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Cancer cell specific delivery of Photosystem I through integrin targeted liposome shows significant anticancer activity Abhijit Saha, Saswat Mohapatra, Gaurav Das, Batakrishna Jana, Subhajit Ghosh, Debmalya Bhunia, and Surajit Ghosh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13352 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 11, 2016

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ACS Applied Materials & Interfaces

Cancer Cell Specific Delivery of Photosystem I Through Integrin Targeted Liposome Shows Significant Anticancer Activity Abhijit Saha,1 Saswat Mohapatra,2 Gaurav Das, 2 Batakrishna Jana,1 Subhajit Ghosh,1 Debmalya Bhunia,1 Surajit Ghosh*1,2 1. Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India. Fax: +91-33-24735197/0284; Tel: +91-33-2499-5872 2. Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700 032, India. * To whom Correspondence should be addressed CORRESPONDING AUTHOR INFORMATION: Fax: +91-33-2473-5197/0284; Tel: +91-33-2499-5872; E-mail: [email protected]

KEYWORDS: Photosystem I, reactive oxygen species, mitochondrial membrane potential, caspase3, apoptosis, cancer, lipopeptide, integrin targeted delivery.

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ABSTRACT: Many anti-cancer drugs are developed for the treatment of cancer from natural sources. Photo System I (PSI), a protein complex present in the chloroplast is involved in photosynthesis and generates ROS in plant. Here, we have used the ROS generation property of PSI for cancer therapy. We have shown that PSI can enter into different kinds of cancer cell like human lung carcinoma (A549) and mouse melanoma (B16F10) cell lines and generates ROS inside the cells. It inhibits the proliferation of cancer cell and causes apoptotic death of cancer cells. We have also shown that PSI induces apoptosis through mitochondria dependent internal pathway, induces caspase3, causes DNA fragmentation and arrests cell cycle at SubG0 phase. We also prepared, using C16-LDV lipopeptide [C16 long chain attached on the N-terminal of the tri-peptide containing amino acids leucine (L), aspartic acid (D) and valine (V) abbreviated as NH2-LDV-COOH], α4β1 integrin targeted liposomal formulation of PSI which specifically kills the cancer cell without affecting normal cells and it is found to be more potent compared to clinically used drug doxorubicin. Finally, we have found that LDV liposomal formulation of PSI inhibits the growth of tumor in C57BL/6J mice model. INTRODUCTION Photosystem I (PSI) is a large multi-protein light-harvesting complex, which generates the most negative redox potential and reactive oxygen species (ROS) in plant.1-2 Recently, various ROS generator has been used as anticancer agent in various cancer cells as well as chemo-sensitive enhancer against drug resistant cancer cell lines.3-7 Medicinal products from nature have been used for therapeutics against various diseases including cancer from ancient days. In modern chemotherapy, around 75% of the anticancer agents were derived from natural products.8-15 Recently, Photosystem I (PSI) from various sources has been extensively studied for developing novel bio-photovoltaic devices or generation of solar fuel.16-25 However, medicinal aspect of PSI


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has not been explored yet. Considering the ROS generation ability of PSI in plant, in this manuscript, we have studied whether PSI is still able to generate ROS inside the cancer cell or not. Interestingly, we found that PSI, isolated from Indian spinach leaves Spinacia Oleracea, generates significant amount of ROS inside the human lung cancer cell (A549) and mouse melanoma cancer cell (B16F10), induces apoptosis through mitochondria dependent internal pathway, induces caspase3, causes DNA fragmentation and arrests cell cycle at SubG0 phase. However, due to non-specificity of PSI, it kills both cancer and normal cells. Therefore, we have made a α4β1 integrin receptor targeted liposomal formulation of PSI which specifically targets and kills B16F10 cancer cell without affecting the normal lung fibroblast cell (WI38) (Scheme 1). Moreover, liposomal formulation of PSI is found to be more potent compared to clinically used drug doxorubicin. Finally, we have found that LDV liposomal formulation of PSI inhibits the growth of tumor in C57BL/6J mice melanona model.

EXPERIMENTAL SECTION Materials Dimethylsulfoxide (DMSO) and MeOH were purchased from Spectrochem. TritonX-100, sucrose and sorbitol were purchased from SRL. 2-[4-(2-hydroxyethyl)-piperazin-1-yl]ethanesulfonic acid (HEPES), glycine and bis-acrylamide solution (30%) were purchased from Himedia. Ammonium persulfate (APS) and coomasie brilliant blue were purchased from Fisher scientific. Sodium dodecyl sulfate, succinic anhydride, triethylamine (Et3N), tricine, N, N´dimethylformamide (DMF) and trifluoroacetic acids (TFA) were purchased from Merck. N, N, N´, N´-Tetramethylethylenediamine (TEMED) was purchased from Fluka. Ethylene-bis(oxyethylenenitrilo)-tetraacetic acid (EGTA), 4-Piperazinediethanesulfonic acid (PIPES), bovine



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serum albumin (BSA), guanosine-5´-triphosphate sodium salt hydrate (GTP), 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dulbecco’s modified eagle’s medium (DMEM), 2, 7-dichlorofluorescein diacetate (DCF-DA), dihydroethidium (DHE), MES, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), bovine serum albumin (BSA), trypsinEDTA solution, Kanamycin sulfate, dimethylsulfoxide for cell culture and formaldehyde solution for molecular biology were purchased from Sigma Aldrich. Penicillin-Streptomycin, neutravidin and fetal bovine serum (FBS) were purchased from Invitrogen. Annexin V apoptosis detection kit was purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-Mad2 antibody, rabbit monoclonal anti-alpha Tubulin (EP1332Y) antibody and Goat polyclonal antiRabbit IgG H&L (Cy3.5 ®) pre-adsorbed were purchased from Abcam. Mouse monoclonal antiBubR1 (human) was purchased from MBL International Corporation. Goat anti-mouse IgG fluorescein conjugated was purchased from Millipore. Bisbenzimide H 33258 (hoechst) was purchased from Calbiochem. Cover glass bottom dishes were purchased from SPL. Wang resin (100-200 mesh) and all amino acids for solid phase synthesis of LDV peptide were purchased from Novabiochem. The peptide was purified in Shimadzu HPLC system (LC 20AP) using reverse phase C18 column (Waters). All compounds were used without further purification. Isolation of PSI from spinach Isolation of PSI from spinach leaves was performed following previously described method.26 Briefly, freshly procured 500 g of Indian spinach leaves were washed with plenty of miliQ water, homogenized in 400 mM sorbitol and 50 mM tricine-KOH (pH=7.8) in water using liquid nitrogen. The mixture was passed through double layer of Mira-cloth. The filtrate was centrifuged at 1000g for 5 minutes. Pellet was re-suspended in 50 mM sorbitol, 5 mM EDTANaOH and 50 mM tricine-KOH (pH=7.8). The mixture was centrifuged at 10000g for 5 minutes.


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Pellet was re-suspended in small volume of water. This solution contains total chlorophyll. We have taken the absorbance (A) of the chlorophyll solution at 664 nm and 648 nm and concentration of total chlorophyll was calculated using the formula [C chlorophyll (a + b) = (5.24 X A664 + 22.24 X A648) µg/mL]. We have added 0.8% (w/v) triton-X-100 to the chlorophyll solution and stirred for 30 minutes at room temperature to separate the PSI from other chloroplast complexes. It was centrifuged at 42000g for 30 minutes and supernatant was collected. Supernatant was loaded on the 25 mL of linear sucrose gradient (0.1M to 2M) and centrifuged at 100000g at 4 oC for 18 hours. A dark green band present at the interface of 1M and 2M sucrose solution was collected. It was homogenized, aliquots were made and stored at 80 oC. PSI concentration was determined from absorbance measurement at 595 nm using Bradford reagent and BSA standard curve. SDS-PAGE gel electrophoresis of PSI Gel electrophoresis was performed in a Bio-Rad gel apparatus using 12% separating gel and a voltage of 40V. We have used 30% acrylamide / bisacrylamide solution for preparation of separating gel and stacking gel along with 10% SDS, 10% APS and TEMED. We have used tris buffer of pH 8.8 and 6.8 for the preparation of separating gel and stacking gel respectively. We have loaded 20 µL of PSI (62.5 µg/mL) into the wells. Gel was stained with coomasie brilliant blue solution and de-stained with 10% acetic acid. Absorption and fluorescence spectra of PSI We have recorded the absorption spectra of PSI in water at room temperature. We have found two absorption maxima near 440 nm and 675 nm. We have also taken the fluorescence spectra of PSI. We have found only one strong peak near 680 nm and a hump near 710 nm when it was



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excited at 675 nm at room temperature. This fluorescence is due to the chlorophyll present in the PSI. Cell culture We have purchased A549, B16F10, A375, HeLa and WI38 cell lines from National Centre for Cell Science (NCCS) Pune, India. Cells were cultured in our lab supplemented with 5% CO2 humidified atmosphere at 37 oC using dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum, kanamycin sulfate (110 mg/L), penicillin (50 units/mL), streptomycin (50 µg/mL) and we have used trypsin-EDTA (1X) solution for cell splitting. Cellular uptake study We have observed the cellular uptake of PSI in different cell lines (A549, B16F10 and WI38). Cells were seeded in a cover glass bottom dish before 24 hours of treatment. We have prepared 15.62 µg/mL solution of PSI in serum free media and cells were incubated with PSI for 4 hours. Cells were washed by media and live cell images were captured through an Andor spinning disc confocal fluorescence microscope with a 60X objective (Olympus) and an Andor iXon3 897 EMCCD camera in DIC, DAPI (405 nm), FITC (488 nm), TRITC (561 nm) and Cy5 channel (638 nm) channels. Live cells were imaged through a Nikon Ti-U eclipse fluorescence microscope with a 40X objective in bright field, DAPI, FITC, TRITC and Cy5 channels. Cellular uptake study of PSI in A549 cell using flow cytometry A549 cells were seeded in a 6-well plate at density of ~5 X 105 cells per well before 24h of treatment. Cells were treated with 15.62 µg/mL solution of PSI in serum free media for 4h. Cells were washed with phosphate buffer and trypsinized. Cellular uptake of PSI in A549 cells was analyzed by FACS using APC-A (Ex - 640 and Em - 633 to 677) with respect to control A549 cells (cells were not treated with PSI).


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Cellular morphology study A549 cells were seeded in a cover glass bottom dish before 24 hours of treatment. We have prepared 15.62 µg/mL solution of PSI in serum free media and cells were incubated with PSI for 4 hours. After that cells were incubated in complete media for 12h, 24h and 48h. Cells were washed by media and live cells were imaged through a Nikon Ti-U eclipse fluorescence microscope with a 40X objective in bright field through an Andor iXon3 897 EMCCD camera. Cell viability assay We have treated A549, B16F10 and WI38 cells with the PSI and cellular viability was checked using MTT assay. It is a colorimetric assay where MTT, a yellow tetrazole compound, is reduced to purple formazan by cellular reductase enzymes of live cells. Dead cells cannot do this type of reduction and are not able to develop the purple colour. Therefore, we can get an idea about the amount of cells that are live after the treatment of PSI with respect to those cells (control) which are not treated with the compound. The live cells are called as viable cells and it is denoted as %viability in the Y-axis. We have used different concentrations of PSI in the X-axis. We have measured the absorbance at 550 nm in Multiskan™ GO Microplate Spectrophotometer and calculated the %viability. Cells were seeded at a density of 10000 cells per well in 96-well plate before 24 hours of treatment. Cells were treated with 0.97, 1.9, 3.9, 7.8, 15.62 and 31.25 µg/mL solutions of PSI in serum free media for 4 hours. After that cells were incubated for 48 hours in complete media. MTT solution (5 mg/mL) was prepared in PBS. 50 µL of MTT solution was added into each well and incubated at 37 oC for 4 hours. Purple coloured formazan was dissolved in 1:1 (v/v) DMSO/MeOH and absorbance of the wells was measured at 550 nm by micro-plate ELISA reader. We have calculated the %viability from this absorbance values. Data shows



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that %viability is decreased with increasing the concentration of PSI i.e. PSI induces the death of cancer cells. %Viability = [(A550Treated Cells - A550 Backgrounds)/ (A550Untreated Cells - A550 Backgrounds)]*100. Cell viability assay with N-acetyl cysteine (NAC) A549 cells were seeded at a density of 10000 cells per well in 96-well plate before 24 hours of treatment. Cells were treated with 6.5 and 13 µg/mL solutions of PSI for 24h with and without pre-incubation with 4 mM solution of NAC for 4h. We have performed MTT assay as described before. Degradation of PSI with time in cancer cells A549 cells were treated with PSI in serum free media for 4h. After that cells were washed with complete media and incubated for 12h, 24h and 48h. To study the amount of PSI present inside the cells, we have imaged the cells in the Cy5 channel in a fluorescence microscope (Nikon Ti-U eclipse). We have found highest Cy5 signal in the cells, which were imaged after 4h and on increasing the time of incubation the signal intensity was decreased, indicating degradation of PSI inside the cell with time. Immunofluorescence microscopy for studying effect of PSI on microtubule networks A549 cells were seeded in a cover glass bottom dish before 24 hours of treatment. We have prepared 15.62 µg/mL solution of PSI in serum free media and cells were treated for 4 hours. After that, cells were incubated in complete media for 48 hours. Then, the cells were washed by PBS and treated with 4% paraformaldehyde solution for 30 minutes for cell fixing. We have treated the cells with 0.1% Triton X-100 in PBS for 10 minutes. Cells were washed with PBS and incubated with 5% BSA in PBS for 30 minutes. After washing with PBS, cells were


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incubated with rabbit monoclonal anti-α-tubulin antibody with dilution 1:300 for 2 hours. After that cells were washed with PBS and incubated with secondary antibody (Cy3.5 pre-absorbed goat anti-rabbit IgG) with dilution 1:600 for 2 hours. Cells were washed with PBS and incubated with Hoechst 33258 (1 µg/mL) for 30 minutes before imaging. Fixed cells were imaged through a Nikon Ti-U eclipse fluorescence microscope with a 40X objective in bright field, DAPI, FITC, and TRITC channels. In another set of experiment we have incubated the cells with PSI for 4h and labelled the microtubule network as described before. Cells were imaged through an Andor spinning disc confocal fluorescence microscope with a 60X objective (Olympus) and an Andor iXon3 897 EMCCD camera in DIC, DAPI (405 nm), FITC (488 nm) and TRITC (561 nm) channels. We have found a well distribution of green PSI inside the A549 cells and some of them were found to be interacting with the microtubule network of the A549 cells. Immunofluorescence microscopy of Mad2 and BubR1 protein expression after treatment with PSI A549 cells were seeded in a cover glass bottom dish before 24 hours of treatment. We have treated the cells with 15.62 µg/mL solution of PSI in serum free media for 4 hours. After that cells were incubated in complete media for 48 hours. Then, the cells were washed by PBS and treated with 4% paraformaldehyde solution for 30 minutes for cell fixing. We have treated the cells with 0.1% Triton X-100 in PBS for 10 minutes. Cells were washed with PBS and incubated with 5% BSA in PBS for 30 minutes. After washing with PBS, cells were incubated with rabbit polyclonal anti-Mad2 anti-body with dilution 1:400 for overnight at 4 oC. After that cells were washed with PBS and incubated with secondary antibody (Cy3.5 pre-absorbed goat anti-rabbit IgG) with dilution 1:400 for 2 hours at 37 oC. Cells were washed with PBS and incubated with



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Hoechst 33258 (1 µg/mL) for 30 minutes before imaging. Fixed cells were imaged through a Nikon Ti-U eclipse fluorescence microscope with a 40X objective in bright field, DAPI, FITC, TRITC and Cy5 channels. In case of immunofluorescence microscopy of BubR1, we have followed the same procedure as like Mad2. Here, we have used mouse monoclonal anti-BubR1 primary antibody (1:400) and goat anti-mouse IgG fluorescence conjugated secondary antibody (1:400). Study of generation of reactive oxygen species (ROS) after treatment of PSI using dichlorodihydrofluorescein diacetate (DCF-DA) and dihydroethidium (DHE) (a) Enhancement of dichlorodihydrofluorescein (DCF) signal in A549 cells after PSI treatment using fluorescence microscopy: A549 cells were seeded in a cover glass bottom dish before 24h of treatment. Cells were treated with 31.25 µg/mL solution of PSI in serum free media for 4h. After that cells were washed with media and incubated for 4h in complete media. To measure the generation of ROS we have treated the cells with freshly prepared DCF (1 µM) in serum free media and cells were incubated for 30 minutes at 37 oC. After that cells were washed with serum free media and imaged in fluorescence microscope in FITC channel. In case of control experiment, cells were not treated with PSI but treated with DCF. (b) Enhancement of dichlorodihydrofluorescein (DCF) signal in A549 cells after PSI treatment using fluorescence assisted cell sorting (FACS): A549 cells were seeded in a 6-well plate at density of ~5 X 105 cells per well before 24h of treatment. Cells were treated with 31.25 µg/mL solution of PSI in serum free media for 4h. After that cells were washed with media and incubated for 24h in complete media. Cells were washed with phosphate buffer and trypsinized. Cells were centrifuged with serum free media at 3000 rpm for 3 minutes. To measure the generation of ROS we have treated the cells with freshly prepared DCF (1 µM) in serum free


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media and cells were incubated for 30 minutes at 37 oC. After that cells were washed with serum free media using centrifugation and analyzed using FITC filter by flow cytometry. (c) Enhancement of dihydroethidium (DHE) signal in A549 cells after PSI treatment using fluorescence microscopy: A549 cells were seeded in a cover glass bottom dish before 24h of treatment. Cells were treated with 31.25 µg/mL solution of PSI in serum free media for 4h. After that cells were washed with media and incubated for 24h in complete media. To measure the generation of ROS we have treated the cells with freshly prepared DHE (5 µM) in serum free media and cells were incubated for 30 minutes at 37 oC. After that cells were washed with serum free media and imaged in fluorescence microscope in TRITC channel. In case of first control experiment, cells were not treated with PSI but treated with DHE. In case of second control experiment, cells were treated with PSI but were not treated with DHE. Second control experiment was used for PSI control, as PSI itself has weak fluorescence in TRITC channel. Fluorescence study of ROS generation after PSI treatment with and without light irradiation A549 cells were seeded in a 96 well plate before 24 hours of treatment. We have prepared 31.25 µg/mL solution of PSI in serum free media and cells were treated with PSI for 4 hours. After that cells were incubated in complete media for 24 hours. During this time a group of cells were irradiated by solar simulator (source of artificial sun light) for 30 min in first lot and 60 min in second lot. Cells were washed and incubated with the 50 µM solution of DCF in serum free media for 60 min at 37 oC. Cells were washed with serum free media and measured the DCF signal in Synergy H1 Multi-Mode Reader fluorimeter (excitation at 485 nm and emission at 530 nm).



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AnnexinV-propidium iodide (PI) assay for apoptosis study after PSI treatment using fluorescence microscope A549 cells were seeded in a cover glass bottom dish before 24 hours of treatment. We have prepared 15.62 µg/mL solution of PSI in serum free media and cells were treated with PSI for 4 hours. After that cells were incubated in complete media for 48 hours. Cells were washed with phosphate buffer (PBS, pH 7.4) and then with assay buffer. Assay buffer was supplied along with the Annexin V apoptosis detection kit (Santa Cruz Biotechnology). We have added 2.5 µL of propidium iodide (PI) and 2.5 µL of annexin V into the 200 µL of assay buffer and live cells were incubated with this solution for 30 minutes at 37 oC inside the cell culture incubator. All those reagents were supplied along with the apoptosis detection kit (Santa Cruz Biotechnology). The stock concentration of Propidium iodide and annexin V was 50 µg/mL and 200 µg/mL respectively. Cells were washed with the assay buffer and immediately live cell were imaged through a Nikon Ti-U eclipse fluorescence microscope with a 40X objective in bright field, FITC, TRITC and Cy5 channels through an Andor iXon3 897 EMCCD camera. FACS analysis for apoptosis study after treatment with PSI Fluorescence activated cell sorting (FACS) experiment was performed for studying the type of cell death. We have studied cell death pathway of A549 and B16F10 cells in FACS using apoptosis detection kit. Cells were seeded at a density of ~5 X 105 cells per well in a 6-well plate before 24 hours of treatment. We have treated the cells with solutions of PSI in serum free media for 4 hours. After that cells were incubated in complete media for 24 and 48 hours. Cells were trypsinized and washed with PBS. Cells were incubated in dark at 37 oC for 30 minutes with a 100 µL solution of assay buffer containing 2.5 µL of Propidium iodide (PI) and 2.5 µL of annexin V. All those reagents were supplied along with the apoptosis detection kit (Santa Cruz


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Biotechnology). The stock concentration of Propidium iodide and annexin V was 50 µg/mL and 200 µg/mL respectively. After that another 400 µL of assay buffer was added to the cells and these total 500 µL of cell solution was analysed by FACS. We have detected the emission of Annexin V and PI in the FITC and PI channels of BD LSRFORTESA flow cytometer using emission filters at 530 and 610 nm respectively. In the represented data, cells in the Q1, Q2 and Q4 quadrants are regarded as necrotic, late apoptotic and early apoptotic cells respectively. Cells in the Q3 quadrant are regarded as normal cells or healthy cells. Data was analyzed using FACS DIVA software. We have found that control cells (cells were not treated with PSI) were mostly populated in Q3 quadrant. Population of cells in the Q4 and Q2 quadrant was increased when cells were treated with PSI. We have compared the percentage of healthy and apoptotic cells after treatment with different concentrations of PSI with the control cells (untreated) using a bar diagram. Cell cycle analysis by flow cytometer after treatment with PSI A549 and B16F10 cells were seeded at a density of ~5 X 105cells per well in a 6-well plate before 24 hours of treatment. We have treated the cells with solutions of PSI in serum free media for 4 hours. After that the cells were incubated in complete media for 48 hours. After incubation, the media containing free cell suspension were taken in micro-centrifuge tube and cells were collected by centrifugation at 3000 rpm for 3 minutes. Then the cells in 6-well plates were trypsinized and collected by centrifugation at 3000 rpm for 3 minutes. All cell pellets were accumulated in phosphate buffer saline (PBS, pH 7.4) and fixed by adding cold ethanol slowly. The final concentration of ethanol was 70% (v/v). Fixed cells were kept in -20 oC. Before analysis fixed cells were washed with PBS (pH 7.4) through centrifugation to remove ethanol. Then cell suspensions in PBS were incubated with PI and RNase A for 45 minutes at room



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temperature. The working concentration of PI and RNase A was 100 µg/mL and 10 µg/mL respectively. Cell suspensions were transferred into FACS tube followed by analysis through FACS. Study of effect of PSI on mitochondrial membrane potential using JC1 dye A549 cells (5× 106 cells/ well) were harvested overnight in 6 well plates and treated with 15.62 µg/mL and 31.25 µg/mL of PS1 for 24h. Subsequently, cells were taken in suspension and intracellular mitochondrial membrane potential has been evaluated using BD™ MitoScreen according to instruction of manufacturer. Briefly, cell suspensions were washed thoroughly and added with working concentration of JC1 in 1X assay buffer and incubated for 15 min at 37 oC in CO2 incubator. Then cells were washed with 1X assay buffer and analyzed under FACS (LSRFORTESA). Immunolocalization study of cytochrome c (Cyc) in fluorescence microscope A549 cells were seeded in a bottom glass cover dish overnight before treatment. Cells were treated with 31.25 µg/mL solution of PSI for 48h with and without pre-incubation with 5 mM Nacetyl cysteine (NAC) for 4h. Subsequently, cells were washed with serum free colorless DMEM media and incubated with 100 nM of mitotracker red for 45 min. After that, cells were washed and fixed with 4% formaldehyde and permeabilised with 0.2% triton X. 5% BSA solution was used for blocking non-specific interaction sites. Next, cells were incubated for overnight with anti-Cyc monoclonal primary antibody. After washing cells were incubated with FITC labelled secondary IgG (H/L) followed by immunocytochemistry study using fluorescence microscope. Cells were imaged through a Nikon Ti-U eclipse fluorescence microscope with a 40X objective in bright field, FITC, TRITC channels. Colorimetric analysis of caspase 3 activity after treatment with PSI


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A549 cells (5× 106 cells/ well) were seeded in 6 well plates and harvested overnight before treatment. Cells were treated with 31.25 µg/mL solution of PSI for 48h with and without preincubation with 5 mM N-acetyl cysteine (NAC) for 4h and doxorubicin. Then we have trypsinized the cells and taken in suspension and cells were lysed using RIPA buffer and total proteins were isolated. Afterwards, protein sample were incubated with 200 µM of Ac-DEVDpNA at incubated 8h in 37 oC water bath. Then, solution was monitored for pNA absorbance by spectrophotometer at 405 nm. Increased in pNA absorbance is linearly proportional to the caspase 3 activity. Tunnel assay for specific detection of apoptosis after PSI treatment A549 and B16F10 cells were seeded in a 6-well plate at density of ~5 X 105 cells per well before 24h of treatment. Cells were treated with 15.62 µg/mL solution of PSI in serum free media for 4h. After that cells were incubated for 24h in complete media. Cells were washed with phosphate buffer and trypsinized. Cells were centrifuged with PBS at 3000 rpm for 3 minutes. Cell pellet was treated with ice cold 1% para formaldehyde solution in PBS and incubated for 20 minutes at 4 oC. Cells were centrifuged at 3500 rpm for 4 minutes. Cell pellet was dissolved in 200 µL of PBS and then cells were fixed with 1000 µL of ice cold EtOH (100%). Cells were stored at -20 o

C before experiment. Fixed cells were centrifuged at 3000 rpm for 5 min. Cell pellet was

incubated with 100 µL of equilibration buffer (supplied along with the kit) for 8 minutes. We have prepared nucleotide mixture on ice tub using 45 µL of equilibration buffer, 5 µL of nucleotide mix and 1 µL of rTdT enzyme for each cell pellet. In case of unstained sample we have not used the rTdT enzyme during preparation of nucleotide mixture. Cell pellet was treated with 50 µL of the nucleotide mixture and incubated at 37 oC water bath for 90 minutes in dark. Cells were treated with 1000 µL of 20 mM EDTA solution (prepared in water) and vortexed.



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Cells were centrifuged at 4000 rpm for 5 minutes. Cell pellet was treated with 400 µL solution of working PI solution containing PI (100 µg/mL) and RNAse A (10 µg/mL) and incubated for 30 minutes at room temperature. Cells were analysed by flow cytometer. Synthesis of long chain attached Leucine-Aspartic Acid-Valine (LDV) lipopeptide A tripeptide NH2-Leu-Asp-Val-COOH (LDV) containing amino acids leucine (L), aspartic acid (D) and valine (V) was synthesized in Microwave Peptide Synthesiser equipped with Liberty 1 system (Discovery, CEM). Wang resin (100-200 mesh) was used as a solid support having substitution level 0.9 mmole/gm. DMF and DCM were used as solvent and all the amino acids, used in the synthesis of LDV were N-terminal fluorenylmethyloxycarbonyl (Fmoc) protected. The side chain protections of all the amino acids were stable in basic medium but were labile in acidic medium. Wang resin was swelled in 1:1 mixture of DMF and DCM solvent for overnight. N, N′-diisopropylethylamine (DIPEA) in DMF having concentration 2M was used as a base and HBTU in DMF having concentration 0.5M was used as an activator. 20% piperidine in DMF was used as deprotection mixture. All the amino acids required for LDV peptide were dissolved in DMF as a concentration of 0.2M separately. After coupling of all the amino acids required for LDV, the N-terminal Fmoc protection was removed by 20% piperidine in DMF. The free -NH2 group of the N-terminal position of the peptide was treated with succinic anhydride and dry triethylamine (Et3N) in dry dichloromethane to introduce a carboxyl group at the N-terminal position of the resin. Finally, this terminal -COOH group was coupled with N, N′-di-nhexadecyl-N-2-aminoethylamine using HOBT and DIC. The resin was dried using DCM, methanol and diethyl ether. Lipopeptide was cleaved from the resin using trifluoroacetic acid (TFA) in DCM. Then, the peptide was precipitated out in cold diethyl ether and purified through reverse phase HPLC on a C18 column using MeOH as mobile phase and characterized by Matrix


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Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) analysis. We have obtained m/z: 936, which correspond to the calculated mass of the LDV lipopeptide (MW = 936). Preparation of LDV liposome containing PSI (LDV-liposome-PSI) Liposome was prepared using DOPC (1 mM), Cholesterol (0.5 mM) and LDV long chain lipopeptide (1 mM) following hydration method. Solutions of DOPC, cholesterol and LDV long chain lipopeptide in organic solvent (CHCl3 and MeOH) were added in glass vial. A thin film of the lipid mixture was prepared by nitrogen flash. It was dried under vacuum for 6h. We have added 1 mL solution of PSI in phosphate buffer saline (having concentration 62.5 µg/mL) into the lipid film for overnight hydration at 4 oC. After that it was vortex for 5 min and then it was sonicated for 5 minutes at 4 oC for 5 times. Liposome was stored at 4 oC refrigerator. We have also prepared LDV liposome without PSI and untagged liposome (without LDV lipopeptide) with PSI as a control liposome. Characterisation of liposome by Cryo Electron Microscope (CryoEM) Liposome solution (2.5 µL) was loaded on the TEM grid. Imaging was performed using TECNAI G2 POLARA, 300KV, equipped with (4KX4K) FEI eagle camera. Measurement of size of LDV-liposome and LDV-liposome-PSI using Dynamic Light Scattering (DLS) experiment The size of LDV-liposome and LDV-liposome-PSI were measured through DLS study. Cellular uptake of LDV-liposome-PSI into B16F10 cells over the untagged-liposome-PSI in the complete media (with serum) B16F10 cells were seeded in a cover glass bottom dish before 24h of treatment. Cells were treated with LDV-liposome-PSI and untagged-liposome-PSI in complete media for 2h and 4h. Cells were washed with media and imaged by fluorescence microscope in the Cy5 channel. We



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have observed the greater cellular uptake of LDV-liposome-PSI into the cells over the untaggedliposome-PSI in 2h and 4h. Targeted delivery of LDV-liposome-PSI into B16F10 cells over the normal WI38 cells (a) Greater cellular uptake of LDV-liposome-PSI into B16F10 cells over the WI38 cells: B16F10 and WI38 cells were seeded in a cover glass bottom dish before 24h of treatment. Cells were treated with LDV-liposome-PSI in complete media for 2h and 4h. Cells were washed with media and imaged by fluorescence microscope in the Cy5 channel. We have observed the greater cellular uptake of LDV-liposome-PSI into the B16F10 cells over the WI38 cells after 2h and 4h of incubation. (b) Effect of LDV-liposome-PSI and untagged-liposome-PSI on B16F10 and WI38 cells: B16F10 and WI38 cells were seeded in a 96-well plate at density of 10000 cells per well before 24h of treatment. Cells were treated with LDV-liposome-PSI (15.62 µg/mL) and untaggedliposome-PSI (15.62 µg/mL) separately in complete media for 24h. After 24h, MTT assay was performed. We have observed higher cell killing by LDV-liposome-PSI for the B16F10 cells whereas lesser cell killing was observed for the WI38 cells in 24h. In case of untagged-liposomePSI we have found similar amount of cell viability for both B16F10 and WI38 cells. Comparison of cell viability of LDV-liposome-PSI with doxorubicin A375 cells were seeded at a density of 10000 cells per well in 96-well plate before 24 hours of treatment. Cells were treated with PSI, LDV-liposome-PSI and doxorubicin for 24 hours. We have performed MTT assay as described before and calculated IC50 value using CompuSyn software. Preparation of 3D multi-cellular tumor spheroid and treatment with PSI


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We have prepared tumor spheroid in a 96-well plate. HeLa cells were seeded in a 96-well plate coated with 1% agarose (w/v). The inoculation concentration was ~5×103 cells/well. The HeLa cells were cultured in RPMI-1640 media supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100 µg/mL) and incubated in a humidified atmosphere with 5% CO2 until the formation of matured spheroid. Spheroid is incubated with 31.25 µg/mL of PSI for 4h and transferred into cover glass bottom dish pre-coated with poly D-lysine. It was incubated with hoechst 33258 for nucleus staining and imaged under fluorescence microscope at 10X objective using 405 nm, 488 nm and 561 nm channels. In case of spheroid growth inhibition study, we have prepared tumor spheroid as describe above. After 4 days of incubation, images of spheroid morphology were captured in DIC mode using inverted Nikon Ti-U eclipse microscope equipped with EMCCD camera and represented as day 0. Then the spheroids were divided in two groups. In one group spheroids were not treated with PSI and it was regarded as control. In another group spheroids were treated with 31.25 µg/mL solution of PSI. Spheroid morphology was assessed for next 5 days using inverted Nikon Ti-U microscope. Volume of the sphere was analysed using following formula as described before. V = 0.5 * Length * Width2 Toxicity study It was performed on melanoma mice model. We have taken 6 weeks old C57BL/6J female mice (5 mice) with average body weight 14-18 g for our study. Animals were housed in pathogen free laboratory environment throughout the experiments and maintained standard conditions according to the rules of animal ethical committee. Mice were treated with 500 µL solution of LDV liposomal PSI solution (concentration of PSI is 62.5 µg/mL) for 90 days. We have observed the behaviour of mice and body weight during the 90 days.



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In vivo study In vivo study was performed on melanoma mice model. We have taken 7 weeks old C57BL/6J female mice with average body weight 16-20 g for our study. Animals were housed in pathogen free laboratory environment throughout the experiments and maintained standard conditions according to the rules of animal ethical committee. Melanoma tumor was generated in the mice by one time subcutaneous injection of B16F10 (mice melanoma) cells (~2 x106 cells/animal) in PBS. Following implantation, tumor growth was measured in millimetres, daily using slide callipers and tumor volume was calculated by the formula: V = 0.5 * Length * Width2. When average tumor volume was nearly 250 mm3, mice were randomly divided in two groups (5 mice/group). Each mice of group 1 was treated with 500 µL solution of LDV liposomal PSI solution (concentration of PSI is 62.5 µg/mL) and each mice of group 2 was taken as control which were treated with PBS. The first treatment day was taken as 0 day. We have continued the treatment upto 14 day. We have measured the tumor volume and mice body weight from 0 day to 14 day. After 14 days treatment mice were sacrificed by cervical dislocation. Tumors were collected and size was measured. Tumors were fixed in 4% formalin in PBS, embedded in paraffin and sectioned with a thickness of 4 µm using Leica Rotary Microtome. The slices were treated with hematoxylin and eosin (HE) staining for histological study and examined by Nikon Ti-U eclipse microscope under 10X objective in bright field. Data Analysis Microscopic images were analysed using Image J software. RESULTS AND DISCUSSION PSI was isolated from spinach leaves, purified and characterized following previously described method with modification in our lab (Figure S1, ESI).27 From SDS-PAGE gel electrophoresis of PSI, we have found that PSI complex contains a set of LHCI polypeptides (20-


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26 kDa), PsaA, PsaB (a doublet near 65 kDa) and low molecular weight peptides bands (10-16 kDa) for PsaD, PsaE, PsaC (Figure 1a). We have also made a reconstituted image of PSI complex using PyMOL Molecular Graphic System (Version 1.7.4 Schrödinger, LLC.) software, which corresponds to the bands found in the SDS PAGE gel (Figure 1b). First, we have studied the cellular uptake of PSI in A549, B16F10 cancer cells and WI38 normal cell (Figure 1c, d, S2 to S4). Fluorescence microscopic images of cells revealed significant fluorescence signals at 405, 488 and 561 nm channels, which indicate that PSI enters into the cytoplasm of the cell and shows fluorescence in these three channels whereas in control we did not observed any fluorescence signal in these channels (Figure S5). Moreover, we have also found that PSI shows fluorescence in Cy5 channel (Figure S6) and intensity in Cy5 channel was higher than other channels. Cellular uptake in A549 cell was also studied by FACS in APC channel (Ex-640 nm and Em-670/14) (Figure S7). We have also found that signal intensity in Cy5 channel (638 nm) was decreased with time indicating degradation of PSI with time inside the cell (Figure S8). Next, we have checked the cell viability of PSI in cancer cells (A549 and B16F10) and normal cell (WI38). Cells were treated with PSI for 48h and found significant cell deaths as we observed cell viability decreases with increasing concentration of PSI (Figure 1e, f, S10). Morphological changes of the cells after treatment with PSI was also observed from microscopic images of PSI treated cancer cells (Figure S9). Morphological changes and cell death by PSI inspired us to find out the pathways involved in cell death of cancer cells. Since we have observed significant morphological change (Figure S9), first we have checked whether PSI interacts with microtubules or not because microtubule is one of the key filaments to maintain the cell structure and space. Thus, we have checked the effect of PSI on microtubule networks inside the A549 cells and found that the microtubule network was not affected by PSI (Figure



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S11). Further, we have also checked that whether PSI has any effect on two spindle checkpoint proteins such as Mad2 and BubR1. We have found that PSI did not activate both the proteins (Figure S12-15). These results clearly indicate that PSI did not perturb the intracellular microtubule organization. Since PSI did not perturb the intracellular microtubule networks but it showed significant anticancer effect, we further investigated the alternative pathways involved in cell killing. As an alternative pathway of killing, we have studied whether PSI can generate intracellular reactive oxygen species (ROS) or not. For that purpose, we have checked the level of ROS present in cancer cells after treatment with PSI (Figure 2). Here, we have used 2, 7dichlorofluorescein diacetate (DCF-DA) and dihydroethidium (DHE) to measure the ROS level inside the cancer cell. Live A549 cells were treated with PSI followed by treatment with DCFDA and DHE for 30 minutes at 37 oC. Interestingly, we have found significant enhancement of green and red signal of DCF-DA and DHE respectively after treatment with PSI compared to control cells (Figure 2, S16 to S20). We have also found the enhancement of green signal of DCF-DA in FITC channel (Ex-488 nm and Em-530/30) in FACS after treatment with PSI with respect to control cells from (Figure 2g,h and S17). The enhancement of green and red signal was due to the oxidation of DCF-DA and DHE respectively by the reactive oxygen species (ROS) generated in A549 cells after treatment with PSI. Amount of ROS generated in A549 cell after treatment with PSI was quantified using Image-J software (Figure 2c, f). To confirm the ROS dependent cell killing we have performed cell viability assay (MTT assay) in presence and absence of N-acetyl cysteine (NAC) which scavenges the free radical produced due to ROS. We have found that % viability increases in presence of NAC which confirm the ROS dependent killing pathway of PSI (Figure 2i). During those experiments we did not perform light irradiation (at 700 nm) to the PSI because light irradiation causes degradation of PSI.28 PSI isolation and all


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the treatments were performed under normal white light. We have also measured the green fluorescence signal of DCF at 530 nm (excitation at 485 nm) from A549 cells with and without irradiation after treatment with PSI (figure S21). The results indicate that PSI can generate ROS without light irradiation. Since, we have observed PSI induced ROS generation and significant cell killing, we further tried to understand the pathway of cell death. Therefore, we have performed annexinVFITC/PI assay in A549 cells after PSI treatment using fluorescence microscopy (Figure S22 to S23). Fluorescence microscopic images showed strong green signal in A549 cells with respect to control A549 cells (Figure S24). Attachment of annexinV-FITC to the cell surface of A549 cells indicates that PSI may cause apoptotic death of A549 cells. To analyze the pathway of the cell death process, we have performed FACS analysis using apoptosis detection kit. From FACS analysis, we have found that PSI causes apoptotic death of A549 and B16F10 cells (Figure 3, S25). Percentage of apoptotic death was increased with increasing concentration and incubation period with PSI (Figure 3). Signal obtained in the PE-Texas Red (Ex-561 nm and Em-610/20) and FITC (Ex-488 nm and Em-530/30) channels was not due to the PSI (data not shown). We have also analyzed the cell cycle of A549 and B16F10 cells after treatment with PSI (Figure S26, S27). We have found the increase of both A549 and B16F10 cell population at subG0 phase after treatment with PSI. It was shown before that enhanced cell population at subG0 phase in cell cycle corresponds to the fragmented DNA resulting apoptotic death.29-30 So, the cell cycle data also supports the apoptotic death of A549 and B16F10 cells after treatment with PSI. Mitochondrial membrane potential has been verified in PS1 treated A549 cells for studying the pathway of apoptosis. Thus, using JC1 dye we observed that JC1 soluble fraction greatly enhanced with dose of PS1 (Figure 4 a-c). This results indicates that apoptosis upon



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treatment with PS1 is probably mediated by mitochondria dependent internal pathway. To further confirm this, we have analyzed the release of cytochrome c from mitochondria. Here, microscopic images clearly indicates that co-localization of cytochrome c with mitotracker was insignificant in PS1 treated cells with respect to the control cells indicating release of cytochrome c (Figure 4d). Thus, we evaluated the cytochrome c release in presence of ROS inhibitor (NAC). Here, we observed significant co-localization of cytochrome c with mitotracker representing no release of cytochrome c. This indicates the apoptotic pathway is ROS dependent. Further, we have analyzed the key player for activation of apoptosis, called caspase3 using colorimetric (Figure 4e) analysis. In colorimetric experiment DEVD PNA, substrate for caspase3 was used. Caspase3 activation has been evaluated relative to control and a known drug doxorubicin (DOX). Active caspase3 breaks peptide substrate after carboxyl group of aspartic acid (D) freeing PNA. Thus, release of PNA has been measured using UV spectrometer indicating active cspase3. Here, we observed significant PNA absorption in PS1 treated cell lysate indicating caspase3 activation (Figure 4e). Further to verify whether the caspase3 activation is ROS dependent, we evaluated PNA absorbance in ROS inhibitor treated cell lysate in presence of PS1. Here, we have observed insignificant change in PNA absorbance with respect to control (Figure 4e). This indicates due to PS1 treatment, ROS plays an important role in caspase3 activation. It was described earlier that DNA fragmentation occurred in apoptotic death of cells.31 Therefore, we have done TUNEL assay with A549 and B16F10 cells after treatment with PSI for further confirmation of apoptotic death of cancer cells. Interestingly, it was found that the amount of fragmented or nicked DNA in A549 and B16F10 cells was enhanced with respect to


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the control cells after treatment with PSI (Figure 5 and S28). This data finally concluded that PSI caused apoptotic death of cancer cells. Next, we have tried to develop targeted delivery of PSI specific to melanoma cancer cells (B16F10), as we have found that PSI can kill both cancer and normal cells (WI38). Generally, cancer cells (B16F10) overexpress specific integrin receptor proteins on the cell surface compared to normal cell (WI38). It has been described before that B16F10 melanoma cells over expressed α4β1 integrin receptor on its cell surface32 and a short tri-peptide LDV can specifically recognize and bind with the α4β1 integrin receptor.33 Therefore, we have synthesized LDV tripeptide conjugated with C16-long chain (Scheme S1 and Figure S29, S30) and prepared LDVliposome for cancer cell specific liposomal delivery of PSI (ESI). Dynamic light scattering measurement shows average size of the LDV-liposomes is 144.7 ± 69.75 nm, which becomes 217 ± 98.08 nm after PSI encapsulation confirming the encapsulation of PSI (Figure S31). We have also performed the Cryo-electron microscopy (Cryo-EM) of the LDV-liposomes and LDVliposome-PSI which shows the liposome formation and it was found that size of liposome was increased after PSI encapsulation (Figure 6 and S32). First, we have studied cellular uptake of LDV-liposome-PSI in B16F10 and WI38 cells (Figure 7a-e). We have found higher amount of cellular uptake of LDV-liposome-PSI into the B16F10 cells compared to WI38 cells after 2 and 4h treatment (Figure 7e). We have also prepared untagged-liposome (without LDV-long chain) containing PSI as control (ESI) and compared the cellular uptake of LDV-liposome-PSI and untagged-liposome-PSI into B16F10 cells (Figure S33, S34). Results indicate higher amount of uptake of LDV-liposome-PSI into the B16F10 cells as compared to untagged-liposome-PSI. Further, we have studied the cell viability assay of both B16F10 and WI38 cells after treatment with LDV-liposome-PSI and untagged-



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liposome-PSI (Figure 7f). Interestingly, we have found significant cell deaths of B16F10 cells while WI38 cells were unaffected in case of treatment with LDV-liposome-PSI and both the cells were less affected by untagged-liposome-PSI. Overall, above results indicate the targeted delivery of PSI into the cancer cell over non-cancerous cell. Further, PSI is a protein from nonhuman origin, so it can elicit adaptive immuno-response generating antibody against it. Our liposomal formulation of PSI help to avoid this problem also. Next, we have compared the effect of LDV liposomal PSI (Lip. PSI) with a known anticancer drug doxorubicin as both generate ROS inside the cells. We have performed cell viability assay with human melanoma cancer cell (A375) and calculated the IC50 values. It was found that LDV liposomal PSI has IC50 value (IC50 6.01 µg/mL) significantly less than that of doxorubicin (IC50 10.19 µg/mL) (Figure 7g-i). This result indicates that PSI is highly potent compared to doxorubicin. We have found that PSI works as an anticancer agent in 2D cancer cell. However, drugs showing activity in 2D cell culture often fail in in vivo study.34 Therefore, before performing in vivo study, we have studied the anti-tumor efficacy of PSI in a tumor mimicking 3D cell culture/spheroid model of HeLa cell (ESI). Interestingly, we have found that PSI enters into the tumor spheroid (Figure S35) and inhibits the growth of spheroid compared to the untreated spheroid (control), which indicates the significant anti-tumor effect of PSI (ESI, Figure 7j). Interesting result in 3D spheroid growth inhibition motivated us to evaluate that whether PSI is effective in in vivo tumor model or not. Here we have chosen melanoma because our primary focus is to develop an ointment based formulation for the treatement of melanoma. For this purpose, in vivo study was performed in C57BL/6J female mice model. We have also found that this formulation is non toxic to the mice (Figure S36) as we have observed increase of mice


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body weight constantly after the treatment with LDV liposomal PSI for 90 days. Next, we have generated melanoma tumor in mice and treated with LDV liposomal PSI solution continuously for 14 days. After 14 days both treated and untreated mice were sacrificed and tumors were removed from the body followed by comparison of sizes of tumors for both the treated and untreated. Further, the tumors were sectioned and immunohistochemical study was performed (Figure S37) following previously described method.35 We have found that significant inhibition of melanoma tumor growth in case of LDV-liposomal PSI treated mice compared to the untreated control mice (Figure 8). Imunohistochemistry result shows that the significant amount of tumor cells, tumor muscles and blood vessels were damaged after treatment of LDVliposomal-PSI. Above result cleary confirms the significant anti-tumor effect of PSI in melanoma.

CONCLUSION In summary, this manuscript reports for the first time that PSI, purified from spinach leaves can serves as an excellent anti-cancer agent. We have shown that PSI can generate significantly high amount of ROS, induces apoptotic death of A549, B16F10 cells through mitochondria dependent internal pathway, induces caspase3 and arrests the cell cycle in subG0 phase. Finally, we have also developed a melanoma cell specific delivery vehicle for PSI, which specifically kills melanoma cells (B16F10) without affecting normal cells (WI38). This formulation is more effective compared to doxorubicin for melanoma and inhibits the growth of melanoma tumor in C57BL/6J mice model. This manuscript opens up a new direction for natural cancer therapy for melanoma from easily available, cheap and edible plant source. ASSOCIATED CONTENT This materials are available free of charge via the internet at http://pubs.acs.org.



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AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We thank NCCS-Pune, for cell lines. AS, SG and BJ thank CSIR, SM thanks UGC, GD thanks ICMR, DB thanks DST-Inspire and SG thanks DST-Ramanujan for their fellowships. SG kindly acknowledges CSIR Network project (BSC0113) for financial assistance and IACS Kolkata for DLS study. Authors wish to Dr. Aditya Konar, for letting us access animal house to carry out in vivo experiments and Dr. Jayati Sengupta for providing us Cryo-EM Polara facility. Supplementary Data Supplementary data associated with this article can be found in the online version.

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Scheme 1. Cartoon represents integrin targeted liposomal delivery of PSI into cancer cell followed by ROS generation and apoptotic death whereas normal cells remain unaffected.



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Figure 1. SDS PAGE gel image of PSI purified from spinach (a). Image shows reconstituted PSI complex (b). Cellular uptake of PSI in B16F10 cells (c) and A549 cells (d). MTT assay shows that cell viability of B16F10 (e) and A549 (f) cells decrease after treatment with PSI. Scale bars correspond to 20 µm.


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Figure 2. ROS generation in A549 cells after treatment with PSI without light irradiation. Image shows the oxidation of DCF after treatment of PSI in bright field (a), in 488 nm channel (b) and comparative bar diagram shows the increase of DCF signal (c). Image shows the oxidation of DHE after treatment of PSI in bright field (d), in 561 nm channel (e) and comparative bar diagram shows the increase of DHE signal (f) in A549 cells after treatment with PSI. (g) Enhancement of DCF signal in FACS after treatment with PSI and (h) the enhancement is showing by a bar diagram. (i) MTT assay in A549 cell with PSI in presence and absence of Nacetyl cysteine (NAC). Scale bars correspond to 20 µm.



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Figure 3. Apoptosis of A549 cells after treatment with PSI. Apoptotic death of A549 cells was increased with increasing the concentration of PSI and time of incubation (a-f). Control A549 cells (g). Comparative bar diagram shows that population of healthy cells were decreased and apoptotic cells were increased with increasing concentration of PSI and with incubation period (h, i).


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Figure 4. FACS data indicates the decrease of mitochondrial membrane potential using JC1 dye (a, b) after PSI treatment. Bar diagram indicating the enhancement of membrane depolarization due to PSI (c). Microscopic images indicate the release of cytochrome c in presence of PSI (d). PNA absorbance in different samples indicates the activation of caspase3 (e). Scale bars correspond to 20 µm.



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Figure 5. Tunnel assay shows that amount of fragmented DNA was increased in B16F10 and A549 cells after treatment with 15.62 µg/mL of PSI (a-d). Bar diagrams show the amount of increase of nicked DNA in B16F10 (e) and in A549 (f) cells after treatment with 15.62 µg/mL PSI.

Figure 6. Cryo-electron microscopic images of the LDV liposome and LDV liposome-PSI.


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Figure 7. Cellular uptake of LDV liposomal PSI into B16F10 cells in 2h (a) and in 4h (c). Cellular uptake of LDV liposomal PSI into WI38 cells in 2h (b) and in 4h (d). Quantitative bar diagram shows that LDV liposomal PSI goes more into B16F10 cells than the WI38 cells (e). LDV liposomal PSI specifically kills B16F10 cells than the WI38 cells whereas normal liposomal PSI cannot kill both the cells (f). Cell viability of LDV liposomal PSI (g) and doxorubicin (h) shows that IC50 value of doxorubicin (DOX) is higher than LDV liposomal PSI (Lip PSI) and it is shown by a comparative bar diagram (i). Graph shows PSI inhibits the growth of 3D-tumor-spheroid compared to the untreated spheroid (control) (j).



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Figure 8. (a) Image of tumor and mice after treatment with LDV liposomal PSI and without PSI treatment (control). Images show the variation of tumor volume (b) and mice body weight (c) with time.


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Cancer Cell Specific Delivery of Photosystem I ... - ACS Publications

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