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. 2018 Mar 27;12(3):2138-2150.
doi: 10.1021/acsnano.7b06995. Epub 2018 Jan 18.

Directing Nanoparticle Biodistribution Through Evasion and Exploitation of Stab2-Dependent Nanoparticle Uptake

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Free PMC article

Directing Nanoparticle Biodistribution Through Evasion and Exploitation of Stab2-Dependent Nanoparticle Uptake

Frederick Campbell et al. ACS Nano. .
Free PMC article

Abstract

Up to 99% of systemically administered nanoparticles are cleared through the liver. Within the liver, most nanoparticles are thought to be sequestered by macrophages (Kupffer cells), although significant nanoparticle interactions with other hepatic cells have also been observed. To achieve effective cell-specific targeting of drugs through nanoparticle encapsulation, improved mechanistic understanding of nanoparticle-liver interactions is required. Here, we show the caudal vein of the embryonic zebrafish ( Danio rerio) can be used as a model for assessing nanoparticle interactions with mammalian liver sinusoidal (or scavenger) endothelial cells (SECs) and macrophages. We observe that anionic nanoparticles are primarily taken up by SECs and identify an essential requirement for the scavenger receptor, stabilin-2 ( stab2) in this process. Importantly, nanoparticle-SEC interactions can be blocked by dextran sulfate, a competitive inhibitor of stab2 and other scavenger receptors. Finally, we exploit nanoparticle-SEC interactions to demonstrate targeted intracellular drug delivery resulting in the selective deletion of a single blood vessel in the zebrafish embryo. Together, we propose stab2 inhibition or targeting as a general approach for modifying nanoparticle-liver interactions of a wide range of nanomedicines.

Keywords: endothelial cells; liposomes; nanomedicine; scavenger receptor; stabilin; targeted drug delivery; zebrafish.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
A zebrafish model for liposome biodistribution. (a) Schematic of liposome injection and quantification in zebrafish. Fluorescently labeled liposomes (1 mM total lipids containing 1 mol % Rhod-PE) were injected into the duct of Cuvier at 54 hpf. Confocal microscopy is performed in a defined region (boxed) caudal to the yolk extension at 1, 8, 24, and 48 h after injection. (b) Whole-embryo view of liposome distribution in kdrl:GFP transgenic embryos, 1 hpi with three different liposome formulations (AmBisome, EndoTAG-1, and Myocet). (c) High-resolution imaging allows quantification of liposomes in circulation (measured in the lumen of the dorsal aorta (white box)) and liposome association with different blood vessel types (see Supporting Information). CHT-EC: caudal hematopoietic tissue endothelial cells, DLAV: dorsal longitudinal anastomotic vessel. ISV: intersegmental vessel. (d) Tissue level view of liposome distribution in kdrl:gfp transgenic embryos, 1 h and 8 h after injection with three different liposome formulations and a single confocal section through the dorsal aorta (DA) at 1 h after injection. (e) Quantification of liposome levels in circulation based on mean rhodamine fluorescence intensity in the lumen of the dorsal aorta at 1, 8, 24, and 48 h after injection (error bars: standard deviation.) n = 6 individually injected embryos per formulation per time point (in two experiments). (f) Quantification of liposome levels associated with venous vs arterial endothelial cells based on rhodamine fluorescence intensity associated with caudal vein (CV) vs DA at 8 h after injection. (g) Quantification of extravascular liposome levels based on rhodamine fluorescence intensity outside of the vasculature between the DLAV and DA at 8 h after injection. (h) Quantification of liposome levels associated with the vessel wall based on rhodamine fluorescence intensity associated with all endothelial cells relative to rhodamine fluorescence intensity in circulation at 1h after injection. (f–h) Bar height represents median values, dots represent individual data points, brackets indicate significantly different values (*: p < 0.05, **: p < 0.01, ***: p < 0.001) based on Kruskal–Wallis and Dunn’s tests with Bonferroni correction for multiple testing. n = 12 individually injected embryos per group (in 2 experiments). (i) Whole-embryo view of liposome distribution in kdrl:GFP transgenic embryos, 1 h after injection with DOPG and DSPC liposomes. Liposome accumulation for both formulations is observed in the primitive head sinus (PHS), common cardinal vein (CCV), posterior cardinal vein (PCV), and caudal vein (CV). (j) Tissue level view of liposome distribution in kdrl:GFP transgenic embryos, 1 h after injection with DOPG and DSPC liposomes at 102 hpf. Liposome accumulation is observed in the entire caudal vein (CV), but only on the dorsal side of the PCV (dPCV, arrows).
Figure 2
Figure 2
Identification of scavenger endothelial cells (SECs) in zebrafish embryos. (a, b) Ex vivo imaging of adult Tie2:GFP transgenic mouse organs, 1 h after injection with DOPG liposomes. (a) Liposome accumulation is observed in liver, but not in the ear skin or heart muscle. (b) Within the liver, DOPG liposomes are observed as punctae within Tie2:GFP+ sinusoidal ECs (arrows) as well as sinusoid-associated cells which based on shape and position were identified as KCs (arrowheads). (c) Tissue level view of lithium carmine distribution in kdrl:GFP and mpeg:GFP transgenic zebrafish embryos, 1 h after injection. Lithium carmine (carminic acid) fluorescence co-localizes both with kdrl:GFP+ endothelial cells in the caudal vein and mpeg:GFP+ monocytes/macrophages (arrowheads) within the CHT. (d) Whole-embryo view of fluorescent oxLDL distribution in kdrl:GFP transgenic embryos, 1 h after injection. Accumulation of oxLDL is observed in the PHS, CCV, PCV, and CV. (e) Whole-embryo view of fluoHA distribution in kdrl:RFP transgenic embryos, 1 h after injection. Accumulation of fluoHA is observed in the PHS, CCV, PCV, and CV. (f) Tissue level view of fluoHA distribution in kdrl:RFP transgenic embryos, 1 h after injection at 102 hpf. FluoHA accumulation is observed in the entire caudal vein (CV), but only on the dorsal side of the PCV (dPCV, arrows). (g) Tissue level view of fluoHA in kdrl:RFP and mpeg:RFP transgenic embryos. Co-localization of RFP expression and fluoHA is observed only within kdrl:RFP endothelial cells, but not mpeg:RFP monocytes/macrophages. (h) Tissue level view of co-injected fluoHA and DOPG liposomes, 1 h after injection reveals co-localization in SECs. Monocytes/macrophages (arrowheads) take up DOPG but not fluoHA. (i) Ex vivo imaging of adult mouse liver, 1 h after injection with fluoHA and DOPG liposomes reveals widespread co-localization within sinusoidal ECs (arrows). KCs (arrowheads) take up DOPG liposomes only.
Figure 3
Figure 3
stab2 is required for anionic liposome uptake by SECs. (a, b) Tissue level view of DOPG (a) and DSPC (b) liposome distribution at 1 hpi in control and dextran sulfate injected embryos, with quantification of liposome levels associated with venous vs arterial endothelial cells based on rhodamine fluorescence intensity associated with CV vs DA. (c) stab2 domain structure predicted to be expressed from the wild-type stab2 and the stab2ibl2 allele. (d) Whole-embryo view of flt1:RFP, flt4:YFP double transgenic embryos at 5 dpf to visualize blood vascular and lymphatic development. No defects were identified during (lymph)angiogenesis and vascular patterning in stab2ibl2 homozygous embryos compared to sibling controls. (e) Fertile adult females (stab2ibl2 homozygous and sibling controls) at 3 months post-fertilization. (f–k) Tissue level view of fluoHA (f) and DOPG (g), DSPC (h), AmBisome (i), EndoTAG-1 (j), and Myocet (k) liposome distribution at 1 hpi in stab2ibl2 and sibling control embryos, with quantification of liposome levels associated with venous vs arterial endothelial cells based on rhodamine fluorescence intensity associated with CV vs DA. (a, b, f–k) Bar height represents median values, dots represent individual data points, and brackets indicate significantly different values (*: p < 0.05, **: p < 0.01, ***: p < 0.001, N.S.: not significant) based on Mann–Whitney test. n = 6–10 per group (in two experiments).
Figure 4
Figure 4
stab2-mediated scavenging of anionic nanoparticles in vivo. (a–i) Tissue level view of DOPS liposome (a, b), PIB-PEG polymersome (c, d), carboxylated polystyrene nanoparticle (e, f), CCMV virus-like particle (g, h), and carboxylated quantum dot (i, j) distribution at 1 hpi in stab2ibl2 and sibling control embryos (a, c, e, g, i) or control and dextran sulfate injected embryos (b, d, f, h, j). Quantification of nanoparticle levels associated with venous vs arterial endothelial cells based on rhodamine fluorescence intensity associated with caudal vein vs DA. (a–j) Bar height represents median values, dots represent individual data points, and brackets indicate significantly different values (*: p < 0.05, **: p < 0.01, ***: p < 0.001, N.S.: not significant) based on Mann–Whitney test. n = 5–12 per group (in two experiments).
Figure 5
Figure 5
Nanoparticle-mediated SEC deletion. (a) Whole-embryo and tissue level views at 48 hpi of the blood vasculature in kdrl:GFP transgenic control embryos, embryos injected with 1 mg/mL clodronic acid, or embryos injected with liposomes containing 1 mg/mL clodronic acid (DSPC or DOPC liposomes). Complete deletion of the caudal vein is observed in embryos injected with DSPC liposomes containing clodronic acid (brackets and asterisks). (b) Schematic representation of blood flow in control embryos or embryos injected with DSPC liposomes containing 1 mg/mL clodronic acid. Blue indicates venous or capillary blood vessels, and red indicates arterial blood vessels. Arrowheads indicate direction of blood flow (based on observations from Movie S1). The removal of the CV (dashed lines) leads to a rerouting of blood flow through the DLAV. (c) Quantification of PCV length in injected embryos. Bar height represents median values, dots represent individual data points, and brackets indicate significant values (**: p < 0.01, ***: p < 0.001) based on Kruskal–Wallis and Dunn’s tests with Bonferroni correction for multiple testing. n = 6 individually injected embryos per group (in two experiments). (d) Progression of SEC deletion. Individual frames from Movie S2 at indicated time points after injection of DSPC liposomes containing 1 mg/mL clodronic acid, injected into kdrl:GFP transgenic embryos. SEC fragmentation in this case is observed mostly between 12 hpi and 16 hpi, followed by a gradual loss of fluorescence or removal of cellular debris. (e) Tissue level view of distribution of DSPC liposomes containing 1 mg/mL clodronic acid at 1 hpi in stab2ibl2 and sibling control embryos. (f) Whole-embryo and tissue level views at 48 hpi of the blood vasculature in kdrl:GFP transgenic stab2ibl2 and sibling embryos. Embryos were injected with DSPC liposomes containing 1 mg/mL clodronic acid. Complete deletion of the caudal vein is observed in sibling control (brackets and asterisks), but not stab2ibl2 mutant embryos. (g) Schematic representation of blood flow in sibling control embryos or stab2ibl2 homozygous mutants, both injected with DSPC liposomes containing approximately 1 mg/mL clodronic acid. Blue indicates venous or capillary blood vessels, and red indicates arterial blood vessels. Arrowheads indicate direction of blood flow (based on observations from Movie S3). The removal of the CV (dashed lines) leads to a rerouting of blood flow through the DLAV in control embryos but not in stab2ibl2 homozygous mutants. (h) Quantification of PCV length in injected embryos. Bar height represents median values, dots represent individual data points, and brackets indicate significant values (***: p < 0.001) based on Mann–Whitney test.

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