RNA interference (RNAi) has immense potential to modulate cell functions. However, effective delivery of siRNA while avoiding deleterious side effects has proven challenging. This study investigates both intended and unintended effects of diblock copolymer nanoparticle (NP) delivery of siRNA delivery to human mesenchymal stem cells (hMSC). Specifically, siRNA delivery was investigated at a range of NP-siRNA:hMSC ratios with a focus on the effects of NP-siRNA treatment on hMSC functions. Additionally, next-generation RNA sequencing (RNA-seq) was used with enrichment analysis to observe side effects in hMSC gene expression. Results show NP-siRNA delivery is negatively correlated with hMSC density. However, higher NP-siRNA:hMSC ratios increased cytotoxicity and decreased metabolic activity. hMSC proliferation was largely unaffected by NP-siRNA treatment, except for a 3-fold reduction in hMSCs seeded at 4,000 cells/cm2. Flow cytometry reveals that apoptosis is a function of NP-siRNA treatment time and seeding density; ~14% of the treated hMSCs seeded at 8,000 cells/cm2 were annexin V+-siRNA+ 24 h after treatment, while 11% of the treated population was annexin V+-siRNA-. RNA-seq shows that NP-siRNA treatment results in transcriptomic changes in hMSCs, while pathway analysis shows upregulation of apoptosis signaling and downregulation of metabolism, cell cycle, and DNA replication pathways, as corroborated by apoptosis, metabolism, and proliferation assays. Additionally, multiple innate immune signaling pathways such as toll-like receptor (TLR), retinoic acid-inducible gene 1 (RIG-I)-like receptor, and nuclear factor-κB (NF-κB) signaling pathways are upregulated. Furthermore, and consistent with traditional siRNA immune activation, cytokine-cytokine receptor signaling was also upregulated. Overall, this study provides insight into NP-siRNA:hMSC ratios that are favorable for siRNA delivery. Moreover, NP-siRNA delivery results in side effects across the MSC transcriptome that suggest activation of the innate immunity that could alter MSC functions associated with their therapeutic potential.
Overall design: 21 total samples were analyzed from 7 experimental groups, with each group performed in triplicate (S1, S2, S3). Groups: (1) Day 0 samples harvested on day of NP-siRNA treatment administration (D0); (2) Untreated samples 1 day post-treatment (D1_NT); (3) NP-siRNA treated samples 1 day post-treatment (D1_Trxt); (4) Untreated samples 5 days post-treatment (D5_NT); (5) NP-siRNA treated samples 5 days post-treatment (D5_Trxt); (6) Untreated samples 14 days post-treatment (D14_NT); (7) NP-siRNA treated samples 14 days post-treatment (D14_Trxt).
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