
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39300124
52607
10.1038/s41467-024-52607-9
Article
Full-course NIR-II imaging-navigated fractionated photodynamic therapy of bladder tumours with X-ray-activated nanotransducers
http://orcid.org/0000-0002-6323-0095
He Liangrui 1
Wang Liyang 2
http://orcid.org/0000-0002-8916-0742
Yu Xujiang yuxuj1017@sjtu.edu.cn

1
Tang Yizhang 1
Jiang Zhao 1
Yang Guoliang ygl0511@126.com

2
http://orcid.org/0000-0002-1629-1039
Liu Zhuang zliu@suda.edu.cn

3
http://orcid.org/0000-0003-3809-0737
Li Wanwan wwli@sjtu.edu.cn

1
1 grid.16821.3c 0000 0004 0368 8293 State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, PR China
2 grid.16821.3c 0000 0004 0368 8293 Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, PR China
3 https://ror.org/05t8y2r12 grid.263761.7 0000 0001 0198 0694 Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu PR China
19 9 2024
19 9 2024
2024
15 824015 1 2024
12 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The poor 5-year survival rate for bladder cancers is associated with the lack of efficient diagnostic and treatment techniques. Despite cystoscopy-assisted photomedicine and external radiation being promising modalities to supplement or replace surgery, they remain invasive or fail to provide real-time navigation. Here, we report non-invasive fractionated photodynamic therapy of bladder cancer with full-course real-time near-infrared-II imaging based on engineered X-ray-activated nanotransducers that contain lanthanide-doped nanoscintillators with concurrent emissions in visible and the second near-infrared regions and conjugated photosensitizers. Following intravesical instillation in mice with carcinogen-induced autochthonous bladder tumours, tumour-homing peptide-labelled nanotransducers realize enhanced tumour regression, robust recurrence inhibition, improved survival rates, and restored immune homeostasis under X-ray irradiation with accompanied near-infrared-II imaging. On-demand fractionated photodynamic therapy with customized doses is further achieved based on quantifiable near-infrared-II imaging signal-to-background ratios. Our study presents a promising non-invasive strategy to confront the current bladder cancer dilemma from diagnosis to treatment and prognosis.

The poor survival rate for bladder cancers is associated with the lack of effective non-invasive theranostic techniques. Here this group reports a lanthanide-doped nanotransducer activated for real-time NIR-II imaging thereby navigates the photodynamic treatment of bladder cancer.

Subject terms

Nanotechnology in cancer
Bladder cancer
Cancer imaging
https://doi.org/10.13039/501100001809 National Natural Science Foundation of China (National Science Foundation of China) 82372089,82272823,82273288 Li Wanwan issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Bladder cancer is a highly prevalent malignancy in elderly individuals1. Conventional approaches for non-muscle-invasive bladder cancer (NMIBC) that occupy more than 75% of total bladder cancers include surgical resection, cystoscopy-assisted photomedicine, and high-dose radiation2,3. Despite their effectiveness, patients with NMIBC are still at high risk of recurrence or tumour progression and live with long-term follow-up. Consequently, the diagnosis, treatment, and 5-year survival rates have not been largely improved since the 1990s4, until the emergence of immunotherapy5. This is mainly because of the failure of conventional technologies in conflating real-time diagnosis, simultaneous treatment, in vivo monitoring, and long-term prognosis1. Therefore, developing non-invasive and full-course imaging-navigated efficient therapeutic approaches are urgently appealing to combat NMIBC.

Photomedicine that has been approved for superficial cancers utilizes visible light to excite photosensitizers and generate reactive oxygen species (ROS), thus enabling simultaneous tumour diagnosis, elimination, and recurrence prevention of NMIBC due to the intrinsic fluorescence, evoked oxidative damage, and immunogenic cell death6–10. Early since 1976, Kelly et al. have witnessed the huge potentials of photodynamic therapy (PDT) for diagnosis and treatment of bladder cancer as they found efficient tumour destruction in a subcutaneous xenograft of human bladder tumour after high intensity illumination of the accumulated hematoporphyrin derivative11. Bladder cancer became the first cancer type that was clinically approved to receive PDT in 1993 and PDT has been currently being evaluated in multiple clinical trials or already used in the clinic for different types of cancers worldwide12,13. However, cystoscopy-assisted photomedicine of photodiagnosis and PDT relies heavily on biopsy for prognostic evaluation and is highly invasive14. A recent innovated technology using a miniaturized implantable powered light-emitting diode to replace cystoscopy as a light source demonstrated wirelessly activation of PDT to combat bladder cancer and the advantages of less-invasive techniques15. Meanwhile, advanced imaging modalities, such as fluorescence imaging, have also been invented to overcome the limitations of current cystoscopy-assisted biopsy to achieve non-invasive diagnosis of bladder cancer16,17. However, current imaging strategies that established mainly based on xenograft models are insufficient to inform treatment decisions, especially follow-up approaches18. Alternatively, radiation non-invasively induces DNA damage and triggers immune responses to destroy tumours19. Fractionated radiotherapy (f-RT) has been established in the clinic to maximize cellular destruction with computed tomography or biopsy for post-treatment monitoring20. Nevertheless, the high doses and lack of imaging for simultaneous and long-term evaluation make it a less efficient strategy for bladder cancer21,22. “Nanomedicine” approaches to package imaging and/or therapeutic moieties have emerged with innovative strategies to ascend cancer treatment23,24. Antosh et al. revealed the promises of X-ray-activated PDT for tumours25, whereas Zhang et al. reported the pioneering work on X-ray-activated persistent luminescent nanomaterials for NIR-II imaging26. Moreover, anti-tumour nanomedicines can be locally administered into the bladder through intravesical instillation instead of systemic administration, which would guarantee biosafety for clinical translations23,27,28. Hence, there exists a huge opportunity for full-course imaging and non-invasive treatment of NMIBC by combining photomedicine, radiation, and nanomedicine.

Herein we revisit the two promising approaches of photomedicine and radiation. In this study, we develop a non-invasive approach for full-course real-time NIR-II imaging-navigated on-demand f-PDT of autochthonous NMIBC in mice based on the engineering of an X-ray-activated nanotransducer. We first investigate the X-ray energy absorption, distribution, and conversion processes in nanoscintillators to yield nanotransducers with concurrent visible radioluminescence for highly efficient PDT and NIR-II luminescence at the biologically transparent window for imaging. Then, tumour angiogenesis-specific peptides are decorated to ensure specific targeting and sufficient accumulation of nanotransducers in autochthonous NMIBC with complex subtypes. As a result, this approach enables enhanced tumour regression, robust recurrence inhibition, improved mouse survival, and NIR-II imaging prognostic monitoring. Finally, we quantify the signal-to-noise ratio of NIR-II luminescence by using lab-made equipment with an X-ray tube, an 808-nm laser, and a fast NIR photon counter, and then examine the functionality of full-course NMIBC monitoring from diagnosis to prognosis to on-demand customize radiation doses.

Results

Engineering and characterization of X-ray-activated nanotransducers

The nanotransducers were yielded from core-shell lanthanide-doped nanoscintillators (Ln-NSs) with biocompatible functionalization. To enable concurrent X-ray-activated visible and NIR-II luminescence, Ln-NSs were engineered by introducing Tb3+ and Nd3+ as the dual activators and Ce3+ as the energy mediator in the low-phonon energy matrix of NaGdF4 with an inert outer layer of NaLuF4 to enhance the photoelectric effect upon X-ray photon absorption29–33 (Fig. 1a). Spherical 9.3-nm NaGdF4:Ce,Tb with optimized molar doping amounts of 20% Ce3+ and 20% Tb3+ were utilized as the core for sequential epitaxial growth of NaGdF4:Nd and NaLuF4 layers via the hot-injection method (Supplementary Fig. 1a–f). Transmission electron microscopy (TEM) images revealed NaGdF4:Ce,Tb@NaGdF4:Nd@NaLuF4 with isotropic epitaxial growth had a monodisperse hexagonal morphology and an average size of 24.6 nm (Fig. 1b and Supplementary Fig. 1f, g). Elemental mapping results revealed the clustered distributions of Gd3+, Ce3+, Tb3+, Nd3+, and Lu3+ (Supplementary Fig. 1h). Intense green emissions at 490, 546, 585, and 620 nm and NIR-II emissions at 865, 895, 1060, and 1340 nm were observed by irradiating Ln-NSs with X-rays (Fig. 1c), which well corresponded to the typical 5D4–7F6, 5D4–7F5, 5D4–7F4, and 5D4–7F3 transitions of Tb3+ and 4F3/2–4I9/2, 4F3/2–4I11/2, and 4F3/2–4I13/2 transitions of Nd3+, respectively26,27. Due to the better passivation effect of isotropic epitaxial growth on diminishing nonradiative relaxation and surface quenching34,35, the hexagonal Ln-NSs were found to exhibit more intense X-ray-activated green and NIR-II luminescence compared to the rod-like Ln-NSs with anisotropic epitaxial growth (Supplementary Figs. 2 and 3) and were hereafter adopted to optimize X-ray-activated luminescent properties and to construct nanotransducers. The more intense X-ray-activated green and NIR-II luminescence of hexagonal Ln-Ns with isotropic epitaxial growth were further confirmed by the absolute fluorescence quantum efficiency estimation (Supplementary Fig. 4). Together with the EDS results (Supplementary Figs. 1h and 2c), it can be inferred that this phenomenon was attributed to the better passivation effect of isotropic epitaxial growth of hexagonal Ln-Ns34,35. The engineered NaGdF4:Ce,Tb@NaGdF4:Nd@NaLuF4 exhibited intrinsic intense NIR-II emission under an 808-nm laser excitation (Fig. 1d), thus allowing an extra stimulus for NIR-II fluorescence activation by less harmful lasers besides X-rays towards specific clinical needs.Fig. 1 X-ray-activated nanoscintillators and nanotransducers.

a Illustration of luminescence mechanism of core-shell lanthanide-doped nanoscintillators with X-ray-activated green and NIR-II emissions (CB conduction band, VB valence band). b Transmission electron microscopy (TEM) image of 24.6-nm NaGdF4:Ce,Tb@NaGdF4:Nd@NaLuF4. Inserts: a high-resolution TEM image (top left) and the corresponding Fourier-transform diffraction pattern (top right). X-ray-activated spectra (45 kV, 190 μA) (c) and excitation spectra and NIR-II emission by an 808-nm laser (d) of the 24.6-nm NaGdF4:Ce,Tb@NaGdF4:Nd@NaLuF4. Excitation spectra monitored at 546 nm (e), fluorescence decay curves monitored at 546 nm (f), and normalized X-ray-activated spectra from 400 to 700 nm (45 kV, 190 μA) (g) of NaGdF4:Ce,Tb (denoted as Ce,Tb) and NaGdF4:Tb (denoted as Tb). Excitation spectra monitored at 1060 nm (h), fluorescence decay curves monitored at 1060 nm (i), and normalized X-ray-activated spectra from 800 to 1400 nm (45 kV, 190 μA) (j) of NaGdF4:Ce,Tb@NaGdF4:Nd (denoted as Ce,Tb@Nd) and NaGdF4:Tb@NaGdF4:Nd (denoted as Tb@Nd). k X-ray-activated visible spectrum of the 24.6-nm NaGdF4:Ce,Tb@NaGdF4:Nd@NaLuF4 (45 kV, 190 μA) and absorption spectrum of RB. l X-ray-activated visible spectra of NSs and NSs-RB (45 kV, 190 μA). m Relative intensities of SOSG incubated with different solutions (n = 6 independent experimental replicates) under X-ray irradiation (45 kV, 190 μA). n X-ray-activated NIR-II spectrum of NSs-RB (45 kV, 190 μA). Inserts: a representative photograph (left) and an NIR-II fluorescent image irradiated by X-rays (right, 70 kV, 0.7 mA). o NIR-II spectrum of NSs-RB excited by an 808-nm laser. Inserts: a representative photograph (left) and an NIR-II fluorescent image excited by an 808-nm laser (right, 5 W). Data in (m) were presented as mean values ± SEM.

The mediation role of Ce3+ on X-ray energy conversion into Tb3+ and Nd3+ was exclusively studied. Compared to the characteristic excitation peak of Tb3+ at 379 nm in NaGdF4:Tb, a strong excitation peak at 250 nm that indexed to the 4f–5d transition of Ce3+ was detected in NaGdF4:Ce,Tb (Fig. 1e). NaGdF4:Ce,Tb exhibited a prolonged fluorescent lifetime under the excitation at 250 nm than that of NaGdF4:Tb at 379 nm (Fig. 1f), indicating energy transfer from Ce3+ to Tb3+ in the host matrix36,37. The intensity of X-ray-activated green emission was enhanced by ~2.2-fold after Ce3+ doping (Fig. 1g). Similar positive mediation effect of Ce3+ on Nd3+ was also disclosed by NaGdF4:Ce,Tb@NaGdF4:Nd and NaGdF4:Tb@NaGdF4:Nd directly (Fig. 1h, i) or through the bridge of Gd3+ (Supplementary Fig. 1c), with a ~12-fold enhancement of NIR-II emission at 1064 nm after Ce3+ doping36 (Fig. 1j). The decreased sizes of NaGdF4:Tb (Supplementary Fig. 1b, I) and NaGdF4:Tb@NaGdF4:Nd (Supplementary Fig. 1e, j) after a 20% Ce3+ dopant indicated the enhanced radioluminescence performance to be contributed by the effective energy transfer of Ce3+ 38,39, thus revealing the fundamental mechanism of the dual-mediation role of Ce3+ in achieving X-ray-activated concurrent visible and NIR-II luminescence. Notably, only a mono-mediation role of Ce3+ has been established among current Ce3+-based nanoscintillators with merely visible radioluminescence but no NIR-II radioluminescence yet29,40. The competitive X-ray energy conversion into co-doped Nd3+ and Tb3+ via Ce3+ was also confirmed by the first-rise-then-fall NIR-II emission intensity and gradually decreased visible emission intensity and the doping concentration of Nd3+ was optimized to be 1.0% (Supplementary Fig. 5a). An inappropriate thickness of Nd3+-layer or a simple spatial switch of adjacent Tb3+-layer and Nd3+-layer would lead to decreases in X-ray-activated visible and NIR-II emissions (Supplementary Fig. 5b–d), which indicated competitive X-ray energy distribution from Ce3+ to Tb3+ and Nd3+ and confirmed the emerged visible and NIR-II radioluminescence to be contributed by the dual-mediation role of Ce3+. Severe adverse cross-relaxation occurred with barely any visible or NIR-II radioluminescence when co-doping Ce3+, Tb3+, and Nd3+ in the host matrix instead of the engineered core-shell structure (Supplementary Fig. 5e). These results confirmed the rationality of our proposed core-shell nanoscintillators with the pivotal dual-mediation role of Ce3+ to achieve concurrent X-ray-activated visible and NIR-II luminescence.

Surface modification of the as-synthesized NSs to convert into hydrophilicity was first conducted by a ligand exchange process using O-phosphorylethanolamine (AEP) with amino groups for further modification. The mass ratio of AEP onto the NSs was determined to be about 4.2:1 (Supplementary Fig. 6). Nanotransducers (denoted as NSs-RB) for f-PDT were then obtained by covalent conjugation of Rose Bengal (RB) as photosensitizers onto the 24.6-nm NaGdF4:20%Ce,20%Tb@NaGdF4:1%Nd@NaLuF4 (denoted as NSs) through the bridge of O-phosphorylethanolamine (AEP). RB is a typical synthetic fluorescein derivative that efficiently generates 1O2 under excitation at 500–600 nm. The energy transfer efficiency of X-ray-activated visible luminescence from NSs to RB reached up to 99.1%, due to the good spectral overlap between visible emissions of NSs and absorption of RB (Fig. 1k, l and Supplementary Fig. 5f). Intense fluorescence signals only occurred under the circumstance of NSs-RB and X-ray irradiation by using Singlet Oxygen Sensor Green (SOSG) as the 1O2 indicator (Fig. 1m), indicating the capability of NSs-RB to promote f-PDT. Besides the therapeutic functions originating from X-ray-activated visible luminescence, NIR-II luminescence excited by either X-rays or an 808-nm laser remained detected with an apparent emission peak at 1064 nm after surface functionalization of RB (Fig. 1n, o), thus indicating potential in vivo bioimaging towards specific clinical needs.

Autochthonous bladder tumour-specific labelling of nanotransducers

To promote our proposed f-PDT as a fit-for-purpose technique in the clinic, female C57BL/6 mice bearing autochthonous bladder tumours induced by N-methyl-N-nitrosourea (MNU), a common carcinogen, were selected (Fig. 2a and Supplementary Fig. 7). The feature of such autochthonous bladder tumours is the highly analogous differential molecular subtypes to human bladder cancer3, compared to MNU-induced orthotopic Sprague-Dawley rat models41, patient- or cell-derived xenograft mice models42,43, or less cancer-type-specific subcutaneous models44. Representative photographs and gold standard hematoxylin and eosin (H&E) staining analysis of the bladders externalized dissected (inner surface exposed) from six randomly selected mice showed NMIBC histopathological features after MNU instillation (Fig. 2b and Supplementary Fig. 8a), in contrast to the smooth and glossy serosa of the bladder from a healthy mouse (Supplementary Fig. 8b). The dissimilarity demonstrated the successful establishment of autochthonous NMIBC mouse models.Fig. 2 Autochthonous bladder tumour specificity and enhanced cellular uptake of F/cRGD-labelled nanotransducers.

a Schematic illustration of the establishment of NMIBC mouse models and experimental protocols for single-f-PDT. b A representative photograph and H&E staining image of the bladder from an NMIBC mouse after MNU instillation. Black arrows: carcinomas. c Surface functionalization of nanotransducers by F/cRGD peptides. Dynamic light scattering (d) and Zeta potentials (n = 4 independent experimental replicates) (e) of NSs-AEP, NSs-RB, and NSs-RB-F/cRGD. f Ex vivo NIR-II fluorescence imaging (ex: 808 nm) of bladders and the corresponding frozen slices (FITC channel, ex/em: 460–500/512–542 nm) that dissected from the NSs-RB-F/cRGD-treated healthy mouse and the NSs-RB-FITC-treated and NSs-RB-F/cRGD-treated NMIBC mice. g Quantitative FITC fluorescence signals with statistical analysis on the corresponding frozen slices (n = 6 independent experimental replicates) in (f). Fluorescence imaging in T24 cells incubated with NSs-RB and NSs-RB-F/cRGD with a concentration of 50 μg mL−1 (h) and quantitative RB (left) and FITC (right) fluorescence intensities with statistical analysis (n = 10 independent experimental replicates) (i). NIR-II fluorescence images (j) and quantitative signal intensities (n = 10 independent experimental replicates) (k) in T24 cells with different treatments (50 μg mL−1). l Flow cytometry analysis (n = 3 independent experimental replicates) of T24 cells after incubation with NSs-RB and NSs-RB-F/cRGD (50 μg mL−1). Data in (e, g, i, and k) were presented as mean values ± SEM. The P values in (g) were calculated by one-way ANOVA and multiple-comparison test. The P values in (i and k) were calculated by paired two-sided Student’s t-test.

Specific targeting capability with the efficient accumulation of nanotransducers in carcinogen-induced autochthonous tumours is highly required to establish a robust non-invasive imaging-navigated therapy for clinical bladder cancer, we thus deliberately labelled our nanotransducers with the tumour-homing cyclic arginine-glycine-aspartate peptide (cRGD, conjugated with fluorescein 5-isothiocyanate (FITC)) and prepared cRGD-FITC (F/cRGD)-labelled nanotransducers (donated as NSs-RB-F/cRGD) (Fig. 2c). The mass ratio of F/cRGD peptides onto NSs-RB was estimated to 7.96:530 (Supplementary Fig. 9). With its high binding affinity to αvβ3 integrins that are highly expressed in tumour neovascularization endothelial cells and cancer cells compared to normal cells24,45–47, F/cRGD and F/cRGD-labelled nanomedicines can specifically target a broad spectrum of tumours (Supplementary Fig. 10). Surface functionalization was confirmed by FTIR spectroscopy (Supplementary Fig. 8c). NSs-RB-F/cRGD exhibited excellent physiological stability with an average diameter of 274.2 nm, whereas the values of NSs-AEP and NSs-RB were 150.8 and 184.8 nm (Fig. 2d). Meanwhile, Zeta potentials varied from +8.9 eV of NSs-AEP and +21.5 eV of NSs-RB to −40.3 eV of NSs-RB-F/cRGD after conjugation of AEP, RB, and F/cRGD (Fig. 2e and Supplementary Figs. 11, 12), demonstrating the successful preparation of NSs-RB-F/cRGD.

Considering the multiple subtypes of carcinogen-induced autochthonous tumours cannot be entirely mimicked by specific cancer cell lines48,49, we estimated specific targeting effect and efficient accumulation of F/cRGD-labelled nanotransducers directly with the mouse models44,50–52. Ex vivo NIR-II fluorescence imaging (excited by an 808-nm laser) was performed with dissected bladders from three anesthetized NMIBC mice infused with FITC-marked NSs-RB (denoted as NSs-RB-FITC) and three with NSs-RB-F/cRGD and three healthy mice infused with NSs-RB-F/cRGD (Fig. 2f and Supplementary Fig. 13). No obvious NIR-II fluorescence signal was detected in the NSs-RB-F/cRGD-treated healthy mouse bladder, indicating no significant non-specific accumulation in normal cells of the bladder by F/cRGD-labelled nanotransducers. Intense NIR-II fluorescence signals observed in the NSs-RB-F/cRGD-treated NMIBC mouse bladder were more than 100 times compared to that of NSs-RB-F/cRGD-treated healthy mouse bladder. Weak NIR-II fluorescence signals were also observed in some regions of the mouse bladder instilled with NSs-RB-FITC with the values to be about 20 times higher than that in healthy mouse bladder (Supplementary Fig. 8d–g), which we speculated to be the passive targeting effect of nanotransducers by the enhanced permeability and retention (EPR) effect. The significantly decreased signal intensity with NSs-RB compared to that with NSs-RB-F/cRGD revealed efficient accumulation was mainly contributed by F/cRGD labelling. In other words, the engineered F/cRGD-labelled nanotransducers were capable of specifically targeting autochthonous bladder tumours with barely any non-specific accumulation in non-tumour cells. Notably, the difference between the bright spots in Fig. 2f and Supplementary Fig. 13 and the diffusely distributed tumour lesions in Fig. 2b was mainly because they were taken from different angles of view of bladders and the overlapping of tumour lesions when inside bladder tissue and the tissue scattering of NIR-II fluorescence.

Cellular specificity and accumulation enhancement of nanotransducers by F/cRGD labelling were further estimated by adopting two human bladder cancer cell lines of T24 and 253J as representatives. Both NSs-RB and NSs-RB-F/cRGD were internalized by the two cells (Fig. 2h and Supplementary Fig. 14a). The endocytosis efficiencies were quantified with ~4.5-fold and ~9.9-fold enhancements in T24 and 253J cells based on the fluorescence intensities of RB with the appearance of intense FITC signals (Fig. 2i and Supplementary Fig. 14b), respectively. Enhanced endocytosis of F/cRGD-labelled nanotransducers was also revealed by in situ monitoring with the NIR-II fluorescent nanotransducers. Strong NIR-II fluorescence signals with ~3.3-fold in T24 cells and ~4.9-fold in 253J cells higher than that with NSs-RB were observed in both cancer cells treated with NSs-RB-F/cRGD (Fig. 2j, k and Supplementary Fig. 14c, d). Normal human urinary tract epithelial cells of SV-HUC-1 were also employed to assess the tumour-targeting effect of NSs-RB-F/cRGD. The RB and FITC intensities detected in SV-HUC-1 cells were estimated to be about one-twentieth as that in T24 cells (Supplementary Fig. 14e–g), which was in good consistency with that obtained by mouse models and further confirmed the enhanced accumulation of nanotransducers by F/cRGD labelling other than non-specific accumulation. Flow cytometry results also revealed the enhanced cellular uptake of NSs-RB-F/cRGD than that of NSs-RB (Fig. 2l and Supplementary Fig. 15). Moreover, the RB and FITC intensities detected in breast cancer cells of MCF-7 with low αvβ3 integrin expression were much lower than those in bladder cancer cells of T24 with high αvβ3 integrin expression (Supplementary Fig. 16), indicating the high affinity of F/cRGD peptides to overexpressed αvβ3 in NMIBC. These results strengthened the rationale of F/cRGD labelling onto nanotransducers towards clinical alike autochthonous bladder tumours.

Cytotoxicity, radiosensitization, and X-ray-activated PDT of F/cRGD-labelled nanotransducers

We then estimated the cytotoxicity and in vitro X-ray-activated PDT capability of nanotransducers with T24 and 253J cells. Greater than 90% of T24, 253J, and SV-HUC-1 cells remained alive after being treated with NSs-RB-F/cRGD and its compositional NSs-AEP and NSs-RB (Fig. 3a and Supplementary Fig. 17a, b), indicating low cytotoxicity of nanoscintillators and nanotransducers with or without F/cRGD labelling. The viabilities of T24 cells treated with NSs-AEP + X-ray, NSs-RB + X-ray, and NSs-RB-F/cRGD + X-ray decreased significantly with a positive relationship with radiation dose (Fig. 3b) or nanotransducer concentration (Fig. 3c), indicating efficient X-ray-activated PDT capability. Severe apoptosis with 27.7%, 23.0%, and 16.3% of T24 cells alive was observed after treated with NSs-RB + X-ray at 1, 2, and 4 Gy, whereas only 20.7%, 19.0%, and 12.6% cells survived when treated with NSs-RB-F/cRGD + X-ray at 1, 2, and 4 Gy. The extra decreases in viabilities were contributed by the enhanced cellular uptake of nanotransducers due to F/cRGD labelling. Similarly, the more efficient X-ray-activated PDT outcomes of NSs-RB-F/cRGD were also observed in 253J cells with viabilities of 20.2%, 6.5%, and 3.3% at 1, 2, and 4 Gy compared to that of NSs-RB with 22.7%, 10.2%, and 5.3% at 1, 2, and 4 Gy (Supplementary Fig. 17c).Fig. 3 Cytotoxicity, radiosensitization effect, and ROS generation assessment.

a Viabilities of T24 cells treated with NSs-AEP, NSs-RB, and NSs-RB-F/cRGD at different concentrations (n = 5 independent experimental replicates). b Viabilities of T24 cells treated with RB, NSs-AEP, NSs-RB, and NSs-RB-F/cRGD with X-ray irradiation at 0, 1, 2, and 4 Gy (nanotransducers: 50 μg mL−1) (n = 5 independent experimental replicates). c Viabilities of T24 cells treated with NSs-RB-F/cRGD at different concentrations and X-ray doses. d Survival fractions of T24 cells incubated with NSs-AEP, NSs-RB, and NSs-RB-F/cRGD under various X-ray doses (50 μg mL−1). Fluorescence images (e) and signal quantification (n = 9 independent experimental replicates) (f) of γ-H2AX immunofluorescence analysis of T24 cells treated with NSs-AEP, NSs-RB, and NSs-RB-F/cRGD (50 μg mL−1) under 1-Gy radiation. Intracellular ROS imaging (DHE) (g) and signal quantification (n = 10 independent experimental replicates) (h) of T24 cells that received different treatments under 1-Gy radiation (nanotransducers: 50 μg mL−1). Intracellular 1O2 imaging (SOSG) (i) and signal quantification (n = 10 independent experimental replicates) (j) in T24 cells with different treatments under 1-Gy radiation (nanotransducers: 25 μg mL−1). Data in (a–d, f, h, and j) were presented as mean values ± SEM. The P values in (b–d, f, h, and j) were calculated by one-way ANOVA and multiple-comparison test.

Moderate decrements in viabilities of T24 and 253J cells were also observed when treated with NSs-AEP + X-ray at 1, 2, and 4 Gy (Fig. 3b and Supplementary Fig. 17c), which were ascribed to be the radiosensitization effect of nanoscintillators in generating cytotoxic hydroxyl radicals (·OH) to destroy cells based on the strong interactions of compositional high-atomic-number elements (Gd, Ce, Tb, Nd, and Lu) with X-rays53,54 (Supplementary Fig. 17d). In vitro clonogenic survival assay confirmed the radiosensitization effect with the enhancement factor at 50% T24 cell survival of NSs-AEP estimated to be 1.47 Gy (Fig. 3d and Supplementary Table 1), in contrast to the high value of 5.32 Gy for X-rays alone55. Despite the recently emerged X-ray-activated PDT process cannot be fitted by classical mathematical models56, NSs-RB-F/cRGD and NSs-RB were found with more severe proliferation decreases under X-rays compared to NSs-AEP, indicating preserved radiosensitization effect with additional PDT effect in nanotransducers. γ-H2AX immunofluorescence analysis on DNA double-strand breaks also revealed the existence of the radiosensitization effect with significantly higher levels of DNA damage and clearer γ-H2AX signals in cell nuclei when treated with NSs-AEP and 1-Gy radiation (Fig. 3e, f). In contrast, no noticeable DNA damage could be observed with the presence of 1-Gy radiation alone. Intense γ-H2AX signals observed with the presence of both X-rays and NSs-RB or NSs-RB-F/cRGD confirmed the positive therapeutic effects of RB functionalization and F/cRGD labelling.

ROS generation of nanotransducers under X-ray irradiation was then estimated by using dihydroethidium (DHE) as a ROS indicator and SOSG as a special 1O2 indicator. The fluorescence signals in T24 cells with NSs-AEP + X-ray + DHE, NSs-RB + X-ray + DHE, and NSs-RB-F/cRGD + X-ray + DHE indicated ·OH generation due to the radiosensitization effect (Fig. 3g, h). Meanwhile, strong fluorescence signals appeared in cells when treated with NSs-RB + X-ray + SOSG and NSs-RB-F/cRGD + X-ray + SOSG, indicating the massive generation of 1O2 through PDT (Fig. 3i, j). Moreover, the fluorescence signals detected in cells treated with NSs-RB-F/cRGD + X-ray + DHE and NSs-RB-F/cRGD + X-ray + SOSG were quantified with the highest values. The radiosensitization effect and PDT individually undergo different mechanisms by consuming water molecules to generate ·OH via electron transfer (type I mechanism) and molecular oxygens to generate 1O2 via energy transfer (type II mechanism)57,58. The generation of ·OH is not affected by the dynamic cellular environment and could remain constant, while 1O2 generation is highly oxygen-dependent and its high reaction rate would gradually decrease along with the consumption of intracellular oxygen molecules. Consequently, the synergistic X-ray-activated PDT of NSs-RB and NSs-RB-F/cRGD on cell death was positively but non-linearly associated with either concentration or radiation dose. Significant differences between cell viabilities of NSs-RB and NSs-RB-F/cRGD could only be observed at relatively low concentrations or radiation doses (Fig. 3b, c). A consistent phenomenon also occurred in 253J cells (Supplementary Fig. 17c), as they are of the same high-grade bladder cancer cell lines59,60. Further investigations confirmed the decreasing incremental rate of cell-killing effect versus concentration by NSs-RB and NSs-RB-F/cRGD with gradually converged cell-killing efficiencies of NSs-RB and NSs-RB-F/cRGD at high concentrations or radiation doses (Supplementary Fig. 17e, f), which not only demonstrated the higher X-ray-activated PDT efficiency of NSs-RB-F/cRGD at a lower concentration or a lower radiation dose compared to that of NSs-RB but also confirmed the positive effect of F/cRGD labelling onto nanotransducers.

Apoptosis analysis on in vitro X-ray-activated PDT of F/cRGD-labelled nanotransducers

We then conducted apoptosis studies by using Annexin V-APC/propidium iodide (PI) assays on cells. To diminish the killing effect of X-rays on cells and disclose the synergistic X-ray-activated PDT of nanotransducers, a low dose of 1 Gy with no detectable cell death was adopted. Severe death was observed in T24 cells after being treated with NSs-RB + X-ray and NSs-RB-F/cRGD + X-ray (Fig. 4a and Supplementary Fig. 18a). Flow cytometry analysis was employed to accurately quantify the early and late apoptosis (Fig. 4b). A low level of total apoptotic cells was observed with a value of 29.41% after being treated with NSs-AEP + X-ray. On the contrary, approximately 44.37% and 55.71% of apoptotic cells were observed when treated with NSs-RB + X-ray and NSs-RB-F/cRGD + X-ray. These qualitative and quantitative results confirmed the efficient X-ray-activated PDT capability of nanotransducers through intrinsic radiosensitization and engineered PDT with enhanced efficiency by cRGD labelling.Fig. 4 Apoptosis analysis and in vitro X-ray-activated PDT assessment.

a Annexin V-APC/PI dual-staining assay of T24 cells with different treatments (nanotransducers: 50 μg mL−1). b Apoptosis of T24 cells conducted by flow cytometry analysis with different treatments under 1-Gy radiation (50 μg mL−1). Western blot analysis (c) and corresponding quantification and statistical analysis (n = 3 independent experimental replicates, the samples derive from the same experiment and that western blots were processed in three parallel experiments) (d) of protein expressions in T24 cells after receiving different treatments under 1-Gy radiation (50 μg mL−1). e Imaging flow cytometry results and representative images of T24 cells incubated for 12 h after different treatments (50 μg mL−1). Data in (d) were presented as mean values ± SEM. The P values in (d) were calculated by one-way ANOVA and multiple-comparison test.

We deduced that the significant cell death under X-ray irradiation by NSs-RB-F/cRGD was contributed by the burst 1O2 and ·OH generation to activate caspase-dependent apoptosis. Western blot assays were thus employed to estimate protein expressions of the B-cell lymphoma-2 (Bcl-2) family and caspase activation61,62. The abound appearance of anti-apoptotic protein of Bcl-2 was observed in T24 cells receiving no treatment or just 1-Gy irradiation (Fig. 4c and Supplementary Fig. 18b). In contrast, cells treated with NSs-AEP, NSs-RB, and NSs-RB-F/cRGD and 1-Gy irradiation expressed high levels of Bcl-2-associated pro-apoptotic proteins of Bax and Bad with low levels of Bcl-2. Individual contributions to X-ray-activated PDT of radiosensitization effect, RB functionalization, and enhanced accumulation by F/cRGD labelling were further revealed by quantified expression values of Bcl-2, Bax, Bad, and cleaved caspase-3 versus β-tubulin (Fig. 4d and Supplementary Fig. 18c). Significant decreases of Bcl-2 could be induced by NSs-AEP + X-ray, NSs-RB + X-ray, and NSs-RB-F/cRGD + X-ray, significant increases in Bax and Bad were observed only after RB functionalization (NSs-RB and NSs-RB-F/cRGD), whereas a significant increase in cleaved caspase-3 occurred only with RB functionalization and cRGD labelling (NSs-RB-F/cRGD). Meanwhile, although upregulation of cleaved caspase-3 was also observed in NSs-RB + X-ray-treated and NSs-RB-F/cRGD + X-ray-treated cells, significant differences were only observed between NSs-RB-F/cRGD and NSs-AEP other than between NSs-RB and NSs-AEP (Supplementary Fig. 18c). These results were in good consistency with that revealed by flow cytometry analysis and demonstrated the benefits and indispensable roles of nanoscintillators, RB, and F/cRGD to engineer nanotransducers.

Imaging flow cytometry (IFC) was used to visualize cellular structure destruction by in vitro X-ray-activated PDT. Compared to the intact cellular morphology with no FITC or RB signals in the control group, T24 cells incubated with NSs-RB-F/cRGD were detected with intense FITC and RB signals and increased side scatter signals (Fig. 4e and Supplementary Fig. 18d), demonstrating successful endocytosis63–65. Upon radiation of 0.5 or 1 Gy with NSs-RB-F/cRGD, signatures of crumpling, membrane blebbing, shrinkage, elongation, and peg-like protrusions with deceased FITC and RB signals appeared in cells with a radiation-dose positively correlated relationship, indicating strong X-ray-activated PDT effects on cellular apoptosis47,66,67. IFC results also revealed a time-dependent cellular structural destruction behaviour of X-ray-activated PDT (Supplementary Fig. 18e, f), which corresponded to the fact that cells damaged by ROS (e.g., 1O2, ·OH) undergo a series of responses (e.g., cell cycle arrest, DNA repair68) before apoptosis finally occurs.

Single-f-PDT achieves robust tumour elimination

We first estimated the anti-tumour potentials of nanotransducers on autochthonous bladder cancer mice models (n = 7 mice each group). This was accomplished by single X-ray exposure (namely, single-fractionated PDT, denoted as single-f-PDT) of bladders in casually placed anesthetized mice that received pre-intravesical instillation of NSs-RB-F/cRGD for 1 h (Fig. 5a). For single-f-PDT, X-ray exposure was performed with an X-ray irradiator (160 kV, 250 mA) at a radiation dose of 4 or 6 Gy (Supplementary Fig. 19a), which have been adopted in the clinic for bladder cancer patients69 or fundamental cancer research70. Surgery-like laser-assisted PDT (650-nm laser, 3 W) with 5-aminolevulinic acid (5-ALA) was employed for comparison (Supplementary Fig. 19b)71,72. Our results showed that NMIBC mice treated by NSs-RB-F/cRGD +6 Gy within 21 days maintained the highest survival rate of 86%. In contrast, NMIBC mice treated with NSs-RB + 6 Gy and laser-assisted PDT (5-ALA + laser) exhibited similar efficiencies with the second highest survival rate of 71% (Fig. 5b, c), decreased survival rates were obtained when omitting RB functionalization (NSs-AEP + 6 Gy), reducing radiation dose (NSs-RB-F/cRGD +4 Gy and NSs-RB-F/cRGD), or receiving other inefficient treatments (RB + 6 Gy and PBS + 6 Gy). Statistically significant differences were only observed between NMIBC mice treated with NSs-RB-F/cRGD + 6 Gy (the highest survival rate) and PBS, indicating robust therapeutic outcomes by proposed single-f-PDT. Notably, with an increased sample number (n = 14 mice each group) by combining the same groups of IX and XI (for long-term monitoring) and groups of X and XII (for long-term monitoring), the survival rates by NSs-RB-F/cRGD + 6 Gy and 5-ALA + laser within 21 days were both found to be significantly different from that with no treatment (Supplementary Fig. 19c), which further confirmed the superior therapeutic capability of non-invasive single-f-PDT.Fig. 5 Tumour regression by non-invasive single-f-PDT.

a Schematic illustration of the experimental protocols for single-f-PDT (n = 7 mice each group). b Survival probability of NMIBC mice receiving different treatments for 21 days. c Photographs of bladders dissected from survived mice at day 21. d Representative H&E staining images of bladders from each group. M muscle, Ur urothelium, Lp lamina propria. Black arrows: NMIBC lesions. Blue arrows: MIBC. Representative immunohistochemistry analysis (e) and quantification of CD4 (f) and CD8 cell proliferation (g) of bladders from healthy mice and NMIBC mice treated with PBS, 5-ALA + laser, and NSs-RB-F/cRGD + 6 Gy. Representative photographs (h) and H&E staining images (i) of spleens from NMIBC mice receiving different treatments. Representative immunohistochemistry analysis (j) and quantification of CD4 (k) and CD8 cell proliferation (l) of spleens from healthy mice and NMIBC mice treated with PBS, 5-ALA + laser, and NSs-RB-F/cRGD + 6 Gy. Data analysis in (b) was performed using a log-rank (Mantel–Cox) test and post-Bonferroni correction (α = 0.05/T, T = 2), 5-ALA + laser versus PBS and NSs-RB-F/cRGD + 6 Gy versus PBS. Data in (f, g, k, and l) were presented as mean values ± SEM. The P values in (f, g, k, and l) were calculated by one-way ANOVA and multiple-comparison test.

The discrepancies in survival rates of NMIBC mice receiving different treatments were suspected to be closely related to tumour regression, inhibition, or progression. H&E and Ki-67 staining analysis revealed only the bladder dissected from mice treated with 5-ALA + laser and NSs-RB-F/cRGD +6 Gy were found with no NMIBC histopathological features (Fig. 5d and Supplementary Fig. 20). In contrast, bladder tumours were detectable among other treated mice in groups III–VIII and severe tumour progression into the muscle-invasive stage (MIBC) occurred in the untreated mice73. Mice treated with NSs-RB-F/cRGD +6 Gy and 5-ALA + laser were also observed with no significant abnormalities in the dissected organs, which indicated no metastasis occurred (Supplementary Figs. 21–23). Together with the survival rates, these results confirmed the robust anti-tumour efficiency of non-invasive single-f-PDT and the essential F/cRGD labelling onto nanotransducers to eventually enhance therapeutic efficiency.

To understand the efficient single-f-PDT capability, immune cell infiltration of the harvest bladders from mice in different groups was then analyzed74,75, given that ROS and radiation have been both reported to remodel tumour microenvironment and/or elicit systemic immunity58,76. Immunohistochemistry analysis revealed comparable proliferation of cytotoxic T lymphocytes (CD4 and CD8 cells) in bladders of NMIBC mice treated with NSs-RB-F/cRGD +6 Gy and 5-ALA + laser to that of healthy mice, with the values quantified to be significantly higher than that of untreated NMIBC mice by using the widely accepted German semi-quantitative scoring system77–79 (Fig. 5e–g and Supplementary Fig. 24). Meanwhile, NMIBC mice receiving insufficient treatments were found with lower proliferation levels of CD4 and CD8 cell proliferation (Supplementary Fig. 19d, e). Severe spleen enlargement was also found in NMIBC mice with insufficient treatments, while in contrast, similar appearances with normal sizes of the spleens were observed between healthy mice and NMIBC mice receiving NSs-RB-F/cRGD +6 Gy and 5-ALA + laser treatments (Fig. 5h). Consistently, histology of the mouse spleen revealed distinct regions of the red pulp and the white pulp in these three groups80,81 (Fig. 5i). Immunohistochemistry analysis also revealed restored immune homoeostasis of both CD4 and CD8 cell proliferation in spleens from NMIBC mice with efficient tumour regression by NSs-RB-F/cRGD + 6 Gy and 5-ALA + laser (Fig. 5j–l and Supplementary Figs. 19f, g, and 25). In supplement of H&E staining, the immune cell infiltration signatures with statistically significant differences also confirmed the robust tumour regression by non-invasive single-f-PDT.

NIR-II imaging-navigated post-operative monitoring of recurrence inhibition

To establish proof-of-concept post-operative monitoring, NIR-II fluorescence images of mice were taken during the treatment period by a conventional small animal living imaging system with an 808-nm laser instead of X-rays to avoid additional X-ray exposure. Re-intravesical instillation of NSs-RB-F/cRGD into bladders was needed at each time interval due to the rapid clearance behaviour (Supplementary Fig. 26a). On the premise of similar fluorescent intensities in all the randomly grouped NMIBC mice at day 0 post-treatment, NIR-II fluorescence signals exhibited opposite treatment-dependent and time-dependent behaviours from days 0 to 21 (Fig. 6a). Similar to untreated NMIBC mice, NMIBC mice treated with mere X-ray irradiation (PBS + 6 Gy and RB + 6 Gy) or nanotransducers (NSs-RB-F/cRGD) were detected with intense signals in bladders with increasing intensities. On the contrary, the intensities gradually decreased when mice were treated with both nanotransducers and X-rays (NSs-AEP + 6 Gy, NSs-RB + 6 Gy, NSs-RB-F/cRGD +4 Gy, and NSs-RB-F/cRGD + 6 Gy) or surgery-like PDT (5-ALA + laser). However, only NSs-RB-F/cRGD + 6 Gy and 5-ALA + laser treatments realized no detectable signals as in healthy mice from days 7 to 21. The slightly increased signal intensities of one mouse of the NSs-RB + 6 Gy group and NSs-RB-F/cRGD +4 Gy group at day 14 revealed the sensitivity and convenience of non-invasive real-time in vivo imaging for tumour development monitoring (Supplementary Fig. 26b). Taken together, the NIR-II fluorescence signals reflected the tendency of tumour regression or progression in mostly NMIBC mice that has been evidenced by the standard H&E staining, thus revealing the huge promises of proposed non-invasive real-time NIR-II imaging to monitor tumour development.Fig. 6 Post-operative monitoring of tumour recurrence inhibition by single-f-PDT.

a Representative in vivo NIR-II fluorescence images of NMIBC mice from each group at days 0, 7, 14, and 21 (n  =  7 mice each group). b In vivo NIR-II fluorescence images of all the remaining mice in groups of 5-AL A+ laser and NSs-RB-F/cRGD + 6 Gy at day 56. c Survival probability of 5-ALA + laser and NSs-RB-F/cRGD + 6 Gy-treated NMIBC mice for 56 days. Photographs (d) and representative H&E and Ki-67 staining images (e) of bladders dissected from the remaining NMIBC mice in groups of 5-ALA + laser and NSs-RB-F/cRGD + 6 Gy at day 56. Representative immunohistochemistry analysis (f) and quantification of CD4 (g) and CD8 cell proliferation (h) of bladders dissected from the remaining mice with 5-ALA + laser and NSs-RB-F/cRGD + 6 Gy treatments at day 56. i Photographs of spleens dissected from the remaining mice in groups of 5-ALA + laser and NSs-RB-F/cRGD + 6 Gy at day 56. Representative immunohistochemistry analysis (j) and quantification of CD4 (k) and CD8 cell proliferation (l) of spleens dissected from the remaining mice with 5-ALA + laser and NSs-RB-F/cRGD + 6 Gy treatments at day 56. m Representative H&E staining images of spleens dissected from the remaining mice in groups of 5-ALA + laser and NSs-RB-F/cRGD + 6 Gy at day 56. Data analysis in (c) was performed using a log-rank (Mantel–Cox) test. Data in (g–l) were presented as mean values ± SEM. The P values in (g–l) were calculated by paired two-sided Student’s t-test.

NIR-II fluorescence imaging navigation was then utilized to estimate the inhibition effect on the high recurrence rate of bladder tumours by single-f-PDT. The two additional groups of NMIBC mice treated with 5-ALA + laser (group XI) and NSs-RB-F/cRGD + 6 Gy (group XII) were raised for another 35 days. Compared to the sharp decrease in the survival rate of mice treated with 5-ALA + laser from 86% at day 21 to 43% at day 56 with highly intense NIR-II fluorescence signals reappeared in bladders, a slight decrease in the survival rate from 86% at day 21 to 71% at day 56 of NSs-RB-F/cRGD + 6 Gy-treated mice was observed with no apparent signals could be detected in the bladders (Fig. 6b, c). Based on the above-established relationship between NIR-II fluorescence signals and tumour conditions, we assured the failure of laser-assisted PDT (5-ALA + laser) but the success of single-f-PDT in inhibiting recurrence.

To verify such a deduction, the long-term monitoring was intervened to end at day 56 with all the remaining mice sacrificed for H&E staining analysis. H&E and Ki-67 staining results confirmed the recurrent NMIBC in all the three survived mice of the 5-ALA + laser group (Supplementary Fig. 26c), whereas merely signatures of inflammatory lesion or seeming carcinoma in situ could be distinguished among all the five survived mice treated with NSs-RB-F/cRGD + 6 Gy (Fig. 6d, e and Supplementary Fig. 26d). Other organs were also detected with no signatures of metastasis (Supplementary Fig. 27). Significantly higher levels of CD4 and CD8 cell proliferation were also observed in the bladders (Fig. 6f–h and Supplementary Fig. 28) and spleens (Fig. 6i–l and Supplementary Fig. 29) from mice treated by non-invasive single-f-PDT with intact splenic regions (Fig. 6m and Supplementary Fig. 26e, f), whereas the surgery-like invasive laser-assisted PDT lacked long-term effectiveness. The consistency of H&E staining results and quantitative immunohistochemistry analysis with real-time NIR-II fluorescence images not only demonstrated the capability of non-invasive imaging in favoring tumour development estimation but also strengthened the robustness of f-PDT against bladder cancers. No significant abnormalities were detected in the dissected urinary tract organ of kidneys (Supplementary Figs. 30 and 31).

Full-course real-time NIR-II imaging enables on-demand f-PDT

We envisaged that the NIR-II fluorescence imaging of nanotransducers should be capable of pre-operative and intra-operative decision-making besides post-operative monitoring and customizing radiation doses for on-demand f-PDT based on tumour development. To demonstrate this, we quantified the signal-to-background ratio (SBR) of NIR-II fluorescence with a fast NIR photon counter equipped in a lab-made system with an X-ray tube (20~80 kV, 0.2~0.7 mA) and an 808-nm laser as the external beam sources (Fig. 7a and Supplementary Fig. 32). SBRs of bladders of four healthy mice were first estimated by our system with the values to be <1.57, we thus established it as the reference standard (Fig. 7b). Proof-of-concept f-PDT process was set in two parallel groups of G1 and G2 with a total of three fractions for each group (n = 5 mice each group) (Fig. 7c). The initial fraction of G1 and G2 were adopted with a relatively high dose of 3 Gy and a relatively low dose of 1 Gy to mimic two divergent clinical therapies. Radiation doses for the following two fractions in G1 and G2 were determined by the quantified real-time NIR-II imaging SBRs compared to the established reference standard. At each time point, mice were first intravesically instilled with NSs-RB-F/cRGD for 1 h and then imaged with the fast NIR photon counter upon immediate irradiation by the X-ray tube and the 808-nm laser. Radiation doses applied at each fraction were realized by simply changing the irradiation time of the X-ray tube.Fig. 7 On-demand f-PDT with full-course real-time monitoring.

a Scheme of our lab-made system (M mirror, D dichroic mirror, GSM Galvo scanning mirror, EM emission). b In vivo NIR-II fluorescence images (top) and corresponding SBRs (bottom) of four healthy mice (n = 4 mice). c Illustration of the experimental protocols for on-demand f-PDT with three fractions. d In vivo NIR-II fluorescence images (top) and corresponding SBRs quantification (bottom) of all the NMIBC mice in G1 at days 0, 7, 14, and 56 (n = 5 mice). e In vivo NIR-II fluorescence images (top) and corresponding SBRs quantification (bottom) of all the NMIBC mice in G2 at days 0, 7, 14, and 56 (n = 5 mice). f Survival probability of mice in G1 and G2 for 56 days (n = 5 mice each group). g Photographs of the bladders dissected at day 56 from all the mice in G1 and G2. h Representative H&E and Ki-67 staining images of bladders dissected from mice in G1 and G2 at day 56 and G2(x) at day 31. Representative immunohistochemistry analysis (i) and quantification of CD4 and CD8 cell proliferation (j) of bladders dissected from mice from G1 and G2 at day 56 and G2(x) at day 31. k Representative H&E staining images of spleens dissected from mice from G1 and G2 at day 56 and G2(x) at day 31. Insert is a photograph of the spleen from G2(x). l Photographs of the bladders dissected on day 56 from mice in G1 and G2. m Quantification of weight of spleens taken at day 56 from all the survived mice in G1 and G2 with that of G2(x) at day 31. Representative immunohistochemistry analysis (n) and quantification of CD4 and CD8 cell proliferation (o) of spleens dissected from mice from G1 and G2 at day 56 and G2(x) at day 31. Data in (d, e, j, m, and o) were presented as mean values +/- SEM. The P values in (d and e) were calculated by one-way ANOVA and multiple-comparison test. Data analysis in (f) was performed using a log-rank (Mantel–Cox) test. The P values in (j, m, and o) were calculated by unpaired two-sided Student’s t-test.

At day 0, the SBRs of randomly grouped NMIBC mice in G1 and G2 were estimated with similar values of about 5.20 and 5.01 (Fig. 7d, e). After imposing an initial 3 Gy for G1 and 1 Gy for G2 upon NSs-RB-F/cRGD administration, the values were quantified to drop to about 2.72 and 3.52 at day 7, respectively. In comparison to the reference standard of 1.57, these quantified SBRs at day 7 indicated efficient tumour inhibition in G1 by NSs-RB-F/cRGD + X-ray (initial 3 Gy) and the less efficient tumour inhibition in G2 by NSs-RB-F/cRGD + X-ray (initial 1 Gy). Under this circumstance, the second fraction of G1 and G2 at day 7 was immediately customized with a low dose of 1 Gy and a high dose of 3 Gy, respectively. The SBRs at day 14 were quantified to be 1.87 for G1 and 2.59 for G2, which reached nearly the reference standard of 1.57. Therefore, the last fraction for both groups was customized with a low dose of 1 Gy. The final SBR estimated at day 56 of 1.49 for G1 and 1.64 for G2 fell within or at the edge of the established criteria, indicating non-invasive tumour development monitoring by real-time quantitative NIR-II imaging SBRs. NMIBC mice in both groups with a smaller total dose of 5 Gy were found with significantly higher survival rates (Fig. 7f), compared to that of single-f-PDT at a larger dose of 6 Gy or invasive laser-assisted PDT (Fig. 6c), demonstrating the feature of on-demand f-PDT. The slightly different SBRs at each time interval and survival rates between G1 and G2 revealed the impacts of divergent f-PDT strategies or clinical therapies on tumour inhibition.

H&E and Ki-67 staining analysis was then adopted to evaluate the reliability of full-course non-invasive real-time NIR-II imaging and on-demand f-PDT. All the survived nine mice were found with no signatures or abnormalities in bladders at day 56 (Fig. 7g, h and Supplementary Fig. 33a), whereas the mouse died at day 31 (marked as G2(x)) was observed with NMIBC and MIBC lesions. Immunohistochemistry analysis revealed higher expression levels of CD4 and CD8 cell proliferation in the bladders of the nine remaining mice with a low level in that of G2(x) (Fig. 7i, j and Supplementary Figs. 33b, 34). Similarly, the nine surviving mice were found with a large amount of intact splenic regions of red pulp and white pulp, while few remained in G2(x) (Fig. 7k and Supplementary Fig. 33c). All the weights of dissected spleens from the survival mice were lower than that of the non-survived G2(x) with no significant differences between G1 and G2(vi–x) (Fig. 7l, m). Restored immune homoeostasis in the nine mice was further evidenced by immunohistochemistry analysis of spleens (Fig. 7n, o and Supplementary Figs. 33d, 35). There were also no signatures of metastasis at day 56 (Supplementary Figs. 36–39). These observations demonstrated the robustness of non-invasive on-demand f-PDT and the reliability of real-time NIR-II imaging as a promising tool for pre-, intra-, and post-operative monitoring and decision-making based on engineered nanotransducers.

The acute and long-term safety of nanotransducers was then estimated. Dissected urinary tract organs of bladder and kidneys from healthy mice with intravesical instillation of NSs-RB-F/cRGD showed no abnormalities (Supplementary Figs. 33e and 40). Urine routine results confirmed all the values to be within normal ranges (Supplementary Table 2). Besides, the administration of NSs-RB-F/cRGD in bladders was also found with no toxicity induced to other organs of the immune system (i.e., spleen) or the reticuloendothelial system (i.e., liver and lung) (Supplementary Fig. 41), and all the measured indexes in blood routine test were analogous to the reference values (Supplementary Fig. 42 and Supplementary Table 3), indicating the biosafety of engineered nanotransducers through intravesical instillation.

Discussion

In this work, aiming at the unmet clinical needs for non-invasive bladder cancer therapy, we report a nanotechnology-assisted approach for non-invasive full-course real-time NIR-II imaging-navigated on-demand f-PDT of autochthonous bladder cancers. Prior to our study, state-of-the-art photomedicine, enduring radiation, and promising nanomedicine were frequently used as supplementary to the dominating surgical resection with biopsy as the gold standard for prediction, monitoring, or follow-up tests, which are highly invasive to be popularized in high-risk aged patients (median age of 73). Besides, none of these conventional approaches can achieve in vivo diagnosis, simultaneous treatment, real-time monitoring, and long-term prognosis. In this study, the key to achieving full-course imaging-navigated treatment is the design and engineering of X-ray-activable Ln-NSs that emits intense green and NIR-II radioluminescence based on the yet unreported dual-mediation role of Ce3+ in modulating X-ray energy conversion. Upon single X-ray irradiation, green radioluminescence can promote robust PDT with restored immune homoeostasis from the dysregulated state to a comparable level to that of healthy mice and eliminate tumours and inhibit recurrence, whereas concurrent NIR-II fluorescence enables real-time imaging for in situ post-operative tumour evaluation. The other key point is the establishment of a lab-made multiple-excitation fast NIR photon counter system, which allows immediate quantification of in vivo NIR-II fluorescence imaging SBRs and timely adjust radiation protocols, namely pre- and intra-operative diagnostic imaging as well as post-operative prognostic monitoring for on-demand treatment decision-making. Current commercial X-ray irradiators or small animal living imaging systems are incapable of such simultaneous non-invasive real-time imaging and on-demand therapy. In addition, with specific targeting capability to autochthonous bladder tumours, the engineered F/cRGD-labelled nanotransducers showed no toxicity via intravesical instillation, which further broadened the scope of nanotransducers towards clinical translation.

Despite these encouraging results, additional efforts are also needed to further promote this strategy. First, laser-activated NIR-II fluorescence imaging can diminish radiation doses for single-f-PDT and is currently adequate for full-course real-time imaging-navigated on-demand f-PDT with X-ray-activated visible and NIR-II luminescence. Mechanistic insights of X-ray absorption and conversion in nanoscintillators or sophisticated engineering of core-shell structure with no elemental migration can be further studied to achieve intense NIR-II radioluminescence for specific clinical needs that require deep penetration and high-contrast in vivo imaging upon excitation of X-rays. Second, as revealed by the improved significant differences between single-f-PDT with increased sample numbers (Supplementary Fig. 19c), a larger number of mice in each group with precise control of autochthonous animal models to achieve minimum clinical important difference (MCID) can be considered. For recurrence inhibition evaluation, mice treated with f-PDT can be raised for a longer period than 56 days. More sophisticated fraction settings and customizations compared to current proof-of-concept demonstrations are also feasible. We envision the results will further improve the robustness and reliability of the proposed imaging-navigated f-PDT with low radiation doses to guarantee biosafety. Third, to establish a more precise and accurate non-invasive imaging modality, the relationship between tumour necrosis or necrotic tissue and fluorescence signals after treatment would also be further investigated by using large animal models or tissues. Fourth, as most autochthonous models display significantly more heterogeneity in terms of time to tumour development and survival after developing tumours, it would be of great significance to disclose information, such as the relationship between signal intensity and pathologic burden of disease, to extend the concept of non-invasive imaging-guided f-PDT towards possible clinical disease course. Last, more evidence at the molecular and cellular levels can be purposely acquired to outline the underlying immunological mechanism beyond current data, given that ROS and radiation have been reported with close associations with innate or adaptive immune responses.

In summary, data presented here already demonstrate the success of NIR-II imaging-navigated f-PDT of clinical alike autochthonous bladder tumours with efficient tumour regression, inhibited recurrence, restored immune homoeostasis, and prolonged survival. We believe that this non-invasive full-course imaging-navigated on-demand therapeutic strategy has great clinical value in revolutionizing bladder cancer treatment paradigms.

Methods

Ethical regulations

The research presented here complies with all relevant ethical regulations. All experiments involving animals were reviewed and approved (O_A2021143) by the guidelines of the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University. The maximal tumour burden permitted by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University was the weight of tumour should not exceed 10% of body weight. In any of animal experiments described in this article, the maximal tumour size of the mouse was never reached.

Materials

Cerium(III) chloride hydrate (CeCl3·7H2O, 99.99%), terbium(III) chloride hydrate (TbCl3·6H2O, 99.99%), gadolinium(III) chloride hydrate (GdCl3·6H2O, 99.99%), neodymium(III) chloride hydrate (NdCl3·6H2O, 99.99%), and lutelium(III) chloride hydrate (LuCl3·6H2O, 99.99%) were purchased from Energy Chemical. Ammonium fluoride (NH4F, 99.99%), methanol (99.9%), dimethyl sulfoxide (DMSO, 99.9%), and ethanol (99.8%) were purchased from Macklin. Cyclohexane (99%), O-phosphorylethanolamine (AEP, 98%), G1-ethyl-3-[3dimethylaminopropyl]carbodiimidehydrochloride (EDC, 97%), MNU (97%), fluorescein-carboxylic acid (FITC-COOH, 95%), and N-hydroxysuccinimide (NHS, 98%) were purchased from Aladdin. Cyclo(-Arg-Gly-Asp-D-Phe-(Lys(FITC))) (cRGD-FITC, 98%) was purchased from Sangon Biotech. Annexin V-APN/PI apoptosis kit was purchased from Adamas Life. Cell Counting Kit-8 (CCK-8) was purchased from Yeasen. SOSG (Invitrogen), DHE, (95%, Abcam), Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gibco), and foetal bovine serum (FBS, Gibco) were purchased from Thermo Fisher. RB (95%), oleic acid (OA, 90%), and 1-octadecene (ODE, 90%) were purchased from Sigma-Aldrich. Ultrapure water was obtained from a Milli-Q Ultrapure Water System.

Characterizations

TEM images and energy dispersive spectrometer (EDS) mapping were captured/conducted using a scanning/transmission electron microscope (STEM, Talos F200X G2). X-ray diffraction patterns were performed using an X-ray diffractometer of D8 DaVinci (Bruker) with Cu Kα radiation (λ = 1.5418 Å). The photoluminescence spectrum, absolute fluorescence quantum yield, and fluorescence lifetime were measured using a photoluminescence spectrometer of FLS-1000 (Edinburgh Instruments) equipped with relevant components including the ultrafast light source. UV−vis absorption spectra were measured using a UV−vis−NIR spectrophotometer (Lamda 950, PerkinElmer). The Fourier transform infrared (FTIR) spectra were obtained by the FTIR spectrometer (Nicolet 6700, Thermo Scientific). Dynamic light scattering (DLS) and Zeta potential (Zetasizer Nano S, UK) were used to determine the distribution of the hydrodynamic size and stability of nanoplatforms. The X-ray source used for the irradiation of cells and the therapy of NMIBC mice was the RS-2000 Pro (Rad Source, USA) with the energy configuration of 160 kV and 25 mA.

Synthesis of NaGdF4:20%Ce,20%Tb@NaGdF4:1%Nd@NaLuF4 (Ce,Tb@Nd@Lu) core-shell-shell Ln-NSs

Synthesis of NaGdF4:20%Ce,20%Tb core nanoparticles. In a typical procedure, GdCl3·6H2O (0.6 mmol), CeCl3·7H2O (0.2 mmol), and TbCl3·6H2O (0.2 mmol) magnetically mixed with OA (10 mL) and ODE (15 mL) in a 100 mL three-neck round-bottom flask. The mixture was heated to 150 °C under vacuum to form a clear solution and then cooled to 50 °C. Methanol (8 mL) containing 4 mmol NH4F and 2.5 mmol NaOH was quickly added into the mixture and stirred for at least 60 min. The solution was heated to 100 °C under vacuum and kept at 100 °C for 15 min to remove methanol. Then, the mixture solution was quickly heated to 280 °C under N2 flow and aged for 60 min. After the solution was cooled to room temperature, ethanol was added to obtain precipitation by centrifugation (5180 × g, 5 min). The precipitation was washed with ethanol and cyclohexane three times and redispersed in 4 mL cyclohexane.

Isotropic epitaxial growth of NaGdF4:1%Nd shell onto NaGdF4:20%Ce,20%Tb core. GdCl3·6H2O (0.99 mmol), NdCl3·6H2O (0.01 mmol) mixed with 10 mL OA and 15 mL ODE in a 100 mL three-neck round-bottom flask. The mixture was heated to 150 °C under vacuum to form a clear solution and then cooled to 50 °C. Methanol (8 mL) containing 4 mmol NH4F and 1.5 mmol NaOH was quickly added into the mixture and stirred for at least 60 min. The solution was heated to 100 °C under vacuum and kept at 100 °C for 15 min to remove methanol, then cooled to room temperature under N2 flow as shell precursors. One millilitre (~0.25 mmol) of NaGdF4:20%Ce,20%Tb core was mixed with 2.5 mL OA and 3.25 mL ODE in a 100 mL three-neck round-bottom flask. The mixture solution was heated to 100 °C under vacuum to remove cyclohexane, then quickly heated to 280 °C under N2 flow. The shell precursor was then added by a syringe pump at a speed of 15 mL h−1. After the hot injection, the solution was maintained at 280 °C for 30 min and cooled to room temperature. Finally, ethanol was added to obtain precipitation by centrifugation (5180 × g, 5 min). The precipitation was washed with ethanol and cyclohexane three times and redispersed in 4 mL cyclohexane. Different NaGdF4:1%Nd shell thicknesses were obtained by controlling the ratio of the shell precursors and core for epitaxial growth.

Isotropic epitaxial growth of NaLuF4 shell onto NaGdF4:20%Ce,20%Tb@NaGdF4:1%Nd core/shell. LuCl3·6H2O (1 mmol) mixed with 10 mL OA and 15 mL ODE in a 100 mL three-neck round-bottom flask. The mixture was heated to 150 °C under vacuum to form a clear solution and then cooled to 50 °C. Methanol (8 mL) containing 4 mmol NH4F and 1.5 mmol NaOH was quickly added into the mixture and stirred for at least 60 min. The solution was heated to 100 °C under vacuum and kept at 100 °C for 15 min to remove methanol, then cooled to room temperature under N2 flow as shell precursors. Four millilitres (~1 mol) of NaGdF4:20%Ce,20%Tb@NaGdF4:1%Nd core/shell was mixed with 10 mL OA and 15 mL ODE in a 100 mL three-neck round-bottom flask. The mixture solution was heated to 100 °C under vacuum to remove cyclohexane, then quickly heated to 280 °C under N2 flow. The shell precursor was then added by a syringe pump at a speed of 15 mL h−1. After the heat injection, the solution was maintained at 280 °C for 30 min and cooled to room temperature. Finally, ethanol was added to obtain precipitation by centrifugation (5180 × g, 5 min). The precipitation was washed with ethanol and cyclohexane three times and redispersed in 4 mL cyclohexane.

Anisotropic epitaxial growth of NaGdF4:1%Nd shell (and NaLuF4 shell) onto NaGdF4:20%Ce,20%Tb core (and NaGdF4:20%Ce,20%Tb@NaGdF4:1%Nd core/shell). The same operation was performed for the shell-to-core (or core/shell) anisotropic epitaxial as for the isotropic epitaxial growth, except that the amount of NaOH was changed from 1.5 to 2.5 mmol, as well as the aging temperature and time to 325 °C and 5 min after injection.

Synthesis of NaGdF4:20%Ce,20%Tb,1%Nd@NaLuF4 core-shell Ln-NSs. The first process was to prepare NaGdF4:20%Ce,20%Tb,1%Nd core nanoparticles by using the same hot-injection method but by directly mixing all the precursors in solution and then washed for isotropic epitaxial shell growth of NaLuF4 with a similar reaction parameter.

X-ray-activated fluorescence test

The X-ray source using a Mini-X2 X-ray tube (AMPTEK) with energy settings of 45 kV and 190 μA was used to measure X-ray-activated fluorescence spectra. The samples were loaded into quartz cuvettes and then placed in tungsten cassettes. After irradiation, the fluorescence penetrates the lead glass and enters the PMT detector.

Preparation of hydrophilic, photosensitizer-loaded, and tumour-specific NSs-RB-F/cRGD conjugates

Preparation of hydrophilic NSs-AEP conjugate: 0.75 g AEP dispersed into 50 mL of ethanol and water (3:2, V/V) in a 100 mL flask. Then 100 mg Ce,Tb@Nd@Lu nanoparticles were added into the mixture, followed by stirring at room temperature for 24 h to exchange the OA ligand. Finally, the conjugate was washed with hexane three times to remove residual OA molecules, followed by three washes with ethanol and hexane (1:3, V/V) and redispersed into DI water for subsequent experiments.

Photosensitizer (RB) loading, NSs-RB: 50 mg of NSs-AEP conjugate, 5 mg of RB, and 600 mg of EDC were mixed in 50 mL DI water. The mixture solution was stirred vigorously overnight. The product was washed with DI water three times and dispersed into 10 mL of PBS buffer (pH = 9.0, 0.1 mol/L).

NSs-RB decorated with FITC label, NSs-RB-FITC: FITC-COOH was covalently conjugated to NSs-RB nanoparticles by using EDC and NHS via an amidation reaction. Briefly, 20 mg of NSs-RB nanoparticles were mixed with 4 mg of FITC-COOH in DMSO. Then, 10 mg of EDC and 25 mg of NHS were added to the mixture and kept stirring in the dark for 24 h. The product was collected and washed with DI water, and finally dispersed into 2 mL of PBS buffer (pH = 7.4).

αvβ3 integrin-targeting peptide cRGD-FITC (F/cRGD) conjugation, NSs-RB-F/cRGD: 10 mg of the F/cRGD peptides was dispersed into 5 mL of PBS buffer (pH = 5.0, 0.1 mol L−1) by stirring for 15 min. Four millimole EDC and NHS were added into the mixture, followed by stirring for 60 min in a dark environment. Then 50 mg of NSs-RB conjugate in 5 mL of PBS buffer (pH = 9.0, 0.1 mol L−1) was added to the F/cRGD mixture and stirred in the dark for 24 h. The product was collected and washed with DI water, and finally dispersed into 5 mL of PBS buffer (pH = 7.4).

X-ray-activated 1O2 generation

NSs-RB (50 μg mL−1), NSs-AEP (50 μg mL−1), and RB (10 μg mL−1) mixed with SOSG (5 μM). DI water was used as a blank control. Hundred microlitres of each group were added separately to a 96-well plate, followed by X-ray irradiation with various doses (0, 1, 2, 4, 6, 8, and 10 Gy) at a dose rate of 1 Gy min−1. The fluorescence intensities (ex/em: 504/525 nm) were quantified by a microplate reader (BioTek). Each group was repeated for five times.

Cell culture

T24 (SCSP-536, Cellosaurus ID:CVCL_0554) human bladder cancer cell line, MCF-7 (SCSP-531, Cellosaurus ID:CVCL_0031) breast cancer cell line, and SV-HUC-1 (TCHu169, Cellosaurus ID:CVCL_3798) human normal bladder epithelial cell line were obtained from Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). 253J (ACL0487, Cellosaurus ID:CVCL_7935) human bladder cancer cell line was obtained from Fengbio company. Two cell lines of T24 and 253J were cultured in RPMI 1640 media with 10% FBS and 1% penicillin/streptomycin. Human normal bladder epithelial cells SV-HUC-1 and breast cancer cells MCF-7 were cultured in DMEM media with 10% FBS and 1% penicillin/streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. All cell lines were authenticated by STR analysis and routinely tested to be free of mycoplasma.

Cytotoxicity

T24 and 253J bladder cancer cells were seeded on 96-well plates (Corning) at 7 × 103 cells/well and cultured for 12 h. The media was replaced with fresh media containing NSs-AEP (0–300 μg mL−1), NSs-RB (0–300 μg mL−1), or NSs-RB-F/cRGD (0–300 μg mL−1), and the cells were further incubated for 24 h. After that, they were washed with PBS and supplemented with fresh media before determining the cell viability by CCK-8 assay using Synergy 2 Multi-Detection Microplate Reader (BioTek).

Cellular uptake of NSs-RB and NSs-RB-F/cRGD

T24, 253J, MCF-7, or SV-HUC-1 cells (5 × 106 cells/well) were seeded in 6-well plates and cultured for 12 h. The media was replaced with fresh media containing NSs-RB (50 μg mL−1) and NSs-RB-F/cRGD (50 μg mL−1) and incubated for 4 h. After washing with PBS twice, 1 mL PBS was added into each well. The fluorescence images and intensity were acquired on a Leica DMi8 fluorescence microscope (ex/em: 460–500/512–542 nm; 540–580/592–668 nm) and flow cytometry (5000 cells, CytoFLEX, Beckman).

NIR-II cellular imaging of NSs-RB and NSs-RB-F/cRGD

T24 and 253J cells (5 × 106 cells/well) were seeded in 6-well plates and cultured for 12 h. The media was replaced with fresh media containing NSs-RB (50 μg mL−1) and NSs-RB-F/cRGD (50 μg mL−1) and incubated for 4 h. After washing with PBS twice, 2.5 mL PBS was added into each well. The NIR-II fluorescence images were acquired on a Micro Vis-600 (NIR OPTICS) fluorescence microscope (ex/em: 808/900–1700 nm).

In vitro X-ray-activated PDT

T24 and 253J cells were seeded on 96-well plates at 7 × 103 cells/well and cultured for 12 h. The media was replaced with fresh media containing NSs-RB-F/cRGD (50 μg mL−1), NSs-RB (50 μg mL−1), NSs-AEP (50 μg mL−1), or RB (10 μg mL−1) and incubated for 4 h. The cells were washed with PBS twice to remove residue materials, then they were exposed to different X-ray doses (0, 1, 2, and 4 Gy) at 1 Gy min−1. The cells were washed with PBS, followed by the supplement of fresh media to allow for a further 24 h incubation. The viability of cells was measured using a CCK-8 assay. PBS group was set as the control. Each group was repeated for five times. Similar procedures were adopted for estimating the cell viabilities of NSs-RB and NSs-RB-F/cRGD from 0 to 200 μg mL−1 at 0, 0.5, 1, 2, and 4 Gy.

Clonogenic assay

T24 cells were seeded on 6-well plates with different amounts (125, 250, 500, 1000, 2000) per well and cultured for 12 h. Fresh media containing NSs-RB-F/cRGD (50 μg mL−1), NSs-RB (50 μg mL−1), and NSs-AEP (50 μg mL−1) were replaced before another incubation for 4 h. Then the cells were exposed to different X-ray doses (0, 1, 2, 4, and 6 Gy) at 1 Gy/min. After that, cells were washed with PBS and incubated with fresh medium every 3 days for 14 days. The colonies were fixed with 4% paraformaldehyde and then stained with Giemsa dye. Only colonies containing at least 50 cells were counted to calculate the colony formation rate. Each group was repeated for three times.

γ-H2AX immunofluorescence analysis

T24 cells were seeded on 6-well plates at 5 × 106 cells/well and cultured for 12 h. Then the cells were incubated with media containing NSs-RB-F/cRGD (50 μg mL−1), NSs-RB (50 μg mL−1), and NSs-AEP (50 μg mL−1), respectively, and followed by 1 Gy dose of X-rays. The cells were incubated for another 30 min and fixed with 4% paraformaldehyde. Then the cells were permeabilized with 0.2% Triton X-100. After that, γ-H2AX rabbit mAb (Abcam, Cat# ab81299, 1:500 dilution) was added and incubated overnight at 4 °C before another incubation with anti-rabbit 488 (Abcam, Cat# ab150113, 1:500 dilution) for 1 h at room temperature. The cells were washed with PBS followed by staining cell nuclei with DAPI. The fluorescence images and intensity of cells were acquired on a Leica DMi8 fluorescence microscope (ex/em: 460–500/512–542 nm; 540–580/592–668 nm).

Quantification of intracellular ROS generation

T24 cells were seeded in 6-well plates and cultured for 12 h. The media was then replaced with fresh media containing NSs-RB-F/cRGD (25 or 50 μg mL−1), NSs-RB (25 or 50 μg mL−1), NSs-AEP (25 or 50 μg mL−1), and RB (10 μg mL−1). After 6 h of incubation and two PBS washes, fresh media containing 10 μM of DHE was added and allowed to incubate for 1 h. After washing with PBS twice, PBS was added, and the cells were exposed to 2 Gy of X-ray. The fluorescence images were acquired on a laser confocal microscope (TCS SP8 STED 3X, Leica). PBS group was set as the control. Each group was repeated three times. For quantification of intracellular 1O2 generation in T24 and 253J, the protocols were the same by just replacing 10 μM of DHE with 5 μM of SOSG.

Imaging flow cytometry

T24 cells (2 × 107 cells) were cultured in petri dishes (Ø = 10 cm) for 12 h and incubated with NSs-RB-F/cRGD (50 μg mL−1) for another 4 h. The cells were exposed to a set dose (0, 0.5, and 1 Gy) of X-rays and then washed with PBS followed by supplementation with fresh medium for further incubation for 6, 12, 18, or 24 h. The cells were then collected and fixed using paraformaldehyde. Imaging flow cytometry (Amnis® ImageStream®XMk II, Luminex) were used to detect cellular structure in health and diseased (1000 cells).

Apoptosis assay

T24 cells (1 × 104 cells) were cultured in confocal dishes (Ø = 15 mm) for 12 h. Cells were incubated with NSs-RB-F/cRGD (50 μg mL−1), NSs-RB (50 μg mL−1), NSs-AEP (50 μg mL−1), and RB (10 μg mL−1) for another 6 h, after which cells were exposed to 0 or 1 Gy of X-ray. The cells were then washed with PBS, followed by the supplement of fresh media to allow for a further 24 h incubation. The cells were then collected and stained using an annexin V-APC/PI dual-staining kit. Cellular apoptosis and necrosis were analyzed using a laser confocal microscope (TCS SP8 STED 3X, Leica) and flow cytometry (10,000 cells, CytoFLEX, Beckman). The PBS group was set as a control.

Western blot

T24 cells were seeded in petri dishes (Ø = 60 mm) and cultured for 12 h, followed by incubating with NSs-RB-F/cRGD (50 μg mL−1), NSs-RB (50 μg mL−1), and NSs-AEP (50 μg mL−1) for another 6 h. Then the cells were exposed to an X-ray of 1 Gy, and they were washed with PBS, followed by the supplement of fresh media to allow for a further 24 h incubation. Cells were lysed in RIPA lysis buffer and the protein concentration of the cell lysates was determined by BCA Protein Assay. Equal amounts of protein were loaded onto SDS-PAGE gel and transferred to PVDF membranes. Western blot was performed using primary antibodies and secondary antibodies conjugated with HRP. For immunoblotting, the following antibodies were used: anti-β-tubulin (Abcam, Cat# ab78078, 1:5000 dilution), anti-cleaved-caspase 3 (Abcam, Cat# ab214430, 1:1000 dilution), anti-Bad (Abcam, Cat# ab32445, 1:1000 dilution), anti-Bax (Abcam, Cat# ab32503, 1:1000 dilution), and anti-Bcl-2 (Abcam, Cat# ab182858, 1:1000 dilution). BIO-RAD GelDoc XR was used to detect the blot bands. Each group was repeated for three times.

Animal experiments

A total of 120 female C57BL/6 mice (a/a, 3 weeks) were provided by Shanghai Lingchang Biotechnology Co., Ltd. All mice were first acclimatized for 1 week and then used for experiments. All mice were housed in SPF-grade facilities, cages with standard conditions (50% relative humidity and 12/12 h light–dark cycle) at 25 °C. All animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (Approved ID: O_A2021143).

Establishment of autochthonous mice bladder tumour model

A total of 120 female C57BL/6 mice (a/a, 3 weeks) were acclimatized for 7 days before the start of the experiment. The mice were anesthetized with 1% pentobarbital sodium. After complete anaesthesia, MNU dissolved in sodium citrate buffer with a concentration of 20 mg mL−1 was administrated through intravesical instillation via epidural catheter within 30 min every other week for a total of four times. Mice needed to be kept sedated for at least 60 min to prevent spontaneous urination and to allow for absorption. Three mice were randomly sacrificed for H&E staining at 7, 14, 21, and 28 days after perfusion to determine the establishment of the MNU-induced bladder tumour mouse models.

Ex vivo NIR-II fluorescence imaging and frozen section

Six female C57BL/6 mice (a/a, 3 weeks) with NMIBC were intravesically instilled with NSs-RB-F/cRGD and NSs-RB-FITC, and three healthy mice were intravesically instilled with NSs-RB-F/cRGD as the control for 1 h. The bladders were then dissected and washed with PBS for in vitro NIR-II fluorescence imaging (808 nm laser) and frozen sections. NIR-II fluorescence of nanoscintillators detected by in vivo imaging systems can be used to evaluate the specific recognition of nanotransducers by bladder tumours in the presence or absence of surface modification. Meanwhile, the green fluorescence of FITC in the frozen section could be acquired by Leica DMi8 fluorescence microscope (ex/em: 460–500/512–542 nm) to show the distribution of nanoscintillators in bladder tumours.

In vivo single-f-PDT

Seventy female C57BL/6 mice (a/a, 3 weeks) with NMIBC were divided into ten groups (n = 7 mice each group). The mice were anesthetized with 1% pentobarbital sodium and instilled intravesically with corresponding solutions. One hour later, PBS buffer was instilled to wash off residual solutions. Then the bladder region of mice was exposed to a predefined X-ray or 650 nm laser dose. Notably, to record the death of mice during the treatment, a combination standard of either real death caused by tumours or the criteria for sacrifice caused by heavy tumour burden with the signatures including being lethargic, poor appetite, limited mobility, and/or weight dropping to about 15 g based on consideration of animal rights and animal welfare and data collection for analysis.

The treatment details of these groups are described as follows:

Group I: healthy mice, PBS (100 μL).

Group II: NMIBC mice, PBS (100 μL).

Group III: NMIBC mice, PBS (100 μL) and irradiated with X-ray (6 Gy).

Group IV: NMIBC mice, RB (1 mg mL−1, 100 μL) and irradiated with X-ray (6 Gy).

Group V: NMIBC mice, NSs-AEP (10 mg mL−1, 100 μL) and irradiated with X-ray (6 Gy).

Group VI: NMIBC mice, NSs-RB (10 mg mL−1, 100 μL) and irradiated with X-ray (6 Gy).

Group VII: NMIBC mice, NSs-RB-F/cRGD (10 mg mL−1, 100 μL).

Group VIII: NMIBC mice, NSs-RB-F/cRGD (10 mg mL−1, 100 μL) and irradiated with X-ray (4 Gy).

Group IX: NMIBC mice, 5-ALA (12 μg mL−1, 100 μL) and irradiated with 650 nm laser (3 W).

Group X: NMIBC mice, NSs-RB-F/cRGD (10 mg mL−1, 100 μL) and irradiated with X-ray (6 Gy).

The X-ray source-to-surface distance was measured as 390 mm with a circular irradiation area (D = 340 mm). A 30 mm-thick lead plate was used to shield the mice with only the pelvis area exposed through a hole to receive X-ray radiation. X-ray beams were generated by the commercially available X-ray irradiator (RS-2000 Pro, Rad Source) at parameters of 160 kV and 250 mA with the beam dose rate to be 1 Gy/min.

In vivo PDT based on 5-ALA

Seven female C57BL/6 mice (a/a, 3 weeks) with NMIBC in Group IX were instilled intravesically with 5-ALA, which could produce endogenous protoporphyrin IX (PpIX) in the tumour. After 4 h, the mice were exposed surgically to the laser by making a small cut to simulate the endoscope techniques in clinical. Bladders were irradiated with laser (650 nm, 3 W) in 15 min. Finally, the incision was surgically sutured and the mice were kept feeding.

In vivo NIR-II fluorescence imaging

The in vivo small animal living imaging system (Suzhou NIR Optics Co., Ltd., China) equipped with a zoom lens assembly (Navitar 6000) was applied for NIR-II fluorescence imaging. An 808 nm diode laser with a power density of 40 mW cm−2 was used as the excitation source. Seventy female C57BL/6 mice (a/a, 3 weeks) with NMIBC in ten treatment groups were instilled intravesically with 100 µL of 10 mg mL−1 NSs-RB-F/cRGD for 1 h, the bladder was flushed with PBS to remove the residual solution. Then NIR-II fluorescence imaging was conducted 30 min after each treatment in the groups of I~X at day 0. Repeated intravesical infusion and NIR-II fluorescence imaging were performed at days 7, 14, and 21. During the whole 4 weeks of the observation period, the body weight and survival rate of all mice were measured and recorded every other day. At the endpoint of the experiment, all ten groups of mice were sacrificed, and the main organs (bladder, heart, liver, spleen, lung, stomach, and kidneys) were collected for subsequent experiments.

Long-term monitoring of bladder tumour recurrence

Fourteen female C57BL/6 mice (a/a, 3 weeks) with NMIBC (n = 7 mice each group, 5-ALA + laser-treated group, XI, like that of group IX; NSs-RB-F/cRGD + 6 Gy-treated group, XII, like that of group X) were set up to monitor the recurrence in various treated mice. During the whole 8 weeks of the observation period, the body weight and survival rate of all mice were measured and recorded every other day. On the 56th day, NIR-II fluorescence imaging was conducted with intravesical instillation of 100 µL of 10 mg mL−1 NSs-RB-F/cRGD. Finally, mice were sacrificed and organs were collected for subsequent experiments.

X-ray-activated NIR-II fluorescence imaging-navigated on-demand fractioned photodynamic therapy

The NIR-II mesoscope was built with the FastFLIM data acquisition unit (Q2, ISS) equipped with an X-ray source (VJ X-ray) and 808-nm laser (MDL-H-808, Changchun New Industries Optoelectronics Technology). The sample was placed on a motorized Z stage and scanned in XY by using a pair of Galvo mirrors (Cambridge Technology). After a 937-nm long pass emission filter (FF01-937/LP-25, Semrock), the emission light was collected by the NIR single photon counting detector (ID220, ID Quantique) via a 62.5 µm multimode fibre. Briefly, ten female C57BL/6 mice (a/a, 3 weeks) with NMIBC (n = 5 mice each group) were administrated by intravesical instillation of 10 mg/mL, 100 µL NSs-RB-F/cRGD. One hour later, PBS buffer was instilled to wash out residual solutions. Then the mice were evaluated by X-ray & 808-nm laser co-activated simultaneous NIR-II fluorescence imaging and fractionated PDT. Fluorescence imaging operation was performed in the 0, 7, 14, and 56 days. Irradiation with different X-ray doses was determined and scheduled according to the quantified SBR of NIR-II imaging. The X-ray source-to-surface distance was measured as 150 mm with a circular irradiation area (D = 100 mm). A 30 mm-thick lead plate was used to shield the mice with only the pelvis area exposed through a hole to receive X-ray radiation. X-ray beams were generated by the commercially available X-ray irradiator (VJ X-ray, China) at parameters of 70 kV and 80 mA with the beam dose rate to be estimated to be 0.025 Gy/min. At the experiment endpoint, all mice were sacrificed and the main organs were collected for subsequent experiments.

Histological and immunohistochemistry analysis

Organs were excised and fixed in paraformaldehyde for H&E staining and immunohistochemistry analysis, followed by examination with a microscope. The pathologic assessment of bladders was analyzed with H&E staining and Ki-67 assessment with the assistance of a trained pathologist based on the criteria of the Health/World International Society of Urological Pathology Organization.

Toxicity assay

Healthy C57BL/6 (5 weeks) mice were divided into two groups (n = 4 mice each group). NSs-RB-F/cRGD (10 mg mL−1) was perfused into mice by intravesical infusion. After 1 and 14 days, blood samples, urine samples, and major organs were dissected from mice for blood routine tests, urine routine tests, and H&E staining analysis. Blood routine test parameters: WBC, white blood cell; Neu, granulocyte; Lym, lymphocyte; Mon, monocytes; Eos, eosinophil; Bas, philicstipplingerythrocyte count; RBC, red blood cell; HGB, haemoglobin; HCT, haematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; RDW-CV, coefficient variation of red blood cell volume distribution width; RDW-SD, standard deviation in red cell distribution width; PLT, platelet count/blood platelet count; MPV, mean platelet volume; PDW, platelet distribution width; PCT, plateletcrit. Urine routine test parameters: WBC, white blood cell; KET, ketone; NIT, nitrite; URO, urobilinogen; BIL, bilirubin; PRO, protein; GLU, glucose; SG, specific gravity; PH, pondus hydrogenii; BLD, blood; CR, creatinine; Ca, calcium; MA, microalbumin.

Immunohistochemistry analysis

Bladders and spleens dissected from NMIBC mice receiving each treatment were excised, fixed in 5% paraformaldehyde, and then subjected to immunohistochemical analysis. Slices were scanned with a digital pathology slide scanner (Aperio GT 450, Leica). The staining intensity and extent in different areas of each slice to estimate the percentage of positive cells (number of positive cells/total number of cells) were scored using the widely accepted German semi-quantitative scoring system that is widely used in literature76–78. Particularly, the intensity of nucleic, cytoplasmic, and membrane is assigned as 0 = no positive staining, 1 = weakly positive in light yellow, 2 = moderately positive in brown, and 3 = strongly positive in sepia. The extent of stained cells is assigned as 0 = 0–5%; 1 = 6–25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100%. The h-score was calculated by the following formulas:1 H−scroe= ∑(pi*i)

pi = percentage of weak/moderate/strong intensity; I = 1, 2, or 3.

Statistics and reproducibility

Origin was used for all data analysis and graph plotting. Data were presented as mean values ± SEM. The results were analyzed by the paired or unpaired two-sided Student’s t-test between two groups. One-way analysis of variance (ANOVA) and multiple comparison was used to determine statistical significance. Exact P values were provided accordingly in the figures or captions. P < 0.05 was used as the threshold for statistical significance; (*) indicates P < 0.05, (**) indicates P < 0.01, and (***) indicates P < 0.001. All statistical analyses were performed with Origin 2018. No statistical method was used to predetermine sample size. The exact number of replicates and statistical tests are indicated in the figure legends. Unless otherwise indicated, n represents the number of independent experimental replicates.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Source data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52607-9.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China, Project No. 82372089 (W.L.), 82272823 (X.Y.), 82273288 (G.Y.) the National Key Research and Development Program of China, Project No. 2017YFA0205304 (W.L.), and the Translational Medicine Research Fund of National Facility for Translational Medicine (Shanghai), Project No. TMSK-2021-117 (W.L.), the Natural Science Foundation of Shanghai (23ZR1434600) (X.Y.). We thank the Instrumental Analysis Center of SJTU for the assistance with optical and TEM characterizations. We also thank Zhejiang Orient Gene Biotech Co., Ltd. for their support.

Author contributions

W.L. supervised and directed the study. L.H., X.Y., G.Y., and W.L. conceived the idea and designed the experiments. L.H. performed nanotransducer synthesis and characterization, and conducted in vitro and in vivo experiments with help from L.W., X.Y., Y.T., and Z.J. The manuscript was written by L.H., X.Y., Z.L., and W.L. G.Y. and L.W. helped polish the manuscript. All authors participated in the discussion and analysis of experimental results and manuscript. L.H. and L.W. contributed equally to this work.

Peer review

Peer review information

Nature Communications thanks Kent Mouw, Kun Wang, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

The authors declare that all relevant data that support the findings of this work are presented in the Article, all remaining data can be found in the Supplementary Information and Source Data files. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Hoogstraten LMC Global trends in the epidemiology of bladder cancer: challenges for public health and clinical practice Nat. Rev. Clin. Oncol. 2023 20 287 304 10.1038/s41571-023-00744-3 36914746
Hoogstraten, L. M. C. et al. Global trends in the epidemiology of bladder cancer: challenges for public health and clinical practice. Nat. Rev. Clin. Oncol. 20, 287–304 (2023).36914746
2. Bosch Svanden Witjes JA Long-term cancer-specific survival in patients with high-risk, non–muscle-invasive bladder cancer and tumour progression: a systematic review Eur. Urol. 2011 60 493 500 10.1016/j.eururo.2011.05.045 21664041
Bosch, S. van den & Witjes, J. A. Long-term cancer-specific survival in patients with high-risk, non–muscle-invasive bladder cancer and tumour progression: a systematic review. Eur. Urol. 60, 493–500 (2011).21664041
3. Kobayashi T Owczarek TB McKiernan JM Abate-Shen C Modelling bladder cancer in mice: opportunities and challenges Nat. Rev. Cancer 2015 15 42 54 10.1038/nrc3858 25533675
Kobayashi, T., Owczarek, T. B., McKiernan, J. M. & Abate-Shen, C. Modelling bladder cancer in mice: opportunities and challenges. Nat. Rev. Cancer 15, 42–54 (2015).25533675
4. Berdik C Unlocking bladder cancer Nature 2017 551 S34 S35 10.1038/551S34a 29117159
Berdik, C. Unlocking bladder cancer. Nature 551, S34–S35 (2017).29117159
5. Tran L Xiao J-F Agarwal N Duex JE Theodorescu D Advances in bladder cancer biology and therapy Nat. Rev. Cancer 2021 21 104 121 10.1038/s41568-020-00313-1 33268841
Tran, L., Xiao, J.-F., Agarwal, N., Duex, J. E. & Theodorescu, D. Advances in bladder cancer biology and therapy. Nat. Rev. Cancer 21, 104–121 (2021).33268841
6. Li X Lovell JF Yoon J Chen X Clinical development and potential of photothermal and photodynamic therapies for cancer Nat. Rev. Clin. Oncol. 2020 17 657 674 10.1038/s41571-020-0410-2 32699309
Li, X., Lovell, J. F., Yoon, J. & Chen, X. Clinical development and potential of photothermal and photodynamic therapies for cancer. Nat. Rev. Clin. Oncol. 17, 657–674 (2020).32699309
7. Burger M Photodynamic diagnosis of non-muscle-invasive bladder cancer with hexaminolevulinate cystoscopy: a meta-analysis of detection and recurrence based on raw data Eur. Urol. 2013 64 846 854 10.1016/j.eururo.2013.03.059 23602406
Burger, M. et al. Photodynamic diagnosis of non-muscle-invasive bladder cancer with hexaminolevulinate cystoscopy: a meta-analysis of detection and recurrence based on raw data. Eur. Urol. 64, 846–854 (2013).23602406
8. Mariappan P Real-life experience: early recurrence with hexvix photodynamic diagnosis–assisted transurethral resection of bladder tumour vs good-quality white light TURBT in new non–muscle-invasive bladder cancer Urology 2015 86 327 331 10.1016/j.urology.2015.04.015 26142924
Mariappan, P. et al. Real-life experience: early recurrence with hexvix photodynamic diagnosis–assisted transurethral resection of bladder tumour vs good-quality white light TURBT in new non–muscle-invasive bladder cancer. Urology 86, 327–331 (2015).26142924
9. Yang B Chen Y Shi J Reactive oxygen species (ROS)-based nanomedicine Chem. Rev. 2019 119 4881 4985 10.1021/acs.chemrev.8b00626 30973011
Yang, B., Chen, Y. & Shi, J. Reactive oxygen species (ROS)-based nanomedicine. Chem. Rev. 119, 4881–4985 (2019).30973011
10. Kwon S Ko H You DG Kataoka K Park JH Nanomedicines for reactive oxygen species mediated approach: an emerging paradigm for cancer treatment Acc. Chem. Res. 2019 52 1771 1782 10.1021/acs.accounts.9b00136 31241894
Kwon, S., Ko, H., You, D. G., Kataoka, K. & Park, J. H. Nanomedicines for reactive oxygen species mediated approach: an emerging paradigm for cancer treatment. Acc. Chem. Res. 52, 1771–1782 (2019).31241894
11. Kelly JF Snell ME Hematoporphyrin derivative: a possible aid in the diagnosis and therapy of carcinoma of the bladder J. Urol. 1976 115 150 151 10.1016/S0022-5347(17)59108-9 1249866
Kelly, J. F. & Snell, M. E. Hematoporphyrin derivative: a possible aid in the diagnosis and therapy of carcinoma of the bladder. J. Urol. 115, 150–151 (1976).1249866
12. Rahman KMM Giram P Foster BA You Y Photodynamic therapy for bladder cancers, a focused review Photochem. Photobiol. 2023 99 420 436 10.1111/php.13726 36138552
Rahman, K. M. M., Giram, P., Foster, B. A. & You, Y. Photodynamic therapy for bladder cancers, a focused review. Photochem. Photobiol. 99, 420–436 (2023).36138552
13. Peng H Unraveling mitochondria-targeting reactive oxygen species modulation and their implementations in cancer therapy by nanomaterials Exploration 2023 3 20220115 10.1002/EXP.20220115 37324035
Peng, H. et al. Unraveling mitochondria-targeting reactive oxygen species modulation and their implementations in cancer therapy by nanomaterials. Exploration 3, 20220115 (2023).37324035
14. Moschini M Characteristics and clinical significance of histological variants of bladder cancer Nat. Rev. Urol. 2017 14 651 668 10.1038/nrurol.2017.125 28895563
Moschini, M. et al. Characteristics and clinical significance of histological variants of bladder cancer. Nat. Rev. Urol. 14, 651–668 (2017).28895563
15. Sun B Wirelessly activated nanotherapeutics for in vivo programmable photodynamic-chemotherapy of orthotopic bladder cancer Adv. Sci. 2022 9 2200731 10.1002/advs.202200731
Sun, B. et al. Wirelessly activated nanotherapeutics for in vivo programmable photodynamic-chemotherapy of orthotopic bladder cancer. Adv. Sci. 9, 2200731 (2022).
16. Hong F Deep NIR-II optical imaging combined with minimally invasive interventional photothermal therapy for orthotopic bladder cancer Chem. Eng. J. 2022 449 137846 10.1016/j.cej.2022.137846
Hong, F. et al. Deep NIR-II optical imaging combined with minimally invasive interventional photothermal therapy for orthotopic bladder cancer. Chem. Eng. J. 449, 137846 (2022).
17. Huang J A renal-clearable macromolecular reporter for near-infrared fluorescence imaging of bladder cancer Angew. Chem. Int. Ed. 2020 59 4415 4420 10.1002/anie.201911859
Huang, J. et al. A renal-clearable macromolecular reporter for near-infrared fluorescence imaging of bladder cancer. Angew. Chem. Int. Ed. 59, 4415–4420 (2020).
18. Dyrskjøt L Bladder cancer Nat. Rev. Dis. Prim. 2023 9 58 10.1038/s41572-023-00468-9 37884563
Dyrskjøt, L. et al. Bladder cancer. Nat. Rev. Dis. Prim. 9, 58 (2023).37884563
19. Chabanon RM Targeting the DNA damage response in immuno-oncology: developments and opportunities Nat. Rev. Cancer 2021 21 701 717 10.1038/s41568-021-00386-6 34376827
Chabanon, R. M. et al. Targeting the DNA damage response in immuno-oncology: developments and opportunities. Nat. Rev. Cancer 21, 701–717 (2021).34376827
20. Petroni G Cantley LC Santambrogio L Formenti SC Galluzzi L Radiotherapy as a tool to elicit clinically actionable signalling pathways in cancer Nat. Rev. Clin. Oncol. 2022 19 114 131 10.1038/s41571-021-00579-w 34819622
Petroni, G., Cantley, L. C., Santambrogio, L., Formenti, S. C. & Galluzzi, L. Radiotherapy as a tool to elicit clinically actionable signalling pathways in cancer. Nat. Rev. Clin. Oncol. 19, 114–131 (2022).34819622
21. Song YP McWilliam A Hoskin PJ Choudhury A Organ preservation in bladder cancer: an opportunity for truly personalized treatment Nat. Rev. Urol. 2019 16 511 522 10.1038/s41585-019-0199-x 31197260
Song, Y. P., McWilliam, A., Hoskin, P. J. & Choudhury, A. Organ preservation in bladder cancer: an opportunity for truly personalized treatment. Nat. Rev. Urol. 16, 511–522 (2019).31197260
22. Thariat J Image-guided radiation therapy for muscle-invasive bladder cancer Nat. Rev. Urol. 2012 9 23 29 10.1038/nrurol.2011.173
Thariat, J. et al. Image-guided radiation therapy for muscle-invasive bladder cancer. Nat. Rev. Urol. 9, 23–29 (2012).
23. Meel R Smart cancer nanomedicine Nat. Nanotechnol. 2019 14 1007 1017 10.1038/s41565-019-0567-y 31695150
Meel, R. et al. Smart cancer nanomedicine. Nat. Nanotechnol. 14, 1007–1017 (2019).31695150
24. Huang J Molecular radio afterglow probes for cancer radiodynamic theranostics Nat. Mater. 2023 22 1421 1429 10.1038/s41563-023-01659-1 37667071
Huang, J. et al. Molecular radio afterglow probes for cancer radiodynamic theranostics. Nat. Mater. 22, 1421–1429 (2023).37667071
25. Shrestha S X-ray induced photodynamic therapy with copper-cysteamine nanoparticles in mice tumors Proc. Natl. Acad. Sci. USA 2019 116 16823 16828 10.1073/pnas.1900502116 31371494
Shrestha, S. et al. X-ray induced photodynamic therapy with copper-cysteamine nanoparticles in mice tumors. Proc. Natl. Acad. Sci. USA 116, 16823–16828 (2019).31371494
26. Pei P X-ray-activated persistent luminescence nanomaterials for NIR-II imaging Nat. Nanotechnol. 2021 16 1011 1018 10.1038/s41565-021-00922-3 34112994
Pei, P. et al. X-ray-activated persistent luminescence nanomaterials for NIR-II imaging. Nat. Nanotechnol. 16, 1011–1018 (2021).34112994
27. Ou X High-resolution X-ray luminescence extension imaging Nature 2021 590 410 415 10.1038/s41586-021-03251-6 33597760
Ou, X. et al. High-resolution X-ray luminescence extension imaging. Nature 590, 410–415 (2021).33597760
28. Bhatia SN Chen X Dobrovolskaia MA Lammers T Cancer nanomedicine Nat. Rev. Cancer 2022 22 550 556 10.1038/s41568-022-00496-9 35941223
Bhatia, S. N., Chen, X., Dobrovolskaia, M. A. & Lammers, T. Cancer nanomedicine. Nat. Rev. Cancer 22, 550–556 (2022).35941223
29. Lei L Manipulation of time-dependent multicolour evolution of X-ray excited afterglow in lanthanide-doped fluoride nanoparticles Nat. Commun. 2022 13 5739 10.1038/s41467-022-33489-1 36180442
Lei, L. et al. Manipulation of time-dependent multicolour evolution of X-ray excited afterglow in lanthanide-doped fluoride nanoparticles. Nat. Commun. 13, 5739 (2022).36180442
30. Hong Z Chen Z Chen Q Yang H Advancing X-ray luminescence for imaging, biosensing, and theragnostics Acc. Chem. Res. 2022 56 37 51 10.1021/acs.accounts.2c00517 36533853
Hong, Z., Chen, Z., Chen, Q. & Yang, H. Advancing X-ray luminescence for imaging, biosensing, and theragnostics. Acc. Chem. Res. 56, 37–51 (2022).36533853
31. Chen Q All-inorganic perovskite nanocrystal scintillators Nature 2018 561 88 93 10.1038/s41586-018-0451-1 30150772
Chen, Q. et al. All-inorganic perovskite nanocrystal scintillators. Nature 561, 88–93 (2018).30150772
32. He L Yu X Li W Recent progress and trends in X-ray-induced photodynamic therapy with low radiation doses ACS Nano 2022 16 19691 19721 10.1021/acsnano.2c07286 36378555
He, L., Yu, X. & Li, W. Recent progress and trends in X-ray-induced photodynamic therapy with low radiation doses. ACS Nano 16, 19691–19721 (2022).36378555
33. Richard C Viana B Persistent X-ray-activated phosphors: mechanisms and applications Light Sci. Appl. 2022 11 123 10.1038/s41377-022-00808-6 35508438
Richard, C. & Viana, B. Persistent X-ray-activated phosphors: mechanisms and applications. Light Sci. Appl. 11, 123 (2022).35508438
34. Fischer S Swabeck JK Alivisatos AP Controlled isotropic and anisotropic shell growth in β-NaLnF4 nanocrystals induced by precursor injection rate J. Am. Chem. Soc. 2017 139 12325 12332 10.1021/jacs.7b07496 28777550
Fischer, S., Swabeck, J. K. & Alivisatos, A. P. Controlled isotropic and anisotropic shell growth in β-NaLnF4 nanocrystals induced by precursor injection rate. J. Am. Chem. Soc. 139, 12325–12332 (2017).28777550
35. Liu D Three-dimensional controlled growth of monodisperse sub-50 nm heterogeneous nanocrystals Nat. Commun. 2016 7 10254 10.1038/ncomms10254 26743184
Liu, D. et al. Three-dimensional controlled growth of monodisperse sub-50 nm heterogeneous nanocrystals. Nat. Commun. 7, 10254 (2016).26743184
36. Peng D Lanthanide-doped energy cascade nanoparticles: full spectrum emission by single wavelength excitation Chem. Mater. 2015 27 3115 3120 10.1021/acs.chemmater.5b00775
Peng, D. et al. Lanthanide-doped energy cascade nanoparticles: full spectrum emission by single wavelength excitation. Chem. Mater. 27, 3115–3120 (2015).
37. Jiang Z Antiangiogenesis combined with inhibition of the hypoxia pathway facilitates low-dose, X-ray-induced photodynamic therapy ACS Nano 2021 15 11112 11125 10.1021/acsnano.1c01063 34170115
Jiang, Z. et al. Antiangiogenesis combined with inhibition of the hypoxia pathway facilitates low-dose, X-ray-induced photodynamic therapy. ACS Nano 15, 11112–11125 (2021).34170115
38. Zhang Y Full shell coating or cation exchange enhances luminescence Nat. Commun. 2021 12 6178 10.1038/s41467-021-26490-7 34702817
Zhang, Y. et al. Full shell coating or cation exchange enhances luminescence. Nat. Commun. 12, 6178 (2021).34702817
39. Gu Y High-sensitivity imaging of time-domain near-infrared light transducer Nat. Photonics 2019 13 525 531 10.1038/s41566-019-0437-z
Gu, Y. et al. High-sensitivity imaging of time-domain near-infrared light transducer. Nat. Photonics 13, 525–531 (2019).
40. Zhong X NaCeF4:Gd,Tb scintillator as an X-ray responsive photosensitizer for multimodal imaging-guided synchronous radio/radiodynamic therapy Nano Lett. 2019 19 8234 8244 10.1021/acs.nanolett.9b03682 31576757
Zhong, X. et al. NaCeF4:Gd,Tb scintillator as an X-ray responsive photosensitizer for multimodal imaging-guided synchronous radio/radiodynamic therapy. Nano Lett. 19, 8234–8244 (2019).31576757
41. Bao Q Hu P Ren W Guo Y Shi J Tumor cell dissociation removes malignant bladder tumors Chem 2020 6 2283 2299 10.1016/j.chempr.2020.06.013
Bao, Q., Hu, P., Ren, W., Guo, Y. & Shi, J. Tumor cell dissociation removes malignant bladder tumors. Chem 6, 2283–2299 (2020).
42. Lee SH Tumor evolution and drug response in patient-derived organoid models of bladder cancer Cell 2018 173 515 528.e17 10.1016/j.cell.2018.03.017 29625057
Lee, S. H. et al. Tumor evolution and drug response in patient-derived organoid models of bladder cancer. Cell 173, 515–528.e17 (2018).29625057
43. Cong Z Magnetic-powered Janus cell robots loaded with oncolytic adenovirus for active and targeted virotherapy of bladder cancer Adv. Mater. 2022 34 e2201042 10.1002/adma.202201042 35452560
Cong, Z. et al. Magnetic-powered Janus cell robots loaded with oncolytic adenovirus for active and targeted virotherapy of bladder cancer. Adv. Mater. 34, e2201042 (2022).35452560
44. An H-W A bispecific glycopeptide spatiotemporally regulates tumor microenvironment for inhibiting bladder cancer recurrence Sci. Adv. 2023 9 eabq8225 10.1126/sciadv.abq8225 36857458
An, H.-W. et al. A bispecific glycopeptide spatiotemporally regulates tumor microenvironment for inhibiting bladder cancer recurrence. Sci. Adv. 9, eabq8225 (2023).36857458
45. Davis GE Affinity of integrins for damaged extracellular matrix: αvβ3 binds to denatured collagen type I through RGD sites Biochem. Biophys. Res. Commun. 1992 182 1025 1031 10.1016/0006-291X(92)91834-D 1540151
Davis, G. E. Affinity of integrins for damaged extracellular matrix: αvβ3 binds to denatured collagen type I through RGD sites. Biochem. Biophys. Res. Commun. 182, 1025–1031 (1992).1540151
46. Desgrosellier JS Cheresh DA Integrins in cancer: biological implications and therapeutic opportunities Nat. Rev. Cancer 2010 10 9 22 10.1038/nrc2748 20029421
Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010).20029421
47. Zhang Y Chen X Gueydan C Han J Plasma membrane changes during programmed cell deaths Cell Res. 2018 28 9 21 10.1038/cr.2017.133 29076500
Zhang, Y., Chen, X., Gueydan, C. & Han, J. Plasma membrane changes during programmed cell deaths. Cell Res. 28, 9–21 (2018).29076500
48. Frese KK Tuveson DA Maximizing mouse cancer models Nat. Rev. Cancer 2007 7 654 658 10.1038/nrc2192
Frese, K. K. & Tuveson, D. A. Maximizing mouse cancer models. Nat. Rev. Cancer 7, 654–658 (2007).
49. Rennick JJ Johnston APR Parton RG Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics Nat. Nanotechnol. 2021 16 266 276 10.1038/s41565-021-00858-8 33712737
Rennick, J. J., Johnston, A. P. R. & Parton, R. G. Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics. Nat. Nanotechnol. 16, 266–276 (2021).33712737
50. Golijanin J Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo Proc. Natl. Acad. Sci. USA 2016 113 11829 11834 10.1073/pnas.1610472113 27688767
Golijanin, J. et al. Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo. Proc. Natl. Acad. Sci. USA 113, 11829–11834 (2016).27688767
51. Wilhelm S Analysis of nanoparticle delivery to tumours Nat. Rev. Mater. 2016 1 16014 10.1038/natrevmats.2016.14
Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 1, 16014 (2016).
52. Liu J Passive tumor targeting of renal-clearable luminescent gold nanoparticles: long tumor retention and fast normal tissue clearance J. Am. Chem. Soc. 2013 135 4978 4981 10.1021/ja401612x 23506476
Liu, J. et al. Passive tumor targeting of renal-clearable luminescent gold nanoparticles: long tumor retention and fast normal tissue clearance. J. Am. Chem. Soc. 135, 4978–4981 (2013).23506476
53. Fowler JF Adams GE Denekamp J Radiosensitizers of hypoxic cells in solid tumours Cancer Treat. Rev. 1976 3 227 256 10.1016/S0305-7372(76)80012-6 1016978
Fowler, J. F., Adams, G. E. & Denekamp, J. Radiosensitizers of hypoxic cells in solid tumours. Cancer Treat. Rev. 3, 227–256 (1976).1016978
54. Gill MR Vallis KA Transition metal compounds as cancer radiosensitizers Chem. Soc. Rev. 2018 48 540 557 10.1039/C8CS00641E 30499573
Gill, M. R. & Vallis, K. A. Transition metal compounds as cancer radiosensitizers. Chem. Soc. Rev. 48, 540–557 (2018).30499573
55. Lv B Structure-oriented catalytic radiosensitization for cancer radiotherapy Nano Today 2020 35 100988 10.1016/j.nantod.2020.100988
Lv, B. et al. Structure-oriented catalytic radiosensitization for cancer radiotherapy. Nano Today 35, 100988 (2020).
56. Sun W Gadolinium-Rose Bengal coordination polymer nanodots for MR-/fluorescence-image-guided radiation and photodynamic therapy Adv. Mater. 2020 32 e2000377 10.1002/adma.202000377 32363649
Sun, W. et al. Gadolinium-Rose Bengal coordination polymer nanodots for MR-/fluorescence-image-guided radiation and photodynamic therapy. Adv. Mater. 32, e2000377 (2020).32363649
57. Diehn M Association of reactive oxygen species levels and radioresistance in cancer stem cells Nature 2009 458 780 783 10.1038/nature07733 19194462
Diehn, M. et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 458, 780–783 (2009).19194462
58. Castano AP Mroz P Hamblin MR Photodynamic therapy and anti-tumour immunity Nat. Rev. Cancer 2006 6 535 545 10.1038/nrc1894 16794636
Castano, A. P., Mroz, P. & Hamblin, M. R. Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer 6, 535–545 (2006).16794636
59. Liu X The CUL4B-miR-372/373-PIK3CA-AKT axis regulates metastasis in bladder cancer Oncogene 2020 39 3588 3603 10.1038/s41388-020-1236-1 32127645
Liu, X. et al. The CUL4B-miR-372/373-PIK3CA-AKT axis regulates metastasis in bladder cancer. Oncogene 39, 3588–3603 (2020).32127645
60. Lee P Chandel NS Simon MC Cellular adaptation to hypoxia through hypoxia inducible factors and beyond Nat. Rev. Mol. Cell Biol. 2020 21 268 283 10.1038/s41580-020-0227-y 32144406
Lee, P., Chandel, N. S. & Simon, M. C. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond. Nat. Rev. Mol. Cell Biol. 21, 268–283 (2020).32144406
61. Karnoub AE Mesenchymal stem cells within tumour stroma promote breast cancer metastasis Nature 2007 449 557 563 10.1038/nature06188 17914389
Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449, 557–563 (2007).17914389
62. Potter DS Dynamic BH3 profiling identifies pro-apoptotic drug combinations for the treatment of malignant pleural mesothelioma Nat. Commun. 2023 14 2897 10.1038/s41467-023-38552-z 37210412
Potter, D. S. et al. Dynamic BH3 profiling identifies pro-apoptotic drug combinations for the treatment of malignant pleural mesothelioma. Nat. Commun. 14, 2897 (2023).37210412
63. Mir MA Tirumkudulu MS A low-cost flow cell for flow cytometry Biosens. Bioelectron. 2022 211 114334 10.1016/j.bios.2022.114334 35588635
Mir, M. A. & Tirumkudulu, M. S. A low-cost flow cell for flow cytometry. Biosens. Bioelectron. 211, 114334 (2022).35588635
64. Rees P Summers HD Filby A Carpenter AE Doan M Imaging flow cytometry Nat. Rev. Methods Prim. 2022 2 87 10.1038/s43586-022-00182-y
Rees, P., Summers, H. D., Filby, A., Carpenter, A. E. & Doan, M. Imaging flow cytometry. Nat. Rev. Methods Prim. 2, 87 (2022).
65. Morrissey P Cell classification in human peripheral blood using the Amnis ImageStream® system Blood 2004 104 3826 10.1182/blood.V104.11.3826.3826
Morrissey, P. et al. Cell classification in human peripheral blood using the Amnis ImageStream® system. Blood 104, 3826 (2004).
66. Nössing C Ryan KM 50 years on and still very much alive: ‘Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics Br. J. Cancer 2023 128 426 431 10.1038/s41416-022-02020-0 36369364
Nössing, C. & Ryan, K. M. 50 years on and still very much alive: ‘Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 128, 426–431 (2023).36369364
67. Hotchkiss RS Strasser A McDunn JE Swanson PE Cell death N. Engl. J. Med. 2009 361 1570 1583 10.1056/NEJMra0901217 19828534
Hotchkiss, R. S., Strasser, A., McDunn, J. E. & Swanson, P. E. Cell death. N. Engl. J. Med. 361, 1570–1583 (2009).19828534
68. Sengupta S Harris CC p53: traffic cop at the crossroads of DNA repair and recombination Nat. Rev. Mol. Cell Biol. 2005 6 44 55 10.1038/nrm1546 15688066
Sengupta, S. & Harris, C. C. p53: traffic cop at the crossroads of DNA repair and recombination. Nat. Rev. Mol. Cell Biol. 6, 44–55 (2005).15688066
69. Hafeez S Protocol for hypofractionated adaptive radiotherapy to the bladder within a multicentre phase II randomised trial: radiotherapy planning and delivery guidance BMJ Open 2020 10 e037134 10.1136/bmjopen-2020-037134 32461298
Hafeez, S. et al. Protocol for hypofractionated adaptive radiotherapy to the bladder within a multicentre phase II randomised trial: radiotherapy planning and delivery guidance. BMJ Open 10, e037134 (2020).32461298
70. Yu X CT/MRI-guided synergistic radiotherapy and X-ray inducible photodynamic therapy using Tb-doped Gd-W-nanoscintillators Angew. Chem. Int. Ed. 2019 58 2017 2022 10.1002/anie.201812272
Yu, X. et al. CT/MRI-guided synergistic radiotherapy and X-ray inducible photodynamic therapy using Tb-doped Gd-W-nanoscintillators. Angew. Chem. Int. Ed. 58, 2017–2022 (2019).
71. Waidelich R Clinical experience with 5-aminolevulinic acid and photodynamic therapy for refractory superficial bladder cancer J. Urol. 2001 165 1904 1907 10.1016/S0022-5347(05)66239-8 11371878
Waidelich, R. et al. Clinical experience with 5-aminolevulinic acid and photodynamic therapy for refractory superficial bladder cancer. J. Urol. 165, 1904–1907 (2001).11371878
72. Yamamoto S Fukuhara H Karashima T Inoue K Real-world experience with 5-aminolevulinic acid for the photodynamic diagnosis of bladder cancer: diagnostic accuracy and safety Photodiagnosis Photodyn. Ther. 2020 32 101999 10.1016/j.pdpdt.2020.101999 32920180
Yamamoto, S., Fukuhara, H., Karashima, T. & Inoue, K. Real-world experience with 5-aminolevulinic acid for the photodynamic diagnosis of bladder cancer: diagnostic accuracy and safety. Photodiagnosis Photodyn. Ther. 32, 101999 (2020).32920180
73. Kaufman DS Shipley WU Feldman AS Bladder cancer Lancet 2009 374 239 249 10.1016/S0140-6736(09)60491-8 19520422
Kaufman, D. S., Shipley, W. U. & Feldman, A. S. Bladder cancer. Lancet 374, 239–249 (2009).19520422
74. Ino Y Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer Br. J. Cancer 2013 108 914 923 10.1038/bjc.2013.32 23385730
Ino, Y. et al. Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br. J. Cancer 108, 914–923 (2013).23385730
75. Goubet A-G Rouanne M Derosa L Kroemer G Zitvogel L From mucosal infection to successful cancer immunotherapy Nat. Rev. Urol. 2023 20 682 700 10.1038/s41585-023-00784-5 37433926
Goubet, A.-G., Rouanne, M., Derosa, L., Kroemer, G. & Zitvogel, L. From mucosal infection to successful cancer immunotherapy. Nat. Rev. Urol. 20, 682–700 (2023).37433926
76. Hu J-J Lei Q Zhang X-Z Recent advances in photonanomedicines for enhanced cancer photodynamic therapy Prog. Mater. Sci. 2020 114 100685 10.1016/j.pmatsci.2020.100685
Hu, J.-J., Lei, Q. & Zhang, X.-Z. Recent advances in photonanomedicines for enhanced cancer photodynamic therapy. Prog. Mater. Sci. 114, 100685 (2020).
77. Xie P The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis Nat. Commun. 2014 5 3733 10.1038/ncomms4733 24821572
Xie, P. et al. The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis. Nat. Commun. 5, 3733 (2014).24821572
78. Paschalis A Prostate-specific membrane antigen heterogeneity and DNA repair defects in prostate cancer Eur. Urol. 2019 76 469 478 10.1016/j.eururo.2019.06.030 31345636
Paschalis, A. et al. Prostate-specific membrane antigen heterogeneity and DNA repair defects in prostate cancer. Eur. Urol. 76, 469–478 (2019).31345636
79. Hu Y Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification Mol. Cancer 2022 21 34 10.1186/s12943-022-01522-y 35114989
Hu, Y. et al. Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol. Cancer 21, 34 (2022).35114989
80. Alexandre YO Mueller SN Splenic stromal niches in homeostasis and immunity Nat. Rev. Immunol. 2023 23 705 719 10.1038/s41577-023-00857-x 36973361
Alexandre, Y. O. & Mueller, S. N. Splenic stromal niches in homeostasis and immunity. Nat. Rev. Immunol. 23, 705–719 (2023).36973361
81. Parkin J Cohen B An overview of the immune system Lancet 2001 357 1777 1789 10.1016/S0140-6736(00)04904-7 11403834
Parkin, J. & Cohen, B. An overview of the immune system. Lancet 357, 1777–1789 (2001).11403834
