1525 system

1D and 2D NMR spectra were obtained on a VARIAN UNITY INOVA 400 spectrometer (Varian, Palo Alto, CA, USA). Mass spectra were determined on a JEOL JMS-AX505WA mass spectrometer (Tokyo, Japan). Column chromatography was carried out over silica gel (40–60 μm, Merck, Kenilworth, NJ, USA) and LiChroprep RP-C18 (40–60 μm, Merck). HPLC (high performance liquid chromatography) was carried out on a Waters 1525 system (Miliford, MA, USA) using reverse-phase column (ODS-2, 5 μm, 4.6 × 15 cm, GL Science, Seoul, Korea). Fractions obtained from column chromatography were monitored by thin layer chromatography (TLC) (RP-C18 F_254S and silica gel 60 F₂₅₄, Merck).

CA4-NPs were synthesized via the Yamaguchi esterification reaction as previously described (Figure 1A) 22 (link). Briefly, PLG-g-mPEG (4.00 g), CA4 (1.00 g, 3.16 mmol), 2, 4, 6-trichlorobenzoyl chloride (1.56 g, 6.40 mmol), DMAP (0.46 g, 3.77 mmol), and triethylamine (TEA, 0.9 mL, 6.46 mmol) were mixed in N, N-Dimethylformamide (DMF) and incubated at 60 °C for 6 h. The reaction mixtures were then precipitated into excess diethyl ether, re-dissolved in DMF, and dialyzed in distilled water (MWCO 3500). CA4-NPs were obtained after lyophilization. High-performance liquid chromatography (HPLC) was performed to measure the drug loading content (DLC, wt%) of CA4. In brief, the CA4-NPs (10.0 mg) were dissolved in 10 mL Milli Q water, and 0.4 mL 1 M NaOH was added, shaking at 37 °C for 1 h to produce free CA4. Next, 0.4 mL of 1.4 M phosphoric acid was added. HPLC (Waters 1525 system equipped with a reverse-phase column Symmetry^® C18) was used to determine the DLC of CA4 by monitoring at 305 nm. Elutions were performed with acetonitrile (or methanol) and water (v/v=4:1) pumped at a flow velocity of 1.0 mL/min at 25 °C. DLC was calculated by the following equation: DLC (%) = (amount of loaded drug/amount of nanoparticles) × 100, and the DLC of CA4-NPs was 15.9 wt%.

The molecular weight of LMP was analyzed by HPGPC with an ultrahydrogel TM Linear column (300 mm × 7.8 mmid × 2). A Waters 1525 system with a RI detector was used to perform the experiment. The mobile phase was 0.1 N NaNO₃ at a flow rate of 0.9 mL/min.

Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. ECD spectra were obtained on a Jasco J-815-150S circular dichroism spectrometer. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets. NMR spectra were measured on an Agilent DD2 500 MHz NMR spectrometer (500 MHz for ¹H and 125 MHz for ¹³C), using TMS as an internal standard. The ESIMS and HRESIMS spectra were obtained from a Micromass Q-TOF spectrometer and a Thermo Scientific LTQ Orbitrap XL spectrometer, respectively. The crystallographic data were collected on a Bruker APEX-II CCD diffractometer equipped with graphite monochromatized Cu Kα radiation. Semi-preparative HPLC was performed on a Waters 1525 system coupled with a Waters 2996 photodiode array detector. A Kromasil C₁₈ semi-preparative HPLC column (250 × 10 mm, 5 μm) was used. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200–300 mesh), Sephadex LH-20 (Amersham Biosciences) and octadecylsilyl Silica gel (Unicorn; 45–60 μm) were used for column chromatography (CC). Precoated Silica gel GF₂₅₄ plates (Yantai Zifu Chemical Group Co., Yantai, China) were used for thin-layer chromatography.

NMR spectra were recorded on a Bruker Advance NEO 400; chemical shifts δ are reported in ppm, using TMS as internal standard, and coupling constants (J) are in Hz. The HRESIMS and ESIMS spectra were obtained using a Micromass Q-TOF mass spectrometer. UPLC-MS was performed on a Waters UPLC^® system (Waters Ltd., Milford, MA, USA) using a C18 column [(Waters Ltd.) ACQUITY UPLC^® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min] and ACQUITY QDa ESIMS scan from 150 to 1000 Da. Column chromatography (CC) was performed on silica gel (Qingdao Haiyang Chemical Group Co., Qingdao, China; 200 to 300 mesh) and Sephadex LH-20 (Amersham Biosciences, Amersham, UK). TLC silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yantai, China; G60, F-254) were used for thin layer chromatography. Semi-preparative HPLC was performed on a Waters 1525 system using a semi-preparative C18 column (Amsterdam, Netherlandish; Kromasil, 5 μm, 10 × 250 mm) equipped with a Waters 2996 photodiode array detector, and the flow rate was 2.0 mL/min.

Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were determined on a Shimadzu double-beam 210 A spectrometer. IR spectra were taken on a Bruker EQUINOX 55 spectrometer using KBr pellets. 1D and 2D NMR spectra were obtained on an Agilent DD2 400 MHz NMR spectrometer or a DRX-500 spectrometer, respectively, with TMS as the internal standard. ESIMS and HRESIMS spectra were obtained from a Micromass Q-TOF spectrometer and a Thermo Scientific LTQ Orbitrap XL spectrometer. Semi-preparative HPLC was performed on a Waters 1525 system using a C18 (Kromasil, 5 μm, 10 × 250 mm) column coupled with a Waters 2996 photodiode array detector. UPLC MS was performed on Waters UPLC^® system using a C18 column [ACQUITY UPLC^® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 mL/min] and ACQUITY QDa ESIMS scan from 150 to 1000 Da. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200–300 mesh), and octadecylsilyl silica gel (Unicorn; 45–60 μm), were used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography.