Single donor human or balb c mouse plasma

To establish the stability of pHLIP(Laa)-STINGa and pHLIP(Daa)-STINGa in plasma, pHLIP-STINGa was mixed with single donor human or BALB/c mouse plasma (Innovative Research) at a concentration of 200 μM, and kept in plasma for 0, 2, 4 or 24 hours at 37°C. Plasma proteins were precipitated by methanol (1:5 volume ratio of plasma to methanol) and centrifugated for 10 min at 13.4 rpm. The supernatant was collected and analyzed by HPLC using a Zorbax SB-C18 4.6 × 250 mm, 5 μm column with a gradient from 10% to 75% acetonitrile in water containing 0.05% TFA. Chromatograms were recorded at 220 nm, 280 nm and 320 nm. As controls pHLIP(Laa), pHLIP(Daa), Pys-PAB-STINGa, SH-PAB-STINGa, where Pys-PAB-STINGa was conjugated with a Cys residue, and diABZI (In vivogen) were analyzed for stability in plasma under the same conditions.

To establish the stability of agents (2pHLIP-dMSA, pHLIP-mMSA, dMSA, mMSA, and pHLIP) in plasma, each agent was mixed with single donor human or BALB/c mouse plasma (Innovative Research) at a concentration of 200 μM, and kept in plasma for 0, 1, 2, 4 or 24 h at 37°C. Plasma proteins were precipitated by methanol (1:5 volume ratio of plasma to methanol) and centrifuged for 10 min at 13.4 rpm. The supernatant was collected and analyzed by HPLC using a Zorbax SB-C18 4.6 × 250 mm, 5 μm column with a gradient from 20% to 80% acetonitrile in water containing 0.05% TFA. Chromatograms were recorded at 220 nm, 280 nm and 325 nm.