Developing natural lipid-based conjugates/prodrugs emerged as a promising topic in pharmaceutical chemistry and biomedicine. Based on our prior works on natural lipid-based gene/drug delivery systems, in this work, a natural antioxidant lipid, α-Tocopherol (vitamin E), was covalently connected with Doxorubicin (Dox, a topoisomerase II inhibitor) to synthesize a Toco-Dox conjugate through two approaches: Triphosgene activation (method A) and 4-nitrophenyl chloroformate (method B) activation methods. Regarding the green chemistry issues, Triphosgene (method A) easily decomposes into extremely lethal phosgene gas (used as a chemical weapon in WWI ), in contrast, 4-Nitrophenyl chloroformate is non-volatile and generates safe-to-handle byproduct 4-nitrophenol (does not release any toxic gas), making it much less hazardous and more eco-friendly. Molecular structure and purity of the Toco-Dox was characterized by ¹H and ¹³C NMR, FT-IR, MALDI-TOF-MS and UHPLC. Toco-Dox could self-assemble into prodrug nanoparticles (NPs) in the DMSO/water mixture, and the related physico-chemical properties of Toco-Dox NPs were characterized by DLS. Moreover, The physicochemical and biological properties of Toco-Dox (Dox as a control) were theoretically calculated or viruallly analyzed. In addition, MCF-7 breast cancer cell inhibition (cytotoxicity) and intracellular localization of the Toco-Dox was preliminarily evaluated. This work provided efficient approaches for developing natural hydrophobic vitamine (lipid)-based prodrug delivery systems.