A R T I C L E S
Lee et al.
nylon-3 copolymers and NIH 3T3 fibroblasts. This approach
enabled us to screen rapidly through a variety of copolymer
compositions in order to identify the most promising systems.
Second, hydrogels formed from PEG bearing some of the most
favorable nylon-3 derivatives are examined as substrates for cell
growth. The performance of these functionalized hydrogels
suggests that they represent promising candidates for tissue
engineering applications.
Figure 1. Comparison of an R-amino acid residue (left) with a ꢀ-amino
acid residue (right).
that serve as cell attachment substrates. The use of perfectly
monodisperse synthetic oligomers to create such substrates is
attractive because, in principle, this approach allows careful
tailoring of the functionality displayed; however, the preparation
of these molecules requires many chemical steps, which makes
these materials costly. Alternatively, cell-attachment substrates
may be generated from self-assembling polymers, which are
necessarily polydisperse. These polymeric materials are often
relatively inexpensive to prepare, but the heterogeneity displayed
by synthetic copolymers in terms of length, subunit composition,
and subunit sequence may limit the extent to which such
materials can promote cellular attachment and growth. Hybrid
approaches are possible as well, in which the self-assembly
function is achieved with a readily prepared polymer that has
no intrinsic signaling capability [e.g., polyethylene glycol
(PEG)], and the signaling function is achieved by appending a
discrete peptide, e.g., a sequence containing the RGD motif, to
the polymer.12
Here we describe an evaluation of nylon-3 polymers as
components for artificial tissue engineering substrates. Very little
is known about the behavior of nylon-3 materials in biological
systems, because until recently only limited functional variation
could be achieved within this polymer class. Our long-term goal
is to develop nylon-3 copolymers containing subunits that direct
self-assembly linked to subunits that convey information leading
to cellular attachment. The initial studies described below,
however, focus exclusively on the latter goal.
Several features of the nylon-3 family render these materials
attractive for biomaterials applications. First, the backbone is
inherently protein-mimetic, since the repeat unit differs from
that in proteins simply by the presence of an additional carbon
(Figure 1; proteins are composed of R-amino acid residues,
while nylon-3 polymers are comprised of ꢀ-amino acid resi-
dues). Second, nylon-3 materials are readily prepared via ring-
opening polymerization (ROP) of ꢀ-lactams.13 This controlled
polymerization provides access to block copolymers as well as
homopolymers and random copolymers. Third, recent develop-
ments in ꢀ-lactam synthesis enable considerable variation in
the side chain functionality that is presented by the nylon-3
chain. In particular, we have generated ꢀ-lactams that allow
incorporation of cationic subunits or polar but uncharged
subunits into the polymer chain.14,15 The limited exploration to
date of biological applications of nylon-3 polymers presumably
reflects the fact that most previous examples contained exclu-
sively hydrophobic subunits and displayed poor solubility.
This report documents a two-stage evaluation of nylon-3
materials as components of cell growth substrates. First, we use
two-dimensional polymer arrays immobilized on functionalized
glass to assess interactions between a small library of cationic
Materials and Methods
Materials. Chemicals, polyethylene glycol (Mn 3400, 202 444),
and chambered coverslips (C7735) were obtained from Sigma-
Aldrich; amine-functionalized glass (SMM2) was from ArrayIt,
collagen (PureCol) was from Inamed Biomaterials; Irgacure 2959
was from Ciba Specialty Chemicals; Dulbecco’s modified Eagle’s
medium (DMEM), cell culture supplements, LIVE/DEAD Viability/
Cytotoxicity Kit (L3224), Quant-iT PicoGreen dsDNA Assay Kit
(P7589), and NanoOrange Protein Quantitation Kit (N6666) were
from Invitrogen; syringe filter with 0.2 µm cellulose acetate
membrane (192-2520) was from Nalgene; round cover glass
(12-545-80) and M-PER lysis buffer (78501) were from Thermo
Fisher Scientific; and NIH 3T3 fibroblast cells were from the
American Type Tissue Collection (ATCC). NIH 3T3 cells were
stained using a LIVE/DEAD kit and scanned using a GeneTAC
UC 4 × 4 scanner (Genomic Solution). All scanned data were
analyzed using the GeneTAC quantitation program or GenePix Pro
6.0 demo version.
Synthetic Procedures for Monomers and PEG-Diacrylate.
ꢀ-Lactams, shown in Figure 2, were synthesized via previously
reported methods.14-18
(()-7-(2-Tritylthioacetyl)-7-azabicyhclo[4,2,0]octan-8-one (I).
To a stirred solution of tritylthioacetic acid (8.9 g, 26.8 mmol) in
dry CH2Cl2 (190 mL) was added dicyclohexylcarbodiimide (DCC)
(2.8 g, 13.4 mmol). After 2 h, the reaction mixture was filtered
through Celite to remove dicyclohexylurea, and the Celite pad was
rinsed with diethyl ether. The combined filtrate was concentrated
in vacuo. To a stirred solution of the concentrated residue and
ꢀ-lactam CH (1) (1.5 g, 12.1 mmol) in dry CH2Cl2 (40 mL) was
added 1 M lithium bis(trimethylsilyl)amide (LHMDS) in THF (12
mL). After 4 h, the reaction mixture was diluted with EtOAc and
washed with 1 N HCl, saturated aqueous NaHCO3, and then brine.
The organic layer was dried (MgSO4) and concentrated in vacuo.
The crude product was purified by silica column chromatography
(6:1 to 3:1 hexane/EtOAc containing 1% Et3N) to give I in 34%
yield as a solid: mp 156-159 °C; 1H NMR (300 MHz, CD3OD) δ
7.48-7.38 (m, 6 H), 7.34-7.16 (m, 9 H), 4.09-4.01 (m, 1 H),
3.49-3.36 (m, 2 H), 3.24-3.14 (m, 1 H), 2.06-1.93 (m, 1 H),
1.91-1.62 (m, 3 H), 1.59-1.38 (m, 4 H); 13C NMR (75 MHz,
CD3OD) δ 168.7, 166.6, 144.2, 129.7, 128.0, 126.9, 67.2, 49.8,
46.5, 36.8, 23.1, 19.4, 18.9, 16.9; HRMS (m/z, ESI) calcd for
C14H19NO4 (M+Na)+ 464.1655, found 464.1676.
PEG-diacrylate. To a stirred solution of polyethylene glycol (2
g, 0.59 mmol) in dry CH2Cl2 was added triethylamine (0.18 g, 1.77
mmol) and acryloyl chloride (0.16 g, 1.77 mmol) at 0 °C. After
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(13) Hashimoto, K. Prog. Polym. Sci. 2000, 25, 1411–1462.
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J. Am. Chem. Soc. 2009, 131, 1589–1597.
(16) Graf, R.; Lohaus, G.; Bo¨rner, K.; Schmidt, E.; Bestian, H. Angew.
Chem., Int. Ed. Engl. 1962, 1, 481–488.
(17) Dener, J.; Fantauzzi, P.; Kshirsagar, T.; Kelly, D.; Wolfe, A. Org.
Proc. Res. DeV. 2001, 5, 445–449.
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16780 J. AM. CHEM. SOC. VOL. 131, NO. 46, 2009