(T1030-01-08) Conjugation Chemistry Used to Construct Protein-Coated Nanoparticles Determines Complement Activation and Associated Efficacy and Toxicity

Purpose: Drug delivery systems are typically constructed by chemically conjugating together multiple therapeutic molecules (e.g., antibody-drug conjugates/ADCs). Many conjugation chemistries, including the recent Nobel Prize-winning ‘click chemistry’, are used to produce these delivery systems. For ADCs, it is well-known that the choice of conjugation chemistry can have a profound impact on the efficacy and safety of the final product. But for newer delivery systems, such as protein-coated nanoparticles, the effect of conjugation chemistry is not well understood. Here we show that the choice of conjugation chemistry primarily affects the complement opsonization of nanoparticles. Complement activation subsequently has dramatic impacts on both safety (via release of anaphylatoxins) and efficacy (via increased clearance by phagocytes) of the final nanoparticle product.

Methods: Non-specific IgG was conjugated to the surface of liposomes using two of the most common conjugation chemistries: DBCO-azide and SATA-maleimide. Liposomes were radiolabeled with I-125 and injected into mice. Radioactivity in each organ was measured via gamma counter.
IgG modified with SATA or DBCO were analyzed for aggregation via HPLC-SEC.
Fluorescent molecules (AF488) and quenchers (TQ2) were conjugated to IgG via NHS-ester reaction. The IgG were then conjugated to the surface of liposomes via DBCO-azide or SATA-maleimide The fluorescence intensity of the liposomes was measured via a fluorometer, with decreased fluorescence meaning that some fluorescence was quenched, and therefore the IgG were located in close proximity on the liposome surface.
Finally, bovine serum albumin (BSA) was fluorescently labeled with AF488 via NHS-ester reaction. Liposome with varying reactive groups (azide, maleimide, or non-reactive lipid) were incubated with AF488-albumin at 37C for 30 minutes. The fraction of liposomes attached to fluorescent albumin was analyzed via NTA (Nanoparticle Tracking Analysis)

Results: IgG-coated liposomes accumulated in the lung, likely due to uptake by marginated neutrophils. The extent of lung uptake varied for different conjugation chemistries (DBCO-azide and SATA-maleimide). Lung uptake was dramatically decreased in C3-knock out mice, and was the same across all conjugation chemistries. This suggests that conjugation chemistries vary in the extent they cause complement activation.
Azide-alkyne chemistry (DBCO-azide) causes aggregation of protein on the nanoparticle surface, as measured by fluorescence quenching. This is likely due to the hydrophobic alkyne functional group. For thiol-maleimide chemistry, maleimide reacts non-specifically with albumin and other plasma proteins, leading to a dense protein-coated surface. In both cases, the dense protein structure and hydrophobic interactions increases the rate of complement opsonization.

Conclusion: The conjugation chemistry used to construct protein-coated nanoparticles dramatically effects the complement opsonization of the nanoparticle. The mechanism of complement activation is different for two of the most common conjugation chemistries. DBCO-azide causes protein aggregation on the surface of the liposome, which leads to complement opsonization. Maleimide functional groups on the liposome reacts non-specifically with proteins in plasma, especially albumin. This leads to a dense protein coating on the surface of the liposome, which increases complement opsonization. This new understanding of the effect of conjugation chemistry on complement activation will enable engineering of new nanoparticles with superior safety and efficacy.


Figure 1. Lung uptake of IgG-liposomes is determined by conjugation chemistry, and depends on complement opsonization.
A) Schematic of DBCO-azide and SATA-maleimide conjugation chemistries. B) Biodistribution of radiolabeled IgG-liposome in mice with systemic inflammation shows significant uptake in lungs. Lung accumulation depends on conjugation chemistry C) Lung uptake is completely absent in complement (C3) knockout mice.


Figure 2. Mechanisms of DBCO-azide induction of complement opsonization
A) Proposed mechanism - DBCO modification causes protein aggregation on liposome surface B) analysis of modified IgG by HPLC-SEC shows little to no protein aggregation due to DBCO modification. C) Schematic of fluorescence quenching method to measure protein aggregation on liposome surface D) Quenching assay shows decreased fluorescence (and therefore protein aggregation) for DBCO-azide chemistry and not SATA-maleimide chemistry.


Figure 3. Mechanisms of SATA-maleimide induction of complement opsonization
A) Proposed mechanism - maleimide reacts with albumin and other proteins in plasma, leading to dense protein surface.
B) Liposome with maleimide functional groups conjugate with fluorescent albumin. PEGylated liposomes with no functional groups or azide functional group do not react with albumin. C) Extend of albumin conjugation increases with increased mol% of maleimide on the liposome and increased incubation time, D) Complement activation, as measured by C3a concentration, increases with increased mol% maleimide on the liposome.