H ₂ S-Activated “One-Key Triple-Lock” Bis-Metal Coordination Network for Visualizing Precise Therapy of Colon Cancer

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2021H2S-Activated “One-Key Triple-Lock” Bis-Metal Coordination Network for Visualizing Precise Therapy of Colon Cancer Cheng Zhang, Jiaqi Li, Chang Lu, Tengxiang Yang, Yan Zhao, Lili Teng, Yue Yang, Guosheng Song and Xiao-Bing Zhang Cheng Zhang State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Jiaqi Li State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Chang Lu State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Tengxiang Yang State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Yan Zhao State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Lili Teng State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Yue Yang State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author , Guosheng Song *Corresponding author: E-mail Address: [email protected] State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author and Xiao-Bing Zhang State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000369 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Colon cancer is the third most common malignancy and the fourth most prevalent cause of death worldwide. Unfortunately, current cancer treatment approaches suffer from low specificity toward colon cancers and lack of facile imaging method to monitor a real-time therapeutic process, usually resulting in severe toxicity to normal tissues. Thus, to achieve precise therapy for colon cancer, we developed a “one-key triple-lock” bis-metal ultrathin coordination network (Gd/Cu-nanosheets) as the first H2S-unlocked molecular dipoles with activatable fluorescence, magnetic resonance imaging (MRI), and photodynamic effect. Notably, within noncancerous tissues, Cu ions can lock up MRI signal, fluorescent emission, and 1O2 generation of molecular dipoles (Gd-porphyrins). Under the trigger of endogenous H2S overexpressed colon cancer model (HCT116), the loss photodynamic effect of the as-disassembled Gd-porphyrin was recovered for efficient and potent killing of colon cancer cells in vivo, with minimization of toxic side effect toward normal tissue. By combining the complementary advantages of bimodal MRI/fluorescence imaging, Gd/Cu-nanosheets enabled high-specificity visualization of colon cancer in living cells and in vivo upon activation of H2S. Moreover, a good correlation between the fluorescence intensity of Gd/Cu-nanosheets and cancer inhibition rates afforded the ability to self-report cancer therapeutic outcomes, thereby providing a potentially powerful tool for personalized and precise treatment of colon cancer. Download figure Download PowerPoint Introduction Colon cancer is ranked the third most common malignancy globally, with an estimated 600,000 colon cancer-related deaths every year.1,2 It is estimated that there were 1.4 million new cases of CRC, accounting for 9.9 % of the global cancer burden. Meanwhile, 693,900 deaths occurred, making it the fourth most common cause of cancer-related mortality.1–4 At present, surgical procedures are routinely used in clinical colon cancer treatment, often combined with radiation therapy or chemotherapy.5,6 However, such treatment approaches suffer the lack of specificity toward tumors, usually resulting in severe damage to normal body tissues, including generalized toxicity and pain.7,8 Thus, it is desirable to develop a highly specific therapeutic strategy for colon cancer. In recent preclinical research, the emerging photothermal or photodynamic therapies were promoted for inhibition of colon cancer, either through hyperthermia- or reactive oxygen species (ROS)-induced light irradiation.3,9,10 However, photothermal or photosensitizing agents administered with high-powered laser irradiation may cause burns or severe damage to normal tissues.11,12 Although these theranostic treatments can be combined with imaging technologies, and such imaging could be employed for only the visualization of tumor location instead of reporting therapeutic response as well.13,14 To personalize cancer treatment and minimize the side effects of overdose agents, the therapeutic process (e.g., exploiting ROS production) should be monitored in real-time by noninvasive imaging technology, to achieve optimal administration of drug doses, irradiation time, or laser power.11,15 Owing to the high expression of the H2S-generating enzyme cystathionine-β-synthase (CBS) in colon cancer typically, the level of H2S in colon cancer tissues (0.3–3.4 mmol L−1) far exceeds that of the surrounding healthy tissues.16,17 In recent years, based on the high expression of H2S, several kinds of organic nanoprobes with H2S-activatable near-infrared (NIR)-I or NIR-II fluorescence emission have been applied for specific imaging in colon cancer.9,18–26 In addition, H2S-activated theranostic platforms have been used for imaging-guided photothermal therapy, chemo-photothermal therapy, and photodynamic therapy (PDT).27–29 Interestingly, photoacoustic imaging is generally well-received due to its high resolution and good tissue penetration.30 Via “turn-on” photoacoustic effect, the in situ sulfidations of Cu2O or [email protected]2O nanoparticles by endogenous H2S were further employed to visualize colon cancer.4,31 Dual-modality optical/photoacoustic probes have also been developed for ratiometric imaging of stimulated H2S upregulation in mice.32 However, these imaging technologies still have limitations for whole-body imaging of in vivo tumors owing to poor tissue penetration of the excitation laser for fluorescence or photoacoustic imaging, even for the emerging NIR-II fluorescence.33 By contrast, magnetic resonance imaging (MRI) possesses unlimited tissue penetration depth and high spatial resolution by detecting the relaxation signals of magnetic contrasts, suitable for detecting deep-seated malignant tumors before surgery.34,35 However, most MR contrast agents were “always on” signals, whether interacting with tumor biomarkers or not. This likely brings about a low signal-to-background ratio, making it difficult to highlight the biological phenomenon of interest.36,37 Our group has pioneered several activatable MnO2-based MRI probes responsive toward reduced glutathione (GSH) or acidity for cancer theranostics.38–40 However, to the best of our knowledge, H2S-responsive MRI probes are rare. Moreover, the unknown local concentration, low sensitivity, and limited dynamic range are also fundamental challenges for most responsive MRI probes.41–44 Considering fluorescence imaging with high sensitivity and real-time detection, the combination of MRI and fluorescence imaging can often offset the shortcomings of each imaging modality used individually to provide dual-modality tool for synergistic diagnosis of malignant tumors.33,41 Although several examples of bimodal MRI/fluorescent probes have been reported, many of them exhibited “always-on” signals, instead of a specific response toward tumor cells.36,37,45,46 This calls for the development of activatable MRI/fluorescent bimodal probes able to produce highly specific responses to H2S for precise therapy for colon cancer. Herein, we developed a bis-metal Gd, Cu two-dimensional coordination network with ultrathin nanostructure (Gd/Cu-nanosheets) for precise PDT and activatable MRI/fluorescence imaging of colon cancer (Scheme 1). Importantly, the introduction of Cu ions could lock up the rotation of molecular dipoles in Gd-porphyrins and the energy of the excited porphyrin. Thereby, our fabricated Gd/Cu-nanosheets exhibited a darker contrast of T2-MRI and lower fluorescence as well as photodynamic effects. In contrast, triggered by H2S, the as-disassembled Gd-porphyrins possessed brighter contrast of T2-MRI, higher fluorescent emission (710 nm), and stronger 1O2 generation. Upon the activation of endogenous H2S secreted by colon cancer, Gd/Cu-nanosheets could also be employed for high-specificity MRI/fluorescence imaging of colon cancer, enabling a high-efficiency PDT of this tumor in vivo, which could be monitored by fluorescence imaging in real-time based on a good correlation between fluorescent emission and 1O2 output. Scheme 1 | Schematic illustration of Gd/Cu-nanosheets for H2S-activatable MRI/fluorescence imaging and high-specificity colon cancer theranostics. MRI, magnetic resonance imaging. Download figure Download PowerPoint Experimental Section Synthesis of Gd/Cu-nanosheets To prepare 5,10,15,20-Tetrakis(4-methoxycarbonylphenyl)porphyrin, 3.5 g of methyl p-formylbenzoate (0.021 mol) into 50 mL of propionic acid and stirring at 140 °C. 2 mL of distilled pyrrole (29 mmol) was added dropwise into the solution mixture. Then the solution was refluxed for 2 h in the dark before cooling down. The crude product was filtrated and purified via column chromatography (petroleum ether/dichloromethane = 1:1, v/ v) to obtain a purple crystalline solid. Proton nuclear magnetic resonance (1H NMR) (400 MHz, Chloroform-d, δ): 8.83 (s, 8H), 8.45 (d, J = 6.2 Hz, 8H), 8.30 (d, J = 6.3 Hz, 8H), 4.12 (s, 12H), −2.81 (s, 2H). Gd-porphyrin was synthesized by adding 20 mg of Tetrakis(4-methoxycarbonylphenyl)porphyrin, 6 g of imidazole, and 100 mg gadolinium chloride hexahydrate into a flask and heating at 180 °C for 2 h under stirring at N2 atmosphere before cooling down. The product was collected via centrifugation (10000 rpm for 10 min) and washed with water, followed by dispersion into 10 mL of a MeOH/tetrahydrofuran (THF) (1∶1) solvent mixture. Next, 5 mL of KOH solution (50 mg·mL−1) was added to the mixture solution and refluxed for 12 h in the dark, followed by evaporation. Subsequently, the crude product was added with 100 mL of water and stirred at 75 °C for 0.5 h. Then the supernatant was collected by centrifugation. Finally, Gd-porphyrin was acidulated to pH 5–6, collected via centrifugation, washed with water, and lyophilized. To prepare Gd/Cu-nanosheets, 200 μL of dimethylformamide (DMF) solution containing Gd-porphyrin (3 mg) was added dropwise into 10 mL of n-octane solution containing sodium bis-(2-ethylhexyl) sulfosuccinate (10 g), followed by stirring at room temperature for 30 min. Then, 200 μL of DMF containing Cu(NO3)2 3H2O (12 mg) and acetic acid (30 μL) was added dropwise to the above solution. After 2 h of stirring, the mixture was transferred into a 50 mL Teflon-lined autoclave and heated at 120 °C for 4 h. After cooling down, the as-prepared Gd/Cu-nanoparticles were collected through centrifugation and washed with ethanol and water. Furthermore, the Gd/Cu-nanoparticles’ solution was sonicated for exfoliation by cell pulverizer for 2 h. Afterward, the solution was centrifuged at 3000 rpm for 5 min, and then the supernatant containing Gd/Cu-nanosheets was collected. Measurement of activatable MRI, fluorescence, and 1O2 generation in solution We measured the longitudinal relaxation rate (r1) and transverse relaxation rate (r2) by incubating varying concentrations of Gd/Cu-nanosheets (0.04, 0.08, 0.16, and 0.32 mM of Gd) with 2 mM of H2S at 37 °C for 10 min, or a negative control setup without H2S. In turn, we measured 1/T2 by incubating Gd/Cu-nanosheets (containing 0.1 mM of Gd and 10% dimethyl sulfoxide [DMSO]) with varying concentrations of H2S at 37 °C for 10 min. To dynamically measure 1/T2, Gd/Cu-nanosheets (0.1 mM of Gd, 10% DMSO) were incubated with 2 mM of H2S at different times. T1 and T2 relaxation times were recorded on a Bruker Minispec analyzer (1.5 T). T2-weighted MRI were acquired with a 7T Biospec MRI scanner (Bruker, Beijing, China). To measure fluorescence, Gd/Cu-nanosheets (optical density at 420 nm = 2.4 [OD420 = 2.4]) were incubated with varying concentrations of H2S (0–200 μM) at 37 °C for 1 h. We tested the selectivity of the reaction by following the experimental procedures described previously.9 Specifically, stock solutions of various chemical species (e.g., NaSH, GSH, Na2SO3, Na2SO4, KSCN, Na2S2O5, Na2SO4, NaClO, H2O2, NaHSO3, cysteine (Cys), and Na2S2O3) were prepared by directly dissolving the corresponding compounds into phosphate buffered saline (PBS). Subsequently, the various species were incubated with Gd/Cu-nanosheets for 1 h at the following concentrations: (1) NaHS (200 μM), (2) GSH (10 mM), (3) SO32− (1 mM), (4) SO42− (1 mM), (5) SCN− (1 mM), (6) S2O52− (1 mM), (7) S2O42− (1 mM), (8) HPO42− (1 mM), (9) H2PO42− (1 mM), (10) HCO3− (1 mM), (11) ClO− (200 μM), (12) H2O2 (200 μM), (13) HSO3− (1 mM), (14) Cys (100 μM), and (15) S2O32− (1 mM). Next, the fluorescence spectra were measured (λex = 650 nm and λem = 680–780 nm). To measure 1O2, the Gd/Cu-nanosheets were incubated with varying concentrations of H2S (0–200 μM) for 1 h, followed by the addition of singlet oxygen sensor green (SOSG; 4 μM). Then the mixture solution was irradiated with 660 nm of laser (9 min, 1.1 W·cm−2). Subsequently, the fluorescence spectra were measured (λex = 494 nm and λem = 510–630 nm). Activatable fluorescence/MRI imaging in living cells Human colon adenocarcinoma cells (HCT116 cells) and mouse breast cancer 4T1 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin−streptomycin) in a humidified incubator containing 5% CO2 at 37 °C. For confocal imaging of H2S, HCT116 cells were preseeded in an optical culture dish and received the following treatments per dish: (1) incubation with 500 μM of aminooxyacetic acid (AOAA) (CBS inhibitor) for 1 h, (2) no treatment, and (3) incubation with 500 μM of Cys for 1 h. Then the treated HCT116 cells were washed three times with Dulbecco’s phosphate- buffered saline (DPBS) and further incubated with Gd/Cu-nanosheets (OD420 = 2.4) for another 4 h at 37 °C. The 4T1 cells were preseeded in an optical culture dish, incubated with Gd/Cu-nanosheets (OD420 = 2.4), and varying concentrations of H2S (0–200 μM) were added to the dishes for 4 h at 37 °C, followed by three times washes with DPBS, and subsequent confocal fluorescence imaging (λex = 640 nm and λem = 663–738 nm). For fluorescence imaging of H2S using the In Vivo Imaging System (IVIS), HCT116 or 4T1 cells preseeded in a black 96-well plate, which received the above treatments. After incubation with Gd/Cu-nanosheets, the IVIS imaging of HCT116 or 4T1 cells were recorded under fluorescence mode with Cy5.5 filter at an acquisition time of 2 s. For confocal imaging of intracellular 1O2, 4T1 cells preseeded in an optical culture dish were incubated with Gd/Cu-nanosheets (OD420 = 2.4) in the presence of varying concentrations of H2S, for 4 h, and then incubated with SOSG fluorescent probe reagent for 0.5 h, with subsequent laser irradiation at 660 nm for 12 min. Next, the treated cells were washed three times with DPBS, followed by confocal fluorescence imaging (λex = 488 nm and λem = 500–550 nm). For T2-MRI of cancer cell pellets, HCT116 or 4T1 cells were incubated with 200 µL of DMEM solution containing Gd/Cu-nanosheets (0.57 mM of Gd) at 37 °C for 24 h. Next, the cells were digested with trypsin and collected by centrifugation into cell pellets for MRI using a 7T Biospec MRI scanner (Bruker). Activatable PDT in living cells For live/dead cell-staining assay, 4T1 cells were seeded in an optical culture dish and incubated with Gd/Cu-nanosheets (OD420 = 2.4) or Gd/Cu-nanosheets (OD420 = 2.4) + H2S (200 μM) for 4 h, followed by irradiation with 660 nm laser (12 min, 1.1 W·cm−2), or medium only control. Subsequently, the cells were incubated with Calcein-AM (2 μmol·L−1) and propidium (PI) (4.5 μmol·L−1) for 30 min at 37 °C and then scanned by confocal microscopy (green channel: λex = 488 nm and λem = 500–550 nm; red channel: λex = 543 nm and λem = 580–650 nm). (Calcein AM and PI) (Cell Viability/ Cytotoxicity Detec) kit was used for the live/dead cell staining assay, which was obtained from the Beyotime (Shanghai, China). For activatable PDT of HCT116 cells, the HCT116 cells were seeded in a 96-well plate and treated with (1) PBS, (2) PBS + laser (12 min, 1.1 W·cm−2), (3) Gd/Cu-nanosheets (OD420 = 2.4), and (4) Gd/Cu-nanosheets (OD420 = 2.4) + laser. After incubation with Gd/Cu-nanosheets for 4 h, the cells from (4) were irradiated with 660 nm laser (12 min, 1.1 W·cm−2). After 12 h of culture, the relative cell viability was determined by the methyl thiazolyl tetrazolium (MTT) assay. For activatable PDT of 4T1 cells, 4T1 cells preseeded in a 96-well plate were incubated with Gd/Cu-nanosheets (OD420 = 2.4) in the presence of varying concentrations of H2S for 4 h at 37 °C. Then the cells received laser irradiation (660 nm, 12 min, 1.1 W·cm−2), or not, and further cultured for 12 h. Subsequently, the relative cell viability was evaluated by the MTT assay. Activatable MRI/fluorescence imaging in vivo All animal operations were performed based on the principles of Laboratory Animal Care and the guidelines of the Institutional Animal Care and Use Committee of Hunan University. Female nude mice and female BALBc white mice (4 weeks) were bought from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, China). Nude mice were subcutaneously implanted with HCT116 cells (2 × 107 cells) to prepare the HCT116 mouse xenograft colon cancer model, with elevated tumor H2S marker levels. BALBc white mice received subcutaneous implantation of 4T1 cells (2 × 106 cells) for the preparation of 4T1 mouse xenograft breast cancer model with negligible tumor H2S levels (negative tumor model control). Next, mice MRI, the mice bearing HCT116 tumor after intratumoral (i.t.) injection with 50 µL of Gd/Cu-nanosheets (0.57 mM of Gd), and with the mice bearing 4T1 tumor received i.t. injection of 50 μL of a mixture of Gd/Cu-nanosheets (0.57 mM of Gd) + H2S (2 mM) or 50 μL of Gd/Cu-nanosheets (0.57 mM of Gd). Then, the mice were scanned at different time points using a 7T-MRI scanner (Bruker) with T2-MRI sequence (size = 256 × 256, field-of-view [FOV] = 3.4 cm × 2.8 cm, slice thickness = 1 mm, repetition time [TR] = 1700 ms, and effective echo time [TE] = 25 ms). For fluorescent imaging of living mice, mice bearing HCT116 tumors were i.t. injected with 50 μL of Gd/Cu-nanosheets (OD420 = 30), and mice bearing 4T1 tumors were i.t. injected with 50 μL of a mixture containing Gd/Cu-nanosheets (OD420 = 30) in the presence of varying concentrations of H2S (0–500 μM). Then fluorescence images were recorded at the indicated times by the IVIS imaging system under fluorescence mode with Cy5.5 filter and an acquisition time of 2 s. Activatable PDT of colon cancer in vivo To perform activatable PDT in mice, mice bearing HCT116 tumors or 4T1 tumors were i.t. injected with 50 μL of Gd/Cu-nanosheets (OD420 = 30) or without the nanosheets, and received 660 nm of laser irradiation (20 min, 1.1 W·cm−2) at 0.1 h postinjection. Then, the tumors were harvested for hematoxylin and eosin (H&E) staining at 24 h posttreatment. To explore the correlation between the fluorescence and anticancer activity, mice bearing the 4T1 tumor were treated as follows: (1) PBS, (2) Gd/Cu-nanosheets (OD420 = 30), or (3–7) Gd/Cu-nanosheets (OD420 = 30), in the presence of varying concentrations of H2S (e.g., 0, 62.5, 125, 250, and 500 μM) at a total i.t. injection volume of 50 μL and 660 nm laser irradiation (1.1 W·cm−2, 12 min) at 0.1 h postinjection. Subsequently, the mice body weights and tumor sizes were measured continuously for 14 days. The tumors from each group were collected on the first day (24 h), and the main organs (heart, liver, kidney, lung, and spleen) were harvested on the 14th day of treatment for H&E staining. Statistical analysis Statistical analysis (p value) was performed using two-tailed Student’s t-tests. Statistically significant differences were considered at a p value < 0.05. Excel (microsoft office, USA) was used for statistical analysis. Results Preparation and characterization of Gd/Cu-nanosheets Gd/Cu-nanosheets were prepared as follows: First, we synthesized tetrakis(4-methoxycarbonylphenyl)porphyrin, subsequently characterized by 1H NMR ( Supporting Information Figure S1). Second, Gd ions were inserted into the central of Finally, was with Cu ions through the group to Gd/Cu-nanoparticles via Gd/Cu-nanoparticles with a good ( Supporting Information Figure The as-prepared Gd/Cu-nanoparticles were characterized by with microscopy Gd and Cu were well with and a of Gd-porphyrin The of Gd/Cu-nanoparticles was ( Supporting Information Figure Figure 1 | Preparation and characterization of and of of of The of the from with the white spectra and fluorescence spectra for and Gd/Cu-nanosheets, Download figure Download PowerPoint To Gd/Cu-nanoparticles into a ( Supporting Information Figure Gd/Cu-nanoparticles were to probe for 2 h. Afterward, the Gd/Cu-nanosheets with a of nm light of Gd/Cu-nanosheets the to nm, with a of and of ( Supporting Information and Moreover, microscopy Gd/Cu-nanosheets with an ultrathin thickness of about nm and Next, we that Gd/Cu-nanosheets were able to in the serum for 24 h ( Supporting Information Figure We also the of Gd/Cu-nanosheets in solution and that of ions were from Gd/Cu-nanosheets after h the of Gd with ( Supporting Information Figure The spectra of Gd/Cu-nanosheets at and nm, instead of with the from the of Gd Notably, after Gd-porphyrin was into Gd/Cu-nanosheets by with Cu the fluorescence of Gd-porphyrin was owing to the effect of Cu ions analysis a loss of a temperature from to °C and a after °C, to the organic and generation of ( Supporting Information Figure Activatable MRI of Gd/Cu-nanosheets in solution Owing to the of H2S with Cu Cu ions were from the Gd/Cu-nanosheets via the of upon reaction with a Gd/Cu-nanosheets were into the of Gd/Cu-nanosheets was as by the microscopy the also after incubation with H2S ( Supporting Information Figure Next, we the MRI of Gd/Cu-nanosheets in response to H2S and to the of Gd Gd/Cu-nanosheets exhibited a and The and were to be and Notably, after incubation with H2S, and of the Gd/Cu-nanosheets were to and and We further the T2-weighted images of Gd/Cu-nanosheets using a 7T-MRI of the the of Gd/Cu-nanosheets darker with Moreover, T2-MRI images of Gd/Cu-nanosheets + H2S at concentrations in contrast to that incubated with no H2S. H2S concentrations from to the 1/T2 value continuously with brighter T2-MRI and Supporting Information Figure Next, the dynamic T2-MRI that 1/T2 were to a after 2 h incubation with H2S (2 mM) To the of H2S on T2-MRI signals, we measured the 1/T2 of different concentrations of H2S in DMSO) and that H2S effect on the 1/T2 value in PBS ( Supporting Information Figure The above indicated that MRI signals of Gd/Cu-nanosheets were responsive toward H2S. Figure 2 | Activatable MRI of Schematic of H2S-activatable of the Gd/Cu-nanosheets after incubation with H2S for 1 h. (r1) and transverse rates (r2) of Gd/Cu-nanosheets treated with 2 mM H2S or T2-weighted images of 1/T2 value of Gd/Cu-nanosheets incubated with various concentrations of H2S. is corresponding to T2-weighted 1/T2 value of Gd/Cu-nanosheets after incubation with H2S. MRI, magnetic resonance Download figure Download PowerPoint In this Gd-porphyrin could be as to the the is considered to a in T1 and T2 relaxation times. The of the is to the following of (1) the of (2) the between (3) the between and (4) the relative Thus, the 1/T2 was by the following 1 2 = 4 6 J ( + 5 J ( + 2 J ( T2 is the transverse relaxation time, is ratio, is the between is the is a density is the Thus, the is to the to the fourth which is to the to the and to the of the density in in 1). the three of molecular and the usually in the of which is to and the effect on the relaxation in a the in a range of that with the it may

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