Sirna Functionalized Targeted Lipid Nanoparticles To Manipulate Exosomal Microrna Packaging In Keratinocytes
Subhadip Ghatak1, Xiaoju Zhou1, Sayak Bhattacharya1, Mohamed S. El Masry1, Amitava Das1, Sashwati Roy1, Robert J. Lee2, Chandan K. Sen1.
1Indiana University, Indianapolis, IN, USA, 2Ohio State University, Columbus, OH, USA.
Background - Bidirectional cell-cell communication via paracrine mechanisms, is critical for wound healing. A new paradigm involving exosome mediated cross-talk has emerged as a predominant mechanism of cellular communication at the site of injury. Exosomes carry a distinctive repertoire of cargo such as miRNAs that are selectively packaged by heterogeneous nuclear ribonucleoprotein (hnRNP). Methods - Keratinocyte-targeted nanocarriers (TLNκ) were designed and loaded with siRNA of hnRNP to selectively inhibit miRNA packaging within exosomes. TLNκ employed a combination of DOTAP/DODMA pH-responsive lipid components to improve endosomal escape. Keratinocyte-targeting was achieved using the peptide sequence ASKAIQVFLLAG. To minimize interference of clearance by non-targeted cells, especially immune cells, surface charge was neutralized. Results - Encapsulation efficiency of siRNA functionalized TLNκ was 94.25%. Mixed culture of human keratinocytes, endothelial cells and fibroblasts showed selective uptake of DiD-labelled TLNκ by keratinocytes. Application of TLNκ encapsulating siRNA of hnRNP (TLNκ/si-hnRNP) to human keratinocytes significantly inhibited the expression of hnRNP by 80% compared to control (TLNκ/si-control). No cytotoxicity of TLNκ was detected even after 48h. Cell culture media were collected after 48h of TLNκ/si-control and TLNκ/si-hnRNP application for isolation of exosomes by differential ultracentrifugation. Histogram of Electron Plasma Resonance Spectroscopy (EPRS) showed lower abundance of small RNA in exosomes isolated from the cell culture media after 48h of TLNκ/si-hnRNP application. Compared to TLNκ/si-control, low abundance of 25 miRNAs containing exomotif sequence was found in TLNκ/si-hnRNP by Nanostring analysis. Protein packaging within exosomes showed no significant change as detected by LC/MS-MS. Conclusion - The nanoparticle reported herein are effective in the inhibition of miRNA packaging within exosomes. Therapeutic significance of these nanoparticles is further enhanced by the translational advantage that all material used for its formulation has prior history of FDA approval for human use.
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