(C-94) Synthesis of Gambogic Acid Conjugated Liposomes for Targeted Drug Delivery

Introduction:: Gambogic acid (GA) is a small molecule derived from Garcinia hanburii that has previously been employed as a chemotherapeutic drug. One reason it has found use in this particular niche is that besides its cytotoxicity, it is a non-competitive ligand for CD71, a membrane transporter responsible for iron uptake that is frequently overexpressed on cancer cells and certain leukocyte populations. However, its application in targeting cancer and drug delivery is somewhat limited by moderate cytotoxicity and high hydrophobicity, which limit its applicability to more complex systems. Liposomes are nanocarriers composed of a phospholipid membrane enclosing an aqueous interior. By containing both hydrophobic (within the membrane) and hydrophilic (within the lumen) regions, they can be used to deliver both hydrophobic and hydrophilic payloads. Their utility is particularly enhanced by the fact that there exist several FDA-approved therapies that rely on liposomal delivery, making the translation of liposome-based technologies relatively straightforward. Knowing the applicability of liposomes for receptor-mediator targets, we synthesized GA-functionalized liposomes to apply Gas targeting effect to a versatile drug delivery system. This work describes the synthesis of GA-functionalized liposomes (GALs).


Materials and Methods:: GA was attached to the lipid DSPE-PEG(2000)-NH₃⁺ (Fig1A). The reaction used dicyclohexylcarbodiimide addition overnight at room temperature to ensure the reaction had finished to completion and we purified the conjugated reaction via column chromatography, also testing the reaction at -4°C to see which temperature optimized the functionalization. We specifically used flash chromatography in a silica column with a two-stage mobile phase (Fig 1B). The first of those stages used a solvent made up of 80% hexane and 20% ethyl acetate. The second stage used a solvent made up of 50% dichloromethane and 50% methanol. The product was dried with sodium sulfate and then GALs were synthesized using thin film rehydration with a 6:3:1 ratio of DPPC: Cholesterol: DSPE-PEG-GA. Unlabeled liposomes (ULs) were synthesized with the same ratio, using DSPE-PEG-Amine instead of DSPE-PEG-GA. The conjugated reaction was then characterized with MALDI-TOF mass spectroscopy. An LDH kit was also used to determine the cytotoxicity of the liposomes to a single-cell suspension of primary splenocytes. Liposomes were extruded to a diameter of 200 nm and were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM).


Results, Conclusions, and Discussions:: Mass spectroscopy data showed a range of peaks corresponding to the mass of DSPE-PEG-GA and DSPE-PEG-Amine. When the reaction was done at room temperature we saw that the peaks corresponding to the GA-adduct dominated the unreacted DSPE-PEG-Amine. Whereas with the 4°C synthesis, the opposite was true. Characterization through TEM and DLS data indicated that both GALs and ULs had a diameter of 200 nm with relatively tight polydispersity. Because it is a chemotherapeutic drug, using GA as a targeting moiety brings up some concerns. To address these, we performed an LDH assay that revealed GA did not kill cells in this formulation.


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