Methods

The PLGA-SeSe-mPEG synthesis process was depicted in Figure 1A. The nuclear magnetic resonance data of the PLGA-SeSe-mPEG was present in Figure 1B.

Schematic diagram for synthesis (A) and Nuclear magnetic resonance spectroscopy (B) of PLGA-SeSe-Mpeg. PLGA, poly(lactic-co-glycolic acid); PEG, polyethylene glycol.

The mPEG_5k-COOH was dissolved in formamide with EDC and NHS (mPEG_5k-COOH: EDC: NHS = 1:5:5). The mixture was stirred for 6 h to mix it thoroughly and then a mixture of selenocystamine and formamide (V/V = 1:9) was dripped at 0°C and allowed to react for another 5 h. Excess precooled acetone was added and allowed to crystallize into crystals, which were filtered through a microporous membrane (0.22 mm). The precipitate products were then dissolved in water and dialysed (MWCO: 3,500) for 48 h. The intermediate product, mPEG-SeSe-NH₂, was obtained by freeze-drying.

PLGA (0.65 g), EDC (80 mg), and NHS (48 mg) was dissolved in 10 mL of dichloromethane, and reacted for 24 h under nitrogen protection. The product was then precipitated by precooled aether and the residue NHS and EDC were removed by washing three times with a mixture of aether/methanol (50/50, v/v). The resulting sediment was vacuum-dried and labeled PLGA-NHS.

Briefly, PLGA-NHS (0.1 g), mPEG-SeSe-NH₂ (0.2 g), and DIPEA (6 uL) were dissolved in a dry solution of dimethyl sulfoxide. The solution was stirred under nitrogen protection for 48 h. The solution was dialysed (MWCO: 8,000–14,000) for 48 h, and the final product PLGA-SeSe-mPEG was obtained by freeze-drying.

PLGA-SeSe-mPEG (50 mg) was added to 1 mL of chloroform and mixed into 6 mL of a 1% PVA and TPGS mixture (PVA: TPGS 1:5). An 80 W ultrasound probe was used for 2 min in an ice bath to form an oil-water emulsion.

The emulsion was then added to 30 mL of 0.3% PVA and stirred overnight to solidify the surface. Ultrafiltration concentration was carried out with a 100 KD ultrafiltration tube. Finally, the volume of the mixture was fixed with pure water to 5 mL, and the samples were collected and stored at 4°C.

DEX, CDMP-1, and PLGA-SeSe-mPEG were dissolved in trichloromethane, respectively. Next, 50 μL of 2 mg/mL DEX, 50 μL of 1 mg/mL CDMP-1, 50 μL of 1 mg/mL rhB, and 500 μg of the PLGA-SeSe-mPEG mixture were evenly and ultrasonically prepared for use. The process for producing drug-loaded particles was the same as that of blank particles described above.