Cascade x-ray energy transfer-based radio-afterglow nanoparticles for cancer theranostics

Abstract

Disclosed herein is a radio-afterglow nanoparticle (RANP) comprising ABCD, wherein: A represents a radioabsorber; B represents a radiosensitizer; C represents a luminescent component; and D represents an amphiphilic polymeric matrix. Also disclosed herein are a method for the detection of cancer in vivo, a method of treating cancer, a method of treating cancer in a patient by administering the aforementioned radio-afterglow nanoparticle, uses of the aforementioned radio-afterglow nanoparticle, and a drug comprising the aforementioned radio-afterglow nanoparticle.

Description

[0001] CASCADE X-RAY ENERGY TRANSFER-BASED RADIO-AFTERGLOW NANOPARTICLES FOR CANCER THERANOSTICS

[0002] The present application claims priority from Singapore patent application no. 10202403287Q filed on 23 October 2024, the entire content of which is incorporated herein by reference.

[0003] Field of Invention

[0004] The present disclosure generally relates to radio-afterglow nanoparticles, with particular, but not exclusive application to cancer theranostics. As such, the present disclosure also relates to the use of such radio-afterglow nanoparticles for one or both of cancer imaging and cancer theranostics, as well as method for the detection of cancer in vivo. Furthermore, the present disclosure relates to method of treating cancer by administering such radio-afterglow nanoparticles, the radio-afterglow nanoparticles for use as a medicament, for use in the treatment of cancer, and in the manufacture of a medicament for the treatment of cancer.

[0005] Background

[0006] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0007] Optical imaging allows for the visualization of physio-pathological processes at molecular level, representing an indispensable tool in fundamental research and clinical diagnostics. However, the dependence on real-time optical irradiation leads to high autofluorescence and low signal to background ratio (SBR), which compromises the diagnostic sensitivity and specificity. Afterglow imaging leverages the long-lasting light emission after the cessation of irradiation on afterglow agents, minimizing the autofluorescence and leading to a higher SBR and deeper tissue penetration compared to fluorescence imaging. Organic afterglow agents store photon energy into chemical defects, which involves photosensitization that generate reactive oxygen species (ROS) and the formation of emissive intermediates to emit afterglow upon decomposition, enabling photodynamic therapy and afterglow imaging for a variety of diseases including cancer, infection, and organ injury. However, due to limited light penetration in biological tissues, they are difficult to be induced for theranostics of deep-seated lesions.

[0008] Although X-rays are able to penetrate deep tissues, X-ray-induced afterglow (radio-afterglow) materials are limited to a few inorganic nanoparticles comprising a host matrix (e.g. metal fluorides) and a luminescence centre (e.g., rare-earth metal ions). Mechanistically, the host matrix contains high-atomic-number (Z) elements that efficiently harvest X-ray photons by down-converting them to secondary electron-hole pairs. The pairs are trapped in the physical defects of matrix that delay their migration towards luminescence centre, where they recombine to emit radio-afterglow. This process could also generate ROS to exert radiodynamic damage on diseased tissues. Since the radiotheranostic functions of inorganic materials are biomarker independent and always active, the diagnostic accuracy and therapeutic efficacies rely heavily on the concentration difference between diseased and normal tissues. By contrast, organic materials have structure versatility for the construction of smart probes with biomarker-activatable radiotheranostic functions; however, they often consist of low-Z elements that have inherently low X-ray deposition, which are difficult to induce radio-afterglow and radiodynamic effects. While leveraging X-ray to initiate prolonged luminescence (radio-afterglow) and stimulate radiodynamic1O2 production from optical agents provide opportunities for diagnosis and therapy at tissue depth which is inaccessible to light, X-ray responsive organic luminescent materials are rare due to their intrinsic low X-ray conversion efficiency.

[0009] Therefore, there exists a need for new radio-afterglow nanoparticles, particularly for cancer theranostics.

[0010] Summary of Invention

[0011] Aspects and embodiments of the invention are provided in the following numbered clauses.

[0012] 1. A radio-afterglow nanoparticle (RANP) comprising ABCD, wherein:

[0013] A represents a radioabsorber;

[0014] B represents a radiosensitizer;

[0015] C represents a luminescent component; and

[0016] D represents an amphiphilic polymeric matrix.

[0017] 2. The radio-afterglow nanoparticle according to Clause 1, wherein the radio-afterglow nanoparticle further comprises E, wherein E represents a targeting moiety.

[0018] 3. The radio-afterglow nanoparticle according to Clause 2, wherein the targeting moiety is a tumor cell targeting moiety, a macrophage-targeting moiety, or an antigen-targeting moiety, optionally wherein the targeting moiety is selected from one or more of the following: hyaluronic acid, mannose, cyclo-RGD (arginine-glycine-aspartic acid peptide) and iRGD (Internalizing RGD), and an antibody.

[0019] 4. The radio-afterglow nanoparticle according to Clause 3, wherein the antibody is selected from one of more of the following: anti-EGFR antibody, anti-VEGF antibody, antitransferrin antibody, Interleukin-2, and anti-CD86 antibody.

[0020] 5. The radio-afterglow nanoparticle according to any preceding clause, wherein the radioafterglow nanoparticle further comprises F, wherein F represents a biomarker cleavable linker.

[0021] 6. The radio-afterglow nanoparticle according to any preceding clause, wherein the radioafterglow nanoparticle further comprises G, wherein G represents an afterglow quencher.

[0022] 7. The radio-afterglow nanoparticle according Clause 6, wherein the afterglow quencher is coupled to the radio-afterglow nanoparticle via a or the biomarker cleavable linker.

[0023] 8. The radio-afterglow nanoparticle according to any preceding clause, wherein A is selected from one or more of:

[0024]

[0025] 9. The radio-afterglow nanoparticle according to any preceding clause, wherein B is selected from one or more of: R « Zn, Cu, Mg R ~ Zri. Cti. Mg

[0026]

[0027] € 7 8 9

[0028] 10. The radio-afterglow nanoparticle according to any preceding clause, wherein C is selected from one or more of:

[0029]

[0030] 11. The radio-afterglow nanoparticle according to any preceding clause, wherein D is selected from one or more of: o A -- °;;. C,:H O O-P-O xx. A LO. x4-p OH H n O

[0031] R = H, -NH2>-COOH. -SH; n = 10 to 50 1 2

[0032] .. NH, [ f f H " M ' 1- -'Ns- " N' ' ■' N ■ - ■: " NH- “ | H J | $ -s N HN ‘ i f, R = H, -NH?, -COOH, -SH; X= 10% to R = H. -NH2, -COOH, -SH; m = 10 to 50; n ~ 4 to 50 90%; m = 10 to 100; n= 10 to 50

[0033]

[0034] 4

[0035] 12. The radio-afterglow nanoparticle according to any preceding clause when dependent on Clause 5, wherein F is selected from a heat-cleavable linker, a hydrogen peroxide-cleavable linker, a Granzyme B-cleavable linker, a glutathione-cleavable linker, a Cathespin B-cleavable linker, and a matrix metalloproteinase-cleavable linker, optionally wherein the biomarker cleavable linker is selected from one or more of:

[0036] Heat-deavabte iinker H;Oj-oSeavable linker Granzyme B-cleavabie tinker

[0037]

[0038] Giutafhione-deavabie linker Cathepsin B-cfeavabte linker Matrix rnetatiaproteinase-cfeava ie iirsket

[0039] 13. The radio-afterglow nanoparticle according to any preceding clause when dependent on Clause 6, wherein G is selected from one or more of: fit= -NH,. -COOK -SH R x -NHj, -CCJOH.: ~SH R » -COOH. -SH

[0040] 3

[0041] R

[0042] j«L:

[0043] R.

[0044] R = rCHjCHjGQQH, R = -GHjGHjNH^. -GHjCH, EOOH.

[0045]

[0046] 5

[0047] 14. The radio-afterglow nanoparticle according to any preceding clause, wherein the radioafterglow nanoparticle is free of inorganic nanoparticles, preferably wherein the radioafterglow nanoparticle is free of metal halides, such as metal fluorides, and is free of rare earth metals or rare earth metal ions.

[0048] 15. The radio-afterglow nanoparticle according to any preceding clause, wherein the radioafterglow nanoparticle comprises only organic molecules.

[0049] 16. A use of a radio-afterglow nanoparticle according to any preceding clause for one or both of cancer imaging and cancer theranostics.

[0050] 17. A method for the detection of cancer in vivo, the method comprising:

[0051] i) administering a radio-afterglow nanoparticle according to any one of Clauses 1 to 15 to a subject; and

[0052] ii) detecting any luminescence, wherein the presence of cancer in vivo is indicated by luminescence.

[0053] 18. A method of treating cancer, the method comprising:

[0054] i) administering a radio-afterglow nanoparticle according to any one of Clauses 1 to 15 to a subject; and

[0055] ii) subjecting the subject to X-ray irradiation.

[0056] 19. A method of treating cancer in a patient by administering a radio-afterglow nanoparticle according to any one of Clauses 1 to 15. 20. A radio-afterglow nanoparticle according to any one of Clauses 1 to 15 for use as a medicament.

[0057] 21. A radio-afterglow nanoparticle according to any one of Clauses 1 to 15 for use in the treatment of cancer.

[0058] 22. Use of a radio-afterglow nanoparticle according to any one of Clauses 1 to 15 in the manufacture of a medicament for the treatment of cancer.

[0059] 23. A drug comprising a radio-afterglow nanoparticle according to any one of Clauses 1 to 15.

[0060] Drawings

[0061] FIG. 1 depicts synthesis of cDTDP.

[0062] FIG. 2 depicts screening of radio-afterglow composition, (a) Radio-afterglow induction from amphiphilic nanoparticles containing a variety of radioabsorbers, radiosensitizers, radioafterglow substrates and amphiphilic polymers, (b, c) Radio-afterglow intensities of DTDP-based nanoparticles (b) and DPAs-based nanoparticles (c) with different radioabsorbers and radiosensitizers. [Radioabsorber] = 100 pg / mL, [Radiosensitizer] = 10 pg / mL, [Radio-afterglow substrate] = 25 pg / mL. F127 as the amphiphilic polymer for all RANPs. (d) Radio-afterglow intensities of nanoparticles assembled by different amphiphilic polymers. [CzTPN] = 100 pg / mL, [NIR775] = 10 pg / mL, [DTDP] = 25 pg / mL. ***p = 0.000559 (DSPE-PEG), *** p = 0.000772. For all experiments, n = 3 independent samples. Data were presented as mean ± s.d. Statistical significance was calculated via one-way ANOVA followed by Tukey’s post hoc test in (d).

[0063] FIG. 3 depicts absorbance spectra of CzTPN, NIR775, and DTDP encapsulated in F127 nanoparticles in PBS (0.01 M, pH 7.4).

[0064] FIG. 4 depicts fluorescence spectra of CzTPN, NIR775, and DTDP encapsulated in F127 nanoparticles in PBS (0.01 M, pH 7.4). Excitation wavelength: 465 nm for CzTPN, 680 nm for NIR775, and 430 for DTDP. FIG. 5 depicts hydrodynamic sizes of RANP assembled by indicated stabilizers. Doping ratio (w / w) for CzTPN: NIR775: DTDP: stabilizer = 10: 1: 2.5: 500. Data were presented as mean ± s.d. (n = 3 independent samples). The significance between multiple groups was analysed by one-way analysis of variance (ANOVA) with T ukey’s post hoc test. *** p = 0.000885 (DSPE-PEG), *** p =0.001320 (PLGA-PEG).

[0065] FIG. 6 depicts radioluminescence energy transfer (RET), (a) Schematic of RET within nanoparticles comprising CzTPN (radioabsorber) and NIR775 (radiosensitizer), (b) Radioluminescence of CzTPN, NIR775 and CzTPN / NIR775 nanoparticles under real-time X-ray irradiation (50 kV, 200 pA). [CzTPN] = 100 pg / mL, [NIR775] = 10 pg / mL, n = 3 independent samples, (c) Radioluminescence spectra of CzTPN, NIR775 and CzTPN / NIR775 nanoparticles in 0.01 M phosphate buffered saline (PBS). [CzTPN] = 100 pg / mL. (d) NIR radioluminescence intensities of CzTPN / NIR775 nanoparticles (in 0.01 M PBS) at different mass doping ratios under real-time X-ray irradiation (50 kV, 200 pA). [CzTPN] = 100 pg / mL, n = 3 independent samples, (e) Electron spin resonance (ESR) spectra of1O2generation from CzTPN, NIR775 and CzTPN / NIR775 nanoparticles. [CzTPN] = 100 pg / mL, [NIR775] = 10 pg / mL. (f) ESR spectra of1O2generation from CzTPN / NIR775 nanoparticles at different mass doping ratios. [NIR775] = 10 pg / mL for all groups, (g) Proposed mechanism of RET in RANP. Radioabsorber (CzTPN) down-coverts X-ray to radioluminescence via photoelectric effect and Compton scattering (scintillation). The radioluminescence is transferred to radiosensitizer (NIR775) to emit NIR radioluminescence and generate1O2. ISC, intersystem crossing. RISC, reverse intersystem crossing. Data were presented as mean ± s.d.

[0066] FIG. 7 depicts fluorescence spectra of NIR775, CzTPN and CzTPN / NIR775 nanoparticles in PBS (0.01 M, pH 7.4). All samples were excited with 420 nm light. [CzTPN] = 100 pg / mL, [NIR775] = 10 pg / mL.

[0067] FIG. 8 depicts representative radioluminescence images (a) and intensities (b) of CzTPN nanoparticles in PBS (0.01 M, pH 7.4) under real-time X-ray irradiation of different tube currents. Tube voltage was fixed at 50 kV. Data were presented as mean ± s.d. (n = 3 independent samples).

[0068] FIG. 9 depicts radioluminescence of CzTPN nanoparticles at different doping ratios of CzTPN to F127. Data were presented as mean ± s.d. (n = 3 independent samples).

[0069] FIG. 10 depicts radioluminescence intensities of CzTPN, polystyrene (PS), and anthracene (AT) nanoparticles (NPs, in 0.01 M PBS) under real-time X-ray irradiation (50 kV). Data were presented as mean ± s.d. (n = 3 independent samples). The significance between multiple groups was analysed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. **** p < 0.0001 versus PS NPs (0.000008) and AT NPs (0.000024).

[0070] FIG. 11 depicts dose-dependent1O2generation, (a)1O2generation from CzTPN / NIR775 nanoparticles after X-ray irradiation of different dosages. [CzTPN] = 100 pg / mL. [NIR775] = 10 pg / mL. (b)1O2generation from CzTPN / NIR775 nanoparticles of different concentrations. The doping ratio of CzTPN to NIR775 was fixed at 10: 1. X-ray, 1 Gy. Data were presented as mean ± s.d. (n = 3 independent samples).

[0071] FIG. 12 depicts ESR spectra of O2·⁻ and ·OH generation from CzTPN / NIR775 nanoparticles.

[0072] [CzTPN] = 100 pg / mL. [NIR775] = 10 pg / mL. X-ray, 1 Gy.

[0073] FIG. 13 depicts radio-afterglow Mechanism, (a) Radio-afterglow from RANP comprising radioabsorber (CzTPN), radiosensitizer (NIR775), and radio-afterglow substrate (DTDP) via cascade intraparticle RET and SOT. (b, c) Quantification (b) and radio-afterglow images (c) of nanoparticles with different formulations in PBS (0.01 M, pH7.4). [CzTPN] = 100 pg / mL, [NIR775] = 10 pg / mL, [DTDP] = 25 pg / mL. 750 LP, 750 nm long pass filter. X-ray dosage, 1 mGy. n = 3 independent samples. **** p < 0.0001 versus formulation 1 (2.5×10-7), formulation 2 (2.6×10-7) and formulation 3 (2.5×10-7). (d) Radio-afterglow spectrum of RANP in PBS (0.01 M pH 7.4) after X-ray pre-irradiation (1 mGy). [DTDP] = 25 pg / mL. (e) Radio-afterglow decay of RANP in PBS after X-ray pre-irradiation (1 mGy). [DTDP] = 25 pg / mL. Dashed line indicated background level, n = 3 independent samples, (f) Size and representative TEM image (inset) of RANP. [DTDP] = 10 pg / mL. n = 3 independent samples, (g) Proposed mechanism of radioafterglow in RANP. Data were presented as mean ± s.d. Statistical significance was calculated via one-way ANOVA followed by Tukey’s post hoc test (b).

[0074] FIG. 14 depicts fluorescence spectra of NIR775, DTDP, NIR775 / DTDP nanoparticles (NPs). All samples were irradiated with 430 nm light. [NIR775] = 10 pg / mL, [DTDP] = 25 pg / mL.

[0075] FIG. 15 depicts X-ray dose-dependent radio-afterglow. S= 1.3, o = 0.02, limit of detection (LOD) was calculated to be 0.046 mGy. Data were presented as mean ± s.d. (n = 3 independent samples).

[0076] FIG. 16 depicts radio-afterglow signals of RANP ([DTDP] = 20 pg / mL, in 0.01 M PBS) irradiated with X-ray for 5 cycles. X-ray, 1 mGy per cycle. Data were presented as mean ± s.d. (n = 3 independent samples). FIG. 17 depicts hydrodynamic sizes of RANPs in number, volume, and intensity modes. Z average = 94.2 nm. Data were presented as mean ± s.d (n= 3 independent samples).

[0077] FIG. 18 depicts Zeta potentials for RANP and tRANP. [DTDP or cDTDP] = 20 pg / mL. Data were presented as mean ± s.d. (n = 3 independent samples).

[0078] FIG. 19 depicts in vitro studies of radio-afterglow imaging depth and radiodynamic cytotoxicity, (a) Schematic showing the induction and detection of radio-afterglow or photoafterglow through chicken breast tissue. CCD, charge-coupled device, (b, c) Representative images (b) and SBRs (c) for radio-afterglow and photoafterglow of RANP ([DTDP] = 100 pg / mL) induced by X-ray (50 kV, 200 n A) or laser (680 nm) through chicken breast tissue of different thicknesses (n = 3 independent samples), (d) Relative1O2generation from RANP ([NIR775] = 40 pg / mL) induced by X-ray (50 kV, 200 pA) or laser (680 nm) through chicken breast tissues of different thicknesses (n = 3 independent samples), (e) Radio-afterglow images of 4T1 cancer cells after indicated treatments. X-ray dosage, 1 mGy. RANP, [DTDP] = 20 pg / mL. Images were captured by microscope without any excitation. Acquisition time: 10 s. (f) Quantification of radio-afterglow intensities in (e) (n = 3 independent samples). **** p < 0.0001 versus PBS (1 ><10’5), RANP (1 *10'5) and X-ray (1 *10'5). (g) Cell viability of 4T1 cancer cells incubated with RANP at different concentrations (equivalent to NIR775) and irradiated with or without X-ray (0.5 Gy) (n = 3 independent samples), (h) Confocal microscopic images of 4T1 cancer cells stained with dichloro-dihydro-fluorescein diacetate (DCFH-DA, green) after indicated treatments. Cells were incubated with or without RANP ([NIR775] = 20 pg / mL) for 12 h, followed by irradiation with or without X-ray (0.5 Gy), (i) Quantification of DCFH-DA fluorescence intensities in (h) (n = 3 independent samples). **** p<0.0001 versus PBS (5.8x1 O'9), RANP (6.1 xi O'9) and X-ray (1 xi0'8). Data were presented as mean ± s.d. Statistical significance was calculated via one-way ANOVA followed by Tukey’s post hoc test (f and i).

[0079] FIG. 20 depicts radio-afterglow induction and detection through tissues, (a) Schematic showing the induction and detection of radio-afterglow or photo-afterglow via chicken breast tissues, (b, c) Representative images (b) and SBRs (c) for radio-afterglow and photoafterglow of RANP ([DTDP] = 100 pg / mL) induced through chicken breast tissue of different thicknesses. Data were presented as mean ± s.d. (n = 3 independent samples).

[0080] FIG. 21 depicts radio-afterglow imaging in MCF-7 cancer cells, (a) Radio-afterglow images of MCF-7 cancer cells after indicated treatments. X-ray dosage, 1 mGy. RANP, [DTDP] = 20 pg / mL. Images were captured by microscope without any excitation. Acquisition time: 10 s. (b) Quantification of radio-afterglow intensities in (a). Data were presented as mean ± s.d. (n = 3 independent samples).

[0081] FIG. 22 depicts radio-afterglow imaging in MCF-7 cancer cells treated with nanoparticles loading single component, (a) Radio-afterglow images of MCF-7 cancer cells after indicated treatments. X-ray dosage, 1 mGy. Images were captured by microscope without any excitation. Acquisition time: 10 s. (b) Quantification of radio-afterglow intensities in (a). Data were presented as mean ± s.d. (n = 3 independent samples).

[0082] FIG. 23 depicts cell viability of 4T1 cancer cells incubated with or without RANP ([NIR775] = 20 pg / mL) and irradiated with X-ray of indicated doses. Data were presented as mean ± s.d. (n = 3 independent samples).

[0083] FIG. 24 depicts viability of 4T1 cancer cells treated with RANP ([NIR775] = 20 pg / mL) and irradiated with X-ray through chicken breast tissues of different thicknesses. CTRL denotes cells treated with RANP but without X-ray irradiation. Data were presented as mean ± s.d. (n = 3 independent samples).

[0084] FIG. 25 depicts RANP-induced apoptosis of 4T1 cancer cells, (a) Gating strategy, (b) Flow cytometric plots of 4T1 cancer cells incubated after indicated treatments for 12 h (n = 3 independent samples). X-ray, 0.5 Gy. RANP, [NIR775] = 20 pg / mL. (c) Quantification of Annexin- fluorescein isothiocyanate (FITC)+propidium iodide (PI)-cell populations (early apoptotic cells) in Figure (b). Data were presented as mean ± s.d. (n = 3 independent samples).

[0085] FIG. 26 depicts in vitro radiodynamic effects, (a) Cell viability of 4T 1 cancer cells pre-treated with or without tryptophan, followed by incubation with RANP at different concentrations (equivalent to NIR775) and irradiation with X-ray irradiation (0.5 Gy), (b) Cell viability of 4T1 cancer cells incubated with PBS, NIR-775 free RANP (with CzTPN and DTDP), and RANP, and irradiated with or without X-ray irradiation (0.5 Gy). [CzTPN] = 200 pg / mL. **** p < 0.0001 for RANP vs PBS plus X-ray and NIR775-free RANP plus X-ray. Data were presented as mean ± s.d. (n = 3 independent samples). The significance between two groups was analyzed by two-tailed Student’s t-test.

[0086] FIG. 27 depicts cell viability of MCF-7 cancer cells incubated with RANP at different concentrations (equivalent to NIR775) and irradiated with or without X-ray irradiation (0.5 Gy). Data were presented as mean ± s.d. (n = 3 independent samples). FIG. 28 depicts RANP-induced apoptosis of MCF-7 cancer cells, (a) Flow cytometric plots of MCF-7 cancer cells incubated after indicated treatments for 24 h (n = 3 independent samples). X-ray, 0.5 Gy. RANP, [NIR775] = 20 pg / mL. (b) Quantification of Annexin-FITC+PI-cell populations (early apoptotic cells) in Figure (a). Data were presented as mean ± s.d. (n = 3 independent samples). The gating strategy was same as that in FIG. 25.

[0087] FIG. 29 depicts RANP-induced ROS in in MCF-7. (a) Confocal microscopic images of MCF-7 cancer cells stained with dichloro-dihydro-fluorescein diacetate (DCFH-DA) after indicated treatments. Cells were incubated with or without RANP ([NIR775] = 20 pg / mL) for 12 h, followed by irradiation with or without X-ray (0.5 Gy). Nuclei was stained with Hoechst 33342 (blue). ROS was shown by DCFH-DA (green), (b) Quantification of ROS fluorescence intensities in (a). Data were presented as mean ± s.d. (n = 3 independent samples). The significance between multiple groups was analysed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. **** p <0.0001 versus PBS (3.6×10-12), RANP (4.4×10-12), and X-ray (5.8×10-12).

[0088] FIG. 30 depicts tumour-specific in vivo radio-afterglow imaging, (a) Molecular mechanism of tRANP for activatable radio-afterglow imaging of cancer. H2O2 overproduced in tumour microenvironment cleaves the phenylboronic ester of caged DTDP (cDTDP), turning on radioafterglow. (b) Radio-afterglow spectra of tRANP ([DTDP] = 10 pM) treated with orwithout H2O2 (20 pM). (c) Schematic of tumour-specific radio-afterglow. tRNAP was subcutaneously and intratumorally injected onto mice, followed by X-ray irradiation at both sides for radio-afterglow imaging, (d) Representative fluorescence and radio-afterglow images of mice locally injected with 10 pL of tRANP ([cDTDP] = 100 pg / mL). NT, normal tissue, (e) SBRs of fluorescence and radio-afterglow signals on injection sites in (d) (n = 3 mice), (f) Schematic of longitudinal imaging of tumour. tRNAP was intravenously injected to subcutaneous 4T1 tumour mice, followed by X-ray pre-irradiation for radio-afterglow imaging, (g) Representative fluorescence and radio-afterglow images of mice intravenously injected with 100 pL of tRANP ([cDTDP] = 250 pg / mL). (h) SBRs of fluorescence and radio-afterglow on tumours in (g) (n = 3 mice), (i) Schematic of radio-afterglow-guided laparotomy of peritoneal metastatic tumours, (j) Representative pre-operative and intraoperative images of peritoneal tumour-bearing mice intravenously injected with 100 pL of tRANP ([cDTDP] = 250 pg / mL) under fluorescence and radio-afterglow imaging, (k) SBRs of fluorescence and radio-afterglow on metastatic tumours of mice during laparotomy in (j) (n = 3 mice). Data were presented as mean ± s.d. FIG. 31 depicts particle size and TEM image (inset) of tRANP ([cDTDP] = 10 pM). Data were presented as mean ± s.d. (n = 3 independent samples).

[0089] FIG. 32 depicts high-performance liquid chromatography (HPLC) spectra of caged DTDP (cDTDP, 10 pM) treated with or without H2O2 (20 pM) under 37 °C for 10 min.

[0090] FIG. 33 depicts fluorescence spectrum of tRANP ([cDTDP] = 10 pM) treated with or without H2O2 (20 pM) measured by fluorometer. Excitation wavelength = 430 nm.

[0091] FIG.34 depicts fold change of radio-afterglow intensity of tRANP ([cDTDP] = 10 pM) incubated with different ROS (20 pM). Data were presented as mean ± s.d. (n = 3 independent samples).

[0092] FIG. 35 depicts tRANP-mediated afterglow imaging of cancer cell, (a) Radio-afterglow images of 4T1 cancer cells incubated with tRANP ([cDTDP] = 20 pg / mL) after indicated treatments observed by microscope. H2O2 (20 pM) and X-ray (1 mGy) were used, (b) Radio-afterglow intensities in Figure (a). Data were presented as mean ± s.d. (n = 3 independent samples).

[0093] FIG.36 depicts ex vivo analysis, (a) Ex vivo images of mice intravenously injected with tRANP. (b) Quantification of fluorescence signals for different organs. Data were presented as mean ± s.d. (n = 3 mice).

[0094] FIG. 37 depicts representative H& E-stained sections of tumours from intraperitoneal metastatic murine model. The bottom row showed the magnified images of corresponding upper figures. Samples from three different mice were analysed.

[0095] FIG. 38 depicts in vivo radiodynamic cancer therapy, (a) Timeline for tRANP-mediated radiodynamic therapy. Mice were intravenously injected with 100 pL of tRANP ([NIR775] = 100 pg / mL) on day 1, 3, and 5, followed by each fraction of X-ray (1 Gy) on tumours on day 2, 4, and 6. (b) Tumour growth curve of mice after indicated treatments (n = 5 mice), (c) Survival rate of mice after indicated treatments (n = 5 mice), (d) Representative confocal images of intratumoral ROS measured by SOSG from mice after indicated treatments, (e) Quantification of intratumoral ROS signals in (d). n = 3 independent samples. ****p < 0.0001 versus PBS (1.6x1 O’8), X-ray (3.0x1 O’8) and tRANP (1.5x1 O’8), (f) Representative images of H& E-stained tumour sections from mice after indicated treatments, (g) Quantification of damaged area in (f). n = 3 independent samples. ****p < 0.0001 versus PBS (6.6x1 O’7), X-ray (2X10’6) and tRANP (8.1x1 O'7), (h) Representative images of anti-caspase-3 antibody-stained tumour sections from mice after indicated treatments, (i) Quantification of caspase-3 signals in (h). n = 3 independent samples. ****p < 0.0001 versus PBS (1×10-6), X-ray (2×10-6) and tRANP (1×10-6), (j) Representative images of H& E-stained lungs sections of mice after indicated treatments. Dashed circles showed the metastatic nodules, (k) Counts of pulmonary metastatic nodules in mice after indicated treatments, n = 3 independent samples. *** p = 0.000132 (versus PBS), *** p = 0.000635 (versus tRANP), ** p = 0.004140. Data were presented as mean ± s.d. Statistical significance was calculated via one-way ANOVA followed by Tukey’s post hoc test (e, g, i and k).

[0096] FIG. 39 depicts optimization of dose regimen for tRANP-mediated radiodynamic therapy, (a, b) Timeline (a) and tumour growth curves (b) of subcutaneous 4T1 tumour bearing mice treated with tRANP and X-ray of different doses. Data were presented as mean ± s.d. (n = 3 mice).

[0097] FIG. 40 depicts tumour inhibition by traditional radiotherapy, (a) Timeline for traditional radiotherapy. Subcutaneous 4T1 tumour-bearing mice were irradiated with X-ray (6 Gy) on day 2, 4, and 6 on tumours, (b) Tumour growth curve of mice treated with or without X-ray (6 Gy) for 3 fractions. Data were presented as mean ± s.d. (n = 3 mice).

[0098] FIG. 41 depicts tumour immunogenic cell death, (a) Representative confocal images of tumours showing calreticulin expression after indicated treatments, (b) Quantification of fluorescence signals of calreticulin in (a). ****p <0.0001 versus PBS (3.0 x 10’9), tRANP (3.7 x 10’9), and X-ray (6.2 x 10'9). (c) Representative confocal images of tumours showing HMGB-1 (high mobility group box 1) expression after indicated treatments, (d) Quantification of fluorescence signals of HMGB-1 in (c). Data were presented as mean ± s.d. (n = 3 independent samples). The significance between multiple groups was analysed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. **** p <0.0001 versus PBS (8.9 x 10’9), tRANP (8.9 x 10’9), and X-ray (2.5 x 10'8).

[0099] FIG.42 depicts representative H& E-stained images of tumour-adjacent skin tissues from mice treated with tRANP-mediated radiodynamic therapy (3 x 1 Gy) or traditional radiotherapy (3 x 6 Gy). The right panel showed the magnified areas. Remarkable inflammatory infiltration and epidermal thickening were caused by traditional radiotherapy rather than by tRANP-mediated therapy. Samples from three different mice were analysed.

[0100] FIG.43 depicts body weight of mice after indicated treatments. Data were presented as mean ± s.d. (n = 5 mice). FIG. 44 depicts representative H& E-stained images of main organs including heart, liver, spleen, lung, kidney of mice treated with tRANP-mediated radiodynamic therapy, n = 3 independent samples.

[0101] FIG. 45 depicts blood urine nitrogen (BUN), creatinine, alanine transaminase (ALT) and aspartate aminotransferase (AST) of mice after treated with PBS or tRANP-mediated radiodynamic therapy. Data were presented as mean ± s.d. (n = 3 mice).

[0102] Description

[0103] It has been surprisingly found that the present invention only switches on its radio-afterglow in the presence of cancer biomarkers for longitudinal tumour detection and precision tumour surgery in vivo. In addition, efficient1O2generation enables the present invention to exert radiodynamic therapy on cancer at a lower X-ray dosage than clinical radiotherapy, which spares normal tissues from ionization-associated damage.

[0104] Thus, in a first aspect of the invention, there is provided a radio-afterglow nanoparticle comprising ABCD, wherein:

[0105] A represents a radioabsorber;

[0106] B represents a radiosensitizer;

[0107] C represents a luminescent component; and

[0108] D represents an amphiphilic polymeric matrix.

[0109] It will be appreciated that A, B and C may be loaded within the polymeric matrix provided by D.

[0110] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of’). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of” or synonyms thereof and vice versa. The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0111] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “a catalyst” includes mixtures of two or more such catalysts, and the like.

[0112] Without wishing to be bound by theory, it is believed that Compound A absorbs X-ray energy and converts it to radioluminescence, which transfers X-ray energy to Compound B to generate a reactive oxygen species (ROS), such as singlet oxygen. ROS is then transferred to Compound C to form an active intermediate, which slowly decomposes to emit radioafterglow. By decompose, it is understood that the active intermediate may relax to a lower-energy state by emitting energy in the form of radio-afterglow. Compound D forms a nanoparticle to load A, B, and C together. That is, A, B and C may be loaded within the amphiphilic polymeric matrix. In certain embodiments to achieve active targeting, a targeting moiety (Compound E) may be conjugated to the nanoparticle to target certain biomolecules or cells. Additionally or alternatively, an afterglow quencher (Compound G) may be anchored onto the nanoparticle via a biomarker cleavable linker (Compound F), leading to silenced radio-afterglow and radiodynamic functions. In such embodiments, only when in the presence of a specific biomarker will Compound F be cleaved to liberate Compound G, thus switching on the radio-afterglow and radiodynamic functions. As will be appreciated, E, F and G may be located on an exterior surface of the nanoparticles.

[0113] When used herein, the term “radioabsorber” refers to a compound that converts X-ray energy to radioluminescence. In particular embodiments of the invention that may be mentioned herein, the radioabsorbers may be organic compounds that display high X-ray conversion efficiencies (e.g. light yield > 104photons Me / V, such as light yield = 4.5 x 104photons / MeV). Examples of such organic compounds include, but are not limited to, anthracene, polystyrene, 9,10-diphenylanthracene, and 2,4,5,6-tetrakis(3,6-di-tert-butylcarbazol-9-yl)-1,3-dicyanobenzene (CzTPN). Without wishing to be bound by theory, the radioabsorber converts X-ray energy to radioluminescence, and the radioluminescence may be transferred to a radiosensitizer to generate singlet oxygen (1O2). For example, the radioluminescence may be transferred to a radiosensitizer via a radioluminescence energy transfer (RET) process to generate singlet oxygen (1O2).

[0114] When used herein, the term “radiosensitizer” refers to a compound that produces singlet oxygen (1O2) when exposed to radioluminescence. In particular embodiments of the invention that may be mentioned herein, the radiosensitizers used may be organic compounds that display fluorescence in the visible to near infrared (NIR) wavelengths and which preferably have high1O2 quantum yields. Examples of suitable radiosensitizers include, but are not limited to, hematoporphyrin, verteporfin, and silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775).

[0115] Without wishing to be bound by theory, the1O2 generated by the radiosensitizer may subsequently be transferred to a luminescent component, thus oxidizing the luminescent component to an active self-luminescent intermediate of high chemical energy. For example, the1O2may be transferred to the luminescent component via a1O2transfer (SOT) process. The self-luminescent intermediate gradually relaxes thus emitting long-lasting luminescence (radio-afterglow). The emitted radio-afterglow may be transferred back to the radiosensitizer, causing a red-shifted radio-afterglow from the radiosensitizer. As will be appreciated, the emitting of radio-afterglow would minimise autofluorescence and lead to a higher signal to background ratio (SBR) and deeper tissue penetration in comparison to fluorescence imaging.

[0116] When used herein, the term “a luminescent component” refers to a component that emits luminescence. In particular embodiments of the invention that may be mentioned herein, the luminescent components used may be organic compounds which form active self-luminescent intermediates that simultaneously decompose to emit radio-afterglow. For example, the organic compounds may be compounds which form dioxetane intermediates that emit bright radio-afterglow after activation. Examples of suitable luminescent components include, but are not limited to dicyanomethylene-4H-benzothiopyran-phenoxyl-adamantylidene (DPAs) and ((2-(3-((E)-4-hydroxy-2-((1r,5R,7S)-4'-methoxyspiro[adamantane-2,3'-[1,2]dioxetane]-4'-yl)styryl)-5,5-dimethylcyclohexan-1-ylidene)malononitrile) (DTDP).

[0117] Any suitable polymeric matrix may be used. In some embodiments that may be mentioned herein, the polymeric matrix may comprise an amphiphilic polymer. Examples of suitable amphiphilic polymers include, but are not limited to poly(ethylene glycol)-block-poly(propylene glycol)-block-poly (PEG-PPG-PEG, Pluronic F127), poly(lactic-co-glycolic acid) (PLGA)-PEG, or 2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG).

[0118] It will be appreciated that an amphiphilic polymer has a hydrophilic head group and a hydrophobic tail. The hydrophobic tails will group together in a polar environment (e.g. water or within a biological system) to form a core polymeric matrix portion, and components A, B and C may reside within this core polymeric matrix portion. It also follows that the hydrophilic head groups will preferentially be exposed to a polar environment. These head groups contain hydrophilic functional groups that may serve to support additional functionality on an exterior surface of the nanoparticles.

[0119] In some embodiments that may be mentioned herein, the radio-afterglow nanoparticle may further comprise E, wherein E represents a targeting moiety. In such embodiments, the targeting moiety may be a tumor cell targeting moiety, a macrophage-targeting moiety, or an antigen-targeting moiety. For example, the targeting moiety may be selected from one or more of the following: hyaluronic acid, mannose, cyclo-RGD and iRGD, and an antibody. In further embodiments, the antibody may be selected from one of more of the following: anti-EGFR antibody, anti-VEGF antibody, anti-transferrin antibody, Interleukin-2, and anti-CD86 antibody. As will be appreciated, the targeting moiety E may help to concentrate the radio-afterglow nanoparticle on or around a suitable biological target, such as those identified above. It will also be appreciated that the above are merely examples and that the technology disclosed herein may be applied to other such biological targets using suitable targeting moieties. As will also be appreciated, for the targeting moiety E to work, it needs to be on an exterior surface of the nanoparticles. As such, E may be attached to a hydrophilic head group portion of an amphiphilic polymer.

[0120] In some embodiments that may be mentioned herein, the radio-afterglow nanoparticle may further comprise F, wherein F represents a biomarker cleavable linker. Any suitable biomarker cleavable linker may be used. For example, F may be selected from a heat-cleavable linker, a hydrogen peroxide-cleavable linker, a Granzyme B-cleavable linker, a glutathione-cleavable linker, a Cathespin B-cleavable linker, and a matrix metalloproteinase-cleavable linker. In such embodiments, the biomarker cleavable linker may be selected from one or more of: Heat-cieavable linker H2O2-cleavable linker Granzyme B-deavable linker

[0121]

[0122] Glutatbione-cteavable linker Cathepsin B-cieavable linker Matrix metalloproteinase- cleavable linker

[0123] As will be appreciated, the purpose of the biomarker cleavable linker is to be cleaved when in the presence of the biomarker in question. That is, the linker is intended to be cleaved when contacted by the relevant biomarker. This may be an enzyme or it may be a chemical produced by certain biological processes. It is noted that these biomarkers may be present in a greater concentration in certain diseases, thereby leading to greater cleavage of the biomarker cleavable linker around sites afflicted by said disease.

[0124] In some embodiments that may be mentioned herein, the radio-afterglow nanoparticle may further comprise G, wherein G represents an afterglow quencher. In such embodiments, the afterglow quencher may be coupled to the radio-afterglow nanoparticle via the biomarker cleavable linker. Any suitable afterglow quencher and any suitable coupling may be used. For example, G may be selected from one or more of:

[0125] R = -COOH -SH R ~ -MHj -COCH-SH R “ -NH;. -SOOH. -8H 1 2 3 R

[0126] A.-A -. N. -- x ■ R

[0127]

[0128] 4 5 As the quencher is attached to the nanoparticle, it prevents luminescence until it is cleaved from the probe. For example, until it is cleaved from the nanoparticle, the nanoparticle does not produce luminescence, but it does when the quencher is cleaved, which may allow for the detection of a particular disease. Thus, the quencher may be conveniently attached to the nanoparticle by way of the biomarker cleavable linker F. While not strictly necessary, it will be appreciated that the addition of F and G may be easier to achieve via functional groups on a hydrophilic head group of an amphiphilic polymer, thereby exposing the quencher and linker on an exterior surface of the probe, which may enable easier cleavage.

[0129] In some embodiments that may be mentioned herein, A may be selected from one or more of:

[0130]

[0131] In some embodiments that may be mentioned herein, B may be selected from one or more of:

[0132]

[0133] In some embodiments that may be mentioned herein, C may be selected from one or more of:

[0134]

[0135] In some embodiments that may be mentioned herein, D may be selected from one or more of:

[0136] 100 65 100 R = H, -NH2, -COOH, -SH; n = 10 to 50 2

[0137] R = H, -NH2, -COOH, -SH; X= 10% to R = H, -NH2, -COOH, -SH; m = 10 to 50; n = 4 to 50 90%; m = 10 to 100; n= 10 to 50

[0138]

[0139] 3

[0140] In some embodiments that may be mentioned herein, the radio-afterglow nanoparticle may be free of inorganic nanoparticles, preferably wherein the radio-afterglow nanoparticle may be free of metal halides, such as metal fluorides, and may be free of rare earth metals or rare earth metal ions.

[0141] When used herein, the term “nanoparticle” is intended to refer to particles that have an average hydrodynamic diameter of from 0.1 to 2,000 nm. For the avoidance of doubt, it is explicitly contemplated that where a number of numerical ranges related to the same feature are cited herein, that the end points for each range are intended to be combined in any order to provide further contemplated (and implicitly disclosed) ranges.

[0142] In some embodiments that may be mentioned herein, the radio-afterglow nanoparticle may comprise only organic molecules.

[0143] In a second aspect of the invention, there is provided a use of a radio-afterglow nanoparticle according to the first aspect of the invention for one or both of cancer imaging and cancer theranostics.

[0144] In a third aspect of the invention, there is provided a method for the detection of cancer in vivo, the method comprising:

[0145] i) administering a radio-afterglow nanoparticle according to the first aspect of the invention to a subject; and

[0146] ii) detecting any luminescence, wherein the presence of cancer in vivo is indicated by luminescence.

[0147] As mentioned above, the radio-afterglow nanoparticles of the present invention may have utility as diagnostic agents for determining the presence of cancer.

[0148] Visualising methods that may be mentioned include spectroscopic detection methods (e.g. fluorescence detection. Specific details of the visualising methods are provided in the examples below.

[0149] In a fourth aspect of the invention, there is provided a method of treating cancer, the method comprising:

[0150] i) administering a radio-afterglow nanoparticle according to the first aspect of the invention to a subject; and

[0151] ii) subjecting the subject to X-ray irradiation. In a fifth aspect of the invention, there is provided a method of treating cancer in a patient by administering a radio-afterglow nanoparticle according to the first aspect of the invention.

[0152] The radio-afterglow nanoparticle may be administered by any suitable route. For example, it may be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0153] The radio-afterglow nanoparticle may be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the nanoparticle and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

[0154] Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0155] The amount of the radio-afterglow nanoparticle in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, as well as the particular patient to be treated. In any event, the amount of the radio-afterglow nanoparticle in the formulation may be determined routinely by the skilled person.

[0156] A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w / w) nanoparticle; and from 50% (w / w) to 99% (w / w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w / w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

[0157] Depending on the disorder, and the patient, to be treated, as well as the route of administration, the radio-afterglow nanoparticle may be administered at varying therapeutically effective doses to a patient in need thereof.

[0158] However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0159] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration in accordance with the third, fourth and fifth aspects of the invention, the dosage can vary from about 0.01 mg to about 1000 mg per day of a radio-afterglow nanoparticle. In some embodiments that may be mentioned herein, the dosage may be administered once per day. In some embodiments that may be mentioned herein, the dosage may be administered once in day one, once in day three, and once in day five, over a period of six days.

[0160] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0161] In a sixth aspect of the invention, there is provided a radio-afterglow nanoparticle according to the first aspect of the invention for use as a medicament.

[0162] In a seventh aspect of the invention, there is provided a radio-afterglow nanoparticle according to the first aspect of the invention for use in the treatment of cancer. In an eighth aspect of the invention, there is provided use of a radio-afterglow nanoparticle according to the first aspect of the invention in the manufacture of a medicament for the treatment of cancer.

[0163] For the avoidance of doubt, in the context of the present invention, the term “treatment’ includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and / or diagnosis of patients which are susceptible to the relevant disease states.

[0164] The terms “patient’ and “patients” include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0165] In a ninth aspect of the invention, there is provided a drug comprising a radio-afterglow nanoparticle according to the first aspect of the invention.

[0166] For the avoidance of doubt, the drug may be administered as described hereinbefore for the radio-afterglow nanoparticle. As such, a full description of the administration of the drug is not included here again for the sake of brevity.

[0167] In some embodiments of the ninth aspect of the invention that may be mentioned herein, the drug may be used to treat cancer.

[0168] The term “cancer” will be understood by those skilled in the art to include conditions such as, but not limited to, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumours, CNS tumours, breast cancer, Castleman disease, cervical cancer, colon cancer, rectum cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal cancer, hypopharyngeal cancer, leukemia (e.g. acute lymphocytic, acute myeloid, chronic lymphocytic, chronic myeloid, chronic myelomonocytic), liver cancer, lung cancer (e.g. small cell or non- small cell), lung carcinoid tumour, lymphoma (e.g. of the skin), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumours, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (basal and squamous cell, melanoma, Merkel cell), small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumour, colorectal cancer, and brain cancer.

[0169] Particular cancers that may be mentioned herein may be in the form of solid tumours (such as adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumours, CNS tumours, breast cancer, Castleman disease, cervical cancer, colon cancer, rectum cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal cancer, hypopharyngeal cancer, liver cancer, lung cancer (e.g. small cell or non-small cell), lung carcinoid tumour, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumours, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (basal and squamous cell, melanoma, Merkel cell), small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumour), colorectal cancer, and brain cancer.

[0170] In particular embodiments that may be mentioned herein, the cancer may be one or more of the following: breast cancer, colorectal cancer, liver cancer, lung cancer (e.g. small cell or non-small cell), brain cancer, and lymphoma.

[0171] Further aspects and embodiments of the invention may relate to the following numbered embodiments.

[0172] 1. In a first aspect of the invention, there is provided a radio-afterglow nanoparticle (RANP) comprising A, B, C, D; and optionally one or more of E, F, and G-:

[0173]

[0174] wherein A represents a radioabsorber, B represents a radiosensitizer, C represents a radioafterglow substrate with chemical defects; D represents an amphiphilic polymer; E represents a targeting moiety; F represents a biomarker cleavable linker; G represents an afterglow quencher.

[0175] 2. Compound A may be selected from but not limited to:

[0176]

[0177] 3. Compound B may be selected from but not limited to:

[0178]

[0179] 4. Compound C may be selected from but not limited to:

[0180]

[0181] 5. Compound D may be selected from but not limited to: 100 65 100 OH R = H, -NH2, -COOH, -SH; n = 10 to 50

[0182] NH2

[0183] R = H, -NH2, -COOH, -SH; X= 10% to R = H, -NH2, -COOH, -SH; m = 10 to 50; n = 4 to 50 90%; m = 10 to 100; n= 10 to 50

[0184]

[0185] 6. Compound E may be selected from but not limited to:

[0186] Hyaluronic acid (targeting tumour cells), mannose (targeting macrophage), cyclo-RGD and iRGD (targeting tumour cells); an antibody.

[0187] In some embodiments that may be mentioned herein, the antibody may be selected from one of more of the following:- anti-EGFR antibody (targeting tumour cells), anti-VEGF antibody (targeting tumour endothelial cells), anti-transferrin antibody (targeting tumour cells), Interleukin-2 (targeting T cells), anti-CD86 antibody (targeting dendritic cells).

[0188] 7. Compound F may be selected from but not limited to:

[0189] Heat-cleavable linker H2O2-cleavable linker Granzyme B-cleavable linker

[0190]

[0191] Glutathione-cleavable linker Cathepsin B-cleavable linker Matrix metalloproteinase-cleavable linker 8. Compound G may be selected from but not limited to:

[0192] 1 2 3 R

[0193] I Xi ”?r ■: 'T. C*te «N<;'NO.,

[0194]

[0195] 4 - 5 9. In a second aspect of the invention, there is provided a use of RANP as described in the first aspect above for cancer imaging and cancer theranostics.

[0196] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0197] Examples

[0198] Materials

[0199] All reagents were purchased from Sigma-Aldrich unless otherwise described. 2, 3,5,6-tetrakis(3,6-di-tert-butylcarbazol-9-yl)-1,4-dicyanobenzene (CzTPN) was ordered from Ossila (U. S. A). Phosphate-buffered saline (PBS), penicillin-streptomycin, high-glucose Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco. Cell counting kit-8 (CCK-8) kit, Hoechst 33342, 4,6-diamidino-2-phenylindole (DAPI), Annexin-fluorescein isothiocyanate (FITC), propidium iodide (PI) were ordered from Beyotime (China). DPAs was synthesized according to our previous reports (Huang, J. et al., Angew Chem Int Ed Engl 2022, 61, e202203235).

[0200] Statistical analysis

[0201] All numeric data are presented as mean ± s.d. unless otherwise indicated. The significance between two groups was analyzed by two-tailed Student’s t-test. The significance between multiple groups was analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Statistical analysis was performed using Graphpad Prism 7.0.4. P values less than 0.05 were considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Example 1. Synthesis of caged ((2-(3-((E)-4-hydroxy-2-((1r,5R,7S)-4'-methoxyspiro[adamantane-2,3'-[1,2]dioxetane]-4'-yl)styryl)-5,5-dimethylcyclohexan-1-ylidene)malononitrile) (cDTDP)

[0202] Compound 1 (936 mg, 4 mmol) and 3 mL of anhydrous tetrahydrofuran (THF) were added into a round bottom flask (RBF) with a stirring bar (FIG. 1). The system was relocated into an ice bath and left to stir for 10 min. Phosphorus tribromide (PBr3) (2160 mg, 8 mmol) was dissolved with 2 mL of anhydrous THF and then injected into the reaction system over 5 min. The reaction was left to react for 2 h, and the system was quenched with the addition of the dichloromethane (DCM, 50 mL) and then washed with concentrated Na2CO3solution twice. The organic phases were collected and dried over Na2SO4, and then concentrated by the rotary evaporation. The liquid product was freeze-dried overnight and directly used for the next step without further purification. DTDP was prepared according to previously reported method (Wei, X. et al., Angew Chem Int Ed Engl 2023, 62, e202213791). DTDP (46.6 mg, 0.1 mmol), compound 2 (118 mg, 0.4 mmol), potassium carbonate (K2CO3) (10 mg, 0.75 mmol), caesium carbonate (Cs2CO3) (24 mg, 0.75 mmol), and 3 mL of anhydrous acetonitrile were added into a round bottom flask with a stirring bar. The reaction was conducted under room temperature and the progression was monitored with thin layer chromatography (TLC). Once no DT could be observed on the TLC plate, the reaction was stopped and purified by column chromatography with DCM / Methanol (100:1), affording DPDT as a yellow solid. Proton-nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker Advance II 400 MHz NMR.

[0203] 1H NMR (400 MHz, CDCl3) δ 7.83 (d, J = 7.6 Hz, 2H), 8.09 (d, J = 8.4 Hz, 1 H), 7.39 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 11.2 Hz, 1 H), 6.87 (s, 1 H), 6.85 (d, J = 11.2 Hz, 1 H), 6.79 (s,1 H), 6.77 (d, J = 10.4 Hz, 1H), 5.21 (s, 2H), 3.33 (s, 1H), 3.29 (s, 3H), 2.60 (s, 2H), 2.43 (s, 2H), 2.08-1.72 (m, 12H), 1.07 (s, 6H). LC-MS analyses were tested with Triple Quadrupole LC / MS (Agilent 1260-6460). MS (ES+): m / z calc, for 682.39; found: 681.54 [M - H]-1.

[0204] Example 2. Composition Screening for radio-afterglow nanoparticles (RANPs)

[0205] Composition screening

[0206] The candidate radioabsorbers, radiosensitizers, and radio-afterglow substrates were dissolved in organic solvents to prepare the stock solution (1 mg / mL), respectively. Amphiphilic polymers (F127, also known as Pluronic F127, a triblock polymer consisting of hydrophobic polypropylene oxide) block flanked by two hydrophilic poly(ethylene oxide) blocks], DSPE-PEG, and poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-PEG) were respectively dissolved in THF to prepare a stock solution (20 mg / mL). Amphiphilic polymer, radioabsorber, radiosensitizer, and radio-afterglow substrate were mixed at a mass ratio of 500: 10: 1: 2.5 in a flask, followed by evaporation of all organic solvents. The dried film was hydrated with aqueous solvent (such as deionized water or 0.01 M pH 7.4 PBS) under vigorous vortex, yielding transparent solution of nanoparticles. [Radioabsorber] = 100 pg / mL (Polystyrene (PS), 2.5 × 10-3pmol / mL; Anthracene (AT), 5.6 × 10-1μmol / mL; 3.0×10-1pmol / mL; CzTPN, 8.1 × 10-2pmol / mL), [Radiosensitizer] = 10 pg / mL (hematoporphyrin (HMP), 1.7 × 10-2pmol / mL; verteporfin (VP), 1.4 × 10-2pmol / mL; NIR775, 7.5 x 10-3pmol / mL), [Radioafterglow substrate] = 25 pg / mL (DPAs, 5.0 × 10-2pmol / mL; DTDP, 5.4 × 10-2μmol / mL). Specifically, RANP was developed by F127, CzTPN, NIR775, DTDP at a mass ratio of 500: 10: 1: 2.5 after optimization. [CzTPN] = 100 pg / mL (8.1 × 10-2pmol / mL), [NIR775] = 10 pg / mL (7.5 x 10-3pmol / mL), [DTDP] = 25 pg / mL (5.4 x 10-2μmol / mL). The excess F127 was removed via ultrafiltration (MWCO 3500) at 4,000 rpm for 10 min. The morphology of RANP was characterized using transmission electron microscope (TEM, JEOL JEM 1400), and hydrodynamic size and zeta potential were determined using Malvern Nano-ZS.

[0207] Optical properties characterization

[0208] CzTPN was dissolved in dichloromethane, NIR775 and DTDP (and caged DTDP) in THF. Nanoparticles were dissolved into PBS (pH 7.4, 0.01 M). The absorbance spectra were recorded using GENESYS™ 50 UV-Vis spectrophotometers (Thermo Fisher, U. S. A.). The fluorescence spectra were recorded using F-4600 fluorescence spectrophotometer (Hitachi, Japan). X-ray was generated using a miniature, lab-standard, portable X-ray tube ordered from Moxtek. Inc. USA. Parameters: target material, tungsten; maximum tube voltage, 70 KV; maximum tube current, 1000 pA; PC I2C control). By adjusting the voltage, current, distance between sample and X-ray, the dose rate of X-ray can be adjusted from pGy / s to Gy / s applied on sample according to manufacturer’s instruction. The dose rate under different conditions were calibrated using an X-ray dosimeter (Raysafe, R / F X2X, FLUKE). For radioluminescence measurement, the portable X-ray tube was equipped with fluorometer, by which the samples were irradiated with real-time X-ray while the emission was recorded using detector of fluorometer. Radioluminescence was also recorded by X-ray tube-incorporated in vivo imaging system (IVIS, Artemis Intelligent Imaging. Inc., China). For radio-afterglow measurement, the emission was recorded after cessation of X-ray irradiation by fluorometer. The radio-afterglow intensities were measured using luminometer (Promega, Glomax™) and IVIS. X-ray responsiveness was tested by radioafterglow measurement under different X-ray dosages (0.1 -2.0 Gy). Limit of detection (LOD) was calculated by 3 o / s, where o denotes the standard deviation of blank samples and s denotes the slope of calibration curve. Results and discussion

[0209] To optimize radio-afterglow, the composition of RANP was screened. Radioabsorbers used were organic agents with high X-ray conversion efficiencies (light yield > 104photons / MeV), including anthracene, polystyrene, 9,10-diphenylanthracene, and 2,4,5,6-tetrakis(3,6-di-tert-butylcarbazol-9-yl)-1,3-dicyanobenzene (CzTPN) (FIG. 2a and Table 1). Radiosensitizers used were hematoporphyrin, verteporfin, and silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), due to their bright visible to NIR fluorescence and high1O2 quantum yields. Radio-afterglow substances used were dicyanomethylene-4H-benzothiopyran-phenoxyl-adamantylidene (DPAs) and ((2-(3-((E)-4-hydroxy-2-((1r,5R,7S)-4'-methoxyspiro[adamantane-2,3'-[1,2]dioxetane]-4'-yl)styryl)-5,5-dimethylcyclohexan-1 -ylidene)malononitrile) (DTDP), which could form dioxetane intermediate emitting bright nearinfrared chemiluminescence (Huang, J. et al., Angew Chem Int Ed Engl 2022, 61, e202203235; and Wei, X. et al., Angew Chem Int Ed Engl 2023, 62, e202213791). The radioabsorber, radiosensitizer and radio-afterglow substate were co-loaded into nanoparticles using amphiphilic polymers as the matrix via nanoprecipitation method, including poly(ethylene glycol)-block-poly(propylene glycol)-block-poly (PEG-PPG-PEG, Pluronic F127), poly(lactic-co-glycolic acid) (PLGA)-PEG, or2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG). Table 1. Optical properties of candidates for radio-afterglow nanoparticles. Radioabsorbers used included anthracene (AT), polystyrene (PS), 9,10-diphenylanthracene (DAT), and CzTPN. Radiosensitizers used included hematoporphyrin, verteporfin, and NIR775. Radio-afterglow substrates used included DPAs and DTDP. Amphiphilic stabilizers used included F127, DSPE-PEG, and PLGA-PEG. Radio-afterglow nanoparticles were constructed via nanoprecipitation methods.

[0210] Radioabsorber Polystyrene (PS) Anthracene (AT) 9,10-Diphenylanthracene (DAT) 4CzTPN-tBu Light yield (photon / MeV) -10000 -15900 -16000 -44900 Radioluminescence / fluorescence -420 -397 -430 -554

[0211] max.

[0212] Reference J. Appt. Phys. Nat. Mater. 2021, Mater. Chem. Front., 2022, 6, Nat. Mater. 2021,

[0213] 2016, 120, 144301 21, 210-216 1470-1476 21, 210-216 Radiosensitizer Hematoporphyrin Verteporfin (VP) NIR775

[0214] (HMP)

[0215] Absorbance (Amax, nm) 382 430, 691 774

[0216] Fluorescence max. (nm) 612 695 780

[0217] Singlet oxygen quantum yield 0.73 0.53 0.29

[0218] Reference J. Photochem. J. Photochem. Photobiol. A: Chem. Photoacoustics,

[0219] Photobiol. B: Biol. 2017, 332, 66-71 2018, 9, 49-61 1997, 37, 131-140

[0220] Radio-afterglow substrate DPAs DTDP Absorbance (Amax) 470 426 Chemiluminescence max. (nm) 760 552 Chemiluminescence yield (10-2einstein / mol) 0.23 4.6

[0221]

[0222] Reference Angew. Chem. Int. Ed. Engl. 2021, Angew. Chem. Int.

[0223] 60, 3999-4003 Ed. 2023, 62, e202213791 Amphiphilic stabilizer PEG-PPG-PEG (F127)

[0224] DSPE-PEG (PEG, Mn 2,000)

[0225] PLGA-PEG (PEG Mn 2,000, PLGA Mn 4,500)

[0226]

[0227] CzTPN-loaded nanoparticles emitted brighter afterglow than those loaded with other radioabsorbers, which could be mainly attributed to its highest light yield (4.5 x 104photons / MeV) (FIGS. 2b and 2c). Moreover, hematoporphyrin-loaded nanoparticles exhibited higher radio-afterglow signals than verteporfin- and NIR775-loaded nanoparticles, in line with their1O2generation yields. Furthermore, DTDP-loaded nanoparticles emitted brighter radioafterglow than DPAs-loaded nanoparticles, probably because of its chemiluminescence quantum yield is ~20 times higher than those of DPAs. Nevertheless, DTDP has a short emission wavelength (peak at 552 nm) that could be attenuated in biological tissues, restraining their deep tissue imaging in vivo (FIGS. 3 and 4). Thus, NIR775 with absorption spectral overlap (with DTDP emission) and fluorescence peaked at 788 nm was chosen as radiosensitizers to ‘relay’ energy from DTDP, despite its moderate1O2generation ability.

[0228] In addition, F127 nanoparticles emit brighter radio-afterglow than in DSPE-PEG and PLGA-PEG nanoparticles under a same mass concentration (FIGS. 2d and 5). This could be attributed to the most compact size of F127 nanoparticles that led to the shortest X-ray energy transfer distances among three nanoparticles. Collectively, RANP comprising CzTPN as radioabsorber, NIR775 as radiosensitizer, DTDP as radio-afterglow substrate, and F127 as vehicle was chosen for subsequent mechanistic experiments.

[0229] Example 3. Cascade X-ray energy converting mechanism

[0230] Radioluminescence experiments were performed by following the optical properties protocol described in Example 2.

[0231] To study the mechanism of radio-afterglow, radioluminescence (light emission under real-time X-ray irradiation) was first tested in F127 nanoparticles loading CzTPN alone (FIGS. 6a and 6b). Under X-ray irradiation, CzTPN nanoparticles emitted bright radioluminescence with the emission maximum at 563 nm, similar to the fluorescence spectrum upon excitation at the maximum absorption of CzTPN at 420 nm (FIG. 7). This suggests that radioluminescence from CzTPN involves ‘Si— > So’ relaxation. Moreover, the radioluminescence intensities were increased with escalating tube current of X-ray (0-0.5 mA) and CzTPN amount in nanoparticles (FIGS. 8 and 9). The highest signal was measured at a mass ratio of CzTPN to F127 at 1:50; precipitation was observed beyond that ratio. At this mass ratio, anthracene- or polystyrene-loaded F127 showed radioluminescence signals 4 and 10 times lower than CzTPN nanoparticles (FIG. 10), respectively. The relatively higher radioluminescence brightness of CzTPN nanoparticles compared to others, coincides with the high light yield of CzTPN, which should be mainly attributed to the low energy gap between Si and Ti (ΔEST< 0.1 eV) that facilitates reverse intersystem crossing and electron-rich donor-acceptor structure that improves intramolecular electron transfer.

[0232] To test the X-ray energy transfer from radioabsorber to radiosensitizer and radiodynamic effect, NIR775 was loaded alone or co-loaded with CzTPN into F127 nanoparticles. Negligible radioluminescence was detected from NIR775 nanoparticles above 750 nm; by contrast, brighter signals were shown in CzTPN / NIR775 nanoparticles and were 30 times brighter than that in CzTPN nanoparticles (FIG. 6b). Moreover, the radioluminescence spectrum of CzTPN / NIR775 nanoparticles resembled the fluorescence spectrum excited at 420 nm (FIG.

[0233] 6c), showing two characteristic peaks corresponding to CzTPN at 563 nm and NIR775 at 788 nm (FIG. 7). With increased doping amount of NIR775, NIR radioluminescence increased at the expense of the visible radioluminescence from CzTPN, showing a highest intensity at a mass ratio of 1: 10 (molar ratio of 1: 10.8, NIR775 to CzTPN) with a radio-afterglow energy transfer (RET) efficiency of 68.9% (FIG. 6d). The sharp decrease of NIR radioluminescence beyond that ratio should be caused by the self-quenching of NIR775.

[0234] Apart from radioluminescence, NIR775 / CzTPN nanoparticles generated1O2that are 100 and 156 times higher than mono-NIR775 or CzTPN nanoparticles under equivalent mass concentrations (FIG. 6e). The highest1O2generation yield was generated at a doping ratio of 1: 10 (NIR775 to CzTPN, w / w), in line with the optimal ratio for radioluminescence (FIG. 6f). Moreover,1O2generation increased with X-ray dosages and concentrations of CzTPN / NIR775 nanoparticles (FIG. 11). However, no superoxide anions or hydroxyl radicals were detected (FIG. 12), indicating that1O2was produced from NIR775, a typical type-ll photosensitizer. Collectively, these data validate the RET process from CzTPN to NIR775, leading to NIR radioluminescence (via ‘Si— > S0’ relaxation) and1O2 production (via ‘Ti— > S0’ relaxation) (FIG.

[0235] 6g).

[0236] After confirming the occurrence of RET and radiodynamic1O2generation, DTDP was coloaded with CzTPN and NIR775 to study the radio-afterglow mechanism (FIG. 13a). After cessation of X-ray irradiation, bright radio-afterglow was observed only for RANP (rather than the mono-system (e.g. DTDP nanoparticles) or the binary system (e.g., CzTPN / DTDP nanoparticles)), indicating that each component is indispensable for radio-afterglow (FIGS.

[0237] 13b and 13c). Moreover, the radio-afterglow spectrum of RANP peaked at 552 and 788 nm, corresponding to DTDP and NIR775, respectively, which resemble its fluorescence spectrum upon excitation of DTDP at 430 nm. The presence of DTDP afterglow emission confirms the formation of chemiluminescent DTDP intermediate induced by cycloaddition reaction with1O2 generated by NIR775; whereas the NIR775 afterglow emission proves the partial chemiluminescent energy transfer from the intermediate DTDP to NIR775, allowing RANP to achieve a record long radio-afterglow at 788 nm for existing organic materials (FIGS. 13d and 14). The radio-afterglow intensities of RANP increased with escalating X-ray dosage with a limit of detection of 0.046 mGy, showing its high X-ray responsiveness (FIG. 15). It also exhibited a half-life of 4.8 min and a lifetime over 12 h, and its intensities maintained after five cycles of X-ray irradiation, indicating rechargeability (FIGS. 13e and 16).

[0238] Based on these observations, the radio-afterglow mechanism via a cascade X-ray energy converting approach was proposed (FIG. 13g). Owing to the high light yield, radioabsorbers attenuate X-ray via photoelectric and Compton effects, resulting in scintillation and radioluminescence. The radioluminescence can then be transferred to radiosensitizers (RET process) to generate1O2.1O2 is subsequently transferred to radio-afterglow substrates (SOT process), oxidizing them to self-luminescent intermediates of high chemical energy. These intermediates gradually decompose to emit the long-last luminescence (radio-afterglow). The yielded radio-afterglow could also be able to transfer back to radiosensitizer, causing the red-shifted radio-afterglow from radiosensitizers. With an optimal hydrodynamic diameter of 94.2 ± 2.6 nm, a slightly negative surface charge of -6.3 ± 1.1 mV, and a spherical shape (transmission electron microscopy) (FIGS. 13f, 17 and 18), RANP comprising CzTPN as radioabsorber, NIR775 as radiosensitizer, DTDP as radio-afterglow substrate, and F127 as vehicle should have ideal blood circulation and tumour accumulation.

[0239] Example 4. In vitro studies of radio-afterglow imaging depth and radiodynamic cytotoxicity

[0240] Deep-tissue penetration assay

[0241] RANP ([DTDP] = 100 pg / mL) in PBS (0.01 M, pH 7.4) was first irradiated with laser (680 nm, 0.3 W / cm2) or X-ray (50kV, 200 pA) without any coverage to obtain the same afterglow signals. Afterwards, RANP solutions were put beneath chicken breast tissues of different thicknesses (2.5, 5, 10 and 15 cm) and irradiated with laser or X-ray, followed by afterglow imaging using an in vivo imaging system after removing the tissues.

[0242] In another set of studies, samples were irradiated, and afterglow signals were detected through chicken breast tissues of different thicknesses (1.5, 3.5 and 5 cm). Exposure time was set at 30 s.

[0243] Radio-afterglow induction on cells 4T1 cancer cells (or MCF-7 cancer cells) were seeded onto glass-bottom dishes (2 × 105per dish) and incubated in complete DMEM (containing 10% FBS and 1% penicillin-streptomycin) for 24 h. Cells were then incubated with or without RANP ([DTDP] = 20 pg / mL) for 12 h, followed by X-ray irradiation (1 mGy) or not. Radio-afterglow signals were immediately captured using inverted microscope (Leica, German) with open filter and any excitation off. Exposure time was set at 10 s. For the observation of cells treated with tRANP, the media was added with H2O2 (20 pM) and incubated for 0.5 h before tRANP incubation.

[0244] In another set of experiment, MCF-7 cells in glass-bottom dishes (2 x 105per dish) were incubated with CzTPN nanoparticles ([CzTPN] = 80 pg / mL), NIR775 nanoparticles ([NIR775] = 8 pg / mL), DTDP nanoparticles ([DTDP] = 20 pg / mL) for 12 h, irradiated with X-ray irradiation (1 mGy), and immediately imaged under inverted microscope with any excitation off.

[0245] In the third set of experiment, MCF-7 cells in glass-bottom dishes (2x105per dish) were preincubated with tryptophan (5 mM) for 6 h and incubated with RANP ([DTDP] = 20 pg / mL) for 12 h, followed by X-ray irradiation (1 mGy) and imaging under inverted microscope.

[0246] In vitro ROS detection

[0247] For1O2measurement, RANP in PBS ([CzTPN] = 100 pg / mL, [NIR775] = 10 pg / mL) was supplemented with 4-amino-2,2,6, 6-tetramethylpiperidine (TEMP, 10 mM), followed by X-ray irradiation (1 Gy). The samples were immediately detected using electron spin resonance (ESR). For superoxide anion and hydroxyl radical detection, RANP in PBS ([NIR775] = 10 pg / mL) was supplemented with 5, 5-dimethyl-1 -pyrroline N-oxide (DMPO, 100 mM). Samples after radiation were immediately analyzed using ESR. RANP in PBS ([NIR775] = 40 pg / mL) was also put beneath chicken breast tissues of different thicknesses prior to X-ray irradiation and1O2 was determined using ESR.

[0248] Cell viability assay

[0249] 4T 1 cancer cells (or MCF-7 cancer cells) were seeded onto 96-well plate (2x 104per well) and incubated for 24 h. Cells were incubated with or without RANP ([NIR775] = 5, 10, 20, 40, 80 pg / mL) for 12 h and irradiated with or without X-ray (0.5 Gy), followed by further incubation for 12 h. Cells without any treatment was set as control. Cells were then incubated with CCK-8 agent (10 pL per well) for 2 h, and the absorbance of each well was determined at 450 nm using microplate reader (BioTek, Lonza, Switzerland). The cell viability was calculated as (mean ODsample– Mean ODblank) / (mean ODcontrol– Mean ODblank) × 100%. In another set, 4T1 cancer cells were treated with RANP at a fixed concentration of [NIR775] = 20 pg / mL for 12 h, and irradiated with different doses of X-ray (0.2, 0.5, 1, 1.5, 2.0 Gy). To mimic cell killing in deep tissues, cells were treated with RANP ([NIR775] = 20 pg / mL) and irradiated with X-ray (0.5 Gy) through chicken tissue of different thicknesses (5, 10, 15 and 20 cm). Cells without any treatment was set as the control group.

[0250] Cell apoptosis study

[0251] 4T1 cells (or MCF-7 cells) were seeded onto 6-well plate and incubated for 24 h. The media were refreshed and supplemented with or without RANP ([NIR775] = 20 pg / mL) and incubated for 12 h. Afterwards, cells were irradiated with X-ray (0.5 Gy) and incubated for another 12 h. Cells were stained with Annexin V-FITC (1:100) and PI (1:100) for 20 min in the dark, according to the manufacturer’s instructions. The stained cells were analyzed using flow cytometry (CytoFLEX, Beckman Coulter, U. S. A.).

[0252] Intracellular ROS detection

[0253] 4T1 cancer cells (or MCF-7 cancer cells) were seeded onto glass-bottom dishes (2*105per dish) and incubated for 24 h. Cells were incubated with or without RANP ([NIR775] = 20 pg / mL) for 12 h, followed by incubation with dichloro-dihydro-fluorescein diacetate (DCFH-DA) for another 2 h. Cells were then irradiated with X-ray (0.5 Gy) or not. Cells were stained with Hoechst 33342 (1: 200), followed by fluorescence observation under confocal laser scanning microscope (CLSM, Leica, German).

[0254] Results and discussion

[0255] To demonstrate deep-tissue radio-afterglow, RANP was irradiated by X-ray or laser with chicken breast tissues of different thicknesses (FIG. 19a) and the signal was then detected after removing the chicken breast tissues. Note that X-ray and laser irradiation conditions were adjusted to afford the same afterglow intensities without chicken breast coverage (FIGS. 19b and 19c). Both photoafterglow and radio-afterglow intensities decreased with the increased tissue thickness, while radio-afterglow was brighter than photoafterglow at all depths, showing 50 times higher in signals at the tissue depth of 2.5 cm. Radio-afterglow was still detectable at 15 cm (signal to background ratio (SBR) 3.7), while photoafterglow was hardly detectable at 5 cm (SBR 2.3). Moreover, when both pre-irradiation and signal acquisition were conducted through tissues, radio-afterglow was detectable at 5 cm (SBR 5.9), in sharp contrast to that of 1.5 cm by photoafterglow (SBR 5.0) (FIG. 20).

[0256] To gain insight into the brighter radio-afterglow in deep tissue relative to photoafterglow,1O2generation from RANP under X-ray or laser irradiation was studied (FIG. 19d). Without tissue coverage, X-ray and laser irradiation conditions were controlled to make1O2 generated by RANP the same. The maximum depth to induce1O2generation was 15 cm and 5 cm for X-ray and laser irradiations, respectively. These data confirm that the superior deep-tissue imaging of radio-afterglow as compared to photoafterglow is due to the higher1O2 yield of the radiodynamic process relative to the photodynamic process in deep tissues.

[0257] The radio-afterglow imaging and radiodynamic effect of RANP was studied in 4T1 and MCF-7 cancer cells. After incubation with RANP for 12 h, radio-afterglow signal from cells was observed, verifying the cellular uptake of RANP (FIGS. 19e and 19f). In contrast, radioafterglow signal was negligible for cells treated with single-component nanoparticles (i.e. CzTPN nanoparticles, NIR775 nanoparticles, and DTDP nanoparticles) (FIGS. 21 and 22). The radiodynamic cytotoxicity of RANP was studied at various incubation concentrations (5-80 pg / mL) by cell counting kit-8 assay (CCK-8) (FIG. 19g). Without X-ray irradiation, cells showed negligible death even at the highest RANP concentration; by contrast, the cell viability decreased with increasing dose of X-ray from 0.01 Gy to 2 Gy (FIG. 23), showing only 10% at 0.5 Gy. Even through 15 cm-thick tissue, 70% of cells-killing could be achieved, verifying the feasibility of RANP-mediated radiodynamic therapy in deep tissue (FIG. 24). Cells after treatment were further stained with fluorescein isothiocyanate (FITC)-tagged annexin and PI, followed by analysis with flow cytometry (FIG. 25). RANP-treated and X-ray-irradiated cells showed the least population of living cells (approximately 20%), consistent with cytotoxicity assay. Note that early apoptotic cells (annexin-FITChigh / PIlow) accounted for approximately 52% of cells, ~ 5 times higher than other groups. This suggests that apoptosis should be a major way of cell death by RANP-mediated radiodynamic therapy, which potentially enhances immunogenicity to benefit tumour immunotherapy.

[0258] To verify the radiodynamic cytotoxic mechanism, cells after different treatments were preincubated with dichloro-dihydro-fluorescein diacetate (DCFH-DA), a turn-on fluorescent sensor for intracellular ROS detection. The CLSM (confocal laser scanning microscope) images (FIGS. 19h and 19i) show that the fluorescence of RANP-treated X-ray-irradiated cells was >9.5 times higher than that of other groups. Moreover, if the cells were pretreated with tryptophan (1O2scavenger) prior to RANP incubation, the cellular viability increased by 4-fold as compared to that without tryptophan treatment (FIG. 26). Cells treated with NIR775-free RANP showed increased viability compared to those treated with RANP. Similar tumor killing effect of RANP was observed for MCF-7 cells (FIGS. 27-29).

[0259] Example 5. In vivo radio-afterglow cancer theranostics H2O2responsiveness of cDTDP and tRANP

[0260] cDTDP ([DTDP] = 10 μM) was incubated with or without H2O2(20 μM) in PBS (0.01 M, pH 7.4) for 10 min followed by analysis with high-performance liquid chromatography (HPLC). tRANP was incubated with or without H2O2(20 μM) in PBS (0.01 M, pH 7.4) for 10 min, followed by X-ray irradiation (1 mGy). The radio-afterglow was recorded using fluorometer with excitation off and using IVIS (exposure time 10 s). RANP was also incubated with other ROS including superoxide anion (O2•−), nitric oxide (NO), and hydroxyl radicals (-OH) at same concentration for comparison.

[0261] Animal models

[0262] All animal experiments were conducted in accordance with the guidelines approved by Ethics Committee of China-Japan Friendship Hospital (China) and Guidelines for Care and Use of Laboratory Animals of the NTU Institutional Animal Care and Use Committee (IACUC, Singapore). For all animal experiments, Balb / c mice (5-6 wks, female) were ordered from GemPharmatech Inc. (China). Mice were kept in ventilated clear plastic cages under appropriate ambient temperature (~22 °C), humidity (50%) and standard 12 h: 12 h light: dark conditions. The subcutaneous tumour model was established by subcutaneous injection of 100 μL of 4T1 cancer cells (2 × 106) into the right flank of mice. Abdominal metastatic model was established by intraperitoneally injecting with 100 μL of 4T1 cancer cells (2 × 106). At 7 days post inoculation, mice were randomly divided into different groups for indicated treatments. X-ray was irradiated solely on tumours (or subcutaneously injected sites) with other parts of body covered with lead shield. Mice were anesthetized with isoflurane / oxygen system during in vivo imaging and surgery. After surgery, mice were additionally treated with meloxicam (2 mg / kg, p.o.) for a week for analgesia. Mice were euthanized when tumour size reaches 2000 mm3(under ethical approval).

[0263] In vivo radio-afterglow imaging

[0264] Subcutaneous 4T1 tumour-bearing mice (n = 3) were shaved to expose both left and right flank (with tumours). 10 μL of tRANP ([cDTDP] = 100 μg / mL) was subcutaneously injected into the left flank and intratumorally injected into the right flank. After 10 min, the two injection sites were irradiated with X-ray (1 mGy) and the afterglow signals were recorded using IVIS with open filter (acquisition time: 30 s). Fluorescence signals were recorded using IVIS equipped with 750 long-pass filter (excitation wavelength: 660 nm, acquisition time: 0.1 s). In another set of imaging study, subcutaneous 4T1 tumour-bearing mice (n = 3) were intravenously injected with 100 μL of tRANP ([cDTDP] = 250 μg / mL) and imaged at indicated time points (0, 3, 6, 12, 24, 36 and 48 h) using IVIS under fluorescence and radio-afterglow modes. For radio-afterglow induction, X-ray (1 mGy) was irradiated on tumours at each time point. Acquisition time: 30 s.

[0265] Imaging-guided laparotomy

[0266] Abdominal 4T1 tumour-bearing mice (n = 3) were intravenously injected with 100 μL of tRANP ([cDTDP] = 250 μg / mL). After 12 h, the abdomen region was irradiated with X-ray (1 mGy), followed by radio-afterglow acquisition using IVIS. Exposure time: 30 s. Afterwards, mice were subjected to laparotomy by opening the abdomen, followed by radio-afterglow acquisition using IVIS without further X-ray irradiation. Tumours were carefully collected under the guidance of radio-afterglow imaging and subjected to histological analysis, and mice were subjected to surgical suture and monitored after surgery. Fluorescence signals were recorded using IVIS (excitation wavelength: 660 nm, acquisition time: 0.1 s).

[0267] Histological analysis

[0268] Main organs including heart, liver, spleen, lung, kidneys, and skin were collected for fixation with 4% PFA for 12 h, 30% sucrose for 24 h, and sectioned for hematoxylin and eosin (H& E) staining. The suspected tumours from laparotomy were also sectioned for H& E staining. Pulmonary metastatic nodules were counted prior to fixation and tissue staining. The sections were observed under an inverted microscope. For immunogenic cell death analysis, tumour sections we further stained with antibody against calreticulin (PA3-900, 1:200, Thermo Fisher Scientific) or high mobility group box 1 (HMGB-1, 3E3, Alexa Fluor 488-labelled, 1:200, Biolegend), as well as DAPI (1: 200). The immunofluorescent sections were observed under CLSM.

[0269] tRANP-mediated radiotherapy in vivo

[0270] On day 1, 4T1 tumour-bearing mice (n = 5 for each group) were divided to four groups and treated with PBS, X-ray only, tRANP only, or tRANP plus X-ray. 100 μL of tRANP ([NIR775] = 100 μg / mL) was intravenously injected with and X-ray (1 Gy) was irradiated on tumours after 24 h. The indicated treatments were conducted on day 3 and 5 as well. The X-ray dosages were optimized with 4T 1 tumour-bearing mice (n = 3 for each group) under 3 fractions of 0.5, 1 and 2 Gy, respectively. For comparison, tRANP-free 4T1 tumour-bearing mice (n = 3) were subjected to X-ray (6 Gyx3 fractions), which mimic clinical radiotherapy. Tumours were measured every two days and volumes were calculated via the formula: V = 1 / 2 ab2, where a is the length and b is the width of tumour mass. Tumour inhibition rate was calculated by (1 -(mean volume of treated tumours ) / (mean volume of PBS-treated tumours)) * 100%. Survival analysis was conducted with Kaplan-Meier estimate using GraphPad Prism 7.0.4. Intratumoral ROS detection

[0271] 4T1 tumour-bearing mice were intravenously injected with 100 μL of tRANP ([NIR775] = 100 μg / mL). At 12 h post injection, singlet oxygen sensor green (SOSG, Thermo Fisher, 1:100) was intratumorally injected into tumours. After 15 min, tumours were irradiated with X-ray (1 Gy), and immediately collected from mice after sacrifice. Fresh tumours were subjected to frozen section and slices were counter-stained with DAPI (1: 200, Beyotime). The tissues slices were observed under confocal microscope.

[0272] Safety profile

[0273] The body weights of mice were measured every two days until the end of experiments. Whole blood samples and sera were collected for the measurement of regular blood parameters and biochemical indicators, respectively, using an automatic analyzer (DRI-CHEM NX700V, Fujifilm, Japan). Blood samples were extracted from healthy mice and tumour-bearing mice treated with RANP. Sera were collected via centrifugation at 1000 rpm for 4 min and determined with commercial kits (Sigma Aldrich, U. S. A.) for blood urine nitrogen (BUN), creatinine, alanine transaminase (ALT) and Aspartate aminotransferase (AST) respectively, according to the manufacturers’ instruction.

[0274] Results and discussion

[0275] To enable precision radio-afterglow imaging, tumour-specific RANP (tRANP) was developed to activate its radio-afterglow only in the presence of H2O2, which is upregulated in tumours (FIGS. 30a and 31). DTDP was caged by phenylborate ester (a moiety specifically oxidized by H2O2), affording cDTDP with diminished intramolecular charge transfer and thus inhibited afterglow emission (FIG. 1 ). In the presence of H2O2, the caging group of cDTDP in tRANP is cleaved, as verified by a new elution peak at 17 min in the HPLC chromatogram, leading to a 40-fold increase of radio-afterglow (FIGS. 30b and 32). Moreover, the radio-afterglow spectral profile of H2O2-treated tRANP resembles its fluorescence (FIG. 33). tRANP demonstrated a superb selectivity towards H2O2over other reactive species such as superoxide anion (O2•−), nitric oxide (NO), and hydroxyl radicals (-OH) (FIG. 34). The biomarker-activatable radioafterglow was observed in 4T1 cells treated with H2O2and X-ray irradiation, showing 8- and 11 -times higher signal than those with X-ray or H2O2treatment alone (FIG. 35).

[0276] To study tumour-specific radio-afterglow in vivo, tRANP was subcutaneously injected into the left flank and intratumorally injected into the 4T 1 tumour on the right flank of mice, respectively (FIG. 30c). At 10 min post-injection, the injection sites were irradiated with X-ray prior to radioafterglow acquisition. The radio-afterglow signal on tumours was 39 times higher than that on subcutaneous sites, with an SBR as high as 169 vs 14 for fluorescence signals (FIGS. 30d and 30e). Next, tRANP was intravenously injected to tumour-bearing mice, and its biodistribution was longitudinally monitored (FIG. 30f). Both fluorescence and radio-afterglow signals on tumours increased over time and peaked at 24 h. At this timepoint, the SBR of radio-afterglow (207) was 18 times higher than fluorescence (11), showing the superiority of radio-afterglow over fluorescence in in vivo cancer detection (FIGS. 30g and 30h). Such a high SBR for subcutaneous tumor detection was one order of magnitude higher than most existing inorganic materials (for example, 16.5 for ZnGa2O4: Cr3+) and comparable to the only reported organic probe (for example, 234 for MRAP) (Table 2) (Huang, J. et al., Nat Mater 2023, 22, 1421-1429; and Chen, Z. Z. etal., Adv Mater 2019, 31, e1905087). Moreover, due to the high responsiveness to ionization, tRANP could be recharged with a minimal X-ray dose of 1 mGy after a single injection, and such dose was at least 20- and 5-times lower than the inorganic materials and the organic probe, respectively (Huang, J. etal., Nat Mater 2023, 22, 1421-1429). Ex vivo imaging showed that tRANP was mostly accumulated in tumour (owing to the optimal size and surface charge), followed by liver and spleen (FIG. 36).

[0277] Table 2. Representative agents for X-ray-excited afterglow in preclinical studies.

[0278] Material Aem Half-life Lifetime X-ray Imaging SBR after Ref.

[0279] dosage depth systemic

[0280] injection

[0281] Inorganic materials

[0282] LGO: Cr -720 Minutes 5 h 0.02 Gy 1. 5 cm N. A. Mater Horiz @mSiC>2 nm 2017, 4,

[0283] 1092-1101 PEG- 525 <1 min 2 h N. A. 2.5 cm N. A. Nanoscale SrAI2O4: Eu2+nm 2017, 9,

[0284] 2718-2722 ZnGa2O4: Cr / 696 Minutes Hours 0.18 Gy 2.0 cm N. A. Adv Fund W nm Mater 2018,

[0285] 28, 1707496 ZnGa2O4: Cr3-690 - 1.6 6 h 0.5 Gy N. A. 16.5 Adv Mater + nm min (HepG2 2019, 31, orthotopic 1905087 tumourbearing

[0286] nude mice)

[0287] Si-Pc@PE- -710 - 2 min 8 h 1 Gy 0.9 cm N. A. Adv Fund mZGGOs nm Mater 2020,

[0288] 30, 2001166

[0289]

[0290] Mn-ZGGOs 696 Minutes 2 h 0.1 Gy 1.0 cm -240 Theranostics nm (U87MG 2021, 11, subcutaneo 7439-7449 us tumourbearing

[0291] Balb / c nude

[0292] mice)

[0293] NaYF4: Er 1525 Minutes > 72 h 200 Gy N. A. 40.9 Nat @NaYF4nm (subcutane Nanotechnol ous 4T1 2021, 16, tumour1011-1018 bearing

[0294] Balb / c

[0295] mice)

[0296] LaGaCh: 750 Minutes >500 h 0.37 Gy N. A. 100 Chem Eng J, Cr3+, Sb3+nm (healthy 2021, 404,

[0297] Kunming 127133 mice)

[0298] Organic materials

[0299] IDPAs 773 18.4 min 25 h 0.005 5 cm -38 Nat Mater nm Gy (U87MG 2023, 22, subcutaneo 1421-1429 us tumourbearing

[0300] Balb / c nude

[0301] mice)

[0302] MRAP 770 N. A. N. A. 0.005 N. A. -234

[0303] nm Gy (U87MG

[0304] orthotopic

[0305] tumourbearing

[0306] Balb / c nude

[0307] mice)

[0308] RANP 788 4.8 min > 12 h 0.001 5 cm 207.1 The present nm Gy (subcutan disclosure eous 4T1

[0309] tumourbearing

[0310] balb / c

[0311] mice)

[0312]

[0313] 121.9

[0314] (abdomenopen

[0315] peritoneall

[0316] y

[0317] metastatic

[0318] tumourbearing

[0319] balb / c

[0320] mice)

[0321]

[0322] SBR: signal-to-background ratio. N. A, not available. LGO, LiGa5O8. MRAP, molecular radio afterglow probe.

[0323] The capability of imaging-guided surgery was tested in a peritoneal metastasis model established by intraperitoneal inoculation of 4T1 cells into mice (FIG. 30i). At 7 days postinoculation, mice were intravenously injected with tRANP for radio-afterglow imaging. Tumours were clearly delineated by radio-afterglow signals in both intact (SBR = 53) and abdomen-open mice (SBR = 122); by contrast, they were indistinguishable from surrounding tissues by fluorescence imaging due to the high background noises (FIGS. 30j and 30k). The stark contrast provided by radio-afterglow imaging enables a complete resection of diminutive tumours as small as 1 mm3, which were confirmed by histological analysis (FIG. 37).

[0324] To study the in vivo radiodynamic therapeutic efficacy, subcutaneous 4T1 tumour-bearing mice were intravenously injected with tRANP, and tumours were irradiated with 3 fractions of X-ray (1 Gy per fraction), which was optimized in pilot assays (FIGS. 38a and 39). Mice with X-ray irradiation or tRANP alone were set for comparison. tRANP plus X-ray irradiation completely suppressed tumour growth with a tumour inhibition rate of as high as 97.2%, and no recurrence even after three weeks post-treatment; by contrast, tRANP alone had negligible tumour inhibition, showing a growth curve similar to PBS-treated group (FIG. 38b). X-ray irradiation alone had a slight inhibitory effect, and tumours were reinvigorated on day 11. The efficient tumour eradication by tRANP coupled with low-dose X-ray contributed to the prolonged survival of tumour-bearing mice up to one-month post-treatment (FIG. 38c). Note that complete tumour eradication could also be achieved by X-ray irradiation alone at a dosage 6 times higher than tRANP-mediated radiodynamic therapy, which is equivalent to the X-ray dosage for clinical radiotherapy (3 fractions of X-ray, 6 Gy per fraction) (FIG. 40) (Lo, S. S. et al., Nat Rev Clin Oncol 2010, 7, 44-54; and Stupp, R. et al., N Engl J Med 2005, 352, 987-996). The mechanism for effective tumour inhibition of tRANP was further analysed by injecting1O2 sensor green (SOSG) into tumours of mice after indicated treatments. Following X-ray irradiation (1 Gy), tumours were collected and sliced for fluorescence imaging. tRANP plus X-ray elicited the highest fluorescence of SOSG in tumours among all groups (FIGS. 38d and 38e), confirming the efficient intra-tumoral generation of1O2. As a result, tRANP-mediated radiodynamic therapy led to the largest area of tumour damage among groups, as characterized by vanished nuclei and broken cell morphology, as well as the highest expression level of caspase-3 (apoptotic biomarker) (FIGS. 38f-i); besides, it elicited the highest expression levels of calreticulin (CRT) and high mobility group box 1 (HMGB-1), suggesting the occurrence of immunogenic cell death (FIG. 41). Consequently, mice after tRANP-mediated radiodynamic therapy possessed the least metastatic nodules in lung (FIGS.

[0325] 38j and 38k).

[0326] Radiodermatitis is a common radiation-associated adverse effect characterized by skin peeling, lupus, and edema. Mice receiving traditional radiotherapy (6 Gy x 3 fractions) showed skin damage and immune cell infiltration in the tumour-adjacent skin at 7 days post treatment despite its ability to inhibit tumour growth; by contrast, tumour-adjacent tissue of mice treated with tRANP-mediated radiodynamic therapy was spared from damage (FIG. 42). This was attributed to the lower doses for tRANP-based therapy, which are also lower than those of most existing radiosensitizers in preclinical tumour radiodynamic therapy (Table 3). No measurable body weight loss nor histological abnormality was observed (FIGS. 43 and 44). Additionally, the indicators for blood biochemistry and hepatic / renal functions were within normal ranges, showing the safety of tRANP-mediated radiodynamic therapy (FIG. 45 and Table 4).

[0327] Table 3. Representative agents for radiodynamic tumour therapy in preclinical studies.

[0328] Material Material X-ray dosage ROS Cancer model Ref.

[0329] dose (dose x

[0330] fraction)

[0331] IDOi@DBP-Hf 0.11 mg 0.5 Gy x 6 •OH, Subcutaneous Nat Biomed nMOF1o2head and neck, Eng 2018, 2, glioblastoma 600-610 and prostate

[0332] cancer

[0333]

[0334] HMOP- 3 mg 8 Gy x 1 •OH, Subcutaneous Nat Comm TBHP / Fe(CO)5CO glioblastoma 2019, 10, 1241 NPs

[0335] NaCeF4: Gd, Tb 0.24 mg 6 Gy x 1 •OH, Subcutaneous Nano Lett ScNPs o2- lung cancer 2019, 19,

[0336] 8234-8244 RGD-ZSM-RB 0.4 mg 1 Gy x 21o2Subcutaneous Adv Mater NPs glioblastoma 2019, 31,

[0337] 1808024 Si-Pc@PEG- 0.4 mg 4 Gy x 21o2Orthotopic liver Adv Funct mZGGOs cancer Mater 2020,

[0338] 30, 2001166 Au@Cii2-xSe 0.07 mg 2 Gy x 4 •OH, Orthotopic Biomaterials NPs o2- glioblastoma 2022, 280,

[0339] 121287 SSCPs 0.4 mg 4 Gy x 1 •OH, Subcutaneous ACS Nano o2- colon cancer 2022, 16,

[0340] 20805-20819 TiO2@MIL 1 mg 1 Gy x 1 •OH, Orthotopic liver Nat o2- cancer Nanotechnol 2023, 18, 1492-1501 PNA 1.2 mg 1 Gy x 41o2Subcutaneous Nano Today breast cancer 2023, 48,

[0341] 101708 TBDCR NP 0.1 mg 8 Gyx21o2, Subcutaneous Adv Sci 2023,

[0342] o2- cervical cancer 10, 2302395 TZM 0.1 mg 6 Gy x 31o2, Subcutaneous Adv Sci 2023,

[0343] •OH osteosarcoma 10, 2206779 FA-Au-CH 0.4 mg 4 Gy x 1 •OH, Subcutaneous ACS Nano NPs1o2colon cancer 2023, 17,

[0344] 25147-25156 tRANP 0.1 mg 1 Gy x 31o2Subcutaneous The present (for breast cancer disclosure CzTPN),

[0345] 0.01 mg

[0346]

[0347] (for

[0348] NIR775)

[0349]

[0350] MOF, metal-organic framework. PNAs, Platinum-based MOF NP nanoassemblies. TiO2@MIL, bacterial membrane coated titanium dioxide nanoparticle. TBDCR NPs, aggregation-induced emission-based nanoscintillator. TZM, tantalum-zirconium co-doped metal-organic frameworks. ITC NPs, 9,9’-(6-iodophenoxy-1,3,5-triazine-2,4-diyl)bis(9H-carbazole) nanoparticles. Au@Cu2-xSe NPs, core-shell gold@copper selenide nanoparticles. Si-PC@PEG-mZGGOs, silicon phthalocyanine doped- polyethylene glycol-Zn3Ga2GeO8:Cr3+, Yb3+, Er3+nanoparticles. HMOP, PEGylated hollow mesoporous organosilica nanoparticles. TBHP, tert-butyl hydroperoxide. CO, carbon monoxide. SCNPs, scintillating nanoparticles. ZSM, Zn- and Mn-incorporated silica. RGD, arginylglycylaspartic acid. RB, rose Bengal. IDOi, indoleamine 2,3-dioxygenase 1 inhibitors. DBP, 5,15-di(p-benzoato)porphyrin. SCNPs, Ce(III)-doped LiYF4@SiO2@ZnO. FA-Au-CH NPs, nanoparticles comprising gold nanoradiosensitizers, photosensitizer chlorin e6, and folic acid. SSCPs, SCNPs@DMSN@CeOx-PEG.

[0351] Table 4. Routine blood analysis of mice treated with tRANP-mediated radiodynamic therapy.

[0352] Parameter Abbreviation Value* Unit Reference White blood cells WBC 5.2±1.4 109 / L 0.8-10.6 Lymphocytes Lymph# 4.6±0.3 109 / L 0.6-8.9 Monocytes Mon# 0.2±0.2 109 / L 0.04-1.4 Granulocytes Gran# 2.3±0.9 109 / L 0.23-3.6 Lymphocyte Lymph% 67.5±18.2 % 40-92 percentage

[0353] Monocyte Mon% 4.9±1.7 % 0.9-18 percentage

[0354] Granulocyte Gran% 28.1 ±5.3 % 6.5-50 percentage

[0355] Red blood cells RBC 8.8±0.3 1012 / L 6.5-11.5 Hemoglobin HGB 138±14.5 g / L 110-165 Hematocrit HCT 44.6±4.7 % 35-55 Average RBC MCV 47.7±4.7 fL 41-55 volume

[0356] Average MCH 15.9±2.1 pg 13-18 hemoglobin content

[0357] of RBCs Mean corpuscular MCHC 327±14.5 g / L 300-360 hemoglobin

[0358] concentration

[0359] RBC distribution RDW 15.5±2.9 % 12-19 width

[0360] Platelets PLT 1074.7±200 10^9 / L 400-1600 Average platelet MPV 5.2±0.8 fL 4.0-6.2 volume

[0361] Platelet distribution PDW 14.5±1.9 12.0-17.5 width

[0362] Platelet hematocrit PCT 0.6±0.1 % 0.100-0.780 * Calculated with blood samples from three independent mice.

[0363] General discussion

[0364] Herein, we report a generic X-ray energy converting approach to develop organic radioafterglow and radiodynamic nanoagents for cancer theranostics. Radio-afterglow nanoparticle (RANP) comprises a radioabsorber that potently converts X-ray to radioluminescence that transfers to a radiosensitizer, leading to the production of1O2.1O2 can then efficiently diffuse to a radio-afterglow substrate to undergo an in-situ reaction to convert it to an active dioxetane intermediate, which simultaneously decomposes and emits long-lasting afterglow after X-ray cessation. Through such as a cascade intraparticle radioluminescence energy transfer (RET) and1O2transfer (SOT) process, RANP can be devised to exhibit tuneable near-infrared (NIR) wavelength, long half-life, and superb brightness of radio-afterglow, allowing for the development of smart activatable nanoparticle that only switches on its radio-afterglow in the presence of cancer biomarkers for longitudinal tumour detection and precision tumour surgery in vivo. Moreover, the efficient1O2generation enables RANP to exert radiodynamic therapy on cancer at a lower X-ray dosage than clinical radiotherapy, which spares normal tissues from ionization-associated damage. Thus, RANP represents a generic approach to develop X-ray-based organic theranostic nanoagent with great translational potential.

[0365] The present disclosure reports a generic and efficient approach via X-ray energy and singlet oxygen cascade transfer process to construct a first example of organic radio-afterglow and radiodynamic nanoparticles (RANPs) and reveals the key parameters governing the radiotheranostic efficacies. The modular composition and well-defined mechanism of RANPs enabled finetuning their radio-afterglow wavelength, brightness, and biomarker responsiveness, leading to a smart cancer-specific radiotheranostic nanoparticle. RANP has the potential not only for ultrasensitive detection of diminutive lesions at excipient stage but also for precise and safe radiotherapy of deep-seated diseases beyond cancer at a minimal dosage. The efficient radiodynamic1O2generation of tRANP permits complete tumour eradication at an X-ray dosage lower than clinical radiotherapy and a drug dosage one to two orders of magnitude lower than most existing inorganic agents, leading to prolonged survival rate with minimized radiation-related adverse effect. Collectively, the present disclosure provides a generic approach to fill in the gap in organic radiotheranostic biomaterials and provides molecular design towards precision radiotherapy.

Claims

Claims1. A radio-afterglow nanoparticle comprising ABCD, wherein:A represents a radioabsorber;B represents a radiosensitizer;C represents a luminescent component; andD represents an amphiphilic polymeric matrix.

2. The radio-afterglow nanoparticle according to Claim 1, wherein the radio-afterglow nanoparticle further comprises E, wherein E represents a targeting moiety.

3. The radio-afterglow nanoparticle according to Claim 2, wherein the targeting moiety is a tumor cell targeting moiety, a macrophage-targeting moiety, or an antigen-targeting moiety, optionally wherein the targeting moiety is selected from one or more of the following: hyaluronic acid, mannose, cyclo-RGD and iRGD, and an antibody.

4. The radio-afterglow nanoparticle according to Claim 3, wherein the antibody is selected from one of more of the following: anti-EGFR antibody, anti-VEGF antibody, antitransferrin antibody, Interleukin-2, and anti-CD86 antibody.

5. The radio-afterglow nanoparticle according to any preceding claim, wherein the radioafterglow nanoparticle further comprises F, wherein F represents a biomarker cleavable linker.

6. The radio-afterglow nanoparticle according to any preceding claim, wherein the radioafterglow nanoparticle further comprises G, wherein G represents an afterglow quencher.

7. The radio-afterglow nanoparticle according Claim 6, wherein the afterglow quencher is coupled to the radio-afterglow nanoparticle via the biomarker cleavable linker.

8. The radio-afterglow nanoparticle according to any preceding claim, wherein A is selected from one or more of:

9. The radio-afterglow nanoparticle according to any preceding claim, wherein B is selected from one or more of:

10. The radio-afterglow nanoparticle according to any preceding claim, wherein C is selected from one or more of:

11. The radio-afterglow nanoparticle according to any preceding claim, wherein D is selected from one or more of:6 R = H, -NH2, -COOH. -SH; n = 10 to 50S H ■; I HN" ' hiR = H, -NH2, -COOH, -SH; X- 10% to R = H, -NH?, -COOH, -SH; m = 10 to 50; n = 4 to 50 90%; m = 10 to 100; n= 10 to 50412. The radio-afterglow nanoparticle according to any preceding claim when dependent on Claim 5, wherein F is selected from a heat-cleavable linker, a hydrogen peroxide-cleavable linker, a Granzyme B-cleavable linker, a glutathione-cleavable linker, a Cathespin B-cleavable linker, and a matrix metalloproteinase-cleavable linker, optionally wherein the biomarker cleavable linker is selected from one or more of:f f"HeBt-cteavahte linker H2O2-cleavable linker Granzvme B-eleavabie linkerGtutathione-cleavable linker Cathepsin B-cteavable linker Matrix metaltoproteinase-cfeavabte linker13. The radio-afterglow nanoparticle according to any preceding claim when dependent on Claim 6, wherein G is selected from one or more of:....... X-.-K;" R R = -NH2, -COOH, -SH R = -NH2, -COOH, -SH R = -COOH, -SH 1 2 3R = -CH2CH2NH2, -CH2CH2COOH, R = -CH2CH2NH2, -CH2CH2COOH, -CH2CH2SH4 514. The radio-afterglow nanoparticle according to any preceding claim, wherein the radioafterglow nanoparticle is free of inorganic nanoparticles, preferably wherein the radioafterglow nanoparticle is free of metal halides, such as metal fluorides, and is free of rare earth metals or rare earth metal ions.

15. The radio-afterglow nanoparticle according to any preceding claim, wherein the radioafterglow nanoparticle comprises only organic molecules.

16. A use of a radio-afterglow nanoparticle according to any preceding claim for one or both of cancer imaging and cancer theranostics.

17. A method for the detection of cancer in vivo, the method comprising:i) administering a radio-afterglow nanoparticle according to any one of Claims 1 to 15 to a subject; andii) detecting any luminescence, wherein the presence of cancer in vivo is indicated by luminescence.

18. A method of treating cancer, the method comprising:i) administering a radio-afterglow nanoparticle according to any one of Claims 1 to 15 to a subject; andii) subjecting the subject to X-ray irradiation.

19. A method of treating cancer in a patient by administering a radio-afterglow nanoparticle according to any one of Claims 1 to 15.

20. A radio-afterglow nanoparticle according to any one of Claims 1 to 15 for use as a medicament.

21. A radio-afterglow nanoparticle according to any one of Claims 1 to 15 for use in the treatment of cancer.

22. Use of a radio-afterglow nanoparticle according to any one of Claims 1 to 15 in the manufacture of a medicament for the treatment of cancer.

25. A drug comprising a radio-afterglow nanoparticle according to any one of Claims 1 to

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