To ensure that mRNA is delivered stably to the target tissue

- Polyplex micelles (PMs) are structurally stable in vitro and in mouse bloodstream.

- RNases degrade mRNA inside PMs in vitro and in mice, even without PM dissociation.

- This study identifies key design factors of PMs for protection from RNases.

- Polycation structure and length are determinants of RNase stability of PMs.

- Enhancing RNase resistance remains challenging for systemic delivery of PMs.

Innovation Center of NanoMedicine (Center Director: Kazunori Kataoka, Location: Kawasaki-Japan, Abbreviation: iCONM) has published a research result on the website of Journal of Controlled Release on June 5, 2025. The paper titled "Structural stability and RNase resistance of mRNA Polyplex micelles for systemic delivery" describes a method for more stable delivery of mRNA drugs, which are prone to losing their activity to degrading enzymes when mounted in micelles, to target tissues.

Polycation-based mRNA delivery systems have an issue with mRNA integrity in the physiological milieu, particularly in blood compartments, despite their vast potential in mRNA therapeutics. Without comprehensive mechanistic analyses, design concepts of polyplexes for in vivo use remain unclear. Herein, we systematically assessed several potential design parameters of polyplex stabilization and provided mechanistic insight into the processes of mRNA degradation loaded in polyplexes, focusing on RNase attack, a process believed to be the leading cause of loss of mRNA integrity loaded into polyplexes. For this purpose, polyplex micelles (PMs) from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymer were used as a platform polyplex system feasible for in vivo application. Elongating PEG from 12-kDa to 42-kDa failed to improve RNase stability despite a plausible increase in the PEG layer thickness on the PM surface. Meanwhile, the elongation of polycation segments and a subtle but critical modulation in the side chain of polycation structure, i.e., changing from poly(l-lysine) to poly(l-ornithine), significantly improved the resistance of cargo mRNA against RNase attack. Nonetheless, nearly 50 % of mRNA was degraded even in the optimal PM formulation after 30 min incubation in 50 % serum. Plausible mechanisms of mRNA degradation include (i) dissociation of PM structure by polyion exchange reaction with anionic biomolecules in serum to release mRNA, followed by RNase attack and (ii) RNase penetration into PM interior to directly attack cargo mRNA without PM dissociation. A series of mechanistic experiments revealed that mRNA was still settled in the PMs even after a loss of mRNA integrity by 50 % serum treatment, indicating the latter to be the main reason for the degradation of cargo mRNA. Further, the integrity of PM structure and cargo mRNA in circulating blood was evaluated separately in mice. Intravital microscopic observation of mRNA complexation status using fluorescence resonance energy transfer (FRET) indicates prolonged mRNA retention in the PM structure even under blood circulation. In contrast, quantitative PCR-based evaluation of mRNA integrity revealed the occurrence of prompt mRNA degradation in the same condition. This study highlights that PM structure is robust enough against dissociation under blood circulation. Yet, the remaining challenge toward optimizing PM-based mRNA delivery systems for systemic application is to build a functionality to prevent RNase invasion into the polyplex core storing cargo mRNA.

Novelty of the Research:

1. RNase penetration into structurally intact polyplex micelles—rather than polyplex dissociation—emerges as the dominant pathway of mRNA degradation. This finding reveals a critical vulnerability in cargo protection despite the structural stability of the carrier, marking a paradigm shift in our understanding of mRNA degradation within polymer-based delivery systems (Figure).

2. Elongation of PEG length from 12-kDa to 42-kDa did not improve RNase resistance, despite a presumed increase in surface shielding by the PEG layer on polyplex micelles.

3. Extending the polycation segment length from 40 to 70 repeating units markedly enhanced the resistance of encapsulated mRNA to RNase-mediated degradation.

4. Subtle yet pivotal modifications in polycation side chains—specifically replacing poly(l-lysine) with poly(l-ornithine)—significantly enhance mRNA resistance to RNase degradation, offering novel structure–function insights of polyplex stabilization.

Future Contributions to Society:

1. Our findings provide a mechanistic foundation for the rational design of next-generation polymer-based mRNA delivery systems. Incorporating anti-RNase strategies—such as core densification or enzymatic shielding—will be essential to prevent RNase infiltration and preserve mRNA integrity. These advancements are pivotal for transforming biologically fragile nucleic acid therapeutics into robust polyplex micelles, thereby accelerating the clinical translation of mRNA-based vaccines, gene therapies, and cancer immunotherapies with broad implications for global health.

2. The demonstrated storage stability of polyplex micelles at room temperature enhances their practical applicability and positions them as promising candidates for real-world clinical deployment.