Nanometer- and angstrom-scale characteristics that modulate complement responses to nanoparticles

Abstract

The contribution of the complement system to non-specific host defence and maintenance of homeostasis is well appreciated. Many particulate systems trigger complement activation but the underlying mechanisms are still poorly understood. Activation of the complement cascade could lead to particle opsonisation by the cleavage products of the third complement protein and might promote inflammatory reactions. Antibody binding in a controlled manner and/or sensing of particles by the complement pattern-recognition molecules such as C1q and mannose-binding lectin can trigger complement activation. Particle curvature and spacing arrangement/periodicity of surface functional groups/ligands are two important parameters that modulate complement responses through multivalent engagement with and conformational regulation of surface-bound antibodies and complement pattern-recognition molecules. Thus, a better fundamental understanding of nanometer- and angstrom-scale parameters that modulate particle interaction with antibodies and complement proteins could portend new possibilities for engineering of particulate drug carriers and biomedical platforms with tuneable complement responses and is discussed here.

Keywords: Antibodies; C1q; Complement system; Dendrimers; Factor H; Mannose-binding lectin; Nanoparticles.

Fig. 1.

Complement activation pathways. The scheme shows sequential enzymatic steps in classical, lectin and alternative complement activation pathways, key complement regulators (e.g., C1-inhibitor, Factor H, Factor I) and selected complement activation products (C4a, C4b, C4d, C3a, C3b, iC3b, C3bc, Bb, C5a, sC5b-9) that can be monitored by different methodologies. Antibodies such as IgM, IgG and IgA, depending on their isotype and conformation, can trigger complement activation through the three pathways. C-reactive protein (CRP) on binding to nanoparticles might also trigger complement activation through the C1 complex arm of the classical pathway. The binding of complement pattern recognition molecules C1q and collectins (MBL, ficolins and collectin-11) to surfaces triggers activation of their associated proenzymes (C1r and C1s in the case of C1q; MASPs in the case of collectins), resulting in complement activation through classical and lectin pathways, respectively. Alternative pathway turnover occurs through autoactivation of soluble C3 that undergoes slow spontaneous hydrolysis [C3(H₂O)], or when nascent C3b undergoes nucleophilic attack; for instance, by hydroxyl or amino groups on nanopartiCle surfaces. The first critical stage in complement cascade is the formation of C3 Convertases (C4bC2a and C3bBb) that cleave C3. This results in the liberation of anaphylatoxin C3a and the opsonic fragments C3b and iC3b. The next step is the assembly of C5 Convertases (C4bC2aC3b and C3bBbC3b), which cleaves C5, resulting in liberation of another anaphylatoxin (C5a) and activation of the terminal pathway of complement. On full complement activation the membrane attack complex C5b-9 is formed. In soluble form, C5b-9 is bound to vitronectin (sC5b-9). The figure and the legend are reproduced with permission from [21] with a slight modification.

Fig. 2.

Schematic representation of factor H showing the locations of the complement and polyanion binding sites. Factor H is composed of 20 domains. Factor H also regulates complement activation by possessing both cofactor activity (CoA) for the factor I-mediated C3b cleavage and decay accelerating activity (DAC) against the alternative pathway C3 convertase through domains 1–4. § = glycosylated region; CR = complement receptor.

Fig. 3.

Role of immunoglobulins in efficiency of C3 deposition on Feraheme^®. (a) Schematic representation of Feraheme structure. CM = Carboxymethyl. (b) Effect of immunoglobulin depletion and reconstitution in plasma of three donors (F28, M61, M56). (c) Effect of immunoglobulin depletion in sera of healthy donors measured with dot blot assay. Each dot represents the mean of three technical replicates per sample. (d) Correlation between levels of immunoglobulins and C3 bound to Feraheme in healthy sera of 12 subjects. (e) Ferahame was incubated in sera with EGTA/Mg²⁺ or EDTA. In (c) and (e) each colour refers to the same individual. Each dot represents the mean of three replicates per sample. (f) Immunoglobulin depletion and reconstitution in lepirudin-anticoagulated plasma from eight breast cancer patients. Each dot represents the mean of three technical replicates per sample. The figure and the legend are reproduced from [30] with permission.

Fig. 4.

Schematic representation of C1q and mannose-binding lectin (MBL). C1q drawing is reproduced with permission from [2].

Fig. 5.

Schematic representation of PAMAM dendrimers and complement activation by dendrimeric platforms. (a–c) Generation 5 PAMAM dendrimers with amine, pyrrolidone and carboxylic acid-Tris end-terminal functionalities. (d) Transmission electron micrograph of generation 4 pyrrolidone-terminated PAMAM dendrimer complexes with phthalocyanine (Pc). Pc is covalently attached to dendrimers. (e & f) Complement responses to pyrrolidone-terminated dendrimer-Pc complexes (Pc-G4 Pyr) (3.5 mgmL⁻¹) in a lepirudin-anticoagulated human plasma through measurements of fluid-phase C5a (e) and sC5b-9 (f). A commercially available sulfated polystyrene nanoparticle suspension (PS) of 60 nm was used as positive control for complement activation (PS concentration = 3.5 mgmL⁻¹). The figure and the legend are reproduced with permission from [55] with a slight modification.

Fig. 6.

Complement responses towards lyotropic non-lamellar liquid crystalline (LLC) nanodispersions. (a) Structures of glyceryl monooleate, citrem and sialic acid. Circles in sialic acid structure denote factor H binding regions. (b) Cryogenic transmission electron micrographs of LLC nanodispersions from a binary lipid mixture of glyceryl monooleate and medium-chain triglycerides, which have been stabilised either with Pluronic F127 (3 wt%) or citrem (1.5 or 3 wt%). Images show nanodispersions suspended in both buffer and human serum. Inset = the fast Fourier transform analysis of particle interiors. Scale bar = 100 nm. (c) Schematic illustration for the effects of citrem and serum on the nanostructural features of the LLC nanodispersions with representative cryogenic electron micrograph enlargements. (d & e) Complement activation by Pluronic F127-stabilised (LD_F127) and 3 wt% citrem-stabilised (LD_citrem3.0) LLC nanodisprsions measured as an elevation of the two end-point complement markers C5a (d) and sC5b-9 (e) in human serum. Zymosan (0.2 mgmL⁻¹) was used as positive control for complement activation. Complement activation is exclusively through calcium-sensitive pathways. The figure and the legend are reproduced with permission from [67].