Abstract: OBJECTIVE To study formulation and process method of large-scale preparation of 5'-cholesteryl-phosphoryl zidovudine(CPZ) self-assemblies freeze-dried powders for injection and their characteristics. METHODS CPZ self-assemblies were prepared with an injection method, where the ethanol solution of CPZ was slowly injected into aqueous solutions followed by rotary evaporation and high-pressure homogeneous process. For the sake of inhibition of CPZ dissociation and improvement of self-assemblies' stability, optimization of formulation and preparation methods involved phosphate buffered solutions with different pH, acetate solutions with different pH, and addition of mannitol. Furthermore, the content of lyoprotectants and sterilization method were explored. CPZ self-assemblies were observed with the transmission electron microscopy (TEM). Size and Zeta potential of them were measured with laser scattering size analyzer. RESULTS The optimal formulation and preparation method included a ratio of mannitol/CPZ (2:1), 0.05% acetic acid solutions as the water phase, which made the large-scale (more than 300 mL) production of CPZ self-assemblies possible. The product could keep long-term stability. The self-assemblies were sterilized by filtrating through 0.45 μm membranes. The re-suspended CPZ self-assemblies had the particle size of 75.17 nm, the PDI was 0.48, and the Zeta potential was -41.2 mV. CPZ self-assemblies were spherical vesicles according to the TEM. CONCLUSION CPZ self-assemblies freeze-dried powders are prepared in large scale with the optimized formulation and preparation method. This study provides a basis for successful development of the novel self-assembled drug delivery system.