Biomedical Engineering Reference
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O
O
Oxalyl chloride
OH
Cl
0 °C to reflux
PEG, DMF
100 °C,
5 days
1
2
EN, DMF
100 °C, 5 days
A
D
B
C
PEI, NMP
r.t., 6 h
Reflux, 24 h
O
O
PABS, DMF
100 °C, 5 days
(
)
OH
n
NH 2
O
H
6
3
H 2 N
) n
O
O
(
N
) n
(
SO 3 H
H
H
4
5
Scheme 6.1 Functionalisation of CNTs with EN, PABS, PEI and PEG.
covalent functionalisation of MWNTs rather than on physisorption (used
in the previous study) 5 to allow transient retention of grafted molecules
on the nanotube surface. MWNTs were irst oxidised using nitric acid,
and the resulting COOH functions introduced on the nanotube ends were
then activated and coupled with either ethylenediamine (EN) or poly-
m -aminobenzene sulphonic acid (PABS), 12 as illustrated in Scheme 6.1
(paths A and B).
At physiological pH used to grow neurons, the MWNTs exhibited different
surface charges, from negatively charged MWNT-COOH ( 1 ), neutral/
zwitterionic MWNT-PABS ( 4 ), to positively charged MWNT-EN ( 3 ). Finally,
MWNT ilms were deposited on glass coverslips coated with PEI, and
hippocampal neuronal cells were cultured on these substrates. In addition to
SEM analysis, the morphological features of live (rather than ixed) neurons,
which directly reflect their potential capability in synaptic transmission,
were characterised by luorescence spectroscopy. The neurons were labelled
with calcein, which is a luorescent dye that stains only live cells. All neurons
that grew on PEI and on MWNT substrates accumulated calcein, which is
indicative of their viability. The number of neurites per neuron remained
the same for the three substrates (Scheme 6.2a). 11-15 However, the average
length of neurites was longer on the positively charged MWNT-EN ( 3 ), while
the number of growth cones was higher on neurons cultured on zwitterionic
MWNT-PABS ( 4 ) or on positive MWNT-EN ( 3 ). Furthermore, the branching of
neurites increased as the substrate became more positive. Thus, by varying
the surface charge of CNTs it was possible to control the number of growth
cones, neurite outgrowth and branching
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