Messenger RNA (mRNA) represents a promising therapeutic tool in the field of tissue engineering for the fast and transient production of growth factors to support new tissue regeneration. However, one of the main challenges to optimizing its use is achieving efficient uptake and delivery to mesenchymal stem cells (MSCs), which have been long reported as difficult-to-transfect. The aim of this study was to systematically screen a range of nonviral vectors to identify optimal transfection conditions for mRNA delivery to MSCs. Furthermore, for the first time, we wanted to directly compare the protein expression profile from three different types of mRNA, namely, unmodified mRNA (uRNA), base-modified mRNA (modRNA), and self-amplifying mRNA (saRNA) in MSCs. A range of polymer- and lipid-based vectors were used to encapsulate mRNA and directly compared in terms of physicochemical properties as well as transfection efficiency and cytotoxicity in MSCs. We found that both lipid- and polymer-based materials were able to successfully condense and encapsulate mRNA into nanosized particles (<200 nm). The overall charge and encapsulation efficiency of the nanoparticles was dependent on the vector type as well as the vector:mRNA ratio. When screened in vitro, lipid-based vectors proved to be superior in terms of mRNA delivery to MSCs cultured in a 2D monolayer and from a 3D collagen-based scaffold with minimal effects on cell viability, thus opening the potential for scaffold-based mRNA delivery. Modified mRNA consistently showed the highest levels of protein expression in MSCs, demonstrating 1.2-fold and 5.6-fold increases versus uRNA and saRNA, respectively. In summary, we have fully optimized the nonviral delivery of mRNA to MSCs, determined the importance of careful selection of the mRNA type used, and highlighted the strong potential of mRNA for tissue engineering applications.

中文翻译：

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优化 mRNA 向间充质干细胞的递送以用于组织工程应用

信使 RNA (mRNA) 是组织工程领域一种有前景的治疗工具，可快速、瞬时产生生长因子以支持新的组织再生。然而，优化其使用的主要挑战之一是实现间充质干细胞（MSC）的有效摄取和递送，长期以来，间充质干细胞一直被报道为难以转染。本研究的目的是系统地筛选一系列非病毒载体，以确定将 mRNA 递送至 MSC 的最佳转染条件。此外，我们第一次想要直接比较 MSC 中三种不同类型 mRNA 的蛋白质表达谱，即未修饰的 mRNA (uRNA)、碱基修饰的 mRNA (modRNA) 和自扩增 mRNA (saRNA)。使用一系列基于聚合物和脂质的载体来封装 mRNA，并直接比较 MSC 的理化特性以及转染效率和细胞毒性。我们发现基于脂质和聚合物的材料都能够成功地将 mRNA 凝结并封装成纳米尺寸的颗粒（<200 nm）。纳米粒子的总电荷和封装效率取决于载体类型以及载体：mRNA 比率。体外筛选时，事实证明，基于脂质的载体在向 2D 单层和 3D 胶原蛋白支架中培养的 MSC 递送 mRNA 方面具有优越性，对细胞活力的影响最小，从而开启了基于支架的 mRNA 递送的潜力。修饰的 mRNA 在 MSC 中始终显示出最高水平的蛋白质表达，与 uRNA 和 saRNA 相比，分别增加了 1.2 倍和 5.6 倍。总之，我们完全优化了 mRNA 向 MSC 的非病毒传递，确定了仔细选择所用 mRNA 类型的重要性，并强调了 mRNA 在组织工程应用中的强大潜力。