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

南福春， 杨 阳， 赵晓智， 葛介超*， 刘卫敏， 任昊慧， 郭宏骞*，汪鹏飞

(1. 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;2. 中国科学院大学 未来技术学院, 北京 100049;3. 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)

1 Introduction

Bladder cancer is one of the top 10 most common cancers for humans with approximately 55 000 new patients diagnosed annually[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[3-5]. 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[6-8]. 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[9-11]. 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 O2under suitable light irradiation to produce highly cytotoxic reactive oxygen species(ROS) that destroys the cancer cells[12-14]. Several PDT photosensitizers for bladder cancer therapy have been approved in Canada and USA[15-17]. However, rapid O2consumption and hypoxia in solid tumors strongly restrict the efficacy of PDT[18-19]. PTT is the other method where photosensitizers produce heat from the light irradiation to induce cellular apoptosis and subsequently achieve tumor ablation[20-21]. 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]: the high temperature in PTT could increase blood flow and provide additional O2for PDT[23]; meanwhile the1O2generated in the PDT process could increase the sensitivity of cells to overheating[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[25-27], catalysis[28-30], and theranostics[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]. Since the development of first CDs with high1O2quantum yield for PDT[36], numerous CDs with multifunctional ability such as for PDT/PTT and multi-model imaging-guided phototherapy have emerged[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,1O2quantum yield of 0.27, and photothermal conversion efficiency as high as 36.2% under a 635 nm laser irradiation[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 theinvitroandinvivoselectivity of nanomedicine to bladder cancer[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.

2 Experiment

2.1 Preparation of PCDs Nanoparticles

CDs were prepared as previously reported[37]. Thiophene benzoic acid(PBA) monomers were prepared through typical Suzuki reaction between thiophene-3-boronic acid and 4-bromobenzoic acid with Pd(PPh3)4as the catalyst. Oxidative polymerization was then conducted to obtain PBA with FeCl3as the catalyst under N2atmosphere. 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-NH2peptide 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(Mw=3 500) for 2 days to remove the nonspecifically bounded PLZ4 peptides and residues[37]. Finally the PCDs were concentrated by ultrafiltration(MWCO: 30 kDa)

2.2 Characterization of PCDs Nanoparticles

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 detect1O2generation 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 intracellular1O2generation was detected by Nikon C1si laser scanning confocal microscope using 2′,7′-dichlorodihydrofluorescein diaceta(DCFH-DA) as the ROS probe.

2.3 In Vitro Imaging and PDT/PTT with PCDs

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% CO2). 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% CO2). For theinvitroPDT 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/cm2. 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.InvitroPTT of PCDs was conducted similarly to the PDT experiment but with the irradiation laser power increased to 1 W/cm2. In all MTT assays, the viability of untreated cells was set as the reference.

2.4 In vivo Fluorescence Imaging and PDT/PTT of PCDs for Orthotopic Bladder Cancer

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[42-43]. The bladder was exposed through a small cut located on the lower abdomen, and MB49 cells(106) 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 mm3. 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 theinvivoimaging system(Maestro 2 Multispectral Small-animal Imaging System).

Treatment experiments were initiated when the bladder tumor size was up to 50-70 mm3(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/cm2) 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 theinvivoinjection 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.

3 Results and Discussion

3.1 Preparation and Characterization of PCDs

Fig.1 (a)Typical TEM images of PCDs(inset is the size distribution of PCDs from the TEM images). (b)High resolution TEM image of PCDs. (c)UV-Vis absorption spectra of CDs and PCDs. (d)Fluorescent spectra of CDs and PCDs. (e)Dispersed stability of PCDs in various solutions(water, PBS, DMEM, and FBS). (f)DLS of PCDs in water. (g)FTIR spectra of CDs, PLZ4 peptide, and PCDs.

Previously we have reported that CDs could produce1O2under 635 nm laser irradiation[34]. In the present work, electron spin resonance(ESR) technology was used to measure the1O2production of PCDs with TEMP as the trapper(Fig.2(a)). The characteristic1O2-induced signal produced by PCDs after irradiation with 635 nm laser for 10 min proves that the PCDs inherit the1O2production ability of CDs. Furthermore, DCFH-DA was applied as the green fluorescent probe to detect the intracellular1O2generation as shown in Fig.2(b). In only laser irradiation and only PCDs groups, negligible green fluorescence could be observed, proving no1O2generation, whereas the strong green fluorescence was observed in laser+PCDs group, confirming the effective1O2generation 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/cm2of 635 nm laser irradiation for 10 min(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), 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/cm2to 1.5 W/cm2to investigate the temperature changes(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/cm2of 635 nm laser for 10 min; hence, the laser power is sufficiently high enough for theinvitrocancer cell killing experiment. As shown in Fig.2(a)-(e), the PCDs could completely inherit the merits of CDs in PDT/PTT therapy.

Fig.2 (a)ESR spectra of PCDs under 635 nm laser irradiation. (b)Fluorescent images of DCFH-DA staining MB49 cells. (c)-(d)Curves of temperature changes of PCDs solution with different concentrations under 635 nm laser for 10 min. (e)Temperature changes of 200 μg/mL PCDs solution with various levels of laser irradiation power.

3.2 In Vitro Imaging and PDT/PTT with PCDs

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 imaginginvitro. 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)), 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.

Fig.3 (a)In vitro imaging of PCDs in MB49 cells and SU-HUV-1 cells under the same condition. (b)Relative cell viability of MB49 cells and SU-HUV-1 cells incubated with different PCDs concentrations in dark or under 635 nm laser irradiation at different power densities of 0.1 W/cm2 for PDT and 1 W/cm2 for PDT/PTT.

The cytotoxicity and PDT/PTT effect of PCDs were quantitatively evaluated by MTT assay. As shown in 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/cm2,the temperature of PCDs solution(200 μg/mL) only increased by 4.7 ℃ as shown in Fig.2(e). Death of cancer cells is impossible to achieve with temperature change under 0.1 W/cm2irradiation. Hence, the laser power was adjusted to 0.1 W/cm2to evaluate the efficacy of PDT. MB49 cell viability decreased to approximately 65% with 200 μg/mL PCDs solution under 0.1 W/cm2power irradiation for 10 min. When the laser power was increased to 1 W/cm2, 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.

3.3 In vivo Imaging of PCDs

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), the relative fluorescence intensities of major organs and tumor site were quantified, and the results are displayed in 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)). 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)). Fluorescence images in 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) indicates that the PCDs mainly accumulated in the bladder tumor site, thus confirming their tumor targeting ability.

Fig.4 (a)Time-dependent FL images of the main organs and bladder after the intravenous injection of PCDs. (b)Quantified FL intensities for the biodistribution of PCDs in major organs and bladder at various intravenous injection time. (c)FL images of main organs and normal bladder at 8 h post intravenous injection. (d)Fluorescent biodistribution of main organs of mice with and without bladder cancer at 8 h post-intravenous injection.

3.4 PDT/PTT of PCDs for Orthotopic Bladder Cancer

A laser power for theinvivoexperiment is not necessary for 1 W/cm2because 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/cm2(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], and the normal tissues are not influenced by this irradiation laser power. Hence, 0.5 W/cm2was used for the phototherapy of bladder tumor.

Fig.5 (a)Survival rate of mice after the intravenous injection different dosages of PCDs(40, 80, 160 mg/kg). (b)Temperature changes of mice normal tissues and bladder cancer tissues at 0.5 W/cm2. (c)Photos of bladder tumor-bearing mice after receiving different kinds of treatment. (d)The tumor volume changes in different groups. (e)H&E staining of bladder tumor sections under different treatments.

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)viatail vein and then irradiated by 635 nm laser with 0.5 W/cm2for 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/cm2) for 10 min(Laser), and only intravenously injected with PCDs(PCDs). As shown in 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)) due to the enormous space occupied by the bladder tumor as compared with the therapeutic groups. 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)). 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)).

Invitroassays 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)). 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). The results indicated no damage or inflammation (no necrosis, edema, inflammatory infiltration, and hyperplasia) and further confirmed the excellent biocompatibility of PCD.

Fig.6 H&E staining images of main mouse organs on different days after the intravenous injection of PCDs(from left to right: heart, liver, spleen, lung, and kidney)

4 结 论

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.Invitroexperiments 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.Invivostudies 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.