Synthesis and characterization of functionalized water soluble cationic poly(ester amide)s

Article abstract of DOI:10.1002/pola.24160

A new family of positively charged, water soluble and functional amino acid-based poly(ester amide)s (Arg-AG PEA) consisting of four building blocks (L-Arginine, DL-2-Allylglycine, oligoethylene glycol, and aliphatic diacid) were synthesized by the soluti

Full text of DOI:10.1002/pola.24160

Synthesis and Characterization of Functionalized Water Soluble
Cationic Poly(ester amide)s
XUAN PANG,1 JUN WU,2 CYNTHIA REINHART-KING,2 CHIH-CHANG CHU1,2
1Department of Fiber Science and Apparel Design, Cornell University, Ithaca, New York 14853-4401
2Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853-4401
Received 27 April 2010; accepted 25 May 2010
DOI: 10.1002/pola.24160
Published online in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: A new family of positively charged, water soluble and
functional amino acid-based poly(ester amide)s (Arg-AG PEA)
consisting of four building blocks (L-Arginine, DL-2-Allylglycine,
oligoethylene glycol, and aliphatic diacid) were synthesized by
the solution copolycondensation. Functional pendant carbon–car-
bon double bonds located in the DL-2-allylglycine unit were incor-
porated into these Arg-AG PEAs, and the double bond contents
could be adjusted by tuning the feed ratio of L-arginine to DL-2-
allylglycine monomers. Chemical structures of this new functional
Arg-AG PEA family were confirmed by Fourier transform infrared
spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spec-
tra. The thermal property of these polymers was investigated;
increasing the methylene chain in both the amino acid and diacid
segments resulted in a reduction in the polymer glass-transition
temperature. All these cationic Arg-AG PEAs had good solubility
in water and polar organic solvents. The cytotoxity of Arg-AG
PEAs was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay. These preliminary MTT results
indicated that Arg-AG PEAs were nontoxic to bovine aortic endo-
C
V
thelial cells (BAECs).
2010 Wiley Periodicals, Inc. J Polym Sci
Part A: Polym Chem 48: 3758–3766, 2010
KEYWORDS: amino acid; arginine; double bond; poly(ester
amide)s
INTRODUCTION Synthetic biodegradable polymers have been
intensively investigated for applications in the fields of bone
fracture fixation devices, drug-controlled release carriers, tis-
sue engineering scaffolds and green plastics for packaging,
and disposal containers because of their biodegradability
and biocompatibility.1–6 In recent years, much research has
focused on absorbable aliphatic polyesters for biomedical
applications, such as polylactide,7,8 poly-e-caprolactone,9
poly(glycolic acid)10 and their copolymers. However, their
broader applications in the biomedical field are limited
because they do not meet certain biological requirements
like water solubility, the lack of functionality along the
polyester chains, and their acidic degradation products are
not desirable.11,12 On the other hand, synthetic poly(amino
acid)s including poly-L-lysine and poly(glutamic acid) have
been explored for various biomedical uses;13–15 however, the
preparation of poly(amino acid)s in bulk still has some dis-
advantages namely, the expensive and unstable N-carboxy-
anhydride monomers.
Researches into PEAs have attracted much attention from
both academic and industrial scientists for their potential in
biomedical and biological fields.17–22
Our lab has successfully established a general methodology
for the synthesis of saturated and unsaturated amino acid-
based PEAs.23–26 The PEAs consisting of well-defined amide
and ester blocks have different thermal, physical, and bio-
logical properties. The unsaturated phenylalanine-based
PEAs are organic solvent soluble and have imbedded double
bonds in the polymer backbone. They were used as pre-
cursors to photo-crosslink with poly(ethylene glycol) di-
acrylate (PEG-DA) to fabricate PEA-based hydrogels for the
control release of paclitaxel.27–30
Of the 20 naturally occurring amino acids, only the hydro-
phobic amino acids, such as L-Valine, L-Leucine, L-Phenylala-
nine, and DL-Methionine16 were selected in synthesis of most
a-amino acids based PEAs. The only natural, positively
charged amino acid ever used was L-lysine, however, in most
cases these PEAs have anionic –COOH pendant functional
groups instead of the cationic amine group because the free
amine group in L-lysine was converted into amide group in
PEA during synthesis.31–33 Gillies et al. recently reported the
synthesis of cationic functional PEA copolymers having
pendant free amine from L-lysine block, but the polymers are
soluble in organic solvent only.22 Most recently, Chu and
As a result, the combination of these two classes of polymers
(polyesters and polyamino acids) into a single polymer
entity, amino acid-based poly(ester amide)s (PEAs), would
seem attractive because these PEAs would have both ester
and amide linkages on their backbones. As such PEAs would
integrate the merits of both polyamides and polyesters.16
Additional Supporting Information may be viewed in the online version of this article. Correspondence to: C.-C. Chu (E-mail: cc62@cornell.edu)
C
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Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 3758–3766 (2010) 2010 Wiley Periodicals, Inc.
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SCHEME 1 Synthetic pathway for the preparation of di-p-nitrophenol esters of dicarboxylic acids (I). x: number of methylene
group in diacid.
coworkers34 reported
a
different method to synthesize
Synthesis of the Monomers
organic solvents soluble PEAs having pendant amine group.
The monomers synthesized could be divided into two cate-
gories: di-p-nitrophenyl ester of dicarboxylic acids (I); di-
p-toluenesulfonic acid salts of bis-L-arginine (or bis-DL-2-allyl-
glycine) esters (II). The synthesis of di-p-nitrophenyl esters
of dicarboxylic acids (I) monomer N-x (x indicated number
of methylene group in diacid) was based on our previously
reported method by reacting dicarboxylic acyl chlorides with
p-nitrophenol (Scheme 1).24 Monomers N-2 and N-8 had the
identical nitrophenol segments but different methylene chain
length in the dichloride segment: A(O)C(CH2)2C(O)A for N-2
and A(O)C(CH2)8C(O)A for N-8.
In this study, we report the synthesis, characterization, and
preliminary biological assessment of a new family of water
soluble and cationic functional PEAs having pendant un-
saturated vinyl group for subsequent photo- or nonphoto
reactions. This new family of water soluble and cationic
functional Arg-AG PEAs having photoreactivity was made
from two amino acid building blocks: L-Arg and a derivative
of L-Gly (DL-2-allylglycine). L-Arg is one of the positively
charged a-amino acids with the highest pI (10.76); in the
protonated form, the guanidino group in arginine is un-
reactive, suggesting the tedious and difficult protection and
deprotection steps normally used by others (including our
laboratory) for the synthesis of functional PEAs can be
eliminated through the protonation of guanidino group
during the synthesis. Incorporation of DL-2-allyglycine (AG)
in the synthesis of this new family of photoreactive, water
soluble, and cationic functionalized PEAs (Arg-AG PEA)
provided photoreactivity to the resulting PEAs by providing
pendant unsaturated carbon-to-carbon double bonds. The
double bond contents in Arg-AG PEAs were tunable by
adjusting the feed ratio of Arg-based to AG-based monomers.
The synthetic procedure of monomer II was described as
below: di-p-toluenesulfonic acid salts of bis-L-arginine esters
Arg-y (y indicated number of methylene group in diol part of
Arg toluenesulfonic acid salt) were prepared as previous
procedure.35 The synthetic pathway of di-p-toluenesulfonic
acid salts of bis-DL-2-allylglycine esters AG-nEG (n indicated
the number of repeating unit in oligoethylene glycol part of
AG toluenesulfonic acid salt) was shown in Scheme 2. DL-2-
allylglycine (0.044mol), p-toluenesulfonic acid monohydrate
(0.05 mol) and oligoethylene glycol (e.g., diethylene glycol or
tetraethylene glycol, at 0.022 mol) in 100 mL of benzene
were placed in a flask equipped with a Dean–Stark appara-
tus, a CaCl2 drying tube, and a magnetic stirrer. Hydro-
quinone was added as inhibitor. The solid–liquid reaction
mixture was heated to reflux for 4 h until 2.0 mL of H2O
evolved. The reaction mixture was then cooled to room tem-
perature, filtered and dried in vacuo at room temperature.
The products, di-p-toluenesulfonic acid salts of bis-DL-2-allyl-
glycine esters (AG-nEG) were purified by dissolving in DMSO
and then recrystallization in ethyl acetate three times. The
final products were white solid.
Herein, the chemical structures, thermal property, and bio-
degradability of these double bond functionalized, water
soluble, cationic Arg-AG PEAs were studied. A preliminary
cytotoxity of Arg-AG PEA was evaluated by MTT assay. The
MTT results indicated that Arg-AG PEAs at the dosage levels
tested were nontoxic to the bovine aortic endothelial cells
(BAECs).
EXPERIMENTAL
Materials
Synthesis of the Water Soluble, Functional, Cationic
Arg-AG PEA Copolymers Having Pendant Photoreactive
Double Bonds
DL-2-Allylglycine (AG), L-Arginine (Arg), p-toluenesulfonic
acid monohydrate (TosOHꢀH2O), diethylene glycol, tetraethyl-
ene glycol, hydroquinone, sebacoyl chloride, succinyl chlo-
ride, 1,4-butanediol, 1,6-hexanediol (Alfa Aesar, Ward Hill,
MA), and p-nitrophenol (J. T. Baker, Phillipsburg, NJ) were
used without further purification. Triethylamine from Fisher
Scientific (Fairlawn, NJ) was dried via refluxing with calcium
hydride and then distilled. Other solvents, such as benzene,
ethyl acetate, acetone, n-butanol, N,N-dimethylacetamide
(DMA), and dimethyl sulfoxide (DMSO), were purchased
from VWR Scientific (West Chester, PA) and were purified by
standard methods before use.
The water soluble, functional, and cationic Arg-AG PEAs
having pendant double bonds were synthesized through the
solution polycondensation of di-p-nitrophenyl ester (N-x) (I)
with a mixture of di-p-toluenesulfonic acid salts of bis-L-argi-
nine (Arg-y) and bis-DL-2-allylglycine esters (AG-nEG) in a
predetermined feed ratio. The combinations used in this
work included x ¼ 2 and 8, y ¼ 4 and 6, n ¼ 2 and 4, and
were listed in Table 1 and illustrated in Scheme 3. The func-
tional Arg-AG PEA copolymers were labeled as x-Arg-y-AG-
nEG, where x was the number of methylene group in the
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
SCHEME 2 Synthetic pathway for the preparation of di-p-toluenesulfonic acid salt of bis-L-arginine/bis-DL-2-allylglycine esters (II). y:
number of methylene group in diol part of Arg; n: number of repeating unit in oligoethylene glycol part of AG toluenesulfonic
acid salt.
diacid unit, y was the number of methylene group in the diol
unit and n was the number of ethylene glycol unit for the
corresponding Arg and AG monomers, respectively.
tance Infrared Fourier Transform Spectroscopy (DRIFTS)
technique was used to collect Infrared (IR) spectra of the
copolymers, because DRIFTS provides a faster and simpler
sample preparation than traditional Fourier transform infra-
red (FTIR) spectroscopy for IR measurement. Samples were
ground into powder and filled into the micro-cup of the dif-
fuse reflectance accessory on a Perkin–Elmer Nicolet Magana
560 FTIR spectrometer (Madison, WI), and IR information of
samples was collected and processed with Omnic software.
Nuclear magnetic resonance (NMR) spectra were recorded
by a Varian Unity INOVA-400 400 MHz spectrometer (Palo
Alto, CA) operating at 400 MHz and 100 MHz for 1H and
13C NMR, respectively. Chemical shifts were given in parts
per million from tetramethylsilane. Deuterated DMSO-d6
(Cambridge Isotope laboratories) was used as the solvents.
A detailed synthetic pathway of Arg-AG PEA 8-Arg-4-AG-4EG
was given as an example to illustrate the procedures: a mix-
ture of monomers (Arg-4, AG-4EG, and N-8) at a predeter-
mined feed ratio in a suitable organic solvent like DMA was
placed to a glass reaction vessel equipped with a magnetic
stirring bar. The reaction vessel was then heated in an oil
ꢁ
bath to 70 C. Triethylamine was added dropwise to the mix-
ture with stirring until the complete dissolution of the three
monomers. The reaction vessel was then kept at 70 ꢁC for
48 h. The resulting functional Arg-AG PEA copolymers were
precipitated from the reaction solution by adding cold ethyl
acetate, followed by filtration, and extraction by ethyl acetate
in a Soxhlet apparatus for 48 h, and finally dried in vacuo at
room temperature.
The number and weight average molecular weight and
molecular weight distribution of the Arg-AG PEAs were
determined with a model 510 gel permeation chromatograph
(Waters Associates, Inc., Milford, CT) equipped with a high-
pressure liquid chromatography pump, a Waters 486 UV
detector, and a Waters 2410 differential refractive index
Chemical Structure Identification
The chemical structures of the Arg-AG PEA copolymers were
characterized by standard chemical methods. Diffuse Reflec-
TABLE 1 List of Polymers Synthesized by Different Monomer Combinations
Monomer I
Monomer II
Polymer
AG-2EG
AG-4EG
Arg-4
Arg-6
Arg-4
Arg-6
N-2
N-8
N-2
N-8
2-Arg-4-AG-2EG
8-Arg-4-AG-2EG
2-Arg-4-AG-4EG
8-Arg-4-AG-4EG
2-Arg-6-AG-2EG
8-Arg-6-AG-2EG
2-Arg-6-AG-4EG
8-Arg-6-AG-4EG
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SCHEME 3 Synthetic pathway for the preparation of functional Arg-AG PEA having pendant carbon-carbon double bonds.
detector. Tetrahydrofuran (THF) was used as the eluent
(1.0 mL/min), and <10 lL of DMF was mixed to increase
the polymer solubility in THF. The columns were calibrated
with polystyrene standards.
Cell Culture
BAECs (primary cells) were purchased from VEC Technolo-
gies. BAECs were maintained at 37 C in 5% CO2 in Medium
ꢁ
199 (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal
Clone III (HyClone, Logan, UT), and 1% each of penicillin–
streptomycin, MEM amino acids (Invitrogen, Carlsbad, CA),
and MEM vitamins (Mediatech, Manassas, VA). BAECs were
used from passages 8–12. Media was changed every two
days. Cells were grown at least to 70% confluence before
splitting or harvesting. The cytotoxicity of Arg-AG PEAs was
performed by MTT assay. The cultured BAEC were seeded at
an appropriate cell density concentration (3000 cells/well)
in 96-well plates and incubated overnight in a 5% CO2
Thermal Property
Thermal properties of the synthesized Arg-AG PEA copoly-
mers were characterized by a differential scanning calori-
metry (DSC) 2920 (TA Instruments, New Castle, DE). The
measurement was carried out from 0 to 300 ꢁC at a scan-
ning rate of 10 ꢁC/min under nitrogen gas at a flow rate of
25 mL/min. TA Universal Analysis software was used for
thermal data analysis. The glass transition temperature (Tg)
was taken at the inflection point.
ꢁ
incubator at 37 C. The cells were then treated with various
Arg-AG PEA solutions (different Arg-AG PEA type and
amounts of solution) for 48 h. Cells exposed to only cell cul-
ture media were used as a control. After 48 h incubation,
15 lL of MTT solution (5 mg/mL) was added to each well,
Solubility
The solubility of the Arg-AG PEAs (50 mg) in H2O and sev-
eral ꢁcommon organic solvents (1.0 mL) at room temperature
(25 C) was evaluated (Table 3).
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
the cell culture plate was incubated for 4 h at 37 ꢁC, 5 %
CO2. The cell culture medium including polymer solution
was then carefully removed and 150 lL of acidic isopropyl
alcohol (with 0.1 M HCl) was added to dissolve the formed
formazan crystal. Optical density was measured at 570 nm
(subtracted from the background reading at 690 nm) using a
microplate reader. Triplicates were used in each experiment.
around 5.17 and 5.69 ppm which were assigned to the methyl-
ene and methine protons of the carbon–carbon double bond
derived from DL-2-allylglycine (Fig. 1).
Polymer Synthesis
The Arg-AG PEA family were prepared through the solution
copolycondensation of monomers (I) and (II) at different
combinations. Eight Arg-AG PEAs with different methylene
chain length [x in the di-p-nitrophenyl ester of diacid (I), y in
the di-p-toluenesulfonic acid salts of bis-L-arginine esters
(II), and n in the bis-DL-2-allylglycine esters (II)] and feed
ratios between (I) and (II) were synthesized to elucidate the
relationship between polymer structures and their chemical
and physical properties as shown in Scheme 3 and Table 1.
Polymerization occurred smoothly in DMA solution and gave
a medium molecular weight ranging from 15.0 to 30.0 kg/mol
and a relatively narrow polydispersity around 1.2.
Statistics
Where appropriate, the data are presented as mean 6 SEM
calculated over at least three data points. Statistically signifi-
cant differences, if any, when compared with control groups
were evaluated by unpaired Student’s t test or Dunnet test,
and between more than two groups by Tukey’s test with or
without one-way ANOVA analysis of variance at p ¼ 0.05.
JMP software (version 8.0, from SAS Company) was used for
statistical analysis.
Chemical Structure Identification
RESULTS AND DISCUSSION
To verify the actual chemical compositions of these new
functional Arg-AG PEAs, their NMR and IR spectra were
obtained. The H NMR spectra of all the Arg-AG PEA samples
Monomer Synthesis
The monomer family (I) (Scheme 1) was synthesized from
readily available starting materials, sebacoyl chloride, succinyl
chloride with p-nitrophenol as described in our previous
publications.16,24
1
showed one set of resonance peak at about 5.07 and
5.74 ppm, which were assigned to the methylene and
methine protons of the carbon–carbon double bond in the
AG block (Fig. 2). The presence of the carbon–carbon double
bond in the functional Arg-AG PEA copolymers was further
confirmed by their 13C NMR spectra as the peaks at 117 and
132 ppm were attributed to the corresponding methylene
and methine carbon atoms. The observation of the peak at
about 1.56 ppm was assigned to the second methylene
protons from the guanidino group in the Arg side chain.
These NMR data confirmed the chemical structure of all the
Arg-AG PEAs shown in Scheme 3. The actual composition
ratios of AG-nEG to Arg-y blocks could be calculated from
The monomer family (II) (Scheme 2) was synthesized from
readily available starting materials, L-arginine and DL-2-allyl-
glycine with diols and oligoethylene glycols. Monomers Arg-4
and Arg-6 had the identical amino acid (L-arginine) but dif-
ferent types of diols: HOA(CH2)4AOH for Arg-4, and
HOA(CH2)6AOH for Arg-6. Monomers AG-2EG and AG-4EG
had the identical amino acid (DL-2-allylglycine) but different
oligoethylene glycol segments: A(CH2CH2AO)2A for AG-2EG
and A(CH2CH2AO)4A for AG-4EG. All the AG-based mono-
mers had the typical peaks in their 1H NMR spectra at
FIGURE 1 1H NMR spectra of di-
p-toluenesulfonic acid salt of
bis-DL-2-allylglycine ester AG-4EG.
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ARTICLE
FIGURE
2
1H NMR spectra of
pendant double bond functional-
ized Arg-AG PEA 8-Arg-4-AG-
4EG.
the integral ratios of the two above mentioned typical peaks
at about 5.07 ppm (for the methylene protons of the double
bond in AG-nEG block) and 1.56 ppm (for the second methyl-
ene protons in the side chain of Arg-y block).
and 24 ꢁC of the 8-Arg-4-AG-2EG-25 vs. 22 ꢁC of the 8-Arg-
4-AG-4EG-25. This x, y, and n structural effect on Tg was
attributed to the higher Arg-AG PEA chain flexibility as the
methylene chain length increases. The increase in the ether
bonds (from 2EG to 4EG) led to a reduction in Tg as well,
because the ether bond was a flexible bond for free rotation
(i.e., increasing the polymer chain flexibility and promoting
the chain segmental movement and hence a lower Tg value).
These Arg-AG PEA structures were further confirmed by
their FTIR spectra (Fig. 3). All the FTIR spectra of the Arg-
AG PEAs had the characteristic absorption bands of NH
stretch (ꢂ3285 cmꢃ1), C¼¼O stretch of ester groups
(ꢂ1740 cmꢃ1), amide I group (ꢂ1642 cmꢃ1) and amide II
group (ꢂ1540 cmꢃ1). Based on these NMR and FTIR data,
we could conclude that the Arg-AG PEAs synthesized
possessed the chemical structure as we anticipated.
It was important to know that the chemical structure of PEA
had a profound effect on Tg. When the Tg of the Arg-AG PEAs
Thermal Properties
The thermal property of the functional Arg-AG PEAs contain-
ing AG unit was obtained by DSC and listed in Table 2. These
Arg-AG PEAs were amorphous because no melting tempera-
ture was detected. The glass transition temperatures (Tg) of
ꢁ
ꢁ
the Arg-AG PEAs ranged from 18 C to 33 C, depending on
x, y, and n as well as the feed ratio of Arg-y to AG-nEG mono-
mers. The change in the material parameters like x, y, and
n would affect the Tg of the resulting functional Arg-AG
PEAs. For example, as the x (number of CH2 group in the
diacid segment of Arg-AG PEAs) increased from C2 (i.e.,
2-Arg-4-AG-4EG-25) to C8 (i.e., 8-Arg-4-AG-4EG-25), the Tg
was reduced from 30 ꢁC of the 2-Arg-4-AG-4EG-25 to 22 ꢁC
of the 8-Arg-4-AG-4EG-25, a 27% reduction. Similar reduc-
tions in Tg were also observed as y (number of CH2 groups
in the Arg diol segment) and n (number of repeating unit in
ꢁ
oligoethylene glycol part of AG) increased, example, 22 C of
the 8-Arg-4-AG-4EG-25 vs. 18 ꢁC of the 8-Arg-6-AG-4EG-25
FIGURE 3 FTIR spectra of Arg-AG PEA 8-Arg-4-AG-4EG.
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
TABLE 2 Polymerization Data of Arg-AG PEA
Polymera
Yield (%)
Mn (kg/mol)
Mw (kg/mol)
Mw/Mn
Tg (ꢁC)
2-Arg-4-AG-2EG
2-Arg-4-AG-4EG
2-Arg-6-AG-2EG
8-Arg-4-AG-2EG
8-Arg-4-AG-4EG
8-Arg-6-AG-4EG
a
72
70
77
81
83
80
23.6
33.5
19.9
24.9
20.3
27.7
29.0
44.2
22.1
30.1
26.6
39.1
1.23
1.32
1.11
1.21
1.31
1.41
33
30
27
24
22
18
Arg-y to AG-nEG feed ratio was kept constant as 3 to 1.
was compared with the Tg of nonpolar amino acids based
PEAs (phenylalanine based PEA-AGs) reported in our previ-
ous study,36 the Tg of the Arg-AG PEAs was generally lower
(as the Tg of phenylalanine-based PEA PEA-AGs ranged from
20 ꢁꢁC to 38ꢁ ꢁC, arginine-based Arg-AG PEAs ranged from
18 C to 33 C). This may suggest that the presence of non-
ionic side chain (i.e., benzyl groups) in Phe could partially
reduce the freedom of polymer chain rotation more than
ionic side chain guanidine group in Arg. Our recent study34
confirmed that the incorporation of pendant amine func-
tional group into Phe-based PEA could also lower their Tg
(as the Tg of Phe-based PEA homopolymers ranged from
40 ꢁC to 55 ꢁC, whereas pendant aminꢁe functional group
shown in Table 3. All the Arg-AG PEAs were soluble in polar
organic solvents like DMSO and H2O, but not soluble in non-
polar organic solvents, such as ethyl acetate, or acetone. An
increase in the methylene chain length in both the diacid (x)
and diol (y) segments of Arg-AG PEA copolymers resulted in
a decrease in their solubility because of the increased co-
polymer hydrophobicity.
Cytotoxicity Evaluation of Arg-AG PEAs
The cytotoxicity evaluation of Arg-AG PEAs was preliminarily
evaluated based on the result from an MTT assay and cellu-
lar morphology. Figure 4 showed BAEC morphology in the
cell culture medium treated with 8-Arg-4-AG-4EG (with
10 lL per well). BAEC morphology treated with Arg-AG PEA
did not show any difference when compared with the con-
trol; BAEC seemed healthy and well-spread without round-
ing, vacuolization, or visible membrane blebbing (indicative
of cell death) in the presence of the Arg-AG PEA medium,
that is, an indication that these new water soluble cationic
Arg-based functional PEAs did not adversely affect the BAEC
proliferation and morphology. The data in Figure 4 also
showed that, after 2 days, the Arg-AG PEA copolymer treated
BAEC reached the same degree of confluency similar to the
control (cells cultured in collagen treated wells).
ꢁ
contained PEA-Lys-NH2 from 18 C to 32 C).
The location of the carbon–carbon double bonds incorporated
into PEA could also have a profound different effect on Tg. In
our previous studies of other generations of PEAs,16,23,24 the
double bonds were introduced into the PEA backbone as un-
saturated PEAs, their Tg increased, because the rigidity of the
carbon–carbon double bond in the PEA backbone made the
PEA chain more difficult to rotate and hence reduced flexibil-
ity and increased Tg. If the double bonds were introduced as
the pendant groups,36 their Tg reduced instead, this suggested
that the presence of AG unit could impart additional chain
flexibility because of the increasing free volume from pendant
double bonds which could act as internal plasticizers, lowered
the intermolecular interaction between PEA chains.
An MTT assay was used to test the total number of actively
metabolizing BAEC present (expressed as OD) as shown in
Figure 5. The data in Figure 5 illustrated that the Arg-AG
PEAs were nontoxic to BAEC cells. According to the statisti-
cal data analysis, there is no significant difference of any
Arg-AG PEA copolymer treatment compared with the control
at the p < 0.05 level using a Dunnet test. Thus, the BAEC
Solubility
The solubility of Arg-AG PEAs in H2O and several common
organic solvents was assessed at room temperature as
TABLE 3 Solubility Data of PEAs
Ethyl
H2O
Acetate
DMSO
DMF
Methanol
Acetone
CHCl3
THF
2-Arg-4-AG-2EG
2-Arg-6-AG-6EG
8-Arg-4-AG-2EG
8-Arg-4-AG-4EG
8-Arg-6-AG-2EG
8-Arg-6-AG-6EG
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ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
þ, soluble; ꢃ, insoluble.
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ARTICLE
FIGURE 4 Microscopic images
(10ꢄ) of BAECs with or without
8-Arg-4-AG-4EG (10 lL) 48 hrs
treatment. (a) blank control; (b) 8-
Arg-AG-4EG-25 treated; (c) 8-Arg-
AG-4EG-33 treated; (d) 8-Arg-AG-
4EG-50 treated.
morphology and viability data suggested that Arg-AG PEA
copolymers were nontoxic at the dosage levels tested (1–
10 lL) and duration (48 h).
solubility in polar solvents. All these functional Arg-AG PEA
had good solubility in polar solvents like water, alcohol,
DMSO, and DMF. The cytotoxity of Arg-AG PEAs was eval-
uated using an MTT assay. The MTT results indicated that
PEA-AGs did not affect BAEC growth at the concentrations
and exposure duration tested. Further research about the
use of the pendant carbon–carbon double bond and their
derivatives are still in progress.
CONCLUSIONS
A series of positively charged water soluble Arg-based func-
tional poly(ester amide)s (Arg-AG PEA) were synthesized by
the solution copolycondensation of amino acid (arginine and
allylglycine)-based monomers (II) and dicarboxylic acid-
based monomers (I). FTIR and NMR were used to confirm
the chemical structures of these functional water soluble
cationic PEAs. The thermal property of these polymers was
investigated; increasing the methylene chain length in both
the diol (y) and dicarboxlic acid (x) segments resulted in a
reduction in the polymer glass-transition temperature and
This study was partially support by the Cornell University
Morgan Tissue Engineering Seed Grant Program. Martha A.
Mutschler’s statistical analysis of the MTT data was very much
appreciated.
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