Roerig Josepha, Mitrach Franziska, Schmid Maximilian, Hause Gerd, Hacker Michael C, Wölk Christian, Schulz-Siegmund Michaela
Pharmaceutical Technology, Institute of Pharmacy, Medical Faculty, Leipzig University, 04275, Leipzig, Germany.
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, 40225, Duesseldorf, Germany.
Small Methods. 2022 Dec;6(12):e2201001. doi: 10.1002/smtd.202201001. Epub 2022 Oct 25.
RNA interference opened new approaches for disease treatment but safe and efficient cell delivery remains a bottleneck. Extracellular vesicles (EVs) are known to naturally shuttle RNA. Due to their potent cell internalization and low-cost scalability, milk-derived EVs in particular are considered promising RNA delivery systems. However, low drug loading currently impedes their use. Here, innovative exogenous loading strategies for small interfering RNA (siRNA) are explored and systematically compared regarding encapsulation efficiency, loading capacity, and loading concentration. Firstly, siRNA is pre-accumulated in liposomes or stabilized calcium phosphate nanoparticles (CaP-NP). The selected systems, which exhibited neutral or negative zeta potentials, are then applied for EV loading. Secondly, EVs are concentrated and applied to protocols known for liposome loading: dehydration-rehydration of vesicles, based on freeze-drying, and mixing by dual asymmetric centrifugation (DAC) after ultracentrifugation. Additionally, DAC after EV ultracentrifugation is combined with CaP-NP leading to a synergistic loading performance. The balance between energy input for siRNA loading and EV integrity is evaluated by monitoring the EV size, marker proteins, and morphology. For the EV-based siRNA formulation via DAC plus CaP-NP, EV properties are sufficiently maintained to protect the siRNA from degradation and deliver cell-death siRNA dose-dependently in Caco-2 cells.
RNA干扰为疾病治疗开辟了新途径，但安全有效的细胞递送仍然是一个瓶颈。细胞外囊泡（EVs）已知可自然转运RNA。由于其强大的细胞内化能力和低成本可扩展性，特别是源自牛奶的EVs被认为是很有前景的RNA递送系统。然而，目前低药物负载量阻碍了它们的应用。在此，探索了用于小干扰RNA（siRNA）的创新外源负载策略，并在封装效率、负载能力和负载浓度方面进行了系统比较。首先，将siRNA预积累在脂质体或稳定的磷酸钙纳米颗粒（CaP-NP）中。然后将表现出中性或负ζ电位的所选系统用于EV负载。其次，将EVs浓缩并应用于已知的脂质体负载方案：基于冷冻干燥的囊泡脱水-再水化，以及超速离心后通过双不对称离心（DAC）进行混合。此外，EV超速离心后的DAC与CaP-NP相结合，产生协同负载性能。通过监测EV大小、标记蛋白和形态来评估siRNA负载的能量输入与EV完整性之间的平衡。对于通过DAC加CaP-NP的基于EV的siRNA制剂，EV特性得到充分维持，以保护siRNA不被降解，并在Caco-2细胞中剂量依赖性地递送细胞死亡siRNA。
