Targeted Exosomes for Drug Delivery: Biomanufacturing, Surface Tagging, and Validation

Abstract

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers need be overcome before widely clinical applications, such as low productivity and ineffective cancer targeting. The present study aimed at creating a new biomanufacturing platform of cancer-targeted exosomes for drugs delivery.Specifically, we created a scalable, robust, high-yield, cell line-based exosomes production process in stirred-tank bioreactor, and developed an efficient surface tagging technique to generate mAbexosomes. The in vitro characterization using transmission electron microscopy, NanoSight nanoparticle tracking analysis and Western blotting confirmed the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy imaging demonstrated mAb-exosomes had strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, we loaded Romidepsin, a histone deacetylase inhibitor, into mAb-exosomes. The in vitro anti-cancer toxicity study showed high cytotoxicity of mAb-exosome-Romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validated the cancer targeting specificity, anti-cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, we developed new techniques enabling targeted exosomes for drug delivery to support large-scale animal studies and facilitate the translation from research to clinics.

Dual-Targeted Extracellular Vesicles to Facilitate Combined Therapies for Neuroendocrine Cancer Treatment

“…First, the EV were produced using our previously published platform [24]. Briefly, 2 L stirred-tank bioreactors (Vineland, NJ, USA) were utilized to conduct EV production.…”

“…The production was performed with parameters controlled at Temp 37 • C, pH 7.0, DO 50% and agitation 70 rpm. The EV were purified following our previously reported procedure [24]. Briefly, when HEK293F cell viability dropped to 80-90% in the bioreactor, the culture broth was collected and centrifuged at 4000 g and 4 • C for 1 h. Then the collected spent medium was filtered to remove cell debris and treated with proteinase K to remove secreted host cell proteins.…”

“…In this study, the fine polishing using Protein A chromatography was not performed. The purified EV were used for surface tagging and drug packing or supplemented with 25 mM trehalose for long-term storage at −80 • C. Second, following the reported procedure [24,36], the anti-SSTR2 mAb was produced by hybridoma cells in 2-L bioreactors and purified using New Generation Chromatography (NGC) system (Bio-Rad, Hercules, CA, USA) equipped with Protein A column. The anti-SSTR2 mAb was titrated using high performance liquid chromatography (HPLC) system (Shimadzu, Columbia, MD, USA) equipped with a UV detector and a viva C-8 column (Phenomenex, Torrance, CA, USA).…”

“…The anti-SSTR2 mAb was titrated using high performance liquid chromatography (HPLC) system (Shimadzu, Columbia, MD, USA) equipped with a UV detector and a viva C-8 column (Phenomenex, Torrance, CA, USA). Third, both anti-SSTR2 mAb and anti-CXCR4 mAb were tagged to EV via 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine polyethylene glycol succinimidyl ester (DSPE-PEG-NHS) linker to generate mAbs-EV, with mPEG-DSPE stabilizer (New York, NY, USA) to improve the circulation stability, as we reported previously [24]. The generated mAbs-EV were titrated with NanoSight (Malvern Panalytical, Malvern, UK) with five captures in 5 min per sample using SCMOS camera at room temperature, pump speed of 25 and detection threshold of 5.…”

“…The anti-SSTR2 mAb or anti-CXCR4 mAb (RD Systems, Minneapolis, MN, USA) was labelled with an Alexa Fluor™ 647 labelling kit to generate fluorescent AF647-mAb. The staining conditions are 1 µg AF647-mAb per million cells at 4 • C for 30 min as reported before [24,36,51,52].…”