Codoping Enhanced Radioluminescence of Nanoscintillators for X

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Codoping Enhanced Radioluminescence of Nanoscintillators for X-RayActivated Synergistic Cancer Therapy and Prognosis Using Metabolomics Farooq Ahmad, Xiaoyan Wang, Zhao Jiang, Xujiang Yu, Xinyi Liu, Rihua Mao, Xiaoyuan Chen, and Wanwan Li ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b04213 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Codoping Enhanced Radioluminescence of Nanoscintillators for X-Ray-Activated Synergistic Cancer Therapy and Prognosis Using Metabolomics Farooq Ahmada#, Xiaoyan Wangb#, Zhao Jianga, Xujiang Yua, Xinyi Liua, Rihua Maoc, Xiaoyuan Chend, Wanwan Lia* State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China a

Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, P. R. China b

Laboratory for Advanced Scintillation Materials & Performance, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 201800, P. R. China c

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States d

#

These authors contributed equally to this work.

* Address correspondence to [email protected]

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ABSTRACT Radio- and Photodynamic therapies are the first line of cancer treatments, which suffers poor light penetration and less radiation accumulation in soft tissues with high radiation toxicity. Therefore, a multifunctional nanoplatform with diagnosis assisted synergistic radio- and photodynamic therapy and tools facilitating early prognosis are urgently needed to fight the war against cancer. Further, integrating cancer therapy with untargeted metabolomic analysis would collectively offer clinical pertinence, through facilitating early diagnosis and prognosis. Here, we enriched scintillation of CeF3 nanoparticles (NPs) through codoping Tb3+ and Gd3+ (CeF3: Gd3+, Tb3+) for viable clinical approach in the treatment of deep-seated tumors. The codoped CeF3: Gd3+, Tb3+ scintillating theranostic NPs were then coated with mesoporous silica, followed by loading with rose bengal (CGTS-RB) for later computed tomography (CT)- and magnetic resonance image (MRI)-guided X-ray stimulated synergistic radio- and photodynamic therapy (RT+XPDT) using low-dose, one-time X-ray irradiation. Results corroborated an efficient tumor regression with synergistic RT+XPDT relative to single RT. Global untargeted metabolome shifts highlighted the mechanism behind this efficient tumor regression using RT, and synergistic RT+XPDT treatment is due to the starvation of non-essential amino acids involved in protein and DNA synthesis, and energy regulation pathways necessary for growth and progression. Our study also concluded that tumor and serum metabolites shift during disease progression and regression served as robust biomarkers for early assessment of disease state and prognosis. From our results, we propose that codoping is an effective and extendable technique to other materials for gaining high optical yield and multifunctionality, and for use in diagnostic and therapeutic applications. Critically, the integration of multifunctional theranostic nanomedicines with metabolomics has excellent

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potential for the discovery of early metabolic biomarkers to aid in better clinical disease diagnosis and prognosis.

KEYWORDS: codoped nanoscintillators, imaging, radiotherapy, x-ray inducible photodynamic therapy, metabolomics Cancer is one of the largest disease threats to the human population1 and state-of-the-art radio2-5 and photodynamic therapies (PDT)6-9 have long been used as front-line treatment for cancer. Unfortunately, the high-infiltration power of ionizing radiation results in energy amassing inside the tumor. This can lead to reduced tumor ablation, along with an enhanced probability of recurrence and radio-resistance with collateral systemic and cumulative toxicity.3,

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Comparatively, PDT is paired with a harmless photosensitizer (PS) to create reactive oxygen species (ROS). Increased ROS directly slow—and even stop—tumor growth by causing inflammation and compromising microvasculature. Critically, this approach results in minimal collateral systemic and cumulative toxicity.11, 12 However, the shallow infiltration of visible light into biological tissue is the prime obstruction in treating tumors located deep within tissues or organs.13 Given the above-said limitations, there is a pressing need for the development of strategies to enhance the accumulation of ionizing radiation inside tumors. Moreover, to also increase the penetration depth of visible light by stimulating and/or amplifying the light deep inside the body using a single energy source. Recently, ionizing radiation has been used to excite nano-scintillators paired with a PS. This leads to ROS generation and subsequent destruction of neoplastic tissue located deep within the body. Collectively this approach is called radio-(photo) dynamic therapy (RDT or XPDT).3, 8-10, 12, 14-19

Indeed, the synergy between RT and XPDT have better apoptotic efficacies—even for cells

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that are refractory to radiotherapy alone and vice versa. The blend of these two treatment modalities (RT+XPDT) allows for a combined attack on both the cell membrane and DNA, resulting in lethal damage that is beyond cellular repair capabilities.4, 20 Therefore, treatment of deeply located tumors using the synergistic treatment approach of RT+XPDT will be a key clinical development in the future. Despite its promising future association, only a few12, 19 studies have demonstrated the synergistic potential of imaging-guided, X-ray-induced RT+XPDT treatment for tumors located deep within the tissue. Given this, the development of multifunctional theranostic nanoplatforms that can be used for combined, real-time diagnosis and treatment of deeply located tumors would be of great value. Rare-earth-doped fluoride nanocrystals have excellent chemical and optical properties that have allowed them to be used in a wide range of applications, including biolabeling, imaging, and low-toxicity therapies9, 21, 22 as well as solar cells and electronics.23-26 CeF3 nanoparticles (NPs) contain scintillating material that converts high-energy, ionizing radiation to low-energy, visible light. Given this broad property, a variety of NPs like CeF3@VP (verteporfin), CeF3@ZnO, CeLaF3/LaF3@Chlorine e6, and CeF3:Tb3+@CTAB-Chlorine e6 in combination with different PSs (e.g., verteporfin, ZnO, and choline e6) were used in X-ray induced photodynamic therapy.27-30 However, all of these approaches showed non-efficient therapeutic output due to their meager scintillation31-33 and resulting low ROS generation. To overcome this problem and allow for the design of efficient nanoscintillators for use in cancer theranostics, we proposed to use codoping to enrich the scintillation of CeF3 NPs. With enriched scintillation, a CeF3 nanoplatform would be able to not only tolerate a diverse biological milieu with minimum systemic toxicity but also retain the sophisticated features of diagnosis and radio-sensitization. It is possible that the lower scintillation yield of conventional CeF3 NPs is due

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to the fact that the Ce 4f orbital lies around 3-4 eV above the valence band; moreover, that the absence of an Auger cascade leads to a poor hole-capture probability.32 From theoretical and practical perspectives, the dominant scintillation mechanism in CeF3 NPs is through efficient energy transfer. However, Tb3+ doping in CeF3 NPs has also not shown enough improvement in the energy transfer efficiency from Ce3+ to Tb3+. This is likely due to the forbidden nature of the intraconfigurational 4f transitions of most lanthanide ions;33 again, resulting in poor scintillation. Codoping has been an effective approach for improving the scintillation of several materials3437

and has yielded up to a three-fold increase in light output.37 In particular, Gd3+ codoping

intensified the light output of Tb3+ by efficiently transferring the excitation energy from the activator in various scintillators (Gd2O3-B2O3-SiO2:Ce3+,Tb3+ and Li2O-BaF2-Al2O3-SiO2– Sb2O3:Gd3+,Tb3+).34, 35 From the aforementioned studies, we hypothesized that codoping with Gd3+ and Tb3+ in a CeF3 NP host would improve scintillation yield. Theoretically, the energy levels of Gd3+ lie between the Ce3+ and Tb3+ in a CeF3:Gd3+, Tb3+ system. Therefore, codoping with Gd3+ may not only promote the efficient migration of trapped energy from the activator (Ce3+) to the sensitizer (Tb3+) but may also render computed tomography (CT) and magnetic resonance imaging (MRI) diagnostics38-40 with radiosensitized39, 41 features due to the presence of a high-Z element (Gd, 64). Unfortunately, no previous work has reported on this hypothesis. Moreover, the dominant, green emission peak of Tb3+ at 543 nm also provide us with the opportunity to use a variety of photosensitizers, such as rose bengal (FDA-approved)42, 43, porphyrin44, or MC540.14 Any of these photosensitizers would allow for good spectral overlap, making this nanoplatform an efficient 1O2 generator with high clinical relevance. Taken together, the improved attributes of enhanced scintillation, radiotherapy, and imaging would qualify CeF3:Gd3+, Tb3+ (CGT) NPs as

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an ideal candidate to act as a suitable theranostic platform for imaging-guided, X-ray induced radio- and photodynamic therapy (RT+XPDT). When compared with preclinical studies, most current anti-cancer nano-medicines have not performed well in clinical trials. This consistent clinical failure is because preclinical studies lack information regarding effective biomarkers for use in diagnosis and targeting, disease progression stratification, and the selection of an optimal treatment strategy.45, 46 Integration with other areas of oncology would solve many of these problems; namely, integrating with metabolomics. This would allow for the design of effective theranostic nanomedicines that would likely have improved chances of a successful clinical translation. It is a well-known fact that cancer is a metabolic disorder47 and exploring ways metabolic reprogramming supports cancer cell survival, growth, and proliferation are of utmost importance for more effective treatments. These metabolic shifts are associated with a complex interplay of intrinsic factors like transcriptional and proteomic pathways48, 49 as well as extrinsic factors like radiation50 and/or nanoparticle51-53 exposure. Given this complexity, analyzing global metabolites would provide invaluable information regarding pathobiological state-based disease screening, selection, and patient categorization for targeted therapies, as well as the therapeutic potential of a given treatment. Additionally, metabolomics has potential as a diagnostic and prognostic tool in the treatment of complex diseases. For example, in the case of glioblastoma, it is difficult to assess treatment outcomes—even after several months.54 Separately, after 2-15 years following initial treatment, breast cancer recurrence can occur locally in either the same or contralateral breast or be more distant.55 Furthermore, the lower sensitivities (54-77%) of standard mammography tests demand a more sensitive and precisely accurate diagnostic tool for breast cancer diagnosis.56 Given these examples, it is clear that there is a pressing need for better solutions for these unresolved diagnostic

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and prognostic issues. Fortunately, cataloging and profiling global metabolome shifts would provide a sharper and more precise picture regarding the cellular physiology, toxicology, and disease progression and regression of cancer. Moreover, it would allow for the prognostication of treatment effectiveness49, 54 as well as the risk and safety assessment of nanomaterials. Here, we report CeF3 NPs codoped with Gd3+, Tb3+ that enjoy stronger scintillation when compared with singly doped Tb3+ with the natural theranostic feature. Furthermore, the intense green emission of Tb3+ allowed us to exploit the biocompatible and FDA-approved rose bengal that had been loaded on mesoporous silica-coated CeF3:Gd3+,Tb3+ (CGTS-RB) NPs. This approach allowed for efficient 1O2 production using X-ray irradiation. As a result, CGTS-RB NPs became a multifunctional tool capable of being used synergistically with dual-modal imaging (CT and MRI) guided non-radioactive radio-/photodynamic therapy (RT+XPDT), which were stimulated by a single X-ray source with minimum dosing. We have also explored and presented the combined use of X-ray-induced, synergistic RT+XPDT treatment modalities with untargeted global metabolomics using gas chromatography time of flight mass spectrometry (GC/TOFMS) for analysis of relevant metabolic tumor and serum biomarkers and their patterns. Critically, how this information is related to the predisposition for tumor shrinkage, therefore providing valuable prognostic information. This study extends prognosis-to-treatment ability beyond the use of imaging-guided, synergistic RT+XPDT by using global metabolome analysis. Crucially, this approach provides another tool to better understand the complex metabolic biomarkers transformations that occur in a tumor, both pre- and post-exposure to ionizing radiation.

RESULTS AND DISCUSSION

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CGTS NPs Synthesis and Characterization. CeF3:Gd3+,Tb3+ (CGT) NPs were synthesized using a hydrothermal process (see Experimental Section). Transmission electron microscopy (TEM) imaging of CeF3:Gd3+,Tb3+ (CGT) NPs (Figure 1a) revealed a particle size of 80±1 nm (SI, Figure S1d) with clear lattice fringes (Figure1b), while dynamic light scattering (DLS) showed the particle size of 89.2 nm (SI, Figure S2). X-ray diffraction (XRD) results (Figure 1e) of CeF3:Gd3+,Tb3+ (CGT) NPs and CeF3:Tb3+ (CT) NPs showed prominent peaks corresponding to the hexagonal phase structure of bulk CeF3 crystal (JCPDS: 08-0045). All diffraction peaks were easily indexed to a pure hexagonal structure of CeF3 (CF), with a shift to slightly higher angle due to the comparatively smaller ionic radius relative to Ce3+ (Figure 1e). CGT NPs were further coated with mesoporous silica (CGTS NPs) with an average size of 91±1 nm (SI, Figure S3), and HRTEM image shows the clear lattice fringes (Figure 1c&d). Furthermore, element mapping confirmed (Figure 1f) successful doping of Gd3+ and Tb3+ into the lattice of CeF3 matrix with mesoporous silica wrapping. The poor therapeutic output of CeF3 NPs was due to weaker photoluminescence (PL) and X-rays excited optical luminescence (XEOL); to overcome this, we optimized the PL by carefully controlling the amounts of Gd3+ and Tb3+ dopants in the CeF3 host (SI, Figure S4). Optimum PL (SI, Figure S5) and XEOL (Figure 1g) was achieved by codoping Gd3+ and Tb3+ in the CeF3 host at 12.3 mol% and 1.24 mol%, respectively. The enhancement in PL intensity due to Gd3+ (12.3 mol%), Tb3+ (1.24 mol%) codoping was confirmed by the increase in fluorescence lifetime of CeF3:Tb3+ (CT) NPs from 1.9 ns to CeF3:Gd3+, Tb3+ (CGT) 5.9 ns (SI, Figure S6).

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Figure 1. Characterization of NPs (a) CeF3: Gd3+, Tb3+ Nanoparticles (b) HRTEM image of CeF3: Gd3+, Tb3+ Nanoparticles (c) mesoporous silica-coated CeF3: Gd3+, Tb3+ nanoparticles (d) HRTEM image of mesoporous silica-coated CeF3: Gd3+, Tb3+ nanoparticles (e) XRD patterns showing the crystalline nature

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of CGTS NPs and that the diffraction peaks were indexed to hexagonal-phased CeF3: Gd3+, Tb3+ (JCPDS No. 08-0045). (f) Elemental mapping of mesoporous silica-coated CeF3: Gd3+, Tb3+ nanoparticles (g) X-ray excited optical luminescence of CGT and CT NPs. (h) X-ray excited optical luminescence spectrum of NPs and absorbance spectrum of rose bengal. (i) Schematic illustration of energy levels of Ce3+, Gd3+, and Tb3+ with possible excitation and emission mechanism.

Generally, each element has a special set of energy levels; in the case of our CeF3:Gd3+, Tb3+ system, the Gd3+ energy levels were between those of Ce3+ and Tb3+(Figure 1i). The excitation energy either directly transferred from Ce3+ to Tb3+ or was transferred through Gd3+ to Tb3+. Thus, Gd3+ acted as a mediator of the energy transfer, transferring trapped energy from Ce3+ to Tb3+ with minimum dissipation. As a result, the CGT NPs emitted intense green XEOL (Figure 1g) and PL (SI, Figure S5) under X-ray and UV irradiation, respectively. Typical Tb3+ emission luminescence peaks at 489, 542, 583, and 620 nm were the characteristic de-excitations from the 5D

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to 7Fn (n = 3, 4, 5 or 6) ground states.42, 57 Results from inductively coupled plasma mass spectrometry (ICP-MS) showed that the

actual doped amounts of Gd3+ and Tb3+ at optimum PL were 5.24 mol% and 1.5 mol% to the combined material mass, respectively (SI, Table S1). For loading PS, the CGT NPs surface was further coated with a layer of mesoporous silica (CGTS) a thickness of 10 nm (Figure 1c). The mesopore size and volume were 3.5 nm and 0.215 cm3/g, respectively (SI, Figure S7). An FDAapproved, water-soluble ophthalmic diagnostic agent—rose bengal—with high 1O2 quantum yield (ø=0.75)43 was chosen because of the excellent overlap between its absorption spectrum and the emission spectrum of CGT NPs (542 nm; Figure 1h). Fourier transform infrared (FTIR) spectra (SI, Figure S8) also verified the amine functionalized silica coating through the presence of SiOand NH2- peaks at 1078 cm-1 and 1637 cm-1,58, respectively. 10


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To improve colloidal stability and dispersion, amine- (NH2-) functionalized CGTS NPs were further PEGylated by conjugating NHS-PEG-COOH (M.W. = 5000) to the surface and confirmed by the C—H stretching peak of —CH2— at 2933 cm-1 (SI, FigureS8). The resulting CGTS NPs were loaded up to 26% with RB (CGTS-RB) (SI, Figure S9). The loading capacity of RB in the CGTS-RB suspension was quantified using UV/vis spectroscopy by measuring absorbance at 550 nm through the standard concentration curve (SI, Figure S10) prepared from free RB (see Experimental Section). Our experiments also showed that only a few RB molecules were released from the CGTS-RB NPs over 48 h in various mediums i-e., water, PBS and 50%FBS solutions (SI, Figure S11), hence proving the stability of the system. The reduction in PL intensity (SI, Figure S12a) and the change in CGTS NP fluorescence lifetime from 4.8 ns to 2.3 ns (SI, Figure S12b) after loading with RB indicated the successful establishment of the Förster resonance energy transfer (FRET) system. Singlet Oxygen Production for XPDT. A singlet oxygen sensor green (SOSG) assay12, 14

then used to investigate the XPDT potential of CGTS-RB NPs by its subsequent 1O2 generation.

We also observed that X-ray, PBS, RB, and CGTS NPs alone were unable to harvest 1O2 (Figure 2a). In comparison, when both CGTS-RB NPs (50 µg/mL) and X-rays were simultaneously applied, a sufficient increase in 1O2 yield was observed with increasing X-ray irradiation dose (Figure 2a). 1O2 production was also examined in vitro using 4T1 cells (Figure 2b). Briefly, CGTS-RB NPs (50 µg/mL) were first incubated with 4T1 cells for 6 h, after which the medium was replenished with a fresh medium that contained 5 µM SOSG. X-ray irradiation (2Gy) and microscopic imaging followed. Green fluorescence was observed in the RT+XPDT(CGTS-RB+XRay) group, indicating significant intracellular harvesting of 1O2 (Figure 2b). Comparatively, there were no notable increases in SOSG signals when 4T1 cells were treated with either CGTS+X-

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ray or X-ray alone. Collectively, these results indicated that the union of RB with CGTS NPs followed by X-ray irradiation yielded operative X-PDT.

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Figure 2. Singlet oxygen production after X-ray irradiation using CGTS NPs as transducers (a) Singlet oxygen production measured using SOSG assay. CGTS-RB system efficiently produces singlet oxygen under irradiation, manifested here as increased fluorescence. As a comparison, there was no singlet oxygen produced in controls. (b) In in vitro SOSG using 4T1 cells, efficient singlet oxygen was produced within cells when X-ray irradiation was applied following cell incubation with CGTS-RB NPs. (c) Viability changes based on a CCK-8 assay for 4T1 cells was performed after 24 h of treatment. X-rays alone and CGTS +X-ray (RT) caused a comparatively less decrease in cellular viability; CGTS-RB+X-ray (XPDT) induced efficient cell death. (d) Impact of tissue depth on XPDT efficiency, XPDT can be stimulated from beneath thick tissue (e.g., 2 and 4 cm) to cause efficient 4T1 cell death. (e) Colonogenic assay showing the survival fraction under CGTS+X-ray (RT) affected the proliferation ability of 4T1 cells; CGTS-RB+X-ray (XPDT) was significantly more effective (* p-value < 0.05). (f) Viability changes based on a CCK-8 assay for Renca cells was performed after 24 h of treatment. (g) Impact of tissue depth on XPDT efficiency, XPDT can be stimulated from beneath thick tissue (e.g., 2 and 4 cm) to cause efficient Renca cell death. (h) Colonogenic assay showing the survival fraction under CGTS+X-ray (RT) affected the proliferation ability of 4T1 cells; CGTS-RB+X-ray (XPDT) was significantly more effective (* p-value < 0.05). (i)Viability changes based on a CCK-8 assay for mgc803 cells was performed after 24 h of treatment. (j) Impact of tissue depth on XPDT efficiency, XPDT can be stimulated from beneath thick tissue (e.g., 2 and 4 cm) to cause efficient mgc803 cell death. (k) Colorogenic assay showing the survival fraction under CGTS+X-ray (RT) affected the proliferation ability of mgc803 cells; CGTS-RB+X-ray (XPDT) was significantly more effective (* p-value < 0.05).

In Vitro X-PDT. We next evaluated the biocompatibility and safety profiles of CGTS and CGTS-RB NPs. This was achieved by incubating each with 4T1, HEK 293T, Renca and Mgc893 cells, followed by a cytotoxicity assessment using a commercially available cell counting kit-8 (CCK-8) assay. Even at high concentrations of either CGTS or CGTS-RB NPs (e.g., 200 µg/mL), there was no notable drop in cell viability in either cell line. These results indicated the low cytotoxicity of both CGTS and CGTS-RB NPs (SI, Figure S13).

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Similarly, 4T1, Renca and Mgc803 cells were incubated separately with either CGTS or CGTS-RB NPs (0 or 50 µg/mL) followed by different doses of X-ray irradiation (0,1, 2, 4, 6, or 8 Gy) had efficient RT (CGTS+X-ray) and synergistic RT+XPDT(CGTS-RB+X-ray) treatment (Figure 2c-k). For instance, 4T1, Renca and Mgc803 cell viability after X-ray irradiation of 1 Gy reduced cell viability to 90%, 84%, 82% and 53%, 49.6%, 50% for the CGTS and CGTS-RB groups, respectively. This indicated the synergy afforded by RT+XPDT treatment was four times more lethal to cells than RT treatment alone and showed much better in vitro therapeutic output at 1Gy as compared to the CeF3-ZnO nanosytem59. X-ray radiation is known to penetrate deep inside the body. Given this, we next sought to assess the treatment efficacies of RT and synergistic RT+XPDT in 4T1, Renca and Mgc803 cells stacked with a thick piece of pork meat (2 cm and 4 cm) (Figure 2d). CGTS and CGTS-RB groups (50 µg/mL) irradiated at 1Gy showed anti-tumor effects for 4T1 tumor cells, with slightly increased cellular viability from 93% and 97% for the CGTS(RT) groups, respectively, to 59% and 72% for the CGTS-RB (RT+XPDT) group, respectively (Figure 2d). While Renca and Mgc803 tumor cells also showed the similar treatment trend with slightly increased cellular viability (Renca, 86% and 90%; Mgc803, 88%, and 92%) for the CGTS(RT) groups, respectively, to (Renca, 55% and 68%; Mgc803, 56% and 70%) for the CGTS-RB (RT+XPDT) group, respectively (Figure f-k). These results were comparable to those samples irradiated without pork interference (90%, 84%, 82% and 53%, 49.6%, 50%), suggesting a relatively trivial impact of tissue depth on treatment efficacy. When compared with RT alone, the viability of synergistic RT+XPDT drops by 24 h, which suggests a different type of RT+XPDT-mediated mechanism of cell death (Figure 2c, 2f, and 2i). Ionizing radiation often does not result in instant cell death, but reduces cellular mitotic capacity;

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this becomes evident in days or weeks.12 Given this, we used a cell colony formation assay to confirm differences of cell death between RT (CGTS+X-ray) and RT+XPDT (CGTS-RB+X-ray) groups at day 9 post-treatment. In contrast with the PBS group (Figure 2e, 2h & 2k) and after Xray irradiation, CGTS-RB NPs showed superior anti-tumor efficacy relative to CGTS NPs. This improved anti-tumor efficiency demonstrated the intensification of ionizing radiation that occurred in the presence of CGTS NPs and CGTS-RB NPs. The sensitization enhancement ratio (SER) for RT (CGTS+X-ray) and RT+XPDT (CGTS-RB+X-ray) groups indicated that the 4 Gy radiation intensification was 1.63, 1.3,1.03 and 2.6, 1.8, 1.7 times higher for 4T1, Renca and Mgc803 cells, respectively, relative to the PBS group (Figure 2e,2h & 2k). Taken together, these results suggest that the inherent synergistic RT and XPDT potential led to the observable, improved, anti-tumor potential of CGTS-RB NPs relative to CGTS NPs. In Vivo Blood Circulation and Tumor Accumulation. Given these positive in vitro results, we next examined the pharmacokinetic behavior and tumor accumulation pattern of CGTSRB NPs (2 mg/mL, 200 µL) in tumor-bearing mice using intravenous (i.v.) administration. For the pharmacokinetic study, we took blood samples at different time points, followed by ICP-MS analysis to measure the amount of Ce. The half-life of CGTS-RB NPs circulating in the blood was approximately 1.12 h (SI, Figure S14a), with a constant, low content of CGTS-RB NPs during a 24 h period. After 24 h, CGTS-RB NPs were bio-distributed in different organs—including the tumor. The tumor uptake of CGTS-RB NPs was measured at 4.8% injected dose per gram (ID g-1) at 24 h (SI, Figure S14b), demonstrating the potent accumulation of CGTS-RB NPs in the tumor. This potent tumor accumulation could be attributed to enhanced permeability and retention effects in solid tumors that possessed tortuous and leaky vasculature.8

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In Vitro and In Vivo Imaging using CT and MRI. The codoping of high-Z elements inherits CGTS-RB NPs with better X-ray attenuation. This, along with their superb spatial resolution coupled with an outstanding capacity for differentiating soft tissues, empowers them to be employed as an effective CT and/or MRI contrast agent. As shown in Figure 3a, graded concentrations of CGTS-RB NPs exhibited a stronger CT imaging ability than commercially available Ultravist-300 and Rose Benagl, with a slope of 6.68 HU mL mg (Gd3+)-1 , 2.42 HU (Figure 3b) and 1.9 HU mL mg (I)-1, respectively (see Figure 3c). The potential of CGTS-RB NPs to function as CT contrast agents was assessed after their intra-tumor (i.t.) injection (2 mg/mL, 25 µL); results indicated a clear, observable signal with HU value of 127 compared with no signal prior to injection (Figure 3d). This observed increase suggests that CGTS-RB NPs alone could be utilized as highly effective CT contrast agents. After assessing CGTS-RB NPs ability for use as a CT contrast agent, we next evaluated T1-weighted MRI contrast. As shown in Figures 3e, 3f, & 3g, the T1-weighted MR images brightness increased with increasing Gd3+ ion concentration, and r1 and r2 value was calculated to be 8.5 and 3.27 mM-1s-1for CGTS-RB NPs and Magnevist, respectively. This was done using the linear relationship of the longitudinal relaxation rate (1/T1) versus Gd3+ ion concentration, which was approximately 2.5-fold higher than that of commercially available Magnevist (3.27 mM-1s-1 ; Figure 3g). Six hours after i.t. (2 mg/mL, 25 µL) and i.v. injections of CGTS-RB NPs (therapeutic dose of 2 mg/mL, 200 µL) tumor distribution (after i.t. injection) and increases tumor accumulation through circulator system (after i.v. injection), we observed an enhanced, tumor MRI signal (Figure 3h). Furthermore, quantitative analysis (SI, Figure S15) of signal enhancement (ΔSNR) in tumors at different time points (0h and 6h) after CGTS-RB NPs administration indicated the notable increase in signal intensity, and the highest ΔSNR in tumors after intratumor and

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intravenous injection of CGTS-RB NPs are 238.07±10.5% and 184.23±9.6%, respectively. Altogether, these results demonstrate the dual-modal CT and MRI imaging capacity of CGTS-RB NPs.

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Figure 3. CGTS-RB NPs potential as imaging contrast agents (a) CT phantom images of CGTS-RB NPs Ultravist-300 and Rose Bengal at different concentrations (b) Corresponding HU values of CGTS-RB NPs and Ultravist-300 solutions (c) Corresponding HU values of Rose Benagl solutions (d) In in vivo CT images of tumors in mice obtained before and after i.t. injection of CGTS-RB NPs (e) T1-weighted images of CGTS-RB NPs

and Magnevist at different concentrations (f) The fitted linear relationship of the

longitudinal relaxation rate versus the Gd3+ ion concentration (g) and Magnevist (h) In vivo MR images of mouse tumor cross sections obtained before and after 6 h of i.t. and i.v. injection of CGTS-RB NPs.

In Vivo RT and XPDT Treatment and Metabolomics. After the in vitro and in vivo therapeutic and diagnostic demonstrations of CGTS and CGTS-RB NPs, the therapeutic potential of these NPs were further assessed in the tumor (4T1)-bearing mice. This was done using i.v. injection of either CGTS or CGTS-RB NPs (2 mg/mL, 200 µL) administered 24 h prior to the start of X-ray treatment. Similar to the PBS group, neither the CGTS NPs- nor CGTS-RB NPs-treated groups had altered tumor growth or proliferation rapidly (Figures 4a and 4b). However, the CGTS and CGTS-RB NPs groups showed evidence of enhanced RT and synergistic RT+XPDT curative outcomes after one-time, X-ray irradiation (160 kV, 25 µA, 6 Gy) that were visualized by tumor regression (Figure 4a). Since the synergistic RT+XPDT+6Gy (CGTS-RB+6Gy) group demonstrated a superior curative outcome relative to the RT+6Gy(CGTS+6Gy) group, we next assessed the synergistic RT+XPDT (CGTS-RB+6Gy) curative potential of half the X-ray dose (3Gy). Tumor regression (Figure 4a) and tumor weight change (Figure 4b) indicated the same curative results for the low dose (3 Gy) used in the synergistic RT+XPDT (CGTS-RB) group relative to the high dose (6 Gy) RT (CGTS+6Gy) group alone. This suggested an excellent, synergistic treatment efficacy—even with reduced radiation exposure (SI, Figure S17).

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Given these results, we assumed the relative curative abilities as follows: CGTS-RB (6Gy)>CGTS-RB(3Gy)~CGTS(6Gy). Further corroborating the synergistic effects of RT+XPDT would provide us better future curative outcome—even at lower X-ray doses (e.g., 3Gy). However, as reported in the literatures, 3 Gy and 6 Gy were either notably less than or equal to the more widely used 6 and 8 Gy.3, 9, 17 Moreover, we have provided here a quantitative and experimental demonstration of the future potential use of synergistic RT+XPDT in improved cancer treatment with reduced radiation exposure and maximum radiation safety. These results also inspired us to discover more potent therapeutic nanomedicines to further minimize X-ray exposure under these and other clinical conditions. We next analyzed the prognosis of synergistic RT+XPDT after 14 d of cancer treatment using the untargeted global metabolomes of tumor and serum by GC/TOFMS analysis. In tumor samples, 16 of 35 metabolites were detected and quantified as having changed after treatment. Serum samples represented a systemic account of changes in the body; of these, 29 out of 46 metabolites changed. This information is useful and could aid in the screening and categorizing of patients based on disease pathology for later targeted treatment regimens. It is also useful as a posttreatment assessment that can be non-invasively applied. Principle component analysis (PCA) scored tumor (SI, Figure S18) and serum (SI, Figure S19) plots, which showed that the metabolite profile was significantly (p

Codoping Enhanced Radioluminescence of Nanoscintillators for X

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