Strategies for High Grafting Efficiency of Functional Ligands to Lipid Nanoemulsions for RGD-Mediated Targeting of Tumor Cells in Vitro

Lipid nanoemulsions (LNEs) are promising nanocarriers for delivering high payloads of lipophilic molecules. Nonetheless, the dynamic nature at their aqueous interfaces results in poor surface chemistry and thus ligand functionalization can be challenging. Herein, two independent strategies, postconjugation and preconjugation, were explored to prepare LNEs grafted covalently with model ligands, fluorescein dye and RGD peptide, respectively. Fluorescein was successfully conjugated with high grafting efficiency to an amine-functionalized lipid nanoemulsion (NH2-LNE) as determined by spectrophotometric analysis. First, we formulated NH2-LNEs by a low-energy spontaneous emulsification technique in the presence of oleylamine (OA) within the oily core of the nanodroplets, thus creating primary amine-reactive sites at the oil/water interface. These amines were used to incorporate fluorescein, yielding fluorescent LNEs with grafting efficiencies of 33, 69, and 69% at NH2-LNEs with [OA]oil = 0.18, 0.34, and 0.49 M, respectively. We also developed RGD-labeled LNEs (RGD-LNEs) and evaluated the nanomaterial with model cell lines that overexpress αVβ3 integrins on their surfaces. To this end, we initially synthesized an RGD-Oleate fatty acid-peptide conjugate by solid-phase synthesis. The lipophilic segment of this conjugate readily embedded into the oily core of the LNE, and the hydrophilic head (RGD moiety) was oriented toward the LNE interface. In vitro cytotoxicity and cellular uptake studies were undertaken on different cancer cell lines including HaCaT human umbilical vein endothelial cells (HUVECs), MCF-7, and U-87 MG and compared to uptake experiments with RAW 264.7 macrophages. Confocal imaging and flow cytometry showed that RGD-LNEs were preferentially taken up by all of the tumor cell lines but showed very slight accumulation in RAW macrophages. Unmodified LNE controls did not show any appreciable cellular uptake. This work provides a simple and reliable methodology for the incorporation of multiple ligands on a single surface to facilitate active tumor targeting with LNE-based drug/imaging carriers for theranostic applications.