Magnetic Resonance Tracking of Stem Cells with Iron Oxide Particles - Intracellular Delivery: Fundamentals and Applications - page 470

of 24 h and a maximum concentration 0.2 mg/ml, as it has been shown that long
duration and high concentration, in particular the combination of both, can result
in cytotoxicity (van den Bos et al. 2003 ; Neri et al. 2008 ).

4.5 Electroporation and Transfection Agents

Inspired by DNA transfection technologies, electroporation and transfection agents
(TA) have been used to improve uptake of particles into cells. When ferumoxide
labelling was assisted by electroporation, iron loading of 11.5 pg/rat MSC was
achieved compared to 3 pg/rat MSC with simple incubation alone (Walczak et al.
2005 ). However, this technique requires careful tuning of the electrical parameters
to avoid affecting cell viability (Daldrup-Link et al. 2005 ).
Poly-L-lysine (PLL), a common cationic TA, binds with DNA to form complexes
of <100 nm in diameters. By complexing PLL with ferumoxide prior to their incu-
bation with hMSC, uptake between 13 pg/cell (Arbab et al. 2003b ) and 16 pg/cell
(Frank et al. 2003 ) have been reported. As an alternative to PLL, protamine sulphate,
an FDA-approved TA, has been complexed with ferumoxide to achieve loading of
11 pg/hMSC (Arbab et al. 2004a ). However, no comparison against the uptake by
simple incubation was done in these studies.
Despite the promising cellular uptake achieved with TA, the method has a few
potential disadvantages. The use of ferumoxide-PLL labelling inhibited chondro-
genesis (Kostura et al. 2004 ), although differentiation overall was unaffected when
hMSC were labelled with ferumoxide-protamine (Arbab et al. 2004b ). It was
suggested that the detrimental effect was a result of using PLL as the TA, rather
than the presence of ferumoxide (Bulte et al. 2004 ), notwithstanding a lack of
detailed study to examine the effect of PLL and protamine sulphate alone on the
chondrogeneicity of MSC.
Despite an apparent increase in iron loading, it has been reported that TA-mediated
labelling may result in only surface attachment to the cell rather than internalisation
of SPIO (Montet-Abou et al. 2007 ). In that study, the use of TA lipofactamine resulted
in SPIO attaching to the surface, in contrast to protamine sulphate which allowed the
complete internalisation of ferumoxide in mouse neural progenitor cells but not in
other cell types. These results suggest that the internalisation of SPIO may be depen-
dent on the type of TA, labelling condition and cell type. Another distinctive feature
of TA-mediated labelling is the cellular uptake with respect to the iron concentration
of the labelling medium. Labelling with naïve ferumoxide or other SPIO results in a
phenomenon where uptake increases with iron concentration of the labelling medium
and saturates at high labelling concentrations. PLL-mediated particle uptake on the
other hand, varies non-linearly with the labelling concentration (Kim 2008 ), and may
present difficulty in optimising cell labelling conditions for different cell types.
This observation may be due to the dynamic nature of the complex formation
between TA and SPIO, which is less understood compared to the complex forma-
tion between TA and DNA. The formation of PLL and DNA complexes, depending