Anti-inflammatory Effect of Self-assembling Glycol-Split Glycosaminoglycan-Stearylamine Conjugates in Lipopolysaccharide-Stimulated Macrophages.

Glycosaminoglycans (GAGs) play important roles in various biological processes such as cell adhesion and signal transduction, as well as promote anti-inflammatory activity. We previously revealed that glycol-split heparin (HP)-aliphatic amine conjugates form self-assembled nanoparticles and suppress the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß in lipopolysaccharide (LPS)-stimulated macrophages much more strongly than native HP (J. CONTROL: Release, 194, 2014, Babazada et al.). Considering that HP is not the only GAG to have anti-inflammatory activity, the present study was initiated to examine whether conjugation of GAGs with aliphatic amines is generally effective in their activity augmentation against LPS-stimulated macrophages. We newly synthesized the stearylamine conjugates of chondroitin sulfate (CS), hyaluronic acid (HA), and low-molecular-weight heparin (LH), and investigated the effect of the position and degree of sulfation and molecular weight of GAGs on their anti-inflammatory activity. All of the conjugates formed self-assembled nanoparticles in aqueous solution. The IC50 value for suppression of TNF-α production from the macrophages was the smallest with the derivative of LH, followed by HP, CS, and HA. The degree of sulfation appeared to be important in determining their anti-inflammatory activity, which would correspond to previous results using the derivatives of site-selectively desulfated HP. Comparison of HP and LH derivatives revealed that fractionated smaller heparin has greater anti-inflammatory activity.

Self-assembling lipid modified glycol-split heparin nanoparticles suppress lipopolysaccharide-induced inflammation through TLR4-NF-κB signaling.

Self-assembling heparin nanoparticles have attracted much attention as promising drug carriers for various drugs, genes and imaging agents. In the present investigation, we found that heparin nanoparticles are selective Toll-like receptor 4 (TLR-4) antagonists and have a much greater anti-inflammatory effect than native heparin. More specifically, we developed self-assembling nanoparticles composed of glycol-split heparin/D-erythro-sphingosine conjugates (NAHNP), characterized their physicochemical properties and anti-inflammatory effect in vitro. Unlike native heparin, NAHNP significantly inhibited lipopolysaccharide-induced activation of MyD88-dependent NF-κB signaling pathway and production of pro-inflammatory cytokines such as TNF-alpha from mouse macrophages with IC50 = 0.019 mg/mL. Furthermore, we investigated the structure-activity relationship of the conjugates and identified the length of attached alkyl chains of d-erythro-sphingosine to be critical for anti-inflammatory effect. Decrease in alkyl chain length of NAHNP resulted in loss of inhibitory activity. In line with these findings, 6-O-sulfate groups of D-glucosamine residue were essential for effective inhibition, while removal of 2-O-sulfo and 3-O-sulfo groups as well as replacement of N-sulfo groups with N-acetyl did not alter anti-inflammatory activity. Therefore, NAHNP would be a promising candidate in acute and chronic inflammatory disorders, in addition to the nature of a drug carrier.

PEG conjugation of a near-infrared fluorescent probe for noninvasive dual imaging of lung deposition and gene expression by pulmonary gene delivery.

Dual imaging of lung deposition and gene expression following the pulmonary delivery of a gene formulation is useful for a precise analysis of gene transfection efficiency in vivo. As a novel probe for evaluating lung deposition, in this study, a poly(ethylene glycol)-conjugated near-infrared fluorescent probe (PEG-NIRF) was newly synthesized, and compared with indocyanine green (ICG), for application to pDNA/polyethyleneimine (PEI) complex. PEG-NIRF had superior characteristics including a larger Stokes shift (absorption maximum, 662 nm; emission maximum, 772 nm) and relatively equivalent fluorescence intensity compared with ICG. ICG affected the physicochemical properties of pDNA/PEI complex with a loss of fluorescence intensity, while PEG-NIRF did not. Experiments in mice demonstrated that PEG-NIRF showed greater lung localization than ICG following pulmonary co-delivery with pDNA/PEI complex, indicating the possibility of accurately evaluating lung deposition. Moreover, it was clarified that the evaluation of lung deposition by PEG-NIRF even at 60 min could be significantly correlated with gene expression in each mouse following pulmonary co-delivery with pDNA/PEI complex. These results suggest that PEG-NIRF is widely applicable to the dual imaging of the lung deposition and gene expression of inhaled gene formulations.