Liposome encapsulation overcomes d-type and k-type CpG ODN dichotomy and induces synergistic immune activation = Lipozoma yüklenmiş D-tipi ve K-tipi ODN'lerin sinerjik immun aktivasyonu
Liposomes are one of the best candidates for the encapsulation of labile bioactive agents due to their safety and high entrapment efficiency. In human, two structurally distinct classes of CpG ODN are capable of activating different signaling pathways, leading to differential immune activation. While K-type ODN triggers plasmacytoid dendritic cells (pDCs) to mature and produce TNFα, D-type ODN leads to IRF-7 dependent IFNα secretion. Strikingly, when K-and D-type ODN are co-incubated in their free forms, K-ODN masks the D-ODN specific immune activation. Identifying proper delivery vehicles that provide both ODN types to display their superior features upon stimulation is of great clinical importance. In this study, first we investigated the synergistic effects of K- and D-ODN upon encapsulating them within five different liposome types. Then with the selected potential liposome combinations, we identified synergistic activation capacities both on human PBMCs and on mice splenocytes. In PBMC cytokine results revealed that D-ODN loaded in all five liposome types stimulated more IFNα than free D-ODN. Similarly, liposomal K-ODN triggered more TNFα than free K-ODN type. While incubation of free K and D- type ODN as expected, abrogated D-specific IFNα production from PBMC, simultaneous incubation with neutral or anionic D-ODN loaded liposomes plus cationic liposomes loaded with K-ODN significantly increased K-specific as well as D-specific effect rather than masking it (i.e. more production of TNFα and IFNα specific for K and D, respectively). This improved synergistic immune activity for both D and K ODN observed with ND+CK combination in 100% of individuals (TNFa) and 90% of individuals for IFNa. Additionally, intracellular cytokine staining findings supported improved TNFα and IFNα, from pDC population of PBMCs. Costimulatory molecule expressions and APC activation also significantly upregulated compared with free treatment. In mice contrary to ND+CK combination, sterically stabilized cationic liposome encapsulated K-ODN combined with i) neutral, ii) anionic, iii) cationic or iv) stealth encapsulated D-ODN increased IL6, IL12 and IFNγ levels, when stimulated simultaneously. Moreover, ex vivo experiments showed that cellular uptake and pro-inflammatory cytokine gene expressions significantly increased with combined liposomal formulations. This study established that by selecting proper liposome type(s) we reverse antagonistic action of K-ODN on DODN and induce a synergistic effect leading to a more robust immunostimulatory activity in both human and mice. This approach could broaden the immunotherapeutic application of these two important CpG ODN classes in clinic.