Journal of Biomaterials and Nanobiotechnology, 2011, 2, 281-292
doi:10.4236/jbnb.2011.23035 Published Online July 2011 (http://www.SciRP.org/journal/jbnb)
281
Novel Biomaterial for NCT—“Rigid” Particles of
(DNA-Gadolinium) Liquid-Crystalline Dispersions
Yuri M. Yevdokimov1, Victor I. Salyanov1, Sergey V. Akulinichev2, Vladimir M. Skorkin2,
Pavel V. Spirin1, Nataliya N. Orlova1,Vladimir I. Popenko1, Vladimir S. Prassolov1
1Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, Russia; 2Institute for Nuclear Research of the
Russian Academy of Sciences, Moscow, Russia.
Received February 11th, 2011; revised March 22nd 2011; accepted April 21st, 2011.
ABSTRACT
The formation and physico-chemical properties of biomaterial, based on double-stranded (ds) DNA molecules and
bearing high concentration of gadolinium, is described. This “rigid” biomaterial demonstrate a few unique properties:
1) the ds DNA molecules forming complexes with gadolinium are fixed in the spatial structure of “rigid” particles, 2) an
abnormal negative band in the circular dichroism spectrum permits to follow the formation of this biomaterial; 3) local
concentration gadolinium in the content of biomaterial can reach 40%. These properties show that we are dealing with
a novel type of biomaterial strongly enriched by gadolinium. This opens a gateway for practical application of this
biomaterial for neutron-capture reactions. A first attempt to apply this material for neutron-capture reaction in combi-
nation with neutron generator of thermal neutron flux was performed. Positive result obtained at destruction of CHO
cells allows one to state that the advantages of this biomaterial are a simple manipulation with it, a possibility to adjust
its gadolinium content, long-term stability of its physico-chemical properties, as well as a reduced cost of neutron-
capture experiment.
Keywords: Liquid-Crystalline DNA Dispersions, Cholesterics, Circular Dichroism Spectrum, Gadolinium,
Neutron-Capture Therapy
1. Introduction
Biological molecules, in particular nucleic acid mole-
cules, are becoming increasingly popular as polyfunc-
tional object for nanobiotechnology [1].
Deliberate and controlled variation in the properties of
these molecules provides possibilities for formation of
various types of nanoconstructions (nanostructures, nano-
biomaterials, etc.) allowing their wide application in bio-
technology and medicine. For instance, nanoconstruc-
tions, formed by double-stranded (ds) DNA molecules
fixed in structure of cholesteric liquid-crystalline disper-
sions (CLCDs) and cross-linked by nanobridges, were
used as biosensing units for biosensor devices [1]. Be-
sides, DNA nanoconstructions may be used as “carriers”
for genetic material or as a “reservoir” for various bio-
logically active compounds embedded in the composition
of these structures.
Among recently studied nanoconstructions, “rigid”
nanoconstruction (“rigid” DNA particles) are of special
practical importance and interest due to their unique phy-
sicochemical properties [2].
Recently possibilities of application of the “rigid” par-
ticles of (ds DNA-Gd) complexes for neutron-capture
therapy (NCT) were hypotesized [3]. Neutron capture the-
rapy (NCT) is a cancer cells treatment that utilizes nuclear
neutron capture reaction (NCR) of radiation producing
elements administrated in vivo by thermal neutron flux
generated, as a rule, by nuclear reactor [4].
The current attention to radiation-producing elements
is essentially focused on gadolinium for its favorable pro-
perties, even though extensive studies have been reported
on the other elements. It is known that naturally existing
gadolinium consists of several stable isotopes. Among
them 157Gd (15.6% in natural sample) has most broad
medical application. Indeed, gadolinium neutron capture
therapy (Gd-NCT) utilizes the following nuclear capture
reaction (NCR) of 157Gd by thermal neutron irradiation
[5]:
157Gd + nth→158Gd + γ-rays + internal conversion
electrons→158Gd + γ-rays + Auger electrons + character-

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 282
istic X-rays.
According to accepted point of view, the success of
gadolinium neutron capture therapy depends, first, on a
high accumulation of Gd in the tumor [6].
Therefore, the first problem here—a sufficient con-
centration of gadolinium could be retained in the tumor
tissue during neutron irradiation after intratumoral injec-
tion. The second problem is the toxicity of free gadolin-
ium, because gadolinium ion is strongly toxic even at
low doses at its administration to tissues [7,8]
For this reason, various kinds of “carriers” (ligands)
capable of forming stable complexes with Gd3+ before it
is administrated to patients have been constructed.
Indeed, the chelate complexes of gadolinium with
polymeric molecules (see, for instance [9]), nanoparticles
of various origin [10,11] have been produced as potent
biomaterials (bioconjugates) for targeting a site and con-
trolled release of a drug. Biodegradable, biocompatible
chitosan nanoparticles [4,11] have recently received con-
siderable attention as systems capable of retaining gado-
linium in the tumor tissue during a Gd-NCT trial.
This facts show that the developers of NCT are inter-
ested in particular molecular constructions (bioconjugates,
biomaterials, etc.), which can retain a sufficient gadolin-
ium concentration.
Thus, a key factor in the success of the current
Gd-NCT trial is the use of biomaterials by means of
which Gd can be delivered efficiently and retained inside
tumor tissues and/or cells during thermal neutron irradia-
tion.
The aim of this study was to investigate whether the
“rigid” particles of the (ds DNA-Gd) complexes can be
practically used as a biomaterial for Gd-NCT. For this
purpose “rigid” ds DNA particles, highly loaded with ga-
dolinium, were obtained and firstly tested in Gd-NCT for
disintegration of the living cells. Our preliminary attempt
showed that this biomaterial has a good potential as a
gadolinium carrier for modern NCT.
2. Materials and Methods
2.1. Preparation of the “Rigid” Particles of
the CLCD of (Double-Stranded
DNA-Gadolinium) Complexes
The formation of the CLCDs based on the phase exclu-
sion of ds DNA from PEG-containing water-salt solu-
tions was performed according to the Method that was
described previously in detail [12]. In the physicochemi-
cal sense, the system under investigation is particles of
the CLCD of the ds DNA that are distributed isotropi-
cally in the water-salt solution of PEG. These particles
do not exist in the absence of high osmotic pressure of
the solvent (induced by high PEG concentrations).
The formed CLCD was treated with GdCl3 solutions to
induce formation of (ds DNA-gadolinium) complexes.
The efficiency of formation of these complexes was
checked by the measuring the CD spectra.
The gadolinium salts (99.99% of purity) were pur-
chased from the Institute “Giredmet” (Moscow, Russia).
The absorption spectra of all solutions were taken on a
spectrophotometer (“Cary100”, Varian, USA) and the
CD spectra were recorded by a portable dichrometer
SKD-2 (manufactured by the Institute of Spectroscopy of
the RAS, Troitzk, Moscow Region). In all cases, the
quartz cells with 1 cm optical path were used.
The morphology of the dsDNA CLCD particles treated
by GdCl3 was examined using Atomic Force Microscope
P47-SPM-MDT (produced by NT-MDT, Russia). To iso-
late these particles, the solution in which they were
formed was filtered through a poly(ethyleneterephtalate)
(PETP) nuclear membrane filter with size of pores of 150
nm (produced by the Institute of Crystallography of the
RAS), that allowed us to immobilize particles; filters
were dried in air for no less than 1 h.
To estimate the number of Gd3+ ions in the content ds
DNA СLCD particles treated by GdCl3, their magnetic
properties have been measured and total magnetic mo-
ment of Gd3+ ions was determined. The magnetic proper-
ties of the samples were measured at magnetic field of
71.29 mT (712.9 Oe) at sample position by the super-
conducting interferometer device (SQUID-magnetometer)
produced by D. Mendeleev University (Moscow, Russia)
[13].
The concentration of the gadolinium in the content of
ds DNA СLCD particles treated with GdCl3 was addi-
tionally checked by the neutron activation analysis [14].
The fluorescence of SYBR Green (an intercalating
fluorescent dye, “Invitrogen” USA, Lot 666062) was
measured by the fluorescence spectrophotometer (“Cary
Eclipse”, Varian, USA).
The particles of CLCD formed by (ds DNA-Gd) com-
plexes were sedimented as a result of low speed centri-
fugation, the pellet was re-suspended in 0.3 ml of H2O
and added to 5 ml of the standard Dulbecco’s MEM me-
dium.
2.2. Gd-NCT with “Rigid” Particles of CLCD
Formed by (ds DNA-Gd) Complexes
2.2.1. Cell Line Cultivation
Chinese hamster ovary (CHO) adherent cells were main-
tained in plastic flasks containing standard Dulbecco’s
MEM medium supplemented with 10% fetal bovine se-
rum (FBS), 4 mM L-glutamine, 1 mM sodium pyruvate,
0.1 mg/ml streptomycin and 100 units/ml penicillin in a
humidified atmosphere containing 5% carbon dioxide at
37˚C.

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 283
For cell line passage cell monolayer was washed with
PBS (10 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl,
2.7 mM KCl, pH 7.4), standard solution of trypsine-
EDTA (Sigma) was added, then plates with cells were
placed in a 5% CO2 atmosphere at 37˚C for 3-5 minutes,
and then medium DMEM with FCS was added, cells
were suspended and were plated in flasks at necessary
concentrations.
CHO cells, forming monolayers, were seed in 4 cul-
tural flasks (set A - N1 and N2; set B- N3 and N4; see
Figure 1) with 25 cm2 square 500000 cells per flask.
(Monolayers formed by these cells were in their original
state within whole time of experiment). In 20 hours me-
dium in two flasks (set A - N2 and set B- N4) was
changed with fresh one, and in two flasks (set A - N1 and
set B - N3) with DMEM medium containing “rigid” par-
ticles of CLCD formed by (ds DNA-Gd) complexes (the
amount of the “rigid” particles per 1 CHO cell in these
flasks was equal to about 5 × 104, that provides “close
enough contacts” between the cells and “rigid” particles).
Then, in 2 hours all four flasks were closed hermetically
and two of them (one with CHO cells plus “rigid” parti-
cles of CLCD formed by (ds DNA-Gd) complexes (in set
A - N1), and the other one - CHO cells only with DMEM
medium (in set A - N2) were irradiated with thermal
neutrons (see Figure 1).
Two flasks (set B-N3 and N4), which were not irradi-
ated (see below), were incubated during 1h in thermo-
stated (37˚C) polymeric box located nearby neutron gen-
erator.
2.2.2. Gd-NCT with “Rigid” Particles of CLCD
In contrast to classical neutron source such as huge nu-
clear reactors, we first attempted to use more smaller in
size, more cheaper neutron generator NG-400 (France)
with the neutron energy about 14 MeV and the total in-
tensity of the order of 1011 neutrons/sec for the thermal
neutron capture inside “rigid” particles the (ds DNA-Gd)
complex and generation of the secondary irradiation out-
side of these particles.
The thermal neutrons were produced by the moderator
system consisting of a tungsten converter and poly (eth-
ylene) block (20 × 20 × 20 cm, called as “phantom”)
assembled in neutron generator. The earlier estimations
showed that the conversion electrons, X-rays and gamma
rays (range in tissue about 5 × 104 nm), which are gener-
ated as a result on the gadolinium thermal neutron cap-
ture reaction, can cause the ds DNA double-strand breaks,
inducing their killing.
Two flasks - one with “rigid” particles and CHO cells
(set A - N1, see above) and the other one - CHO cells
with DMEM medium (set A - N2) were placed in the
rectangular hole (10 × 5 × 5 cm) inside a “phantom”.
Figure 1. Principal scheme of the Gd-NCT experimental
set-up with the use of the “rigid” DNA particles as carriers
for gadolinium. Set B was used as a control. The numbers
from 1 to 4 in sets A and B denote the monolayers of initial
CHO cells.
These flasks (samples) were fixed at the depth of 5 cm
inside the “phantom” and exposed at 37˚C to thermal
neutrons generated by NG-400.
The irradiation time was about 1 h and the thermal
neutron fluence was about 51011 neutron/сm2. The ther-
mal and fast neutron fluxes were estimated by method of
activation analysis [15].
3. Results
3.1. Formation and Properties of “Rigid”
Particles of CLCD of (ds DNA-Gd)
Complexes
Figure 2 compares the CD spectrum of the CLCD (curve
1) formed by initial ds DNA molecules in water-salt
PEG-containing solution to the CD spectra for CLCDs
(curves 2-5) treated with GdCl3 solution.
The formation of ds DNA dispersion is clearly ac-
companied by an appearance of an intense negative band
in the CD spectrum in the region of the spectra, where
the DNA nitrogen bases absorb.
One can remind that every particle of CLCD contains
about 104 ds DNA molecules fixed on distances within
2.5 - 5.0 nm (depending on osmotic pressure of the sol-
vent [12]. Note that the particles of the low-molecular
mass ds DNA dispersions are “microscopic droplets of
concentrated DNA solution”, which cannot be “taken in
hand” or “directly seen”.
According to theoretical calculations, the appearance

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 284
Figure 2. The CD spectra of the ds DNA CLCD in the ab-
sence (curve 1) and in the presence (curves 2-5) of GdCl3 in
solution: Curve 1 – gadolinium rtotal = 0; 2 – 1.65; 3 – 16.5; 4
– 32.7; 5 – 95; CDNA = 0.01 mg/ml; CNaCl = 0.3 M; Cinitial GdCl3
= 0.1 M; CPEG = 170 mg/ml; A = AL – AR (× 10–3 opt. units).
rtotal – is the ratio of total GdCl3 molar concentration to the
molar concentration of the DNA nitrogen bases.
of this band unequivocally testifies the macroscopic cho-
lesteric twist of neighboring DNA molecules in particles
of dispersion. The negative sign of the band in the CD
spectrum proves the left-handed cholesteric twist of the
right-handed DNA molecules (B-form) in these particles.
The intense band in the CD spectrum (Figure 2, curve 1)
located in the absorption region of the nitrogen bases of
the DNA molecule, in our case negative, is the direct
evidence for the formation of the CLCD characterized by
helically twisted spatial structure [16-18], or a so-called
cholesteric structure of particles of LCD and the term
CLCD (cholesteric liquid-crystalline dispersion) was
used to signify these particles.
The gadolinium-concentration dependence of the am-
plitude (ΔA270) of an abnormal negative band in the CD
spectrum of the ds DNA CLCD particles is shown in
Figure 3. One can see that the amplitude of the abnormal
band in the CD spectrum of particles of the CLCD is
changed, albeit slightly, at low concentrations of GdCl3
(gadolinium rtotal ≤ 0.5).
Insert to Figure 3 compares both the diminishing in
the amplitude of low-intensity band in the CD spectra of
original linear ds DNA (curve 1) and the decrease in the
amplitude of an intense negative band in the CD spec-
trum of ds DNA CLCD at low concentration of gadolin-
ium (curve 2).
The state of the DNA secondary structure under condi-
tions of high concentration of gadolinium cations was
checked by application of an “external chomophore”
approach based on theoretical consideration of the pecu-
liarities of the CLCDs CD spectra [19,20]. As an “exter-
nal chromophore” we have used SYBR Green—an in-
tercalating fluorescent dye [21].
Figure 4 demonstrates the obtained results. One can
see that in agreement with theoretical predictions [19] the
Figure 3. The dependence of the amplitude of the abnormal
negative band in the CD spectrum of the CLCD formed by
(ds DNA-Gd) complexes upon gadolinium rtotal value. CDNA
= 0.01 mg/ml; CNaCl = 0.3 M; CPEG = 170 mg/ml; A ( 270
nm) = AL – AR (× 10–3 opt. units). Insert: Curve 1. The de-
pendence of the relative amplitude of the band (λ 280 нм) in
the CD spectra of the linear ds DNA treated with GdCl3
upon gadolinium rtotal value. CDNA = 0.01 mg/ml; CNaCl =
0.003 M. Curve 2. The dependence of the relative amplitude
of the abnormal negative band (λ 280 nm) in the CD spectra
of the CLCD formed by (ds DNA-Gd) complexes upon
gadolinium rtotal value. CDNA = 0.01 mg/ml; CNaCl = 0.3 M;
CPEG = 170 mg/ml. Curve 3. The dependence of the Bragg
peak relative amplitude of the CLCD formed by (ds DNA-
Gd) complexes upon gadolinium rtotal value. CNaCl = 0.3 M;
CPEG = 170 mg/ml; gadolinium rtotal – is the ratio of total
GdCl3 molar concentration to the molar concentration of
the DNA nitrogen bases.
Figure 4. The comparison of the CD spectra of the ds DNA
CLCD treated with SYBR Green (SG; curves 1 - 4) to the
CD spectra of CLCD formed initially by (ds DNA-Gd)
complexes and then treated with SG (curves 1I - 4I). Dotted
curve 5I- the expected CD spectrum of the CLCD formed by
(ds DNA-Gd) complexes and treated with SG under condi-
tion of the homogeneity in the DNA secondary structure.
SG rtotal in the case of curves 1 and 1I = 0; in the case of 2
and 2I – 0.033; 3 and 3I – 0.2; 4 and 4I – 0.66; 5I – 0.66; CDNA
= 0.01 mg/ml; CPEG = 170 mg/ml; CNaCl = 0.3 M; CGdCl3 =
0.003 M; A = AL – AR (× 10–3 opt. units). SG rtotal – is the
ratio of total SG molar concentration to the molar concen-
tration of the DNA nitrogen bases.
treatment of CLCD particles formed by (ds DNA-Gd)
complexes with SYBR Green, used as an “external
chromophore”, is accompanied by an appearance of an
additional abnormal negative band in the CD spectrum.

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 285
Figure 5 compares the changes in the emission inten-
sity of SYBR Green intercalated between nitrogen bases
of initial, linear ds DNA with homogeneous secondary
structure (curve 1), to ds DNA in the content of CLCD
(curve 2) as well as ds DNA in the content of CLCD
treated with gadolinium (curve 3).
Figure 6(a) displays the 2D-AFM images of “rigid”
CLCD particles formed by (ds DNA - Gd) complexes
immobilized onto the surface of a nuclear membrane
filter.
It is evident that these particles exist as independent
individual objects, which are easy to visualize. Figure
6(b) demonstrates the size distribution of these particles
as well as the pores in the filter.
3.2. Gd-NCT with “Rigid” Particles of CLCD
Formed by (ds DNA-Gd) Complexes
We used biomaterial, i.e., “rigid”(ds DNA-Gd) particles
as a potential platform for Gd-NCT. For this purpose
effect of radiation as a result of Gd-NCR on culturated
CHO cells was measured. These cells were exposed to
thermal neutrons flow from neutron generator in the
presence and in the absence of “rigid” (ds DNA-Gd) par-
ticles (see Figure 1).
After irradiation of the cells in the flasks in set A (see
Figure 1) all four flasks from set A and set B were
maintained in a 5% CO2 atmosphere at 37˚C within 16
hours. Then medium in all flasks was changed with fresh
one without “rigid” particles, and the images of CHO
cells in monolayers were analyzed by light microscope
(Leica DMI4000). The images were taken in 20, 60 and
120 hours after irradiation
One can remind that the time of experiment with
Gd-NCT was about 3 hours. Hence, the first important
question to be answered is the penetration of “rigid” par-
ticles containing Gd into CHO cells within this time.
Figure 5. The fluorescence spectra of the initial, linear ds
DNA and CLCD formed by ds DNA as well as the spectrum
of CLCD of (ds DNA-Gd) complexes treated by SYBR
Green (SG, curves 1, 2, and 3, respectively). CDNA = 0.01
mg/ml; CNaCl = 0.3 M; CPEG = 170 mg/ml; CSG = 9.73 × 10–6
M; CGdCl3 = 0.003 M.
(a)
(b)
Figure 6. (a) 2-D AFM image of the “rigid” particles immo-
bilized onto the surface of the nuclear membrane filter
(PETP). CDNA = 0.001 mg/ml; CNaCl = 0.03 M; CPEG = 17
mg/ml; CGdCl3 = 0.0023 M. (The dark spots are “pores” in
the nuclear membrane filter); (b) Size distribution of the ds
DNA CLCD particles treated by GdCl3 (1) and the pores (2)
in the membrane filter. CDNA = 0.001 mg/ml; CNaCl = 0.03 M;
CPEG = 17 mg/ml; CGdCl 3 = 0.0023 M.
Control experiment showed, that before irradiation, the
penetration of “rigid” (ds DNA-Gd) particles into CHO
cell was practically absent. This means that in our case
the “rigid” particles, which are located outside the CHO
cells, were used for NCT.
Figure 7(a) shows the image typical of initial CHO
cells without irradiation (set B N4).
Figure 7(b) represents the image of CHO cells in
monolayers corresponding to flask N2 (set A), i.e., the
image for CHO cells irradiated by thermal neutrons in
the absence gadolinium carrier.
Figure 7(c) shows the image for CHO cells irradiated
by thermal neutrons in the presence of “rigid” gadolin-
ium carrier (set A N1).
4. Discussion
4.1. Formation and Morphology of “Rigid”
Particles of CLCD of (ds DNA-Gd)
Complexes
Figure 2 shows that a quite sharp increase in the ampli-

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 286
(a) (b)
(c)
Figure 7. The images of CHO cells monolayers taken after
120 hours of cell processing by light microscope (Leica
DMI4000). (a) the image without the thermal neutron irra-
diation and without gadolinium carrier; (b) the image with
thermal neutron irradiation without gadolinium carrier; (c)
the image after 1 hour of the thermal neutron irradiation
with gadolinium carrier.
tude of the intense negative band in the CD spectrum of
the CLCD of the (ds DNA-Gd) complexes occurs at a
large concentration of gadolinium cations in the solution
(rtotal > 20) and its maximum is shifted by 10 nm toward
long wavelengths (inset to Figure 2, curves 1-5).
As it was shown previously [3,22-24] when rare earth
cations, and in particular, Gd cations, are bonded to lin-
ear ds DNA, noticeable alterations in the CD spectra of
these molecules were observed at gadolinium rtotal values
close to 0.5 [22,24]. This effect was explained as a de-
formation (alteration) of the ds DNA secondary structure
[25,26]. This deformation can be associated with the
conformational transition of B  Z type [25,27]. Besides,
the junctions between B-DNA and Z-DNA fragments
contain extruded bases providing the sites with modified
local properties. Combination of these two effects is ac-
companied by breaking of the regular, homogeneous
character of the ds DNA secondary structure. The “modi-
fied” ds DNA molecules can be separated into alternating
fragments differing in conformations, for instance, of B
 Z – Z  B – B  Z  B  –Z– type. Hence, even
low concentrations of gadolinium cations are capable of
inducing the breaking of regular character of the ds DNA
secondary structure of initial linear ds DNA molecules.
Because formation of ds DNA CLCD does not result in
change in the parameters of the DNA secondary structure,
one can expect that the breaking process happens in the
case of ds DNA molecules packed in CLCD particles.
To check the character of the secondary structure of ds
DNA molecules ordered in the particles of CLCD treated
with various concentrations of gadolinium salt, the small-
angle X-ray scattering (SAXS) curves of these objects
were recently obtained [28].
The most important observation detected by SAXS is
that structural changes in the ds DNA molecules in the
content of CLCD particles occur while the concentrations
of the gadolinium salt are very low. At gadolinium rtotal =
0.66 the characteristic Bragg peak in X-ray scattering
curves has completely disappeared (Figure 3, curve 3).
Therefore, if the fragments of the neighboring molecules
of the complexes of the DNA with gadolinium or even
all molecules packed in particles of CLCD acquire an
inhomogeneous secondary structure, the translational
order of these fragments (molecules) is broken and the
small-angle reflection on X-ray scattering curves must
disappear and this disappearance is observed experimen-
tally [28]. Hence, the analysis of the SAXS spectra. con-
firms the assumption that the treatment of particles of the
CLCD by gadolinium cations leads to the appearance of
the modified secondary structure of the DNA molecules.
Taking into account the suggestion above that “modi-
fied” DNA molecules are separated into alternating
fragments differing in conformations (e.g., B – Z – B – B
– Z – Z etc.), the existence of ds DNA fragments with the
B-form was checked by the method of “external chro-
mophore”. As an external chromophore we have used
intercalating drug—SYBR Green, which is highly selec-
tive for native B-form of ds DNA molecules with regular
secondary structure.
Figure 4 shows that in the CD spectrum of (ds DNA-
Gd) complexes there are two bands. One occurs in the
absorption region of the DNA nitrogen bases (
 ~ 270
nm) and the other lies in the absorption region of SYBR
Green chromophores (  ~510 nm). Under binding of
SYBR Green with ds DNA molecules in content of
CLCD particles formed by (ds DNA-Gd) complexes both
bands have negative signs despite of SYBR Green con-
centration. The identical signs of two bands in the CD
spectra unequivocally mean that SYBR Green molecules
are fixed in quasinematic DNA layers. The amplitude of
the band in the CD spectrum in the region of SYBR
Green absorption grows with increasing number of its
molecules bound to DNA, although the amplitude of the
band in the region of DNA absorption remains practi-
cally constant. The shown CD spectra mean that the ori-
entation of SYBR Green molecules coincides with the
orientation of the nitrogen base about the DNA axis and
SYBR Green molecules intercalate into DNA so that the
angle between SYBR molecule and the long axes of the
DNA is ~90˚.

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 287
However, the experimentally measured amplitudes
(compare, for instance, curve 4’ to theoretically calcu-
lated curve 5’) are 2 times smaller then expected ones (if
one can take into account the correlation between the
amplitudes of abnormal bands in the DNA and “external
chromophores” absorption regions [19]. The detected
difference shows that, indeed, that at high gadolinium
concentration the breaking of the ds DNA secondary
structure in the CLCD particles of (ds DNA-Gd) com-
plexes takes place.
The measurements of the SYBR Green fluorescence in
the content of CLCD particles formed by (ds DNA-Gd)
complexes speak in favor of this point of view. Figure 5
demonstrates that the fluorescence intensity of SYBR
Green is decreased in the case of both ds DNA CLCD
and (ds DNA-Gd) CLCD. The most important facts con-
sist in the following: 1) condensation of ds DNA mole-
cule and formation of the ds DNA CLCDs is accompa-
nied by drop in the intensity of fluorescence of SYBR,
and 2) there is difference in fluorescence of SYBR Green
intercalated between nitrogen bases pairs in the content
of ds DNA molecules ordered in initial CLCD and in the
content of CLCD treated with gadolinium. Since the sol-
vent used for measurements of curves (2) and (3) was not
changed and because SYBR Green molecules bind with
regular B-form of ds DNA, the difference between
curves (2) and (3) confirms ones more the statement that
neighboring ds DNA molecules, packed in particles of
the CLCD (ds DNA-Gd) complexes, acquire an inhomo-
geneous secondary structure.
Figure 3 shows that at high gadolinium concentration
in solution (rtotal > 20) the CD spectrum of CLCD formed
by ds DNA changes dramatically. The observed increase
in the amplitude of the negative band and the change in
the shape of the CD spectrum of the DNA CLCD are
similar to changes in the CD spectra of this CLCD upon
cross-linking of neighboring DNA molecules due to the
formation of nanobridges between them [29]. The forma-
tion of such nanobridges leads both to decrease in solu-
bility of initial CLCD structure and to the disappearance
of the “liquid” character in the location of DNA mole-
cules in the particles of the CLCD [1].
This allows one to suppose, that interaction of gado-
linium with ds DNA molecules is accompanied by de-
crease in solubility of these molecules as well. In addi-
tion, the inhomogeneous chemical nature of the nitrogen
bases in ds DNA molecules leads to the fact that the in-
teraction between gadolinium cations and ds DNA mo-
lecules is accompanied by nanoscale conformational
changes (similar to the changes that are appeared at B 
Z transition) only in the fragments of these molecules.
Because separation of chains of the ds DNA molecules in
the content of particles of the LCD is impossible due to
the sterical reasons [30,31], the alteration of the ds DNA
secondary structure, induced by gadolinium treatment,
can be “transformed” into the change in the mode of spa-
tial packing of the neighboring DNA molecules in these
particles. Because ds DNA molecules cannot “leave” the
physical volume of CLCD particles, due to the fixed os-
motic pressure of PEG-containing solution, the loss of
solubility of individual neighboring ds DNA molecules
combined with an increase in the interaction between
their fragments with different conformations initiates the
transition of the overall structure of CLCD particles from
a “liquid” to a “rigid” state [32].
It is known, that a “liquid” mode of spatial location of
ds DNA molecules in the particles of CLCD dispersions
prevents their immobilization on the surface of mem-
brane filters. However, if poorly soluble CLCD particles
consisting of molecules of the (ds DNA–Gd) complexes
are formed, the immobilization of particles on the surface
of the nuclear membrane filter becomes possible and the
size and shape of these particles can be investigated.
Figure 6(a) shows the AFM image of ds DNA CLCD
particles after their treatment with GdCl3 and immobili-
zation on nuclear membrane filter. Figure 6(b) demon-
strates the size distribution of these particles as well as
the pores in the filter.
One can see that these particles exist as independent,
individual objects.
The presence of single particles (Figure 6(a)) testifies
that at treatment of particles of ds DNA CLCD by GdCl3,
the “liquid” character of the DNA packing in these parti-
cles is disappeared and the particles have a rigid spatial
structure.
Therefore, particles of the CLCD of the ds DNAs
whose phosphate groups are neutralized by gadolinium
ions become poorly soluble and can exist in the absence
of osmotic pressure of the PEG-containing solution and
the osmotic pressure of the water-salt PEG-containing
solution is not required for supporting the spatial struc-
ture of CLCD particles formed by (DNA-gadolinium)
complexes.
The mean size of particles makes 4500 - 5000 A˚, i.e.
the mean diameter of the ds DNA CLCD particles after
gadolinium treatment coincides with the mean diameter
of initial ds DNA CLCD particles [12,33]. The particles
have shape of the spherocylinders and the diameter of
particles is close to their height. The obtained result is
important, because it allows one to suggest that the av-
erage packing density of the DNA molecules in particles
of the CLCD of the (ds DNA–Gd) complex is quite close
to the packing density of the DNA molecules in particles
of the CLCD formed from initial DNA molecules. In this
case, the mean concentration of chromophores (nitrogen
bases) of the DNA in particles of the CLCD of the

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 288
DNA–gadolinium complex must also retain not only high
[34], but sufficient for holding the abnormal optical ac-
tivity of these particles.
The visualization of single particles indicates that,
when particles of the CLCD of ds DNA are treated by the
GdCl3 solution, the liquid character of packing of DNA
molecules in these particles is indeed disappeared and
particles acquire a rigid spatial structure. Such a structure
is presented not only the decrease in the solubility of
DNA molecules, but also the presence of strong interac-
tion between the fragments of neighboring DNA mole-
cules, because gadolinium ions can be nonuniformly dis-
tributed.
Hence, the treatment of the ds DNA CLCD by GdCl3,
is accompanied not only by neutralization of phosphate
groups of the DNA molecules by Gd3+ ions, but by a sig-
nificant attraction between the neighboring DNA mole-
cules. Disappearance of the fluidity of the ds DNA
CLCD particles proves a short-range attractive interac-
tion between the charged DNA molecules arising from
interlocking Gd3+ ions, sometimes called as “counterion
cross-links”. An existence of independent particles speaks
in favor of an appearance of noncompensated positive
surface charge on the CLCD particles. This, in turn, pro-
hibits the coalescence of these particles.
In addition, nonuniform distribution of gadolinium
ions over the surface of DNA molecules having the in-
homogeneous secondary structure is accompanied by
irregular interaction between the fragments of the neigh-
boring DNA molecules in quasinematic layers. Because
each phosphate group of the DNA molecules, carrying
one “effective” negative charge, is neutralized by Gd3+
ion [35], that carries three positive charges, this means
that the altered surface charge distribution makes an ad-
ditional contribution to the chiral interaction between
adjacent (ds DNA-Gd) complexes in the particles [22,19].
Under these conditions, the interaction between the
modified DNA molecules can induce change in the
twisting of the cholesteric helical structure of the DNA
molecules. In this case one can expect that the pitch (P)
of the spatial twist of cholesteric structure formed by the
ds DNA is changed as a result of interaction of gadolin-
ium ions with these molecules.
Indeed, the change in the twist angle between neighbor-
ing quasinematic layers of CLCD is supported by results
of the theoretical calculations [3,32,36] according to
which an increase in the twist angle (decrease in P value)
of the spatial structure of (ds DNA-Gd) CLCD particles
[36] determines a drastic increase in the amplitude of an
abnormal negative CD band in the absorption region of
DNA nitrogen bases, when the CLCD particles are
transformed from “liquid” to into “rigid” state.
The results above allows one to suggest the scheme of
“liquid-rigid” structural transition of ds DNA CLCD
shown in Figure 8.
One can see, that here the spatial ordering of neigh-
boring DNA molecules in quasi-nematic layers is practi-
cally absent; besides, under these conditions the twist
angle between neighboring DNA quasi-nematic layers is
increased (the P value is decreased, right structure). Ac-
cording to [32,37] in the presence of large excess of
gadolinium cations, these cations can displace the so-
dium ions, initially bounded to the phosphate groups of
the ds DNA. The gadolinium ions neutralizing the nega-
tive charges of the phosphate groups of the ds DNAs
make particles of the CLCD of (ds DNA-Gd) insoluble in
PEG-salt-aqueous solutions. It is worth noting that, when
Gd3+ ions are bonded to polyphosphates, poorly soluble
Gd-polyphosphate is formed (solubility constant is equal
to about 10–12 M) [38,39]. Since ds DNA molecules have
polyphosphate nature, these molecules in the presence of
saturating gadolinium concentrations become poorly
soluble in poly(ethyleneglycol)-water salt solutions. Un-
der high concentration of gadolinium, the stable spatial
structure of dispersion particles is formed and the pres-
ence of poly(ethyleneglycol) is not required to stabilize
the structure of particles of the CLCD. Moreover, gado-
linium ions, neutralizing the charges of the phosphate
groups of the DNAs, create an excess positive surface
charge on particles of the CLCD and aggregation of these
particles. This behavior is corroborated by the atomic
force microscopic data according to which these gado-
linium-ion-treated particles of the CLCD of the DNAs
are existing as single independent objects (see Figure 6).
According to this scheme the amplification of the ab-
normal negative band (λ ~ 270 nm) in the CD spectrum
of CLCD particles formed by ds DNA molecules treated
with high concentration of gadolinium cations confirms
the formation of “rigid” CLCD particles of (ds DNA-Gd)
Figure 8. Sheme of transition from “liquid” to “rigid”
structure of the particle of the CLCD induced by high Gd3+
concentration.

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 289
complex (Figures 2 and 3) under used conditions.
In addition, the results of low-temperature magne-
tometric study and neutron activation analyses showed [3,
15] that in the case of “rigid” particles formed at rtotal >
20, one gadolinium cation is bounded approximately to
one DNA phosphate group. The evaluations showed that
local concentration gadolinium in the content of these
particles can reach 40%, i.e., we are dealing with a novel
type of biomaterial containing a very high local concen-
tration of gadolinium.
The obtained “rigid” CLCD particles strongly enriched
by gadolinium open a possibility for various manipula-
tions with them, for instance, their application as “Gd-
carriers” at neutron capture therapy (NCT).
4.2. Gd-NCT with “Rigid” Particles of CLCD
Formed by (ds DNA-Gd) Complexes
A potential of the “rigid” (ds DNA-Gd) particles as bio-
material for Gd-NCT, is based on a few facts:
1) the formation of these particles can be easily checked
by the CD spectroscopy or by the AFM;
2) a long-term stability of the physicochemical proper-
ties of these particles allows one to manipulate with these
particles;
3) this biomaterial has a higher concentration of 157Gd
compared to other known Gd-carriers such as, for in-
stance, Gd-chelate complexes [4,6,9,11].
Comparison of Figure 7(b) to Figure 7(a) shows that
irradiation of CHO cells by the thermal neutron fluence
(5 × 1011 neutron/сm2) within 1 h results only in minor (if
any!) changes in the CHO cells and does not influence
their ability to grow. Even after 120 hours of cell proc-
essing, these cells, irradiated in absence of (ds DNA-Gd)
particles grow as initial cells and form monolayer. This
signifies that thermal neutron irradiation of intact CHO
cells under our conditions does not influence strongly the
proliferation ability of these cells.
Figure 7(c) shows the image for CHO cells irradiated
by thermal neutrons in the presence of “rigid” gadolin-
ium carrier (set A N1). It can be seen, that after 120
hours of cell processing, the image of CHO cells differs
from that of cells irradiated without “rigid” gadolinium
carrier. The efficiency of cells proliferation is reduced
and cells don’t form colonies. The growth of CHO cells
administrated with the “rigid” (ds DNA-Gd) particles and
then irradiated with thermal neutrons was significantly
suppressed compared to that in control cells. Besides, the
cell debris in cultural medium begins to appear after 60
hours of cell processing (data not shown). The amount of
cell debris after 120 hours shows that significant part of
cells was disintegrated. Finally, the irradiation of CHO
cells in presence of “rigid” particles results in full ab-
sence of alive CHO cells.
Taking into account that flasks N1 and N2 (set A)
were irradiated by thermal neutrons simultaneously the
difference in the cell killing efficacy for these samples
(Figure 1, set A) might be due to the thermal NCR in-
duced only by gadolinium in the content of “rigid” parti-
cles containing (ds DNA-Gd) complexes. Hence, CHO
cells in the sample with gadolinium were killed, while
the cells in control samples survived under the same
conditions of irradiation [40].
The presence of strongly deformed cells (collapsed
cells) Figure 7(c) allows one to suppose, that although
concrete reasons for an appearance of these cells were
not investigated carefully, that irradiation of CHO cells
in presence of (ds DNA-Gd) “rigid” particles is accom-
panied by a few processes:
1) disintegration of genetic material of these cells;
2) deformation and destruction of lipoprotein mem-
brane as a result of possible retention of Gd-containing
particles in lipoprotein membrane of CHO cells and
3) penetration of small fraction of (ds DNA-Gd) “rigid”
particles into the CHO cells inducing additional destruc-
tion of these cells.
It is necessary to stress that according to known data
[5,7,8,40] in the case of other Gd-carries the cell growth
was inhibited until to 10 days after the neutron irradia-
tion.
Considering the reasons for the effects shown in Fig-
ure 7(c), one can remind the following. When bom-
barded with thermal neutrons, 157Gd releases photons and
electrons with energies up to 7.9 MeV [5,41]. Previous
studies on CHO cells have shown a significant enhance-
ment of lethal effects induced by Gd-NCR [5]. During
the Gd-NCR, the emission of γ-rays is followed by the
internal conversion and subsequent emission of Auger
electrons [41] and these electrons play an important role
in cell killing. As the range of these electrons is ex-
tremely limited in tissue, gadolinium distribution in the
cells, particularly with respect to the genetic material, is
crucial in determining the extent of biological effects
induced by Gd-NCT. Indeed, it was shown in [42,43]
that in the thermal neutron irradiation of plasmid DNA/
gadolinium mixture the extent of double-strand breaks to
be considerably reduced by sequestering the gadolinium
from DNA, suggesting the effect to have been mainly
due to Auger electrons.
However, the ranges of high-energy electrons and
photons produced in the gadolinium NCR [41] are suffi-
ciently long within tissue for cell inactivation to occur
even if Gd is present in the vicinity of the cell. Indeed, it
was demonstrated that mouse ascites cells in the perito-
neal cavity to be inactivated by the radiation released
from 157Gd contained in microcapsules, suggesting the
proximity of 157Gd to cellular genome not to be critical.

Novel Biomaterial for NCT—“Rigid” Particles of (DNA-Gadolinium) Liquid-Crystalline Dispersions 290
Taking into account the fact that gadolinium in micro-
capsules was effective in suppressing murine ascites tu-
mor, most of the effects observed apparently resulted
from the radiation of high-energy internal-conversion
electrons having adequate ranges and photons from Gd.
This result permits to suppose that the intracellular pres-
ence of Gd is, indeed, not critical in Gd-NCT.
It is known as well that the energy of conversion elec-
trons is equivalent to the path inside biological tissue
more then 50 m [44]. It was demonstrated earlier [45],
that the conversion electrons (the energy  7 KeV) and
 -rays are not significantly absorbed inside the initial
“rigid” (ds DNA-Gd) CLCD particles. Besides, the cal-
culations showed that more than 70% of the conversion
electrons can penetrate into the CHO cells and create the
radiation dose, requested for cell destruction. Taking into
account that the (ds DNA-Gd) CLCD particle density
was about of 5·104 particles per cell and the thermal neu-
tron fluence was of the order of 5 × 1011 neutron/сm2,
this results in 100 neutron captures i in n each particle. In
that case the secondary electrons and photons, with the
probability of about 100%, can induce the DNA dou-
ble-strand breaks in the cell nucleus if the (ds DNA-Gd)
CLCD particles located nearby the surface of CHO cells.
This means that we can expect that the secondary irradia-
tion from “rigid” (ds DNA-Gd) particles can hit the ma-
terial of cell nucleus, inducing its disintegration. Indeed,
comparison of the images shown in Figure 7 in combi-
nation with the value of neutron fluence equals to 5 ×
1011 neutron/cm2 and high Gd-concentration in “rigid”
particles used in our study permits one to state, that
highly-gadolinium-loaded DNA “rigid” particles have
potential usefulness for Gd-NCT.
We are clearly understanding that for practical medical
application, the optimization of physico-chemical pa-
rameters of “rigid” (ds DNA-Gd) particles, including con-
centration of Gd, as well as the time before irradiation
and the time of irradiation is required. One can suppose
that the efficiency of application of these particles in
Gd-NCT can be even increased in the case of forced
penetration of DNA “rigid” particles inside CHO cells.
Besides, it is necessary to perform “the size of “rigid”
particles—the dose” calculations [46,47] because, as it
was shown in [46,47], the doses from neutrons, prompt
γ-rays and internal conversion electrons, and the dose
distribution to be a function of strongly-interrelated pa-
rameters such as tumor size, gadolinium concentration
and its spatial distribution through the tumor, tumor-to-
normal tissue concentration ration and tumor site.
In conclusion, one can say that the obtained results
(despite of their noncomplete character) showed that
“rigid” (ds DNA-Gd) particles have a potential as a novel
biomaterial with high concentration of gadolinium for
NCT. In this case appears a possibility to use simpler (in
comparison to nuclear reactors) devices for NCT realiza-
tion. Our results demonstrate as well that the intracellular
presence of Gd-carrier is not an obligatory condition for
effective disintegration of CHO cells at Gd-NCT.
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