Using mixed self-assembled monolayers presenting RGD and (EG)3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces

Article abstract of DOI:10.1021/ja972467o

This paper describes surfaces that promote the ligand-directed binding of cells and resist the cellular deposition of adhesive proteins. These surfaces are based on self-assembled monolayers (SAMs) of alkanethiolates on gold that present mixtures of argin

Full text of DOI:10.1021/ja972467o

6548
J. Am. Chem. Soc. 1998, 120, 6548-6555
Using Mixed Self-Assembled Monolayers Presenting RGD and
(EG)3OH Groups To Characterize Long-Term Attachment of Bovine
Capillary Endothelial Cells to Surfaces
Carmichael Roberts,† Christopher S. Chen,‡ Milan Mrksich,† Valerie Martichonok,†
Donald E. Ingber,*,‡ and George M. Whitesides*,†
Contribution from the Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138, and Department of Surgery and Pathology, Children’s Hospital and
HarVard Medical School, Enders 1007, 300 Longwood AVenue, Boston, Massachusetts 02115
ReceiVed July 21, 1997
Abstract: This paper describes surfaces that promote the ligand-directed binding of cells and resist the cellular
deposition of adhesive proteins. These surfaces are based on self-assembled monolayers (SAMs) of
alkanethiolates on gold that present mixtures of arginine-glycine-aspartate (RGD), a tripeptide that promotes
cell adhesion by binding to cell surface integrin receptors, and oligo(ethyleneglycol) moieties, groups that
resist nonbiospecific adsorption of proteins and cells. Surface plasmon resonance (SPR) spectroscopy was
used to measure the adsorption of carbonic anhydrase and fibrinogen to mixed SAMs comprising RGD groups
((EG)6OGRGD) and tri(ethylene glycol) groups ((EG)3OH); SAMs having values of the mole fraction of RGD
(øRGD) e 0.05 adsorbed nearly undetectable levels of carbonic anhydrase or fibrinogen. Bovine capillary
endothelial cells attached and spread on SAMs at øRGD g 0.00001, with spreading of cells reaching a maximum
at øRGD g 0.001. These mixed SAMs reduced the deposition of proteins by attached cells relative to both
fibronectin adsorbed on SAMs of hexadecanethiolate on gold and RGD peptide coated on glass. After allowing
cells to attach for 2 or 4 h to any of these surfaces presenting RGD groups, addition of soluble GRGDSP to
the medium contacting the adherent cells rapidly released them from the surfaces. However, if cells were
allowed to attach to surfaces for 24 h, only those cells attached to the mixed SAM presenting (EG)6OGRGD
and (EG)3OH groups could be released using the soluble GRGDSP at a rate comparable to cells attached to
fibronectin for 2 h. These results demonstrate that RGD alone is sufficient for adhesion and survival of cells
over 24 h.
Introduction
surface plasmon resonance (SPR) spectroscopy7 to study at-
tachment and spreading of bovine capillary endothelial cells on
-Gly-Arg-Gly-Asp (GRGD) presented in a background of
(EG)3OH groups that resists the deposition of extracellular
matrix by the cell (Figure 1).
In the mid 1980s, Pierschbacher and Ruoslahti found that
many extracellular matrix proteins contain the tripeptide RGD
and that this short peptide is an important ligand for cell
adhesion.8,9 It was subsequently found that cells employ a large
family of transmembrane proteins called the integrins to
recognize this and other ligands of the ECM.10-12 This finding
was followed by many reports that demonstrated substrates
modified with the RGD peptide alone supported the adhesion
and spreading of mammalian cells. Brandley and Schnaar, for
example, used polyacrylamide gel substrates that were modified
with the RGD peptide to study cell adhesion.13 This early work
demonstrated a model system for studies of cell adhesion but
had the limitation that the immobilized ligands were presented
Adhesion of cells to the extracellular matrix (ECM) influences
the shape, growth, viability, differentiation, migration, and
metabolism of cells.1-6 It has been difficult, however, to
characterize the biological activities of specific constituents of
the ECM (e.g., fibronectin, laminin, vitronectin, collagens, and
proteoglycans), primarily because within hours after plating cells
onto substrates presenting specific ECM proteins, cells can
deposit a new, uncharacterized layer of ECM; this process of
depositing adhesive proteins is called “remodeling”. We
hypothesized that mixed self-assembled monolayers (SAMs) that
presented specific ECM moieties in an “inert”, nonadsorbing
interface would provide a surface that would promote attachment
through specific cell adhesion receptors while preventing the
remodeling of the substrate. Here, we used mixed SAMs and
† Harvard University.
‡ Children’s Hospital and Harvard Medical School.
(1) Ingber, D. E. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3579-3583.
(2) Boudreau, N. C.; Sympson, J.; Werb, Z.; Bissell, M. J. Science 1995,
267, 891.
(7) Raether, H. Physics of Thin Films; Hass, G., Francombe, M. Hoffman,
R. Eds.; Academic Press: New York, 1977; Vol. 9, pp 145-261.
(8) Pierschbacher, M. D.; Ruoslahti, E. Nature 1984, 309, 30-33.
(9) Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 491-497.
(10) Tamkun, J. W.; DeSimone, D. W.; Fonda, D.; Patel, R. S.; Buch,
C.; Horwitz, A. F.; Hynes, R. O. Cell 1986, 46, 271-282.
(11) Hynes, R. O. Cell 1992, 69, 11-25.
(12) Clark, E. A.; Brugge, J. S. Science 1995, 2368, 233.
(13) Brandley, B. K.; Schnaar, R. L. Anal. Biochem. 1988, 172(1), 270-
278.
(3) Watt, F. M.; Jordan, P. W.; O’Neill, C. H. Proc. Natl. Acad. Sci.
U.S.A. 1988, 85, 5576-5580.
(4) Flaumenhaft, R.; Rifkin, D. B. Curr. Opin. Cell Biol. 1991, 3, 817-
823.
(5) Basson, C. T.; Kocher, O.; Basson, M. D.; Asis, A.; Madri, J. A. J.
Cell. Phys. 1992, 153, 118-128.
(6) Salomon, D. S.; Liotta, L. A.; Kidwell, W. R. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 382-386.
S0002-7863(97)02467-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/17/1998

Attachment of BoVine Capillary Endothelial Cells to Surfaces
J. Am. Chem. Soc., Vol. 120, No. 26, 1998 6549
moieties prevent the adsorption of protein.19-21 In this study
we develop surfaces that promote cell attachment by the specific
interaction of RGD with cell surface integrin receptors and resist
significant deposition of cell-derived matrix components. We
prepare the mixed SAMs using solutions that contain both
(EG)6OGRGD and (EG)3OH terminated alkanethiolates. By
using this approach, we generate structures that are well-defined
surfaces; the most important unresolved issues concern the 2-D
distribution of the RGD groups and the nature and distribution
of defects in the SAMs. The important point is that these
surfaces present RGD groups at an otherwise “inert”sthat is,
nonadsorbingsinterface. Using this model system, we dem-
onstrate that RGD alone is sufficient to maintain long-term
biospecific attachment and survival of cells; the deposition of
proteins expressed by cells during remodeling is not essential
to the cells in the first 24 h.
Experimental Procedure
Preparation of Alkanethiol 1 (see Figure 2):
Synthesis. A convergent synthetic strategy was used to prepare
alkanethiol 1. Each of the amide linkages was achieved using
diphenylphosphoryl azide (DPPA) to activate the terminal carboxylic
acids in situ.
Alcohol 5, converted to the sodium alkoxide, was reacted with tert-
butylbromoactetate to give ester 6. The ester was then transformed
into carboxylic acid 7 using trifluoroacetic acid (TFA).
The GRGD segment 4 was prepared from dipeptides 2 and 3.
Hydrogenation of 4 provided the terminal amine which was subse-
quently coupled with 7 to afford alkene 8 and complete the block portion
of the synthesis.
The conversion of alkene 8 to thioester 9 was achieved by the radical
addition of thioacetic acid across the terminal olefin. Finally, all
protecting groups were removed using an aqueous mixture of TFA,
ethanedithiol, and thioanisole to give the target molecule 1.
Materials and Methods. Reactions were monitored by thin-layer
chromatography (TLC) using 0.25-mm silica gel plates (E. Merck).
Column chromatography was performed using silica gel-60 (particle
size 0.040-0.063) (E. Merck). All reactions in nonaqueous solvents
were executed under nitrogen.
N-[(Phenylmethoxy)carbonyl]glycyl-N5-[[[(3,4-dihydro-2,2,5,7,8-
pentamethyl-2H-1-benzopyran-6-yl)sulfonyl]amino]iminomethyl] -L-
ornithyl Acid (2b). To a solution of N-[(phenylmethoxy)carbonyl]-
glycyl-N-hydroxysuccinimide (3.81 g, 12.4 mmol) in DMF (40 mL)
cooled to 0 °C was added N5-[[[(3,4-dihydro-2,2,5,7,8-pentamethyl-
2H-1-benzopyran-6-yl)sulfonyl]amino]iminomethyl]-L-ornithyl acid 2a
(5.0 g, 11.3 mmol) followed by dropwise addition of diisopropylethyl-
amine (DIPEA) (7.48 mL, 43 mmol). The reaction mixture was stirred
at 0 °C for 1 h, allowed to warm to room temperature and stirred at
room temperature, for an additional 3 h. Product 2b precipitated from
cold H2O after acidification with 1 N aqueous HCl (26 mL). The
obtained precipitate was filtered, washed with cold H2O, and dried in
vacuo to afford 2b (6.7 g, 94%). 1H NMR (500 MHz, CDCl3): δ
1.27 (s, 6 H), 1.55 (bs, 2 H), 1.75 (m, 3 H),1.85 (bs, 1 H), 2.06 (s, 3
H), 2.49 (s, 3 H), 2.50 (s, 3 H), 2.56 (bs, 2 H), 3.14 (bs, 2 H), 3.89 (bs,
2 H), 4.48 (bs, 1 H), 5.00 (s, 2 H), 6.15-6.50 (m, 4 H), 7.24 (m, 5 H),
7.61 (bs, 1 H).
L-Aspartic Acid, Glycyl,-Bis(1,1-dimethylethyl) Ester (3b). To
a solution of Z-NH-G-ONHS (6.87 g, 22.4 mmol) in DMF (40 mL)
cooled to 0 °C was added H2ND(OtBu)-OtBu 3a (5.0 g, 20.4 mmol)
followed by dropwise addition of DIPEA (7.48 mL, 43 mmol). The
reaction mixture was stirred at 0 °C for 1 h, allowed to warm to room
temperature, and stirred at room temperature for an additional 3 h. The
reaction mixture was added dropwise to cold H2O and left on ice for
2 h. The obtained precipitate was filtered, washed with cold H2O, and
Figure 1. Diagram of self-assembled monolayer of alkanethiolates on
gold presenting (EG)6OGRGD and (EG)3OH groups.
in the heterogeneous environment of the gel; this heterogeneity
made it impossible for the authors to determine the density of
functional ligand. Massia and Hubbell prepared alkylsiloxanes
presenting RGD peptides for studies of cell adhesion and
spreading.14 Unlike the polyacrylamide gels, the modified
alkylsiloxane surfaces presented the RGD ligands in a homo-
geneous environment. Consequently, Massia and Hubbell were
able to quantitate the relationship between the density of peptide
ligands and the efficiency of cell attachment and spreading. The
modified alkylsiloxane surfaces, however, were prepared using
a synthetic strategy that employed the random attachment of
the peptides fragments directly to the siloxane surface. As a
result, the characterization of these modified alkylsiloxane
surfaces was nontrivial. Drumheller and Hubbel improved the
biospecificity of cell-substrate interactions by developing a
surface made of poly(ethylene glycol) in a cross-linked matrix
of acrylic acid and trimethylolpropane triacrylate grafted with
RGD peptide.15 The adhesive properties of this surface
remained unchanged by the presence of adhesion proteins in
solution. However, these studies did not address the issue of
whether these modified surfaces prevented cells from remodeling
the interface.
Surfaces fabricated to date have not been shown to resist the
deposition of additional adhesive ligands expressed by the
attached cells; within hours, the molecular composition of the
surface, and therefore the spectrum of cell surface receptors
being engaged, change uncontrollably. Although the use of
protein synthesis inhibitors to block synthesis and deposition
of new ECM proteins may be useful for a few hours,16 this
approach severely impairs many processes within cells and is
lethal to them within 8-24 h.17,18 As a result it is difficult to
assess the effects of specific ligand-cell interactions on many
biological processes, including adhesion itself.
Using SAMs of alkanethiolates on gold, we have previously
demonstrated that surfaces presenting oligo(ethylene glycol)
(14) Massia, S. P.; Hubbell, J. A. J. Cell Biol. 1991, 114, 1089-1100.
(15) Drumheller, P. D.; Hubbell, J. A. Anal. Biochem. 1994, 222, 380-
388.
(16) Aznavoorian, S.; Stracke, M. L.; Krutzsch, H.; Schiffmann, E.;
Liotta, L. A. J. Cell Biol. 1990, 110, 1427-38.
(17) Lewis JG. Adams DO. Fan S. J. Leukocyte Biol. 1995, 57, 635-
42.
(18) Lord, M. J.; Roberts, L. M.; Robertus, J. D. FASEB J. 1994, 8,
201-208.
(19) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G.
M. J. Am. Chem. Soc. 1991, 113, 12-20.
(20) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167.
(21) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115,
10714-10721.

6550 J. Am. Chem. Soc., Vol. 120, No. 26, 1998
Roberts et al.
Figure 2. Synthesis of GRGD hexaethyleneglycol alkanethiol (1). (i) ZNHCH2CONHS, DIPEA; (ii) ZNHCH2CONHS, DIPEA, DMF; (iii) DPPA,
DIPEA, DMF; (iv) NaH, DMF then BrCH2COOt-Bu; (v) TFA, CH2Cl2; (vi) 10% Pd/C, EtOH on 4 then DPPA, DIPEA, DMF; (vii) CH3COSH,
AIBN, THF with UV irradiation; (viii) TFA, PhSMe, HS(CH2)2SH, CH2Cl2.
dried in vacuo to afford 3b (7.3 g, 82%). 1H NMR (400 MHz,
CDCl3): δ 1.41 (s, 9 H), 1.43 (s, 9 H), 2.71 (dd, J ) 3.48, 17.11 Hz,
1H), 2.88 (dd, J ) 4.17, 17.11 Hz, 1H), 3.92 (m, 2 H), 4.68 (dt, J )
4.29, 8.36 Hz, 1H), 5.12 (s, 2 H), 5.38 (bs, 1 H), 6.85 (d, J ) 7.90 Hz,
1H), 7.29-7.36 (m, 5 H).
was used in the next step without further purification. The flask
containing crude amine from 3b was purged with N2, acid 2b (2.3 g,
3.65 mmol) and dry DMF (20 mL) were added, and the stirred solution
was cooled to 0 °C. Diphenylphosphoryl azide (DPPA) (0.94 mL, 4.38
mmol) was added, followed by a solution of DIPEA (0.76 mL, 4.38
mmol) in DMF (5 mL), and the stirring was continued at 0 °C for 10
h. The mixture was diluted with EtOAc (100 mL) and washed
successively with H2O (3 × 20 mL), 5% aqueous NaHCO3 (20 mL),
and brine (2 × 20 mL). The organic phase was dried (MgSO4), and
the solvent was removed in vacuo to give the residue which was
chromatographed (eluting with 10% EtOH in EtOAc) to give product
4 (2.23 g, 71%). 1H NMR (500 MHz, CDCl3): δ 1.26 (s, 6 H), 1.36
L-Aspartic Acid, N-[(Phenylmethoxy)carbonyl]glycyl-N5-[[[(3,4-
dihydro-2,2,5,7,8-pentamethyl-2H-1-benzopyran-6-yl)sulfonyl]ami-
no]iminomethyl]-L-ornithylglycyl-, Bis(1,1-dimethylethyl) Ester (4).
Compound 3b (1.46 g, 3.35 mmol) was hydrogenated in EtOH (30
mL) over 10% Pd/C (0.3 g) until the TLC (33% EtOAc in hexanes)
indicated that 3b had been consumed. The reaction mixture was filtered
through Celite and concentrated in vacuo to give the crude amine that

Attachment of BoVine Capillary Endothelial Cells to Surfaces
J. Am. Chem. Soc., Vol. 120, No. 26, 1998 6551
(s, 9 H), 1.38 (s, 9 H), 1.54 (m, 2 H), 1.63 (m, 2 H), 1.75 (t, J ) 6.78
Hz, 2 H), 1.85 (m, 2 H), 2.05 (s, 3 H), 2.50 (s, 3 H), 2.51 (s, 3 H), 2.57
(t, J ) 6.72 Hz 2 H), 2.65 (dd, J ) 3.36, 17.00 Hz, 1 H), 2.78 (dd, J
) 4.20, 17.00 Hz, 1 H), 3.18 (bs, 2 H), 3.81-4.01 (m, 4 H), 4.50 (m,
1 H), 4.63 (m, 1 H), 5.03 (s, 2 H), 6.10-6.40 (m, 4 H), 7.25 (m, 5 H),
7.43 (m, 1 H), 7.77 (m, 1 H). HRMS (FAB) calcd for C44H65N7O12-
SNa (M + Na) 938.4310, found 938.4310.
3,6,9,12,15,18,21-Heptaoxadotriacont-31-enoic Acid, 1,1-Di-
methylethyl Ester (6). To a solution of alcohol 520,21 (1.04 g, 2.4
mmol) in dry DMF (5 mL) cooled to 0 °C was added NaH (144 mg of
60% suspension in oil, 3.6 mmol). The mixture was stirred at 0 °C
for 10 min, tert-butylbromoacetate (532 µL, 3.6 mmol) was added in
one portion, and the mixture was allowed to warm to room temperature.
After the mixture was stirred for 6 h additional tert-butylbromoacetate
(532 µL, 3.6 mmol) was added, and the stirring was continued at 40
°C for 10 h. After the mixture cooled to room temperature, EtOAc
(50 mL) was added, organic phase was washed with H2O and brine,
dried (MgSO4), and concentrated in vacuo. Column chromatography
(3% MeOH in CH2Cl2) afforded product 6 (0.81 g, 62%). 1H NMR
(400 MHz, CDCl3): δ 1.25 (bs, 10 H), 1.29-1.41 (m, 2 H), 1.45 (s, 9
H), 1.55 (m, 2 H), 2.01 (m, 2 H), 3.42 (t, J ) 6.80 Hz, 2H), 3.56 (m,
2 H), 3.60-3.73 (m, 22 H), 4.00 (s, 2 H), 4.89 (m, 2 H), 5.73-5.84
(m, 1 H). MS (FAB) calcd for C29H56O9Na (M + Na) 571, found
571.
L-Aspartic Acid, N-[(1-oxo-3,6,9,12,15,18,21-heptaoxatriacont-31-
en-1-yl)glycyl-N5-[[[(3,4-dihydro-2,2,5,7,8-pentamethyl-2H-1-ben-
zopyran-6-yl)sulfonyl]amino]iminomethyl]-L-ornithylglycyl-, Bis-
(1,1-dimethylethyl) Ester (8). To a solution of 6 (376 mg, 0.69 mmol)
in CH2Cl2 (5 mL), was added TFA (5 mL) and the mixture was stirred
for 3 h. After concentration in vacuo and column chromatography (10%
MeOH in CH2Cl2) heptaoxadotriacont-31-enoic acid 7 (290 mg, 0.59
mmol, 86%) was obtained. Z-Protected tetrapeptide 4 (507 mg, 0.65
mmol) was hydrogenated in EtOH (10 mL) over 10% Pd/C (0.1 g).
After filtration and concentration in vacuo the crude amine was
obtained. The amine and carboxylic acid 7 were combined, the flask
was purged with N2, dry DMF (3 mL) was added, and the stirred
solution was cooled to 0 °C. DPPA (150 µL, 4.38 mmol) was added,
followed by a solution of DIPEA (120 µL, 4.38 mmol) in DMF (1
mL), and the stirring was continued at 0 °C for 10 h. The mixture
was diluted with EtOAc (10 mL) and washed successively with H2O,
5% aqueous NaHCO3, and brine. The organic phase was dried
(MgSO4), and the solvent was removed in vacuo to give a residue which
was chromatographed (5% MeOH in CH2Cl2 f 10% MeOH in CH2Cl2)
to give product 8 (355 mg, 48%). 1H NMR (500 MHz, CDCl3): δ
1.25 (s, 10 H), 1.28 (s, 6 H), 1.30-1.36 (m, 2 H), 1.39 (s, 9 H), 1.40
(s, 9 H), 1.48-1.63 (m, 4 H), 1.68-1.75 (m, 1 H), 1.78 (t, J ) 6.80
Hz, 2 H), 1.89-1.95 (m, 1 H), 2.01 (q, J ) 6.85 Hz, 2 H), 2.07 (s, 3
H), 2.53 (s, 3 H), 2.55 (s, 3 H), 2.60 (t, J ) 6.70 Hz, 2 H), 2.67 (dd,
J ) 4.75, 16.94 Hz, 1 H), 2.79 (dd, J ) 5.01, 16.99 Hz, 1 H), 3.22 (m,
2 H), 3.40 (t, J ) 6.85 Hz, 2 H), 3.52-3.55 (m, 2 H), 3.58-3.64 (m,
22 H), 3.66-3.71 (m, 2 H), 3.88 (dd, J ) 5.68, 16.62 Hz, 1 H), 3.95
(dd, J ) 5.64, 14.47 Hz, 1 H), 3.98-4.05 (m, 2 H), 4.01 (s, 2 H), 4.46
(q, J ) 7.61 Hz, 1 H), 4.63 (dt, J ) 4.86, 8.04 Hz, 1 H), 4.88-5.00
(m, 2 H), 5.73-5.83 (m, 1 H), 6.36 (bs, 2 H), 7.11 (d, J ) 8.06 Hz, 1
H), 7.43 (bs, 1 H), 7.63 (bt, J ) 5.29 Hz, 1 H), 7.90 (t, J ) 5.76 Hz,
1 H). MS (FAB) calcd for C61H105N7O18SNa (M + Na) 1278, found
1278.
L-Aspartic Acid, N-[(1,34-Dioxo-3,6,9,12,15,18,21-heptaoxa-33-
thiapentatriacont-1-yl)glycyl-N5-[[[(3,4-dihydro-2,2,5,7,8-pentamethyl-
2H-1-benzopyran-6-yl)sulfonyl]amino]iminomethyl]-L-ornithyl-
glycyl-, Bis(1,1-dimethylethyl) Ester (9). A mixture of 8 (650 mg,
0.52 mmol), thioacetic acid (742 µL, 10.4 mmol), and AIBN (20 mg)
in THF (10 mL) was irradiated by UV. After 2 h additional AIBN
(20 mg) was added, and irradiation was continued for 3 h. The mixture
was concentrated in vacuo and chromatographed (5% MeOH in CH2Cl2
f 10% MeOH in CH2Cl2) to give thioacetate 9 (641 mg, 93%). 1H
NMR (500 MHz, CDCl3): δ 1.26 (bs, 12 H), 1.28 (s, 6 H), 1.28-1.33
(m, 2 H), 1.39 (s, 9 H), 1.40 (s, 9 H), 1.47-1.64 (m, 6 H), 1.68-1.75
(m, 1 H), 1.78 (t, J ) 6.80 Hz, 2 H), 1.88-1.96 (m, 1 H), 2.07 (s, 3
H), 2.28 (s, 3 H), 2.53 (s, 3 H), 2.54 (s, 3 H), 2.60 (t, J ) 6.68 Hz, 2
H), 2.68 (dd, J ) 4.80, 16.94 Hz, 1 H), 2.78 (dd, J ) 5.10, 16.96 Hz,
1 H), 2.82 (t, J ) 7.36, 2 H), 3.23 (m, 2 H), 3.40 (t, J ) 6.85 Hz, 2 H),
3.52-3.55 (m, 2 H), 3.58-3.64 (m, 22 H), 3.66-3.71 (m, 2 H), 3.87
(dd, J ) 5.70, 16.64 Hz, 1 H), 3.95 (dd, J ) 5.68, 14.48 Hz, 1 H),
3.98-4.05 (m, 2 H), 4.01 (s, 2 H), 4.45 (bq, J ) 7.52 Hz, 1 H), 4.63
(dt, J ) 4.90, 8.18 Hz, 1 H), 6.36 (bs, 2 H), 7.14 (d, J ) 8.07 Hz, 1
H), 7.52 (bs, 1 H), 7.71 (bs, 1 H), 7.94 (t, J ) 5.78 Hz, 1 H). MS
(FAB) calcd for C63H109N7O19S2Na (M + Na) 1354, found 1354.
L-Aspartic Acid, N-[(32-Mercapto-1-oxo-3,6,9,12,15,18,21-hep-
taoxatriacont-1-yl)glycyl-L-arginylglcyl- (1). A mixture of CF3COOH/
PhSMe/HS(CH2)2SH/H2O 37:1:1:1 (v/v) was used for cleavage of the
protective groups in 9. A solution of 9 (320 mg, 0.25 mmol) in CH2Cl2
(2 mL) was cooled 0 °C, and the cleavage mixture (10 mL) was added.
The obtained solution was stirred at 0 °C for 1 h, allowed to warm to
room temperature, and stirred for 2 h at room temperature. The mixture
was concentrated in vacuo, CH2Cl2 (10 mL) was added, and the mixture
was concentrated in vacuo again. The residue was dissolved in a
minimum amount of CH2Cl2, cold Et2O (50 mL) was added dropwise
into the flask with vigorous stirring, and the obtained precipitate was
filtered and dried in vacuo to afford AcS(CH2)11(OCH2CH2)6OCH2C-
(O)NHGRGDOH (195 mg, 0.20 mmol, 80%) as a white solid. MS
(FAB) calcd for C41H75N7O16SNa (M + Na) 976, found 976. The
obtained thioacetate was dissolved in absolute MeOH (10 mL), and
the solution was cooled 0 °C. NaOMe (1.54 mL of 0.39 M in MeOH,
0.60 mmol) was added, and the mixture was stirred at 0 °C for 3 h.
The mixture was neutralized with Dowex exchange resin (H-form),
filtered, and concentrated in vacuo. Repeated precipitation (3 times)
from CH2Cl2 with cold Et2O afforded pure 1 (63 mg, 34%). 1H NMR
(500 MHz, CD3OD): δ 1.27-1.46 (m, 12 H), 1.52-1.61 (m, 4 H),
1.63-1.71 (m, 2 H), 1.72-1.81 (m, 2 H), 1.92-2.00 (m, 2 H), 2.48 (t,
J ) 7.15 Hz, 2 H), 2.82 (t, J ) 5.41 Hz, 2 H), 3.20 (t, J ) 6.76 Hz,
2 H), 3.46 (t, J ) 6.62 Hz, 2 H), 3.57 (m, 2 H), 3.61-3.66 (m, 18 H),
3.68 (m, 2 H), 3.71 (m, 2 H), 3.84-4.02 (m, 4 H), 4.06 (s, 2 H), 4.40
(t, J ) 7.32 Hz, 1 H), 4.63 (t, J ) 5.50 Hz, 1 H); 13C NMR (125 MHz,
CDCl3): δ 24.97, 25.94, 27.20, 29.41, 30.22, 30.57, 30.65, 30.72, 35.22,
37.62, 42.02, 43.13, 43.39, 50.77, 54.33, 71.14, 71.19, 71.33, 71.43,
71.47, 71.52, 72.08, 72.37, 158.63, 171.06, 171.58, 173.74, 174.17,
174.92, 175.02. HRMS (FAB) calcd for C39H73N7O15SNa (M + Na)
934.4783, found 934.4805.
Preparation of SAMs. Substrates were prepared as previously
described,20,21 beginning with evaporation of titanium (1 nm) and gold
onto glass slides (38 nm of Au for SPR experiments; and 12 nm of Au
for cell culture).22 The slides were immersed in ethanolic solutions
containing mixtures of HSC11(EG)6OGRGD and HSC11(EG)3OH (2 mM
total thiol) in ethanol for 4 h. Ellipsometric measurements of mixed
SAMs of HSC11(EG)6OGRGD and HSC11(EG)3OH resulted in thick-
nesses ranging from 22.8 to 33.7 Å; these values are in good agreement
with those expected for a well-packed SAM containing trans-extended
alkanethiolates.21 In reporting the composition of the system in terms
of the parameter, øRGD, we assume the relative mole fractions of
(EG)6OGRGD and (EG)3OH groups on the surface and in solution to
be the same (eq 1). This assumption may be incorrect in detail, but
the relative trends will certainly be very similar.23
[EG6OGRGD]
øGRGD
)
(1)
[EG6OGRGD] + [EG3OH]
Fibronectin (FN) coated substrates, used as a positive control for
adhesion assays, were prepared by immersing metallized substrates in
hexadecanethiol (2 mM) in ethanol for 4 h; then, coated substrates were
placed in a solution of fibronectin in PBS (Collaborative Biomedical,
50 µg/mL) for 1 h. Another control substrate using a 20 amino acid
fragment containing RGD (Telios) coated on glass (RGD-glass) was
prepared by immersing glass coverslips in a solution of the peptide
(50 µg/mL) in sodium bicarbonate (100 mM, pH 9.4) overnight to
generate maximal coating density.
(22) SPR spectroscopy requires a reflective gold surface (38 nm of Au).
In contrast, a transparent surface (12 nm of Au) is required for the study of
cells.
(23) Prime and Whitesides (refs 17 and 18) found that the thickness of
mixed SAMs of (EG)n alkanethiolates increases almost linearly with
composition.

6552 J. Am. Chem. Soc., Vol. 120, No. 26, 1998
Roberts et al.
Surface Plasmon Resonance Spectroscopy. We used the Biacore
1000 for all studies described here. We modified the manufacturer’s
chips to accept our substrates, as described previously.21 Phosphate-
buffered saline (P3813), fibrinogen (F4883; 94% clottability), and
carbonic anhydrase (C3934) were purchased from Sigma and used as
received. Solutions of proteins were filtered through 0.22 µm filters
immediately before use.
Cell Culture. Bovine capillary endothelial (BCE) cells were isolated
from adrenal cortex and cultured as described previously.24 Cells were
dissociated with trypsin-EDTA, washed in Dulbecco’s Modified Eagle’s
Medium (DMEM) containing 1% bovine serum albumin (1% BSA/
DMEM), and plated onto substrates in chemically defined media (10
µg/mL high-density lipoprotein, 5 µg/mL transferrin, 5 ng/mL basic
fibroblast growth factor in 1% BSA/DMEM).1 Cells were incubated
in 10% CO2 at 37 °C.
Assessment of Efficiency of Cell Attachment. A fixed number of
cells were plated onto substrates (15000 cells/cm2) containing varying
amounts of RGD peptide. After 4 h, substrates were gently washed in
PBS and fixed with 4% paraformaldehyde in PBS for 30 min. The
number of cells attached per field was determined from photographs
taken of samples on a Nikon Axiophot microscope at 200X magnifica-
tion.
Measurement of de noWo Matrix Deposition. Cells were pre-
incubated in 95% cysteine-free, methionine-free medium (DMEM/cm-)
containing 1% dialyzed fetal calf serum for 24 h. After dissociation
with trypsin-EDTA, cells were washed in DMEM/cm containing 1%
BSA and plated for 4 or 24 h onto substrates in the presence of 35S-
methionine and 35S-cysteine (50 µCi/mL, Amersham: Promix). Sub-
strates were washed three times with PBS, and cells were gently
extracted with 0.1% ammonium hydroxide, leaving the deposited matrix
on the substrate as previously described.23 Deposited matrix was then
removed from substrates with radioimmunoprecipitate assay (RIPA)
buffer (1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl
sulfate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.2).26 Sodium hydroxide
and hydrogen peroxide were added (to 1 M and 2% w/w respectively)
and incubated at 37 C for 10 min. Samples were precipitated in ice
cold 25% trichloroacetic acid containing 2% casein proteolytic frag-
ments (Amersham: casamino acids) and filtered through glass fiber
filters (Whatman). After rinsing the precipitate on the filters three times
in 5% trichloroacetic acid and once in acetone, samples were dried
and counted in a scintillation counter (Wallac).
Detachment of Cells Using Soluble GRGDSP Peptide. Cells were
plated onto substrates for 2, 4, or 24 h, after which the samples were
transferred to an Omega RTD 0.1 stage heating ring mounted on a
Nikon Diaphot inverted microscope. Cells were then immersed in
defined media containing soluble GRGDSP27-29 (1 mg/mL, Peninsula
Laboratories). Photographs were taken of the cells over time to
document the effects of the soluble peptide. Projected cell areas were
determined using these photographs with image analysis software
(Oncor:BDS).
Figure 3. Percentage of a monolayer of protein irreversibly adsorbed
to mixed SAMs presenting RGD groups and tri(ethylene glycol) groups.
SPR response curves were obtained for the adsorption of carbonic
anhydrase and fibrinogen to SAMs containing different mole fractions
of RGD groups (top, middle panels, respectively). From these curves,
steady-state adsorption of proteins was determined as a function of
ø
RGD. Steady state changes in resonance angles (∆θ) were normalized
to previously measured values for a full monolayer of these proteins
(∆θ of 0.15° and 0.45°, respectively);27 values of øRGD are indicated on
the plot.
Results
Mixed SAMs of (EG)3OH and (EG)6OGRGD Resist the
Nonspecific Adsorption of Proteins. We used SPR to measure
the amount of carbonic anhydrase and fibrinogen that adsorbed
to mixed SAMs containing different mole fractions of
(EG)6OGRGD and (EG)3OH (eq 1). SPR is an optical technique
that measures the angle of minimum reflectivity of polarized
light incident on a gold-coated glass slide; the displacement in
the angle is related linearly to the change in refractive
indexsand therefore the mass of proteinsin the interfacial
region. Because the protein-containing solution has a higher
refractive index than does the buffer, this solution will cause
an increase in ∆θ that is not due to adsorption. Previous studies
using SPR have determined that a monolayer of carbonic
anhydrase and fibrinogen adsorbed on the surface of the SAMs
of hexadecane alkanethiolates gives ∆θ values of 0.15° and
0.45°, respectively.29 In the experiments reported here, buffer
was allowed to flow over the monolayer followed by a solution
of detergent to remove adsorbed impurities, and then a solution
of protein was allowed to flow over the monolayer to observe
adsorption followed by a solution of buffer to observe any
desorption. Our results, repeated twice on separate occasions,
show that SAMs having values of the mole fraction of RGD
(øRGD) e 0.05 adsorb nearly undetectable amounts of carbonic
(24) Ingber, D. E.; Folkman, J. J. Cell Biol. 1989, 109, 317-330.
(25) Ingber, D. E.; Madri, J. A.; Jamieson, J. D. Am. J. Pathol. 1986,
122, 129-139.
(26) Plopper, G.; Ingber, D. E. Biochem. Biophys. Res. Comm. 1993,
193, 571-578.
(27) Dejana, E.; Colella, S.; Languino, L. R.; Balconi, G.; Corbascio,
G. C.; Marchisio, P. C. J. Cell Biol. 1987, 104, 1403-11.
(28) Sims, J. R.; Karp, S.; Ingber, D. E. J. Cell Sci. 1992, 103, 1215-
1222.
(29) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11,
4383-4385.

Attachment of BoVine Capillary Endothelial Cells to Surfaces
J. Am. Chem. Soc., Vol. 120, No. 26, 1998 6553
Figure 4. Adhesion of endothelial cells to mixed SAMs presenting RGD groups and tri(ethylene glycol) groups; nominal values of
are
øRGD
indicated on the plot. The number of cells attached per field are indicated on the vertical axis. The photographs above the bar graph illustrate the
amount of cell attachment and cell spreading observed for a given SAM: (A) cells on fibronectin-coated on (CH2)15 CH3 SAM; (B) cells are
attached and spread; (C) cells are attached and spread; (D) cells are attached but do not spread; (E) cells are unable to attach. Error bars indicated
standard error of the mean.
anhydrase or fibrinogen (<0.5% of maximum, fully adsorbed
monolayer of each protein, respectively), while mixed SAMs
having values of øRGD > 0.05 adsorbed less than 5% of a
monolayer of these proteins (Figure 3).
peptide nonspecifically adsorbed onto glass (Figure 5).30
Surfaces containing øRGD of 0.001 significantly reduced protein
deposition by severalfold (p < 0.01). We presume that
deposition of radiolabeled matrix onto the fibronectin-coated
SAM and the peptide-coated glass involved exchange of the
radiolabeled protein with adsorbed protein. Because the mech-
anisms for exchange of soluble and adsorbed proteins are
complicated and not well understood, we cannot provide an
explanation for the greater amount of deposition on the peptide-
coated glass substrate. While SAM substrates do not completely
resist matrix deposition, the reduction of protein accumulation
on SAM substrates may increase the window of time during
which the effects of specific ligand-receptor interactions on
cellular processes can be studied.
Cells Adhere Biospecifically to Surfaces Presenting RGD
Ligand. The previous experiment demonstrates that a newly
deposited layer of ECM forms on traditional surfaces within
several hours after attachment of cells, possibly allowing cells
to attach via mechanisms that do not involve binding to RGD.
To test whether cells, over time, continued to attach to surfaces
via RGD moieties, or if multiple interactions developed, cells
were allowed to spread on substrates for up to 24 h and
challenged with soluble GRGDSP peptide. Cells were first
allowed to attach to the RGD-presenting substrates. By 2 h,
they spread equally on the four different substratessmixed
SAMs containing either 0.1% or 1% RGD, fibronectin nonspe-
Cells Adhere and Spread to RGD SAMs in a Concentra-
tion Dependent Manner. Figure 4 shows the density of cells
attaching to SAMs presenting different mixtures of (EG)6OGRGD
and (EG)3OH groups. SAMs having øRGD e 1 × 10-6
effectively resisted attachment of endothelial cells (approxi-
mately 3 ( 1 cells per field versus 150 ( 10 for øRGD ) 1).
Cells readily attached to SAMs having øRGD g 1 × 10-5, but
maximal cell spreading was only observed when øRGD g 1 ×
10-3. On SAMs having øRGD g 1 × 10-4, for example, cells
attached but remained rounded and did not spread. At least
six fields were counted per experiment, and the experiment was
repeated on three different occasions.
Engineered Surfaces Reduce the Deposition of Extra-
cellular Matrix by Cells. Although SAMs composed of RGD
between øRGD ) 0.001 and øRGD ) 0.01 promote complete cell
adhesion and spreading, SPR established that they resist
nonspecific protein adsorption (Figure 3). These results suggest
that such surfaces allow cells to attach but resist the deposition
of new extracellular matrix by cells. To examine directly
whether cells can deposit ECM on surfaces of mixed SAMs,
attached cells were cultured in the presence of radioactive amino
acids, followed by direct measurement of de noVo deposition
of radioactive proteins onto the surfaces. Results show that
mixed SAMs of RGD can reduce deposition of new proteins
as compared to surfaces presenting fibronectin nonspecifically
adsorbed onto hexadecanethiolate SAMs or RGD-containing
(30) SAMs of pure (EG)3OH alkanethiolate also resulted in similarly
low (<500 cpm) nonspecific adsorption of radioactive amino acids in the
absence of cells. Since cells presented with an (EG)3OH SAM do not attach,
they rapidly die. The (EG)3OH surface is, therefore, not strictly a control
substrate in the experiment.

6554 J. Am. Chem. Soc., Vol. 120, No. 26, 1998
Roberts et al.
When cells were attached for 4 h before challenging with
soluble GRGDSP, the rate of cell retraction and subsequent
detachment was already slower for cells attached to FN-coated
SAMs and RGD-coated glass (Figure 6A; 6C top versus middle
panel). In contrast, cells continued to rapidly detach from mixed
SAMs upon addition of soluble GRGDSP (Figure 6A; 6C
middle panel). By 24 h, all substrates show an increased ability
to resist cell detachment by the soluble peptide (Figure 6B; 6C
bottom panel). Cells on FN-coated substrates barely retracted
and could not be detached even after 4 h of incubation with the
soluble peptide. In contrast, cells on RGD mixed SAMs readily
detached with soluble peptide, at rates equal to or greater than
cells attached to FN-coated substrates for only 2 h. The increase
in cellular retraction and detachment rate from RGD-coated glass
between 4 and 24 h suggests that complex remodeling changes
in the surface may be occurring, consistent with the markedly
high rate of radioactive protein deposition found on this surface
(Figure 5).
The significance of these results is to suggest that only the
specific interactions of RGD with cellular receptors are involved
in initial adhesion (i.e., less than 2 h) of cells to RGD-containing
substrates. Events are more complex, however, with cells
allowed to attach for 24 h to these systems. Within 4 h, cells
were able to deposit a functional non-RGD matrix on FN-coated
SAMs and RGD-coated glass substrates such that cell adhesion
was partially mediated through binding of other cell-surface
adhesive receptors to this matrix. By 24 h these undefined cell-
matrix interactions altered the adhesion and spreading interac-
tions of cells entirely, as evidenced by the changes in the ability
of soluble GRGDSP to detach cells from these substrates. In
contrast, adhesion of cells to the mixed SAMs presenting
(EG)3OH and (EG)6OGRGD lost specificity at significantly
slower rates such that even at 24 h, the detachment rate of cells
attached to these surfaces remained equal or better than rates
of cells attached to FN-coated surfaces for only 2 h. The cellular
interactions with RGD remained important for much longer
times.
The rapidity with which cells detached from the RGD mixed
SAMs is remarkable and has not previously been reported. Past
studies describe the average time of detachment of these cells
to be between 20 and 60 min,28 whereas most cells detached
from SAM substrates in e2 min. These data suggest that
substrates that are traditionally used to present RGD also contain
additional unknown adhesive factors in the background of the
substrate. These results cannot rule out, however, that the faster
detachment of cells from the RGD-terminated SAMs is due to
a lower affinity of the immobilized peptide for cellular integrin
receptors. Nonetheless, substrates such as these mixed SAMs
might be useful in experiments that require rapid release of
attached cells without the damage to cell surfaces caused by
treatment with proteases or by other, nondiscriminate methods
of releasing cells.
Figure 5. Direct measurement of de noVo deposition of radioactive
proteins onto mixed SAMs presenting RGD groups and tri(ethylene
glycol) groups. Cells were cultured on mixed SAMs presenting RGD
groups and tri(ethylene glycol) groups, fibronectin-coated SAMs of
hexadecanethiolate (C16-SAM) or RGD-coated glass for 4 or 24 h in
the presence of radioactive (35S) amino acids. (A) Measurement of
radioactivity (cpm) of substrate after removal of cells at 4 or 24 h,
indicating amount of de noVo deposition of proteins. (B) Measurement
of radioactivity (cpm) of control substrates immersed in radioactive
medium in the absence of cells for 4 or 24 h, indicating amount of
passive adsorption of the labeled amino acids. Error bars indicated
standard error of the mean.
These results define sharply the molecular interaction involved
in adhesion between these cells and mixed SAMs presenting
RGD and (EG)3OH moieties and eliminate the ambiguity in
interpretation that has obscured results obtained with surfaces
that are less carefully tailored than these to limit adhesion to
only one type of interaction.
cifically adsorbed to SAMs of hexadecanethiolate, and RGD-
containing peptide nonspecifically adsorbed to glass. Addition
of the soluble peptide caused the rapid retraction and subsequent
release (2-30 min) of cells from all substrates (1%, 0.1% RGD,
FN, and RGD coated directly on glass). These results support
previous findings that cell attachment via RGD is reversible
and demonstrates that it is only necessary to antagonize
interactions with binding sites for RGD to cause release of an
attached cell from a surface presenting this ligand.
Conclusions
Fundamental studies of mechanisms of cell adhesion have
been limited in the past by the inability to design and generate
surfaces with defined molecular structure. Using SAMs to
address these technical limitations, we have demonstrated a

Attachment of BoVine Capillary Endothelial Cells to Surfaces
J. Am. Chem. Soc., Vol. 120, No. 26, 1998 6555
Figure 6. Retraction of cells caused by soluble GRGDSP peptide. Bovine capillary endothelial cells were allowed to attach to these substrates:
mixed SAMs presenting RGD and tri(ethylene glycol) groups, fibronectin-coated SAMs of hexadecanethiolate, and RGD-coated glass. After allowing
attachment to surfaces for 2, 4, or 24 h, soluble GRGDSP peptide to a concentration of 0.5 mg/mL. (A, B) Phase contrast micrographs of cells
(attached 4 or 24 h, respectively) immediately before and 10 min after addition of peptide. (C) Plots of projected cell area over time after the
addition of peptide. Between 25 and 50 cells were analyzed for each condition and time.
general approach to engineer surfaces with precise molecular
and chemical compositions to study specific cell-substrate
interactions.
of new matrix secreted by the attached cells onto the surface.
As a result, the specificity of the cell-substrate interaction was
maintained for at least 24 h. Thus, by using substrates that
present RGD in an interface that is otherwise inert to adsorption
and attachment of biological entities (proteins and cells), we
demonstrate that integrin-RGD interactions alone are sufficient
for long-term attachment and survival of cells.
Previous studies have shown that the binding of specific cell-
surface integrins to the RGD peptide can mediate adhesion of
cells to substrates.8-15 Interpretation of these studies was,
however, limited because it was difficult to determine the initial
density of RGD moieties required for efficient cell attachment
and subsequent spreading and to study the changes of the surface
over time that result from the active degradation and redeposition
of extracellular matrix onto the substrate by the cells.
By engineering a surface presenting RGD peptide in an
interface that is otherwise resistant to protein adsorption (because
it comprises (EG)3OH groups), we have created a substrate that
allows biospecific adsorption of cells directly to the SAM.
Furthermore, the synthetic strategy used to generate these SAMs
presents a surface that is well-defined at a molecular scale. The
use of an ethylene glycol in the SAM also reduces the deposition
Acknowledgment. This work was supported by the National
Institutes of Health (GM 30367 to G.M.W. and CA 55833 to
D.E.I.), the Office of Naval Research, the Advanced Research
Projects Agency, and the National Science Foundation (DMR-
94-00369). C.S.R. and M.M. are grateful to the National Science
Foundation and the American Cancer Society, respectively, for
postdoctoral fellowships. C.S.C. was partially supported by the
Harvard-M.I.T. Division of Health Sciences and Technology
Medical Engineering and Medical Physics Program.
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