Enhancement of Cytotoxicity and Inhibition of Angiogenesis in Oral

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Enhancement of cytotoxicity and inhibition of angiogenesis in oral cancer stem cells by a hybrid nanoparticle of bioactive quinacrine and silver: Implication of base excision repair cascade Shakti Ranjan Satapathy, Sumit Siddharth, Dipon Das, Anmada Nayak, and Chanakya Nath Kundu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00461 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 10, 2015

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Molecular Pharmaceutics

Enhancement of cytotoxicity and inhibition of angiogenesis in oral cancer stem cells by a hybrid nanoparticle of bioactive quinacrine and silver: Implication of base excision repair cascade

Shakti Ranjan Satapathy, Sumit Siddharth, Dipon Das, Anmada Nayak, Chanakya Nath Kundu*

Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa, 751024, India.

Running title: QAgNP caused apoptosis in OSCC-CSCs

For reprints and all correspondence: *Chanakya Nath Kundu Postal address: KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Odisha, India, Pin-751024. E-mail: [email protected] Phone : +91-0674-272-5466, Fax: +91-0674-272-5732 The authors declare no conflict of interest.

1 ACS Paragon Plus Environment


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Table of contents (TOC)

A PLGA based monodispersed hybrid nanoparticle (QAgNP) was prepared by single emulsion solvent evaporation method with quinacrine (QC) in organic phase and silver (Ag) in aqueous phase. QAgNP exhibited antitumor activity in various cancer cell lines by damaging DNA and induced apoptosis in OSCC-cancer stem cells by inhibiting efficient DNA BER pathway. ( indicates the down-regulation.


)

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Abstract A PLGA based uniform (50-100 nm) hybrid nanoparticle (QAgNP) with positive zeta potential (0.52 ± 0.09 mV) was prepared by single emulsion solvent evaporation method with bioactive small molecule quinacrine (QC) in organic phase and silver (Ag) in aqueous phase. Physiochemical properties established it as a true hybrid nanoparticle and not a mixture of QC and Ag. Anti-tumor activity of QAgNP was evaluated by using various cancer cell lines including H-357 oral cancer cells and OSCC-cancer stem cell in vitro model system. QAgNP caused more cytotoxicity in cancer cells than normal epithelial cells by increasing BAX/BCLXL, cleaved product PARP-1, arresting the cells at S phase along with DNA damage. In addition, QAgNPs offered greater ability to kill the OSCC-CSCs in compared to NQC and AgNPs. QAgNP offered anti-cancer action in OSCC-CSCs by inhibiting the base excision repair (BER) within the cells. Interestingly, alteration of BER components (Fen-1 and DNA polymerases (β, δ and ε) and unalteration of NHEJ (DNA-PKC) or HR (Rad-51) components were noted in QAgNP treated OSCC-CSC cells. Furthermore, QAgNP significantly reduced angiogenesis in compare to physical mixture of NQC and AgNP in fertilized eggs. Thus, these hybrid nanoparticles caused apoptosis in OSCC-CSCs by inhibiting the angiogenesis and BER in cells.

Keywords: Quinacrine, Silver, Hybrid Nanoparticle, Oral cancer, Oral cancer stem cells.



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1. Introduction Silver based nanoparticles (AgNPs) are widely used in medicine, industry, agriculture as well as environmental monitoring. Recently, by using chemical and plant based silver nanoparticles, we have shown that AgNPs exhibit anti-cancer potentiality in colon cancer cells. AgNP caused apoptosis in cells by increasing p53 level, DNA damage while reducing NF-κB as well as disrupting the mitochondrial membrane potential in colon cancer cells.1,

2

Inspite of

multiple advantages as anti-cancer agent, AgNPs suffer from serious limitations that include toxicity to normal cells at higher doses and non-target oriented delivery. Apart from that it also get bind to cells and don’t act better in free form than conjugated, which has restricted the study of silver in xenograft models as well as in clinic.3 Quinacrine (QC) is a 9-amino acridine derivative initially known as an ideal anti-malarial agent but recently has been repurposed against cancer with successful outcomes in breast, colon, pancreas, lung and renal cell carcinoma.4,

5

Currently, it is in phase-II clinical trial against

hormone refractory prostate cancer.6 QC exhibits anti-cancer potentiality in cells by disrupting different signalling pathways such as p53-NF-κB, PI3K-mTOR-AKT, etc.6 along with DNA damage, topoisomerase-II inhibition and induction of autophagy.4, 5 The synergistic anti-breast cancer activity has also been reported with a natural bioactive compound lycopene.7 Despite of these promising pharmaceutical advantages, QC, owe to its low bioavailability, localized deposition followed by developing yellow pigmentation in skin, skin rash and some immunological complication in patient.8, 9 Combinational drug treatments have played a fundamental role in the treatment of various cancers.10, 11 It has been shown that most of the anticancer drugs exert either synergistic or additive or even antagonistic effects with other compounds in various cell line and tumor


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models. Combination treatment offers the advantage in target oriented delivery along with different toxicity profiles in comparison to the individual administration to improve therapeutic efficacy with reduced toxicity. Recently, Ganta et al. have reported the efficacy of combinational delivery of flaxseed based nanoemulsion containing paclitaxel and curcumin for enhancement of anti-cancer potentiality in human ovarian adeno-carcinoma cells.10 Patil et al. have also shown the co-delivery of tariquidar along with paclitaxel in PLGA-NPs that overcomes multidrug resistance and increased the therapeutic efficiency of paclitaxel in drug resistant breast cancer cells.12 Reports suggest that nano-materials carrying the anticancer agents exhibit prolonged drug retention in the tumors that decrease the tumor growth and hence increases the life span of the tumor bearing animals.13 Yang et al. reported that nanoform of silver possess the ability to control the release of antitumor drug as shown in HeLa cells.14 Oral cancer is one of the most fatal ailments faced by mankind today. Oral squamous cell carcinoma (OSCC) is considered to be the sixth most common malignancy worldwide that accounts for 30-40% of all the malignant tumors in India while it’s only 2-4% in western countries. Cultural diversity, geographic parameters and popularity of addictive habits of tobacco based compounds results in high occurrence of oral cancer in India.15 Diverse treatment strategies along with the variable natural behavior of this cancer make it difficult to cure.16 Higher death rate and post-treatment complications lead to poor prognosis of the disease with lowest (maximum 5 years) survival rates among all other cancers.17 However, early diagnosis could remarkably increase the survival rate for this cancer compared to others. Recent studies provide the important evidences that, solid tumors contain a subpopulation of cancer stem cells (CSCs) which posses self renewal potentiality and major responsible for relapse of the cancer.18,

19

CSCs play a pivotal role in tumor initiation, progression, invasion,



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metastasis and drug resistance.20-22 Extensive characterization of putative OSCC-CSCs is yet to be investigated. Reports suggest the characteristics of CSCs which include highly expressed drug transporters, quiescent cell-cycle arrest, high DNA damage repair machinery and resistance to programmed cell death which play vital role for its resistance towards conventional chemo as well as radiotherapy.23, 24 Researchers reported the delivery of doxorubicin to breast CSCs by conjugation with gold nanoparticle.25 Similar report from Wang et al also showed the efficient delivery of doxorubicin to multicellular spheroids by conjugating with the chitosan nanoparticles surface functionalized with a tumor penetrating peptide iRGD.26 Study of Lim et al suggest the efficacy of brain CSCs targeted polymer encapsulated curcumin nanoparticle formulation with increased bioavailability.27 In addition, Ke et al reported the breast cancer as well as breast CSCs targeted co-delivery of two drugs; doxorubicin and thioridazine using polymeric micelle as a carrier system that is considered to be effective against breast cancer.28 But no such efficacy has been reported for oral cancer or oral CSCs. With this background, we hypothesize that, by encapsulating the nano formulated anticancer drug QC and nano form of metallic silver in a biocompatible and biodegradable polymer, PLGA, we can not only overcome drawbacks associated with both silver and QC but also increase the target oriented anti-cancer activity. Thus, here we investigate the anti-cancer potentiality of a single hybrid nano formulation (QAgNPs) containing silver and QC in different cancer cell lines as well as oral cancer stem cell model system. We also studied the antiangiogenic potentiality of QAgNP in fertilized eggs.

2. Experimental section 2.1.

Materials and methods 6 ACS Paragon Plus Environment

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2.1.1. Cell culture and chemicals The A-549 (Lung cancer), A-431 (Skin cancer), HEK-293 (Kidney cancer), HeLa (Cervical cancer), HCT-116 (Colon cancer), MCF-7 (Breast cancer), MDA-MB-231 (Breast cancer), and VERO (Normal kidney epithelial) cells were maintained in DMEM with 1% antibiotic (100 units of penicillin and 10 mg streptomycin per mL in 0.9% normal saline) and 10% fetal bovine serum (HIMEDIA, India) while H-357 (Oral cancer) cells were grown in DMEM-F12 (50:50, v/v) medium supplemented with 10% FBS, 1% antibiotic (100 U/mL of penicillin, 10 mg/mL of streptomycin in 0.9% normal saline), 0.5 mg/mL of hydrocortisone. Normal breast epithelial cells, MCF-10A were grown in DMEM-F12 (50:50, v/v) medium supplemented with 10% FBS, 1% antibiotic (100 U/mL of penicillin, 10 mg/mL of streptomycin in 0.9% normal saline), 0.5% µg/mL of hydrocortisone, 100 ng/mL of cholera toxin, 10 µg/mL of insulin, 10 ng/mL of epidermal growth factor and 1% (w/v) of L-glutamine. Normal human colon cells, FHC, were grown in DMEM with 1% antibiotic (100 units of penicillin and 10 mg streptomycin per mL in 0.9% normal saline) and 10% fetal bovine serum (HIMEDIA, India) but additionally supplemented with 10 mM HEPES, 10 ng/mL cholera toxin, 0.005 mg/mL insulin, 0.005 mg/mL transferrin, and 100 ng/mL hydrocortisone. All the cell lines were maintained in a humidified atmosphere in 5% CO2 at 37°C. A-549 (CCL-247), A-431 (CRL-1555), HEK-293 (CRL-1573), HeLa (CCL-2), HCT-116 (CCL-247), MCF-7 (HTB-22), MDA-MB-231 (HTB26), VERO (CCL-81), FHC (CRL-1831) and MCF-10A (CRL-10317) cells were procured from ATCC, Manassas, VA, USA, while H-357 was obtained from Sigma Aldrich, USA. Quinacrine (QC), PLGA used for this study were purchased from Sigma Chemicals (Sigma, St Louis, MO, USA) while silver nitrate was procured from HiMedia (Mumbai, India). Anti-DNA Ligase-I, anti-DNA Pol-β antibodies were purchased from Novus Biological. Anti-DNA Pol- δ, anti-DNA



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Pol- ε and anti-lamin B1 antibodies were obtained from Abcam. All other antibodies used were procured from Cell Signaling Technologies, MA, USA.

2.1.2 Synthesis of hybrid nanoparticles of QC and Ag Hybrid NPs containing QC and Ag were formulated by using modified oil-in-water single emulsion solvent evaporation technique. Briefly, 45 mg of PLGA polymer was dissolved in 5 mL of chloroform. To this, 1 mg of QC was added and sonicated for 2 min in ice bath with 55W of energy output to prepare a uniform PLGA-QC organic solution. Then this organic solution was emulsified with equal volume of 1 mM aqueous solution of silver nitrate to form an oilwater emulsion.29 The emulsification was carried out using a microtip probe sonicator at 55W of energy output (Hielscher UP200S, Hielscher, USA) for 8 min over an ice bath. The emulsion was stirred overnight at room temperature on a magnetic stir plate which allowed the evaporation of organic solvent and formation of NPs. NPs were recovered by ultracentrifugation (Beckman Coulter Ultracentrifuge Optima L-90K, USA) at 35,000 rpm for 20 min at 4°C and followed by overnight lyophilization (Free Zone Bench top Freeze Dry System, Lanconco, MO, USA) to get the powdered NPs. These particles were termed as QAgNPs. Single drug nano-formulations (either NQC or AgNPs) were also prepared separately by using a similar procedure.

2.1.3. Characterization of NPs 2.1.3.1.

UV-Vis Spectra analysis

The absorption spectra of the nanoformulations were measured by EPOCH multivolume spectrophotometer (BioTek Instruments, USA) in regular intervals to determine the stability and quality of the nanoparticles by the procedure described earlier.1 Post formulation of QAgNP, NQC and AgNP, spectral analysis was performed in 300 nm to 700 nm wavelength and continued for one month with a regular interval of 24 hr. The characteristic peaks obtained for


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NQC, AgNP and QAgNP were plotted and compared in order to conclude the uniqueness of the formulation. The analysis was based on Surface Plasmon Resonance (SPR).

2.1.3.2.

Transmission Electron Microscopy

Nanoformulations were evaluated for size using Transmission Electron Microscopy (TEM) (JEOL-JEM 2100, Japan). Nanoparticle samples (0.5 mg/mL) were suspended in water and sonicated for 30 sec. Then one drop of the suspension was placed on the carbon films supported by copper grid. Images were visualized at 120 kV under the microscope.

2.1.3.3.

Particle size analysis & Zeta potential measurement

Particle size, size distribution (Polydispersity index) and zeta potential of the formulations were determined by using a Zetasizer Nano ZS (ZEN3600, Malvern Instrument, UK) following method described earlier.1 Briefly, 1 mg/mL of the NQC, AgNPs and QAgNPs were prepared in MilliQ water and sonicated for 30 sec at 55 W in ice bath using a sonicator (Hielscher UP200S, Hielscher, USA). 100 µL of the nanoparticle suspension was diluted in 1mL of MilliQ water and used for measurement.

2.1.3.4.

Fourier Transform Infrared (FTIR)

FT-IR measurements were carried out to find out the chemical interactions occurred during the NP formulation according to procedure described earlier.1 In brief, lyophilized samples were mixed and crushed with KBr and the mixture was pressed into a pellet with a pressure of 300 kg/cm2. The pellet was subjected to FT-IR analysis (Nicolet™ iS™5 FT-IR Spectrophotometer, Thermo Scientific, MA, USA).

2.1.3.5.

Differential Scanning Calorimetry (DSC)

Phase behavior of NQC, AgNP, QAgNP and physical mixture of NQC & AgNP was studied by differential scanning calorimetry using DSC-60 instrument. The instrument was



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comprised of calorimeter (DSC 60), flow controller (FCL60) and thermal analyzer (TA60) and operating software TA60 from Shimadzu Corporation, Kyoto, Japan. Approx. 2 mg of each sample were placed on aluminum pan and were crimped followed by heating under nitrogen flow (30 mL/min) at a scanning rate of 5°C/min from 25°C to 250°C.30 Aluminum pan containing same quantity of indium was used as reference. The heat flow as a function of temperature was measured for all four samples.

2.1.4. Co-localization of QAgNP in cells A cell based microscopic analysis was performed to check the presence of QC and silver in PLGA capped QAgNPs in NP exposed cells. Briefly, H-357 cells were seeded in 6-well tissue culture plates and incubated for 24 hr. Cells were exposed to QAgNPs for 6 hr post incubation. Presence of QC and Ag was visualized in GFP (as QC provides auto green florescence) and DAPI (Ag provides the blue florescence) filter respectively and the individual images were merged to get the localization of the molecules. Analysis was performed using a fluorescence microscope (Evos fl Fluorescence Microscope, Thermo Fisher Scientific, MA, USA).

2.1.5. MTT cell viability Assay To test the effect of the indicated compounds on growth of cells with short term exposure, an MTT assay was carried out as described earlier.1 The MTT assay is a quantitative and rapid colorimetric method for measuring the viability of cells. Briefly, 8000-10000 cells/well were seeded in a 96 well tissue culture plate in triplicate and incubated for 24 hr. Cells were treated with different concentrations of indicated compounds and incubated for 48 hr. Then MTT reagent was added and OD was measured at 570 nm after dissolving the purple formazan crystals with detergent. Data was calculated and represented as percent viability vs. concentrations. Experiment was repeated atleast thrice.


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2.1.6. Clonogenic cell survival assay The clonogenic cell survival assay is a long-term cell viability assay that determines the ability of a single cell to proliferate indefinitely, thereby retaining its reproductive ability to form a colony or a clone. This assay has been carried out according to protocol described earlier.1, 7 Briefly, cells (800-1000 cells/well) were seeded for 24 hr, and then exposed to the indicated compounds for 48 hr. Media was replaced in every 2 days with fresh medium and cells were allowed to grow for 5-6 doublings. Crystal violet stained colonies were counted using a Gel documentation system (UVP, CA, USA) and data was calculated. Finally, data was represented as percent survival vs. concentrations. Assay was performed at least thrice.

2.1.7. Cell cycle analysis with FACS Regulation of cell cycle profile and apoptosis was analyzed by FACS to test the effect of indicated compounds on H-357 cells according to protocol described earlier.1 70-80% confluent cells cultured in 60 mm tissue culture dishes were exposed to fixed dose of indicated compounds and incubated for 48 hr. Cells were trypsinized and washed twice with 1X PBS containing 0.05% RNase-A. Then cells were fixed with pre-chilled 70% ethanol and kept at -20°C overnight followed by staining with Propidium iodide (50 µg/ml) containing RNase (200 µg/mL RNase in PBS). Stained cells were sorted using FACS (FACS Canto II, Becton and Dickinson, CA, USA) and 10,000 events were analyzed. Different phases of the cell cycle were determined using Cell Quest Software (Becton and Dickinson, CA, USA).

2.1.8. Alkaline single cell gel electrophoresis assay (Comet assay) A comet assay was performed to determine the degree of oxidative DNA damage.7, 31 H357 cells were exposed to fixed concentrations of indicated compounds and incubated for 48 hr before harvesting. Treated cells were trypsinized, washed and suspended in 40 µL ice cold



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1XPBS. Roughly, 5000-6000 cells were re-suspended in 25 µL of 1X PBS and mixed with 75 µL of low melting agarose (A9414, Sigma-Aldrich, Bangalore, India) at 37°C and then spreaded uniformly over a preheated (37°C) comet slide. After solidifying, the slides were immersed in pre-chilled lysis solution (10 mM Tris, 100 mM EDTA, 2.5 M NaCl, 1% Triton X-100, 10% DMSO and pH 10.0) for 1 hr at 4°C. After lysis, electrophoresis was performed in freshly prepared alkaline electrophoresis buffer at 30V for 15 min. Slides were then dipped in neutralization buffer (0.4 M Tris HCl and pH 7.5) and washed twice with distilled water followed by 70% ethanol. After drying, 40 µL of SYBR green (Sigma-Aldrich, Bangalore, India) was added to the slides and incubated in the dark for 30 min at room temperature. DNA migration was observed using a fluorescence microscope (Nikon, Tokyo, Japan) at 40X magnification. We have taken the data from minimum eight to ten focus points with average cell number of 20-30. The comet tail length was measured as a function of DNA damage and analyzed using TriTek CometScore™ software (TriTek Corporation, VA, USA).

2.1.9. Immunoblotting analysis Approximately 1×105 H-357 and H-357-PEMT cells/well were seeded in 100 mm tissue culture plates and treated with indicated compounds for required time period. After incubation, whole cell lysates (for checking the BAX, BCL-XL, PARP-1, etc.) and nuclear lysates (for measuring the DNA repair proteins) were prepared according to protocol described earlier using RIPA lysis buffer and protein was separated by SDS-PAGE. Immunoblotting was performed as per the protocol described earlier.1, 32

2.1.10.Development of metastasis model Multicellular tumor spheroids were formed using liquid overlay method.16, 33, 34 Briefly, 12-well tissue culture plate were coated with 0.5% agarose, prepared with MilliQ water and


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allowed to air-dry for 30 min in sterile condition. Approximately 40,000 H-357 cells were overlayed on the agarose coated wells and incubated for 3-4 days with serum free medium for the formation of tumor spheroids. Uniform spheroids observed in all the wells after 6-7 days of incubation. Spheroids were isolated by centrifugation at 850 rpm for 5 mins followed by dissociation with trypsin-EDTA. Dissociated single cells of squamospheres were incubated in serum containing medium (10% FBS). Floating single cells of spheroids got adhered with the availability of serum and formed a monolayer of cell, which is designated as H-357-PEMT. Uniform spheroids and H-357-PEMT were further used for the experiments.

2.1.11.Preparation of conditioned media of the metastatic cells Conditioned media was prepared according to the protocol mentioned earlier with minor modification.35 Approx. 1×106 number of H-357 PEMT cells were seeded in 60 mm cell culture dishes and incubated for 24 hr. After the incubation period, the supernatant containing the consumed media was centrifuged at 1800 rpm at 4°C for 5 min. The supernatant was collected in a fresh tube and concentrated using Eppendorf Concentrator plus (Eppendorf, Hamburg, Germany). Final product was considered as conditioned media (CM), aliquoted accordingly and stored for further use.

2.1.12.Matrigel invasion assay Tumor invasion of basement membrane has been considered to be one of the major steps that lead to the successful formation of a metastasis.36 Matrigel invasion assay was performed to study the ability of cells to attach to the matrix, invade through the matrix and to migrate forward. This assay was performed in a 24-well transwell plate with a pore size of 8 µm and inserts coated with 20 µL of matrigel (BD Biosciences, 356234). Approx. 3×105 H-357, H-357squamospheres, H-357-PEMT cells were suspended in 100 µL of serum free media and seeded



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on the matrigel coated inserts. Complete media was added to the lower chamber, incubated for 24 hr. After incubation, non-invaded cells were removed while invaded cells were fixed with 4% paraformaldehyde followed by DAPI staining. Stained cells were counted at 40X under the fluorescence microscope (Nikon, Tokyo, Japan).

2.1.13.In direct ELISA In direct ELISA was performed to detect CD-133, a OSCC-CSC marker in in vitro condition following the protocol mentioned earlier.37,

38

Briefly, protein antigen was mixed in

coupling buffer, coated on to 96 well microplate and incubated overnight at 4°C followed by washing with wash buffer and then blocking with super cocktail buffer. Then primary antibody was added and incubated for 2 hr at RT. After washing with TBST, the wells were then incubated with HRP conjugated secondary antibody for 1 hr. After washing twice with TBST, 2, 2’-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) substrate solution was added. Finally, absorbance of the coloured product formed was detected at 405 nm using microplate reader.

2.1.14.QAgNP mediated damage of squamospheres and OSCC-PEMT NPs were considered to lead the cells to death not only by inducing the apoptotic pathways but also damaging the cellular morphology after treatment. So we tried to check the effect of QAgNP on the morphology in squamosphere as well as PEMT. H-357-squamospheres and H-357-PEMT were exposed to fixed doses of NQC, AgNPs and QAgNPs for 24 hr and the effect was visualized with the aid of a fluorescence microscope (Evos Fluorescence Microscope, Thermo Fisher Scientific, MA, USA).

2.1.15.In vivo base excision repair (BER) assay In vivo BER assay is a rapid, sensitive and quantitative method which provides information regarding repair capacity of cells; we have used a known type of DNA damage


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introduced in a p21p promoter whose activity can be monitored to assess the efficiency of the repair in the cell. The modified plasmid when transfected into cells would show a poor promoter activity as compared to the unmodified p21p plasmid. However, the promoter activity can be restored if the modified DNA is allowed to go through DNA repair process within the cell. We randomly modified C residues of the p21P promoter to a reduced abasic site (R-p21P) plasmid by chemical and enzymatic modification.32, 39, 40 Briefly, a closed circular DNA of p21 (pGL2-p21) promoter downstream of the luciferase-reporter gene, was determined by 3M sodium bisulfate in the presence of 50 mM hydroquinone. Deamination modifies cytosine into a uracil-residue (U-p21P) that is a suitable substrate for SP-BER. U-p21P was further exposed to uracil-DNA glycosylase (UDG) followed by reduction with 0.1M sodium borohydride to generate reduced AP-sites (R-p21P) for LP-BER substrate. H-357-PEMT cells were grown in 40 mm tissue culture dishes till 60-70 % confluence. Then cells were transfected with 2.0 µg/mL of U-p21P or R-p21P cDNA and 0.5 mg/mL of pCMV-β-galactosidase (β-gal) plasmid using 7 µL/mL of Lipofectamine 2000 reagent. The pCMV-β-gal served as an internal control to check the transfection efficiency of cells. Post transfection, the medium was replaced with complete medium supplemented with 10% FBS and the cells were exposed to NQC, AgNP and QAgNP for 48 hr. After incubation, cells were harvested and SP-BER and LP-BER activities were measured by determining the luciferase gene-reporter activity of cellular lysate using a DLR luciferase assay instrument (Berthold, Germany). 2.1.1.6. In ovo assay (Chick Chorioallantoic Membrane (CAM) Assay) CAM assay, an in ovo assay is used for the study of angiogenesis, cancer cell invasion and metastasis. Chick Chorioallantoic Membrane (CAM) is available outside the embryo for the



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study of the complex biological system; CAM provides an extraembryonic membrane with dense capillary network and is used for the study of the angiogenesis and angiogenic inhibitors. We performed this assay to study the inhibition of regular as well as PEMT conditioned media (CM) induced angiogenesis by QAgNP. CAM assay was performed according to the protocol referred earlier.41 Briefly, fertilized eggs were incubated in a humidified atmosphere at 37°C for 10 days. On the 10th day, under aseptic conditions, a window was made in the eggshell to monitor the growth of the embryo. The window was further resealed with porous autoclavable adhesive tape. On the 11th day, one set of eggs were exposed to 0.7 µg/mL of QAgNP onto an autoclaved sterilized filter paper inserted into the eggshell though the window and incubated for 48 hr to check its effect on regular angiogenesis. Similar set of eggs showing regular angiogenesis were treated with NQC (1.8 µg/mL), AgNP (3.6 µg/mL) and physical mixture of the both (NQC (1 µg/mL) + AgNP (1.4 µg/mL)) for 48 hr in order to check the effect of individual nanoform as well as physical mixture of both the components of QAgNP. Another set of eggs were added with 500 µg protein of the conditioned media (CM) of the H-357-PEMT cells inserted similarly and incubated for 24 hr to induce angiogenesis. After incubation, these eggs were further exposed to 0.7 µg/mL of QAgNP and incubated for 48 hr. The control set of eggs were incubated without any insert. The progressive increase of vascularity in CAM was studied and pictures were captured photographically.

2.2.

Results

2.2.1. Synthesis & physicochemical characterization of nanoformulations QAgNPs, the PLGA capped hybrid nanoparticles containing QC and Ag were formulated using single emulsion solvent evaporation method.29 QC and Ag were efficiently encapsulated inside the PLGA and showed high degree of stability as indicated from UV-Vis


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spectrophotometric analysis (Figure 1a). The different pattern of UV-Vis spectra of AgNP, NQC and QAgNP (encapsulated combined drug of QC and Ag) revealed that all the three nanoformulations are different and QAgNP is not a mixture of NQC and AgNP. Interestingly, QAgNP showed a characteristic peak at 420 nm while NQC and AgNP exhibited their corresponding peaks at 400 and 430 nm, respectively. It is interestingly to note that the nanoparticle didn’t form any aggregation/ precipitation during storage till 120 days (data not shown) which indicate the hybrid nanoparticles are very stable at room temperature. Furthermore, we checked the stability of the physical mixture containing NQC and AgNP (50:50) and interestingly we found two distinct peaks at 420 and 430 nm indicating presence of two individual NPs, which again confirmed the fact that QAgNP is a unique and single particle exhibiting the properties of both QC and Ag but not a mixture of two individual nano forms. Arrows indicate the respective peak point of NQC, AgNP and QAgNP. This clearly represents QAgNP is a unique formulation. Moreover, the curve area of QAgNP spectra covers the respective curve areas of NQC as well as AgNP. This observation infers that QC and Ag are not only just present inside the polymer capsule but are in their nano form and stable. However, the nature of binding between QC and Ag is unknown and need further investigation. AgNO3, PLGA and QC did not show any peak in the range of 400 nm to 450 nm (Data not shown). Size and morphology of NPs were determined by TEM analysis. QAgNP, AgNP and NQC showed spherical polydispersed nanoparticles with size ranging 50-100, 100 and 20 nm, respectively (Figure 1b). Data obtained from dynamic light scattering (DLS) showed the QAgNP have an average diameter of 382.4 ± 0.11 nm with a positive zeta potential of 0.523 ± 0.09 mV which were higher than that of AgNP and NQC. The average diameter of AgNP and NQC were 172.9 ± 0.29, 259.9 ± 0.18 but zeta potential were 0.096 ± 0.04 and 0.136 ± 0.01, respectively



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(Figure 1c). PDI values for NQC, AgNP and QAgNP were found out to be 0.121, 0.201 and 0.122 respectively. The low PDI value of QAgNP shows that it contains mostly uniform monodispersed particles with very few polydispersed particles. This proves its pharmaceutical efficacy, where a low PDI is always of priority. FT-IR study was performed to identify any chemical changes that occurred in the polymer due to the addition of drug during synthesis reaction.42 Figure 1d shows the FT-IR spectra of NQC, AgNP and QAgNP. Spectral analysis of PLGA based AgNPs showed peaks at 873 cm-1, 1551 cm-1 and 2981 cm-1 representing C-H, N-O asymmetric stretch and O-H stretch respectively. Spectral analysis of NQC exhibited characteristic bands due to different functional groups. NQC showed a peak at 1904 cm-1 that represents C=O stretch. However, the peak at 1630 cm-1 for PLGA-AgNPs is slightly shifted to 1688 cm-1 due to some chemical interaction happened between QC, Ag and polymer matrix. Apart from that, the peak at 1688 cm-1 for QAgNP is a completely unique peak, absent in NQC and PLGA-AgNP indicating formation of a new nanoformulation. Thus data presented here appeared that QAgNP is not the mixture of AgNP and NQC but a single individual compound. The physical status of individual nano-forms (NQC & AgNP) was compared with that of QAgNP by DSC analysis. DSC thermogram of each nanoformulation was presented in figure 1e. Thermal analysis has always been considered as a useful tool for determining that whether the particle of interest has been properly dispersed in the polymeric capsule. DSC analysis revealed the lowering in glass transition temperature in QAgNP (281.64°C) in compared to that of NQC (273.22°C) and AgNP (294.97°C). However, the physical mixture of NQC and AgNP has exhibited the two glass transitions at 221.23 and 239.57°C indicating the presence of two components in their individual state. Hence it can not only be inferred that QAgNP is a unique


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formulation not a physical mixture of NQC and AgNP but also a nanoformulation containing uniformly dispersed particles.

Figure 1. Physico-chemical characterization of QAgNPs. (a) UV-Vis spectral analysis of NQC, AgNP, NQC + AgNP (50:50) and QAgNP. (b) Particle size and shape analysis by TEM. (c) Table showing size distribution and zeta potential analysis of NQC, AgNP and QAgNP. (d) FT-



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IR analysis of NQC, AgNP and QAgNP. (e) DSC thermogram of NQC, AgNP, QAgNP and physical mixture of NQC + AgNP.

2.2.2. QAgNP decrease the anchorage dependent cancer cell growth Next, we checked whether QAgNP released QC as well as Ag inside the cells after uptake and offered anti-cell growth properties in cancer cells, first we carried out a microscopy based co-localization study after treating QAgNP in H-357 oral cancer cells. Figure 2a showed the fluorescence of AgNP, NQC inside the cells. Presence of silver emits blue fluorescence (observed in DAPI filter as Ag emits auto fluorescence in the range of 357 nm to 447 nm) while QC auto fluoresce green (GFP range; 470 nm to 510 nm). The merged image showed the colocalization of both QC and Ag (Figure 2a). Then, we measured the anti-cell proliferative activity after short term and long term treatment. For short term anchorage dependent cell proliferation, we have used MTT assay, a widely accepted colorimetric based assay. We treated eight different cancer cells (Breast-MCF-7 and MDA-MB-231, Cervical-HeLa, Colon-HCT-116, Lung-A-549, Skin-A-431, Kidney-HEK293, OSCC-H-357) along with two normal epithelial cells (Breast-MCF-10A, Kidney-Vero) with increasing dose of NQC, AgNP, QAgNP for 48 hr and then cell viability was measured. Each of the NP offered a characteristic cell viability profile in each cell line. Figures 2b represent IC50 (Concentration of the agent needed to reduce fifty percent cells in culture) value of each compound in different cell lines. It was noted that NPs were 10-15 fold more cytotoxic in cancer cells in comparison to normal cells. It was also observed that QAgNP showed IC50 at 2-5 fold lower in comparison to NQC and AgNP. Interestingly, it was noted that QAgNP offered fifty percent cell death in OSCC cells as low as 0.7 µg/mL concentration (Figure 2b). Clonogenic cell


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survival assay was performed to check the long term effect of nanoparticle exposure on proliferating capacity of the cells (Figure 2c). QAgNPs showed an LC50 of 0.7, 1.2, 0.8 µg/mL while that of NQC and AgNP are 1.8, 4, 2 µg/mL and 3.6, 10, 4 µg/mL respectively in H-357, HCT-116, and HeLa cell lines, respectively (Figure 2c (I, II, III)). This indicates that QAgNPs account almost 50% reduction at a more than 2 fold and 5 fold lower dose in compared to NQCs and AgNPs, respectively. In the other hand, QAgNP didn’t show any significant effect on proliferation of normal colon epithelial cells (FHC) as well as kidney epithelial cells (VERO) upto 10 µg/ml concentration (Figure 2c (IV)). Hence conclude that NPs induced death is more pronounced in highly proliferative cancer cells than in normal epithelial cells. To check the effect of cell survival activity in the mixture of NQC and AgNP, we have carried out a separate experiment. The H-357 cells were pre-treated with fixed concentration of either NQC (1 µg/mL) or AgNP (1 µg/mL) followed by increasing dose of AgNP or NQC, respectively for 48 hr prior to clonogenic assay. Figure 2 c (V) showed that QAgNP offered more H-357 killing efficiency in compare to the mixture of NQC and AgNP. QAgNP caused fifty percent cell death at 0.7 µg/mL while same amount of cell death occurred after the treatment with 1 µg/mL NQC plus 1.4 µg/mL AgNP or 1 µg/mL AgNP plus 1.8 µg/mL NQC.



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Figure 2. (a) QAgNPs showed both the characteristic of Ag and QC. Auto fluorescence of Ag and QC in H-357 cells as observed after QAgNPs exposure. QAgNPs caused an anchorage dependent cell death. (b) MTT cell viability assay. Cell viability assay was performed in NQCs, AgNPs and QAgNPs exposed different cancer and normal cells. Cells were exposed to varying dose of the NPs for 48 hr. Table representing the IC50 values obtained in different cell lines. (c) Clonogenic cell survival assay was performed in (I) H-357, (II) HCT-116 and (III) HeLa. (IV) 22 ACS Paragon Plus Environment

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FHC and VERO cells after treatment with NQC, AgNP & QAgNP. (V) Comparative analysis of clonogenic cell survival in physical mixture of NQC & AgNP and QAgNP exposed H-357 cells. Data includes mean ± S.D. of atleast three different experiments. Statistical significance was determined by two way ANOVA ***p

Enhancement of Cytotoxicity and Inhibition of Angiogenesis in Oral

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