<p>Diphenylalanine and its analogs cause many concerns owing to their perfect self-assembly properties in the fields of biology, medicine, and nanotechnology. Experimental research has shown that diphenylalanine-based analogs with ethylenediamine linkers (PA, P = phenylalanine, and A = analog) can self-assemble into spherical assemblies, which can serve as novel anticancer drug carriers. In this work, to understand the assembly pathways, drug loading behavior, and formation mechanism of PA aggregates at the molecular level, we carried out dissipative particle dynamics (DPD) simulations of PA molecule systems. Our simulation results demonstrate that PA molecules spontaneously assemble into nanospheres and can self-assemble into drug-loaded nanospheres upon addition of the cancer chemotherapeutic agent doxorubicin (DOX). We also found that the hydrophobic side chain beads of PA molecules exhibited a unique onion-like distribution inside the nanospheres, which was not observed in the experiment. The onion-like nanospheres were verified by calculating the radial distribution function (RDF) of the DPD beads. Furthermore, based on the analysis of the percentages of different interaction components in the total nonbonded energies, main chain-side chain interactions between PA molecules may be important in the formation of onion-like nanospheres, and the synergistic effects of main chain-side chain, main chain-drug, side chain-drug, and main chain-solvent interactions are significant in the formation of drug-loaded nanospheres. These findings provide new insights into the structure and self-assembly pathway of PA assemblies, which may be helpful for the design of efficient and effective drug delivery systems.</p>