EP0909165A1

EP0909165A1 - Organised assemblies containing entrapped negatively charged polyelectrolytes

Info

Publication number: EP0909165A1
Application number: EP97927347A
Authority: EP
Inventors: Kenneth Adrian Dawson; Alexander Vladimirovich Gorelov; Jury Inst.of Theoretical and Experimental Rochev
Original assignee: University College Dublin
Current assignee: University College Dublin
Priority date: 1996-07-02
Filing date: 1997-07-01
Publication date: 1999-04-21
Also published as: IE960485A1; WO1998000112A1; AU3187597A

Abstract

Organised assemblies of zwitterionic lipids and negatively charged polyelectrolytes formed from repeating monomer units, in which the polyelectrolyte is substantially uniformly distributed, are described. The assemblies 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. The negatively charged polyelectrolytes can be oligomers such as oligonucleotides, including anti-sense oligomers or polymers such as nucleic acids, polysaccharides and proteins with a net negative charge.

Description

Organised assemblies containing entrapped negatively charged polyelectrolytes

Technical Field

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.

Background Art

Multilamellar vesicles are known and have been used to deliver small drug molecules. The advantage of using such vesicles, which are also commonly and interchangeably referred to as liposomes, is their resemblance to biological membranes.

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. In the case of 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.

There is a need for 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.

Encapsulation of negatively charged oligomers by unilamellar vesicles is known (R.R.C. New(editor) "Liposomes a Practical Approach", IRL Press (1994) 57,91). However, a very low encapsulation efficiency was reported. The use of unilamellar vesicles formed of cationic lipids is known for encapsulating negatively charged polyelectrolytes which naturally associate with such cationic lipids (Gershon, H. et al. (1993) Biochemistry, 32 7143). However, cationic lipids because of their net charge cause membrane disruption. For example, they are found to merge with the membrane bilayer structure by forming bilayers themselves. Also they form pores which can lead to bursting or lysing of the cells. Thus, 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) Biυchimica 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.

Feigner, J.H. et al ((1994) The Journal of Biological Chemistry 269 4, 2550-2561) have formulated cationic and neutral phospholipids together as large multilamellar vesicles or small sonicated unilamellar vesicles in water and found that this mixture of lipid types improved

DNA uptake. These workers apparently continued to use cationic lipids because this appeared to be the only way to entrap in an effective way the charged species in neutral lipid.

To date it has only been possible to encapsulate or entrap modest amounts (-40% efficiency) of active negatively charged polyelectrolytes with uncharged lipid systems by an "entrapment" technique, whereby vesicles are formed in a solution containing DNA while at the same time trapping the DNA contained in the solution about which the vesicles are formed. The efficiency of incorporation is based on the fraction of the initial DNA sample which is incorporated.

In the case of cationic lipids unilamellar vesicles formed of such cationic lipids are laid down along the DNA strands so that significantly higher efficiencies are possible with this technique than with the technique involving uncharged lipids.

DNA has been trapped between layers of cationic lipids in a Langmuir-Blodgett film (Okahata, Y., et al., (1996) Langmuir 12 1326-1330).

There are no reports in the literature of DNA or other negatively charged polyelectrolytes being encapsulated between the layers of a multilamellar vesicle formed from cationic or mixed cationic and neutral lipids.

However, in the case of the two last mentioned systems, unilamellar liposomes are found to be the most efficient because extra wall thickness does not increase the amount of material that liposomes can hold.

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.

Disclosure of Invention

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. By "negatively charged polyelectrolytes formed from repeating monomer units" as used herein is meant negatively charged oligomers and polyelectrolytes. Thus, the term embraces oligomers, such as oligonucleotides, including anti-sense oligomers. The term also embraces 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.

However, 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

(lecithin) and phosphatidyl ethanolamine.

The organised assemblies according to the invention can be in the form of multilamellar vesicles with the polyelectrolyte entrapped between lipid bilayers. Alternatively, 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. These structures are further described and illustrated in the Examples and accompanying Figures.

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;

ii) adding a solution containing a multivalent cation to the mixture of lipid vesicles and polyelectrolyte;

iii) removing substantially all water from the particles obtained in step ii);

iv) adding an amount of a membrane-disrupting solvent to the mixture;

v) removing the solvent; and

vi) reconstituting the solid material in an aqueous medium so as to generate said organised assemblies.

In the formation of the assemblies according to the invention 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, as hereinabove defined, 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. However, in practice 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. When a divalent cation is used 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. Thus, depending on the nature of the polyelectrolyte used and, indeed, the multivalent cation one can determine how much lipid can be attached to a single oligomer or polymer.

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.

Preferably, an excess of the multivalent cation is used. The cations used are derived from suitable salts.

Suitably the concentration of cation varies in a range from 0 to 50 mmoles/litre until the polyanion-liposome-cation complex is obtained. However, in practice almost any concentration of cation can be used and any excess electrolyte can be washed out.

Water is preferably removed in step iii) by freeze-drying.

The structures that result from 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.

Preferably, the membrane-disrupting solvent is removed by evaporation of the solvent under vacuum or under a stream of inert gas.

Preferably, 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.

Brief Description of Drawings:

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; and

Fig. 5 is a fluorescent image of the cells of Fig. 4.

Modes for Carrying Out the Invention

The invention will be further illustrated by the following Examples.

Example 1

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 length of the fragments, as determined by electrophoresis, was in the range 300-500 base pairs (bp).

The DNA fragments (1 mg/ml) were mixed with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC)

(obtained from Avanti) obtained by sonication. A variety of DNA to lipid rations were used as indicated below.

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.

Vortexing the resulting hydrated lipid-DNA film at 45-50°C yielded a suspension of large multilamellar vesicles (LMV).

Addition of the Ca²⁺ complexing agent EDTA and sedimentation of LMV by centrifugation allowed separation of DNA which had been entrapped from DNA remaining in solution or bound to the outside of the LMV. The ratio of DNA in the LMVs to that in the supernatant or bound to the outside of the LMVs was 7:3. The complexes were kept for about 30 min. at 18°C before differential scanning calorimetry (DSC) measurement.

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.

Freeze-fracture electron microscopy (EM) of the DPPC LMV revealed fracture surfaces characteristic for multilamellar vesicles as shown in Fig 2.

Key to Fig. 2:

A) Multilamellar vesicles without DNA B) Multilamellar vesicles with DNA, wherein the ratio of DNA to lipid is 1:2 mole/mole;

C) Small unilamellar vesicles obtained by sonication;

D) Unknown particles in bilayer;

E) Complexes of small unilamellar vesicles with DNA and calcium before freeze-drying and treatment with chloroform; and

F) Irregular rod-like structures appear on the fracture surface.

In the case of Fig. 2E, it will be observed that despite the absence of multilamellar structures the vesicles are larger than the vesicles depicted in Fig. 2C at the same magnification, which indicates the DNA-induced fusion of vesicles.

The above results indicate that the DNA-lecithin complexes, mediated by Ca²⁺ or Mg²⁺ cations, is a rather useful model of DNA-lipid interactions. The DSC thermograms of DNA-Ca²⁺-DPCC complex (Fig. 1) 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. There is a correlation between the increase of the molar proportion of DNA in samples and the increase of the second peak, which indicates that the high temperature transition corresponds to the formation of DNA- Ca²⁺-DPPC complexes. 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 2

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- Ca²⁺-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.

Fracture and carbon-platinum shadowing were performed at -150°C in a JEOL system as previously described (Borovyagin, V.L., et al., (1987) J. Membran. Biol. 100, 229-242). For negative contrast the samples were stained by 2% uranil acetate and then applied to carbon coated grids. 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. 3B and 3C the photo negatives are presented. Rod - like fibrils on the hydrophobic fracture surface of membranes are apparent in Fig. 3D. Regular bundles of fibrils similar to those demonstrated in Figs. 3A and 3B are revealed also by staining with uranil acetate (see Fig. 3E).

Thus, we have observed two types of structures: round and rod-like particles located on the hydrophobic fracture surface of some liposomes and regular bundles of fibres residing free in the suspension (Fig 3.). 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 (Figs. 3 A, B, C and E) 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.

It is likely that the fibrillar bundles correspond to hexagonal organisation of lipid tubes filled with DNA in the central core. The revealed repeat distance of about 6θA is typical for hexagonal tubes of phospholipids (De Kruijff, B., et al. in the Enzymes of Biological Membranes. V.l, Membrane Structure and Dynamics. (Ed. Martonosi A.N.) 131-204 (Plenum Press, New York & London 1985). As indicated above DNA fragments of about 300-500 bp were used which corresponds to a DNA length of about 100-150 nm. However, the length of the observed bundles was much higher which indicates that in lipid tubes the DNA fragments might be oriented tail-to-tail.

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.

Thus, 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-Ca²⁺ complexes to favour the polymorphic behaviour of lecithins and initiate the formation of inverted lipid tubes with DNA in the central cores.

Example 3

The delivery of fluorescently-labelled oligonucleotides to a suspension of leukemia cells with the aid of multilamellar vesicles according to the invention has been studied. 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 CaCl₂ 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 CaCl₂ 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 Ca²⁺ 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).

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.

Claims

Claims:-

1. An organised assembly of a zwitterionic lipid and a negatively charged polyelectrolyte formed from repeating monomer units in which the polyelectrolyte is substantially uniformly distributed.

2. An assembly according to Claim 1 , which is in the form of a multilamellar vesicle with the polyelectrolyte entrapped between lipid bilayers.

3. An assembly according to Claim 1 , which is in the form of substantially tubular structures.

4. An assembly according to Claim 2, which includes an amount of tubular structures according to Claim 3.

5. An assembly according to any preceding claim, wherein the oligomeric polyelectrolyte is a nucleic acid.

6. An assembly according to any one of Claims 1-4, wherein the oligomeric polyelectrolyte is a polysaccharide.

7. A process for preparing organised assemblies according to any one of Claims 1-6, which 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;

iii) removing substantially all water from the particles obtained in step ii); iv) adding an amount of a membrane-disrupting solvent to the mixture;

v) removing the solvent; and

8. A process according to Claim 7, wherein the multivalent cation is selected from calcium, magnesium, manganese and zinc.

9. A process according to Claim 7 or 8, wherein the membrane-disrupting solvent is selected from alcohols, aldehydes, amides, amines, ethers, halogenated hydrocarbons, ketones, nitriles, sulphoxides, thiols and thioethers or a mixture thereof.

10. A delivery system comprising organised assemblies according to any one of Claims 1-6.