Paramagnetic Surface Active Ionic Liquids: Interaction with DNA and MRI Application

Colloid and Interface Science Communications 26 (2018) 14–23

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Paramagnetic Surface Active Ionic Liquids: Interaction with DNA and MRI Application Praveen Singh Gehlota, Hariom Guptaa,b,1, Arvind Kumarb,

T

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a Academy of Scientific and Innovative Research (AcSIR)-Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), G. B. Marg, Bhavnagar 364002, Gujarat, India b CSIR-Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research, G. B. Marg, Bhavnagar, 364002, Gujarat, India.

A R T I C LE I N FO

A B S T R A C T

Keywords: Paramagnetic surface active ionic liquids DNA MRI Contrast agents

Paramagnetic surface active ionic liquids (PMSAILs) have been synthesized wherein long alkyl chain bearing imidazoilium, pyridinium or isoquinolinium are cationic and bromotrichloroferrate (III) is paramagnetic counterion constituents. PMSAILs have been investigated for their aggregation properties using surface tension, conductivity, and dynamic light scattering (DLS). Interaction of PMSAILs with DNA in aqueous solutions has been examined through CD, fluorescence, ITC, DLS, zeta potential, and agarose gel electrophoresis. DNA compaction has been observed upon interaction with PMSAILs in dilute solutions. PMSAIL with isoquinolinium cation was found more efficient towards the DNA compaction. Decompaction of DNA could be achieved simply with the addition of NaCl. Further, the application of PMSAILS as MRI contrast materials has been explored. All the investigated PMSAILS exhibited excellent dual mode T1 & T2 contrast property in low concentration regime. To improve the real-time practical importance, MRI results have been compared with clinically available GdBOPTA based MRI contrast agent.

1. Introduction In the surface science, especially in the colloid branch, ionic liquids (ILs) have taken unique position due to their greener aspects and better performance compared to conventional surfactants. Inherent amphiphilic nature of desired constituent ions in ILs has rendered superior surface active properties viz. adsorption efficiency, effectiveness in surface tension reduction, low CAC, and ability to form various nanostructure etc. [1]. ILs with long alkyl chains in either cation or anion or in both have been termed as a surface active ionic liquids (SAILs) [2] and exhibit combined properties of ILs and surfactants [3]. Like, conventional surfactants, SAILs also form various self-assembled structures such as micelles, [4] elongated, spherical, cylindrical, disk-like micelles, [5] worms like micelles, [6] vesicles, [2] tubules and ribbon shaped vesicles [7] and multilamellar structures of vesicles [8] etc. These aggregates resembles to biological compartment and bilayer membranes, specially the vesicles [9]. The variations in morphologies of aggregates have found uses in industrial [10], chemical [11], and pharmaceutical [12, 13] applications. Hayashi, and Hamaguchi introduced the ILs with magnetic properties by simply exchange of Cl− anion with [FeCl4]− [14]. Such

paramagnetic ionic liquids (PMILs) have found their multiple uses in desulfurizations [15], organic synthesis [16], microextraction [17–19], electro-catalysis [20], probe for vesicles [21], self-assemble media for surfactants [22], acidic catalysis [23], density measurement [24], paramagnetic polymer synthesis [25, 26], microemulsion formulation [27], synthesis of chitosan supported magnetic IL based catalysis [28], CO2 separation [29], application in analytical [30, 31] and other various applications [32], Similar to SAILs, a new type of paramagnetic ionic liquid has been introduced by Paul Brown and Julian Eastoe group which are magnetoresponsive and have been called as Paramagnetic Surface Active Ionic Liquids (PSAILs), also termed as a “Magnetic Surfactant”. This PSAILs may have symmetric [FeCl4]−or asymmetric anion [FeCl3Br]− [33]. ILs [34], conventional surfactants [35] and SAILs [36] have been studied with genomic DNA at deep understanding level in pharmacokinetic and biotechnology field. Similarly PMILs have also been studied with DNA and explored for their utility in DNA preservation and stability [37], extraction of DNA [38] etc. The PMSAILs with azo group along with magnetic responsive moiety have been used in controlled DNA release process [39]. These PMSAILs have shown the ability to form aggregates or nanosphere upon interaction with DNA, and these

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Corresponding author. E-mail address: [email protected] (A. Kumar). 1 Present address: Analytical Chemistry Division, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP) Lucknow, India. https://doi.org/10.1016/j.colcom.2018.07.004 Received 13 June 2018; Received in revised form 18 July 2018; Accepted 20 July 2018 2215-0382/ © 2018 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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acetonitrile solvent.

self-assembled structures have been further used in drug delivery [40]. Paul Brown et al. demonstrated the migration of DNA and proteins molecule under low magnetic field strength with keeping their native nature constant by using PMSAILs [41]. It is clear that PMSAILs have significant importance as biotechnological tool. Carla I. Danielhas et al. shows the enhancement in r1 relaxation rate of magnetic IL [P66614] [FeCl4] in mixture of ionic liquids ([P66614][Cl] + [P66614][FeCl4]) with DMSO solvent [42]. They also explored the future advantages of PMILs in contrast application. We have taken the indication from their work and studied the self-assembly of newly synthesized PMSAILs and their MRI properties for potential use as contrast reagent. In this report we have showed dual response (T1 and T2 relaxation) in MRI from PMSAILs which is rare and found in hybrid nanomaterials only [43, 44]. These PMSAILs may eliminate the adverse effect of Gd metal ion on cell functionality [45] or harmful effect from metallic nanoparticles on cell and its physiology [46–48]. Studies on the physical interaction of PMSAILs with DNA also showed that after certain concentration of PMSAILs DNA precipitates due to compactness. This ability is more pronounced with isoquinolinium head group then imidazolium and pyridinium.

2.3.2. Conductivity Measurements Conductivity was measured on digital Eutech auto temperature conductivity meter model PC 2700, Thermo Scientific, US, assemble with a conductivity cell and temperature probe. Cell constant for the cell used was 1.0. The solution temperature was maintained by using Julabo thermostat at 25 °C (with accuracy ± 0.1 °C). The degree of counterion binding (β) has been calculated from equation provided in Annexure 2 of supporting information. 2.3.3. Tensiometry Tensiometry was employed to measure the critical micellar concentration (CMC) and surface parameters. The surface tension measurements were done using automated Attension force Tensiometer Sigma 700, Biolin Scientific, Sweden with Du Noüy ring method. The surface tension values have collected in triplicate and the average value of them has been considering for further calculation. Uncertainty which was calculated by means of standard deviation in measurements of each reading and was found to be ± 0.1 mN·m−1.

2. Materials and Methods

2.3.4. Dynamic Light Scattering (DLS) DLS was used to measure the size of aggregates formed by PMSAILs in aqueous solution by using NaBiTec Spectro-Size300 light scattering apparatus (NaBiTec, Germany) with a He-Ne laser (660–670 nm, 4 mW) at angle of 90. Zetasizer Nano ZS light scattering apparatus (Malvern Instruments, U.K.) with a He-Ne laser (633 nm, 4 mW) at angle of 90 was used for measure the hydrodynamic diameter of DNA in absence and presence of PMSAILs at various concentration.

2.1. Material Isoquinoline (> 98% purity), pyridine (> 98% purity), 1-bromododecane (> 98% purity) were purchased from TCI Chemical (India) Pvt. Ltd. 1-Methyl imidazole was purchased from Spectrochem, India. ACS reagents such as ferric chloride hexahydrate (97% purity), ethidium bromide, trizma base (99.9% purity), and sodium salt of deoxyribonucleic acid (DNA) from salmon testes were purchased from Sigma-Aldrich. Methanol, acetonitrile, ethyl acetate, and n-hexane solvents of AR grade were procured from SD-fine chem. Ltd., India. All the chemicals were of AR grade and were used as received. Millipore grade water with specific conductivity 3 μS·cm−1 and surface tension 71 mN·m−1 was used for the solutions preparation.

2.3.5. Far-UV Circular Dichroism Spectroscopy Far-UV circular dichroism (CD) spectra of DNA in Tris.HCl buffer at pH 7.4 was recorded in triple measurement at the wavelength range 200–400 nm in a Jasco J-815 CD spectrometer, US under N2 environment with temperature 298.15 K. Experiments were carried out in a quartz cuvette having a path length of 1 mm. The spectra were collected at a scan rate 100 nm/min. The response time and the bandwidths were 2 s and 0.2 nm, respectively.

2.2. Synthesis of Paramagnetic Surface Active Ionic Liquids

2.3.6. Ethidium bromide exclusion assay According to previous report [49], 50 μL solution of 0.1 mM EB was mixed with 2 mL of buffer, and the fluorescence spectra of water-EB were recorded in the absence of DNA and the presence of DNA from 500 to 700 nm at an excitation wavelength (λex) of 530 nm using a Fluorolog horiba Jobin Yvon fluorescence spectrophotometer. The PMSAILs solution was added successively up to 10 mM Fe concentration in 2 mL cuvette containing EB-DNA complex and recorded the spectra at 298.15 K. The percentage of EB binding was observed due to the replacement of EB by PMSAILs from DNA was calculated using Eq. (9) given in Annexure 2 of supporting information.

Bromide precursors (a2, b2, and c2, Fig. S1) and ferric chloride hexahydrate (1:1 mol eq.) were taken in methanol solvent and reaction mixtures were kept for reflux over the day. After completion of the reaction, the solvent was removed by rota-evaporator and product was dried under vacuum. The product was washed with a little volume of water to remove unreacted ferric chloride. Products were completely dried and stored in a vacuum desiccator to make it free from any moisture issue. Characterization was done with UV–visible and Raman spectra, CHNS, ICP-OES (Inductive coupled plasma optical emission spectroscopy for % Fe) and is given in supporting information. Scheme 1. shows the molecular structure of synthesized PMSAILs. DNA stock solution was prepared by dissolving an appropriate amount of DNA in 50 mL 80 mmol.L−1 trizma base hydrochloride (TE.HCl) buffer solution and kept it overnight for complete solubilisation. The actual concentration of DNA was determined using NanoDrop® Spectrophotometer ND-1000. 90–92 ng.μL−1 DNA concentration was used in whole study. 2.3. Methods

2.3.7. Determination of Zeta Potential (ζ) The surface charge of negative DNA in the presence of PMSAILs were measured from Zeta potential (ζ) experiment using a Zetasizer Nano ZS light scattering apparatus (Malvern Instruments, U.K.) with a He-Ne laser (633 nm, 4 mW) at 298.15 K. For measurements, solution was transferred through 0.25 μm membrane filter into ‘DTS 1060’ cell possessing gold-coated electrode. The Smoluchowski approximation was selected during measurements.

2.3.1. UV–Visible and Raman Spectroscopy The synthesized asymmetric anion [FeCl3Br]− was verified by using Shimadzu UV-2700 UV–VIS spectrophotometer, Japan and LabRAM HR Evolution Horiba Jobin Yvon Raman spectrometer, Japan at 298.15 K. For UV measurement, samples were prepared in

2.3.8. Isothermal Titration Calorimetry (ITC) Enthalpy changes (dH) due to micellization and the interaction of DNA in successive injections with PMSAILs in buffer solution were measured using MicroCal ITC200 microcalorimeter, U.K. with an instrument controlled Hamiltonian syringe having volume capacity of

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Scheme 1. Chemical structure of investigated PMSAILs.

40 μL. 2μL aliquots of stock solution were added into the sample cell containing 200 μL of water or DNA solution with continuous stirring (at 500 rpm). The parameters like temperature, time of addition and duration between each additions were controlled by automated software.

each phantom were calculated using Eq. (2).

3. Result and discussion 3.1. Thermal Phase Behavior of PMSAILs

2.3.9. Agarose Gel Electrophoresis 2μL loading dye for tracking path (0.25% w/v bromophenol Blue, 0.25% w/v xylene cyanol FF and 30% glycerol in water) mixed with 5 μL PMSAILs-DNA solution and these mixtures were loaded into wells. Electrophoresis was carried out at 50 V in TBE (pH 8.3) buffer for 60 min after 3 h incubation at room temperature using Electrophoresis Power supply BGPS 300/400. The DNA bands were visualized and images were taken from Gel Doc™ XR+ with imageLab™ software (molecular imager) BIO-RAD.

Thermal behavior of synthesized PMSAILs has been investigated using NETZSCH TG 209 F1 Libra TGA and NETZCH DSC 204 F1 Phoenix DSC. DSC thermogram is given in Fig. 1. Glass transition temperature for PMSAILs was found below 100 °C. Due to significant symmetric nature of pyridinium and isoquinolinium cationic head group, these PMSAILs have little bit higher Tg value than imidazolium cation containing PMSAIL. The Tg values are < 100 °C and fulfilling the criteria of conventional definition of an Ionic liquid [51] and hence can termed as a paramagnetic surface active ionic liquids (PMSAILs). Change in % mass of PMSAILs with the action of temperature has been recorded from Thermal Gravimetry Analysis and observed that initial loss in mass is due to moisture or water elimination at near to 120200 °C. After that maximum loss in mass has occurred between 300 and 400 °C. Tonset, Tstart, and Tg values of PMSAILs are given in supporting information (Table S2). TGA and DTG thermograms are also provided in supporting information (Fig. S3).

2.3.10. 1H NMR and Magnetic Resonance Imaging All the precursors have been characterized via 1H NMR using BRÜKER AVANCE 500 MHz, Germany. Magnetic resonance imaging (MRI) and relaxation rate experiments were carried out according to earlier report [50]. Magnetic resonance images and relaxometric contrasting characteristics of PMSAILs and Gd-BOPTA were acquired with BRÜKER AVANCE 500 MHz (11.7 T) NMR instrument using microimaging probe and Paravision imaging software. T1 and T2 weighted MRI images of aqueous phantoms of each PMSAIL having different concentration (0.3, 0.5, 0.7 and 1.0 mM) measured in terms of Fe concentration were acquired 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, and FOV = 0.5 cm, TR = 250 ms, TE = 8 ms, 128 × 128 matrix and FOV = 0.5 cm, TR = 2000 ms, TE = 72, 128 × 128 matrix respectively for Gd-BOPTA. T1 and T2 relaxation times of aqueous phantoms having different concentration of PMILs (0.3, 0.5, 0.7 and 1.0 mM Fe) and GdBOPTA (0.3, 0.5, 0.7, 1.0 mM Gd), were measured using of 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/T1and1/T2) versus metal concentration using Paravision software. T1 and T2 relaxation times for

Fig. 1. DSC profile of PMSAILs. 16

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Fig. 2. (A) Trends in surface tension and (B) specific conductivity along with increasing concentration of PMSAILs.

values are low and Πcmc and Гmax are higher contrary to [C12mim] [FeCl3Br] and [C12iQ][FeCl3Br] at air/water interface. This may be due to symmetric nature of the pyridinium (absence of alkyl group and ring residue) which play an important role in assembling the molecules compactly at air-water interface with maximum numbers [53]. Therefore, it has higher value of maximum surface excess concentration and lower value of covered area occupied by a single molecule. The negative Gibbs free energy values for aggregation of PMSAILs derived from conductivity experiments (Fig. 2B, Table 2) indicate that the self-assembling process is spontaneous. Low β value has been observed for all the investigated PMSAILs and is indicating their lower counter ion binding tendency. This is likely due to bigger and tetrahedral nature of anion which have less tendency to bind with the cationic head group of micelles. ITC enthalpograms for PMSAILs in water are given in supporting in formation (Fig. S4). Endothermic peaks in ITC data are revealing endothermic nature of micellization which is driven by enthalpy process [54]. Intensity weighted size distributions for the formed aggregates were determined from DLS measurements. The size distribution of aggregates for all three PMSAILs at a solution concentration of 10 mmol.L−1 has been shown in Fig. 3. The radius of the aggregates ranges between 10 and 30 nm which is comparatively bigger than conventional surfactant micelles and could be due to hydrophobic large sized counter ion.

3.2. Aggregation Behaviour and Thermodynamic Parameters Aggregation behavior including CMC and related parameters for PMSAILs in aqueous solutions were examined from three independent techniques, namely, surface tension measurement, electrical conductivity measurement, and isothermal titration calorimetry (ITC). The plots of surface tension (γ) and specific conductivity (κ) at 25 °C against concentration are depicted in Fig. 2A. The surface tension of aqueousPMSAILs solutions decreased polynomialy with the logarithm of concentration because of the synergic adsorption of PMSAILs molecules at the air/water interface, and attained a CMC value, above which a nearly constant value of surface tension (γcmc) is observed. The surface activity and related parameters at air/solution interface of any amphiphiles are always discussed in terms of adsorption efficiency (πCMC), effectiveness reduction in surface tension (pC20), surface excess concentration (Γmax), covered area by single molecule (Amin) and packing parameter (P). The applicable and well-reported relations to calculate these parameters are well-described in the Supporting Information (Annexure 2). The CMC and summary of the surface active parameters for PMSAILs are listed in Table 1. The CMC values for PMSAILs are relatively lower with respect to their precursor analogue [52]. The hydrophobic nature of anion [FeCl3Br]− is likely responsible for lower CMC. As can be seen from the Table 1, for [C12Py][FeCl3Br] the pC20 and Amin

Table 1 Critical micelle concentration (CMC), surface tension at CMC (γCMC), effective surface tension reduction (πCMC), adsorption efficiency (pC20), maximum surface excess concentration (Γmax), and area occupied by a single molecule at the air−water interface (Amin) of PMSAILs at 298.15 K. Name

[C12mim] [FeCl3Br] [C12Py][FeCl3Br] [C12iQ][FeCl3Br]

SFT

πCMC

pC20

Γmax

Amin

CMC in mmol.L−1

mN.m−1

mol.L−1

μmol.m−2

Å

2.2

37.8

3.3

3.98

41.8

5.2 2.4

41.2 39.2

3.0 3.2

7.26 3.94

22.94 42.30

Table 2 Critical aggregation concentration (CAC), Gibbs free energy of aggregation (ΔGaggo), Gibbs free energy of adsorption at air-water interface (ΔGadso), Free energy on ionic surfactant to polyelectrolyte interaction (ΔGs→po) for PMSAILs at 298.15 K. Name

[C12mim] [FeCl3Br] [C12Py] [FeCl3Br] [C12iQ][FeCl3Br]

17

CMC mmol.L−1

β

ΔGaggo

ΔGadso

ΔGs→po

kJ.mol−1

kJ.mol−1

kJ.mol−1

Cond.

ITC

2.5

2.1

0.25

−31.2

−40.7

−2.24

5.6

5.3

0.38

−31.32

−37.1

−4.29

2.4

2.6

0.33

−32.4

−42.2

−4.35

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of common salt NaCl. The excess amount of NaCl was added in precipitated DNA solution which converted condense DNA into soluble form likely due to efficient ion exchange process. Resoluble DNA was further confirmed by CD spectroscopy and gel electrophoresis which showed same bands as that of native DNA without any degradation (Fig. S6). The binding capability of this PMSAILs with DNA was examined using intercalated dye ethidium bromide (EB) though fluorescence spectroscopy. The 20 to 25 fold enhancement in fluorescence intensity of the EB was observed when it was bound with DNA due to the migration of ethidium cation into hydrophobic microenvironment of double strand DNA from aqueous solution. Here water plays a role of quencher [59, 60]. The addition of PMSAILs solution into this DNA-EB complex, fluorescence intensity started to decrease due to the formation of DNA-surfactant complex via displacement of intercalated EB. [61]. Fig. 4B shows the reduction in fluorescence intensity due to displacement of EB from the DNA-EB complex upon increasing the Fe concentration of [C12mim][FeCl3Br]. The fluorescence spectra for other PMSAILs are given in supporting information (Fig. S5). It has been found that [C12iQ][FeCl3Br] has the maximum ability to displace EB from DNA over other PMSAILs. The 3D surface plot (Fig. S7) reveals that % EB binding drops when N/P ratio increases. The area of the 3D plot has been divided into several colored zones, wherein the light blue region depicts negligible EB displacement or 100% EB binding when no PMSAIL was added. The % of EB binding for [C12iQ][FeCl3Br] is about 58.82% at an N/P charge ratio of 0.02, whereas % of EB binding for [C12mim][FeCl3Br] and [C12Py][FeCl3Br] is about 41.89% and 55.75% at 0.6 N/P ratio respectively (Fig. S7). [C12mim] or [C12Py] PMSAILs have weaker capability to interact and displace EB from lipoplex compared to [C12iQ] based PMSAIL. It can be assumed that near to CMC, thermodynamically stable lipoplex is disable to accept more amphiphilic molecules for displacement of EB for imidazolium and pyridinium based paramagnetic surfactant, so no longer intensity of EB fall at higher N/P ratio or higher Fe concentration [62]. Binding Isotherms of PMSAILs-DNA aqueous systems were recorded using isothermal titration microcalorimetry (ITC). The isotherms were used to investigate the thermodynamic behavior associated with the interaction between PMSAILs and DNA. Fig. 5 illustrates the representative ITC enthalpograms for the titration of 50 mmol.L−1 Fe concentration of [C12mim][FeCl3Br] with 90 ng.μL−1 DNA concentration. The actual binding isotherms of these PMSAILs have been corrected by subtraction of the corresponding dilution heat derived from the injection of identical amounts of the PMSAILs into buffer alone [34]. ITC profile for [C12Py][FeCl3Br] is given in supporting information, Fig. S8 whereas for [C12iQ][FeCl3Br] ITC profile could not be recorded because of precipitation of the complex. Further, the titration was carried out only up to 1 mmol.L−1 Fe of [C12mim][FeCl3Br] and [C12Py][FeCl3Br] due to aggregation of surfactants on DNA to form insoluble precipitate or surfactant-DNA lipoplex beyond this

Fig. 3. Intensity weighted size distribution of PMSAILs in aqueous medium.

3.3. Compaction of DNA via Physical Interaction With PMSAILs Interaction of PMSAILs with Animal Double Stranded DNA (Salmon Fish) in Aqueous Solutions Were Studied Using CD, Fluorescence, Zeta Potential, DLS and Agarose Gel-Electrophoresis. The Systematic Studies Revealed the Compaction of DNA after Certain Concentration of PMSAILs Fig. 4A shows a representative CD spectra of DNA upon interaction with [C12mim][FeCl3Br] PMSAILs. CD spectra of pure DNA exhibiting a negative band, crossover point and a positive band of pure B conformer of DNA is provided in supporting information with other PMSAILs (Fig. S5) [55]. These bands are retained and are unchanged up to 1 mmol.L−1 Fe concentration of [C12Py][FeCl3Br] or [C12mim] [FeCl3Br] whereas for [C12iQ][FeCl3Br] the bands are retained and unchanged up to 0.5 mmol.L−1 Fe concentration. In these concentration ranges, band position and intensity of corresponding bands are closely superimpose to pure B-DNA which is indicating its secondary structural constancy. On further addition of PMSAILs, DNA has gone in compaction phenomenon which is indicated by distortion in intensity and shifting in band position. A cationic surfactant-complex (lipoplex [56]) is probably the reason for DNA Compaction. According to a previous report from Grueso et al. and Xu et al., a cationic surfactant which has Br− as counterion condensed the DNA effectively at high concentrations [57, 58]. Because of the hydrophobic nature of magnetic [FeCl3Br]− anion compared to conventional Br− or Cl−, it is less likely to bind the cationic head group effectively. Therefore, the cationic head are free to interact with negatively charged surface of DNA and condense the DNA potentially. More exposed positive head groups interact with DNA negative surface and condense the DNA even at low PMSAILs concentrations (monomeric) [40]. However, the DNA decompaction could not be observed in complex magnetic anion like [FeCl3Br]− [58]. Herein we have observed the decompaction of DNA structure with the assistance

Fig. 4. A representative CD spectra of DNA (A) and Fluorescence spectra of EB-DNA complex (B) at various Fe concentration of [C12mim][FeCl3Br] PMSAILs. 18

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Fig. 7. A representative profile for Zeta potential. Fig. 5. A representative ITC Binding Isotherm for DNA with [C12mim] [FeCl3Br].

have different auto correlation function and decay time. The zeta potential measurement experiments were performed to validate the concentration regimes where DNA negative surface become saturated. Fig. 7 represents the variations in zeta potential values for [C12mim][FeCl3Br]/DNA complexes with Fe concentration. With increasing Fe conc., zeta potential increased linearly, followed by a slight decrease, and then increased rapidly to reach a plateau value. When concentration is sufficiently low, there is little interaction of [C12mim][FeCl3Br] with DNA. With increasing concentration, a few molecules of [C12mim][FeCl3Br] have started to interact onto the DNA and some hidden core charges of the DNA chains exposed outside leading to the decrease of zeta potential [65]. With a further increase in concentration, the DNA charges are saturated and negative charge is neutralized gradually by [C12mim][FeCl3Br] which is accompanied by the PMSAILs[C12mim][FeCl3Br]/DNA complex formation. For [C12Py] [FeCl3Br] and [C12iQ][FeCl3Br], the zeta potentials of the complexes are negative at low concentration of Fe and attains a minimum value. With a further increase of Fe concentration the zeta potential increases and becomes positive at certain higher concentration (Fig. S10). Positive value of zeta potential indicate the existence of free micelles formed by PMSAILs in the bulk instead of binding onto the existing stable complexes. These results represent the electrostatic interaction between negative phosphate groups and positive head groups of PMSAILs. Agarose gel electrophoresis experiments were carried out to understand DNA degradation (Fig. 8). Initial bands are similar to pure BDNA in low concentration range which is indicating the presence of unbound DNA molecules. The band becomes notably vague when concentrations reaches 2 mmol.L−1 for [C12mim][FeCl3Br] and [C12Py] [FeCl3Br] (Fig. S11), and 0.3 mmol.L−1 for [C12iQ][FeCl3Br]. This may be due to lesser availability of unbound DNA in system as most of the molecules forms complex with PMSAILs. Further increase of concentration leads to the disappearance of the illuminated bands,

concentration. This concentration has been further assumed as a critical aggregation concentration (CAC) [36]. Gibbs free energy for ionic surfactant to polyelectrolyte interaction (ΔGs→p) has been calculated using equation (annexure II, Eq. (8)) and found that isoquinolinium based PMSAIL shows maximum interaction with DNA in contrast to rest two PMSAILs [63]. Dynamic light scattering (DLS) measurements were carried out for size changes in DNA with increasing Fe concentration of PMSAILs. Fig. 6 indicate the intensity weighted distribution DLS profile with increasing concentration of Fe. As can be seen from DLS profiles, the size distribution of the pure DNA solution gives three peaks with average hydrodynamic diameters (Dh nm) of about (a) 19 nm, (b) 103 and (c) 570 nm. The peaks at low concentration of Fe (up to to 1 mM) indicate a size similar to that of pure DNA. However, when concentration reaches 1 mmol.L−1, a broad peak with the average Dh of ~452 nm appears, which indicates the aggregation of PMSAIL molecules on DNA surfaces. At still higher concentration (10 mmol.L−1), a larger aggregate with average Dh ~1436 nm made through DNA compaction with PMSAIL molecules is observed. The variations of DNA size upon interaction with PMSAILs in aqueous solutions have similarity to the previous reports [64]. When concentration of PMSAILs is sufficiently high (25 mmol.L−1), in addition to the large peak, a third peak with an average Dh ~32 nm and ~27 nm was observed for [C12Py][FeCl3Br] and [C12iQ][FeCl3Br] respectively due to micelles formation (Fig. S9). At the same concentration, these values are comparable in absence of DNA (Fig. 3) confirming the existence of free micelles with larger aggregate in the system. Auto correlation function (Fig. S9) which is technique to extract time dependence signals in presence of noise represent the confirmation of larger stable complex in system where larger and smaller aggregate

Fig. 6. A representative Intensity weighted Size distribution of DNA in presence of [C12mim][FeCl3Br] at below and above CMC. Red dotted circle rendered bigger aggregate and black circle indicate DNA with monomers. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. A representative agarose gel electrophoresis pattern for (A) [C12mim][FeCl3Br] and (B) [C12iQ][FeCl3Br] at various Fe concentration of PMSAILs.

confirming the complex has formed by all DNA molecules with PMSAILs and there are no free DNA molecules left in the systems. Here we also found that the appearance of the single band instead of multiple bands like as ladder which is indicating that DNA did not enter into any degradation process in the presence of PMSAIL molecules [36].

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 a PMSAILs obey a linear relationship with Fe concentration which can be represented mathematically by the following expression [66]:

3.4. Relaxivity and MRI study in aqueous medium

1 1 = + ri C Ti, C Ti,0

(1)

Ti,C and Ti, 0 (i = 1 or 2) are relaxation time of sample at C concentration and in absence of contrast reagent, ri is the relaxivity of the contrast agent. T1 and T2 weighted imaging are obtained via using spin echo (SE) MRI pulse sequence. The signal intensity for SE pulse sequence can be expressed as [67]:

Gd metal has been replaced with Fe as a magnetic source for imaging due to kidney related issues in a report by Whitney A High et al. [45]. Even the nanoparticles which are frequently investigated by a number of researchers have shown to create harmful issues with the living cell [46]. Here, we have explored the possibility of PMSAILs as MRI contrast agent with some advantage over existing CA as these are neither containing the metallic nanoparticles nor bulky metal- ligand complex, and do not degrade DNA up to desired concentrations of Fe. Therefore, MRI and relaxation studies were undertaken to demonstrate the utility and effectiveness of PMSAILs as an MRI contrast agent (CA) for diagnosis purpose. The MRI image intensity depends on the population of 1H nuclei of the biological tissue or cell or tested solvent, relaxation time and their relaxation rate also. Relaxation rate 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,

I = I0 (1 − e−TR / T1 )(e−TE / T2)

(2)

Intensity of T1 and T2 weighted image are purely T1 and T2 dependent; T1 and T2 weighting can be achieved by eliminating the T2 term (T2 term→1) for T1 weighting and T1 term (T1 term→1) for T2 weighting with the selection of the appropriate combination of TE (time of echo) and TR (time of repetition) value. It can be noticed from Eqs. (1) and (2) that intensity increases in T1 weighted image but decreases in T2 weighted images with the increase of Fe concentration. In order to investigate the contrast property, T1 and T2 weighted MRI images of aqueous phantoms of PMSAILs making four different Fe concentration (0.3, 0.5, 0.7 and 1.0 mM Fe) were measured in terms of 20

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Fig. 9. T1 and T2 weighted MR images of PMSAILs at various Fe concentrations.

Fig. 10. (A) Relaxivity of MRI responsive PMSAILs at various Fe concentration and (B) Relaxivity of Gd-BOPTA for comparison.

images with Fe concentration reveals contrast property of aqueous PMSAILs qualitatively and suggests that these PMSAILs have negative as well as positive contrast behavior (T1 and T2 contrast). Fig. 10 shows a linear relationship of r1, r2, and r2* relaxivity on Fe concentration of PMSAILs (values are given in Table 3) similar to the literature result available for dehydroascorbic acid coated Fe3O4

Fe concentration and their relaxation times were determined by using Eq. (2) for T1 and T2. T1, and T2 weighted MRI images of PMSAILs along with a standard Gd based CA are shown in Fig. 9. A drastic intensity reduction in T2 weighted images and a slight intensity enhancement in T1 weighted image is observed with the increase of Fe concentration. This intensity variation observed in MRI

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Table 3 r1, r2 and r2/r1 parameters for PMSAILs from T1 and T2 measurements. Name

[C12mim][FeCl3Br] [C12Py][FeCl3Br] [C12iQ][FeCl3Br] Gd-BOPTA

r1

r2

mM−1.s−1

mM−1.s−1

6.73 6.51 6.47 4.31

35.56 34.41 34.30 4.95

[4]

r2/r1 [5] 5.28 5.28 5.30 1.15

[6] [7]

nanoparticles [50]. Tegafaw et al. reported the r2/r1 value for Gd-Dy oxide hybrid nanoparticles ~6 and claimed its dual nature [43]. Similarly Fe2O3 + Fe3O4 nanoparticles exhibiting dual contrast have shown r2/r1 value near to 5.9 [68]. As can be seen from Table 3, the investigated PMSAILs have been also able to generate dual contrast with intermediate r2/r1 values ~5 similar to that exhibited by metal nanoparticles based CAs which is greater than commercially utilized MRI CA, Gd-BOPTA (r2/r1 is 1.1).

[8] [9] [10] [11] [12] [13]

4. Conclusion [14]

PMSAILs have been synthesized and investigated for their aggregation behavior. CMC values of PMSAILs have been found lower than their respective precursors due to the hydrophobic nature of anion. The compounds are shown more effective in surface reduction and adsorption phenomenon. Higher surface excess concentration and less Amim found for isoquinolinium based PMSAIL is due to its symmetric nature and π-π interactions. Interaction of PMSAILs with DNA in aqueous solutions shows that at low concentration these do not alter the conformation of DNA and beyond CMC, molecular aggregation made complex with DNA. At higher concentrations, DNA becomes compact and phase separation occurs. Ability to compactness is found in order with respect to head group [C12iQ] > [C12Py] ≈ [C12mim]. DNA decompaction can be achieved simply with the addition of a common electrolyte NaCl. Utility of PMSAILs for MRI application have been studied. The signal intensity of the T1-weighted and T2-weighted images indicate that these can be utilized as potential MRI contrast agents with dual mode. These PMSAILs have several additional advantages over Gd-based MRI contrast agent like non-toxic nature of Fe constituent, easy to synthesis, and dual mode in single molecule.

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

Acknowledgment

[23]

Financial support through a Senior Research Fellowship to P.S.G. from the University Grant Commission. Department of Science and Technology (DST), India (No. EMR/2016/004747) is gratefully acknowledged for financial support. The analytical division & central instrumentation facility of CSMCRI is acknowledged for helping with sample characterization. Authors are thankful to Managl Singh Rathore and Kusum Khatri for gel electrophoresis experiments. BDIM is acknowledged for providing PRIS number CSIR-CSMCRI-094/2018.

[24]

[25] [26]

[27]

Appendix A. Supplementary data

[28]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.colcom.2018.07.004.

[29]

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