WO1997031624A1 - Systeme d'apport de liposome - Google Patents

Systeme d'apport de liposome Download PDF

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Publication number
WO1997031624A1
WO1997031624A1 PCT/US1997/003077 US9703077W WO9731624A1 WO 1997031624 A1 WO1997031624 A1 WO 1997031624A1 US 9703077 W US9703077 W US 9703077W WO 9731624 A1 WO9731624 A1 WO 9731624A1
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Prior art keywords
liposome
receptor
folate
binding
liposomes
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PCT/US1997/003077
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English (en)
Inventor
David H. Thompson
Philip S. Low
Yuanjin Rui
Susan Wang
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Purdue Research Foundation
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Priority to AU23169/97A priority Critical patent/AU2316997A/en
Publication of WO1997031624A1 publication Critical patent/WO1997031624A1/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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • the present invention is directed to liposomes, and more particularly, a liposomal delivery system and method for transporting materials such as drugs, nucleic acids, and proteins to a targeted population of cells.
  • the liposomes of the present invention comprise modified lipids that enhance the delivery of exogenous molecules encapsulated therein to the cytoplasm of cells.
  • Liposomes are microscopic lipid bilayer vesicles that enclose a cavity.
  • the liposomal vesicles can contain a single phospholipid bilayer (unila ellar vesicle) or multiple phospholipid bilayers (multilamellar vesicle) .
  • Liposome technology has been applied to the formulation and delivery of pharmaceutics, diagnostic imaging, clinical analysis, cosmetics, food processing and cellular transfection.
  • U.S. Pat. No. 3,993,754 discloses an improved chemotherapy method for treating malignant tumors in which an anti-tumor drug is encapsulated within liposomes and the liposomes are injected into an animal.
  • encapsulation of pharmaceuticals in liposomes can reduce drug side effects, improve pharmacokinetics of delivery to a target site, and improve the therapeutic index of a drug.
  • the delivery of administered liposomal carriers to a cell can be enhanced by attaching or adsorbing various ligands to the exterior surface of the liposomal vesicle (For an overview see Martin, F.J., et al. Liposomes a Practical Approach (New, R.R.C., Ed) pages 163-182, IRL Pres, Oxford (1990) .
  • the ligand can be attached (through covalent, hydrogen or ionic bonds) to the phospholipids forming the liposome either by direct linkage or by connection through intermediary linkers, spacer arms, bridging molecules.
  • the ligand can be anchored into the liposome bilayer through hydrophobic interactions.
  • a specified ligand is chemically conjugated by covalent, ionic or hydrogen bonding to the liposomal surface of a liposome by forming a conjugate having a moiety (the ligand portion) that is still recognized in the conjugate by a target receptor.
  • the phototoxic compound psoralen has been conjugated to insulin and internalized by the insulin receptor endocytotic pathway (Gasparro, Biochem. Biophys. Res. Comm. 141(2), pp. 502-509, Dec.
  • hepatocyte specific receptor for galactose terminal asialoglycoproteins has been utilized for the hepatocyte-specific transmembrane delivery of asialoorosomucoid-poly-L-lysine non-covalently complexed to a DNA plasmid (Wu, G.Y., J. Biol. Chem., 262(10), pp.
  • the cell receptor for epidermal growth factor has been utilized to deliver polynucleotides covalently linked to EGF to the cell interior
  • EGF epidermal growth factor
  • Myers European Patent Application 86810614.7, published June 6, 1988
  • the intestinally situated cellular receptor for the organo etallic vitamin B 12 -intrinsic factor complex has been used to mediate delivery to the circulatory system of a vertebrate host a drug, hormone, bioactive peptide or immunogen complexed with vitamin B 12 and delivered to the intestine through oral administration
  • the mannose-6-phosphate receptor has been used to deliver low density lipoproteins to cells (Murray, G.
  • Vitamins such as thiamin, folate, biotin, and riboflavin have also been used to enhance the uptake of exogenous molecules (US Patent No. 5,108,921 and 5,416,016) .
  • Liposome Preparation General methods of making liposomes are known.
  • Liposomes may be produced by a wide variety of methods.
  • Multilamellar vesicles are formed by simple hydration of dry lipid powders. The particles formed are typically quite large (>10 ⁇ m) and are often oligolamellar (i.e., possessing more than one bilayer membrane) . This method is most commonly used to produce giant, unilamellar liposomes for micropipet measurements to determine the mechanical properties of bilayer membranes.
  • Ultrasonication with probe type sonicators or processing through a French press produces small, unilamellar vesicles (SUV) with average diameters in the 25-50 range.
  • SUV unilamellar vesicles
  • LDL low-density lipoprotein
  • Extrusion techniques are the most widely used methods for SUV liposome production for in vi tro and in vivo studies due to their ease of production, readily selectable particle diameters (dictated by the nominal pore size of the track-etch membranes used for extrusion, typically between 50-120 nm for in vi vo experiments) , batch-to-batch reproducibility, and freedom from solvent and/or surfactant contamination.
  • Solvent injection and detergent dialysis techniques for liposome production give heterogeneous distributions of particle sizes and are not commonly used for biophysical or biochemical experimentation due to the retention of membrane impurities in these particles.
  • Materials to be encapsulated may be passively entrapped or "remote" loaded.
  • liposomes Despite many years of investigation, selective targeting and membrane translocation of compounds to cells in the body remains problematic.
  • One limitation to the widespread use of liposomes derives from the rapid accumulation of intravenously administered liposomes in the reticuloendothelial system.
  • liposomes Even with targeting entities bound to the liposome surface, liposomes accumulate rapidly in organs with fenestrated capillaries, such as the liver, spleen, and bone marrow.
  • the uptake of liposomes by the reticuloendothelial system can be limited by the inclusion of glycolipids such as monosialoganglioside (GM1) or hydrogenated Phosphatidylinositol (HPI) in the lipid bilayer (Litzinger, D.C. and Huang, L. (1992) Biochim.
  • GM1 monosialoganglioside
  • HPI hydrogenated Phosphatidylinositol
  • lipids can be derivatized with polyethyleneglycol (PEG) , see for example, Moghimi, S.M. and Patel, H.M. (1992) Bi atm Bi ophycs . Acta, 1135, 269-274.
  • PEG polyethyleneglycol
  • the PEG coating is believed to inhibit nonspecific adsorption of serum proteins and thereby prevent nonspecific recognition of the liposomes by macrophages (Papahadjopoulos, D., Allen, T.M., Gabison, A., Mayhew, E., Matthay, K., Huang, S.K., Lee, K.-D., Woodle, M.C., Lasic, D.D., Redemann, C. and Martin, F.J. (1991) Proc . Na tl . Acad. Sci . USA, 88, 11460-11464) .
  • PEG derivatization is now commonly used to prevent liposome phagocytosis by the reticuloendothelial system. Such "stealth liposomes" are reported to survive more than 24 hours in circulation compared to only ⁇ 2 hours observed for their unprotected counterparts (Klibanov, A.L., Maruyama, K., Beckerleg, A.M., Torchilin, V.P. and Huang, L. (1991) Bi atm Biophycs . Acta , 1062, 142-148) .
  • the targeting ligands can be attached to the ends of the polymeric chains that render the liposomal resistant to uptake by the reticuloendothelial system (Kilanov, A.L., and Huang, L., Long Circulating Liposomes: Development and Perspectives, Journal of Liposome Research, 2(3), P. 321- 334 (1992).
  • 4,882,164 similarly discloses a light sensitive liposome which undergoes a trans to cis isomerization upon irradiation with an appropriate wave ⁇ length of light (ultraviolet light) to allow the fluid contents of the liposome to escape through the membrane into the surrounding environment.
  • GB Patent 2,209,468 discloses liposomes with an incorporated photosensitizing agent that absorbs light and alters the lipid membrane to release a drug from the liposome. The development of liposomes that could be targeted to a population of cells and induced to release their payload upon activation by a metabolic or externally applied trigger would greatly improve the efficacy of liposomes as a delivery vehicle.
  • the present invention is directed to a novel composition, and method of using that novel composition, for improving the delivery of exogenous molecules to the cytoplasm of cells.
  • the novel delivery system comprises an exogenous molecule entrapped by a liposome vesicle, wherein a targeting ligand is complexed (either directly or indirectly) to the surface of the liposome, and the liposome comprises a triggerable membrane fusion lipid.
  • the liposomal membrane comprises triggerable lipids and lipids complexed to a ligand, wherein the ligand is capable of interacting with cellular membranes to enhance the uptake of the ligand and attached liposome.
  • the triggerable lipid contains a vinyl ether functionality which is cleaved in response to a reduction in pH to produce a local disruption in the liposomal membrane.
  • Fig. 1 Graphic representation of the percent calcein released relative to the percent DPlsC hydrolyzed from DPlsC:DHC liposomes at pH 4.5 as a function of DHC content.
  • Fig. 2 Graphic representation of the percent calcein released per time from DPlsC:DHC liposomes at pH 4.5 as a function of DHC content.
  • Fig. 3 Graphic representation of the propidium iodide release kinetics in KB cells using folate-targeted DPlsC:DHC liposomes.
  • Fig. 4 Graphic representation of the release of PI from liposomal vessicles into the cytoplasm of cultured KB cells.
  • Fig. 5 Graphic representation of the cytotoxicity of arabinofuranosylcytocine (Ara-C) in KB cell cultures.
  • Cells were plated to 50% confluence in 24-well culture plates before treatment with free Ara-C (diamonds) , Ara-C encapsulated in EPC:folate liposomes (squares), or DPlsC:folate liposomes (triangles) for 4 h. The cells were then washed, incubated in fresh FDMEM, and analyzed for DNA synthesis after 24 h.
  • Fig. 6 Graphic representation of total PI bound to cultured KB cells after incubation of the cells with targeted and non-targeted PI encapsulated lyosomes.
  • a triggerable lipid is defined herein as a lipid that undergoes a chemical or conformational change upon exposure to a predetermined condition.
  • a pH sensitive lipid is defined herein as a lipid that undergoes a chemical or conformational change upon exposure to a decreased pH.
  • a targeting lipid is defined herein as a lipid ligand complex, wherein the ligand is capable of being internalized by receptor mediated uptake by the cell.
  • Actively and passively targeted liposomes have attracted a great deal of attention as drug delivery vehicles due to their favorable biocompatibility, high drug:lipid ratios, and blood clearance characteristics.
  • Methods for efficiently, transporting the liposomal contents to the target cell cytoplasm have not been generally available in the form of a plasma-stable liposome. This obstacle is especially problematic for the cytoplasmic delivery of peptides, antisense oligonucleotides, and gene constructs.
  • the present invention is directed to an improved liposome that enhances the delivery of exogenous molecules to the cytoplasm of a targeted population of cells.
  • the enhanced delivery can be quantitated in terms of selectivity, speed of uptake, and as the percentage of material delivered to the cytoplasm.
  • the hybrid liposome system of the present invention obviates these problems by incorporating both ligand receptor-mediated targeting moieties and a cytoplasmic release mechanism.
  • the ligand enhances the cellular uptake of the liposome by the targeted cells and the cytoplasmic release mechanism (for example, vinyl ether-based triggerability upon exposure to the low pH environment of the endosome) enhances the delivery of exogenous molecules to the cytoplasm of cells.
  • phospholipids suitable for the formation of liposomes are modified by complexing a ligand to the phospholipid headgroup using techniques know to those skilled in the art. These modified lipids are combined with additional lipids, including triggerable lipids, to prepare a liposomal complex in accordance with the present invention.
  • phospholipids suitable for the formation of liposomes are modified by covalently linking a spacer (for example, a PEG molecule) to the phospholipid headgroup and linking (through a covalent, ionic or hydrogen bond) the opposite end of the linker to a ligand, wherein the ligand is subject to receptor mediated cellular uptake.
  • a spacer for example, a PEG molecule
  • linking through a covalent, ionic or hydrogen bond
  • modified lipids are combined with additional lipids, including for example, pH sensitive lipids such as diplasmenylcholine lipid (1,2- di-O- (Z-l'-hexadecenyl)-sn-glycero-3-phosphatidylcholine or DPlsC) , to prepare a targeted liposomal complex in accordance with the present invention.
  • pH sensitive lipids such as diplasmenylcholine lipid (1,2- di-O- (Z-l'-hexadecenyl)-sn-glycero-3-phosphatidylcholine or DPlsC)
  • the liposome complex is loaded with an exogenous molecule using methods known to those of ordinary skill in the art.
  • receptor mediated transmembrane transport is initiated thus internalizing the complex within the cell.
  • Ligands useful in accordance with the present invention include any compound that mediates uptake of that compound by a cell.
  • the ligand interacts with a particular cell type or tissue, and thus linking the ligand to the liposome enables the preferential uptake (i.e. targeting) of liposomes by that particular cell type or tissue.
  • Suitable ligands useful for mediating the uptake of a liposome include antibodies and/or compounds capable of binding to a receptor and being internalized by receptor mediated endocytosis.
  • Vitamins and other essential minerals and nutrients can be utilized to enhance the uptake of exogenous molecules.
  • a vitamin ligand can be selected from the group consisting of folate, folate receptor-binding analogs of folate, and other folate receptor-binding ligands, biotin, biotin receptor-binding analogs of biotin and other biotin receptor-binding ligands, riboflavin, riboflavin receptor-binding analogs of riboflavin and other riboflavin receptor-binding ligands, and thiamin, thiamin receptor-binding analogs of thiamin and other thiamin receptor-binding ligands.
  • the liposomal carrier system of the present invention can be utilized to deliver a variety of exogenous molecules to the cytoplasm of cells, including diagnostic agents and molecules capable of modulating or otherwise modifying cell function, such as pharmaceutically active compounds. These compounds can be entrapped by the liposome vesicles of the present invention either by encapsulating water-soluble compounds in their aqueous cavities, or by carrying lipid soluble compounds within the membrane itself.
  • Exogenous molecules for use in accordance with the present invention can include, but are not limited to: peptides, oligopeptides, proteins, apoproteins, glycoproteins, antigens and antibodies thereto, haptens and antibodies thereto, receptors and other membrane proteins, retro-inverso oligopeptides, protein analogs in which at least one non-peptide linkage replaces a peptide linkage, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, hormones, lipids, phospholipids, liposomes; toxins such as aflatoxin, digoxin, xanthotoxin, rubratoxin; antibiotics such as cephalosporins, penicillin, and erythromycin; analgesics such as aspirin, ibuprofen, and acetaminophen, bronchodilators such theophylline and albuterol; beta-blockers such as propranolol, metoprolol, atenolol
  • nucleotides include nucleotides; oligonucleotides; polynucleotides; and their art-recognized and biologically functional analogs and derivatives including, for example; methylated polynucleotides and nucleotide analogs having phosphorothioate linkages; plasmids, cosmids, artificial chromosomes, other nucleic acid vectors; antisense polynucleotides including those substantially complementary to at least one endogenous nucleic acid or those having sequences with a sense opposed to at least portions of selected viral or retroviral genomes; promoters; enhancers; inhibitors; other ligands for regulating gene transcription and translation.
  • Table 1 summarizes the various physical and chemical phenomena that can be used as a basis for liposome triggering. Many of these approaches have, in fact, been explored for unloading liposomes upon application of an external stimulus.
  • Photo thermal stimulation (e.g., light, microwaves, bulk heating, etc.)
  • Membrane fusion rates depend on both the molecular properties of the membrane bilayer (e.g., lipid headgroup charge, lateral mobility, and intrinsic curvature), as well as its supramolecular properties (e.g., hydration layer thickness, bilayer composition, membrane asymmetry, lateral phase separation, and thermally induced density fluctuations) .
  • Content leakage is less well understood since the inherent leakage properties of a liposomal membrane will be dependent on the physical state and composition of the membrane bilayer, the presence of transient vs. persistent defects (pores) size and surface density of the defects, as well as the properties of the contents that are effusing from it.
  • a novel liposomal composition for enhancing delivery of an exogenous molecule to the cytoplasm of a cell.
  • the composition comprises a liposome, wherein said liposome membrane contains amphipathic lipids, preferably phospholipids, having a polar head group and two lipophilic chains that allow the lipid to pack into a bilayer structure. At least a portion of the phospholipids comprising the liposome membrane have lipophilic chains containing a vinyl ether functionality. In one preferred embodiment both lipophilic chains contain a vinyl ether functionality.
  • a specific phospholipid (pH sensitive lipid) that fulfills this requirement is a plasmalogen having the formula:
  • R X and R 2 are independently C 12 -C 24 alkyl or C 12 -C 24 alkenyl and R 3 is a bilayer forming phosphoryl ester of the formula -CH 2 OP0 2 OR, wherein R is selected from the group comprising 2-aminoethyl, 2-(trimethylamino)ethyl, 2-(N,N- dimethylamino)ethyl, 2- (trimethylammonium)ethyl, 2-carboxy- 2-aminoethyl, succinamidoethyl, or inosityl.
  • q and p are each 1, and R, and R 2 are each (CH 2 ) n CH 3 , where n is 12-24.
  • one of R ⁇ or R 2 is 12-16 carbons long, and the other chain is at least 16 carbons long, more preferably 18 carbons.
  • a novel liposomal composition is provided for enhancing delivery of an exogenous molecule to the cytoplasm of a cell.
  • the composition comprises an exogenous molecule encapsulated in a liposome, wherein said liposome comprises liposome- forming phospholipids, at least a portion of which are complexed to a ligand, and a portion of which comprise vinyl ether phospholipids of the formula:
  • the ligand of the phospholipid-ligand complexes is subject to receptor mediated cellular uptake, and in one embodiment the ligand is selected from the group consisting of folate, folate receptor-binding analogs of folate, and other folate receptor-binding ligands, biotin, biotin receptor-binding analogs of biotin and other biotin receptor-binding ligands, riboflavin, riboflavin receptor-binding analogs of riboflavin and other riboflavin receptor-binding ligands, and thiamin, thiamin receptor-binding analogs of thiamin and other thiamin receptor-binding ligands.
  • the liposome comprises multiple types of vinyl ether phospholipids.
  • the liposome comprises a vinyl ether phospholipid of the formula:
  • R 3 is a phosphoryl ester and n and m are independently 12-24.
  • a plasma-stable liposome comprising a naturally-occurring vinyl ether linked phospholipid, diplasmenylcholine (1, 2-di-O- (Z-l '-hexadecenyl) -sn-glycero- 3-phosphatidylcholine or DPlsC) .
  • DPlsC liposomes Acid-catalyzed hydrolysis of DPlsC liposomes produces glycerophosphatidylcholine, fatty acids and aldehydes, and permeability of the liposome membrane increases significantly when ⁇ 20% of the DPlsC lipids are hydrolyzed. Unlike many pH-sensitive liposome formulations, DPlsC liposomes possess remarkable plasma stability characteristics at 37 C C and neutral pH. Pure DPlsC liposomes do not leak calcein upon exposure to 10% heat- inactivated fetal calf serum (HIFC) for up to 48 h.
  • HIFC heat- inactivated fetal calf serum
  • DPlsC + 40% DHC 0 0 0 a ' b Liposomes were mixed with pure heat-inactivated fetal calf serum at 1:1 and 9:1 ratios, respectively. % calcein release values are ⁇ 5%.
  • the liposomes of the present invention are utilized in an improved method for delivering an exogenous molecule to the cytoplasm of a targeted living cell.
  • This method can be performed either in vivo or in vi tro .
  • the method comprises the step of contacting a cell with a liposome complex, wherein the complex includes a liposome, having the exogenous molecule encapsulated therein.
  • R ⁇ and R 2 are C 12 -C 24 alkyl and R 3 is a bilayer forming phosphoryl ester of the formula -CH 2 OP0 2 OR, wherein R is selected from the group comprising 2-aminoethyl, 2- (trimethylamino) ethyl, 2- (N,N-dimethylamino) ethyl, 2- (trimethylammonium) ethyl, 2-carboxy-2-aminoethyl, succinamidoethyl, or inosityl.
  • q and p are each 1, and Rj and R 2 are each (CH 2 ) n CH 3 , where n is 12-24.
  • one of R 2 or R 2 is 12-16 carbons long, and the other chain is at least 16 carbons long, more preferably 18 carbons.
  • the ligand associated with the surface of the liposome is preferably linked to the phospholipid headgroups via covalent, ionic or hydrogen bonds and the ligand is selected from the group consisting of folate, folate receptor-binding analogs of folate, and other folate receptor-binding ligands, biotin, biotin receptor-binding analogs of biotin and other biotin receptor-binding ligands, riboflavin, riboflavin receptor-binding analogs of riboflavin and other riboflavin receptor-binding ligands, and thiamin, thiamin receptor-binding analogs of thiamin and other thiamin receptor-binding ligands.
  • the liposome complex comprises a liposome encapsulating an exogenous molecule, wherein the liposome comprises a targeting lipid and a pH sensitive lipid having the formula:
  • the targeting lipid in accordance with one embodiment, is a lipid of the formula DSPE-linker- ligand and one preferred linker is a polyethyleneglycol spacer arm.
  • the liposome comprises about 0.1% to about 1.5% of the targeting lipid, about 20% to about 99.5% of the pH sensitive lipid with the remainder being any amphipathic lipid having a polar head group and two lipophilic chains that allow the lipid to pack into a bilayer structure.
  • the liposome optimally comprises about 0.1% to about 1.5%, more preferably about 0.1% to about 0.5%, DSPE-PEG3350- folate, about 60% to about 99.5%, more preferably about 80% to about 99.5% DplsC, and 0 to about 20%, more preferably about 10% or less, DHC.
  • Living cells which can serve as the target for the method of this invention include prokaryotes and eukaryotes, including yeasts, plant cells and animal cells. The present method can be used to modify cellular function of living cells in vi tro, i.e., in cell culture, or in vivo, where the cells form part of, or otherwise exist in plant tissue or animal tissue.
  • Exogenous molecules encapsulated within the disclosed liposomal delivery vehicles can be used to deliver effective amounts of diagnostic, pharmaceutically active, or therapeutic agents through parenteral or oral routes of administration to human or animal hosts.
  • the present method can be performed on any cells in any manner which promotes contact of the liposome complex with the targeted cells having the requisite receptors.
  • the liposomal compositions can be administered generally to an animal or human to target cells that form part of the tissue of the animal or human.
  • the target cells can include, for example, the cells lining the alimentary canal, such as the oral and pharyngeal mucosa, the cells forming the villi of the small intestine, or the cells lining the large intestine.
  • Such cells of the alimentary canal can be targeted in accordance with this invention by oral administration of a composition comprising an exogenous molecule encapsulated by the liposome of the present invention.
  • cells lining the respiratory system (nasal passages/lungs) of an animal can be targeted by inhalation of the present compositions; dermal/epidermal cells and cells of the vagina and rectum can be targeted by topical application of the present compositions; and cells of internal organs including cells of the placenta and the so-called blood/brain barrier can be targeted particularly by parenteral administration of the present compositions.
  • compositions for therapeutic use in accordance with this invention contain effective amounts of the exogenous molecule encapsulated in the presently described liposomes, admixed with art-recognized excipients and pharmaceutically acceptable carriers appropriate to the contemplated route of administration.
  • Folate-PEG-NH 2 was synthesized by reacting 500 mg polyoxyethylene-jbis-amine with an equimolar quantity of folic acid in 5 ml dimethylsulfoxide containing one molar equivalent of dicyclohexycarbodiimide and 10 ⁇ l pyridine. The reaction mixture was stirred overnight in the dark at room temperature. At this point, 10 ml water was added and the insoluble by-product, dicyclohezylurea, was removed by centrifugation. The supernatant was then dialyzed against 5 mM NaHC0 3 buffer (pH 9.0) and then against deionized water to remove the dimethylsulfoxide and unreacted folic acid in the mixture.
  • the trace amount of unreacted polyoxyethylene-2?is-amine was then removed by batch- adsorption with 5 g of cellulose phosphate cation-exchange resin pre-washed with excess 5 mM phosphate buffer (pH 7.0).
  • the trace amount of PEG-bis- folate may be removed by anion-exchange chromatography on a DEAE-trisacryl Sepharose column.
  • Folate-PEG-amine can be easily eluted with 10 mM NH 4 HC0 3 (pH 8.0).
  • the produced folate-PEG-NH 2 was then lyophilized and analyzed for folate content by absorbance at 363 nm and -NH 2 content by the ninhydrin assay.
  • the ratio of folate to free -NH 2 groups in this product was ⁇ 1 .
  • iV-Succinyl-DSE was synthesized by reacting overnight 1.1 molar equivalent of succinic anhydride with 100 mg DSPE in 5ml chloroform containing 10 ⁇ l pyridine. The product was precipitated with cold acetone and verified by thin-layer chromatography. N-Succinyl-DSPE was re ⁇ dissolved in chloroform and its carboxyl group was activated by reacting with one molar equivalent of dicyclohexyl-carbodii ide for 4 h at room temperature. An equimolar amount of the above synthesized folate-PEG-NH 2 dissolved in chloroform was then added.
  • DPlsC Diplasmenylcholine lipid
  • DPlsC Diplasmenylcholine lipid
  • 13.6 mg of DPlsC was dissolved in 0.5 ml CHC1 3 and 15 ⁇ l of folate-PEG-DSPE conjugate solution (6.7 mM in CHC1 3 ) was added.
  • the mixture was evaporated with a stream of dry N 2 to form a thin lipid film; this film was evaporated further by lyophilization for 3 hours in a 1 ⁇ vacuum.
  • the dried thin film was then hydrated with 1.0 ml of propidium iodide solution (10 mg/ml in pH 7.4 HEPES buffer containing 150 mM NaCl) using five freeze-thaw-vortex cycles to disperse the lipid as multilamellar liposomes (MLV) .
  • the MLV were extruded 10 times through two stacked 0.1 ⁇ m polycarbonate membranes at 55 °C.
  • the unencapsulated propidium, iodide was removed by gel chromatography using a Sephadex G-50 column and HEPES buffer, pH 7.4 as eluent.
  • KB cells a human nasopharyngeal epidermal carcinoma cell line were maintained in a medium containing physiological concentrations of folate, i.e., minimum essential medium minus the folic acid additives and supplemented with 10% heat-inactivated fetal calf serum.
  • the cells were grown at 37°C in a humidified atmosphere containing 5% C0 2 .
  • the folate content of the fetal calf serum supplement brings the folate concentration of the medium to a near physiological value for human serum. Liposome preparation.
  • DPlsC Liposomes were prepared by hydration of thin lipid films in the presence of analyte (50mM calcein solution or 10 mg/ml propidium iodide in phosphate buffered saline) , followed by extrusion at 55°C through two lOOnm Nuclepore filters. Extraliposomal analytes were removed by Sephadex G-50 gel filtration.
  • analyte 50mM calcein solution or 10 mg/ml propidium iodide in phosphate buffered saline
  • Calcein fluorescence dequenching was monitored by diluting 50 ⁇ l aliquots of the hydrolysis mixtuwre into 2 ml of 150 mM NaCl/20 mM HEPES, pH 7.4 prior to measurement of the calcein fluorescence spectrum; leackage rates were determined using a ratio method described below (under the heading: Assay) .
  • Folate- targeted DPlsC liposomes were prepared as descrived above, except that 0.5% DSPE-PEG3350-folate was incorporated in the lipid film prior to hydration in the presence of 10 mg/ml propidium iodide (PI) .
  • Extraliposomal propidium iodide was removed by gel filtration using 20 mM pphosphate buffered saline, pH 7.4 (PBS) as eluent.
  • % release (flu x -flu lnitial /flu max -flu lnitlal ) 100, where flu, was the fluorescence at each time point, and flu max was the fluorescence of maximum release at the same time point.
  • Fluorescence assay of KB cells treated with DPlsC:folate liposomes containing encapsulated propidium iodide (PI) indicate that acidification of these folate- targeted liposomes within the endosomal compartment leads to rapid and efficient release of PI into the cytoplasm (83% PI release within 8 h) .
  • the ability of folate- targeted DPlsC:DHC liposomes to promote endosomal release in KB cells was evaluated by fluorometric assay (540 nm excitation, 615 nm emission) using PI as a fluorescent probe.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • PI release kinetics revealed that 83% of the encapsulated PI escaped both the liposomal and endosomal compartments within 8 hours when ⁇ IO mol% DHC was present in the DPlsC membrane; 36% release occurred within 8 h (50% after 24 h) when the DHC content was increased to 20 mol% (Fig. 3) .
  • EALA folate-targeted egg phosphatidyleholine
  • DPlsC (33.4 mg in 2.0 ml CHC1 3 ) was combined with 35 ⁇ l of folate-PEG-DSPE conjugate solution (6.7 mM in CHC1 3 ) .
  • the mixture was evaporated with a stream of dry N 2 , the resulting thin film was lyophilized in a l ⁇ vacuum for 4 hours.
  • the lipid film was then hydrated with 1.0 ml of Ara-C solution (2.0 M in pH 7.4 PBS buffer) for 4 hours, freeze-thaw-vortexed five times, and extruded 10 times through two stacked 0.1 urn polycarbonate membranes at 55°C.
  • the extravesicular Ara-C was removed by gel filtration using a Sephadex G-50 column and phosphate buffered saline (PBS), pH 7.4 as eluent.
  • PBS phosphate buffered saline
  • the same procedure as described immediately above was used to prepare the control empty DPlsC:folate liposomes, except that the lipid was hydrated with PBS buffer containing no Ara-C.
  • KB cells were plated in 24-well culture plates and grown for 24 h to approximately 50% confluence before treatment with free Ara-C, Ara-C encapsulated in egg phosphatidylcholine (EPC):folate liposomes, and Ara-C encapsulated in DPlsC:folate liposomes.
  • the cells were then washed to remove the unbound drug and incubated in fresh media in the presence of 2 ⁇ Ci/well [ 3 H]thymidine. After 24 h, cells were lysed, and the DNA precipitated with trichloroacetic acid. The DNA was then dissolved in 2 N NaOH and the [ 3 H]thymidine incorporation measured by scintillation counting.
  • DPlsC:folate liposomes is 0.49 ⁇ M in KB cell cultures compared to an IC 50 value of 2.6mM for free Ara-C.
  • folate-targeted DPlsC liposomes exhibit a remarkable 6000- fold enhancement of inhibition relative to free Ara-C in KB cell cultures.
  • the IC 50 value of Ara-C encapsulated in EPC:folate liposomes is 40.0 ⁇ M in KB cell cultures, thus DPlsC:folate liposomes exhibit an approximate 100-fold enhancement over non-triggerable targeted liposomes.
  • DPlsC:10 mol%DHC-folate liposomes containing Ara-C represent an improvement over transferrin-conjugated, Ara-C containing pH-sensitive PE liposomes by a factor of greater than sixty (the IC 50 value for the transferin- liposomes is 30.0 ⁇ M) and pH-sensitive immunoliposomes by a factor exceeding 1000.
  • No inhibition of DNA synthesis was observed in KB cells treated with empty DPlsC-folate liposomes (control) , indicating that neither the lipid nor its degradation products have a significant effect on cellular function at the lipid concentrations used.
  • Fig. 6 shows total PI bound to KB cells. After KB cells were incubated with free PI or the various targeted (DPlsC:folate + DOPC:folate) and non-targeted (DPlsC) liposomes the cells were wshed and then lysed to determined the total ng PI bound to the cells. The data shows a significant increse in the numer of targeted liposomes bound to the KB cells relative to non-targeted liposomes and free PI.

Abstract

Procédé et liposome améliorés permettant l'apport d'une molécule hexogène au cytoplasme d'une cellule. La membrane liposomique comprend des lipides ciblables et des lipides complexés à un ligand, le ligand pouvant avoir une interaction avec la membrane cellulaire afin d'augmenter l'absorption du ligand et du liposome fixé.
PCT/US1997/003077 1996-02-27 1997-02-26 Systeme d'apport de liposome WO1997031624A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1319667A2 (fr) * 2001-12-07 2003-06-18 Development Center For Biotechnology Procédé pour la synthèse en phase solide de conjugués peptide-espaceur-lipide, les conjugués synthésisée par ledit procédé, et liposomes cibles contenant ledit conjugués
EP1588706A2 (fr) 1998-12-23 2005-10-26 Novartis AG Utilisation d'un antagoniste du récepteur d'AT-1 ou d'un modulateur du récepteur d'AT-2 pour traiter des affections associées à une augmentation des récepteurs d'AT-1 ou d'AT-2
JP2008115147A (ja) * 2006-10-31 2008-05-22 Inst Nuclear Energy Research Rocaec 脂質−スペーサ−官能基−ペプチドを製造する方法
US7641914B2 (en) 2000-10-11 2010-01-05 Gencell, S.A. Acid-sensitive compounds, their preparation and uses
US11185505B2 (en) 2010-05-28 2021-11-30 Purdue Research Foundation Delivery of agents to inflamed tissues using folate-targeted liposomes

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5277913A (en) * 1991-09-09 1994-01-11 Thompson David H Liposomal delivery system with photoactivatable triggered release
US5399331A (en) * 1985-06-26 1995-03-21 The Liposome Company, Inc. Method for protein-liposome coupling

Patent Citations (2)

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US5399331A (en) * 1985-06-26 1995-03-21 The Liposome Company, Inc. Method for protein-liposome coupling
US5277913A (en) * 1991-09-09 1994-01-11 Thompson David H Liposomal delivery system with photoactivatable triggered release

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1588706A2 (fr) 1998-12-23 2005-10-26 Novartis AG Utilisation d'un antagoniste du récepteur d'AT-1 ou d'un modulateur du récepteur d'AT-2 pour traiter des affections associées à une augmentation des récepteurs d'AT-1 ou d'AT-2
US7641914B2 (en) 2000-10-11 2010-01-05 Gencell, S.A. Acid-sensitive compounds, their preparation and uses
EP1319667A2 (fr) * 2001-12-07 2003-06-18 Development Center For Biotechnology Procédé pour la synthèse en phase solide de conjugués peptide-espaceur-lipide, les conjugués synthésisée par ledit procédé, et liposomes cibles contenant ledit conjugués
EP1319667A3 (fr) * 2001-12-07 2003-07-02 Development Center For Biotechnology Procédé pour la synthèse en phase solide de conjugués peptide-espaceur-lipide, les conjugués synthésisée par ledit procédé, et liposomes cibles contenant ledit conjugués
US7153933B2 (en) 2001-12-07 2006-12-26 Development Center For Biotechnology Solid phase method for synthesis peptide-spacer-lipid conjugates, conjugates synthesized thereby and targeted liposomes containing the same
JP2008115147A (ja) * 2006-10-31 2008-05-22 Inst Nuclear Energy Research Rocaec 脂質−スペーサ−官能基−ペプチドを製造する方法
US11185505B2 (en) 2010-05-28 2021-11-30 Purdue Research Foundation Delivery of agents to inflamed tissues using folate-targeted liposomes

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