Magnetic nanoparticles with chelating shells prepared by RAFT/MADIX polymerization

Article abstract of DOI:10.1039/c6nj01938b

In this study, the preparation of multifunctional materials based on magnetic nanoparticles (MNP) and original thiosemicarbazide derivatives is presented. The synthesized nanohybrids consist of an iron oxide core and a polymeric shell, which possesses the ability to complex metal ions. They exhibit superparamagnetic properties and can thus be easily separated from complex mixtures using an external magnetic field (facile separation, purification, and recyclability). Synthesized carbamohydrazonothioate derivatives were polymerized on dithiocarbonate-coated MNPs using the RAFT/MADIX (reversible addition-fragmentation transfer/macromolecular design by interchange of xanthates) method. Three types of nanohybrids were obtained and their physicochemical properties were investigated. The ability of nanohybrids to complex palladium(ii) ions and the spectroscopic properties of the obtained materials were studied. In addition, the hemolytic activity tests of polymer-coated particles were performed in order to confirm their potential in biomedical applications.

Full text of DOI:10.1039/c6nj01938b

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Magnetic nanoparticles with chelating shells prepared by
RAFT/MADIX polymerization
Karolina H. Markiewicz,a† Paula Zembko,a Katarzyna Półtorak,a Iwona Misztalewska,a Sławomir
Wojtulewski,a Anna M. Majcherb, Emilia Fornalc and Agnieszka Z. Wilczewskaa†
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In this paper is presented the preparation of multifunctional materials based on magnetic nanoparticles (MNP) and
original thiosemicarbazide derivatives. Synthesized nanohybrids consist of iron oxide core and polymeric shell which
possess ability to complex metal ions. They exhibit superparamagnetic properties and can thus be easily separated from
complex mixtures by using an external magnetic field (facile separation, purification and recyclability). Synthesized
carbamohydrazonothioate derivatives were polymerized on dithiocarbonate-coated MNP using RAFT/MADIX (reversible
addition-fragmentation transfer/macromolecular design by interchange of xanthates) method. Three types of nanohybrids
were obtained and their physicochemical properties were investigated. The ability of nanohybrids to complex palladium(II)
ions and spectroscopic properties of the obtained materials were studied. Additionally, hemolytic activity tests of polymer-
coated particles were performed in order to confirm their potential in biomedical applications.
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they exhibit interesting biological properties (i.e. antibacterial,
antiviral, and antineoplastic activity) related to their ability to
diffuse through a semipermeable cell membranes.13–17 Low costs
Introduction
Magnetic nanoparticles (MNP) are an emerging class of materials
for applications in catalysis, nanomedicine, and environment
protection.1–3 Bare MNP tend to agglomerate and phase separate in
solution, thus, it is essential to stabilize them with a protecting
shell. Mostly, polymers are favored over small molecules as they
can ensure an efficient stabilization of nanoparticles and give the
possibility to control chemical composition (homopolymer,
copolymer) and properties (e.g. hydrophobic, hydrophilic) of a
shell.4 On the other hand, anchoring polymers to MNP ensures fast
separation of such nanohybrids, good recovery after using and also
enables reapplication of the material. Additionally, a suitably
designed polymeric shell can offer some extra functions (e.g.
biocompatibility, resistance to physiological conditions (pH or
enzymes), complexing properties or biological activity) leading to a
multifunctional polymer-magnetic nanohybrids.
and facility of their synthesis are additional factors justifying their
wide applications in medicinal18–20 and analytical chemistry21–24. As
thiosemicarbazide-based ligands are broadly used in various fields,
recovery of their metal complexes is of great importance. Anchoring
this type of ligands on magnetically separable solid phase is a
promising solution.
In this study, RAFT/MADIX (reversible addition-fragmentation
transfer/macromolecular design by interchange of xanthates)
polymerization was employed to form shells around MNP.
RAFT/MADIX technique is
a
type of controlled radical
polymerization which uses xanthates/dithiocarbonates (chain
transfer agents) to mediate polymerization process.25,26 This
method enables an excellent control of growth, composition and
architecture of polymers.27 It can be easily performed in mild
conditions and is compatible with a huge variety of monomers.4,27,28
In ‘grafting from’ approach initial immobilization of chain transfer
Herein we describe the preparation of functional (complexing and
easily separable) polymer-magnetic nanohybrids using iron oxide
nanoparticles and original thiosemicarbazide-based monomers.
Thiosemicarbazide and its derivatives are excellent chelating ligands
for transition metal ions due to their mixed hard-soft donor
character and versatile coordination behavior.5–13 Furthermore,
agents (CTA) on
a nanoparticle surface is followed by
polymerization of vinyl monomer. CTA is anchored to a nanoparticle
surface by its R-group and consequently act as propagating leaving
group (polymer chains are grown directly from a nanoparticle
surface).29 There are only several reports describing covalent
modification of MNP with dithiocarbonates, including our previous
results.2,30–33
Three types of polymer-magnetic nanohybrids were obtained using
thiosemicarbazide-based monomers. Their metal complexing
abilities were studied. Additionally, hemocompatibility of monomer
and polymer-coated MNP was evaluated to confirm the interest of
using such nanohybrids in biomedical fields. It is worth nothing that
there are only a few reports describing polymers containing
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thiosemicarbazide moiety,16,34,35 and none of them concerns
polymerization at the surface of magnetic nanoparticles.
BMS is given in the Table S1. CCDC-1049864 (BM) and CCDC-
1049967 (BMS) contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Results and discussion
Synthesis of bifunctional monomers. The synthesis of ligand
involved two reactions. Initially, terminal amine group of
thiosemicarbazide (TSC) was protected in the reaction with
benzaldehyde (Figure S1-S2). Next, purified benzaldehyde
thiosemicarbazone (BTSC) was treated with 4-chloromethyl styrene
(CMS). The whole process is presented in Figure 1. Operating
conditions in the second reaction were optimized to obtain the
highest yield of the product. In order to avoid undesirable
polymerization reaction of vinyl functions the reaction was
performed in room temperature without the access of light and in
the presence of polymerization inhibitor.
Surface initiated RAFT/MADIX polymerization. RAFT/MADIX
polymerization technique was employed to conduct polymerization
of the obtained monomers on the surface of dithiocarbonate-
coated magnetic nanoparticles (Figure 1). In this study, iron oxide
core-aminosilane shell nanoparticles were used. Their synthesis,
characterization and applications have been previously described.37
Ethyl dithiocarbonate (xanthate) - RAFT/MADIX chain transfer agent
- was covalently bonded to the surface of nanoparticles according
to the reported method.32 Polymerization was realized by simple
introduction of a dithiocarbonate-coated magnetic nanoparticles
(MNP@SC(S)OEt) to a conventional free-radical system (monomer
and initiator). Prior reaction RAFT/MADIX agent, monomer, initiator
and solvent were sonicated until a homogeneous suspension was
formed. The reactions were carried at the constant temperature for
24h under argon gas protection. Due to the differences in the
solubility of monomers (BM and BMS) polymerization reactions
were performed in different solvents. Initially, several solvents were
investigated e.g. toluene, THF, methanol, and ethanol. The best
results i.e. the highest thicknesses of polymeric shells (determined
by TG analysis) were obtained using THF (BM) and ethanol (BMS) as
the solvents. AIBN was used as initiator and it was added in 4
portions, at the beginning of the polymerization, after 2 hours, 4
hours and 16 hours of stirring. After reactions, the cycle of
redispersion in solvent and magnetic separation was used several
times to rinse unreacted monomer, free polymer chains and obtain
‘pure’ polymer-grafted nanoparticles. In each case mass increase
after polymerization was observed. As a result, two types of
nanohybrids were obtained: MNP@PBM and MNP@PBMS.
Figure 1. Synthesis of bifunctional thiosemicarbazone-based monomer and
its RAFT/MADIX polymerization on dithiocarbonate-coated MNP.
The obtained product was purified and characterized by NMR (1H,
13C, 2D), FT IR, LC MS/MS spectroscopies (Figure S3-S7) and
elemental analysis (CHNS). An X-ray diffraction analysis was also
taken to determine the molecular structure of the product (Figure
2).
Free radical polymerizations and RAFT/MADIX polymerizations
(using O-ethyl-S-(1-methoxycarbonyl)ethyldithiocarbonate as CTA26
)
of both monomers were also carried out in solution. In all cases, in
the course of reactions, formation of poorly soluble solid fractions
was observed. Apparently, growing chains were excluded during
reaction due to decrease of solubility. 1H NMR spectra of these
fractions confirmed polymerization. The signals of vinyl protons (at
5.2, 5.7 and 6.7 ppm) disappeared, the peaks of aromatic protons
(7.3, 7.8 ppm) become broader, and also new broad signals related
to CH and CH2 protons in polymer chains appeared in the range 1-
1.7 ppm. Additionally, in the cases of RAFT/MADIX polymerization,
the absence of CTA agent signals in the NMR spectra of soluble
residuals confirmed participation of CTA in polymerization process.
It is worth of pointing out that during polymerization performed on
MNP@SC(S)OEt particles size exclusion, which is inevitable during
polymerization carried out in solution, does not take place. In this
regard, polymerization on MNP@SC(S)OEt surface is not limited by
the solubility of growing polymer chains.
Figure 2. Molecular structures (a’), (b’) and crystal structures (a’’) (b’’) of BM
and BMS respectively (hydrogen bonds - dashed lines). The figure was
prepared with OLEX236
.
In order to prepare monomer which could be polymerized in less
toxic polar solvents the obtained bifunctional monomer was
transformed into its hydrochloric salt (BMS) (Figure S9-S13). The
changes in NMR and FT IR spectra of BMS in respect to BM indicate
differences in the structure of both products. Additionally,
determined by XRD molecular structure of BMS is different (Figure
2). A summary of relevant crystallographic data for both BM and
The modifications of the MNP@SC(S)OEt surface were investigated
by ATR FT IR spectroscopy. The spectra of the obtained monomers
and polymer-grafted nanoparticles are presented in the Figure 3.
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likely related to the decomposition of carbamohydrazonothioate
part, whereas the third one, with the maximum at 800°C, is
attributed to the degradation of the polymer chain as well as silica
shell. This assumption is based on TG studies of pure monomers
which decompose at a broad temperature range between 150/200-
650°C (Figure S8 and S14). In the thermograms of the MNP@PBMS
sample (Figure 4a and 4b) similar curves characteristics can be
observed. However, the rate of mass change is different in this case
and the maxima in DTG curve are harder to distinguish comparing
to MNP@PBM sample. Two first degradation stages merge into
broad peak (150-510°C) with two maxima at 256 and 354°C
whereas the third one is slightly shifted to a lower temperature
range (650-830°C) with maximum at 710°C. The differences in
thermograms of MNP@PBM and MNP@PBMS indicate diverse
chemical nature of the polymers surrounding MNP. Comparison of
the total weight losses revealed higher polymerization efficiency for
BM than for BMS under the applied conditions.
Figure 3. ATR FT IR spectra of obtained monomers and polymer-magnetic
nanohybrids.
In each spectrum of nanoparticles a similar set of bands can be
observed. The existence of magnetic core is indicated by broad
band at 550-570 cm−1 which corresponds to the Fe-O stretching
modes. The band at around 1000-1150 cm-1 can be ascribed to the
Si-O, Si-O-Si and Fe-O-Si bonds vibrations (these bonds created by
sol gel method with (3-aminopropyl)trimethoxysilane are present in
the starting material - MNP@SC(S)OEt).32 The bands at around 2900
cm−1 corresponding to C–H stretching modes and broad band with
the maximum around 3300 cm-1 related to N-H vibrations are also
present in each spectrum. In the spectrum of MNP@SC(S)OEt
nanoparticles, the absorption modes at 1647 cm−1 and 1537 cm-1
correspond to the vibrations of amide moieties (C=O stretching, N-H
bending and C-N stretching). The peaks at 1199 cm−1 and at 1043
cm−1 can be assigned to the C-O-C and S-C-S stretching vibrations of
ethyl xanthate groups. After polymerization reactions changes in
the spectra of MNP@PBM and MNP@PBMS samples are observed,
primarily, in the fingerprint region. For example, at 660-690 cm-1 C-
H bending vibrations of aromatic rings can be seen. Furthermore,
additional (in comparison to MNP@SC(S)OEt) bands around 1200-
1450 cm-1 appeared which can be ascribed to vibrations of CH and
CH2 groups in polymer chains. The stretching modes of C=N, and
C=CAr groups showed up at 1500-1670 cm-1 changing intensity and
shape of bands in this region.
Thermogravimetric studies were performed in order to investigate
thermal properties of the obtained nanoparticles. Figure 4 shows
TG (4a) and DTG (4b) curves of MNP@SC(S)OEt, MNP@PBM, and
MNP@PBMS samples. TG curve refers to the temperature-
dependent mass change in percent, whereas DTG curve refers to
the rate of mass change. The TG curve of the MNP@SC(S)OEt shows
total weight loss around 10% in the entire temperature range. This
weight loss is related to the decomposition of ethyl xanthate as well
as aminosilane shell which is present on MNP surface. After
polymerization reactions total weight loss in both samples is higher
(51% for MNP@PBM and 30% for MNP@PBMS). TG and DTG curves
of MNP@PBM (Figure 4a and 4b) show three stages of weight loss
at temperature ranges of 190-320°C, 320–500°C and 700–880°C.
The first two ones, with the maxima at 280°C and 390°C are most
Figure 4. (a) TG and (b) DTG curves of magnetic nanohybrids.
Figure 5 shows TEM and SEM images of MNP@PBM particles. Bare
iron oxide nanoparticles are likely to agglomerate and create
clusters. Thus, in TEM micrographs, aggregates of magnetic cores
(with a relatively narrow size distribution and the average diameter
of 10 nm) covered with a shell are observed. The shell surrounding
MNP@PBM particles consist of aminosilane and polymer layers and
is around 2-3 nm. Similar morphologies were observed in the case
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of MNP@PBMS, however, the thickness of the shell is lower. These
results are in a good agreement with the data obtained by TG
analysis.
with transition metal ions by bonding through the sulphur and
hydrazinic nitrogen atom. Obtained in this study monomers are S-
substituted, therefore, their chelating abilities are different.
However, these monomers (and thus their polymers) possess
binding sites such as –NH or –CH=N– able to form metal complexes.
To study complexing abilities of the obtained monomers and
polymer-coated nanoparticles Pd(II) metal ions were used. Initially,
pure monomers were examined in complexation procedures. The
solution of metal salt (2.5·10-5M) was added dropwise to the
solution of monomer (2.5·10-5M). The resulting mixture was tested
by UV-Vis spectroscopy to investigate binding action of monomers
with metal ions. The spectra of pure monomers show two different
transitions with the λmax values around 250 and 300 nm. Slight shifts
of the band at 250 nm and vanishing of the band at 300 nm were
observed in both cases (Figure S15). Next, in order to investigate
their chelating properties, both types of polymer-coated
nanoparticles (MNP@PBM and MNP@PBMS) have been tested in
the complexation procedures. The suspensions of nanoparticles and
solutions of metal ions were shaken under argon atmosphere in
room temperature for 24h. The products were magnetically
separated and carefully washed few times to rinse all uncomplexed
metal salts. UV-Vis analyses performed after this step revealed no
significant changes in spectra. It can be related to the fact that
Figure 5. (a) TEM image and (b) SEM image of MNP@PBM particles.
Zeta potential measurements were performed to determine
colloidal stability of the obtained nanohybrids. The values of zeta
potential for MNP@PBM (11.3 ± 0.6 mV) and MNP@PBMS (37.4 ±
1.1 mV) nanoparticles show that the latter ones are more stable in
aqueous dispersion.
Magnetization versus field measurements within
a range of
temperatures revealed that bare nanoparticles exhibit
superparamagnetic behaviour at room temperature. In the case of
modified MNP increasing the temperature up to the experimentally nanoparticles themselves (MNP@SC(S)OEt) absorb strongly in the
entire tested range.
Thiosemicarbazones have been reported as fluorescent sensors for
available maximum of 400 K was necessary to approach the
superparamagnetic region and to obtain closing of the hysteretic
loops which are still visible at 300 K. Law of approach to saturation
(LA) was fitted to the data in high magnetic fields to obtain the
values of the saturation magnetisation.38 Figure 6 shows that the
value of the saturation magnetization (MS) for polymer-coated MNP
is reduced in comparison to bare and aminosilane-coated MNP (e.g.
62.9 and 48.6 emu g-1, for MNP and MNP@PBM, respectively).
These results are consistent with our previous studies which
revealed that MS value mostly depends on the thickness of a shell
and barely depends on its composition.39
recognition of transition metal ions.23,24 Therefore, the fluorescence
response of obtained monomers towards metal ions was
investigated. Free monomers do not show emission bands whereas
their metal complexes show emission maxima around 312/315 nm
(Figure S16).
Figure 7. Fluorescence spectra of polymer-modified MNP and their
complexes with Pd(II) (normalized to 1).
Figure 7 presents emission spectra of MNP modified with polymers
and their complexes with Pd(II). Emission maxima for MNP@PBM
and MNP@PBMS samples are observed at 352 and 340 nm,
respectively. Introduction of palladium(II) ions caused changes in
the emission spectra of nanohybrids, emission maxima in both
cases are blue shifted to 310 nm. Additionally, in the spectrum of
MNP@PBMS-Pd sample the second maximum at 400 nm appeared.
Figure 6. Magnetization as a function of external magnetic field measured
for MNP, MNP@NH2 and MNP@PBM at 400 K. Solid lines represent LA fits
to the data.
Complexation of palladium(II) ions. Thiosemicarbazide and their
derivatives (e.g. thiosemicarbazones) react as complexing ligands
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examined by FT IR, TGA and SEM/EDX methods and compared to
MNP@PBM-Pd sample.
Thermogravimetric analyses of MNP@PBM-Pd and MNP@PBMS-Pd
samples were performed to estimate the amount of Pd in these
samples (Figure 8). In both cases, lower weight loss is observed for
complexes than for ‘free’ polymer-magnetic nanoparticles. It is
associated with metal residuals present in these samples. The
difference of total weight losses for MNP@PBM and MNP@PBM-Pd
is higher than for MNP@PBMS and MNP@PBMS-Pd (20% and 12%)
suggesting higher amount of complexed Pd(II) in the former case.
In figure 9a similar set of bands in both spectra as in the
MNP@PBM spectrum (Figure 3) can be observed. Pd-Cl stretching
frequencies cannot be seen because they normally occur in far
infrared region (below 400 cm-1). However, the ratio of intensities
of polymer bands (above 1000 cm-1) to magnetic core bands (550
cm-1) is much higher in MNP@PBM-Pd(2) sample. This indicates
thicker polymer shell in the case of MNP@PBM-Pd(2) particles and
suggests higher efficiency of metal-monomer complex
polymerization than free monomer polymerization. TG
measurements (Figure 9b) confirmed this data showing higher total
weight loss for MNP@PBM-Pd(2) sample (50%) than for
MNP@PBM-Pd (30%). SEM/EDX analysis of MNP@PBM-Pd(2)
sample revealed 16.59 (±0.53) mass percent of palladium in the
sample.
Figure 8. TGA/DTG curves of MNP@PBM, MNP@PBM-Pd, MNP@PBMS, and
MNP@PBMS-Pd.
SEM/EDAX analyses were performed to further confirm the
presence and estimate the amount of metal ions in MNP@PBM-Pd
and MNP@PBMS-Pd samples. According to the results, amount of
palladium in MNP@PBM-Pd (19.6±2.1%) sample is roughly twice as
high as it is in MNP@PBMS-Pd (9.7±0.5%). It can be related to a
different amount of complexing shells (PBM and PBMS) surrounding
magnetic cores as well as to different complexing abilities of these
layers. Our previous results showed that MNP coated with
aminosilane shell do not show complexing activity towards
palladium(II).37
To study polymerization process of synthesized monomer on
MNP@SC(S)OEt nanoparticles a second approach was used. The
strategy relied on initial preparation of a ligand-metal complex (BM-
Pd) followed by its polymerization on MNP@SC(S)OEt.
Polymerization was conducted in the same conditions as in the case
of free monomer. Obtained MNP@PBM-Pd(2) particles were
Figure 9. (a) ATR FT IR spectra and (b) TGA/DTG curves of MNP@PBM-Pd
and MNP@PBM-Pd(2).
Hemocompatibility of bifunctional monomer and obtained
nanoparticles.
Thiosemicarbazide-based
polymer-magnetic
nanohybrids can be used in a range of biomedical applications.
However, as nanoparticles may generate various toxic effects in
host cells,40 risk associated with the exposure to these materials has
to be evaluated. The hemolytic activity of BM, MNP@SC(S)OEt and
MNP@PBM was tested following standard protocols using human
red blood cells (RBCs) as the samples.41 Figure 10 shows results of
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the hemolytic activity measurements. None of tested materials
disrupt RBCs membranes at concentrations of 1 to 100 μg/mL
(hemolysis rates were lower than 5%).
Experimental
Materials and methods
Thiosemicarbazide, benzaldehyde, 4-chloromethyl styrene,
hydroquinone, and palladium(II) chloride were purchased from
Sigma-Aldrich and used as received. Initiator 2,2′-azobis(2-
methylpropionitrile) (AIBN) was obtained from MERCK and was
recrystallized from chloroform. Triethylamine and all solvents were
purchased from Avantor Performance Materials Poland S.A. and
were distilled prior use.
The 1H and 13C NMR spectra were recorded on a Bruker Avance
spectrometer (400 and 100 MHz, respectively) as solutions in CDCl3,
CD3OD or DMSO. Chemical shifts are expressed in parts per million
(ppm, δ) downfield from tetramethylsilane (TMS). FT IR spectra
were recorded using Thermo Scientific Nicolet 6700 FT IR
spectrophotometer. A thin layer of sample was placed in direct
contact with an infrared attenuated total reflection (ATR) diamond
crystal. All FT IR spectra were collected in the wavenumber range
of 4000 to 500 cm-1 by co-adding 32 scans with a resolution of 4
cm-1. Melting points were determined on a Mettler Toledo MP70
system. Elemental analyses were carried out using a VarioMICRO
Figure 10. Hemolysis percentages measured at the concentration range of 1-
100 μg/mL for BM, MNP@SC(S)OEt, and MNP@PBM incubated with RBCs at Cube analyzer from Elementar Analysensysteme GmbH. High-
37°C for 1 h.
resolution mass spectrometry experiments were carried out using a
liquid chromatograph (series 1200, Agilent Technologies) coupled to
a tandem mass spectrometer (Agilent Technologies 6538 UHD
Accurate Mass Q-TOF LC/MS) equipped with a HPLC-chip-cube.
Crystals of BM and BMS suitable for single crystal X-ray structural
measurement were obtained by slow evaporation of the methanol
solution at the room temperature. The X-Ray data were collected
on a SuperNova diffractometer (Agilent) with a CCD detector and
MoKα radiation at 100K. The crystal structures were solved with
Conclusions
Herein is presented the formation of new type of chelating-
magnetic nanohybrids. These nanohybrids consist of polymers
covalently attached to the surface of iron oxide nanocores,
therefore, they are stable and resistant to washout. In order to
obtain these materials, new polymerizable and complexing
monomer based on thiosemicarbazide was synthesized and fully
characterized. Next, surface-initiated RAFT/MADIX polymerization
reactions of the monomer and its hydrochloric salt (BM and BMS)
were performed on xanthate-coated iron oxide nanoparticles
(MNP@SC(S)OEt). In both cases formation of polymer coating was
documented by FTIR, TEM and TG analyses. To study complexing
abilities of MNP@PBM and MNP@PBMS nanohybrids Pd(II) ions
were used. The results of SEM/EDX microscopy and TG analysis
showed that the content of metal varies depending on the type of
shell and it is roughly twice as high in MNP@PBM-Pd as in
MNP@PBMS-Pd samples. Additionally, the second approach to
polymerization on the surface of dithiocarbonate-coated MNP was
olex2.solve and refine with olex2.refine42 (BM) and ShelXL (BMS)43
.
All hydrogen atoms were located in electron-density difference
maps. The non-hydrogen atoms were refined anisotropically.
Transmission electron microscopy photographs were taken on
Tecnai G2 X-TWIN microscope. SEM/EDAX measurements were
performed on a SEM FEI FP2017/12 Inspect instrument equipped
with an Ametek energy dispersive X-ray EDAX Octane Pro system
operate at an accelerating voltage of 25 kV. A thin layer of sample
was placed on carbon tape and coated with 5 nm layer of gold.
Magnetic properties of the nanoparticles were studied using a
Quantum Design MPMS 5XL SQUID-type magnetometer. The
sample was placed in a standard gelatin capsule and immobilized
with Varnish glue for the measurement. All the data were carefully
corrected for the diamagnetic contribution of the holder.
Thermogravimetric analysis (TGA) was performed on a Mettler
Toledo Star TGA/DSC unit. Nitrogen was used as a purge gas (20
mL·min-1). Samples between 2 and 10 mg were placed in aluminum
pans and heated from 50°C to 800°C (TGA) with a heating rate of
20K/min. Colloidal stability of the obtained materials was studied
applied
- bifunctional monomer-metal complex (BM-Pd) was
polymerized on MNP@SC(S)OEt. Relatively comparable content of
palladium ions was estimated for its MNP@PBM complexes
obtained by both approaches. However, the second strategy
resulted in higher polymerization efficiency.
The obtained materials due to their combined properties
(magnetism and metal affinity) are promising candidates for
application in heavy metal ions removal or organic catalysis. It is using the Zetasizer Nano-ZS (Malvern Instruments, Worcestershire,
worth nothing that bactericidal properties of the obtained
monomers were successfully tested (in-house data). Additionally,
hemolytic activity test results confirmed the possibility of using
these nanohybrids in biomedical applications (hemolysis rates were
lower than 5%).
UK) equipped with a 4-mW helium/neon laser (λ = 633 nm) and a
thermoelectric temperature controller. Particle suspensions
prepared in deionized water (0.05 wt%) were preincubated at 25°C
for 5 min before measurement. UV-Vis spectra were taken on Jasco
V-670 Spectrophotometer and fluorescence analyses were done on
Hitachi F7000 Fluorescence Spectrophotometer (exciation
6 | J. Name., 2012, 00, 1-3
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wavelength 254 nm). Hemolytic activity was investigated using Synthesis of vinyl derivative of benzaldehyde thiosemicarbazone
human red blood cells (RBCs) suspended in phosphate-buffered salt (BMS). HCl was dropped to BM solution in CHCl3. Precipitation
saline (PBS) (hematocrit ~5%) with a concentration of tested of yellow powder was observed. The product (1E,2E)-1-{amino[(4-
antibacterial agents ranging from 0-100 μg/ml. RBCs were ethenylbenzyl)sulfanyl]methylidene}-2-benzylidenehydrazinium
1
incubated with tested agents for 1 h at 37°C. Relative hemoglobin chloride was recrystallized from methanol. mp = 176.1-177.9°C. H
concentration in supernatants after centrifugation at 2500 rpm for NMR δH(400 MHz, CD3OD, Me4Si): 8.28 (s, 1H, HC=N), 7.78 (m, 2H,
15ꢀmin was monitored by measuring optical absorbance at 540 nm. H-Ar), 7.38 (m, 7H, HAr), 6.78 (m, 1H, CH=), 5.77 (d, J=17.65 Hz, 1H,
100% hemolysis was taken from samples in which 1% Triton X-100 CH2=), 5.21 (d, J=10.88 Hz, 1H, CH2=), 4.29 (s, 2H, CH2-S). 13C NMR
was added to disrupt all cell membranes.
δC(100 MHz, CD3OD, Me4Si): 162.7 (C-S), 152.2 (CH), 136.6 (C),
136.1 (C), 133.4 (CH), 131.0 (CH), 129.3 (CH), 128.7 (CH), 126.4 (CH),
114.6 (CH2), 34.1 (CH2). ATR FTIR (νmax, cm-1): 3362 (NH), 3038
(CHAr), 2971 (CH), 1645 (C=N), 1581, 1511 (NH), 1447, 1408 (C=CAr),
1135 (CN), 1051, 758, 688 (CHAr), 617(CS). Elemental analysis.
Found: C, 59.48; H, 5.57; N, 11.50; S, 9.06. C18H22N3SClO requires C,
59.41; H, 6.09; N, 11.55; S, 8.81.
Ethics Statement. Hemolytic activity of tested agents was evaluated
in blood samples from adult healthy volunteers under IRB approval:
R-I-002/382/2012. This study was approved by the institutional
review board (IRB) of The Medical University of Bialystok. All
subjects provided informed written consent and collected samples
were anonymous.
General procedure for free radical polymerization. Monomer (BM
or BMS) and solvent (THF or ethanol) were placed in a flask
Synthetic procedures
Synthesis
of
benzaldehyde
thiosemicarbazone
(BTSC). equipped with a magnetic stirrer and were degassed for 15 min by
Benzaldehyde (6.1 mL, 60 mmol, 1.1 equiv.) was dropped to the bubbling argon. Then, AIBN was added and solution was heated at
solution of thiosemicarbazide (5 g, 55 mmol, 1 equiv.) in anhydrous 60°C/80°C for 24h under argon gas protection. During reactions
ethanol (200 mL). The mixture was stirred for 60 h at room precipitation of yellow poorly soluble fractions was observed. PBM
temperature under light shielding conditions. Then, the solvent was 1H NMR δH(400 MHz, CDCl3, Me4Si): 8.07-8.01 (HC=N), 7.60-7.00
removed under reduced pressure and obtained crude product was (HAr), 4.39-4.11 (CH2-S), 1.81-1.08 (CH-CH2). PBMS 1H NMR δH(400
purified by crystallization from ethyl acetate to give yellowish MHz, CD3OD, Me4Si): 8.04-8.01 (HC=N), 7.50-6.50 (HAr), 4.36-4.34
1
powder (4.1 g, 82%). H NMR δH(400 MHz, DMSO, Me4Si): 11.42 (s, (CH2-S), 1.81-1.26 (CH-CH2).
1H, =N-NH), 8.19 (d, J=42 Hz, 2H, NH2), 8.04 (s, 1H, -CH=N-), 7.81 General procedure for RAFT/MADIX polymerization with O-ethyl-
(m, 2H, HAr), 7.38 (t, J=3.29 Hz, 3H, HAr). 13C NMR δC(100 MHz, S-(1-methoxycarbonyl)ethyldithio-carbonate. O-ethyl-S-(1methoxy
DMSO, Me4Si): 178.0 (C=S), 142.3(CH), 134.2 (C), 129.8 (CH), 128.7 carbonyl)ethyldithiocarbonate, monomer (BM or BMS) and solvent
(2CH), 127.3 (2CH). ATR FTIR (νmax, cm-1): 3399, 3229, 3140 (NH), (THF or ethanol) were placed in a flask equipped with a magnetic
3017 (CHAr), 2975 (=CH), 1599, 1583(NH), 1523, 1468 (SCN), 1446 stirrer and were degassed for 15 min by bubbling argon. Then, AIBN
(C=CAr), 1372 (CH), 1282, 1224, 1098 (CN), 1054 (C=S), 948, 868, 817 was added and solution was heated at 60°C/80°C for 24h under
(CHAr), 759, 686, 611, 539 (CS). Elemental analysis. Found: C, 53.88; argon gas protection. During reactions precipitation of yellowish
H, 5.01; N, 23.11; S, 18.03. C8H9N3S requires C, 53.61; H, 5.06; N poorly soluble fractions was observed. PBM-SC(S)OEt 1H NMR
23.44; S, 17.89.
δH(400 MHz, DMSO, Me4Si): 8.10-7.97 (HC=N), 7.50-7.30 (HAr), 4.41-
Synthesis of vinyl derivative of benzaldehyde thiosemicarbazone 4.37 (CH2-O), 3.77-3.76 (CH-COO), 3.17-3.14 (CH2-S), 1.55-1.00 (CH2,
(BM). Benzaldehyde thiosemicarbazone (4g, 22 mmol, 1 equiv.) was CH). PBMS-SC(S)OEt 1H NMR δH(400 MHz, CD3OD, Me4Si): 7.90-7.80
dissolved in anhydrous ethanol (200 mL), then, 4-chloromethyl (HC=N), 7.60-7.30 (HAr), 4.50-4.43 (CH2-O), 3.70-3.60 (CH-COO),
styrene (6.3 mL, 45 mmol, 2 eq) was added. The reaction was 3.20-3.10 (CH2-S), 1.55-1.10 (CH2,CH).
General procedure for polymerization on MNP@SC(S)OEt.
A
performed in the presence of Et3N and hydroquinone at room
temperature for 24h under light shielding conditions. The solvent
was evaporated under reduced pressure. The product was purified
by medium pressure liquid chromatography (MPLC) and
crystallization from n-hexane and dichloromethane. Yellowish
powder (4-ethenylbenzyl N'-[(E)-phenylmethylidene]carbamohydra
typical polymerization procedure was as follows: a mixture of
MNP@SC(S)OEt, monomer (BM or BMS), solvent and AIBN was
sonicated under argon gas protection until
a homogeneous
suspension was formed. Next, it was transferred into an oil bath
and stirred at the constant temperature at 60°C/80°C. After 48h,
the polymerization was stopped by diluting the mixture in solvent
and magnetic decantation. The cycle of redispersion and magnetic
separation was repeated several times to rinse free polymer chains
and obtain “pure” polymer-grafted nanoparticles. MNP@PBM ATR
FTIR (νmax, cm-1): 3300 (NH), 3059 (CHAr), 2927 (CH), 1666 (NH),
1
zonothioate) was obtained (5.4 g, 83%). mp = 78.4-79.5°C. H NMR
δH(400 MHz, CDCl3, Me4Si): 8.43 (s, 1H, HC=N), 7.78 (m, 2H, H-Ar),
7.42 (m, 7H, H-Ar), 6.78 (m, 1H, CH=), 5.79 (d, J=10.87 Hz, 1H, CH2=),
5.25 (d, J=10.85 Hz, 1H, CH2=), 4.36 (s, 2H, CH2-S). 13C NMR δC(100
MHz, CDCl3, Me4Si): 161.1 (C-S), 154.6 (CH), 136.9 (C), 136.7 (C),
136.3 (C), 135.0 (C), 130.0 (CH), 129.2 (CH), 128.5 (CH), 127.7 (CH),
126.4 (CH), 113.9 (CH2), 34.1 (CH2). ATR FTIR (νmax, cm-1): 3473, 3362
(NH), 3079, 3057, 3025 (CHAr), 2936, 2895 (CH), 1966, 1917, 1813
(Ar), 1604 (C=N), 1577, 1523 (NH), 1489, 1446 (C=CAr), 1355, 1305
(CH), 1197, 1172 (CN), 902, 825, 756, 698 (CHAr), 643 (CS).
Elemental analysis. Found: C, 69.03; H, 5.66; N, 13.40; S, 10.84.
C17H17N3S requires C, 69.12; H, 5.8; N, 14.22; S, 10.85. LC MS/MS
(ESI) m/z found 296.1220 [C17H17N3S+H]+; calculated 296.1216.
1416 (CH) 1112, 1015 (SiO), 557 (FeO). MNP@PBMS ATR FTIR (νmax
cm-1): 3332 (NH), 2921, 2850 (CH), 1646 (NH), 1444 (CH), 1113,
,
1042 (SiO), 565 (FeO).
Acknowledgements
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This work was financially supported by the National Science 24 S. Suganya, D. Udhayakumari and S. Velmathi, Anal. Methods,
Centre, Poland, grants no. NCN/2011/03/B/ST5/02691 (AZW) and 2013, 5, 4179–4183.
2014/13/N/ST5/01563 (KHM). The equipment in The Center of 25 M. Destarac, C. Brochon, J.-M. Catala, A. Wilczewska and S. Z.
Synthesis and Analysis BioNanoTechno of University of Bialystok Zard, Macromol. Chem. Phys., 2002, 203, 2281–2289.
was funded by EU, project no: POPW.01.03.00–20–034/09–00 and 26 D. Taton, A.-Z. Wilczewska and M. Destarac, Macromol. Rapid
POPW.01.03.00-004/11. The authors gratefully acknowledge use of Commun., 2001, 22, 1497–1503.
the mass spectrometry services and facilities of the Center for 27 S. Perrier and P. Takolpuckdee, J. Polym. Sci. Part Polym. Chem.,
Interdisciplinary Research of The John Paul II Catholic University of 2005, 43, 5347–5393.
Lublin, funded by POPW.01.03.00-06-003/09-00. The authors are 28 M. Destarac, Macromol. React. Eng., 2010, 4, 165–179.
grateful to Dr Katarzyna Niemirowicz for hemolysis tests.
29 Y. Zhao and S. Perrier, in Controlled Radical Polymerization at
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Complexing and easily separable polymer-magnetic nanohybrids based on iron oxide nanoparticles
and original carbamohydrazonothioate derivatives.