Targeted nanoparticle mediated delivery of nucleic acids into muscle stem cells for prevention of critical illness myopathy (B06#)

尽管许多方法已经建立并在体外研究，但体内细胞的遗传操作仍然具有挑战性。一个原因是靶向纳米颗粒必须克服静脉给药后的不同障碍。必须避免免疫原性识别，在纳米颗粒可以将其有效载荷转移到作用部位之前，必须克服内皮屏障的穿越和内化到靶细胞中的障碍。在核酸作为有效载荷的情况下，必须考虑到进一步的困难：它们很容易降解，并且由于它们的亲水性而不能穿过膜。纳米颗粒保护遗传物质并促进其内体摄取并以活性形式释放到靶细胞的细胞质中。为了将新的信息导入细胞，信使RNA（mRNA）可以更充分，因为mRNA不必进入细胞核。因此，不需要穿过核膜递送。具有阳离子电荷的聚合物可用于结合、保护和转移带负电荷的遗传物质。这一原则几十年来一直为人所知。然而，许多基因载体在细胞培养实验中虽然有效，但在体内却失败了。为了有效地将基因递送到靶细胞，将开发用于递送编码GFP的mRNA的多功能纳米颗粒。所使用的聚合物不仅通过静电相互作用结合遗传物质，由于它们的疏水性质，它们还形成颗粒。对于体内应用，将引入第二功能层以防止副反应和清除。阳离子颗粒将被这些嵌段共聚物屏蔽，其中一个嵌段含有pH值依赖性的阴离子电荷以结合到阳离子颗粒，并且第二嵌段引入隐形部分。功能性末端基团允许引入靶向部分以增强靶向基因递送。将在先进的细胞培养装置中使用流体方法结合免疫细胞和内皮细胞的共培养来测试颗粒。不同的方面，如纳米颗粒的细胞靶向和摄取，内体释放和mRNA的细胞间递送以及纳米颗粒介导的基因递送的效率将得到解决。设计参数，如大小，表面电荷，粒子密度，以及主动和被动目标之间的平衡将在该项目中进行研究。“

The genetic manipulation of cells in vivo is still challenging although numerous approaches are well established and investigated in vitro. One reason is the fact that targeted nanoparticles have to overcome different barriers subsequent to intravenous administration. Immunogenic recognition must be avoided, crossing of the endothelial barrier and internalization into the targeted cells are hurdles to be taken before the nanoparticles can transfer their payload to the site of action. In the case of nucleic acids as payload, further difficulties have to be taken into account: They are easily degraded and unable to cross membranes because of their hydrophilicity. Nanoparticles protect the genetic material and promote its endosomal uptake and release to the cytoplasm of the targeted cell in an active form. To import new information into cells, messenger RNA (mRNA) can be more sufficient as mRNA does not have to enter the cell nucleus. Thereby, delivery across the nuclear membrane is not necessary. Polymers with cationic charges can be used to bind, protect and transfer the negatively charged genetic material. This principle has been known since decades. However, a lot of gene carrier failed in vivo although being efficient in cell culture experiments. For an efficient gene delivery to targeted cells, multi-functional nanoparticles for the delivery of mRNA encoding GFP will be developed. The used polymers not only bind the genetic material by electrostatic interactions, due to their hydrophobic nature they also form particles. For in vivo applications, a second functional layer will be introduced to prevent side reactions and clearance. The cationic particles will be shielded by these block copolymers where one block contains pH value dependent anionic charges to bind to the cationic particle and the second block introduces stealth moieties. Functional end groups allow the introduction of targeting moieties to enhance the targeted gene delivery. The particles will be tested in an advanced cell culture setup using a fluidic approach combined with co-cultivation of immune as well as endothelial cells. Different aspects, such as cellular targeting and uptake of nanoparticles, endosomal release and intercellular delivery of mRNA as well as efficiency of nanoparticle mediated gene delivery will be addressed. Design parameters like size, surface charge, particle density, and the balance between active and passive targeting will be investigated within the project. “