Biomedical Engineering Reference
In-Depth Information
latory domains, (ii) limited specificity among integrin adhesion receptors,
and (iii) inability to bind certain receptors due to conformational differences
compared to the native ligand. These limitations are critical shortcomings
because specific integrin receptors trigger different signaling pathways and
cellular programs [48, 68-72]. Consequently, “second-generation” bioligands
have been pursued to address the limitations associated with short bioadhe-
sive oligopeptides.
3.3
Second-Generation Biomimetic Adhesive Supports:
Ligands with Integrin Specificity
Engineered ligands, both short oligopeptides and recombinant protein frag-
ments, incorporating additional residues or/and structural characteristics
mimicking the native ligand have been developed to convey receptor speci-
ficity among RGD-binding integrins (Fig. 5). As discussed in Sect. 2.3, binding
of
specific
integrin receptors can be exploited to regulate distinct cellular out-
comes. Inclusion of flanking residues and constraining the conformation of
the RGD motif to a loop via cyclization improve ligand specificity for inte-
grins [73-75]. Nevertheless, these short peptides are limited in their ability to
support specific integrin binding. For example, RGD domains in a loop con-
formation similar to FN bind
α
β
3
but support poor
α
β
1
binding when com-
V
5
pared to native FN [76]. Binding of
1
requires both the PHSRN sequence
in the 9th type III repeat and RGD motif in the 10th type III repeat of FN [77].
Each domain independently contributes little to binding, but in combination,
they synergistically bind to
α
β
5
α
5
β
1
[78, 79]. In efforts to include this essential
PHSRN synergy site outside the RGD binding motif in fibronectin, mixtures
of RGD and PHSRN peptides, either independently or within the same back-
bone, have been tethered onto nonfouling supports [80, 81]. Although these
ligands support integrin binding and cell adhesion, their activity has not been
directly compared to FN. Due to the high sensitivity of
α
5
β
1
-FN binding to
small perturbations in the structural alignment of these domains [70, 82], re-
constitution of the proper binding structure using short peptides remains
a challenging task. As an alternative to these synthetic routes, recombinant
FN fragments spanning the 9th and 10th type III repeats have been teth-
ered onto supports or incorporated into peptide backbones [83, 84]. These
engineered ligands support robust
α
5
β
1
-mediated adhesion and focal ad-
hesion assembly at levels comparable to native FN (Fig. 5). In addition to
providing increased specificity over linear RGD peptides, the use of recombi-
nant fibronectin fragments offers several advantages compared to whole FN,
including reduced antigenicity, elimination of domains that may elicit unde-
sirable reactions, and enhanced cost efficiency. Recombinant fragments also
provide flexibility in the engineering of specific characteristics on the frag-
ment via site-directed mutagenesis in order to enhance tethering and activity.
