Biomedical Engineering Reference
In-Depth Information
7.4.2.2 Preparation and Characterization of OVA Adsorbed
Aquasomes
The OVA loaded hydroxyapatite NMs were prepared with slight modifications
of a reported process.
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The process is described below.
(1)
Mix 15 mg of hydroxyapatite into a beaker with 30 mL of 29.2 mM treha-
lose with vigorous stirring.
(2)
Sonicate the suspension for 10 min at approximately 20 kHz at 25 °C using
a probe sonicator (Soniweld India Ltd., Mumbai, India).
(3)
Centrifuge the dispersion at 10,000 rpm for 20 min at 4 °C.
(4)
Discard any remaining pellets.
(5)
Lyophilize the supernatant at a condenser temperature of −82 °C and
pressure of less than 10−1 mbar using trehalose as stabilizer.
(6)
Dissolve in 20 mL of 25 mM phosphate buffer, pH 7.4.
(7)
Remove excess trehalose centrifugation.
(8)
Take 10 mg of the trehalose coated NMs (ceramic/carbohydrate based
biocompatible and biodegradable nanoparticulate system called “aqua-
somes”) and disperse in 8 mg/mL OVA in PBS pH 7.4 OVA.
(9)
Incubate the solution at 4 °C overnight.
(10)
Wash the OVA adsorbed aquasomes with DI water.
(11)
Centrifuge at 10,000 rpm for 30 min.
(12)
Repeat the washing three times and store at 4 °C.
(13)
Characterize as in the previous section.
(14)
Establish the sugar content on surface of hydroxyapatite following the
anthrone method
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using glucose as standard.
(15)
Establish the integrity of the OVA by SDS-PAGE under non-denaturing
condition.
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Extract the OVA out by dissolving the aquasomes in 2 mL of
5% (w/v) SDS in 0.1 N HCl. Use Coomassie blue staining for the protein
bands.
The results indicated that the aquasome formulations were spherical and elon-
gated in shape. Photon correlation spectroscopy (PCS) confirmed the nanometer
FIGURE 7.4
X-ray diffraction pattern of hydroxyapatite core particles obtained from the mixture
of calcium nitrate and di-ammonium hydrogen phosphate.
From Pandey, 2011.
