Gpc system

The characterization of the synthesized MPITC-R8 conjugate was obtained using 1H NMR of the synthesized conjugate and size exclusion chromatography (SEC). 1H NMR was performed by dissolving the compound (10 mg) in deuterated methanol (CD3OD) (1 mL) using a Bruker spectrometer (300 MHz, Bruker, Billerica, MA, USA) operating at 300 mega Hz at 25 °C. The SEC was performed to estimate the molecular weight of the synthesized MPITC-R8 conjugate. The samples were eluted through an Ultrahydrogel™ linear SEC column (7.8 mm × 300 mm) in a gel permeation chromatography (GPC) system (Waters Corporation, St. Louis, MO, USA). Milli-Q water was used as a mobile phase with a flow rate of 0.7 mL/min. The SEC standards were run before analyzing the conjugates.

The structures of the synthesized compounds were identified by ¹H NMR spectroscopy (JEOL AL400, Tokyo, Japan). The weight-average molecular weight (M_w), number-average molecular weight (M_n), and the distribution (M_w/M_n) of the polymers were measured on a Waters GPC system, which was equipped with a Waters 1515 HPLC solvent pump, a Waters 2414 refractive index detector, and two Waters Styragel high resolution columns, at 40°C using HPLC grade THF as eluent at a flow rate of 0.35 mL/min. Monodispersed polystyrenes were used to generate the calibration curve. Absorption spectra were measured using a JASCO V-550 UV/VIS spectrophotometer. Fluorescence spectra were measured using a JASCO FCT-133 spectrometer.

¹H NMR spectra were measured on a Bruker ARX 400 MHz spectrometer using DMSO-d6 (for TA-Br), CDCl₃ (for TA-PGMA) and D₂O (for TP, TP-E, and TP-ColIV) as the solvents with tetramethylsilane (Me₄Si) as an internal standard. Thermogravimetric analysis measurement of TA-Br-Gd was performed on a Thermal Gravimetric Analyzer (TG 209, NETZSCH). GPC measurement of TA-PGMA was performed on a Waters GPC system with DMSO as the eluent. The concentrations of Gd³⁺ in TP and TP-Gd were investigated using inductively coupled plasma mass spectrometry (ICP-MS, Thermo Scientific iCAP 6000 series). The fluorescence intensities of rhodamine B modified ColIV and TP-ColIV-RhB were characterized using a fluorescence spectrophotometer (Hitachi F-7000). Dynamic light scattering (DLS) measurements of polycation/miRNA complexes were performed with a Zetasizer Nano ZS equipped with a laser of wavelength 633 nm at a 173° scattering angle (Malvern Instruments, Southborough, MA). Atomic force microscopy (AFM) studies were carried out with the Dimension Icon model with a Nanoscope IIIa controller (Bruker, Santa Barbara, CA), where samples were imaged using the ScanAsyst mode. Gel electrophoresis was implemented in a Sub-Cell system (Bio-Rad Laboratories), and then a UV transilluminator and BioDco-It imaging system (UVP Inc.) was employed to record miRNA bands.