Multifunctional Bioconjugate for Cancer Cell-Targeted Theranostics

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A Multifunctional Bioconjugate for Cancer Cell-Targeted Theranostics Wei Du, Yue Yuan, Lin Wang, Yusi Cui, Hui Wang, Huiqin Xu, and Gaolin Liang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.5b00570 • Publication Date (Web): 18 Nov 2015 Downloaded from http://pubs.acs.org on November 20, 2015

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Bioconjugate Chemistry

A Multifunctional Bioconjugate for Cancer Cell-Targeted Theranostics ⊥

Wei Du,† Yue Yuan,† Lin Wang, Yusi Cui,† Hui Wang,‡ Huiqin Xu,*,‡ and Gaolin Liang*,† †

CAS Key Laboratory of Soft Matter Chemistry, National Synchrotron Radiation Laboratory, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡ Department of Nuclear Medicine, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei, Anhui 230022, China ⊥

School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China

ABSTRACT: Cancer cell-targeted imaging and drug delivery remain a chanlleg for precise cancer theranostics. MUC1 is a large transmembrane glycoprotein that may potentially serve as a target for cancer theranostics. Herein, using a MUC1-targeting aptamer (APT) as the “warhead”, we rationally designed and constructed a hybrid nanoparticle 1-NPs-QDs-hAPT (Vehicle) that could be applied for MUC1-targeted cell uptake and imaging. By intercalating different Vehicle amounts with the anticancer drug doxorubicin (DOX), we obtained the multifunctional bioconjugate Vehicle-DOX with a maximized drug payload and DOX fluorescence quenching capability. Confocal microscopy cell imaging indicated that Vehicle-DOX could be used to track MUC1-targeted drug release. A cytotoxicity study indicated that Vehicle-DOX could be applied for MUC1-targeted cytotoxicity. We anticipate that our multifunctional bioconjugate Vehicle-DOX could be applied for in vivo tumor-targeted theranostics.

INTRODUCTION Cancer remains one of the leading causes of death worldwide. According to statistics from the World Health Organization (WHO), cancer caused 8.2 million deaths in 2012. Despite rapid developments in cancer diagnostics and treatments, the overall cancer survival rate has not substantially improved in recent decades.1 Selective targeting and delivery efficiency remain two major challenges facing the development of therapeutic and diagnostic agents for cancer theranostics.2,3 Multifunctional nanoparticles (NPs) possess unique advantages for the cancer-specific delivery of imaging and therapeutic agents.4,5 Due to their high surface area-to-volume ratio, NPs possess a large payload, a high signal intensity and stability, and the capacity to enact multiple and simultaneous applications.6,7 In addition, NPs preferentially accumulate at tumor sites due to the pathophysiologic characteristics of tumor blood vessels, resulting in the enhanced permeability and retention (EPR) of NPs.8,9 Thus, multifunctional NPs, which can function in targeting, imaging, and therapy, have been intensively studied to overcome the conventional limitations of cancer diagnosis and therapy.10-14 MUC1 is a well-characterized, large transmembrane glycoprotein that acts as a target in cancer therapy. In most malignant adenocarcinomas, including breast cancer, lung cancer, and colon cancer, the expression of MUC1 is increased by at least ten-fold, making it an attractive biomarker for cancer-targeted therapies.15 The use of MUC1-targeting aptamers for theranostic applications such as cell labeling,16 cancer diagnostics and imaging,17-19 and targeted cancer therapy20,21 has been reported. Among MUC1-targeting aptamers, S2.2 (5’-GCA GTT GAT CCT TTG GAT ACC CTG G-3’), has been reported to bind MUC1 proteins with high affinity and specificity.22, 23

Scheme 1. Schematic illustration of the procedures for preparing the bioconjugate Vehicle-DOX.

Inspired by these poineering studies, we rationally constructed, as shown in Scheme 1, a type of multifunctional bioconjugate (referred to as Vehicle-DOX) for MUC1-targeted imaging and cytotoxicity of the human breast cancer cell line MCF-7. Vehicle-DOX is composed of four components: the backbone nanoparticle 1-NPs, the imaging agent [CdTe/CdS quantum dots (QDs)], the targeting “warhead” hairpin aptamer (hAPT), and the anti-cancer drug doxorubicin (DOX). In detail, the backbone 1-NPs is a type of organic, oligomeric nanoparticle that self-assembled from the monomer Cys(SEt)Lys[Cys(SEt)]CBT (1). Upon treatment with tris(2-carboxyethyl)phosphine (TCEP), 1 undergoes a “click” condensation reaction24-27 to yield

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amphiphilic oligomers, which rapidly self-assemble into 1-NPs with abundant hydrophilic reactive groups (-SH and -NH2) on its surface. Then, the highly luminescent, carboxylated, NIR-emitting CdTe/CdS core/shell QDs reacted with the -NH2 groups of 1-NPs to prepare 1-NPs-QDs via classical 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS) activation chemistry. Subsequently, the maleimide motifs on the 5’-NH2C6APT derivative (Mal-APT) react with the -SH groups on the 1-NPs via the thiol-ene click reaction,28 resulting in 1-NPs-QDs-APTs. To encapsulate a drug for delivery, the linear aptamer must be renatured into a hairpin confirmation. Thus, the APTs on 1-NPs-QDs-APTs were renatured to yield 1-NPs-QDs-hAPTs (Vehicle), whose hairpin APTs (hAPTs) retained the ability to target MUC1. Physical loading of DOX is an efficient way for its delivery.29-31 DOX is known to preferentially intercalate double-stranded 5’-GC-3’ or 5’-CG-3’ sequences32, 33 and its fluoresence is quenched after intercalation.34, 35 We therefore chose DOX for preparation of the multifunctional bioconjugate Vehicle-DOX for cancer celltargeted imaging and therapy.

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efficiently washed off (Figure S6b). Thus, the molar ratio 1/100 between Mal-APT and 1-NPs was employed for the preparation of 1-NPs-QDs-APT for all subsequent experiments.

RESULTS AND DISCUSSION Preparation and Characterization of 1-NPs-QDs-APT (Vehicle). We began this study by synthesizing of monomer 1 (Figure S1) and by performing the reduction-controlled condensation of 1 and the self-assembly of 1-NPs. At a concentration of 1 mM, 1 was dissolved in water containing 5% methanol. Five min after the addition of 4 equivalent amounts of TCEP and adjusting the pH to 7.4 with sodium carbonate, we observed that the clear 1 solution became a turbid dispersion and its UV-vis absorbance at 500–700 nm increased markedly (Figure S2), suggesting the formation of nanoparticles (i.e., 1-NPs). Observation by transmission electron microscopy (TEM) indicated that 1-NPs tended to crosslink with each other (Figure 1a), probably due to the formation of disulfide linkages among the nanoparticles during TEM sample preparation. Further characterization by cryo-TEM suggested that 1-NPs were roundish nanoparticles but not polymorphic aggregates (Figure S4). Statistical analysis indicated that 1-NPs have an average diameter of 46.4 ± 6.7 nm (Figure S3a). We next prepared the 1-NPs-QDs conjugates. First, QDs-NHS was freshly prepared by mixing CdTe/CdS QDs (500 μL, 0.2 mg/mL) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 10 μL, 0.2 M) and Nhydroxysuccinimide (NHS, 10 μL, 0.2 M), and the mixture was then stirred for 30 min. The mixture was desalted by ultrafiltration using a Microcon 3-kDa molecular weight cut-off (MWCO) ultrafilter (Millipore), followed by washing (3 × 1 mL H2O). The freshly prepared QDs-NHS was resuspended in PBS buffer to prepare serial dilutions at 0.01, 0.05, 0.1, 0.5, and 1 mg/mL. Then, 50 μL of freshly prepared monodispersed 1-NPs was added to 50 μL of each above QDs-NHS dilution and stirred overnight in the dark. After centrifugation and washing (3 × 100 μL PBS buffer) to remove unconjugated QDs-NHS, 1-NPs-QDs with different QD payloads were obtained. Mal-APT was prepared with 4-maleimidobutyric acid (Mal) and 5’NH2C6 APT using EDC·HCl and NHS as the coupling agents (Figure S5). Before the addition of Mal-APT to 1-NPsQDs to prepare 1-NPs-QDs-APT, we optimized the reaction ratio between 1-NPs and Mal-APT. Mal-APT was reacted with 1-NPs at the molar ratios 1/1, 1/20, 1/40, 1/100, or 1/140 (note that the molarity of 1-NPs here was calculated in 1), and the reaction mixtures were subjected to agarose gel electrophoresis. The results indicated that at a Mal-APT to 1-NPs molar ratio of 1/100, there was maximal binding of Mal-APT using a minimal amount of 1-NPs (Figure S6a). In addition, the unbound aptamers were

Figure 1. TEM images of 1-NPs (a), 1-NPs-QDs (b), 1-NPsQDs-APT (c), and NPs-QDs-hAPT (Vehicle) (d). Insets: HRTEM images showing the crystal lattice of CdTe/CdS QDs. (e) Fluorescence spectra of as-prepared 1-NPs, 1-NPs-QDs, 1-NPsQDs-APT, 1-NPs-QDs-hAPT (Vehicle), and QDs at 0.1 mg/mL, respectively. Excitation: 500 nm. Four equivalents of TCEP were added into each of the aforementioned serial dilutions of 1-NPs-QDs. Five min later, the pH value was adjusted to 7.4 using sodium carbonate, followed by the addition of Mal-APT (1/100 of 1-NPs). Then, the reaction mixtures were ultrasonicated for 30 min and oscillated overnight in the dark to yield 1-NPs-QDs-APT dilutions after centrifugation. Then, the dilutions were washed with 3 × 50 μL PBS buffer (pH 7.4). Next, the 1-NPs-QDs-APT dilutions were renatured in PBS buffer (pH 7.4) to yield 1-NPs-QDs-hAPT (Vehicle) stocks using the following protocol: the dilutions were heated to 94 °C at 2.5 °C/s, held at 94 °C for 5 min, cooled to 4 °C at 0.1 °C/s, and held at 4 °C. Then, the 5 Vehicle stocks containing APT hairpin structures and different amounts of QDs were used for cell imaging to identify the most suitable QDs/1-NPs (or QDs/hAPT) ratio for use in cancer cell-targeted theranostics (i.e., the minimum amount of QDs for satisfactory imaging balanced with maximum hAPT for DOX encapsulation). Each of the 5 Vehicle stocks was dissolved in 1 mL serum-free culture medium and then incubated with MCF-7 cells for 1.5 h at 37 °C. The cells were washed with PBS several times to remove the free Vehicle prior to imaging. As shown in Figure S7, the amount of QDs on the Vehicle increased and the red fluorescence from the QDs was enhanced, while the blue fluorescent signal from 1-NPs weakened. This result indicates that as more QDs were loaded onto the 1-NPs, less Vehicle entered the cells. This finding is consistent with the fact


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Figure 2. Testing binding specificity. Fluorescence (top, Texas Red channel) and overlay (bottom, Differential interference contrast (DIC) with fluorescence) images of MCF-7 (left) or HepG2 (middle left) cells after incubation with 50 μM Vehicle at 37 °C for 2 h. Fluorescence and overlay images of MCF-7 (middle right) or HepG2 (right) cells after incubation with 50 μM 1-NPs-QDs at 37 °C for 2 h. Scale bar: 20 μm. that a single QD is much larger than a singer hAPT;36 with an increase in QDs on the surface of the Vehicle, fewer interactions occurred between the hAPT and MUC1 on the cell membrane. To achieve a maximum drug payload while maintaining satisfactory imaging capacity, we chose the Vehicle containing 0.1 mg/mL CdTe/CdS QDs for the following experiments. Using the optimized conditions, we successfully prepared 1NPs-QDs, 1-NPs-QDs-APT, and 1-NPs-QDs-hAPT (Vehicle) and characterized them using TEM, cryo-TEM, and fluorescence spectra. Figure 1b shows a TEM image of 1-NPs-QDs, which has an average diameter of 35.5 ± 6.2 nm (Figure S3b). High resolution (HR) TEM imaging clearly indicates the crystal lattice structure of CdTe/CdS QDs, suggesting that the covalent conjugation of QDs to 1-NPs was successful (inset of Figure 1b). Compared to the fluorescence emission peak at 728 nm of the free QDs (intensity 3586), as-prepared 1-NPs-QDs have a 61.0% relative fluorescence emission peak at 732 nm (intensity 2188) (Figure 1e), suggesting that approximately 39.0% of the QDs were not conjugated to 1-NPs and were washed out during the preparation. Figure 1c shows a TEM image of 1-NPs-QDs-APT, and the inset HR-TEM image shows the crystal lattice structure of the QDs on these conjugates. These images suggested that conjugation of Mal-APT to 1-NPs-QDs induced neither the release of QDs from the conjugates nor a size change of the conjugates. Compared with 1-NPs-QDs, the fluorescence of 1NPs-QDs-APT (intensity 1594) decreased approximately 17.2% (Figure 1e), likely due to the loss of conjugates during the preparative centrifugation and washing procedures. Similarly, renaturing 1-NPs-QDs-APT to 1-NPs-QDs-hAPT (Vehicle) did not induce the release of QDs from the conjugates (indicated by the inset HR-TEM image in Figure 1d). However, the preparative procedures resulted in some loss of the conjugate, as indicated by the fluorescence spectra in Figure 1e. Cryo-TEM images of as prepared 1-NPs-QDs, 1-NPs-QDs-APT, and 1-NPs-QDs-hAPT (Vehicle) showed that they are monodispersive, roundish nanoparticles with average diameter less than 50 nm, echoing with above TEM observations (Figure S4). Binding Specificity of the Vehicle. After preparation, we tested the targeting specificity of our Vehicle to MUC1overexpressing cells. Western blotting indicated that MUC1 is overexpressed in MCF-7 cells but not in HepG2 cells (Figure S8). MCF-7 or HepG2 cells were incubated with 50 μM Vehicle or 1-

NPs-QDs (calculated in 1) in serum-free culture medium at 37 °C for 2 h. Subsequently, the cells were repeatedly washed with PBS to remove excessive Vehicle or 1-NPs-QDs prior to imaging. As shown in Figure 2, the Vehicle was effectively taken up by MCF7 cells (left lane) but not HepG2 cells (middle left lane), indicating that the uptake of Vehicle by MCF-7 cells was primarily mediated by active targeting of hAPT on the Vehicle surface to the MUC1 protein on the cell membrane. Furthermore, 1-NPs-QDs, which do not have hAPT for targeting, were barely taken up by MCF-7 or HepG2 cells (middle right and right lanes), suggesting that the cell uptake efficiency of 1-NPs-QDs via passive endocytosis is very low. We therefore reasoned that the active targeting of Vehicle to MCF-7 cells could be employed to enhance the delivery of DOX to MCF-7 cells. Intercalation of DOX with the Vehicle to Prepare the Bioconjugate Vehicle-DOX. The intercalation of DOX into the CG sequence in the hAPT of the Vehicle results in the quenching of DOX fluorescence via “donor-quencher” fluorescence resonance energy transfer (FRET) effect.37 By this mechanism, we prepared a Vehicle-DOX conjugate with both maximum drug payload and DOX fluorescence quenching. At a fixed DOX concentration of 1.5 μM, we gradually increased the amount of the Vehicles incubated with DOX at room temperature for 40 min. As shown in Figure 3a, with the increase of Vehicles (i.e., the increase of the molar ratio of 1 to DOX), the QDs fluorescence intensity at 724 nm in the incubation mixtures consistently increased, as expected. Up to a 1/DOX molar ratio of 400, the fluorescence intensity of DOX at 593 nm decreased with the increase of the molar ratio, suggesting that DOX fluorescence was gradually quenched by hAPT on the Vehicle (Figure 3a & b). However, when the 1/DOX molar ratio exceeded 400, the fluorescence intensity at 593 nm increased along with the increasing 1/DOX molar ratio (Figure 3a & b). Thus, we used a 1/DOX molar ratio of 400 in the preparation of Vehicle-DOX. Under this condition, the loading efficiency (LE) of DOX (DOXLE (%) = 100 × (weight of DOX loaded into products)/weight of total DOX) was calculated to be 77.7 ± 7.1%. The fluorescence spectra of DOX, Vehicle, and Vehicle-DOX are shown in Figure 3c. For comparison, we incubated DOX with different amounts of 1-NPs-QDs and measured the fluorescence spectra. The results indicated that at a 1/DOX molar ratio 100, the fluorescence intensity of DOX at 593 nm was practically unaffected by



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Figure 3. (a) Fluorescence spectra of 1.5 μM DOX in PBS (pH 7.4) with different amounts of 1-NPs-QDs-hAPT (Vehicle) (i.e., the increase in the molar ratio of 1 to DOX at 0, 1, 10, 100, 400, 700, 1000, and 1300). (b) A hill plot of the fluorescence intensity data in (a) at 593 nm. Experiments were performed in triplicate. (c) Fluorescence spectra of 1.5 μM DOX (black), Vehicle (red), and Vehicle-DOX at a 1/DOX molar ratio of 400 (blue). Excitation: 480 nm.

Figure 4. (a) Confocal laser scanning microscopic images of MCF-7 cells after treatment with 400 μM Vehicle-DOX (calculated in 1) at 37°C for 15 min, 30 min, 60 min, or 120 min. (b) Confocal laser scanning microscopy images of MCF-7 cells pre-incubated with 40 μM hAPT at 37 °C for 30 min and then incubated with 400 μM Vehicle-DOX (calculated in 1) at 37 °C for 15 min, 30 min, 60 min, or 120 min. DOX and QD fluorescence is shown in green and red, respectively. Cell images were obtained using an excitation wavelength of 488 nm and two band-pass (573-614 nm for DOX and 687-743 nm for QDs) emission filters. Scale bar: 20 μm. 1-NPs-QDs (Figure S9a). At a 1/DOX molar ratio of 400, the fluorescence intensity of DOX at 593 nm decreased by approximately 25.2% in the presence of 1-NPs-QDs (Figure S9b), obviously lower than the 46.3% change induced by the Vehicle (Figure 3b). This result suggests that the fluorescence quenching of DOX by Vehicle was induced between DOX and hAPT via the FRET effect rather than electrostatic interactions. Time-dependent drug release profile of DOX from Vehicle-DOX was carried out via dialysis against PBS buffer (pH 7.4) and shown in Figure S10. While most of free DOX (ca. 92%) diffused through the dialysis membrane within 1 h, only about 35% of DOX was released from Vehicle-DOX after 4 h dialysis. This result suggested that our Vehicle-DOX bioconjugate was stable. Cryo-TEM image of asobtained Vehicle-DOX showed that they are monodispersive, roundish NPs (Figure S4). Tracking Drug Delivery Using Bioconjugate Vehicle-DOX. As previously mentioned, DOX fluorescence at 593 nm is efficiently quenched by approximately 46.3% in the VehicleDOX conjugate. Upon binding to MUC1, the hairpin conformation of hAPT on the Vehicle-DOX conjugate unfolds and DOX is uncaged,38 restoring fluorescence emission at 593 nm. Thus, we were able to use a 687-743 nm band-pass emission filter to capture QDs fluorescence to trace the cellular uptake of

Vehicle-DOX, and a 573-614 nm band-pass emission filter to record DOX fluorescence enhancement to trace drug release. In detail, we incubated MUC1-overexpressing MCF-7 cells with 400 μM Vehicle-DOX (calculated in 1) at 37 °C and washed them several times prior to confocal fluorescence microscopic imaging at different time points. As shown in Figure 4a, with increased incubation time, the DOX fluorescence signal (filter setting: 573614 nm) gradually increased, indicating that DOX was continuously released from the Vehicle-DOX conjugate. Interestingly, within a 60 min incubation time, the DOX fluorescence signal colocalized with that of QDs (filter setting: 687-743 nm), suggesting that DOX was released at Vehicle-DOX sites. Thus, we speculate that during the 60 min incubation, Vehicle-DOX was targeted to MUC1, a process that involves cell membrane translocation, endosome formation, and/or lysosome formation, during which DOX was released but did not escape the cytosome. At 120 min, we could still observe strong fluorescence signals for DOX but some of these signals were not colocalized with QDs signals, suggesting that some DOX molecules escaped from lysosomes and entered the cytoplasm. Thus, Vehicle-DOX could be utilized for MUC1-targeted cell imaging and drug release. To validate our hypothesis, we pre-incubated the cells with 40 μM hAPT for 30 min to block MUC1 sites, and we then incubated the


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cells with Vehicle-DOX prior to microscopic imaging. Figure 4b shows that compared with the experimental groups, far fewer Vehicle-DOX conjugates were taken up by MUC1-blocked MCF-7 cells. During a 120 min incubation, we did not observe DOX fluorescence signals colocalized with those of the Vehicle (i.e., fluorescence signals from QDs). This finding suggests that prior to being taken up by the cells, DOX molecules were physically dissociated from the Vehicle-DOX bioconjugate in the culture medium.

viability by approximately 10% upon incubation with VehicleDOX. Thus, we conclude that Vehicle-DOX resulted in MUC1targeted cytotoxicity. We also investigated the MUC1-targeted cytotoxicity of our Vehicle-DOX on another MUC1-positive cell line A549.20 As shown in Figure S12, at same DOX concentration, Vehicle-DOX showed stronger cell-growth-inhibitory effect on A549 cells than DOX. This was probably due to the antimultidrug resistance ability of our nanocarrier.25

CONCLUSIONS

Figure 5. The dose response results of MCF-7 cells and MUC1blocked MCF-7 cells (i.e., pre-incubated with 40 μM hAPT) against DOX and Vehicle-DOX, respectively. The cells were incubated with DOX or Vehicle-DOX for 4 h followed by another 44 h of cell growth prior to the Alamar Blue cell viability assay. All experiments were performed in triplicate. *, P < 0.05. The Targeting Cytotoxicity of Vehicle-DOX on MCF-7 cells. Before evaluating the targeting cytotoxicity of Vehicle-DOX, we first tested the cytotoxicity of the Vehicle. After a 4 h incubation with MCF-7 cells, treatment with 5 μM DOX and Vehicle-DOX (at 5 μM DOX concentration) resulted in 33.7 ± 0.3% and 71.3 ± 2.8% viabilities, respectively. In contrast, cells in the Vehicle group had a viability of 105.2 ± 3.9% (Figure S11). This suggests that the Vehicle is biocompatible with the cells at this concentration. DOX is reported to be cell membrane-permeable and can be taken up by cells through a passive diffusion mechanism, rapidly transported to the nucleus, and readily bound to chromosomal DNA upon which it induces cell death via apoptosis pathway.39 Our imaging results indicate that VehicleDOX was taken up by MCF-7 cells via MUC1-mediated cell membrane translocation. Since the drug resistance ability between MCF-7 cells and HepG-2 cells was quite different (Figure S11), HepG-2 cells were not considered as the effective control for MUC1-targeted cytotoxicity study. Therefore, in this study, we used MCF-7 cells with or without hAPT pretreatment (i.e., with or without blocking MUC1) to study the targeting cytotoxicity of Vehicle-DOX. As shown in Figure 5, cell growth inhibition was positively associated with DOX concentration in all groups. In free DOX-treated groups, the viabilities of both MCF-7 cells and hAPT-pretreated MCF-7 cells showed a similar dose response. However, in the Vehicle-DOX-treated groups, pretreatment with free hAPT obviously lowered the cytotoxicity of Vehicle-DOX at all tested concentrations (Table S2). In detail, from DOX concentrations ranging from 0.62 μM to 10 μM, the viabilities of of cells in the Vehicle-DOX group decreased from 92.7 ± 5.8% to 59.8 ± 1.6%, whereas those of the hATP-pretreated Vehicle-DOX group decreased from 100.6 ± 4.8% to 70.3 ± 3.3%, respectively. Pretreatment of the MCF-7 cells with hAPT increased cell

Using an AB2-type small molecule 1 and a reductioncontrolled condensation reaction, we prepared oligomeric nanoparticles 1-NPs, which exhibit an abundance of –NH2 and – SH groups on their surfaces. 1-NPs were easily and reproducibly conjugated with NIR-emitting CdTe/CdS QDs and Mal-APT to prepare the oligomeric/inorganic hybrids 1-NPs-QDs and 1-NPsQDs-APT, respectively. Renaturing 1-NPs-QDs-APT yielded the 1-NPs-QDs-hAPT (Vehicle) used for drug loading. The molar ratio 1/100 of Mal-APT to 1-NPs and 0.1 mg/mL CdTe/CdS QDs were determined to be optimal and were used to prepare the Vehicle, which exhibited maximal drug payload and cell uptake efficiency. TEM images and fluorescence spectra were employed to characterize the obtained serial conjugates. Fluorescence microscopic imaging indicated that the Vehicle could be applied for MUC1-targeted cell uptake and imaging. Confocal microscopic cell imaging indicated that the bioconjugate VehicleDOX could be applied for the tracking of MUC1-targeted drug release. Cytotoxicity studies indicated that Vehicle-DOX could be used for MUC1-targeted cytotoxicity. We anticipate that our multifunctional nanoparticle Vehicle-DOX could be used for in vivo tumor-targeted theranostics and are currently investigating this application.

EXPERIMENTAL PROCEDURES Materials and Methods. Commercially available reagents were used without further purification, unless noted otherwise. All other chemicals were reagent grade or higher. 1-ethyl-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), Nhydroxysuccinimide (NHS), and doxorubicin hydrochloride (DOX·HCl) were purchased from Sigma-Aldrich. 4maleimidobutyric acid was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The 5’NH2-C6 spacer GCA GTT GAT CCT TTG GAT ACC CTG G-3’ (5’NH2C6APT)) and tablets for the preparation of PBS buffer (0.01 M, pH 7.4) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Monomer 1 was synthesized according to the literature,40 and its characterizations were shown in the Supporting Information (Scheme S1 and Figure S1). NIR-emitting carboxyl core-shell CdTe/CdS quantum dots were synthesized according to the literature.41 Ultrapure water (18.2 MΩ. cm) was used throughout the experiments. The cells were obtained from the Cell Bank of Chinese Academy of Sciences. Eelectrospray ionization-mass spectrometry (ESI-MS) spectra were recorded on a LCQ Advantage MAX ion trap mass spectrometer (Thermo Fisher). HPLC analyses were performed using an Agilent 1200 HPLC system equipped with a G1322A pump and an in-line diode array UV detector using an Agilent Zorbax 300SB - C18 RP column with CH3CN (0.1% of trifluoroacetic acid (TFA) and water (0.1% of TFA) as the eluent. The fluorescence microscopic images in Figure 2 were taken under a OLMPUS IX71 fluorescence microscope. Confocal laser scanning microscopy images in Figure 4 were obtained on a Zeiss LSM 710 laser scanning confocal microscope. The fluorescent intensity of Alamar Blue assay was read by a Thermo Scientific Varioskan Flash 3001 instruments.



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The transmission electron microscopy (TEM) images in Figure 1 were obtained on a JEOL 2100 electron microscope, operating at 200 kV. Cryo transmission electron microscopy (cryo-TEM) images were obtained on a Tecnai F20 transmission electron microscope from FEI company, operating at 120 kV. UV-vis absorbance spectra were recorded on a lambda 25 UV-visible spectrophotometer (PerkinElmer, America) at room temperature. The fluorescence spectra were recorded on a F-4600 fluorescence spectrophotometer (Hitachi High-Technologies Corporation, Japan). Cell Culture. MCF-7 human breast adenocarcinoma epithelial cells and HepG2 hepatoma carcinoma cells were cultured in Dulbecco’s modified eagle medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO). The cells were expanded in tissue culture dishes and maintained in a humidified atmosphere of 5% CO2 at 37 °C. The medium was changed every other day. Western Blot. Lysates were prepared from subconfluent cells. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. The immunoblots were probed with an anti-MUC1 antibody. The immunocomplexes were detected with a horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) system. Agarose Gel Electrophoresis. Gel electrophoresis was carried out using 2% agarose gels in 0.5× Tris/borate/EDTA (TBE) buffer at 100 V for 40 min. After staining with ethidium bromide, gels were imaged with a Gene Genius Bio-imaging system (SynGene). Fluorescent Microscopic Imaging of Cells (Figure 2). MCF7 and HepG2 cells were seeded into a 6-well plate at 1 × 105 cells per well. After culturing for 24 h at 37 °C with 5% CO2, the cells were washed with pre-warmed PBS buffer and incubated with pre-warmed serum-free fresh medium at 37 °C for 1 h. Afterwards, MCF-7 cells or HepG2 cells were incubated with 50 μM Vehicle or 1-NPs-QDs (calculated in 1) in serum-free culture medium at 37 °C for 2 h, respectively. Next, the cells were washed with PBS for many times to remove excessive Vehicle or 1-NPs-QDs prior to imaging. In Vitro DOX Release. Vehicle-DOX was suspended in phosphate-buffered saline (PBS, pH 7.4) at a concentration of 4 mM (calculated in 1, corresponding DOX concentration was 10 mM). 400 μL Vehicle-DOX or 10 μM DOX was introduced into a dialysis bag (MWCO 1000), respectively, and then immersed into 25 mL release medium (PBS, pH 7.4) in an incubator shaker set at 150 rpm at 37 °C. At the predetermined time intervals, samples (100 μL for each) were withdrawn from the medium for fluorescence intensity measurement. The cumulative release percentages of DOX from Vehicle-DOX or free DOX were calculated as follows and plotted against time: Cumulative DOX release = amount of released DOX / amount of total DOX × 100%. Confocal Laser Scanning Microscopic Imaging of MCF-7 Cells (Figure 4). MCF-7 cells were allowed to adhere to a glass cover slip in a 24-well plate at 3 × 104 cells per well (300 μL). After a 24 h incubation period at 37 °C with 5% CO2, the cells were washed with pre-warmed PBS buffer and incubated with pre-warmed serum-free fresh medium at 37 °C for 1 h before the addition of 40 μM renatured APT (hAPT) in PBS buffer (for experimental groups) or PBS buffer alone (for control groups). After a 30 min incubation, the cell culture medium was removed and the cells were washed with PBS buffer. Then, the cells were incubated with 400 μM Vehicle-DOX (calculated in 1, corresponding DOX concentration was 1 μM) for 15 min, 30 min, 60 min, or 120 min. After incubation, the culture medium was removed and the cells were washed with PBS buffer 3 times, fixed with 4% formaldehyde in PBS buffer (pH 7.4) for 30 min. After washing with PBS buffer three times, the cells were mounted in a non-fluorescent mounting medium prior to confocal microscopic

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imaging (Car Zeiss LSM 710, a 573-614 nm filter set was used for DOX imaging, and a 687-743 nm filter set was used for QDs imaging). Alamar Blue Cell Viability Assay. The cytotoxicity of free DOX and Vehicle-DOX was tested using a standard Alamar Blue assay. The assay is based on the ability of living cells to convert blue redox dye (resazurin) into bright red resorufin, which can be read by a spectrophotometric reader. Non-viable cells rapidly lose metabolic capacity and thus do not generate a color signal. Thus, the intensity of the color is proportional to cell viability. MCF-7 cells were seeded into a black 96-well plate at 5 × 103 cells per well and incubated for 24 h at 37 °C with 5% CO2. Then, the cells were washed with pre-warmed PBS buffer and incubated with pre-warmed serum-free fresh medium for 1 h before the addition of 40 μM renatured APT (hAPT) in PBS buffer (for experimental groups) or PBS buffer (for control groups). After a 30 min incubation, the culture medium was removed and the cells were washed with PBS buffer 3 times. Then, the cells were further incubated with either DOX or Vehicle-DOX containing 0.62, 1.2, 2.5, 5.0, or 10 μM DOX or cell culture medium for 4 h. Then, the culture medium was removed and replaced with fresh cell culture medium. The Alamar Blue assay was conducted after another 44 h of cell growth. At 44 h, culture medium was removed and fresh medium containing 10% Alamar Blue dye was added (100 μL). The cells were then incubated for 4 h. The data were obtained using a Thermo Scientific Varioskan Flash 3001 to detect fluorescence with the excitation and emission wavelengths set at 560 nm and 590 nm, respectively. The following formula was used to calculate the viability of cell growth: Cell Viability = (sample - background)/(untreated - background) × 100%. Statistical Analysis. Two-tailed unpaired t-test was used to evaluate statistical significance between DOX- and DOX + hAPTtreated cells, Vehicle-DOX- and hAPT + Vehicle-DOX-treated cells in Figure 5. The results were recognized as statistically significant if p-value is less than 0.05. They are marked with asterisk symbol (*) in the graphics. All p-values can be found in Table S2 in Supporting Information.

ASSOCIATED CONTENT Supporting Information Synthetic routes, Mass spectra, UV-vis spectra, and HPLC traces of 1 and Mal-APT; cyo-TEM of as prepared nanoparticles; stability of Vehicle-DOX; cell cytotoxicity. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (H.X.). Tel: (+86)-551-62923194. *E-mail: [email protected] (G.L.). Tel: (+86)-551-63607935; fax: (+86)-551-63600730. Author Contributions W. D. performed the syntheses, characterizations, cell culture, cell imaging, and cytotoxicity assay. Y. Y. prepared the NIR-emitting carboxyl core-shell CdTe/CdS quantum dots. L.W. helped with agarose gel electrophoresis and Western blotting. Y. C. helped with the synthesis of 1. H. W. helped with cell imaging. H. X. helped with project design. G. L. designed this project and wrote the paper. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT


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This work was supported by the Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major program of Development Foundation of Hefei Center for Physical Science and Technology, and the National Natural Science Foundation of China (Grants 81371587, U1532144, and 21375121).

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