Shape Engineering Boosts Magnetic Mesoporous Silica Nanoparticles

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Shape Engineering Boosts Magnetic Mesoporous Silica Nanoparticlesbased Isolation and Detection of Circulating Tumor Cells Zhimin Chang, Zheng Wang, Juan Yue, Hao Xing, Li Li, Mingfeng Ge, Mingqiang Li, Huize Yan, Hanze Hu, Dan Shao, Qiaobing Xu, Dayong Jin, and Wen-Fei Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19325 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 23, 2018

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ACS Applied Materials & Interfaces

Shape

Engineering

Boosts

Magnetic

Mesoporous

Silica

Nanoparticles-based Isolation and Detection of Circulating Tumor Cells

Zhi-min Chang,† Zheng Wang,†,‡ Juan Yue,†,‡ Hao Xing,†,‡ Li Li,† Mingfeng Ge,† Mingqiang Li,⊥ Huize Yan,⊥ Hanze Hu,⊥ Dan Shao,*,†,⊥ Qiaobing Xu,$ Dayong Jin,# and Wen-fei Dong*,†

†

CAS Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical

Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, China. ‡

University of Chinese Academy of Sciences, Beijing 100049, China

⊥

Department of Biomedical Engineering, Columbia University, New York, NY 10027,

United States $

Department of Chemistry, University of Massachusetts, 710 North Pleasant Street,

Amherst, Massachusetts 01003, United States #

Laboratory of Advanced Cytometry, ARC Centre of Excellence for Nanoscale

BioPhotonics, Department of Physics and Astronomy, Macquarie University, Sydney, New South Wales 2109, Australia

1

ACS Paragon Plus Environment

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Abstract Magnetic mesoporous silica nanoparticles (M-MSNs) are attractive candidates for the immuno-magnetic isolation and detection of circulating tumor cells (CTCs). Understanding of the interactions between the effect of the shape of M-MSNs and CTCs is crucial to maximize the binding capacity and capture efficiency as well as to facilitate the sensitivity and efficiency of detection. In this work, fluorescent M-MSNs were rationally designed with sphere and rod morphologies while retaining their robust fluorescence and uniform surface functionality. After conjugation with the antibody of epithelial cell adhesion molecule (EpCAM), both of the different shaped M-MSNs-EpCAM

obtained

achieved

efficient

enrichment

of

CTCs

and

fluorescent-based detection. Importantly, rod-like M-MSNs exhibited faster immuno-magnetic isolation as well as better performance in the isolation and detection of CTCs in spiked cells and real clinical blood samples than their sphere-like counterparts. Our results showed that shape engineering contributes positively towards immuno-magnetic isolation, which might open new avenues to the rational design of magnetic-fluorescent nanoprobes for the sensitive and efficient isolation and detection of CTCs.

Keywords: magnetic mesoporous silica nanoparticles, circulating tumor cells, immuno-magnetic isolation, fluorescent detection, EpCAM

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1. Introduction Circulating tumor cells (CTCs), which have escaped from the tumor site into the circulating blood, play an important role in cancer metastasis, and the level of the CTCs also crucially impacts the prognosis and survival of patients.1-3 Continuous efforts have been made towards the development of approaches that enable the isolation and noninvasive detection of CTCs, providing the information needed to advance our understanding of the biologic profiles and clinical significance of these rare cells.4,5 Among numerous approaches for CTCs isolation and enrichment, magnetic nanoparticle-based immuno-magnetic isolation has received substantial attention due to its fast magnetic response combined with the advantages of the nanoscale properties, including a high surface area, good suspendability and biocompatibility.6-15 Nevertheless, immunomagnetic nanoparticles require rational design with multiple functions to maximize the binding capacity and capture efficiency as well as facilitate the sensitivity and efficiency of detection. In this regard, magnetic mesoporous silica nanoparticles (M-MSNs) have been developed as multifunctional magnetic-fluorescent nanoprobes for the isolation and detection of CTCs due to their large mesoporous volume for fluorescent probe loading and facile surface functionalization for the cross-linking of biological targets.15-19 In our design, M-MSNs are not only functionalized with antibody to target biomarkers over-expressed on the surfaces of CTCs for magnetic separation but also are endowed with enhanced fluorescent ability for further bioimaging and analysis.

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The rising application of M-MSNs in biomedical applications is strongly correlated to their key properties including their shape, size, surface chemistry, roughness and magnetic material parameters.20-24 For immunomagnetic isolation, various studies have reported that biological targets can be selectively captured for therapeutic and diagnostic studies through manipulating the surface chemical properties of M-MSNs.25,26 Particularly, the magnetic property is crucial for the throughput

and

separation

yield

in clinical applications.27,28

Besides

the

above-mentioned parameters, shape is also considered to be associated with nano-bio interactions and biological behaviors, including proliferation, apoptosis, adhesion, migration, biodistribution and biocompatibility.24,29,30 Furthermore, the mechanisms of the interactions between cell membranes and differently shaped nanoparticles have been widely investigated.31,32 Although we have previously reported that rod-like M-MSNs exhibited higher cellular internalization and tumor accumulation than sphere-like M-MSNs, resulting in better magnetic targeting abilities, magnetic hyperthermia performance and MRI properties,20,21 there is still limited information regarding the effects of the shape of M-MSNs on the isolation and detection of CTCs. Therefore, an improved understanding of the interactions between shape and CTCs is crucial to establish optimal standards for the design of M-MSNs. In the present paper, the architecture of M-MSNs is tailored through controlling the growth of the fluorescent mesoporous silica portion on the magnetic core (Scheme 1). These magnetic-fluorescent nanoprobes with sphere and rod shapes share similar physicochemical properties and surface functionality. For the selective capture and

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accurate detection of CTCs, magnetic-fluorescent nanoprobes are attached to the antibody of epithelial cell adhesion molecule (EpCAM), which can target MCF-7 breast cancer cells and CTCs from clinical blood samples. The effects of the shape of EpCAM-M-MSNs on the magnetic isolation and fluorescent detection of CTCs are investigated in detail to optimize the most promising candidates for the sensitive and efficient isolation and detection of CTCs.

2. Experimental 2.1. Reagents and materials The chemicals iron (III) chloride anhydrous (FeCl3), polyacrylic acid (PAA), diethylene glycol (DEG), tetraethyl orthosilicate (TEOS, 98%), fluorescein isothiocyanate

(FITC),

cetyltrimethylammonium

3-aminopropyltriethoxysilane

(APS),

bromide

(CTAB),

N,N-dimethylbenzamide

(DMF),

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 99%), and N-hydroxysuccinimide (NHS, 98.5%) were obtained from the Sigma-Aldrich Co. (St Louis, MO, USA). Sodium hydroxide (NaOH), ammonium nitrate (NH4NO3), ammonium hydroxide (NH4OH, 28%), succinic anhydride and anhydrous ethanol were purchased from the Beijing Chemical Reagent Co. (Beijing, China). Trypsin was supplied by the Sangon Biotech Co. Ltd (Shanghai, China). The antibody of epithelial cell adhesion molecule (EpCAM) (PE594 labeled) was purchased from Novus Biologicals. PE-labeled anti-cytokeratin was purchased from BD Biosciences. 2.2. Synthesis of fluorescent M-MSNs

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Fluorescent M-MSNs with different morphologies were prepared through a modified sol-gel method using PAA-stabilized Fe3O4 NPs as a substrate and CTAB as a template.20,21 In brief, FITC-APS was first prepared through mixing FITC with APS (10% w/w) in the dark overnight. Then, 1 mL of water solution of Fe3O4 magnetic nanospheres (10 mg/mL) was dispersed in 10 mL of CTAB solution under sonication. The mixture was added into a tree-neck bottle at 40 ℃, and co-silica sources containing TEOS and FITC-APS solution was dropwise injected into the mixture with 0.5 mL NH4OH. After 20 min of fast mechanical stirring, a certain amount of alcohol was added to terminate the reaction. The products were collected and washed three times through magnetic separation. Differently shaped M-MSNs were synthesized through tuning the CTAB/TEOS+FITC-APS ratio as follows: sphere-like MSNs (S-M-MSNs) = 120 mg/30 µL+6 µL and rod-like MSNs (R-M-MSNs) = 60 mg/10 µL+2 µL. 2.3. Surface functionalization of M-MSNs To functionalize the carboxyl groups, 10 mg of R-M-MSNs was added into 50 mL of DMF solution containing succinic anhydride (2 wt%) and reacted with stirring at 40 ℃ for 24 h. After magnetic separation and washing, carboxyl-functionalized MSNs were obtained. To modify the M-MSNs-COOH with antibody, a modified EDC/NHS reaction was carried out according to a previous report.33,34 In brief, 5 µL of 0.5 M EDC and 5 µL of 1 M NHS were added into the 10 mL of PBS solution containing R-M-MSNs-COOH (1 mg/mL). Then, 1 mg of epithelial cell adhesion

6


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molecule (EpCAM) antibodies was added into the mixture. After stirring for 4 h, the antibody-conjugated M-MSNs (M-MSNs-EpCAM) were obtained. 2.4. Characterization The morphologies of M-MSNs were observed by transmission electron microscopy (JEOL Ltd, Japan), and their magnetic measurements were obtained by a TDM-B vibrating sample magnetometer (VSM) at 300 K. The Z-potentials and average sizes of the M-MSNs were determined by a Zetasizer Nano ZS (Malvern Instruments, USA). UV-visible absorption spectra and fluorescence spectra of two types of M-MSNs at same concentration were measured by a U-3310 spectrophotometer (Hitachi, Japan) and a RF-5301 PC spectrophotometer (Shimadzu, Japan), respectively. The pore size distributions and surface properties of the M-MSNs were determined by the Barrett-Joyner-Halenda (BJH) method and the Brunauer-Emmett-Teller (BET) method, respectively. The iron oxide content of the M-MSNs was measured via inductively coupled plasma mass spectrometer (ICP-MS) (Xseries II; Thermo Scientific, Waltham, MA, USA) 2.5. Cell culture and capture efficacy of tumor cells The human breast cancer cell line (MCF-7), human hepatocellular carcinoma cell line (HepG2), human colon cancer cell line (HCT-116) and Jurkat T leukemia cells were cultured at 37 ℃ under 5% CO2 in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL). To test the capture efficacy of M-MSNs-EpCAM, R-M-MSNs-EpCAM and S-M-MSNs-EpCAM with various concentrations (6.25,

7



12.5, 50, 100, 150 µg/mL) were mixed with 1 × 105 of MCF-7 or Jurkat T cells. After 10 min of incubation, the cells coupled with M-MSNs-EpCAM were separated by an external magnetic field, and the number of cells in the supernatant and that were captured were both measured by a hemocytometer, thus allowing the calculation of their capture efficacy. To explore the specificity of M-MSNs-EpCAM, differently shaped M-MSNs (100 µg/mL) with or without anti-EpCAM were respectively treated with EpCAM-overexpressed MCF-7 cells and EpCAM-low expressed Jurkat T cells, and their respective capture efficacies were calculated. To study the fluorescence intensity of M-MSNs-EpCAM-targeted MCF-7 cells, R-M-MSNs-EpCAM or S-M-MSNs-EpCAM with a concentration of 100 µg/mL were incubated with MCF-7 cells (5 x 104) and Jurkat T cells (5 x 104). The cells were collected by an external magnetic field and then separated through centrifugation (1000 r/min), followed by redistribution in PBS solution. Then, the fluorescence intensity was measured by a spectrophotometer. 2.6. Capture of spiked tumor cells from mimic CTC samples MCF-7 cells (5 × 104) and Jurkat T cells (5 × 104) were cultured in 24-well plates, respectively. Then, MCF-7 cells and Jurkat T cells were dissociated by trypsin and mixed in PBS solution. R-M-MSNs-EpCAM or S-M-MSNs-EpCAM (100 µg/mL) were added into the cell suspension and co-cultured for 10 min. Then MCF-7 cells were stained with PE594-labeled anti-EpCAM monoclonal antibody. Fluorescent images were observed by a confocal laser scanning microscope (CLSM, Olympus

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FV1000 Japan), and the mean fluorescence intensity was analyzed by flow cytometry (FACS, Becton Dickinson Biosciences, Drive Franklin Lakes, U.S.). To investigate the specificity of the immunomagnetic assay, we spiked MCF-7 cells with Jurkat T cells at different ratios. In these synthetic samples, the concentration of Jurkat T cells is the same (106 cells per mL), and the concentration of Hoechst33342-stained MCF-7 cells ranged from 25 to 500 cells per mL. M-MSNs-EpCAM (100 µg/mL) was added into the cell suspension and co-cultured at 37 ℃. Then, the cells coupled with M-MSNs-EpCAM were separated by an external magnetic field, and the number of T cells in the supernatant and that were captured were both measured by a confocal laser scanning microscope, allowing the calculation of their capture efficacy. 2.7. Capture efficiency of spiked tumor cells in mimic clinical samples MCF-7 cells were dispersed over whole blood with the concentration of Hoechst33342-stained MCF-7 cells ranging from 25 to 500 cells per mL to mimic clinical samples. Whole blood samples were obtained from healthy humans, placed into EDTA-coated vacutainer tubes and kept at 4 ℃, and they were used within 24 h. M-MSNs-EpCAM were added into the samples and co-cultured at 37 ℃ for 10 min. The cells adhered to M-MSNs-EpCAM were magnetically separated, and the number of captured cells and uncaptured cells were measured by confocal laser scanning microscope, allowing their capture efficacy to be calculated. Additionally, M-MSNs-EpCAM and MCF-7 cells in whole blood (100 cells per mL) were incubated for 5, 10, 20, and 30 min, respectively, and at the certain incubation times,

9



The cells coupled with M-MSNs-EpCAM were magnetically separated for 10 min, the capture efficiency was determined as mentioned above. The capture efficiencies from whole blood of three other types of tumor cells (HepG2, HCT-116 and Jurkat T cells) were also investigated to confirm the specific isolation of M-MSNs-EpCAM. 2.8. Detection of CTCs in breast cancer patient peripheral blood samples Blood samples from 20 CTC-containing breast cancer patients and 20 healthy controls were collected. The differently shaped M-MSNs-EpCAM were treated with blood samples from CTC-containing breast cancer patients and healthy controls, respectively, for 10 min. The magnetically isolated cells were investigated by confocal laser scanning microscope. CTCs were characterized as CK19 positive and DAPI positive. 2.9. Statistical analyses Data are expressed as mean ±SD values. The difference between groups was analyzed using a one-way analysis of variance and Bonferroni post hoc test was used to analyze the differences among the two groups. P < 0.05 was considered to represent a statistically significant difference.

3. Results and Discussion Fluorescent M-MSNs with various morphologies were synthesized by a one-pot synthesis with Fe3O4 NPs as a substrate, TEOS and FITC-linked APS as co-silica sources and CTAB as a template using a modified sol-gel method.20,21 In this method, two differently shaped M-MSNs were first obtained by controlling the hydrolysis and

10


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co-condensation of the fluorescent silica source in different ratios of surfactants. Then, carboxyl-functionalized fluorescent M-MSNs were conjugated to EpCAM antibodies through the previously reported EDC/NHS-mediated amidation reaction.33,34 As observed in transmission electron microscopy (TEM) images (Figure 1a and b), S-M-MSNs-EpCAM exhibited a core-shell structure with a suitable size of approximately 200 nm, while R-M-MSNs-EpCAM possessed a Janus structure with a rod-like shape and overall dimensions of 300 nm × 100 nm (length × width) (Supplementary Figure S1). The obtained M-MSNs-EpCAM also had a similar diameter and negative surface charge in water (Supplementary Figure S2 and 3). The mass fractions of iron oxide nanoparticles in the S-M-MSNs and R-M-MSNs was 45% and 56%, respectively. Magnetization hysteresis loops of M-MSNs-EpCAM (Figure 1c) demonstrated the good superparamagnetism, while rod-like M-MSNs (62.3 emu/g) exhibited higher saturation magnetization than the sphere-like M-MSNs (38.2 emu/g). These phenomenons might be attributed to the high mass fraction of iron oxide, as well as more magnetic field was diminished by the silica coating in the sphere-like M-MSNs than that by silica conjugation in the rod-like M-MSNs.20 To obtain M-MSNs with robust fluorescence, the hybrid organic/inorganic method has been employed to embed into the mesoporous silica framework. Compared to surface-linked fluorescent NPs, framework-encapsulated fluorescent NPs not only exhibited chemical and mechanical stability, they also improved the photophysical properties of the fluorescent probes.35,36 As shown in Figure 1d, the fluorescence spectra of FITC-embedded M-MSNs-EpCAM demonstrated similar fluorescent

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properties to those of pure FITC, which were suitable for fluorescence detection. Moreover, S-M-MSNs and R-M-MSNs at the same concentration showed identical fluorescence intensity, making them suitable to compare the capture efficacy via ﬂuorescent tracking. The peak at 280 nm on the absorption spectrum indicated that the EpCAM antibody was conjugated onto the surface of M-MSNs (Supplementary Figure

S4).

Furthermore,

the

surface

area

and

pore

volume

of

the

R-M-MSNs-EpCAM were determined to be 618.5m2/g and 0.39 cm3/g, respectively, which were higher than the corresponding values of 423.7 m2/g and 0.30 cm3/g for the S-M-MSNs-EpCAM. The concentration of M-MSNs-EpCAM used in the magnetic capture was first optimized. With the increase of the M-MSNs-EpCAM concentration, the capture efficiency increased until the concentration reached 100 µg/mL, at which 77.5% (for S-M-MSNs-EpCAM) and 88.9% (for R-M-MSNs-EpCAM) of MCF-7 cells were captured (Figure 2a). To investigate specificity of M-MSNs-EpCAM to capture epithelial cancer cells, MCF-7 cells (EpCAM-overexpressed human breast cancer cells) were selected as a positive control, while Jurkat T (EpCAM-low expressed human peripheral blood leukemia T cells) were taken as a negative control. As shown in Figure 2b, over 80% of MCF-7 cells can be enriched through the M-MSNs-EpCAM-based immunomagnetic assay, whereas the capture ratio was merely less than 10% in the case of Jurkat T cells. There was no difference in the lower capture efficiency of M-MSNs without anti-EpCAM between the two types of cells. These results demonstrated the highly specific binding nature of the

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EpCAM-modified M-MSNs. Importantly, R-M-MSNs-EpCAM (89.6%) exhibited a significantly

higher

capture

efficiency

for

MCF-7

cells

than

that

of

S-M-MSNs-EpCAM (79.7%). This phenomenon might be attributed to the higher surface area for specific binding, strong endocytic behavior and better magnetic properties of the rod-like structures.20 We then evaluated the sensitivity of the detection of fluorescent CTCs by measuring the fluorescence intensity of M-MSNs-EpCAM. Consistent with the capture efficiency from Figure 2b, R-M-MSNs-EpCAM-targeted MCF-7 cells showed a higher fluorescent intensity than S-M-MSNs-EpCAM-targeted MCF-7 cells (Figure 2c). Taken together, the presence of the EpCAM antibody and robustly fluorescent FITC molecules enabled an excellent CTCs capture efficiency of M-MSNs, while rod-shaped NPs were more suitable for the selective enrichment and fluorescence detection of CTCs. Then, the capability of M-MSNs-EpCAM to capture CTCs in synthetic samples was investigated. We spiked MCF-7 cells with Jurkat T cells to mimic the clinical conditions for further assessment of their potential in practice. As observed in Figure 3, PE594 labeled-MCF-7 cells exhibited much a higher fluorescence of M-MSNs-EpCAM than Jurkat T cells, indicating that the binding between M-MSNs-EpCAM and MCF-7 cells was effective and specific. The co-localized fluorescence displayed that both of the M-MSNs-EpCAM could exhibit targeted binding to the PE594 labled-MCF-7 cells without interference from surrounding Jurkat T cells. As expected, R-M-MSNs-EpCAM exhibited a stronger fluorescence intensity in MCF-7 cells than S-M-MSNs-EpCAM (Figure 4a), further demonstrating

13



that the shape boosted the capture of CTCs in spiked conditions. On the basis of the above-mentioned fluorescence detection, the efficiency of the immunomagnetic assay can be determined by spiking MCF-7 cells with Jurkat T cells at different ratios. As shown in Figure 4b, given concentrations ranging from 25 to 500 cells per mL, over 95% and 85% of cells were captured by R-M-MSNs-EpCAM in PBS buffer, which is higher than the corresponding values of 90% and 78% for S-M-MSNs-EpCAM. Collectively, these results indicated that rod-like M-MSNs had better performance in the isolation and detection CTCs in a spiked experiment. We then spiked MCF-7 cells with whole blood to detect the capture efficiency at different cell numbers. As shown in Figure 5a, all M-MSNs-EpCAM still exhibited a high capture efficiency of MCF-7 cells with the increased cell number. Similar to the results in the PBS condition, rod-like M-MSNs had better performance in comparison to sphere-like M-MSNs in whole blood. Since the incubation and isolation time have always been crucial factors for real sample detection, we first investigated the efficiency to capture rare MCF-7 cells in whole blood while varying the incubation time. As shown in Figure 5b, 10 min of incubation enabled both types of M-MSNs to catch more than 80% of MCF-7 cells in mimic clinical samples. Importantly, rod-like M-MSNs exhibited a higher capture efficiency than sphere-like M-MSNs. To further confirm and compare the specific isolation of M-MSNs-EpCAM, we selected two other types of EpCAM-positive cancer cells and one type of EpCAM-negative cancer cells for spiking with whole blood (Figure 5c). The capture efficiencies of both M-MSNs-EpCAM in HepG2 and HCT-116 cells reached greater than 70%, while

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reaching less than 10% in HeLa cells. As expected, R-M-MSNs-EpCAM exhibited a higher capture efficiency than S-M-MSNs-EpCAM in EpCAM-positive cancer cells. It is worth to note that our M-MSNs have a good biocompatibility on different cells including cancer cells and normal cells,18,20,21 the capture concentration (less than 100 µg/mL) have no toxic effect on MCF-7 cells, which might not affect the metabolism of captured cells. On the basis of the efficient, rapid, and specific capture and isolation of CTCs in mimic conditions, we employed differently shaped M-MSNs-EpCAM to real clinical blood samples from CTC-containing breast cancer patients and the healthy population. As shown in Figure 6a and Table S1, CTCs in the cancer patients’ blood samples could be captured and detected, while there were no CTCs found in any healthy samples, suggesting that M-MSNs-EpCAM were successfully applied to real patient blood samples. Importantly, the detection rate of R-M-MSNs-EpCAM (85%) for CTCs was higher than that of S-M-MSNs-EpCAM (75%), which demonstrated the better sensitivity and efficiency of the isolation of CTCs in the rod-like M-MSN-based immunomagnetic assay. In addition, CTCs detected with DAPI+ and CK19+ are shown in Figure 6b and Supplementary Figure S5, further confirming the results of our analysis as well. Taken together, all of these findings demonstrated that our M-MSNs-EpCAM can capture CTCs with a high sensitivity and have comparable results with current reports using other type of magnetic NPs.7,37,38

15



4. Conclusion In summary, two fluorescent M-MSNs with different morphologies were fabricated, and the effects of their shape on the isolation and detection of CTCs were investigated and compared. We showed that both differently shaped M-MSNs enabled good sensitivity for the isolation and fluorescence detection of CTCs with the aid of the EpCAM antibody. Compared to sphere-like M-MSNs, rod-like M-MSNs was found to have faster enrichment and more efficient detection of CTCs in spiked cells and real clinical blood samples. These findings demonstrated that the morphology of M-MSNs could have a great impact on their interaction with CTCs and control the performance of immuno-magnetic isolation. Our study provides novel insight into the immuno-magnetic isolation potency of M-MSNs, which holds promise for the development of next generation magnetic-fluorescent nanoprobes with flexible design options for the sensitive and efficient isolation and detection of CTCs.

ASSOCIATED CONTENT Supporting Information. SEM, size distribution, Zeta potential, absorption spectra, CTC detection and fluorescent images of M-MSNs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *

Corresponding author.

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Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences (CAS), 88 Keling RD, Suzhou, Jiangsu, China , 215163 Tel: +86-512-6958-8307 Fax: +86-512-6958-8088 E-mail addresses: [email protected](D. Shao), [email protected] (W.-F. Dong)

Conflict of Interest The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the the National Natural Science Foundation of China (Grand No. 81601609, 81771982, 61535010, 81371681, and 8160071152), National Key Research and Development Program of China (Grand No. 2017YFF0108600 and 2016YFF0103800), the Key Research Program of the Chinese Academy of Sciences (No. KFZD-SW-21), the Natural Science Foundation of Jiangsu Province (No. BE2015601) and the Science and Technology Department of Suzhou City (No. SS201539 and ZXY201434).

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References 1.

Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J.

M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W., Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. N. Engl. J. Med. 2004, 2004, 781-791. 2.

Paterlini-Brechot, P.; Benali, N. L., Circulating Tumor Cells (CTC) Detection: Clinical

Impact and Future Directions. Cancer Lett. 2007, 253, 180-204. 3.

Alix-Panabières, C.; Pantel, K., Circulating Tumor Cells: Liquid Biopsy of Cancer. Clin.

Chem. 2013, 59, 110-118. 4.

Alix-Panabières, C.; Pantel, K., Technologies for Detection of Circulating Tumor Cells: Facts

and Vision. Lab Chip 2014, 14, 57-62. 5.

Joosse, S. A.; Pantel, K., Biologic Challenges in the Detection of Circulating Tumor Cells.

Cancer Res. 2013, 73, 8-11. 6.

Wang, C.; Ye, M.; Cheng, L.; Li, R.; Zhu, W.; Shi, Z.; Fan, C.; He, J.; Liu, J.; Liu, Z.,

Simultaneous Isolation and Detection of Circulating Tumor Cells with a Microfluidic Silicon-nanowire-array Integrated with Magnetic Upconversion Nanoprobes. Biomaterials 2015, 54, 55-62. 7.

Niu, M.; Du, M.; Gao, Z.; Yang, C.; Lu, X.; Qiao, R.; Gao, M., Monodispersed Magnetic

Polystyrene Beads with Excellent Colloidal Stability and Strong Magnetic Response. Macromol. Rapid Commun. 2010, 31, 1805-1810. 8.

Tu, C.; Yang, Y; Gao, M., Preparations of Bifunctional Polymeric Beads Simultaneously

Incorporated with Fluorescent Quantum Dots and Magnetic Nanocrystals. Nanotechnology 2008, 19, 105601. 9.

Hoshino, K.; Huang, Y.-Y.; Lane, N.; Huebschman, M.; Uhr, J. W.; Frenkel, E. P.; Zhang, X.,

Microchip-based Immunomagnetic Detection of Circulating Tumor Cells. Lab Chip 2011, 11, 3449-3457. 10. Shen, Z.; Wu, A.; Chen, X., Current Detection Technologies for Circulating Tumor Cells. Chem. Soc. Rev. 2017, 46, 2038-2056. 11. Poudineh, M.; Aldridge, P. M.; Ahmed, S.; Green, B. J.; Kermanshah, L.; Nguyen, V.; Tu, C.; Mohamadi, R. M.; Nam, R. K.; Hansen, A., Tracking the Dynamics of Circulating Tumour Cell

18


Page 18 of 29

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Phenotypes Using Nanoparticle-mediated Magnetic Ranking. Nat. Nanotechnol. 2017, 12, 274-281. 12. Wu, L.-L.; Wen, C.-Y.; Hu, J.; Tang, M.; Qi, C.-B.; Li, N.; Liu, C.; Chen, L.; Pang, D.-W.; Zhang, Z.-L., Nanosphere-based One-step Strategy for Efficient and Nondestructive Detection of Circulating Tumor Cells. Biosens. Bioelectron. 2017, 94, 219-226. 13. Chen, L.; Wu, L.-L.; Zhang, Z.-L.; Hu, J.; Tang, M.; Qi, C.-B.; Li, N.; Pang, D.-W., Biofunctionalized Magnetic Nanospheres-based Cell Sorting Strategy for Efficient Isolation, Detection and Subtype Analyses of Heterogeneous Circulating Hepatocellular Carcinoma Cells. Biosens. Bioelectron. 2016, 85, 633-640. 14. Kwak, B.; Lee, J.; Lee, D.; Lee, K.; Kwon, O.; Kang, S.; Kim, Y., Selective Isolation of Magnetic Nanoparticle-mediated Heterogeneity Subpopulation of Circulating Tumor Cells Using Magnetic Gradient Based Microfluidic System. Biosens. Bioelectron. 2017, 88, 153-158. 15. Kim, J. H.; Chung, H. H.; Jeong, M. S.; Song, M. R.; Kang, K. W.; Kim, J. S., One-Step Detection of Circulating Tumor Cells in Ovarian Cancer Using Enhanced Fluorescent Silica Nanoparticles. Int. J. Nanomed. 2013, 8, 2247–2257. 16. Zhang, L.; Dong, W.-F.; Sun, H.-B., Multifunctional Superparamagnetic Iron Oxide Nanoparticles: Design, Synthesis and Biomedical Photonic Applications. Nanoscale 2013, 5, 7664-7684. 17. Knežević, N. Ž.; Ruiz-Hernández, E.; Hennink, W. E.; Vallet-Regí, M., Magnetic Mesoporous Silica-based Core/Shell Nanoparticles for Biomedical Applications. RSC Adv. 2013, 3, 9584-9593. 18. Shao, D.; Li, J.; Zheng, X.; Pan, Y.; Wang, Z.; Zhang, M.; Chen, Q.-X.; Dong, W.-F.; Chen, L., Janus “Nano-Bullets” for Magnetic Targeting Liver Cancer Chemotherapy. Biomaterials 2016, 100, 118-133. 19. Chang, Z.; Wang, Z.; Lu, M.; Li, M.; Li, L.; Zhang, Y.; Shao, D.; Dong, W., Magnetic Janus Nanorods for Efficient Capture, Separation and Elimination of Bacteria. RSC Adv. 2017, 7, 3550-3553. 20. Wang, Z.; Chang, Z.; Lu, M.; Shao, D.; Yue, J.; Yang, D.; Zheng, X.; Li, M.; He, K.; Zhang, M., Shape-Controlled Magnetic Mesoporous Silica Nanoparticles for Magnetically-Mediated Suicide Gene Therapy of Hepatocellular Carcinoma. Biomaterials 2017, 154, 147-157. 19



Page 20 of 29

21. Shao, D.; Lu, M.-m.; Zhao, Y.-w.; Zhang, F.; Tan, Y.-f.; Zheng, X.; Pan, Y.; Xiao, X.-a.; Wang, Z.; Dong, W.-f., The Shape Effect of Magnetic Mesoporous Silica Nanoparticles on Endocytosis, Biocompatibility and Biodistribution. Acta Biomater. 2017, 49, 531-540. 22. Li, L.; Liu, T.; Fu, C.; Tan, L.; Meng, X.; Liu, H., Biodistribution, Excretion, and Toxicity of Mesoporous SilicaNanoparticles After Oral Administration Depend on Their Shape. Nanomedicine 2015, 11, 1915-1924. 23. Hao, N.; Li, L.; Tang, F., Shape Matters When Engineering Mesoporous Silica-Based Nanomedicines. Biomater. Sci. 2016, 4, 575-591. 24. Chu, Z.; Zhang, S.; Zhang, B.; Zhang, C.; Fang, C.-Y.; Rehor, I.; Cigler, P.; Chang, H.-C.; Lin, G.; Liu, R., Unambiguous Observation of Shape Effects on Cellular Fate of Nanoparticles. Sci. Rep. 2014, 4, 4495. 25. Borsa, B. A.; Tuna, B. G.; Hernandez, F. J.; Hernandez, L. I.; Bayramoglu, G.; Arica, M. Y.; Ozalp,

V.

C.,

Staphylococcus

Aureus

Detection

in

Blood

Samples

by

Silica

Nanoparticle-Oligonucleotides Conjugates. Biosens. Bioelectron. 2016, 86, 27-32. 26. Sen, T.; Sebastianelli, A.; Bruce, I. J., Mesoporous Silica-Magnetite Nanocomposite: Fabrication and Applications in Magnetic Bioseparations. J. Am. Chem. Soc. 2006, 128, 7130-7131. 27. Patel, P.; Mahmud, D.; Park, Y.; Yoshinaga, K.; Mahmud, N.; Rondelli, D., Clinical Grade Isolation of Regulatory T Cells from G-CSF Mobilized Peripheral Blood Improves with Initial Depletion of Monocytes. Am. J. Blood Res. 2015, 5, 79-85. 28. Esmaeilsabzali, H.; Beischlag, T. V.; Cox, M. E.; Dechev, N.; Parameswaran, A. M.; Park, E. J., An Integrated Microfluidic Chip for Immunomagnetic Detection and Isolation of Rare Prostate Cancer Cells from Blood. Biomed. Microdevices 2016, 18, 22. 29. Toy, R.; Peiris, P. M.; Ghaghada, K. B.; Karathanasis, E., Shaping Cancer Nanomedicine: the Effect of Particle Shape on the in vivo Journey of Nanoparticles. Nanomedicine 2014, 9, 121-134. 30. Barua, S.; Yoo, J.-W.; Kolhar, P.; Wakankar, A.; Gokarn, Y. R.; Mitragotri, S., Particle Shape Enhances Specificity of Antibody-Displaying Nanoparticles. Proc. Natl. Acad. Sci. 2013, 110, 3270-3275. 31. Meng, H.; Yang, S.; Li, Z.; Xia, T.; Chen, J.; Ji, Z.; Zhang, H.; Wang, X.; Lin, S.; Huang, C., Aspect Ratio Determines the Quantity of Mesoporous Silica Nanoparticle Uptake by a Small 20


Page 21 of 29 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


GTPase-Dependent Macropinocytosis Mechanism. ACS Nano 2011, 5, 4434-4447. 32. Vácha, R.; Martinez-Veracoechea, F. J.; Frenkel, D., Receptor-mediated Endocytosis of Nanoparticles of Various Shapes. Nano Lett. 2011, 11, 5391-5395. 33. Shao, D.; Zeng, Q.; Fan, Z.; Li, J.; Zhang, M.; Zhang, Y.; Li, O.; Chen, L.; Kong, X.; Zhang, H., Monitoring HSV-TK/Ganciclovir Cancer Suicide Gene Therapy Using CdTe/CdS Core/Shell Quantum Dots. Biomaterials 2012, 33, 4336-4344. 34. Shao, D.; Li, J.; Xiao, X.; Zhang, M.; Pan, Y.; Li, S.; Wang, Z.; Zhang, X.; Zheng, H.; Zhang, X., Real-time Visualizing and Tracing of HSV-TK/GCV Suicide Gene Therapy by Near-infrared Fluorescent Quantum Dots. ACS Appl. Mater. Interfaces 2014, 6, 11082-11090. 35. Montalti, M.; Prodi, L.; Rampazzo, E.; Zaccheroni, N., Dye-Doped Silica Nnanoparticles as Luminescent Organized Systems for Nanomedicine. Chem. Soc. Rev. 2014, 43, 4243-4268. 36. Huang, X.; Li, L.; Liu, T.; Hao, N.; Liu, H.; Chen, D.; Tang, F., The Shape Effect of Mesoporous Silica Nanoparticles on Biodistribution, Clearance, and Biocompatibility in vivo. ACS Nano 2011, 5, 5390-5399. 37. Green, B. J.; Saberi Safaei, T.; Mepham, A.; Labib, M.; Mohamadi, R. M.; Kelley, S. O., Beyond the Capture of Circulating Tumor Cells: Next-Generation Devices and Materials. Angew. Chem., Int. Ed. 2016, 55, 1252-1265. 38. Myung, J. H.; Tam, K. A.; Park, S. j.; Cha, A.; Hong, S., Recent Advances in Nanotechnology-Based Detection and Separation of Circulating Tumor Cells. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2016, 8, 223-239.

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Figure Captions

Scheme 1. Schematic diagram for the comparison of differently shaped M-MSNs-EpCAM in the magnetic isolation and fluorescence detection of circulating tumor cells.

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Figure 1. Characterization of differently shaped M-MSNs. TEM images of (a) sphere-like M-MSNs and (b) rod-like M-MSNs. (c) Magnetization curve of M-MSNs. (d) Fluorescence emission spectra of FITC, S-M-MSNs and R-M-MSNs.

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Figure 2. Shape engineering boosted the capture efficiency of breast cancer cells. (a) Efficiencies

of

capturing

MCF-7

cells

with

different

concentrations

of

S-M-MSNs-EpCAM or R-M-MSNs-EpCAM. (b) Efficiencies of capturing MCF-7 cells and Jurkat T cells with various M-MSNs. (c) Relative fluorescence intensities of the isolated cells. These data represent three separate experiments and are presented as the mean value ± SD and *P < 0.05 versus the R-M-MSNs-EpCAM group.

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Figure 3. Fluorescence images of captured MCF-7 cells (EpCAM+) from mimic CTC samples. The red fluorescence represents MCF-7 cells, and the green fluorescence represents M-MSNs-EpCAM, while the scale bars represent 10 µm.

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Figure 4. Capture, detection, and isolation of spiked MCF-7 cells from mimic CTC samples. (a) Relative fluorescence intensity of the MCF-7 or Jurkat T cells. (b) The capture efficiency of MCF-7 cells using differently shaped M-MSNs-EpCAM in solutions of Jurkat T cells spiked with different numbers of MCF-7 cells. These data represent three separate experiments and are presented as the mean value ± SD and *P < 0.05 versus the R-M-MSNs-EpCAM group.

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Figure 5. Shape engineering boosted the capture efficiency of MCF-7 cells from mimic CTC samples. (a) The capture efficiency of MCF-7 cells using differently shaped M-MSNs-EpCAM in whole blood spiked with different numbers of MCF-7 cells. (b) Efficiencies to capture MCF-7 cells in whole blood at different incubation times. (c) Capture efficiencies from whole blood spiked with four different types of tumor cells: MCF-7, HepG2, HCT-116 and Jurkat T cells. These data represent three separate experiments and are presented as the mean value ± SD and *P < 0.05 versus the R-M-MSNs-EpCAM group.

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Figure 6. Shape engineering boosted the capture efficiency of CTCs from clinical blood samples. (a) CTCs detection from 20 CTCs-containing blood from breast cancer patients. (b) Fluorescent images of CTCs captured with differently shaped M-MSNs-EpCAM from clinical blood samples of CTC-containing breast cancer patients. The red fluorescence represents CTCs (CK19+) cells, and the green fluorescence represents M-MSNs-EpCAM, while the scale bars represent 5 µm.

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Shape Engineering Boosts Magnetic Mesoporous Silica Nanoparticles

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