Biomedical Engineering Reference
In-Depth Information
cells to confluent sheets on these surfaces at 37
C,
which is above the LCST of the polymer. When the
PNIPAAm collapses, the interface becomes hydrophobic
and leads to adsorption of cell adhesion proteins, en-
hancing the cell culture process. Then when the tem-
perature is lowered, the interface becomes hydrophilic as
the PNIPAAm chains rehydrate, and the cell sheets re-
lease from the surface (along with the cell adhesion
proteins). The cell sheet can be recovered and used in
tissue engineering, e.g., for artificial cornea and other
tissues. Patterned surfaces have also been prepared
(
Yamato
et al.
, 2001
). Smart polymers may also be
grafted to surfaces to provide surfaces of gradually
varying hydrophilicity and hydrophobicity as a function
of the polymer composition and conditions. This phe-
nomenon has been applied by Okano, Kikuchi, and co-
workers to prepare chromatographic column packing,
leading to eluate-free (''green'') chromatographic sepa-
rations (
Kobayashi
et al.
, 2001; Kikuchi and Okano,
2002
).
Ishihara
et al.
(1982
, 1984b) developed photo-
responsive coatings and membranes that reversibly
changed surface wettability or swelling, respectively, due
to the photoinduced isomerization of an azobenzene-
containing polymer.
proteins, and NHS attachment chemistry is most often
utilized. Other possible sites include -COOH groups of
aspartic or glutamic acid, -OH groups of serine or tyro-
sine, and -SH groups of cysteine residues. The most
likely attachment site will be determined by the reactive
group on the polymer and the reaction conditions, es-
pecially the pH. Because these conjugations are generally
carried out in a nonspecific way, the conjugated polymer
can interfere sterically with the protein's active site or
modify its microenvironment, typically reducing the
bioactivity of the protein. On rare occasions the conju-
gation of a polymer increases the activity of the protein.
(e.g.,
Ding
et al.
, 1998
).
Biomedical uses of smart polymers in solution have
mainly been as conjugates with proteins. Random conju-
gation of temperature-sensitive (mainly) and pH-sensitive
(occasionally) polymers to proteins has been extensively
investigated, and applications of these conjugates have
been focused on immunoassays, affinity separations,
enzyme recovery, and drug delivery (
Schneider
et al.
,
1981; Okamura
et al.
, 1984; Nguyen and Luong, 1989;
Ta n i g u c h i
et al.
,1989
,1992;
Chen and Hoffman, 1990;
Monji
et al.
, 1990; Pecs
et al.
,1991
; Park and Hoffman,
1992;
Ta k e i
et al.
,1993b
, 1994a;
Galaev and Mattiasson,
1993; Fong
et al.
, 1999; Anastase-Ravion
et al.
, 2001
). In
some cases the ''smart'' polymer is a polyligand, such as
polybiotin or poly(glycosyl methacrylate), which is used
to phase separate target molecules by complexation to
multiple binding sites on target proteins, such as strepta-
vidin and Concanavalin A, respectively (
Larsson and
Mosbach, 1979; Morris
et al.
, 1993; Nakamae
et al.
,
1994
).
Wu
et al.
(1992, 1993)
have synthesized
PNIPAAm-phospholipid conjugates for use in drug de-
livery formulations as components of thermally sensitive
composites and liposomes.
Site-specific smart polymer
bioconjugates on surfaces
Conjugation of a responsive polymer to a specific site
near the ligand-binding pocket of a genetically engi-
neered protein is a powerful new concept. Such site-
specific protein-smart polymer conjugates can permit
sensitive environmental control of the protein's recog-
nition process, which controls all living systems.
Stayton and Hoffman
et al.
(
Stayton
et al.
, 2000
) have
designed and synthesized smart polymer-protein con-
jugates where the polymer is conjugated to a specific
site on the protein, usually a reactive -SH thiol group
from cysteine that has been inserted at the selected site
(
Fig. 3.2.6-5
). This is accomplished by utilizing cassette
mutagenesis to insert a site-specific mutation into the
DNA sequence of the protein, and then cloning the
mutant in cell culture. This method is applicable only
to proteins whose complete peptide sequence is known.
The preparation of the reactive smart polymer is similar
to the method described above, but now the reactive
end or pendant groups and the reaction conditions are
specifically designed to favor conjugation to -SH groups
rather than to -NH
2
groups. Typical SH-reactive poly-
mer end groups include maleimide and vinyl sulfone
groups.
The specific site for polymer conjugation can be lo-
cated far away from the active site (
Chilkoti
et al.
, 1994
),
in
Smart polymers on surfaces
One may covalently graft a polymer to a surface by ex-
posing the surface to ionizing radiation in the presence of
the monomer (and in the absence of air), or by preirra-
diating the polymer surface in air, and later contacting
the surface with the monomer solution and heating in the
absence of air. (See also Section 3.1.4 on surface prop-
erties of materials.) These surfaces exhibit stimulus-
responsive changes in wettability (
Uenoyama and
Hoffman, 1988; Takei
et al.
, 1994b; Kidoaki
et al.
, 2001
).
Ratner and co-workers have used a gas plasma discharge
to deposit temperature-responsive coatings from
a NIPAAm monomer vapor plasma (
Pan
et al.
, 2001
).
Okano and Yamato and co-workers have utilized the ra-
diation grafting technique to form cell culture surfaces
having a surface layer of grafted PNIPAAm. (
Yamato and
Okano, 2001; Shimizu
et al.
, 2003
). They have cultured
order
to
avoid
interference
with
the
biological
