Comparative analysis of metallic nanoparticles as exogenous soft tissue contrast for live in vivo micro-computed tomography imaging of avian embryonic morphogenesis

Abstract

Background: Gestationally survivable congenital malformations arise during mid-late stages of development that are inaccessible in vivo with traditional optical imaging for assessing long-term abnormal patterning. MicroCT is an attractive technology to rapidly and inexpensively generate quantitative three-dimensional (3D) datasets but requires exogenous contrast media. Here we establish dose-dependent toxicity, persistence, and biodistribution of three different metallic nanoparticles in day 4 chick embryos.

Results: We determined that 110-nm alkaline earth metal particles were nontoxic and persisted in the chick embryo for up to 24 hr postinjection with contrast enhancement levels at high as 1,600 Hounsfield units (HU). The 15-nm gold nanoparticles persisted with x-ray attenuation higher than that of the surrounding yolk and albumen for up to 8 hr postinjection, while 1.9-nm particles resulted in lethality by 8 hr. We identified spatial and temporally heterogeneous contrast enhancement ranging from 250 to 1,600 HU. With the most optimal 110-nm alkaline earth metal particles, we quantified an exponential increase in the tissue perfusion vs. distance from the dorsal aorta into the flank over 8 hr with a peak perfusion rate of 0.7 μm(2) /s measured at a distance of 0.3 mm.

Conclusions: These results demonstrate the safety, efficacy, and opportunity of nanoparticle based contrast media in live embryos for quantitative analysis of embryogenesis. Developmental Dynamics 245:1001-1010, 2016. © 2016 Wiley Periodicals, Inc.

Keywords: biodistribution; development; dorsal aorta; embryo; nanoparticles; perfusion; toxicity.

Figure 1

(A) Acute heart rate analysis of day 3 chicks following treatment with metallic nanoparticle contrast media injected at 10% blood volume with EBSS serving as a negative control. A significant heart rate decline was observed immediately post injection (0+) across all contrast medias and EBSS controls (P<0.05) but heart rates returned to their initial values within 1 hour post injection (n = 10, 10, 4). (B) Anatomical analysis one week post exposure to metallic nanoparticle contrast media (day 10 embryos) reveals no morphological defects for either the 15nm AuNP or the 110nm EXTN particles when compared to EBSS controls (P<0.05, n = 9, 9, 4). (C) Modeled particle contrast degradation versus to the amount of metal (mg) present in the solution determined through a dilution series ranging from 100% particle solution to 0% (1X PBS) with inset representative media diluted at 12.5% concentration from the stock solution (n=3).

Figure 2

(A) Comparison of peak contrast enhancement between three different metallic nanoparticle contrast media microinjected into day 4 chick embryos at 10% blood volume. Contrast enhancement was measured within the heart, dorsal aorta, head, limb, and allantoic sac (n = 3). Contrast was significantly higher in embryos treated with 110nm EXTN particles (P<0.05) for the heart, dorsal aorta, and limb. (B) Representative 2D grayscale cross section images through the sagittal plane labeled with developing organ systems of day 4 chick embryos. 110nm EXTN particles demonstrate higher x-ray attenuation and soft tissue contrast enhancement as compared to the gold nanoparticles. Furthermore, a size dependent extravasation is suggested due to larger particles retained in the vasculature whereas the smallest 1.9nm AuNP particles have extravasated into the surrounding tissue. H = heart, He = head, DA = dorsal aorta, L = limb, and A = allantois

Figure 3

Representative 3D renderings of day 4 chick embryos microinjected with different metallic nanoparticle contrast media and imaged at 50um resolution with 800 projection scans. (A) Maximum intensity projections (MIP) of day 4 chicks calibrated to a single contrast enhancement color map given in the image denoting localized higher and lower contrast values measured in HU and (B) volumetric renderings of day 4 chicks injected with different contrast media. Supplement: 3D MIP movie

Figure 4

Temporal contrast enhancement comparison (A) Contrast enhancement of the 1.9nm AuNP particles across multiple organ systems. Contrast extravasated quickly from the vasculature into the surround tissue and all samples died within 8 hours post injection. (B) Contrast enhancement of the 15nm AuNP particles over 8 hours post injection. Cardiovascular structures were visualized readily immediately post injection and by 8 hours contrast enhancement was the same across the embryo. Embryos survived for 24 hours post injection but contrast enhancement at 24 hours was not different from the background resulting in zero visualization of the embryo. (C) Contrast enhancement in day 4 embryos treated with 110nm EXTN particles produced the highest levels of contrast with strong attenuation in structures with high vascularity but enhancement across the embryo equalized by 8 hours post injection but tissue boundaries were still readily visualized. At 24 hours post injection, tissue boundaries were not readily seen but the embryonic body was visualized as compared to background.

Figure 5

Direct metallic nanoparticle comparison as compared to Visipaque. Contrast enhancement in each organ system considered for this study was compared across all three particles types presented against the previously published VP. The 110nm EXTN particles outperformed VP particularly in the cardiovascular structures, maintaining significantly high attenuation at 8 hours post injection in the hearts, dorsal aorta, and limb. At 24 hours post injection, the 110nm EXTN particles outperformed the VP demonstrating high levels of contrast enhancement in the embryonic body.

Figure 6

(A) Modeled particle diffusion over an 8 hour period of time from the dorsal aorta, sampled above the vitelline vein. Contrast enhancement was measured at known distances away from the dorsal aorta at 8 hours post injection. Concentration and amount of metal values were derived from standard curves previously established (Figure 1). Fick’s first law of diffusion was used to interpolate the diffusion coefficient as a function of distance from the dorsal aorta 8 hours post injection. (B) representative maximum intensity projections of immediately post injection (0 hours) and 8 hour post injection chick embryos with magnified insets of the dorsal aorta (black dashed line) and surrounding tissue. Color map based on contrast enhancement (HU) given in the legend.