br In this study in order to realize the controlled
In this study, in order to realize the controlled and targeted delivery of DOX, a light-responsive amphiphile, HA-NB-SC, was synthesized, and was used as a vehicle for targeted cancer therapy (Scheme 1). HA-NB-SC was characterized by nuclear magnetic resonance (NMR), fourier transform infrared (FTIR) spectrometer, X-ray diﬀraction (XRD), ele-ment analysis and UV–vis spectrophotometer. HA functioned as the main chain owing to its excellent hydrophilicity and specific targeting to tumor cells via overexpressed CD44 receptors. DOX was loaded in the core of HA-NB-SC nanomicelles. The physicochemical characterization, in vitro drug release upon UV irradiation, in vitro cytotoxicity, in vitro cellular uptake, and competitive inhibition assay of the advanced na-nomicelles have been investigated in detail herein.
2. Materials and methods
Sodium hyaluronate (10 kDa) was purchased from QuFu GuangLong Biochem Co., Ltd. N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), 4-Dimethylaminopyridine (DMAP), tetra-butylammonium hydroxide (TBAH), stearyl alcohol (SC), 4 Å molecular sieves, 3-nitro-4-(bromomethyl)benzoic Kainic acid (NB), doxorubicin hydro-chloride (DOX·HCl), fluorescein diacetate (FDA), nile red (NR), 4′,6-Diamidino-2-phenylindole (DAPI) were purchased from Aladdin Chemical Reagent Company, China. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide), Hoechst 33342 and propidium iodide (PI) was obtained from Sigma Co. (St. Louis, MO, USA). Ion-exchange resin, Amberlite IR-120 was purchased from Alfa Aesar, China. Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco Ltd. (Grand Island, NY, USA) and fetal bovine serum (FBS) was from Carbohydrate Polymers 206 (2019) 309–318
Hyclone (Logan, UT, USA). Dichloromethane (DCM) was dried by re-fluxing over CaH2 and distilled prior to use. Dimethyl sulfoxide (DMSO) was dried over molecular sieves and other reagents were purchased locally and all of analytical grade.
2.2. Synthesis of the amphiphilic unit
HA-NB-SC was synthesized according to a reported procedure with minor modifications (Sun et al., 2017, 2018; Vazquez et al., 2009). Sodium hyaluronate was dissolved in distilled water, and then passed through a cationic-exchange resin (H+ form). The above solution was neutralized by TBAH up to pH 7 obtaining HA salt named HA-TBAH. After freeze-drying, HA-TBAH (500 mg) was dissolved in dry DMSO, followed by dropping of NB-SC (10 mol% compared to the disaccharide units of HA) in dried DMSO. The mixture was stirred at 40 °C for 48 h, and dialysis against 0.1 M NaCl, hot ethanol and distilled water, re-spectively. HA-NB-SC sponge was fabricated by freeze-drying.
The characterization is described in Supplementary data in detail.
2.4. Determination of critical micelle concentration (CMC)
The CMC was determined using NR as a fluorescence probe. A stock solution of NR in acetone was added to a vial and the solvent was al-lowed to evaporate to form a thin film at the bottom of the vial. Polymeric micelle solutions at diﬀerent concentrations were added to the vials and the final NR concentration was 5 × 10−6 M in water. The concentrations of polymer nanomicelles varied from 1 × 10-4 mg/mL to 6.0 mg/mL. The solutions were kept on a shaker at room temperature for 12 h to reach equilibrium prior to fluorescence measurement. Fluorescence spectra were recorded on a fluorescence spectro-photometer at room temperature. The excitation spectra were scanned from 580 to 750 nm at the emission wavelength of 550 nm. The fluor-escence intensity was analyzed as a function of micelle concentrations. r>