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United States Patent |
6,114,169
|
Bridenbaugh
,   et al.
|
September 5, 2000
|
Methods for preparing polynucleotide transfection complexes
Abstract
Methods are provided for the preparation of transfection complexes of
polynucleotides and polycations, especially cationic lipids, suitable for
delivering polynucleotides to cells. In particular, methods are provided
for preparing transfection complexes using a reduced-volume, dual-feed
process. Complexes are formed upon the collision of two feed stream,
containing polynucleotides and polycation, respectively, under conditions
resulting in turbulent mixing conditions in minimal volume, and removal of
transfection complexes under laminar flow conditions. Alternatively, the
components are mixed in a static mixer. The process is easily scaleable
and highly reproducible.
Inventors:
|
Bridenbaugh; Robert (Millbrae, CA);
Dang; Warren (Alameda, CA);
Koe; Gary (San Mateo, CA)
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Assignee:
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Valentis, Inc. (Burlingame, CA)
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Appl. No.:
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178371 |
Filed:
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October 23, 1998 |
Intern'l Class: |
C12N 015/64 |
Field of Search: |
435/455,468
|
References Cited
U.S. Patent Documents
4450103 | May., 1984 | Konrad et al. | 530/351.
|
4462940 | Jul., 1984 | Hanisch et al. | 530/351.
|
4623723 | Nov., 1986 | Keller et al. | 536/25.
|
4900677 | Feb., 1990 | Hewitt | 435/259.
|
4997932 | Mar., 1991 | Reardon et al. | 536/25.
|
5096818 | Mar., 1992 | DeBonville | 435/270.
|
5208160 | May., 1993 | Kikyotani et al. | 435/270.
|
Foreign Patent Documents |
WO 97/23601 | Jul., 1997 | WO.
| |
Other References
Theodossiou et al., "The processing of a plasmid-based gene from E. Coli.
Primary recovery by filtration", Bioprocess Engineering, 16:173-183
(1997).
Marquet et al., "Characterization of Plasmid DNA Vectors for Use in Human
Gene Therapy, Part 1", BioPharm, 42-50 (1997).
Papamichael et al., "Aqueous Phase Extraction of Proteins: Automated
Processing and Recycling of Process Chemicals", J. Chem. Tech Biotechnol
54:47-55 (1992).
Veide et al., "Continuous extraction of .beta.-D-galactosidase from
Escherichia coli in an aqueous two-phase system: effects of biomass
concentration on partitioning and mass transfer", Enzyme Microb. Technol.,
6:325-330 (1984).
|
Primary Examiner: Brusca; John S.
Attorney, Agent or Firm: Townsend and Townsend and Crew LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Ser. No. 60/063,126 filed Oct.
24, 1997, and U.S. Ser. No. 60/094,437, filed Jul. 28, 1998, both of which
are incorporated herein by reference.
Claims
We claim:
1. A method of preparing a polynucleotide transfection complex, said method
comprising:
providing a first feed stream comprising a polynucleotide in solution and a
second feed stream comprising a polycation in solution;
mixing the first and second feed stream using a static mixer whereby
polynucleotide transfection complexes are formed in solution; and
removing the solution of polynucleotide transfection complexes.
2. The method according to claim 1 wherein the polynucleotide is DNA.
3. The method according to claim 1 wherein the polycation is selected from
the group consisting of cationic lipid, polylysine, polyarginine, and
polyhistidine.
4. The method according to claim 3 wherein the polycation comprises a
cationic lipid.
5. The method according to claim 4 wherein the polycation further comprises
a neutral lipid.
6. A method of preparing a polynucleotide transfection complex comprising:
providing a first feed stream comprising polynucleotides in solution and a
second feed stream comprising cationic liposomes in solution;
mixing the first and second feed stream using a static mixer whereby
polynucleotide transfection complexes are formed in solution; and
removing the solution of polynucleotide transfection complexes.
7. The method according to claim 6 wherein the mixing of the first and
second feed streams occurs in a static mixer.
8. The method according to claim 6 wherein the polynucleotide is DNA.
Description
FIELD OF THE INVENTION
This invention relates to preparation of polynucleotide transfection
complexes and their use in delivering polynucleotides to cells. In
particular, the invention relates to methods for preparing complexes of
polynucleotides and polycations suitable for transfecting eukaryotic cells
in vivo and in vitro.
BACKGROUND OF THE INVENTION
A number of methods exist for introducing exogenous genetic material to
cells, which methods have been used for a wide variety of applications
including, for example, research uses to study gene function, and ex vivo
or in vivo genetic modification for therapeutic purposes. Ex vivo genetic
modification involves the removal of specific cells from an animal,
including humans, introduction of the exogenous genetic material, and then
re-introduction of the genetically modified cells into the animal. By
contrast, in vivo genetic modification involves the introduction of
genetic material directly to the animal, including humans, using an
appropriate delivery vehicle, where it is taken up by the target cells.
Generally, the various methods used to introduce nucleic acids into cells
have as a goal the efficient uptake and expression of foreign genes. In
particular, the delivery of exogenous nucleic acids in humans and/or
various commercially important animals will ultimately permit the
prevention, amelioration and cure of many important diseases and the
development of animals with commercially important characteristics. The
exogenous genetic material, either DNA or RNA, may provide a functional
gene which, when expressed, produces a protein lacking in the cell or
produced in insufficient amounts, or may provide an antisense DNA or RNA
or ribozyme to interfere with a cellular function in, e.g., a
virus-infected cell or a cancer cell, thereby providing an effective
therapeutic for a disease state.
Engineered viruses are commonly used to deliver genes to cells. Viral
vectors are generally efficient in gene delivery but have certain
drawbacks, for example stimulation of an immune response when delivered in
vivo. As a result, therefore, a number of non-viral nucleic acid delivery
systems have been and continue to be developed. Thus, for example,
cationic lipids are commonly used for mediating nucleic acid delivery to
cells. See, for example, U.S. Pat. No. 5,264,618, which describes
techniques for using lipid carriers, including the preparation of
liposomes and pharmaceutical compositions and the use of such compositions
in clinical situations. Other non-viral gene delivery systems likewise
involve positively-charged carrier molecules, for example, peptides such
as poly-L-lysine, polyhistidine, polyarginine, or synthetic polymers such
as polyethylimine and polyvinylpyrrolidone.
Nucleic acids are generally large polyanionic molecules which, therefore,
bind cationic lipids and other positively-charged carriers through charge
interactions. It is believed that the positively charged carriers (or
polycations), form tight complexes with the nucleic acid, thereby
condensing it and protecting it from nuclease degradation. In addition,
polycationic carriers may act to mediate transfection by improving
association with negatively-charged cellular membranes by giving the
complexes a positive charge, and/or enhancing transport from the cytoplasm
to the nucleus where DNA may be transcribed.
For cationic lipid-mediated delivery, the cationic lipids typically are
mixed with a non-cationic lipid, usually a neutral lipid, and allowed to
form stable liposomes, which liposomes are then mixed with the nucleic
acid to be delivered. The liposomes may be large unilamellar vesicles
(LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles
(SUVs). The liposomes are mixed with nucleic acid in solution, at
concentrations and ratios optimized for the target cells to be
transfected, to form cationic lipid-nucleic acid transfection complexes.
Alterations in the lipid formulation allow preferential delivery of
nucleic acids to particular tissues in vivo. See PCT patent application
numbers WO 96/40962 and WO 96/40963.
With respect to any of the polycationic nucleic acid carriers, transfection
efficiency is highly dependent on the characteristics of the
polycation/nucleic acid complex. The nature of the complex that yields
optimal transfection efficiency depends upon the mode of delivery, e.g. ex
vivo or in vivo; for in vivo delivery, the route of administration, e.g.,
intravenous, intramuscular, intraperitoneal, inhalation, etc.; the target
cell type, etc. Depending on the use, therefore, different carriers will
be preferred. In addition to the choice of polycationic carrier,
transfection efficiency will depend on certain physical characteristics of
the complexes as well, such as charge and size. These characteristics
depend largely on the method by which the complexes are prepared.
Particularly for human therapeutic purposes, therefore, it is desirable to
have a method of forming the nucleic acid/polycationic carrier complexes
in a highly controllable manner. Further, it is desirable to have a
process for preparing the complexes which is highly reproducible and
scaleable.
The present invention provides these and related advantages as well.
Relevant Literature
Cationic lipid carriers have been shown to mediate intracellular delivery
of plasmid DNA (Felgner et al., (1987) Proc. Natl. Acad. Sci. (USA),
84:7413-7416); mRNA (Malone et al., (1989) Proc. Natl. Acad. Sci. (USA)
86:6077-6081); and purified transcription factors (Debs et al., (1990) J.
Biol. Chem. 265: 10189-10192), in functional form. Literature describing
the use of lipids as carriers for DNA include the following: Zhu et al.,
(1993) Science, 261:209-211; Vigneron et al., (1996) Proc. Natl. Acad.
Sci. USA, 93:9682-9686; Hofland et al., (1996) Proc. Natl. Acad. Sci. USA,
93:7305-7309; Alton et al., (1993) Nat. Genet. 5:135-142; von der Leyen et
al., (1995) Proc. Natl. Acad. Sci. (USA), 92:1137-1141; See also Stribling
et al., (1992) Proc. Natl. Acad. Sci (USA) 89:11277-11281, which reports
the use of lipids as carriers for aerosol gene delivery to the lungs of
mice. For a review of liposomes in gene therapy, see Lasic and Templeton,
(1996) Adv. Drug Deliv. Rev. 20:221-266.
The role of helper lipids in cationic lipid-mediated gene delivery is
described in Felgner et al., (1994) J. Biol. Chem. 269(4): 2550-2561
(describing improved transfection using DOPE); and Hui et al., (1996)
Biophys. J. 71: 590-599. The effect of cholesterol on liposomes in vivo is
described in Semple et al., (1996) Biochem. 35(8): 2521-2525.
The use of cationic peptides and proteins in DNA delivery is described in
Emi et al., (1997) Biochem Biophys Res. Comm. 231(2):421-424
(polyarginine); Fritz et al., (1996) Hum. Gene Ther. 7(12):13951404
(histone H1 and SV40 large T antigen nuclear localizing signal); Gao and
Huang (1996) Biochemistry 35(3) 1027-1036 (poly-L-lysine, protamine);
Legendre and Szoka (1993) Proc. Natl. Acad. Sci USA 90(3):893-897
(gramicidin S); and Niidome et al., (1997) J. Biol. Chem.
272(24):15307-15312 (cationic alpha-helical oligopeptides). Additional
transfection facilitating agents are described in Ibanez, et al., (1996)
Biochem Cell Biol 74(5):633-643 (spermidine); Budker et al., (1997)
Biotechniques 23(1):139 (histone H1 and amphipathic polyamines); and
Barthel et al., (1993) DNA Cell Biol. 12(6):553-560 (lipospermine).
A method of preparing cationic lipid/nucleic acid transfection complexes by
first forming lipid micelles in the presence of detergent is described in
WO 96/37194. Methods of preparing DNA-lipid complexes using polyethylene
glycol-phospholipid conjugates and polyamines are described in Hong et
al., (1997) 400(2):233-237.
SUMMARY OF THE INVENTION
The invention provides a method of preparing a polynucleotide transfection
complex, the method comprising providing a feed stream containing a
polynucleotide in solution and a second feed stream containing a
polycationic carrier in solution, mixing the two feed streams by flowing
the mixture through a static mixer. Preferably, the two feed streams
converge at a junction and flow through a static mixer located at a
minimal distance from the junction and thereby produce polynucleotide
transfection complexes.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing the dual feed stream method of polynucleotide
transfection complex preparation.
FIG. 2 is a plot of particle size against DNA:cationic lipid ratio.
Addition of DNA and liposome to the point of precipitation was performed
at increasing concentrations of both components. The target DNA:cationic
lipid ratio (Img:6(mole) remained constant for each plot. DNA
concentrations are presented as the target concentrations for this ratio.
The addition of liposome to DNA, and DNA to liposome are shown on the left
and right sides of charge neutrality, respectively.
FIG. 3 shows the density gradient profiles of DNA:cationic lipid complexes
(1:6 ratio). Profiles are measured by flow-cell UV spectrophotometer at
237 nm. The contents of the centrifuged samples (approx. 13 ml) are pumped
through the flow-cell at a rate of 1 ml/min. The ordinate represents the
approximate location within the centrifuge tube.
FIG. 4 shows the density gradient profiles of DNA:cationic lipid complexes
(1:12 ratio). Profiles are measured by flow-cell UV spectrophotometer at
237 nm. The contents of the centrifuged samples (approx. 13 ml) are pumped
through the flow-cell at a rate of 1 ml/min. The ordinate represents the
approximate location within the centrifuge tube.
FIG. 5 shows the density gradient profile of DNA-cationic lipid complexes
prepared according to the dual feed stream method. Profiles are measured
by flow-cell UV spectrophotometer at 237 nm. The contents of the
centrifuged samples (approx. 13 ml) are pumped through the flow-cell at a
rate of 1 ml/min. The ordinate represents the approximate location within
the centrifuge tube.
FIG. 6 is a histogram showing the levels of transfection obtained in lung
tissue, as measured by CAT expression, resulting from transfection of a
CAT reporter plasmid using complexes prepared by the methods described in
the Examples that follow.
FIG. 7 is a diagram showing the methods of the invention using a static
mixer.
DESCRIPTION OF SPECIFIC EMBODIMENTS
The physical nature of nucleic acid:polycationic carrier transfection
complexes is highly dependent on the method in which they are prepared.
Typically, transfection complexes are prepared by adding one solution to
the other, i.e. nucleic acid to polycation or polycation to nucleic acid,
with constant stirring. For in vivo uses, it is desirable to prevent the
formation of macroaggregates or precipitation during the complexation
process.
The method of preparing polynucleotide transfection complexes described
herein is based on a number of observations that have not been previously
appreciated. For example, the nature of the transfection complexes is
dependent on the concentrations of the nucleic acid and polycation
solutions, and larger complexes are formed as the nucleic acid:polycation
ratios approach charge neutrality. Also, the kinetics of complex formation
are very fast. The complexes so formed are capable of interaction with the
starting components. Thus, the nature of interaction between the starting
components may be altered by the presence of complexes in the solution.
The interference by complexes becomes increasingly significant throughout
the complexation process as the concentration of complexes increases
within the mixing fluid. In effect, each new addition of starting
component (either nucleic acid or polycation) "sees" a different
environment of complex/starting component solution.
Accordingly, the method of the present invention allows the nucleic acid
and polycationic carrier molecules to react and form complexes in a
minimal volume. The complexes thus formed are removed immediately, thereby
limiting the interference of complex with the process of complex
formation. The process assures adequate mixing of nucleic acid and
polycation, while controlling the concentrations of each species in the
mixing volume.
In one embodiment, the reduced-volume, dual feed stream process involves
the collision of two feed streams (nucleic acid and polycation) in a
minimal volume, and the exit of the complex stream away from the site of
interaction. The process is highly controllable, reproducible and easily
scaleable.
In preferred embodiments, the mixture is flowed through a static mixer to
ensure complete mixing of the nucleic acid and polycationic carrier
molecule. Static mixers are advantageous because substantially complete
mixing can be obtained while minimizing shear of the nucleic acid. In
addition, static mixers allow continuous flow, and are readily scalable,
allowing for economical preparation of nucleic acid transfection complexes
on large scale.
For the purposes of this document, the term "static mixer" refers to any
flow-through device which provides enough contact time between two or more
liquids to allow substantially complete mixing of the liquids. Typically,
static mixers contain an internal helical structure which allows the
liquids to come in contact in an opposing rotational flow and causes them
to mix in a turbulent or laminar flow. Such mixers are described, for
instance, U.S. Pat. No. 3,286,922.
"Transfection" as used herein means the delivery of exogenous nucleic acid
molecules to a cell, either in vivo or in vitro, whereby the nucleic acid
is taken up by the cell and is functional within the cell. A cell that has
taken up the exogenous nucleic acid is referred to as a "host cell",
"target cell" or "transfected cell." A nucleic acid is functional within a
host cell when it is capable of functioning as intended. Usually, the
exogenous nucleic acid will comprise an expression cassette which includes
DNA coding for a gene of interest, with appropriate regulatory elements,
which will have the intended function if the DNA is transcribed and
translated, thereby causing the host cell to produce the peptide or
protein encoded therein. DNA may encode a protein lacking in the
transfected cell, or produced in insufficient quantity or less active
form, or secreted, where it may have an effect on cells other than the
transfected cell. Other examples of exogenous nucleic acid to be delivered
include, e.g., antisense oligonucleotides, mRNA, ribozymes, or DNA
encoding antisense RNAs or DNA/RNA chimeras. Nucleic acids of interest
also include DNA coding for a cellular factor which, when expressed,
activates the expression of an endogenous gene.
"Transfection efficiency" refers to the relative number of cells of the
total within a cell population that are transfected and/or to the level of
expression obtained in the transfected cells. It will be understood by
those of skill in the art that, by use of appropriate regulatory control
elements such as promoters, enhancers and the like, the level of gene
expression in a host cell can be modulated. The transfection efficiency
necessary or desirable for a given purpose will depend on the purpose, for
example the disease indication for which treatment is intended, and on the
level of gene expression obtained in the transfected cells.
"Polycation" refers to any molecular entity having multiple positive
charges, which, when combined with nucleic acid, interacts by ionic
interactions with the nucleic acid. "Polycationic carrier" refers to a
polycation which, when combined with a polynucleotide, forms a complex
suitable for transfecting eukaryotic cells. For example, cationic lipids
have been shown to be efficient polycationic carriers for nucleic acid
delivery to cells. Typically, cationic lipid carriers are in the form of
liposomes having both cationic and non-cationic lipid (usually neutral
lipid) components. Thus, a "lipid carrier" or "cationic lipid carrier"
refers to a lipid composition of one or more cationic lipids and,
optionally, one or more non-cationic lipids for delivering agents to
cells. The lipid carrier may be in any physical form including, e.g.,
liposomes, micelles, interleaved bilayers, etc.
The term "cationic lipid" is intended to encompass lipids that are
positively charged at physiological pH, and more particularly,
constituitively positively charged lipids comprising, for example, a
quaternary ammonium salt moiety. Cationic lipids used for gene delivery
typically consist of a hydrophilic polar head group and lipophilic
aliphatic chains. Similarly, cholesterol derivatives having a cationic
polar head group may also be useful. Farhood et al., (1992) Biochim.
Biophys. Acta 11 11:239-246; Vigneron et al., (1996) Proc. Natl. Acad.
Sci. (USA) 93:9682-9686.
Lipid carriers usually contain a cationic lipid and a neutral lipid,
usually in approximately equimolar amounts. The neutral lipid is helpful
in maintaining a stable lipid bilayer in liposomes, and can significantly
affect transfection efficiency. The liposomes may have a single lipid
bilayer (unilamellar) or more than one bilayer (multilamellar). They are
generally categorized according to size, where those having diameters up
to about 50 to 80 nm are termed "small" and those greater than about 80 to
1000 nm, or larger, are termed "large." Thus liposomes are typically
referred to as large unilamellar vesicles (LUVs), multilamellar vesicles
(MLVs) or small unilamellar vesicles (SUVs). Methods of producing cationic
liposomes are known in the art. See, e.g., Liposome Technology (CFC Press,
NY 1984); Liposomes by Ortro (Marcel Schher, 1987); Methods Biochem Anal.
33:337462 (1988).
Cationic lipids of interest include, for example, imidazolinium derivatives
(WO 95/14380), guanidine derivatives (WO 95/14381), phosphatidyl choline
derivatives (WO 95/35301), and piperazine derivatives (WO 95/14651).
Examples of cationic lipids that may be used in the present invention
include DOTIM (also called BODAI) (Solodin et al., (1995) Biochem. 34:
13537-13544), DDAB (Rose et al., (1991) BioTechniques 10(4):520-525),
DOTMA (U.S. Pat. No. 5,550,289), DOTAP (Eibl and Wooley (1979) Biophys.
Chem. 10:261-271), DMRIE (Felgner et al., (1994) J. Biol. Chem. 269(4):
2550-2561), EDMPC (commercially available from Avanti Polar Lipids,
Alabaster, Ala.), DCChol (Gau and Huang (1991) Biochem. Biophys. Res.
Comm. 179:280-285), DOGS (Behr et al., (1989) Proc. Natl. Acad. Sci. USA,
86:6982-6986), MBOP (also called MeBOP) (WO 95/14651), and those described
in WO 97/00241. Particularly preferred are EDMPC for aerosolized delivery
to airway epithelial cells, and DOTIM, DOTAP or MBOP for intravenous
delivery to vascular endothelial cells of various organs, particularly the
lung. In addition, cationic lipid carriers having more than one cationic
lipid species may be used to produce complexes according to the method of
the present invention.
Neutral lipids of use in transfection complexes are known, and include, for
example, dioleoyl phosphatidylethanolamine (DOPE), Hui et al., (1996)
Biophys. J. (71):590-599; cholesterol, Liu et al., (1997) Nat. Biotech.
(15):167-173; and dilauroyl phosphatidylethanolamine (DLPE) (co-pending
patent application Ser. No. 08/832,749). Normally, cationic lipid and
non-cationic lipids are used in approximately equimolar amounts.
Additional polycationic carriers include positively charged peptides and
proteins, both naturally occurring and synthetic, as well as polyamines,
carbohydrates or synthetic polycationic polymers. Examples include
polylysine, polyarginine, protamine, polybrene, histone, cationic
dendrimer, and synthetic polypeptides based on viral peptides, e.g.,
having cell binding, endosomal release or nuclear localizing functions,
etc. For certain applications, polycationic carriers may include cationic
lipid as well as peptide moieties. See, e.g., WO 96/22765.
The nucleic acid may be in any physical form, e.g., linear, circular or
supercoiled; single-stranded, double-, triple-, or quadruple-stranded; and
further including those having naturally occurring nitrogenous bases and
phosphodiester linkages as well as non-naturally occurring bases and
linkages, e.g. for stabilization purposes. Preferably it is in the form of
supercoiled plasmid DNA. Plasmid DNA is conveniently used for DNA
transfections since there are no size constraints on the DNA sequences
that may be included, and it can be produced in large quantity by growing
and purifying it from bacterial cells.
"Transfection complex" or "polynucleotide transfection complex" refers to a
combination of a polycationic carrier and a nucleic acid, in any physical
form, for use in transfecting eukaryotic cells. A transfection complex may
include additional moieties, e.g., targeting molecules such as receptor
ligands or antibody fragments, or other accessory molecules. For example,
nuclear localizing peptides may be included for facilitating transport of
the polynucleotide to the cell nucleus. Kalderon et al., (1984) Cell
39:499-509; Chelsky et al., (1989) Mol. Cell Biol. 9:2487-2492; Dingwall &
Laskey (1991) Trends Biochem. Sci. 16:478-481. Proteins or peptides may be
included in the transfection complex to facilitate release of the
transfection complex from the endosome after internalization. Raja-Walia
et al., (1995) Hum. Gene Therap. 2:521-530; Bai et al., (1993) J. Virol.
67:5198-5205. In addition, enzymes involved in transcription and/or
translation may be included to facilitate gene expression in the cell
cytoplasm without transport to the cell nucleus. Gao & Huang (1993) Nucl
Acids Res. 21:2867-2872.
The transfection complexes may also be prepared to include a targeting
moiety, to target delivery of the complex to the desired target cell in
vivo. Thus, strategies are known in the art for including receptor ligands
for delivery to cells expressing the appropriate receptor, or using
antibodies or antibody fragments to target transfection complexes to cells
expressing a specific cell surface molecule. See WO 96/37194; Ferkol et
al., (1993) J. Clin. Invest. 92:2394-2400.
The polycationic carriers and polynucleotide molecules are mixed, resulting
in polynucleotide transfection complexes. In addition to the mixing
conditions, the physical structure of such complexes depends on the
polycationic carrier and nucleic acid components, the ratios between them,
concentrations of each, buffer ionic strength, and the like. The
polycationic carriers are mixed with nucleic acids in aqueous solution, at
concentrations and ratios optimized for the target cells to be
transfected.
For preparation of cationic lipid/polynucleotide complexes, the cationic
lipids will typically be in the form of liposomes. The lipid mixtures
typically are prepared in chloroform, dried, and rehydrated in, e.g., 5%
dextrose in water or a physiologic buffer to form liposomes. Low ionic
strength solutions are preferred. Liposomes may be LUVs, MLVs, or SUVs.
Usually, the liposomes formed upon rehydration are predominantly MLVs, and
SUVs are formed from them by sonication or by extrusion through membranes
with pore sizes ranging from 50 to 600 nm to reduce their size. Most
preferably, the liposomes are extruded through a series of membranes with
decreasing pore sizes, e.g., 400 nm, 200 nm and 50 nm.
The nucleic acid will usually be plasmid DNA, prepared in a low ionic
strength solution to prevent interference by additional ions with the
complexation process. A low-ionic strength solution means a solution
having a conductivity less than about 35 mS, preferably less than about 10
mS, and most preferably less than about 1 mS. Desirably, the DNA solution
will contain no salts. Preferably the DNA is in a solution of about 5%
dextrose in 5 mM Tris-HCl (pH 8.0).
The nucleic acid and polycationic carrier solutions are prepared separately
at the desired concentrations, and provided in two feed streams. In one
embodiment, illustrated in FIG. 1, the two feed streams 10, 20 collide at
a Tee junction 30. The complexes formed upon the mixing of the two
solutions within the Tee junction 30, exit away from the site of
interaction.
The tubing size and flow rate are chosen to provide adequate mixing at the
Tee. Turbulent flow is determined by the Reynold's number, Re, calculated
according to the equation:
Re=Dv.rho./.mu.
where D is the diameter of the tubing (cm), v is the flow velocity
(cm/sec), .rho. is the density of the solution (g/ml), and .mu. is the
viscosity of the solution (centipoise). The transition regime from laminar
to turbulent flow exists at 2,100<Re<3,000. Bird, Stewart, and Lightfoot,
Transport Phenomena (John Wiley & Sons, Inc., NY, 1960), p. 108.
The parameters chosen should provide for mixing under turbulent conditions.
Thus, the input feed streams may be provided under turbulent flow
conditions, or they may be provided under laminar flow conditions,
provided that turbulent mixing results from the colliding of the streams.
In some embodiments flow rate is selected to provide laminar flow in the
input streams, but turbulent flow conditions at the Tee junction.
Generally, tubing sizes and inlet flow rates for both nucleic acid and
polycationic carrier solutions, are selected such that the outlet velocity
is at least about 7.5 cm/sec,, usually at least about 10 cm/sec, and often
at least about 20 cm/sec. When expressed in terms of Reynolds values, the
outlet solution preferably has Re at least about 180 and usually at least
about 250, and often greater than about 500. When the solutions are
provided under turbulent flow conditions, the Re value for the nucleic
acid solution will have an upper limit at the point where the nucleic acid
exhibits degradation due to shearing. Re values up to at least about 7100
do not cause degradation of the nucleic acid.
One of skill will recognize that the flow rates or Reynolds values of the
polycationic carrier solutions need not be the same as those of the
nucleic acid solutions. Examples of flow rates suitable for use in the
present invention are provided in Example 4, below. The parameters listed
below produce Re which lie within the laminar flow range, Re=1870, and
turbulent mixing results from the colliding streams. The tube diameter and
velocity correspond to a flow rate of approximately 70 ml/min.
D=1/32"=0.079 cm
v=235.77 cm/sec
.rho..congruent.1.00 g/cc
.mu..congruent.1.00 centipoise
The product stream, 40, is designed such that laminar flow develops as a
consequence of a larger tube diameter. In the above example, a tube
diameter of 3/32"=0.238 cm results in laminar flow conditions. Laminar
flow reduces intra-stream mixing and the interaction between thc formed
polycation/nucleic acid complexes. The product stream will settle into
fully-developed laminar flow and the turbulent effects of mixing will
diminish when the tube length exceeds Le, equivalent length of discharge
pipe. Perry and Green, Perry's Chemical Engineering Handbook, Sixth
Edition (McGraw-Hill Inc., NY 1984), pp. 5-34. The correlation between Re,
D, and Le is given by the following equation:
Le=0.035DRe
In the above example, the product stream tube, 40, length is 25.4 cm, which
exceeds the required Le of approximately 10.4 cm. Product is collected
after laminar flow has developed. This ensures that product interaction is
minimized immediately after it is formed.
The feed streams may be provided in other orientations, besides the Tee
junction in the above example, as long as the polynucleotide and
polycation are thoroughly mixed. For example, the feed streams may be
provided in a Y-junction, or as concentric cylinders, or feed into a
static mixer. In addition, more than two feed streams may be provided, if
desired, for example, where the final transfection complexes will contain
three or more components.
As noted above, static mixers can also be used to prepare the complexes. In
these embodiments, the static mixer is connected at a minimal distance
downstream of a junction of the nucleic acid solution and the polycationic
carrier solution. Adequate mixing, important in preventing the formation
of large particulates (>1(m), becomes limiting when the volume of the
mixing container is increased. A static mixer is employed to allow
sufficient mixing of the nucleic acid and polycationic carrier components,
while reducing shear stress and thus nucleic acid degradation associated
with rigorous mixing conditions. Static mixers are particularly preferred
when large volumes, e.g. volumes greater than one liter, are mixed.
The degree of mixing is controlled by varying the flow rate of the
solutions through the mixer, the type of mixer used, the diameter of the
mixer, and the number of elements in the mixer. A laminar flow static
mixer is preferred. For instance, in the preparation of transfection
complexes of the invention a Kenics laminar flow static mixer (7 inches
long, 21 element, 0.250 inch outer diameter, 0.187 inch inner diameter,
316L stainless steel) is connected down stream of a junction where the two
feed streams converge. Feed stream #1, comprised of a polynucleotide
solution and feed stream #2, comprised of a polycationic carrier solution,
or dispersion, flow into the junction at typical linear velocities of 0.17
to 0.77 feet per second, corresponding to flow rates between about 50 and
250 ml/min, and preferably between about 100 and 180 ml/min. The static
mixer should contain at least 21 elements, and may have up to 36 elements.
The combined streams are immediately fed into the static mixer to enhance
mixing between the two streams and formation of the transfection
complexes. The resulting mixture containing the polynucleotide
transfection complexes is collected from the static mixer exit stream.
Alternatively a turbulent flow static mixer may be used (e.g., a Komax
static mixer with 21 elements, 5 inches long, 1/4 inch outer diameter,
0.194 inch inner diameter, 316L Stainless steel). The linear velocity,
however, must be reduced significantly to avoid shearing the nucleic acid
when using a turbulent flow static mixer. Alternative static mixers may be
obtained from Statomix (Conprotec, Inc., Salem, N.H.), ranging in length
from 6 to 101/2 inches, outer diameters from 0.188 to 0.25 inches, and
inner diameters from 0.132 to 0.194 inches, and 24 to 36 elements. By
using the analytical methods described herein, the nucleic acid integrity
can be monitored through a range of linear velocities to determine the
conditions allowing acceptable throughput and acceptable product quality.
FIG. 7 shows an exemplary system in which a static mixer is used to provide
complete mixing of the nucleic acid and the polycation.
Tank 1 contains the polycationic carrier solution or dispersion and Tank 2
contains the nucleic acid solution. The pump is started and flow moves
simultaneously through both lines and through the Tee junction. The two
resultant streams are radially mixed by the static mixer helical elements
and polynucleotide complexes are formed and collected in a sterile
container. Initial concentrations and flow rates from Tank 1 and Tank 2
can be adjusted to achieve the desired ratio of polycationic carrier to
nucleic acid in the resulting transfection complexes.
A number of analytical methods are known for characterizing the complexes
prepared according to the method of the invention. Visual inspection may
provide initial information as to aggregation of the complexes.
Spectrophotometric analysis may be used to measure the optical density,
giving information as to the aggregated status of the complexes; surface
charge may be determined by measuring zeta potential; agarose gel
electrophoresis may be utilized to examine the amounts and physical
condition of the polynucleotide molecules in the complexes; particle
sizing may be performed using commercially available instruments; HPLC
analysis will give additional information as to resulting component
ratios; and dextrose or sucrose gradients may be used to analyze the
composition and heterogeneity of complexes formed.
It will be appreciated that using the method of complex preparation
described herein, polynucleotide transfection complexes may be prepared in
a variety of formulations depending of the desired use. Uses contemplated
for the complexes of the invention include both in vivo and in vitro
transfection procedures corresponding to those presently known that use
cationic lipid and other cationic carriers, including those using
commercial cationic lipid preparations, such as Lipofectin, and various
other published techniques using conventional cationic lipid technology
and methods. See, generally, Lasic and Templeton (1996) Adv. Drug Deliv.
Rev. 20: 221-266 and references cited therein. Thus, the ratios of each
component in the complexes, final concentrations, buffer solutions, and
the like are easily adjusted by adjusting the starting components. The
method allows the resulting transfection complexes to the prepared in a
highly controlled fashion, efficiently using starting materials and
yielding active transfection complexes.
Cationic lipid-nucleic acid transfection complexes can be prepared in
various formulations depending on the target cells to be transfected. See,
e.g., WO 96/40962 and WO 96/40963. While a range of lipid-nucleic acid
complex formulations will be effective in cell transfection, optimum
conditions are determined empirically in the desired experimental system.
Lipid carrier compositions may be evaluated by their ability to deliver a
reporter gene (e.g. CAT which encodes chloramphenicol acetyltransferase,
luciferase, alkaline phosphatase or .beta.-galactosidase) in vitro, or in
vivo to a given tissue in an animal, such as a mouse.
For in vitro transfections, the various combinations are tested for their
ability to transfect target cells using standard molecular biology
techniques to determine DNA uptake, transcription and/or protein
production, including Southern blot analysis, Northern blot analysis,
Western blot analysis, PCR, RT-PCR, ELISA and reporter gene activity
assays. Typically, in vitro cell transfection involves mixing nucleic acid
and lipid, in cell culture media, and allowing the lipid-nucleic acid
transfection complexes to form for about 10 to 15 minutes at room
temperature. The transfection complexes are added to the cells and
incubated at 37(C for about four hours. The complex-containing media is
removed and replaced with fresh media, and the cells incubated for an
additional 24 to 48 hours.
In vivo, particular cells can be preferentially transfected by the use of
particular cationic lipids for preparation of the lipid carriers, for
example, by the use of EDMPC to transfect airway epithelial cells (WO
96/40963) or by altering the cationic lipid-nucleic acid formulation to
preferentially transfect the desired cell types (WO 96/40962). Thus, for
example, in circumstances where a negatively charged complex is desired,
relatively less cationic lipid will be complexed to the nucleic acid
resulting in a higher nucleic acid to cationic lipid ratio. Conversely, in
circumstances where a positively charged complex is desired, relatively
more cationic lipid will be complexed with the nucleic acid, resulting in
a lower nucleic acid to cationic lipid ratio.
The lipid mixtures are complexed with DNA in different ratios depending on
the target cell type, generally ranging from about 6:1 to 1:20 (g
DNA:nmole cationic lipid. For transfection of airway epithelial cells,
e.g., via aerosol, intratracheal or intranasal administration, net
negatively charged complexes are preferred. Thus, preferred DNA:cationic
lipid ratios are from about 10:1 to about 1:20, preferably about 3:1. For
intravenous administration, preferred DNA:cationic lipid ratios range from
about 1:3.5 to about 1:20 (g DNA: nmole cationic lipid, most preferably,
about 1:6 to about 1:15 (g DNA: nmole cationic lipid. Additional
parameters such as nucleic acid concentration, buffer type and
concentration, etc., will have an effect on transfection efficiency, and
can be optimized by routine experimentation by a person of ordinary skill
in the art
Delivery can be by any means known to persons of skill in the art, e.g.,
intravenous, intraperitoneal, intratracheal, intranasal, intramuscular,
intradermal, etc. PCT patent application WO 96/40962 describes the
preparation and use of cationic lipid carriers for in vivo DNA delivery.
For aerosol administration, via intranasal or intraoral delivery, the
cationic lipid-nucleic acid transfection complex will withstand both the
forces of nebulization and the environment within the lung airways and be
capable of transfecting lung cells. Techniques for delivering genes via
aerosol administration of cationic lipid-DNA transfection complexes is
described in U.S. Pat. No. 5,641,662.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLES
Example 1
Preparation of DNA and Liposomes
Plasmid p4119 containing the CAT reporter gene under the control of the
HCMV promoter was prepared at a concentration of 5 mg/ml in 10 mM
Tris-HCl. Cholesterol (Sigma, St. Louis, Mo.) and DOTIM (Sigma) were
dissolved in chloroform (EM Sciences, Gibbstown, N.J.) at a 1:1 mole
ratio, and lipid films were formed with a rotary evaporator. The films
were hydrated with 5% (w/v) dextrose in water (D5W) at room temperature
and extruded through a series of membranes having pore sizes of 400 nm,
200 nm, and 50 nm. Liposomes were prepared in a concentration of 40 mM in
D5W. The final concentration of DOTIM and cholesterol in the liposome
dispersion was determined by HPLC to be 16.4 mM DOTIM and 16.9 mM
cholesterol.
Approximately 25 mg of DNA was dialyzed overnight into 2 L of 2.5 mM
histidine (Sigma) in D5W (pH 6.0). The concentration of DNA was determined
by absorbance at 237 nm and adjusted to 0.625 mg/ml.
Example 2
Titration of DNA and Liposomes to the Point of Precipitation
1 ml of liposome solution (3.75 mM DOTIM) was aliquoted into a 4 ml vial
and stirred gently. A 50 .mu.l aliquot of DNA solution was added and
allowed to stir for approximately 5 min. A 10 .mu.l sample was removed and
diluted 30.times. in D5W for particle sizing information. DNA addition and
sampling were repeated until aggregates were visible to the naked eye.
Particle size data was obtained using a NiComp 370 sub-micron particle
sizer (Particle Sizing Systems Inc., Santa Barbara, Calif.). The titration
procedure was repeated using initial DOTIM concentrations of 1.875 mM and
7.5 mM. The concentration of the DNA was varied to maintain a constant
DNA:lipid ratio. Titration of liposome to DNA was performed in the same
manner.
FIG. 2 shows the average vesicle size produced by titration of starting
components. We observe that the onset of precipitation begins at vesicle
sizes of approximately 240 nm. As the DNA/liposome ratios approach 1:3 mg
DNA:.mu.mol cationic lipid (theoretical charge neutrality), the complexes
begin to precipitate. We hypothesize that the reduced ionic interaction
allows shorter range van der Waals forces to dominate, causing complex
particles to aggregate. In order to prevent the formation of large
aggregates and eventually precipitation, DNA/liposome complexes must
either carry net ionic charges to repel one another, or steric constraints
beyond which van der Waals forces are no longer effective (e.g., Stealth
liposomes, Lasic, 1997).
Titration experiments show that the DNA/liposome complexes begin to
precipitate much earlier than the theoretical 1:3 ratio. Even with
formulations designed to carry a net positive charge to prevent
precipitation, the driving force toward charge neutrality can cause the
formation of near neutral complexes in local environments. DNA's affinity
for complexes competes with its affinity for free liposomes. This
interference by the end product becomes increasingly significant as more
product is formed. The complexes formed using this method leads to
inefficient use of liposome, and may result in aggregate-prone complexes.
A condition that promotes the formation of more uniform complexes
minimizes the interference of product on the interaction between starting
components.
Example 3
Comparison of Different DNA/Liposome Complexation Methods
Method 1. DNA at a concentration of 0.625 mg/ml was added to a mixing
solution of DOTIM/cholesterol liposomes (DOTIM concentration 3.75 mM),
stirred in a glass vial at 500 rpm. An equal volume of DNA was dispensed
at a rate of 37.5 ml/min, and allowed to mix for 20 seconds. The final DNA
and DOTIM concentrations were 0.3125 mg/ml and 1.875 mM respectively.
Final DNA:cationic lipid ratio was 1:6 (.mu.g DNA:nmole cationic lipid).
Complexes were also prepared by the same method at a final DNA:cationic
lipid ratio of 1:12 (.mu.g DNA:nmole cationic lipid).
Method 2. Complexes were prepared as described for Method 1, except that
the DNA was added to the liposome solution at the rate of 6 ml/min.
Method 3. A 10 ml solution of DNA (0.625 mg/ml) was loaded into a 60 ml
Becton-Dickinson plastic syringe. A 10 ml liposome solution (DOTIM
concentration 3.75 mM) was prepared and loaded into a separate syringe.
The two syringes were connected through PTFE tubing (ID=1/32"; OD=1/16")
to opposite ends of a PTFE Tee. The Tee outlet was connected to a 25.4 cm
long section of PTFE tubing (ID=3/32"; OD=5/32"). The syringes were loaded
into a programmable multi-syringe pump (Cole Parmer Instrument Co., Vernon
Hills, Ill.). The contents of the syringes were simultaneously pumpled at
a rate of 70 ml/min through the Tee and collected from the outlet tube
into a sterile container. The holdup contents were discarded. The final
DNA and DOTIM concentrations were 0.3125 mg/ml and 1.875 mM respectively
(1:6 ratio (.mu.g DNA:nmole cationic lipid)). Using this method,
DNA/liposome complexes were also prepared at final concentrations of
0.3125 mg/ml DNA and 3.75 mM DOTIM (1:12 ratio (.mu.g DNA:nmole cationic
lipid)).
Methods for characterization of DNA/liposome complexes include
spectrophotometric analysis at 400 nm, zeta potential analysis using a
Brookhaven Zetaplus (Brookhaven Instruments, Holtsville, N.Y.), particle
sizing using a NiComp 370, and dextrose density centrifugation (described
below). In addition, in vivo transfection activity of the complexes was
determined by CAT expression in the lungs of ICR mice 24 hr after a 200
.mu.l IV tail vein injection. CAT expression was determined by ELISA assay
and normalized to the amount of total protein (ng CAT/mg total protein).
Dextrose gradients (5% w/v to 20% w/v) were prepared using the BioComp
Gradient Master (BioComp Instruments, Inc., New Brunswick, Canada). At
room temperature, centrifuge tubes (12 ml) were half-filled with 5%
dextrose followed by careful addition of 6 ml of 20% dextrose to the
bottom of the tube with a syringe and canula. The tubes were placed in the
Gradient Master and programmed to produce the linear gradients (time=2 min
25 sec., angle=81.5.degree., speed setting=15). The gradients were allowed
to equilibrate to 5.degree. C. for 1-2 hrs. Approximately 200 .mu.l sample
was loaded to the top of the gradient, and spun for 1 hr at 40,000 rpm and
4.degree. C. using a Beckman -70 ultracentrifuge with a SW-41 rotor. The
centrifuged gradients were loaded into a tube piercing apparatus
(Brandell) and 30% w/v dextrose was pumped at 1 ml/min into the bottom of
the tube. The contents of the tube were forced through an on-line UV/VIS
spectrophotometer (Rainin) and absorbance was measured at 237 nm (DOTIM
absorbance).
Table 1 compares the optical density (OD.sub.400), particle size and zeta
potential of the complexes prepared using the three methods. Method 2
complexes have higher OD.sub.400 values, larger average particle sizes,
and lower zeta-potential values compared with methods 1 and 3. This
suggests that complexes produced using method 2 are more aggregated and
have a lower net positive charge, consistent with formation of near
neutral complexes. There is no apparent difference in the characteristics
of complexes prepared using methods 1 and 3.
TABLE 1
______________________________________
DNA: Particle
cationic lipid ratio OD.sub.400 size Zeta-potential
Method (mg/.mu.mol) (absorbance) (nm) (mV)
______________________________________
1 1:6 0.183 238 45.8
2 1:6 0.204 338 39.7
3 1:6 0.172 227 45.4
1 1:12 0.171 207 45.9
2 1:12 0.206 274 39.0
3 1:12 0.160 206 39.6
______________________________________
Characterization: optical density (OD.sub.400), particle size, and zeta
potential of complexes. Optical density is expressed as the absorbance at
400 nm wavelength for a sample diluted 1:20 in 5% w/v dextrose. Particle
size is represented by the mean diameter of a complex solution diluted
1:30 in 5% w/v dextrose. Zetapotential is obtained from a 1:10 dilution o
a complex solution in purified water.
FIG. 3 shows the profiles of DNA/liposome complexes (1:6 DNA/lipid ratio)
prepared by the three methods. These profiles show significant differences
in the types of DNA/liposome populations. Free liposomes settle at the top
of the gradient, and generally do not penetrate further into it. The data
show that, for methods 1 and 2, a large peak associated with free
liposomes resides at the top of the gradient, while method 3 produces
significantly less free liposomes. It is likely that the lower quantity of
free liposomes resulting from method 3 is due to the increased interaction
of free liposome and DNA, closer to the predicted 1:6 ratio.
The profile resulting from method 3 complexes also shows populations
residing a much lower densities than those produced by methods 1 and 2,
further supporting the notion of greater DNA/liposome association in
method 3 complexes. In addition, method 2 complexes, which are shown in
Table 1 to have the largest mean diameter, also produce profiles with high
density populations and a significant amount of free liposome. This method
was designed to induce the interaction between product (complexes) and
starting material (DNA and liposomes) by slower addition of DNA. Complexes
produced in this manner tend toward the 1:3 charge neutrality ratio and,
therefore, are closer to the point of precipitation.
The profile for complexes produced by method 3 also shows several different
populations of DNA/liposome complexes. The number of different populations
produced by this method may reflect a relatively high rate of attraction
between the cationic lipid and negatively charged DNA. Though the method
of complexation was designed to reduce the interaction between product and
starting material, apparently the rates of attraction between DNA,
liposome and complex are sufficiently high to produce several distinct
populations.
FIG. 4 shows the density gradient profiles for complexes produced at a 1:12
DNA:cationic lipid ratio. For each of the three methodologies, there does
not appear to be significant differences in the DNA-containing populations
as compared to the 1:6 complexes. The quantity of free liposome, however,
is significantly higher for the 1:12 complexes.
In a separate experiment, DNA-lipid complexes were prepared at a 1:6 ratio
according to Method 3. The resulting complexes were analyzed by glucose
density gradient centrifugation after 4 days at 5.degree. C. The resulting
profile is shown in FIG. 5. The density gradient profile of these
complexes shows a more homogeneous population of DNA-lipid complexes than
obtained using Methods 1 and 2.
The data in Table 1 show slight variations in OD400, size and zeta
potential between the 1:6 and 1:12 complexes. Since each of these
measurements are based on mean values of entire populations, however, the
differences may simply be due to the presence of excess free liposome.
Centrifugation profiles show similar results with the exception of the
additional free liposome. Populations associated with DNA/liposome
complexes appear to be the same in both formulations.
In vivo expression. FIG. 6 shows the expression level of CAT detected in
the lung of ICR mice (n=6), 24 hr post-injection. Though the variability
of CAT expression is high in each of the test variables, we do observe
significant levels of CAT expression compared with naked DNA. All methods
and DNA: cationic lipid ratios appear to produce similar levels of
expression.
Example 4
Comparison of Feed Stream Parameters on Preparation of DNA/Liposome
Complexes
The following experiments were performed to determine feed stream
parameters for preparation of DNA/liposome complexes as described above.
Successful preparation was determined by particle size analysis using a
NiComp 370 sub-micron particle sizer (Particle Sizing Systems Inc., Santa
Barbara, Calif.) and visual inspection for precipitation. The data in
Table 2 shows the system configurations and the parameters tested, where
Re is the calculated Reynolds' number for flow through a smooth tube.
TABLE 2
__________________________________________________________________________
Inlet
Outlet
Inlet Outlet
tubing tubing flow Inlet flow Outlet
size size rates velocity Inlet rate velocity Outlet
System (I.D.) (I.D.) (ml/min) (cm/sec) Re (ml/min) (cm/sec) Re
__________________________________________________________________________
1 0.8 mm
2.4 mm
10-70
33-233
264-
20-140
7.4-52
178-
1864 1248
2 0.3 mm 2.4 mm 5-15 118-353 354- 10-30 3.7-11 89-
1059 265
3 0.3 mm 0.8 mm 2.3-13.5 54-318 162- 4.6-27 15.3-89.5 122-
954 716
__________________________________________________________________________
The results shown in Table 2 indicate that a correlation exists between the
outlet Re and the ability to produce complexes without precipitation. From
these data we can define a lower limit parameter of an outlet Reynolds'
number no less than 180-250. It must be emphasized that the calculated
Reynolds' number is defined for a smooth tube. Any alterations to the
interior of the tube to increase "roughness" can create sufficient mixing
conditions at lower Reynolds' numbers (calculated as above).
TABLE 3
______________________________________
Inlet Flow
Outlet Visual
System rate velocity Outlet Re Particle size Inspection
______________________________________
1 10 7.4 178 N/A precipitated
15 11.1 266 421 .+-. 286 cloudy
20 14.8 355 282 .+-. 126 opalescent
30 22.2 533 211 .+-. 61 opalescent
40 30 720 210 .+-. 54 opalescent
50 37 888 209 .+-. 53 opalescent
70 52 1248 212 .+-. 56 opalescent
2 15 11 267 239 .+-. 99 opalescent
10 7.3 178 N/A precipitated
5 3.7 89 N/A precipitated
3 13.5 89.5 716 217 .+-. 68 opalescent
7 45 358 221 .+-. 77 opalescent
2.3 15.3 122 N/A precipitated
______________________________________
The upper limit flow rate was determined by the limitations of the syringe
pump used in these experiments. Using an outlet tubing size of 0.3 mm I.D.
and an inlet flow rate of 50 ml/min, an outlet flow velocity of 2358
cm/sec was generated and was capable of producing DNA/liposome complexes
without precipitation. The calculated inlet Reynolds' number for this
system was 7074. The integrity of the DNA for this system was determined
by quantitation of super-coiled and open-circled forms before and after
processing. The data show that no significant degradation of DNA occurs
during these processes. Although an upper failure limit cannot be defined
at this time, the data suggest that complexes can be produced at high
Reynolds' numbers (at least 7100) without DNA damage.
Example 5
Preparation of DNA and Liposomes Using a Static Mixer
This study compares the formation of DNA/cationic liposome complexes using
a small-scale diluter (10 ml) and a static mixer (>7 ml capacity).
Plasmid DNA was provided at a concentration of approximately 5 mg/ml in 10
mM Tris-HCl, pH 8.0. The DNA was diluted to a concentration of 0.5 mg/ml
using 5% w/v dextrose. Liposomes were prepared at a concentration of 20 mM
ethyl-dimyristoyl-phosphatidylcholine (EDMPC)/20 mM
Diphytanoyl-phosphatidylethanolamine (DipPE). The liposomes were diluted
to a concentration of 4 mM EDMPC/4 mM DipPE. Using the procedures
described below, DNA/cationic liposome complexes were prepared by mixing
equal volumes of the diluted DNA and liposome solutions.
A. Diluter Method
The diluter method of DNA/liposome complex production is by equal volume
addition of DNA to a continuously mixed dispersion of liposomes. The
addition rate, type of mixer, and mixing speeds were optimized to produce
the desired particle size for the specific geometry of the vessel used.
With this procedure, the parameters must be re-optimized as the vessel
geometry is changed with scale. Mixing configurations are important in
production and must be tightly controlled.
Five ml of the diluted liposome dispersion was added to a sterile 24 ml
glass vial. A stirbar, of defined geometry, was added and rotated at speed
of approximately 800 rpm. An equal volume of DNA (5 ml) was added at a
rate of 1.25 ml/min using a Hamilton microlab diluter, Model #500 series
(Reno, Nev.).
B. Static Mixer Method
Equal volumes of 0.5 mg/ml DNA and 4 mM EDMPC/4 mM DipPE liposomes were
combined into a single feed stream and run through a 21 element, Kenics
static mixer yes (Chemineer, North Andover, Mass.) at inlet flow rates of
80 ml/min, corresponding to a linear flow rate of 0.45 feet per second.
The final complexes were collected in a 50 ml sterile centrifuge tube.
Following preparation of the complexes, several physical and chemical
parameters were tested to analyze the differences in the two methods.
Among these tests were particle size, turbidity, zeta potential, pH, and
DNA and lipid integrity tests (HPLC, thin layer chromatography, and
agarose gel electrophoresis). There were no significant differences in the
physical characteristics of the complexes. No significant difference in
chemical composition or degradation was observed. The particle size range
of the complexes made by the static mixer was tighter than those made by
the diluter method as shown in Table 4. Importantly, complexes prepared by
either method showed equivalent transfection efficiencies when tested in
vivo by intraperitoneal injection of 250 .mu.l of complex into SKOV-3
tumor-bearing Balb/C nude mice. Tumors were remove 24 hours post injection
and assayed for the presence of chloramphenicol acetyltransferase (CAT)
reporter protein.
TABLE 4
______________________________________
Assay Diluter Method
Static Mixer Method
______________________________________
Particle size (nm)
134
Average 162
Minimum 161
Maximum 162
Std. Dev. 0.6
Count 3
In vivo expression (pg/mg 40 .+-. 30 100 .+-. 150
total protein)
______________________________________
Example 6
This experiment shows the effect of increasing flow rate on turbidity
(optical density at 400 nm), complex size (nm), cationic carrier integrity
and DNA integrity. Complexes were prepared using the components and the
static mixer method described above, at the flow rates shown in Table 5.
These data show that production of a polynucleotide transfection complex
using the static mixer can be accomplished across a wide range of flow
rates with minimal effect on the physical and chemical characteristic of
the starting material, The Reynold's number of the feed streams associated
with the tested flow rates indicate flow within the laminar flow regime in
most cases (Note Re above 1000 with static mixer is consider to be
turbulent flow). The risk of damage to the starting material is,
therefore, lower than that resulting from the dual-feed stream method
described in Example 5. The data are summarized in Table 5.
TABLE 5
______________________________________
Linear flow Turbidity Complex
velocity Reynold's (400 nm 1:20 particle size DNA integrity
(ft/sec) number dilution) (nm) (Agarose gel)
______________________________________
0.17 245 0.10 269 .+-. 143
minimal
degradation
0.31 446 0.08 189 .+-. 82 minimal
degradation
0.56 803 0.08 189 .+-. 86 minimal
degradation
0.81 1164 0.08 188 .+-. 91 minimal
degradation
______________________________________
All publications and patent applications cited herein are hereby
incorporated by reference to the same extent as if fully set forth herein.
The invention now being filly described, it will be apparent to one of
ordinary skill in the art that many changes and modifications can be made
thereto without departing from the spirit or scope of the appended claims.
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