Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

INTRODUCTION: Selective suppression of RAPTOR by small interfering RNA (siRNA)-mediated RNA interference (RNAi) inhibits the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) as well as enhances autophagy and Akt through a feedback loop, thereby providing protective effects against intervertebral disc degeneration. However, siRNA is not easily internalized in cells in vivo, requiring effective transfection methods. Frequent injection can also accelerate degeneration. Therefore, a safe and long-lasting drug delivery system (DDS) is necessary. In this study, we focused on cationized gelatin nanospheres (cGNS) as a biodegradable DDS for the controlled release of Raptor siRNA to disc nucleus pulposus (NP) cells, aiming to elucidate the safety and efficiency of cGNS in RNAi, compared with lipofection.

METHODS: cGNS were prepared by chemically introducing spermine into gelatin, cross-linked with glutaraldehyde. By incorporating Raptor siRNA at a ratio of 20 pmol per 1 µg of cGNS, cGNS_Rap was obtained. Meanwhile, lipofection_Rap was achieved using Lipofectamine^TM RNAiMAX according to the manufacturer protocol. In vitro, (1) Disc NP cells harvested from 12-week-old male Sprague–Dawley rats were used. Following cGNS_Rap and lipofection_Rap transfection, to determine the maximum non-toxic concentration, cytotoxicity was assessed by the water-soluble tetrazolium salt-8 assay. (2) Using fluorescently labeled Raptor siRNA and cGNS, the intracellular localization and time-course maintenance were compared between cGNS_Rap and lipofection_Rap post-transfection. (3) Knockdown efficiency of cGNS_Rap versus lipofection_Rap was measured by Western blotting, focusing on the extent and duration of RAPTOR protein expression. In addition, the severity of autophagy induction by cGNS_Rap versus lipofection_Rap was monitored with autophagy markers, LC3-II and p62/SQSTM1. In vivo, (4) Using fluorescently labeled siRNA, 2 µl of cGNS_Rap or lipofection_Rap were injected into rat tail discs, the efficiency of which was compared in cryosections.

RESULTS: In vitro, (1) The maximum non-toxic concentration of Raptor siRNA was 400 pmol/ml using cGNS_Rap, compared with 60 pmol using lipofection_Rap. (2) The cGNS_Rap transfection presented the percentage of siRNA-positive cells as 99.7%±0.6% at 24 h, while it was 52.4%±7.3% at 14 d even after 1 passaging at 7 d (84.4%±2.8%) (Figure 1). Compared with 60pmol/ml lipofection, cGNS_Rap at both 400 and 60 pmol/ml consistently showed a higher siRNA-positive cell percentage at all time points (all p<0.05). (3) When using cGNS_Rap, the maximum knockdown efficiency was observed at 48 h compared to the control (53.9%±5.1%, p<0.01). Significant suppression of RAPTOR protein was maintained up to 7 d (64.7%±19.8%, p=0.01). In contrast, lipofection showed the highest knockdown efficiency at 24 h (48.5%±14.5%, p<0.001), which diminished at 48 h. Furthermore, the application of cGNS_Rap displayed a sustained induction of autophagy, as evidenced by increased LC3-II and decreased p62/SQSTM1 expression (Figure 2). In vivo, (4) the percentage of siRNA-positive cells was 100.0%±0.0% by cGNS_Rap but 26.6%±5.2% by lipofection_Rap at 28-d post-transfection (p<0.01) (Figure 3).

DISCUSSION: The cGNS delivers a larger amount of siRNA into cells without cytotoxicity, compared with lipofection. In addition, this facilitates more sustained intracellular siRNA maintainance and longer-term knockdown efficiency. The gelatin used for cGNS is safe and easily accessible, potentially providing a cost-effective DDS approach for siRNA-mediated RNAi.