2 n morpholino ethanesulfonic acid mes buffer

According to Ekmekci et al. (2009) (link) the pH of the solution can affect the co-aggregation ability. Hence, 2-(N-Morpholino) ethanesulfonic acid (MES) buffer (69892, Sigma-Aldrich) with a pH adjusted according to the pH of the skin (pH 5) was used instead of PBS for cell suspension and otherwise carried out as in section “Absorbance Detection of Co-aggregation (Secondary Screen).”

We purchased (E/Z)-2-buten-1-ylsuccinic anhydride (product) and (E/Z)-2-hexenyl-1-ylsuccinic anhydride (product of competitor 2) from TCI chemicals. Succinic acid (competitor 1), succinic anhydride (product of competitor 1), Nile Red, 1-ethyl-3-(3 dimethyl-aminopropyl)carbodiimide hydrochloride (EDC), trifluoroacetic acid (TFA) and 2-(N-morpholino)ethane sulfonic acid (MES buffer) were purchased from Sigma-Aldrich. All chemicals were used without any further purification unless otherwise indicated. High performance liquid chromatography (HPLC) grade acetonitrile (ACN) was purchased from VWR.

To conjugate FITC to HAMA, we first dissolved 1.0 g of HAMA (100 kDa) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH = 5.6, Sigma-Aldrich). Then, the 0.1-M N-(3-dimethylamino propyl)-N-ethylcarbodiimide (EDC, Sigma-Aldrich) and 0.2-M N-hydroxysuccinimide (NHS, Sigma-Aldrich) mixture was added to activate the carboxylate groups on HAMA. 10-µg mL⁻¹ FITC-poly(ethylene glycol)-amine (FITC-PEG-NH₂, M_w = 2 kDa, Nanocs, USA) was added and reacted overnight at 4 °C in dark. The reacted solution was dialyzed against DI water for 5 days and then lyophilized for use.

Chondroitin sulfate (CS) sodium salt from shark cartilage, N-Hydroxysuccinimide (NHS) (molecular weight (Mw) 115.09 g/mol, purity 98%), 2-(N-Morpholino)ethanesulfonic acid (MES) buffer (Mw 195.24 g/mol, purity 99%), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) (Mw 191.70 g/mol, purity 98%), Hydroxylamine solution 50 wt% in H₂O (Mw 33.03 g/mol), and Formalin solution (neutral buffered, 10%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrazine-Amine (Mw 187.09, purity 95%) was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). We purchased 8-Arm PEG-Norbornene (8A-PEG-N) (Mw 20 kD and 40 kD) and 4A-PEG-N (Mw 20 kD) from Creative PEG Works (Durham, NC, USA). Dialysis membrane (MwCO 3.5 KD) was purchased from Spectrum (Waltham, MA, USA).

Figure 2a and b show the synthesis of antibiotic conjugated MNBs. Vancomycin and allantoin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MNBs (100 nm in diameter) were sonicated for about 40 s to prevent aggregation. MNBs (200 mg) were dispersed in 40 mL of 1 M HCl and stirred at room temperature (RT) for 1 h. The MNBs were separated over 2 min using a magnetic rack (Bioneer Co., Ltd, Daejeon, Korea), and residual HCl was removed by washing in 40 mL of PBS three times followed by dispersal in 10 mL PBS. PEG (25 mg) (Sigma-Aldrich) dissolved in 25 mL of Tris buffer (pH 8.5) was mixed with 50 mg MNBs overnight at RT. To conjugate Van to MNBs@PEG-COOH, 5 mg MNBs@PEG-COOH was added to 500 µL of 2-(N-morpholino) ethane sulfonic acid (MES) buffer (0.1 M, pH 6.0) (Sigma-Aldrich); 4 mg ethyl carbodiimide hydrochloride (EDC) (Sigma-Aldrich), 7 mg N-hydroxy succinimide (NHS) (Sigma-Aldrich), and 10 mg Vancomycin dissolved in 1 mL of MES buffer were added and the mixture was stirred at RT for 15 min. Then, the MNBs@PEG-COOH suspension was dispersed in the Vancomycin-EDC-NHS suspension via continuous stirring at RT for 2 h. MNBs@PEG-Van were separated using the magnetic rack and washed with 500 µL of PBS. Finally, MNBs@PEG-Van were resuspended in 1 mL of PBS and stored at 4 °C. MNBs@PEG-Al were similarly prepared.