2.6. Characterization of MSN@DOX

DOX was selected as a drug model for applying MSNs to drug delivery systems. Here, DOX-loaded MSNs (MSN@DOX) were developed and evaluated by assessing the drug loading, in vitro drug release, cytotoxicity, and cellar uptake of MSN@DOX.

HPLC analysis of DOX was performed with the Agilent 1100 HPLC system (Agilent Technology, Santa Clara, USA) equipped with a UV detector. The column used was XterraTM RP C18, 5 μm × 4.6 mm × 250 mm, and the column temperature was maintained at 40 °C. The mobile phase was prepared with ACN and 10 mM NaH₂PO₄ (pH 4.0, phosphoric acid) at a ratio of 70:30. The flow rate was set to 1 mL/min and the injected volume was 20 μL. The detection wavelength was set to 480 nm, respectively. The encapsulation efficiency (EE) and loading capacity (LC) of DOX were calculated through the following equation.

DOX was loaded into the MSNs using a diffusion–filling–precipitation method [26]. Briefly, various amounts of DOX and 10 mg of MSNs were mixed in 5 mL of distilled water, and stirred slowly for 24 h under protection from light at RT. The mixture was adjusted to pH 7.8 by adding a dibasic sodium phosphate solution (0.1 mol/L), and a desalination process was induced. The DOX molecules adsorbed through the desalination process were precipitated and adsorbed into MSNs.

Each sample was centrifuged at 12,000 rpm for 10 min to collect MSN@DOX, and then washed 3 times with distilled water to remove unreacted material. Then, MSN@DOX powder was obtained through a lyophilization process. The amount of DOX in MSN@DOX was determined by measuring the supernatant obtained through centrifugation in the washing process by the HPLC method.

To confirm the release profile of DOX from MSN@DOX, an in vitro drug release test was conducted in PBS solutions with pH values of 5.0, 6.8, and 7.4. Briefly, a 1 mg/mL MSN@DOX suspension containing 2 mg of DOX was added to a 6–8 kDa dialysis bag, and stirred in a tube including 40 mL of PBS at 37 °C at 100 rpm. The drug release test was conducted for 48 h, and at predetermined time points, 0.5 mL of the sample was collected by filtration through a 0.45 μm filter. The same volume of fresh PBS was supplied to maintain the sink condition. The DOX concentration of the sample was determined by the HPLC method as in the Section 2.6.1.