Click grafting of alkyne-containing vinyl polymers onto biosynthesized extracellular matrix protein containing azide functionality and adhesion control of human umbilical vein endothelial cells

Article abstract of DOI:10.1039/c5ra04643b

In vivo incorporation of a phenylalanine (Phe) analogue, p-azidophenylalanine (p-N₃Phe) into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket, in which the Ala294Gly/Thr251Gly mutant PheRS (PheRS) was expressed under the control of T7 promoters. In this study, biosynthesized aECM-CS5-ELF containing p-N₃Phe (aECM-CS5-ELF-N₃) was coupled with alkyne-containing vinyl polymers prepared via controlled radical polymerization of three vinyl monomers, (styrene, acrylamide, and N-isopropylacrylamide) using a trithiocarbonate as the RAFT agent. Grafting of the vinyl polymers onto the aECM was accomplished via a copper-catalyzed alkyne-azide click reaction. The lower critical transition temperature (LCST) was evaluated, as well as the solubility in aqueous and organic media, which are dependent on the incorporation ratio of p-N₃Phe and species of graft chains, in which the LCST behavior was altered remarkably when poly(N-isopropylacrylamide) moieties were attached as side chains. Circular dichroism measurements indicate conformational change was not induced by the grafting. Specific adhesion of human umbilical vein endothelial cells (HUVECs) onto the (aECM-CS5-ELF-N₃)-graft-poly(N-isopropylacrylamide) composite surface and subsequent temperature-sensitive detachment were also demonstrated.

Full text of DOI:10.1039/c5ra04643b

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PAPER
Click grafting of alkyne-containing vinyl polymers
onto biosynthesized extracellular matrix protein
containing azide functionality and adhesion control
of human umbilical vein endothelial cells†
Cite this: RSC Adv., 2015, 5, 41445
Tomoki Yamada and Akinori Takasu*
3
In vivo incorporation of a phenylalanine (Phe) analogue, p-azidophenylalanine (p-N Phe) into an artificial
extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that
harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket, in which the
Ala294Gly/Thr251Gly mutant PheRS (PheRS**) was expressed under the control of T7 promoters. In this
study, biosynthesized aECM-CS5-ELF containing p-N
containing vinyl polymers prepared via controlled radical polymerization of three vinyl monomers,
styrene, acrylamide, and N-isopropylacrylamide) using a trithiocarbonate as the RAFT agent. Grafting of
3 3
Phe (aECM-CS5-ELF-N ) was coupled with alkyne-
(
the vinyl polymers onto the aECM was accomplished via a copper-catalyzed alkyne–azide click reaction.
The lower critical transition temperature (LCST) was evaluated, as well as the solubility in aqueous and
3
organic media, which are dependent on the incorporation ratio of p-N Phe and species of graft chains,
in which the LCST behavior was altered remarkably when poly(N-isopropylacrylamide) moieties were
attached as side chains. Circular dichroism measurements indicate conformational change was not
induced by the grafting. Specific adhesion of human umbilical vein endothelial cells (HUVECs) onto the
Received 16th March 2015
Accepted 30th April 2015
DOI: 10.1039/c5ra04643b
3
(aECM-CS5-ELF-N )-graft-poly(N-isopropylacrylamide) composite surface and subsequent temperature-
www.rsc.org/advances
sensitive detachment were also demonstrated.
Either when bronectin coats a plate or is present in the
extracellular matrix, interaction with HUVEC are mediated by
Introduction
7
8,11
Tissue engineering has become a key therapeutic tool in the integrin receptors such as a5b1, and a4b1.
The sequence
treatment of damaged or diseased organs and tissues, such as motif REDV, found within the CS5 domain, is thought to be the
8
b
blood vessels (small-diameter vascular gra) and urinary blad- shortest sequence capable of a4b1 integrin binding. In Tir-
1,2
6c,6e
ders. The extracellular matrix (ECM), which includes protein rell's studies on aECM-CS5-ELF,
HUVEC adhesion and
ligands, such as elastin and bronectin, is an insoluble spreading were dependent of the presence of the CS5 domain of
macromolecular complex that forms an environmental bronectin. Nonetheless, major challenges still need to be
3
–5
substrate outside the cells of organs and tissues. The cell-to- overcome, in particular, the construction of tissues with high
ECM contact plays an important role in the regulation of bio- cell densities and the prevention of post-transplant inamma-
logical processes such as wound healing, organogenesis, and tion. Furthermore, the poor solubility and processability of
4,5
metastasis. Tirrell et al. have reported the results of studies on aECM-CS5-ELF is inhibiting the expansion of the range of
articially designed analogues of ECM proteins (aECMs) con- further applications, and must be addressed.
taining an elastin sequence and the bronectin cell binding
When designing new advanced biomaterials to be used in
6
domain (CS5), aECM-CS5-ELF. They also examined how these tissue engineering, facile chemical modication of cell–matrix
aECMs affect the adhesive behavior of human umbilical vein interactions is essential. One approach is incorporation of non-
6
9
endothelial cells (HUVECs). The elastin-like sequences give natural amino acids containing a reactive functional group into
6b,6d
these materials elasticity and mechanical integrity,
while an aECM for post-translational chemical modication while
cell adhesion and cell spreading on bronectin are maintained. maintaining the structural integrity of the protein. For example,
incorporation of phenylalanine (Phe) analogues [including
3
p-N Phe, p-bromophenylalanine (p-BrPhe), and p-iodophenyla-
Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of
Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan. E-mail: takasu.akinori@ lanine (p-IPhe) having an orthogonal functional group] into a
nitech.ac.jp; Fax: +81-52-735-5342; Tel: +81-52-735-7159
recombinant protein enables chemical modication via “click”
reactions, Heck reactions, or Sonogashira couplings, and has
1
†
Electronic supplementary information (ESI) available: H NMR, UV, MALDI-TOF
mass, and IR spectra. See DOI: 10.1039/c5ra04643b
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Fig. 1 Incorporation of p-azido-L-phenylalanine into the aECM-CS5-ELF-F protein and coupling with vinyl polymers prepared via RAFT radical
polymerization of vinyl monomers.
been accomplished using a bacterial expression host that In the present study, biosynthesized aECM-CS5-ELF containing
harbors a mutated phenylalanyl-tRNA synthetase (PheRS) with p-N
an enlarged binding pocket.
3
Phe (aECM-CS5-ELF-N
3
) was prepared using PheRS** and a
Tirrell et al. previously identi- T7 promoter. Next, alkyne-containing trithiocarbonate was
ed mutations which enlarge the Phe binding cavity of E. coli generated for use as the RAFT agent for controlled radical
10,11
11

10a
10b
PheRS, Ala294Gly and Thr251Gly, and produced the cor- polymerization of three vinyl monomers: styrene, acrylamide,
10a
10b
responding Ala294Gly (PheRS*) and Thr251Gly/Ala294Gly
and N-isopropylacrylamide. Hybridization of the aECM with
(
PheRS**) mutants, respectively. The Thr251Gly/Ala294Gly vinyl polymers is accomplished via a copper-catalyzed alkyne–
18
double mutant, PheRS**, effectively incorporates even p-ace- azide click reaction based on a “graing onto” procedure
tylphenylalanine (p-AcPhe) into mouse dihydrofolate reductase (Fig. 1). The lower critical transition temperature (LCST) was
when the T5 system is used. Although the amount of material evaluated, as well as the solubility in aqueous and organic
10b
produced is insufficient due to the moderate strength of the T5 media, which is dependent on the incorporation ratio of
promoter, we previously reported that this limitation can be p-N
3
Phe as well as the length and type of gra chains. Notably,
overcome using a combination of T7 promoter and T7 RNA the LCST behavior was altered signicantly when PNIPAM was
11
polymerase. The T7 RNA polymerase has greater activity and is attached as the side chains. From the circular dichroism
12–14
more selective than the endogenous E. coli RNA polymerase.
measurements, a conformational change (ex. from random coil
The T7 expression system (pET system) is capable of producing to a-helix) was investigated before and aer the graing.
ꢀ
1
large amounts of protein (up to 100 mg L
of culture Specic adhesion of HUVECs onto the aECM-CS5-ELF/vinyl
11
medium). We also reported PEGylation of aECM using a click polymers composite surface was also demonstrated.
strategy, and showed that the increased hydrophilicity upon
PEGylation of the elastin domain results in lowered non-specic
Experimental section
Materials
adhesion of human umbilical vein endothelial cells
11
(
HUVECs). We hypothesize that in order to control the
hydrophilicity of the elastin-type sequences within the aECM by p-N
Phe was obtained from Bachem AG (Hauptstrasse 114/4416
3
conjugation of the vinyl polymers, poly(acrylamide) (PAAm) and Bubendorf, Switzerland). The twenty natural amino acids
poly(N-isopropylacrylamide) (PNIPAM), further advanced and 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid
biomaterials should be provided. Particularly, these new mate- (DDMAT) were obtained from Aldrich. The cell culture medium
rials can be utilized in a promising tissue engineering approach and trypsin/EDTA solution were obtained from Lonza. Crystal
relying on the use of cell culture surfaces graed to PNIPAM, violet was obtained from Wako. The GREDVY peptide was
which is based on the idea that cell adhesion/detachment on prepared via Fmoc solid-phase peptide synthesis in our labo-
PNIPAM-modied substrates can be achieved via a simple ratory and characterized by matrix-assisted laser desorption/
1
5
temperature switch, the LCST.
ionization time-of-ight (MALDI-TOF) mass spectroscopy
+
Recently, chemical modication of several proteins via (H
graing from procedure has been studied using the reversible
N-GREDVY-COOH + Na : m/z ¼ 762.2).
2
addition fragmentation chain transfer (RAFT) radical polymer- Measurements
1
6
ization technique. However, until now, few reports dealing
1
ꢁ
The H NMR spectra of the aECMs were acquired at 27 C using
a Bruker DPX200 spectrometer (200 MHz) or a Bruker DPX-600
17
with graing onto procedure was reported, as far as we know.
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Scheme 1 Preparation of alkyne-containing RAFT agent 1.
(
600 MHz) spectrometer (Bruker Japan, Japan). The number- (ncIts857 ind 1 Sam7 nin5 lacUV5-T7 gene 1) pheA], was used as
average molecular weight (M and polydispersity index the expression host. The AF strain was transformed with the
/M ) of the polymers were estimated by size-exclusion repressor plasmid pLysS-IQ and with pET28-CS5-ELF-PheRS** to
n
)
(M
w
n
chromatography (SEC) using a Tosoh DP8020 pump system, a afford the expression strain AF-IQ[pET28-CS5-ELF-PheRS**]. We
refractive index (RI) detector (Tosoh RI-8020), and a TSKgel have already reported the in vivo translational activities of the Phe
3
SuperMultiporeHZ-M column (eluent, chloroform; ow rate, analogues, (p-N Phe, p-BrPhe, p-IPhe, p-ethynylphenylalanine,
ꢀ
1
ꢁ
11
0
(
.35 mL min ; temperature, 40 C; Tosoh Corp.) using poly- p-cyanophenylalanine, and p-AcPhe). M9 minimal medium (1 L)
ꢀ
1
styrene)s as the calibration standard. MALDI-TOF mass spectra supplemented with 0.4% glucose, 35 mg L thiamine, 0.1 mM
were measured with a Voyager RN using dithranol as a matrix MgSO , 0.1 mM CaCl , the 20 naturally occurring amino acids
4
2
ꢀ
1
ꢀ1
reagent. NaI was used as the cationization salt to generate (at 40 mg L each), and antibiotics (kanamycin 25 mg L
,
+
ꢀ1
sodium-cationized ions ([M + Na] ). The purity and molecular chloramphenicol 20 mg L ) was inoculated with 300 mL of
weight of the puried aECM-CS5-ELF-N were conrmed using culture containing the expression strain. When the optical
sodium dodecyl sulfate-polyacrylamide gel electrophoresis density at 600 nm reached 0.7–1.0, a medium shi was per-
3
(
SDS-PAGE). FT IR spectra (KBr disc) were acquired using a formed. The cells were sedimented via centrifugation for 7 min at
ꢁ
JASCO FTIR-430 spectrometer.
20 100 ꢂ g and 4 C, and the supernatant liquid was removed.
The cells were resuspended in supplemented M9 medium de-
cient in Phe and Trp. Protein expression was induced aer the
medium shi using isopropyl-b-D-1-thiogalactopyranoside (IPTG)
Preparation of alkyne-containing RAFT Agent
19
Following the procedure reported by Brittain et al.,
to a nal concentration of 1 mM. Ten minutes later, a Phe
DDMAT (0.5 g, 1.37 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC, 0.39 g, 2.0 mmol), 4-dimethylaminopyr-
idine (DMAP, 0.25 g, 2.0 mmol), and 5 mL of dichloromethane
were added to a round-bottomed ask and stirred for a few
minutes under a nitrogen atmosphere (Scheme 1). Into the
mixture, 0.31 mL (4.11 mmol) of 3-butyn-1-ol was added, and
stirred overnight at room temperature. Aer diluting 30 mL of
dichloromethane in a round-bottomed ask, the product was
washed with 30 mL of aqueous HCl (0.1 M), 30 mL of water, and
ꢀ1
11
analogue was added at a concentration of 250 mg L
. The cells
were cultured for four hours post-induction, and protein
expression was monitored via SDS-polyacrylamide gel electro-
phoresis (PAGE) using a normalized OD600 of 0.5 per sample.
Protein purication (for 1 L of culture)
The purication scheme, which takes advantage of the inverse
temperature transitions of aECMs, has been reported else-
3
0 mL of 10% aqueous NaCl three times, and dried under
6b,11
where.
Briey, the wet cell mass was dispersed in TEN buffer
reduced pressure. The expected product 1 was obtained as a
yellow syrup (0.49 g, 85% yield) without further purication.
1
H NMR (200 MHz, CDCl
CH ), 1.65 (6H, s, C(CH
.53 (2H, dt, CH C^C), 3.28 (2H, t, –CH
3
, d, ppm): 0.87 (3H, t, CH
), 1.97 (1H, t, HC^C),
S), 4.2 (2H, t, OCH C).
2 3
CH ), 1.2–1.4
(20H, m, CH
2
2
3 2
)
2
2
2
2
Plasmid construction
The incorporated polylinker encodes a T7-tag, a hexahistidine
tag (His tag), and an enterokinase cleavage site (Fig. 1). This
linker was cloned into a pET28 plasmid between the Nco I and
10c
Xho I sites. The pET28-CS5-ELF-PheRS* plasmid was used as a
template for a polymerase chain reaction (PCR) mutagenesis to
generate the coding sequence for PheRS**. This new plasmid
11
was designated pET28-CS5-ELF-PheRS**.
Incorporation of p-N Phe into the aECM-CS5-ELF
3
1
Fig. 2 Expanded H NMR spectra (in DMSO-d
6
at ambient tempera-
using PheRS** (red line)
as the mutated phenylalanyl-tRNA synthetases and aECM-CS5-ELF-F
Buffer and media were prepared according to standard protocols.
A phenylalanine auxotrophic derivative of E. coli BL21(DE3),
ture, 600 MHz) of purified aECM-CS5-ELF-N
3
constructed in our laboratory and designated AF [HsdS gal (blue line).
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ꢀ
1
ꢁ
ꢀ1
at a concentration of 1 g mL , frozen, and defrosted at 4 C with molecular weight (M
n
¼ 4800 g mol ) and polydispersity (1.6).
ꢀ
1
ꢀ1
1
addition of 1.0 mg mL
DNase, 10 ng mL
RNase, and
H NMR (200 MHz, CDCl , d, ppm): 0.88 (u-terminus, t,
3
ꢀ
1
5
0 ng mL phenylmethylsulfonyl uoride (PMSF). The bio- CH CH ), 1.14 (6H, br, NHCH(CH ) ), 1.25 (u-terminus, m,
2
3
3 2
synthesized aECMs, aECM-CS5-ELF-N s, which were partitioned CH ), 1.65 (2H, br, CH CH), 2.09 (1H, br, CH CH), 2.54
3
2
2
2
into the pellet of the whole-cell lysate aer centrifugation (a-terminus, br, CH
2
C^C), 3.33 (u-terminus, t, –CH
2
SC]S),
ꢁ
(
20 100 ꢂ g, 60 min, 37 C), were resuspended in 4 M urea. The 4.01 (1H, br, NCH), 4.16 (a-terminus, br, COOCH
2
), 5.78–6.94
ꢁ
solutions were centrifuged (20 100 ꢂ g, 60 min, 4 C) to remove (br, NH).
non-protein cellular debris and then dialyzed against water
ꢁ
(3 days, 4 C) using cellulose tubing (cut off 14 000). The
Preparation of tris-(3-hydroxypropyltriazolylmethyl)amine
precipitates were removed via centrifugation (20 100 ꢂ g, 60 min,
(
THPTA, ligand for click reaction)
ꢁ
2
C), and the claried supernatant were then lyophilized. The
Synthesis of 3-azido-1-propanol. Sodium azide (1.43 g,
2.0 mmol) was added to a solution of 3-bromo-1-propanol
purity of the proteins and the uniformity of their molecular
weights were conrmed via SDS-PAGE. The relative amounts of
2
(1.54 g, 11.1 mmol) in DMF (6 mL) and stirred at room
3
p-N Phe analogues incorporated were derived from the areas of
temperature for 48 hours. The mixture was diluted with 30 mL
of CH Cl and washed 5 times with 30 mL of 10% aqueous NaCl.
1
the 600 MHz H NMR spectra aromatic peaks (Fig. 2).
2
2
The organic layer was dried, ltered, and concentrated to give
RAFT radical polymerization of styrene using RAFT agent 1
1
the product. Yield: 57%. H NMR (CDCl
.6 Hz), 3.45 (t, 2H, 6.8 Hz), 2.55 (br, 1H), 1.83 (m, 2H).
Synthesis of THPTA. Tripropargylamine (0.13 g, 1 mmol) in
3
) d (ppm): 3.73 (t, 2H,
19
Following the reported procedure, styrene (1.385 g, 13 mmol),
5
0
RAFT agent 1 (125 mg, 0.3 mmol), a,a -azoisobutyronitrile
(
AIBN, 3 mg, 0.018 mmol) and 1.5 mL of toluene were added to a
an acetonitrile/methanol solution (3 mL) was treated sequen-
round-bottom ask. Aer degassing, the reaction mixture was
stirred at 90 C for 18 hours under an inert atmosphere. The
reaction was stopped by quenching in an ice bath. Polystyrene
tially with 3-azido-1-propanol (0.4 g, 4 mmol), 2,6-lutidine
ꢁ
ꢀ2
(
0.11 g, 1 mmol), and Cu(I)Br (14.3 mg, 1 ꢂ 10 mmol). Upon
addition of the copper salt, the reaction was cooled in an ice
(
PSt) was obtained aer precipitation in cold methanol. SEC was
bath. Aer the mixture was stirred at room temperature for
ꢀ
1
used to calculate molecular weight (M
n
¼ 1700 g mol ) and
3
days, the reaction mixture was evaporated under reduced
1
polydispersity (1.2). (0.55 g, 36% yield). H NMR (200 MHz,
CDCl , d, ppm): 0.88 (u-terminus, t, CH CH ), 1.26 (u-terminus,
m, CH
1
3
pressure and dissolved in methanol. The crude product was
1
3
2
3
precipitated in acetonitrile. Yield: 71%. H NMR (DMSO-d
ppm): 8.03 (s, 3H), 4.66 (t, 3H, 4.9 Hz), 4.44 (t, 6H, 6.8 Hz), 3.62
) d
6
2
), 1.5 (a-terminus, s, C(CH
3
)
2
2
), 1.3–1.7 (2H, br, CH CH),
(
.7–2.0 (1H, br, CH CH), 2.20 (a-terminus, s, CH C^CH),
2
2
(s, 6H), 3.40 (q, 6H, 5.4 Hz), 1.97 (m, 6H) (see also Fig. S1†).
.25 (u-terminus, t, –CH SC]S), 6.31–6.87 (br, aromatic
2
protons), 6.89–7.23 (br, aromatic protons).
Graing of poly(acrylamide) onto aECM-CS5-ELF
ꢀ
4
aECM-CS5-ELF-N
(
3
ꢀ
[10 mg, 2.5 ꢂ 10 (azido unit) mmol], PAAm
RAFT radical polymerization of acrylamide using RAFT agent 1
3
ꢀ2
12 mg, 3.8 ꢂ 10 mmol), THPTA (14.3 mg, 3.3 ꢂ 10 mmol),
19
Following the reported procedure, RAFT agent 1 (10 mg,
.024 mmol), acrylamide (100 mg, 1.4 mmol), 1 mL of dime-
ꢀ3
Cu(I)Br (1.43 mg, 1 ꢂ 10 mmol) and 4 mL of phosphate buffer
0
(
pH 7.4) were added to a round-bottom ask and stirred at room
thylsulfoxide (DMSO), and AIBN (0.13 mg, 0.014 mmol) were
temperature for 4 days under an inert atmosphere. The aqueous
solution was dialyzed water (3 days, 4 C) and lyophilized. IR
2964 (nC–H), 1665 [nC]O (amideI)], 1542
dNH (amideII)], 1455, 1418 (dC–H).
combined in a Schlenk ask. Aer degassing, the reaction
ꢁ
ꢁ
mixture was heated to 70 C for 2 days under N
2
. The poly-
ꢀ1
(KBr) cm
:
merization was quenched by cooling the ask in an ice bath,
and PAAm was obtained aer precipitation in cold methanol.
[
1
The molecular weight of PAAm was calculated via H NMR
LCST measurements
ꢀ
1
1
spectroscopy (91 mg, 82% yield, M ¼ 3200 g mol ). H NMR
n
The LCST values were measured using aECM aqueous solutions
1 mg/1 mL). The temperature was increased at a rate of 1 C
min while the percent transmission at 350 nm of the solution
was measured using a V-550 spectrometer (JASCO, Tokyo,
Japan).
(
200 MHz, D O, d, ppm): 0.57 (u-terminus, t, CH CH ), 0.76,
2
2
3
ꢁ
(
1
.14 (u-terminus, br, CH
2
CH
2
), 1.62 (2H, br, CH
2
CH), 2.15
ꢀ
1
(
1H, br, CH
2
CH), 2.53 (a-terminus, br, CH
2
C^C), 2.86 (u-
).
terminus, t, –CH
2
S), 4.11 (a-terminus, br, COOCH
2
RAFT radical polymerization of N-isopropyl acrylamide using
RAFT agent 1
Coating of aECM-CS5-ELF-N
3
having PAAm and PNIPAM side
chains onto a commercially-available cultivation dish
RAFT agent 1, (8.3 mg, 0.02 mmol), N-isopropylacrylamide
(
(
56.6 mg, 0.5 mmol), 1 mL of tetrahydrofuran (THF), and AIBN Physical mixtures of aECM-CS5-ELF-N
3
/(aECM-CS5-ELF-N
3
)-
1.6 mg, 0.01 mmol) were mixed in a Schlenk ask. Aer gra-PAAm (2 mg/2 mg) and aECM-CS5-ELF-N
/(aECM-CS5-
3
ꢁ
degassing, the reaction mixture was heated to 60 C for 2 days ELF-N )-gra-PNIPAM (2 mg/2 mg) were dissolved in water
3
under N . The polymerization was quenched by cooling the (1 mL), respectively. Small amounts of the solutions (80 mL)
2

ask in an ice bath, and PNIPAM was obtained (65 mg, 24% were dropped and spread onto 40 mm ꢂ 15 mm Petri dishes.
ꢁ
yield) aer precipitation in n-hexane. SEC was used to calculate The dishes were dried at 40 C for 4 h. The dried dishes were
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irradiated using an ASAHI SPECTRA MAX-303 (ASAHI SPECTRA, pET-CS5-ELF-PheRS** to generate the expression system AF-IQ
Tokyo, Japan) (l ¼ 250–400 nm) for 30 min to induce cross- [pET-CS5-ELF-PheRS**]. Expression of aECM-CS5-ELF-N3 was
linking via nitrene chemistry of azido group. The dishes were under the control of an inducible T7 promoter. The expression
immersed in cold 8 M urea buffer for 3 min and rinsed with 10% system was induced with IPTG and cultivated for an additional
aqueous NaCl. Then, they were immersed in ethanol and dried four hours to allow for the biosynthesis of aECM-CS5-ELF-N
3
.
ꢁ
at 40 C for 1 hour.
The whole cell lysates and puried aECM proteins were
analyzed via SDS-PAGE, and the gels show a signicant amount
of aECM protein with a calculated molecular weight of 42.6 kDa,
the expected aECM proteins were biosynthesized. Using
Cell culture
The HUVECs (Lonza Walkersville, Inc.) were maintained in a
ꢀ1
PheRS**, p-N Phe (250 mg L culture) was readily incorporated
ꢁ
3
3
7 C, 5% CO , humidied environmental chamber. The cells
2
into aECM-CS5-ELF, and in most cases produced more than
were grown in endothelial growth medium-2 (EGM-2, 2% FBS)
Lonza Walkersville), which was replaced every 2 days. Near
conuent HUVEC cultures were passaged by treatment with
.025% trypsin/0.01% EDTA (Lonza Walkersville).
1
1
5
0 mg of the target proteins from 1 L culture. Analysis of the
(
1
H NMR spectrum of aECM-CS5-ELF-N
poration level of pN Phe was 67% when PheRS** was used
Fig. 2, red line). This incorporation level is derived from the
ratio of the area of the aromatic signal at 7.20 ppm ascribed to
Phe and the signal at 6.98 ppm (d, 8.1 Hz) ascribed to p-N Phe.
3
shows that the incor-
3
0
(
Cell spreading
3
HUVECs in EGM-2 were added to 40 mm ꢂ 15 mm Petri dishes
at a concentration of 5000 cells per Petri dish. Petri dish was
made of polystyrene, we used aer washing with ethanol. Aer Alkyne-containing vinyl polymers prepared via RAFT radical
days, the plates were removed from the environmental polymerization technique
3
chamber and the cells were imaged using a 10ꢂ phase contrast
objective on a Nikon Eclipse TS100/TS100-F epiuorescence
microscope (Tokyo, Japan).
DDMAT was esteried using 3-butyn-1-ol to afford the expected
alkyne-containing trithiocarbonate RAFT agent 1 for use in the
controlled radical polymerization of vinyl monomers
(
Scheme 1). Trithiocarbamates are also known to be effective
Cell resistance to detachment
RAFT agents for mediating the polymerization of conjugated
16,17
HUVECs incubated for 3 days in EGM-2 were added to a Petri vinyl monomers.
The results of the RAFT radical polymeri-
dish. A solution of the GREDVY peptide in phosphate buffered zations of styrene are summarized in Table 1. The trithio-
saline (PBS) (1.5 mM) was added. Aer 30 min of incubation at carbamate 1 seemed to induce reversible chain transfer
ꢁ
3
7 C and 5% CO , non-adhered cells were removed by inver- reactions. When styrene was used as the vinyl monomer, the
2
sion of the plate and rinsing with PBS. The cells were stained prepared trithiocarbamate 1, was effective for the control of
with crystal violet, and thoroughly rinsed with water. The dye molecular weight (runs 1–3). In run 1, the molecular weight
3
3
was solubilized using a 1% SDS solution. The absorbance was (M
measured at 595 nm with a Jasco J820K spectropolarimeter. At and the molecular weight distribution (M
least three independent experiments were carried out in order runs 2 and 3, the feed monomer ratio was increased
n
¼ 4.2 ꢂ 10 ) was similar to the calculated value (5.2 ꢂ 10 ),
w
/M ) was only 1.3. In
n
to verify reproducibility.
([M]
0
/[1]
0
/[AIBN]
0
¼ 300/8/1 and 700/8/1) and the molecular
3 3
weights increased to M
n
¼ 11.0 ꢂ 10 and 24.0 ꢂ 10 , respec-
tively. When the monomer was changed to acrylamide (run 4)
and N-isopropylacrylamide (run 5), the expected a-alkynyl
PAAm and PNIPAM were prepared, respectively.
Results and discussion
Incorporation of pN
3
Phe into aECM-CS5-ELF-F
Incorporation of p-N
3
Phe into a protein can be used to fabricate
The MALDI-TOF mass spectrum of the poly(styrene)
a chemoselective access point for alkyne-initiated vinyl poly- produced by RAFT radical polymerization using the trithio-
mers prepared via RAFT radical polymerization. For this work, carbamate 1 as the RAFT agent was used to determine the
the phenylalanine auxotrophic E. coli strain (AF) carrying the absolute molecular weights and the structure of the poly-
repressor plasmid pLysS-IQ, AF-IQ, was transformed with (styrene) (Fig. 3). In the spectrum, there is one set of peaks with
Table 1 RAFT radical polymerization of vinyl monomers using trithiocarbamate 1
a
b
Monomer/CTA/AIBN
(molar ratio)
Temperature
( C)
Time
(h)
M
(ꢂ 10
n(calculated)
M
n
Conv.
(%)
ꢁ
ꢀ3
ꢀ3
a
Run
Vinyl monomers
)
(ꢂ 10
)
w n
M /M
1
2
3
4
5
Styrene
Styrene
Styrene
Acrylamide
N-Isopropylacrylamide
100/8/1
300/8/1
700/8/1
100/2/1
100/2/1
90
90
90
70
70
20
40
48
48
24
5.2
4.2
1.3
1.3
1.4
—
75
54
72
83
76
15.6
36.5
3.6
11.0
24.0
3.6
5.7
4.8
1.7
a
b
1
3
SEC measurements in CHCl relative to poly(styrene) standards; before reprecipitation. Percent conversion determined by H NMR spectroscopy.
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Fig. 3 Expanded MALDI-TOF mass spectrum of alkyne-containing PSt.
18
a repeat of 104.3 m/z, which corresponds to the molecular were coupled, with Cu(I)Br as the catalyst (Scheme 2). The click
weight of the styrene unit. When the peak at 1664 was exam- reaction proceeds in DMSO at room temperature for 3 days,
ined, it was found to coincide with the calculated peak for 1665 affording the expected product in high yield (90–99%). The
[
styrene (104 Da) ꢂ 12 + a-(R-) and u-[Z-(C]S)S] terminal results are summarized in Table 2. Aer the PSt was graed onto
groups (417 Da) ¼ 1665]. This suggests that the RAFT radical the aECM-CS5-ELF-N , the solubility was altered, and the
polymerizations were initiated by a fragmented R group from protein-based gra copolymer was soluble in DMF, DMSO, and
the trithiocarbamate 1; the pattern for a product initiated by even in chloroform (aECM-CS5-ELF-F and aECM-CS5-ELF-N are
not soluble in chloroform). In the H NMR spectrum of (aECM-
CS5-ELF-N )-gra-PSt in CDCl (Fig. 4), the signals ascribed to
3
3
1
AIBN was not observed.
3
3
the aECM-CS5-ELF-N3 backbone are clearly visible, although
these signals were not detected in the spectrum of aECM-CS5-
Graing polystyrene onto aECM-CS5-ELF
ELF-N
3
(data not shown). The modied solubility in CDCl
3
also
To demonstrate a graing onto aECM protein, aECM-CS5-ELF-
supports the expected graing onto the aECM-CS5-N
3
protein.
3
N (67% incorporation) and a-alkynyl PSt (alkene at a terminus)
Scheme 2 Grafting of alkyne-containing vinyl polymers prepared via RAFT radical polymerization onto extracellular matrix protein containing
azide functionality (aECM-CS5-ELF-N ).
3
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a
3
Table 2 Grafting of vinyl polymers onto aECM-CS5-ELF-N
c
d
Vinyl polymer
[Vinyl polymer]
(molar ratio)
0
/[p-N
3
Phe]
0
Temperature
( C)
Time
(day)
Yield
(%)
Conv.
(%)
b
ꢀ3
ꢁ
Run
(M
n
ꢂ 10
)
Solvent
1
2
3
PSt (11.0)
PAAm (3.2)
PNIPAM (4.8)
20/1
10/1
15/1
DMSO
PBS
PBS
27
27
27
4
4
4
99
99
99
>99
>99
>99
a
b
aECM-CS5-ELF-N
3
(67% incorporation), in PBS for 4 days. SEC measurements in CHCl relative to poly(styrene) standards; before
3
c
d
reprecipitation. Calculated based on aECM-CS5-ELF-N
3
backbone was quantitative (90–99%). Percent conversion estimated by FT-IR
spectroscopy.
3
Aer graing of PSt onto aECM-CS5-N , the thermal prop-
erties were also changed (Fig. 5). In DSC measurements of the
protein-based gra copolymer, the heat capacity has an inec-
ꢁ
tion point ascribed to the T at 84 C. On the other hand, the
g
ꢁ
alkyne-containing PSt (green line) has a Tg at 62 C. These
results also indicate that the expected (aECM-CS5-ELF-N )-gra-
3
poly(styrene) was synthesized.
Graing of poly(acrylamide) onto aECM-CS5-ELF
In order to demonstrate graing of hydrophilic vinyl polymer,
aECM-CS5-ELF-N (67% incorporation) was also coupled with
3
a-alkynyl PAAm (alkyne at a terminus) using Cu(I)Br and THPTA
3
Fig. 5 DSC trace of (aECM-CS5-ELF-N )-graft-PSt (red line), alkyne-
containing PSt (green line), and aECM-CS5-ELF-N
3
(black line) (heating
ꢁ
rate: 10 C min).
as the catalyst and ligand, respectively (Table 2). The click reac-
tion proceeds in PBS at room temperature for 4 days, also
affording the expected product in excellent coupling efficiencies that (aECM-CS5-ELF-N )-gra-PAAm does not show electropho-
3
(
99%). The graing was conrmed by disappearance of IR resis. We speculated that it was due to interaction of PAAm gras
ꢀ
1
absorbance at 2118 cm ascribed to azido group. Subsequently, and electrophoretic gel matrix for SDS-PAGE (made of PAAm).
the aminolysis was performed using n-propylamine to remove From these results, it was concluded that (aECM-CS5-ELF-N )-
the long alkyl chain (C12
25SC]S–), in which UV absorbance gra-poly(acrylamide) was successfully prepared.
due to C12
The goal was to produce a protein with an acceptable LCST in
3
H
H
25SC]S– (l ¼ 305 nm) disappeared (Fig. S2†). In
order to cross-check the coupling, both the puried (aECM-CS5- order to produce aECM with reversible adhesion. A property of
ELF-N )-gra-PAAm and aECM-CS5-ELF-N3 proteins were elastin-like polypeptides which distinguishes them from other
3
analyzed via SDS-PAGE (Fig. 6). The gels show a signicant polypeptides is that they are miscible with water at all concen-
amount of aECM-CS5-ELF-N
ular weight of 42.6 kDa. On the other hand, in the SDS-PAGE of Furthermore, this miscibility behavior is reversible. For aECM-
protein with a calculated molec- trations below the lower critical solution temperature (LCST).
3
11
ꢀ
1
puried (aECM-CS5-ELF-N
3
)-gra-PAAm, no bands ascribed to CS5-ELF-N
3
(2 mg mL ; Fig. 7, green line), the LCST value is
ꢁ
proteins (including the parent protein) were observed, indicating 36 C. Aer graing of PAAm onto aECM-CS5-ELF-N
3
, no LCST
1
ꢁ
3 3
Fig. 4 H NMR spectrum of (aECM-CS5-ELF-N )-graft-PSt (red line) and alkyne-containing PSt (green line) (200 MHz, CDCl 27 C).
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appears to be an increased amount of a-helices (41 and 43%,
respectively) (as demonstrated by the presence of signals at
ꢁ
2
06 nm and 222 nm) at low temperature (20 C) in comparison
11
with aECM-CS5-ELF-F. In aECM-CS5-ELF-N , the random coil
3
ꢁ
signal (43% at 20 C) is remarkably decreased (17%) as the
temperature increases to 60 C (Fig. 8a), and thus, the CD
ꢁ
spectra no longer resemble proteins having random coil/b-turn
11
secondary structures. Instead, the proportion of b-turns
increased to 47%, inducing LCST behavior (Fig. 8a). Aer
graing (Fig. 8b), the spectrum was similar to that of
aECM-CS5-ELF-N3 and showed double minima at 204 and
2
22 nm, indicating an increased proportion of a-helices (43%)
ꢁ
at 20 C. However, the spectra were unaffected by temperature
(Fig. 8b), in that a high proportion of random coils (42%) and
a-helices (42%) and a low proportion of b-turns (16%) persist
ꢁ
even at 60 C. These results indicate that the introduction of
Fig. 6 SDS-PAGE of aECM-CS5-ELF-N
3
(middle) and (aECM-CS5- PAAm side chains does not alter the secondary structure of the
3
ELF-N )-graft-PAAm (right). Left lane is marker.
parent protein (aECM-CS5-ELF-N ) and the disappearance of
3
the LCST response is ascribed not to the conformation of the
backbone but the increased hydrophilicity ascribed to the
behavior was observed (Fig. 7, blue line). The secondary struc- PAAm side chains.
tures of elastin-like polypeptides are characterized by a high
proportion of random coils and a low proportion of b-turns at
low temperatures. The fraction of the secondary structure was
Graing poly(N-isopropylacrylamide) onto aECM-CS5-ELF
calculated by a curve-tting method using a linear combination The ultimate goal is the temperature-dependent detachment of
of typical CD spectra for a-helical, b-sheet and random coil cultivated and spread HUVECs, because the (aECM-CS5-ELF-
20
conformations. This distribution of random coils and b-turns N )-gra-PNIPAM has LCST as well as a cell binding site. Okano
3
11
gradually inverts with increasing temperature. These struc- et al. already reported the temperature-dependent detachment
tural features are reected in the CD spectra of aECM-CF5-ELF-F of cultivated and spread cells using a PNIPAM surface, in which
at low temperatures by a distinct minimum at 197 nm and a less the hydration/dehydration of the PNIPAM segment switched the
1
5
pronounced minimum at 218 nm, which are associated with the detachment/adhesion.
presence of random coils (75%) and b-turns (25%), respec- described above, graing onto aECM-CS5-ELF-N3 was per-
Following the similar procedure
11
ꢁ
tively. As expected, with increasing temperature to 60 C, the formed to afford the expected (aECM-CS5-ELF-N )-gra-PNI-
3
random coil signal becomes less pronounced (50%), while that PAM. Similar to (aECM-CS5-ELF-N )-gra-PAAm, the graing
3
ꢀ1
of the b-turn increases (50%), indicating a transition from a less was conrmed by disappearance of IR absorbance at 2118 cm
11
ordered to a more ordered secondary structure state.
The CD spectra of the two aECMs, aECM-CS5-ELF-N3 performed using n-propylamine to remove the long alkyl chain
Fig. 8a) and (aECM-CS5-ELF-N )-gra-PAAm (Fig. 8b) demon- (C H SC]S–), in which UV absorbance due to C H SC]S–
ascribed to azido group and successively the aminolysis was
(
3
1
2
25
12 25
strate that they retain the characteristics associated with (l ¼ 305 nm) disappeared (Fig. S2†). Aer graing, (aECM-CS5-
ꢁ
proteins having mixed random coil (43 and 47%, respectively)/ ELF-N )-gra-PNIPAM showed LCST behavior at 22 C ascribed
3
b-turns (16 and 10%, respectively), although in both cases there to PNIPAM segments (Fig. 7, please see also Fig. S3†), while
(
(
aECM-CS5-ELF-N
aECM-CS5-ELF-N
3
3
)-gra-PAAM did not (Fig. 7). The structure of
1
)-gra-PNIPAM was also conrmed by
H
NMR in DMSO-d (Fig. S4†).
6
3
Photo cross-linking of (aECM-CS5-ELF-N )-gra-PAAm
As (aECM-CS5-ELF-N
functionality because the click graing was completed, the
coated lm (a physical mixture of aECM-CS5-ELF-N and
aECM-CS5-ELF-N
3
)-gra-PAAm has no remaining azide
3
(
3
)-gra-PAAm) was irradiated (l ¼ 250–400
nm) to demonstrate photo cross-linking to afford the coated
blend lm. Aer UV irradiation, the coated lm was not soluble
in organic or aqueous media. Photo cross-linking of a physical
Fig.
7
LCST measurements on aECM-CS5-ELF-N
3
(green, 67%
mixture of aECM-CS5-ELF-N3 and (aECM-CS5-ELF-N )-gra-
PNIPAM was also performed and similar coated lm was
obtained.
3
incorporation), (aECM-CS5-ELF-N
blue), and (aECM-CS5-ELF-N
LCST ¼ 23 C) at concentrations of 1 mg mL in H
3
)-graft-PAAm (after aminolysis,
3
)-graft-PNIPAM (after aminolysis, red
ꢁ
ꢀ1
2
O.
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ꢀ1
ꢀ1
Fig. 8 CD spectra of aECM-CS5-ELF-N
3
(a), and (aECM-CS5-ELF-N
3
)-graft-PAAm (b) at a concentration of 0.01 mg mL (a) and 0.05 mg mL
(
2
b) in H O.
Cell spreading
(Nunc, Inc.), Fig. 9b], the percentage of well spread cells on
6
b,6c,11
cross-linked (aECM-CS5-ELF-N
3
)-gra-PAAm (>99% graing)
According to previous papers,
cell spreading on cross-
(Fig. 9d) was signicantly higher at all times, indicating that the
linked aECM lm is dependent on the presence of REDV
domains. HUVECs plated on cross-linked aECM-CS5-ELF-N3/
aECM-CS5-ELF-N )-gra-PAAm blend lms were monitored at
day intervals by phase contrast microscopy and were found to
be dark (spread) (Fig. 9). The number of adhered cells was
actually counted using UV measurements. Aer microscopic
observation, the cells were stained with 0.5% aqueous crystal
for 10 min in order to quantify the adhered cells.
Following the reported procedure,
REDV segment acts as a cell-binding segment. On the other
hand, (aECM-CS5-ELF-N )-gra-PNIPAM, prepared via click
3
(
3
reaction, also showed adhesion of HUVECs (Fig. 9e), but
introduction of the PNIPAM content resulted in a signicant
reduction in the extent of cell spreading (Fig. 9e). We speculated
that extent of cell spreading upon graing of PAAm was higher
than that of PNIPAM, because at 37 C the former is more
swollen with cultivation media than latter.
1
ꢁ
11,21
violet
11,21
the dishes were washed
with water to remove the bright, rounded (not spread) cells,
ꢁ
which were then dried at 37 C. Subsequently, 1% sodium
Competitive peptide inhibition
dodecyl sulfate was added, the cells were dissolved for 1 h, and
the absorbance at 595 nm (ascribed to crystal violet from spread
cells) was estimated using UV/vis spectroscopy (Fig. 10). On
To test the hypothesis that the HUVECs adhere to the cross-
linked lm through the REDV cell-binding domain and that
non-specic cell adhesion can be suppressed by graing of
PAAm or PNIPAM, competitive peptides were used to inhibit
adhesion. Tirrell et al. previously showed that cell adhesion to
absorbed aECM proteins is dependent upon the presentation of
3
blended lms, in which the p-N Phe unit acts as the photo-
sensitive cross linker via nitrene chemistry, the cells spread
within 1 day (Fig. 9). When compared with a positive control
cultured on a commercially available cultivation dish [Cell Desk
6
authentic cell-binding domains. However, cross-linked protein
Fig. 9 Cell morphology after 10 min to 4 days on (a) a blank, (b) a positive control [commercially available cultivation dish], (c) cross-linked
aECM-CS5-ELF-N
3
, (d) a cross-linked blend of aECM-CS5ELF-N
)-graft-PNIPAM.
3 3
/(aECM-CS5-ELF-N )-graft-PAAm, and (e) a cross-linked blend of aECM-C-
S5ELF-N /(aECM-CS5-ELF-N
3
3
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GRDEVY (not GREDVY) had no signicant effect on the number
of cells adhered to aECM. Furthermore, increasing the GREDVY
concentration from 1.5 mM to 3.0 mM decreased the number of
HUVECs adhered to the lm. These results demonstrate that
some HUVECs specically adhere to the REDV cell-binding
domain in the cross-linked lm, and that non-specic adhe-
sion is suppressed by the introduction of PNIPAM side chains
via a “graing onto” procedure.
Cell detachment with temperature control
For the cross-linked blend of aECM-CS5ELF-N /(aECM-CS5-
3
ELF-N )-gra-PNIPAM, cell detachment was also evaluated by
3
ꢁ
decreasing the temperature below the LCST (15 C) aer incu-
ꢁ
ꢁ
bation at 37 C for 4 days. The HUVECs were observed at 15 C
for 60 min (Fig. 11). The results indicate that the surfaces
Fig. 10 Competitive peptide inhibition of HUVEC adhesion onto
aECMs with 1.5 mM GREDVY.
prepared from cross-linked aECM-CS5-ELF-N
3
and a cross-
)-gra-
linked blend of aECM-CS5ELF-N /(aECM-CS5-ELF-N
3
3
PNIPAM were effective at triggering cell detachment when the
temperature fell below the LCST (Fig. 11). On the aECM-
CS5ELF-N /(aECM-CS5-ELF-N )-gra-PAAm surface, few cells
6

lms exhibit signicant nonspecic adhesion. Aer the
3
3
HUVECs were incubated on the cross-linked lm, subsequent
washing with the competitive GREDVY peptide (1.5 mM in
PBS) was carried out. GREDVY peptide was prepared in our
laboratory and the structure was conrmed by MALDI-TOF
became round aer the observation period (60 min, Fig. 11c),
which coincides well with the lack of thermosensitivity (the
LCST behavior shown in Fig. 7). Fig. 11b shows a set of phase
contrast images of an area of HUVECs in real-time from 0 to 60
21
21
+
mass spectrum (H
(
2
N-GREDVY-COOH + Na : m/z ¼ 762.2)
ꢁ
min at ca. 15 C. At rst, most cells adopted a spindle shape
Fig. S5†). The number of cells adhered to cross-linked aECM-
with the formation of lamellipodia. Aer 60 min, most cells
adopted a round morphology as they had lost their anchoring
points. It should be noted that some HUVECs failed to detach
from the surface because of the absence of intracellular inter-
actions (as observed in cell sheets), but they could be detached
by gently rinsing with the culture medium. Similar to described
above, 1% sodium dodecyl sulfate was added, the cells were
dissolved for 1 h, and the absorbance at 595 nm (ascribed to
crystal violet from spread cells) was estimated using UV/vis
spectroscopy (Fig. 12). Thus, the rounded cells observed were
3
CS5-ELF-N (HUVECs were incubated on the cell culture Petri
dish) was counted and shown in Fig. 10, in which in the absence
of peptide to normalize (expressed as 100%) for passage-to-
passage variations. Aer washing, signicant non-specic
adhesion was observed in the cross-linked aECM-CS5-ELF-N
86% on average) and (aECM-CS5-ELF-N )-gra-PAAm (90% on
3
(
3
average) lms, but non-specic adhesion was apparently
decreased (12% on average) in the cross-linked (aECM-CS5-ELF-
N )-gra-PNIPAM lm (Fig. 10). The negative control peptide
3
Fig. 11 Time lapse microscopic imaging of HUVECs detaching from (a) cross-linked aECM-CS5-ELF-N
ELF-N /(aECM-CS5-ELF-N )-graft-PNIPAM, and (c) a cross-linked blend of aECM-CS5-ELF-N /(aECM-CS5-ELF-N
dip coating. A progressive cell shrinking and detaching process is observed over 1 h.
3
, (b) a cross-linked blend of aECM-CS5-
3
3
3
3
)-graft-PAAm prepared by
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2
3
S. M. Schoenwaelder and K. Burridge, Curr. Opin. Cell Biol.,
1
999, 11, 274–286.
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4
5
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6
(a) A. Panitch, T. Yamaoka, M. J. Fournier, T. L. Mason and
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Fig. 12 HUVEC detachment from (left) cross-linked aECM-CS5-ELF-
3 3
N , (middle) a cross-linked blend of aECM-CS5ELF-N /(aECM-CS5-
7 E. Dejana, S. Colella, G. Conforti, M. Abbadini, M. Gaboii
ELF-N )-graft-PNIPAM, and (right) a cross-linked blend of aECM-
3
and P. C. Marchisio, J. Cell Biol., 1988, 107, 1215–1223.
3 3
CS5ELF-N /(aECM-CS5-ELF-N )-graft-PAAm prepared by dip
8
(a) M. J. Humphries, S. K. Akiyama, A. Komoriya, K. Olden
and K. M. Yamada, J. Cell Biol., 1986, 103, 2637–2647; (b)
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(a) J. C. M. van Hest and D. A. Tirrell, Chem. Commun., 2001,
coating.
also counted as detached cells in this work (Fig. 12). Fig. 12
indicated the HUVEC cells adhered counted by UV measure-
9
1897–1904; (b) K. L. Kiick, C. M. van Hest and D. A. Tirrell,
ment and that the cross-linked blend of aECM-CS5ELF-N
3
/
Angew. Chem., Int. Ed., 2000, 39, 2148; (c) P. Wang, Y. Tang
and D. A. Tirrell, J. Am. Chem. Soc., 2003, 125, 6900; (d)
I. Kwon, K. Kirshenbaum and D. A. Tirrell, J. Am. Chem.
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D. A. Tirrell, Polym. Prepr., 2003, 44(1), 1065.
(
aECM-CS5-ELF-N )-gra-PNIPAM was the most sensitive for
3
temperature-triggered cell detachment in this experimental
condition.
1
0 (a) K. Kirshenbaum, I. S. Carrico and D. A. Tirrell,
ChemBioChem, 2002, 3, 235–237; (b) D. Datta, P. Wang,
I. S. Carrico, S. L. Mayo and D. A. Tirrell, J. Am. Chem. Soc.,
2002, 124, 5652; (c) I. S. Carrico, S. A. Maskarinec,
S. C. Heilshorn, M. L. Mock, J. C. Liu, P. J. Nowatzki,
C. Franck, G. Ravichandran and D. A. Tirrell, J. Am. Chem.
Soc., 2007, 129, 4874–4875.
Conclusions
The results presented herein provide a new tool for the de novo
design of “intelligent aECMs” that may be used in cell cultiva-
tion scaffolds as well as small-diameter vascular gra
construction. Vinyl polymers chemically coupled to Phe analogs
incorporated into aECM-CS5-ELF-N via click reactions can ne-
3
tune the adhesion behavior of HUVEC/aECM-CS5-ELF systems. 11 A. Takasu, S. Kondo, A. Ito, Y. Furukawa, M. Higuchi,
These fundamental results provide new guidelines for the
T. Kinoshita and I. Kwon, Biomacromolecules, 2011, 12,
design of cultivation scaffolds based on hydration/dehydration
3444–3452.
(LCST) behavior.
12 F. W. Studier and B. A. Moffatt, J. Mol. Biol., 1986, 189, 113–
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1
1
1
1
3 A. H. Rosenberg, B. N. Lade, D. Chui, S. Lin, J. J. Dunn and
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Acknowledgements
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A.T. is grateful to Prof. David A. Tirrell for the opportunity to
work in his laboratory at California Institute of Technology. The
authors acknowledge support from the Nagoya Institute of
Technology's Research Promotion Program, from NIH grants
EB1971 and GM 62523, and from the NSF Center for the Science
and Engineering of Materials at the California Institute of
Technology. We thank Dr Hideo Yoshizato for technical
assistance.
1
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