amazonensis antigen (TLA) was prepared while described in Cargnelutti et al. were heavily loaded with IMQ at ~44 g IMQ/mg phospholipids [~20 folds higher than the non-SR-A1 ligand soyPC liposomes loaded with IMQ (LIPO-IMQ)]. immune response. contain ligands of SR-A1. With this work we hypothesize that nanoARC prepared with lipids extracted from may constitute service providers that solve the problem of formulating the TLR7 ligand imiquimod (IMQ). IMQ is an immune response modifier, authorized by the FDA for the treatment of actinic keratosis, superficial basal-cell carcinoma lesions, besides of various off-label uses on precancerous and cancerous skin lesions (Papadavid et al., 2007). Its use in preclinical Rabbit Polyclonal to OR4A15 transcutaneous immunization is limited however, since in the presence of a cytotoxic T lymphocyte (CTL) epitope, the elicited immune response decays within a few days, requiring co-stimulation via CD40 for instance, to enhance the primary CTL response and effective formation of memory space CTL (Warger et al., 2007). Aldara, the same as additional preclinical particulate service providers for IMQ so far developed, such as solid lipid nanoparticles (Zhou et al., 2007), liposomes (Fox et al., 2014), nanogels (Stein et al., 2014), or emulsions (Lopez et al., 2017), launch IMQ into the software site microenvironment upon structural degradation of JNJ-7706621 the carrier particle (Chollet et al., 1999). The IMQ distributing beyond the application site has been identified as a source of systemic toxicity (Steinhagen et al., 2011; Mifsud et al., 2014). Here we propose to modify the pharmacodynamics of IMQ by loading it within nanoARC. We hypothesize that nanoARC, by being naturally targeted to SRA-1 expressing macrophages, would deliver the carried IMQ into the endo-lysosomal pathway and therefore concentrate its effect into a specific intracellular site. IMQ induces a moderate TLR7-self-employed inhibition on adenylyl cyclase activity, impairing a negative feedback mechanism that normally limits inflammatory reactions (Sch?n et al., 2006; Sch?n and Sch?n, 2007). The avoidance of this inhibition by SR-A1 targeted delivery of IMQ and the lack of isostearic acid responsible for additional TLR7-self-employed inflammatory effects (Walter et al., 2013), would lower the chances of systemic swelling a potential toxicity (Heikkinen and Susitaival, 2011). Switching from a diffusion mediated cell entering mechanism for free IMQ, to an endocytic uptake of nanoARC transporting IMQ (nanoARC-IMQ) by SR-A1+ cells would lead to a massive delivery IMQ to the endo-lysosomal system, concentrating its activity only on SR-A1+ and TLR7 expressing cells. This would improve IMQ immunogenicity, reducing at the same time its systemic bioavailability, and therefore it’s adverse effects. Formulating the fragile foundation IMQ [(1-(2-methylpropyl)-1 -imidazo [4,5-c] quinolin-4-amine] however, results a demanding task (Gogoll et al., 2012). There are a few organic providers (Chollet et al., 1999) capable of efficiently dissolve IMQ, such as the isostearic acid (ISA), a relatively toxic constituent of the poorly stable commercial cream Aldara (Chollet et al., 1999). The unique structural features of archaeolipid bilayers may help to partition the IMQ, ruling out the need for ISA. To the best of our knowledge, this is the 1st approach where a ligand indicated on the surface of nanoARC is definitely combined with an immunomodulator, to render double targeted nanoARC. The lack of complex manufacture methods needed JNJ-7706621 to covalent labeling is an additional benefit that may facilitate analytical characterization, batch reproducibility and adaptation to higher level production of such formulation (Korsmeyer, 2016; Landesman-Milo and Peer, 2016). Here we will prepare and structurally characterize nanoARC-IMQ and test its and immunomodulatory overall performance upon 3 subcutaneous doses mixed with model mucocutaneous leishmania parasite proteins in Balb/C mice. Materials and methods Materials Soybean phosphatidylcholine (SPC, purity 90%) was a gift from Lipoid (Ludwigshafen, Germany). Imiquimod (purity 98%) was a gift from Laboratorio Lazar (Buenos Aires, Argentina). MONTANIDE? ISA 763 A VG was from Seppic (Puteaux, France). Ficoll was from GE Healthcare (Munich, Germany). Hypaque was from Winthrop Products (Buenos Aires, Argentina). Roswell Park Memorial Institute (RPMI) 1640 medium was from Gibco, Existence Technologies (New York, USA). Antibiotic/antimycotic remedy (penicillin 10,000 IU/mL, streptomycin sulfate 10 mg/mL, amphotericin B 25 g/mL), glutamine, and trypsin/ethylenediamine tetraacetic acid were from PAA Laboratories GmbH (Pasching, Austria). Fetal bovine serum (FBS) was from Internegocios, Cordoba, JNJ-7706621 Argentina. Concanavalin A (ConA), Phorbol 12-myristate 13-acetate (PMA), Laurdan, saponin, Carboxy fluorescein succimidil ester (CFSE), 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS).