U.S. patent application number 10/015078 was filed with the patent office on 2002-08-01 for macromolecule-lipid complexes and methods for making and using.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Koltover, Ilya, Raedler, Joachim Oskar, Safinya, Cyrus R..
Application Number | 20020102297 10/015078 |
Document ID | / |
Family ID | 22306574 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102297 |
Kind Code |
A1 |
Safinya, Cyrus R. ; et
al. |
August 1, 2002 |
Macromolecule-lipid complexes and methods for making and using
Abstract
The invention provides novel compositions involving
macromolecule-lipid complexes and methods for making them. These
compositions and methods of the invention are significant
improvements in the field of macromolecule-lipid complex
processing, macromolecule targeting and delivery to various
biological systems.
Inventors: |
Safinya, Cyrus R.; (Santa
Barbara, CA) ; Raedler, Joachim Oskar; (Garching,
DE) ; Koltover, Ilya; (Pasadena, CA) |
Correspondence
Address: |
MANDEL & ADRIANO
35 NORTH ARROYO PARKWAY
SUITE 60
PASADENA
CA
91103
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
22306574 |
Appl. No.: |
10/015078 |
Filed: |
October 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10015078 |
Oct 26, 2001 |
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09105571 |
Jun 26, 1998 |
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09105571 |
Jun 26, 1998 |
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08985625 |
Dec 5, 1997 |
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60032163 |
Dec 6, 1996 |
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Current U.S.
Class: |
424/450 ;
702/19 |
Current CPC
Class: |
A61K 9/1272 20130101;
B82Y 5/00 20130101; A61K 47/6911 20170801 |
Class at
Publication: |
424/450 ;
702/19 |
International
Class: |
A61K 009/127; G06F
019/00 |
Goverment Interests
[0002] This invention was made with Government support under NSF
grants DMR-9624091 and DMR-9632716. The Government has certain
rights in this invention.
Claims
What is claimed is:
1. A method for regulating the structure of a macromolecule-lipid
complex having: a. a selected characteristic or multiple selected
characteristics from the group of macromolecule interaxial distance
(d.sub.M), membrane thickness of the lipid combination
(.delta..sub.m), macromolecule area (A.sub.M), macromolecule
density (.rho..sub.M), lipid density (.rho.L), and the ratio (L/D)
between the weight of the lipid combination (L) and the weight of
the macromolecule (D); and b. a charged macromolecule and lipid
combination; the method comprising selecting the characteristic or
characteristics from the group and modulating one or more of the
non-selected characteristics from the group so as to produce the
macromolecule-lipid complex having the desired structure.
2. A method for regulating the structure of a macromolecule-lipid
complex comprising: a. selecting a charged macromolecule; b.
selecting a charged lipid combination; the charge of the lipid
combination being opposite of the charge of the macromolecule; c.
determining an amount of the macromolecule and the lipid
combination sufficient to regulate the structure of the complex by:
i. selecting a characteristic or multiple characteristics of the
complex from a group of characteristics consisting of macromolecule
interaxial distance (d.sub.M), membrane thickness of the lipid
combination (.delta..sub.m), macromolecule area (A.sub.M),
macromolecule density (.rho..sub.M), lipid density (.rho..sub.L),
and the ratio (L/D) between the weight of the lipid combination (L)
and the weight of the macromolecule (D); and ii. modulating any of
the characteristics not selected in (i) so as to achieve the
selected characteristic thereby determining the amount of the
macromolecule and lipid combination sufficient to regulate the
structure of the complex; and d. combining the macromolecule with
the lipid combination in the amount so determined thereby resulting
in the complex having the desired structure.
3. A method for regulating the interaxial distance of adjacent
macromolecules within a macromolecule-lipid complex comprising: a.
selecting a charged macromolecule; b. selecting a charged lipid
combination; the charge of the lipid combination being opposite of
the charge of the macromolecule; c. determining an amount of the
macromolecule of (a) and the lipid combination of (b) sufficient to
regulate the structure of the complex by: i. selecting a desired
macromolecule interaxial distance (d.sub.M); and ii. modulating any
of membrane thickness of the lipid combination (.delta..sub.M),
macromolecule area (A.sub.M), macromolecule density (.rho..sub.M),
lipid density (.rho..sub.L), and the ratio (L/D) between the weight
of the lipid combination (L) and the weight of the macromolecule
(D) so as to achieve the desired macromolecule interaxial distance;
and d. combining the macromolecule with the lipid combination in
the amounts so determined so as to produce the complex having the
desired structure.
4. A method for regulating the density of macromolecules within a
macromolecule-lipid complex comprising: a. selecting a charged
macromolecule; b. selecting a lipid combination; the charge of the
lipid combination being opposite of the charge of the
macromolecule; c. determining an amount of the macromolecule of (a)
and the lipid combination of (b) sufficient to regulate the
structure of the complex by: i. selecting a desired macromolecule
density; and ii. modulating any of membrane thickness of the lipid
combination (.delta..sub.M), macromolecule area (A.sub.M),
macromolecule interaxial distance, lipid density (.rho..sub.L), and
the ratio (L/D) between the weight of the lipid combination (L) and
the weight of the macromolecule (D) so as to achieve the desired
macromolecule density, d. combining the macromolecule with the
lipid combination in the amount so determined so as to produce the
complex having the desired structure.
5. The method of claim 1 or 2, wherein the characteristic so
selected from group is macromolecule interaxial distance or
macromolecule density.
6. The method of claim 1 or 2, wherein the characteristics so
selected from the group are macromolecule interaxial distance and
macromolecule density.
7. The method of claim 1, 2, 3, or 4, wherein modulating is
effected using the formula: d.sub.M=(L/D)
(A.sub.M.rho..sub.M)/(.delta..sub.m.rho.L).
8. The method of claim 1 or 2, wherein the macromolecule is a
charged macromolecule and the charge of the lipid combination is
opposite of the charge of the macromolecule.
9. The method of claim 1, 2, 3, or 4, wherein the macromolecule is
a nucleic acid molecule.
10. The method of claim 1, 2, 3, or 4, wherein the macromolecule
may be linear, circular, nicked circular or supercoiled.
11. The method of claim 10, wherein the nucleic acid molecule is a
DNA or RNA.
12. The method of claim 1, 2, 3, or 4, wherein the macromolecule is
a peptide, protein, polysaccharide, a combination of a protein and
carbohydrate moiety.
13. The method of claim 1, 2, 3, or 4, wherein the lipid
combination comprises a neutral lipid component and a charged lipid
component.
14. The method of claim 1, 2, 3, or 4, wherein the lipid
combination and the macromolecule are associated so as to form a
complex in an isoelectric point state.
15. The method of claim 1, 2, 3, or 4, wherein the lipid
combination and the macromolecule are associated so as to form a
complex in a positively charged state.
16. The method of claim 1, 2, 3, or 4, wherein the lipid
combination and the macromolecule are associated so as to form a
complex in a negatively charged state.
17. The method of claim 13, wherein the neutral lipid is dioleoyl
phosphatidyl cholin (DOPC) or
1,2-dioleoyl-sn-glycero-3-phosphoethanolami- ne (DOPE).
18. The method of claim 13, wherein the charged lipid is
1,2-diacyl-3-trimethylammonium-propane (DOTAP).
19. The method of claim 1, 2, 3, or 4, wherein the
macromolecule-lipid complex is a multilamellar structure wherein
the lipid combination forms alternating lipid bilayers and
macromolecule monolayers.
20. The method of claim 1, 2, 3, or 4, wherein the
macromolecule-lipid complex forms either an inverted hexagonal
complex phase or a regular hexagonal complex phase.
21. A macromolecule-lipid complex produced by the method of claim
1, 2, 3, or 4.
22. The macromolecule-lipid complex of claim 21, wherein the
macromolecule comprises: i. a lipid combination having a charged
lipid component and a neutral lipid component; and ii. a charged
macromolecule; the charge of the lipid combination being opposite
of the charge of the macromolecule; the lipid and the macromolecule
being associated so as to form a complex in an isoelectric point
state, wherein lipid combination forms a bilayer membrane to which
the charged macromolecules are associated in an isoelectric point
state, wherein the relative amounts of the neutral lipid component
relative to the charged lipid component generates the lipid bilayer
membrane having a thickness of between 25 and 75 angstroms.
23. A macromolecule-lipid complex of claim 21, wherein the complex
comprises: i. a charged lipid combination; and ii. a charged
macromolecule; the charge of the lipid combination being opposite
of the charge of the nucleic acid molecule; the lipid and the
macromolecule being associated so as to form a complex in an
isoelectric point state, wherein: a. the lipids form a bilayer
membrane to which the macromolecule is associated, wherein the
relative amounts of the lipid components generate the lipid bilayer
membrane having a thickness of between 25 and 75 angstroms; and b.
the conformation of the complex has macromolecule exhibiting
interaxial spacing of a range between 50 and 75 angstroms.
24. A method for creating a pattern on a surface comprising: a.
selecting a charged macromolecule; b. selecting a lipid
combination; c. determining an amount of the macromolecule of (a)
and the lipid combination of (b) sufficient to regulate the
structure of the complex by: i. selecting a desired macromolecule
density or interaxial distance; and ii. modulating any of membrane
thickness of the lipid combination (.delta..sub.m), macromolecule
area (A.sub.M), lipid density (.rho..sub.L), and the ratio (L/D)
between the weight of the lipid combination (L) and the weight of
the macromolecule (D) so as to achieve the desired macromolecule
density or interaxial distance thereby determining the amount of
the macromolecule and lipid combination sufficient to regulate the
structure of the complex; d. applying the lipid combination on the
surface in amount so determined; and e. applying the macromolecule
over the lipid combination of (a) in the amount so determined,
wherein the macromolecule self assembles onto the lipid combination
thereby forming a complex and creating a pattern on the
surface.
25. The method of claim 24, wherein the pattern is used to create a
mask.
26. A method for creating a material having desired properties
comprising: a. applying a macromolecule-lipid complex to a surface
by the method of claim 24; b. applying molecules which make up the
material onto the complex of (a), wherein the molecules
self-assemble based on its interactions with the complex; and c.
removing the complex from the surface thereby creating the material
having the regulated structure.
27. The method of claim 26, wherein the complex is in a
multilamellar, regular hexagonal, or inverted hexagonal phase.
28. The method of claim 26, wherein the material so created is a
molecular sieve for separating molecules based on size.
29. A molecular sieve produced by the method of claim 26.
30. The method of claim 24, wherein modulating is effected using
the formula: d.sub.M=(L/D)
(A.sub.M.rho.M)/(.delta..sub.m.rho.L).
31. A macromolecule-lipid complex, the complex comprising a
macromolecule(s), lipid or lipid combination, and a cosurfactant(s)
molecule.
32. The macromolecule-lipid complex of claim 31, wherein the charge
of the macromolecule is opposite to the charge of the lipid.
33. The macromolecule-lipid complex of claim 31, wherein the lipid
is a neutral lipid and is selected from a group consisting of
dioleoyl phosphatidyl cholin,
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,
1,2-dicaproyl-sn-glycero-3-phosphoethanolamine,
1,2-dioctanoyl-sn-glycero- -3-phosphoethanolamine,
1,2-dicapryl-sn-glycero-3-phosphoethanolamine,
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,
1,2-dimyristoyl-sn-glycer- o-3-phosphoethanolamine,
1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolam- ine,
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine,
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine,
1,2-dipalmitoleoyl-sn-g- lycero-3-phosphoethanolamine,
1,2-distearoyl-sn-glycero-3-phosphoethanolam- ine,
1,2-dipretrselinoyl-sn-glycero-3-phosphoethanolamine,
1,2-dielaidoyl-sn-glycero-3 -phosphoethanolamine,
1,2-dilauroyl-sn-glycer- o-3-phosphoethanolamine,
1,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1
,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine,
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine- ,
1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine,
1,2-myristoleoyl-sn-glycero-3-phosphocholine,
1,2-dimyristelaidoyl-sn-gly- cero-3 -phosphocholine,
1,2-palmitoleoyl-sn-glycero-3-phosphocholine,
1,2-palmitelaidoyl-sn-glycero-3-phosphocholine,
1,2-petroselinoyl-sn-glyc- ero-3-phosphocholine,
1,2-dioleoyl-sn-glycero-3-phosphocholine, 1
,2-dielaidoyl-sn-glycero-3-phosphocholine,
1,2-dilinoleoyl-sn-glycero-3-p- hosphocholine,
1,2-linolenoyl-sn-glycero-3-phosphocholine,
1,2-eicosenoyl-sn-glycero-3-phosphocholine,
1,2-arachidonoyl-sn-glycero-3- -phosphocholine,
1,2-erucoyl-sn-glycero-3-phosphocholine,
1,2-nervonoyl-sn-glycero-3-phosphocholine,
1,2-propionoyl-sn-glycero-3-ph- osphocholine,
1,2-butyroyl-sn-glycero-3-phosphocholine,
1,2-valeroyl-sn-glycero-3-phosphocholine,
1,2-caproyl-sn-glycero-3-phosph- ocholine,
1,2-heptanoyl-sn-glycero-3-phosphocholine,
1,2-capryloyl-sn-glycero-3-phosphocholine,
1,2-nonanoyl-sn-glycero-3-phos- phocholine,
1,2-capryl-sn-glycero-3-phosphocholine,
1,2-undecanoyl-sn-glycero-3-phosphocholine,
1,2-lauroyl-sn-glycero-3-phos- phocholine,
1,2-tridecanoyl-sn-glycero-3-phosphocholine,
1,2-myristoyl-sn-glycero-3-phosphocholine,
1,2-pentadecanoyl-sn-glycero-3- -phosphocholine,
1,2-palmitoyl-sn-glycero-3-phosphocholine,
1,2-phytanoyl-sn-glycero-3-phosphocholine,
1,2-heptadecanoyl-sn-glycero-3- -phosphocholine,
1,2-stearoyl-sn-glycero-3-phosphocholine,
1,2-bromostearoyl-sn-glycero-3-phosphocholine,
1,2-nonadecanoyl-sn-glycer- o-3-phosphocholine,
1,2-arachidoyl-sn-glycero-3-phosphoeholine,
1,2-heneicosanoyl-sn-glycero-3-phosphocholine,
1,2-behenoyl-sn-glycero-3-- phosphocholine,
1,2-tricosanoyl-sn-glycero-3-phosphocholine,
1,2-lignoceroyl-sn-glycero-3-phosphocholine.
34. The macromolecule-lipid complex of claim 31, wherein the lipid
is a charged lipid and is selected from a group consisting of
1,2-diacyl-3-trimethylammonium-propane,
1,2-dimyristoyl-3-trimethylammoni- um-propane,
1,2-dipalmitoyl-3-trimethylammonium-propane,
1,2-distearoyl-3-trimethylammonium-propane,
1,2-diacyl-3-dimethylammonium- -propane,
1,2-dimyristoyl-3-dimethylammonium-propane,
1,2-dipalmitoyl-3-dimethylammonium-propane,
1,2-distearoyl-3-dimethylammo- nium-propane, and
1,2-dioleoyl-3-dimethylammonium-propane.
35. The macromolecule-lipid complex of claim 31, wherein the
cosurfactant molecule is an alcohol and the alcohol is selected
from a group consisting of butanol, pentanol, hexanol, heptanol,
octanol, nonanol, and geraniol.
36. The macromolecule-lipid complex of claim 31, wherein the
macromolecule is selected from the group consisting of nucleic acid
molecules, proteins, peptides, immunomodulating compounds,
glycoproteins, lipoproteins, hormones, neurotransmitters,
tumoricidal agents, growth factors, toxins, analgesics,
anesthetics, monosaccharides, polysaccharides, narcotics,
catalysts, enzymes, antimicrobial agents, anti-inflammatory agents,
anti-parasitic agents, dyes, radiolabels, radio-opaque compounds,
and fluorescent compounds.
37. A method for creating a macromolecule-lipid complex in an
hexagonal phase, the method comprising: a. determining an amount of
the lipid or lipid combination by selecting a lipid or lipid
combination where the sum of the products of the spontaneous
curvature for each lipid and the volume fraction for each lipid is
greater than zero, and b. adding a macromolecule to the lipid or
lipid combination determined in step a under sufficient conditions
to create the macromolecule-lipid complex in the hexagonal
phase.
38. A method for creating a macromolecule-lipid complex in an
inverted hexagonal phase, the method comprising: a. determining an
amount of the lipid or lipid combination by selecting a lipid or
lipid combination where the sum of the products of the spontaneous
curvature for each lipid and the volume fraction for each lipid is
less than zero, and b. adding a macromolecule to the lipid or lipid
combination determined in step a under sufficient conditions to
create the macromolecule-lipid complex in the inverted hexagonal
phase.
39. A method for creating a macromolecule-lipid complex in a
lamellar phase, the method comprising: a. determining an amount of
the lipid or lipid combination by selecting a lipid or lipid
combination where the sum of the products of the spontaneous
curvature for each lipid and the volume fraction for each lipid is
zero, and b. adding a macromolecule to the lipid or lipid
combination determined in step a under sufficient conditions to
create the macromolecule-lipid complex in the lamellar phase.
40. The method of claim 37, 38, or 39, wherein the volume fraction
of the lipid is determined from FIG. 3 for each phase.
41. The method of claim 37 or 38, wherein the volume fraction of
the lipid is greater than 0.6.
42. The method of claim 39, wherein the volume fraction of the
lipid is greater than 0.7 and less than 0.85.
43. The method of claim 39, wherein the volume fraction of the
lipid is less than 0.4.
44. The method of claim 37, 38, or 39 further comprising the step
of adding a cosurfactant molecule to the complex so created.
45. A method for making a macromolecule-lipid complex in any of a
lamellar, hexagonal, or inverted hexagonal phase, the complex
having a lipid or lipid combination, a macromolecule(s), and a
cosurfactant(s), the method comprising: a. selecting the lipid or
lipid combination and macromolecule(s); and b. determining the
membrane bending rigidity of the lipid or lipid combination and
macromolecule(s) so that the cosurfactant(s) and its amount can be
determined, such that the cosurfactant so determined will result in
an alteration to the membrane bending rigidity, so as to result in
any of the lamellar, hexagonal, or inverted hexagonal phase and the
spontaneous curvature of the membrane zero or non-zero.
46. A macromolecule-lipid complex produced by the method of claim
37, 38, 39 or 45.
47. A method for transferring the macromolecule in the
macromolecule-lipid complex of claim 21, 31, or 46 to a cell
comprising contacting the complex with the cell under sufficient
conditions so that the macromolecule releases from the complex and
transferring the macromolecule to the cell.
48. A lubricant composition comprising the macromolecule complex of
claim 21, 31 or 46 and an acceptable carrier.
49. The lubricant composition of claim 48, wherein the lubricant
exhibits liquid crystalline properties.
50. A method for reducing friction between two surfaces comprising
contacting the surfaces with the liquid lubricant of claim 48 so as
to reduce friction between the two surfaces when the surfaces are
put in contact.
51. A method for creating a pattern on a surface comprising
applying the macromolecule-lipid complex of claim 21, 31, or 46 on
the surface thereby creating a pattern on the surface.
52. The method of claim 51, wherein the pattern is used to create a
mask.
53. A method for creating a material having desired properties
comprising: a. applying a macromolecule-lipid complex to a surface
by the method of claim 51; b. applying the material onto the
complex of (a), wherein the molecules self-assemble based on its
interactions with the complex; and c. removing the complex from the
surface thereby creating the material having the regulated
structure.
54. The method of claim 53, wherein the complex is in a
multilamellar, regular hexagonal, or inverted hexagonal phase.
55. The method of claim 53, wherein the material so created is a
molecular sieve for separating molecules based on size.
Description
[0001] This application is a continuation in part of U.S. Ser.
No.08/985, 625 filed Dec. 5, 1997, which was a continuation in part
of U.S. Ser. No. 60/032,163, filed Dec. 6, 1996.
[0003] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
BACKGROUND OF THE INVENTION
[0004] Conventional macromolecule delivery and release
technologies, which in the past have concentrated on improvements
in mechanical devices such as implants or pumps to achieve more
targeted and sustained releases of drugs, is now advancing on a
microscopic and even molecular level. Recombinant technology has
produced a variety of new potential therapeutics in the form of
nucleic acids, proteins and peptides and these successes have
driven the search for newer and more flexible macromolecule
delivery and targeting methods and systems.
[0005] Microencapsulation of different molecules within
biodegradable polymers and lipid complexes has achieved successes
in improving the targeting and delivery of a variety of molecules
including nucleic acids and various chemotherapeutic agents. For
example, lipid complexes are currently used as delivery vehicles
for a number of molecules where sustained release or target release
to specific biological sites is desired. In the case of nucleic
acids, charged nucleic acid-lipid complexes are utilized to enhance
transfection efficiencies in somatic gene transfer by facilitating
the attachment of nucleic acids to the targeted cells.
[0006] Success in somatic gene therapy depends on the efficient
transfer and expression of extracellular DNA to the nucleus of
eucaryotic cells, with the aim of replacing a defective or adding a
missing gene (1). Viral-based carriers of DNA are presently the
most common method of gene delivery, but there has been a
tremendous activity in developing synthetic nonviral vectors. In
particular, cationic liposomes (CLs), in which the overall positive
charge of the cationic liposome-DNA (CL--DNA) complex enhances
transfection by attaching to anionic animal cells, have shown gene
expression in vivo in targeted organs, and human clinical protocols
are ongoing (2-4). Cationic liposome transfer vectors exhibit low
toxicity, nonimmunogenicity, and ease of production, but their
mechanism of action remains largely unknown with transfection
efficiencies varying by up to a factor of 100 in different cell
lines (2-6).
[0007] This unpredictability, which is ubiquitous in gene therapy
(7) and in particular in synthetic systems, may be attributed to a
lack of knowledge regarding the interactions between DNA and CLs
and the resulting structures of CL--DNA complexes. DNA membrane
interactions might also provide clues for the relevant molecular
forces in the packing of DNA in chromosomes and viral capsids.
Studies show regular DNA condensed morphologies induced by
multivalent cations (8) and liquid-crystalline (LC) phases at high
concentrations of DNA both in-vitro (9) and in-vivo in bacteria
(10). More broadly, the nature of structures and interactions
between membranes and polymers, either adsorbed (11) or tethered to
the membranes (12), is currently an active area of research.
[0008] Feigner et al. (3) originally proposed a "bead-on-string"
structure of the CL--DNA complexes picturing the DNA strand
decorated with distinctly attached liposomes. Electron microscopy
(EM) studies have reported on a variety of structures including
string-like structures and indications of fusion of liposomes in
metal-shadowing EM (13), oligolamellar structures in cryo-TEM (14),
and tube-like images possibly depicting lipid bilayer-covered DNA
observed in freeze-fracture EM (15).
[0009] A variety of modifications of the lipid membranes have been
attempted with limited success, including polymerizing or
crosslinking the molecules in the bilayer to enhance stability and
reduce permeation rates, and incorporating polymers into the
bilayer to reduce clearance by macrophages in the bloodstream.
While these modifications have proved beneficial, without means to
overcome the inherent unpredictability of these complexes by
controlling crucial factors such as lipid membrane thickness and
the intermolecular spacing of the encapsulated molecules, the use
of these molecules is severely limited. The present invention is
directed to overcoming this limitation.
SUMMARY OF THE INVENTION
[0010] The invention provides novel compositions involving
macromolecule-lipid complexes and methods for making them. These
compositions and methods of the invention are significant
improvements in the field of macromolecule-lipid complex synthesis,
macromolecule targeting and delivery to various biological
systems.
[0011] The present invention provides methods for making
macromolecule-lipid complexes and methods for controlling
components of the macromolecule-lipid complexes such as the
membrane thickness and intermolecular spacing of the complex
constituents.
[0012] In one embodiment for making macromolecule-lipid complexes,
the method comprises mixing a lipid combination (e.g., a neutral
lipid and a charged lipid) in a sufficient amount with a
macromolecule so as to form a complex with specific geometric and
charge qualities. By varying the relative amounts of (1) the
charged and neutral lipids, (2) the weight amount and/or the
macromolecule and (3) the assembly solution, conditions distinct
complexes can be generated having desired isoelectric point or
charged states. By utilizing this process for controlling both the
exterior lipid structure and interior macromolecular ordering, an
extremely versatile molecular targeting and delivery system can be
developed for a variety of applications. The invention has
applications in the numerous methods which utilize lipids and
various macromolecules such as gene therapy, nucleic acid based
vaccine development and peptide and protein delivery.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1(A) is a series of high resolution differential
interference contrast microscopy images of cationic liposome-DNA
complexes showing the formation of distinct condensed globules in
mixtures of different lipid to DNA weight ratios. The scale bar is
10 .mu.m.
[0014] FIG. 1(B) is a plot of the average size of the lipid-DNA
complexes measured by dynamic light scattering.
[0015] FIG. 2(A) is a series of small-angle x-ray scattering scans
in water as a function of different lipid to DNA weight ratio
(L/D). (Inset is under extreme dilute conditions).
[0016] FIG. 2(B) is plot of the spacing d and dDNA as a function of
L/D.
[0017] FIG. 2(C) is a series of small-angle x-ray scattering scans
of the lamellar L.alpha. phase of DOPC/DOTAP water mixtures done at
lower resolution (rotating anode x-ray generator).
[0018] FIG. 3(A) is a schematic picture of the local arrangement in
the interior of lipid-DNA complexes.
[0019] FIG. 3(B) is a micrograph of the DNA-lipid condensates under
bright light.
[0020] FIG. 3(C) is a micrograph of DNA-lipid condensates under
crossed polarizers.
[0021] FIG. 4(A) is a series of small-angle x-ray scattering scans
of CL--DNA complexes at approximately the isoelectric point.
[0022] FIG. 4(B) is d.sub.DNA and d from FIG. 4(A) plotted as a
function of L/D.
[0023] FIG. 4(C) the average domain size of the 1D lattice of DNA
chains derived from the width of the DNA peaks shown in 4(B).
[0024] FIG. 5(A) is a schematic representation showing the
macromolecule-lipid complex formation from the negatively charged
DNA and positively charged liposomes. Schematics of lamellar
I.sub..alpha..sup.c and inverted hexagonal complex
H.sub.II.sup.c.
[0025] FIG. 5(B) is the powder X-ray diffraction patterns of two
distinct (H.sub.II.sup.c and L.sub..alpha..sup.c)
liquid-crystalline phases of CL--DNA complexes.
[0026] FIGS. 6(A-D) are video-microscopy images of CL--DNA
complexes in H.sub.II.sup.c and L.sub..alpha..sup.c.
[0027] FIG. 7 are two SAXS scans obtained following the
transformation from L.sub..alpha..sup.c to H.sub.II.sup.c phase in
the case when the macromolecule is DNA (Left) or a polynucleotide T
(right).
[0028] FIG. 8 shows the variation of structural parameters in
L.sub..alpha..sup.c and H.sub.II.sup.c complexes with the three
different types of polyelectrolytes and correlative schematic
diagrams showing the structure of a unit cell in the three
H.sub.II.sup.c complexes (with DNA, Poly-T, or PGA as the
macromolecule).
[0029] FIG. 9 is a schematic of DNA-lipid complex oriented in
microchannels with applications in nanolithograph and separations
(or in oriented multilayers).
[0030] FIG. 10 is a schematic of two distinct pathways from the
lamellar (L.sub..alpha..sup.C) phase to the columnar inverted
hexagonal (H.sub.II.sup.C)phase of cationic liposome-DNA (CL--DNA)
complexes.
[0031] FIG. 11 are Synchrotron SAXS graphs showing the patterns of
the lamellar (L.sub..alpha..sup.C)and columnar inverted hexagonal
(H.sub.II.sup.C) phases of positively charged CL--DNA
complexes.
[0032] FIG. 12 is a graph representation of the variation of the
unit cell parameters in the lamellar (L.sub..alpha..sup.C) and
hexagonal (H.sub.II.sup.C) complexes as a function of .PHI..sub.PE
in .lambda.--.
[0033] FIG. 13 (A-D) are video-microscopy image of positively
charged CL--DNA complexes in the H.sub.II.sup.C(a) and
L.sub..alpha..sup.C(b) phases, viewed in
Differential-Interference-Contrast (DIC) (left), lipid fluorescence
(middle), and DNA fluorescence (right).
[0034] FIG. 14 is a schematic of three common shapes of lipid
molecules (surfactants).
DETAILED DESCRIPTION OF THE INVENTION
[0035] DEFINITIONS:
[0036] As used in this application, the following words or phrases
have the meanings specified.
[0037] As used herein, the term "surfactant" means any of various
substances that are surface-active (Handbook of Lipids Research
Book #4, Physical Chemistry of Lipids for Alkanes &
Phospholipid, Plenum Press, London, Donald N. Small, Editor,
1988).
[0038] As used herein, the term "lipid" means any surfactant both
biologically and non-biologically derived.
[0039] As used herein, the term "lipid combination" means any
mixture of two or more lipids.
[0040] As used herein, the term "sufficient amount" means a
concentration of a given component that is determined to be
adequate to produce the desired effect or characteristic.
[0041] As used herein, the term "making" means constructing in a
systematic manner.
[0042] As used herein, the term "complex" means a substance
composed of two or more molecules, components, or parts.
[0043] As used herein, the term "isoelectric point state" means the
set of conditions under which the electric charge of the complex is
approximately zero.
[0044] As used herein, the term "negative state" means the set of
conditions under which the electric charge of the complex has a net
negative charge.
[0045] As used herein, the term "positive state" means the set of
conditions under which the electric charge of the complex has a net
positive charge.
[0046] As used herein, the term "charged state" means the set of
conditions under which the electric charge of the complex has some
net charge or zero charge.
[0047] As used herein, the term "the macromolecule interaxial
distance (d.sub.M)" means the perpendicular distance between the
cylinder axis of neighboring macromolecules or the average distance
between macromolecules.
[0048] As used herein, the term "membrane thickness of the lipid
combination (.delta..sub.m)" means the thickness of a bilayer of
lipid molecule made up of a particular lipid combination.
[0049] As used herein, the term "macromolecule area (AM)" means the
cross section area of the macromolecule.
[0050] As used herein, the term "area per lipid chain (A.sub.L)"
means the cross section area of the lipid chain.
[0051] As used herein, "macromolecule density (.rho.M)" means the
density of the macromolecule.
[0052] As used herein "lipid density (PL)" means the density of the
lipid combination.
[0053] As used herein "inverted hexagonal complex phase" means the
phase wherein the lipid combination forms a monolayer around the
macromolecule (i.e., with lipid tails pointing outward); thereby
creating a lipid monolayer macromolecule tube which then assembles
into a hexagonal lattice. Also referred to herein as a cone shaped
molecule (FIG. 14).
[0054] As used herein "regular hexagonal complex phase" means the
phase wherein the lipid combination assembles into a cylindrical
rod (i.e. with lipid tails pointing inward) and macromolecule
attached to the outer surface of the rod; thereby creating
cylindrical rods with attached macromolecules which then assemble
in a hexagonal lattice. Also referred herein as an inverted cone
shaped molecule (FIG. 14).
[0055] As used herein "modulating" means determining the amounts of
the macromolecule and lipid combination sufficient produce a
macromolecule-lipid complex having a desired structure.
[0056] As used herein "co-surfactant" is a membrane altering agent,
i.e., an agent that reduces membrane rigidity or changes the
spontaneous curvature of the membrane (i.e., the stiffness
modulus). An example includes, but is not limited to, an alcohol.
There are a wide variety of alcohols that will serve to produce a
flexible membrane (e.g., in the range of
1k.sub.BT<K<20k.sub.BY. Medium chain alcohols from butanol to
nonanol will function in this context, with pefitanol, heptanol and
hexanol being preferred. Additionally, biologically derived
alcohols such as geraniol will also function in this manner.
[0057] As used herein ".kappa." is the lipid monolayer
rigidity.
[0058] As used herein "R" the radius of curvature.
[0059] As used herein "R.sub.o" is the natural radius of
curvature.
[0060] As used herein the natural curvature of cationic DOTAP is
defined as C.sub.o.sup.DOTAP=1/R.sub.o.sup.DOTAP=0. This expresses
the fact that membranes of pure DOTAP are known to favor the
lamellar L.sub..alpha. phase.
[0061] As used herein the natural curvature of DOPE is defined as
C.sub.o.sup.DOPE=1/R.sub.o.sup.DOPE<0. This expresses the fact
that membranes of pure DOPE have a negative natural curvature and
that DOPE has a larger area per 2 chains than area per head
group.
[0062] As used herein .PHI..sub.PE.sup.V is the volume fraction of
DOPE in the lipid mixture monolayer.
[0063] As used herein the natural curvature of the monolayer
mixture of DOTAP and DOPE is expressed as
C.sub.o=1/R.sub.o=.PHI..sub.PE.sup.VC.sub.- o.sup.DOPE.
[0064] In order that the invention herein described may be more
fully understood, the following description is set forth.
METHODS OF THE INVENTION
[0065] The invention provides methods for regulating the structure
of a charged macromolecule-lipid complex having a selected
characteristic or multiple characteristics. These characteristics
include interaxial distance (dM), membrane thickness of the lipid
combination (.delta..sub.m), macromolecule area (A.sub.M),
macromolecule density (.rho..sub.M), lipid density (.rho..sub.L),
and the ratio (L/D) between the weight of the lipid combination (L)
and the weight of the macromolecule (D). The benefit of being able
to precisely control the micromolecular structure of
macromolecule-lipid complexes is that it will be possible to tailor
make specific structures which have defined chemical and biological
activities. For example specific structural attributes of cationic
lipid-DNA structures are known to impact transfection efficiencies
in different biological systems. By being able to manipulate these
structural attributes, the chance of success in somatic gene
therapy, which depends on the efficient transfer and expression of
extracellular DNA to the nucleus of eucaryotic cells, will be
greatly improved.
[0066] The complex comprises a macromolecule and lipid combination.
Preferably, both the macromolecule and lipid combination are
charged. Further, the charge of the lipid combination or lipid is
preferably opposite of the charge of the macromolecule.
[0067] Preferably, the lipid combination comprises a neutral lipid
component and a charged lipid component. By varying the relative
amount of the charged and neutral lipid, and the weight of the
macromolecule, distinct complexes can be generated having selected
isoelectric point or charged states. For example, the lipid
combination and the macromolecule can be associated so as to form a
complex in an isoelectric point state, Alternatively, the lipid
combination and the macromolecule can be associated so as to form a
complex in a positively charged state. Further alternatively, the
lipid combination and the macromolecule can be associated so as to
form a complex in a negatively charged state.
[0068] Additionally, in accordance with the practice of the
invention, the ratio of the neutral lipid component relative to the
charged lipid component can be 70/30, 50/50, 0/100, or 10/90. It
clear that in the embodiment, wherein the ratio of the neutral
lipid component relative to the charged lipid component is 0/100, a
lipid combination is not used but only a single lipid component is
used.
[0069] Examples of suitable macromolecules include nucleic acid
molecules, peptides, proteins, polysaccharides, combinations of a
protein and carbohydrate moiety and a synthetic macromolecule of
non-biological origin, e.g., doped polyacetylene macromolecules
(J.G.S. Cowie "Polymers Chemistry and Physics of Modem Materials",
Chapter 7, (Blackie Academic & Professional Press) (1993)).
[0070] Examples of suitable neutral lipids include but are not
limited to: dioleoyl phosphatidyl cholin,
1,2-dioleoyl-sn-glycero-3-phosphoethanolami- ne,
1,2-dicaproyl-sn-glycero-3-phosphoethanolamine,
1,2-dioctanoyl-sn-glycero-3-phosphoethanolamine,
1,2-dicapryl-sn-glycero-- 3-phosphoethanolamine,
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine,
1,2-dipentadecanoyl-sn-- glycero-3-phosphoethanolamine,
1,2-dipalnitoyl-sn-glycero-3-phosphoethanol- amine,
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine,
1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine,
1,2-dipretrselinoyl-sn-g- lycero-3-phosphoethanolamine,
1,2-dielaidoyl-sn-glycero-3-phosphoethanolam- ine,
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinolenoyl-sn-gly- cero-3-phosphoethanolamine,
1,2-diarachidonoyl-sn-glycero-3-phosphoethanol- amine,
1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine,
1,2-myristoleoyl-sn-glycero-3-phosphocholine, 1
,2-dimyristelaidoyl-sn-gl- ycero-3-phosphocholine,
1,2-palmitoleoyl-sn-glycero-3-phosphocholine,
1,2-palmitelaidoyl-sn-glycero-3-phosphocholine,
1,2-petroselinoyl-sn-glyc- ero-3-phosphocholine,
1,2-dioleoyl-sn-glycero-3-phosphocholine,
1,2-dielaidoyl-sn-glycero-3-phosphocholine,
1,2-dilinoleoyl-sn-glycero-3-- phosphocholine,
1,2-linolenoyl-sn-glycero-3-phosphocholine,
1,2-eicosenoyl-sn-glycero-3-phosphocholine,
1,2-arachidonoyl-sn-glycero-3- -phosphocholine,
1,2-ericoyl-sn-glycero-3-phosphocholine,
1,2-nervonoyl-sn-glycero-3-phosphocholine,
1,2-propionoyl-sn-glycero-3-ph- osphocholine,
1,2-butyroyl-sn-glycero-3-phosphocholine,
1,2-valeroyl-sn-glycero-3-phosphocholine,
1,2-caproyl-sn-glycero-3-phosph- ocholine,
1,2-heptanoyl-sn-glycero-3-phosphocholine,
1,2-capryloyl-sn-glycero-3-phosphocholine,
1,2-nonanoyl-sn-glycero-3-phos- phocholine,
1,2-capryl-sn-glycero-3-phosphocholine,
1,2-undecanoyl-sn-glycero-3-phosphocholine,
1,2-lauroyl-sn-glycero-3-phos- phocholine,
1,2-tridecanoyl-sn-glycero-3-phosphocholine,
1,2-myristoyl-sn-glycero-3-phosphocholine,
1,2-pentadecanoyl-sn-glycero-3- -phosphocholine,
1,2-palmitoyl-sn-glycero-3-phosphocholine,
1,2-phytanoyl-sn-glycero-3-phosphocholine,
1,2-heptadecanoyl-sn-glycero-3- -phosphocholine,
1,2-stearoyl-sn-glycero-3-phosphocholine,
1,2-bromostearoyl-sn-glycero-3-phosphocholine,
1,2-nonadecanoyl-sn-glycer- o-3-phosphocholine,
1,2-arachidoyl-sn-glycero-3-phosphocholine,
1,2-heneicosanoyl-sn-glycero-3-phosphocholine,
1,2-behenoyl-sn-glycero-3-- phosphocholine,
1,2-tricosanoyl-sn-glycero-3-phosphocholine,
1,2-lignoceroyl-sn-glycero-3-phosphocholine.
[0071] Examples of suitable charged lipids include, but are not
limited to, 1,2-diacyl-3-trimethylammonium-propane,
1,2-dimyristoyl-3-trimethylam- monium-propane,
1,2-dipalmitoyl-3-trimethylammonium-propane,
1,2-distearoyl-3-trimethylammonium-propane,
1,2-diacyl-3-dimethylammonium- -propane, 1
,2-dimyristoyl-3-dimethylammonium-propane,
1,2-dipalmitoyl-3-dimethylammonium-propane,
1,2-distearoyl-3-dimethylammo- nium-propane, and
1,2-dioleoyl-3-dimethylammonium-propane.
[0072] In accordance with the practice of the invention, the
nucleic acid molecule can be single stranded, double stranded,
triple stranded or quadruple stranded. Further, the nucleic acid
molecule can be DNA or RNA. The DNA or RNA can be naturally
occurring or recombinantly-made. Alternatively, it can be a
synthetic polynucleotide. The polynucleotides include nucleic acid
molecules having non-phosphate backbones which improve binding. The
macromolecule may be linear, circular, nicked circular, or
supercoiled.
[0073] In one embodiment of the invention, the method comprises
selecting a selected characteristic or characteristics described
above and modulating one or more of the non-selected
characteristics from the group so as to regulate the structure of
the macromolecule-lipid complex having the selected characteristic.
Preferably, modulation is effected using the formula: dM=(L/D)
(A.sub.M.rho..sub.M)/(.delta..sub.m.rho.L). The relationship
d.sub.M=(A.sub.M/.rho..sub.m)/(.delta..sub.m/.rho..sub.1) (L/D)
equates the cationic charge density (e.g., due to the cationic
membrane) with the anionic charge density (e.g., due to the
macromolecule). Here, PM=density of macromolecule (g/cc) and
.rho.L=densities of membrane, d.sub.m the membrane thickness, and
A.sub.M the macromolecule area.
[0074] In another embodiment of the invention, the method comprises
modulating any of the characteristics (i.e., a single
characteristic or multiple characteristics) associated with the
charged macromolecule-lipid complex as described above so as to
regulate the structure of the macromolecule-lipid complex having
the selected characteristic.
[0075] The method further comprises determining amounts of the
macromolecule and the lipid combination so selected which would be
sufficient to achieve the selected characteristic or
characteristics thereby regulating the structure of the complex. In
one embodiment this can be accomplished by selecting a selected
characteristic or multiple characteristics to be achieved. These
characteristics are macromolecule interaxial distance (d.sub.M),
membrane thickness of the lipid combination (.delta..sub.m),
macromolecule area (A.sub.M), macromolecule density (.rho..sub.M),
lipid density (.rho..sub.L), and the ratio (L/D) between the weight
of the lipid combination (L) and the weight of the macromolecule
(D). Then the characteristics not selected can be modulated so as
to achieve the selected characteristic. After determining the
proper amounts, the method provides mixing the macromolecule with
the lipid combination in the amount so determined.
[0076] For example, when the selected characteristic is a specific
value of the interaxial distance of adjacent macromolecules within
the macromolecule-lipid complex, the method provides selecting a
charged macromolecule and lipid combination, wherein the charge of
the lipid combination is opposite of the charge of the
macromolecule. The amounts of the macromolecule and lipid
combination sufficient to regulate the structure of the complex is
then determined using the formula d.sub.M=(L/D)
(A.sub.M.rho.M)/(.delta..sub.m.rho.L) . In one example the
interaxial distance is in a range between 24.5 and 60 angstroms. In
another example, the interaxial distance is about 60 angstroms. By
regulating the interaxial distance of adjacent macromolecules in a
complex, the distance between macromolecules within the complex or
phase is necessarily regulated. Therefore, this invention also
encompasses methods for regulating the distance between
macromolecules.
[0077] Alternatively, when the selected characteristic is a
specific value for the average density of macromolecules within a
macromolecule-lipid complex, the amounts of the macromolecule and
lipid combination sufficient to regulate the structure of the
complex is determined using the formula, d.sub.M=(L/D)
(A.sub.M.rho.M)/(.delta..sub.m.rho.L).
[0078] Further, the macromolecule-lipid complex can be a
multilamellar structure wherein the lipid combination forms
alternating lipid bilayers and macromolecule monolayers.
Alternatively, the macromolecule-lipid complex can form either an
inverted hexagonal complex phase or a regular hexagonal complex
phase. The complex, whether part of a multilamellar or hexagonal
structure, comprises macromolecules associated with the lipid in an
arrangement that can be regulated and controlled in accordance with
the method of the invention.
[0079] In another embodiment, the lipid combination and the
macromolecule are associated so as to form a complex in an
isoelectric point state and the complex has macromolecules
exhibiting interaxial spacing of greater than 24.5 angstroms. The
resulting complex can have a charge of about zero. In another
embodiment, the lipid and the macromolecule is associated so as to
form a complex in an isoelectric point state, wherein the amount of
the neutral lipid component relative to the charged lipid component
ranges from 2 to 95 percent. The resulting complex can have a
charge of about zero. Further, the lipid and the macromolecule can
associate so as to form a complex in a charged state, wherein the
amount of the neutral lipid component relative to the charged lipid
component ranges from 55 to 95 percent. The resulting complex can
have a net charge.
[0080] Additionally, the lipid combination can form a bilayer
membrane to which charged macromolecules are associated, and
wherein the relative amounts of the lipid components generate the
lipid bilayer membrane having a thickness of between 25 and 70
angstroms. Alternatively, the lipid combination can form a bilayer
membrane to which charged macromolecules are associated and wherein
the relative amounts of the lipid components generate the lipid
bilayer membrane having a thickness of between 41 and 60 angstroms.
Further, the lipid combination can form a bilayer membrane to which
charged macromolecules are associated, and wherein the relative
amounts of the lipid components generate the lipid bilayer membrane
having a thickness of between 32 and 48 angstroms.
[0081] Also, the lipid combination can form a monolayer membrane to
which charged macromolecules are associated, and wherein the
relative amounts of the lipid components generates the lipid
monolayer membrane having a thickness of between 12 and 40
angstroms.
[0082] In addition to the bilayer membrane form (also referred to
herein as lamellar or multilamellar), the resulting complex can
form a monolayer (also referred to herein as being in a hexagonal
phase, e.g. inverted hexagonal or regular hexagonal). For example,
the lipid combination can form a monolayer membrane to which
charged macromolecules are associated and wherein the relative
amounts of the lipid components generate the lipid monolayer
membrane having a thickness of between 15 and 35 angstroms.
Alternatively, the lipid combination can form a monolayer membrane
to which charged macromolecules are associated, wherein the
relative amounts of the lipid components generate the lipid
monolayer membrane having a thickness of between 16 and 30
angstroms.
[0083] The invention further provides a macromolecule-lipid complex
produced by the methods of the invention described above.
[0084] In one embodiment, the resulting macromolecule-lipid complex
comprises a lipid combination having a charged lipid component and
a neutral lipid component; and a charged macromolecule. The charge
of the lipid combination being opposite of the charge of the
macromolecule. The lipid combination and the macromolecule
associate thereby forming a complex in an isoelectric point state.
In this state, the lipid combination forms a bilayer membrane to
which the charged macromolecule is associated and the relative
amounts of the neutral lipid component relative to the charged
lipid component generates a lipid bilayer membrane having a
thickness of between 25 and 75 angstroms.
[0085] In another embodiment, in the resulting macromolecule-lipid
complex, the lipids form a bilayer membrane to which the
macromolecule is associated, wherein the relative amounts of the
lipid components generate a lipid bilayer membrane having a
thickness of between 25 and 75 angstroms; and the conformation of
the complex has macromolecules exhibiting interaxial spacing of a
range between 50 and 75 angstroms.
[0086] The invention further provides a process for generating
formulations which form the basis for the processing of templates
(e.g., during a lithography process) and for producing molecular
sieves with precise control over pore size for sizing
molecules.
[0087] For example, the invention provides a process for creating a
pattern on a surface (e.g., during a lithography process) using
complexes having regulated structures made using the methods
described above. The process comprises applying a lipid combination
on the surface and applying macromolecules over the lipid
combination. Alternatively, the macromolecule can be applied on the
surface and the lipid combination applied over the macromolecules.
The amounts of the macromolecule and lipid combination is
determined by the formula: d.sub.M=(L/D)
(A.sub.M.rho.M)/(.delta..sub.m.rho.L). Mixing the amounts so
determined results in macromolecules which self assemble onto the
lipid combination (or vice versa) thereby forming a complex and
creating a pattern created by the complex on the surface. In one
embodiment, the pattern can be used to create a mask, e.g., for
lithography.
[0088] Additionally, the invention provides a process for creating
a material having selected properties such as optical, mechanical,
electronic, optoelectronic, or catalytic characteristics not
previously realized from bulk components of the material. This
process comprises applying a macromolecule-lipid complex to a
surface. The complex must have a regulated structure created by the
methods of the invention. The process further provides applying
molecules which make up the material onto the complex, wherein the
molecules self-assemble based on its interactions with the complex.
The complex is then removed from the surface thereby creating the
material having a selected property. The complex can be in a
multilamellar, regular hexagonal phase, or inverted hexagonal
phase. The resulting material can function as a molecular sieve
having precise pore size. The invention further provides a
molecular sieve produced by the process above.
[0089] The invention also provides methods for creating a
macromolecule-lipid complex in an hexagonal phase (also referred to
herein as a regular hexagonal phase) (See pathway I of FIG. 10). In
one embodiment the method comprises determining an amount of the
lipid or lipid combination. This can be done by selecting a lipid
or lipid combination where the sum of the products of the
spontaneous curvature for each lipid and the volume fraction for
each lipid is greater than zero (Biochemistry of Lipids and
Membranes, edited by J. E. Vence, Benjamin Cummings Publishing
Company, Menlo Park, 1985; (Israel Achvili, Intermolecular and
Surface Forces, 2nd Ed., 1991, Academic Press Limited). Further,
the method provides adding a macromolecule to the lipid or lipid
combination determined under sufficient conditions thereby creating
the macromolecule-lipid complex in the hexagonal phase.
[0090] The invention also provides methods for creating a
macromolecule-lipid complex in an inverted hexagonal phase (See
pathway I of FIG. 10). In this instance, the method comprises
determining an amount of the lipid or lipid combination by
selecting a lipid or lipid combination where the sum of the
products of the spontaneous curvature for each lipid and the volume
fraction for each lipid is less than zero. Additionally, a
macromolecule or macromolecules is added to the lipid or lipid
combination so selected so as to create the macromolecule-lipid
complex in the inverted hexagonal phase.
[0091] Additionally the invention provides methods for creating a
macromolecule-lipid complex in a lamellar phase (See pathway I of
FIG. 10). This method comprises determining an amount of the lipid
or lipid combination by selecting a lipid or lipid combination
where the sum of the products of the spontaneous curvature for each
lipid and the volume fraction for each lipid is approximately zero.
Additionally, a macromolecule or macromolecules can be added to the
lipid or lipid combination so determined so as to create the
macromolecule-lipid complex in the lamellar phase.
[0092] In accordance with the practice of the invention, the volume
fraction of the lipid can be determined from FIG. 3 for each of the
desired phase. Once the phase is selected, the required volume
fraction to achieve that phase can be determined as demonstrated in
Example 4 because the spontaneous curvature of the lipid is known
or a constant.
[0093] In one embodiment of the invention, when the complex is in
hexagonal or inverted hexagonal phase, the volume fraction of the
lipid is greater than 0.6. In another embodiment, when the complex
is in hexagonal or inverted hexagonal phase, the volume fraction of
the lipid is greater than 0.7 and less than 0.85. Additionally, in
one embodiment, when the complex is in lamellar phase, the volume
fraction of the lipid is less than 0.4.
[0094] Additionally, the invention provides a further step to each
of the invention above, namely, the step of adding a cosurfactant
molecule to the complex so created. The cosurfactant molecules
alters the rigidity of the lipid membrane thus allowing
modifications to the membrane. Example 4 teaches this altered
membrane can provide a molecule delivery system superior to those
known in the art.
[0095] The present invention also provides additional embodiments
for methods of making a macromolecule-lipid complex in the desired
phase, e.g., lamellar, hexagonal, or inverted hexagonal phase. In
one embodiment, the complex comprises a lipid or lipid combination,
a macromolecule or macromolecules, and a cosurfactant or
cosurfactants (See pathway II of FIG. 10).
[0096] In this embodiment, the method comprises selecting the lipid
or lipid combination and macromolecule(s) appropriate for making
the desired phase. This is done by determining the membrane bending
rigidity of the lipid or lipid combination and macromolecule(s)
(lipid/macromolecule combination). Additionally, the spontaneous
curvature of the lipid/macromolecule combination is determined. One
can determine the type and the amount of cosurfactant necessary to
achieve the desired phase by determining the membrane bending
rigidity of the lipid or lipid combination and macromolecules, the
cosurfactant(s). Example 4 discloses how such a determination can
be done.
[0097] Once the cosurfactant is selected, the addition of the
surfactant to the lipid/macromolecule combination will result in an
alteration to the membrane bending rigidity and the spontaneous
curvature of the membrane is zero or non-zero.
[0098] The invention also provides macromolecule-lipid complexes
produced by the method of the invention.
[0099] Additionally, the invention provides methods for
transferring the macromolecule or macromolecules in the
macromolecule-lipid complexes of the invention to a cell or desired
surface. This comprises contacting the complex with the cell or
surface under sufficient conditions so that the macromolecule or
macromolecules are released from the complex thereby resulting in
transfer. The chosen cosurfactant can enhance or deter the ability
of the complex to transfer the macromolecule therein. The lipid or
lipid combination selected also effects the transfer ability.
[0100] Also, the invention provides lubricant compositions
comprising any of the macromolecule-lipid complexes of the
invention and an acceptable carrier. The lubricant exhibits liquid
crystalline properties. The structure of these lubricants is only
weakly temperature dependent and is changed primarily by changing
the composition of surfactants(e.g.
lipids)/cosurfactants/macromolecules.
[0101] The lubricants are processed to be either water or oil
soluble. The major phases are (1) the lamellar L.alpha., (2) the
hexagonal H.sub.I, and (3) the inverted hexagonal H.sub.II.sup.C.
The L.alpha. consists of layers of surfactants (with or without
cosurfactants) separated by solvent (oil or water). The H.sub.I
consists of cylindrical surfactant micelles (with or without
cosurfactant) with water in between. The H.sub.II, consists of
inverse surfactant monolayers (with or without cosurfactant) with
oil in between. Block copolymers (e.g., diblock, or triblock) can
be used instead of surfactants.
[0102] A second class of lyotropic L.sup.Cs that were created with
the methods of the invention include "hybrid" L.sup.C phases
comprising surfactants, e.g., lipids, (or block copolymers)
complexed with macromolecules (e.g. polyelectrolytes such as DNA,
RNA, polypeptides). Initial phase diagram containing such
structures are shown in FIGS. 12 and 11b.
[0103] The L.sub..alpha..sup.c, the H.sub.I.sup.c, and
H.sub.II.sup.c structures can contain an additional macromolecular
component, e.g., a cosurfactant. The addition of the cosurfactant
changes the mechanical properties of the lubricants at the
molecular level; e.g. by changing the diameter and elastic
(torsional, bending) moduli of the macromolecules.
[0104] These lubricants would be useful in methods to reduce
friction between two surfaces. This method comprises contacting the
surfaces with the lubricant of the invention so as to reduce
friction between the two surfaces when the surfaces are put in
contact.
[0105] The invention also provides methods for creating a pattern
on a surface. In one embodiment, the method comprises applying the
macromolecule-lipid complexes of the invention on the surface so as
to create a pattern thereon. In accordance with the practice of the
invention, the pattern is used to create a mask.
[0106] The present invention further provides methods for creating
a material having desired properties. In one embodiment, the method
comprises applying a macromolecule-lipid complex to a surface by
the method of above. Additionally, the material can be applied to
the complex so that the molecules of the material can self-assemble
based on its interactions with the complex. The complex is then
removed from the surface thereby creating the material having the
regulated structure. In accordance with the practice of the
invention, the complex can be in a multilamellar, regular
hexagonal, or inverted hexagonal phase. Additionally, the material
so created can be used as a molecular sieve for separating
molecules based on size.
[0107] COMPOSITIONS OF THE INVENTION
[0108] The present invention provides nucleic acid-lipid complexes
comprising a charged lipid combination and a charged nucleic acid
molecule. In one embodiment of the invention, the charge of the
lipid combination is opposite of the charge of the nucleic acid
molecule. Further, the resulting complex has a desired isoelectric
point state and nucleic acids exhibiting interaxial spacing of
greater than 24.5 angstroms. In another embodiment, the interaxial
spacing range is about between 24.5 and 60 angstroms. In yet
another embodiment, the interaxial spacing is about 60 angstroms.
In accordance with the practice of the invention, the conformation
of the resulting complex can be a multilamellar structure with
alternating lipid bilayers and nucleic acid monolayers.
[0109] Suitable examples of nucleic acid molecules include, but are
not limited to, deoxyribonucleic acid (DNA), ribonucleic acid
(RNA). The macromolecules may be linear, circular, nicked circular
or supercoiled. The nucleic acid molecules can have phosphate
backbones but not necessarily so. Alternatively, nucleic acid
molecules having non-phosphate backbones which improve binding are
also encompassed within this invention.
[0110] In one embodiment, the complex comprises a charged lipid
combination; and a charged nucleic acid molecule. The charge of the
lipid combination can be opposite of the charge of the nucleic acid
molecule. Further, the lipid and the nucleic acid molecule are
associated so as to form a complex in an isoelectric point state.
In this state, the relative amounts of the lipid components
generates the lipid bilayer membrane having a thickness of between
25 and 75 angstroms. Additionally, the conformation of the complex
has nucleic acids exhibiting interaxial spacing of a range between
50 and 75 angstroms.
[0111] The present invention further provides macromolecule-lipid
complexes comprising a charged lipid combination; and a charged
macromolecule. Examples of suitable macromolecules include, but are
not limited to, nucleic acid molecules such as single or double
stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or
hybrids thereof, or modified analogs thereof of varying lengths. In
addition, the macromolecule can be a peptide, a protein (or
modified analogs thereof). Further, the macromolecule may be a drug
such as a chemotherapeutic agent or a modified analog thereof.
[0112] In one embodiment of the macromolecule-lipid complex the
charge of the lipid combination is opposite of the charge of the
nucleic acid molecule. Also, the lipid and the nucleic acid
molecule are associated so as to form a complex in an isoelectric
point state.
[0113] The lipid combination can have a charge lipid component and
a neutral lipid component. The amount of the neutral lipid
component relative to the charged lipid component can range from 2
to 95 percent.
[0114] Alternatively, in another embodiment of the
macromolecule-lipid complex, the amount of the neutral lipid
component relative to the charged lipid component ranges from 55 to
95 percent. Also, in accordance with the practice of the invention,
the ratio of the neutral lipid component relative to the charged
lipid component can be 70/30.
[0115] Suitable lipids include, but are not limited to, dioleoyl
phophatidyl choline or dioleoyl phophatidyl ethanolamine and
dioleoyl triethylammonium propane combination.
[0116] In a further embodiment of the macromolecule-lipid complex,
the lipid combination can be a charged lipid combination and the
macromolecule can be a charged macromolecule. The lipids form a
bilayer membrane in the complex to which the charged macromolecule
can be associated. In this embodiment, the charge of the lipid
combination can be opposite of the charge of the nucleic acid
molecule. Further, the lipid and the nucleic acid molecule are
associated so as to form a complex in an isoelectric point state.
Additionally, the relative amounts of the lipid components
generates the lipid bilayer membrane having a thickness of between
25 and 75 angstroms.
[0117] In another embodiment of the macromolecule-lipid complex,
the lipid and the nucleic acid molecule are associated so as to
form a complex in a positively charged state, wherein the lipids
form a bilayer membrane to which charged macromolecule is
associated, and the relative amounts of the lipid components
generates the lipid bilayer membrane having a thickness of between
41 and 75 angstroms.
[0118] Also, in another embodiment of the macromolecule-lipid
complex, the lipid and the nucleic acid molecule are associated so
as to form a complex in a negatively charged state, wherein the
lipids form a bilayer membrane to which charged macromolecule is
associated, and the relative amounts of the lipid components
generates the lipid bilayer membrane having a thickness of between
32 and 75 angstroms.
[0119] In accordance with the practice of the invention, the lipid
can be dioleoyl phophatidyl cholin or dioleoyl phophatidyl
ethanolamine and dioleoyl triethylammonium propane. In this
embodiment, the charge of the lipid combination in the complex can
be opposite of the charge of the nucleic acid molecule. The
dioleoyl phophatidyl cholin or dioleoyl phophatidyl ethanolamine
and dioleoyl triethylammonium propane form a bilayer membrane to
which the charged macromolecule is associated in an isoelectric
point state, wherein the relative amounts of dioleoyl phophatidyl
cholin or dioleoyl phophatidyl ethanolamine lipids relative to the
dioleoyl triethylammonium propane generates the lipid bilayer
membrane having a thickness of between 25 and 75 angstroms.
[0120] In accordance with the practice of this invention, in the
macromolecule-lipid complex, the amount of the neutral lipid
component relative to the charged lipid component ranges from 0 to
95 percent and whose charge is approximately zero. Alternatively,
the amount of the neutral lipid component relative to the charged
lipid component ranges from 55 to 95 percent and which has either a
positive or negative charge.
[0121] There is a great flexibility in the structure of these
complexes, which may vary greatly in their molecular ordering.
These complexes may be relatively simple or may consist of a highly
ordered structure. For example the conformation of such a complex
can include a multilamellar structure with alternating lipid
bilayers and nucleic acid monolayers.
[0122] The invention further provides formulations which form the
basis for the processing of templates and for producing molecular
sieves with precise control over pore size.
[0123] The invention provides a macromolecule-lipid complex having
as components of the complex (1) a macromolecule or macromolecules,
(2) a lipid or lipid combination, and (3) a cosurfactant or
cosurfactants. The addition of the cosurfactant reduces the elastic
cost and decrease the membrane rigidity thus allowing a more
favorable environment for the transition from lamellar phase to
hexagonal or inverted hexagonal phase. In accordance with the
practice of the invention, the lipid can be substituted by any
surfactant. Although, lipids are preferred.
[0124] Also in accordance with the practice of the invention, the
macromolecule and lipid can be charged. For example, when the
macromolecule is charged, the lipid can be neutral. Preferably, the
charge of the macromolecule is opposite to the charge of the
lipid.
[0125] Suitable examples of cosurfactant molecules include but is
not limited to an alcohol. The alcohol can be butanol, pentanol,
hexanol, heptanol, octanol, nonanol, and geraniol. Other
biologically derived alcohols is acceptable.
[0126] The lipids can be cationic, anionic or neutral. Examples of
suitable cationic lipids include but are not limited to DOTMA,
DDAB, CTAB, and DOTAP. A suitable lipid is a phospholipid, e.g,
lecithin, phophatidylinositol, sphingomyelin, cardiolipin,
phosphatidic acid and the cerebrosides. Other lipids include
stearylamine, dicetyl phosphate, cholesterol and tocopherol.
[0127] Examples of suitable noncationic lipids include phosphatidyl
choline, cholesterol, phosphatidylehtanolamine,
dioleoylphosphatidyl choline, dioleoylphophatidyl glycerol., and
dioloeoylphosphatidyl ethanolarnine.
[0128] Examples of suitable macromolecule include nucleic acid
molecules (DNA, RNA, hybrids thereof, or nucleoside), proteins,
peptides, immunomodulating compounds, glycoproteins, lipoproteins,
hormones, neurotransmitters, tumoricidal agents, growth factors,
toxins, analgesics, anesthetics, monosaccharides, polysaccharides,
narcotics, catalysts, enzymes, antimicrobial agents,
anti-inflammatory agents, anti-parasitic agents, dyes, radiolabels,
radio-opaque compounds, and fluorescent compounds.
[0129] The following examples are presented to illustrate the
present invention and to assist one of ordinary skill in making and
using the same. These examples are not intended in any way to
otherwise limit the scope of the invention.
EXAMPLE 1
[0130] Cationic liposomes complexed with DNA (CL--DNA) are
promising synthetically based nonviral carriers of DNA vectors for
gene therapy. The solution structure of CL--DNA complexes was
probed on length scales from subnanometer to micrometer by
synchrotron x-ray diffraction and optical microscopy. The addition
of either linear .lambda.-phage or plasmid DNA to CLs resulted in
an unexpected topological transition from liposomes to optically
birefringent liquid crystalline condensed globules. X-ray
diffraction of the globules reveals a novel multilamellar structure
with alternating lipid bilayer and DNA monolayers. We discovered
that .lambda.-DNA chains form a one-dimensional lattice with
distinct interhelical packing states. Remarkably, in the
isoelectric point state, the .lambda.-DNA interaxial spacing
expands between 24.5 and 60 angstroms upon lipid dilution and is
indicative of a long-range electrostatic-induced repulsion possibly
enhanced by chain undulations.
[0131] We have carried out a combined in situ optical microscopy
and x-ray diffraction (XRD) study of CL--DNA complexes (an
embodiment of a macromolecule-lipid complex). On semi-macroscopic
length scales, the addition of linear or circular plasmid DNA to
binary mixtures of cationic liposomes induces a topological
transition from liposomes into collapsed condensates in the form of
optically birefringent LC globules with size on the order of 1
.mu.m.
[0132] The solution structure of the globules was revealed on the 1
to 100 nm length scale by high-resolution synchrotron XRD studies.
Unexpectedly, the complexes consist of a higher ordered
multilamellar structure with DNA sandwiched between cationic
bilayers.
[0133] We have discovered distinct interhelical packing states for
linear %-phage DNA, above and below, and at the isoelectric point
of the complex by varying the concentrations of DNA and the lipid
components comprising the complex. Remarkably, in the isoelectric
state of the CL--DNA complex the DNA interaxial distance dDNA
increases from 24.5 to 60 .ANG. as a function of lipid dilution and
is quantitatively consistent with an expanding one-dimensional (1D)
lattice of DNA chains. Thus, the DNA chains confined between
bilayers form a novel 2D smectic phase.
[0134] DNA molecules can be readily labeled and imaged by
fluorescence microscopy (16). Free .lambda.-DNA in aqueous solution
appears as a highly dynamic blob of 1 .mu.m in diameter, in
agreement with a classical random coil configuration, while the
contour length of .lambda.-phage DNA is 16.5 .mu.m. The CLs
consisted of binary mixtures of lipids which contained either DOPC
(dioleoyl phosphatidyl cholin) or DOPE (dioleoyl phosphatidyl
ethanolamine) as the neutral co-lipid and DOTAP (dioleoyl
trimethylammonium propane) as the cationic lipid. A mixture of
DOPE/DOTAP (1:1, wt:wt) was prepared in a 20 mg/ml chloroform stock
solution. 500 ml was dried under nitrogen in a narrow glass beaker
and desiccated under vacuum for 6 hours. After addition of 2.5 ml
Millipore water and 2 hr incubation at 40.degree. C. the vesicle
suspension was sonicated by clarity for 10 minutes. The resulting
solution of liposomes, 25 mg/ml was filtered through 0.2 .mu.m
Nucleopore filters. For optical measurements the concentration of
SUV used was between 0.1 mg/ml and 0.5 mg/ml. All lipids were
purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.).
[0135] The DOTAP/DOPC and DOTAP/DOPE CLs had a size distribution
ranging between 0.02 to 0.1 .mu.m in diameter, with a peak around
0.07 .mu.m (the liposome and complex sizes were measured by dynamic
light scattering (Microtrac UPA 150, Leeds and Northrup). We used
highly purified linear k-phage DNA (48,502 bp) in most of the
experiments but some were carried out with Escherichia coli DNA and
pBR322 plasmid DNA (4361 bp); the latter, consisted of a mixture of
nicked circular and supercoiled DNA. Purified .lambda.-phage DNA
and pBR322 plasmid were purchased from Biolabs, New England.
Optical and x-ray data were taken with linear .lambda. prepared in
2 ways: (1) used as delivered, and (2) by heating to 65.degree. C.
and reacting with a surplus of a 12-base oligo complementary to the
3'COS end. Subsequently the DNA was ligated (T4 DNA ligase,
Fischer). The methods gave the same result. For the optical
experiments the DNA concentration used was between 0.01 mg/ml and
0.1 mg/ml. Condensation of CLs with )-DNA was directly observed
using differential interference microscopy (DIC) and fluorescence
microscopy. A Nikon Diaphot 300 equipped for epifluorescence and
high resolution DIC was used.
[0136] We show in FIG. 1A a series of DIC images 30 min after
preparation in CL--DNA mixtures as a function of the total lipid to
.lambda.-DNA weight ratio L/D, where L=DOTAP+DOPE denotes the
weight of lipid and D the weight of DNA. FIG. 1A shows
high-resolution DIC images of CL--DNA complexes forming distinct
condensed globules in mixtures of different lipid to DNA weight
ratio (L/D); scale bar is 10 .mu.m.
[0137] Similar images were observed with .lambda.-DNA replaced by
the pBR322 plasmid DNA or DOPE replaced by DOPC. At low DNA
concentrations (FIG. 1A, L/D=50), in contrast to the pure liposome
solution where no objects >0.2 .mu.m were found, 1 .mu.m large
globules are observed. The globules coexist with excess liposomes.
As more DNA is added, the globular condensates form larger chain
like structures (FIG. 1A, L/D =10). The Brownian motion of these
globules suggests that they are linked by an invisible thread. At
L/D =5 the chain-like structures flocculate into large aggregates
of distinct globules. For L/D <5, the complex size was smaller
and stable in time again (FIG. 1A, L/D =2), and coexisted with
excess DNA. Fluorescence-labeled DNA and lipid can be detected on
each globule, indicating that the globules are DNA-lipid
condensates. Sonicated DOPE-DOTAP (1:1) liposomes were prepared at
0.1 mg/ml with 0.2 mol % DHPE-Texas Red fluorescence label. DNA
stained by YOYO (Molecular Probes) was added under gentle mixing at
different lipid-to-DNA ratios (L/D). Polarized microscopy also
shows that the distinct globules are birefringent indicative of
their LC nature.
[0138] The size dependence of the complexes as a function of L/D
(FIG. 1B) was independently measured by dynamic light scattering
(the liposome and complex sizes were measured by dynamic light
scattering (Microtrac UPA 150, Leeds and Northrup). The large error
bars represent the broad polydispersity of the system. The size
dependence of the aggregates can be understood in terms of a
charge-stabilized colloidal suspension. The charge of the complexes
was measured by their electrophoretic mobility in an external
electric field. For L/D >5 (FIG. 1A; L/D=50 or 10) the complexes
are positively charged, while for L/D <5 (FIG. 1A; L/D=2) the
complexes are negatively charged. The charge reversal is in good
agreement with the stoichiometrically expected charge balance of
the components DOTAP and DNA at L/D=4.4 where L=DOTAP+DOPE in equal
weights. Thus, the positively and negatively charged globules at
L/D=50 and L/D=2 respectively, repel each other and remain
separate, while as L/D approaches 5, the nearly neutral complexes
collide and tend to stick due to van der Waals attraction.
Remarkably, the size of the globules appears to be only weakly
dependent on the length of the DNA in similar experiments carried
out with Escherichia coli DNA or pBR322 plasmid (4361 bp).
[0139] FIG. 2A shows a series of SAXS scans of CL--DNA complexes in
excess water as a function of different lipid to DNA weight ration
(L/D). The Bragg reflections at q.sub.001=0.096 .ANG..sup.-1 and
q.sub.002=0.1.92 .ANG..sup.-1 result from the multilamellar
L.sub..alpha. structure with intercalated monolayer DNA (see FIG.
3A). The intermediate peak at q.sub.DNA is due to the
DNA-interaxial spacing d.sub.DNA as described in the text. Inset:
SAXS scan of an extremely dilute (lipid+DNA=0.014% volume in water)
%-DNA-DOPE/DOTAP (1:1) complex at L/D=10, which shows the same
features as the more concentrated mixtures and confirms the
multilamellar structure (with alternating lipid bilayer and DNA
monolayers) of very dilute mixtures typically used in gene therapy
applications.
[0140] The XRD experiments revealed unexpected structures for
mixtures of CLs and DNA. FIG. 2B shows the spacings d and d.sub.DNA
as a function of L/D show that (i) d is nearly constant and (ii)
two distinct states of DNA packing, one where the complexes are
positive (L/D>5, d.sub.DNA approximately 46 .ANG.) and the other
state where the complexes are negative (L/D>5, dDNA
approximately 35 .ANG.) FIG. 2C shows SAXS scans of the lamellar
L.sub..alpha. phase of DOPC/DOTAP (cationic)-water mixtures done at
lower resolution (rotating-anode x-ray generator). A dilution
series of 30% (d=57.61 .ANG.), 50% (d=79.49 .ANG.), and 70%
(d=123.13 .ANG.) H.sub.2O by weight is shown. High resolution
synchrotron x-ray scattering were performed at the Stanford
Synchrotron Radiation Laboratory. Lower resolution XRD experiments
were performed using a rotating anode source.
[0141] Small angle x-ray scattering (SAXS) data of dilute
(.PHI..sub.w=the volume fraction of water=98.6%.+-.0.3%) DOPC/DOTAP
(1:1)-.lambda.-DNA mixtures as a function of L/D (L=DOPC+DOTAP)
(FIG. 2A) are consistent with a complete topological rearrangement
of liposomes and DNA into a multilayer structure with DNA
intercalated between the bilayers (23) (FIG. 3A). The DNA-lipid
condensates were prepared from a 25 mg/ml liposome suspension and a
5 mg/ml DNA solution. The solutions were filled in 2 mm diameter
quartz capillaries with different ratios L/D respectively and mixed
after flame sealing by gentle centrifugation up and down the
capillary.
[0142] FIG. 3A shows a schematic picture of the local arrangement
in the interior of lipid-DNA complexes (shown at two different
concentrations in FIG. 1A and in FIG. 3B below. The semiflexible
DNA molecules are represented by rods on this molecular scale. The
neutral and cationic lipids comprising the membrane are expected to
locally demix with the cationic lipids (red) more concentrated near
the DNA. Micrographs of DNA-lipid condensates under (B) bright
light and (C) crossed polarizers showing LC-like defects. Two sharp
peaks at q=0.0965.+-.0.003 and 0.193.+-.0.006 .ANG..sup.-1
correspond to the (001) peaks of a layered structure with an
interlayer spacing d(=.delta..sub.m+.delta..sub.w) which is in the
range 65.1.+-.2 .ANG. (FIG. 2B, open squares). The membrane
thickness and water gap are denoted by am and .delta..sub.m and
.delta..sub.w, respectively (FIG. 3A). The middle broad peak qDNA
arises from DNA-DNA correlations and gives
d.sub.DNA=2.pi./q.sub.DNA (FIG. 2B, solid circles). The
multilamellar structure with intercalated DNA is also observed in
CL--DNA complexes containing supercoiled DNA both in water, and
also in Dulbecco's Modified Eagle Medium used in transfection
experiments in gene therapy applications. This novel multilamellar
structure of the CL--DNA complexes are observed to protect DNA from
being cut by restriction enzymes. The intercalation of .lambda.-DNA
between membranes in CL--DNA complexes was found to protect it
against a HindIII restriction enzyme which cuts naked .lambda.-DNA
at 7 sites (21).
[0143] In the absence of DNA, membranes comprised of mixtures of
DOPC and the cationic lipid DOTAP (1:1) exhibit strong long-range
interlayer electrostatic repulsions that overwhelm the van der
Waals attraction (26, 27). In this case, as the volume fraction
.PHI..sub.w of water is increased, the L.sub..alpha. phase swells
and d is given by the simple geometric relation
d=.delta..sub.m/(1-.PHI..sub.w) (26). The SAXS scans in FIG. 2C
shows this behavior with the (001) peaks moving to lower q as Dw
increases. From d (=2.pi./q.sub.(001)) at a given .PHI..sub.w we
obtain .delta..sub.m=39.+-.0.5 .ANG. for DOPC/DOTAP (1:1).
Liposomes made of DOPC/DOTAP (1:1) with .PHI..sub.w=98.5% do not
exhibit Bragg diffraction in the small wave-vector range covered in
FIG. 2A.
[0144] The DNA that condenses on the CLs strongly screens the
electrostatic interaction between lipid bilayers and leads to
condensed multilayers. The average thickness of the water gap
.delta..sub.w=d-.delta..sub.m=65.1 .ANG.-39 .ANG.=26.1 .ANG.2.5
.ANG. is, just sufficient to accommodate one monolayer of B-DNA
(diameter=20 .ANG.) including a hydration shell (28). We see in
FIG. 2B that d is almost constant as expected, for a monolayer DNA
intercalate (FIG. 3A). In contrast, as L/D decreases from 18 to 2,
dDNA suddenly decreased from=44 .ANG. in the positively charged
state just above L/D=5 (near the stoichiometric charge neutral
point) to =37 .ANG. for the negatively charged state (FIG. 2B). In
these distinct states, lamellar condensates coexist with excess
giant liposomes in the positive state, and with excess DNA in the
negative state. The multilamellar structure of the complex (with
.lambda.-DNA) and the distinct DNA interhelical packing states was
also found in SAXS data in binary mixtures of cationic lipids which
contained DOPE [which has a high transfection efficiency (2)] as
the neutral co-lipid. However, the complexes showed a
phase-separation into two lamellar phases.
[0145] The driving force for higher order self-assembly is the
release of counterions. DNA carries 20 phosphate groups per helical
pitch of 34.1 .ANG., and due to Manning condensation 76% of these
anionic groups are permanently neutralized by their counterions,
which leads to a distance between anionic groups=the Bjerrum
length=7.1 .ANG. (30). During condensation, the cationic lipid
tends to fully neutralize the phosphate groups on the DNA in effect
replacing and releasing the originally condensed counterions (both
those bound to the 1D DNA and to the 2D cationic membranes) in
solution.
[0146] To improve on the signal-to-background intensity ratio the
synchrotron XRD experiments were carried out at concentrations
(lipid+DNA=1.4.+-.0.3% volume in water), which, although dilute,
were nevertheless greater than the concentrations used in the
microscopy work. The DNA-lipid condensates were prepared from a 25
mg/ml liposome suspension and a 5 mg/ml DNA solution. The solutions
were filled in 2 mm diameter quartz capillaries with different
ratios LID respectively and mixed after flame sealing by gentle
centrifugation up and down the capillary.
[0147] A typical SAXS scan in mixtures at the optical microscopy
concentrations (FIG. 1A) is shown in FIG. 2A (inset) which exhibits
the same features and confirms that the local multilayer and DNA
structure (FIG. 3A) is unchanged between the two concentrations.
The x-ray samples consisted of connected yet distinct globules
(FIG. 3B). What is remarkable is the retention of the globule
morphology consistent with what was observed at lower
concentrations in DIC (FIG. 1A). Under crossed polarizers (FIG. 3C)
LC defects, both focal conics and spherulites (31), resulting from
the smectic-A-like layered structure of the DNA-lipid globules are
evident. The globules at the lower concentrations (FIG. 1A) show
similar LC defects.
[0148] We further probed the nature of .lambda.-DNA-packing within
the lipid layers by conducting a lipid dilution experiment in the
isoelectric point state of the complex. The total lipid
(L=DOTAP+DOPC) was increased while the charge of the overall
complex, given by the ratio of cationic DOTAP to DNA, was kept
constant at DOTAP/DNA=2.40.+-.0.1. The projected charge density of
DNA (two anionic charges per 68 .ANG..sup.2) is very nearly matched
by two cationic head groups on DOTAP of=70 .ANG..sup.2 each and
thus permits near complete neutralization of the complex (FIG.
3A).
[0149] FIG. 4A shows a series of SAXS scans of CL--DNA complexes at
DOTAP/DNA=2.4+0.1 (approximately the isoelectric point) which shows
the DNA peak (arrow) moving toward smaller q as L/D increases (that
is, increasing the DOPC to DOTAP ratio at a constant DOTAP/DNA;
L=DOTAP+DOPC, D=DNA). FIG. 4B shows dDNA and d from (A) plotted as
a function of L/D (see FIG. 2A for notation). Circles are
synchrotron data, and triangles are rotating anode. The solid line
is the prediction of a packing calculation (with no adjustable
parameters) where the DNA chains form a space-filling ID lattice.
FIG. 4C shows the average domain size of the ID lattice of DNA
chains derived from the width of the DNA peaks shown in (B)
[corrected for resolution and powder averaging broadening effects].
The SAXS scans in FIG. 4A, (arrow points to the DNA peak) show that
d.sub.DNA=2.pi./q.sub.DNA increased, with lipid dilution from 24.54
.ANG. to 73.5 .ANG. as L/D increased with lipid dilution between
2.45 and 13.8 (FIG. 4B). The most compressed interaxial spacing of
24.55 .ANG. at L/D=2.45 approaches the short-range repulsive
hard-core interaction of the B-DNA rods containing a hydration
layer (28).
[0150] The DNA interaxial spacing can be calculated rigorously from
simple geometric considerations. If we assume that all of the DNA
is adsorbed between the bilayers and that the orientationally
ordered DNA chains separate to fill the increasing lipid area as
L/D increases, while maintaining a ID lattice (FIG. 3A), then: 1 d
DNA = A d D m L ( L / D ) ( 1 )
[0151] Here, .pi..sub.D=1.7 (g/cc) and .rho.L=1.07 (g/cc) denote
the densities of DNA and lipid respectively, .delta..sub.m the
membrane thickness, and A.sub.D the DNA area.
A.sub.D=Wt(.lambda.)/(.rho..sub.DL(X- ))=186 .ANG..sup.2,
Wt(.lambda.)=weight of .lambda.-DNA=31.5.times.10.sup.-
6/(6.022.times.10.sup.23) g and L(.lambda.)=contour length of
.lambda.-DNA=48502.times.3.4 .ANG.. The solid line in FIG. 4B is
then obtained from Eq. 1 with no adjustable parameters and clearly
shows a remarkable agreement with the data over the measured
interaxial distance from 24.5 to 73.5 .ANG.. The observed deviation
from linear behavior both in the data and the solid line arises
from the slight increase in .delta..sub.m as L/D increases. The
variation in the interlayer spacing d
(=.delta..sub.w+.delta..sub.m) (FIG. 4B) arises from the increase
in the membrane bilayer thickness .delta..sub.m as L/D increases
(each DOPC molecule is =4 .ANG. to 6 .ANG. longer than a DOTAP
molecule). .delta..sub.m was obtained at each L/D by measuring d in
the L.sub..alpha. phase multilayer membranes at the corresponding
DOTAP to DOPC ration and using the relation .delta..sub.m=d
(1-.PHI..sub.w), .PHI..sub.w=water volume fraction. The measured
.delta..sub.m and d, gave .delta..sub.w=25 .ANG..+-.1.5 .ANG. close
to the spacing for the DNA monolayer (see FIG. 3A).
[0152] The existence of a finite-sized ordered lattice is made
unambiguous from the line widths of the DNA peaks (FIG. 4A) where
we find that the ID lattice of DNA chains has a correlated domain
size extending to near 10 unit cells (FIG. 4C). Thus, the DNA
chains form a ID ordered array adsorbed between 2D membranes; that
is, they form a novel finite-sized 2D smectic phase.
[0153] The lattice expansion at the isoelectric point covering
interaxial distances with negligible short-range hydration forces
(28) (B-DNA diameter.apprxeq.20 .ANG.) is indicative of a
long-range repulsion. The distribution of the counterion lipid
(DOTAP) concentration according to the Poisson-Boltzmarm equation
along the top and bottom monolayer which bound the DNA molecules
(FIG. 3A) will lead to a long-range electrostatic-induced
interhelical interaction from the counterion lipid pressure (due to
the expected local demixing of the cationic and neutral lipids) and
the electric field. Preliminary salt dependent experiments which
show shifts in the DNA peak indicate that long-range electrostatic
induced interactions are present. Additionally, because of the
semi-flexible nature of .lambda.-DNA [consisting of between 170 and
340 persistence lengths (.xi..sub.p) in dilute solution
(.xi..sub.p.apprxeq.between 500 and 1000 .ANG.)], we expect the
long-range repulsions to be further enhanced by chain-undulation
interactions. A similar enhancement has been observed in a
hexagonal lattice of DNA (28, 34). This phase of ID DNA chains is
the lower dimensional analog of 2D fluid membranes in that it may
either be dominated by electrostatic-induced forces (26, 27) or the
interplay between electrostatics and undulations (35-37).
[0154] Further experiments are needed to elucidate the precise
nature of the intermolecular forces and the interplay between
electrostatic and chain undulation interactions (38). Future
studies may also reveal states with 3D correlations between the DNA
chains from layer to layer in analogy to recent theoretical
findings in highly condensed DNA phases (39). The observed
quantitative control over the structural nature of the DNA packing
in CL--DNA complexes may lead to a better understanding of the
important structural parameters relevant to transfection
efficiencies in gene therapy; in particular, they should be
directly relevant to our understanding of the interactions of the
complex with cellular lipids and the mechanism of DNA transfer
across the nuclear membrane.
EXAMPLE 2
[0155] This example provides the hexagonal phase of a cationic
lipid-polyelectrolyte complex (an embodiment of a
macromolecule-lipid complex). This embodiment is a LC structure of
the complex achieved by varying the lipid composition. It is a
novel LC phase with DNA double-strands surrounded by lipid
monolayers arranged on a regular hexagonal lattice. This embodiment
interacts differently with giant negatively charged liposomes,
compared to the lamellar phase, and represents the simplest model
of outer cellular membranes. We demonstrate the generality of the
lamellar-hexagonal transformation by observing it in complexes of
cationic lipid with two other negatively charged
biopolymers--polyglutamic acid (PGA), a model polypeptide and
poly-thymine (polyT), a model single-stranded oligonucleotide. We
identify the interactions leading to the transformations between
the two complex phases for the three different biological
polyelectrolytes. Aside from the significance for gene therapy, our
findings suggest new pathways for controlling structural parameters
of polyelectrolyte-surfactant complexes, which has been suggested
as templates for the formation of new soft materials.
[0156] Example 1 shows that mixing linear DNA with liposomes of
DOPC/DOTAP mixtures leads to a topological transition into CL--DNA
complexes of lamellar structure L.sub..alpha..sup.c, where DNA
monolayers are sandwiched between lipid bilayers (41). In this
example, the existence of a completely different inverted hexagonal
H.sub.II.sup.c, liquid-crystalline state in complexes of linear
1-DNA with liposomes of DOPE/DOTAP mixtures is unambiguously
demonstrated for the first time using synchrotron small-angle x-ray
diffraction and optical microscopy. We show how changing the ratio
of cationic DOTAP to neutral DOPE lipid in the liposomes leads to
CL--DNA complexes with lamellar or hexagonal structure (FIG.
5a).
[0157] The use of cationic lipids can be extended to deliver other
negatively charged biopolymers into cells, in particular
polypetide-based drugs and single-stranded oligonucleotides for
antisense therapy (22, 23). We show that these polyelectrolytes
also form complexes with cationic lipids of lamellar and hexagonal
structure, similar to the CL--DNA complexes. Comparison of the
three types of complexes allows to gain an insight on how the
polyelectrolyte charge density and diameter tune the interactions
between lipids and polymer, shifting the phase boundaries between
L.sub..alpha..sup.c and H.sub.II.sup.c complexes.
[0158] FIG. 5A shows the formation pathway of a complex from the
free DNA and liposomes. l-DNA in solution has a random-coil
configuration of .about.1 .mu.m diameter. The Cls consisting of
binary DOPE/DOTAP mixture have an average size of 0.06 .mu.m. In
order to reduce the electrostatic free energy, both DNA and lipid
charges are partially neutralized by their respective counterions.
During the CL--DNA complex formation cationic lipids replace DNA
counterions, releasing the [Na.sup.+] and [Cl.sup.--] ions into
solution with a very large entropic free energy gain (of order
k.sub.BT per released counterion). The result is a close
association between DNA and lipid in a compact complex of
.about.0.2 .mu.m size. The overall charge of the complex is
determined by the weight ratio r of cationic lipid and DNA. The
complexes are positive for r>2.2 and negative for r<2.2,
indicating that charge reversal occurs when complexes are
stoichiometrically neutral with one positive lipid per each
negatively charged nucleotide base.
[0159] Surprisingly, the internal structure of the complex changes
completely with DOPE/DOTAP ratio. Defining the volume fraction of
DOPE as .phi..sub.IIE as the fraction of neutral DOPE in the lipid
mixture, the complex is lamellar L.sub..alpha..sup.c for
.phi..sub.PE<0.41 and has inverted hexagonal h.sub.II.sup.c
structure for .phi..sub.PE>0.7. In complexes with
0.41<.phi..sub.PE<0.7 the two structures coexist. Small-angle
x-ray scattering (SAXS) data of complexes with .phi..sub.PE=0.41
and 0.75 (FIG. 5b) clearly shows the presence of two completely
different structures. The two sharp peaks at q=0.099 .ANG..sup.-1
and 0.198 .ANG..sup.-1 correspond to (001) and (002) peaks of a
lamellar structure with interlayer spacing d=63.4 .ANG.. Since
DOPE/DOTAP bilayer has thickness .delta..sub.m=40 .ANG. at
.phi..sub.PE=0.41 .sup.13, the water gap between bilayers
d.sub.w=d-d.sub.m=23.4 .ANG. is just large enough to accommodate a
monolayer of DNA with a hydration shell of water. This structure is
analogous to the one previously reported in DOPC/DOTAP-DNA
complexes (Example 1). The middle broad peak at q.sub.DNA arises
from regular 2D-smectic arrangement of DNA, giving the spacing
between the DNA strands d.sub.DNA=2.sup.II/qDNA.
[0160] For .phi..sub.PE>0.7 the peaks of the SAXS scan index
perfectly on a hexagonal lattice with a repeat spacing of
.alpha.=4.pi./{square root}{square root over (3 )}.sub.q10=67.8
.ANG.. We were able to observe Bragg peaks up to 7th order,
indicating a high degree of regularity of the structure. Schematic
of the new H.sub.II.sup.c phase is shown in FIG. 5a. Each of the
DNA molecules is surrounded by a monolayer of lipid and the unit
cells of DNA/lipid inverted cylindrical micelles are arranged in a
hexagonal lattice. The structure resembles that of the inverted
hexagonal (H.sub.II) phase of pure DOPE in excess water (30), with
the water space inside the lipid micelle filled by DNA. The higher
electron density of DNA with respect to water leads to the relative
suppression of (22) and Bragg peak intensities compared with that
in pure lipid H.sub.II phase. Assuming again an average bilayer
thickness of 40 .ANG., the diameter of micellar void in the
H.sub.II.sup.c phase is .about.28 .ANG., again sufficient for a DNA
molecule with approximately two hydration shells. To improve the
signal/background ratio, samples for synchrotron SAXS experiments
were prepared at lipid and DNA concentrations about 100 times
greater then typically used in optical microscopy and transfection
studies. SAXS scans of mixtures at typical transfection
concentrations, also shown in FIG. 5b, have Bragg peaks at exactly
the same positions as in corresponding more concentrated samples.
This confirms that the internal L.sub..alpha..sup.c and
H.sub.II.sup.c structures of the complexes and the phase boundaries
between them are independent of the overall DNA and lipid
concentrations.
[0161] In either of the condensed phases the complexes appear as
highly dynamic birefringent aggregates when viewed with
video-enhanced optical microscopy (FIG. 6A, B). Each complex
consists of several connected blobs close to charge neutrality,
with the aggregates becoming smaller and eventually dissociating
into individual blobs with the increasing complex charge.
Interestingly, the shape of aggregates is different in the two
complex phases: the L.sub..alpha..sup.c phase forms linear
structures, while in the H.sub.II.sup.c, phase the aggregates are
predominantly branched. Microscopy of DNA and lipids with
appropriate fluorescent labels allows us to image their respective
distributions in the complex. This observations show that the
complex is indeed a compact object, with a close association of
lipid and DNA, since in both phases the complexes exhibit
fluorescence in DNA and lipid modes. The complexes coexist with
excess DNA for r<2.2 and with excess lipid when r>2.2.
However, we never observe presence of macroscopic lipid aggregates,
proving that the only condensed liquid crystalline structures in
the CL--DNA mixtures are complexes. On a larger length-scale and at
higher lipid and DNA concentrations, bigger LC aggregates are
observed (FIG. 6a), with very different defect structures in the
two phases. H.sub.II.sup.c phase never exhibits the spherrulites
characteristic of the L.sub..alpha..sup.c phase. The spherulites
are an unmistaking signature of lamellar liquid-crystalline
structure (32), and are not present in hexagonal phases.
[0162] The membrane of giant anionic liposome is a good model of
the outer cell membrane--the first barrier to the complex on its
way to DNA delivery. There is a striking difference in the way
H.sub.II.sup.c and L.sub..alpha..sup.c complexes interact with
model anionic lipid membranes. We show in FIG. 6C, D typical
micrographs of slightly positively charged (r=4) complexes attached
to the fluid membranes of giant liposomes. The L.sub..alpha..sup.c
complexes attached to anionic membrane remain stable for many
hours. The compact complex morphology can be seen in DIC as well as
in DNA and lipid fluorescence. Clearly there is no fusion between
the complex and the giant liposome. A strikingly different behavior
is observed with H.sub.II.sup.c complexes, They lose their compact
structure immediately upon attaching to the liposome, spreading and
fusing with it. Since the amount of lipid in the complex is
comparable with that in liposome, and since the fusion occurs very
quickly, it results in formation of a local multilamellar structure
on the giant liposome surface. The loss of the compact complex
structure and the subsequent spreading of the DNA fluorescence are
clear indications of fusion and the first observed example of the
effect of complex structure on its interaction with a membrane.
This finding unambiguously demonstrates the importance of complex
internal structure for the efficiency of CL--DNA vectors.
[0163] The presence of H.sup.II.sup.c and L.sub..alpha..sup.c
phases is universal in complexes of DOPE/DOTAP mixtures with other
anionic polyelectrolytes. FIG. 7 shows SAXS scans of complexes with
DNA and oligonucleotide polyT (100 bases long) as a function of
.phi..sub.PE. As .phi..sub.PE increases, the complexes undergo a
first order phase transition from lamellar to hexagonal structure
with a broad range of .phi..sub.PE were the two phases coexist. The
same structures are also observed in complexes of DOPE/DOTAP with
anionic polypeptide PGA (MW=81,000). The only difference in the
structure of complexes between DNA and the shorter polyelectrolytes
is the absence of polymer-polymer correlation peak in the Lc,
phase. We attribute this difference to the difference in length and
rigidity between very long and stiff DNA and shorter, more flexible
polyT and PGA.
[0164] We compare the phase diagrams of CL-polyelectrolyte
complexes for the three different polymers in FIG. 8, which also
shows the variation of repeat distances of complex structure as a
function of .phi..sub.PE. To understand the phase sequence in
complexes it is useful to consider structure of DOPE/DOTAP mixtures
without the polyelectrolytes. These phase boundaries are indicated
on top of FIG. 8. Pure lipids also form lamellar L.sub.a and
inverted hexagonal H.sub.II structures, although the phase
boundaries are very different from CL-polymer systems and the
H.sub.II phase is only present in coexistence with La structure.
Therefore the phase sequence in the CL-polyelectrolyte mixtures
mimics the ones preferred by the pure lipids, with stabilization of
the pure inverted hexagonal phase. DOPE forms stable H.sub.II
phases, whereas DOTAP has stable lamellar structures. Once the
complex is formed and lipid and polymer counterions are released,
the internal structure of the complex will be affected by several
comparable free energy contributions. Since DOPE monolayers have
negative spontaneous curvature and bending energy of only a few
k.sub.BT.sup.16, increasing .phi..sub.IIE will allow the lipid
layers to curve around the polyelectrolites, forming the
H.sub.II.sup.c structure. Additionally, the lipid head-group area
and correspondingly chain length will adjust itself so as to
further minimize the free energy of the system, since the
stretching energy of the lipid chain is only slightly greater then
the bending energy of the monolayers. The three polyelectrolites
which we have studied have different diameters (20 .ANG. DNA, 13
.ANG. PGA which has a-helix conformation inside the complex,
.about.10 .ANG. poly-T) and different linear charge densities
(1=2e.sup.--/3.4 .ANG. DNA, 1e.sup.--/1.5 .ANG. PGA,
.about.1e.sup.--/3.4 .ANG. poly-T). This changes the relative
magnitude of electrostatic interaction in the complex, as well as
the required amount of lipid monolayer bending in the
H.sub.II.sup.c phase, thus shifting the phase boundaries and
structure of a unit cell in the complex.
[0165] Further insight into the relative phase boundaries and
structures in the three CL-polymer complexes may be gained if one
considers that the charge densities of polyelectrolyte and lipid
monolayers have to match within the H.sub.II.sup.c unit cell 2 = D
A ( 1 - PE ) ,
[0166] were A is the lipid head-group area and D is the radius of
lipid monolayer, which may be larger then polyelectrolyte diameter.
Let us assume first that the lipid layer thickness remains fixed at
d.sub.m=40 .ANG. in the H.sub.II.sup.c complex. Then in CL--DNA
complex D=24 .ANG. and A=65 .ANG. (normal value), giving
.phi..sub.PE=0.5, close to experimentally observed lower boundary
of the H.sub.II.sup.c phase. This implies closely matched diameters
of DNA and lipid monolayers in the complex unit cell (FIG. 8). In
CL-pT complex D=25 .ANG. and A=65 .ANG., giving at
.phi..sub.PE=0.75, again close to the experimentally observed
value. This corresponds to a loosely bound unit cell, as shown in
FIG. 8. Higher H.sub.II.sup.c phase boundary and greater difference
between polymer and monolayer diameters arise because of the weaker
electrostatic interaction and larger monolayer bending in CL-pT
complex compared with CL--DNA. In CL-PGA H.sub.II.sup.c phase, a
reasonable phase boundary may be only achieved if the head-group
area is substantially smaller, resulting in stretching of the lipid
chains and increase in lipid layer spacing. With A=40 .ANG. and
D=20 .ANG. one obtains .phi..sub.PE=0.6, in reasonable agreement
with experiment. Here stronger electrostatic interaction and small
polymer diameter result in crowding of lipid heads. The additional
free energy of stretching the chains may be the cause of the very
narrow region of stability of pure H.sub.II.sup.c phase in CL-PGA
system.
[0167] We have provided a first demonstration for the existence of
distinctly different lamellar and hexagonal LC structures of
CL--DNA complexes. These structures are formed at different lipid
compositions and interact differently with model anionic membranes.
The two LC phases also form in other Cl-biopolyelectrolyte
complexes used for intra-cellular delivery. Comparison between the
complexes in three different systems also improves the
understanding of interactions shaping complex structure. This will
be important for controlled design of the new class of
surfactant-polyelectrolyte materials (46), of which our complexes
are examples.
[0168] FIG. 5(A) shows the schematic of the complex formation from
the negatively charged DNA and positively charged liposomes.
Complete topological rearrangement of lipids and DNA in this
process is driven by release of partially-bound counterions from
the diffuse screening layers into bulk solution, which lowers the
electrostatic free energy of the system. However, once the
counterions are released and the lipids are bound to DNA, the
liquid-crystalline structure of the complex will depend on the
interplay of various comparable contributions to the complex free
energy. These vary with the lipid composition of the complex,
resulting in two different observed structures: the lamellar
complex LC when the volume fraction of neutral DOPE lipid
(.phi..sub.PE) is .phi..sub.PE<0.41 and the inverted hexagonal
complex H.sub.II.sup.c for .phi..sub.PE>0.7. The two structures
coexist for intermediate .phi..sub.PE.;
[0169] FIG. 5(B) provides the powder X-ray diffraction patterns of
the two distinct liquid-crystalline phases of CL--DNA complexes.
Scan of the H.sub.II.sup.c complex at .phi..sub.PE=0.75 (open
circles, top) shows the first three order Bragg peaks of the
hexagonal DNA/lipid lattice at q.sub.10=0.107 .ANG..sup.-1,
q.sub.11=0.185 .ANG..sup.-1 and q.sub.20=0.214 .ANG..sup.-1. Scan
of the lamellar L.sub..alpha..sup.c complex at .phi..sub.PE=0.41
(filled circles, bottom) shows the peaks at q.sub.001=0.099
.ANG..sup.-1 and q.sub.002=0.198 .ANG..sup.-1 resulting from the
lamellar periodic structure with DNA intercalated between lipid
bilayers and a peak at q.sub.DNA=0.172 .ANG..sup.-1 due to the
smectic structure of the intercalated DNA. In both cases the
samples were prepared by mixing concentrated deionized water
solutions of DNA (5 mg/ml) and lipid (25 mg/ml) directly in a 1.5
mm diameter quartz x-ray capillary with r=3. Because these
concentrations are higher then typically used in preparation of
CL--DNA complexes for cell transfection, we have also recorded SAXS
patterns of complexes made from dilute DNA (0.01 mg/ml) and lipid
(0.1 mg/ml) solutions (solid lines). The peak positions are the
same for experiments done with concentrated and dilute complexes,
indicating that the complex phases remain the same at lipid and DNA
concentrations typically used for cell transfection.
[0170] FIGS. 6(A-B) provides video-microscopy images of CL--DNA
complexes in (a) H.sub.II.sup.c and (b) L.sub..alpha..sup.c phases.
In all cases complexes were viewed in DIC (left), lipid
fluorescence (middle) and DNA fluorescence (right). For
fluorescence experiments cationic lipids were labeled with 0.2 mol
% of DHPE-TexasRed and DNA was labeled with YoYo-1 iodide at 1 dye
molecule/15bP ratio. The complex morphology is different in the two
phases: branched in the H.sub.II.sup.c and linear in the
L.sub..alpha..sup.c phase. In both phases the lipid is closely
associated with DNA, as evidenced by the exactly same morphology of
complexes in the two fluorescence modes. Complexes were prepared by
gently mixing DNA (0.01 mg/ml) and lipid (0.1 mg/ml) stock
solutions with .phi..sub.PE=0.73 (a) and .phi..sub.PE=0.3 (b) to
yield the r=3 weight ratio (slightly positively charged complexes).
The complexes were further diluted with deionized water for
observation. Scale bar is 2 .mu.m in DIC and 4 .mu.m in
fluorescence images. FIGS. 6(C-D) provides video microscopy of
positively charged H.sub.II.sup.c (c) and L.sub..alpha..sup.c (d)
complexes that interact differently with the negatively charged
giant liposomes. The lamellar complexes simply stick to the
liposomes and remain stable for many hours, retaining their
blob-like morphology. The blobs are localized in DIC as well as
lipid and DNA fluorescence modes. The hexagonal complexes break-up
and spread immediately after attaching to giant liposomes,
indicating a fusion process between the complex and the liposome
lipid bilayer. Spreading of the complex is evident in both lipid
and DNA fluorescence modes. Giant unilamellar liposomes were
prepared from the mixtures of 90% DOPC (neutral) and 10% DOPG
(negatively charged) lipids. CL--DNA complexes were prepared as
described above with r=4. Scale bar is 10 .mu.m in both DIC and
fluorescence images.
[0171] FIG. 7 provides SAXS scans following the transformation from
L.sub..alpha..sup.c to H.sub.II.sup.c phase with increasing amount
of DOPE for complexes with DNA (i) and poly-Thymine (ii). The
dashed line indicates L.sub..alpha..sup.c phase peaks. At very high
DOPE content (.phi..sub.PE>0.85) the H.sub.II.sup.c complexes
coexist with the excess H.sub..alpha..sup.c, phase of pure DOPE
(peaks marked with arrows). In both (i) and (ii) r=3, slightly
above charge-neutrality.
[0172] FIG. 8 shows variation of structural parameters in
L.sub..alpha..sup.c and H.sub.II.sup.c complexes with the three
different types of polyelectrolites (i) 1-DNA, (ii) poly-Thymine
(polyT), (iii) polyglutamic acid (PGA). In all cases a
.apprxeq.{square root}{square root over (3)}/2 d, were a is the
repeat distance of pure H.sub.II.sup.c and d is the membrane repeat
distance in pure L.sub..alpha..sup.c complex. Thus
L.sub..alpha..sup.c and H.sub.II.sup.c phases are always
epitaxially matched, but this condition is not satisfied for the
regions of phase coexistence. The arrows on top of the figure
indicate the phase boundaries in the mixtures of DOPE and DOTAP
lipids, indicating that the presence of polyelectrolites stabilizes
the pure lamellar and hexagonal phases. Schematic representations
show the structure of a unit cell in the three H.sub.II.sup.c
complexes, demonstrating that the thickness of water layer and the
stretching of the lipid chains should be different in the three
polyelectrolyte-lipid complexes.
EXAMPLE 3
[0173] Recently we have found that cationic liposomes (CL)
complexed with DNA (CL--DNA) form a novel self-assembled structure
consisting of a higher ordered multilamellar structure with DNA
sandwiched between cationic lipid bilayers shown schematically in
FIG. 5. These series of x-ray diffraction experiments lead to the
observation of a variation in the DNA interaxial distance as a
function of the lipid to DNA (L/D) weight ratio in multilayers
which unambiguously showed that the x-ray technique was directly
probing the DNA structure in multilayer assemblies. It was found
that the linear DNA confined between bilayers forms an expanding
one-dimensional lattice of chains with the center to center
distance between DNA varying in a controlled manner in the
nanometer range 25 .ANG.<d.sub.DNA<60 .ANG..
[0174] Microstructures with submicron linewidths as substrates for
confining and orienting this multilamellar CL--DNA structure is
shown schematically in FIG. 9. The oriented multilamellar structure
would have many important technological applications. For example,
in developing nano-scale masks in lithography and molecular sieves
with nanometer scale cylindrical pores (FIG. 9).
EXAMPLE 4
[0175] We have discovered a novel two-dimensional (2D) columnar
phase in mixtures of DNA complexes with cationic liposomes (CL) in
a concentration regime empirically known to be significantly more
efficient at transfecting mammalian cells in culture compared to
the lamellar (L.sub..alpha..sup.c) structure of CL--DNA complexes.
The structure derived from synchrotron x-ray diffraction consists
of DNA coated by cationic lipid monolayers and arranged on a 2D
hexagonal lattice (H.sub.II.sup.c). Two membrane-altering pathways
induce the L.sub..alpha..sup.C to the H.sub.II.sup.C transition:
one where the spontaneous curvature of the lipid monolayer is
driven negative, and another, where the membrane bending rigidity
is lowered using a new class of helper-lipids. Significantly,
optical microscopy has revealed that in contrast to the
non-transfectant L.sub..alpha..sup.C complexes which bind stably to
anionic vesicles (models of cellular membranes), the transfectant
HI.sub.II.sup.C complexes are unstable, rapidly fusing and
releasing DNA upon adhering to anionic vesicles. The observations,
underscore the importance of structure to "early-stage" gene
delivery events, and provide support for a mechanism of DNA escape
from anionic endosomal vesicles known to be a major barrier to
transfection.
[0176] There is now a surge in interest in elucidating the
structures in complexes consisting of DNA mixed with oppositely
charged cationic liposomes (CLs) (closed bilayer membrane shells of
lipid molecules). The interest in complexes arises because they
mimic natural viruses in their ability to act as synthetic carriers
of extracellular DNA across outer cell membranes and nuclear
membranes for gene delivery (47, 48, 49, 50, 51, 52). The principle
advantages of nonviral over viral methods for gene delivery include
nonimmunicity and, in particular, the potential of transferring
large pieces of DNA into cells. This was dramatically demonstrated
when the first-generation human artificial chromosome (HAC) of
order 10 Mega base pairs was transferred into cells using CLs
although extremely inefficiently (53, 54). The low transfection
efficiencies (a measure of the efficiency in transferring exogenous
DNA into cells and its expression) with nonviral delivery methods
results from a poor understanding of transfection-related
mechanisms at the molecular and self-assembled levels, including, a
general lack of knowledge of structures of CL--DNA complexes, their
interactions with cell membranes, and events leading to cell entry
and DNA delivery.
[0177] It is known that transfection efficiency mediated by
mixtures of cationic lipids and so-called neutral "helper-lipids"
varies widely and unpredictably (47, 51, 55). The choice of the
helper-lipid has been empirically established to be important. For
example, transfection of mammalian cells in culture is efficient in
mixtures of the univalent cationic lipid DOTAP (dioleoyl
trimethylammonium propane) and the neutral helper-lipid DOPE
(dioleoyl-phosphadtidylethanolamine), and not in mixtures of DOTAP
and a similar helper-lipid DOPC (dioleoyl-phosphatidylc- holine)
(while DOPE, DOTAP and DOPC were used in this example, one skilled
in the art would know that other lipids could be substituted) (56,
57). We demonstrated that DNA mixed with cationic liposomes
comprised of DOPC/DOTAP leads to a topological transition into
condensed CL--DNA complexes with a multilamellar structure
(L.sub..alpha..sup.c) with DNA monolayers sandwiched between
cationic lipid bilayers (12) similar to the schematic in FIG. 10
(left).
[0178] In this invention, the existence of a completely different
columnar inverted hexagonal H.sub.II.sup.C liquid-crystalline state
in CL--DNA complexes is unambiguously demonstrated for the first
time using synchrotron small-angle x-ray diffraction and optical
microscopy (FIG. 10; right). We elucidate the role of the commonly
used helper-lipid DOPE in inducing the L.sub..alpha..sup.C to
H.sub.II.sup.C structural transition by controlling the spontaneous
curvature C.sub.o=1/R.sub.o of the lipid monolayer (FIG. 10;
pathway I). Further, an entirely new class of helper molecules are
introduced which control the membrane bending rigidity .kappa. and
give rise to a distinctly different pathway to the H.sub.II.sup.C
phase (FIG. 10; pathway II). The importance of the precise
self-assembled structures to biological function is underscored,
first in the demonstration that DOPE containing CL--DNA complexes,
which are empirically known to transfect, exhibit the
H.sub.II.sup.C rather than the L.sub..alpha..sup.C structure, and
second in optical imaging experiments which demonstrate that
interactions with model cell membranes mimicking the early stages
of transfection are structure-dependent.
[0179] We show in FIG. 11(A) synchrotron small angle x-ray
scattering (S-AXS) scans in positively charged CL--DNA complexes
for .rho.=DOTAP/DNA (wt./wt.)=3 as a function of increasing
.phi..sub.PE (weight fraction of DOPE) in the DOPE/DOTAP cationic
liposome mixtures along pathway I. The SAXS experiments were
carried out at the Stanford Synchrotron Radiation Laboratory at 8
keV. CL--DNA complexes were prepared by mixing deionized water
solutions of highly purified linear .lambda.-phage DNA (5mg/ml;
48502 bp; contour length of 16.5 .mu.m) and cationic liposomes of
mixed lipids (25 mg/ml) directly in a 1.5mm diameter quartz x-ray
capillary with p=DOTAP/DNA=3 (wt./wt.) which yielded positive
complexes. The CLs consisting of binary DOPE/DOTAP mixtures have an
average size of 0.06 .mu.m. During the CL--DNA complex formation
cationic lipids replace DNA counterions, releasing the Na.sup.+ and
Cl.sup.- ions into solution with a very large entropic free energy
gain (of order k.sub.BT per released counterion). The result is a
close association between DNA and lipid in a compact complex with
an average size of 0.2 .mu.m size (59) . The complexes are positive
for .rho.>2.2 and negative for .rho.<2.2, indicating that
charge reversal occurs when complexes are stoichiometrically
neutral with one positive lipid per each negatively charged
nucleotide base. We find that the internal structure of the complex
changes completely with increasing DOPE/DOTAP ratios. SAXS data of
complexes with .PHI..sub.PE=0.41 and 0.75 clearly shows the
presence of two different structures. At .PHI..sub.PE=0.41, SAXS of
the lamellar L.sub..alpha..sup.C complex (filled circles) shows
sharp peaks at q.sub.001=0.099 .ANG..sup.-1 and q.sub.002=0.198
.ANG..sup.-1 resulting from the lamellar periodic structure
(d=2.pi./q.sub.001=63.47 .ANG.) with DNA intercalated between
cationic lipid (FIG. 10, left). Since the DOPE/DOTAP bilayer
thickness at .PHI..sub.PE=0.41 is .delta..sub.m=40 .ANG.(59), the
water gap between bilayers .delta..sub.w=d-.delta..sub.m=2- 3.47
.ANG. is just large enough to accommodate a monolayer of DNA with a
hydration shell of water. The middle broad peak at q.sub.DNA=0.172
.ANG. is due to the ID array of DNA chains with the spacing between
the DNA strands d.sub.DNA=2/q.sub.DNA. This structure found in
CL--DNA complexes with .PHI..sub.PE<0.41 is analogous to the one
reported in recent studies of the structure and interactions in
DOPC/DOTAP-DNA complexes (58, 60).
[0180] For 0.7<.PHI..sub.PE<0.85 the peaks of the SAXS scans
of the CL--DNA complexes are indexed perfectly on a two-dimensional
(2D) hexagonal lattice with a unit cell spacing of
a=4.pi./[(3).sup.0.5q.sub.1- 0]=67.4 .ANG. for .PHI..sub.PE=0.75.
We were able to observe Bragg peaks up to the 7th order because of
the high brilliance of the synchrotron source, indicating a high
degree of regularity of the structure. FIG. 2(A) at
.PHI..sub.PE=0.75 shows the first four order Bragg peaks of this
hexagonal structure at q.sub.10=0.107 .ANG..sup.-1, q.sub.11=0.185
.ANG..sup.-1, q.sub.20=0.214 .ANG..sup.-1, and q.sub.21=0.283
.ANG..sup.-1. The structure is consistent with a 2D columnar
inverted hexagonal structure shown in FIG. 10 (right) which we
refer to as the H.sub.II.sup.C phase of CL--DNA complexes. The DNA
molecules are surrounded by a lipid monolayer with the DNA/lipid
inverted cylindrical micelles arranged on a hexagonal lattice. The
structure resembles that of the inverted hexagonal H.sub.II phase
of pure DOPE in excess water (61), with the water space inside the
lipid micelle filled by DNA. The larger electron density of DNA
with respect to water leads to the relative suppression of the (57)
and (69) Bragg peak intensities compared with that in the lipid
H.sub.II, phase (59). Assuming again an average lipid monolayer
thickness of 20 .ANG., the diameter of micellar void in the
H.sub.II.sup.C phase is close to 28 .ANG., again sufficient for a
DNA molecule with approximately two hydration shells. For
0.41<.PHI..sub.PE<0.7 the L.sub..alpha..sup.C and
H.sub.II.sup.C structures coexist as shown at .PHI..sub.EF=0.65 and
are nearly epitaxially matched with a.apprxeq.d. For
.PHI..sub.PE>0.85 the H.sub.II.sup.C phase coexists with the
H.sub.II phase of pure DOPE which has peaks at q.sub.10=0.0975
.ANG..sup.-1, q.sub.11=0.169 .ANG..sup.-1, q.sub.20=0.195
.ANG..sup.-1 (arrows in FIG. 11(A) at .PHI..sub.PE=0.87) with a
unit cell spacing of a=74.41 .ANG..
[0181] We also plot in FIG. 11(A) at .PHI..sub.PE=0.41 and 0.75
(solid lines), SAXS scans of CL--DNA complexes at 0.01%
concentrations typically used in cell transfection studies (56,
57). We see that the complexes have their first order Bragg peaks
at exactly the same positions as in the corresponding more
concentrated samples. This demonstrates that in this range of
concentrations the internal structures of the complexes are
independent of the overall DNA and lipid concentrations. For most
of the SAXS experiments we prepared CL--DNA at more concentrated
lipid and DNA concentrations (.apprxeq. 1%) to improve the
signal/background intensity ratio. These mixtures appear as
aggregates of the individual complexes shown in FIG. 13(A and B)
and retain a similar globular morphology.
[0182] The L.sub..alpha..sup.C to H.sub.II.sup.C phase transition
can be induced along a second pathway II (FIG. 10) by the use of a
novel new "helper-lipid mixture" that we introduce in this
invention. To demonstrate this pathway we consider complexes
containing mixtures of DOPC and DOTAP which are always found to
exhibit the lamellar L.sub..alpha..sup.C structure (12) as the SAXS
scan shows in FIG. 11(B) (bottom; .PHI..sub.PC=0.7) with an
interlayer spacing of d=2.pi./q.sub.001=66.84 .ANG.. As a function
of increasing hexanol, a membrane soluble co-surfactant, to the
helper-lipid DOPC we find a structural transition to the
H.sub.II.sup.C phase. This is shown in SAXS scans of complexes
containing DOPC/DOTAP/hexanol (.PHI..sub.PC=0.7, mole ratio of
hexanol to total lipid is 3:1) where the first four diffraction
peaks (01), (11), (20), and (21) of the hexagonal lattice are
clearly indexed with a unit cell size a=62.54 .ANG.. In FIG. 11(C)
we find that in CL--DNA complexes of pure cationic lipid DOTAP the
addition of hexanol does not induce the transition and we always
find the L.sub..alpha..sup.C structure. In this case, the only
effect of the addition of hexanol is to thin the cationic bilayer
membrane (consisting of hexanol:DOTAP at a 3:1 mole ratio) from
d=57.91 .ANG. to d=54.17 .ANG.. The interaxial DNA-DNA spacing is
also observed to increase from d.sub.DNA=27.1 .ANG. to 28.82 .ANG.
consistent with a decrease in the membrane charge density with the
addition of hexanol.
[0183] To understand the L.sub..alpha..sup.C to H.sub.II.sup.C
transition qualitatively along the two pathways (I and II of FIG.
10) we consider the interplay between the electrostatic and
membrane elastic interactions in the complexes. Pure electrostatic
interactions alone are expected to favor the H.sub.II.sup.C phase
which minimizes the charge separation between the anionic groups on
the DNA chain and the cationic lipids (47, 62). The electrostatic
interaction may be resisted by the Helfrich elastic cost (per unit
area) of forming a cylindrical monolayer membrane around DNA:
F/A=0.5 .kappa.(1/R-1/R.sub.o).sup.2 (1)
[0184] Here, .kappa. is the lipid monolayer rigidity, R the radius
of curvature, and R.sub.o the natural radius of curvature. Along
pathway I (FIG. 10) the membrane consists of the two components
DOTAP and DOPE. Cationic DOTAP has a natural (also referred to
herein as spontaneous) curvature
C.sub.0.sup.DOTAP=1/R.sub.0.sup.DOTAP=0/ that is, membranes of pure
DOTAP are known to favor the lamellar L.sub..alpha. phase. However,
DOPE has a negative natural curvature
C.sub.o.sup.DOPE=1R.sub.o.sup.DOPE&- lt;0; that is, DOPE has a
larger area per 2 chains than area per head group (FIG. 10 center
top). Pure DOPE in water forms the inverted hexagonal H.sub.II
phase (61). Thus, along pathway I the natural curvature of the
monolayer mixture of DOTAP and DOPE is driven negative with
C.sub.o=1/R.sub.o=.PHI..sub.PE.sup.VC.sub.o.sup.DOPE, where
.PHI..sub.PE.sup.V is the volume fraction of DOPE in the lipid
mixture monolayer. Hence, as a function of increasing .PHI..sub.PE
we expect a softening of the elastic cost of monolayer deformation
and the transition to the H.sub.II.sup.C phase favored by the
electrostatic interactions as observed experimentally (FIG.
11(A)).
[0185] Pathway II (FIG. 10) involves a subtle mechanism and
introduces an entirely new class of helper-lipids to the field of
nonviral gene therapy. Along this pathway the membrane bending
rigidity .kappa. is reduced significantly because of the addition
of the membrane-soluble cosurfactant molecule hexanol. Cosurfactant
molecules, while not able to stabilize an interface separating
hydrophobic and hydrophilic regions, when mixed in with longer
chain "true" surfactants can lead to dramatic changes in interface
elasticities. Experimental studies have shown that the addition of
hexanol to membranes of lamellar phases with a mole ratio of
between two to four will lead to a significant decrease of the
bending rigidity .kappa. from .apprxeq.20 k.sub.BT to between 2 to
5 k.sub.BT (63). Simple compressional models of surfactant chains
show that .kappa. scales with chain length 1.sub.n
(.varies..delta..sub.m, membrane thickness, n=number of carbons per
chain) and the area per lipid chain A.sub.L as
.kappa..varies.1.sub.n.sup.3/A.sub.L.sup.5(64). Hexanol affects
both 1.sub.n and A.sub.L shown schematically in FIG. 10 (center
bottom). First, the membrane thickness .delta..sub.m decreases upon
addition of the shorter tail cosurfactant molecule hexanol (C.sub.6
chain) to the mixture of DOPC and DOTAP (C.sub.18 chains). Second,
the addition of a significant amount of short hexanol chains to the
long chains (from DOPC and DOTAP) effectively results in a sudden
excess free volume and significantly larger area per lipid chain.
This will lead to a further strong suppression of K making the
membrane highly flexible. Thus, we expect a reduction of the
elastic cost (determined by (1)) of curving the membrane due to the
reduction of .kappa. to lead to the formation of the H.sub.II.sup.C
phase favored by the electrostatic interactions. This was observed
experimentally (FIG. 11(B), open squares). We have further observed
that the transition to the H.sub.II.sup.C phase along pathway II
occurs only in CL--DNA complexes with low enough charge
density-DOTAP/DOPC<0.5 (59). FIG. 11(C) shows SAXS data in this
regime where the L.sub..alpha..sup.C structure is retained in
complexes with pure DOTAP with and without added hexanol consistent
with theory which predicts a renormalized increase in K with
increasing surface charge density (65).
[0186] It is important to note that in the absence of DNA, lipids
formed from a mixture of DOPC and DOTAP with or without hexanol
form stable lamellar L.sub..alpha. phases (with C.sub.o=0) in the
lipid mixtures studied in this work with no tendency of forming the
inverted H.sub.II phase (59). This then is a clear distinction
between the two classes of helper-lipids used along the two
pathways where DOPE/DOTAP/water mixtures do form coexisting
H.sub.II.sup.C and L.sub..alpha..sup.C phases.
[0187] We demonstrate the generality of the lamellar
L.sub..alpha..sup.C to hexagonal H.sub.II.sup.C transformation by
observing it in complexes of DOPE/DOTAP mixtures with two other
negatively charged polyelectrolytes--polyglutamic acid (PGA), a
model polypeptide, and poly-thymine (poly-T), a model of
single-stranded oligo-nucleotides which are used in antisense
delivery applications (66, 67). The phase diagram of
CL-polyelectrolyte complexes is plotted in FIG. 12 showing the
variation of the unit cell parameters in the L.sub..alpha..sup.C
and H.sub.II.sup.C complexes as a function of .PHI..sub.PE for DNA,
a 100 bp poly-T, and PGA. The phase sequence in DOPE/DOTAP mixtures
without the polyelectrolytes is indicated at the top by horizontal
arrows. Pure lipids also form L.sub..alpha. and H.sub.II
structures, although, the H.sub.II is present only in coexistence
with the L.sub..alpha. phase which indicates that the
polyelectrolytes stabilize the H.sub.II.sup.C single phase. The
observed different phase boundaries most likely originate from
differences in diameter and linear charge density between the
polyelectrolytes which in turn leads to different required amounts
of lipid monolayer bending around the polyelectrolyte in the
H.sub.II.sup.C complex. This demonstrates the interplay between
electrostatics and membrane elasticities in these hybrid systems
(59).
[0188] In both condensed phases the complexes appear as highly
dynamic birefringent aggregates when viewed with video-enhanced
optical microscopy in differential-interference-contrast (DIC) and
fluorescence configurations as shown in FIG. 13(A) for
H.sub.II.sup.C ((.PHI..sub.PE=0.73) and FIG. 13(B) for
L.sub..alpha..sup.C ((.PHI..sub.PE=0.3) complexes along pathway I.
For fluorescence experiments cationic lipids were labeled with 0.2
mol % of DHPE-TexasRed and DNA was labeled with YoYo-1 iodide at a
1 dye molecule/i 5bP ratio. Complexes were prepared by gently
mixing DNA (0.01 mg/ml) and lipid (0.1 mg/ml) stock solutions. The
complexes were further diluted with deionized water for
observation. Giant unilamellar vesicles were prepared from mixtures
of 90% DOPC (neutral) and 10% DOPG (negatively charged) lipids.
Positively charged CL--DNA complexes were prepared. The SAXS
experiments were carried out at the Stanford Synchrotron Radiation
Laboratory at 8 keV. CL--DNA complexes were prepared bi mixing
deionized water solutions of highly purified linear X-phage DNA (5
mg/ml; 48502 bp; contour length of 16.5 .mu.m) and cationic
liposomes of mixed lipids (25 mg/ml) directly in a 1.5 mm diameter
quartz x-ray capillary with .kappa.=DOTAP/DNA=3 (wt./wt.) which
yielded positive complexes. The CLs consisting of binary DOPE/DOTAP
mixtures have an average size of 0.06 .mu.m. During the CL--DNA
complex formation cationic lipids replace DNA counterions,
releasing the Na.sup.+ and Cl.sup.- ions into solution with a very
large entropic free energy gain (of order k.sub.BT per released
counterion). The result is a close association between DNA and
lipid in a compact complex with an average size of 0.2 pm size
(59).
[0189] The positive complexes (with .rho.=3) are seen to form
aggregates consisting of connected blobs with the aggregates
becoming smaller and eventually dissociating into individual blobs
with increasing complex charge. Interestingly, the shape of
aggregates is different in the two complex phases: the
L.sub..alpha..sup.C phase forms linear structures, while in the
H.sub.II.sup.C phase the aggregates are predominantly branched
indicating an inherent anisotropic shape to the H.sub.II.sup.C
complexes (59). FIG. 13(A) shows the distribution of Lipid
fluorescence (middle) and DNA fluorescence (right) in the same
CL--DNA complex in the H.sub.II.sup.C phase and FIG. 13(B) shows it
for a CL--DNA complex in the L.sub..alpha..sup.C phase. The
observed overlap of lipid and DNA distributions and the precisely
identical morphologies in the two fluorescence modes shows that the
complexes are indeed highly compact objects with a close
association of lipid and DNA consistent with the SAXS data of these
extremely dilute samples (FIG. 11(A)). At these concentrations and
volume fractions of DOPE the complexes coexist with excess DNA for
.rho.<2.2 and with excess lipid when .rho.>2.2 and we have
not observed the presence of macroscopic lipid aggregates, which
indicates that the only condensed liquid crystalline structures in
the CL--DNA mixtures are complexes.
[0190] To understand the effect of structure on the early stages of
transfection we studied the interaction of CL--DNA complexes with
giant anionic vesicles (G-vesicles) which are models of CL--DNA
complex--anionic endosomal vesicles of cells. Experiments indicate
that the main entry route to mammalian cells is endocytosis where a
local inward deformation of the cell plasma membrane leads to
budding off of an internal vesicle forming the early stage endosome
(68, 69, 70, 71). Thus, at the early stages of cell transfection,
an intact CL--DNA complex is captured inside an endosomal vesicle
which is anionic due to the anionic lipids of the plasma
membrane.
[0191] There is a striking difference between positively charged
H.sub.II.sup.C and L.sub..alpha..sup.C complexes in their
interaction with model anionic lipid membranes even when both types
of structures contain DOPE. We show in FIG. 13(C and D) typical
micrographs of positively charged (.rho.=4) complexes attached to
the fluid membranes of G-vesicles. The L.sub..alpha..sup.C
complexes attach to the G-vesicles and remain stable (C). The
compact complex morphology can be seen in DIC (left) as well as in
the lipid (C, middle) and DNA (C, right) fluorescence. Clearly
there is no fusion between the complex and the G-vesicle.
H.sub.II.sup.C complexes behave dramatically differently upon
attaching to the G-vesicle, rapidly fusing and spreading with it
and losing their compact structure (FIG. 13(D), left, DIC). Since
the amount of lipid in the complex is comparable with that in the
G-vesicle, and since the fusion occurs very quickly, it results in
the formation of multiple free lamella which are observed to
undergo bilayer fluctuations. The loss of the compact complex
structure and the subsequent desorption of DNA molecules from
membrane and their brownian motion between the lamella are seen in
fluorescence (FIG. 13(D), right). This behavior is expected
following fusion which results in the mixing of cationic-lipid
(from the H.sub.II.sup.C complex) with anionic lipid (from the
G-vesicle) effectively "turning off" the electrostatic interactions
(which gave rise to the compact CL--DNA complexes) and releasing of
DNA molecules inside the space between the lamnellae and the
G-vesicle bilayer. Since the geometry is the inverse of CL--DNA
complexes inside anionic endosomal vesicles an expected result is
that upon fusion the inverse geometry will occur with DNA released
and expelled outside the endosome within the cytoplasm.
Fluorescence microscopy studies show similar behavior in mouse
fibroblast cell cultures where L.sub..alpha..sup.C complexes appear
intact in the cell for two hours after endocytic uptake, whereas,
H.sub.II.sup.C complexes show fusion after endocytic uptake.
[0192] The findings unambiguously establish a correlation between
the self-assembled structure of CL--DNA complexes and transfection
efficiency: the empirically established transfectant complexes in
mammalian cell cultures exhibit the H.sub.II.sup.C structure rather
than the L.sub..alpha..sup.C. The reported behavior is in complexes
containing univalent cationic lipids; multivalent cationic lipids
may behave differently. Further, optical microscopy reveals a most
likely origin for why different structures transfect cells with
varying efficiency: in contrast to L.sub..alpha..sup.C complexes,
H.sub.II.sup.C complexes are found to fuse and release DNA when in
contact with anionic vesicles which are cell free models of
cellular organelle membranes, in particular, anionic endosomal
vesicles. Thus, the data suggest a simple direct mechanism of DNA
release into the cytoplasm from endosomal vesicles containing
H.sub.II.sup.C complexes. This then paves the way for a fundamental
understanding of the early-stage events following the endocytic
uptake of CL13 DNA complexes by mammalian cells in nonviral gene
delivery applications.
[0193] FIG. 10 shows a schematic of two distinct pathways from the
lamellar L.sub..alpha..sup.C phase to the columnar inverted
hexagonal H.sub.II.sup.C phase of cationic liposome-DNA (CL--DNA)
complexes. Along Pathway I the natural curvature
(C.sub.o=1/R.sub.o) of the cationic lipid monolayer is driven
negative by the addition of the helper-lipid DOPE. This is shown
schematically (middle top) where the cationic lipid DOTAP is
cylindrically shaped while DOPE is cone-like leading to the
negative curvature. Along pathway II the L.sub..alpha..sup.C to
H.sub.II.sup.C transition is induced by the addition of a new class
of helper-lipids consisting of mixtures of DOPC and the
cosurfactant hexanol which reduces the membrane bending
rigidity.
[0194] FIG. 11 shows synchrotron SAXS patterns of the lamellar
L.sub..alpha..sup.C and columnar inverted hexagonal H.sub.II.sup.C
phases of positively charged CL--DNA complexes. FIG. 11(A) shows
SAXS scans of CL--DNA complexes as a function of increasing weight
fraction .PHI..sub.PE(=DOPE/[DOPE+DOTAP)]) along pathway I of FIG.
10. At .PHI..sub.PE=0.41, the SAXS results from a single phase with
the lamellar L.sub..alpha..sup.C structure shown in FIG. 10(left).
At .PHI..sub.PE=0.75, the SAXS scan results from a single phase
with the columnar inverted hexagonal H.sub.II.sup.C structure shown
in FIG. 10(right). At .PHI..sub.PE=0.65, the SAXS shows coexistence
of the L.sub..alpha..sup.C (dotted line) and H.sub.II.sup.C phases.
At .PHI..sub.PE=0.87, the SAXS shows coexistence of the
H.sub.II.sup.C phase and the inverted hexagonal H.sub.II phase of
pure DOPE (Arrows). SAXS patterns of complexes made from extremely
dilute DNA (0.01 mg/ml) and lipid (0.1 mg/ml) solutions are plotted
as solid lines for .PHI..sub.PE=0.41 and 0.75. FIG. 10(B) shows
SAXS scans of CL--DNA at a constant DOPC weight fraction
.PHI..sub.PC(=DOPC/[DOPC+DOTAP)]) with no hexanol (a co-surfactant)
and at a hexanol to total lipid mole ratio of 3:1 along pathway II
of FIG. 10. With no hexanol (filled squares), the structure is
lamellar L.sub..alpha..sup.C whereas the complexes with hexanol
(open squares) exhibit the hexagonal H.sub.II.sup.C structure. FIG.
10(C) shows SAXS scans of CL--DNA complexes with DOPC weight
fraction .PHI..sub.PC=0. The complexes remain in the
L.sub..alpha..sup.C phase with and without added hexanol.
[0195] FIG. 12 shows variation of the unit cell parameters in the
lamellar L.sub..alpha..sup.C (open symbols denote the interlayer
spacing d) and hexagonal H.sub.II.sup.C (filled symbols denote the
hexagonal unit cell dimension a) complexes as a function of
.PHI..sub.PE in .lambda.-DNA (circles, open and filled),
poly-Thymine (triangles, open and filled), and polyglutamic acid
(squares, open and filled; PGAtween dashed and dotted lines), the
coexisting L.sub..alpha..sup.C and H.sub.II.sup.C (between the
solid and dashed lines), and H.sub.II.sup.C and H.sub.II regimes
(beyond dotted lines). The arrows on top of the figure indicate the
phase boundaries in the lamellar phase in mixtures of DOPE and
DOTAP.
[0196] FIG. 13(A), and (B) show video-microscopy images of
positively charged CL--DNA complexes in the H.sub.II.sup.C (A) and
L.sub..alpha..sup.C (B) phases. In all cases complexes were viewed
in Differential-Interference-Contrast (DIC) (left), lipid
fluorescence (middle), and DNA fluorescence (right). Scale bar is 3
.mu.m in DIC and 6 .mu.m in fluorescence images. FIG. 13(C), and
(D) show positively charged H.sub.II.sup.C and L.sub..alpha..sup.C
complexes interact differently with the negatively charged giant
vesicles (G-vesicles). The L.sub..alpha..sup.C complexes simply
stick to the G-vesicle and remain stable for many hours, retaining
their blob-like morphology (C). The blobs are localized in DIC as
well as lipid and DNA fluorescence modes. The H.sub.II.sup.C
complexes break-up and spread immediately after attaching to
G-vesicles, indicating a fusion process between the complex and the
vesicle lipid bilayer (D). The loss of the compact structure of the
complex is evident in both lipid and DNA fluorescence modes. Scale
bar is 20 .mu.m in both DIC and fluorescence images.
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