Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel

Abstract

Small interfering RNAs (siRNAs) are powerful tools for post-transcriptional gene silencing, which offers enormous opportunities for tissue engineering applications. However, poor serum stability, inefficient intracellular delivery, and inevitable toxicity of transfection reagents are the key barriers for their clinical translation. Thus, innovative strategies that allow safe and efficient intracellular delivery of the nucleic acid drugs at the desired site is urgently needed for a smooth clinical translation of therapeutically appealing siRNA-based technology. In this regard, we have developed an innovative siRNA transfection protocol that employs a short incubation time of just 5 min. This allows easy transfection in suspension followed by transplantation of the cells in a hyaluronic acid (HA) hydrogel system. We also report here the unique ability of siRNA to bind HA that was quantified by siRNA release and rheological characterization of the HA-hydrogel. Such interactions also showed promising results to deliver functional siRNA in suspension transfection conditions within 30 min using native HA, although removal of excess HA by centrifugation seem to be essential. In the 2D experiments, suspension transfection of hMSCs with RNAiMAX resulted in ≈90% gene silencing (with or without removal of the excess reagent by centrifugation), while HA demonstrated a modest ≈40% gene silencing after removal of excess reagent after 30 min. Transplantation of such transfected cells in the HA-hydrogel system demonstrated an improved knockdown (≈90% and ≈60% with RNAiMAX and HA respectively after 48 h), with lower cytotoxicity (up to 5-days) as determined by PrestoBlue assay. The gene silencing efficiency in the 2D and 3D conditions were also confirmed at the protein levels by Western blot analysis. We postulate this novel transfection method could be applied for in vivo applications as it allows minimal manipulation of cells that are to be transplanted and reduce toxicity.

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non‐Viral Transfection Method

“…We have also recently demonstrated that this HA-mediated transfection method could be implemented in suspension cultures,p rovided the free HA is removed by centrifugation. [20] To ascertain the CD44 dependence of the siRNAuptake and gene KD,C D44-blocking studies were performed. [11] In these experiments,t he plated MG-63 cells were first incubated with low molecular weight HA (5 kDa) for 2hand the medium was then replenished with fresh medium containing 200 kDa HA (64 mm)and anti-GAPDH siRNA(200 nm).…”

Influence of ions to modulate hydrazone and oxime reaction kinetics to obtain dynamically cross-linked hyaluronic acid hydrogels

“…HA is known to play a significant role in cell proliferation and migration 26 and in maintaining the stem cell niche within our body. 27 Therefore hydrogels using HA components have been extensively used for several biomedical applications, such as cell delivery, 28 and drug delivery 29 and in the field of regenerative medicine. 30 We have previously developed a hydrazone-cross-linked HA hydrogel 29 and used it to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to regenerate bone in vivo.…”

“…30 We have previously developed a hydrazone-cross-linked HA hydrogel 29 and used it to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to regenerate bone in vivo. 8,31 We have also utilized such materials to deliver small molecule drugs 32 and large nucleic acid drugs 28,33,34 and for tuning the differential release of proteins. 35 One of the key aspects of hydrogel preparation is the reaction kinetics of gel formation.…”

The Application of Hyaluronic Acid-Based Hydrogels in Bone and Cartilage Tissue Engineering

“…At the same time, aldehyde-modified hydrogels combined with acylhydrazide polyvinyl alcohol (PVA) can effectively deliver nontoxic siRNAs that can be applied to MSCs to block the inhibition of the clinically related gene PLEKHO1. is effect was demonstrated in siRNA release experiments and through the rheological properties of siRNA-encapsulated hydrogels [66].…”