Inhibition of Thioredoxin Reductase by Targeted Selenopolymeric

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Inhibition of Thioredoxin Reductase by Targeted Selenopolymeric Nanocarriers Synergizes the Therapeutic Efficacy of Doxorubicin in MCF7 Human Breast Cancer Cells Mahaveer Prasad Purohit, Neeraj Kumar Verma, Aditya Kumar Kar, Amrita Singh, Debabrata Ghosh, and Satyakam Patnaik ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07056 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 25, 2017

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ACS Applied Materials & Interfaces

Inhibition of Thioredoxin Reductase by Targeted Selenopolymeric Nanocarriers Synergizes the Therapeutic Efficacy of Doxorubicin in MCF7 Human Breast Cancer Cells Mahaveer P. Purohit1, 2, Neeraj K. Verma1, 3, Aditya K. Kar1, 2, Amrita Singh1, Debabrata Ghosh2, 4 and Satyakam Patnaik1, 2*

1

Water Analysis Laboratory, Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian

Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow-226001, Uttar Pradesh, India 2

Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Toxicology

Research Campus, Lucknow-226001, Uttar Pradesh, India 3

BBD University, School of Dental Sciences, Faizabad Road, Lucknow-226028, Uttar Pradesh,

India 4

Immunotoxicology laboratory, Food Drug and Chemical Toxicology Group, CSIR-Indian

Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, 31, Mahatma Gandhi Marg, Lucknow-226001, Uttar Pradesh, India.

*

Author for correspondence: +918960420042

E-mail address: [email protected], [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT Increasing evidences suggest Selenium nanoparticles (Se NPs) as potential cancer therapeutic agents and emerging drug delivery carriers, yet, the molecular mechanism of their anticancer activity still remains unclear. Recent studies indicate, Thioredoxin Reductase (TrxR), a selenoenzyme, as a promising target for anticancer therapy. The present study explored the TrxR inhibition efficacy of Se NPs as a plausible factor impeding tumor growth. Hyaluronic acid (HA) functionalized selenopolymeric nanocarriers (Se@CMHA NPs) were designed wielding chemotherapeutic potential for target specific Doxorubicin (DOX) delivery. Se@CMHA nanocarriers are thoroughly characterized asserting their chemical and physical integrity and possess prolonged stability. DOX loaded selenopolymeric nanocarriers (Se@CMHA-DOX NPs) exhibited enhanced cytotoxic potential towards human cancer cells compared to free DOX in an equivalent concentration eliciting selectivity towards cancer cells. In first of its kind findings, selenium as Se NPs in these polymeric carriers progressively inhibit TrxR activity, further augmenting the anticancer efficacy of DOX through a synergistic interplay between DOX and Se NPs. Detailed molecular studies on MCF7 cells also established that upon exposure to Se@CMHA-DOX NPs, MCF7 cells endure G2/M cell cycle arrest and p53 mediated caspase independent apoptosis. To gauge the relevance of the developed nanosystem in in vivo settings, 3D tumor sphere model mimicking the overall tumor environment was also carried out and the results clearly depict the effectiveness of our nanocarriers in reducing tumor activity. These findings are reminiscent of the fact that our Se@CMHA-DOX NPs could be a viable modality for effective cancer chemotherapy. KEYWORDS: Selenopolymeric nanocarriers; Drug delivery; Cancer therapy; Thioredoxin reductase; Doxorubicin; Apoptosis 2 ACS Paragon Plus Environment

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1. INTRODUCTION Cancer is one of the top killer diseases all over the world accounting for almost 8.2 million deaths in 2012 as per World Cancer Report 2014 by WHO1. Although, the mortality and morbidity rate reduced because of successive improvement in traditional therapies and better diagnostic technology, yet the challenge to develop newer strategies to combat various cancers is wide open. Use of nanotherapeutics in cancer prevention is a paradigm shift advancement wherein the shortcomings of conventional chemotherapy such as lack of aqueous solubility, poor pharmacokinetics, inadequate specificity etc. are circumvented by multifaceted approach2. In recent years a lot of efforts concerted on developing novel nanosystems, having a multipronged therapeutic approach. These systems embodied more than one biologically and/or therapeutically active materials which synergistically enhance the efficacy of the payload. In this context of combinatorial therapy, Selenium (Se), owing to its excellent biological property, particularly the effects related to the immune response and cancer prevention activity has garnered attentions of researchers over the past few years towards the development of de novo anticancer drug delivery systems3-4. Micronutrient Se, as a constituent of selenoproteins, plays an essential structural, enzymatic and catalytic role in human physiology with several distinct health benefits5. Reports based on evidence from epidemiologic, clinical, and experimental studies clearly support that the trace element, Se, in its various organic and inorganic forms and dosages appears to have chemopreventive,

chemotherapeutic

and

chemoprotective

properties6-8.

These

reports

substantiate the fact that, seleno-compounds are highly effective amplifier of efficacy and selectivity of chemotherapeutic drugs and therefore preferred as supplements/adjuvant in cancer chemotherapy. However, its clinical application is fettered due to the low therapeutic index9.



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Recent studies have revealed that Se at nanoformulation exhibited comparable efficacy as Selenomethionine (SeM) and Methylselenocysteine (MSC) in upregulating selenoenzymes and tissue Se levels, but was less toxic10-11.The reduced toxicological profile of Se NPs makes it useful as a promising candidate in designing newer cancer therapeutic agents. For instance, Luo et al. reported that Se NPs inhibit the growth of human cervical cancer cells, HeLa and human breast cancer cells, MDA-MB-231 through induction of S phase cell cycle arrest12. Jiang et al. reported Gracilaria lemaneiformis polysaccharide decorated Se NPs demonstrated strong cytotoxicity toward glioblastoma by inhibiting αvβ3 integrin in a targeted fashion13. Similarly, a large number of prior art elucidated that Se NPs in various formulations are able to cause cancer cell death14-16. Cyclic peptide-capped Se NPs reported improving the antiproliferative activities of several anticancer drugs doxorubicin (DOX), gemcitabine, clofarabine, etoposide, camptothecin, irinotecan, epirubicin, fludarabine, dasatinib, and paclitaxel in human ovarian adenocarcinoma (SK-OV-3) cells17. In a recent report, SiO2-coated ultra-small Se particles (Se@SiO2 nanospheres) was used as a nanoplatforms for delivery of DOX towards treatment of cancer wherein the Se and drug together exert an enhanced activity through a synergistic mode18. Additionally, Liu et al demonstrated Se NPs as potential drug carrier enhancing the therapeutic ability of anticancer drug 5-fluorouracil through a synergistic action19. Similarly, Huang et al. used transferrin conjugated Se NPs to show the enhanced cellular uptake and anticancer efficacy of DOX in MCF7 cells20. In a more recent report, Curcumin potentiates the chemotherapeutic efficacy of Se NPs by a combinatorial effect, compared to their bulk counterparts21. However, molecular mechanisms behind the anticancer activity of Se NPs and its synergistic interplay with chemotherapeutic drugs are yet to be appreciated. One of the plausible modes of action is presumably by regulation of Thioredoxin reductase (TrxR), an antioxidant selenoenzyme22.


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Thioredoxin system, comprising thioredoxin (Trx) and thioredoxin reductase (TrxR) along with NADPH, is one of the major thiol dependent electron donor systems that play a critical role in various cellular activities including cell proliferation, protection against oxidative stress, cellular redox control, and apoptosis23. Mammalian TrxR is a Se containing oxidoreductase which catalyzes the NADPH dependent reduction of oxidized Trx and various other substrates, thereby maintaining the intracellular redox balance24. Se as selenocysteine unit is present at the active site of all the three mammalian TrxR isoenzymes; TrxR1 (cytosol), TrxR2 (mitochondrial) and TGR (testis) and essential for their catalytic activity25. Recently, TrxR was discovered as a potential molecular target for cancer therapy, owing to it’s over expression in various cancer cells26. The high expression of this redox enzyme is correlated to neoplasticity, development of drug resistant phenotype and consequently providing a protective effect against induced cell death by chemotherapeutic agents. Many reports suggested that TrxR inhibition impairs the Trx redox system leading to alteration in various intracellular signaling cascades, mitochondrial associated factors, transcriptional factors, tumor suppressor genes and heightened oxidative stress, resulting loss of cellular function and ultimately cell death27. Emerging experimental evidences suggested that Se compounds exert their cancer preventive activities by inhibiting the TrxR activity via formation of more stable di-selenide bond in essential selenocysteine unit at the conserved active site of the protein28. On the basis of the above findings, we postulate, Se at the nanoscale has the potential to inhibit TrxR activity and thereby synergizing the efficacy of anticancer drugs. In this study, Hyaluronic acid (HA) functionalized tumor targeted seleno-polymeric nanoparticles were developed and explored their application as potential nano carriers for the anticancer drug, DOX delivery. Glycosaminoglycan HA, a natural polysaccharide and a ligand



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for CD44 receptors29-30 was opted as targeting moiety owing to the fact that nearly all cancerous cells like pancreatic, breast and lung cancer etc., have over expressions of CD4431. Experiments were conducted to assess the therapeutic potential of DOX in the free form compared to that of when delivered through our designed selenopolymeric nano carriers in breast cancer MCF7 cell line. The results obtained confirmed synergism between the anticancer effect of Se NPs present within seleno-polymeric nanocarriers and DOX. We further tried to deduce the molecular basis of our observations and interestingly, we found that Se NP’s role in the enhanced therapeutic activity of DOX is modulated by means of inhibiting TrxR. Moreover, to the best of our knowledge, this study is first of its kind substantiating this under explored mechanism of action of Se NPs, i.e. as a potential TrxR inhibitor. The findings of this study will further aid in understanding Se NP's chemoprevention properties against cancer and Se@CMHA-DOX NPs could be a viable modality for effective cancer chemotherapy. 2. EXPERIMENTAL SECTION 2.1. Materials. All chemicals and reagents were of highest purity grade. Sodium selenite (Na2SeO3), Ascorbic acid, Poly(ethylene glycol) bisamine (Mw 2000 kDa), Triton-X-100, Doxorubicin Hydrochloride, Hyaluronic acid sodium salt (HA, MW 250 kDa; from rooster comb),

Cetyl

alcohol,

1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide

Hydroxysuccinimide (NHS), Dialysis bag (12000 MW.CO), Coumarin 6,

(EDC),

N-

3-(4, 5-di

methylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), 2’, 7’-dichlorofluorescein diacetate (DCFDA), RPMI, Phosphate buffer saline, recombinant rat TrxR, human Trx1, 5,5′-Dithiobis (2nitrobenzoic acid) (DTNB), guanidine hydrochloride, protease inhibitor cocktail, glycerol, propidium iodide, 10X Western blocking buffer and QuantiProTM BCA Assay Kit for protein determination were purchased from Sigma-Aldrich. AnnexinV/PI apoptosis kit was obtained


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from BD Pharmingen (BD Biosciences, San Diego, CA, USA). Fetal Bovine Serum (FBS) procured from cell clone. Trypsin-EDTA (0.25%), antifade mounting medium, Alexa Fluor antibodies and Cascade Biologics Medium 171 (M171) and Mammary Epithelial Growth Supplements (MEGS) for tumor sphere formation were obtained from Life Technologies. PVDF membrane and chemiluminescence substrate were obtained from Merck-Millipore (USA). Other primary and secondary antibodies used in the present study were purchased from Santa Cruze. All other chemicals used in the study were of AR or GR grade available in India. 2.2. Synthesis of Native Se NPs and amine functionalized Se NPs (Se@NH2 NPs). Preparation of amine functionalized Se NPs (Se-NH-[PEG]n-NH2), here onwards written as Se@NH2, were accomplished as follows. As a preliminary step towards the synthesis of Se@NH2 NPs, stock solutions of sodium selenite (Na2SeO3, 5 mM) and ascorbic acid (vitamin C, 20 mM) were prepared by dissolving 8.7 mg of Na2SeO3 and 35.2 mg of Vitamin C in 10 mL of Milli-Q water individually. To a 5 mL aliquot of Na2SeO3 stock solution was added 10 mL of 1 mg/mL PEG-bisamine solution (final PEG concentration 0.05%) and left to stir for 1 h to obtain a homogeneous mixture. Subsequently, 5 mL of ascorbic acid stock solution was added drop wise to the above solution to initiate NP's nucleation. The formation of Se NPs is ascertained by visualizing the color change of the reaction mixture from colorless to orangishred. After 24 h stirring at room temperature, the resulting solution was dialyzed against Milli-Q water with intermittent water changes to remove the unreacted chemicals and finally, freezedried to obtain fine powders of Se@NH2 NPs. At this time, the color of the dispersed particles changes from orangish-red to dark gray, indicating the phase transformation of Se from amorphous Se to hexagonal phase crystallinity. Native Se NPs were prepared similarly without using any stabilizer (PEG- bisamine).



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2.3. Quantification of amine density on the surface of Se@NH2 NPs. The density of free amine groups on the surface of Se@NH2 NPs was determined by a colorimetric titration method according to the procedure adopted by Campo et al32. Described method consisted of the incubation of amine functionalized nanomaterial with an excess of UV sensitive reagent 4nitrobenzaldehyde. The number of hydrolyzed aldehyde molecules is proportional to the number of the free amine groups present on the surface of NPs. In a typical experiment, the Se@NH2 NPs (5 mg) were washed three times with 1 mL coupling solution (0.8% glacial acetic acid in dry methanol, (v/v). Thereafter, 1 mL of 4-nitrobenzaldehyde solution (7 mg in 10 mL coupling solution) was added and the suspension was allowed to react for 3h over a shaker. After removing the supernatant, 4-nitrobenzaldehyde reacted NPs were washed repeatedly with coupling solution. Subsequently, 1 mL of hydrolysis solution (mixture of 75 mL H2O, 75 mL methanol and 0.2 mL glacial acetic acid) was added to the particles and the tube was shaken further for 1 h following which the mixture was centrifuged and the absorbance of the supernatant was measured by using spectrophotometer at 282 nm. The number of hydrolyzed molecules were calculated by previously constructed calibration curve of 4-nitrobenzaldehyde in hydrolysis solution. 2.4. Synthesis of cetyl modified Hyaluronic acid (CMHA). CMHA was prepared using published procedure described elsewhere with some modifications33. Briefly, the chemical synthesis of CMHA divided in Part A and B. 2.4.1. Part A- Preparation of 6-O-(3-hexadecyloxy)-1-chloropropane-2-ol (1). To an equimolar reaction mixture of hexadecane-1-ol (cetyl alcohol) and epichlorohydrin in 1,2dichloroethane (75 mL) was added 100 µL of concentrated HCl followed by 48 h refluxing of the resulting solution. After usual aqueous work up, the solvent was evaporated using rotavapor


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to obtained fluffy white substance, 1, in ∼96% quantitative yield. Chemical characterizations of 1 was in good agreement with the published data in the literature and discussed in supplementary section. 2.4.2. Part B- Reaction of 1 with Hyaluronic acid (HA) to form cetyl modified HA (CMHA). For modifying HA with 1 following synthetic procedure was used. In brief, to an aqueous solution of HA sodium salt (100 mg in 5 mL water) was added 2 mL of sodium hydroxide solution and stirred at room temperature for 5 min. To the above mixture was added a solution of 1 (29.1 mg dissolved in 5 mL THF) and the temperature of reaction mixture was allowed to rise to 50 ºC with continuous stirring. After 24 h, the resulting solution was concentrated on a rotary evaporator and washed thrice with diethyl ether (10 mL) to get rid of unreacted 1. Subsequently, the collected aqueous phase was acidified with dilute HCl, concentrated and dialyzed against Milli-Q water for 48 h and freeze dried to yield a white fluffy material (CMHA) in ~70% quantitative yield. The chemical integrity of CMHA was established by FT-IR and 1H NMR spectroscopy and literature and discussed in supplementary section. 2.5. Conjugation of Se@NH2 NPs with CMHA (Se@CMHA). Conjugation of CMHA with Se@NH2 NPs was achieved using standard carbodiimide chemistry following the protocol described elsewhere34 for coupling of carboxyl groups of CMHA to free primary amines of Se@NH2 NPs. Briefly, to an aqueous solution of CMHA (10 mL of 1 mg/mL solution) was added 1 mL solution of EDC (20 mg in 1 mL water) to activate the carboxyl groups of the modified polymer. To the above mixture, a solution of NHS (20 mg in 2 mL THF) was added dropwise and stirred for 6 h maintaining the pH at 8. Subsequently, to the above activated CMHA solution was added a dispersed solution of Se@NH2 NPs (10 mg, 5 mL water) and the reaction mixture was left to stir for 24 h at room temperature. Following completion of the



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reaction, the resulting solution was finally dialyzed against Milli-Q water for 48 h with an intermittent water change and lyophilized to obtain selenium impregnated CMHA NPs (here onwards Se@CMHA NPs). The Se@CMHA NPs are stored at 4 ºC for subsequent studies. 2.6. Drug encapsulation on Se@CMHA NPs. Doxorubicin (DOX) as a model anticancer drug was physically entrapped in Se@CMHA NPs to form Se@CMHA-DOX NPs following the protocol below. Briefly, to a dispersed solution of Se@CMHA NPs (40 mg) in Milli-Q water (8 mL) was slowly added an aqueous solution of DOX (2 mL, 2 mg/mL) over a period of 20 min and stirred gently for 24 h at room temperature in dark keeping the final drug to NPs ratio to 1:10 (w/w). Subsequently, the solution was dialyzed against Milli-Q water to remove surface adsorbed and unbound DOX, and subsequently lyophilized to obtain Se@CMHA-DOX NPs as fluffy red product in ~82% (36 mg) quantitative yield revealing drug encapsulation. 2.7. Fluorescent labeling of NPs. Fluorescent labeled Se@CMHA NPs were prepared using the method described by Liu et al.19 with some modifications. Coumarin 6 (1 mg) dissolved in ethanol (1 mL) was added dropwise to an aqueous dispersed suspension of Se@CMHA NPs (25 mg) at room temperature. The resulting suspension was stirred for 24 h in the dark followed by dialysis against 30% ethanol/H2O mixture over 24 h to remove the unreacted and surface adsorbed dye. Finally, the resulting solution was lyophilized and used for cell uptake study. 2.8. Characterization of Se@CMHA NPs. Se@CMHA NPs were characterized using various microscopic and spectroscopic techniques. Briefly, zeta potential and mean diameter of the nanoparticles were measured on a Nano-ZSP instrument (Malvern Instruments, UK). Transmission Electron Microscope (TEM) samples were prepared by placing a drop of dispersed Se@CMHA NPs solution onto carbon coated copper grid for determining morphology and


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polydispersity in particle size. The micrographs were obtained on TECNAI G2 Spirit (FEI, Netherland) equipped with Gatan digital camera operated at an accelerating voltage at 80 kV. Elemental composition of the Se@CMHA NPs was analyzed using Field Emission Scanning Electron Microscope (FE-SEM) coupled with Energy Dispersive X-ray analysis (EDAX) on Quanta FEG 450 (FEI, Netherland). FT-IR spectra of the samples were recorded on a single beam Nicolet™ iS™5 FT-IR Spectrometer. XRD technique was used for phase analysis and to determine crystallinity of selenium in the Se@CMHA NPs. Nanoparticles were exposed to a generator voltage of 35 kV at 30 mA using CuK radiation source (λ = 1.5406 Å) in a PHILIPS Panlytical model PW 3040/60 X’Pert Pro-diffractometer. The diffraction angle (2) range of observation was 20 - 100º in a continuous scan mode at a scan rate of 0.5º min-1 at constant temperature of 22 ± 1 ºC. 2.9. Drug loading efficiency and drug release behavior of Se@CMHA-DOX NPs. In order to determine DOX loading efficiency, 3 mg of Se@CMHA-DOX NPs were dispersed in 1 mL dimethyl sulfoxide (DMSO) by mild sonication. The suspension was vortexed thoroughly, centrifuged at 12,000 rpm for 5 min and the collected supernatant was subjected to UV analysis at 480 nm. The amount of drug present was calculated using a standard calibration curve (conc. vs absorbance) of the drug alone in DMSO. The encapsulation efficiency (% EE) and the percentage drug loading (% DL) of the drug on Se@CMHA NPs were estimated following formula given below. % DL =

Weight of DOX present in Se@CMHA − DOX NPs × 100 Weight of Se@CMHA − DOX NPs

% EE =

Weight of DOX present in Se@CMHA − DOX NPs × 100 Weight of DOX used



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To examine drug release at different physiological pH, 5 mg of Se@CMHA-DOX NPs are dispersed in 2 mL of 10 mM PBS (pH 5.5 and 7.4 separately), poured into a dialysis bag suspended in 20 mL respective PBS in a screw cap glass bottle. The glass bottle was gently shaken at 37 ºC in an incubator shaker and at pre-determined time intervals, aliquots were taken out followed by UV analysis. The outer medium was replenished with an equal amount of fresh buffer each time the aliquots were withdrawn. The cumulative amounts of DOX in samples were determined by a pre-drawn calibration curve of drug alone in PBS (pH 7.4 and 5.5 separately). 2.10. Cell culture. Different mammalian cell lines, MCF7 breast adenocarcinoma cells, MCF 10A human mammary epithelial cells, A549 lung carcinoma cells, BEAS-2B lung epithelial cells, A431 epidermoid carcinoma cells and HaCaT human keratinocyte cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and subcultured as per supplier’s protocol in a humidified incubator at 37 ºC with 5% CO2 atmosphere. 2.11. Cell viability assay. Cytotoxicity was determined by measuring the ability of viable cells to transform MTT to a purple formazan dye. For this, cells were seeded in 96 well culture plates (1×104 cells/well) and incubated overnight in a CO2 incubator at 37 ºC for cell attachment followed by treatment with doses of different NPs and other chemicals according to the experiments for 48 h. Following treatment, 10 µL (For 100µL culture medium) of MTT solution (5 mg/mL in PBS) was added to the each well and incubated for another 4 h. To dissolve the formazan crystals, the medium was aspirated and replaced with 100 µL/well DMSO and the absorbance was read at 570 nm on a microplate reader (Ώ fluostar, BMG labtech). The cell viability was expressed as the percentage of live cells relative to the control. All experiments performed in triplicate.


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2.12. Synergy analysis. The synergistic interaction of Se NPs and DOX was evaluated by using the Chou-Talalay method35. For this, an Isobologram was plotted according to the data generated by viability assay by using Compusyn Software. The connected line between the ED50 (effective doses for the 50 % cell death) of Se NPs and DOX represent the additive line. When the ED50 data point of drugs near or on the additive line it represents an additive treatment effect, while the data points below or above the additive line represents the synergism or antagonism effect respectively. The synergism was also identified in terms of combination index (CI) value. CI value of 1 meant an additive effect between Se NPs and DOX, while CI value < 1 represents synergism and CI value > 1 indicates antagonism. 2.13. Tumor sphere formation assay. MCF7 cells were seeded in ultra-low attachment 6well plates at a density of 50×103 cells/well in M171 medium supplemented with MEGS. The cells were incubated at 37 ºC in a CO2 incubator for 7 days with intermittent addition of fresh medium for the formation of tumor spheres36. Thereafter, the tumor spheres were treated with Se NPs, DOX and Se@CMHA-DOX NPs for 48 h. After the completion of the treatment duration, the morphological changes in the tumor spheres were examined under the microscope. The effect of the treatment was represented as a change in the average diameter of tumor spheres (µm), calculated using ImageJ software. 2.14. Cellular uptake studies. The cellular uptake of cancer targeted Se@CMHA NPs was monitored qualitatively and quantitatively. For these experiments, Se@CMHA NPs were tagged with coumarin 6 fluorophore. MCF7 cells were seeded on chamber slide at a density of 5×104 cells/chamber and allowed to adhere for 12 h for qualitative analysis. Thereafter, the media was replaced with fresh media containing 50 µg/mL of coumarin 6 labeled NPs and exposed for various time points. The cells were then washed repeatedly with PBS to remove the residual NPs



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outside the cells. Afterward, cells were fixed with chilled methanol, dried, mounted with antifade mounting medium supplemented with DAPI followed by visualization under a Nikon ﬂuorescence microscope equipped with a camera. Images were merged using ImageJ software. For quantitative measurement of cellular uptake, MCF7 cells were seeded into 96-well plates at 104 cells/well (0.1 mL) and allowed to adhere overnight. Subsequently, the medium of the wells was replaced with fresh media containing 50 µg/mL of coumarin 6 labeled Se@CMHA NPs and cells were further incubated for different time points. Following incubation, the medium was discarded and the cells were washed repeatedly with chilled PBS to remove the residual NPs outside the cells. Further, cells were lysed with 100 mL of 0.5% Triton X-100 in 0.2 M NaOH solution and fluorescence from coumarin 6 tagged NPs was measured using a microplate reader (Ώ fluostar, BMG labtech) with excitation and emission wavelengths set at 430 and 485 nm, respectively. The cellular uptake efficiency was calculated using a calibration curve of respective fluorescent NPs previously drawn by NPs treated alike, without cells and expressed as the amount of NPs internalized by 104 cells. Cellular uptake of Se@CMHA-DOX NPs was monitored by using flow cytometer. MCF7 cells were seeded in 6 well culture plates at a density of 3×105 cells/well and allowed to adhere for 12 h. Thereafter, the media was replaced with fresh media containing 50 µg/mL of Se@CMHA-DOX NPs and exposed for various time points (0, 2, 4, 6 h). Subsequently, the cells were washed repeatedly with PBS to remove the residual NPs outside the cells, harvested, suspended in PBS and subjected to flow cytometric analysis of NP’s cellular internalization. The degree of cellular uptake of Se@CMHA-DOX NPs was represented by the increase in the mean fluorescence intensity of DOX. 2.15. Hyaluronan competing assay. Competitive binding of HA receptors between Se@CMHA NPs and HA in excess was performed on MCF7 cells according to the procedure


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described by Huang et al. with some modification20. Briefly, a density of 104 cells/well was seeded in 96-well plates and allowed to adhere overnight. Attached cells were pretreated with an excess amount of HA (0 and 50 µg/mL) for 1 h in a CO2 incubator at 37 ºC with exception of control wells followed by addition of coumarin 6 labeled Se@CMHA NPs (50 µg/mL) to the wells and were incubated for various time points. After completion of the incubation, the medium was removed from the wells and the cells were rinsed thrice with cold PBS to remove the residual nanoparticles which were not internalized by the cells and subsequently lysed by adding 100 µL of 0.5 % Triton X-100 in 0.2 M NaOH solution. Finally, the fluorescent intensity of the coumarin 6 loaded Se@CMHA NPs internalized by cells was measured using plate reader with excitation and emission wavelengths set at 485 and 525 nm, respectively. A standard curve for the fluorescent labeled Se@CMHA NPs was constructed by suspending different concentrations of NPs and treating them in a similar way to calculate the amount of NPs internalized by 104 cells. 2.16. Quantification of intracellular DOX using UHPLC: To quantify the intracellular DOX we have followed method describe elsewhere38 with modification. Briefly, MCF7 cells were plated in 6 well plates at 1×106 cells per well and left for 12 h for cells to adhere. Thereafter, cells were incubated with Se@CMHA-DOX NPs (5 µg/mL) and free DOX (0.205 µg/mL, equivalent content in Se@CMHA-DOX NPs) for different time points (3, 6, 12 and 24 h). After the completion of the incubation, cells were washed with PBS, trypsinized, counted and lysed in Milli Q water under sonication. These sonicated samples were centrifuged at 12000 rpm to remove the cell debris and the supernatants were subjected to UHPLC for quantification of DOX.



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Analyses

were

performed

with

a

Nexera

X2

Ultra

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High-Performance

Liquid

Chromatography (UHPLC) system, equipped with a binary pump system, column thermostat, SIL-30AC autosampler, and an RF-20Axs fluorescence detector connected with C18 Column (Kinetex, EVO, 1.7µm, 100 x 2.1 mm) operating at 30 °C. The mobile phase consisted of a mixture of pH 2.5, 0.05 M trichloro-acetic acid (TCA) and acetonitrile (60/40, v/v). Fluorescence detection was carried out at an excitation wavelength (λex) of 480 nm and an emission wavelength (λem) of 558 nm. The eluted peak of DOX was identified and quantified using an external standard curve of known concentrations. 2.17. In-vitro Thioredoxin reductase (TrxR) activity assay. Recombinant active rat TrxR 2.5 µg (1.17 U) was incubated with Se NPs, DOX and Se@CMHA-DOX NPs separately, for set time period (0, 5, 10, 20, 30, 40, 50, 60 min) and enzyme activity was determined by 5,5Dithiobis-(2-nitrobenzoic acid) (DTNB) reduction assay in the 100 µL solution containing 50 mM Tris HCl, pH 7.5, 200 mM NADPH, 5 mM DTNB, and 1 mM EDTA following standard protocol. The TrxR activity was calculated by recording the change in the absorbance at 412 nm withthe help of a microplate reader (Ώ fluostar, BMG labtech). 2.18. TrxR activity assay in cell lysate. TrxR activity in the MCF7 cell lysate was determined by endpoint insulin assay described elsewhere37. In brief, MCF7 cells were treated with Se NPs, DOX and Se@CMHA-DOX NPs for 24 and 48 h separately. After the completion of exposure time, the cells were washed with PBS and lysed in lysis buffer (25 mM Tris-HCl; pH 7.5, 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 20 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, 0.5% Triton X-100) supplemented with protease inhibitor cocktail. The obtained protein was quantified by QuantiProTM BCA Assay Kit (Sigma). An activity assay buffer was prepared by mixing 55 mM HEPES (pH 7.6),


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0.2 mM insulin, 0.4 mM NADPH, 2 mM EDTA and 2 µM human Trx1 (for control without Trx1). Subsequently, 25 µg cell lysate was mixed with 50 µL of activity assay buffer in 96 well plate and left for 20 min at 37 ºC. Thereafter, 200 µL of 1 mM DTNB in 6 M guanidine hydrochloride solution was added to quench the reaction and the amount of free thiols generated from insulin were determined by DTNB reduction at 412 nm (extinction coefficient for 2-nitro5-thiobenzoic acid 13.6 mM−1cm−1). 2.19. Reactive Oxygen Species generation. The effect of Se@CMHA-DOX NPs and free drug on intracellular ROS generation in MCF7 cells were monitored by DCFH-DA assay. Briefly, cells were seeded (5×104 cells/well/200 µl) in 96 well culture plate and grown overnight followed by discarding the old medium and replenishing with serum free media. Thereafter, cells were allowed to further incubate for 2h in a CO2 incubator following which treatment exposure with Se NPs, DOX and Se@CMHA-DOX NPs was performed. Subsequently, 100 µM DCFDA was added and the changes in the fluorescence (λex = 500 nm and λem = 530 nm) were recorded using microplate reader (Ώ fluostar, BMG labtech) every 15 minutes up to 90 min. Values were expressed as a percentage of the control. 2.20. Western blot analysis. Cells were treated with Se@CMHA-DOX NPs (0, 2.5, 5 and 7.5 µg/mL) for 24 h and processed for western blot analysis. Total cell extracts were prepared by cell lysis using RIPA buffer (50 mM Tris-HCl; pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF and 2 mM EDTA) supplemented with protease inhibitor cocktail and centrifuged at 15,000×g for 20 min at 4 ºC. To observe the Cytochrome C (Cyt.C) release from mitochondria to cytoplasm followed by Se@CMHA-DOX NPs treatment, cytosolic extract was prepared by incubating the cells with hypotonic buffer (20 mM HEPES; pH 7.4, 10 mM KCl, 250 mM sucrose, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA) supplemented



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with protease inhibitor cocktail for 30 min in cold condition. Thereafter, the cells were disrupted in a homogenizer by optimal gentle strokes and centrifuged at 1000×g for 5 min at 4 ºC to remove unbroken cells and nuclei. To separate the mitochondria and cytosolic fraction, the collected homogenate was centrifuged again at 15,000×g for 20 min at 4 ºC. Protein concentrations in cell lysates were estimated using the QuantiProTM BCA Assay Kit (Sigma). 100 µg of protein mixed with 5X loading buffer (Tris 62.5 mM, SDS 4%, glycerol 20%, βmercaptoethanol 5% and bromophenol blue 0.02%), and samples were denatured for 5 min at 100 ºC. An equal amount of samples were subjected to 10% SDS-PAGE gel, separated by electrophoresis and transferred to a PVDF membrane in cold condition. Thereafter, the membrane was blocked 1 h at room temperature with 1X blocking buffer (Sigma) and probed with primary antibodies at 1:1000 dilutions in 5% blocking buffer containing Tris buffered saline (TBS) overnight at 4 ºC. The PVDF membrane was washed 3 times for 15 min each with TBST buffer (TBS and 0.05% Tween-20) and incubated with appropriate horseradish peroxidase conjugated secondary antibody (1:3000). After 4 h of incubation at room temperature, the membrane was thoroughly washed to remove the unreacted antibodies and the proteins were visualized by chemiluminescence substrate in Versa gel documentation system (BioRad). To minimize the error due to the loading difference in each lane, membranes were stripped and reprobed with β-actin. Image-J software was used to quantify the band intensities. 2.21. Immunofluorescence. Immunocytochemical experiments were performed to check the Se@CMHA-DOX NPs induced Cyt.C release from mitochondria to the cytoplasm and nuclear translocation of mitochondrial apoptogenic factors, AIF and Endo-G. Briefly, cells were seeded at a density of 50,000 cells/chamber in chamber slides and left overnight for cell attachment following which cells were exposed to different doses of Se@CMHA-DOX NPs concentration


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(0 and 5 µg/mL). After 24 h, cells on slides were washed with PBS, incubated with Mito Mito tracker for 30 min and fixed with chilled methanol. Similarly, for the nuclear translocation study, cells were treated with various nanoformulations for 30 h and fixed with chilled methanol. These fixed cells were blocked with PBS containing 2% FBS and 0.05% Tween-20 for 1 h following incubation with primary antibodies (1:200; Cyt.C, AIF, and Endo-G) overnight at 4 ºC. After incubation, cells were washed repeatedly with PBST (PBS with 0.05% Tween-20) and further incubated with appropriate fluorophore conjugated secondary antibody (1:500). Following incubation, cells were washed with PBST, stained with DAPI or PI, mounted in antifade mounting medium and subjected to the microscopic analysis. The nuclear translocation of AIF and Endo-G was quantified by using Image J (Intensity Ratio Nuclei Cytoplasm Tool) software. 2.22. Cell cycle analysis. For cell cycle analysis we have followed the protocol described in our recently published article39. Cells treated with Se@CMHA-DOX NPs (5 µg/mL) for 30 h along with control cells were harvested, washed with PBS, and fixed with 70% ethanol overnight at -20 ºC. Further, fixed cells were washed twice with PBS, treated with RNase and permeabilized with Triton X-100. Thereafter, cells were stained with 50 µg/mL propidium iodide (PI) and analyzed by flow cytometer. 10,000 events were recorded for each sample. 2.23. Apoptosis study. The apoptotic rate of Se@CMHA-DOX NPs treated MCF7 cells was analyzed by phosphatidylserine externalization employing Annexin V-FITC/Propidium Iodide (PI) binding assay following the protocol described previously40. Briefly, cells were seeded at a density of 3×105 cells/well in 6 well plates and left overnight for cell attachment. Thereafter, cells were treated with Se@CMHA-DOX NPs (0 and 5 µg/mL) for 30 h followed by trypsinization. The cell pellets were suspended in 500 µL of 1X Annexin V binding buffer



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containing Annexin V-FITC (5 µL) and PI (5 µL), followed by incubation for 15 min at room temperature in the dark and analyzed by flow cytometer (BD Inc, USA) without further delay. 2.24. Statistical Analysis. All data presented as the mean ± standard deviation (SD) and a pvalue of

Inhibition of Thioredoxin Reductase by Targeted Selenopolymeric

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