Jennica L. Zaro

Exosomes or small extracellular vesicles are considered a new generation of bioinspired-nanoscale drug delivery system (DDS). Endogenous exosomes function as signalosomes since they convey signals via ligands or adhesion molecules located on the exosomal membrane, or packaged inside the exosome. Recently, exosome membrane modification, therapeutic payloads encapsulation, and modulation of in vivo disposition of exosomes have been extensively investigated, among which significant advances have been made to optimize exosome-mediated delivery to solid tumors. Exosomes, specifically tumor cell-derived exosomes, are presumed to have tumor-preferential delivery due to the homotypic features. However, quality attributes that dictate the tissue distribution, cell type-selective uptake, and intracellular payload release of the administered exosomes, as well as the spatiotemporal information regarding exosome fate in vivo, remain to be further investigated. This review summarizes recent advances in developing exosomes as drug delivery platforms with a focus on tumor targeting. The pharmacokinetic features of naive exosomes and factors influencing their intracellular fate are summarized. Recent strategies to improve tumor targeting of exosomes are also reviewed in the context of the biological features of tumor and tumor microenvironment (TME). Selected approaches to augment tumor tissue deposition of exosomes, as well as methods to enhance intracellular payload delivery, are summarized with emphasis on the underlying mechanisms (eg, passive or active targeting, endosomal escape, etc.). In conclusion, this review highlights recently reported tumor-targeting strategies of exosome-based drug delivery, and it's in the hope that multiple approaches might be employed in a synergistic combination in the development of exosome-based cancer therapy.

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.