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Development of Multifunctional Fluorescent-Magneto Nanoprobes for Selective Capturing and Multicolor Imaging of Heterogeneous Circulating Tumor Cells Avijit Pramanik, Aruna Vangara, Bhanu Priya Viraka Nellore, Sudarson Sekhar Sinha, Suhash Reddy Chavva, Stacy J. Jones, and Paresh Chandra Ray ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03262 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 7, 2016

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ACS Applied Materials & Interfaces

Development of Multifunctional Fluorescent-Magneto Nanoprobes for Selective Capturing and Multicolor Imaging of Heterogeneous Circulating Tumor Cells Avijit Pramanik, Aruna Vangara, Bhanu Priya Viraka Nellore, Sudarson Sekhar Sinha, Suhash Reddy Chavva, Stacy Jones and Paresh Chandra Ray * 1

Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS, USA; Email:[email protected]; Fax: +16019793674

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ABSTRACT: Circulating tumor cells (CTC) are highly heterogeneous in nature due to epithelial-mesenchymal transition (EMT), which is the major obstacle for exploitation of CTC analysis as "liquid biopsy". Driven by the need, current article reports the development of new class of multifunctional fluorescent-mganetic multicolor nanoprobes for targeted capturing and accurate identification heterogeneous CTCs. A facile design approach for synthesis and characterization of bio-conjugated multifunctonal nanoprobes that exhibit excellent magnetic properties and emit very bright and photo stable multicolor fluorescence at red, green and blue under 380 nm excitation is reported. Experimental data presented show that multifunctional multicolor nanoprobe can be used for targeted capturing and multicolor fluorescence mapping of heterogenous CTCs and it can distinguish targeted CTCs from non-targeted cells. KEYWORDS: multifunctional fluorescent-mganetic nanoprobes, multicolor fluorescence nanodots, heterogenous circulating tumor cells capturing, mapping of epithelial, mesenchymal and stem cells simultaneously ----------------------------------------------------------------------------------------------------------------------------------------11

1. INTRODUCTION Even in 21st century, due to the tumor metastasis, cancer is the second most common cause of death in USA 1-4. It is now well documented that circulating tumor cells (CTCs) are the main vehicles of the metastatic relapse 5-11. Since CTCs are extremely rare cells (1-10 cells/mL) in blood containing millions of leukocytes and erythrocytes cells, detecting CTCs without separation from blood is highly challenging in clinics6-15. Reported clinical data show CTC concentration in blood can be as low as one per 107 cells, effective separation, enrichment and identification steps are necessary, even for patient with advanced cancer1-8. Since naturally all untreated biological materials are diamagnetic, in clinics magnetic cell separation is highly popular for the separation CTCs from clinical blood sample using antibody attached magnetic bead1-8. CTCs separation from blood is also very important to avoid huge light scattering and autofluorescence from millions of leukocytes and erythrocytes cells1-8. The next challenge medical doctors are facing is CTCs are highly heterogeneous. Due to epithelialmesenchymal transition (EMT), CTCs are undetected for more than one-third of metastatic cancer patients 5-

. Driven by the need, current article reports for the first time the development of multicolor nanodots conjugated magnetic nanoparticle based multifunctional fluorescent-magnetic nanoprobes which have capability to capture and identify heterogeneity of CTCS. In our design, highly magnetic properties of multifunctional nanoprobes have been used for the separation of epithelial, mesenchymal and stem cell CTCs from whole blood sample. On the other hand, multicolor fluorescence nanodots at the surface of multifunctional nanoprobes have been used for multicolor imaging of heterogeneous CTCs selectively and simultaneously. Last few years, nanodots including graphene quantum dots (GQDs), carbon dots (CDs), polymer dots (PDs) and gold cluster dots (GCDs) have emerged as a new type of bright fluorescent probes for biological imaging due to very good photo-stability and biocompatibility with cells and tissues 12-26. Since the size of gold clusters dots (GCDs) is comparable to the Fermi wavelength, the free electrons in GCDs generates discrete electronic transitions, which allow them to exhibit strong photoluminescence properties 27-35. Similarly, the backbone of polymer dots (PDs) which made from conjugated polymer structure exhibit very

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high optical cross section, as a result, PDs display huge fluorescence which can be tuned from visible to NIR by changing the size 12-18. On the other hand, tunable surface functional group in CDs exhibits huge photoluminescence which can be attributed due to the presence of surface energy traps. These nanodots' photoluminescence can be varied by the intrinsic inner structure and surface chemical groups 19-26. Since organic fluorescent dyes have poor solubility in aqueous solutions and undergoes photobleaching 2-4, nanodots will be better bio-molecular probes for fluorescent mapping. Although recently there are good advances on developing different nanodots with tunable optical properties15-22, finding multicolor fluorescent GQDs, GCDs, CDs or PDs at single wavelength excitation is rare. As a result, for mapping heterogeneous CTCs, we have used blue color fluorescence PDs, green fluorescence CDs and red color fluorescence GCDs, where using 380 nm excitation, one can perform multicolor imaging of different subpopulation of CTCs using these materials. Due to the absence of magnetic properties, nanodots will not be able to separate CTCs from blood sample and as a result, we have designed multicolor nanodots attached magnetic nanoparticle based fluorescent-magnetic nanoprobe for selective separation and mapping of epithelial, mesenchymal and stem cells CTCs simultaneously. For selective capture and accurate identification of heterogeneous CTCs, blue fluorescence PDs conjugated fluorescent-magnetic nanoprobes were attached with epithelial markers (anti-EpCAM or anti-HER2 antibody) which can target SK-BR-3 epithelial cancer cell. On the other hand, green fluorescence CDs conjugated fluorescent-magnetic nanoprobes were attached with mesenchymal marker (anti- twist antibody), which will be able to capture CAL-120 breast cancer cell having high levels of mesenchymal markers and it will be green in color in a fluorescence image. Similarly, red fluorescence GCDs conjugated fluorescent-magnetic nanoprobes were attached with CSC marker (anti- CD34 antibody), and as a result, captured CSC bone marrow CD34+ stem cells will be red color in fluorescence image. Our reported result shows that nanodot decorated multifunctional nanoprobes are capable of capturing and accurately identifying the subpopulations of CTCs from whole blood sample.

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Blue fluorescence polymer dots attached magnetic nanoplatforms were synthesized using a multistep process as shown in Figure S1A-B in the supporting information. Synthesis details have been reported in the supporting information. In the first step, blue fluorescence polymer dots (PDs) were synthesized using amphiphilic polymer solvent evaporation technique 16. For this purpose, amphiphilic copolymer was constructed by conjugating polyethyleneimine and D.Llactide using ring-opening polymerization method. In the next step, for the development of polymer dots, PEI-PLA copolymer was dissolved in dichloromethane and 1%(w/v) of PVA. The mixture was kept at 35°C in vacuum chamber. At the end the purified particles were characterized by high-resolution tunneling electron microscopy (TEM) and dynamic light scattering (DLS) measurement , as reported in Figure S1 and Table S1 in supporting information. Figure S1C shows the TEM image of polymer dots. Inserted high-resolution image shows that the size of polymer dots is about 2-3 nm. Table S1, indicate that the average size is about 3 nm for polymer dots. Next, the carboxy acid functionalized magnetic nanoparticle were prepared from ferric chloride and 1,6hexanedioic acid using co-precipitation method as shown in Scheme 1B. Synthesis details has been reported in the supporting information. After finishing the process, black precipitate of Fe3O4 nanoparticles were separated from supernatant using neodymium magnet. As shown in Figure S1D, high-resolution SEM image shows that the average particle size is about ~30 nm. DLS measurement, as reported in Table S1 also indicate that the average size is about 30 nm for magnetic nanoparticle. Inserted EnergyDispersive X-ray (EDX) spectroscopy elemental mapping in Figure S1D clearly shows the presence of Fe in the developed magnetic nanoparticle. The magnetic properties determined using a superconducting quantum interference device (SQUID) magnetometer at room temperature indicates superparamagnetic behavior with specific saturation magnetization of 39.3 emu g−1 for the aminefunctionalized magnetite nanoparticle. At the end, we have used EDC/NHS esterification to produce PDs coated magnetic nanoprobe, as shown in Scheme 1B in the supporting information.

2. RESULTS AND DISCUSSIONS 2.1. Development and characterization of multifunctional blue fluorescent magneto-PDs nanoprobes

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Figure 1: A) SEM image shows the morphology of magneto-PDs nanoprobe. Inserted EDX elemental mapping shows the presence of Fe, C and O in fluorescent-magnetic nanoplatform, B) Fluorescence image under UV light; B1: blue fluorescence from PDs coated fluorescent-magnetic nanoplatform. B2: Fluorescence disappears after magnetic separation. C) Emission spectra from magneto-PDs nanoprobe at 380 nm excitation shows the λem is around 440 nm. D) Photograph shows that the magneto-PDs nanoprobe is highly magnetic, and as a result, we can separate them by using a bar magnet. For this purpose, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Nhydroxysulfosuccinimide (NHS) and 4(dimethylamino) pyridine (DMAP) were added to PDs and acid functionalized Fe3O4 nanoparticles. After that the esterified PDs attached Fe3O4 nanoparticles were separated using neodymium magnet several times and washed with distilled water to remove the excess reactants. Figure 1A shows the SEM image which indicates that the size of PDs coated magnetic nanoplatform is about ~40 nm for magnetic nanoplatform, which is about 10 nm more than only magnetic nanoparticle. Inserted EDX elemental mapping clearly shows the presence of Fe, C and O. We have also performed DLS measurement in the solution phase,

as reported in Table 1 in the supporting information, which indicate that the average size is about 40 nm for PDs coated magnetic nanoplatform. Figure 1D shows that magneto-PDs nanoprobes are highly magnetic and as a result, we can separate them very quickly using a bar magnet. SQUID magnetometer properties measurement indicates super-paramagnetic behavior with specific saturation magnetization of 32.6 emu g−1 for the polymer dots coated magnetite nanoplatforms. Figure 1B1 shows the blue emitted fluorescence from magneto-PDs nanoprobes in the presence of UV light. Figure 1B2 shows that no fluorescence is observed from the solution after magnetic separation, which

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indicates that almost all magneto-PDs nanoprobes were separated by the magnet. Figure 1C shows the emission spectra which clearly indicate the λmax for emission for magneto-PDs nanoprobes are around 440 nm, as a result it shows blue color fluorescence. The photoluminescence quantum yield (QY) for magnetoPDs nanoprobe was determined by counting the integrated luminescence intensities using quinine sulfate as a standard (QY 54%) 13-16. Quantum yield was calculated with respect to standard quinine sulfate using equation 114-25,

Φnd = Φref

(1)

where the nanoprobe is denoted as nd and the standard quinine sulfate is denoted as ref. In the equation 1, Φ is the quantum yield at 380 nm excitation, A is the absorbance, I is the fluorescence intensity and η is the refractive index. From the experimental photoluminescence and theoretical equation 1, we determined the quantum yield for PDs is 0.68 at 380 nm light excitation. 2.2. Development and chracterization of multifunctional red fluorescent magneto-GCDs nanoprobes Red fluorescence magneto-GCDs nanoprobes were synthesized using a multistep process as shown in Figure S2A. Synthesis details has been reported in the supporting information. In the first step, red fluorescent gold cluster dots capped with a bidentate ligand, dihydrolipoic acid (DHLA) were synthesized by mixing sodium hydroxide, α-lipoic acid, HAuCl4.3H20 and NaBH4 with constant stirring using reported method 31. The solid residue was collected in a 20 ml scintillation vial, diluted to a final volume of 5 mL with distilled water, and stored at 4°C for future use. Figure 2B shows the TEM image of freshly prepared GCDs. Inserted HRTEM indicate that GCDs are about 4 nm in size. DLS data as reported in the Table S2 also indicate that the average size is about 3 nm for GCDs. Next, amine functionalized magnetic nanoparticles were synthesized by dissolving FeCl3 in ethylene glycol, sodium acetate and 1,6-hexadiamine, as we have reported before 31. The mixture was sealed in a teflon-lined stainless steel autoclave and was heated at 230 °C for 8 h. Then the product was washed with hot water and ethanol. Figure S2B show the SEM images of amine functionalized magnetic nanoparticles, which indicate that the particle size is

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about 40 nm. Inserted EDX mapping in Figure S2B clearly shows the presence of Fe. The magnetic properties determined using SQUID magnetometer indicates super-paramagnetic behavior with specific saturation magnetization of 43.6 emu g−1 for the aminefunctionalized magnetite nanoparticles. In the final step, we have synthesized red fluorescence magnetoGCDs nanoprobes. For the formation of fluorescentmagnetic nanoprobes, we have used coupling chemistry between -CO2H group of α-Lipoic acid attached GCDs and -NH2 group of amine-functionalized magnetic nanoparticle via amide linkages, as shown in Figure S2A. Synthetic details have been described in the supporting information. The purified particles were characterized by various spectroscopic techniques like Fourier transform infrared spectroscopy (FTIR), TEM and EDX analysis, as reported in Figure S2 and Figure 2. Figure S2C shows the FTIR data obtained from magneto-GCDs nanoprobes. Reported FTIR spectrum shows a very strong Amide A band which is due to the N-H stretching vibration of amide. The spectrum also shows strong amide I band which is mainly associated with the C=O stretching vibration related to the backbone conformation and amide II band which is mainly due to the N-H bending vibration coupled with the C-N stretching vibration. We have also noted amide III band. The high-resolution TEM data, as shown in Figure 2B, shows that the size of magneto-GCDs nanoprobes is about 55 nm, which has been confirmed using DLS measurement in solution phase, as reported in Table S2. EDX elemental mapping, as reported in the inserted Figure in Figure 2B confirms the presence of Fe and Au in the magnetic nanoplatform. SQUID magnetometer properties measurement indicates super-paramagnetic behavior with specific saturation magnetization of 36.6 emu g−1 for the GCDs coated magnetite nanoplatform. Figure 2G shows the color of GCDs coated magnetic nanoplatform in the absence of UV ight. Figure 2C show the red emitted fluorescence from GCDs coated magnetic nanoplatform in the presence of UV light. Figure 2D shows the emission spectra, which clearly indicate that the λmax for emission for magneto-GCDs nanoprobes are around 680 nm.

2.3. Development and characterization of multifunctional green fluorescent magneto-CDs nanoprobes

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Figure 2: A) TEM image shows the morphology of GCDs. Inserted HRTEM image indicate that the particle size is around 4 nm. It also indicate the crystalline structure for gold dots. B) TEM image shows the morphology of magneto-GCDs nanoprobes. Inserted EDX elemental mapping shows the presence of Fe, Au in magneto-GCDs nanoprobes, G) magneto-GCDs nanoprobes in the absence of UV light. C) Fluorescence image under UV light for magneto-GCDs nanoprobes, which clearly shows red color fluorescence at UV light excitation. D) Emission spectra from the mixture of magneto-PDs and magneto-GCDs nanoprobes at 380 nm excitation shows the λem is around 440 nm due to magneto-PDs and λem is around 680 nm due to magneto-GCDs. We have used 83 ppm magneto-GCDs and 12 ppm magneto-PDs, for the fluorescence measurement. The fluorescence intensity is in arbitrary unites (a.u.). Green fluorescence carbon dots (CDs) attached magneto-CDs nanoprobes were synthesized using a multistep process, as shown in Figure S3. Synthesis details have been reported in the supporting information. Initially, the green fluorescence carbon dots (CDs) was synthesized using the literature method.20 In brief, ortho-phenylenediamine was dissolved in pure ethanol and then the solution was transferred into a stainless steel autoclave with a teflon liner and heated at 180oC for 12 hours. The autoclave was cooled to room temperature and the reaction mixture was evaporated using rotary evaporator. The orange color carbon dots were further purified with a silica column

chromatography using mixtures of CH2Cl2 and MeOH as eluents. Figure 3A shows the TEM image of freshly prepared CDs which are about 9 nm size. Figure 3B shows the histogram of size distribution for carbon dots measured by DLS, which indicates that the average size is about 10 nm for GCDs. Next, the acid functionalized magnetic nanoparticle were prepared from ferric chloride as shown in Figure S3.. For the formation of fluorescent-magnetic nanoprobes, we have used EDC coupling chemistry. The purified particles were characterized by SEM and EDX analysis, as reported in Figure 3C, which indicate that the size of magneto-CDs nanoprobes is about 55 nm.

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Figure 3: A) TEM image shows the morphology of CDs. Inserted HRTEM image indicate that the particle size is around 9 nm. B) Figure shows the histogram of size distribution for carbon dots measured by DLS C) SEM image shows the morphology of magneto-CDs nanoprobes. EDX elemental mapping shows the presence of Fe, C and O. D) Fluorescence image under UV light magneto-CDs nanoprobes, which clearly shows green color fluorescence. DLS measurement data reported in Table S3 indicates that the average size is about 55 nm for magneto-CDs nanoprobes. EDX elemental mapping, as reported in the inserted Figure in Figure 3C, confirms the presence of Fe, C and O in the magneto-CDs nanoprobes. SQUID magnetometer properties measurement indicates super-paramagnetic behavior with specific saturation magnetization of 34.9 emu g−1 for the magneto-CDs nanoprobes. Figure 3D shows the green emitted fluorescence from the magneto-CDs nanoprobes in the presence of UV light. Figure 4A shows the emission spectra which clearly indicate the λmax for emission for magneto-CDs nanoprobes are around 550 nm, and as a result, it shows blue color fluorescence. The photoluminescence quantum yield (QY) for magneto-CDs nanoprobe was determined to be

0.23 with respect to quinine sulfate as a standard (QY 54%). 2.4. Developing antibody-conjugated nanoprobe and finding their biocompatibility and photostability Figure 4A shows that our PDs based, GCDs based and CDs based magnetic nanoprobes exhibit three distinguished fluorescence in blue, green and red color range respectively when they were excited at 380 nm excitation light. As a result, we will be able to use them to target epithelial, mesenchymal and stem cells selectively and simultaneously. For targeted capture and imaging of SK-BR-3 epithelial cancer cell, blue fluorescence magneto-PDs nanoprobe were attached with epithelial markers (anti-HER2 antibody). To accomplish this, initially magneto-PDs nanoprobe were

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coated by amine modified polyethylene glycol (NH2PEG). After PEGylation, anti-HER2 antibody was conjugated with amine functionalized PEG coated magneto-PDs nanoprobe using our reported method9,10,24. Similarly, to capture CAL-120 breast cancer cells having high levels of twist mesenchymal markers, green fluorescence magneto-CDs nanoprobes were conjugated with anti-twist antibody. Also to target bone marrow CD34+ stem cells, red fluorescence magneto-GCDs nanoprobes were conjugated with anti-CD34 antibody. Since biocompatibility is very important for imaging, at first we have found out the biocompatibility of the antibody attached fluorescent-magneto nanoprobes. For this purpose, different epithelial, mesenchymal and CSC cells as well as for normal skin HaCaT cells (7.8 x 104 cells/mL) were incubated separately with magneto-PDs nanoprobes for 24 hours. After that, the cell viability was measured using MTT test. Figure 4B clearly shows that even after 24 hours of incubation, more than 98% cell viability was observed. We have performed the same experiment for magnetoGCDs nanoprobes and magneto-CDs nanoprobes. We have not observed cytotoxicity from any of our developed fluorescent-magneto nanoprobes reported here. All the cytotoxicity results clearly show very good biocompatibility of our newly developed fluorescentmagneto nanoprobes. Next, to understand the photostability of the multifunctional fluorescent-magneto nanoprobes, we performed time-dependent intensity change experiments upon exposure of 380 nm light for one hour. As shown in Figure 4B, the luminescence signals from fluorescent-magneto nanoprobes remain almost unchanged (decrease maximum 6%) even after 1 h of illumination. Our reported photostability data clearly show very good photo-stability of the multifunctional fluorescent-magneto nanoprobes developed by us.

2.5. Targeted separation and mapping of epithelial, mesenchymal and stem cell CTCs from whole blood sample Next, to find out whether magnetic nanoplatform can be used for capturing SK-BR-3 epithelial cancer cell, CAL-120 mesenchymal cancer cell and CD34+ stem cells selectively and simultaneously from whole blood sample, 10 cells/mL of tumor cells and 106 cells/mL peripheral blood mononuclear cells (PBMC) were spiked into 15 mL suspensions of citrated whole rabbit blood purchased from Colorado Serum Company. Since in the actual spiked blood sample from patients, CTC coexist with the several million peripheral blood

mononuclear cells, we have spiked 106 cells/mL peripheral blood mononuclear cells (PBMC) with cancer cells in the spiked whole blood. kit. Amount of HER2, twist or CD34+ present in different cells were measured using the enzyme-linked immunosorbent assay (ELISA). Using ELISA analysis, we find HER2, twist or CD34+ are absent in whole rabbit blood or peripheral blood mononuclear cells (PBMC). For the control experiment, citrated whole rabbit blood was spiked by HaCaT normal skin cells. Using ELISA analysis we found that HER2, EpCAM, twist or CD34+ are absent in HaCaT cells. We have kept the concentration of each cells in the mixture in such a way that after mixing, the epithelial, mesenchymal or stem cell CTCs concentration is 10 cells/mL in the spiked whole blood sample. After 90 minutes of gentle shaking of 10 cells/mL of tumor cells, 106 cells/mL PBMC and 15 mL suspensions of citrated whole rabbit blood mixture, we have used the spiked blood for targeted capturing and imaging experiment. In the next step, anti CD34 antibody atatched magneto-GCDs nanoprobes at different concentrations were mixed with spiked whole blood containing 10 cells/mL of tumor cells, 106 cells/mL PBMC for 30 minutes at room temperature before performing the magnetic separation experiment. After that, targeted cells bound magneto-GCDs nanoprobes were separated using a bar magnet. At the end, targeted CTCs captured by magneto-GCDs nanoprobes and the amount of CTCs in the supernatant after magnetic separation were characterized using enzyme-linked ELISA kit and fluorescence mapping analysis as shown in Figures 4. ELISA experimental data as reported in Figure 4F shows that the CD34+ stem cells capture efficiency by anti-CD34 antibody conjugated magnetic nanoplatforms is more than 98%. Since red fluorescent GCDs are decorated on anti-CD34 antibody conjugated magneto-GCDs nanoprobes, which binds to bone marrow CD34+ stem cells, we have used single photon imaging to visualize the capture bone marrow CD34+ stem cells. Figure 4D shows the red luminescence image of bone marrow CD34+ stem cells, which shows that anti-CD34 antibody conjugated magneto-GCDs nanoprobes can be used for very bright red emission imaging of cancer cells. Figures 4E shows that the anti-CD34 antibody conjugated magneto-GCDs nanoprobes does not bind with peripheral blood mononuclear cells or rabbit blood cells due to the lack of antigen-antibody interaction and as a result, we have not observed any luminescence image from the supernatant after magnetic separation. TEM image reported in Figure 4C shows GCDs are captured by stem cell.

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Figure 4: A) Emission spectra from magneto-PDs nanoprobes, magneto-GCDs nanoprobes and magnetoCDs nanoprobes at 380 nm excitations, which exhibit three distinguish fluorescence in blue, green and organce range. We have used 20 ppm magneto-GCDs, 8.8 ppm magneto-CDs and 2.9 ppm magneto-PDs, for the fluorescence measurement. The fluorescence intensity is in arbitrary unites (a.u.). B) Plot demonstrates the biocompatibility of our magneto-PDs nanoprobes. C) TEM image shows that antibody-attached GCDs are attached on CD34+ stem cells surface. D) Single-photon luminescence image shows that huge amount of bone marrow CD34+ stem cells are captured by magneto-GCDs nanoprobes. E) Single-photon fluorescence image from supernatant indicates that almost all CD34+ stem cells are separated by the magnet. Also peripheral blood mononuclear cells and rabbit blood cells do not bind with anti CD34 antibody attached

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magneto-GCDs nanoprobes. F) Percentage of CD34+ stem cells captured by anti CD34 antibody atatched magneto-GCDs nanoprobes, when whole blood was spiked by 10 cells/mL CD34+ stem cells and 106 cells/mL peripheral blood mononuclear cells (PBMC). G) Percentage of HER2+ cancer cells captured by anti- CD34 antibody attached magneto-GCDs nanoprobes,. Our results clearly show that anti- CD34 antibody attached magneto-GCDs nanoprobes are highly selective to capture CD34+ stem cells and as a result, it does not bind with HER2 (+) SK-BR-3 cancer cells. H) Percentage of cells captured by anti -CD34 antibody attached magneto-GCDs nanoprobes when i) whole blood was spiked by 10 cells/mL CD34+ stem cells and 106 cells/mL PBMC ii) whole blood was spiked by 10 cells/mL CD34+ stem cells and 105 cells/mL HaCaT normal cells, iii) whole blood was spiked by 4.5 x 105 cells/mL SK-BR-3 cells and 106 cells/mL PBMC, and iv) whole blood was spiked by 4.5 x 105 cells/mL HaCaT normal cells and 106 cells/mL PBMC. All the reported data clearly show that anti- CD34 antibody conjugated GCDs coated magnetic nanoplatforms are highly selective to capture CD34+ stem cells. I) Percentage of HER2 positive cells captured by anti-HER2 antibody attached magneto-PDs nanoprobes, when i) whole blood was spiked by 10 cells/mL HER2 positive SK-BR-3 cells and 106 cells/mL PBMC, ii) whole blood was spiked by 105 cells/mL CD34+ stem cells and and 106 cells/mL PBMC, iii) whole blood was spiked by 105 cells/mL HaCaT cells and 106 cells/mL PBMC. All the reported data clearly show that anti- HER2 antibody conjugated magnetoPDs nanoprobes are highly selective to capture HER2 positive SK-BR-3 cells. All the above reported results show that anti-CD34 antibody conjugated marrow CD34+ stem cells, which shows that anti-CD34 antibody conjugated magneto-GCDs nanoprobes can be used to separate and map bone marrow CD34+ stem cells from the whole blood sample. To find out how selective the cell capturing and mapping for CD34+ stem cells from spiked blood using anti-CD34 antibody conjugated magneto-GCDs nanoprobes, we have performed cell capturing and fluorescence mapping experiment using CD34 (-) SK-BR-3 breast tumor cells and HaCaT normal skin cells. For this purpose, we have used spiked blood containing 4.5 x 105 cells/mL SK-BR-3 tumor cells and spiked blood containing 4.5 x 105 cells/mL HaCaT normal cells separately. Figure 4H show that the anti-CD34 antibody conjugated magneto-GCDs nanoprobes do not bind with CD34 (-) SKBR-3 breast tumor cells or HaCaT normal skin cells. As a result, cell capture efficiency was less than 1%. Similarly, we have also performed capture efficiency experiment with anti-HER2 antibody attached magneto-PDs nanoprobes for CD34+ stem cells spiked blood. As shown in Figure 4G, our experimental data clearly show that anti-HER2 antibody attached magneto-PDs nanoprobes does not bind with CD34+ stem cells and as result, capture efficiency was less than 1%. On the other hand, as shown in Figures 4I and 5A, the capture efficiency is more than 98% for HER2 (+) SK-BR-3 cells by anti-HER2 antibody attached magneto-PDs nanoprobes. All the above reported experimental data clearly show that anti-CD34 antibody conjugated magneto-GCDs nanoprobes are highly selective for capturing and mapping of CD34+ stem cells. Since blue fluorescence PDs are decorated

on anti-HER2 antibody conjugated magneto-PDs nanoprobes, which binds with SK-BR-3 breast cancer epithelial cells, as shown in Figure 5B, we have observed very bright blue emission from SK-BR-3 cancer cells. Figures 5C shows that the a anti-HER2 antibody attached magneto-PDs nanoprobes does not bind with blood cells due to the lack of antigenantibody interaction and as a result, we have not observed any luminescence from the supernatant after magnetic separation. All the above reported results show that anti-HER2 antibody attached magneto-PDs nanoprobes can be used to separate and map epithelial SK-BR-3 cells from whole blood sample. Next, to demonstrate that the versatile multicolor fluorescent- magneto nanoprobes can be used for capturing of mesenchymal and stem cells CTCs simultaneously, we have performed experiments with mesenchymal and stem cells spiked blood sample. For this purpose, at first we have used whole blood spiked by 10 cells/mL CD34+stem cells, 10 cells/mL twist (+) CAL-120 mesenchymal cells and 106 cells/mL PBMC. For capturing and mapping mesenchymal and stem cells CTCs simultaneously, we have added antiHER2 antibody attached magneto-PDs nanoprobes, anti-CD34 antibody attached magneto-GCDs nanoprobes in 15 mL spiked blood sample. After 30 minutes of shaking, we have captured CTCs by a bar magnet. ELISA data, as shown in Figures 5D, indicate that our multicolor fluorescent- magneto nanoprobes have the capability to capture multiple types of mesenchymal and stem cells CTCs from spiked blood sample and capturing efficiency can be about 97%.

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Figure 5: A) Percentage of HER2 (+) SK-BR-3 epithelial cells that are captured by anti-HER2 antibody attached magneto-PDs nanoprobes, when whole blood was spiked by 10 cells/mL SK-BR-3 epithelial cells and 106 cells/mL PBMC. B) Single-photon luminescence image shows that huge amount of HER2 (+) SKBR-3 epithelial cells are captured by magneto-PDs nanoprobes. Blue color of the observed fluorescence is

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due to the presence of magneto-fluorescent PDs nanoprobes on cancer cell surface. C) Single-photon fluorescence image from supernatant shows no observable fluorescence image from supernatant, which indicates that blood cells do not bind with anti-HER2 antibody attached magneto-PDs nanoprobes and also all HER2 (+) SK-BR-3 epithelial cells are captured by the magneto-PDs nanoprobes. D, E) Figures demonstrate capturing stem cells and mesenchymal cells simultaneously using nanoprobes. D) ELISA data show percentage of CD34(+) stem cells and twist (+) CAL-120 mesenchymal cells are captured simultaneously by anti-CD34 antibody attached magneto-GCDs nanoprobes and anti-twist antibody attached magneto-CDs nanoprobes. E) Fluorescence image shows that antibody conjugated multicolor fluorescent magnetonanoprobes are capable of capturing stem and mesenchymal cells simultaneously from the spiked blood.. F, G, H) Figures demonstrate capturing of epithelial, stem and mesenchymal cells simultaneously using nanoplatforms. F) ELISA data show the percentage of HER2 (+) epithelial cells, CD34(+) stem cells and twist (+) CAL-120 mesenchymal cells captured simultaneously by anti-HER2 antibody attached magneto-PDs nanoprobes, anti-CD34 antibody attached magneto-GCDs nanoprobes and anti-twist antibody attached magneto-CDs nanoprobes. G) Fluorescence image from supernatant shows that about all epithelial, stem and mesenchymal cells are separated by magnet. H) Fluorescence image shows that multicolor nanodots decorated antibody conjugated nanoprobes are capable of capturing epithelial, stem and mesenchymal cells simultaneously from spiked blood.

Multi-color fluorescence image, as shown in Figure 5E shows that nanodots decorated antibody conjugated multicolor fluorescent- magneto nanoprobes are capable of capturing stem and mesenchymal cells simultaneously from spiked blood. As reported in Figure 5E, red color fluorescence cells are CD34(+) stem cells and it is due to the presence of anti-CD34 antibody attached magneto-GCDs nanoprobes on stem cell surface. Similarly due to the presence of anti-twist antibody attached CDs coated magnetoCDs nanoprobes on mesenchymal cell surface, green color fluorescence cells are CAL-120 mesenchymal cells. Next, for capturing and mapping epithelial, mesenchymal and stem cells simultaneously, we have added anti-HER2 antibody attached magneto PDs nanoprobes, anti-CD34 antibody attached magnetoGCDs nanoprobes and anti-twist antibody attached magneto-CDs nanoprobes in 15 mL spiked blood sample. For this purpose, we have spiked the whole blood sample by 10 cells/mL HER2 (+) epithelial cells, 10 cells/mL CD34+stem cells, 10 cells/mL twist (+) CAL-120 mesenchymal cells and 106 cells/mL PBMC. After 30 minutes of shaking, we have captured CTCs subpopulations by a bar magnet. Figure 5F shows the ELISA data, which clearly indicate that our bio-conjugated multicolor fluorescent- magneto nanoprobes have the capability to capture epithelial, mesenchymal and stem cell CTCs from spiked blood sample and capturing efficiency from spiked blood sample can be about 97%. Figure 5H shows the multi-color fluorescence image, which indicates that nanodots decorated antibody conjugated multicolor fluorescent- magneto nanoprobes

are capable of capturing epithelial, stem and mesenchymal cells simultaneously from spiked blood. As shown in Figure 5H, blue color fluorescence cells are HER2 (+) SK-BR-3 cells and it is due to the presence of anti-HER2 antibody attached magneto-PDs nanoprobes on stem cell surface. On the other hand, red color fluorescence cells are CD34(+) stem cells and it is due to the presence of anti-CD34 antibody attached magneto-GCDs nanoprobes on stem cell surface. Similarly, due to the presence of anti-twist antibody attached magneto-CDs nanoprobes on mesenchymal cell surface, green color fluorescence cells are CAL120 mesenchymal cells. Fluorescence image from supernatant, as shown in Figure 4G, indicates that almost all epithelial, stem and mesenchymal cells are captured by the magnet. Also, peripheral blood mononuclear cells and rabbit blood cells do not bind antibody attached nanodots coated multicolor fluorescentmagneto nanoprobes and as a result, we have not observed any fluorescence image from the supernatant. All the above experimental data clearly show that different antibody attached multicolor fluorescent- magneto nanoprobes can be used for capturing epithelial, stem and mesenchymal cells simultaneously from the spiked blood and it is highly selective for capturing targeted tumor cells from the spiked blood.

3. CONCLUSIONS In conclusion, in this article we have reported the design of bio-conjugated multifunctional nanoprobes that exhibit excellent magnetic and multicolor fluorescencent properties with targeted capturing and mapping capability for epithelial, mesenchymal and stem cells simultaneously. We have shown the new

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means of capturing and analyzing epithelial, mesenchymal and stem cell CTCs from spiked blood using multicolor nanodots attached antibody coated nanoprobe, which has the capability to find a solution for the characterization of CTC heterogeneity found in clinical samples. We have demonstrated that our nanoprobes are capable of selectively and simultaneously detect different subpopulation CTCs containing SK-BR-3 epithelial, CAL-120 mesenchymal and bone marrow CD34+ stem cells in spiked whole blood. Our reported data show that the nanorobes are highly selective for capturing targeted tumor cells and the capture efficiency can be as high as 97% for epithelial, mesenchymal and stem cells simultaneously. Reported data demonstrate that multicolor fluorescence imaging can be used for mapping epithelial, mesenchymal and stem cells CTCs simultaneously, which indicates that nanoprobes are capable of finding circulating tumor cells heterogeneity by accurately identifying the multiple subpopulations of CTCs from blood sample. Although the sensitivity of ELISA for CTC detection is comparable with the reported nanodot based assay, ELISA had to be coupled to a magnetic bead for enrichment of CTCs from blood sample, since the concentration of CTCs in blood can be as low as one per 107 cells. Whereas, due to the presence of magnetic nanoparticles in the nanoprobes developed by us, it can be used for enrichment, separation and detection via fluorescence imaging. Since nanoprobes exhibit narrow emission bands, it can be used simultaneous separation and detection of multiple CTCs together, which has been demonstrated here. We anticipate that the nanoprobe design reported here has the capability to provide EMT profiling in CTCs from clinical sample after proper engineering design. Although we have performed CTC detection after spiked into 15 mL whole blood containing 10 cells/mL CTCs, since in clinical setting only 1-10 CTCs/mL are present for cancer patients and CTCs need to be detected in 7.5 mL whole blood, a better design is necessary to enhance the sensitivity. 4. SUPPORTING INFORMATION AVAILABLE Detailed synthesis and characterization of nanoprobes and other experiments are available as a supporting information. This information is available free of charge via the internet at http://pubs.acs.org/. 5. ACKNOWLEDGEMENTS

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Dr. Ray thanks NSF-PREM grant # DMR-1205194 for their generous funding. We are grateful for use of the JSU Analytical Core Laboratory –RCMI facility supported by NIH grant # G12MD007581 6. REFERENCES 1. Yoon, H. J.; Kozminsky, M.; and Nagrath, S.; Emerging Role of Nanomaterials in Circulating Tumor Cell Isolation and Analysis, ACS Nano, 2014, 8, 1995-2017 2. Yu, M.; Bardia, A.; Aceto, N.; Bersani, F.; Madden, M. W.; Donaldson, M. C.; Desai, R.; Zhu,H.; Comaills, V.; Zheng, Z.; Wittner, B. S. A. Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility Science, 2014, 345, 216–220 3. Chaffer, C. L.; Weinberg, R. A.A Perspective on Cancer Cell Metastasis, Science 2011, 331,1559– 1564 4. Krebs M. G.; Metcalf, R. L.; Carter, L.; Brady, G.; Blackhall, F. H.; Dive, C..; Molecular analysis of circulating tumour cells-biology and biomarkers. Nat Rev Clin Oncol, 2014, 11, 129-144. 5.Halo, T. L.; McMahon, K. M.; Angeloni, N.L.; Xu, Y.; Wang, W.; Chinen, A. B.; Malin, D.;Strekalova, E.; Cryns, V. L.; Cheng, C. Mirkin, C. A.; Thaxton,, Nano Flares for the detection, isolation, and culture of live tumor cells from human blood, Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 17104–17109.

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7. Table Of Contents (TOC) Graphic

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