Within the last decade, the introduction of checkpoint inhibitors proposed to boost the patients’ anti-tumor immune response has proven the efficacy of immunotherapeutic approaches for tumor therapy. Furthermore, especially in the context of the development of biocompatible, cell type targeting nano-carriers, nucleic acid-based drugs aimed to initiate and to enhance anti-tumor responses have come of age. This review intends to provide a comprehensive overview of the current state of the therapeutic use of nucleic acids for cancer treatment on various levels, comprising (i) mRNA and DNA-based vaccines to be expressed by antigen presenting cells evoking sustained anti-tumor T cell responses, (ii) molecular adjuvants, (iii) strategies to inhibit/reprogram tumor-induced regulatory immune cells e.g., by RNA interference (RNAi), (iv) genetically tailored T cells and natural killer cells to directly recognize tumor antigens, and (v) killing of tumor cells, and reprograming of constituents of the tumor microenvironment by gene transfer and RNAi. Aside from further improvements of individual nucleic acid-based drugs, the major perspective for successful cancer therapy will be combination treatments employing conventional regimens as well as immunotherapeutics like checkpoint inhibitors and nucleic acid-based drugs, each acting on several levels to adequately counter-act tumor immune evasion.

Carriers for RNA delivery must be dynamic, first stabilizing and protecting therapeutic RNA during delivery to the target tissue and across cellular membrane barriers and then releasing the cargo in bioactive form. The chemical space of carriers ranges from small cationic lipids applied in lipoplexes and lipid nanoparticles, over medium-sized sequence-defined xenopeptides, to macromolecular polycations applied in polyplexes and polymer micelles. This perspective highlights the discovery of distinct virus-inspired dynamic processes that capitalize on mutual nanoparticle–host interactions to achieve potent RNA delivery. From the host side, subtle alterations of pH, ion concentration, redox potential, presence of specific proteins, receptors, or enzymes are cues, which must be recognized by the RNA nanocarrier via dynamic chemical designs including cleavable bonds, alterable physicochemical properties, and supramolecular assembly–disassembly processes to respond to changing biological microenvironment during delivery.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH‐responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence‐defined molecules. The pH‐dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH‐dependent structural and physical changes. This “molecular chameleon character” turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T‐shapes, U‐shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100‐fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra‐low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1–3 µg luciferase‐encoding mRNA in mice.

Nanomedicine has a great potential to revolutionize the therapeutic landscape. However, up-to-date results obtained from in vitro experiments predict the in vivo performance of nanoparticles weakly or not at all. There is a need for in vitro experiments that better resemble the in vivo reality. As a result, animal experiments can be reduced, and potent in vivo candidates will not be missed. It is important to gain a deeper knowledge about nanoparticle characteristics in physiological environment. In this context, the protein corona plays a crucial role. Its formation process including driving forces, kinetics, and influencing factors has to be explored in more detail. There exist different methods for the investigation of the protein corona and its impact on physico-chemical and biological properties of nanoparticles, which are compiled and critically reflected in this review article. The obtained information about the protein corona can be exploited to optimize nanoparticles for in vivo application. Still the translation from in vitro to in vivo remains challenging. Functional in vitro screening under physiological conditions such as in full serum, in 3D multicellular spheroids/organoids, or under flow conditions is recommended. Innovative in vivo screening using barcoded nanoparticles can simultaneously test more than hundred samples regarding biodistribution and functional delivery within a single mouse.