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Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.ORCID iD: 0000-0003-2896-2765
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.ORCID iD: 0000-0002-7256-0758
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.ORCID iD: 0000-0001-8872-9928
2019 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 20, no 3, p. 1317-1324Article in journal (Refereed) Published
Abstract [en]

Small interfering RNAs (siRNAs) are powerful toolsfor post-transcriptional gene silencing, which offers enormousopportunities for tissue engineering applications. However, poorserum stability, inefficient intracellular delivery, and inevitabletoxicity of transfection reagents are the key barriers for their clinicaltranslation. Thus, innovative strategies that allow safe and efficientintracellular delivery of the nucleic acid drugs at the desired site isurgently needed for a smooth clinical translation of therapeuticallyappealing siRNA-based technology. In this regard, we havedeveloped an innovative siRNA transfection protocol that employsa short incubation time of just 5 min. This allows easy transfection insuspension followed by transplantation of the cells in a hyaluronicacid (HA) hydrogel system. We also report here the unique ability ofsiRNA to bind HA that was quantified by siRNA release andrheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA insuspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to beessential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with orwithout removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removalof excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improvedknockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) asdetermined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the proteinlevels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allowsminimal manipulation of cells that are to be transplanted and reduce toxicity.

Place, publisher, year, edition, pages
2019. Vol. 20, no 3, p. 1317-1324
National Category
Polymer Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-379682DOI: 10.1021/acs.biomac.8b01712ISI: 000461270500019PubMedID: 30642167OAI: oai:DiVA.org:uu-379682DiVA, id: diva2:1297391
Funder
Swedish Foundation for Strategic Research , 2009-1035Swedish Foundation for Strategic Research , SBE13-0028EU, FP7, Seventh Framework Programme, FP7/2007-2013/607868Available from: 2019-03-19 Created: 2019-03-19 Last updated: 2019-04-12Bibliographically approved

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Paidikondala, MaruthibabuNawale, Ganesh N.Varghese, Oommen P.

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