64Cu‑Labeled AGuIX Nanoprobes: PET‑Guided Radiotherapy and Radiosensitization in HepG2 Tumor‑Bearing Mice

Background

Hepatocellular carcinoma (HCC) ranks among the world’s most common malignancies, with 782,500 new cases and 745,500 deaths reported in 2012, of which 70–90 % were HCC. Most patients present at an advanced stage, limiting curative options to 20–25 % of cases. Consequently, radiotherapy has become a key component of multidisciplinary HCC management. However, normal liver tissue limits dose escalation, as radiation‑induced liver disease (RILD) poses a severe risk. Radiosensitizers that preferentially accumulate in tumour tissue can increase tumour cell kill at lower doses, thereby mitigating normal‑tissue toxicity.

AGuIX, a novel gadolinium‑based nanoparticle (~5 nm diameter) first described in 2013, is rapidly renally cleared and can be functionalised with radioactive or fluorescent labels for SPECT, PET, MRI, or fluorescence imaging. The high gadolinium content endows AGuIX with tumour‑sensitising capabilities, demonstrated in vitro across diverse cell lines with sensitisation enhancement ratios (SER) ranging from 1.1 to 2.5. Because AGuIX shows low hepatic background yet high tumour uptake—thanks to the enhanced permeability and retention (EPR) effect—it is an attractive candidate for HCC radiosensitisation.

Despite extensive preclinical work on AGuIX for MRI‑guided radiotherapy, its pharmacokinetics remain incompletely characterised. Precise biodistribution data are essential to optimise dosing schedules and predict therapeutic windows. The positron emitter ⁶⁴Cu (T_1/2 = 12.4 h) offers the temporal flexibility to image both rapid and slow‑clearing agents. Here we report the first in vivo biodistribution and PET imaging of ⁶⁴Cu‑labeled AGuIX in HepG2 tumour‑bearing nude mice, and we evaluate its radiosensitising effect using ¹⁸F‑FDG PET/CT.

Methods

General Information

AGuIX nanoparticles (sub‑5 nm, dehydrated, spherical) were sourced from Nano‑H (Lyon, France) and rehydrated in sterile DEPC‑treated water. Human HCC HepG2 cells (ATCC) were cultured in MEM supplemented with 10 % FCS. The ⁶⁴Cu isotope was obtained from Wisconsin University. Male BALB/c athymic nude mice (6 weeks, 16–18 g) were used, with all protocols approved by the Institutional Animal Care and Use Committee at the University of Virginia.

Transmission Electron Microscopy (TEM)

AGuIX (0.5 mM) was incubated with HepG2 cells for 1 h, followed by washing with 0.1 M PBS and centrifugation. Cell pellets were fixed in 4 % formaldehyde and 1 % glutaraldehyde for TEM imaging.

⁶⁴Cu Radiolabeling

AGuIX (10 µmol) was mixed with 100 µl 0.5 M NH₄OAc (pH 5.5) and incubated for 5 min. 1–3 mCi of ⁶⁴CuCl₂ (0.1 N HCl) was added, and the mixture was heated at 37 °C for 1 h. The product was purified by 3 kDa Amicon filtration. Radiochemical purity was confirmed by iTLC (20 mM citric acid) and consistently exceeded 98 %. Specific activity ranged from 3–10 MBq µmol^–1, with final yields of 50–100 MBq per synthesis.

Tumor Models

HepG2 cells (5 × 10⁶) were resuspended in 0.1 ml HBSS and injected subcutaneously into the right flank of each nude mouse. Tumours became palpable after ~2 weeks.

Biodistribution of ⁶⁴Cu‑AGuIX

Ten mice (5 male, 4 female) received intraperitoneal injections of ~0.9 MBq ⁶⁴Cu‑AGuIX (0.2 ml). At 9, 21, and 40 h post‑injection, mice were euthanised, and organs (heart, muscle, lung, kidney, spleen, liver, tumour) were harvested, weighed, and counted in a γ‑counter. Data were expressed as % injected dose per gram (% ID/g).

Micro‑PET Imaging

Each tumour‑bearing mouse received 22.2 MBq ⁶⁴Cu‑AGuIX intraperitoneally. PET scans were acquired at 9 h and 21 h under isoflurane anaesthesia, using a SuperArgus system (Sedecal, Spain).

Irradiation and ¹⁸F‑FDG PET/CT

Twelve mice were divided into three groups (n = 4). Baseline PET imaging was performed 30 min after tail‑vein injection of 16.4 ± 4.7 MBq ¹⁸F‑FDG. Groups received either saline, 1 mg, or 10 mg AGuIX intraperitoneally 1 h before irradiation. Irradiation was delivered at 250 kV, 8 mA, 2‑mm Al filter (1.2 Gy min^–1) for a total of 6 Gy; the protocol was repeated the following day. 24 h after the second dose, a second ¹⁸F‑FDG PET scan (11.1 ± 1.0 MBq) was performed. Standardised uptake values (SUV_max) were measured in tumours and normalised to liver SUV_ave to obtain T/L ratios.

Statistical Analysis

All experiments were performed in triplicate. Data are presented as mean ± SE. Significance was assessed by two‑tailed unpaired t tests or one‑way ANOVA (p < 0.05 considered significant).

Results and Discussion

While contrast‑enhanced MRI can detect AGuIX, PET offers superior sensitivity and quantitative accuracy, enabling detection of nanomolar concentrations that are below MRI’s threshold. Here we demonstrate that ⁶⁴Cu‑labeled AGuIX provides clear tumour delineation and prolonged retention in HepG2 xenografts, supporting its use for PET‑guided radiotherapy.

TEM Study

Incubation of HepG2 cells with 0.5 mM AGuIX for 1 h resulted in cytoplasmic uptake, as visualised at ×6 500 and ×52 000 magnification (Figure 1). The nanoparticles maintained a well‑dispersed morphology, indicating cellular stability and compatibility.

Localization of AGuIX within HepG2 cells. a. TEM images (× 6500) depicting uptake. b. Magnified view (× 52000) showing cytoplasmic distribution.

Radiolabeling

One‑step chelation of ⁶⁴Cu to AGuIX via the built‑in DOTA achieved >98 % radiochemical yield. The resulting specific activity was 3–10 MBq µmol^–1, with final product activity of 50–100 MBq per synthesis.

Biodistribution Studies

After intraperitoneal injection, ⁶⁴Cu‑AGuIX exhibited robust tumour uptake (7.82 ± 1.50, 8.43 ± 6.23, 6.84 ± 1.40 % ID/g at 9, 21, and 40 h, respectively). Non‑target organs retained <1 % ID/g, confirming rapid clearance. Kidney uptake was notably lower (~5 % ID/g) than reported for intravenous administration, likely due to continuous peritoneal absorption. These data support the use of ⁶⁴Cu‑AGuIX as a quantitative tool to guide radiosensitiser dosing.

Biodistribution of ⁶⁴Cu‑AGuIX in tumour‑bearing mice (% ID/g at 9, 21, and 40 h). Mean ± SD, n = 3.

Micro‑PET Imaging

Clear tumour signal was observed at 9 h, with even higher contrast at 21 h as background activity declined (Figure 3). The data corroborate the biodistribution findings and demonstrate the practical feasibility of PET‑guided treatment planning.

Micro‑PET images of tumour‑bearing mice. Upper panel (coronal view) and lower panel (transverse view) at 9 h and 21 h post‑injection. Tumour indicated by red arrow.

¹⁸F‑FDG PET/CT Evaluation of Irradiated Xenografts

All irradiated mice displayed reduced tumour ¹⁸F‑FDG uptake. SUV_max ratios (B/A) were 1.03 ± 0.03 (saline), 1.04 ± 0.04 (1 mg AGuIX), and 1.24 ± 0.02 (10 mg AGuIX). The 10 mg group showed a statistically significant increase in tumour‑to‑liver uptake ratio compared to the 1 mg and saline groups (p < 0.001), confirming dose‑dependent radiosensitisation. No difference was observed between 1 mg AGuIX and saline, indicating that higher doses are required for clinical benefit.

Pre‑ and post‑irradiation ¹⁸F‑FDG PET images for saline (left), 1 mg AGuIX (middle), and 10 mg AGuIX (right). Images share identical colour scales.

Quantitative analysis: T/L (B) – tumour to liver SUV_ave before irradiation; T/L (A) – after irradiation; T/L (B/A) – ratio of the two. 1 mg and 10 mg AGuIX groups shown.

While renal uptake of radiolabelled AGuIX can be high after intravenous injection, intraperitoneal delivery mitigates this exposure, resulting in sustained tumour retention with minimal kidney exposure. Future dosimetry studies will clarify organ‑specific radiation tolerance and support clinical translation.

Conclusions

We have successfully radiolabelled AGuIX with ⁶⁴Cu, achieving >98 % purity and substantial tumour uptake that persists for >40 h in HepG2 xenografts. PET imaging demonstrates clear tumour delineation and provides a quantitative readout for treatment planning. Radiosensitisation studies using ¹⁸F‑FDG PET confirm that a 10 mg intraperitoneal dose of AGuIX enhances the therapeutic effect of 6 Gy radiotherapy in HCC models. These findings validate AGuIX as a dual‑function theranostic nanoprobe for image‑guided radiotherapy and highlight the need for comprehensive dosimetry before clinical application.