br The route of FA CS
The route of [email protected] NP preparation is illustrated in Scheme 1. Firstly, the FA-CS conjugate was prepared as previously described . A mixture of FA, EDC and NHS (1.0 M, molar ratio 1:1:1) was prepared in 15 mL anhydrous DMSO, and continuously stirred at room temperature until the FA was fully dissolved (ca. 1 h). Thereafter, the activated FA was slowly added to a 1.0% (w/v) solution of CS (5.0 g in 500 mL of acetate buffer, Moniliformin 4.7). The resulting mixture was stirred at room temperature overnight in the dark. The product was dialyzed against 1.0 mM aqueous NaOH with a 3500 Da molecular weight cutoff (MWCO) membrane for 72 h to remove unreacted FA. Finally, the resultant FA-CS was isolated by lyophilization.
Subsequently, OA was grafted to FA-CS via an esterification reac-tion. Briefly, 0.50 g of FA-CS was dissolved in 12 mL of pyridine in the presence of DMAP (0.05 g), and 0.90 g OA was added. The molar ratio of OA –COOH groups to FA-CS –OH groups was 2:1. The reaction was kept at room temperature for 3 h and then the solvent evaporated under reduced pressure. The precipitate was dissolved in acetone, and washed three times with water (3 × 5 mL). The resultant solid FA-CS-g-OA was then collected (yield 1.02 g, 72.9%). Analogous CS-g-OA particles were prepared by reacting OA with non-functionalized CS.
The FA-CS-g-OA and CS-g-OA NPs were seen to self-assemble to nanospheres in an aqueous medium, and the antitumor drug DOX was loaded via the solvent dialysis method. In this, freeze-dried NPs (100 mg) and DOX (20 mg) were dissolved in DMSO (10 mL), sealed in a dialysis bag with a 8000–10,000 Da Mw cutoff, and dialyzed against water for 48 h at 25 °C. The resultant suspension was centrifuged at 6000 rpm for 30 min to remove any unencapsulated drug.
2.5. Characterization of NPs
The mean diameters, polydispersity indices (PDI) and zeta poten-tials (ZP) of CS, FA-CS, FA-CS-g-OA, and [email protected] NPs were determined on a Zetasizer Nano ZS90 instrument (Malvern Instruments, Westborough, MA, USA) using a dynamic light scattering (DLS) method. Measurements were performed in triplicate in three in-dependent experiments. For structure confirmation, Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet Nexus 670 spec-trometer (Thermo Fisher, Waltham, MA, USA) using powdered NPs (ca. 2 mg) dispersed in KBr (ca. 200 mg). Scans were recorded over the range 3500–1125 cm−1 with a resolution of 2 cm−1 and 64 scans taken per sample. 1H NMR measurements were performed on a DRX 400 nuclear magnetic resonance spectrometer (Bruker, Faellanden, Switzerland) using D2O or d6-DMSO as the solvent. Ultraviolet (UV) spectra were measured using a UV-2012 spectrophotometer (UNICO, Shanghai, China). The morphologies of the [email protected] NPs were examined using a scanning electron microscope (SEM; Nova TM Nano SEM, FEI, Hillsboro, OR, USA) and transmission electron micro-scope (TEM; JEM 1200EX, JOEL, Tokyo, Japan). For TEM experiments, a drop of dispersed NPs was placed onto a carbon-coated grid, and the excess removed. Phosphotungstic acid hydrate (0.5%, 10 μL) was then added, followed by air drying for 30 min. Thermogravimetric analysis (TGA) was performed using a DTG-60H analyzer (Shimadzu, Kyoto, Japan).
To determine the percentage encapsulation efficiency (EE) and drug loading (DL) of DOX in [email protected], a dispersion of the NPs was digested with DMSO (100 mL). After centrifugation to precipitate any solid material, the amount of unencapsulated drug in the supernatant was determined by UV/vis spectroscopy. The DOX concentration was determined with reference to a pre-determined calibration curve. The EE and DL were calculated as follows: %EE = (DOXt – DOXf) / DOXt × 100%