Internalization of targeted quantum dots by brain capillary endothelial cells in vivo

Production and purification of mAbs in vitro

Hybridoma cell lines Ri7217.1.4 (Ri7) and 8d3 secreting rat mAbs specific for the mouse TfR1 were obtained from Drs Jayne Lesley (Salk Institute, via Dr Pauline Johnson, U. of British Columbia, Canada) and Britta Engelhardt (U. of Bern, Bern, Switzerland). The antibodies were designed to target an exofacial epitope on the TfR. Hybridoma cell lines were cultured in CELLine bioreactors in serum-free mAb medium (BD Biosciences, Mississauga, ON, Canada). Supernatants were harvested weekly. mAbs were purified using HiTrap protein G columns and the Akta Prime Plus system (GE Healthcare, Baie d’Urfé, QC, Canada) according to the manufacturer’s recommendations. Purified antibodies were concentrated with Amicon ultracentrifugal filter units (Millipore, Billerica, MA; molecular weight cutoff, MWCO = 30 kDa) and subsequently dialyzed against 1X phosphate-buffered saline (PBS) (pH 7.4) using Slide-A-Lyzer dialysis cassettes (MWCO = 10 kDa; Pierce, Rockford, IL). All protein concentrations were determined based on bicinchoninic acid (BCA) assays (Pierce). The weekly yield of purified mAbs averaged 3 mg.

mAb conjugation to Qdot 625 nanocrystals

Ri7, 8d3, and control IgG (BioXCell, West Lebanon, NH, USA) (300 µg) were thiolated for 1 h with a 40:1 molar excess of freshly prepared 2-iminothiolane (Traut’s reagent) in 0.05 M sodium borate/0.1 mM EDTA, pH 8.5. Thiolated mAbs were transferred to a buffer containing 0.05 M HEPES/0.01 mM EDTA (pH 7.0) by centrifugation in a Vivaspin (Sartorius Stedim Biotech, Aubagne, France) filter unit (MWCO 30 kDa). This step was repeated twice to discard 2-iminothiolane and reduce the final volume to 40 µl. Conjugation of mAbs to Qdot 625 nanocrystals (Life Technologies, Burlington, ON, Canada) was performed according to the manufacturer’s indications. Briefly, SMCC-activated Qdots and thiolated mAbs were desalted and then mixed together and incubated for 1 h at room temperature to allow conjugation. To quench the conjugation reaction, 10 µl of fresh 10 mM 2-mercaptoethanol solution were added to the mixture and incubated for 30 min. Finally, samples were concentrated to a final volume of 20 µl and conjugated mAbs were then purified using a size-exclusion chromatography column to discard unconjugated material. We estimated the number of antibodies attached to a single Qdot by measuring the protein concentration divided by the Qdot concentration calculated from the absorbance measured at 612 nm. Between three and four antibodies on average were conjugated to a single Qdot following these experimental procedures. Conjugated Qdots were characterized by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK). Parameters recorded included the hydrodynamic diameter, polydispersity index, and ζ-potential. Size distribution of Qdots was also assessed from the direct diameter measurement of 50 particles on TEM images, using ImageJ sofware.

In vitro competition studies

Murine neuroblastoma (N2A, #CCL-131, ATCC, Manassas, VA) and brain endothelioma (bEnd5, #96091930, HPACC, Salisbury, UK) cells were seeded in 96-well plates containing DMEM supplemented with 10% FBS at 4 × 10⁴ and 3 × 10⁴ cells/well, respectively. One day later, the medium was removed and cells were pretreated for 15 min with unlabeled Ri7 or control IgG (0, 10, 50, or 200 nM). Ri7-Qdots were then added to a final mAb concentration of 8.3 nM and incubation was resumed for 15 min. After washing twice with cold PBS, internalization of Ri7-Qdots was measured with a Synergy HT (Biotek, Winooski, VT) plate reader (440 nm (λ_exc)/645 nm (λ_em)). A similar protocol was used with µ-Slide VI^0.4 chamber slides (IBIDI, Munich, Germany), where N2A (1.2 × 10⁴ cells/well) and bEnd5 (2 × 10⁴ cells/well) cells were pretreated with 50 nM unconjugated Ri7, 8d3, or control IgG and then incubated with 16.7 nM Ri7-Qdots. The washing steps were performed with 1X D-PBS containing Mg²⁺ and Ca²⁺. After two washes, cells were fixed for 20 min with 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. Qnuclear™ Deep Red stain was used to label nuclei, and slides were mounted with Qmount™ Qdot mounting media (Life Technologies).

Animals

Adult ♂ Balb/c mice (Charles River, Wilmington, MA, USA) weighing 20–30 g were used. They had ad libitum access to food and water. All procedures were performed in accordance with the Canadian Council on Animal Care standards and Animal Research: Reporting In Vivo Experiments guidelines and were approved by the Animal Ethics Committee of Université Laval.

Tissue preparation for postmortem analysis

For microscopy experiments, following a single intravenous (caudal vein) injection of 300 µg (15 mg/kg) of mAbs conjugated to Qdots, animals were sacrificed 0.5, 1, 4, or 24 h postinjection by terminal transcardiac perfusion under deep anesthesia with ketamine/xylazine. The injected dose of ketamine/xylazine was calculated based on the mouse weight (0.1 ml/10 g of a ketamine/xylazine solution at 87.5/12.5 mg/ml). Terminal perfusions were performed with 25 ml of 1X PBS, 50 ml of 3.5% acrolein in 0.1 M sodium phosphate buffer, pH 7.4, and/or 50 ml of 4% PFA in the same buffer. Brains were rapidly dissected, postfixed in 4% PFA for 4 h and cut with a vibratome (Leica Microsystems, Richmond Hill, ON, Canada) in transverse sections of 55 µm, which were collected in cold PBS. Tissues used for colocalization analysis were cut in coronal sections of 12 µm with a cryostat (Leica Microsystems). For Qdot biodistribution analysis, mice were sacrificed 1 h postinjection by decapitation under deep anesthesia with ketamine/xylazine (87.5/12.5 mg/ml), excepted for the capillary depletion experiment where mice were sacrificed by intracardiac perfusion with PBS 1X. Brain, heart, kidney, liver, lungs, and plasma were collected, weighed, and rapidly frozen until homogenization.

Capillary depletion

Brains were homogenized in 1.0 ml of physiological cold HEPES buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, and 10 mM glucose at pH 7.4). Afterward, 1.0 ml of a cold 37% dextran solution was added to the homogenate to obtain a final concentration of 18% and all samples were centrifuged at 5400g for 20 min at 4℃. The resulting postvascular supernatant and capillary pellet were separated manually and kept for further cadmium determination by ICP-MS. In the brain, the enzyme γ-GT is specifically expressed by BCECs and its activity can be used to confirm the success of the capillary depletion and to determine the contamination of the supernatant by BCECs content. γ-GT activity (Teco Diagnostics, Anaheim, CA) was assayed according to the manufacturer’s indications. Briefly, 100 µl of working reagent was added to 20 µl of each sample. Absorbance at 405 nm was recorded at 37℃ every minute, during 10 min.

Biodistribution analysis of Qdots

All samples were digested in 0.5 ml of an acidic solution (HNO₃ (67–70%, #870003-261, VWR Canada, Montreal, CA) and H₂O₂ (30–32%, #H325-500, Fisher Scientific, Ottawa, CA) 1:1) at 60℃ for 12 h using heating blocks. Samples were homogenized every hour using a vortex mixer and tube caps were opened frequently during the process to equilibrate pressure. After complete digestion, samples were transferred to a 15 ml conical tube, diluted to a final 10 ml volume with ultratrace analysis water (#14211-1 L-F, Sigma-Aldrich, St Louis MO, USA). Then, all samples were filtered through 0.45 µm syringe filters (#SLHVM33RS, EMD Millipore, Etobicoke, CA) and transferred to sampling tubes. Cadmium determination was carried out in an ICP-MS (Agilent 8000 Triple Quad ICP-MS, Agilent technologies, Santa Clara CA, USA) equipped with a fully automated sampling unit (ASX-520, Agilent technologies). Cadmium signal (m/z = 111) was measured following experimental parameters and instrument settings proposed by the manufacturer handbook. Standard solutions in the µg/l (ppb) range were prepared from a 1000 ± 4 mg/l Cd²⁺ stock solution (#20895, Sigma-Aldrich). Blank sample analysis has shown no significant sign of signal interference and the limit of detection was evaluated at ∼0.2 µg/l using the (3σ_blank/m) approximation. Calibration standards were measured before, after, and midway through sample analysis and no significant drift of the analytical signal was observed (RSD ≅ 7%). A Qdot-free brain sample spiking at 10 µg/l Cd²⁺ was used as a reference material and was determined at 9.3 ± 0.7 µg/l giving the method an accuracy of ∼8%. Protein concentrations were determined based on BCA assays (Pierce).

Distribution volume (Vd) values in each organ were calculated as follows:

Vd = ( organcadmiumconcentration / plasmacadmiumconcentration )

Immunofluorescence

Washing steps were performed using 1X PBS, pH 7.4, between each step of the immunofluorescence protocol (see below). Brain sections from mice injected with Qdot-mAbs were blocked for 1 h in a PBS solution containing 5% horse serum (Life Technologies) and 0.2% Triton X-100. Sections were then incubated overnight at 4℃ with primary antibodies in the blocking solution: goat anti-type IV collagen (Chemicon/Millipore, Temecula, CA, 1:500), mouse anti-feminizing locus on X-3 (Fox-3/neuronal nuclei (NeuN)), a neuronal marker (NeuN, Chemicon/Millipore, 1:1000), mouse anti-glial fibrillary acidic protein (GFAP, Sigma-Aldrich, Oakville, ON, Canada, 1:1000), and rabbit anti-laminin (Sigma-Aldrich, 1:1000). Following incubation with primary antibodies, slices were exposed to Alexa Fluor-conjugated donkey anti-goat and anti-mouse secondary antibodies (Life Technologies, 1:1000). Slides were then coverslipped with Mowiol™ mounting media (Sigma-Aldrich).

Immunogold labeling of unconjugated Ri7 mAb

Sections were washed in 1X (pH 7.4) PBS and blocked for 90 min with the same PBS solution containing 2% normal goat serum and 0.5% gelatin. Sections were incubated with a junction antibody (rabbit anti-rat IgG (H + L), 1:500, Vector Laboratories, Burlington, ON, Canada) for 90 min, washed with PBS, and incubated for 20 h at 4℃ in a solution containing a Nanogold goat anti-rabbit Fab’ secondary antibody conjugated to 1.4 nm colloidal gold particles (Nanoprobes, Yaphank, NY). These sections were washed in 3% (w/v) sodium acetate (pH 7.0) and then treated with HQ Silver Intensification kit (Nanoprobes) to increase the effective size of the immunogold particles and processed for TEM as described below. All incubations were performed in the blocking solution.

Ultrastructural analysis of mAb internalization

Sections were then osmicated, dehydrated in ethanol and propylene oxide, and flat-embedded in Durcupan (Fluka, Buchs, Switzerland). Quadrangular pieces from each section were removed, glued to the tip of resin blocks, and cut in ultrathin sections (∼80 nm) with a Leica EM UC7 ultramicrotome. ¹⁸ Ultrathin sections were collected on bare 150-mesh copper grids, stained with lead citrate, and examined with a Tecnai 12 transmission electron microscope (100 kV; Philips Electronic) with an integrated MegaView II digital camera (SIS, Schwentinental, Germany).

Data and statistical analysis

Quantification of Qdots was performed on 35–50 sections per animal (n = 3–4), using Image J software. In vitro experiments were performed in triplicate. ANOVA followed by a Bonferroni post hoc test and analyses of contingency (chi-squared test) were used to detect significant differences when appropriate. Statistical significance was set at *p < 0.05, ***p < 0.001, and ****p < 0.0001. All statistical analyses were performed using Prism 5 for Macintosh (GraphPad Software Inc., San Diego, CA).

Unconjugated anti-TfR mAbs prevent binding of Ri7-Qdot complexes

To estimate the size of Ri7-Qdot conjugates, we first took advantage of the electron density of Qdot nanocrystals. By measuring the diameter of 50 nanoparticles, we estimated the Qdots size on TEM images to 9.15 ± 0.87 nm (Figure 1(a) and (b)). As measured with DLS, Ri7-Qdots had a mean size of 26.1 ± 0.4 nm with a polydispersity index of 0.162 ± 0.047 and a ζ-potential of -12.7 mV (Figure 1(c)). Internalization of Ri7-Qdot conjugates was confirmed in vitro using cultured bEnd5 murine BCECs and N2A mouse neuroblastoma cells, which both express the TfR (Figure 1(d) to (k)). To verify that the internalization of Ri7-Qdots was mediated by the TfR, we pretreated N2A and bEnd5 cells with two anti-TfR mAb (Ri7 and 8d3) or a control rat IgG for 15 min. Unlabeled Ri7, unlike control IgG, reduced the uptake of Ri7-Qdots in both cell lines by up to 80% within 15 min (Figure 1(d) and (e)). Qdot binding to TfR was suppressed by the addition of either Ri7 or 8d3 in both N2A (Figure 1(f) to (h)) and bEnd5 cells (Figure 1(i) to (k)), supporting the potential of Ri7-Qdot conjugates to target BCECs. OPEN IN VIEWER

Adding antibodies targeting the TfR leads to increased distribution of Qdots in the brain

To determine the extent by which Ri7 changes Qdot distribution after a systemic administration, we performed a biodistribution experiment in the mouse. Cadmium determination by ICP-MS was used to evaluate the relative distribution of Qdots (conjugated with either Ri7 or control IgG) in the brain, liver, spleen, kidneys, lung, and the plasma, 1 h after i.v. injection. The apparent volumes of distribution (Vd) were computed and are shown in Figure 2. Vd in the brain was fourfold higher for Ri7-Qdots compared to control 2a3-Qdots, consistent with a significant TfR-targeted delivery. Moreover, cadmium concentrations were approximately 10-fold higher in isolated capillaries compared with remaining brain tissue, suggesting that Ri7-Qdots remained mainly sequestered in BCECs (Figure 2(f)). The relative purity of the capillary pellet was confirmed by assessing the enzymatic activity of the endothelial specific enzyme γ-GT (pellet: 10.0 ± 3.7 mmol/min/mg, supernatant: 1.0 ± 0.1 mmol/min/mg). OPEN IN VIEWER

Intravenously injected Ri7-Qdots selectively and extensively label cerebral blood vessels

To determine the brain distribution of Ri7-Qdot complexes in vivo, we first conducted fluorescence microscopy analyses after intravenous injection to Balb/c mice. Data shown in Figure 3 indicate that Ri7-Qdot complexes were extensively bound to cerebral blood vessels, including small-diameter (<12 µm) capillaries. The observed signal was strong as early as 30 min postinjection, remained at peak up to 4 h postinjection, and then declined significantly 24 h postinjection ((a) to (c)). No signal was detectable for control IgGs conjugated with Qdots (d). We also observed qualitative changes over time. Between 4 and 24 h, the fluorescence signal had a granular-like appearance, suggesting a progressive concentration of Ri7-Qdot complexes into intracellular vesicles. To determine whether Ri7-Qdot complexes had reached neurons or glial cells, we performed immunofluorescence colocalization studies using NeuN and GFAP antibodies to label neuronal cells and astrocytes, respectively. However, no Ri7-Qdot signal was found in either neurons or astrocytes in any of the ∼30 sections investigated, 1 h after systemic injection ((f) and (g)). On the other hand, the specific distribution of Ri7-Qdot complexes in brain microvessels was confirmed by their colocalization with immunolabeled collagen IV, a marker of the basal lamina (H). OPEN IN VIEWER

Endocytosis of Ri7-Qdot complexes following intravenous injection

To determine whether Ri7-Qdot complexes enter BCECs via endocytosis, undergo transcytosis through the BBB, or simply bind to adluminal TfR without being internalized into BCECs, we performed TEM experiments. The analysis of brain sections from animals sacrificed 30 min, 1, 4, and 24 h following i.v. injection of Ri7-Qdots showed evidence of endocytosis of the complexes into BCECs (Figure 4). At 30 min, Qdots located within BCECs were distributed mainly in tubular structures (∼60–80 nm o.d.) and small vesicles (∼100 nm o.d.). A small population of Ri7-Qdots was located in the luminal compartment of the blood vessels on the adluminal side of endothelial cells, suggesting binding of the complex to TfR (Figure 4(a) to (d)). No Qdots conjugated to control IgG were detected in vessels (Figure 4(e)). OPEN IN VIEWER

To further determine the fate of anti-TfR mAbs after endocytosis, we also performed analyses of brain sections from mice sacrificed 1, 4, and 24 h after i.v. injection. After 1 h (Figure 4(f) to (j)), 4 h (Figure 5(a) to (f)), and 24 h (Figure 5(g) to (l)) the vast majority of Ri7-Qdot complexes were concentrated into multivesicular bodies (MVBs), but a significant fraction was still present in tubular structures, small vesicles, as well as on the basal lamina. At 24 h, fusion of large vesicles containing Qdots with lysosome was observed (Figure 5(i)). Some Qdot complexes also reached the abluminal side of BCECs and the brain parenchyma (Figure 5(b) and (l)). However, those observations were very rare, and Qdots found in the parenchyma were generally located in the vicinity of the basal lamina (∼100–300 nm). Finally, a few Qdots were occasionally observed within pericytes (Figure 5(f)). Immunofluorescence analyses were also conducted at 24 h (Figure 6). Ri7-Qdots complexes were almost exclusively bound to brain microvessels, no fluorescence signal was visible beyond the basal lamina (Figure 6(a) and (b)), and no colocalization with neurons (Figure 6(c)) or astrocytes (Figure 6(d)) was observed. OPEN IN VIEWER

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Figure 6.

Labeling of brain blood vessels 24 h after intravenous injection of TfR mAbs. Mice were injected with mAbs conjugated with Qdot nanocrystals and sacrificed 24 h postinjection. (a) Colocalization of Ri7-Qdots (red) with the basal lamina immunolabeled with type IV collagen (green) and (b) laminin marker (green). (c) Immunofluorescence against the neuronal marker NeuN showed absence of Qdot-labeled Ri7 (red) within neurons (green). (d) Ri7-Qdot signal (red) was not detected in astrocytes labeled with GFAP (green). Photomicrographs were taken in the cerebral cortex of mice (n = 3–4 animals per group). Scale bar = 20 µm.

Adding a large moiety to anti-TfR mAb does not interfere with BBB crossing

To validate whether the conjugation of Qdot nanocrystals to mAbs altered the capacity of Ri7 to be exported to the brain parenchyma, we injected unlabeled antibodies and performed immunogold labeling using the appropriate secondary antibodies (Figure S1). We observed that unlabeled anti-TfR mAbs were distributed similarly to Qdot-conjugated Ri7 (Figure S1(a) to (d)), suggesting that Qdot conjugation to an anti-TfR mAb does not affect distribution of the latter within brain cells.

Qdot count analysis

Next, we performed a semi-quantitative determination of the subcellular distribution of Qdots (n = 3–4) in brain capillaries (Figure 7). The total number of internalized Qdots reached 8 ± 1 at 30 min post injection, peaked at 34 ± 7 at 1 h, and decreased thereafter (16 ± 3 and 9 ± 1 at 4 and 24 h, respectively). At all time points, the vast majority (70–90%) of Qdots remained localized within intracellular compartments of BCEC, along with subpopulations of Qdots found on the luminal or abluminal side of brain microvessels (Figure 7(a)). Nonetheless, the proportion of Qdots in the luminal compartment of brain blood vessels declined over time while the percentage of Qdots on the abluminal side increased, suggesting that Ri7-Qdot complexes migrated toward the abluminal side over a 24 h period (Figure 7(a)). However, as stated above, the fraction of Qdots apparently reaching the brain parenchyma (i.e. beyond the basal lamina) remained relatively low (Figure S2). Supplementary Figure S3 (Figure S3(a) to (c)) illustrates the delimitations used to define the luminal, endothelial, and abluminal compartments. The abluminal compartment included the basal lamina, pericytes, astrocytic, and neuronal projections. OPEN IN VIEWER

Overall, Qdots were mostly found in small vesicles (58 ± 3.5%) at all time intervals examined (Figure 7(b)). The proportion of Qdots found in tubular structures was the highest 30 min (∼30%) after injection and decreased over time (24 h: ∼8%) while the proportion of Qdots found in MVBs increased over time (30 min: ∼6%) to reach its maximum 4 h postinjection (∼40%), suggesting a progressive routing of Qdots toward MVBs via the late endocytic pathway. Electron micrographs shown in supplementary Figure 3(d) to (f) illustrate typical examples of the subcellular structures analyzed.

In summary, qualitative and quantitative TEM analyses have revealed an extensive internalization of systemically administered Ri7-Qdots into BCECs, with progressive accumulation into MVBs, consistent with their incorporation via an endocytotic process.