Method details

GO (2 mg/mL) dissolved in PBS (pH 8.0), and SPDP (100 mM) dissolved in DMSO were prepared respectively. After adding 20 μL of SPDP solution into 2 mL GO solution, the mixture was gently shaken for 2 h at room temperature, followed by purification with an ultrafiltration tube (30 kDa MWCO, Merck Millipore). The as-prepared GO-SPDP was diluted with PBS (pH 8.0) to 1.8 mg/mL (of equivalent GO concentration), and allowed to react with TCEP solution (60 mM) for 30 min to generate thiol-modified GO (GO-SH). After ultrafiltration with PBS (pH 8.0), 400 μL BiBOEDS (10 mM) was added dropwise to GO-SH (1 mL, 2.7 mg/mL) and gently shaken for 2 h. The crude product was dialyzed against PBS for 24 h at 4°C using a dialysis tube (3500 Da MWCO, Yuanye Biotech) to obtain GO-Br.

NIPAm (200 mg), PMDETA (1.2 mg), CuCl₂⋅2H₂O (1.2 mg) were dissolved with H₂O (2 mL) and methanol (MeOH) (1 mL) (Wu et al., 2019) to obtain a blue monomer solution in a Schlenk flask at NIPAm/PMDETA/CuCl₂ = 255/1/1 (molar ratio). The flask was sealed carefully, frozen with liquid nitrogen, and deoxygenated with nitrogen for 15 min 200 μL Vc (3.1 mg/mL for Entries 4∼6; 3.7 mg/mL for Entries 7∼9; 4.3 mg/mL for Entries 10∼12; 6.2 mg/mL for Entries 1∼3 and 16∼18; 9.3 mg/mL for Entries 13∼15, Tables S1 and S2) was then added into the thawed monomer solution under nitrogen atmosphere, deoxygenated for 15 min, and mixed with 1 mL deoxygenated GO-Br solution (2 mg/mL). After another freeze-deoxygenation-thaw cycle, the Schlenk flask was placed on a magnetic stirrer at room temperature for different periods of time (solution color changed into light green). The reaction was quenched by exposing to the atmosphere (solution color turned blue). GO-PN (1∼3) (Entries 1∼3, Table S1) were prepared using Vc (6.2 mg/mL), with polymerization time for 1, 2, 3 h, respectively. The crude products were then ultrafiltrated (100 kDa MWCO, Merck Millipore) with PBS at 4°C to generate GO-PNs. To demonstrate the dependence of polymerization on the presence of exogenous Cu^II, Vc and PMDETA, several control synthesis experiments were performed with polymerization time for 3 and 12 h, followed by ultrafiltration (30 kDa MWCO).

According to Amplex^TM Red Galactose/Galactose Oxidase Assay Kit protocol, GO catalyzes the oxidation of Gal at the C6 position to produce hydrogen peroxide (H₂O₂). H₂O₂ reacts with Amplex Red reagent under catalysis by horseradish peroxidase (HRP) to produce resorufin, a red fluorescent oxidation product. Stock solutions including 200 U/mL GO, 20 mM Gal, 10 mM Amplex Red reagent, and 100 U/mL HRP were firstly prepared. 50 μL of Amplex Red, 10 μL of HRP, 50 μL of Gal and 4.89 mL of 1× reaction buffer (AmplexTM Red Galactose/Galactose Oxidase Assay Kit, Thermo Fisher Scientific Inc.) were mixed to prepare the 2× working solution buffer. GO solutions with various concentrations in 1× reaction buffer were added to a 96-well plate (50 μL per well), followed by addition of 50 μL of 2× working solution buffer. The mixture was incubated at room temperature for 1 h, and then analyzed on a multiskan FC microplate photometer (Thermo Fisher Scientific). The GO activity calibration curve was obtained by recording the absorbance value at 560 nm.

To investigate the effects from PN preparation process on GO activity, GO, GO-SPDP, GO-SPDP + TCEP (This refers to 0.005 mg/mL GO-SPDP solution after co-incubation with 5 mM TCEP for 1 h and then removal of TCEP, hereafter), GO-Br, GO-PN1 and GO-PN1+TCEP (This refers to 0.005 mg/mL GO-PN1 solution after co-incubation with 5 mM TCEP for 1 h and then removal of TCEP, hereafter) were diluted to 0.005 mg/mL of equivalent GO concentration using 1× reaction buffer, respectively. 50 μL of each sample was transferred into 96-well plate, mixed with 50 μL of 2× working solution buffer. After reaction at room temperature for 1 h, the absorbance value was recorded.

To determine the activity of GO-PN1 towards different monosaccharides, different 2× working solution buffer counterparts were prepared by mixing 50 μL of Amplex Red reagent, 10 μL of HRP, 4.89 mL of 1× reaction buffer with 50 μL of different monosaccharides (Gal, GalNAc, Glc, Man, Sia, Suc, Rib, Xyl and Fru, 20 mM). Then 50 μL of GO-PN1 (0.005 mg/mL of equivalent GO concentration) with and without TCEP (5 mM TCEP) in 1× reaction buffer were transferred into 96-well plate, followed by addition of 50 μL of different 2× working solution buffer counterparts. After reaction at room temperature for 1 h, the absorbance value was recorded.

To evaluate the activity of PN (1∼3) towards Gal, 50 μL of GO, PN1, PN2 or PN3 in 1× reaction buffer (0.005 mg/mL) was transferred into 96-well plate respectively, and then mixed with 50 μL of 2× working solution buffer. After reaction at room temperature for 1 h, the absorbance value was recorded.

To obtain time course of absorbance for Gal oxidation under catalysis by GO or GO-PN (1∼3), 50 μL of GO or GO-PN (1∼3) (0.005 mg/mL of equivalent GO concentration) in 1× reaction buffer was added into 96-well plate respectively, and then mixed with 50 μL of 2× working solution buffer. The absorbance value was recorded for 1 h at 10 min intervals.

According to the protocol of Cell Counting Kit-8, MCF-7 cells (1×10⁴, 100 μL per well) were cultured in 96-well plates for 24 h. After washing and blocking, the cells were subjected to incubation with 100 μL of GO, PN1 (0.05 mg/mL), GO-PN1, and the mixture of GO-PN1 and TCEP (GO or GO-PN1: 0.05 mg/mL of equivalent GO concentration; TCEP: 5 mM), respectively, at 4°C for 1 h. After washing, the cells were allowed to incubate with RPMI-1640 medium containing 10% FBS and 10% CCK8 at 37°C for 4 h. Then the absorption at 450 nm was measured, and the cell viability (%) was calculated based on the equation: cell viability (%) = (average OD value of treated cells/average OD value of control cells) × 100%.

To investigate the effect of TCEP on cellular viability, according to the protocol of Cell Counting Kit-8, MCF-7 cells (1×10⁴, 100 μL per well) were cultured in 96-well plates for 24 h. After washing with PBS, the cells were incubated with 100 μL TCEP at different concentrations (1, 2.5, 5, 7.5, 10 mM) at 4°C for 1 h, then rinsed with PBS again. RPMI-1640 medium containing 10% FBS and 10% CCK8 was then added into each well and allowed to incubate at 37°C for 4 h. The absorption at 450 nm was measured, and the cell viability (%) was calculated as mentioned above. The control cells were those without TCEP incubation.

To modulate GO remodeling activity on live cells, a series of GO-PN composites were synthesized with different conditions, including five feed molar ratios and various polymerization time. MCF-7 or HeLa cells were washed with PBS for three times, and blocked using PBS containing 10% goat serum at 37°C for 30 min. The cells were then washed three times with PBS, and incubated with GO or different GO-PNs (0.05 mg/mL of equivalent GO concentration) at 4°C for 1 h. After gentle washing with PBS for three times, the cells were subjected to reaction with 100 μL PBS containing 10 mM aniline, 100 μM FTZ and 5% fetal bovine serum (FBS) at 4°C for 1 h, followed by PBS washing for three times. Then the cells were imaged with an SP8 STED confocal laser scanning microscopy (Leica). All images were analyzed by Leica Application Suite Advanced Fluorescent (LAS AF) software.

For freely diffused substrates, different 2× working solution buffers (5 mL) were prepared by mixing 50 μL of Amplex Red reagent, 10 μL of HRP, and different substrates (50 μL 20 mM Gal, 100 μL 10 mM GM1, 160 μL 20 mg/mL MUC2, or 160 μL 20 mg/mL desialylated fetuin) in 1× reaction buffer (Amplex^TM Red Galactose/Galactose Oxidase Assay Kit, Thermo Fisher Scientific Inc.). 50 μL of GO or GO-PN (1∼3) (0.005 mg/mL of equivalent GO concentration) in 1× reaction buffer was transferred into 96-well plate, followed by addition of 50 μL of different 2× working solution buffers. After reaction at room temperature for 1 h, the absorbance value was recorded.

Different 2× working solution buffer counterparts (1 mL) were prepared by mixing 10 μL of Amplex Red reagent, 2 μL of HRP, and different samples (GO or GO-PN (1∼3), 100 μL, 0.05 mg/mL of equivalent GO concentration) in 1× reaction buffer. To each substrate solution (Gal, GM1 or MUC2, 50 μL) with a given concentration, 50 μL of different 2× working solution buffer counterparts were respectively added. The concentration series for substrates were as follows: Gal (μM), 100, 200, 300, 400, 500; GM1 (μM), 400, 500, 600, 700, 800; MUC2 (μg/mL), 400, 500, 600, 700, 800. After reaction at room temperature for 30 min, the absorbance value was recorded.

Different 2× working solution buffer counterparts (1 mL) were prepared by mixing 10 μL of Amplex Red reagent, 2 μL of HRP, and different samples (GO or GO-PN (1∼3), 100 μL, 0.05 mg/mL of equivalent GO concentration) in 1× reaction buffer. PBS containing GM1 (200 μM) and CTxB (1.6 μM) was incubated at room temperature for 1 h. A control sample was also prepared by incubating GM1 (200 μM) and BSA (8 μM) in PBS at room temperature for 1 h. The above-mentioned samples (50 μL) were individually added into 96-well plate, and then mixed with 50 μL of different 2× working solution buffer counterparts. After reaction at room temperature for 1 h, the absorbance value was recorded. A five-fold concentration of BSA compared to CTxB was added to the system due to the assumption that each CTxB has five subunits for binding with GM1.

After washing and blocking as mentioned above, the MCF-7 cells were subjected to incubation with the mixture of GO (or GO-PN1, 0.05 mg/mL of equivalent GO concentration) and MUC2 (640 μg/mL) at 4°C for 1 h. The supernatants and cells were both collected. The supernatants were centrifuged at 4°C at 10,000 rpm for 6 min to remove suspended cells, and were subjected to reaction with PBS containing 10 mM aniline, 100 μM FTZ, and 100 mM K₄[Fe(CN)₆] (to preclude further Gal/GalNAc remodeling during this labeling step) at 4°C for 1 h. After ultrafiltration (100 kDa MWCO) to remove small molecules, samples were dialyzed against PBS for 24 h at 4°C using a dialysis tube (300 kDa MWCO) to remove GO (or GO-PN1). The obtained MUC2 was diluted to 0.1 mg/mL and subjected to fluorescence detection (F-7100 fluorescence spectrophotometer, Hitachi) under excitation wavelength at 488 nm. The cells were gently washed with PBS for three times, and subjected to reaction with 100 μL PBS containing 10 mM aniline, 100 μM FTZ, and 5% FBS at 4 ^oC for 1 h. After washing with PBS for three times, the cells were imaged with an SP8 CLSM.

GO, GO-Br, GO-PN1, GO-PN2 and GO-PN3 (0.005 mg/mL of equivalent GO concentration) were diluted in 1× reaction buffer, respectively. Each sample of 50 μL was added into 96-well plate, and then mixed with 50 μL of different 2× working solution buffer containing 100 μM Amplex Red reagent, 200 mU/mL HRP, and 200 μM Gal (or GM1). After reaction at 25 or 37°C for 1 h, the absorbance value was recorded.

After washing and blocking, MCF-7 cells were subjected to incubation with GO (0.05 mg/mL), GO-PN1 (0.05 mg/mL of equivalent GO concentration), or the mixture of GO-PN1 (0.05 mg/mL of equivalent GO concentration) and TCEP (5 mM) at 4°C for different periods of time (30, 40, 50, 60 min). After washing with PBS for three times, the cells were subjected to reaction with 100 μL PBS containing 10 mM aniline, 100 μM FTZ, and 5% FBS at 4°C for 1 h. After washing with PBS for three times, the cells were imaged with an SP8 CLSM.

After washing and blocking, MCF-7 cells were subjected to incubation with GO (0.05 mg/mL) in the presence of TCEP at different concentrations (1, 5, 10, 15, 20, 25 mM) at 4°C for 1 h. After washing with PBS for three times, the cells were subjected to reaction with 100 μL PBS containing 10 mM aniline, 100 μM FTZ, and 5% FBS at 4°C for 1 h. After washing with PBS for three times, the cells were imaged with an SP8 CLSM.

To exclude the influence of extracellular GSH on glycan remodeling on live cells, after washing and blocking, MCF-7 cells were respectively subjected to incubation with GO, the mixture of GO-PN1 and TCEP, and the mixture of GO-PN1 and GSH (GO or GO-PN1: 0.05 mg/mL of equivalent GO concentration; TCEP: 5 mM; GSH: 20 μM) at 4°C for 1 h. After washing with PBS for three times, the cells were subjected to reaction with 100 μL PBS containing 10 mM aniline, 100 μM FTZ, and 5% FBS at 4°C for 1 h. After washing with PBS for three times, the cells were imaged with an SP8 CLSM.

GO, GO-SPDP and GO-Br were firstly desalted by ultrafiltration with water (30 kDa MWCO). The matrix, saturated SA solution, was prepared using a solvent containing 50% ultrapure water (Millipore Simplicity System, Bedford), 50% acetonitrile and 0.1% trifluoroacetic acid. 1 μL of matrix solution and 1 μL of sample solution (5.6 mg/mL for GO, 6.5 mg/mL for GO-SPDP, and 4.6 mg/mL for GO-Br) were successively applied onto the MALDI target plate. After solvent evaporation, another 1 μL of matrix solution was spotted onto the plate and allowed to dry at 37°C. Samples were then analyzed by MALDI-TOF mass spectrometer (MALDI-7090 MALDI TOF mass spectrometer, Shimadzu).

Samples dissolved in PBS with equivalent GO concentration of 0.67 mg/mL were shaken at room temperature for 1 h, respectively. Then each sample solution was diluted with LDS sample buffer to 0.5 mg/mL of equivalent GO concentration. 10 μL of the mixture was loaded into hand casting 10% TGX Stain-Free polyacrylamide gels for electrophoresis separation using a standard Tris-glycine running buffer system (Electrophoresis Analyzer, Bio-Rad) at 200 V for 40 min. Gels were stained with Coomassie brilliant blue at room temperature for 30 min, decolorized with decolorizing solutions, and imaged on a Bio-Rad ChemDoc XRS facility.

The prepared GO-PN (1∼3) were further purified by heating at 40°C for 5 min, and centrifuged at 40°C at 10,000 rpm for 6 min. The resulting GO-PN pellet was resuspended with water to wash, and then heated and recentrifuged as above for three times. Each type of GO-PNs (0.9 mg/mL of equivalent GO concentration) was mixed with TCEP (60 mM), and shaken at room temperature for 1 h to cleave the PNs. The mixture was incubated at 40°C for 5 min and centrifuged at 40°C at 10,000 rpm for 6 min to remove the GO in the supernatant. The resulting PN pellet was resuspended with water, heated and recentrifuged as above for three times, and then lyophilized. The PNs were redissolved in THF (10 mg/mL), filtered with 0.22 μm filter, and the M_n, M_w, and PDI were measured on Waters gel permeation chromatography (GPC) system at a flow rate of 1.0 mL/min. The GPC is equipped with a Waters 1515 HPLC pump, a Waters 2414 refractive index detector, and a tandem column system with Waters Styragel HR 2, Styragel HR 4 and Styragel HR 5 columns.

GO (0.1 mg/mL), PN (1∼3) (1 mg/mL) and GO-PN (1∼3) (of equivalent GO concentration of 0.1 mg/mL) were dissolved in PBS, respectively. LCST curves were measured at 490 nm by a UV-VIS spectrophotometer (Cary 3500, Agilent). Samples were heated from 25 to 45°C at 1°C/min. The LCST was defined as the temperature at which the transmittance of the solution was 50% of the maximum.

PN (1∼3), GO, GO-SPDP, GO-Br, GO-PN (1∼3) and mixtures of GO-PN (1∼3) and TCEP were shaken at room temperature for 1 h, respectively. GO and its conjugates were dissolved with an equivalent GO concentration of 0.2 mg/mL, and the concentrations for PN (1∼3) and TCEP were 0.2 mg/mL and 5 mM, respectively. The absorbance of each sample was measured from 200 to 600 nm by a UV-VIS-NIR spectrophotometer (UV-3600, Shimadzu).

GO, GO-SPDP, GO-Br and GO-PN (1∼3) (of equivalent GO concentration of 0.5 mg/mL) were respectively dissolved in 20 mM Tris-HCl for CD measurement from 180 to 260 nm using Applied Photophysics Circular Dichroism (Applied Photophysics).