Water insoluble cationic poly(ester amide)s: Synthesis, characterization and applications

Article abstract of DOI:10.1039/c2tb00070a

We reported a new family of water insoluble, biocompatible and biodegradable cationic arginine and phenylalanine based poly(ester amide)s (Arg-Phe-PEAs) for potential biomedical applications. The Arg-Phe-PEA consists of 3 building blocks: amino acids, dio

Full text of DOI:10.1039/c2tb00070a

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Water insoluble cationic poly(ester amide)s: synthesis,
characterization and applications†
Cite this: J. Mater. Chem. B, 2013, 1,
353
a
ab
*
Jun Wu and Chih-Chang Chu
We reported a new family of water insoluble, biocompatible and biodegradable cationic arginine and
phenylalanine based poly(ester amide)s (Arg-Phe-PEAs) for potential biomedical applications. The Arg-
Phe-PEA consists of 3 building blocks: amino acids, diols, and dicarboxylic acids and was prepared from
both L-arginine and L-phenylalanine amino acids by solution polycondensation. The structure and
properties of Arg-Phe-PEAs were characterized by standard physicochemical methods. In vitro biological
tests of the Arg-Phe-PEAs included enzymatic biodegradation, cell attachment and proliferation, and
macrophage inflammation tests. The enzymatic biodegradation tests showed that the biodegradation
rate of Arg-Phe-PEAs can be controlled by regulating the polymer composition (the ratio of Arg to Phe).
The cellular tests indicated that the bovine aortic endothelial cells (BAECs) have very good cell
attachment and proliferation on the Arg-Phe-PEA surface. The in vitro inflammation data showed that
the Arg-Phe-PEAs induced far lower inflammatory response than FDA approved biomaterials. By a
nano-precipitation method, Arg-Phe-PEAs were formulated into nanoparticles (NPs) with particle sizes
below 200 nm. The protein release profiles from Arg-Phe-PEA NPs showed that the introduction of
arginine significantly enhanced the protein encapsulation efficacy and changed the protein release rate
compared with pure phenylalanine based poly(ester amide)s (Phe-PEAs).
Received 4th September 2012
Accepted 15th October 2012
DOI: 10.1039/c2tb00070a
www.rsc.org/MaterialsB
polymers helps to improve the cellular interaction performance
of materials, such as cell attachment; (3) for drug delivery
1 Introduction
Cationic water insoluble non-biodegradable polymers have
been developed for commercial uses mainly as the coating for
papers, and textiles to x anionic dyes and inks, i.e., providing
printable surface. These commercially available cationic water
insoluble polymers have been synthesized via free-radical
copolymerization with cationic monomers like allylic quater-
nary ammonium salt monomer and other cationic mono-
mers.1–4 However, these polymers are not suitable for
biomedical applications due to their biodegradability and
biocompatibility problems.
Water insoluble biocompatible and biodegradable polymers
with cationic groups5–7 are important for biomedical applica-
tions like drug delivery and tissue engineering applications due
to the following reasons: (1) compared with water soluble
cationic polymers, water insoluble cationic polymers can be
fabricated into microspheres/microparticles, nanoparticles
(NPs), nanobers and drug eluting coating formulations
without loss of structural integrity through dissolution; (2) for
tissue engineering applications, the cationic property of
applications, the positive charge of the carrier could help to
enhance the loading efficiency and sustained release of nega-
tively charged drugs (such as nucleic acids, some proteins/
peptides and small molecular drugs).8,9 However, few of the
existing commercial cationic water-insoluble polymers are
biodegradable and biocompatible, and the recent fast devel-
opment of biotechnology demands new cationic water insoluble
biodegradable polymers with improved biocompatibility and
controllable physical–chemical properties.5–7 Our objective for
this study is to develop a new family of biocompatible, biode-
gradable and water insoluble cationic arginine based polymers,
to serve as potential tissue engineering scaffolds and protein
delivery vehicles.
In this report, we described the development of a new family
of cationic water insoluble biodegradable amino acid based
poly(ester amide)s (AA-PEAs, Scheme S1 (ESI†)) that exhibited
excellent biocompatibility, particularly biomaterial-induced
inammatory response and supporting cell proliferations. This
new family of cationic AA-PEA is based on 2 amino acids: a
cationic hydrophilic L-arginine (Arg) and a neutral hydrophobic
amino acid like L-phenylalanine (Phe) (Arg-Phe-PEAs). In vitro
biological tests of the Arg-Phe-PEAs were conducted to examine
their enzymatic biodegradation, cell attachment and prolifera-
tion, and inammation properties. The negatively charged
proteins were encapsulated into the Arg-Phe-PEA NPs to
aDepartment of Biomedical Engineering, Cornell University, Ithaca, NY, 14853-4401,
USA. E-mail: cc62@cornell.edu; Fax: +1-607-2551093
bDepartment of Fiber Science and Apparel Design, Cornell University, Ithaca, NY,
14853-4401, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2tb00070a
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investigate the protein loading efficacy and release proles of
NPs. When compared with other cationic water insoluble
biodegradable polymers,5–7 this new Arg-Phe-PEA polymer
family has the following signicant advantages: (1) many amino
acid-based poly(ester amide)s (AA-PEAs) have been proven
in vitro and in vivo to have good biocompatibility, biodegrad-
ability, processing and mechanical properties;7,10–22 (2) Arg and
Phe are the natural essential amino acids for humans, and the
side guanidinium group of Arg has a strong pKa of 12.5, which
could offer stronger electrical interactions with negatively
charged molecules; (3) unlike those cationic block copolymers
(e.g., polylactide–poly-L-lysine block copolymer5,6) in which the
cationic group is concentrated in one block of the copolymer
(e.g., Lys), the cationic charge unit (Arg) in the Arg-Phe-PEA can
be evenly distributed along the Arg-Phe-PEA backbone; (4) the
positive charge density in the Arg-Phe-PEA can be easily tuned
within a wide range by adjusting the polymer composition.
The synthesis of the Arg-Phe-PEA polymer involves the
solution polycondensation reaction of the mixture of 3 different
monomers at predetermined feed ratios: (1) di-p-toluene-
sulfonic acid salts of bis-(L-phenylalanine) a,u-alkylene diesters
(as bis-nucleophiles I); (2) tetra-p-toluenesulfonic acid salts of
bis-(L-arginine) a,u-alkylene diesters (as bis-nucleophiles II);
and (3) di-p-nitrophenyl ester of dicarboxylic acids (as bis-elec-
trophiles). The percentage of Arg of the resulting Arg-Phe-PEAs
Scheme 1 Synthesis of monomers and polymers.
can be controlled by tuning of the feed ratio of di-p-toluene- depending on the type of diols: di-p-toluenesulfonic acid salt of
sulfonic acid salts of bis-(L-Phe) a,u-alkylene diesters to tetra-p- bis(L-phenylalanine) butane diesters, Phe-4, y ¼ 4; tetra-p-tolue-
toluenesulfonic acid salts of bis-(L-Arg) a,u-alkylene diesters. nesulfonic acid salt of bis(L-arginine) butane diesters, Arg-4-S,
Therefore, the Arg-Phe-PEA properties, such as hydrophobicity, y ¼ 4; tetra-p-toluenesulfonic acid salt of bis(L-arginine) hexane
charge property, thermo property and enzymatic biodegrada- diesters, Arg-6-S, y ¼ 6, where y is the number of methylene
tion rate, can be precisely tuned to meet a wide range of groups in the diol, and S indicated that the L-arginine diester
different requirements. This synthesis approach can be used as monomer was in the p-toluenesulfonic acid salt form.
a universal and simple synthetic strategy with a high potential
Arg-Phe-PEAs were prepared by the solution polycondensation
utility for the preparation of hybrid AA-PEAs having different of the above I and II monomers (NA, NS and Phe-4, Arg-4-S, Arg-
pendant functional groups.
6-S) at different combinations and the prepared polymers are
summarized inTableS1 (ESI†). These Arg-Phe-PEAsarelabeled as
x-Arg-y1-S-x-Phe-y2-z%, where x and y1/y2 are the numbers of
methylene groups in diacid and diol, respectively, and z% is the
molar percent of L-arginine diester monomer in the mixture of
2 Experimental
2.1 Synthesis of monomers and polymers
The general scheme of the synthesis of Arg-Phe-PEAs is divided L-arginine diester monomer and L-phenylalanine diester mono-
into the following three major steps (Scheme 1): the preparation mer. y1isfromargininemonomerIIandy2isfromphenylalanine
of di-p-nitrophenyl ester of dicarboxylic acids (I); the prepara- monomer II. For example, the 8-Arg-6-S-8-Phe-4-20% sample
tion of tetra-p-toluenesulfonic acid salts of bis(L-arginine) a,u- indicates that x of this copolymer is 8, and y1 (in Arg-block) is 6
alkylene diesters and di-p-toluenesulfonic acid salts of bis(L- and y2 (in Phe-block) is 4, and the molar% of L-Arg diester
phenylalanine) a,u-alkylene diesters (II); and the synthesis of monomer II in the copolymer is 20%. In order to make the types
Arg-Phe-PEAs (III) via solution polycondensation of I and II. All and numbers of the resulting Arg-Phe-PEA copolymer samples
the details for the synthesis of monomers (I and II) can be found manageable for the study, we chose to vary y at a xed x in each
in our previous reports.10,23
copolymer series, i.e., at each monomer I (NA or NS), monomer II
Di-p-nitrophenyl esters of dicarboxylic acids (I) were prepared (Arg-y or Phe-y) of different y was used to synthesize the Arg-Phe-
by reacting dicarboxylic acyl chloride varying in methylene PEAs. One pure Phe-PEA (8-Phe-4) and two pure Arg-PEAs (4-Arg-
length with p-nitrophenol as previously reported.10,23 In this 4-Sand 8-Arg-6-S) were also prepared here as the polymer controls
study, two types of monomers (I) were made: di-p-nitrophenyl and their synthesis/characterization steps have been reported
adipate (NA), x ¼ 4 and di-p-nitrophenyl sebacate (NS), x ¼ 8, previously.10,23 All the prepared monomers and polymers were
where x is the number of methylene groups in the diacid. The characterized by standard physicochemical methods.
monomers (II) were prepared via a solid–liquid reaction at
An example of the synthesis of 8-Arg-6-S-8-Phe-4-20% via a
high temperature as previously reported.10,23 Three types of L- solution polycondensation is given here. Monomer I (NS,
phenylalanine or L-arginine based monomers (II) were made, 1.0 mmol) and monomer II (Arg-6-S of 0.2 mmol and Phe-4
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of 0.8 mmol) in 1.5 mL of dry DMSO were mixed well by vor- CO2 incubator. Cell media were changed every day. Aer the
texing. The mixture solution was heated to 70 ꢀC with stirring to predeterminedperiods (48hand96h),thecellcultureplateswere
obtain a uniform mixture. Triethylamine (0.31 mL, 2.2 mmol) taken from the incubator. The cell morphology was recorded
was added drop by drop to the mixture at 70 ꢀC with a vigorous under a microscope. The media from the wells were then aspi-
stirring until complete dissolution of the monomers. The rated, and 0.5 mL fresh media were added to each well. Aer that,
solution color turned yellow within several minutes. The reac- 40 mL of MTT solution (5 mg mLꢁ1) was subsequently added to
ꢀ
tion vial was then kept for 12 h at 70 C in a thermostat bath each well, followed by 4 h incubation at 37 ꢀC, 5% CO2. The cell
without stirring. The resulting polymer product was precipi- culture medium was carefully removed and 400 mL of acidic iso-
tated out by adding cold ethyl acetate, and puried by a Soxhlet propyl alcohol (with 0.1 M HCl) was added to dissolve the formed
extractor using ethyl acetate as a solvent for 24 h. The nal dried formazan crystal. The plate was slightly shaken for 30 min and
Arg-Phe-PEAs were yellow or pale yellow solid and dried in vacuo 100 mL of solution was transferred from each well to a 96 well cell
at room temperature.
culture plate. The optical density (OD) of each well was measured
at 570 nm (subtract background reading at 690 nm) by using a
microplate reader. Triplicates were used in each experiment.
2.2 In vitro enzymatic biodegradation of Arg-Phe-PEA
The following polymers were selected for in vitro enzymatic
biodegradation tests: 8-Arg-6-S-8-Phe-4-S-10%, 8-Arg-6-S-8-Phe-
4-S-20% and 8-Phe-4. The polymer lm sample (around 200 mg
each, same size and thickness, round shape) was immersed in a
small vial containing 10 mL of PBS buffer (pH 7.4, 0.1 M) with a-
chymotrypsin (0.2 mg mLꢁ1). The vials were then incubated at
37 ꢀC with a constant reciprocal shaking (ca. 100 rpm). The
immersion media were refreshed every 2 days in order to main-
tain the enzymatic activity. Aer predetermined immersion
durations, the polymer lm samples were removed by ltration,
washed with distilled water 3 times, and then dried under
vacuum at 30 ꢀC for 24 h to completely remove the residual water.
The degree of biodegradation was calculated from the weight
loss of the polymer lm based on the following equation:
2.4 In vitro measurement of inammatory response of Arg-
Phe-PEA
The in vitro inammatory response of the Arg-Phe-PEAs was
studied according to the published protocol.23 J774 macro-
phages were seeded onto 12 mm polymer-coated glass cover-
slips in 24-well tissue culture plates at a cell concentration of
10 000 cells per well. The cover glasses were coated by dipping a
polymer DMF solution (2 wt%) on one side of the cover glass
surface and vacuum drying for 24 h at room temperature.
Positive controls were glass coverslips in media containing
lipopolysaccharide (LPS, from E. coli 0111:B4, Sigma-Aldrich, St.
Louis, MO) at nal concentrations of 1.25 mg mLꢁ1 and 5.00 mg
mLꢁ1. A plain glass coverslip in media alone was used as a cell-
free negative control. 8-Arg-6-S-8-Phe-4-20% was selected for
this inammatory response test. PBMA and PCL were used as
commercial FDA-approved polymer controls. Macrophage acti-
vation aer 48 h incubation was measured using an ELISA kit
(Invitrogen, Carlsbad, CA) to measure released mouse TNF-a
according to the manufacturer’s protocol. Sample TNF-a
concentrations were calculated from a standard calibration
curve using a 4-parameter standard curve-tting algorithm
(Gen5 soware, BioTek Instruments, Winooski, VT). All testing
samples were run in triplicate (N ¼ 3). All samples and stan-
dards were read in duplicate on a 96-well plate reader at 450 nm
and referenced against a chromogen blank.
Wt (%) ¼ (Wo ꢁ Wt)/Wo ꢂ 100
where Wo is the original weight of the dry polymer lm sample
before immersion and Wt is the dry polymer lm sample weight
aer incubation for t hours/days (with or without a-chymo-
trypsin enzyme). The average weight loss of three specimens
was measured for each type of sample (N ¼ 3).
2.3 Cell attachment and proliferation assay on the Arg-Phe-
PEA coating surface
The evaluation of BAEC attachment and proliferation capability
on the Arg-Phe-PEA coating surface was performed by cell
attachment assay followed by a MTT assay. The round micro-
2.5 NP preparation and protein loading/release study
cover glasses (diameter, 12 mm, no. 2, VWR, West Chester, PA) Arg-Phe-PEA and Phe-PEA NPs were prepared via the self-
were coated with polymer DMF solution (2 wt%) and dried under assembly of PEA in aqueous solutions through an optimized
vacuum for 24 h at room temperature. The following polymers single-step nanoprecipitation method. Briey, the PEA polymer
were tested: 8-Arg-6-S-8-Phe-4-20%, 8-Phe-4 and non-biode- was dissolved in DMSO with concentrations ranging from 1 to
gradable poly(n-butyl methacrylate) (PBMA). Commercially 5 mg mLꢁ1. The resulting PEA solution was then added into the
available PBMA has been widely used in stent coatings and other 1% PVA aqueous solution dropwise under gentle magnetic bar
medical devices and was used here as the positive control.24 A cell stirring (1000 rpm) at room temperature. The remaining
culture plate coated with collagen was used as a negative control. organic solvent and free molecules were removed by washing
Aer drying, the polymer-coated glass coverslips were placed the NP solution using an Amicon Ultra-4 centrifugal lter
onto the bottom of cell culture plates and were sterilized over- (Millipore, Billerica, MA) with a molecular weight cutoff of
night under UV irradiation in the cell culture hood before use.
100 000 Da. To prepare protein encapsulated NPs, BSA DMSO
BAECs at an appropriate cell density concentration (20 000 solution (2 mg mLꢁ1) was mixed with the PEA DMSO solution
cellsper well)wereseededontoeach test well in24-well plates(BD with designed volume ratios before the nanoprecipitation
Falconꢀ, polystyrene treated) and then incubated in a 37 ꢀC, 5% process. The NP size (diameter, nm) and surface charge (zeta
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potential, mV) were obtained from three repeated measure-
ments by quasi-elastic laser light scattering with a ZetaPALS
dynamic light scattering detector (15 mW laser, incident beam
676 nm; Brookhaven Instruments Corporation, Holtsville, NY).
NP images were obtained by transmission electron microscopy
(TEM). For TEM tests, the NPs were rst incubated with negative
staining solution of uranyl acetate. Aer washing and drying,
the NPs were then imaged by TEM (Tecnaiꢀ G2 Spirit
BioTWIN).
In this study, BSA was selected as a model protein to measure
the protein loading yield and release prole from each type of
NPs. Briey, 10.0 mL of protein encapsulated NP PBS solutions
at a concentration of 1.0 mg mLꢁ1 were transferred into an
Amicon Ultra-4 centrifugal lter tube with a molecular weight
cutoff of 100 000 Da. The protein loading yield was calculated
using the original total amount of proteins mixed with polymers
to subtract the amount of free BSA in the buffer solutions
immediately aer the NP formulation. The free BSA solutions
were collected by centrifuging at 4000 rpm for 5–10 minutes.
Aer that, the solutions were incubated in a 37 ꢀC water bath for
the release study. At each time point, the released free BSA
solutions were collected separately from the NP solutions in
three lter tubes by centrifuging at 4000 rpm. The resulting free
BSA content in each lter tube was assayed using a BCA assay.
Fig. 1 1H-NMR spectrum of 8-Arg-6-S-8-Phe-4-20%.
(ESI†) and the number average molecular weight (Mn) of this
new Arg-Phe-PEA family ranged from 15.0 to 25.0 kg molꢁ1 with
a polydispersity of 1.10–1.30.
The solubility of Arg-Phe-PEAs can greatly affect their
potential biomedical applications. In this report, 2.0 mg mLꢁ1
was selected as a standard to determine whether a polymer is
soluble or not. Table S2 (ESI†) shows the polymer solubility in
several types of solvents. Arg-Phe-PEAs synthesized in this study
were water insoluble, but they all dissolved in DMSO and DMF
within the studied Arg contents in the Arg-Phe-PEA copolymers
(ranging from 10% to 20%). The solubility data in Table S2†
showed some signicant solubility difference from the pure
Arg-PEA and Phe-PEA homopolymers. When compared with a
pure Arg-PEA, the Arg-Phe-PEA copolymers were water insol-
uble; when compared with a pure Phe-PEA, the Arg-Phe-PEAs
were insoluble in THF and chloroform, which would dissolve a
pure Phe-PEA well.
In this report, only 10% and 20% arginine were introduced
into Phe-PEA. If a greater percentage of arginine was intro-
duced, the new Arg-Phe-PEA physicochemical properties would
change signicantly, including hydrophobicity, thus changing
the overall processing feasibility of the formulations. Our
results indicated that Arg-Phe-PEAs with more than 40% of
arginine were not able to form good coating lms. In this report,
the introduction of 10% and 20% arginine was used to
demonstrate the new cationic property and test the proposed
hypothesis.
3 Results and discussion
Overall, this work is a continuation of our work in developing
biocompatible cationic poly(ester amide)s and in this report we
have shown signicant improvements and encouraging appli-
cations with these novel polymers in comparison to our previ-
ously published work.7,20,22,23,25 For example, compared to our
recently reported water soluble arginine based poly(ester
amide)s,20,22,23 the polymers reported in this work are water
insoluble, which could be formulated into nanoparticles,
microspheres, coatings, lms and scaffolds; compared to our
recently reported water insoluble lysine based poly(ester amide)
s,7,25 this work signicantly simplies the synthesis strategies.
The introduced guanidinium groups of arginine have signi-
cantly higher pKa than the primary amine groups of lysine, and
will have stronger interactions with negatively charged proteins,
nucleic acids and small molecular drugs.
3.1 Synthesis and characterization of Arg-Phe-PEAs
In this study, Arg-Phe-PEAs were prepared according to the
reaction scheme in Scheme 1. All the Arg-Phe-PEAs were
prepared with high yields (>80%) under the optimized reaction
conditions. The chemical structure of the prepared Arg-Phe-
PEAs was conrmed by 1H-NMR spectra. As a representative, the
1H-NMR spectrum of 8-Arg-6-S-8-Phe-4-20% is shown in Fig. 1.
3.2 Static contact angle
The contact angles of Phe-PEA and Arg-Phe-PEA were measured
and compared with poly(3-caprolactone) (PCL) (Table 1). Among
1
1
All the H-NMR peaks of the polymer were identied. The H-
NMR peaks marked with numbers from 1 to 24 are assigned to
the corresponding protons as shown in Fig. 1. The actual molar
Table 1 Static contact angle of polymer coatings
Polymer
name
8-Arg-6-S-
8-Phe-4-10% 8-Phe-4-20%
8-Arg-6-S-
1
percentage of Arg (z%) calculated from the integration of H-
PCL
8-Phe-4
NMR peaks is around 24% for 8-Arg-6-S-8-Phe-4-20%. The
molecular weight of polymers was measured by a GPC method
Static angle (ꢀ) 90.86 ꢃ 0.77 79.62 ꢃ 1.03 42.51 ꢃ 0.91 29.17 ꢃ 1.47
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356 | J. Mater. Chem. B, 2013, 1, 353–360