Summary of chemistry doctoral thesis: Study on the fabrication of magnetic fluids based on superparamagnetic iron oxide nanoparticles (SPIONs) applied to magentic resonance imaging (MRI)

The goad of the thesis is to build the manufacture process of nano-sized magnetic fluids based on iron oxide (uniform particle size and high magnetic saturation) with stable technology; Characteristic research of magnetic properties of magnetic nanoparticles; assessment of toxicity and test of effects on cells, aiming to make contrast medicine in imaging diagnosis by magnetic resonance imaging (MRI), application on accurately identifying cancer.

MINISTRY OF EDUCATION AND TRAINING

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

GRADATE UNIVERSIY OF SCIENCE AND TECHNOLOGY

This thesis was done at:

Laboratory of Biomedical Nanomaterials, Institute of Materials and Sciene, Vietnam Academy of Science and Technology

Laboratory of Electronic-Electrical Engineering, Institute for tropical technology, Vietnam Academy of Science and Technology

Centre for Pratices and Experimences, Vinh University

Supervisor: Prof., Dr Tran Dai Lam Assoc.Prof., Dr Nguyen Hoa Du

INTRODUCTION

Recent applications of magnetic nanoparticles in biomedical applications, especially in imaging diagnostics using MRI Magnetic Resonance Imaging engineering have attracted the attention of scientists around the world Currently in imaging diagnostics using MRI magnetic resonance imaging, Tl contrast agents have become a traditional commodity, which is a complex of paramagnetic ions with a large torque value like Gd3+ (7 unpaired electrons) These Gd3+ ions are combined with molecules such as DTPA (diethylentriamine penta acetic acid) and create Gd-DTPA chelate round complex structures During the recovery process, the interaction between the magnetic moment of the proton and the magnetic moment of the paramagnetic ions causes the T1 time to be reduced, so the recovery rate R1 increases The concentration of agents is different in each cell tissue region, thus providing an effective contrasting on MRI images For nearly 10 years now, along with the development of nanotechnology iron oxide (IO) nanoparticle having been strongly researched and actual many commercial products that increase MRI contrast levels using this iron oxide material, proving that iron oxides-MRI can give better quality of contrast level than Gd-DTPA because iron oxide particles have a higher magnetic induction coefficient IO-MRI substances can reduce both T1 and T2, increasing MRI recovery rates in both Tl and T2 MRI modes The important requirements for MRI contrast increasing products are that magnetic nanoparticles must have a relatively uniform particle distribution and magnetic saturation enough large, and the coating materials must have good biological compatibility While some commercial products in the world, such as Resovist, use dextran as a coating material, with a 65 nm core particle created from saturation of about 65 emu/g Products with particle sizes in the 20-40nm region such as AMI-227: Sinerem/Combidex are suitable for lymph and bone In the last 10 years, people have been studying to create superparamagnetic nanoparticles with a particle size smaller than 20 nm (also known as microscopic if the particle size is D<10 nm) and especially iron oxide particles, marked with magnetic markers is intended for MRI targeted imaging

In Vietnam, up to now the fabrication of nanoparticles in general and magnetic nanoparticles in particular has been focused to research in accordance with two aspects: basic research and application-orientation research The profound research results are published mainly from large research institutions such as: Hanoi National University, Hanoi University

of Science and Technology and Vietnam Academy of Science and Technology The synthesis

of magnetic nanomaterials is mostly carried out in water and by synthetic methods such as co-precipitation methods, hydrothermal methods, microwave methods and ultrasonic electrochemical synthesis methods Due to the synthesis in the water environment, the fabricated magnetic nanoparticles have not high quality, the particles are uneven in size and heterogeneous in shape and therefore they are restricted for use in vivo printing applications

in biomedicine such as used as a contrast drug in imaging diagnosis by MRI magnetic resonance imaging, magnetic induction heating, etc in addition, the such unevenness even

affects the research results of their magnetic properties Therefore, up to now, the selection

of conditions in the fabrication of Fe3O4 nano magnetic fluid to produce particles with small particle size, uniform distribution, homogeneous shape and high durability, high magnetism and high biocompatibility, thus making it possible to apply as contrast medicine in imaging diagnostics using MRI magnetic resonance imaging, creating optimal values of impulses TR,

TE when taking with T1, T2 mode, and determining recovery coefficient r1, r2 to assess the quality of magnetic fluids as contrast medicine in imaging diagnosis by MRI magnetic resonance imaging is still asking for continuing and systematic research

Derived from the research on nanomaterials in the world as well as in Vietnam, based

on the research and Doctor training potential of the Institute of Science and Technology, Vietnam Academy of Science and Technology, under the guidance of a group of experienced

scientists, we select the topic "Study on the fabrication of magnetic fluids based on

superparamagnetic iron oxide nanoparticles (SPIONs) applied to magentic resonance imaging (MRI) application" to make this thesis content

Research object of the thesis:

Magnetic fluid system based on superparamagnetic iron oxide

Research targets of the thesis:

The goad of the thesis is to build the manufacture process of nano-sized magnetic fluids based on iron oxide (uniform particle size and high magnetic saturation) with stable technology; Characteristic research of magnetic properties of magnetic nanoparticles; assessment of toxicity and test of effects on cells, aiming to make contrast medicine in imaging diagnosis by magnetic resonance imaging (MRI), application on accurately identifying cancer

Scientific and practical meaning of the thesis:

The implementation organization of the topic itself has important implications for developing a multi-disciplinary science and technology direction as Nanotechnology for Medicine There will be academic exchange, mutual learning between research groups in the industries deem as independent Scientifically, the magnetization of magnetic particle systems for biomedical applications is strongly influenced by many factors, but the mechanism of these effects is still a problem that has not been studied fundamentally

For the application of cancer diagnosis and treatment, nanotechnology in general is creating a great expectation that is able to contribute to solve the problem of early disease diagnosis and drugs to target or intervention areas localized at the destination The subject has

a goal of using magnetic fluid improving the contrast of nuclear magnetic resonance imaging (MRI), which can contribute to the analysis of early-stage cancer tissue

Research methodology:

The thesis is conducted by experimental method combined with numerical calculation techniques The research sample is fabricated by hydrothermal and thermal decomposition methods Study the structure of the sample by X-ray diffraction techniques (XRD), electron microscopy (FESEM, TEM and HRTEM) The magnetic properties of the materials are surveyed by magnetic measurements on the vibrating sample magnetometer (VSM) system Using Fourrier Transformation InfraRed (FTIR), Thermal gravimetric analysis (TGA) to evaluate the presence of functional groups on the particle surface and the mass reduction of polymer-coated magnetic particle layer Dynamic Light Scattering (DLS) technique determines the hydrodynamic size and durability of magnetic fluids Experimental assessment

of toxicity through in-vitro test MRI imaging method T1, T2 for studies of contrast enhancement of material samples for manufacturing (on 1.5T MRI scanner, SIEMENS MAGNETOM, Germany)

Research contents of the thesis:

1 Successful summary of Fe3O4 magnetic nanoparticles with uniform particle size and high saturation magnetization by hydrothermal and thermal decomposition methods

2 Successfully fabrication of high-strength magnetic fluids on Fe3O4 particles synthesized by the above two methods

3 Research on the toxicity and durability of magnetic fluids

4 Study the applicability of image contrast enhancer in MRI magnetic resonance imaging

The layout of the thesis:

The thesis has 137 pages (not including references, appendices), including the introduction, 5 chapters of content and conclusions

The main results of the thesis are published in 09 published projects, including 01 article published under SCI list, 01 article sent from SCI list submitted and reviewing, 05 articles on National magazine, 01 article published in the Proceedings of the National Science Conference, and registered 01 intellectual property (SC) has been published in the volume A Industrial Ownership Gazette

Main results of the thesis:

The influence and optimization of technological conditions on the structure and magnetic properties of chitosan-coated Fe3O4 nanoparticles (CS) were investigated using hydrothermal method

Successfully fabricated magnetic fluids based on Fe3O4 particles by thermal decomposition method by phase transformation and coating by polymer PMAO

Fe3O4@PMAO liquid samples are highly durable in different conditions, single-dispersed, uniform particles

Evaluation of the toxicity of magnetic fluids on typical samples with different cell lines,

results are good IC50 index Manufactured fluid Samples are not cytotoxic, which is the basis for conducting subsequent experiments on animals

Determined the relaxation rate of nuclear magnetic resonance imaging (MRI) of 2 systems Fe3O4@CS, Fe3O4@PMAO showed that the uniform fabrication systems have high

r2 values of over 150 mM-1s-1 for samples of Fe3O4@PMAO, higher than commercial products Resovist These substances, when given MRI imaging tests, show good potential for applications to increase contrast

In vitro, ex-vivo and in-vivo studies of MRI contrast enhancement showed that many of

the magnetic fluids of the manufacturing subject group exhibited good contrast enhancement Applying the Fe3O4@PMAO system to solid tumors under the skin and liver tumors, shows the potential for observing the detailed shape and structure of the tumor, supporting diagnosis and treatment

CHAPTER 2 REVIEW OF SPINEL FERRITE MATERIAL AND MAGNETIC RESONANCE IMAGING METHOD BY MRI SHOOTING ENGINEERING 1.1 The structure and magnetic properties of spinel ferrite materials

1.1.1 Structure of spinel ferrite

Ferrite spinel is the term used to refer to a material with a two-subnetwork structure of which interactions are antiferromagnetic or magnetic ferrite A basic cell unit of spinel ferrite (with crystal lattice constant a ~ 8.4 nm) is formed by 32 atoms O2- and 24 cation (Fe2+, Zn2+,

Co2+, Mn2+, Ni2+, Mg2+, Fe3+ và Gd3+) In a base cell there are 96 positions for cations (64 in octahedral position, 32 in tetrahedral position) The number of cations is more octahedral in the tetrahedral position (A), in particular there are 16 cations occupied in the octahedral position (B) while in the tetrahedral position there are only 8 cations (including valency cation

2+ or 3+)

1.1.2 Magnetic properties of spinel ferrite materials

According to molecular field theory, the magnetic origin in spinel ferrite materials is due to the indirect exchange interaction between metal ions (magnetic ions) in two subnetworks A and B through oxygen ions

1.1.3 Magnetism of nanometer-sized particle magnetic materials

The superparamagnetic phenomenon (or status) occurs for ferromagnetic materials composed of small crystalline particles When the particle size is large, the system will be in the multidomain state (i.e each particle will be composed of many magnedomain particles) When the particle size decreases, the substance will turn into a mono-state, which means that each particle will be a dime When the particle size decreases too small, the directional energy (which predominantly dominates are that the crystal magnetic anisotropic energy is much smaller than the thermal energy, then the thermal energy will break the parallel orientation of magnetic moments) and then the magnetic moment of the particle system will orient chaotically as in paramagnetic material

1.2 The studying situation of nanomaterials in the domestic and abroad

In Vietnam, a number of research groups at the Institute of Materials Science, International Training Institute for Materials Science - Hanoi University of Science and Technology and Hanoi National University (VNU) have also announced their manufacture of magnetic nanoparticles for biomedical applications and for basic research

1.3 Manufacture methods of magnetic fluids

1.3.1 Synthesizing methods of magnetic nanomaterials

For biomedical applications, the material is often made by a number of chemical methods such as co-precipitation, solgel, microemulsion, hydrothermal, thermal decomposition, microstorage, etc Chemical methods can create nanoparticles with a quite high uniformity and facilitate to be able to coat particles and transfer phase of the particles from oil to water Each of the above methods has different characteristics

1.3.2 Particle coating technologies in water solvent

For nanoparticles synthesized by chemical methods in water solvents, the coating of the particles or the functionalization of the nanoparticle surface after fabrication is a very important factor to ensure both magnetic properties and biological compatibility When the surface is coated and functionalized, the nanoparticles easily disperse in a suitable solvent and become homogeneous colloidal particles called magnetic fluids

1.3.3 The process to transfer the phase from organic solvents to water solvents

In order to obtain high-quality magnetic nanoparticles, sample fabrication is usually carried out in organic solvents at high boiling temperatures such as: benzyl ther, phenyl ether, octadecene Therefore, before being able to be used in Biomedical, these magnetic nanoparticles need to be transferred from organic solvents to water solvents through phase transfer processes

1.4 Application of magnetic nanoparticle systems in biomedical

Magnetic nanoparticles have the potential to be applied in many different fields In biomedical, magnetic nanoparticles can be used to extract biological molecules using magnetism, nano curcumin, and substances increasing contrast in magnetic resonance imaging (MRI) and magnetic burning application for cancer treatment However, this thesis focuses on researching magnetic nanoparticle application orientation Fe3O4 to enhance nuclear magnetic resonance imaging (MRI) affect

CHAPTER 2 EXPERIMENTAL ENGINEERING 2.1 Summary of magnetic fluid system Fe 3 O 4 @CS by hydrothermal method

Magnetic nanoparticles Fe3O4@CS is synthesized by hydrothermal method according

to Figure 2.1 diagram

Figure 2.1 Fabrication process of magnetic fluids Fe 3 O 4 @CS

2.2 Summary of nanoparticle system Fe 3 O 4 @OA/OLA by thermal decomposition method

Nanoparticle system Fe3O4@OA/OLAis synthesized by thermal decomposition method according to Figure 2.2 diagram

Figure 2.2 Fabrication process of magnetic nanoparticles Fe 3 O 4 @OA/OLA

2.3 Transfer the phase of nanoparticles slowly from organic solvents to water solvents

The phase transfer process of magnetic nanoparticles from organic solvent to water is carried out according to the diagram of Figure 2.3

2.4 Experimental planning method

In experimental chemical and chemical technology studies, there are many experimental problems described as extreme problems: determining the optimal conditions of the process, the optimal composition of the mixture Experimental planning allows to simultaneously change all the factors that affect the process and allow quantitative evaluation of basic effects and simultaneous interaction effects of the elements, thereby optimizing chemical technologies

2.5 Evaluate the toxicity of fluids from cancer cells

Evaluate potentially lethal the cancer cells and healthy cells and intact cells of fabricated magnetic fluid

2.6 Testing the ability to contrast agent in MRI imaging techniques

MRI imaging experiment in T1, T2 is used for the researches of image contrast enhancement of images of manufacturing materials (on 1.5T MRI scanner, SIEMENS MAGNETOM, Germany)

CHAPTER 3 RESEARCH MAGNETIC FLUID BASED ON MAGNETIC IRON

OXIDE SYTHETISED BY HYDROTHERMAL METHOD 3.1 Implement the optimal three-level quadratic experimental planning

Table 3.1 Levels of independent variables and experimental conditions

Run Variable levels Temperature

Time (h)

Concentration

Fe 3+ (M)

Ms (emu/g)

Figure 3.3 Surface plot and contour plot of the combined effects of A and B (a); A and C

(b) on the yield of Ms at another coded level of zero

The suitability analysis of the model and the significance of the model assessed by ANOVA analysis (Figure 3.2) and correlation indicators The significance of the regression coefficients is tested by standard F, with values p<0.05 indicating significant regression coefficients Thus, in Figure 3.2 found that the value of "Model F-value" is 35.75, the model

is completely statistically significant with 99.08% reliability With all the factors of sample incubation temperature, time, Fe3+ concentration and each pair of these factors have a value

of p<0.05, indicating that each of these factors also interact with each other and are meaningful (Figure 3.2), this is illustrated more clearly when observing the response surface

in Figure 3.3

Figure 3.4 describes the fit line according to the Langevin function of the samples at optimal conditions M11 - M13 at the magnetism at the magnetism 10 kOe Throught this figure, the experimental data on the M(H) base line of all samples measured at 300K temperature closely matched according to Langevin function with high accuracy (R2> 0.998)

Figure 3.4 Experimental and fitting hysteresis curves of the Fe 3 O 4 @CS nanoparticles (inset

is the enlarged hysteresis curve)

3.2 Structural and morphological characteristics of magnetic nanoparticles Fe 3 O 4 @CS

(a) (b) (c)

Figure 3.5 X-ray diffractions (a) and và FTIR spectrum (b) and TGA analysis (c) of Fe 3 O 4 ,

CS and Fe 3 O 4 @CS samples

Figure 3.5a shows all the diffraction lines of the samples coincide with the standard lines

of Fe3O4 with spinel structure The combination with Chitosan makes the pics of the Fe3O4@CS nano sample more noise than the pics of pure Fe3O4 samples but does not change the crystal structure Figure 3.5b (a) shows that, on the infrared spectrum of the sample Fe3O4@CS, there are characteristic oscillations related to functional groups of Fe3O4 particles and CS cover This proves that Fe3O4 nanoparticles were covered by CS

On TEM image (Figure 3.6), the spherical shaped particles with small size from 12-18 nm and relatively uniform, the particles after covering Fe3O4@CS are larger than those of Fe3O4

before covering However, the particles when dispersed in water have not been dispersed simple shape, the groups have shinking phenomenon with the organic chitosan shell about 21.5% (Figure 3.5c)

Figure 3.6 TEM images of the magnetic fluids Fe 3 O 4 @CS prepared by optimization

3.3 Characteristics of Fe 3 O 4 @CS magnetic fluid system durability

4.5, 7.4, 11.5 and 12 and b) different NaCl concentrations of 0, 50, 100, 200, and 300 mM

By determining the strength of a sample of Fe3O4@CS fluid in a physiological field, the fluid sample has a high durability in a wide pH range, long time and high strength when the salt content is up to 300 mM This shows that the samples of Fe3O4@CS fluid have suitable properties in biomedical conditions

3.4 Test and evaluation of the toxicity of chitosan-coated Fe 3 O 4 magnetic fluid system

Figure 3.8 Sarcoma 180 cell’s viability after incubating with different concentration of

Fe 3 O 4 @CS MNPs after 48h: (C1): 500 µg/ml, (C2): 250 µg/ml, (C3): 125 µg/ml, (C4): 62,5 µg/ml, (C5): 30,25 µg/ml and (C6): 15,125 µg/ml All presented values were expressed as mean±standard deviation (SD) (a) and Observation of Sar.180 cells morphology under the different concentrations of Fe 3 O 4 @CS using inverted microscope C A : control with culture

medium C B : control with DMSO Objective lens: 20X, zoom: 5.6

fluids are not toxic to Sarcoma 180 cells with 83% - 106% of survival cells after 48 hours of incubation with nanomagnetic fluids with concentrations of 15.125 µg/ml and 30.25 µg/ml When increasing the concentration of nanoparticles up to 62.5 µg/ml, 125 µg/ml, 250 and 500 µg/ml, the rate of survival cells decreased respectively to 66.1%, 38.4%, 17.8% and 2.8135%

when the increase in magnetic fluid concentration leads the reduction of cell growth From the in vitro test results and comparing with recent publications, it can be concluded that

cancer cell line

CHAPTER 4: STUDYING THE MAGNETIC FLUID SYSTEM BASED ON IRON OXITE SYNTHETIZED BY THE THERMAL DECOMPOSITION METHOD 4.1 Effect of solvent and temperature on properties of Fe 3 O 4 particle

Figure 4.1 TEM images of Fe 3 O 4 synthesized at different reactions solvent and temperature

of 1 hours

By surveying the effect of temperature, it was found that the sample was made at a temperature lower than the solvent temperature for particles of small, uneven, and grainy size Dibenzyl ether solvents give heterogeneous particles in shape, uneven size compared to octadecene solvents Particles of uniform size with grain boundaries are more clear when the reaction temperature is increased to 300 to 310 oC and 320 oC This indicates that the temperature and nature of the solvents are important factors in the formation and development

of particles

Figure 4.1 The M(H) curves of Fe 3 O 4 synthesized at different reactions solvent (inset is the

enlarged hysteresis curve)

The value from Ms saturation increased from 51 emu/g DIO1) to 59 emu/g DIO2) and 62 emu/g (OIO-DIO3) when changing the reaction temperature from 270oC đến

(OIO-310oC (Figure 4.1c)

4.2 Effect of reaction time on magnetic structure and properties