During cancer therapy, phagocytic clearance of dead cells plays a vital role in immune homeostasis. The nonapoptotic form of cell death, ferroptosis, exhibits extraordinary potential in tumor treatment. However, the phagocytosis mechanism that regulates the engulfment of ferroptotic cells remains unclear. Here, we establish a novel pathway for phagocytic clearance of ferroptotic cells that is different from canonical mechanisms by using diverse ferroptosis models evoked by GPX4 dysfunction/deficiency. We identified the oxidized phospholipid, 1-steaoryl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), as a key eat-me signal on the ferroptotic cell surface. Enriching the plasma membrane with SAPE-OOH increased the efficiency of phagocytosis of ferroptotic cells by macrophage, a process that was suppressed by lipoprotein-associated phospholipase A2. Ligand fishing, lipid blotting, and cellular thermal shift assay screened and identified TLR2 as a membrane receptor that directly recognized SAPE-OOH, which was further confirmed by TLR2 inhibitors and gene silencing studies. A mouse mammary tumor model of ferroptosis verified SAPE-OOH and TLR2 as critical players in the clearance of ferroptotic cells in vivo. Taken together, this work demonstrates that SAPE-OOH on ferroptotic cell surface acts as an eat-me signal and navigates phagocytosis by targeting TLR2 on macrophages.

To carry doxorubicin (DOX) on breast cancer site effectively, halloysite nanotubes conjugated with poly­(ethylene glycol) and folate (HNTs-PEG-FA) is designed as a targeted drug delivery system. Halloysite nanotubes (HNTs) are shortened to ∼200 nm by ultrasonic scission and functionalized with amide groups to conjugate with N-hydroxylsuccinimide-polyethylene glycol carboxylic acid (NHS-PEG-COOH) and folate (FA). DOX@HNTs-PEG-FA is prepared by loading DOX on HNTs-PEG-FA via physical adsorption. The sustained and controlled release of DOX from DOX@HNTs-PEG-FA is up to 35 h in an acidic environment (pH 5.3). DOX@HNTs-PEG-FA, performed as a new nanodelivery system, shows significant inhibition of proliferation and induction of death in MCF-7 cells with positive FA receptor but not in L02 cells with negative FA receptor. Results of acridine orange/ethidium bromide and flow cytometric assay indicate that DOX@HNTs-PEG-FA induces cell death through apoptosis. Compared to the same dose of DOX, DOX@HNTs-PEG-FA generates more reactive oxygen species (ROS) in MCF-7 cells, which lead to mitochondrial damage and apoptosis. Furthermore, with fluorescence images and transmission electron microscopy, uptake of DOX@HNTs-PEG-FA by tumor cells is both through endocytosis and direct penetration mechanism. The in vivo antibreast cancer activity of DOX@HNTs-PEG-FA is further confirmed in 4T1-bearing mice. In contrast to DOX, DOX@HNTs-PEG-FA effectively reduces heart toxicity and inhibits solid tumor growth with higher cleaved caspase-3 protein level in tumor tissue of 4T1-bearing mice. DOX@HNTs-PEG-FA reveals a higher DOX fluorescence intensity in tumor tissue than in other normal tissues including heart, spleen, lung, and kidney at different time points. All these results suggest that FA-conjugated HNTs may be designed to be a novel drug delivery system for targeted therapy of breast cancer via intravenous injection.

Halloysite is weakly toxic to cells and can be excreted out through the gastrointestinal metabolism of zebrafish.

Here, polyamidoamine grafted halloysite nanotubes (PAMAM- g-HNTs) were synthesized for loading of siRNA in order to intracellular delivery of siRNA and treat of breast cancer via gene therapy. The successful grafting of PAMAM on HNTs was confirmed by various analytical methods. The size, zeta potential, and grafting ratio of PAMAM- g-HNTs is ∼206.2 nm, +19.8 mV, and 3.04%, respectively. PAMAM- g-HNTs showed good cytocompatibility toward HUVECs (84.7%) and MCF-7 cells (82.3%) even at high concentration of 100 μg/mL. PAMAM- g-HNTs/siRNA exhibited enhanced cellular uptake efficiency of 94.3% compared with Lipofectamine 2000 (Lipo2000)/siRNA (83.6%). PAMAM- g-HNTs/small interfering RNA-vascular endothelial growth factor (siVEGF) led to 78.0% knockdown of cellular VEGF mRNA and induced 33.6% apoptosis in the MCF-7 cells, which is also much higher than that of Lipo2000/siVEGF. In vivo anti-cancer results demonstrated that PAMAM- g-HNTs/siVEGF treated 4T1-bearing mice showed enhanced anti-cancer efficacy than Lipo2000/siVEGF group. Also, the nanocarrier system showed negligible toxic effects toward the major organs of mice. In vivo fluorescence imaging studies showed that there is a slight decrease in the fluorescence signal of PAMAM- g-HNTs/cy5-siVEGF after 72 h post-injection. Therefore, PAMAM- g-HNTs show promising application as novel nanovectors for siRNA delivery and gene therapy of cancer.

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