Immobilized tcep disulfide reducing gel

Nanoparticles were prepared via single-step nanoprecipitation as previously described with modifications. Briefly, 0.75 mg of PLGA, 0.15 mg of drug cocktail, 0.375 mg of TPGS, 0.04 mg of DOPC, 0.04 mg cholesterol and 0.08 mg DSPE-PEG(2000)-maleimide in 40 μl of DMSO were mixed as the oil phase and each time 10 μl of oil phase was added to 280 μl of deionized water dropwise under gentle stirring. The NPs were self-assembled with continuous stirring for 30 minutes at room temperature. The ratio of the volumes of the oil and water phases was fixed at 1/7 (v/v) throughout all of the experiments. For peptide conjugation, peptide was reduced using immobilized TCEP disulfide reducing gel (Thermo Scientific) according to manufacturer’s recommendations. Twenty-five μl of 1 mg/ml of CTCE-9908 peptides or scramble peptides were added into the emulsion where it reacted with DSPE-PEG(2000)-maleimide. Four hours later, the unreacted maleimide groups were quenched by adding l-cysteine. The solution was centrifuged at 16,220 rpm for 30 min at 25 °C to collect the NPs, and the NPs were then resuspended in water in the volume equal to the emulsion for characterization. The same procedure was used to synthesize C6-loaded NPs.

Pre-reduced uncoupled roGFP2 and Mrx1-roGFP2 (1 µM) were incubated with mycothiol solutions (1 mM total) containing increasing fractions of MSSM. The total concentration of MSH (MSH _total) refers to MSH equivalents i. e MSH_total = [MSH]+2[MSSM]. OxD_MSH is the fraction of MSH _total that exists as [MSSM] and can be conveniently calculated using the following formula: Reduced from of mycothiol (MSH) was obtained by reducing MSSM with immobilized TCEP disulfide reducing gel (Thermo Scientific) under anaerobic conditions as per manufacturer's instructions.