Calcium-siRNA Nanocomplexes Optimized by Bovine Serum Albumin Coating Can Achieve Convenient and Efficient siRNA Delivery for Periodontitis Therapy

Purpose: Reducing toxicity, immunogenicity, and costs of small interfering RNAs (siRNA) carrier materials are key goals for RNA interference (RNAi) technology transition from bench to bed. Recently, calcium ions (Ca²⁺) have garnered attention as a novel, alternative material for delivering siRNA to cells. However, the tolerance for Ca²⁺ concentration varies in different cell types, which has limited its applications in vivo. Bovine serum albumin (BSA) can bind to Ca²⁺ through chelation. Moreover, BSA is a favorable coating material for nanoparticles owing to its excellent biocompatibility. Therefore, we hypothesized that coating Ca²⁺-siRNA with BSA helps buffer Ca²⁺ toxicity in vivo.

Methods: BSA-Ca²⁺-siRNA nanoparticles were prepared, and the size, shape, encapsulation, and release efficiency were characterized using atomic force microscopy, scanning electronic microcopy, and gel electrophoresis. Binding nanoparticles were evaluated using attenuated total reflection-Fourier-transform infrared spectroscopy. The cellular uptake, intracellular release, cytotoxicity, and gene knockdown of nanoparticles were evaluated in periodontal ligament stem cells (PDLSCs) using laser-scanning confocal microscope, flow cytometry, and real-time quantitative polymerase chain reaction.

Results: BSA and Ca²⁺-siRNA could form a stable nano-scale complex (~140 nm in diameter). The nanocomplexes could maintain siRNA release for more than 1 week in neutral phosphate-buffered saline (PBS) and could induce accelerated degradation in acidic PBS (pH 5.0). The nanoparticles were taken up by the cells, primarily through macropinocytosis, and were then released intracellularly through the acidification of endosomes/lysosomes. Importantly, the BSA-Ca²⁺ carrier had high transfection efficiency and biocompatibility both in vitro and in vivo. To demonstrate the therapeutic potential of our BSA coating-optimized Ca²⁺-siRNA technology, we showed that BSA-Ca²⁺-siWWP1 complexes strongly enhanced the osteogenic differentiation of inflammatory PDLSCs.

Conclusion: BSA-Ca²⁺ could potentially be used for siRNA delivery, which is not only highly efficient and cost-effective but also biocompatible to host tissues owing to the BSA coating.