Full-course NIR-II imaging-navigated fractionated photodynamic therapy of bladder tumours with X-ray-activated nanotransducers

Liangrui He; Liyang Wang; Xujiang Yu; Yizhang Tang; Zhao Jiang; Guoliang Yang; Zhuang Liu; Wanwan Li

doi:10.1038/s41467-024-52607-9

. 2024 Sep 19;15:8240. doi: 10.1038/s41467-024-52607-9

Full-course NIR-II imaging-navigated fractionated photodynamic therapy of bladder tumours with X-ray-activated nanotransducers

Liangrui He ¹, Liyang Wang ², Xujiang Yu ^1,^✉, Yizhang Tang ¹, Zhao Jiang ¹, Guoliang Yang ^2,^✉, Zhuang Liu ^3,^✉, Wanwan Li ^1,^✉

PMCID: PMC11413067 PMID: 39300124

Abstract

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.

Subject terms: Nanotechnology in cancer, Bladder cancer, Cancer imaging

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.

Introduction

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 radiation^2,3. 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 death^6–10. 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 worldwide^12,13. 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 cancer^16,17. 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 cancer^21,22. “Nanomedicine” approaches to package imaging and/or therapeutic moieties have emerged with innovative strategies to ascend cancer treatment^23,24. 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 translations^23,27,28. 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 absorption^29–33 (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 ⁴F_3/2–⁴I_9/2, ⁴F_3/2–⁴I_11/2, and ⁴F_3/2–⁴I_13/2 transitions of Nd³⁺, respectively^26,27. Due to the better passivation effect of isotropic epitaxial growth on diminishing nonradiative relaxation and surface quenching^34,35, 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-Ns^34,35. 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 — 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 matrix^36,37. 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 yet^29,40. 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 models^42,43, 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 — 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 α_vβ₃ integrins that are highly expressed in tumour neovascularization endothelial cells and cancer cells compared to normal cells^24,45–47, 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 lines^48,49, we estimated specific targeting effect and efficient accumulation of F/cRGD-labelled nanotransducers directly with the mouse models^44,50–52. 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 α_vβ₃ integrin expression were much lower than those in bladder cancer cells of T24 with high α_vβ₃ integrin expression (Supplementary Fig. 16), indicating the high affinity of F/cRGD peptides to overexpressed α_vβ₃ 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).

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-rays^53,54 (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 lines^59,60. 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 — 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 activation^61,62. 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 endocytosis^63–65. 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 apoptosis^47,66,67. 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 — 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 analyzed^74,75, given that ROS and radiation have been both reported to remodel tumour microenvironment and/or elicit systemic immunity^58,76. 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 system^77–79 (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 groups^80,81 (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 — 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.

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).

α_vβ₃ 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 literature^76–78. 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:

H - scroe = \sum (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^{(23.9MB, pdf)}

Peer Review File^{(24.9MB, pdf)}

Reporting Summary^{(3MB, pdf)}

Source data

Source data^{(2.1MB, xlsx)}

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.

Footnotes

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

Contributor Information

Xujiang Yu, Email: yuxuj1017@sjtu.edu.cn.

Guoliang Yang, Email: ygl0511@126.com.

Zhuang Liu, Email: zliu@suda.edu.cn.

Wanwan Li, Email: wwli@sjtu.edu.cn.

Supplementary information

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

References

1.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). [DOI] [PubMed] [Google Scholar]
2.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). [DOI] [PubMed] [Google Scholar]
3.Kobayashi, T., Owczarek, T. B., McKiernan, J. M. & Abate-Shen, C. Modelling bladder cancer in mice: opportunities and challenges. Nat. Rev. Cancer15, 42–54 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Berdik, C. Unlocking bladder cancer. Nature551, S34–S35 (2017). [DOI] [PubMed] [Google Scholar]
5.Tran, L., Xiao, J.-F., Agarwal, N., Duex, J. E. & Theodorescu, D. Advances in bladder cancer biology and therapy. Nat. Rev. Cancer21, 104–121 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
6.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). [DOI] [PubMed] [Google Scholar]
7.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). [DOI] [PubMed] [Google Scholar]
8.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. Urology86, 327–331 (2015). [DOI] [PubMed] [Google Scholar]
9.Yang, B., Chen, Y. & Shi, J. Reactive oxygen species (ROS)-based nanomedicine. Chem. Rev.119, 4881–4985 (2019). [DOI] [PubMed] [Google Scholar]
10.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). [DOI] [PubMed] [Google Scholar]
11.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). [DOI] [PubMed] [Google Scholar]
12.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). [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Peng, H. et al. Unraveling mitochondria-targeting reactive oxygen species modulation and their implementations in cancer therapy by nanomaterials. Exploration3, 20220115 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Moschini, M. et al. Characteristics and clinical significance of histological variants of bladder cancer. Nat. Rev. Urol.14, 651–668 (2017). [DOI] [PubMed] [Google Scholar]
15.Sun, B. et al. Wirelessly activated nanotherapeutics for in vivo programmable photodynamic-chemotherapy of orthotopic bladder cancer. Adv. Sci.9, 2200731 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
16.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). [Google Scholar]
17.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). [DOI] [PubMed] [Google Scholar]
18.Dyrskjøt, L. et al. Bladder cancer. Nat. Rev. Dis. Prim.9, 58 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Chabanon, R. M. et al. Targeting the DNA damage response in immuno-oncology: developments and opportunities. Nat. Rev. Cancer21, 701–717 (2021). [DOI] [PubMed] [Google Scholar]
20.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). [DOI] [PMC free article] [PubMed] [Google Scholar]
21.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). [DOI] [PubMed] [Google Scholar]
22.Thariat, J. et al. Image-guided radiation therapy for muscle-invasive bladder cancer. Nat. Rev. Urol.9, 23–29 (2012). [DOI] [PubMed] [Google Scholar]
23.Meel, R. et al. Smart cancer nanomedicine. Nat. Nanotechnol.14, 1007–1017 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Huang, J. et al. Molecular radio afterglow probes for cancer radiodynamic theranostics. Nat. Mater.22, 1421–1429 (2023). [DOI] [PubMed] [Google Scholar]
25.Shrestha, S. et al. X-ray induced photodynamic therapy with copper-cysteamine nanoparticles in mice tumors. Proc. Natl. Acad. Sci. USA116, 16823–16828 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Pei, P. et al. X-ray-activated persistent luminescence nanomaterials for NIR-II imaging. Nat. Nanotechnol.16, 1011–1018 (2021). [DOI] [PubMed] [Google Scholar]
27.Ou, X. et al. High-resolution X-ray luminescence extension imaging. Nature590, 410–415 (2021). [DOI] [PubMed] [Google Scholar]
28.Bhatia, S. N., Chen, X., Dobrovolskaia, M. A. & Lammers, T. Cancer nanomedicine. Nat. Rev. Cancer22, 550–556 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
29.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). [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Hong, Z., Chen, Z., Chen, Q. & Yang, H. Advancing X-ray luminescence for imaging, biosensing, and theragnostics. Acc. Chem. Res.56, 37–51 (2022). [DOI] [PubMed] [Google Scholar]
31.Chen, Q. et al. All-inorganic perovskite nanocrystal scintillators. Nature561, 88–93 (2018). [DOI] [PubMed] [Google Scholar]
32.He, L., Yu, X. & Li, W. Recent progress and trends in X-ray-induced photodynamic therapy with low radiation doses. ACS Nano16, 19691–19721 (2022). [DOI] [PubMed] [Google Scholar]
33.Richard, C. & Viana, B. Persistent X-ray-activated phosphors: mechanisms and applications. Light Sci. Appl.11, 123 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
34.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). [DOI] [PubMed] [Google Scholar]
35.Liu, D. et al. Three-dimensional controlled growth of monodisperse sub-50 nm heterogeneous nanocrystals. Nat. Commun.7, 10254 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Peng, D. et al. Lanthanide-doped energy cascade nanoparticles: full spectrum emission by single wavelength excitation. Chem. Mater.27, 3115–3120 (2015). [Google Scholar]
37.Jiang, Z. et al. Antiangiogenesis combined with inhibition of the hypoxia pathway facilitates low-dose, X-ray-induced photodynamic therapy. ACS Nano15, 11112–11125 (2021). [DOI] [PubMed] [Google Scholar]
38.Zhang, Y. et al. Full shell coating or cation exchange enhances luminescence. Nat. Commun.12, 6178 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Gu, Y. et al. High-sensitivity imaging of time-domain near-infrared light transducer. Nat. Photonics13, 525–531 (2019). [Google Scholar]
40.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). [DOI] [PubMed] [Google Scholar]
41.Bao, Q., Hu, P., Ren, W., Guo, Y. & Shi, J. Tumor cell dissociation removes malignant bladder tumors. Chem6, 2283–2299 (2020). [Google Scholar]
42.Lee, S. H. et al. Tumor evolution and drug response in patient-derived organoid models of bladder cancer. Cell173, 515–528.e17 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
43.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). [DOI] [PubMed] [Google Scholar]
44.An, H.-W. et al. A bispecific glycopeptide spatiotemporally regulates tumor microenvironment for inhibiting bladder cancer recurrence. Sci. Adv.9, eabq8225 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Davis, G. E. Affinity of integrins for damaged extracellular matrix: α_vβ₃ binds to denatured collagen type I through RGD sites. Biochem. Biophys. Res. Commun.182, 1025–1031 (1992). [DOI] [PubMed] [Google Scholar]
46.Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer10, 9–22 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Zhang, Y., Chen, X., Gueydan, C. & Han, J. Plasma membrane changes during programmed cell deaths. Cell Res.28, 9–21 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Frese, K. K. & Tuveson, D. A. Maximizing mouse cancer models. Nat. Rev. Cancer7, 654–658 (2007). [DOI] [PubMed] [Google Scholar]
49.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). [DOI] [PubMed] [Google Scholar]
50.Golijanin, J. et al. Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo. Proc. Natl. Acad. Sci. USA113, 11829–11834 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater.1, 16014 (2016). [Google Scholar]
52.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). [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Fowler, J. F., Adams, G. E. & Denekamp, J. Radiosensitizers of hypoxic cells in solid tumours. Cancer Treat. Rev.3, 227–256 (1976). [DOI] [PubMed] [Google Scholar]
54.Gill, M. R. & Vallis, K. A. Transition metal compounds as cancer radiosensitizers. Chem. Soc. Rev.48, 540–557 (2018). [DOI] [PubMed] [Google Scholar]
55.Lv, B. et al. Structure-oriented catalytic radiosensitization for cancer radiotherapy. Nano Today35, 100988 (2020). [Google Scholar]
56.Sun, W. et al. Gadolinium-Rose Bengal coordination polymer nanodots for MR-/fluorescence-image-guided radiation and photodynamic therapy. Adv. Mater.32, e2000377 (2020). [DOI] [PubMed] [Google Scholar]
57.Diehn, M. et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature458, 780–783 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Castano, A. P., Mroz, P. & Hamblin, M. R. Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer6, 535–545 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Liu, X. et al. The CUL4B-miR-372/373-PIK3CA-AKT axis regulates metastasis in bladder cancer. Oncogene39, 3588–3603 (2020). [DOI] [PubMed] [Google Scholar]
60.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). [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature449, 557–563 (2007). [DOI] [PubMed] [Google Scholar]
62.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). [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Mir, M. A. & Tirumkudulu, M. S. A low-cost flow cell for flow cytometry. Biosens. Bioelectron.211, 114334 (2022). [DOI] [PubMed] [Google Scholar]
64.Rees, P., Summers, H. D., Filby, A., Carpenter, A. E. & Doan, M. Imaging flow cytometry. Nat. Rev. Methods Prim.2, 87 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Morrissey, P. et al. Cell classification in human peripheral blood using the Amnis ImageStream® system. Blood104, 3826 (2004). [Google Scholar]
66.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. Cancer128, 426–431 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Hotchkiss, R. S., Strasser, A., McDunn, J. E. & Swanson, P. E. Cell death. N. Engl. J. Med.361, 1570–1583 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
68.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). [DOI] [PubMed] [Google Scholar]
69.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 Open10, e037134 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
70.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). [DOI] [PubMed] [Google Scholar]
71.Waidelich, R. et al. Clinical experience with 5-aminolevulinic acid and photodynamic therapy for refractory superficial bladder cancer. J. Urol.165, 1904–1907 (2001). [DOI] [PubMed] [Google Scholar]
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.32, 101999 (2020). [DOI] [PubMed] [Google Scholar]
73.Kaufman, D. S., Shipley, W. U. & Feldman, A. S. Bladder cancer. Lancet374, 239–249 (2009). [DOI] [PubMed] [Google Scholar]
74.Ino, Y. et al. Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br. J. Cancer108, 914–923 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
75.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). [DOI] [PubMed] [Google Scholar]
76.Hu, J.-J., Lei, Q. & Zhang, X.-Z. Recent advances in photonanomedicines for enhanced cancer photodynamic therapy. Prog. Mater. Sci.114, 100685 (2020). [Google Scholar]
77.Xie, P. et al. The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis. Nat. Commun.5, 3733 (2014). [DOI] [PubMed] [Google Scholar]
78.Paschalis, A. et al. Prostate-specific membrane antigen heterogeneity and DNA repair defects in prostate cancer. Eur. Urol.76, 469–478 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Hu, Y. et al. Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol. Cancer21, 34 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
80.Alexandre, Y. O. & Mueller, S. N. Splenic stromal niches in homeostasis and immunity. Nat. Rev. Immunol.23, 705–719 (2023). [DOI] [PubMed] [Google Scholar]
81.Parkin, J. & Cohen, B. An overview of the immune system. Lancet357, 1777–1789 (2001). [DOI] [PubMed] [Google Scholar]

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Data Availability Statement

[CR1] 1.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). [DOI] [PubMed] [Google Scholar]

[CR2] 2.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). [DOI] [PubMed] [Google Scholar]

[CR3] 3.Kobayashi, T., Owczarek, T. B., McKiernan, J. M. & Abate-Shen, C. Modelling bladder cancer in mice: opportunities and challenges. Nat. Rev. Cancer15, 42–54 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Berdik, C. Unlocking bladder cancer. Nature551, S34–S35 (2017). [DOI] [PubMed] [Google Scholar]

[CR5] 5.Tran, L., Xiao, J.-F., Agarwal, N., Duex, J. E. & Theodorescu, D. Advances in bladder cancer biology and therapy. Nat. Rev. Cancer21, 104–121 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.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). [DOI] [PubMed] [Google Scholar]

[CR7] 7.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). [DOI] [PubMed] [Google Scholar]

[CR8] 8.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. Urology86, 327–331 (2015). [DOI] [PubMed] [Google Scholar]

[CR9] 9.Yang, B., Chen, Y. & Shi, J. Reactive oxygen species (ROS)-based nanomedicine. Chem. Rev.119, 4881–4985 (2019). [DOI] [PubMed] [Google Scholar]

[CR10] 10.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). [DOI] [PubMed] [Google Scholar]

[CR11] 11.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). [DOI] [PubMed] [Google Scholar]

[CR12] 12.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). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Peng, H. et al. Unraveling mitochondria-targeting reactive oxygen species modulation and their implementations in cancer therapy by nanomaterials. Exploration3, 20220115 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Moschini, M. et al. Characteristics and clinical significance of histological variants of bladder cancer. Nat. Rev. Urol.14, 651–668 (2017). [DOI] [PubMed] [Google Scholar]

[CR15] 15.Sun, B. et al. Wirelessly activated nanotherapeutics for in vivo programmable photodynamic-chemotherapy of orthotopic bladder cancer. Adv. Sci.9, 2200731 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.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). [Google Scholar]

[CR17] 17.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). [DOI] [PubMed] [Google Scholar]

[CR18] 18.Dyrskjøt, L. et al. Bladder cancer. Nat. Rev. Dis. Prim.9, 58 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Chabanon, R. M. et al. Targeting the DNA damage response in immuno-oncology: developments and opportunities. Nat. Rev. Cancer21, 701–717 (2021). [DOI] [PubMed] [Google Scholar]

[CR20] 20.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). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.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). [DOI] [PubMed] [Google Scholar]

[CR22] 22.Thariat, J. et al. Image-guided radiation therapy for muscle-invasive bladder cancer. Nat. Rev. Urol.9, 23–29 (2012). [DOI] [PubMed] [Google Scholar]

[CR23] 23.Meel, R. et al. Smart cancer nanomedicine. Nat. Nanotechnol.14, 1007–1017 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Huang, J. et al. Molecular radio afterglow probes for cancer radiodynamic theranostics. Nat. Mater.22, 1421–1429 (2023). [DOI] [PubMed] [Google Scholar]

[CR25] 25.Shrestha, S. et al. X-ray induced photodynamic therapy with copper-cysteamine nanoparticles in mice tumors. Proc. Natl. Acad. Sci. USA116, 16823–16828 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Pei, P. et al. X-ray-activated persistent luminescence nanomaterials for NIR-II imaging. Nat. Nanotechnol.16, 1011–1018 (2021). [DOI] [PubMed] [Google Scholar]

[CR27] 27.Ou, X. et al. High-resolution X-ray luminescence extension imaging. Nature590, 410–415 (2021). [DOI] [PubMed] [Google Scholar]

[CR28] 28.Bhatia, S. N., Chen, X., Dobrovolskaia, M. A. & Lammers, T. Cancer nanomedicine. Nat. Rev. Cancer22, 550–556 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.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). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Hong, Z., Chen, Z., Chen, Q. & Yang, H. Advancing X-ray luminescence for imaging, biosensing, and theragnostics. Acc. Chem. Res.56, 37–51 (2022). [DOI] [PubMed] [Google Scholar]

[CR31] 31.Chen, Q. et al. All-inorganic perovskite nanocrystal scintillators. Nature561, 88–93 (2018). [DOI] [PubMed] [Google Scholar]

[CR32] 32.He, L., Yu, X. & Li, W. Recent progress and trends in X-ray-induced photodynamic therapy with low radiation doses. ACS Nano16, 19691–19721 (2022). [DOI] [PubMed] [Google Scholar]

[CR33] 33.Richard, C. & Viana, B. Persistent X-ray-activated phosphors: mechanisms and applications. Light Sci. Appl.11, 123 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.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). [DOI] [PubMed] [Google Scholar]

[CR35] 35.Liu, D. et al. Three-dimensional controlled growth of monodisperse sub-50 nm heterogeneous nanocrystals. Nat. Commun.7, 10254 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Peng, D. et al. Lanthanide-doped energy cascade nanoparticles: full spectrum emission by single wavelength excitation. Chem. Mater.27, 3115–3120 (2015). [Google Scholar]

[CR37] 37.Jiang, Z. et al. Antiangiogenesis combined with inhibition of the hypoxia pathway facilitates low-dose, X-ray-induced photodynamic therapy. ACS Nano15, 11112–11125 (2021). [DOI] [PubMed] [Google Scholar]

[CR38] 38.Zhang, Y. et al. Full shell coating or cation exchange enhances luminescence. Nat. Commun.12, 6178 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Gu, Y. et al. High-sensitivity imaging of time-domain near-infrared light transducer. Nat. Photonics13, 525–531 (2019). [Google Scholar]

[CR40] 40.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). [DOI] [PubMed] [Google Scholar]

[CR41] 41.Bao, Q., Hu, P., Ren, W., Guo, Y. & Shi, J. Tumor cell dissociation removes malignant bladder tumors. Chem6, 2283–2299 (2020). [Google Scholar]

[CR42] 42.Lee, S. H. et al. Tumor evolution and drug response in patient-derived organoid models of bladder cancer. Cell173, 515–528.e17 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.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). [DOI] [PubMed] [Google Scholar]

[CR44] 44.An, H.-W. et al. A bispecific glycopeptide spatiotemporally regulates tumor microenvironment for inhibiting bladder cancer recurrence. Sci. Adv.9, eabq8225 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Davis, G. E. Affinity of integrins for damaged extracellular matrix: α_vβ₃ binds to denatured collagen type I through RGD sites. Biochem. Biophys. Res. Commun.182, 1025–1031 (1992). [DOI] [PubMed] [Google Scholar]

[CR46] 46.Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer10, 9–22 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR47] 47.Zhang, Y., Chen, X., Gueydan, C. & Han, J. Plasma membrane changes during programmed cell deaths. Cell Res.28, 9–21 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR48] 48.Frese, K. K. & Tuveson, D. A. Maximizing mouse cancer models. Nat. Rev. Cancer7, 654–658 (2007). [DOI] [PubMed] [Google Scholar]

[CR49] 49.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). [DOI] [PubMed] [Google Scholar]

[CR50] 50.Golijanin, J. et al. Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo. Proc. Natl. Acad. Sci. USA113, 11829–11834 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR51] 51.Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater.1, 16014 (2016). [Google Scholar]

[CR52] 52.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). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR53] 53.Fowler, J. F., Adams, G. E. & Denekamp, J. Radiosensitizers of hypoxic cells in solid tumours. Cancer Treat. Rev.3, 227–256 (1976). [DOI] [PubMed] [Google Scholar]

[CR54] 54.Gill, M. R. & Vallis, K. A. Transition metal compounds as cancer radiosensitizers. Chem. Soc. Rev.48, 540–557 (2018). [DOI] [PubMed] [Google Scholar]

[CR55] 55.Lv, B. et al. Structure-oriented catalytic radiosensitization for cancer radiotherapy. Nano Today35, 100988 (2020). [Google Scholar]

[CR56] 56.Sun, W. et al. Gadolinium-Rose Bengal coordination polymer nanodots for MR-/fluorescence-image-guided radiation and photodynamic therapy. Adv. Mater.32, e2000377 (2020). [DOI] [PubMed] [Google Scholar]

[CR57] 57.Diehn, M. et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature458, 780–783 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR58] 58.Castano, A. P., Mroz, P. & Hamblin, M. R. Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer6, 535–545 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR59] 59.Liu, X. et al. The CUL4B-miR-372/373-PIK3CA-AKT axis regulates metastasis in bladder cancer. Oncogene39, 3588–3603 (2020). [DOI] [PubMed] [Google Scholar]

[CR60] 60.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). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR61] 61.Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature449, 557–563 (2007). [DOI] [PubMed] [Google Scholar]

[CR62] 62.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). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR63] 63.Mir, M. A. & Tirumkudulu, M. S. A low-cost flow cell for flow cytometry. Biosens. Bioelectron.211, 114334 (2022). [DOI] [PubMed] [Google Scholar]

[CR64] 64.Rees, P., Summers, H. D., Filby, A., Carpenter, A. E. & Doan, M. Imaging flow cytometry. Nat. Rev. Methods Prim.2, 87 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR65] 65.Morrissey, P. et al. Cell classification in human peripheral blood using the Amnis ImageStream® system. Blood104, 3826 (2004). [Google Scholar]

[CR66] 66.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. Cancer128, 426–431 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR67] 67.Hotchkiss, R. S., Strasser, A., McDunn, J. E. & Swanson, P. E. Cell death. N. Engl. J. Med.361, 1570–1583 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR68] 68.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). [DOI] [PubMed] [Google Scholar]

[CR69] 69.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 Open10, e037134 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR70] 70.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). [DOI] [PubMed] [Google Scholar]

[CR71] 71.Waidelich, R. et al. Clinical experience with 5-aminolevulinic acid and photodynamic therapy for refractory superficial bladder cancer. J. Urol.165, 1904–1907 (2001). [DOI] [PubMed] [Google Scholar]

[CR72] 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.32, 101999 (2020). [DOI] [PubMed] [Google Scholar]

[CR73] 73.Kaufman, D. S., Shipley, W. U. & Feldman, A. S. Bladder cancer. Lancet374, 239–249 (2009). [DOI] [PubMed] [Google Scholar]

[CR74] 74.Ino, Y. et al. Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer. Br. J. Cancer108, 914–923 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR75] 75.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). [DOI] [PubMed] [Google Scholar]

[CR76] 76.Hu, J.-J., Lei, Q. & Zhang, X.-Z. Recent advances in photonanomedicines for enhanced cancer photodynamic therapy. Prog. Mater. Sci.114, 100685 (2020). [Google Scholar]

[CR77] 77.Xie, P. et al. The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis. Nat. Commun.5, 3733 (2014). [DOI] [PubMed] [Google Scholar]

[CR78] 78.Paschalis, A. et al. Prostate-specific membrane antigen heterogeneity and DNA repair defects in prostate cancer. Eur. Urol.76, 469–478 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR79] 79.Hu, Y. et al. Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification. Mol. Cancer21, 34 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR80] 80.Alexandre, Y. O. & Mueller, S. N. Splenic stromal niches in homeostasis and immunity. Nat. Rev. Immunol.23, 705–719 (2023). [DOI] [PubMed] [Google Scholar]

[CR81] 81.Parkin, J. & Cohen, B. An overview of the immune system. Lancet357, 1777–1789 (2001). [DOI] [PubMed] [Google Scholar]

PERMALINK

Full-course NIR-II imaging-navigated fractionated photodynamic therapy of bladder tumours with X-ray-activated nanotransducers

Liangrui He

Liyang Wang

Xujiang Yu

Yizhang Tang

Zhao Jiang

Guoliang Yang

Zhuang Liu

Wanwan Li

Abstract

Introduction

Results

Engineering and characterization of X-ray-activated nanotransducers

Fig. 1. X-ray-activated nanoscintillators and nanotransducers.

Autochthonous bladder tumour-specific labelling of nanotransducers

Fig. 2. Autochthonous bladder tumour specificity and enhanced cellular uptake of F/cRGD-labelled nanotransducers.

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

Fig. 3. Cytotoxicity, radiosensitization effect, and ROS generation assessment.

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

Fig. 4. Apoptosis analysis and in vitro X-ray-activated PDT assessment.

Single-f-PDT achieves robust tumour elimination

Fig. 5. Tumour regression by non-invasive single-f-PDT.

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

Fig. 6. Post-operative monitoring of tumour recurrence inhibition by single-f-PDT.

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

Fig. 7. On-demand f-PDT with full-course real-time monitoring.

Discussion

Methods

Ethical regulations

Materials

Characterizations

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

X-ray-activated fluorescence test

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

X-ray-activated 1O2 generation

Cell culture

Cytotoxicity

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

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

In vitro X-ray-activated PDT

Clonogenic assay

γ-H2AX immunofluorescence analysis

Quantification of intracellular ROS generation

Imaging flow cytometry

Apoptosis assay

Western blot

Animal experiments

Establishment of autochthonous mice bladder tumour model

Ex vivo NIR-II fluorescence imaging and frozen section

In vivo single-f-PDT

In vivo PDT based on 5-ALA

In vivo NIR-II fluorescence imaging

Long-term monitoring of bladder tumour recurrence

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

Histological and immunohistochemistry analysis

Toxicity assay

Immunohistochemistry analysis

Statistics and reproducibility

Reporting summary

Supplementary information

Source data

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Competing interests

Footnotes

Contributor Information

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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