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.

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 glycolsplit 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-1β 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 IC 50 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 siteselectively desulfated HP. Comparison of HP and LH derivatives revealed that fractionated smaller heparin has greater anti-inflammatory activity.Key words glycosaminoglycan; self-assembled nanoparticle; anti-inflammatory activity; Toll-like receptor 4; macrophage; structure-activity relationship Glycosaminoglycans (GAGs) are linear polysaccharides that play important roles in processes such as cell adhesion and signal transduction. 1) Heparin (HP) is one of the highly sulfated GAGs that consist of repeats of alternating uronic acid and D-glucosamine.2) HP is a well-known anticoagulant that activates antithrombin, leading to anticoagulation 3) ; moreover, it has a variety of additional biological effects such as antiangiogenesis and anti-inflammation. Chondroitin sulfate (CS), consisting of repeats of alternating D-glucuronic acid and N-acetyl-D-galactosamine, 2,4) is used clinically to reduce pain and improve articular function in patients with osteoporosis.5,6) Hyaluronic acid (HA) consists of repeats of alternating D-glucuronic acid and N-acetyl-D-glucosamine, but characteristically lacks sulfo groups. 2,4) HA has been used to protect synovial membranes and heal injuries. [7][8][9] Although the biological activity varies for each GAG, a common feature is the presence of an anti-inflammatory effect. Several in vitro [10][11][12][13][14] and in vivo 15,16) studies have shown that HP, CS, and HA suppress the lipopolysaccharide (LPS)-induced production of inflammatory cytokines by inhibiting the activation of MyD88, p38 mitogen-activated protein kinase (MAPK), an...

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.

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