br The surfactant CTAC was extracted using methanolic acidic
The surfactant CTAC was extracted using methanolic acidic condi-tions. 1.8 g of [email protected] was dispersed in 300 mL of anhydrous methanol with 16 mL (37.4 wt%) of concentrated hydrochloric acid. After vigorously stirring overnight, the mixture was refluxed at 60 °C for 48 h to eﬀectively extract CTAC and obtain MSN.
1.2 g of [email protected] was dispersed in 150 mL of methanol with 4 mL of 3-mercaptopropyltrimethoxysilane and the mixture was stirred vigorously for 24 h at ambient temperature. [email protected] was ob-tained after centrifugation (10,000 r/min, 10 min), washing several times with DI water and methanol and drying under vacuum. The method for removing surfactant of [email protected] was ac-cording to the above methanol-acidic extraction method.
First, S-(2-aminoethylthio)-2-thiopyridine hydrochloride was syn-thesized manually according to previous literature . Colloids and Surfaces B: Biointerfaces 175 (2019) 65–72
Then, 800 mg of MSN-SH was dispersed in 160 mL of methanol with 800 mg of S-(2-aminoethylthio)-2-thiopyridine hydrochloride and the mixture was stirred vigorously for 24 h at ambient temperature. MSN-SS-NH2 was obtained after centrifugation (8000 r/min, 10 min), washing with DI water and methanol and drying in vacuum.
2.5. Preparation of MSN-SS-alkyne
600 mg of MSN-SS-NH2 was dispersed in 160 mL of methanol. After adding 6 mL of bromopropyne and 6 drops of triethylamine, the mix-ture was vigorously stirred at ambient temperature for 24 h. By cen-trifuging (8000 r/min, 10 min), washing with DI water and anhydrous methanol and drying in vacuum, MSN-SS-alkyne was obtained.
2.6. Preparation of MSN-SS-KLA
100 mg of MSN-SS-alkyne was dispersed in 10 mL of methanol. Then 100 mg of KLA-N3, 100 mg of CuSO4 • 5H2O and 158.5 mg of sodium ascorbate were added in the solution. The reaction is carried out under the protection of nitrogen Artesunate at ambient temperature for 3 days. After centrifugation (8000 r/min, 5 min), washing with DI water and methanol and drying in vacuum, MSN-SS-KLA was obtained.
2.7. Preparation of [email protected]
50 mg of MSN-SS-KLA was dissolved in 50 mL of anhydrous me-thanol and sonicated. Then 8 mg of DOX was added and the mixture was further sonicated for 1 h. [email protected] was obtained after the mixture being stirred vigorously overnight, and dialyzed for several days.
2.8. Preparation of [email protected]/BSA
10 mg of [email protected] was dissolved in 10 mL of DI water, then 10 mL of 1 mg/mL BSA was added. The mixture was stirred at ambient temperature overnight. After centrifugation and washing, [email protected]/BSA was obtained.
2.9. In vitro release study
In vitro release experiments were carried out under diﬀerent con-ditions: PBS without dithiothreitol (DTT) at pH 7.4, PBS with 0.5 mM DTT at pH 7.4, PBS with 5 mM DTT at pH 7.4, PBS without trypsin at pH 7.4, PBS with 30 U trypsin at pH 7.4, PBS with 60 U trypsin at pH 7.4. Commonly, 5 mg of the corresponding nanoparticles ([email protected]/BSA) was suspended in 1 mL of diﬀerent PBS solution. The solution was put into a dialysis bag (MWCO: 3500 Da) which was di-rectly immersed into 5 mL of PBS buﬀer solution and then placed on a shaker (SHZ-82 A) at 37 °C under oscillating slowly (150 rpm). After particular time intervals, fluorescence intensity of the PBS buﬀer so-lution was analyzed by FL-4600 spectrofluorophotometer (Hitachi) (λex = 488 nm, slit width = 5 nm). The cumulative release of DOX was calculated according to the respective standard curve.
Cells co-culture with diﬀerent materials was carried out using HeLa cells (Human cervicalcancer cells). Firstly, HeLa cells were seeded into a 24-well cell culture plate at a density of 5 × 104 cells/well, and 1 mL of DMEM medium containing 10% FBS and 1% penicillin was added to each well. Then the cells were incubated in a 5% CO2 incubator at 37 °C for 24 h. Thereafter, 1 mL DMEM medium containing [email protected]/BSA (10 μg/mL) was added into the cell culture plate. After in-cubating for 4 h, the medium in the each well was discarded and the nucleus of cells were stained with 100 μL of Hochest 33,258 staining solution. After further incubating for 20 min, the staining solution in