Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Cristina Bardita; Dan Predescu; Sanda Predescu

doi:10.3791/50316

Method Article

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

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DOI:

10.3791/50316

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June 21st, 2013

Cristina Bardita^*¹ , Dan Predescu^*¹^,² , Sanda Predescu¹^,²

¹Department of Pharmacology, Rush University, ²Pulmonary and Critical Care Department, Rush University

^* These authors contributed equally

Summary

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Abstract

Previous studies showed that knockdown of ITSN-1s (KD_ITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition¹. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNA_ITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce². Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration³Here, efficient and specific KD_ITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNA_ITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KD_ITSN throughout the study⁴. Mouse tissues collected at several time points post-siRNA_ITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KD_ITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Introduction

Naked siRNA cannot penetrate the cell membrane, being negatively charged, and it is easily degraded by enzymes in blood, tissues, and cells. Even with recently structural modifications to improve stability, siRNA accumulation at the target site after administration is extremely low and requires an efficient intracellular vehicle⁵. Cationic liposomes emerged as safe nucleic acids carriers with the potential to transfer large pieces of DNA/RNA into cells by encapsulating and protecting the nucleic acids from enzymatic degradation⁶. Also, cationic liposomes spontaneously interact with DNA/RNA, thus promoting gene transfer to the cells^2<....