Biomedical Engineering Reference
In-Depth Information
cles. Generation of these particles only in HCO
3
-
-, but not in Hepes-buffered media or solution
(pH 7.5) containing the same amount of exogenous Ca
2+
and phosphate (0.9 mM), indicates
the possible involvement of carbonate along with phosphate and Ca
2+
in particle formation.
Elemental analysis proved the existence of C (3%), P (17%) and Ca
2+
(32%) and FT-IR spectra
(Fig. 1a) identified carbonate, as evident from the peaks between 1410 and 1540 cm
-1
and at
approximately 880 cm
-1
, along with phosphate in the particles, as shown by the peaks at
1000-1100 cm
-1
and 550-650 cm
-1
. X-ray diffraction patterns (Fig. 1b) indicated less crystalline
nature, represented by broad diffraction peaks of the particles, compared to that of hydrox‐
yaptite (Fig. 1c) - an intrinsic property of carbonate apatite [12].
Figure 1.
Infrared spectra of generated carbonate apatite (A) and X-ray diffraction patterns of carbonate apatite (B)
and hydroxyapatite (C).
3.2. Influences of pH and temperature on generation of effective particles of carbonate
apatite
We have investigated a long range of pH (7.0 to 7.9) of the HCO
3
−
-buffered medium as well
as incubation temperatures (25 °C to 65 °C) in order to make particles by exogenously added
Ca
2+
and subsequently transfect HeLa cells using the generated particles. Interestingly, the
optimal Ca
2+
concentrations required for generation of effective number of DNA/carbonate
apatite particles leading to the high transfection efficiency, were inversely related to the pHs
of the media (Fig. 2-a) and the incubation temperatures (Fig. 2-b). Thus, while 4 mM Ca
2+
was sufficient to induce particle formation at pH 7.4 by incubating the Ca
2+
-supplemented
buffered medium for 30 min at 37 °C, only 1 mM
2+
was enough to stimulate particle genera‐
tion to the similar level at pH 7.9. Like pH, incubation temperatures have also profound and
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