BIOCOMPATIBILITY STUDIES OF NANOCAPSULES OF EPOXY
NETWORK POLYMER CONTAINING QUANTUM DOT AND
SUPERPARAMAGNETIC NANOPARTICLES FOR CANCER
CELL LABELLING AND THERAPY
Vinícius Fortes de Castro1, Alvaro Antonio Alencar de Queiroz1
1
Centro de Estudos e Inovação em Materiais Biofuncionais Avançados, Universidade Federal de Itajubá,
Av. BPS, 1303, 37.500-903, Itajubá (MG), Brasil.
E-mail: vfc_mg@yahoo.com.br, alencar@unifei.edu.br
Abstract. The purpose of this study was to explore the physics and biologic properties of polymer
nanospheres carrying Y3Fe5-xAlxO12(YFeAl) nanoparticles. Magnetic and fluorescent polymer
nanospheres containing the photoactive and superparamagnetic YFeAl-ZnS nanoparticles were prepared
by inter-facial polymerization of epoxidic polymer based on the ether diglycidic of bisphenol A (DGEBA).
The microstructure and size distribution of the polymer nanospheres were determined by transmission
electron microscopy (TEM) and atomic force microscopy (AFM). The fluorescence of the polymer
nanospheres was observed using the epifluorescence microscopy. In-vitro experiments about the
biocompatible properties of polymer nanospheres carrying the hybrid YFeAl-ZnS nanoparticles were
accessed in mammalian cells (CHO). It was observed that the polymer nanospheres did not affect the
viability of mammalian cells or the growth rate of cell culture. The biophysical properties of the polymer
nanospheres carrying the hybrid YFeAl-ZnS ceramic indicated that this can be a highly versatile
nanobiomaterial for therapeutic and cancer diagnosis.
Keywords: Quantum dots, ZnS, Magneto fluid hyperthermy.
1.
INTRODUCTION
The prevalence of cancer worldwide continues to drive the scientists to search
new techniques for improving cancer treatment. Cancer is a group of diseases
characterized by uncontrolled growth and spread of abnormal cells. According to the
most recent edition of the World Health Organization’s (WHO) World Cancer Report,
the global cancer burden doubled in the last thirty years and is estimated to double again
between 2000 and 2020 and nearly triple by 2030 [INCA, 2010]. It was estimated that
by 2020, an estimated 60 percent of all new cancer cases will occur in the least
developed nations. The continued population growth and aging, affect significantly the
impact of cancer around the world. This impact will fall primarily on the countries of
medium and low development.
In Brazil, cancer represents the second cause of death in adults, and that,
according to projections from the National Cancer Institute are expected in 2012 a total
of 257.870 new cases for males and 260.640 for females. It is confirmed that the
estimate of the type of skin cancer no melanoma (134.000 new cases) will be more
frequent in the Brazilian population, followed by prostate tumors (60.000), female
breast (53.000), colon and rectum (30.000), lung (27.000), stomach (20.000) and
cervical cancer (18.000).
The evolution of medicine, by means of nanotechnology and nanoscience
(N&N) has introduced new levels of knowledge, providing significant impacts of
science and technology in various areas of human activity, allowing a large percentage
of cancer patients currently have the opportunity to cure or significant increase of
quality of life significantly as a viable reality. Nanotechnology in turn, is the ability to
manipulate individual atoms and molecules to produce nanostructured materials with
real health care applications.
The application of nanomaterials to tumor and biomarker imaging as well as
delivery and targeting of therapeutics for cancer treatment have received significant
attention in this century. A wide range of nanomaterials have been used to construct
nanoparticles for encapsulation of chemotherapeutics to increase the capability of
delivery or to provide unique optical, magnetic, electrical and structural properties for
imaging and therapy [Solanki, 2008].
Currently, the nanospheres represent a very important milestone for medicine
because they can be used as drug carriers, radioactive isotopes and fluorescent
compounds with important applications as chemotherapeutic photonic systems for use
in photodynamic therapy. The use of nanospheres for design of bioactive medical
devices implies the achievement of specific targets aimed at the human body, these
being the fields of a new field of medicine called nanomedicine.
Touted as a new technological revolution in medicine, nanomaterials called
quantum dots are being used as biological markers for fluorescent imaging of cancer
cells [Yong, 2007]. A variety of quantum dots based on CdS and ZnS has been tested as
fluorescent markers for mapping cancer. Currently our research group has been
dedicated to the development of processes for obtaining ZnS quantum dots using
polymers microstructure digitized and highly branched molecules called dendrimers
[Queiroz, 2006].
Although the application of quantum dots for medical imaging area is promising,
the biocompatibility of these materials has not only aroused the concern of scientists of
biomaterials, but also the medical community itself. Recently, our research group has
developed techniques for encapsulation of magnetic nanoparticles with organic
polymers for use as hemocompatible nanomaterials in magnetohyperthermia [Castro,
2010]. Recently, the use of superparamagnetic iron oxide (SPIO) nanoparticles has
received special attention for use in cancer treatment by magnetic fluid hyperthermia
(MFH) technique [Lock, 2003].
Hyperthermia belongs to the list of conventional treatments accepted by
"American Cancer Society" and remains one of the most powerful therapeutic
modalities to improve the outcome of patients with cancer [Overgaard, 1989]. It is also
one of the best adjuvant that enhances the effectiveness of radiotherapy and
chemotherapy. In this sense, the Phase III clinical trials show that, when the
hyperthermia was associated with radiotherapy, it has improved local control of
melanoma from 28% to 46% at 2 years follow-up, increased the total remission of the
cancer recurring breast cancer from 38% to 60%, increased the rate of complete
remission of advanced cervical cancer from 57% to 82%, and glioblastoma multiforme
increased the survival of 2 years from 15% to 31% [Tartaj, 2003].
Hyperthermia is a therapeutic procedure used to provide temperature rise in a
body region that is affected by a tumor, in order to cause lyses’ of cancer cells. Its
fundaments is based on the fact that the temperature of 41°C to 42°C has the effect of
directly destroying the tumor cells, since they are less resistant to sudden increases in
temperature than the surrounding normal cells. MFH is based on a defined transfer of
power onto magnetic nanoparticles in an alternate magnetic field that is determined by
the type of SPIO particles, frequency and magnetic field strength and which results in
local generation of heat. Depending on the equilibrium temperature set in the tumor
tissue, this heat may either destroy the tumor cells directly (thermoablation) or result in
a synergic reinforcement of radiation efficacy (hyperthermia).
In MFH the production of localized heat for tumor treatment has been hindered
due to certain factors such as heterogeneous temperature distribution and the inability of
heating smaller visceral masses. In order to achieve better control of local heating
effects without damaging normal cells, magneto nanoparticles may be designed with a
desired Curie temperature range (TC).
Overall, magnetic nanoparticles have received attention for use in cancer
treatment technique hyperthermia because they can be guided or localized in a specific
target by external magnetic fields. The possibility of vectoring magnetic nanoparticles
by magnetic field gradient led to the development of various packaging techniques of
magnetic particles so obtained systems become effective carriers of drugs with tumor
specificity for the controlled release of chemotherapeutic agents.
An important prerequisite for the clinical use of the nanoparticles in
magnetohyperthermia technique is the low toxicity and a high moment of magnetic
saturation of nanoparticles that will minimize the doses required for the treatment. In
this context, the nanoparticles composition of ferrites based on yttrium-iron-aluminum
oxides (Y3Fe5-xAlxO12, YFeAl) appear as a promising candidate because they have a
high Curie temperature (TC) and high magnetic saturation moment [Sun, 2002].
However, such particles are highly cytotoxic and it is necessary to develop techniques
for encapsulation of ferromagnetic YFeAl nanoparticles to the desirable biocompatible
properties for clinically safe application in MFH.
Today, we have interested in the encapsulation of the superparamagnetic YFeAl
nanoparticles and ZnS quantum dots in diglycidilether of bisphenol A (DGEBA)
networks (Figure 1) for a number of reasons [Queiroz, 2004]: i) to make the YFeAl
biocompatible, ii) increase thermal, mechanical or chemical stability, iii) decrease
friction and iv) inhibit corrosion by biological fluids. The encapsulation of YFeAl in
epoxidic networks prevents the aggregation in physiological fluids as compared to the
bare particles and provides a biofunctional surface for modification and subsequent
bioconjugation with protein or drug receptors.
Figure 1: Illustration of the DGEBA nanospheres carriers of YFeAl and ZnS.
In this work is described the preparation of new biocompatible nanocapsules
containing the hybrid fluorescent and magnetic nanoparticles based on ZnS quantum
dots (QD) and YFeAl, respectively. While the application of quantum dots for medical
imaging area is promising, the biocompatibility of these materials has not only aroused
the concern of scientists of biomaterials, but also on the medical community. The
system obtained is an important step in the development of intelligent nanospheres
expanding significantly the potential therapeutic and diagnostic medicine in cancer
treatment.
2.
MATERIALS AND METHODS
The superparamagnetic YFeAl nanoparticles were obtained by the wet method
in three-necked reactor equipped with reflux condenser from precursor solution of iron
chloride (FeCl2), yttrium (YCl3) and aluminum (AlCl3). The YFeAl nanoparticles were
coated with ZnS quantum dots by using polyglycerol dendrimers (PGLD) as imprinting
matrix.
The mean diameter size and size distribution of fluorescent YFeAl-ZnS were
analyzed using a Laser Diffraction Particle Size Analyzer (Zeta Sizer Nano ZSMalvern). The XRD diffractometer used in this study was a Shimadzu model XRD
6000. Analyses were performed with monochromatic radiation of Cu-Ka (1.5406 Å) at
a voltage of 40 kV and 40 mA, scan synchronized with the step of 0.05° in the range 2
of 20-80° and 5 min exposure to 1 and 0.3° for the incident slit, and programmable
divergent, respectively. The samples in powder form were deposited on a glass
substrate.
The morphology of fluorescent YFeAl-ZnS nanospheres was studied using a
transmission electron microscope (TEM, H800-3 Hitachi) operating at 100–150 kV
accelerating voltage. Samples were mixed in 1% OsO4 for 1 hour, dehydrated in acetone
and then embedded in liquid epoxy resin. After hardening, the resin blocks were
sectioned in 50 nm sections and stained before photographed.
For the synthesis of nanoparticles of ZnS, Zn stoichiometric mixture of a
micrometer-sized metal (Aldrich) and powdered sulfur (S) in a 2:1 ratio were introduced
into a stainless steel reactor temperature 25°C. The reactor was purged with N2 for 1
hour and then the temperature was raised to 700°C at heating rate of 20°C.min-1. The
temperature of 700 °C was maintained for 1 hour and then the system was cooled to
room temperature in the cooling rate of 5 °C min-1. The reaction yield was
approximately 90% for the attainment of ZnS.
The microspheres containing DGEBA and YFeAl of ferromagnetic particles,
quantum dots of ZnS were obtained by the technique of reverse micelles, starting from a
reaction between the DGEBA epoxy group and an initiating agent, dietilenotetramina
(DETA ) also known as crosslinking agent.
The magnetization property of the fluorescent YFeAl-ZnS nanospheres was
studied by using a vibrating sample magnetometer (VSM, Model 7407, Lake Shore
Cryotronics, Inc., OH, USA). Saturation magnetization, coercive force and remnant
magnetization were obtained from the hysteresis loops. Each sample was measured in
triplicate.
The zeta potential of nanospheres was determined from the electrophoretic
mobility by Laser Doppler Anemometry in an electric field of 150 V/cm. The
measurements were performed on equipment Nanosizer/Zetasizer Nano ZS® Model
ZEN 3600 (Malvern Instruments - USA) using the theory of O''Brien and White
[Lakowicz, 1983]. For the determination of zeta potential, the microspheres were
suspended in a phosphate buffered saline solution (PBS-NaCl), 1 mM, pH 7.4 and
analyzed in triplicate measurements. The Laser Doppler Anemometry was used to
determine the flow rate of a fluid flow of the nanospheres. The observation of the
particles suspended in a fluid transforms the problem of measuring velocity of a fluid in
a problem of measuring speed of a solid body. All existing methods using nonuniformity introduces the particle in a fluid. This disturbance can be seen by appropriate
means. The anemometer the laser beam with an appropriate optical assembly, creates a
system of interference fringes can see the displacement of solid particles.
The fluorescence of the obtained polymer nanospheres carrying the hybrid
YFeAl-ZnS nanoparticles was observed by epifluorescence microscopy (Zeiss
Axioplan).
The study of cell viability after incubation of the culture medium of cell lines
with the nanospheres containing DGEBA/YFeAl, DGEBA/YFeAl/ZnS was performed
according to ISO 10993-5 [International Organization for Standardization, 1999]. The
extract of the YFeAl-ZnS nanoparticles was used in the cytotoxicity assay with a culture
of Chinese hamster ovary cells (CHO K1). CHO cells were cultured in RPMI 1640
medium containing 10% fetal calf serum (FCS) and 1% antibiotics
(penicillin/streptomycin). A cell culture plate with 96 wells were prepared with
increasing dilutions of extract of the YFeAl-ZnS nanoparticles (50 µL/well, four wells
/each dilution). Then 50 µL of cell suspension (3.000 cells) were dispensed into the
wells. Four columns of control wells were prepared with medium without cells (white)
and half cells (negative control). The microplate was incubated in a humidified
atmosphere of 5% CO2. After 72 hours, 20 µL of a mixture (20:1) 0.2% of MTS and
PMS 0.09% in PBS (phosphate buffered solution) was added to the wells and left for 2
hours. The incorporation of the dye was measured by reading absorbance at 490 nm in
microplate reader against the blank column. The level of cytotoxicity (IC50%) was
estimated by interpolation as the concentration of the extracts resulting in 50%
inhibition of the uptake of MTS, after the construction of a graph of average percentage
of living cells against the concentration of the extract (%). Parallel to the test, a solution
of phenol and 0.3% polymer HDPE were used respectively as positive and negative
controls.
The hemolytic activity of nanoparticles DGEBA/YFeAl, DGEBA/YFeAl/ZnS
was tested against human erythrocytes. Blood was collected in the presence of citrate
buffer (150 mM, pH 7.4) was centrifuged for 15 min at 700xg, washed three times and
resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, KH2PO4 1,76
mM, pH 7.4). A suspension of the nanospheres of 100 mL was tested in triplicate. After
incubation the suspension of nanoparticles at 37°C with a 4% solution (v/v)
erythrocytes in PBS the system was centrifuged for 5 min at 700 xg, aliquots of
supernatant were used to measure the absorbance at a wavelength 414 nm on a Cary 50
spectrophotometer (Varian). Measures the absorbance of the supernatant to 0 and 100%
hemolysis was obtained with 100 mL of the solution of the red blood cells in PBS and
4% Triton X-100 01% (v/v), respectively.
The in-vitro interaction of the YFeAl-ZnS nanoparticles with proteins were
assayed with human serum albumin (HSA) and human serum fibrinogen (HFB). Human
albumin leads to a predominant protein in blood abundantly exceeding the rest of the
plasma proteins [Bradford, 1976]. Solutions of 10 mg.mL-1 HSA (Aldrich 99% pure)
and 10 mg.mL-1 of HFB (Aldrich 99% pure) were separately prepared in phosphate
buffered solution pH 7.2 and ionic strength of 0,01 M (PBS). Samples of the
nanospheres were placed in Teflon® tube with 6 mL of PBS solution for 2 hours at 37
°C. The amount of protein adsorbed was determined by using a spectrophotometer
UV/VIS (Cary 50, Varian) by the difference between the concentration of HSA and
HFB before and after contact with the polymer. Protein concentration was assessed
according to the Bradford method at 595 nm. [Bradford, 1976].
3.
RESULTS AND DISCUSSIONS
The transmission electron micrographs (TEM) of the YFeAl-ZnS nanoparticles
are shown in Figure 2. The hybrid magneto and fluorescent nanoparticles showed little
size dispersion, with diameters in the range of 20-30 nm.
(A)
(B)
Figure 2: TEM micrograph of the hybrid YFeAl-ZnS nanoparticles (A) and particle
size distribution (B).
The TEM micrographs of as prepared DGEBA nanospheres carrying the
magneto-fluorescent YFeAl-ZnS ceramic shows uniform sphere and a homogeneous
size distribution around 60-80 nm (Figure 3A). The homogeneous distribution of the
size of the nanoparticles facilitates the process of conversion of magnetic energy into
heat once that provides a homogeneous temperature distribution within the tumor tissue.
Figure 3: TEM micrograph of the polymer nanospheres carrying the hybrid YFeAl-ZnS
ceramics (A) and the size distribution of the nanospheres (B).
One of the most important factors for the treatment of tumors by hyperthermia is
a magnetic particle size distribution. A homogeneous size distribution facilitates the
hyperthermia therapy since it provides a homogeneous distribution of temperature
within the tumor tissue [Castro, 2010].
Figure 4 illustrates the size distribution of nanospheres DGEBA/YFeAl ZnS
encapsulated with the epoxy biocompatible polymer. There is an average distribution of
particle size in the range 100-125 nm for the polymer nanospheres of the pure epoxide
(DGEBA) and 225 nm for the nanospheres loaded nanoparticles optical/magnetically
active. Since the average diameter of the capillaries lies between 4 to 16µm, the
diameter of the polymer nanospheres of carriers DGEBA epoxy/ZnS-YFeAl obtained in
this study appears to be convenient for the diagnosis and treatment processes involving
tumor angiogenesis. Additionally it’s may be observed in SEM micrographs (Fig. 4)
that microspheres surface are apparently smooth without the presence of pores
indicating that the nanocomposite DGEBA/ZnS-YFeAl fills the free volume between
the epoxy polymer chains without causing deformation of the nanospheres.
Figure 4: SEM micrographs of the systems: DGEBA/YFeAl (A,B), DGEBA/ZnS (C)
DGEBA/YFeAl (D) and DGEBA (E,F).
The behavior of magnetic nanospheres DGEBA/YFeAl and DGEBA/YFeAl/
ZnS prepared is of fundamental importance for the analysis of how these materials will
behave in front of applying a particular magnetic field. The desired behavior for the
nanospheres and their application in magneto-hyperthermia is to paramagnetism or
superparamagnetism, characterized by the alignment of magnetic dipoles of the material
only by the presence of a magnetic field.
The magnetization curves of the nanospheres carriers DGEBA/YFeAl and
DGEBA/YFeAl/ZnS are shown in Figure 5. It can be seen that there is very little
variation in the magnetic hysteresis for both materials, characterizing the behavior of
paramagnetism, when the external magnetic field is removed from the nanoparticles do
not retain magnetism. However, there was a difference in the values of magnetic
saturation with 53.7 emu.g-1 for the nanospheres DGEBA/YFeAl/ZnS emu.g-1 and 60.7
emu.g-1 for the DGEBA/YFeAl nanospheres. The magnetic saturation of the
nanospheres corresponds to the point where the increase of the intensity of applied
magnetic field does not promote the magnetization of the material increased. The
remanent magnetization, the corresponding magnetization of the material that remains
after removal of the external magnetic field was 3.4 emu.g-1 for the DGEBA/ YFeAl
nanospheres and 3.1 emu.g-1 for the DGEBA/YFeAl/ZnS nanospheres. The results
show that the DGEBA nanospheres remain magnetized only in the presence of an
external magnetic field.
The small difference in the magnetization of the DGEBA/YFeAl/ZnS
nanospheres may be associated with variation in mass of nanoparticles during the
encapsulation of the hybrid nanocomposite YFeAl/ZnS.
Figure 5: Magnetization curve of the nanospheres of DGEBA/YFeAl and
DGEBA/YFeAl/ZnS at room temperature (27°C).
Considering the area under the curve in Figure 5, we calculated the magnetic
energy present in the nanospheres when they were subjected to a magnetic field. Figure
6 shows the energy of magnetization as a function of iron concentration in YFeAl
nanoparticles. There is a strong dependence of the magnetic energy stored in function of
iron concentration (mol%) in both samples of nanospheres, DGEBA/YFeAl and
DGEBA/YFeAl/ZnS.
Figure 6: Magnetic Energy (J.m3) as a function of iron concentration in nanospheres
DGEBA/YFeAl (●) and DGEBA/YFeAl/ZnS (○).
The treatment by hyperthermia requires an increase in temperature of the system
when it is subjected to a magnetic field. The Figure 7 shows the temperature evolution
when the nanospheres DGEBA/ZnS-YFeAl are subjected to an oscillating magnetic
field. It is observed that the ideal temperature for the treatment magnetichyperthermia,
41°C, is reached when the stoichiometry of Fe is equal to 1,7 (x = 1,70).
280
240
200
TC (ºC)
160
120
80
40
0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Al (mol%)
Figure 7: Curie transition temperature (Tc) as a function of the Al content (x)
mol(%) in the ceramic Y3Fe 5-x Al X O12.
Figure 8 shows the epifluorescence micrograph of the DGEBA/YFeAl-ZnS
nanospheres. The micrographs were obtained in solid form and, in phosphate buffersaline (PBS) pH 7.0 to simulate the human physiological fluid. The samples were
excited with light of wavelength exceeding 350 nm.
Figure 8: SEM micrograph of the nanosphere epifluorescence
DGEBA/YFeAl/ZnS in saline PBS pH 7.4. The micrographs were taken on an
epifluorescence inverted microscope (Diaphot-TMD, Nikon, Tokyo, Japan) equipped
with epifluorescence illumination. ND32 filter was used to reduce the light intensity and
objective with increased 1000X. The samples were excited with light of wavelength 350
nm (DGEBA/YFeAl/ZnS) and 550 nm.
It was observed that both the nanospheres DGEBA/YFeAl/ZnS exhibit an
intense fluorescence (Fig. 8). Since the polymer nanospheres of the pure epoxide are not
fluorescent, the fluorescent properties correspond to those observed ZnS quantum dot
on the surface of the ceramic ferromagnetic. It was also observed in epifluorescence
images that there is significant extinction luminance of nanospheres loaded with
ceramic magneto-optically active due to the dielectric constant of the medium indicating
that the nanospheres loaded with DGEBA/ZnS-YFeAl can be used as an effective
biomarker for applications in diagnostic medicine. Biomarkers show the intensity of
luminescence observed at room temperature (27°C) of the polymer nanospheres loaded
with DGEBA epoxy/YFeAl ZnS.
The use of biocompatible nanoparticles for the detection of tumor cells still in its
early stage may allow the diagnosis and treatment of disease is still in its early stages
which can benefit the patient about the possibility of cancer control. Many in vitro
methods to test for cytotoxicity of biomaterials were standardized using cell cultures.
These cytotoxicity tests consist in subjecting the material directly or indirectly by
contact with a culture of mammalian cells, verifying the cellular changes and cell lysis
(cell death), inhibition of cell growth and other effects caused by the biomaterial and/or
their extracts [ Queiroz, 2004].
Figure 9 presents the cell viability of the nanospheres DGEBA/YFeAl and
DGEBA/YFeAl/ZnS.
Figure 9: Cytotoxicity assay with mammalian cells of the nanospheres of nanoparticles
YFeAl carriers, ZnS. C + and C - represent the positive and negative controls,
respectively.
The emphasis on interaction between proteins and synthetic surfaces is generally
regarded as a key step for the successful implementation of any biomaterial. After
contact with physiological fluids, many proteins adsorbed to the nanoparticles
circulating in the human body could induce indirect interaction of blood cells with the
material.
It was observed the occurrence of a higher adsorption of HSA compared to
fibrinogen adsorption for the nanospheres DGEBA/YFeAL, DGEBA/YFeAl/ZnS, as
illustrated in Figure 10, showing a tendency toward such areas not trombogeneicidade
synthetic. The adsorption of fibrinogen plays a significant role in the phenomenon of
hemocompatibility of polymeric materials since it is a coagulation factors facilitates
platelet adhesion, participating in exchange reactions with other proteins important in
blood clotting mechanism [Keating, 2001].
Figure 10: Adsorption of serum proteins albumin (HSA) and fibrinogen (HFB)
on the surface of nanospheres DGEBA/YFeAl (●), DGEBA/YFeAl/ZnS (●).
Once the DGEBA nanocarriers YFeAl, ZnS should be used in contact with
living organisms, is of particular importance to study their reaction when in contact with
blood. For this reason was analyzed the ability of the nanospheres in inducing the lysis
of erythrocytes (hemolysis) [Queiroz, 2004].
Hemolysis is a process which gives the breakdown of red blood cells with the
resulting hemoglobin release into the medium. With hemolysis test is intended to
determine the reaction of the nanocapsules carriers hemolytic, that is, to know what
degree of lysis of erythrocytes, knowing the amount of hemoglobin that is released into
plasma. This study is important in that it is possible to determine the fragility of the
membranes of red blood cells when in contact with the nanocapsules. After some time
the nanoparticles in direct contact with the diluted blood is made by reading the plasma
by spectrophotometric analysis UV/Vis and through a calibration curve is calculated as
the concentration of hemoglobin. Since the initial concentration of hemoglobin in blood
and plasma are previously known through a number of calculations obtain the percent of
hemolysis [Shiohara, 2004].
Figure 11 shows the percentage of hemolysis of the DGEBA/YFeAl and
DGEBA/YFeAl/ZnS nanospheres. It is may be observed that the percentage of
hemolysis is very close to the medical grade silicon used as a negative control which
shows good hemocompatibility of the nanospheres conditions in vitro assay.
Figure 11: Analysis of the hemocompatibility of DGEBA carriers YFeAl
nanospheres (●), ZnS (). The negative and positive controls are medical grade silicone
(●
CONCLUSIONS
Photoactive and magnetic polymeric DGEBA nanospheres containing hybrid
YFeAl-ZnS nanoparticles were successfully prepared and exhibited size distribution,
fluorescence and magnetic properties adequate for use in magnetic fluid hyperthermia.
The in vitro cell culture experiment proved that the polymer nanospheres carrying the
YFeAl-ZnS shows good biocompatibility for use in mammalian cells. All of these
results indicate that the polymer nanospheres containing YFeAl-ZnS nanoparticles may
have promising medical applications in tumor therapy and diagnostic.
REFERENCES
1. Bradford, M. M. A Rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical Biochem., 72, p.248-254, 1976.
2. Castro, V. F. et al, Propriedades magnéticas e biocompatíveis de nanocompósitos para utilização em
magneto-hipertermia. Revista Brasileira de Física Médica (RBFM). 2010;4(1):79-82.
3. INCA, Incidência de câncer no Brasil. Estimativa 2010 at http://www.inca.gov.br/estimativa/2010/
4. International Organization for Standardization (1999 E). “Biological evaluation of medical devices
tests for citotoxicity: in vitro methods”, ISO 10993-5.
5. Keating, J.F.; MCQUEEN, M.M., Substitutes for autologous bone graft in orthopaedic trauma. J. Bone
Joint Surg. [Br], 82: B3-8, (2001).
6. Lock I, Jerov M, Scovith S (2003) Future of modeling and simulation, IFMBE Proc. vol. 4, World
Congress on Med. Phys. & Biomed. Eng., Sydney, Australia, 2003, pp 789–792
7. Overgaard, J.; The current and potential role of hyperthermia in radiotherapy. Intern J Rad Onc Biol
Phys 1989; 6: 535-49.
8.Queiroz AAA, Passos ED, Silva MR, Higa OZ, Bressiani AHA, Bressiani JC. Biocompatible
superparamagnetic nanospheres for the cancer treatment. Apresentado no III Congresso Latino
Americano de Órgãos Artificiais e Biomateriais (COLAOB). Campinas (SP), 2004, p. 182.
9. Shiohara A, Hoshino A, Hanaki K, Suzuki K, Yamamoto K, 2004. On the cytotoxicity caused by
quantum dots. Microbiol Immunol 48 (9) : 669-75.
10. Solanki A, Kim JD, Lee K-B (2008). Nanotechnology for regenerative medicine: nanomaterials for
stem cell imaging. Nanomedicine 3(4): 567-578.
11.Sun S, Zeng H. Size-controlled synthesis of magnetite nanoparticles. J Am Chem Soc.
2002;124(28):8204-5.
12. Tartaj P, Morales MP, Veintemillas-Verdaguer S, Gonzales-Carreño T, Serna JC. The preparation of
magnetic nanoparticles for applications in biomedicine. J Phys D: Appl Phys 2003;36:R182-R197.
13.Yong, K. et al., Quantum rod bioconjugates as targeted probes for confocal and two-photon
fluorescence imaging of cancer cells. Nano Letters, v. 3, n.3, p. 761-765, 2007.
BIOCOMPATIBILITY STUDIES OF NANOCAPSULES OF EPOXY
NETWORK POLYMER CONTAINING QUANTUM DOT AND
SUPERPARAMAGNETIC NANOPARTICLES FOR CANCER
CELL LABELLING AND THERAPY
Vinícius Fortes de Castro1, Alvaro Antonio Alencar de Queiroz1
1
Centro de Estudos e Inovação em Materiais Biofuncionais Avançados, Universidade Federal de Itajubá,
Av. BPS, 1303, 37.500-903, Itajubá (MG), Brasil.
E-mail: vfc_mg@yahoo.com.br, alencar@unifei.edu.br
Abstract. The purpose of this study was to explore the physics and biologic properties of polymer
nanospheres carrying Y3Fe5-xAlxO12(YFeAl) nanoparticles. Magnetic and fluorescent polymer
nanospheres containing the photoactive and superparamagnetic YFeAl-ZnS nanoparticles were prepared
by inter-facial polymerization of epoxidic polymer based on the ether diglycidic of bisphenol A (DGEBA).
The microstructure and size distribution of the polymer nanospheres were determined by transmission
electron microscopy (TEM) and atomic force microscopy (AFM). The fluorescence of the polymer
nanospheres was observed using the epifluorescence microscopy. In-vitro experiments about the
biocompatible properties of polymer nanospheres carrying the hybrid YFeAl-ZnS nanoparticles were
accessed in mammalian cells (CHO). It was observed that the polymer nanospheres did not affect the
viability of mammalian cells or the growth rate of cell culture. The biophysical properties of the polymer
nanospheres carrying the hybrid YFeAl-ZnS ceramic indicated that this can be a highly versatile
nanobiomaterial for therapeutic and cancer diagnosis.
Keywords: Quantum dots, ZnS, Magneto fluid hyperthermy.