Multimodal Fluorescence and Bioluminescence Imaging Reveals Transfection Potential of Intratracheally Administered Polyplexes for Breast Cancer Lung Metastases.

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.

Fluorescence- and computed tomography for assessing the biodistribution of siRNA after intratracheal application in mice.

Pulmonary delivery of nucleic acids opens the possibility for direct treatment of lung diseases, like fibrosis, cancer, and infections. Lung retention and biodistribution of nucleic acids remain important issues for the development of suitable therapeutic approaches. Moreover, monitoring the dynamic biodistribution processes of siRNA after aerosol delivery can help in identifying bottlenecks and optimizing therapeutic concepts. We investigated dynamic biodistribution events after intratracheal application of chemically stabilized siRNA labelled with near infrared emitting dye AlexaFluor750 (AF750). Epifluorescence imaging was combined with spectral unmixing to improve the signal to noise ratio. Transillumination imaging has been utilized for quantitative fluorescence imaging tomography (FLIT) together with contrast agent enhanced X-ray absorption computed tomography (CT). Spectral unmixing allowed unambiguous detection of AF750 signals, which could be clearly distinguished from food derived autofluorescence. After successful delivery to the lung, fluorescent signals were also observed in kidneys and bladder, indicating renal excretion of AF750-siRNA. Gel electrophoresis of urine samples showed presence of intact siRNA, at least to a considerable extent. FLIT/CT allowed signal quantification and precise allocation to anatomical structures.