Diosmin-Capped Silver Nanoparticles Promote Osteogenic Differentiation of Human Periodontal Ligament Stem Cells: Box–Behnken Optimization and in vitro Evaluation

Roxane Abdel Gawad,1 Shymaa Hatem,2 Nehal I Rizk,3 Shereen N Raafat,4,5 Dalia E Ali,6 Riham A El-Shiekh,7 Doaa H Shakshak8 1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; 2Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Future University in Egypt, Cairo, Egypt; 3Department of Biochemistry, Faculty of Pharmacy and Drug Technology, Egyptian Chinese University, Cairo, Egypt; 4Department of Pharmacology (Medical Sciences), Faculty of Dentistry, The British University in Egypt, El Sherouk City, Cairo, Egypt; 5Dental Science Research Group, Health Research Centre of Excellence, The British University in Egypt, El Sherouk City, Cairo, Egypt; 6Department of Pharmacognosy and Natural Products, Faculty of Pharmacy, Pharos University in Alexandria, Alexandria, Egypt; 7Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt; 8Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, The British University in Egypt, El-Sherouk City, Cairo, EgyptCorrespondence: Doaa H Shakshak; Shymaa Hatem, Email [email protected]; [email protected]: Diosmin is a bioactive flavonoid with anti-inflammatory, anti-oxidant, and osteogenic properties; however, its poor water solubility limits its therapeutic use. To overcome this, the study developed Diosmin-loaded silver nanoparticles (AgNPs), leveraging the bone-regenerative potential of silver, as a unique strategy to improve the osteogenic differentiation of periodontal ligament stem cells (PDLSCs).Methods: The nanoparticles loaded with the extracted Diosmin were prepared using a scalable green reduction method and optimized using the Box–Behnken design (BBD) and response surface methodology. Key factors (silver nitrate concentration, Diosmin concentration, and reaction temperature) were assessed for their effects on the particle size, zeta potential, and polydispersity index. This study also evaluated the effects of Diosmin-loaded AgNPs on the viability and osteogenic differentiation of human PDLSCs. Alizarin Red staining and alkaline phosphatase activity were used to assess osteogenic differentiation. Moreover, real-time quantitative polymerase chain reaction (RT-qPCR) was used to track the expression of osteogenic-associated markers, osteoprotegerin (OPG) and runt-related transcription factor 2 (RUNX2), as well as proinflammatory markers, TNF-α and IL-1β.Results: The optimized Diosmin-loaded AgNPs identified by the BBD showed high desirability, with particle size of 181.0± 8.45 nm using dynamic light scattering technique (DLS) and a negative surface charge of − 21.45± 1.27 mV, confirmed by transmission electron microscopy (TEM) photomicrographs. Moreover, energy-dispersive X-ray (EDX) analysis showed the preparation of AgNPs through showing their elemental peaks. Stability studies demonstrated that formulations remained stable at 4.0± 2.0°C for up to 3 months. The results showed that, in contrast to silver nitrate (AgNO3), Diosmin-loaded AgNPs promoted osteogenic differentiation and modulated the expression of inflammatory markers, indicating a potential role in the regulation of the local inflammatory microenvironment associated with osteogenesis.Conclusion: Green AgNPs have proven their efficacy as a potential approach for osteogenic differentiation.Keywords: diosmin, silver nanoparticles, green synthesis, box-behnken design, osteogenic differentiation, periodontal ligament stem cells