Variable Antibody-dependent Activation of Complement by Functionalized Phospholipid Nanoparticle Surfaces

Lynne M. Mitchell,Gregory M. Lanza,Christopher M. Lubniewski,Otto F. Schall,Dipanjan Pan,Jennifer L. Huang,J. Kendall Killgore,Christine T.N. Pham,Dennis E. Hourcade,Samuel A. Wickline

doi:10.1074/jbc.m110.180760

Lynne M. Mitchell, Gregory M. Lanza

Open Access

https://doi.org/10.1074/jbc.m110.180760

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Abstract

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.

Highlights

Lipid-encapsulated particles such as liposomes, micelles, and emulsions represent classes of NPs of varying sizes that have been introduced into the clinical setting
The CP is activated by antigen-antibody complexes and certain molecular patterns, the lectin pathway gadolinium DOTA-NH3-caproyl-O-methyl-phosphatidylethanolamine; GdDOTA-DAG, gadolinium DOTA-amide-caproyl-diacylglycerol; EA, IgMsensitized sheep erythrocytes; NHS, normal human serum; C, complement; fB, factor B; fH, factor H; fI, factor I
Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift C activation that is initiated by antibody, propagated via the classical pathway, and is independent of charge effects

Summary

EXPERIMENTAL PROCEDURES

Nanoparticle Synthesis—The paramagnetic emulsions were comprised of 20% (v/v) perfluoro-octylbromide, 2.0% (w/v) of a surfactant co-mixture, and 1.7% (w/v) glycerin in distilled, deionized water. CH50 Hemolysis Assay—To determine residual C activity [20], NPs (10% v/v) were incubated in 20% human serum in DGVB2ϩ buffer (150 ␮l total) for 5 min at 37 °C. Resulting supernatants were mixed with DGVB2ϩ buffer to a total of 800 ␮l, and titration curves were constructed from a series of reactions, each composed of diluted supernatant (150 ␮l) plus 5 ϫ 107 (100 ␮l) of IgM-sensitized sheep erythrocytes (EA, CompTech). A modified CH50 hemolysis assay was used to measure residual C activity of serum collected from NP-treated and control mice [21]. In this assay, highly sensitized sheep erythrocytes were prepared by adsorbing additional IgG to commercially prepared EAs (CompTech). 1.25 ϫ 109 EA cells were resuspended in 1.25 ml DGVB2ϩ buffer and rabbit anti-sheep erythrocyte polyclonal IgG diluted 1:30 in 1.25 ml

Nanoparticle surfactant components and physical data

Findings

DISCUSSION