Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer‐targeted exosomes for drug delivery. Specifically, a scalable, robust, high‐yield, cell line based exosome production process is created in a stirred‐tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)‐exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb‐exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb‐exosomes. The in vitro anti‐cancer toxicity study shows high cytotoxicity of mAb‐exosome‐romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti‐cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large‐scale animal studies and to facilitate the translation from research to clinics. 相似文献
Apoptosis occurs in many autoimmune diseases. Excess iodine induces thyrocyte apoptosis and increases the incidence and prevalence of autoimmune thyroiditis (AIT). However, the sequence of events between the appearance of thyrocyte apoptosis and the occurrence of thyroiditis remains uncharacterized. Furthermore, few studies have investigated the role of macrophage phagocytosis in the development of AIT. Therefore, we evaluated the relationship between apoptosis and inflammatory infiltration in NOD.H-2h4 mouse thyroids by comparing the sequence of events in tissue samples. We also investigated the role of macrophages by comparing macrophage phagocytosis function in BALB/c, C57BL/6, and NOD.H-2h4 mice treated with different levels of iodine. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and thyroid inflammatory scores revealed that apoptosis (2 weeks) occurred before inflammatory infiltration (4 weeks). Phosphatidylserine (PS) expression on the extracellular surface of the cell membrane and double-stranded DNA fragments associated with apoptosis appeared at 2 and 8 weeks, respectively. Additionally, although apoptosis was enhanced in the thyroids of mice supplemented with excess iodine (0.05 ± 0.12 vs 1.63 ± 0.82% for BALB/c, 0.09 ± 0.14 vs 1.51 ± 0.34% for C57BL/6, and 0.07 ± 1.11 vs 4.72 ± 0.62% for NOD.H-2h4 mice), only NOD.H-2h4 mouse thyroids presented with inflammation. Furthermore, macrophages from NOD.H-2h4 mice (44.46 ± 1.79%) exhibited decreased phagocytotic activity relative to that in BALB/c (54.21 ± 4.58%) and C57BL/6 (58.96 ± 4.04%) mice. There were no differences in phagocytosis function between NOD.H-2h4 mice supplemented with excess iodine or left untreated (24.50 ± 2.66 vs 21.71 ± 1.79%, p = 0.06). In conclusion, deficiencies in the apoptosis clearance of macrophages in NOD.H-2h4 mice may constitute an early pathogenic mechanism in AIT that is not influenced by iodine intake.
The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7–MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress. 相似文献