Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Peptide-Targeted Polyplexes for Aerosol-Mediated Gene Delivery to CD49f-Overexpressing Tumor Lesions in Lung.

Peptide ligands can enhance delivery of nucleic acid-loaded nanoparticles to tumors by promoting their cell binding and internalization. Lung tumor lesions accessible from the alveolar side can be transfected, in principle, using gene vectors delivered as an aerosol. The cell surface marker CD49f (Integrin α6) is frequently upregulated in metastasizing, highly aggressive tumors. In this study, we utilize a CD49f binding peptide coupled to linear polyethylenimine (LPEI) promoting gene delivery into CD49f-overexpressing tumor cells in vitro and into lung lesions in vivo. We have synthesized a molecular conjugate based on LPEI covalently attached to the CD49f binding peptide CYESIKVAVS via a polyethylene glycol (PEG) spacer. Particles formed with plasmid DNA were small (<200 nm) and could be aerosolized without causing major aggregation or particle loss. In vitro, CD49f targeting significantly improved plasmid uptake and reporter gene expression on both human and murine tumor cell lines. For evaluation in vivo, localization and morphology of 4T1 murine triple-negative breast cancer tumor lesions in the lung of syngeneic BALB/c mice were identified by MRI. Polyplexes applied via intratracheal aerosolization were well tolerated and resulted in measurable transgene activity of the reporter gene firefly luciferase in tumor areas by bioluminescence imaging (BLI). Transfectability of tumors correlated with their accessibility for the aerosol. With CD49f-targeted polyplexes, luciferase activity was considerably increased and was restricted to the tumor area.

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance - often due to insufficient drug delivery - is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

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.