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Received date: 03/10/2022 Published date: 24/10/2022 Ebook

Gold (III)-Gadolinium (II) Complex – Hybrid Nanoparticles: From Synthesis to Nanomedicine and Imaging Applications

Fatima Aouidat¹, Memona Khan¹, Xiaowu Li^3-4, Frederik Tielens⁵, Jolanda Spadavecchia^1-4

¹CNRS, UMR 7244, CSPBAT, Laboratoire de Chimie, Structures et Propriétés de Biomatériaux et d’Agents Thérapeutiques Université Paris 13, Sorbonne Paris Cité, Bobigny, France.
³Department of hepato-biliary surgery, Shenzhen University General Hospital,Shenzhen, China
⁴Department of hepato-biliary surgery, Shenzhen University Clinical Medical Academy, Shenzhen, China
⁵General Chemistry (ALGC), Vrije Universiteit Brussel (Free University BrusselsVUB), Pleinlaan 2, 1050 Brussel, Belgium.

*Corresponding author

*Jolanda Spadavecchia, 1CNRS, UMR 7244, CSPBAT, Laboratoire de Chimie, Structures et Propriétés de Biomatériaux et d’Agents Thérapeutiques Université Paris 13, Sorbonne Paris Cité, Bobigny, France

DOI: 10.55920/JCRMHS.2022.02.001069

ABSTRACT

The development of biopolymers as building blocks, for the synthesis of Gd (II) nanoparticles, as therapeutic, could play a key role in nanomedicine. Biopolymers are not only designed to complex monovalent biomolecules, but also for building into multivalent active targeting materials such as diagnostic and/or therapeutic hybrid nanoparticles.
In this paper we report, for the first time, a novel synthesis of Gd (III)-biopolymer –Au (III)-complex, acting as key ingredient of hybrid gold nanoparticles: Gd(II)AuNPs. Physical chemical hybrid nanoparticles evaluation by spectroscopic analytical techniques (Raman spectroscopy, UV-Visible and TEM) was carried out, and stability under pH and physiological conditions were achieved. The theoretical characterization by DFT (Density Functional Theory) analysis, to investigate the interaction between the Au and the Gd precursors, during the first nucleation step, under specific conditions was performed. Magnetic features with relaxivity measurements at 7T were also performed as well as cytotoxicities studies on hepatocytes cell lines. In vivo biodistribution studies in mice to characterize the potential applications for biology as MRI contrast agents were then achieved. These results will strengthen the role of gadolinium as complex to gold in order to tune Gd (II)AuNPs, as real diagnostic agent in the field of nanomedicine.

Introduction

In recent decades, multifunctional nanomaterials have been intensively investigated in the area of nanomedicine[1],diagnosis[2], and imaging[3]. The consolidation of different functional materials into a nanocomposites, generates new opportunities in order to improve a variety of emerging applications of hybrid nanomaterials[4] [5].The application area in which nanoparticles have obtained remarkable profit is in biomedical field and in particular in diagnostic imaging[6]. Many kinds of nanoparticles have been inquired for several imaging applications, including biomolecules[7], metals[8],metal oxides[9],and semiconducting nanomaterials[10].
The nanoparticles selected, is dependent upon the required imaging modality; Gadolinium (Gd), own a wide magnetic moment and unpaired electrons in the external shell, carry out as a clinical positive contrast agent for MRI as chelates[11]. Although, clinical contrast agents for CT (cancer treatment) are principally based on tri-iodobenzene, which can effectively absorb X-rays. Unfortunately, the short circulation time of the Gd chelates and iodinated compounds, due to the nature of the small molecules, can avoid the relative imaging technique from collecting the prerequisite information. Moreover, it is laborious to promote the grafting of small molecules for active targeting [11b].
Gold nanoparticles (AuNPs) have been recently a common choice as contrast and therapeutic agents due to their superior optical properties, good biocompatibility and bioconjugation with biomarkers to create nanosized contrast agents with molecular specificity[12] [13] [14].
Nanoprobes based on Gd (III) and AuNPs have recently exhibited significantly increased relaxivity values in Magnetic Resonance Imaging (MRI) compared to those of chelated Gd (III) complexes[15].
The payload of gadolinium, particularly under its Gd (II) form in NPs structures, has a notable effect in plenty of research fields from plasmonics to nanomedicine applications[16]. Nevertheless, strategies to load NPs as contrast agents used in MRI, in harsh or poor loading conditions, result in NP surface modifications that alter targeting in vivo[17].To provide a safe application in biological media, and to understand the importance of cationic metals in biological systems (charge balancing, stabilizing structures, catalyzing reactions, …), gadolinium ions must be chelated to prevent metal release in the body. Another option could be the incorporation of the cations in “safe” nanostructures, and keeping in mind that also the final design of the complex-hybrid NP might combine both options after being assembled together. In the recent literature a fast synthesis method to develop polymer-modified AuNPs using biopolymers as stabilizers in order to obtain high stability and efficiency under biological conditions[18]have been described. Other authors have synthetized and described, hybrid nanoparticles based on sugar stabilizers for different applications in nanomedicine[19].
In the present study, three bimetallic nano formulations named NP1, NP2, and NP3, including Au-Gd complex wrapped into a biopolymer structure were designed and synthetized. In the first step, gadolinium (III) was introduced as “free” cation, complexed with gold (III) ion in aqueous solution. Then, the stacking process with mixed biopolymer chains, favoring the initial reduction of the bimetallic complex and the migration of them into polymeric chains and final reduction to hybrid – bimetallic nanoparticles (Au0-Gd (II)) was performed followed by the characterization of the NPs structure. Furthermore, these nanovectors including paramagnetic Gd (III) ions provided potential interesting magnetic features for MRI imaging. The core of the study is the development of innovative molecular imaging methods based on dynamic recording of the uptake, remanence and clearance of novel imaging probes. Indeed, the quantitative pharmacokinetics of theranostic nanovectors allowed to study the in vivo behavior of the probes necessary to determine the stealthiness and targeted property of the probes, and supply the final biomolecular mapping for the determination of the diagnosis and the efficiency of a therapy. It is clear that these novel perspectives will have a decisive impact on the evolution of high hybrid nanomaterials used in nanomedicine.

Experimental Section

Materials

Tetrachloroauric acid (HAuCl₄^*3H₂O), Gadolinium chloride hexahydrate (GdCl₃^*6H₂O),Sodium borohydride (NaBH₄), Phosphate Buffer Solution (PBS, pH4.0, 7.2-9.0), Sodium Chloride (Na Cl),culture media cell (DMEM) ,Dicarboxylic PolyEthylene Glycol (PEG)-600 (PEG), Collagen Type I from calf skin (COL), Chitosan (Deacetylated chitin, Poly(D-glucosamine) (CHIT),were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). All chemicals were used as such without further purification. Milli Q water was used throughout the experiments. All solvents without any further purification were used. Experiments were carried out at room temperature if not specified otherwise.

Physical chemical characterization

All the measurements were performed in triplicate in order to validate the reproducibility of the synthetic and analytical procedures.
UV-Vis absorption spectroscopy: Absorption spectroscopy measurements were carried out by means of a double-beam Varian Cary 500 UV-Vis spectrophotometer (Agilent, France). Absorption spectra were recorded in the 350-900 nm spectral range in water at AuNPs concentration equal to 10-4 M.
Transmission Electron microscopy (TEM): TEM images were acquired with a JEOL JEM 1011 microscope (JEOL, USA) at an accelerating voltage of 100 kV. TEM specimens were prepared after separating the biopolymer from the metal particles by centrifugation.
Typically, 1 ml of Gd (II)AuNPs was centrifuged for 10 min at a speed of 6,000 rpm. The upper part of the colorless solution was removed and the solid portion was re-dispersed in 1 ml of water. 2 μL of this re-dispersed particle suspension was placed on a carbon coated copper grid (manufactured by Smethurst High-Light Ltd and marketed exclusively by Agar Scientific) and dried at room temperature.
Scanning Electron Microscopy-Energy dispersive x-Ray analysis (SEM-EDX):Scanning electron microscopy (SEM) investigation was performed on an environmental SEM (ESEM, Quanta 200 FEG, FEI Company Hillsboro, OR) equipped with an (EDX) spectrometer (Genesis 2000, XMS System 60 with a Sapphire Si/Li Detector from EDAX Inc., Mahwah, NJ).
Raman Spectroscopy: The Raman experiments have been performed on an Xplora spectrometer (Horiba Scientifics-France). The Raman spectra have been recorded using an excitation wavelength of 785 nm (diode laser) at room temperature. For measurements in solution, a macro-objective with a focal length of 40 mm (NA = 0.18) was used in backscattering configuration. The achieved spectral resolution is close to 2 cm-1.
Dynamic light scattering (DLS): The size measurements were performed using a Zetasizer NANOZS (Malvern Instruments, Malvern, UK) equipped with a He-Ne laser (633 nm, fixed scattering angle of 173°) at room temperature.
Zeta potential measurements: The zeta potential of Gd (II)AuNPs dispersed in water was measured using the electrophoretic mode of a Zetasizer NANOZS (Malvern Instruments Ltd, UK).

Release kinetics under acidic conditions

Release of the Gd(II)AuNPs were carried out in buffers of pH 4.0 under specific conditions^[²⁰^] . In a glass vial, 1mL of Gd (II)AuNPs were dispersed in 50 µL of phosphate buffer of desired pH and kept on docile stirring at 37°C of temperature. After regular pre-determined time intervals, 1 mL of dispersion was withdrawn and centrifuged to separate the dye released from the particles. Amount of dye released from the NPs was calculated by measuring its absorbance at 549 nm in supernatant. The solution was transferred back to the glass vial after noting the absorbance and raman signal.

Cytotoxicity assay

The cytotoxicity was determined on TIB-75 hepatocytes using the Alamar blue test. The cells were cultured at 37°C in DMEM + Glutamax containing 10 % fetal bovine serum (FBS, Gibco Life Technologies), 100 µM of streptomycin, and 100 units/mL of penicillin. They were maintained in a 5 % CO₂-humified atmosphere and passaged twice a week by removing the adherent cells with 0.05 % Trypsin- EDTA. Fluorescence measurements at 570 nm were performed and IC50 was assessed from the normalized values using Graphpad Prism software.

In vitro relaxivities studies

Samples of MNPs and UMLs were diluted in injection buffer (0.108 M NaCl, 0.02 M sodium citrate and 0.01 M HEPES) at various concentrations of Gd (0, 0.1, 0.5, 1.0, 3 mM).

In vitro relaxivity experiments were carried out by recording T₁ and T₂ maps with a 7T MR imaging vertical spectrometer fitted with an ultra-shielded refrigerated magnet (300WB, Bruker, Avance II, Wissembourg, France), and equipped with a nominative 200 mT/m actively shielded gradient. The software Paravision 5.1 allowed the acquisitions with the following parameters: for T₁ map: RARE images ; TE = 13ms ; TR = 15 s, 8 s, 3 s, 1.2 s, 0.8 s, 0.594 s, 0.3 s, 0.144 s, 0.05 s, 0.033s, RARE factor 2; for T₂ map: multi-echo MSME images: hermitian pulse, TR/TE = 15 s/11 ms, 32 echos. Fields of view of 3 x 3cm², a matrix size of 128 x 64 and a slice with a thickness of 1.5mm were used for T₁ and T₂ maps. Relaxation times T₁, T₂ and T₂^* of each sample were calculated by fitting (for ).

Molar relaxivities r₁, and r₂ in mM^-1.s^-1 were obtained using the following equation: with y: 1 or 2.

Biodistribution studies were conducted in vivo by MRI^[²¹^]to assess the uptake and clearance from kidney, muscle, spleen, liver more easily observable by MRI.

In vivo biodistribution studies by MRI

All animal work was performed in accordance with the institutional animal protocol guidelines in place at the University Paris Descartes, saisine CEEA34.JS.142.1 and approved by the Institute’s Animal Research Committee.

Wild-type female 8 weeks BALB/c mice were anaesthetized by Isoflurane (1.5% air/O₂ 0.5/0.25 L.min^-1) inhalation and placed in a dedicated contention cradle. 100 µL of Gadolinium Au nanoparticles in saline 0.9% with an optimized concentration of 10 mM of Gd were intravenously injected via the tail vein while the mouse was in the scanner. One agent (NP3) was imaged representative of the series of agent with n=6 mice for reproducibility.

Images were acquired at 7 Tesla (300 MHz) micro imaging spectrometer (Bruker, Karlsruhe Germany) previously described.

The scanning protocol was developed using Paravision 5.1 software.

After positioning anatomic slice recorded in axial directions to locate the different organ of inter DCE Dynamic contrast Enhanced sequence was recorded using Intragate Flash multislices for motion free artifacts T1 weighted sequence. The final images have a spatial resolution of 117 µm x 117 µm in plane. The total scan time was in the order of 3 min 14 s per images. The dynamic follow-up is measured during a scan time of 40 min, then at 3h, 6h, 24h and 48 hours post injection.

To study the biodistribution of the nanoparticules, several regions of interest (ROI) were monitored, the liver, muscle, spleen and the kidney. The corresponding MRI intensity related to the amount of the nanoparticles contrast agent was plotted against time to visualize the uptake and clearance of scaffolds in the organs. Comparison with commercial DOTA-Gd (Guebert, France) as a reference was also performed at the corresponding concentration of Gd (10 mM). Uptake, remanence and clearance times were visually assessed from the dynamic curves.

Stability of Gd(II)-AuNPs

The stability of nanoparticles was detected by UV VIS. All nanoparticles were dissolved in culture media (DMEM +0.3% FBS) solution during 72 h (Figure S2 Supporting Informations).

Synthesis Procedures

Synthesis of NP1 (Gd(II)AuPEG NPs)

10 ml of Gd solution (0.86mM) was mixed with 20 ml of 0.0001M aqueous HAuCl₄ solution for 10 min. After this time, 250 µl of Polyethylene glycol 600 Diacid (PEG) was added under stirring for 5 min. After 10 min, 1.2 ml of NaBH₄ (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2h. The resulting red-rose solution was centrifuged and purified at the same conditions.

Synthesis of NP2 (Gd (II)AuPEG-COLNPs)

NP2 were prepared under same protocol of NP1.

10 ml of Gd solution was mixed with 20 ml of 0.0001M aqueous HAuCl₄ solution for 10 min. After this time, 250 µl of Polyethylene glycol 600 Diacid (PEG) was added under stirring for 5 min; then 500µl of Collagen (COL) and 1mlwas added under stirring at room temperature. After 10 min, 1.2 ml of NaBH₄ (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2h. The resulting violet solution was centrifuged and purified at the same conditions.

Synthesis of NP3 (Gd (II) AuPEG-COL-CHIT NPs)

NP3 were prepared under same protocol of NP2.

10 ml of Gd solution was mixed with 20 ml of 0.0001M aqueous HAuCl₄ solution for 10 min. After this time, 250 µl of Polyethylene glycol 600 Diacid (PEG) was added under stirring for 5 min; Then 500µl of Collagen and 1ml of CHIT solutions were added under stirring at room temperature. After 10 min, 3mL (and/or 1.2 ml ) of NaBH₄ (0.01M) was added drop-wise followed by rapid stirring and kept without agitation for 2h. The resulting red-wine solution was centrifuged and purified at the same conditions.

Computational Details– DFT study

The interaction energies were calculated using the ab initio plane-wave pseudopotential approach as implemented in the VASP code^[²²^]. The Perdew-Burke-Ernzerhof (PBE) functional^[²³^] was chosen to perform the periodic DFT calculations with accuracy on the overall convergence tested elsewhere^[²⁴^]. The valence electrons were treated explicitly and their interactions with the ionic cores are described by the Projector Augmented-Wave method (PAW),²⁸^,^37-38 which allows to use a low energy cut off equal to 400 eV for the plane-wave basis. The integration over the Brillouin zone was performed on the G-point, in all calculations.

In the geometry optimizations at 0 K, the positions of all atoms in the supercell are relaxed in the potential energy determined by the full quantum mechanical electronic structure until the total energy differences between the loops is less than 10^-4 eV.

In order to account for the dispersion interaction in system, DFT-D3^[²⁵^] was used, as implemented in VASP, which consists in adding a semi-empirical dispersion potential to the conventional Kohn-Sham DFT energy.

In order to compare the results with the experiment, water was implicitly included in the calculations, by means of the PCM correction as implemented in VASP.^[²⁶^]

Results and discussion

Formation mechanism of Gd- Au complex

Several molecular interactions between organic compounds and metal chlorides were accomplished for medical and bio-physical applications^[²⁷^]^[²⁸^]. The key of this study, is to apply a gadolinium-gold chloride complex as building blocks, of hybrid nanoparticles under specific conditions of reaction. For this aim, in the first step, GdCl₃^*6H₂O and HAuCl₄^*3H₂O were mixed in ultrapure water solution, at room temperature (scheme 1- panel s1).

The formation of complex Gd (III)-Au (III) (Figure 1) showed the UV-Vis fingerprint of each solution (GdCl₃^*6H₂O , HAuCl₄^*3H₂O) and the mixed of them (Gd-Au). The UV-Vis Spectra showed a typical spectra of HAuCl₄ (black line) with 2 prominent peaks at 256 nm and 290 nm (black line in the Figure 1A). When GdCl₃ was added to HAuCl₄ solution, a chemical-physical modification appeared, in the UV Vis Spectra (blue line in Figure 1A). The optical modification in the absorption spectra, was characterized by a intensity decrease and blue shift of the peak from 303 nm to 280 nm are due to the electronic transition associated to GdAuCl₂^- ions upon complexation. The evolution of Gd-Au complex was still monitored under acidic and basic conditions by localized surface plasmon (LSP).Figure S1 in Supporting Information, evidences two absorption peaks at 242 nm and 308 nm under acidic conditions (PBS; pH 4) compared to a singular peak at 235 nm under basic conditions (PBS;pH 9). This spectroscopic behavior, is principally originates from the simultaneous participation of protonic and/or anionic groups into electronic transition bands conferring the appearance and/or disappearance of plasmon peaks in the UV –VIS spectra confirming the hybrid complex arrangement (Gd- Au)^[^28a^].

Figure 1 : P(A) UV-Vis absorption of HAuCl4*3H2O (black line), GdCl3*6H2O (red line); Au-Gd complex (blue line) in the range 200-900 nm (B) Raman spectra of Gd-Au complex (blue line) in the range 200-3000 cm-1, compared to HAuCl4*6H2O (black line), GdCl3*6H2O (red line) as controls. Experimental conditions: λexc = 785 nm; laser power 20 mW; accumulation time 180s./em>

Raman Spectroscopy (Figure 1B) also display a peak at 477 cm^-1 due to Gd-Au-Cl and the disappearance of the peaks at 655 cm^-1 and 550 cm^-1 due to Gd-OH and Au-OH stretching. This spectroscopic behavior was associated to π-π* electronic transitions due to interactions between GdCl₃ and AuCl₂^-ions^[²⁹^] and gives a better evidence of the complex formation. confirming the electronic delocalization throught the bimetallic complex ^[^28a^].

Formation mechanism of hybrid Gd(II)Au NPs and role of polymers as stabilizing agent

Many authors^[³⁰^] have realized the association between Au nanoparticles and Gd chelates in order to realize multimodal MRI/CT contrast agents^[³¹^]. For this purpose, other scientific investigations , based onto Gd-chelate-embedded gold nanorods ^[³²^],Gd-enriched DNA AuNPs conjugates^[³³^], and grafting Gd chelates on gold nanostructures as multimodal MRI/CT contrast agents, have been carried out. Thanks to the small sizes of Gd chelates, a low magnetic center Gd (III) payload for the particle, prevents further bioconjugation and active targeting of AuNPs in biomedical applications^[^2b^]. In contrast with previous works, a novel Au(III)-Gd (III)complex, entrapped in polymeric chains, used as building blocks to form hybrid bimetallic nanoparticles (Gd-Au) in which gadolinium was reduced to Gd (II).

The synthesis of Gd(II)AuNPs (NP1-NP2-NP3) was carried out by reducing tetraclororoauric acid (HAuCl₄^*3H₂O) in the presence of GdCl₃^*6H₂O and different bio-polymers (PEG-diacid COL, CHIT) using sodium borohydride (NaBH₄) as a reducing agent.

When biopolymers (PEG, COL, CHIT) were stacking onto Gd (III)-Au(III) complex , (Figure 2A), an increase and shift of the peak from 303 nm to 308 nm due to PEG and COL (blue and red lines in Figure 2 A1) was observed. A consequently decrease of the same peak at 303 nm and broadening of the peak at 246 nm in the presence of CHIT (green line in Figure 2A1) was remarkable .We suppose that this phenomenon is due to electronic delocalization through polymer chains-metal complex and HOMO-LUMO transition, confirming that polymers ( PEG, COL, CHIT) were effectively involved into the nucleation process and creates an interaction with Gd-Au. Indeed, during the polymer-staking process, an initial reduction of hybrid complex biopolymer was observed with appearance of a broadened plasmon in the range 580-590 nm depending on the nature of the polymer (Figure 2 A2).

Gd-Au-polymer modification onto the surface of AuNPs solutions was prepared to this end (scheme 1). Particles formation and growth were controlled by the amphiphilic character of the polymers and includes mains steps: (1) Complexation between GdCl₃and AuCl₄^-to form gold-gadolinium clusters (Gd (III)-Au (III)) (scheme 1- panel s1). (2) adsorption of COOH-terminated polymer molecules onto Gd (III)-Au (III) complex and initial reduction to Gd (III)-Au (II) (3) interaction of collagen (COL) and Chitosan (CHIT) molecules respectively by electrostatic forces (scheme 1- panel s2).(4) complete reduction of metal ions (Gd (II)) -Au⁰ in that vicinity growth of gadolinium-gold particles, and colloidal stabilization (scheme 1- panel s3). In the latter case, Au-Gd complex molecules are expected to be involved in the nucleation process and may, thus, influence the final shape and size of nanoparticles.

Scheme 1: Schematic of proposed mechanism of GdCl₃-AuCl₄^-reduction by complexation and particle formation (Gd(II) AuNPs) in the presence of polymer and sugar as surfactants (Please note that drawings are not in scale and are not intended to be representative of the full samples composition and stoichiometry).

Figure 2 :(A) UV-Vis absorption of Gd- Au complex (purple line) and after staking with PEG diacide (red line), COL (blue line) and CHIT (green line) and NP3 (black line) in the range 200-900nm. (A1) zoom oft he range 200-400 nm ; (A2) zoom oft he range 200-400 nm.

Physico-Chemical characterization of Gd(II)AuNPs

Previously G.Boyes et al.^[³⁴^] have grafted a thiol-terminated polymer chains onto Gd nanocomposite through the coordination of the thiolate end group with Gd (III) ions^[³⁵^].

The employment of polymer have showed a strong interaction between Gd composite and AuNPs^[³⁴^,³⁶^]. In our study, it was demonstrated that,the investment of biopolymer as chelating surfactant, play a key role in the final form of bimetallic hybrid nano formulation.

TEM images of NP1embedded in a shell of PEG, showed a metal core of diameter around 16.8 ± 1nm (Figure 3-A left); if we calculate the hydrodynamic layer of polymer and gold core, we obtained a diameter of about 184,5 nm (see histogram size in Supporting Information). Unlike nanostructures were obtained with NP2. They exhibit a polyedric-like shape, embedded in a shell of PEG and COL, in which metal nanoparticles showed a diameter around 58.9 ± 1 nm (Figure 3-A middle panel; see histogram size in Supporting Information ). Similar polyedric nanostructures have previously obtained thanks to the grow of macromolecules onto [110] gold facet^[³⁷^]. In our case, it was supposed that during grow process, COL and PEG assumes a conformational chemical arrangement responsible for adsorption onto preferentially [110] gold facet, with consequently achievement of polyhedric shape.TEM images of NP3 display a snow-like shape, embedded in a shell of PEG, COL, and CHIT with a similar diameter around 63,1 nm ± 2 nm (Figure 3-A see histogram size in Supporting Information). Previously we reported the synthesis of similar nanostructures using dicarboxylic PEG, Chitosan^[^18c^] ^[^18a^] ^[³⁸^] and drugs^[³⁹^] ^[⁴⁰^] ^[⁴¹^], while characteristic snowflakes nanoparticles were obtained by incorporating protoporphyrin molecules in the growth solution of AuNPs^[³⁷^]. On the basis of previous studies^[^18a^,^41-42^], this chemical behavior, is due to a different adsorption onto gold facets [110] of CHIT onto complex PEG-COL-GdAuCl₂^-, based on their different steric conformation of chemical groups during nucleation and growth process of Gd (II) AuNPs. Hence, it was assumed that , when the polymer ligands were added to the Gd -Au solution, the carboxylic group of polymer, being initially bound to Gd(III)-Au (III) clusters electrostatically, was embedded to dicarboxylic PEG in a mushroom conformation^[⁴³^]^,^[^18c^] and then bounded with COL and CHIT, respectively, in order to form hybrid nanoparticles. Accordingly, it is possible to obtain different shape and size varying the concentrations of reagent and the order of added mixture in the reaction. In this way, the behavior of each polymers in the formation of bimetallic micelles during the growth process of nanoparticles can be modulated.

Figure 3B, report absorption spectra of hybrid Gd (II) -AuNPs, all characterized by peaks at 300 nm and 238 nm and a surface plasmon band in the range 530-560 nm. The slow shift of the band position depends on the ratio of the gold salt and the capping materials during the reaction processes. NP1 ( red line Figure 3 B) shows a plasmon peak at 560 nm. This peak is assigned to collective surface plasmon oscillation, of the metal electrons in the conduction band, due to interaction of electrons with light of that wavelength. PEG can be used as stabilizing polymers for AuNPs because of the dispersed solutions could be obtained due to the formation of coordination bands between Au (III) and Gd (III) ions with carboxylic groups. This chelation evenly better dispersed Au ions and Gd which were reduced to form single hybrid Gd (II)Au NPs of relatively uniform size. NP2 (blue line) andNP3 (green line) in Figure 3 B, shows a strong resonance band at around 222 nm, a weaker one at ca 300 nm and a strong decrease and shift of the plasmon peak to 534 nm, due to steric arrangement for NP2 and 555 nm for NP3 of PEG, COL, and CHIT as stabilizers during synthetic process with the amine or carboxylic groups, respectively.

Figure 3: (A) TEM images of Gd (II) AuNPs (B) normalized UV-Vis absorption of NP1 (red line), NP2 ( blue line) and NP3 ( green line ) in the range 300-900 nm and (C) Raman spectra products compared to free GdCl₃*6H₂0 as control. (A) Scale bars: 200 nm; 50 nm ; 20 nm. (D) Raman spectra. Experimental conditions: λ_exc = 785 nm; laser power 20 mW; accumulation time 180s.

The NP sizes were confirmed by DLS measurements. Zeta potential measurements show that Gd (II)AuNPs were colloidally stable at physiological pH. The synthesized Gd (II)Au NPs (NP1-NP2-NP3) did show an almost negligible change in the LSP band position over a period of 72h in cell culture media (DMEM+0.3% FBS) (Figure S2 in Supporting Information).Raman spectroscopy and surface enhanced Raman scattering, were used to confirm the influence of Gd ions in Au-polymers nanoparticles. As described previously^[⁴⁴^], the presence of Gd ions provide to enhance the Raman signal of biomolecules (protein, natural polymer) capped onto gold nanoparticles (AuNPs).

Raman spectra for powder GdCl₃as control_,and Gd (II)Au NPs at 4.5 × 10⁻⁴M are shown in figure 3C. The region from 1200 to 1550 cm⁻¹ corresponds to vibrations of N-H bending and C-N stretching, while the region between 1550 and 1750 cm⁻¹corresponds to the C=O stretching mode. The band observed near 836 cm⁻¹, corresponds to vibrations of the aromatic ring. This enhancement, is the result of the electric field around the gold nanoparticles that interact with collagen molecules, in the presence of gadolinium. The characteristic vibration mode for the amine group is located at 1630 cm⁻¹(highlighted band in Figure 3). This band shows a Raman signal enhancement factor ∼40 times higher than that for free collagen^[⁴⁵^]. The increment in the Raman signal of collagen could be due to a combination of three factors: (i) formation of well-defined hot-spots, where Gd (II) ions works as spacers between gold nanoparticles; (ii) the refractive index contrast produced by the presence of Gd (II) ions can cause an increment in the electric field around the nanoparticle producing, a higher enhancement factor, and (iii) a charge transfer effect between gold nanoparticles and Gd (II) ions^[⁴⁶^]. Gd (II)-Au nanoparticles were characterized by EDX microanalysis, in which the simultaneous presence of Gd and Au of all nanoparticles were qualitatively proven (Figure S3 Supporting Information).

Kinetic release and disaggregation

Release kinetics is a critical parameter for biomedical application of NP^[²⁰^] ^[⁴⁷^]. The release of Gd complex from AuNPs (NP3) in buffer solutions, under acidic (PBS; pH 4) and temperature conditions (37°C) were investigated by LSP and Raman spectroscopy. At pH 4.0, release of 2% was noticed after 18 h and remained almost same even after 180 h (Figure S4-A in the Supporting Information). The Raman band at 260 cm^-1 was monitored to evaluate Gd (III) release from polymer-AuNPs (NP3) . We monitored our experiments each hour, noting a remarkable spectral change after 24 h, until completely increasing after 48 h (Figure S4-B in the Supporting Information). These results demonstrated that Gd (III) release was pH- and time- dependent. We assume that during incubation at pH 4, Gd-Au complex migrates in the PEG and CHIT chains and is released upon CHIT-PEG-GdAuCl₂^-.This hypothesis can explain a depolymerization and the change of size observed at different pH^[³⁹^] (Figure S4-C in the Supporting Information) . We also observed a gradual change of shape and size from nanoflowers of about 50 nm (as synthetized) (Figure S4-C panel a, t=1h) to small nanospheres of about 5 nm (pH 4.0) (Figure S4-C panel d, t=72h).Between these two states, we observe an intermediate one in which a morphological change as well as a depolymerization from the original structure was more evident (Figure S4-C panel b, c). We conclude that, under pH conditions, a dramatical change of shape and size of NP3 were made and Gd (III) were released as gold complex, as previously described for other experiments^[³⁹^] ^[⁴¹^] .

Computational results

In order to investigate the interaction between the Au and the Gd precursors, being considered as the very first nucleation step of the particles studied, a molecular geometry study at the DFT level corrected for dispersion interactions was performed.

The precursors contain Cl^- and OH^- ligands. The ligand stoichiometry depends on the pH at which the synthesis is performed (Figure 4). As was show in one of our earlier studies on the Au precursor, the presence of Cl^- has an impact on the interaction that drives the nucleation and the interaction with its environment.^[⁴⁸^]

Figure 4: The square planar (AuX₄)⁺ precursor and the GdX₃ precursor with X = Cl or OH.

Both precursor molecules were positioned in each other’s neighborhood (Au-Gd distance » 7 Å) followed by a geometry optimization. Different start geometries were investigated and at every metastable converged geometry an extra geometry optimization was performed until the total energy of the complex could not be lowered (stabilized) more. This procedure was undertaken for every combination of Cl^-/OH^- and this for every relative position around the Au and the Gd center. The interaction energy is calculated as follows:

DE_int= E(HAu(OH)_4-xCl_x···Gd(OH)_3-yCl_y) – E(HAu(OH)_4-xCl_x) – E(Gd(OH)_3-yCl_y) (1)

A graphical representation of the interaction energies (see eq. 1) between both metal complexes for every value of x and y is presented in Figure 5.

Figure 5: (graph right) Interaction energy DE_int as a function of the Cl substitution around Au and Gd for the complex HAu(OH)_4-xCl_x and Gd(OH)_3-yCl_y. Energies in eV. The values on the x axes correspond to the x values while the numbers in the legend correspond to the y values. The DE_intfor y = 1 are connected with a red line. a) Geometry of the most stable HAu (OH)_4-xCl_x···Gd(OH)_3-yCl_y complex. b)Geometry of the least stable HAu(OH)_4-xCl_x···Gd(OH)_3-yCl_y complex, showing a distorted trigonal geometry for Gd and a hydrogen bond between the Au and the Gd complex. The distance between both metal centers is 2.35 Å – 3.06 Å for structure a and b, respectively.

It can be seen that the presence of chlorine influences the interaction energy in a non-linear way (See e.g. red line in Figure5). Indeed, the position, and the number of Cl^- around each metal center, influences the stabilization of the complex. Moreover, it is interesting to note that one can divide the interactions in the following types: OH-OH, OH-Cl, Cl-Cl, and Gd-Au.

The most stable configuration is found for GdCl₃ interacting with HAu(OH)₄, (i.e. x = 0, y = 3; -4.19 eV) (Figure 5 a). The presence of one Cl^- on the Au metal complex destabilizes it with 0.18 eV. From this result one has to conclude that the H-bond interaction, that is expected to be formed between the complexes containing OH-groups do not compete with the metal-metal interaction in the complex, and that Cl-Cl halogen bonding is, as expected weaker than an hydrogen bond.

The destabilizing effect after the addition of one Cl^- on the Au center, is tempered after addition of a second and a third Cl^-. Nevertheless, the complexes do not reach the stabilization found for the HAu(OH)₄…GdCl₃ complex containing no Cl^- on Au.

It is clear that this form of the precursor is the best for the Au precursor to interact with the Gd-precursor. Just for the sake of completeness, the HAu(OH)₃Cl…Gd(OH)Cl₂is found the least stable one. However, two questions emerges: What is the stabilizing effect between the OH-Cl groups? And, what is the effect of the relative distribution of the Cl groups around the metal center on the interaction energy?

The geometry of this complex shows us the penta-coordination of Gd, the presence of bridging OH groups between Gd and Au and the shift of the H atom on a Au-OH group. Gd shows a high affinity for Cl, whereas Au prefers OH groups in our calculations. In general, the geometrical feature that destabilizes the complex is the presence of a Cl between the metal centers.

Having these stability trends in mind, what about the Cl content of the complexes in experimental conditions? Especially, the role of the pH on the Cl content of the complexes. For the Au-precursor it is known that the optimal nucleation conditions for the formation of gold nano clusters is in the range of 6 – 9.

The gold complex at this optimal pH, is shown in Figure 5b which corresponds to HAu(OH)Cl₃. Surprisingly, it is this precursor type that has the strongest destabilizing effect.

The gold complex was found to be decrease its affinity for the Gd complex with increasing number of Cl^- groups.^[⁴⁸^] However, in the actual case of interaction with a Gd complex, the opposite trend is observed, i.e. GdCl₃ interacts more strongly to Au(OH)₃Cl than the Gd hydroxides (See Figure 3 in ref. ^[⁴⁸^]).

The explanation to this contrasting finding can be explained by electronegativity softness (polarizability) competition^[⁴⁹^]. From the very low electronegativity of Gd compared with the very high electronegativity of Au, i.e. 1.20 and 2.54 in the scale of Pauling, respectively, it is clear that the polarity of the ligands is largely affected. Which can explain this opposite trend compared with Au or another more electronegative elements, such as Si, as was found in the case of silica (Si-OH groups).

The hydroxylated Gd complex is found to more strongly interact than the chlorinated ones in accordance with the higher electronegativity of the oxygen atoms. In contrast, the interaction decreases upon chlorination resulting from the higher polarizability, charge capacity or softness of Cl^-.

The Cl^- surrounding Gd can attract more charge and thus interact more strongly. Earlier, soft rare earth metal ions strongly bonded with hard donor oxygen atoms has been explained by Pearson’s HSAB principle.^[⁵⁰^] With this trend in mind one can predict the optimal conditions to form Gd-Au clusters; i.e. the hypothetical pH conditions in which Gd is surrounded by Cl and Au by OH groups, i.e. basic conditions.

In vitro cytotoxicity

Viabilities of TIB-75 (hepatocytes) cells were evaluated using increasing concentrations of nanoparticles. Figure 6 A shows a mild toxicity at Gd concentration above 0,2 mM concentration to 3.8 mM. The in vivo injected amount should therefore be inferior respectively from 2 mM to 38 mM of Gd.

Relaxivities in solution were measured at 7 Teslasand were compared to Dotarem Gd complex commercial MRI contrast agent. Values of longitudinal and transverse relaxivities r₁ and r₂ are given for these three types of objects in figure 6 B and Table 1. Gd (III)complex contrast agents are well known for their contrast agent properties in T₁ MR Imaging. They provide a hyper signal in appropriate T₁ weighted MRI acquisition sequences. Gd (II)Au nanoparticles longitudinal relaxivity r₁increases, compared to the one of Gd (III)Dotarem and to free Gd (III) ions.

Figure 6: A) Viability by Alamar Blue assay of TIB-75 (hepatocytes) cell lines after incubation with Gd -Au nanoparticles (NP1-NP2-NP3); B) Examples of MRI images of T1 and T2 weighting recorded at 7T and the corresponding r1 and r2(respectively 20.0 and 8,8 mM^-1.s^-1), r1/r2 = 2.3) relaxivities values showing the efficient T1 type features of the contrast agent for MRI at 7T.

Table 1:Values of relaxivities r1 and r2 are expressed in (mM^-1.s^-1) measured at 7T, and corrected (second lines) from ICP AES elementary analysis (IPG ICP AES Facility, Paris); r2/r1 ratio are calculated to evidence the T1 type MRI contrast agent (>1).

In vivo biodistribution by MRI

In vivo biodistribution studies were then performed to assess the kinetic behavior of the nanoparticles in mice and assess their stealthiness property after intravenous injection. For further nanomedecine application in vivo, nanovectors should indeed circulate long enough superior to 30 min into the bloodstream before being captured by the liver and spleen. The NP size superior to 20 nm predicts a hepatic uptake as the second uptake and clearance pathway for exogeneous scaffolds of a small size around 5 nm is the kidney. Dynamic Contrast Enhanced MR imaging at 7T with specific duration adjustment from min to days allows following the organs uptake by kinetic acquisitions to obtain the whole process of nanoparticle elimination: liver uptake, remanence and clearance^[²¹^,⁵¹^]. Figure 8 and Table 2 summarizes the kinetic of uptake and clearance of NP3 with examples of MRI pictures illustrating the evolution of signal versus time at each remarkable point (A: initial signal, B: decreasing slope, C: plateau, D: increasing signal). The NP uptake by the liver results ina increase of signal in the liver up to 20% and a remanence time superior to 24h up to 48h.

Figure 8: Example of Dynamic Contrast Enhanced (DCE) MRI biodistribution in vivo before and after injection of v=100ul of solution of Gd -Au nanoparticules (NP3) during 48h post injection. Enhancement of the signal visualized the uptake of the NP in the organ and the disappearance of the signal showed the clearance.

The biodistribution of others organs (spleen and kidney) were recorded by In vivo DCE MRI at 7T and showed in figure S5 in Supporting Informations.

Table 2: Timings of biodistribution of the NP3 measured in vivo from DCE MRI in mice.

Conclusion

In this paper the development of new chemical polymeric hybrid nanostructures, whose optical and morphological properties are optimized for their application in therapeutic targeting, diagnosis and therapy. In contrast to previous works, a new strategy of gadolinium complexed to gold ions, and then stacking with biopolymer matrix was employed. Chemical-Physical characterization studies were conducted extensively and fully elucidated the formation mechanism, of the nanostructure as well as, the conformational changes associated with such processes. We demonstrated that Gd (II) AuNPs have several advantages for the visualization of hepatocytes in the liver. Specifically, these nanoconjugates provide an efficient cellular uptake of large quantities of Gd (III) into cells, while preserving a T1 contrast inside cells that affords an robust in vivo detection using T1-weighted MR images.

After in vitro assays of cytotoxicity and imaging, in vivo assays of dynamic biodistribution were recorded. Given these encouraging results, this bimetallic hybrid-nanomaterial system constitutes a concrete promise as a therapeutic entity in in the field of medicinal applications.

ACKNOWLEDGMENT

With the support of SATT IDF Innov, J.S. has filed a patent application on the nanoparticles presented in this manuscript. Nanomaterial and method of production of a nanomaterial for medical applications, such as MRI or SERS - Inventor : Jolanda Spadavecchia, European Patent Application number EP17305087.3, filed January 27, 2017 and PCT application PCT/EP2018/051988 filed January, 26, 2018.”

This work has been partly performed on the CNanoMat platform of the University Paris 13 and the National natural science foundation of China under grant 81430063.Computational resources and services were provided by the Shared ICT Services Centre funded by the VrijeUniversiteit Brussel, the Flemish Supercomputer Center (VSC) and FWO.The MRI experiments were performed at the LIOPA/uMRI imaging facility belonging to the consortium Plateformesd’Imageries du Vivant of Paris Descartes, PIV and UTCBS/SEISAD/ENSCP team. The IDEX SPC Sorbonne Paris Cité IDV Imageries du Vivant is also acknowledged. We also thank the Ecole Doctorale of UPMC, U406 for financing SB’s thesis and KawtherFeddag, MS student for the biological assays.

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