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Nucleic acids with bacterial and viral origins are recognized by toll like receptors (TLRs) that trig-gers mammalian innate immune system to secrete proinflammatory or inflammatory cytokines. Even though these ligands have a high potency to be used in clinical applications as a vaccine adjuvant or as an immunotherapeutic agent, the rapid clear-ance from the body by serum proteins, in vivo deg-radation via nucleases, consequently lead to poor in
vivo stability and performance thus hampers their
clinical applications.
Drug delivery systems are promising tools to in-crease the stability of therapeutic agents and provide the targeting and internalization of the incorporated cargo. Among many, liposomes that are nano-sized lipid vesicles are one of the most commonly used drug delivery vehicles. Due to safety and high en-trapment efficiency of bioactive agents liposomes are one of the best candidates to be utilized in the encapsulation of very labile and valuable bioactive agents. In this study, different liposomes were used to encapsulate TLR ligands in order to understand their immune stimulatory effects.
Mouse splenocytes and bone marrow derived mDCs were stimulated with different liposome formula-tions with varying physiochemical features loaded with CpG (A or B-types) and pI:C (TLR9 and TLR3 ligands, respectively). Production of proinflamma-tory cytokines were analyzed by ELISA and the maturation level and expression of co-stimulatory molecules of DCs was studied by FACS.
Additionally, thioglycollated peritoneal exudate cells (PECs) were isolated and treated with differ-ent liposome formulations in order to investigate inflammasome inducing ability of unencapsulated free liposomes or together with their cargo. The cells were studied by FACS, while the supernatants were analyzed for IL1b production by ELISA. Our data revealed that when DCs were treated with liposomal formulations, maturation signals along weith co-stimulatory molecule expression of DCs were improved up to 50 fold compared to free CpG or pIC induced activations. The IL6, IL12 and IFNȖ production from splenocytes and DCs were signifi-cantly boosted by liposomes.
This improvement was highest for CpG-A loaded anionic liposomes, whereas stealth
cationic liposomes were the best formulation for CpG-B type. Neutral and stealth cationic liposomes were very effective formulations for pIC mediated cytokine production and DC maturation. Surpris-ingly, formulations containing cholesterol were generally very effective inflammasome activators as evidenced by IL-1b production of stimulated PECs.
In conclusion, it was demonstrated that liposome formulations are efficient vehicles to increase the immune stimulation potency of TLR ligands by increasing DC maturation, pro-inflammatory cy-tokine secretion and inflammasome activation.
