【公開日：2024.07.25】【最終更新日：2024.06.29】
課題データ / Project Data
課題番号 / Project Issue Number
23UT0006
利用課題名 / Title
Messenger RNA loaded delivery systems
利用した実施機関 / Support Institute
東京大学 / Tokyo Univ.
機関外・機関内の利用 / External or Internal Use
外部利用/External Use
技術領域 / Technology Area
【横断技術領域 / Cross-Technology Area】（主 / Main）計測・分析/Advanced Characterization（副 / Sub）-
【重要技術領域 / Important Technology Area】（主 / Main）次世代ナノスケールマテリアル/Next-generation nanoscale materials（副 / Sub）次世代バイオマテリアル/Next-generation biomaterials
キーワード / Keywords
電子顕微鏡/ Electronic microscope,生分解性材料/ Biodegradable material,ナノ粒子/ Nanoparticles
利用者と利用形態 / User and Support Type
利用者名（課題申請者）/ User Name (Project Applicant)
Dirisala Anjaneyulu
所属名 / Affiliation
公益財団法人川崎市産業振興財団 ナノ医療イノベーションセンター（iCONM）
共同利用者氏名 / Names of Collaborators in Other Institutes Than Hub and Spoke Institutes
ARIM実施機関支援担当者 / Names of Collaborators in The Hub and Spoke Institutes
Ayumi Kimura
利用形態 / Support Type
（主 / Main）技術補助/Technical Assistance（副 / Sub）-
利用した主な設備 / Equipment Used in This Project
報告書データ / Report
概要（目的・用途・実施内容）/ Abstract (Aim, Use Applications and Contents)
Polycation-based mRNA delivery systems have an issue with integrity in physiological conditions, 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. 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 tolerability 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. The cryo-transmission electron microscopic (cryo-TEM) visualization of mRNA packaging within the polyion complex core of PMs helped us understand the morphologies of PMs and calculate the PEG density. 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 FRET (fluorescence resonance energy transfer) 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.
実験 / Experimental
To calculate the surface area of packaged mRNA, the morphology of PMs (N/P ratio 2) was visualized by the cryo-TEM instrument. Briefly, each PM solution (3 µL, 100 ng/mL of GLuc mRNA in 10 mm HEPES buffer, pH 7.3) was separately applied onto a 300-mesh copper grid coated with carbon film (QUANTIFOIL R 3.5/1.0). The excessive solution was blotted for 3 s with filter paper using an automatic plunge freezer (EM GP, Leica microsystems, Wetzlar, Germany) to stretch a thin film of PM solution over the grid (95% humidity, 24 ℃). The sample deposited grid was quickly plunged into a cryogen (liquid ethane at –175 ℃) to shock-frozen the sample. The grid was then set into a cryo-TEM instrument (JEM-2100F, JEOL, Tokyo) using a side-entry cryo-transfer holder (Gatan 914, Gatan Inc., Pleasanton, CA) for imaging. The major axis length (l) and radius (r) (= minor axis length/2) of packaged mRNA were measured from cryo-TEM images using Image Processing and Analysis in Java 1.5.3 software (NIH, Bethesda, MD).
結果と考察 / Results and Discussion
We fixed the PEG length to 12-kDa to systematically explore the contribution of other factors, including polycation species and their lengths. For this purpose, four block copolymers possessing poly(l-lysine) (PLL) or poly(l-ornithine) (PLO) in the polycation segment with an approximate degree of polymerization (DP) of 40 or 70 were prepared; PLL DP was 43 and 67 in PEG-PLL, and PLO DP was 42 and 69 in PEG-PLO. These block copolymers are denoted as PEG-PLL(12-43), PEG-PLL(12-67), PEG-PLO(12-42), and PEG-PLO(12-69). PMs prepared from these block copolymers are 12-PLL43, 12-PLL67, 12-PLO42, and 12-PLO69 PMs. In agarose gel electrophoresis, the effective mRNA complexation was observed at an N/P ratio of 2, irrespective of polycation species and lengths. In z-potential measurements, all four PMs exhibited a near-neutral charge of +1.2 – +2.0 mV, indicating successful PM preparation. In dynamic light scattering measurements, all PMs displayed unimodal size distribution histograms with a hydrodynamic diameter of 54 – 62 nm and a polydispersity index of 0.15 – 0.19. The cryo-transmission electron microscopic (cryo-TEM) visualization of mRNA packaging within the polyion complex core of PMs revealed a mixture of different core morphologies between the DP40 (12-PLL43 and 12-PLO42 PMs) and DP70 series (12-PLL67 and 12-PLO69 PMs) (Figure 1). Both 12-PLL43 and 12-PLO42 PMs (DP40 series) mostly showed filamentous-like structures and a few spherical and ellipsoidal structures. In contrast, both 12-PLL67 and 12-PLO69 PMs (DP70 series) mostly manifested spherical and ellipsoidal packaging of mRNA. PMs of the DP40 series showed longer major axis lengths (48 – 60 nm) than those prepared from the DP70 series (29 – 31 nm). At the same time, PMs of the DP40 series showed slightly thinner minor axis lengths (10 – 12 nm) than those of the DP70 series (16 – 19 nm), irrespective of polycation species, either PLL or PLO. We calculated PEG density and normalized it to reduced tethering density (RTD). RTD is defined as the number of PEG chains occupying the area of an isolated unperturbed PEG chain (piRg2) [RTD = <sigma>piRg2, where Rg is the radius of gyration of an isolated unperturbed PEG chain and <sigma> is the number-averaged PEG density (<sigma>, chains/nm2)]. Notably, PMs of the DP70 series RTD values far below 1 (RTD 0.77 for 12-PLL67 PM and 0.69 for 12-PLO69 PM), suggesting minimal steric repulsive forces between adjacent PEG chains situated on the mRNA, apparently minimizing the surface area of packaged mRNA. This observation indeed agreed with the spherical and ellipsoidal packaging of mRNA to reduce the interfacial free energy of a charge-neutralized polyion complex. On the contrary, PMs of the DP40 series exhibited RTD values close to 1 (RTD 0.9 for 12-PLL43 PM and 0.89 for 12-PLO42PM), where the tethered PEG chains start to contact with the neighboring PEG chain (contact mushroom) with weak or no appreciable lateral interactions, thus hindering the mRNA from collapsing into spherical or ellipsoidal structures and eventually packaged into rod structures in a regulated manner.
図・表・数式 / Figures, Tables and Equations
Figure 1. Packaging structure analyses of mRNA within polyion complex cores of polyplex micelles using cryo-TEM. (A,B,C) 12-PLL43 PM, (D,E,F) 12-PLL67 PM, (G,H,I) 12-PLO42 PM, and (J,K,L) 12-PLO69 PM. (A,D,G,J) Representative images. (B,E,H,K) The major axis length distribution of the polyplex micelle cores. (C,F,I,L) The minor axis length distribution of the PM cores.
その他・特記事項（参考文献・謝辞等） / Remarks(References and Acknowledgements)
成果発表・成果利用 / Publication and Patents
論文・プロシーディング（DOIのあるもの） / DOI (Publication and Proceedings)
口頭発表、ポスター発表および、その他の論文 / Oral Presentations etc.
特許 / Patents
特許出願件数 / Number of Patent Applications：1件
特許登録件数 / Number of Registered Patents：1件