The idea of using virus-like particles as nanocarriers for heterologous cargo transport and delivery requires controlling the stability of the container–cargo system. In particular, the conditions of the cargo delivery entail tailoring the escape of the molecular payload when the virus particle is disassembled. What happens to the internalized molecules when the nanocage is opened? To this end, it is necessary to control the cargo–container interaction which, in turn, would tune the retention of cargo when the disassembly of the nanocarrier takes place. Thus, it is necessary to develop nanocarrier systems that facilitate the control of the cargo retention conditions as a function of its interaction with the nanocontainer. We designed three mutants of human picobirnavirus where the RNA–coat protein interaction, observed previously via cryo-electron microscopy, is modified by changing the N-terminal end of the coat protein. Here, we use atomic force microscopy for inducing the mechanical unpacking of the RNA internalized in particles of each mutant. Our experiments crack-opened individual particles in real time to monitor the cargo release. Among other results, we have measured that an increment in the N-terminal length by just 8% increases the cargo retention of partially disrupted particles by a factor of 10 with respect to the wild type. Our study elucidates the interplay between the RNA–coat protein interaction of each mutant and their capacity for cargo retention during disassembly. [Full article]