Abstract

Background

A. TiO2 Nanoparticles smaller than 20 nm contain “corner defects” which can bind enediol bidentate ligands such as dopamine and Alizarin Red S. Binding dopamine to the corner defects allows for binding DNA or PNA to the nanoparticles, thus creating nanoconjugates.

B. TiO2-Nanoconjugates undergo inducible charge separation from exposure to electromagnetic radiation above 3.2eV. This charge separation created electropositive holes. These electropositive holes travel through the dopamine linker through the backbone of PNA, and can lead to the scission of DNA backbone. [Diagram produced by Eric Brown, modified from Gayle Woloschak]

C. Plasmid DNA or bacterial DNA) is used to measure DNA damage in our experiment. It normally takes on a supercoiled configuration but when the supercoiled configuration is cleaved on one strand the plasmid will take on a nicked configuration and if both strands are cleaved, then the plasmid will take on a linear configuration.

A. B. C.

Hypothesis and ObjectivesHYPOTHESES• Coating Fe3O4@TiO2 with Quercetin and Doxorubicin will enhance DNA

cleavage in vitro• Fe3O4@TiO2 nanoconjugates will be effective in inducing in vitro photocatlytic

DNA cleavageOBJECTIVES • Determine the primary mechanism by which Fe3O4@TiO2 nanoparticles

cause cleavage of plasmid DNA

Results

A. Effect of Alizarin Red S on cleavage of plasmid DNA by Fe3O4@TiO2 At concentrations as low as 35% coverage, Alizarin Red S causes enhanced amounts of plasmid DNA cleavage by Fe3O4@TiO2 nanoparticles. However, it is not necessary to cause cleavage of plasmid DNA.

Effect of Alizarin Red S, Quercetin, and Doxorubicin on cleavage of plasmid DNA by Fe3O4@TiO2. Cleavage of plasmid DNA by Fe3O4@TiO2 nanoparticles is more efficient when the nanoparticles are coated with Alizarin Red S. Coating nanoparticles with 35% Doxorubicin had a negligible affect on the cleavage of plasmid DNA. Quercetin appears to enhance the cleavage of plasmid DNA as we see multiple bands that were not seen with other coatings.

A. B.

C.

Results

A. Supercoiled DNA levels were decreased by ~30%in all non DMSO samples. The addition of DMSO caused cleavage in all samples to return back to control levels.

B. Supercoiled DNA levels were decreased by ~30%in all non DMSO samples. The addition of DMSO caused cleavage in all samples to return back to control levels.

Quercitin Enhances Fe3O4@TiO2 Cleavage of Plasmid DNA In Vitro

Quantification of Supercoiled DNA Following Light Induced DNA Cleavage Assay Utilizing Fe3O4@TiO2 Nanoconjugates Using Volume Analysis

A. B.

Conclusions

Acknowledgements

Materials and Methods

Exploring the Affects of Supplemental Coatings and Determining the Main Mechanism of In Vitro DNA Cleavage Utilizing Fe3O4@TiO2 Nanoparticles and Nanoconjugates

Derrius L. Quarles1*, Felix Richter2, Caroline B. Doty2, Tatjana Paunesku Ph.D2,3, and Gayle E. Woloschak Ph.D2,3,4

1Morehouse College 830 Westview Drive, Atlanta, Georgia 303142Department of Radiation Oncology, Northwestern University, 303 E. Chicago Avenue,

Chicago, Illinois 606113Department of Radiology, Northwestern University, 303 E. Chicago Avenue, Chicago, Illinois 60611

4Department of Cellular and Molecular Biology, Northwestern University, 303 E. Chicago Avenue, Chicago, Illinois 60611* Corresponding author: Derrius Quarles, E-mail: derriusquarles@gmail.com

[1] Paunesku et al., Biology of TiO2-oligonucleotide nanoconjugates. Nature Materials, 2003 May, 2(5):343-6.[2] Thurn et al. Labeling Ti02 Nanoparticles with Dyes for Optical Fluorescence Microscopy and Determination of Tio2-DNA Nanoconjugate Stability. NIH Author Manuscript, 2009 June; 5(11):1318-1325[3] Doty et al. Effects of Fluorescent Dye Coating of Metal Oxide nanoparticles on DNA Scission. Journal of the Robert H. Lurie Cancer Center of Northwestern University, 2009 8:72-76

•Coating Fe3O4@TiO2 nanoparticles with Alizarin Red S enhances cleavage ofplasmid DNA•Coating Fe3O4@TiO2 nanoparticles with Quercetin greatly enhances cleavage of

plasmid DNA•Fe3O4@TiO2 nanoconjugates induce a significant amount of in vitro DNA

cleavage •Production of ROS by Fe3O4@TiO2 nanoparticles is the main mechanism that

causes cleavage of plasmid DNA.

In Vitro Light induced Cleavage Assay w/ Fe3O4@TiO2 Nanoparticles 6nm Fe3O4@TiO2 nanoparticles at a concentration of 564nM were coated with 1.5mM Alizarin Red S in dark tubes. 6.8nM pKaede plasmid DNA was added to the Alizarin coated nanoparticles and half of the samples were pipetted onto parafilm for light induction. Light was applied to the samples for 5 minutes at an intensity of 75 watts by a Fiber-Lite© High Intensity Illuminator Series 180. Samples were then loaded onto a 1.2% agarose gel stained with Gel Star. DNA gels were visualized using a Typhoon Fluorescence Scanner.In Vitro Light induced Cleavage Assay w/ Fe3O4@TiO2-PNA Nanoconjugates6nm Fe3O4@TiO2 nanoconjugates were covalently linked, via a dopamine linker, to a Peptide Nucleic Acid (PNA) oligonucleotide. The PNA linked to the nanoparticle was a 17 base pair sequence that matches a sequence in the HPV viral DNA genome. 6.8nM Plasmid DNA was then added to these nanoconjugates in dark tubes. Half of the the sample was then pipitted onto parafilm for light induction. Light was applied to the samples for 5 minutes at an intensity of 75 watts by a Fiber-Lite© High Intensity Illuminator Series 180. Samples were then loaded onto a 1.2% agarose gel stained with Gel Star. DNA gels were visualized using a Typhoon Fluorescence Scanner.Quantitative Analysis of Cleavage AssayAfter visualization via Typhoon Fluorescence Scanner, gels were then quantitatively analyzed utilizing ImageQuant volume analysis. Volume analysis utilizes the amount of fluorescence exhibited from each band of DNA within a defined area. The amounts of fluorescence (volume) from each band is then compared and specific quantities of supercoiled, nicked, and linear DNA are computed..

BACKGROUNDNanoparticles are defined as particles with a size in the range of 1-100nm. Within this size range, many compounds exhibit unique chemical and surface properties that do not exist in the bulk form. These properties can be manipulated to form biomaterials that can deliver drugs to targeted sites and to kill cancerous cells directly. Titanium Dioxide (TiO2), is such a material. In bulk form it is virtually non-reactive and non photocatalytic, however, at the nano level, when exposed to photon energies above 3.2eV (e.g white light) in an aqueous environment electrons in the valence shell jump to the conduction band causing the production of electropositive holes on its surface that lead to the production of reactive oxygen species (ROS). These ROS have the ability to induce cleavage of DNA in vitro. HYPOTHESISWe speculated that binding Doxorubicin and Quercetin Dihydrate to the surface of Fe3O4@TiO2

nanoparticles would increase their DNA cleaving capabilities in vitro. The objectives of this investigation was to evaluate the ability of the antiproliferative compounds Quercetin and Doxorubicin to enhance the cleavage of DNA in vitro in conjunction with Titanium Dioxide (Ti02) shell - Iron Oxide (Fe3O4) core (Fe3O4@TiO2) nanoparticles. We also wanted to investigate the main mechanism in which Fe3O4@TiO2 nanoconjugates (Fe3O4@TiO2 with a dopamine linker and a PNA attached) cleave DNA. METHODSTo evaluate the effectiveness of the antiproliferative compounds ability to enhance DNA cleavage we utilized light induced cleavage assays. To investigate the main mechanism in which Fe3O4@TiO2 nanoconjugates induce DNA cleavage we also used light induced cleavage assays, however, we modified the assays with the use of Fe3O4@TiO2 conjugated to a dopamine linker bound to a 17 base pair PNA and DMSO (dimethyl sulfoxide), which sequesters ROS.RESULTSQuercetin was found to enhance the cleavage of DNA in vitro while Doxorubicin had minimal affects. The addition of DMSO to samples of nanoconjugates and plasmid DNA greatly reduced the amount of cleavage observed, allowing us to conclude that ROS formation is the main mechanism of cleavage.

Fe3O4@TiO2 Nanoconjugates w/ 50% DMSO and GIE

B/C.

LADDER

TiO2-PNA Nanoconjugates Synthesis

TiO2 Light Induced ROS Formation

Plasmid DNA Electrophoresis Migration

A.Fe3O4@TiO2 Nanoconjugates w/ 50% DMSO no GIE DNA Cleavage seen with all nanoconjugates, however, cleavage was significantly reduced after addition of 50% DMSOB.Fe3O4@TiO2 Nanoconjugates w/ 50% DMSO and GIE DNA Cleavage seen with all nanoconjugates, however, cleavage was significantly reduced after addition of 50% DMSO

Fe3O4@TiO2 Nanoconjugates w/ 50% DMSO no GIE

Results