Note: Descriptions are shown in the official language in which they were submitted.
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SOLID SURFACE WITH IMMOBILIZED DEGRADABLE CATIONIC POLYMER
FOR TRANSFECTING EUKARYOTIC CELLS
Related Applications
This application claims priority to U.S. provisional application no.
60/637,344, filed
December 17, 2004, which is incorporated herein by reference.
Background of the Invention
Field of the Invention
Embodiments of the invention relate to devices and methods for cell
transfection.
In particular, einbodiinents of the invention are directed to a cell
transfection formula and to
a cell culture device that has been treated with the transfection formula. The
treated cell
culture device can be stored at room temperature and provides a transfection
method that is
simple and quick.
Description of the Related Art
Gene transfection methods can be used to introduce nucleic acids into cells
and are
useful in studying gene regulation and function. High throughput assays that
can be used to
screen large sets of DNAs to identify those encoding products with properties
of interest
which are particularly useful. Gene transfection is the delivery and
introduction of
biologically functional nucleic acids into a cell, particularly a eukaryotic
cell, in such a way
that the nucleic acid retains its function witlun the cell. Gene transfection
is widely applied
in studies related to gene regulation, gene function, molecular therapy,
signal transduction,
drug screening, and gene therapy studies. As the cloning and cataloging of
genes from
higher organisms continues, researchers seek to discover the function of the
genes and to
identify gene products with desired properties. This growing collection of
gene sequences
requires the development of systematic and high-throughput approaches to
characterizing
gene products and analyzing gene function, as well as other areas of research
in cell and
molecular biology.
Both viral and non-viral gene carriers have been used in gene delivery. Viral
vectors have been shown to have higher transfection efficiency than non-viral
carriers, but
the safety of viral vectors hampers applicability (Verma I.M and Somia N.
Nature 389
(1997), pp. 239-242; Marhsall E. Science 286 (2000), pp. 2244-2245). Although
non-viral
transfection systems have not exhibited the efficiency of viral vectors, they
have received
significant attention, because of their theoretical safety when compared to
viral vectors. In
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addition, viral vector preparation is a complicated and expensive process,
which limits the
application of viral vectors in vitro. The preparation of non-viral carriers
is simpler and
more cost effective in comparison to preparation of viral carriers, making
synthetic gene
carriers desirable as transfection reagents, particularly for in vitro
studies.
Most non-viral vectors mimic important features of viral cell entry in order
to
overcome cellular barriers, which are meant to prevent infiltration by foreign
genetic
material. Non-viral gene vectors, based on a gene carrier backbone, can be
classified as a)
lipoplexes, b) polyplexes, and c) lipopolyplexes. Lipoplexes are assemblies of
nucleic
acids with a lipidic component, which is usually cationic. Gene transfer by
lipoplexes is
called lipofection. Polyplexes are complexes of nucleic acids with cationic
polymer.
Lipopolyplexes comprise both a lipid and a polymer component. Often such DNA
complexes are further modified to contain a cell targeting or an intracellular
targeting
moiety and/or a membrane-destabilizing component, for example, a viral protein
or peptide
or a membrane-disruptive synthetic peptide. Recently, bacteria and phages have
also been
described as shuttles for the transfer of nucleic acids into cells.
Most non-viral transfection reagents are synthetic cationic molecules and have
been
reported to "coat" the nucleic acid by interaction of the cationic sites on
the cation and
anionic sites on the nucleic acid. The positively-charged DNA-cationic
molecule complex
interacts with the negatively charged cell membrane to facilitate the passage
of the DNA
through the cell membrane by non-specific endocytosis. (Schofield, Brit.
Microencapsulated. Bull, 51(1):56-71 (1995)). In most conventional gene
transfection
protocols, the cells are seeded on cell culture devices 16 to 24 hours before
transfection.
The transfection reagent (such as a cationic polymer carrier) and DNA are
usually prepared
in separate tubes, and each respective solution is diluted in medium
(containing no fetal
bovine serum or antibiotics). The solutions are then mixed by carefully and
slowing adding
one solution to the other while continuously vortexing the mixture. The
mixture is
incubated at room temperature for 15-45 minutes to allow complex fonnation
between the
transfection reagent and the DNA and to remove residues of serum and
antibiotics. Prior to
transfection, the cell culture medium is removed and the cells are washed with
buffer. The
solution containing the DNA-transfection reagent complexes is added to the
cells, and the
cells are incubated for about 3-4 hours. The medium containing the
transfection reagent is
then be replaced with fresh medium. The cells are finally analyzed at one or
more specific
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time point(s). This is obviously a time consuming procedure, particularly when
the number
of samples to be transfected is very large.
Several major problems exist in conventional transfection procedures. First,
conventional procedures are time-consuming, particularly when there are many
cell or gene
samples to be used in transfection experiments. Also, the results derived from
common
transfection procedures are difficult to reproduce, due to the number of steps
required. For
instance, the DNA-transfection reagent complex formation is influenced by
concentration
and volume of nucleic acid and reagents, pH, temperature, type of buffer(s)
used, length
and speed of vortexing, incubation time, and other factors. Although the same
reagents and
procedure may be followed, different results may be obtained. Results derived
from multi-
step procedures are often influenced by human or mechanical error or other
variations at
each step. In addition, refreshing the cell culture medium following
transfection disturbs
the cells and may cause them to detach from the surface on which they are
cultured, thus
leading to variation and unpredictability in the final results. Due to all the
factors noted,
conventional transfection methods require a highly skilled individual to
perform the
transfection experiment or assay.
Researchers require an easier and more cost effective method of transfecting
cells,
and a high-throughput method of transfectiing cells is needed in order to
transfect large
sample numbers efficiently.
Sabatini (U.S. 2002/0006664A1) describes a composition containing DNA which is
deposited on a glass slide. However the system only allows transfection with
the previously
deposited DNA. This is a major disadvantage of this system. As it only
provides for
transfecting with previously deposited DNA, every researcher cannot use his or
her desired
nucleic acids.
U.S. Publication No. 2004/0138154A1, which is incorporated herein by
reference,
describes a cell culture/transfection device where the transfection is
mediated by a lipid
polymer. U.S. Publication No. 2005/0176132A1, also incorporated herein by
reference,
describes a calcium salt mediated transfectable cell culture device.
US Publication No. 2003/0215395A1, incorporated herein by reference, describes
degradable polymers which can be used for gene delivery.
As discussed above, conventional transfection is a lengthy and technically
difficult
procedure. Generally, three steps are required: 1) cells are seeded in a cell
culture plate or
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dish and incubated until sufficient confluence is achieved; 2) transfection
reagent / nucleic
acid complexes are prepared; and 3) nucleic acids of interest are added along
witli the
transfection reagent and further incubation is carried out. Two incubation
periods are
needed and typically it talces more than two days to complete all the steps.
In contrast,
embodiments of the present invention provide a simple procedure that involves
only a
single incubation step. A cell culture device, which has previously been
coated with a
transfection reagent, allows transfection by adding the nucleic acid of
interest and the cell
culture in succession. The transfected cells may then be cultured in the same
device. Thus
the cells may be transfected and cultured in the cell culture device without
the need for
further manipulation of the cells immediately after the transfection step.
Transfection
efficiency is comparable to regular transfection, but the time required for
the operation is
reduced by more than one day. Embodiments of the invention include a
transfectable cell
culture device which greatly reduces the labor of transfection assays, and
enables
transfection with any nucleic acid of interest in an easy meth.od with low
cytotoxicity.
Also, the transfectable cell culture device of the invention is stable for
long temi storage at
room temperature.
Suinmary of the Invention
Embodiments of the invention are directed to a device which includes a solid
support coated with a transfection reagent mixture. Preferably, the
transfection reagent in
the coating is not complexed with a biomolecule, such as a nucleic acid.
Preferably, the
solid support is polystyrene resin, epoxy resin or glass. Preferably, the
coating is on the
surface of the solid support. Preferably, the coating amount of the
transfection reagent is
from about 0.1 to about 100 g/cm2. Preferably, the transfection agent is a
polymer. More
preferably, the polyiner is a cationic polymer. Preferably, the transfection
agent comprises
a degradable cationic polyiner. More preferably, the degradable cationic
polymer is made
by linking cationic compounds or oligomers with degradable linkers. The
transfection
agent may comprise both a degradable cationic polymer and a non-degradable
cationic
polymer. Preferably, the ratio of the non-degradable cationic polymer to the
degradable
cationic polymer is 1:0.5 to 1:20 (non-degradable : degradable) by weight.
In preferred embodiments, the transfection reagent includes a plurality of
cationic
molecules and at least one degradable linker molecule connecting said cationic
molecules
in a branched arrangement, wherein said cationic molecules are selected from :
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(i) a cationic compound of formula (A) or (B) or a combination thereof:
~ R2 Ra
R~ N~ R6-~NH-R,-)-R$
R3 R5 Formula B
Formula A
wllerein R' is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another
Formula A, or
Formula B;
R2 is a straight chain alkylene group of the formula: -(CHZ)a wherein a is an
integer
number from 2 to 10;
R3 is a straight or branched chain alkylene group of the formula: -(CbH2b)-
wherein b is an
integer number from 2 to 10;
R4 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or
Formula B;
RS is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or
Formula B;
R~ is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, Formula A, or another
Formula B;
R7 is a straight or branched chain alkylene group of the formula: -(CcH2c)- in
which c is an
integer number from 2 to 10; and
R8 is a hydrogen atom, an allcyl of 2 to 10 carbon atoms, Formula A, or
another Formula B;
(ii) a cationic dendritic or branched polyamidoamine (PAMAM) with terminated
primary or
secondary amino groups;
(iii) a cationic polyamino acid; or
(iv) a cationic polycarbohydrate;
and wherein said degradable linker molecule is represented by the formula:
A(Z)d
wherein A is a spacer molecule having at least one degradable bond, Z is a
reactive residue
which reacts with amino group, and d is an integer equal to or more than two
and wherein
A and Z are bound covalently.
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In preferred embodiments, the cationic compound or oligomer is poly(L-lysine)
(PLL), polyethyleneimine (PEI), polypropyleneimine (PPI), pentaethyleneamine,
N,N'-
bis(2-aminoethyl)-1,3-propanediamine, N,N'-bis(2-aminopropyl)-ethylenediamine,
spermine, spermidine, N-(2-aminoethyl)-1,3-propanediainine, N-(3-aminopropyl)-
1,3-
propanediamine, tri(2-aminoethyl)amine, 1,4-bis(3-aminopropyl)piperazine, N-(2-
aminoethyl)piperazine, dendritic polyamidoamine (PAMAM), chitosan, or poly(2-
dimethylamino)ethyl methacrylate (PDIVIAEMA).
In preferred embodiments, the linker molecule is di- and multi-acrylates, di-
and
multi-acrylamides, di- and multi-isothiocyanates, di- and multi-isocyanates,
di- and multi-
epoxides, di- and multi-aldehydes, di-and multi-acyl chlorides, di- and multi-
sulfonyl
chlorides, di- and multi-halides, di- and multi-anhydrides, di- and multi-
maleimides, di- and
multi-N-hydroxysuccinimide esters, di- and multi-carboxylic acids, or di-and
multi-a-
haloacetyl groups.
In preferred embodinients, the linlcer molecule is 1,3-butanediol diacrylate,
1,4-
butanediol diacrylate, 1,6-hexanediol diacrylate, 2,4-pentanediol diacrylate,
2-methyl-2,4-
pentanediol diacrylate, 2,5-dimethyl-2,5-hexanediol diacrylate, poly(ethylene
glycol)
diacrylate, trimethylolpropane triacrylate, pentaerythritol tetraacrylate,
di(trimethylolpropane) tetraacrylate, dipentaerythritol pentaacrylate, or a
polyester with at
least three acrylate or acrylamide side groups.
In preferred embodiments, the molecular weight of the polymer is from 500 da
to
1,000,000 da. More preferably, the molecular weight of the polymer is from
2000 da to
200,000 da.
In preferred embodiments, the molecular weight of the cationic compound or
oligomer is from 50 da to 10,000 da. In preferred embodiments, the molecular
weight of
the linker molecule is from 100 da to 40,000 da.
Preferably, the solid support is a dish bottom, a multi-well plate, or a
continuous
surface.
In some preferred embodiments, the transfection agent is covalently associated
with
a nucleic acid. In other preferred embodiments, the transfection agent is non-
covalently
associated with a nucleic acid.
In preferred embodiments, the device can be stored at room temperature for at
least
5 months without significant loss of transfection activity.
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Einbodiments of the invention are directed to a method of cell transfection
which
includes the steps of adding a solution including a nucleic acid to be
transfected to a device
which includes a solid support coated with a transfection reagent mixture,
adding
eukaryotic cells to the solution; and incubating the cells and the nucleic
acid solution to
allow cell transfection. Preferably, the incubation is for 5 min. to 3 hours.
More
preferably, the incubation is for 10 min. to 90 min.
Preferably, the nucleic acid is DNA, RNA, DNA/RNA hybrid or chemically-
modified nucleic acid. More preferably, the DNA is circular (plasmid), linear,
fragment or
single strand oligonucleotide (ODN). More preferably, the RNA is single strand
(ribozyme) or double strand (siRNA).
In some preferred embodiments, the cell is a mammalian cell. In some preferred
embodiments, at least some of the cells undergo cell division. In some
preferred
embodiments, the cell is a transformed or primary cell. In some preferred
embodiments,
the cell is a somatic or stem cell. In some preferred embodiments, the cell is
a plant cell.
Further aspects, features and advantages of this invention will become
apparent
from the detailed description of the preferred einbodiments which follow.
Brief Description of the Drawings
These and other features of this invention will now be described with
reference to
the drawings of preferred embodiments which are intended to illustrate and not
to limit the
invention.
Figure 1 shows the cell shape of transfected 293 cells. The transfection agent
treatments were linear polyethyleneimine (L-PEI) based polymer, lipid based
polymer,
degradable cationic polymer, and no treatment (intact 293 cells).
Figure 2 shows percentage of EGFP- positive cells.
Figure 3 shows cell condition after transfection.
Figure 4 shows the stability of a transfectable cell culture device in a mylar
bag with
02 absorber.
Figure 5 shows the stability of a transfectable cell culture device in a mylar
bag with
CO2 absorber.
Figure 6 shows the stability of a transfectable cell culture device in a mylar
bag with
02 and CO2 absorber.
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Figure 7 shows the stability of a transfectable cell culture device in a mylar
bag.
Detailed Description of the Preferred Embodiment
While the described embodiment represents the preferred einbodiment of the
present invention, it is to be understood that modifications will occur to
those skilled in the
art witllout departing from the spirit of the invention. The scope of the
invention is
therefore to be detennined solely by the appended claims.
Embodiments of the invention are directed to a transfection device and method
which is simple, convenient, and efficient compared to conventional
transfection assays. A
transfection device is made according to methods described herein by affixing
a
transfection reagent on the solid surface of a cell culture device. By using
this device,
researchers need only add a nucleic acid or other biomolecule to be
transfected and cells to
the surface of the cell culture device. There is no need to pre-mix the DNA or
biomolecule
with a transfection reagent. This removes a key timing-consuming step, which
is required
by conventional transfection procedures. Only approximately 40 minutes is
required to
complete the entire transfection process for 10 samples, compared to 2 to 5
hours or more
required by current methods. This is particularly advantageous for high
throughput
transfection assays, in which hundreds of samples are tested at a time.
As coinpared to conventional transfection, there are several advantages to the
method described herein. It provides a transfection device that is very easy
to store, and it
provides a simple method for biomolecule delivery or gene transfection in
which no
biomaterial/transfection reagent mixing step is required. The transfection
procedure
described herein can be finished in a short period of time, for instance
approximately 5 min.
to 3 hours, and it provides a high throughput inetlzod for transfection or
drug delivery in
which large numbers of samples may be transfected at a time.
In preferred embodiments, transfection reagents are simply coated onto the
surface
of a cell culture device, which can be easily commercialized and mass-
produced.
Customers, researchers for instance, need only add a bioniolecule, such as a
nucleic acid of
interest, directly to the surface of a cell culture device in order to prepare
the device prior to
addition of cells. An incubation period for a predetermined time allows the
biomolecule
and the transfection reagent(s) to form a complex for uptake by cells in the
next step. Cells
are then seeded on the surface of the cell culture device and incubated,
without the
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necessity of changing the medium, and the cells are analyzed. Changing medium
during
the transfection procedure is unnecessary. The methods described herein
dramatically
reduce the risk of error, by reducing the number of steps involved, thus
increasing
consistency and accuracy of the system.
The composition containing the transfection agent can be affixed to any
suitable
surface. For example, the surface can be glass, plastics (such as
polytetrafluoroethylene,
polyvinylidenedifluoride, polystyrene, polycarbonate, polypropylene), silicon,
metal (such
as gold), membranes (such as nitrocellulose, methylcellulose, PTFE or
cellulose), paper,
biomaterials (such as protein, gelatin, agar), tissues (such as skin,
endothelial tissue, bone,
cartilage), or minerals (such as hydroxylapatite, graphite). According to
preferred
embodiments the surfaces may be slides (glass or poly-L-lysine coated slides)
or wells of a
inulti-well plate.
For slides, such as a glass slide coated with poly-L-lysine (e.g., Sigma,
Inc.), the
transfection reagent is fixed on the surface and dried, and then a nucleic
acid of interest or a
nucleic acid to be introduced into cells is introduced. Generally, the nucleic
acid is spotted
onto the glass slide in a microarray. The slide is incubated at room
temperature for 30
minutes to form nucleic acid/transfection reagent complexes on the surface of
the
transfection device. The nucleic acid/transfection reagent complexes create a
medium for
use in high throughput microarrays, which can be used to study hundreds to
thousands of
nucleic acids, or other biomolecules at the same time. In an alternative
embodiment, the
transfection reagents can be affixed on the surface of the transfection device
in discrete,
defined regions to form a microarray of transfection reagents. In this
embodiment,
biomolecules, such as nucleic acids, which are to be introduced into cells,
are spread on the
surface of the transfection device and incubated with the transfection reagent
microarray.
This method can be used in screening transfection reagents or other delivery
reagents from
thousands of compounds. The results of such a screening method can be
exarnined through
computer analysis.
In another embodiment of the invention one or more wells of a multi-well plate
may
be coated with one or more transfection reagent(s). Plates commonly used in
transfection
are 96-well and 384-well plates. The transfection reagent can be evenly
applied to the
bottom of each well in the multi-well plate. Generally, the transfection
reagent is applied to
the bottom of plate in the range of about 0.1 to about 100gg/cma. Further, the
coating
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amount of the transfection reagent may be varied depending on the type of well
plate to be
used. For example, for a 6-well plate, 12-well plate or 96-well plate, the
coating
concentration of the transfection reagent is preferably from about 0.5 to
about 50 g/cm2,
and more preferably from about 1 to 20 g/cm2. In the case of a 384-well plate,
the coating
concentration of the transfection reagent is preferably from about 0.5 to
about 50gg/cm2,
and more preferably from about 1 to 30gg/cm2. In another embodiment of the
invention, a
10-cm cell culture dish is coated with a transfection reagent. The
transfection reagent can
be evenly applied to the bottom of dish. The transfection reagent may be
applied to the
bottom of dish in the range of about 0.1 to about 100gg/cm2, more preferably
about 0.2 to
about 20 g/cma.
Hundreds of nucleic acids or other biomolecules are then added into the
well(s) by,
for instance, a multichannel pipette or automated machine. Results of
transfection are then
determined by using a microplate reader. This is a very convenient method of
analyzing the
transfected cells, because microplate readers are commonly used in most
biomedical
laboratories. The multi-well plate coated with transfection reagent can be
widely used in
most laboratories to study gene regulation, gene function, molecular therapy,
and signal
transduction. Also, if different kinds of transfection reagents are coated on
the different
wells of multi-well plates, the plates can be used to screen many transfection
or delivery
reagents efficiently. Recently, 1,536 and 3,456 well plates have been
developed, which
may also be used according to the methods described herein.
In preferred embodiments, the transfection device is stored in a material
suitable for
packaging which may be plastic (e.g., cellophane), an elastomeric material,
thin metal,
Mylar , or other polyester film material. The storage may be with or without
oxygen
and/or carbon dioxide absorbers. The transfection plates prepared as described
herein may
be stored for at least 5 months at room temperature with retention of
significant cell-
transfecting activity.
The transfection reagent is preferably a cationic compound which can introduce
biomolecules, such as nucleic acids into cells. Preferred embodiments use
cationic
oligomers, such as low molecular weight polyethyleneimine (PEI). More
preferably, the
transfection agent is a degradable cationic polymer. Optionally, the
transfection agent
includes a cell-targeting or an intracellular-targeting moiety and/or a
membrane-
destabilizing component, as well as delivery enhancers.
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In general, delivery enhancers fall into two categories. These are viral
carrier
systems and non-viral carrier systems. As human viruses have evolved ways to
overcome
the barriers to transport into the nucleus discussed above, viruses or viral
components are
useful in transport of nucleic acid into cells. Additionally, the degradable
polymers may be
conjugated to or associated with a viral or non-viral protein to enhance
transfection
efficiency. For example, vesicular stomatitis virus G protein (VSVG) and other
peptides or
proteins which are known to those of skill in the art may be added to the
polymers in order
to improve transfection efficiency.
Another example of a viral component useful as a delivery enhancer is the
hemagglutinin peptide (HA-peptide). This viral peptide facilitates transfer of
biomolecules
into cells by endosome disiuption. At the acidic pH of the endosome, this
protein causes
release of the biomolecule and carrier into the cytosol.
Non-viral delivery enhancers may be either polymer-based or lipid-based. They
are
generally polycations which act to balance the negative charge of the nucleic
acid.
Polycationic polymers have shown significant promise as non-viral gene
delivery enhancers
due in part to their ability to condense DNA plasmids of unlimited size and to
safety
concerns with viral vectors. Examples include peptides with regions rich in
basic amino
acids such as oligo-lysine, oligo-arginine or a coinbination thereof and
polyethylenimine
(PEI). These polycationic polymers facilitate transport by condensation of
DNA. Branched
chain versions of polycations such as PEI and Starburst dendrimers can mediate
both DNA
condensation and endosome release (Boussif, et al. (1995) Proc. Natl. Acad.
Sci USA vol.
92: 7297-7301). PEI is a highly branched polymer with terminal amines that are
ionizable
at pH 6.9 and internal amines that are ionizable at pH 3.9 and because of this
organization,
can generate a change in vesicle pH that leads to vesicle swelling and
eventually, release
from endosome entrapment.
Another means to enhance delivery is to design a ligand on the transfection
reagent.
The ligand must have a receptor on the cell that has been targeted.
Biomolecule delivery
into the cell is then initiated by receptor recognition. When the ligand binds
to its specific
cell receptor, endocytosis is stimulated. Examples of ligands which have been
used with
various cell types to enhance biomolecule transport are galactose,
transferrin, the
glycoprotein asialoorosomucoid, adenovirus fiber, malaria circumsporozite
protein,
epidermal growth factor, human papilloma virus capsid, fibroblast growth
factor and folic
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acid. In the case of the folate receptor, the bound ligand is internalized
through a process
tenned potocytosis, where the receptor binds the ligand, the surrounding
membrane closes
off from the cell surface, and the internalized material then passes through
the vesicular
membrane into the cytoplasm (Gottschalk, et al. (1994) Gene Ther 1:185-191).
Various agents have been used for endosome disruption. Besides the HA-protein
described above, defective-virus particles have also been used as
endosomolytic agents
(Cotten, et al. (July 1992) Proc. Natl. Acad. Sci. USA vol. 89: pages 6094-
6098). Non-
viral agents are either amphiphillic or lipid-based.
The release of biomolecules such as DNA into the cytoplasm of the cell can be
enhanced by agents that either mediate endosome disruption, decrease
degradation, or
bypass this process all together. Chloroquine, which raises the endosomal pH,
has been
used to decrease the degradation of endocytosed material by inhibiting
lysosomal hydrolytic
enzyines (Wagner, et al. (1990) Proc Natl Acad Sci USA vol. 87: 3410-3414).
Branched
chain polycations such as PEI and starburst dendrimers also promote endosome
release as
discussed above.
To completely bypass endosomal degradation, subunits of toxins such as
Diptheria
toxin and Pseudomonas exotoxin have been utilized as components of chimeric
proteins
that can be incorporated into a gene/gene carrier complex (Uherek, et
al.(1998) J Biol.
Chem. vol. 273: 8835-8841). These components promote shuttling of the nucleic
acid
through the endosomal membrane and back through the endoplasmic reticulum.
Once in the cytoplasm, the nucleic acid must find its way to the nucleus.
Localization to the nucleus may be enhanced by inclusion of a nuclear
localization signal
on the nucleic acid-carrier. A specific amino acid sequence that functions as
a nuclear-
localization signal (NLS) is used. The NLS on a cargo-carrier complex
interacts with a
specific nuclear transport receptor protein located in the cytosol. Once the
cargo-carrier
complex is assembled, the receptor protein in the complex is thought to malce
multiple
contacts with nucleoporins, thereby transporting the complex through a nuclear
pore. After
a cargo-carrier complex reaches its destination, it dissociates, freeing the
cargo and other
components.
Subsequences from the SV40 large T-antigen has been used for transport into
nuclei. This short sequence from SV40 large T-antigen acts as a signal that
causes the
transport of associated macromolecules into the nucleus.
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Biodegradable cationic polymers typically exhibit low cytotoxicity, but also
low
transfection efficiency due to rapid degradation, making them less competitive
against other
carriers for gene transfer and otller applications. These degradable cationic
polymers
improve transfection efficiency by linking low molecular weight cationic
compounds or
oligomers together with degradable linkers. The linker molecules may contain
biologically,
physically or cheinically cleavable bonds, such as hydrolysable bonds,
reducible bonds, a
peptide sequence with enzyme specific cleavage sites, pH sensitive, or sonic
sensitive
bonds. The degradation of these polymers may be achieved by methods including,
but not
limited to hydrolysis, enzyme digestion, and physical degradation methods,
such as optical
cleavage (photolysis).
One of the advantages of the degradable cationic polymers described herein is
that
degradation of the polymers is controllable in terms of rate and site of
polymer degradation,
based on the type and structures of the linlcers.
lii preferred embodiments, the transfection reagent includes a plurality of
cationic
molecules and at least one degradable linker molecule connecting said cationic
molecules
in a branched arrangement.
Cationic oligomers, such as low molecular weight polyetllyleneimine (PEI), low
molecular weight poly(L-lysine) (PLL), low molecular weight chitosan, and low
molecular
weight PAMAM dendrimers, can be used to make the polymers described herein.
Furthermore, any molecule containing amines with more than three reactive
sites can be
used.
Cationic oligomers may be selected from, but are not limited to:
(i) a cationic compound of formula (A) or (B) or a combination thereof:
/R2 R4
R, N\ R6-~NH-R,+R$
R3 R5 Formula B
Formula A
wherein Rl is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another
Formula A, or
Formula B;
R2 is a straight chain alkylene group of the formula: -(CH2)a- wherein a is an
integer
number from 2 to 10;
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R3 is a straight chain alkylene group of the formula: -(CbH2b)- wherein b is
an integer
number from 2 to 10;
R4 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or
Formula B;
R5 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, another Formula A, or
Formula B;
R6 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, Formula A, or another
Formula B;
R7 a straight or branched chain allcylene group of the formula: -(CH2c)- in
which c is an
integer number from 2 to 10; and
R8 is a hydrogen atom, an alkyl of 2 to 10 carbon atoms, Formula A, or another
Formula B;
(ii) a cationic dendritic or branched polyamidoamine (PAMAM) with terininated
primary or
secondary amino groups;
(iii) a cationic polyamino acid; and
(iv) a cationic polycarbohydrate.
Examples of such cationic molecules include, but are not limited to, the
cationic
molecules shown in Table 1.
Table 1: Structures of cationic com~ounds and oligomers according to preferred
embodiments of the invention
Symbol Name Structure
Pentaethylenehexamine H
Cl H2N'---- N~~NH2
C2 Linear polyethylenimine ( N
Mw=423) ~ ~n
Branched 151
C3 polyethylenimine NH H2N
(Mw=600) r--j
H2NI/-N N
Branched H ~-N
C4 polyethylenimine H N~ NH
(Mw=1200) 2 H2N~
N,N' -Bis(2-aminopropyl)-H
C5 ethylenediamine H2N-'-~~H~-iN~~NH2
Spermine N H
NH2 N C6 H2N~~H~~i
C7 N-(2-aminoethyl)-1,3- H2N--"~N,-,,~,NH2
propanediamine H
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Symbol Name Structure
N-(3-aminopropyl)-1,3- HzNNNH2
C8 propanediamine H
N,N'-Bis(2-aminoethyl)-
C9 1,3-propanediamine H2N NN"-"---'NH2
C10 Poly(amidoamine) PAMAM
Dendrimer
Cll Po1y(propyleneimine) DAB-Ain-16
dendrimer
C12 Spermidine H2N'--\H-,,-,,_,,NH2
C13 1,4-Bis(3-aminopropyl) H2N1,,,~,,,N N~,,-~NH2
piperazine
1-(2-
p,
C14 minoethyl) iperazine H vN~~ NH2
H2N-\-- N ~,,NH2
C15 Tri(2-aminoethyl)amine
NH2
C16 Poly(L-lysine)
Cationic polymers used herein may include priinary or secondary amino groups,
which can be conjugated with active ligands, such as sugars, peptides,
proteins, and other
molecules. In a preferred embodiment, lactobionic acid is conjugated to the
cationic
polymers. The galactosyl uiiit provides a useful targeting molecule towards
hepatocyte
cells due to the presence of galactose receptors on the surface of the cells.
In a fiuther
embodiment, lactose is conjugated to the degradable cationic polymers in order
to introduce
galactosyl units onto the polymer.
Degradable linking molecules include, but are not limited to, di- and multi-
acrylates, di- and multi-methacrylates, di- and multi-acrylamides, di- and
multi-
isothiocyanates, di- and multi-isocyanates, di- and multi-epoxides, di- and
inulti-aldehydes,
di- and multi-acyl chlorides, di- and multi-sulfonyl chlorides, di- and multi-
halides, di- and
multi-anhydrides, di- and multi-malemides, di- and multi-carboxylic acids, di-
and multi-a-
haloacetyl groups, and di- and multi-N-hydroxysuccinimide esters, which
contain at least
one biodegradable spacer. The following formula describes a linker which may
be used
according to preferred embodiments:
A(Z)d
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wherein A is a spacer molecule having at least one degradable bond, Z is a
reactive residue
which reacts with amino group, and d is an integer equal to or more than two
and wherein
A and Z are bound covalently.
Several embodiments of reactive residues of the linker molecules have been
illustrated in Table 2, however these examples are not limiting to the scope
of the
invention. Reactive residues may be selected from, but are not limited to,
acryloyl,
maleimide, halide, carboxyl acylhalide, isocyanate, isothiocyanate, epoxide,
aldehyde,
sulfonyl chloride, and N-hydroxysuccinimide ester groups or combinations
thereof.
Table 2: Structures of biodegradable linker molecules used in preferred
embodiments of the invention
Symbol Name Structure
1,3-Butanediol ~ ~
L1 diacrylate H2C=HC-C-O-CHCH2CH2-O-C-CH=CH2
CH3
2-Methyl-2,4- ~ CH3 ~
L2 pentanediol diacrylat H2C=HC-C-O-CHCH2C-O-C-CH=CH2
CH3 CH3
Trimethylolpropane / ~
L3 triacrylate CH3CH2C-IOCHZ-C-CH=CH~)
' 3
2,4-Pentanediol O O
L4 diacrylate H2C=HC-C- -CHCH2 CH-O-C-CH=CH2
CH3 CH3
Pentaerythritol 0
11
L5 tetraacrylate C~CH20-C-CH=CH2 )
4
Dipentaerythritol 0
if
H2C=HC-C-OH2C H2 ~
pentaacrylate 2( 2 ~ -(CH O-C-CH=CH2) L6 HOH2C ~c-c-o-c-c ~ 3
Di(trimethylolprop o 0
C H
ane) tetraacrylate H2C=HC-C-OHz ~ 2 i CH20-C-CH=CH2
L7 C2H5 C-CH2-O-C-C C2H5
H2C=HC-C-OH2C CH20-C-CH=CH2
O 0
1,4-Butanediol ~ o
L8 diac late H2C=HC-C-O-CH2CH2CH2CH2-O-C-CH=CH2
1,6-Hexanediol ~ O
L9 H C=HC-C-0-CH CH CH 0-C-CH=CH
diacrylate a 2( z)a a- z
2,5-Dimethyl-2,5- o cH3 cH3 0
L10 hexanediol H2C=HC-C-O-CCHZCH2C-O-C-CH=CH2
CH3 CH3
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Symbol Name Structure
diacrylate
The degradation rates of the polymers can be controlled by changing the
polymer
composition, feed ratio, and the molecular weight of the polymers. For
example, when
linkers with bulkier alkyl groups are used, the polymers will degrade slower.
Also,
increasing molecular weight will cause a decrease in the degradation rate in
some cases.
Degradation rates of the polymers may be controlled by adjusting the ratio of
cationic
polymer to linker or by changing the various degradable linlcer molecules.
Acrylate linkers are much cheaper than disulfide-containing linkers, because
synthesis of the disulfide-containing linlcers is more difficult. Acrylate
linkers can be
hydrolysable in any water solution. Therefore a polynler containing acrylate
linkers can be
degraded in various environments as long as it contains water. Tlius, polymers
containing
acrylate linkers have broad applications compared to disulfide-linker-
containing polymers.
In addition, the degradation rate of polymers with disulfide-linkers are
usually the same, but
the degradation rate of polymers synthesized with acrylate linkers can vary
depending on
the different acrylate linkers used.
In some embodiments, the transfection reagent can be mixed with a matrix, such
as
proteins, peptides, polysaccharides, or otlier polymers. The protein can be
gelatin, collagen,
bovine serum albumin or any other protein that can be used in affixing
proteins to a surface.
The polymers can be hydrogels, copolymers, non-degradable or biodegradable
polymers
and biocompatible materials. The polysaccharide can be any compound that can
form a
membrane and coat the delivery reagent, such as chitosan. t11er reagents,
such as
cytotoxicity reductive reagents, cell binding reagents, cell growing reagents,
cell
stimulating reagents or cell inhibiting reagents and the compounds for
culturing specific
cells, can be also affixed to the transfection device along with the
transfection or delivery
reagent. The transfection agent may comprise both a degradable cationic
polyiner and a
non-degradable cationic polymer. The ratio of the non-degradable cationic
polymer to the
degradable cationic polymer is preferably from 1:0.5 to 1:20 (non-degradable :
degradable)
by weight, and more preferably from 1:2 to 1:10 by weight.
According to another embodiment, a gelatin-transfection reagent mixture,
comprising transfection reagent (e.g., lipid, polymer, lipid-polymer or
membrane
destabilizing peptide) and gelatin that is present in an appropriate solvent,
such as water or
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double deionized water, may be affixed to the transfection device. In a
further einbodiment
a cell culture reagent (e.g., fibronectin, collagen, salts, sugars, protein,
or peptides) may also
be present in the gelatin-transfection reagent mixture. The inixture is evenly
spread onto a
surface, such as a slide or multi-well plate, thus producing a transfection
surface bearing the
gelatin-transfection reagent mixture. In alternative embodiments, different
transfection
reagent-gelatin mixtures may also be spotted in discrete regions on the
surface of the
transfection device. The resulting product is allowed to dry completely under
suitable
conditions such that the gelatin-transfection reagent mixture is affixed at
the site of
application of the mixture. For example, the resulting product can be dried at
specific
temperatures or humidity or in a vacuum-dessicator.
The concentration of transfection reagent present in the mixture depends on
the
transfection efficiency and cytotoxicity of the reagent. Typically there is a
balance between
transfection efficiency and cytotoxicity. At concentrations in which a
transfection reagent
is most efficient, while keeping cytotoxicity at an acceptable level, the
concentration of
transfection reagent is at the optimal level. The concentration of
transfection reagent will
generally be in the range of about 1.0 g/ml to about 1000 g/ml. In preferred
embodiments, the concentration is from about 10 g hnl to about 600 g/ml.
Similarly, the
concentration of gelatin or another matrix depends on the experiment or assay
to be
performed, but the concentration will generally be in the range of 0.01% to
0.5% (w/v) of
the transfection reagent solution.
In preferred embodiinents, the molecules to be introduced into cells are
nucleic
acids. The nucleic acid can be DNA, RNA, DNA/RNA hybrid, peptide nucleic acid
(PNA),
etc. If the DNA used is present in a vector, the vector can be of any type,
such as a plasmid
(e.g., plasinid carrying green fluorescence protein (GFP) gene and/or
luciferase (luc) gene)
or viral-based vector (e.g. pLXSN). The DNA can also be linear fragment with a
promoter
sequence (such as CMV promoter) at the 5' end of the cDNA to be expressed and
a poly A
site at the 3' end. These gene expression elements allow the cDNA of interest
to be
transiently expressed in mammalian cells. If the DNA is a single strand
oligodeoxynucleotide (ODN), for example antisense ODN, it can be introduced
into cells to
regulate target gene expression. In embodiments using RNA the nucleic acid may
be single
stranded (antisense RNA and ribozyme) or double stranded (RNA interference,
SiRNA). In
most cases, the RNA is modified in order to increase the stability of RNA and
improve its
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function in down regulation of gene expression. In peptide nucleic acid (PNA),
the nucleic
acid backbone is replaced by peptide, which makes the molecule more stable.
The methods
described herein can be used to introduce nucleic acids into cells for various
purposes, for
example molecular therapy, protein function studies, or molecule mechanism
studies.
Under appropriate conditions, a nucleic acid solution is added into the
transfection
device, which has been coated with the transfection reagent, to form a nucleic
acid
transfection reagent complex. The nucleic acids are preferably dissolved in
cell culture
medium without fetal bovine serum and antibiotics, for example Dulbecco's
Modified
Eagles Medium (DMEM). However, any appropriate cell culture media may be used
including, but not limited to, Minimum Essential Eagle, F-12 Kaighn's
Modification
medium, or RPMI 1640 medium. If the transfection reagent is evenly affixed on
the slide,
the nucleic acid solution can be spotted onto discrete locations on the slide.
Alternatively,
transfection reagents may be spotted on discreet locations on the slide, and
the nucleic acid
solution can simply be added to cover the whole surface of the transfection
device. If the
transfection reagent is affixed on the bottom of multi-well plates, the
nucleic acid solution
is simply added into different wells by inulti-channel pipette, automated
device, or other
delivery methods which are well lciown in the art. The resulting product
(transfection
device coated with transfection reagent and desired nucleic acid) is incubated
for
approximately 5 min. to 60 min., more -preferably, from 25-30 minutes at room
temperature to form the nucleic acid/transfection reagent complex. In some
einbodiments,
for example, if different nucleic acid samples are spotted on discrete
locations of the slide,
the DNA solution will be removed to produce a surface bearing the nucleic acid
samples in
complex with the transfection reagent. In other alternate embodiments, the
nucleic acid
solution can be kept on the surface. Secondly, cells in an appropriate medium,
such as
DMEM, and appropriate density are plated onto the surface. The resulting
product (a
surface bearing biomolecules and plated cells) is maintained under conditions
that result in
entry of the nucleic acids of interest into the plated cells. In alternate
embodiments, the
cells are mixed with the biomolecule or nucleic acid. The cell/biomolecule
mixture is then
added to the transfection device and incubated, at room temperature.
Suitable cells for use according to the methods described herein include
prokaryotes, yeast, or higher eukaryotic cells, including plant and animal
cells, especially
mammalian cells. In preferred embodiments, eulcaryotic cells, such as
mammalian cells
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(e.g., human, inonkey, canine, feline, bovine, or murine cells), bacterial,
insect or plant
cells, are plated onto the transfection device, which is coated with
transfection reagent and
nucleic acids of interest, in sufficient density and under appropriate
conditions for
introduction/entry of the nucleic acids of interest into the eukaryotic cells
and either
expression of the DNA or interaction of the biomolecule with cellular
components. In
particular embodiments the cells may be selected from hematopoietic cells,
neuronal cells,
pancreatic cells, hepatic cells, chondrocytes, osteocytes, or myocytes. The
cells can be fully
differentiated cells or progenitor/stem cells.
In preferred embodiments, eukaryotic cells are grown in Dulbecco's Modified
Eagles Medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS)
with
L-glutamine and penicillin/streptomycin (pen/strep). It will be appreciated by
those of skill
in the art that certain cells should be cultured in a special medium, because
some cells need
special nutrition, such as growth factors and amino acids. Appropriate media
for culture of
particular cell types are known to those of skill in the art. The optimal
density of cells
depends on the cell types and the purpose of experiment. For example, a
population of 70-
80% confluent cells is preferred for gene transfection, but for
oligonucleotide delivery the
optimal condition is 30-50% confluent cells. For example, if 5x 104 293
cells/well were
seeded onto a 96 well plate, the cells would reach 90% confluency at 18-24
hours after cell
seeding. For HeLa 705 cells, only 1 ac 104 cells/well are needed to reach a
similar confluent
percentage in a 96 well plate.
After the cells are seeded on the surface containing the nucleic acid
samples/transfection reagent, the cells are incubated under optimal conditions
for the cell
type (e.g. 37 C, 5-10% C02). The culture time is dependent on the purpose of
experiment.
Typically, the cells are incubated for 24 to 48 hours for cells to express the
target gene in
the case of gene transfection experiments. In the analysis of intracellular
trafficking of
biomolecules in cells, minutes to several hours of incubation may be required
and the cells
can be observed at defined time points.
The results of biomolecule delivery can be analyzed by different methods. In
the
case of gene transfection and antisense nucleic acid delivery, the target gene
expression
level can be detected by reporter genes, such as green fluorescent protein
(GFP) gene,
luciferase gene, or (3-galactosidase gene expression. The signal of GFP can be
directly
observed under a microscope, the activity of luciferase can be detected by a
luminometer,
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and the blue product catalyzed by (3-galactosidase can be observed under a
microscope or
determined by a microplate reader. One of skill in the art is fainiliar with
how these
reporters function and how they may be introduced into a gene delivery system.
The
nucleic acid and its product, or other biomolecules delivered according to
methods
described herein and the target modulated by these biomolecules can be
determined by
various methods, such as detecting immunofluorescence or enzyme
immunocytochemistry,
autoradiography, or in situ hybridization. If immunofluorescence is used to
detect
expression of an encoded protein, a fluorescently labeled antibody that binds
the target
protein is used (e.g., added to the slide under conditions suitable for
binding of the antibody
to the protein). Cells containing the protein are then identified by detecting
a fluorescent
signal. If the delivered molecules can modulate gene expression, the target
gene expression
level can also be determined by metlzods such as autoradiography, in situ
hybridization, and
in situ PCR. However, the identification method depends on the properties of
the delivered
biomolecules, their expression product, the target modulated by it, and/or the
final product
resulting from delivery of the biomolecules.
Example 1
Preparation of DeQradable Cationic Polyiner
The synthesis of a polymer which is derived from polyethylenimine oligomer
with
molecular weight of 600 (PEI-600) and 2,4-pentandiol diacrylate (PDODA) is
provided as a
general procedure for preparation of a degradable cationic polymer. To a vial,
4.32 g of
PEI-600 in 25 ml of methylene chloride were added by using pipette or syringe.
2.09 g of
PDODA was quickly added to the above PEI-600 solution with stirring. The
reaction
mixture was stirred for 4 hours at room temperature (201 C). Then, the
reaction mixture was
neutralized by adding 50 ml of 2M HCI. The white precipitate was centrifuged,
washed
with methylene chloride, and dried at room temperature under reduced pressure.
Example 2
Prgparation of Transfectable Cell Culture Device with Degradable Cationic
Polymer
Degradable cationic polymer was prepared as indicated in Example 1. Linear
polyethyleneimine (L-PEI) based polymer and lipid based polymers were used for
transfecting plasmid DNA into mammalian cells in vitro to evaluate the
transfection
efficiency. For L-PEI based polymer, jet PEI (Qbiogene) transfection reagent
was used.
Lipofectamine2000 (Invitrogen) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-
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trimethylammonium salts (DOTAP; Sigma-Aldrich) were employed as lipid based
polymers. Degradable cationic polymer and DOTAP were dissolved in methanol,
and jet
PEI and Lipofectamine2000 were diluted by deionized water. Flat bottom 96-well
cell
culture plates (bottom surface: 0.32 cm2 per each well; BD Biosciences) were
treated with
these polymer solutions. The actual amounts affixed on the bottom were as
follows: (a)
Degradable cationic polymer; 3 g per well, tllus 9.4 g/cm2, (b) jet PEI; 1
l per well, (c)
Lipofectamine2000; 0.375 gg per well, (d) DOTAP; 2 and 4 pmole per well. These
plates
were dried at room temperature under reduced pressure and sealed in a vacuum
pack until
use.
Example 3
Transfection witli Transfectable Cell Culture Device for 293 Cells
25 or 50 ng of pEGFP-N1 plasmid (purchased from Clontech) in 25 l of opti-
MEM I (Invitrogen) was added in each well and kept at room temperature for 25
minutes.
Then, 5 X 104 of 293 cells in 100 l of Dulbecco's modified Eagle Medium
(DMEM)
(Invitrogen) with 10% calf serum (Invitrogen) were added and incubated at 37 C
in 7.5% of
CO2. After 24 to 36 hrs. incubation, transfection efficiency was estimated by
observing
EGFP fluorescence by using epifluorescent microscope (1X70, Olympus).
Transfection efficiencies are shown in Table 3. Degradable cationic polpner
and jet
PEI, i.e. L-PEI based polymer showed high transfection efficiency.
Teble 3
Polymer EGFP-positive cells
C7egradable cationic polymer 60-70%
Jet PEI 50%
LipQfectarrmine2000 Less than 10%
DOTAP 4 pn7ole%.rell 0%
DOTAP 2 pmoleFwell 0%
Example 4
Evaluation of Cytotoxicity
Cytotoxicity of the described method was evaluated. Cell shape of 293 cells,
transfected as indicated in Example 3, were compared by microscopic
observation (Figure
1). Cells transfected by using degradable polymer showed normal shape, which
was similar
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to intact 293 cells. However, those transfected by using L-PEI based polymer
(jet PEI) and
lipid based polymer (Lipofectamine2000) were rounded. We concluded that the
degradable
cationic polymer can deliver genes without damaging cells.
Example 5
Optimization of Degradable Cationic Polymer Amount
Various amounts of degradable cationic polymer were affixed on the cell
culture
devices, and transfection efficiency was evaluated. 96-well cell culture
plates were coated
with degradable cationic polymer by the saine protocol as shown in Example 2.
The actual
amount of polymer was as follows: 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10 and 20 }ag
per well.
Then, transfection was carried out as described in Example 3 and transfected
cells were
incubated at 37 C in 7.5% of COZ. Amount of plasmid DNA added before seeding
cells
was 0.13, 0.25, 0.50 or 1.0 -pg per well. After 40 hours incubation,
percentage of
fluorescing cells and cell condition were estimated by epifluorescent
microscopy. Figure 2
shows percentages of EGFP-positive cells after transfection. High transfection
efficiencies
were allocated between 2.5 to 5.0 ug per well (thus, 7.8 to 16 gg/cm) of
degradable
cationic polymer witli 0.25 and 0.5 pg per well (thus, 0.78 to 1.6 g/cm) of
plasmid DNA.
Cell condition in these experiments is shown in Figure 3. Cells transfected in
the
plates with L-PEI and lipid based polymers had rounded shape and had
aggregated. The
morphology was due to cytotoxicity. Cell condition was acceptable when the
amount of
degradable cationic polymer affixed on the bottom of the plate was from 2.5 to
5.0 pg per
well. Also, all the plasmid DNA conditions that we tested gave us good cell
condition with
degradable cationic polymer if the amount was from 2.5 to 5.0 pg per well.
Example 6
StabilitYSti:udy
There are products in the marlcet, in which there is a coating on the surface
of cell
culture devices for a special purpose, for example, to assist cell growth.
Normally, the
coating material is a kind of protein, lilce collagen or fibronectin. As they
are temperature-
sensitive, these cell culture devices require refrigerated storage which is a
disadvantage,
especially if they are bulky. For this reason, stability at room teinperature
is an important
feature.
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The cell culture/transfection devices of this invention were tested to study
their
stability after long-term storage. The cell transfection devices were prepared
as described
in Example 2, and vacuum-sealed in Mylar Bags (Dupont Corp.), which is a film
with an
oxygen barrier material and aluminum foil with or without oxygen and carbon
dioxide
absorbers. Storage was at 25 C. Then, transfection efficiency with plasmid DNA
carrying
luciferase gene (pCMV-LUC) was tested periodically. The procedure for
transfectable cell
culture devices was as described in Example 3 except the plasmid DNA was
different.
Luciferase activity of cells were determined by using a Dynex MLX Microtiter
plate
luminometer and Luciferase Assay System (Promega Corp. Madison, WI USA) to
determine transfection efficiency.
Figures 4, 5 and 6 show change of transfection efficiencies after storage at
251C
with 02 and/or C 2 absorbing materials in Mylar Bags. There was no obvious
decrease of
transfection efficiency after 5 month storage. Moreover, even when cell
culture devices
were kept at 25 C in Mylar Bags without 02 and/or CO2 absorbing materials,
transfection
efficiency was stable after 5 month and still quite high (Figure 7). The cell
culture devices
of this invention are quite stable at room temperature. The device can be
stored without
special storage conditions.
Example 7
Preparation of Non-degradable Cationic Pol n~er
Non-degradable polymer was prepared as follows: Approximately 5 g of
polyethlenimine (Aldrich, product number: 408727) was dissolved in 50 ml of
dichloromethane, then 100 ml of 2.OM hydrogen chloride in diethyl ether
(Aldrich, product
number: 455180) was added and mixed well to form polymer hydrochloride. Then,
the
polymer hydrochloride was collected by centrifuge, and rinsed with 150 ml of
diethyl ether.
This rinse with diethyl ether was carried out twice. The resultant
precipitation after the
rinse was dried under vacuum condition at room temperature for 3 hours. Then,
the dried
powder was stored at 4 C with desiccant until use.
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Example 8
Preparation of 96-well Transfectable Cell Culture Device with Degradable
Cationic
Polymer and Non-degradable Cationic Polylner
Degradable cationic polymer was prepared as indicated in Example 1. Non-
degradable cationic polymer was obtained as described in Example 7. Both
polymers were
dissolved in methanol and mixed together to make a coating solution. The final
concentration of each polymer was: Degradable cationic polymer; 40 g/ml, and
Non-
degradable cationic polymer; 10 g/ml. Then, flat bottom 96-well cell culture
plates
(bottom surface: 0.32 cm2 per each well; BD Biosciences) were treated with the
coating
solution. Actually, 25 l of the coating solution was dispensed in each well,
and dried
under vacuum condition to remove methaiiol. Under these coating conditions, 1
g of
degradable cationic polymer was affixed on each well of a 96-well plate;
therefore the
density of the degradable cationic polymer was 3.1 g/cm'. Also, 0.25 g of
non-
degradable cationic polymer was affixed on each well of the 96-well plate so
that the
density of the non-degradable cationic polymer was 0.78 g/cm2. In total, 1.25
g of
polymer was affixed on each well of the 96-well plate; therefore the density
of polymer was
3.9 g/cmz. The cell culture devices prepared in this example were vacuum
sealed in Mylar
Bags with desiccant, and stored at room temperature until further use.
Example 9
Preparation of 12-well Transfectable Cell Culture Device with Degradable
Cationic
Polymer and Non-degradable Cationic Pol)mj
Degradable cationic polymer was prepared as indicated in Example 1. Non-
degradable cationic polymer was obtained as described in Example 7. Both
polymers were
dissolved in methanol and mixed together to malce a coating solution. The
final
concentration of each polymer was: Degradable cationic polymer; 80 g/ml, and
Non-
degradable cationic polymer; 10 g/ml. Then, flat bottom 12-well cell culture
plates
(bottom surface: 3.8 cm2 per each well; BD Biosciences) were treated with
these polymer
solutions. 100 l of the coating solution was dispensed in each well, and
dried under
vacuum condition to remove methanol. Under these coating conditions, 8.0 g of
degradable cationic polymer was affixed on each well of a 12-well plate so
that the density
of the degradable cationic polymer was 2.1 g/cmzand 1.0 g of non-degradable
cationic
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polyiner was affixed on each well of the 12-well plate so that the density of
the non-
degradable cationic polymer was 0.26 g/cm2. In total, 9.0 g of polymer was
affixed on
each well of the 12-well plate; therefore the density of polymer was 2.4
g/cm2. The cell
culture devices prepared in this exainple were vacuuin sealed in Mylar Bags
with desiccant,
and stored at room temperature until further use.
Example 10
Preparation of 6-well Transfectable Cell Culture Device with Degradable
Cationic Polymer
and Non-degradable Cationic PolM
Degradable cationic polymer was prepared as indicated in Example 1. Non-
degradable cationic polyiner was obtained as described in Exainple 7. Both
polymers were
dissolved in methanol and mixed together to make a coating solution. The final
concentration of each polymer was: Degradable cationic polymer; 80 g/ml, and
Non-
degradable cationic polymer; 10 g/ml. Then, flat bottom 6-well cell culture
plates (bottom
surface: 9.6 cm2 per each well; BD Biosciences) were treated with the coating
solution. 200
l of the coating solution was dispensed in each well, and dried under vacuum
condition to
remove methanol. Under these coating conditions, 16 g of degradable cationic
polymer
was affixed on each well of a 6-well plate so that the density of the
degradable cationic
polymer was 1.7 g/cm2 and, 2.0 g of non-degradable cationic polymer was
affixed on
each well of the 6-well plate so that the density of the non-degradable
cationic polymer was
0.21 g/cm2. In total 18 g of polymer was affixed on each well of the 6-well
plate;
therefore the density of polymer was 1.9 g/cmZ. The cell culture devices
prepared in this
example were vacuum sealed in Mylar Bags with desiccant, and stored at room
temperature
until further use.
Example 11
Transfection with 96-well Transfectable Cell Culture Devices Prepared with
Degradable
and Non-degradable Cationic Pol n ers
Mammalian cells were incubated in 10-cm cell culture dishes, rinsed with
phosphate-buffered saline, and treated with trypsin solution. Then, the
trypsinized cells
were diluted in appropriate cell culture medium with serum to prepare a cell
suspension.
The cell density used in this example is shown in Table 4.
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pEGFP-Nl plasmid was diluted in opti-MEM, and the final concentration was
adjusted to 10 g/ml. Then, 25 l of the plasmid solution was added in each
well of the 96-
well transfectable cell culture device prepared as indicated in Example 8, and
kept at room
temperature for 25 minutes. Then, 100 gl of the cell suspension was added in
the well, and
incubated at 37 C in 7.5% of CO2. After 36 to 48-hour incubation, transfection
efficiency
was estimated by observing EGFP fluorescence by using epifluorescent
microscope (IX70,
Olympus).
Table 4 indicates the percentage of the cells with EGFP fluorescence in
various
mammalian cell lines. The 96-well transfectable cell culture device in this
invention
transfected various mammalian cell lines with high efficiency.
Example 12
Transfection with 12-well Transfectable Cell Culture Devices Prepared with
Degradable
and Non-degradable Cationic Polymers
Marmnalian cells were incubated in 10-cm cell culture dishes, rinsed with
phosphate-buffered saline, and treated with trypsin solution. Then, the
trypsinized cells
were diluted in appropriate cell culture medium with serum to prepare cell
suspension. The
cell density used in this example is shown in Table 4.
pEGFP-Nl plasmid was diluted in opti-MEM, and the final concentration was
adjusted to 5 gghnl. Then, 200 l of the plasmid solution was added in each
well of the 12-
well transfectable cell culture device prepared as indicated in Example 9, and
kept at room
temperature for 25 minutes. Then, 1 ml of the cell suspension was added in the
well, and
incubated at 37 C in 7.5% of COZ. After 36 to 48-hour incubation, transfection
efficiency
was estimated by observing EGFP fluorescence by using epifluorescent
microscope (IX70,
Olympus).
Table 4 indicates the percentage of the cells with EGFP fluorescence in
various
mammalian cell lines. The 12-well transfectable cell culture device in this
inventiontransfected various mammalian cell lines with high efficiency.
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Example 13
Transfection with 6-well Transfectable Cell Culture Devices Prepared with
Degradable and
Non-degradable Cationic Pol ers
Mammalian cells were incubated in 10-cm cell culture dishes, rinsed with
phosphate-buffered saline, and treated with trypsui solution. Then, the
trypsinized cells
were diluted in appropriate cell culture medium with serum to prepare cell
suspension. The
cell density used in this example is shown in Table 4.
pEGFP-N1 plasmid was diluted in opti-MEM, and the final concentration was
adjusted to 5 g/ml. Then, 400 gl of the plasmid solution was added in each
well of the 6-
well transfectable cell culture device prepared as indicated in Example 10,
and kept at room
temperature for 25 minutes. Then, 2 ml of the cell suspension was added in the
well, and
incubated at 37 C in 7.5% of CO2. After 36 to 48-hour incubation, transfection
efficiency
was estimated by observing EGFP fluorescence by using epifluorescent
microscope (IX70,
Olyinpus).
Table 4 indicates the percentage of the cells with EGFP fluorescence in
various
mammalian cell lines. The 6-well transfectable cell culture device in this
invention
transfected various mammalian cell lines with high efficiency.
Tablo4 Percentage of cells with fluorescence, and initial cell density
% EGFP Initial Cell Density (eells/ml)
Cell Line 6-well 12-well 96-well 6-well 12-vvell 96-well
293 80 80 80 2.5x105 2.5x105 2.5x105
705 80 80 80 1.5x105 1.5x105 1.5x105
COS-7 70 70 70-80 1.5-2.0x105 1.5x105 1.5x105
HT-1080 70-80 70 70 0.5-1.0x105 O.5x105 1.0x105
HeLa 70 80 70 1.0-2.0x105 1.0x105 0.5x105
MDCK 50 60 1.0x105 1.5x105
CHO-K1 30-40 50 50 1.5x105 2.0x105 2.0x105
DU145 30-40 40 '30-40 1.5-2.0x105 1.5x105 1.5x105
A549 20-30 20-30 30-40 2.0x105 2.0x105 2.0x105
CV-1 20-30 30 20-30 1.0x105 1.5x105 1.5x105
HepG2 20 30 10-20 1.0-2.0x105 1.5x105 1.5x10$
It will be understood by those of skill in the art that numerous and various
modifications can be made without departing from the spirit of the present
invention.
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Therefore, it should be clearly understood that the forms of the present
invention are
illustrative only and are not intended to limit the scope of the present
invention.
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