Department of Chemical and Biomolecular Engineering, University of Cantabria, 39011 Santander, Spain.
Health Research Institute Valdecilla (IDIVAL), 39011 Santander, Spain.
Biomolecules. 2022 Jul 22;12(8):1012. doi: 10.3390/biom12081012.
In this work, we performed a methodological comparative analysis to synthesize polyethyleneimine (PEI) nanoparticles using (i) conventional nanoprecipitation (NP), (ii) electrospraying (ES), and (iii) coaxial electrospraying (CA). The nanoparticles transported antisense oligonucleotides (ASOs), either encapsulated (CA nanocomplexes) or electrostatically bound externally (NP and ES nanocomplexes). After synthesis, the PEI/ASO nanoconjugates were functionalized with a muscle-specific RNA aptamer. Using this combinatorial formulation methodology, we obtained nanocomplexes that were further used as nanocarriers for the delivery of RNA therapeutics (ASO), specifically into muscle cells. In particular, we performed a detailed confocal microscopy-based comparative study to analyze the overall transfection efficiency, the cell-to-cell homogeneity, and the mean fluorescence intensity per cell of micron-sized domains enriched with the nanocomplexes. Furthermore, using high-magnification electron microscopy, we were able to describe, in detail, the ultrastructural basis of the cellular uptake and intracellular trafficking of nanocomplexes by the clathrin-independent endocytic pathway. Our results are a clear demonstration that coaxial electrospraying is a promising methodology for the synthesis of therapeutic nanoparticle-based carriers. Some of the principal features that the nanoparticles synthesized by coaxial electrospraying exhibit are efficient RNA-based drug encapsulation, increased nanoparticle surface availability for aptamer functionalization, a high transfection efficiency, and hyperactivation of the endocytosis and early/late endosome route as the main intracellular uptake mechanism.
在这项工作中，我们进行了方法学比较分析，使用（i）常规的纳米沉淀法（NP）、（ii）电喷射（ES）和（iii）同轴电喷射（CA）合成聚乙烯亚胺（PEI）纳米颗粒。这些纳米颗粒输送反义寡核苷酸（ASOs），要么被包裹（CA 纳米复合物），要么被静电绑定在外部（NP 和 ES 纳米复合物）。合成后，PEI/ASO 纳米缀合物被肌肉特异性 RNA 适体功能化。使用这种组合配方方法，我们获得了纳米复合物，进一步将其用作 RNA 治疗药物（ASO）的纳米载体，特别是递送到肌肉细胞中。特别是，我们进行了详细的基于共聚焦显微镜的比较研究，以分析富含纳米复合物的微米级区域的整体转染效率、细胞间均匀性和每个细胞的平均荧光强度。此外，使用高倍电子显微镜，我们能够详细描述纳米复合物通过网格蛋白非依赖的内吞作用途径的细胞摄取和细胞内运输的超微结构基础。我们的结果清楚地表明，同轴电喷射是合成治疗性基于纳米颗粒的载体的一种很有前途的方法。同轴电喷射合成的纳米颗粒具有一些主要特点，包括有效包裹 RNA 药物、增加纳米颗粒表面的适体功能化可用性、高转染效率以及作为主要细胞内摄取机制的内吞作用和早期/晚期内体途径的超激活。
