Tracing Boron with Fluorescence and PET Imaging of Boronated

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Biological and Medical Applications of Materials and Interfaces

Tracing Boron with Fluorescence and PET Imaging of Boronated Porphyrin Nanocomplex for Imaging Guided Boron Neutron Capture Therapy Yaxin Shi, Jiyuan Li, Zizhu Zhang, Dongban Duan, Zhengchu Zhang, Hui Liu, Tong Liu, and Zhibo Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14682 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Applied Materials & Interfaces

Tracing Boron with Fluorescence and PET Imaging of Boronated Porphyrin Nanocomplex for Imaging Guided Boron Neutron Capture Therapy Yaxin Shi1, Jiyuan Li1, Zizhu Zhang3, Dongban Duan1, Zhengchu Zhang1, Hui Liu1, Tong Liu3, Zhibo Liu1,2* 1Radiochemistry

and Radiation Chemistry Key Laboratory of Fundamental Science,

Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China 2Peking

University-Tsinghua University Center for Life Sciences, Beijing, 100871,

China 3Beijing

Capture Tech Co., Ltd., Beijing, 102413, China

Corresponding Author*E-mail: [email protected] KEYWORDS: boron neutron capture therapy; theranostics; micelle; copper-64; positron emission tomography

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Abstract Boron neutron capture therapy (BNCT) induces high-energy radiation within cancer cells while avoiding damage to normal cells that without uptake of BNCT drugs, which is holding great promise to provide excellent control over locally invasive malignant tumors. However, lack of quantitative imaging technique to determine local boron concentration has been a great challenge for nuclear physicians to apply accurate neutron irradiation during the treatment, which is a key factor that has limited BNCT’s application in clinics. To meet this challenge, this study describes coating boronated porphyrins

with

a

biocompatible

Poly(lactide–co-glycolide)–monomethoxy-

poly(polyethylene-glycol) (PLGA-mPEG) micelle for selective tumor accumulation and reduced toxicity comparing with previously reported boronated porphyrin drugs. Fluorescence imaging and PET imaging were performed, unveiling the potential imaging properties of this boronated porphyrin nanocomplex (BPN) to locate tumor region and to determine tissue-localized boron concentration which facilitates treatment planning. By studying the pharmacokinetics of BPN with Cu-64 PET imaging, the treatment plan was adjusted from single bolus injection to multiple times of injections of smaller doses. As expected, high tumor uptake of boron (125.17±13.54 ppm) was achieved with an extraordinarily high tumor to normal tissue ratio: tumor to liver, muscle, fat and blood were 3.24±0.22, 61.46±20.26, 31.55±10.30 and 33.85±5.73, respectively. At last, neutron irradiation with BPN showed almost complete tumor suppression, demonstrating that BPN holds a great potential for being an efficient boron delivery agent for imaging-guided BNCT.

Introduction Boron neutron capture therapy (BNCT) is a binary, biochemically-targeted radiotherapy1 which provided excellent tumor control over locally invasive malignant tumors such as melanoma,2-3 glioblastoma4-5 and recurrent head and neck cancer.1, 6-8 The damage of BNCT to cancer cells is based on the reactions that occur when irradiating low-energy thermal neutrons to boron-10 to yield high relative biological effectiveness (RBE) and high linear energy transfer (LET) radiations, including alpha particles and recoiling lithium-7 nuclei, within the radius of one single cell.6, 9 In spite


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of the fact that BNCT treat cancer by “cellular-level nuclear reactions”, several barriers still stand in the way of BNCT to become precision therapy.10 Lack of quantitative imaging technique to trace boron has been a long-standing challenge to apply accurate neutron irradiation during BNCT treatment.11-13 The therapeutic efficacy of BNCT is directly related to the local boron concentration of tumor and the tumor to normal tissue ratio.9 Ideally, the intensity of neutron irradiation needs to be dynamically tuned according to the pharmaceutical variation of boron distribution in patient.12,

14-15

However, this two essential information is usually

obtained from indirect estimation by continuously running blood tests of patients by inductively coupled plasma-optical emission spectrometry (ICP-OES) for boron concentration analysis during treatment in clinics.16 The patient is still a “black-box” and no imaging technique of mapping boron in the patient is available.17 Furthermore, no systematic research has been contributed to study whether boron prefers to locate within the cell nucleus, which is of great importance to achieve irreparable damage to cancer cells.18 As for mapping boron within cell and patient, a theranostic complex unifies optical imaging, PET imaging, and tumor-selective boron delivery needs to be established for imaging-guided NCT. Among all the attempts have been made to develop new boron delivery agents19-22 fulfilled the clinical requirements of BNCT, boronated porphyrins23-26 are particularly promising due to: (1) Their demonstrated high accumulation and long retention in cancer cells;27 (2) Possibility to combine photodynamic therapy (PDT) with BNCT;2829

(3) Most essentially, the potential imaging properties such as fluorescence imaging23,

30

and PET imaging31 by labeling with Cu-64 to locate tumor region and carry out

quantification of local boron concentration which facilitates treatment planning. The early reported boronated porphyrins BOPP25, 32-33 and CuTCPH34-35 shown excellent tumor control during preclinical biological investigations which were placed in great expectation. However, clinical applications of boronated porphyrins are held back by low tumor-blood ratio and direct toxic effect on platelets found in phase I clinical research.36 In the condition of the great advantages that boronated porphyrins hold as BNCT agents, approaches to selectively deliver boronated porphyrins to tumor region with long retention and low toxicity is needed urgently. To meet the challenges mentioned above, this study presented a novel boronated porphyrin loaded PLGA-mPEG nanocomplex (BPN) for dual PET and fluorescence



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imaging and therapeutic boron delivery for BNCT (Figure 1A). BPN was able to perform efficient delivery of boron both in vitro and in vivo by incorporating tetraboronated porphyrin (TBPP) with PLGA-mPEG micelles. Direct contact between boronated porphyrins and blood cells is avoided to reduce off-target toxicity while BPN is more selective tumor accumulation as the result of the enhanced permeability and retention (EPR) effects.37 Administration of BPN (or

64Cu-BPN)

allowed for clear

visualization of tumor xenografts in mice, suggesting a unique advantage of multimodal molecular imaging targeting tumor for the guidance of BNCT. Most importantly, PET imaging revealed that single bolus injection of boron agent did not provide either enough boron accumulation in tumor or good tumor to non-tumor ratio. Instead, multiple injections with lower boron dose were applied prior to neutron irradiation, ICP-OES analysis demonstrated excellent tumor uptake of boron (>100 ppm) and optimal tumor to non-tumor contrast (tumor-to-muscle ratio >50).38 As expected, BNCT with BPN almost completely cured melanoma tumor in the tumor-bearing mouse, the median survival time of mice treated with BPN and neutron irradiation was remarkably elongated than those treated only with neutron irradiation. In all, BPN overcomes the disadvantages of traditional boronated porphyrin drugs and holds a great potential for being an efficient theranostic agent for clinical BNCT. RESULTS AND DISCUSSION Characterization of BPN Characteristic IR absorbance peaks of PLGA-mPEG and BPN showed that the structure of BPN was almost consistent with that of PLGA-mPEG, indicated the successful encapsulating with TBPP (Figure 1B). Synthesized BPN exhibited as monodispersed spherical micelles without agglomeration according to TEM measurement (Figure 1C and Figure S1, S2). The size of micelles increased slightly after encapsulating TBPP, with the individual size of approximately 100 nm and polydispersity index (PDI) of approximately 0.1 which was in favor of EPR effect (Figure 1D and Figure S3). GPC traces showed that the relative molecular weight of the micelles was more than 8000, demonstrating the successful synthesis of the corresponding polymers (Figure 1E). Zeta-potential of BPN and PLGA-mPEG was measured at the concentrations of 1 μg/ml. Found that zeta-potential of PLGA-mPEG was -1.4 mV while BPN was -39 mV indicated that BPN were moderately stable during circulation, which is of great importance for high tumor accumulation (Figure 1F). Excellent stability was validated


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by monitoring the size of BPN (Figure S4). Fluorescence was also observed and the fluorescence spectrum of BPN showed that the wavelength of excitation light was 420 nm while the wavelength of emission light to be 680 nm (Figure 1G). Fluorescence spectrum of TBPP was shown in Figure S5. No significant cellular cytotoxicity of BPN was observed Besides the extraordinarily high demand about boron concentration in the tumor, low cytotoxicity is another key factor for developing a successful boron delivery agent. The cytotoxicity of BPN, PLGA-mPEG and TBPP was conducted using a basic CCK8 assay. As shown in Figure 2A, the PLGA-mPEG micelles alone showed little cytotoxicity to B16-F10 cells when their concentration was below 600 μg/ml. The cytotoxicity of boronated porphyrin drug was observed with rather severe toxicity (Figure 2B). As expected, upon encapsulated with PLGA-mPEG to form nanocomplex, BPN showed notably less toxicity compared with naked boronated porphyrin (Figure 2C). Additionally, over 77.3% of B16-F10 cells survived after treated with 90 μg/ml BPN, showing that BPN exhibited excellent biocompatibility. BPN exhibited intense uptake in both cytoplasm and nucleus BNCT is a single cell-targeted radiotherapy.39 Therapeutic efficacy of BNCT treatment relys on the cellular boron concentration which needs to be at least 25 ppm per 109 cells.40 While ICP analysis to determine cellular uptake of boron agents has been routinely used, but very few studies about boron distribution in different cytoplasmic organelles have been reported. The cellular uptake of BPN was evaluated both by ICPOES and confocal microscopy by incubating TBPP encapsulated PLGA-mPEG nanocomplex with B16-F10 cells (Figure 2D, E). First, boron concentration was assessed by ICP-OES. The concentration of boron in B16-F10 cells was reached up to 100 ppm after 24 h of incubation, and finally reached to 250 ppm after 48 h. To further confirm the cellular uptake, microscope images showed an evident uptake of BPN in the cytoplasm of tumor cells which was correlated with the previous data from ICP-OES. Notably, besides cytoplasm, high BPN uptake was observed in the nucleus as well, indicated that BPN-BNCT was able to deliver very localized energy for breaking the DNA double-strain helix.41

Mapping boron with microscope imaging

showed that BPN successfully delivered boron into the nucleus, where boron neutron capture could cause DNA double strand break with the greatest efficiency.



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Optical imaging exhibited notable accumulation of BPN in xenografts The fluorescent properties are another advantages of porphyrin derivatives to boron delivery, BPN showed the fluorescent property which inherited from the encapsulating TBPP molecules inside. Therefore, optical imaging techniques42 was used to image boron in vivo with BPN. However, 4T1 xenografts was applied instead of B16-F10 due to the rather strong fluorescent quenching effect of the later. BPN was applied to 4T1 tumor-bearing mice intravenously and the optical images were obtained with fluorescence imaging system. The tumor regions in mice started to exhibit contrast over other normal tissues surrounding. And until the time of 24 h post administration, the malignat regions in mice shown enhanced contrast, whereas there was a low uptake of other normal tissues surrounding (Figure 3A). The fluorescent imaging result showed early tumor accumulation and long tumor retention of BPN. At the time of 24 h post administration, the mice were sacrificed and the fluorescent images of major organs were exhibited in Figure 3B, which showed that the fluorescent intensity of tumor was the highest. PET imaging illustrated in vivo pharmacokinetics and biodistribution of

64Cu-

BPN in tumor-bearing mice To quantitatively analyze the dynamic biodistribution of boron, PET imaging43-45 was applied to investigate the pharmacokinetics of BPN. Indeed, one of the most essential advantages which boronated porphyrins hold was the potential ability to chelate Cu-64 for PET imaging.46 Cu-64 is a widely used radionuclide for both experimental and clinical application. [64Cu]BPN was obtained through chelating Cu-64 in high purity and high specific activity with BPN and [64Cu]Cu2+ according to the method reported before.46 [64Cu]BPN was injected intravenously to B16-F10 tumor-bearing mice, and the PET-images were recorded with a microPET system. Representative PET image and biodistribution were presented in Figure 4A & 4B and Table S1, showing high accumulation and long retention in tumor through EPR effect. Skin cancer related normal tissues were taken into particular consideration, the tumor to muscle, tumor to blood, tumor to fat ratios were up to 6.54 ± 1.81, 2.50 ± 0.25 and 7.18 ± 1.04, respectively (Figure 4C). The remaining radioactivity then gradually excreted from the mouse through the hepatobiliary system. As illustrated in Figure S6, the liver uptake declined in a time-dependent manner, and the major radioactivity cleared from the body in the form of feces, demonstrating good biocompatibility and low toxicity. In summary,


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BPN provided the accurate location of tumors for imaging-guided BNCT, avoiding unnecessary irradiation to normal tissues.

Multiple injections of a small amount of BPN provided intense and very selective tumor accumulation of boron In order to trace the boron distribution during the treatment, [64Cu]BPN was co-injected with “cold” BPN which contained about 5 mg of boron (Figure 5A). According to the PET imaging and biodistribution showed in Figure 4, the boron concentration in B16F10 xenografts was 80 ppm (~16 ppm of Boron-10, Table S2), which was about enough to achieve a successful BNCT treatment (as shown in Figure 5B). However, the ratios of tumor to liver, muscle, fat and blood were only 0.18 ± 0.01, 5.87 ± 0.83, 6.65 ± 0.69 and 2.31 ± 0.24, respectively, which were at the same level to previous boron delivery agents47 but still unsatisfactory. PET imaging illustrated that the tumor uptake of BPN was consistent after reaching to the maximum value, while the uptake in non-target organs gradually declined after reaching to the peak value at a time point soon after intravenous injection. In addition, taking the inspiration from previous publications, injection plan was altered from one-time injection of 5 mg of boron to five-times injection of 1 mg of boron48. The assumption has been made that the tumor uptake would be at least equally high as single bolus injection while the background uptake would be only one fifth, resulting in significantly increased selectivity for boron accumulation in tumor. This hypothesis was tested as shown in Figure 5C and analyzed by ICP-OES measurements. As expected, the tumorous boron concentration was up to 130 ppm, 163% higher than single bolus groups, while the background uptake was much lower after the application of the new injection plan (Table S3), the ratios of tumor to liver, muscle, fat and blood were 3.24 ± 0.22, 61.46 ± 20.26, 31.55 ± 10.30 and 33.85 ± 5.73, respectively, which was used latter in neutron irradiation experiments (Figure 5C, 5D). Comparing to the well-studied boronated porphyrin for BNCT, BOPP, tumor to blood ratio of BPN is about 5 fold more than those of BOPP while tumorous boron concentration of BPN is about 2 fold more than those of BOPP under similar injection dose, indicating the enhanced boron delivery efficiency of BPN33. BNCT with BPN exhibited excellent tumor control on B16-F10 bearing mice



BPN at 250 mg TBPP/kg body weight based on the previous tests for BNCT in subcutaneous B16-F10 tumor-bearing mice. BPN was administrated intravenously 24 hours prior to BNCT (Figure 6A). Thermal neutron beam at the dose of 1.0*1012 neutron per cm2 was performed on B16-F10 tumors for 10 min. BPN did not affect the tumor growth without neutron irradiation. Thermal neutron irradiation did not affect the tumor growth without BPN administration as well. Tumor volumes increased by 20-fold in 2 weeks. With thermal neutron irradiation, tumor growth rate was suppressed obviously in mice administrated with BPN 14 days after BNCT (Figure 6B). In addition, compared to the other groups, the average survival time of mice in the treatment group (BPN and neutron) was significantly prolonged (Figure 6C). Then we investigated the biological safety of BPN-mediated BNCT, as shown in Figure 6D and 6E , no obvious weight changes or abnormity of major organs were observed, indicating side effect of BPN-mediated BNCT was neglectable. Finally, as shown in the pictures of tumors at different days post BNCT (Figure 6F), the efficacy of BNCT treatment was remarkable, the average tumor volume of mice with BPN injection and neutron irradiation was smaller than the other groups, which indicates that BPN could significantly enhance the therapeutic effect of BNCT. CONCLUSION Taking the advantage of nano drug delivery system, BPN, a novel boronated porphyrin loaded PLGA-mPEG was developed, providing a successful approach in boronated porphyrins delivery for imaging-guided BNCT. Fluorescent imaging showed that BPN preferred to locate within the cell nucleus. Both optical imaging and PET imaging provided accurate locations of malignant areas meanwhile proved that BPN holds the advantages of high tumor accumulation and high T/N ratio. Moreover, injection plan was optimized based on the understanding of BPN biodistribution by in vivo PET imaging, increasing the ratio of tumor to healthy tissue by at least one magnitude. In expectation, mice bearing B16-F10 tumor administrated with BPN demonstrated almost complete tumor suppression after neutron irradiation, suggesting that BPN was holding a great clinical potential in cancer management with imaging-guided BNCT.


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MATERIALS AND METHODS Materials All chemicals were used directly without further purification. D,L-Lactide (LA) were purchased from D&B (China) and glycolide (GA) were purchased from J&K (China).

o-Carborane

were

purchased

from

Zhengzhou

Alfa

(China).

Monomethoxypoly (ethylene-glycol) (mPEG, molecular weight 2000) was obtained from TCI (China). Meso-Tetra (4-carboxyphenyl) porphine (TCPP) were purchased from Energy Chemicals (China). Other relative chemicals were obtained from J&K (China). Cell Counting Kit-8 (CCK-8) was purchased from Biyuntian Biotechnology Institute. 64Cu (3.7 MBq/μL) was supplied by Beijing Cancer Hospital (Beijing, China). Synthesis of PLGA-mPEG copolymer PLGA-mPEG was synthesized through ring-opening polymerization: PLGAmPEG was synthesized with LA, GA and EO in the composition of 3:1:1 (LA, GA, and EO represent lactic acid, glycolic acid, and ethylene oxide components, respectively). LA, GA, and mPEG were placed in a round-bottomed flask, and 0.05% (w/w) of the stannous octoate as a catalyst were added to the vacuum sealed system in an oil bath at the temperature of 180 °C.49 The reaction was carried out for 4 hours to obtain a crude material which was cooled to room temperature. After that, the resulting mixture was dissolved in a certain volume of methylene chloride, precipitated in ice-cold ether, and taken into suction-filtration under reduced pressure, which was dried in the vacuum to obtain the desired product.50 Preparation of BPN nanoparticles BPN were prepared by dialysis method. Briefly, 20 mg of TBPP and 100 mg of PLGA-mPEG copolymer were dissolved N,N-Dimethylformamide (DMF) (5 ml) and stirred for 30 min.51 The resulting solution was added dropwise to water (100 ml) following by stirring for 4 h at room temperature. Then the crude product was taken into dialysis tubing (MWCO=7 kDa) (purchased from Shanghai yuan ye BioTechnology Co., Ltd) and dialyzed extensively in deionized water to remove the unincorporated reactant. The desired product was separated and concentrated by ultracentrifugation at high speed (4000 rpm).52



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Characterization of PLGA-mPEG and BPN The 1H NMR spectrum of TBPP and PLGA-mPEG were measured at 25 °C using tetramethylsilane as the internal standard and deuterated chloroform as the solvent. The relative molecular mass and composition were calculated based on the integral value. PLGA-mPEG and BPN were dissolved in methylene chloride and dripped on KBr pellets, dried to obtain the IR spectrum. PLGA-mPEG was dissolved in tetrahydrofuran at the concentration of 10 mg/ml, and flowed through a Styragel® HT3 (7.8 mm × 300 mm, 10 μm, molecular weight: 500 to 30000) column using tetrahydrofuran as eluent, and the molecular mass was measured at the column temperature of 35°C. The molecular weight and polydispersity index of PLGA-mPEG used for GPC measurement were M.W.=9856 and P.I.=1.553, respectively. Photon correlation spectroscopy and microelectrophoresis were used to determine the size and zeta potential of BPN in a Malvern Z-sizer 5000 instrument. The morphological examination of BPN was performed with TEM (JEM-2100, JEOL, Japan). A drop of BPN solution was added to a copper mesh covered with an ultra-thin carbon film and dried at room temperature for 30 min. The excess solution was removed before negatively staining with phosphotungstic acid solution (1% w/v). BPN solution was prepared as 0.02 μg/ml in PBS (0.01 mol/L, pH 7.4), and fluorescence excitation and fluorescence

emission

spectra

of

BPN

were

measured

by

fluorescence

spectrophotometer. Drug-loading rate and encapsulation efficiency determination Encapsulation efficiency of TBPP were calculated by measuring UV-Vis spectra (Figure S7). TBPP was dissolved in DMF at concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 0.1 μg/ml. The maximum absorption wavelength of TBPP by UV scanning is 420 nm, and the block copolymer has no UV absorption at this wavelength, so the 420 nm is determined as the characteristic absorption wavelength of TBPP. The absorbance of TBPP solution at different concentrations was measured by UV spectrophotometry. The absorbance (A) was linearly regressed with TBPP concentration (C). The standard curve was obtained at 0.02-0.1 mg/ml: Y=212.08X-3.3641 R2 = 0.9994. The results show that there is a good linear relationship between TBPP concentration and absorbance in this range. The BPN was dried to give a powder sample which was completely dissolved in DMF. The absorption peak at 420 nm was measured with UV spectrophotometer (Figure S8). The


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concentration of TBPP in BPN was calculated by standard concentration curves. Each experiment was conducted in triplicate. TBPP loading (19.06 ± 0.7)% and encapsulation efficiency (95.3 ± 3.7)% were calculated from the following equations.5354

TBPP loading (%) =

amount of loaded TBPP in mg ×100% (1) amount of polymer in mg

Encapsulation efficiency (%) =

amount of loaded TBPP in mg ×100% (2) amount of TBPP added in mg

Cells culture The mouse melanoma cell line B16-F10 was maintained in DMEM cell culture medium (Corning, Manassas, USA) supplemented with 10% fetal bovine serum (FBS). The mouse breast cancer cell line 4T1 was maintained in PRMI 1640 cell culture medium (Corning, Manassas, USA) supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37 °C in a humidified incubator containing 5% CO2 and adherently grown in cell culture flasks. Tumor-bearing animal models 6-week-old female C57BL/6 mice and BABL/c mice were ordered from Vital River Laboratories and maintained in SPF status (Beijing, China) with free access to water and standard food. All animal experiments were conducted in accordance with standards approved by the Peking University Ethics Committee. Approximately 1×106 B16-F10 cells suspended in 60 μl of PBS were subcutaneously implanted into the right shoulder of C57BL/6 mice. Similarly, approximately 1×106 4T1 cells suspended in 60 μl of PBS were subcutaneously implanted into the right shoulder of BABL/c mice.55 The mice were kept under specific pathogen-free conditions, handled and maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee. All animal experiments were conducted in accordance with the regulations of the Experimental Animal Center of Peking University. Cell uptake Studies Cell uptake studies of BPN were performed on B16-F10 cells. Briefly, B16-F10 cells (1×105 per well) were seeded in 24-well plates (triplicate for different groups) and incubated overnight at 37 °C. Incubated cells for different times (6, 12, 24,48 h)



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with the boron concentration reached 50 ppm in serum-free cell culture medium, respectively.27 At specified time points, cells were carefully washed 3 times with icecold PBS and lysed with 0.1 M HNO3 for 10 minutes at room temperature. We determined the cellular boron concentration by ICP-OES: we continued to digest the crudely treated cells obtained in the previous step using a microwave accelerated reaction system (Mars; CEM), followed by dilution with deionized water. The boron concentration was determined by ICP-OES on a PerkinElmer Optima 7000 DV according to the published method.38 Cellular toxicity assay of TBPP and BPN and PLGA-mPEG 3

B16-F10 cells were seeded in 96-well plates at a concentration of 5 × 10 cells per well and incubated overnight in a cell culture incubator. After 24 hours, TBPP, BPN, and PLGA-mPEG at different concentrations were added into each well and incubated for another 24 hours. The cell viability was assessed with the Cell Counting Kit-8 (CCK-8). In vivo fluorescence imaging of BPN Fluorescent images of 4T1 tumor-bearing mice were recorded after anesthetized by inhalation of a mixture of 2% isoflurane/oxygen. After intravenous injection of 200 μL of BPN solution (TBPP=5 mg/ml) into the mice, whole body fluorescence scanning was performed at specific time points (1, 2, 4, 12, 24 h) on mice using an IVIS Lumina III In Vivo Imaging System with an excitation wavelength of 420 nm and emission monitored at 680 nm. (IVIS® Imaging Systems with the CCD ranges from 1024×1024 to 2048×2048 pixels in size, the image exposure time is 10s). The mice were sacrificed 24 hours after the intravenous injection of BPN. Tumor, spleen, large intestine, liver, kidney, heart, lung, fat, muscle and brain were collected after dissection for ex vivo imaging. 56-57 64Cu

Labeling and Animal Model for Dynamic PET Imaging 64CuCl in 2

0.01 M hydrochloric acid was provided from Peking University Cancer

Hospital. Briefly, the BPN (TBPP=5 mg/ml) solution was transferred to 1.5 ml DNA low-binding tubes (Eppendorf) and made up to 1 ml of NaAc buffer (pH 5.5, 0.2 M), Radiolabeling of TBPP-loaded nanoparticles was accomplished by reacting 5 mCi of 64CuCl 2

with 1ml of BPN at 37 ℃ for 2 h with constant shaking. 64Cu-BPN was purified


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by using ultrafiltration centrifugation tubes. Phosphate buffered saline (PBS) was used as eluent to remove free 64CuCl2, 64Cu-BPN was centrifuged at 40000 r/min for 10 min and supernatant was collected for in vivo studies, radiochemical yield was 96.13% and the stability of radiolabeled products were determined by thin layer chromatography at specific time points. (Figure S9).46 Small-Animal PET/CT Imaging PET imaging was conducted on the micro-PET/CT scanner (Mediso Medical Imaging Systems).44 300 µCi of 64Cu-BPN was intravenously injected to the B16-F10 tumor-bearing mice and 4T1 tumor-bearing mice when the tumor volume reached 403

70 mm ; images were recorded at 2, 4, 12, 24 h after injection (Figure 4, Figure S10). Mice were anesthetized by inhalation of a 2% isoflurane/oxygen mixture and placed on a scanner bed approximately 10 minutes prior to PET/CT image acquisition. After the last scan, the mice were sacrificed. Organs of interest were collected and weighed, the radioactivity was measured by a gamma counter. Based on the prepared standards, the average percent injection dose (%ID/g) of 64Cu uptake in each tissue was calculated as a percentage of the injected dose per gram of tissue (Figure 4B, Figure S11).55 Biodistribution Studies A total dose of 250 mg/kg based on TBPP of BPN was intravenously injected to the B16-F10 tumor-bearing mice in five times when the tumor volume reached 40-70 3

mm . Mice given five times injection were euthanized at 24 h after the last injection. At specific time points, the boron content in blood and organs were assessed: brain, lung, heart, liver, kidney, spleen, tumor, blood and tail were weighed and subsequently digested with 68% HNO3 using a microwave accelerated reaction system (Mars; CEM). Boron concentration of different tissues was determined by ICP-OES.38 In vivo BNCT A total dose of 250 mg/kg based on TBPP of BPN were intravenously injected to the B16-F10 tumor-bearing mice in five times 3 to 1 days before the BNCT experiment. The mice were directly irradiated with a thermal neutron beam for 10 min with the neutron flux in 1.0 × 1012 neutron/cm2 at In-Hospital Neutron Irradiator (IHNI).5 Tumor volume of all mice was measured daily with the same caliper and the same observer throughout the experiment for 30 days using the equation volume = (length × width2)/2,



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and the same scale was used to measure the change in body weight of the mice. Data were collected and the relationship between tumor volume and time was plotted, the therapeutic effects of different groups were evaluated from the experimental results. When the tumor size reached 2800 mm3 or the loss was > 20% of total body weight, the mice were removed from the experimental group and euthanized. Histology Histopathological sections were used for further toxicity studies, two weeks days after various treatments, the mice from different experimental groups were randomly selected, dissected, and harvested organs such as heart, liver, spleen, lungs, and kidneys which were fixed in 4.0% paraformaldehyde. It was embedded in paraffin and stained with hematoxylin and eosin (H&E) after cutting into sections. H&E staining images of different groups of mice were obtained by bright field microscopy. Supporting Information Synthesis of TBPP, TEM images of BPN, STEM-HAADF elemental mapping of BPN, GPC curve of PLGA-mPEG, hydrodynamic diameter of BPN in cell culture medium with 10% FBS, fluorescence spectrum of TBPP, PET-CT images, UV-Vis absorption spectra of TBPP, UV-Vis absorption spectra of BPN, RTLC spectrums of 64Cu-BPN,

64Cu

and

and Bio-distribution of BPN.

Statistical Analysis The data in the experiment was statistically analyzed using Prism, version 6.0 (GraphPad Software, Inc.), Excel (Microsoft), and Origin 8 (OriginLab). Financial Support The source of funding for this work is the National Natural Science Foundation of China (NSFC 21778003) and the Ministry of Science and Technology of the People's Republic of China (2017YFA0506300). Acknowledgments We thank doctor Yang Chen for technical assistance. This work was founded by National Natural Science Foundation of China (NSFC 21778003) and the Ministry of Science and Technology of the People's Republic of China (2017YFA0506300).


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Figure 1. Characterization of BPN. (A) Schematic illustration of imaging-guided BNCT with BPN. (B) FT-IR spectra of BPN, PEG-PLGA, and TBPP. (C) Representative TEM image of BPN. (D) Size distribution of of PEG-PLGA micelle and BPN. (E) Gel permeation chromatograms (GPC) of copolymer PEG-PLGA. (F) Zeta-potential of PEG-PLGA micelle alone and BPN. (G) Fluorescence spectrum of BPN (TBPP concentration 5 μg/mL).



Figure 2. Cellular uptake and cytotoxicity of BPN. (A) Surviving fraction (n=3) of B16-F10 cells under various concentrations of PLGA-PEG. (B) Surviving fraction (n=3) of B16-F10 under various concentrations of TBPP. (C) Surviving fraction (n=3) of B16-F10 cells under various concentrations of BPN. (D) Boron concentration (n=3) of cells incubated with BPN (boron concentration=50 ppm). High accumulation in B16F10 cells was observed. (E) Confocal microscopy images of B16-F10 cells incubated with BPN for 24 h. Scale bar =10 μm.


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Figure 3. Fluorescence imaging showed accumulation of BPN in tumor region. (A) Fluorescence images of 4T1 tumor bearing mice 0 h, 1 h, 2 h, 4 h, 12 h, 24 h post intravenous injection of 1 mg BPN. (B) Fluorescence images of variant organs in 4T1 bearing mice 24 h post injection of BPN (TBPP=1 mg):1 tumor, 2 brain, 3 kidney, 4 spleen, 5 fat, 6 heart, 7 lung, 8 muscle, 9 liver, 10 large intestine.



A tt

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% ID /g

30 20

10

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L In K iv e i te d r In s t n e te in y e S s t /s to in m e/ a l c P h a n F c at r T ea u s M m u or s B cl lo e B od ra in

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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T /B

T /M

T /F

Figure 4. BPN showed high-specific accumulation in the tumor and also exhibited notably long tumor retention. (A) Whole-body maximum intensity projection PET images of showing the uptake of BPN. Tumor(t) were indicated by white arrows. (B) Corresponding Biodistribution of

64Cu-BPN

in B16-F10 bearing mice 24 h post

injection. Data are means ± SD ( n= 4 mice ). (C)Tumor to blood (T/B), tumor to muscle (T/M), tumor to fat (T/F) of %ID/g ratios respectively.


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Figure 5. The optimization of injection method enhanced boron delivery efficiency of BPN. (A) One-time injection schematic representation of the timeline of the experimental period. (B) Biodistribution of BPN in B16-F10 bearing mice 24 h post once injection of BPN (TBPP=5 mg). Data are means±SD (n=4 mice). (C) Five times injection schematic representation of the timeline of the experimental period. (D) Boron concentrations in extracted organs after five-times injection of BPN (TBPP=5 mg) into B16-F10 bearing mice (mean±SE, μg/g).



Figure 6. Comparing with other groups, mice pre-injected with BPN followed by neutron-irradiation showed significantly more effective tumor suppression without exhibiting systemic toxicity. (A) Experimental time flow chart. (B) Tumor growth curves (n=6) after treatments (0-28 days). The injection dose of BPN was 250 mg TBPP/kg, and the irradiation time was 10 mins (full power at IHNI). (C) Survival curves indicated that only BPN-neutron group provided effective tumor control. (D) Weight growth curves of certain groups of mice (0-28 days). (E) H&E-stained of heart, liver, spleen and lung slices collected from different groups of mice two weeks day after various treatments. (a) Control group. (b) BPN+neutron group. (c) Neutron only group. (d) BPN only group. (F) Representative photographs of each group of B16-F10 tumor bearing mice days post treatment (n=4). (a) Control group. (b) BPN+neutron group. (c) Neutron only group. (d) BPN only group.


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TOC Graphic:


Tracing Boron with Fluorescence and PET Imaging of Boronated

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