And the pH-responsiveness of the liposomes was evaluated by measuring the amount of pyranine released from them. After testing different ratios, we decided to use the POPC and DSPE-PG8MG ratio of 85 and15. To investigate cellular uptake and intracellular localization of pH-liposomes, we synthesized Rhodamine-labelled pH-sensitive empty liposomes as described in the Materials and Methods section. Pancreatic cancer cells MiaPaCa-2 were incubated with Rhodamine-labelled liposomes for 4 hours and liposomal MCE Chemical NAN-190 (hydrobromide) fluorescence was examined with a fluorescent microscope. As shown in Fig 2A, punctate fluorescence was observed at a perinuclear region indicative of lysosomal localization. This point was confirmed by the use of Lysosensor Green DND-189. Lysosensor Green DND-189 exhibits green fluorescence only when inside intracellular acidic compartments such as lysosomes. Co-localization of red Rhodamin fluorescence and green Lysosensor fluorescence was observed, confirming lysosomal localization of the liposomes. Thus, liposomes appear to accumulate in (R,S)-Ivosidenib lysosomes when taken up by cells. Liposome��s loading efficiency of drugs and dyes was tested by mixing with liposomes, using Sepharose 4B column to remove free drugs and releasing the content from liposomes by Triton X-100. GGTI concentration was measured by comparing with a standard GGTI sample using LC-MS.We estimated that the loading capacity of GGTI into liposomes is approximately 30 of total GGTI. To examine the size of reconstituted liposomes, we subjected GGTI-loaded liposomes to dynamic light scattering measurement. As can be seen in Fig 2B, we found that the size of the GGTI-loaded liposomes ranged from 46 to 65 nm in diameter with average size of 54.9 nm. Thus, our preparation has a relatively narrow size distribution. Release of GGTI from liposomes was examined by loading GGTI, collecting GGTI-loaded liposomes and releasing the content by exposing to PBS buffer with various pH values ranging from 4.5 to 8. As shown in Fig 2C, liposomes retained GGTI inside tightly at neutral or basic buffers. Little release of GGTI was observed at a pH range 6�C8. However, when pH was below 6, a sig
