Elucidating dynamic morphological changes of gene-carrying vectors and their nucleic acid release under varying intracellular conditions has been a technical challenge. Atomic force microscopy (AFM) was used to observe nucleic acid/polymer polyplexes under endosomal and reducible cytosolic conditions. Both ketalized (acid-degradable) and unmodified (nondegradable) polyethylenimine (PEI) in linear and branched forms were used to prepare plasmid DNA- or siRNA-complexing polyplexes. Then, the polyplexes' complexation and disassembly were observed by in situ AFM in various differentially changing buffers that represent intracellular conditions. Results demonstrated obvious morphological destruction of DNA/ketalized linear PEI (KL-PEI) polyplexes under mildly acidic endosomal conditions, whereas no morphological changes were observed by DNA/ketalized branched PEI (KB-PEI) under the same conditions. In addition, siRNA was more efficiently dissociated from KL-PEI than KB-PEI under the same conditions. Nondegradable PEI did not show any evidence that DNA or siRNA was released. Anionic biomacromolecules (e.g., heparan sulfate), which was hypothesized to dissociate nucleic acids from cationic polymers, did not successfully disassemble polyplexes but appeared to be adsorbed on cationic polymers. The in situ AFM results combined with in vitro cellular transfection and gene silencing indicated that efficient endosomal escape of plasmid DNA in a compact polyplex form is required for efficient gene expression. On the contrary, rapid dissociation of siRNA from its cationic carrier is crucial for efficient gene silencing. The findings of this study may provide new insightful information for designing stimuli-responsive nonviral gene vectors as well as expanding tools for investigating nonviral vectors in nanoscales under biologically inspired conditions.

The dynamic and versatile nature of diseases such as cancer has been a pivotal challenge for developing efficient and safe therapies. Cancer treatments using a single therapeutic agent often result in limited clinical outcomes due to tumor heterogeneity and drug resistance. Combination therapies using multiple therapeutic modalities can synergistically elevate anti-cancer activity while lowering doses of each agent, hence, reducing side effects. Co-administration of multiple therapeutic agents requires a delivery platform that can normalize pharmacokinetics and pharmacodynamics of the agents, prolong circulation, selectively accumulate, specifically bind to the target, and enable controlled release in target site. Nanomaterials, such as polymeric nanoparticles, gold nanoparticles/cages/shells, and carbon nanomaterials, have the desired properties, and they can mediate therapeutic effects different from those generated by small molecule drugs (e.g., gene therapy, photothermal therapy, photodynamic therapy, and radiotherapy). This review aims to provide an overview of developing multi-modal therapies using nanomaterials ("combo" nanomedicine) along with the rationale, up-to-date progress, further considerations, and the crucial roles of interdisciplinary approaches.