1 Laboratory of MacroMolecular Cancer Therapeutics (MMCT), Center of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Vienna , Vienna, Austria .
2 Institutes of Molecular Immunology and Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany .
Hum Gene Ther. 2017 Dec;28(12):1202-1213. doi: 10.1089/hum.2017.137. Epub 2017 Oct 26.
Local delivery of anticancer agents or gene therapeutics to lung tumors can circumvent side effects or accumulation in non-target organs, but accessibility via the alveolar side of the blood-air barrier remains challenging. Polyplexes based on plasmid and linear polyethylenimine (LPEI) transfect healthy lung tissue when applied intravenously (i.v.) in the mouse, but direct delivery into the lungs results in low transfection of lung tissue. Nevertheless, LPEI could offer the potential to transfect lung tumors selectively, if accessible from the alveolar side. This study combined near infrared fluorescent protein 720 (iRFP720) and firefly luciferase as reporter genes for detection of tumor lesions and transfection efficiency of LPEI polyplexes, after intratracheal microspraying in mice bearing 4T1 triple negative breast cancer lung metastases. Simultaneous flow cytometric analysis of iRFP720 and enhanced green fluorescent protein expression in vitro demonstrated the potential to combine these reporter genes within transfection studies. Polyplex biophysics was characterized by single nanoparticle tracking analysis (NTA) to monitor physical integrity after microspraying in vitro. 4T1 cells were transduced with iRFP720-encoding lentivirus and evaluated by flow cytometry for stable iRFP720 expression. Growth of 4T1-iRFP720 cells was monitored in Balb/c mice by tomographic near infrared imaging, tissue and tumor morphology by computed tomography and magnetic resonance imaging. In 4T1-iRFP720 tumor-bearing mice, intratracheal administration of luciferase-encoding plasmid DNA by LPEI polyplexes resulted in successful tumor transfection, as revealed by bioluminescence imaging.
局部递送至肺部肿瘤的抗癌剂或基因治疗药物可以避免副作用或在非靶器官中积累，但通过气-血屏障的肺泡侧进行递送至仍然具有挑战性。基于质粒和线性聚乙烯亚胺（LPEI）的多聚物在静脉内（i.v.）应用于小鼠时会转染健康的肺组织，但直接递送至肺部会导致肺组织的转染效率低。然而，如果能够从肺泡侧进入，LPEI 可能具有选择性转染肺部肿瘤的潜力。本研究结合近红外荧光蛋白 720（iRFP720）和萤火虫荧光素酶作为报告基因，用于检测携带 4T1 三阴性乳腺癌肺转移的小鼠经气管内微喷雾后的肿瘤病变和 LPEI 多聚物的转染效率。体外同时进行 iRFP720 和增强型绿色荧光蛋白表达的流式细胞术分析表明，有可能在转染研究中结合这些报告基因。通过单纳米颗粒跟踪分析（NTA）对多聚物的生物物理性质进行了表征，以监测体外微喷雾后的物理完整性。4T1 细胞用 iRFP720 编码的慢病毒转导，并通过流式细胞术评估稳定的 iRFP720 表达。通过断层近红外成像监测 4T1-iRFP720 细胞在 Balb/c 小鼠中的生长情况，通过计算机断层扫描和磁共振成像监测组织和肿瘤形态。在 4T1-iRFP720 肿瘤荷瘤小鼠中，通过 LPEI 多聚物递送至气管内的荧光素酶编码质粒 DNA 导致成功的肿瘤转染，如生物发光成像所示。
