Biomedical Engineering Reference
In-Depth Information
Micropatterning peptides on surfaces that support protein physisorption immediately raises
the question of whether cells will, sooner or later, attach to ECM proteins present in the medium
or secreted by the cells. Increased control over cell-substrate interactions may be achieved by
immobilizing cell-adhesion peptides on PEG copolymers or PEG-functionalized SAMS. In
1998, Molly Shoichet's group at the University of Toronto, in Canada, devised a simple method to
micropattern cell attachment peptide sequences on a PEG background. First, PEG-aldehyde was
immobilized on an aminosilane SAM on glass. A microfabricated grid was then placed in con-
tact with the PEG surface and used as a stencil mask for gold sputtering, resulting in gold islands
on a PEG background. Finally, commercially available peptide sequences labeled with cysteine
(which contains a gold-reactive thiol group) were immobilized on the gold areas. Hippocampal
neurons seeded under serum-free conditions were observed to attach selectively to the peptide-
modiied areas only and displayed neurite extension statistics similar to or greater than those
found in neurons seeded on laminin. Similarly, a team led by George Whitesides at Harvard
University and Alexander Rich at MIT microstamped hexa(ethylene glycol)-terminated alka-
nethiol SAMs on gold; next, they chemisorbed (on the nonstamped areas) a cysteine-ended oli-
gopeptide containing a cell-adhesion peptide sequence to demonstrate the selective attachment
of cells (human epidermal carcinoma cells, 3T3 mouse embryo ibroblasts, and bovine aortic
endothelial cells) onto the peptide areas in medium containing 10% serum (
Figure 2.44
).
2.6.3.2 Selective Photochemical Immobilization of Peptides
In 1995, Patrick Aebischer's group, then at Lausanne University, coupled the laminin fragment
CDPGYIGSR to a photosensitive benzophenone or diazirin group. hus, the peptide could
be immobilized onto various materials, such as hydroxylated luorinated ethylene propylene,
poly(vinyl alcohol), and glycophase glass, by exposure to UV light from a high-pressure mercury
lamp. Benzophenone is a particularly convenient photosensitive group because, unlike azide-
based compounds, it is reversibly excitable and stable in ambient light. Peptide micropatterns
were created by selective exposure through a mask, and selective neuroblastoma cell attachment
onto the peptide areas was demonstrated. Later, this photochemistry was applied by laser to an
agarose gel to form 3-D patterns of the laminin fragment in agarose.
Similarly, in 1995, Takashi Sugawara and Takehisa Matsuda from the National Cardiovascular
Center Research Institute in Osaka, Japan, reacted the amine terminus of synthetic peptide
sequences with 4-azidobenzoyloxysuccinimide, a photoreactive and amino-reactive cross-linker;
on UV exposure, the peptide (now linked to a photosensitive group) could be immobilized onto
poly(vinyl alcohol) in micropatterns by selective exposure through a chrome mask. Bovine endothe-
lial cells attached selectively onto the peptide patterns in medium containing 15% fetal calf serum.
In 1997, a large collaborative team led by Wei-Shou Hu from the University of Minnesota
also created micropatterns of synthetic cell-adhesive peptides that had been photosensitized
by coupling to benzophenone. As a nonadherent background surface, they used a SAM of
hexaethyleneglycol-undecanethiolate on gold, a surface designed to repel cell and protein adhe-
sion. When this surface was UV-illuminated under a solution of photosensitive oligopeptides, it
became cell-adherent. By scanning a UV laser beam or by UV-illuminating the surface through
a chrome mask, they were able to create oligopeptide (adhesive) micropatterns on a nonadhe-
sive background. Because the density of immobilized RGD groups (i.e., the adhesiveness of the
surface) can be ine-tuned by adjusting the exposure, they were able to create
gradients
of adhe-
siveness by varying the duration of exposure to the UV laser beam. Fibroblasts attached to these
surfaces at a density that increased with increasing oligopeptide density.
2.6.3.3 Selective Microluidic Delivery of Peptides
In 1998, a collaborative team led by Kevin Shakeshef from the University of Nottingham in
the United Kingdom and by Robert Langer from MIT used microluidic patterning to create
peptide templates for cell attachment (
Figure 2.45
). First, amine-terminated PEG was cou-
pled to NHS-biotin, and lactide was polymerized from the hydroxy end of PEG. he obtained
