Biosynthesis and characterization of copper oxide nanoparticles from indigenous fungi and its effect of photothermolysis on human lung carcinoma

Accepted Manuscript Biosynthesis and characterization of copper oxide nanoparticles from indigenous fungi and its effect of photothermolysis on human lung carcinoma

Kandasamy Saravanakumar, Sabarathinam Shanmugam, Nipun Babu Varukattu, Davoodbasha MubarakAli, Kandasamy Kathiresan, Myeong-Hyeon Wang PII: DOI: Reference:

S1011-1344(18)30695-X https://doi.org/10.1016/j.jphotobiol.2018.11.017 JPB 11407

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

29 June 2018 12 October 2018 23 November 2018

Please cite this article as: Kandasamy Saravanakumar, Sabarathinam Shanmugam, Nipun Babu Varukattu, Davoodbasha MubarakAli, Kandasamy Kathiresan, Myeong-Hyeon Wang , Biosynthesis and characterization of copper oxide nanoparticles from indigenous fungi and its effect of photothermolysis on human lung carcinoma. Jpb (2018), https://doi.org/10.1016/j.jphotobiol.2018.11.017

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ACCEPTED MANUSCRIPT

Biosynthesis and characterization of copper oxide nanoparticles from indigenous fungi and its effect of photothermolysis on human lung carcinoma Kandasamy Saravanakumara, Sabarathinam Shanmugamb, Nipun Babu Varukattuc, Davoodbasha

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MubarakAlid, Kandasamy Kathiresane and Myeong-Hyeon Wanga,* [email protected] Department of Medical Biotechnology, College of Biomedical Sciences, Kangwon National

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University, Chuncheon, Gangwon, 24341, Republic of Korea

Bioprocess and Biomaterials Laboratory, Department of Microbial Biotechnology, Bharathiar

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University, Coimbatore, 641046, India. Present Address: Department of Biology, Shantou

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University, Shantou, Guangdong, China

Proteomics & Molecular Cell Physiology Lab, Department of Zoology, Bharathiar University,

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Coimbatore-641 046, Tamil Nadu, India. Present Address: Shantou University Medical College,

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Guangdong, China

School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology,

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Chennai, Tamil Nadu, India e

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University,

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Parangipettai 608 502, Tamil Nadu, India *

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Corresponding author at: Department of Medical Biotechnology, College of Biomedical Science,

Kangwon National University, Chuncheon, Gangwon, 24341, Republic of Korea. Abstract

In this report, copper oxide nanoparticles (TA-CuO NPs) were synthesized using cell-free extract of Trichoderma asperellum and assessed their photothermal induced anticancerous activity. The fungal mediated TA-CuO NPs was confirmed by the surface plasmon resonance at 285-295 nm. The amide (C=O) and aromatic (C=C) groups in secondary metabolites of the extract was found to

ACCEPTED MANUSCRIPT be an encapsulating or reducing agents for TA-CuO NPs, as indicated by IR spectra. Crystalline nature by cubic face-centered structure of the TA-CuO NPs was confirmed by XRD and their size ranges from 10 to 190 nm and an average of 110 nm by particle size analyzer (PSA). The Ultra HRSEM study revealed spherical shaped TA-CuO NPs. The FETEM results were also in strong

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agreement with PSA and UHR SEM. The survey-scan spectrum of XPS indicated the presence of

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C1s (47.83%), Cu2p (16.11%), Na1s (2.2%) and O1s (33.86%). The cell death was significantly

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found higher in photothermal induced by near-infrared laser (TA-CuO NPs-NIR) treated than that of TA-CuO NPs treatment. The level of ROS (35.62 %) was higher in the treated cells than that of the

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untreated control, in accordance with the nucleus damage and losses in the mitochondrial membrane

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potential (ΔΨm). the upregulation of Bcl-2 in untreated cells and Cas-3 in TA-CuO NPs-NIR treated cells was confirmed by western blot analysis. This work agreed the potential biogenic TA-CuO NPs

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for promising in vitro photothermolysis of cancer cells, for the development of anticancer

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nanotherapeutics.

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anticancer, apoptosis.

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Keywords: Trichoderma, copper oxide nanoparticles, photothermal activity, human lung carcinoma,

1. Introduction

Cancer is an abnormal growth of cells and recognized as a life-threatening human disease with an increased rate of morbidity and mortality worldwide [1, 2]. The chemotherapeutic agents including alkylating metabolites exhibiting poor specificity on targeting cancer cells, which cause the adverse effects to the normal cells [3]. The discovery of cancer drugs in combination with smart controlled drug delivery system targeting the cancer cells is more effective and biocompatible [4]. In the

ACCEPTED MANUSCRIPT modern drug discovery, nanotechnology received much attention on the manufacture of smart biomaterials that can revolutionize as nanomedicine with versatile properties of increased drug release and efficiency for diagnosis or treatment of various human ailments [5, 6]. Several metallic materials including gold [7, 8], silver [9, 10], iron [11]

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are reported to be

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such as Hep2 [12], HT-29 [13], MCF-7 [14], and A549 [4] respectively.

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antimicrobials or anticancer agents against various bacterial and fungal pathogens and cancer cells

Photothermal therapy (PTT) is advantageous over other conventional cancer therapy such as

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chemotherapy or radiotherapy due to its less or no toxicity, cancer-site specificity, minimal invasion, least side effects and fast recovery [15, 16]. PTT agents are inserted in cancer cells to absorb and

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convert the light sources (UV, visible or NIR) into thermal energy and to generate the heat and

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reactive oxygen species (ROS) for ablating the cancer cells without harming the normal cells [17]. inorganic nanomaterials such as gold, silver, copper sulfide/oxide, palladium, titanium oxide

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nanoparticles, composite metallic nanostructures and carbon nanotubes are reported to be efficient

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PTT agents to convert the photo energy to heat as ablation therapy against cancer cells [15, 18]. Among these nanomaterials, CuO NPs received much attention on their bioactive applications due to

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its versatile functions such as catalysis, photoconductivity and photothermal [19], antimicrobial [20],

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anticancer [1, 21], biocidal [22] [23]. In addition, CuO NPs are less-toxic, high chemical and physical stability [19] and long self-life, as compared to organic antimicrobial agents [24]. Fungal genus, Trichoderma is notorious to produce various novel metabolites with bioactivities [25] and has the ability to synthesis the anti-pathogenic silver nanoparticles [26]. To the best of our knowledge, there is no report on the utilization of Trichoderma strains on the synthesis of CuO NPs and that of these nanoparticles as PTT agent for cancer therapy. The present work, therefore, was attempted for the first time to synthesis CuO NPs using the Trichoderma asperellum

ACCEPTED MANUSCRIPT (TA-CuO NPs) and as a photothermal agent to convert the near-infrared light energy into heat and ROS for ablating the cancer cells. 2. Materials and Methods

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2.1. Chemicals, cell lines, and fungal strains

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Copper nitrate (Cu(NO3)2. 3H2O, 99.0 %), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), - 4′,6-

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diamidino-2-phenylindole (DAPI), and Rhodamine-123 were purchased from Sigma-Aldrich,

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Yongin, Republic Korea, Water-soluble tetrazolium (WST)-1 assay kit (EZ-CyTox, Daeil Lab Service, Republic of Korea), Fetal bovine serum (FBS, Gibco), antibiotics, penicillin and

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streptomycin (Gibco), Dulbecco’s modified eagle medium (DMEM, Gibco) were procured from

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Thermo Fisher scientific, Republic of Korea. All the other analytical grade of chemicals and reagents

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were procured from SUNtek scientific utility network, Chuncheon, Republic of Korea. Trichoderma asperellum (SKCGW003) GenBank (NCBI) accession number MG552071,

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used in the study was previously isolated from the sediment collected from coastal wetland,

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Gangwan do, the Republic of Korea using the potato dextrose agar medium (PDA). Trichoderma strains were preserved in glycerol (20%) in a cryovial at -80°C for future use. The human lung

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carcinoma, A549 cell line (KCLB-10185) was obtained from Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and cultured in DMEM containing 10% FBS at 37°C in a 5% CO2 humidified incubator for 24 h to achieve 70-80% confluent followed by preservation in Cell Freezing Media (DMSO, BCS) in liquid nitrogen in a cryogenic vial for the experimental use. 2.2. Preparation of mycelial extract

ACCEPTED MANUSCRIPT Trichoderma asperellum strain SKCGW003 was grown in the growth medium as described previously [27], containing (g.L-1) KH2PO4 (7), K2HPO4 (2), MgSO4.7H2O (0.1), (NH2)SO4 (1), yeast extract (0.6), glucose (10) in an orbital shaker at 28°C, 180 rpm for four days. After the incubation period, the mycelial biomass was harvested by filtration using Whatman No.1 filter paper.

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The medium debris in the mycelia was removed through washing with distilled water for several

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times. To obtain the mycelial water extract, a weight of 20 g mycelial biomass was added into an

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Erlenmeyer flask containing 100 mL of double distilled water and mixed well, then it was kept in the agitator at 180 rpm in room temperature for 2 h. Finally, the mycelial-free water extract (TA-CFE)

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2.3. Synthesis and characterization of CuO NPs

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was obtained by filtration and used for the synthesis of TA-CuO NPs.

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To prepare the TA-CuO NPs, 5 mM of Cu (NO3)2.3H2O was dissolved in 100 mL of TA-CFE in a

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200 mL Erlenmeyer flask and then the solution was stirred at 40 °C for overnight in dark condition followed by heating at 75-80 °C for 120 min. Finally, the product was cooled at room temperature

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then the dark brown TA-CuO NPs was harvested by centrifugation at 16000 rpm for 15 min, and

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then washed with distilled water. Finally, the product of TA-CuO NPs was dried at 80°C for 2 h and then subjected to the biomaterial characterization such as UV spectrophotometer (Optizen 2120UV,

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Korea) ranging from 300-700 nm. The shifting of molecules due to formation of TA-Cu NPs was observed by Fourier-transform infrared spectroscopy (FTIR, PerkinElmer Paragon 500, USA), and X-ray diffractometer (X’pert-pro MPD- PANalytical, Netherland), operated at 40 keV with Cu κα radiation in θ-2θ, The particle size was analyzed using particle size analyzer (PSA, Malvern Mastersizer 2000, Britain), Ultra high resolution scanning electron microscope (UHR-SEM:S-4800; Hitachi, Japan), equipped with energy-dispersive spectroscopy (EDS) and transmission electron

ACCEPTED MANUSCRIPT microscope (TEM, JEOL-JSM 1200EX, Japan). X-ray photoelectron spectroscopy (XPS) was used for chemical analysis (Thermo Scientific™ K-Alpha™). 2.4. Photothermal based anticancer activity

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Effect of photothermal induced TA-CuO NPs by near-infrared laser (NIR) and non photothermal

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induced TA-CuO NPs on cytotoxicity of A549 cells was analyzed using the WST-1[28]. The human

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lung carcinoma A549 cells (1 x 105 cells.mL-1) were seeded in 96 well plates (coster) containing the DMEM culture medium incorporated with 10% FBS and 1% of antibiotic solutions and incubated in

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a 5% CO2 humidified incubator for 24 h to reach 80-90% confluence. Then the culture medium was

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replaced with fresh culture medium of DMEM with various concentrations of TA-CuO NPs (3.625, 6.25, 125, 25, 100,125, 250, 500, 1000 µg.mL-1) and incubated in the humidified atmosphere as

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mentioned above. Then the cells were irradiated with NIR at 2W/cm2 of 808 nm for 5 min then the

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cells were kept in 5% CO2 incubator for 24 h. The cell viability was determined in heat-shocked with TA-CuO NPs treated and untreated control cells by the WST-1 method according to manufacturer’s

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instructions. The experiments were contacted in three independent trials with three replicates for

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each trial and the cell viability results are presented as a mean and standard error. Further, the effect of photothermal induced TA-CuO NPs treatment on A549 cells was determined for morphological

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changes using the methods described previously [9, 29]. The ROS generation was observed using the DCFH-DA stain with excitation of 495 nm and emission of 529 nm. The nucleus damage was observed by DAPI with excitation of 358 nm and emission of 461 nm, and then the apoptosis index was calculated based on mitochondrial membrane potential (ΔΨm) using the Rhodamine-123 with excitation of 488 nm and emission of 525 nm. All the fluorescence images were taken using the fluorescence microscope (Olympus, CKX53 culture microscope, Japan).

ACCEPTED MANUSCRIPT 2.5. Western blot analysis The effect of the photothermal induced TA-CuO NPs was studied on the expression of apoptosis-related protein (Bcl 2 and Cas 3) in A549 cells, treated with TA-CuO NPs (IC50 concentration) in comparison with untreated (control) using the western blot analysis according to

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the protocols described earlier [9]. In detail, A549 cells (1 x 105) were seeded in a T25 flask

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containing DMEM medium and incubated in 5% CO2 incubator for 24 h. Then, the medium was removed and washed with phosphate buffer saline (PBS) and then added with freshly prepared

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DMEM incorporated with an IC50 concentration of TA-CuO NPs (24.7 µg.mL-1). The plates were kept under NIR irradiation (laser at 2W/cm2 of 808 nm) for 5 min. Then, the treated and untreated

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cells were again incubated for 24 h at the same humidified atmosphere. The treated and untreated

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cells were collected and dissolved in lysis buffer (including cocktail inhibitor, Thermo Fishers Scientific Korea Ltd, Republic of Korea) with 30s sonication and centrifuged at 12000 rpm at 4°C

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for 15 for the extraction of protein. Then the concentration of protein was estimated by Bradford

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assay [40]. For protein separation, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) was used and the SDS-PAGE gel was transferred to 0.2 µm immune-Blot PVDF

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membrane (Bio-Rad Laboratories, CA, USA). The membrane was immersed in blocking buffer (5%

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BSA, Tris-buffer saline with 0.1% Tween 20 (TBST)) for 1 hour at ambient temperature. The primary antibody was sprayed on the membrane and incubated at 4°C for 12 h and then the TBST was used to remove the primary antibody. Followed by this, the secondary antibody (1:2000 in TBST) was sprayed and incubated at 4°C for 1 h. Finally, the protein band intensity was measured using ECL reagent kit (Bio-Rad, USA). 3. Results and discussion

ACCEPTED MANUSCRIPT Bionanomaterials mediated photothermal therapy is relatively less known for the treatment of cancers [30, 31]. Presence of the flavonoids, glycosides, amino acids, phenolics, saponins and tannins in the plant or microbial cell-free extracts are involved as catalysts or capping or reducing agents for the biosynthesis of nanoparticles [32]. Trichoderma strains are known to produce a wide

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verity of secondary metabolites including peptaibols, polyketides, pyrones, terpenes, and

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diketopiperazine [33], enzymes and glycolipids [34], but they are not utilized in the synthesis of

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CuO NPs. The present work successfully synthesized the TA-CuO NPs using the TA-CFE,

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characterized and also studied in vitro photothermal induced therapy for A549 cancer cells.

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3.1. Characterization of TA-CuONPs

TA-CFE mediated synthesis of TA-CuO NPs were confirmed through color changes from light

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yellow to dark brown. The UV-Vis scanning (200-500 nm) indicated the strong surface plasmon

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resonance peak between 285-295 nm whereas no significant peak with TA-CFE (Fig.1a), which confirmed the presence of TA-CuO NPs in accordance with earlier reports [21, 35]. In general,

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copper reacts with hydroxyl anion (OH-) of water to form the copper hydroxide and then the input of

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TA-CFE containing compounds or enzymes and proteins converts the copper hydroxide into copper nanoparticles in oxide form, which is similar to the aqueous plant extracts mediated synthesis of

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nanomaterials [36].

FTIR analysis of the biogenic synthesized TA-CuO NPs is shown in Fig.1b. This enables to predict functional molecules involved in the synthesis process [21, 37]. The TA-CFE showed the IR peaks in 3109 cm-1 (C-H, stretch, alkene), 1669 cm-1 (C=O, amide, strong), 1064 cm-1 (C=O, stretch, ester), 981 cm-1 (C=C, strong), 865 cm-1 (C-H, strong), 605 cm-1 (C-Br, halo compounds), and 463 cm-1 (C-I). Whereas the TA-CuO NPs peaked at 3305 cm-1 (O-H, strong H bonded), 2923 cm-1 (C-H,

ACCEPTED MANUSCRIPT stretch, alkane, strong), 2163 cm-1 (C≡C, stretch), 2031 cm-1 (C=C=C, allene, medium), 1640 cm-1 (C=C, amide, stretch), 1428 cm-1 (O-H, bending, carboxylic group), 1033 cm-1 (C-N, amine, stretch), 877 cm-1 (C-H bending), and 471 cm-1 shifts and these confirmed vibration of the Cu-O in TA-CuO NPs [1, 21, 38]. In addition, the presence of the amide groups (C=O) and aromatic secondary

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metabolites (C=C) [1] in TA-CFE served as encapsulating or reducing agent for the synthesis of TA-

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CuO NPs.

X-ray diffraction patterns based characterization of the TA-CuO NPs exhibited the planes

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(110), (002), (111), (-112), (-202), (020), (-202), (-113), (022), (220), (311), and (004) (Fig.1c) based on the d spacing [A] values (Table.S1) and it confirmed the crystalline nature and cubic face-

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centered structure in agreement with the joint committee on powder diffraction standards (JCPDS.

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96-901-5925) [1, 21]. The size of the synthesized TA-CuO NPs ranged from 10 to 190 nm with an average size of 110 nm were revealed by particle size analysis (Fig.1d). The UHR SEM observation

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revealed the spherical shaped, evenly agglomerated TA-CuO NPs (Fig. 2a). EDS results confirmed

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the presence of copper (31.71%) and oxide (14.83 %) (Fig.2b, c,d). This is in accordance with the

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earlier report of the EDS analysis of CuO NPs [21]. The FE TEM analysis revealed the evenly agglomerated TA-CuO NPs (Fig. 2e,f) and this was also

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in agreement with the PSA and UHR SEM analysis of the present study. The XPS analysis of TACuO NPs based on survey-scan spectrum indicated the quantification of elemental atoms as C1s (47.83%), Cu2p (16.11%), Na1s (2.2%) and O1s (33.86%) (Fig.3a) in accordance with highresolution spectra of O1s (Fig. 3b). The Cu2p scan indicated the strong fitting peak in 932 eV and 933 eV for Cu 2p3/2, 952 eV, 940 eV, 949 eV, 956 eV and 961 eV for Cu2p the earlier reports [36, 39].

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(Fig. 3c), similar to

ACCEPTED MANUSCRIPT 3.2. Photothermal induced the anticancer activity of TA-CuO NPs The nanomaterials based therapy are extensively used in the treatment of cancers due to its permeability, biocompatibility, and biodegradability [40]. The present work assessed the TA-CuO NPs (NIR) mediated photothermolysis and cytotoxicity of TA-CuO NPs of cancer cells by WST-1

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assay followed by ROS generation, and various cellular observations in A549 cancer cells. The cell death was observed to be significantly increased with the concentration of TA-CuO NPs. Further, the

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IC50 was found lower in TA-CuO NPs-NIR (24.7 µg.mL-1) than TA-CuO NPs (40.625 µg.mL-1)

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treated cells alone (Fig.4a) due to the photothermal induced heat mediated ablation of cancer cells [15, 18]. Further studies on the morphological and ROS generation using the fluorescent signal of

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DCFH-DA were attempted [41] and the results exhibited that TA-CuO NPs-NIR treated A549 cells

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showed higher fluorescence intensity (Fig.4b,c) with increased level of ROS (35.62 %) than the untreated control cells with ROS level of 8.6 % (Fig. 4d). This result was also in accordance with the

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nucleus staining of DAPI and mitochondrial membrane potential (ΔΨm) loss. There was a

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significant loss of ΔΨm in treated cells as compared to untreated cells (Fig.5a) as evident by the loss

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of probe Rhodamine-123 binding capacity [42, 43]. The apoptosis index was found to be 15.26 for untreated cells and 79.82 for TA-CuONPs-

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NIR (Fig. 5b). The present results indicated that photothermal application of the TA-CuO NPs-NIR induced the ablation of cancer cells through activation of ROS generation followed by alteration of ΔΨm function that led to cancer cell death [44]. Moreover, the pathways involved in cancer cell division and cell death [45], in which the pro-apoptosis and anti-apoptosis related proteins are the hallmarks for predication of drug-induced cell death [46]. Hence, to further confirm the present results, the western blot analysis of the apoptosis-related proteins expression was made. This analysis revealed that the expression of Bcl-2 (protein % of 30.12) was higher in untreated cells than

ACCEPTED MANUSCRIPT that in treated cells, whereas the Caspase 3 expression was found higher (protein % of 48.95) in TACuO NPs-NIR treated cells (Fig.5c, d). The Bcl-2 and Cas-3 are used as signaling proteins that regulate the apoptosis, in which the Bcl-2 that occurs in outer mitochondrial membrane promotes the cell survival and inhibits the pro-apoptosis related proteins [47, 48], whereas the Cas-3 is a pro-

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apoptosis protein that triggers the cancer cell death [49]. The western blot results revealed the up-

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regulation of Bcl-2 in untreated cells and Cas-3 in TA-CuO NPs treated cells, which is in accordance

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with the study reported previously [9].

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4. Conclusion

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In the present study, TA-CuO NPs were synthesized using the extract of Trichoderma asperellum (SKCGW003) and characterized adopting high throughput techniques, UHR SEM with

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EDS, FE-TEM, XPS, XRD, and PSA. It was found that TA-CuO NPs shown a crystalline and

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spherical shaped particles with an average size of 110 nm. TA-CuO NPs induced photothermolysis of A549 cancer cells by ROS generation, nucleus damage, mitochondrial membrane potential (ΔΨm)

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and regulatory protein expression. On the whole, this work reporting anticancer property of biogenic

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TA-CuO NPs through photothermolysis for the first time, which could be used further as therapeutics as CuO NPs in modern medicine.

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Fig.1. Characterization of TA-CuONPs synthesized by Trichoderma asperellum: Ultraviolet-visible spectroscopy (a), Fourier-transform infrared spectroscopy spectrum (b), 2(c), particle size analysis (d). Fig.2. Characterization of TA-CuONPs synthesized by Trichoderma asperellum: Ultra highresolution scanning electron microscopy analysis SEM (a), EDS mapping of Cu (b), EDS mapping of O (c), EDS spectrum of Cu and O (d), Filed Emission Transmission Electron microscopy images of TA-CuONPs (e,f) Fig.3. Survey-scan XPS spectrum (a) high resolution spectra of O1s (b) and cu2p(c) scan for TACuONPs

ACCEPTED MANUSCRIPT Fig.4. Effect of TA-CuONPs and NIR induced TA-CuO NPs on cell viability human lung cancer cells A549 (a) ROS generation, untreated control kits (b), TA-CuONPs (NIR) treated cells (c), determination of relative ROS level (d) Fig.5. DAPI and Rh123 staining to observe the nucleus and mitochondrial damage in A549 cells (a) determination of apoptosis index based Rh123 staining intensity (b), western blot analysis of gene expression (c), determination of protein (d)

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Conflict of interest

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The authors declare that they have no conflict of interest Acknowledgments

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This work was supported by Korea Research Fellowship Program through the National Research

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Foundation of Korea (NRF) funded by the Ministry of Science, ICT (2017H1D3A1A01052610). This work was partially supported by Brain Korea 21 PLUS. We also thankful to the Central

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laboratory, Kangwon National University, Chuncheon, Korea for material characterization and K.K

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thankful to the UGC, New Delhi.

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CuO NPs were synthesized from water extracts Trichoderma sp.

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High through-put techniques were applied to characterize CuO NPs

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The synthesized CuO NPs were used for photothermolysis of cancer cells

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CuO NPs exhibited the photothermolysis of A549 cancer cells

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