Glycofunctionalization of Liposomes via Chemoselective Linkers and in-vitro/in-vivo Applications

The role of carbohydrates in cellular communication and biological targeting has been a major topic of study in recent years. Different pathologies, including cancer, tend to overexpress lectin receptors that bind specifically to selected sugar moieties. Based on this fact, simple and complex glycans have become interesting candidates for specific cellular targeting. This study aimed to find a procedure providing an easy and efficient way to functionalize nanoparticles with simple and complex glycans, exploiting commercial deprotected sugars. In order to do so we developed 6-Carbon linkers displaying an aminooxy- group, or a phenylhydrazine- group, able to spontaneously and chemoselectively attach a reducing sugar. The other end of each linker contains a thiol group that can attach to PEGylated liposomes via maleimide-thiol coupling. The design of the linkers can therefore be considered a straightforward method for the decoration of nanoparticles with an array of simple and complex glycans Results: liposomes functionalized with two different linkers, aminooxy- or phenylhydrazine, were each distinctively coupled with multiple simple sugars including glucose, mannose, and galactose as well as a complex sugar Sialyl Lewis X and they were fully characterized. Traditional methods of detection and quantification of carbohydrates expressed on the outer surface of the liposome have been modified and optimized to generate new protocols of nanosight based agglutination assay using corresponding lectins, and direct/indirect detection/quantification methods specific to glycoliposomes using the Phenol-Sulfuric assay. Additionally, the relevance of glycofunctionalization strategies on Nanoparticle-Cell communication and interaction was verified via 1) functionalization of liposomes with glucose or mannose and the testing of cytotoxicity/selectivity of uptake using macrophages, 2) the functionalization of liposomes with galactose or Sialyl Lewis X to target receptors overexpressed in hepatocellular carcinoma for in-vitro/in-vivo studies. Briefly, Macrophages express C-type lectins CD206 (Cluster of differentiation 206) that are specific for mannose. In the first study, glucose functionalized liposomes were directly compared to mannose functionalized liposomes to test for targeting properties via cell-uptake using RAW 264.7 (murine macrophage cell line). Meanwhile, in the second study, Asialoglycoprotein receptor (ASGPR), a hepatic lectin specific for galactose, and selectin, a sialic acid specific receptor overexpressed in proinflammatory tissues, have been used for targeted delivery of MRI compatible liposomes encapsulated with Gadolinium (MRI contrast agent) and Sorafenib (a first line defense drug of hepatocellular carcinoma). In-vitro uptake studies were performed using HepG2, and the cells were further inoculated into nude mice to monitor biodistribution and efficacy via MRI. Conclusion: This study presents new methods for the glyco-functionalization of liposomes along with novel characterization techniques specific for carbohydrates, thus creating a complete glyco-model with great potential in translational applications and drug delivery displayed by the in-vitro/in-vivo studies conducted.