Calcium/Aluminum-Cored Asymmetric Bilayer Nanoparticles for Codelivery of Ziyuglycoside II and PD-L1 siRNA Exert Anti-Breast Tumor Effects

<b>Objectives</b> Breast cancer remains a major cause of female cancer-related deaths, with current therapies limited by poor tumor targeting and an immunosuppressive microenvironment. This study designed CA/ZYII-siP-c-L-an asymmetric lipid bilayer-coated calcium/aluminum (CA)-core nanoparticle-to co-deliver PD-L1 siRNA (siP) and ziyuglycoside II (ZYII) to boost therapeutic efficacy. <b>Methods</b> CA/ZYII-siP-c-L was fabricated through modified microemulsification to first construct the CA cores, followed by thin-film hydration for encapsulation of ZYII within the hydrophobic domain, and via hybridization of the outer lipid layer with DSPE-PEG1000-PAMAM to finally enable specific adsorption of siP. The characterization of CA/ZYII-siP-c-L was performed to get size distribution, zeta potential and in vitro release behavior. In vitro cytotoxicity of the nanoparticles to NIH3T3 and 4T1 cells was detected by the CCK-8 method. The uptake capacity to 4T1 breast cancer cells was determined using inductively coupled plasma optical emission spectrometry and high-performance liquid chromatography. Pharmacokinetic studies and tissue distribution experiments were performed. In BALB/c mice bearing orthotopic 4T1 tumors, efficacy evaluations were conducted with the detection of tumor immune microenvironment; meanwhile, organ damage was evaluated by hematoxylin-eosin staining of major organs and detection of routine biochemical indicators. <b>Results</b> CA/ZYII-siP-c-L was characterized by dynamic light scattering (mean size ~185.7 nm) and zeta potential analysis (~9.35 mV). In vitro, the nanoparticle exhibited low cytotoxicity in NIH3T3 normal cells, high uptake by 4T1 breast cancer cells, and pH-responsive release. For the pharmacokinetic study, CA nanoparticle system could significantly enhance the systemic exposure of ZYII, compared to free ZYII suspension. In BALB/c mice with orthotopic 4T1 tumors, CA/ZYII-siP-c-L accumulation in tumors was 3.5-fold higher than that of free drugs, significantly enriching helper T cells and cytotoxic T lymphocytes while reducing regulatory T cells and suppressive dendritic cells in the tumor immune microenvironment; this immunomodulatory effect, combined with PD-L1 silencing at protein levels, contributed to ~62% inhibition of tumor growth with no organ damage (confirmed by hematoxylin and eosin staining of major organs and normal biochemical indices). <b>Conclusions</b> CA/ZYII-siP-c-L integrates safety, targeting, and codelivery capabilities, offering a promising strategy for breast cancer treatment by combining siP-mediated immunity regulation and the antitumor effects of ZYII.