Nghiên cứu quy trình chế tạo và đánh giá hiệu quả tác động của hệ nano đa chức năng (polymer drug fe3o4 folate) lên tế bào ung thư tosm tat tieng anh

MINISTRY OF EDUCATION
AND TRAINING

VIETNAM ACADEMY
OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

……..….***…………

LE THI THU HUONG

Study on fabrication and effectiveness evaluation of multifunctional
nanosystem (polymer-drug-Fe3O4-folate) on cancer cells

Major: Electronic materials
Code: 9440123

SUMMARY OF MATERIALS SCIENCE DOCTORAL THESIS

Hanoi – 2018


This thesis was finished at Institute of Mataerials Science and
Graduate university of Science and Technology, Vietnam Academy
of Science and Technology

Supervisor 1: Dr. Ha Phuong Thu
Supervisor 2: Prof. Dr. Nguyen Xuan Phuc

Reviewer 1: …

Reviewer 2: …
Reviewer 3: ….

This thesis will be defended against Board of thesis defense at Graduate
university of Science and Technology – Vietnam Academy of Science
and Technology at … …, Date ………………..

It can be found at:
-

Library of Graduate university of Science and Technology
Vietnam National Library


INTRODUCTION
1. The urgency of the thesis
Today, the development of science and technology has made great
strides in biomedical science but human beings are still facing many
diseases, most notably cancer. There are many cancer drugs available on the
market. However, the biggest disadvantage of many cancer drugs is that they
are less soluble in water or more readily excreted. Besides, the selectivity of
these drugs is not high, and more or less affects healthy tissues and results in
side effects including nausea, diarrhea, anemia or reduced immunity of the
body. This is due to most treatments not only affect the tumor locally, but
also affect a large part of the body's normal tissues and organs. To overcome
the shortcomings of the method above, researchers have applied
nanotechnology, using nanometer-sized materials as a vehicle to deliver
cancer-specific drugs such as Curcumin, Paclitaxel, Doxorubicin. to the
tumor safely. In addition, magnetic nanomaterials have been studied
extensively for cancer screening, cancer diagnosis by magnetic resonance

imaging (MRI), thermotherapy by increasing tumor temperature under
magnetic field, and especially tumor targeting by magnets... Magnetic
nanoparticles and anti-cancer drugs could be encapsulated in the shells of
natural or synthetic polymers such as dextran, modified dextran, chitosan,
modified chitosan, alginate, PLA-TPGS, PLA-PEG ... to become nano stable
systems. The surface of these system can be added a number of target factors
such as folate, aptamer, tranferin, lectin and antibody. Such a multifunctional
nanosystem will increase the effect on certain cancer cells, partly addressing
the need for chemotherapy to be highly selective for cancer cells. The
benefits of the material utility are: reducing the dose of the drug, focusing on
the tumor position, avoiding to affect the healthy cells and therefore
minimizing adverse side effects on patients. From the above mentioned
issues, it is possible to use a multifunctinal nanosystem consisting of Fe3O4
nanoparticles coated with modified chitosan, modified dextran, alginate or
copolymers and attached folate as a vehicle for Curcumin (Cur) or
Doxorubicin (Dox) to safely target the cancerous tumor. Based on that fact,
the thesis "Research and make the effect of polyunsaturated (polymer-drugFe3O4-folate) on cancer cells" was done.
1


2. The objectives of the thesis
- Manufacturing multifunctional nanoparticles including: Fe3O4
nanoparticles (magnetical properties) coated with biocompatible polymers,
attaching drugs (Cur, Dox) and targeted folate factor (optical properties))
that are well dispersed in water, able to target the cancer.
- Experiment and evaluate the effect of the nanoparticles on cancer cell lines
such as HT29; HeLa; HepG2 ... and on experimental animals
3 . The main contents of the thesis
- Synthesis of multifunctional nanocomposite materials containing curcumin
and doxorubicin based on Fe3O4 nanoparticles coated with natural polymers

(O-carboxylmethylchitosan and alginate).
- Characterization of the materials by modern physicochemical methods:
FTIR, UV-VIS, fluorescence spectrum, XRD, VSM, TGA, SEM, TEM ...
- Determine the effect of multifunctional nanoparticles on cancer cell lines:
Hep-G2, HeLa, LU-1, ... and in mice.
Chapter 1. OVERVIEW
In this chapter, we review the issues involved in the synthesis of
multifunctional nanoparticles and the effect assessment of these systems on
cancer cells. Multifunctional systems consist of Fe3O4 nanoparticles coated
with polymer, drugs loading and folate attaching. In details, this part
provides an overview of the properties, synthesis methods and applications
of Fe3O4 nanoparticles. Especially, the issues that need to be addressed in
order to use Fe3O4 nanoparticles in biomedical field were clearly shown. The
nature and applicability of natural polymers commonly used (O-carboxyl
methyl chitosan, alginate, dextran) were discussed while characteristics and
some studies using the drug substances: curcumin and Doxorubicin were
presented. In addition, the method of folate attachment to the nanoparticles
and the targeted effect of this agent were overviewed.
Chapter 2. CONDITION AND EXPERIMENTAL METHOD
2.1. Synthesis of multifunctional nanosystems
Multifunctional nanomaterials were synthesized through the procedures
shown in Figure 2.1. The magnetic nanoparticles (Fe3O4) were synthesized
by co-precipitation of Fe2+ and Fe3+ at 1:2 molar ratio with normal apparatus
2


[41] or using microwave technique on Sineo-Uwave 1000 apparatus. Fe3O4
nanoparticles were then coated with OCMCS (1 mg/ml) or alginate at
different concentrations. In the next step, Curcumin or Doxorubicin was
introduced into the system by adsorption interaction with the magnetic core

or reaction with the polymer shell. Ultimately, the optimized drug delivery
system was chosen to incorporate folate-targeting factor or CdTe quantum
dots.

Figure 2.1: Synthesis procedures of multifunctional nanosystems

2.2. Characterization
The characteristics of the systems were determined by modern
methods: X-ray diffraction (XRD), infrared spectroscopy (FTIR), UV-Vis
spectroscopy, fluorescence spectroscopy, thermal analysis, scanning electron
microscopy (SEM), tranmittance electron microscopy (TEM). Drug
encapsulating efficacy, drug loading content, and drug release profiles were
determined by UV-Vis spectroscopy.
The cytotoxicity of the samples was determined according to the method of
Skehan and Likhiwitayawuid [171, 172].
Atomic Absorption Spectrum (AAS) method was used to quantify Fe
present in mouse tissues.
In vivo experiments:
3


7-10 mm tumor – bearing mice were divided into 4 groups, each group
of 6 mice, including: control group (mice with untreated tumors) and groups
treated with FA, FAD, FADF, respectively. In each treatment cycle, the drug
was injected directly into the tumor at 50 l/mouse. At 40 minutes post
injection, the mouse was fixed in a plastic tube and put into a RDO-HFI coil
of a magnetic field with frequency of 178 kHz and strength of 90 Oe for 30minute time. Two consecutive cycles separated by 3 days. Changes in tumor
size were recorded before each treatment. These information was used to
assess the therapeutic effects of Doxorubicin loading magnetic nanoparticles
on model mouse with lung cancer.

Data analysis: Excel 2010, OriginPro 8 hoặc SPSS 22.0.
Chapter 3: CURCUMIN LOADING OCMCS COATED Fe3O4
NANOPARTICLES
3.1. Synthesis of nano Fe3O4 nanoparticles (NPs)
Fe3O4 nanoparticles ware successfully synthesized by co-precipitation
method (Fe-O bond characterized by absorption peaks at 575 cm-1 on
infrared spectra), reverse spinel structure (with typical peaks in XRD
diagram), saturation magnetization of 70.5 emu/g, superparamagnetic
property with Mr and Hc  0 and average size of 15 nm.
3.1.2. Microwave synthesized Fe3O4 NPs
3.1.2.1. Magnetic properties
Magnetic remanance Mr and coercivity Hc of fabricated samples were 
0, indicating that the material were superparamagnetic (Table 3.1). Thus,
microwave technique did not change this property of the materials. The
saturation of the M5 sample was the highest compared to the other samples,
reaching 69 emu/g. This value is not much different than the Fe3O4 sample
prepared under normal conditions (70.5 emu/g).
Table 3.1: Magnetic parameters of microwave synthesized Fe3O4 NPs

Sample M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11
Ms
53,9 56,2 56,7 63,0 69,0 64,6 59,6 60,6 60,7 62,7 64,5
(emu/g)
Hc
2,5 14 4 2,5 0 20 0 18 2 2 21
4


(Oe)
Mr

0,5 1,0 0,2 0,4
(emu/g)

0

1,7

0

2

0,1 0,2 1,9

Thus, M5 is the best magnetic sample.
3.1.2.2. X-ray diffraction
The XRD diagriam of the M5 sample showed full series of Fe3O4 typical
peaks and no strange peaks, indicated that M5 formed with a single-phase
spinel structure. This result confirms that M5 is the best sample in term of
crystal structure.
4.2.1.3. IR spectra
In Fig. 3.4, it can be seen that the microwave-assisted synthesized Fe3O4
samples showed the characteristic peak for the Fe-O bond at about 570 cm-1.
In some samples, however, a lower intensity peak at 630 cm-1 was observed,
corresponding to the presence of Fe2O3 in these samples [174]. The spectra
reveal that M5 is the highest purity sample with only one characteristic peak
with high intensity.
Thus, through the magnetometry, crystal structure and infrared spectra,
we selected the M5 sample for further studies.
3.2. Effect of curcumin amount on curcumin loading systems (FOC1FOC5)
The amount of curcumin varying from 20 to 100 mg was investigated

to determine the effect of the curcumin amount on magnetic properties as
well as the stability of the systems (evaluated by measuring the zeta potential
of the systems). The results are presented in Table 3.2.
Table 3.2: Properties of curcumin loading systems

Sample
FOC1 FOC2 FOC3 FOC4 FOC5
Mass of curcumin (mg)
20
40
60
80
100
Ms (emu/g)
54,9 52,9 49,0 35,3 25,8
Zeta potential(mV)
40,2 32,6 30,4 18,2 8,1
The saturation magnetization measurement of FOC1-5 showed that when
the amount of curcumin increased from 20 to 100 mg, the saturation
magnetization of the samples decreased, especially in the samples FOC4 and
FOC5. The Zeta potiential of FOC1-5 samples were positive because the
magnetic particles were coated with O-carboxylmethyl chitosan polymer
5


with many NH2 functional groups on the surface. The change in Zeta
potential of these samples is similar to that in saturation magnetization. Zeta
potential values of FOC1-3 are greater than 30 mV showing that these
samples could maintain stable state [170]. Meanwhile, Zeta potential values
of FOC4 and FOC5 are significantly lower than those of above samples (less

than 20 mV). In order to ensure that the multifunctional system carries the
largest range of curcumin loaded and retains its magnetic properties, we use
a curcumin mass of 60 mg for other related synthesis procedure. This
curcumin amount is also used to prepare FOCF system. The actual curcumin
contents of the systems are quantified by thermal analysis (Section 3.3.4).
3.3. FOC and FOCF NPs
3.3.1. IR spectra
Infrared spectra of FOC and FOCF were compared with the infrared
spectra of each component: Fe3O4, OCMCS, Curcumin and folic acid. The
transfer of characteristic peaks proves that the system has been successfully
synthesized.
3.3.2. Flourescence spectra
Curcumin is a natural fluorescence compound. After receiving
stimulation by radiation at 442 nm, the FOC solution emits fluorescence
spectrum at a maximum wavelength of 515 nm.In comparison with the
fluorescence spectrum of curcumin in ethanol/water (1:1) with a maximum at
542 nm, fluorescence of FOC exhibits a blue shift (27 nm shift towards short
wavelength region). This is due to the interaction of the curcumin molecule
with Fe3O4/OCMCS. In terms of intensity, FOC solution fluoresces much
less weakly than free curcumin does. This is due to the presence of Fe 3O4 in
the sample which reduces the fluorescent ability of curcumin [132].
3.3.3. FeSEM
The surface morphology of the FOC and FOCF systems was
determined through SEM images. The results show that the size of these
particles is about 30 nm, which is larger than the size of the original Fe 3O4
particle (about 20 nm), suggesting that curcumin and folic acid adsorbed
onto the surface of Fe3O4 nanoparticles.
3.3.4. Thermal analysis
Figure 7 show the DrTGA, TGA and DTA curves of FOC and FOCF
samples. The TGA curves showed wto steps of weight loss of FOC and three

6


steps of weight loss of FOCF sample and then there was no change in weight
of samples when continuing increase temperature. The weight loss for the
first step of each sample at around 100oC is attributed to quantitative mass
losses of water present in the samples. All the other steps are endothermal,
that can be explained by the decomposition of OCMCS, curcumin or folic
acid. As mentioned above, the weight of OCMCS in the sample was very
small, so the weight loss was almost attributed to the weight of curcumin or
folic acid in the samples. The second step for weight loss of FOC
corresponds to the third steps of FOCF at temperature range of 360 and
430oC and can be assigned as the decomposition of curcumin. Therefore, the
second weight loss step at around 299oC of Fe3O4/OCMCS/Cu/Fol must be
the loss due to the decomposition of folate. The result also show that in the
first sample the mass of curcumin and Fe3O4 account for 45% and 48% the
total mass while the mass of folic acid, curcumin and Fe3O4 are 26%, 25%
and 46%, repectively total mass of Fe3O4/OCMCS/Cu/Fol sample. Based on
this data, curcumin-loading capicity was calculated and found to be about
0.95 mg and 0.54 mg per mg of Fe3O4 in FOC and FOCF NPs. Despite of the
decrease, FOCF is a good loader of curcumin as compared to other studies
[134, 176, 177] and can be used as selective orientation drug deliverer.
Figure 3.14 shows the structure of FOC and FOCF, in which curcumin is
adsorbed on the surface of Fe3O4 particles.

Figure 3.1: Structural models of FOC and FOCF

3.3.5. XRD diagrams and magnetic properties

7

80

(a)Fe3O4
(440)
(b) Fe3O4/OCMCS/Cur
(c) Fe3O4/OCMCS/Cur/Fol
(511)
(400)
(422)

(200)

(a)

60
40

Ms (emu/g)

(311)

(c)

(b)

20

1.5

(b)

1.0

(c)

0.5
0.0

-25

-20

-15

-10

-5

0

0
-20

(c) Fe3O4/OCMCS/Cur/folic
(b) Fe3O4/OCMCS/Cur

-40
(a)

(a) Fe3O4

-60

30

40

50
o
2theta ( )

60

70

Figure 3.15: XRD diagrams of (a)
Fe3O4, (b) FOC and (c) FOCF

-80
-15000 -10000

-5000

0

5000

10000

15000

H (Oe)

Figure 3.16: Hysterisis loops of (a) Fe3O4,
(b) FOC and (c) FOCF

The XRD patterns of FOC and FOCF show no difference from that of
the Fe3O4 nanoparticles (Figure 3.15). It was clear that there were six
diffraction peaks corresponding to six faces of (200), (311), (400), (422),
(511) and (440) which were characteristic for single phase spinel structure of
Fe3O4. These facts indicate that the two systems have not changed their
crystal line structure during the encapsulation process. Magnetization
measurements also provided evidence that the Fe3O4 nanoparticle
encapsulated in maintained its crystalline structure (Figure 3.16). The
magnetic properties of FOC and FOCF NPs was measured by VSM. The
saturated magnetization of the FOC and FOCF NPs was about 53 emu/g,
which was about 20 emu/g lower than that of free Fe 3O4 due to the
adsorption of curcumin or folic acid in the surface of Fe3O4. Although the
magnetism has decreased, nanoparticles can still be adsorbed quickly and
firmly by the magnet. On the other hand, it is well known that magnetic
particles less than 30 nm will demonstrate the characteristic of
superparamagnetism, which can be verified by the magnetization curve. The
remanence (Mr) and coercivity (Hc) for FOC and FOCF NPs in the figure
were close to zero, exhibiting the characteristic of superparamagnetism
[169].
3.3.6. Magnetic inductive heating effect
The results of induction heating are presented in Table 3.4. When the
iron oxide concentration decreases, both the saturated Ts temperature and the

initial heating rate dT/dt (determined at t = 0) decrease. Particles
concentrations of 0.3 mg/ml or more resulted in saturated temperatures of up
to 42 °C and higher after 10 minutes.
8


Retention time of 10 minutes and possibly longer can be established by
maintaining the magnetic field conditions. Because cancer cells can undergo
apoptosis within the range of 42-46 °C [73], FOCF can be used to treat
cancer by thermotherapy.
Table 3.4: Induction heating parameters of curcumin loading samples

Concentration
FOC
(mg/ml)
Ts (1500 s)
0.1
45.5

dT/dt
0.02

FOCF
Ts (1500 s)
38.6

dT/dt
0.01

0.3

50.0

0.03

44.2

0.02

0.5

54.6

0.04

54.7

0.03

0.7

58.6

0.06

58.9

0.04

1

64.3

0.09

67.5

0.06

3.3.9. Cytotoxicity
Curcumin

Combination: Red - actine; blue –
cell nucleus; green – curcumin

Figure 3.21: Fluorescence of HT29 cells under normal conditions
(control) and in 15 hour incubation with FOC

Fluorescent images showed cellular uptake of curcumin into HT29 cell
(green color) when incubated with FOC (Fig. 3.21). The cause of the green
signal here is that curcumin is capable of spontaneous fluorescence when
9


stimulated with Argon lasers. There is no signal in control sample. This
finding also demonstrates that incorporating curcumin into the nano-carrier
does not affect the ability of the curcumin to enter the cell, the nanoparticle
that ensures the release of curcumin into the cell.
3.3.10. Biodistribution
Biodistribution of FOC and FOCF on different mouse organs are shown

in figure 3.23. In Sarcoma 180 tumors, FOCF was present significantly
higher than FOC after injection of 2.5 h. After 5 h, the folate attached system
was still present higher than that of FOC without folate. From the heating
curves, it is possible to reveal that the higher the Fe3O4 content, the higher
the heat and saturated temperature, so as the Fe3O4 concentration increases
with the folate-targeting element, the FOCF system can be more efficiently
used to cure cancer. In addition, when a magnet was applied to the back of
the mouse treated with FOCF, after 5 hours, the amount of magnetic
nanoparticles appearing in the mouse kidney and spleen remained higher
than in organs of the mouse treated with FOCF. This suggests that folate and
magnetic fields may contribute to prolong the retention time of magnetic
particles in the body, and may therefore be more likely to be transported to
the tumor.
Chapter 4: DOXORUBICIN LOADING ALGINATE COATED
Fe3O4 NANOPARTICLES
4.1. Effect of alginate concentration on Dox loading capacity and system
properties
Unlike curcumin, Dox is a good soluble drug in water. Therefore, Dox
is difficult to interact with the hydrophobic surface of Fe3O4. To encapsulate
Dox on the multi-functional system, it is necessary to attach Dox to the
particle surface by chemical bonding. Research has shown that the polymer
shell is the decisive factor in the ability of Dox to carry Fe 3O4 nanoparticles
[143].
4.1.1. IR and flourescence spectra
The Fe-O bond of Fe3O4 in FA4, FA10, and FA4D samples is
characterized by the peak in the 570 cm-1 region. Compared with pure Fe3O4
(575 cm-1), the wave number of Fe-O oscillations in alginate-coated samples
decreased (566, 574 and 563 cm-1, respectively, on the spectrum of FA4,
10

FA10, FAD). The coating process of alginate on the surface of Fe 3O4 has
been achieved. In addition, the shift in the wave numbers of characteristic
peak for organic bonds proves that doxorubicin has been encapsulated into
the nanoparticles. Figure 4.2 is the FA4D fluorescence spectrum versus that
of free Dox. Chemical bonding between Dox and the nanoparticles may be
confirmed by fluorescence peak position of FA4D (17 nm shift compared to
free Dox) [131]. In addition, the fluorescence intensity of FA4D decreased
sharply because of the fluorescence suppression effect of Fe3O4 present in
the sample [180].
4.1.2. Loading content (LC) and Encapsulation efficiency (EE)
The drug encapsulating efficiency (EE) of the samples is shown in Table
4.1. The data show that alginate concentration is an important factor
influencing Dox loading performance. The higher the alginate concentration,
the greater the Dox EE. This phenomenon can be explained by the formation
of chemical bonds between Dox and alginate on the surface of Fe 3O4
nanoparticles. However, due to increased alginate content from FA2D to
FA10D, the total mass increase so the drug loading content (LC) does not
increase continuously. The maximum drug loading content was reach at
FA4D, so we chose this sample for further bioassay.
Table 4.1: EE and LC values

Sample
EE (%)

FA2D
61,2±0,5

FA4D
78,5±0,3

FA6D
85,0±0,9

FA8D
87,2±0,8

FA10D
90,8±0,7

LC (%)

17,89±0,15 18,96±0,07 17,49±0,19 15,62±0,14 14,41±0,11

4.1.3. Size distribution and TEM images
The size distribution of the nanosystems determined by the DLS
spectrum depends a great deal on the alginate concentration. Prior to carrying
Dox, Fe3O4 particles coated with FA4 and FA8 alginate had the
hydrodynamic sizes of 18 nm and 91 nm. High concentration of alginate in
FA8 forms a thicker coating layer and expands the hydrodynamic size of the
FA8 particles due to the hydrophilicity of alginate. Loading Dox in FA4D
and FA8D significantly increased the particle size (255 and 480 nm
respectively), while the size distribution was also wider than FA4 and FA8.
Corresponding to the amount of Dox, FA8D contains more Dox than FA4D
11


Lin (cps)

(400)

(440)

(422)

-10000

-5000

40
20
0

H (Oe)
0

5000

10000
-1

(511)

60

Ms (emu g )

(220)

FA2

FA4
FA6
FA8
FA10
FA4D
FA8D

Fe3O4
FA4D

-1

(311)

Ms (emu g )

so the increase in its particel size is also greater than FA4D. FA4D is more
appropriate than FA8D for biomedical applications. The TEM image shows
that the particle size varies from 6 to 13 nm with the average size about 9.3
nm corresponding to the size calculated from the X-ray diffraction method
(Figure 4.5) according to the Scherrer equation (D = K / (cos) 8 nm)
[161].
4.1.4. XRD diagrams and magnetic properties

-20

4

3

-40
30

40

50
o
2theta ( )

60

2

1

70

-60

0

H (Oe)

Figure 4.5: XRD diagram of Fe3O4 and
FA4D

-40

-20

0

Figure 4.6: Tính chất từ của các hệ hạt

Characteristic peaks of Fe3O4 crystals in FA4D samples are fully
present. These peaks have relatively low intensity compared to free Fe 3O4
due to the presence of organic components (alginate and dox) in the system.
Table 4.2: Magnetic parameters of alginate-coated samples

Sample

FA2

Ms
61.2
(emu/g)
Hc (Oe) 18
Mr
1.5
(emu/g)

FA4 FA6 FA8 FA10

FA4D

FA8D

69.5 65.3 65.8 63.9

51.6

33.9

Fe3O4 Fe3O4
[176] [177]
43
25

14
1.4

50
4.1

52
3.2

45
2

12
1.0

13
1.1

17
1.6

108

6

In figure 4.6, both magnetic remanance and coercivity of the samples
are approximately zero, demonstrating that the nanoparticle systems are
superparamagnetic. In addition, the Hc and Mr values of FA4D and FA8D
were significantly higher than that of non-Dox samples. This may have been
due to Dox presence that altered the magnetic anisotropy [186]. Saturation
magnetization decrease as alginate concentration increase (from FA4 to
12


FA10). The saturation magnetization of FA4 are the highest in this sample
range. The cause of this phenomenon is due to the alginate content, a nonmagnetic substance, increasing in samples. For FA2, the lowest saturation in
the range observed in this sample can be explained by the fact that at low
alginate concentrations this polymer does not fully cover the magnetic
particle surface, so that they can partially oxidize by the air during the
sample drying and becomes Fe2O3 which is lower in magnetization [164].
The saturation magnetization of the 2 Dox loading samples, FA4D and
FA8D, significantly decreased compared with the non-drug samples (51.6
and 33.9 emu/g respectively), indicating that Dox was present in the samples
with significant amount. The deeper reduction of the FA8D vs. FA4D is a
result of the greater Dox loading in this system. However, the values of the
saturation magnetization of FA4D and FA8D are large enough to be easily
and quickly separated from the reaction media by external magnetic fields.
4.1.5. Magnetic inductive heating effect
The magnetic inductive hyperthermia results of samples with different
concentrations of Fe3O4 particles (range from 0.5 to 3.0 mg ml-1 in term of
Fe3O4) of FA4 and FA4D and the same concentration (3.0 mg ml -1 in term of
Fe3O4) of FA8 and uncoated Fe3O4 in deionized water measured in the same
field conditions, namely of a frequency of 178 kHz and amplitude of 80 Oe

are shown on Table 4.3. The magnetic induction heating characteristics
observed for the material (figure 4.7 (a) and (b)) are concentration
dependences of saturation temperature Ts (estimated at heating time of t =
1500 s). It is found that upon decreasing of NPs concentration by adding
more and more water, both Ts and dT/dt of the sample decrease. All the
samples can reach the temperature up to 42°C and even higher for 20 min.
The temperature retention could prolong when the heating conditions were
held. Because cancer cells may be killed in the temperature range of 42–
46oC [73], we therefore note that the systems, both DOX loading
nanoparticles FA4D and FA4 is able to act as a good thermoseed for cancer
hyperthermia application. The heating characteristics of FA4 and FA4D
shows little change while the saturation magnetizations of the 2 samples are
so different (as shown in figure 4.6). This can be explained by the interaction
between the particles in the aqueous medium (as magnetic induction heated)
is altered to become solid form (for magnetization measurements). Thus,
13


FA4D can become a potential combination of chemotherapeutic and
hyperthermia cancer treatment.
Table 4.3: Magnetic induction heating of FA4, FA4D, FA8 and Fe3O4 samples

Sample

FA4

FA4D

FA8
Fe3O4

Conc.
(mg/ml)
0.5
1
2
3
0.5
1
2
3
3
3

T1500s
(oC)
49.7
52.1
62.1
68.4
48.1
50.5
59.2
65.1
62.1
60.1

dT/dt
0.03
0.03

0.04
0.06
0.02
0.03
0.04
0.05
0.05
0.07

SAR
(W/g)
225.7
129.6
85.7
85.0
150.5
112.9
73.2
66.9
64.1
103.1

ILP
(nHm2.kg-1)

11.6

9.2
8.8
14.1

4.1.6. Thermal analysis
Figure 4.9 shows the thermal analysis results of FA4 and FA4D. It can
be seen from the figure that both samples lose mass in the temperature range
of 80 to 550oC. Around 100oC, there is the same small mass loss (about 2%)
of water present in the 2 samples while the next steps of mass loss are
extremely different. FA4 shows two exothermal peaks but loses only 12% its
mass during the heating process. This can be explained by the decomposition
of alginate in the sample. On the other hand, FA4D shows only one peak in
Heat flow diagram at 380oC corresponding to a mass loss of 24%, that is
double to those of FA4. This mass loss step of FA4D also can be matched
with the disappearance of organic components in the sample and can support
for the formation of a complex between DOX and Alginate in the sample.
Figure 4.10 shows the general structure of Fe3O4 nanoparticles coated
with alginate with doxorubicin loading (FA4D or FAD) and folate attached
(FADF). In these systems, Dox is binded to the alginate shell by chemical
interaction

14


(a)

TG (/%)

479 C 581 C

-10

o

16
12
8

-15
-20

4

-25

0
100 200 300 400 500 600 700 800
Furnace temperature (/°C)

380 C

-5

16

-10

12

-15

TG% FA4D
Heatflow FA4D

-20
-25

(b)

20

8
4

HeatFlow (/µV)

o

o

0

HeatFlow (/µV)

-5

20

TG (/%)

TG% FA4
Heatflow FA4

0

0
100 200 300 400 500 600 700 800
Furnace temperature (/°C)

Figure 4.9: Thermal Analysis Diagrams of FA4 (a) and FA4D (b)

.
Figure 4.1: Structural models of FAD and FADF

4.1.7. In vitro drug release
The in vitro release process of DOX from the FA4D in neutral (pH 7.4)
and acidic (pH 5) medium (shown in figure 4.11) are both gradual release. In
the first 12 hours, the rate of drug release is maximal and reaches 21% and
29.5% at 12h, at pH 7.4 and pH 5 respectively. The drug release from
nanoparticles was slower at pH 7.4 than at pH 5.0. After 120 hours,
approximately 61% of the total drug was released in pH 5.0 conditions, in
comparison with a 42% release rate in pH 7.4 conditions. The DOX release
from the FA4D nanoparticles may be achieved by the degradation of alginate
layer through hydrolysis process. The hydrolysis process increases in acidic
solutions leading to a higher percentage of release at pH 5 compared to that
at neutral conditions. Because the environment in cancer tumors is acidic,
this indicates that the nano drug system is suitable for tumor treatment.
4.1.8. Cytotoxicity
Because DOX is highly toxic [140], the released amount of DOX is
enough to treat cancer cells, as indicated by low IC50 values of FA4D on the
15

cell lines (figure 4.12(b)). All the IC50 values are smaller than 5 g ml-1, and
much less than those of DOX loaded PLA-TPGS nanoparticles that we
reported before. Difference in cytotoxicity of FA4D and DOX loaded PLATPGS can be resulted from the synergic effect of Fe3O4 nanoparticles and
anticancer activity of DOX on the cell accumulation. Recently, sodium
alginate–polyvinyl alcohol–bovine serum albumin coated Fe3O4
nanoparticles were synthesized and used as DOX delivery system. However,
this complicate system exhibits toxicity on Hep-G2 cell lines only at high
range of DOX concentration (200-1000 g ml-1). This range is much larger
than DOX concentrations used in this study indicating that our optimized
drug delivery system show better anticancer activity in this cell line. In
addition, cytotoxicity of FA4D and free DOX on different cell lines were
compared. The IC50 values of FA4D are higher than those of free DOX can
be a result of the slow release process of DOX from the nanoparticles [147].
It was also reported that the impact of nanoparticles loaded with doxorubicin
on cell survival depended on just a certain extent of DOX concentration and
the main factor that affects the toxicity is the time. In another report, the IC 50
on some cancer cells of DOX loaded chitosan coated Fe3O4 also decrease
with time. The lower IC50 values of the Hep-G2, LU-1, RD and FL cell lines
compared to that of normal cells (Vero cell line) indicate that the
nanoparticles express higher toxic effect on cancer cells than healthy cells.
Therefore, the drug delivery systems suggest a safer chemotherapy for
cancer treatment in the way of decrease the toxicity for normal cells.
(a)
(b)
1.5

Hep-G2
LU-1
RD
FL

Vero

80
60
40

0

0.72

01
05
Concentration (g/ml)

25

0.60

0.6
0.39

0.0

0.2

1.20

1.41
1.30

0.96

0.9

0.3

20

FA4D
DOX

1.2

IC50 (g/ml)

Cell survival (%)

100

0.21
Hep-G2

0.11
LU-1

RD
Cell line

0.16
FL

Vero

Figure 4.12: Dose-response curve and comparisons of FA4D and free Dox IC50

4.2. Effect of microwave-assisted synthesized Fe3O4 core on system
properties
16


Microwave technology is used to synthesize Fe3O4 nanoparticles with
many advantages, most notably that this technique allows shortening of
reaction time and in large scale. In this section, we investigated the
microwave-assisted synthesis of Fe3O4 nanocore at different conditions with
microwave technique and compared the cell killing efficiency of
multifunctional systems with the microwave-assisted synthesized Fe3O4 core
with normal coprecipitated Fe3O4 particles. To assess induction magnetic
heating effect and interaction of the nanosystems with biological systems, we
fabricated FA and FAD samples with components and methods similar to
those of FA4 and FA4D from the Fe3O4 M5 particles fabricated by
microwave technique.
4.2.1. Material characteristics and magnetic inductive heating efffect
Both FA and FAD samples are highly stable, exhibiting a large zeta
potential value. The heating curve exhibits a similar trend compared to the
sample in the conventional co-precipitation conditions. Comparison of
saturation temperature (determined at 1500 s) of FA and FAD (table 4.6)
with corresponding results of FA4 and FA4D (Table 4.3) found no
significant difference in heating effect of microwave assisted synthesized
sample compared to conventional synthetic conditions.
4.2.2. Cytotoxicity

Comparison results of the IC50 values of microwave assisted
synthesized systems and conventional co-precipitate systems are presented in
Table 4.7 .
Table 4.7: IC50 of the microwave samples and conventional samples

Cell line HepG2
Dox1
0,21
2
Dox
0,18
FA4D
0,72
FAD
0,67
FADF
0,44
1
2

LU-1

RD

FL

Vero

HeLa

0,39
0,35
0,96
1,02
0,87

0,11
0,60
-

0,16
1,20
-

1,30
1,34
1,41
1,43
1,39

0,25
0,81
0,68

A control sample was used to determine the IC50 of FA4D
A control sample was used to determine the IC50 of FAD and FADF

The results in Table 4.7 show that the FAD affecting pattern on Hep-G2,
LU-1 and Vero cell lines was not significantly different from that of FA4D.
17

This shows that the use of microwave technique to fabricate Fe3O4 meets the
material requirement as well as interaction with the biological system. The
preservation of this material or biological interaction of FAD versus FA4D
may be due to the intrinsic nature of the microwave technique used is still
coprecipitation. The advantage of this technique is simple operation, and
short reaction time. Especially, this technique allows the synthesis of Fe 3O4
nanoparticles in large scale. Therefore, in subsequent studies to synthesize
Dox loading folate or quantum dot attaching nanosystem and in vivo test
specimens, we used Fe3O4 particles prepared by microwave technique.
4.3. Folate attached (FADF) or CdTe loaded (FADQ) NPs
4.3.1. IR spectra
Infrared spectra demonstrate the existence of folic acid in the FADF
system.
4.3.2. Flourescence spectra
The fluorescence spectrum of FADF versus folic acid has a clear shift in
the peak of emission (from 420 nm to 428 nm). The peak at 428 nm is far
shifted from the peak of Dox, suggesting that in the two fluorescents, folic
acid predominates in FADF samples. This result again confirms the presence
of folic acid in the system. On the other hand, while the fluorescence
spectrum of the FAD sample is much lower than that of Dox, the
fluorescence intensity of FADF does not change much compared to either
folic acid or pure dox. In the case of FADF, the fluorescence intensity of this
sample was slightly lower than that of folic acid, allowing FADF to be used
as a fluorescence probe to observe the interaction of the nanosystems with
biological systems.
50000

cuong do

420
428

30000

70000
60000
50000
40000
30000
20000
10000
0
450
20000

10000

10000

450

500

550

600

buoc song (nm)

650

0

700

b)

580 nm

FAQ 0.05 mg Fe3O4/ml
FAQ 0.1 mg Fe3O4/ml
FAQ 0.2 mg Fe3O4/ml
FAQ 0.4 mg Fe3O4/ml
FAQ 0.8 mg Fe3O4/ml

20000

0
400

a)

S1 (Counts)

40000

folic
FADF

FAD
DOX

450

500

550

FAQD 0.05 mg Fe3O4/ml
FAQD 0.1 mg Fe3O4/ml
FAQD 0.2 mg Fe3O4/ml
FAQD 0.4 mg Fe3O4/ml
FAQD 0.8 mg Fe3O4/ml

500

550

600

650

700

612 nm

600

Wavelength (nm)

650

700

Figure 4.17: Fluorescence spectra of FAD, FADF compared to folic acid and dox
(a) and samples containing CdTe quantum dots (b)

18


The FAQ and FADQ samples that contain CdTe have a fluorescent
emission at 580 nm of CdTe. In addition, FADQ samples consisting Dox
exhibit fluorescence at 612 nm, similar to FAD.
4.3.6. Passive and active Dox release by magnetic inductive heating effect
The passive Dox release profile at 37 ° C from FADF was performed
and gave the similar results as the FAD. Changing the pH of the solution
almost does not affect the FADF heating ability in the magnetic field. At just
the same temperature as the body temperature (about 37 oCm), Dox is
released from 4.7 to 11.2% from FAD or FADF. In magnetic field of 80 Oe,
nanoparticles generate more heat (or reach higher temperature) than in
magnetic field of 70 Oe. The release of Dox from the nanoparicles also
occurs faster, and the amount of Dox released larger. Unlike conventional
drug release, magnetic inductive heating effect makes the particles heated
from inside of the particle and hence accelerates the release of the drug.
Thus, the magnetic field can be adjusted to control the speed and amount of
drug release. Some other studies have shown that it is possible to release the
drug in an active way.
Table 4.9: Dox release profile when heated with different magnetic fields

Time
(s)
0
750
1500
2250
3000
750
1500
2250
3000

FAD pH 5
FAD pH7.4
t
% Dox
t
% Dox
o
o
( C) release ± ( C) release ±
SD
SD
30
0
30
0
70 Oe
39,27 11.2±1.3 40.23 6.0±0.7
43.08 28.8±0.8 44.52 25.3±1.3

45.32 49.5±0.9 46.05 37.9±1.8
45.87 56.3±1.5 46.81 41.5±1.2
80 Oe
46.13 30.1±1.5 45.56 21.5±0.5
51.25 53.5±0.6 50.78 38.8±2.1
51.98 67.6±0.9 51.43 49.4±1.7
52.16 78.1±1.6 51.83 56.4±1.2

FADF pH 5
FADF pH 7.4
t
% Dox
t
% Dox
o
o
( C) release ± ( C) release ±
SD
SD
30
0
30
0
37.63
42.35
43.68
44.30

10.9±0.4
29.1±0.6

46.7±1.7
51.6±1.0

38.11
43.48
44.99
45.71

4.7±1.0
24.4±0.7
37.2±1.3
39.4±2.0

45.87
50.19
51.16
51.24

28.2±2.0
48.0±1.4
62.7±0.9
74.2±1.8

46.22
51.74
52.10
52.31

20.6±0.9
35.4±0.9

45.1±1.7
51.0±1.5

As a result, both FADF and FADQ have suitable material properties for
use in biological objects.
4.3.7. Cytotoxicity
19


4.3.7.1. Cytotoxicity of FADF
Compared to pure Dox, FADF is toxic on four Hep-G2, LU-1, Vero, and
HeLa-lower cell lines (larger IC50). The reason can be that Dox was
chemically binded to the surface of the nanoparticles, which slows down the
action of the Dox on the cell. Compared to FAD, FADF exhibited higher
toxicity due to the fact that in this sample, the folate factor helped the sample
to better target the cell. However, both FAD and FADF with low IC 50 (less
than 2 μg/ml) showed good chemotherapy for the studied cell lines.
1.4

CdTe
FAQ
FAQD

4.0
3.5

1.0
0.8
0.6
0.4

0.2
0.0

IC50>5 ug/ml

4.5

IC50 (g/ml)

IC50 (gml)

1.2

DOX
FAD
FADF

3.0
2.5
2.0
1.5
1.0

Hep-G2

LU-1
Vero
Dong te bao

HeLa

Figure 4.24: Cytotoxicity of Dox
loading systems

0.5
0.0

Hep-G2

LU-1

RD

Vero

Figure 4.26: Cytotoxicity of CdTe loading
systems

4.3.7.2. Cytotoxicity of CdTe loaded NPs
For each cell line, the IC50 value of the FAQ is slightly higher than
CdTe, meaning that when CdTe is attached to the Fe3O4 nanoparticles by the
alginate polymer matrix, the CdTe toxicity decreases (Figure 4.26). The
sample consisting of CdTe and Dox concurrently (FADQ) was the most
effective in cancer treatment (IC50 values for Hep-G2, LU-1, RD and Vero
were 1.34, 3.83, 1.35 and 2.13 μg/ml, respectively) thanks to the
combination of cancer drug Dox and CdTe quantum dots. As such, FAD,
FADF, and FADQ can perform a variety of functions such as hyperthermia,
fluorescence or chemotherapy. FADQ demonstrates good cell killing ability
and at the same time shows high toxicity to experimental animals (white
mice) bearing tumors (dead or very weak rats after injection of 50 l/mouse).

Therefore, only FA, FAD and FADF were further in vivo investigated on
mice.
4.3.8. Stability of FAD, FADF and FADQ in biophysical environment

20


FAD, FADF and FADQ were determined the stability in a solution
containing 0.2 M salts and different pH values by Zeta potential
measurement. The results are shown in Table 4.12.
Table 4.12: The Zeta potential (mV) of FAD, FADF and FADQ in solution of NaCl 0.2
M and different pH values

pH
FAD
FADF
FADQ

3
-19.2 ± 1.9
-18.5 ± 2.1
-9.4 ± 2.1

5
-32.0 ± 2.6
-30.4 ± 1.7
- 13.8 ± 2.3

7
-33.2 ± 1.5

-32.2 ± 2.9
-15.6 ± 1.8

9
-43.1 ±1.2
-39.0 ± 2.5
-19.4± 1.4

The results in Table 4.12 show that Zeta potential of Dox loading
alginate coated Fe3O4 samples were negative, even at pH 3. This can be
explained by the fact that the alginate shell of the magnetic particles is rich in
COO- groups. Similar results concerning alginate coated nanoparticles were
also previously published [194]. The Zeta potential of FADF is not
significantly different from that of FAD. In the pH range of 5 to 9, FAD and
FADF have good stability (with Zeta potential over 30 mV). The FADQ is
less stable than FAD and FADF.
4.3.9. In vivo evaluation
4.3.9.2. Distribution of Fe on mice’ organs
Quantification of Fe in mice’ tissues and organs revealed that the levels
of Fe in the tissues and in particular in the tumors (except in the blood) of the
FADF mice were higher than those of the control group. However,
statistically significant differences were not observed. Comparison of Fe
content from the FAD group compared with the control group tended to be
similar (p = 0.08).
4.3.9.3. In vivo treatment results by nanosystems associated with magnetic
induction heating
During treatment, the weight of the mice groups was not significantly
different at each measurement point.
The mean tumor size at the time of group deviding was not
significantly different between groups. After 3 treatment cycles, the tumor

size of FADF group has been shown to be undeveloped. The size of the
tumor tends to go flatly. This group had significantly smaller tumors than the
other 3 groups after 4 cycles, corresponding to the fifth measurement point
21


(p <0.05). At the eighth measurement, almost all tumors in the FADF mice
were reduced as tumor size of the first measurement point. The FA tumor
size was significantly lower than the control group at the time of the sixth
measurement onwards (p <0.05). The FAD-treated group had a smaller
tumor size than the other two treatment groups (FA and FADF). At the
eighth measurement, the mean tumor size was significantly smaller than the
control group. Tumors in untreated mice were very large, affecting the mice
'movement. The treated mice, especially those treated with FADF, had a
gradual decrease in tumor size, some of the rats lost tumors at the end of the
experiment. This result shows the encouraging effect of the heat treatment
method. With non-drug FA samples, thermal effects contribute to the reduce
of cancer cells.
Magnetic heating

Figure 4.31: Changes in tumor size during in vivo treatment

As noted in Section 4.2.1, the FA sample has a better thermal effect than
FAD. This may be the reason for the fact that mice treated with the FA
reduced tumors faster than treated with FAD. Thus, in FA or FAD samples,
ferromagnetic iron oxide nanoparticles exhibit the role of heat generation
that kill cancer cells. Comparison of the measured size tumor at the 7th and
8th measurements also indicates the superiority of the thermal therapy
combined with chemotherapy in the FAD. After 8 treatments, tumor size in
FAD mice was significantly reduced. It has been shown that combination can

produce synergistic effects that increase the therapeutic effect of both
chemotherapy and thermal therapy [195]. The mechanism of this synergistic
effect is that under the heat effect, cancer cells become more sensitive to the
toxicity of chemotherapy drugs (increased absorption into cells, inhibition of
DNA repair, and enhancement of toxic reactions). At the same time, this
combination increases the level of release and drug accumulation in the
tumor [77]. The results of active release in Section 4.3.6 in this thesis also
22


confirm the explanation. This combination regimen may also reduce tumor
recurrence after treatment [195]. On the other hand, although the heat
generated in the magnetic field is lower than the FA, the FADF gives the
best performance compared to the other two systems, possibly due to the role
of the folate present in the system. Folate is capable of binding closely to
folate receptors on cancer cells [196, 150], so it is possible to retain the
FADF nano system and increase the magnetic concentration at the tumor,
thereby increasing the in vivo therapeutic effect and at the same time
promoting the chemo effect of Dox released from the system. Some studies
have also shown that folate incorporation on magnetic particles allows
effective targeting, decreases toxicity, and improves therapeutic efficacy in
in vivo models [197].
CONCLUSION
In this thesis, we have implemented the following contents:
1. Synthesis of Fe3O4 magnetic nanoparticles by co-precipitation and coprecipitation using microwave technique. Optimal microwave conditions are:
temperature of 70oC, microwave time of 15 minutes with stirring speed of
600 rpm.
2. Synthesis of OCMCS coated curcumin loading Fe3O4 nanoparticles
synthesized from co-precipitation Fe3O4. The curcumin content was
investigated and the optimum ratio of curcumin was determined to be 60 mg

for 50 ml of Fe3O4/OCMCS solution.
3. FOC and FOCF have a high absorption capacity of curcumin: 0.95 and
0.54 mg/mg Fe3O4. In addition, these systems have the ability to generate
heat, allowing to kill cancer cells by heat. In particular, the folate attached
FOCF system demonstrated the ability to target the Sarcoma 180 tumor well,
allowing for improved efficacy of the drug delivery system.
4. Synthesis of Dox nanoparticles from conventional co-precipitated and
microwave-assistanted synthesized Fe3O4 nanoparticles. The optimized
alginate coating was 4 mg/ml in the FA4D sample. This system is 78.5% in
Dox encapsulating effeciency, 51.6 emu/g in saturation magnetization.
FA4D solution with concentrations ranging from 0.5-3 mg/ml can reached
the temperature of kill cancer cells (42oC) after 20 minutes of heating with
23