135 Exosomes are naturally occurring, membranous
nanovesicles of 40-100 nm in diameter. They arise from the endocytic cellular pathway through three stages: (i) formation of endocytic vesicles ii) mul-tivesicular bodies (MVBs) formation in the cytosol and (iii) fusion of the MVBs with the plasma mem-brane to release their nanovesicular cargoes (1, 2). As well as functioning as natural vectors of inter-cellular signaling within a given tissue or between differenT-cells and tissues, exosomes could be ex-ploited for the delivery of therapeutic cargoes (3). To explore the therapeutic potential, we investi-gated whether cell line-derived exosomes could be loaded with ss/ds DNA or RNA. The nano-delivery vehicle is expected to provide better protection of the cargo, more effective delivery to immune sites while facilitating better internalization that lead to enhanced activity.
Exosomes were isolated from RAW264.7 superna-tants by i) differential centrifugation, ii) filtration and iii) ultra-centrifugation. Purified exosomes were loaded with Cy5- labeled K3 and D35 CpG oligos via dehydration-rehydration method and labeled with lipophilic dye, SP-DiOC(18). SP-DiOC labeled, CpG ODN loaded exosomes were incubated with RAW 264.7 cells at 10:1 ratio (Exosome:Cell) for 24h for cytokine production assays or incubated for several hours for confocal studies. For exosome internalization studies, cells were analyzed by FACS or by confocal microscopy. Exosome treated cell supernatants were used for cytokine ELISA assays.
Different CpG DNA loaded SP-DiOC labeled ex-osomes were (i.e. Exo(K3-Cy5)SP-DiOC or Exo(D35-Cy5)SP-DiOC) analyzed by FACS to assess the level
of cellular internalization. Data revealed that K3 exosomes loaded cells were 80% positive for both Cy5 and SP-DiOC signal. RAW cells were found to be 100% positive for Cy5 and SP-DiOC in the case of D35-loaded exosomes. Co-localization of exosomes and ODN signals in the cells were confirmed by confocal microscopy. Supernatants from CpG ODN loaded nanovesicle treated cells were used to detect TNFĮ, and IL6 secretion profiles by ELISA. This data implicated that K and D-type CpGODNs can be effectively encapsulated into exosomes by lyophilization. Encapsulation of these particles into exosomes enhanced their stimulatory activity as evidenced by their increased IL-6 and TNF-Į se-cretion by RAW264.7 cells. This effect was dose de-pendent for both K3 and D35 containing exosomes. Internalization kinetics and magnitude of ODNs when loaded into exosomes were much higher than free ODN.
Collectively, our findings strongly demonstrated that exosome-loaded DNA led to formation of ef-fective nanovesicle drug delivery system.
This technique promises to offer a biocompatible and personalized therapeutic approach with a great potential to overcome the poor performance mainly due to pre-mature elimination of synthetic particles since they are recognized as non-self upon in vivo administration.
References: [1] Trajkovic K, Hsu C, Chiantia S, Rajendran L, et al. 2008. Science 319:1244–7. , [2] Chaput N, Théry C. 2011. Semin Immunopathol. 33:419-40. , [3] Lakhal S, Wood MJ. 2011. Bioessays 33:737-41.
Acknowledgment: This work was supported by a TUBITAK SBAG grant #: 111S316.
