Optimization of Polyarginine-Conjugated PEG Lipid Grafted Proliposome Formulation for Enhanced Cellular Association of a Protein Drug.

The purpose of this study was to develop an oral proliposomal powder of protein using poly-l-arginine-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) (PLD) for enhancing cellular association upon reconstitution and to compare its effects with a non-grafted and PEGylated formulation. Cationic proliposome (CATL), PLD-grafted CATL (PLD-CATL), PEGylated CATL (PEG CATL), and PLD grafted-PEG CATL (PLD-PEG CATL) were prepared and compared. Successful conjugation between poly-l-arginine and DSPE-PEG was confirmed by H NMR and FT-IR. PLD was successfully grafted onto the proliposomal powder during the slurry process. Although reconstituted liposomal sizes of CATL and PLD-CATL were increased by agglomeration, PEGylation reduced the agglomeration and increased the encapsulation. The viabilities of cells treated with both CATL and PLD-CATL formulations were low but increased following PEGylation. With regard to cellular association, PLD-CATL enhanced cellular association/uptake more rapidly than did CATL. Upon PEGylation, PEG CATL showed a lower level of cellular association/uptake compared with CATL while PLD-PEG CATL did not exhibit the rapid cellular association/uptake as seen with PLD-CATL. However, PLD-PEG CATL still enhanced the higher cellular association/uptake than PEG CATL did without PLD. In conclusion, proliposomes with PLD could accelerate cellular association/uptake but also caused high cellular toxicity. PEGylation reduced cellular toxicity and also changed the cellular association pattern of the PLD formulation.