Summary

Background: Radiation nanomedicines are described here for glioblastoma multiforme (GBM) composed of gold nanoparticles (AuNPs) that integrate the Auger electron-emitter, ¹⁹⁷Hg. [¹⁹⁷Hg]Hg-AuNPs were conjugated to anti-epidermal growth factor receptor (EGFR) panitumumab or were non-targeted. The aim was to compare the cytotoxicity and DNA-damaging properties in vitro of panitumumab-[¹⁹⁷Hg]Hg-AuNPs and non-targeted [¹⁹⁷Hg]Hg-AuNPs on U251-Luc human GBM cells and estimate their cellular dosimetry. Additional aimwas to compare the biodistribution in vivo of panitumumab-[¹⁹⁷Hg]Hg-AuNPs and [¹⁹⁷Hg]Hg-AuNPs after convection-enhanced delivery (CED) in NRG mice with U251-Luc tumours in the brain and estimate the absorbed doses in the tumour and surrounding margins of healthy brain.

Results: [¹⁹⁷Hg]Hg-AuNPs (26.8 ± 6.4 nm) were produced with a radiochemical yield of 98 ± 1% by incorporating ¹⁹⁷Hg into the Turkevich synthesis method, forming a mercury-gold amalgam. Panitumumab-[¹⁹⁷Hg]Hg-AuNPs exhibited high affinity binding to EGFR-positive U251-Luc cells. The binding of panitumumab-[¹⁹⁷Hg]Hg-AuNPs to U251-Luc cells was 15-fold higher than [¹⁹⁷Hg]Hg-AuNPs, and internalization and nuclear uptake were 12-fold and 18-fold greater, respectively. Panitumumab-[¹⁹⁷Hg]Hg-AuNPs caused 84-fold more DNA double-strand breaks (DSBs) in U251-Luc cells than [¹⁹⁷Hg]Hg-AuNPs. Panitumumab-[¹⁹⁷Hg]Hg-AuNPs were ninefold more effective at reducing the clonogenic survival of U251-Luc cells than [¹⁹⁷Hg]Hg-AuNPs. Panitumumab-[¹⁹⁷Hg]Hg-AuNPs were twofold more cytotoxic than non-radioactive panitumumab-AuNPs and fivefold more cytotoxic than panitumumab. The absorbed doses in the nucleus of U251-Luc cells treated in vitro with panitumumab-[¹⁹⁷Hg]Hg-AuNPs or [¹⁹⁷Hg]Hg-AuNPs were 8.8 ± 2.9 Gy and 0.6 ± 0.1 Gy, respectively. SPECT/CT imaging showed that panitumumab-[¹⁹⁷Hg]Hg-AuNPs and [¹⁹⁷Hg]Hg-AuNPs were strongly retained at the infusion site in the brain after CED up to 7 d in NRG mice with orthotopic U251-Luc tumours. Uptake of panitumumab-[¹⁹⁷Hg]Hg-AuNPs in the tumour-bearing right hemisphere was 172-fold and 579-fold greater than in the healthy left hemisphere and cerebellum, respectively. The uptake of [¹⁹⁷Hg]Hg-AuNPs in the tumour-bearing right hemisphere was 85-fold and 64-fold higher than the left hemisphere and cerebellum, respectively. Most normal tissue uptake was < 1% ID/g, except for kidneys, spleen and liver. Dosimetry showed that 58% of the tumour received > 190 Gy for CED of 1.0 MBq of panitumumab-[¹⁹⁷Hg]Hg-AuNPs vs. 0.6% of the tumour for non-targeted [¹⁹⁷Hg]Hg-AuNPs, but both agents deposited > 50 Gy in 95% of the tumour. Doses decreased dramatically to 1.7 and 3.3 Gy at 1–3 mm from the tumour edge for panitumumab-[¹⁹⁷Hg]Hg-AuNPs and [¹⁹⁷Hg]Hg-AuNPs, respectively.

Conclusion: Radiation nanomedicines incorporating the AE-emitter, ¹⁹⁷Hg administered by CED are a promising approach to treatment of GBM. Panitumumab-[¹⁹⁷Hg]Hg-AuNPs are particularly attractive due to their EGFR-mediated binding, internalization and nuclear importation in GBM cells, which amplifies their in vitro cytotoxicity.

Results from nanoScan® SPECT/CT

• strong retention at the infusion site: SPECT/CT imaging demonstrated that both panitumumab-[¹⁹⁷Hg]Hg-AuNPs (EGFR-targeted) and non-targeted [¹⁹⁷Hg]Hg-AuNPs remained strongly localized at the convection-enhanced delivery (CED) site in the brains of NRG mice with orthotopic U251-Luc glioblastoma tumors up to 7 days post-infusion.
• Minimal redistribution elsewhere: The images showed very low activity outside the tumor infusion site — there was no detectable activity in other brain regions or major normal organs on SPECT/CT, indicating that the nanoparticles were largely confined locally after delivery.
• Greater diffusion of non-targeted particles: Closer examination of the SPECT/CT images revealed that non-targeted [¹⁹⁷Hg]Hg-AuNPs appeared to diffuse farther from the infusion site than the panitumumab-targeted nanoparticles, which remained more tightly localized, suggesting potential effects of target binding and cellular uptake on particle distribution.
• High uptake in tumor-bearing hemisphere: Quantitative biodistribution data derived in part from SPECT/CT corroborated that the tumor-bearing hemisphere had very high uptake of both targeted and non-targeted particles compared with the normal left hemisphere and cerebellum, with hundreds-fold higher % injected dose per gram in tumor tissue versus normal brain.

Figure 5. SPECT/CT images of NRG mice with orthotopic U251-Luc human GBM tumours in the right hemisphere of the brain administered a panitumumab-[197Hg]Hg-AuNPs or b non-targeted [197Hg]Hg-AuNPs by CED (arrows) at selected times up to 168 h post-infusion (p.i.). Intensity scales (MBq/mL) are shown for each timepoint post-infusion. Magnified view of the head showing differences in local diffusion from the infusion site in the brain for c panitumumab-[197Hg]Hg-AuNPs or d non-targeted [197Hg]Hg-AuNPs

Figure 6. Biodistribution of panitumumab-[¹⁹⁷Hg]Hg-AuNPs or non-targeted [¹⁹⁷Hg]Hg-AuNPs at 168 h p.i. The tumour could not be excised from the brain and was included in the right hemisphere tissue while the left hemisphere and cerebellum were non-tumour bearing. Values shown are the mean ± SD. There were no significant differences in the biodistribution of panitumumab-[¹⁹⁷Hg]Hg-AuNPs and non-targeted [¹⁹⁷Hg]Hg-AuNPs at this timepoint

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