(G-278) Sequence Arrangement of Cationic Amphiphilic Polymers Affect Protonation Degree and their mRNA Translation

Introduction:: Cationic polymers have been widely investigated to deliver in vitro transcribed (IVT) mRNA for vaccination, genome editing, and cancer immunotherapy. The state-of-the-art cationic polymers contains two functional moieties: ionizable amino moiety and hydrophobic moiety. Ionizable amino moiety is responsible for IVT mRNA binding and endosomal escape and hydrophobic moiety provides colloidal stability of mRNA-loaded nanoparticles in physiological buffers. Most amphiphilic polymers were randomly modified (or arranged) with these two moieties because of their easy synthesis. However, random-arranged polymers encounter difficulty of quality of controls for batch-to-batch mass production, becoming bottle neck of clinical translation. Here, we prepared sequence-arranged alternative amphiphilic polymers by using solid-phase synthesis and evaluated their functionality (protonation degree and mRNA binding) of ionizable amino moiety. For this purpose, we synthesized sequence-arranged polymers of polyaspartamide derivative polymers, which were previously prepared by random introduction of diethylenetriamine (DET) and octylamine (OCT). DET moiety has two repeating aminoethylene units with pKa 8.9 and 6.2, thus inducing high endosome escape at acidic pH. OCT moiety enhances polyplex stability in vitro and in vivo by optimizing hydrophobicity in the polyplex. We prepared three different sequence-arranged alternative polymers and one control polymers by solid phase synthesis to investigate protonation degree, particle formation, and mRNA translation efficacy.


Materials and Methods:: A series of sequence-arranged amphiphilic polymers were prepared by solid phase peptide synthesis with tert-butyl protected aspartic acid and octyl modified aspartic acid. Four 8mer polymers with different sequences were synthesized to have DDDDDDDD, ODODODOD, OODDOODD, and OOOODDDD, which D and O indicated Asp(DET) and Asp(OCT), respectively. Successful synthesis of 8mers polymers were confirmed in Maldi-Tof and GPC. A series of 8mer peptides were compared to evaluate the impacts of sequence arrangements on protonation degree and solubility in buffer solution. Protonation degree of DET groups in the side chain of four different 8mer polymers were investigated by using automatic potentiometric titration, which was performed in the pH ranges between 2.5 and 12.0 in the presence of 150 mM NaCl. Critical association concentration of amphiphilic polymers was determined by static light scattering using a Zetasizer Pro (Malvern Instruments, Worcestershire, UK), equipped with a He–Ne laser (λ = 633 nm), at a temperature of 25 °C and a detection angle of 173°. The samples in HEPES buffer were analyzed by using a low volume quartz cuvette (ZEN2112, Malvern Instruments) at various polymer concentration from 0.002 mg/mL to 4 mg/mL.


Results, Conclusions, and Discussions:: We successfully synthesized sequence-arranged amphiphilic polymers by solid phase synthesis, confirmed by Malfi-Tof and GPC. First, protonation degree of DET moieties in four different sequences were compared to check whether sequence arrangement affects proton assess in DET moiety, which is responsible for mRNA binding, endosomal escape, and solubility of polymers. Interestingly, OODDOODD showed similar protonation profiles compared to DDDDDDD, indicating two DET moieties in a row easily accept proton. In contrast, ODODODOD and OOOODDDD exhibited retarded protonation profiles probably due to hindered proton access. Critical association concentration (CAC) of four amphiphilic polymers were also investigated by light scattered intensities. DDOODDOO (CAC = 0.304 mg/mL) was 2-3 folds higher than DODODODO (CAC = 0.155 mg/mL) and DDDDOOOO (CAC = 0.083 mg/mL). This indicated that sequence arrangements of the polymers also determined aqueous solubility at pH 7.3 due to protonation degree. Based on these results, we decided DDOODDOO for proper sequence arrangements of DET and OCT moieties and then further synthesized polymers with longer lengths. 12mer, 16mer, and 20mer DDOOs were successfully synthesized via solid phase synthesis. Three amphiphilic polymers were associated with luciferase-coded IVT mRNA and formed hundreds nanometer-sized polyplexes. Each polyplexes were evaluated for luciferase expression levels in cultured cancer cells and showed that 20mer DDOO polyplexes showed the highest mRNA expression. This was due to their enhanced cellular uptake of mRNA toward cancer cells, measured by flow cytometry. We demonstrated that two moiety arrangements of amphiphilic polymers dramatically affected protonation degree, aqueous solubility, polyplex formation, and ultimately mRNA transfection efficiency. Indeed, we newly developed synthesis protocols of sequence arranged polyaspartamide derivative via solid phase synthesis, leading to one step closer to clinical translation of the polymers and increase potential for IVT mRNA mediated therapy.


Acknowledgements (Optional): : H. J. Kim was supported by the National Research Foundation of Korea (NRF) grant (No. 2021R1A2C1007668 and 2022M3E5F1017719).


References (Optional): : 1. H. J. Kim, S. Ogura, T. Otabe, R. Kamegawa, M. Sato, K. Kataoka, K. Miyata, “Fine-Tuning of Hydrophobicity in Amphiphilic Polyaspartamide Derivatives for Rapid and Transient Expression of Messenger RNA Directed Toward Genome Engineering in Brain” ACS Central Science 5, 1866-1875 (2019).

2. J. Yum, B. S. Kim, S. Ogura, R. Kamegawa, M. Naito, Y. Yamasaki, H. J. Kim, K. Miyata "Fine-tuning of polyaspartamide derivatives with alicyclic moieties for systemic mRNA delivery" Journal of Controlled Release, 342, 148-156 (2022).