Surface engineering of quantum dots for in vivo vascular imaging

Abstract

Quantum dot-antibody bioconjugates (QD-mAb) were synthesized incorporating PEG cross-linkers and Fc-shielding mAb fragments to increase in vivo circulation times and targeting efficiency. Microscopy of endothelial cell cultures incubated with QD-mAb directed against cell adhesion molecules (CAMs), when shielded to reduce Fc-mediated interactions, were more specific for their molecular targets. In vitro flow cytometry indicated that surface engineered QD-mAb labeled leukocyte subsets with minimal Fc-mediated binding. Nontargeted QD-mAb nanoparticles with Fc-blockade featured 64% (endothelial cells) and 53% (leukocytes) lower nonspecific binding than non-Fc-blocked nanoparticles. Spectrally distinct QD-mAb targeted to the cell adhesion molecules (CAMs) PECAM-1, ICAM-1, and VCAM-1 on the retinal endothelium in a rat model of diabetes were imaged in vivo using fluorescence angiography. Endogenously labeled circulating and adherent leukocyte subsets were imaged in rat models of diabetes and uveitis using QD-mAb targeted to RP-1 and CD45. Diabetic rats exhibited increased fluorescence in the retinal vasculature from QD bioconjugates to ICAM-1 and VCAM-1 but not PECAM-1. Both animal models exhibited leukocyte rolling and leukostasis in capillaries. Examination of retinal whole mounts prepared after in vivo imaging confirmed the fluorescence patterns seen in vivo. Comparison of the timecourse of retinal fluorescence from Fc-shielded and non-Fc-shielded bioconjugates indicated nonspecific uptake and increased clearance of the non-Fc-shielded QD-mAb. This combination of QD surface design elements offers a promising new in vivo approach to specifically label vascular cells and biomolecules of interest.

Figure 1

Evaluation of Fc-blocked QD-mAb conjugate specificity toward TNF-α stimulated (TNF+) or untreated (TNF−, image insets) rat endothelial cells (YPEN-1). (A–C) Fluorescence micrographs of QD655-anti-PECAM (A), QD655-anti-VCAM (B), and QD655 (C). Insets show incubation of TNF− YPEN-1 with matched bioconjugates. (D) Mean image intensities from immunofluorescence analysis of TNF ± YPEN-1 incubated with QD655-mAb bioconjugates (reported as mean + S.D., n = 3 images per sample). Intensities for each TNF+ and TNF− pair were compared by t-test for statistical significance (P < 0.05 indicated by asterisk).

Figure 2

Comparison of Fc-blocked and non-Fc-blocked QD655-mAb conjugate specificity toward TNF-α-stimulated YPEN-1 (TNF+). (A–B) TNF+ YPEN-1 incubated with non-Fc-blocked (A) and Fc-blocked (B) QD655-anti-ICAM bioconjugates (magnification 100×). Arrowheads indicate possible Fc receptor capping, which was substantially reduced when using Fc-blocked conjugates. (C,D) TNF+ YPEN-1 incubated with non-Fc-blocked (C) and Fc-blocked (D) QD655-IgG₁ isotype control bioconjugates, with capping sites labeled with arrowheads (magnification 400×). (E) Mean image intensity analysis reveals lower nonspecific binding of bioconjugates featuring Fc blockade (reported as mean + S.D., n = 3 images per sample). Intensities for each matched Fc-blocked and non-Fc-blocked bioconjugate were compared by t-test for statistical significance (P < 0.05 indicated by asterisks).

Figure 3

FACS analysis of leukocytes labeled with QD-mAb bioconjugates in vitro. (A) QD585-anti-RP-1 (Fc-blocked) specific labeling of neutrophils in peripheral blood. (B) QD585-anti-CD45 (Fc-blocked) labeling of all leukocyte subsets, with arrow from B to D indicating fluorescence histogram of gated leukocytes (D). (C) Gated neutrophil populations from rat peripheral blood (arrow from A to C shows a gated neutrophil population used for analysis of 1 sample), (488 nm Ar laser excitation, phycoerythrin (PE) emission filter (585/42 nm)), with mean fluorescence intensity of each sample indicated in bold. Light blue: PE-anti-RP-1 dye-labeled positive control mAb. Purple: QD585-anti-RP-1 (Fc-blocked). Blue: QD585 quenched with l-cysteine. Brown: QD-Ms-IgG2a isotype non-Fc blocked. Green: QD-Ms-IgG2a isotype Fc blocked. Red: Unlabeled rat peripheral blood. (D) Rat leukocytes labeled with QD585-anti-CD45 (red, gated leukocytes from (B)), QD585-Ms IgG1 Fc blocked isotype control (blue) or unlabeled (green).

Figure 4

Frames from in vivo digital videos of STZ treated and untreated rat retinas. Pre-injection digital videos using the appropriate fluorescence filters for the spectrally distinct biomarkers of interest (left column; A, D, G, J) were acquired prior to bioconjugate injection. QD585-anti-ICAM, QD655-anti-VCAM, QD525-anti-PECAM, and QD565-Ms IgG₁ were systemically injected in STZ-treated rats. Untreated control rat fundi (middle column; B, E, H, K) and STZ-treated rat fundi (right column; C, F, I, L) were imaged either 30 min (ICAM) or 90 min (VCAM, PECAM, IgG₁) postinjection under identical acquisition settings used for background autofluorescence measurements for each biomarker channel. Fluorescence enhancement due to target binding in untreated controls (B, E, H, K) was primarily observed for PECAM (H). The retinal vasculature of STZ-treated rats (C, F, I, L) showed a marked increase in fluorescence due to binding of QD-mAb conjugates to ICAM (C) and VCAM (F), with at least 5-fold increases in fluorescence intensity over background measured for labeled vasculature. Injection of conjugates to PECAM (I) produced similar levels of fluorescence under both conditions (H, I). Fluorescence signal from nonspecific IgG₁ mAb bioconjugates (J–L) was not detected in either control or STZ retinas. Images shown were acquired at imaging intervals resulting in optimal signal-to-noise ratio.

Figure 5

In vivo imaging of endogenously QD-mAb-labeled leukocyte trafficking in two rat models of inflammation. The CCD acquired images with exposure times ranging from 50 to 200 ms with 110 ms between exposures. Flowing of QD585-RP-1 labeled cells in vessels (A,B) or leukocyte rolling (C) in STZ model as detected by four pulses of a xenon flashlamp at 50 ms intervals within 30 min of probe injection. Leukostasis was frequently observed in the STZ-treated rat model (D in right panel). Stagnant neutrophil appears as a hyperfluorescent dot in the microcirculation. Stagnant neutrophils were continuously visible in the retinal vasculature for over 1 h of in vivo imaging. Stagnant leukocytes labeled in vivo with QD655-anti-CD45 were present at high densities in EIU rat models (E) relative to untreated controls (F). Stagnant neutrophils labeled in STZ-streated diabetic animals were visible in postmortem retinal flat mounts (G).

Figure 6

Comparison of Fc-blocked (A–D, I) and non-Fc-blocked (E–H, J) QD-IgG₁ mAb bioconjugates in vivo. (A) Pre-injection autofluorescence in QD655 emission channel. (B–D) Fc-blocked QD655-IgG₁ in retina observed 12 s postinjection (B) where the first pass of bolus through retinal circulation is observed, 1 h postinjection (C), and 2 h postinjection (D), by which time Fc-blocked QD655-induced fluorescence is still above background levels (I, dashed line). (E) Autofluorescence levels in QD565 channel. (F–H) non-Fc-blocked QD565-IgG₁ bolus was not qualitatively visible at 12 s postinjection (F), and subsequent image acquisitions at 1 h (G) and 2 h (H) revealed eventual return of image intensity to pre-injection levels (J, solid line), suggesting a relatively rapid clearance and/or uptake of non-Fc-blocked QD-mAb.

Figure 7

In vivo microcirculatory VCAM-1 expression (A) can be confirmed using immunofluorescence analysis postmortem (B), as shown for QD655-anti-VCAM-1 labeled vasculature. While QD655-VCAM-1 (Fc-blocked) conjugates were shown to specifically stain vasculature in retinal histological sections (C), no background staining due to QD-IgG₁ (Fc-blocked) was observed above background autofluorescence levels (D).

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