Purpose: To develop an anti-influenza prophylactic and therapeutic chitosan nanoplex that is viral strain independent. The chitosan nanoplex has two modes of action: stimulation of Type I interferon (IFN-I) through the cytosolic RNA sensor RIG-I, and suppression of the influenza virulence factor, Non-Structural Protein-1 (NS1), by gene silencing.
Methods: Chitosan nanoplexes are formed by the ionic complexation between the cationic component: chitosan (1 mg/ml; 800 µl); and the anionic components: tripolyphosphate (1 mg/ml; 320 µl), and anti-NS1 shRNA (300 µg/ml 112 µl). The anionic solution is added dropwise to chitosan, stirred for 30 minutes, allowed to stand for 60 minutes, and purified by centrifugation at 15000 x g for 30 minutes. The resulting nanoparticles are redispersed by sonication in 150 mM NaCl. The nanoplexes are characterized by: size (dynamic light scattering); charge; encapsulation efficiency (UV-VIS); and efficacy assessed at 24 and 48 h by stimulation of IFN-I in B16-Blue™ (InvivoGen, San Diego, CA) cells stably transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene, as well as viral titers from a mouse trachea epithelial cell culture (MTEC) system that mimics the air-liquid interface found in the lung’s bronchial epithelium.
Results: Chitosan nanoplexes are ~100 nm in size, and have a positive zeta potential. Encapsulation efficiency/nanoplex recovery is >50% and is dependent upon the concentration of shRNA used. The IC50 is 21 ng/l and is only 8.4 fold higher than the positive control Lyovec™ (InvivoGen). Peak IFN-I production occurs by 48 h. MTEC cultures treated with nanoplexes reduce (p=0.0118) viral titers 24 h post infection compared to non-treated controls.
Conclusion: We have successfully developed a chitosan nanoplex that can deliver anti-influenza shRNA to cells and stimulate IFN-I production in a concentration dependent manner. Future work involves decreasing the time to maximal IFN-I production, as well as determining efficacy in a murine model of influenza. As influenza continues to develop resistance to the current armament of antiviral drugs and vaccination contraindications ensue, our nanoplexes offer a promising therapeutic modality for protection against future epidemic, pandemic, and pathologic strains of influenza.
This work is supported by the Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant 1T32GM099607 and the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR001412. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Prince Bonsu– University at Buffalo, New York
Siavash Sedghi– University at Buffalo
Ravi Alluri– University at Buffalo
Barbara Mullan– University at Buffalo
Suryaprakash Sambhara– Chief, Center for Disease Control and Prevention
Paras Prasad– SUNY Distinguished Professor, Institute for Lasers, Photonics and Biophotonics, University at Buffalo, New York
Bruce Davidson– Research Associate Professor, Department of Anesthesiology, University at Buffalo, New York
Paul Knight– SUNY Distinguished Professor, Department of Anesthesiology, University at Buffalo, New York