Theranostic Nanoprobes Mediated Simultaneous Monitoring and

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Theranostic Nanoprobes Mediated Simultaneous Monitoring and Inhibition of P-glycoprotein Potentiates Multidrug-Resistant Cancer Therapy Yuanqing Wei, Hongping Xia, Fen Zhang, Kan Wang, Peicheng Luo, Yafeng Wu, and Songqin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02118 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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Analytical Chemistry

Theranostic Nanoprobes Mediated Simultaneous Monitoring and Inhibition of P-glycoprotein Potentiates Multidrug-Resistant Cancer Therapy Yuanqing Wei,†,‡ Hongping Xia,§,‡ Fen Zhang,† Kan Wang,† Peicheng Luo,† Yafeng Wu,*, † Songqin Liu†

†

Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, School

of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China. §

Department of Pathology, School of Basic Medical Sciences & The Affiliated Sir Run

Run Hospital, Nanjing Medical University, Nanjing 21116, China ‡ These authors contributed equally.

*Corresponding

author: Tel.: 86-25-52090613; Fax: 86-25-52091098. E-mail:

[email protected] (Y.F. Wu).

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Multidrug resistance is a major cause of failure in the clinical cancer therapy, in which the overexpression of P-glycoprotein (P-gp) plays a crucial role. Herein, we fabricate a theranostic nanoprobe with the function of simultaneous detection and inhibition of P-gp to diagnose and combat multidrug-resistant cancer in vitro and in vivo. For constructing nanoprobe, elacridar modified quantum dots (QDs-Ela), acting as gatekeeper, are grafted onto the doxorubicin (DOX) loaded, folic acid (FA) decorated mesoporous silica nanoparticles (MSNs). Upon targeted uptake by multidrug-resistant cancer cells, Bel-7402/ADR are used as a model, the acidic environment results in QDs-Ela removing from nanoprobe and subsequent DOX releasing. The removed QDs-Ela could specifically combine with P-gp in the cancer cell membrane and inhibit their active sites, which prevents the efflux of intracellular DOX and increases the retention of DOX. Another way, the fluorescence intensity of the binding QDs-Ela quantifies the P-gp expression level. Subsequently, in vitro and in vivo experiments both demonstrate the enhanced multidrug-resistant cancer therapy efﬁcacy, i.e. nanoprobe has 10 times better curative effect than free DOX. In addition, due to the conjugation of FA, the nanoprobe exhibits selective cell targeting ability to Bel-7402/ADR cells. This nanoplatform paves a new avenue for the accurate treatment of multidrug-resistant cancers.

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INTRODUCTION Multidrug resistance (MDR) is the main barrier for effective cancer therapy, in which the overexpression of P-glycoprotein (P-gp) plays a vital role.1,2 P-gp is a transmembrane glycoprotein with a molecular weight of 170 kDa, is thought to act as an energy-dependent drug efflux pump for reducing intracellular concentration of anticancer drugs.3 The expression level of P-gp is also a significant prognostic indicator for the diagnosis of multidrug-resistant cancer.4,5 Thus, sensitive detection and inhibition of P-gp is a key step for early diagnosis and efficient treatment of multidrugresistant cancer. Up to now, the strategies for detecting6-9 or inhibiting10-14 P-gp have been widely reported. For P-gp detection, there are mainly including three aspects: in cellular level, fluorescent dyes can be used to assess the functional activity of P-gp by flow cytometry, since both drugs and some fluorescent dyes can be excreted out of cells by P-gp;6 in protein level, immunohistochemistry,15 western blot16 and flow cytometry analysis17 are more widely used. The principles of these methods are basically the same, anti-P-gp monoclonal antibody was incubated with P-gp containing samples, then different reagent labeled secondary antibodies were combined to conduct different strategies, i.e., biotin labeled for immunohistochemistry, enzyme-labeled for western blot and fluorescence labeled for flow cytometry analysis; in molecular level, P-gp related MDR1 gene can be detected by in situ hybridization,18 Northern blot,19 Slot blot20 and RT-PCR.21 For P-gp inhibition, there are commonly three approaches: firstly, small molecule inhibitors or competing substrates combine with the active site of P-gp 3



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directly;22,23 secondly, some small molecules like curcumin,24 buthionine sulfoximine4 or specific siRNA13,25 have the ability inhibiting the expression of P-gp by knockdown of MDR1 gene’s translation; lastly, some polymers prevent the P-gp ATPase from hydrolyzing ATP by blocking the ATP binding sites, thereby inhibiting P-gp function.14 However, the integration of P-gp detection and inhibition into one nanoprobe has not been accomplished yet, which can significantly improve the therapeutic effect of multidrug-resistant cancers and reduce the side effects. Herein, we fabricate a nanoprobe for simultaneously detecting and inhibiting P-gp to accomplish early diagnosis and efficient treatment of multidrug-resistant cancer. As shown in Scheme 1, for the construction of nanoprobe, the chemotherapeutic drug DOX is physically adsorbed in the pore of the mesoporous silica nanoparticles (MSNs) and the pores are gated by elacridar (a kind of P-gp inhibitor, which can block the P-gp active sites) modified quantum dots (QDs-Ela). For targeted delivery, folic acid (FA) is modified on the MSN. Upon targeted uptake by cancer cells, the acidic environment results in QDs-Ela removing from nanoprobe due to the cleavable of hydrazone bonds, and subsequent DOX releasing. The removed QDs-Ela could specifically combine with P-gp in the cancer cell membrane and inhibit their active sites, which prevents the efflux of intracellular DOX and increases the retention of DOX. Another way, the fluorescence intensity of the binding QDs-Ela quantifies the P-gp expression level. The strategy remarkably enhances multidrug-resistant cancer therapy effect in vitro and in vivo. The nanoprobe also exhibits high drug loading rate, low biotoxicity and high biocompatibility. To the best of our knowledge, this is the first time to realize 4


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simultaneous detection and inhibition of P-gp using one nanoprobe.

EXPERIMENTAL SECTION Materials and reagents. (Folic acid)-poly(ethylene glycol)-carboxy succinimidyl ester (FA-PEG-NHS, Mw = 10 kDa) was obtained from Ponsure Biotechnology (Shanghai, China). Amino modified mesoporous silica nanoparticles (MSNs-NH2) were purchased from So-Fe Biomedicine (Shanghai, China). Amino modified CdSe/ZnS quantum dots (QDs-NH2) were purchased from Xingzi New-Material Technology (Shanghai, China). Tris-(hydroxymethyl)aminomethane (TRIS), cesium carbonate (Cs2CO3), sodium iodoacetate (CH2ICO2Na), phosphoric acid (H3PO4), monosodium

phosphate

(NaH2PO4),

disodium

phosphate

(Na2HPO4),

3-

mercaptopropionic acid, hydrazine hydrate, doxorubicin (DOX), curcumin, elacridar (Ela), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Nhydroxysuccinimide (NHS), ethanol (EtOH), methanol (MeOH), RIPA lysis buffer and d6-dimethyl sulfoxide (d6-DMSO) were purchased from Sigma-Aldrich (USA). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), RPMI 1640 medium, red membrane tracer DiD, MTT cell proliferation and cytotoxicity assay kit were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). All of the chemicals were used as received without further purification. Ultrapure water (18.2 M cm) was got through Thermo purification system. Room temperature denotes 25 °C in all experiments, unless otherwise stated. Apparatus. The morphology of the materials was observed with a transmission 5



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electron microscope (TEM, JEM-2010, JEOL, Japan) and a scanning electron microscope (SEM, JSM-7001F, JEOL, Japan). The particle size and zeta potential were measured at 25 °C on a Malven Zetasizer NanoZS instrument (Britain). UV-vis spectra were performed on UV-visible spectrometer (Shimadzu UV-2450, Kyoto, Japan). Fluorescence spectra were carried out on a FluoroMax-4 spectrofluorometer with xenon discharge lamp excitation (HORIBA, USA). Confocal laser microscopy images and differential interference contrast (DIC) images were carried out by a confocal laser scanning microscopy (CLSM, FluoViewTM FV1000, Olympus, Japan). MTT assay was measured with microplate reader (Multiskan GO, Thermo Fisher Scientific, China). Intracellular DOX detection was carried out on a flow cytometry (BD FACSCalibur, BD Biosciences, USA). Synthesis of elacridar derivatives. A solution of elacridar (2.5 mg, 4.42 μmol) in ethanol (5 mL) was heated under reflux for 12 h in the presence of cesium carbonate (1.63 mg, 5.0 μmol) and sodium iodoacetate (1.04 mg, 5.0 μmol). The product was condensed and purified with column chromatography (ethyl acetate:methanol = 1:1) to obtain 1.2 mg of yellow solid (yield 42%). NMR (D6 DMSO): δ 8.53 (1H, s), 8.43 (1H, dd, J = 7.14, 1.87), 8.37 (1H, dd, J = 7.14, 1.87), 8.03 (2H, d, J = 8.37), 7.78 (1H, dd, J = 8.12, 1.33), 7.28 (2H, d, J = 8.46), 7.02 (2H, m), 6.92 (1H, t, J = 7.74), 6.87 (1H, s), 6.79 (1H, s), 4.88 (1H, d, J = 15.52), 4.65 (1H, d, J = 15.52), 3.76 (3H, s), 3.74 (3H, s), 3.46 (3H, s), 3.06 (6H, m), 2.61 (1H, m), 2.54 (2H, s), 2.39 (1H, m). MS (EI): m/z [M + H]+ calcd for C36H34N3NaO7: 643.2294. Found: 644.3. Synthesis of QDs-Ela and nanoprobe. For QDs-Ela synthesis, EDC (19.2 μg, 0.1 6


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μmol), NHS (11.6 μg, 0.1 μmol) and elacridar derivatives (64 μg, 0.1 μmol) were dissolved in 0.1 mL of methanol. After shaking for 30 min, 1 mL QDs−NH2 aqueous solution (2 μM, 1 mg mL−1) was added and shaking at room temperature for another 12 h. The product was collected by centrifugation (10000 g, 10 min) and washed three times with ultrapure water, monodisperse elacridar modified QDs (QDs-Ela) are obtained. For nanoprobe synthesis, MSNs-NH2 methanol solution (10 mL, 0.29 mg mL−1) was stirred with FA-PEG-NHS (0.7 mg, MW 10 kDa) for 30 minutes, then 1 mL DMF solution with CDI (5 mg, 0.031 mmol) and levulinic acid (3.16 μL, 0.03 mmol) was added and stirring for 12 h. The residue was collected by centrifugation (5000 g, 5 min) and washed three times and re-dispersed in methanol. Then a pre-reacted methanol solution (1 mL) of 3-mercaptopropionic acid (10 μL, 0.11 mmol) and hydrazine hydrate (5.5 μL, 0.11 mmol) was added into MSN solution. After 12 h, the product was collected and re-dispersed into Tris buffer (pH 8.5) with DOX (0.58 mg, 1.06 μmol) and stirring for 12 h, followed by adding QDs-Ela (0.58 mg, 1.16 nmol) for another 12 h. The final product was collected by centrifugation (5000 g, 5 min) and washed with PBS (pH 7.4) for thrice. Simultaneous monitoring and inhibition of P-gp potentiates multidrugresistant cancer therapy with as-prepared theranostic nanoprobes. The fluorescence intensities of QDs were measured with a FluoroMax-4 spectrofluorometer at an excitation wavelength of 350 nm. For monitoring the P-gp expression, Bel7402/ADR cells were seeded at ∼5 × 105 cells per well into 12-well cell culture cluster 7



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(Thermo Fisher Scientific, China) and incubated for 24 h. Then, different concentrations of QDs-Ela/QDs were incubated with cells for 4 h. For comparison, 2 clusters were pre-incubated with curcumin at 0, 15, 30 and 45 μM for 2 days. After rinsing with PBS twice, the cells were lysed by RIPA lysis buffer and the fluorescence intensities of cells were acquired by fluorescence spectroscopy. The same protocol for Bel-7402 cells was conducted as control. To further investigate P-gp detecting ability of nanoprobes, Bel-7402/ADR cells were pre-treated with different concentrations of curcumin (0, 15, 30, 45 μM) for 2 days, followed by incubating with nanoprobes for 4 h. Afterward, the cells were visualized with CLSM. To obtain the level of intracellular DOX, Bel-7402/ADR cells were incubated with DOX and nanoprobe for 1 h, followed by rinsing with PBS twice and trypsinizing to perform the fluorescence detection of DOX with the flow cytometer. The same protocol for L-O2 and Bel-7402 cells were performed as controls. All experiments detected at least 10000 cells. For cell imaging, Bel-7402/ADR (FR+, P-gp+), Bel-7402 (FR+, P-gp−) or L-O2 (FR−, P-gp−) cells were incubated with 40 µL 1.25 mg mL−1 nanoprobes for 0.5, 2 and 4 h at 37 °C, respectively, then observed by CLSM. MTT assay. MTT assay was carried out to investigate the material toxicity of probe. Bel-7402/ADR cells were firstly seeded to 6 plates of 96-well cell culture clusters at a seeding density of ∼5 × 104 cells per well in 200 µL complete medium, which was incubated at 37 °C for 24 h. After rinsing with PBS, 3 plates of Bel7402/ADR cells were incubated with 200 µL culture media containing serial 8


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concentrations of DOX, MSNs and probe for 20 h, respectively. Another 3 pates of Bel7402/ADR cells were incubated with 200 µL culture media containing 10 μg mL−1 of DOX, 125 μg mL−1 of MSNs and nanoprobe for different time periods, respectively. Then, 50 µL MTT solution in binding buffer from MTT cell proliferation and cytotoxicity assay kit was added to each well. After 4 h incubation, the medium containing unreacted MTT was removed carefully, and 150 µL DMSO was added to each well to dissolve the produced formazan. After shaking, the optical density (OD) at a wavelength of 570 nm was measured with microplate reader (Multiskan GO, Thermo Fisher Scientific, China). Animal study in the orthotopic HCC models. All experiments using mice were approved by the Institutional Animal Care and Use Committee (IACUC). Bel7402/ADR luciferase expression cells were firstly subcutaneous injected into 4-6 weeks old nude mice to generate tumors. Then the mice were anesthetized with Hypnorm/Midazolam and the tumor were cut into similar size small pieces. Similar sized tumoroids were picked up and transplanted into the liver of mice by operation to establish the orthotopic xenograft 7402/ADR tumor models. The mice were imaged and grouped based on equivalent signal intensity at one week after the surgery. The mice were then treated with PBS, free DOX, nanoprobe without DOX or nanoprobe (2 mg kg−1) via semiweekly intravenous injections for 4 weeks. Tumor growth was examined weekly by bioluminescence imaging using the Xenogen IVIS Lumina system (Xenogen Corporation, Hopkinton, MA). Briefly, following anesthesia with 2% isoflurane, mice were IP injected with D-Luciferin (100 mg kg−1; Caliper Life Sciences, Inc, Hopkinton, 9



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MA) and received 1 min scans to assess the bioluminescent signal. The tumor tissues were collected for immunohistochemistry analysis using a primary antibody; anti-P-gp (1:100)

(Abcam)

or

an

isotype-matched

IgG

as

a

negative

control.

Immunohistochemical staining was performed with the Dako Envision Plus System (Dako, Carpinteria, CA) according to the manufacturer’s instructions. To study DOX concentration in the liver tumor tissues of mice treated with different materials, tumor tissues are homogenized and mixed with 900 μL of extraction buffer (10% Triton X100, deionized water, and isopropanol at volumetric ratio of 1:2:15) and centrifuged for 10 minutes at 3,000 rpm. The supernatants are collected and evaporated to dryness. The obtained dry residues are then dissolved in methanol and further centrifuged at 15,000 rpm for 5 minutes to remove the undissolved materials. The samples are analyzed via HPLC method. DOX concentration is expressed as the amount of DOX per gram of the tumor tissues. Statistical Analysis. Data were expressed as means ± SD from at least three experiments. Statistical analyses were carried out using an Origin 85 software. CLSM images were analyzed by Olympus FluoView Ver.3.1a software. Flow cytometer data were analyzed with a FlowJo software.

RESULTS AND DISCUSSION Synthesis and Characterization of the Theranostic Nanoprobes. The synthesis of designed theranostic nanoprobes includes three steps. Firstly, QDs-Ela is synthesized by EDC coupling chemistry (Scheme S1a). Prior to conjugation, elacridar is modified 10


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with carboxyl groups using sodium iodoacetate as electrophile and cesium carbonate as proton taker,26 which is characterized by mass spectrometry (Figure S1) and 1H NMR (Figure S2). After Ela coupling, the zeta potential of QDs slightly changes from −6.0 to −7.1 mV (Figure 1a). The TEM image shows good monodispersity and clear lattice fringes of the as-prepared QDs-Ela with an average diameter of 7 nm (Figure S3a). Secondary, MSN-NH2 with an average size of 200 nm (Figure S3b, S4, Table S1) is used as a reservoir to encapsulate the drugs, which is modified with folic acid (FA) through reactive ester substitution to enhance the targeting ability to folate receptor (FR) in the cancer cell membrane. Then hydrazone bond and mercapto groups are introduced on the surface of MSN-NH2-FA for QDs-Ela coupling (Scheme S1b). Thirdly, QDsEla (~7 nm) are capped onto the nanopores (~4 nm) of the DOX loaded MSNs to prevent DOX leaking through the formation of Zn-S bonds (Scheme S1c).27 The ratio of MSNs-NH2 to QDs-Ela is optimized (Table S2, Figure S5), when the ratio is 5:1, QDs are densely gathered around MSNs and the lattice of QDs (yellow cycles) is observed from high resolution TEM image (Figure 1b). Zeta potentials and DLS also confirm the successful combination of MSNs and QDs, the zeta potential of MSN-NH2 is +16.6 mV due to the large existence of amino groups, for MSN-DOX, the zeta potential changes to −11.4 mV due to the substitution of amino groups. Upon capping with negative charged QDs-Ela, the zeta potential turns down to −15.8 mV (Figure 1a) and DLS changes from 192 nm to 198 nm (Table S1, Figure S4). The DOX loading capacity and efficiency are optimized in Tris buffer (pH 8.5) (Figure S6). DOX loading capacity increases slowly and DOX loading efficiency 11



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decreases sharply over 0.21 mg mg−1. For overall consideration, DOX to MSNs ratio of 0.21 mg mg−1 is selected. The DOX release performance is evaluated by incubation of the as-prepared nanoprobes with Tris buffer at different pH values and designated time points (Figure S7). In the physiological condition, there is negligible DOX release (9 μg mL−1) from nonspecific adsorption are obtained for Bel-7402 (P-gp−), demonstrating the high specificity between Ela and Pgp. Furthermore, the proposed strategy can be used to monitor dynamic changes of Pgp expression levels in the cell membrane, Bel-7402/ADR cells are pre-treated with curcumin, an inhibitor against P-gp expression,24 at 0, 15, 30 and 45 μM for 2 days, then incubated with QDs-Ela/nanoprobe for fluorescence spectrometer and CLSM analysis, respectively. With the increasement of curcumin’s concentration, the observed fluorescent signal gradually decreases, which means that P-gp expression levels in Bel13



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7402/ADR cells are reduced to 64%, 48% and 20% at curcumin’s concentration of 15, 30 and 45 μM, respectively (Figure 2d). The corresponding CLSM images show the same trend, the bright QDs fluorescent cycles vanish gradually with the increasement of curcumin’s concentration, while DOX remains almost no change (Figure 3). We then investigate the inhibition of P-gp in Bel-7402/ADR cells with the proposed therapeutic nanoprobes by CLSM (Figure 4). The nanoprobes are firstly phagocytized by cells through folate receptor and trapped into the intracellular lysosome within 30 minutes, where QDs-Ela are removed from the nanoprobes and DOX is then released. At this time, the fluorescent signals of QDs and DOX are relatively low due to the acidic environment inhibits 480 nm cyan light emission of QDs partially (Figure S9) and little amount of DOX is released (Figure 4a-e).28 After that, the QDs-Ela move to and combine with P-gp in the cell membrane gradually, strong QDs signals are observed around the cell membrane (Figure 4f). On the other hand, DOX releasing is increased with time period and finished within 2 hours (Figure 4g). It is noted that the DOX signals at 4 hours are as strong as the signals at 2 hours (Figure 4l). The corresponding OD values of the fluorescence in QDs channel increase as bleeding duration according with the track of QDs inside cells, while the OD value of DOX channel increases before 2 hours, then stays at the same level for another 2 hours (read with Image Pro software, Figure S10), verifying DOX is released from nanoprobes in lysosomes and maintained inside cells due to the inhibition of P-gp. Free DOX, MSNs loaded with DOX (MSN-DOX), and MSN-DOX modified with QDs without elacridar (MSN-DOX-QDs) are used as controls for head-to-head comparison. 14


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As shown in Figure S11 and S12, when Bel-7402/ADR cells are incubated with different materials for 4 h, DOX signals for DOX, MSN-DOX and MSN-DOX-QDs groups are obviously lower than nanoprobe group. This is contributed to the inhibition of P-gp prevents the efflux of intracellular DOX, which in turn increases the retention of DOX. To evaluate the FA targeting ability, another three drug-resistant cancer cell lines, A2780/ADR with FR (FR+, P-gp+), MCF-7/ADR without FR (FR−, P-gp+) and HepG2/ADR without FR (FR−, P-gp+) are employed. FR positive cells (A2780/ADR and Bel-7402/ADR) exhibit stronger QDs and DOX signals than FR negative cells (MCF-7/ADR and HepG2/ADR) (Figure S13), this is ascribed to the FR targeting ability of A2780/ADR and Bel-7402/ADR cells, leading to more nanoprobes can be phagocytized. Non drug-resistant cancer cell line, Bel-7402 cells (FR+, P-gp−) and normal cell line, L-O2 cells (FR−, P-gp−) are also studied. For Bel-7402 (FR+, P-gp−) cells, QDs-Ela are retained in cells without forming QDs cycles and the DOX remains the same fluorescent intensity as the prolonged incubation time (Figure S14), confirming that there is no P-gp expression in the cell membrane and no DOX excreting. While for L-O2 (FR−, P-gp−) cells, few QDs and DOX are observed in cells after 2 h incubation, which are ascribed to no FR and P-gp expression in the cell membrane (Figure S15). In vitro Therapeutic Efficacy Evaluation. To investigate therapeutic effect of nanoprobes against different cell lines in vitro, cytotoxicity assessments are accomplished by MTT assay. Bel-7402/ADR cells (FR+, P-gp+), possessing drug15



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resistant ability on account of P-gp overexpression, they can easily survive in 20 μg mL−1 DOX with more than 90% of cell viability after 20 h (Figure 5a, e). When incubated with nanoprobes, the cell viability of Bel-7402/ADR cells is about 44% at nanoprobe dose of 250 μg mL−1, equivalent to releasing 20 μg mL−1 DOX. In the aspect of IC50, the curative effect of nanoprobe (IC50 = 150 μg mL−1, equivalent to releasing 12 μg mL−1 DOX) is 10 times better than that of DOX (IC50 = 110 μg mL−1) on the treatment of Bel-7402/ADR cells. The same approach is conducted to MCF-7/ADR without FR (FR−, P-gp+), Bel-7402 cells (FR+, P-gp−) and L-O2 cells (FR−, P-gp−). For MCF-7/ADR cells (FR−, P-gp+), the high cell viability of 90.8% for DOX is ascribed to the drug efflux of drug-resistant cells and high cell viability of

82.5% for

nanoprobes is attributed to few nanoprobes entered into cells without FR targeting (Figure 5b, f). For Bel-7402 cells (FR+, P-gp−), the cell viability is 41.3 % and 52.7% for DOX and nanoprobes (Figure 5c, g), the similar viability is attributed to no P-gp expression in non drug-resistant cancer cells. For L-O2 cells (FR−, P-gp−), the cell viability is 20.9 % and 65.8% for DOX and nanoprobes (Figure 5d, h) due to no P-gp and FR expression in normal cells L-O2. Therefore, the therapeutic effect differences with DOX and nanoprobes are more obvious for drug resistant cancer cells with FR. We deduce that the good therapeutic effect against MDR cancer cells with FR is ascribed to the maintained intracellular DOX concentration in the cells, flow cytometer is used to confirm the deduction (Figure 5i, l). Intracellular DOX concentration in Bel7402/ADR cells incubated with nanoprobes are obviously higher than that incubated with DOX. While for Bel-7402 cells, intracellular DOX concentrations are basically 16


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the same whatever cells are incubated with DOX or nanoprobes. And for L-O2 cells, intracellular DOX concentration in cells incubated with nanoprobes is even slightly less than that incubated with DOX. In vivo Imaging and Therapeutic Efficacy. The succeeding in P-gp detection, bioimaging and good therapeutic efficacy of nanoprobes in vitro encourage us to further investigate the in vivo performance for treating MDR cancers,29-31 which is carried out on nude mice bearing orthotopic Bel-7402/ADR xenograft. Based on our long time experience to generate the orthotopic HCC models, it is more consistent tumor size by transplanting a similar size of tumor tissue comparing to directly injection of tumor cells to the mice liver. Because the tumor cells will diffuse to everywhere of the liver or outside of the liver even if injection tumor cells with matrigel. In Figure 6a, the luminescence signal originates from the D-luciferin catalyzed by luciferase, reflecting tumor region and size in mice.32-33 After materials are administered to the mice intravenously via the tail vein treatment, mice injected with nanoprobes exhibit the smallest tumor size compared with control groups (PBS, DOX and nanoprobe without DOX), DOX concentration in the tumor tissues of mice treated with nanoprobe is the highest (Figure S16). Quantitative analysis of bioluminescence signals exhibits tumor growth rate of nanoprobe treatment group is the lowest (Figure 6b), demonstrating the remarkable therapeutic efficacy of our nanoprobe on MDR cancers tentatively. To evaluate the tumor targeting and P-gp monitoring capabilities, nanoprobes without DOX and nanoprobes are visualized through fluorescence images (Figure 6c), apparent fluorescence signals are only observed at the tumor regions, indicating the 17



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accumulation of nanoparticles at the tumor sites. Since the fluorescence of nanoprobes comes from the QDs which combine with P-gp in the cell membrane, which also reflects the P-gp expression level, the nanoprobes without DOX treatment group exhibits high fluorescence while the nanoprobe treatment group exhibits weak fluorescence since the P-gp expression is inhibited during therapy. The representative images of H&E organs staining (Figure S17) show that the nanoprobe treatment has no side effect to normal tissues e.g. kidney and liver. To further discover the antitumor mechanism, the tumors of free DOX group and nanoprobe group are sectioned for H&E staining and immunohistochemical (IHC) assay. The representative images of H&E tumor staining show that most of the cells in nanoprobe group are lysed and destroyed, indicating that nanoprobe treatment induces much more necrosis in vivo compared to DOX treatment (Figure 6d). P-gp immunohistochemical analysis is further employed to monitor the P-gp expressions after the different treatments of Bel-7402/ADR tumor-bearing mice. The images show that nanoprobe treatment but not DOX treatment inhibits the expression of P-gp in the tumors of mice (Figure 6e), suggesting that nanoprobe can reverse the MDR of carcinoma cells through inhibiting P-gp functions.

CONCLUSIONS In summary, we have developed a theranostic nanoprobe with the function of simultaneous detection and inhibition of P-gp to diagnose and combat multidrugresistant (MDR) cancer in vitro and in vivo for the first time. The nanoprobes are able 18


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to efficiently transport DOX into the MDR cancer cells and maintain intracellular DOX concentration, thus the therapeutic efficacy is significantly enhanced. Because in acidic environment, QDs-Ela are removed from nanoprobes and DOX is releasing, the removed QDs-Ela can specifically combine with P-gp and inhibit the active sites of Pgp on the cell membrane. The nanoprobes also exhibit a series of advantages, such as pH-responsiveness, non-toxicity and biological specificity. Therefore, this work proposes a new strategy to deliver DOX to drug-resistant cancers without drug efflux, which could have prospect for clinical application against drug-resistant tumor.

ASSOCIATED CONTENT Supporting Information Available: Mass spectrum and 1H NMR of Ela derivatives; TEM images and DLS detection of nanoparticles; optimization of nanoprobe synthesis and DOX loading; DOX releasing behavior; UV-vis spectrums of nanoprobes; CLSM images of nanoprobes in different cell lines; H&E organs staining for bio-safety; the in vivo DOX concentration in the tumor tissues of mice group. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Y.F. Wu) Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest. 19



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ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 21635004, 21627806, 21705018), the Fundamental Research Funds for the Central Universities (2242017K3DN11) and State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1913). REFERENCES (1) Krishna, R.; Mayer, L. D., Eur. J. Pharm. Sci. 2000, 11, 265-283. (2) Santos, S. A.; Paulo, A., Curr. Cancer Drug Targets 2013, 13, 814-828. (3) Ambudkar, S. V.; Kimchi-Sarfaty, C.; Sauna, Z. E.; Gottesman, M. M., Oncogene 2003, 22, 7468-7485 (4) Nobili, S.; Landini, I.; Giglioni, B.; Mini, E., Curr. Drug Targets 2006, 7, 861-879. (5) Pajeva, I. K.; Hanl, M.; Wiese, M., ChemMedChem 2013, 8, 748-762. (6) Ludescher, C.; Thaler, J.; Drach, D.; Drach, J.; Spitaler, M.; Gattringer, C.; Huber, H.; Hofmann, J., Br. J. Haematol. 1992, 82, 161-168. (7) Sukhanova, A.; Devy, M.; Venteo, L.; Kaplan, H.; Artemyev, M.; Oleinikov, V.; Klinov, D.; Pluot, M.; Cohen, J. H. M.; Nabiev, I., Anal. Biochem. 2004, 324, 60-67. (8) Deng, B.; Tia, Y.; Yu, X.; Song, J.; Guo, F.; Xiao, Y. X.; Zhang, Z. L., Anal. Chim. Acta 2014, 820, 104-111. (9) Zhou, H.; Lin, C. W.; Zhang, Y.; Zhang, X. Z.; Zhang, C.; Zhang, P. B.; Xie, X. W.; Ren, Z. Q., Cell Prolif. 2017, 50, 10. (10) Batrakova, E. V.; Li, S.; Vinogradov, S. V.; Alakhov, V. Y.; Miller, D. W.; Kabanov, A. V., J. Pharmacol. Exp. Ther. 2001, 299, 483-493. 20


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ChemMedChem 2013, 8, 1701-1713. (23) Giacone, D. V.; Carvalho, V. F. M.; Costa, S. K. P.; Lopes, L. B., J. Pharm. Sci. 2018, 107, 698-705. (24) Choi, B. H.; Kim, C. G.; Lim, Y.; Shin, S. Y.; Lee, Y. H., Cancer Lett. 2008, 259, 111-118. (25) Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J. I.; Nel, A. E., ACS Nano 2010, 4, 4539-4550. (26) Lowden, C. T.; Bastow, K. F., J. Med. Chem. 2003, 46, 5015-5020. (27) Wang, Q.; Xu, Y.; Zhao, X.; Chang, Y.; Liu, Y.; Jiang, L.; Sharma, J.; Seo, D. K.; Yan, H., J. Am. Chem. Soc. 2007, 129, 6380-6381. (28) Tang, D. D.; Zhang, J. Y.; Zhou, R. X.; Xie, Y. N.; Hou, X. D.; Xu, K. L.; Wu, P., Nanoscale 2018, 10, 8477-8482 (29) Li, Y.; Tang, J.; He, L.; Liu, Y.; Liu, Y.; Chen, C.; Tang, Z., Adv. Mater. 2015, 27, 4075-4080. (30) Hou, K.; Fixler, D.; Han, B.; Shi, L.; Feder, I.; Duadi, H.; Wang, X.; Tang, Z., ChemNanoMat 2017, 3, 736-739. (31) Li, Y.; Jin, J.; Wang, D.; Lv, J.; Hou, K.; Liu, Y.; Chen, C.; Tang, Z., Nano Research 2018, 11, 3294-3305. (32) hang, C.; Yan, Z.; Arango, M. E.; Painter, C. L.; Anderes, K., Clin. Cancer Res. 2009, 15, 238-246. (33) Jenkins, D. E.; Oei, Y.; Hornig, Y. S.; Yu, S.-F.; Dusich, J.; Purchio, T.; Contag, P. R., Clin. Exp. Metastasis 2003, 20, 733-744. 22


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Scheme 1. Schematic illustration of nanoprobe preparation and cell entry for enhanced multidrug-resistant cancer therapy. DOX is encapsulated into FA modified MSNs-NH2, then QDs-Ela are capped onto the nanopores of the DOX loaded MSNs to prevent DOX leaking through the formation of Zn-S bonds. After FR-mediated endocytosis, nanoprobes enter into lysosomes, in the acid environment, QDs-Ela are removed from the nanoprobes the due to the hydrazone bonds on the MSNs are cleaved, following by the release of the DOX. The removed QDs-Ela will specifically combine with P-gp in the cell membrane, the expression level of P-gp can be quantitatively measured due to the fluorescent properties of QDs. Moreover, the inhibition of P-gp prevents DOX excreting and maintains the intracellular DOX concentration. The strategy remarkably enhances multidrug-resistant cancer therapy effect in vitro and in vivo.

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Figure 1. Characterization of nanoprobe. (a) Zeta potential characterization of MSNNH2, MSN-DOX, nanoprobe, QDs and QDs-Ela, error bars show the standard deviation of three experiments. (b) TEM image shows QDs-Ela are distributed around MSNs, and the lattice of QDs (yellow cycles) is observed under high resolution TEM image. (c) BET analysis of MSN-NH2, nanoprobe and removing of QDs-Ela on nanoprobe in acidic environment.

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Figure 2. Detection of P-gp in the cell membrane. (a) Linear curve of QDs’ concentration against fluorescent intensity, λex = 350 nm. (b) The change of binding QDs’ concentration on the Bel-7402/ADR cell membrane with the increased concentration of QDs-Ela. QDs-Ela are incubated with Bel-7402/ADR cells till QDsEla are saturated binding to P-gp in the cell membrane. After rinsing with PBS to remove excess nanoprobes, cells are lysed by RIPA lysis buffer to conduct fluorescence detection. (c) The specific study of QDs-Ela and P-gp, nanoprobes are incubated with Bel-7402/ADR (P-gp+) or Bel-7402 (P-gp−) cells, discovering that QDs-Ela can specifically bind with P-gp in Bel-7402/ADR cells. (d) The quantitative monitoring of P-gp expression in Bel-7402/ADR cells, Bel-7402/ADR cells are incubated with curcumin, an inhibitor against P-gp expression, at 0, 15, 30 and 45 μM for 2 days. With the increase of curcumin’s concentration, the fluorescent intensity decreases, consist with P-gp concentrations expressed by Bel-7402/ADR cells. Error bars show the standard deviation of three experiments.

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Figure 3. CLSM images of Bel-7402/ADR cells pre-treated with curcumin at concentrations of 0 µM (a-e), 15 µM (f-j), 30 µM (k-o) and 45 µM (p-t) for 2 days, respectively, then incubated with 40 µL 1.25 mg mL−1 nanoprobes for 4 h. CLSM analysis shows that different concentrations of curcumin will limit the P-gp protein translation in varying degrees, and the QDs cycles vanish gradually with combined QDs decrease. Scale bars are 15 μm.

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Figure 4. CLSM analysis of nanoprobe internalization and movement process in Bel7402/ADR cells, Bel-7402/ADR cells are incubated with 40 µL 1.25 mg mL−1 nanoprobes for 30 min, 2 h and 4 h, respectively. (a-e) Nanoprobes are phagocytized by cells through folate receptor and trapped into the intracellular lysosome in 30 minutes. (f-j) The removed QDs-Ela combine with P-gp in the cell membrane gradually, DOX is releasing and finished in 2 h. (k-o) The inhibition of P-gp level prevents the efflux of intracellular DOX, which in turn increases the retention of DOX. At 4 h, the DOX signals are as strong as the signals at 2 h. Scale bars are 15 μm.

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Figure 5. The investigation of therapeutic effect of DOX/nanoprobes against different cell lines, cytotoxicity assessments are accomplished by MTT assay. Viability of (a) Bel-7402/ADR (FR+, P-gp+), (b) MCF-7/ADR (FR−, P-gp+), (c) Bel-7402 (FR+, P-gp−) and (d) L-O2 (FR−, P-gp−) cells incubated with nanoprobes (black), or DOX (red) under different concentrations for 20 h; viability of (e) Bel-7402/ADR, (f) MCF-7/ADR, (g) Bel-7402 and (h) L-O2 cells treated with 10 μg mL−1 DOX or 125 μg mL−1 nanoprobes for different time periods, and all the experiments are repeated three timesand all the MTT assays are repeated three times; flow cytometer is used to detect intracellular DOX concentration in the (i) Bel-7402/ADR, (j) MCF-7/ADR, (k) Bel-7402 and (l) LO2 cells incubated with DOX or nanoprobe for 1 h, followed by rinsing with PBS twice and trypsinizing to perform the fluorescence detection of DOX.

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Figure 6. The therapy effect in vivo. (a) Representative images show bioluminescence signals of the orthotopic liver tumor xenografts at the therapeutic end point of different treatments. (b) Quantitative analysis of bioluminescence signals of all mice in the four treatment groups measured on a weekly basis. (c) The evaluation of the tumor targeting and P-gp monitoring capabilities of nanoprobes. The nanoparticles accumulation 29



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images in the tumor site observed by the QDs signal. (d) The representative images of H&E tumor staining show that nanoprobe treatment induced much more necrosis compared to DOX treatment (10X, scale bar, 100 μm). (e) The representative immunohistochemistry (IHC) staining images show that nanoprobe treatment but not DOX treatment inhibits the expression of P-gp in the tumors of mice (20X, scale bar, 20 μm).

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For TOC only

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Theranostic Nanoprobes Mediated Simultaneous Monitoring and

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