Calcium Phosphate Nanoparticle Delivery of siTRIB3 Inhibits EMT and Stemness in NSCLC

Abstract

Background: TRIB3 is upregulated in non-small cell lung cancer (NSCLC) and associates with worse survival, yet targeted therapeutics remain lacking. Calcium phosphate (CaP) nanoparticles offer biocompatible, serum-stable delivery for siRNA therapy. Here, we developed CaP nanoparticles loading siRNA targeting TRIB3 (NP CaP /si Trib3 ) and evaluated antitumor efficacy and mechanisms in vitro and in vivo. Methods: NP CaP /si Trib3 was synthesized via biomineralization, and RNase resistance was verified by PAGE. Mouse KP-1 NSCLC cells were used for transfection, apoptosis (Annexin V/7-AAD), migration (scratch), invasion (Matrigel Transwell), qRT-PCR, and Western blot of epithelial–mesenchymal transition (EMT) and stemness markers. C57BL/6 subcutaneous tumor models received tail-vein injections for efficacy and biosafety assessment. Results: TRIB3 was significantly upregulated in NSCLC tissues compared with normal lung tissues and was associated with poorer overall survival. NP CaP /si Trib3 formed nanosheets with an average size of approximately 159.4 nm and exhibited a more negative zeta potential than bare CaP, indicating successful siRNA loading. The CaP shell effectively protected si Trib3 from RNase degradation. NP CaP /si Trib3 efficiently silenced TRIB3 protein expression in KP-1 cells, with gene knockdown efficacy comparable to Lipo8000 but with lower cytotoxicity. Functionally, NP CaP /si Trib3 significantly promoted apoptosis and inhibited cell migration and invasion. In vivo, intravenously administered NP CaP /si Trib3 , which primarily accumulated in tumors through the enhanced permeability and retention (EPR) effect, markedly suppressed tumor growth without affecting body weight. Mechanistically, NP CaP /si Trib3 reversed EMT by upregulating E-cadherin and downregulating N-cadherin and Snail, and reduced cancer stemness by decreasing the expression of Sox2, Nanog, Pou5f1, Klf4, and c-Myc. Biosafety evaluations demonstrated negligible hemolysis, normal serum biochemistry and hematology, and no histopathological damage in major organs. Conclusion: NP CaP /si Trib3 is an effective and biocompatible siRNA nanoplatform that suppresses NSCLC progression through dual inhibition of EMT and cancer stemness, supporting TRIB3 as a promising therapeutic target. Keywords: non-small cell lung cancer, the pseudokinase tribbles homolog 3, calcium phosphate nanoparticles, siRNA delivery, epithelial–mesenchymal transition