Here DNA has been used as templating and self-assembling reagent to grow the chain like nanostructure. We have designed the composite in such a fashion that we obtained optical and magnetic properties together in a single biological material. Optical properties characterized by UV–visible absorption, Circular Dichroism (CD) and their analysis show no denaturization of DNA. Transmission electron micrographs (TEM) indicate formation of chain like structure of the nanoparticles. Particles were functionalized with folic acid for labeling and treatment of cancer cell.
Journal of Advanced Research (2012) 3, 359–363 Cairo University Journal of Advanced Research SHORT COMMUNICATION Engineering of DNA templated tri-functional nano-chain of Fecore–Aushell and a preliminary study for cancer cell labeling and treatment Madhuri Mandal a b a,* , Ayan Bandyopadhyay b Material Science Division, S.N Bose National Centre for Basic Sciences, Sector-III, Block – JD, Salt Lake, Kolkata 700098, India Department of Physics, West Bengal State University, Barasat, North 24 Paraganas, India Received 26 December 2011; revised April 2012; accepted April 2012 Available online 16 May 2012 KEYWORDS Fecore–Aushell–DNA nanochain; Trifunctional; Biomaterial; Cancer cell Abstract Here DNA has been used as templating and self-assembling reagent to grow the chain like nanostructure We have designed the composite in such a fashion that we obtained optical and magnetic properties together in a single biological material Optical properties characterized by UV–visible absorption, Circular Dichroism (CD) and their analysis show no denaturization of DNA Transmission electron micrographs (TEM) indicate formation of chain like structure of the nanoparticles Particles were functionalized with folic acid for labeling and treatment of cancer cell ª 2012 Cairo University Production and hosting by Elsevier B.V All rights reserved Introduction Biological molecules such as protein, DNA, enzymes, cancer cell targeting folic acid, etc with very small size are very active in a very small range Therefore manufacturing of various small substances combining with biological molecules is very * Corresponding author Tel.: +91 33 2335 5706/8; fax: +91 33 2335 3477 E-mail address: madhuri@bose.res.in (M Mandal) 2090-1232 ª 2012 Cairo University Production and hosting by Elsevier B.V All rights reserved Peer review under responsibility of Cairo University http://dx.doi.org/10.1016/j.jare.2012.04.002 Production and hosting by Elsevier important and recently these kinds of work getting much interest to the scientific community But proper engineering is necessary during synthesis such systems to develop the properties like magnetic, electrical as well as optical for their possible applications in brain research, neuro-computation, prosthetics, biosensors, bio-machines, etc For these we need to understand how bio molecule works with attachment of tiny magnetic and optical materials or nanoparticles DNA-metal nanocomposite consisting of combined electrical and magnetic properties has the potentiality to be used in molecular electronic devices, but its electrical and magnetic properties vary widely with change of different structures That is why nowadays the creation of three-dimensional, ordered structures of metal nanoparticles incorporated into DNA and their studies are big challenge to the researchers [1–3] Mirkin and co-workers [4] described a method of assembling colloidal gold nanoparticles into macroscopic aggregates using DNA as the linking element Nanowires of noble metals like gold [4], silver [5], palladium [6], platinum [7], and copper 360 Experimental The work was done in the Material Science Division, S.N Bose National Centre for Basic Sciences, Sector-III, Block – JD, Salt Lake, Kolkata 700098, India All the reagents used were 99.9% pure and purchased from Sigma–Aldrich Ultrapure distilled water (UPD water) DNAse, RNAse free was used in all synthesis procedures A Stock DNA solution (1 g/ L) was prepared by mixing appropriate amounts of DNA with Tris–EDTA buffer (pH 7.4) and was stirred overnight Source of DNA was herring sperm and it was purchased from Sigma– Aldrich The buffer solution helps to prepare a homogeneous DNA solution without any pop off of A and G bases in DNA and was stored in a refrigerator A stock solution of ferric chloride of 0.1 (M) and a stock solution of 0.05 (M) aqueous gold chloride (HAuCl4) were made The stock solution of ferric chloride was mixed with stock DNA solution at ratio 2:1 in volume ratio respectively and the mixture was stirred for 30 using a magnetic stirrer The UV–visible spectra were taken after mixing the solution well The resulting solution was then reduced by g of sodium borohydride taking in ml of water The solution color was turned to black, which indicates formation of iron nanoparticles by the reduction of iron ion to iron metal particles The 500 L stock aqueous gold chloride (HAuCl4) solution of 0.05 (M) was added to this dispersion Iron and gold ratio was taken in 30:1 atomic ratio The formation of gold coating was evident by appearance of a blackish pink coloration of the solution Gold was added just to prevent the oxidation Then the solution was functionalized with 0.1 M folic acid solution The cancer cell was injected to the mice to cause cancer artificially in mice After few days the cancer cells were collected from mice and treated with these nanoparticles after and before functinalization with folic acid In this case we have done a control experiment We have taken only cancer cell in 0.1% sodium chloride solution, cancer cell in 0.1% sodium chloride solution treated with nonfunctionalized nanoparticles and cancer cell in 0.1% sodium chloride solution treated with folic acid functionalized nanoparticles They are kept for h then all of them treated with the dye trypan-blue to check the viability of cancer cells Results and discussions The UV–visible spectra were taken at different stages during synthesis of material as shown in Fig The buffer solution has no characteristic absorption peak as shown in curve a, Fig The aqueous DNA solution taken in buffer medium has an absorption band at 260 nm (curve b, Fig 1) After formation of Fe nanoparticles on DNA chain resulted no significant red shift of absorption peaks for DNA (at 260 nm) (curve c, Fig 1) which indicates no aggregation of DNA strands But a significant amount of increase of absorbance value of DNA at about 260 nm is observed It is because an interaction between Fe atom and DNA Due to such interaction number of base pair per turn in DNA changes and an increase in absorption takes place After addition of HAuCl4 the solution turned to blackish pink color with appearance of an additional hump at 540 nm (as shown in Fig 2) due to the surface plasmon resonance (SPR) mode of gold nanoparticles Surface Plamon Resonances (SPR) are collective electron charge oscillations in metallic nanoparticles when they are excited by light They exhibit enhanced near-field amplitude at the resonance wavelength This field is highly localized at the nanoparticles and the resonance wavelength depends on the size of the nanoparticles We have previously prepared gold coated silver nanoparticles, Pd nanoparticles, etc where also after gold coating similar kind of SPR band was appeared [16–18] This indicates formation of core shell like structure of Fecore–AuShell onto DNA chain Circular Dichroism (CD) spectra were taken at all the steps of solution preparation and addition of different reagents for particles formation The CD spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to asymmetric molecules present in DNA molecule Dextrorotation and levorotation refer, respectively, to the properties of rotating plane polarized light clockwise (for dextrorotation) or anticlockwise (for levorotation) If a chiral molecule is dextrorotary, its enantiomer will be levorotary Such rotation takes place due to asymmetric structure of the molecules In case of DNA both Intensity (a u.) [8] have been deposited on DNA by chemical reduction method It has recently been demonstrated that metal nanoclusters can be formed through a DNA templated process that uses the chemical reduction of DNA-complexed metal ions [9] Onedimensional parallel and two-dimensional crossed palladium nanowires [10], copper nanowire [11], and silver nanorod [12] were fabricated on DNA template with direct reduction of metal ion Recently, Wei et al synthesized silver nanoparticles, nanorods and nanowires on the surface of DNA network [13] Synthesis methods, however, required long processing times and high temperatures with multiple steps Conductive gold have been synthesized on DNA scaffold [14] Synthesis of DNA templated chain like magnetic and optical material will provide new breakthrough in the nanotechnology research We have synthesized wire of gold coated Fe nanoparticles by DNA directing method Our results indicate that DNA serves as template for the growth of nano-wires For the first time we have reported synthesis of this kind of material in our previous work [15] Previous material was consists of Ni and as Ni is toxic so we switch to Fe instead of Ni In this paper we have reported some more studies on interaction of these particles with cancer cell before and after functionalization by folic acid M Mandal and A Bandyopadhyay a b c d 200 300 400 500 600 700 Wavelength (nm) Fig UV–visible spectra taken at different stages during the synthesis of material (a) For buffer solution, (b) after the addition of DNA to buffer solution, (c) after the formation of Fenanoparticles attached to DNA, (d) after complete formation of FecoreAushell DNA nanocomposite Intensity (A.U.) An easy process of cancer cell labeling 0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 -0.01 400 500 361 600 700 Wavelength (nm) Fig UV–visible spectra taken after Au-shell formation on Fe nanoparticles attached to DNA chain concentrating on SPR peak for Au CD (mdeg) a b c d e Fig TEM images (a) After formation of Fe nanoparticles on DNA template and (b) after formation of FecoreAushell-nanoparticles attached to DNA -1 -2 -3 220 240 260 280 300 320 340 Wavelength (nm) Fig Circular dichroism spectra taken for the solution at different stages of the material synthesis (a) For buffer solution, (b) after the addition of DNA to buffer solution, (c) after the addition of ferric chloride to the solution, (d) after the formation of Fe nanoparticles on DNA chain, e: after gold coating on Fe nanoparticles attached to DNA chain the dextrorotary and levorotary molecules are present Those are base pairs Therefore it gives both the negative and positive peaks in CD spectra and intensity of peaks is associated with number of base pair per turn The absence of asymmetry in structure results in zero CD intensity Secondary structure of DNA can be determined by CD spectroscopy in the ‘‘UVabsorption’’ spectral region At these wavelengths the chromophore is the peptide group The chromophore is the group present in a molecule which absorbs light and reaches to the excited state and within very short time again goes to ground state by releasing energy The chromophore group is responsible for giving absorption spectroscopy CD spectroscopy is one kind of absorption spectroscopy and for DNA molecule the peptide groups are the active chromophore in UV-absorption spectral region CD spectra taken for solution at different stages of synthesis of material are shown in Fig as curves a, b, c, d and e Where (a) is for buffer solution, (b) is for DNA in buffer solution, (c) is after addition of ferric chloride to the solution and (d) is after Fe nanoparticles formation on DNA chain and (e) is after gold coating on Fe nano attached to DNA chain A positive peak at 275 nm indicates B-confor- mation of DNA and change in intensity at this position indicates change in number of base pairs per turn After addition of iron salts, intensity at 275 nm increases which indicate number of base per turn increases for B-conformation After addition of salts and formation of nanoparticles the peak at 275 nm does not disappear This indicates that no denaturization or melting of DNA takes place after attachment of metal nanoparticles onto it but some change in intensity in both positive and negative signals takes place which indicates a minor change in asymmetric structure with change of number of base pair per turn in the B-conformation This structural change takes place due to electrostatic bond formation between DNA and ferric chloride A study has been done for such change of structure by addition of NaCl salts [19] TEM image before and after gold coating on iron nanoparticles attached on DNA chain is shown in Fig 4a and b The image indicates a clear chain like nanoparticles The particle size is of 20 nm diameter and more than 100 nm of length of the chain before gold coating but after gold coating particle diameter increases to 25 nm These chains like particles are formed due to attachment of these metallic nanoparticles on DNA chain Similar kind of particles morphology was also obtained in our previous work [15] DNA consists of negatively charged phosphate and amino groups that are good binding agents of metal ions of positive charge Due to such binding of metal ions on DNA chain, a chain like metal–DNA composite structure formation is possible but without use of DNA such chain like structure is not formed The XRD spectra taken for the Fe–Au–DNA sample are shown in Fig Indexed peak indicates both the Fe and Au are present But as the particles size is very small the peak broadening takes place which causes overlapping of peaks 362 M Mandal and A Bandyopadhyay plate and is able to functionalize the particles by folic acid for cancer cell labeling and it will be useful to kill the cancer cells Our next experiment is to work with mice model to check the feasibility of these particles for cancer treatment and to make a statistical study on it 350 Absorbance (a.u.) Fe-Au-DNA 300 Fe (110) Au (111) 250 200 Acknowledgements 150 20 30 40 50 60 70 80 Theta Fig Powder XRD spectra taken for FecoreAushell–DNA nanocomposite Authors are thankful to the Department of Science and Technology, Government of India for the funding under the project SR/FT/CS-090/2009 and thanks to Dr Manju Ray, IACS, Kolkata for her help and suggestions and thanks to Moumita for her assistance References Fig Image of cancer cells after treatment with folic acid functionalized FecoreAushell nanoparticles for Au(1 1) at about 38.2 and for Fe (1 0) at about 44.6 We have taken EDS data also which again authenticate presence of both Fe and Au in the sample in the ratio 70:30 We have observed that folic acid functionalized particles are attached to cancer cell but nonfunctionalized particles are not attached to cancer cell Another important phenomenon we have observed here that most of the cancer cells in 0.1% sodium chloride solution after treating with nonfunctionalized nanoparticles are alive (more than 70% cells are alive) but the cells treated with folic acid functionalized nanoparticles are died, almost 80% of cancer cells are died in this case The image of cancer cells after treatment with folic acid is shown in Fig Particles nonfunctionalized with folic acid are not getting attached to cancer cells so they cannot contribute in recognition or killing the cancer cells The particles after funtionalization by folic acid are attached to cancer cells and most of the cells are died in this case The reason may be that those nanoparticles may cause some changes in life cycle of cancer cells or cause some interruption in life cycle or may be after folic acid treatment the particles can go inside the cancer cells causing cell death Conclusion We have synthesized nanochain of Fecore–Aushell–DNA by simple chemical process where DNA successfully acts as tem- [1] Dillenback LM, Goodrich GP, Keating CD Temperatureprogrammed assembly of DNA:Au nanoparticle bioconjugates Nano Lett 2006;6:16–23 [2] Goodrich GP, Helfrich MR, Overberg JJ, Keating CD Effect of macromolecular crowding on DNA:Au nanoparticle bioconjugate assembly Langmuir 2004;20:10246–51 [3] Mirkin CA Programming the assembly of two- and threedimensional architectures with DNA and nanoscale inorganic building blocks Inorg Chem 2000;39:2258–72 [4] Mirkin CA, Letsinger RL, Mucic RC, Storhoff JJ A DNAbased method for rationally assembling nanoparticles into macroscopic materials Nature 1996;382:607–9 [5] Braun E, Eichen Y, Sivan U, Ben-Yoseph G DNA templated assembly and electrode attachment of conducting silver wire Nature 1998;391:775–8 [6] Richter J, Seidel R, Kirsch R, Mertig M, Pompe W, Plaschke J, et al Nanoscale palladium metallization of DNA Adv Mater 2000;12:507–10 [7] Seidel R, Ciacchi LC, Weigel M, Pompe W, Mertig M Synthesis of platinum cluster chains on DNA templates: conditions for a template controlled cluster growth J Phys Chem B 2004;108:10801–11 [8] Monson CF, Woolley AT DNA-templated construction of copper nanowires Nano Lett 2003;3:359–63 [9] Petty JF, Zheng J, Hud NV, Dickson RM DNA-templated ag nanocluster formation J Am Chem Soc 2004;126: 5207–12 [10] Deng Z, Mao C DNA-templated fabrication of 1D parallel and 2D crossed metallic nanowire arrays Nano Lett 2003;3: 1545–8 [11] Cho YS, Huh YD Synthesis of ultralong copper nanowires by reduction of copper-amine complexes Mater Lett 2009;63:227–9 [12] Becerril HA, Stoltenberg RM, Monson CF, Woolley AT Ionic surface masking for low background in single- and doublestranded DNA-templated silver and copper nanorods J Mater Chem 2004;14:611–6 [13] Wei G, Zhou H, Liu Z, Song Y, Wang L, Sun L, et al One-step synthesis of silver nanoparticles, nanorods, and nanowires on the surface of DNA network J Phys Chem B 2005;109: 8738–43 [14] Kundu S, Liang H Microwave synthesis of electrically conductive gold nanowires on DNA scaffolds Langmuir 2008;24:9668–74 [15] Mandal M, Mandal K Synthesis of DNA templated trifunctional electrically conducting, optical, and magnetic nanochain of Nicore–Aushell for biodevices J Appl Phys 2009;106:26101–3 An easy process of cancer cell labeling [16] Mallik K, Mandal M, Pradhan N, Pal T Seed mediated formation of bimetallic nanoparticles by UV irradiation: a photochemical approach for the preparation of ‘‘core-shell’’ type structures Nano Lett 2001;1:319–22 [17] Mandal M, Ghosh SK, Kundu S, Esumi K, Pal T UV photoactivation for size and shape controlled synthesis and coalescence of gold nanoparticles in micelles Langmuir 2002;18:7792–7 363 [18] Mandal M, Kundu S, Ghosh SK, Pal T Micelle mediated UV-photoactivation route for the evolution of Pdcore–Aushell and Pdcore Agshell bimetallics from photogenerated Pd nanoparticles J Photochem Photobiol A: Chem 2004;167: 17–22 [19] Baase WA, Johnson WC Circular dichroism and DNA secondary structure Nucl Acids Res 1979;6:797–814 ... broadening takes place which causes overlapping of peaks 362 M Mandal and A Bandyopadhyay plate and is able to functionalize the particles by folic acid for cancer cell labeling and it will be useful... Fe nanoparticles attached to DNA chain concentrating on SPR peak for Au CD (mdeg) a b c d e Fig TEM images (a) After formation of Fe nanoparticles on DNA template and (b) after formation of FecoreAushell-nanoparticles... formation on DNA chain and (e) is after gold coating on Fe nano attached to DNA chain A positive peak at 275 nm indicates B-confor- mation of DNA and change in intensity at this position indicates