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

Abstract

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.

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

“…of 15 kDa, Nacalai Tesque, Kyoto, Japan), LH (5 kDa, Kissei, Tokyo, Japan), CS (chondroitin 6-sulfate, 15 kDa, Nacalai Tesque, Kyoto, Japan), and HA (20 kDa, Lifecore, U.S.A.) as described previously. 17) Next, 20 mg of each glycolsplit GAG was dissolved in 1.5 mL formamide containing 10 mg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and mixed with 5 mg of stearylamine (Wako Pure Chemical Industries, Ltd.) dissolved in 1 mL tetrahydrofran. The reaction mixture was stirred at room temperature for 24 h. The reaction mixtures were then precipitated in ethanol, centrifuged at 15000×g for 10 min, and decanted.…”

“…Cell Isolation and in Vitro Assay for Cytokines Mouse peritoneal macrophages were isolated as described previously. 17) All animal experiments were performed in accordance with the Principles of Laboratory Animal Care as directed by the U.S. National Institutes of Health and the Guidelines for Animal Experiments of Kyoto University. Macrophages were harvested with ice-cold RPMI 1640 from 5-week-old female ICR mice (SLC, Shizuoka, Japan) 4 d after intraperitoneal injection of 1 mL 2.9% thioglycolate medium (Nissui Pharmaceutical, Tokyo, Japan).…”

“…Our previous study 17) revealed that desulfation of the 6-O-sulfo group of the HP conjugates remarkably lowers their anti-inflammatory effect, but desulfation at either 2-O-or N-sulfo groups does not. This prompted us to hypothesize that the structure of GAGs, including the position and degree of sulfation, greatly affects their anti-inflammatory effect.…”

“…18) The present study was initiated to examine whether conjugation with hydrophobic amines is effective for not only HP but also other GAGs in augmentation of anti-inflammatory activity against LPS-stimulated macrophages. Our previous study 17) revealed that desulfation of the 6-O-sulfo group of the HP conjugates remarkably lowers their anti-inflammatory effect, but desulfation at either 2-O-or N-sulfo groups does not. This prompted us to hypothesize that the structure of GAGs, including the position and degree of sulfation, greatly affects their anti-inflammatory effect.…”

“…Since HP conjugates prevented phosphorylation of IL-1 receptorassociated kinase-1 (IRAK-1) and degradation of inhibitor of NF-κB (IκBα), its anti-inflammatory effect was concluded to occur via inhibition of the Toll-like receptor 4 (TLR4)-mediated MyD88-dependent NF-κB pathway. 17) A subsequent study demonstrated that the HP-D-erythro-sphingosine conjugate delayed the progression of arthritis in type II collagen-induced arthritis mouse models. 18) The present study was initiated to examine whether conjugation with hydrophobic amines is effective for not only HP but also other GAGs in augmentation of anti-inflammatory activity against LPS-stimulated macrophages.…”