【公開日：2025.06.16】【最終更新日：2025.06.16】
課題データ / Project Data
課題番号 / Project Issue Number
22UT0006
利用課題名 / Title
Development of messenger RNA-loaded nanomachines
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）次世代バイオマテリアル/Next-generation biomaterials（副 / Sub）-
キーワード / Keywords
ナノ粒子/ Nanoparticles,高分子系生体材料/ Polymer materials,電子顕微鏡/Electron microscopy,DDSマテリアル/ DDS material
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)
Anjaneyulu Dirisala
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)
Polyplex micelles (PMs) were prepared by mixing poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) with varying PEG lengths (molecular weight; Mw) and messenger (m)RNA at a fixed molar ratio of amines (N) in PEG-PLL to phosphates (P) in mRNA (N/P ratio) of 2. PEG-PLL block copolymers were prepared to possess a PEG length of 12-kDa or 42-kDa with a PLL length fixed to approximately 70; PLL DP was 67 in PEG-PLL with 12-kDa PEG and 75 in PEG-PLL with 42-kDa PEG. These block copolymers are denoted as PEG-PLL(12-67) and PEG-PLL(42-75), respectively. PMs were prepared by mixing these block copolymers and mRNA, denoted as 12-PLL67 PM and 42-PLL75 PM. Cryogenic transmission electron microscopy (cryo-TEM) imaging of PMs reveals the different mRNA packaging between 12-PLL67 and 42-PLL75 PMs. Cryo-TEM visualizes nearly rod-shaped structures (based on aspect ratio = 1.9) for 12-PLL67 PM (Fig. 1A) and elongated rod-shaped structures (aspect ratio = 5.6) for 42-PLL75 PM (Fig. 1B). Indeed, 42-PLL75 PM possessed a longer major axis in cryo-TEM than12-PLL67 PM (Fig. 1C, and 1D).
実験 / Experimental
The estimation of the number-average PEG density (σ, chains/nm2) of PMs requires two characteristics; the number-average core surface area (A, nm2) of packaged mRNA and the binding number of PEG-PLL to mRNA (chains). As two different PEG Mw (12- and 42-kDa) were used in this study, the obtained σ was normalized to reduced tethering density (RTD). RTD is defined as the number of PEG chains occupying the area of an isolated unperturbed PEG chain (πRg2) [RTD = σπRg2, where Rg is the radius of gyration of an isolated unperturbed PEG chain]. For calculating the surface area of packaged mRNA, the morphology of PMs (N/P ratio 2, 12-PLL67, and 42-PLL75) was visualized by cryo-TEM device. Briefly, each PM solution (3 µL, 100 ng/mL of GLuc mRNA in10 mm HEPES buffer, pH 7.3) was separately applied onto a copper grid containing carbon film (QUANTIFOILTM R 3.5/1.0) with circular holes of 3.5 μm diameter. 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 3.5 μm holes (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 device (JEM-2100F, JEOL, Tokyo) using a side-entry cryo-transfer holder (Gatan 914, Gatan Inc., Pleasanton, CA) for imaging. 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). The surface area (A) of packaged mRNA inside the PM was calculated based on its appearance as a rod-shaped structure (treating it as a cylinder) from cryo-TEM images using an equation: A = [l × 2πr] + 2[πr2]. The A was estimated by dividing the sum of the A of all rod structures by the total count of measurements.
結果と考察 / Results and Discussion
For characterizing the PEG shielding of these PMs, their σ(chains/nm2) were quantified by dividing the PEG chain number of each PM by the surface area of the PM core. In PEG chain number measurement, free polymers and polymers attached to mRNA were separated using ultracentrifugation, followed by quantifying free polymers in the supernatant via a fluorescence-based method. The number-averaged surface area (A) was determined from cryo-TEM images. Note that packaged mRNA-loaded core was selectively observed in the cryo-TEM images owing to high electron density in the core than PEG shell (Fig. 1A and 1B). Based on these measurements, σ was calculated and then normalized using PEG Mw, as a longer PEG chain covers a larger area of PM cores. The normalized PEG density, namely reduced tethering density (RTD = σπRg2), was a widely used parameter of the PEG crowdedness, designating the number of tethered PEG chains that occupy an area covered by an isolated unperturbed PEG chain (πRg2). Using Rg (radius of gyration) of an isolated PEG chain, i.e., 4.7 nm for 12-kDa PEG and 9.7 nm for 42-kDa PEG, the RTD was calculated to be 0.47 for 12-PLL67 and 1.07 for 42-PLL75 PM. Notably, PEG covered almost all surface areas of the 42-PLL75 PM core, with RTD above 1. Closer observation of cryo-TEM images reveals the different core structures between 12-PLL67 and 42-PLL75 PMs. Cryo-TEM visualizes nearly rod-shaped structures (aspect ratio = 1.9) for 12-PLL67 PM (Fig. 1A) and elongated rod-shaped structures (aspect ratio = 5.6) for 42-PLL75 PM (Fig. 1B). Indeed, 42-PLL75 PM possessed a longer major axis in cryo-TEM than 12-PLL67 PM (Fig. 1C, and 1D). The extended structure of 42-PLL75 PM may be attributed to the steric repulsion of long 42-kDa PEG chains competing with the complexation force of the polycation.
図・表・数式 / Figures, Tables and Equations
Figure 1. Cryo-TEM imaging of 12-PLL67 and 42-PLL75 PMs. (A,B) Representative images. (C,D) The major axis length distribution of the PM cores. (A,C) 12-PLL67 PM, (B,D) 42-PLL75 PM (D).
Figure 1. Cryo-TEM imaging of 12-PLL67 and 42-PLL75 PMs. (A,B) Representative images. (C,D) The major axis length distribution of the PM cores. (A,C) 12-PLL67 PM, (B,D) 42-PLL75 PM (D).
その他・特記事項（参考文献・謝辞等） / Remarks(References and Acknowledgements)
This work was supported by Grants-in-Aid for Early-Career Scientists [18K18393 and 21K18062 to A.D.] from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). The authors sincerely thank Ayumi Kimura for her valuable suggestions and assistance in conducting cryo-TEM observations.
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)
-
Pengwen Chen, Nanocarriers escaping from hyperacidified endo/lysosomes in cancer cells allow tumor-targeted intracellular delivery of antibodies to therapeutically inhibit c-MYC, Biomaterials, 288, 121748(2022).
DOI: 10.1016/j.biomaterials.2022.121748
口頭発表、ポスター発表および、その他の論文 / Oral Presentations etc.
- DOI：10.1002/marc.202100754
特許 / Patents
特許出願件数 / Number of Patent Applications：0件
特許登録件数 / Number of Registered Patents：0件