Cell Surface Engineering Using a Layer-by-Layer Nanofilm for Biomedical Applications - Cell Surface Engineering: Fabrication of Functional Nanoshells - page 223

solutions. 11,12 The preparation of nanometer-sized multilayer films com-
posed of ECM components on the surface of the first layer of cells will
provide a cell-adhesive surface for the second layer of cells. Rajagopalan
et al. demonstrated a bilayer structure composed of hepatocytes and other
cells by preparing a polyelectrolyte (PE) multilayer consisting of chitosan and
DNA onto the hepatocyte surface. 101 However, chitosan cannot dissolve in
neutral buffer, and the use of PE films as a cell-adhesive material is limited
due to the cytotoxicity of polycations. 102,103 The appropriate choice of natural
ECM components for preparation of the nanofilms is important to avoid
cytotoxicity, and the typical ECM presents cell-adhesive moieties such as RGD
(arginine-glycine-aspartic acid) and other functional moieties. 104 We selected
FN and G to prepare nano-ECM films on the cell surface. FN is a flexible
multifunctional glycoprotein, and plays an important role in cell attachment,
migration, differentiation, and so on. 2 FN is well known to interact not only
with a variety of ECM proteins such as collagens (gelatins) and glycosami-
noglycans but also with the a 5 b 1 integrin receptor on the cell surface. 105 Al-
though FN and G have a negative charge under physiological conditions, they
interacted with each other because FN has a collagen binding domain, 2 in-
dicating different driving force of PE films using polycations. Thus, the FN-G
nanofilms are expected to provide a suitable cell-adhesive surface similar to
the natural ECM for the second layer of cells, without any cytotoxicity.
The fabrication of 3D-cellular multilayers composed of cells and FN-G
nano-ECM films was performed according to the process shown in
Figure 11.4. The LbL assembly of FN and G onto the cell surface was ana-
lyzed quantitatively using a QCM as the assembly substrate, and with a
phospholipid bilayer membrane as a model cell membrane. 78 A phospho-
lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine
(DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) was prepared
onto the base layer, a 4-step assembly of poly(diallyldimethylammonium
chloride) (PDDA) and PSS, according to Krishna et al.'s report. 106 The mean
thickness of the LbL assembly after 1, 7, and 23-steps was calculated to be
2.3, 6.2, and 21.1 nm, respectively. The top and cross sections of the confocal
laser scanning microscopy (CLSM) 3D-merged images indicated a homo-
geneous assembly of fluorescently labeled FN-G nanofilms on the surface of
mouse L929 fibroblasts. For the quantitative studies on the thickness of the
multilayers on the cell surface, the fluorescence intensity of rhodamine-la-
beled FN (Rh-FN) was estimated by a line scan. The fluorescence intensity of
the Rh-FN-G nanofilms increased linearly on increasing the LbL assembly
step number, similar to the frequency shift of the QCM analysis, indicating a
clear increase in the film thickness on the cell surface. These results dem-
onstrated the fabrication of FN-G nanofilms on the cell surface.
We fabricated a bilayer of mouse L929 fibroblast cells with or without
FN-G nanofilms by using a cover glass as a substrate. When the 7-step as-
sembled FN-G nanofilms were prepared on the surface of the first L929 cell
layer, the second layer cells were then observed on the first cell layer.
However, when the nanofilm was not prepared or the 1-step-assembled

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