Charles University, Faculty of Science
Department of Physical and Macromolecular Chemistry Univerzita Karlova, Přírodovědecká fakulta Katedra fyzikální a makromolekulární chemie
Doctoral study program: Macromolecular chemistry Doktorský studijní program: Makromolekulární chemie
Summary of the PhD. thesis Autoreferát disertační práce
Preparation of uniform superparamagnetic particles with polymer coating for biomedical applications
Příprava uniformních superparamagnetických částic s polymerním povlakem pro biomedicínské aplikace
Mgr. Vitalii Patsula
Supervisor/Školitel:
Ing. Daniel Horák, CSc.
Institute of Macromolecular Chemistry, AS CR Ústav Makromolekulární Chemie, AV ČR, v.v.i.
Prague / Praha 2018
2 Abstract
Aim of this thesis was to design and prepare polymer-coated monodisperse Fe3O4
nanoparticles as a safe and non-toxic contrast agent for magnetic resonance imaging (MRI) and heat mediator for hyperthermia. Uniform superparamagnetic Fe3O4 nanoparticles were synthesized by thermal decomposition of Fe(III) oleate, mandelate, or glucuronate in high- boiling solvents at temperature >285 °C. Size of the particles was controlled in the range of 8- 27 nm by changing reaction parameters, i.e., temperature, type of iron precursor, and concentration of stabilizer (oleic acid and/or oleylamine), while preserving uniformity of the nanoparticles. Because particles contained hydrophobic stabilizer on the surface, they were dispersible only in organic solvents. To ensure water dispersibility, oleic acid on the particle surface was replaced by hydrophilic and biocompatible methoxy-poly(ethylene glycol) (PEG) and poly(3-O-methacryloyl-α-D-glucopyranose) by ligand exchange. Polymers were previously terminated with anchoring-end groups (hydroxamic or phosphonic) to provide firm bonding to iron atoms on the particle surface. Fe3O4 nanoparticles were also hydrophilized by encapsulation into a silica shell by reverse microemulsion method. Tetramethyl orthosilicate was used to prepare Fe3O4@SiO2 nanoparticles, which were further functionalized with amino groups using (3-aminopropyl)triethoxysilane. Finally, the amino groups were used to introduce PEG on the particle surface to ensure colloidal stability in physiological medium.
Magnetic Fe3O4 nanoparticles were characterized by a variety of techniques, including dynamic light scattering and transmission electron microscopy to determine hydrodynamic size and morphology, respectively. X-ray powder diffraction and vibrating sample magnetometry were used to investigate particle composition and magnetic properties. The content of iron, iron oxide, and presence of polymer shell were determined by atomic absorption spectroscopy, thermogravimetric analysis, and attenuated total reflectance Fourier- transform infrared spectroscopy (ATR FTIR), respectively. Chemical structure of the modified polymers was confirmed by 1H and 31P NMR, and ATR FTIR spectroscopies.
Toxicity of newly developed polymer-coated nanoparticles was tested in vitro on selected cell lines; non-toxicity of the particles was confirmed. Relaxivity measurements showed good imaging properties of particles compared to commercially available agents.
Moreover, calorimetrically measured specific absorption rate of the particles revealed their potential applicability as a heat mediator for hyperthermia treatment.
Keywords: polymer; superparamagnetic; monodisperse; nanoparticle; iron oxide; toxicity.
3 Abstrakt
Cílem této práce bylo navrhnout a připravit polymerem pokryté monodisperzní Fe3O4
nanočástice jako bezpečné a netoxické kontrastní činidlo pro magnetické rezonanční zobrazení a mediátor pro hypertermii. Uniformní superparamagnetické Fe3O4 nanočástice byly syntetizovány teplotním rozkladem oleátu, mandelátu a glukuronátu železitého ve vysokovroucích rozpouštědlech při teplotách >285 °C. Velikost částic byla regulována v rozmezí 8–27 nm změnou reakčních parametrů, např. teplotou, typem organického prekurzoru a koncentrací stabilizátoru (olejové kyseliny a/nebo oleylaminu) tak, aby byla zachována uniformita nanočástic. Částice připravené teplotním rozkladem obsahovaly hydrofobní stabilizátor a byly proto dispergovatelné pouze v organických rozpouštědlech.
Aby byly dispergovatelné ve vodě, byla olejová kyselina na poverchu částic nahrazena hydrofilním a biokompatibilním methoxy-poly(ethylenglykolem) (PEG) and poly(3-O- methakryloyl-α-D-glukopyranosou) pomocí metody výměny ligandů. Navázání obou polymerů k atomům železa na povrchu nanočástic bylo dosaženo díky vhodným koncovým skupinám (hydroxamovým nebo fosfonovým). Fe3O4 nanočástice byly také hydrofilizovány enkapsulací do siliky, hydrolýzou tetramethyl ortoxysilikátu, tzv. reverzní mikroemulzní metodou. Takto připravené Fe3O4@SiO2 nanočástice byly dále funkcionalizovány aminovými skupinami pomocí (3-aminopropyl)triethoxysilanu. Na tyto skupiny byl poté navázán PEG, aby byla zajištěna koloidní stabilita ve fyziologickém mediu.
Hydrodynamická velikost a morfologie Fe3O4 nanočástic byla stanovena různými technikami zahrnujícími dynamický rozptyl světla a transmisní elektronovou mikroskopii.
Složení a magnetické vlastnosti částic byly zkoumány rentgenovou práškovou difrakcí a vibračním magnetometrem. Obsah železa či jeho oxidů a přítomnost polymerního povlaku byla detekována atomovou absorpční spektroskopií, termogravimetrickou analýzou a infračervenou spektroskopií s Fourierovou transformací se zeslabeným úplným odrazem (ATR FTIR). Chemická struktura modifikovaných polymerů byla potvrzena 1H NMR, 31P NMR, a ATR FTIR spektroskopií.
Toxicita nově vyvinutých polymerem pokrytých nanočástic byla testována in vitro na vybraných buněčných liniích a bylo potvrzeno, že částice jsou netoxické. Měřením relaxivity byl demonstrován dobrý kontrast pro magnetické rezonanční zobrazení ve srovnání s komerčně dostupnými činidly. Kalorimetricky stanovený specifický absorpční výkon částic rovněž ukázal na jejich možné použití jako mediátoru v hypertermii.
Klíčová slova: polymer; superparamagnetický; monodisperzní; nanočástice; oxid železa;
toxicita.
4 Abbreviations
AAS ACVA ATR FTIR BA b.p.
CPTA Dh
Dn
Fe3O4@SiO2
Fe3O4@SiO2-PEG Fe3O4@PEG-HA Fe3O4@PEG-PA Fe3O4@PMG-P IS
MS
MRI mNSCs MSCs OA OD OLA PEG PEG-HA PEG-PA PMDG PMDG-NH2
PMDG-PEt PMG PMG-P SAR SiO2
SQ TEM TGA TMOS TOA Ð
Atomic absorption spectroscopy 4,4’-Azobis(4-cyanovaleric acid)
Attenuated total reflectance Fourier-transform infrared spectroscopy Butylamine
Boiling point
4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid Hydrodynamic diameter
Number-average particle diameter Silica-coated Fe3O4
PEG-functionalized silica-coated Fe3O4 PEG-HA-coated Fe3O4
PEG-PA-coated Fe3O4 PMG-P-coated Fe3O4 Icosane
Saturation magnetization Magnetic resonance imaging Murine neural stem cells Mesenchymal stem cells Oleic acid
Octadec-1-ene Oleylamine
Poly(ethylene glycol)
Hydroxamic acid-terminated methoxy-poly(ethylene glycol) Phosphonic acid-terminated methoxy-poly(ethylene glycol) Poly(3-O-methacryloyl-1,2:5,6-di-O-isopropylidene-α-D- glucofuranose)
Amino-terminated poly(3-O-methacryloyl-1,2:5,6-di-O- isopropylidene-α-D-glucofuranose)
α-Carboxyl-ω-bis(ethane-2,1-diyl)diethylphosphonate poly(3-O- methacryloyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose) Poly(3-O-methacryloyl-α-D-glucopyranose)
Bis(ethane-2,1-diyl)phosphonic acid-terminated poly(3-O- methacryloyl-α-D-glucopyranose)
Specific absorption rate Silica
Squalene
Transmission electron microscopy Thermogravimetric analysis Tetramethyl orthosilicate Trioctylamine
Dispersity
5 Contents
1. Introduction……… 6
2. Aims………... 7
3. Results and discussion………... 7
3.1. Preparation of monodisperse superparamagnetic Fe3O4 nanoparticles with controlled size……….. 7
3.2. Preparation of polymers for particle modification………. 10
3.3. Phase-transfer of Fe3O4 nanoparticles in water………... 12
3.4. Biological experiments……… 14
4. Conclusions……… 16
5. References……….. 17
6. Curriculum Vitae…………...………... 19
7. Selected publications and conferences……….. 20
6 1. Introduction
A special place among all kinds of the nanomaterials belongs to magnetic nanoparticles due to their ability to be manipulated by an external magnetic field. Generally, magnetic particles consist of ferromagnetic elements, such as iron, cobalt, and nickel. Because the last two elements can induce toxicity1, they are not good candidates for applications in biomedicine. In contrast, iron-based nanoparticles exhibit minor toxicity and were approved by US Food and Drug Administration as contrast agents for magnetic resonance imaging (MRI)2,3. Among all known forms of iron oxide, maghemite (γ-Fe2O3) and magnetite (Fe3O4) are the most appropriate for medical uses due to their superior magnetic properties, good chemical and colloidal stability and excellent biocompatibility4. Therefore, surface-modified iron oxide nanoparticles have been suggested for cell labeling and separations, drug delivery, hyperthermia, and as contrast agents for MRI5-8. Biomedical applications of iron oxides require nanoparticles with specific size, surface and magnetic properties, which are determined by selection of the synthetic method9. In the last few decades a considerable attention has been paid to development of new methods of iron oxide nanoparticle fabrication.
These techniques include co-precipitation, sol-gel reaction, microemulsion, flame spray pyrolysis, thermal decomposition, sonolysis or biosynthesis10-14.
In this work, the thermal decomposition method was selected for the particle synthesis, because it allows fabrication of monodisperse magnetic iron oxide nanoparticles, control of their size, and use of inexpensive non-toxic iron (III) carboxylates as an iron precursor. The main disadvantage of the method consists in usage of high temperature during the synthesis and hydrophobic nature of coating on the particles, which requires post-synthesis modification to make them water-dispersible10. Superparamagnetic iron oxide nanoparticles have a large surface-to-volume ratio and surface energy. To minimize excessive energy, particles naturally tend to aggregate. In addition, uncoated nanoparticles exhibit high nonspecific adsorption of proteins, which is undesirable for biomedical applications. Coating of the nanoparticle surface can prevent these unwanted physical and chemical processes.
Polymer coatings can be divided into natural and synthetic ones. From the vast variety of natural polymers, the most commonly used are dextran, chitosan, alginate, heparin, hyaluronic acid, and starch15,16. From the synthetic polymers, great attention is paid to poly(ethylene glycol) (PEG), poly(ethyleneimine), poly(N-vinylpyrolidone), and poly(N,N- dimethylacrylamide)17,18. The coating material should possess some specific characteristics, such as biocompatibility, hydrophilicity, presence of functional groups, and strong repulsion forces. Moreover, polymer shell controls interaction of the particles with living cells in a well- defined and controllable way. Increased affinity of the particles to the cell surface is needed for cell labeling applications in regenerative medicine or preparation of drug carriers for cancer treatment. On the other hand, low affinity to cell membrane will be beneficial for preparation of long-time circulating contrast agents for MRI.
In this thesis, influence of different reaction parameters (i.e., temperature, type and concentration of stabilizer) on the formation of uniform magnetic Fe3O4 nanoparticles was studied. PEG, poly(3-O-methacryloyl-α-D-glucopyranose) (PMG), and silica were investigated as coating materials for surface modification of the magnetic nanoparticles.
Polymer-functionalized particles were examined as a possible cellular marker or contract
7 agent for MRI, and as heat mediator for hyperthermia. The toxicity and cellular uptake of surface functionalized superparamagnetic nanoparticles were tested on different cell cultures.
2. Aims
The main goal of the thesis was to design and synthesize monodisperse superparamagnetic iron oxide nanoparticles with polymer-modified surface to combine superior properties of the particles (monodispersity, superparamagnetism, biocompatibility, and large surface area-to-volume ratio) with excellent hydrophilicity and repulsive characteristic of polymer coating. This will make them a powerful theragnostic tool for applications as contrast agent or cellular label for MRI and heat mediator for magnetic hyperthermia.
The specific aims of the thesis were as follows:
1. Synthesis of monodisperse superparamagnetic Fe3O4 nanoparticles with controlled size by thermal decomposition of Fe(III) carboxylates.
2. Investigation of the effect of various reaction parameters on particle morphology.
3. Coating of the particles with hydrophilic and biocompatible shells using - poly(ethylene glycol),
- poly(3-O-methacryloyl-α-D-glucopyranose), - silica.
4. Physico-chemical characterization of the particles.
5. Determination of biocompatibility and preliminary usage of the nanoparticles in biomedical applications.
3. Results and discussion
3.1. Preparation of monodisperse superparamagnetic Fe3O4 nanoparticles with controlled size
Selection of a proper iron organic precursor and a short nucleation time play an important role in preparation of monodisperse superparamagnetic nanoparticles. In this thesis, Fe(III) oleate, mandelate, and glucuronate were investigated as a non-toxic alternative to commonly used iron pentacarbonyl for preparation of such particles. The synthesis involved thermal decomposition of iron complexes in high-boiling solvents in the presence of a stabilizer. The mechanism of the particle formation was explained by ketonic decarboxylation reaction19,20. Thermal decomposition resulted in breaking of Fe-O and FeO-C bonds and generation of radicals that collide and form intermediate species, namely poly(iron oxo) clusters. When the temperature reached a critical value, the clusters served as building blocks of the nanoparticles via a burst nucleation process followed by growth21,22. The nanoparticle size, its distribution characterized by dispersity (Ð), and morphology depended mainly on the thermal stability of the intermediates, rather than on instability of chosen iron organic precursor22. Hence, the intermediates had to be additionally stabilized by the surfactant and the nucleation had to be fast to generate nanoparticles of uniform size.
Evolution of the particle size and Ð were studied by changing several reaction parameters, e.g., type and concentration of the stabilizer (oleic acid - OA and/or oleylamine - OLA), and reaction temperature. When increasing concentrations of OA (0.008-
8 0.08 mmol/ml) were used during decomposition of Fe(III) oleate at 320 °C, the particle size decreased from 12 to 8 nm and the uniformity was preserved (Ð ~1.06) according to transmission electron microscopy (TEM). This can be explained by both formation of stable intermediates and large number of nuclei stabilized at high OA concentrations. As a result, the particle size decreased. In the second set of the experiments, a mixture of OA/OLA (0.08 mmol/ml) was used instead of OA and OA/OLA ratio was changed from 0.23 to 4.3 mol/mol. Monodisperse Fe3O4 nanoparticles (Ð = 1.04) were formed only at high OA/OLA ratios (3 and 4.3 mol/mol) and no clear dependence of the particle size on the amount of the stabilizer was observed. Such a result can be explained by poor stabilization efficiency of OLAand prolongation of the nucleation stage. Improved stabilization by OA, in contrast to OLA, can be attributed to enhanced affinity of carboxyl groups to the metal ions, which strongly bind to the nanoparticle surface during the nucleation and growth process23. Similar effect of OA/OLA ratio on the particle size and Ð was observed during decomposition of Fe(III) mandelate under the same reaction conditions.
In the next set of the experiments, the reaction temperature was controlled by selection of high-boiling solvents with different boiling point (b.p.), i.e., squalene (SQ; 285 °C), octadec-1-ene (OD; 320 °C), icosane (IS; 343 °C), and trioctylamine (TOA; 365 °C).
Decomposition of Fe(III) oleate, mandelate, and glucuronate at 285 °C and 0.3 mmol of OA/ml produced polydisperse nanoparticles (Ð ˃1.1) with number-average particle diameter (Dn) reaching 18, 71, and 20 nm, respectively. Broadening of the particle size distribution can be attributed to relatively low reaction temperature (285 °C). In this case, effective separation between the nucleation and growth stage, which is crucial for formation of monodisperse particles, was obviously not achieved. Magnetic nanoparticles prepared at ≥320 °C were monodisperse (Ð ~1.03). According to TEM, the particle size increased from 12 to 19 nm and from 12 to 27 nm with increasing reaction temperature from 320 to 365 °C for particles prepared from Fe(III) oleate and mandelate, respectively. Similar data were obtained for the particles synthetized from Fe(III) glucoronate (Figure 1). As a result, with increasing b.p. of the solvent, diameter of Fe3O4 nanoparticles increased (with the exception of SQ), which was explained by the faster growth of the nanoparticles at high temperature. Size of the monodisperse magnetic nanoparticles could be thus controlled from 8 to 27 nm by changing different reaction parameters (Table 1). ATR FTIR spectroscopy confirmed presence of oleic acid on the surface of the nanoparticles independently of the used precursor type. Amounts of coating determined by both atomic absorption spectroscopy (AAS) and thermogravimetric analysis (TGA) were comparable. With increasing reaction temperature, OA amount on the particle surface decreased from ~80 to 4 wt.%, which can be attributed to higher decomposition rate of OA at a higher temperature (Table 1).
According to X-ray powder diffraction, the crystal structure of the obtained magnetic nanoparticles corresponded to magnetite. Appearance of Fe(II) in spinel phase of the nanoparticles was attributed to generation of CO and H2 during the decomposition reaction reducing Fe(III) to Fe(II), which is in agreement with earlier published data22. Magnetic properties of the particles were characterized by vibrating sample magnetometry.
Magnetization curves showed absence of hysteresis and remanent magnetization, confirming superparamagnetic behavior of the Fe3O4 nanoparticles. When their size increased from 8 to 12 nm, the saturation magnetization (MS) also increased from 40.8 to 62 Am2/kg. Resulting
9 MS values were lower than that of bulk magnetite (MS = 92-100 Am2/kg)24, but comparable with earlier reported data25. The difference can be explained by presence of a low-magnetic layer (substituted antiferromagnet) in the particle structure as a result of imperfect decomposition reaction.
Figure 1. TEM micrographs of oleic acid-stabilized Fe3O4 nanoparticles prepared from (a-c) Fe(III) oleate, (d-f) Fe(III) mandelate, and (g-h) Fe(III) glucoronate at (a, d, g) 320 °C, (b, e, h) 343 °C, and (c, f) 365 °C.
Table 1. Preparation and characterization of Fe3O4 nanoparticles.
Precursor T (°C)
Stabilizer
Dn
(nm) Ð
Coating (wt.%) OA
(mmol/ml) OLA
(mmol/ml) OA/OLA
(mol/mol) AAS TGA
Fe(III) oleate 320 0.08 - - 8 1.04 82.3 77.5 Fe(III) oleate 320 0.06 - - 9 1.03 84.8 78.2 Fe(III) oleate 343 0.08 - - 10 1.03 27.3 - Fe(III) glucuronate 320 0.3 - - 11 1.03 83.2 - Fe(III) mandelate 320 0.3 - - 12 1.02 80.8 -
Fe(III) oleate 320 - - - 13 1.06 78.1 -
Fe(III) oleate 320 0.065 0.015 4.3 14 1.04 74 - Fe(III) glucuronate 365 0.3 - - 15 1.06 50 - Fe(III) oleate 343 0.3 - - 17 1.02 35.4 37 Fe(III) glucuronate 343 0.15 - - 18 1.03 69 -
Fe(III) oleate 365 0.3 - - 19 1.02 6.6 7.3
Fe(III) mandelate 320 0.15 0.15 1 21 1.04 78.1 - Fe(III) oleate 285 0.08 - - 22 1.03 73.2 - Fe(III) mandelate 343 0.3 - - 23 1.06 2.6 -
Fe(III) oleate 285 - - - 25 1.04 68.6 -
Fe(III) mandelate 365 0.3 - - 27 1.03 3.2 -
T - temperature; OA - oleic acid; OLA – oleylamine; Dn - number-average particle diameter;
Ð - dispersity; AAS - atomic absorption spectroscopy; TGA - thermogravimetric analysis.
10 3.2. Preparation of polymers for modification of particles
Chemical attachment of hydrophilic polymers to Fe3O4 particles was preferred for preparation of ferrofluids with a long-term colloidal stability. To achieve this, polymers have to contain anchoring groups, e.g., phosphonate or hydroxamate, capable of formation of strong complexes with iron atoms26,27.
Modification of PEG. Phosphonic acid-terminated PEG (PEG-PA) was prepared by a two-step procedure (Figure 2). First, Michaelis-Arbuzov reaction28 of α-methoxy-ω-bromo- PEG (Mn = 750 g/mol) with trimethyl phosphite resulted in dimethyl ester of phosphonic acid- terminated methoxy-PEG. In the second step, the ester was hydrolyzed in the presence of HCl to PEG-PA.
CH3O O n Br
CH3O P OCH3
OCH3 CH3O O n P
O OCH3
OCH3 CH3Br
H+
P CH3O O n
O OH
OH
+
- 130 °CPEG-PA
Reflux
Figure 2. Preparation of phosphonic acid-terminated methoxy-PEG (PEG-PA).
Hydroxamic acid-terminated methoxy-PEG (PEG-HA) was prepared by reaction of N-hydroxysuccinimide-activated methoxy-poly(ethylene glycol) (NHS-PEG; Mn = 750 g/mol) with hydroxylamine hydrochloride in the presence of sodium hydrogen carbonate to yield PEG-HA (Figure 3).
CH3O O
NH
n
O NH H
O O
N O CH3O O
NH
n
O O O
O
N
H2 OH HCl NaHCO3 NaCl
PEG-HA
+ ·
- ,0 °CNHS-PEG
Figure 3. Preparation of hydroxamic acid-terminated methoxy-PEG (PEG-HA).
Bis(ethane-2,1-diyl)phosphonic acid-terminated poly(3-O-methacryloyl-α-D- glucopyranose) (PMG-P) was obtained according to Figure 4. 3-O-methacryloyl-1,2:5,6-di- O-isopropylidene-α-D-glucofuranose (MDG) monomer was synthesized by reaction of 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose with methacryloyl chloride in the presence of triethylamine and then polymerized by 4,4’-azobis(4-cyanovaleric acid) (ACVA)-initiated RAFT reaction to poly(3-O-methacryloyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose) (PMDG) with 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPTA) as a transfer agent.
The weight-average molecular weight of the polymer was 4,600 g/mol with Ð = 1.06. RAFT-
11 end groups in PMDG were aminolyzed by butylamine (BA) and the remaining thiols were coupled with N-(2-aminoethyl)maleimide to yield amino-terminated PMDG (PMDG-NH2). In the next step, PMDG-NH2 reacted with diethyl vinylphosphonate via aza-Michael addition, resulting in α-carboxyl-ω-bis(ethane-2,1-diyl)diethylphosphonate derivative of PMDG (PMDG-PEt). Diethylphosphonate groups of PMDG-PEt were deprotected using trimethylsilyl bromide. As a result, phosphonic acid was introduced as an efficient anchoring group to provide strong covalent linkage with iron atoms on the Fe3O4 nanoparticle surface.
Finally, α-carboxyl-ω-bis(ethane-2,1-diyl)phosphonic acid-terminated PMDG was treated with 80 % formic acid to form water-soluble bis(ethane-2,1-diyl)phosphonic acid-terminated poly(3-O-methacryloyl-α-D-glucopyranose) (PMG-P). Structures of prepared PEG- derivatives, PMDG, PMDG-NH2, PMDG-PEt, and PMG-P were confirmed by 1H NMR and ATR FTIR spectroscopies.
O O O
O O
O O
O
H S
O O
O O O
O O
n
NC O
S
N NH2 O
H S
O O
O O O
O O NC n
O O
O P O
O O O N
H S
O O
O O O
O O
n
NC
O O
O
P OO O P
OO N O
O N
H S
O O
O O O
O O NC n
O O
O
P OHOH O P
OHOH
N O N
O
H S
O NC n
O O
O
P OHOH O P
OHOH N O
O
O O
H OH
O OH H O O
O O O O
H N(Et)3
N NH2
O O
Si Br
H+
Cl O ACVA
+
CPTAMDG PMDG
PMDG-NH2 PMDG-PEt
PMDG-P PMG-P
BA
Figure 4. Synthesis of bisphosphonic acid-terminated PMG (PMG-P).
12 3.3. Phase-transfer of Fe3O4 nanoparticles in water
PEG-coated magnetic particles. Due to superior physico-chemical and biological properties, PEG was selected for surface modification of the magnetic nanoparticles to ensure their colloidal stability under different environmental conditions (e.g., pH and ionic strength) and to make them applicable in biomedicine as MRI contrast agent or in drug delivery systems. Monodisperse OA-stabilized magnetic nanoparticles with Dn = 12 nm (Ð = 1.06) were modified with PEG-PA and PEG-HA via ligand exchange in toluene at 70 °C.
According to ATR FTIR spectroscopy, both PEG-derivatives successfully exchanged OA on the particle surface. According to TGA, Fe3O4@PEG-PA and Fe3O4@PEG-HA particles contained 78 and 75 wt.% of the polymer shell, respectively. Colloidal stability of the PEGylated particles was evaluated at different NaCl concentrations (1-1,000 mmol/l) using DLS (Figure 5). The hydrodynamic diameter (Dh) of Fe3O4@PEG-PA nanoparticles was almost constant (~45 nm) in all range of salt concentrations, confirming perfect colloidal stability. Fe3O4@PEG-HA particles were stable at a moderate salinity (1-10 mmol NaCl/l); Dh
was ~68 nm. Higher NaCl concentration increased Dh to 121 nm due to aggregation. After several hours of storage, the particles precipitated on the bottom of vials (100 and 1,000 mmol NaCl/l). Nevertheless, PEG-HA coating provided good stabilization of the nanoparticle dispersion under physiological conditLRQV ȗ-potential of both Fe3O4@PEG-PA and Fe3O4@PEG-HA nanoparticles dropped to 0 mV at 1,000 mmol of NaCl/l, which was attributed to the formation of counter ion layer29. Data also suggested that the particle stability was mostly achieved by steric repulsion of PEG chains.
Figure 5'HSHQGHQFHRIȗ-potential (full line) and hydrodynamic diameter Dh (dashed line) of Fe3O4@PEG-HA and Fe3O4@PEG-PA nanoparticles on concentration of NaCl.
PMG-P-coated magnetic particles. In an attempt to design a new cellular labeling agent for MRI applications, surface of the Fe3O4 particles was modified with phosphonic acid-terminated poly(3-O-methacryloyl-Į-D-glucopyranose). PMG-P displaced OA on the magnetic nanoparticles by ligand exchange in a toluene/water mixture at 70 °C. Presence of PMG-P coating on the iron oxide surface was confirmed by ATR FTIR spectroscopy.
According to TGA, particles contained 72 wt.% of coating. Colloidal stability of the particles in water was measured at different NaCl concentrations (1-1,000 mmol/l). Dh DQGȗ-potential
13 increased from 140 to 890 nm and from -15 to -5 mV, respectively, as the NaCl concentration increased. As Fe3O4@PMG-P ferrofluid was stable for 1 week at 1 and 10 mmol NaCl/l, PMG-P can be considered as a promising modification agent to maintain colloidal stability of the iron oxide nanoparticles in aqueous media.
Silica-coated magnetic nanoparticles. Silica shell was introduced on the Fe3O4
particles by water-in-oil reverse microemulsion technique, involving hydrolysis and condensation of tetramethyl orthosilicate (TMOS) inside reverse micelles under basic catalysis30. This approach provides easy control of the shell thickness by simply changing the TMOS/Fe3O4 ratio during the reaction. Characteristics of the core-shell Fe3O4@SiO2 particles prepared from 10-nm OA-stabilized Fe3O4 nanoparticles are presented in Table 2. Silica shell thickness increased from 4 to 13 nm (TEM) with increasing TMOS/Fe3O4 ratio, while the particle uniformity was preserved (Ð ~1.01; Figure 6). Because saturation magnetization is a function of mass, increase of the thickness of diamagnetic silica shell decreased MS of the final Fe3O4@SiO2 nanoparticles. Particles with a thin shell are preferred in the biomedical applications, due to good magnetic response and reduced dosage. Presence of silica shell on the iron oxide surface was confirmed by energy-dispersive X-ray analysis and ATR FTIR spectroscopy. Fe3O4@SiO2 nanoparticles demonstrated good colloidal stability in water (Dh ~110 nm) attributed to negative surface charge (-30 mV) due to the presence of silanol groups on the particle surface. Amino-functionalized particles were prepared by modification of the Fe3O4@SiO2 nanoparticles with (3-aminopropyl)triethoxysilane. The amino groups were then used to introduce PEG on the particle surface to ensure colloidal stability even in physiological media. Moreover, PEGylation minimized toxicity of the silica-coated particles and made them invisible to the reticuloendothelial system, i.e., prevented their uptake by different cells (mainly leucocytes), increasing thus the blood circulation time. ATR FTIR spectroscopy confirmed presence of PEG on the Fe3O4@SiO2 nanoparticles, which maintained colloidal stability in water (Dh = 43 nm), although the total negative surface charge was -5 mV.
Table 2. Characterization of Fe3O4@SiO2 nanoparticles.
TMOS/Fe3O4
(µl/mg)
Dn
(nm) Ð SiO2 thickness
(nm)
40/80 18 1.01 4
20/30 20 1.01 5
40/30 28 1.01 9
60/30 36 1.02 13
TMOS - tetramethyl orthosilicate; Dn - number-average particle diameter; Ð – dispersity.
14 Figure 6. TEM micrographs of core-shell Fe3O4@SiO2 nanoparticles with (a) 4-, (b) 5-, (c) 9-, and (d) 13-nm thick silica shell.
3.4. Biological experiments
Careful evaluation of iron oxide nanoparticle cytotoxicity is important for identifying non-toxic concentrations needed in different biomedical applications. Toxicity of PEG-PA- and PEG-HA-coated nanoparticles (Dn = 12 nm) at different concentrations and exposure times was tested on the human peripheral blood cells. No significant cytotoxic effect of Fe3O4@PEG-PA and Fe3O4@PEG-HA particles (0.12-ȝJ/cm2) on the cells was found after 24 and 72 h of incubation. According to AAS, the content of internalized iron in human peripheral blood cells after 24 h of incubation with Fe3O4@PEG-PA and Fe3O4@PEG-HA nanoparticles was 25 and 72 pg of Fe/cell, respectively. This indicates that phosphonic or hydroxamic functional groups in the polymer coating significantly influenced uptake of the particles by the cells. Highly internalized Fe3O4@PEG-HA nanoparticles can be therefore suggested as a new cellular labeling agent for MRI. On the other hand, low internalization of Fe3O4@PEG-PA particles by the cells prolongs their circulation and in the blood stream, which is beneficial for application as a contrast agent in MRI.
In additional application, differently sized PEG-PA-functionalized particles (10, 14, and 24 nm) were studied in terms of their prospective use as a heat mediator for hyperthermia.
Specific absorption rate (SAR) in an alternating magnetic field was determined calorimetrically (Figure 7). Larger ferrimagnetic Fe3O4@PEG-PA particles (Dn = 24 nm) showed higher heating performance than small superparamagnetic ones, which makes them promising for magnetic hyperthermia.
Figure 7. Dependence of specific absorption rate (SAR) on Fe3O4@PEG-PA nanoparticle size; measured at 400 kHz and 24 kA/m.
15 Transplantation of stem cells for regeneration of damaged tissues requires noninvasive monitoring by MRI, using suitable cell labels with sufficient NMR relaxivity. For this purpose, we developed Fe3O4@PMG-P nanoparticles and tested their cytotoxicity on mesenchymal stem cells (MSCs). Viability of the cells treated with Fe3O4@PMG-P (0.1- 0.5 mM) nanoparticles for 48 h was similar to that of control cells (without particles), confirming non-toxicity of the particles. Relaxivity of Fe3O4@PMG-P particles was 264 and 339 s−1/mM of Fe3O4 at 0.5 and 4.7 T, respectively, which was similar to that of commercially available contrast agents based on iron oxides31. Fe3O4@PMG-P nanoparticles thus represent a safe alternative to recently used cell labels.
Cytotoxicity of core-shell Fe3O4@SiO2 and Fe3O4@SiO2-PEG nanoparticles was tested on murine neural stem cells (mNSCs) in terms of cell viability and survival/mortality.
Fe3O4@SiO2 particles at concentrations ≤2 mg/l did not affect mNSCs survival and viability.
When 20 mg of the particles/l was used, the cell viability decreased by more than 50 % compared to the control, while the cell mortality increased to 20 %. At 200 mg of Fe3O4@SiO2/l, survival of mNSCs reached ~50 % of control. In contrast, Fe3O4@SiO2-PEG particles at all used concentrations did not significantly affect the survival or the viability of mNSCs. Moreover, labelling of mNSCs with Fe3O4@SiO2 was highly efficient and dose- depended, while Fe3O4@SiO2-PEG particles were not internalized by the cells (Figure 8).
Consequently, Fe3O4@SiO2-PEG nanoparticles can be used as a long-time circulating contrast agent for MRI of tissues, while Fe3O4@SiO2 particles can serve as a cellular label. It is a big advantage that the Fe3O4@SiO2 particles labelled more than 50 % of the mNSC population at 20-fold lower concentration (200 mg/l) than the commercial dextran-coated nanomag®-D-spio agent32.
0 10 20 30 40 50
2.7 mg Fe/l
2 mg Fe/l
0.23 mg Fe/l
0.8 mg Fe/l 0.23 mg Fe/l 0.23 mg Fe/l
Labeling efficiency (%)
Fe3O4@SiO2-PEG (mg/l) Fe3O4@SiO2 (mg/l)
2 20 200
200 20
2
Figure 8. Labeling efficiency of mNSCs by Fe3O4@SiO2 and Fe3O4@SiO2-PEG nanoparticles analyzed by flow cytometry. mNSCs were exposed to different concentrations of particles for 24 h. Labeling efficiency, expressed as the mean of three independent experiments conducted in five replicates, was calculated as the percentage of the increase of the side-scattered light of the laser beam relative to negative controls. Error bars represent standard deviations. Fe content in mNSCs was measured by inductively coupled plasma mass spectrometry. Non- treated cells (control) contained 0.23 ± 0.03 mg Fe/l.
16 4. Conclusions
(i) Small, smart, and safe magnetic particles were developed for the biomedical applications.
(ii) Hydrophobic superparamagnetic Fe3O4 nanoparticles with uniform size were prepared by the thermal decomposition approach. The diameter of the particles was controlled in the range of 8-27 nm by variation of the reaction conditions. The particles were manipulatable by a magnet and dispersible in organic solvents in the absence of external magnetic field. Monodispersity of the particles ensured their uniform physical, chemical, and biological properties.
(iii) Poly(ethylene glycol) and poly(3-O-methacryloyl-α-D-glucopyranose) were terminated with phosphonic or hydroxamic acid providing strong interactions with iron atoms.
(iv) Hydrophobic Fe3O4 particles were successfully transferred to water via ligand exchange with the above mentioned polymers. Resulting dispersions were stable even in the physiological media. Presence of coating on the particle surface was confirmed by a range of physico-chemical methods.
(v) Monodisperse core-shell Fe3O4@SiO2 nanoparticles were obtained by hydrolysis and condensation of Si precursors in reverse emulsion system. Thickness of the silica shell was controlled in range of 4-13 nm by changing the reaction parameters. SiO2
prevented leakage of Fe ions in media and made functionalization with reactive groups possible, which is important for future attachment of biomolecules, e.g., proteins, drugs, or antibiotics.
(vi) Toxicity of all synthetized superparamagnetic nanoparticles, which always has to be considered when novel systems are developed for biomedical applications, was evaluated on different cell cultures, including macrophages, human peripheral blood cells, murine neural stem cells, and mesenchymal stem cells. As a result, surface- functionalized particles were non-toxic and biocompatible.
(vii) Newly developed surface-modified monodisperse magnetic nanoparticles are appropriate candidates for medical applications, such as long-time circulating agents and/or cellular labels for MRI or drug delivery systems. In addition, 24-nm Fe3O4
nanoparticles showed promising properties as a heat mediator for magnetic hyperthermia.
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19 6. Curriculum Vitae
Education
08.2012 – to date PhD. student of macromolecular chemistry, Charles University in Prague and Department of Polymer Particles of the Institute of Macromolecular Chemistry AS CR, v.v.i.
10.2011 – 07.2012 Scholarship holder of UNESCO/IUPAC postgraduate course in Advanced Polymer Science, Intitute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, with topic “Functional magnetic nanoparticles“ (mentor Ing. Daniel Horák, CSc)
09.2009 – 11.2010 Lviv Polytechnic National University, Institute of Chemistry and Chemical Technologies, magister degree (specialization
„Biotechnology of biologically active substances”). Diploma thesis title: “Synthesis and properties of acylaminomethylbenzenthiosulfo acids esters”
09.2005 – 06.2009 Lviv Polytechnic National University, Institute of Chemistry and Chemical Technologies, bachelor diploma (specialization
“Biotechnology”).
09.1994 – 06.2005 Secondary school in Biskovychi, Lviv region, Ukraine Scientific experience
10.2008 – 11.2010 Research in the Department of Technology of Biologically Active Substances, Pharmacy and Biotechnology at Lviv Polytechnic National University.
Name: Vitalii Patsula
Date and place of birth: 30.04.1988, Ralivka, Ukraine
Address: Volavkova 1845/11, Praha, 162 00, Czech Republic
Nationality: Ukrainian
Telephone: 00420 777 510 636
E-mail: patsula@imc.cas.cz
Marital status: Married
20 7. List of publications and contributions at conferences
1. Patsula V., Petrovský E., Kovářová J., Konefal R., and Horák D., Monodisperse superparamagnetic nanoparticles by thermolysis of Fe(III) oleate and mandelate complexes, Colloid. Polym. Sci. 292, 2097–2110 (2014). IF = 1.865
2. Patsula V., Kosinová L., Lovrić M., Ferhatovic Hamzić L., Rabyk M., Konefal R., Paruzel A., Šlouf M., Herynek V., Gajović S., and Horák D., Superparamagnetic Fe3O4
nanoparticles: Synthesis by thermal decomposition of iron(III) glucuronate and application in magnetic resonance imaging, ACS Appl. Mater. Interfaces 8, 7238−7247 (2016). IF = 7.504 3. Kostiv U., Patsula V., Šlouf M., Pongrac I. M., Škokić S., Dobrivojević Radmilović M., Pavičić I., Vinković Vrček I., Gajović S., and Horák D., Physico-chemical characteristics, biocompatibility, and MRI applicability of novel monodisperse PEG-modified magnetic Fe3O4&SiO2 core-shell nanoparticles, RSC Adv. 7, 8786−8797 (2017). IF = 3.108
4. Patsula V., Moskvin M., Dutz S., and Horák D., Size-dependent magnetic properties of iron oxide nanoparticles, J. Phys. Chem. Solids 88, 24–30 (2016 ). IF = 2.059
5. Patsula V., Tulinská J., Trachtová S., Kuricová M., Liskova A., Španová A., Ciampor F., Vavra I., Rittich B., Ursinyova M., Dusinska M., Ilavská S., Horvathova M., Masanova V., Uhnakova I., and Horák D., Toxicity evaluation of monodisperse PEGylated magnetic nanoparticles for nanomedicine, Nanotoxicology, under revision. IF = 6.428
Contributions on conferences Oral presentation
Patsula V. and Horák D., Monodisperse superparamagnetic nanoparticles with controlled size, Career in Polymers IV, Prague, Czech Republic 2012, Abstract Book, L5.
Poster presentations
1. Patsula V. and Horák D., Monodisperse superparamagnetic nanoparticles with controlled size for biomedical applications, Career in Polymers V, Prague, Czech Republic 2013, Abstract Book, P1.
2. Patsula V., Horák D., Tulinská J., Líšková A., and Kuricová M., Monodisperse superparamagnetic nanoparticles for biomedical applications, Frontiers of Polymer Colloids:
From Synthesis to Macro-Scale and Nano-Scale Applications, 78th Prague Meeting on Macromolecules, Prague, Czech Republic 2014, Abstract Book, p. 152.
3. Patsula V. and Horák D., Uniform superparamagnetic Fe3O4 nanoparticles:
Preparation, characterization and surface modification, Career in Polymers VI, Prague, Czech Republic 2014, Abstract Book, P5.
4. Horák D., Babič M., Patsula V., Moskvin M., and Zasońska B., Superparamagnetic iron oxide nanoparticles and their surface modifications, RADIOMAG meeting Coating Requirements for Magnetic Particles to be Used for Magnetic Hyperthermia, Limasol, Cyprus 2015.
5. Patsula V. and Horák, D., Poly(3-O-methacryloyl-α-D-glucopyranose)-coated superparamagnetic Fe3O4 nanoparticles: Synthesis characterization and application in magnetic resonance imaging, Career in Polymers VIII, Prague, Czech Republic 2016, Abstract Book, P3.
