Nucleic acid nanoparticles (NANPs) are promising immune modulators due to their well-established structural properties and distinct structure-activity relationship with the immune system. We previously identified that NANPs' size, shape, composition, and type of delivery vehicle define their uptake by immune cells and subsequently induced cytokine profile. In this work, we examined the delivery efficiencies and immunological impacts of two representative NANPs─DNA cubes and RNA cubes─complexed with a benchmark delivery vehicle, Lipofectamine 2000 vs. different generations of amine-terminated poly(amidoamine) dendrimers. Using molecular dynamics simulations, we modeled dendrimer interactions with nucleic acid cargos. Next, we used traditional 2D and more recently established 3D cell cultures to assess dendrimers' influence on NANPs uptake. Immune activation was evaluated in several cell lines engineered with reporter genes driven by key immune signaling pathways. Specifically, HEK-lucia reporter cells were used to evaluate RIG-I activation, while THP1-Dual cells provided quantitative readouts for both IRF and NF-κB transcription factor activity. Our findings demonstrate that both dendrimer generation and NANP composition influence cellular uptake and immune responses. This study underscores the importance of formulation in shaping NANPs' biological properties and further advances the understanding of their immunological properties critical for the development of NANPs-based adjuvants.