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Phospholipid nanoparticles (PNs) encapsulating vitamin C and E derivatives, 3-O-cetyl ascorbic acid (CA) and tocopherol acetate (TA), respectively, were examined using the film rehydration and extrusion method. PN formulations (TA-Cassome) were prepared by mixing CA, soya phosphatidylcholine (Soya PC), sodium cholate, and TA at a molar ratio of 20/80/5/6. Glycerol (GL) or diglycerol (DG) were also added to improve the skin accumulation of CA and TA. Three TA-Cassome formulations were evaluated using a dynamic light scattering (DLS), NMR, TEM, skin accumulation test for CA and TA, and small-angle X-ray diffraction (SAXD) analysis. TA-Cassome formulations (150nm) were stable for two weeks and they encapsulated 1.8mg/mL of TA. TEM and SAXD analysis revealed that the nanoparticles formed a spherical multilayer structure. 1H and 31P NMR indicated that GL and DG enhanced the proton mobility of choline groups of soya PC molecules located on the membrane surface of TA-Cassome. Accumulation of CA and TA in the dermis was increased by adding GL and DG. SAXD analysis revealed that GL and DG promoted the formation of new lamellar structures on the stratum corneum, which contributed to improving the skin accumulation of CA and TA.