Abstract
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Introduction: Exosomes and exosome-mimetic nanovesicles (EV) have found to be effective therapeutics or drug delivery vehicles in a wide range of human diseases including cancer and neurodegenerative diseases (1). In a recent paper, EV derived from pro-inflammatory M1 macrophages could effectively induce antitumor immune response and enhance the efficacy of immune checkpoint inhibitors (2). However, in multiple previous studies, exosomes and EV were rapidly cleared by reticuloendothelial system and therefore their tumor targeting efficiencies were limited. Herein, we have developed a simple surface modification method of EV derived from M1 macrophages using polyethylene glycol (PEG) to enhance in vivo tumor targeting efficiency. Method: The macrophage cell line RAW264.7 was used for preparing EV. RAW264.7 cells were incubated with 100 ng/mL of lipopolysaccharide and 20 ng/mL of IFN-γ for M1 macrophage induction. M1 macrophage induction was investigated by flow cytometry with different kinds of macrophage factors (M1: TNFa, iNOS, IL6, M2: CD163, Arginase1). After 24 hours later, 1.0 x 108 M1 macrophages were sequentially extruded through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm. The sample was ultracentrifuged for precipitating and washing out the EVs. After discarding supernatant, EV pellet were resuspended in phosphate buffer solution (PBS). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG) was added to obtain surface modified EVs (PEGylated EV). Dynamic light scattering was used to measure the size, intensity-weighted z-average diameter and zeta potential of two kinds of EV samples. 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DiR) was labeled to EV for in vivo imaging. DiR stained bare EV and PEGylated EV were purified from free dye and PEG by elution using a PD-10 column. Eluted samples were intravenously injected to CT26 tumor bearing Balb/c mouse and the mice were imaged using an IVIS spectrum. Result: Successful M1 induction was confirmed by increased expression of TNFa and iNOS in induced macrophages compared to uninduced macrophages. The PEGylated EV and bare EV showed hydrodynamic sizes of 191.7 ± 62.74 nm and 179.6 ± 51.35 nm, respectively. Both EVs were stable in PBS for 7 days (Fig.2). Transmission electron microscopy (TEM) examination demonstrated that both EVs are similar in size and shape. PEGylated EV showed a more neutral surface charge than bare EV (-5.91 ± 4.02 mV vs -43.4 ± 7.04 mV, respectively), which is a favorable feature for a longer in vivo circulation (Fig.3). In the in vivo IVIS images, the tumor bearing mice injected with bare EV showed high signal in the liver region, but no significant tumor region uptake. However, mice injected with PEGylated EV showed remarkable tumor uptake. Ex vivo imaging of major organs and tumor confirmed the findings of in vivo imaging. Strikingly, tumor to liver uptake ratio of PEGylated EV was almost 7 times higher than that of bare EV (Fig.3). Conclusion: We developed PEGylated EVs from M1 macrophages that have significantly higher tumor targeting efficiency than bare EVs. Based on this excellent tumor targeting ability, our modified EVs could be used for an effective immunomodulation of tumor microenvironment. Furthermore, this simple method of modifying EVs can be applied to various types of exosomes or EVs for cancer therapeutics. Reference (1) Antimisiaris, S. et al. (2018). Pharmaceutics, 10(4), 218. (2) Choo, Y. W et al. (2018). ACS nano, 12(9), 8977-8993.
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