Ac 80

A ¹H NMR spectrometer (AC-80, Bruker Biospin, Germany) was used to obtain ¹H NMR spectra of chemicals. Celastrol was dissolved in deuterated DMSO, while CSOSA, NH₂-PEG₂₀₀₀-NH₂, and TET-CSOSA polymers were dissolved in heavy water (D₂O) at the concentration of 10 mg/mL. The degree of amino substitution (SD%) of CSO on micelles was detected by 2,4,6-trinitrobenzenesulfonic acid (TNBS) test. A Zetasizer (3000HS, Malvern Instruments Ltd, UK) was used to examine the hydrodynamic diameters and zeta potentials of blank and Celastrol-loaded micelles in DI water at the concentration of 1 mg/mL. A transmission electron microscope (TEM;JEOLJEM-1230, Japan) was used to observe the morphological characters of Celastrol-loaded micelles. The samples were placed on copper grids with films and stained with 2% (w/v) phosphotungstic acid for TEM viewing. Pyrene was used as a probe to determine the critical micelle concentration (CMC) of CSOSAand TET-CSOSA by fluorescence measurement. The intensity ratio of the first peak (I₁, 374 nm) to the third peak (I₃, 384 nm) and the polymer concentration were analyzed to calculate CMC.

Chitosan at molecular weight of 18.8kDa was gained by degrading 95% deacetylated chitosan (Mw=450kDa, Yuhuan, China) with enzymatic 25 (link). The glycolipid polymer (GLPM) was synthesized on the basis of method as reported before with modification 26 (link). Totally, 0.159 g SA and 0.535 g EDC were mixed into 7.5 mL ethanol for 1h at 60 ℃, 0.3 g chitosan (Mw=18kDa) were added into 15 mL water at 60 ℃ for 30 min. Then, the solutions were mixed dropwise and stirred at 60 ℃ for 12h. Subsequently, mixtures were dialyzed against water by dialysis bag method (Mw=7 kDa, Spectrum Laboratories, Laguna Hills, CA) for 3 days and freeze-dried. The primary products were purified by ethanol for three times to remove the residual substances. The collected solutions were re-suspended into water and collected by lyophilization. The structure of glycolipid polymers was confirmed by ¹H NMR spectrometer (AC-80, Bruker Biospin, Germany).

Acetylated UA 1.7 g (3.75 mmol) and PEG-SA 7.5 g (3.75 mmol) were weighed into a round bottom flask. Next, 150 mL of toluene was added, and the mixture refluxed until no water droplets appeared in the water separator (125 °C for 5 h, magnetic stirred). The mixture was dried under reduced pressure. After that, DCC (1.5 g), DMAP (0.1 g), and dichloromethane were added and stirred for two days at 25 °C. The filtrate was washed thrice with saturated NaHCO₃ and NaCl. An appropriate amount of anhydrous sodium sulfate was added to the dichloromethane layer and soaked overnight. The filtrate was decompressed to remove the solvent and purified further on a silica gel column (CH₂Cl₂:CH₃OH = 25:1, v/v). The single point R_f=0.68 (CH₂Cl₂:CH₃OH = 10:1) analyzed by TLC was visualized with sulfuric acid. Finally, UA-PEG-SA was vacuum dried at room temperature and recovered as a waxy solid. The acquired solid was dissolved in DMSO-d₆ and analyzed by ¹H NMR (AC-80, Bruker Biospin Co., Ettlingen, Germany).

NMR spectrometer (AC-80, Bruker Biospin, Germany) was used to obtain the ¹H NMR spectra of chemicals. Dimethyl sulfoxide-d₆ was the solvent for NMR measurement.
The critical micelle concentration (CMC) of Dex-PLGA and A54-Dex-PLGA was evaluated by fluorescence measurement using pyrene as a probe. The excitation wavelength was set at 337 nm, the excitation slit at 10 nm and emission slit at 2.5 nm. The intensities of the emission at a wavelength range of 300–470 nm were recorded on a fluorescence spectrophotometry (F-2500, Hitachi Co., Japan). The concentration of polymer solution was ranging from 10⁻³ to 1.0 mg mL⁻¹. Then the intensity ratio of the first peak (I₁, 374 nm) to the third peak (I₃, 385 nm) was analyzed for determination of CMC.
The size and distribution of polymer micelles were determined by dynamic light scattering using a ZETASIZER (3000HS, Malvern Co.,UK). The morphological examinations were performed by a transmission electron microscopy (TEM, JEOL JEM-1230, Japan). The samples were dropped on copper grids and stained with 2% (w/v) phosphotungstic acid for viewing.
The solution of SPIO-loaded micelles were lyophilized and magnetic properties of the powders were measured on a vibrating sample magnetometer (a Physical Property Measurement System from Quantum Design, MPMS-XL-5, CA, USA) with a maximum applied field of 1.8 T at room temperature.