pH-Sensitive Nanocrystals of Carbonate Apatite- a Powerful and Versatile Tool for Efficient Delivery of Genetic Materials to Mammalian Cells - Advances in Biomaterials Science and Biomedical Applications - page 272

expression level for carbonate apatite-mediated transfection was over 25-fold higher than
for lipofection and CaP co-precipitation method (Fig. 2c). Nano gram level of DNA was
even sufficient for efficient transgene expression (Fig. 2c). Transfection efficiency was also
significantly high in HepG2 (Fig. 2d), NIH 3T3 cells (Fig. 2e) and mouse primary hepato‐
cytes (Fig. 2f). We performed MTT assay in HeLa cells (not shown here) to clarify that high
transfection efficiency was accompanied by high viability of the cells [12].
3.4. Estimation of particle sizes and cellular uptake of particle-associated plasmid DNA
To explore why carbonate apatite is so efficient as a vector for gene delivery, we investigat‐
ed two basic properties of carbonate apatite [12]. Carbonate, when present in the apatite
structure, limits the size of the growing apatite crystals and increases the dissolution rate
[12]. We carried out scanning electron microscopic observation of generated carbonate apa‐
tite (Fig. 3A) which revealed reduced growth of the crystals, most of which had diameters of
50 to 300 nm. We verified this size limiting effect of carbonate by observing cellular uptake
of the PI (propidium iodide)-labeled plasmid DNA adsorbed to the apatites, since large par‐
ticles are phagocytosed less efficiently than small ones [12]. DNA was carried into the cells
by carbonate apatite (Fig. 3B-c) at least 10 times more efficiently than hydroxyapatite, gener‐
ated by 1 min incubation (Fig. 3B-d). Longer period (30 min) incubation resulted in large hy‐
droxyapatite particles [12], showing significantly reduced transfection efficiency [12] (Fig.
2A) due to extremely low cellular uptake of DNA [12]. Our findings, therefore, clearly sug‐
gest that carbonate apatite is superior over hydroxyapatite for its intrinsic property of pre‐
venting crystal growth, leading to high efficiency cellular uptake of DNA.
Figure 3. A, scanning electron microscopy, showing limited growth of generated carbonate apatite cryatals. Scale bar,
600 nm. B, cellular uptake of PI-labeled plasmid DNA associated with carbonate apatite and hydroxyapatite. a, no up‐
take of DNA (control), since endocytosis was blocked by energy depletion (50 mM 2-deoxy glucose and 1 mM Na-
azide). DNA/carbonate apatite particles were prepared in 1 ml serum-free media (described in legend to Fig. 3) using
6 mM Ca 2+ and 2 μg DNA. 40 ng (b) and 200 ng (c) of DNA in 20 μl and 100 μl of 1ml suspension respectively, were
allowed for cellular uptake for 4 hr. d, 2 μg of DNA adsorbed to hydroxyapatite (described in experimental protocol)
was allowed for uptake for the same period of time. Bar indicates 50 μM.