Avance dmx 500 nmr spectrometer

Chitosan oligosaccharide (CSO) was prepared by enzymatic degradation of chitosan according to the previous study [36 (link)]. The molecular weight of CSO with 18.4 kDa molecular weight used in this paper was determined by gel permeation chromatography (GPC) with TSK-gel column (G3000SW, 7.5 mm i.d. × 30 cm). CSSA was then synthesized via a reaction of the carboxyl group of SA with the amine group of the CSO in the presence of EDC. Briefly, 400 mg CSO was dissolved in 40 mL deionized water, and 100 mg SA and 380 mg EDC were dissolved in 20 mL ethanol by sonicating treatment (Sonic Purger CQ250, Academy of Shanghai Shipping Electric Instrument) for 30 s, respectively. The CSO solution was heated to 60 °C under stirring accompanied by dropwise addition of SA solution. The reaction mixtures were stirred for 6 h and then dialyzed against deionized water using a dialysis membrane (molecular weight cut-off (MWCO): 7 kDa, Spectrum Laboratories, Laguna Hills, CA, USA) for 48 h. Finally, the product was collected by lyophilization followed by a further wash with ethanol to remove products. The washed product was then collected by lyophilization again.
CSO, SA, and CSSA were dissolved in Deuterium oxide (D₂O), and the chemical structures were characterized by ¹H NMR with an AVANCE DMX 500 NMR spectrometer (Bruker, Rheinstetten, Germany).

The high-performance liquid chromatography (HPLC) system used was composed of a Waters 717 plus Autosampler, a Waters 600 Controller, a Waters 996 Photodiode Array Detector, and a Waters Millog workstation (Waters, Shinagawa, Tokyo, Japan). Optical rotations were measured in methanol on a PerkinElmer-341 polarimeter. The IR spectra were run on a NicoletAvatar-360FT-IR spectrometer. ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were measured at 25°C on a Bruker AVANCE DMX 500 NMR spectrometer with TMS as internal standard. CD spectra were measured on a JASCO J-715 (JASCO) spectropolarimeter. UV spectra were also recorded in methanol on a Shimadzu UV2550. ESIMS were recorded on an Agilent 6460 Triple Quad LCMS. Preparative HPLC was performed on a ChuangXinTongHeng system equipped with a Venusil MP-C18 column (10 mm × 250 mm, Agela Technologies, Tianjin, China). The organic solvents used in chromatographic separation were of analytical grade purchased from Sayfo Technology (Tianjin China) and chromatographic grade for HPLC analysis purchased from Tedia, United States. Deionized water was prepared by reverse osmosis Milli-Q water (18 MW; Millipore, Bedford, MA, United States) and used for all solutions and dilutions. Agar powder for plate culture and cobalt chloride was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

To synthesize the fluorinated PAMAM, the heptafluorobutyric anhydride was used as the fluorinated reagent^{38 (link)}. Briefly, heptafluorobutyric anhydride (54.12 mg) and PAMAM (50.00 mg) were dissolved in 8 ml of methanol. The mixture was stirred at room temperature for 48 h, and then dialyzed against distilled water for another 48 h (MWCO: 3500 Da). The product was obtained through lyophilization and subjected to structural characterization using ¹⁹F NMR on AVANCE III spectrometer (Bruker, Rheinstetten, Germany). The molecular weight values of PAMAM and FP were measured by AB SCIEX 5800 MALDI-TOF mass spectrometer (Framingham, Massachusetts), in which 2,5-dihydroxybenzoic acid was used as a matrix. The butyric acid-modified PAMAM was synthesized as described above except for the usage of butyric anhydride (20.88 mg), and characterized using ¹H NMR on AVANCE DMX 500 NMR spectrometer (Bruker, Rheinstetten, Germany). The morphology of FP/miR-23b nanoparticles were assayed by Hitachi H800 transmission electron microscope (TEM) at an accelerating voltage of 200 kV. The hydrodynamic diameter and zeta potential of nanoparticles were measured by Malvern Nano ZS90 Zetasizer (Malvern, UK) and analyzed by Malvern Zetasizer software 7.11.