Enhancement of Membrane Rigidity by Chain-Length Dependent Doxorubicin Embedment in PEGylated Liposomal Lipid Bilayers

Jia-Jhen Kang^1*, Zhih-Chen Huang², Li-Wen Tang³, Chun-Jen Su¹, Hua-De Gao², Hsien-Ming Lee², U-Ser Jeng^1,3,4

¹National Synchrotron Radiation Research Center, Hsinchu, Taiwan
²Institute of Chemistry, Academia Sinica, Taipei, Taiwan
³Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan
⁴College of Semiconductor Research, National Tsing Hua University, Hsinchu, Taiwan

* Presenter:Jia-Jhen Kang, email:kang.jj@nsrrc.org.tw

Poly(ethylene glycol)-grafted (PEGlyated) liposomes receive increasingly more attention due to their practical applications in delivering vaccines, nutrients, and drug molecules such as doxorubicin (DOX). PEGlyated liposomes have been well-demonstrated for their capability in carrying DOX in terms of rod-like crystallites enclosed inside the unilamellar vesicles. An unresolved issue of whether DOX also intercalates inside the liposome bilayers is addressed in this study using simultaneous small- and wide angle X-ray scattering (SWAXS), complementary with an integrated system of an asymmetric flow field-flow fractionation instrument coupled with multi-angle light scattering, dynamics light scattering, UV-vis absorption, and refractometer (AF4-MALS-DLS-UV-RI). The DOX-loaded PEGlyated liposomes used are comprised of phosphocholine lipids diCn:1PC, of different lipid chain lengths of n = 18, 20, and 22, and a fixed molar ratio of lipid:cholesterol:DSPE-PEG2000 of 45:50:5. The SAXS data were fitted with a 5-layered model to describe the membrane structure of the pristine liposomes, and the result indicates that the membrane thickness increases with increasing lipid chain length. As for the DOX-loaded liposomes, the rod-like nanocrystallites formed by DOX are found within all the three types of PEGlyated liposomes. Moreover, the membrane structure shows variation from that of the pristine liposome, implying that the uptake of DOX influences the alignment between lipids forming the vesicle. Thus, our results indicate that DOX may intercalate within the liposomes' unilamellar bilayers. Particularly, DOX is found to best intercalate into the diC22:1PC bilayer of a relatively thickest hydrophobic chain region, resulting in a largest liposome deformation to a faceted spherical shell from the ideal spherical shell with no DOX, as consistently evidenced by the cryo-TEM images and the liposome shape analysis of AF4-MALS. A composition analysis is further established to deduce the overall lipid:DOX ratios of 5.2, 4.6, and 10.3 for the three types of PEGlyated liposomes, respectively.

Keywords: liposome, small-angle X-ray scattering, wide-angle X-ray scattering, flow field-flow fractionation