Orthotopic Bladder Tumor Targeted Carbon Dots for Fluorescence Imaging-guided Phototherapy Enhanced Publication

Orthotopic Bladder Tumor Targeted Carbon Dots for Fluorescence Imaging-guided Phototherapy 碳点用于荧光成像介导的原位膀胱癌靶向光动力/光热治疗 1 2 first-author 南福春(1992-)，男，山东潍坊人，博士研究生，2017年于青岛科技大学获得硕士学位，主要从事新型碳纳米材料在肿瘤诊疗中的研究。E-mail: nanfuchun@mail.ipc.ac.cn http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725008&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725009&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725022&type= 南福春 (1992-)，男，山东潍坊人，博士研究生，2017年于青岛科技大学获得硕士学位，主要从事新型碳纳米材料在肿瘤诊疗中的研究。E-mail: nanfuchun@mail.ipc.ac.cn nanfuchun@mail.ipc.ac.cn 3 3 1 2 corresp *E-mail: jchge2010@mail.ipc.ac.cn *E-mail: jchge2010@mail.ipc.ac.cn 葛介超(1970-)，男，山东临沂人，博士，研究员，2008年于山东师范大学获得博士学位，主要从事新型光响应纳米材料的设计及其在抗肿瘤、病毒、细菌、炎症或创伤修复等领域应用的研究。E-mail: jchge2010@mail.ipc.ac.cn http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725012&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725024&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725027&type= 葛介超 (1970-)，男，山东临沂人，博士，研究员，2008年于山东师范大学获得博士学位，主要从事新型光响应纳米材料的设计及其在抗肿瘤、病毒、细菌、炎症或创伤修复等领域应用的研究。E-mail: jchge2010@mail.ipc.ac.cn jchge2010@mail.ipc.ac.cn 1 2 1 3 corresp dr.ghq@nju.edu.cn dr.ghq@nju.edu.cn 郭宏骞(1965-)，男，吉林长春人，博士，教授，1999年于北京大学获得博士学位，主要从事泌尿生殖系肿瘤、肾移植、微创泌尿外科的治疗研究。E-mail: dr.ghq@nju.edu.cn http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725029&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725017&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725035&type= 郭宏骞 (1965-)，男，吉林长春人，博士，教授，1999年于北京大学获得博士学位，主要从事泌尿生殖系肿瘤、肾移植、微创泌尿外科的治疗研究。E-mail: dr.ghq@nju.edu.cn dr.ghq@nju.edu.cn 1 2 Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China 中国科学院理化技术研究所　光化学转换与功能材料重点实验室，北京　100190 中国科学院理化技术研究所　光化学转换与功能材料重点实验室，北京　100190 School of Future Technology, University of Chinese Academy of Sciences, Beijing 100049, China School of Future Technology, University of Chinese Academy of Sciences, Beijing 100049, China 中国科学院大学　未来技术学院，北京　100049 中国科学院大学　未来技术学院，北京　100049 Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, China Department of Urology, Drum Tower Hospital, Medical School of Nanjing University, Institute of Urology, Nanjing University, Nanjing 210008, China 南京大学医学院附属鼓楼医院　泌尿外科(南京大学泌尿外科学研究所)，江苏　南京　210008 南京大学医学院附属鼓楼医院　泌尿外科(南京大学泌尿外科学研究所)，江苏　南京　210008 Bladder cancer has become one of the most frequent malignant tumors in the urinary system. Chemotherapy and radical cystectomy could not effectively cure this illness and even lead to side effects and low-quality life, which seriously threaten the lives of patients. Phototherapy provides a new method for bladder cancer treatment. Here, multifunctional carbon dots theranostics was developed and modified by a bladder cancer-specific peptide(PLZ4). PLZ4-carbon dots(PCDs) integrate targeted delivery, photodynamic therapy(PDT), and photothermal therapy(PTT) in one procedure. In vitro fluorescence imaging revealed that PCDs tend to aggregate in tumor cells than in normal cells, thus allowing them to effectively ablate MB-49 bladder cancer cells under a 635 nm laser. In vivo biodistribution showed that the PCDs specifically accumulated in bladder cancer tissues but exhibited negligible fluorescence in normal bladder tissues. After combined PDT/PTT, the orthotopic bladder tumor can be completely killed, thereby proving that PCDs have a potential as a candidate for bladder cancer treatment. 膀胱癌是泌尿系统中最常见的肿瘤之一。目前，化疗与手术切除等治疗手段不能有效治愈该肿瘤，并且上述两种治疗方式会产生副作用，降低患者的生活质量。光疗则为膀胱癌提供了一种新的治疗手段。在本工作中，以靶向膀胱癌细胞的小肽PLZ4修饰多功能碳点，得到了兼具靶向膀胱癌细胞及光动力/光热治疗功能的PLZ-4碳点(PCDs)。细胞实验表明该PCDs能够用于膀胱癌细胞靶向成像，并且在635 nm激光照射下，能高效杀灭膀胱癌细胞。活体实验表明经过尾静脉注射后，PCDs能够有效地聚集在原位膀胱癌处，在635 nm激光照射下，原位膀胱癌能够被完全清除，表明该PCDs在荧光成像介导的光动力/光热治疗原位膀胱癌中有潜在的应用价值。 carbon dots phototherapy orthotopic bladder cancer targeted tumor therapy 碳点 光疗 原位膀胱癌 肿瘤靶向治疗 Introduction 1 Bladder cancer is one of the top 10 most common cancers for humans with approximately 55 000 new patients diagnosed annually [ 1 1 - 2 2 1-2 ] . In most cases, this illness progresses following two distinct pathways: one is low-grade non-muscle invasive bladder cancers (NMIBCs) that are not immediately life-threatening, account for approximately 70% of tumor incidence, and have high propensity for recurrence; and the other is muscle invasive bladder cancers(MIBCs) that progress rapidly and account for the bulk of patient mortality [ 3 3 - 5 5 3-5 ] . NMIBCs are often associated with polypoid tumors that are invisible to the naked eye and associated with poor prognosis. Regardless of the experience of the chirurgeon, transurethral resection(TUR) could not fully excise the bladder cancer tissues, and the residue cancer cells have the chance to develop to MIBCs. To date, radical cystectomy is the gold-standard treatment for inhibiting the growth of non-metastatic MIBCs. However, this method is not suitable for cancer survivors after cystectomy because it could cause high mortality rate and give the patients the worst health-related quality of life [ 6 6 - 8 8 6-8 ] . Hence, effective methods that inhibit MIBC growth and preserve the bladder must be developed. Phototherapy is an efficient tumor therapy due to its minimally invasive, relatively tumor selectivity, fine biocompatibility, low dark toxicity, and low risk for resistance development [ 9 9 - 11 11 9-11 ] . This treatment has two types, namely, photodynamic therapy(PDT) and photothermal therapy(PTT). PDT is a typical representative of phototherapy where photosensitizers could react with the surrounding O 2 under suitable light irradiation to produce highly cytotoxic reactive oxygen species(ROS) that destroys the cancer cells [ 12 12 - 14 14 12-14 ] . Several PDT photosensitizers for bladder cancer therapy have been approved in Canada and USA [ 15 15 - 17 17 15-17 ] . However, rapid O 2 consumption and hypoxia in solid tumors strongly restrict the efficacy of PDT [ 18 18 - 19 19 18-19 ] . PTT is the other method where photosensitizers produce heat from the light irradiation to induce cellular apoptosis and subsequently achieve tumor ablation [ 20 20 - 21 21 20-21 ] . However, PTT usually requires relatively high laser irradiation power, thus increasing the risk of damaging normal tissues. Combined PDT/PTT provides an efficient method to overcome their individual drawbacks [ 22 22 ] : the high temperature in PTT could increase blood flow and provide additional O 2 for PDT [ 23 23 ] ; meanwhile the 1 O 2 generated in the PDT process could increase the sensitivity of cells to overheating [ 24 24 ] . To date, PDT/PTT combined therapy has achieved high efficiency in subcutaneous tumor-bearing mice; however, its effectiveness as a therapy for MIBCs is seldom reported. As a new member of carbon nanomaterials, carbon dots(CDs) show great potential in many fields, such as bioimaging [ 25 25 - 27 27 25-27 ] , catalysis [ 28 28 - 30 30 28-30 ] , and theranostics [ 31 31 - 34 34 31-34 ] , because they possess many merits such as excellent optical properties( e.g ., high photostability and tunable emission), versatility, outstanding biocompatibility, facile preparation, and modification flexibility [ 35 35 ] . Since the development of first CDs with high 1 O 2 quantum yield for PDT [ 36 36 ] , numerous CDs with multifunctional ability such as for PDT/PTT and multi-model imaging-guided phototherapy have emerged [ 37 37 - 39 39 37-39 ] and consequently achieved excellent therapeutic effect on subcutaneous tumor-bearing mice and laid a great foundation for MIBC phototherapy with CDs as the photosensitizer. Among the several kinds of CDs with phototherapy efficiency developed by our group, one has a PDT and PTT synergetic effect for tumor therapy, 1 O 2 quantum yield of 0.27, and photothermal conversion efficiency as high as 36.2% under a 635 nm laser irradiation [ 37 37 ] . However, the inability of the current CDs for active targeting causes low selectivity and poor bioavailability during the tumor phototherapy. Moreover, CDs based phototherapy for MIBCs treatment have not been reported so far. As a bladder cancer specific peptide, PLZ4 peptide (amino acid sequence: cQDGRMGFc) has been utilized as a targeting agent to improve the in vitro and in vivo selectivity of nanomedicine to bladder cancer [ 40 40 - 41 41 40-41 ] . In this paper, PLZ4 peptide modified CDs(PCDs) were firstly used as a phototherapy agent for orthotopic bladder tumor targeting treatment. Among of the PLZ4-CDs, CDs possess dual PDT and PTT effects under 635 nm laser irradiation. Meanwhile, the PLZ4 peptide endows CDs with active targeting ability towards MB49 bladder cancer cells. These collective properties enable PCDs to be applied as a targeting nanomedicine for PDT/PTT treatment. As far as we know, this study is the first to illustrate the capability of CDs for MIBCs treatment. This study provides a potential candidate material based on CDs for orthotopic bladder cancer treatment. Experiment 1 Preparation of PCDs Nanoparticles 2 CDs were prepared as previously reported [ 37 37 ] . Thiophene benzoic acid(PBA) monomers were prepared through typical Suzuki reaction between thiophene-3-boronic acid and 4-bromobenzoic acid with Pd(PPh 3 ) 4 as the catalyst. Oxidative polymerization was then conducted to obtain PBA with FeCl 3 as the catalyst under N 2 atmosphere. The CDs were obtained through the hydrothermal treatment of PBA dispersed in the alkaline solution and heated for 24 h at 180 ℃ in an autoclave. After cooling to room temperature, the CDs solution was filtered through 220 nm to remove large particles. Amino-modified PLZ4 peptides were purchased from Hangzhou Dangang Biotechnology Co., Ltd. The CDs(5 mg) were then added with 1 mL of water, stirred, and added with 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride(EDCI)(25 μmol, 4.8 mg) and N-hydroxysulfosuccinimide sodium salt(25 μmol, 5.4 mg) for 24 h to form CDs-NHS ester. Afterward, 20 mg of PLZ4-NH 2 peptide was added into the mixture and stirred for another 24 h to synthesize PCDs. Finally, the obtained PCDs solution was dialyzed against water using a dialysis membrane( M w =3 500) for 2 days to remove the nonspecifically bounded PLZ4 peptides and residues [ 37 37 ] . Finally the PCDs were concentrated by ultrafiltration(MWCO: 30 kDa) Characterization of PCDs Nanoparticles 2 PCDs morphology was characterized by transmission electron microscopy(TEM) (JEOL JEM-2100), and CDs elaboration was obtained by high-resolution TEM(JEOL JEM-2100F). The diameter of PCDs was measured as more than 40 nanoparticles according to the TEM images through the software of Nano Measurer 1.2. Hydrodynamic diameter and zeta potential were detected using Zetasizer Nano ZS provided by Malvern Panalytical. UV-Vis and fluorescence spectra were obtained by Hitachi U-3000 and F-4500 spectrophotometers, respectively. The Fourier transform infrared(FTIR) spectra of materials in KBr were obtained by the Varian Excalibur 3100 FTIR spectrometer. Electron spin technique(ESP 3000E, Bruker) was employed to detect 1 O 2 generation for PCDs with 2,2,6,6-tetramethylpiperidine(TEMP) as the trapping agent. The photothermal effect of PCDs under a 635 nm laser irradiation was measured according to a previous method. The intracellular 1 O 2 generation was detected by Nikon C1si laser scanning confocal microscope using 2',7'-dichlorodihydrofluorescein diaceta(DCFH-DA) as the ROS probe. In Vitro Imaging and PDT/PTT with PCDs 2 MB49 bladder cancer cells and normal bladder epithelial cells(SU-HUV-1) were cultured with RM1640 medium and high Dulbecco’s modified Eagle medium(DMEM), respectively, which contained 10% fetal bovine serum(FBS) and 1% penicillin at 37 ℃(5% CO 2 ). PCDs cellular uptake was investigated through confocal microscopy. MB49 cells and SU-HUV-1 cells were incubated with 50 μL(1 mg/mL) of PCDs solution in 1 mL of culture media at 37 ℃ for 4 h. The cells were then washed with phosphate buffer saline(PBS) twice to remove unloaded PCDs. Finally, the PCDs cellular fluorescent images were obtained using a Nikon C1si laser scanning confocal microscope under the same conditions. The dark cytotoxicity and PDT/PTT efficacy of PCDs were investigated through MTT assay. For the dark cytotoxicity, MB49 cancer cells and SU-HUV-1 normal cells were respectively seeded into 96 plates and incubated with different concentrations of PCDs solution (0, 50, 100, 150, 200 μg/mL) for 24 h at 37 ℃(5% CO 2 ). For the in vitro PDT efficacy, MB49 cells and SU-HUV-1 cells were respectively incubated with PCDs(0, 50, 100, 150, 200 μg/mL) for 4 h at 37 ℃ and then replaced with new culture media and irradiated by a 635 nm laser for 10 min with 0.1 W/cm 2 . After irradiation, the cells were incubated for another 24 h, the culture media were replaced by 80 μL of dimethylsulfoxide(DMSO), and the absorbance at 570 nm was measured and analyzed by ANOVA. In vitro PTT of PCDs was conducted similarly to the PDT experiment but with the irradiation laser power increased to 1 W/cm 2 . In all MTT assays, the viability of untreated cells was set as the reference. In vivo Fluorescence Imaging and PDT/PTT of PCDs for Orthotopic Bladder Cancer 2 All animal experiments were approved by the China Committee for Research and Animal Ethics in compliance with the law on experimental animals. C57BL/6 mice were chosen to establish the orthotopic bladder cancer model [ 42 42 - 43 43 42-43 ] . The bladder was exposed through a small cut located on the lower abdomen, and MB49 cells(10 6 ) were then carefully injected into the bladder wall by using an insulin syringe. The tumor in the bladder was confirmed 7 days after the injection. The PCDs(2 mg/mL, 50 μL) were injected into the mice through the tail vein until the volume of bladder tumor reached 60 mm 3 . Fluorescent images of mean organs and bladder were obtained at various time points(0, 4, 8, 12, 24 h) after the intravenous injection of PCDs with the in vivo imaging system(Maestro 2 Multispectral Small-animal Imaging System). Treatment experiments were initiated when the bladder tumor size was up to 50-70 mm 3 (4 mice in each group). For treatment group, the mice were intravenous injected with 50 μL of PCDs(2 mg/mL), and the tumor tissues were irradiated with a 635 nm laser(0.5 W/cm 2 ) for 10 min. Control groups contained only PBS injection, only laser irradiation, and only PCDs injection. The mice were sacrificed after 12 h therapy, and the bladders were obtained for hematoxylin and eosin(H & E) staining. After 14 days, the mice were sacrificed and the tumor volumes were measured. The mice were intravenously injected with PCDs solution at different dosages(40, 80, 160 mg/mL, n =4) to measure the acute toxicity of PCDs and determine the survival of mice. The mice were sacrificed at 7 d and 14 d after the in vivo injection of PCDs(50 μL, 2 mg/mL). The major organs of the mice were obtained for H & E staining to further investigate the toxicity of PCDs. Results and Discussion 1 Preparation and Characterization of PCDs 2 CDs containing carboxyl groups were prepared and bounded to the PLZ4 peptide containing amide groups through amidation to obtain PCDs. PLZ4 peptide could specifically target to bladder cancer cells due to the overexpression of αvβ3 integrin [ 40 40 - 41 41 40-41 , 44 44 ］ . Fig.1(a) Fig.1(a) shows the transmission electron microscopy(TEM) image of PCDs with the diameters ranging from 20 to 40 nm(inset in Fig.1(a) Fig.1(a) ). The high-resolution transmission electron microscopy(HRTEM) image in Fig.1(b) Fig.1(b) reveals high crystallinity with the interlayer space 0.21 nm due to the (100) lattice spacing of graphene [ 45 45 ] . After modification by PLZ4 peptide, the UV-Vis absorption of PCDs shows slightly redshift compared to CDs, a broad absorption from 350 to 650 nm( Fig.1(c) Fig.1(c) ). The emission peak of PCDs had redshifted from 600 nm to 615 nm under a 480 nm excitation( Fig.1(d) Fig.1(d) ). These property changes are ascribed to the J-aggregates of the aromatic structure of individual CDs [ 46 46 ] . Fig.1(e) Fig.1(e) shows that the PCDs were stable in physiological mediums including water, PBS, DMEM, and FBS. The hydrodynamic diameter of PCDs was ~132 nm( Fig.1(f) Fig.1(f) ), which is larger than the value from TEM images. The FTIR was applied to confirm the preparation of PCDs. As shown in Fig.1(g) Fig.1(g) , the PLZ4 peptide exhibited obvious FTIR absorption of amino (—NH 2 ) at about 3 220 cm -1 , whereas the corresponding FTIR absorption in PCDs was found to decrease, indicating the successful amidation reaction between —NH 2 on PLZ4 peptide and carboxyl groups on CDs [ 47 47 ] . And compared with CDs, the PCDs exhibited obvious FTIR absorption at about 1 650 cm -1 ascribed to the vibrations of aromatic C http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725049&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725054&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=26725051&type= O stretching of amide groups [ 48 48 ] , further proving the successfully preparation of PLZ4 peptide modified CDs. After modification by PLZ4 peptide, the zeta potential changed from -47.7 mV of CDs to -38.5 mV of PCDs due to the depletion of carboxyl groups on CD surface, as shown in Fig.S1. These results are beneficial for the intravenous injection and accumulation of PCDs in solid tumor through the enhanced permeation and retention phenomenon(EPR) effect. Previously we have reported that CDs could produce 1 O 2 under 635 nm laser irradiation [ 34 34 ] . In the present work, electron spin resonance(ESR) technology was used to measure the 1 O 2 production of PCDs with TEMP as the trapper( Fig.2(a) Fig.2(a) ). The characteristic 1 O 2 -induced signal produced by PCDs after irradiation with 635 nm laser for 10 min proves that the PCDs inherit the 1 O 2 production ability of CDs. Furthermore, DCFH-DA was applied as the green fluorescent probe to detect the intracellular 1 O 2 generation as shown in Fig.2(b) Fig.2(b) . In only laser irradiation and only PCDs groups, negligible green fluorescence could be observed, proving no 1 O 2 generation, whereas the strong green fluorescence was observed in laser+PCDs group, confirming the effective 1 O 2 generation capacity of PCDs. Photothermal effect is another important property for the initial CDs and hence was investigated for PCDs. The temperature changes of different PCDs concentrations(0-200 μg/mL) were investigated under 1.5 W/cm 2 of 635 nm laser irradiation for 10 min( Fig.2(c) and (d) Fig.2(c) and (d) ). The temperature of pure water (without PCDs) only increased by 5.6 ℃, whereas the PCDs solution with regulated concentration of 20-200 μg/mL rapidly increased from 14.4 ℃ to 34.9 ℃. These results indicated that PCDs addition could effectively increase the temperature of the solution under a 635 nm laser irradiation. As shown in Fig.2(d) Fig.2(d) , the temperature changes do not exhibit a linear relationship with PCDs concentration because the heat transfer rate with the surrounding environment increases with the temperature difference. The PCDs solution(200 μg/mL) was also exposed to 635 nm laser with various power densities from 0.1 W/cm 2 to 1.5 W/cm 2 to investigate the temperature changes( Fig.2(e) Fig.2(e) ). The results exhibited that the increasing irradiation power rapidly increased the temperature of the PCDs solution. Cancer cells could be killed in 4-6 min when the temperature is higher than 50 ℃. The temperature of PCDs solution(200 μg/mL) increased to 25.9 ℃ under 1 W/cm 2 of 635 nm laser for 10 min; hence, the laser power is sufficiently high enough for the in vitro cancer cell killing experiment. As shown in Fig.2(a)-(e) Fig.2(a)-(e) , the PCDs could completely inherit the merits of CDs in PDT/PTT therapy. In Vitro Imaging and PDT/PTT with PCDs 2 Owing to PLZ4 peptide modification, PCDs are endowed with bladder cancer cell targeting ability. The uptake of PCDs in bladder cancer cells(MB49) and normal bladder epithelial cells(SU-HUV-1) was compared through fluorescent imaging in vitro . The red fluorescent intensity of PCDs in MB49 cells was stronger than that in SU-HUV-1 cells under the same condition ( Fig.3(a) Fig.3(a) ), indicating that the PCDs could be selectively taken up by bladder cancer cells. Hence, PLZ4 peptide on CD surfaces could remarkably enhance the internalization of PCDs in bladder cancer cells. The cytotoxicity and PDT/PTT effect of PCDs were quantitatively evaluated by MTT assay. As shown in Fig.3(b) Fig.3(b) , MB49 and SU-HUV-1 cells did not exhibit apoptosis with the increasing PCDs concentration, indicating that PCDs possess low cytotoxicity even at 200 μg/mL. When the irradiation power was 0.1 W/cm 2 ,the temperature of PCDs solution(200 μg/mL) only increased by 4.7 ℃ as shown in Fig.2(e) Fig.2(e) . Death of cancer cells is impossible to achieve with temperature change under 0.1 W/cm 2 irradiation. Hence, the laser power was adjusted to 0.1 W/cm 2 to evaluate the efficacy of PDT. MB49 cell viability decreased to approximately 65% with 200 μg/mL PCDs solution under 0.1 W/cm 2 power irradiation for 10 min. When the laser power was increased to 1 W/cm 2 , the viability of MB49 cell drastically decreased to ~19%, revealing the good PDT/PTT killing efficacy for MB49 cells. However, the viability of SU-HUV-1 remained at 70% under the same laser power irradiation. These MTT results indicated that PCDs could effectively kill MB49 cells but do not show high toxicity to normal SU-HUV-1 cells. In vivo Imaging of PCDs 2 The PCDs dispersed in PBS were injected into the mice with orthotopic bladder cancer. The mice were killed at different time points before and after the injection, the obtained major organs( i.e ., heart, liver, spleen, lung, and kidneys) and bladder with tumor were imaged by small-animal imaging system to evaluate PCDs biodistribution through fluorescence intensities. As shown in Fig.4(a) Fig.4(a) , the relative fluorescence intensities of major organs and tumor site were quantified, and the results are displayed in Fig.4(b) Fig.4(b) . The PCDs mainly accumulated in liver, lung, kidneys, and bladder tumor. The PCDs achieved the maximum value in bladder tumor 8 h post injection as indicated through the fluorescence changes( Fig.4(b) Fig.4(b) ). Meanwhile, the PCDs were also injected to normal mice, and the major organs and bladder were imaged at 8 h post injection( Fig.4(c) Fig.4(c) ). Fluorescence images in Fig.4(a) and 4(c) Fig.4(a) and 4(c) show that the fluorescence in cancerous bladder was higher than that in normal bladder. Quantitative analysis in Fig.4(d) Fig.4(d) indicates that the PCDs mainly accumulated in the bladder tumor site, thus confirming their tumor targeting ability. PDT/PTT of PCDs for Orthotopic Bladder Cancer 2 A laser power for the in vivo experiment is not necessary for 1 W/cm 2 because the heat capacity of biological organisms is lower than that of water. The temperature changes of tumor and normal bladder tissues were measured and recorded 8 h after the intravenous injection with exposure to 635 nm laser with 0.5 W/cm 2 ( Fig.5(b) Fig.5(b) ). The temperature increased by approximately 5.6 ℃ after 10 min irradiation. By contrast, the temperature of bladder tumor tissues easily increased to > 48 ℃. The bladder tumor tissues are easily ablated at this temperature [ 49 49 ] , and the normal tissues are not influenced by this irradiation laser power. Hence, 0.5 W/cm 2 was used for the phototherapy of bladder tumor. Fig.5(c) and 5(d) Fig.5(c) and 5(d) show the therapeutic results of PCDs for orthotopic bladder cancer. Four groups of MB49 orthotopic bladder tumor-bearing C57BL/6 mice were used in our experiment. The mice were injected PCDs(5 mg/kg) via tail vein and then irradiated by 635 nm laser with 0.5 W/cm 2 for 10 min after 8 h in the therapeutic group(PCDs+Laser). The control groups include the following: intravenously injected with PBS(PBS), only irradiated by 635 nm laser(0.5 W/cm 2 ) for 10 min(Laser), and only intravenously injected with PCDs(PCDs). As shown in Fig.5(c) Fig.5(c) , the bladder tumor of therapeutic groups was totally destroyed after 14 days; however, the bladder tumor grew rapidly in all control groups. The control groups had larger bladder volume( Fig.5(c) Fig.5(c) ) due to the enormous space occupied by the bladder tumor as compared with the therapeutic groups. Fig.5(d) Fig.5(d) shows the changes of the tumor volume size in different groups. No significant changes were observed in the control groups, but the tumors were totally ablated in the treatment groups. The bladder tumors of different groups were sliced and stained with H & E at 12 h post-treatment( Fig.5(e) Fig.5(e) ). The tumor cells in the phototherapy treatment group were totally destroyed, but those in the control groups did not show evident cell damage ( Fig.5(e) Fig.5(e) ). In vitro assays indicated that PCDs have no evident cytotoxicity to cells under dark environment, but do not necessarily imply their safety for mice. Acute toxicity test for PCDs was conducted in mice to evaluate their potential toxicity( Fig.5(a) Fig.5(a) ). The survival rate of mice remained at 100% even when the dosage of PCDs was increased to 80 mg/kg, which is much higher than the therapeutic dosage of 5 mg/kg. When the intravenous dosage of PCDs was up to 160 mg/kg, one-half of the mice died after 3 days. These results indicated that PCDs exhibit excellent safety to living mice even when the dosage is 16 times the value used for phototherapy. The major mouse organs(hearts, livers, spleens, lungs, and kidneys) were sliced after the intravenous injection of PCDs and stained by H & E to investigate the potential toxicity of PCDs at 7 and 14 days post-injection( Fig.6 Fig.6 ). The results indicated no damage or inflammation (no necrosis, edema, inflammatory infiltration, and hyperplasia) and further confirmed the excellent biocompatibility of PCD. 结论 1 In this study, PLZ4 peptide modified CDs were firstly used as a phototherapy agent for orthotopic bladder tumor targeting treatment. Among of the PCDs, CDs possess dual PDT and PTT effects under 635 nm laser irradiation. Meanwhile, PLZ4 peptide endows CDs with active targeting ability towards MB49 bladder cancer cells. In vitro experiments show that the PCDs can be specifically taken up by MB49 cells resulting in the high accumulation of PCDs in MB49 cells, and thus achieving the accurate killing effect. In vivo studies indicate that the vein tail injected PCDs could highly accumulate at the orthotopic bladder tumor site. Furthermore, the orthotopic bladder tumor could be totally ablated by PCDs phototherapy. Meanwhile, the PCDs exhibit excellent biocompatibility and safety for living mice. This study provides a new carbon nanomaterial candidate for the phototherapy of bladder cancer(especially for the MIBCs) aiming at bladder preservation. Response Letter is available for this paper at:http://cjl.lightpublishing.cn/thesisDetails#10.37188/CJL. 20220017. References ANTONI S , FERLAY J , SOERJOMATARAM I , et al . Bladder cancer incidence and mortality: a global overview and recent trends [J]. Eur. Urol . , 2017 , 71 ( 1 ): 96 - 108 . Bladder cancer incidence and mortality: a global overview and recent trends RICHTERS A , ABEN K K H , KIEMENEY L A L M . The global burden of urinary bladder cancer:an update [J]. World J. 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