Bridging the In Vitro to In Vivo gap: Using the Chick Embryo Model to Accelerate Nanoparticle Validation and Qualification for In Vivo studies

Abstract

We postulate that nanoparticles (NPs) for use in therapeutic applications have largely not realized their clinical potential due to an overall inability to use in vitro results to predict NP performance in vivo. The avian embryo and associated chorioallantoic membrane (CAM) has emerged as an in vivo preclinical model that bridges the gap between in vitro and in vivo, enabling rapid screening of NP behavior under physiologically relevant conditions and providing a rapid, accessible, economical, and more ethical means of qualifying nanoparticles for in vivo use. The CAM is highly vascularized and mimics the diverging/converging vasculature of the liver, spleen, and lungs that serve as nanoparticle traps. Intravital imaging of fluorescently labeled NPs injected into the CAM vasculature enables immediate assessment and quantification of nano-bio interactions at the individual NP scale in any tissue of interest that is perfused with a microvasculature. In this review, we highlight how utilization of the avian embryo and its CAM as a preclinical model can be used to understand NP stability in blood and tissues, extravasation, biocompatibility, and NP distribution over time, thereby serving to identify a subset of NPs with the requisite stability and performance to introduce into rodent models and enabling the development of structure-property relationships and NP optimization without the sacrifice of large populations of mice or other rodents. We then review how the chicken embryo and CAM model systems have been used to accelerate the development of NP delivery and imaging agents by allowing direct visualization of targeted (active) and nontargeted (passive) NP binding, internalization, and cargo delivery to individual cells (of relevance for the treatment of leukemia and metastatic cancer) and cellular ensembles (e.g., cancer xenografts of interest for treatment or imaging of cancer tumors). We conclude by showcasing emerging techniques for the utilization of the CAM in future nano-bio studies.

Keywords: chick embryo; chorioallantoic membrane; drug development; intravital imaging; nanoparticle bioavailability; nanoparticle development; structure−function analysis.

Figure 1

Developmental timeframe of the chick embryo (CE). Highlighted stages of CE development relevant to their usage in biomedical research and NP formulation assessment. This is not an exhaustive list and contains biological events relevant to possible experimental procedures.⁻ Images are adapted with permission from ref (24). Copyright 2014 Academic Press.

Figure 2

Chicken embryo and chorioallantoic membrane (CAM) models for biomedical research. (A) In ovo chicken embryo model showing an early stage post fertilization. Reprinted with permission from ref (31). Copyright 2020 Elsevier. (B) Ex ovo chicken embryo model with visible CAM overlaying the embryo. Adapted with permission from ref (6). Copyright 2010 Nature Publishing Group. (C) The CAM vascular network visualized by assembly of single photographic frames. Open arrows indicate major venous vessels, and filled arrows indicate major arterial vessels. (D–F) Scanning electron microscope (SEM) images of the CAM vessel network. The CAM is inverted in the SEM preparations to allow clear visualization of the capillary plexus (*) both fed and drained from below by large vessels. Arrowheads signify feeding arterioles into the CAM or departing venules from the CAM plexus into a larger artery or vein, respectively. Reprinted with permission from ref (44). Copyright 2016 The American Physiological Society (APS).

Figure 3

Using intravital microscopy in the CAM to understand nanoparticle behavior. (A, B) Screen captures from a video of 150 nm PEGylated MSNPs in the CAM vasculature over 60 min. The artery is denoted with * and the vein with #. (A) Under 10 min, the MSNPs remain in circulation. (B) By 60 min, the particles have begun to accumulate on the vessel walls in the capillary plexus. (C) MSNPs coated with a lipid bilayer circulating 30 min postinjection. Inset: CryoTEM image of the MSNP with the lipid bilayer highlighted with arrows. Adapted with permission from ref (84). Copyright 2016 American Chemical Society. (D) Human erythrocyte membrane coated MSNPs arrest within the CAM within minutes of injection. Images in (A)–(C) were adapted with permission from ref (222). (E–H) Comparison of the vascular interaction of size and charge-matched MSNPs in the CAM. MSNs coated with PEG-PEI (orange) and PEG-NMe₃⁺ (green) were imaged in the CAM 10 min postinjection. Images in (E)–(H) adapted with permission from ref (86). Copyright 2016 American Chemical Society. (E) Merged image, (F) PEG-PEI-coated MSNs arrest on the vessel walls while (G) PEG-NMe³⁺ coated MSNs remain in circulation. (H) Magnification highlighting (arrow) the arrest of PEG-PEI-coated MSNs on the endothelial cells.

Figure 4

Comparison of plasmid delivery by lipid nanoparticles (LNPa) with different helper lipids. LNPs loaded with a plasmid encoding GFP were directly injected into the forelimb bud of chicken embryos. The efficacy of delivery of LNPs formulated with the helper lipids DOPE (A, E), DOPC (B, F), DSPC (C, G), and SOPC (D, H) were compared using fluorescence microscopy of the intact embryo. The DOPE-based LNPs were the most effective, and imaging thin sections of the embryo forelimb (I) showed delivery throughout the forelimb and vascular infiltration leading to delivery to the heart (J). Reprinted with permission from ref (106). Copyright 2017 Elsevier.

Figure 5

CAM blood vessel response to liposomal mTHPC photodynamic therapy. Liposomal mTHPC was injected i.v. into the CAM and the illuminated with a 652 nm diode laser 15 min (A–C), 1 h (D–-F) and 3 h (G–I) after i.v. injection. Twenty-four hours postillumination, 200 nm fluorescent polystyrene spheres were i.v. injected to highlight the asculature (C, F, I). Reprinted with permission from ref (162). Copyright 2014 Taylor & Francis Ltd.

Figure 6

Xenografts in the CAM for nanomaterial studies. (A) Xenografts recruit blood vessels when they are implanted in the CAM. Both the xenograft and the blood vessels can be visualized, allowing a rapid study of the effects of therapeutic nanomaterials targeted to both the tumor and the associated vasculature. (B, C) Tumors can be retrieved from the CAM and studied using common histology techniques such as H&E staining. Untreated breast cancer xenografts show rapid cell proliferation, while tumors treated with liposomes and photodynamic therapy show extensive necrosis. Images in (B) and (C) were reprinted with permission from ref (162). Copyright 2014 Taylor & Francis Ltd. (D, E) Intravital imaging of fluorescently labeled CPMV-based viral nanoparticles which allow direct visualization of the vasculature both immediately (E) and 24 h postinjection (D). (F, G) Intravital imaging can reveal tumor neovascularization. Fibrosarcoma xenograft (magenta) with endothelium labeled with fluoresceine lectin (green) with (G) and without (F) CPMV-based NPs to visualize tumor neovascularization. Images in (A) and (D)–(G) were adapted with permission from ref (6). Copyright 2010 Nature Publishing Group.

Figure 7

Intravital microscopy showing binding and cargo delivery by antibody-targeted lipid-coated MSNPs to leukemia cells. Leukemia cells were injected into the CAM vasculature and imaged 4 h (A–C) and 16 h post injection (D–F). Fluorescence microscopy showed that the leukemia cells (blue) within the vasculature (lectin vascular stain, lavender) were bound by lipid-coated MSNPs (red) loaded with cell impermeant cargo (YO-PRO-1, green) at 4 h postinjection. By 16 h postinjection, the membrane impermeant cargo has been released from the lipid-coated MSNP within the leukemia cell. Adapted with permission from ref (84).

Figure 8

Intravital and fluorescence-based imaging in the CAM, xenografts, and avian embryo. (A) Intravital imaging of single melanoma cells (yellow) extravasating in the CAM capillary network (green). (B) Monitoring the spread of GFP-labeled epidermoid carcinoma micrometastases through the CAM over time. Adapted with permission from ref (189). Copyright 2011 Journal of Visualized Experiments. (C) Monitoring the real-time expression of conditionally expressed E-cadherin (green) in extravasated (yellow) breast cancer cells in response to chemical induction. Images in (C) were reproduced with permission under a Creative Commons CC-BY license from ref (191). (D) Optical imaging of fluorescent metastatic melanoma cells in the liver of the avian embryos 5 days post vascular injection. Images in (A) and (D) were reprinted in part with permission under a Creative Commons CC-BY license from ref (71). (E) Phase contrast and optical imaging of FITC-labeled PMO nanoparticles accumulated in the tumor 3 days post vascular injection. Images in (E) were reproduced with permission under a Creative Commons CC-BY license from ref (95).

Figure 9

Intravital and fluorescence-based imaging in the CAM, xenografts. and avian embryo. (A) Quantification of tumor perfusion using laser-speckle perfusion imaging (LSPI). Glioblastoma xenografts were imaged without treatment (upper left), treatment with antiangiogenic therapy (upper right), lactate uptake blocker (lower left), or a combination of both therapies. Reprinted with permission under a Creative Commons CC-BY license from ref (193). (B) Ultrasound monitoring of xenograft growth in the CAM. Adapted with permission under a Creative Commons CC-BY license from ref (194). (C) Conventional vascular imaging using ultrasound (left) and optimized ultrasound microvessel imaging (right). Images in (C) were adapted with permission from refs ( and 228). Copyright 2022 C. Jeffrey Brinker. (D) MRI imaging of chick embryo for development of MRI contrast agents. The liver is highlighted as the region of interest (green), and the background is chosen outside of the egg. Reprinted with permission under a Creative Commons CC-BY license from ref (202). (E) SPECT/CT images of chick embryo and mice after injection of [^99mTc]-DMSA contrast. Arrows highlight the kidneys, demonstrating matching bioaccumulation in chick embryo and mice. Adapted with permission from ref (207). Copyright 2015 Elsevier. (F–H) PET/CT images of the CE and CAM. (F) Two-dimensional coregistered PET and CT images of avian embryo and glioblastoma (arrow) after ¹⁸F-FDG injection. (G) Visual comparison of the photographed avian embryo with glioblastoma xenograft and PET/CT image at the level of the CAM. (H) 3D overlay of PET and CT images of ¹⁸F-FDG uptake in the embryo and glioblastoma xenograft on the CAM (arrow). Images in (F)–(H) were reprinted with permission from ref (208). Copyright 2013 Journal of Nuclear Medicine.

Figure 10

Role of the chick embryo (CE) model for bridging experimental efforts toward clinical modeling of NPs. (A) The CE can be a cost-effective, humane, and rapid means of evaluating many NP candidates prior to experimentation in rodent models. (B) The CE can also be used to optimize the dose for a lead candidate NP that already has a known antitumor effect. The CE offers the ability to perform longitudinal imaging of NP distribution at a low cost for various NP concentrations. After optimization, the determined dose can then be applied in rodent models to confirm therapeutic potential. Image of culture plate adapted with permission under a Creative Commons CC-BY license from ref (226). Copyright 2020 TOGO.