Delivery of Immunostimulatory RNA Oligonucleotides by Gelatin Nanoparticles Triggers an Efficient Antitumoral Response

Carole Bourquin, Cornelia Wurzenberger, Simon Heidegger, Sebastian Fuchs, David Anz, Sarah Weigel, Nadja Sandholzer, Gerhard Winter, Conrad Coester, Stefan Endres
2010 Journal of immunotherapy  
RNA oligonucleotides have emerged as a new class of biologicals that can silence gene expression but also stimulate immune responses through specific pattern-recognition receptors. The development of effective delivery systems remains a major challenge for the therapeutic application of the RNA oligonucleotides. In this study, we have established a novel biodegradable carrier system that is highly effective for the delivery of immunostimulatory RNA oligonucleotides. Formulation of RNA
more » ... otides with cationized gelatin nanoparticles potentiates immune activation through the Toll-like receptor 7 (TLR7) in both myeloid and plasmacytoid dendritic cells. Further, nanoparticle-delivered RNA oligonucleotides trigger production of the antitumoral cytokines IL-12 and IFN-a. Binding to gelatin nanoparticles protects RNA oligonucleotides from degradation by nucleases, facilitates their uptake by dendritic cells, and targets these nucleic acids to the endosomal compartment in which they are recognized by TLR7. In these effects, the nanoparticles are superior to the conventional transfection reagents lipofectamine, polyethylenimine, and DOTAP. In vivo, the delivery of TLR7-activating RNA oligonucleotides by gelatin nanoparticles triggers antigen-specific CD8 + T-cell and antibody responses. Indeed, immunization with RNA-loaded nanoparticles leads to an efficient antitumoral immune response in two different mouse tumor models. Thus, gelatin-based nanoparticles represent a novel delivery system for immunostimulatory RNA oligonucleotides that is both effective and nontoxic. FIGURE 2. Nanoparticle formulation of immunostimulatory RNA oligonucleotides promotes their uptake into intracellular compartments. Murine bone marrow cells were stimulated for 6 hours with 10 mg/mL fluorescein-labeled 9.2dr RNA oligonucleotides complexed to different carriers. A, Histograms illustrate uptake of fluorescently labeled RNA by bone marrow cells analyzed by flow cytometry (bold line: RNA with indicated carrier; fine line: free RNA; dashed line: nanoparticles without RNA). B, Graph shows percentage of FITC-RNA-positive cells as mean ± SEM of triplicate samples. Asterisks without brackets indicate comparison with unstimulated cells. ***P < 0.001. C, Confocal microscopy of RNA oligonucleotide-stimulated cells shows intracellular localization of the fluorescently labeled RNA after 3 hours. Left panel shows fluorescence images, right panel shows differential interference contrast (DIC) pictures merged with fluorescence. All results are representative of 2 independent experiments.
doi:10.1097/cji.0b013e3181f5dfa7 pmid:20948443 fatcat:sihl3qlhlre6foywwxoqflxlni