Biomedical Engineering Reference
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Figure 14.
Comparison of luciferase expression for differentially formulated apatite particles and liposomes. Particles
were prepared by addition of 3 μl of 1 M CaCl
2,
2 μg of luciferase plasmid DNA and 2 μg of either fibronectin, E-cad-Fc
or both to 1 ml bicarbonate-buffered DMEM and incubation for 30 min at 37
0
C. Embryonic stem cells were incubated
with the generated particles for 4 hr and after replacement of particle-containing media with fresh media, further in‐
cubated for 1 day in order to quantitate luciferase expression. Transfection efficiency was normalized after estimation
of total proteins in cell lysate. Transfection by lipofectamine was performed according to the instructions provided by
Invitrogen.
3.10. DNA binding with differentially formulated cell adhesive protein-embedded
particles
Since direct mixing of DNA and cell-adhesive proteins in Ca
2+
and PO
4
3-
-containing medi‐
um prior to induction of particle formation by incubation at 37
0
C for 30 min, could interfere
with maximum DNA loading due to the competitive binding of the proteins to the growing
crystals, we investigated DNA binding efficiency by first adding DNA to the particle-prepa‐
ration medium prior to time-dependent addition of the proteins [22]. As shown in Fig. 15, in
the direct mixing process (control), DNA binding is much higher for E-cadherin-Fc com‐
pared to fibronectin, indicating that E-cadherin-Fc facilitates DNA loading probably by accel‐
erating particle growth because turbidity of particle suspension was higher for E-cadherin-
Fc than for fibronectin (not shown). It is worth mentioning that only particles have also higher
affinity towards DNA (almost 40%) that the particles associated with fibronectin which showed
lower turbidity than the particles (mentioned before), suggesting again that particle growth
has a significant role in the observed DNA binding efficiency. When cell adhesive proteins
were added after 5, 10 and 20 min from the start of incubation of DNA-containing particle-
preparation medium, followed by incubation for an additional 25, 20 and 10 min respective‐
ly, DNA binding to the particles was enhanced to a significant extent for fibronectin, E-
cadherin and fibronectin/E-cadherin-Fc compared to the control, suggesting than a competitive
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