- Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization | Chemical Reviews
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- NIR area array CCD-based singlet oxygen luminescence imaging for photodynamic therapy
The excitation and emission monochromator slits were both set to 2 nm, 3 nm, respectively. For the fluorescence emission spectra, the excitation wavelength for RhoSSCy was set to nm and nm, respectively. The cells were maintained in an exponential growth phase by periodic subcultivation. The cell density was determined using a hemocytometer, and this was performed prior to any experiments.
Illustration of synthesis and functions of RhoSSCy. By contrast, in the groups without biothiols, the cells were pre-treated with N-ethylmaleimide NEM, as a thiol-reactive reagent that can block cellular thiols for 30 min before being incubated with the probe.
Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization | Chemical Reviews
Rho channel and Cy channel were excited at nm and nm, respectively, and collected in the ranges of nm and nm, respectively. Following the incubation, the cells were washed three times with PBS, gently dissociated from the wells with trypsin, and then resuspended in PBS. Infrared channel APC-Cy7 was used with excitation at nm and collection in the ranges of nm. Briefly, 0. After shaking the plates for 10 min, absorbance values of the wells were read with a microplate reader at nm.
The cell viability of treated cells was then obtained by comparison with the incubated but non-exposed control. They were weeks old at the start of each experiment and weighed g. Tumors were then allowed to grow to cm in diameter for days. All animal operations were in according with institutional animal use and care regulations, approved by the Laboratory Animal Center of Guangdong. A band pass filter nm and a nm long pass filter were selected to be used as the excitation filter and the emission filter, respectively. For in vivo PA imaging, the mice were placed in supine position in the animal holder.
Cross-sectional multispectral optoacoustic image datasets were acquired through the tumor at single wavelengths in the NIR window nm using whole body multispectral optoacoustic tomography MSOT system iThera Medical, Inc. The raw signals were reconstructed and specific signals were spectrally resolved by the pseudo-inverse unmixing method as implemented in the ViewMSOT software.
For laser treatment groups, the tumors of mice were irradiated by the nm laser at mW cm -2 for 30 min, and the tumor volume or body weight of the mice was recorded. To further detect the PDT effect in vivo , tumors at 48 h after treatment were stained with hematoxylin and eosin. On 30 d, livers and kidneys which had a major accumulation of RhoSSCy were stained with hematoxylin and eosin for biosafety evaluation. The disulfide linker and pH tunable amino-group allowed probe respond to both thiols and pH in tumor microenvironments. It means the conjugation of Rho group with Cy group resulted in blue shift of absorption peak of Rho group at nm.
In addition, the protonation and lower pH resulted in a red shift of the absorption peak from nm to near infrared region Figure S4 B. One of them came from Rho group and the other came from Cy group. Optical properties of RhoSSCy. It was necessary to take into account the fluorescent intensity of the probe at nm at the same time. The calibration curves of fluorescence intensity at nm versus GSH concentration were total overlapped at different pH values as shown in Figure S5 D.
The fluorescence intensity at nm was directly proportional to the GSH concentration regardless of the variation of pH value. However, when other amino acids and some possible coexisting organics were added to the solution of probe, the changes of RhoSSCy fluorescence intensity were negligible Figure S5 E. RhoSSCy with the amino connected to Cy displayed 'switching on' of fluorescence upon protonation as the proton-induced suppression of photo-induced electron transfer PET led to fluorescence as the predominant deactivation pathway [ 57 - 59 ].
Most notably, the probe displayed an excellent stability and reversibility after 6 cycles between pH 5 and pH 9 Figure 2 F. Since the tumor microenvironment has low extracellular pH and at the same time our probe has good response to pH in NIR region [ 60 ], it is reasonable and feasible to use our probe to imaging tumor. E The linear calibration plot of fluorescent intensity and its corresponding pH value based on the flow cytometry analysis in D.
By contrast, the Cy fluorescence intensity at nm Cy channel declined obviously with pH increasing. So in the merge channel obtained based on above two channels, the cells without GSH at pH 6. The statistical results constructed according to these fluorescence images displayed a characteristic pH-dependent and thio-dependent signal change Figure 3 B and 3 C , implying that RhoSSCy had excellent pH and biothiols dual-stimuli response to living cells in vitro. Flow cytometric analysis is a reliable and convenient method for quantifying the cellular fluorescence intensity.
When the pH values were stepwisely reduced from 8. Plotting this fluorescence change against each pH unit, we observed a total of ten folds fluorescence enhancement in every pH signal Figure 3 E , which further confirmed that the fluorescent intensity of RhoSSCy at nm was highly correlated with pH value. In order to investigate the molecular targeting and accumulation ability of RhoSSCy in tumor cells, we compared the accumulation of RhoSSCy in tumorigenic and nontumorigenic cell models in vitro.
The confocal fluorescence imaging from Figure 4 A showed that the red fluorescence come from RhoSSCy was significantly higher in tumor cells A cells and MCF-7 cells than in normal cells T cells , which indicated that RhoSSCy high-selectively accumulated in tumor cells. The results that the cellular uptake of RhoSSCy was also significantly higher in tumor cells than normal cells were also confirmed by flow cytometry Figure 4 B. The mechanism of RhoSSCy for tumor targeting is not clear, it is possibly associated with glycolytic metabolic patterns, which are preferentially utilized by tumor cells for energy supply, or organic-anion transporting polypeptide OATP transporters, which are well-recognized to be over-expressed in a variety of tumor cells [ 30 , 61 ].
As shown in Figure 5 A and 5 B, the fluorescence signal firstly appeared in liver at 0. Thereafter, the signal at the tumor site began to drop, but was maintained even after 48 h postinjection, demonstrating that the tumor targeted delivery and long retention of RhoSSCy in tumor tissue. Cellular targeting and uptake of RhoSSCy. B Semiquantitative fluorescent intensity Fluo. D Semiquantitative biodistribution of RhoSSCy in tumor determined by the averaged fluorescent intensity. F Semiquantitative fluorescent intensity of dissected tumor and organs at indicated time-points.
As shown in Figure 5 E and 5 F, the most RhoSSCy were accumulated in the liver and tumor tissue, and the spleen and kidneys showed relative weak fluorescence, while the fluorescence in the other regions of the mouse body was weaker compared with the fluorescence of RhoSSCy in tumor tissue. These results clearly indicated the RhoSSCy molecules preferentially accumulated in the tumor, which was in agreement with the tumor cells targeting in vitro mentioned above.
After intravenous injection, fluorescence in some vital organs, such as liver, kidney, and spleen increased gradually. After 10 h, fluorescence in these organs decreased gradually Figure 5 A. The changes of the fluorescence signal indicated that RhoSSCy eliminated in hepatobiliary and renal excretion pathways from the animal body, which was similar with the metabolic pathway of other indocyanine dyes [ 62 ]. Photoacoustic imaging PA is a novel, hybrid, and noninvasive imaging modality that combines the merits of both optical and ultrasonic methods [ 63 , 64 ], and also can evaluate the drug delivery efficiency and therapeutic effects with a relatively high spatial resolution in deep tissue.
RhoSSCy has a strong NIR absorption band between nm allowing deeper tissue penetration without causing significant heating.
Thus it may be an ideal probe for PA imaging. We carried out in vivo PA imaging of nude mice bearing MCF-7 tumor injected with RhoSSCy by tail vein and measured the tumor signal before and various time points after injection. Figure 6 C showed the corresponding ex vivo cryoslice, which come from the software of whole body MSOT system and displayed the possible anatomic construction of the PA imaging in Figure 6 D. Figure 6 D and 6 E indicated that the PA signals in tumor tissue increased with time and distributed around with the tumor microvessels.
The results suggested that the RhoSSCy molecules were greatly accumulated at tumor. The time-dependent curve of PA intensity showed that the PA signal in tumor tissue was clearly visible at 4h and reached maximum near 15 h post injection, which was in high agreement with the in vivo NIRF imaging behavior. It was clear that PA imaging provided a high spatial resolution, which was prone to understand the biodistribution of RhoSSCy in tumor tissue and got the delivery information on tumor microstructure. PDT has emerged as an effective treatment for tumor with minimal nonspecific damage to adjacent healthy tissues.
It has been reported that some of the cyanine dyes have photosensitizing characteristics for cancer PDT [ 65 , 66 ]. For instance, indocyanine green ICG has been extensively investigated as a potential photosensitizer by many research groups [ 67 , 68 ]. A The linear relationship between PA intensity P. B The photo of nude mouse bearing MCF-7 tumor. White dash line indicated the position of PA imaging. D PA imaging of a tumor bearing nude mouse.
Pink circle indicated the location of tumor. E Semiquantitative fluorescent intensity of the tumor at indicated time points. Photodynamic therapy using RhoSSCy in vitro. B Fluorescent images of MCF-7 cells under the different conditions. Viable cells were stained green with calcein-AM. To study the photodynamic efficacy of RhoSSCy in vitro , the PDT-induced cell death was examined through measuring fluorescence of calcein-AM, which stained viable cells green and dead cells show no fluorescence.
As shown in Figure 7 B, most of the cells treated with RhoSSCy plus laser irradiation were colorless, indicating apparent cell death after 24 h, while the cells treated with RhoSSCy only and the cells treated with laser irradiation only were almost entirely alive. The dark cytotoxicity is presumably ascribed to the water-insolubility and specific chemical structure of RhoSSCy. RhoSSCy was intravenously injected into tumor-bearing mice when the tumor size grew to mm 3. Compared with other groups, the mice treated with RhoSSCy plus laser irradiation showed remarkable tumor growth suppression and valuable tumor regression after two weeks.
In contrast, RhoSSCy-treated mice without laser irradiation showed no apparent change of tumor size which indicated that the therapeutic effect was highly depended on light-triggering PDT. During the treatments, the body weight was monitored, which suggested the treatments-induced toxicity Figure 8 C. The body weight of these experimental groups was not significantly different from the control group, indicating that those treatments were reasonably well-tolerated. The results above clearly demonstrated that tumor-targeted photodynamic therapy of RhoSSCy was highly effective and safe in vivo.
B Survival rate of mice bearing MCF-7 tumors of disparate groups within 30 d after corresponding treatments. C Body weight change of mice under different conditions. Biothiols and acid-base equilibrium play critical roles in biological processes, so it is necessary to develop efficient methods to monitor biothiols and pH level in living cells. When two or more molecules need to be examined in a system at the same time, a general method is to use two different probes simultaneously. However, this strategy often suffers from uneven probes loading, nonhomogeneous distribution, uncontrollable localization, larger invasive effects, metabolisms, and spectral overlap interference between probes.
Compared with the single response probes, the dual-response probes which can selectively respond to dual-species should be more appropriate for visualizing variation of objects under test for biological events. To the best of our knowledge, a small-molecule probe that could display differential fluorescence OFF-ON dual-response to thiols and pH simultaneously has not been covered in the literature. Additionally, RhoSSCy probe had the ability to high-selectively accumulate into the tumor cells and tissue. Moreover, the solid tumor microenvironment is generally acidic and the fluorescence of RhoSSCy probe was enhanced in the near infrared region under the acidic condition.
So it was suitable for tumor fluorescence imaging. At the same time, RhoSSCy had a strong absorption band nm allowing deeper tissue penetration without causing significant heating and so it was also appropriate for PA imaging. Traditional imaging techniques are frequently limited by narrow penetration, low sensitivity, low specificity, and poor spatial resolution. After the laser-triggered PDT treatment, the tumor size was highly suppressed and the mice survival was greatly promoted.
The superiority of tumor targeting dual-modal imaging and PDT treatment was integrated into a small-molecule probe of RhoSSCy, which could provide the complementary information for accurate imaging-diagnosis, imaging-guided phototherapy and curative effect assessment of tumor. RhoSSCy probe was synthesized by conjugating Rho with Cy using a disulfide linker and a pH tunable amino-group, which resulted in ratiometric detection of free thiols and directly sensing intracellular change of pH and thiol concentration together.
Meanwhile, RhoSSCy molecules entered tumor cells easily and preferentially accumulated in the tumor. With great efforts in overcoming the drawbacks of NIR dyes and integrating fantastic theranostics, there is no doubt that this well-defined small-molecule probe RhoSSCy was valuable not only to multi-modality imaging application, but also to target-specific molecular diagnosis and phototherapy treatment of cancers.
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