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Bioconjugated Quantum Rods as Targeted Probes for Efficient Transmigration Across an in Vitro Blood-Brain Barrier Gaixia Xu,†,§ Ken-Tye Yong,† Indrajit Roy,† Supriya D. Mahajan,‡ Hong Ding,† Stanley A. Schwartz,‡ and Paras N. Prasad*,† Institute For Lasers, Photonics And Biophotonics, The State University of New York, Buffalo, New York 14260-3000, Department of Medicine, Division of Allergy, Immunology, and Rheumatology, Buffalo General Hospital, Buffalo, New York 14203 and Institute of Optoelectronics, ShenZhen University, China, 518060. Received December 21, 2007; Revised Manuscript Received March 25, 2008
We report here, what we believe to be the first time, the successful transport of bioconjugated quantum rods (QRs) across an in vitro blood-brain barrier (BBB) model via a receptor-mediated transport, as well as the use of QR multiplexing technique to compare simultaneously the transmigration efficiency of different biomolecules across the BBB. The migration rate of bioconjugated QRs crossing the in vitro BBB was found to be concentrationand time-dependent. This work illustrates a nanoparticle-based platform that will not only allow a direct visualization of the transmigration ability of various kinds of biomolecules across the BBB, but also facilitate the development of novel diagnostic and therapeutic nanoprobes for early diagnosis and therapy of various disorders of the brain following systemic administration.
INTRODUCTION The blood-brain barrier (BBB) is a dynamic interface between the body and the brain, which is actively engaged in regulatory functions of the central nervous system (CNS), in addition to its primary role of protecting the brain from harmful substances and fluctuations in blood (1–3). Brain microvascular endothelial cells (BMVECs) composing the BBB are underlined by a continuous basement membrane, reinforced by pericytes and astrocytic endfeet, and are connected with adjacent endothelial cells (4–9). Owing to the presence of continuous strands of tight junctions between adjacent lateral endothelial membranes, the BBB is almost completely sealed, and therefore, it strictly limits the entrance of systemically circulating endogenous and exogenous compounds into the CNS (3, 10, 11). Thus, while the BBB constitutes a natural defense mechanism that safeguards the brain against the invasion of various circulating toxins and infected cells, it also offers a significant impediment toward the delivery of various diagnostic and therapeutic agents in the brain via the systemic route (4, 12). Therefore, targeted delivery across the BBB is one of the most challenging fields of research dealing with the treatment of various neurological disorders (13, 14).1 In general, transport mechanisms across the BBB can be broadly divided into three types, namely, passive, carriermediated, and vesicular transport (6). For example, lipid-soluble, nonpolar substances can enter the brain by passive diffusion across the BBB. On the other hand, polar substances and small peptides can be transported across the endothelium by carriermediated influx. Lastly, the vesicular transport is by either receptor-mediated or absorptive-mediated transcytosis via cat* Corresponding author. E-mail:
[email protected]. † The State University of New York. ‡ Buffalo General Hospital. § ShenZhen University. 1 Abbreviations: CNS, central nervous system; BBB, blood-brain barrier; QD, quantum dot; QR, qauntum rod; Tf, transferrin; TfR, transferrin receptor; aC4, anti-Claudin 4; BMVECs, brain microvascular endothelial cells; NHAs, normal human astrocytes.
ionic proteins. However, to date, there are still many unknown and undiscovered parameters that are affecting the permeability of the BBB (10, 15). Thus, it is of great importance to systematically investigate the molecular composition, structure, and transport kinetics of the BBB under a variety of conditions in order to comprehend the underlying mechanisms of BBB permeability, which will help us to devise novel strategies for enhanced delivery of various neuropharmaceuticals into the brain. Currently, nanotechnology is being investigated for overcoming the limited penetration of drugs through the BBB, which is an extremely important and evolving area in CNS pharmacology (16–20). To date, various types of nanoparticles and liposomes have been synthesized and used as drug carriers or contrast agents for targeted drug delivery and diagnosis of brain tumors across the BBB (17, 21–24). It is generally understood that drugloaded nanoparticles can cross over the BBB as an intact entity, which will then release the drugs within the CNS. Though few liposomal and polymeric nanoparticles have been used for targeted delivery across the BBB, there have been no reports of using high-quality quantum dots (QDs) or quantum rods (QRs) as targeted probes to monitor the transmigration of specific molecules through BBB. The use of QD/QRs as luminescence probes for numerous biological and biomedical applications has become an area of intense research focus over the past few years (25–28). They offer several advantages over organic dyes, including increased brightness, stability against photobleaching, excitation within a broad spectral range, and a tunable and narrow emission spectrum. The QD/QR surface can be functionalized with a variety of biomolecules and have shown the potential to dramatically outperform conventional organic dyes in imaging of cellular and subcellular structures and in a variety of bioassays (29–31). Recently, targeted QD/QR-based probes have been modified to carry therapeutic molecules such as anticancer drugs and short interfering (si) RNA (32, 33). Recently, our group has shown that the QR/QD bioconjugates can serve as targeted optical probes for two-photon fluorescence imaging of cancer cells (34, 35). Here, we further extended the application of QR bioconjugates as efficient targeted probes for
10.1021/bc700477u CCC: $40.75 2008 American Chemical Society Published on Web 05/13/2008
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Figure 1. TEM images of CdSe/CdS/ZnS QRs cast from aqueous dispersions (a,c). The red- (a) and orange- (b) emitting QRs have average lengths of 26 and 14 nm and diameters of 6.5 and 4 nm, respectively. UV-vis-NIR absorption (green) and PL emission (red) spectra of monodispersed red- (b) and orange- (d) emitting CdSe/CdS/ZnS QRs.
transmigration across the BBB. A validated endothelial and astrocytic coculture BBB model was used to examine the transmigration of QR bioconjugates across the BBB (36–38). It is known that the transferrin receptor (TfR) is a kind of specific BBB transporter that allows selected biomolecules to move across the BBB (39). Transferrin (Tf)-conjugated QRs were synthesized and used for transport across the in vitro BBB model via a receptor-mediated transport mechanism. It was found that the migration rate of Tf-conjugated QRs crossing the in vitro BBB is concentration- and time-dependent. Further, another tight-junction protein Claudin-4 was used concurrently to validate the multiplex imaging technique and also to evaluate quasi-quantitatively the comparative transmigration efficiency and specificity of Tf and anti-Claudin 4 antibody (aC4), by conjugating them with red and orange emitting QRs, respectively. The results showed higher transmigration efficiency of Tf-conjugated QRs over the aC4-conjugated ones across the BBB. To the best of our knowledge, this is the first time that the QR multiplexing technique is employed to compare the targeting efficiency of different specific molecules across the BBB. These results illustrate a nanoparticle-based platform that will not only allow a direct visualization and quantification of the transmigration ability of various kinds of biomolecules across the BBB, but also facilitate the development of novel diagnostic and therapeutic nanoprobes for early diagnosis and therapy of various disorders of the brain following systemic administration.
RESULTS AND DISCUSSION Figure 1a,c shows TEM images of red- and orange-emitting CdSe/CdS/ZnS QRs cast from aqueous media, respectively. The red- and orange-emitting QRs have lengths of 26 and 14 nm,
Figure 2. Time-dependent hydrodynamic diameter of lysine coated QRs dispersed in PBS buffer.
and diameters of 6.5 and 4 nm, respectively. The absorption and PL spectra of the red and orange QRs are also shown in Figure 1b,d, respectively. Absorption spectra of the QRs show the expected structure with absorption onsets of 615 and 589 nm for red- and orange-emitting QRs, respectively. The PL spectra of the red- and the orange-emitting monodispersed QRs show band edge emission at 658 and 608 nm. Thus, we refer to the red- and orange-emitting QRs as 658 nm QRs and 608 nm QRs, respectively. These stored QRs can be directly used for cell staining without any sign of quenching effects. The photoluminescence quantum yields (QY) of 608 and 658 nm
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Figure 3. Confocal microscopic images of BMVEC and NHA cells, treated with (a,d) lysine-coated CdSe/CdS/ZnS QRs. (c,f) Tf-conjugated CdSe/CdS/ZnS QRs. Cells in (b) and (e) were saturated with free Tf for 2 h, before treatment with QR-Tf. Confocal microscopy images were obtained with laser excitation at 405 nm.
QRs are estimated to be 30% and 50%, respectively, which are sufficiently high for bioimaging. In this study, dynamic light scattering (DLS) was also used to determine the colloidal stability of the QRs dispersed in PBS (pH ) 7.4). The time-dependent profile of the effective diameter of the QRs is shown in Figure 2. Over the time range from 0 to 600 min, the effective diameter varies by less than 10%, suggesting that their colloidal stability is not affected under physiological pH. It is worth mentioning that the size of the QRs is ∼26 nm in length and ∼6.5 nm in width and their total hydrodynamic size is further increased by the MUA and lysine coating. Therefore, light scattering data show a total hydrodynamic diameter of ∼60 nm. The light scattering analysis assumes that particles are spherical. Thus, the actual sizes determined by light scattering in our case cannot be taken literally. Therefore, we want to stress that the light scattering technique used here is solely to prove the nonaggregation of the QRs under physiological conditions. To employ QRs as an efficient targeted contrast agent for traversing the BBB, the QRs must be conjugated with specific biorecognition molecules. To date, it is well-known that the transferrin receptor (TfR) is highly localized on the endothelial surfaces of the brain. TfR will generally trigger the receptormediated transport across the BBB because it undergoes trancytosis through the BBB. Thus, transferrin (Tf), an irontransporting protein, was chosen as one of the specific ligands for facilitating transport across the BBB. Prior to pursuing the in vitro BBB model studies, primary human BMVEC and normal human astrocyte (NHA) cells were individually studied to confirm the selective uptake of the TfQR biocojugates. Figure 3c,f represents confocal images showing robust staining of BMVEC and NHA cells, respectively, following treatment with Tf-QRs conjugates. Minimal uptake was observed with unconjugated QRs (Figure 3a,d). This result suggests that the Tf-conjugated QRs are transported within BMVEC and NHA cells through the transferrin receptor-
Figure 4. Fluorescence signal distribution collected from BMVEC and NHA cells following treatment with non-bioconjugated (QR only) and bioconjugated (QR-Tf) QRs. The effect of saturating cells with free Tf prior to treatment with QR-Tf is also represented. The error bars represent the range that was obtained for each point from a total of four points.
mediated endocytosis pathway. To further confirm the targeted delivery of QR-Tf bioconjugates is receptor-mediated, the BMVEC and NHA were presaturated with free Tf in order to block the available Tf receptors on the cell surface. The results indicated significant reduction in the uptake of QR-Tf bioconjugates (Figure 3b,e), suggesting the specific nature of the uptake (34, 35). In addition to these experiments, we have also performed local spectral analysis in order to quantify the cellular uptake of the nanoparticles in the various conditions given above. The local spectra were plotted as an average of spectral intensity obtained from different random points within the confocal fluorescence images. Figure 4 shows that the fluorescence signal obtained from the cells (both BMVECs and NHAs)
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Figure 5. Confocal microscopic image of upper and bottom layer of BBB model treated with Tf-conjugated red QRs. In all cases, green represents emission the autofluorescence and red represents emission from 658 nm QRs.
following treatment with the QR-Tf bioconjugates is significantly higher than that obtained from cells treated with nonbioconjugated QRs and QR-Tf bioconjugates following presaturation with free Tfs. This result presents a semiquantitative estimation that reflects the superiority of the receptormediated cellular uptake over passive uptake. On the basis of the successful in vitro experiment on the individual cell lines, we hypothesized that these bioconjugates could be used as efficient targeted probes for crossing the BBB. In this work, the bioconjugates were used to study the transmigration activity across an in vitro BBB model, which is established using primary human BMVEC and NHA cells. A schematic drawing of the BBB model is presented in the Supporting Information (Scheme 1). This in vitro BBB model consists of two-compartment wells in a culture plate. After formation of the model, Tf-QR bioconjugates were introduced into the upper chamber (blood end of the model) and incubated for different time points and bioconjugate concentrations. Next, the media from upper and bottom (brain end of the model) chambers were separately collected and the photoluminescence (PL) of the media was measured using a spectrofluorimeter in order to estimate the transmigration rate of the bioconjugates across the BBB. Confocal imaging technique was used to study and examine the BMVEC-PET-NHA cell layers (both upper and bottom sides) labeled with the QR bioconjugates. Figure 5 shows the confocal images of upper and lower sides of the BBB model incubated with the Tf-QR bioconjugates at 6, 26, and 42 h, respectively. The autofluorescence signal (green) from the cells was used as the background for comparison. The upper panel shows confocal images of the BMVEC layer and the bottom panel is for the NHA layer. As shown in Figure 5, the uptake of Tf-QRs in both upper and bottom cell layers increases with the incubation time. This suggests that the migration of the Tf-QR conjugates is driven by both receptor mediated and kinetic processes. To confirm the receptor-mediated uptake process, control experiments were performed using unconjugated QRs incubated
Figure 6. Relationship between fluorescence intensity changes of medium and the incubation time.
with the BBB model. After 26 h, the upper and bottom layers were carefully rinsed with PBS solution and examined under a confocal microscope. As expected, minimal uptake was observed in the BBB model treated with unconjugated QRs upon comparison to the Tf-QRs (Supporting Information, Figure S1). To further probe efficiency of permeability of Tf-QRs across the BBB, the PL intensities of the media from the upper and bottom chambers of the BBB model were systematically measured as a function of treatment time and analyzed. Figure 6 shows the band edge emission of the Tf-QRs in the upper and bottom chambers of the BBB model treated for various incubation times. Although there is clear time dependence, the upper medium retains more than 70% of their initial PL intensity, even at 26 h. Over the time range of 26 to 42 h, the PL intensity varies by more than 30%. This suggests that there is a critical period for significant passage of Tf-QRs through the BBB that is greater than 26 h.
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Figure 7. Confocal microscopic image of upper and bottom layers of the BBB model treated with ∼0.56, 2.3, and 9.2 pmol of Tf-conjugated red QRs. In all cases, green represents emission of the autofluorescence and red represents emission from 658 nm Tf-QRs.
To better understand the effects of amount of Tf-QR bioconjugates on the permeability and transport kinetics across the in vitro BBB model, three different concentrations of TfQRs were used. Figure 7 presents the confocal images of the upper and lower sides of the BBB model incubated with ∼0.56, 2.3, and 9.2 pmol of Tf-QRs at 26 h. From the confocal microscopy analysis, we find that the quantities of Tf-QRs labeled on both BMVEC (upper) and NHA (lower) cell layers increase with the increment of the Tf-QR concentration. However, when the Tf-QR concentration exceeded 9.2 pmol, a large amount of morphologically damaged cells was observed, suggesting that this concentration is the maximum tolerated dose prior to the onset of cytotoxicity. In addition to conjugation of Tf, we have also conjugated aC4 with QRs for evaluating the specificity of different targeting molecules for BMVECs and NHAs. To compare the targeting specificity of aC4 and Tf on the cells, a multiplexed imaging technique was used by employing two different colors of QRs. More specifically, the 608 nm QRs were conjugated with aC4 and the 658 nm QRs were conjugated with Tf for specific targeting to Claudin 4 and transferrin receptors, respectively, on the BBB (and its constituent cells). Figure S2 (Supporting Information) shows confocal images of BMVEC and NHA cells colabeled with 4.0 pmol aC4-QRs (608 nm) and 0.46 pmol TfQRs (658 nm). Although the aC4-QRs (608 nm) is added about in a concentration 8.7 times than that for Tf-QRs (658 nm), the uptake of the 658 nm QR bioconjugates is slightly higher than that for the 608 nm QRs, as seen from the localized spectral analysis. The uptake concentration of Tf-QR bioconjugates was estimated to be twice the amount of aC4-QRs for both BMVEC and NHA cells (see Supporting Information Table S1). This experiment clearly demonstrates the Claudin 4 antigen also mediates targeting on BMVEC and NHA cells, but the targeting efficiency is significantly lower than that using the transferrin receptor. The same formulation was further employed to compare the transmigration efficiency of aC4 and Tf for the in vitro BBB model. Figure 8 shows confocal images and spectra of the upper and bottom cell layers of the intact BBB model, again
demonstrating superior staining/transmigration of the Tf-QRs (658 nm) over the aC4-QRs (608 nm). Local spectral analysis of the overall labeling of bioconjugates on the cells confirms that the fluorescence signal from the Tf-QR (658 nm) bioconjugates is more intense than that from the aC4-QR (608 nm) bioconjugates. Here, it was found that the uptake concentration of Tf-QR bioconjugates was three times the concentration of aC4-QRs at the bottom layer (see Supporting Information Table S1). From these results, it appears that it is more efficient to use the Tf-conjugated QRs for transport across the BBB, which agrees with the fact that the transferrin receptors are overexpressed in the BBB cells. Therefore, upon comparing the targeting molecules used in this work, Tf has higher affinity for individual cells lines composing the BBB and better transmigration efficiency across the in vitro BBB model. In comparison the aC4-QRs (608 nm), although can be targeted to the individual cells, are less suited as a nanocarrier to pass through the BBB owing to their lower tansmigration ability. This multiplexing technique can provide a novel nanophotonic method to screen a number of potential ligands for targeted delivery of diagnostics and therapeutics across the BBB, which will shorten the research cycle of CNS drug discovery and screening. Besides using the QRs as biomarkers, we have also systematically investigated their cyctotoxicity effects on the BMVEC and NHA cell lines. Figure S3 (Supporting Information) shows the in vitro cytotoxicity effects of lysine-coated QRs, Tf-QRs, and aC4-QRs on the BMVEC and NHA cell lines. All the bioconjugates maintained greater than 80% cell viability, even at particle concentration as high as 7.9 pmol. This indicates that such bioconjugates can be used as efficient nanoprobes at noncytotoxic dosages. In conclusion, we have used photostable, water-dispersible lysine-coated CdSe/CdS/ZnS QRs as efficient targeted probes for monitoring transport across an in vitro BBB model. The lysine-coated QRs can be readily conjugated with Tf and aC4 for specific targeting of the BBB cells. The receptor-mediated uptake of Tf-QR bioconjugates into the BMVEC and the NHA cells was confirmed by presaturating the cells with free Tf,
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Figure 8. Confocal microscopic images and the local spectra of upper and bottom layer of the BBB model treated with multiplexed bioconjugates. The BBB model was treated with both aC4-conjugated 608 nm QRs and Tf-conjugated 658 nm QRs for 26 h incubation. In all cases, blue represents emission of the autofluorescence, green represents emission from 608 nm QRs, and red represents emission from 658 nm QRs. Confocal microscopy images were obtained with laser excitation at 405 nm. Scale bar: 47.62 µm.
thereby drastically lowering the uptake of the Tf-QR bioconjugates. This work demonstrates a nanoparticle-based platform that will not only allow a direct visualization of the transmigration ability of various kinds of biomolecules across the BBB, but also facilitate the development of novel diagnostic and therapeutic nanoprobes for early diagnosis and therapy of various disorders of the brain, following systemic administration. QRs, owing to their high surface area, can be coincorporated with other diagnostic and therapeutic molecules for targeted therapy of CNS disorders. Development of such multifunctional QRs with combined diagnostic, targeting, and therapeutic molecules are in progress.
ACKNOWLEDGMENT This study was supported by grants from the NCI RO1CA119397 and the John R. Oishei Foundation. Support from the Center of Excellence in Bioinformatics and Life Sciences at the University at Buffalo is also acknowledged. Supporting Information Available: Description of the materials, experimental methods, supplementary figures and
table. This material is available free of charge via the Internet at http://pubs.acs.org.
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