Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Biomaterials. 2011 Dec;32(34):9128-35. doi: 10.1016/j.biomaterials.2011.08.024. Epub 2011 Aug 25.
In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.
在癌症免疫疗法中，应该触发免疫系统特异性识别和消除患者体内的肿瘤细胞。这可以通过负载树突状细胞（DC）与肿瘤相关抗原（TAA）来实现。这可以通过用编码肿瘤相关抗原的信使 RNA 转染 DC 来实现。在这里，我们通过与微泡结合的 mRNA-脂质体来证明树突状细胞的瞬时转染。通过施加超声将微泡附着的脂质体引入细胞。我们的数据表明，超声介导的 mRNA 复合物的递送导致 DC 的有效转染。当使用编码荧光素酶的 mRNA 时，在超声应用后 8 小时检测到最大水平的酶活性。随着孵育时间的延长，蛋白表达逐渐下降。这种处理不会影响细胞的活力。通过使用荧光标记的脂质体，通过流式细胞术确定 DC 中 mRNA-脂质体的细胞内定位。超过 50%的 DC 含有荧光标记的 mRNA 复合物。在没有额外的成熟信号的情况下，用负载有 mRNA-脂质体的微泡转染未成熟的 DC 并施加超声仅诱导成熟标志物（CD40 和 CD86）表达水平的微小变化。然而，在存在激活刺激（LPS）的情况下，细胞能够进一步成熟，如 CD40 表达的显著上调所示。因此，我们的结果表明，负载有 mRNA-脂质体的微泡可以作为一种适用且安全的工具，用于培养的 DC 的高效 mRNA 转染。
