lipid-a and pentaerythritol

Immune adjuvants are important components of current and prospective cancer vaccines. In this study, we aimed at evaluating the use of a synthetic lipid A derivative, pentaerythritol lipid A (PET lipid A), loaded into poly(lactic-co-glycolic acid) particles, as a potential cancer vaccine adjuvant. Poly(lactic-co-glycolic acid) particles (size range: 250-600 nm) were successfully formulated to include PET lipid A and/or the model tumor antigen, chicken ovalbumin (OVA). It was shown that particulated PET lipid A had a distinct advantage at promoting secretion of the immune potentiating cytokine, IL-12p70, and upregulating key costimulatory surface proteins, CD86 and CD40, in murine dendritic cells in vitro. In a murine tumor model, involving prophylactic vaccination with various permutations of soluble versus particulated formulations of OVA with or without PET lipid A, modest benefit was observed in terms of OVA-specific cell-mediated immune responses when PET lipid A was delivered in particles. These findings translated into a corresponding trend toward increased survival of mice challenged with OVA-expressing tumor cells (E.G7). In terms of translation of safe adjuvants into the clinic, these results promote the concept of delivering toll-like receptor-4 agonists in particles because doing so improves their adjuvant properties, while decreasing the chances of adverse effects due to off-target uptake by nonphagocytic cells.

Topics: Adjuvants, Immunologic; Animals; Antigens; Biocompatible Materials; Cancer Vaccines; Chemistry, Pharmaceutical; Cytokines; Dendritic Cells; Female; Lactic Acid; Lipid A; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Propylene Glycols; Toll-Like Receptors

The present study had two main objectives. First, was to compare the immune stimulatory effect of two synthetic lipid A analogues (7-acyl lipid A and pentaerythritol-based lipid A (PET lipid A)) on maturation/stimulation of bone marrow derived dendritic cells (DCs). Our second objective was to develop a liquid chromatography/mass spectrometry (LC-MS) method for the quantitative analysis of lipid A-based vaccine adjuvants. Treatment of immature DCs with 7-acyl lipid A and PET lipid A up regulated the surface expression of CD86 and CD40 molecules, and also induced similar profile of pro-inflammatory cytokine secretion. LC-MS analyses were performed using a Waters Micromass ZQ 4000 spectrometer, coupled to a Waters 2795 separations module with an autosampler. Calibration curves with R(2)>0.999 were constructed over the concentration range of 1.25-20 microg/ml for the solution of 7-acyl lipid A and PET lipid A. The method was tested in a 3 day validation protocol. The accuracy of the assay at different concentrations tested ranged from 89 to 108% and from 92 to 107% for 7-acyl lipid A and PET lipid A, respectively. The limit of quantification for both 7-acyl lipid A and PET lipid A was 1.25 microg/ml (signal/noise (S/N)) ratio >15:1. The sensitivity of the method (the limit of detection) was 0.35 and 0.15 ng for 7-acyl lipid A and PET lipid A, respectively (S/N ratio between 4:1 or 3:1). As a preliminary application, this method has been successfully applied to the determination of 7-acyl lipid A and PET lipid A content in poly (D,L-lactic-co-glycolic acid) nanoparticles (PLGA-NP).

Topics: Adjuvants, Immunologic; Animals; B7-2 Antigen; Bone Marrow Cells; CD40 Antigens; Chemistry, Pharmaceutical; Chromatography, Liquid; Cytokines; Dendritic Cells; Drug Compounding; Flow Cytometry; Lactic Acid; Lipid A; Mass Spectrometry; Mice; Mice, Inbred C57BL; Nanoparticles; Phenotype; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Propylene Glycols; Reference Standards; Reproducibility of Results; Solutions; Up-Regulation