PH responsive hydrogel nanocomposites for targeted Shikonin delivery enhance anti PDL1 immunotherapy in osteosarcoma.

[OBJECTIVE] To enhance the therapeutic effect of atezolizumab on osteosarcoma (OS) by constructing a pH-responsive hydrogel nanocomplex (Gel@PLGA@FA) as a delivery platform for Shikonin.

[METHODS] First, Shikonin was initially employed to analyze the GSE14359 dataset, leading to the identification of 28 differentially expressed genes (DEGs). Based on this, a risk score model was constructed and molecular dynamics simulations were performed to assess the binding ability between Shikonin and cyclin-dependent kinase 1 (CDK1). In addition, the in vitro antiproliferative effect of Shikonin on MG63 and Saos-2 OS cell lines and its selective toxicity on normal cells were assessed. In order to overcome the disadvantages of poor water solubility and normal cytotoxicity towards Shikonin, a complex loaded with Shikonin by pH-responsive intelligent hydrogel nanomaterials was synthesized and its anti-programmed death ligand-1 (PD-L1) therapeutic effect on OS cells was evaluated.

[RESULTS] Molecular dynamics simulation showed that Shikonin showed strong binding ability to CDK1, showing stable conformation, enhanced structural stability and other characteristics. In vitro experiments showed that Shikonin had a significant anti-proliferative effect on OS cells, while it had selective toxicity on normal liver, kidney and osteoblasts. The pH-responsive hydrogel nanomaterial (Gel@PLGA@FA) loaded with Shikonin showed good drug release characteristics at different pH conditions, especially in the tumor microenvironment to achieve controllable drug release. Combined use of Gel@PLGA@Shikonin@FA and atezolizumab effectively down-regulated CDK1 and PD-L1 expression, inhibited cell proliferation and promoted apoptosis, significantly enhancing the anti-PD-L1 therapeutic effect on OS cells. JC-1 staining experiments further confirmed that this combination therapy could perturb mitochondrial membrane potential and lead to stronger apoptosis.

[CONCLUSION] This study reveals the unique mechanism of action of Shikonin as a potential anticancer drug and demonstrates the potential of pH-responsive hydrogel nanomaterials as efficient and safe delivery systems for targeted cancer therapeutics. The strategy of Gel@PLGA@Shikonin@FA combined with atezolizumab provides a new idea and experimental basis for OS treatment.