Estradiol Hemisuccinate-Modified Surface-Engineered Carbon Dots

The present work highlights design and development of noncovalently surface-modified carbon dots by 17β-estradiol hemisuccinate that selectively stai...
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Research Article pubs.acs.org/journal/ascecg

Estradiol Hemisuccinate-Modified Surface-Engineered Carbon Dots: Target-Specific Theranostic Agent Saheli Sarkar, Krishnendu Das, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Jadavpur, Kolkata − 700 032, India S Supporting Information *

ABSTRACT: The present work highlights design and development of noncovalently surface-modified carbon dots by 17β-estradiol hemisuccinate that selectively stains estrogen receptor (ER)-rich cancer cells as well as kill ER (+) cancer cells by target-specific delivery of the anticancer drug doxorubicin. Positively surface-charged blue-emitting and green-emitting cationic carbon dots (CCDs) were prepared. Blue-emitting cationic carbon dots (CCDb) were prepared by the thermal coupling of tris(hydroxymethyl)aminomethane and betaine hydrochloride, while green-emitting cationic carbon dots (CCDg) were prepared by the thermal coupling of citric acid and ehylenediamine. Negatively charged estradiol hemisuccinate (E2) was synthesized from 17β-estradiol. Both CCDb and CCDg were noncovalently coupled with E2 through electrostatic interaction to prepare CCDb-E2 and CCDg-E2 hybrids, respectively. These surface-modified carbon dots were characterized by microscopic and spectroscopic techniques. Both CCD-E2 hybrids were highly water soluble. CCDb-E2 and CCDg-E2 exhibited enhanced emissions more than those of the respective native CCDs. Consequently, these CCDb-E2 and CCDg-E2 hybrids were utilized as selective cellular markers of estrogen receptor-rich (ER+) MCF7 cells over estrogen receptor negative (ER-) MDA-MB-231 cells and noncancerous CHO cells. Moreover, the anticancer drug doxorubicin (dox)-loaded CCDb-E2 and CCDg-E2 (CCDb-E2-dox and CCDg-E2-dox, respectively) showed selective killing of ER(+) MCF7 cells through a late apoptotic pathway by 2-fold higher efficacy compared to ER(−) MDA-MB-231 cells and noncancerous CHO cells. KEYWORDS: Carbon dot, 17β-Estradiol, Estrogen receptor, Surface engineering, Bioimaging, Theranostic agent

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properties.32−34 Ease of synthesis and tailor-made surface modification of carbon dots with different functional motifs can make it task specific as required.35−37 Surface-functionalized carbon dots have been reported to be used separately as cell staining probes and cargo delivery vehicles.38−42 However, target-specific theranostic applications using carbon dots are really scarce. Feng et al. used charge-convertible polymer-coated carbon dots for theranostic application by exploiting the pH difference between the extracellular and intracellular domains of tumors.43 To the same end, Mewada et al. reported folic acidfunctionalized bovine serum albumin (BSA)-wrapped carbon dots for target-specific delivery of doxorubicin.44 However, BSA is known to form hydrophobic aggregates which may affect the stability of the carbon dots in aqueous domains.45 In both instances, either polymers or biopolymers (protein) have been used to impart hydrophilicity within the nanohybrids with the risk of precipitation with respect to the long-term stability of aqueous carbon dot solutions.45,46 Hence, it would be highly

INTRODUCTION Multifarious approaches are being developed and are also on the rise for cancer treatment, which is one of the major health problems globally. For the past many years, chemotherapy has been the primary method of cancer treatment.1,2 In this context, liposomes, polymeric micelles, nanogels, carbon nanotubes, graphene oxides, and various nanoparticles have got extensive exploitation for selective delivery of chemotherapeutic drugs to cancer cells.3−11 Recent research investigations are focused on early detection of cancer with a motivation of “prevention is better than cure”.12,13 Thus, preparation of theranostic agents, i.e., the combination of diagnostics and therapy, is a budding field to fight against this deadly disease.14−16 Nanoparticle-based theranostic agents like quantum dots, iron oxide, and silica nanoparticles have gotten considerable attention.17−20 However, these nanoparticles often suffer from low water solubility, poor cytocompatibility, and nonbiodegradability.21,22 To this end, inherently fluorescent carbon dots with high photostability and potential biocompatibility are gaining noteworthy significance in biomolecular sensing, bioimaging, biocatalysis, etc.23−31 Carbon dots are an emerging class of zero-dimensional allotropes of carbon having outstanding physical and chemical © 2017 American Chemical Society

Received: June 28, 2017 Revised: July 24, 2017 Published: August 3, 2017 8356

DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

Research Article

ACS Sustainable Chemistry & Engineering

furnace and allowed to cool to ambient conditions. Brownish-black material was extracted with 1 N HCl (25 mL). The suspension was centrifuged for 30 min at 12000 rpm, and the residue was discarded. The supernatant was lyophilized to get the CCDg with a yield of ∼65%. Synthesis of Estradiol Hemisuccinate (E2). 17β-Estradiol (500 mg, 1.8 mmol) was dissolved in dry toluene (20 mL). To this solution, succinic anhydride (900 mg, 9.0 mmol) and pyridine (3 mL) were added.48 The mixture was subjected to reflux for 24 h. The reaction mixture was cooled to room temperature followed by the removal of excess succinic anhydride through filtration. The filtrate (estradiol disuccinate) was concentrated in a rotary evaporator and dissolved in methanol followed by stirring with an excess of sodium bicarbonate overnight. After the hydrolysis of phenolic ester, filtration was done to remove excess sodium bicarbonate. Unreacted estradiol was extracted out by diethyl ether three times from the aqueous suspension of the residue. The pH of the aqueous suspension was adjusted to 7.0 using 1 N HCl. The resulting mixture was poured into crushed ice in the presence of 0.1 N HCl with constant scratching. The obtained product was separated by filtration as a white crystalline solid and washed thoroughly with water to remove excess acid followed by drying in air. The obtained estradiol hemisuccinic acid was crystallized from boiling toluene and characterized by 1H NMR and mass spectra (detailed characterizations are given in Supporting Information). Estradiol hemisuccinic acid was suspended in water, and NaHCO3 was added with a 1:1 molar ratio to get a clear solution of estradiol hemisuccinate. The solution was lyophilized to get solid estradiol hemisuccinate (E2). The synthetic scheme is given in Scheme S1, Supporting Information. Preparation of Estradiol Hemisuccinate-Modified CCD (CCDE2). Both CCDs (CCDb and CCDg, 1 mg, taken separately in 1 mL water) were mixed with estradiol hemisuccinate (2 mg) (1:2, w/w) and bath sonicated for 15 min to get a clear solution of estradiol hemisuccinate-modified CCD (CCD-E2). CCD with the concentration above and below 1 mg as well as E2 with proportionately above and below 2 mg in 1 mL of water became turbid after 15 min of sonication. Characterization. To prepare samples for transmission electron microscopy (TEM), 4 μL of the synthesized CCDs solution was cast on a Cu-coated TEM grid (300-mesh), and these Cu-grids were subjected to drying under vacuum for 4 h prior to imaging. JEOL JEM microscopy (2100F UHR) was used for taking TEM images. For X-ray photoelectron spectroscopy (XPS), the CCD solution (10 μL) was placed on a rectangular Cu plate followed by drying under vacuum for 8 h. XPS analysis was carried out in an X-ray photoelectron spectrometer (Omicron, series 0571). A drop of the CCD solution was cast on a freshly cleaved mica surface to prepare samples for atomic force microscopy (AFM), and samples were air-dried overnight. A Veeco AP0100 microscope was used for AFM imaging in noncontact mode. Xray diffraction (XRD) was carried out on a Bruker D8 Advance diffractometer, and the source of the X-ray was Cu Kα radiation (α = 0.15406 nm) with a 40 kV voltage and 30 mA current. Thermal gravimetric analysis (TGA) of the CCD-E2 hybrids was carried out in TA SDT Q600 under a N2 atmosphere at a heating rate of 20 °C min. Quantum Yield Measurement. The absorbance of CCDb, CCDg, CCDb-E2, and CCDg-E2 solutions was measured restricting the absorbance value less than 0.01.39 Integrated emission intensities of the solutions were measured in a luminescence spectrophotometer. The quantum yield was obtained using following equation

important to develop water-soluble surface-modified carbon dots with desired functionality for receptor-mediated theranostic applications. To impart target specificity, 17β-estradiol was chosen as a surface-modifying moiety for carbon dots. 17β-Estradiol is an endogenous ligand of estrogen receptors (ER), which are wellknown intracellular steroid receptors.47 It mainly gets expressed in estrogen-responsive organs.48 17β-Estradiol binds to the estrogen receptors during its genomic activity and controls cell proliferation in cancer cells (for example, breast carcinoma) and therefore becomes the primary target for breast cancer treatment.47 We have prepared two positively surface-charged cationic carbon dots (CCDs) and negatively charged estradiol hemisuccinate (E2) from 17β-estradiol. These CCDs were noncovalently coupled with E2 simply by electrostatic interaction. The successful functionalization of CCDs with E2 (CCD-E2) was investigated by microscopic and spectroscopic techniques. CCD-E2 nanohybrids were highly water soluble and exhibited improved emitting behavior more than that of native CCDs. Consequently, CCD-E2 hybrids were utilized as selective cellular-staining agents for estrogen receptor-rich (ER+) MCF7 cells over ER negative (ER-) MDA-MB-231 cells and non-cancerous CHO cells. Moreover, doxorubicin (dox, anticancer drug)-loaded CCD-E2 nanohybrids (CCD-E2-dox) selectively killed ER(+) MCF7 cells through late apoptotic pathways by 2-fold higher efficacy compared to ER(−) MDAMB-231 cells and noncancerous CHO cells.

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EXPERIMENTAL SECTION

Materials. Tris(hydroxymethyl)aminomethane, betaine hydrochloride, 17β-estradiol, an Annexin V-FITC apoptosis kit, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were procured from Sigma-Aldrich Pvt. Ltd. Citric acid, ethylenediamine, succinic anhydride, NaHCO3, and all other reagents and solvents were bought from SRL, India. All experiments were carried out using Milli-Q water. A live/dead viability kit was obtained from Genetix, India. 1H NMR spectroscopy was carried out in an AVANCE 500 MHz (Bruker) spectrometer. A Varian Cary Eclipse luminescence spectrometer was used to record fluorescence spectra. A PerkinElmer Lambda 25 and Spectrum 100 were used to record UV−vis and FTIR spectra, respectively. Centrifugation was performed using a Thermo Scientific Espresso centrifuge. Nano-ZS of Malvern Instruments Limited was used to measure zeta potential. A Telsonic bath sonicator was used to perform bath sonication. Heat-inactivated fetal bovine serum (FBS), Dulbecco’s Modified Eagles’ Medium (DMEM), trypsin (from porcine pancreas), and a lactate dehydrogenase (LDH) assay kit were procured from Himedia. MCF7 and MDA-MB-231 cells were received from NCCS, Pune, India. Synthesis of Blue-Emitting Cationic Carbon Dot (CCDb). To synthesize blue-emitting cationic carbon dots (CCDb), an aqueous solution (5 mL) of both betaine hydrochloride (2.0 g, 14 mmol) and tris(hydroxymethyl)aminomethane (1.2 g, 14 mmol) was mixed with shaking until complete dissolution. This mixed solution was dried by lyophilization. The obtained dried mass was collected and heated for 2 h at 200 °C in a furnace and then allowed to cool to ambient conditions. The obtained mass was extracted with 25 mL of water. The suspension was then centrifuged for 30 min at 12,000 rpm, and the residue was discarded. The supernatant was lyophilized to get the CCDb with a yield of ∼72%. Synthesis of Green-Emitting Cationic Carbon Dot (CCDg). To synthesize green emitting cationic carbon dots (CCDg), an aqueous solution (5 mL) of both citric acid (3.0 g, 14 mmol) and ethylenediamine (0.84 g, 14 mmol) was mixed with shaking until complete dissolution. This mixed solution was dried by lyophilization. The obtained dried mass was collected and heated for 2 h at 200 °C in a

Q = Q st(Ism/Ist)(ODst /ODsm )(ηsm 2/ηst 2) where Q is the quantum yield, I represents integrated emission intensity measured at excitation maxima 340 nm, OD represents optical density, and η represents refractive index. The subscript “sm” stands for sample, and “st” indicates standard fluorescence of a known fluorophore. Here, we have used quinine sulfate as a standard dissolved in 0.1 M H2SO4. The quantum yield of quinine sulfate is 0.54. Media Stability of CCD-E2. The media stability of the CCDb-E2 and CCDg-E2 solutions was investigated using [CCD] = 100 μg/mL and [E2] = 200 μg/mL. CCDb-E2 and CCDg-E2 solutions were added to DMEM media having 0−75% of FBS, and it was kept undisturbed for 48 h. The final concentration of CCDs in the media was 1 mg/mL. 8357

DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

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Cells were then washed with PBS buffer, followed by being fixed with paraformaldehyde (4%) for half an hour. Then, a glycerol solution (50%) was used to mount the cells on the slide. A coverslip was used to cover the slide and was kept for 24 h. A bioimaging experiment was carried out in fluorescence microscope under an exposure of 90% (Figure S6). Visual observations also showed that both CCDb-E2 and CCDg-E2 were stable in cell culture media with increasing FBS content up to 75% (Figure 4a,c). Next, we investigated the cytocompatibility of CCDb-E2 and CCDg-E2 against mammalian cells by MTT assay. Varying concentrations of CCDs (100−500 μg/mL) were added to

quantum yields of CCDb and CCDb-E2 were 6.1% and 11.1%, respectively, while those of CCDg and CCDg-E2 were found to be 6.9% and 12.0%, respectively, with respect to quinine sulfate. So, CCDb-E2 and CCDg-E2 showed 1.8- and 1.7-fold higher quantum yield compared to the corresponding native CCDs. The energy traps present on the carbon dots surface possibly become more emitting upon stabilization due to the surface passivation.32 The deconvoluted XPS spectra of the C 1s orbitals of CCDb-E2 and CCDg-E2 showed similar peaks that were observed in the case of native CCDb and CCDg (Figure S4a,c). However, deconvoluted XPS spectra of N 1s orbitals of CCDbE2 and CCDg-E2 (Figure S4b,d) are quite different from that of corresponding native CCDs. Peaks corresponding to pyridinic N, pyrrolic N, and quaternary N of CCDb-E2 got shifted to 397.8, 398.6, and 399.4 eV, respectively, compared to that of pure CCDb (Figure S4b). Moreover, two new peaks appeared at 400.0 and 397.1 eV due to the presence of both the higher energy and lower energy of the N−O linkage, respectively.52 Similarly, in the case of CCDg-E2, peaks corresponding to pyridinic N, pyrrolic N, and quaternary N of CCDb-E2 got shifted to 397.6, 398.4, and 399.3 eV. Here, also two new peaks observed at 399.8 and 396.9 eV correspond to the presence of both higher energy and lower energy of the N−O linkage, respectively (Figure S 4d). The successful formation of CCDb-E2 and CCDg-E2 nanohybrids were further investigated through thermogravimetric (TGA) analysis. TGA plots of both CCDb-E2 and CCDg-E2 8362

DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

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Figure 6. Brightfield and fluorescence microscopic images of cells after 6 h incubation with CCDg-E2 (100 μg/mL): (a,b) MCF7, (c,d) MDA-MB-231, and (e,f) CHO cells. Scale bars correspond to 20 μm.

regard, both CCDs (100 μg/mL) were incubated separately with ER(+)MCF7, ER(−)MDA-MB-231, and noncancerous CHO cells for 6 h, followed by all three cells being observed under a fluorescence microscope. Interestingly, in the case of CCDb-E2, MCF7 cells showed bright blue fluorescence, whereas MDAMB-231 and CHO cells did not exhibit any notable fluorescence under similar experimental conditions (Figure 5). Similarly, in the case of CCDg-E2, MCF7 cells exhibited bright green fluorescence, whereas MDA-MB-231 and CHO cells did not exhibit any notable fluorescence (Figure 6). E2-modified CCDs could easily get internalized into ER-expressing breast cancer MCF7 cells and illuminate the cells with its intrinsic blue or green emission. On the other hand, MDA-MB-231 devoid of ER and CHO (noncancer cell) possibly could not uptake the CCDb-E2 or CCD g -E2 efficiently, and consequently, no notable fluorescence was observed under a fluorescence microscope. Thus, surface-engineered CCDb-E2 and CCDg-E2 can selectively label ER-expressing cancer cells as bioimaging probes. Doxorubicin Loading on CCD-E2. With the aim of exploiting these CCDb-E2 and CCDg-E2 bioprobes in targeted therapeutics, the well-known anticancer drug doxorubicin (dox) was loaded on both CCDb-E2 and CCDg-E2. Doxorubicin has been widely used in a range of cancer treatments because of its intercalation ability with DNA that leads to the killing of the cells. However, free doxorubicin displays low antitumor activity and also cannot discriminate between cancer and noncancerous cells.

ER(+) MCF7 and ER(−) MDA-MB-231 breast cancer cells and noncancerous CHO cells. Interestingly, after 24 h incubation, it was found that the cell viability of both CCDb-E2 and CCDg-E2 was 90−95% toward ER(+), ER(−) and noncancerous cells (Figure 4b,d). The cell viability of CCDb-E2 and CCDg-E2 at 100 and 500 μg/mL was further investigated against all three cell lines by LDH assay which exhibited >95% cells were alive after 24 h incubation with surface-modified carbon dots (Figure S7). Furthermore, MCF7, MDA-MB-231, and CHO cells were separately incubated with 500 μg/mL of CCDb-E2 and CCDg-E2 for 24 h. Cells were treated with the live/dead viability kit (for eukaryotic cells) and investigated under a microscope (Figures S8 and S9). Fluorescence images showed that green cells were present predominantly, while the presence of red cells was very negligible in MCF7, MDA-MB-231, and CHO. Moreover, in flow cytometry, these samples exhibited significant green fluorescence (Figures S8 and S9). Both microscopic and flow cytometry investigations indicate the considerable cell viability of both CCDb-E2 and CCDg-E2 which is in concurrence with the MTT and LDH assay. Hence, CCDb-E2 and CCDg-E2 fulfilled both prerequisites for their utilization in theranostic applications. Bioimaging of ER(+) Cancer Cells. High cell viability and intrinsic fluorescence properties obviously justify the possible utility of CCDb-E2 and CCDg-E2 as bioimaging probes. Moreover, successful surface modification with E2 makes these CCDs potentially target specific to ER(+) cancer cells. In this 8363

DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

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Figure 7. (a) UV−visible and (b) fluorescence spectra of free doxorubicin (dox) and doxorubicin-loaded CCDb-E2 (CCDb-E2-dox). (c) UV−visible and (d) fluorescence spectra of free doxorubicin (dox) and doxorubicin-loaded CCDg-E2 (CCDg-E2-dox). [dox] = 5 μg/mL and [CCD-E2] = 10 μg/ mL.

native CDs) got loaded on the surfaces of both native CCDb and CCDg. Cancer Cell Selective Delivery of Doxorubicin. Doxorubicin-loaded CCDb-E2 and CCDg-E2 were explored for targetspecific therapeutic applications toward the ER(+) breast cancer cell, MCF7. CCDb-E2-dox and CCDg-E2-dox hybrids having 10 μg/mL of CCDb-E2 or CCDg-E2 and 5 μg/mL of doxorubicin were incubated with MCF7 cells for 6 h. In the case of both CCD-E2-dox, a bright red fluorescence under fluorescence microscopy confirmed the successful delivery of doxorubicin within the ER+ MCF7 cells (Figures 8a,b and 9a,b). Similarly, the CCD-E2-dox hybrid was incubated with ER(−) MDA-MB-231 and noncancerous CHO cells. Interestingly, no notable internalization of doxorubicin was found under fluorescence microscopy (Figures 8d,e,g,h and 9d,e,g,h). Thus, both surface-engineered CCDb-E2 and CCDg-E2 nanohybrids modified with estradiol moiety selectively delivered cargo inside the cancer cells by targeting the estrogen receptors. Selective delivery of dox inside ER(+) cancer cells by CCDb-E2 and CCDg-E2 was also analyzed by flow cytometry. A corresponding flow cytometric plot for MCF7 cells exhibited significantly high fluorescence intensity in the order of 104−105 (mean fluorescence = 65,211, Figure 8c). On the contrary, the flow cytometric plot of ER(−) MDA-MB231 and noncancerous CHO cells showed significantly lower fluorescence intensity in the order of 102−104 having mean fluorescence of 1119 and 1023 (Figure 8f,i). Similar flow cytometric experiments performed with CCDg-E2-dox in MCF7 cells exhibited high emission intensity in the order of 104−105 with a mean fluorescence of 55,223. However, the flow cytometric plot of ER(−) MDA-MB-231 and noncancerous

Doxorubicin was loaded at 2:1 w/w (CCD-E2:dox), where dox (500 μg) was mixed with 1 mg of a CCDb-E2 or CCDg-E2 (1 mg CCDb or CCDg and 2 mg E2) solution for maximizing the uploading of doxorubicin on the surface of E2-modified carbon dots. A red shift of 14 nm (488 to 502 nm) in the UV−visible spectra of both doxorubicin-loaded CCDb-E2 (CCDb-E2-dox) and doxorubicin-loaded CCDg-E2 (CCDg-E2-dox) was observed with respect to free doxorubicin (488 nm) indicating the possible loading of the drug on the CCDb-E2 surface (Figure 7a,c).7 Also, the sharp quenching in the emission intensity of doxorubicin at 590 nm (λex= 488 nm) confirmed the drug loading on E2modified CCDs surfaces (Figure 7b,d). Moreover, the aqueous solution of free dox was titrated with both CCDb-E2 and CCDgE2 which resulted in the quenching of dox fluorescence (Figure S10a,c). With increasing concentrations of CCDb-E2 and CCDgE2, the intrinsic fluorescence of dox gradually decreased. The Stern−Volmer plot of I0/I versus the concentration of CCDb-E2 indicates a binding constant of 74 L/g (Figure S10b), while that for CCDg-E2 was 72 L/g (Figure S10d). These higher binding constant53 values further confirmed the successful formation of CCDb-E2-dox and CCDg-E2-dox conjugates. Noncovalent grafting via π−π interactions between the sp2-carbon network of CCDs and the aromatic moieties of doxorubicin possibly facilitated its loading on the surface of carbon dots.48,54 The amount of loaded doxorubicin was found to be 431 μg on 1 mg of CCDb-E2; i.e., almost 86% of the drug got loaded on the surface modified CCDb. Similarly, 401 μg of doxorubicin was found to be loaded on 1 mg of CCDg-E2 (almost 80% drug loading). Doxorubicin was also loaded on native CCDb and CCDg, and it was found that 82% of the drug (408 μg the drug on 1 mg of 8364

DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

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Figure 8. Brightfield and fluorescence microscopic images of cells after 6 h incubation with CCDb-E2-dox, where [dox] = 5 μg/mL and [CCDb-E2] = 10 μg/mL. (a,b) MCF7 cells, (d,e) MDA-MB-231 cells, and (g,h) CHO cells. Corresponding flow cytometric plots of (c) MCF7 cells, (f) MDA-MB-231 cells, and (i) CHO cells. In all the flow cytometric plots, the x-axis denotes the doxorubicin fluorescence intensity. The mean fluorescence values are given in the insets. Scale bars correspond to 20 μm.

[CCDb-E2] = 100 μg/mL and [dox] = 50 μg/mL showed 81 ± 3% killing of MCF7 cells. In contrast, under similar experimental conditions, only 40 ± 3% of MDA-MB-231 and 38 ± 3% of CHO cells were found to be dead (Figure 10a). Similarly, CCDg-E2dox having [CCDg-E2] = 100 μg/mL and [dox] = 50 μg/mL showed 85 ± 2% killing of MCF7 cells, whereas under similar experimental conditions only 38 ± 2% of MDA-MB-231 and 35 ± 3% of CHO cells were killed (Figure 10a). The killing efficiency of CCDb-E2-dox and CCDg-E2-dox conjugates was further studied by LDH assay. MCF7, MDA-MB-231, and CHO cells were incubated with fixed CCDb-E2-dox and CCDg-E2-dox concentrations having 100 μg/mL of CCDb-E2 or CCDg-E2 and 50 μg/mL of doxorubicin for 12 h. The LDH assay of CCDb-E2dox showed 78 ± 3% killing of ER(+) MCF7 cells compared to 37 ± 2% and 38 ± 2% killing of ER(−) MDA-MB-231 cells and noncancerous CHO cells, respectively (Figure S12a). Similarly, the LDH assay of CCDg-E2-dox showed 80 ± 2% killing of ER(+) MCF7 cells in comparison to 35 ± 2% and 37 ± 3% killing of ER(−) MDA-MB-231 cells and noncancerous CHO cells, respectively (Figure S12a). Therefore, both CCDb-E2-dox and CCDg-E2-dox exhibited ∼2-fold better killing efficacy toward ER(+) cancer cells compared to both ER(−) cancer cells and normal cells. Moreover, in the case of free doxorubicin, only ∼30% killing of MCF7, MDA-MB-231, and CHO cells was observed at 50 μg/mL of free drug upon 12 h of incubation (Figure 10a). This indicates the inefficient and nonselective killing ability of doxorubicin without CCDb-E2 and CCDg-E2 cargo transporters. Moreover, the killing efficiency of doxorubicin-loaded native CCDs (CCDb-dox and CCDg-dox) were

CHO cells showed significantly lower emission intensity in the order of 102−104 with a mean fluorescence of 1090 and 1103, respectively (Figure 9c,f,i). This observation confirms the high content of doxorubicin inside ER(+) cancer cells, while both CCDb-E2 or CCDg-E2 cannot deliver the drug with same efficiency inside MDA-MB-231 and CHO cells. Moreover, the internalization ability of free dox was also investigated by microscopic and flow cytometric techniques. However, no notable fluorescence was observed under fluorescence microscopy in case of all three cells: MCF7, MDA-MB-231, and CHO (Figure S11a,b,d,e,g,h). These results were also corroborated with flow cytometric plots of MCF7, MDA-MB-231, and CHO that showed significantly lower fluorescence of 1183, 1152, and 1123, respectively (Figure S11c,f,i). This indicates the inefficient and nonselective internalization ability of doxorubicin without CCDb-E2 and CCDg-E2 hybrids. Hence, microscopic and flow cytometric analyses clearly delineate that CCDb-E2 and CCDgE2 are highly efficient for estradiol-assisted selective delivery of doxorubicin inside the ER(+) cancer cells. Selective Killing of Cancer Cells. To investigate the selective killing of cancer cells by delivered doxorubicin, we studied the % killing of ER (+) MCF7, ER (−) MDA-MB-231, and noncancerous CHO cells by MTT assay. MCF7, MDA-MB231, and CHO cells were treated for 12 h with varying concentrations of CCD-E2-dox (10−100 μg/mL) at 2:1 w/w. Killing efficiency by both doxorubicin-loaded CCDb-E2 and CCDg-E2 steadily increased from 30% to higher with increasing amount of CCDb-E2-dox or CCDg-E2-dox from 5 μg/mL and above (Figure 10a). A drug-loaded nanohybrid comprising 8365

DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

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ACS Sustainable Chemistry & Engineering

Figure 9. Brightfield and fluorescence microscopic images of cells after 6 h incubation with CCDg-E2-dox, where [dox] = 5 μg/mL and [CCDg-E2] = 10 μg/mL. (a,b) MCF7 cells, (d,e) MDA-MB-231 cells, and (g,h) CHO cells. Corresponding flow cytometric plots of (c) MCF7 cells, (f) MDA-MB-231 cells, and (i) CHO cells. In all the flow cytometric plots, the x-axis denotes the doxorubicin fluorescence intensity. The mean fluorescence values are given in the insets. Scale bars correspond to 20 μm.

Figure 10. (a) % Killing of MCF7, MDA-MB-231, and CHO cells determined by MTT assay incubated with varying concentrations of CCDb-E2-dox, CCDg-E2-dox, and free dox, where [dox] = 5−50 μg/mL and [CCD-E2] = 10−100 μg/mL for 12 h. IC50 determination of (b) CCDb-E2-dox and (c) CCDg-E2-dox after 12 h incubation for MCF7, MDA-MB-231, and CHO cells. The experimental errors were in the range of 3−5% in triplicate experiments.

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DOI: 10.1021/acssuschemeng.7b02130 ACS Sustainable Chem. Eng. 2017, 5, 8356−8369

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Figure 11. Flow cytometric analysis of apoptosis in (a) untreated MCF7 cells. MCF7 cells treated with (b) free doxorubicin, (c) CCDb-E2-dox hybrid, and (d) CCDg-E2-dox hybrid for 6 h, where [dox] = 10 μg/mL and [CCD-E2] = 20 μg/mL.

negative, PI positive) shows the population of necrotic cells, Q2 (both Annexin V-FITC and PI positive) shows the population of late apoptotic cells, Q3 (Annexin V-FITC positive, PI negative) shows the population of early apoptotic cells, and Q4 (both Annexin V-FITC and PI negative) shows the population of intact cells. MCF7 cells were separately incubated with 10 μg/mL of free doxorubicin, CCDb-E2-dox, and CCDg-E2-dox comprising [CCDb-E2] = 20 μg/mL or [CCDg-E2] = 20 μg/mL and [doxorubicin] = 10 μg/mL for 6 h. The detached cells were then incubated with the AnnexinV-FITC/PI apoptotic kit and analyzed by flow cytometry. The MCF7 cells treated with free doxorubicin showed a maximum population at Q4 (both Annexin V-FITC and PI negative), almost similar to that of the control cells (Figure 11a,b). Interestingly, cells treated with CCDb-E2-dox and CCDg-E2-dox nanohybrids showed a comparatively higher population at Q2 (both Annexin V-FITC and PI positive), indicating the late apoptotic pathway of killing of MCF7 cells (Figure 11c,d). Thus, estrogen receptor-mediated cellular transportation of doxorubicin by CCDb-E2 and CCDgE2 nanohybrids selectively killed MCF7 cells through a late apoptotic pathway. This enhanced late apoptotic efficacy of both CCDb-E2-dox and CCDg-E2-dox more than that of the unbound drug is befitting for enhancing the therapeutic effect and reducing the toxicity of doxorubicin.

also investigated by MTT assay. Interestingly, for both CCDbdox and CCDg-dox having [CCD] = 100 μg/mL and [dox] = 50 μg/mL, ∼80% killing of all three cells ER(+) MCF7, ER(−) MDA-MB-231, and noncancerous CHO cells was observed (Figure S12b). This confirmed the nonselective nature of native CCDs, and noncovalent modification by E2 converted these nonselective native CCDs to target-specific CCDs with a potential of theranostic application. The IC50 (half inhibitory concentration) calculated for both CCDb-E2-dox and CCDg-E2-dox for MCF7 was 12 μg/mL and (Figure 10b,c). However, we could not measure the IC50 value for CCDb-E2-dox and CCDg-E2-dox in the case of ER(−) MDAMB-231 cells and normal cell CHO within the investigated experimental concentration of doxorubicin. On the other hand, the IC50 value could not be measured for free dox in the case of all three cells, ER(+) MCF7, ER(−) MDA-MB-231, and noncancerous CHO, within the investigated experimental concentrations of doxorubicin (Figure S12c). Thus, it can be concluded that both CCDb-E2-dox and CCDg-E2-dox hybrids are highly selective in therapeutic efficacy to estrogen receptor-rich cancer cells. Estradiol moiety in the CCD-E2 nanohybrids possibly assisted its interaction with estrogen receptors of breast cancer cells, and consequently, target-specific delivery of doxorubicin inside MCF7 cells resulted in efficient and selective killing of cancer cells. Cell Apoptosis. To investigate the killing pathway of ER+ MCF7 cells by CCDb-E2-dox and CCDg-E2-dox, an Annexin VFITC/PI-based flow cytometric assay was carried out.55 In this procedure, the scatter plot of double variable (Annexin V-FITC and PI) comprises four distinct quadrants: Q1 (Annexin V-FITC

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CONCLUSION In summary, we have developed 17β-estradiol-modified surfaceengineered carbon dots toward estrogen receptor-mediated selective staining as well as targeted delivery of the anticancer drug doxorubicin inside cancer cells. These 17β-estradiol-based 8367

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carbon nanovectors showed superior stability in the biological milieu and significant biocompatibility toward the mammalian cells. These newly developed nanohybrids selectively stained estrogen receptor-rich MCF7 cells over estrogen receptor negative MDA-MB-231 cells and normal CHO cells. Moreover, these nanohybrids were found highly efficient for uploading the anticancer drug doxorubicin. These drug-loaded nanohybrids exhibited target-specific killing of ER(+) MCF7 cells through a late apoptotic pathway by 2-fold better efficacy in contrast to ER(−) MDA-MB-231 cells and noncancerous CHO cells. Hence, these 17β-estradiol-tailored noncovalently surfacemodified carbon dots may serve as promising candidates as cancer-targeting theranostic agents.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02130. Synthetic scheme of estradiol hemisuccinate (E2), characterization of estradiol hemisuccinic acid, FTIR, NMR, XRD, XPS spectra of CCDb and CCDg, TGA plots of CCDb-E2 and CCDg-E2, suspension stability index of CCDb-E2, CCDg-E2 with varying FBS concentration in DMEM and number of days in 10% FBS-DMEM media, cell viability of CCDb-E2 and CCDg-E2 by LDH assay, live/dead fluorescence microscopic images and flow cytometric data for MCF7 and MDA-MB-231, CHO cells, spectra for determination of binding constant of CCDb-E2-dox and CCDg-E2-dox, fluorescence microscopic images and flow cytometric plots for internalization of free dox into MCF7 and MDA-MB-231, CHO cells, % killing of MCF7 and MDA-MB-231, CHO cells determined by LDH assay for CCDb-E2-dox and CCDbE2-dox, % killing of MCF7 and MDA-MB-231, CHO cells determined by MTT assay for CCDb-dox and CCDb-dox, IC50 calculation of free dox. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Prasanta Kumar Das: 0000-0002-0203-8446 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.K.D. is thankful to the Council of Scientific and Industrial Research (CSIR), India (ADD, CSC0302) for financial assistance. S.S. acknowledges DST, India, and K.D. acknowledges CSIR, India, for Research Fellowships.

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