Short
peptide-based supramolecular assemblies have drawn much attention in the field of drug delivery. However, the progress still remains limited owing to the inefficient drug loading capacity of conventional short
peptide-based materials. In this study, based on coordinated intramolecular π-π stacking, we customize a
dipeptide-based
rhein derivative (
rhein-
diphenylalanine peptide, RDP), which could spontaneously form spherical nanoassemblies for drug delivery. A structure-based virtual screening of a library of small molecules is conducted to identify the suitable compounds which could be effectively delivered by this nanocarrier. Sorted by binding energy results, fifteen superior and five inferior molecules are found. Subsequently, the co-assembly capacity of high-affinity molecules (
camptothecin,
CPT) and low-affinity molecules (
norcantharidin, NCTD) with the
dipeptide-based carrier is predicted via dissipative particle dynamics (DPD) simulation. Consistent with computational results, the in vitro experimental results show that
CPT-encapsulated nanoassemblies have significant advantages in the particle size distribution and recrystallization-inhibitory effect compared with NCTD. Furthermore, in vivo experiments were conducted to determine whether
CPT is precisely delivered to
tumor sites by using the
dipeptide-based nanoassemblies. The
CPT-loaded nanoassemblies show better effects in terms of drug biodistribution and in vivo anti-
tumor efficacy compared to free
CPT. The cooperative computational and experimental strategies (in vitro and in vivo) used in this work lay a good foundation to systematically understand short
peptide-based assemblies for precise drug delivery.