In this work, we examined the detailed mechanisms that affect the interaction between large DNA constructs with multifunctional units and SWCNTs coated with the positively charged PEI polymers, the toxicity of the conjugates, release of DNA from the SWCNT surface, and the expression of functional cassettes of different sizes in plant cells. Because PEI is a highly charged polymer, we used chitosan, a moderately charged polymer, as a control to validate our results. In addition, DNA can be released from chitosan at the basic pH of chloroplast but not inside the acidic pH of the nucleus. Thus, chitosan can serve as negative control for nucleus-based expression of the transformed DNA. Our results demonstrated that once condensed on the PEI-CNT, plasmids are not released from the complex, and most of the bounded plasmid part is not accessible for the cellular transcription machinery. Our results highlight the limitations of PEI-CNT mediated plasmid DNA delivery for practical application in plants and provide clear-cut guidelines for further technological development to establish nanoparticles as carriers for multifunctional DNA plasmids towards genome engineering applications in diverse plant species.