Brain Uptake of a Fluorescent Vector Targeting the ... - ACS Publications

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Brain Uptake of a Fluorescent Vector Targeting the Transferrin Receptor: A Novel Application of in Situ Brain Perfusion Wael Alata,†,‡ Sarah Paris-Robidas,†,‡ Vincent Emond,‡ Fanchon Bourasset,§ and Frédéric Calon*,†,‡ †

Faculty of Pharmacy, Université Laval, Québec, QC G1V 0A6, Canada Neurosciences Axis, Centre de recherche du CHU de Québec, Québec (QC), QC G1V 4G2, Canada § Department of Pharmacokinetics, INSERM U705, CNRS UMR 8206, Faculty of Pharmacy, Université Paris Descartes, Université Paris Diderot, Sorbonne Paris Cité, Paris, France ‡

ABSTRACT: Monoclonal antibodies (mAbs) targeting blood− brain barrier (BBB) transporters are being developed for brain drug targeting. However, brain uptake quantification remains a challenge, particularly for large compounds, and often requires the use of radioactivity. In this work, we adapted an in situ brain perfusion technique for a fluorescent mAb raised against the mouse transferrin receptor (TfR) (clone Ri7). We first confirmed in vitro that the internalization of fluorolabeled Ri7 mAbs is saturable and dependent on the TfR in N2A and bEnd5 cells. We next showed that the brain uptake coefficient (Clup) of 100 μg (∼220 nM) of Ri7 mAbs fluorolabeled with Alexa Fluor 750 (AF750) was 0.27 ± 0.05 μL g−1 s−1 after subtraction of values obtained with a control IgG. A linear relationship was observed between the distribution volume VD (μL g−1) and the perfusion time (s) over 30−120 s (r2 = 0.997), confirming the metabolic stability of the AF750-Ri7 mAbs during perfusion. Co-perfusion of increasing quantities of unlabeled Ri7 decreased the AF750-Ri7 Clup down to control IgG levels over 500 nM, consistent with a saturable mechanism. Fluorescence microscopy analysis showed a vascular distribution of perfused AF750-Ri7 in the brain and colocalization with a marker of basal lamina. To our knowledge, this is the first reported use of the in situ brain perfusion technique combined with quantification of compounds labeled with near-infrared fluorophores. Furthermore, this study confirms the accumulation of the antitransferrin receptor Ri7 mAb in the brain of mice through a saturable uptake mechanism. KEYWORDS: blood−brain barrier, in situ brain perfusion, transferrin receptor, fluorescence

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was later applied to mice by Dagenais et al. in 200011 (Figure 1). Besides high sensitivity,12 ISBP displays several other advantages, such as complete control over the perfusate content and flow rate while avoiding peripheral metabolism and the possibility of directly assessing the transport mechanisms.13,14 However, the use of ISBP is generally limited to compounds for which a radiolabeled version is available. Because of cost, growing concerns regarding radioprotection, and the relative simplicity and versatility of recently developed fluorolabeling procedures, the need to develop a fluorescence-based ISBP technique is becoming more evident. The recent development of far-red and near-infrared (NIR) fluorescent dyes with spectroscopic characteristics that allow a differentiation between the true signal and autofluorescence has rendered the quantification of total fluorescence in brain homogenates possible.15

INTRODUCTION Poor brain bioavailability has been recognized as one of the leading causes of failure in drug development.1−3 Such low brain access to drugs is mainly attributed to the presence of the blood−brain barrier (BBB), which protects the brain from most bloodborne hydrophilic molecules. The BBB consists of brain capillary endothelial cells (BCECs), which are distinct from other endothelial cells by virtue of the presence of tight junctions and the expression of multiple influx and efflux transport systems.4 The basal lamina, pericytes, and astrocytic end-feet located near the BCECs also contribute to the cohesion of the BBB structure.5 Even as of today, many compounds enter phase II and phase III clinical trials with brain bioavailability issues unresolved. Thus, it is essential to develop valid methods to determine the capacity of drug candidates to cross the BBB as early as possible in the drug development process.2,6 Among the few techniques developed to assess the brain uptake of molecules, in vivo methods involving an injection into the carotid artery, such as the in situ brain perfusion technique (ISBP), present key quantitative advantages.7−9 ISBP came to light in 1984 when it was applied to rats by Takasato et al.10 and © XXXX American Chemical Society

Received: July 19, 2013 Revised: October 29, 2013 Accepted: November 11, 2013

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dx.doi.org/10.1021/mp400421a | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Animal Ethics Committee of the Centre Hospitalier de l’Université Laval. Reagents. [14C]sucrose (0.6 Ci mmol−1) was purchased from Moravek Biochemicals (Brea, CA, USA). SOLVABLE solubilizer and Ultima Gold scintillation cocktail were purchased from PerkinElmer (Boston, MA, USA). Alexa Fluor (AF) dyes, 4′,6-diamidino-2-phenylindole dilactate (DAPI), and culture reagents were purchased from Life Technologies (Burlington, ON, Canada). Production and Purification of mAbs in Vitro. Hybridoma cell lines were cultured in CELLine bioreactors in mAb serum-free 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 (Millipore Corporation, Billerica, MA, USA) ultracentrifugal devices [molecular mass cutoff (MMCO) 30 kDa] and subsequently dialyzed against 0.01 M phosphate-buffered saline (PBS), pH 7.4, using 10 kDa MMCO Slide-A-Lyzer dialysis cassettes (Pierce Chemical, Rockford, IL, USA). Protein concentrations were determined using bicinchoninic acid assays (Pierce Chemical). The weekly yield of purified mAbs averaged 3 mg. Hybridoma cell line Ri7.217.1.4 (Ri7) secreting the rat mAbs specific for the mouse TfR was obtained from Dr. Jayne Lesley (Salk Institute, La Jolla, CA, USA) via Dr. Pauline Johnson (University of British Columbia, Vancouver, BC, Canada). Purified rat IgG2a isotype control (clone 2A3) was acquired from Bio X Cell (West Lebanon, NH, USA). mAb Conjugation to Alexa Fluor Dyes. mAbs (Ri7, control IgG) were thiolated with a 40:1 M excess of freshly prepared 2-iminothiolane (Traut’s reagent, Sigma-Aldrich, Oakville, ON, Canada) after a 1 h incubation in 0.15 M sodium borate/0.1 mM EDTA, pH 8.5. Thiolated mAbs were diluted in 0.05 M HEPES/0.1 mM EDTA, pH 7.0, and then concentrated using a 30 kDa MMCO Vivaspin filter device (Sartorius Stedim Biotech, Aubagne, France). To conjugate thiolated mAbs, AF750 or AF488 C5-maleimide (125 nM) was added and incubated overnight in 2 mL glass bottles under an inert nitrogen atmosphere. Vivaspin devices were then used to discard unbound AF maleimide and concentrate the AFconjugated mAbs. Volumes were completed to have the desired concentration (5 μg/μL) with HEPES/EDTA buffer before the perfusion. Cell Culture Studies. Murine Neuro-2a (N2A) cells (CCL131, ATCC, Manassas, VA, USA) and murine bEnd5 cells (96091930, ECACC, Public Health England, Salisbury, UK) were used for in vitro internalization studies. The N2A cell line has been established from a spontaneous neuroblastoma tumor of a strain A albino mouse. These cells have a neuronal and amoeboid stem cell morphology and express acetylcholinesterase and tubulin.26 The bEnd5 cell line has been established from brain endothelial cells of BALB/c mice. These cells have an endothelial-like morphology and are positive for PECAM-1, endoglin, -32, and Flk-1.27−29 Cells were seeded in 96-well plates (40 000 and 30 000 cells per well for N2A and bEnd5, respectively) containing 100 μL of Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). The next day, cells were incubated with the specified concentration (0.4, 2, or 10 nM) of either AF488-Ri7 or AF488-IgG for 5, 15, or 45 min at 37 °C, unless specified otherwise. Alternatively, for competition studies, N2A and bEnd5 cells were first pretreated

Figure 1. Illustrative schema of the in situ brain perfusion technique. The right common carotid artery and the external carotid artery are ligated at the heart side and the level of bifurcation, respectively. The right common carotid artery is then catheterized with a polyethylene catheter filled with heparin. Before perfusion, the thoracic cavity is opened and the heart is cut, and the perfusion is started immediately via the internal carotid artery. (Adapted from ref 10.)

As the brain requires bloodborne molecules for its normal function, the BBB expresses transporters, such as the transferrin receptor (TfR) and insulin receptor. Vectors targeting these transporters have thus been developed using monoclonal antibody (mAb) technology.16,17 Because of the high expression of the TfR on BCECs,18 reports suggest that mAbs recognizing rat (clone OX-26) and mouse (clones 8D3 and Ri7) TfR are uptaken by the brain after systemic injections.19−25 However, the quantitative evidence reported was obtained using 8D3 and Ri7 mAbs labeled with the γ-rayemitting radioisotope 125I.21 We recently discovered that NIRfluorescent dyes such as Alexa Fluor 750 allow quantification of vector penetration in brain homogenates after IV injection as a result of the relatively low brain autofluorescence levels with the absorption/emission parameters selected.22 A window of opportunity was thus opened for using ISBP with fluorescent compounds. Therefore, the goal of this study was to develop a nonradioactive, fluorescence-based ISBP method to quantify and characterize the brain uptake of a fluorolabeled protein in vivo. Because of the importance of TfR in brain drug delivery, the Ri7 mAb vector targeting the TfR was selected as the main test compound.

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EXPERIMENTAL SECTION Animals. Adult (4 week old) male BALB/c mice (Charles River Laboratories Inc., Wilmington, MA, USA) weighing 20 to 30 g were used. They had free access to food and water. All procedures were performed in accordance with the Canadian Council on Animal Care standards and were approved by the B



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and centrifuged at 10000g for 20 min at 4 °C to generate a fraction containing all of the detergent-soluble proteins, including cytosolic, extracellular, nuclear, and membranebound proteins. Samples of the perfusate and brain homogenates were added to black 96-well plates, and fluorescence was measured with a Kodak 4000MM image station with filters appropriate for AF750 (excitation, 720 nm; emission, 790 nm). Images were analyzed and pseudocolored with the Kodak MI software (Carestream Health, Woodbridge, CT, USA). The final fluorescence values were normalized according to exposure time, total fluorescence dose administered, and brain weight. Calculations of Transport Parameters. In AF750-mAb perfusion experiments, the calculations were similar to those for previously published data,11,30 but there were some modifications. The distribution volume (VD, μL g−1) of a fluorolabeled vector was calculated using the following equation:

for 15 min with unlabeled Ri7 or IgG (0, 10, 50, or 250 nM) and then exposed for a further 15 min to AF488-Ri7 (5 nM). Cells were then washed once with cold 0.1 M PBS and then with 0.025% trypan blue to quench noninternalized fluorescence. Measures were taken with a Synergy HT plate reader (filters: excitation, 485/20 nm; emission, 528/20 nm) from Biotek (Winooski, VT, USA). To visualize AF488-Ri7 accumulation, N2A and bEnd5 cells were seeded as mentioned, and 10 nM AF488-Ri7 was added to the medium at 4 °C. Incubation was resumed for 30 min at either 4 or 37 °C. Cells were then washed with PBS, fixed in 4% paraformaldehyde, stained with DAPI, washed, and imaged using an EVOS FL Auto Cell Imaging System (Life Technologies) with DAPI and GFP filter cubes. Z-stack images (12 μm depth, 60 photos, 0.366 μm step) were acquired, and deconvolution was processed with the DeconvolutionLab plugin (Biomedical Imaging Group, EPFL, Lausanne, Switzerland) for ImageJ (National Institutes of Health, Bethesda, MD, USA). Surgical Procedure and Perfusion Technique. The surgery and conditions of perfusion were done as previously described.11,30 Mice were anesthetized by intraperitoneal injection of xylazine/ketamine (8/140 mg kg−1). Next, the right common carotid artery was catheterized following ligation of the external branch (Figure 1). Before perfusion, the thorax of the animal was opened and the heart was cut, and perfusion was immediately started with a flow rate of 2.5 mL min−1. The perfusion fluid consisted of bicarbonate-buffered physiological saline (mM solute: 128 NaCl, 24 NaHCO3, 4.2 KCl, 2.4 NaH2PO4, 1.5 CaCl2, 0.9 MgCl2, 9 D-glucose). The solution was gassed with 95% O2/5% CO2 to obtain a pH of 7.4 and heated to 37 °C. Unless specified otherwise, a fixed quantity (100 or 200 μg) of either AF750-Ri7 or AF750-IgG were perfused during 1 min, followed by a 4 min washout with 10 mL of the same buffer. The washout time was included (1) to remove unbound AF750-labeled mAbs from brain vessels and capillaries and (2) to allow enough time for receptor-mediated uptake, which was expected to require a few minutes.12 Such a long washout carries the risk of release of AF750-Ri7 from BCECs back to the capillary lumen in the last minutes of perfusion. However, this is unlikely given the high affinity of Ri7 to the TfR31 and the fact that the linear relationship between Clup and time (see Figure 5 in the Results) was consistent with unidirectional uptake of AF750-Ri7. BBB Integrity during the Perfusion. In a subset of animals, co-perfusion with 0.3 μCi/μL [14C]sucrose (a vascular space marker that does not cross the BBB) during the last minute of the washout provided a measure of the vascular volume and an evaluation of the physical integrity of the BBB. Quantification of [14C]sucrose was performed by digesting the perfused hemisphere with 1 mL of SOLVABLE at 50 °C overnight and then adding 9 mL of Ultima Gold scintillation cocktail to the mix. Total [14C] was determined in a Packard Tri-Carb model 1900TR liquid scintillation analyzer. The calculations of vascular volumes were done as described previously.11,30 Brain Homogenates. The hemispheres perfused with AF750-mAbs were homogenized in 4 volumes of lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1% Triton X-100, 0.5% sodium dodecyl sulfate, and 0.5% sodium deoxycholate) containing Complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) with phosphatase inhibitors (50 mM sodium fluoride and 1 mM sodium pyrophosphate). Samples were sonicated briefly (3 × 10 s)

VD = Xbrain /Cperf where Xbrain (fluo g−1) is the amount of fluorescence in the right brain hemisphere and Cperf (fluo μL−1) is the concentration of fluorescence in the perfusate. The apparent brain uptake coefficient (Clup, μL g−1 s−1) was calculated using the following equation:

Clup = VD/Tperf where Tperf is the perfusion time (s). The quantity of fluorolabeled vector per gram of brain (Q, μg g−1) was calculated using the following equation: Q = Xbrain /Fperf

where Fperf (fluo μg−1) is the fluorescence per microgram of fluorolabeled vector, which was calculated by dividing Cperf (fluo μL−1) by the quantity of fluorolabeled vector per microliter (μg μL−1). Immunohistofluorescence. Tissue preparation for brain localization of Ri7 was performed on hemispheres perfused with 100 μg of AF750-Ri7, postfixed in 4% paraformaldehyde for 2 days, and transferred into a 20% sucrose/0.5% sodium azide solution for cryoprotection. Coronal brain sections with a thickness of 25 μm were cut with a freezing microtome (Leica Microsystems, Richmond Hill, ON, Canada). Experiments were performed on three animals and on at least five sections per animal. Washes in 0.1 M PBS, pH 7.4, were performed between each step. Free-floating brain sections from mice perfused with AF750-Ri7 were blocked for 1 h in a PBS solution containing 5% horse serum (Invitrogen) and 0.2% Triton X-100. Sections were then incubated overnight at 4 °C with primary antibodies in the blocking solution: goat anti-type IV collagen (Coll IV, 1:500; Millipore Bioscience Research Reagents, Temecula, CA) and mouse antineuronal nuclei (NeuN, 1:1000; Millipore Bioscience Research Reagents). After incubation with primary antibodies, slices were exposed to AF488 donkey anti-goat and AF568 anti-mouse secondary antibodies (1:1000; Invitrogen). Finally, slices were mounted onto SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA) and placed under coverslips with Mowiol mounting medium. Different appropriate filters were used for Ri7 (excitation, 710/75 nm; emission, 810/90 nm), Coll IV (excitation, 480/30 nm; emission, 535/40 nm), and NeuN (excitation, 540/40 nm; C



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Figure 2. Accumulation of AF488-Ri7 in murine neuroblastoma (N2A) and brain endothelial (bEnd5) cells. (A) N2A and (B) bEnd5 cells were seeded in 96-well plates containing DMEM with 10% FBS. One day later, they were exposed to AF488-Ri7 or AF488-IgG (0.4, 2, or 10 nM) for 5, 15, or 45 min. Cells were then washed once with cold PBS and 0.025% trypan blue to quench noninternalized fluorescence (n = 3 experiments in triplicate). Fluorescence was measured with a Synergy HT plate reader (485/528 filters). (C, E, G) N2A and (D, F, H) bEnd5 cells were cultured as mentioned above and exposed for 30 min to 10 nM AF488-Ri7 at either 4 °C (C, D) or 37 °C (E, F) or to 10 nM AF488-IgG at 37 °C (G, H). Cells were then washed with cold PBS, fixed with 4% paraformaldehyde, and stained with DAPI. AF488-Ri7 can be seen in green and DAPI-stained nuclei in blue. Vesicular-like staining is visible in (E) and (F). The images represent compressed, deconvolved Z-stacks. Data are shown as means ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Abbreviations: AF488, Alexa Fluor 488; Ri7, Ri7.217.1.4; IgG, control rat IgG2a; mAb, monoclonal antibody; RFU, relative fluorescence units. D



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Imaging Station 4000 MM Digital Imaging System (Molecular Imaging Software version 4.0.5f7, Carestream Health, Rochester, NY, USA). Data and Statistical Analysis. Data are shown as mean ± standard error of the mean (SEM). Student’s unpaired t test was used to identify significant differences between two groups when appropriate. Statistical differences between three groups were determined using the appropriate one-way analysis of variance (ANOVA) and posthoc tests for comparison between groups. All of the tests were two-tailed, and statistical significance was set as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

emission, 600/50 nm). Photomicrographs were acquired using Simple PCI version 5.0 software (Hamamatsu, Sewickley, PA, USA) linked to a Nikon Eclipse 90i microscope (Nikon Instruments, Toronto, ON, Canada). Capillary Depletion. A capillary depletion technique was used to isolate brain microvessels by density gradient centrifugation as adapted from a previous study.32 Four male BALB/c mice were anesthetized deeply and then perfused transcardially with 50 mL of ice-cold 0.1 M PBS. Next, brains were collected and transferred into ice-cold 0.1 M PBS, where cerebellum, meninges, brain stem, and large superficial blood vessels were removed. The brain was gently homogenized in ice-cold DMEM containing 10% FBS using Potter homogenizer, and a fraction of this homogenate was kept and identified as the total brain homogenate fraction (H). The remaining homogenate was centrifuged at 500g for 10 min at 4 °C. The supernatant was excluded, and the pellet was homogenized in 5 mL of ice-cold DMEM containing 25% bovine serum albumin (BSA) and centrifuged at 1500g for 20 min at 4 °C. The supernatant was excluded, and the remaining pellet was gently homogenized in 1 mL of ice-cold DMEM containing 10% FBS. The homogenate was filtered through a 60 μm filter to eliminate larger vessels, and the filtrate was centrifuged at 12000g for 45 min at 4 °C. The pellet containing the microvessels was washed in ice-cold 0.1 M PBS and centrifuged again at 12000g for 20 min at 4 °C. The supernatant was discarded, and the pellet was identified as the capillary fraction (C). The two fractions (H and C) were stored at −80 °C until processed for Western blot analysis. Tissue Processing. N2A and bEnd5 cells and the H and C brain fractions were homogenized in 8 volumes of lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1% Triton X-100, 0.5% SDS, and 0.5% deoxycholate) containing Complete protease inhibitors, 10 mg/mL pepstatin A, and phosphatase inhibitors (1 mM sodium pyrophosphate, 50 mM sodium fluoride). The obtained suspension was sonicated briefly (3 × 10 s) and centrifuged at 100000g for 20 min at 4 °C. The protein concentration was determined with the BCA assay in the supernatant, which was then stored at −80 °C until Western blotting. Western Blot Analysis. Equal amounts of proteins were added to Laemmli’s loading buffer, heated to 95 °C for 5 min before loading, and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Proteins were electroblotted onto PVDF membranes (Immobilon, Millipore, MA, USA) before blocking in 5% nonfat dry milk, 0.5% BSA, and 0.1% Tween 20 in 0.1 M PBS for 1 h at room temperature. The membrane was washed three times for 10 min in 0.1% PBS containing 0.1% Tween 20. The membrane was then incubated overnight at 4 °C with primary antibodies diluted in 0.1 M PBS containing 0.1% Tween 20, 5% nonfat dry milk, and 0.5% BSA. The dilutions of primary antibodies were 1:5000 for mouse anti-TfR (Abmart) and 1:10000 for mouse anti-GAPDH (ABM, Richmond, BC, Canada). The next day the membrane was washed three times for 10 min in 0.1% PBS containing 0.1% Tween 20 and then incubated for 1 h at room temperature with horseradish peroxidase-labeled goat antimouse (Jackson, West Grove, PA, USA) diluted 1:100000 in 0.1 M PBS containing 0.1% Tween 20 and 1% BSA. The membrane was again washed three times for 10 min in 0.1% PBS containing 0.1% Tween 20 and then probed with chemiluminescence reagents (Lumiglo Reserve, KPL, Gaithersburg, MD, USA). Immunoblots were analyzed with a KODAK

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RESULTS Cellular Uptake of AF488-Ri7 in N2A and bEnd5 Cells. To first confirm the specific targeting and uptake of the Ri7 mAb by mouse TfR, AF488-Ri7 accumulation was evaluated in vitro using two mouse-TfR-expressing cell lines, N2A and bEnd5. As shown in Figure 2A,B, a time- and concentrationdependent (0.4 to 10 nM) accumulation of AF488-Ri7 was observed in both cell lines. The observed curves were consistent with a TfR-dependent saturable mechanism. To verify whether the cellular accumulation of AF488-Ri7 was due to endocytosis rather than receptor binding, we performed uptake experiments at 4 and 37 °C in both cell lines. At 4 °C, the signal was limited to the cell surface (Figure 2C,D), suggesting binding of Ri7 to TfR without endocytosis. On the other hand, vesicular-like staining indicated that internalization of AF488-Ri7 occurred at 37 °C (Figure 2E,F), suggesting that endocytic mechanisms were activated after Ri7 binding to TfR in both N2A and bEnd5 cells. In contrast, uptake of the negative control AF488-IgG remained negligible compared with AF488-Ri7 (10 nM) in the two cell lines after a 30 min incubation at 37 °C (Figure 2G,H). Competition of Unlabeled Ri7 with Cellular Uptake of AF488-Ri7 by N2A and bEnd5 Cells. To establish whether Ri7 uptake into N2A and bEnd5 cells was a saturable process involving the mouse TfR, we carried out a competition study using increasing concentrations of unlabeled Ri7. In these experiments, a 15 min pre-exposition with three escalating concentrations of unlabeled Ri7 (10, 50, and 250 nM) was performed to compete with AF488-Ri7 (5 nM) for binding sites on TfR. As demonstrated in Figure 3A,B, unlabeled Ri7 was highly potent to prevent binding of AF488-Ri7 in the two cell lines, decreasing its cellular accumulation by 75% at 250 nM. As expected, addition of unlabeled IgG (10 to 250 nM) did not compete with AF488-Ri7 internalization. To document the level of TfR expression in the two cell lines, a Western blot analysis was performed. Relative to GAPDH, a higher concentration of mouse TfR was found in N2A cells than in bEnd5 cells (Figure 3 C). Accumulation of Fluorescent Anti-TfR mAb (AF750Ri7) in the Brain after in Situ Brain Perfusion. Because of autofluorescence of brain tissue in the visible spectrum, the proposed experimental paradigm was developed using an NIRfluorescent dye. To quantify Ri7 brain uptake, we thus coupled Ri7 with AF750.22 The fluorescence in brain homogenates was first measured after the perfusion of 100 or 200 μg of AF750mAb (Ri7 or control IgG), equivalent to concentrations of ∼220 and ∼440 nM, respectively, in the perfusate. The degrees of fluorolabeling per microgram of proteins for Ri7 and IgG were comparable. The fluorescence retrieved in brain homogenates from animals perfused with either 100 or 200 E



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Figure 4. Brain uptake of mAb targeting the TfR (AF750-Ri7) when the physical integrity of the BBB is maintained. (A, B) Shown are examples of pseudocolored brain homogenates in a 96-well plate as detected with 720/790 filters along with brain distribution volumes VD (μL g−1) after in situ brain perfusion of (A) 100 or (B) 200 μg of AF750-mAb (n = 9−10 per group). The brain uptake coefficient ClupRi7 (μL g−1 s−1) was calculated as (VD,Ri7 − VD,IgG)/perfusion time. Black column, group perfused with AF750-IgG; white column, group perfused with AF750-Ri7. Data were normalized by subtraction of background values (Bckd) obtained from mice perfused with perfusion buffer alone. (C) The BBB is intact after the perfusion of Ri7. Distribution volumes of the vascular space marker [14C]sucrose were measured by in situ brain perfusion. The vascular volume was unchanged for the group perfused with Ri7 relative to the group perfused with buffer alone (n = 6). Black column, group perfused with buffer; white column, group perfused with Ri7. Data are shown as means ± SEM. **, p < 0.01; ***, p < 0.001. Abbreviations: AF750, Alexa Fluor 750; Ri7, Ri7.217.1.4; IgG, control rat IgG2a (2A3); mAb, monoclonal antibody; TfR, transferrin receptor.

Figure 3. Decrease of accumulation of AF488-Ri7 in N2A and bEnd5 cells and higher expression of murine transferrin receptor (TfR) in N2A cells compared with bEnd5 cells. (A, B) N2A and bEnd5 cells were seeded in 96-well plates containing DMEM with 10% FBS. One day later, pretreatment for 15 min with unlabeled Ri7 or unlabeled IgG (0, 10, 50, or 250 nM) competed against the binding of AF488-Ri7. After further exposure to 5 nM AF488-Ri7 for 15 min, the cells were washed once with cold PBS and 0.025% trypan blue to quench noninternalized fluorescence (n = 2 experiments in triplicate). Fluorescence was measured with a Synergy HT plate reader (485/ 528 filters). (C) TfR content was measured by Western blotting in cultured N2A and bEnd5 cells and normalized with GAPDH. n = 4 dishes per cell line, 48 μg of proteins per lane. Data are shown as means ± SEM. Control values (without unlabeled mAb) were adjusted to 100%. *, p < 0.05; ***, p < 0.001. Abbreviations: AF488, Alexa Fluor 488; Ri7, Ri7.217.1.4; IgG, control rat IgG2a (2A3); RFU, relative fluorescence units; mAb, monoclonal antibody.

μg of AF750-Ri7 was significantly higher than that in animals perfused with control AF750-IgG (Figure 4A,B) indicating a significant accumulation of Ri7 vectors in the brain. On the basis of our in vitro data indicating that the signal associated with the IgG control was nonspecific, distribution volume (VD) values obtained in mice perfused with control AF750-IgG were subtracted from the data obtained with AF750-Ri7 to determine more accurately the VD of Ri7. From these values, the brain uptake coefficient (Clup) values of AF750-Ri7 were

estimated to be 0.27 and 0.26 μL g−1 s−1, for perfused doses of 100 and 200 μg, respectively. We verified the physical integrity of the BBB to ensure that Ri7 accumulation in the brain was not due to leakage of the BBB during perfusion. To that aim, we measured the brain vascular volume calculated by co-perfusing [14C]sucrose (Vvasc, μL g−1), a compound that does not cross the BBB and remains in the vascular space. All groups displayed Vvasc values of F



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approximately 15 μL g−1 (Figure 4C), consistent with an intact BBB. These data further confirm that the ISBP technique used here with AF750-Ri7 does not impair the integrity of the BBB. Linearity of the Brain Uptake of AF750-Ri7 over Time. To determine the appropriate perfusion time and to ensure that no metabolic changes occurred during the perfusion, it was important to investigate the linearity of AF750-Ri7 brain uptake over time. The time course of AF750-Ri7 brain uptake is presented in Figure 5. The distribution volume of AF750-Ri7

Figure 6. Decrease in brain uptake of mAb targeting the TfR (AF750Ri7). The brain uptake coefficient, Clup (μL g−1 s−1), of 100 μg of fluorolabeled Ri7 (AF750-Ri7) was determined after in situ brain perfusion. Co-perfusion with 400 or 900 μg of unlabeled Ri7 during 60 s impeded AF750-Ri7 uptake. Black column, AF750-Ri7; gray column, AF750-Ri7 + 400 μg of unlabeled Ri7; white column, AF750-Ri7 + 900 μg of unlabeled Ri7. Data were normalized by subtraction of background values (Bckd) obtained from mice perfused with perfusion buffer alone. Data are shown as means ± SEM of eight or nine mice per group. ***, p < 0.001. Also shown is an example of pseudocolored brain homogenates in a 96-well plate as detected with 720/790 filters. Abbreviations: AF750, Alexa Fluor 750; Ri7, Ri7.217.1.4; mAb, monoclonal antibody; TfR, transferrin receptor.

Figure 5. Time course of 100 μg of mAb targeting the TfR (AF750Ri7) uptaken by the right cerebral hemisphere after in situ brain perfusion, expressed as distribution volume VD (μL g−1). Regression analysis of individual data gave r2 = 0.997. Data were normalized by subtraction of background values (Bckd) obtained from mice perfused for 60 s with perfusion buffer alone. Data are shown as means ± SEM of nine mice per data point. Also shown is an example of pseudocolored brain homogenates in a 96-well plate as detected with 720/790 filters. Abbreviations: AF750, Alexa Fluor 750; Ri7, Ri7.217.1.4; mAb, monoclonal antibody; TfR, transferrin receptor.

the cerebral parenchyma, a fluorescence microscopy analysis was performed on sections of brain perfused with 100 μg of AF750-Ri7 in situ. As shown in Figure 8A, fluorescence from AF750-Ri7 was restricted to cerebral microvessels. Immunolabeling of anti-collagen IV, a basal lamina marker, confirmed the colocalization and accumulation of AF750-Ri7 in BCECs. Colocalization analyses with the neuronal marker NeuN were also performed, and no colocalization of AF750-Ri7 and neurons was observed. Finally, using the capillary depletion technique followed by Western blotting, we found that the TfR was concentrated in capillaries, compared with total brain homogenates from four mice (Figure 8B).

was linear over all of the perfusion time points (30, 60, and 120 s), confirming the stability of AF750-Ri7 and indicating that the transport rate remained unchanged over time and that the brain uptake was unidirectional33 from blood to brain over 120 s of brain perfusion. A perfusion time of 60 s was thus chosen for subsequent single-time-point experiments. Saturation of AF750-Ri7 Brain Uptake. To determine whether the brain uptake of AF750-Ri7 is saturable, increasing quantities of unlabeled Ri7 were co-perfused with a constant dose of AF750-Ri7. The brain uptake coefficient (Clup) of AF750-Ri7 was reduced significantly with co-perfusion of 400 μg or 900 μg of unlabeled Ri7 (Figure 6), down to the residual control values of AF750-IgG. To further confirm this result and to estimate the saturation point, increasing doses (50−1000 μg) of AF750-Ri7 were used next. The quantity of AF750-Ri7 per gram of brain tissue increased until a plateau was reached at approximately 300 μg (Figure 7A). The saturating perfused dose associated with 50% TfR occupancy (SD50) corresponds to half of the graphically determined maximum receptor population (Bmax). After subtraction of control values (nonspecific binding of AF750-IgG), curve fitting analysis resulted in an SD50 of approximately 226 μg (∼500 nM). Accordingly, the Clup was reduced progressively from 1.67 ± 0.15 μL g−1 s−1 for perfusion of 50 μg of AF750-Ri7 down to a plateau at ∼0.20 μL g−1 s−1 with a perfused dose above 400−500 μg (Figure 7B). Confinement of AF750-Ri7 in BCECs. Fluorescence quantification from brain homogenates does not discriminate parenchymal penetration from retention in BCECs. To verify whether the AF750-Ri7 signal originated from BCECs and/or

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DISCUSSION The results of this series of studies are consistent with the following conclusions. First, in vitro determination of AF488Ri7 uptake in mouse N2A and bEnd5 cells showed that the uptake mechanism involves a site-specific and saturable process through the TfR. An IgG control did not compete with the cellular accumulation of AF488-Ri7. Second, ISBP experiments demonstrated the accumulation of AF750-Ri7 into the brain, confirming the feasibility of combining NIR-fluorescent detection with ISBP. Third, we showed that the brain uptake of AF750-Ri7 is a saturable process, as the Clup values plummeted after the total perfused dose was increased. The saturating dose (SD50) of AF750-Ri7 was estimated to be 226 μg, equivalent to a concentration of ∼500 nM in the perfused buffer. Fourth, fluorescence microscopy analysis demonstrated that BCECs retained most of the AF750-Ri7 after ISBP. Quantitative assessment of the brain uptake of large molecules is an essential prerequisite for their development into clinical applications for central nervous system (CNS) diseases. Since fluorolabeling can be readily performed in large proteins, the present fluorescence-based ISBP technique stands as a method of choice for routine assessment of brain uptake of large G



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Figure 7. Saturation of brain uptake of mAb targeting the TfR (AF750-Ri7) after perfusion of increasing doses. (A) Cerebral concentrations (μg g−1) of AF750-Ri7 (n = 3−4) and (B) brain uptake coefficients Clup (μL g−1 s−1) (n = 3−4) were determined using in situ brain perfusion after normalization by subtracting the autofluorescence background obtained from mice perfused with perfusion buffer alone. Data are shown as means ± SEM. Also shown is an example of pseudocolored brain homogenates in a 96-well plate as detected with 720/790 filters. Abbreviations: SD50, perfused dose associated with 50% TfR occupancy; Bmax, maximum transferrin receptor population; AF750, Alexa Fluor 750; Ri7, Ri7.217.1.4; mAb, monoclonal antibody; TfR, transferrin receptor.

Figure 8. (A) Brain distribution of AF750-Ri7 after in situ brain perfusion of mAb targeting the TfR (AF750-Ri7). Mice were perfused with 100 μg of AF750-Ri7 targeting murine TfR during 60 s. The examples shown are representative of results obtained from three animals. Microscopy analysis showed that AF750-Ri7 (white) colocalized with the basal lamina marker collagen IV (green) on cerebral microvessels, whereas no signal was found in neurons immunostained with NeuN (red). Scale bars represent 50 μm. Abbreviations: AF750, Alexa Fluor 750; Ri7, Ri7.217.1.4; mAb, monoclonal antibody; TfR, transferrin receptor. (B) Western blots were performed to measure TfR expression in brain homogenates (H) and isolated capillaries (C) fractions from four mice. Data are shown as means ± SEM. **, p < 0.01; ***, p < 0.001.

biotherapeutics, provided that the fluorolabeling does not change the properties of the targeted biotherapeutic. Because of its large expression on microvessels arborizing throughout the brain and its capacity to ferry iron-bound Tf into the brain, the TfR has attracted a lot of interest for brain drug delivery.17,23,34 Our data confirmed that TfR expression is relatively higher in brain capillaries compared with other brain compartments. Vectors targeting the TfR include the murine OX-26 mAb for brain transport through the rat BBB20,35 and rat mAbs targeting the murine TfR, such as 8D3 and Ri7.21,36 These vectors have been conjugated to therapeutic peptide/ protein or liposomes for preclinical assays in various models of human diseases.16,25,37−40 Strong evidence of brain drug delivery has been gathered with OX-26 in the rat using various techniques,25,41−43 with the exception of reports using histochemistry and brain capillary depletion techniques.44,45 In the mouse, evidence of transport of Ri7 or 8D3 anti-TfR antibodies across the BBB21,36 has not been confirmed by a confocal microscopy report in which intravenously administered AF647-Ri7 remained confined to BCECs in the mouse.22 The disparity between these results may reflect differences in

experimental approaches, such as the method of terminal perfusion or the relative dilution of the anti-TfR mAb in a large postendothelial volume, hindering its detection in brain parenchyma cells. It should be kept in mind that the present ISBP method alone is not sufficient to conclude that full transport across the BBB occurred. Complementary microscopic data are necessary to assess the cellular brain distribution of a perfused fluorolabeled compound. Although the immunohistofluorescence data provided here corroborate the view that the total increase of Ri7 mAbs in the brain is mostly mediated by binding and endocytosis into BCECs, we cannot rule out the possibility that undetectable amounts of the mAb crossed BCECs and reached the brain parenchyma. H



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of Tf is spatially different from that of TfR-targeting mAbs.57,58 Another intriguing possibility raised recently is that the BCEC confinement of AF750-Ri7 observed here results from a strong affinity with the TfR. According to this hypothesis, only TfR mAbs with low affinity are ferried through BCECs and subsequently released across the BBB.59 Here, an SD50 of 500 nM is consistent with a relatively high binding affinity of Ri7 for the TfR, possibly explaining why AF750-Ri7 remains confined in BCECs after perfusion.22,59,60 The use of fluorescence-coupled ISBP as shown here circumvents the safety issues associated with radioactivity. Exposure to external radiation, even for medical purposes, increases the risk for cancer, and the American National Research Council concluded that no exposure level can be assumed to be truly safe.61 Despite drawbacks such as unavoidable background autofluorescence in tissues, limited stability, and light sensitivity, fluorescence detection is an analytical technique that is relatively simple to implement. Conjugation between fluorophores and amine or thiol groups on a protein can be done in the laboratory by application of routine-use protocols. Operating costs are also modest compared with those for radioactivity, allowing the use of larger quantities of fluorolabeled molecules without particular safety provisions. Finally, coupling of vectors to different fluorophores offers the possibility to detect several wavelengths simultaneously and combine homogenate quantification with microscopy.

The specificity of the Ri7 mAb for the TfR has been further confirmed in the present study on the basis of our in vitro and in vivo data. The Ri7 mAb was initially chosen in this study because of its specific uptake by the brain compared with 8D3, which is also significantly taken up by the liver and kidney.21 In vitro experiments confirmed that accumulation of AF488-Ri7 in the two cell lines N2A and bEnd5 was inhibited by increasing concentrations of unlabeled Ri7, with no interference from the isotype control (IgG), indicating that the cellular uptake of Ri7 requires specific binding with its receptor. This result was consistent with in vitro work using micelles complexed to Tf46 or transfection experiments with liposomes conjugated to 8D3 or Ri7.47 Endocytosis is an active process that is slowed at reduced temperatures.48−50 Our findings that the vesicular-like signal associated with AF488-Ri7 accumulates in cells at 37 °C but is confined to the cell surface at 4 °C argues in favor of an endocytotic process. In vivo competition, as assessed with ISBP, further supports specific binding of Ri7 to the luminally exposed TfR in brain microvessels. From a quantitative perspective, the brain uptake rate of Ri7 reported here remained relatively low, at least at high concentrations. Perfusion of 100 μg of AF750-Ri7 led to a Clup of 0.27 μL g−1 s−1 after correction for nonspecific signal. The relevance of such a correction is supported by in vitro and in vivo data showing that IgG binding was nonspecific. In contrast, the uptake of AF750-Ri7 reached 1.47 ± 0.15 μL g−1 s−1 when 50 μg was perfused (∼110 nM), consistent with a saturable mechanism. Nevertheless, our data stand in relative agreement with the results of previous studies using comparable perfusion techniques after conversion into similar units. In the rat brain, the Clup of the radiolabeled anti-TfR vector OX-26 was approximately 0.07 μL g−1 s−1 using transcardial perfusion,44 whereas the Clup of OX-26 and Tf itself ranged between 0.08 and 0.18 μL g−1 s−1 using ISBP.41 Still, these values remain relatively low compared with those for fully diffusible compounds such as diazepam and fatty acids, which display Clup values of up to 40 μL g−1 s−1.11,51 In contrast, a routine-use CNS drug such as morphine, which is partially pumped out of the brain by the ABC-transporter Pglycoprotein (Pgp), displays a Clup of 0.3 μL g−1 s−1.52 Thus, the measured brain uptake of Ri7 is consistent with previous reports and stays within an acceptable range for drug delivery purposes. Our present in vitro and in vivo data argue for a saturable mechanism underlying AF750-Ri7 accumulation in brain cells. The transport rate of AF750-Ri7 became negligible at concentrations over 500 nM, evidencing complete saturation of TfR-mediated transport in vivo. Such an interpretation is in line with previous data on saturable transport of perfused Tf or OX-26 in the rat brain.41,53 Similarly, uptake of radiolabeled 8D3 or AF750-Ri7 was fully inhibited by intravenous coinjection of a saturating dose of unlabeled mAb.21,22 Our ISBP approach allowed the calculation of a saturating perfused dose (SD50), which was estimated to be 226 μg, corresponding to a concentration of ∼500 nM; this value is not very far from the dissociation constant of human Tf to the TfR, which has been estimated to be slightly above 700 nM.54 Still, these numbers remain below the physiological concentration of Tf (25 μM or 2000 μg/mL),55 suggesting that TfR at the BBB is fully saturated under physiological conditions.34 Nevertheless, systemically administered fluorolabeled/radiolabeled Ri7 or Ri7-loaded nanocarriers in rodent accumulate in the brain,21,22,56 supporting the hypothesis that the binding site

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CONCLUSION In summary, this study confirms the accumulation of the antiTfR mAb AF750-Ri7 into BCECs of mice within minutes after intracarotid perfusion. Taken together, the results are consistent with TfR-specific unidirectional brain uptake of AF750-Ri7 through a fully saturable mechanism. The present work thus offers a novel application of ISBP to provide quantitative data on the brain transport of large fluorolabeled molecules, avoiding the use of radioactivity.

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AUTHOR INFORMATION

Corresponding Author

*Address: Neurosciences Axis, Centre de recherche du CHU de Québec, 2705 Laurier Blvd., Room T2-05, Québec, QC G1V 4G2, Canada. Tel: (418) 656-4141 ext. 48697. Fax: (418) 6542761. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Melissa Ouellet for the illustrative schema of the in situ brain perfusion technique and Eric Béliveau for the Z-stack deconvolution. Financial support was provided by the Canadian Institute of Health Research (CIHR, MOP84251) and the Canada Foundation for Innovation.

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