U.S. patent application number 13/189082 was filed with the patent office on 2012-07-12 for stable lipid-comprising drug delivery complexes and methods for their production.
This patent application is currently assigned to University of Pittsburgh. Invention is credited to Xiang Gao, Leaf HUANG, Frank L. Sorgi.
Application Number | 20120178702 13/189082 |
Document ID | / |
Family ID | 23486103 |
Filed Date | 2012-07-12 |
United States Patent
Application |
20120178702 |
Kind Code |
A1 |
HUANG; Leaf ; et
al. |
July 12, 2012 |
STABLE LIPID-COMPRISING DRUG DELIVERY COMPLEXES AND METHODS FOR
THEIR PRODUCTION
Abstract
Novel stable, concentrated, biologically active and ready-to-use
lipid-comprising drug delivery complexes and methods for their
production are described. The biological activity of the complexes
produced are comparable to the formulations prepared according to
the prior art admixture method and upon purification, the complexes
produced by the method of this invention are 50 to 500 fold more
concentrated than the complexes formed by admixture. The method
described herein provides for the large scale production of
lipid-comprising drug delivery systems useful for gene therapy and
other applications.
Inventors: |
HUANG; Leaf; (Durham,
NC) ; Gao; Xiang; (Nashville, TN) ; Sorgi;
Frank L.; (Sonoma, CA) |
Assignee: |
University of Pittsburgh
Pittsburgh
PA
|
Family ID: |
23486103 |
Appl. No.: |
13/189082 |
Filed: |
July 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12637544 |
Dec 14, 2009 |
7993672 |
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13189082 |
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11943434 |
Nov 20, 2007 |
7655468 |
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12637544 |
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11014528 |
Dec 15, 2004 |
7335509 |
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11943434 |
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09376395 |
Aug 18, 1999 |
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11014528 |
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08939874 |
Sep 29, 1997 |
6008202 |
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09376395 |
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08751888 |
Nov 18, 1996 |
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08939874 |
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08376701 |
Jan 23, 1995 |
5795587 |
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08751888 |
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Current U.S.
Class: |
514/20.9 ;
435/375; 530/322; 530/395 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 47/645 20170801; A61K 47/543 20170801; C12N 15/88
20130101 |
Class at
Publication: |
514/20.9 ;
530/322; 530/395; 435/375 |
International
Class: |
A61K 38/14 20060101
A61K038/14; C12N 5/071 20100101 C12N005/071; C07K 14/00 20060101
C07K014/00 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] This invention was made with Government support under Grant
No. DK44935, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1: A noncovalent nucleic acid/lipid/polycation complex having a net
positive charge at pH 6.0-8.0, the complex comprising a nucleic
acid, at least one cationic lipid and a polycation, wherein the
polycation is a cationic peptide or a polypeptide comprising
polylysine, polyornithine, or polyarginine, and wherein the
noncovalent complex is formed by adding a nucleic acid to a
solution comprising the at least one cationic lipid and the
polycation.
2: The complex of claim 1, wherein the ratio of lipid to nucleic
acid ranges from about 0.1 nmol to 200 nmol lipid per 1 .mu.g
nucleic acid.
3: The complex of claim 1, wherein the ratio of lipid to nucleic
acid ranges from about 1 nmol to about 25 nmol lipid per 1 .mu.g
nucleic acid.
4: The complex of claim 1, wherein the polycation:nucleic acid
ratio is about 0.01 .mu.g to about 100 .mu.g polycation to 1 .mu.g
nucleic acid.
5-11. (canceled)
12: A noncovalent nucleic acid/lipid/polycation complex having a
net positive charge at pH 6.0-8.0, the complex comprising a nucleic
acid, at least one cationic lipid and a polycation, wherein the
polycation is a cationic peptide or polypeptide comprising spermine
or spermidine.
13: The complex of claim 12, wherein the complex is formed by
adding a nucleic acid to a solution comprising the at least one
cationic lipid and the polycation.
14: The complex of claim 12, further comprising a neutral
lipid.
15: The complex of claim 14, wherein the neutral lipid is
cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
16: The complex of claim 12, wherein the at least one cationic
lipid is present as a cationic liposome, wherein the at least one
cationic lipid is present in the liposome at a concentration of
from about 10 to about 100 mole % of total liposomal lipid.
17: The complex of claim 16, wherein the at least one cationic
lipid is present in the liposome at a concentration of from about
20 to about 80 mole % of total liposomal lipid.
18: The complex of claim 16, wherein the at least one cationic
lipid is present in the liposome at a concentration of from about
20 to about 60 mole % of total liposomal lipid.
19: The complex of claim 12, wherein the polycation is up to about
500 amino acids in length.
20: The complex of claim 12, wherein the polycation is about 20 to
about 100 amino acids in length.
21: The complex of claim 12, wherein the polycation is about 20 to
about 35 amino acids in length.
22: The complex of claim 12, wherein the polycation is about 25 to
about 50 amino acids in length.
23: The complex of claim 1, wherein the polycation has a molecular
weight of from 300 to 200,000 Daltons.
24: A method for delivering nucleic acid to cells comprising
contacting the cells with a noncovalent nucleic
acid/lipid/polycation complex having a net positive charge at pH
6.0-8.0, the complex comprising a nucleic acid, at least one
cationic lipid and a polycation, wherein the polycation is a
cationic peptide or a polypeptide comprising polylysine,
polyornithine, or polyarginine, and wherein the noncovalent complex
is formed by adding a nucleic acid to a purified solution
comprising the at least one cationic lipid and the polycation.
25-28. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/637,544, filed on Dec. 14, 2009, which is a
continuation of U.S. patent application Ser. No. 11/943,434, filed
on Nov. 20, 2007, now U.S. Pat. No. 7,655,468, issued Feb. 2, 2010,
which is a continuation of U.S. patent application Ser. No.
11/014,528, filed on Dec. 15, 2004, now U.S. Pat. No. 7,335,509,
issued Feb. 26, 2008, which is a continuation of U.S. patent
application Ser. No. 09/376,395, filed on Aug. 18, 1999, now
abandoned, which is a continuation of U.S. patent application Ser.
No. 08/939,874, filed on Sep. 29, 1997, now U.S. Pat. No.
6,008,202, issued Dec. 28, 1999, which is a continuation-in-part of
U.S. patent application Ser. No. 08/751,888, filed on Nov. 18,
1996, now abandoned, and a continuation-in-part of U.S. patent
application Ser. No. 08/376,701, filed on Jan. 23, 1995, now U.S.
Pat. No. 5,795,587, issued Aug. 18, 1998, the disclosures of each
of which are hereby incorporated herein by reference in their
entirety.
FIELD OF INVENTION
[0003] The present invention relates to cationic lipids and their
use as vehicles for the transfer of nucleic acids or other
macromolecules, such as proteins, into cells. More specifically,
this invention relates to lipid-comprising drug delivery complexes
which are stable, biologically active, capable of being
concentrated, and to methods for their production.
BACKGROUND OF INVENTION
[0004] The development of new forms of therapeutics which use
macromolecules such as proteins or nucleic acids as therapeutic
agents has created a need to develop new and effective means of
delivering such macromolecules to their appropriate cellular
targets. Therapeutics based on either the use of specific
polypeptide growth factors or specific genes to replace or
supplement absent or defective genes are examples of therapeutics
which may require such new delivery systems. Clinical application
of such therapies depends not only on the efficacy of new delivery
systems but also on their safety and on the ease with which the
technologies underlying these systems can be adapted for large
scale pharmaceutical production, storage, and distribution of the
therapeutic formulations. Gene therapy has become an increasingly
important mode of treating various genetic disorders. The potential
for providing effective treatments, and even cures, has stimulated
an intense effort to apply this technology to diseases for which
there have been no effective treatments. Recent progress in this
area has indicated that gene therapy may have a significant impact
not only on the treatment of single gene disorders, but also on
other more complex diseases such as cancer. However, a significant
obstacle in the attainment of efficient gene therapy has been the
difficulty of designing new and effective means of delivering
therapeutic nucleic acids to cell targets. Thus, an ideal vehicle
for the delivery of exogenous genes into cells and tissues should
be highly efficient in nucleic acid delivery, safe to use, easy to
produce in large quantity and have sufficient stability to be
practicable as a pharmaceutical.
[0005] Non-viral vehicles, which are represented mainly by the
cationic liposomes, are one type of vehicle which have, for the
following reasons, been considered for use in gene therapy. First,
the plasmid DNA required for liposome-mediated gene therapy can be
widely and routinely prepared on a large scale and is simpler and
carries less risk than the use of viral vectors such as
retroviruses. Second, liposome-mediated gene delivery, unlike
retroviral-mediated gene delivery, can deliver either RNA or DNA.
Thus, DNA, RNA, or an oligonucleotide can be introduced directly
into the cell. Moreover, cationic liposomes are non-toxic,
non-immunogenic and can therefore be used repeatedly in vivo as
evidenced by the successful in vivo delivery of genes to
catheterized blood vessels (Nabel, E. G., et al. (1990) Science,
249: 1285-1288), lung epithelial cells (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), and
other systemic uses (Zhu, N., et al. (1993) Science, 261: 209-211,
Philip, R., et al. (1993) Science, 261: 209-211; Nabel, G. et al
(1994) Hum. Gene Ther., 5:57-77) of cationic liposomes.
[0006] Although a variety of cationic liposome formulations,
including the commercially available cationic liposome reagent
DOTMA/DOPE (N-1,-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium
chloride/dioleoyl phosphatidylethanolamine), are known in the art
(Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:
7413-7417), a cationic liposome formulation designated DC-Chol/DOPE
(3.beta.N--(N',N'-dimethylaminoethane)-carbamoyl
cholesterol/(dioleoyl phosphatidylethanolamine) has been shown in
in vitro studies (Gao, X., and Huang, L. (1991) Biochem. Biophys.
Res. Commun., 179: 280-285) to be relatively non-toxic and more
efficient than DOTMA/DOPE. Moreover, following extensive in vivo
studies (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) in which DC-Chol/DOPE was demonstrated to be
both safe and efficacious as a nucleic acid delivery system, this
formulation was approved by the U.S. Food and Drug Administration
(FDA) and the U.K. Medicines Control Agency (MCA), and has been
used in two separate gene therapy clinical trials (Nabel, G. J., et
al. (1993) Proc. Natl. Acad. Sci. U.S.A., 90: 11307-11311, Caplen,
N. J., et al. (1995) Nature Medicine, 1: 39-46).
[0007] However, the use of DC-Chol/DOPE and other currently
existing cationic liposomes as vehicles for delivering nucleic
acids to cellular targets are inconvenient for large scale
therapeutic applications for a number of reasons. First, the ratios
of liposome to nucleic acid utilized to form nucleic acid/liposome
complex in the prior art admixture method results in the formation
of complexes which are large in diameter and relatively unstable.
Thus, none of the presently utilized cationic liposome
formulations, including DC-Chol/DOPE, are designed as stable and
ready-to-use pharmaceutical formulations of nucleic acid/liposome
complex. This limitation of the admixture method requires that the
user prepare the complex prior to each use, an inconvenience which
requires special training of personnel. In addition, the
preparation of the complex by admixture prior to each use
introduces a possible source of dosage variability which hinders
evaluation of treatments utilizing these complexes due to possible
over- or under-dosing of the recipient.
[0008] Second, the prior art admixture method of preparing nucleic
acid/cationic liposome complexes prior to each use requires that a
dilute nucleic acid solution (less than 4 .mu.g/ml) and a dilute
liposome dispersion (less than 50 .mu.M) be used to prepare the
nucleic acid/liposome complex in order to reduce the chance of
forming large and less active aggregates. This limitation makes it
difficult to make small biologically active complexes without using
less than optimal conditions, such as reducing the amount of
liposomes (which causes reduced nucleic acid transfer activity) or
increasing the amount of liposome (which causes enhanced toxicity).
Moreover, the fact that the complex must be made in dilute
concentrations is a significant drawback to clinical applications,
particularly in the case of intratumor injection of the complex,
since only a small volume of the complex can be injected in each
site (Nabel, G. J., et al. (1993) Proc. Natl. Acad. Sci. U.S.A.,
90: 11307-11311).
[0009] Accordingly, an object of this invention is to provide
stable, biologically active, lipid-comprising drug delivery
complexes which are capable of being formulated in a high
concentration as well as methods of producing such complexes.
SUMMARY OF INVENTION
[0010] This invention provides methods for producing
lipid-comprising drug delivery complexes having a net positive
charge and/or a positively charged surface. By "drug" as used
throughout the specification and claims is meant any molecular
entity, which is either monomeric or oligomeric, and which, when
complexed with lipid or with lipid and polycation, is being
administered to an individual for the purpose of providing a
therapeutic effect to the recipient. Thus, macromolecules having an
overall net negative charge or regions of negativity would be
expected to be capable of forming the delivery complexes of this
invention. Macromolecules which are particularly suitable for use
with the complexes of this invention are for example, DNA, RNA,
oligonucleotides or negatively charged proteins. However,
macromolecules having a positive charge (e.g., large cationic
protein) would also be expected to be capable of forming the
complexes of this invention by sequentially complexing the cationic
macromolecule with anionic molecule or polymer and then with
cationic lipid.
[0011] The complexes of the invention comprise a drug/lipid complex
formed by mixing the drug to be delivered with cationic liposomes
in a drug to lipid ratio such that the drug/lipid complex formed
has a net positive charge and a drug/lipid/polycation complex
formed by mixing drug with cationic liposomes and polycation in a
drug to lipid to polycation ratio such that the
drug/lipid/polycation complex formed has a net positive charge. By
"net positive charge" as applied to the drug/lipid complex is meant
a positive charge excess of lipid to drug. By "net positive charge"
as applied to the drug/lipid/polycation complex is meant that the
positive charges of the cationic lipid and the polycation exceed
the negative charge of the drug. However, it is to be understood
that the present invention also encompasses drug/lipid and
drug/lipid/polycation complexes having a positively charged surface
irrespective of whether the net charge of the complex is positive,
neutral or even negative. A positively charged surface of a complex
may be measured by the migration of the complex in an electric
field by methods known to those in the art such as by measuring
zeta potential (Martin, A., Swarick, J., and Cammarata, A.,
Physical Pharmacy & Physical Chemical Principles in the
Pharmaceutical Sciences, 3rd ed. Lea and Febiger, Philadelphia,
1983), or by the binding affinity of the complex to cell surfaces.
Complexes exhibiting a positively charged surface have a greater
binding affinity to cell surfaces than complexes having a neutral
or negatively charged surface. Furthermore, the positively charged
surface could be sterically shielded by the addition of non-ionic
polar compounds, of which polyethylene glycol is an example.
[0012] The invention therefore relates to methods for producing
these drug/lipid and drug/lipid/polycation complexes comprising
mixing the drug to be delivered with cationic liposomes, and
optionally polycation, in a ratio such that the complex formed has
a net positive charge and/or a positively charged surface.
[0013] In another embodiment of this invention, the methods for
producing drug lipid or drug/lipid/polycation complexes may further
comprise the step of purifying said complexes from excess free
components (drug, lipid, polycation) following their
production.
[0014] The drug/lipid and drug/lipid/polycation complexes of this
invention are generally stable, capable of being produced at
relatively high concentration, and retain biological activity over
time in storage. Such complexes are of utility in the delivery of
nucleic acids, proteins and other macromolecules to cells and
tissues.
[0015] In another embodiment of this invention complexes are found
comprising polycationic polypeptides having a high arginine
content.
DESCRIPTION OF FIGURES
[0016] FIG. 1 shows a typical size distribution (mean diameter) of
nucleic acid/liposome complexes prepared as an admixture from
DC-Chol/DOPE (3:2 mol/mol) liposomes and pRSVL plasmid DNA (2
.mu.g) at the indicated lipid to DNA ratios.
[0017] FIGS. 2A and 2B show the distribution of the liposome marker
.sup.3H-cholesteryl hexadecyl ether (.largecircle.) and the
.sup.32P-DNA marker (.box-solid.) among sucrose gradient fractions.
The location of each fraction in the sucrose gradients of both
FIGS. 2A and 2B is indicated at the top of FIG. 2A.
[0018] FIG. 2A shows the distribution of the .sup.3H and .sup.32P
markers following ultracentrifugation of free liposomes (10
.mu.moles of DC-Chol/DOPE (2:3) in 2 ml volume) or free DNA (50
.mu.g pRSVL DNA in a 2 ml volume) through a sucrose density
gradient. FIG. 2B shows the distribution of the .sup.3H and
.sup.32P markers following ultracentrifugation of the DNA-lipid
complex (formed via mixing of 20 .mu.moles DC-Chol/DOPE (2:3)
liposomes and 0.4 mg pRSVL DNA in 2 ml volume) through a sucrose
density gradient.
[0019] FIG. 3 shows the transfection activities in CHO cells of
admixture DNA/liposome complex (.largecircle.), admixture
DNA/liposome/poly-L-lysine (PLL) complex (.quadrature.) DNA/lipid
complex ( ) and DNA/lipid/PLL complex (.box-solid.). The
DC-Chol/DOPE liposomes used to form the above complexes contained
varying mole % of DC-Chol as indicated at the bottom of FIG. 3. The
DNA/lipid ( ) and DNA/lipid/PLL (.box-solid.) complexes were
purified on a sucrose density gradient prior to being assayed for
transfection activity. Transfection activity is indicated on the
vertical axis as relative light units of luciferase activity.
[0020] FIG. 4 shows the transfection activities of admixture
DNA/liposome complex (.largecircle.) and admixture DNA/liposome/PLL
complex (.quadrature.) compared to the transfection activities of
DNA/lipid ( ) and DNA/lipid/PLL complexes stored at 4.degree. C.
for 130 days following their purification on a sucrose density
gradient. The DC-Chol/DOPE liposomes used to form the above
complexes contained varying mole % of DC-Chol as indicated at the
bottom of FIG. 4. Transfection activity is indicated on the
vertical axis as relative light units of luciferase activity.
[0021] FIG. 5 shows the concentration of extractable protein from
CHO cells, 36 hours after the cells were treated with admixture
DNA/liposome complex (.largecircle.); admixture DNA/liposome/PLL
complex (.box-solid.); DNA/lipid complex ( ); or DNA/lipid/PLL
complex (.box-solid.). The DNA/lipid and DNA/lipid/PLL complexes
were purified on a sucrose density gradient prior to being assayed
for transfection activity. The DC-Chol/DOPE liposomes used to form
the above complexes contained varying mole % of DC-Chol as
indicated at the bottom of FIG. 5.
[0022] FIG. 6 shows the results of CAT assays of tumor extracts
prepared from mice having ovarian tumors. 2.times.10.sup.6 human
ovarian carcinoma cells were subcutaneously injected into SCID mice
at day 0. On day 14, 100 .mu.l solutions containing pUCCMVCAT DNA
(contains the chloramphenicol acetyl transferase gene of E. coli)
30 .mu.g) complexed with DC-Chol liposomes (30 nmoles) in the form
of admixture (lanes 1 and 2; duplicate samples) or the same amount
of DNA in the form of purified complex (prepared from DNA:DC-chol
liposome at ratio 1 .mu.g/25 nmoles, lanes 3 and 4; duplicate
samples) were directly injected into tumors. 48 hours following
transfection, the mice were sacrificed and tumor extract containing
100 .mu.g protein was assayed for CAT activity. Lane 5 shows
positive control CAT activity for standard E. coli CAT.
[0023] FIGS. 7A-7C show the transfection activities of admixture
DNA/lipid complex and purified and unpurified DNA/lipid/PLL
complexes in 293 cells (FIG. 7A) C3 cells (FIG. 7B) and BL6 cells
(FIG. 7C). Transfection activity is indicated on the vertical axis
of FIGS. 7A-7C as relative light units of luciferase activity.
[0024] FIGS. 8A and 8B show a comparison of the ability of
protamine sulfate, USP (PS) and poly-L-Lysine (PLL) to increase the
transfection activity in CHO and 293 cells, respectively. Varying
amounts of PS ( ) or PLL (.largecircle.) were added to 1 .mu.g of
pUK-21 CMV LUC DNA prior to complexation, with 7.5 nmol of DC-Chol
liposomes per well. Transfection, luciferase, and protein assays
were performed as described in Example 9 and each data point
represents the mean (with standard variation) of three data points
and are normalized to protein content.
[0025] FIG. 9 shows the effect on transfection activity of
different types of protamine where 2 .mu.g of each indicated type
of protamine or 1 .mu.g poly-L-lysine were added to 1 .mu.g of
pUK-21 CMV LUC DNA prior to complexing with 7.5 nmol of DC-Chol
liposomes per well. Transfection, luciferase, and protein assays
were performed as described in Example 10 and each point represents
the mean (with standard deviation) of four data points and are
normalized to protein.
[0026] FIG. 10 shows two different formulations which were prepared
by mixing pCMV-Luc DNA with DOTAP (25.38 .mu.g/.mu.g DNA) or DNA
with Protamine Sulfate, USP (PS) and complexing this mixture with
DOTAP (0.8 .mu.g PS/.mu.g DNA/23.27 .mu.g DOTAP). Dextrose was
added to both formulations to make a final concentration of 5%, and
were subsequently injected into mice (CD 1 female, 4-6 weeks old)
through the tail vein (50 .mu.g of DNA/mouse). Twenty-four hours
following injection, the mice were sacrificed and the major organs
collected. Tissues were homogenized in a lysis buffer, centrifuged
at 14,000 g for 10 min and the supernatant was analyzed for
luciferase activity and protein content. The results are expressed
as relative light units (RLU) per mg of protein (n=3). No activity
was found with DNA complexed with PS (0.8 .mu.g PS/.mu.g DNA) in
the absence of DOTAP (data not shown). Complexation of DOTAP with
DNA gave a variable level of expression, mainly in the lung.
Inclusion of Protamine Sulfate, USP into the DNA/DOTAP complex
resulted in a more consistent and higher level of gene
expression.
[0027] FIG. 11 shows in vivo gene expression of PS/DNA/DOTAP
complexes as a function of DOTAP concentration. DNA was mixed with
PS (0.8 .mu.g PS/.mu.g DNA) followed by complexation with
increasing amounts of DOTAP. Injection of these complexes and assay
of gene expression were performed as described in FIG. 10. The
results indicate increasing amounts of DOTAP were associated with
increases in gene expression in the lungs and spleen (n=3).
[0028] FIG. 12 shows in vivo gene expression of PS/DNA/DOTAP
complexes as a function of the dose of the complex delivered. DNA
was mixed with PS followed by complexation with DOTAP (0.8 .mu.g
PS/.mu.g DNA/23.27 .mu.g DOTAP) at increasing doses, as expressed
by the dose of DNA. Increasing doses of the complex were injected
into mice and gene expression was assayed 24 hours later (n=3). The
results indicate increasing doses of the PS/DNA/DOTAP complex were
associated with an increase in gene expression. However, at the
dose above 75 .mu.g (of DNA) per mouse, toxicity was noticed and
one mouse died at a dose of 100 .mu.g (of DNA).
[0029] FIG. 13 shows in vivo gene expression of PS/DNA/DOTAP
complexes as a function of time. PS/DNA/DOTAP complexes were
prepared as described in FIG. 12 and were injected into mice at a
DNA dose of 50 .mu.g per mouse. At different times following
injection, the mice were sacrificed and the major organs were
assayed for gene expression (n=3). It can be seen that high levels
of gene expression was observed in the lung as early as 6 hours
following injection and persisted for 24 hours, declining
thereafter. High levels of gene expression were also detected in
the spleen 6 hours after injection but declined with time.
[0030] FIG. 14 shows the in vitro transfection activity of
increasing amounts of PS/DNA (2:1 w/w ratio) at a fixed amount (7.5
nmol) of DC-Chol liposomes in CHO cells. Optimal activity was seen
at a dose of 2 .mu.g of DNA, 4 .mu.g PS and 7.5 nmol of DC-Chol
liposomes.
[0031] FIG. 15 shows the in vitro transfection activity of
increasing amounts of DC-Chol liposomes at a fixed amount of PS/DNA
(2 .mu.g PS; 1 .mu.g DNA) in CHO cells. Optimal activity was seen
at 2.5 nmol of DC-Chol liposomes and remained constant thereafter,
indicating that 2.5 nmol of DC-Chol liposomes was sufficient to
adequately deliver 2 .mu.g of PS and 1 .mu.g of DNA.
[0032] FIGS. 16-19 show the ability of PS to augment gene
expression when complexed to DNA with Clonfectin (Clontech) (FIG.
16), DC-Chol liposomes (FIG. 17), Lipofectin (Gibco BRL) (FIG. 18),
and DOTAP/DOPE (1:1 m/m) (Avanti Polar Lipids) (FIG. 19)
formulations. CHO cells were transfected with various amounts of
liposomes complexed with 1 .mu.g of DNA with (.box-solid.) or
without (.quadrature.) the inclusion of 2 .mu.g of PS. With each
liposome formulation, significant increases in transfection
activity are noted in complexes containing PS when compared to
complexes consisting of DNA alone with lipid. These results
indicate that the increase in transfection activity is not limited
to DC-Chol liposomes.
[0033] FIG. 20 shows the relationship between Lipid:Protamine
Sulfate:DNA ratios and particle sizes. The X axis describes the
Protamine Sulfate:DNA ratio. The Y axis describes the Lipid:DNA
ratio. Particle sizes (nm) corresponding to the respective ratios
at Day 0 and Day 7 appear in the matrix cell at the intersection of
the X and Y axes. Particle sizes around 200 nm are thought to be
favorable since this represents optimal sizing for coated pit
internalization.
[0034] FIG. 21 shows the in vitro transfection activities of
different LPD formulations in HeLa cells (a human cervical
carcinoma cell line). The Protamine sulfate concentration of each
formulation used is shown on the X axis. Transfection activity is
indicated on the Y axis as relative light units of luciferase
activity normalized to the amount of extracted protein.
[0035] FIG. 22 shows the in vitro transfection activities of
different LPD formulations in SKOV-3 cells (a human ovarian
carcinoma cell line). The various DC-Chol and LPD delivery vehicles
used in the study are indicated on the X axis. Transfection
activity is indicated on the Y axis as relative light units of
luciferase activity normalized to the amounts of extracted protein.
The n value represents the number of individual experiments which
were averaged for the data table. Each individual experiment
consisted of replicates where n=3 or 6 for each formulation
represented.
[0036] FIG. 23 shows the relationship between the Lipid:Protamine
Sulfate:DNA formulation used in vivo to deliver pCMV-Luc
intraperitoneally in an SKOV-3 murine model and the observed
Luciferase expression activity. The composition of the delivery
formulation is indicated on the X axis. The nomenclature used to
describe the formulations follows the pattern of nanomoles of
Lipid:m Protamine Sulfate:.mu.g DNA. Transfection activity is
indicated on the Y axis as relative light units of luciferase
activity. The graph shows the RLU/mg values obtained for each
individual mouse (circles) and the average value per formulation
(squares).
[0037] FIG. 24 shows a dose response graph for the treatment of
Nude mice injected intraperitoneally with 2.times.10.sup.6
SKOV-3ip1 cells at Day 0. On Day 5 mice were injected with
different L:P:D formulations comprising an E1A expression plasmid.
Group 1 animals were repeatedly injected with the 5% Dextrose
vehicle only; Group 2 animals were repeatedly injected with the
equivalent amount of DC-Chol/DOPE (6:4 mol:mol ratio) and Protamine
sulfate as one would find in the high dose 15:2:1 group but without
the E1A expression plasmid present; Group 3 animals were repeatedly
injected with 15 .mu.g/dose of naked E1A expression plasmid, which
was equivalent to the highest dose injected in formulated vehicles;
Group 4 animals were repeatedly injected with 15 .mu.g/dose of E1A
expression plasmid compacted with Protamine sulfate at a ratio of 2
.mu.g Protamine/1 .mu.g DNA, which was equivalent to the highest
dose injected in the formulated vehicles; Group 14 animals were
repeatedly injected with a 1.5 .mu.g/dose 15:2:1 LPD formulation;
Group 15 animals were repeatedly injected with a 5 .mu.g/dose
15:2:1 LPD formulation; Group 16 animals were repeatedly injected
with a 15 .mu.g/dose LPD formulation. The time course of the
experiment in days is shown on the X axis. The number of surviving
mice is shown on the Y axis.
DESCRIPTION OF INVENTION
[0038] This invention relates to lipid-comprising drug delivery
complexes having a net positive charge and/or a positively charged
surface at pH 6.0-8.0. These complexes comprise lipids, drugs, and
optionally further comprise polycations. The invention further
relates to a method for producing these complexes where the method
may optionally include the step of purifying these formulations
from excess individual components. For the production of the
drug/lipid complexes of this invention, inclusion of the
purification step is a preferred embodiment. It should be
understood that where the purification step is applied to the
drug/lipid/polycation complexes, the recovery of these complexes in
a pure state free from excess components following purification is
lower than the recovery of drug/lipid complexes following their
purification since the peak containing the drug/lipid/polycation
complex following sucrose purification via density centrifugation
is broader than the peak containing drug/lipid complexes and hence,
overlaps with the peaks of the free components.
[0039] The lipid-comprising drug delivery complexes of this
invention are stable, capable of being produced at relatively high
concentrations, and retain biological activity of the drug
component over time in storage. The method of producing these
complexes is based on a binding model between two oppositely
charged polymers (e.g. negatively charged nucleic acid and
positively charged lipids) in which the formation of large unstable
aggregates is avoided by neutralizing the negative charge of the
drug via the use of an excess amount of positive charge in the form
of cationic liposomes or cationic liposomes and polycation. The
complexes of this invention have been observed to retain their
initial diameter and bioactivity over 4 months in storage in 10%
sucrose buffer.
[0040] The "drug" which is contained in the lipid-comprising drug
delivery complexes of the present invention may be nucleic acids,
polyanionic proteins, polysaccharides and other macromolecules
which can be complexed directly with cationic lipids. However,
cationic drugs (eg large cationic protein) can be directly
complexed with an anionic lipid or sequentially complexed first
with anionic lipid or polymer followed by cationic lipid. The use
of this process permits delivery of positive or neutral charged
drug to cells by the complexes of the present invention.
[0041] To produce drug/lipid and drug/lipid/polycation complexes
with a net positive charge, the positive charge excess of lipid to
drug or of lipid and polycation to drug may be up to about a
30-fold positive charge excess in the complex of total lipids to
drug or of lipid and polycation to drug, preferably about a 2 to
10-fold charge excess and most preferably about a 2 to 6-fold
charge excess. Complexes which possess a positive charge on their
surface may have similar preferred ranges of surface charge excess
to drug. To produce a nucleic acid/lipid complex having a positive
charge excess of lipid to nucleic acid, mole amounts of cationic
liposomal lipid to be mixed with 1 .mu.g of nucleic acid to produce
a nucleic acid/lipid complex which has positive charge excess of
lipid to nucleic acid at pH 6.0-8.0 may range from about 0.1 nmol
to about 200 nmol of lipid, preferably about 5 nmol to about 100
nmol lipid, depending on the positive charge content of the
cationic liposome. Of course, if the drug were a protein, the
amount of lipid to be mixed with 1 .mu.g of negatively charged
protein would be at least 10-fold less than the amount of lipid to
be mixed with 1 .mu.g of DNA as shown above since proteins are less
charge dense than nucleic acids. Those of ordinary skill in the art
would readily understand that depending upon the positive charge
content of the cationic liposomes, different mole amounts of
different cationic liposomes would have to be mixed with an
equivalent amount of drug to produce a positive charge excess of
lipid to drug.
[0042] When a drug/lipid/polycation complex having a net positive
charge and/or a positively charged surface is to be produced, the
inclusion of the polycation reduces the amount of lipid which must
be mixed with drug to the extent that the positive charge from the
lipid may be less than the negative charge from the drug. This
reduction in the amount of lipid reduces the toxicity of the
polycation-containing formulations. Mole amounts of cationic
liposomes to be used in formulating nucleic acid/lipid/polycation
complexes may range from about 0.1 nmol to about 200 nmol lipid per
1 .mu.g nucleic acid, more preferably from about 1 to about 25
nmoles lipid per 1 .mu.g nucleic acid depending on the positive
charge content of the cationic liposomes. It is to be generally
understood that in producing the nucleic acid/lipid and nucleic
acid/lipid/polycation complexes of the present invention, the mole
amount of liposomes required to produce these complexes will
increase as the concentration of nucleic acid mixed with the
liposomes is increased.
[0043] Those of ordinary skill in the art would readily understand
that when the complexes of the present invention are purified, the
positive charge excess of cationic liposomes to drug or of cationic
liposomes and polycation to drug immediately prior to mixing will
be greater than the positive charge excess in the purified
complexes of lipid to drug or of lipid and polycation since the
purification step results in the removal of excess free lipids
and/or free polycation.
[0044] In order to illustrate how the charges attributed to
cationic lipid, drug and polycation may be determined at pH 6.0-8.0
the following example is provided. Assuming the drug to be
delivered is DNA, one determines the negative charge of the DNA to
be delivered by dividing the amount of DNA to be mixed, or the
amount of DNA in the complex, by 330, the molecular weight of a
single nucleotide where one nucleotide equals one negative charge.
Thus, the negative charge for 1 .mu.g of DNA is 3.3 nmols.
[0045] For 10 nmol of DC-Chol/DOPE (2:3) liposomes one calculates
the effective charge of the lipid by multiplying the amount of
total liposomal lipid (10 nmol) by 0.4 (40% of the total liposomal
lipid is the cationic lipid DC-Chol) to yield 4 nmol DC-Chol lipid
in the liposomes. Since at pH 6-8, one molecule of DC-Chol has one
positive charge, the effective positive charge of liposomal lipid
at the time of mixing, or in the complex, is 4.0 nmol. Of course,
those of skill in the art would readily understand that other
cationic lipids may have a lesser or greater amount of positive
charge per molecule of cationic lipid at pH 6-8.0 than DC-Chol.
[0046] Assuming the polycation to be mixed to form the complex is a
bromine salt of poly-L-lysine (PLL), the positive charge of PLL at
the time of mixing is obtained by dividing the amount of PLL to be
mixed by 207, the molecular weight of one lysyl residue where one
lysyl residue equals one positive charge. Thus, the positive charge
for 1 .mu.g of PLL is approximately 5.0 nmols. To calculate the
positive charge contributed by lysyl residues in a formed complex,
the amount of lysine present in the complex is divided by the
molecular weight of one lysyl residue taking into account the
weight of a counterion, if present.
[0047] Application of the above calculations to data presented in
Table 1 herein (see Example 3) illustrates how a positive to
negative charge ratio can be calculated both at the time of mixing
of DNA and liposome and, after purification of the complex produced
by the mixing of DNA and liposome. In Table 1 of Example 3, 0.4 mg
of DNA is mixed with 20 .mu.mols of cationic DC-Chol/DOPE liposomes
to produce DNA/lipid complex. For cationic liposomes having a
DC-Chol/DOPE ratio of 4:6, the positive charge content of the
liposomal lipid is calculated to be 8000 nmol and the negative
charge content of the 0.4 mg DNA to be mixed with liposomes is
calculated to be 1320 nmols based on the sample calculations
presented in the above paragraphs. Therefore, the positive to
negative charge ratio at the time of mixing is 6.06 (8000 divided
by 1320). However, after the complex was purified, the lipid to DNA
ratio of this purified complex was 12.7 nmol lipid/.mu.g DNA as
shown in Table 1 (see the "4:6 row"). This 12.7 ratio translates to
a positive to negative charge ratio of 1.5 thus showing that
purification removed excess positive charge of free liposomes.
[0048] Also in Table 1, where DNA/lipid/PLL complex was prepared by
mixing 4 .mu.mol of liposomes (4:6 DC-Chol/DOPE) and 1 mg PLL with
0.4 mg DNA, one can calculate the positive to negative charge ratio
at the time of mixing as follows. Based on the sample calculations
presented in the above paragraphs, the 4 .mu.mol liposomal lipid
contributes 1600 nmol of positive charge, the 1 mg of PLL
contributes 5000 nmol of positive charge and the 0.4 mg DNA
contributes 1,320 nmol of negative charge. Thus, the positive to
negative charge ratio at the time of mixing
liposomes , PLL and DNA is 5 ( 1600 + 5000 ) 1320 .
##EQU00001##
[0049] It is further to be understood by those skilled in the art
that the net charge of the complex may be determined by measuring
the amount of DNA, lipid and when present, polycation in the
complex by the use of an appropriate analytical technique such as
the use of radioisotopic labeling of each component or by elemental
analysis. Once the amounts of each component (DNA, lipid and when
present, polycation) in a complex at a given pH are known, one
could then calculate the approximate net charge of that complex at
the given pH taking into account the pK's of the components which
may be known or determined analytically.
[0050] In a preferred embodiment, the drug is a nucleic acid
sequence, preferably a nucleic acid sequence encoding a gene
product having therapeutic utility.
[0051] In one embodiment of the invention, a method for producing
nucleic acid/lipid complexes having a net positive charge and/or
positively charged surface at pH 6-8.0, comprises, combining
nucleic acids with cationic liposomes in a nucleic acid to lipid
ratio such that the nucleic acid/lipid complex formed has a
positive charge excess of lipid to nucleic acid.
[0052] In an alternative embodiment, nucleic acid and cationic
liposome may be mixed with a polycation in a nucleic acid to lipid
to polycation ratio such that the nucleic acid/lipid/polycation
complexes formed have a positive charge excess of lipid and
polycation to nucleic acid at pH 6-8.
[0053] In a preferred embodiment, the nucleic acid/lipid and
nucleic acid/lipid/polycation complexes are produced by slowly
adding nucleic acid to the solution of liposome or liposome plus
polycation and mixing with a stirring bar where the mixing is
allowed to proceed second after addition of DNA. Alternatively, the
liposome or liposome/polycation mix can be added into a single
chamber from a first inlet at the same time the nucleic acid is
added to the chamber through a second inlet. The components are
then simultaneously mixed by mechanical means in a common chamber.
The complexes may also be produced by first mixing the nucleic acid
with the polycation and then adding the liposome suspension.
[0054] The cationic liposomes mixed with drug or with drug and
polycation to form the complexes of the present invention may
contain a cationic lipid alone or a cationic lipid in combination
with a neutral lipid. Suitable cationic lipid species include, but
are not limited to: 3.beta.[.sup.4N--(.sup.1N,.sup.8N-diguanidino
spermidine)-carbamoyl] cholesterol (BGSC);
3.beta.[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol
(BGTC); N,N.sup.1,N.sup.2,N.sup.3 Tetra-methyltetrapalmitylspermine
(cellfectin);
N-t-butyl-N'-tetradecyl-3-tetradecyl-aminopropion-amidine
(CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB);
1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
(DMRIE);
2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-p-
ropanaminium trifluorocetate) (DOSPA);
1,3-dioleoyloxy-2-(6-carboxyspermyl)-propyl amide (DOSPER);
4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM)
N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3
dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); 1, 2
bis(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)-alkyl-N,N,N-trisubstituted
ammonium surfactants; 1, 2 dioleoyl-3-(4'-trimethylammonio)
butanol-sn-glycerol (DOBT) or cholesteryl (4' trimethylammonia)
butanoate (ChOTB) where the trimethylammonium group is connected
via a butanol spacer arm to either the double chain (for DOTB) or
cholesteryl group (for ChOTB); DORI
(DL-1,2-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammonium)
or DORIE
(DL-1,2-O-dioleoyl-3-dimethylaminopropyl-.beta.-hydroxyethylammoniu-
m) (DORIE) or analogs thereof as disclosed in WO 93/03709;
1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC);
cholesteryl hemisuccinate ester (ChOSC); lipopolyamines such as
dioctadecylamidoglycylspermine (DOGS) and dipalmitoyl
phosphatidylethanolamylspermine (DPPES) or the cationic lipids
disclosed in U.S. Pat. No. 5,283,185,
cholesteryl-3.beta.-carboxyl-amido-ethylenetrimethyl-ammonium
iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl
carboxylate iodide, cholesteryl-3.beta.-carboxyamidoethyleneamine,
cholesteryl-3.beta.-oxysuccinamido-ethylenetrimethylammonium
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'-dimethylaminoethane)carbamoyl cholesterol
(DC-chol), and
3.beta.-N-(polyethyleneimine)-carbamoylcholesterol.
[0055] Examples of preferred cationic lipids include
N-t-butyl-N'-tetradecyl-3-tetradecyl-aminopropion-amidine
(CLONfectin),
2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium trifluoroacetate (DOSPA),
1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP),
N-[1-(2,3,dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride)
(DOTMA), cholesteryl-3.beta.-carboxyamidoethylenetri-methylammonium
iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl
carboxylate iodide, cholesteryl-3.beta.-carboxyamidoethyleneamine,
cholesteryl-3.beta.-oxysuccin-amidoethylenetrimethyl-ammonium
iodide,
1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3.beta.-oxysuc-
cinate iodide, 2-(2-trimethylammonio)ethylmethylamino
ethyl-cholesteryl-3.beta.-oxysuccinateiodide,
3.beta.N--(N',N'dimethyl-aminoethane)-carbamoyl-cholesterol
(DC-chol), and 3.beta.N-(polyethyleneimine)-carbamoyl
cholesterol.
[0056] Since an attribute of the complexes of the invention is
their stability during storage (i.e., their ability to maintain a
small diameter and retain biological activity over time following
their formation); it will be understood by those of ordinary skill
in the art that preferred cationic lipids are those lipids in which
bonds between the lipophilic group and the amino group are stable
in aqueous solution. While such bonds found in cationic lipids
include amide bonds, ester bonds, ether bonds and carbamoyl bonds,
preferred cationic lipids are those having a carbamoyl bond. An
example of a preferred cationic lipid having a carbamoyl bond is
DC-Chol. Those of skill in the art would readily understand that
liposomes containing more than one cationic lipid species may be
used to produce the complexes of the present invention. For
example, liposomes comprising two cationic lipid species,
lysyl-phosphatidylethanolamine and .beta.-alanyl cholesterol ester
have been disclosed (Brunette, E. et al. (1992) Nucl. Acids Res.,
20:1151).
[0057] It is to be further understood that in considering cationic
liposomes suitable for use in mixing with drug and optionally with
polycation, to form the complexes of this invention, the methods of
the invention are not restricted only to the use of the lipids
recited above but rather, any lipid composition may be used so long
as a cationic liposome is produced.
[0058] Thus, in addition to cationic lipids, cationic liposomes
used to form the complexes of the invention may contain other
lipids in addition to the cationic lipids. These lipids include,
but are not limited to, lyso lipids of which
lysophosphatidylcholine (1-oleoyl lysophosphatidylcholine) is an
example, cholesterol, or neutral phospholipids including dioleoyl
phosphatidyl ethanolamine (DOPE) or dioleoyl phosphatidylcholine
(DOPC) as well as various lipophylic surfactants, containing
polyethylene glycol moieties, of which Tween-80 is one example. The
lipid complexes of the invention may also contain negatively
charged lipids as well as cationic lipids so long as the net charge
of the complexes formed is positive and/or the surface of the
complex is positively charged. Negatively charged lipids of the
invention are those comprising at least one lipid species having a
net negative charge at or near physiological pH or combinations of
these. Suitable negatively charged lipid species include, but are
not limited to, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutaryl
phosphatidlylethanolanine), phosphatidyl glycerol and phosphatidic
acid or a similar phospholipid analog.
[0059] It is further contemplated that in the cationic liposomes
utilized to form the complexes of the invention, the ratio of
lipids may be varied to include a majority of cationic lipids in
combination with cholesterol or with mixtures of lyso or neutral
lipids. When the cationic lipid of choice is to be combined with
another lipid, a preferred lipid is a neutral phospholipid, most
preferably DOPE.
[0060] Methods for producing the liposomes to be used in the
production of the lipid-comprising drug delivery complexes of the
present invention are known to those of ordinary skill in the art.
A review of methodologies of liposome preparation may be found in
Liposome Technology (CFC Press NY 1984); Liposomes by Ostro (Marcel
Dekker, 1987); Methods Biochem Anal. 33:337-462 (1988) and U.S.
Pat. No. 5,283,185. Such methods include freeze-thaw extrusion and
sonication. Both unilamellar liposomes (less than about 200 nm in
average diameter) and multilamellar liposomes (greater than about
300 nm in average diameter) may be used as starting components to
produce the complexes of this invention.
[0061] In the cationic liposomes utilized to produce the drug/lipid
complexes of this invention, the cationic lipid is present in the
liposome at from about 10 to about 100 mole % of total liposomal
lipid, preferably from about 20 to about 80 mole % and most
preferably about 20 to about 60 mole %. The neutral lipid, when
included in the liposome, may be present at a concentration of from
about 0 to about 90 mole % of the total liposomal lipid, preferably
from about 20 to about 80 mole %, and most preferably from 40 to 80
mole %. The negatively charged lipid, when included in the
liposome, may be present at a concentration ranging from about 0
mole % to about 49 mole % of the total liposomal lipid, preferably
from about 0 mole % to about 40 mole %. In a preferred embodiment,
the liposomes contain a cationic and a neutral lipid, most
preferably DC-Chol and DOPE in ratios between about 2:8 to about
6:4. It is further understood that the complexes of the present
invention may contain modified lipids, protein, polycations or
receptor ligands which function as a targeting factor directing the
complex to a particular tissue or cell type. Examples of targeting
factors include, but are not limited to, asialoglycoprotein,
insulin, low density lipoprotein (LDL), folate and monoclonal and
polyclonal antibodies directed against cell surface molecules.
Potential targets include, but are not limited to, liver, blood
cells, endothelial cells and tumor cells. Furthermore, to enhance
the circulatory half-life of the complexes, the positive surface
charge can be sterically shielded by incorporating lipophilic
surfactants which contain polyethylene glycol moieties.
[0062] It is to be further understood that the positive charge of
the complexes of this invention may be affected not only by the
lipid composition of the complex but also by the pH of the solution
in which the drug/lipid complexes are formed. For example,
increasing pH (more basic) will gradually neutralize the positive
charge of the tertiary amine of the cationic lipid DC-Chol. In a
preferred embodiment, the complexes of the present invention are
produced, and stored, at a pH such that the complexes have a net
positive charge and/or positively charged surface. A preferred pH
range is pH 6.0-8.0, most preferably pH 7.0-7.8.
[0063] When a polycation is to be mixed with nucleic acid and
cationic liposomes, the polycation may be selected from organic
polycations having a molecular weight of between about 300 and
about 200,000. These polycations also preferably have a valence of
between about 3 and about 1000 at pH 7.0. The polycations may be
natural or synthetic amino acids, peptides, proteins, polyamines,
carbohydrates and any synthetic cationic polymers. Nonlimiting
examples of polycations include polyarginine, polyornithine,
protamines and polylysine, polybrene (hexadimethrine bromide),
histone, cationic dendrimer, spermine, spermidine and synthetic
polypeptides derived from SV40 large T antigen which has excess
positive charges and represents a nuclear localization signal. In
one embodiment, the polycation is poly-L-lysine (PLL).
[0064] In another more preferred embodiment, the polycation is a
polycationic polypeptide having an amino acid composition in which
arginine residues comprise at least 30% of the amino acid residues
of the polypeptide and lysine residues comprise less than 5% of the
amino acid residues of the polypeptide. In addition, preferably
histidine, lysine and arginine together make up from about 45% to
about 85% of the amino acid residues of the polypeptide and serine,
threonine and glycine make up from about 10% to about 25% of the
amino acid residues of the polypeptide. More preferably, arginine
residues constitute from about 65% to about 75% of the amino acid
residues of the polypeptide and lysine residues constitute from
about 0 to about 3% of the amino acid residues of the
polypeptide.
[0065] In addition to the above recited percentages of arginine and
lysine residues, the polycationic polypeptides of the invention may
also contain from about 20% to about 30% hydrophobic residues, more
preferably, about 25% hydrophobic residues. The polycationic
polypeptide to be used in producing drug/lipid/polycation complexes
may be up to 500 amino acids in length, preferably about 20 to
about 100 amino acids in length; more preferably, from about 25 to
about 50 amino acids in length, and most preferably from about 25
to about 35 amino acids in length.
[0066] In one embodiment, the arginine residues present in the
polycationic polypeptide are found in clusters of 3-8 contiguous
arginine residues and more preferably in clusters of 4-6 contiguous
arginine residues.
[0067] In another embodiment, the polycationic polypeptide is about
25 to about 35 amino acids in length and about 65 to about 70% of
its residues are arginine residues and 0 to 3% of its residues are
lysine residues.
[0068] The polycationic polypeptides to be used in formulating the
complexes of the invention may be provided as naturally occurring
proteins, particularly certain protamines having a high arginine to
lysine ratio as discussed above, as a chemically synthesized
polypeptide, as a recombinant polypeptide expressed from a nucleic
acid sequence which encodes the polypeptide, or as a salt of any of
the above polypeptides where such salts include, but are not
limited to, phosphate, chloride and sulfate salts.
[0069] In one embodiment, a drug such as DNA could be complexed
with an excess of polycationic polypeptide such that a net
positively charged complex is produced. This complex, by nature of
its positive charge, could favorably interact with negatively
charged lipid (s) to form a DNA/lipid/polycationic polypeptide
complex.
[0070] The transfection activity of a polycationic
polypeptide/DNA/lipid complex of the invention in CHO cells, is
preferably equal to or greater than the transfection activity of a
poly-L-lysine/DNA/lipid complex in CHO cells when each polycation
is complexed with the same cationic liposome and plasmid construct
containing a reporter gene where reporter genes include, but are
not limited to, the chloramphenicol acetyl transferase gene, the
luciferase gene, the .beta.-galactosidase gene and the human growth
hormone gene, the alkaline phosphatase gene and a green fluorescent
protein gene.
[0071] In producing nucleic acid/lipid/polycation complexes of the
present invention, the ratio of polycation to nucleic acid is kept
fixed while varying the amount of liposome. However, those of skill
in the art would recognize that the ratio of polycation to nucleic
acid will be affected by the charge density of the liposome to be
mixed with the nucleic acid and polycation. For example, if the
charge density of liposomes is decreased as a result of changes in
the lipid composition of the liposome (eg decreasing the ratio of
cationic lipid:neutral lipid in the liposome), the amount of
polycation to be mixed with nucleic acid and liposome may be
increased to compensate for the decrease in positive charge
contributed by the liposomes. However, when polycation is utilized,
it is preferred to use subsaturating amounts of polycation (i.e.,
amounts which will not saturate all the negative charge of the
nucleic acid) in order to allow the cationic lipids to complex with
the nucleic acid. Thus, in a preferred embodiment of the invention,
a positive charge excess of lipid to nucleic acid is used even when
polycation is mixed with lipid and nucleic acid. Amounts of
polycation which may be mixed with 1 .mu.g of nucleic acid and
varying amounts of cationic liposomes in the present invention
range from about 0.01 .mu.g to about 100 .mu.g of polycation per
.mu.g of nucleic acid, preferably from about 0.1 .mu.g to about 10
.mu.g of polycation per .mu.g of nucleic acid.
[0072] Where purification of nucleic acid/lipid and nucleic
acid/lipid/polycation complexes from excess free DNA, free
liposomes and free polycation is desired, purification may be
accomplished by centrifugation through a sucrose density gradient
or other media which is suitable to form a density gradient.
However, it is understood that other methods of purification such
as chromatography, filtration, phase partition, precipitation or
absorption may also be utilized. In a preferred method,
purification via centrifugation through a sucrose density gradient
is utilized. The sucrose gradient may range from about 0% sucrose
to about 60% sucrose, preferably from about 5% sucrose to about 30%
sucrose. The buffer in which the sucrose gradient is made can be
any aqueous buffer suitable for storage of the fraction containing
the complexes and preferably, a buffer suitable for administration
of the complex to cells and tissues. A preferred buffer is pH
7.0-8.0 Hepes.
[0073] It is understood that in the present invention, preferred
nucleic acid sequences are those capable of directing protein
expression. Such sequences may be inserted by routine methodology
into plasmid expression vectors known to those of skill in the art
prior to mixing with cationic liposomes or liposomes and polycation
to form the lipid-comprising drug delivery complexes of the present
invention. The amount of nucleic acid mixed together with cationic
liposomes or with cationic liposomes and polycation may range from
about 0.01 .mu.g to about 10 mg, preferably from about 0.1 .mu.g to
about 1.0 mg. It is understood that where the nucleic acid of
interest is contained in plasmid expression vectors, the amount of
nucleic acid recited above refers to the plasmid containing the
nucleic acid of interest.
[0074] The purification of the nucleic acid/lipid and nucleic
acid/lipid/polycation complexes of the present invention serves to
concentrate the nucleic acids and lipids contained in the resultant
complexes from about 50-fold to about 500-fold such that the lipid
content contained in the complexes may be as high as about 40
.mu.mol/ml and the nucleic acid content may be as high as about 2
mg/ml.
[0075] The nucleic acid/lipid/polycation complexes of the present
invention produce particles of varying diameters upon formulation.
As pointed out in the Background of Invention smaller particles
tend to show greater size stability than larger particles.
Furthermore, smaller particles may be more suitable for use as
nucleic acid delivery vehicles. Particle diameters can be
controlled by adjusting the nucleic acid/lipid/polycation ratio in
the complex. FIG. 20 illustrates this fact for Lipid:Protamine
Sulfate:DNA complexes. The diameter of the complexes produced by
the methods of the present invention is less than about 400 nm,
preferably less than about 200 nm, and more preferably less than
150 nm.
[0076] Nucleic acid/lipid/polycation ratios in the present
invention further affect the biological activity of the complexes.
The in vitro and in vivo transfection of mammalian cells can be
facilitated by adjusting the relative amounts of nucleic acid,
lipid and polycation. FIGS. 21, 22 and 23 present data
demonstrating this effect for the Lipid/Protamine Sulfate/DNA
mediated transfection of HeLa and SKOV-3 cells. Where the
polycation is Protamine Sulfate and the nucleic acid is DNA, the
nucleic acid/polycation ratio for the complex is between 1:0.01 and
1:100. The nucleic acid/polycation ratio is preferably between
1:0.1 and 1:10, more preferably between 1:0.5 and 1:5 and most
preferably between 1:1 and 1:3.
[0077] The complexes formed by the methods of the present invention
are stable for up to about one year when stored at 4.degree. C. The
complexes may be stored in 10% sucrose or a 5% dextrose solution
upon collection from the sucrose gradient or they may be
lyophilized and then reconstituted in an isotonic solution prior to
use. In a preferred embodiment, the complexes are stored in
solution. The stability of the complexes of the present invention
is measured by specific assays to determine the physical stability
and biological activity of the complexes over time in storage. The
physical stability of the complexes is measured by determining the
diameter and charge of the complexes by methods known to those of
ordinary skill in the art, including for example, electron
microscopy, gel filtration chromatography or by means of
quasi-elastic light scattering using, for example, a Coulter N4SD
particle size analyzer as described in the Examples. The physical
stability of the complex is "substantially unchanged" over storage
when the diameter of the stored complexes is not increased by more
than 100%, preferably by not more than 50%, and most preferably by
not more than 30%, over the diameter of the complexes as determined
at the time the complexes were purified.
[0078] Assays utilized in determining the biological activity of
the complexes vary depending on what drug is contained in the
complexes. For example, if the drug is nucleic acid encoding a gene
product, the biological activity can be determined by treating
cells in vitro under transfection conditions utilized by those of
ordinary skill in the art for the transfection of cells with
admixtures of DNA and cationic liposomes. Cells which may be
transfected by the complexes includes those cells which may be
transfected by admixture DNA/liposome complexes. The activity of
the stored complexes is then compared to the transfection activity
of complexes prepared by admixture. If the drug is a protein, then
activity may be determined by a bioassay suitable for that
protein.
[0079] It is further understood by those of skill in the art that
the complexes of the present invention may be used in vivo as
vectors in gene therapy.
[0080] Therapeutic formulations using the complexes of the
invention preferably comprise the complexes in a physiologically
compatible buffer such as, for example, phosphate buffered saline,
isotonic saline or low ionic strength buffer such as 10% sucrose in
H.sub.2O (pH 7.4-7.6) or in Hepes (pH 7-8, a more preferred pH
being 7.4-7.6). The complexes may be administered as aerosols or as
liquid solutions for intratumoral, intravenous, intratracheal,
intraperitoneal, and intramuscular administration.
[0081] Any articles or patent referenced herein are incorporated by
reference. The following examples illustrate various aspects of the
invention but are intended in no way to limit the scope
thereof.
EXAMPLES
Materials
[0082] DOPE was purchased from Avanti Polar Lipid, Inc. (Alabaster,
Ala.). pRSVL, a plasmid which encodes the luciferase gene under the
control of Rous sarcoma virus long terminal repeat, (De Wet, J. R.
et al. (1987) Mol. Cell. Biol., 7: 725-737) was amplified in E.
coli and purified using the standard CsCl-EtBr ultracentrifugation
method (Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular
Cloning: A Laboratory Manual (2d ed) Cold Spring Harbor Laboratory
Press: New York (1989)). All the tissue culture media were obtained
from Gibco BRL (Gaithersburg, Md.). Human embryonic kidney 293
cells, CHO (Chinese Hamster Ovary cells), BL6 and BHK (Baby Hamster
Kidney cells) cells were from American Type Culture Collection
(Rockville, Md.). Mouse lung cells (MLC) are primary culture cells
originally derived from the lung of a Balb/c mouse by Dr. S. Kennel
(Oak Ridge National Laboratory, TN). 293, BL6, BHK and MLC cells
were cultured with DMEM media, CHO cells were cultured with F12
media, and C3 Hela cells were cultured in RPMI-1640 medium. All
media was supplemented with 10% fetal bovine serum (Hyclone
Laboratories, Inc., Logan, Utah) and 100 unit/ml penicillin and 100
.mu.g/ml streptomycin. Poly-L-lysine hydrobromide (MW 3000 and MW
25,600) and other chemicals were from Sigma (St Louis, Mich.).
[0083] DC-Chol was synthesized according to the method of Gao and
Huang (1991) (Gao, X., and Huang, L. (1991) Biochem. Biophys. Res.
Commun., 179: 280-285) with modifications in the purification steps
as follows: after the reaction, 10 ml hexane was added and the
mixture was extracted three times with 10 ml water. The organic
phase was collected and dried under vacuum at 4.degree. C. The
resulting solid was dissolved in a minimal amount of absolute
ethanol with heat, and recrystallized in acetonitrile at 0/C. The
purity of DC-Chol was at least >95%, as analyzed by TLC method
and .sup.1H-NMR and the yield of about 70% was a significant
improvement over that of the previously reported method of Gao, X.,
and Huang, L. ((1991) Biochem. Biophys. Res. Commun., 179:
280-285).
Methods
Preparation and Purification of Complexes
[0084] Cationic liposomes at a 20 mM total lipid concentration were
prepared from DC-Chol and DOPE at various ratios by a sonication
method, according to a published procedure (Gao, X., and Huang, L.
(1991) Biochem. Biophys. Res. Commun., 179: 280-285). Trace amount
of .sup.3H cholesteryl hexadecyl ether (Amersham, Arlington
Heights, Ill.) was included for quantitation purpose. The size of
these liposomes was between 100 to 150 nm in diameter, as
determined by quasi-elastic light scattering using a Coulter N4SD
particle sizer (Coulter Electronics, Inc., Hialeah, Fla.). Unless
indicated otherwise in the following Examples, DNA/lipid complexes
were prepared at a typical laboratory scale by adding amounts of
free DC-Chol/DOPE liposomes as indicated in each Example in a
volume of 1 ml of a 2 mM Hepes buffer (pH 7.6) to a 15.times.7.5
polystyrene culture tube (Baxter, McGraw Pare, Ill.), a
micro-magnetic stirrer was placed in the tube, and the solution was
well mixed. Amounts of pRSVL DNA as indicated in each Example were
then added dropwise from a stock solution (0.2 mg/ml, in 2 mM Hepes
buffer, pH 7.6) to the liposome solution over a period of 3 min.
Trace amounts of pRSVL labeled with .sup.32P using a nick
translation kit (Promega, Madison, Wis.) and .sup.32P dCTP
(Amersham, Arlington Heights, Ill.) was included for the purpose of
quantitation.
[0085] To prepare purified lipid/PLL/DNA complexes, an amount of
the above 0.2 mg/ml DNA solution as indicated in each Example was
added to 1 ml PLL/liposome mixture containing amounts of liposomes
and PLL as indicated in each Example. DNA/lipid complexes were
loaded on the top of a sucrose step gradient composed of 0.5 ml
each of 5%, 7.5%, 10% and 15% sucrose (w/w) and DNA/lipid/PLL
complexes were loaded on top of a sucrose step gradient composed of
0.5 ml each of 5%, 10%, 15%, 20%, 25% and 30% sucrose (w/w). The
DNA/lipid and DNA/lipid/PLL complexes were then purified from free
lipid and PLL by ultracentrifugation at 100,000 g for 30 min at
4.degree. C. After centrifugation, fractions of 200 .mu.l were
taken from the top to the bottom of the tube. Aliquots from each
fraction were assayed for both .sup.3H and .sup.32P radioactivity
using a scintillation counter. Fractions that contained peak value
of the .sup.32P were collected and pooled. These pooled fractions
were then assayed for particle size and for transfection
activity.
In Vitro Transfection Assay
[0086] The biological activity of the above complexes were assayed
by in vitro transfection of cells in the Examples as follows.
Briefly, cells grown in 48 well plate were incubated with DNA/lipid
complex diluted in 0.5 ml CHO-S-SFM (Gibco BRL) or with admixture
DNA/liposome complex prepared according to Gao and Huang (1991)
(Gao, X., and Huang, L. (1991) Biochem. Biophys. Res. Commun., 179:
280-285). For transfection of pRSVL DNA using DC-Chol liposomes in
the presence of PLL, liposomes were first mixed with PLL, then
complexed with DNA. All transfections were performed for 4 hours at
37.degree. C. After transfection, cells were further cultured for
36 hours in the appropriate media containing 10% fetal bovine
serum. Cells were then washed with PBS and lysed with 100 .mu.l of
1.times. lysis buffer provided by a luciferase assay kit (Promega,
Madison, Wis.). A 4 .mu.l sample of the lysate was assayed for
luciferase activity using 100 .mu.l substrate solution from the
reconstituted luciferase assay kit and an AutoLumat LB953
luminometer (Berthold, Germany). Protein concentration from each
lysate was assayed by a Coomassie blue dye method according to the
manufacturer's protocol (Pierce, Rockford, Ill.).
Example 1
The Size of the DNA/Lipid Complex is Determined by the Ratio of DNA
to Lipid
[0087] This experiment was conducted to show that the size of the
DNA-lipid complex formed by admixture changed as the ratio of DNA
mixed with liposome varied. In brief, pRSVL plasmid DNA (2 .mu.g)
was mixed with varying amounts of DC-Chol/DOPE (3:2) liposomes in 2
mM Hepes buffer at pH 7.6 in a final volume of 500 .mu.L and after
7 minutes, the size of the complex was determined with a Coulter
N4SD laser light scattering particle sizer operating in the
unimodel mode.
[0088] As shown in FIG. 1, large aggregates did not form when DNA
was in excess (ratios of liposome to DNA less than 7) but at ratios
of lipid to DNA that were charge neutral (.about.10), the size of
the complex reached a maximum. In addition, when the ratio of
liposome-to-DNA was kept constant at 10 nmoles/.mu.g, the size of
the complex increased as both DNA and liposome concentration
increased and eventually formed precipitates. However, when the
ratio of liposomes to DNA was increased, the size of the complex
was progressively reduced until the size of the complex became
constant (250-300 nm) when the ratio of liposome-to-DNA exceeded 25
nmoles lipid/.mu.g DNA. This result may be due to the fact that the
DNA was perhaps coated by excess liposomes and therefore
aggregation between the complexes did not occur.
[0089] Based on the data presented in FIG. 1, lipid/DNA complexes
were prepared using a liposome-to-DNA ratio of 50 nmoles/.mu.g by
slowly adding a DNA solution of 200 .mu.g/ml to an excess amount
(10 .mu.mols) of liposome. The size of the complex formed was about
250 nm. When the ratio of liposome-to-DNA was changed to 25
nmoles/.mu.g, the size of the complex increased to about 350 nm.
The complexes formed using either the 25 nmole/.mu.g or 50
nmole/.mu.g ratios appeared to be physically stable since no
precipitates formed during storage for four weeks at 4.degree.
C.
Example 2
Purification of DNA/Lipid Complexes
[0090] When DNA was mixed with liposomes at a ratio of 1 .mu.g/50
nmoles, an excess of free liposomal lipids was observed to co-exist
with the DNA/lipid complex. Since excess free lipids are toxic to
cells, an experiment was conducted to determine if free liposomal
lipids could be separated from the DNA/lipid complex by a density
gradient ultracentrifugation method. In brief, free liposomes (10
.mu.moles of DC-Chol/DOPE (2:3) in a volume of 2 ml); free DNA (50
.mu.g pRSVL in a volume of 2 ml) and DNA/lipid complex formed by
mixing 20 .mu.moles DC-Chol/DOPE (2:3) and 0.4 mg pRSVL plasmid DNA
(50 nmoles/.mu.g) were each centrifuged for 30 minutes at 4.degree.
C. at 100,000 g over a gradient consisting of 0.5 ml each of 5%,
7.5%, 10% and 15% sucrose (W/W). Fractions of 200 .mu.l were then
collected from the top to the bottom of the tube and assayed for
the distribution of DNA marker (.sup.32P, .box-solid.) and lipid
marker (.sup.3H, .largecircle.). FIGS. 2A and 2B show the results
of typical separations of free liposomal lipid, free DNA (FIG. 2A)
and DNA/lipid complex (FIG. 2B) on the sucrose gradient. The
results presented in FIG. 2B show that after centrifugation, the
complex formed a major band at the 10% sucrose layer. By
comparison, FIG. 2A shows that most of the radioactivity of the
free DNA or free liposomal lipids distributed at the top half of
the tube and did not enter the sucrose gradient. In addition,
although the peak of .sup.3H and the peak of .sup.32P in FIG. 2B
co-existed at fraction number 16, there was a significant amount of
the .sup.3H distributed in fractions 1 to 10 indicating that the
excess free liposomal lipids were well separated from the DNA/lipid
complex.
Example 3
Physical Stability of Purified Lipid/DNA and Lipid/PLL/DNA
Complexes
[0091] DNA/lipid complexes were formed by mixing 20 .mu.moles of
liposomes of various DC-Chol/DOPE compositions (see Table 1) with
0.4 mg pRSVL plasmid DNA at a ratio of 1 .mu.g DNA/50 nmoles lipid.
Lipid/PLL/DNA complexes were formed by mixing 4 .mu.moles of
liposomes of various DC-Chol/DOPE compositions with 1 mg PLL
(MW=3000) and 0.4 mg pRSVL plasmid DNA. Both complexes were then
purified from free lipids, free DNA and free PLL by sucrose
gradient centrifugation as described in the Methods section. The
peak fractions were collected and pooled. Pooled samples were then
assayed for diameter immediately after collection (0 days) or after
storage in 10% sucrose at 4.degree. C. for 120 days. Table 1 shows
the results of these assays.
TABLE-US-00001 TABLE 1 Physical stability of purified lipid/DNA and
lipid/PLL/DNA pre-complexes Liposome Purified composition Size (nm)
complex Ratio Recovery (DC-chol/ PLL Day Day of lipid/DNA of DNA
DOPE) (.mu.g/.mu.gDNA) 0 120 (nmoles/.mu.g) (% Total) 2:8 0 168 280
23.2 51 3:7 0 187 252 14.0 66 4:6 0 175 195 12.7 73 5:5 0 174 210
13.2 70 6:4 0 198 232 10.1 69 2:8 2.5 165 287 20.8 17 3:7 2.5 99
101 19.2 22 4:6 2.5 138 132 38.3 29 5:5 2.5 184 178 22.4 27
[0092] The data presented in Table 1 shows that purified lipid/DNA
and lipid/PLL/DNA complexes were small (under 200 nm) in size at
day 0 and that their size did not increase dramatically with
storage. Further, the ratios of DNA-to-lipid in the purified
complexes was between 10 to 23 nmoles lipid/.mu.g DNA depending on
the composition of the liposomes used and this ratio did not change
after storage for 120 days. A reciprocal relationship between the
concentration of DC-Chol in the liposomes and the amount of the
lipid in the complex was also observed indicating that liposomes
enriched with DC-Chol show stronger DNA binding or charge
neutralizing activity than liposomes less enriched with DC-Chol.
The far-right hand columns shows recovery of .sup.32P-labeled DNA
in the DNA/lipid and DNA/lipid/PLL complexes following their
purification on the sucrose density gradient. The results show that
recovery of DNA in the non-PLL containing complexes was higher than
that observed for the PLL-containing complexes.
Example 4
Biological Activity of the Purified Complexes in Various Cells
[0093] Since the DNA/lipid complexes formed by a mixture of
liposomes to DNA having a high lipid to DNA ratio were both small
and stable over time, experiments were conducted to compare the
transfection activity of these complexes to the activity of
DNA/liposome complex prepared by the admixture method.
[0094] In one experiment, CHO cells cultured in 48 well-plates were
treated for 4 hours with an admixture of either 1 .mu.g pRSVL and
10 nmoles DC-Chol/DOPE liposomes of different lipid compositions
alone (.largecircle.) or together with 1 .mu.g PLL (MW=3,000)
(.quadrature.) or, the cells were treated with purified DNA/lipid
complex ( ) or purified DNA/lipid/PLL complex (.box-solid.) formed
by mixing 1 .mu.g DNA with 50 nmoles DC-Chol/DOPE liposomes
(DNA/lipid complex) or with 10 nmoles DC-Chol/DOPE liposomes and 1
.mu.g PLL (DNA/lipid/PLL complex) followed by centrifugation
through a sucrose density gradient as described in the Methods
section. 36 hours after treatment, cells were lysed in 100 .mu.l
lysis buffer and 4 .mu.l of the lysate was assayed for luciferase
activity using 100 .mu.l of luciferase substrate solution.
Luciferase activity was then counted over a period of 20 seconds.
The results presented in FIG. 3 show that the most preferred
liposome composition for transfecting CHO cells was 40% DC-Chol and
60% DOPE. In addition, in the presence of additional 1 .mu.g
poly-L-lysine (PLL, MW=3,000), a 2-7 fold enhancement of the
transfection activity was seen in most cases. Of particular
interest, the activity of the purified DNA/lipid complex was
similar to that of the admixture DNA/lipid complex when the same
amount of DNA was added to cells. However, the transfection
activity of the purified DNA/lipid/PLL complex was about 30% to 50%
lower than that of the DNA/liposome/PLL complex prepared by the
admixture procedure.
[0095] In order to determine that the results obtained in CHO cells
were not cell-specific, the transfection activities of the purified
DNA/lipid and DNA/lipid/PLL complexes in two other cells, BHK and
mouse lung cells (MLC), were compared to that of DNA/liposome
complexes formed by admixture.
[0096] In brief, cells (either BHK or MLC) grown in 48-well plates
at 60% confluency were transfected with 1 .mu.g pRSVL complexed
with 10 nmoles of DC-Chol liposomes (admixture complex), with the
same amount of DNA mixed with liposomes at a DNA/liposome ratio of
1 .mu.g/50 nmols to produce purified DNA/lipid complex or with
purified DNA/lipid/PLL complex prepared at a DNA/liposome/PLL ratio
of 1 .mu.g/10 nmols/2 .mu.g. Cells were then harvested at 36 hours
post-transfection, and the luciferase activity of the transfected
cell lysates was determined as described in the Methods section.
The results of these experiments are shown in Tables 2 and 3.
TABLE-US-00002 TABLE 2 Expression of luciferase gene in BHK cells
transfected with pRSVL Luciferase Activity (Relative Light Units
.times. 10.sup.-3) Liposome Admixture Purified Purified composition
DNA/liposome DNA/lipid DNA/lipid/PLL (DC-Chol/DOPE) complex complex
complex 2:8 91.8 .+-. 9.5 110.1 .+-. 5.2 214.6 .+-. 41.1 3:7 61.2
.+-. 19.9 1886.8 .+-. 266.7 151.7 .+-. 62.9 4:6 438.2 .+-. 14.4
1638.8 .+-. 63.9 446.3 .+-. 16.9 5:5 837.8 .+-. 8 1015.0 .+-. 41.2
234.2 .+-. 46.4
TABLE-US-00003 TABLE 3 Expression of luciferase gene in mouse lung
cells transfected with pRSVL Luciferase Activity (Relative Light
Units .times. 10.sup.-3) Liposome Admixture Purified Purified
composition DNA/liposome DNA/lipid DNA/lipid/PLL (DC-Chol/DOPE)
complex complex complex 2:8 1.1 .+-. 0.7 0.4 .+-. 0.2 0.3 .+-. 0.1
3:7 1.5 .+-. 1.0 0.3 .+-. 0.0 4.1 .+-. 1.3 4:6 3.1 .+-. 0.2 2.0
.+-. 0.3 14.6 .+-. 3.1 5:5 0.1 .+-. 0.0 1.5 .+-. 1.2 10.1 .+-.
2.3
[0097] Interestingly, for the BHK cell line, the transfection
activity of the purified DNA/lipid complex was significantly higher
than that of the DNA/liposome complex formed by admixture. For
cells such as MLC, which are difficult to transfect, purified
complexes made from DNA/liposome/PLL mixtures were apparently
superior to admixture complexes and to purified DNA/lipid complexes
made without PLL.
[0098] In order to determine whether lipid/PLL/DNA complexes could
be made using different ratios of lipid and nucleic acid and a
different molecular weight PLL than that used in the previous
examples, the following experiment was conducted.
Lipid/poly-L-lysine/DNA complex was prepared from 20 .mu.g pRSVL
plasmid DNA, 10 .mu.g poly-L-lysine (MW 25,600), and DC-chol/DOPE
liposomes (4.5/5.5 molar ratio) at the ratios of lipid to DNA shown
in Table 4. The resulting complexes were then purified by sucrose
gradient ultracentrifugation as described in the methods section.
An aliquot of the purified complex containing 0.5 .mu.g of DNA was
used to transfect CHO cells, and luciferase activity was then
measured. The results of this experiment are shown below in Table
4.
TABLE-US-00004 TABLE 4 Effect of lipid/DNA ratio on purified
complex containing poly- L-lysine (MW 25,600) Ratio Composition of
(nmoles purified complex lipid/ (nmoles lipid/ Size of purified
Transfection activity.sup.b .mu.g DNA) .mu.g DNA) complex (nm)
(counts (SD) .times. 10.sup.-3) 3.3 1.1 89 108 (5) 6.6 2.5 98 6,065
(604) 12.5 4.3 101 5,846 (668) 20.0 9.6 35 7,633 (977)
[0099] The results show that in the presence of increased amounts
of polycation, lower ratios of lipid to DNA may be used to produce
DNA/lipid/polycation complexes having appreciable transfection
activity.
Example 5
Transfection Activity of Stored Complexes
[0100] CHO cells cultured in 48 well-plates were treated for 4
hours with admixture of 1 .mu.g pRSVL and 10 nmoles of DC-Chol/DOPE
liposomes of different DC-Chol/DOPE compositions alone
(.largecircle.) or together with 1 .mu.g PLL (mw=3,000)
(.quadrature.), or with purified DNA/lipid ( ) or DNA/lipid/PLL
(.box-solid.) complexes stored at 4.degree. C. for 130 days in 10%
sucrose. The purified complexes had been formed by mixing 1 .mu.g
pRSVL and 50 nmoles of DC-Chol/DOPE liposomes of different
DC-Chol/DOPE compositions alone (DNA/lipid) or with 10 nmole of
DC-Chol/DOPE liposomes and 1 .mu.g PLL (DNA/lipid/PLL complex)
followed by centrifugation through a sucrose density gradient as
described in the Methods section. The results presented in FIG. 4
show that the luciferase activity of cell lysates prepared from
cells transfected with the stored DNA/lipid and DNA/lipid/PLL
complexes was comparable with the luciferase activity observed in
cell lysates of cells transfected with the corresponding complexes
prepared by admixture.
Example 6
Comparative Cytotoxicity of DNA/Liposome Complexes Prepared by
Admixture to that of Purified DNA/Lipid Complexes
[0101] Cell toxicity of the different complexes was studied in CHO
cells as follows. CHO cells were treated with admixture
DNA/liposome complex (.largecircle.), admixture/liposome/PLL
complex (.quadrature.); purified DNA/lipid complex ( ); or purified
DNA/lipid/PLL complex (.box-solid.). The admixture complexes were
formed by mixing 1 .mu.g pRSVL DNA with 10 nmoles DC-Chol/DOPE
liposomes of different DC-Chol/DOPE compositions alone or together
with 1 .mu.g PLL (mw=3,000). The purified complexes were formed by
mixing 1 .mu.g pRSVL DNA with 50 nmoles DC-Chol/DOPE liposomes
alone (DNA/lipid complex) or with 10 nmoles DC-Chol/DOPE liposomes
and 1 .mu.g PLL (DNA/lipid/PLL complex) followed by centrifugation
through a sucrose density gradient as described in the Methods
section. 36 hours after treatment, the cells were lysed, protein
was extracted and then quantitated by a Coomassie blue dye
method.
[0102] FIG. 5 shows the results of this experiment where the amount
of total extractable protein recovered at the end of the experiment
serves as an indicator of the portion of the cells which survived
after the indicated treatment. The data presented shows that while
the purified complex appeared to be slightly more toxic to the
cells than the admixture DNA/liposome complex, morphologically, the
transfection did not cause any serious cytotoxic effects in cells
treated with either admixture complexes or the purified complexes,
except that the cells treated with purified complexes containing
high mole % DC-Chol were less confluent at the end of the
experiment.
Example 7
In Vivo Transfection of Tumors by Purified DNA/Lipid Complexes
[0103] 3.times.10.sup.6 human ovarian carcinoma cells were injected
subcutaneously into SCID mice at day 0.14 days later, 100 .mu.l
solutions containing pUCCMVCAT DNA (30 .mu.g) complexed with
DC-Chol (3:2 DC-Chol:DOPE) liposomes (30 nmoles) in the form of
admixture (lanes 1 and 2) or the same amount of DNA in the form of
purified DNA/lipid complex (prepared from DNA and DC-Chol liposomes
at ratios of 1 .mu.g DNA/25 nmoles lipid) were directly injected
into tumors. Animals were sacrificed 2 days later and tumor
extracts containing 100 .mu.g protein were assayed for CAT activity
at 37.degree. C. according to Ausubel, et al. (1991) Current
Protocols in Molecular Biology (Wiley, Boston), Vol. 1, pp.
9.6.2-9.6.5). The results show that purified complex, while
prepared under non-optimal conditions, exhibited in vivo
transfection activity.
Example 8
Comparative Transfection Activity of Purified and Unpurified
DNA/Lipid/PLL Complexes with Admixture DNA/Lipid Complex
[0104] The transfection activities of purified and unpurified
DNA/Lipid/PLL complexes and admixture DNA/lipid complex in three
cell lines (293, BL6 and C3) were measured as follows:
[0105] Purified DNA/lipid/PLL complex was formed by mixing 1 .mu.g
pRSVL DNA with 10 nmoles DC-Chol/DOPE liposomes (2:3 mol/mol) and 1
.mu.g PLL (MW=25,600) followed by purification via centrifugation
through a sucrose density gradient as described in the Methods
section.
[0106] Unpurified DNA/lipid/PLL complexes were formed by mixing 100
.mu.g pRSVL DNA with 80 .mu.g PLL (MW=25,600) and 1702 nmol
DC-Chol/DOPE (2:3 mol/mol) liposomes (i.e. a DNA/lipid/PLK ratio of
1 .mu.g DNA/17 nmol lipid/0.8 .mu.g PLL) in a final volume of 500
.mu.l of water. 20 .mu.l of the unpurified DNA/lipid/PLL complex
(i.e. 4 .mu.g DNA, 3.2 .mu.g PLL and 68.1 nmol lipid) was then
added to 780 .mu.l of serum free medium appropriate for the cell
line to be transfected.
[0107] Admixture DNA/lipid complex was formed by mixing 1 .mu.g
pRSVL DNA with 10 nmols of DC-Chol/DOPE (3:2 mol/mol)
liposomes.
[0108] 293, C3 and BL6 cells grown to 80% confluence in 24-well
plates were transfected for four hours at 37.degree. C. with 1
.mu.g of DNA in the form of purified DNA/lipid/PLL complex,
admixture DNA/lipid complex or unpurified DNA/lipid/PLL complex.
After transfection, cells were further cultured for 36-48 hours in
the appropriate media containing 10% fetal calf serum. Luciferase
activity was then measured as described in the Methods section.
[0109] The results presented in FIGS. 7A (293 cells) 7B (C3 cells)
and 7C (BL6 cells) demonstrate that the unpurified DNA/lipid/PLL
complex exhibited the highest transfection activity in all three
cell lines tested.
Materials and Methods for Examples 9-14 and in FIGS. 8-19
Plasmid DNA
[0110] The plasmid DNA used for all experiments consisted of a
luciferase (LUC) reporter gene driven by the human cytomegalovirus
(CMV) immediate early promoter and cloned into a pUK21 backbone.
Plasmid DNA was prepared by growing bacterial stocks in a glycerol
enriched Terrific Broth (TB) media. The bacteria was subject to
detergent and alkaline lysis, propanol precipitation and treatment
by high salt to remove high molecular weight RNA. The resulting
supernatant was subjected to size exclusion chromatography to
separate low molecular RNA from plasmid DNA. The DNA was shown to
be absent of chromosomal DNA or RNA by gel electrophoresis and had
an A.sub.260/280 ratio between 1.80-1.90 as determined
spectrophotometrically.
Liposomes
[0111] DC-Chol was synthesized as previously described (Gao and
Huang, (1991) Biochem. Biophys. Res. Commun., 179:280-285)). DOPE
was purchased from Avanti Polar Lipids, Inc. DC-Chol liposomes were
produced by microfluidization at a 6:4 molar ratio (DC-Chol to
DOPE) and to a concentration of 2 .mu.mol/ml (1.2 mg/ml of total
lipid) as previously described in Int. J. Pharm. 14:34:30 1996.
Polycations
[0112] Poly-L-Lysine (MW 18,000-19,200), Protamine Free Base (Grade
IV), Protamine Phosphate (Grade X), Protamine Chloride (Grade V),
Protamine Sulfate (Grade II), Protamine Sulfate (Grade III), and
Protamine Sulfate (Grade X) were all obtained from Sigma Chemical
Co. Protamine Sulfate, USP was obtained from Elkins-Sinn, Fujisawa
or from Eli Lilly and Company. All of the protamines were from
salmon sperm except the grade III which was from herring.
Transfection of Mammalian Cells In Vitro
[0113] CHO cells were seeded into a 48-well plate and grown in F-12
media supplemented with 10% Fetal Bovine Serum (FBS) in a 37/C
incubator in a 5% CO.sub.2 atmosphere. The cells were exposed to
DNA complexed to DC-Chol liposomes with or without a polycation
such as protamine or poly-lysine and allowed to transfect for 5.5-6
hours after which the transfection media (Hanks buffer) was
replaced with fresh F-12 media supplemented with 10% Fetal Bovine
Serum (FBS). Typically, 8 .mu.g of Protamine (or 4 .mu.g of
poly-lysine) were mixed with 4 .mu.g of DNA in a 1 ml volume of
Hanks buffer in a 4 ml tube for approximately 5-15 run at room
temperature. A second 4 ml tube containing 30 nmol of DC-Chol
liposomes in 1 ml of Hanks buffer was added to the 1 ml sample of
DNA/polycation and allowed to complex for 5-15 minutes at room
temperature. After mixing, 0.5 ml aliquots (containing 1 .mu.g DNA,
2 .mu.g protamine or 1 .mu.g poly-1-lysine, and 7.5 nmol of DC-Chol
liposomes) were added to each well of the 48-well plate. The cells
were allowed to incubate for a total of 35.5-38.5 hrs prior to
being assayed for luciferase activity. Each point shown in FIGS. 8
and 9 represents the mean (with standard deviation) luciferase
activity of three to four data points and are normalized to protein
context.
[0114] In vitro transfection assays for HeLa cells (available from
ATCC) and the SKOV-3 human ovarian carcinoma cell line (available
from ATCC) were run under the same conditions as described above
for CHO cells.
Luciferase Assay
[0115] CHO cells were allowed to transfect for 4-8 hours in
transfection media and then allowed to incubate in F12+10% FBS for
an additional 32-44 hours. The media was then aspirated and the
cells were washed once with a 0.9% sodium chloride solution. The
cells were lysed with 100 .mu.l of lysis buffer (2 mM EDTA, 100 mM
Tris, 0.05% Triton X-100) per well and subjected to one cycle of
freeze-thaw. The cell lysates were collected, briefly centrifuged
to pellet the cellular debris, and 10 .mu.l was used for the
luciferase assay. The samples were loaded into an AutoLumat LB953
(Berthold) Luminometer, 100 .mu.l of the luciferase substrate
(Promega) was added to the cell lysate, and the relative light
units (RLU) of each sample were counted for 20 seconds. Samples
were also assayed by Coomassie Blue Plus Protein Reagent (Pierce)
and normalized to the protein content in each sample.
Example 9
Comparison of the Ability of Protamine Sulfate USP (Elkins-Sinn)
and Poly-L-Lysine to Increase Transfection Activity in CHO
Cells
[0116] Protamines are naturally occurring cationic proteins which
are characterized as being small (mw=4,000), extremely basic, and
containing a large amount of arginine (Ando et al., (1973) in
Protamines--Isolation, Characterization, Structure and Function
(Springer-Verlay, New York) pp. 3-87)). Protamines are only found
in the head of mature sperm and their sole function is to condense
DNA such that it can be efficiently packaged and ultimately
expressed within the nucleus of an egg. Protamines are typically
purified from fish (salmon or herring) sperm. These proteins are
approximately 30-35 amino acids in length and about two thirds
(2/3) of these residues are arginine. The arginine residues are
found in clusters of 4-6 in four distinct regions. About one fourth
of the remaining residues are hydrophobic, resulting in evenly
spaced hydrophobic and hydrophilic regions. The result of this
unique arrangement of amino acids is that the protamine molecule is
forced to assume an .alpha.-helical structure in the presence of
nucleic acids. The spacing of the amino acids in protamine
parallels the spacing of the bases in DNA. As a result, the
protamine .alpha.-helical structure aligns along the major groove
of DNA and associates strongly with the DNA without changing its
conformational state. It can also cross-link with adjacent DNA
strands producing a highly ordered, condensed DNA/protamine complex
(Warrant and Kim, (1978) supra). While poly-lysine and
poly-arginine have also been shown to condense DNA, they lack the
fine structural characteristics found in protamine. As a result,
the condensation by poly-lysine or poly-arginine results in a
complex which is structurally different. This results in a change
in the conformational and crystalline state of DNA from the
biologically active B-form (as found in protamine) to an inactive
crystalline A-form (Subirana, (1983) in the Sperm Cell ed. Andre,
J. (Martainui, Nizeroff, the Hague), pp. 197-213)).
[0117] Accordingly, an experiment was conducted in which increasing
amounts of salmon sperm protamine sulfate, USP or poly-L-Lysine
were added to 1 .mu.g of DNA and allowed to associate prior to the
addition of 7.5 nmol of DC-Chol liposomes.
[0118] As shown in FIGS. 8A and 8B, increasing amounts of
poly-L-Lysine resulted in an increase in transfection activity
reaching a constant level of activity at 1 .mu.g of poly-L-lysine
per 1 .mu.g of DNA.
[0119] Increasing amounts of Protamine Sulfate, USP also resulted
in an increase in expression, reaching a constant level of
expression at 2 .mu.g of Protamine Sulfate, USP per 1 .mu.g of DNA
(see FIGS. 8A and 8B). However, in sharp contrast to the previously
published report of Gao and Huang ((1996) Biochem., 35:1027-1036),
in which transfection activity with protamine free base was
consistently lower than that observed with poly-L-Lysine, the level
of expression achieved with Protamine Sulfate, USP/DNA/liposome
complexes was unexpectedly 4-5 fold higher than the levels seen
with poly-L-lysine and 40-fold higher than that observed for
DNA/liposome complexes without polycation.
Example 10
Effect of Different Types of Protamine on Transfection Activity
[0120] In order to further explore this unexpected difference in
the ability of protamine free base and protamine sulfate USP to
potentate transfection activity, the ability of various types of
protamine to potentiate gene expression in CHO cells was compared
to standard DNA/liposome complexes as well as complexes
supplemented with poly-L-Lysine.
[0121] As shown in FIG. 9, the potentiation of gene expression by
different types of protamine varied considerably with protamine
phosphate and free base showing minimal if any increase in gene
expression over that seen in the absence of any polycation while
protamine sulfates showed a 3-5 fold increase in gene expression
over poly-L-lysine and increased gene expression by 60-fold over
background (DNA/lipid complex without a polycation (poly-L-lysine
or protamine sulfate)). In addition, protamine chloride also showed
an increase in gene expression over poly-L-lysine, albeit not as
great an increase as that observed for protamine sulfates
Example 11
Amino Acid Analysis of Various Types of Protamine
[0122] In an effort to explain the large variation in transfection
activity of the various protamines described above, the relative
percentages of amino acid residues (i.e., amino acid composition)
present in protamine phosphate, chloride, free base, sulfate (grade
III), and sulfate USP (Lilly and Elkins-Sinn) were determined for
each compound. The results of this analysis are presented in Table
5. Predicted values are calculated from a published sequence of
salmon sperm (Warrant and Kim, (1978) Nature, 271:130-135).
TABLE-US-00005 TABLE 5 Amino Acid Composition of Various Salts of
Protamine (expressed as percentage of composition) Sulfate Sulfate,
USP Sulfate, USP Amino Acid Free Base Phosphate Chloride (Grade
III) (Lilly) (Elkins-Sinn) Predicted * Aspartate 0.23 0.55 0 0.12
0.50 0.03 0 Threonine 0.10 0.35 0 0 0 0 0 Serine 5.95 6.60 8.91
8.46 8.95 8.23 12.5 Glutamate 0.07 0.01 0 0 0.09 0 0 Glycine 6.69
7.07 7.04 6.56 7.30 6.36 6.25 Alanine 1.44 1.76 1.49 0.94 1.73 1.32
0 Valine 4.69 4.57 4.61 4.93 4.43 4.31 6.25 Methionine 0.56 0.77
0.70 0.64 0.75 0.64 0 Isoleucine 1.26 1.23 1.33 0.81 1.31 1.20 0
Leucine 0.17 0.20 0 0 0.22 0 0 Histidine 0.10 0.11 0 0 0 0 0 Lysine
8.14 8.84 1.49 0.47 0.21 0.23 0 Arginine 61.82 59.61 70.37 68.25
65.90 69.27 65.63 Proline 8.79 8.35 8.60 8.81 8.27 8.42 9.38
[0123] As can be seen, the amino acid composition of protamine free
base is very similar to that of the protamine phosphate. Strong
similarity in amino acid composition is also observed among the
three different protamine sulfates and protamine chloride. Of
particular interest are the differences in amino acid composition
between the protamine sulfates and protamine chloride and the
protamine free base and phosphate. The most striking difference is
a 6-32 fold increase in lysine content in the phosphate and free
base form of protamine relative to the sulfates and the chloride.
An additional difference is a 1.5 fold increase in the amount of
serine in the chloride and the sulfates relative to the phosphate
and free base. Also observed is a decrease in the percentage of
arginine present in the free base and phosphate in comparison to
the sulfates and chloride. Of particular interest, the observed
differences in lysine content correlate with the variations in
activity observed in FIG. 9.
[0124] Of clinical interest, protamines are naturally occurring
compounds which elicit rare, if any immune responses in the host
and have been used clinically for several decades in insulin
delivery systems whereas all current delivery systems utilize
synthetic polymers having unknown safety profiles in humans.
Example 12
In Vivo Transfection by DNA/Protamine Sulfate/Lipid Complexes
[0125] Use of PS/DNA/liposome formulations in vivo has been
demonstrated by administering various formulations intravenously to
mice and assaying for reporter gene based on luciferase activity. A
formulation containing PS/DNA/DOTAP (0.8 .mu.g/1 .mu.g/23.27 .mu.g)
in 5% dextrose has been shown to have a greater transfection
activity than either PS/DNA (0.8 .mu.g/1 .mu.g) (data not shown) or
complexes produced in the absence of PS (23.27 .mu.g DOTA/.mu.g
DNA) (FIG. 10). Gene expression was highest in lung tissue compared
to kidney, spleen, liver, and heart. In vivo luciferase activity
was positively correlated with increasing concentrations of DOTAP
and was maximal at a level of 11:1 (DOTAP/DNA mol/mol) (FIG. 11).
Increasing the concentration of DNA also resulted in increased gene
expression but was associated with some toxicity above a dose of 75
.mu.g of DNA and the death of one mouse at a dose of 100 .mu.g of
DNA (FIG. 12). In vivo gene expression appeared to be maximal at 6
hours following injection and subsequently declined thereafter
(FIG. 13).
Example 13
In Vitro Optimization of Amounts of Protamine Sulfate, DNA, and
DC-Chol Liposomes for Maximal Transfection
[0126] Optimization of the protamine sulfate/DNA/DC-Chol liposome
complexes were examined in order to establish parameters for the
complex formulation. As shown in FIGS. 8A and 8B, maximal activity
was seen when 2 .mu.g of protamine sulfate was complexed with 1
.mu.g of DNA and 7.5 nmol of DC-Chol liposomes. In order to
establish the optimal amount of DNA/Protamine sulfate which can be
delivered by a fixed amount of DC-Chol liposomes, increasing
amounts of DNA/PS (at a 1:2 w/w ratio) were complexed with 7.5 nmol
of DC-Chol liposomes. As can be seen in FIG. 14, optimal
transfection activity was achieved when 2 .mu.g of DNA and 4 .mu.g
of PS were complexed with 7.5 nmol of DC-Chol liposomes.
[0127] Having established that 2 .mu.g of PS is required to
optimally deliver 1 .mu.g of DNA (FIGS. 8A and 8B), the amount of
DC-Chol liposomes required to efficiently deliver this quantity of
DNA/PS was determined. As shown in FIG. 15, using 1 .mu.g of DNA
complexed with 2 .mu.g of PS, efficient delivery was established
with as little as 2.5 nmol of DC-Chol liposomes. Prior experiments
were performed at this DNA/PS ratio by using 7.5 nmol of DC-Chol
liposomes. Since it is thought that cellular toxicity is a function
of liposome concentration, it is preferential to use as minimal a
dose of lipids as needed to deliver a specified quantity of genetic
material. FIG. 16 suggests that as little as 2.5 nmol of DC-Chol
liposomes is required to deliver a complex consisting of 1 .mu.g of
DNA and 2 .mu.g of PS. The results of FIG. 15 suggest that
quantities of 2 .mu.g of DNA and 4 .mu.g of PS can be delivered
efficiently. This finding suggests that 2 .mu.g of DNA complexed
with 4 .mu.g of PS would best be delivered by 5 nmol of DC-Chol
liposomes.
Example 14
The Ability of Protamine Sulfate to Increase the Transfection
Activity of Several Different Cationic Liposome Formulations In
Vitro
[0128] Having established the ability of PS to augment the
transfection activity of DC-Chol liposomes (FIGS. 8A and 8B),
several other commercially available liposome formulations were
also tested. FIG. 16 shows the ability of Clonfectin (Clontech) to
transfect CHO cells with 1 .mu.g of pUK 21 CMV LUC (.quadrature.)
and with a complex consisting of 2 .mu.g of PS and 1 .mu.g of DNA
(.box-solid.). The addition of PS results in a 5-20 fold increase
in luciferase activity over the concentrations tested. FIG. 17
shows the ability of DC-Chol liposomes to transfect CHO cells with
1 .mu.g of DNA (.quadrature.) and with a complex consisting of 2
.mu.g of PS and 1 .mu.g of DNA (.box-solid.). The addition of PS
results in a 10-85 fold increase in luciferase activity over the
concentrations tested. FIG. 18 shows the ability of Lipofectin
(Gibco BRL) to transfect CHO cells with 1 .mu.g of pUK 21 CMV LUC
(.quadrature.) and a complex consisting of 2 .mu.g of PS and 1
.mu.g of DNA (.box-solid.). The addition of PS results in a 15-105
fold increase in luciferase activity over the concentrations
tested. FIG. 19 shows the ability of DOTAP/DOPE (1:1 mol/mol)
liposomes to transfect CHO cells with 1 .mu.g of pUK 21 CMV LUC
(.quadrature.) and with complex consisting of 2 .mu.g of PS and 1
.mu.g of DNA (.box-solid.). The addition of PS results in a 10-220
fold increase in luciferase activity over the concentrations
tested.
[0129] While we have hereinbefore described a number of embodiments
of this invention, it is apparent that the basic constructions can
be altered to provide other embodiments which utilize the methods
and devices of this invention. Therefore, it will be appreciated
that the scope of this invention is defined by the claims appended
hereto rather than by the specific embodiments which have been
presented hereinbefore by way of example.
Example 15
Relationship Between Lipid:Protamine Sulfate:DNA Ratios and Average
Particle Size
[0130] A number of lipid:protamine sulfate:DNA formulations were
made and analyzed to establish the relationship between
Lipid:Protamine Sulfate:DNA ratios and particle size. Complexes
were formed between PS and pCMV-Luc plasmid DNA in incremental
ratios varying from 0:0 to 4:0. Aliquots of DC-Chol HCl/DOPE
(Aronex) suspended in 10% dextrose were added to the PS/DNA complex
to produce Lipid:DNA ratios from 1:1 to 10:1. The particle sizes of
the resulting LPD complexes were taken at room temperature on a
Malvern Zetasizer when the complexes were first formed (Day 0) and
again after storage at 4.degree. C. for 7 days. The data generated
from this study are shown in FIG. 20.
[0131] The data from FIG. 20 indicate a correlation between
Lipid:Protamine Sulfate:DNA ratios and particle size.
[0132] Particle sizes around 200 nm are preferable for coated pit
internalization.
Example 16
In Vitro Transfection Efficiency of Different LPD Formulations on
HeLa and SKOV-3 Cells
[0133] HeLa and SKOV-3 cells were transfected with different LPD
formulations according to the protocol presented in the Materials
and Methods section. Transfection efficiency was measured using the
Luciferase Assay 48 hrs post transfection.
[0134] As depicted in FIG. 21, the addition of 2 .mu.g Protamine
Sulfate to a 1:1 lipid-DNA complex increases luciferase activity in
HeLa cells by 500 fold. The addition of Protamine Sulfate to a 10:1
lipid-DNA complex to produce a 10:2:1 LPD formulation increases
luciferase by 50 fold. No improvement in transfection activity is
observed beyond a ratio of 2:1 Protamine:DNA.
[0135] FIG. 22 presents a comparative study of the transfection
efficiency of SKOV-3 cells using LPD and DC-Chol delivery vehicles.
The n value represents the number of individual experiments which
were averaged for this data table. LPD formulations produced
consistently higher luciferase activity than did the DC-Chol
formulations, with the 15:2:1 LPD complex proving 8-10 fold more
efficient that the best LD complex.
Example 17
Transfection of Mammalian Cells In Vivo
[0136] Nude mice between 6 and 12 weeks of age were inoculated
intraperitoneally with 2.times.10.sup.6 SKOV-3 human ovarian
carcinoma cells in a total injection volume of 0.5 mls of PBS.
After 6-7 weeks of tumor cell engraftment animals were injected
with different formulations of pCMV-luc plasmid DNA and DNA/Lipid
in a total volume of 1.0 ml (5% Dextrose final, isotonic solution).
Animals were sacrificed 16 hours post formulation injection. Tumor
nodules were removed and lysed. Protein concentrations were
determined according to the Luciferase Assay described above.
[0137] FIG. 23 shows data comparing the in vivo transfection
efficiency of vehicle plus naked DNA to following LPD formulations:
0:0:1, 0:2:1, 3:0:1, 3:2:1, 10:0:1, 10:2:1, 15:0:1 and 15:2:1. The
RLU/mg values obtained for each individual mouse are denoted by
circles, while the average RLU/mg values are shown as squares. Data
show that the 15:2:1 LPD complex produced the highest average
expression level of the formulations tested, yielding a 3-4 fold
higher expression value.
Example 18
LPD E1A Survivability Study
[0138] Nude mice were injected intraperitoneally with
2.times.10.sup.6 SKOV-3ip1 cells (a human ovarian carcinoma cell
line selected as a subline from SKOV-3 due to its more rapid growth
and higher HER2/neu expression). At five days post implantation, a
treatment regimen involving delivery of the E1A expression plasmid
comprising the Ad5 E1A gene with its native promoter (see, e.g.,
Mien-Chie Hung et al., U.S. Pat. No. 5,651,964). Animals were
injected intraperitoneally on days 5, 6, and 7 with 1 ml of a 5%
Dextrose vehicle comprising the formulations presented in FIG. 24:
Group 1 animals were repeatedly injected with the 5% Dextrose
vehicle only; Group 2 animals were repeatedly injected with the
equivalent amount of DC-Chol/DOPE (6:4 ratio) and Protamine sulfate
as one would find in the high dose 15:2:1 group but without the E1A
expression plasmid present; Group 3 animals were repeatedly
injected with 15 .mu.g/dose of naked E1A expression plasmid, which
was equivalent to the highest dose injected in formulated vehicles;
Group 4 animals were repeatedly injected with 15 .mu.g/dose of E1A
expression plasmid compacted with Protamine sulfate at a ratio of 2
.mu.g Protamine/1 .mu.g DNA, which was equivalent to the highest
dose injected in the formulated vehicles; Group 14 animals were
repeatedly injected with a 1.5 .mu.g/dose 15:2:1 LPD formulation;
Group 15 animals were repeatedly injected with a 5 .mu.g/dose
15:2:1 LPD formulation; Group 16 animals were repeatedly injected
with a 15 .mu.g/dose LPD formulation. Animals were subsequently
injected once a week with the same formulation for the time listed
on the X axis. Animals either survived, died due to tumor burden,
or were sacrificed prior to death if death was imminent.
[0139] FIG. 24 shows that DNA must be formulated in a lipid vehicle
to work properly upon intraperitoneal injection (Groups 3 and 4).
The data further demonstrate a positive correlation between the
dose of E1A given as a lipid formulation and the mouse survival
rate (Groups 14, 15 and 16).
* * * * *