Inmunoadyuvant effect of oligodeoxynucleotids with cpg motifs (CpG - ODN) loaded in nanostructureted systems (coagels)

SANCHEZ VALLECILLO, MARIA FERNANDA; PALMA SANTIAGO; ULLIO GAMBOA, GABRIELA; ALLEMANDI DANIEL; MORON GABRIEL; PISTORESI M. C.; MALETTO, BELKYS

Córdoba

Congreso; 1era Reunión Internacional de Ciencias Farmacéuticas; 2010

Facultad de Ciencias Químicas - UNC

INTRODUCTION CpG-ODN has shown very promising inmunoadyuvant activity. Despite successes demonstrated in early clinical trials, the clinical use of free CpG-ODN remains still uncertain because some troubles (enzymatic degradation, unfavorable pharmacokinetics, a lack of specificity for target cells and poor cellular uptake) (1, 2). In order to improve the biodisponibility of CpG-ODN, we used CpG-ODN formulated in nanostructures (coagels) of 6-O- ascorbyl palmitate (Coa-ASC16), wich has the ability to form supramolecular aggregate that are produced by phase cooling below a critical micellar temperature(3). MATERIALS AND METHODS Mice: 3- months old- female BALB/c, C57BL/6 or TLR4-/- mice. The mice were housed in our animal facility until use under specific pathogen-free conditions. Our animal facility meet the terms of the Guide to the Care and Use of Experimental Animals, published by the Canadian Council on Animal Care and has the assurance number A 5802-01 delivered by the Office of Laboratory Animal Welfare (NIH). Antigen: OVA was purchased from (grade V) Sigma-Aldrich (St. Louis,MO). Oligodeoxynucleotides: CpG-ODN, the sequence 1826: TCCATGACGTTCCTGACGTT. CpG-ODN was synthesized with a nuclease-resistant, phosphorothioate backbone, (Operon Technologies-Alameda, CA). Coa-ASC16, was prepared by heating ASC16 in isotonic dextrose (with or without CpG-ODN) to above the phase transition temperature and allowing the temperature to fall to room temperature[3]. Immunization: Mice were immunized on day 0, 7 and 15 with OVA mixed with CpG-ODN (OVA/CpGODN) or OVA mixed with CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16). Each mouse was injected subcutaneously in the tail, in the neck region, and in both hind limbs. The dose of OVA was 60Hg/animal/dose. CpG-ODN was administered at a dose of 75Hg per animal. Antibody assays: specific antibody against OVA were determined by enzyme-linked immunosorbent assay (ELISA). Cytokine-specific ELISA: level of IL-6 was measured by capture ELISA. Flow cytometry analysis: cells were pre-incubated for 20 min at 4°C with anti CD32/CD16 monoclonal antibody and then stained with primary monoclonal antibodies conjugated with an appropriate fluorochrome for 30 min at 4°C. Cells were acquired on a FACS Canto II cytometer and data were analyzed using FlowJo software. Statistical analysis: Data were analyzed by Graph Pad Prism4 software (Graph Pad Software. San Diego, CA). All data were considered statistically significant if p values were