U.S. patent application number 12/645931 was filed with the patent office on 2011-06-23 for biodegradable polymers, complexes thereof for gene therapeutics and drug delivery, and methods related thereto.
Invention is credited to Kazuki FUKUSHIMA, James HEDRICK, Yi Yan YANG.
Application Number | 20110151566 12/645931 |
Document ID | / |
Family ID | 44151665 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110151566 |
Kind Code |
A1 |
HEDRICK; James ; et
al. |
June 23, 2011 |
BIODEGRADABLE POLYMERS, COMPLEXES THEREOF FOR GENE THERAPEUTICS AND
DRUG DELIVERY, AND METHODS RELATED THERETO
Abstract
A biodegradable cationic polymer is disclosed, comprising first
repeat units derived from a first cyclic carbonyl monomer by
ring-opening polymerization, wherein more than 0% of the first
repeat units comprise a side chain moiety comprising a quaternary
amine group; a subunit derived from a monomeric diol initiator for
the ring-opening polymerization; and an optional endcap group. The
biodegradable cationic polymers have low cytotoxicity and form
complexes with biologically active materials useful in gene
therapeutics and drug delivery.
Inventors: |
HEDRICK; James; (Pleasanton,
CA) ; FUKUSHIMA; Kazuki; (San Jose, CA) ;
YANG; Yi Yan; (Singapore, SG) |
Family ID: |
44151665 |
Appl. No.: |
12/645931 |
Filed: |
December 23, 2009 |
Current U.S.
Class: |
435/455 ;
435/375; 525/461; 525/54.1; 525/54.2; 528/203; 528/272;
528/292 |
Current CPC
Class: |
C08G 2261/126 20130101;
C08G 63/64 20130101; C08G 64/0241 20130101; C08G 64/30 20130101;
C08G 64/0208 20130101; C08G 64/38 20130101; C08G 64/18 20130101;
C08G 63/06 20130101; C08G 64/42 20130101; C12N 2533/30 20130101;
C08G 63/912 20130101; C12N 15/88 20130101; A61K 47/34 20130101;
C12N 5/0068 20130101; Y02P 20/582 20151101 |
Class at
Publication: |
435/455 ;
528/272; 528/292; 528/203; 525/461; 525/54.2; 525/54.1;
435/375 |
International
Class: |
C12N 15/11 20060101
C12N015/11; C08G 63/02 20060101 C08G063/02; C08F 283/02 20060101
C08F283/02; C08G 63/91 20060101 C08G063/91; C12N 5/00 20060101
C12N005/00 |
Claims
1. A biodegradable cationic polymer, comprising: first repeat units
derived from a first cyclic carbonyl monomer by ring-opening
polymerization, wherein more than 0% of the first repeat units
comprise a side chain moiety comprising a quaternary amine group; a
subunit derived from a monomeric diol initiator for the
ring-opening polymerization; and an optional endcap group.
2. The cationic polymer of claim 1, wherein the more than 0% of the
first repeat units comprise a side chain moiety comprising a
quaternary amine group and a tertiary amine group.
3. The cationic polymer of claim 1, wherein the side chain moiety
of the first repeat units further comprise a side chain carboxylic
acid group.
4. The cationic polymer of claim 1, wherein the cationic polymer
further comprises a second repeat unit derived from a second cyclic
carbonyl monomer, and the second repeat unit comprises a side chain
acetal ester group.
5. The cationic polymer of claim 1, wherein the cationic polymer is
a polycarbonate.
6. The cationic polymer of claim 1, wherein the cationic polymer is
a block copolymer.
7. The cationic polymer of claim 1, wherein the cationic polymer is
amphiphilic and self-assembles in water to form nanoparticles
having an average particle size of 10 nm to 500 nm at a pH of from
5.0 to 8.0.
8. A method of forming a biodegradable cationic polymer,
comprising: forming a first mixture comprising a first cyclic
carbonyl monomer, a catalyst, an accelerator, a monomeric diol
initiator, and an optional solvent, wherein the first cyclic
carbonyl monomer comprises a monovalent leaving group capable of
reacting with a tertiary amine to form a quaternary amine; forming
a first polymer comprising first repeat units derived from the
first cyclic carbonyl monomer by ring-opening polymerization;
optionally endcapping the first polymer to form a precursor
polymer; and treating the precursor polymer with the tertiary amine
to form the cationic polymer, wherein more than 0% of the first
repeat units derived from the first cyclic carbonyl monomer
comprise a side chain moiety comprising a quaternary amine.
9. The method of claim 8, wherein the tertiary amine is a
bis-tertiary amine and the more than 0% of the first repeat units
derived from the first cyclic carbonyl monomer comprise the side
chain moiety comprising the quaternary amine and a tertiary
amine.
10. The method of claim 9, wherein the bis-tertiary amine is
selected from the group consisting of
N,N,N',N'-tetramethyl-1,2-ethanediamine (TMEDA),
N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA),
N,N,N',N'-tetramethyl-1,4-butanediamine (TMBDA),
N,N,N',N'-tetraethyl-1,2-ethanediamine (TEEDA),
N,N,N',N'-tetraethyl-1,3propanediamine (TEPDA),
1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),
N,N,N',N'-tetraethyl-1,4-butanediamine (TEBDA),
4-dimethylaminopyridine (DMAP),
4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),
4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations
thereof.
11. The method of claim 8, wherein the tertiary amine comprises a
carboxy group and the more than 0% of the first repeat units
derived from the first cyclic carbonyl monomer comprise the side
chain moiety comprising the quaternary amine and a carboxylic
acid.
12. The method of claim 8, wherein the first mixture comprises a
hydrophobic second cyclic carbonyl monomer, and the cationic
polymer is a random copolymer comprising a second repeat unit
derived from the second cyclic carbonyl monomer by ring-opening
polymerization; wherein the second cyclic carbonyl monomer does not
comprise a monovalent leaving group capable of reacting with the
tertiary amine to form a side chain moiety comprising any
quaternary amine.
13. The method of claim 12, wherein the second repeat unit
comprises a side chain acetal ester group.
14. A method of forming a biodegradable cationic block copolymer,
comprising: forming a reaction mixture comprising a catalyst, an
accelerator, a monomeric diol initiator, and an optional solvent;
sequentially adding to the reaction mixture and reacting by
ring-opening polymerization a first cyclic carbonyl monomer
followed by a second cyclic carbonyl monomer, thereby forming a
first block copolymer, wherein the first cyclic carbonyl monomer
comprises a monovalent leaving group capable of reacting with a
tertiary amine to form a quaternary amine, and the second cyclic
carbonyl monomer is not capable of reacting with the tertiary amine
to form any quaternary amine; optionally endcapping the first block
copolymer, thereby forming a precursor block copolymer; and
treating the precursor block copolymer with a tertiary amine to
form the cationic polymer, wherein the cationic polymer comprises
first repeat units derived from the first cyclic carbonyl monomer,
and more than 0% of the first repeat units comprise a side chain
moiety comprising the quaternary amine.
15. The method of claim 14 wherein the sequential reaction is
performed in reverse order to form the first block copolymer.
16. The method of claim 14, wherein the first block copolymer is
endcapped using a carboxylic anhydride, thereby forming a terminal
ester group.
17. The method of claim 14, wherein the first cyclic carbonyl
monomer is a compound of formula (1): ##STR00099## wherein: t is an
integer from 0 to 6; each Y is a divalent radical independently
selected from the group consisting of ##STR00100## and ##STR00101##
and each Q.sup.1 is a monovalent radical independently selected
from the group consisting of hydrogen, halides, carboxy groups,
alkyl groups comprising 1 to 30 carbons, aryl groups comprising 6
to 30 carbon atoms, and groups having the structure ##STR00102##
wherein M.sup.1 is a monovalent radical selected from the group
consisting of --R.sup.1, --NHR.sup.1, --NR.sup.1R.sup.1, and --SW;
and each R.sup.1 is a monovalent radical independently selected
from the group consisting of alkyl groups comprising 1 to 30
carbons, and aryl groups comprising 6 to 30 carbons; and wherein
one or more of the Q.sup.1 groups of the first cyclic carbonyl
monomer comprises a monovalent leaving group capable of reacting
with a tertiary amine to form a quaternary amine.
18. The method of claim 14, wherein the first cyclic carbonyl
monomer is a compound of formula (2): ##STR00103## wherein: each
Q.sup.2 is a monovalent radical independently selected from the
group consisting of hydrogen, a halides, carboxy groups, alkyl
groups comprising 1 to 30 carbons, aryl groups comprising 6 to 30
carbon atoms, and groups having the structure ##STR00104## wherein
M.sup.1 is a monovalent radical selected from the group consisting
of --R.sup.1, --OR.sup.1, --NHR.sup.1, --NR.sup.1R.sup.1, or
--SR.sup.1, and each R.sup.1 is a monovalent radical independently
selected from the group consisting of alkyl groups comprising 1 to
30 carbons, and aryl groups comprising 6 to 30 carbons; R.sup.2 is
a monovalent radical independently selected from the group
consisting of alkyl groups comprising 1 to 30 carbons, and aryl
groups comprising 6 to 30 carbons; Q.sup.3 is a monovalent radical
selected from the group consisting of hydrogen, alkyl groups having
1 to 30 carbons, and aryl groups having 6 to 30 carbons; and
wherein the R.sup.2 group of the first cyclic carbonyl monomer
comprises a monovalent leaving group capable of reacting with a
tertiary amine to form a quaternary amine.
19. The method of claim 14, wherein the first cyclic carbonyl
monomer is a compound of formula (3): ##STR00105## wherein u is an
integer from 1 to 8; each Q.sup.4 is a monovalent radical
independently selected from the group consisting of hydrogen,
halides, carboxy groups, alkyl groups comprising 1 to 30 carbons,
aryl groups comprising 6 to 30 carbon atoms, and groups having the
structure ##STR00106## where M.sup.1 is a monovalent radical
selected from --R.sup.1, --OR.sup.1, --NHR.sup.1,
--NR.sup.1R.sup.1, or --SR.sup.1; each R.sup.1 is a monovalent
radical independently selected from the group consisting of alkyl
groups comprising 1 to 30 carbons, and aryl groups comprising 6 to
30 carbons; optionally, a ##STR00107## group of formula (3)
independently represents a --O--, --S--, --NHR.sup.1, or
--NR.sup.1R.sup.1; optionally, a ##STR00108## group of formula (3)
independently represents a ##STR00109## group; and wherein one or
more of the Q.sup.4 groups of the first cyclic carbonyl monomer
comprises a monovalent leaving group capable of reacting with a
tertiary amine to form a quaternary amine.
20. The method of claim 14, wherein the first cyclic carbonyl
monomer is a compound of formula (4): ##STR00110## wherein each
Q.sup.5 is a monovalent radical independently selected from the
group consisting of hydrogen, halides, carboxy groups, alkyl groups
comprising 1 to 30 carbons, aryl groups comprising 6 to 30 carbon
atoms, and groups having the structure ##STR00111## wherein M.sup.1
is a monovalent radical selected from --R.sup.1, --OR.sup.1,
--NHR.sup.1, --NR.sup.1R.sup.1, or --SR.sup.1; each R.sup.1 is a
monovalent radical independently selected from the group consisting
of alkyl groups comprising 1 to 30 carbons, and aryl groups
comprising 6 to 30 carbons; each Q.sup.6 is a monovalent group
independently selected from the group consisting of hydrogen, alkyl
groups having 1 to 30 carbons, and aryl groups having 6 to 30
carbons; and each v independently represents an integer from 1 to
6; and wherein one or more of the Q.sup.5 and/or a Q.sup.6 groups
of the first cyclic carbonyl monomer comprises a monovalent leaving
group capable of reacting with a tertiary amine to form a
quaternary amine.
21. The method of claim 14, wherein the cationic block copolymer
comprises a second repeat unit derived from the second cyclic
carbonyl monomer, and the second repeat unit comprises a side chain
acetal ester group.
22. The method of claim 14, wherein the initiator is BnMPA.
23. The method of claim 14, wherein the tertiary amine is a
bis-tertiary amine selected from the group consisting of
N,N,N',N'-tetramethyl-1,2-ethanediamine (TMEDA),
N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA),
N,N,N',N'-tetramethyl-1,4-butanediamine (TMBDA),
N,N,N',N'-tetraethyl-1,2-ethanediamine (TEEDA),
N,N,N',N'-tetraethyl-1,3propanediamine (TEPDA),
1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),
N,N,N',N'-tetraethyl-1,4-butanediamine (TEBDA),
4-dimethylaminopyridine (DMAP),
4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),
4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations
thereof.
24. A polymer complex, comprising: a negatively charged
biologically active material; and a biodegradable cationic polymer,
comprising: first repeat units derived from a first cyclic carbonyl
monomer by ring-opening polymerization, wherein more than 0% of the
first repeat units comprise a side chain moiety comprising a
quaternary amine group; a subunit derived from a monomeric diol
initiator for the ring-opening polymerization; and an optional
endcap group.
25. The polymer complex of claim 24, wherein the more than 0% of
the first repeat units further comprise a side chain tertiary amine
group.
26. The polymer complex of claim 24, wherein the more than 0% of
the first repeat units further comprise a side chain carboxylic
acid group.
27. The polymer complex of claim 24, wherein the cationic polymer
is a polycarbonate.
28. The polymer complex of claim 24, wherein the cationic polymer
further comprises a second repeat unit derived from a hydrophobic
second cyclic carbonyl monomer by ring opening polymerization,
wherein the second repeat unit comprises a side chain acetal ester
group.
29. The polymer complex of claim 24, wherein the negatively charged
biologically active material is selected from the group consisting
of genes, nucleotides, proteins, peptides, drugs, and a combination
thereof.
30. The polymer complex of claim 24, wherein the cationic polymer
is a block copolymer.
31. A method of forming a polymer complex, comprising: contacting a
first aqueous mixture comprising a biodegradable cationic polymer
with a second aqueous mixture comprising a negatively charged
biologically active material to form a third aqueous mixture
comprising the polymer complex, wherein the biodegradable cationic
polymer comprises: first repeat units derived from a first cyclic
carbonyl monomer by ring-opening polymerization, a subunit derived
from a monomeric diol initiator for the ring-opening
polymerization, and an optional endcap group, wherein more than 0%
of the first repeat units comprise a side chain moiety comprising a
quaternary amine group.
32. A method of treating a cell, comprising: contacting the cell
with nanoparticles of a polymer complex comprising a biodegradable
cationic polymer and a negatively charged biologically active
material; wherein the biodegradable cationic polymer comprises:
first repeat units derived from a first cyclic carbonyl monomer by
ring-opening polymerization, a subunit derived from a monomeric
diol initiator for the ring-opening polymerization, and an optional
endcap group, wherein more than 0% of the first repeat units
comprise a side chain moiety comprising a quaternary amine
group.
33. The method of claim 32, wherein said contacting induces 0% to
15% hemolysis.
34. The method of claim 32, wherein the nanoparticles have 0% to
20% cytotoxicity.
35. The method of claim 32, wherein the biologically active
material is a gene, and the cell expresses the gene.
36. A biodegradable amphiphilic cationic polymer, comprising: two
polymer chains comprising respective first ends, respective second
ends, and respective first repeat units; a monomeric linking group
comprising two backbone heteroatoms independently selected from the
group consisting of oxygen, nitrogen, and sulfur; wherein i) the
respective first ends of the two polymer chains are each linked to
one of the two backbone heteroatoms, ii) the respective second ends
optionally comprise respective endcap groups, and iii) the
respective first repeat units comprise respective first backbone
functional groups independently selected from the group consisting
of ester, carbonate, carbamate, urea, thiocarbamate, thiocarbonate,
or dithiocarbonate, and more than 0% of the respective first repeat
units comprise respective side chain quaternary amine groups.
37. The cationic polymer of claim 36, wherein the two backbone
heteroatoms are oxygen atoms.
38. The cationic polymer of claim 36, wherein the cationic polymer
in aqueous solution at a pH of 5.0 to 8.0 self-assembles into
nanoparticles having an average particle size of 10 nm to 500 nm,
as measured by dynamic light scattering using a He--Ne laser beam
at 658 nm and a scattering angle of 90.degree..
39. The cationic polymer of claim 36, wherein the cationic polymer
is biodegradable in accordance with ASTM D6400.
40. The cationic polymer of claim 36, wherein the first side chains
further comprise respective tertiary amine groups.
41. The cationic polymer of claim 36, wherein the two polymer
chains comprise respective second repeat units comprising i)
respective second backbone functional groups independently selected
from the group consisting of ester, carbonate, carbamate, urea,
thiocarbamate, thiocarbonate, or dithiocarbonate, and ii)
respective second side chains comprising respective latent
carboxylic acid groups capable of forming carboxylic acid groups in
an endosomal environment.
42. The cationic polymer of claim 36, wherein the two polymer
chains are block copolymer chains.
43. A polymer complex, comprising: a negatively charged
biologically active material; and the biodegradable amphiphilic
cationic polymer of claim 36.
44. A method of forming a polymer complex, comprising: contacting a
first aqueous mixture comprising the biodegradable amphiphilic
cationic polymer of claim 36 with a second aqueous mixture
comprising a negatively charged biologically active material to
form a third aqueous mixture comprising the polymer complex.
45. A method of treating a cell, comprising: contacting the cell
with nanoparticles of the polymer complex of claim 43.
46. A method of forming a biodegradable cationic polymer,
comprising: forming a first mixture comprising i) a first cyclic
carbonyl monomer, ii) an organocatalyst, iii) an accelerator, iv) a
monomeric initiator comprising two nucleophilic initiator groups,
the two nucleophilic initiator groups independently selected from
the group consisting of alcohols, amines, and thiols, and v) an
optional solvent, wherein the first cyclic carbonyl monomer
comprises a monovalent leaving group capable of reacting with a
tertiary amine to form a quaternary amine; agitating the first
mixture, thereby forming a first polymer comprising first repeat
units derived from the first cyclic carbonyl monomer by
ring-opening polymerization; optionally endcapping the first
polymer; and treating the first polymer or the endcapped first
polymer with the tertiary amine, thereby forming the cationic
polymer, wherein more than 0% of the first repeat units derived
from the first cyclic carbonyl monomer comprise a side chain moiety
comprising a quaternary amine.
Description
BACKGROUND
[0001] The present invention relates to biodegradable polymers, and
more specifically, complexes thereof with biologically active
molecules, for use in gene therapeutics and drug delivery.
[0002] Recent advances in gene therapeutics have lead to a critical
need for a broader selection of less expensive, non-cytotoxic
carrier materials for gene transfection. The carrier materials
should package and protect a genetic cargo in the extracellular
environment, transport it across the cell membrane, and release the
cargo within the cell at a suitable point. Carrier materials are
typically classified into viral vectors and non-viral vectors.
Viral vectors have excellent efficiencies in delivery and
expression of genes. However, they often cause immunologically
adverse responses, are expensive to produce, and are limited with
respect to gene encapsulation.
[0003] Non-viral vectors include cationic polymers and cationic
lipids (lipoplex). These materials have drawn increasing attention
as alternatives to viral vectors owing to their potentially low
production costs and flexibility with respect to molecular design.
Cationic polymers and cationic lipids can bind electrostatically to
negatively charged genetic material to form a carrier-gene complex
having decreased net charge. These complexes can potentially
facilitate effective transfection of genetic material into a
cell.
[0004] Ideally the polymer-gene complex should enter the cytoplasm
of the cell by endocytosis. The complex must escape from the acidic
endosomal environment into the cytosol before infusing into
lysosomes where enzymes can decompose the genes. A key design
feature for such behavior is the tuning of amine residues of the
polymer carrier to provide buffering capacity, which leads to
osmotic swelling and rupture of the endosome, freeing the
bio-active cargo and/or the bio-active cargo-carrier complex (i.e.,
polyplex) into the cytoplasm (proton sponge hypothesis), and thus
enabling gene transfection. For example, the free amine residues on
the cationic polymer such as poly(ethylene imine) (PEI) can
effectively buffer the protons in the endosome, causing osmotic
swelling and rupture of the endosome. PEI is recognized as a good
non-viral vector having superior transfection efficiency. This is
attributed to its high charge density, which facilitates formation
of a compacted complex, and its high density of near-neutral
secondary amine sites, which provide buffering capacity. However,
PEI is not biodegradable and it is also considered highly
cytotoxic. Thus, a challenge in the design of synthetic
transfection vectors based on cationic polymers is achieving low
cytotoxicity and high transfection efficiency. Poly(beta-amino
ester)s (PBAEs), modified PEIs, poly(amino acid)s,
poly(beta-aminosulfonamide)s (PBASs), dendrimers based on
poly(L-lysine) (PLL), and poly(amidoamine) (PAMAM) dendrimers have
been reported as efficient non-viral vectors for safe gene
delivery. These polymers, however, have several issues to be
solved, especially in the synthetic process. PBAEs and PBASs
prepared by polycondensation have relatively broad molecular weight
distributions, PEI and its derivatives usually contain some
branched structures that are difficult to control, dendrimers
require multiple steps to generate desirable molecular weight, and
polymers based on amino acids have high production costs due to
expensive starting materials.
[0005] Polymer-gene complexation via electrostatic interactions is
an effective transporting and protection strategy for genetic
material, however this tight packing also presents a problem in the
unpacking of genes. Numerous strategies have been explored to
facilitate the release of the bio-active cargo including degradable
systems, reversible crosslinking, modestly charged cationic
polymers, hierarchical self-assembled pH-responsive terpolymers and
a number of charge shifting strategies.
[0006] A continued need exists for less expensive, less cytotoxic,
more biodegradable synthetic carriers for gene transfection and
drug delivery. The carriers should have predictable molecular
weight and low polydispersity, and form reversible extracellular
complexes with bio-active molecules, particularly nucleotides.
SUMMARY
[0007] Accordingly, in an embodiment, a biodegradable cationic
polymer is disclosed, comprising:
[0008] first repeat units derived from a first cyclic carbonyl
monomer by ring-opening polymerization, wherein more than 0% of the
first repeat units comprise a side chain moiety comprising a
quaternary amine group;
[0009] a subunit derived from a monomeric diol initiator for the
ring-opening polymerization; and
[0010] an optional endcap group.
[0011] In another embodiment, a method of forming a biodegradable
cationic polymer is disclosed, comprising:
[0012] forming a first mixture comprising a first cyclic carbonyl
monomer, a catalyst, an accelerator, a monomeric diol initiator,
and an optional solvent, wherein the first cyclic carbonyl monomer
comprises a monovalent leaving group capable of reacting with a
tertiary amine to form a quaternary amine;
[0013] forming a first polymer comprising first repeat units
derived from the first cyclic carbonyl monomer by ring-opening
polymerization;
[0014] optionally endcapping the first polymer to form a precursor
polymer; and
[0015] treating the precursor polymer with the tertiary amine to
form the cationic polymer, wherein more than 0% of the first repeat
units derived from the first cyclic carbonyl monomer comprise a
side chain moiety comprising a quaternary amine.
[0016] In another embodiment, a method of forming a biodegradable
cationic block copolymer is disclosed, comprising:
[0017] forming a reaction mixture comprising a catalyst, an
accelerator, a monomeric diol initiator, and an optional
solvent;
[0018] sequentially adding to the reaction mixture and reacting by
ring-opening polymerization a first cyclic carbonyl monomer
followed by a second cyclic carbonyl monomer, thereby forming a
first block copolymer, wherein the first cyclic carbonyl monomer
comprises a monovalent leaving group capable of reacting with a
tertiary amine to form a quaternaray amine, and the second cyclic
carbonyl monomer is not capable of reacting with the tertiary amine
to form any quaternary amine;
[0019] optionally endcapping the first block copolymer, thereby
forming a precursor block copolymer; and
[0020] treating the precursor block copolymer with a tertiary amine
to form the cationic polymer, wherein the cationic polymer
comprises first repeat units derived from the first cyclic carbonyl
monomer, and more than 0% of the first repeat units comprise a side
chain moiety comprising the quaternary amine.
[0021] In another embodiment, a polymer complex is disclosed,
comprising:
[0022] a negatively charged biologically active material; and
[0023] a biodegradable cationic polymer, comprising: [0024] first
repeat units derived from a first cyclic carbonyl monomer by
ring-opening polymerization, wherein more than 0% of the first
repeat units comprise a side chain moiety comprising a quaternary
amine group; [0025] a subunit derived from a monomeric diol
initiator for the ring-opening polymerization; and [0026] an
optional endcap group.
[0027] In another embodiment, a method of forming a polymer complex
for treating a cell is disclosed, comprising:
[0028] contacting a first aqueous mixture comprising a
biodegradable cationic polymer with a second aqueous mixture
comprising a negatively charged biologically active material to
form a third aqueous mixture comprising the polymer complex,
wherein the biodegradable cationic polymer comprises: first repeat
units derived from a first cyclic carbonyl monomer by ring-opening
polymerization, a subunit derived from a monomeric diol initiator
for the ring-opening polymerization, and an optional endcap group,
wherein more than 0% of the first repeat units comprise a side
chain moiety comprising a quaternary amine group.
[0029] In another embodiment, a method of treating a cell is
disclosed, comprising:
[0030] contacting the cell with nanoparticles of a polymer complex
comprising a biodegradable cationic polymer and a negatively
charged biologically active material; wherein the biodegradable
cationic polymer comprises: first repeat units derived from a first
cyclic carbonyl monomer by ring-opening polymerization, a subunit
derived from a monomeric diol initiator for the ring-opening
polymerization, and an optional endcap group, wherein more than 0%
of the first repeat units comprise a side chain moiety comprising a
quaternary amine group.
[0031] The above-described and other features and advantages of the
present invention will be appreciated and understood by those
skilled in the art from the following detailed description,
drawings, and appended claims.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0032] FIG. 1 is a photographic image agarose gel electrophoresis
experiments at different pH and at various N/P ratios of polyplexes
prepared with the cationic polymer of Example 15 and DNA.
[0033] FIG. 2 is a bar chart showing the relationship between N/P
ratio and particle size of the polyplex prepared at pH 7.0, 6.0 and
5.0.
[0034] FIG. 3 is a bar chart showing the relationship between N/P
ratio and zeta potential of the polyplex prepared at pH 7.0, 6.0
and 5.0.
[0035] FIG. 4 is a bar chart comparing luciferase expression levels
in HepG2 human liver carcinoma cell line for polyplexes fabricated
at different pH and N/P ratios. DNA and PEI/DNA controls are also
shown.
[0036] FIG. 5 is a bar chart comparing cell viability at different
pH and N/P ratios.
DETAILED DESCRIPTION
[0037] Biodegradable cationic polymers are disclosed that form
nano-sized complexes with biologically active molecules and are
less cytotoxic than other carriers such as poly(ethylene imine)
(PEI). The cationic polymers are derived by ring-opening
polymerization (ROP) of a first cyclic carbonyl monomer having a
monovalent leaving group, such as an alkyl halide or sulphonate
ester, which is capable of reacting with a tertiary amine to form a
moiety comprising a quaternary amine. Other cyclic carbonyl
monomers are selected as diluents for the former, to provide
hydrophobicity, and/or to provide charge shifting capability to the
polymer. The cationic polymers can be densely charged and thus
freely soluble in water, or possess amphiphilic properties, forming
nanoparticles in aqueous solution. The ring-opening method allows
precise control of the molecular weight of the polymer, achieves a
low polydispersity, and is compatible with a variety of functional
groups. The reaction with the tertiary amine to form the moiety
comprising a quaternary amine can be performed before or after the
ring-opening polymerization, more particularly after the
polymerization. The quaternization is accompanied by minimal, if
any, crosslinking of the cationic polymer. Examples of cyclic
carbonyl monomers include cyclic carbonate monomers and lactones,
including lactides, that ring-open to form polymers comprising
carbonate and ester repeat units, respectively. The quaternary
amine is located on the polymer side chain, and if desired can be
linked directly to the polymer backbone. The positively charged
quaternary amine groups provide binding strength to negatively
charged biologically active materials. In an embodiment, the
tertiary amine is a bis-tertiary amine, and the cationic polymer
comprises a side chain moiety comprising a quaternary amine and a
tertiary amine. The side chain tertiary amine groups provide
buffering capacity to facilitate release of the bio-active material
from the polymer complex. Other functional groups can be used to
facilitate the release of the bio-active material from the polymer
complex, such as secondary amine groups, citraconic amide groups,
ester groups, and imine groups. The release of a bio-active
material can also be facilitated by cationic polymers capable of
charge-shifting. In charge shifting, the net positive charge of the
cationic polymer is reduced by the conversion of a non-charged
group on the cationic polymer side chain into a negatively charged
group after the polymer complex has entered the cell. A cationic
polymer capable of charge-shifting can comprise, for example, a
latent carboxylic acid group, such as an acetal ester, in addition
to the quaternary amine. Acetal esters can be hydrolyzed under the
mildly acidic conditions of the endosomal environment to form a
carboxylic acid group. In the more basic environment of the
cytosol, the carboxylic acid groups become ionized, thereby
lowering the net positive charge of the cationic polymer and
allowing the release of the negatively charged bio-active material.
The ring-opening polymerization of the cyclic carbonyl monomers
allows for tuning of the charge-shifting capability of the cationic
polymers for a specific bio-active material. The cationic polymers
can be linear or branched, and can be easily modified to tune the
charge and the buffering strength. With a gene, the cationic
polymer forms a complex (i.e., polyplex) having an average particle
size of from about 10 nm to about 250 nm.
[0038] The term "biodegradable" is defined by the American Society
for Testing and Materials as a degradation caused by biological
activity, especially by enzymatic action, leading to a significant
change in the chemical structure of the material. For purposes
herein, a material is "biodegradable" if it undergoes 60%
biodegradation within 180 days in accordance with ASTM D6400.
[0039] For purposes herein, a "cargo" can be any biologically
active substance that forms a reversible, nano-sized complex with
the disclosed cationic polymers, with the proviso that the complex
enters a cell by endocytosis, and the complex releases the
biologically active substance at a desired stage within the cell.
Biologically active substances include biomolecules (e.g., DNA,
genes, peptides, proteins, enzymes, lipids, phospholipids, and
nucleotides), natural or synthetic organic compounds (e.g., drugs,
dyes, synthetic polymers, oligomers, and amino acids), inorganic
materials (e.g., metals and metal oxides), radioactive variants of
the foregoing, and combinations of the foregoing. "Biologically
active" means the substance can alter the chemical structure and/or
activity of a cell in a desirable manner, or can selectively alter
the chemical structure and/or activity of a cell type relative to
another cell type in a desirable manner. As an example, one
desirable change in a chemical structure can be the incorporation
of a gene into the DNA of the cell. A desirable change in activity
can be the expression of the transfected gene. Another change in
cell activity can be the induced production of a desired hormone or
enzyme. Alternatively, a desirable change in activity can be the
selective death of one cell type over another cell type. No
limitation is placed on the relative change in cellular activity
caused by the biologically active substance, providing the change
is desirable and useful. Moreover, no limitation is placed on the
so-called "cargo," providing the cargo induces a useful cellular
response when released from the complex.
[0040] In the following description of general formulas for cyclic
carbonyl monomers, a "first cyclic carbonyl monomer" refers to a
first category of cyclic carbonyl monomers comprising a monovalent
leaving group capable of reacting with a tertiary amine to form a
moiety comprising a quaternary amine. The term "second cyclic
carbonyl monomer" refers to a second category of cyclic carbonyl
monomer, that contains no monovalent leaving group capable of
reacting with the tertiary amine to form a moiety comprising any
quaternary amine. Otherwise, the first and second cyclic carbonyl
monomers can have a structure selected from any of following
described formulas.
[0041] The cyclic carbonyl monomers can be selected independently
from compounds of the general formula (1):
##STR00001##
wherein t is an integer from 0 to 6, and when t is 0 carbons
labeled 4 and 6 are linked together by a single bond. Each Y is a
divalent radical independently selected from
##STR00002##
and
##STR00003##
where the dash "--" indicates the point of attachment. The latter
two groups are also expressed herein as --N(Q.sup.1)-- and
--C(Q.sup.1).sub.2-. Each Q.sup.1 is a monovalent radical
independently selected from hydrogen, halides, carboxy groups,
alkyl groups comprising 1 to 30 carbons, aryl groups comprising 6
to 30 carbon atoms, or groups having the structure
##STR00004##
where M.sup.1 is a monovalent radical selected from --R.sup.1,
--OR.sup.1, --NHR.sup.1, --NR.sup.1R.sup.1, or --SR.sup.1, where
the dash represents the point of attachment, and each R.sup.1 is a
monovalent radical independently selected from alkyl groups
comprising 1 to 30 carbons, or aryl groups comprising 6 to 30
carbons. One or more Q.sup.1 groups can further comprise a
monovalent leaving group capable of reacting with a tertiary amine
to form a moiety comprising a quaternary amine (i.e., a positive
charged quaternary ammonium ion bonded to four carbons).
Non-limiting examples of monovalent leaving groups include halides
in the form of an alkyl halide (e.g., alkyl chloride, alkyl
bromide, or alkyl iodide), sulphonate esters (e.g., tosylate or
mesylate esters), and epoxides. Each Q.sup.1 group can
independently be branched or non-branched. Each Q.sup.1 group can
also independently comprise additional functional groups, including
a ketone group, aldehyde group, alkene group, alkyne group,
cycloaliphatic ring comprising 3 to 10 carbons, heterocylic ring
comprising 2 to 10 carbons, ether group, amide group, ester group,
and combinations of the foregoing additional functional groups. The
heterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two
or more Q.sup.1 groups can together form a ring. A first cyclic
carbonyl monomer of formula (1) comprises one or more Q.sup.1
groups comprising a monovalent leaving group capable of reacting
with a tertiary amine to form a moiety comprising a quaternary
amine. A second cyclic carbonyl monomer of formula (1) comprises no
functional group capable of reacting with the tertiary amine to
form a moiety comprising any quaternary amine.
[0042] A more specific cyclic carbonyl monomer capable of
ring-opening polymerization has the general formula (2):
##STR00005##
wherein Q.sup.2 is a monovalent radical independently selected from
the group consisting of hydrogen, halides, carboxy groups, alkyl
groups comprising 1 to 30 carbons, aryl groups comprising 6 to 30
carbon atoms, and groups having the structure
##STR00006##
wherein M.sup.1 is a monovalent radical selected from --R.sup.1,
--OR.sup.1, --NHR.sup.1, --NR.sup.1R.sup.1, or --SR.sup.1, wherein
each R.sup.1 is a monovalent radical independently selected from
the group consisting of alkyl groups comprising 1 to 30 carbons,
and aryl groups comprising 6 to 30 carbons; R.sup.2 is a monovalent
radical independently selected from the group consisting of alkyl
groups comprising 1 to 30 carbons, and aryl groups comprising 6 to
30 carbons; and Q.sup.3 is a monovalent radical selected from the
group consisting of hydrogen, alkyl groups having 1 to 30 carbons,
and aryl groups having 6 to 30 carbons. In an embodiment, each
Q.sup.2 is hydrogen, Q.sup.3 is a methyl or ethyl group, and
R.sup.2 is an alkyl group comprising 1 to 30 carbons. A first
cyclic carbonyl monomer of formula (2) comprises an R.sup.2 group
comprising a monovalent leaving group capable of reacting with a
tertiary amine to form a moiety comprising a quaternary amine. A
second cyclic carbonyl monomer of formula (2) comprises no
functional group capable of reacting with the tertiary amine to
form a moiety comprising any quaternary amine.
[0043] Another more specific cyclic carbonyl monomer has the
general formula (3):
##STR00007##
wherein each Q.sup.4 is a monovalent radical independently selected
from the group consisting of hydrogen, halides, carboxy groups,
alkyl groups comprising 1 to 30 carbons, aryl groups comprising 6
to 30 carbon atoms, and groups having the structure
##STR00008##
where M.sup.1 is a monovalent radical selected from --R.sup.1,
--OR.sup.1, --NHR.sup.1, --NR.sup.1R.sup.1, or --SR.sup.1, wherein
each R.sup.1 is a monovalent radical independently selected from
the group consisting of alkyl groups comprising 1 to 30 carbons,
and aryl groups comprising 6 to 30 carbons; and u is an integer
from 1 to 8. The lactone ring can optionally comprise a
carbon-carbon double bond; that is, optionally, a
##STR00009##
group of formula (3) can independently represent a
##STR00010##
group. The lactone ring can also comprise a heteroatom such as
oxygen, nitrogen, sulfur, or a combination thereof; that is,
optionally a
##STR00011##
group of formula (3) can independently represent a --O--, --S--,
--NHR.sup.1, or an --NR.sup.1R.sup.1 group, wherein R.sup.1 has the
same definition as above. A first cyclic carbonyl monomer of
formula (3) comprises one or more Q.sup.4 groups comprising a
monovalent leaving group capable of reacting with a tertiary amine
to form a moiety comprising a quaternary amine. A second cyclic
carbonyl monomer of formula (3) comprises no functional group
capable of reacting with the tertiary amine to form a moiety
comprising any quaternary amine. In an embodiment, u is an integer
from 1 to 6 and each Q.sup.4 is hydrogen.
[0044] Another more specific cyclic carbonyl monomer is a dioxane
dicarbonyl of the general formula (4):
##STR00012##
wherein each Q.sup.5 is a monovalent radical independently selected
from the group consisting of hydrogen, halides, carboxy groups,
alkyl groups comprising 1 to 30 carbons, aryl groups comprising 6
to 30 carbon atoms, and groups having the structure
##STR00013##
where M.sup.1 is a monovalent radical selected from --R.sup.1,
--OR.sup.1, --NHR.sup.1, --NR.sup.1R.sup.1, or --SR.sup.1, wherein
each R.sup.1 is a monovalent radical independently selected from
the group consisting of alkyl groups comprising 1 to 30 carbons,
and aryl groups comprising 6 to 30 carbons; each Q.sup.6 is a
monovalent group independently selected from the group consisting
of hydrogen, alkyl groups having 1 to 30 carbons, and aryl groups
having 6 to 30 carbons; and each v is independently an integer from
1 to 6. A first cyclic carbonyl monomer of formula (4) comprises
one or more Q.sup.5 groups and/or a Q.sup.6 groups comprising a
monovalent leaving group capable of reacting with a tertiary amine
to form a moiety comprising a quaternary amine. A second cyclic
carbonyl monomer of formula (4) comprises no functional group
capable of reacting with the tertiary amine to form a moiety
comprising any quaternary amine. In an embodiment, each v is 1,
each Q.sup.5 is hydrogen, and each Q.sup.6 is an alkyl group
comprising 1 to 6 carbons.
[0045] The cyclic carbonyl compounds can have one or more
asymmetric carbon centers that can be present in isomerically
enriched form, either as an R-isomer or an S-isomer. Further, each
asymmetric carbon center can independently be present in an
enantiomeric excess of 80% or more, more specifically 90%.
[0046] Examples of cyclic carbonyl monomers of formulas (1) or (2)
having a monovalent leaving group in the form of an alkyl halide
include the cyclic monomers of Table 1.
TABLE-US-00001 TABLE 1 ##STR00014## ##STR00015## ##STR00016##
[0047] Additional examples of cyclic carbonyl monomers of formula
(2) include the compounds of Table 2. These can be used, for
example, as co-monomers in the ring-opening polymerization of the
halide monomers of Table 1, to form random copolymers or block
copolymers.
TABLE-US-00002 TABLE 2 ##STR00017## m = 1,Trimethylene carbonate,
(TMC) m = 2,Tetramethylene carbonate, (TEMC) m = 3,Pentamethylene
carbonate, (PMC) ##STR00018## R = hydrogen, (MTCOH) R = methyl,
(MTCOMe) R = t-butyl, (MTCO.sup.tBu) R = ethyl, (MTCOEt)
##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034## R = methyl R = iso-propyl
[0048] Examples of cyclic carbonyl monomers of formula (3) include
the compounds of Table 3.
TABLE-US-00003 TABLE 3 ##STR00035## R= H: n = 1: beta-
Propiolactone, (b-PL) R= H; n = 2: gamma- Butyrolactone, (g-BL) R=
H; n = 3: delta- Valerolactone, (d-VL) R = H; n = 4: epsilon-
Caprolactone, (e-CL) R = CH.sub.3; n = 1: beta- Butyrolactone,
(b-BL) R = CH.sub.3; n = 2: gamma- Valerolactone, (g-VL)
##STR00036## Pivalolactone (PVL) ##STR00037## 1,5-Dioxepan-2-one
(DXO) ##STR00038## 5-(Benzyloxy)oxepan-2-one ##STR00039##
7-Oxooxepan-4-yl 2-bromo-2- methylpropanoate (BMP-XO) ##STR00040##
5-Phenyloxepan-2-one (PXO) ##STR00041## 5-Methyloxepan-2-one (MXO)
##STR00042## 1,4,8-Trioxa(4,6)spiro-9-undecane (TOSUO) ##STR00043##
5-(Benzyloxymethyl)oxepan-2-one (BOMXO) ##STR00044##
7-Oxooxepan-4-yl 3-hydroxy- 2-(hydroxymethyl)-2- methylpropanoate
(OX-BHMP) ##STR00045## (Z)-6,7-Dihydrooxepin-2(3H)-one
[0049] Examples of cyclic carbonyl monomers of formula (4) include
the compounds of Table 4.
TABLE-US-00004 TABLE 4 ##STR00046## D-Lactide, (DLA), L-Lactide,
(LLA), or racemic Lactide, 1:1 D:L forms, (DLLA) ##STR00047##
meso-Lactide, (MLA) (two opposite centers of asymmetry, R and S)
##STR00048## Glycolide (GLY)
[0050] As stated above, complexation via electrostatic interactions
is an effective packaging strategy for genetic material; however,
post-transfection release is often difficult and hence transfection
rates can be low. To circumvent this problem a charge-shifting
strategy can be utilized, wherein the cyclic carbonyl monomer
comprises a latent carboxylic acid group. By this is meant that the
cationic polymer comprises a pendant protected carboxylic acid that
can be converted to a carboxylic acid at about pH 5, corresponding
to the pH of the endosomal environment. An example of a latent
carboxylic acid group is an acetal-protected carboxylic acid group,
herein also referred to as an acetal ester group. The acetal ester
group has the general formula (5)
##STR00049##
wherein *-- represents the bond to a cyclic carbonyl moiety, and
R.sup.c and R.sup.d are monovalent radicals independently
comprising from 1 to 20 carbons. In an embodiment, R.sup.c is
methyl and R.sup.d is ethyl. In another embodiment, the second
cyclic carbonyl monomer is MTCOEE:
##STR00050##
[0051] When copolymerized into the cationic polymer, repeat units
derived from MTCOEE comprise a side chain acetal ester that is
readily deprotected in the acidic endosomal environment. Once
released into the cytoplasm, the resulting carboxylic acid groups
of the cationic polymer can be deprotonated, thus neutralizing the
net charge on the carrier and potentially facilitating the release
of the bio-active material. Cationic polymers derived from MTCOEE
are capable of binding DNA to form self-assembled nanoparticles
having an average diameter of between 90 and 110 nm.
[0052] Another strategy for facilitating endosomal release involves
non-covalent interactions to stabilize a bio-active cargo, for
example, using cyclic carbonyl monomers comprising a fluorinated
tertiary alcohol group. Fluorinated tertiary alcohol groups are
known to bind to phosphates and related structures, but with
interaction energies that are lower than electrostatic
interactions, and hence more easily released.
[0053] The above monomers can be purified by recrystallization from
a solvent such as ethyl acetate or by other known methods of
purification, with particular attention being paid to removing as
much water as possible from the monomer. The monomer moisture
content can be from 1 to 10,000 ppm, 1 to 1,000 ppm, 1 to 500 ppm,
and most specifically 1 to 100 ppm, by weight of the monomer.
[0054] The above-described cyclic carbonyl monomers, at least one
of which comprises a monovalent leaving group capable reacting with
the tertiary amine to form a quaternary amine, undergoes
ring-opening polymerization to form a first polymer. The first
polymer is a living polymer capable of initiating chain growth with
the same or a different cyclic carbonyl monomer, or a mixture of
cyclic carbonyl monomers, to form a block copolymer. The first
polymer is optionally treated with an endcapping agent to prevent
further chain growth and to stabilize the reactive end groups. The
resulting precursor polymer is then treated with a tertiary amine
to form the cationic polymer. The first polymer, the precursor
polymer, and the cationic polymer can be produced in atactic,
syndiotactic or isotactic forms. The particular tacticity depends
on the cyclic monomer(s), isomeric purity, and the reaction
conditions.
[0055] Alternatively, the cationic polymer can be obtained by
ring-opening polymerization of a cyclic carbonyl monomer comprising
a quaternary amine group and a tertiary amine group. However, these
monomers are more difficult to prepare, are less stable, and the
corresponding polymers tend to be more polydisperse. Therefore, the
quaternization reaction is performed after the ring-opening
polymerization.
[0056] In the simplest example, the first polymer is a homopolymer
prepared from a reaction mixture comprising a first cyclic carbonyl
monomer comprising a monovalent leaving group capable of reacting
with the tertiary amine to form a moiety comprising a quaternary
amine, a catalyst, an accelerator, a monomeric diol initiator, and
an optional solvent. In an embodiment, the catalyst and the
accelerator are the same material. For example, some ring opening
polymerizations can be conducted using
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) alone, with no another
catalyst. The ring-opening polymerization is generally conducted in
a reactor under inert atmosphere such as nitrogen or argon. The
polymerization can be performed by solution polymerization in an
inactive solvent such as benzene, toluene, xylene, cyclohexane,
n-hexane, dioxane, chloroform and dichloroethane, or by bulk
polymerization. The ROP reaction temperature can be from about
ambient temperature to 250.degree. C. Generally, the reaction
mixture is heated at atmospheric pressure for 0.5 to 72 hours to
effect polymerization, forming a second mixture comprising the
first polymer. A subunit derived from the monomeric diol initiator
is attached to an end of each ring opened polymer chain grown
therefrom. The first polymer is then optionally endcapped to form a
precursor polymer. The precursor polymer is then treated with a
tertiary amine to form the cationic polymer, wherein more than 0%
of repeat units derived from the first carbonyl monomer comprise a
side chain moiety comprising a quaternary amine.
[0057] The first polymer can also be a random copolymer formed by
the ring opening polymerization of a mixture comprising, for
example, a first cyclic carbonyl monomer and a hydrophobic second
cyclic carbonyl monomer. The random first polymer can be endcapped
to form a random precursor copolymer. In this case, the subunit
derived from the initiator, and the terminal repeat unit comprising
an endcap group, can be linked to a repeat unit derived from either
monomer. The random precursor copolymer is then treated with a
tertiary amine to form a random cationic copolymer, wherein more
than 0% of the repeat units derived from the first cyclic carbonyl
monomer comprise a moiety comprising a quaternary amine. The repeat
units derived from the second cyclic carbonyl monomer do not
comprise a monovalent leaving group capable of reacting with the
tertiary amine to form any quaternary amine. It is understood that
the reaction mixture can include additional cyclic carbonyl
monomers if desired, of the first category and/or the second
category.
[0058] More particularly, the first polymer is a block copolymer,
formed by the sequential ring-opening polymerization of, for
example, a first cyclic carbonyl monomer and a hydrophobic second
cyclic carbonyl monomer, to form a first block copolymer. The first
block copolymer is then optionally endcapped to form a precursor
block polymer. The precursor block polymer is then treated with a
tertiary amine to form a cationic block copolymer, wherein more
than 0% of the repeat units derived from the first cyclic carbonyl
monomer comprise a side chain moiety comprising a quaternary amine.
As before, the repeat units derived from the second cyclic carbonyl
monomer not comprise a monovalent leaving group capable of reacting
with the tertiary amine to form a quaternary amine. Depending on
the sequence order of the ring opening polymerizations, the subunit
derived from the monomeric diol initiator can be attached to first
repeat units derived from the first cyclic carbonyl monomer, or to
second repeat units derived from the second carbonyl monomer. In
one example, the first cyclic carbonyl monomer is polymerized first
to form a first block of the block copolymer, and the second cyclic
carbonyl monomer is polymerized second to form a second block of
the block copolymer. In this example, the cationic block copolymer
comprises a hydrophilic core block derived from the first cyclic
carbonyl monomer, which is attached to the subunit derived from the
monomeric diol initiator, and a hydrophobic outer block derived
from the second carbonyl monomer, which is optionally endcapped. In
another example, the second cyclic carbonyl monomer is polymerized
first, and the first cyclic carbonyl monomer is polymerized second.
In this example, the cationic block copolymer comprises a
hydrophobic core block comprising second repeat units attached to
the subunit derived from the diol initiator, and a hydrophilic
outer block, which is optionally endcapped. If desired, additional
blocks can be grown from the living ends of the non-endcapped
chains using the first cyclic carbonyl monomer, the second cyclic
carbonyl monomer, a different cyclic carbonyl monomer, or
combinations thereof. The block copolymers are amphiphilic, forming
self-assembled nano-sized particles in aqueous solution.
[0059] Exemplary catalysts for the ROP polymerization include metal
oxides such as tetramethoxy zirconium, tetra-iso-propoxy zirconium,
tetra-iso-butoxy zirconium, tetra-n-butoxy zirconium,
tetra-t-butoxy zirconium, triethoxy aluminum, tri-n-propoxy
aluminum, tri-iso-propoxy aluminum, tri-n-butoxy aluminum,
tri-iso-butoxy aluminum, tri-sec-butoxy aluminum,
mono-sec-butoxy-di-iso-propoxy aluminum, ethyl acetoacetate
aluminum diisopropylate, aluminum tris(ethyl acetoacetate),
tetraethoxy titanium, tetra-iso-propoxy titanium, tetra-n-propoxy
titanium, tetra-n-butoxy titanium, tetra-sec-butoxy titanium,
tetra-t-butoxy titanium, tri-iso-propoxy gallium, tri-iso-propoxy
antimony, tri-iso-butoxy antimony, trimethoxy boron, triethoxy
boron, tri-iso-propoxy boron, tri-n-propoxy boron, tri-iso-butoxy
boron, tri-n-butoxy boron, tri-sec-butoxy boron, tri-t-butoxy
boron, tri-iso-propoxy gallium, tetramethoxy germanium, tetraethoxy
germanium, tetra-iso-propoxy germanium, tetra-n-propoxy germanium,
tetra-iso-butoxy germanium, tetra-n-butoxy germanium,
tetra-sec-butoxy germanium and tetra-t-butoxy germanium;
halogenated compound such as antimony pentachloride, zinc chloride,
lithium bromide, tin(IV) chloride, cadmium chloride and boron
trifluoride diethyl ether; alkyl aluminum such as trimethyl
aluminum, triethyl aluminum, diethyl aluminum chloride, ethyl
aluminum dichloride and tri-iso-butyl aluminum; alkyl zinc such as
dimethyl zinc, diethyl zinc and diisopropyl zinc; tertiary amines
such as triallylamine, triethylamine, tri-n-octylamine and
benzyldimethylamine; heteropolyacids such as phosphotungstic acid,
phosphomolybdic acid, silicotungstic acid and alkali metal salt
thereof; zirconium compounds such as zirconium acid chloride,
zirconium octanoate, zirconium stearate and zirconium nitrate. More
particularly, the catalyst is zirconium octanoate, tetraalkoxy
zirconium or a trialkoxy aluminum compound.
[0060] Other ROP catalysts include metal-free organocatalysts that
can provide a platform to polymers having controlled, predictable
molecular weights and narrow polydispersities. Examples of
organocatalysts for the ROP of cyclic esters, carbonates and
siloxanes are 4-dimethylaminopyridine, phosphines, N-heterocyclic
carbenes (NHC), bifunctional aminothioureas, phosphazenes,
amidines, and guanidines. In an embodiment the catalyst is
N-(3,5-trifluoromethyl)phenyl-N'-cyclohexyl-thiourea (TU):
##STR00051##
[0061] Another metal-free ROP catalyst comprises at least one
1,1,1,3,3,3-hexafluoropropan-2-ol-2-yl (HFP) group. Singly-donating
hydrogen bond catalysts have the formula (6):
R.sup.2--C(CF.sub.3).sub.2OH (6).
R.sup.2 represents a hydrogen or a monovalent radical having from 1
to 20 carbons, for example an alkyl group, substituted alkyl group,
cycloalkyl group, substituted cycloalkyl group, heterocycloalkyl
group, substituted heterocycloalklyl group, aryl group, substituted
aryl group, or a combination thereof. Exemplary singly-donating
hydrogen bonding catalysts are listed in Table 5.
TABLE-US-00005 TABLE 5 ##STR00052## ##STR00053## ##STR00054##
##STR00055## ##STR00056##
[0062] Doubly-donating hydrogen bonding catalysts have two HFP
groups, represented by the general formula (7):
##STR00057##
wherein R.sup.3 is a divalent radical bridging group containing
from 1 to 20 carbons, such as an alkylene group, a substituted
alkylene group, a cycloalkylene group, substituted cycloalkylene
group, a heterocycloalkylene group, substituted heterocycloalkylene
group, an arylene group, a substituted arylene group, and a
combination thereof. Representative double hydrogen bonding
catalysts of formula (7) include those listed in Table 6. In a
specific embodiment, R.sup.2 is an arylene or substituted arylene
group, and the HFP groups occupy positions meta to each other on
the aromatic ring.
TABLE-US-00006 TABLE 6 ##STR00058## ##STR00059## ##STR00060##
##STR00061##
[0063] In one embodiment, the catalyst is selected from the group
consisting of 4-HFA-St, 4-HFA-Tol, HFTB, NFTB, HPIP, 3,5-HFA-MA,
3,5-HFA-St, 1,3-HFAB, 1,4-HFAB, and combinations thereof.
[0064] Also contemplated are catalysts comprising HFP-containing
groups bound to a support. In one embodiment, the support comprises
a polymer, a crosslinked polymer bead, an inorganic particle, or a
metallic particle. HFP-containing polymers can be formed by known
methods including direct polymerization of an HFP-containing
monomer (for example, the methacrylate monomer 3,5-HFA-MA or the
styryl monomer 3,5-HFA-St). Functional groups in HFP-containing
monomers that can undergo direct polymerization (or polymerization
with a comonomer) include acrylate, methacrylate, alpha, alpha,
alpha-trifluoromethacrylate, alpha-halomethacrylate, acrylamido,
methacrylamido, norbornene, vinyl, vinyl ether, and other groups
known in the art. Typical examples of such polymerizeable
HFP-containing monomers may be found in: Ito et al., Polym. Adv.
Technol. 2006, 17(2), 104-115, Ito et al., Adv. Polym. Sci. 2005,
172, 37-245, Ito et al., US20060292485, Maeda et al. WO2005098541,
Allen et al. US20070254235, and Miyazawa et al. WO2005005370.
Alternatively, pre-formed polymers and other solid support surfaces
can be modified by chemically bonding an HFP-containing group to
the polymer or support via a linking group. Examples of such
polymers or supports are referenced in M. R. Buchmeiser, ed.
"Polymeric Materials in Organic Synthesis and Catalysis,"
Wiley-VCH, 2003, M. Delgado and K. D. Janda "Polymeric Supports for
Solid Phase Organic Synthesis," Curr. Org. Chem. 2002, 6(12),
1031-1043, A. R. Vaino and K. D. Janda "Solid Phase Organic
Synthesis: A Critical Understanding of the Resin", J. Comb. Chem.
2000, 2(6), 579-596, D. C. Sherrington "Polymer-supported Reagents,
Catalysts, and Sorbents: Evolution and Exploitation--A Personalized
View," J. Polym. Sci. A. Polym. Chem. 2001, 39(14), 2364-2377, and
T. J. Dickerson et al. "Soluble Polymers as Scaffold for
Recoverable Catalysts and Reagents," Chem. Rev. 2002, 102(10),
3325-3343. Examples of linking groups include C.sub.1-C.sub.12
alkyl, a C.sub.1-C.sub.12 heteroalkyl, ether group, thioether
group, amino group, ester group, amide group, or a combination
thereof. Also contemplated are catalysts comprising charged
HFP-containing groups bound by ionic association to oppositely
charged sites on a polymer or a support surface.
[0065] The ROP reaction mixture comprises at least one catalyst
and, when appropriate, several catalysts together. The ROP catalyst
is added in a proportion of 1/20 to 1/40,000 moles relative to the
cyclic carbonyl monomers, and preferably of 1/1,000 to 1/20,000
moles.
[0066] The ring-opening polymerization is conducted in the presence
of an accelerator, in particular a nitrogen base. Exemplary
nitrogen base accelerators are listed below and include pyridine
(Py), N,N-dimethylaminocyclohexane (Me.sub.2NCy),
4-N,N-dimethylaminopyridine (DMAP), trans
1,2-bis(dimethylamino)cyclohexane (TMCHD),
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),
7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (-)-sparteine,
(Sp) 1,3-bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1),
1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2),
1,3-bis(2,6-di-1-propylphenyl(imidazol-2-ylidene (Im-3),
1,3-bis(1-adamantyl)imidazol-2-ylidene (Im-4),
1,3-di-1-propylimidazol-2-ylidene (Im-5),
1,3-di-t-butylimidazol-2-ylidene (Im-6),
1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene
(Im-7),
1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene,
1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8)
or a combination thereof, shown in Table 7.
TABLE-US-00007 TABLE 7 ##STR00062## Pyridine (Py) ##STR00063##
N,N-Dimethylaminocyclohexane (Me.sub.2NCy) ##STR00064##
4-N,N-Dimethylaminopyridinc (DMAP) ##STR00065## trans
1,2-Bis(dimethylamino) cyclohexane (TMCHD) ##STR00066##
1,8-Diazabicyclo[5.4.0]undec- 7-ene (DBU) ##STR00067##
7-Methyl-1,5,7-triazabicyclo [4.4.0]dec-5-ene (MTBD) ##STR00068##
1,5,7-Triazabicyclo[4.4.0] dec-5-ene (TBD) ##STR00069##
(-)-Sparteine (Sp) ##STR00070## 1,3-Bis(2-propyl)-4,5-
dimethylimidazol- 2-ylidene (Im-1) ##STR00071##
1,3-Bis(2,4,6-trimethylphenyl)imidazol-2- ylidene (Im-2)
##STR00072## 1,3-Bis(2,6-di-i-propylphenyl (imidazol-2-ylidene
(Im-3) ##STR00073## 1,3-Bis(1-adamantyl)imidazol-2-yliden) (Im-4)
##STR00074## 1,3-Di-i-propylimidazol-2-ylidene (Im-5) ##STR00075##
1,3-Di-t-butylimidazol-2-ylidene (Im-6) ##STR00076##
1,3-Bis(2,4,6-trimethylphenyl)-4,5- dihydroimidazol-2-ylidene
(Im-7) ##STR00077## 1,3-Bis(2,6-di-i-propylphenyl)-4,5-
dihydroimidazol-2-ylidene (Im-8)
[0067] In one embodiment, the accelerator has two or three
nitrogens, each capable of participating as a Lewis base, as for
example in the structure (-)-sparteine. Stronger bases generally
improve the polymerization rate.
[0068] The ROP reaction mixture also comprises an initiator.
Initiators generally include nucleophiles such as alcohols, amines
and thiols. The initiator can be monofunctional, difunctional or
multifunctional such as dendritic, polymeric or related
architectures. Monofunctional initiators can include nucleophiles
with protected functional groups that include thiols, amines, acids
and alcohols. The alcohol initiator can be any suitable alcohol,
including mono-alcohol, diol, triol, or other polyol, with the
proviso that the choice of alcohol does not adversely affect the
polymerization yield, polymer molecular weight, complexation with a
bio-active material, and/or the desirable mechanical and physical
properties of the product polymer. The alcohol can also be
multi-functional comprising, in addition to one or more hydroxyl
groups, a halide, an ether group, an ester group, an amide group,
or other functional group. Exemplary alcohols includes methanol,
ethanol, propanol, butanol, pentanol, amyl alcohol, capryl alcohol,
nonyl alcohol, decyl alcohol, undecyl alcohol, lauryl alcohol,
tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl
alcohol, heptadecyl alcohol, stearyl alcohol, nonadecyl alcohol and
other aliphatic saturated alcohols, cyclopentanol, cyclohexanol,
cycloheptanol, cyclooctanol and other aliphatic cyclic alcohols;
phenol, substituted phenols, benzyl alcohol, substituted benzyl
alcohol, benzenedimethanol, trimethylolpropane, a saccharide,
poly(ethylene glycol), propylene glycol, alcohol functionalized
block copolymers derived from oligomeric alcohols, or alcohol
functionalized branched polymers derived from branched alcohols, or
a combination thereof. In an embodiment, the ROP initiator is a
monomeric diol selected from the group consisting of ethylene
glycols, propylene glycols, hydroquinones, and resorcinols. Even
more specifically, the initiator is BnMPA, a precursor used in the
preparation of cyclic carbonate monomers:
##STR00078##
[0069] The ring-opening polymerization reaction can be performed
with or without the use of a solvent. Optional solvents include
dichloromethane, chloroform, benzene, toluene, xylene,
chlorobenzene, dichlorobenzene, benzotrifluoride, petroleum ether,
acetonitrile, pentane, hexane, heptane, 2,2,4-trimethylpentane,
cyclohexane, diethyl ether, t-butyl methyl ether, diisopropyl
ether, dioxane, tetrahydrofuran, or a combination comprising one of
the foregoing solvents. When a solvent is present, a suitable
monomer concentration is about 0.1 to 5 moles per liter, and more
particularly about 0.2 to 4 moles per liter. In a specific
embodiment, reaction mixture for the ring-opening polymerization is
free of a solvent.
[0070] The ring-opening polymerization can be performed at a
temperature that is about ambient temperature or higher, more
specifically a temperature from 15.degree. C. to 200.degree. C.,
and more particularly 20.degree. C. to 200.degree. C. When the
reaction is conducted in bulk, the polymerization is performed at a
temperature of 50.degree. C. or higher, and more particularly
100.degree.C. to 200.degree. C. Reaction times vary with solvent,
temperature, agitation rate, pressure, and equipment, but in
general the polymerizations are complete within 1 to 100 hours.
[0071] Whether performed in solution or in bulk, the
polymerizations are conducted in an inert (i.e., dry) atmosphere
and at a pressure of from 100 to 500 MPa (1 to 5 atm), more
typically at a pressure of 100 to 200 MPa (1 to 2 atm). At the
completion of the reaction, the solvent can be removed using
reduced pressure.
[0072] The catalyst is present in an amount of about 0.2 to 20 mol
%, 0.5 to 10 mol %, 1 to 5 mol %, or 1 to 2.5 mol %, based on total
moles of cyclic carbonyl monomer.
[0073] The nitrogen base accelerator is present in an amount of 0.1
to 5.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2 to 0.5 mol
%, based on total moles of cyclic carbonyl monomer. As stated
above, in some instances the catalyst and the nitrogen base
accelerator can be the same compound, depending on the particular
cyclic carbonyl monomer.
[0074] The amount of initiator is calculated based on the
equivalent molecular weight per hydroxyl group in the alcohol
initiator. The hydroxyl groups are present in an amount of 0.001 to
10.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, and 0.2 to 0.5 mol
%, based on total moles of cyclic carbonyl monomer. For example, if
the molecular weight of the initiator is 100 g/mole and the
initiator has 2 hydroxyl groups, the equivalent molecular weight
per hydroxyl group is 50 g/mole. If the polymerization calls for 5
mol % hydroxyl groups per mole of monomer, the amount of initiator
is 0.05.times.50=2.5 g per mole of monomer.
[0075] In a specific embodiment, the catalyst is present in an
amount of about 0.2 to 20 mol %, the nitrogen base accelerator is
present in an amount of 0.1 to 5.0 mol %, and the hydroxyl groups
of the initiator are present in an amount of 0.1 to 5.0 mol % based
on the equivalent molecular weight per hydroxyl group in the
initiator.
[0076] As stated above, the first polymer is a living polymer. The
first polymer comprises a terminal hydroxyl group, terminal thiol
group, or terminal amine group, each of which can initiate ROP
chain growth. Herein, the first polymer is endcapped to prevent
further chain growth and/or otherwise stabilize the backbone.
Endcapping materials and techniques are well established in polymer
chemistry. These include, for example materials for converting
terminal hydroxyl groups to esters, such as carboxylic acid
anhydrides, carboxylic acid chlorides, or reactive esters (e.g.,
p-nitrophenyl esters). In an embodiment, the first polymer is
treated with acetic anhydride to endcap the chains with acetyl
groups, forming the precursor polymer.
[0077] The first polymer and/or the precursor polymer can have a
number average molecular weight M.sub.n as determined by size
exclusion chromatography of at least 2500 g/mol, more specifically
4000 g/mol to 150000 g/mol, and even more specifically 10000 g/mol
to 50000 g/mol. In an embodiment, the first polymer and/or the
precursor polymer has a number average molecular weight M.sub.n of
10000 to 20000 g/mole. The first polymer and/or the precursor
polymer also has a narrow polydispersity index (PDI), generally
from 1.01 to 1.35, more particularly 1.10 to 1.30, and even more
particularly 1.10 to 1.25. The first polymer and/or the precursor
polymer can be a homopolymer, a random copolymer, or a block
copolymer. In an embodiment, the cationic polymer is a polyester
homopolymer, random polyester copolymer, a polycarbonate
homopolymer, random polycarbonate copolymer, or a random
polyestercarbonate copolymer.
[0078] The catalysts can be removed by selective precipitation or
in the case of the solid supported catalysts, simply by filtration.
The first polymer can comprise residual catalyst in an amount
greater than 0 wt. % (weight percent), based on total weight of the
first polymer and the residual catalyst. The amount of residual
catalyst can also be less than 20 wt. %, less than 15 wt. %, less
than 10 wt. %, less than 5 wt. %, less than 1 wt. %, or most
specifically less than 0.5 wt. % based on the total weight of the
first polymer and the residual catalyst. Similarly, the precursor
polymer can comprise a residual catalyst in an amount greater than
0 wt. %, based on total weight of the precursor polymer and the
residual catalyst. The amount of residual catalyst can also be less
than 20 wt. %, less than 15 wt. %, less than 10 wt. %, less than 5
wt. %, less than 1 wt. %, or most specifically less than 0.5 wt. %
based on the total weight of the precursor polymer and the residual
catalyst.
[0079] The precursor polymer comprises first repeat units derived
from the first cyclic carbonyl monomer. The first repeat units
comprising a side chain moiety comprising a reactive monovalent
leaving group, which when treated with a tertiary amine, produces a
cationic polymer comprising a moiety comprising a quaternary amine.
No limitation is placed on the structure of the tertiary amine,
with the proviso that the tertiary amine is capable of reacting
with more than 0%, 10% or more, 20% or more, 30% or more, 40% or
more, 50% or more, 60% or more, 70% or more, or more particularly
80% or more of the monovalent leaving groups of the first repeat
units to form a side chain moiety comprising a quaternary
amine.
[0080] The tertiary amine can comprise a single nitrogen such as a
trialkylamine, including but not limited to trimethylamine,
triethylamine, tripropylamine, and the like. The tertiary amine can
further comprise additional functional groups, in particular a
carboxylic acid group, for example 3-(N,N-dimethylamino)propionic
acid. In such instances, the cationic polymer will comprise first
repeat units comprising a side chain moiety comprising a quaternary
amine and a carboxylic acid group.
[0081] The tertiary amine can also comprise isotopically enriched
versions of the tertiary amine, such as trimethylamine-.sup.14C,
trimethylamine-.sup.15N, trimethylamine-.sup.15N,
trimethyl-.sup.13C.sub.3-amine, trimethyl-d.sub.9-amine, and
trimethyl-d.sub.9-amine-.sup.15N. The tertiary amine can also
comprise a radioactive moiety suitable for targeting a specific
cell type, such as a cancer cell. The radioactive moiety can
comprise a heavy metal radioactive isotope.
[0082] More particularly, the tertiary amine is a bis-tertiary
amine of the general formula (8):
##STR00079##
where L'' is a divalent linking group comprising 2 to 30 carbons,
and each monovalent R.sup.b group is independently selected from
alkyl groups comprising 1 to 30 carbons or aryl groups comprising 6
to 30 carbons. Each R.sup.b group can independently be branched or
non-branched. Each R.sup.b group can independently comprise
additional functional groups such as a ketone group, aldehyde
group, hydroxyl group, alkene group, alkyne group, cycloaliphatic
ring comprising 3 to 10 carbons, heterocylic ring comprising 2 to
10 carbons, ether group, amide group, ester group, and combinations
of the foregoing additional functional groups. The heterocyclic
ring can comprise oxygen, sulfur and/or nitrogen. Two or more
R.sup.b groups can also together form a ring. Representative L''
groups include --(CH.sub.2).sub.z'-- where z' is an integer from 2
to 30, --(CH.sub.2CH.sub.2O).sub.z--CH.sub.2CH.sub.2-- where z'' is
an integer from 1 to 10, --CH.sub.2CH.sub.2SCH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2SSCH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2SOCH.sub.2CH.sub.2--, and
--CH.sub.2CH.sub.2SO.sub.2CH.sub.2CH.sub.2--. L'' can further
comprise a monovalent or divalent cycloaliphatic ring comprising 3
to 20 carbons, a monovalent or divalent aromatic ring comprising 6
to 20 carbons, a ketone group, aldehyde group, hydroxyl group,
alkene group, alkyne group, a heterocylic ring comprising 2 to 10
carbons, ether group, amide group, ester group, and combinations of
the foregoing functional groups. The heterocyclic ring can comprise
oxygen, sulfur and/or nitrogen. The bis-tertiary amine can also
comprise isotopically enriched forms of the bis-tertiary amine,
such as deuterium, carbon-13, and/or nitrogen-15 enriched forms
thereof.
[0083] More specific bis-tertiary amines include
N,N,N',N'-tetramethyl-1,2-ethanediamine (TMEDA),
N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA),
N,N,N',N'-tetramethyl-1,4-butanediamine (TMBDA),
N,N,N',N'-tetraethyl-1,2-ethanediamine (TEEDA),
N,N,N',N'-tetraethyl-1,3propanediamine (TEPDA),
1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),
N,N,N',N'-tetraethyl-1,4-butanediamine (TEBDA),
4-dimethylaminopyridine (DMAP),
4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),
4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations
thereof. In an embodiment, the bis-tertiary amine is TMEDA.
[0084] The precursor polymer is treated with the tertiary amine in
a suitable organic solvent such as dimethylsulfoxide (DMSO) to form
the cationic polymer. The reaction is conducted under anhydrous
conditions, at ambient or elevated temperature using excess
tertiary amine relative to the monovalent leaving group. In
general, the tertiary amine is used in an amount of from 2 to 10 to
moles per mole of monovalent leaving group in the precursor
polymer, more particularly 3 to 8 moles per mole of monovalent
leaving group in the precursor polymer, even more particularly 3 to
5 moles per mole of monovalent leaving group in the precursor
polymer. The positive charged quaternary amine forms a salt with
the displaced leaving group, which becomes a negatively charged
counterion. Alternatively, the negatively charged counterion can be
ion exchanged with another more suitable negatively charged
counterion using known methods, if desired.
[0085] The cationic polymer is isolated by one or more
precipitations in an organic solvent such as tetrahydrofuran,
followed by filtration and drying in vacuo. More than 0% of the
first repeat units comprise a side chain moiety comprising a
quaternary amine group. When the precursor polymer is treated with
a bis-tertiary amine, more than 0% of the first repeat units
comprise a side chain moiety comprising a quaternary amine group
and a tertiary amine group. When the precursor polymer is treated
with a tertiary amine comprising a carboxy group, more than 0% of
the first repeat units derived from the first cyclic carbonyl
monomer comprise the side chain moiety comprising the quaternary
amine and a carboxylic acid. The quaternary amine group is present
in the cationic polymer in an amount of from more than 0% of the
side chain monovalent leaving groups derived from the first cyclic
carbonyl monomer. More particularly, the quaternary amine group is
present in the cationic polymer in an amount of from 10 to 100%, 20
to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to
100%, or 80 to 100% of the side chain monovalent leaving groups
derived from the first cyclic carbonyl monomer. When the precursor
polymer is treated with a bis-tertiary amine, the tertiary amine
group can be present in the cationic polymer in an amount of from
more than 0% of the monovalent leaving groups in the first repeat
units of the precursor polymer, more particularly from 10 to 100%,
from 20 to 100%, from 30 to 100%, from 40 to 100%, from 50 to 100%,
from 60 to 100%, from 70 to 100%, or from 80 to 100% of the
monovalent leaving groups in the first repeat units of the
precursor polymer.
[0086] The cationic polymer can have a number average molecular
weight M.sub.n as determined by size exclusion chromatography of at
least 2500 g/mol, more specifically 4000 g/mol to 150000 g/mol, and
even more specifically 10000 g/mol to 50000 g/mol. In an
embodiment, the cationic polymer has a number average molecular
weight M.sub.n of 10000 to 20000 g/mole. The cationic polymer also
has a narrow polydispersity index (PDI), generally from 1.01 to
1.35, more particularly 1.10 to 1.30, and even more particularly
1.10 to 1.25.
[0087] More particularly, the cationic polymer is an amphiphilic
block copolymer comprising derived by sequential ring opening
polymerization of a first cyclic carbonyl monomer and a second
cyclic carbonyl monomer. The cationic polymer can comprises two or
more block copolymer chains linked to the subunit derived from a
polyol initiator. Each of the two or more block copolymer chains
comprises a hydrophobic block and a hydrophilic block, and each of
the two or more block copolymer chains can be optionally endcapped.
In an embodiment, the cationic block copolymer comprises a
hydrophilic core block comprising first repeat units derived from
the first cyclic carbonyl monomer, wherein the first repeat units
are linked to the subunit derived from a polyol initiator, and a
hydrophobic outer block comprising second repeat units derived from
a second cyclic carbonyl monomer. In another embodiment, the
sequential polymerization of the cyclic carbonyl monomers is
reversed. That is, the cationic block copolymer comprises a
hydrophobic core block comprising second repeat units derived from
the second cyclic carbonyl monomer, wherein the second repeat units
are linked to the subunit derived from a polyol initiator, and a
hydrophilic outer block comprising first repeat units derived from
the first cyclic carbonyl monomer linked to the hydrophobic core
block. In an embodiment, the polyol initiator is a monomeric
diol.
[0088] In aqueous solution the cationic polymers self-assemble into
nanoparticles having an average particle size, for example, of from
10 nm to 500 nm, 10 nm to 250 nm, 50 nm to 200 nm, 50 nm to 150 nm,
50 nm to 120 nm, and even more particularly from 50 nm to 100 nm,
as measured by dynamic light scattering (Brookhaven Instrument
Corp., Holtsville, N.Y., U.S.A.) equipped with a He--Ne laser beam
at 658 nm (scattering angle: 90). The particle size measurements
are repeated for 5 runs for each sample, and the particle size are
reported as the average of 5 readings. For the foregoing particle
sizes, the aqueous solution can have a pH of from 5.0 to 8.0.
[0089] A method of forming a biodegradable cationic polymer
comprises forming a first mixture comprising a first cyclic
carbonyl monomer, a catalyst, an accelerator, a monomeric diol
initiator, and an optional solvent, wherein the first cyclic
carbonyl monomer comprises a monovalent leaving group capable of
reacting with a tertiary amine to form a quaternary amine; forming
a first polymer comprising first repeat units derived from the
first cyclic carbonyl monomer by ring-opening polymerization;
optionally endcapping the first polymer to form a precursor
polymer; and treating the precursor polymer with the tertiary amine
to form the cationic polymer, wherein more than 0% of the repeat
units derived from the first cyclic monomer comprise a side chain
moiety comprising a quaternary amine. In an embodiment, the
tertiary amine is a bis-tertiary amine and the side chain moiety
comprises the quaternary amine and a tertiary amine. In another
embodiment, the bis-tertiary amine is selected from the group
consisting of N,N,N',N'-tetramethyl-1,2-ethanediamine (TMEDA),
N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA),
N,N,N',N'-tetramethyl-1,4-butanediamine (TMBDA),
N,N,N',N'-tetraethyl-1,2-ethanediamine (TEEDA),
N,N,N',N'-tetraethyl-1,3propanediamine (TEPDA),
1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),
N,N,N',N'-tetraethyl-1,4-butanediamine (TEBDA),
4-dimethylaminopyridine (DMAP),
4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),
4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations
thereof. In another embodiment, the tertiary amine comprises a
carboxy group and the first repeat unit comprises a side chain
moiety comprising the quaternary amine and a carboxylic acid. In
another embodiment, the first mixture comprises a hydrophobic
second cyclic carbonyl monomer, and the cationic polymer is a
random copolymer comprising a second repeat unit derived from the
second cyclic carbonyl monomer by ring-opening polymerization;
wherein the second cyclic carbonyl monomer does not comprise a
monovalent leaving group capable of reacting with a tertiary amine
to form a quaternary amine. In another embodiment, the second
repeat unit comprises a side chain acetal ester group.
[0090] Another method of forming a biodegradable cationic block
copolymer comprises forming a reaction mixture comprising a
catalyst, an accelerator, a monomeric diol initiator, and an
optional solvent; sequentially adding to the reaction mixture and
reacting by ring-opening polymerization a first cyclic carbonyl
monomer followed by a second cyclic carbonyl monomer, thereby
forming a first block copolymer, wherein the first cyclic carbonyl
monomer comprises a monovalent leaving group capable of reacting
with a tertiary amine to form a quaternaray amine, and the second
cyclic carbonyl monomer is not capable of reacting with the
tertiary amine to form the quaternary amine; optionally endcapping
the first block copolymer, thereby forming a precursor block
copolymer; and treating the precursor block copolymer with the
tertiary amine to form the cationic polymer, wherein the cationic
polymer comprises first repeat units derived from the first cyclic
carbonyl monomer, and more than 0% of the first repeat units
comprise a side chain moiety comprising the quaternary amine. In an
embodiment, the sequential reaction is performed in reverse order
to form the first block copolymer. In another embodiment, the first
block copolymer is endcapped using a carboxylic anhydride, thereby
forming a terminal ester group. In another embodiment, the cationic
block copolymer comprises a second repeat unit derived from the
second cyclic carbonyl monomer, and the second repeat unit
comprises a side chain acetal ester group. In another embodiment,
the initiator is a monomeric diol selected from the group
consisting of ethylene glycols, propylene glycols, hydroquinones,
and resorcinols. In another embodiment, the initiator is BnMPA. In
another embodiment, the monovalent leaving group is selected from
the group consisting of halides, sulphonate esters, and epoxides.
In another embodiment, the tertiary amine is a bis-tertiary amine
and the side chain moiety comprises the quaternary amine and a
tertiary amine. In another embodiment, the bis-tertiary amine is
selected from the group consisting of
N,N,N',N'-tetramethyl-1,2-ethanediamine (TMEDA),
N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA),
N,N,N',N'-tetramethyl-1,4-butanediamine (TMBDA),
N,N,N',N'-tetraethyl-1,2-ethanediamine (TEEDA),
N,N,N',N'-tetraethyl-1,3propanediamine (TEPDA),
1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),
N,N,N',N'-tetraethyl-1,4-butanediamine (TEBDA),
4-dimethylaminopyridine (DMAP),
4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),
4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations
thereof.
[0091] In general, the cationic polymer obtained by ring-opening
polymerization comprises as many branches as the number of
initiating sites on the initiator. Further, the cationic polymer
comprises as many blocks as the number of sequential ring-opening
polymerizations prior to endcapping, with the understanding that
successive ring-opening polymerizations are performed using
different cyclic carbonyl monomer compositions.
[0092] The cationic polymers form complexes (polyplexes) with a
negatively charged bio-active material such as a gene, a
nucleotide, a protein, a peptide, a drug, or a combination thereof.
In aqueous solution at a pH of from 5.0 to 8.0, the complexes
self-assemble into nanoparticles having an average particle size,
for example, of from 10 nm to 500 nm, 10 nm to 250 nm, 50 nm to 200
nm, 50 nm to 150 nm, 50 nm to 120 nm, and even more particularly
from 50 nm to 100 nm, as measured by dynamic light scattering
(Brookhaven Instrument Corp., Holtsville, N.Y., U.S.A.) equipped
with a He--Ne laser beam at 658 nm (scattering angle: 90.degree.).
The polymer/DNA complexes are prepared at various N/P ratios by
gently mixing. Prior to particle size analysis, the complex
solutions are allowed to stabilize for 30 minutes. The particle
size measurements are repeated for 5 runs for each sample, and the
particle size are reported as the average of 5 readings. In an
embodiment, the bio-active material is a negatively charged genetic
material, and the polyplex is tightly packed due to the strong
interaction of oppositely charged groups on the cationic polymer
and the negatively charged genetic material. The nano-sized
complexes induce 0 to 15% hemolysis, more particularly no
hemolysis, and have a cytotoxicity of 0 to 20%, or more
particularly no cytotoxicity. In another embodiment, the bio-active
material is a drug.
[0093] Also disclosed is a method of preparing a polymer complex
for treating a cell, comprising contacting a first aqueous mixture
comprising a biodegradable cationic polymer with a second aqueous
mixture comprising a negatively charged biologically active
material to form a third aqueous mixture comprising the polymer
complex, wherein the biodegradable cationic polymer comprises:
first repeat units derived from a first cyclic carbonyl monomer by
ring-opening polymerization, a subunit derived from a monomeric
diol initiator for the ring-opening polymerization, and an optional
endcap group, wherein more than 0% of the first repeat units
comprise a side chain moiety comprising a quaternary amine group.
The polymer complex has a particle size of 50 to 500 nm at a pH of
from 5.0 to 8.0.
[0094] Further disclosed is a method of treating a cell, comprising
contacting the cell with a nanoparticles of a polymer complex
comprising a biodegradable cationic polymer and a negatively
charged biologically active material; wherein the biodegradable
cationic polymer comprises: first repeat units derived from a first
cyclic carbonyl monomer by ring-opening polymerization, a subunit
derived from a monomeric diol initiator for the ring-opening
polymerization, and an optional endcap group, wherein more than 0%
of the first repeat units comprise a side chain moiety comprising a
quaternary amine group. In an embodiment, the biodegradable polymer
is an amphiphilic block copolymer. The nanoparticles can have a
particle size of 50 to 500 nm at a pH of from 5.0 to 8.0. In an
embodiment, the negatively charged biologically active material is
a gene. The cells can be exposed to the polymer complex in vitro,
ex vivo and then subsequently placed into an animal, or in vivo
(for example, an animal or human). In another embodiment, the
negatively charged biologically active material is a molecular drug
or a protein. In another embodiment, the polymer complex induces no
hemolysis. In another embodiment, the nanoparticles have no
cytotoxicity.
[0095] Exemplary commercially available drugs include
13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine,
5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine,
Abraxane, Accutane.RTM., Actinomycin-D, Adriamycin.RTM.,
Adrucil.RTM., Afinitor.RTM., Agrylin.RTM., Ala-Cort.RTM.,
Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ.RTM.,
Alkeran.RTM., All-transretinoic Acid, Alpha Interferon,
Altretamine, Amethopterin, Amifostine, Aminoglutethimide,
Anagrelide, Anandron.RTM., Anastrozole, Arabinosylcytosine, Ara-C,
Aranesp.RTM., Aredia.RTM., Arimidex.RTM., Aromasin.RTM.,
Arranon.RTM., Arsenic Trioxide, Asparaginase, ATRA, Avastin.RTM.,
Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene,
BEXXAR.RTM., Bicalutamide, BiCNU, Blenoxane.RTM., Bleomycin,
Bortezomib, Busulfan, Busulfex.RTM., C225, Calcium Leucovorin,
Campath.RTM., Camptosar.RTM., Camptothecin-11, Capecitabine,
Carac.TM., Carboplatin, Carmustine, Carmustine Wafer, Casodex.RTM.,
CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine.RTM., Cetuximab,
Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone,
Cosmegen.RTM., CPT-11, Cyclophosphamide, Cytadren.RTM., Cytarabine,
Cytarabine Liposomal, Cytosar-U.RTM., Cytoxan.RTM., Dacarbazine,
Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin,
Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal,
DaunoXome.RTM., Decadron, Decitabine, Delta-Cortef.RTM.,
Deltasone.RTM., Denileukin Diftitox, DepoCyt.TM., Dexamethasone,
Dexamethasone Acetate, Dexamethasone Sodium Phosphate Dexasone,
Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil.RTM., Doxorubicin,
Doxorubicin Liposomal, Droxia.TM., DTIC, DTIC-Dome.RTM.,
Duralone.RTM., Efudex.RTM., Eligard.TM., Ellence.TM., Eloxatin.TM.,
Elspar.RTM., Emcyt.RTM., Epirubicin, Epoetin Alfa, Erbitux,
Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol,
Etopophos.RTM., Etoposide, Etoposide Phosphate, Eulexin.RTM.,
Everolimus, Evista.RTM., Exemestane, Fareston.RTM., Faslodex.RTM.,
Femara.RTM., Filgrastim, Floxuridine, Fludara.RTM., Fludarabine,
Fluoroplex.RTM., Fluorouracil, Fluorouracil (cream),
Fluoxymesterone, Flutamide, Folinic Acid, FUDR.RTM., Fulvestrant,
G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar,
Gleevec.TM., Gliadel.RTM. Wafer, GM-CSF, Goserelin,
Granulocyte--Colony Stimulating Factor, Granulocyte Macrophage
Colony Stimulating Factor, Halotestin.RTM., Herceptin.RTM.,
Hexadrol, Hexylen.RTM., Hexamethylmelamine, HMM, Hycamtin.RTM.,
Hydrea.RTM., Hydrocort Acetate.RTM., Hydrocortisone, Hydrocortisone
Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortone
Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan
Idamycin.RTM., Idarubicin, Ifex.RTM., IFN-alpha Ifosfamide, IL-11
IL-2 Imatinib mesylate, Imidazole Carboxamide Interferon alfa,
Interferon Alfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11,
Intron A.RTM. (interferon alfa-2b), Iressa.RTM., Irinotecan,
Isotretinoin, Ixabepilone, Ixempra.TM., K Kidrolase (t),
Lanacort.RTM., Lapatinib, L-asparaginase, LCR, Lenalidomide,
Letrozole, Leucovorin, Leukeran, Leukine.TM., Leuprolide,
Leurocristine, Leustatin.TM., Liposomal Ara-C, Liquid Pred.RTM.,
Lomustine, L-PAM, L-Sarcolysin, Lupron.RTM., Lupron Depot.RTM.,
Matulane.RTM., Maxidex, Mechlorethamine, Mechlorethamine
Hydrochloride, Medralone.RTM., Medrol.RTM., Megace.RTM., Megestrol,
Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex.TM.,
Methotrexate, Methotrexate Sodium, Methylprednisolone,
Meticorten.RTM., Mitomycin, Mitomycin-C, Mitoxantrone,
M-Prednisol.RTM., MTC, MTX, Mustargen.RTM., Mustine Mutamycin.RTM.,
Myleran.RTM., Mylocel.TM., Mylotarg.RTM., Navelbine.RTM.,
Nelarabine, Neosar.RTM., Neulasta.TM., Neumega.RTM., Neupogen.RTM.,
Nexavar.RTM., Nilandron.RTM., Nilutamide, Nipent.RTM., Nitrogen
Mustard, Novaldex.RTM., Novantrone.RTM., Octreotide, Octreotide
acetate, Oncospar.RTM., Oncovin.RTM., Ontak.RTM., Onxal.TM.,
Oprevelkin, Orapred.RTM., Orasone.RTM., Oxaliplatin, Paclitaxel,
Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin.RTM.,
Paraplatin.RTM., Pediapred.RTM., PEG Interferon, Pegaspargase,
Pegfilgrastim, PEG-INTRON.TM., PEG-L-asparaginase, PEMETREXED,
Pentostatin, Phenylalanine Mustard, Platinol.RTM.,
Platinol-AQ.RTM., Prednisolone, Prednisone, Prelone.RTM.,
Procarbazine, PROCRIT.RTM., Proleukin.RTM., Prolifeprospan 20 with
Carmustine Implant, Purinethol.RTM., Raloxifene, Revlimid.RTM.,
Rheumatrex.RTM., Rituxan.RTM., Rituximab, Roferon-A.RTM.
(Interferon Alfa-2a) Rubex.RTM., Rubidomycin hydrochloride,
Sandostatin.RTM., Sandostatin LAR.RTM., Sargramostim,
Solu-Cortef.RTM., Solu-Medrol.RTM., Sorafenib, SPRYCEL.TM.,
STI-571, Streptozocin, SU11248, Sunitinib, Sutent.RTM., Tamoxifen,
Tarceva.RTM., Targretin.RTM., Taxol.RTM., Taxotere.RTM.,
Temodar.RTM., Temozolomide, Temsirolimus, Teniposide, TESPA,
Thalidomide, Thalomid.RTM., TheraCys.RTM., Thioguanine, Thioguanine
Tabloid.RTM., Thiophosphoamide, Thioplex.RTM., Thiotepa, TICE.RTM.,
Toposar.RTM., Topotecan, Toremifene, Torisel.RTM., Tositumomab,
Trastuzumab, Treanda.RTM., Tretinoin, Trexall.TM., Trisenox.RTM.,
TSPA, TYKERB.RTM., VCR, Vectibix.TM., Velban.RTM., Velcade.RTM.,
VePesid.RTM., Vesanoid.RTM., Viadur.TM., Vidaza.RTM., Vinblastine,
Vinblastine Sulfate, Vincasar Pfs.RTM., Vincristine, Vinorelbine,
Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon.RTM.,
Xeloda.RTM., Zanosar.RTM., Zevalin.TM., Zinecard.RTM.,
Zoladex.RTM., Zoledronic acid, Zolinza, and Zometa.
[0096] Any cell that can be transfected by a non-viral vector can
be treated with the above-described complexes. In particular the
cells are eukaryotic cells, mammalian cells, and more particularly
rodent or human cells. The cells can be derived from various
tissues, including extraembryonic or embryonic stem cells,
totipotent or pluripotent, dividing or non-dividing, parenchyma or
epithelium, immortalized or transformed, or the like. The cell may
be a stem cell or a differentiated cell. Cell types that are
differentiated include adipocytes, fibroblasts, myocytes,
cardiomyocytes, endothelium, dendritic cells, neurons, glia, mast
cells, blood cells and leukocytes (e.g., erythrocytes,
megakaryotes, lymphocytes, such as B, T and natural killer cells,
macrophages, neutrophils, eosinophils, basophils, platelets,
granulocytes), epithelial cells, keratinocytes, chondrocytes,
osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine
or exocrine glands, as well as sensory cells.
[0097] The above-described complexes can be used as non-viral
transfection vectors. The target gene is not limited to any
particular type of target gene or nucleotide sequence. For example,
the target gene can be a cellular gene, an endogenous gene, an
oncogene, a transgene, a viral gene, or translated and
non-translated RNAs. Exemplary possible target genes include:
transcription factors and developmental genes (e.g., adhesion
molecules, cyclin-dependent kinase inhibitors, Wnt family members,
Pax family members, Winged helix family members, Hox family
members, cytokines/lymphokines and their receptors,
growth/differentiation factors and their receptors,
neurotransmitters and their receptors); oncogenes (e.g., ABLI,
BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, ERBB2, ETSI, ETV6,
FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC,
MYCLI, MYCN, NRAS, PIMI, PML, RET, SKP2, SRC, TALI, TCL3, and YES);
tumor suppressor genes (e.g., APC, BRAI, BRCA2, CTMP, MADH4, MCC,
NFI, NF2, RB1, TP53, and WTI); and enzymes (e.g., ACP desaturases
and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol
dehydrogenases, amylases, amyloglucosidases, catalases,
cyclooxygenases, decarboxylases, dextrinases, DNA and RNA
polymerases, galactosidases, glucose oxidases, GTPases, helicases,
integrases, insulinases, invertases, isomerases, kinases, lactases,
lipases, lipoxygenases, lysozymes, peroxidases, phosphatases,
phospholipases, phosphorylases, proteinases and peptidases,
recombinases, reverse transcriptases, telomerase, including RNA
and/or protein components, and topoisomerases).
[0098] The following examples demonstrate that the biodegradable
polycarbonate and poly(estercarbonate) copolymers produced by
organocatalytic ring-opening polymerization are effective non-viral
gene carriers. The combination of biodegradable halogen-containing
carbonate and a simple quaternization reaction with amines provides
a versatile pathway to forming cationic polymers for gene carriers
having diverse functionality. The halide on the precursor polymers
can be varied depending on the target architectures and the types
of application. The precursor polymers, particularly those bearing
chloride residues, form packed complexes with genes, and the
polyplexes show low cytotoxicity. The cationic polycarbonates can
self-assemble into micellar nanoparticles having a hydrophobic core
and a positively charged surface. Therefore, they can also be used
for delivery of small molecular drugs and proteins, and for
simultaneous delivery of drugs and genes, or drugs and
proteins.
EXAMPLES
Materials for Polymer Synthesis
[0099] THF, DMF, and methylene chloride used in the reaction were
obtained by a solvents drying system (Innovative).
N-(3,5-trifluoromethyl)phenyl-N'-cyclohexyl-thiourea (TU) was
prepared as reported by R. C. Pratt, B. G. G. Lohmeijer, D. A.
Long, P. N. P. Lundberg, A. Dove, H. Li, C. G. Wade, R. M.
Waymouth, and J. L. Hedrick, Macromolecules, 2006, 39 (23),
7863-7871, and dried by stirring in dry THF over CaH.sub.2,
filtering, and removing solvent under vacuum. BisMPA benzylester
(BnMPA) was prepared as described below, and further dried by
dissolving in dry THF, stiffing with CaH.sub.2, filtering, and
removing the solvent in vacuo. Acetic anhydride, DMSO,
N,N,N',N'-tetramethyl-1,2-ethanediamine (TMEDA),
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and (-)-sparteine were
stirred over CaH.sub.2, vacuum distilled, then stored over
molecular sieves (3 .ANG.). L-lactide and D-lactide (Purac, 99%)
were recrystallized from dry toluene 3 times prior to use.
Trimethylenecarbonate (TMC) was azeotropically dried from toluene
prior to use. Other reagents were used as received.
Materials for Physicochemical and Biological Characterizations of
Polymers.
[0100] Acetic acid, sodium acetate, polyethylenimine (PEI,
branched, weight average molecular weight M.sub.w 25 kDa), agarose,
ethidium bromide and 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyl
tetrazolium bromide (MTT) were all purchased from Sigma-Aldrich and
used as received. Ultra pure water of HPLC grade was obtained from
J. T. Baker (U.S.A.). Phosphate-buffered saline (PBS) and
tris-boric acid-EDTA (TBE) buffers were purchased from 1st BASE
(Malaysia) and diluted to the intended concentration before use.
Reporter lysis buffer and luciferin substrate were purchased from
Promega (U.S.A.). DMEM growth medium, fetal bovine serum (FBS),
penicillin and streptomycin were all purchased from Invitrogen
Corporation (U.S.A.). Plasmid DNA encoding the 6.4 kb firefly
luciferase (pCMV-luciferase VR1255C) driven by cytomegalovirus
(CMV) promoter was kindly provided by Car Wheeler, Vical (U.S.A.),
which was amplified in E. coli DH5.alpha. and purified with
Endofree Giga plasmid purification kit supplied by Qiagen (Dutch).
HepG2 cell line was obtained from ATCC (U.S.A.) and grown under the
recommended conditions according to the supplier.
Monomer Syntheses.
[0101] A particularly useful synthon for functional biodegradable
monomers is so-called MTC family of cyclic carbonate monomer
derived from 2,2-bis(methylol)propionic acid (bis-MPA). Bis-MPA
provides a facile route to 5-methyl-5-carboxyl-1,3-dioxan-2-one
(MTCOH) and derivative thereof, as shown in Scheme 1.
##STR00080##
[0102] This approach parallels that of (meth)acrylate
derivatization and has been demonstrated to create a wide selection
of functional monomers capable of undergoing ring-opening
polymerization. 2,2-Bis(methylol)propionic acid (BisMPA) is first
converted (i) to a benzyl ester BnMPA (herein also used as an
initiator for the polymerizations), followed by reaction (ii) of
BnMPA with triphosgene to form a cyclic carbonyl monomer, MTCOBn.
MTCOBn is debenzylated (iii) to produce the cyclic carbonyl
carboxylic acid, MTCOH. Two pathways are shown for forming an ester
from MTCOH. In the first pathway, (iv), MTCOH is treated with a
suitable carboxy activating agent, such as dicyclohexylcarbodiimide
(DCC), which reacts with ROH to form MTCOR in a single step.
Alternatively, MTCOH can be converted first (v) to the acid
chloride MTCCl followed by treatment (vi) of MTCCl with ROH in the
presence of a base to form MTCOR. Both pathways are illustrative
and are not meant to be limiting. The following conditions are
typical for the reactions shown in Scheme 1: (i) Benzylbromide
(BnBr), KOH, DMF, 100.degree. C., 15 hours, 62% yield of the benzyl
ester of bis-MPA; (ii) triphosgene, pyridine, CH.sub.2Cl.sub.2,
-78.degree. C. to 0.degree. C., 95% yield of MTCOBn; (iii) Pd/C
(10%), H2 (3 atm), EtOAc, room temperature, 24 hours, 99% yield of
MTCOH; (iv) ROH, DCC, THF, room temperature, 1 to 24 hours; (v)
(COCl).sub.2, THF, room temperature, 1 hour, 99% yield of MTCCl;
(vi) ROH, NEt.sub.3, RT, 3 hours yields MTCOR.
[0103] Using the above scheme, MTCCl was reacted with
3-bromopropanol, 3-choloropropano, 2-iodoethanol, and ethanol to
form the corresponding MTCOPrBr, MTCOPrCl, MTCOEtI, and MTCOEt. The
haloesters were purified by either recrystallization or by flash
chromatography (ethyl acetate/hexane) in high yields (>85%).
MTCOEt was used as a non-functional counterpart for dilution
effects and to introduce hydrophobic blocks to the polymer for
self-assembly.
Example 1
Preparation of
5-methyl-5-(3-chloropropyl)oxycarboxyl-1,3-dioxan-2-one,
(MTCOPrCl), mw 236.65
##STR00081##
[0105] A catalytic amount (3 drops) of DMF was added to a THF
solution (200 mL) of MTCOH (11.1 g, 69 mmol), followed by a
solution of oxalyl chloride (7.3 mL, 87 mmol) in THF (100 mL),
gently added over 20 min under N.sub.2 atmosphere. The solution was
stirred for 1 hour, bubbled with N.sub.2 flow to remove volatiles,
and evaporated under vacuum to give the intermediate.
[0106] A mixture of 3-chloro-1-propanol (5.4 mL, 76 mmol) and
pyridine (6.2 mL, 65 mmol) in dry THF (50 mL) was added dropwise to
a dry THF solution (100 mL) of the intermediate over 30 min, while
maintaining a solution temperature below 0.degree. C. with an
ice/salt bath. The reaction mixture was kept stirring for another 3
hours at room temperature before it was filtered and the filtrate
evaporated. The residue was dissolved in methylene chloride and
washed with 1N HCl aqueous solution, saturated NaHCO.sub.3 aqueous
solution, brine and water, stirred with MgSO.sub.4 overnight, and
the solvent evaporated. The crude product was passed through a
silica gel column by gradient eluting of ethyl acetate and hexane
(50/50 to 80/20) to provide the product as a colorless oil that
slowly solidified to a white solid (9.8 g, 60%).
Example 2
Preparation of
5-methyl-5-(3-bromopropyl)oxycarboxyl-1,3-dioxan-2-one, (MTCOPrBr),
mw 281.10
##STR00082##
[0108] MTCOPrBr was prepared by the procedure of Example 1 on a 45
mmol scale using 3-bromo-1-propanol as the alcohol. The product was
purified by column chromatography, and subsequently recrystallized
to yield white crystals (6.3 g, 49%). .sup.1H NMR (400 MHz,
CDCl.sub.3): delta 4.69 (d, 2H; CH.sub.2OCOO), 4.37 (t, 2H;
OCH.sub.2), 4.21 (d, 2H; CH.sub.2OCOO), 3.45 (t, 2H; CH.sub.2Br),
2.23 (m, 2H; CH.sub.2), 1.33 (s, 3H; CH.sub.3). .sup.13C NMR (100
MHz, CDCl.sub.3): delta 171.0, 147.3, 72.9, 63.9, 40.2, 31.0, 28.9,
17.3.
Example 3
Preparation of
5-methyl-5-(2-iodoethyl)oxycarboxyl-1,3-dioxan-2-one, (MTCOEtI), mw
314.08
##STR00083##
[0110] MTCOEtI was prepared by the procedure of Example 1 on a 45
mmol scale, using 2-iodoethanol as the alcohol, and was purified by
column chromatography and subsequent recrystallization to yield
yellowish crystals (7.7 g, 54%). .sup.1H NMR (400 MHz, CDCl.sub.3):
delta 4.73 (d, 2H; CH.sub.2OCOO), 4.45 (t, 2H; OCH.sub.2), 4.22 (d,
2H; CH.sub.2OCOO), 3.34 (t, 2H; CH.sub.2I), 1.38 (s, 3H; CH.sub.3).
.sup.13C NMR (100 MHz, CDCl.sub.3): delta 170.5, 147.3, 72.8, 65.6,
40.3, 17.5, -0.3.
Organocatalytic Ring-Opening Polymerizations.
[0111] Ring-opening polymerizations were conducted using benzyl
2,2-bis(methylol)propionate (BnMPA) as an initiator in the presence
of organocatalysts,
N-(3,5-trifluoromethyl)phenyl-N'-cyclohexyl-thiourea (TU) and
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), in methylene chloride at
room temperature (1-2 hours) to yield pre-cationic polymers
comprising pendant 3-halopropyl esters with molecular weight
consistent with the feed ratio ([M].sub.0/[I].sub.0), narrow
polydispersities (1.1-1.2), and end group fidelity. To avoid
scission of polymer chain by the back-biting stemming from the
terminal hydroxyl group in the presence of amine during the
reaction, the precursors were acetylated with acetic anhydride for
24 hours to 48 hours.
[0112] The ROP polymers prepared below have the general formula
(9):
##STR00084##
wherein L' is the subunit derived from the initiator, w is the
number of initiating groups on L', M.sup.1 is a cyclic carbonyl
monomer, M.sup.2 is a another cyclic monomer, E' is an endcap
group, and a:b is the M.sup.1:M.sup.2 mole ratio. The initial ROP
polymer is also referred to as a precursor polymer. In the
preparation of block copolymers, is M.sup.1 added first, followed
by M.sup.2. For random copolymers, it is understood that either
monomer M.sup.1 or M.sup.2 can be attached to the initiator L'.
Each polymerization was initiated with BnMPA, a diol; therefore w=2
and two polymer chains are formed that are linked by the subunit
derived from the initiator. Each polymer chain was endcapped with
acetyl groups using acetic anhydride.
Polycarbonates
Example 5
Polymerization of MTCOPrCl
##STR00085##
[0114] MTCOPrCl (501 mg, 2.1 mmol), BnMPA (4.7 mg, 0.02 mmol,
initiator), and TU (37.2 mg, 0.1 mmol) were dissolved in methylene
chloride (1 mL), and this solution was transferred to a vial
containing DBU (15.2 mg, 0.1 mmol) to start polymerization at room,
temperature ([M].sub.0/[I].sub.0=100). After 2 hours, acetic
anhydride (72.4 mg, 0.71 mmol) was added into the mixture and the
mixture was stirred for 48 hours (conversion .about.95%). The
solution was then precipitated into cold methanol twice and the
precipitate was centrifuged and dried in vacuum. Yield: 466 mg
(93%), GPC (THF): M.sub.n 12200 g/mol, PDI 1.17, .sup.1H NMR (400
MHz, CDCl.sub.3): delta 7.39-7.29 (m, 5H; Ph), 5.16 (s, 2H;
PhCH.sub.2), 4.38-4.19 (br, .about.350H; CH.sub.2OCOO, OCH.sub.2
polymer), 3.64-3.55 (m, .about.117H; CH.sub.2Cl polymer), 2.15-2.07
(m, .about.114H; CH.sub.2 polymer), 2.06 (s, 6H; OCH.sub.3 acetyl
end), 1.27 (br, .about.169H; CH.sub.3 polymer).
Example 6
Polymerization of MTCOPrBr
##STR00086##
[0116] MTCOPrBr (288 mg, 1.0 mmol), BnMPA (4.4 mg, 0.01 mmol,
initiator), and TU (9.8 mg, 0.03 mmol) were dissolved in methylene
chloride (1 mL), and this solution was transferred to a vial
containing DBU (3.3 mg, 0.02 mmol) to start polymerization at room,
temperature ([M].sub.0/[I].sub.0=52). After 2 hours, acetic
anhydride (96.9 mg, 0.95 mmol) was added into the mixture and
stirred for 2 nights (conversion 94%). The solution was then
precipitated into cold methanol twice and the precipitate was
centrifuged and dried in vacuum. Yield: 265 mg (92%), GPC (THF):
M.sub.n 11700 g/mol, PDI 1.11, .sup.1H NMR (400 MHz, CDCl.sub.3):
delta 7.38-7.28 (m, 5H; Ph), 5.17 (s, 2H; PhCH.sub.2), 4.40-4.17
(m, .about.348H; CH.sub.2OCOO, OCH.sub.2 polymer), 3.53-3.36 (m,
.about.111H; CH.sub.2Br polymer), 2.23-2.15 (m, .about.111H;
CH.sub.2 polymer), 2.06 (s, 6H; OCH.sub.3 acetyl end), 1.30-1.24
(br, .about.169H; CH.sub.3 polymer).
Example 7
Polymerization of MTCOEtI
##STR00087##
[0118] MTCOEtI (312 mg, 1.0 mmol), BnMPA (4.4 mg, 0.02 mmol,
initiator), and TU (9.4 mg, 0.03 mmol) were dissolved in methylene
chloride (1 mL), and this solution was transferred to a vial
containing DBU (3.3 mg, 0.02 mmol) to start polymerization at room
temperature ([M].sub.0/[I].sub.0=51). After 2 hours, acetic
anhydride (107.2 mg, 1.05 mmol) was added into the mixture and
stirred for 2 nights (conversion 94%). The solution was then
precipitated into cold methanol twice and the precipitate was
centrifuged and dried in vacuum. Yield: 268 mg (86%), GPC (THF):
M.sub.n 10500 g/mol, PDI 1.22, .sup.1H NMR (400 MHz, CDCl.sub.3):
delta 7.37-7.31 (m, 5H; Ph), 5.17 (s, 2H; PhCH.sub.2), 4.44-4.36
(m, .about.92H; OCH.sub.2 polymer), 4.36-4.24 (m, .about.178H;
CH.sub.2OCOO polymer), 3.35-3.27 (m, .about.89H; CH.sub.2I
polymer), 2.07 (s, 6H; OCH.sub.3 acetyl end), 1.34-1.24 (br,
.about.144H; CH.sub.3 polymer).
Example 8
Block Polymerization of TMC and MTCOPrCl
##STR00088##
[0120] TMC (108 mg, 1.0 mmol, designated MD, BnMPA (11 mg, 0.05
mmol), and TU (17.5 mg, 0.05 mmol) were dissolved in methylene
chloride (1 mL), and this solution was transferred to a vial
containing DBU (7.3 mg, 0.05 mmol) to start polymerization at room
temperature ([M.sub.1].sub.0/[I].sub.0=20). After complete
consumption of the first monomer (M.sub.1) was confirmed by NMR (3
hours, conversion 97%), the reaction mixture was transferred to a
vial containing MTCOPrCl (603 mg, 2.55 mmol), the second monomer
M.sub.2, for the second polymerization
([M.sub.2].sub.0/[I].sub.0=50) and stirred for another 18 hours
(conversion 96%). Acetic anhydride (117 mg, 1.15 mmol) was then
added into the mixture and stirred for 2 nights. The solution was
then precipitated into cold methanol twice and the precipitate was
centrifuged and dried in vacuum. Yield: 640 mg (90%), GPC (THF):
M.sub.n 12000 g/mol, PDI 1.19, .sup.1H NMR (400 MHz, CDCl.sub.3):
delta 7.38-7.30 (m, 5H; Ph), 5.17 (s, 2H; PhCH.sub.2), 4.33-4.26
(m, .about.208H; CH.sub.2OCOO, OCH.sub.2P(MTCprCl)), 4.26-4.20 (m,
.about.70H, CH.sub.2OCOO.sub.PTMC), 3.63-3.56 (m, .about.73H;
CH.sub.2Cl.sub.P(MTCprCl)), 2.15-2.00 (m, .about.111H;
CH.sub.2P(MTCprCl), CH.sub.2PTMC, OCH.sub.3 acetyl end), 1.27 (br,
.about.107H, CH.sub.3P(MTCprCl)).
Polyester-Polycarbonate Block Copolymers
Example 9
Block Polymerization of LLA and MTCOPrBr
[0121] In the following preparation, the stereochemistry of
L-lactide (LLA) is not shown.
##STR00089##
[0122] L-lactide (146 mg, 1.0 mmol) (LLA), BnMPA (12 mg, 0.05
mmol), and TU (9.0 mg, 0.024 mmol) were dissolved in methylene
chloride (1 mL), and this solution was transferred to a vial
containing (-)-sparteine (3.0 mg, 0.013 mmol) to start
polymerization at room, temperature ([M.sub.1].sub.0/[I].sub.0=20).
After complete consumption of the first monomer was confirmed on
NMR (1.5 h, conversion 96%), the reaction mixture containing the
polyester was transferred to a vial containing MTCOPrBr (427 mg,
1.52 mmol), which was further transferred to a vial containing TU
(9.7 mg, 0.026 mmol) and DBU (4.1 mg, 0.027 mmol) for the second
polymerization ([M.sub.2].sub.0/[I].sub.0=29). The second reaction
mixture was stirred for another 1 hour (conversion 97%). Acetic
anhydride (205 mg, 2.01 mmol) was then added into the mixture and
stirred for 2 nights. The solution was then precipitated into cold
methanol twice and the precipitate was centrifuged and dried in
vacuum to provide the polyester-polycarbonate block copolymer.
Yield: 524 mg (90%), GPC (THF): M.sub.n 12200 g/mol, PDI 1.14,
.sup.1H NMR (400 MHz, CDCl.sub.3): delta 7.38-7.28 (m, 5H; Ph),
5.22-5.09 (m, .about.35H; PhCH.sub.2, CH.sub.PLA), 4.38-4.19 (m,
.about.158H; CH.sub.2OCOO, OCH.sub.2P(MTCprBr)), 3.48-3.41 (m,
.about.56H, CH.sub.2Br), 2.23-2.14 (m, .about.55H; CH.sub.2), 2.06
(s, 6H; OCH.sub.3 acetyl end), 1.61-1.52 (m, .about.106H;
CH.sub.3PLA), 1.32-1.27 (br, .about.86H, CH.sub.3P(MTCprBr)).
Example 10
Block Polymerization of DLA and MTCOPrBr
[0123] This polymer was prepared by the same procedure as Example
9, adding D-lactide (DLA) as the first monomer instead of L-lactide
(LLA). Yield: 503 mg (87%), GPC (THF): M.sub.n 12400 g/mol, PDI
1.13. .sup.1H NMR (400 MHz, CDCl.sub.3): delta 7.38-7.28 (m, 5H;
Ph), 5.22-5.09 (m, .about.39H; PhCH.sub.2, CH.sub.PLA), 4.38-4.19
(m, .about.195H; CH.sub.2OCOO, OCH.sub.2P(MTCprBr)), 3.48-3.41 (m,
.about.63H, CH.sub.2Br), 2.23-2.14 (m, .about.62H; CH.sub.2), 2.06
(s, 6H; OCH.sub.3 acetyl end), 1.61-1.52 (m, .about.119H;
CH.sub.3PLA), 1.32-1.27 (br, .about.97H, CH.sub.3P(MTCprBr))
Random Polycarbonate Copolymer
Example 11
Random Polymerization of MTCOEt and MTCOPrBr
##STR00090##
[0125] The vertical brackets in the above structure indicate that
either of the repeat units derived from MTCOPrBr or MTCOEt can be
bonded to the subunit derived from the initiator, as well as the
acetyl group.
[0126] MTCOPrBr (282 mg, 1.0 mmol), MTCOEt (188 mg, 1.0 mmol),
BnMPA (9.0 mg, 0.04 mmol), and TU (18.7 mg, 0.05 mmol) were
dissolved in methylene chloride (1 mL), and this solution was
transferred to a vial containing DBU (7.8 mg, 0.05 mmol) to start
polymerization at room, temperature ([M].sub.0/[I].sub.0=50). After
2 hours, acetic anhydride (194 mg, 1.90 mmol) was added into the
mixture and stirred for 2 nights (conversion 93%). The solution was
then precipitated into cold methanol twice and the precipitate was
centrifuged and dried in vacuum. Yield: 370 mg (77%), GPC (THF):
M.sub.n 11400 g/mol, PDI 1.20, .sup.1H NMR (400 MHz, CDCl.sub.3):
delta 7.37-7.31 (m, 5H; Ph), 5.16 (s, 2H; PhCH.sub.2), 4.35-4.24
(m, .about.247H; CH.sub.2OCOO, OCH.sub.2PMTC(prBr)), 4.23-4.14 (m,
.about.56H; OCH.sub.2PMTC(Et)), 3.48-3.41 (m, .about.47H;
CH.sub.2Br), 2.23-2.14 (m, .about.47H; CH.sub.2PMTC(prBr)), 2.06
(s, 6H; OCH.sub.3 acetyl end), 1.30-1.20 (m, .about.227H; CH.sub.3,
CH.sub.2CH.sub.3PMTC(Et)).
Preparation of Cationic Polymers
[0127] The pre-cationic halo-functional polymers (i.e., initial ROP
polymers) were reacted with N,N,N',N'-tetramethylethylenediamine
(TMEDA) in DMSO to provide the corresponding cationic polymers.
Several bis-amines were surveyed, but only tertiary amines were
chosen as feasible reagents because the primary and secondary
amines led to a significant reduction in the polycarbonate
backbone.
Example 12
##STR00091##
[0129] The homopolymer of Example 5 (427 mg, [Cl]=1.77 mmol) was
dissolved in DMSO (8 mL) and mixed with TMEDA (1.1 mL, 7.22 mmol),
and stirred for 6 h at 90.degree. C. The mixture was then
precipitated into THF twice and the precipitate was collected by
centrifugation and dried in vacuum. Yield: 546 mg (86%), GPC (DMF):
M.sub.n 11300 g/mol, PDI 1.27, .sup.1H NMR (400 MHz, MeOH-d.sub.4):
delta 7.42-7.32 (br, 5H; Ph), 5.19 (s, 2H; PhCH.sub.2), 4.45-4.17
(m, .about.252H; CH.sub.2OCOO, OCH.sub.2 polymer), 3.63-3.44 (br,
.about.149H; CH.sub.2N.sup.+ polymer), 3.27-3.18 (br, .about.210H;
N.sup.+CH.sub.3 polymer), 2.85-2.76 (br, .about.73H; CH.sub.2N
polymer), 2.36-2.30 (br, .about.213H; NCH.sub.3 polymer), 2.28-2.17
(br, .about.70H; CH.sub.2 polymer), 2.06 (s, 3H; OCH.sub.3 acetyl
end), 1.34-1.25 (br, .about.119H; CH.sub.3 polymer), 1.22 (s, 3H;
CH.sub.3 end group).
Example 13
##STR00092##
[0131] TMEDA (0.38 mL, 2.5 mmol) was added to a DMSO solution (3
mL) of the polymer formed in Example 6 (177 mg, [Br]=0.62 mmol).
The solution was stirred overnight at room temperature and
precipitated into THF twice, and the precipitate was centrifuged
and dried in vacuum. Yield: 220 mg (88%), .sup.1H NMR (400 MHz,
MeOH-d.sub.4): delta 7.42-7.30 (br, 5H; Ph), 5.20 (s, 2H;
PhCH.sub.2), 4.46-4.13 (m, .about.266H, CH.sub.2OCOO, OCH.sub.2
polymer), 3.66-3.42 (br, .about.168H; CH.sub.2N.sup.+ polymer),
3.28-3.17 (br, .about.243H; N.sup.+CH.sub.3 polymer), 2.87-2.75
(br, .about.84H; NCH.sub.2 polymer), 2.37-2.29 (br, .about.251H;
NCH.sub.3 polymer), 2.30-2.16 (br, .about.85H; CH.sub.2 polymer),
2.07 (s, 6H; OCH.sub.3 acetyl end), 1.37-1.23 (br, .about.133H;
CH.sub.3 polymer).
Example 14
##STR00093##
[0133] This cationic polymer was prepared using the same procedure
described in Example 13 except with the polymer prepared in Example
7, on a 201 mg scale. Yield: 211 mg (77%), .sup.1H NMR (400 MHz,
D.sub.2O): delta 7.49-7.31 (m, 5H; Ph), 5.22 (s, 2H; PhCH.sub.2),
4.69-4.56 (br, .about.68H; OCH.sub.2), 4.47-4.23 (m, .about.176H;
OCOCH.sub.2), 3.90-3.76 (br, .about.74H; N.sup.+CH.sub.2),
3.66-3.51, (br, .about.78H; OCH.sub.2CH.sub.2N.sup.+), 3.29-3.15
(br, .about.220H; N.sup.+CH.sub.3), 2.93-2.82 (br, .about.76H;
NCH.sub.2), 2.33-2.23 (br, .about.222H; NCH.sub.3), 2.07 (s, 6H;
CH.sub.3 acetyl), 1.38-1.20 (br, .about.124H; CH.sub.3).
Example 15
##STR00094##
[0135] To a DMSO solution (10 mL) of the polymer formed in Example
8 (578 mg, [Cl]=1.93 mmol), TMEDA (1.27 mL, 8.5 mmol) was added.
The reaction mixture was stirred for 6 h at 90.degree. C. and
precipitated into THF twice. The precipitate was centrifuged and
dried into vacuum. Yield: 735 mg (92%), GPC (DMF): M.sub.n 15700
g/mol, PDI 1.27, .sup.1H NMR (400 MHz, MeOH-d.sub.4): delta
7.41-7.32 (br, 5H; Ph), 5.19 (br, 2H; PhCH.sub.2), 4.48-4.13 (br,
.about.388H; CH.sub.2OCOO, OCH.sub.2 polymer), 3.65-3.45 (br,
.about.179H; CH.sub.2N.sup.+ polymer), 3.28-3.18 (br, .about.270H;
N.sup.+CH.sub.3 polymer), 2.87-2.77 (br, .about.88H; NCH.sub.2
polymer), 2.38-2.30 (br, .about.272H; NCH.sub.3 polymer), 2.28-2.16
(br, .about.88H; CH.sub.2 polymer), 2.08-1.98 (m, .about.44H;
CH.sub.2 polymer, OCH.sub.3 acetyl end), 1.35-1.25 (br,
.about.149H, CH.sub.3 polymer).
Example 16
##STR00095##
[0137] The polymer formed in Example 9 (406 mg, [Br]=1.07 mmol) and
TMEDA (0.65 mL, 4.3 mmol) were mixed in DMSO (4.0 mL), stirred
overnight at room temperature and precipitated into THF twice. The
precipitate was centrifuged and dried into vacuum. Yield: 515 mg
(97%), .sup.1H NMR (400 MHz, MeOH-d.sub.4): delta 7.42-7.30 (br,
5H; Ph initiator), 5.29-5.11 (m, .about.42H; PhCH.sub.2 initiator,
CH.sub.PLA), 4.49-4.15 (br, .about.204H, CH.sub.2OCOO, OCH.sub.2
polymer), 3.67-3.43 (br, .about.123H, CH.sub.2N.sup.+ polymer),
3.29-3.15 (br, .about.177H, N.sup.+CH.sub.3 polymer), 2.85-2.74
(br, .about.61H, NCH.sub.2 polymer), 2.37-2.28 (br, .about.189H,
NCH.sub.3 polymer), 2.29-2.15 (br, .about.62H, CH.sub.2 polymer),
2.06 (s, 6H, OCH.sub.3 acetyl end), 1.60-1.50 (m, .about.128H;
CH.sub.3PLA), 1.35-1.24 (br, .about.103H, CH.sub.3).
Example 17
[0138] This polymer from Example 10 was treated with TMEDA
according to the procedure used in Example 16 to obtain a cationic
polymer, the difference being the subunit derived from DLA rather
than LLA. Yield: 497 mg (96%), .sup.1H NMR (400 MHz, MeOH-d.sub.4):
delta 7.42-7.31 (br, 5H; Ph initiator), 5.24-5.13 (m, .about.41H;
PhCH.sub.2 initiator, CH.sub.PLA), 4.46-4.18 (m, .about.206H,
CH.sub.2OCOO, OCH.sub.2 polymer), 3.66-3.45 (br, .about.124H,
CH.sub.2N.sup.+ polymer), 3.28-3.18 (br, .about.173H,
N.sup.+CH.sub.3 polymer), 2.84-2.75 (br, .about.57H, NCH.sub.2
polymer), 2.35-2.28 (br, .about.175H, NCH.sub.3 polymer), 2.28-2.16
(br, .about.59H, CH.sub.2 polymer), 2.06 (s, 6H, OCH.sub.3 acetyl
end), 1.59-1.52 (m, .about.121H; CH.sub.3PLA), 1.35-1.25 (br,
.about.110H, CH.sub.3).
Example 18
##STR00096##
[0140] TMEDA (0.40 mL, 2.69 mmol) was added to a DMSO solution (3
mL) of the polymer from Example 11 (342 mg, [Br]=0.67 mmol). The
solution was stirred overnight at room temperature and precipitated
into the mixture of THF/hexane (3:1) twice, and the precipitate was
centrifuged and dried in vacuum. Yield: 377 mg (90%), .sup.1H NMR
(400 MHz, MeOH-d.sub.4): delta 7.41-7.35 (br, 5H; Ph), 5.19 (s, 2H;
PhCH.sub.2), 4.42-4.23 (m, .about.253H, CH.sub.2OCOO,
OCH.sub.2PMTC(prBr--N)), 4.28-4.13 (m, .about.56H;
OCH.sub.2PMTC(Et)), 3.64-3.49 (br, .about.96H; CH.sub.2N.sup.+),
3.28-3.19 (br, .about.142H; N.sup.+CH.sub.3), 2.84-2.75 (br,
.about.52H; NCH.sub.2), 2.35-2.28 (br, .about.145H; NCH), 2.29-2.17
(br, .about.49H; CH.sub.2PMTC(prBr--N)), 2.06 (s, 6H; OCH.sub.3
acetyl end), 1.35-1.19 (m, .about.234H; CH.sub.3 polymer).
Charge Shifting Polymers
Example 19
##STR00097##
[0142] 5-methyl-5-(1-ethoxyethyl)oxycarboxyl-1,3-dioxan-2-one
(MTCOEE; 62 mg, 0.27 mmol), MTCOPrBr (212 mg, 0.75 mmol), BnMPA
(4.6 mg, 0.02 mmol), and TU (19.4 mg, 0.05 mmol) were dissolved in
methylene chloride (1 mL), and this solution was transferred to a
vial containing DBU (7.4 mg, 0.05 mmol) to start polymerization at
room, temperature ([M].sub.0/[I].sub.0=50). After 2.5 h, the
solution was precipitated into cold methanol and the precipitate
was centrifuged and dried in vacuum. Yield: 241 mg (87%), GPC
(THF): M.sub.n 11800 g/mol, PDI 1.19, .sup.1H NMR (400 MHz,
acetone-d.sub.6): delta 7.45-7.32 (m, 5H; Ph), 5.96 (q, .about.12H;
CH.sub.(OEE)), 5.20 (s, 2H; PhCH.sub.2), 4.42-4.22 (m, .about.333H;
CH.sub.2OCOO, OCH.sub.2polymer), 3.75-3.48 (m, .about.128H;
OCH.sub.2(OEE), CH.sub.2Br), 2.27-2.16 (m, .about.87H;
CH.sub.2(OPrBr)), 1.35 (d, .about.44H; CHCH.sub.3(OEE)), 1.33-1.23
(m, .about.182H; CH.sub.3polymer) 1.22-1.08 (m, .about.69H;
CH.sub.3(OEE). a:b=1.0:3.1.
Example 20
Quaternization of Example 19
##STR00098##
[0144] Trimethylamine gas (394 mg, 6.7 mmol) was charged to an
acetonitrile solution (4 mL) of the polymer of Example 19 (202 mg,
[Br]=0.56 mmol) immersed in a dry-ice/acetone bath. The solution
was then allowed to warm to room temperature and kept stirring for
18 h before acetonitrile and excess gasses were removed under
vacuum. The concentrated residue was dried in vacuum (.about.90%
aminated). Yield: 200 mg (85%), .sup.1H NMR (400 MHz,
MeOH-d.sub.4): delta 7.43-7.32 (m, 5H; Ph), 6.02-5.93 (m,
.about.6H; CH.sub.(OEE)), 5.21 (s, 2H; PhCH.sub.2), 4.48-4.11 (m,
.about.267H; CH.sub.2OCOO and CH.sub.2O.sub.polymer), 3.75-3.64 (m,
.about.15H; OCH.sub.2CH.sub.3(OEE)), 3.63-3.45 (m, .about.78H;
N.sup.+CH.sub.2(PAB)), 2.29-2.15 (b, .about.298H;
N.sup.+CH.sub.3(PAB)), 2.32-2.15 (b, .about.68H; CH.sub.2(PAB)),
1.41-1.35 (d, .about.19H; CHCH.sub.3(OEE)), 1.35-1.23 (m,
.about.122H; CH.sub.3polymer), 1.24-1.10 (m, .about.46H;
CH.sub.2CH.sub.3 (OEE)). M.sub.n (NMR)=14700 g/mol.
[0145] The polymer preparations are summarized in Table 8 for
precursor polymers (Examples 5 to 11, and 19) and their
corresponding cationic polymers (Examples 12 to 18, and 20)
TABLE-US-00008 TABLE 8 Precursor Polymer.sup.a,b Random/ Cationic
Polymer.sup.c Example Block M.sup.1 M.sup.2 Example 5 MTCOPrCl 12 6
MTCOPrBr 13 7 MTCOEtI 14 8 Block TMC MTCOPrCl 15 9 Block LLA
MTCOPrBr 16 10 Block DLA MTCOPrBr 17 11 Random MTCOEt MTCOPrBr 18
19 Random MTCOEE MTCOPrBr 20 .sup.aEach polymerization was
initiated with BnMPA. .sup.bM.sup.1 was added first for block
copolymerizations. .sup.cQuaternizations were performed with
TMEDA.
[0146] Table 9 summarizes the analytical data (number average
molecular weight M.sub.n, polydispersity index (PDI), % yield, %
conversion of the halide X to quaternary amine) obtained on the
precursor polymers (Examples 5 to 11, and 19) and their
corresponding cationic polymers (Examples 12 to 18, and 20).
TABLE-US-00009 TABLE 9 Initial ROP Polymer Cationic Polymer Example
M.sub.n.sup.a PDI.sup.a Yield (%) Example M.sub.n.sup.b Yield (%)
N.sup.+b,c (%) X 5 12200 1.17 93 12 13900 86 85 Cl 6 11700 1.11 92
13 17500 88 93 Br 7 10500 1.22 86 14 17400 77 90 I 8 12000 1.19 90
15 18100 92 91 Cl 9 12200 1.14 90 16 16500 97 90 Br 10 12400 1.13
87 17 16100 96 85 Br 11 11400 1.20 77 18 15300 90 ~100 Br 19 11800
1.19 87 20 14700 85 90 Br .sup.aDetermined by GPC (THF) using
polystyrene standards. .sup.bCalculated from integral ratios on NMR
spectra. .sup.cConversion of halogenated residues into quaternary
amines.
[0147] The utility of the organocatalytic system (TU/DBU) was
demonstrated through the synthesis of narrowly dispersed
homopolymers, random polymers, and block copolymers having
predictable molecular weights. The polydispersity ranged from 1.11
to 1.22. The precursor polymers had a number average molecular
weight M.sub.n of 10500 to 12400. The cationic polymers had a
number average molecular weight M.sub.n of 13100 to 19433. The
conversion of halide to quaternary amine was about 84% to 100%.
[0148] The reactivity of the precursor polymer with an amine
depends on the halide on the side chain. Although the polymer of
Example 5 (X.dbd.Cl) can form quaternary amine easily with
trimethylamine in acetonitrile at room temperature, it needed more
polar solvent such as DMSO and heating (90.degree. C.) to produce
the cationic polymer of Example 12 with TMEDA (4 equivalents TMEDA
per equivalent of [Cl]). In comparison, the precursor polymers of
Example 6 (X.dbd.Br) and Example 7 (X.dbd.I) were converted at room
temperature to the corresponding cationic polymers of Example 13
and 14 respectively, using TMEDA in DMSO or acetonitrile. Little
difference was found between the reactivity of bromide and iodide
in the reaction rate with TMEDA.
[0149] The difference in the reactivity between chlorine, bromine
and iodine can be helpful in the design of block copolymers,
especially amphiphilic block copolymers to form micelles containing
the cationic polycarbonate segments. As shown above, a cationic
block copolymer can be formed comprising a cationic hydrophilic
segment at both ends (Examples 15 to 17) and a hydrophobic core.
The hydrophobic core comprises repeat units derived from
trimethylene carbonate (TMC) or a lactide (LLA or DLA). However,
the hydrophobic core derived from LLA and DLA was found to be
thermally labile during TMEDA quaternization reactions that
required heat, in particular with polycarbonate subunits bearing a
chloride leaving group. Consequently, these monomers were employed
to form precursor polymers having bromide or iodide leaving groups,
which could react with TMEDA at room temperature. For precursor
polymers comprising chloride leaving groups, a hydrophobic block
comprising poly(trimethylene carbonate) derived from TMC was
relatively stable at the elevated temperature used in the TMEDA
quaternization reaction.
[0150] The reactivity of halogens may also affect the stability of
the charged polymers. Although around 90% of halogen residues are
converted, it is difficult to convert all side chain halide groups
to quaternary amine, owing to the reaction equilibrium, steric
hindrance, and charge repulsion, even when excess TMEDA is used.
The unreacted alkyl halide groups are potential crosslinking sites
for reaction with the tertiary amine at the very end of the side
chain. The cationic polymers derived from chloride-containing
precursor polymers are quite stable because of their low
reactivity, whereas the cationic polymers derived from bromide or
iodide containing precursor polymers included a small amount of
insoluble material. However, no crosslinking was observed in the
reaction to produce the cationic polymer of Example 18 derived from
the random precursor polymer of Example 11. The cationic polymer of
Example 18 showed good solubility in water when the comonomer molar
ratio MTCOEt:MTCOPrBr was 1:1.
Physicochemical and Biological Tests.
General Procedure for Preparation of Polymer/DNA and PEI/DNA
Complexes.
[0151] The polymer was dissolved in ultra pure water (HPLC grade,
pH 7.0) or 20 mM sodium acetate buffer (pH 5.0 or 6.0) and PEI was
dissolved in ultra pure water. The complexes were formed directly
by mixing equal volume of polymer or PEI and DNA solutions to
achieve the intended N/P ratios (the ratio of moles of the amine
groups of the cationic polymer to those of the phosphate groups of
DNA). To allow for complete electrostatic interaction between the
polymer or PEI and DNA molecules, the solution was equilibrated at
room temperature for 30 minutes upon mixing before being used for
further studies.
Gel Retardation Assay.
[0152] Various formulations of polymer/DNA complexes were prepared
with different N/P ratios. Post-equilibration, the complexes were
electrophoresed on 1% agarose gel (stained with 4 microliters of
0.5 microgram/mL ethidium bromide per 50 mL of agarose solution) in
0.5.times.TBE buffer at 80 mV for 60 minutes. A stock solution of
TBE buffer contains 53 g of TRIS base, (HOCH.sub.2).sub.3CNH.sub.2;
27.5 g of boric acid, and 20 ml of 0.5 M ethylenediamine
tetraacetic acid (EDTA) (pH 8.0). The TBE stock solution was
diluted 0.5.times. before use. The gel was then analyzed on a UV
illuminator (Chemi Genius, Evolve, Singapore) to show the position
of the complexed DNA relative to that of the naked DNA.
Particle Size and Zeta Potential Analyses.
[0153] The polymer/DNA complexes were prepared according to N/P 1,
5, 10, 20, 30, 40 and 50. After 30 minutes of incubation, 100
microliter of the complex solutions were diluted 11 times with 1 mL
of ultra pure water or 20 mM sodium acetate buffer (pH 5.0 or pH
6.0). Naked DNA, diluted to the same concentration with ultra pure
water or the sodium acetate buffer, was employed as control. Prior
to analysis, the diluted complex solutions were allowed to
stabilize for 30 minutes. The particle size of the polymer/DNA
complexes were measured using dynamic light scattering (Brookhaven
Instrument Corp., Holtsville, N.Y., U.S.A.) equipped with a He--Ne
laser beam at 658 nm (scattering angle: 90.degree.) and Zetasizer
(Malvern Instrument Ltd., Worchestershire, UK), respectively. The
particle size and zeta potential measurements were repeated for 5
runs for each sample, and the data were reported as the average of
5 readings.
Cytotoxicity Test.
[0154] HepG2 cells were maintained in DMEM growth medium
supplemented with 10% FBS (fetal bovine serum), 100 microgram/mL
penicillin and 100 units/mL streptomycin at 37.degree. C., under
the atmosphere of 5% CO.sub.2. To assess the cytotoxicity of
polymer/DNA complexes in HepG2 cells, a standard MTT (Dimethyl
thiazolyl diphenyl tetrazolium salt) assay protocol was employed.
On a 96-well plate, cells were seeded at a density of
1.times.10.sup.4 cells/well and allowed to grow for 24 hours to
reach 60% to 70% confluence. The DNA complexes were prepared
according to the protocol described above. Naked DNA solutions were
also prepared to the same concentration as that of the complex
solutions. Each well was replaced with 100 microliters of fresh
growth medium and treated with 10 microliters of the complex
solution. The cytoxicity test was performed in replicates of 8
wells per N/P ratio. After 4 hours of incubation, the wells were
replaced with fresh medium and incubated further for 68 hours. Upon
replacing the wells with 100 microliters of fresh medium and 20
microliters of MTT solution (5 mg/mL in PBS buffer), the cells were
incubated for another 4 hours. Finally, the used media were removed
and the internalized purple formazan crystals in each well were
dissolved with 150 microliters of DMSO. A 100 microliter aliquot of
the formazan/DMSO solution was transferred from each well to a new
96-well plate, and the absorbance (A) was measured using a
microplate spectrophotometer (BioTek Instruments Inc, Winooski,
Vt., U.S.A.) at the wavelength of 550 nm and 690 nm. To measure the
relative cell viability in different N/P ratios, the absorbance of
formazan solution in the treated cells were compared to that of the
control cells:
Cell
viability=[(A.sub.550-A.sub.690)sample/(A.sub.550-A.sub.690)control-
].times.100%
The data were statistically analyzed for significant differences,
based on the Student's t-test at p<0.05.
In Vitro Gene Expression.
[0155] The in vitro gene transfection of the polymer/DNA complexes
was studied in HepG2 cells. Cells were seeded onto a 24-well plate
at a density of 8.times.10.sup.4 cells/well and cultivated with 0.5
mL of DMEM (Dulbecco's modified Eagle's medium) growth medium for
24 hours until 60% to 70% confluent. The cells in each well were
replaced with fresh growth medium, and subsequently transfected
with 50 microliters of the complex solution (containing 2.5
micrograms of DNA). After 4 hours of transfection, the used media
were replaced with fresh media and the cells were incubated
further. The culture media were removed after 68 hours and the
cells were washed with PBS buffer (phosphate buffered saline,
containing 137 mM NaCl, 2.7 mM KCl, 10 mM podium phosphate dibasic,
2 mM potassium phosphate monobasic, pH of 7.4) before being added
with 0.2 mL of 1.times. reporter lysis buffer. After being
subjected to two cycles of freezing (-80.degree. C. for 30 minutes)
and thawing, the cell lysates were centrifuged at 14,000 rpm and
4.degree. C. for 10 minutes to remove cell debris. The supernatant
(20 microliters) was mixed with 100 microliters of luciferin
substrate buffer, and its fluorescence intensity (in terms of
relative light units RLU) was immediately measured using a
luminometer (Lumat LB9507, Mandel Scientific Inc, Ontario, Canada).
The RLU readings were normalized against protein concentration of
the supernatant, determined by BCA protein assay, to give the
overall expression efficiency.
[0156] In all in vitro gene expression experiments, naked DNA was
used as negative control. PEI/DNA complexes were used as positive
control, and were prepared at the optimal N/P ratio (i.e., 10), at
which PEI induced high gene expression yet provided more than 50%
cell viability. The luciferase expression efficiency at each N/P
ratio was expressed as an average of 6 replicate wells. Statistical
analysis was performed using the Student's t-test. Differences were
considered statistically significant at p<0.05.
[0157] The above prepared amphiphilic polymers (Examples 12 to 18)
can form micellar nanoparticles in aqueous solutions. As a typical
example, cationic polymer of Example 15 formed nanoparticles with
size of 370 nm and zeta potential of 34 mV by direct dissolution of
the polymer in 20 mM sodium acetate buffer (pH 6.0). Polyplexes
based on the cationic polymer of Example 15 exhibited strong
binding ability to DNA. FIG. 1 is a photograph showing the results
of agarose gel electrophoresis of polyplexes prepared at different
pH and at various N/P ratios using the cationic polymer of Example
15. For polyplexes prepared at pH 7 complete retardation of DNA
mobility was observed at N/P=4. For polyplexes prepared at pH 6 or
pH 5, complete retardation of DNA mobility was observed at N/P=3.
At lower pH more tertiary amines are protonated, enhancing DNA
binding ability of the cationic polymer. Moreover, the polyplexes
had a particle size less than 150 nm, and a positive zeta potential
at high N/P ratios, which are advantageous for cellular uptake.
[0158] FIG. 2 is a bar chart showing the relationship between N/P
ratio and particle size of the polyplex prepared at pH 7.0, 6.0 and
5.0. Particle size decreased with increasing N/P ratios (5 to 50)
for pH 5.0 (from 110 nm to about 80 nm) and for pH 6.0 (about 250
nm to about 100 nm). For pH 7.0 the particle size remained
relatively constant or slightly increased (about 120 nm to 150 nm)
for the N/P ratios 1 to 50.
[0159] FIG. 3 is a bar chart showing the relationship between N/P
ratio and zeta potential of the polyplex prepared at pH 7.0, 6.0
and 5.0. The zeta potential was highest for high N/P ratios and
lower pH values, indicating that the polyplex has higher positive
charge density at lower pH, and therefore greater DNA binding
capacity.
[0160] FIG. 4 is a bar chart comparing luciferase expression levels
in HepG2 human liver carcinoma cell line for polyplexes fabricated
at different pH and N/P ratios. DNA and PEI/DNA controls are also
shown. Among three pH conditions, pH 6.0 yielded the highest gene
expression level, which was 1.1.times.10.sup.7 RLU/mg protein at
N/P 30. As shown in FIGS. 2 and 3, although the polyplexes had
similar particle size at high N/P ratios (e.g., 30 to 50) when
prepared at pH 7.0 and pH 6.0, the polyplexes fabricated at pH 6.0
had higher zeta potential. Without being bound by theory, the
higher zeta potential might enhance the interaction of the
polyplexes with negatively charged cell membrane, promoting
cellular uptake of the polyplexes and thus increasing gene
expression efficiency. The tertiary amines were designed to provide
proton sponge effect for endosomal escape of the polyplexes.
Compared to pH 6.0, more tertiary amines are protonated at pH 5.0,
which might weaken the proton sponge effect and caused the observed
lower gene expression levels.
[0161] Cell viability data is shown in the bar chart of FIG. 5.
Although the highest gene expression level induced by the
polyplexes (at pH 6.0 and N/P 30) was lower than that mediated by
PEI at its optimal N/P ratio (i.e., N/P 10), the polyplexes had
much lower cytotoxicity than the PEI/DNA complexes. For example,
the cell viability was 96% for the disclosed polyplexes, but only
64% for PEI/DNA complexes. The improved viability is attributed to
the greater biodegradability of the disclosed polymers.
[0162] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms "a", "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise. It will be further understood
that the terms "comprises" and/or "comprising," when used in this
specification, specify the presence of stated features, integers,
steps, operations, elements, and/or components, but do not preclude
the presence or addition of one or more other features, integers,
steps, operations, elements, components, and/or groups thereof.
[0163] The corresponding structures, materials, acts, and
equivalents of all means or step plus function elements in the
claims below are intended to include any structure, material, or
act for performing the function in combination with other claimed
elements as specifically claimed. The description of the present
invention has been presented for purposes of illustration and
description, but is not intended to be exhaustive or limited to the
invention in the form disclosed. Many modifications and variations
will be apparent to those of ordinary skill in the art without
departing from the scope and spirit of the invention. The
embodiments were chosen and described in order to best explain the
principles of the invention and their practical application, and to
enable others of ordinary skill in the art to understand the
invention.
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