Waters chromatographic system

Free polyamines were extracted and measured as described previously (Chen et al., 2013 (link)). Leaves (0.5 g) were extracted in 5 ml of 5% (v/v) cold perchloric acid (PCA) and incubated on ice for 1 h. The homogenate was centrifuged at 20,000 ×g for 30 min. Aliquots (0.5 ml) of supernatant were mixed with 1 ml of 2 M NaOH and 7 μl of benzoyl chloride and incubated at 37°C for 20 min in dark for benzoylation. The benzoylated polyamines were extracted to diethyl ether, resuspended in 1 ml of mobile phase solution (64% methanol in an isocratic elution), and filtered (4.5 μm filter) before HPLC analysis. Twenty μl of sample was injected into a Waters chromatographic system (Waters, Mildford, MA, United States), supplied with a C18 column (4.6 mm × 250 mm), and detected at 254 nm using a 2487 dual UV detector (Waters, Milford, MA, United States). Polyamine levels were calculated based on standard curves of commercial standards in combination with a recovery of the extraction procedure.

High-performance liquid chromatography (HPLC) was used to measure the amount of doxorubicin. A Waters chromatographic system (Waters Technologies, USA) was recruited and the chromatographic separation was performed on a Kinetex C₁₈ column (4.6 × 100 mm, 2.6 μm particle size; Phenomenex, USA) at 35 °C (mobile phase: acetonitrile and water (32: 68, v/v) at pH 2.6 by adjusting with 85% orthophosphoric acid; flow rate: 1 mL/min; excitation and emission wavelengths: 475 nm and 555 nm, respectively; daunorubicin hydrochloride, from Solarbio Science & Technology Co. (Shanghai, China), was used as an internal standard). The EE and DL were calculated as the following equations: EE (%) = W/W_t × 100% and DL (%) = Q/Q_t × 100%, where W and Q are the amount of drug (Dox) loaded in vesicles, whereas W_t and Q_t are the total amount of the feeding doxorubicin and the feeding materials (Dox, vesicles, etc.), respectively.

Polyamines were extracted from leaves (0.5 g) with 4 ml of freshly prepared 5% (v/v) perchloric acid and extracted at 4°C, followed by centrifugation at 15,000 g for 30 min. The supernatants were used for detection of free polyamines using high-performance liquid chromatography (HPLC) as previously described (Chen et al., 2013 (link); Guo et al., 2014 (link)). Aliquots (0.5 ml) were benzoylated as described by Flores and Galston (1982) (link). The benzoylated polyamines were re-suspended in 1 ml of mobile phase solution and filtered (4.5 μm filter) before HPLC analysis. Twenty microliter of sample was injected into a Waters chromatographic system (Waters, Mildford, MA, USA), supplied with a C18 column (Dalian Elite Analytical Instruments Co., Ltd., Dalian, China, 4.6 mm × 250 mm). The mobile phase was 64% methanol in an isocratic elution, at a flow rate of 0.7 ml min^-1 and ambient temperature. Identification and quantification of Put, Spd, and Spm in each sample were achieved by comparing each peak retention time and peak area with the standard polyamines, being detected at 254 nm using a 2487 dual UV detector (Waters, Milford, MA, USA). Put, Spd, and Spm contents were calculated using standard curves with commercial standards and a correction for recovery after the extraction procedure. Three pots of plants were used for measurement as replications.