3 Results and discussion
The synthesized magnetic colloids and fabricated thin films of colloids
by LBL-SAMU were studied using different characterization techniques.
The MRI studies along with characterization techniques are described
below.
3.1 Characterization of the
epoxy-functionalized magnetic colloidal
particles
Magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal
particles were successfully prepared using a seeded emulsion
polymerization technique. The chemical composition of the prepared
magnetic latex particles was investigated using FTIR spectroscopy
(Figure 2). As clearly seen from the FTIR spectrum (Fig.2a), magnetic
emulsion (ME) emulsion shows two characteristic peaks at 570 and 630
cm-1 which related to Fe-O vibration bands. After
polymerization (Fig.2b), new absorption bands at 1600
cm-1 (C=C stretching) and 3000-3100
cm-1 (C=CH stretching) of the aromatic benzene ring
appeared, indicating the formation of poly(divinylbenzene) in presence
of Fe3O4 nanoparticles. Additionally, a
very characteristic peak at 1725 cm-1 (C=O carbonyl
vibration), two absorption bands at 1255 and 1180 cm-1(C-O stretching) which related to the methacrylate ester vibrations of
GMA units, and two characteristic peaks at 837 and 909
cm-1 (CH stretching) of the oxirane ring of GMA also
appeared. These results prove the successful copolymerization of the
functional GMA monomer with DVB forming the epoxy-functionalized
magnetic colloidal particles [2].
Morphology of the prepared magnetic polymer particles was investigated
using TEM, as shown in Figure 3. TEM photos show the formation of
well-defined core-shell morphology upon coating the magnetic emulsion
droplets with the crosslinked poly(divinylbenzene-co-glycidyl
methacrylate) copolymers. The average hydrodynamic diameter
(D h) of the prepared magnetic particles was
measured using the DLS technique. After polymerization, the particle
size of the magnetic emulsion droplets (450 nm) increased to be 527 nm
due to the introduction of an organic part represented by the
crosslinked poly(divinylbenzene-co-glycidyl methacrylate) polymer shell.
Moreover, the prepared colloidal particles have uniform and narrow size
distribution (i.e. monodisperse) as evidenced by polydispersity index
(PDI=0.23) and TEM images
More interestingly, chemical structure, physical properties, and the
amount of the used inorganic “magnetic” material in the final particle
are the driven parameters controlling their physical separation under
the magnetic field. Then, the good control of the magnetic properties
before and after encapsulation is of paramount importance. In this
regard, the magnetic content of the prepared magnetic latex particles
was evaluated by using the TGA technique. As seen in Figure 4, TGA
thermogram of Fe3O4 magnetic emulsion
shows two main slopes; a very moderate one (4 % weight loss) between 25
and 200 °C, which attributed to release of moisture entrapped by the
emulsified Fe3O4 nanoparticles, and a
larger one (20 % weight loss) between 220 and 500 °C due to the
decomposition of organic layer (oleic acid and SDS surfactants) treated
Fe3O4 nanoparticles. This indicates that
magnetic emulsion contains ≈ 76 % of magnetic iron nanoparticles. After
polymerization of DVB and GMA, the magnetic content of the crosslinked
poly(divinylbenzene-co-glycidyl methacrylate) coated
Fe3O4 nanoparticles reduced to be ≈ 66
wt% due to the incorporation of organic polymer shell during the
polymerization process.
On the other hand, Figure 5 shows the magnetization curve of the
magnetic emulsion (ME) before and after coating with
poly(divinylbenzene-co-glycidyl methacrylate). As clearly seen from
magnetization versus magnetic field (Fig. 5), the saturation
magnetization of the seed magnetic emulsion (43.85 emu
g-1) was reduced to be 35.49 emu.g-1after coating with poly(divinylbenzene-co-glycidyl methacrylate). More
importantly, the superparamagnetic property was conserved which explains
that the polymerization conditions have no significant effect on the
intrinsic magnetic property of the used iron oxide nanoparticles.
Colloidal stability of the prepared magnetic polymer colloidal particles
is of great interest especially when be applied in the biomedical
diagnostic domain. Therefore, Zeta potential (ζ) measurements were
performed to study the electro-kinetic phenomenon based on the surface
charge of the prepared magnetic polymer particles in different pH
environments. As shown in Figure 6, the prepared magnetic colloidal
particles have a negative charge over the pH range investigated. In the
case of magnetic emulsion seed (ME), the observed negative zeta
potential, irrespective of the investigated pH domain, can be attributed
to the negatively charged sulfate group
(SO4-) of SDS used during (o/w)
magnetic emulsion formation. Regarding the prepared magnetic polymer
particles, the negative zeta potential can be attributed to the
negatively charged sulfate group
(SO4-) originating from the KPS
initiator. Consequently, both systems (seed magnetic emulsion and
prepared magnetic polymer particles) exhibit good colloidal stability
over the pH range.
3.2 Characteristics of Fabricated
Films
3.2.1 Fabrication of Self-assembled Thin
Films
In the current work, the LBL-SAMU technique presents several advantages
over other techniques such as Langmuir Blodgett, organo-sulfur compounds
chemisorbed on the metal surface, self-assembled monolayers, and
multilayers of colloids. Relative to LBL-SAMU these techniques offer
several problems in surface characterization control, the limitation for
the material to be adsorbed, high yield surface reactions, and the
molecules not firmly trapped. LBL-SAMU a typical technique involves the
alternate layering of cationic and anionic charges on the substrate
outer surface. It’s a robust, stable, and environmentally friendly
technique. Substrate topology and size does not affect the mechanism of
the mentioned technique. Repeating the cycle several times, LBL-SAMU can
be easily used to form multilayers with better control over the
thickness, functionality, composition, uniformity, and molecular
architecture [21-24].
3.2.2 Morphology and growth studies of thin
films
Magnetic colloidal particles (with the average size of 527 nm) were
employed to prepare thin films bearing negative charge groups on their
surface. Optika B-600MET optical microscopy (voltage range:
90V-240V~85VA-50/60Hz) provides direct information about
the uniformity of particle distribution, surface properties, and
morphology. The representative optical microscopic micrographs of 5, 10,
15 bilayers thin films are presented in Figure 7. An increasing trend
for the particles adsorbed on the prepared substrate surface with an
increased number of bilayers is obtained via self-assembly. Minimal
particle’s spreading was noticed on 5 bilayer micrograph due to less
availability of charged ions. It is possible to happen that during
alternate layering of cationic- anionic charges, adsorption may result
in enhancement of deposition of particles [19, 21, 25, 26]. In
addition, particle distribution appeared to be more and more uniform on
the prepared substrate with increasing the number of bilayers.
To assess the constituting atoms relative composition within the
synthesized biothin films of colloids, EDS analysis was done. The
results of EDS analysis (Table 1) confirms that an increased number of
bilayers provide large time for ions availability and their adsorption
on the substrate that in turn leads to an increase in iron content. To
obtain further insight into the growth of prepared biothin films and to
support further optical micrographs, scanning electron microscopy (SEM)
was performed. The SEM images of films reported that colloids particles
may form agglomerates during adsorption. There are several regions where
agglomerates are observed in SEM images. Figure 8 represents interesting
morphology and growth patterns by SEM images. For example, a comparison
of 5, 10 and 15 bilayers thin films depicting that colloid adsorption
have significantly and gradually increased by increasing the number of
bilayers with a 20% increment in iron contents imparting more magnetic
characteristics. Particles are deposited as such in spherical geometry.
Regarding the growth of films, most of the interesting information is
significant in the present biothin film preparation technique. One can
easily tune the parameters of this technique to control the thickness
and magnetic characteristics imparted during the fabrication procedure.
The fabricated films can find externally applied magnetic field tunablein vitro applications (e.g. therapy, and sensitive diagnostics
such as protein DNA detection) [27].
3.2 In-Vitro MRI of Magnetic
Colloids
MRI testing was performed to acquire two basic types of scan images
based on T1 and T2 relaxation times. In
MRI scanners, samples are placed under strong externally applied
magnetic field area; an RF pulse is then applied to cause the protons of
hydrogen atoms to spin with Larmour frequency and then relax after the
removal of RF pulse. Both T1 and T2images are different from each other in the sense that
T2 is characterized by the transverse relaxation time
(Spin-spin relaxation time), a time constant determining the signal
decay, while T1 is characterized by the longitudinal
relaxation time (Spin-lattice relaxation time). The positive MR contrast
agents (e.g. gadolinium) can reduce the spin-lattice relaxation time
“T1”. They are bright on MRI. It is observed that Gd
is a well positive T1contrast agent, while colloids based on ferrofluids
are a better negative T2 contrast agent. Both
T1 and T2 relaxation mechanisms show
different times for particular tissue in this way one can easily
differentiate between tissues. Taking the importance of
T1 and T2 images in MRI, magnetic
colloids are studied for their potential to be used as MR contrast
agents [10, 19, 28-32]. Table 2 summarized the results of the
acquired T1 and T2 weighted images for
their contrast performance. Mean intensity values are evident that the
colloids in this study are relatively better performing as MR
T2 contrast agents. The T2 relaxation
mechanism happens because of the energy exchange between the hydrogen
atoms in water [30, 31, 33-35]. It seems that the signal intensities
for each sample are not identical.
Contrast agents are the collection of contrast-enhancing media that are
used in magnetic resonance imaging to increase contrast and to improve
visualization of the internal body. After oral or intravenous
administration of these agents, they interact with the atoms of the body
and alter the relaxation times under the influence of a strong magnetic
field. In MRI scanners during analysis; segments of the samples are
exposed to a main (very strong) magnetic field. The relaxation process
emits protons of energy that are detected by the scanner and recorded to
get image after mathematical conversions.
The relaxation properties of the magnetic colloids were tested by
measuring the T2 relaxation time of the water protons in
the dispersion of colloids. The colloids create dephasing of protons
moments and perturb the magnetic relaxation processes with shortened the
water proton spin-spin relaxation times in the surrounding environment
resulting in a reduced MRI signal intensity and consequent darkening of
MR images. The resultant MR images (T1 and
T2 weighted) are presented in Figure 9.
Figure 10 exhibits the differences in contrast generation during
T2 and T1 relaxation mechanisms. The MRI
image can be acquired by different relaxation processes giving a lower,
higher, or medium signal level. In the current work, it looks that
T2 has strong relaxivity which points to a strong
variation in contrast and signals intensity on T2weighted images. Under the influence of externally applied higher
magnetic field, to record the T2 relaxation time for
each reported sample, ST: 5.1mm were taken at various TE (echo time TE:
45, 75, 105, 135 and 165 ms) with a repetition time (TR) of 5,000 ms.
Similarly, the T1 relaxation time of reported samples
was measured at TR: 40, 225, 425, and 825 ms while keeping constant echo
time at 12 ms. One more important observation is that with the increase
in Fe concentration, MRI contrast becomes better that is due to the
enhanced magnetic character within the sample. It might be a possible
combination of iron oxides with functionalities that may cause an
increase in hydrophilic character that allows nearer proximity of the
protons with contrast agent molecules which leads to shorter
spin-lattice relaxation time [36] [37]. Furthermore, with
increased Fe concentration in colloidal suspension, the MR signal
intensities are most significantly decreases giving steeper relaxation
curve and providing dark manifestation by reduced spin-spin relaxation
time. The variation in signal intensities with Fe concentrations also
provide informational data about the relaxation rates due to energy
exchange between the atoms. Since the MR contrast agents are those
materials that are used to improve the visibility of body tissues during
the scan. These agents can produce sufficient contrast to differentiate
between normal area and diseased tissue by altering the relaxation time
[34, 38-42]. This shows the efficient potential of the developed
colloids as negative MR contrast agents.
4
Conclusions
Biopolymer core-shell magnetic colloids of the average size of 527 nm
were successfully prepared. The prepared magnetic polymer particles have
good colloidal stability and possess superparamagnetic properties which
makes them suitable candidates to be used as contrast agents in magnetic
resonance imaging (MRI). The detection and diagnostic applications of
magnetic colloids were studied and both T1 and
T2 weighted images were taken. The molecular imaging
modalities showed that the colloids perform well as T2contrast agents. With an increase in magnetic modalities within water
dispersion, the contrast became darker, while the MR signal intensities
decreased significantly forming a steeper relaxation curve. Fabrication
of biothin films of the magnetic colloids was carried out effectively
and colloids were adsorbed by retaining their characteristic properties
to communicate with the biological systems. The film’s growth was
controlled by the number of bilayers deposited on the substrate.
Particles formed agglomerates during adsorption. When comparing the
bilayer thin films (5-15), 20% of iron contents became rise which may
intensify the strength of magnetic character. Thus, our biopolymer
core-shell magnetic colloids can be potentially developed with the
magnetic functionality having significant medical applications,
predominantly in diagnostic as well as in monitoring the effects of
chemotherapy on tumor suppression.
Acknowledgement
This research was supported by the National University of Sciences and
Technology, Islamabad, Pakistan (NUST).
Conflict of
interest
There is no conflict of interest.
References
[1] Sun, C., Sze, R., Zhang, M., Folic acid‐PEG conjugated
superparamagnetic nanoparticles for targeted cellular uptake and
detection by MRI. J. Biomed. Mater. Res. Part A 2006, 78 ,
550-557.
[2] Barick, K., Aslam, M., Prasad, P. V., Dravid, V. P., Bahadur,
D., Nanoscale assembly of amine-functionalized colloidal iron oxide.J. Magn. Magn. Mater. 2009, 321 , 1529-1532.
[3] Hu, F., Jia, Q., Li, Y., Gao, M., Facile synthesis of ultrasmall
PEGylated iron oxide nanoparticles for dual-contrast T1-and T2-weighted
magnetic resonance imaging. Nanotechnol. 2011, 22 , 245604.
[4] Yamada, K., Sorensen, A. G., BASICS OF MRI.Neuro-oncology: The Essentials 2000, 56.
[5] Hornak, J. P., JP Hornak 1996.
[6] Martina, M.-S., Fortin, J.-P., Ménager, C., Clément, O.,
et al. , Generation of superparamagnetic liposomes revealed as highly
efficient MRI contrast agents for in vivo imaging. J. Am. Chem.
Soc. 2005, 127 , 10676-10685.
[7] Eissa, M. M., Polymer Encapsulation of Magnetic Iron Oxide
Nanoparticles for Biomedical Applications. J. Colloid Sci.
Biotechnol. 2014, 3 , 201-226.
[8] Naseer, N., Fatima, H., Asghar, A., Fatima, N., et al. ,
Magnetically Responsive Hybrid Polymer Colloids for Ultrasensitive
Molecular Imaging. J. Colloid Sci. Biotechnol. 2014, 3 ,
19-29.
[9] Hashemi, R. H., Bradley, W. G., Lisanti, C. J., MRI: the
basics , Lippincott Williams & Wilkins 2012.
[10] Rabias, I., Pratsinis, H., Drossopoulou, G., Fardis, M.,
et al. , In vitro studies on ultrasmall superparamagnetic iron oxide
nanoparticles coated with gummic acid for T2 MRI contrast agent.Biomicrofluidics 2007, 1 , 044104.
[11] Fanun, M., Colloids in drug delivery , CRC Press 2010.
[12] Rabias, I., Fardis, M., Devlin, E., Boukos, N., et al. ,
No aging phenomena in ferrofluids: the influence of coating on
interparticle interactions of maghemite nanoparticles. ACS nano2008, 2 , 977-983.
[13] Zhong, Y., Whittington, C. F., Zhang, L., Haynie, D. T.,
Controlled loading and release of a model drug from polypeptide
multilayer nanofilms. Nanomed- Nanotechnol., Bio. Med. 2007,3 , 154-160.
[14] Pankhurst, Q. A., Connolly, J., Jones, S., Dobson, J.,
Applications of magnetic nanoparticles in biomedicine. J. Phy. D:
App. Phy. 2003, 36 , R167.
[15] Ahmed, N., Ahmad, N. M., Fessi, H., Elaissari, A., In Vitro MRI
of biodegradable hybrid (Iron oxide/polycaprolactone) magnetic
nanoparticles prepared via modified double emulsion evaporation
mechanism. Colloids Surface. B 2015.
[16] Jada, A., A Special Issue on Inorganic Colloidal Particles,
Synthesis, Surface Properties and Applications. J. Colloid Sci.
Biotechnol. 2014, 3 , 1-2.
[17] Gossuin, Y., Gillis, P., Hocq, A., Vuong, Q. L., Roch, A.,
Magnetic resonance relaxation properties of superparamagnetic particles.Wiley Interdisciplinary Reviews: Nanomed. Nanobiotechnol. 2009,1 , 299-310.
[18] Berry, C. C., Wells, S., Charles, S., Aitchison, G., Curtis, A.
S., Cell response to dextran-derivatised iron oxide nanoparticles post
internalisation. Biomater. 2004, 25 , 5405-5413.
[19] Hassan, M. A., Saqib, M., Shaikh, H., Ahmad, N. M., Elaissari,
A., Magnetically Engineered Smart Thin Films: Toward Lab-on-Chip
Ultra-Sensitive Molecular Imaging. J. Biom. Nanotechnol. 2013,9 , 467-474.
[20] Ahmad, N. M., Ali, S. J., Saqib, M., Stimuli‐responsive
self‐assembled multilayer azo thin films: Effect of aggregates and salt
on significant spectral shifts. J. Polym. Sci. Pol. Chem. 2012,50 , 1881-1889.
[21] Decher, G., Hong, J., Schmitt, J., Buildup of ultrathin
multilayer films by a self-assembly process: III. Consecutively
alternating adsorption of anionic and cationic polyelectrolytes on
charged surfaces. Thin solid films 1992, 210 , 831-835.
[22] Arys, X., Fischer, P., Jonas, A. M., Koetse, M. M., et
al. , Ordered polyelectrolyte multilayers. Rules governing layering in
organic binary multilayers. J. Am. Chem. Soc. 2003, 125 ,
1859-1865.
[23] Decher, G., Schlenoff, J. B., Multilayer thin films:
sequential assembly of nanocomposite materials , John Wiley & Sons
2006.
[24] Ulman, A., Formation and structure of self-assembled
monolayers. Chem. Rev. 1996, 96 , 1533-1554.
[25] Ahmad, N. M., Saqib, M., Barrett, C. J., Novel
Azobenzene-Functionalized Polyelectrolytes of Different Substituted Head
Groups 1: Synthesis, Characterization and Absorption Spectroscopy
Studies. J. Macromol. Sci. A 2009, 47 , 106-118.
[26] Peyratout, C. S., Dähne, L., Tailor‐made polyelectrolyte
microcapsules: from multilayers to smart containers. Angewandte
Chemie International Edition 2004, 43 , 3762-3783.
[27] Häfeli, U., Scientific and clinical applications of
magnetic carriers , Springer 1997.
[28] Sinibaldi, E., Pensabene, V., Taccola, S., Palagi, S., et
al. , Magnetic nanofilms for biomedical applications. J.
Nanotechnol. Eng. Med. 2010, 1 , 021008.
[29] Lee, D., Cohen, R. E., Rubner, M. F., Antibacterial properties
of Ag nanoparticle loaded multilayers and formation of magnetically
directed antibacterial microparticles. Langmuir 2005, 21 ,
9651-9659.
[30] Laurent, S., Forge, D., Port, M., Roch, A., et al. ,
Magnetic iron oxide nanoparticles: synthesis, stabilization,
vectorization, physicochemical characterizations, and biological
applications. Chem. Rev. 2008, 108 , 2064-2110.
[31] Shultz, M. D., Calvin, S., Fatouros, P. P., Morrison, S. A.,
Carpenter, E. E., Enhanced ferrite nanoparticles as MRI contrast agents.J. Magn. Magn. Mater. 2007, 311 , 464-468.
[32] Caravan, P., Strategies for increasing the sensitivity of
gadolinium based MRI contrast agents. Chem. Soc. Rev. 2006,35 , 512-523.
[33] Schweiger, C., Pietzonka, C., Heverhagen, J., Kissel, T., Novel
magnetic iron oxide nanoparticles coated with poly (ethylene
imine)-< i> g</i>-poly
(ethylene glycol) for potential biomedical application: Synthesis,
stability, cytotoxicity and MR imaging. Int. J. Pharm. 2011,408 , 130-137.
[34] Jain, T. K., Richey, J., Strand, M., Leslie-Pelecky, D.
L., et al. , Magnetic nanoparticles with dual functional
properties: drug delivery and magnetic resonance imaging.Biomater. 2008, 29 , 4012-4021.
[35] Arsalani, N., Fattahi, H., Nazarpoor, M., Synthesis and
characterization of PVP-functionalized superparamagnetic Fe3O4
nanoparticles as an MRI contrast agent. Exp. Polym. Lett. 2010,4 , 329-338.
[36] Jaganathan, H., Gieseck, R. L., Ivanisevic, A., Transverse
relaxivity changes after layer-by-layer encapsulation of multicomponent
DNA templated nanostructures. J. Phys. Chem. C 2010, 114 ,
22508-22513.
[37] Rahman, M. M., Elaissari, A., Multi-stimuli responsive magnetic
core–shell particles: synthesis, characterization and specific RNA
recognition. J. Colloid Sci. Biotechnol. 2012, 1 , 3-15.
[38] Mohammad-Taheri, M., Vasheghani-Farahani, E., Hosseinkhani, H.,
Shojaosadati, S. A., Soleimani, M., Fabrication and characterization of
a new MRI contrast agent based on a magnetic dextran–spermine
nanoparticle system. Iran. Polym. J. 2012, 21 , 239-251.
[39] Jaganathan, H., Gieseck, R. L., Ivanisevic, A., Characterizing
proton relaxation times for metallic and magnetic layer-by-layer-coated,
DNA-templated nanoparticle chains. Nanotechnol. 2010, 21 ,
245103.
[40] Jaganathan, H., Hugar, D. L., Ivanisevic, A., Examining MRI
contrast in three-dimensional cell culture phantoms with DNA-templated
nanoparticle chains. ACS App. Mater. Interfaces 2011, 3 ,
1282-1288.
[41] Zhang, Z., Sun, Q., Zhong, J., Yang, Q., et al. ,
Magnetic resonance imaging-visible and pH-sensitive polymeric micelles
for tumor targeted drug delivery. J. Biomed. Nanotechnol. 2014,10 , 216-226.
[42] Wu, C., Lin, L., Lin, L., Huang, H., et al. ,
Biofunctionalized magnetic nanoparticles for in vitro labeling and in
vivo locating specific biomolecules. App. Phys. Lett. 2008,92 , 142504.