Iron-Based Ionic Liquids for Magnetic Resonance Imaging Application

Abstract

In the biomedical treatment, identification of diseases and their diagnosis are running with help of many biomedical techniques including imaging such as magnetic resonance imaging (MRI). MRI technique requires an identification of targeted cell or lesion area which can be achieved by contrast agent. For clinical use, T1 positive MRI contrast agents and T2 negative MRI contrast agents are being used. However, these contrast agents have several drawbacks such as toxic effect of metal centre, poor resolution, weak contrast, low intensity image and short signal for long-term in vivo measurement. Therefore, development of new contrast agents is imperative. Ionic liquids with their unique properties have been tried as novel contrasting materials. Particularly, iron-containing amino-acid-based ionic liquids or amino-acid-based paramagnetic ionic liquids (PMILs) have been reported and demonstrated as MRI contrast agents. These PMILs have shown superior features over reported contrast agents such as dual-mode contrast, biofriendly nature, involvement of non-toxic magnetic centre (Fe), stable aqueous solution, better image intensity at low concentration level and easy to synthesis. PMILs have been characterized well and studied with animal DNA using various techniques. The result revealed that animal DNA is remain safe and stable structurally up to 5 mmol.l−1. These cost-effective PMILs opened the greater opportunity in the field of contrast-based biomedical applications.

1. Introduction

It is well known that, among various biomedical imaging modalities, magnetic resonance imaging (MRI) is one of the most powerful, ionizing-radiation-free and non-invasive imaging technique, and principally it resembles NMR (Nuclear Magnetic Resonance) technique. Image is formed by spatially encoding NMR signals received from the proton relaxation of the molecules under applied magnetic field [1, 2]. During imaging, the identification of targeted cell or lesion can be visualized by using some of tracking or sensing agent. In the X-ray techniques, BaSO₄ will be used to enhance the contrasting level and make difference in brightness of background to target or lesions area. Similarly, in the magnetic resonance imaging (MRI) technique, MRI contrasting agents are widely used to boost up the image sensitivity and achieve anatomical differentiation or detection accuracy by enhancing the contrast of the image. According to the nature of generating contrast, MRI contrast agents used clinically are T₁ MRI contrast agents and T₂ MRI contrast agents. T₁ contrast (positive contrast) enhances brightness in T₁-weighted images; however, T₂ contrast (negative contrast) enhances darkness in T₂-weighted images [2]. Clinically used contrast agent is mostly composed of gadolinium metal ion. These contrast agents are metal–ligand structure. Gadolinium-based contrast agents (GBCAs) show T₁ positive contrast during imaging process [3]. Most commonly, a contrast agent exhibits either T₁ or T₂ contrasting nature in domination, but recently dual-contrasting nature in single agent with significant T₁ and T₂ relaxivity values has been reported. Most of these dual-contrasting agents are either multi-layered core-shell nanoparticle or nanoparticle-Gd-chelate complexes which need a highly precise multistep and sophisticated synthesis procedure [4, 5]. But at present, none of the dual (T₁ and T₂) contrasting agents are commercially available for MRI diagnosis. For GBCAs, it is reported that a sever nephrogenic systemic fibrosis (NSF) complication is recently recognized. Deposited Gd metal ion can induce critical clinical problems such as chronic kidney disease (CKD) and acute kidney injury, etc. to the patient [6]. Thus, in short, there are adverse effects of such contrast agents which are dominant and need to remove or overcome these obstacles either in performance or heath issues. For sake of knowledge, the contrast agents can be grouped broadly into three categories depending upon their function and nature of contrast. T₁ contrast agent: gadolinium (Gd)-chelate-based contrast agents (GBCAs) such as Magnevist®, Dotarem® and Omniscan™ are commonly used in daily present clinical practice for T₁ contrast in most of clinical aspects [5]. The Magnevist is ionic contrast agent and has N-methylglucamine counter positive ion. It is used for the visualization of abnormal vascularity. However, these GBCAs have higher osmolality values [7]. Theoretically, the origin of T₁ response is related to the longitudinal relaxivity (r₁) of aqueous solutions metal–ligand-bearing contrast agent. The main parameters such as the number of water molecules in the first coordination sphere of the metal ion(q), their residence time in the first coordination sphere (t_M) and the molecular tumbling time(t_R) are optimized to determine the value of r1. These parameters are related to water exchanging process between the first coordination sphere of paramagnetic metal ions-ligand moiety and the surrounding water [8]. Most of GBCAs for which q value falls in the range of 1–3 have sufficient thermodynamic and kinetic stability [9, 10]. T₂ contrast agents: superparamagnetic iron oxide nanoparticles (SPIONs)-based contrast agents such as Resovist®, Feridex® and Gastromark™ are commercially available and clinically approved T₂ contrasting agents. They have higher relaxivity value and considered as negative contrast agent due to enhancement of darkness in T₂ images [11, 12]. To prevent agglomeration, either each of these particles is covered with a core-shell or magnetic crystallite embedded in a coating. For example, ferumoxide is made from dextran, whereas ferumoxsil is made of siloxanes. Size of the core determines the relaxivity property of particle. Here also, parameters such as to r₁ are governed transverse relaxivity (r₂), which is further related to T₂ contrast. The relaxation induced by superparamagnetic particles can be explained by the classic outer-sphere relaxation theory [13]. According to this theory, the relaxation rates of water protons diffusing nearby the unpaired electrons present in paramagnetic ions are responsible for the particle’s magnetization [14]. Enhancement in T₂ relaxation increases with the particle size [15, 16]. Therefore, SPIOs were firstly developed as T₂ contrast agents due to their larger size [17]. According to the overall size of the particles, superparamagnetic iron oxides are classified [7]. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles [18] have a diameter less than 50 nm, whereas small superparamagnetic iron oxide (SSPIO) nanoparticles have size between 1 mm and 50 nm. Micron-sized particles of iron oxide (MPIO) nanoparticles are large particles with a diameter of several microns. Since T₁- and T₂-weighted contrast agents exhibit great response and possess unique qualities, but there are some reports which have described their limitations. Therefore, synergic integration of these two functions (T₁ and T₂) for MRI is expected to get more comprehensive and cooperative diagnostic information over the single T₁ or T₂ contrast agent [19, 20, 21]. The development of dual-mode contrast agent of MRI in a single instrumental system could proficiently eliminate certain difficulties. It could also improve the diagnostic accuracy for most of diseases. It is reported that some functionalized or mixed nanomaterials exhibit intrinsic dual-contrast effects in magnetic resonance imaging. The FeCo nanoparticles (NPs) reported by H. Dai show high T₁/T₂ contrast effects, but there is a lack of understanding of the dual-mode contrast mechanism [22]. Many researchers have reported the Gd³⁺-containing magnetite (Fe₃O₄) NPs, MnO-containing nanoparticles and SPIONs as dual-MRI contrast agents [4, 5, 23, 24, 25, 26, 27, 28, 29]. A recent patent shows pH-sensitive nano-formulates (PMNs) contrast agent comprising extremely small iron oxide nanoparticles (ESIONs) [30]. Songjun Zeng and Jianhua Hao have been used a hybrid lanthanide nanoparticle as a dual-mode contrast agent for imaging-directed tumor diagnosis [31]. The iron oxide nanoparticles coated with Gd-DTPA and fibrin-binding peptides have also been reported by Xu et al. for the detection and localization of thrombosis [32]. Similarly, the cyclic RGD functionalized liposomes (cRGD@MLP-Gd) encapsulated with gadolinium diethylenetriamine penta-acetic acid (Gd-DTPA) and superparamagnetic iron oxide (SPIO) are prepared by Fang Yang and Chun-Jian Li and used for thrombus-targeted imaging activity [33].

Each and every researcher is familiar with Ionic Liquids (ILs) and its functionality. Due to its tuneable nature, ionic liquids have gained special attention. Ionic liquids are compounds comprising entirely of ions where at least one ion should be asymmetric organic ion and melts below the 100°C [34]. It is already reported that the physicochemical properties of ILs depend on the nature of cation, anion and alkyl substitutions [35], and properties can be altered by varying the nature of ions, thus making them task-specific [36]. Task-specific ionic liquids with characteristic physicochemical properties produced many advantages and have been used in various applications widely [37, 38, 39, 40]. Researchers have introduced the inherent magnetic properties by using transition metal at molecular level. First time paramagnetic magnetic ionic liquid (PMIL) has been reported by Hayashi Satoshi et al. and their magnetic property explained. These ionic liquids are composed of iron metal halide (FeCl₄⁻ ion) [41]. Due to inherent paramagnetic character, these ionic liquids were termed as paramagnetic ionic liquids (PMILs). PMILs are made up of a distinct group with versatile properties such as magnetic character and widely used in a various applied fields. For example, the PMILs have been used in desulfurizations [42], organic synthesis [43], microextraction [44, 45], electro-catalysis [46], probe for vesicles [47], self-assembling media for surfactants [48], acidic catalysis [49, 50], density measurements [51], paramagnetic polymer synthesis [52, 53], microemulsion formulation [54], synthesis of chitosan supported magnetic ionic liquid based catalysis [55], CO₂ separation [56], application in analytical science [57] and other various applications [58]. Kumar et al. have reported paramagnetic surface-active ionic liquids (PMSAILs), another class of PMILs. Paramagnetic surface-active ionic liquids (PMSAILs) are long chain bearing those ionic liquids which have amphiphilic nature and have ability to form nano-aggregates such as micelle and vesicles in their solution. The PMSAILs are demonstrated as contrast agent in aqueous solution for MRI application and also studied with animal DNA to check its structural stability [59]. Many ionic liquids have been prepared using amino acid, and their biocompatible and biofriendly nature [60, 61] have been checked. Since L-amino acid is biological monomer that is the building block of proteins. L-amino-acid-based chiral PMILs have been synthesized and studied by Isiah M. Warner et al. [62]. Here, the iron-containing amino-acid-based PMILs are studied and first time explored the application as contrast agents which more promisingly interact with essential biological molecules and surprisingly enhance the contrast intensity with retention time. These PMILs-based contrast agents were made of biofriendly amino acid and iron halide. Authors have studied broadly and investigated its interaction with DNA through various techniques including CD, fluorescence, ITC, zeta and gel electrophoresis. MRI property of these PMILs is also investigated and claimed their superior contrast activity. Since these are made of amino acid and iron moiety, therefore, they are non-hazardous, toxic-metal-free and biofriendly contrast agent (Table 1) [63]. This work is patented in Indian patent office [64].

Table 1.

Name of amino acid and chemical structures of used in the study as contrast agents.

Reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

2. Paramagnetic ionic liquids as contrast agents

As it has been already mentioned in introduction, ionic liquids (ILs) have gained special attention due their flexibility and versality. Thus, ionic liquids have been used in various applications, and ionic liquid is still being used. In the continuation of the applications, Carla I. Daniel et al. have mentioned the possibility of low-toxic magnetic ionic liquid as contrast agent at end of conclusion. They have conducted a proton nuclear magnetic relaxation dispersion ¹H NMRD study of the molecular dynamics in mixtures of phosphonium-based magnetic ionic liquid[P₆₆₆₁₄][FeCl₄] with [P₆₆₆₁₄][Cl] ionic liquid and mixtures of [P₆₆₆₁₄][FeCl₄] with dimethyl sulfoxide (DMSO). The enhancement in r₁ relaxation rate of MIL in mixture of ILs + [P₆₆₆₁₄][FeCl₄]) with DMSO solvent [65]. The relaxation enhancement is directly linked with contrast property as we know. The proton spin–lattice relaxation dispersion rate (r₁), was measured for both these systems, and the rate indicates a much larger paramagnetic relaxation enhancement for [P₆₆₆₁₄][FeCl₄] with [P₆₆₆₁₄][Cl], in comparison with that observed for the mixtures of [P₆₆₆₁₄][FeCl₄] with DMSO. This difference has reflected that in this mixture, the proton spin–lattice relaxation does not depend on the concentration of paramagnetic ions [FeCl₄]⁻ linearly. The paramagnetic ions [FeCl₄]⁻ seem to disturb the local molecular organization, molecular order, dynamics and packing in the [P₆₆₆₁₄][Cl] ionic medium as compared with non-ionic organic solvent DMSO. Thus, here, aqueous solution of iron-containing PMILs is examined for imaging, and their relaxivity rate at various concentrations is studied.

Kumar et al. have synthesized amino-acid-based PMILs [63]. These amino acids are used in two forms—esterified and without esterified. Esterified amino acids are prepared as reported earlier in the literature [61, 66]. Chloro halide salt of amino acid is simply mixed with ferric chloride in equimolar ratio in the ethanol solvent. Dark brown PMILs are obtained at end of process after solvent evaporation. So, these PMILs are easy to synthesize and can be cost-effective. These PMILs are characterized well, and structural verification of [FeCl₄]⁻ ion is done by UV and Raman shift using Shimadzu UV-2700 UV–VIS spectrophotometer, Japan, and LabRAM HR Evolution Horiba Jobin Yvon Raman spectrometer, Japan, at 298.15 K (Figure 1). The Raman shift value for [FeCl₄]⁻ ion should be near 334 cm⁻¹ [67, 68]. The paramagnetic nature of PMILs is confirmed by the EPR spectrum using mt-MiniScope MS5000 ESRStudio by Freiberg instrument at 298.15 K. The EPR spectrum of PMILs in solution phase indicates a single isotropic EPR line which appeared due to mixed ⁶S₁ state. EPR spectrum of magnetic centre, here Fe³⁺ ion, strongly depends on its tetrahedral environment [69]. The amount of Fe in each PMILs is measured and calculated using Perkin Elmer ICP optima 2000 DV ICP-OES (Inductively Coupled Plasma–Optical Emission Spectroscopy) analyzer. In the terms of Fe, aqueous solutions from 0.1 to 5 mmol.l⁻¹ are prepared for further experiments including interaction with DNA and magnetic resonance imaging.

Figure 1.

Representative UV and Raman spectrum of PMIL (ProC₁[FeCl₄]). Reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

3. Physiochemical properties of PMILs

Degradation and glass transition temperature of PMILs was investigated using NETZSCH TG 209 F1 Libra TGA and NETZCH DSC 204 F1 Phoenix DSC, respectively. A presentative differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) thermograms are given in Figure 2. Combinedly, these are thermal analyses, and it is the technique in which physical properties of a substance or a mixture of substances are measured against either of temperature or time, wherein the substances are subjected to a controlled temperature programme system. If the physical property is weight, then it is termed as a TGA. In thermogravimetric analysis (TGA), weight changes or % of the mass loss of PMILs is measured as a function of temperature. Thermogravimetric curves (graph between % mass loss versus temperature) allow an evaluation of thermal stabilities. Degradation temperature (T_g) was marked where maximum mass changes occur [70, 71, 72, 73]. In the figure, initial loss in mass is due to moisture or water elimination at near 120–200°C. After that maximum loss in mass was observed at 200–300°C. For the TGA thermogram, onset temperature (T_onset) is the intersection of the baseline of weight (after the loss of the water) and the tangent of the weight versus temperature curve or simply, a temperature at which the sample loses weight with fastest rate. The start temperature (T_start) is beginning of decomposition [72]. For ionic liquid, glass transition temperature is measured due to its amorphous nature. Glass transition temperature does not have sharp changes like melting point. Glass transition temperatures (T_g) for PMILs are found below 100°C. There is assumption that lowering the melting point of ionic liquids is achieved due to distortion in the lattice of crystal, and these disturbances generate low lattice enthalpy and weak ionic attraction between asymmetric ions. However, like branching or enlargement of substituent is majorly responsible of disruption in crystal packing [74]. In DSC, the sample and reference are kept at the same temperature, and the energy d(∆q)/dt required to preserve zero temperature differential (∆T = 0) between the sample and the reference is measured on the function of temperature during a thermal event in the sample. As a result, the endothermic peak indicates absorption of heat, and the exothermic peak will rise when heat is released. Depending on the nature of peaks, glass transition temperature (T_g), melting point (T_m), crystallization transition (T_c) and heat capacity can be calculated [71, 75, 76, 77]. Since, T_g values are less than 100°C and fulfilled the criteria of an IL [78], therefore our product will be called as paramagnetic ionic liquids (PMILs).

Figure 2.

Representative DSC and TGA thermogram (ProC₁[FeCl₄]). Reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

4. Interaction of PMILs with animal DNA

To explore the biofriendly nature and structural stability of animal DNA, the physical interaction of this PMILs has been investigated. For this whole investigation, 90–92 ng ul⁻¹ concentration of DNA was prepared in buffer solution using NanoDrop® Spectrophotometer ND-1000. After that, PMILs are studied with animal (salmon fish) double-stranded β-DNA (ds-β-DNA) and studied the conformational and structural stability of DNA in aqueous medium. For this, PMILs were examined with DNA using various techniques including circular dichroism (CD), fluorescence, isothermal thermal calorimetry (ITC), zeta potential and Agarose gel-electrophoresis, and the concentration regime where DNA remains safe in native form is identified. It is found that long-chain-bearing PMILs undergo complex formation (DNA-PMILs) in the form of precipitate at higher concentration [59].

4.1 Circular dichroism (CD) and fluorescence

To investigate the structural and conformational stability, CD (Jasco J-815 CD spectrometer under the N₂ environment at temperature 298.15 K) and fluorescence spectra of DNA are recorded in the presence of aqueous solution of PMILs, and the spectra are shown in Figure 3. It is well known that the presence of a positive band at about 276 nm and a negative band near 245 nm with crossover point at nearby 258 nm collectively indicates the existence of native pure DNA in buffer solution. These values indicate that used pure DNA is the fully hydrated double-helix β form and dextrorotatory in nature [79]. The secondary structure of DNA remained same as native DNA within the concentration range (0.1–5 mmol.l⁻¹ Fe); after that, at higher concentrations (above 3 mmol.l⁻¹ Fe), PMIL underwent interaction process with negative sites of DNA, and consequently, bands are distorted. Thus, at low concentration of PMILs, CD bands of DNA unchanged and are likely to superimpose on native bands representing the structural and conformational stability of DNA in these concentration ranges. For tertiary structure confirmation, interaction of PMILs with DNA was observed by Ethidium Bromide (EB) exclusion assay using a Fluorolog horiba Jobin Yvon fluorescence spectrophotometer. Fluorescence intensity of intercalating dye EB abruptly enhanced when dye intercalate at minor grooves of DNA with order of 20–25 times with respect to alone EB in buffer medium. Here, water is responsible which acts as a strong quencher [80, 81, 82, 83]. From Figure 3, it can observed that intensity peak height is similar to EB-Native DNA complex at low concentration (0.1–3 mmol.l⁻¹) that means PMILs did not bind to the EB-DNA complex efficiently. But at higher concentration, intensity peak height reduces due to involvement of cationic counterpart to the EB-DNA complex. Positive counter ion of PMILs started to interact with the minor negative groove of DNA via strong electrostatic interaction after complete removal of the spine of hydration, and consequently, EB dislocates effectively from its hydrophobic environment [84, 85]. It is assumed that more hydrated small cation-containing PMILs are incompetent to dislocate the EB efficiently due to weak electrostatic interactions, but these interactions become dominant when concentration is increased. In the case of long-chain-containing PMILs, DNA showed compaction phenomenon at higher concentration [59]. DNA compaction is confirmed by distortion in intensity and shifting in band position due to formation of cationic surfactant complex (lipoplex) [86]. So, from figure it can be judged that CD spectra and fluorescence spectra confirmed conformational and structural stability of DAN, which remains similar to native DNA at low concentration of PMILs.

Figure 3.

Representative CD spectra, fluorescence spectra of ED-DNA complex, ITC binding enthalpogram and zeta potential at various concentrations (ProC₁[FeCl₄]). Reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

4.2 Isothermal titration calorimeter and zeta potential

To investigate the interaction of PMILs with DNA thermodynamically, binding isothermal enthalpogram is measured from Isothermal Titration Calorimetry (ITC) experiments using MicroCal ITC200 microcalorimeter instrument with controlled Hamiltonian syringe. Measured enthalpy in the ITC experiment is a combination of overall heat produced from various phenomena involving the binding of PMILs on DNA through the electrostatic and hydrophobic interactions, hydration of PMILs and the change in the conformation of hydrated DNA [87]. From Figure 3, it is observed that variation in enthalpy (ΔH) of DNA-PMILs interaction process is less at low concentration and shows negligible DNA-PMILs binding. But, at higher concentration, a characteristic large enthalpic peak appeared which revealed significant DNA-PMILs interactions or can say complex formation [59, 88], which means PMILs have a significant impact on DNA after certain critical concentration. In the continuation, the effect of PMILs on DNA negative surface is observed by measuring zeta potential (ζ) using a Zetasizer Nano ZS light scattering apparatus (Malvern Instruments, U.K.) with a He-Ne laser (633 nm, 100 mW) at 298.15 K. The trend of changes in zeta potential with respect to concentration of PMILs is negative to positive. Initially it is negative due to surface charge of DNA alone or less binding of PMILs at low concentration. After that, it is passed through neutral point to positive value at higher concentration. It can be understood like, initially a few molecules of PMILs interact with DNA surface and at this time, some hidden negative charges of core area of the DNA chains become exposed outside; consequently, zeta value initially decreases [89]. After that, more positive counter ions of PMILs started to bind with exposed negative surface of DNA when overall exposed negative surface of DNA was neutralized, and then, subsequently, zeta value shifted to its positive values and then finally reached its maximum positive value. The neutralized negative surface of DNA makes PMIL-DNA complex formation, and such a complex occurs when all negative phosphate groups bind with available positive cations [90, 91, 92, 93]. This observation also concedes with ITC enthalpogram.

4.3 Agarose gel electrophoresis

Whether DNA breaks down or not in the presence of PMILs, with this objective, the agarose gel electrophoresis experiment was performed using Electrophoresis Power supply BGPS 300/400. In Figure 4, initial bright bands are similar to pure DNA at low concentration (up to 5 mM Fe) which indicates the presence of unbound DNA molecules. It was ensured that DNA degradation does not happen in lower concentration regimes after that the band becomes remarkably vague at higher concentration; further these illuminated vague bands disappear when concentration increases. Disappearance of band tells that all DNA molecules have been bound with PMILs, and there are no free DNA molecules available. Moreover, the absence of multiple bands like a ladder confirmed that DNA did not degrade or break in the presence of PMILs molecules [94].

Figure 4.

Representative agarose gel-electrophoresis electrophoresis pattern of DNA in the presence of PMILs at various concentrations (ProC₁[FeCl₄]). Reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

Therefore, collectively it can be summed that these PMILs are safer in terms of biofriendly nature, structural stability and degradation of DNA at certain range of concentration. These PMILs are safe and free from any adverse effect on animal DNA. This concentration range is selected for magnetic resonance imaging experiment.

5. Relaxation study of PMILs and its outcomes

Magnetic resonance imaging and relaxation study has been carried to demonstrate the utility and effectiveness of PMILs as an MRI contrast agent (CA). MR imaging experiment and relaxivity measurement were carried out using 11.7 Tesla MRI instrument (Brücker Advance 500 MHz proton NMR) using a micro-imaging probe and Paravision imaging software at CSIR CSMCRI, India. T₁- and T₂-weighted MRI images of aqueous phantoms of each PMIL at different concentration (0.3, 0.5, 0.7 and 1.0-mM Fe) are measured using the spin-echo pulse sequences (RAREVTR and MSME) with acquisition parameters FOV = 0.4 cm, TR = 350 ms, TE = 8 ms, 128 × 128 matrix and FOV = 0.4 cm, TR = 2000 ms, TE = 36, 128 × 128 matrix respectively for PMSAILs. T₁ and T₂ relaxation times of aqueous phantoms are measured using Bruker RAREVTR (FOV = 0.4 or 0.5 cm, TE = 8 ms, TR = 250 to 2850 ms and 128 × 128 matrix) and MSME (FOV = 0.4 or 0.5 cm, TE = 12 to 72 ms, TR = 2000 ms and 128 × 128 matrix) MRI pulse sequences respectively. T₁ and T₂ relaxivity values were calculated through Eq. (1) by linear curve fitting of relaxation rate (1/T₁ and 1/T₂) versus Fe concentration using Paravision software.

The study has explored its utility and estimated the effectiveness of the PMILs as MRI contrast agents and can be used for diagnosis. Generally, the intensity of MRI image depends on the population of ¹H nuclei of water molecules present in the biological tissue or cell or solvent, relaxation time and their relaxation rate also. Relaxation rate belongs to spin–spin relaxation and spin–lattice relaxation. Relaxation rate greatly influenced by environment of water molecules that are going to exchange process between magnetic centre or surrounding. Thus, relaxation rate (r) can vary with the variation of the local magnetic field and magnetic field inhomogeneity around the ¹H nuclei of the sample. Incorporation of a magnetic entity such as gadolinium chelates and superparamagnetic nanoparticles of Gd, Fe, Mn, into the samples will bring changes in relaxation rate via generating a variation in the local magnetic field and magnetic field inhomogeneity around the ¹H nuclei of the concerned sample.

The relaxation rate of PMILs obeys a linear relationship with Fe concentration and can be represented mathematically by the following expression [95].

$$\frac{1}{T_{i,C}}=\frac{1}{T_{i,0}}+r_{i}C$$E1

T_i,_C and T_i,₀ (i = 1 or 2) are relaxation time of sample at C concentration and absence of contrast reagent respectively, and r_i is the relaxivity of the contrast agent. T₁- and T₂-weighted images are recorded via using spin echo (SE) MRI pulse sequence, and the signal intensity for SE pulse sequence can be expressed as [2].

$$I=I_0\left(1-e^{-\mathit{TR}/T_1}\right)\left(e^{-\mathit{TE}/T_2}\right)$$E2

Intensity of T₁- and T₂-weighted image is totally T₁- and T₂-dependent; T₁ and T₂ weighting can be achieved by eliminating one term in the presence of other. For T₁-weighted image, T₂ term should be eliminated and the same for T₂-weighted image, T₁ term should be eliminated with the selection of the appropriate combination of TE (time of echo) and TR (time of repetition) values. Further, it can be seen from Eqs ($1$) and ($2$) that the intensity increases in T₁-weighted image but decreases in T₂-weighted images with the increase of Fe concentration and vice versa too. Local magnetic field inhomogeneity generated by Fe constituent also affects the transverse relaxation time (T₂), and due to this involvement of local field inhomogeneity, T₂ converts into T₂*. T₂* relaxation time is a combination of true T₂ relaxation time with relaxation rate generated due to local magnetic field inhomogeneity, and its value is always larger than T₂ relaxation time and can be mathematically expressed as [96].

$$\frac{1}{T_{2,C}^{\ast }}=\frac{1}{T_{2,C}}+\mathit{\gamma \Delta B}$$E3

where γ is the gyromagnetic ratio, ∆B is magnetic field inhomogeneity across the voxel and 1/T₂ is the relaxation rate contribution of magnetic field inhomogeneity. T₂*-weighted MRI images are obtained with gradient echo pulse sequence by choosing appropriate values of TE, TR and flip angle (α) of the excitation pulse to minimize the T₁ effect in the images. Signal intensity under this pulse sequence can be illustrated as

$$I=I_0\frac{\left(1-e^{-\mathit{TR}/T_1}\right)\mathit{sin}\alpha }{\left(1-e^{-\mathit{TR}/T_1}\right)\mathit{cos}\alpha }{e}^{-\mathit{TE}/T_2^{\ast }}$$E4

In order to investigate the contrast property of PMILs, five different concentrations (0.0, 0.3, 0.5, 0.7 and 1.0 mM Fe) were prepared, and T₁, T₂ and T₂*-weighted MRI images of aqueous phantoms were obtained. Their relaxation times were determined by using Eq. ($2$) for T₁, T₂, and Eq. ($4$) for T₂*. T₁ and T₂-weighted MRI images of PMILs are shown in Figure 2. An intensity reduction in T₂ and T₂*-weighted images and simultaneously an intensity enhancement in T₁-weighted image were observed along with Fe concentration. This intensity variation found in MRI images with respect to Fe concentration reveals contrast property of aqueous PMILs qualitatively and further suggests that these PMILs have negative as well as positive contrast behavior or can say PMLs can be used as T₁ and T₂ contrast agents.

Figure 5 represent a linear relationship of relaxation rate (r) with Fe concentration of PMILs which is found similar as reported in the literature available for various contrast agents [97]. This contrast property of PMILs is determined quantitatively for PMILs; r₁, r₂ and r₂* relaxivity measured by a linear curve fitting of their corresponding curves (Eq. ($1$)). The measured r₁ and r₂ values are given in Table 2.

Figure 5.

T₁- and T₂-weighted MR images and relaxivity pattern of amino-acid-based PMILs at various Fe concentrations. Reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

PMILs	Osmolality^a; Osmol.Kg^−1	r1; (mM^−1.s^−1)	r2; (mM^−1.s^−1)	r2/r1
Pro[FeCl4]	2.7	11.04	58.32	5.28
ProC1[FeCl4]	3.1	8.29	44.93	5.42
Glu[FeCl4]	2.8	11.06	55.15	4.99
GluC1[FeCl4]	3.3	10.98	63.16	5.75
AlaC1[FeCl4]	3.2	6.64	37.27	5.61
ValC1[FeCl4]	3.2	6.81	38.43	5.64
Gd-BOPTA	1.9	4.31	4.95	1.20

Table 2.

Osmolality, relaxivity values (r₁ and r₂) and ratio of r₂ and r₁ (r₂/r₁) for PMILs and Gd-BOPTA.

Data reproduced from Ref. [63] with permission from Royal Society of Chemistry respectively.

It is also found that these PMILs are able to generate dual contrast with intermediate r₂/r₁ value like metal nanoparticles. Low value of r₂/r₁ represents the positive contrast, whereas high value indicates negative, but intermediate value of this represents dual nature of contrast agent.

Tegafaw et al. reported that the r₂/r₁ value for Gd-Dy oxide made hybrid nanoparticles is near 6 and claimed its dual nature [5]. Fe₂O₃ + Fe₃O₄ nanoparticles (Resovist) also have r₂/r₁ value near 5.9 [3]. Similarly, r₂/r₁ value for these PMILs is also observed near 5.2 and which is greater than Gd-BOPTA (r₂/r₁ ≈ 1.1). Therefore, it can be summarized that the synthesized nanoparticle-free and biofriendly PMILs are potential T₂ and T₁ dual-mode contrast agents. These PMILs can take position in the list of newly discovered or clinically used contrast agents.

6. Conclusion and future prospect

PMILs has been explored as a dual-responsive (T₁ and T₂) contrast agents for magnetic resonance imaging, and such dual-behavior contrast agents are reported by rare researchers [4, 5]. PMILs have green nature (use of biofriendly amino acid moiety) and magnetic property (use of biocompatible non-toxic Fe (III) ion). Use of Fe eliminated the adverse effect of toxic metal ion like Gd(III) on cell functionality [98] or any Gd metal concerned diseases [99]. However, it is also reported that metallic nanoparticles used for single as well as dual contrast agents have also showed the harmful effect on cell and its physiology [100, 101, 102]. For the sake of knowledge, Maureen R. Gwinn and Val Vallyathan have reviewed and reported the toxic effect of nanoparticles (NPs)-based contrast agents which similarly act as air-borne UFPs (Ultra-Fine Particles) and cause diseases with long latency [102]. Similarly, Meng Tang et al. have also reviewed the various adverse effects of NPs on cell organelles and reported that NPs create mitochondrial dysfunction, endoplasmic reticulum stress and lysosomal rupture [103]. Even clinically used Gd-based contrast agents (GBCAs) are also under the question mark with their health-related issues and lengthy synthesis. The report from FDA drug safety newsletter also has criticized the GBCAs and explained the drastic effect of Gd on kidney-related issues such as fibrosing disease, NSF (Nephrogenic Systemic Fibrosis) with acute or chronic severe renal (kidney) insufficiency and renal dysfunction. Some other reports are also available where researchers have reported the accumulation of gadolinium in the renal tissue of patients suffering from NSF [98]. In this study, PMILs have been studied, and their possible use in imaging techniques such as MRI contrast agent with some better advantage has been revealed. Most frequently used Gd metal has been replaced with Fe as a magnetic source in PMILs for imaging due to kidney-related issues. The efficacy of these PMILs is also compared with clinically approved ongoing Gd-based contrast agent. If we compare, PMILs neither contain any metallic nanoparticles nor metal with bulky ligand complex. These PMILs are found to be free from the issue of metal leaching, and any adverse or side effect which is common for nanomaterials when they are used as contrast agents. These PMILs are easy to synthesize and cost-effective, and at certain concentration, they are safe in reference to animal DNA. However, for real effect on cell or health, there is research still ongoing. So, targeted and specific uses of these PMILs need further deep investigations with living cells or organisms.

The comparative study of PMILs with commercially available and FDA-approved Gd-based contrast agents is also completed to examine the comparative performance. Gadobentae Diglumine (Gd-BOPTA) is used as a model contrast agent for this studied. All the experiments were carried out under the similar conditions, and it was found that PMILs-based contrast agents have remarkable properties with significant imaging responses and relaxivity values. The relaxivity values (r₁ and r₂) for Gd-BOPTA are much less compared with PMILs and wherein the ratio of r₂/r₁ is found around 1.2 for Gd-BOPTA which makes it positive contrast agent (T₁ mode) only. The relaxivity values (r₁ and r₂), ratio of r₂/r₁ and osmolality value of PMILs with Gd-BOPTA are given in Table 2. The measured osmolarity values are found in range of 3 Osmol.Kg⁻¹ for PMILs which is comparable to Gd-BOTA contrast agent. Low osmolality helps to reduce the pain and other contrary effect during injection [104]. From the results discussed above, it can be concluded that PMILs have several advantages over existing contrast agents (NPs and GBCAs). The PMILs have following advantages:

1. The synthesis of PMILs is easy, less tedious, green and cost-effective.

2. There is no need of a long and lengthy pre-synthesis of ligands moiety, costly precursors, reactants, toxic reagents and volatile organic solvents for synthesis like other MRI contrast agents.

3. The problems related to thermodynamic instability and metal leaching problem can be avoided in PMILs.

4. The use of Fe metal in PMILs eliminates the severe health problems which occur due to toxic metals such as Gd.

It can be said that any biological moiety including proteins, vitamin, nitrogen contain sugar moiety, nitrogen-containing heterocycles or drug molecules or any molecule which can be turned into positive ions may be used to synthesize the PMILs via given synthetic procedure in the literature [63]. This study opens other new potential applications of PMILs in the fields of medical, pharma and analytical science. Moreover, such study also triggered further investigations into cytotoxicity, effect on biological process, cancer cell growth, tracking of drug molecules in body and its other uses in physical constant verification and measurement. In the field of imaging, PMILs may become most potential and promising contrast agent with suitable modification. However, research is still ongoing to explore new molecules with better advantages of such iron (III)-containing ionic-liquid-based contrast agents.

Acknowledgments

Praveen Singh Gehlot gratefully acknowledges the Commissioner of Higher Education Department, Government of Gujarat, India, and his former fellowship (JRF & SRF) granted by University Grant Commission, India. Authors also acknowledge financial support through Department of Science and Technology (DST), India. Authors are thankful to CSIR-CSMCRI, India, for providing the place for research work and additional assistance.

Conflict of interest

The authors declare no conflict of interest.