U.S. patent application number 10/436950 was filed with the patent office on 2003-11-13 for emulsion and micellar formulations for the delivery of biologically active substances to cells.
Invention is credited to Huang, Leaf, Liu, Dexi, Liu, Feng, Yang, Jing-Ping.
Application Number | 20030211143 10/436950 |
Document ID | / |
Family ID | 24129008 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211143 |
Kind Code |
A1 |
Liu, Dexi ; et al. |
November 13, 2003 |
Emulsion and micellar formulations for the delivery of biologically
active substances to cells
Abstract
New emulsion and micelle formulations are described as are
complexes of these formulations with biologically active
substances. The novel formulations are different from cationic
lipid vectors such as cationic liposomes in that the complexes
formed between biologically active substances and the emulsion and
micellar formulations of this invention are physically stable and
their transfection activity is resistant to the presence of serum.
These novel formulations are disclosed to be useful in areas such
as gene therapy or vaccine delivery.
Inventors: |
Liu, Dexi; (Pittsburgh,
PA) ; Liu, Feng; (Pittsburgh, PA) ; Yang,
Jing-Ping; (Pittsburgh, PA) ; Huang, Leaf;
(Wexford, PA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
24129008 |
Appl. No.: |
10/436950 |
Filed: |
May 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10436950 |
May 12, 2003 |
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10038417 |
Jan 2, 2002 |
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6586003 |
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10038417 |
Jan 2, 2002 |
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09595385 |
Jun 15, 2000 |
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09595385 |
Jun 15, 2000 |
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08534180 |
Sep 26, 1995 |
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6120794 |
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Current U.S.
Class: |
424/450 ;
435/458; 514/44R |
Current CPC
Class: |
Y10S 514/938 20130101;
A61K 9/1075 20130101 |
Class at
Publication: |
424/450 ; 514/44;
435/458 |
International
Class: |
A61K 048/00; A61K
009/127; C12N 015/88 |
Claims
1. An oil-in-water emulsion formulation comprising lipid components
and an aqueous carrier, wherein the lipid components comprise an
oil component, a cationic amphiphile component and a nonionic
surfactant component.
2. The emulsion formulation of claim 1 wherein the lipid components
further comprise a neutral phospholipid component.
3. The emulsion formulation of claim 1 wherein the oil component is
present in an amount from about 10 to about 80 weight % of the
total lipid components in the formulation, the amphiphile component
is present in an amount from about 5 to about 80 weight % of the
total lipid components and the nonionic surfactant component is
present in an amount from about 5 to about 50 weight % of the total
lipid components.
4. The emulsion formulation of claim 3 further comprising a neutral
phospholipid component present in an amount from about 5 to about
25 weight % of the total lipid components in the formulation.
5. The emulsion formulation of claim 4, wherein the amphiphile
component is a cationic lipid.
6. A micellar formulation comprising lipid components and an
aqueous carrier, wherein the lipid components comprise a cationic
amphiphile component and a nonionic surfactant component.
7. The micellar formulation of claim 6, wherein the lipid
components further comprise a neutral phospholipid component.
8. The micellar formulation of claim 6 wherein the amphiphile
component is present in an amount from about 10 to about 90 weight
% of the total lipid components and the nonionic surfactant
component is present in an amount from about 90 to about 10 weight
% of the total lipid components.
9. The micellar formulation of claim 8, further comprising a
neutral phospholipid component present in an amount from about 5 to
about 40 weight % of the total lipid components.
10. The micellar formulation of claim 9, wherein the amphiphile
component is a cationic lipid.
11. A complex for facilitating the delivery of a negatively charged
biologically active substance to cells, said complex comprising the
negatively charged biologically active substance and the emulsion
formulation of claim 1.
12. The complex of claim 11, wherein the negatively charged
substance is a nucleic acid.
13. The complex of claim 12, wherein the weight ratio of nucleic
acid to total lipid components in the emulsion formulation in said
complex is about 1:1 to about 1:50.
14. A complex for facilitating the delivery of a negatively charged
biologically active substance to cells, said complex comprising
said negatively charged substance and the micellar formulation of
claim 6.
15. The complex of claim 14, wherein the negatively charged
substance is a nucleic acid.
16. The complex of claim 15, wherein the weight ratio of nucleic
acid to total lipid components in the micellar formulation in said
complex is from about 1:1 to about 1:50.
17. A method for delivering a negatively charged biologically
active substance to cells comprising exposing the cells to the
complex of claim 11 thereby facilitating the delivery of the
negatively charged biologically active substance to the cells.
18. The method of claim 17 wherein the cells are mammalian cells
exposed to the complex in the presence of serum.
19. A method for delivering a negatively charged biologically
active substance to cells comprising exposing the cells to the
complex of claim 14 thereby facilitating the delivery of the
negatively charged biologically active substance to the cells.
20. The method of claim 19 wherein the cells are mammalian cells
exposed to the complex in the presence of serum.
21. The methods for delivering a negatively charged biologically
active substance to cells according to claims 17 and 19 wherein the
cells are exposed to the complex in vivo by administering the
complexes to an animal or human in an amount effective to
facilitate the delivery of the negatively charged substance to the
cells of the animal or human.
22. The method of claim 21, wherein the negatively charged
substance is a nucleic acid.
23. The methods for delivering a negatively charged biologically
active substance to cells according to claims 17 and 19 wherein the
cells are exposed to the complex in vitro.
24. The method of claim 23, wherein the negatively charged
substance is a nucleic acid.
25. A method of producing an oil-in-water emulsion formulation
comprising: (a) combining an oil component, a cationic amphiphile
component and a nonionic surfactant component; and (b) adding
aqueous carrier to produce said emulsion formulation.
26. A method of producing a micellar formulation comprising: (a)
combining a cationic amphiphile component and a nonionic surfactant
component; and (b) adding aqueous carrier to produce said micellar
formulation.
27. The methods of claims 25 and 26 further comprising combining
the components of step (a) with a neutral phospholipid
component.
28. The method of claim 27, wherein the components of step (a) are
combined in an organic solvent and the solvent is removed to leave
a lipid film prior to step (b).
29. A method of producing a lipid film having an oil component, a
cationic amphiphile component and a nonionic surfactant component;
said method comprising: (a) combining an organic solvent with the
oil component, the amphiphile component and the nonionic surfactant
component; and (b) removing the organic solvent to leave said lipid
film.
30. A method of producing a lipid film having a cationic amphiphile
component and a nonionic surfactant component; said method
comprising: (a) combining an organic solvent with the amphiphile
component and the nonionic surfactant component; and (b) removing
the organic solvent to leave said lipid film.
31. A lipid film capable of forming an oil-in-water emulsion upon
suspension in an aqueous carrier, said film having an oil
component, a cationic amphiphile component and a nonionic
surfactant component.
32. A lipid film capable of forming a micelle upon suspension in
solution, said film having a cationic amphiphile component and a
nonionic surfactant component.
33. The lipid films of claims 32 and 33, said films further having
a neutral phospholipid component.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of lipid
dispersions to deliver biologically active substances to cells. In
particular, the present invention relates to emulsion and micellar
formulations and to the ability of these formulations to form
stable complexes with biologically active substances and thereby
facilitate the delivery of these substances to cells.
BACKGROUND OF THE INVENTION
[0002] Cationic liposomes are of interest as a non-viral vehicle
for the delivery of biologically active substances such as drugs,
hormones, enzymes, nucleic acids and antigens, including viruses,
to cells both in vitro and in vivo. Indeed, cationic liposomes have
been demonstrated to deliver genes in vivo (Nabel, E. G., et al.
(1990) Science, 249: 1285-1288), (Brigham, K. L., et al. (1989) Am.
J. Respir. Cell Mol. Biol., 195-200, Stribling, R., et al. (1992)
Proc. Natl. Acad. Sci. U.S.A., 89: 11277-11281), (Plautz, G. E., et
al. (1993) Proc. Natl. Acad. Sci. U.S.A., 90: 4645-4649, Stewart,
M. J., et al. (1992) Hum. Gene Ther., 3: 267-275). However, the
inhibition by serum components of the transfer of nucleic acids by
cationic liposomes limits the application of liposomes as a vector
for nucleic acids in vivo to regional administrations which avoid
exposure to serum.
[0003] In addition, stability is a major problem limiting the use
of liposomes, both in terms of shelf life and after administration
in vivo. Thus, it is desirable to explore the use of other types of
lipid dispersions as delivery systems utility for biologically
active substances.
[0004] U.S. Pat. No. 4,610,888 refers to the use as a drug-delivery
system of water-in-oil emulsions in which the volume of aqueous
phase ranges from about 0.7% to about 10.25% of the volume of the
lipid components used. However, such water-in-oil emulsions are
unsuitable for delivering substances in blood or in other aqueous
body tissues.
SUMMARY OF INVENTION
[0005] The present invention relates to novel emulsion and micellar
formulations useful for delivering biologically active substances
to cells. The emulsion and micellar formulations of this invention
are compatible with blood, retain activity in the presence of serum
and are stable in storage. The emulsions of this invention comprise
lipid components and an aqueous carrier, where the lipid components
comprise an oil component, a cationic amphiphile component,
preferably a cationic lipid, and a nonionic surfactant component.
The micellar formulations comprise lipid components and an aqueous
carrier, where the lipid components comprise a cationic amphiphile
component and a nonionic surfactant component. The lipid components
of the emulsion and micellar formulation of the present invention
may further comprise a neutral phospholipid component.
[0006] "Component" as used throughout the specification and claims
is defined as: comprising at least one cationic amphiphile or a
mixture of amphiphiles when used in the phrase "amphiphile
component"; comprising at least one oil or a mixture of oils when
used in the phrase "oil component"; comprising at least one
nonionic surfactant or a mixture of nonionic surfactants when used
in the phrase "nonionic surfactant component"; comprising at least
one neutral phospholipid or a mixture of neutral phospholipids when
used in the phrase "neutral phospholipid component".
[0007] The invention further relates to complexes formed by
combining biologically active substances and the above-identified
emulsion and micellar formulations. These biologically active
substance:emulsion and biologically--active substance:micelle
complexes are stable over time and may have therapeutic and/or
prophylactic utility in vivo depending on the activity of the
biologically active substance contained in the complex.
[0008] This invention also provides a method for delivering a
biologically active substance to cells by exposing cells to the
complexes of this invention. In one embodiment, a method of
exposing cells to a biologically active substance is provided, said
method comprising culturing said cells in the presence of a
biologically active substance:emulsion complex or a biologically
active substance:micelle complex thereby facilitating delivery of
the biologically active substance to cells.
[0009] The invention further provides a method of delivering a
biologically active substance to cells in vivo comprising
administering to an animal or human the complexes of this
invention. It is to be understood that the complexes used for the
delivery of biologically active substances to cells in vitro or in
vivo may be freshly prepared by admixture or may be prepared
earlier and stored prior to their use.
[0010] The invention further relates to a kit containing an
emulsion or micellar formulation of the present invention. The
invention also provides a kit containing a complex formed between a
biologically active substance and an emulsion or micellar
formulation of the present invention.
[0011] Methods for producing emulsion and micellar formulations
according to the invention are also'provided herein.
[0012] In one embodiment, a method for producing an emulsion
formulation of this invention comprises
[0013] a) combining an organic solvent with an oil component, a
cationic amphiphile component and a nonionic surfactant
component;
[0014] b) removing the organic solvent to leave a lipid film;
and
[0015] c) suspending the lipid film in an aqueous carrier to
produce said emulsion formulation miscible in aqueous solution.
[0016] In an alternative embodiment, the oil may serve as the
organic solvent in step (a) such that the method for producing an
emulsion formulation of this invention comprises
[0017] a) combining an oil component, a cationic amphiphile
component and a nonionic surfactant component; and
[0018] b) adding an aqueous carrier to the combination of step
(a).
[0019] When a neutral phospholipid component is to be included in
the emulsion, the neutral phospholipid component is combined with
the above components in step (a).
[0020] The method for producing a micellar formulation miscible in
aqueous solution comprises:
[0021] a) combining an organic solvent with a cationic amphiphile
component and a nonionic surfactant component;
[0022] b) removing the organic solvent to leave a lipid film;
and
[0023] c) suspending the lipid film in an aqueous carrier to
produce said micellar formulation miscible in aqueous solution.
[0024] When a neutral phospholipid component is to be included in
the micellar formulation, the neutral phospholipid component is
combined with the above components in step (a).
DESCRIPTION OF FIGURES
[0025] FIG. 1 shows the optimization of transfection of BL6 cells
with complexes formed between pCMV-Luc DNA and formulations #21
(.circle-solid.-.circle-solid.), #27 (.box-solid.-.box-solid.), #28
(.tangle-solidup.-.tangle-solidup.) or #30
(.largecircle.-.largecircle.) (see Table 3 for compositions of
formulations) by mixing 6 .mu.l of each formulation (6 .mu.l of
each formulation contained 4.5 .mu.g of DC-Chol) with varying
amounts of pCMV-Luc DNA as indicated on the horizontal axis.
[0026] FIG. 2 shows the optimization of transfection of BL6 cells
with complexes formed between pCMV-Luc DNA and varying amounts of
formulations #21 (.circle-solid.-.circle-solid.), #27
(.box-solid.-.box-solid.), #28 (.tangle-solidup.-.tangle-solidup.)
or #30 (.largecircle.-.largecircle.) as indicated on the horizontal
axis (see Table 3 for compositions of formulations). The amount of
pCMV-Luc DNA was fixed at 2 .mu.g for formulations #27 and #30 and
at 1.5 .mu.g pCMV-Luc DNA for formulations #21 and #28 and ".mu.g
formulation" on the horizontal axis refers to .mu.gs of total lipid
components present in the amount of formulation combined with
pCMV-Luc DNA to form complex.
[0027] FIG. 3 shows the stability of the complexes formed between
DNA and the indicated emulsion or micellar formulations (see Table
3 for composition of formulations). Complex was prepared with 2
.mu.g of pCMVCAT and 16 .mu.l of the indicated formulations
containing the same amount of DC-Chol (12 .mu.g) in a final volume
of 250 .mu.l, except for the DC-Chol/DOPE liposome:DNA complexes
which were prepared with 1 .mu.g pCMVCAT and 6 .mu.g liposome in a
final volume of 250 .mu.l.
[0028] FIG. 4 shows the effect of varying the concentration of
Tween 80 in emulsions containing 0.25 mg oil, 0.25 mg DOPE, 0.75 mg
DC-Chol and x mg Tween 80 per ml on the average diameter of
concentrated ((.circle-solid.-.circle-solid.) and diluted
(.largecircle.-.largecircle.- ) pCMV-Luc DNA/emulsion
complexes.
[0029] FIGS. 5A and 5B show the effect of Tween 80 on the
transfection/activity of concentrated (FIG. 5A) and diluted (FIG.
5B) pCMV-Luc DNA/emulsion complexes in BL6 cells in medium
containing either 0 or 20% serum. The emulsion formulations used to
produce the diluted and concentrated DNA:emulsion complexes
contained 0.25 mg oil, 0.25 mg DOPE, 0.75 mg DC-Chol and varying
amounts of Tween 80 per ml. Concentrated DNA/emulsion complex was
formed by adding 2 .mu.l of solution containing 8 .mu.g of pCMV-Luc
DNA to 72 .mu.l of emulsion and diluted DNA/emulsion complex was
formed by combining 2 .mu.g of pCMV-Luc DNA in 125 .mu.l with 18
.mu.l of emulsion diluted to 125 .mu.l.
[0030] FIG. 6 shows CAT reporter gene expression in mice injected
via the tail vein with DNA:emulsion or DNA:micelle complexes. The
complexes were formed as follows: 200 .mu.l each of 4.times.
concentrates of formulations #21 (1100 .mu.g total lipid
components), #28 (1000 .mu.g total lipid components), #34 (900
.mu.g total lipid components) and #31 (700 .mu.g total lipid
components) were mixed with 6 .mu.l of 5M NaCl to a final
concentration of NaCl of 0.15M and then combined with 25 .mu.l of 4
.mu.g/.mu.l pCMV-CAT DNA (100 .mu.g). The amount of DC-Chol
contained in each DNA:emulsion and DNA:micelle complex was 600
.mu.g. After two days, organs were excised and protein was
extracted. CAT activity was measured by using 0.1 .mu.Ci [.sup.14C]
chloramphenicol as substrate. Each bar represents the mean of two
mice.
DETAILED DESCRIPTION OF INVENTION
[0031] The present invention relates to emulsion and micellar
formulations which form stable complexes with biologically active
and thereby facilitate the delivery of the biologically active
substances to cells.
[0032] The emulsion formulations of this invention are oil-in-water
emulsions which comprise an aqueous carrier and the following lipid
components, an oil component, a cationic amphiphile component, a
nonionic surfactant component and optionally, a neutral
phospholipid component.
[0033] Preferably, the total lipid components are present in the
emulsion formulation in an amount from about 0.001 to about 20% by
weight, more preferably from about 0.01 to about 10% by weight and
most preferably from about 0.05 to about 2% by weight, with the
remainder of the emulsion by weight being aqueous carrier. Thus,
for example, for formulation #1 in Table 1 where 0.625 mg of total
lipid components are present in 0.5 ml of PBS, the weight % of
total lipid components in formulation #1 can be calculated as
follows: Assuming 1 ml of PBS, like 1 ml of water, weighs
approximately 1000 mg, then 0.5 ml of PBS weighs 500 mg and the
weight % of total lipid components contained in formulation #1 is 1
0.625 mg 0.625 mg + 500 mg .times. 100 = 0.125 % .
[0034] Of the total lipid components present in the emulsion
formulations of this invention, preferably, the amphiphile
component is present in an amount from about 5 to about 80 weight %
of the total lipid components in the emulsion formulation; the oil
component is present in an amount from about 10 to about 80 weight
% of the total lipid components; the nonionic surfactant component
is present in an amount from about 5 to about 50 weight % of the
total lipid components, and optionally, the neutral phospholipid
component is present in the formulation in an amount from about 5
to about 25 weight % of the total lipid component.
[0035] More preferably, the oil component is present in an amount
from about 10-60 weight % of the total lipid components in the
emulsion formulation; the amphiphile component is present in an
amount from about 20-60 weight % of the total lipid components; the
nonionic surfactant component is present in an amount from about
10-50 weight % of the total lipid components and optionally, a
neutral phospholipid component is present in amount from about
10-40 weight % of the total lipid components.
[0036] Most preferably, the emulsion formulation comprises the oil
component in amount from about 10-20 weight % of the total lipid
components; the amphiphile component in an amount from about 40-60
weight % of the total lipid components; the nonionic surfactant
component in an amount from about 20-50 weight % of the total lipid
components and optionally, the neutral phospholipid component in an
amount from about 10-20 weight % of the total lipid components. A
particularly preferred emulsion formulation contains an oil, a
cationic amphiphile, a nonionic surfactant and a neutral
phospholipid in a weight ratio of about 2:6:1:2.
[0037] The micellar formulations of this invention are compatible
with blood. The micellar formulations comprise an aqueous carrier
and the following lipid components: a cationic amphiphile
component, a nonionic surfactant component and optionally, a
neutral phospholipid component.
[0038] Preferably, the total lipid components are present in the
micellar formulation in an amount ranging from about 0.0001 to
about 70% by weight, more preferably from about 0.001 to about. 60%
by weight and most preferably from about 0.001 to about 50 by
weight, with the remainder by weight of the micellar formulation
being aqueous carrier. Thus, for example, for formulation #15 in
Table 2 where 1.25 mg of total lipid components are present in 1 ml
of PBS, the weight % of total lipid components in formulation #15
can be calculated as follows: Assuming 1 ml of PBS, like 1 ml of
water, weighs approximately 1000 mg, then the weight % of total
lipid components in formulation #15 is 2 1.25 mg 1.25 mg + 1000 mg
.times. 100 = 0.125 % .
[0039] Of the total lipid components contained in the micellar
formulations of this invention, preferably, the amphiphile
component is present in an amount from about 10 to about 90 weight
0% of the total lipid components in the micellar formulation, the
nonionic surfactant-component is present in an amount from about 10
to about 90 weight % of the total lipid components; and optionally,
the neutral phospholipid component is present in an amount from
about 5 to about 40 weight % of the total lipid components.
[0040] More preferably, the amphiphile component is present in an
amount from about 30 to about 90 weight % of the total lipid
components in the micellar formulation, the nonionic surfactant
component is present in an amount from about 10 to about 70 weight
% of the total lipid components and optionally, the neutral
phospholipid component is present in an amount from about 5 to
about 30 weight % of the total lipid components.
[0041] Most preferably, the amphiphile component is present in an
amount from about 50 to about 90 weight %C of the total lipid
components in the micellar formulation, the nonionic surfactant
component is present in an amount from about 10 to about 50 weight
% of the total lipid components and optionally, the neutral
phospholipid component is present in an amount from about 10 to
about 20 weight % of the total lipid components. A particularly
preferred micellar formulation contains a cationic amphiphile, a
nonionic surfactant and a neutral phospholipid in a weight ratio of
about 6:1:2.
[0042] By oil component as used herein is meant any water
immiscible component that is conventionally referred to as an oil.
It is understood that the oil component may include mixtures of two
or more oils. Examples of oils which can be used to produce the
emulsion formulations of the present invention include, but are not
limited to, natural oils such as almond oil, coconut oil, cod liver
oil, corn oil, cottonseed oil, castor oil, olive oil, palm oil,
peanut oil, peppermint oil, rose oil, safflower oil, sesame oil,
soybean oil, sunflower oil and vegetable oil and synthetic oils
such as triethylglycerol and diethylglycerol. A preferred oil is
castor oil.
[0043] The cationic amphiphile component of the formulations of
this invention may be any cationic amphiphile or mixture of
amphiphiles which is effective for use in liposomes or for
producing lipid complexes capable of delivering a biologically
active substance to cells. For example, the amphiphiles described
in Bolcsak et al U.S. Pat. No. 5,100,662, which is incorporated
herein by reference, would be suitable for use in this invention.
Additional examples of cationic amphiphiles suitable for
formulating the emulsion and micellar formulations of this
invention include, but are not limited to, cationic lipids such as
1,2bis(oleoyloxy)-3-(trimethylammonio) propane (DOTAP);
N-[1,-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium chloride
(DOTMA) or other N-(N,N-1-dialkoxy)-alklyl-N,N,N-trisubstituted
ammonium surfactants; 1,2dioleoyl-3-(4'-trimethylammonio)
butanoyl-sn-glycerol (DORT) or cholesteryl (4'trimethylammonia)
butanoate (ChOTB) where the trimethylammonium group is connected
via a butanoyl spacer arm to either the double chain (for DOTB) or
cholesteryl group (for ChOTB); DORI
(DL-1,2-dioleoyl-3-dimethylaminopropyl-B-hydroxyethylammonium) or
DORIE
(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammonium)
(DORIE) or analogs thereof as disclosed in WO 93/03709,
incorporated herein by reference;
1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC);
cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as
doctadecylamidoglycylspermine (DOGS) and dipalmitoyl
phosphatidyesthanolamidospermine (DPPES) or the cationic lipids
disclosed in U.S. Pat. No. 5,283,185, incorporated herein by
reference,
cholesteryl-3.beta.-carboxyl-amido-ethylenetrimethylammonium
iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl
carboxylate iodide, cholesteryl-3.beta.-carboxyamidoethyleneamine,
cholesteryl-3.beta.-oxysuccinamidoethylenetrimethylammonium iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3.beta.-oxysuc-
cinate iodide, 2-[(2-trimethylammonio)-ethylmethylamino]
ethyl-cholesteryl-3.beta.-oxysuccinate iodide,
3.beta.[N-(N',N'-dimethyla- minoethane) carbamoyl] cholesterol
(DC-Chol), and 3.beta.-[N-(polyethylene-
imine)-carbamoyl]cholesterol.
[0044] Examples of preferred amphiphiles include
cholesteryl-3.beta.-carbo- xyamidoethylenetri-methylammonium
iodide, 1-dimethylamino-3-trimethylammon-
io-DL-2-propyl-cholesteryl carboxylate iodide,
cholesteryl-3.beta.-carboxy- amidoethyleneamine,
cholesteryl-3.beta.-oxysuccinamidoethylenetrimethylamm- onium
iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-
.beta.-oxysuccinate iodide,
2-[(2-trimethylammonio)ethylmethylamino]ethyl--
cholesteryl-3.beta.-oxysuccinateiodide, 3.beta.[N-(N',
N'dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol), and
3.beta.[N-(polyethyleneimine)-carbamoyl]cholesterol.
[0045] Since an attribute of the emulsion and micellar formulations
of the present invention is their stability when stored alone or as
complexes with biologically active substances, it will be
understood by those of ordinary skill in the art that preferred
cationic amphiphiles are cationic lipids in which bonds between the
lipophilic group and the amino group are stable in aqueous
solution. Such bonds include amide bonds, ester bonds, ether bonds
and carbamoyl bonds. A more preferred cationic lipid is
DC-Chol.
[0046] The nonionic surfactant component of the formulations of
this invention includes at least one nonionic surfactant of a
molecular weight between 200 and 20,000. In one embodiment, these
surfactants may be formed by reacting a hydrophobic
hydroxyl-containing compound (e.g., an alcohol or phenol) with
ethylene oxide where the number of ethylene oxide groups may be
added to any desired extent. However, those of ordinary skill in
the art would understand that the ability to stabilize the
emulsions or micelles of this invention may depend on the relative
amount of ethylene oxide added to a given hydrophobic group. It is
further understood that surfactants having branched chain ethylene
oxide moieties cover more surface area than surfactants having
single chain ethylene oxide moieties and that therefore, the single
chain surfactants may have to be used in larger amounts than the
branched chain surfactants to produce the emulsion and micellar
formulation of this invention.
[0047] Examples of nonionic surfactants of this invention include,
but are not limited to, polyethylene glycol, derivatives of
phosphatidylethanolamine and synthetic detergents commercially
available under the brand names Span, 1
[0048] Preferred surfactants are branched chain surfactants such as
Tween 20, Tween 40, Tween 60 and Tween 80.
[0049] When optionally added to the emulsion and micellar
formulations of this invention, the neutral phospholipid component
way be a single neutral phospholipid or a mixture of neutral
phospholipids. Examples of neutral phospholipids which may be
optionally added to the formulations of this invention include, but
are not limited to, phosphatidylcholine (PC) or
phosphatidylethanolamine (PE) or fully saturated or partially
hydrogenated phosphatidylcholines (PC) or phosphatidylethanolomines
(PE) having aliphatic chains between 6 and 24 atoms in length such
as dioleoyl-PC (DOPC) and dioleoyl-PE (DOPE). A preferred neutral
phospholipid is DOPE.
[0050] Methods for producing the emulsion and micellar formulations
of the present invention are also provided.
[0051] One method for producing emulsion formulations of this
invention comprises:
[0052] (a) combining an organic solvent with an oil component, a
cationic amphiphile component, a nonionic surfactant component and
optionally, a neutral phospholipid component;
[0053] (b) removing the organic solvent to leave a lipid film;
and
[0054] (c) suspending the lipid film in an aqueous carrier to
produce said emulsion formulation.
[0055] An alternative method for producing the emulsion
formulations of this invention comprises:
[0056] (a) combining an oil component, a cationic amphiphile
component, a nonionic surfactant component and optionally, a
neutral phospholipid component; and
[0057] (b) adding an aqueous carrier to the combination of
components in step (a) to produce said emulsion.
[0058] Preferably, average diameters of the emulsion formulations
are less than about 1000 nm, more preferably less than 800 nm, and
most preferably less than 500 nm.
[0059] Preferred components of the emulsions of the present
invention include phosphate-buffered saline (PBS) as the aqueous
carrier, castor oil as the oil component, DC-Chol as the amphiphile
component, Tween 80 as the nonionic surfactant component and
optionally, phosphatidylcholine or DOPE as the neutral phospholipid
component.
[0060] A method for producing the micellar formulations of this
invention comprises:
[0061] (a) combining an organic solvent with a cationic amphiphile
component, a nonionic surfactant component and optionally a neutral
phospholipid component;
[0062] (b) removing the organic solvent to leave a lipid film;
and
[0063] (c) suspending the lipid film in an aqueous carrier to form
said micellar formulation.
[0064] Preferably, average diameters of the micellar formulations
are less than about 1000 nm, more preferably less than about 800
rim; and most preferably less than about 500 nm.
[0065] Preferred components of the micellar formulations of the
present invention include phosphate-buffered saline as the aqueous
carrier, DC-Chol as the amphiphile component, Tween 80 as the
nonionic surfactant component and optionally, PC or DOPE as the
neutral phospholipid component.
[0066] When an organic solvent is used in the above methods to
produce the micellar and emulsion formulations of this invention,
any organic solvent which does not leave a toxic residue following
removal and which solubilizes the lipid components of the emulsion
and micellar formulations of this invention is suitable for use.
Examples of suitable solvents include lower alcohols,
dimethoxyethane, dioxane, tetrahydrofuran, tetrahydropyran,
diethylether, acetone, dimethylsulfoxide (DMSO), dimethylformamides
(DMF), and halogenated hydrocarbons, such as chloroform,
acetonitrile, or mixtures thereof. A preferred organic solvent is
chloroform.
[0067] The organic solvent may be removed by drying the combination
of step (a) under a suitable gas such as argon or nitrogen and/or
under a vacuum. The dried film may then be lyophilized and stored
at about -80 to about 37.degree. C. or may be resuspended in a
suitable aqueous carrier. Aqueous carriers suitable for use in this
invention are non-toxic to cells and may or may not be buffered.
When the carriers are buffered, suitable buffers include buffers
such as citrate, carbonate, bicarbonate, acetate, Tris, glycinate
and maleate. Aqueous carriers which may be used in the formulations
of this invention include, but are not limited to, distilled water,
normal saline solution and phosphate-buffered saline. It is to be
understood that a preferred pH range for the emulsion and micellar
formulations of this invention is a pH range in which the
particular cationic amphiphile component present in a formulation
is positively charged. Those of ordinary skill in the art would
readily be able to determine such a pH range from the pKa of the
cationic amphiphile component present in a particular
formulation.
[0068] It is further understood that the aqueous carrier in which
the lipid film is suspended may include ingredients such as
stabilizers, antibiotics, or antifungal and antimycotic agents.
[0069] Once formed, the micellar and emulsion formulations may be
mixed with biologically active substances to produce complexes
which are stable in storage as reflected by a retention of the
activity of the biologically activity substance over time or by
retention of the diameter of the emulsion or micellar formulation
over time.
[0070] In one embodiment, the ability of an emulsion or micellar
formulation of this invention to deliver a biologically active
substance to a cell may be tested by exposing cells to complexes
formed between an emulsion or micellar formulation and a plasmid
construct containing a reporter gene as the biologically active
substance. Such reporter genes are known to those of ordinary skill
in the art and include, but are not limited to, the chloramphenicol
acetyltransferase gene, the luciferase gene, the
.beta.-galactosidase gene and the human growth hormone gene.
[0071] By "biologically active substance" as used throughout the
specification and claims is meant a molecule, compound, or
composition, which, when present in an effective amount, reacts
with and/or affects living cells and organisms. It is to be
understood that depending on the nature of the active substance,
the active substance may either be active at the cell surface or
produce its activity, such as with DNA or RNA, after being
introduced into the cell.
[0072] Examples of biologically active substances include, but are
not limited to, nucleic acids such as DNA, cDNA, RNA (full length
mRNA, ribozymes, antisense RNA, decoys), oligodeoxynucleotides
(phosphodiesters, phosphothioates, phosphoramidites, and all other
chemical modifications), oligonucleotide (phosphodiesters, etc.) or
linear and closed circular plasmid DNA; carbohydrates; proteins and
peptides, including recombinant proteins such as for example
cytokines (is interleukins), trophic and growth or naturation
factors (eg NGF, G-CSF, GM-CSF), enzymes, vaccines (eg HBsAg,
gp120); vitamins, prostaglandins, drugs such as local anesthetics
(e.g. procaine), antimalarial agents (e.g. chloroquine), compounds
which need to cross the blood-brain barrier such as anti-parkinson
agents (e.g. leva-DOPA), adrenergic receptor antagonists (e.g.
propanolol), anti-neoplastic agents (e.g. doxorubicin),
antihistamines, biogenic amines (e.g. dopamine), antidepressants
(e.g. desipramine), anticholinergics (e.g. atropine),
antiarrhythmics (e.g. quinidine), antiemetics (e.g.
chloroprimamine) and analgesics (e.g. codeine, morphine) or small
molecular weight drugs such as cisplatin which enhance transfection
activity, or prolong the life time of DNA in and outside the
cells.
[0073] When the biologically active substance is an antigenic
protein or peptide, the complexes formed by the emulsion or
micellar formulations of the present invention may be utilized as
vaccines. In this embodiment, the presence of oil in the emulsion
formulation may enhance an adjuvant effect of the complex.
[0074] Preferred biologically active substances are negatively
charged substances such as nucleic acids, negatively charged
proteins and carbohydrates including polysaccharides, or negatively
charged drugs.
[0075] In a more preferred embodiment the biologically active
substances are nucleic acids and in a most preferred embodiment,
the nucleic acids are nucleic acids which encode a gene or a gene
fragment or which effect transcription and/or translation.
[0076] The complexes of the present invention may be utilized to
deliver biologically active substances to cells in vitro or in
vivo.
[0077] When the biologically active substance is a nucleic acid, it
is believed that the cationic amphiphile binds to the negatively
charged nucleic acid. Preferably, nucleic acid:emulsion complexes
of this invention to be used in vitro or in vivo have a weight
ratio of nucleic acid:total lipid components in the emulsion of
about 1:1 to about 1:50, more preferably a weight ratio of nucleic
acid:total lipid components in the emulsion of about 1:1 to about
1:30 and most preferably, a weight ratio of nucleic acid:total
lipid components in the emulsion of about 1:1 to about 1:20. Thus
for example, in Example 11 where a DNA:emulsion complex was formed
by combining 100 .mu.g of DNA with a volume of emulsion formulation
#21 containing 1100 .mu.g total lipid components, the weight ratio
of DNA:total lipid components of emulsion #21 in the complex was
100 .mu.g/1100 .mu.g or 1:11.
[0078] Preferably, nucleic acid:micelle complexes of this invention
to be used in vitro or in vivo have a weight ratio of nucleic
acid:total lipid components in the micelle of about 1:1 to about
1:50, more preferably a weight ratio of nucleic acid:total lipid
components in the micelle about 1:1 to about 1:30 and most
preferably a weight of nucleic acid:total lipid components of the
micelle ratio of about 1:1 to about 1:20. Thus for example, in
Example 11 where a DNA:micelle complex was formed by combining 100
.mu.g of DNA with a volume of micellar formulation #31 containing
700 .mu.g total lipid components, the weight ratio of DNA:total
lipid components of micelle #31 in the complex was 100 .mu.g/700
.mu.g or 1:7.
[0079] It is to be understood that the combining of emulsion or
micellar formulations with a nucleic acid to form the nucleic
acid:emulsion or nucleic acid:micellar complexes of this invention
may be carried out for at least 5 minutes in the presence or
absence of serum at a temperature from about 4.degree. C. to about
37.degree. C. The resultant nucleic acid:emulsion and nucleic
acid:micelle complexes may then be immediately used in vitro or in
vivo or may be stored prior to use.
[0080] Preferably, average diameters of nucleic acid:emulsion or
nucleic acid:micelle complexes to be used in vitro or in vivo are
100-4000 nm, more preferably 100-2000 nm and most preferably
100-1000 nm.
[0081] In one embodiment, the nucleic acid micelle and nucleic
acid:emulsion complexes of this invention may be used to transfect
cells with nucleic acid. Cells suitable for transfection in vitro
include eukaryotic cells, including all mammalian cell lines
suitable for transfection by cationic-lipids, cells put into
primary culture from a host, or cells resulting from passage of the
primary culture.
[0082] When, for example, 10.sup.5 cells are transfected in vitro,
transfection is carried out by exposing the cells to preferably
from about 0.1 to about 5 .mu.gs of nucleic acid:emulsion complex,
more preferably to about 0.5 to from about 2 .mu.gs of nucleic
acid:emulsion complex.
[0083] When 10.sup.5 cells are to be transfected with nucleic
acid:micelle complex, transfection is carried out by exposing the
cells to preferably from about 0.1 to about 20 .mu.gs of nucleic
acid:micelle complex; more preferably to about 1 to about 10 .mu.gs
of nucleic acid:micelle complex.
[0084] As used herein, .mu.g of nucleic acid:emulsion complex or
.mu.g of nucleic acid:micelle complex refers to the sum of the
.mu.g amount of nucleic acid and the .mu.g amount of total lipid
components in the emulsion or micellar formulation contained in the
complex. For example, 5 .mu.g of nucleic acid:emulsion complex
might contain 0.5 .mu.g of nucleic acid and 4.5 .mu.g of total
lipid components of emulsion formulation.
[0085] Those of ordinary skill in the art would readily understand
that the total amount of nucleic acid:emulsion or nucleic
acid:micelle complex to be used varies directly with the number of
cells to be transfected. One advantage of the emulsion and micellar
formulations of this invention over prior art cationic lipid
vectors is that the emulsions and micelles of the invention, when
complexed with nucleic acid, are more effective for transfecting
cells cultured in serum-containing medium.
[0086] The present invention therefore relates to the use of the
nucleic acid:emulsion and nucleic acid:micelle complexes of this
invention to deliver nucleic acids to cells in an animal or human
in vivo. Thus, the present invention also relates to the use of
nucleic acid:emulsion or nucleic acid:micelle complexes as delivery
systems in gene therapy.
[0087] Suitable routes of administration of the nucleic acid
containing complexes of this invention to an animal or human
include inoculation or injection by, for example, intravenous,
oral, intraperitoneal, intramuscular, subcutaneous, intra-aural,
intraarticular or intra-mammary routes, topical application, for
example on the skin, scalp, ears or eyes and by absorption through
epithelial or mucocutaneous linings, for example, nasal, oral,
vaginal, rectal and gastrointestinal among others, and as an
aerosol. Those of ordinary skill in the art would readily
understand that the mode of administration may determine the sites
in the organism to which the biologically active substance will be
delivered and may effect the amount of complex to be
administered.
[0088] Since as shown in Example 11, administration of
approximately 4 .mu.g of nucleic acid:emulsion or nucleic
acid:micelle complex/gram of body weight to a 25 gram mouse
produced transfection activity in vivo, those of ordinary skill in
the art using this ratio of 4 .mu.g of complex/gram of mouse body
weight could obtain other ratios of .mu.g of complex/gram of body
weight which are optimized for transfection activity in other
animals or humans.
[0089] In an alternative embodiment, the emulsion and micellar
formulations themselves may bind with biomacromolecules (i.e.
molecules produced by the animal or human) in situ after systematic
or topical administrations and behave as a local depot for
endogenous bioactive substances.
[0090] All articles or patents referenced herein are incorporated
by reference. The following examples illustrate various aspects of
the invention but are in no way intended to limit the scope
thereof.
EXAMPLES
Material and Methods
[0091] Materials:
[0092] DC-Chol cationic lipid was synthesized according to Gao and
Huang (Biochem. Biophys. Res. Commun., 179:280-285, 1991). Tween 80
and castor oil were obtained from Fisher, pluronic co-polymer L63
was obtained from BASF. Brij, Span, and pluronic F68 and F127
surfactants were purchased from Sigma. Dioleoyl
phosphatidylethanolamine (DOPE) and egg phosphatidylcholine (PC)
were obtained from Avanti Polar Lipids. LipofectAMINE liposomes
(DOSPA (2,3-dioleyloxy-N-[2(spermine
carboxamido)ethyl]-N,N,-dimethyl-1-propanaminium) and DOPE) in a
weight ratio of 3:1) were obtained from Life Technologies, Inc.
[0093] Preparation of Emulsions and Micelles:
[0094] Tween 80 diluted in chloroform was combined with DC-Chol
(micelles) and, where indicated DOPE or phosphatidylcholine; or
with castor oil, DC-Chol and, where indicated, DOPE or
phosphatidylcholine (emulsions) at different weight ratios. The
organic solvent was then evaporated under a stream of nitrogen gas
and the lipid film was vacuum desiccated at 4.degree. C. overnight
to remove residual organic solvent. One ml of phosphate buffered
saline (PBS, pH 7.4) was then added and the mixture was allowed to
hydrate for 1 h. The lipid suspension was then mixed with a vortex
mixer and subsequently homogenized for 3-4 min using a tissue
tearer at a speed of about 20,000 rpm. Average diameters of the
emulsion of micelle formulations and of the DNA:emulsion or
DNA:micelle complexes were measured by laser light scattering using
a Coulter N4SD submicron particle sizer.
[0095] Preparation of DC-Chol/DOPE Liposomes:
[0096] Unilamellar small liposomes of approximately 100 nm in
diameter were prepared by microfluidization of hydrated mixture of
DC-Chol and DOPE (weight ratio of 1:1) and filter sterilized. The
final lipid concentration of the DC-Chol/DOPE liposomes used in the
transfection experiments was 1.2 .mu.g/.mu.l of PBS.
[0097] Tissue Culture:
[0098] Murine melanoma BL6 cells were cultured in RPMI medium
supplemented with 10% fetal bovine serum. Human embryonic kidney
293 cells and F.sub.o were cultured in DMEM medium supplemented
with 10% fetal bovine serum. CHO cells were cultured in F12 medium
supplemented with 10% fetal bovine serum.
[0099] Plasmid DNA:
[0100] A pCDNA.sub.3 plasmid, pCMV-Luc, containing the luciferase
gene under the control of cytomegalovirus (CMV) immediate early
promoter was used to assess the efficiency of transfection. A
similar plasmid, pRSV-Luc, containing the same luciferase gene
under the control of a Rous sarcoma virus promoter was also used to
assess transfection efficiency. Plasmid pCMV-CAT is a pUC18 based
plasmid containing the E. coli chloramphenicol acetyltransferase
(CAT) gene downstream from the CMV promoter. The preparation and
purification of plasmid DNA was carried out according to standard
procedure (Sambrook, J., Fritsch, E. F., & Maniatis, T.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab
Press: Plainview, 1: pp 21-52, 1989.).
[0101] Transfection:
[0102] Cells cultured in 48 well plates (about 70-80% confluent)
were used for transfection and 3 wells were transfected with each
formulation. The pCMV-Luc or pRSV-Luc plasmid DNAs were diluted in
125 .mu.l of serum free CHO-S-SFM medium (Life Technologies, Inc.).
The emulsion, micelle, DC-Chol/DOPE liposomes or lipofectAMINE
liposomes were diluted in 125 .mu.l of Hank's balanced salt
solution (HBSS). The diluted DNA and formulations or liposomes were
combined, with or without the addition of fetal bovine serum to
20%, and incubated at room temperature for 5-10 min, before being
added to the cells. The cells were incubated at 37.degree. C. for 5
h. Transfection medium was replaced with growth medium containing
10% fetal bovine serum, and cells were then cultured for 2 days
before the luciferase assay was performed.
[0103] Luciferase Assay:
[0104] Cells were washed twice with PBS and incubated at room
temperature for 10 min in the presence of 100 .mu.l lysis buffer
(0.1M Tris-HC-1, pH 7.8/0.05% Triton X-100/2 mM EDTA) and then
centrifuged at 12,000.times.g. Ten .mu.l of supernatant was taken
for luciferase assay using the luciferase assay system (Promega) in
a luminometer (AutoLumat LB953 from EG&G, Berthhold).
Luciferase activity is given in relative light units (RLU).
[0105] Animal Studies:
[0106] Female CD 1 mice, 5 weeks old, were purchased from Charles
River Breeding Laboratories. Animal care was according to the
institutional guidelines. 200 .mu.l each of 4.times. concentrates
(for example, the 4.times. concentrate of formulation #21 contained
1.0 mg oil, 1.0 mg PC, 0.5 mg Tween 80 and 3.0 mg DC-Chol per ml of
PBS) of formulations #21 (1100 .mu.g total lipid components), #28
(1100 .mu.g total lipid components), #34 (900 .mu.g total lipid
components) and #31 (700 .mu.g total lipid components) (600 .mu.g
DC-Chol per formulation) were mixed with 5M NaCl to a final
concentration of NaCl of 0.15M and then combined with 25 .mu.l of 4
.mu.g/.mu.l pCMV-CAT DNA (100 .mu.g). The total mixture (231 .mu.l)
was injected into mice by tail vein. Two days later, mice were
killed and liver, spleen, kidney, lung and heart were excised for
CAT assay.
[0107] Chloramphenicol Acetyltransferase(CAT) Assay:
[0108] The organs excised from animals were homogenized in 40 mM
Tris-HCl, pH 7.5;10 mM EDTA; 150 mM NaCl. After homogenization,
cells were lysed by three freeze-thaw cycles, and the lysate was
heated at 65.degree. C. for 10 min to inactive deacetylases and
centrifuged for 10 min. The protein concentration of the
supernatant extracts was measured with a Coomassie blue G 250-assay
(Pierce). Protein was extracted from each organ and 200 .mu.g of
extract was then assayed for the CAT activity using [.sup.14C]
chloramphenicol as a substrate as previously described (Ausubel, F.
M., Breht, R., Kingstone, R. E., et al. Current Protocols in
Molecular Biology (Wiley, Boston), Vol. 1, pp 962-965, 1991.).
Example 1
Physical Stability of Emulsion Formulations and Transfection
Ability of DNA:Emulsion Complexes
[0109] To test which components of the emulsion formulations are
important for physical stability and transfection ability, 9
different emulsion formulations containing different amounts of
castor oil, egg phosphatidylcholine (PC), Tween 80 and DC-Chol were
formulated. The average diameter of the formulations was measured
as was their ability to transfect 293 cells by combining 6 .mu.l of
each emulsion (7.5 .mu.g total lipid components) with 0.5 .mu.g
RSV-Luc. The resulting data are presented in Table 1.
1TABLE 1 Transfection activity of DNA:emulsion complexes formed by
combining DNA with different emulsion formulations Composition (mg)
Average of formulation Diameter (nm) Transfection Activity.sup.b
Formulation Oil PC Tween 80 DC-Chol PBS (ml).sup.a of formulation
(RLU .times. 10.sup.6/well) #1 0.125 0.250 0.125 0.125 0.50 170 118
.+-. 22 #2 0.250 0.250 0.250 0.750 1.20 151 444 .+-. 4 #3 0.750
0.250 0.025 0.250 1.02 212 265 .+-. 46 #4 0.125 0.125 0.025 0.750
0.82 218 332 .+-. 89 #5 0.250 0.125 0.125 0.250 0.60 181 220 .+-. 7
#6 0.750 0.125 0.250 0.125 1.00 154 68 .+-. 1 #7 0.125 0.500 0.250
0.250 0.90 165 65 .+-. 20 #8 0.250 0.500 0.025 0.125 0.72 201 1
.+-. 0.6 #9 0.750 0.500 0.125 0.750 1.70 161 261 .+-. 47
DC-Chol/DOPE 122 445 .+-. 2 (1:1 ratio by weight) LipofectAMINE
(DOSPA and 100 427 .+-. 25 DOPE in a 3:1 weight ratio) .sup.aThe
final concentration of the emulsion was 1.25 .mu.g total lipid
components/.mu.l. Lipid concentrations of DC-Chol/DOPE liposome and
LipofectAMINE were 1.2 .mu.g/.mu.l and 2.0 .mu.g/.mu.l
respectively. .sup.b293 cells were transfected with complexes
formed by combining 0.5 .mu.g of pRSV-Luc and 6 .mu.l of emulsion
(7.5 .mu.g total lipid components) or with complexes formed by
combining 0.5 .mu.g of pRSV-Luc and 2.5 .mu.l of DC-Chol/DOPE
liposomes (3 .mu.g lipid components) or with complexes formed by
combining 0.5 .mu.g of pRSV-Luc and #2.25 .mu.l of LipofectAMINE
liposomes (4.5 .mu.g lipid components). Each well contained
approximately 70.80 .mu.g extractable protein.
[0110] The data show that the emulsions are physically stable with
size ranging from 150 to 218 nm in average diameter as measured by
laser light scattering. Further, complexes of DNA with those
emulsions with high content of DC-Chol (0.750 mg), the only
cationic component in the emulsion, showed high transfection
activity comparable to that of the cationic DC-Chol/DOPE and
LipofectAMINE liposomes.
Example 2
Transfection Activity of DNA:Emulsion and DNA:Micelle Complexes
[0111] Emulsion and micellar formulations which contained high
content of the cationic amphiphile DC-Chol (0.75 mg or more) were
complexed with pRSV-Luc DNA and examined for transfection activity
in BL6 and 293 cells. The data presented in Table 2
2TABLE 2 Transfection activity of DNA:emulsion and DNA:micelle
complexes Average Composition (mg) Diameter Transfection
Activity.sup.b of formulation (nm) of BL6 cells 293 cells
Formulation Oil PC Tween 80 DC-Chol DOPE Stearylamine PBS
(ml).sup.a formulation (RLU .times. 10.sup.3/well) (RLU .times.
10.sup.6/well) #10 0.250 0.250 0.250 0.750 -- -- 1.2 129 13 .+-. 8
530 .+-. 78 #11 0.125 0.125 0.250 0.375 -- -- 0.7 143 15 .+-. 4 546
.+-. 62 #12 0.125 0.125 0.375 0.375 -- -- 0.8 127 4 .+-. 2 239 .+-.
15 #13 0.125 0.125 0.125 0.500 -- -- 0.7 153 54 .+-. 34 708 .+-.
213 #14 0.125 0.125 0.125 0.750 -- -- 0.9 152 112 .+-. 37 880 .+-.
13 #15 -- 0.250 0.250 0.750 -- -- 1.0 165 19 .+-. 2 861 .+-. 22 #16
0.250 -- 0.250 0.750 -- -- 1.0 161 78 .+-. 14 710 .+-. 47 #17 0.250
-- 0.250 0.750 0.250 -- 1.2 155 23 .+-. 10 463 .+-. 90 #18 0.250
0.250 0.250 -- -- 0.400 0.90 193 3 .+-. 1 9 .+-. 1 #19 -- -- 0.250
0.750 -- -- 0.8 186 262 .+-. 104 806 .+-. 71 #20 0.250 0.250 --
0.750 -- -- 1.0 160 19 .+-. 9 840 .+-. 20 DC-Chol/DOPE 0.600 0.600
1.0 122 81 .+-. 40 221 .+-. 35 .sup.aThe final concentration of the
formulations was 1.25 .mu.g total lipid components/.mu.l. Lipid
concentration of DC-Chol/DOPE liposome was 1.2 .mu.g/.mu.l.
.sup.b293 cells were transfected with complexes formed by combining
0.5 .mu.g of pRSV-Luc and 6 .mu.l of the indicated emulsion or
micellar formulation (7.5 .mu.g total lipid components) or with
complexes formed by combining 0.5 .mu.g of pRSV-Luc and 2.5 .mu.l
DC-Chol/DOPE liposomes (3.0 .mu.g lipid components). Each well
contained #approximately 70-80 .mu.g extractable protein except for
#18 which contained 50 .mu.g protein.
[0112] show that complexes of DNA with the emulsions and the two
micellar formulations (#15 and #19) were active. However, the
complex formed by combining DNA with formulation #18 that contained
stearylamine instead of DC-Chol showed low transfection activity
which may be due to the toxicity of stearylamine to cells as
evidenced by the fact that the amount of protein extractable from
wells transfected with complex containing formulation #18 was less
than the amount of protein extractable from wells transfected with
complexes containing all other formulations (see Table 2, footnote
b). Of interest, the activities of a micellar (#19) and an emulsion
(#14) DNA complex were high, and indeed more active, than the
cationic liposome formulation (Dc-Chol/DOPE) in both cell
lines.
Example 3
Transfection Activity of More DNA:Emulsion and DNA:Micelle
Complexes
[0113] The physical diameter of additional emulsion and micellar
formulations was measured as was the transfection activity of
complexes formed between DNA and these formulations. The results
are shown in Table 3.
3TABLE 3 Transfection activity of more DNA:emulsion and DNA:micelle
complexes Composition (mg).sup.a of formulation Average
Transfection Activity.sup.b Pluronic Diameter (nm) BL6 293
Formulation Oil PC Tween 80 DC-Chol DOPE L63 of formulation (RLU
.times. 10.sup.5/well) (RLU .times. 10.sup.7/well) #21 0.250 0.250
0.125 0.750 -- -- 137 228 .+-. 40 -- #22 0.250 0.250 0.250 0.750 --
-- 218 235 .+-. 67 101 .+-. 10 #23 0.250 0.250 0.500 0.750 -- --
133 2 .+-. 0.4 11 .+-. 16 #24 0.250 0.250 0.250 1.000 -- -- 152 322
.+-. 76 104 .+-. 4 #25 0.250 0.250 0.250 1.500 -- -- 152 98 .+-. 28
34 .+-. 14 #26 -- 0.250 0.250 0.750 -- -- 168 450 .+-. 11 122 .+-.
40 #27 0.250 -- 0.125 0.750 -- -- 163 426 .+-. 83 130 .+-. 26 #28
0.250 -- 0.125 0.750 0.250 -- 169 645 .+-. 219 34 .+-. 1 #29 0.250
0.250 -- 0.750 -- -- 146 117 .+-. 44 56 .+-. 4 #30 -- -- 0.250
0.750 -- -- 202 759 .+-. 65 94 .+-. 58 #31 -- -- 0.125 0.750 -- --
179 324 .+-. 22 188 .+-. 40 #32 -- -- -- 0.750 -- 0.250 204 150
.+-. 45 88 .+-. 20 #33 -- -- -- 0.750 -- 0.500 212 107 .+-. 32 82
.+-. 16 #34 -- -- 0.125 0.750 0.250 -- 200 890 .+-. 35 --
DC-Chol/DOPE 122 1033 .+-. 204 40 .+-. 13 .sup.a1 ml of PBS was
added to each formulation. The concentration of DC-Chol in each
formulation was 0.75 mg/ml except #24 and #25 in which the DC-Chol
concentrations were 1.0 mg/ml and 1.5 mg/ml respectively. However,
the total lipid concentration of each formulation was different.
.sup.bCells were transfected with complexes formed by combining 0.5
.mu.g of pCMV-Luc and 6 .mu.l of each formulation (4.5 .mu.g
DC-Chol per 6 .mu.l of each formulation) or with complexes formed
by combining 0.5 .mu.g pCMV-Luc and 2.5 .mu.l of DC-Chol/DOPE
liposomes (3.0 .mu.g lipid components). Each well contained
approximately 70-80 .mu.g extractable protein.
[0114] The results show that complexes of DNA and micelles
containing DC-Chol and Tween 80 (#30 and #31), were again quite
active and replacing Tween 80 with pluronic L63 (#32 and #33) did
not significantly alter the average diameters of the formulations
or their transfection activity in 293 cells. Another micelle
containing DC-Chol, Tween 80 and PC (phosphatidylcholine) (#26) was
also active. The remaining formulations (#22-25, 27-29) were
emulsions which contained castor oil. Complexes of DNA and these
emulsion formulations were fairly active in transfection. When
complexes of DNA and these micellar and emulsion formulations were
tested in another cell-line (BL6 mouse melanoma cells),
qualitatively similar results were obtained except the activity in
this cell line was generally lower than that of the 293 cells. The
transfection activities of complexes of DNA and these emulsions and
micelles were comparable to that of the cationic liposome
formulation (DC-Chol/DOPE) in 293 cells, but somewhat lower in BL6
cells.
Example 4
[0115] Optimization of Transfection Conditions:
[0116] To optimize the transfection activity of the DNA:emulsion
and DNA:micelle complexes, the amount of DC-Chol in each emulsion
or micelle formulation was kept constant at 4.5 .mu.g (6 .mu.l of
formulations #s 21, 27, 28 and 30, respectively were used; refer to
Table 3 for compositions of formulations) and the amount of
pCMV-Luc DNA was varied from 0 to 5 .mu.g. As shown in FIG. 1,
complexes of DNA and formulations #27 and #30 (refer to Table 3 for
compositions) showed a maximal transfection activity in BL6 cells
at 2 .mu.g DNA while complexes of DNA and formulations #21 and #28
showed a maximum activity at 1.5 .mu.g DNA.
[0117] Next, the amount of pCMV-Luc DNA was fixed at 2 .mu.g for
formulations #27 and #30 and at 1.5 .mu.g for formulations #21 and
#28 and complexes of DNA and varying amounts of emulsion or micelle
formulation as indicated on the horizontal axis of FIG. 2 (where
.mu.g formulation on the horizontal axis refers to .mu.g total
lipid components present in the volume of formulation combined with
pCMV-Luc DNA to form complex) were produced. The results presented
in FIG. 2 show that complexes of DNA and formulations #27, #28 and
#30 exhibited a relatively broad peak of activity in BL6 cells with
the optimal amount of total lipid components present in the volume
of formulation combined with DNA to produce complex being about 18
.mu.g. However, complexes of DNA and formulation #21 exhibited a
narrower peak of activity with the optimal amount of total lipid
components present in the volume of formulation combined with DNA
to produce complex being about 13 .mu.g.
Example 5
Sensitivity of Transfection Activity of DNA:Emulsion and
DNA:Micelle Complexes to Serum
[0118] All the above described transfection experiments were
carried out in a serum-free medium. Therefore, to determine the
sensitivity of transfection activity of DNA/emulsion and
DNA/micelle complexes to the presence of serum, BL6 cells were
transfected in the medium containing 0 or 20% fetal bovine serum
with complexes of DNA and 10 different emulsions or micelles (or
DC-Chol/DOPE liposomes) as follows.
[0119] Different formulations of the compositions shown in Table 4
were prepared in a total volume of 1 ml PBS (pH 7.4). BL6 cells in
a 48 well plate were then transfected with 2 .mu.g of pCMV-Luc and
16 .mu.l of each formulation (containing 12 .mu.g DC-Chol), or with
2 .mu.g of pCMV-Luc and 16 .mu.l of DC-Chol/DOPE liposomes (1.2
.mu.g/.mu.l), in medium containing either 0 or 20% fetal bovine
serum and luciferase activity was detected. Each well contained
approximately 70-80 .mu.g extractable protein. The results are
shown in Table 4
4TABLE 4 Transfection Activity Of Additional DNA:emulsion and
DNA:micelle complexes COMPOSITION (mg).sup.a LUCIFERASE ACTIVITY of
formulation (RLU/well .times. 10.sup.7) FORMULATION Oil PC DOPE
Tween 80 DC-Chol -Serum +Serum (20%) #35 0.25 0.25 -- 0.125 0.75 14
.+-. 3 49 .+-. 2 #36 -- 0.25 -- 0.125 0.75 32 .+-. 6 98 .+-. 6 #37
0.25 -- -- 0.125 0.75 170 .+-. 7 5 .+-. 3 #38 0.25 0.25 -- -- 0.75
24 .+-. 0.8 56 .+-. 13 #39 0.25 0.25 -- 0.125 -- 0 0 #40 0.25 --
0.25 0.125 0.75 50 .+-. 3 151 .+-. 6 #41 0.25 -- 0.25 -- 0.75 40
.+-. 5 85 .+-. 8 #42 -- -- 0.25 0.125 0.75 140 .+-. 5 267 .+-. 44
#43 -- -- -- 0.125 0.75 156 .+-. 13 298 .+-. 42 #44 -- -- -- 0.25
0.75 259 .+-. 12 307 .+-. 8 DC-Chol/DOPE liposomes -- -- 0.60 --
0.60 107 .+-. 21 47 .+-. 9 .sup.aThe average diameter of each
formulation was 100-250 nm.
[0120] demonstrate that the transfection activity of DC-Chol/DOPE
liposomes was quite sensitive to serum (only about 33% activity
remained in the presence of serum) while of the 10 formulations
tested, only complex of DNA and formulation #37 showed serum
sensitivity where serum sensitivity is a reduction in transfection
activity in the presence of serum relative to the level of activity
observed in the absence of serum. In addition, the fact that
complex of DNA and formulation #39 showed no activity in the
presence or absence of serum demonstrated that the presence of
cationic amphiphile is critical to transfection activity.
Particularly interesting are complexes of DNA and formulations #35,
#36 and #40 (corresponding in composition to formulations 21, #34
and #28 respectively in Table 3) which, of the formulations tested,
exhibited the greatest enhancement of transfection activity in the
presence of serum.
Example 6
Sensitivity of Transfection Activity of Complexes of DNA and
Selected Formulations to Serum in Different Cell Lines
[0121] To determine if the serum sensitivity observed in BL6 cells
in Table 4 was observed in other cell lines, F.sub.o cells, CHO
cells and 293 cells were transfected with 16 .mu.l of selected
formulations (each containing 12 .mu.g DC-Chol/16 Al) from Table 4
combined with 2 .mu.g of pCMV-Luc DNA or, with 16 .mu.l
DC-Chol/DOPE liposomes (1.2 .mu.g/.mu.l) combined with 2 .mu.g of
pCMV-Luc DNA, in medium containing 0 or 20% serum as in Example 5.
The results of these experiments are shown in Table 5. The data
show that complexes of DNA and all formulations are active in
transfecting cells and are in general, serum-resistant.
5TABLE 5 Transfection Activity of Complexes Of DNA And Selected
Formulations in Different Cell Lines. LUCIFERASE ACTIVITY (RLU/Well
.times. 10.sup.7) FORMULATION F.sub.0 Cells CHO Cells 293 Cells
(w/w) -Serum +Serum -Serum +Serum -Serum +Serum #35
(Oil/PC/Tw/DC-Chol 8.3 .+-. 3.7 13.2 .+-. 7.0 8.0 .+-. 0.7 5.9 .+-.
0.4 33.0 .+-. 7.8 37.0 .+-. 3.4 (2:2:1:6).sup.a #37
(Oil/Tw/DC-Chol) 2.7 .+-. 0.2 1.6 .+-. 0.9 1.6 .+-. 1.6 10.4 .+-.
1.3 33.3 .+-. 11.3 44.2 .+-. 1.2 (2:1:6:).sup.b #40
(Oil/DOPE/Tw/DC-Chol) 8.7 .+-. 2.1 12.0 .+-. 2.1 0.4 .+-. 0.0 7.5
.+-. 0.4 27.9 .+-. 7.0 34.7 .+-. 2.1 (2:2:1:6).sup.c #44
(Tw/DC-Chol) 22.0 .+-. 3.3 13.2 .+-. 1.1 0.4 .+-. 0.1 1.5 .+-. 0.3
68.0 .+-. 17.0 168.0 .+-. 17.0 (2:6).sup.d DOPE/DC-Chol liposomes
2.7 .+-. 0.2 0.5 .+-. 0.1 1.7 .+-. 0.0 2.8 .+-. 0.5 88.0 .+-. 3.9
67.0 .+-. 3.9 (1:1).sup.e .sup.a2:2:1:6 = 0.25 mg/0.25 mg/0.125
mg/0.75 mg per ml of solution. .sup.b2:1:6 = 0.25 mg/0.125 mg/0.75
mg per ml of solution. .sup.c2:2:1:6 = 0.25 mg/0.25 mg/0.125
mg/0.75 mg per ml of solution. .sup.d2:6 = 0.25 mg/0.75 mg per ml
of solution. .sup.e1:1 = 0.6 mg/0.6 mg per ml of solution.
Example 7
[0122] Stability of DNA:Emulsion and DNA:Micelle Complexes
[0123] Five different formulations (#'s 26, 27, 28, 29 and 34 of
Table 3) were tested for the stability of their complex with DNA.
Complex was prepared by combining 2 .mu.g pCMV-CAT DNA and 16 .mu.l
of the indicated emulsion or micelle formulation (where 16 .mu.l of
each formulation contained the same amount of DC-Chol, 12 .mu.g) or
by combining 1 .mu.g pCMV-CAT DNA and 6 .mu.g of DC-Chol/DOPE
liposomes. As can be seen in FIG. 3, formulations #26 #28 and #29
formed relatively small complexes with DNA; the average diameter of
the complex as measured by laser light scattering ranged from
200-300 nm, and remained small even after 10 days at 4.degree. C.
Formulation #34 and #27, on the other hand, formed larger complexes
with DNA with average diameters of 600 and 900 nm, respectively. In
contrast, DC-Chol/DOPE liposomes had formed large aggregates (1,800
nm on day 1) which had grown to even larger ones (>4,000 nm) on
day 3 and subsequently precipitated out of solution (data not
shown). Thus, all new formulations could form complexes with DNA
that had physical Stability better than that of complexes formed
with the DC-Chol/DOPE liposomes.
Example 8
Effect of Different Surfactants on the Transfection Activity of
Complexes of DNA and Emulsions Composed of
Oil/DOPE/DC-Chol/Surfactant in a Weight Ratio of 2:2:6:x
[0124] Emulsions containing different surfactants were prepared in
1 ml of PBS containing 0.25 mg of oil, 0.25 mg of DOPE, 0.75 mg of
DC-Chol and different amounts of the indicated surfactants where
the total amount of surfactant used in each formulation was
approximately the same by mole. BL6 cells were then transfected
with 2 .mu.g of pCMV-Luc DNA combined with 16 .mu.l of formulation
(12 .mu.g DC-Chol/16 .mu.l of each formulation) and assayed for
luciferase activity. The results of this experiment are shown in
Table 6.
6TABLE 6 Effect Of Different Surfactants On The Transfection
Activity Of Complexes Of DNA and Emulsions Composed Of OIL/DOPE/DC-
Chol/surfactant (0.25 mg:0.25 mg:0.75 mg:X mg per ml of PBS)
LUCIFERASE ACTIVITY (RLU/Well) .times. 10.sup.7 Surfactant X (mg)
-Serum +Serum (20%) Tween 20 0.117 1.9 .+-. 0.4 4.9 .+-. 1.1 40
0.122 1.9 .+-. 0.3 5.3 .+-. 0.7 60 0.125 3.2 .+-. 0.3 5.6 .+-. 2.0
Brij 72 0.034 7.0 .+-. 0.7 10.0 .+-. 2.0 74 0.068 9.4 .+-. 1.6 6.5
.+-. 0.3 76 0.110 5.4 .+-. 0.8 0.08 .+-. 0.03 100 0.446 0.3 .+-.
0.2 0.003 .+-. 0.006 Span 20 0.033 1.2 .+-. 0.0 0.1 .+-. 0.0 40
0.038 1.9 .+-. 0.1 0.1 .+-. 0.0 60 0.041 1.4 .+-. 0.4 0.3 .+-. 0.1
80 0.041 1.4 .+-. 0.4 0.3 .+-. 0.1 pluronic F 0.802 7.8 .+-. 0.3
9.0 .+-. 1.4 68 pluronic F 1.202 7.9 .+-. 0.9 9.9 .+-. 1.1 127
[0125] Of the surfactants tested, complexes formed from emulsions
containing Tween 20, Tween 40, Tween 60, Brij 72, Brij 74, F68 or
F127 demonstrated transfection activity that was not sensitive to
the presence of 20% serum and complexes formed from formulations
containing the Tween series of detergents showed the greatest
increase in transfection activity in the presence of serum relative
to that observed in the absence of serum.
Example 9
Effect of Tween 80 Concentration in Emulsions on the Average
Diameters of Concentrated and Diluted DNA/Emulsion Complexes
[0126] Concentrated DNA/emulsion complex was formed by adding 2
.mu.l of solution containing 8 .mu.g of DNA (pCMV-Luc) directly to
72 .mu.l of emulsions containing 0.25 mg Oil/0.25 mg DOPE/0.75 mg
DC-Chol and varying mg amounts of Tween 80 per ml. As in the prior
examples diluted DNA/emulsion complex was formed by combining 2
.mu.g of DNA in 125 .mu.l with 18 .mu.l of the same emulsions used
in the concentrated complex but diluted to 125 .mu.l. The average
diameters of the concentrated and diluted complexes were measured 1
hour after incubation at room temperature. The results shown in
FIG. 4 demonstrate that increasing amounts of Tween 80 reduced the
size of the concentrated complexes but had no effect on the size of
the diluted complexes.
Example 10
Effect of the Amount of Tween 80 in an Emulsion on Transfection
Activity of Concentrated and Diluted DNA/Emulsion Complexes in the
Presence or Absence of 20% Serum
[0127] Emulsions and concentrated and diluted pCMV-Luc DNA/emulsion
complexes were prepared as in Example 9 and the transfection
activity of the complexes was measured in BL6 cells in the presence
or absence of 20% serum. The results of these experiments are shown
in FIGS. 5A (concentrated complex) and 5B (diluted complex). While
the diluted complexes appear to show better activity than the
concentrated complexes, the need to keep the volume of complex
administered to an animal small may favor the use of more
concentrated complexes in vivo.
Example 11
Animal Studies With DNA:Emulsion and DNA:Micelle Complexes
[0128] Formulations #21, 28, 31 and 34 (refer to Table 3 for
compositions) were tested for gene transfer activity in mice. 200
.mu.l each of 4.times. concentrates of formulations #21 (1100 .mu.g
total lipid components), #28 (1000 .mu.g total lipid components),
#34 (900 .mu.g total lipid components) and #31 (700 .mu.g total
lipid components)) were mixed with 6 .mu.l of 5M NaCl to a final
concentration of 0.15M NaCl and then combined with 4 .mu.g/.mu.l
pCMV-CAT DNA (100 .mu.g). The complexes were then injected i.v. via
the tail vein of the mouse (each mouse weighed approximately 25
grams) and CAT activity was measured in major organs two days after
injection. The data presented in FIG. 6 clearly demonstrates that
complexes of DNA and formulations #28 (emulsion), #31 (micelle) or
#34 (micelle) could transfect various organs with relatively high
activity while complex of DNA and formulation #21 (emulsion), on
the other hand, was weak and comparable to that of DC-Chol/DOPE
liposomes. In addition, the activity of complex of DNA and
formulation #31 seems to be lung specific, as no other organs were
significantly transfected; complex of DNA and formulation #28 could
transfect all organs quite well with only weak transfection of the
kidney, and complex of DNA and formulation #34 showed a high
activity in the heart with very low activity in the kidney.
* * * * *