A Simple and Effective "Elimination" Approach for Selective Cancer Therapy to Reveal the Role of H2O2.

Fluorescence imaging capability for visualizing tumor microenvironments can play a role in advancing drug discovery efforts, exploring therapeutic efficacy, and thus significantly improving the prognosis. Specifically, we present the design, synthesis, spectroscopic properties, and the targeting diagnosis of a single boronate-appended benzorhodol. Drug release from the prodrug LHX-B-CPT was detected by tackling the fluorescent signal after the addition of H2O2 to the target cancer cells. The prodrug entered the cells via endocytosis mechanisms due to the folate unit, highlighting that the final delivery locations can minutely influence drug efficacy. Thus, this theranostic system is a new therapeutic agent, based on the reasoning that the depletion of H2O2 would be readily detected at the subcellular level by the fluorescence changes.

Introduction

Currently, targeted drug delivery systems have become the method of choice in cancer treatments, especially in conjunction with surgery.[1−3] Small conjugate-based systems have been extensively investigated for cancer treatment regimens and diagnostics.[4−8] The ability to monitor drug release is used as a single property that can function for both therapy and diagnosis. Therefore, theranostics have made tremendous contributions to the assessment of chemotherapy and minimized adverse side effects on normal cells. Moreover, cancer cells have higher replication, apoptosis, angiogenesis, and proliferation rates, resulting in the excessive production of reactive oxygen species (ROS).[9−12]

As a typical ROS, hydrogen peroxide (H2O2) is an important marker for various physiological processes including oxidative stress, immune responses, and cellular signal transduction.[13−16] Indeed, at the cell organelle level, H2O2 serves beneficial and essential functions for cell survival, migration, differentiation, signaling pathways, and immune system function.[17] However, aberrant or excessive production of H2O2 is strongly associated with several human diseases including stroke, cancer, and neurodegeneration, as well as diabetes.[18−21] The intrinsic enhancement or suppression in the intracellular levels of H2O2 induces a malignant phenotype and cell malignant transformation of cancer cells. As noted above, in conjunction with achieving novel molecular agents for targeting, imaging, and therapy, great efforts have been devoted to the development of activatable fluorescent probes for personalized drug discovery.

Thus far, the theranostic strategy has been performed by introducing fluorophores as optical reporting elements into the prodrug.[22−25] However, anticancer drugs frequently have multiple limitations, including drug resistance and lack of specificity, which should be overcome to efficiently eliminate the cancer cells.[26] Moreover, the off-target delivery of these types of drugs results in undesired secondary malignancies, greatly increasing undesirable side effects.[27] On the basis of these design considerations, we report a novel H2O2-activatable multifunctional theranostic prodrug with minimized complexity in terms of size, containing an anticancer drug, a cleavable linker, and a benzorhodol moiety (Scheme ). The caged anticancer drug CPT is a therapeutic agent used to treat various carcinomas, and is one of the most efficient drugs that is expected to be released upon being triggered by high intracellular H2O2 concentrations during receptor-mediated endocytosis. The malignancy-dependent power of theranostics can be further improved by introducing 1,4-elimination into the benzorhodol ring, with demonstrated preferential release of the therapeutic agent at the diseased site, exhibiting increased intracellular concentrations of H2O2 versus normal cells.

Scheme 1

Schematic Drawing Illustrating the Tracking Principle for Active Drug Release in Living Cells

Results and Discussion

H2O2-Sensing Performance of LHX-B-CPT in Aqueous Buffer

To show that the prodrug LHX-B-CPT was effectively activated by the breakage of a boric acid ester bond, which in turn stimulated the LHX-OH fluorophore, the fluorescence enhancement of the probe, upon the addition of 200 equiv of H2O2, was examined under optimum conditions, as can be seen in Figure a. The effect of probe concentration in phosphate-buffered saline (PBS, pH = 7.4) was studied by treating with a H2O2 solution (200 μM), and the fluorescence intensities (λex = 470 nm, λem = 552 nm) after and before reaction with H2O2were evaluated at different H2O2concentrations. The results show that concentration-dependent enhanced fluorescence was observed when a H2O2 concentration of 5 μM was used. From Figure b, it can be observed that the intensity of the absorption spectra gradually increases upon the addition of H2O2. The emission behaviors of the prodrug in the absence and presence of H2O2 were supported by buffered (10 mM PBS, pH = 7.4), with the results given in Figure c. The prodrug LHX-B-CPT itself leads to almost no emission, but the addition of hydrogen peroxide produces a large spectroscopic enhancement at 552 nm in 10 mM PBS at pH 7.4 (Figure c), and its intensity increased linearly with an increase in the concentration of H2O2 from 0 to 200 μM (Figure d). In addition, it should be noted that the detection limit was calculated as 0.026 μM (3δ/κ, in which δ is the standard deviation of blank measurements and κ is the slope for the range of the linearity) under the experimental conditions (Figure S9). These promising results clearly demonstrate the potential use of the prodrug LHX-B-CPT for the quantitative detection of H2O2 under simulated physiological conditions.

Figure 1

(a) Effect of probe concentration (H2O2 concentration: 200 μM) in PBS (pH = 7.4). (b) Absorption spectra of LHX-B-CPT (5 μM) before and after reaction with H2O2 at pH 7.4 (PBS) containing 8% (v/v) of dimethyl sulfoxide (DMSO); λex = 470 nm. (c) Fluorescence emission spectra of LHX-B-CPT (5 μM) before and after reaction with H2O2. (d) Fluorescence intensity of LHX-B-CPT (5 μM) at 552 nm versus H2O2 concentration (0–200 μM) in aqueous solutions.

pH Activatability and Selectivity of LHX-B-CPT

The effect of pH as a significant influencing factor on the recognition behavior of the prodrug LHX-B-CPT in sensing H2O2 was also investigated. From Figure a, we observed that the probe can exist stably over a wide pH range and the prodrug LHX-B-CPT itself is not pH sensitive. Accordingly, the pH variation will not produce isomerization in this pH range. However, upon addition of H2O2 to the employed probe, it showed spectroscopic changes over the pH range from 6 to 8. In this sense, the experiment demonstrates that the prodrug can be assessed under physiological pH environments and, therefore, has practical applications in biological systems.

Figure 2

(a) Fluorescence spectra of 5 μM LHX-B-CPT versus pH in the absence and presence of 200 μM H2O2 in the buffer solution. (b) Fluorescence changes of LHX-B-CPT (5 μM) after reaction with H2O2 for different periods of time. (c, d) Emission changes of LHX-B-CPT (5 μM) in the presence of various biologically relevant ROS and foreign species (200 μM) in aqueous solutions; λex = 470 nm.

The potential specificity of the H2O2 prodrug is another very crucial parameter for its potential application in understanding the diverse biological roles played by H2O2. Therefore, the fluorescence responses of the prodrug toward other biologically related species were then assessed under the same conditions. As shown in Figure c,d, the 5 μM prodrug LHX-B-CPT solution in PBS (pH 7.4, 10 mM) exhibited nonfluorescence at 552 nm excitation. Gratifyingly, upon addition of 200 μM H2O2, a remarkable fluorescence enhancement was observed. In contrast, other analytes caused negligible changes even at much higher concentrations in the prodrug LHX-B-CPT solution. Thus, the probe exhibited selectivity toward H2O2 over other analytes. The above results confirm that the probe possesses the capability of highly selective detection of H2O2 in complex cellular milieu.

Time Course of LHX-B-CPT

The time-dependent fluorescence responses show that the signal increased quickly after the addition of H2O2 (ca. 6-fold at 20 min and 12.6-fold at 40 min), and it was observed that 80% of fluorescence quenching takes place within 40 min of addition of 120 μM of H2O2 at a time (Figure b). Overall, considering the unique reactivity, fast degradation, and extremely low background signal of intracellular H2O2, such a quick response also demonstrates the potential of LHX-B-CPT to evaluate H2O2 imaging essentially in living systems.

Mechanisms of Systems

At this point, the mechanisms by which the hydrogen peroxide-induced presumed reactions are proposed to take place are illustrated in Scheme . Complementary high-performance liquid chromatography (HPLC) analysis of the H2O2-treated prodrug LHX-B-CPT solution demonstrated the generation of the final fluorescent product LHX-OH. As shown in Figure S8, upon the addition of excess hydrogen peroxide, the HPLC chromatogram clearly displays the effective dissociation of LHX-B-CPT after 8.9 min, with the disappearance of the peak with a retention time of 2.6 min. Instead, a new peak with an identical retention time to CPT (t = 6.7 min) was observed; the peaks were identified by electrospray ionization-mass spectrometry (ESI-MS) measurements (Figure S9). All of these results confirm that H2O2 triggers the borate cleavage of LHX-B-CPT and produces the drug CPT, which consequently undergoes rapid intramolecular cyclization to release the prodrug for fluorescence signal activation.

Scheme 2

Structure and Fluorescence Response Mechanism of LHX-B-CPT

Fluorescence Imaging of H2O2 in Living Cells

Subsequently, to evaluate the lysosome-targeting ability of the prodrug LHX-B-CPT in cellular environments, we carried out a colocalization experiment with KB cells (Figure ). Costaining with the commercially effective lysosome-specific sensor LysoTracker was employed to further identify the subcellular location. The cells were loaded with 1 μM prodrug for 1 h at 37 °C. As might be expected, both the Tracker and the prodrug displayed strong localized fluorescence within lysosomes; the yellow merged images show that the signal of the prodrug LHX-B-CPT fits well with that of LysoTracker. To gain insight into the intracellular location of LHX-B-CPT release, colocalization experiments were carried out for the mitochondria (MitoTracker). As shown in Figure , such overlaps were not observed with MitoTracker. These results are thus in agreement with the probe LHX-B-CPT-induced significant cell membrane permeability and general relevance of particular labeling of lysosomes in biological investigations.

Figure 3

Intracellular location of LHX-B-CPT (1.0 μM) using LysoTracker (colocalization Pearson correlation coefficient 0.93) and MitoTracker in KB cells. The bottom panels show the enlarged views of the white frames.

To further verify the possibility of monitoring the therapeutic efficiency of LHX-B-CPT, time-dependent (5, 20, and 60 h) fluorescence responses were recorded. As illustrated in Figure , when the HeLa cells were treated with LHX-B-CPT, they exhibited negligible fluorescence in their corresponding channels; however, remarkable fluorescence was observed with increasing treatment time. Thus, based on the results shown in Figures and 4, we conclude from cell imaging that the substrate LHX-B-CPT is taken up by KB and HeLa cells through folate-mediated endocytosis accompanied by borate bond cleavage.

Figure 4

Colocalization experiments involving the probe LHX-B-CPT in HeLa cells. The cells were incubated with LHX-B-CPT (1.0 μM) at 37 °C and then the images were obtained at each time point (5, 20, and 60 min).

Finally, the cytotoxicity of the prodrug LHX-B-CPT was evaluated through KB cells (cancer cells) and WI38 cells (normal cells), which are folate receptor-positive and -negative cell lines, respectively. The results are evaluated in relation to cell viability and are shown in Figure . Cell viability is a contrast to living dead cells, based on a total sample; and cell practicability measurement usually enabled to confirm the death life of cells. Notably, for the probe, even at the high concentration of 5 μM, the cell viability is still more than 70%; overall, the sensor provides lower cytotoxicity to KB cells under the experimental conditions, which may be due to the good biocompatibility of LHX-B-CPT.

Figure 5

Viability of KB cells and WI38 cells after incubation with LHX-B-CPT of different concentrations.

Conclusions

In summary, we have presented a prodrug platform to deliver drugs specifically to the lysosomal matrix through a combination of folate targeting and ROS-induced uncaging. The system rapidly activated increased fluorescence in living cells, which was employed for real-time monitoring of the therapeutically active CPT and application of chemotherapeutic treatments. More importantly, LHX-B-CPT displayed high anticancer activity in the targeted cancer cells and staged the diseased state via imaging modalities. Thus, we hope that the novel strategy, by adaptation, application for the use of probes in the construction of the screening of new potential diagnosis with precise control over the drug release.

Experimental Section

Materials and Apparatus

All reagents were of analytical grade, purchased from major suppliers, and applied directly in the experiment without further drying or purification. Deionized water was used throughout all of the experiments, and the pH was adjusted using a dilute sodium hydroxide solution or hydrochloric acid. All chemicals were obtained from major suppliers such as Sangon (Shanghai), Alfa Aesar (Tianjin), Sigma-Aldrich (Beijing), and J&K (Guangzhou) and used as received. All fluorescence spectra were measured using an F-7000 fluorescence spectrophotometer (Hitachi). The 1H nuclear magnetic resonance (NMR) spectra were obtained at 500 MHz on a Bruker Advance-500 spectrometer with tetramethylsilane (TMS) as the internal standard. Mass spectral analysis was performed with a Bruker Esquire 3000 plus mass spectrometer. All of the measurements were made at room temperature (25 °C). Fluorescence images of KB cells were taken on an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). The efficient synthesis of compound LHX-B-CPT following the synthetic methodology is summarized in the Supporting Information.

Synthesis of Compounds

Rhodamine derivatives were used as organic fluorophores because of the attractive and desirable features of ease of chemical modification through the spirocycle and the very high chemical stability as scaffold fluorophores. With this design, we envisaged that the “smart” probe LHX-B-CPT can selectively explore an analyte with a turn-on fluorescence response. LHX-B-CPT is a straightforward benzorhodol that is obtained by converting the hydrogen atom substituent into an aldehyde group to accomplish its multiple functions; thus, the excellent guiding molecule was monitored based on fluorescence. The detailed synthetic pathway and product characterizations are described in the Supporting Information.

Cell Culture

KB cells and WI38 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (high glucose) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (penicillin: 10 000 U·mL–1, streptomycin: 10 000 U·mL–1) at 37 °C in a humidified atmosphere of 5% CO2. Cells were passaged at about 80% cell confluency using a 0.25% trypsin solution.