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.
Trang 1SHORT COMMUNICATION
Engineering of DNA templated tri-functional nano-chain
cell labeling and treatment
Madhuri Mandal a,* , Ayan Bandyopadhyay b
a
Material Science Division, S.N Bose National Centre for Basic Sciences, Sector-III, Block – JD, Salt Lake, Kolkata 700098, India
bDepartment of Physics, West Bengal State University, Barasat, North 24 Paraganas, India
Received 26 December 2011; revised 2 April 2012; accepted 9 April 2012
Available online 16 May 2012
KEYWORDS
Fe core –Au shell –DNA
nano-chain;
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 mag-netic properties together in a single biological material Optical properties characterized by UV–vis-ible absorption, Circular Dichroism (CD) and their analysis show no denaturization of DNA Transmission electron micrographs (TEM) indicate formation of chain like structure of the nanopar-ticles Particles were functionalized with folic acid for labeling and treatment of cancer cell
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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
important and recently these kinds of work getting much inter-est to the scientific community But proper engineering is nec-essary 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 electri-cal 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 assem-bling 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
* 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
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Peer review under responsibility of Cairo University.
http://dx.doi.org/10.1016/j.jare.2012.04.002
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Cairo University
Journal of Advanced Research
Trang 2[8] have been deposited on DNA by chemical reduction
meth-od 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]
One-dimensional parallel and two-One-dimensional crossed palladium
nanowires[10], copper nanowire[11], and silver nanorod[12]
were fabricated on DNA template with direct reduction of
me-tal 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
re-search We have synthesized wire of gold coated Fe
nanopar-ticles 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
mate-rial 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
functionali-zation by folic acid
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
Ultra-pure 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
fer-ric chloride of 0.1 (M) and a stock solution of 0.05 (M)
aque-ous 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 min using a magnetic stirrer The UV–visible spectra were
taken after mixing the solution well The resulting solution
was then reduced by 1 g of sodium borohydride taking in
5 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
dis-persion 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
nonfunctional-ized nanoparticles and cancer cell in 0.1% sodium chloride
solution treated with folic acid functionalized nanoparticles
They are kept for 1 h then all of them treated with the dye try-pan-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 inFig 1 The buffer solution has no characteristic absorption peak as shown in curve a, Fig 1 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 sig-nificant red shift of absorption peaks for DNA (at260 nm) (curve c, Fig 1) which indicates no aggregation of DNA strands But a significant amount of increase of absorbance va-lue of DNA at about 260 nm is observed It is because an inter-action between Fe atom and DNA Due to such interinter-action number of base pair per turn in DNA changes and an increase
in absorption takes place After addition of HAuCl4the solu-tion turned to blackish pink color with appearance of an addi-tional hump at540 nm (as shown inFig 2) due to the surface plasmon resonance (SPR) mode of gold nanoparticles Surface Plamon Resonances (SPR) are collective electron charge oscil-lations 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 nano-particles 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 differ-ences 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 levo-rotation 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
0 1 2
3
a b c d
Wavelength (nm) Fig 1 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 Fe-nanoparticles attached to DNA, (d) after complete formation of
Fe Au DNA nanocomposite
Trang 3the 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
‘‘UV-absorption’’ spectral region At these wavelengths the
chromo-phore is the peptide group The chromochromo-phore 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
responsi-ble 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 inFig 3as 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 indi-cates change in number of base pairs per turn After addition of iron salts, intensity at 275 nm increases which indi-cate 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 denatur-ization or melting of DNA takes place after attachment of me-tal nanoparticles onto it but some change in intensity in both positive and negative signals takes place which indicates a
min-or 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 be-tween 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 nanopar-ticles attached on DNA chain is shown inFig 4a and b The image indicates a clear chain like nanoparticles The particle size is of20 nm diameter and more than 100 nm of length
of the chain before gold coating but after gold coating particle diameter increases to25 nm These chains like particles are formed due to attachment of these metallic nanoparticles on DNA chain Similar kind of particles morphology was also ob-tained 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 com-posite 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 inFig 5 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
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Wavelength (nm)
Fig 2 UV–visible spectra taken after Au-shell formation on Fe
nanoparticles attached to DNA chain concentrating on SPR peak
for Au
-3
-2
-1
0
1
2
3
4
5
6
Wavelength (nm)
a b c d e
Fig 3 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
Fig 4 TEM images (a) After formation of Fe nanoparticles on DNA template and (b) after formation of FecoreAushell -nanopar-ticles attached to DNA
Trang 4for Au(1 1 1) at about 38.2 and for Fe (1 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 ratio70:30
We have observed that folic acid functionalized particles
are attached to cancer cell but nonfunctionalized particles
are not attached to cancer cell Another important
phenome-non we have observed here that most of the cancer cells in
0.1% sodium chloride solution after treating with
nonfunc-tionalized nanoparticles are alive (more than 70% cells are
alive) but the cells treated with folic acid functionalized
nano-particles are died, almost 80% of cancer cells are died in this
case The image of cancer cells after treatment with folic acid
is shown inFig 6 Particles nonfunctionalized with folic acid
are not getting attached to cancer cells so they cannot
contrib-ute 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
can-cer cells causing cell death
Conclusion
We have synthesized nanochain of Fecore–Aushell–DNA by
simple chemical process where DNA successfully acts as
tem-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
Acknowledgements Authors are thankful to the Department of Science and Tech-nology, 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
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