PO.CL07.02 · 临床研究
聚合物递送TRAIL mRNA诱导非小细胞肺癌细胞凋亡
Polymeric delivery of TRAIL mRNA for inducing apoptosis in non-small cell lung cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
肿瘤坏死因子相关凋亡诱导配体（TRAIL）激活死亡受体通路以触发caspase依赖性凋亡，然而重组TRAIL在非小细胞肺癌（NSCLC）中因快速清除和凋亡抵抗而表现出有限的活性。以信使RNA（mRNA）形式递送TRAIL或可克服这些局限，但mRNA疗法在肿瘤学中因递送效率低下、不稳定和胞质释放不完全而仍受限制。本研究评估了用于TRAIL mRNA递送的可生物降解阳离子脂质聚合物，并考察了将TRAIL mRNA与靶向抗凋亡介质的小干扰RNA（siRNA）联合是否能在抵抗性NSCLC环境中增强凋亡反应。基于递送特征筛选了可生物降解的阳离子脂质聚合物。通过流体动力学直径、zeta电位和透射电子显微镜（TEM）对纳米颗粒进行了表征。使用A549肺腺癌细胞进行机制研究，包括多日活力测定、采用Caspase-Glo法检测caspase-3/7活性（24-72小时）、采用四甲基罗丹明乙酯（TMRE）进行线粒体膜电位分析、通过Hoechst染色和共聚焦显微镜观察核形态、通过碱性彗星实验评估DNA损伤，以及用于检测TRAIL蛋白的酶联免疫吸附测定（ELISA）。另外还使用了NSCLC细胞系：Calu-3、H1975 GFP⁺和H1299 GFP⁺进行基于活力的生物测定，以支持mRNA诱导的TRAIL活性。所选聚合物形成了尺寸为100-200 nm、zeta电位为+15-40 mV的紧凑纳米颗粒。在A549细胞中，TRAIL mRNA诱导了明显的时间依赖性凋亡反应：活力逐步下降，在较高mRNA剂量下于较晚时间点达到约40-50%。caspase-3/7活性在24小时时增加约2倍，并在48-72小时时仍可检测到。共聚焦成像显示线粒体去极化以及核浓缩和碎裂，彗星实验证实了DNA链损伤。单独使用siRNA可降低活力，但未能达到TRAIL mRNA的效果。在联合研究中，siRNA使较低的TRAIL mRNA剂量即可实现与较高剂量单用mRNA相当的活力降低，并在整个时间进程中进一步维持了活力的降低，从而改善了反应的效力和持续时间。此处评估的递送平台支持了TRAIL mRNA在A549细胞中的有效活性，而加入siRNA在降低mRNA剂量的同时进一步强化并延长了凋亡反应。这些发现凸显了一条切实可行的路径，即设计核酸联合策略以对抗NSCLC中的凋亡抵抗。本摘要部分内容在作者的监督和全面科学验证下借助AI起草。
查看英文原文 English abstract
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates death-receptor pathways to trigger caspase-dependent apoptosis, yet recombinant TRAIL has demonstrated limited activity in non-small cell lung cancer (NSCLC) because of rapid clearance and apoptotic resistance. Delivering TRAIL as messenger RNA (mRNA) may overcome these limitations, but mRNA therapeutics remain limited in oncology due to delivery inefficiencies, instability, and incomplete cytosolic release. This study evaluated biodegradable cationic lipopolymers for TRAIL mRNA delivery and examined whether combining TRAIL mRNA with small interfering RNAs (siRNAs) targeting anti-apoptotic mediators could strengthen apoptotic responses in resistant NSCLC settings. Biodegradable cationic lipopolymers were screened based on their delivery profiles. Nanoparticles were characterized by hydrodynamic diameter, zeta potential, and transmission electron microscopy (TEM). A549 lung adenocarcinoma cells were used for mechanistic studies, including multi-day viability assays, caspase-3/7 activity using the Caspase-Glo assay (24-72 hours), mitochondrial membrane potential analysis using tetramethylrhodamine ethyl ester (TMRE), nuclear morphology by Hoechst staining and confocal microscopy, DNA damage assessment by alkaline comet assay, and enzyme-linked immunosorbent assay (ELISA) for TRAIL protein detection. Additional NSCLC cell lines: Calu-3, H1975 GFP⁺, and H1299 GFP⁺ were used for viability-based bioassays to support mRNA-induced TRAIL activity.The selected polymer formed compact nanoparticles with 100-200 nm size and+15-40 mV zeta-potential. In A549 cells, TRAIL mRNA induced a clear time-dependent apoptotic response: viability declined progressively and reached approximately 40-50% at later time points with higher mRNA doses. Caspase-3/7 activity increased by ~2-fold at 24 hours and remained detectable at 48-72 hours. Confocal imaging showed mitochondrial depolarization together with condensed and fragmented nuclei, and comet assay confirmed DNA strand damage. siRNAs alone reduced viability but did not match the effect of TRAIL mRNA. In combination studies, siRNAs allowed lower TRAIL mRNA doses to achieve viability reductions comparable to higher-dose mRNA alone and maintained reduced viability further into the time course, improving both potency and duration of response.The delivery platform evaluated here supported effective TRAIL mRNA activity in A549 cells, and incorporating siRNAs further reinforced and prolonged the apoptotic response at reduced mRNA doses. These findings highlight a practical path toward combination nucleic acid strategies designed to counter apoptotic resistance in NSCLC.Portions of this abstract were drafted with the assistance of AI under the supervision and full scientific verification of the authors.
利益披露 Disclosure
G. Sandhu, None..
H. Uludag, None.