Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Summary

Here, we present a protocol to obtain ⁶⁸Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T₁)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Abstract

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with ⁶⁸Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl₃ and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 ± 1.1 nm and a hydrodynamic size of 7.5 ± 2.1 nm. Moreover, they have a high longitudinal relaxivity (r₁) value of 11.9 mM^-1·s^-1 and a modest transversal relaxivity value (r₂) of 22.9 mM^-1·s^-1, which results in a low r₂/r₁ ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a ⁶⁸GaCl₃ elution from a ⁶⁸Ge/⁶⁸Ga generator is added to the starting materials, a nano-radiotracer doped with ⁶⁸Ga is obtained. The product is obtained with a high radiolabeling yield (> 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Introduction

The combination of imaging techniques for medical purposes has triggered the quest for different methods to synthesize multimodal probes^1,2,3. Due to the sensitivity of positron emission tomography (PET) scanners and the spatial resolution of MRI, PET/MRI combinations seem to be one of the most attractive possibilities, providing anatomical and functional information at the same time⁴. In MRI, T₂-weighted sequences can be used, darkening the tissues in which they accumulate. T₁-weighted sequences may also be used, producing the brightening of the specific accumulation location⁵. Among them, positive contrast is often the most adequate option, as negative contrast makes it much harder to differentiate signal from endogenous hypointense areas, including those often presented by organs such as the lungs⁶. Traditionally, Gd-based molecular probes have been employed to obtain positive contrast. However, Gd-based contrast agents present a major drawback, namely their toxicity, which is critical in patients with renal problems^7,8,9. This has motivated research in the synthesis of biocompatible materials for their use as T₁ contrast agents. An interesting approach is the use of iron oxide nanoparticles (IONPs), with an extremely small core size, that provide positive contrast¹⁰. Due to this extremely small core (~2 nm), most of the Fe³⁺ ions are on the surface, with 5 unpaired electrons each. This increases longitudinal relaxation time (r₁) values and yields much lower transversal/longitudinal (r₂/r₁) ratios compared to traditional IONPs, producing the desired positive contrast¹¹.

To combine IONPs with a positron emitter for PET, there are two key issues to take into account: radioisotope election and nanoparticle radiolabeling. Regarding the first issue, ⁶⁸Ga is an alluring choice. It has a relatively short half-life (67.8 min). Its half-life is suitable for peptide labeling since it matches common peptide biodistribution times. Moreover, ⁶⁸Ga is produced in a generator, enabling the synthesis in bench modules and avoiding the need for a cyclotron nearby^12,13,14. In order to radiolabel the nanoparticle, surface-labeling radioisotope incorporation is the prevalent strategy. This can be done using a ligand that chelates ⁶⁸Ga or taking advantage of the affinity of the radiometal toward the surface of the nanoparticle. Most examples in the literature concerning IONPs use a chelator. There are examples of the use of heterocyclic ligands such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)¹⁵, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)^16,17, and 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)¹⁸, and the use of 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD), a tetradentate ligand ¹⁹. Madru et al.²⁰ developed a chelator-free strategy in 2014 to label IONPs using a chelator-free method used by another group posteriorly²¹.

However, major drawbacks of this approach include a high risk of in vivo transmetalation, low radiolabeling yields, and lengthy protocols unsuitable for short-lived isotopes^22,23,24. For this reason, Wong et al.²⁵ developed the first example of core-doped nanoparticles, managing to incorporate ⁶⁴Cu in the core of the IONPs in a 5-min synthesis using microwave technology.

Here, we describe a rapid and efficient procedure to incorporate the radionuclide into the core of the nanoparticle, eluding many of the drawbacks presented by traditional methods. For this purpose, we propose the use of a microwave-driven synthesis (MWS), which reduces reaction times considerably, increases yields, and enhances reproducibility, critically important parameters in IONP synthesis. The refined performance of MWS is due to dielectric heating: rapid sample heating as molecular dipoles try to align with the alternating electric field, being polar solvents and reagents more efficient for this type of synthesis. In addition, the use of citric acid as a surfactant, together with microwave technology, results in extremely small nanoparticles, producing a dual T₁-weighted MRI/PET²⁶ signal, herein denoted as ⁶⁸Ga Core-doped iron oxide nanoparticles (⁶⁸Ga-C-IONP).

The protocol combines the use of microwave technology, ⁶⁸GaCl₃ as positron emitter, iron chloride, sodium citrate, and hydrazine hydrate, resulting in dual T₁-weighted MRI/PET nanoparticulate material in hardly 20 min. Moreover, it yields consistent results over a range of ⁶⁸Ga activities (37 MBq, 111 MBq, 370 MBq, and 1110 MBq) with no significant effects on the main physicochemical properties of the nanoparticles. The reproducibility of the method using high ⁶⁸Ga activities extends the field of possible applications, including large animal models or human studies. In addition, there is a single purification step included in the method. In the process, any excess of free gallium, iron chloride, sodium citrate, and hydrazine hydrate are removed by gel filtration. Total free isotope elimination and the purity of the sample ensure no toxicity and enhance imaging resolution. In the past, we have already demonstrated the usefulness of this approach in targeted molecular imaging^27,28.

Protocol

1. Reagent Preparation

1. 0.05 M HCl
   1. Prepare 0.05 M HCl by adding 208 µL of 37% HCl to 50 mL of distilled water.
2. High-performance liquid chromatography eluent
   1. Prepare high-performance liquid chromatography (HPLC) eluent by dissolving 6.9 g of sodium dihydrogen phosphate monohydrate, 7.1 g of disodium hydrogen phosphate, 8.7 g of sodium chloride, and 0.7 g of sodium azide in 1 L of water. Mix well and check the pH. Pass the eluent through a 0.1-µm cutoff sterile filter and degas before use. Acceptance range: pH 6.2 - 7.0 (if not, adjust with NaOH [1 M] or HCl [5 M]).

2. Synthesis of Citrate-coated Iron Oxide Nanoparticles

1. Dissolve 75 mg of FeCl₃·6H₂O and 80 mg of citric acid trisodium salt dihydrate in 9 mL of water.
   NOTE: These quantities provide 12 mL of final purified nanoparticles ([Fe] ~1.4 mg·mL^-1). Quantities can be scaled down to obtain a final volume of 2.5 mL.
2. Put the mixture in the microwave-adapted flask.
3. Load a dynamic protocol in the microwave. Set the temperature to 120 °C, the time to 10 min, the pressure to 250 psi, and the power to 240 W.
4. Add 1 mL of hydrazine hydrate to the reaction.
   NOTE: Hydrazine hydrate starts iron reduction. Therefore, a change in the appearance of the solution, from light yellow to brown, is observed.
5. Start the microwave protocol.
6. Meanwhile, rinse a gel filtration desalting column with 20 mL of distilled water.
7. Once the protocol has finished, allow the flask to cool at room temperature.
8. Pipette 2.5 mL of the final mixture onto the column and discard the flow-through.
   NOTE: The microwave stops the protocol at 60 °C; the nanoparticles can be added directly to the gel filtration column at 60 °C.
9. Add 3 mL of distilled water to the column and collect the nanoparticles in a glass vial.
   NOTE: Nanoparticles can be stored at room temperature for 1 week. After this time, nanoparticle aggregation appears, increasing their hydrodynamic size.

3. Synthesis of ⁶⁸Ga Core-doped Iron Oxide Nanoparticles (⁶⁸Ga-C-IONP)

1. Put 75 mg of FeCl₃·6H₂O and 80 mg of citric acid trisodium salt dihydrate into the microwave-adapted flask.
2. Elute the ⁶⁸Ge/⁶⁸Ga generator using the recommended volume and concentration of HCl, according to the vendor (in our case, 4 mL of 0.05 M HCl). After the injection of that volume in the self-shielded generator, (4 mL of) ⁶⁸GaCl₃ is obtained, ready to use without further processing.
   NOTE: Follow the corresponding radioactivity safety measures for steps 3.2 - 3.12. ⁶⁸Ga is a positron and gamma emitter isotope. The use of the appropriate safety measures to avoid exposure to radiation by the operator is crucial. Researchers must follow an ALARA (as low as reasonably achievable) protocol using typical shielding and radionuclide-handling procedures. Moreover, the use of a ring, body badges, and a contamination detector is mandatory.
3. Add 4 mL of ⁶⁸GaCl₃ to the microwave-adapted flask. This volume can be smaller, depending on the generator activity and desired activity of final nanoparticles.
4. Pipette 5 mL of distilled water into the flask and mix well.
5. Load a dynamic protocol in the microwave. Set the temperature to 120 °C, the time to 10 min, the pressure to 250 psi, and the power to 240 W.
6. Add 1 mL of hydrazine hydrate to the reaction.
   NOTE: Hydrazine hydrate starts iron reduction. Therefore, a change in the appearance of the solution, from light yellow to brown, is observed.
7. Start the microwave protocol.
8. Meanwhile, rinse a gel filtration desalting column with 20 mL of distilled water.
9. Once the protocol has finished, allow the flask to cool at room temperature.
10. Pipette 2.5 mL of the final mixture onto the column and discard the flow-through.
    NOTE: The microwave stops the protocol at 60 °C; the nanoparticles can be directly added to the gel filtration column at 60 °C.
11. Add 3 mL of distilled water to the column and collect the nanoparticles in a glass vial.
12. Calculate radiolabeling efficiency using a NaI well-type detector. This parameter typically measures the activity of the ⁶⁸Ga incorporated in the reaction. After synthetic and purification processes, the activity of the purified sample is measured. Because of the short half-life of ⁶⁸Ga, the initial activity has to be corrected at time (t). Normalization with time follows the standard equation:
    N_T = N₀ · e^-λt
    Here,
    N_T: Counts at time (t)
    N₀: Counts at time (t) = 0
    λ: Decay constant
    t: Elapsed time
    
    NOTE: Radiolabeling efficiency should be between 90% - 95%.

4. Analysis of ⁶⁸Ga Core-doped Iron Oxide Nanoparticles (⁶⁸Ga-C-IONP)

1. Dynamic light scattering
   1. Use dynamic light scattering (DLS) to measure the hydrodynamic size of ⁶⁸Ga-C-IONP. Pipette 60 µL of the sample into a cuvette and perform three size measurements per sample. To ensure reproducibility, this should be repeated with several nanoparticle batches.
2. Colloidal stability
   1. Assess the colloidal stability of ⁶⁸Ga-C-IONP by measuring the hydrodynamic size of the sample after incubation in different buffers (PBS, saline, and mouse serum) for different times, ranging from 0 to 24 h. Incubate 500 µL of the sample in each buffer at 37 °C. At the selected times, take 60-µL aliquots and pipette them into DLS cuvettes to measure their hydrodynamic size.
3. Electron microscopy
   1. Analyze the core size of ⁶⁸Ga-C-IONP using transmission electron microscopy (TEM) and annular dark-field imaging (STEM-HAADF) (ref TEM protocol: NIST - NCL Joint Assay Protocol, PCC-X, Measuring the Size of Nanoparticles Using Transmission Electron Microscopy).
4. Gel filtration radio-chromatogram
   1. Fractionate the elution into 500-µL aliquots during the gel-filtration purification step and measure the radioactivity present in each one using an activimeter; thus, rendering a gel-filtration chromatogram.
5. Radiochemical stability of ⁶⁸Ga-C-IONP
   1. Incubate ⁶⁸Ga-C-IONP in mouse serum for 30 min at 37 °C (repeated 3x). After that time, purify the nanoparticles by ultrafiltration and measure the radioactivity present in the nanoparticles and filtrate. No activity should be detected in the different filtrates.
6. Relaxometry
   1. Measure longitudinal (T₁) and transverse (T₂) relaxation times in a relaxometer at 1.5 T and 37 °C. Four different concentrations of ⁶⁸Ga-C-IONP (2 mM, 1 mM, 0.5 mM, and 0.25 mM) should be measured. Plot relaxation rates (r₁=1/T₁, r₂=1/T₂) against iron concentration. The slope of the curve obtained renders r₁ and r₂ values.
7. MR and PET phantom images
   1. Acquire in situ MR (T₁-weighted sequence) and PET phantom images for a series of dilutions of ⁶⁸Ga-C-IONP (0 mM, 1 mM, 6.5 mM, and 9.0 mM) to observe the increasing signal in correlation with the PET activity and MRI.

Results

⁶⁸Ga-C-IONP were synthesized by combining FeCl₃, ⁶⁸GaCl₃, citric acid, water, and hydrazine hydrate. This mixture was introduced into the microwave for 10 min at 120 °C and 240 W under controlled pressure. Once the sample had cooled down to room temperature, the nanoparticles were purified by gel filtration to eliminate unreacted species (FeCl₃, citrate, hydrazine hydrate) and free ⁶⁸Ga (Figure 1

Discussion

Iron oxide nanoparticles are a well-established contrast agent for T₂-weighted MRI. However, due to the drawbacks of this type of contrast for the diagnosis of certain pathologies, T₁-weighted or bright contrast is many times preferred. The nanoparticles presented here not only overcome these limitations by offering positive contrast in MRI but also offer a signal in a functional imaging technique, such as PET, via ⁶⁸Ga incorporation in their core. Microwave technology enhances t...

Materials

Name	Company	Catalog Number	Comments
Iron (III) chloride hexahydrate	POCH	2317294
Citric acid, trisodium salt dihydrate 99%	Acros organics	227130010
Hydrazine hydrate	Aldrich	225819
Hydrochloric acid 37%	Fisher Scientific	10000180
Sodium dihydrogen phosphate monohydrate	Aldrich	S9638
Disodium phosphate dibasic	Aldrich	S7907
Sodium chloride	Aldrich	746398
Sodium Azide	Aldrich	S2002
Sodium dihydrogen phosphate anhydrous	POCH	799200119
68Ga Chloride	ITG Isotope Technologies Garching GmbH, Germany	68Ge/68Ga generator system
Microwave	Anton Paar	Monowave 300
Centrifuge	Hettich	Universal 320
Size Exclusion columns	GE Healthcare	PD-10