Peg 2k

About 100 mg of PLGA copolymer (PLG4:PLGA 80/20) and 50 mg of TPGS (Sigma, Bangalore, India) were dissolved in 5 mL of acetone (Fischer Scientific). The solution was sonicated for 60 seconds using a probe sonicator (Hielscher sonicator, ultrasonic processor UP 100H, Berlin, Germany [100 W, 30 kHz, 0.8 cycle, 80% amplitude]), and it was stirred continuously at 4°C. To this, 2 mL of Huminsulin (100 IU/mL, Eli-Lilly, Haryana, India) was added while stirring the solution and it was further sonicated for 60 seconds. The solution was kept in an ice bath and it formed a water-in-oil (W/O) emulsion. It was again sonicated for 180 seconds with 10 mL of aqueous PEG 2000 (PEG 2K; 1% w/v, Sigma, India) solution to form a double emulsion (W/O/W), and it was stirred continuously for 6 hours to remove the organic solvent. Then, it was centrifuged (Remi high-speed centrifuge C-24, Remi House, Mumbai, India) at 12,000 rpm (4°C) for 15 minutes and the ISTPPLG4 NPs were formed (Figure 1). The residue was freeze-dried (Christ Alpha 2–4 LSC freeze dryer, Martin Christ Gefriertocknungsanlagen GmbH, Osterode am Harz, Germany) for 12 hours, and the pellet obtained was stored at 4°C for further studies. The same protocol was also followed for the synthesis of copolymer PLG6 (PLGA 70/30).