Unambiguous detection of atherosclerosis using bioorthogonal nanomaterials.

The importance of atherosclerosis is driving research to create improved diagnostic tools based on molecular imaging. Pretargeted imaging is the use of bioorthogonal probes that selectively accumulate upon reaction with a pre-modified biomolecule in vivo. To date, this very promising approach has not been applied to atherosclerosis. Neither has been the use of a single nano-radiomaterial for PET / T1-MR imaging of atherosclerosis. Here, we synthesized bioorthogonal nano-radiomaterials for in vivo pretargeted molecular imaging in a mouse model of atherosclerosis. Based on tetrazine-ligation, these functionalized 68Ga iron oxide nano-radiomaterials provide simultaneous PET and T1-MRI signals and selectively accumulate in atherosclerotic plaques in mice sequentially injected with trans-cyclooctene-modified antibodies against oxidized LDL followed by the hybrid nano-radiomaterial. Our results demonstrate the ability of this approach to unambiguously detect atherosclerosis. Furthermore, we show the first example of how hybrid imaging can be used for pretargeted bioorthogonal molecular imaging with nanomaterials.


A C C E P T E D M A N U S C R I P T
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Background
Atherosclerosis is a complex chronic inflammatory disease of the blood vessel wall in which plaques build up inside the arteries. The generation of oxidized low-density lipoprotein (oxLDL) plays a key role in the initiation, progression and destabilization of atherosclerotic lesions. 1 OxLDL has therefore been widely used as a target for molecular imaging of atherosclerosis. OxLDL is an important target not only due to its role in plaque pathobiology, but also because it is a possible marker of plaque vulnerability. Molecular imaging studies have focused on antibody-labeled magnetic resonance contrasts 2,3 and more recently on fluorescence. 4,5 In the field of nuclear imaging, there are a few examples of single-photon emission computed tomography (SPECT) [6][7][8] and a recent study using positron emission tomography. 9 The methodology in all these studies share the need to match the lengthy biodistribution of the different antibodies in vivo by using long half-life isotopes, such as 125 I (t 1/2 = 59 days) or 64 Cu (t 1/2 = 16 hours).
The development of probes for hybrid molecular imaging is particularly appealing for the early diagnosis and characterization of complex diseases like atherosclerosis. The combined use of positron emission tomography (PET) and magnetic resonance imaging (MRI) technology can provide combined structural and functional information, and this approach is beginning to show its potential, particularly in the cardiovascular area. 10,11 There is a growing number of contributions where atherosclerosis is characterized by hybrid PET/MR imaging. This is normally done by using a PET radiotracer and, after radioactive decay, carrying out the MRI experiments without any contrast agent, [12][13][14] or by separated PET and T 2 -weigthed MRI experiments by the consecutive injection of two different probes, 15 or one surface-functionalized probe. 16 To our knowledge, the characterization of atherosclerosis with one probe for hybrid PET/T 1 -MRI, in one A C C E P T E D M A N U S C R I P T 4 experiment, has not been done before. We recently developed a new kind of nanoradiomaterial in which short half-life 68 Ga isotopes are incorporated in the core of extremely small iron oxide particles, combining the size-dependent properties of nanotechnology with the unparalleled sensitivity of nuclear imaging techniques. This nano-radiomaterial (NRM) provides simultaneous signals for PET and T 1 -MRI. 17,18 Bioorthogonal chemistry enables the selective conjugation of two compounds in the challenging in vivo environment. Since the ground-breaking work that initiated the use of this type of chemistry for biomedical applications, the field has expanded tremendously. [19][20][21] Bioorthogonal or pretargeted molecular imaging is based on the complementary labelling of the biomolecule (typically an antibody) and the probe; after injection of the biomolecule, the tracer is administered and selectively accumulates at the site of the bioorthogonal reaction. This type of chemistry has many advantages in molecular imaging and is particularly appealing since it allows the use of short half-life isotopes to detect biomolecules with long biodistribution times. [22][23][24] This approach has already been applied to the characterization of cancer, using small chelators such as tracers, [25][26][27] antibodies, 28 or nanoparticles. [29][30][31] Among reactions showing bioorthogonal features, tetrazine ligation is probably the best suited for in vivo imaging. The extremely rapid reaction between trans-cyclooctene (TCO) and tetrazine (TZ) derivatives and the variety of compounds available make this reaction the first choice for many applications. Ostfildern-Scharnhausen, Germany). The reaction mixture was then cooled to 60°C and the 68 Ga-NRM product was purified by passing the mixture through a PD-10 column to eliminate excess small reagents, including all unincorporated radiotracer. This purification process provided 9 mL of 68 Ga-NRM with a total activity of 781 MBq (measured 40 minutes after starting the reaction), a radiolabeling yield of 92%.  shaking. After this, solution was discarded and membrane was washed three times with TBS + 3% BSA for 10 minutes. NRM accumulation was studied by naked-eye.

Animals and ethics.
All animal experiments conducted in this work were approved by the ethics and animal welfare committee at CNIC and were developed according to the Spanish and UE legislation. Experimental protocol approved by Madrid regional government, PROEX 277/16.

Synthesis and characterization of the nano-radiomaterial.
The general hybrid approach followed in this study is summarized in Figure 1 Radioactive elution profile shows a large peak due to the 68 Ga incorporated in the core of the particles and a small peak due to free 68 Ga (Figure 2b). We next incorporated the tetrazine moiety to the 68 Ga-NRM surface through amide formation between benzylamino-tetrazine and the carboxylic acid groups in citrate ( 68 Ga-NRM-TZ, Figure   1b). Radioactive labelling stability was confirmed by incubation with mouse serum at

A C C E P T E D M A N U S C R I P T
12 Biodistribution results (Figure 3a) show a large accumulation in spleen and liver, typical of these NRM and of nanoparticles with this size 18 . Importantly, the percentage of dose is very large in the aorta, almost the same as in the liver, suggesting a successful accumulation with the pretargeted approach. The rest of the organs show very low levels of radioactivity, as expected.
For the clearance studies the same conditions were followed but including two groups does not elicit any deleterious effect in the animals, even considering that the main accumulation organ is the liver and that ApoE -/can suffer from altered liver function.

Pretargeted hybrid detection of atherosclerosis.
Having completed the characterization of the Ab-TCO and 68 Ga-NRM-TZ components of the pretargeted approach, we proceeded to the in vivo imaging, using the strategy depicted in Figure 1c. In order to have a complete study we used the following 5 different conditions ( Figure 5 (Fig 5b and 5c). For condition d) PET/CT signal was also absented from ApoE -/-HFD mice injected with unmodified 68 Ga-NRM ( Figure   5d). Finally, in the blocking experiment, condition e), no signal was found.

A C C E P T E D M A N U S C R I P T
16 The non-invasive diagnosis of atherosclerosis is a hot topic in nanomedicine and molecular imaging. Although many imaging probes have been studied, it is not easy to find one providing clear in vivo detection. Among the many targets present in the disease we focused in oxidized phospholipids, key compounds in the initiation and progression of atherosclerosis. In this study we have used combination of biorthogonal chemistry and nano-radiomaterials for the detection of the disease in animal models.
The rationale of using pretargeted imaging is based on three factors: the use of short half-life radioisotopes, the higher selectivity of the radiotracer, thus avoiding healthy tissue, and the rapid excretion of the radiotracer. 21  A C C E P T E D M A N U S C R I P T Figure 1. The approach followed in this study. a) IgM 3D structure and the conjugation of TCO molecules targeting primary amino groups in lysines (highlighted in blue in the structure, not to scale); b) Synthesis of 68 Ga core-doped iron oxide nano-radiomaterials (NRM) and their functionalization with a tetrazine derivative compound. c) Imaging protocol. Mice received injections of Ab-TCO followed 24 hours later by injection of 68 Ga-NRM-TZ. After 1 hour, in vivo PET scans were recorded of atherosclerotic lesions, and aortas were then excised and examined by ex vivo T 1 -MRI to detect localized regions of NRM accumulation.