Safety Evaluation and Imaging Properties of Gadolinium-Based Nanoparticles in nonhuman primates

In this article, we report the safety evaluation of gadolinium-based nanoparticles in nonhuman primates (NHP) in the context of magnetic resonance imaging (MRI) studies in atherosclerosis bearing animals and healthy controls. In healthy NHP, the pharmacokinetics and toxicity profiles demonstrated the absence of dose, time, and sex-effects, as well as a suitable tolerance of intravenous administration of the nanoparticles. We investigated their imaging properties for arterial plaque imaging in a standard diet or a high cholesterol diet NHP, and compared their characteristics with clinically applied Gd-chelate. This preliminary investigation reports the efficient and safe imaging of atherosclerotic plaques.


Imaging properties of the Gd-NPs in control (Cont) healthy monkeys. The T1-MRI properties
of Gd-NPs were first studied in healthy monkeys to observe the general biodistribution of the particles. After the intravenous injection of Gd-NPs, the main vascular network was clearly identified, and the main organs, i.e. heart, liver, and kidneys. One should note that there was a marked enhancement of blood vessels at first-pass (Fig. 2, see also Figure S1) and the bolus injection was very well tolerated without any changes in hemodynamic, cardiac, or ventilation parameters. Within the first 30 minutes, most of the nanoparticles were eliminated by the kidney route, as observed in Fig. 2A (last panel). Low T1 contrast was persistent in the muscles, liver, and kidneys. The contrast enhancement indicated a rapid renal washout of the nanoparticles: the T1 contrast enhancement strongly increased in the ureters within the first 150 seconds, before it was drastically reduced during the next minute (Fig. 2B). Moreover, at 35 min post-administration, the T1 contrast of the ureters was once more very intense, indicating a continuous washout of Gd-NPs. The main MRI findings were in accordance with the pharmacokinetics' profiles, which indicated a Gd-NPs blood half-life of ≈ 2 hours at the administrated dose (i.e. 200 mg/kg for MRI investigations).

Imaging properties of Gd-NPs in old animals under a high cholesterol (HC) diet for 24 months.
Similarly to the previous investigation, MRI was performed on old monkeys under a 24-months HC diet (referred as HC ++ animal, Fig. 3A). The contrast enhancements were similar to healthy animals except for the liver, which indicated a highest enhancement (Fig. 3B). Similar to healthy animals, the Gd-NPs were well tolerated without any changes in hemodynamic, cardiac or ventilation parameters.  Table S1.
of contrast agent may occur due to the leaky wall's endothelial layer and the inflammation, which recruits highly active macrophages 21 . In our condition, the 24-months HC diet induced moderate and more advanced atherosclerosis, as indicated by the ultrasound and biochemical parameters recorded from the treated animals (Supplementary information Table S2). As indicated by pre-contrast T1 MRI (Fig. 4), the carotid walls were not observable before the administration of any contrast agent. In absence of vulnerable plaque, both Gd-DOTA and Gd-NPs allowed very minimal carotid wall delineation. In presence of vulnerable plaque, both Gd-DOTA and Gd-NPs delineated the carotid wall with similar contrast properties. Gd-NPs appeared to have similar imaging properties as compared to Gd-DOTA. In the case of a well-established pathology (Fig. 4, HC ++ ), the vulnerable plaques were better identified by Gd-NPs, in comparison to Gd-DOTA. The T1-contrast obtained after Gd-NPs was measured with time and the elimination kinetics of Gd-NPs were determined (Fig. 5). In healthy animals, Gd-NPs were rapidly washed out, whereas a significant retention was observed in HC animals. Gd-NPs retention might be proportional with the stage of the pathology, as the highest retention was observed for the most developed pathology (right carotid of HC ++ animal).

Discussion -Conclusion
The use of nanoparticles as a contrast imaging agent requires their specific distribution in the body after intravenous injection, a rapid clearance from the body without undesired accumulation, a safe profile, and good contrast properties. Gd-NPs present the above mentioned properties with a fine distribution within the entire body starting at the first heartbeats following the intravenous administration, as well as a fast renal clearance as demonstrated with healthy NHP. After high-dose repeated IV administration, the particles were well tolerated, without modification of the antemortem and post-mortem parameters as compared to untreated animals. In particular, H&S staining indicated a safe renal elimination of Gd-NPs. Transient and minimal vacuolations of the proximal convoluted tubules was observed in rodents as previously reported 18,19 , but the NHP did not present such transient alteration for similar equivalent doses, indicating a strong tolerance and safe elimination in NHP.       Mean blood half-life measured in NHP was very similar to the one measured in rats for equivalent doses, with 2.35 hours versus 2.31 hours, respectively 18 . Altogether, the safety profile indicated a NOEL of 450 mg/kg/administration, which corresponds to 145 mg/kg/administration for Humans 22,23 .
In contrast to intravascular ultrasound which accurately image the vessel wall at high resolution 24 , MRI is noninvasive. Combined to a T1-weighted contrast agent, MRI also allows to assess the morphological plaque characteristics. In particular, the local lesion and its evolution could be monitored using a carotid MRI protocol, considering the vessel wall permeability on gadolinium-enhanced MRI 5 . Under high cholesterol diet, old NHP had at-risk plasmatic profile (high LDL/HDL ratio, high triglycerides (see Table S3) and high hsCRP levels) and developped atherosclerosis lesions similar to human plaques at the same vascular sites 25 . As shown by MRI, our animals had carotid plaques with the same advanced and vulnerable characteristics as in patients.
For similar contrast properties 16,20 , Gd-DOTA is a cyclic ionic chelate, and Gd-NPs possess DOTA-derivatives. Both compounds possess very strong complexation for Gd (logβ 110 = 25.58 for Gd-DOTA, and logβ 110 = 25.58 for Gd-NPs), preventing the release of free Gd 20 . Safe administration was observed for old atherosclerosis NHP. Imaging of the vessel's wall in this pathologic animal was demonstrated using both chelates and chelates bound to NPs. The signal measured in the vessel wall was correlated to the plaque development for Gd-NPs investigations; the former agent possesses a longer circulation time as compared to Gd-DOTA that is rapidly cleared from the body which may have favored its retention in the plaque (13.2 min vs. 6.8 min in mice, respectively) 19 . Therefore, this agent has more suitable properties to quantify neovessels leakiness using DCE-MRI, another important functional parameters to define vulnerable plaques 10 . Thanks to the high safety of Gd-NPs, this could be studied longitudinally in the near future using kinetic modeling in order to assess the vessel's wall permeability over time and correlate it with the plaque's evolution and downstream clinical events. Altogether, this study demonstrates the safety of the Gd-NPs, in particular for old pathologic animals. The T1-contrast properties were previously reported for vessel wall and plaque imaging. Further studies will indicate the potential of Gd-NP for such investigations.

Nonhuman primate (NHP) studies. All animal studies and experiments were approved by the French
Ministry of Agriculture and carried out in accordance with the official regulation of the French Ministry of Agriculture after approval by the local Ethical Committee (No 1367 & 1239).
Pharmacokinetics and toxicity study. A total of 24 healthy cynomolgus monkeys (macaca fascicularis) (n = 3/sex/group) were assigned to 4 groups: control, low-, moderate-, and high-dose of AGuIX series, i.e. 0, 150, 300, and 450 mg/kg/administration, respectively (Wil Research, France). The doses were administered intravenously, once per week for 2 weeks (day 0 and day 7). Blood samples were collected following each administration at 5 and 30 min, 1, 2, 6 and 24 h. The blood plasma distribution kinetics was analyzed based on a non-compartment model (Kinetica 4.4.1, Thermo Fisher). All animals were observed for mortality, clinical signs, ophthalmology, body weight, food consumption, hematology, biochemistry, pathology, toxicokinetics, and urinary parameters. Animals from the control group received a sterile solution of pure water containing calcium chloride (1.5 mM) and NaOH to mimic the adjuvant at pH 7.4. Hematoxylin and eosin (HES) staining were performed on tissue sections excised from the heart, lung, kidneys and liver to visualize the toxicity induced by AGuIX, 2 weeks after the second administration.
Atherosclerosis NHP model. Atherosclerosis animals were evaluated after being 24 months under a high cholesterol diet. The model is developed by Cynbiose (Marcy L'Etoile, France), and the animals of the present study are part of a larger study to evaluate plaque vulnerability using imaging biomarkers in this translational NHP model. Briefly, after ovariectomy, old females were fed a high cholesterol (HC) diet (Energy 19.3 MJ/kg, with 45 kJ% from fat, containing 0.5% cholesterol, 23% lipids, 11.3% from saturated fatty acids and 38% sugars, mainly sucrose) (V3944-000, Ssniff, Germany). The food ration was adapted according to the animal's body weight (100 g for animals under 5 kg and 200 g for animals over 5 kg). The study duration was 24 months. One fruit was provided daily to each animal. Delicacies were also occasionally given to the animals at the end of the day as part of the Testing Facility environmental enrichment program. The control animals were maintained under the same housing conditions, but with the regular NHP diet (Energy 13.7 MJ/kg, with 51 kJ% from carbohydrate, and 11 kJ% from fat containing 4% fat, and 10.2% sugars) (Ssniff, Germany). Total cholesterol and lipidic profiles were evaluated in plasma and high sensitive C reactive protein (hsCRP) was measured in serum. Expert cardiologist and veterinarian under general anesthesia performed ultrasonography, at 12 and 18 months to evaluate plaque development in different vascular beds: the carotids, aorta, and iliac arteries. Gene expression and cytokines were measured in the heart, carotids, and aorta at the end of the atherosclerosis inflammatory profile study.
Magnetic Resonance Imaging. The biodistribution study was carried out in cynomolgus monkeys under a normal diet (macaca fascicularis, control animals 5 and 8 y.o., n = 2) and in high cholesterol fed NHPs (HC +/++ animals 16 and 17 y.o., n = 2) at 200 mg/mL of AGuIX i.e. 0.1 mmol of Gd/kg equiv. Magnetic resonance imaging (MRI) was performed with a 3T scanner (Magnetom Prisma, Siemens, Erlangen, Germany) equipped with high performance gradients (80 mT/m, slew rate 200 T/m/s). All images were acquired with a set of standard multi-channels received MRI coils delivered by the manufacturer. These coils were installed once during the positioning of the animal in the scanner and activated according to the field of view. First, the monkeys were installed in supine position on the lower part of the 64-channel Head/Neck coil as well as the 32-channel Spine coil. Then two additional 4-channel flexible surface coils were placed on top on the neck (SC coil, size of 14 cm by 9 cm) and the thorax (FS coil, size of 37 cm by 7 cm) respectively. Whole body and high resolution imaging of the carotids were performed under general anesthesia (xylazine/ketamine, 100-200 mg/kg) with continuous monitoring of temperature, O 2 saturation and cardiac/respiration rate. The induction was performed by intramuscular injections of ketamine at 10 mg/kg (Ketamine 1000, Virbac, Carros, France) and midazolam at 0.2 mg/kg (Midazolam Aguettant, Lyon, France) followed by anesthetic maintenance by intravenous infusion of ketamine at 12 mg/kg/h. Dynamic images were acquired with the TWIST (Time resolved angiography With Interleaved Stochastic Trajectories) pulse sequences. TWIST sequence was applied in the coronal orientation and acquired with a separation of 5 seconds between frames. The imaging parameters were: TR/TE/Flip angle = 2.95 msec/1.10 msec/25° and voxel size 0.8 × 0.8 × 1.2 mm 3 . For comparison, a bolus of gadolinium chelate (0.1 mmol Gd/kg, Gd-DOTA, DOTAREM ® Guerbet, Aulnay-sous-Bois, France) was administered intravenously at 0.1 mL/sec followed by a saline flush. After renal excretion of standard gadolinium chelate, the sequence was repeated with the Gd-NPs at an adjusted dose for a similar angiographic T1 effect (0.1 mmol Gd/ kg Gd-NPs). High-resolution images of the carotids were acquired with an ECG-gated Turbo Spin Echo sequence with black-blood module. The parameters were TR/TE = 466 ms/5.8 ms, TSE factor = 9, reconstructed voxel size of 0.3 × 0.3 × 2 mm and 9 slices planed perpendicular to the common carotid using a TOF acquisition.
Scientific RepoRts | 6:35053 | DOI: 10.1038/srep35053 MRI data treatment. MRI data analysis was performed using the Inveon ® Research Workplace 4.1 software (Siemens, Erlangen, Germany). The regions of interest were drawn in the carotid vessel wall and in the artery's lumen to obtain the dynamic contrast information during the contrast agent first-pass, and in the different organs of interest for the contrast distribution at a steady state. Statistical analysis. Statistical analysis was performed using unpaired T-Test (Excel software) for the vulnerable plaque contrast.