Abstract
Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation.
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Data availability
Uncropped gels and dot blots for Supplementary Figs. 1, 14 and 16 are provided in the Supplementary Information. Additional raw data are available from the corresponding author upon request. Source data are provided with this paper.
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Acknowledgements
The study was supported by the NIH grants R01 CA257958 and R01 AI154959 to D.S., and R01 AR051749 to V.M.H. (principal investigator) and N.K.B. (co-investigator).
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Y.L., S.J., R.I.S., S.M.M. and D.S. designed the experiments. Y.L., S.J., G.W., H.G. and D.S. performed the experiments. N.K.B., V.M.H. and S.T. provided the critical materials and reagents and valuable advice. R.I.S., D.S. and S.M.M. analysed the data. D.S. wrote the manuscript. All authors discussed the results and assisted in the preparation of the manuscript.
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Extended data
Extended Data Fig. 1 IC50 of targeted regulators in different human donors.
a, Human donors with different efficiencies of C3 opsonization of large SPIO NWs. Labels show sex and age of each donor. b, IC50 is in high picomolar to low nanomolar range for all donors regardless of the complement activation.
Extended Data Fig. 2 IC50 of targeted regulators in different species.
a, Mouse ortholog CR2-Crry shows mid-nanomolar IC50 in BALB/c strain sera. b, Human CR2-CR1 shows low nanomolar inhibition in sera from 3 rat strains, and is more potent than human CR1. Each experiment was repeated twice.
Extended Data Fig. 3 CR2-CR1 labels a subpopulation of nanoparticles in rats in vivo.
a, Sprague Dawley rats were i.v. injected with 1.5 mg/kg of large Cy5-CLIO NWs together with 0.3 mg/kg AF488-CR2-CR1. Flow cytometry analysis of NWs recovered 5 min post injection shows ~1% of particles positive for CR2-CR1. b, Rats were injected i.v. with NWs only. Flow cytometry analysis of NWs recovered after 5 min and stained for C3 shows ~10% of NWs positive for C3. Repeated twice. c, Confocal images of recovered NWs that were post-stained for C3. Both CD2-CR1 and C3 proteins colocalize on nanoparticles. Representative images from 2 independent experiments. Size bar = 2 µm.
Extended Data Fig. 4 CR2-CR1 labels a subpopulation of liposomes recovered from a patient infused with LipoDox.
PEGylated liposomal doxorubicin (LipoDox) nanoparticles were recovered from a cancer patient’s plasma 30 min after the start of infusion and stained with: a, FITC-anti human C3. b, AF488-CR2-CR1. Only a few percent of liposomal nanoparticles are positive. Notably, C3-positive and CR2-CR1-positive liposomes often appear to be larger than non-opsonized liposomes (arrows). Representative images are shown. The experiment was repeated in 2 patients. Size bar = 1 µm.
Supplementary information
Supplementary Information
Supplementary Figs. 1–17, Discussion and uncropped gels and dot blots for Supplementary Figs. 1, 14 and 16.
Supplementary Video 1
Trimmed representative clip showing mobility of saline-injected rat.
Supplementary Video 2
Trimmed representative clip showing mobility of NW-injected rat.
Supplementary Video 3
Trimmed representative clip showing mobility of NW + CR2-CR1-injected rat.
Source data
Source Data Fig. 1
Uncropped gel.
Source Data Fig. 2
Uncropped dot blot.
Source Data Fig. 3
Uncropped dot blot.
Source Data Fig. 4
Uncropped dot blot.
Source Data Fig. 5
Uncropped dot blot.
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Li, Y., Jacques, S., Gaikwad, H. et al. Inhibition of acute complement responses towards bolus-injected nanoparticles using targeted short-circulating regulatory proteins. Nat. Nanotechnol. 19, 246–254 (2024). https://doi.org/10.1038/s41565-023-01514-z
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DOI: https://doi.org/10.1038/s41565-023-01514-z
