Uniform Lipopolysaccharide (LPS)‐Loaded Magnetic Nanoparticles for the Investigation of LPS–TLR4 Signaling

The interaction of highly conserved microbial constituents with the innate immune system contributes greatly to the recognition and reaction against intruding pathogens by mammals. Examples of such microbial constituents include the lipopolysaccharides (LPS) and lipooligosaccharides (LOS), also known as endotoxin (E), of Gram-negative bacteria. These potent pro-inflammatory molecules include a hydrophilic oligosaccharide chain of variable length and a hydrophobic, membrane anchoring moiety, termed lipid A.[1–3] The interaction of E with cells of the innate immune system leads to the formation and release of endogenous mediators initiating inflammatory and immune responses essential for optimal antibacterial defense.[4,5] Such a cascade of events is triggered by activation of the Toll-like receptor 4 (TLR4) by E. This is the last step of a sequential process of E recognition and interaction with extracellular and cell surface host proteins, including LPS-binding protein (LBP), soluble and membrane-associated CD14, secreted and TLR4-associated MD-2, and TLR4 itself.[6–8] The amphiphilic character of both LPS and LOS results in the formation of micelles in aqueous environment above their critical micellar concentration (CMC) (Scheme 1, upper).[9] CMC values between 10−8 M and 10−7 M for deep rough mutant LPS Re, [10,11] and between 1.3 and 1.6 μM for E. coli LPS, [12] were reported. In balanced salts solutions containing physiologic extracellular concentrations of Mg2+ and Ca2+, CMC values of 1 nM or lower are likely.[13,14] From these data and from the fact that LPS aggregates are usually highly stable, aggregated forms of LPS should predominate in the concentration range relevant for biological responses. In physiological fluids, LPS aggregates were also found as membrane “blebs”, which are constitutively released from growing Gram-negative bacteria.[15] Transmission electron microscopy revealed that blebs exist predominantly as vesicles with an average size of 40–80 nm.[15] The current view of mammalian LPS sensing and signaling is that it is initiated by the LBP-catalyzed extraction and transfer of an LPS monomer from aggregates[16] to CD14[17] and subsequent transfer of the LPS monomer from CD14 to MD-2 and to the MD-2-TLR4 heterodimer.[7] While monomeric E-CD14 and E-MD-2 complexes are therefore the proximal vehicles for activation of MD-2-TLR4 and TLR4, respectively, by E, preceding interactions of host E-binding proteins that preferentially interact with E-rich interfaces and either promote (LBP) or preclude (BPI) transfer of E to CD14 play key roles in determining the potency of TLR4 activation by E.[18] Accordingly, variables in the aggregation state and the 3D form of E aggregates may directly influence the kinetics and potency of TLR4 activation and signaling.[9]