Synthesis and Characterization of
Iron Composite Nanoparticles for Cancer Therapy
Amanda Huey
Dr. Guandong Zhang
Dr. Daniel Cullen
PI: Dr. Ian Baker
Center for Nanomaterials Research at Dartmouth
Research Experience for Undergraduates Program
August 15, 2008
1
1. Introduction
Magnetic nanoparticles can be used for many applications, including magnetic recording
media, catalysis, MRI contrast enhancement, drug delivery and hyperthermia.
1,2 Hyperthermia is
a promising form of cancer therapy that locally heats tissue to greater than 42ºC for to destroy
tissue, particularly tumors.
1 The difficulty of this therapy lies in selectively heating cancer
tumors.
1 By localizing magnetic nanoparticles to the desired area and applying an alternating
magnetic field (AMF), the particles can generate heat by hysteresis loss, thus killing cancer cells
with minimal injury to normal tissues. Highly hysteretic particles are desirable because they will
give off the most heat with the lowest dose to a patient.
1,2 Iron, which has a very high magnetic
saturation value (220 emu/g), is ideal for hyperthermia application.
Other criteria necessary for magnetic nanoparticle hyperthermia application include:
biocompatibility, non-toxicity, high accumulation in target tissue and good dispersion in aqueous
solution.
1,3 Yet iron is reactive to both oxygen and water and can eventually be oxidized into
weakly ferromagnetic and antiferromagnetic species respectively, rendering particles less useful
to hyperthermia.
1 Therefore, it is critical to utilize protection strategies that chemically stabilize
naked nanoparticles against oxidation.
2 Such strategies include: passivation to form a strongly
adherent magnetite (Fe
3O4) shell around the iron core and coating particles with coatings that
protect the iron core.
1,2 It is hypothesized that hydrophobic coatings and bilayer coatings protect
the iron core of nanoparticles better than hydrophilic and monolayer coatings. Coatings can be
attached to nanoparticles via van der Waals forces, charge attraction, or covalent bonding
directly to the nanoparticle surface. Protective layers can then be used for further
functionalization, depending on the desired application for the nanoparticles.
2 For hyperthermia,
biocompatible outermost coatings must be used to help particles avoid opsonization and
phagocytosis by the reticulendothelial system (the body’s mechanism to destroy foreign
substances) so that can be implemented.
3
This project focuses on synthesizing iron nanoparticles and then using surface
modification to protect the iron core and achieve a biocompatible coating. The particles’
magnetic properties, surface coating and heating properties were then characterized using:
Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD), Vibrating Sample
Magnetometry (VSM), Hyperthermia/Specific Absorption Rate (SAR), Infrared Spectroscopy
(IR) and Thermogravimetry/Differential Thermal Analysis (TG/DTA). The long-range goal of
this research is to be able to synthesize biocompatible iron composite nanoparticles for cancer
therapy.
2. Experimental
2.1 Synthesis of Fe/Fe
3O4 Nanoparticles and Surface Modification
2.1.1 Synthesis of Fe/Fe
3O4 Nanoparticles
Fifteen nm Fe/ Fe
3O4 core-shell nanoparticles were synthesized using the microemulsion
method, in which NaBH
4 reduces aqueous FeCl3. Two microemulsion solutions were prepared.
Each microemulsion solution contained the same oil phase (n-octane), surfactant (cetyl trimethyl
ammonium bromide [CTAB]) and co-surfactant (n-butanol), but the water phase differed, with
one microemulsion containing FeCl
3 and the other NaBH4.
To prepare a microemulsion, 75 mL n-octane, 14.3g CTAB and 12.8 mL n-butanol were
agitated in an Erlenmeyer flask. Separately, 30 mL of 0.20M FeCl
3 solution and 30 mL of 0.85M
2
NaBH
4 solution were prepared and then each was added to a separate oil phase containing flask.
The microemulsions were then agitated under Argon gas protection for 45 minutes. Then, the
NaBH
4 microemulsion was added drop-by-drop to the FeCl3 microemulsion over the course of
about 10 minutes under high speed agitation and the mixture was left to react for 10 more
minutes. Afterwards, under Ar protection, the fresh nanoparticles were separated on a magnetic
field and then washed with de-gassed deionized (DI) water, then methanol, then acetone as
quickly as possible to prevent oxidation. The particles were then passivated in an Ar-filled
desiccator for two days to form the protective magnetite (Fe
3O4) shell.
2.1.2 Fe/Fe
3O4 Nanoparticles Stabilized with Trimethylamine N-oxide
To best preserve the iron core, directly after nanoparticle synthesis130 mg freshly
synthesized particles were added to a solution of 15 mg trimethylamine N-oxide in 2 mL of
isopropyl alcohol (IPA). The mixture was sonicate and left to react for 40 minutes, then rinsed
twice with methanol and dried with Ar.
2.1.3 Phase Transfer of Fe/Fe
3O4 Nanoparticles Using Tetramethyl Ammonium Hydroxide
(TMAH)
Fifteen mg Fe/Fe
3O4/CTAB (original) nanoparticles were dispersed in 5 mL hexane and
then the solution was added to 5 mL 12% tetramethyl ammonium hydroxide (TMAH) solution.
The mixture was sonicated and shaken until the particles transferred to the bottom (aqueous)
layer.
2.1.4 Fe/Fe
3O4 Nanoparticles Modified by Hydrophobic Silanes: Hexamethyldisilazane
(HMDS) and Octadecyltrichlorosilane (OTS)
HMDS modification: Directly following synthesis, after the methanol wash, about 50 mg
of Fe/Fe
3O4 nanoparticles were washed with TMAH, then methanol again, then with 9 mL of 2:1
toluene: methanol solution, then with toluene. Then particles were dispersed in 9 mL 3.0 %
hexamethyldisilazane (HMDS) toluene solution in a glass vial filled with Ar. The sample was
then discontinuously sonicated at 50
°C for 4 hours. Finally, nanoparticles were separated and
dried at 100
°C for 2 minutes under Ar protection.
OTS modification: 30 mg Fe/Fe
3O4/CTAB particles were added to a solution of 3 mL
IPA and .3 mL TMAH. The solution was sonicated 25 minutes, then rinsed twice with IPA and
once with toluene. The particles were then dispersed in 3 mL toluene plus 30 μL OTS and
sonicated 20 minutes. The particles were then rinsed once with toluene and dried on a hot plate at
50ºC under Ar protection.
2.1.5 Fe/Fe
3O4 Nanoparticles Modified by Phosphatidylcholine (PC)
For a 30% PC coating, 80 mg Fe/Fe
3O4/HMDS (or Fe/Fe3O4/OTS) modified particles
were added to a solution of 0.24 mL PC and 0.12 mL chloroform. The solution was sonicated 25
minutes at room temperature and then the chloroform was evaporated under Ar protection.
2.1.6 Fe/Fe
3O4 Nanoparticles Modified by Pluronic® Copolymers
Pluronic
® F-127: 26.1 mg Fe/Fe3O4/CTAB particles were dispersed in a solution of 7.9
mg F-127 in .15 mL chloroform and sonicated for 50 minutes, then dried under Ar protection.
Pluronic
® P-123: 15.4 mg Fe/Fe3O4/CTAB particles were dispersed in a solution of 5.3
mg P-123 in .1 mL chloroform, sonicated for 50 minutes, and dried under Ar protection.
3
2.1.7 Fe/Fe
3O4 Nanoparticles Modified by Covalent Bonding: Dopamine-Dicarboxylterminated
Polyethylene Glycol
First, 6 mL PBS and 14 mL DI water were mixed and sonicated, then 0.30g N-(3-
Dimethylaminopropyl)-N
′-ethylcarbodiimide hydrochloride (EDC) and 0.30g NHydroxysuccinimide
(NHS) were added to the solution and sonicated. Then 0.30g dopamine
(DA) hydrochloride was added and the solution was sonicated. Finally, 0.2 mL dicarboxylterminated
polyethylene glycol (PEG) was added and the solution was sonicated 20 minutes and
left to react overnight (about 18 hours) to ensure amide bond formation due to the EDC reaction.
The next day, a solution of 0.1g KOH in 2 mL DI water was used to change the pH of 5.5
mL EDC reaction solution to about 6.5. Then, 23.8 mg Fe/Fe
3O4/CTAB (or Fe/Fe3O4/oxidizer)
particles were added to solution, sonicated 25 minutes, then rinsed twice with methanol,
centrifuged for 5 minutes, then dried with Ar.
2.2 Characterization of Nanoparticles
The crystallinity and structure of iron composite nanoparticles were determined by using
X-ray diffraction (XRD). Transmission electron microscope (TEM) was used to verify the shape
and size of particles, as well as the presence of an iron core. The magnetic saturation and
coercivity of the nanoparticles were determined by examining the hysteresis loop produced by
the vibrating sample magnetometer (VSM). The surface coating on particles was examined by
using infrared spectroscopy (IR) and Thermogravimetry/Differential Thermal Analysis
(TG/DTA). Finally, the heating effect of the Fe/Fe
3O4 nanoparticles was examined under an
alternating magnetic field measured by a heating setup in the lab.
3. Results & Discussion
Iron nanoparticles were synthesized using the microemulsion method because this
method yields monodisperse iron nanoparticles without requiring high temperature or high
pressure conditions. It is easy to control particle size and thus the magnetic properties of the
nanoparticles by changing the oil to water ratio of the solution. In the microemulsion, the water
droplets act as a mini-reactor site for equation (1) to occur:
2FeCl
3 + 6NaBH4 + 18H2O → 2Fe0 ↓ + 21H2 ↑ + 6B(OH)3 + 6NaCl (1)
The iron core of freshly synthesized particles must be preserved by formation of a thin
magnetite (Fe
3O4) shell. One method of Fe3O4 shell formation is passivation using Argon gas;
another uses an oxidizer, such as Trimethylamine N-oxide, which helps transfer oxygen to the
iron core to form magnetite, rather than a less magnetic iron oxide, such as hematite (
α-Fe2O3) or
goethite (
α-FeOOH). Figure 1 shows VSM results demonstrating the benefit of using oxidizer
directly after particle synthesis in order to preserve the iron core and thus the magnetic properties
of the particles. Particles preserved with oxidizer have a magnetic saturation (Ms) of 90 emu/g,
while the original particles only have an Ms of 58 emu/g.
4
Figure 1: VSM results showing the effect of oxidizer on iron core and magnetic property
preservation in Fe/Fe
3O4 nanoparticles
Freshly synthesized particles also have a layer of surfactant (CTAB) coating, which must
be modified in order to better preserve the iron core. New coatings can replace old coatings due
to stronger bonds with nanoparticles. In addition, new coatings can be used to achieve a phase
transfer of the nanoparticles from the oil phase to the water phase, which is necessary for
biocompatibility. Phase transfer is important because even though CTAB is an amphiphilic
compound, Fe/Fe
3O4/CTAB particles disperse in the oil phase. Figure 2 shows a schematic of
surfactant replacement, with tetramethyl ammonium hydroxide (TMAH) replacing CTAB and
Figure 3 shows the resulting phase transfer of the nanoparticles from oil to water phase.
Figure 2: Schematic of surfactant replacement with TMAH replacing CTAB on Fe/Fe
3O4
nanoparticle surface
5
Figure 3: Phase transfer of Fe/Fe
3O4 particles from oil to water phase
5
One type of coating that helps preserve the iron core is a hydrophobic silane coating, such
as hexamethyldisilazane (HMDS) or octadecyltrichlorosilane (OTS), since hydrophobic coatings
impede oxidation better than hydrophilic coatings. Figure 4 shows the schematic of HMDS
modification.
Figure 4: HMDS silanization on the Fe/Fe
3O4 nanoparticle surface5
Figure 5 shows the benefit of using a hydrophobic coating, rather than a hydrophilic one,
to preserve the iron core of nanoparticles. The original particles (CTAB coated) have an Ms
value of 104 emu/g and the hydrophobic HMDS coated particles have a Ms of 83 emu/g. The
decrease in the Ms value is due to the oxidation of iron that occurs during the modification
process. However, the hydrophilic TMAH coated particles only have an Ms value of 73 emu/g,
showing that nanoparticles with a hydrophilic coating are more easily oxidized.
Figure 5: VSM results for original (CTAB), HMDS, and TMAH coated Fe/Fe
3O4
nanoparticles
OTS silanization works analogously to HMDS silanization, but the longer hydrophobic
tail of OTS (18 carbons) functions to better protect the iron core of the particles. Figure 6 shows
the heating results showing the heating effects of particles coated with OTS and
phosphatidylcholine (PC, a biocompatible coating, to be discussed later) versus particles coated
with HMDS and PC. Since both solutions consist of 0.6% of some iron-containing compounds,
the higher heating effect of the OTS/PC particles must be due to better preservation of the iron
core thanks to the longer hydrophobic tail on OTS.
6
Figure 6: Heating effect results comparing hydrophobic silane coatings of
Fe/Fe
3O4/OTS/PC vs. Fe/Fe3O4/HMDS/PC particles
Since silanes are not biocompatible, a biocompatible coating must be added over the
silane, usually via van der Waals forces. Figure 7 shows a mechanism of the addition of a
biocompatible phospholipid, phosphatidylcholine (PC), to a silanized nanoparticle.
Figure 7: Mechanism of PC assembly via van der Waals forces to form a biocompatible
outermost layer
5
Pluronic
® copolymers, which are also added via van der Waals forces (Figure 9), provide
a unique way of preserving the iron core and providing a biocompatible coating due to their
hydrophobic and hydrophilic chain sections (Figure 8) and temperature dependent properties.
7
8. a) b)
Figure 8: a) General structure of Pluronic
® copolymers b) Structure of the two
copolymers used to modify nanoparticles
5
Figure 9: Schematic of Fe/Fe
3O4 nanoparticle modification with Pluronic® copolymer5
Below critical micelle temperature (CMT), Pluronic
® is hydrophilic; above it, the PPO
chain dehydrates and becomes hydrophobic and micelles self-assemble. At even higher
temperatures, the entire chain becomes hydrophobic and micelles self-associate.
4 It is
hypothesized that these properties might help produce better hyperthermia heating effects.
Another way to preserve the iron core and provide a biocompatible coating is through
covalent bonds to the nanoparticle and between coating layers; these tighter bonds better
preserve the iron core and hold coatings in place. According to Yunyu Shi, 2-(3,4-
dihydroxyphenyl)ethylamine (dopamine, or DA) provides good, strong covalent bonds to Fe
3O4
in nanoparticles.
3 The NH2 group of dopamine can then be used for further functionalization,
such as formation of an amide bond with biocompatible carboxyl-group terminated polyethylene
glycol (PEG), yet with two carboxyl groups on PEG, cross-linking between particles becomes a
problem.
3 Cross-linking was avoided by maintaining a low concentration of particles in solution,
thus linking both carboxyl groups of PEG to a single particle. Dopamine and PEG were bonded
together first via amide bond formation by the EDC reaction, as seen in Figure 10a. Particles
were then added to solution to form a bond between the hydroxyl group of dopamine and the
nanoparticle (Figure 10b). The particles were added second in order to avoid prolonged exposure
to aqueous conditions, which leads to oxidation of the iron core.
8
No comments:
Post a Comment