Anti-CD133 monoclonal antibody conjugated immunomagnetic nanosensor for molecular imaging of targeted cancer stem cells

Accepted Manuscript Title: Anti-CD133 monoclonal antibody conjugated immunomagnetic nanosensor for molecular imaging of targeted cancer stem cells Authors: Xueqin Wang, Bo Li, Ruifang Li, Yan Yang, Huiru Zhang, Baoming Tian, Liuqing Cui, Haibo Weng, Fang Wei PII: DOI: Reference:

S0925-4005(17)31841-5 https://doi.org/10.1016/j.snb.2017.09.175 SNB 23266

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

10-5-2017 28-8-2017 25-9-2017

Please cite this article as: Xueqin Wang, Bo Li, Ruifang Li, Yan Yang, Huiru Zhang, Baoming Tian, Liuqing Cui, Haibo Weng, Fang Wei, Anti-CD133 monoclonal antibody conjugated immunomagnetic nanosensor for molecular imaging of targeted cancer stem cells, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.09.175 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-CD133 monoclonal antibody conjugated immunomagnetic nanosensor for molecular imaging of targeted cancer stem cells

Xueqin Wang*a, Bo Lia, Ruifang Li a, Yan Yang b, Huiru Zhanga Baoming Tian b, Liuqing Cui a, Haibo Weng b, Fang Wei b*

a

College of Bioengineering, Henan University of Technology, Zhengzhou, Henan 450001, P.R .China

b

School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001,P.R.China

* Corresponding authors: Xueqin Wang, E-mail address: [email protected], College of Bioengineering, Henan University of Technology, Zhengzhou, Henan 450001, P.R .China. Tel.: + 86 371 67756928; fax: + 86 371 67756928. Fang Wei, E-mail address: [email protected], School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001,P.R.China. Tel.: + 86 371 67739513; fax: + 86 371 67739513.

Highlights about the present work



◆ A smart immunomagnetic nanosensor was developed based on 1

nanoparticles (NPs) grafted with

the raised monoclonal antibody (mAb) by

the marker antigen CD133 of glioblastoma CSCs. 

◆ The anti-CD133 mAb-conjugated nanosensor was endowed with biological specificity and high activity.



◆ The immunomagnetic nanosensor could be used for molecular imaging of specifically targeted glioblastoma CSCs.



◆ The immunomagnetic nanosensor was a promising nanovehicle for human brain tumor diagnosis and therapy.

Abstract Magnetic nanosensors are considered as highly attractive platforms for effective treatments in cancer diagnosis and therapy. Herein a smart immunomagnetic nanosensor is novelly developed for molecular imaging of targeted glioblastoma cancer stem cells (CSCs), based on specific interaction between cell-membrane marker antigen CD133 of glioblastoma CSCs and its raised anti-CD133 monoclonal antibody (mAb). Superparamagnetic iron oxide (SPIO) γ-Fe2O3 nanoparticles (NPs) were fabricated as nanosensor cores with approximately 10-15 nm in size, and coated with carboxymethyl chitosan (CMCS) via sodium tripolyphosphate (TPP) crosslinking, and then chemically modified with polyethylenimine (PEI). Anti-CD133 2

mAb, specific affinity with the cancer stem biomarker CD133 expressed on the membrane surface of glioblastoma CSCs, was subsequently conjugated with the PEImodified SPIO NPs to form anti-CD133 mAb conjugated immunomagnetic nanosensor.

The

prepared

immunomagnetic

nanosensor

i.e.

anti-CD133

mAb-conjugated nanoscale magnetic sensor (mAb-nano-MSN) was biologically assayed with cellular toxicity, cell cycle and specificity, and finally delivered to the targeted CSCs for fluorescence imaging and magnetic resonance imaging (MRI) within human brain glioblastoma CSCs. The results demenstrated that the developed immunomagnetic nanosensor displayed preferable properties such as excellent biocompatibility, non-toxicity and high specificity. The glioblastoma CSCs treated with anti-CD133 mAb-nano-MSN displayed a strong red fluorescence signal and a negative

contrast

enhancement

compared

with

the

cells

treated

with

non-mAb-functionalized magnetic nanopartitles, probably due to efficient endocytosis mediated by anti-CD133 mAb grafted onto the prepared magnetic nanosensor. Therefore, these results indicated the fabricated anti-CD133 mAb-nano-MSN could be used as a promising nanovehicle for molecular imaging for the targeted CSCs in human brain tumor diagnosis and therapy.

Keywords Immunomagnetic nanosensor; Molecular imaging; Cancer stem cells (CSCs); Anti-CD133 monoclonal antibody

1. Introduction Cancer stem cells (CSCs) are tumorigenic, and are proposed to persist in 3

heterogeneous tumors as a distinct subpopulation, which cause tumor relapse and metastasis by giving rise to new tumors [1,2]. CSCs play a vital role in carcinogenesis and tumor progression, and are currently becoming one of the prime targets for molecular/cellular exploration and clinical development of various types of cancers [3-5]. Magnetic nanoparticles are highly attractive platform materials for bioapplications owing to their unique properties including uniform size [6,7], biocompatibility [8-10], superior imaging characteristics [11,12], and facile surface modification to introduce additional cell-specific targeting coordinated molecules [13-15], has enabled these systems to be employed as smart nanomedicines that incorporate combinations of different components [16,17]. Recent progress in magnetic nanosystems has greatly improved many notable and important biomedical and clinical applications such as cell/protein detection and screening [18, 19], gene/drug delivery [20-22], biological sensor construction [23, 24], medical imaging and cell tracking [25, 26], and son on. Cell-specific targeting strategies for high-performance magnetic nanosystems could be achieved by immobilization of target-specific moieties, such as receptor ligand [27-29], antibodies [30, 31], small peptides [32, 33], and aptamers [34, 35]. In particular, the antibody-conjugated magnetic nanoparticles are able to specifically bind with their antigens, which are overexpressed in certain cancer cells, and internalize into the cells by receptor-mediated endocytosis [36]. Emerging therapeutic strategies in cancers have been focusing on the use of monoclonal antibodies to refine delivery of cytotoxic agents. 4

Glioblastoma is one of the most common primary brain tumors in adults without efficient therapy for now. Low efficacy in glioblastoma treatments is usually caused by challenges in delivering agents efficiently targeted to glioblastoma stem cells. The lack of such progresses in the treatments reflects the need for new diagnostic and therapeutic approaches in human brain tumor treatments. CD133 has recently been identified as a common marker of CSCs for many cancer types, which is a novel cell-membrane protein used as a targeting antigen because it is expressed on the membrane surface of glioblastoma CSCs, as a prognostic indicator for tumor regrowth and malignant progression [37]. In this present study, we aimed to fabricate a smart immunomagnetic nanosensor based on nanoparticles (NPs) grafted with the raised monoclonal antibody (mAb) by the marker antigen CD133 of glioblastoma CSCs for molecular imaging of specifically targeted glioblastoma CSCs. To achieve this goal, the SPIO NPs were firstly synthesized with approximately 10-15 nm in size, and coated with carboxymethyl chitosan (CMCS) via sodium tripolyphosphate (TPP) crosslinking, and then chemically modified with polyethylenimine (PEI) to prepare the PEImodified magnetic nanoparticles (PEI-MNNs). Assisted with coupling reagents sulfosuccinimidyl- 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) and Traut’s Reagent, the PEI-MNNs were successfully conjugated with anti-CD133 mAb to construct anti-CD133 mAb-conjugated immunomagnetic nanosensor, which was specifically targeted for antigenic protein CD133 expressed on the membrane surface of glioblastoma CSCs. This study demonstrated that the fabricated 5

anti-CD133 mAb-conjugated immunomagnetic nanosensor was provided with biological specificity and high activity, and could be used as a promising nanovehicle for molecular imaging of the targeted glioblastoma CSCs in brain tumor diagnosis and therapy. 2. Experimental Section 2.1. Materials and reagents Recombinant human epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) were purchased from Peprotech (Rocky Hill, NJ, USA). Leukemia inhibitory factor (LIF) was purchased from Chemicon (Temecula, CA, USA). B27 was received from Invitrogen (Carlsbad, CA, USA). Vincristine was obtained from Haizheng

Pharmaceutical

Co.,

Ltd.

(Zhejiang,

China).

CMCS,

sodium

tripolyphosphate (TPP), dimethyl sulfoxide (DMSO), RBITC, 3-(4,5-dimethylthiazlo2-diphenyl-tetrazolium) bromide (MTT), Hoechst 33258, sulfo-SMCC, and Traut’s Reagent, propidium iodide (PI), and fluorescein diacetate (FDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The human glioblastoma cell line U251 was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell culture medium and the fetal bovine serum (FBS) were acquired from Gibco (Invitrogen, CA, USA). The specific primary antibodies against CD133 (mouse monoclonal IgG) were obtained from Abcam (Cambridge, UK, USA). The fluorescence-labeled secondary antibodies goat anti-mouse antibody (IgG-RBITC) and goat anti-mouse antibody (IgG-Alexa flour 594) were obtained from Boster

6

(Wuhan, China). All the solvents and other chemicals were purchased from local commercial suppliers and were of analytical reagent grade, unless otherwise stated. All solutions were prepared using ultra-purified water supplied by a Milli-Q water system (Millipore, Billerica, MA). 2.2. Assay principle of the prepared immunomagnetic nanosensor for molecular imaging of targeted CSCs The general principle of the immunomagnetic nanosensor for real-time molecular imaging of targeted glioblastoma CSCs (Scheme 1) mainly includes: 1) the isolation and enrichment of targeted CSCs through vincristine induced cell apoptosis from glioblastoma U251 cells; and 2) the fabrication of immunomagnetic nanosensor by conjugating the PEI modified magnetic nanoparticles with anti-CD133 mAb assisted with coupling reagents sulfo-SMCC and Traut’s Reagent; and 3) real-time imaging via specifically delivering the prepared immunomagnetic nanosensor into glioblastoma CSCs. 2.3. Synthesis of magnetic PEI-MNNs In the present study, superparamagnetic γ-Fe2O3 nanoparticles (SPIO NPs) were synthesized as magnetic nanosensor cores through the chemical coprecipitation method [38, 39], and more information about synthesis of SPIO NPs are provided in the Supplementary Information (SI). The CMCS modified magnetic nanoparticles (CMCS@SPIO NPs) were synthesized through TPP crosslinking. Briefly, 50 mL of TPP (1 mg/mL) were mixed with SPIO NPs (10 mg/mL), and vigorously stirred for

7

30 min at 60 °C. The mixture was kept for 12 h at room temperature (RT), and washed three times with DI water to obtain TPP@SPIO NPs. 10 mL of CMCS solution (1% w/v, dissolved in acetic acid) was added into the above TPP@SPIO NPs and allowed to react for 30 min in ultrasonic emulsifier. The prepared MNNs were washed three times with DI water, and stored in 4°C. The prepared MNNs were then modified with PEI mediated by EDC/NHS. In brief, aqueous PEI solution were added to activated MNNs solution (V/V,1:2) in MES buffer, magnetically stirred for 24 h. Then the formed PEI-MNNs were washed three times under magnetic field using phosphate-buffered saline (PBS, pH 7.4), and finally suspended in PBS (pH 7.4) and stored at 4 °C until use. The amount of PEI coupled with the MNNs was measured with the ninhydrin chromogenic method as previously described elsewhere [40]. 2.4. Fabrication of anti-CD133 mAb conjugated immunomagnetic nanosensor and RBITC labeling Anti-CD133 mAb conjugated immunomagnetic nanosensor was prepared according to the following process, in which the PEI-MNNs were firstly reacted with amine reactive succinimidyl ester-activated carboxy group of anti-CD133 mAb by using the cross linker sulfo-SMCC. In a typical reaction, the anti-CD133 mAb was first thiolated by mixing 240 μL of anti-CD133 mAb (1 mg/mL) in borate buffer (pH 8, containing 2 mM EDTA) followed by addition of 30 μL of Traut’s Reagent (2 mg/mL), and then the mixture was reacted under stirring for 60 min at RT to obtain anti-CD133 mAb-SH. 250 μL of Sulfo-SMCC solution (5 mg/mL) dissolved in PBS (pH 7.4) was 8

added into 5.5 mL of PEI-MNNs suspension (10 mg/mL), and the mixture was reacted for 30 min at RT to form sulfo-SMCC-PEI-MNNs. The anti-CD133 mAb-SH was

finally

added

into

the

sulfo-SMCC-PEI-MNNs

solution

to

obtain

anti-CD133-PEI-MNNs, i.e. anti-CD133 mAb-conjugated nanoscale magnetic sensor (mAb-nano-MSN).

Finally,

the

prepared

anti-CD133

mAb-conjugated

immunomagnetic nanosensor was washed three times with PBS (pH 7.4), re-dispersed in PBS (pH 7.4), and stored at 4 °C for future use. RBITC was considered as a probe with excellent fluorescence properties. The isothiocyanato group of RBITC could bind directly to the amino group of the anti-CD133 mAb-conjugated mAb-nano-MCs to produce a fluorescently labeled immunomagnetic nanosensor. 10 mg anti-CD133 mAb-conjugated immunomagnetic nanosensor were suspended in 1000 μL PBS (pH 9.0) solution, and then 100 μL RBITC solution was added into suspension, and kept on the shaker at dark for 12 h to prepare RBITC-labeled anti-CD133 mAb-conjugated immunomagnetic nanosensor. The residual substance was washed away under a magnetic field, and then the RBITC-labeled anti-CD133 mAb-conjugated immunomagnetic nanosensor were observed with an inverted fluorescence microscope. 2.5. Activity assay of the prepared immunomagnetic nanosensor The activity assay of the prepared immunomagnetic nanosensor was performed using an indirect fluorescence immunoassay [41]. Briefly, 100 µL anti-CD133 mAb-nano-MSN (1 mg/mL) were loaded into 0.5 mL polystyrene cuvettes pre-coated

9

with 1% (w/v) bovine serum albumin (BSA) [42] and magnetically separated to remove the upper suspension. The fluorescence-labeled secondary antibody goat anti-mouse antibody (5 μL, IgG-Alexa flour 594) was diluted (1:20, v/v) with PBS (pH 7.4), and pipetted into each cuvette, and then incubated at 37 °C for 30 min in a shaker incubator. The post-immunoassay nanoparticles were magnetically separated, and washed with the PBS containing 0.05% (v/v) Tween-20, and observed under an inverted fluorescence microscope (Eclipse TE 2000-U). 2.6. Cancer stem cells culture Human glioblastoma cell line U251 was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The culture method was followed as previously described [43]. The U251 cells were routinely cultured in RPMI 1640 medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, and then kept in a humidified atmosphere with 5% CO2 at 37 °C. During the exponential growth phase, the U251 cells were harvested and seeded in a serum-free neural stem cell medium containing Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12), B27 (1×), recombinant human epidermal growth factor (20 ng/mL), basic fibroblast growth factor (20 ng/mL), and leukemia inhibitory factor (10 ng/mL), and 8 ng/mL vincristine were added into culture medium to induce apoptosis, and yield the glioblastoma CSCs. 2.7. Immunocytochemistry staining The isolated glioblastoma CSCs were firstly seeded in 24-well plates that was 10

pre-coated with PLL[43], and cultured for 3 h in an RPMI-1640 medium supplemented with 10% FBS, and then fixed using 4% paraformaldehyde for 30 min at room temperature after washing with PBS thrice. The fixed cells were permeabilized with Triton X-100 (0.1%) solution for 30 min at room temperature, and then pre-treated with 5% newborn calf serum for 30 min at 37 °C to block nonspecific binding, and incubated with specific primary antibodies against CD133 (mouse monoclonal IgG1, 1:100) overnight at 4 °C. After rinsing thrice with PBS, the fluorescence-labeled secondary goat anti-mouse antibodies (IgG-RBITC, 1:20) were added and incubated for 1 h at 37 °C. The cell were counterstained with DAPI (100 nM, Sigma-Aldrich Co.) to reveal the nuclei. 2.8. SEM analysis of glioblastoma CSCs The glioblastoma CSCs were seeded in the PLL-coated cover slip, cultured in RPMI-1640 medium containing 10% FBS for 24 h, and then fixed with 2.5% glutaraldehyde at 4 °C overnight. After rinsing four times with PBS (pH 7.4), the sample was treated with 1% osmium tetroxide for 20 min, and then dehydrated with a series of ethanol gradients (30%, 50%, 75%, 100%) [44]. After drying with CO2, the CSCs were observed under a scanning electron microscope (SEM, JSM-6360LV, Japan). 2.9. Cellular viability assay The viability of the glioblastoma CSCs, as well as their differentiated progeny, was assessed according to the FDA and PI double-staining protocol [45, 46]. The isolated 11

CSCs were then resuspended in the RPMI-1640 medium supplemented with 10% FBS and cultured for 4 h, 24 h, 72 h, 96 h, and 7 d after being washed with PBS three times. Subsequently, 1 μg/mL FDA solution and 20 μg/mL PI solution were successively introduced into the culture plates. The living cells were stained green by FDA, whereas the dead cells were stained red by fluorescent dye PI. The viability of treated cells was analyzed under an inverted fluorescence microscope (Eclipse TE 2000-U, Nikon, Kyoto, Japan) equipped with a high-resolution CCD camera (CV-S3200, JAI Co., Japan). 2.10. Assay of cell proliferation capacity The proliferation capacity of the treated glioblastoma CSCs was assayed using a standard 3-(4, 5)- dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide (MTT) method [47,48]. The glioblastoma CSCs were incubated with 100 µL of the anti-CD133 mAb-nano-MSN solution (100 μg/mL) for 24h, 48h, 72h and 96h, and then added with 200 μL MTT solution (final concentration: 0.5 mg/mL). The sample was incubated for 4 h, and added with 150 μL dimethyl sulfoxide (DMSO) solution, and analyzed on a microplate reader (Bio-Rad 680, Japan) at 570 nm after incubation in a shaking incubator for 15 min. 2.11. Cellular uptake The cellular uptake was assayed by co-culturing glioblastoma CSCs with the RBITC labeling anti-CD133 mAb-conjugated mAb-nano-MSN. Briefly, the CSCs were seeded into a 24-well plate and cultured for 72h, and then incubated with the 12

RBITC labeling anti-CD133 mAb-conjugated mAb-nano-MSN (100 μg/mL) for 4 h. The rinsed residual nanoparticles were rinsed with PBS, and the prepared samples were finally evaluated by fluorescence microscopy (Eclipse TE 2000-U, Nikon, Japan) equipped with a high-resolution CCD camera (CV-S3200, JAI Co., Japan). 2.12. Transmission electron microscopy (TEM) The glioblastoma CSCs were incubated with anti-CD133 mAb-nano-MSN (200 μg/mL) for 24 h in 6-well plate (1 × 10

6

cells/ well), and pre-treated with 2.5%

glutaraldehyde in PBS (pH 7.4), and then fixed in the solution with 2% osmium tetroxide and 3% potassium ferrocyanide for 1 h. The treated cells were then stained with a 2% aqueous uranyl acetate solution and dehydrated in a graded series of alcohol. The dehydrated sample was embedded in propylene oxide, and sectioned in 70 nm thickness with a Leica UC6 ultramicrotome, and finally observed on a transmission electron microscope (Hitachi, HT7700, Tokyo, Japan). 2.13. Cell cycle analysis The glioblastoma CSCs were treated with anti-CD133 mAb-nano-MSN (100 μg/mL), and collected to dissociate into a single-cell suspension in 500 μL PBS buffer (pH 7.4), and then fixed with 500 μL of 70% ice-cold ethanol at 4 °C overnight. The prepared cell suspension was then centrifuged to discard the fixative, and then resuspended with 2 mL of PBS solution. Finally the cell suspension was stained with 1 mL of 200 μg/ml PI solution (propidium iodide) containing 20 µg/mL RNase, and analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA), and 13

analyzed using CELLQUEST software (BD Biosciences). 2.14. MRI analysis The glioblastoma CSCs were treated with different concentrations of anti-CD133 mAb-nano-MSN (0, 20, 40, 100, 200, 400 µg/mL), and rinsed thrice with PBS, and trypsinized and centrifuged, and then resuspended in 1.5 mL PBS (containing 0.5% agarose) in 5 mL Eppendorf tubes for MRI. The T2-weighed images were carried out using a MesoMR60 scanner (Niumag, Shanghai, China) at RT. The T2 mapping sequence (TR: 3000 ms, TE: 180 ms, slice width: 5.0 mm, K matrix 192 × 256) was used to measure the transverse relaxation time. 2.15. Image acquisition and analysis Bright-field and fluorescence images were acquired using an inverted microscope (Eclipse TE 2000-U) equipped with a CCD camera (CV-S3200). Software Image-Pro Plus® 6.0 (Media Cyternetics) and SPSS 17.0 (SPSS Inc.) were used to perform image analysis and statistical data analysis, respectively. The quantitative data were presented as means ± standard deviation (SD) for each experiment. All experiments were performed with three replicates, and the results presented were from representative experiments. 3. Results and Discussion 3.1. Synthesis and characterization of immunomagnetic nanosensor In the present study, the anti-CD133 mAb-nano-MSN were prepared as illustrated

14

in Scheme 2, including (i) synthesis of superparamagnetic γ-Fe2O3 core (SPIO NPs) following with CMCS modification mediated with TPP (CMCS-TPP@SPIO NPs) i.e. MNNs; and (ii) PEI modification of MNNs via EDC/NHS crosslinking (PEI-MNNs); and (iii) sulfo-SMCC modification of PEI-MNNs and thiolation of anti-CD133 mAb with Traut’s Reagent; and (iv) formation of immunomagnetic nanosensor by conjugating the PEI-MNNs with anti-CD133 mAb. The as-prepared MNNs and PEI-MNNs were characterized after CMCS and PEI modifications (Fig. 1). The TEM analyses showed that the prepared SPIO NPs were morphologically uniform, and the diameter ranged from 10 nm to 15 nm (SI, Fig.S1), and the CMCS-TPP@SPIO NPs i.e. MNNs also displayed a mono-dispersed sphere with a uniform size of approximately 40 nm (Fig. 1 B). Besides, as graphed in Fig. 1C, the FT-IR data showed that the C–N stretching vibration was at 1633.2 cm−1 for amide group, and the C–O bond vibration of ether group was at 1079.5 cm−1, and the characteristic band (Fe–O) of SPIO NPs was located at 595.7cm−1. In addition, the peak at 2952.7 cm−1 was attributed to the stretching vibration for methylene residuals, and the characteristic band of –COOH was also observed at 3424.5 cm−1. Overall, these data indicated that the SPIO NPs were prepared and successfully modified with CMCS and PEI respectively. The XRD analysis showed six characteristic peaks corresponding to (220), (311), (400), (422), (511), and (440) were observed in the prepared SPIO NPs, MNNs, and PEI-MNNs, which distinctly match the standard γ-Fe2O3 reflections. These data showed that incorporation of PEI and CMCS to SPIO NPs did not influence γ-Fe2O3 15

crystallization (SI, Fig. S2 A-C).

As shown in Fig.2 (A), the magnetization curve of the prepared NPs was a symmetrical hysteresis loop, indicating that these NPs would be promptly magnetized in the presence of a magnetic field and the removal of magnetic field results in a residual minimal magnetization within the particles [49]. Moreover, the saturation magnetization of SPIO NPs, MNNs, and PEI-MNNs were estimated about 56.5, 47.8, and 45.5 emu/g, respectively. The coercivity of these NPs was nearly zero. In the presence of polymer shell, the MS of SPIO NPs was apparently higher than that of MNNs, and PEI- MNNs (Fig. 2A). Ninhydrin could react with amino residuals in PEI molecule, which could be detected with a spectrophotometer at 570 nm, and the absorbance of reaction was positively correlated with the amount of PEI molecules. Thus, the PEI content in NPs was calculated as 29.5%, in the PEI-MNNs and 27.2% in anti-CD133 mAb-conjugated magnetic nanoparticles respectively (Fig. S3 A-C). 3.2. Stability and magnetic responsiveness assay The stability of PEI-MNNs suspended in different solutions, including DI water, PBS (pH 7.4), and RPMI-1640 culture medium, was determined at different set times. The results showed that suspension of PEI-MNNs remained relatively stable in DI water and RPMI-1640 culture medium, but become sediments after about 15 min in 16

PBS buffer (Fig. 2 B). The magnetic responsiveness of PEI-MNNs in different liquids was also analyzed under an external magnetic field, and the results demonstrated that PEI-MNNs exhibited excellent responsiveness in DI water, PBS (pH 7.4), and RPMI-1640 culture medium (Fig. 2 C), which indicated that the fabricated PEI-MNNs were suitable as MRI imaging agents in the following study. 3.3. Activity assay of the prepared immunomagnetic nanosensor The indirect fluorescence immunoassay was used to detect activity of anti-CD133 mAb-nano-MSN, and the results showed a much stronger red fluorescence was observed in the immunomagnetic nanoparticles (Fig.3 B &D), which indicated that the anti-CD133 mAb was successfully conjugated with magnetic nanoparticles as a immunomagnetic nanosensor, in contrast with the unconjugated magnetic nanoparticles that did not display any detectable fluorescence (Fig.3C& E). 3.4. SEM and viability analysis of glioblastoma CSCs To isolate of glioblastoma CSCs, the glioblastoma U251 cells were first cultured in serum-free medium supplemented with both growth factors and an anticancer drug vincristine (VCR), which was a kind of mitotic inhibitor that irreversibly binds to microtubules and spindle proteins during the mitotic S phase, interfering with mitotic spindle assembly and thereby restraining cancer cell growth [50]. Thus, cancer cells died rapidly, and the primary spheres of glioblastoma CSCs formed (Fig. S4). As showed in Fig. 4 (A), a typical morphology of CSCs and their differentiated 17

progeny was observed with optical phase contrast image. The glioblastoma CSCs differentiated into monolayer adherent cells after culture for 7d in RPMI-1640 medium with 10% FBS, which indicated an ability of multiple differentiation potential. Meantime, The ultrastructure of CSCs and their differentiated progenies was observed under scanning electron microscopy (SEM), the SEM analysis demonstrated that the initiated spheres of glioblastoma CSCs displayed a smooth surface with few protrusions that were tightly connected the spheres in RPMI-1640 medium with 10% FBS, and gradually an increasing number of progeny cells with visible protrusions grew out of the secondary spheres and migrated from the sphere surface of glioblastoma CSCs (Fig. 4 B). Besides, the cellular viability of glioblastoma CSCs and their differentiated progeny were assessed using the PI and FDA double-staining protocol [45, 46], and the assaying results showed that both glioblastoma CSCs and their differentiated progeny exhibited an intact viability (Fig.4 C). The immunocytochemistry staining also showed that the cancer stem biomarker CD133 (as an antigen) was extensively expressed by glioblastoma CSCs (Fig. 4D). 3.5. Cytotoxicity assay of immunomagnetic nanosensor The cytotoxicity of the fabricated anti-CD133 mAb-nano-MSN was evaluated with glioblastoma CSCs by MTT assaying. As shown in Fig. 5, the data showed that cellular proliferation of treated glioblastoma CSCs was not affected at the determined concentration 100 μg/mL.

18

3.6. Cellular uptake and real-time localization of immunomagnetic nanosensor The cellular uptake of anti-CD133 mAb-nano-MSN by glioblastoma CSCs was evaluated using fluorescence microscopy (Fig. 6 A-D). The results showed that the glioblastoma CSCs treated with anti-CD133 mAb-conjugated magnetic nanosensor displayed a strong red fluorescence signal, which accumulated inside cell endosomes. However, glioblastoma CSCs treated with non-mAb-functionalized magnetic nanopartitles including MNNs and PEI-MNNs displayed relatively weak red fluorescence, and no fluorescence was detected in the untreated glioblastoma CSCs. This analysis indicated that the prepared anti-CD133 mAb-conjugated magnetic nanosensor could be efficiently internalized by glioblastoma CSCs. Meanwhile, as in the Fig. 6 (E- H’), the TEM analysis also demonstrated the anti-CD133 mAb-conjugated magnetic nanopartitles was localized within the cytoplasm and trapped in single membrane organelles, and approached to cell nuclei. Taken together, these observations indicated the fabricated anti-CD133 mAb-conjugated magnetic nanopartitles could be efficiently targeted to glioblastoma CSCs. 3.7. Cell cycle analysis of treated glioblastoma CSCs Theoretically, the DNA synthesis phase (S phase) is a specific stage during cell cycle in which DNA is replicated for cell division [51]. As shown in Fig.7 and Fig.S7, the flow cytometric analysis indicated that the number of glioblastoma CSCs at the S-phase was increasing as a higher concentration of anti-CD133 mAb-nano-MSN were applied, but the number of cells at the G1 phase was decreasing. Therefore, these

19

results indicated the immunomagnetic nanosensor could specifically track glioblastoma CSCs that intactly maintained highly proliferative after treatments. 3.8. MRI Glioblastoma CSCs were cultured with different concentrations of anti-CD133 mAb-nano-MSN (0, 20, 40, 100, 200, 400 µg/mL) to evaluate the potential as a targeted MR contrast agent, and the T2-weighted MR phantom imaging was illustrated in the Fig. 8. The results showed that the treated glioblastoma CSCs and U251 cells demonstrated comparatively a significantly negative contrast enhancement (signal darkening) after incubation with anti-CD133 mAb-nano-MSN, although the cells incubated with PEI-MNNs also showed a contrast enhancement due to nonspecific interaction of cationic PEI with CSCs and U251 cells at the begin. However, owing to endocytosis

mediated

by

CD133,

the

cells

incubated

with

anti-CD133

mAb-nano-MSN gradually demonstrated a distinguishable darkening of MR images compared with those incubated with the CD133-unconjugated NPs. Moreover, the treated glioblastoma CSCs displayed much more signal darkening due to high expression CD133 on the membrane surface of glioblastoma CSCs than U251 cells.

4. Conclusion In this study, we have fabricated a novel and smart immunomagnetic nanosensor decorated with anti-CD133 mAb for real-time molecular imaging in the targeted glioblastoma CSCs. The results demonstrated that the immunomagnetic nanosensor was efficiently uptaken and internalized into glioblastoma CSCs, in which the 20

targeting antigen CD133 was preferentially expressed as a novel cell-membrane protein. The conjugation of anti-CD133 mAb to magnetic nanoparticles exhibited very low cytotoxicity, and could greatly improve their targeting efficacy into CD133-overexpressing CSCs. Besides, the immunomagnetic nanosensor could be specifically monitored real-time in the targeted glioblastoma CSCs as a fluorescence nanoprobe and distinguished contrast agents for MRI imaging. Overall, the present study demonstrated that the fabricated immunomagnetic nanosensor could be used as a promising nanovehicle for molecular imaging for the targeted glioblastoma CSCs in human brain tumor diagnosis and therapy. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 314 008 55), the Science and Technology Research Project in Henan Province of China (No. 162 102 310 404), the Technology Research and Development Support Funds Project of Zhengzhou City (No. 153 PXX CY 184), the Basal Research Fund of Henan University of Technology (No. 2014 YWQ Q15).

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Ren, M. Li, H. Wu, J. Wang, L. Zhang, Conjugation of biomolecules with magnetic protein microspheres for the assay of early biomarkers associated with acute myocardial infarction, Anal. Chem. 81 (2009) 6210–6217. [50] H. Thomadaki, K. V. Floros, A. Scorilas, Molecular response of HL-60 cells to mitotic inhibitors vincristine and taxol visualized with apoptosis-related gene expressions, including the new member BCL2L12, Ann. N.Y. Acad. Sci. 1171 (2009) 276–283. [51] J. E. Celis, A. Celis, Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase, Proc. Natl. Acad. Sci. USA. 82 (1985) 3262–3266. Author Biographies Xueqin Wang is an associate professor at College of Bioengineering, Henan University of Technology. She obtained her Ph.D. on College of Veterinary Medicine from Northwest A&F University in 2012. Her main research interests are in two areas: (1) Design and fabrication of biosensors and their application in bioanalysis; and (2) synthesis of targeted-drug carriers and their application in tumor treatment. 29

Bo Li is a Master student focusing on synthesis and application of magnetic nanoparticles at College of Bioengineering, Henan University of Technology. Ruifang Li is a professor at College of Bioengineering, Henan University of Technology. She obtained her Ph.D. at School of Life Sciences, Sun Yat-sen University in 2004. She is currently conducting research in synthesis of drug delivery system and its application in tumor targeted treatment. Yan Yang is a Ph.D. student focusing on the synthesis and application of magnetic nanoparticles at School of Life Sciences, Zhengzhou University. She was awarded a M.S. degree in pharmacy from the same university in 2016. Huiru Zhang is a professor at College of Bioengineering, Henan University of Technology. She obtained her Ph.D. on Veterinary Medicine from Northwest A&F University in 2005. Her main research interest is synthesis and application of drug delivery system. Baoming Tian is a professor at School of Life Sciences, Zhengzhou University. He received his Ph.D. from Huazhong Agricultural University. His research interest is in the area of biosensors. Haibo Weng is an associate professor at School of Life Sciences, Zhengzhou University. He received his Ph.D. from Nanjing University. His research interests include synthesis and application of the nanoscaled magnetic materials. Fang Wei is an associate professor at School of Life Sciences, Zhengzhou University. He obtained his Ph.D. at School of Life Sciences from Northwest A&F University in 2010. His main research interest is synthesis and application of magnetic 30

nanoparticles in biology.

Fig. 1. Schematic of PEI-MNNs (A). TEM image of as-prepared CMCS-TPP@SPIO NPs i.e. MNNs (B). FT-IR spectra of SPIO NPs, MNNs, PEI-MNNs, CMCS and PEI (C).

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Fig. 2. (A)The magnetic hysteresis loop of different NPs at 300 K. (B) Stability of PEI-MNNs in different solutions including DI water, PBS and RMPI-1640 with 10% FBS. (C) Response of NPs to an external magnetic field by adding an external magnetic field in different medium: DI water (1), PBS (2) and RMPI-1640 with 10% FBS (3).

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Fig. 3. (A) Schematic drawing of an anti-CD133 mAb-conjugated immunomagnetic nanosensor. (B) Fluorescence of immunomagnetic nanosensor (with Alexa flour 594-labeled goat anti-mouse IgG) was visualized at the post-immunoassay. (C) no fluorescence was observed for the unconjugated magnetic nanoparticles at the post-immunoassay. (D and E) Fluorescence intensity was quantitatively calculated according to the lined graphs in (B) and (C) using Image-Pro® Plus 6.0 software.

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Fig. 4. Glioblastoma CSCs formed by the vincristine- induced apoptosis of U251 cells and serum-free culture. (A) Phase contrast image of CSCs and their differentiated progeny. (B) Scanning electron micrograph of CSCs and their differentiated progeny. (C) Viability assay of the CSCs and their differentiated progeny using the FDA and PI double-staining protocol, and the chemo-resistant cancer cells remianed viable. (D) Immunocytochemistry staining of the CSCs with anti-CD133 mAb (in red). Cells were located by counterstaining with DAPI (blue). Scale bar = 100 μm.

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Fig.5. The growth of glioblastoma CSCs after treatment of anti-CD133 mAb-nano-MSN at a concentration of 100 μg/mL.

Fig. 6. Cellular uptake of anti-CD133 mAb-conjugated immunomagnetic nanosensor analyzed with fluorescence microscopy and transmission electron microscopy (TEM). 35

(A-D): images of CSCs incubated for 4 h with RBITC-labeling nanocomposites (100 μg/mL) under the fluorescence microscopy. A: controls (untreated CSCs). B: MNNs. C: PEI-MNNs. (D) anti-CD133 mAb-nano-MSN. The fluorescent nanocomposites (were located around DAPI-stained nuclei within cells. Scale bar = 100 μm. Typical TEM images of U251 cells (E-H) and CSCs (E’-H’) incubated with MNNs, PEI-MNNs and anti-CD133 mAb-nano-MSN (100 μg/mL). The red arrows showed the nanosensors enter the cell, Scale bar = 200 nm.

Fig. 7. Assay of cell cycle distribution of the treated CSCs after culture for 72 h with 100 μg/mL anti-CD133 mAb-nano-MSN. The percentage of cells in S phase increased along with culture time and G1 phase decreased, suggesting that the treated CSCs were active and proliferative.

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Fig. 8. T2-weighted MR images and their color maps of CSCs and U251 cells after incubation with the various concentrations of anti-CD133 mAb-nano-MSN i.e. anti-CD133-PEI-MNNs and PEI-MNNs (0, 20, 40, 100, 200, 400 µg/mL) for 4 h.

Scheme 1. Schematic representation of the isolation CSCs from heterogeneous tumor population by chemical apoptosis-induced enrichment, and the anti-CD133 mAb37

conjugated immunomagnetic nanosensor aided molecular imaging for specific targeted CSCs. Apoptosis was firstly induced with vincristine treatment, and serum-free culture was then used to enrich the target glioblastoma CSCs.

Scheme 2. Schematic illustration for chemical formation of the anti-CD133 mAbconjugated immunomagnetic nanosensor. (A) SPIO NPs were synthesized and 38

modified with CMCS through TPP to prepare CMCS@SPIO NPs i.e. MNNs, (B) MNNs were modified with PEI via EDC/NHS, (C) PEI-MNNs were crosslinked with sulfo-SMCC, (D) anti-CD133 mAb was thiolated through Traut’s Reagent in borate buffer to form anti-CD133-SH, and anti-CD133 mAb-conjugated immunomagnetic nanosensor were finally synthesized (E).

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