Tailoring the efficacy of multifunctional biopolymeric-graphene oxide

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Tailoring the efficacy of multifunctional biopolymeric-graphene oxide quantum dot based nanomaterial as nanocargo in cancer therapeutic application Sriparna De, Kartik Patra, Debatri Ghosh, Koushik Dutta, Aditi Dey, Gunjan Sarkar, Jyotirmay Maiti, Arijita Basu, Dipak Rana, and Dipankar Chattopadhyay ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00689 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

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ACS Biomaterials Science & Engineering

Tailoring the efficacy of multifunctional biopolymeric-graphene oxide quantum dot based nanomaterial as nanocargo in cancer therapeutic application

Sriparna De a, Kartick Patra b, Debatri Ghosh c,Koushik Duttaa Aditi Dey d, Gunjan Sarkar a, Jyotirmay Maiti b, Arijita Basu a, Dipak Rana e and Dipankar Chattopadhyay *,a

a

Department of Polymer Science and Technology, University of Calcutta, 92, A.P.C. Road,

Kolkata 700 009, India, E-mail: [email protected] b

Department of Zoology, West Bengal State University, Barasat 700 126, West Bengal, India

c

Institute of Post Graduate Medical Education & Research (IPGMER), SSKM Hospital, Kolkata

700 020, India d

Immunology and Microbiology Laboratory, Department of Human Physiology with

Community Health, Vidyasagar University, Midnapore 721 102, West Bengal, India e

Department of Chemical and Biological Engineering, Industrial Membrane Research Institute,

University of Ottawa, 161 Louis Pasteur St, Ottawa, Canada

* CORRESPONDING AUTHOR FOOTNOTE Tel.:+91-94333790340; Fax: +91-33-2351-9755; E-mail:[email protected]

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ABSTRACT Nanotechnology has acquired an immense recognition in cancer theranostic plethora. Considerable progress has been made in the development of targeted drug delivery system for potent delivery of anti-cancer drugs to tumour specific site. Recently multifunctional nanomaterials are being explored and used as nanovehicles to carry drug molecules with enhanced therapeutic efficacy. In this present work, graphene oxide quantum dot (GOQD) was conjugated with folic acid functionalized chitosan (FA-CH) to develop a nanocargo (FA-CHGOQD) for drug delivery in cancer therapy. The synthesized nanomaterials were characterized using Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). Photoluminescence spectroscopy (PL) was also employed to characterize the formation of GOQD. To validate the efficacy of FA-CH-GOQD as nanocarriers, doxorubicin (DOX) drug was chosen for encapsulation. The in-vitro release pattern of DOX was examined in various pH ranges. The drug release rate in a tumour cell microenvironment at pH 5.5 was found higher than that under a physiological range of pH 6.5 and 7.4. A MTT assay was performed to understand the cytotoxic behavior of GOQD and FA-CH-GOQD/DOX. Cytomorphological micrographs of the A549 cell exhibited the various morphological arrangements subject to apoptosis of the cell. Cellular uptake studies manifested that FA-CH-GOQD could specifically transport DOX within a cancerous cell. Further anti-cancer efficacy of this nanomaterial was corroborated in a breast cancer cell line and demonstrated through 4’,6-diamidino-2phenylindole dihydrochloride (DAPI) staining micrographs. KEYWORDS: Chitosan, graphene oxide quantum dot, doxorubicin, controlled release, targeted delivery, cytomorphology 2


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INTRODUCTION The enormous exploration of nanoscience and nanotechnology endeavors new corridors for the advancement of novel materials in the nano scale range. Multifunctional nanoparticles are now becoming extensively identified and proving their versatility as drug delivery vehicles in cancer chemotherapeutics. However most of the drugs utilized in cancer therapy fail to secure significant clinical outcome because they do not have the propensity to trigger the cancer site.1-4 Moreover, a significant percentage of administered drug is accumulated over tumour specific tissue leading to severe side effects and thus constraint their clinical use and therapeutic efficacy.5 A variety of nanoparticles offer opportunities for developing drug-delivery system owing to their fascinating properties such as facile synthesis, tunable size,6 well-defined optical and surface properties and excellent biocompatibility.7,8 Among these carbon based nanocarriers including carbon nanospheres, nanodiamonds, graphene and its derivatives have been widely acknowledged for cancer therapy and diagnosis. Recently polymeric nanocarriers have rendered versatile platforms for the delivery of pharmacological drug with enhanced therapeutic efficacy and the ability to overcome multidrug resistance in cancer diagnosis.9-12 Simultaneously in conventional drug delivery systems, each drug functions within an optimal concentration range above which it is toxic and below which it is ineffective. Hence current research demands a new drug delivery system with a favorable therapeutic window after administration of a single dose, and targeted drug delivery to the acute site of interest.13-16 In this juncture biopolymers have often been adopted as precursor materials for designing a new drug delivery formulations owing

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to their fascinating properties like non-toxicity, excellent biodegradability, biocompatibility, and environmental sensitivity, etc.17-19 Since its first magnificent historical breakthroughs by British scientists in 2004, graphene has acquired pronounced attention among researchers from diverse regimes of science for venturing many of its distinctive properties such as single-atom-layered structure, superb heat and electrical conductivity,20-22 good mechanical flexibility23 and low toxicity.24 Graphene oxide and its derivative have also unveiled significant avenues for various biological applications including cellular bioimaging,25-27 targeted drug delivery,28 and photothermal therapy,29 particularly in cancer diagnostic plethora. Liu et al.30,31 first reported nano-graphene oxide as carrier for doxorubicin (DOX) loading in cellular bioimaging and drug delivery applications. Consequently, Zhang’s group32 and Wang et al.33 introduced a functionalized graphene oxide (GO) and quantum dot conjugated graphene probe respectively for bio-imaging and drug delivery applications. Recently, theoretical and experimental studies have focused on the quantum confinement which could tailor the finite size emerging graphene oxide quantum dot ‘GOQD’ which is a highly promising candidate for biomedical application. GOQD possess intrinsic fluorescence and high surface area with delocalized electrons suitable for efficient loading of drug molecule through π-π stacking.34 Meanwhile, the native hydrophilic functionality, tunable size and chemical inertness of GOQD makes it a versatile material as a drug carrier substance. Also it can deliver chemotherapeutics to tumour specific site inducing DNA cleavage with improved efficacy. Despite this fact, the cytotoxicity of GOQD still needs to be addressed and requires a major recognition for further advancement. Furthermore, polysaccharides have received the utmost scientific appreciation because of their extraordinary physicochemical and biological features. Among them Chitosan (CH), α (1– 4


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4)2-amino-2-deoxy-β D glucan, has now drawn immense recognition due to its fascinating properties including pH sensitivity, biocompatibility, biodegradability, mucoadhesivity, low toxicity, low immunogenicity and high cell membrane permeability.35 CH nanocarriers can offer passive drug delivery that unzip a new avenue by boosting therapeutic effects with a good survival rate, high drug loading efficiency and prolonged circulation time. These favorable features promote it as one of the most avant-garde material for the designing of drug delivery vehicles in cancer therapeutic applications.35 Likewise, to tune the cell targeting efficiency and the engulfment of nanocarrier through receptor mediated endocytosis, certain modification of CH is highly desirable. Thus CH has been tailored with folic acid (FA), wherein FA is a folate receptor ligand of cell membrane and favors the triggering of membrane integrity and enhanced nanoparticle internalization.36,37 It is also a stable, reasonable and low immunogenic compound with high affinity towards folate receptors which are overexpressed in various cancerous cell surfaces.38 Subsequently, FA can penetrate into the cells and transport through many organelles by vasicular trafficking inducing intracellular uptake of anticancer drug as well as its targeting ability.39 Hitherto, cancer remains a challenging global threat towards the entire human community. To address this threat, several strategies have been extensively exploited, which includes the development of new anticancer drugs and potent drug delivery systems with enhanced therapeutic index. Encouraged by these aforementioned problems, a new anticancer nanocarrier system has been designed with the abilities of targeted and controlled release of drug to the tumour site. GOQD was first synthesized using the modified Hummers method followed by hydrothermal cutting of GO nanoparticles. To tune its cytotoxicity, FA functionalized CH has been used for encapsulation which can efficiently load and deliver the anticancer drug DOX to 5



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the cancerous tissue. The resulting FA-CH-GOQD nanomaterial was characterized using FTIR, UV, SEM, TEM and DLS analysis. The loading of DOX and its controlled release in various pH ranges has also been examined through UV-Vis spectroscopy. The anticancer efficacy of synthesized nanomaterial and potential ability as drug carrier substance was monitored in cancerous cell lines. Further, after administering different dosages of FA-CH-GOQD/DOX, the morphological alteration of the cells has been scrutinized using SEM micrographs and bright field images. Additionally the nuclear DNA condensation and its damage pattern have also been evaluated through DAPI staining and comet assay technique, respectively.

EXPERIMENTAL SECTION Materials Pristine graphite powder (92% pure) was procured from Sigma-Aldrich Inc. (MW 12.01 g mol−1, mp 3652–3697ºC). CH (Medium molecular wt, 200-800 cp, 75-85% deacetylated form) and FA was bought from Sigma Aldrich Inc., and used without any further purification. Nhydrosuccinamide (NHS), 1-(3-Dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride (EDC) and DOX were also procured from Sigma-Aldrich Inc. Concentrated sulphuric acid (98% H2SO4, GR grade), phosphoric acid, potassium permanganate (KMnO4 purified), hydrogen peroxide solution (30% H2O2) and all other chemicals of analytical grade were procured from Merck. Dulbecco′s Modified Eagle′s Medium (DMEM) media, streptomycin and penicillin were collected from Sigma-Aldrich Inc. Fetal bovine serum (FBS) was obtained from GIBCO/Invitrogen. DAPI was purchased from Sigma-Aldrich. 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-tetrazolium bromide (MTT) reagent was acquired from Himedia, India. All other used

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chemicals were of highest purity grade (SRL Pvt. Ltd. & Merck Ltd.). Sequentially solvents were distilled following the standard protocol prior to use.40

Harvesting of cell lines and its maintenance A549 cell (Human lung carcinoma) SH-SY5Y (Neuroblastoma cell) and MDA-MB 231 (Breast cancer cell) were procured from NCCS, Pune (India). These cells were cultured and maintained in DMEM supplemented with 10% FBS (ATCC) and 1% penicillin/streptomycin (ATCC). The cell suspension has been poured into 25 cm2 vials and kept in an incubator (37oC, 5% CO2) followed by trypsinization. Simultaneously, the cultured cells were utilized for various experiments until the number of cells count is ~2×104 per well.

Isolation of peripheral blood lymphocytes In 5 ml heparin-coated Vacutainers (Becton, Dickinson and Company, India), the blood samples were stored by veni-puncture method according to Hudson and Hay. 5ml blood samples were diluted with PBS in a ratio of 1:1. Histopaque 1077 was used for density gradient centrifugation. Histopaque was mixed with blood sample and centrifuged at 400 g for 40 mins at room temperature. Thereafter the upper monolayer buffy coat was separated which contains lymphocytes and placed in a culture plate. The suspension was cultured in a RPMI 1640 complete media. The lymphocytes were kept in a 5% CO2 and 95% humidified CO2 incubator at 37oC temperature for further array.

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Synthesis of GOQD based FA conjugated CH nanomaterial At first GO was synthesized following the modified Hummer’s method.41 GOQD was prepared according to the method reported by Sun et al.42 In brief, 20 mL of GO suspension (0.5 mg/mL) was first carefully mixed with concentrated HNO3 (10 mL) and H2SO4 (4 mL). Then the mixture was subjected to heat treatment and refluxed. The resulting product consists of a brown transparent dispersion along with black colored precipitates. Afterwards the whole solution was subjected to hydrothermal treatment in a Teflon coated autoclave and heated at 120oC for 6 h. The reaction mixture was cooled to room temperature followed by mild ultrasonication for a few hours. The pH was adjusted to 8 with Na2CO3 in an ice bath. Subsequently the suspension was filtered through a 0.22 µm microporous membrane to eliminate the large artifacts of GO, and a yellow colored solution was collected. The filtrate was then dialyzed in a dialysis bag (retained molecular weight: 1000 Da) for 3 days and GOQD was obtained. Simultaneously, a solution of FA and EDC was prepared in DMSO followed by magnetic stirring for 2 h at room temperature. Afterwards the dropwise addition of the resulting solution into CH solution (1% w/v) in acetic acid was stirred for at least 10-12 h in the dark followed by sequential dialysis with phosphate buffer saline (PBS) and distilled water. Thereafter, the product was obtained post freeze drying process. Encapsulation of GOQD was achieved by adding FA–CH (1% w/v in acetic acid, pH 5.5) to an equal volume of water dispersed GOQD followed by rapid stirring at 12,000 rpm for 24 h. Finally the product was lyophilized prior to do characterization. The schematic diagram of synthesis of FA-CH-GOQD has been cited in Scheme 1. Loading of DOX onto FA-CH-GOQD nanomaterial The antitumour drug was successively loaded by adding 20 mL (2 mg/mL) of DOX into the dispersion of FA-CH-GOQD nanomaterial (20 mg dispersed in 10 mL water) under stirring 8


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for 48 h in the dark at room temperature. Centrifugation was performed with the resulting suspension to remove unbound DOX molecule. The obtained solution was frequently dialyzed using dialysis membrane (mol. wt cut off of 2000 Da) against PBS buffer for 24 h. The bath solution was replaced continuously with fresh PBS buffer every 4 h. The drug loading efficiency of DOX was measured and calculated using UV-Vis spectroscopy at an absorption wavelength of 480 nm. Based on the underlying equation the drug loading efficiency was examined and calculated. Drug loading (%) = (weight of incorporated drug−weight of free drug in the bath solution)/weight of nanocarriers) ×100.

Release pattern of DOX from FA-CH-GOQD nanomaterial The release behavior of DOX from FA-CH-GOQD nanomaterial was monitored in different pH buffer range such pH 7.4, 5.5 and 6.5, respectively. The FA-CH-GOQD/DOX (3.0 mg) was dispersed in different PBS (3.0 mL) buffers of pH ranges of 7.4, 5.5 and 6.5, respectively and was poured in a dialysis bag, with MWCO of 12-14 kDa. The dialysis bag was then dipped in 100 mL of corresponding release medium at different pH. Afterwards the samples were incubated in a shaker incubator and maintained at 37°C in the dark for the whole tenure of the experiment. A measured quantity (3 mL) of solution from the release medium was decanted after every time step and corresponding fresh PBS buffer was replenished into the medium to keep the constant volume. The released amount of DOX was monitored at different time points from 0 to 50 h by a UV–Vis spectrophotometer (480 nm). Hemolytic assay Blood was collected from lab volunteers according to an approved guideline by the Institutional Ethical Committee, Department of Zoology, West Bengal state University Kolkata, 9



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India, with proper consent of the volunteer. Fresh blood was collected and centrifuged at 4000Xg for 10 min. The cell was pellet down and washed thrice followed by re-suspension in 10 mM PBS at a pH 7.4. The final concentration was obtained as 1.6×109 erythrocytes/ml. Afterwards equal volumes of erythrocytes were incubated with different concentrations of capsaicin and eugenol followed by shaking at 37ºC for 1 h. Samples were then centrifuged at 3500Xg for 10 min at 4ºC. The lysis of RBC was measured at varying concentration of DOX conjugated GOQD based nanomaterial by taking absorbance at an OD of 540 nm. Complete hemolysis (100%) was examined using 1% Triton X 100 as a control. Thus the hemolytic activity index of the spice active components was carried out in percentage based on the following equation: H=100 X (OpOb)/(Om-Ob) wherein, Op is the measured optical density of used nanomaterial concentration, Ob is the optical density of the buffer solution and Om is the optical density of Triton X 100.

In-vitro cell viability assessment through MTT assay Cytotoxicity studies and probable cellular interaction has been critically employed to interpret the anticancer efficacy of the synthesized GOQD based nanomaterial. The MTT assay has been conducted to investigate the in-vitro cytotoxicity. Both cell lines (A549 and SHSY5Ycells) were employed and harvested in a DMEM medium supplemented with penicillin (100 units/mL), streptomycin (100 g/mL), and 10% (v/v) heat-inactivated FBS. To assess the cell viability, 200 µL cells were seeded into 96-well plates at a density of 8×103 cells/well. After ◦

overnight incubation at 37 C in a humidified 5% CO2-containing atmosphere the cells reached confluence level. The cells were then removed and fresh medium comprised of different concentrations of DOX (5-30 µg/mL), FA-CH-GOQD/DOX nanomaterial were added into each 96 well and incubated for another 24 h. Consequently 20 µL of MTT solution was introduced to 10


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each well and the cells were incubated for an additional 4 h. After removal of the medium 150 µL DMSO was mixed followed by continuous shaking for 20 min in a shaker incubator. Finally, the optical density (OD) value of every well was recorded on an Elisa microplate reader at wavelength of 480 nm. The relative cell viability was examined by comparing the control cells. All experiments were conducted in triplicate set to investigate the influential effect of the GOQD based nanomaterial on the proliferation of cells and quantified as the % of cell viability using the formula described below:

% cell viability = [ODsample - ODcontrol] × 100/ODcontrol

Characterizations GOQD and FA-CH-GOQD have been characterized using FTIR. The test was carried out in PerkinElmer spectrum Express Version 1.03.00 instrument within the range of 500-4500 cm-1. FTIR spectrum has also been utilized to confirm the successful loading of drug molecule on the GOQD based nanomaterial by identifying the characteristic peak of primary amine groups and N-H stretching vibration. UV-Vis spectroscopy is a resourceful tool to evaluate the optical properties of the synthesized nanoproducts. UV-Vis spectra of the prepared materials were documented in Perkin Elmer Lambda 25 by taking dispersed nanomaterials in double distilled water. Additionally the presence of conjugated FA (wt %) and GOQD (wt %) in FA-CH and FA-CH-GOQD based nanomaterial were determined by UV-Vis spectroscopy respectively. Briefly, FA-CH and GOQD (0.05 mg/mL) in water: DMSO (1/1: v/v) mixture was prepared and corresponding

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absorbance was recorded at λmax 363 and 227 nm respectively.43 The calibration curve of FA and GOQD has been plotted and depicted in Figure 1a & 1b.

Figure 1. Linear plot of standard curve of (a) FA and (b) GOQD To explore the morphology of the synthesized GOQD and its functionalized nanomaterial, scanning electron microscopy (ZEISS EVO-MA 10) was employed. The cytomorphological changes of the cells after incubating with DOX loaded GOQD based nanomaterial have also been monitored by SEM analysis. For cellular morphology analysis, first the coverslips were cleaned thoroughly using ultrasonic treatment for at least 30 min followed by washing with 70% ethanol for 1 h. Thereafter, the coverslips were dried for subsequent experiments. The cells were seeded to the wells containing the coverslips at 1×103 cells per well. The desired concentrations of DOX loaded nanomaterials were added in to each well and incubated for different time intervals. Afterwards fixation of the cells was done with 200 ߤL methanol and 200 ߤL distilled water for 15 minutes and then washed successively followed by dehydration with 70%, 90%, and absolute alcohol for 10 min each. Following fixation and dehydration, coverslips containing cells were coated with gold using an EMS850 sputter-coating 12


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device (Electron Microscopy Sciences, Hatfield, PA, USA). SEM micrographs were captured and analyzed using Smart-SEM software. Furthermore, the morphology of the synthesized sample was speculated using TEM (JEOL-JEM-2100 with a 200 kV accelerating voltage). To correlate with TEM analysis the Field Emission Scanning Electron Microscopy (FE-SEM) images of FA-CH-GOQD nanomaterial was monitored using a JEOL JSM-7600F instrument. For TEM study the nanomaterial suspension was dropped onto a copper grid and dried overnight. Thereafter the TEM images of prepared samples were inspected. The topology of FA-CHGOQD nanomaterial and DOX loaded nanomaterial was also monitored through Atomic Force microscopy (AFM) (Agilent Technologies Pvt. Ltd).

Assessment of nuclear morphological alteration and cellular uptake by DAPI staining To screen the nuclear morphology of the cells, DAPI staining was performed by seeding the desired cells (2×105 cells/mL) within six well plates. The harvested cells were incubated with GOQD based nanomaterial (5, 10, 15 and 20 µg/ml) for 24 h. For comparison, the untreated cells were kept as control. Then isolated cells were stained with DAPI dye as per the methodology of Lin et al (2006) reported earlier prior to certain modification.44 After incubation, the fixation of the cells was performed with 2.5% glutaraldehyde for 15 min, permeabilized with 0.1% Triton X-100 and stained with 1 mg/mL DAPI for 5 min at 37oC. Afterwards the cells were then washed with PBS buffer and scrutinized by fluorescence Microscopy (Nikon Eclipse Lv100pol). The cellular uptake FA-CH-GOQD/DOX nanomaterial has been examined in a A549 cell using fluorescence microscopy analysis. At first cells were cultured in 24-well culture plates containing 5×104 cells/well. After 24 h incubation at 37oC, cells were incubated with free DOX, 13



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FA-CH-GOQD/DOX or PBS for 2 h. Then the medium was decanted followed by PBS buffer wash. For cellular imaging study the cells in the rest one well of individual sample were prefixed with 500 µL of 4% formaldehyde for 15 min at 4◦C and washed two times with PBS followed by counterstaining with DAPI (1 µg/mL) for 15 min at 37oC sequentially. The cells were thoroughly monitored under fluorescence microscope (EVOS® FL cell imaging system).

Study of cellular morphology by acridine orange (AO)–ethidium bromide (EtBr) double staining method To monitor the probable pattern of cell death, we explored the cells using the EtBr-AO double staining method. A number of 2×104 cancerous cells were harvested into each well of a 6well plate and incubated for 24 h at 37oC in a humidified 5% CO2 atmosphere. GOQD based nanomaterial (20 µg/mL) were simultaneously poured into the well for 24 h. Thereafter incubation was done and cells were properly washed once with PBS buffer. The cell suspension (10 µL) were then transferred on a glass slide and stained with 10 ml AO (50 µg/mL) and EtBr (50 µg/mL). Subsequently, the cells were scrutinized under a fluorescence microscope (Nikon Eclipse Lv100pol) with 400×magnification and analyzed accordingly.

Comet assay FA-CH-GOQD/DOX nanomaterial induced DNA damage was inspected using comet single cell electrophoresis (SCGE) assay.45 Cells were seeded in 6 well plates (0.3 x 106 cells) and then incubated with different concentrations of nanomaterials for 24 h. Afterward the drug incubated cells were isolated from each plate to form a single cell suspension. 100 µL of drug treated single cells were re-suspended in 1% low melting agarose (LMA) (200 µL). These cells 14


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suspension were layered onto the microscopic slides pre-coated with 1% normal melting agarose (NMA). Cells were lysed by immersing the slide in cold lysis buffer solution (10 mM Na– EDTA, 2.5 M NaCl, 10 mM Tris, 1% Triton × 100 and 10% DMSO in pH 10) followed by incubation for 1 h at 4ºC in dark condition. Then slides were immersed in an unwinding buffer for 20 minutes and electrophoresis was carried out for 20 min (voltage 25 V and 3000 mA current) to pull the negatively charged DNA towards the anode. Thereafter all the slides were washed three times in neutralizing buffer (0.4 M Tris-buffer, pH 7.5) and DNA was stained using EtBr (10µg/mL). The images of comets were employed to evaluate the DNA content of each nucleus of the disrupted cells at 5, 10 and 20 µg/ml concentration. The slides were monitored under a fluorescence microscope. The DNA damaging parameters such as Comet tail length, Tail DNA percentage, tail moment and OTM were assessed and analyzed using CASP Lab software.

RESULTS AND DISCUSSION Fourier transform infrared spectroscopy (FTIR) To elucidate the interaction between the FA-CH, GOQD and conjugation of the drug with drug carrier molecule, FTIR analysis has been employed by inspecting the absorption bands of the constituent’s material. The FTIR spectra of GOQD,FA, FA-CH-GOQD nanomaterial, free DOX and drug conjugated GOQD based nanomaterial are depicted in Figure 2a and 2b, respectively. The assigned FTIR absorption peaks derived from Figure 2a are tabulated in Table 1.

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Figure 2. (a) FTIR spectrum of GOQD, FA, FA-CH and FA-CH-GOQD based nanomaterial; (b) FTIR spectrum of DOX and DOX conjugated FA-CH-GOQD based nanomaterial.

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Table 1. Assigned Peak of FTIR spectrum

Sample Name

FA

FA functionalized CH composite

FA-CH-GOQD nanomaterial

IR absorbtion band (cm-1)

Description

3555 3420, 3327 2925, 2842 1692 1633 1602 1489 1417 3328 2925,2866 1664 1586 1418 1169 1066

ν(O-H) band ν(N-H….H) band νas (C-H) and νs(C-H) of –CH2 ν(C=O) from COOH ν(C=O) from -CONH2 δ (NH2) Phenyl ring O-H deformation peak of Phenyl ring ν(N-H….H) band νas (C-H) and νs(C-H) of –CH2 ν(C=O-) from amide I ν(N-H) amide II O-H deformation peak of Phenyl ring νas (C-O-C) and νs(C-O-C) νas (C-O-C) and νs(C-O-C)

3293 2935, 2889 1648 1151 1087

ν(N-H….H) band νas (C-H) and νs(C-H) of –CH2 ν(C=O-) from amide I νas (C-O-C) and νs(C-O-C) νas (C-O-C) and νs(C-O-C)

ν = stretching vibration; νs = symmetric stretching vibration; νas = asymmetric stretching vibration; δ = bending vibration.

It is evident from Figure 2a that FA shows several characteristic peaks occurring at 3555, 3420, 3327, 2925, 2842, 1692, 1633, 1602, 1489, and 1417 cm-1. A hydroxyl stretching region (3500-3000 cm-1) is observed due to the stretching of hydroxyl and NH- vibrational bands. The distinct band at 1692 cm-1 corresponds to the C=O stretching vibration of the carbonyl group whereas the C=O stretching vibration of CONH2 group appears at 1633 cm-1. Further, two well defined peaks of the bending mode of NH-vibration and -OH deformation band of phenyl 17



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skeleton are noticed at 1602 and 1417 cm-1, respectively. Additionally, the band at 1489 cm-1 is ascribed to the distinct absorption band of a phenyl ring. In the case of FA-CH, the C=O stretching vibrational bond shifted to a lower wavelength along with typical absorption peak of both molecules. This indicates the good interactive nature within FA and CH molecules imparting the formation FA functionalized CH molecule. Besides that in case of FA-CH-GOQD nanomaterial all the peaks of FA-CH composite is present. Subsequently, the peaks at 1737, 1619 and 1392 cm-1 of GOQD are overlapped with the more intense peaks of FA-CH composites46. Therefore, no such significant peak is identified from the FTIR spectrum of FACH-GOQD nanomaterial. More interestingly, the peak at 1664 cm-1 assigned for C=O stretching band and the peak at 3328 cm-1 corresponds to the N-H stretching shifting to lower wavelength in FA-CH-GOQD nanomaterial. Also the broadening of the N-H stretching band is noted in the FA-CH-GOQD based nanomaterial as compared to FA-CH composite material. This signifies the better interaction between GOQD and FA-CH composite material.

As observed from Figure 2b, DOX exhibits various characteristic peaks at 3381, 2924, 1727, 1628, 1421, and 1070 cm-1. These peaks are due to the different quinone and ketone carbonyl groups of the DOX molecule. The assigned broad peak at 1564 cm-1 is responsible for the N-H stretching band. Further DOX shows two distinct peaks at 880 and 763 cm-1, wherein 880 cm-1 may be ascribed to the primary amine NH2 wag and the peak at 763 cm-1 signifies the N-H deformation bonds. This is in good agreement with previous literature reported by Yuan et al.47 Further the successful loading of DOX onto FA-CH-GOQD nanomaterial is also evident from Figure 2b, wherein some additional peaks are found at 1537, 1385, 1418 and 1188 cm-1 corresponding to the bending vibrational band and absorption band of DOX molecule. 18


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Compositional analysis To determine the wt% of FA and GOQD present in FA-CH and FA-CH-GOQD, UV-Vis spectroscopy analysis was conducted and data was tabulated in Table 2. 43,48 Table 2. Compositional analysis of FA-CH composite and FA-CH-GOQD based nanomaterial Initial wt. of materials

Yield of product

Absorbance

Concentration

wt % of FA

CH (1g)

FA (1.5g)

1760 mg

FA-CH (at λmax 363 nm)

FA-CHGOQD (at λmax 227 nm)

FA

GOQD

in FACH composit e

attache d with CH

CH-FA (1g)

GOQD (50mg)

1021 mg

0.0315

0.0150

827.2 mg

35.735 Mg

47

55.15

wt % of GOQD present in FACHGOQD nanocomp osites 3.5

attached with FACH

71.47

From the Table 2, it is apparent that 55.15 wt% of FA is attached with CH moiety as well as in the synthesized product (FA-CH), where 47 wt% FA is present. Whereas 3.5 wt% GOQD is present in the FA-CH-GOQD nanomaterial and around 71.47 wt% of GOQD is conjugated with FA-CH composite. Hence, it can be concluded that 55.15 wt% FA is attached with CH and 28.53 wt% of FA-CH is attached with GOQD.

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Scheme 1. Pictorial representation of formation of FA-CH-GOQD based nanomaterial

Transmission electron microscopy (TEM) The morphology of synthesized GOQD has been monitored using a high resolution transmission electron microscopy (HRTEM) and shown in Figure 3. The crystalline structure and electron diffraction pattern are witnessed in the HRTEM images showing a fringe lattice spacing of ~ 0.252-0.249 nm (Figure 3b).These are in well accordance with the data of graphite (0.246 nm) within the limit of error.49 The size of GOQD is found within the range of 2-5 nm. Subsequently after functionalization with FA conjugated CH molecule, the size of GOQD based nanomaterial is appeared with spherical structural morphology within a range of 5-20 nm. This is perhaps due to the encapsulation of FA and CH moiety onto GOQD nanoparticle implying the formation of GOQD based nanomaterial. 20


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Figure 3. (a) HRTEM images, (b) selected area diffraction pattern (SAED) of GOQD, and (c) HRTEM image of FA-CH-GOQD. UV-Visible and Photoluminescence (PL) spectroscopy The PL property and UV-Visible absorption spectra of GOQD have been examined and are shown in Figure 4.

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Figure 4. (a) Optical property and (b) PL spectra of GOQD.

As evident from the UV-Vis spectrum (Figure 4a), a well-defined peak is found at around ~229 nm corresponding to the π-π* vibration of aromatic groups.50,51 Moreover the PL property of GOQD (Figure 4b) is noticed in an excitation dependent manner wherein a red shift is occurred with the excitation wavelength within a range of 320-440 nm. Also with increase in excitation wavelength the fluorescence emission position of the GOQDs is shifted to a higher wavelength region. As the excitation wavelength is increased from 320 to 440 nm, the emission fraction is shifted within 620 to 740 nm, which is in accordance with other reported carbonaceous quantum dots.52,53

Scanning electron Microscopy (SEM) The morphology of FA-CH-GOQD nanomaterial and DOX loaded GOQD based nanomaterial have been monitored using SEM analysis and are depicted in Figure 5.

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Figure 5. SEM micrographs of (a) FA-CH, (b) FA-CH-GOQD nanomaterial, and (c) DOX conjugated FA-CH-GOQD based nanomaterial d) FE-SEM image of FA-CH-GOQD based nanomaterial

The FA-CH (Figure 5a) reveals the appearance of chitosan microstructure showing a wrinkled and flake like morphological arrangement which is attributed to the self-aggregation tendency of the CH molecule. Further incorporation of GOQD within functionalized CH matrix displays comparatively porous and interconnected architecture (Figure 5b). This indicates that FA-CH-GOQD nanomaterial offers a suitable microenvironment upon which the drug molecules can be entrapped efficiently. As observed from Figure 5c, after loading of DOX drug, the rough and highly porous surface texture changed into a comparatively homogeneous morphology with less porosity which is possibly due to the electrostatic interaction between FA-CH-GOQD nanomaterial and DOX molecule. More importantly some agglomeration of DOX (Figure 5c, highlighted with red circles) is also clearly noted, implying the successful loading of drug 23



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molecule within the functionalized CH matrix. Besides that the FESEM image (Figure 5d) of GOQD based nanomaterial also reveals the aggregated spherical morphological architecture (indicated by arrow) which is highly corroborated with the TEM micrograph of nanomaterial.

Topographical studies The surface topology of FA-CH-GOQD nanomaterial and DOX loaded FA-CH-GOQD based nanomaterial has been investigated and their phase topography is illustrated in Figure 6.

Figure 6. 2D and 3D AFM images of (a) FA-CH-GOQD nanomaterial and (b) DOX loaded FACH-GOQD based nanomaterial. As observed from the AFM images (Figure 6a), GOQD based nanomaterial shows a porous surface morphology with a surface roughness value of ~190 µm. The resulting porous microstructure provides a favorable microenvironment for efficient drug loading. However, after DOX loading onto FA-CH-GOQD nanomaterial (Figure 6b), relatively smooth and 24


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homogeneous surface topology was observed, wherein the surface roughness value is decreased to ~20 nm. These reflect successful entrapment of drug molecule within the nanomaterials. These findings can be highly corroborated with the SEM micrographs described above.

Loading of DOX onto the GOQD based nanomaterial using UV-vis spectroscopy The UV-Vis spectrum of DOX loaded FA-CH-GOQD nanomaterial is represented in Figure 7. The characteristics peak around ~500 nm signifies the presence of DOX molecule.

Figure 7. UV-Vis absorption spectra of FA-CH-GOQD nanomaterial, DOX loaded nanomaterial, and DOX molecule The absorption peak is bathochromic shifted by ~20-30 nm for DOX loaded GOQD as compared to bare DOX which is perhaps due to the π-π stacking and hydrophobic interaction of DOX with FA-CH-GOQD nanomaterial.54 The loading efficiency of DOX onto FA-CH-GOQD nanomaterial is around 72±6 wt%. Inspired by this fact, it can be revealed that the drug molecule 25



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(DOX) is efficiently loaded onto FA-CH-GOQD which modulate its usage in chemotherapeutics and cellular bio-imaging applications.

Dynamic light scattering (DLS) measurement The surface functionalization and stability of the synthesized nanoproducts are further corroborated using zeta potential mesurement studies as elabortaed in Figure 8. The zeta potential is endowed to be -39.1 mV for DOX loaded FA-CH-GOQD nanomaterial which signifies good stability in solution phase. This may possibly be due to the availability of the free COO- group in the solution phase. After modification with FA-CH, the surface potential of pristine GOQD is found to be -30.2 mV revealing a negetive surface charge which is a crucial factor for in-vivo application.55 The synthesized FA-CH-GOQD based nanocarrier exhibits a narrow particle size distribution showing a polydispersity index value of 0.323. It is intriguing to note that the measured hydrodynamic sizes of both particles are quite larger than that of TEM micrographs. This can be attributed to the fact that DLS provides large aggregates of particles and the distribution pattern in its hydrated state while TEM just measures individual GOQD based nanomaterial and GOQD nanoparticles with distinct morphology. Additionally the presence of FA along with CH moiety accumulating water molecule onto the surface of nanomaterial which in turn result in increased hydrodynamic volume.56 Likewise the above synthesized formulation is highly promising for therapeutic applications in cancer diagnostics field.

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Figure 8. DLS spectra showing zeta potential of (a) DOX loaded FA-CH-GOQD nanomaterial, (b) FA-CH-GOQD nanomaterial, particle size distribution of (c) FA-CH-GOQD nanomaterial and GOQD.

In-vitro drug release To explore the wide utilization of DOX as a chemotherapeutic anticancer drug, the release behavior in in-vitro system has been monitored at different pH values~ 7.4, 6.5 and 5.5 which manifest a normal physiological range and an acidic tumor microenvironment, respectively. Prolonged doses of DOX exhibit severe chemotherapeutic side effects.

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Figure 9. In-vitro DOX release profile under different physiological pH.

By virtue of the distinct structure of GOQD based nanomaterial, this drawback can be deciphered adequately by exploring multi-functionalized GOQD as an effective drug delivery vehicle. In this context, DOX has been adopted as a model drug to insight drug release pattern of FA-CH-GOQD based nanomaterial.57,58,59

As depicted in Figure 9, at pH~7.4, the release profile of DOX is very slow from synthesized nanomaterial and only about ~12% of the total bound DOX drug has been released within 48 h. This can be ascribed to the deprotonation of the DOX molecule which is quite hydrophobic in nature, inducing π-π interaction and exhibiting inefficacious release of drug from 28


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the GOQD based nanomaterial system. It is also clearly seen from Figure 9 that in a pH~6.5 about ~22% of the drug released within 48 h from synthesized nanomaterial, which is a very common release pattern at this particular pH. In contrast under an acidic environment (pH~5.5), the release efficacy of DOX has been increased relatively. The release rate progressively decreased after 20 h whereas about ~ 57% of the total bound DOX was released within 48 h. This behavior is due to the inadequate interaction with the GOQD based nanomaterial under an acidic condition despite of the substantial drug release noticed in our FA-CH-GOQD based nanocarrier system. This is probably due to the initial burst release of a drug molecule from the domain of the saturated drug loaded nanomaterial. Further, this appreciable release can also be attributed to the presence of FA in the carrier molecule which possibly weakens the electrostatic interaction between the FA-CH-GOQD based nanomaterial and DOX molecule. Consequently based on these above facts it can be conferred that by tailoring GOQD, under physiological condition, DOX could be effectively loaded on it and released at a lower pH medium. This typical feature is highly valued for efficient drug delivery in cancerous tissues, intracellular lysosomes and endosomes. Thus, these above findings offer a mechanistic approach for drug release study which is highly desirable for cancer therapeutic application.

Effect of nanomaterial towards PBL and hemolysis The toxicity profile of the DOX loaded FA-CH-GOQD has been investigated in Figure 10 using hemolytic assay and DAPI staining micrographs of the blood lymphocyte cell. The hemolytic assay shows up to 30 µg/mL of nanomaterials concentration and no significant hemolysis is observed indicating no toxic response towards red blood cells. Upon treatment with various concentrations of nanomaterial the percentage of hemolysis is appeared within a range of 29



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5. Hence these used doses of nanomaterial are non-toxic and can be further utilized to monitor the therapeutic efficacy in cancer research.

Figure 10. (a) Hemolytic assay of various concentrations of DOX loaded FA-CH-GOQD based nanomaterial, and (b) Morphology of lymphocyte treated with (I) Control, (II) DOX, (III) FACH-GOQD, and (IV) DOX/FA-CH-GOQD.

Further to assess the cytotoxicity towards normal cells, DAPI staining micrographs of PBL cells have been performed and represented in Figure 10b. As observed no such prominent alteration of nuclear morphology is noticed in the case of FA-CH-GOQD based nanomaterial treated cells. In contrast, after incubating with DOX, the integrity of cell nuclei is marginally hampered which is perhaps due to the high loading of DOX molecule. Thus this synthesized DOX loaded nanomaterial can be used as a potent nanocarrier system for cancer diagnosis and therapy.

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Therapeutic efficacy of FA-CH-GOQD/DOX complexes To assess the cytotoxicity of DOX loaded FA-CH-GOQD based nanomaterial towards lung cancer cells (A549) and neoroblastoma (SH-SY5Y) cell line, MTT assay was employed and depicted in Figure 11. Both cells have been incubated with free DOX, FA-CH-GOQD based nanomaterial and DOX/FA-CH-GOQD nanomaterial for 24 h exposure. As shown in Figure 11a after incubating with FA-CH-GOQD no such prominent cytotoxicity has been noted even if at relatively high concentrations. Whereas in the case of SH-SY5Y cells DOX loaded FA-CH-GOQD exhibits a significant loss of cell viability as compared to A549 cells which implies that DOX can effectively deliver and possess notable selectivity to trigger the tumor site. Nonetheless, DOX exhibited higher cytotoxicity to SH-SY5Y cells facilitating a drastic drop in the cell viability. This distinctive phenomenon can also be justified due to the inadequate release of DOX molecule from the FACH-GOQD nanocarrier.

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Figure 11. In-vitro cytotoxicity assessment of free DOX, FA-CH-GOQD based nanomaterial and DOX loaded FA-CH-GOQD towards A549 and SH-SY5Y cell line: (a) FA-CH-GOQD based nanomaterial incubated cell lines, (b) A549 cell incubated with different concentration of free DOX and DOX/FA-CH-GOQD nanomaterial, and (c) SH-SY5Y cell treated with different concentration of free DOX and DOX/FA-CH-GOQD nanomaterial.

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Figure 12. Fluorescence microscopy images of SH-SY5Y and A549 cells incubated with FACH-GOQD/DOX nanomaterial. PBS treated cells were used as control. Each panel depicts 20 µm scale bar.

Further to apprehend the behavior of the synthesized nanomaterial in cell culture medium and their cellular drug delivery profile can be monitored through DAPI staining and assessed in fluorescence microscopy studies. Figure 12 elucidates the fluorescence images of the SH-SY5Y cell and A549 cell after being incubated with FA-CH-GOQD/DOX nanomaterial and free DOX using the red fluorescence to locate the trace of DOX molecule. A significant red fluorescence signal is inspected in the FA-CH-GOQD/DOX nanomaterial treated SH-SY5Y cells implying that DOX conjugated nanomaterial has been more effectually internalized by this cell than that of the A549 cells. However in the case of free DOX treated cell exhibits much less prominent fluorescence signal. This is possibly due to the futile absorption of the DOX molecule within the cell resulting in obvious quenching of fluorescence intensity. Consequently, from the above observation, it can be conferred that FA-CH-GOQD nanomaterial offers a suitable domain to load DOX and trigger its cellular internalization with higher efficacy. 33



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Cytomorphological analysis (in-vitro targeting) The cytomorphological changes of A549 cells treated with different nanoparticles and GOQD based nanomaterial has been explored and illustrated in Figure 13. As observed from Figure 13a, cells exposed to nanofillers show distinct morphological alteration. It reveals that GOQD based nanomaterial incubated cells lead to confluent cell aggregates with cell blebbing and subsequently cells appeared as clustered form. However no such prominent effect has been noticed in GOQD treated cell culture medium. Only the alteration of regular cell morphology with loss in cell membrane integrity is noted (Figure 13b)

Figure 13. Bright field images of cytomorphological changes of A549 cell treated with (a) control, (b) GOQD, (c) DOX, and (d) FA-CH-GOQD/DOX. Moreover a significant structural variation has been observed in DOX loaded GOQD based nanomaterial (Fig 13d) resulting in a total disrupted cell surface with the formation of 34


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floating apoptotic bodies. This can be attributed to the high toxic nature of the drug molecule (DOX).

Evaluation of cellular alteration induced by GOQD based nanomaterial To exploit the time dependent cytomorphological changes of A549 cells treated with synthesized DOX loaded FA-CH-GOQD based nanomaterial has also been monitored using SEM micrographs and illustrated in Figure 14.

Figure 14. Morphological changes of A549 cells at different incubation time (a) control, (b) 12 h, (c) 24 h, and (d) 48 h. As observed from Figure 14a, the A549 cell reveals a polygonal shape in control interphase, which exhibits the distinctive features of normal cell surface such as multiple 35



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microvilli along with extended lamellipodia and interconnected plasma membrane. The GOQD based nanomaterial treatment shows altered cellular morphology manifesting membrane shrinkage, rounding as well as significant blebbing of the plasma membrane. Besides that the condensation along with posterior cellular disintegration leads to the formation of apoptotic cell bodies which is evident from SEM micrographs (Figure 14c).60 Cells undergoing apoptotic protocols suffered a noticeable alteration of cellular morphology which can be advantageous to understand the cell death mechanism. The nucleus of apoptotic cells appear as bumpy shaped and exhibit structural remodeling with nodule type protrusion. Further Figure 14d, insights the late necrotic process as seen in the rupture of plasma membrane caused by the detachment of bubbles resulting in the gradual liberation of cytoplasmic content.61 The schematic diagram of cytomorphological alteration of the cancerous cell and the membrane rupture pattern has been shown in scheme 2.

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Scheme 2. Schematic representation of multifunctional GOQD based nanomaterial induced cell apoptosis.

Screening of cell morphology through EtBr-AO double staining Apoptotic morphology of the cancerous cells has been scrutinized and shown in Figure 15. The staining pattern deciphers a viable cell with intact green colored DNA showing as almost round in shape.

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Figure 15. Study of apoptotic morphology a) Control cancer cell line, b) DOX/FA-CH-GOQD based nanomaterial (5 µg/mL), and c) DOX/FA-CH-GOQD based nanomaterial (20 µg/mL) treated cells; yellow arrow indicates early apoptosis and red arrow indicates late apoptosis of the cell.

From Figure 15b, it is worthy to note that the early apoptotic cell appears as fragmented and is stained a slight orange in color. A typical characteristic morphology is noticed in the early apoptotic cell which includes plasma membrane blebbing with floating apoptotic bodies. Additionally after staining, most of the cells display a deep orange in color with quenched structure which imparts that the cells are undergoing a late apoptotic phase. Therefore, these

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above findings are highly impressive and highlight the cell death pattern of cancerous tissues, which suggest cell death occurs primarily through a apoptosis process.62

DNA damage inducing effect of FA-CH-GOQD/DOX nanomaterial The DNA damaging assessment has been carried out using comet assay which is a highly sensitive, rapid and reliable method to identify DNA damage within cells. To understand DNA fragmentation and its distortion pattern, this assay has been conducted focusing on parameters such as olive tail movement (OTM) and tail movement (TM). Control cells exhibit very small olive tail movement (OTM) that conveys the presence of unaltered DNA moiety with uniform membrane integrity.63 Interestingly, upon FA-CH-GOQD/DOX nanomaterial treatment, the DNA strand gets separated and released. Thereafter the detached DNA strands are migrated upon electrophoresis resulting in an increase in OTM. As observed in Figure 16 with increasing concentration of FACH-GOQD/DOX nanomaterial the tail DNA percentage is noticed to be appreciably higher than control cells whereas the percentage of head DNA is found to be decreasing successively. In the case of 20 µg/mL treated cells, the tail DNA percentage is reached at a maximum level (89.22%) indicating an obvious DNA damage and denaturation. Conversely 5 and 10 µg/mL nanomaterial treated cells show 38.16 and 44.56% of tail DNA whereas only 0.84% tail DNA is found in the control one.

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Figure 16. DNA damage inducing effect of DOX conjugated GOQD based nanomaterial on A549 cells (40×magnification) using comet assay: (a) control, (b) 5 µg/mL, (c) 10 µg/mL, and (d) 20 µg/mL.

Further TM and OTM are two important parameters to assess the comet score which is responsible for the disrupted DNA morphology. A highest TM (95.47) and OTM (61.29) are observed in 20 µg/mL treated cells providing significant comet score for DNA damage (Table 3).

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Hence from the aforementioned, it can be concluded that, after incubation with FA-CHGOQD/DOX, the double helix DNA strand splits into a single strand which leads to cell death of tumour tissues by inducing oxidative damage.

Table 3. Parameters of comet assay Comet assay parameters

Control

5 µg/mL

10 µg/mL

20 µg/mL

Comet head length

55

51

53

39

Comet tail length

4

41

67

107

Comet length

59

92

120

146

Head DNA (%)

99.15

61.8

55.40

10.77

Tail DNA (%)

0.84

38.16

44.59

89.22

Tail movement (TM)

0.03

15.64

29.87

95.47

Olive Tail movement (OTM)

0.23

15.92

20.02

61.29

41



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Scheme 3. Schematic pathway for morphology alteration of cancer cell after incubating with DOX loaded nanomaterial.

Anti-cancer efficacy of DOX loaded FA-CH-GOQD based nanomaterial Consistent with the above observations and to emphasize the potency of FA-CH-GOQD nanomaterial loaded DOX in chemotherapeutics, nuclear morphology has been speculated in the breast cancer cell line (MDA-MB-231).

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Figure 17. Cell incubated with a) 0 µg/mL, b) 5 µg/mL, c) 10 µg/mL, and d) 20 µg/mL of FACH-GOQD/DOX nanomaterial.

The nuclear morphological feature of different DOX loaded nanomaterial (5, 10 and 20 µg/mL) treated cells has been thoroughly scrutinized and illustrated in Figure 17. As evident in Figure 17, with increase in concentration of FA-CH-GOQD/DOX nanomaterial the nucleus gets fragmented and the integrity of the nuclear membrane is entirely disrupted. This is primarily due to the chromatin marginalization, inducing DOX release within the cellular system. In particular most of the cell nuclei appear as a round shape and are irregular in nature with ruptured cellular architecture which can also be validated from DAPI stain micrographs in Figure 17d. This additionally signifies the substantial adaptability of FA-CH-GOQD nanomaterial, as a promising 43



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nanocarrier for passive cellular internalization and release of DOX molecule to the targeted cancer cell (Scheme 3). Based on these above findings, the detailed study is in progress with this cell line. Further future research will also emphasize the prospect of this formulated nanocarrier system and tuning to its therapeutic efficacy in cancer therapy.

CONCLUSION In summary we have successfully designed a graphene oxide quantum dot based nanoparticle which can act as a good nanocarrier and efficiently deliver drug (DOX) in cancer chemotherapy. To tune its biocompatibility and cellular internalization GOQD has been tailored with CH and a tumor targeting moiety like FA respectively. FTIR study confirms the successful synthesis of FA-CH-GOQD based nanomaterial. UV-Vis study reveals the effective loading of DOX onto this nanomaterial through π-π stacking interaction. The zeta potential of GOQD based nanoparticle is found to be -39.1 mV showing appreciable stability. The synthesized GOQD based nanoformulation exhibits greater pH responsive DOX drug release behavior in acidic environment. Further this DOX loaded nanoparticle is efficiently internalized by A549 cells through receptor mediated endocytosis and shows potential cytotoxicity by a inducing cell death mechanism. The anticancer efficacy of this nanomaterial can also be validated from DAPI stain images of breast cancer cells indicating DNA fragmentation and loss in nuclear membrane integrity. Also DNA comet assay has been explored which signifies that DNA oxidative damage is alleviated in a dose dependent manner by FA-CH-GOQD/DOX nanomaterial. The findings herein infer that FA-CH-GOQD is a promising nanocarrier for targeted drug delivery of an anticancer drug with enhanced therapeutic efficacy. Finally we propose that this synthesized nanoparticle can serve as a versatile biocompatible nanovehicle to trigger the treatment of 44


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cancerous tissue and will be impressively accessible to clinics to offer a new theranostic window for better quality of life for cancer patients.

AUTHOR INFORMATION Corresponding Author * Tel: +91-9433379034; Fax: +91-33-2351-9755; E-mail: [email protected] Author Contributions S. De and K. Dutta synthesized the designed the nanocargo, experiments and wrote the paper. Invitro cell culture has been performed interpreted by K. Patra, D. Ghosh, A. Basu and A Dey. D. Chattopadhyay conceived and supervised the work. G. Sarkar, J. Maiti contributed to preparing manuscript. D.Rana, D. Chattopadhyay made intellectual contributions. All authors contributed to critical reading and thorough revision of this manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors Sriparna De gratefully acknowledge DST-SERB, Govt. of India, for providing Post-Doctoral fellowship under NPDF Scheme. We also acknowledge the Center for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta, for instrumental facilities. For AFM analysis we are highly grateful to Prof. Madhusudan Roy and Dr. Ramkrishna Dev Das, Saha Institute of Nuclear Physics, Kolkata.

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For Table of Contents Use Only Tailoring the efficacy of multifunctional biopolymeric-graphene oxide quantum dot based nanomaterial as nanocargo in cancer therapeutic application Sriparna De a, Kartik Patra b, Debatri Ghosh c,Koushik Duttaa Aditi Dey d, Gunjan Sarkar a, Jyotirmoy Maity b, Arijita Basu a, Dipak Rana e and Dipankar Chattopadhyay

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Tailoring the efficacy of multifunctional biopolymeric-graphene oxide

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