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Preparation of Microkernel-Based Mesoporous (SiO2-CdTe-SiO2)@SiO2 Fluorescent Nanoparticles for Imaging Screening and Enrichment of Heat Shock Protein 90 Inhibitors from Tripterygium Wilfordii Yue Hu, Zhao Yi Miao, Xiaojing Zhang, Xiaotong Yang, Yingying Tang, Sheng Yu, ChenXiao Shan, Hongmei Wen, and Dong Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05295 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Analytical Chemistry

Preparation of Microkernel-Based Mesoporous (SiO2-CdTeSiO2)@SiO2 Fluorescent Nanoparticles for Imaging Screening and Enrichment of Heat Shock Protein 90 Inhibitors from Tripterygium Wilfordii Yue Hu,† Zhao-Yi Miao,† Xiao-Jing Zhang,† Xiao-Tong Yang,† Ying-Ying Tang,† Sheng Yu, † ChenXiao Shan, † Hong-Mei Wen, † and Dong Zhu*†‡§ †

School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023, P. R. China.

‡

Jiangsu Key Laboratory for Functional Substance of Chinese Medicine.

§

State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine.

* Corresponding author. E-mail: [email protected]; Fax: +86 2585811839; Tel: +86 25 85811839

ABSTRACT: The currently utilized ligand fishing for bioactive molecular screening from complex matrixes cannot perform imaging screening. Here, we developed a new solid-phase ligand fishing coupled with in situ imaging protocol for the specific enrichment and identification of heat shock protein 90 (Hsp 90) inhibitors from Tripterygium wilfordii, utilizing a multiple-layer and microkernel-based mesoporous nanostructure composed of a protective silica coating CdTe quantum dots (QDs) core and a mesoporous silica shell, i.e. microkernel-based mesoporous (SiO2-CdTe-SiO2)@SiO2 fluorescent nanoparticles (MMFNPs) as extracting carries and fluorescent probes. The prepared MMFNPs showed highly uniform spherical morphology, retained fluorescence emission and great chemical stability. The fished ligands by Hsp 90α-MMFNPs were evaluated the preliminary bioactivity based on real-time cellular morphology imaging by confocal laser scanning microscopy (CLSM), and then identified by mass spectrometry (MS). Celastrol was successfully isolated as Hsp 90 inhibitors, and other two specific components screened by Hsp 90α-MMFNPs, i.e. demecolcine and wilforine, were preliminarily identified as potential Hsp 90 inhibitors through the verification of strong affinity to Hsp 90 and antitumor bioactivity. The approach based the MMFNPs provides a strong platform for imaging screening and discovery of plant-derived biologically active molecules with high efficiency and selectivity.

Semiconductor quantum dots (QDs) have achieved great success in biolabeling and bioimaging,1-4 although they have still some disadvantages including their unsteadiness in the special environments and potential biotoxicity.5-8 Plenty of reports have indicated that coating QDs with a silica could partly overcome the problems.9-13 However, majority of the coating silica are solid and cannot provide large surface area while the mesoporous structure could be widely applied for the specific adsorption, catalysis and cargo delivery due to their high surface area and large pore volume. Although a few researches reported on mesoporous silica coating,11,12,14 the QDs were largely dispersed in the scope of silica coating, which induces to the formation of relatively large silica/QDs core and small silica shell (generally two thirds of nucleus and a third of shell), easily resulting in not only cytotoxicity due to leakage of cadmium but also chemical instability caused by fluorescence fluctuation response to the external environment. Therefore, it is urgent to fabricate microkernel mesoporous fluorescent nanoparticles responsible for protecting QDs in further bio-application. Ligand fishing based on protein target-ligands binding, has been proposed as an efficient technique with high-selectivity in early stage pharmacologic molecule discovery,15-19 which is applicable for fishing specific ligands from complex matrix. Compared with solution phase protein targets, immobilized

protein targets on solid surface are more steadfast and resistant to environmental changes.20,21To date, various solid supporters, especially represented by gold nanoparticles,22 magnetic nanoparticles23-26 and silica nanospheres,27 have been applied for the discovery of potential active molecules, but few of them have been reported on imaging screening and enrichment of active molecules from complicated herbal extracts. Hsp 90 has been considered as an attractive cancer targeted protein,28-30 and Hsp 90 inhibitors are attracting incremental attention on anti-cancer research.31-34 Many Hsp 90 inhibitors are compounds derived from herbal plants,35-41 obviously, natural products continue to be highly interested in the discovery of Hsp 90 inhibitors. Tripterygium wilfordii, a radicular xylem of the Celastraceae family, has been used to treat with rheumatoid arthritis and psoriasis due to its abundant bioactive components such as alkaloids, diterpenoids, triterpenoids and sesquiterpenes,42 but few researches systematically reported bioactive components which can inhibit Hsp 90.43,44 We previously developed a fluorescent ligand fishing approach for the identification of Hsp 90 inhibitors from complex matrixes, Curcuma longa L. based on InP/ZnS

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quantum dots on the surface of mesoporous nanoparticles.45 Nevertheless, the developed fluorescent ligand fishing also Scheme 1. Schematic representation of the preparation of Hsp 90α-MMFNPs and its application based on solid-phase ligand fishing for screening Hsp 90 inhibitors from Tripterygium wilfordii.

remained challenges such as the susceptible fluorescence to external environment due to the QDs only coating the surface of the mesoporous nanoparticles. Herein, a multiple-layer mesoporous nanostructure composed of a protective silica coating CdTe QDs core and a mesoporous silica shell, i.e. microkernel-based mesoporous (SiO2-CdTe-SiO2)@SiO2 fluorescent nanoparticles (MMFNPs), was successfully fabricated for in situ biomedical imaging and enrichment of bioactive comments. As depicted in Scheme 1, this type of fluorescent nanoparticle was functionalized with Hsp 90α in the presence of crosslinking agent to obtain Hsp 90α-(SiO2CdTe-SiO2)@SiO2 nanoparticles (Hsp 90α-MMFNPs) for screening specific ligands based on affinity extraction targeted Hsp 90 from complex herbal extracts, Tripterygium wilfordii crude extraction. Ligands loaded Hsp 90α-MMFNPs were performed bioactivity-induced cellular morphology imaging to investigate the preliminary bioactivity and the ligands were further identified by HPLC-TOF/MS and GC-MS analysis. To further verify the validity of this proposed method, molecule docking, proliferation inhibition test and wound scratch assay were also carried out.

EXPERIMENTAL SECTION Fabrication of Hsp90 functionalized (SiO2-CdTeSiO2)@SiO2 nanoparticles. Preparation of thiolfunctionalized SiO2 core. The thiol-functionalized silica was synthesized to form the innermost core via the reverse microemulsion method.13 Briefly, cyclohexane (7.0 mL), Triton X-100 (1.65 mL), n-pentanol (1.70 mL), ammonia solution (25 wt%, 220 µL) and ultrapure water (400 µL) were added to a flask and sealed under stirring for 30 min. Then, TEOS (100 µL) was introduced for three days, after which MPS (10 µL) was added for another 24 h. Isopropanol was added to terminate the reaction. The resultant precipitate was centrifuged, washed with isopropanol, ethanol and water, respectively, and dispersed in pure water to obtain silica core. Synthesis of SiO2@CdTe QDs nanoparticles. Fluorescent CdTe QDs was then fabricated onto the surface of SiO2 nanoparticles.13 In brief, 50 mL of 0.04 mol/L CdCl2 (aq), trisodium citrate dehydrate (800 mg) and silica core were successively added into a three-necked flask and stirred for 15 min. Cd ions were absorbed on the surface of small silica in

virtue of the interaction of thiol with Cd2+. Then, MSA (0.2 g) was added and the pH of the reaction system was regulated to 10.5. Under the circulation of nitrogen, NaBH4 (0.1 g) and 10 mL of 0.01 mol/L Na2TeO3 (aq) were tardily added, refluxing at 100 °C for 24 h. Suspensions of different time intervals were sparingly collected for characterizations of QDs. The reaction mixture was centrifuged and washed with ethanol to give core-satellite SiO2@CdTe suspensions. Preparation of MMFNPs. The (SiO2-CdTe-SiO2)@SiO2 nanoparticles were further synthesized according to our previous report.27 Briefly, CTAB (0.5 g), TMB (3.5 mL) and SiO2@CdTe suspensions were diluted to a solution containing deionized water (240 mL) and 2 mL of 2 mol/L NaOH (aq), stirring at 80 °C for 4 h. TEOS (2.5 mL) was quickly added for another 2 h. The resultant precipitate was treated by filtration, washing and drying in a vacuum at 45 °C, and then the dried precipitate was refluxed in an ethanol solution of ammonium nitrate (NH4NO3/C2H5OH, 10 mg/mL) to remove the reagent-residues. The products were aminated by APS under refluxing in anhydrous ethanol at 80 °C, and then filtered, washed and dried to obtain aminated MMFNPs. Immobilization of Hsp 90α via amidation reaction. Cterminus of Hsp 90α was immobilized on the surface of MMFNPs by the typical amidation reaction.29 In detail, MMFNPs (10 mg) were rinsed with potassium phosphate buffer (10 mM, pH 5.5) three times. Then, human Hsp 90α protein (20 µg) was suspended in 40 µL of potassium phosphate buffer and the suspension was added to the MMFNPs followed with vortex-mixing for 5 min. 20 µL of EDAC solution (10 mg/mL) was added and oscillated with rotation at 200 rpm for 24 h at 4 °C. At room temperature, the mixture was centrifuged and the supernatant was discarded. The resultant products, i.e. Hsp 90α-MMFNPs, were washed three times with Tris-HCl buffer (10 mM, pH 7.4) and then dried with nitrogen. As-treated MMFNPs without Hsp 90α were synthesized with the same procedures for control. Preparation of Tripterygium wilfordii extraction. Approximate 20 g powders of the crude rhizomes of Tripterygium wilfordii were soaked overnight with 200 mL of 95% (v/v) ethanol, followed by ultrasound for 45 min, and the filtrates were concentrated on a rotary evaporator to yield

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Analytical Chemistry dried residue (1.0 g). A stock solution (10 mg/mL) of the

residue was then stored at 4°C for further experiments.

Figure 1. TEM images of (A) thiol-functionalized silica nanoparticles, (B) the SiO2@CdTe nanoparticles and (C) the MMFNPs . (D) UV–vis absorption and (E) the corresponding PL spectrum of SiO2@CdTe supernatant under refulx for 0.5 (a), 4 (b), 8 (c), 12 (d), 16 (e), 20 (f) and 24 h (g). (F) PL spectra of SiO2 (a), SiO2@CdTe (b) and MMFNPs (c). (G) N2 adsorption–desorption isotherms and pore-size distribution of prepared MMFNPs. (H) PL spectrum of MMFNPs in boiling water for 5 (a), 20 (b), 40 (c), 60 (d), 80 (e), 100 (f) and 120 min (g). The inset is the photographs of the MMFNPs boiled suspension under room light and UV light at 365 nm. (I) UV-Vis spectra of suspension of Hsp 90α (a), MMFNPs (b) and Hsp 90α-MMFNPs (c). Ligand fishing with functionalized Hsp 90 nanocomposites. Hsp 90α-MMFNPs (10 mg) were incubated with 2 mL of 10 mg/mL Tripterygium wilfordii extraction and vortex-mixed at 4°C for 0.5 h. After centrifugation, the complexes were washed with ammonium acetate buffer (10 mM, pH 7.4) to remove non-specific absorbents. Subsequently, the ligands fished by nanocomposite were eluted by methanol, and the supernatant with ligands was collected for further MS analysis. The same incubation with as-treated MMFNPs with un-related protein of bovine serum albumin (BSA) was carried out as a negative control, while astreated MMFNPs without Hsp 90α were performed as a blank control. Optimization of ligand fishing. Ursolic acid, designed for negative control, was an unexpected Hsp 90 inhibitor from Tripterygium wilfordii and chosen to assess veracity and specificity of the proposed method. Celastrol was utilized as positive control for optimizing factors during the fishing procedure and further validating the method. The HPLC chromatograms were used to estimate the efficiency by comparing the quantity of celastrol bound to MMFNPs. Cell imaging by CLSM. Hsp 90α-MMFNPs loaded with ligands were dispersed in complete DMEM and filtered to form a 200 µg/mL stocking solution of nanoparticles. MCF-7 cells were seeded at 5000 cells/well in a sterilized 4-well plate overnight, and then cultured by stocking solutions of nanoparticles for a period of time in an incubator at 37 °C under 5% CO2 atmosphere, followed by washing with PBS and fixing with 4% paraformaldehyde for 15 min at 37 °C. The fixed cells were stained with Hoechst 33342 for 5 min. Position determination microscopy with fluorescence of astreated cells was performed with the 40× objective of CLSM.

Cytotoxicity study of nanoparticles/ligands. MCF-7 cells were seeded in a 96-well plate at 6×103 cells/well to achieve adherence. Then the medium was replaced with fresh DMEM containing various concentrations of nanoparticles/ligands for different time intervals. Cells were washed with PBS and cultured in 100 µL of DMEM containing 10 µL of MTT solutions (5 mg/mL) for 4 h. Then, the medium was replaced with 150 µL of DMSO. All samples were assayed in triplicate. In vitro scratch wound-healing assay. MCF-7 cells were placed at 5×105 cells/ml in 6-well dishes and incubated overnight adhering confluent monolayers for wounding. Scratches were performed using a 200-µL tip and photographs were taken immediately by microscope (time zero). Then, different compounds in fresh complete culture medium were added to dishes for 48 h, and photographs of scratches were also taken by microscope. During this time period, the distance migrated by the cell monolayer to close the wounded area was measured. Experiments were carried out in triplicate. HPLC-MS analysis. HPLC analysis was carried out on a Thermo Acclaim LC system, while spectra were scanned from 190 to 500 nm with a Thermo AcclaimTM RSLC120 C18 column (100 mm × 3.0 mm i.d., 2.2 µm) at temperature of 20 °C. The mobile phase was a mixture of acetonitrile (A) and 1.0% aceticacid solution (B) at a flow rate of 0.5 mL/min, and the following gradient programme was used: 20 % A for 4 min, linear gradient to 35 % A in 1 min, linear gradient to 65% A in 1 min, 65 % A for 29 min, linear gradient to 75 % A in 10 min, linear gradient to 90 % A in 5 min, 90% A for 10 min, and return to initial conditions in the wake of equipoise for 2 min before the next sample injection. Sample injection volume was 10.0 µL. MS data in full scan mode from m/z 50 to 1000 were analysed by PeakView 1.2@Software.



GC-MS analysis. GC separation was achieved on an Agilent HP-5 capillary column (30 m×0.25 mm i.d., 0.25 µm) with split injection. Helium (purity 99.999%) was used as a carrier gas at a flow rate of 1.6 mL/min and the injection volume was 1.0 µL. The temperatures of injection port, interface and ion source were respectively set at 280 °C, 220 °C and 230 °C, using the following oven programme: firstly at 40 °C for 1 min, 20 °C/min to 200 °C, 4 °C/min to 280 °C and finally held for 10 min. Identification of the compounds was acquired by the NIST database combined with measured spectral data in the full scan mode. Virtual ligands docking. All the inferred ligands participated in binding the virtual 3D coordinates of the X-ray crystal structures of Hsp 90 (PDB: 3IED) to evaluate the attended mode and the stability of connection. Discovery Studio 3.0@Software was used to analyse interaction between ligands and Hsp 90, which has an advantage in utilizing the interior algorithm to generate the whole poses inside known or predicted binding sites for ligands. Briefly, water molecules in the protein 3D model were removed and all hydrogen atoms were added. Initial structures of investigated ligands were minimized by applying the force field. Docking parameters consisted of setting the number of hotspots to 100, docking tolerance to 0.25 and the max number of hits to 100, while the max hits to save were set to 10 for saving running time.

RESULTS AND DISCUSSION Characterization of Hsp 90 functionalized MMFNPs. Figure 1A showed the TEM image of silica nanoparticles grafted thiol groups as seeds with a diameter of 2.6±0.3 nm. CdTe nanocrystals were in situ formed on the surface of the silica nanoparticles as shown in Figure 1B, and the average size of the SiO2@CdTe nanoparticles was 5.6 ±0.5 nm, larger than that of the core silica nanoparticles, indicating the successful growth on the silica surface. In order to restrain the release of Cd, enhance the surface area and retain the fluorescence of CdTe QDs, another mesoporous silica was then coated onto the surface of the SiO2@CdTe nanoparticles. Figure 1C showed highly uniform spherical morphology of the monodispersed nanoparticles with radial mesopore channels and a mean diameter of 105 ± 2.5 nm. As shown in Figure 1D and 1E, the UV-Vis absorption and photoluminescence (PL) spectrum of the SiO2@CdTe nanoparticles demonstrated that a set of fluorescent nanoparticles with different emission peaks can be synthesized by adjusting the refluxing time. The corresponding photographs were also shown in Supporting Information Figure S1 with 365 nm UV lamp. By comparing the fluorescence integrated areas of the SiO2@CdTe nanoparticles and MMFNPs, 82% of the original QDs fluorescence can be retained after encapsulation onto the SiO2@CdTe nanoparticles as shown in Figure 1F. The prepared MMFNPs exhibited a high surface area of 327 m2•g-1 and pore diameter of 5 nm by the characteristic type of I–V curves with a hysteresis loop generated by capillary condensation according to the IUPAC classification as shown in Figure 1G, indicating their mesoporous structure which could be beneficial for the subsequent specific adsorption. According to the atomic absorption spectrometry (AAS) analysis results (Figure S2), no signals of Cd were detected in the supernate of MMFNPs solution with 2 hours boiling, while 57.4 µg/L of Cd was detected in SiO2@CdTe, indicating the

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formation of SiO2@CdTe and further the completely coating of SiO2 layer. The fluorescence of the bare SiO2@CdTe nanoparticles decreased and even disappeared under UV irradiation in boiling water within 120 min (Figure S3). In contrast, the prepared MMFNPs were stable during 40 min, and the PL intensity in Figure 1H remained at 72% after 120 min boiling. Therefore, MMFNPs show a significantly improved PL stability compared with SiO2@CdTe nanoparticles. Figure S4 exhibited the PL changing of MMFNPs under different pH solutions. The MMFNPs were almost stable in a wide pH range of 3-13, indicating considerable chemical stability, however, the fluorescence of SiO2@CdTe nanoparticles was remarkably decreased under acidic conditions and easily influenced even in minor pH variations. After the bond of the Hsp 90α, Hsp 90α-MMFNPs exhibited an apparent UV absorbance at 265 nm attributing to proteins, as shown in Figure 1I, suggesting the successful conjugation of Hsp 90α on fluorescent nanocomposites. The amount of immobilized Hsp 90α on the surface of MMFNPs was about 210 µg/mg calculated by typical bicinchoninic acid approach (BCA) kit. Optimization of the ligand fishing based on Hsp 90αMMFNPs. Celastrol, reported as a typical Hsp 90 inhibitor from Tripterygium wilfordii,46,47 was selected to test the feasibility of the Hsp 90α-MMFNPs based ligand fishing assay and optimize fishing conditions, for instance, temperature would affect the activity of Hsp 90 while incubation time and eluent would have effect on the quantity of ligands bound to Hsp 90. Hence, incubation temperature (4 °C, 25 °C, 37 °C), incubation time (from 0.5 h to 4 h), concentration (v/v from 50 % to 100 %) and volume of the eluent were optimized during ligand fishing process. Celastrol was highly enriched by the Hsp 90α-MMFNPs, in contrast, scarcely was bound to as-treated nanoparticles without Hsp 90α (Figure S5). When the standard solution of ursolic acid was mixed into that of celastrol, celastrol was alone absorbed onto the Hsp 90α-MMFNPs (Figure S6). All of these results obviously proved the high feasibility and specificity of the assay for isolating Hsp 90 inhibitors. Finally, the optimal incubation conditions were obtained as following: incubation temperature at 4 °C consistent with previous reports,15,48 incubation time for 0.5 h, methanol (v/v 100%, 0.5 mL) as eluant to dissociate bound components (Figure S7-10). In situ cell imaging. In vitro cytotoxicity of MMFNPs suspensions was firstly assessed by cell viability through standard MTT assay as shown in Figure S11. At the increasing concentrations (from 50 µg/mL to 500 µg/mL), the cells viability after incubation by MMFNPs for 24 h still kept greater than 80%. When cells were treated with 200 µg/mL of MMFNPs at different time intervals, more than 90 % cell viability was still obtained, which exhibits low biotoxicity. As shown in Figure 2A, when 200 µg/mL solutions of MMFNPs incubated cells for 1 h, very bright red fluorescence was observed around the nucleus, suggesting MMFNPs could be efficiently internalized by the cells via passive endocytosis within a short time attributing to favorable biocompatibility. The results of the cytotoxicity profile already demonstrated that this dosage of MMFNPs for cell imaging did not induce obvious cytotoxicity within 24 h. Figure 2B and 2C displayed the imaging of MCF-7 cells incubated with 200 µg/mL solutions of Hsp 90α-MMFNPs loaded ligands (Experimental group) and the pure Hsp 90α-MMFNPs (Control group) for 4 h. The cells in experimental group were condensed to turn


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Analytical Chemistry round and trended to form a mass of apoptotic bodies in comparison with control group, which indicated that the fished ligands could have a potential therapeutic efficacy for inhibiting the cancer proliferation and inducing apoptosis. As shown in Figure S12, the pure Hsp 90α-MMFNPs still maintained observable fluorescence signals, proving excellent chemical stability and photostability of the nanocomposites.

GC-MS analysis of ligands fished from Tripterygium wilfordii by Hsp 90α-MMFNPs. Figure 4 displayed the GCMS spectrums of Tripterygium wilfordii extraction (Figure 4A), elution fractions from Hsp 90α-MMFNPs (Figure 4B), eluent from as-treated MMFNPs without Hsp90α (Figure 4C) and eluent from BSA-MMFNPs (Figure 4D) in full scan mode. The MS signals of direct Tripterygium wilfordii extraction detection were highly complexity due to matrix effect,

Figure 2. CSLM images of MCF-7 cells after incubation with (A) MMFNPs for 1.0 h, (B) Hsp 90α-MMFNPs loaded ligands and (C) the pure Hsp 90α-MMFNPs for 4 h with corresponding dark field and merge field. Arrows indicate representative apoptotic nuclei with the nuclear membrane rupturing. Scale bar: 50 µm. HPLC-TOF/MS analysis of ligands fished from Tripterygium wilfordii by Hsp 90α-MMFNPs. As shown in Figure 3, direct injection of Tripterygium wilfordii extract, elution after ligand fishing assay by Hsp 90α-MMFNPs, astreated MMFNPs without Hsp90α and BSA-MMFNPs were analysed by HPLC-TOF/MS in sequence. Compared with the direct analysis without fishing procedures, the MS signal of specific components could be easily detected after MMFNPs based ligand fishing experiment in virtue of the preliminary removal of ion interference and non-specific components. Eighteen components in elution from the Hsp 90α-MMFNPs were inferred and listed in Table 1, twelve of which were not detected in Figure 3C, predicting that these twelve kinds of specific ligands might be probable selective Hsp90α-targeted components. Meanwhile, only two obvious peaks were detected in Figure 3D, which indicated the high selectivity of Hsp 90α-MMFNPs. For further proof, four components were exactly identificated as demecolcine (Component 1), wilforine (Component 9), triptotriterpenic acid (Component 11), celastrol (Component 18) by hit formula with peak view1.2@software combined with MS spectrograms of reference samples, as shown in Figure 3E. Quantitative amounts of demecolcine, wilforine and celastrol enriched from Tripterygium wilfordii by Hsp 90αMMFNPs were respectively 1.176, 3.136 and 11.321 mg/g, calculated by an external standard method. Other eight fractions have no report in the previous study of Tripterygium wilfordii, suggesting that they could be new entities with a strong affinity to Hsp 90α. Component 18, celastrol, screened from the elution by Hsp 90α-MMFNPs, had been confirmed to be a novel inhibitor of Hsp 90, in which case it could be regarded as strong proof to verify the feasibility and specificity of our ligand fishing method based on Hsp 90αMMFNPs.

Figure 3. HPLC-TOF/MS spectrograms of (A) the direct injection of Tripterygium wilfordii extract, (B) the elution after ligand fishing assay by Hsp 90α-MMFNPs, (C) as-treated MMFNPs without Hsp90α and (D) BSA-MMFNPs, (E) the reference samples of compound 1, compound 9 and compound 18. Red-labeled peaks represent the references standard.



moreover, they were often drowned by the interferences of non-active components. In contrast, the Hsp 90α samples after ligand fishing assay exhibited much cleaner mass spectra owing to the elimination of the interferences. In Figure 4B, eleven fished ligands in elution from the Hsp 90α-MMFNPs were inferred by EI-MS spectra databases, the results of which are listed in Table 2. Among these, eight kinds of fished ligands were selectively detected compared with control groups as shown in Figure 4C and 4D, predicting that they have selective affinity with target protein Hsp 90. Table 1. Precursor ions [M+H] + of identified peaks in eluent and hit formula with Peak View1.2@software in the LC-TOF/MS spectrogram. Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Rt (min) 6.700 16.031 17.348 18.112 20.990 23.205 25.470 27.193 30.021 31.761 33.275 33.444 34.687 44.412 49.514 52.506 55.278 55.763

Compound name

Hit formula

m/z, [M+H] +

Demecolcine

C21H25NO5 C27H43NO4 C21H45N C24H36O2 C21H30O4 C35H49NO3 C32H34O4 C38H34O C43H49NO18 C40H43N9O9S C30H48O4 C30H58O2 C24H34O C25H40O4 C25H38O2 C23H46O8 C28H40O C29H38O4

371.1805 446.3265 312.3625 357.2788 347.2217 532.3785 483.2530 507.2682 868.3117 826.2977 473.3625 451.4510 371.2945 405.2999 371.2945 450.3551 393.3152 451.2843

Wilforine Triptotriterpenic acid -

Celastrol

Virtual docking of specific ligands to Hsp 90. Twelve specific components by LC-MS and GC-MS analysis were chosen to dock with the whole binding sites of Hsp 90 (PDB: 3IED) for screening specific ligands with strong affinity to Hsp 90. As shown in Supporting Information Figure S13-14, three of all docking ligands, i.e. celastrol, wilforine and demecolcine, preferentially bound to the largest binding loop, making a potential cation-π interaction between the cationic and aromatic/conjugated systems which widely exists in biological systems and plays an important role in the recognition of ligands.49 In detail, celastrol could form an obvious π-interaction with the positively charged amino acid residues of Hsp 90 (LYS 184 and LYS 188) owing to its conjugated structure, accompanied by hydrogen bonds between the oxygens of the carboxylic acid moiety and LYS 190, in which case it could be suggested that celastrol shows more sufficient affinity with Hsp 90 compared with other pentacyclic triterpenoids such as ursolic acid. Both of wilforine and demecolcine interacted with the positively charged amino acid chains of Hsp90 (LYS 188), and the former was coordinated with two hydrogen bonds (LYS 188 and LYS 190), the latter cooperating with three hydrogen bonds (LYS 184, GLY 296, SER 297). Interactions of two-dimensional planar graph between Hsp 90 and ligands are shown in Supporting Information Figure S14. Docking results further revealed that celastrol, wilforine and demecolcine have strong affinity with Hsp 90, indicating a promising Hsp 90 inhibitor. Verification of bioactivity of fished ligands. Figure 5A displayed the cell proliferation inhibition of MCF-7 cells treated by the culture medium of dried Tripterygium wilfordii

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crude extractions, eluent fractions with fished ligands by Hsp 90α-MMFNPs (Experimental group) and as-treated MMFNPs without Hsp90α (Control group). Obviously, experimental group showed potent cancer cell inhibitory activity compared with control group, which indicated that eluent fractions from the proposed Hsp 90α-MMFNPs were rich of bioactive compounds targeted Hsp 90. As shown in Figure 5B, the final screened ligands by docking results, i.e. celastrol, demecolcine and wilforine, were further investigated to verify the bioactivity, which respectively exhibited cancer cell growth inhibitory activity with IC 50 value at 4.183, 16.725, 34.833

Figure 4. GC-MS spectrograms of (A) the direct injection of Tripterygium wilfordii extract, (B) the elution after ligand fishing assay by Hsp 90α-MMFNPs, (C) as-treated MMFNPs without Hsp90α and (D) BSA-MMFNPs. Numbers indicate


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Analytical Chemistry main fractions in elution and hit structural formula with NIST database, which are listed in Table 2.

wound scratch assay. Apparently, celastrol had the highest inhibitory effects on cancer cell migration, followed by demecolcine and wilforine. These results confirmed that the bioactivity of fished ligands from the Hsp 90α-MMFNPs could be highly attributed to the enrichment of these three compounds targeted Hsp 90.

µmol/L, and 17-AAG, a known Hsp 90 inhibitor,50 was chosen as positive control with IC 50 value at 0.401 µmol/L. Moreover, Figure 5C displayed the microscope images of MCF-7 cells incubated with different compounds through Table 2. Precursor ions of main fractions in elution and hit structural formula with NIST database in GC-MS spectrogram. Number

Rt (min)

1

2.688

R1-Barrigenol

Name

Hit structural formula with NIST C30H50O6

506, 439, 334, 298

m/z, ions

2

9.725

24R,25-dihydroxycholecalciferol

C27H44O3

416, 383, 253, 221

3

14.619

Ursodeoxycholic acid

C24H40O4

392, 374, 356, 302

4

14.726

8,14-Seco-3,19-epoxyandrostane-8,14-dione, 17acetoxy-3β-methoxy-4,4-dimethyl-

C24H36O6

420, 360,304,265

5

18.077

Spirost-8-en-11-one

C27H40O4

420, 356, 314, 281

6

19.635

Propanoic acid

C27H42O4

430, 415, 355, 337

7

21.780

Demecolcine

C21H25NO5

371, 342, 312, 207

8

32.799

Bufa-20,22-dienolide

C24H34O4

386, 350, 325, 281

9

34.284

Ergosta-5,22-dien-3-ol

C30H48O2

440, 380, 327, 255

10

35.902

β-Sitosterol

C29H50O

414, 396, 329, 255

11

37.384

Betulinaldehyde

C30H48O2

440, 411, 207, 189

Table 3. Advantages and disadvantages of comparison with different screening methods. Screening Methods Gold nanoparticles Magnetic beads Mesoporous SiO2/TiO2 nanocomposites Mesoporous silica-InP/ZnS QDs nanocomposites Mesoporous (SiO2-CdTe-SiO2)@SiO2 nanocomposites

Specificity + + + + +

In vitro Hsp 90α inhibition assay. Figure 6 displayed the inhibition of Hsp 90α in MCF-7 cells treated by the culture medium of dried eluent fractions with fished ligands from Hsp 90α-MMFNPs (Experimental group), as-treated MMFNPs without Hsp90α (Control group), celastrol, demecolcine and wilforine, respectively. Compared with the untreated cells, celastrol had the maximum inhibition of Hsp 90α, followed by demecolcine and wilforine, which positively correlated with proliferation inhibition. Experimental group showed higher inhibitory effects on Hsp 90α than control group, suggesting the inhibition of fished ligands by Hsp 90α-MMFNPs to Hsp 90. Superiority of the proposed ligand fishing. Current

Imaging + +

Fluorescence stationary controllable

Applications Designations Plant extracts Proteins Complexes Complexes

References [22] [23-26] [27] [45] This work

Figure 5. (A) Cell viability of MCF-7 cells after incubation of Tripterygium wilfordii crude extractions, eluent fractions with fished ligands by experimental and control group. (B) Cell growth inhibition of MCF-7 cells in incubation of different concentrations of compounds. (C) Microscope images of MCF-7 cells incubated with different compounds through wound scratch assay (Scale bar: 500 µm). fishing techniques based on other nanoparticles were compared with our developed fluorescent ligand fishing in Table 3. The Hsp 90α-MMFNPs-based ligand fishing is superior in the high efficiency and specificity for fishing ligands from herbal extraction, allowing easy adaptation to complex matrixes. Moreover, as-prepared MMFNPs as fluorescent tracers showed impressive chemical stability and excellent optical targeting function, which could be helpful to show real-time screening results.

Figure 6. Expression inhibition of Hsp 90α in MCF-7 cells treated by different samples for 24 h.

CONCLUSIONS In summary, a highly fluorescent, lower cytotoxicity and super stable microkernel-based mesoporous nanoparticle with an average diameter of 105 ± 2.5 nm was fabricated. By using



the nanoparticles as sorbents and fluorescent tracers, a ligand fishing approach coupling with in situ imaging for screening and identification of Hsp 90 inhibitors from natural products was developed. Twelve components were selectively screened from Tripterygium wilfordii, four of them including celastrol were identified by HPLC/MS analysis and eleven components were inferred by GC/MS analysis. Molecular docking further revealed the strong affinity of three fished components, i.e. celastrol, wilforine and demecolcine, with Hsp 90, indiacating a promising Hsp 90 inhibitor. Their respective bioactivity was verified by MTT assay and wound scratch assay. We believe that this proposal could provide a facile and effective platform for Hsp 90 inhibitors from complex matrix.

ASSOCIATED CONTENT Supporting Information Chemicals; Apparatus; Hsp 90α ELISA; Photographs and PL spectrum of SiO2@CdTe; HPLC chromatograms; Docking diagram. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Corresponding author. E-mail: [email protected]; Fax: +86 2585811839; Tel: +86 25 85811839

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by Project of National Natural Science Foundation of China (81573388). This work was also sponsored by “Qing Lan Project of Jiangsu province” and “Six talent peaks project of Jiangsu Province (YY-032)”. We also greatly appreciate sponsorship of “A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions” (PAPD).

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