Hydrogen Sulfide-Specific and NIR-Light-Controllable Synergistic Activation of Fluorescent Theranostic Prodrugs for Imaging-Guided Chemo-Photothermal Cancer Therapy

Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2020Hydrogen Sulfide-Specific and NIR-Light-Controllable Synergistic Activation of Fluorescent Theranostic Prodrugs for Imaging-Guided Chemo-Photothermal Cancer Therapy Ge Xu†, Wei Guo†, Xianfeng Gu†, Zhijun Wang, Rongchen Wang, Tianli Zhu, He Tian and Chunchang Zhao Ge Xu† Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 , Wei Guo† Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203. , Xianfeng Gu† Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203. , Zhijun Wang Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203. , Rongchen Wang Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 , Tianli Zhu Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 , He Tian Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 and Chunchang Zhao *Corresponding author: E-mail Address: [email protected] Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237 https://doi.org/10.31635/ccschem.020.201900072 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Theranostic prodrugs are promising for cancer medicine; however, the inability to activate these systems exclusively at the desired tumor location compromises the specificity and efficacy of cancer treatment. Here, we developed a fluorescent theranostic nanoprodrug with synergistic hydrogen-sulfide-specific and near-infrared (NIR)-light-controllable activation for imaging-guided chemo-photothermal cancer therapy. This nanoprodrug system was fabricated by the inclusion of hydrogen sulfide (H2S)-activatable small molecule to the theranostic prodrug and a photothermal transducer in the interior of a NIR-light-responsive container. The resultant nanoprodrug could be activated specifically, evidenced by a "turn on" of NIR fluorescence in H2S-rich cancers; thus, enabling accurate cancer location and identification of when and where to implement NIR irradiation for efficient conversion of photoenergy into heat. Such a prominent photothermal effect guarantees photothermal ablation of cancers and the release of the H2S-preactivated DOX for cancer chemotherapy. By utilizing this H2S-responsive and NIR-light-controllable nanoprodrug, complete suppression of tumors was realized under the guidance of the NIR fluorescence imaging. Thus, our study provides a novel strategy toward theranostic prodrug systems that enable precision cancer treatment with high specificity and efficacy. Download figure Download PowerPoint Introduction Although cancer is known to be one of the major life-threatening diseases, successful treatment is still an unsolved issue.1,2 In the clinic, drug therapies, involving the administration of cytotoxic agents to cause cellular damage, have been widely used for treatments of cancers.3 However, the drugs that are currently available often possess low therapeutic efficacy and off-target toxicity, inevitably, leading to adverse effects on the patient's normal tissues.4,5 These challenges have inspired the development of theranostic prodrug systems with integrated diagnostic and therapeutic functions for precision cancer medicine.6–12 Such systems could respond to cancer-associated stimuli to activate diagnostic imaging and release therapeutic drugs at desired cancer sites.13–27 However, despite the overexpression of certain factors in a tumor, endogenous stimuli generally feature nonspecific systemic distribution,28,29 thus, unable to activate the prodrugs exclusively at the desired tumor location. Recently, near-infrared (NIR)-light-responsive nanoplatforms have been explored to fulfill on-demand drug release.30,31 In these nanocomposites, photothermal transducers are able to convert photoenergy into heat for induction of conformational changes of a thermosensitive matrix, enabling controllable on-demand drug release under NIR-light irradiation.32–34 In particular, the therapeutic outcome might be optimized by the diagnostic-imaging-guided spatial location of the thermosensitive nanocomposites at the tumors and the timing of implementation of the light illumination.35 We envisioned that a smart nanoplatform could be fabricated by encapsulation of theranostic prodrugs into the interiors of a light-responsive matrix, wherein the therapeutic performance would require cancer-stimuli-specific and NIR-light-controllable synergistic activation to enable cancer treatment exclusively at the desired time and location. However, no such synergistic nanoprodrugs have been explored for precision cancer medicine. Herein, we developed a fluorescent theranostic nanoprodrug (FTNpd), shown in Figure 1, with hydrogen sulfide (H2S)-specific and NIR-light-responsive synergistic activation for imaging-guided chemo-photothermal (chemo-PTT) cancer therapy. Since increased H2S production has been implicated in various cancers,36–44 it could be regarded as a promising drug target for cancer theranostics. Surprisingly, most available H2S-responsive probes have focused on the detection of H2S levels either in vitro or in vivo, under numerous pathological conditions, including acute inflammatory and vascular disorders, rather than their investigation as potential targets for cancer therapy.45–48 Thus, we sought to develop a H2S-responsive theranostic prodrug (BSO–DOX) that could combine detection, imaging, and therapy of H2S-rich cancers. BSO–DOX was derived from a boron-dipyrromethene (BODIPY) core fluorescent dye, conjugated with the chemotherapeutic DOX via a H2S cleavable linker. A three-diethylene glycol monomethyl ether chain–functionalized aza-BODIPY (Aza-BOD) dye that was inert to H2S was prepared as the NIR photothermal transducer. Nanoplatform FTNpd was feasibly fabricated through coencapsulation of BSO–DOX and Aza-BOD into thermoresponsive nanoparticles, consisting of the natural-phase transition material (PCM) based on lauric acid.32 BSO–DOX in FTNpd showed H2S-dependent NIR fluorescence light up, along with the liberation of DOX into the PCM matrix due to H2S-initiated cleavage of a sulfoxide linkage. The specific NIR emission enabled accurate identification of H2S-rich human colorectal cancer (CRC) HCT116 cells and guided the implementation of NIR irradiation of Aza-BOD. The exposure of Aza-BOD to NIR light caused a highly efficient conversion of photoenergy into heat for not only photothermal ablation of the cancers, but also melting the PCM matrix to trigger the release of active DOX for chemotherapy. Therefore, the theranostic nanoprodrug, FTNpd, possessed CRC-specific, and photo-controllable synergistic therapeutic characteristics to facilitate CRC precision treatment with high specificity and efficacy. Figure 1 | Schematic illustration of the fluorescent theranostic nanoprodrug, FTNpd, with hydrogen sulfide (H2S)-specific and NIR-light-responsive synergistic activation for imaging-guided chemo-photothermal cancer therapy. Download figure Download PowerPoint Experimental Methods Detailed methods, including organic synthesis, experimental protocols, the characterizations, and the supplementary figures are available in the Supporting Information. Preparation of FNTpd PCM containers composed of lauric acid, DSPE-mPEG-2000, and lecithin were prepared using a nanoprecipitation technique. Briefly, a mixture of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxy poly(ethylene glycol) DSPE-mPEG-2000 (18 mg) and lecithin (6 mg) in water (3 mL), containing 4% ethanol, was heated to 50 °C in an oil bath under stirring for 10 min, followed by the addition of lauric acid (600 μL, 4 mg/mL) in methanol. Afterward, BSO–DOX (48 μL, 7.4 mg/mL) and Aza-BOD (48 μL, 6.0 mg/mL) in DMSO were added to the preheated aqueous solution of the phospholipid derivative polymer and stirred vigorously for 2 min. The resultant mixture was cooled down quickly on an ice bath, then stirred at room temperature for 2 min. The mixture obtained was filtered through a polyvinylidene fluoride (PVDF) membrane (0.45 μm) into a dialysis membrane bag of molecular weight of 1000 and dialyzed against deionized water for 8 h at room temperature to remove PEG and other unreacted small ion molecules from the FNTpd product. Afterward, the FNTpd obtained was filtered again through a PVDF membrane (0.22 μm) and diluted for testing. Results and Discussion Synthesis of H2S-activatable small-molecule theranostic prodrug As shown in Scheme 1 and Supporting Information Scheme S1 and S2, BSO–DOX was synthesized readily via a five-step synthetic route commencing from our previously reported compound 1.49 Compound 3 with H2S-responsive sulfoxide function was afforded through the aromatic nucleophilic substitution (SNAr) between monochlorinated boron-dipyrromethene BODIPY 1 fluorescent dye and 4-mercaptobenzyl alcohol, followed by oxidation of the corresponding compound 2 using meta-chloroperoxybenzoic acid (m-CPBA). Then compound 3 and 1,2-dimethyl-1H-imidazol-5(4H)-one were combined under a Knoevenagel condensation reaction to generate compound 4 in anhydrous EtOH, which underwent carbonation with 4-nitrophenyl chloroformate to provide compound 5 in high yield. Subsequently, the target prodrug BSO–DOX was obtained from a reaction between compound 5 and DOX. Scheme 1 | Synthetic route for theranostic prodrug BSO–DOX. Download figure Download PowerPoint H2S-Mediated cleavage of sulfoxide linker to liberate BSH and DOX We investigated H2S-induced activation of the prodrug, BSO–DOX, by confirming its optical properties in the absence and presence of H2S using phosphate-buffered saline/acetonitrile solution (CH3CN/PBS, 1∶1, v/v, 20 mM pH 7.4) at room temperature by real-time spectrometry. The free BSO–DOX exhibited two strong optical absorption bands at 384 and 496 nm in the UV–visible optical absorption spectra (Varian Cary 100, America). An emission band at 590 nm was observed when the excitation wavelength was 465 nm. Exposure to NaHS (100 μM) led to the generation of a new absorption band at ∼ 667 nm and attenuation of the original 496 nm band (Figure 2a), exhibiting a significant redshift of 171 nm. In the fluorescence emission spectra (Varian Cary Eclipse, America), the treatment of BSO–DOX with sodium hydrosulfide (NaHS) activated a significant NIR fluorescence at 713 nm (Figure 2b), which was intensified by ∼ 1000-fold upon excitation at 600 nm. This fluorescence intensity enhancement exhibited a good linear correlation with increasing NaHS concentration (0–20 μM), affording a sensitive detection limit of 19 nM ( Supporting Information Figure S1). These optical changes were attributable to the H2S-mediated cleavage of sulfoxide linker to liberate BSH and Thio-DOX,40 which subsequently underwent 1,6-elimination reaction to release DOX (Figure 2c). The translation process was demonstrated by high-resolution mass spectrometry (HRMS) analyses. As shown in Supporting Information Figure S2, HRMS spectrum of BSO–DOX solution in the presence of NaSH afforded peaks of 544.1910 (corresponding to [DOX + H]+) and 479.2655 (corresponding to [BSH + H]+). As such, the activated fluorescence signal was an indication of DOX release from the prodrug BOD-DOX. Figure 2 | Time-dependent spectra. UV–vis optical absorption (a) and fluorescence emission (b) changes of BSO–DOX (10 μM) upon activation by NaHS (100 μM) in buffer solution (CH3CN/PBS, 1∶1, v/v, 20 mM, pH 7.4, room temperature). (c) H2 S-Mediated cleavage of sulfoxide linker and the subsequent 1,6-elimination reaction to liberate BSH and drug DOX. Download figure Download PowerPoint H2S-Activatable and NIR-light-controllable synergistic activation of FTNpd in PBS After we established that BSO–DOX is indeed capable of releasing NIR imaging fluorophore and DOX through H2S activation, we fabricated a smart theranostic nanoprodrug, FTNpd, and tested if the coencapsulation of BSO–DOX and Aza-BOD into the interior of the light-responsive PCM matrix was achieved by the nanoprodrug to enable precision cancer treatment through cancer-stimuli-specific and NIR-light-controllable synergistic action. To produce FTNpd, PCM containers, composed of lauric acid, DSPE-mPEG-2000, and lecithin were prepared using a nanoprecipitation technique.32 Afterward, BSO–DOX and Aza-BOD at a molecular ratio of 1∶1 were entrapped within the core. Aza-BOD was used as a photothermal transducer because it possessed a strong absorption at 730 nm, which guaranteed a highly efficient light-to-heat energy conversion under NIR laser irradiation.50 Transmission electron microscopy (TEM) images showed a uniformly distributed FTNpd with a diameter of 60 nm ( Supporting Information Figure S3), while the hydrodynamic diameter was 68 nm, revealed by dynamic light scattering (DLS) experiments. Besides, the hydrodynamic diameter of FTNpd remained at ∼ 68 nm in aqueous solution, and no apparent aggregation was observed for 7 days ( Supporting Information Figure S3), indicative of high colloidal stability. Then free access of H2S to BSO–DOX, locked in the solid matrix of FTNpd, was investigated for efficient activation. As expected, FTNpd showed a dramatic optical response to H2S in PBS buffer solutions. Upon the addition of NaHS (100 μM), a new absorption at 686 nm was elicited, accompanied by an absorption reduction at 507 nm (Figure 3a). Such treatment with NaHS, once again, induced a 179 nm redshift. This observation was in good accordance with the absorption changes of free BSO–DOX treated with NaHS in CH3CN/PBS, which demonstrated that H2S was able to diffuse freely into the interior of FTNpd for efficient reaction with BSO–DOX. Specifically, the addition of NaHS led to a robust NIR fluorescence enhancement at 720 nm when the excitation wavelength was set at 600 nm (Figure 3b), making FTNpd preferable for visualization of H2S in vivo, as NIR light possessed good spatial resolution and deep tissue penetration.51 Like BSO–DOX in CH3CN/PBS, the time-dependent fluorescence enhancement exhibited a direct and good correlation with the liberation of DOX into the PCM matrix. Favorably, negligible changes in fluorescence were observed with biologically interfering analytes ( Supporting Information Figure S4), including reactive sulfur (RSS) and oxygen (ROS). Thus, suggesting that the inclusion of BSO–DOX in FTNpd unambiguously afforded highly selective responsiveness to H2S. Of note, FTNpd was found to be H2S-activatable within a physiological decreasing pH range from 9 to 5 ( Supporting Information Figure S5). Figure 3 | Time-dependent optical UV–vis absorption spectra (a) and NIR fluorescence (b) changes of FTNpd (20 μM BSO–DOX) upon activation by NaHS (100 μM) in PBS buffer. (c) Temperature change profiles of FTNpd (20 μM BSO–DOX) upon continuous exposure to 785 nm laser irradiation for 10 min. (d) Cumulative DOX release curves from FTNpd after various treatments. 785 nm laser with a power density of 1.57 W/cm2. Download figure Download PowerPoint As the thermoresponsive gating material, lauric acid has a well-defined melting point at 44 °C; hence, FTNpd must exhibit prominent photothermal effect in response to NIR irradiation to increase the temperature beyond 44 °C for the release of the diagnostic cargoes encapsulated in the nanoprodrug. Accordingly, we examined the capability of Aza-BOD in FTNpd for converting photoenergy into heat in an effective way. Under continuous exposure to laser irradiation at 785 nm, the temperature of FTNpd increased gradually (Figure 3c). For example, after 10 min irradiation at 4 W/cm2, the maximum photothermal temperature of FTNpd approached ∼ 73 °C, which was much higher than the melting point of lauric acid, and therefore, facilitated the drug release. Repeated heating and cooling operation indicated that no changes were noted in the maximal temperature for at least three cycles ( Supporting Information Figure S6), suggesting good photothermal stability of FTNpd. The photothermal conversion efficiency when exposed to a 785 nm laser at the power density of 1.57 W/cm2 was calculated to be 34.6%. The combined high photoenergy to heat conversion efficiency and the outstanding photothermal stability suggested that FTNpd indeed was potent for photothermal therapy (PTT), and the melting of the PCM matrix triggered the release of the cytotoxic payloads to ensure chemotherapy of targeted cancer cells. Next, we investigated the H2S and NIR-light synergetic control over the DOX release from FTNpd. As shown in Figure 3d, continuous irradiation of FTNpd with NIR laser in the presence of H2S afforded a gradual release of DOX. In contrast, treatments with H2S or laser irradiation alone resulted in negligible detection of drug release from FTNpd. Thus, these results verified that the efficiency in the release of the active DOX required H2S activation of the prodrug, BSO–DOX, along with NIR laser irradiation. Such an H2S-activatable and NIR-light-controllable synergistic drive of the therapeutic action of FTNpd represent a platform for an exclusive precision cancer treatment at the desired tumor location and time. Intracellular DOX release from FTNpd controlled by H2S and NIR irradiation Since BSO–DOX in FTNpd showed H2S-dependent NIR fluorescence light up, the capability of FTNpd for fluorescent trapping of cellular H2S was studied. As shown in Supporting Information Figure S7, FTNpd-stained CRC HCT116 cells (H2S-rich cancer cells) showed time-dependent enhancement of red fluorescent signals, while the treatment was prolonged in H2S-deficient, human liver carcinoma HepG2 cells, showing lack of red fluorescent signals. These imaging results demonstrated clearly that FTNpd could be used in the identification of H2S-rich cancer cells via tracking of high levels of endogenous H2S. Cellular release of DOX from BSO–DOX and FTNpd in H2S-rich CRC HCT116 cells with or without NIR laser irradiation were analyzed using confocal laser scanning microscopy (CLSM). As shown in Figure 4a, a gradual enhancement of red fluorescence in the nuclei was observed upon prolonged treatment of CRC HCT116 cells with BSO–DOX, indicating that the intracellular H2S indeed activated BSO–DOX to release DOX, which overlapped well with the blue fluorescence of the nuclei tracker Hoechst dye.52 In the case of FTNpd, DOX release was only initiated when NIR irradiation and H2S coexisted. In the absence of NIR irradiation, the red fluorescence was apparent in the cytoplasm rather than the nuclei, despite the pretreatment of CRC HCT116 cells with FTNpd at various time intervals (Figure 4b), indicative of profuse inclusion of DOX within the FTNpd core. In marked contrast, FTNpd-pretreated cells showed a prominent generation of red fluorescence in the nuclei when subjected to a 785 nm NIR laser with a power density of 1.57 W/cm2 for 10 min (Figure 4c), validating that Aza-BOD converted the photoenergy in FTNpd into heat to induce the solid–liquid transition of the thermosensitive nano prodrug to controllable drug release. Notably, the fluorescence signal intensity in the nuclei correlated well with the preincubation time, which aroused from the H2S-promoted efficient breakage of the sulfoxide linkage to generate an elevated level of DOX, released through NIR irradiation. These results confirmed further the intracellular DOX release from FTNpd under precise control by cellular H2S, along with NIR irradiation. Also, we demonstrated the H2S-activatable and NIR-light-controllable synergistic activation of FTNpd by performing control experiments with the H2S-deficent HepG2 cells. As shown in Supporting Information Figure S8, in BSO–DOX or FTNpd-treated HepG2 cells, no active DOX was released to the nuclei, as shown in the images, regardless of whether HepG2 cells were irradiated with NIR light or not. Additionally, analysis by flow cytometry demonstrated the H2S and NIR-light synergetic control over DOX release from FTNpd in cells ( Supporting Information Figure S9). These results, undoubtedly, revealed the essential role of H2S in triggering the liberation of DOX from BSO–DOX. Figure 4 | (a) Representative confocal fluorescence images of CRC HCT116 cells after treatment with BSO–DOX (10 μM) for 5, 15, 30, and 60 min (red channel). (b) Images of CRC HCT116 cells after treatment with FTNpd (20 μM BSO–DOX) for 15, 30, and 60 min (red channel). No NIR-light irradiation was applied. (c) Images of CRC HCT116 cells stained with FTNpd for 15, 30, and 60 min (red channel), followed by NIR irradiation for 10 min. The laser power was 1.57 W/cm2. Scale bars: 20 μm. The nuclei red fluorescence was generated by free DOX, while the blue cellular nuclei were stained by Hoechst 33342 (blue channel). Download figure Download PowerPoint Given that FTNpd contained coloaded therapeutic agents, we evaluated the capability of FTNpd for chemo-PTT of cancer cells under laser irradiation. We performed cell proliferation, MTT, assay in CRC HCT116 cells with FTNpd in the absence or presence of light irradiation to investigate the performance specificity of our synergistic nanoprodrug system further. Our results demonstrated that in the absence of light irradiation, FTNpd exhibited negligible cytotoxicity to CRC HCT116 cells even at a concentration as high as 40 μM of BSO–DOX. In contrast, the cell viabilities were found to decrease with increased concentrations of FTNpd along with laser irradiation. For example, the CRC HCT116 cell viability of FTNpd-loaded cells (BSO–DOX 40 μM) was attenuated at ∼ 10% upon 785 nm laser irradiation for 10 min ( Supporting Information Figure S10). These results suggested the high therapeutic efficacy of FTNpd in the presence of light irradiation. In the case of H2S-deficent HepG2 cells, even though there was no release of active DOX for chemotherapy, the efficiency of the PTT guaranteed high phototoxicity of FTNpd toward HepG2 cells ( Supporting Information Figure S11). In vivo therapeutic application of FTNpd We evaluated the in vivo biodistribution of FTNpd after intravenous injection into CRC HCT116 tumor-bearing mice, followed by in vivo and ex vivo fluorescence imaging. We observed bright fluorescent signals in the hypogastrium 1 h postinjection of the probe, while no detectable fluorescence was noted yet in the tumor ( Supporting Information Figure S12a). Additionally, ex vivo fluorescence imaging showed an efficient accumulation of FTNpd in the liver and negligible localization in the tumor, as well as other major organs ( Supporting Information Figure S12b). Notably, the ex vivo fluorescence imaging at 3 and 12 h post-FTNpd treatment displayed barely detectable fluorescence in the liver, which indicated a rapid elimination rate of FTNpd from living mice. To identify the right time point to implement NIR irradiation of FTNpd for chemo-PTT in vivo, H2S-rich and H2S-deficient tumor-bearing mice models were constructed and administrated with FTNpd by intratumoral injection. Subsequently, NIR fluorescence images were recorded, as described above, at various time intervals. As shown in Figure 5a, bright NIR fluorescence signals in the CRC HCT116 tumor gradually increased and achieved a maximal intensity at 2 h postinjection due to the efficient activation in the CRC HCT116. The fluorescence signal at 2 h showed a 6.1-fold enhancement, compared with the initial value at 15 min. In contrast, with the robust fluorescence enhancement observed in CRC HCT116 tumor-bearing mice, there were no apparent time-dependent fluorescence changes in HepG2 tumor-bearing mouse model, when images were recorded at parallel various time intervals after the injection. The relatively weak NIR fluorescence that appeared in the H2S-deficient tumor might be attributable to the always-on Aza-BOD trapped in FTNpd. Thus, our results demonstrated that H2S-activated NIR imaging provided the optimal temporal and spatial point for therapeutic administration. As the NIR imaging showed that the CRC HCT116 tumors had the highest fluorescence intensities at 2 h postinjection, 785 nm NIR laser irradiation was conducted at this time point for the observation of an in vivo cancer therapy. As presented in Figure 5b, the tumor temperature of the mice treated with FTNpd rapidly increased to approximately 74.3 °C under continuous irradiation for 10 min with a laser of 1.57 W/cm2, which was 18.9 °C higher than that of FTNpd-treated mice when exposed to a 785 nm NIR laser of 0.98 W/cm2 (Figure 5c). By contrast, the tumors from mice without FTNpd exhibited only a slight temperature elevation, reaching 36.2 °C after 10 min of NIR-light exposure (Figure 5d). These results suggested that FTNpd is an outstanding agent for tumor PTT in vivo. Notably, upon NIR-light (1.57 W/cm2) exposure for merely 1.5 min, the tumor temperature was significantly higher than the melting point of lauric acid, which facilitated the conformational changes of the PCM matrix for the liberation of DOX. These results indicated that FTNpd also represents a good candidate for combined photothermal therapy and chemotherapy of cancers. Figure 5 | (a) NIR fluorescence imaging of tumor-bearing mice at various times after the intratumoral injection of FTNpd (100 nmol BSO–DOX). The red dashed circles indicated the tumor location. (b–d) Infrared thermal images of CRC HCT116 tumor-bearing mice upon exposure to laser irradiation for different times: (b) FTNpd-treated mice, showing the tumor regions after 785 nm laser of 1.57 W/cm2 exposure; (c) FTNpd-treated mice showing the tumor regions after 785 nm laser of 0.98 W/cm2 exposure; (d) no FTNpd administration. Download figure Download PowerPoint Then we evaluated the in vivo therapeutic outcome by monitoring the tumor volumes after treatment of 5 mice groups (n = 4) with the following: Group 1: PBS; Group 2: 785 nm laser irradiation only; Group 3: FTNpd only; Group 4: Aza-BOD plus NIR light; Group 5: FTNpd plus NIR light, for 15 days. As outlined in Figure 6a and b, negligible tumor inhibition was obtained in mice treated with 785 nm laser irradiation alone, indicating that NIR irradiation had no antitumor efficacy. Also, no significant tumor growth suppression was observed after FTNpd treatment without NIR-light exposure, suggesting that FTNpd alone has very weak antitumor activity. We found that the treatment of Aza-BOD plus NIR light (0.98 W/cm2) showed apparent suppression of the tumor growth due to the PTT effect. Importantly, the tumor growth of mice group treated with both FTNpd, followed by laser irradiation (0.98 W/cm2), exhibited substantial suppression, indicative of the synergetic PTT and chemotherapy performance enabling amplification of the therapeutic efficacy. The corresponding intuitive photographs provided insight into the excellent antitumor effectiveness. Among all of the five treatment groups, the mice treated with FTNpd and NIR laser irradiation scored the smallest tumor size measurement, further confirming the synergistic effect of chemo-PTT on inhibition of tumor growth. Notably, in the tumor mice groups treated with FTNpd or Aza-BOD, the tumors could be suppressed entirely after exposure to a 785 nm NIR laser with a higher power density of 1.57 W/cm2 ( Supporting Information Figure S13), indicative of the potent PTT effect of the photothermal transducer. Finally, hematoxylin and eosin (H&E) staining experiments using the tumor tissue sections obtained from