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Doxorubicin loaded 17β-estradiol based SWNT dispersions for target specific killing of cancer cells
Colloids and Surfaces B: Biointerfaces 142 (2016) 367–376
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Doxorubicin loaded 17-estradiol based SWNT dispersions for target specific killing of cancer cells Moumita Ghosh, Prasanta Kumar Das ∗ Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
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Article history: Received 14 October 2015 Received in revised form 29 February 2016 Accepted 1 March 2016 Available online 4 March 2016 Keywords: Amphiphiles Carbon nanotube Non-covalent Dispersion Estradiol Doxorubicin Target specific delivery
a b s t r a c t The present work reports the synthesis of a 17-estradiol based amphiphiles comprising of polyethylene glycol (PEG) moiety linked through succinic acid that non-covalently dispersed (76%) the single walled carbon nanotubes (SWNTs) in water. The superior exfoliation of carbon nanotubes was characterized by microscopic and spectroscopic studies. Significant stability of these SWNT dispersions was observed in the presence of protein in cell culture media and the nanohybrids were highly biocompatible toward mammalian cells. Anticancer drug doxorubicin loaded on these nanohybrids was selectively delivered within estrogen receptor rich cancer cells, MCF7 (breast cancer cell) and A549 (lung cancer cell). Microscopic studies showed the localization of doxorubicin within the cancer cell nucleus whereas no such localization was observed in ER negative cells. Both these ER positive cancer cells were killed by ∼3 fold higher efficiency than that of ER negative MDA-MB-231 (advanced breast cancer cell) and HeLa cells that are deprived of estrogen receptors. Thus, judiciously designed estradiol based nanohybrids proved to be excellent tool for SWNT dispersion and also for selectively killing of ER positive cancer cells. To the best of our knowledge, for the first time non-covalently modified SWNTs by estradiol based amphiphilic dispersing agent have been used for selective killing of ER positive cancer cells by doxorubicin loaded on dispersed SWNTs. It holds immense promise to be exploited as a cancer therapeutic agent. © 2016 Elsevier B.V. All rights reserved.
1. Introduction “Finding the cure starts with hope” is a tagline associated with the widespread disease cancer. Over decades researchers across the world have focused in this “finding” so that maximum therapeutic activity can be achieved against this deadly disease [1–11]. ‘Target specific delivery’ is one of the challenging tasks in cancer treatment. Otherwise it is often associated with killing of normal cells, rapid aging, cardiovascular problems etc. [12]. Target specific drug delivery strategies involve the tagging of a particular ligand to conventional drug delivery vehicles like liposomes, polymeric micelles, nanogels, etc., which deliver the uploaded cargo to a specific cancer cells upon recognizing the complementary receptor of the ligand [13–18]. Most widely applicable tumor-targeting ligands are monoclonal antibodies, polysaccharides like hyaluronic acid, peptide like RGD, polyunsaturated fatty acids, and vitamins like folic acid and biotin [13–22]. To this end, estrogen receptor (ER) is a class of the nuclear hormone receptor more specifically intracellular steroid receptors,
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (P.K. Das). http://dx.doi.org/10.1016/j.colsurfb.2016.03.005 0927-7765/© 2016 Elsevier B.V. All rights reserved.
which are overexpressed mostly in estrogen responsive organs like ovary, uterus and mammary glands etc. [23–27]. 17-estradiol is well known to bind the estrogen receptors resulting in their conformational changes and simultaneous release of molecular chaperones. This causes transcription of various genes that stimulate proliferation of mammary cells leading to increase in cell division, DNA replication and mutations [28–31]. Consequently, 17-estradiol mediated targeted cancer therapy has been an interesting research domain where delivery agents like gels, liposomes, have emerged as promising vectors [23–28]. However, these vectors are often associated with compromised estrogen binding affinity due to modification in the steroid structure of 17-estradiol. As a result along with the inherent drug resistant phenomenon countered by cancer cells, the therapeutic activity of these cargo carriers also gets affected as their binding specificity is hampered. At the same time the intranuclear transport of the cargo by these delivery vehicles from the plasma membrane is not well established [28]. To this end, carbon nanotube (CNT), a cylindrical pseudo one dimensional allotrope of carbon having huge surface area has emerged as a potent intracellular as well as intranuclear cargo transporter [2,14,21,32–35]. However it is to be noted that for suitable utilization of CNTs as cargo transporters in biological milieu, the most fundamental prerequisite remains in its solubiliza-
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Fig. 1. Structure of the estradiol based amphiphiles.
tion in aqueous medium. Amphiphobic CNTs have been modified by both covalent and non-covalent methods for its exfoliation in water [2,14,32–36]. Covalent approach involves functionalization in aromatic backbone while non-covalent approach deals with the dispersion of CNTs by amphiphilic molecules. Interestingly noncovalent approach is favored as it does not perturb the surface properties of CNTs [32–36]. Previously, estradiol based covalently functionalized multiwalled carbon nanotubes have been utilized for intranuclear drug delivery [28]. However, non-covalently modified CNT surface by simple estradiol based dispersing agents has not been explored in target specific drug delivery within cancer cells. Thus developing a simple approach where the inherent properties of both CNT and steroid structure of estradiol is preserved and that would also selectively deliver cargo into cancer cells still remains a challenging task. Herein we report the synthesis of a 17-estradiol based amphiphiles comprising of polyethylene glycol (PEG) moiety linked through succinic acid (Fig. 1) that efficiently dispersed the single walled carbon nanotubes (SWNTs) in water. The nanohybrids were successfully utilized as a platform for loading anticancer drug doxorubicin (DOX). DOX-loaded nanohybrids were selectively delivered within estrogen receptor rich cancer cells, MCF7 and A549. Both these ER positive cancer cells were killed by ∼3 fold higher efficiency than that of MDA-MB-231 and HeLa cells that are deprived of estrogen receptors. To the best of our knowledge, till date selective killing of ER positive cancer cells by doxorubicin loaded non-covalently dispersed SWNTs using estradiol based amphiphile has not been reported. It holds immense potential to be used as a cancer therapeutic agent. 2. Experimental procedures 2.1. Materials Silica gel of 100–200 mesh, N,N-dicyclohexylcarbodiimide (DCC), 4-N,N-dimethylamino)pyridine (DMAP), Nhydroxybenzotriazole (HOBt), succinic anhydride, solvents and all other reagents were procured from SRL, India. Milli-Q water was used throughout the study. Thin layer chromatography was performed on Merck precoated silica gel 60-F254 plates. Doxorubicin was extracted from doxorubicin hydrochloride. 17-estradiol, poly(ethylene glycol) methyl ether (Mw = 550) and polyoxyethylene bis (amine) (Mw = 3350), CDCl3 for NMR
experiments were obtained from Aldrich Chemical Co. The single walled carbon nanotubes (SWNT, diameter 1.2–1.5 nm) were purchased from Sigma Aldrich and used as received. All materials used in the cell culture study such as Dulbecco’s Modified Eagles’ Medium (DMEM), heat inactivated fetal bovine serum (FBS), trypsin from porcine pancreas, LDH kit were obtained from Himedia and MTT was obtained from Sigma Aldrich Chemical Company. UV–vis spectra were taken in Perkin Elmer Lambda 25 spectrophotometer and the UV–vis-NIR spectra of the conjugates were monitored using Varian Cary 5000 spectrophotometer. Raman spectra was recorded using laser light (514.5 nm, scattering angle: 908, integration time: 10 s, 20 scans, 75 mW) on a Horiba Jobin Yvon instrument (Model T64000). Transmission Electron Microscopy (TEM) measurements were performed on JEOL JEM 2010 microscope. Atomic force microscopy (AFM) was performed on Veeco, model AP0100 microscope in non-contact mode. Probe sonication was done using Omni Sonic Ruptor 250. Bath sonication was performed with a Telsonic Ultrasonics bath sonicator. Sorvall RC 90 was used for ultracentrifugation. 2.2. Synthesis of the 17ˇ-estradiol based amphiphiles Estradiol hemisuccinate was synthesized following a previously reported protocol [28]. At first 17-estradiol (500 mg, 1.8 mmol) was dissolved in anhydrous toluene (20 mL). To it 5-fold molar excess of succinic anhydride (900 mg, 9.0 mmol) was added and refluxed thoroughly for 24 h in the presence of pyridine (3 mL). The entire reaction mixture was cooled to room temperature when excess succinic anhydride was precipitated out and removed by filtration. The filtrate was evaporated and concentrated under reduced pressure in a rotary evaporator. The obtained residue, estradiol disuccinate was then dissolved in methanol and stirred overnight with excess of sodium bicarbonate. Selective hydrolysis of phenolic ester was taken place in the resulted solution. The complete hydrolysis was ensured from TLC using a 1:1 (v/v) mixture of dichloromethane and methanol. Excess sodium bicarbonate was removed by filtration. Followed by water (10 mL) and diethyl ether (30 mL) was added to the reaction mixture. It was vigorously stirred and extracted with diethyl ether. The procedure was repeated three times to ensure the removal of unreacted estradiol. Then the aqueous phase was adjusted to pH 7.0 with 1 N HCl and the reaction mixture was poured into a mixture of 0.1 N HCl and crushed ice with continuous scratching with a glass rod. A white crystalline prod-
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Fig. 2. (a) Raman spectrum of SWNT-E2, (b) TEM image of dispersed SWNT-E2, (c) AFM image of dispersed SWNT-E2.
uct was obtained which was separated by filtration and washed thoroughly with water and air dried. The obtained product, estradiol hemisuccinate was crystallized from toluene under boiling conditions. The acid terminal of estradiol hemisuccinate was then coupled with either PEG550 or mono-Boc protected PEG3350 using DCC (1 equiv.) in presence of a catalytic amount of DMAP and 1 equiv. of HOBt in dry DCM. Pegylated compound 1 was then purified by column chromatography using 100–200 mesh silica gel and chloroform-methanol as the eluent. For compound 2, the Boc protected E2-PEG3350 was treated with requisite amount of trifluoroacetic acid to get the free amine (Compound). The detailed synthetic scheme is given in Supplementary scheme S1. 2.3. SWNT dispersion Compound 1 was found to be insoluble in water. Compound 2 was dissolved in water (4 mL) having a concentration of 2.5 mg/mL. SWNT (1 mg) was added to it. The mixture was subjected to probe sonication for 10 min followed by bath sonication for 2 h and again probe sonication for 10 min. The resulting suspension was centrifuged for 1.5 h at 6000 rpm and the supernatant was collected as aqueous dispersion of SWNT (SWNT-E2). The amount of the dispersed SWNT in the supernatant was calculated from the previously reported absorbance vs. concentration linear plot equation at 550 nm [32,33]. 2.4. Sample preparation for UV–vis-NIR, raman, AFM and TEM The supernatant obtained after centrifugation at 6000 rpm was used for spectroscopic and microscopic studies of the dispersed nanotubes. In case of the UV–vis-NIR and Raman spectroscopic studies a background correction was performed with the aqueous solution of the amphiphile. Raman laser wavelength was 514 nm. For AFM, a drop of the supernatant was cast on a freshly cleaved mica surface and the samples were air-dried overnight before imag-
ing. In a similar fashion, for TEM images a drop of the supernatant was placed on a 300-mesh Cu-coated TEM grid and dried under vacuum for 4 h before taking the image. The bundle sizes were measured using 5 images for SWNT-E2 dispersion. A statistical analysis of the bundle diameter was done by plotting histograms.
2.5. Media stability of the dispersion The media stability of the SWNT-E2 dispersion was tested after removal of the excess dispersing agent, which is present in addition to the optimum amount required for the stable dispersion [32,33]. This was done by ultracentrifugation of the nanotube dispersion at 45,000 rpm for 30 min. This resulted in the formation of a pellet of the mono-dispersed nano-hybrids. Water was added to the pellet, briefly probe sonicated and ultracentrifugation was performed again. The supernatant was discarded to ensure complete removal of excess surfactants. The residue was then dispersed in minimum quantity of water and added to FBS media-solution having a varying concentration range of FBS (0–75%) and the solutions were kept for 48 h. The final concentration of SWNT was 25 g/mL in all the solutions. The long time stability of SWNT-E2 was tested by addition of the nanohybrid (25 g/mL) to 10% FBS-DMEM media and the solution was kept undisturbed for 10 days. The supernatant of the solution was collected at different time intervals and the absorbance of the supernatant was recorded at 550 nm to measure the suspension stability index. SuspensionStabilityIndex(SSI) = At /A0 × 100 where At = Absorbance of the supernatant after a specific time interval at 550 nm, A0 = Initial absorbance of the solution at 550 nm. Media stability was also checked at 100 g/mL SWNT-E2 concentration in 10% FBS-DMEM media for 24 h.
Fig. 3. (a) Media stability of SWNT-E2 (25 g/mL) with increasing FBS concentration, (b) cell viability of SWNT-E2 with MDA-MB-231 and MCF 7 cells, the error bars represent (SD, n = 5).
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Fig. 4. LIVE/DEAD fluorescence microscopic images (a, b, e, f) of the cells incubated for 24 h with SWNT-E2 (a and b) MCF7 cells, (c and d) corresponding flow cytometric data for MCF7, (e and f) A549 cells, (g and h) corresponding flow cytometric data for A549 cells.
2.6. Cell culture
2.8. Modified LDH-cell viability assay
Breast cancer cells MCF7, MDA-MB-231, lung cancer cell A549 and cervical cancer cell HeLa were obtained from National Center for Cell Science (NCCS), Pune (India), and maintained in DMEM medium supplemented with 10% FBS, 100 mg/L streptomycin and 100 IU/mL penicillin. Cells were grown in a 25 mL cell culture flask and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 to approximately 70–80% confluence. A subculture was performed every 2–3 days. After 48–72 h, media was removed to eliminate the dead cells. Next, the adherent cells were detached from the surface of the culture flask by trypsinization. Cells were now in the exponential phase of growth for checking the viability of the SWNTamphiphile hybrid as well as these cells were used for checking the ability to deliver drug inside the cells as well as killing of cancer cells by SWNT-amphiphile nanohybrid.
It is being reported that the absorbance of SWNT might cause an interference with the absorbance of formazan at 570 nm in MTT study [38,39]. Thus to further verify the viability results obtained from MTT study, modified LDH assay was performed [38]. At first as depicted above all the four cell lines MCF7, A549, MDA-MB231 and HeLa cells were seeded at a density of 20,000 cells per well in a 96-well microtiter plate for 18–24 h before the assay and were incubated with SWNT-E2 dispersion (100 g/mL) for 24 h at 37 ◦ C under 5% CO2 . After 24 h the media containing SWNT-E2 and was removed leaving the alive cells adhered to the bottom of the well. Now 10 L of cell lysate solution along with buffer provided with the LDH kit was added to each well and kept for 45 min for complete cell lysis. Now this mixture was centrifuged to completely remove the SWNT-E2 and dead cells which precipitated down. The supernatant was collected having lactate dehydrogenase enzyme from originally live cells in a separate microtiter plate. This supernatant was then incubated with LDH substrate and kept in dark for 30 min after which stop solution was added to stop the kinetics. The absorbance of the solutions at 490 nm was measured using BioTek® Elisa Reader. The number of live cells were expressed as percent viability = [A490 (treated cells) − background/A490 (untreated cells) − background] × 100.
2.7. MTT assay The cell viability of the SWNT-E2 dispersion was tested by microculture MTT reduction assay as reported earlier [37]. This assay is derived from the reduction of a soluble tetrazolium salt to an insoluble colored formazan product by the mitochondrial dehydrogenase enzyme present only in live cells. The quantitative estimation of the formazan product was done spectrophotometrically after dissolution of the dye in DMSO. The enzyme activity and the amount of the formazan produced are proportional to the number of alive cells. The decrease in absorbance value is due to the killing of the cells or inhibition of the cell proliferation by the nanostructures. MCF7, A549, MDA-MB-231 and HeLa cells were seeded at a density of 20,000 cells per well in a 96-well microtiter plate for 18–24 h before the assay and were incubated with SWNT-E2 dispersion for 24 h at 37 ◦ C under 5% CO2 . Then, 10 L MTT stock solution (5 mg/mL) in phosphate buffer saline was added to the above mixture and the cells were further incubated for another 4 h. The precipitated formazan was dissolved thoroughly in DMSO and absorbance at 570 nm was measured using BioTek® Elisa Reader. The number of live cells were expressed as percent viability = [A570 (treated cells) − background/A570 (untreated cells) − background] × 100.
2.9. Cell viability determined by live dead viability kit To examine the cell viability of SWNT-amphiphile dispersion, LIVE/DEAD Viability/Cytotoxicity Kit for eukaryotic cells was used. The kit consists of a mixture of two nucleic acid binding stains, Calcein AM (aceto methoxy) (component A) and ethidium homodimer-1 (component B). The former has the ability to pass through the cell membrane while the later can only get internalized into cells with compromised cell membrane. After the transportation of Calcein AM into the cell, the esterase enzyme present only in live cells cleaves the acetoxy group. This active form of calcein intercalates with the DNA and results in bright green fluorescence. On the other hand ethidium homodimer-1 upon intercalation with DNA exhibits red fluorescence in the dead cells. Just prior to assay, the kit was thawed to room temperature and 4 L of the supplied EthD-1 stock solution (2 mM) was added to 2 mL of autoclaved, tissue culture-grade PBS buffer and the mixture was vortexed. This
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Fig. 5. Bright field and fluorescence microscopic images of cells after 6 h incubation with SWNT-E2-DOX, (a, b) MCF7 cells, (d, e) MDA-MB-231 cells (g, h) A549 cells, and (j, k) HeLa cells. Corresponding flow cytometric histogram plots of (c) MCF7 cells, (f) MDA-MB-231 cells, (i) A549 cells and (l) HeLa cells. In all the flow cytometry plots the x-axis denotes the doxorubicin fluorescence intensity. The mean fluorescence values are given in the inset.
gave an approximately 4 M EthD-1 solution. 1 L of the supplied 4 mM calcein AM stock solution was then added to the 2 mL EthD-1 solution and vortexed. The resulting mixture of calcein AM (2 M) and EthD-1 (4 M) was then added to SWNT-E2 pre-treated (for 3 h at 37 ◦ C) MCF7 and A549 cells and incubated for 30 min. The cells were subsequently observed under the Olympus IX51 inverted microscope using an excitation filter of BP460–495 nm and a band absorbance filter covering wavelength below 505 nm at 10x magnification. The bright green color resulting from the enhanced fluorescence of oligonucleotide intercalated calcein indicated the presence of live cells. The images of ethidium homodimer-1 intercalation were taken using the excitation filter BP530-550 and a
band absorbance filter covering wavelength below 570 nm, where negligible red fluorescence were observed. 2.10. Flow cytometry experiments For flow cytometry experiments MCF7, A549 cells were stained with the LIVE/DEAD kit (Calcein and Ethidium Homodimer) for 30 min after treatment with 100 g/mL of SWNT-E2 dispersion. The cells were analyzed using a flow cytometer, BD Accuri C6 operating at 488 nm excitation wavelength and emission wavelength using 533 ± 30 nm for live cells and 585 ± 40 nm bandpass filter for dead cells.
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Fig. 6. % Cell killing of ER positive and ER negative cancer cells incubated with SWNT-E2-DOX for 12 h (a) MCF 7 and MDA-MB-231 cells, (b) A549 and HeLa cells. IC50 determination of the SWNT-E2-DOX after 12 h incubation for (c) MCF 7 and MDA-MB-231 cells and (d) A549 and HeLa cells, the error bars represent (SD, n = 7).
2.11. Extraction of doxorubicin from doxorubicin hydrochloride Anticancer drug doxorubicin (DOX) was extracted from a commercially available doxorubicin hydrochloride solution using a mixture of chloroform and pH 8 Tris EDTA buffer. Doxorubicin hydrochloride (5 mg) was dissolved in 50 mL pH 8.0 Tris EDTA buffer having a composition of 0.1 M KCl, 40 mM tris(hydroxymethyl) aminomethane (Tris), 100 mM EDTA. To this solution, 100 mL of chloroform was added and the biphasic mixture was stirred vigorously for 30 min. The entire mixture was taken in a separating funnel and the red colored chloroform layer was separated and evaporated in vacuum to get pure DOX. The entire procedure was repeated 3 times to ensure complete extraction of doxorubicin from doxorubicin hydrochloride. 2.12. Doxorubicin loading The dispersed nanotubes were subsequently utilized for the loading of the DOX. DOX was loaded onto SWNT-E2 following a protocol reported earlier [13–18]. DOX was added to a dispersed solution of SWNT-E2 at pH 8.0 PBS buffer maintaining a weight ratio of 2:1 for SWNT and DOX and the mixture was stirred overnight. UV and fluorescence spectra of this hybrid material were checked with respect to control solutions having same SWNT and DOX concentration. Initially DOX (50 g mL−1 ) was mixed with SWNT-E2 (100 g mL−1 ) dispersion at pH 8.0 PBS buffer and stirred overnight. The drug-loaded nanohybrid was then centrifuged at 178,000g
for 30 min. The supernatant obtained was discarded to remove any unbound drug. The pellet was further washed with pH 8.0 buffer and centrifuged to remove unbound drug before cellular study. Finally, the pellet obtained was re-dispersed in 1(N) HCl and centrifuged again at 178,000g for 30 min. Hereby absorbance of the supernatant was measured and this process was repeated until no absorbance of DOX was obtained in the supernatant. Finally, the concentration of the drug in the supernatant was calculated (40 g mL−1 ) from the standard DOX calibration curve of absorbance vs. concentration having the equation. y = 0.00567x + 0.19183 where y denotes absorbance of the solution, and x denotes the concentration of the DOX. In a separate set of experiments 200 g mL−1 DOX was mixed with 400 g mL−1 SWNT-E2 dispersion at pH 8.0 PBS buffer and stirred overnight. The amount of the drug loaded on SWNT-E2 surface was detached completely by the procedure mentioned above in 1(N) HCl and the entire material was centrifuged to precipitate out SWNT-E2. The supernatant was collected and lyophilized to get the amount of drug loaded. It was found that 158 g was loaded on SWNT-E2 surface indicating an overall 80% loading. When the conjugates for cellular experiments were prepared, initially 120 g/mL of drug was added to a dispersed solution having 200 g/mL SWNTE2 and excess drug was removed by the process described above. The quantity of excess DOX was found to be around 20 g/mL. Thus
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100 g/mL of DOX was loaded to 200 g/mL SWNT-E2 maintaining a weight ratio of 2:1 (SWNT/drug) for all the experiments. 2.13. Imaging of cells SWNT-E2-DOX was added into 24 well chambered plates containing MCF7, A549, MDA-MB-231 and HeLa cells in 10% FBSDMEM culture media for 6 h at 37 ◦ C in 5% CO2 atmosphere. The concentration of SWNT in each well was 10 g/mL and the DOX concentration was fixed at 5 g/mL. The final volume of the solution was 300 L in each well. After 6 h of incubation, the cells were repeatedly washed with PBS buffer to ensure complete removal of the extracellular hybrids. The cells were then observed under the Olympus IX51 inverted microscope using an excitation filter BP530-550 and a band absorbance filter covering wavelength below 570 nm. Bright red images were observed when DOX was internalized inside the cells. The images were taken at 40× magnification. 2.14. Flow cytometry experiments For flow cytometry experiments, MCF7, A549, MDA-MB-231 and HeLa cells were treated with SWNT-E2-DOX for 6 h having the concentrations of 10 g/mL SWNT and 5 g/mL DOX. After 6 h of incubation, the cells were repeatedly washed with PBS buffer to ensure complete removal of the extracellular hybrids. The treated cells were then trypsinized, centrifuged and re-suspended in 500 L of PBS. The cells were analyzed using a flow cytometer, BD Accuri C6 operating at 488 nm excitation wavelength and emission wavelength using a 585 ± 40 nm (FL-2) bandpass filter for DOX. Initially, 50,000 cells were taken for seeding and the data was acquired from 10,000 cells. 2.15. MTT assay with SWNT-E2-DOX DOX loaded SWNT-E2 suspensions were added into 96 well chambered plates containing confluent MCF7, A549, MDA-MB231 and HeLa cells in 10% FBS-DMEM culture media. The SWNT and DOX concentrations were varied from 5 to 25 g mL−1 and 2.5–12.5 g mL−1 , respectively, maintaining the weight ratio of 2:1 and MTT was done by usual procedure as described above. The percentage killing of the DOX loaded nanohybrid SWNT-E2-DOX after 12 h of incubation was examined. 2.16. Modified LDH assay with SWNT-E2-DOX Modified LDH assay with SWNT-E2-DOX was performed in the same procedure as described above in Section 2.8 with all the four cell lines MCF7, A549, MDA-MB-231 and HeLa cells. The cells were incubated with fixed concentrations of SWNT-E2-DOX (25 g/mL/12.5 g/mL) for 12 h and the modified LDH assay was carried out in the same way as described above to determine% of live cells. 2.17. Nucleus staining experiment Transportation of doxorubicin inside cancer cell nucleus by SWNT-E2-DOX was further studied by nucleus staining experiments. The SWNT-E2-DOX incubated MCF7 cells were subsequently incubated with nucleus staining agent Hoechst dye (2.5 g/mL) for 30 min. The blue color originates from the intercalation of Hoechst dye with the DNA in nucleus and the red color at the same region corresponds to the already intercalated doxorubicin clearly indicates the nuclear localization of doxorubicin in cancer cells.
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3. Results and discussion 3.1. Development of SWNT dispersions by 17ˇ-estradiol-based amphiphiles Estradiol based SWNT dispersing agent (Fig. 1) was synthesized where estradiol was chosen as the hydrophobic unit while PEG moiety was attached to estradiol via a succinic acid spacer to bring in hydrophilicity to the amphiphile (Supplementary scheme S1). Estradiol would facilitate the binding of the nanohybrids with the desired specificity to its complementary ER on cancer cells. On the other hand PEG moiety is expected to impart hydrophilicity and stealth property to the dispersed nanohybrids. Initially low molecular weight PEG (Mw ∼ 550) was incorporated to the amphiphile’s structure. However, compound 1 (Fig. 1) was found to be insoluble in water. To increase the hydrophilicity of the dispersing agent, high molecular weight PEG (Mw ∼ 3350) was attached to the estradiol moiety by succinic acid spacer. As to our expectation compound 2 (Fig. 1) was found to be soluble in water. Amphiphile 2 efficiently dispersed SWNT up to 76% which was correlated with a standard calibration plot. The aqueous SWNT dispersion by estradiol based amphiphile 2 (SWNT-E2) was characterized by Raman spectroscopy (Fig. 2a). The sharp G-band arising at 1580 cm−1 in the Raman spectrum is due to the tangential C C stretching transitions in carbon nanotubes and Radial breathing mode at 100–350 cm−1 corroborates with the Raman spectra of pristine solid SWNT (Supplementary Fig. S1) [35]. Further the SWNT-E2 dispersion was characterized by UV-vis-NIR spectroscopy which showed peaks in the range of 550–900 nm (S22 transition) and 800–1600 nm (S11 transition) arising as a result of interband transitions between the mirror image spikes in the density of states of SWNTs (Supplementary Fig. S2) [32–36]. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images showed dispersed nanotubes having a diameter of around 4–5 nm and length of about 400–500 nm, respectively (Fig. 2b,c, Supplementary Fig. S3). Notably, no precipitation of carbon nanotube was observed from the SWNT-E2 dispersions for more than 7 days. 3.2. Media stability and cell viability of SWNT dispersions In order to suitably utilize the carbon nanotube dispersion of SWNT-E2 as cellular transporters, primarily it must satisfy the two most important prerequisites, i) the amphiphile-nanotube dispersion has to be stable in biological milieu over a sufficient span of time and ii) SWNT-E2 dispersion needs to be biocompatible to mammalian cells. The stability of the SWNT-E2 nanohybrid was studied in cell culture media (comprising of DMEM, salts and antibiotics) with increasing concentration of fetal bovine serum (FBS) protein up to 75%. The residue obtained after removal of the excess surfactant was then taken in minimal quantity of water and added to FBS media-solution and kept for 48 h. The final concentration of SWNT was 25 g/mL. Importantly the SWNT dispersion was found to be stable without any precipitation for more than 48 h (Fig. 3a). Furthermore for continual delivery of cargo inside the cells, circulation of the nanoconjugate within biological milieu over a sufficient span of time is also essential. Thus the long time stability of SWNT-E2 was tested by addition of the nanohybrid (25 g/mL) to 10% FBS-DMEM media and kept undisturbed for 10 days. Visual observations showed that the dispersions were stable up to 10 days in 10% FBS-media solutions. SWNT-E2 dispersion was stable up to 100 g/mL concentration in 10% FBS-media solutions for 24 h (Supplementary Fig. S4). The suspension stability index of the nanohybrid with increasing concentration of FBS as well as up to 10 days was found to be more than 90% (Supplementary Fig. S5). However pristine SWNT was found to be precipitated out from the cell culture media probably due to its insolubility in absence
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Fig. 7. (a) Internalization of doxorubicin into the nucleus of MCF7 cells upon 6 h incubation with SWNT-E2-DOX (b) MCF7 cells stained with Hoechst dye after incubation of cells for 6 h with SWNT-E2-DOX (c) superimposed image of a and b.
of dispersing agent (Supplementary Fig. S6a). Upon satisfying the first requirement, it was important to determine whether SWNT-E2 nanohybrid is sufficiently biocompatible to mammalian cells. Thus varying concentrations of dispersed SWNT-E2 (10–100 g mL−1 ) was added to both previously cultured ER positive breast cancer cells (MCF7), lung cancer cells A549 and the corresponding control (ER negative) cells, MDAMB-231 (advanced breast cancer cell) and HeLa (cervical cancer cells) cells. Interestingly the cell viability of the SWNT-E2 dispersion was found to be considerably high (80–96%) after 24 h of incubation at 37 ◦ C and 5% CO2 atmosphere toward both ER positive and ER negative cell lines (Fig. 3b, Supplementary Fig. S6b). The cell viability of all the four cell lines at 100 g mL−1 of SWNT-E2 was further confirmed from LDH assay which showed ∼85–95% cells were viable 24 h after incubation (Supplementary Fig. S7). To confirm the cytocompatiblity of nanohybrids toward mammalian cells, both MCF7 and A549 cells were incubated with 100 g mL−1 of SWNT-E2 for 24 h. The cells were then treated with the LIVE/DEAD viability kit and observed under fluorescence microscope (Fig. 4). Fluorescence images showed predominant presence of green cells and absence of red cells in both MCF7 and A549 cells. Further these samples were analyzed by flow cytometry studies where significant presence of green fluorescence and absence of red stained cells indicated sufficient cell viability of the nanohybrid (Fig. 4). Thus, SWNTs dispersed by estradiol based dispersing agent have no detrimental effect on mammalian cells and can be suitably utilized for cargo delivery across the cell membrane.
3.3. Drug loading on the dispersed SWNT and quantification of loaded DOX on SWNT surface Accordingly anticancer drug doxorubicin (DOX) was loaded on the aqueous SWNT-E2 dispersion. DOX was loaded at pH 8.0 in a weight ratio of 2:1 (SWNT:DOX) where 50 g of DOX was used for loading on 100 g of SWNT-E2 so that the solubility of DOX in water could be minimized and its adsorption on dispersed SWNT surface could be maximized [40]. A red shifting (∼10 nm) in the UV-absorbance peak of DOX at 500 nm in SWNT-E2 dispersion indicated that the drug was loaded on dispersed nanotubes (Supplementary Fig. S8a). Moreover the drastic quenching in the intrinsic fluorescence of DOX at 590 nm (ex = 480 nm) further validated the loading of the DOX on SWNT surface (Supplementary Fig. S8b) [40,41]. The SWNT backbone of the nanovector allowed the binding of the drug possibly through - interaction between the aromatic backbone and anthracyclic moiety of DOX. The amount of loaded DOX was found to be 40 g on 100 g of SWNT-E2 indicating around 80% loading of drug on nanotube surface. Importantly,
DOX loaded SWNT-E2 dispersion was stable at pH 8.0 and precipitation of CNT or revival of DOX fluorescence in the solution was not observed even after 48 h. DOX was chosen as cargo because of its intrinsic fluorescent character which would help in its imaging inside the cells as well as due to its ability to kill the cancerous cells through its known mechanism upon intercalation with DNA.
3.4. Selective internalization of DOX within cancer cells Herein the drug loaded SWNT-E2 was utilized for the estradiol induced target specific delivery of the DOX to ER positive breast cancer cell, MCF7. SWNT-E2-DOX hybrid having 10 g mL−1 of SWNT-E2 and 5 g mL−1 of DOX, respectively was incubated with MCF7 cells for 6 h. The incubation was followed by washing of the cells with PBS buffer in order to remove the excess nanohybrid from the medium. Internalization of DOX inside MCF7 cells is evident from the red fluorescence in the corresponding fluorescence microscopic images of the cells (Fig. 5a,b). Thus SWNT-E2 nanohybrid is proficient in delivering cargo inside ER positive cells. Now to address the selectivity of SWNT-E2 nanohybrid, it is important to verify that whether it can deliver the cargo into ER negative cells. Hence the same experiment was carried out using ER negative cancer cell, MDA-MB-231. Encouragingly, no detectable internalization of DOX was observed in the corresponding fluorescence microscopic image (Fig. 5d,e). Estradiol based selective delivery of drug inside ER positive cancer cells by SWNT dispersion was further verified using another estrogen receptor rich cancer cell, A549. Here too, the respective fluorescence microscopic image confirmed the successful delivery of DOX inside the ER positive A549 cancer cells (Fig. 5g,h). In another control experiment, we used cancerous HeLa cell which is devoid of any overexpressed estrogen receptors. However, no prominent internalization of DOX was observed inside this cell upon incubation with SWNT-E2-DOX nanohybrid (Fig. 5j,k). Target specific internalization of DOX within ER positive cancer cells was also monitored by flow cytometric analysis. The corresponding flow cytometric analysis of MCF7 and A549 cells showed high fluorescence intensity in the order of 104 –105 having mean fluorescence of 62907 and 47577, respectively (Fig. 5c,l, Supplementary Fig. S9a, S10a). This confirms high population of fluorophore DOX inside the ER positive cancer cells upon successful internalization [40,41]. It is being well reported that the fluorescence intensity of DOX increases when it intercalates with DNA within nucleus of cells [10]. In case of ER negative cancer cells, the corresponding flow cytometric plots showed markedly lower fluorescence intensity in the order of 103 –104 having mean fluorescence of 2770 for MDAMB-231 cells and the intensity was further lowered to 102 –103 with mean fluorescence of 1112 for HeLa cells
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(Fig. 5c, i, Supplementary Fig. S9b, S10b). Hence It is obvious that in case of MDA-MB-231 and HeLa cells notable delivery of DOX was not observed by SWNT-E2 dispersion due to lack of estrogen receptors. Thus, both fluorescence microscopic images and the enhanced fluorescence intensity in flow cytometric analysis for MCF7 and A549 cells clearly delineate the estradiol assisted target specific delivery of DOX inside these ER positive cancer cells by SWNT-E2 dispersion. 3.5. Selective killing of cancer cells To investigate whether this target specific internalization of DOX resulted in selective killing of estrogen receptor rich cancer cells, we studied the% killing of all the four cell lines by MTT assay. MCF7, A549, MDA-MB-231 and HeLa cells were incubated with varying concentration of SWNT-E2-DOX hybrid having SWNT (5–25 g mL−1 ) and DOX (2.5–12.5 g mL−1 ), respectively maintaining the weight ratio of 2:1 for 12 h. The killing of ER positive cells steadily increased with increasing concentration of SWNT-E2DOX hybrid (Fig. 6a,b). Encouragingly, at [SWNT] = 25 g mL−1 and [DOX] = 12.5 g mL−1 , 76 ± 3% MCF7 cells and 66 ± 4% A549 cells were killed by SWNT-E2-DOX hybrid. In contrast, with the same concentration of SWNT and DOX, only 22 ± 5% of MDA-MB-231 cells and 24 ± 5% of HeLa cells were killed after 12 h (Fig. 6a,b). The killing results were further confirmed from the LDH assay at fixed SWNT-E2-DOX concentration having 25 g mL−1 of SWNT-E2 and 12.5 g mL−1 of DOX in all the four cell line, MCF7, A549, MDA-MB231 and HeLa after 12 h incubation. LDH assay showed 78 ± 3% and 74 ± 2% killing of ER positive MCF7 and A549 cells compared to only 23 ± 2% of MDA-MB-231 cells and 29 ± 2% of HeLa cells which are ER negative (Supplementary Fig. S11). Importantly SWNT-E2 and DOX alone was not efficient enough to kill the cancer cells (∼4–6% cell killing with 25 g mL−1 SWNT-E2 without DOX and ∼30-35% cell killing with free DOX without SWNT-E2 after 12 h incubation) (Supplementary Fig. S12 and Fig. S13). Therefore SWNT-E2-DOX showed ∼3 fold higher killing efficiency specifically toward ER positive cancer cells compared to that of ER negative cancer cells. The absence of estradiol receptor in MDA-MB-231 and HeLa cells made the SWNTE2-DOX hybrid inefficient to deliver the drug inside these cells leading to the poor killing of ER negative cancer cells. Thus it can be inferred that SWNT-E2-DOX are selective enough to deliver drug to estradiol receptors rich cancer cells. The presence of estradiol based amphiphile in SWNT-E2-DOX hybrid facilitated its binding with the counter receptor in MCF7 and A549 cells resulting in the selective killing of MCF7 cells and A549 upon intercalation of the anticancer drug DOX with DNA [10]. The IC50 (Half Inhibitory Concentration) values obtained for SWNT-E2-DOX for MCF7 and A549 cells were 9 g mL−1 and 11 g mL−1 , respectively (Fig. 6c,d). However, IC50 value could not be measured for SWNT-E2-DOX in the ER negative cells within the mentioned experimental concentration of DOX (Fig. 6c,d). Also the IC50 values of only DOX and SWNT-E2 in MCF7, MDA-MB-231, A549 and Hela cell lines were not measurable due to the similar reason as described above (Supplementary Fig. S12 and S13). 3.6. Imaging of DOX within cells DOX intercalation within the ER positive cell nucleus is further verified from nucleus staining experiment [33,34]. After a pretreatment of MCF7 cells with SWNT-E2-DOX for 6 h the cells were subsequently incubated with nucleus staining agent Hoechst dye (2.5 g mL−1 ) for 30 min. The red colored region (Fig. 7a) corresponds to the already intercalated DOX delivered by SWNT-E2. Hoechst dye is known to intercalate with nuclear DNA thereby originating bright blue color (Fig. 7b) [33]. The simultaneous coexistence of blue and red colour (Fig. 7c) at the same region
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(nucleus) confirms the successful internalization of DOX within the nucleus of MCF7 cells. 4. Conclusion In conclusion, 17-estradiol based amphiphiles have been synthesized to prepare non-covalent dispersion of single walled carbon nanotubes, which is stable and biocompatible. Estradiol moiety was exploited for specific binding of the nanovector to the estrogen receptors rich cancer cells, MCF7 and A549. Anticancer drug DOX loaded on this receptor specific estradiol fabricated SWNTs was successfully delivered within MCF7 and A549 cells and resulted in target specific killing of ER positive cancer cells compared to that of ER negative cells like MDA-MB-231 and HeLa. Such ‘target specific’ nanovector can be utilized in future to develop effective delivery platforms in biomedicine. Acknowledgements P.K.D. is thankful to CSIR, India (ADD, CSC0302) for financial assistance. M.G. acknowledges CSIR, India for Research fellowships. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.03. 005. References [1] K.D. Fowers, J. Kopecek, Macromol. Biosci. 12 (2012) 502. [2] A.A. Bhirde, V. Patel, J. Gavard, G. Zhang, A.A. Sousa, A. Masedunskas, R.D. Leapman, R. Weigert, J.S. Gutkind, J.F. Rusling, ACS Nano 3 (2009) 307. [3] R. Malhotra, V. Patel, B.V. Chikkaveeraiah, B.S. Munge, S.C. Cheong, R.B. Zain, M.T. Abraham, D.K. Dey, J.S. Gutkind, J.F. Rusling, Anal. Chem. 84 (2012) 6249. [4] R.I. Pakunlu, Y. Wang, M. Saad, J.J. Khandare, V. Starovoytov, T. Minko, J. Control. Release 114 (2006) 153. [5] R. Qi, H. Xiao, S. Wu, Y. Li, Y. Zhang, X. Jing, J. Mater. Chem. B 3 (2015) 176. [6] H. Xiao, G.T. Noble, J.F. Stefanick, R. Qi, T. Kiziltepe, X. Jing, B. Bilgicer, J. Control. Release 173 (2014) 11. [7] Y. Zheng, Y. Wen, A.M. George, A.M. Steinbach, B.E. Phillips, N. Giannoukakis, E.S. Gawalt, W.S. Meng, Biomaterials 32 (2011) 249. [8] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Control. Release 65 (2000) 271. [9] K. Raemdonck, J. Demeester, S.D. Smedt, Soft Matter 5 (2009) 707. [10] V. Bagalkot, O.C. Farokhzad, R. Langer, S. Jon, Angew. Chem. Int. Ed. 45 (2006) 8149. [11] S. Nazir, T. Hussain, A. Ayub, U. Rashid, A.J. MacRobert, Nanomedicine 10 (2014) 19. [12] H. Maeda, H. Nakamura, J. Fang, Adv. Drug Deliv. Rev. 65 (2013) 71. [13] Z.L. Cheng, A.A. Zaki, J.Z. Hui, V.R. Muzykantov, A. Tsourkas, Science 338 (2012) 903. [14] Z. Liu, X. Sun, N.N. Ratchford, H. Dai, ACS Nano 1 (2007) 50. [15] X. Zhang, L. Meng, Q. Lu, Z. Fei, P.J. Dyson, Biomaterials 30 (2009) 6041. [16] J. Chen, S. Chen, X. Zhao, L.V. Kuznetsova, S.S. Wong, I. Ojima, J. Am. Chem. Soc. 130 (2008) 16778. [17] V. Bagalkot, O.C. Farokhzad, R. Langer, S. Jon, Angew. Chem. Int. Ed. 45 (2006) 8149. [18] L. Meng, X. Zhang, Q. Lu, Z. Fei, P.J. Dyson, Biomaterials 33 (2012) 1689. [19] S. Dhar, Z. Liu, J. Thomale, H. Dai, S.J. Lippard, J. Am. Chem. Soc. 130 (2008) 11467. [20] R.P. Feazell, N.N. Ratchford, H. Dai, S.J. Lippard, J. Am. Chem. Soc. 129 (2007) 8438. [21] S. Chen, X. Zhao, J. Chen, J. Chen, L. Kuznetsova, S.S. Wong, I. Ojima, Bioconjug. Chem. 21 (2010) 979. [22] C. Zhang, S. Gao, W. Jiang, S. Lin, F. Du, Z. Li, W. Huang, Biomaterials 31 (2010) 6075. [23] R. Schiff, S. Massarweh, J. Shou, C.K. Osborne, Clin. Cancer Res. 9 (2003) 447s. [24] J. Kurebayashi, T. Otsuki, H. Kunisue, K. Tanaka, S. Yamamoto, H. Sonoo, Clin. Cancer Res. 6 (2000) 512. [25] K.B. Horwitz, W. McGuire, J. Biol. Chem. 253 (1978) 2223. [26] B.S. Reddy, R. Banerjee, Angew. Chem. Int. Ed. 44 (2005) 6881. [27] K.L. Dao, R.N. Hanson, Bioconjug. Chem. 23 (2012) 2139. [28] M. Das, R.P. Singh, S.R. Datir, S. Jain, Mol. Pharm. 10 (2013) 3404. [29] S. Rai, R. Paliwal, B. Vaidya, P.N. Gupta, S. Mahor, K. Khatri, A.K. Goyal, A. Rawat, S. Vyas, Curr. Med. Chem. 14 (2007) 2095.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Doxorubicin loaded 17-estradiol based SWNT dispersions for target specific killing of cancer cells Moumita Ghosh, Prasanta Kumar Das ∗ Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
a r t i c l e
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Article history: Received 14 October 2015 Received in revised form 29 February 2016 Accepted 1 March 2016 Available online 4 March 2016 Keywords: Amphiphiles Carbon nanotube Non-covalent Dispersion Estradiol Doxorubicin Target specific delivery
a b s t r a c t The present work reports the synthesis of a 17-estradiol based amphiphiles comprising of polyethylene glycol (PEG) moiety linked through succinic acid that non-covalently dispersed (76%) the single walled carbon nanotubes (SWNTs) in water. The superior exfoliation of carbon nanotubes was characterized by microscopic and spectroscopic studies. Significant stability of these SWNT dispersions was observed in the presence of protein in cell culture media and the nanohybrids were highly biocompatible toward mammalian cells. Anticancer drug doxorubicin loaded on these nanohybrids was selectively delivered within estrogen receptor rich cancer cells, MCF7 (breast cancer cell) and A549 (lung cancer cell). Microscopic studies showed the localization of doxorubicin within the cancer cell nucleus whereas no such localization was observed in ER negative cells. Both these ER positive cancer cells were killed by ∼3 fold higher efficiency than that of ER negative MDA-MB-231 (advanced breast cancer cell) and HeLa cells that are deprived of estrogen receptors. Thus, judiciously designed estradiol based nanohybrids proved to be excellent tool for SWNT dispersion and also for selectively killing of ER positive cancer cells. To the best of our knowledge, for the first time non-covalently modified SWNTs by estradiol based amphiphilic dispersing agent have been used for selective killing of ER positive cancer cells by doxorubicin loaded on dispersed SWNTs. It holds immense promise to be exploited as a cancer therapeutic agent. © 2016 Elsevier B.V. All rights reserved.
1. Introduction “Finding the cure starts with hope” is a tagline associated with the widespread disease cancer. Over decades researchers across the world have focused in this “finding” so that maximum therapeutic activity can be achieved against this deadly disease [1–11]. ‘Target specific delivery’ is one of the challenging tasks in cancer treatment. Otherwise it is often associated with killing of normal cells, rapid aging, cardiovascular problems etc. [12]. Target specific drug delivery strategies involve the tagging of a particular ligand to conventional drug delivery vehicles like liposomes, polymeric micelles, nanogels, etc., which deliver the uploaded cargo to a specific cancer cells upon recognizing the complementary receptor of the ligand [13–18]. Most widely applicable tumor-targeting ligands are monoclonal antibodies, polysaccharides like hyaluronic acid, peptide like RGD, polyunsaturated fatty acids, and vitamins like folic acid and biotin [13–22]. To this end, estrogen receptor (ER) is a class of the nuclear hormone receptor more specifically intracellular steroid receptors,
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (P.K. Das). http://dx.doi.org/10.1016/j.colsurfb.2016.03.005 0927-7765/© 2016 Elsevier B.V. All rights reserved.
which are overexpressed mostly in estrogen responsive organs like ovary, uterus and mammary glands etc. [23–27]. 17-estradiol is well known to bind the estrogen receptors resulting in their conformational changes and simultaneous release of molecular chaperones. This causes transcription of various genes that stimulate proliferation of mammary cells leading to increase in cell division, DNA replication and mutations [28–31]. Consequently, 17-estradiol mediated targeted cancer therapy has been an interesting research domain where delivery agents like gels, liposomes, have emerged as promising vectors [23–28]. However, these vectors are often associated with compromised estrogen binding affinity due to modification in the steroid structure of 17-estradiol. As a result along with the inherent drug resistant phenomenon countered by cancer cells, the therapeutic activity of these cargo carriers also gets affected as their binding specificity is hampered. At the same time the intranuclear transport of the cargo by these delivery vehicles from the plasma membrane is not well established [28]. To this end, carbon nanotube (CNT), a cylindrical pseudo one dimensional allotrope of carbon having huge surface area has emerged as a potent intracellular as well as intranuclear cargo transporter [2,14,21,32–35]. However it is to be noted that for suitable utilization of CNTs as cargo transporters in biological milieu, the most fundamental prerequisite remains in its solubiliza-
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Fig. 1. Structure of the estradiol based amphiphiles.
tion in aqueous medium. Amphiphobic CNTs have been modified by both covalent and non-covalent methods for its exfoliation in water [2,14,32–36]. Covalent approach involves functionalization in aromatic backbone while non-covalent approach deals with the dispersion of CNTs by amphiphilic molecules. Interestingly noncovalent approach is favored as it does not perturb the surface properties of CNTs [32–36]. Previously, estradiol based covalently functionalized multiwalled carbon nanotubes have been utilized for intranuclear drug delivery [28]. However, non-covalently modified CNT surface by simple estradiol based dispersing agents has not been explored in target specific drug delivery within cancer cells. Thus developing a simple approach where the inherent properties of both CNT and steroid structure of estradiol is preserved and that would also selectively deliver cargo into cancer cells still remains a challenging task. Herein we report the synthesis of a 17-estradiol based amphiphiles comprising of polyethylene glycol (PEG) moiety linked through succinic acid (Fig. 1) that efficiently dispersed the single walled carbon nanotubes (SWNTs) in water. The nanohybrids were successfully utilized as a platform for loading anticancer drug doxorubicin (DOX). DOX-loaded nanohybrids were selectively delivered within estrogen receptor rich cancer cells, MCF7 and A549. Both these ER positive cancer cells were killed by ∼3 fold higher efficiency than that of MDA-MB-231 and HeLa cells that are deprived of estrogen receptors. To the best of our knowledge, till date selective killing of ER positive cancer cells by doxorubicin loaded non-covalently dispersed SWNTs using estradiol based amphiphile has not been reported. It holds immense potential to be used as a cancer therapeutic agent. 2. Experimental procedures 2.1. Materials Silica gel of 100–200 mesh, N,N-dicyclohexylcarbodiimide (DCC), 4-N,N-dimethylamino)pyridine (DMAP), Nhydroxybenzotriazole (HOBt), succinic anhydride, solvents and all other reagents were procured from SRL, India. Milli-Q water was used throughout the study. Thin layer chromatography was performed on Merck precoated silica gel 60-F254 plates. Doxorubicin was extracted from doxorubicin hydrochloride. 17-estradiol, poly(ethylene glycol) methyl ether (Mw = 550) and polyoxyethylene bis (amine) (Mw = 3350), CDCl3 for NMR
experiments were obtained from Aldrich Chemical Co. The single walled carbon nanotubes (SWNT, diameter 1.2–1.5 nm) were purchased from Sigma Aldrich and used as received. All materials used in the cell culture study such as Dulbecco’s Modified Eagles’ Medium (DMEM), heat inactivated fetal bovine serum (FBS), trypsin from porcine pancreas, LDH kit were obtained from Himedia and MTT was obtained from Sigma Aldrich Chemical Company. UV–vis spectra were taken in Perkin Elmer Lambda 25 spectrophotometer and the UV–vis-NIR spectra of the conjugates were monitored using Varian Cary 5000 spectrophotometer. Raman spectra was recorded using laser light (514.5 nm, scattering angle: 908, integration time: 10 s, 20 scans, 75 mW) on a Horiba Jobin Yvon instrument (Model T64000). Transmission Electron Microscopy (TEM) measurements were performed on JEOL JEM 2010 microscope. Atomic force microscopy (AFM) was performed on Veeco, model AP0100 microscope in non-contact mode. Probe sonication was done using Omni Sonic Ruptor 250. Bath sonication was performed with a Telsonic Ultrasonics bath sonicator. Sorvall RC 90 was used for ultracentrifugation. 2.2. Synthesis of the 17ˇ-estradiol based amphiphiles Estradiol hemisuccinate was synthesized following a previously reported protocol [28]. At first 17-estradiol (500 mg, 1.8 mmol) was dissolved in anhydrous toluene (20 mL). To it 5-fold molar excess of succinic anhydride (900 mg, 9.0 mmol) was added and refluxed thoroughly for 24 h in the presence of pyridine (3 mL). The entire reaction mixture was cooled to room temperature when excess succinic anhydride was precipitated out and removed by filtration. The filtrate was evaporated and concentrated under reduced pressure in a rotary evaporator. The obtained residue, estradiol disuccinate was then dissolved in methanol and stirred overnight with excess of sodium bicarbonate. Selective hydrolysis of phenolic ester was taken place in the resulted solution. The complete hydrolysis was ensured from TLC using a 1:1 (v/v) mixture of dichloromethane and methanol. Excess sodium bicarbonate was removed by filtration. Followed by water (10 mL) and diethyl ether (30 mL) was added to the reaction mixture. It was vigorously stirred and extracted with diethyl ether. The procedure was repeated three times to ensure the removal of unreacted estradiol. Then the aqueous phase was adjusted to pH 7.0 with 1 N HCl and the reaction mixture was poured into a mixture of 0.1 N HCl and crushed ice with continuous scratching with a glass rod. A white crystalline prod-
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Fig. 2. (a) Raman spectrum of SWNT-E2, (b) TEM image of dispersed SWNT-E2, (c) AFM image of dispersed SWNT-E2.
uct was obtained which was separated by filtration and washed thoroughly with water and air dried. The obtained product, estradiol hemisuccinate was crystallized from toluene under boiling conditions. The acid terminal of estradiol hemisuccinate was then coupled with either PEG550 or mono-Boc protected PEG3350 using DCC (1 equiv.) in presence of a catalytic amount of DMAP and 1 equiv. of HOBt in dry DCM. Pegylated compound 1 was then purified by column chromatography using 100–200 mesh silica gel and chloroform-methanol as the eluent. For compound 2, the Boc protected E2-PEG3350 was treated with requisite amount of trifluoroacetic acid to get the free amine (Compound). The detailed synthetic scheme is given in Supplementary scheme S1. 2.3. SWNT dispersion Compound 1 was found to be insoluble in water. Compound 2 was dissolved in water (4 mL) having a concentration of 2.5 mg/mL. SWNT (1 mg) was added to it. The mixture was subjected to probe sonication for 10 min followed by bath sonication for 2 h and again probe sonication for 10 min. The resulting suspension was centrifuged for 1.5 h at 6000 rpm and the supernatant was collected as aqueous dispersion of SWNT (SWNT-E2). The amount of the dispersed SWNT in the supernatant was calculated from the previously reported absorbance vs. concentration linear plot equation at 550 nm [32,33]. 2.4. Sample preparation for UV–vis-NIR, raman, AFM and TEM The supernatant obtained after centrifugation at 6000 rpm was used for spectroscopic and microscopic studies of the dispersed nanotubes. In case of the UV–vis-NIR and Raman spectroscopic studies a background correction was performed with the aqueous solution of the amphiphile. Raman laser wavelength was 514 nm. For AFM, a drop of the supernatant was cast on a freshly cleaved mica surface and the samples were air-dried overnight before imag-
ing. In a similar fashion, for TEM images a drop of the supernatant was placed on a 300-mesh Cu-coated TEM grid and dried under vacuum for 4 h before taking the image. The bundle sizes were measured using 5 images for SWNT-E2 dispersion. A statistical analysis of the bundle diameter was done by plotting histograms.
2.5. Media stability of the dispersion The media stability of the SWNT-E2 dispersion was tested after removal of the excess dispersing agent, which is present in addition to the optimum amount required for the stable dispersion [32,33]. This was done by ultracentrifugation of the nanotube dispersion at 45,000 rpm for 30 min. This resulted in the formation of a pellet of the mono-dispersed nano-hybrids. Water was added to the pellet, briefly probe sonicated and ultracentrifugation was performed again. The supernatant was discarded to ensure complete removal of excess surfactants. The residue was then dispersed in minimum quantity of water and added to FBS media-solution having a varying concentration range of FBS (0–75%) and the solutions were kept for 48 h. The final concentration of SWNT was 25 g/mL in all the solutions. The long time stability of SWNT-E2 was tested by addition of the nanohybrid (25 g/mL) to 10% FBS-DMEM media and the solution was kept undisturbed for 10 days. The supernatant of the solution was collected at different time intervals and the absorbance of the supernatant was recorded at 550 nm to measure the suspension stability index. SuspensionStabilityIndex(SSI) = At /A0 × 100 where At = Absorbance of the supernatant after a specific time interval at 550 nm, A0 = Initial absorbance of the solution at 550 nm. Media stability was also checked at 100 g/mL SWNT-E2 concentration in 10% FBS-DMEM media for 24 h.
Fig. 3. (a) Media stability of SWNT-E2 (25 g/mL) with increasing FBS concentration, (b) cell viability of SWNT-E2 with MDA-MB-231 and MCF 7 cells, the error bars represent (SD, n = 5).
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Fig. 4. LIVE/DEAD fluorescence microscopic images (a, b, e, f) of the cells incubated for 24 h with SWNT-E2 (a and b) MCF7 cells, (c and d) corresponding flow cytometric data for MCF7, (e and f) A549 cells, (g and h) corresponding flow cytometric data for A549 cells.
2.6. Cell culture
2.8. Modified LDH-cell viability assay
Breast cancer cells MCF7, MDA-MB-231, lung cancer cell A549 and cervical cancer cell HeLa were obtained from National Center for Cell Science (NCCS), Pune (India), and maintained in DMEM medium supplemented with 10% FBS, 100 mg/L streptomycin and 100 IU/mL penicillin. Cells were grown in a 25 mL cell culture flask and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 to approximately 70–80% confluence. A subculture was performed every 2–3 days. After 48–72 h, media was removed to eliminate the dead cells. Next, the adherent cells were detached from the surface of the culture flask by trypsinization. Cells were now in the exponential phase of growth for checking the viability of the SWNTamphiphile hybrid as well as these cells were used for checking the ability to deliver drug inside the cells as well as killing of cancer cells by SWNT-amphiphile nanohybrid.
It is being reported that the absorbance of SWNT might cause an interference with the absorbance of formazan at 570 nm in MTT study [38,39]. Thus to further verify the viability results obtained from MTT study, modified LDH assay was performed [38]. At first as depicted above all the four cell lines MCF7, A549, MDA-MB231 and HeLa cells were seeded at a density of 20,000 cells per well in a 96-well microtiter plate for 18–24 h before the assay and were incubated with SWNT-E2 dispersion (100 g/mL) for 24 h at 37 ◦ C under 5% CO2 . After 24 h the media containing SWNT-E2 and was removed leaving the alive cells adhered to the bottom of the well. Now 10 L of cell lysate solution along with buffer provided with the LDH kit was added to each well and kept for 45 min for complete cell lysis. Now this mixture was centrifuged to completely remove the SWNT-E2 and dead cells which precipitated down. The supernatant was collected having lactate dehydrogenase enzyme from originally live cells in a separate microtiter plate. This supernatant was then incubated with LDH substrate and kept in dark for 30 min after which stop solution was added to stop the kinetics. The absorbance of the solutions at 490 nm was measured using BioTek® Elisa Reader. The number of live cells were expressed as percent viability = [A490 (treated cells) − background/A490 (untreated cells) − background] × 100.
2.7. MTT assay The cell viability of the SWNT-E2 dispersion was tested by microculture MTT reduction assay as reported earlier [37]. This assay is derived from the reduction of a soluble tetrazolium salt to an insoluble colored formazan product by the mitochondrial dehydrogenase enzyme present only in live cells. The quantitative estimation of the formazan product was done spectrophotometrically after dissolution of the dye in DMSO. The enzyme activity and the amount of the formazan produced are proportional to the number of alive cells. The decrease in absorbance value is due to the killing of the cells or inhibition of the cell proliferation by the nanostructures. MCF7, A549, MDA-MB-231 and HeLa cells were seeded at a density of 20,000 cells per well in a 96-well microtiter plate for 18–24 h before the assay and were incubated with SWNT-E2 dispersion for 24 h at 37 ◦ C under 5% CO2 . Then, 10 L MTT stock solution (5 mg/mL) in phosphate buffer saline was added to the above mixture and the cells were further incubated for another 4 h. The precipitated formazan was dissolved thoroughly in DMSO and absorbance at 570 nm was measured using BioTek® Elisa Reader. The number of live cells were expressed as percent viability = [A570 (treated cells) − background/A570 (untreated cells) − background] × 100.
2.9. Cell viability determined by live dead viability kit To examine the cell viability of SWNT-amphiphile dispersion, LIVE/DEAD Viability/Cytotoxicity Kit for eukaryotic cells was used. The kit consists of a mixture of two nucleic acid binding stains, Calcein AM (aceto methoxy) (component A) and ethidium homodimer-1 (component B). The former has the ability to pass through the cell membrane while the later can only get internalized into cells with compromised cell membrane. After the transportation of Calcein AM into the cell, the esterase enzyme present only in live cells cleaves the acetoxy group. This active form of calcein intercalates with the DNA and results in bright green fluorescence. On the other hand ethidium homodimer-1 upon intercalation with DNA exhibits red fluorescence in the dead cells. Just prior to assay, the kit was thawed to room temperature and 4 L of the supplied EthD-1 stock solution (2 mM) was added to 2 mL of autoclaved, tissue culture-grade PBS buffer and the mixture was vortexed. This
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Fig. 5. Bright field and fluorescence microscopic images of cells after 6 h incubation with SWNT-E2-DOX, (a, b) MCF7 cells, (d, e) MDA-MB-231 cells (g, h) A549 cells, and (j, k) HeLa cells. Corresponding flow cytometric histogram plots of (c) MCF7 cells, (f) MDA-MB-231 cells, (i) A549 cells and (l) HeLa cells. In all the flow cytometry plots the x-axis denotes the doxorubicin fluorescence intensity. The mean fluorescence values are given in the inset.
gave an approximately 4 M EthD-1 solution. 1 L of the supplied 4 mM calcein AM stock solution was then added to the 2 mL EthD-1 solution and vortexed. The resulting mixture of calcein AM (2 M) and EthD-1 (4 M) was then added to SWNT-E2 pre-treated (for 3 h at 37 ◦ C) MCF7 and A549 cells and incubated for 30 min. The cells were subsequently observed under the Olympus IX51 inverted microscope using an excitation filter of BP460–495 nm and a band absorbance filter covering wavelength below 505 nm at 10x magnification. The bright green color resulting from the enhanced fluorescence of oligonucleotide intercalated calcein indicated the presence of live cells. The images of ethidium homodimer-1 intercalation were taken using the excitation filter BP530-550 and a
band absorbance filter covering wavelength below 570 nm, where negligible red fluorescence were observed. 2.10. Flow cytometry experiments For flow cytometry experiments MCF7, A549 cells were stained with the LIVE/DEAD kit (Calcein and Ethidium Homodimer) for 30 min after treatment with 100 g/mL of SWNT-E2 dispersion. The cells were analyzed using a flow cytometer, BD Accuri C6 operating at 488 nm excitation wavelength and emission wavelength using 533 ± 30 nm for live cells and 585 ± 40 nm bandpass filter for dead cells.
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Fig. 6. % Cell killing of ER positive and ER negative cancer cells incubated with SWNT-E2-DOX for 12 h (a) MCF 7 and MDA-MB-231 cells, (b) A549 and HeLa cells. IC50 determination of the SWNT-E2-DOX after 12 h incubation for (c) MCF 7 and MDA-MB-231 cells and (d) A549 and HeLa cells, the error bars represent (SD, n = 7).
2.11. Extraction of doxorubicin from doxorubicin hydrochloride Anticancer drug doxorubicin (DOX) was extracted from a commercially available doxorubicin hydrochloride solution using a mixture of chloroform and pH 8 Tris EDTA buffer. Doxorubicin hydrochloride (5 mg) was dissolved in 50 mL pH 8.0 Tris EDTA buffer having a composition of 0.1 M KCl, 40 mM tris(hydroxymethyl) aminomethane (Tris), 100 mM EDTA. To this solution, 100 mL of chloroform was added and the biphasic mixture was stirred vigorously for 30 min. The entire mixture was taken in a separating funnel and the red colored chloroform layer was separated and evaporated in vacuum to get pure DOX. The entire procedure was repeated 3 times to ensure complete extraction of doxorubicin from doxorubicin hydrochloride. 2.12. Doxorubicin loading The dispersed nanotubes were subsequently utilized for the loading of the DOX. DOX was loaded onto SWNT-E2 following a protocol reported earlier [13–18]. DOX was added to a dispersed solution of SWNT-E2 at pH 8.0 PBS buffer maintaining a weight ratio of 2:1 for SWNT and DOX and the mixture was stirred overnight. UV and fluorescence spectra of this hybrid material were checked with respect to control solutions having same SWNT and DOX concentration. Initially DOX (50 g mL−1 ) was mixed with SWNT-E2 (100 g mL−1 ) dispersion at pH 8.0 PBS buffer and stirred overnight. The drug-loaded nanohybrid was then centrifuged at 178,000g
for 30 min. The supernatant obtained was discarded to remove any unbound drug. The pellet was further washed with pH 8.0 buffer and centrifuged to remove unbound drug before cellular study. Finally, the pellet obtained was re-dispersed in 1(N) HCl and centrifuged again at 178,000g for 30 min. Hereby absorbance of the supernatant was measured and this process was repeated until no absorbance of DOX was obtained in the supernatant. Finally, the concentration of the drug in the supernatant was calculated (40 g mL−1 ) from the standard DOX calibration curve of absorbance vs. concentration having the equation. y = 0.00567x + 0.19183 where y denotes absorbance of the solution, and x denotes the concentration of the DOX. In a separate set of experiments 200 g mL−1 DOX was mixed with 400 g mL−1 SWNT-E2 dispersion at pH 8.0 PBS buffer and stirred overnight. The amount of the drug loaded on SWNT-E2 surface was detached completely by the procedure mentioned above in 1(N) HCl and the entire material was centrifuged to precipitate out SWNT-E2. The supernatant was collected and lyophilized to get the amount of drug loaded. It was found that 158 g was loaded on SWNT-E2 surface indicating an overall 80% loading. When the conjugates for cellular experiments were prepared, initially 120 g/mL of drug was added to a dispersed solution having 200 g/mL SWNTE2 and excess drug was removed by the process described above. The quantity of excess DOX was found to be around 20 g/mL. Thus
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100 g/mL of DOX was loaded to 200 g/mL SWNT-E2 maintaining a weight ratio of 2:1 (SWNT/drug) for all the experiments. 2.13. Imaging of cells SWNT-E2-DOX was added into 24 well chambered plates containing MCF7, A549, MDA-MB-231 and HeLa cells in 10% FBSDMEM culture media for 6 h at 37 ◦ C in 5% CO2 atmosphere. The concentration of SWNT in each well was 10 g/mL and the DOX concentration was fixed at 5 g/mL. The final volume of the solution was 300 L in each well. After 6 h of incubation, the cells were repeatedly washed with PBS buffer to ensure complete removal of the extracellular hybrids. The cells were then observed under the Olympus IX51 inverted microscope using an excitation filter BP530-550 and a band absorbance filter covering wavelength below 570 nm. Bright red images were observed when DOX was internalized inside the cells. The images were taken at 40× magnification. 2.14. Flow cytometry experiments For flow cytometry experiments, MCF7, A549, MDA-MB-231 and HeLa cells were treated with SWNT-E2-DOX for 6 h having the concentrations of 10 g/mL SWNT and 5 g/mL DOX. After 6 h of incubation, the cells were repeatedly washed with PBS buffer to ensure complete removal of the extracellular hybrids. The treated cells were then trypsinized, centrifuged and re-suspended in 500 L of PBS. The cells were analyzed using a flow cytometer, BD Accuri C6 operating at 488 nm excitation wavelength and emission wavelength using a 585 ± 40 nm (FL-2) bandpass filter for DOX. Initially, 50,000 cells were taken for seeding and the data was acquired from 10,000 cells. 2.15. MTT assay with SWNT-E2-DOX DOX loaded SWNT-E2 suspensions were added into 96 well chambered plates containing confluent MCF7, A549, MDA-MB231 and HeLa cells in 10% FBS-DMEM culture media. The SWNT and DOX concentrations were varied from 5 to 25 g mL−1 and 2.5–12.5 g mL−1 , respectively, maintaining the weight ratio of 2:1 and MTT was done by usual procedure as described above. The percentage killing of the DOX loaded nanohybrid SWNT-E2-DOX after 12 h of incubation was examined. 2.16. Modified LDH assay with SWNT-E2-DOX Modified LDH assay with SWNT-E2-DOX was performed in the same procedure as described above in Section 2.8 with all the four cell lines MCF7, A549, MDA-MB-231 and HeLa cells. The cells were incubated with fixed concentrations of SWNT-E2-DOX (25 g/mL/12.5 g/mL) for 12 h and the modified LDH assay was carried out in the same way as described above to determine% of live cells. 2.17. Nucleus staining experiment Transportation of doxorubicin inside cancer cell nucleus by SWNT-E2-DOX was further studied by nucleus staining experiments. The SWNT-E2-DOX incubated MCF7 cells were subsequently incubated with nucleus staining agent Hoechst dye (2.5 g/mL) for 30 min. The blue color originates from the intercalation of Hoechst dye with the DNA in nucleus and the red color at the same region corresponds to the already intercalated doxorubicin clearly indicates the nuclear localization of doxorubicin in cancer cells.
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3. Results and discussion 3.1. Development of SWNT dispersions by 17ˇ-estradiol-based amphiphiles Estradiol based SWNT dispersing agent (Fig. 1) was synthesized where estradiol was chosen as the hydrophobic unit while PEG moiety was attached to estradiol via a succinic acid spacer to bring in hydrophilicity to the amphiphile (Supplementary scheme S1). Estradiol would facilitate the binding of the nanohybrids with the desired specificity to its complementary ER on cancer cells. On the other hand PEG moiety is expected to impart hydrophilicity and stealth property to the dispersed nanohybrids. Initially low molecular weight PEG (Mw ∼ 550) was incorporated to the amphiphile’s structure. However, compound 1 (Fig. 1) was found to be insoluble in water. To increase the hydrophilicity of the dispersing agent, high molecular weight PEG (Mw ∼ 3350) was attached to the estradiol moiety by succinic acid spacer. As to our expectation compound 2 (Fig. 1) was found to be soluble in water. Amphiphile 2 efficiently dispersed SWNT up to 76% which was correlated with a standard calibration plot. The aqueous SWNT dispersion by estradiol based amphiphile 2 (SWNT-E2) was characterized by Raman spectroscopy (Fig. 2a). The sharp G-band arising at 1580 cm−1 in the Raman spectrum is due to the tangential C C stretching transitions in carbon nanotubes and Radial breathing mode at 100–350 cm−1 corroborates with the Raman spectra of pristine solid SWNT (Supplementary Fig. S1) [35]. Further the SWNT-E2 dispersion was characterized by UV-vis-NIR spectroscopy which showed peaks in the range of 550–900 nm (S22 transition) and 800–1600 nm (S11 transition) arising as a result of interband transitions between the mirror image spikes in the density of states of SWNTs (Supplementary Fig. S2) [32–36]. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images showed dispersed nanotubes having a diameter of around 4–5 nm and length of about 400–500 nm, respectively (Fig. 2b,c, Supplementary Fig. S3). Notably, no precipitation of carbon nanotube was observed from the SWNT-E2 dispersions for more than 7 days. 3.2. Media stability and cell viability of SWNT dispersions In order to suitably utilize the carbon nanotube dispersion of SWNT-E2 as cellular transporters, primarily it must satisfy the two most important prerequisites, i) the amphiphile-nanotube dispersion has to be stable in biological milieu over a sufficient span of time and ii) SWNT-E2 dispersion needs to be biocompatible to mammalian cells. The stability of the SWNT-E2 nanohybrid was studied in cell culture media (comprising of DMEM, salts and antibiotics) with increasing concentration of fetal bovine serum (FBS) protein up to 75%. The residue obtained after removal of the excess surfactant was then taken in minimal quantity of water and added to FBS media-solution and kept for 48 h. The final concentration of SWNT was 25 g/mL. Importantly the SWNT dispersion was found to be stable without any precipitation for more than 48 h (Fig. 3a). Furthermore for continual delivery of cargo inside the cells, circulation of the nanoconjugate within biological milieu over a sufficient span of time is also essential. Thus the long time stability of SWNT-E2 was tested by addition of the nanohybrid (25 g/mL) to 10% FBS-DMEM media and kept undisturbed for 10 days. Visual observations showed that the dispersions were stable up to 10 days in 10% FBS-media solutions. SWNT-E2 dispersion was stable up to 100 g/mL concentration in 10% FBS-media solutions for 24 h (Supplementary Fig. S4). The suspension stability index of the nanohybrid with increasing concentration of FBS as well as up to 10 days was found to be more than 90% (Supplementary Fig. S5). However pristine SWNT was found to be precipitated out from the cell culture media probably due to its insolubility in absence
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Fig. 7. (a) Internalization of doxorubicin into the nucleus of MCF7 cells upon 6 h incubation with SWNT-E2-DOX (b) MCF7 cells stained with Hoechst dye after incubation of cells for 6 h with SWNT-E2-DOX (c) superimposed image of a and b.
of dispersing agent (Supplementary Fig. S6a). Upon satisfying the first requirement, it was important to determine whether SWNT-E2 nanohybrid is sufficiently biocompatible to mammalian cells. Thus varying concentrations of dispersed SWNT-E2 (10–100 g mL−1 ) was added to both previously cultured ER positive breast cancer cells (MCF7), lung cancer cells A549 and the corresponding control (ER negative) cells, MDAMB-231 (advanced breast cancer cell) and HeLa (cervical cancer cells) cells. Interestingly the cell viability of the SWNT-E2 dispersion was found to be considerably high (80–96%) after 24 h of incubation at 37 ◦ C and 5% CO2 atmosphere toward both ER positive and ER negative cell lines (Fig. 3b, Supplementary Fig. S6b). The cell viability of all the four cell lines at 100 g mL−1 of SWNT-E2 was further confirmed from LDH assay which showed ∼85–95% cells were viable 24 h after incubation (Supplementary Fig. S7). To confirm the cytocompatiblity of nanohybrids toward mammalian cells, both MCF7 and A549 cells were incubated with 100 g mL−1 of SWNT-E2 for 24 h. The cells were then treated with the LIVE/DEAD viability kit and observed under fluorescence microscope (Fig. 4). Fluorescence images showed predominant presence of green cells and absence of red cells in both MCF7 and A549 cells. Further these samples were analyzed by flow cytometry studies where significant presence of green fluorescence and absence of red stained cells indicated sufficient cell viability of the nanohybrid (Fig. 4). Thus, SWNTs dispersed by estradiol based dispersing agent have no detrimental effect on mammalian cells and can be suitably utilized for cargo delivery across the cell membrane.
3.3. Drug loading on the dispersed SWNT and quantification of loaded DOX on SWNT surface Accordingly anticancer drug doxorubicin (DOX) was loaded on the aqueous SWNT-E2 dispersion. DOX was loaded at pH 8.0 in a weight ratio of 2:1 (SWNT:DOX) where 50 g of DOX was used for loading on 100 g of SWNT-E2 so that the solubility of DOX in water could be minimized and its adsorption on dispersed SWNT surface could be maximized [40]. A red shifting (∼10 nm) in the UV-absorbance peak of DOX at 500 nm in SWNT-E2 dispersion indicated that the drug was loaded on dispersed nanotubes (Supplementary Fig. S8a). Moreover the drastic quenching in the intrinsic fluorescence of DOX at 590 nm (ex = 480 nm) further validated the loading of the DOX on SWNT surface (Supplementary Fig. S8b) [40,41]. The SWNT backbone of the nanovector allowed the binding of the drug possibly through - interaction between the aromatic backbone and anthracyclic moiety of DOX. The amount of loaded DOX was found to be 40 g on 100 g of SWNT-E2 indicating around 80% loading of drug on nanotube surface. Importantly,
DOX loaded SWNT-E2 dispersion was stable at pH 8.0 and precipitation of CNT or revival of DOX fluorescence in the solution was not observed even after 48 h. DOX was chosen as cargo because of its intrinsic fluorescent character which would help in its imaging inside the cells as well as due to its ability to kill the cancerous cells through its known mechanism upon intercalation with DNA.
3.4. Selective internalization of DOX within cancer cells Herein the drug loaded SWNT-E2 was utilized for the estradiol induced target specific delivery of the DOX to ER positive breast cancer cell, MCF7. SWNT-E2-DOX hybrid having 10 g mL−1 of SWNT-E2 and 5 g mL−1 of DOX, respectively was incubated with MCF7 cells for 6 h. The incubation was followed by washing of the cells with PBS buffer in order to remove the excess nanohybrid from the medium. Internalization of DOX inside MCF7 cells is evident from the red fluorescence in the corresponding fluorescence microscopic images of the cells (Fig. 5a,b). Thus SWNT-E2 nanohybrid is proficient in delivering cargo inside ER positive cells. Now to address the selectivity of SWNT-E2 nanohybrid, it is important to verify that whether it can deliver the cargo into ER negative cells. Hence the same experiment was carried out using ER negative cancer cell, MDA-MB-231. Encouragingly, no detectable internalization of DOX was observed in the corresponding fluorescence microscopic image (Fig. 5d,e). Estradiol based selective delivery of drug inside ER positive cancer cells by SWNT dispersion was further verified using another estrogen receptor rich cancer cell, A549. Here too, the respective fluorescence microscopic image confirmed the successful delivery of DOX inside the ER positive A549 cancer cells (Fig. 5g,h). In another control experiment, we used cancerous HeLa cell which is devoid of any overexpressed estrogen receptors. However, no prominent internalization of DOX was observed inside this cell upon incubation with SWNT-E2-DOX nanohybrid (Fig. 5j,k). Target specific internalization of DOX within ER positive cancer cells was also monitored by flow cytometric analysis. The corresponding flow cytometric analysis of MCF7 and A549 cells showed high fluorescence intensity in the order of 104 –105 having mean fluorescence of 62907 and 47577, respectively (Fig. 5c,l, Supplementary Fig. S9a, S10a). This confirms high population of fluorophore DOX inside the ER positive cancer cells upon successful internalization [40,41]. It is being well reported that the fluorescence intensity of DOX increases when it intercalates with DNA within nucleus of cells [10]. In case of ER negative cancer cells, the corresponding flow cytometric plots showed markedly lower fluorescence intensity in the order of 103 –104 having mean fluorescence of 2770 for MDAMB-231 cells and the intensity was further lowered to 102 –103 with mean fluorescence of 1112 for HeLa cells
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(Fig. 5c, i, Supplementary Fig. S9b, S10b). Hence It is obvious that in case of MDA-MB-231 and HeLa cells notable delivery of DOX was not observed by SWNT-E2 dispersion due to lack of estrogen receptors. Thus, both fluorescence microscopic images and the enhanced fluorescence intensity in flow cytometric analysis for MCF7 and A549 cells clearly delineate the estradiol assisted target specific delivery of DOX inside these ER positive cancer cells by SWNT-E2 dispersion. 3.5. Selective killing of cancer cells To investigate whether this target specific internalization of DOX resulted in selective killing of estrogen receptor rich cancer cells, we studied the% killing of all the four cell lines by MTT assay. MCF7, A549, MDA-MB-231 and HeLa cells were incubated with varying concentration of SWNT-E2-DOX hybrid having SWNT (5–25 g mL−1 ) and DOX (2.5–12.5 g mL−1 ), respectively maintaining the weight ratio of 2:1 for 12 h. The killing of ER positive cells steadily increased with increasing concentration of SWNT-E2DOX hybrid (Fig. 6a,b). Encouragingly, at [SWNT] = 25 g mL−1 and [DOX] = 12.5 g mL−1 , 76 ± 3% MCF7 cells and 66 ± 4% A549 cells were killed by SWNT-E2-DOX hybrid. In contrast, with the same concentration of SWNT and DOX, only 22 ± 5% of MDA-MB-231 cells and 24 ± 5% of HeLa cells were killed after 12 h (Fig. 6a,b). The killing results were further confirmed from the LDH assay at fixed SWNT-E2-DOX concentration having 25 g mL−1 of SWNT-E2 and 12.5 g mL−1 of DOX in all the four cell line, MCF7, A549, MDA-MB231 and HeLa after 12 h incubation. LDH assay showed 78 ± 3% and 74 ± 2% killing of ER positive MCF7 and A549 cells compared to only 23 ± 2% of MDA-MB-231 cells and 29 ± 2% of HeLa cells which are ER negative (Supplementary Fig. S11). Importantly SWNT-E2 and DOX alone was not efficient enough to kill the cancer cells (∼4–6% cell killing with 25 g mL−1 SWNT-E2 without DOX and ∼30-35% cell killing with free DOX without SWNT-E2 after 12 h incubation) (Supplementary Fig. S12 and Fig. S13). Therefore SWNT-E2-DOX showed ∼3 fold higher killing efficiency specifically toward ER positive cancer cells compared to that of ER negative cancer cells. The absence of estradiol receptor in MDA-MB-231 and HeLa cells made the SWNTE2-DOX hybrid inefficient to deliver the drug inside these cells leading to the poor killing of ER negative cancer cells. Thus it can be inferred that SWNT-E2-DOX are selective enough to deliver drug to estradiol receptors rich cancer cells. The presence of estradiol based amphiphile in SWNT-E2-DOX hybrid facilitated its binding with the counter receptor in MCF7 and A549 cells resulting in the selective killing of MCF7 cells and A549 upon intercalation of the anticancer drug DOX with DNA [10]. The IC50 (Half Inhibitory Concentration) values obtained for SWNT-E2-DOX for MCF7 and A549 cells were 9 g mL−1 and 11 g mL−1 , respectively (Fig. 6c,d). However, IC50 value could not be measured for SWNT-E2-DOX in the ER negative cells within the mentioned experimental concentration of DOX (Fig. 6c,d). Also the IC50 values of only DOX and SWNT-E2 in MCF7, MDA-MB-231, A549 and Hela cell lines were not measurable due to the similar reason as described above (Supplementary Fig. S12 and S13). 3.6. Imaging of DOX within cells DOX intercalation within the ER positive cell nucleus is further verified from nucleus staining experiment [33,34]. After a pretreatment of MCF7 cells with SWNT-E2-DOX for 6 h the cells were subsequently incubated with nucleus staining agent Hoechst dye (2.5 g mL−1 ) for 30 min. The red colored region (Fig. 7a) corresponds to the already intercalated DOX delivered by SWNT-E2. Hoechst dye is known to intercalate with nuclear DNA thereby originating bright blue color (Fig. 7b) [33]. The simultaneous coexistence of blue and red colour (Fig. 7c) at the same region
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(nucleus) confirms the successful internalization of DOX within the nucleus of MCF7 cells. 4. Conclusion In conclusion, 17-estradiol based amphiphiles have been synthesized to prepare non-covalent dispersion of single walled carbon nanotubes, which is stable and biocompatible. Estradiol moiety was exploited for specific binding of the nanovector to the estrogen receptors rich cancer cells, MCF7 and A549. Anticancer drug DOX loaded on this receptor specific estradiol fabricated SWNTs was successfully delivered within MCF7 and A549 cells and resulted in target specific killing of ER positive cancer cells compared to that of ER negative cells like MDA-MB-231 and HeLa. Such ‘target specific’ nanovector can be utilized in future to develop effective delivery platforms in biomedicine. Acknowledgements P.K.D. is thankful to CSIR, India (ADD, CSC0302) for financial assistance. M.G. acknowledges CSIR, India for Research fellowships. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.03. 005. References [1] K.D. Fowers, J. Kopecek, Macromol. Biosci. 12 (2012) 502. [2] A.A. Bhirde, V. Patel, J. Gavard, G. Zhang, A.A. Sousa, A. Masedunskas, R.D. Leapman, R. Weigert, J.S. Gutkind, J.F. Rusling, ACS Nano 3 (2009) 307. [3] R. Malhotra, V. Patel, B.V. Chikkaveeraiah, B.S. Munge, S.C. Cheong, R.B. Zain, M.T. Abraham, D.K. Dey, J.S. Gutkind, J.F. Rusling, Anal. Chem. 84 (2012) 6249. [4] R.I. Pakunlu, Y. Wang, M. Saad, J.J. Khandare, V. Starovoytov, T. Minko, J. Control. Release 114 (2006) 153. [5] R. Qi, H. Xiao, S. Wu, Y. Li, Y. Zhang, X. Jing, J. Mater. Chem. B 3 (2015) 176. [6] H. Xiao, G.T. Noble, J.F. Stefanick, R. Qi, T. Kiziltepe, X. Jing, B. Bilgicer, J. Control. Release 173 (2014) 11. [7] Y. Zheng, Y. Wen, A.M. George, A.M. Steinbach, B.E. Phillips, N. Giannoukakis, E.S. Gawalt, W.S. Meng, Biomaterials 32 (2011) 249. [8] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Control. Release 65 (2000) 271. [9] K. Raemdonck, J. Demeester, S.D. Smedt, Soft Matter 5 (2009) 707. [10] V. Bagalkot, O.C. Farokhzad, R. Langer, S. Jon, Angew. Chem. Int. Ed. 45 (2006) 8149. [11] S. Nazir, T. Hussain, A. Ayub, U. Rashid, A.J. MacRobert, Nanomedicine 10 (2014) 19. [12] H. Maeda, H. Nakamura, J. Fang, Adv. Drug Deliv. Rev. 65 (2013) 71. [13] Z.L. Cheng, A.A. Zaki, J.Z. Hui, V.R. Muzykantov, A. Tsourkas, Science 338 (2012) 903. [14] Z. Liu, X. Sun, N.N. Ratchford, H. Dai, ACS Nano 1 (2007) 50. [15] X. Zhang, L. Meng, Q. Lu, Z. Fei, P.J. Dyson, Biomaterials 30 (2009) 6041. [16] J. Chen, S. Chen, X. Zhao, L.V. Kuznetsova, S.S. Wong, I. Ojima, J. Am. Chem. Soc. 130 (2008) 16778. [17] V. Bagalkot, O.C. Farokhzad, R. Langer, S. Jon, Angew. Chem. Int. Ed. 45 (2006) 8149. [18] L. Meng, X. Zhang, Q. Lu, Z. Fei, P.J. Dyson, Biomaterials 33 (2012) 1689. [19] S. Dhar, Z. Liu, J. Thomale, H. Dai, S.J. Lippard, J. Am. Chem. Soc. 130 (2008) 11467. [20] R.P. Feazell, N.N. Ratchford, H. Dai, S.J. Lippard, J. Am. Chem. Soc. 129 (2007) 8438. [21] S. Chen, X. Zhao, J. Chen, J. Chen, L. Kuznetsova, S.S. Wong, I. Ojima, Bioconjug. Chem. 21 (2010) 979. [22] C. Zhang, S. Gao, W. Jiang, S. Lin, F. Du, Z. Li, W. Huang, Biomaterials 31 (2010) 6075. [23] R. Schiff, S. Massarweh, J. Shou, C.K. Osborne, Clin. Cancer Res. 9 (2003) 447s. [24] J. Kurebayashi, T. Otsuki, H. Kunisue, K. Tanaka, S. Yamamoto, H. Sonoo, Clin. Cancer Res. 6 (2000) 512. [25] K.B. Horwitz, W. McGuire, J. Biol. Chem. 253 (1978) 2223. [26] B.S. Reddy, R. Banerjee, Angew. Chem. Int. Ed. 44 (2005) 6881. [27] K.L. Dao, R.N. Hanson, Bioconjug. Chem. 23 (2012) 2139. [28] M. Das, R.P. Singh, S.R. Datir, S. Jain, Mol. Pharm. 10 (2013) 3404. [29] S. Rai, R. Paliwal, B. Vaidya, P.N. Gupta, S. Mahor, K. Khatri, A.K. Goyal, A. Rawat, S. Vyas, Curr. Med. Chem. 14 (2007) 2095.
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