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Genomic DNA Interactions Mechanize Peptidotoxin-mediated Anti-cancer Nanotherapy Santosh K. Misra, Aaron S. Schwartz-Duval, and Dipanjan Pan Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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Molecular Pharmaceutics

Genomic DNA Interactions Mechanize Peptidotoxin-mediated Anti-cancer Nanotherapy Santosh K. Misra, Aaron S. Schwartz-Duval, Dipanjan Pan*, Departments of Bioengineering, Materials Science and Engineering and Beckman Institute, University of Illinois at Urbana-Champaign, Mills Breast Cancer Institute, and Carle Foundation Hospital, Urbana, Illinois 61801, USA. Corresponding author. Email: *[email protected] KEYWORDS. Peptidotoxins, nanoparticles, nanomedicine, cancer therapy, DNA interference

ABSTRACT. Host defense peptides (HDPs) are a class of evolutionarily conserved substances of the innate immune response that has been identified as major players in the defense system in many living organisms. Some of the HDPs are also referred as peptidotoxins which offer immense potential for anti-cancer therapy. However, their therapeutic potential is yet to be fully translated mainly due to their off-target toxicity. Here we show that their nano-enabled delivery may become beneficial in controlling their delivery in intra-cellular space. We introduced an amphiphilic polymer to synthesize a well-defined, self-assembled, rigid-cored polymeric nanoarchitecture for controlled delivery of three model peptidotoxins, i.e. melittin, TSAP-1 and a negative control peptide of synthetic origin. Interestingly, our results revealed strong interaction

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of peptidotoxins with duplex plasmid DNA. Extensive biophysical characterization (UV-vis spectroscopy, gel electrophoresis, MTT assay and flow assisted cell sorting) experimentally verified that peptidotoxins were able to interact with genomic DNA in vitro and in turn influence the cancer cell growth. Thus, we unraveled that through genomic DNA regulation, peptidotoxins can play a role in cell cycle regulation and exert their anticancer activities.

INTRODUCTION

Host defense peptides (HDPs) are recognized as chief players in the defense system found among all classes of life. They are usually amphipathic, have a net positive charge (generally +2 to +9) and are short in sequence length (10-100 aa). The anticancer property of HDPs has recently been explored in human cancer model.1-12 This class of peptides generally represent high water solubility, a broad spectrum of cytotoxicity, and show ability to overcome multidrug resistance developed in cancer cells treated with conventional chemotherapy drugs.13-18 Several biophysical studies have shown that HDPs with 20-40 amino acid residues can penetrate the cell membranes of microorganisms. However, controlling their efficiency towards desired site and cellular space is a major challenge in effectively realizing their potential. For improving selectivity and reducing off target toxicity, we lay emphasis on a fundamental chemical strategy to design anano-vehicle. Three model peptides were selected including Melittin(Peptide-I), TsAP-1 (Peptide-II) and a non-potent amino acid sequence (Peptide-III termed ‘CtrlPT’), to validate preparation of nano-toxins and their improved efficiency. Melittin is an amphipathic peptide made up of 26 amino acid (aa) residues


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(GIGAVLKVLTTGLPALISWIKRKRQQ), has been found to be a potent component of bee venom Apis mellifera.19-23

Figure 1.(A)Strategy of anti-cancer therapy by peptide-DNA interaction inhibiting DNA unwinding required for transcription and in turn translation process on cell growth and division and (B) Hydrogen bonded peptide-I with duplex DNA having 17 H-bond interactions and low docking score of -12.12 mmol/kcal.

TsAP-1 is a non-disulphide-bridged toxin 17-mer peptide (FLSLIPSLVGGSISAFK) derived from the venom of the Brazilian yellow scorpion, Tityusserrulatus, with anti-microbial and anticancer characteristics.24-26Melittin and TsAP-1 are known to exert their toxic activity by disrupting plasma membranes following pore formation.19 Amino acid residues of melittin and TsAP-1 interact directly with anionic cellular membranes via electrostatic interactions and hydrophobic regions; this interaction is responsible for membrane permeation and disruption. Our in silico results indicated that the fate of released peptido-toxin from nano-carrier can be rationalized in regard to its interaction with genomic pool cellular components as interaction of


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peptidotoxin with DNA (Figure 1A). Specifically, the interactions of melittin with DNA have been explored in-silico by performing docking studies. In this representative study it was found that melittin intertwines well and remains within a close proximity of DNA structures, through 17 H-bond interactions and 3 hydrophobic interactions with quite low docking energy due to multi-interactions and stable docked structures (Figure 1B). The role of melittin in its interactions with DNA has been investigated but not evaluated experimentally against genomic DNA in cellular systems. Herein, we present a methodology to prepare nano-assemblies of PRCM (polymeric rigid core micelles) and their application in generating nano-peptido-toxins by incorporating peptidotoxins. We also evaluated interaction of Nano peptidotoxins with genomic DNA and its structural mimic (plasmid DNA). We utilized UV-Vis spectroscopy, gel electrophoresis and flow assisted cell scanning as study tools to determine interactions of nano-peptido-toxins with DNA. Finally, cell growth inhibition assay (MTT) was used to evaluate improved efficiency of nano-peptido-toxins against MCF-7 as a model breast cancer cell line.

MATERIALS AND METHODS Materials All the suspensions/solutions were made using deionized water (dH2O), buffers at a defined pH, and analytical grade reagents were used. All Nano-toxin preparations were done in a germ-free sterile condition. Preparation and storage of all reagents were maintained at temperatures required for each specific experiment. All produced waste material was managed in accordance with federal, state and local regulations. Tetrahydrofuran (THF); ≥99.9%, inhibitor-free, Potassium chloride, molecular biology grade, ≥99.0%, Tris(hydroxymethyl)aminomethane,


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≥99.8%, Ethylenediaminetetraacetic acid, 99.4-100.6%, powder, Ethidium Bromide, molecular biology grade, powder, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 98% Uranyl acetate dehydrate puriss. p.a., ACS reagent, ≥98.0% (T) (Sigma-Aldrich). Poly(styrene) 67-block-poly (acrylic acid) 27

(PS67-b-PAA27,Mn 1,600–1,950 (poly(acrylic acid)), Mn

6,500–7,000 (polystyrene), Mn 8,100–9,100, average Mn 8,700, mp: 192–197°C; Mw/Mn = 1.2), Peptide-I [Melittin (Melittin from honey bee venom, ≥85% (HPLC)), Sequence: Gly-IleGly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-LysArg-Gln-Gln-NH2 , Peptide-III [CtrlPT 96%, {Ahx}-Leu-Pro-Cys-Asp-Tyr-Tyr-Gly-Thr-CysLeu-Asp] were purchased from Sigma-Aldrich. Fetal bovine serum was procured from GIBCOTM. pBR322 plasmid DNA, Chain Length: 4,361 bp (ClonTech). Peptide-II [TsAP-1 (Venom of the Brazilian yellow scorpion, Tityusserrulatus; 96.7 %, Phe-Leu-Ser-Leu-Ile-ProSer-Leu-Val-Gly-Gly-Ser-Ile-Ser-Ala-Phe-Lys) (Novoprolabs). The 6× DNA loading dye (30% v/v glycerol, 0.25% w/v bromophenol blue, 0.25% w/v xylene cyanol FF), TE buffer (mix 1 mL of 1 M Tris-HCl, pH 8.0, and 200 µL of 0.5 M EDTA were purchased from Thermo Fisher Scientific. The 50× TAE buffer (242 g of Tris to 57.1 mL of glacial acetic acid (CH3COOH) and 100 mL of 0.5 M EDTA was prepared before use by making up to 1 L with dH2O. EtBr staining buffer was prepared using 300 µg of EtBr to 1 L of 1× TAE. 1× lysis buffer was purchased from Promega, Fitchburg, WI. 1% (w/v) agarose gel was prepared by dissolving 100 mg of agarose (Sigma Aldrich) in 100 mL of 1× TAE buffer, then heat the solution by means of a microwave for 1-2 min.

DNA duplex-peptide interaction. Docking studies were performed considering the most favorable interactions between amino acids and nucleobases as arginine and lysine with guanine and also for lysine with thymine.


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Interactions between DNA bases and amino acid residues were introduced by H-bonding and hydrophobic packing interactions in the major groove. It was found that amino acid residues Glu and Asp were H-bond acceptors and only interacted with C or A bases. Ser, Cys, and Thr amino acids were found to play dual role as both donor and acceptor, and Arg and Lys played H-bond donor but not acceptors. Some extent of hydrophobic interaction was retained in the major groove of DNA due to presence of the single methyl group of the T base. Hydrophobic interactions for Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Thr with three methyl groups were found with the T base. In similar interaction pattern, two ring CH groups of the C bases interacted with hydrophobic residues whereas Ala appeared to be insufficiently hydrophobic to contact the C base.

MCF-7 (ATCC® HTB-22™) culture. Cell culture medium was made from high-glucose Dulbecco’s Modified Eagle Medium (DMEM) stored at 4 °C and used after thawing at 37 °C. Complete cell culture medium was made using cell culture medium containing 10% (v/v) FBS and 1X penstrep (100 U/mL). Dulbecco’s phosphate buffered saline (DPBS) was used without calcium chloride and magnesium chloride added in autoclaved water with sterile-filtered, suitable for cell culture, pH = 7.4. 1× trypsin solution with EDTA (0.25%).

Preparation of PRCM. In a 4 ml scintillation vial polyethylene glycol cetyl ether (1 mg) was melted at 65°C for 5 min. Once melted, 2 ml of water was added dropwise with a 22 gauge needle (~1 drop/sec) with magnetic stirring (1150 RPM). After allowing the solution to stir from approximately 20


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minutes, 250 µL of a 2 mg/ml PS67-b-PAA27/THF solution was added dropwise (0.1 drop/sec) with continued stirring for an additional 24 hours to evaporate THF. After overnight THF evaporation, total volume was made up to 2 ml by adding autoclaved nanopure water (0.2 µM), stirred for 10 min at room temperature and purified by dialysis against nanopure (0.2 µM) water (10,000 Da MWCO cellulose membrane) for a 24h. Once purified, PCRM should be stored at 4 °C till next use.

Preparation of Nano-peptidotoxin. In 1.5 ml polystyrene sterile centrifuged tubes, 20 mM peptide (I,II, or III) solutions in 100 mM KCl were prepared. Before combining with PCRM to form nano-peptidotoxins, the 20 mM peptide solutions were diluted to a concentration of 5 mM in ultra-pure water. Once diluted to 5 mM, 50 µl portions of the peptide (I, II, or III) solutions were added to 100 µl of the as prepared PRCMs and made up to 1 mL with ultra-pure water. To promote the interactions between the PCRMs and peptidotoxins, the mixtures were vortexed at room temperature for 5 min before incubating at 37 °C for 30 min. Following this, the nanopeptidotoxins were purified through dialysis against nanopure (0.2 µM) water using a 10,000 Da MWCO cellulose membrane for a 12h time point by replacing dialyzing water after every 4h at RT. Purified nano-peptidotoxins were stored 4 °C till use.

Determination of Peptide-I loading percentage in Nano-peptidotoxins. In 1.5 ml polystyrene sterile centrifuged tubes, 20 mM peptide I solutions in 100 mMKCl were prepared. Before mixing with PCRM to form nano-peptidotoxins, the 20 mM peptide solutions were diluted to a concentration of 5 mM in ultra-pure water. Once diluted to 5 mM, 50 µl


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portions of the peptide I solutions were added to 100 µl of the as prepared PRCMs and made up to 1 mL with ultra-pure water. The mixed suspension was then vortexed at room temperature for 5 min before incubating at 37 °C for next 30 min. The nano-peptidotoxins were pelleted out of solution through centrifugation at 120K rpm for 1h at 4 °C. From this, the supernatant was collected for UV-Vis spectroscopic analysis. Using varied known concentrations of Peptide-I, a standard UV-Vis absorption spectra was generated in order to correlate the unknown concentration suspended peptide from the supernatant collected from NanoPeptide-I with the measured absorbance values. The loading percentage was calculated using the following formula: % Peptide-I Loading = ((Total amount of peptide used -amount of Peptide-I from collected supernatant after centrifugation)/(Total amount of Peptide-I loaded in NanoPeptide-I)) x100

Characterization of Nanopeptidotoxins evaluation of the hydrodynamic diameter. As prepared Nanopeptidotoxin samples were diluted to 1 µM concentration preceding DLS measurement. Following the manufacturer’s instructions, DLS machine was set to match the materials refractive index and absorptivity of the Nanopeptidotoxin. For each DLS measurement there were >10 measurements resulting in the characteristic peaks and polydispersity indexes (PDI). These peaks represent the population distribution of particles.

Evaluation of the anhydrous diameter. Nanotoxin suspension (2.5 µL) was drop-coated of on carbon-coated copper grids and allowed 2 minutes for the particles to settle onto the grid. Uranyl acetate stain in dH2O (0.1% w/v) was then drop-coated (5 µL) into the cationic liposome suspension, and incubated for 2 min at RT. After 2


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min-incubation at RT, the excess liquid was wicked off the grid and samples were allowed to air dry under a covered lid for ~2 h preceding TEM imaging.

Evaluation of zeta potential of nanopeptidotoxins. As prepared Nanopeptidotoxin samples were diluted to 5 µM concentration preceding Zeta potential measurement. Add samples in zeta sizing cuvettes. Run samples on zeta sizer machine (MALVERN) following the manufacturer’s instructions.

Gel electrophoresis for DNA binding assay. To aliquots of Peptide-III, Peptide-II or Peptide-I of various concentrations, 2 µL of pDNA (0.1 µg/µL) was admixed for a range of molar ratios from 0.01-2.5 (pDNA: peptide mole ratio). These mixtures were then incubated for ~20 min at RT to allow complexation. Gel electrophoresis cocktail was prepared by adding 3 µL of DNA loading dye with the DNA: Peptide mixture, to prepare a total of 20 µL of complexes made up with dH2O. Samples were loaded in a 1% (w/v) agarose gel along with an uncomplexed DNA control sample and run at 100 V for 20-30 min in 1× TAE buffer. After completion of DNA migration, the gel was removed, rinsed with diH2O, and then stain with EtBr in running buffer (1× TAE solution of 300 µg/L EtBr) for ~5 min. After staining the excess EtBr was washed with washing buffer (1× TAE buffer) for 5 min before imaging. The gel was exposed with light of λ = 362 nm for 0.5-2 sec to visualize and photograph bands of EtBr intercalated DNA or peptidoplex. The DNA band density was determined using analysis software GelQuantNET. The DNA binding (percentage) was calculated using the following formula:


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DNA binding (%) = (Band intensity from lipoplex-background intensity)/(band intensity from un-complexed DNA-background intensity) × 100

UV-Vis spectroscopic study for nanopeptidotoxin-DNA interaction. The UV-Vis absorption ranging from 200 to 700 nm was recorded for solutions of 1 µL of pBR322 (1 µg/µL; 3 µmol) admixed with 1 mL PBS buffer after 5 min incubation at RT. To this solution, 0.5 µL aliquots of 1 mM Peptide-I, Peptide-II or Peptide-III to pDNA solution was added and incubate for 5 min before acquiring UV absorption. Further additions of 0.5 µL aliquots were added with UV-Vis measurements to finally reach a peptide concentration of 7.5 µM.

Interaction of Nano-peptidotoxins with genomic DNA in vitro. In a 6-well cell culture plate, MCF-7 cells were seeded at a density of 4 × 105 cells/well, with 2 mL/well of antibiotic-free complete cell culture medium. Cells were incubated for 24h at 37 °C, 99% humidity with regular supply of 5% CO2 till ~80% confluence. Cell treatments were prepared by adding Peptide-I, NanoPeptide-I, Peptide-II, NanoPeptide-II, Peptide-III, and NanoPeptide-III formulations in reconstituted cell growth medium (200 nM peptide).Preceding treatment the entire old medium from wells of growing cells was removed and replaced with 2 ml of freshly prepared treatment formulations. Cells were then incubated for 6h and 48h at 37 °C, 99% humidity with regular supply of 5% CO2. After incubation for 4h, entire spent medium was collected. After incubations were completed, cells were trypsinized (0.5% Trypsin (500 µL) after washing it with 500 µL of DPBS and incubating for 3 min at 37 °C. Trypsinized cells were collected and pelleted via centrifugation at 4000 g for 10 min at 4 °C. Collected cell pellet was


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resuspended in 100 µL of reconstituted medium then fixed with 75% chilled EtOH with continuous vortexing. Fixed cells were stored for 12h at -20 °C. At the end fixed cells were spun at the 4000 g for 10 min and produced cell pellet was washed with 1 mL of DPBS three times. Cells were re-suspended in 1 mL of RNAse I (2 µg/mL) and incubated at 37 °C for 12h. Propidium iodide (PI, 2 µg/mL) was added to cell suspensions and incubated at RT for 20 min. A flow assisted cell scanning was performed on collected PI rich cell population. Cell numbers were plotted against fluorescence intensity of stained cells.

Functional activity of Nano-peptidotoxins by cell growth inhibition. In a 96-well cell culture plate, MCF-7 cells were seeded at a density of 10 × 103 cells/well, with 200 µL/well of antibiotic-free complete cell culture medium. Cells were incubated for 24h at 37 °C, 99% humidity with regular supply of 5% CO2 till ~80% confluence. Cell treatments were prepared by adding Peptide-I, Nano-Peptide-I, Peptide-II, NanoPeptide-II, Peptide-III, and NanoPeptide-III formulations in reconstituted cell growth medium (200 and 100 nM peptide). Preceding treatment the entire old medium from wells of growing was removed and replaced with 200 µl of freshly prepared treatment formulations. Cells were then incubated for 44 h at 37°C, 99% humidity with regular supply of 5% CO2. After incubation of 44h, 10 % (v/v) of MTT (5 mg/mL) was added to growing cells for 4 h at 37 °C. After this 4 h incubation, the entire spent medium removed and the formazan crystals dissolved by adding 200 µL/well of DMSO to all the wells with 5 min RT incubation with gentle rocking. After the formazan crystals were dissolved the absorption was recorded at λ = 592 nm to evaluate the cell viability. Cell viability (percentage) was calculated according to the following formula:


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Cell viability (%) = (A592Lipoplex treated cells-A592background)/(A592untreated cells-A592background) × 100

Statistical Analysis: The tests were done in triplicate and the results were expressed as the mean ± standard deviation. The data were analyzed on the GraphPad Prism 6.0 software using ONE WAY analysis of variance (ANOVA) Bonferroni correction for post hoc. The p

Genomic DNA Interactions Mechanize ... - ACS Publications

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