Grafting of anti-nucleolin aptamer into preformed and remotely loaded liposomes through aptamer-cholesterol post-insertion

A new combination strategy of an active loading and active targeting approach was applied in this work. The liposomes actively loaded with Curcumin (CRM) (Lip(CRM)) were decorated with cholesterol tagged-anti-nucleolin AS1411 aptamer (NCL) via a new post-insertion approach, utilizing the cholesterol as a wedge to incorporate aptamer into the surface of the liposome bilayer. A successful NCL post-insertion was verified by agarose gel electrophoresis and dynamic light scattering (DLS). The cellular uptake of Apt(NCL)-Lip was investigated using flow cytometry and Confocal Laser Scanning Microscopy (CLSM) on two different human breast cancer cell lines (MCF-7 and MDA-MB-231). The uptake and cytotoxicity of loaded CRM were investigated using flow cytometry and MTT assay. Our results showed successful post insertion of NCL aptamer to the surface of Lip. Also, higher cellular uptake was noted for Apt(NCL-Alexa)-Lip(Rhod) compared to blank Lip(Rhod) in both cell lines. Moreover, CLSM showed prominent endocytosis and uptake of Apt(NCL-Alexa)–Lip(Rhod) into the cytoplasm of breast cancer cells. Furthermore, the results showed a significant increase in the uptake and cytotoxicity of Apt(NCL)-Lip(CRM) compared to Lip(CRM) in both cell lines. Overall, our results demonstrate a successful post-insertion of cholesterol-tagged aptamer into liposomes and the possible combination between active loading and active targeting.