A biocompatible arginine-based polycation

Article abstract of DOI:10.1002/adfm.201000969

Self assembly between cations and anions is ubiquitous throughout nature. Important biological structures such as chromatin often use polyvalent assembly between a polycation and a polyanion. The biomedical importance of synthetic polycations arises from their affinity to polyanions such as nucleic acid and heparan sulfate. However, the limited biocompatibility of synthetic polycations hampers the realization of their immense potential. By examining biocompatible cationic peptides, we hypothesize that a biocompatible polycation should be biodegradable and made from endogenous cations. We design an arginine-based biodegradable polycation and demonstrate that it is more compatible by several orders of magnitude than conventional polycations in vitro and in vivo. This biocompatibility diminishes when L-arginine is substituted with D-arginine or when the biodegradable ester linker is changed to a biostable ether linker. We believe that this design can lead to many biocompatible polycations that can significantly advance a wide range of applications including controlled release, tissue engineering, biosensing, and medical devices. The design of PAGS and the control polymers that probe the importance of endogenous cations and their degradability in terms of biocompatibility is studied. The biocompatibility is shown to diminish when L-arginine is substituted with D-arginine or when the biodegradable ester linker is changed to a biostable ether linker.

Full text of DOI:10.1002/adfm.201000969

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A Biocompatible Arginine-Based Polycation
Blaine J. Zern, Hunghao Chu, Adeboye O. Osunkoya, Jin Gao, and Yadong Wang*
peptides are a series of amino acids linked
through peptide bonds yielding a polymer
that is hydrolyzable in the presence of
appropriate enzymes. We hypothesized
that mimicry of the essential structure of
cationic peptides would result in a biocom-
patible polycation. Firstly, natural cationic
peptides undergo hydrolysis catalyzed by
their respective proteases. Secondly, the
positive charges of cationic peptides arise
from endogenous amino acids such as
L-arginine and L-lysine. A minimalistic
chemical mimicry of these cationic pep-
tides is a synthetic hydrolyzable polycation
based on cationic amino acids.
Specifically, we chose to use ester func-
tional groups in the polymer to introduce
hydrolyzability because of the well-defined
synthetic routes and similarity with bio-
materials.[7] We chose to use L-arginine
because it is the most basic natural amino
acid and it is prevalent in endogenous
polyanion-binding domains.[4,8] The incor-
Self assembly between cations and anions is ubiquitous throughout
nature. Important biological structures such as chromatin often use poly-
valent assembly between a polycation and a polyanion. The biomedical
importance of synthetic polycations arises from their affinity to polyanions
such as nucleic acid and heparan sulfate. However, the limited biocompat-
ibility of synthetic polycations hampers the realization of their immense
potential. By examining biocompatible cationic peptides, we hypothesize that
a biocompatible polycation should be biodegradable and made from endog-
enous cations. We design an arginine-based biodegradable polycation and
demonstrate that it is more compatible by several orders of magnitude than
conventional polycations in vitro and in vivo. This biocompatibility diminishes
when L-arginine is substituted with D-arginine or when the biodegradable
ester linker is changed to a biostable ether linker. We believe that this design
can lead to many biocompatible polycations that can significantly advance a
wide range of applications including controlled release, tissue engineering,
biosensing, and medical devices.
1. Introduction
poration of L-arginine facilitates ionization of the resultant poly-
cations at a neutral pH. We chose to integrate L-arginine into a
polyester using a ring-opening reaction between the α-amino
group of arginine and the epoxy ring of diglycidyl succinate
(Figure 1). The resultant polycation is referred to as poly(L-
argininate glyceryl succinate), or PAGS. Besides arginine, the
other building blocks of PAGS are derived from succinic acid
and glycerol. Succinic acid is readily found in the body, and
plays a key role in the citric acid cycle in cellular respiration.[9]
Glycerol derivatives are prevalent throughout the body in lipids
and many signaling molecules. We anticipate that using deriva-
tives of endogenous building blocks is necessary to design a
polycation with good biocompatibility. The following data sug-
gests that the biocompatibility of PAGS is orders of magnitude
higher than polyethyleneimine (PEI) both in vitro and in vivo.
We chose PEI as a control because it is a polycation most scien-
tists are familiar with. To probe the necessity of the key design
features, we synthesized control polymers with D-arginine and a
biostable backbone.[10] These perturbations on the endogenous
building blocks and degradability significantly reduced the bio-
compatibility of PAGS.
The application of synthetic polycations in biomedicine and bio-
technology is limited by their unsatisfactory biocompatibility.[1]
To design a biocompatible polycation, we seek inspiration from
nature. Cationic peptides are important mediators in a variety of
homeostatic processes including histone, which regulates DNA
replication and gene expression,[2] hepcidin, which balances
iron concentrations,[3] heparin-binding domains, which control
growth-factor stability and activity,[4] and defensins, which act
as anti-microbial agents in host defense.[5] These polypeptides
are composed of hydrophobic and positively charged domains
that control their physiological functions.[6] Structurally, cationic
Dr. B. J. Zern
Institutes for Translational Medicine and Therapeutics
and Environmental Medicine
University of Pennsylvania School of Medicine
3620 Hamilton Walk, Philadelphia, PA 19104, USA
H. Chu, Dr. J. Gao, Prof. Y. Wang
Department of Bioengineering and the McGowan Institute
of Regenerative Medicine
University of Pittsburgh
300 Technology Drive, Pittsburgh, PA 15219, USA
E-mail: yaw20@pitt.edu
2. Results and Discussion
Prof. A. O. Osunkoya
Department of Pathology and Laboratory Medicine
Emory University School of Medicine
Atlanta, GA 30322, USA
Polycondensation between L-arginine ethyl ester and digly-
cidyl succinate in N,N-dimethylforamide yielded PAGS as a
pale yellow powder, which was soluble in water. The polymer
was purified by repetitive cycles of evaporation under vacuum
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the toxicity from different perspectives and at progressively
more severe levels. We examined the effects of PAGS on 1) the
cell-membrane integrity using a lactate dehydrogenase (LDH)
assay, 2) the metabolic activity using a 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 3) the apop-
tosis using a caspase-3 assay, and 4) the cell viability using cal-
cein AM (the acetomethoxy derivative of calcein) and ethidium
homodimer-1 staining followed by fluorescence measurements
(live/dead assay). Many existing polycations disrupt cell mem-
branes within minutes of contact, and consequently the extracel-
lular concentration of LDH, an intracellular enzyme, increases
significantly.[14] The MTT metabolic activity assay provides a
good indication to the level of stress that the cells experience.[15]
If the stress crosses a threshold, cell apoptosis will occur and
the caspase-3 levels correlate with the severity of apoptosis.[16]
Finally, the differences in cell viability resulting from apoptosis
and necrosis were evaluated by a live/dead assay.[17]
We examined the in vitro biocompatibility of PAGS by sub-
jecting the cells to media with increasing concentrations of
either PAGS or PEI. The PAGS concentrations used were 1, 2,
5, and 10 mg mL−1 , and the PEI concentrations used were 0.05,
0.5, and 5 mg mL−1 . The cell incubation time with polycation-
supplemented media was either 4 h or 24 h. The 4 h incubation
time was used to analyze earlier stresses and the 24 h incuba-
tion was used to assess cell death. A 4 h incubation time dem-
onstrated that PAGS was non-toxic up to at least 10 mg mL−1
(Figure 2A) as determined by LDH, MTT, and caspase-3 assays.
Live/dead assay indicated that even after a 24 h exposure to
a PAGS concentration of 10 mg mL−1 , cells were as viable as
control populations subjected to normal culture medium. The
majority of cells were alive and displayed a normal morphology
(Figure S4, Supporting Information). In contrast, PEI induced
significant cell toxicity as indicated by LDH and MTT assays
at concentrations as low as 0.05 mg mL−1 (Figure 2B). Further-
more, extensive cell death was observed by the expression of
ethidium homodimer-1 with 0.05 mg mL−1 PEI (Figure S5,
Supporting Information). The morphology of cells exposed to
PEI was consistent with the results of the viability assay indi-
cating massive cell death (Figure S6, Supporting Information).
These assays revealed that PAGS has excellent in vitro biocom-
patibility and represents at least a 200-fold improvement over
PEI.
When exogenous D-arginine was used to prepare the
polymer, the resultant material caused significant metabolic
reduction at all concentrations and decreased the cell viability
above the concentration of 2 mg mL−1 (Figure 2C). The sub-
stitution of the biodegradable ester linkage with a more stable
ether linkage led to a significant loss of cell membrane integrity
at a concentration of 10 mg mL−1 and decreased the cell viability
above 5 mg mL−1 (Figure 2D). It should be noted that polycations
can coat the cells and affect cell division.[18] Representative
live/dead images of cells cultured in the presence of the poly-
cations showed that PAGS is more biocompatible than D-PAGS
and Et-PAGS (Figure 2E–G). These observations are consistent
with the hypothesis on the importance of using endogenous,
degradable cations for the biocompatibility of polycations.
The difference in degradation of the 3 PAGS variations was
observed by the change in the elution time in gel permeation
chromatography (GPC) measurements after the polymers were
Figure 1. The design of PAGS and the control polymers that probe the
importance of endogenous cations and their degradability. All polymers
were synthesized by polycondensation between equimolar amounts of
arginine ethyl ester and the diglycidyl starting material. Endogenous
L-Arg and degradable diglycidyl succinate made PAGS; substituting L-Arg
with the exogenous D-Arg yielded D-PAGS, and substitution of diglycidyl
succinate with the biostable 1,4-butanediol diglycidyl ether produced
Et-PAGS.
and the solvent was washed using methanol and ethyl acetate.
Nuclear magnetic resonance spectroscopy (NMR) revealed a
change in chemical shift from approximately 3.2 ppm in the dig-
lycidyl ester to approximately 4.0 ppm in PAGS (Figure S1, Sup-
porting Information). This shift corresponds to the opening of
the epoxy ring in the diglycidyl ester. An intense C=O stretching
peak at 1735 cm−1 was found in the Fourier-transform infrared
spectroscopy data (FTIR, Figure S2, Supporting Information)
which confirms the formation of ester bonds, and an intense
band at 1673 cm−1 with a shoulder at 1635 cm−1 indicates the
presence of the guanidinium side chain of arginine.[11] PAGS
has a glass transition temperature of 42.8 °C and a melting
temperature of 88.1 °C, as was revealed by differential scanning
calorimetry. pH titration revealed that the pKa for PAGS was
approximately 10.5 (Figure S3, Supporting Information), which
is comparable to PEI with a pKa range of 9–10.[12]
We investigated the cytocompatibility of PAGS in vitro using
non-immortalized baboon smooth muscle cells (SMCs). In
vitro biocompatibility assays of polycations often use cell lines,
which tend to be more robust than primary cells. We chose
non-immortalized cells to be able to predict the in vivo biocom-
patibility of PAGS (MW = 10 500 Da) more accurately. PEI of
nearly identical molecular weight PEI (MW = 10 000 Da) served
as the control because it is a well-studied polycation that has
been used in a wide variety of biomedical applications.[12,13]
There is no standardized procedure to determine a polycation’s
biocompatibility in vitro, so we chose four assays that evaluate
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The potential effect of degradation products
on the cell metabolic activity was investigated
by conducting MTT assays using SMCs in the
presence of conditioning medium containing
polymer-degradation products. D-PAGS deg-
radation products are the only ones that
showed mild cytotoxicity (Figure 3B).
Many state-of-the-art polycations have been
tested in vitro at concentrations <0.1 mg mL−1 .
To the best of our knowledge, none have
displayed a biocompatibility close to that of
PAGS. It should be noted that cytotoxicity
is cell dependent.[20] The literature values
cited in this discussion were obtained with
a wide variety of cell lines. Polycations are
known to disrupt cell membranes, resulting
in the leakage of LDH. Polycations such as
polylysine have been shown to release sig-
nificant amounts of LDH within 30 minutes
of exposure.[21] For PAGS, no difference in
LDH level was observed between the normal
culture medium and the PAGS media after
a 4 h exposure. Several propositions sug-
gested that polycation toxicity increases with
increasing MW and charge density [expressed
as (number of charge carriers)/(molecular
weight of the polymer repeating unit)] and
decreasing order of amines respectively.[21b,22]
PAGS exhibited a biocompatibility that is
several orders of magnitude higher than
existing polycations regardless of which pro-
posed criterion was used. 1) Relative to PEI
(10 000 Da), which was cytotoxic at a con-
centration as low as 0.05 mg mL−1 , PAGS
(10 500 Da) exhibited no toxicity up to at least
10 mg mL−1 . Thus, PAGS was at least 200 times
more compatible than PEI of near identical
MW. 2) There is a charge density difference
between PEI and PAGS. Each PEI repeating
unit (43 g mol−1 ) has one positive-charge
carrier (amine). This leads to a charge den-
sity of 0.0233. Each PAGS repeating unit
(418 g mol−1 ) has two positive-charge carriers
(guanidine and amine). This leads to a charge
density of 0.00478. Thus the charge density of
PEI is approximately 5 times that of PAGS. A
synthetic polycation with a similar charge den-
sity to PAGS is poly(vinyl pyridinium bromide)
(PvPBr, charge density: 0.0054), which was
shown to be cytotoxic to L929 murine fibrob-
last cell lines at 0.1 mg mL−1 .[21] Thus PAGS
was at least 100 times more compatible than a
polycation with approximately the same charge
density. 3) Concerning the order of amines,
which was the third proposed criterion, PAGS
Figure 2. In vitro cytocompatibility of PAGS tested with non-immortalized baboon SMCs using
lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), caspase-3 assay, and live/dead assays. A) PAGS experimental groups are normalized
to the control (SMCs exposed to normal cell culture medium). The assays showed that the
cells had an intact membrane, exhibited normal metabolic activities, and were viable when
exposed to up to at least 10 mg mL−1 of PAGS. Cells were incubated for 4 h except in live/dead
assay, when they were incubated for 24 h. B) PEI exhibited toxicity at concentrations as low as
0.05 mg mL−1 under identical conditions using identical assays. C) When the endogenous L-Arg
is substituted by D-Arg, the resultant polymer displayed a significantly higher toxicity as indicated
by all 4 assays. D) When the degradable ester linkage is replaced by a much more stable ether
bond, the resultant polymer, Et-PAGS, exhibited a significantly higher toxicity as indicated by
more cell membrane leakage and increased cell death. The multicomparison ANOVA, Tukey
method, p < 0.05 was considered statistically significant. Any statistically significant difference
between the experimental and the control group is noted by an “∗”. e–g) Fluorescent (100×)
images of live (calcein AM, green) and dead (ethiduim homodimer-1, red) cells incubated
for 24 h with 10 mg mL−1 of E) PAGS, F) D-PAGS, and G) Et-PAGS indicate that endogenous
building blocks and biodegradability are necessary for cytocompatibility.
contains tertiary amines and guanidiniums. We could not find
a synthetic polycation that contained both. Thus, we compared
PAGS with polyarginine and polyamidoamine (PAMAM). These
two polycations contain guanidiniums and tertiary amines,
exposed to cholesterol esterase in phosphate-buffered saline
(PBS).[19] The ether linkage in Et-PAGS rendered the polymer
effectively non-degradable, whereas the change from L- to
D-arginine did not affect the polymer degradation (Figure 3A).
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respectively. Polyarginine was cytotoxic to
HeLa cells at 0.03 mg mL−1 .[23] Other arginine-
rich polymers, such as an arginine-modified
PAMAM, exhibited cytotoxicity to HeLa cells
at 0.04 mg mL−1 .[24] Poly(3-guanidinopropyl
methacrylate),
a
guanidinium-containing
polymer, was toxic to COS-7 cells at 0.03 mg
mL−1 .[25] Finally, PAMAM exhibited a toxicity
to L929 murine fibroblasts at 1 mg mL−1 .[21b]
Again, this demonstrates that PAGS pos-
sesses unprecedented biocompatibility relative
to existing polycations.
In vitro assays can provide valuable indica-
tions of biocompatibility and help determine
Figure 3. The effect of structural variations on the degradation of PAGS. A) Polyether Et-PAGS
did not degrade within 14 days. Polyesters with either L- or D-arginine showed significant the mechanism of toxicity. However, complex
degradation. B) The degradation products of D-PAGS exhibited mild cytotoxicity, whereas the
L-arginine-containing PAGS and the relatively biostable Et-PAGS showed no cytotoxicity. ∗
Multicomparison ANOVA, Tukey method, p < 0.05.
responses at the organ and whole body level
can only be obtained through in vivo anal-
yses. We investigated the in vivo biocompat-
ibility of PAGS by intraperitoneal injection of
150 μL of polycation solution in saline in six-week old BALB/c
mice weighing approximately 20 g. Each mouse was weighed
and injected with either a PAGS solution (8 mg per 20 g body
weight), PEI solution (0.2 mg per 20 g body weight), or saline
solution. Organs including heart, liver, lungs, spleen, kidneys,
and bladder were collected on day 1, 5, and 30 after injection.
Tissues were fixed and stained by hematoxylin and eosin (H&E)
and terminal deoxynucleotidyl transferase dUTP nick end labe-
ling (TUNEL) staining. The organs were analyzed for tissue
damage (H&E) and cell apoptosis (TUNEL).
The major organs of animals injected with PAGS showed
normal tissue architecture relative to the saline control for the
different time points (liver: Figure 4A and B; heart, lung, kidney,
and spleen: Figure S7, Supporting Information). Mice injected
with PAGS showed no observable changes in apoptosis com-
pared to control mice (Figure 4C). Mice injected with PEI were
the only animals that showed signs of toxicity. Of the organs har-
vested, only liver tissues harvested on day 1 suffered significant
damage (Figure 4E and F). On average, these liver tissues exhib-
ited 15% cell death. Similarly, significant apoptosis was observed
in livers of the day-1 PEI group only. All other PEI tissues col-
lected at other time points did not exhibit apoptosis. None of
the tissues in the PAGS group at any time point showed signs
of tissue damage (Figure 4G). The tissues from all the organs
except the livers in the day-1 PEI group were indistinguishable
histopathologically from healthy controls in terms of inflamma-
tion and necrosis. The day-1 PEI group liver exhibited 15% focal
centrilobular necrosis on average. Hepatocytes surrounding the
necrotic areas were characterized by grade 2 (out of 3 grades of
severity) inflammation. The inflammation was predominantly
Figure 4. In vivo biocompatibility of PAGS. Mice were either injected with
acute in nature with infiltrate containing mostly neutrophils
8 mg of PAGS or 0.2 mg of PEI intraperitoneally. Of all the organs isolated,
and expression of a small number of lymphocytes. Bile ducts
the liver was the only one that showed any significant histological changes.
were spared from the inflammatory responses. There was no
significant bile stasis, bridging fibrosis, or cirrhosis identified.
No micro- or macro-vesicular steatosis was noted. Also, there
was no associated atypical or malignant transformation of hepa-
tocytes. Hepatocytes in the uninvolved areas were normal.
The amount of PAGS used for the in vivo experiments was
40 times higher than that of PEI. Even so, animals exposed to
PAGS displayed no toxicity and tissues maintained their normal
A representative image of liver tissue in the PAGS group, H&E staining
(A, 100× and B, 200×) revealed a normal tissue architecture and TUNEL
(C, green fluorescence) revealed no apoptosis. D) DAPI staining revealed
nuclei in the same tissue section as C. For PEI at 0.2 mg per animal, H&E
staining (E, 100× and F, 200×) revealed that the liver tissue suffered approxi-
mately 15% necrosis. G) TUNEL staining showed extensive apoptosis in the
livers of the PEI groups. H) DAPI staining revealed nuclei in the same tissue
section as G. All images were from samples isolated on day 1 post injection.
All image acquisition parameters are identical for PAGS and PEI.
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histological architecture. The charge density
of PAGS is around 1/5 of that of PEI, thus,
the total charge in the PAGS samples was at
least 8 times that of PEI. Therefore, PAGS is
more biocompatible by several orders of mag-
nitude than PEI when looking at matching
MW and total charge. Because of arginine’s
size (MW = 174), it was impossible to create
an arginine-based polycation that equates the
charge density of PEI (monomer MW = 43),
however, the in vitro and in vivo evaluations
demonstrate clearly that PAGS possesses
excellent biocompatibility, which is not found
in existing polycations.
Figure 5. The interaction between PAGS and biological polyanions. A) PAGS conjugates heparin
to form a fibrillar matrix as revealed by scanning electron microscopy (SEM) at a magnification
of 10 000×. B) Titration of heparin with PAGS as analyzed by zeta potential measurements.
We set out to investigate the importance
of biodegradability and use of endogenous
building blocks for the biocompatibility of
polycations. Many polycations satisfy one of the two param-
eters. To the best of our knowledge, none meet both. Poly-
lysine (11.2 kDa) has been shown to be highly toxic (0.1 mg
mL−1 ) and induce apoptosis in a wide range of cells.[21a,26] Pol-
yarginine has shown toxicity to HeLa cells at a concentration of
0.03 mg mL−1 .[23] These polycations are built from endogenous
amino acids but are orders of magnitude less biocompatible
than PAGS. The ester bonds in PAGS are more susceptible to
hydrolysis than the amide bonds in polyarginine and polylysine.
This difference likely contributes to the significant improve-
ment of PAGS’s biocompatibility. To improve their biocom-
patibility many synthetic polycations have been designed to be
hydrolyzable.[1,27] These polycations have in general displayed a
higher biocompatibility than non-degradable ones. The highest
reported concentration for in vitro tests of this type of polyca-
tion was 1 mg mL−1 . To the best of our knowledge, there has
been no report on the in vivo biocompatibility of these existing
hydrolyzable polycations. The essential difference between
these hydrolyzable polycations and PAGS is that the cations
in PAGS are derived from L-arginine, supporting the hypoth-
esis that endogenous cations are necessary for a biocompatible
polycation.
Synthetic polycations can be used for many biomedical appli-
cations. Control of the synthesis process should allow control
over its interaction with biological polyanions. One can take
advantage of these interactions between oppositely charged
macromolecules to precipitate biological polyanions, such as
nucleic acids and glycosaminoglycans. As a preliminary test, we
explored the potential use of PAGS by investigating the binding
between PAGS and heparin, one of the most negatively charged
glycosaminoglycans. The resultant [polycation:heparin] complex
could be used to deliver heparin-binding growth factors. The
interaction between heparin and heparin-binding growth factors
is expected to stabilize and potentially activate the growth fac-
tors upon binding with the corresponding growth factor recep-
tors.[28] Furthermore, it was possible to control the release of the
growth factors by adjusting the polyvalency between the poly-
cation and heparin. PAGS complexed heparin and precipitated it
out of PBS as a fibrillar matrix (Figure 5A). The diameter of the
fibrils ranged from approximately 1 μm to sub-micrometer in
diameter and sheets ranged from 5–20 μm in diameter. Glob-
ular structures were also observed being dispersed among the
fibrils. The addition of PAGS to the heparin solution increased
the zeta potential following a sigmoidal curve (Figure 5B).
Synthetic polycations can be used in many other applications
including cell encapsulation, polyelectrolyte multilayers, bio-
sensing, and coating of medical devices and implants. Polycation
toxicity has been a great concern in these applications and
a more biocompatible polycation would significantly advance
their clinical translation.[29] A polycation can interact with algi-
nate, a polymer widely used for cell encapsulation, to form a
hydrogel with increased bioactivity and improved mechanical
properties.[30] Polycations can also stabilize and control the
molecular weight cut-off of the alginate microcapsule mem-
brane.[31] Additionally, polycations can mediate cell encapsula-
tion by forming multilayers through Coulomb interactions with
polyanions. Polyelectrolyte multilayers are advantageous for
cell encapsulation because the layer-by-layer assembly allows
control of the local biochemical environment.[32]
3. Conclusions
We believe that the design principle employed in this research
is applicable to other building blocks and can lead to a variety
of novel biomaterials. Other cationic amino acids, such as
lysine, and diacids, such as fumaric acid, may yield polycations
with additional functional groups and unique properties. The
reported design allows structural flexibility and functional diver-
sity including backbone rigidity, hydrophilicity, charge density,
total charge, and side chain length. These inherent controls
of the polymer properties can be used to direct the interaction
between the polycations and polyanions, an important deter-
minant in many biomedical applications. The availability of
biocompatible polycations may broaden the applications of bio-
logical polyanions and change their methods of administration.
4. Experimental Section
D-Arginyl Ethyl Ester Synthesis: D-Arginyl ethyl ester was synthesized by
vigorously stirring D-arginine monohydrochloride and excess anhydrous
ethanol in an ice bath. Acetyl chloride was added dropwise to the cold
solution. The mixture was refluxed overnight. The reactants and side
products were evaporated under reduced pressure to obtain the product.
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Diglycidyl Succinate: Diallyl succinate was synthesized by esterification
of succinic acid and allyl alcohol under the catalysis of sulfuric acid. The
reaction mixture was refluxed overnight and then sodium bicarbonate was
added to neutralize the acid. The organic phase was extracted using ethyl
acetate and brine. The product was then exposed to anhydrous sodium
sulfate to remove residual water. Diglycidyl succinate was synthesized
by epoxidation of diallyl succinate with meta-chloroperoxybenzoic
acid (mCPBA) in dichloromethane. The reaction was refluxed at 40 °C
overnight. The reaction mixture was then run through an ionic resin
column containing tertiary amine beads to remove unreacted mCPBA.
Diglycidyl succinate was further purified using flash chromatography
and stored at −20 °C.
Polymer Synthesis: The arginine-based polymers were synthesized
via polycondensation reaction of a 1:1 molar ratio of L- or D-arginine
ethyl ester and diglycidyl succinate or 1,4-butanediol diglycidyl ether in
N,N-dimethylformamide (DMF) under N2. The reaction mixture was
stirred and kept at 60 °C for 7 days. The resultant polymers were purified
by repeat precipitation in ethyl ether or ethyl acetate until the nuclear
magnetic resonance (NMR) showed the products were pure enough.
Fourier-transform infrared (FTIR) spectroscopy and gel-permeation
chromatography (GPC) were used to further characterize the polymers.
pH Titration: Acid titrations were performed by using 10 mL of
1 mg mL−1 PAGS solution. 0.1 M HCl was added sequentially while the
solution was being stirred. The pH values were recorded by a SevenEasy
pH meter (Metter-Toledo, Columbus, OH).
dropped directly on a SEM sample stud, lyophilized, and sputtered with
gold for SEM observation.
Polymer Degradation: Degradation of PAGS, Et-PAGS, and D-PAGS
were performed following established protocols.[19] Briefly, the polymers
were dissolved in PBS. Bovine pancreatic cholesterol esterase (Sigma-
Aldrich, St. Louis, MO) was added to reach a final concentration of
4 units per milligram of polymer. The reaction was carried out at 37 °C
for 7 or 14 days. The retention time of the polymers before and after
the enzymatic treatment was determined by a Viscotek GPCmax VE2001
equipped with a VE 3580 RI detector (Malvern, Westborough, MA). A
Suprema Max 3000 Å column (PSS, Warwick, RI) and PBS were used as
the stationary and mobile phases respectively.
MTT Assay of Degradation Products: The degradation products were
collected by passing through an Amicon Ultra centrifugal dialysis
membrane with a cut-off molecular weight of 3 kDa (Millipore, Billerica,
MA). The fraction that had a MW less than 3 kDa was collected and
lyophilized overnight. The degradation products were added to the cell
culture medium (conditioning medium) in which the MTT assay was
performed using the same protocol as described above.
Statistical Analysis: For each condition tested, the results of four
replicates were reported as a mean value with the standard deviation.
The multicomparison ANOVA, Tukey Method, was used as a statistic tool
to compare the different experimental values; p < 0.05 was considered
significantly different.
In Vitro Biocompatibility: Baboon smooth muscle cells (BaSMCs) were
obtained from carotid arteries harvested from 17–20 kg juvenile male
baboons and thoroughly characterized. Cells were cultured in MCDB
131 medium (Mediatech, Manassas, VA) supplemented with 10% fetal
bovine serum (FBS), 1.0% L-glutamine, 50 μg mL−1 ascorbic acid and
20 μg mL−1 gentamycin at 37 °C with 5% CO2. BaSMCs (passages 6–14)
were used for in vitro biocompatibility. To test the lactate dehydrogenase
(LDH) activity, metabolic activity, and cell viability, 8 × 103 cells were
seeded on 96-well plates one day before assay. For apoptotic assay,
5 × 104 cells were seeded on 6-well plates. Different concentrations of
polymer solution were prepared by dissolving the polymers in MCDB
131 culture medium and incubated with cells for 4 h to test the LDH
activity, metabolic activity, and apoptotic assay or 24 h to test cell
viability. The extracellular LDH activity was measured using CytoTox 96
Non-Radioactive Cytotoxicity (Promega Madison, WI). The metabolic
activity, caspase-3 activity, and cell viability were measured using a
Vybrant® MTT Cell Proliferation Assay Kit, EnzCheck Caspase-3 Assay
Kit, and Live/Dead Viability/Cytotoxicity Kit (Molecular Probes, Eugene,
OR), respectively. All data were normalized to the control, which was a
culture medium without any polymer.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
This research is supported by an NSF grant # DMR-1005766. We thank
Anh Nguyen for experimental assistance. We greatly appreciate the
Nils Kröger laboratory for assistance with the dynamic light scattering
experiments.
Received: May 14, 2010
Revised: July 21, 2010
Published online: December 14, 2010
In Vivo Biocompatibility: Male Balb/C mice weighing 19–21 g were
injected intraperitoneally with PAGS/saline solution (n = 10), PEI/saline
solution (n = 9), or saline (n = 3) as control. The animals were cared
for in compliance with protocols approved by the Committee on Animal
Care of the Georgia Institute of Technology following NIH guidelines
for the care and use of laboratory animals (NIH publication No. 85–23
rev. 1985). Major organs including the heart, liver, lung, kidney, spleen,
and bladder with prostate gland were harvested at day 1, 5, and 30 post-
injection. The organs were rinsed with PBS, fixed with 10% formalin, and
then submersed in 30% sucrose at 4 °C. For cryosection, all samples
were embedded in Tissue-Tek OCT Compound (Sakara Finetek USA,
Torrance, CA). Cross-sections (10 μm thick, the longitudinal axis cut)
were stained with the standard hematoxylin and eosin (H&E) staining
method to examine inflammation and fibrosis. Sections were also
stained for nuclear DNA fragmentation using DeadEnd Fluorometric
TUNEL System (Promega, Madison, WI) to examine apoptosis. All slides
were analyzed blindly by Dr. Adeboye Osunkoya at Emory University.
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