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WO1998000112A1 - Systemes organises contenant des polyelectrolytes pieges et a charge negative - Google Patents

Systemes organises contenant des polyelectrolytes pieges et a charge negative Download PDF

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Publication number
WO1998000112A1
WO1998000112A1 PCT/IE1997/000044 IE9700044W WO9800112A1 WO 1998000112 A1 WO1998000112 A1 WO 1998000112A1 IE 9700044 W IE9700044 W IE 9700044W WO 9800112 A1 WO9800112 A1 WO 9800112A1
Authority
WO
WIPO (PCT)
Prior art keywords
polyelectrolyte
organised
lipid
dna
negatively charged
Prior art date
Application number
PCT/IE1997/000044
Other languages
English (en)
Inventor
Kenneth Adrian Dawson
Alexander Vladimirovich Gorelov
Original Assignee
University College Dublin
Rochev, Jury
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University College Dublin, Rochev, Jury filed Critical University College Dublin
Priority to AU31875/97A priority Critical patent/AU3187597A/en
Priority to EP97927347A priority patent/EP0909165A1/fr
Publication of WO1998000112A1 publication Critical patent/WO1998000112A1/fr

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Classifications

    • 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
    • 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
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases

Definitions

  • This invention relates to delivery systems for delivering active agents to target sites and, in particular, to organised assemblies for delivering negatively charged polyelectrolytes intracellularly.
  • Multilamellar vesicles are known and have been used to deliver small drug molecules.
  • U.S. Patent No. 5,173,219 covers a method for making multilamellar liposomes having a spherical configuration and adjustable size. These liposomes are indicated in Example 5 to be capable of incorporating both lipid soluble and water soluble substances with an efficiency of approximately 74% and 56%, respectively.
  • a water soluble material such as doxorubicin
  • the drug must be dissolved in a 5% glucose aqueous phase and/or in a high strength aqueous phase at the evaporation step.
  • multilamellar vesicles which can incorporate with high efficiency negatively charged polyelectrolytes, including negatively charged oligomers, such as oligonucleotides for use, for example, in the transfection of cells.
  • cationic lipids are found to be toxic above about 5 nanomolar amounts. Accordingly, they cannot be used, for example, in sufficiently high concentrations to ensure significant transfection to be of practical use in vivo (Behr, J-P., (1994) Bioconjugate Chem., 5 No. 5 383). Van der Woude, I. et al ((1995) Bi ⁇ chimica et Biophysica Acta 1240 34-40) describe the use of vesicles of synthetic cationic amphiphiles as carrier systems for DNA in the transfection of mammalian cells and show how at high concentrations such amphiphiles become toxic as reflected by an enhanced degree of hemolysis.
  • DNA has been trapped between layers of cationic lipids in a Langmuir-Blodgett film (Okahata, Y., et al., (1996) Langmuir 12 1326-1330).
  • Phospholipids cannot directly interact with DNA because of the repulsive forces that arise between similarly charged species. This interaction can be mediated, however, by the use of cationic molecules and also by the use of divalent metal cations (Budker, V.G. et al. (1980) Nucl. Acid Research 8 2499-2515). However these workers studied only the binding of the lipids to the DNA and did not report any attempts at encapsulation.
  • the invention provides organised assemblies of zwitterionic lipids and negatively charged polyelectrolytes formed from repeating monomer units in which the polyelectrolyte is substantially uniformly distributed.
  • the assemblies according to the invention can entrap the polyelectrolyte with an efficiency of the order of 70% or greater and, can, therefore, be used as an effective means for the delivery of active polyelectrolytes to eucaryotic and procaryotic cells.
  • negatively charged polyelectrolytes formed from repeating monomer units as used herein is meant negatively charged oligomers and polyelectrolytes.
  • the term embraces oligomers, such as oligonucleotides, including anti-sense oligomers.
  • polymers such as the nucleic acids DNA and RNA, polysaccharides and proteins with a net negative charge.
  • the zwitterionic lipids for use in forming the organised assemblies according to the invention are preferably naturally occurring phospholipids selected from phosphatidylcholine, phosphatidylethanolamine, cardiolipin, sphyngomyelin, lysophosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid.
  • any natural or synthetic membrane-forming lipid bearing at least one pair of negative and positive charges can be used.
  • Especially preferred phospholipids include phosphatidyl choline
  • the organised assemblies according to the invention can be in the form of multilamellar vesicles with the polyelectrolyte entrapped between lipid bilayers.
  • the organised assemblies can be in the form of substantially fibrillar or tubular structures, hereinafter referred to collectively as tubular structures, except where specific mention is made of such fibrillar structures.
  • the multilamellar vesicles can also include an amount of such tubular structures.
  • the invention also provides a process for preparing the organised assemblies according to the invention, which process comprises the following steps: i) mixing unilamellar vesicles of a zwitterionic lipid material with a negatively charged polyelectrolyte formed from repeating monomer units in an aqueous medium;
  • the lipid monomers are bound to the polyelectrolyte by the bridging cation and form structures where the monomers wrap around said polyelectrolyte.
  • the size and lamellarity of the resultant vesicles can be varied by selecting a suitable ratio of polyanion to lipid, the initial concentration of both lipid and polyanion and the concentration of cation.
  • the unilamellar vesicles of zwitterionic lipids are prepared in a manner known per se, such as by sonication.
  • the multivalent cation is preferably a divalent or trivalent cation, more especially a divalent cation selected from calcium, magnesium and zinc.
  • any non-toxic, multivalent cation can be used, for example Fe +++ ,which would have the capability of binding to three negatives charges, for example two lipids to one DNA side chain.
  • a divalent cation a negative charge on the zwitterionic lipid binds to one of the positive charges and a negative charge on the polyelectrolyte binds to the other positive charge so as to form a bridge between the lipid and the polyelectrolyte so that highly organised assemblies or packages are formed.
  • the amount of polyelectrolyte that can be used relative to a given amount of zwitterionic lipid is determined stoichiometrically.
  • the amount of polyelectrolyte that can be used relative to a given amount of zwitterionic lipid is determined stoichiometrically.
  • Step ii) can be carried out in a wide range of media.
  • Typical media include distilled water, pure water and almost any aqueous solution, including buffer solutions with and without electrolytes such as sodium chloride.
  • the concentration of the multivalent cation used is also determined stoichiometrically.
  • an excess of the multivalent cation is used.
  • the cations used are derived from suitable salts.
  • the concentration of cation varies in a range from 0 to 50 mmoles/litre until the polyanion-liposome-cation complex is obtained.
  • concentration of cation can be used and any excess electrolyte can be washed out.
  • Water is preferably removed in step iii) by freeze-drying.
  • step iii) involve a strong bonding of lipid and polyelectrolyte, but are of undetermined form.
  • the membrane-disrupting solvent is preferably selected from organic solvents such as alcohols, aldehydes, amides, amines, ethers, halogenated hydrocarbons, ketones, nitriles, sulphoxides, thiols and thioethers or a mixture thereof.
  • Preferred halogenated hydrocarbons are chlorinated alkanes and fluorinated alkanes, more especially chloroform.
  • the membrane-disrupting solvent is removed by evaporation of the solvent under vacuum or under a stream of inert gas.
  • the reconstitution of the solid material is carried out by resuspending the material by shaking, vortexing or ultrasound treatment.
  • Suitable aqueous media include pure water and almost any aqueous solution and buffer solutions with and without electrolytes, such as sodium chloride.
  • the invention also provides a delivery system comprising organised assemblies according to the invention for the purposes of transfection or drug delivery.
  • the organised assemblies according to the invention can be formulated in various forms and administered to a subject in many ways. For example, they can be administered parenterally or intravenously as suspensions or in a form suitable for inhalation. Alternatively, they can be formulated for topical application.
  • Fig. 1 is a series of DSC thermograms for DNA-calcium-DPPC complexes prepared in accordance with Example 1 ;
  • Figs. 2A - 2F are electron micrographs of the vesicles and other organised assemblies of DPPC described in Example 1 ;
  • Figs. 3A-3D are freeze fracture micrographs and
  • Fig. 3E a negative contrast micrograph of DNA-calcium-EggPC complexes prepared in accordance with Example 2;
  • Fig. 4 is a bright-field image of cells after incubation for 2 hours following treatment as described in Example 3.
  • Fig. 5 is a fluorescent image of the cells of Fig. 4.
  • Short fragments of calf thymus DNA (Sigma) were obtained by a standard sonication procedure.
  • the calf thymus DNA was additionally purified by phenol and chloroform (the value A 260 A 280 was more than 1.9) and mildly treated by ultrasound until rather homogeneous native fragments were formed.
  • the DNA fragments (1 mg/ml) were mixed with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC)
  • the medium used was a 0.5 mM HEPES buffer solution having a pH of 7.5.
  • CaC was added slowly with rapid stirring, as stock solution (100 mM) in the same buffer, to this mixture to a final concentration of 20 mM.
  • the resulting cloudy mixture was freeze-dried and resuspended in chloroform. After removing the chloroform by rotary evaporation, water was added to the volume of the initial mixture before freeze-drying.
  • DSC showed that at high DNA to lipid ratio (1 :2 mole/mole) only one transition with temperature, well above the transition temperature of pure DPPC, occurred. At lower DNA to lipid ratio a second peak emerged with the transition temperature of pure lipid as shown in Fig 1.
  • DSC thermograms were obtained of the DNA-calcium-DPPC complexes in the presence of the specified molar proportion (n) of DNA:lipid as shown in Fig. 1. Measurements were carried out by means of a differential scanning microcalorimeter DASM-4 with a rate of heating of 0.25 °K/min. The results obtained confirm that DNA is highly associated with the lipid and is spread relatively uniformly in LMV.
  • EM Freeze-fracture electron microscopy
  • the DSC thermograms of DNA-Ca 2+ -DPCC complex reveal the appearance of a distinct maximum at a temperature of about 43.3°C in addition to the main maximum at 41.6°C.
  • the total enthalpy of both transitions for all the scans we have performed was found to be about 7 ⁇ 0.6 kcal/mol.
  • the thermograms show that in the conditions of this experiment, a large part of the lipid was involved in the formation of DNA-lipid complexes with new thermotropic properties.
  • Example 1 was repeated except that egg lecithin (EggPC) was used in place of DPPC.
  • Electron microscopy revealed tubular-like structures in bilayers as depicted in Figs. 3A-3E corresponding to freeze-fracture (Figs. 3A-3D) and negative contrast (Fig. 3E) micrographs of DNA- Ca 2+ -EggPC complexes prepared as described for complexes with DPPC in Example 1. Circled arrowheads in the corners of all freeze- fracture micrographs mark the shadow direction. Bars represent lOOnm. The samples were quenched in propane using the sandwich technique. No cryoprotectors or chemical fixators were used.
  • FIG. 3 A shows intramembrane particles and rods on the fracture surface of membrane vesicles (asterisk) and free regular fibrils in suspension (arrowheads)
  • Fig 3B shows fibrils for Fig 3A at higher magnification.
  • Fig 3C shows another type of fibril for which the capability to form coils and branches is demonstrated. It should be noted that in Figs.
  • Rod-like fibres (Figs. 3A and 3D) on the hydrophobic fracture surface are rather similar to "spaghetti" structures found in complexes of DNA with synthetic cationized lipids (Sternberg, B., et al. (1994) FEBS Lett. 336, 361-366) and are believed to represent inverted tubes of lipid surrounding DNA molecules in the membrane bilayer.
  • the DNA-cation complexes could modify the structural organization of the hydrophobic region of membranes formed by natural lipids and initiate formation of rod-like intramembrane particles.
  • the regular bundles of fibrils represent another kind of DNA-lipid complex. Most of the bundles were not connected to membranes and existed free in suspension. The visual appearance of the bundles revealed both by freeze-fracture (Figs. 3 A, B, C) and negative staining (Fig. 3E) microscopy was similar. Electron microscopy revealed the electron dense strips separated by unstained white strips of lipid. Although not wishing to be bound by any theoretical explanation of the invention we believe that the regular bundles are formed by lipid tubes filled by DNA in the central hole. The affinity of DNA for uranyl acetate could be responsible for the formation of dark strips.
  • Lecithins usually form stable bilayer structures and are not inclined to polymorphic behaviour except for cases where some specific biological active modulators are present. It seems reasonable to postulate, therefore, that complexes of DNA with polyvalent cations could occupy a prominent place among the known modulators of polymorphic transitions in lecithins.
  • freeze-fracture electron microscopy of DNA-calcium-lecithin complexes revealed the formation of rather specific regular bundles of fibrils with repeat distance of about 6nm. These structures have never, as far as we are aware, been observed before. Similar structures were revealed also by staining of samples with uranyl acetate. The presented results demonstrate the capability of DNA-Ca 2+ complexes to favour the polymorphic behaviour of lecithins and initiate the formation of inverted lipid tubes with DNA in the central cores.
  • Multilamellar vesicles with oligonucleotides incorporated therein were prepared by a procedure corresponding to that described in Example 1.
  • Small unilamellar vesicles (SUV) were prepared from dipalmitoylphosphatidylcholine and dipalmitoylphosphatidyl-ethanolamine by sonication.
  • the oligonucleotides were phosphorothioate oligonucleotides labelled with fluorescein isothiocyanate.
  • the suspension of SUV was mixed with the oligonucleotides in the ratio 1 :20 mole of bases per mole of lipid in the presence of 5mM CaCl 2 in sterile twice, distilled water. The mixture was first freeze-dried and then small amounts of chloroform were added. Chloroform was then evaporated by a stream of nitrogen and then a sterile 5 mM solution of CaCl 2 was added. The resuspension of lipid-oligonucleotide complexes was facilitated by short (5- 10 min.) sonication. The resulting suspension was added to the cells in the ratio 100 nM of oligonucleotide to 250,000 cells.
  • the final concentration of Ca 2+ ions in the medium was 0.5 mM. Following incubation in a serum-free medium for 1 hour, serum was added and the cells were incubated for another 1 to 3 hours. Prior to investigation by flow- cytometry and fluorescent microscopy, the cells were washed twice with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Flow-cytometry showed that 70-100% of cells were fluorescent indicating a high level of uptake. Fluorescent microscopy showed that all cells were fluorescent with dead cells being brighter which indicates higher uptake. The results are shown in Figs. 4 and 5.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
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Abstract

Cette invention concerne des systèmes organisés de lipides zwitterioniques et de polyélectrolytes à charge négative formés par la répétition d'unités monomères, systèmes dans lesquels le polyélectrolyte est réparti de manière globalement uniforme. Ces systèmes sont capables de piéger le polyélectrolyte avec une efficacité de l'ordre de 70 %, ou plus, et peuvent ainsi être utilisés comme un moyen efficace d'administration de polyélectrolytes actifs à des cellules eucaryotes et procaryotes. Ces polyélectrolytes à charge négative peuvent consister en des oligomères tels que des oligonucléotides, y compris des oligomères anti-sens ou des polymères tels que des acides nucléiques, des polysaccharides et des protéines possédant une charge nette négative.
PCT/IE1997/000044 1996-07-02 1997-07-01 Systemes organises contenant des polyelectrolytes pieges et a charge negative WO1998000112A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU31875/97A AU3187597A (en) 1996-07-02 1997-07-01 Organised assemblies containing entrapped negatively charged polyelectrolytes
EP97927347A EP0909165A1 (fr) 1996-07-02 1997-07-01 Systemes organises contenant des polyelectrolytes pieges et a charge negative

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IE960485A IE960485A1 (en) 1996-07-02 1996-07-02 Organised assemblies containing entrapped negatively charged¹polyelectrolytes
IE960485 1996-07-02

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WO1998000112A1 true WO1998000112A1 (fr) 1998-01-08

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PCT/IE1997/000044 WO1998000112A1 (fr) 1996-07-02 1997-07-01 Systemes organises contenant des polyelectrolytes pieges et a charge negative

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AU (1) AU3187597A (fr)
IE (1) IE960485A1 (fr)
WO (1) WO1998000112A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059638A3 (fr) * 1998-05-15 2000-02-24 Chiron Corp Compositions et methodes d'administration de molecules d'acide nucleique
KR100399412B1 (ko) * 2001-01-19 2003-09-26 삼성전자주식회사 서로 다른 크기의 2개 이상의 내부 뱅크를 가진 반도체메모리 장치

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4078052A (en) * 1976-06-30 1978-03-07 The United States Of America As Represented By The Secretary Of Health, Education And Welfare Large unilamellar vesicles (LUV) and method of preparing same
DE2747378A1 (de) * 1976-10-23 1978-04-27 Choay Sa Liposomen, verfahren zu ihrer herstellung und sie enthaltende arzneimittel
US4394448A (en) * 1978-02-24 1983-07-19 Szoka Jr Francis C Method of inserting DNA into living cells
US4942036A (en) * 1988-08-25 1990-07-17 Blair Geho W Therapy by vesicle delivery to the hydroxyapatite of bone
FR2667072A1 (fr) * 1990-09-24 1992-03-27 Bioetica Sa Complexe ternaire de chitosane, d'ions calcium et de lipides, procede de preparation et leurs applications.
WO1992013524A1 (fr) * 1991-02-07 1992-08-20 A. Nattermann & Cie. Gmbh Produit pharmaceutique de traitement de maladies virales
US5512295A (en) * 1994-11-10 1996-04-30 The Board Of Trustees Of The Leland Stanford Junior University Synthetic liposomes for enhanced uptake and delivery

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4078052A (en) * 1976-06-30 1978-03-07 The United States Of America As Represented By The Secretary Of Health, Education And Welfare Large unilamellar vesicles (LUV) and method of preparing same
DE2747378A1 (de) * 1976-10-23 1978-04-27 Choay Sa Liposomen, verfahren zu ihrer herstellung und sie enthaltende arzneimittel
US4394448A (en) * 1978-02-24 1983-07-19 Szoka Jr Francis C Method of inserting DNA into living cells
US4942036A (en) * 1988-08-25 1990-07-17 Blair Geho W Therapy by vesicle delivery to the hydroxyapatite of bone
FR2667072A1 (fr) * 1990-09-24 1992-03-27 Bioetica Sa Complexe ternaire de chitosane, d'ions calcium et de lipides, procede de preparation et leurs applications.
WO1992013524A1 (fr) * 1991-02-07 1992-08-20 A. Nattermann & Cie. Gmbh Produit pharmaceutique de traitement de maladies virales
US5512295A (en) * 1994-11-10 1996-04-30 The Board Of Trustees Of The Leland Stanford Junior University Synthetic liposomes for enhanced uptake and delivery

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059638A3 (fr) * 1998-05-15 2000-02-24 Chiron Corp Compositions et methodes d'administration de molecules d'acide nucleique
KR100399412B1 (ko) * 2001-01-19 2003-09-26 삼성전자주식회사 서로 다른 크기의 2개 이상의 내부 뱅크를 가진 반도체메모리 장치

Also Published As

Publication number Publication date
EP0909165A1 (fr) 1999-04-21
AU3187597A (en) 1998-01-21
IE960485A1 (en) 1998-01-14

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