Lysosome-escaping and nucleus-targeting nanomedicine for enhanced cancer catalytic internal radiotherapy.

PURPOSE

Radio-nanomedicine, such as I-labelled TiO nanoparticles (I-TiO NPs), presents a promising tumour treatment approach. By leveraging I as an electron donor to activate TiO NPs and promote γ-ray-induced HO radiolysis, I-TiO generates reactive oxygen species (ROS) and induces DNA damage, thus enabling catalytic internal radiotherapy (CIR). Since DNA is a key target of radiation and ROS-induced damage, enhancing nuclear delivery is critical. However, lysosomal entrapment of I-TiO NPs greatly restricts the efficacy of CIR. To overcome this limitation, we conjugated I-TiO with transactivator of transcription/hemagglutinin-2 (I-TiO-TAT/HA2), hypothesising that TAT/HA2-mediated lysosomal escape and nuclear accumulation could improve the anti-tumour effects of I-TiO.

METHODS

TiO NPs and TiO-TAT/HA2 were synthesised and labelled with I. Subcellular localisation was observed by confocal microscopy and biological transmission electron microscopy (bio-TEM). The effects of I-TiO-TAT/HA2 on PANC-1 cells were assessed by the CCK-8 assay (cell viability), flow cytometry (apoptosis and ROS generation), proliferating cell nuclear antigen (PCNA) staining (proliferation), γ-H2AX immunofluorescence (DNA damage), and western blotting (DNA repair protein expression). In a subcutaneous pancreatic cancer mouse model, the intratumoural intra-tumoural retention of cyanine 5-labelled NPs (Cy5-NPs) was tracked via fluorescence imaging, whereas I-TiO-TAT/HA2 was monitored by single-photon emission computed tomography (SPECT). Mice received intra-tumoural injections of DMEM, I, I-TiO, or I-TiO-TAT/HA2, and tumour volume and mouse weight and survival were recorded. Tumour glycometabolism was evaluated using F-fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT) before and after treatment. Haematoxylin and eosin (H&E), TdT-mediated dUTP nick end labelling (TUNEL), and immunohistochemical staining (Ki-67, DNA repair proteins) were performed.

RESULTS

The labelling rates of I-TiO and I-TiO-TAT/HA2 averaged 89.0% and 90.1%, respectively. Confocal microscopy and bio-TEM confirmed the nucleus-targeting ability of TiO-TAT/HA2. In vitro, I-TiO-TAT/HA2 significantly increased apoptosis and DNA damage and reduced DNA repair protein expression (RAD51and 53BP1) versus I-TiO (all p < 0.05). I-TiO-TAT/HA2 additionally reduced PCNA expression and increased ROS production (both p > 0.05). In vivo, Cy5-NPs, I-TiO, and I-TiO-TAT/HA2 exhibited prolonged intra-tumoural retention. Tumours treated with I-TiO-TAT/HA2 displayed a significantly smaller volume, enhanced necrosis and apoptosis (H&E, TUNEL), and downregulated DNA repair protein expression (all p < 0.05) and reduced Ki-67 expression (p > 0.05) compared with the effects of I-TiO.

CONCLUSION

I-TiO-TAT/HA2 exhibited markedly enhanced efficacy, likely through the precise nuclear delivery of ROS rather than increased ROS production. This strategy presents a promising method to improve tumour therapy, and it could be adapted for other macromolecular therapeutics.