Alkyl-malonate-substituted thiacalix[4]

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May 16, 2017 - pudding” morphology, where the Gd(III) complexes form hard small (1.5-4 nm) cores ...... quantum chemical modelling was applied to reveal the difference in ... laboratory “Transmission electron microscopy” of Kazan National .... Cloud point extraction of lanthanide(III) ions via use of Triton X-100 without.
Accepted Manuscript Original article Alkyl-malonate-substituted thiacalix[4]arenes as ligands for bottom-up design of paramagnetic Gd(III)-containing colloids with low cytotoxicity Alexey Stepanov, Irek Nizameev, Rustem Amirov, Sofia Kleshnina, Gulshat Khakimullina, Svetlana Solovieva, Alexandra Voloshina, Anastasya Strobykina, Aidar Gubaidullin, Ramil Nugmanov, Asiya Mustafina PII: DOI: Reference:

S1878-5352(17)30105-3 http://dx.doi.org/10.1016/j.arabjc.2017.05.017 ARABJC 2103

To appear in:

Arabian Journal of Chemistry

Received Date: Accepted Date:

22 February 2017 16 May 2017

Please cite this article as: A. Stepanov, I. Nizameev, R. Amirov, S. Kleshnina, G. Khakimullina, S. Solovieva, A. Voloshina, A. Strobykina, A. Gubaidullin, R. Nugmanov, A. Mustafina, Alkyl-malonate-substituted thiacalix[4]arenes as ligands for bottom-up design of paramagnetic Gd(III)-containing colloids with low cytotoxicity, Arabian Journal of Chemistry (2017), doi: http://dx.doi.org/10.1016/j.arabjc.2017.05.017

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Alkyl-malonate-substituted thiacalix[4]arenes as ligands for bottom-up design of paramagnetic Gd(III)-containing colloids with low cytotoxicity Alexey Stepanova,*, Irek Nizameeva,b, Rustem Amirovc, Sofia Kleshninaa, Gulshat Khakimullinac, Svetlana Solovievaa, Alexandra Voloshinaa , Anastasya Strobykinaa, Aidar Gubaidullina, Ramil Nugmanovc, Asiya Mustafinaa a

A.E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center,

Russian Academy of Sciences, Arbuzov str., 8, 420088, Kazan, Russia b c

Kazan National Research Technological University, Kazan, 420015, Russia

Kazan (Volga region) Federal University, Kremlyovskaya str., 18, 420008, Kazan, Russia

Abstract The present work introduces thiacalix[4]arene adopting 1,3-alternate conformation with alkyl-malonate terminal substituents as ligands for Gd(III) ions. pH-dependent complex formation of Gd(III) ions via alkyl-malonate substituents in aqueous DMSO solutions results in a precipitation. The precipitated complexes were converted into hydrophilic colloids of “plumpudding” morphology, where the Gd(III) complexes form hard small (1.5-4 nm) cores included into larger (about 180 nm) soft PSS shells. The precipitate-to-colloid transformation is facilitated by polystyrolsulfonate (PSS) for Gd(III) complexes with thiacalix[4]arene bearing propyl-malonate groups, while the presence of PSS triggers a dissolution of the precipitated complexes for thiacalix[4]arenes with pentyl-malonate substituents. To a lesser extent the similar tendency disturbs the formation of PSS-stabilized colloids on the basis of butyl-malonate substituted thiacalix[4]arene. The PSS-stabilized colloids exhibit high longitudinal and transverse relaxivities (r1=23.8 and r2=29.4 mM-1s-1 at 0.47 T, respectively), while the recoating of the PSS-stabilized colloids with polyethyleneimine is accompanied by the dissolution of the hard cores. High relaxivity along with low cytotoxicity of PSS-stabilized colloids indicates their applicability as contrast agents in MRI. Keywords-

Malonate

thiacalix[4]arenes;

Longitudinal

relaxivity;

Paramagnetic

colloids;

Hydrophilic colloids; Gd(III) ions; Cytotoxicity *Corresponding author. Address: A.E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, Kazan 420088, Russia. Tel.: +7 843 273-93-65; fax: +7 843 273-18-72. E-mail address: [email protected] (Alexey Stepanov)

Alkyl-malonate-substituted thiacalix[4]arenes as ligands for bottom-up design of paramagnetic Gd(III)-containing colloids with low cytotoxicity Alexey Stepanova,*, Irek Nizameeva,b, Rustem Amirovc, Sofia Kleshninaa, Gulshat Khakimullinac, Svetlana Solovievaa, Alexandra Voloshinaa , Anastasya Strobykinaa, Aidar Gubaidullina, Ramil Nugmanovc, Asiya Mustafinaa a

A.E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center,

Russian Academy of Sciences, Arbuzov str., 8, 420088, Kazan, Russia b c

Kazan National Research Technological University, Kazan, 420015, Russia

Kazan (Volga region) Federal University, Kremlyovskaya str., 18, 420008, Kazan, Russia

Abstract The present work introduces thiacalix[4]arene adopting 1,3-alternate conformation with alkyl-malonate terminal substituents as ligands for Gd(III) ions. pH-dependent complex formation of Gd(III) ions via alkyl-malonate substituents in aqueous DMSO solutions results in a precipitation. The precipitated complexes were converted into hydrophilic colloids of “plumpudding” morphology, where the Gd(III) complexes form hard small (1.5-4 nm) cores included into larger (about 180 nm) soft PSS shells. The precipitate-to-colloid transformation is facilitated by polystyrolsulfonate (PSS) for Gd(III) complexes with thiacalix[4]arene bearing propyl-malonate groups, while the presence of PSS triggers a dissolution of the precipitated complexes for thiacalix[4]arenes with pentyl-malonate substituents. To a lesser extent the similar tendency disturbs the formation of PSS-stabilized colloids on the basis of butyl-malonate substituted thiacalix[4]arene. The PSS-stabilized colloids exhibit high longitudinal and transverse relaxivities (r1=23.8 and r2=29.4 mM-1s-1 at 0.47 T, respectively), while the recoating of the PSS-stabilized colloids with polyethyleneimine is accompanied by the dissolution of the hard cores. High relaxivity along with low cytotoxicity of PSS-stabilized colloids indicates their applicability as contrast agents in MRI. Keywords-

Malonate

thiacalix[4]arenes;

Longitudinal

relaxivity;

Paramagnetic

colloids;

Hydrophilic colloids; Gd(III) ions; Cytotoxicity

*Corresponding author. Address: A.E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, Kazan 420088, Russia. Tel.: +7 843 273-93-65; fax: +7 843 273-18-72. E-mail address: [email protected] (Alexey Stepanov)

1. Introduction Gd(III) complexes have attracted significant attention in recent decades due to their use in MR imaging [Burtea et al., 2008; Lauffer et al., 1988; Platzek et al., 1997]. Many commercial MRI contrast agents are based on Gd(III) complexes [Burtea et al., 2008; Platzek et al., 1997]. It is worth noting that longitudinal relaxivity and stability of molecular MRI contrast agents can be easily tuned by variation of both hydration number and ligand environment of Gd(III). Nevertheless, the shortcomings of the molecular complexes, such as toxicity and adverse effects, gives rise to the development of nanoparticulate approach, where nanosized species of water insoluble salts [Na et al., 2009; Na et al., 2007; Hifumi et al., 2006; Evanics et al., 2006; Xu et.al., 2016; Zhou et al., 2010; Dong et al., 2014; Rodriguez-Liviani et al., 2013; Ren et al., 2012; Zairov et al., 2016; Zairov et al., 2017], oxides [Zhou et al., 2014; Majeed at al., 2014; Bridot et al., 2007; Wang et al., 2015; Park et al., 2009; Luo et al., 2013; Cho et al., 2014; Chen et al., 2004; Fang et al., 2014; Li et al., 2016; Babic-Stojica et al., 2016; Cho et al., 2014; Gu et al., 2014; Heinz, 2016; Heinz et al., 2017] or complexes [Aime et al., 1996; Morcos et al., 2008;] of Gd(III) are stabilized with polyethyleneoxides or polyelectrolytes. Literature data highlight main factors affecting magnetic relaxation rates in aqueous dispersions of Gd(III) nanoparticles, although no theoretical framework for accurate interpretation of relaxivities in Gd(III)-based colloids is available to date. It is wellknown that nanoparticulate form of Gd(III) complexes improves longitudinal relaxivity due to slowed down translational movement of Gd(III) centres [Perrier et al., 2013; Caravan et al., 2009]. The size of nanoparticles greatly affects the relaxivity. In particular, the size should be about 2 nm for successful hydration of the majority of Gd(III) centres within nanoparticles [Reiter et al., 2006; Pereira et al., 2010; Carne-Sanchez et al. 2013]. Thus, any external factors guiding a transformation of Gd(III) complexes into Gd(III)-based nanoparticles are of great importance in rising of longitudinal relaxivity. From this point of view Gd(III) complexes are more suitable for both phase and size optimization through changing their water solubility than Gd(III)-based nanoparticles. Coordination chemistry of lanthanide complexes with polycarbonic acids indicates many examples, where precipitated electroneutral complex tends to dissolve upon further deprotonation of carboxylic groups, which are not participated in coordination with Gd(III) [Ramamoorthy et al., 1972]. It is worth noting that the development of nanoparticulate Gd(III) contrast agents requires specific solubility of Gd(III) complexes. The solubility should be rather poor to form hard Gd(III)containing dispersions, but not too poor to avoid their uncontrollable growth. Ligands with hydrophilic chelating groups embedded to hydrophobic macrocyclic backbone, such as calix[4]arene, calix[4]resorcinarene or thiacalix[4]arene are well-known versatile platforms for metal complexes [Shamsutdinova et al., 2016; Stepanov et al., 2010; Sliwa et al. 2010]. For example, Schühle et al. has reported Gd-DOTA complexes conjugated with calix[4]arene with high

longitudinal relaxivity of 31.2 mM -1s-1 [Schühle et al., 2010]. The present work introduces synthesis of three thiacalix[4]arene derivatives bearing alkyl-malonate terminal moieties with different length of alkyl linkers (Scheme 1) as ligands for Gd(III) complexes. The facile procedure for stabilisation of water insoluble complexes in aqueous solutions in form of soft-hard colloids is presented herein in correlation with magnetic relaxation and cytotoxicity of the colloids.

2. Materials and methods 2.1. Materials Commercial chemicals Gd(NO3)3·6H2O (99.9%), K2CO3 (99.9%), NaOH (pellets, 98.5%), KOH (pellets, 98.5%), HNO3 (70%), HCl (37%), H2SO4 (96%), dimethylsulfoxide (DMSO) (99.8%), dimethylformamide (DMF) (99%), chloroform (99.9%), NaH (60% suspension in oil), MgSO 4 (99%), glacial acetic acid (99%), xylenol orange (o-cresolsulfonphthaleindi-(methyl-iminodiacetic acid) sodium salt), poly(sodium-p-styrenesulfonate) (Mw ~70000) were obtained from Acros Organics. Malonic ether was purchased from Alfa Aesar (99%). Deuterated chloroform and (99.8%), DMSO (99.8%), polyethylenimine (PEI) (Mw ~25000) were purchased from Sigma Aldrich. DMSO and DMF were distilled twice over P2O5 under reduced pressure prior to use. Chloroform was purified as follows: a certain volume CHCl3 was washed a few times with doubly distilled water and concentrated H2SO4, dried over K2CO3 and finally was distilled at atmospheric pressure. All other chemicals were used as received without further purification. Cell culture WI-38 VA 13 subline 2RA (human embryo lung) was obtained from collection of the institute of cytology of Russian Academy of Sciences. The standard nutrient medium “Igla” with an addition of 10% calf serum and 1% of indispensable amino acids was purchased from Institute of poliomyelitis and viral encephalitis named after M.P. Chumakov (Moscow, Russia). 2.2. Synthesis of PSS and PSS-PEI-stabilized paramagnetic aqueous colloids A series of DMSO solutions containing invariant concentrations of ligands 1, 2, 3 (1.2·10-3 M) and Gd(NO3)3 (1.2·10-3 M) at various concentrations of Et 3N have been prepared. Et 3N was varied to be at 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12 ligand:Et 3N concentrations ratios. After that 1 ml of solution containing Gd(III), calixarene and Et 3N has been added dropwise with the help of syringe pump (adding rate 1 ml/min) to the 5 ml of aqueous solution of PSS (1 g/L, 0.5 M NaCl) under vigorous stirring (1400 rpm). The prepared aqueous-DMSO solution was then sonicated for 30 minutes with use of ultrasound water bath and centrifuged (13500 rpm, 20 min). The separated colloids were then washed with doubly distilled water by four centrifugation/redispersion steps. The supernatant solution

was

decanted,

and

Gd(III)

concentration

in

supernatant

was

analyzed

spectrophotometrically at 579 nm using Xylenol Orange as an indicator at pH 6.1 in acetate buffer according to well-known procedure [Serdyuk et al., 1964]. The extraction efficiency of Gd(III) from

aqueous-DMSO solutions into colloids separated by centrifugation (E, %) was calculated through equation (1) [Mustafina et al., 2006; Favre-Reguillon et al., 2004] with the error less than 3%. E=[(Ci-Ca)/Ci]100,

(1)

where Ci is the initial concentration of Gd(III), Ca is the concentration of Gd(III) in the aqueousDMSO phase after the phase separation.

2.2.1. Determination of Gd(III) concentration in the obtained colloids After being washed with water the real concentration of Gd(III) in the obtained suspensions was determined as follows: 50 µl of 1M HNO3 were added to 6 ml of the studied suspension and shaked vigorously for 1 min to yield virtually transparent solution. Then this solution was analyzed for Gd(III) quantity spectrophotometrically with use of Xylenol Orange as alluded to above.

2.3. Methods 2.3.1. Characterization X-ray powder diffraction (XRPD) measurements were performed on a Bruker D8 Advance diffractometer equipped with Vario attachment and Vantec linear PSD, using Cu radiation (40 kV, 40 mA) monochromated by the curved Johansson monochromator (λ Cu Kα1 1.5406 Å). Roomtemperature data were collected in the reflection mode with a flat-plate sample. Sample was applied in liquid form on the surface of standard zero diffraction silicon plate. After drying the layer applied on top of it a few more layers were placed to increase the total amount of sample. The sample was kept spinning (15 rpm) throughout the data collection. Patterns were recorded in the 2θ range between 3o and 100o, in 0.008o steps, with a step time of 0.1–4.0s. Dynamic light scattering (DLS) measurements were performed by means of the Malvern Mastersize 2000 particle analyzer. A He–Ne laser operating at 633 nm wavelength and emitting vertically polarized light was used as a light source. The measured autocorrelation functions were analyzed by Malvern DTS software and the second -order cumulant expansion methods. The effective hydrodynamic radius (R H) was calculated by the Einstein–Stokes relation from the first cumulant: D = k BT/6πηRH, where D is the diffusion coefficient, kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity. The diffusion coefficient was measured at least three times for each sample. The average error in these experiments is approximately 4%. The samples for DLS study were prepared from deionized water, sonicated for 60 minutes and equilibrated at 25.0±0.1°C before DLS and zeta-potential measurements. The UV-vis measurements were conducted on Lambda 35 spectrophotometer (PerkinElmer, USA) using a 10 mm cuvettes at room temperature.

pHs of the solutions were controlled with Microprocessor pH meter «pH 212» (Hanna Instruments, Germany) The pH-meter was calibrated with standard aqueous buffer solutions (pHs 7.01 and 4.01). 1

H NMR spectra of the synthesized calix[4]arenes were recorded on a DRX-Bruker MSL-400

(400 MHz) (Bruker Avance, Germany) spectrometer at room temperature. Deuterated dimethylsulfoxide was utilized as the solvent for 1H NMR spectra recording. The concentration of the calix[4]arenes was 1 mM. 13

C NMR spectra of the synthesized calix[4]arenes were recorded on a Bruker (400 MHz)

spectrometer at room temperature. Deuterated dimethylsulfoxide was utilized as the solvent for

13

C

NMR spectra recording. The concentration of the calix[4]arenes was 1 mM. The IR spectra were recorded on a Bruker Vector 22 Fourier transform infrared spectrometer (Germany) in KBr pellets at 1 cm−1 resolution with accumulation of 64 scans in the wavenumber range of 400-4000 cm−1 at 25 ◦C. The electrospray ionization (ESI) mass spectra were obtained on a ThermoQuest TRACE MS quadrupole mass spectrometer using a water cooled direct inlet system. The preparation of the samples for TEM analysis was as follows: the obtained aqueous colloids were twentyfold diluted with deionized water and sonicated for 30 min. Then, 15 µl of the dispersion was dispersed on 200 mesh copper grids with continuous formvar support film and H 2O was evaporated in a muffle furnace at 70°C. Next, the TEM images were obtained by use of Hitachi HT7700 (Japan) at an accelerating voltage of 100 kV.

2.3.2. Cell viability evaluation Cell viability of human embryo lung cells (WI-38 VA 13 subline 2RA) towards nanoparticles was determined by means of multifunctional system Cytell Cell Imaging (GE Healthcare Life Sciences, Sweden) using application Cell Viability BioApp. The cells were dispersed on a panel with 96 holes (cell conc. 200000 cells per ml) and cultivated in CO 2incubator at 37°C. Next, the culture medium was sampled in 24 h and 150 ml of the studied dispersions were introduced into holes. The experiments were repeated three times. Intact cells cultivated simultaneously with the studied ones served as a reference. The fraction of the grown-up cells was expressed in % vs. reference cells.

2.3.3. Relaxometric measurements The prepared samples (0.5 ml) with different concentrations of paramagnetic colloids were transferred in 10 mm NMR sample tubes, sonicated using a sonicator water bath and equilibrated at 25.0±0.1 ˚C for 10 minutes by use of circulating water bath before relaxometric measurements. The transverse relaxation times T2 of water molecule protons in studied dispersions were measured

using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence [Meiboom, Gill 1958] using a Bruker minispec 20 NMR analyzer (Bruker, Germany). The longitudinal relaxation times T 1 of water molecule protons were defined with the use of the analyzer’s inversion-recovery pulse sequence with 20 data collected for fitting [Henoumont et al., 2009]. The experiments were performed at 19.65 MHz proton resonance frequency. The relative measurement deviation for transverse and longitudinal relaxation times does not exceed 3%. Transverse R2=1/(T2CGd), mM-1s-1 and longitudinal R1=1/(T1CGd), mM-1s-1 relaxivities were calculated from the measured relaxation times T2 and T1, respectively; CGd signifies gadolinium concentrations. Quantum chemical calculations were done in several steps: optimal conformers selection; initial complex geometry organization; complex optimization procedure. Due to complexity of the molecules the conformers search procedure has been done only for calix[4]arene’s substituents. Conformers of substituents generated with cxcalc plug-in (ChemAxon) [ChemAxon JChem. https://chemaxon.com/download/jchem-suite] using MMF94 force field, followed by the duplicates discard using in house tool and geometry optimization with PM7 [Stewart, 2013] semi-empirical method implemented in the MOPAC 2016 program [Stewart, 2016] with repeated duplicates discard. Unique conformers found by PM7 are combined with calix[4]arene core and optimized by DFT calculations. Then lowest energy conformers of calix[4]arene derivatives are selected for further calculations of Gd(III) complexes as initial guess of geometry. DFT calculations of the most stable conformer of calix[4]arene derivative were made with Priroda 16 program [Laikov, 2005] with build-in PBE functional on L2 basis level [Laikov, 2005].

3. Results and Discussions 3.1. Synthesis of ligands 1, 2, 3 Firstly, ω-bromoalkyl derivatives of n-t-butylthiacalix[4]arenes (containing 3, 4 and 4 methylene groups) have been synthesized via our previously published procedure [Tyuftin et al., 2009] (Scheme 1).

Scheme 1 Synthesis and structural formulae of the ligands 1 (n=3), 2 (n=4), 3 (n=5).

Then, malonic ester derivatives of tetra-p-tert-butyl-tetrathiacaix[4]arenes were obtained through interaction of ω-bromoalkyl derivatives of n-t-butylthiacalix[4]arenes with malonic ether and NaH (60% suspension in oil) with their further hydrolysis to malonic acid derivatives of tbutylthiacalix[4]arenes (Scheme 1). The detailed synthetic procedure is described in the supplementary materials for this article along with their spectral characterization.

3.2. Complex formation of ligands 1, 2, 3 with Gd(III) and colloid stabilization of the precipitated complexes The high enough solubility of ligands 1, 2, 3 is observed in DMSO, which is the reason for choosing the latter as the solvent for complex formation with Gd(III), where triethylamine (TEA) was added to deprotonate malonic acid moieties. The complex formation in DMSO turns the solutions to turbidity, which complicates accurate detection of the complex stoichiometry. The turbidity is followed by precipitation, when the DMSO solution is mixed with water in 1:5 volume ratio. The phase separation by centrifugation of the obtained colloids results in true aqueous-organic supernatant solution and solid phase. The latter can be washed and re-dispersed in water, while the aqueous DMSO supernatants can be analyzed in order to detect the extraction of Gd(III) ions from solution into the solid phase. The aqueous colloids obtained through re-dispersion of the solid phase in water suffer from instability. Colloid stabilization through hydrophilic coating is well known route to obtain core-shell colloids. For these purposes saline solution of polysodium polystyrolsulfonate (PSS) was used instead of pure water. The extraction of Gd(III) is represented in Fig.1 by E% (the equation for E% calculation see in the Exp. Section) versus TEA:ligand concentration ratio at 1:1 (Gd:ligand). The E-values (Fig.1) depend on TEA concentration, although this dependence is affected by the nature of ligand and the presence of PSS. The E% value for ligand 1 tends to increase up to 83.0% at specific TEA:ligand (1:6) and Gd:ligand (1:1) concentration ratios in the solution of PSS, while the E-value reaches only 56.8% in the absence of PSS. Thus, the phase separation in these specific conditions enables to separate the complexes, which will be further designated as Gd x(L)y (L=1, 2 and 3) from aqueous-organic solutions. The E% value does not increase at 1:2 versus 1:1 (Gd:ligand) ratio at the same TEA:ligand ratio, which points to 1:1 stoichiometry of the complex formation for ligand 1. The separation, followed by multiple washing and redispersion results in stable along time aqueous colloids, which can be characterized with different physico-chemical methods.

Figure 1 The extraction efficiency of Gd(III) (0.2 mM) from H2O/DMSO mixture vs. Et3N/calix[4]arene molar ratio (Gd:L=1:1). The colourless square and circle indicate the E-value at 2:1 (Gd:1) and 1:2 (Gd:1) molar ratio, respectively. The UV-Vis spectra of compounds 1, 2, 3 are characterized by the band at 270 nm (Fig.S1 in Supplementary Material). It is rather anticipated that the length of alkylmalonate substituent does not affect the spectral mode. Moreover, both deprotonation of alkylmalonic moieties and their chelating with metal ions result in negligible changes in the UV-Vis spectra of the ligands. Taking into account the spectral properties of PSS (Panel D in Fig.2), UV-Vis spectra of the colloids can reveal the presence of ligand and polyelectrolyte in the synthesized aqueous colloids (Fig.2). For this reason, the aqueous colloids based on ligands 1-3 were obtained in the same concentration conditions (for more details see Exp. Section). The UV-Vis spectra of the colloids (Fig.2) have the similar band at 270 nm, while its intensity depends on both the nature of the ligand and the synthetic conditions (with or without PSS). This tendency indicates that both synthetic conditions and a nature of the ligand affect its concentration in the studied colloids. The PSS-induced two-fold enhancement of absorption intensity for ligand 1 indicates that the presence of PSS facilitates the complex formation in the heterogeneous conditions, while the opposite tendency is observed for ligands 2 and 3 (Fig.2). The difference between spectral patterns of [Gdx(1)y] and PSS-[Gdx(1)y] is the increased absorption at 260 nm for the latter. This can be explained by the contribution of PSS to the electronic absorption (Fig.2D), while no detectable difference between the spectral patterns of Gdx(L)y and PSS-Gdx(L)y is revealed for ligands 2 and 3. Thus, PSS both enhances the phase separation of Gdx(1)y complex and stabilizes the obtained colloids. The opposite tendency is observed for complexes with ligands 2 and 3, which can be explained by greater losses of complexes Gdx(L)y (L=2, 3) after washing procedures. Moreover, the colloids based on ligand 3 look like true solutions (Fig.3). DLS measurements of the synthesized colloids were performed for more detailed characterization of their colloid properties.

Figure 2 UV-Vis spectra of the obtained dispersions with and without PSS: panel A (2PSS-[Gdx(1)y], 1-[Gdx(1)y]); panel B (1-[Gdx(2)y], 2-PSS-[Gdx(2)y]); panel C (1-[Gdx(3)y], 2-PSS[Gdx(3)y]); panel D displays the UV-Vis spectra of PSS aqueous solution (7.15·10-6 М). Table 1 presents the averaged sizes (d), electrokinetic potential (ζ) values and polydispersity indices (PDI) of the aqueous colloids. These data (Table 1) reveal significant effects of ligand nature and synthetic conditions on colloid properties. The DLS data for the colloids obtained by precipitation of [Gdx(2)y] and [Gdx(3)y] in aqueous DMSO solutions agree well with their colloid instability, while better monodispersity and negative electrokinetic potential values is revealed for [Gdx(1)y] colloids (Table 1). The negative surface charge of [Gdx(1)y]-based colloids can be explained by the negative charge of [Gdx(1)y] complexes, where the charge originates from the noncoordinated anionic alkyl-malonate groups (Scheme 1). It is worth noting that the colloids obtained in the presence of PSS are characterized by increased negative ζ-values (Table 1). Thus, the effect of PSS on the formation of [Gdx(1)y]-based cores and their colloid stabilization is worth discussing. It is well known that stabilization of any hard colloids with polyelectrolytes is driven by their adsorption onto a surface of hard template, which in turn is mainly contributed by electrostatic attraction. Nevertheless, the results pointing to stabilization of [Gdx(1)y]-based cores by PSS are worth discussing. In particular, both negative ζ-values and colloid stability of PSS-[Gdx(1)y] colloids are greater versus the same colloids obtained without PSS (Table 1). This tendency along with the effect of PSS on the concentration of [Gd x(1)y]-based colloids revealed from the extraction and UV-Vis data (Figs.1, 2A) disclose the participation of PSS in colloid stabilization of [Gd x(1)y]-

based cores. Thus, the “salting out” effect of PSS and sodium chloride is the reason for the enhanced phase separation of [Gdx(1)y]-colloids. The PSS-induced colloid stabilization of negatively charged [Gdx(1)y]-based cores (Table 1) is not common phenomenon and should be greatly facilitated by counter-ions binding.

Figure 3 The appearance of PSS-[Gdx(1)y], PSS-[Gdx(3)y] and PEI-PSS-[Gdx(1)y] dispersions in water (1, 2, 3, respectively).

Table 1. Dynamic light scattering data and losses of Gd(III) after washing for the synthesized colloids at 25°Ca. Colloid

db, nm

Losses after washing, %

PDI

ζ, mV

pH

[Gdx(1)y] [Gdx(2)y]

182±13.6 1307 (76.1%) 225 (23.9%) 551.6 (62.4%) 150.2 (37.6%) 170±5.5

-

0.11 0.56

-22.6±6.01 -14.4±5.24

6.03 3.09

-

0.928

c

3.70

-

0.12

-32.1±3.2

6.55

205.9±5.3

51.2

0.14

- 28.4±5.4

6.63

182.5±5.30

-

0.12

-24.80±4.35

6.0

244.3±16

30.9

0.14

+42.8±5.5

6.58

329.3±21.5

0.18

+37.9±5.8

6.48

259.7±21.50

0.19

+25.60±6.20

5.92

[Gdx(3)y]

PSS[Gdx(1)y] PSS[Gdx(2)y] PSS[Gdx(3)y] PEI-PSS[Gdx(1)y] PEI-PSS[Gdx(2)y] PEI-PSS[Gdx(3)y] a

No buffer and electrolytes were added to the suspensions for size and ζ-potential measurements By intensity c Can not be precisely determined due to too high polydispersity b

As it was mentioned above effect of PSS on [Gdx(2)y]- and [Gdx(3)y]-based colloids is quite different from that of [Gdx(1)y]-colloids. In particular, the DLS measurements reveal negatively charged colloids with the averaged size and electrokinetic potential values similar to those of PSSstabilized [Gdx(1)y]-colloids (Table 1), while the soft PSS-colloids contain smaller concentrations of [Gdx(2)y]- and [Gdx(3)y]-based cores. The insignificant concentration of [Gdx(3)y]-based cores is the reason for the specific appearance of PSS-[Gdx(3)y] colloids (Fig.3). Moreover, the analysis of Gd(III) concentration in the colloids after multiple washing procedures confirms greater losses of [Gdx(2)y] versus [Gdx(1)y]. Transmission electronic microscopy (TEM) images were obtained in order to visualize nanoparticulate form of Gd(III) complexes. The TEM images of PSS-[Gdx(1)y], PSS-[Gdx(2)y] and PSS-[Gdx(3)y] are presented in Fig.4.

Figure 4 TEM images of PSS-[Gdx(1)y] (Panel A), PSS-[Gdx(2)y] (Panel B), PSS-[Gdx(3)y] (Panel C) and PSS-PEI-[Gdx(1)y] (Panel D).

Comparison of the TEM images indicates the obvious nanoparticulate form of PSS[Gdx(1)y] and PSS-[Gdx(2)y] colloids, which turns to dark spots for PSS-[Gdx(3)y]. So, no hard cores are apparent from the TEM images of PSS-[Gdx(3)y] colloids, which distinguishes them from PSS-[Gdx(1)y] and PSS-[Gdx(2)y]. The image in Fig.4C reveals no

hard cores, which in turn explains why PSS-[Gdx(3)y] colloids look like true solutions (Fig. 3). The hard Gd(III)-based complex cores may be of crystalline or amorphous nature. XRD measurements of PSS-[Gdx(1)y)] were performed to reveal their nature. The data (Fig.5) point to amorphous nature of the hard cores of PSS-[Gdx(1)y] colloids, which is confirmed by the similar data for the dried sample on the silicon plate surface. In particular, the liquid type diffraction pattern with strong diffusive peaks at the background of decreasing scattering curve is observed for PSS-[Gdx(1)y)] sample (Fig.5). The intensive small-angle scattering of the sample points to its microheterogeneous structure, which is in good confirmation with the corresponding TEM images of the dried colloids (Fig.4). Moreover, the insignificant number of interference peaks indicates the lack of separate crystalline phases of the individual components of the sample.

Figure 5. The experimental XRPD pattern of PSS-[Gdx(1)y] colloid.

3.3. Magnetic relaxation properties of the colloids The small concentration of [Gdx(3)y] in PSS-based colloids (Fig.2) makes them poor basis for any applications.

Thus, longitudinal and transverse relaxation rates (1/T1 and 1/T2,

respectively) were measured for PSS-[Gdx(2)y] and PSS-[Gdx(1)y] at various Gd(III) concentrations. Fig.6 presents the linear plots of 1/T 1 and 1/T2 versus Gd(III) concentration at 0.47 T (25°C).

Figure 6. 1/T1 (panels A, C) and 1/T 2 (panels B, D) vs. Gd(III) concentration for PSS[Gdx(1)y] (panels A, B) and PSS-[Gdx(2)y] (panels C, D).

The longitudinal and transverse relaxivity values (r1 and r2, respectively) of the colloids were calculated by the change of the relaxation per unit concentration of Gd(III) and represented in Fig.6. The relaxivity values (r1 and r2) are rather similar for PSS-[Gdx(1)y] and PSS-[Gdx(2)y] colloids, although the transformation of ligand 1 to the colloids is better than for ligand 2 (Fig.2, Table 1). Thus, longitudinal relaxation rates of PSS-[Gdx(2)y] colloids are restricted by their concentration, which does not exceed 0.06 mM of Gd(III) (see panels C, D in Fig.6). The greater concentration of PSS-[Gdx(1)y] colloids is the reason for the higher 1/T 1 values (panels A, B in Fig.6). An adsorption of oppositely charged polyelectrolytes onto charged colloids is well-known route of their recharging. Polyethyleneimine (PEI) is a widely-used polyelectrolyte for recoating of negatively charged colloids. The recoating carried out through the common procedure results in changing from minus to plus of the measured electrokinetic potential values indicating surface recharging of the PEI-PSS-coated colloids (Table 1). Moreover, these colloids tend to change their appearance as illustrated in Fig.3. The comparison of TEM images also reveals the difference between the PSS- and PEI-PSS-[Gdx(1)y] colloids (Figs. 4A, D). In particular, insignificant amounts of hard cores are apparent for PEI-PSS-[Gdx(1)y] colloids from the TEM images, which points to their dissolution under the PEI deposition. The dissolution results from increased basicity arisen from amino groups of PEI, while pH of the bulk solution remains virtually unchanged after the

deposition of PEI. The dissolution of [Gd x(1)y]-based hard cores under the PEI deposition onto the PSS-[Gdx(1)y] colloids is the reason for the decreased [Gdx(1)y] concentration, which in turn makes the PEI-PSS-[Gdx(1)y] colloids less important from the viewpoint of magnetic relaxation rates. The similar tendency is observed for PEI-PSS-[Gdx(2)y] colloids. Thus, the results point to compound 1 with propyl-malonate substituents as the optimal ligand for the nanoparticulate form of Gd(III) complexes. The reasons for the impact of alkyl-malonate length in the coordination mode of the ligands with Gd(III) ions are worth discussing. 3.4. Structure impact in coordination of Gd(III) ions with malonate groups of ligands 1, 2 and 3 Literature data indicate that stability of lanthanide complexes with malonates results from the specific coordination mode, where lanthanide ions are coordinated via two oxygens of carboxylates linked by methylene group [Hernandez-Molina et al., 2003; Canadillas-Delgado et al., 2006; Fang et al., 2008; Hernandez-Molina et al., 2002]. Embedding of four alkyl-malonate moieties onto thiacalix[4]arene platform adopting 1,3-alternate conformation provides an opportunity to bind lanthanides via two malonate groups. Taking into account the amorphous nature of the complexes quantum chemical modelling was applied to reveal the difference in coordination modes of the ligands. The results indicate that the most efficient coordination mode of Gd(III) requires planar arrangement of the malonate groups as shown in Scheme 2, while the length of alkyl linkers of alkylmalonate groups should be of some impact in such arrangement. Indeed, the alkyl linker lengths are enough for bis-chelate coordination of Gd(III) via two malonate groups of the ligands. Nevertheless, the coordination mode is more symmetrical with small deviation between the Gd-O bond lengths for ligands 3 and 2, while the greater deviation between the Gd-O bond lengths is observed for ligand 1. Scheme 2 illustrates the difference in the coordination modes for ligands 1 and 2. In particular, the Gd-O bond distances in [Gd(L)] are 2.14 and 2.20 Å when L=1, while they are 2.16 and 2.18 Å when L=2. The revealed difference is not the reason for the above mentioned favourable transformation of ligand 1 into the PSS-stabilized Gd(III)-based colloids versus ligands 2 and 3. Nevertheless, literature data [Fang et al., 2008; Hernandez-Molina et al., 2002] point to the possibility of bridge-like coordination mode of malonate anions with two lanthanide ions. The quantum chemical calculations indicate that the bis-chelate coordination mode (Scheme 2) is favourable versus the bridge-like coordination, although the difference in Gibbs free energy values (1.7 kcal/mol) is rather small. Thus, the observed difference in colloid properties of the nanosized precipitated complex species for complex [Gdx(1)y] versus [Gdx(2)y] and [Gdx(3)y] points to a predominance of kinetic versus thermodynamic factors in the specific heterogeneous conditions.

Scheme 2 (A) The most efficient coordination mode of Gd(III) with two malonate units, (B) optimized structure of Gdx(1)y complex, (C) optimized structure of Gdx(2)y complex, (D) bridgelike coordination mode of Gd(III) with thiacalix[4]arene 1.

3.5. Effect of the colloids on cell viability The results indicate the that PSS-[Gd(1)] colloids are promising for their use as contrast agents in MRI. Thus, their effect on cell viability should be determined. The cell viability of embryo lung cells was measured in the presence of the synthesized colloids at Gd(III) concentration 0.09 M ensuring high 1/T1 value. The cell viability measurements presented in Fig.7 indicate negligible cytotoxicity of PSS-[Gd(1)] and PSS-[Gd(2)]. The observed tendency indicates that Gd(III) complexes with the synthesized ligands in the nanoparticulate form are stable enough to minimize the toxic effect of Gd(III) ions.

Figure 7 Cell viability assay of human embryo lung cells (WI-38 VA 13 subline 2RA) without (Ref) and after addition of 8.55·10-5 M Gd(III): 1- PSS-[Gdx(1)y], 2- PSS-[Gdx(2)y]. Error bars signify standard deviations of the cell viability test determined from 3 measurements.

4 Conclusion The work introduces one-pot synthesis of Gd(III)-based aqueous colloids from water insoluble propyl-malonate-substituted thiacalix[4]arene adopting 1,3-alternate conformation and Gd(III) ions facilitated by polystyrolsulfonate (PSS). The synthesized colloids are of significant practical importance due to their magnetic relaxation properties with r 1=23.8 and r2=29.4 mM-1 s-1 at 0.47 T, exceeding those for commercial Gd(III)-based contrast agents. The experimental data indicate great effect of PSS on both concentration and stability of the colloids based on propylmalonate-substituted thiacalix[4]arene, while the presence of PSS enhances the dissolution of the Gd(III) complexes with pentyl-malonate-substituted thiacalix[4]arene. The significant structure effect on the colloid behaviour can’t be explained by the difference in the coordination modes of the corresponding Gd(III) complexes. The results point to significant influence of kinetic along with thermodynamic factors on the complex formation of alkyl-malonate-substituted thiacalix[4]arenes with Gd(III) in the specific microheterogeneous conditions.

Acknowledgement We thank RSF (grant no 17-13-01013) for financial support. TEM images were obtained in the laboratory “Transmission electron microscopy” of Kazan National Research Technological University.

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