, Z1 et Z2 étant identiques ou différents, et la teneur en phospholipides du gel ou du liquide contenant des particules de gel de l'étape (II) n'étant pas comprise dans une fourchette 15-30 % en poids du gel ou du liquide contenant les particules de gel.
PCT/US2002/041846 2001-12-31 2002-12-31 Encapsulation efficace dans des liposomes WO2003057191A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006076891A2 (fr) * 2005-01-18 2006-07-27 Abnoba Heilmittel Gmbh Procede et dispositif d'encapsulage de substances dans des liposomes ayant une structure membrane librement ajustable
CN110250495A (zh) * 2019-06-12 2019-09-20 浙江工商大学 一种Vc脂质体水凝胶及其制备方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741513A (en) * 1990-02-08 1998-04-21 A. Natterman & Cie. Gmbh Alcoholic aqueous gel-like phospholipid composition, its use and topical preparations containing it

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741513A (en) * 1990-02-08 1998-04-21 A. Natterman & Cie. Gmbh Alcoholic aqueous gel-like phospholipid composition, its use and topical preparations containing it

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006076891A2 (fr) * 2005-01-18 2006-07-27 Abnoba Heilmittel Gmbh Procede et dispositif d'encapsulage de substances dans des liposomes ayant une structure membrane librement ajustable
WO2006076891A3 (fr) * 2005-01-18 2006-11-09 Abnoba Heilmittel Gmbh Procede et dispositif d'encapsulage de substances dans des liposomes ayant une structure membrane librement ajustable
CN110250495A (zh) * 2019-06-12 2019-09-20 浙江工商大学 一种Vc脂质体水凝胶及其制备方法
CN110250495B (zh) * 2019-06-12 2023-09-08 浙江工商大学 一种Vc脂质体水凝胶及其制备方法

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