Needa Brown
email: ne.brown@northeastern.edu

Paul Rocchi #

Léna Carmès

Romy Guthier

Meghna Iyer

Léa Seban

Toby Morris

Stephanie Bennett

Michael Lavelle

Johany Penailillo

Ruben Carrasco

Chris Williams

Elizabeth Huynh

Zhaohui Han

Evangelia Kaza

Tristan Doussineau

Sneh M Toprani

Xingping Qin

Zachary D Nagel

Kristopher A Sarosiek

Agnès Hagège

Sandrine Dufort

Guillaume Bort

François Lux

Olivier Tillement

Ross Berbeco
email: ross_berbeco@dfci.harvard.edu

Paul Rocchi

Tuning ultrasmall theranostic nanoparticles for MRI contrast and radiation dose amplification

Keywords: nanoparticle, gadolinium, bismuth, theranostics, magnetic resonance, radiation therapy

The introduction of magnetic resonance (MR)-guided radiation treatment planning has opened a new space for theranostic nanoparticles to reduce acute toxicity while improving local control. In this work, second-generation AGuIX ® nanoparticles (AGuIX-Bi) are synthesized and validated to maintain MR positive contrast while further amplifying the radiation dose by replacing some Gd 3+ cations with higher Z Bi 3+ . Next-generation nanoparticles are based on the AGuIX ® platform, which is currently being evaluated in multiple Phase II clinical trials in combination with radiotherapy. In this clinically scalable methodology, AGuIX ® is used as an initial chelation platform to exchange Gd 3+ for Bi 3+ . AGuIX-Bi nanoparticles are synthesized with three ratios of Gd/Bi, each maintaining MR contrast while further amplifying radiation dose relative to Bi 3+ . In a non-small cell lung cancer model, increased Bi 3+ is associated with more DNA damage and improves in vivo efficacy with a statistically significant delay in tumor growth and 33% complete regression. The addition of Bi 3+ by our synthetic method leads to nanoparticles that present slightly

altered pharmacokinetics with lengthening of the period of high tumor accumulation with no observed evidence of toxicity. We demonstrate the safety and enhanced efficacy of AGuIX-Bi at the selected ratio of 30Gd/70Bi, both crucial steps toward patient translation.

Introduction

The first gadolinium (Gd)-based contrast agent, Magnevist  , entered the global clinics in 1988 [START_REF] Lohrke | 25 years of contrastenhanced MRI: developments, current challenges and future perspectives[END_REF]. Since then, advances in contrast agents and Magnetic Resonance Imaging (MRI) capabilities have allowed several novel formulations to enter the clinic including the theranostic nanoparticle (NP) AGuIX ® (Activation and Guidance of Irradiation by X-Ray). AGuIX ® NPs were developed by NH TherAguix and first described in 2011 [START_REF] Lux | Ultrasmall rigid particles as multimodal probes for medical applications[END_REF]. They are sub-5 nm NPs composed of polysiloxane and a high concentration of Gd (~15 Gd per NP) for MR guidance and local radiation dose amplification. Gadolinium chelates within AGuIX ® serve a dual role as a MR-contrast agent and a local amplifier, emitting more low-energy photoelectrons and Auger electrons during radiation therapy. The small size enables high tumor-specific accumulation, taking advantage of the enhanced permeation and retention (EPR) effect and quick renal clearance. AGuIX ® has shown promise in phase I dose escalation studies on 15 patients with continuation into multiple phase II trials (NCT03818386 & NCT04789486) for the treatment of brain metastases, lung cancer, and pancreatic cancer in combination with radiation therapy [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Bort | EPR-mediated tumor targeting using ultrasmall-hybrid nanoparticles: From animal to human with theranostic AGuIX nanoparticles[END_REF]. AGuIX ® is one of only two radiosensitizer nanoparticles to make it into clinical trials, the other being hafnium oxide (ZHf = 72) nanoparticles, NBTXR3, developed by Nanobiotix. Compared to AGuIX ® , NBTXR3 relies on intratumoral injections thus limiting therapies to only superficially accessible tumors and providing limited diagnostic capabilities [START_REF] Bonvalot | NBTXR3, a first-in-class radioenhancer hafnium oxide nanoparticle, plus radiotherapy versus radiotherapy alone in patients with locally advanced soft-tissue sarcoma (Act. In. Sarc): a multicentre, phase 2-3, randomised, controlled trial[END_REF].

Although AGuIX ® NPs have demonstrated promising theranostic potential, treatments do not lead to complete regression. A push to improve the therapeutic potential of conventional AGuIX ® NPs while still maintaining MR contrast motivated the formulation of second-generation bismuth (Bi)-gadolinium nanoparticles (AGuIX-Bi). In this proof-of-concept study, we specifically chose to design AGuIX-Bi NPs to address the unique imaging and therapeutic challenges associated with centrally-located non-small cell lung cancer (NSCLC) in alignment with objectives of the ongoing Nano-SMART clinical trial (NCT04789486). Being the third most common type of carcinoma, lung tumors account for approximately 25% of cancer related deaths and are estimated to lead to over 238,340 new cases in 2023 alone with 85% consisting of NSCLCs [START_REF]American cancer society: Cancer facts and figures[END_REF]. A unique challenge for NSCLC radiation therapy is the constant motion associated with the cardiac and respiratory cycles within the lungs [START_REF] Sim | A review of the role of MRI in diagnosis and treatment of early stage lung cancer[END_REF]. This motion can lead to inaccurate tumor dose delivery and increased risk for surrounding healthy tissue damage, especially in centrally-located lesions with high rates of proximal critical organ (central airways, esophagus, and vascular structures) toxicity. Stereotactic body radiation therapy (SBRT), which involves a high dose per fraction (>6 Gy fraction -1 ), is a proven method for the treatment of early-stage NSCLC patients, achieving local control rates of 90% [START_REF] Dosoretz | Radiation therapy in the management of medically inoperable carcinoma of the lungs: Results and implications for future treatment strategies[END_REF][START_REF] Parashar | Radiation therapy for early stage lung cancer[END_REF]; however, dose-limiting toxicities are a major obstacle restricting this treatment option for thousands of patients each year. Breath-holds are often used to mitigate motion artifacts and proximal toxicities; however, NSCLC patients often have other comorbidities that make breath-holds difficult. Alternatively, image guidance via MRI is being integrated into treatment workflows, improving tumor dose delivery while reducing patient burden [START_REF] Sim | A review of the role of MRI in diagnosis and treatment of early stage lung cancer[END_REF]. Although MRI provides excellent soft tissue contrast at most body sites, the unique high air content and low tissue density within the lungs dramatically reduces the signal-to-noise ratio. This coupled with motion due to the cardiac and respiratory cycles leads to susceptibility artifacts and image degradation [START_REF] Sim | A review of the role of MRI in diagnosis and treatment of early stage lung cancer[END_REF].

Recent literature with tumor specific gadolinium-based nanoparticles [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF] has shown potential for improved contrast and signal-to-noise ratios when coupled with MRI. The introduction of MRguided radiation therapy within the clinic has opened up a new space for theranostic nanoparticles like AGuIX ® that will enable better tumor delineation, local radiation amplification, and sparing of healthy tissue.

In this paper, second-generation NPs have been formulated which maintain MR positive contrast while further amplifying the radiation dose effect with additional chelation of bismuth.

Compared to AGuIX ® , these AGuIX-Bi NPs incorporate higher atomic number (Z) Bi 3+ (ZBi = 83) cations in addition to Gd 3+ (ZGd = 64) enhancing their theranostic applications. The probability of the photoelectric effect increases proportional to the atomic number of the atom (Z 3 ) and decreases when the energy of the incident photon increases (E 3 ). In soft tissue, the photoelectric effect has a relatively small contribution at energies >30 keV, however, it is the dominant interaction for high-Z nanoparticles leading to the deposition of localized focal ionizing radiation. The secondary photoelectrons and Auger electrons have a short range, a few nanometers to micrometers, leading to a higher local radiation dose effect in tissues with NP uptake [START_REF] Mesbahi | A review on gold nanoparticles radiosensitization effect in radiation therapy of cancer[END_REF]. High energy (≥6 MV) photon beams are conventionally used in the clinic to increase skin sparing, however, these beams are comprised of a spectrum of energies with substantial amounts of lower energy photons (<150 kV) which can interact with high Z NPs leading to local dose amplification. This is particularly true for modern flattening filter-free (FFF) beams. In addition to the inherent low energy photons within the spectrum, scatter within the tissue as a function of depth, field size, and distance from the central axis as well as removal of the flattening filter can further contribute to increased lower energy photons [START_REF] Detappe | Key clinical beam parameters for nanoparticle-mediated radiation dose amplification[END_REF]. Although physical dose amplification by the photoelectric effect is important, in almost all experiments the degree of observed biological dose enhancement of high Z NPs is much greater than the predicted physical dose, highlighting unknown cellular radiobiological responses that are not driven simply by increasing radiation dose [START_REF] Rosa | Biological mechanisms of gold nanoparticle radiosensitization[END_REF][START_REF] Butterworth | Radiosensitization by gold nanoparticles: effective at megavoltage energies and potential role of oxidative stress[END_REF]. Other chemical and biological contributions are still being investigated including a recent study with AGuIX ® suggesting several biological mechanisms including increased cellular DNA damage due to inhibition of homologous recombination repair pathway proteins, increased apoptosis via activation of caspase-3, and regulation of ferroptotic cell death by inhibiting the NRF2-GSH-GPX4 signaling pathway [START_REF] Sun | AGuIX nanoparticles enhance ionizing radiation-induced ferroptosis on tumor cells by targeting the NRF2-GPX4 signaling pathway[END_REF].

In this work, three different gadolinium/bismuth ratio nanoparticles have been synthesized respectively 70Gd/30Bi, 50Gd/50Bi, and 30Gd/70Bi (molar ratio). The formulation methodology developed is a stable and clinically scalable process in which AGuIX ® is seen as a chelation platform NP initially filled with Gd that is replaced with Bi at varying ratios. Similar to AGuIX ® , the ultrasmall size of AGuIX-Bi nanoparticles facilitates EPR-directed tumor-specific accumulation and kidney clearance. The addition of Bi 3+ maintains similar physio-chemical characteristics to AGuIX ® , retains MRI positive contrast due to the remaining Gd 3+ cations while amplifying DNA damage and therapeutic efficacy, and increases in vivo pharmacokinetics thereby lengthening the period of high tumor accumulation. Given the promise of AGuIX ® in phase I and ongoing phase II clinical trials, these preliminary studies are necessary to lay the foundation for the translation of these next-generation AGuIX-Bi nanoformulations for MR-guided radiation therapy.

Results and Discussion

AGuIX ® as a tunable chelation platform for next-generation nanoparticles

AGuIX ® are ultrasmall nanoparticles composed of a polysiloxane core with covalently grafted gadolinium chelates. DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1yl) pentanedioic acid), a derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), is a macrocyclic molecule known for strong binding constants of complexes with trivalent cations, e.g. Bi 3+ (log10(K) = 30.3) [START_REF] Baranyai | The use of the macrocyclic chelator DOTA in radiochemical separations[END_REF][START_REF] Csajbók | Equilibrium, 1H and 13C NMR spectroscopy, and X-ray diffraction studies on the complexes Bi (DOTA)-and Bi (DO3A-Bu)[END_REF] or Gd 3+ (log10(K) = 25.6) [START_REF] Baranyai | The use of the macrocyclic chelator DOTA in radiochemical separations[END_REF][START_REF] Sherry | Primer on gadolinium chemistry[END_REF]. DOTAGA was chosen due to the conservation of the DOTA structure that ensures strong Gd 3+ chelation even after grafting of the inorganic matrix (log10(K) = 24.78) [START_REF] Mignot | A Top-Down synthesis route to ultrasmall multifunctional Gd-Based silica nanoparticles for theranostic applications[END_REF]. The high stability comes from the macrocyclic nature of the ligand and the ability of the carboxylic acid groups to chelate in a deprotonated state [START_REF] Sherry | Primer on gadolinium chemistry[END_REF]. During formulation, gadolinium dissociation from the covalently grafted DOTAGA@(Gd 3+ ) groups on AGuIX ® was performed by protonating the chelate in highly acidic conditions. AGuIX ® NPs were placed in a 1 M HCl acidic medium driving competitive chelate protonation leading to the release of some Gd 3+ cations [START_REF] Sherry | Primer on gadolinium chemistry[END_REF][START_REF] Pniok | Thermodynamic and kinetic study of scandium (III) complexes of DTPA and DOTA: a step toward scandium radiopharmaceuticals[END_REF]. The solution was further heated to 50°C during the release step, favoring the dissociation kinetics. Using a reverse phase C4 HPLC column, the free Gd 3+ was easily separated from the remaining mixture components. As illustrated in Figure 1, the released gadolinium was monitored via Inductively coupled plasma-Mass spectrometry (ICP-MS) all along the process. At the end of the first release step, the amount of gadolinium initially released should be directly correlated with the amount of free DOTAGA in the final product. To obtain NPs with 70% of the initial DOTAGA(Gd) being replaced by DOTAGA(Bi), the gadolinium release had to be pushed to at least 70% of the initial Gd amount at the time the reaction was stopped. This Gd release level was reached after 19 h of reaction. At this point, the area of the free Gd signal recovered was 78.5% of the reference area (Figure 1A) indicating the end of the release step. To stop the freed Gd 3+ from re-chelating, purification was performed using the Vivaflow 200 ultrafiltration system (5 kDa cutoff). High-performance liquid chromatography-ICP/MS (HPLC-ICP/MS) confirmed the absence of free Gd 3+ after purification drying.

Before moving to the bismuth chelation step, a free DOTAGA titration was performed to confirm the real amount of free DOTAGA in the starting material. The result showed a free DOTAGA level of 0.64 µmol mg -1 of the product (Figure S1) which represented 86% of the initial DOTAGA(Gd 3+ ) content of the AGuIX ® product as can be seen in Figure 1B. The bismuth chelation was divided into three steps to reach the three different Bi targets. After each step, the free DOTAGA content was measured (Figure S2-S4). We observed that the experimental chelation followed closely with the expected chelation yield moving from 86% free DOTAGA to 56%, from 56% to 34%, and from 34% to 14% (Figure 1A). To obtain the 3 final products, each intermediate product was chelated with the right amount of missing Gd 3+ (Figure 1C) as a final step. The resulting products, AGuIX-Bi 70Gd/30Bi, 50Gd/50Bi, and 30Gd/70Bi, have been purified using a Vivaflow 200 ultrafiltration system (5 kDa cutoff) to ensure the absence of remaining free Gd 3+ and Bi 3+ cations in solution.

Previous preliminary work demonstrated the efficacy of bismuth incorporation [START_REF] Detappe | Ultrasmall silica-based bismuth gadolinium nanoparticles for dual magnetic resonance-computed tomography image guided radiation therapy[END_REF], however optimization of Gd/Bi ratios was not investigated. In the Detappe et al. work, the gadoliniumbismuth particles were obtained through a post-functionalization process of the AGuIX ® in which DOTA-NHS was used to react with free amino functional groups at the surface of the nanoparticle.

The bismuth was then added on the newly grafted DOTA, but the ratio of Bi was limited by the grafting of DOTA-NHS on the polysiloxane matrix and could not exceed 30-40% due to steric hindrance and the lack of amino functions at the surface of the polysiloxane. Moreover, tuning the ratio of Bi required changing the number of DOTA grafted at the surface resulting in changes in surface charge and size which complicates comparisons between NPs with different Gd/Bi ratios.

In the current work, AGuIX ® was used as a chelation platform NP initially filled with Gd, thus providing a more scalable formulation for clinical translation. Through an acidic treatment, we were able to remove the initial Gd and regenerate free DOTAGA. The newly generated free DOTAGA was used to achieve the desired complexation of different elements at different ratios making this a more tunable and scalable platform. Figure 1. A) Increase of free gadolinium in the reaction mixture depending on the reaction time (in HCl 1 M at 50°C) followed by HPLC-ICP/MS and compared to gadolinium standard (dash plot) corresponding to 100% release of initial gadolinium. The lower part is a schematic view of the process starting from AGuIX ® to obtain a particle devoid of about 80% of its initial gadolinium content. B) Results of the free DOTAGA titration at each step of the bismuth chelation show the diminishment of free DOTAGA amounts all along the chelation process. The lower part is an illustration of the bismuth chelation process beginning with 20Gd/0Bi, to generate 3 different batches of particles: 20Gd/30Bi, 20Gd/50Bi, and 20Gd/70Bi. C) Schematic view of the final gadolinium chelation to obtain the 3 final AGuIX-Bi products with desired Gd/Bi ratios: 70Gd/30Bi, 50Gd/50Bi, and 30Gd/70Bi (molar ratio).

Characterization of AGuIX-Bi nanoformulations

The size of a nanoparticle impacts its pharmacokinetics [START_REF] Blanco | Principles of nanoparticle design for overcoming biological barriers to drug delivery[END_REF] and its elimination pathway [START_REF] Blanco | Principles of nanoparticle design for overcoming biological barriers to drug delivery[END_REF][START_REF] Du | Transport and interactions of nanoparticles in the kidneys[END_REF].

Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter (DH) of the NPs.

Volume distributions of the DH for the three final products are displayed in Figures S5-S7. The measured DH for the three Gd/Bi ratios are similar: 4.5 ± 1.6 nm for 70Gd/30Bi, 5.1 ± 1.9 nm for 50Gd/50Bi, and 4.8 ± 1.9 nm for 30Gd/70Bi (Table 1). All NP formulations have diameters below the glomerular cutoff of < 8 nm [START_REF] Du | Transport and interactions of nanoparticles in the kidneys[END_REF], suggesting potential for renal clearance from the body similar to previously published AGuIX ® NPs [START_REF] Sancey | Long-term in vivo clearance of gadolinium-based AGuIX nanoparticles and their biocompatibility after systemic injection[END_REF]. The overall hydrodynamic diameter of the AGuiX-Bi NPs is close to the diameter of AGuIX ® NPs with a DH = 4.6 ± 1.6 nm (Table 1). The volume distribution is displayed in Figure S8.

The chromatograms (HPLC-UV, High-performance liquid chromatography-Ultraviolet) at 295 nm agree with the maximum absorption of the original AGuIX ® [START_REF] Truillet | Coupling of HPLC with electrospray ionization mass spectrometry for studying the aging of ultrasmall multifunctional gadolinium-based silica nanoparticles[END_REF] nanoparticle, as well as a high absorption area of DOTAGA(Bi) [START_REF] Lima | H2Me-do2pa: an attractive chelator with fast, stable and inert nat Bi 3+ and 213 Bi 3+ complexation for potential α-radioimmunotherapy applications[END_REF]. The retention times (Tr) of the three final products 70Gd/30Bi, 50Gd/50Bi, and 30Gd/70Bi respectively 10.6 min, 10.6 min, and 10.4 min; comparable to the AGuIX ® retention time of 10.9 min (Table 1). These results confirm the low impact on final NP formulation characteristics, making clinical scalability of the NPs with various ratios of Gd and Bi more feasible as well as providing confidence in the ease of nanoformulation tunability to incorporate other heavy metals, drugs, or isotopes for additional multimodal diagnosis and therapeutics compared to the previous methodology [START_REF] Detappe | Ultrasmall silica-based bismuth gadolinium nanoparticles for dual magnetic resonance-computed tomography image guided radiation therapy[END_REF]. A high purity of 92% is obtained for the three final products measured directly from the HPLC-UV chromatogram absorbance ratio at 295 nm (% 𝑝𝑢𝑟𝑖𝑡𝑦 = 𝐴𝐵𝑆 295 [10-15 𝑚𝑖𝑛] 𝐴𝐵𝑆 295 [0-15 𝑚𝑖𝑛 ] ) (Figure S9). We observe an overall increase in peak absorbance for all nanoparticles compared to AGuIX ® . This increase is due to the obtention of DOTAGA(Bi) complexes [START_REF] Lima | H2Me-do2pa: an attractive chelator with fast, stable and inert nat Bi 3+ and 213 Bi 3+ complexation for potential α-radioimmunotherapy applications[END_REF] and is proof of the chelation process efficiency. This efficacy was further confirmed through the HPLC-SEC-ICP-MS (SEC, size-exclusion chromatography) chromatogram (Figure S10A) where we observe the co-localization of Bi, Gd, and Si (Silicone) of the nanoparticle core. The Gd/Bi ratio evolution can also be seen through the ICP-MS intensity variation on each chromatogram (Figure S10B).

The ζ-potentials show that the pH is equal to 7.11 at the iso-electric point (IEP) (Table 1) and is the same for all three final products. As the three products come from the same starting material, those equal IEPs are a sign of the same final complexation levels. Moreover, their IEP is close to the IEP of the initial AGuIX ® at 7.15 (Table 1), indicating that the surface of the three final products and the original AGuIX ® particle should be similar. In addition, the release and the chelation process can also be followed by ζ-potential measurement and IEP change. As shown in Figure S11, the pH at IEP is decreasing after gadolinium release (5.58 vs. 7.15 for AGuIX ® ) which is a sign of the presence of free DOTAGA at the surface. During the Bi chelation step, we observe an increase after each step moving from 5.58 at the beginning of the process to 6.95 after the final Bi chelation. This increase is due to the formation of DOTAGA(Bi) at the particle surface.

An elemental analysis of the three final products can be found in Table 1. The Gd and Bi massic content of each final product is in perfect accordance with their desired Gd/Bi molar ratio. The product AGuIX-Bi (70Gd/30Bi) contains 479 µmol of Gd and 203 µmol of Bi per mg of product which corresponds to 70.3% of Gd and 29.7% of Bi. The product AGuIX-Bi (50Gd/50Bi) contains 342 µmol of Gd and 327 µmol of Bi per mg of product which correspond s to 51.1% of Gd and 48.9% of Bi. The product AGuIX-Bi (30Gd/70Bi) contains 201 µmol of Gd and 477 µmol of Bi per mg of product which corresponds to 29.6% of Gd and 70.4% of Bi. The three products have a similar total metal content of 682 µmol mg -1 , 669 µmol mg -1 , and 678 µmol mg -1 , respectively. This total metal is lower than the initial AGuIX ® metal content equal to 744 µmol mg -1 which can be the consequence of some hydrolysis of the amide bond linking the DOTAGA groups to the nanoparticle core (due to acidic conditions) resulting in a decrease of the total DOTAGA content or to the elimination of some smaller nanoparticles during the purification steps.

AGuIX-Bi (30Gd/70Bi) longitudinal relaxivity (r1) at 7 T was 7 s -1 Gd mM -1 compared to 5 s -1 Gd mM -1 for AGuIX ® . A similar trend was seen at 1.5 T with r1 increasing from 18.7 s -1 Gd mM -1 for AGuIX ® to 23.7, 20.1, 30.2 s -1 Gd mM -1 for AGuIX-Bi 70Gd/30Bi, 50Gd/50Bi, and 30Gd/70Bi respectively. Similar to previous literature for Gd 3+ compounds, r1 decreased with increasing magnetic field strength. At higher field strengths, the paramagnetic property of a contrast agent is enhanced leading to stronger T2-related signal decay and lower r1 relaxivity [START_REF] Hagberg | Effect of r1 and r2 relaxivity of gadolinium-based contrast agents on the T1-weighted MR signal at increasing magnetic field strengths[END_REF]. Consequently, for clinical MR-Linac systems operating at even lower magnetic fields of 0.35 -1 T, there is potential for further improved contrast for Gd 3+ compounds such as AGuIX-Bi compared to the 7 T and 1.5 T presented. A recent study comparing Gd 3+ concentration per nanoparticle and r1 relaxivity demonstrated that at low Gd 3+ concentrations, r1 relaxivity increased until a maximum was reached. Further addition of Gd 3+ caused a quenching effect with decreasing r1 relaxivity relative to concentration [START_REF] Marangoni | Enhancing T1 magnetic resonance imaging contrast with internalized gadolinium (III) in a multilayer nanoparticle[END_REF]. This quenching effect was attributed to the packing of Gd 3+ into a limited volume, potentially restricting access of H2O molecules to the Gd 3+ coordination sphere. Similar quenching effects have been observed in other Gd 3+ -based nanoparticle systems [START_REF] Yong | Multifunctional nanoparticles as biocompatible targeted probes for human cancer diagnosis and therapy[END_REF][START_REF] Xu | Increasing surface area of silica nanoparticles with a rough surface[END_REF][START_REF] Tóth | The role of water exchange in attaining maximum relaxivities for dendrimeric MRI contrast agents[END_REF] and may explain the increase in relaxivity as the ratio of gadolinium within AGuIX-Bi was decreased thus reducing packing. Studies have also suggested that increased Gd 3+ payload may lead to disproportionately increased T2 effects which could negatively impact r1 relaxivity. The increased T2 contribution is primarily attributed to Gd 3+ geometric confinement which increases dipolar interactions between neighboring gadolinium ions [START_REF] Marangoni | Enhancing T1 magnetic resonance imaging contrast with internalized gadolinium (III) in a multilayer nanoparticle[END_REF][START_REF] Yong | Multifunctional nanoparticles as biocompatible targeted probes for human cancer diagnosis and therapy[END_REF][START_REF] Xu | Increasing surface area of silica nanoparticles with a rough surface[END_REF][START_REF] Tóth | The role of water exchange in attaining maximum relaxivities for dendrimeric MRI contrast agents[END_REF][START_REF] Liberman | Synthesis and surface functionalization of silica nanoparticles for nanomedicine[END_REF][START_REF] Barisik | Size dependent surface charge properties of silica nanoparticles[END_REF]. We observed a decrease in the r1/r2 ratios as the concentration of Gd 3+ increased suggesting a disproportional increase in r2 relaxivity contribution. To confirm retention of MRI signal and compare differences in Gd/Bi ratios in clinically relevant MRI systems, agar phantoms of varying nanoparticle concentrations were imaged in a 0.35T MRIdian MRI-Linac (Figure S12A) and 3T Siemens MR scanner (Figure S12B). As expected AGuIX ® maintained the strongest MRI signal followed by AGuIX-Bi (70Gd/30Bi), and equivalent signal for AGuIX-Bi (50Gd/50Bi) and AGuIX-Bi (30Gd/70Bi).

Overall minimal loss of MRI signal was seen with increasing ratios of Bi confirming strong contrast was maintained even at the highest Bi concentration in multiple clinical scanners.

T 7 T

Product Gd

(w%)

Bi

(w%)

D H (nm) pH IEP r 1 (s -1 Gd mM -1 ) r 2 (s -1 Gd mM -1 ) r 1 /r 2 r 1 (s -1 Gd mM -1 )
AGuIX

Biological characterization of AGuIX-Bi

Studies to assess toxicity and treatment efficacy were conducted in human NSCLC cells (A549), derived from a 58 y male epithelial carcinoma patient with a KRAS mutant and EGFR wild-type [START_REF] Swain | Assessment of cell line models of primary human cells by Raman spectral phenotyping[END_REF]. An MTT assay was used to assess mitochondrial metabolism as an indicator of nanoparticle toxicity and cellular viability. MTT reagent permeates live cell membranes and is reduced by oxidoreductase, dehydrogenase enzymes, and NAD(P)H (nicotinamide adenine dinucleotide phosphate) during different stages of the mitochondrial electron transport chain [START_REF] Ghasemi | The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis[END_REF]. When comparing the cellular metabolism of the various AGuIX-Bi formulations (Figure 2A), minimum toxicity was seen up to 3 mg mL -1 following 24 h post-incubation, similar to AGuIX ® (Figure S13). We calculated logIC50 values of 1.6, 1.5, 1.4, and 1.5 mg mL -1 for AGuIX ® , AGuIX-Bi (70Gd/30Bi), AGuIX-Bi (50Gd/50Bi), and AGuIX-Bi (30Gd/70Bi), respectively. Breaks in double-stranded DNA (dsDNA) following radiation were assayed using γH2AX, a well-established marker for double-strand breaks (DSBs); histone H2AX is phosphorylated by ATM (ataxia-telangiectasia mutated) at or near sites of DSBs to recruit repair machinery [START_REF] Martin | Involvement of H2AX in the DNA damage and repair response[END_REF][START_REF] Lee | Development of a high-throughput γ-H2AX assay based on imaging flow cytometry[END_REF][START_REF] Martinez-Pastor | Assessing kinetics and recruitment of DNA repair factors using high content screens[END_REF]. Quantification of DSBs is an indicator of nanoparticle radiation dose amplification and was evaluated for the different ratios of Gd/Bi nanoparticles (Figure 2B, Figure S14A) and AGuIX ® (Figure S14B). As the ratio of Bi within the formulation increased, the number of DSBs increased at 4 Gy and 6 Gy confirming the predicted impact of including a higher Z atom within the formulation. This correlation between damage and an increase in the Bi ratio was further confirmed with clonogenic assays comparing the three AGuIX-Bi formulations (Figure S15A). Given that increased Bi content amplified damage without impacting cellular viability, all following biological characterizations were conducted with AGuIX-Bi (30Gd/70Bi) NPs.

Clonogenic assays assessed the radiation dose amplification efficacy of AGuIX ® versus

AGuIX-Bi (30Gd/70Bi) confirming decreased viability with a sensitizer enhancement ratio (SER50%) of 1.28 for AGuIX-Bi (30Gd/70Bi) (Figure 2C, Figure S15B). Although SER values can be heavily impacted by various confounding factors including concentration and cellular uptake of sensitizer, tumor model, and radiation energy (keV vs MeV) and source (photons vs protons), an SER of 1.28 is comparable to other clinical and experimental radiosensitizers such as gemcitabine [START_REF] El Shafie | In vitro evaluation of photon and raster-scanned carbon ion radiotherapy in combination with gemcitabine in pancreatic cancer cell lines[END_REF][START_REF] Morgan | Improving gemcitabine-mediated radiosensitization using molecularly targeted therapy: a review[END_REF], hafnium oxide nanoparticles [START_REF] Maggiorella | Nanoscale radiotherapy with hafnium oxide nanoparticles[END_REF], and gold nanoparticles [START_REF] Sung | Dependence of gold nanoparticle radiosensitization on cell geometry[END_REF][START_REF] Penninckx | Gold nanoparticles as a potent radiosensitizer: A transdisciplinary approach from physics to patient[END_REF]. Further comparison of AGuIX ® and AGuIX-Bi (30Gd/70Bi) DNA damage was measured directly using the alkaline CometChip assay (Figure S16) following 6 Gy and 10 Gy radiation. Damage was quantified as the percentage of DNA in the comet tail region. Higher levels of basal DNA damage were observed in mock-irradiated cells treated with NPs, consistent with previous reports [START_REF] Sun | AGuIX nanoparticles enhance ionizing radiation-induced ferroptosis on tumor cells by targeting the NRF2-GPX4 signaling pathway[END_REF] (Figure 2D). At 10 Gy, a higher level of DNA damage was induced with AGuIX-Bi (30Gd/70Bi) compared with AGuIX ® . The alkaline CometChip assay thus corroborates the finding of enhanced radiation-induced damage with the addition of Bi to the nanoparticle formulation. To determine how NP and radiation treatment would impact cell death, we co-treated cells with either AGuIX ® or AGuIX-Bi (30Gd/70Bi) and ionizing radiation at 6 Gy and 10Gy to measure apoptosis induction after 24 and 48 hours. We found that AGuIX-Bi (30Gd/70Bi) at 10 Gy induced more apoptosis compared to AGuIX ® (Figure 2E). Also, we noticed cells became positive for annexin V (AxV), which binds to externalized phosphatidylserine in the early stages of apoptosis, before becoming positive for propidium iodide (PI), which only enters cells in the later stages of apoptosis when the plasma membrane is compromised [START_REF] Montero | Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy[END_REF] (Figure S17). This indicates that cells co-treated with these nanoparticles and ionizing radiation were undergoing apoptotic cell death. Interestingly when comparing DNA damage and clonogenic survival of AGuIX-Bi (30Gd/70Bi) to AGuIX ® at the same massic concentration, AGuIX-Bi (30Gd/70Bi) had more DNA damage and apoptosis induction even though the total molar metal content is 9.7% lower than AGuIX ® , further highlighting the impact of the higher atomic number of Bi on the photoelectric cross-section and subsequent therapeutic effect. No differences in nanoparticle cellular internalization were identified in NSCLC (Figure 2F and Figure S18) cells suggesting the decrease in cellular viability was due to the addition of high Z Bi rather than changes in nanoparticle uptake. AGuIX-Bi (30Gd/70Bi) relative injected dose within all organs including tumors was higher compared to AGuIX ® , likely due to increased blood circulation time. A Bi/Gd molar ratio of 2.3  0.56 was observed across all extracted tissues, aligning with the predicted theoretical value of 2.33, thus suggesting in vivo Gd/Bi co-localization of AGuIX-Bi (30Gd/70Bi) NPs (Figure S19). Further confirmation of in vivo stability needs to be considered in future translational work. A recent study [START_REF] Labied | Biodegradation of metal-based ultra-small nanoparticles: A combined approach using TDA-ICP-MS and CE-ICP-MS[END_REF] assessed the degradation of AGuIX  in PBS, human blood serum, and urine to determine if free gadolinium is released, which is considered harmful for patients [START_REF] Grobner | Gadolinium-a specific trigger for the development of nephrogenic fibrosing dermopathy and nephrogenic systemic fibrosis?[END_REF]. This study confirmed that the primary product of AGuIX  degradation is not free Gd but rather APTES-DOTAGA-Gd (APTES, (3-Aminopropyl)triethoxysilane), a chelated form of Gd. Given that AGuIX-Bi (30Gd/70Bi) is a derivative of the AGuIX  nanoparticles which have demonstrated success in phase I dose escalation studies and ongoing phase II clinical trials, we expect similar chelated degradation products that minimize risk to patients.

AGuIX ® had high tumor accumulation 1h post-injection followed by rapid clearance, while AGuIX-Bi (30Gd/70Bi) was retained within the tumor 24 h post-injection (Figure 3C). AGuIX ® had a shorter blood circulation time with rapid clearance between 1-4 h while AGuIX-Bi (30Gd/70Bi) had slower kinetics and high blood concentrations persisted 4 h post-injection with clearance finally observed at 24 h. Both AGuIX ® and AGuIX-Bi (30Gd/70Bi) were primarily eliminated via renal clearance (40 -60% ID g -1 ), however 10 -15% ID g -1 of AGuIX-Bi (30Gd/70Bi) cleared through the liver compared to only 2 -3% ID g -1 of AGuIX ® . Nanoparticles sub-5 nm can quickly clear via renal excretion as seen with AGuIX ® , however, a longer circulation time is associated with increased surface serum protein accumulation which enhances opsonization and phagocytosis via the mononuclear phagocyte system (MPS) associated with increased liver and splenic filtration [START_REF] Alexis | Factors affecting the clearance and biodistribution of polymeric nanoparticles[END_REF][START_REF] Di | Size, shape, charge and "stealthy" surface: Carrier properties affect the drug circulation time in vivo[END_REF]. AGuIX ® and AGuIX-Bi (30Gd/70Bi) MRI tumor accumulation and retention mirrored ICP-MS biodistribution results with AGuIX ® peaking 30 mins post-injection and AGuIX-Bi (30Gd/70Bi) peaking at 30 mins and sustaining accumulation for 24 h (Figure 3D).

AGuIX ® accumulation correlated with previously reported work in orthotopic lung, pancreatic, hepatic, and brain cancer models supporting EPR-based preferential accumulation [START_REF] Bort | EPR-mediated tumor targeting using ultrasmall-hybrid nanoparticles: From animal to human with theranostic AGuIX nanoparticles[END_REF][START_REF] Bianchi | Targeting and in vivo imaging of non-small-cell lung cancer using nebulized multimodal contrast agents[END_REF][START_REF] Dufort | Nebulized gadolinium-based nanoparticles: a theranostic approach for lung tumor imaging and radiosensitization[END_REF].

Although there is a difference in peak contrast enhancement for AGuIX ® compared to AGuIX-Bi (30Gd/70Bi) at 30 mins post-injection, this variance can be attributed to the difference in total Gd content in each NP (Figure 3E, Figure S20), specifically, AGuIX ® has approximately 3-fold more Gd per nanoparticle compared to AGuIX-Bi (30Gd/70Bi). Even at the lower Gd content, sufficient T1-weighted contrast was present to identify the tumor compared to surrounding healthy tissue for AGuIX-Bi (30Gd/70Bi). To further explore the differences in biodistribution, Taylor Dispersion Analysis (TDA) ICP-MS was used to better characterize the mean hydrodynamic radii of AGuIX-Bi (30Gd/70Bi) nanoparticles (Figure S21). Although DLS is a common technique to determine the hydrodynamic diameter of nanoparticles, the scattered light is proportional to the sixth power of the radius, thus often strongly biasing the results towards a larger particulate presence [START_REF] Hawe | Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates[END_REF].

Compared to DLS, TDA can better resolve smaller species and is not biased by aggregates or any larger species that may be present. Previous work using TDA-ICP-MS confirmed that the average size of AGuIX ® nanoparticles is 5.6  0.2 nm (87  5%) [START_REF] Labied | Biodegradation of metal-based ultra-small nanoparticles: A combined approach using TDA-ICP-MS and CE-ICP-MS[END_REF]. The same protocol was applied to AGuIX-Bi (30Gd/70Bi) confirming an increase in the overall size with a diameter of 7.1  0.3 nm (86  1%). This increase in size may have contributed to the longer blood circulation, tumor retention, and difference in clearance of the AGuIX-Bi (30Gd/70Bi) compared to AGuIX ® . We predict this increase in size may be due to the preferential elimination of smaller nanoparticles during the acidic Gd release and subsequent purification steps, however further studies will be necessary to confirm this hypothesis. It should be noted that in both sets of data, we see a second population of smaller diameter particles (1.2  0.7 nm, 13% AGuIX ® and 1.7  0.1 nm, 14%, AGuIX-Bi (30Gd/70Bi)), which may correspond to nanoparticle fragments due to the biodegradability of the nanoparticle in a highly diluted medium [START_REF] Sancey | Long-term in vivo clearance of gadolinium-based AGuIX nanoparticles and their biocompatibility after systemic injection[END_REF]. Based on ongoing clinical trials with AGuIX ® , this smaller population does not seem to impact overall efficacy or toxicity and is eliminated first and rapidly as confirmed by extensive animal studies on AGuIX  NPs [START_REF] Sancey | Long-term in vivo clearance of gadolinium-based AGuIX nanoparticles and their biocompatibility after systemic injection[END_REF].

AGuIX-Bi toxicity and therapeutic efficacy

Short-term and long-term toxicity of AGuIX-Bi (30Gd/70Bi) was assessed via histopathology and serum toxicology. Short-term (Figure 4A & S22-S23) and long-term (Figure S24) toxicity were evaluated 24 h and 1 mo post-treatment, respectively. No evidence of toxicity was identified in any treatments, which was further confirmed by the evaluation of histopathology slides by a trained pathologist. Differences in histology, cell size, and color intensities among the different treatments were related to differences in tissue processing and fixation. Serum toxicity was evaluated 24 h post-radiation therapy for blood urea nitrogen (BUN), an indicator of kidney dysfunction since primary clearance of AGuIX-Bi (30Gd/70Bi) occurs via the kidney (Figure 4B). Evaluation of Alanine aminotransferase (ALT), Aspartate transaminase (AST), and Gamma-Glutamyl transferase (GGT) indicated no liver damage in treated mice. Compared to AGuIX ® , AGuIX-Bi (30Gd/70Bi) had a significantly higher clearance of NPs via the liver, but no additional damage was identified. The systemic inflammatory response was assayed via Lactate dehydrogenase (LDH) to assess the impact of injecting foreign nanoformulations, however, no indication of damage was determined. Due to the difference in metabolic rates of mice versus humans, a direct comparison of dose and pharmacokinetics cannot be accurately measured [START_REF] Huh | Interspecies scaling and prediction of human clearance: comparison of small-and macro-molecule drugs[END_REF], however the dose of 300 mg kg -1 used in this study was three times higher than the clinically safe dose of 100 mg kg - 1 used in the NANO-RAD (NCT02820454) phase I dose escalation clinical trial for AGuIX ® [START_REF] Verry | Theranostic AGuIX nanoparticles as radiosensitizer: A phase I, dose-escalation study in patients with multiple brain metastases (NANO-RAD trial)[END_REF] and comparable to AGuIX ® dose used in other animal models [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Bort | EPR-mediated tumor targeting using ultrasmall-hybrid nanoparticles: From animal to human with theranostic AGuIX nanoparticles[END_REF][START_REF] Dufort | The high radiosensitizing efficiency of a trace of gadolinium-based nanoparticles in tumors[END_REF] showing no signs of toxicity. Therapeutic studies with radiation dose effect amplification were performed at the optimum time when NP tumor accumulation was high relative to healthy tissue, taking into account the substantial difference in biodistribution and pharmacokinetics for each NP. Optimum AGuIX-Bi (30Gd/70Bi) nanoparticle uptake occurred starting at 30 mins and was maintained for 24 h, however, nanoparticle blood concentration also remained high for the first 1-4 h with clearance at 24 h. To maximize tumor nanoparticle-mediated dose amplification and minimize non-specific toxicity, radiation was delivered 24 h post-AGuIX-Bi (30Gd/70Bi) injection. In contrast, optimum AGuIX ® uptake occurred between 30 mins -1 h with rapid tumor clearance as early as 1 h postinjection, similar to previously established literature in various murine tumor models [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Bort | EPR-mediated tumor targeting using ultrasmall-hybrid nanoparticles: From animal to human with theranostic AGuIX nanoparticles[END_REF][START_REF] Dufort | The high radiosensitizing efficiency of a trace of gadolinium-based nanoparticles in tumors[END_REF][START_REF] Du | Radiosensitization effect of AGuIX, a gadolinium-based nanoparticle, in nonsmall cell lung cancer[END_REF]. In the NANO-RAD (NCT02820454) phase I clinical trial, AGuIX ® plasma half-life was determined to be 1.3 h (range 0.8-3 h) [START_REF] Verry | Theranostic AGuIX nanoparticles as radiosensitizer: A phase I, dose-escalation study in patients with multiple brain metastases (NANO-RAD trial)[END_REF]. To compare the efficacy of AGuIX ® at peak tumor accumulation and mirror previous literature treatment plans [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Bort | EPR-mediated tumor targeting using ultrasmall-hybrid nanoparticles: From animal to human with theranostic AGuIX nanoparticles[END_REF][START_REF] Dufort | The high radiosensitizing efficiency of a trace of gadolinium-based nanoparticles in tumors[END_REF][START_REF] Du | Radiosensitization effect of AGuIX, a gadolinium-based nanoparticle, in nonsmall cell lung cancer[END_REF], radiation was delivered 30 mins post-injection. Mice treated with AGuIX-Bi (30Gd/70Bi) and radiation therapy had significantly delayed tumor growth (Figure 5A) with 33% complete regression (Figure 5B). In clinical trials, radiation is delivered 4 h post-AGuIX ® injection allowing sufficient time for peak tumor accumulation (plasma half-life 1.3 h, range 0.8-3 h) while minimizing the impact on healthy tissues [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF][START_REF] Verry | Theranostic AGuIX nanoparticles as radiosensitizer: A phase I, dose-escalation study in patients with multiple brain metastases (NANO-RAD trial)[END_REF]. However, counterintuitively a recent study in a glioblastoma rat model compared radiation 1 h and 24 h post-AGuIX ® injection and found better efficacy at 24 h even though the total AGuIX ® content within the tumor was 36x lower compared to 1 h. It was suggested that rapid clearance of the bulk of the nanoparticles between 1 -24 h indicated weak interactions between the cancerous cells and the NPs, however AGuIX ® that remained within the tumor at 24

Mice treated with

h were allowed sufficient time to interact with the cells and adhere to the surface or even be internalized [START_REF] Dufort | The high radiosensitizing efficiency of a trace of gadolinium-based nanoparticles in tumors[END_REF]. As previously stated, secondary photoelectric and Auger electrons have a short tissue interaction distance [START_REF] Mesbahi | A review on gold nanoparticles radiosensitization effect in radiation therapy of cancer[END_REF] suggesting that smaller distances from cells and specifically cellular nuclei may sustain a stronger radiosensitization impact at even lower doses. The longer tumor retention of high concentrations of AGuIX-Bi (30Gd/70Bi) suggests the possibility of greater clinical impact due to the potential for stronger interactions of nanoparticles and the surrounding cancerous tissue.

Conclusion

The idea of heavy metal nanoparticles as radiation dose amplifiers has been explored preclinically for over four decades with little advancement due to challenges in scalable cGMP formulation processes, desirable pharmacokinetics, and concerns of non-specific toxicity. In this work, we have developed a formulation methodology to synthesize next-generation theranostic bismuthgadolinium nanoparticles using cGMP AGuIX ® , which are currently in clinical trials, as a tunable chelation platform for ease of clinically scalable production. In a NSCLC model, we have demonstrated the impact of adding higher Z Bi 3+ to the nanoformulation leading to increased levels of DNA damage and decreased cellular viability. In vivo, we observed no short-term or long-term toxicity associated with the nanoformulation and improved therapeutic efficacy with 33% complete regression while maintaining MR contrast. Given the promise of AGuIX ® in phase I and ongoing phase II clinical trials, these preliminary studies synthesizing and validating the theranostic application of AGuIX-Bi (30Gd/70Bi) are necessary to lay the foundation for the translation of these next-generation nanoparticles.

Experimental Section/Methods

Chemical materials

The precursor NPs AGuIX ® were provided as a lyophilized powder by NH TherAguix (Grenoble, France). A 2 M HCl solution was created by diluting HCl (extra pure, 37%, CarlRoth). A 1 M NaOH solution was created from NaOH pellets (Fischer Chemical). The Cu 2+ titration solution was created by dissolving CuCl2 (powder, 99%, Sigma Aldrich, France) in pH 2 aqueous solution. BiCl3

and GdCl3(6H2O) used for chelation were purchased at reagent grade (>98%, Sigma Aldrich).

HNO3 (69%, supra grade) used for organ digestion was purchased from CarlRoth. Acetonitrile (CH3CN, ACN, > 99.9%) from Sigma Aldrich (France) and trifluoroacetic acid (TFA) from Fischer Chemical (USA) were used for the HPLC-UV phase preparation. TRIS, NaCl, and HCl obtained from Sigma were used for the preparation of the mobilization solution in TDA-ICP-MS.

AGuIX ® and AGuIX-Bi synthesis

The AGuIX-Bi NPs have been synthesized starting from the original AGuIX ® NPs. The synthesis has been performed in 3 steps. The first step consists of Gd 3+ release from the initial AGuIX ® NPs to obtain free DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl) pentanedioic acid) ready to chelate new metal ions. The second step focuses on the Bi 3+ complexation of newly regenerated DOTAGA groups. The last step is the final Gd 3+ complexation to obtain the desired products with three different Gd/Bi molar ratios respectively 70Gd/30Bi, 50Gd/50Bi, and 30Gd/70Bi.

Step 1 -Gd 3+ release AGuIX ® NPs were dispersed in 200 g L -1 in Milli-Q H2O (ρ > 18 M Ω cm -1 ). The AGuIX ® solution was combined with a HCl (2 M) solution 30 mins after dispersion leading to a final composition of 1 M HCl and 100 g L -1 AGuIX ® , which was left stirring at 50°C. The mixture was sampled to assess the Gd 3+ release by HPLC-UV and ICP-MS. The amount of released Gd 3+ was compared to a standard Gd 3+ reference corresponding to the initial Gd 3+ content. As different Gd/Bi ratio NPs will be synthesized (70Gd/30Bi, 50Gd/50Bi, 30Gd/70Bi), Gd 3+ release is pushed to a sufficient level to produce a product ready to create the 3 different final ratios. In our case, the release was pushed to at least 70% to obtain the product 30Gd/70Bi. After 19 h of reaction, the released level was reached. The mix was isolated and the free Gd 3+ was removed via purification with a Vivaflow to ensure the complexation of any possible free Gd 3+ remaining in the medium. Then a measure of the free DOTAGA amount through Cu 2+ titration was performed to confirm the presence of sufficient amounts of free DOTAGA and preparation of the following chelation steps.

Step 2 -Bi 3+ chelation

The bismuth complexation is challenged by the low solubility product of Bi (Ks (BiOH3) = 3.2 x 10 -40 ) [START_REF] Martell | Critical Stability Constants: First Supplement[END_REF] and fast precipitation into BiOH3, resulting in a white solution (Figure S25). To ensure a good complexation, this part was divided into 3 steps. In the first step, 30% of the total DOTAGA amount was chelated. It was followed by two 20% complexation steps to reach 70% of DOTAGA chelated with Bi 3+ . After each Bi 3+ complexation step, a part of the mixture was isolated to carry out Gd 3+ complexation and obtain the corresponding final product with the expected Gd/Bi ratio.

This second step ended with 3 different products having different Gd/Bi ratios equal to 20Gd/30Bi, 20Gd/50Bi, and 20Gd/70Bi.

During a complexation step, the required amount of BiCl3 was added to the mixture, then the pH was adjusted to 6-7 and the mixture was placed at 80°C under stirring. The mixture was left at 80°C for two days with regular pH readjustment to 6-7. After two days, the initial precipitated white solution was clear (Figure S25) and the pH was stable. To ensure the end of the complexation step, a sample of the mix was extracted to perform a DOTAGA titration.

Step 3 -Gd 3+ complexation Each batch with their respective amount of Bi (30%, 50%, 70%) followed a Gd 3+ complexation step to reach the final expected Gd/Bi proportion in the particle: 70Gd/30Bi, 50Gd/50Bi and 30Gd/70Bi. The process was the same as for step 2, except that the right amount of GdCl3(6H2O) was added to the mixture taking into account the already chelated Gd. In the end, each solution was purified with the Vivaflow 200 ultrafiltration system to remove the excess chloride and sodium ions. Finally, the solution was sterile filtered through a 0.2 µm filter, removing any large impurities.

NPs were freeze-dried using the Christ-Alpha 1-2 lyophilizer and stored.

HPLC-ICP/MS to follow gadolinium release

Throughout the process, reaction mixture samples were analyzed to follow the Gd 3+ release.

Analysis was performed with a Perkin -Elmer Nexion 2000B combined with a Perkin-Elmer Flexar LC. Separation was accomplished by a C4 reverse phase HPLC column (Jupiter®, 5 µm, 300 A, 150 x 4.6 mm) and measurements were conducted in isocratic mode using the phase composition H2O / ACN / TFA (98.9 / 1 / 0.1% v) at 1 mL min -1 speed flow. Gd signal was monitored using 152 Gd. The operating conditions for the ICP-MS were as follows: auxiliary gas flow rate = 1.2 L min -1 ; plasma gas flow rate =15 L min -1 ; nebulizer gas flow rate = 0.84 L min -1 ;

RF power = 1600 W for the plasma. The remaining parameters were tuned for maximization of the Gd signal. Syngistix software (version 2.3) controlled the ICP-MS and Empower (version 7.3) was used to acquire the Gd signal.

Measurement of gadolinium and bismuth in the final product by ICP-AES

The measure of Gd and Bi amount in the final products was conducted by Crealins laboratory from the 6NAPSE group (ISA, Lyon, France). The measurements were performed through ICP-AES iCAP 65000 Duo device (Thermo Scientific).

HPLC-UV

Gradient HPLC analysis was performed using the Shimadzu Prominence series UFLC system with a LC-20 AD liquid chromatograph, a CBM-20A controller bus module, a CTO-20A column oven, and an SPD-20A UV-visible detector. UV-visible absorption was measured at 295 nm. Separation was performed at a flow rate of 1 mL min -1 using C4 reverse phase HPLC column (Jupiter®, 5 µm, 300 A, 150 x 4.6 mm). The gradient initial solution was 95% solvent A -5% solvent B (A = H2O / ACN / TFA: 98.9 / 1 / 0.1% v, B = ACN / H2O / TFA: 89.9 / 10 / 0.1% v) over 5 min. During the next step, samples were eluted over 15 mins by a gradient developed from 5 to 90% of solvent B in solvent A. The concentration of solvent B was maintained over 5 min and then the concentration of solvent B was decreased to 5% over a period of 5 min to re-equilibrate the system, followed by an additional 5 min at this final concentration.

Free DOTAGA measurement

Measurement of the amount of free DOTAGA groups was performed at different steps of the process either to evaluate the initial amount or to follow the complexation steps. Samples with increasing amounts of copper (Cu 2+ ) were prepared in acetate buffer (pH 4.5) and left to react for 30 min before being analyzed by HPLC-UV (method above). Free DOTAGA groups were measured by recording the increase in absorbance at 295 nm due to the formation of DOTAGA@(Cu 2+ ) during titration. The amount of uncomplexed DOTAGA was directly related to the breaks in the slope of the absorbance graph.

ζ-potential and dynamic light scattering (DLS) measurements

NPs were diluted in an aqueous solution containing 0.01 M NaCl to 10 g L -1 and size distribution along with ζ-potential was measured with a Zetasizer NanoS DLS (Dynamic Light Scattering, laser He-Ne 633 nm, Malvern Instrument).

Relaxivity Measurements

Relaxivity measurements were performed on a Bruker Minispec mq60 NMR analyzer (Bruker, USA) at 37°C, 1.5 T (60 MHz) with NPs at a concentration of 100 or 50 g L -1 .

Agar phantoms clinical MRI

Agar phantoms (1%) of various concentrations of AGuIX  or AGuIX-Bi (70Gd/30Bi, 50Gd/50Bi, or 30Gd/70Bi) were prepared by dissolving noble agar (Sigma-Aldrich) in boiling deionized water.

While still liquid, NPs were added to 1 mL of agar solution in microvials and allowed to cool and solidify creating phantoms with concentrations of 0.0039 -2 mg mL -1 NPs. Phantoms were imaged using a T1-weighted clinical sequence on the 0.35T MRIdian MRI-Linac and 3T Siemens MR.

Images were quantified using 3D Slicer.

Cell culture studies

Human non-small cell lung cancer cells (A549, ATCC CCL-185, Manassas, VA) were cultured in 10% fetal bovine serum (FBS; Invitrogen, USA) and 1% pen/strep (10,000 U mL -1 penicillin; 10,000 µg mL -1 streptomycin; Invitrogen, US) supplemented Roswell Park Memorial Institute medium (RPMI 1640; Gibco, Invitrogen, USA) at 37°C, 5% CO2 and optimal humidity.

MTT cell viability assay

A549 cells (10,000 cells well -1 ) were seeded in a 96-well plate and allowed to grow for 24 h.

Varying concentrations of each formulation (0 -25 mg mL -1 ) of AGuIX-Bi or AGuIX ® NPs were diluted in 100 µL culture media and incubated with cells for 24 h. Cells were then washed and 100 µL of 10% MTT solution (5 mg mL -1 ) was added and incubated for 4 h at 37°C. MTT crystals were dissolved in 100 µL of DMSO and absorbance was quantified at 570 nm.

Clonogenic assay

A549 cells were seeded at 250 -1000 cells per well and incubated with 1 mg mL -1 AGuIX ® or AGuIX-Bi (70Gd/30Bi, 50Gd/50Bi, or 30Gd/70Bi) for 24 h. Following incubation, cells were irradiated at 2, 4, and 6 Gy using a 220 kVp Small Animal Research Radiation Platform (SARRP, Xstrahl, Suwanee, GA) at a dose rate of 4 Gy per minute. The cells were allowed to grow for approximately 7 -10 days or until control well colonies reached 50 cells per colony. Cells were fixed with 10% neutral buffered formalin overnight and stained with 1% crystal violet overnight.

Quantification was conducted using an in-house ImageJ (v 1.52p) colony counter macro. The sensitizer enhancement ratio (SER) was calculated as the ratio of doses at 50% survival fraction with and without nanoparticles (𝑆𝐸𝑅 50% = 𝐷 𝑁𝑃+𝑅𝑎𝑑

𝐷 𝑅𝑎𝑑

).

γH2AX assay

Cells were seeded at a density of 10,000 cells on microscopy slides overnight. A549 cells were incubated with each nanoparticle formulation (1 mg mL -1 ) overnight followed by irradiation on the SARRP to 2, 4, and 6 Gy. One hour post-treatment, cells were fixed, blocked, and permeabilized with 10% FBS and 0.3% Triton X-100. Cells were stained with anti-γH2AX-AlexaFlour 488 overnight and then mounted with DAPI and Flouromount-G. Images were taken using a Zeiss AxioObserver microscope 100x and analyzed using an in-house ImageJ (v 1.52p) foci counter macro (Figure S26).

Alkaline CometChip assay

To quantitate the amount of DNA damage induced by AGuIX ® or AGuIX-Bi (30Gd/70Bi) NPs, alkaline CometChip assay was performed. A549 cells were treated with 1 mg mL -1 of AGuIX ® or AGuIX-Bi (30Gd/70Bi) along with untreated controls for 24 h. CometChip preparation and cell loading were performed as described previously [START_REF] Ge | CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed cells[END_REF][START_REF] Toprani | High-throughput screening platform for nanoparticle-mediated alterations of DNA repair capacity[END_REF]. In brief, 10,000 cells per 100 μL were loaded in each well of 96-well plates for 30 min at 37 o C and 5% CO2. After gravity settling, the chips were rinsed with 1X DPBS (Gibco) to wash excess cells. Chips were then overlaid with 1% (w/v) molten low-melting agarose (Gold Biotechnology) prepared in 1X DPBS and allowed to gelate for 2 min at room temperature. All chemical reagents used for CometChip assays were from Sigma or VWR unless otherwise stated. The chips were irradiated using a Small Animal Radiation Research Platform (SARRP, Xtrahl, Inc., Suwanee, GA) at 6 Gy or 10 Gy. A mock-irradiated chip was prepared and handled identically except without radiation exposure. Post irradiation, the chips were immediately submerged in ice-cold alkaline lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10 with 1% Triton X-100) and kept overnight at 4°C. Alkaline DNA unwinding was performed using alkaline buffer (0.3 M NaOH and 1 mM Na2EDTA in distilled water) at 4°C for 40 min followed by electrophoresis in the same DNA unwinding buffer at 4°C for 30 min at 1V cm -1 and a constant current of 300 mA. Chips were neutralized using 0.4 M Tris-HCl buffer (pH 7.5) and stained using 1X (0.01% (v/v) of 10,000X stock) SYBR gold nucleic acid gel stain (Invitrogen, cat.

No. S11494) for 10 min at room temperature. Fluorescence comet images were captured at 10X magnification using an inverted LED fluorescence motorized microscope (Zen 3.2 pro blue edition, Zeiss Apotome 2, Carl Zeiss microscopy, GmbH). At least 500 cells were scored for each condition using Trevigen comet analysis software (R&D systems, MN, USA).

Chemosensitivity assay

Cells were seeded at a density of 0.1 × 10 6 cells mL -1 in 96-well plates with 0.1 mL of culture medium. The cells were treated with the following conditions: 1 mg mL -1 AGulX ® , 1 mg mL -1 AGulX-Bi (30Gd/70Bi), and radiation at 6 Gy or 10 Gy for 24 and 48 hours. After the designated treatment period, the cells were stained with Alexa Fluor 488-conjugated annexin V (AxV) and propidium iodine (PI) in 10X Annexin V binding buffer (100 mM HEPES, 40 mM KCl, 1.4 M NaCl, 7.5 mM MgCl2, 25 mM CaCl2, 7.4) at a dilution of 1:500. The staining solution was then added to the cells at a 1:10 dilution, and the cells were incubated on ice for 20 minutes.

AxV/PI positivity was subsequently measured using an Attune NxT flow cytometer (Thermo Fisher).

Internalization study

Cells were seeded at a density of 50,000 cells on microscopy slides overnight. A549 cells were incubated with each Cy5.5 tagged NP formulation (1 mg mL -1 ) overnight. Cells were fixed, blocked, and permeabilized with 10% FBS and 0.3% Triton X-100. Cells were stained with Cellmask Orange Plasma membrane stain (ThermoFisher) and then mounted with DAPI and Flouromount-G. Images were taken using a Zeiss AxioObserver microscope 63x. . Mice were given retroorbital, intravenous injections of 300 mg kg -1 of either AGuIX ® or AGuIX-Bi (30Gd/70Bi) followed by 10 Gy radiation either 30 mins or 24 h post-injection, respectively (n = 6 -9). Control mice that did not receive nanoparticles were given saline. Mice were euthanized if tumor volume surpassed 10% of total animal body weight or if any other signs of distress were identified (ulcerations > 10 mm, lethargy, etc).

Animal tumor model and in vivo studies

ICP-MS biodistribution and pharmacokinetics

ICP-MS measurements were performed to confirm the presence and quantify each organ's Gd and Bi content at different times after retro-orbital intravenous administration. Mice were injected retroorbitally with 300 mg kg -1 (~6 mg animal -1 ) of AGuIX ® or AGuIX-Bi (30Gd/70Bi) and sacrificed at 1 h, 4 h, 24 h, and 7 days (n = 3 animals timepoint -1 ). Tumors, blood, and other vital organs

TDA-ICP-MS

Experiments were performed on a P/ACE MDQ capillary electrophoresis instrument coupled to an ICP-MS (Agilent 7700) for Gd and Bi detection. Following a previously established protocol [START_REF] Labied | Biodegradation of metal-based ultra-small nanoparticles: A combined approach using TDA-ICP-MS and CE-ICP-MS[END_REF],

TDA-ICP-MS experiments were conducted in 75 µm i.d. x 64 cm hydroxypropylcellulose modified capillaries. NP solutions (1 g L -1 ) were prepared just before analysis and hydrodynamically injected (0.3 psi, 3 s). Samples were mobilized by applying a mobilization pressure of 0.7 psi using TRIS 10 mM, NaCl 125 mM, pH 7.4. Detection was performed by ICP-MS at m/z= 158 et 209.

Assuming populations of different sizes in the sample, (e.g. small molecules and nano-objects), the taylorgram obtained by Taylor Dispersion Analysis can be assimilated to the sum of several Gaussian signals. Deconvolution and integration of the peak were performed using the Origin 8.5

software to obtain molecular diffusion coefficients and then mean hydrodynamic diameters.

SARRP radiation treatment planning and delivery

A Small Animal Radiation Research Platform (SARRP, Xtrahl, Inc., Suwanee, GA) was used to perform image-guided radiation studies in vitro and in vivo. The DFCI SARRP therapy beam was

initially measured with an ADCL-calibrated ion chamber, and the output was checked monthly.

Mice were anesthetized with a 1 -3% isoflurane mixture and an airflow of 1.5 L min -1 . A conebeam computed tomography (CBCT) image was acquired at 60 kVp (0.8 mA current with a 1 mm Al filter) to identify the tumor isocenter in MuriSlice. A single posterior-anterior 220 kVp (13 mA current with a 0.15 mm Cu filter) beam delivered a dose of 10 Gy to the isocenter with a 1 x 1 cm 2 collimated field. Dosimetry studies were carried out in the MuriPlan treatment planning software to determine the radiation dose distribution in the tumor (Figure S27).

Toxicology

Mice bearing A549 subcutaneous tumors were treated with either saline or 300 mg kg -1 AGuIX-Bi (30Gd/70Bi) followed by 10 Gy irradiation 24h post-injection. Tumors and other vital organs (liver, lungs, spleen, kidney, and heart) were collected either 24 h or 1 mo post-therapy and histologically stained (H&E). Liver and kidney sections were additionally stained for caspase-3 to evaluate damage. A pathologist assessed each sample to identify any short-or long-term toxicity effects.

Blood was collected and allowed to clot before collection of serum. Serum was analyzed for kidney (BUN), liver (ALT, AST, GGT), and systemic inflammatory damage (LDH) at the Beth Israel Deaconess Medical Preclinical Murine Pharma Core.

Magnetic resonance imaging

Statistical analysis

Results are reported as the mean  standard error of the mean (SEM) as indicated in figure legends.

All statistical analysis was performed with GraphPad Prism (version 8). A Log-rank

Figure 2 .

 2 Figure 2. A) In vitro cytotoxicity of AGuIX-Bi nanoformulations as a function of concentration

Figure 3 .

 3 Figure 3. Biodistribution performed by ICP-MS in three animals per time point after retroorbital,

Figure 4 .

 4 Figure 4. A) Histopathology of mice treated with either saline or AGuIX-Bi (30Gd/70Bi) and/or

  AGuIX-Bi (30Gd/70Bi) plus radiation had delayed mean tumor growth of 58 days compared to 22 and 18 days for AGuIX ® plus radiation and radiation-only groups, respectively (Figure5C). When combined with radiation therapy, both NPs maintained tumor growth delay up to day 27 post-treatment compared to the radiation-only group but eventually succumbed to exponential regrowth, indicating the potential need for repeat cycles of treatment to better mimic clinical treatment workflows that include administration of radiation therapy. Given that a single treatment dose had no impact on mouse weight indicating no systemic toxicity with either NP alone or in combination with radiation therapy (Figure5D), multiple cycles of NP and radiation can be an option for sustained tumor delay and eventual regression. In the NanoCOL Phase Ib (NCT03308604) clinical trial, AGuIX ® NPs were delivered two times during the 5 weeks tumor volume doubling relative to Day 0. Complete tumor regression was observed in 33% of mice treated with AGuIX-Bi (30Gd/70Bi) plus radiation. C) Mean time-to-tumor doubling shows that mice treated with AGuIX-Bi (30Gd/70Bi) plus radiation had significantly longer tumor delay compared to control groups. D) No change in mouse body weight was observed following any treatment indicating no systemic toxicity. (* p< 0.05, ** p<0.01, and *** p<0.001)

  200 ultrafiltration system (PES, molecular weight threshold = 5 kDa, Sartorius Stedim Biotech, Germany). The final solution pH was adjusted to 7.4 by NaOH (1 M) and stored overnight at 50°C

  Animal studies were approved by Dana Farber Cancer Institute (DFCI) Institutional Animal Care and Use Committee (IACUC, Protocol Number 14-032) and conducted in full compliance with the Association for the Assessment and Accreditation of Laboratory Animal Care, governmental and institutional regulations and principals outlined in the United States Public Health Service Guide. Female 4 -6 week athymic Fox1 nu mice were used (Charles River Laboratories, Wilmington, USA) weighing 16-20 g. Mice were fed ad libitum with standard food pellets and water. Mice were inoculated subcutaneously with 3 x 10 6 A549 cells in 100 µL -1 into the dorsolateral right flank and allowed to grow for 2 -3 weeks or until tumors reached 3 -4 mm in diameter. Tumor volume was determined with calipers using the formula: 𝑣𝑜𝑙𝑢𝑚𝑒 = 𝑙𝑒𝑛𝑔𝑡ℎ 𝑥 𝑤𝑖𝑑𝑡ℎ 2 2

(

  liver, kidney, spleen, lungs, and heart) were collected for inductively coupled plasma mass spectrometry (ICP-MS). Tumors and organs were dissected, weighed, and digested in HNO3 (69%) through microwave Multiwave 5000 (Anton Paar, Austria) and analyzed by ICP-MS Nexion 2000 B (Perkin-Elmer).

  Mantel-Cox test with the Bonferroni correction for multiple comparisons was used to assess median tumordoubling time. A two-way ANOVA with Tukey's multiple comparisons test was used to assess tumor growth and alkaline CometChip assay results. A one-way ANOVA was used to compare three or more means with Kruskal-Wallis nonparametric test (non-Gaussian population) for mean tumor doubling time and H2AX in vitro assay. All in vitro studies were conducted in triplicates. Differences of p < 0.05 were considered statistically significant.

Figure S6 :

 S6 Figure S6: A) Schematic representation of AGuIX-Bi (50Gd/50Bi) NP with detailed structure of

Figure S7 :

 S7 Figure S7: A) Schematic representation of AGuIX-Bi (30Gd/70Bi) NP with detailed structure of

Figure S8 :

 S8 Figure S8: A) Schematic representation of AGuIX ® NP with detailed structure of chelated

Figure S9 :

 S9 Figure S9: HPLC-UV chromatograms of AGuIX ® particle (10 µL, 100 g L -1 ) and the three final

Figure S10 :

 S10 Figure S10: A) HPLC-SEC-ICP-MS chromatograms of the three final Gd/Bi following Gd, Bi and

Figure S11 :

 S11 Figure S11: IEP evolution all along the process. The decrease from one step to another is related

Figure S12 :

 S12 Figure S12: Agar phantom MRI images and signal intensity quantification of AGuIX ® and various

Figure S13 :

 S13 Figure S13: In vitro cytotoxicity of AGuIX ® as a function of concentration 24 h post-incubation

Figure S14 :

 S14 Figure S14: A) Representative single cell nuclei (blue) showing increasing γH2AX foci (red dots)

Figure S15 :

 S15 Figure S15: A) Clonogenic assay comparing the impact of increasing Bi ratio on cell survival. B)

Figure S17 :

 S17 Figure S17: Representative flow cytometry data of cells positive for annexin V (AxV, orange),

Figure S18 :

 S18 Figure S18: Representative nanoparticle (red) internalization images of A549 cells (blue) treated

Figure S19 :

 S19 Figure S19: Raw ICP-MS nanomolar amount of A) gadolinium and B) bismuth in extracted tissue

Figure S20 :

 S20 Figure S20: Raw ICP-MS nanomolar amount of gadolinium in extracted tumor tissue.

Figure S21 .

 S21 Figure S21. A) Representative taylorgram and corresponding bimodal fit for gadolinium (left) and

Figure S22 :

 S22 Figure S22: Histopathology of mice treated with either saline or AGuIX-Bi (30Gd/70Bi) and/or

Figure S23 :

 S23 Figure S23: Caspase-3 staining of liver and kidney, which have the highest accumulation of

Figure S24 :

 S24 Figure S24: Histopathology of mice treated with either saline or AGuIX-Bi (30Gd/70Bi) and/or

Figure S25 :

 S25 Figure S25: A) Image of the solution after addition of the BiCl3. At high concentrations of BiCl3,

Figure S26 :

 S26 Figure S26: Representative example of ImageJ automated macro steps for H2AX foci

Figure S27 :

 S27 Figure S27: Representative SARRP treatment plan for a mouse given 10 Gy radiation. A cone-

Table 1 .

 1 Physical and chemical characteristics comparison of AGuIX

	®	11.7	-	4.6  1.6 7.15	18.7	30.3	1.62	5.0
	AGuIX-Bi (70Gd/30Bi)	7.54 4.24 4.5  1.6 7.11	23.7	41.4	1.75	-
	AGuIX-Bi (50Gd/50Bi)	5.38 6.84 5.1  1.9 7.11	20.1	36.0	1.79	-
	AGuIX-Bi (30Gd/70Bi)	3.16 9.97 4.8  2.0 7.11	30.2	56.7	1.88	7.0

® and AGuIX-Bi 70Gd/30Bi, 50Gd/50Bi and 30Gd/70Bi products.

Acknowledgments

We would like to thank Dr. Thomas Brichart for his help in creating the nanoparticle illustration through the Blender software, and his advice throughout the project. The research reported in this publication was partially funded by the CNRS (French National Centre for Scientific Research) IRP Radioboost. Research reported in this publication was supported by the National Cancer Institute of the National Institute of Health under award number R01CA240804. The research reported in this publication was partially supported by NCI award number 5R25CA174650.

Research reported in this publication was partially supported by pilot project grant from Harvard-NIEHS Center for Environmental Health (P30ES000002). We thank Dr. Bevin Engelward for sharing some of the materials needed to perform the CometChip assay. The content is solely the

of conventional radiation therapy (45 Gy, 25 sessions) and once again before two weeks of brachytherapy (15 Gy) for effective clinical impact in cervical cancer patients [START_REF] Lux | AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine[END_REF]. Nanoparticles were given at a massic concentration of 300 mg kg -1 followed by radiation 30 mins post-AGuIX ® or 24 h post-AGuIX-Bi (30Gd/70Bi) injection. Relative tumor growth for each group is presented until the day the first animal within that group dropped out. B) Time-to-tumor-doubling is indicated with endpoint failure defined as MRI experiments were conducted on a Bruker BioSpec 7T/30 cm USR horizontal bore Superconducting Magnet System (Bruker Corp., Billerica, MA) outfitted with the B-GA12S2 gradient and integrated with up to a 2 nd order room temperature shim system that provides a slew rate of 3440 T m -1 s -1 and a maximum gradient amplitude of 440 mT m -1 and. The Bruker 35 mm ID birdcage volume radiofrequency (RF) coil was utilized for RF excitation and receiving.

Experiments were performed with animals anesthetized at 1.5% isoflurane mixed in medical air and a flow rate of about 2 L min -1 . Body temperature was maintained using a warm air fan at 37°C. Animal respiration and temperature were regulated by the 1025T SAII (Sa Instruments Inc., Stony Brook, NY) monitoring and gating system. A pressure-transducer for respiratory gating was placed on the abdomen. Bruker Paravision 6.0.1 was used for MRI data acquisition, T1 data analysis, and T1 map generation. T1 weighted tumor images were obtained using a fast spin echo (RARE) sequence with fat suppression using the following parameters: TR = 650 ms, TE = 16 ms, FOV =

Supplementary Material

Supplementary Material is available online at Theranostics or from the author. responsibility of the authors and does not necessarily represent the official views of the National Institute of Health.

Conflicts of Interest