U.S. patent application number 10/829513 was filed with the patent office on 2004-10-07 for chelating systems for use in the delivery of compounds to cells.
Invention is credited to Budker, Vladimir G., Hagstrom, James E., Monahan, Sean D., Slattum, Paul M., Trubetskoy, Vladimir, Wolff, Jon A..
Application Number | 20040197318 10/829513 |
Document ID | / |
Family ID | 22237841 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197318 |
Kind Code |
A1 |
Wolff, Jon A. ; et
al. |
October 7, 2004 |
Chelating systems for use in the delivery of compounds to cells
Abstract
Chelator containing compounds are utilized in the delivery of
molecules, polymers, nucleic acids and genes to animal cells. At
least one chelator such as crown ether is attached to a polymer and
then associated with another polymer such as DNA. An ion is then
added to the mixture thereby forming condensed DNA. In condensed
form and in complex with the chelator, DNA can be delivered to a
cell.
Inventors: |
Wolff, Jon A.; (Madison,
WI) ; Monahan, Sean D.; (Madison, WI) ;
Hagstrom, James E.; (Middleton, WI) ; Slattum, Paul
M.; (Madison, WI) ; Budker, Vladimir G.;
(Middleton, WI) ; Trubetskoy, Vladimir;
(Middleton, WI) |
Correspondence
Address: |
Mark K. Johnson
Mirus Corporation
505 S. Rosa Rd.
Madison
WI
53719
US
|
Family ID: |
22237841 |
Appl. No.: |
10/829513 |
Filed: |
April 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10829513 |
Apr 22, 2004 |
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09234606 |
Jan 21, 1999 |
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60093230 |
Jul 17, 1998 |
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Current U.S.
Class: |
424/93.21 ;
435/455 |
Current CPC
Class: |
C12N 15/87 20130101;
A61K 48/0025 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/093.21 ;
435/455 |
International
Class: |
A61K 048/00; C12N
015/85 |
Claims
We claim:
1) A process for the delivery of a compound to a cell, comprising:
a) associating a chelator with a polymer; b) delivering the polymer
to the cell.
2) The process of claim 1 wherein the polymer comprises a first
polymer and a second polymer.
3) The process of claim 2 wherein the first polymer and the second
polymer comprise nucleic acids, proteins, genes, antisense
polymers, DNA/RNA hybrids, synthetic polymers.
4) The process of claim 3 wherein the chelator comprises a crown
ether system.
5) The process of claim 4 wherein the crown ether system comprises
covalently binding the crown ether to the second polymer.
6) The process of claim 2 wherein the first polymer comprises a
nucleic acid.
7) The process of claim 6 wherein the second polymer comprises a
net positive charge.
8) The process of claim 7 wherein the second polymer comprises
polyamine.
9) The process of claim 1 further comprising associating a chelator
with a polymer and a signal.
10) A process for compacting a nucleic acid for delivery to a cell,
comprising: a) associating a polychelator with a nucleic acid; b)
delivering the nucleic acid to the cell.
11) The complex of claim 10 wherein associating a polychelator
further comprises associating a polychelator and a salt and a
nucleic acid.
12) A complex for delivering a compound to a cell, comprising: a) a
nucleic acid; b) a polychelator; and, c) an ion.
13) The complex of claim 12 wherein the complex is less than 500
nanometers in diameter.
Description
FEDERALLY SPONSORED RESEARCH
[0001] N/A
FIELD OF THE INVENTION
[0002] he invention relates to chelator-containing systems for use
in biologic systems. More particularly, chelators and polychelators
are utilized in the delivery of molecules, polymers, nucleic acids
and genes to cells.
BACKGROUND
[0003] Polymers are used for drug delivery for a variety of
therapeutic purposes. Polymers have also been used in research for
the delivery of nucleic acids (polynucleotides and
oligonucleotides) to cells with an eventual goal of providing
therapeutic processes. Such processes have been termed gene therapy
or anti-sense therapy. One of the several methods of nucleic acid
delivery to the cells is the use of DNA-polycation complexes. It
has been shown that cationic proteins like histones and protamines
or synthetic polymers like polylysine, polyarginine, polyomithine,
DEAE dextran, polybrene, and polyethylenimine may be effective
intracellular delivery agents while small polycations like spermine
are ineffective. The following are some important principles
involving the mechanism by which polycations facilitate uptake of
DNA:
[0004] Polycations provide attachment of DNA to the target cell
surface. The polymer forms a cross-bridge between the polyanionic
nucleic acids and the polyanionic surfaces of the cells. As a
result the main mechanism of DNA translocation to the intracellular
space might be non-specific adsorptive endocytosis which may be
more effective then liquid endocytosis or receptor-mediated
endocytosis. Furthermore, polycations are a convenient linker for
attaching specific receptors to DNA and as result, DNA-polycation
complexes can be targeted to specific cell types.
[0005] Polycations protect DNA in complexes against nuclease
degradation. This is important for both extra- and intracellular
preservation of DNA. Gene expression is also enabled or increased
by preventing endosome acidification with NH.sub.4Cl or
chloroquine. Polyethylenimine, which facilitates gene expression
without additional treatments, probably disrupts endosomal function
itself. Disruption of endosomal function has also been accomplished
by linking to the polycation endosomal-disruptive agents such as
fusion peptides or adenoviruses.
[0006] Polycations can also facilitate DNA condensation. The volume
which one DNA molecule occupies in a complex with polycations is
drastically lower than the volume of a free DNA molecule. The size
of a DNA/polymer complex is probably critical for gene delivery in
vivo. In terms of intravenous injection, DNA needs to cross the
endothelial barrier and reach the parenchymal cells of interest.
The largest endothelia fenestrae (holes in the endothelial barrier)
occur in the liver and have an average diameter of 100 nm. The
trans-epithelial pores in other organs are much smaller, for
example, muscle endothelium can be described as a structure which
has a large number of small pores with a radius of 4 nm, and a very
low number of large pores with a radius of 20-30 nm. The size of
the DNA complexes is also important for the cellular uptake
process. After binding to the target cells the DNA-polycation
complex should be taken up by endocytosis. Since the endocytic
vesicles have a homogenous internal diameter of about 100 nm in
hepatocytes and are of similar size in other cell types, DNA
complexes smaller than 100 nm are preferred.
[0007] Condensation of DNA
[0008] A significant number of multivalent cations with widely
different molecular structures have been shown to induce
condensation of DNA.
[0009] Two approaches for compacting (used herein as an equivalent
to the term condensing) DNA:
[0010] 1. Multivalent cations with a charge of three or higher have
been shown to condense DNA. These include spermidine, spermine,
Co(NH.sub.3).sub.6.sup.3+,Fe.sup.3+, and natural or synthetic
polymers such as histone H1, protamine, polylysine, and
polyethylenimine. Analysis has shown DNA condensation to be favored
when 90% or more of the charges along the sugar-phosphate backbone
are neutralized.
[0011] 2. Polymers (neutral or anionic) which can increase
repulsion between DNA and its surroundings have been shown to
compact DNA. Most significantly, spontaneous DNA self-assembly and
aggregation process have been shown to result from the confinement
of large amounts of DNA, due to excluded volume effect.
[0012] Depending upon the concentration of DNA, condensation leads
to three main types of structures:
[0013] 1) In extremely dilute solution (about 1 ug/mL or below),
long DNA molecules can undergo a monomolecular collapse and form
structures described as toroid.
[0014] 2) In very dilute solution (about 10 ug/mL) microaggregates
form with short or long molecules and remain in suspension.
Toroids, rods and small aggregates can be seen in such
solution.
[0015] 3) In dilute solution (about 1 mg/mL) large aggregates are
formed that sediment readily.
[0016] Toroids have been considered an attractive form for gene
delivery because they have the smallest size. While the size of DNA
toroids produced within single preparations has been shown to vary
considerably, toroid size is unaffected by the length of DNA being
condensed. DNA molecules from 400 bp to genomic length produce
toroids similar in size. Therefore one toroid can include from one
to several DNA molecules. The kinetics of DNA collapse by
polycations that resulted in toroids is very slow. For example DNA
condensation by Co(NH.sub.3).sub.6Cl.sub.3 needs 2 hours at room
temperature.
[0017] The mechanism of DNA condensation is not clear. The
electrostatic force between unperturbed helices arises primarily
from a counterion fluctuation mechanism requiring multivalent
cations and plays a major role in DNA condensation. The hydration
forces predominate over electrostatic forces when the DNA helices
approach closer then a few water diameters. In a case of
DNA--polymeric polycation interactions, DNA condensation is a more
complicated process than the case of low molecular weight
polycations. Different polycationic proteins can generate toroid
and rod formation with different size DNA at a ratio of positive to
negative charge of 0.4. T4 DNA complexes with polyarginine or
histone can form two types of structures; an elongated structure
with a long axis length of about 350 nm (like free DNA) and dense
spherical particles. Both forms exist simultaneously in the same
solution. The reason for the co-existence of the two forms can be
explained as an uneven distribution of the polycation chains among
the DNA molecules. The uneven distribution generates two
thermodynamically favorable conformations.
[0018] The electrophoretic mobility of DNA-polycation complexes can
change from negative to positive in excess of polycation. It is
likely that large polycations don't completely align along DNA but
form polymer loops that interact with other DNA molecules. The
rapid aggregation and strong intermolecular forces between
different DNA molecules may prevent the slow adjustment between
helices needed to form tightly packed orderly particles.
SUMMARY
[0019] Described in this specification is a process for the
delivery of a compound to a cell, comprising associating a chelator
with a polymer, then delivering the polymer to the cell. The
polymer may comprise a first polymer and a second polymer. The
first polymer and the second polymer may comprise nucleic acids,
proteins, genes, antisense polymers, DNA/RNA hybrids, synthetic
polymers.
[0020] In another embodiment, a process is described for compacting
a nucleic acid for delivery to a cell, comprising associating a
polychelator with a nucleic acid and delivering the nucleic acid to
the cell. The process may additionally include associating a
polychelator and a salt and and a nucleic acid.
[0021] In another embodiment, a complex is disclosed for delivering
a compound to a cell, comprising a nucleic acid, a polychelator and
an ion.
[0022] Reference is now made in detail to the preferred embodiments
of the invention, examples of which are illustrated in the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a visual depiction illustrating an example of one
polychelator beginning to associate with a non-compacted DNA
strand.
[0024] FIG. 2 is a visual depiction illustrating a example of the
addition of an ion to one polychelator associating with a DNA
strand thereby depicting a condensed DNA strand.
DETAILED DESCRIPTION
[0025] Gene therapy research may involve the biological pH gradient
that is active within organisms as a factor in delivering a
polynucleotide to a cell. Different pathways that may be affected
by the pH gradient include cellular transport mechanisms, endosomal
disruption/breakdown, and particle disassembly (release of the
DNA).
[0026] Gradients that can be useful in gene therapy research
involve ionic gradients that are related to cells. For example,
both Na.sup.+ and K.sup.+ have large concentration gradients that
exist across the cell membrane. Systems containing chelators (see
FIG. 1) can utilize such gradients to influence delivery of a
polynucleotide to a cell. DNA can be compacted using chelator
containing systems by adding cations that interact with the
chelator. By interacting an appropriate cation with a DNA/chelator
containing system, DNA condensation can take place. Since the
cation utilized for compaction may exist in higher concentration
outside of the cell membrane compared to inside the cell membrane,
this natural ionic gradient can be utilized in delivery
systems.
[0027] The interaction of the cations with the chelator will
install positive charge to the system and therefore increase or
initiate the interaction/compaction between the system and DNA (see
FIG. 2). If the system containing the chelator already contains
positive charge, for example poly L-lysine containing crown ether
moieties, the interaction of the cations with the crown ether ring
would serve to increase the positive charge of the system, and may
influence the interaction/compaction with DNA. Once the complex is
delivered to and associated with or incorporated into the cell
(endocytosis), the complexed cation is in low concentration since
the internal portion of the cellular ionic gradient contains lower
cation concentration. Therefore, decomplexation of the cation from
the crown ether takes place and decreases binding of the crown
ether containing system to the DNA, facilitating the release of DNA
from the complex. The chelator system described provides a
reversible binding system for DNA compaction and delivery.
[0028] Polymers
[0029] A polymer is a molecule built up by repetitive bonding
together of smaller units called monomers. In this application the
term polymer includes both oligomers which have two to about 80
monomers and polymers having more than 80 monomers. The polymer can
be linear, branched network, star, comb, or ladder types of
polymer. The polymer can be a homopolymer in which a single monomer
is used or can be copolymer in which two or more monomers are used.
Types of copolymers include alternating, random, block and
graft.
[0030] To those skilled in the art of polymerization, there are
several categories of polymerization processes that can be utilized
in the described process. The polymerization can be chain or step.
This classification description is more often used that the
previous terminology of addition and condensation polymer. "Most
step-reaction polymerizations are condensation processes and most
chain-reaction polymerizations are addition processes" (M. P.
Stevens Polymer Chemistry: An Introduction New York Oxford
University Press 1990). Template polymerization can be used to form
polymers from daughter polymers.
[0031] Step Polymerization:
[0032] In step polymerization, the polymerization occurs in a
stepwise fashion. Polymer growth occurs by reaction between
monomers, oligomers and polymers. No initiator is needed since
there is the same reaction throughout and there is no termination
step so that the end groups are still reactive. The polymerization
rate decreases as the functional groups are consumed.
[0033] Typically, step polymerization is done either of two
different ways. One way, the monomer has both reactive functional
groups (A and B) in the same molecule so that
A-B yields -[A-B]-
[0034] Or the other approach is to have two difunctional
monomers.
A-A+B-B yields -[A-A-B-B]-
[0035] Generally, these reactions can involve acylation or
alkylation. Acylation is defined as the introduction of an acyl
group (--COR) onto a molecule. Alkylation is defined as the
introduction of an alkyl group onto a molecule.
[0036] If functional group A is an amine then B can be (but not
restricted to) an isothiocyanate, isocyanate, acyl azide,
N-hydroxysuccinimide, sulfonyl chloride, aldehyde (including
formaldehyde and glutaraldehyde), ketone, epoxide, carbonate,
imidoester, carboxylate, or alkylphosphate, arylhalides
(difluoro-dinitrobenzene), anhyderides or acid halides,
p-nitrophenyl esters, o-nitrophenyl pentachlorophenyl esters, or
pentafluorophenyl esters. In other terms when function A is an
amine then function B can be acylating or alkylating agent or
amination.
[0037] If functional group A is a sulfhydryl then function B can be
(but not restricted to) an iodoacetyl derivative, maleimide,
aziridine derivative, acryloyl derivative, fluorobenzene
derivatives, or disulfide derivative (such as a pyridyl disulfide
or 5-thio-2-nitrobenzoic acid{TNB} derivatives).
[0038] If functional group A is carboxylate then function B can be
(but not restricted to) a diazoacetate or an amine in which a
carbodiimide is used. Other additives may be utilized such as
carbonyldiimidazole, DMAP, N-hydroxysuccinimide or alcohol using
carbodiimide and DMAP.
[0039] If functional group A is an hydroxyl then function B can be
(but not restricted to) an epoxide, oxirane, or an amine in which
carbonyldiimidazole or N,N'-disuccinimidyl carbonate, or
N-hydroxysuccinimidyl chloroformate or other chloroformates are
used.
[0040] If functional group A is an aldehyde or ketone then function
B can be (but not restricted to) an hydrazine, hydrazide
derivative, amine (to form a Schiff Base that may or may not be
reduced by reducing agents such as NaCNBH.sub.3) or hydroxyl
compound to form a ketal or acetal.
[0041] Yet another approach is to have one difunctional monomer so
that
[0042] A-A plus another agent yields -[A-A]-.
[0043] If function A is a sulfhydryl group then it can be converted
to disulfide bonds by oxidizing agents such as iodine (12) or
NaIO.sub.4 (sodium periodate), or oxygen (O.sub.2). Function A can
also be an amine that is converted to a sulfhydryl group by
reaction with 2-Iminothiolate (Traut's reagent) which then
undergoes oxidation and disulfide formation. Disulfide derivatives
(such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid{TNB}
derivatives) can also be used to catalyze disulfide bond
formation.
[0044] Functional group A or B in any of the above examples could
also be a photoreactive group such as aryl azides, halogenated aryl
azides, diazo, benzophenones, alkynes or diazirine derivatives.
[0045] Reactions of the amine, hydroxyl, sulfhydryl, carboxylate
groups yield chemical bonds that are described as amide, amidine,
disulfide, ethers, esters, enamine, urea, isothiourea, isourea,
sulfonamide, carbamate, carbon-nitrogen double bond (imine),
alkylamine bond (secondary amine), carbon-nitrogen single bonds in
which the carbon contains a hydroxyl group, thio-ether, diol,
hydrazone, diazo, or sulfone.
[0046] Chain Polymerization: In chain-reaction polymerization
growth of the polymer occurs by successive addition of monomer
units to limited number of growing chains. The initiation and
propagation mechanisms are different and there is usually a
chain-terminating step. The polymerization rate remains constant
until the monomer is depleted.
[0047] Monomers containing vinyl, acrylate, methacrylate,
acrylamide, methaacrylamide groups can undergo chain reaction which
can be radical, anionic, or cationic. Chain polymerization can also
be accomplished by cycle or ring opening polymerization. Several
different types of free radical initiatiors could be used that
include peroxides, hydroxy peroxides, and azo compounds such as
2,2'-Azobis(-amidinopropane) dihydrochloride (AAP). A compound is a
material made up of two or more elements.
[0048] Types of Monomers: A wide variety of monomers can be used in
the polymerization processes. These include positive charged
organic monomers such as amines, imidine, guanidine, imine,
hydroxylamine, hydrozyine, heterocycles (like imidazole, pyridine,
morpholine, pyrimidine, or pyrene. The amines could be pH-sensitive
in that the pKa of the amine is within the physiologic range of 4
to 8. Specific amines include spermine, spermidine,
N,N'-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and
3,3'-Diamino-N,N-dimethyldipropylammonium bromide.
[0049] Monomers can also be hydrophobic, hydrophilic or
amphipathic. Amphipathic compounds have both hydrophilic
(water-soluble) and hydrophobic (water-insoluble) parts.
Hydrophilic groups indicate in qualitative terms that the chemical
moiety is water-preferring. Typically, such chemical groups are
water soluble, and are hydrogen bond donors or acceptors with
water. Examples of hydrophilic groups include compounds with the
following chemical moieties carbohydrates; polyoxyethylene,
peptides, oligonucleotides and groups containing amines, amides,
alkoxy amides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic
groups indicate in qualitative terms that the chemical moiety is
water-avoiding. Typically, such chemical groups are not water
soluble, and tend not to hydrogen bond. Hydrocarbons are
hydrophobic groups. Monomers can also be intercalating agents such
as acridine, thiazole organge, or ethidium bromide.
[0050] Other Components of the Monomers and Polymers: The polymers
have other groups that increase their utility. These groups can be
incorporated into monomers prior to polymer formation or attached
to the polymer after its formation. These groups include: Targeting
Groups--such groups are used for targeting the polymer-nucleic acid
complexes to specific cells or tissues. Examples of such targeting
agents include agents that target to the asialoglycoprotein
receptor by using asiologlycoproteins or galactose residues. Other
proteins such as insulin, EGF, or transferrin can be used for
targeting. Protein refers to a molecule made up of 2 or more amino
acid residues connected one to another as in a polypeptide. The
amino acids may be naturally occurring or synthetic. Peptides that
include the RGD sequence can be used to target many cells. Peptide
refers to a linear series of amino acid residues connected to one
another by peptide bonds between the alpha-amino group and carboxyl
group of contiguous amino acid residues. Chemical groups that react
with sulfhydryl or disulfide groups on cells can also be used to
target many types of cells. Folate and other vitamins can also be
used for targeting. Other targeting groups include molecules that
interact with membranes such as fatty acids, cholesterol, dansyl
compounds, and amphotericin derivatives.
[0051] After interaction of the supramolecular complexes with the
cell, other targeting groups can be used to increase the delivery
of the drug or nucleic acid to certain parts of the cell. For
example, agents can be used to disrupt endosomes and a nuclear
localizing signal (NLS) can be used to target the nucleus.
[0052] A variety of ligands have been used to target drugs and
genes to cells and to specific cellular receptors. The ligand may
seek a target within the cell membrane, on the cell membrane or
near a cell. Binding of ligands to receptors typically initiates
endocytosis. Ligands could also be used for DNA delivery that bind
to receptors that are not endocytosed. For example peptides
containing RGD peptide sequence that bind integrin receptor could
be used. In addition viral proteins could be used to bind the
complex to cells. Lipids and steroids could be used to directly
insert a complex into cellular membranes.
[0053] The polymers can also contain cleavable groups within
themselves. When attached to the targeting group, cleavage leads to
reduce interaction between the complex and the receptor for the
targeting group. Cleavable groups include but are not restricted to
disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,
acetals, ketals, enol ethers, enol esters, enamines and imines.
[0054] Reporter or marker molecules are compounds that can be
easily detected. Typically they are fluorescent compounds such as
fluorescein, rhodamine, texas red, cy 5, cy 3 or dansyl compounds.
They can be molecules that can be detected by UV or visible
spectroscopy or by antibody interactions or by electron spin
resonance. Biotin is another reporter molecule that can be detected
by labeled avidin. Biotin could also be used to attach targeting
groups.
[0055] A polycation is a polymer containing a net positive charge,
for example poly-L-lysine hydrobromide. The polycation can contain
monomer units that are charge positive, charge neutral, or charge
negative, however, the net charge of the polymer must be positive.
A polycation also can mean a non-polymeric molecule that contains
two or more positive charges. A polyanion is a polymer containing a
net negative charge, for example polyglutamic acid. The polyanion
can contain monomer units that are charge negative, charge neutral,
or charge positive, however, the net charge on the polymer must be
negative. A polyanion can also mean a non-polymeric molecule that
contains two or more negative charges. The term polyion includes
polycation, polyanion, zwitterionic polymers, and neutral polymers.
The term zwitterionic refers to the product (salt) of the reaction
between an acidic group and a basic group that are part of the same
molecule. Salts are ionic compounds that dissociate into cations
and anions when dissolved in solution. Salts increase the ionic
strength of a solution, and consequently decrease interactions
between nucleic acids with other cations.
[0056] Chelators
[0057] A Chelator is a polydentate ligand, a molecule that can
occupy more than one site in the coordination sphere of an ion,
particularly a metal ion, primary amine, or single proton. Examples
of chelators include crown ethers, cryptates, and non-cyclic
polydentate molecules.
[0058] A crown ether is a cyclic polyether containing
(--X--(CR.sub.1-2).sub.n).sub.m units, where n=1-3 and m=3-8. The X
and CR.sub.1-2 moieties can be substituted, or at a different
oxidation states. X can be oxygen, nitrogen, or sulfur, carbon,
phosphorous or any combination thereof. R can be H, C, O, S, N, P.
The crown ether ring system is named as [(n+1)m crown m] for
X=oxygen, as [(n+1)m azacrown m] when X=nitrogen, as [(n+1)m
thiocrown m] when X=sulfur. In the case of two or more heteroatoms
present in the ring the heteroatom positions are specified. For
example, 12-crown-4, 4-aminobenzo-12-crown-4,
4-formylbenzo-12-crown-4, 4-hydroxybenzo-12-crown-4,
4-acryloylamidobenzo-12-crown-4, 4-vinylbenzo-12-crown-4,
15-crown-5, 4-aminobenzo-15-crown-5, 4-formylbenzo-15-crown-5,
4-hydroxybenzo-15-crown-5, 4-acryloylamidobenzo-15-crown-5,
4-vinylbenzo-15-crown-5, 18-crown-6, benzo-18-crown-6,
4-aminobenzo-18-crown-6, 4-formylbenzo-18-crown-6,
4-hydroxybenzo-18-crown-6, 4-acryloylamidobenzo-18-crown-6,
4-vinylbenzo-18-crown-6, (18-crown-6)-2,3,11,12-tetracarboxcylic
acid, 2-hydroxymethyl-18-crown-6, 2-aminomethyl-18-crown-6,
1-aza-18-crown-6, 16-crown-4, 20-crown-4, and 18-crown-6,
polyvinylbenzo 15-crown-5.
[0059] A subset of crown ethers described as a cryptate contain a
second (--X--(CR.sub.1-2).sub.n).sub.z strand where z=3-8. The
beginning X atom of the strand is an X atom in the
(--X--(CR.sub.1-2).sub.n).sub.m unit, and the terminal CH.sub.2 of
the new strand is bonded to a second X atom in the
(--X--(CR.sub.1-2).sub.n).sub.m unit.
[0060] Non-cyclic polydentate molecules containing
(--X--(CR.sub.1-2).sub.- n).sub.m unit(s), where n=1-4 and m=1-8.
The X and CR.sub.1-2 moieties can be substituted, or at a different
oxidation states. X can be oxygen, nitrogen, or sulfur, carbon,
phosphorous or any combination thereof. For example di(ethylene
glycol), hexa(ethylene glycol) and other polyglycols, tri(propylene
glycol), ethylene diamine, N,N,N',N'-tetramethyldiethyldiam- ine,
N,N,N',N'-ethylenediaminetetraacetic acid, spermine, spermidine,
diethylenetriamine, 1,3-diaminopropane, phenanthroline,
1,2-bis(dimethylphosphino)-ethane,
1,4-bis(dicyclohexylphosphino)butane,
1,2-bis(phenylphosphino)-ethane,
1,4-bis(phenylphosphino)-butane.
[0061] A polychelator is a polymer associated with a plurality of
chelators by an ionic or covalent bond and can include a spacer. A
spacer is any linker known to those skilled in the art to enable
one to join one moiety to another moiety. The moieties can be
hydrophilic or hydrophobic. Preferred spacer groups include, but
are not limited to C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl,
C6-C18 aralkyl, C6-C18 aralkenyl, C6-C18 aralkynyl, ester, ether,
ketone, alcohol, polyol, amide, amine, polyglycol, polyamine,
thiol, thio ether, thioester, phosporous containing, and
heterocyclic. The polymer can be cationic, anionic, zwitterionic,
neutral, or contain any combination of cationic, anionic,
zwitterionic, or neutral groups with a net charge being cationic,
anionic or neutral, and may contain steric stabilizers, peptides,
proteins, signals, or amphipathic compound for the formation of
micellar, reverse micellar, or unilamellar structures. Preferably
the amphipathic compound can have a hydrophilic segment that is
cationic, anionic, or zwitterionic, and can contain polymerizable
groups, and a hydrophobic segment that can contain a polymerizable
group.
[0062] A Steric stabilizer is a long chain hydrophilic group that
prevents aggregation of final polymer by sterically hindering
particle to particle electrostatic interactions. Examples include:
alkyl groups, PEG chains, polysaccharides, hydrogen molecules,
alkyl amines. Electrostatic interactions are the non-covalent
association of two or more substances due to attractive forces
between positive and negative charges.
[0063] Signals
[0064] In a preferred embodiment, a chemical reaction can be used
to attach a signal to a nucleic acid complex. The signal is defined
in this specification as a molecule that modifies the nucleic acid
complex and can direct it to a cell location (such as tissue cells)
or location in a cell (such as the nucleus) either in culture or in
a whole organism. By modifying the cellular or tissue location of
the foreign gene, the expression of the foreign gene can be
enhanced.
[0065] The signal can be a protein, peptide, lipid, steroid, sugar,
carbohydrate, nucleic acid or synthetic compound. The signals
enhance cellular binding to receptors, cytoplasmic transport to the
nucleus and nuclear entry or release from endosomes or other
intracellular vesicles.
[0066] Nuclear localizing signals enhance the targeting of the gene
into proximity of the nucleus and/or its entry into the nucleus.
Such nuclear transport signals can be a protein or a peptide such
as the SV40 large T ag NLS or the nucleoplasmin NLS. These nuclear
localizing signals interact with a variety of nuclear transport
factors such as the NLS receptor (karyopherin alpha) which then
interacts with karyopherin beta. The nuclear transport proteins
themselves could also function as NLS's since they are targeted to
the nuclear pore and nucleus.
[0067] Signals that enhance release from intracellular compartments
(releasing signals) can cause DNA release from intracellular
compartments such as endosomes (early and late), lysosomes,
phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans
golgi network (TGN), and sarcoplasmic reticulum. Release includes
movement out of an intracellular compartment into cytoplasm or into
an organelle such as the nucleus. Releasing signals include
chemicals such as chloroquine, bafilomycin or Brefeldin A1 and the
ER-retaining signal (KDEL sequence), viral components such as
influenza virus hemagglutinin subunit HA-2 peptides and other types
of amphipathic peptides.
[0068] Cellular receptor signals are any signal that enhances the
association of the gene or particle with a cell. This can be
accomplished by either increasing the binding of the gene to the
cell surface and/or its association with an intracellular
compartment, for example: ligands that enhance endocytosis by
enhancing binding the cell surface. This includes agents that
target to the asialoglycoprotein receptor by using
asiologlycoproteins or galactose residues. Other proteins such as
insulin, EGF, or transferrin can be used for targeting. Peptides
that include the RGD sequence can be used to target many cells.
Chemical groups that react with sulfhydryl or disulfide groups on
cells can also be used to target many types of cells. Folate and
other vitamins can also be used for targeting. Other targeting
groups include molecules that interact with membranes such as
lipids fatty acids, cholesterol, dansyl compounds, and amphotericin
derivatives. In addition viral proteins could be used to bind
cells.
[0069] Systems
[0070] The present invention provides systems for the transfer of
polynucleotides, oligonucleotides, and other compounds into
association with cells within tissues in situ and in vivo,
utilizing chelators such as crown ether. Crown ether containing
polymers are constructed by the reductive amination of
4'-formylbenzo-15-crown-5 with poly L-lysine to the extent of
approximately 40-85% modification of the polycation primary amines.
In a preferred embodiment, poly L-lysine containing
benzo-15-crown-5 moiety (with primary amines acylated with acetic
anhydride) is utilized for the compaction of pDNA upon addition of
cations such as Li.sup.+ and NH.sub.4.sup.+. Poly L-lysine
containing the benzo-15-crown-5 moiety is effective in a
transfection of 3T3-Swiss albino mouse fibroblasts.
[0071] In other preferred embodiments,
4-acryloylamidobenzo-18-crown-6 and 4'-vinylbenzo-18-crown-6 were
polymerized for use in compacting DNA and delivering the compacted
DNA to cells. The polymer backbone that contains the crown ether
can be made from cationic, neutral, anionic, or zwitterionic
monomers, or any combination thereof. Additionally, systems can be
constructed by mixing polyions with another polymer containing the
crown ether system.
[0072] In addition to polynucleotide condensation utilizing crown
ether containing polymers and polycation systems containing crown
ethers, related systems containing atoms other than carbon in the
ring of a heterocyclic compound (heteroatoms) may be employed
towards the same result. Related systems include: cryptands
(including mono and poly-cyclic cryptates), aza and thia analogs of
crown ethers, crown ether systems in which the heteroatoms may be
selected from oxygen, nitrogen, sulfur, or any combination
thereof.
[0073] Polychelators for gene therapy and gene therapy research can
involve anionic systems as well as charge neutral or
charge-positive systems. The anionic polymer containing chelators
and related hosts can be utilized in "recharging" (another layer
having a different charge) the polychelator-polynucleotide complex.
The resulting recharged complex can be formed with an appropriate
amount of negative charge such that the resulting complex has a net
negative charge. The interaction between the polycation and the
polyanion can be ionic, can involve the ionic interaction of
chelator systems of the two polymer layers with shared cations
(FIG. 1), or can be crosslinked between cationic and anionic sites
with a crosslinking system (including cleavable crosslinking
systems, such as those containing disulfide bonds). The interaction
between the chelators located on the two polymer layers can be
influenced with the use of added cations to the system. With the
appropriate choice of cation, the layers can be made to
disassociate from one another as the cation diffuses from the
complex into the cell in which the concentration of the cation is
low (use of a cation gradient).
[0074] The polychelators described can contain additional chemical
moieties. Additional chemical moieties include (but are not limited
to) cell recognition an targeting systems, gene transfer enhancing
signals, signals that enhance cellular binding to receptors,
cytoplasmic transport to the nucleus and nuclear entry or release
from endosomes or other intracellular vesicles. Cations that can be
used include (but are not limited to) Li.sup.+, Na.sup.+,
Mg.sub.2.sup.+, K.sup.+, Ca.sup.2+, Cs.sup.+, Fe.sup.3+,
NH.sub.4.sup.+, and primary amines.
[0075] Chelator condensed polynucleotide, complex containing a
chelator-polyion system, or combination thereof may be delivered
intravasculary, intrarterially, intravenously, orally,
intraduodenaly, via the jejunum (or ileum or colon), rectally,
transdermally, subcutaneously, intramuscularly, intraperitoneally,
intraparenterally, via direct injections into tissues such as the
liver, lung, heart, muscle, spleen, pancreas, brain (including
intraventricular), spinal cord, ganglion, lymph nodes, lymphatic
system, adipose tissues, thyroid tissue, adrenal glands, kidneys,
prostate, blood cells, bone marrow cells, cancer cells, tumors, eye
retina, via the bile duct, or via mucosal membranes such as in the
mouth, nose, throat, vagina or rectum or into ducts of the salivary
or other exocrine glands. "Delivered" means that the polynucleotide
becomes associated with the cell. The polynucleotide can be on the
membrane of the cell or inside the cytoplasm, nucleus, or other
organelle of the cell.
[0076] The process of delivering a polynucleotide to a cell has
been commonly termed "transfection" or the process of
"transfecting" and also it has been termed "transformation". The
polynucleotide could be used to produce a change in a cell that can
be therapeutic. The delivery of polynucleotides or genetic material
for therapeutic and research purposes is commonly called "gene
therapy". The polynucleotides or genetic material being delivered
are generally mixed with transfection reagents prior to
delivery.
[0077] A biologically active compound is a compound having the
potential to react with biological components. More particularly,
biologically active compounds utilized in this specification are
designed to change the natural processes associated with a living
cell. For purposes of this specification, a cellular natural
process is a process that is associated with a cell before delivery
of a biologically active compound. In this specification, the
cellular production of, or inhibition of a material, such as a
protein, caused by a human assisting a molecule to an in vivo cell
is an example of a delivered biologically active compound.
Pharmaceuticals, proteins, peptides, polypeptides, hormones,
cytokines, antigens, viruses, oligonucleotides, and nucleic acids
are examples of biologically active compounds.
[0078] The term nucleic acid is a term of art that refers to a
polymer containing at least two bases. Bases include purines and
pyrimidines that further include natural compounds guanosine,
cytosine, inosine, guanine, thymidine, uracil and synthetic
derivatives of purines and pyrimidines or natural analogs. (A
polynucleotide is distinguished here from an oligonucleotide by
containing more than 120 monomeric units.) Nucleotides are the
monomeric units of nucleic acid polymers. The term includes
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form
of an oligonucleotide messenger RNA, anti-sense, plasmid DNA, parts
of a plasmid DNA or genetic material derived from a virus (viral
DNA) linear DNA, or chromosomal DNA or derivatives of these groups.
RNA may be in the form of a tRNA (transfer RNA), snRNA (small
nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA),
anti-sense RNA, and ribozymes. Anti-sense is a polynucleotide that
interferes with the function of DNA and/or RNA. The term nucleic
acid further refers to a string of at least two
base-sugar-phosphate combinations. Natural nucleic acids have a
phosphate backbone, artificial nucleic acids may contain other
types of backbones and bases. In addition these forms of DNA and
RNA may be single, double, triple, or quadruple stranded. The term
also includes PNAs (peptide nucleic acids), phosphothionates, and
other variants of the phosphate backbone of native nucleic
acids.
[0079] Ionic (electrostatic) interactions are the non-covalent
association of two or more substances due to attractive forces
between positive and negative charges, or partial positive and
partial negative charges.
[0080] Condensed Nucleic Acids: A method of condensing a polymer is
defined as decreasing its linear length, also called compacting.
Condensing a polymer also means decreasing the volume that the
polymer occupies. An example of condensing nucleic acid is the
condensation of DNA that occurs in cells. The DNA from a human cell
is approximately one meter in length but is condensed to fit in a
cell nucleus that has a diameter of approximately 10 microns. The
cells condense (or compacts) DNA by a series of packaging
mechanisms involving the histones and other chromosomal proteins to
form nucleosomes and chromatin. The DNA within these structures is
rendered partially resistant to nuclease DNase) action. The process
of condensing polymers can be used for delivering them into cells
of an organism.
[0081] A delivered polymer can stay within the cytoplasm or nucleus
apart from the endogenous genetic material. Alternatively, the
polymer could recombine (become a part of) the endogenous genetic
material. For example, DNA can insert into chromosomal DNA by
either homologous or non-homologous recombination.
[0082] Intravascular: An intravascular route of administration
enables a polymer or polynucleotide to be delivered to cells more
evenly distributed and more efficiently expressed than direct
injections. Intravascular herein means within a tubular structure
called a vessel that is connected to a tissue or organ within the
body. Within the cavity of the tubular structure, a bodily fluid
flows to or from the body part. Examples of bodily fluid include
blood, lymphatic fluid, or bile. Examples of vessels include
arteries, arterioles, capillaries, venules, sinusoids, veins,
lymphatics, and bile ducts. The intravascular route includes
delivery through the blood vessels such as an artery or a vein.
[0083] An administration route involving the mucosal membranes is
meant to include nasal, bronchial, inhalation into the lungs, or
via the eyes.
[0084] Association
[0085] In a preferred embodiment, an ion is added to the
polychelator. Preferably, the ion is a Li.sup.+, Fe.sup.3+,
Na.sup.+, M.sup.2+, K.sup.+, Ca.sup.2+, Mn.sup.2+, Ni.sup.2+,
Cu.sup.2+, Cu.sup.3+, Zn.sup.2+, Al.sup.3+, or NH.sub.4.sup.+ salt,
or a primary amine, or polymer containing one or more primary
amine(s) for example, polylysine, histone, or polyethylenimine.
Then the charged polychelator is put in contact with another
polyion such as an oligonucleotide or nucleic acid forming a
complex. One or more polyions may be added to the complex to
recharge, attach signals, increase stability, and add
protection.
[0086] In a preferred embodiment, a polychelator is put in contact
with another polyion. An ion is added to the polychelator--polyion
mixture. Preferably, the ion is a Li.sup.+, Fe.sup.3+, Na.sup.+,
Mg.sup.2+, K.sup.+, Ca.sup.2+, Mn.sup.2+, Ni.sup.2+, Cu.sup.2+,
Cu.sup.3+, Zn.sup.2+, Al.sup.3+, or NH.sub.4.sup.+ salt, or a
primary amine, or polymer containing one or more primary amine(s)
for example, polylysine, histone, or polyethylenimine. The polyion
may comprise an oligonucleotide or nucleic acid. One or more
polyions may then be added to the complex.
[0087] In another preferred embodiment, a polychelator is put in
contact with another polyion. The polyion comprises a polycation,
for example polylysine, histone, or polyethylenimine, or a
polyanion, for example polyglutamic acid, a oligonucleotide, a
nucleic acid, a zwitterionic polymer, a neutral polymer, or a
polychelator. One or more polyions may then be added to the
complex.
[0088] In another preferred embodiment, a polycation is associated
with a polyanion. Preferably, the polycation comprises a polyamine.
More preferably, the polyamine comprises a polymer containing
primary amines, for example polylysine, histone, or
polyethylenimine. Preferably, the polyanion comprises a nucleic
acid. A chelator or polychelator is then added to the
polycation--polyanion complex. The chelator is associated with a
steric stabilizer, peptide, protein, signal, or amphipathic
compound for the formation of micellar, reverse micellar, or
unilamellar structures. The amphpathic compound can have a
hydrophilic segment that is cationic, anionic, or zwitterionic, and
can contain polymerizable groups, and a hydrophobic segment that
can contain a polymerizable group.
[0089] In another preferred embodiment, a polycation is associated
with a polyanion. Preferably, the polycation comprises a polyamine.
More preferably, the polyamine comprises a polymer containing
primary amines, for example (but not limited to) polylysine,
histone, or polyethylenimine. Preferably, the polyanion comprises a
nucleic acid. A polyion is then added to the complex. Preferably
the polyion contains primary amines but possesses a net negative
charge. A chelator or polychelator is then added to the
polyion--polycation--polyanion complex. Preferably, the chelator is
associated with a steric stabilizer, peptide, protein, signal, or
amphipathic compound for the formation of micellar, reverse
micellar, or unilamellar structures. Preferably the amphpathic
compound can have a hydrophilic segment that is cationic, anionic,
or zwitterionic, and can contain polymerizable groups, and a
hydrophobic segment that can contain a polymerizable group.
[0090] In another preferred embodiment, a polycation is associated
with a polyanion. Preferably, the polycation comprises a polyamine.
More preferably, the polyamine comprises a polymer containing
primary amines, for example (but not limited to) polylysine,
histone, or polyethylenimine. Preferably, the polyanion comprises
an oligonucleotide, more preferably being a nucleic acid. A
polychelator is then added to the polycation--polyanion complex.
Preferably, the polychelator has a net negative charge.
Example 1
[0091] Synthesis of Polyacrylamidobenzo-18-crown-6 (PAA18C6)
[0092] To a solution of 4-Acryloylamidobenzo-18-crown-6 (100 mg,
0.262 mmol, Acros Chemical Company) in toluene (0.2 mL) was added
2, 2'-azobisisobutyronitrile (200 .mu.g, 1 .mu.mol, Aldrich
Chemical Company). The resulting solution was heated to 90.degree.
C. for about 6 hrs DMSO (0.1 mL) was added, the solution was
stirred for about 12 hrs, and concentrated under reduced pressure.
The resulting residue was dissolved in DMSO at a concentration of
10 mg/mL, diluted with water and purified by column chromatography
on Sephadex G-50. High molecular weight material (>30,000) was
collected, washed with H.sub.2O (3.times.), and concentrated using
Centricon Plus-20 filters (cutoff 10,000, Millipore). The maximum
adsorption of the PAA18C6 was determined to be 255 nm with a
extinction coefficient of 8177 1 mol.sup.-1 cm.sup.-1 (Beckman DU-7
Spectrophotometer).
Example 2
[0093] Cation Binding to Polyacrylamidobenzo-18-crown-6 Based on
Auramine O Fluorescence
[0094] When added to an aqueous solution of PAA18C6, a cation that
can complex to the benzo-18-crown-6 ligand can convert the neutral
polymer into a polycation. The conversion of the polymer to a
polycation should decrease the binding of 4,
4'-(imidocarbonyl)bis(N,N-dimethylaniline) monohydrochloride
(Auramine O, a cationic dye) to the polymer. The binding affinity
of Auramine O can therefore be used for estimating the binding
affinity of different cations for the PAA18C6.
[0095] To a solution of 4,
4'-(imidocarbonyl)bis(N,N-dimethylaniline) monohydrochloride
[Auramine O] (3.times.10.sup.-5M, Sigma Chemical Company) in 10 mM
HEPES, 0.1 mM EDTA, pH 7.5, was added PAA18C6 and the fluorescence
intensity was recorded (Hitachi Fluorescence Spectrophotometer,
model F-3010, excitation wavelength=458 nm, emission wavelength=510
nm). The fluorescence intensity was increased substantially in the
presence of PAA18C6. The inverse fluorescence intensity is linearly
related to the inverse of the PAA18C6 concentration at a constant
concentration of Auramine O. The binding constant can be estimated
as 0.14.times.10.sup.6M.sup.-1 (Smid, J. Pure and Appl. Chem., 54,
2129-2140, 1982). It has been shown that Auramine O can be bounded
by polyvinylbenzo-cown ethers resulting in an enhanced fluorescence
quantum yield (Smid, J. Pure and Appl. Chem., 54, 2129-2140,
1982).
[0096] To a solution of PAA18C6 (2.times.10.sup.-5M) and Auramine O
(1.times.1.sup.-5M) in 1 mL of 10 mM triethanolamine (TEA), 0.5 mM
EDTA, pH 7.5, was added various salts. The solutions were mixed for
5 min. and the fluorescence was determined (excitation
wavelength=458 nm, emission wavelength=515 nm).
[0097] Based on the replacement of Auramine O from the PAA18C6,
dissociation constants for the various cations were estimated:
1 Cation K.sub.d .times. 10.sup.3 Li.sup.+ >100 NH.sub.4.sup.+
16 Na.sup.+ 7.6 1,3-Diaminopropane.sup.2+ 6.2 Rb.sup.+ 2.2 K.sup.+
1.0 Cs.sup.+ 0.5 Decylamine 0.3 Poly-L-lysine (PLL) <0.04
[0098] The PAA18C6 appears to bind monovalent cations with some
selectivity. The divalent cation Mg.sup.2+ and the trivalent cation
Tb.sup.3+ do not displace Auramine O from the PAA18C6. Polylysine
is shown to have a very high affinity for PAA18C6.
Example 3
[0099] Interaction of Polyacrylamidobenzo-18-crown-6 with Labeled
Poly-L-Lysine.
[0100] To poly-L-lysine (PLL) (4 mg, Sigma Chemical Company) in
potassium phosphate buffer (pH 8, 0.1 mL) was added
7-Chloro-4-nitrobenz-2-oxa-1,3-- diazole (NBD-Cl) (0.4 mg, Sigma
Chemical Company) The solution was heated at 37.degree. C. for 2 h,
cooled, and purified by gel-filtration on Sephadex G-25. The
fluorescence was determined (Hitachi Fluorescence
Spectrophotometer, model F-3010, excitation wavelength=466 nm,
emission wavelength=540 nm), and the level of modification was
estimated to be 5%.
[0101] To a solution of NBD-PLL (80 .mu.g) in 1 mL of 10 mM TEA,
0.5 mM EDTA, pH 8, was added increasing amounts of PAA 18C6 and the
fluorescence was determined (Hitachi Fluorescence
Spectrophotometer, model F-3010). The interaction of NBD-PLL with
PAA18C6 substantially decreased the quantum yield of fluorescence.
As the amount of PAA18C6 was increased, a corresponding decrease in
signal intensity was observed.
2 PAA 0 2.62 6.5 13.1 19.6 26.2 18C6 (.mu.g) I.sub.540 49 47 43 39
35 31
[0102] The relationship between fluorescence intensity and PAA18C6
complex formation indicates that fluorescence can be utilized to
estimate the stability of the PAA18C6 complex.
Example 4
[0103] Displacement of Poly-L-Lysine from
Polyacrylamidobenzo-18-crown-6 with Potassium Chloride.
[0104] To a solution of NBD-PLL (80 .mu.g) in 1 mL of 10 mM TEA,
0.5 mM EDTA, pH 8, was added PAA18C6 (26 .mu.g). The solution was
mixed for 5 min. and the fluorescence was determined (Hitachi
Fluorescence Spectrophotometer, model F-3010, excitation
wavelength=466 nm, emission wavelength=540 nm). Increasing amounts
of KCl were added, and the fluorescence was again determined. As
the amount of KCl was increased, a corresponding increase in the
fluorescence intensity was observed, indicating a dissociation of
the NBD-PLL--PAA18C6 complex.
3 KCl (mM) 0 2 4 8 10 12 30 40 I.sub.540 31 32 34 37 41 46 48
49
[0105] The results indicate that a large excess of K can replace
PLL from the complex with PAA18C6 even though PLL has a lower
dissociation constant.
Example 5
[0106] Polyacrylamidobenzo-18-crown-6 Complexes with pDNA
[0107] Complexes of pDNA and PAA18C6 were prepared by the rapid
mixing of 5 .mu.g of plasmid DNA (Zhang, G., Vargo, D., Budker, V.,
Armstrong, N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8,
1763-1772, 1997) with varying amounts of PAA18C6 in 1 mL of 10 mM
TEA buffer, 0.5 mM EDTA, pH 7.5, both with and without KCl (30 mM).
The mixtures were held at room temperature for 3 minutes and
measured for light scattering (Hitachi Fluorescence
Spectrophotometer model F3010, 500 nm for both the incident beam
and detection beam, slits for both beams were fixed at 5 nm, 90
degrees). Various amounts of a 2M KCl solution were added to each
sample, the mixtures were held at room temperature for 5 minutes,
and the light scattering was again determined.
[0108] Experiments conducted without the addition of KC; indicated
that PAA18C6 interacts with DNA at high concentrations of the
PAA18C6. However, addition of KC; to the solution dramatically
increases the ability of PAA18C6 to bind to the DNA. In 30 mM KC;
the polymer containing the crown ether is a polycation (crown
ether--potassium complex) which binds DNA efficiently. At a
DNA/PAA18C6 ratio of 18/20.5 (close to equimolar) aggregation
occurs. Further increasing the PAA18C6 concentration results in a
decrease in the sample turbidity, supposedly as result of the
decreasing size of the particle. This result is similar to that
found in the case of polylysine--DNA interactions. However, in
contrast to polylysine--DNA complexes, no aggregation of the
PAA18C6--DNA particles could be induced upon increasing salt
concentrations to 150 mM KCl in the presence of excess PAA18C6.
4 PAA 150 mM 18C6 (.mu.M) no KCl (I) 30 mM KCl (I) KCl (I) 0 2 13
14 10.3 7 203 352 20.5 16 2126 1201 34.2 35 653 632 51.3 73 456 313
102.6 200 396 326 171 415 400 --
[0109] Agarose gel analysis of the PAA18C6--DNA complexes was
performed (1% agarose). The concentration of DNA was 16.4 .mu.M,
while the concentration of PAA18C6 was varied from 0 to 51 .mu.M.
The buffer used for both sample preparation and the agarose gel
electrophoresis was 10 mM TEA buffer, 0.5 mM EDTA, pH 7.5, with KCl
(30 mM). At PAA18C6 concentrations of 20.4 .mu.M or greater, all
the DNA remained in the well. The intensity of ethidium bromide
fluorescence was also observed to be substantially decreased.
[0110] The results from both the light scattering experiments and
the DNA retardation in agarose gel indicates the formation of
PAA18C6--DNA complexes. The ability of PAA18C6 to bind DNA is
greatly enhanced by the addition of KCl to the solution.
Example 6
[0111] Condensation of DNA with Polyacrylamidobenzo-18-crown-6
[0112] Fluorescein labeled DNA was used for the determination of
DNA condensation in complexes with PAA18C6. pDNA was modified to a
level of 1 fluorescein per 100 bases using Mirus' Labellt
Fluorescein kit. The fluorescence was determined using a
fluorescence spectrophotometer (Hitachi, Model F-3010), at an
excitation wavelength of 497 nm, and an emission wavelength of 520
nm.
[0113] To fluorescein labeled DNA (8.8 .mu.M) in 1 mL 10 mM TEA
buffer, 0.5 mM EDTA, pH 7.5, and 30 mM KCl was added varying
amounts of PAA18C6. The mixtures were held at room temperature for
5 minutes and the fluorescence was determined. (see: Trubetskoy, V.
S., Slattum, P. M., Hagstrom, J. E., Wolff, J. A., Budker, V. G.,
"Quantitative Assessment of DNA Condensation," Anal. Biochem (1999)
incorporated by reference) Since fluorescence intensity is
decreased by DNA condensation, results indicate that PAA18C6
compacts DNA.
5 PAA18C6 0 3.4 6.8 10.2 13.6 17 (.mu.M) I.sub.520 149 110 61 42 25
22
Example 7
[0114] Particle Size of pDNA--Polyacrylamidobenzo-18-crown-6
Complexes.
[0115] To a solution of pDNA (16 .mu.g, 47 .mu.M) in 1 mL H.sub.2O
was added varying amounts of PAA18C6. The solutions were mixed for
2 minutes and particle sized (Particle Sizer, Brookhaven
Instruments Corporation). To a solution of pDNA (16 .mu.g) in 1 mL
of 10 mM HEPES, 50 mM KCl, pH 7.4, were added varying amounts of
PAA18C6. The solutions were mixed for 2 minutes and particle sized
(Particle Sizer, Brookhaven Instruments Corporation). It should be
noted that HEPES contains sodium, which will condense DNA in the
presence of the PAA18C6.
6 PAA H.sub.2O HEPES, 50 mM KCl 18C6 (.mu.M) nm(%) nm(%) 0 -- --
13.7 -- 38(74), 194(26) 27.4 -- 151(35), 580(65) 51.3 -- 316(25),
5623(75) 102.6 34(31), 75(56), 304(13) 86(30), 6935(70) 171 46(82),
110(18) 45(98) 273.6 194(100) 63(80), 165(20)
[0116] At a monomer ratio of PAA18C6--DNA of 60-47 (equimolar),
strong aggregation occurs in the sample. However, as the molar
excess of PAA18C6 is increased, small particles dominate the
sample. The particles are stable in solutions of high ionic
strength. For example, particles formed in 10 mM HEPES, 50 mM KCl,
pH 7.4, with a PAA18C6--DNA ratio of 274-47 (.mu.M) exhibit a size
(% of particles) profile of 63 nm (80) and 165 nm (20). As the
ionic strength of the solution is increased to 150 mM KCl, greater
than 90% of the particles have a diameter of less than 80 nm.
Example 8
[0117] Complexes Comprised of pDNA and
Poly-L-Lysine/Polyacrylamidobenzo-1- 8-crown-6.
[0118] To poly-L-lysine (PLL) (10 mg, Sigma Chemical Company) in
potassium phosphate buffer (pH 8, 0.1 mL) was added
5-(dimethylamino)naphthalene-1-- sulfonyl chloride (Dansyl
Chloride) (0.5 mg, Sigma Chemical Company) The solution was heated
at 37.degree. C. for 2 h, cooled, and purified by gel-filtration on
Sephadex G-25 to afford DANS-PLL. The fluorescence was determined
(Hitachi, model F-3010, excitation wavelength=329 nm, emission
wavelength=515 nm). The level of modification was estimated to be
1.2%. To a solution of DANS-PLL (191 .mu.M) in 1 mL of 10 mM TEA
buffer, 0.5 mM EDTA, pH 7.5, was added varying amounts of PAA18C6,
and the fluorescence was again determined.
7 PAA18C6 (.mu.M) 0 17 34 51 68 I.sub.515 3.8 8.3 10.9 13.2
15.1
[0119] As the amount of PAA18C6 was increased, the fluorescence of
the sample increased. This increase in fluorescence is attributed
to hydrophobicity of the sample since dansyl is heavily influenced
by sample hydrophobicity. In contrast, DNA has a marginal effect on
DANS-PLL fluorescence.
[0120] The particle sizing data indicates that particles prepared
from solution 3 exhibit stability in salt solutions (150 mM NaCl).
Particles prepared from PLL--p DNA showed increased size upon
addition of NaCl indicating instability in salt. Particles prepared
from PAA18C6--pDNA with NaCl exhibited increased size upon the
addition of PLL, again indicating particle instability. Particles
prepared from either solution 1 (to which PLL was added) or
solution 2 (to which PAA18C6 was added) also were shown to be
unstable, indicating that complexes formed in these cases is
different in nature to the complexes formed from solution 3.
[0121] The fluorescence of a complex prepared from DANS-PLL (25
.mu.g) and PAA18C6 (33 .mu.g) decreased from 47 to 11 after the
addition of KCl (40 mM). In contrast, a complex prepared from DNA
(100 .mu.g), DANS-PLL (25 .mu.g), and PAA18C6 (33 .mu.g) exhibited
a decrease in fluorescence from 48 to 24 in presence of KCl (40
mM). Therefore, the PAA18C6 interacts with the DANS-PLL--DNA
complex more strongly then with free DANS-PLL.
Example 9
[0122] Mouse Portal Vein Injection of
DNA--Polyacrylamidobenzo-18-crown-6 Complexes
[0123] Plasmid delivery into the portal vein of ICR mice was
performed as described (Zhang, G., Vargo, D., Budker, V.,
Armstrong, N., Knechtle, S., Wolff, J. A. Human Gene Therapy, 8,
1763-1772, 1997, incorporated by reference). To pCILuc DNA (100
.mu.g) in 1 mL H.sub.2O containing heparin (2.5 .mu.g/mL, Lypho-Med
Inc.), mannitol (15%), NaCl (100 mM), and KCl (50 mM), was added
various amounts of PAA18C6. The mixture was incubated for 5
minutes, and injected over 30 sec utilizing a 30 gauge, 0.5 inch
needle. A micro vessel clip was applied during the injection at the
junction of the hepatic vein and caudal vena cava. One day after
the injection, the animal was sacrificed, and a luciferase assay
was conducted. The amount of luciferase protein (pg) was calculated
according to the equation LP(pg)=5.1.times.10.sup.-5xRLU+3.683.
8 PAA18C6 (.mu.g/animal) Luciferase (ng per liver) 78 120.4 .+-.
25.5 156 90.7 .+-. 59.2
[0124] The experiment indicates that DNA--PAA18C6 complexes show
successful transfection activity of pCILuc DNA.
Example 10
[0125] Synthesis of Polyvinylbenzo-18-crown-6 (PV18C6)
[0126] To a solution of 4-Vinylbenzo-18-crown-6 (63.0 mg, 0.186
mmol, Acros Chemical Company) in toluene (0.2 mL) was added 2,
2'-azobisisobutyronitrile (130 .mu.g, 0.79 .mu.mol, Aldrich
Chemical Company). The resulting solution was heated to 95.degree.
C. for 18 hrs. at which time an additional portion of 2,
2'-azobisisobutyronitrile (50 .mu.g, 0.3 .mu.mol, Aldrich Chemical
Company) was added. The resulting solution was heated an additional
18 hrs. and concentrated under reduced pressure. The resulting
residue was dissolved in DMSO at a concentration of 10 mg/mL,
diluted with water and purified by column chromatography on
Sephadex G-25. High molecular weight material was collected and
further purified by column chromatography on Sephadex G-50. High
molecular weight material (>30,000) was collected, washed with
H.sub.2O (3.times.), and concentrated using Centricon Plus-20
filters (cutoff 10,000, Millipore).
Example 11
[0127] Complexes Comprised of pDNA and
Polyvinylbenzo-18-crown-6
[0128] To a solution of pDNA (23 .mu.g) in 1 mL of 10 mM HEPES, pH
7.4, both with and without CsCl (20 mM), was added various amounts
of PV18C6. After 2 min. the particle size was measured (Particle
Sizer, Brookhaven Instruments Corp.).
9 HEPES without CsCl HEPES, 20 mM CsCl PV18C6 (.mu.g) nm(%) nm(%) 0
-- -- 2.5 -- 32(91), 177(3) 5 -- 32(90), 169(10) 10 60(94), 246(6)
32(69), 81(27), 1500(4) 20 28(93), 168(7) 54(92), 243(8) 30 56(90),
253(10) 362(88), 2318(12) 50 73(100) 111(74), 9000(26)
[0129] When a near equimolar amount of PV 18C6 (around 30 .mu.g)
and pDNA was tested, aggregation occurred in the sample. In this
example, the majority of particles were 362 nm in size. However, as
the molar excess of PV18C6 is increased, small particles dominate
the sample. The particles formed were stable in solutions of high
ionic strength.
Example 12
[0130] Condensation of pDNA with Polyvinylbenzo-18-crown-6 in the
Presence of Cations
[0131] Rhodamine labeled pDNA was used for the determination of
pDNA condensation in complexes with PV18C6. pDNA was modified to a
level of 1 Rhodamine per 100 bases using Mirus' Labellt Rhodamine
kit. The fluorescence was determined using a fluorescence
spectrophotometer (Hitachi, Model F-3010), at an excitation
wavelength of 590 nm, and an emission wavelength of 608 nm.
[0132] To Rhodamine labeled pDNA (5 .mu.g) in 1 mL 10 mM HEPES
buffer, 0.5 mM EDTA, pH 7.5, was added of PV18C6 (15 .mu.g).
Various concentrations of Li.sup.+, Na.sup.+, K.sup.+, and Cs.sup.+
were added to the solutions. The resulting mixtures were held at
room temperature for 5 minutes and the fluorescence was
determined.
10 Cation F/F.sub.o I.sub.608 Concentration (mM) Li.sup.+ Na.sup.+
K.sup.+ Cs.sup.+ 0 94 93 93 87 2 90 89 85 79 4 88 83 79 70 8 83 80
73 63 12 80 79 64 59 20 78 77 57 54 28 75 75 53 50 40 75 72 53 50
52 75 70 49 45 70 74 65 48 43
[0133] As noted previously, the labeled DNA fluorescence decreases
with DNA compaction. The experimental results for the DNA--PV18C6
complex demonstrate a decrease in fluorescence of the DNA as the
concentration of cation is increased in the sample. Thus, the DNA
is compacted by PV18C6 in the presence of Li.sup.+, Na.sup.+,
K.sup.+, and Cs.sup.+.
Example 13
[0134] Rat Femoral Artery Injection of
DNA--Polyvinylbenzo-18-crown-6 Complexes
[0135] PV18C6 (500 .mu.g) was added to a solution of pCILuc DNA
(500 .mu.g) in 10 mL NaCl (100 mM) and KCl (50 mM). After 5 min.,
the solution was injected into the femoral artery of adult
Sprague-Dawley rats. The blood inflow and outflow were blocked by
putting micro vessel clips on the external iliac, the internal
iliac, the deferent duct, and the caudal epigastric arteries and
veins. Papaverine (5.5 .mu.g) in 3 mL of normal saline solution was
injected into the iliac artery 5 min. before the DNA injection.
After two days, the animals were sacrificed, and a luciferase assay
was performed. (See Budker, V., Zhang, G., Danko, I., Williams, P.,
and Wolff, J., "The Efficient Expression Of Intravascularly
Delivered DNA In Rat Muscle," Gene Therapy 5, 272-6(1998) which is
incorporated herein by reference.)
11 PV18C6 (.mu.g/animal) Luciferase (.mu.g/leg) 400 1.804 .+-.
0.933
[0136] The experiment indicates that DNA--PV18C6 complexes show
successful transfection activity of pCILuc DNA.
Example 14
[0137] Condensation of pDNA with
Polyacrylamidobenzo-18-crown-6/Poly-L-Lys- ine.
[0138] Three solutions were prepared:
[0139] 1) PAA18C6 (1.057 .mu.mol) was taken up in 300 .mu.L of 10
mM TEA buffer (pH 7.4).
[0140] 2) Poly-L-lysine (1.148 .mu.mol) was taken up in 300 .mu.L
of 10 mM TEA buffer (pH 7.4).
[0141] 3) To a solution of PAA18C6 (1.057 .mu.mol) in 300 .mu.L of
10 mM TEA buffer (pH 7.4) was added poly-L-lysine (1.148
.mu.mol).
[0142] Various amounts of solution 3 was added to a solution of
pDNA (24 .mu.g, 0.070 .mu.mol) in 1 mL of 10 mM TEA buffer (pH
7.4). Various amounts of solutions 1 and 2 were added to a solution
of pDNA (24 .mu.g, 0.070 .mu.mol) in 1 mL of 10 mM HEPES/TEA buffer
(pH 7.1). The solutions were mixed for 5 minutes and particle sized
(Particle Sizer, Brookhaven Instruments Corporation). NaCl was
added to each sample (final concentration of 150 mM), the solutions
were mixed for 5 minutes and again particle sized (Particle Sizer,
Brookhaven Instruments Corporation).
[0143] To preparations derived from solution 1 was added PLL using
concentrations equivalent to samples derived from solution 3. To
preparations derived from solution 2 was added PAA18C6 using
concentrations equivalent to samples derived from solution 3. The
solutions were mixed for 5 minutes and again particle sized
(Particle Sizer, Brookhaven Instruments Corporation).
12 PLL PAA18C6 Particle Size (nm (%)) (.mu.mol) (.mu.mol) no NaCl
150 mM NaCl +PAA18C6 or PLL 0.038 0.035 137(58), 621(42) 144(7),
479(93) 0.076 0.070 10000 10000 0.153 0.141 50(78), 175(22) 84(74),
212(26) 0.229 0.211 27(71), 93(29) 102(70), 588(30) 0.344 0.317
45(82), 120(18) 89(98) 0.038 -- 93 30(66), 333(34) 56(11), 377(89)
0.076 -- 62(9), 1558(77), 6000(14) 10000(100) 10000(100) 0.153 --
50(70), 135(30) 700(66), 9000(34) 10000(100) 0.229 -- 80(96),
195(4) 2135 386(57), 8782(43) 0.344 -- 37(86), 103(14) 80(7),
1672(82), 750(30), 5623(70) 9000(11) -- 0.035 -- 40(27), 351(78)
32(13), 562(87) -- 0.070 -- 25(13), 360(87) 35(15), 370(17), 2380(
68) -- 0.141 -- 353(100) 352(100) -- 0.211 -- 353(100) 301(33),
625(67) -- 0.317 -- 869(100) 842(100)
[0144] The particle sizing data indicates that particles prepared
from solution 3 exhibit stability in salt solutions (150 mM NaCl).
Particles prepared from PLL--p DNA showed increased size upon
addition of NaCl indicating instability in salt. Particles prepared
from PAA18C6--pDNA with NaCl exhibited increased size upon the
addition of PLL, again indicating particle instability. Particles
prepared from either solution 1 (to which PLL was added) or
solution 2 (to which PAA18C6 was added) also were shown to be
unstable, indicating that complexes formed in these cases is
different in nature to the complexes formed from solution 3.
Example 15
[0145] Cell Transfection of
pDNA--Polyacrylamidobenzo-18-crown-6/Poly-L-Ly- sine Complexes
[0146] NIH 3T3 cells (mouse fibroblast) were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum and split one day prior to transfection. Confluent cultures
(50-60%) were washed once in Opti-MEM followed by addition of 1.5
mL of Opti-MEM to each 35 mm well. To half of the wells was added
Chloroquine (Sigma Chemical Company, 25 .mu.M) 1 minute prior to
transfection. Complexes were prepared by adding pCILucDNA (3 .mu.g)
to various amounts of PLL or PLL/PAA18C6 (1/1) in 24 .mu.L of 10 mM
TEA buffer(pH 7.4). The solutions were mixed for 5 minutes and
added to the cells. The plates were incubated at 37.degree. C. in
5% CO.sub.2 for 4 hours, and the serum removed. DMEM supplemented
with 10% fetal calf serum (2 mL) was added to each well. The cells
were incubated at 37.degree. C. in 5% CO.sub.2 for 40-48 hours,
harvested, and the expression of luciferase was determined as
previously described.
13 PLL PLL (RLU) PLL/PAA18C6 (RLU) (.mu.g) no Chloroquine
+Chloroquine no Chloroquine +Chloroquine 3.2 1234 .+-. 441 28396
.+-. 24384 51747 .+-. 23859 68987 .+-. 21487 4.8 12664 .+-. 5754
36694 .+-. 12947 29718 .+-. 8230 15783 .+-. 7790 7.2 12125 .+-.
10161 28501 .+-. 16436 3347 .+-. 2650 3494 .+-. 76
[0147] The data indicates an increase in transfection for the
PLL/PAA18C6--pDNA samples relative to the corresponding PLL--pDNA
samples at both 3.2 and 4.8 .mu.g of PLL. Additionally, chloroquine
was not benificial in transfections with PLL/PAA18C6--pDNA samples,
in contrast to transfections with PLL--pDNA samples. There was
strong cytotoxicity observed in wells with a high PLL/PAA18C6--pDNA
concentration resulting in decresed expression relative to the
corresponding PLL--pDNA sample.
Example 16
[0148] Mouse Intramuscular Injection of
DNA--Polyvinylbenzo-18-crown-6 Complexes
[0149] To a solution of pCILuc DNA(40 .mu.g) in 200 .mu.L KCl (50
mM)/glycene (200 mM), was added various amounts of PV18C6. After 5
min., the solution was injected into the exposed quadriceps of ICR
male mice utilising a 30 qauge, 0.5 inch needle. Twenty four hours
post injection, the animals were sacrificed, and the muscle
harvested. Samples were homogenized in lux buffer (1 mL), and
centrifuged for 15 minutes at 4000 RPM. Following centrifugation,
10 .mu.L of supernatant was removed and added to 90 .mu.L
additional lux buffer. The amount of luciferase protein was
calculated according to the equation RLU per
animal.times.5.1.times.1- 0(-5).
14 pCILuc DNA .mu.g PV18C6 (.mu.mol) Volume (.mu.L) RLU 40 150 3294
40 0.085 150 23839 40 0.17 150 5486
[0150] The experiment indicates that DNA--PV18C6 complexes show
successful transfection activity of pCILuc DNA.
[0151] The foregoing is considered as illustrative only of the
principles of the invention. Further, since numerous modifications
and changes will readily occur to those skilled in the art, it is
not desired to limit the invention to the exact construction and
operation shown and described. Therefore, all suitable
modifications and equivalents fall within the scope of the
invention.
* * * * *