Modulated Protonation of Side Chain Aminoethylene Repeats in N-Substituted Polyaspartamides Promotes mRNA Transfection

Abstract

Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

Rapid Exchange Between Free and Bound States in RNA–Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release

“…Studies have shown correlations of knockdown/transfection efficiencies of therapeutic RNA polyplexes with modulations of polymer properties. 14–16 However, there is lack of molecular level understanding of polyplex structure and dynamics and how these properties vary as a function of key vector properties such as molecular weight, hydrophobicity, and charge density. 17 Polyplexes are often difficult to characterize in terms of molecular stoichiometry, thermodynamic constants, exchange kinetics, and dynamics.…”

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…To date, naked, chemically modified, or protamine-complexed mRNA have shown promise in phase I/II cancer trials (24)(25)(26). Recently, preclinical development of materials specific for mRNA delivery has resulted in cationic polymers such as polymethacrylates (27)(28)(29), poly(aspartamides) (30,31), and polypeptides (32), as well as multicomponent cationic lipid or lipid-like formulations (21,(33)(34)(35)(36). In many of these examples, however, transfection efficiencies can be quite low, ranging 20-80% in cells (18), with likely much lower efficiencies in vivo, which requires either high mRNA doses or hydrodynamic injections (32,37).…”

Structurally Programmed Assembly of Translation Initiation Nanoplex for Superior mRNA Delivery

“…This phenomenon, along with a potentiometric titration, has been utilized to predict the protonation structures of N1 (EDA), N2 (DET), N3 (TET) and N4 (TEP) at pH 7.4, with N2 (DET) and N4 (TEP) bearing two and three protonation structures in equilibrium, respectively (Scheme 1). 25–26 Using the same approach, we found that N5 (PEH) assumes a ~56% degree of protonation at pH 7.4. This implies that nearly three out of five amine groups on the side chain are protonated.…”