Unbiased discovery of in vivo imaging probes through in vitro profiling of nanoparticle libraries

Abstract

In vivo imaging reveals how proteins and cells function as part of complex regulatory networks in intact organisms, and thereby contributes to a systems-level understanding of biological processes. However, the development of novel in vivo imaging probes remains challenging. Most probes are directed against a limited number of pre-specified protein targets; cell-based screens for imaging probes have shown promise, but raise concerns over whether in vitro surrogate cell models recapitulate in vivo phenotypes. Here, we rapidly profile the in vitro binding of nanoparticle imaging probes in multiple samples of defined target vs. background cell types, using primary cell isolates. This approach selects for nanoparticles that show desired targeting effects across all tested members of a class of cells, and decreases the likelihood that an idiosyncratic cell line will unduly skew screening results. To adjust for multiple hypothesis testing, we use permutation methods to identify nanoparticles that best differentiate between the target and background cell classes. (This approach is conceptually analogous to one used for high-dimensionality datasets of genome-wide gene expression, e.g. to identify gene expression signatures that discriminate subclasses of cancer.) We apply this approach to the identification of nanoparticle imaging probes that bind endothelial cells, and validate our in vitro findings in human arterial samples, and by in vivo intravital microscopy in mice. Overall, this work presents a generalizable approach to the unbiased discovery of in vivo imaging probes, and may guide the further development of novel endothelial imaging probes.

Figure 1

Binding of small molecule-modified nanoparticles to macrophages (heatmap columns demarcated by orange bar) and endothelial cells (columns demarcated by green bar). The log₁₀ of bound nanoparticle concentrations (in pM) are plotted in each cell of the heatmap. Structures of four highly ranked small molecule-modified nanoparticles, along with their asymptotic permutation p-values, are shown at right. Act MP and R MP = activated and resting macrophages, respectively. Ao = aorta, IA = iliac artery, MV = microvascular, PA = pulmonary artery, SV = saphenous vein, UV = umbilical vein. FITC-CLIO = CLIO-NH₂-FITC.

Figure 2

Relative nanoparticle binding to endothelial cells (left) or macrophages (right) for conjugates 16–3 and 16–6 (relative to binding of the starting material CLIO-NH₂-FITC (CLIO)). The concentration of 16–3 and 16–6 bound to each cell type has been normalized by dividing by the concentration of CLIO-NH₂-FITC that bound to the same cell type; binding of CLIO-NH₂-FITC has been normalized to 1 in each graph. Cell line abbreviations are the same as in Figure 1

Figure 3

Nanoparticle localization in human carotid endarterectomy samples. The leftmost column shows nanoparticle localization (NP) via staining with antibodies against the FITC moiety on nanoparticles (additional 2x magnification views are shown in inserts); the middle column shows staining against CD31 (part A) or CD68 (part B); and the rightmost column shows a merged image that also includes DAPI nuclear staining. A. 16–6 and 16–3 show markedly increased (relative to CLIO-NH₂-FITC) immunofluorescence that co-localizes with CD31 signal, consistent with endothelial localization. B. CLIO-NH₂-FITC, 16–6 and 16–3 all show comparable uptake into macrophages that co-localizes with CD68 staining. L = lumen of carotid artery.

Figure 3

Nanoparticle localization in human carotid endarterectomy samples. The leftmost column shows nanoparticle localization (NP) via staining with antibodies against the FITC moiety on nanoparticles (additional 2x magnification views are shown in inserts); the middle column shows staining against CD31 (part A) or CD68 (part B); and the rightmost column shows a merged image that also includes DAPI nuclear staining. A. 16–6 and 16–3 show markedly increased (relative to CLIO-NH₂-FITC) immunofluorescence that co-localizes with CD31 signal, consistent with endothelial localization. B. CLIO-NH₂-FITC, 16–6 and 16–3 all show comparable uptake into macrophages that co-localizes with CD68 staining. L = lumen of carotid artery.

Figure 4

In vivo intravital microscopy of 16-6 and CLIO-NH₂-FITC in mouse ear vessels following intravenous injection. A. (Left) Near-infrared channel: Angiosense-IVM 750 (red) circulates in the vascular space, and defines vessels; (Right) Merge of FITC channel (probe 16–6 localization; green) and near-infrared channel (Angiosense-IVM 750 intravascular reference; red). White arrowheads indicate regions of 16–6 localization immediately adjacent to the vessel lumen consistent with endothelial localization. B. Same as part A, but with unmodified CLIO-NH₂-FITC as the injected probe, showing that CLIO-NH2-FITC does not accumulate adjacent to vessels. CLIO = CLIO-NH₂-FITC. Scale bar = 20 microns.

Scheme 1

Conjugation of FITC and anhydrides to nanoparticle surfaces consisting of cross-linked, aminated dextran