SOX9 siRNA Loaded Lipid Nanoparticles Actively Targeted: Formulation, Delivery, and Antitumor Effect on Colorectal
Zhen Zhang1, Hongbo Wang2, Zhijun Zhang1
1College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Abstract:
Small interfering RNA (siRNA)-based therapy, which silences disease-associated genes, has emerged as a potential therapeutic strategy for various disorders, including cancer. Lipid nanoparticles (LNPs) have become a leading platform for efficient siRNA delivery. SOX9, a family member of SRY-related high-mobility-group box (SOX) transcription factors, plays an important role in the pathogenesis and progression of colorectal cancer (CRC). Here, we developed cRGDfK peptide-modificed LNPs (R-LNPs) composed of DLin-MC3-DMA, DMG-PEG, DSPC, DSPE-PEG-cRGDfK, and cholesterol for the targeted delivery of SOX9 siRNA (siSOX9) in CRC treatment. The formulation, delivery, and antitumor effect on CRC in vitro and in vivo were explored. The optimized R-LNPs exhibited favorable physicochemical properties including a uniform particle size (159.6 ± 0.93 nm), low polydispersity index (PDI = 0.207 ± 0.016), near-neutral zeta potential (2.74 ± 0.35 mV), and high encapsulated efficiency (90.71 ± 1.63%). Additionally, R-LNPs demonstrated sustained and controlled in vitro release and good serum stability. The prepared R-LNPs were efficiently uptaken by HCT-116 via clathrin, lipid rafts, and caveolae dependent endocytosis and macropinocytosis. Subsequently, R-LNPs were colocalized with lysosomes before escaping into the cytoplasm, ensuring effective siRNA release. In vivo distribution studies confirmed tumor-specific accumulation for R-LNPs in HCT-116 xenograft models. Functionally, R-LNPs loading siSOX9 inhibited the proliferation, migration, and invasion of HCT-116 cells and Caco-2 cells (P < 0.05 or 0.01) and inhibited tumor growth and proliferation in tumor-bearing mice (P < 0.05 or 0.01). The antitumor effects were attributed to the silence of SOX9, which subsequently downregulated key oncogenic mediators, including β-catenin, cyclin D1, and c-Myc (P < 0.05 or 0.01). Furthermore, R-LNPs demonstrated favorable safety in in vivo application. In conclusion, our cRGDfK-modified LNPs loading siSOX9 represent a promising targeted therapeutic strategy for CRC, offering both efficacy and biocompatibility.
Insights
Targeted lipid nanoparticles (LNPs) carrying SOX9 siRNA effectively inhibit colorectal cancer (CRC) progression. This novel therapy demonstrates significant antitumor effects and favorable safety profiles in preclinical models.
Area of Science:
- Nanomedicine
- Molecular Biology
- Oncology
Background:
- Small interfering RNA (siRNA) therapy offers potential for treating diseases like cancer by silencing specific genes.
- Lipid nanoparticles (LNPs) are a key platform for delivering siRNA effectively.
- SOX9 is implicated in the development and progression of colorectal cancer (CRC).
Purpose of the Study:
- To develop and evaluate cRGDfK peptide-modified LNPs (R-LNPs) for targeted delivery of SOX9 siRNA (siSOX9) in CRC treatment.
- To assess the physicochemical properties, in vitro and in vivo delivery, and antitumor efficacy of the R-LNPs.
- To investigate the safety and mechanism of action of the R-LNP/siSOX9 formulation.
Main Methods:
- Formulation of R-LNPs using DLin-MC3-DMA, DMG-PEG, DSPC, DSPE-PEG-cRGDfK, and cholesterol.
- Characterization of R-LNPs for particle size, PDI, zeta potential, and encapsulation efficiency.
- In vitro studies involving cell uptake, endosomal escape, and cytotoxicity assays on CRC cell lines (HCT-116, Caco-2).
- In vivo studies including biodistribution in HCT-116 xenograft models and assessment of antitumor effects in tumor-bearing mice.
- Analysis of SOX9 gene silencing and downstream oncogenic mediator downregulation (β-catenin, cyclin D1, c-Myc).
Main Results:
- Optimized R-LNPs showed desirable physicochemical properties: 159.6 nm size, 0.207 PDI, 2.74 mV zeta potential, and 90.71% encapsulation efficiency.
- R-LNPs exhibited sustained in vitro release, good serum stability, and efficient cellular uptake via multiple endocytic pathways.
- Tumor-specific accumulation of R-LNPs was confirmed in vivo.
- R-LNPs loaded with siSOX9 significantly inhibited CRC cell proliferation, migration, and invasion, and suppressed tumor growth in mice.
- SOX9 silencing led to downregulation of key oncogenic mediators, with favorable in vivo safety.
Conclusions:
- cRGDfK-modified LNPs effectively deliver siSOX9 to CRC cells and tumors.
- The R-LNP/siSOX9 formulation demonstrates significant in vitro and in vivo antitumor efficacy by targeting the SOX9 pathway.
- This targeted nanotherapy presents a promising, biocompatible therapeutic strategy for colorectal cancer treatment.
