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.

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