Highly Hydrated Paramagnetic Amorphous Calcium Carbonate Nanoclusters as a Superior MRI Contrast Agent

1 Amorphous calcium carbonate (ACC) plays a key role as transient precursor in the early stages of biogenic 2 calcium carbonate formation in nature. However, due to its instability in aqueous solution, there is still 3 rare success to utilize ACC in biomedicine. Here, we report the mutual effect between paramagnetic 4 gadolinium ions and ACC, resulting in ultrafine paramagnetic amorphous carbonate nanoclusters (ACNC) 5 in the presence of both gadolinium occluded highly hydrated ACC-like environment and poly(acrylic acid). 6 Gadolinium is confirmed to enhance the water content in ACC, and the high water content of ACNC (23 7 molecules H 2 O per 1 Gd) contributes to the much enhanced magnetic resonance imaging (MRI) contrast 8 efficiency compared with commercially available gadolinium-based contrast agents. Furthermore, the 9 enhanced T 1 weighted MRI performance and biocompatibility of ACNC are further evaluated in various 10 animals including rat, rabbit and beagle dog, in combination with promising safety in vivo . Overall, 11 exceptionally facile mass-productive ACNC exhibits superb imaging performance and impressive stability, 12 which provides a promising strategy to design MR contrast agents. 13


Introduction
calcium carbonate is discovered, which contributes to the maximized hydrated content in the as-prepared 1 amorphous composite nanoclusters and an extremely high longitudinal relaxation. The final amorphous 2 nanoclusters possessed an extremely high water to Ca ratio (water/Ca = 7.2) compared with the normal 3 ACCs (ratios remained constant at about 0.4-1.9). The longitudinal relaxivity of ACNC (37.93 ± 0.63 4 mM -1 ·s -1 under 3.0 T) has also benefited from the high water content, which is ten times higher than that 5 of the commercially available MR contrast agent gadopentetic acid (Gd-DTPA) and highly resistant to 6 ion leakage so it can serve as a potential MR contrast agent.
Highly hydrated content of ACNC. In order to investigate the contribution of each component to the  Attractively, there was still a higher water content held in ACC-Gd as compared to pure ACC even in 10 deionized water for a long time. The weight loss before 300 ºC of ACC-Gd and ACC powder exposed to 11 the air over 6 months was more than 10% and only 0.1%, respectively (fig. S11). In both TGA and 12 volumetric Karl Fischer titration measurement results, the weight loss before 300 ºC could be assigned to 13 the loss of water, and ACNC exhibited the highest water content of calculative 20 wt.-% among the 14 products with different composition ( Fig. 2A). According to previous reports, the water contents per mole 15 of calcium in additive-free ACC and PAA-stabilized ACC were typically located in the range of 0.4-1.58 16 and 1. 33-1.93, respectively 20, 24 . In contrary, both ACC-Gd and ACC-PAA showed a higher water to Ca 17 ratio, and the highest ratio was obtained in ACNC (water/Ca = 7.2) (Fig. 2B), indicating a synergetic 18 enhancement of water binding by PAA and Gd in these amorphous nanoparticles. More importantly, to 19 compare the ratio of water to Gd as listed in Fig. 2C, the hydrated content per mole gadolinium in the 20 presence of both ACC-like environment and PAA in ACNC is more than four times that of PAA-occluded 21 gadolinium carbonate (PAA-AGC). Compared with AGC, the presence of ACC in the Gd-ACC composite 22 also resulted in a two-fold increase of the hydrated content per gadolinium ion. 1 To investigate the origin of the hydration, analytical ultracentrifugation (AUC) was applied to determine 2 the hydration water of ACNC via the frictional ratio and Perrin function assuming a spherical shape seen 3 in the electron microscopy images (table S4). The samples show sedimentation coefficients in the order 4 of 10 -13 s, which is typical for prenucleation clusters (PNCs) that have similar sizes 25 . The ACNCs have a 5 diameter of 1.5 nm, which is even smaller than the one found by SAXS, though still in reasonable 6 agreement. Their molar mass of 2200 g/mol indicates that they consist of approximately 20 CaCO3 ion 7 pairs including the Gd 3+ ions. The determined amount of bound hydration water in solution of 8.8 mol 8 H2O per CaCO3 (and 23.0 mol H2O per Gd 3+ ), is considerably higher than in common ACCs (Fig. 2,B 9 and C, table S5). Notably, the water content of ACNC determined by AUC is in reasonable agreement 10 with the results from TGA, considering that the hydration was determined in wet and dry states, 11 respectively 6 . 12 Stable high relaxivity of ACNC. Besides the contribution of the dopant of the gadolinium ion to the 13 highly hydrated ACC content, a typical paramagnetic behavior was achieved in the amorphous carbonate 14 nanoclusters as shown in the M-H curve (Fig. 3A). Both the high hydration and confinement of the 15 gadolinium ions, the paramagnetic ACNC was conducive to increasing MRI contrast performance. The T1 16 relaxation times of ACNC dispersed in normal saline at varying concentrations were measured using a 3.0 17 Tesla MR scanner. As shown in the T1 map (Fig. 3B), the longitudinal relaxivity (r1) of ACNC reached 18 37.21 mM -1 ·s -1 (Fig. 3C), which was ten times higher than that of commercially used Gd-DTPA (3.19 mM -19 1 ·s -1 ). Besides the contribution from the large size of macromolecules and nanoparticles, ACNC possessed 20 four times higher longitudinal relaxivity compared to PAA modified amorphous gadolinium carbonate 21 (AGC-PAA) (7.91 mM -1 ·s -1 ), which was attributed to the high hydration content of ACC-like 22 environments. This high content of water may be primarily attributed to the stronger hydration ability of 1 trivalent Gd 3+ as compared to divalent Ca 2+ ions 19 , which reasonably enhanced inner-sphere and outer-2 sphere relaxations of the ACC like environment occluded ACNC 14 . After three tests, the r1 value of ACNC 3 was calculated to be approximately 37.93 ± 0.63 mM -1 ·s -1 (Fig. 3D).

4
In addition, r1 and r2 values of ACNC were measured under different magnetic fields (3.0 T and 0.5 T), 5 and its corresponding r2/r1 ratio was calculated (Fig. 3, E and F). The r1 value of ACNC measured on 3.0 6 T was as high as 38.19 mM −1 s −1 , and the corresponding r2 value and r2/r1 ratio were 72.49 mM −1 s −1 and 7 1.90, respectively. Using a low field MR scanner system (0.5 T), the corresponding r1 and r2 values of 8 ACNC were 66.37 mM −1 s −1 and 78.04 mM −1 s −1 , respectively, and the r2/r1 ratio was 1.18.

9
High stability is essential for gadolinium-based contrast agents, as transmetallation will lead to the 10 release of dissociated gadolinium ion with reported toxicity such as nephrogenic systemic fibrosis 14, 26 . 11 Laurent and Muller reported the poor kinetic inertness against transmetallation for linear gadolinium-12 based contrast agents such as commercially used Gd-DTPA 27 . As shown in Fig. 3G, the PAA-Ca/Gd 13 chelate exhibited an even worse inertness than Gd-DTPA after exposure to Zn 2+ in PBS for 48h. On the 14 contrary, ACNC demonstrably enhanced the stability against transmetallation, due to the gadolinium 15 confinement in ACC-like environments.

16
Stability and biocompability of ACNC. The absorption spectra of arsenazo III is generally used to detect 17 the leakage of gadolinium ion from Gd-based nanocomposite 28,29 . When the Arsenazo III aqueous solution 18 was mixed with Gd 3+ , pink solution turned blue due to the formation of arsenazo-Gd 3+ complex (Fig. 4A).

19
As shown in Fig. 4B, free gadolinium ion at a low concentration of 1 µg/mL was detectable by arsenazo 20 III mediated absorption spectra. However, in the normal saline dispersion of ACNC, no leakage of free 21 gadolinium ions in one week dialysis was detected using this colorimetric analysis, which was further 22 confirmed by ICP-MS (Fig. 4C). In sharp contrast to ACNC, PAA-Ca/Gd chelate showed an obvious 1 leakage compared with ACNC, further confirming the confinement of gadolinium ions by carbonate.  To determine whether ACNC cause hemolysis, ACNC at different concentrations were incubated with 9 human blood serum at 37 °C for a hemolysis test. According to the standard, ACNC have no 10 haemocylolysis even at a high concentration of 0.5 mg (Gd) /mL, suggesting a good blood compatibility    The hydration level plays a significant role in determining the contrasting performance of a MR contrast 21 agent 14 . Unfortunately, the hydration content of Gd-based nanocrystals is limited by traditional high temperature synthesis processes 15 . Worth mentioning is that the high moisture content, including interior 1 water and deeply located structural water is the most distinctive feature of ACC 7-11 . However, the potential 2 application of unstable hydrated ACC is largely ignored. Interestingly, the ionic radius of the gadolinium 3 ion is very close to that of the calcium ion, implying a possible interaction between gadolinium ions and 4 calcium ions that can be exploited by us.

5
In summary, our study confirms mutual effects between the paramagnetic lanthanide gadolinium ion 6 and amorphous calcium carbonate, which is beneficial to maximizing the water content in the obtained 7 amorphous composite nanoclusters. The material is synthesized through a facile one-pot process at room 8 temperature, enabling the large scale and cost effective production. Importantly, this high water content 9 contributes to the transparent MRI contrasting enhancement of gadolinium-based nanoclusters. In 10 combination with their low toxicity, partial renal clearance and easy potential for mass production, our 11 work enables further identification of the biomedical potential of ACC composites, and we anticipate that  Sample preparation. In a typical procedure to synthesize additive-free amorphous calcium carbonate 8 (ACC), 10 mL Na2CO3 aqueous solution (0.1 mol·L -1 , 10 mL) was poured into calcium chloride (0.1 9 mol·L -1 , 10 mL) aqueous solutions under vigorous magnetic stirring for 15 seconds. The aqueous 10 suspension after adding 20 mL ethanol were centrifuged at 3000 rpm for 2 min immediately, then the 11 precipitates were washed by ethanol, centrifuged for 5 min at 8000 rpm twice.

12
In a typical procedure to synthesize ACNC, anhydrous calcium chloride (CaCl2, 0.1 mol·L -1 ), 13 gadolinium chloride hexahydrate (GdCl3, 0.02 mol·L -1 ) and PAA (0.45 g) were dissolved in 10 mL DIW 14 with magnetic stirring. Anhydrous sodium carbonate (Na2CO3, 0.1 mol·L -1 , 10 mL) was poured into the 15 above solution with vigorous magnetic stirring for 1 min. 20 mL of ethanol was added to terminate the 16 reaction. The mixture was immediately centrifuged for 2 min at 3000 rpm, and precipitates were re-17 suspended by DIW, centrifuged for 5 min at 8000 rpm twice. For liter-scale synthesis, the reaction volume 18 was scaled up 50 times and proceeded under robust mechanical agitation.

19
For the synthesis of ACC stabilized by PAA (ACC-PAA), 0.45 g PAA was used in the synthesis process 20 without the addition of gadolinium salt, and the product was washed by ethanol. For the preparation of ACC-Gd, PAA was absent in the synthesis process and anhydrous CaCl2 (0.1 mol·L -1 ) and GdCl3 (0.02 1 mol·L -1 ) were mixed in 10 mL DIW. Then the obtained ACC-Gd was washed by ethanol.

2
In a typical procedure to synthesize amorphous gadolinium carbonate (AGC) and AGC-PAA, Na2CO3 3 (0.1 mol·L -1 , 10 mL) was poured into 10 mL GdCl3 aqueous solution (0.067 mol·L -1 , mol[GdCl3]: 4 mol[Na2CO3] = 2: 3) under vigorous magnetic stirring for 15 seconds, then 20 mL ethanol was poured 5 into the aqueous suspension. After centrifuged at 3000 rpm for 2 min immediately, the precipitates were 6 washed by ethanol, centrifuged for 5 min at 8000 rpm twice. In addition, in the presence of 0.45 g PAA 7 in the GdCl3 aqueous solution, AGC-PAA was prepared in the same procedure. frictional coefficient f to that of an equivalent spherical molecule of the same anhydrous mass and density 1 f0), ̅ is the partial specific volume (cm 3 /g) of the solute and ̅ is the hydrated specific volume (the 2 volume occupied by the solute and associated solvent per unit mass of the anhydrous solute) and ̅ 2 = 3 specific volume of water given as: If all excess friction is due to the hydration and the solute is a sphere, then P is 1 54 and the hydration 6 can be calculated as: carried out on Sysmex XE2100 and Vitros 5600, respectively.

8
In vivo metabolic study. ACNC normal saline solutions were intravenously injected into twenty mice 9 (male, 20 g) at 3 mg Gd/kg bodyweight (0.5mg Gd/mL, 120 µL) via rapid manual injection for 10 a simulated bolus injection study. These mice were randomly divided into four groups, and various organs 11 were resected from the mice in each group 1, 7, 15 and 30 days after intravenous injection, respectively.

22
Signal-noise ratio (SNR) was calculated in a single image (κ) based on two separate regions of interest 1 (ROIs). One in the vessel of interest (ROIvessel) were measured by placing a signal at the same ROI in the 2 same slice both in Gd-DTPA and ACNC group, which was recorded as the mean signal intensities of vessel 3 (Svessel). One in the image background (ROIbackground) was located in an artifact-free homogeneous area,