A sequential enzyme-activated and light-triggered pro-prodrug nanosystem for cancer detection and therapy†

Zelin Chen‡ , Bowen Li‡ , Xin Xie , Fang Zeng * and Shuizhu Wu *
State Key Lab of Luminescent Materials & Devices, College of Materials Science & Engineering, South China University of Technology, Guangzhou 510640, P. R. China. E-mail: shzhwu@scut.edu.cn; mcfzeng@scut.edu.cn; Fax: +86 20 22236363; Tel: +86 20 22236262

First published on 5th September 2017

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1. Introduction

Traditional chemotherapy has shown accredited efficacy for cancer treatments but is often hindered by several side-effects (high toxicity and non-specificity) on normal cells.^1–4 On-demand treatment upon effective cancer detection or discrimination of cancer cells from normal cells would greatly reduce the high toxicity of traditional chemotherapy resulting from the non-specificity of drugs. In addition, a new strategy is needed to reduce the side-effects of prodrugs, which can be activated by the unique microenvironment in a tumor.^5–10 The traditional pursuit of prodrugs has largely focused on special internal stimuli in the tumor, such as high levels of biothiols,^11–17 lower pH,^18–24 reactive oxygen species,^25–32 and various overexpressed enzymes.^33–41 However, recent research revealed that the activation of prodrugs in cancer cells may be successively controlled by internal and external stimuli,^42–45 and this type of prodrug is also described as a “pro-prodrug”. A new strategy for the pro-prodrug is that it is first activated by the differential expression of internal stimuli, thus forming the prodrug, and then this prodrug can discriminate tumor cells from normal cells and release anticancer drugs via external stimuli.^46–52

DT-diaphorase, an important detoxifying enzyme, is widely distributed in a variety of tissues and can catalyze an obligatory two electron reduction of a broad range of substrates (quinones) using either nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as electron donors.^53–55 Many literature reports indicate that DT-diaphorase is overexpressed in many tumor cells/tissues, such as breast cancer, non-small cell lung cancer, colorectal cancer and cervical cancer.⁵⁶ All these cancer cells have been found to exhibit strongly elevated DT-diaphorase concentrations (up to 50-fold compared to normal cells).^57,58 On account of the huge difference in the DT-diaphorase level between tumor and normal tissues, the enzyme has attracted considerable attention as a potential candidate for cancer diagnosis.^59–62 Our group has demonstrated that a DT-diaphorase responsive prodrug is beneficial for cancer chemotherapy; however, there are no reports on the exploitation of DT-diaphorase for developing a pro-prodrug for imaging cancer cells and possible therapy.^63,64

Methotrexate (MTX), as a folate reductase inhibitor, mainly inhibits dihydrofolate reductase, thus preventing dihydrofolic acid from being reduced to tetrahydrofolic acid (a cofactor in the anabolism of amino acids and nucleic acids), which accordingly leads to the inhibition of DNA biosynthesis. It is one of the most widely used drugs in medical oncology for the treatment of several types of cancer.^65,66 However, many obstacles including toxic side effects toward normal cells, drug resistance, and acute and chronic toxicity remain that impede its clinical application. Besides, its poor water solubility constitutes a hurdle in terms of wide-ranging clinical application of MTX.^67–69 Methotrexate-based prodrugs are an effective approach to overcoming these shortcomings, as well as improving therapeutic efficacy, but their poor water solubility is still a major issue for cancer treatment.^70,71

With the aim of overcoming the above limitations, we developed a novel theranostic pro-prodrug for cancer detection/imaging and therapy in DT-diaphorase over-expressed tumor cells; the MTX-based pro-prodrug nanosystem features enzyme-activated fluorescence and subsequent light-triggered on-demand drug release. This pro-prodrug contains three parts: the anticancer drug methotrexate (MTX), coumarin and quinone propionic acid (Scheme 1). In this pro-prodrug, the prodrug (coumarin derivatives) serves as the fluorophore to assess the DT-diaphorase level upon enzymatic reaction (enzyme activated), which would be conducive to cancer detection/imaging, and also as a phototrigger (upon unlocking) to release the active drug. The quinone propionic acid unit, an electron accepter, is a key part of the pro-prodrug that can lock the coumarin phototrigger and switch off its fluorescence. In this design, as for the locked state, the coumarin phototrigger and fluorescence are banned via photoinduced electron transfer (PET) to the quinone propionic acid unit. On the contrary, the unlocked state, which is activated only by DT-diaphorase and monitored by coumarin fluorescence, can release the MTX in response to either one-photon visible light or two-photon near infrared (NIR) light. The pro-prodrug was also loaded into the liposomes, which could improve the biocompatibility and reduce the side-effects, particularly to enhance the aqueous solubility of MTX.^72–74 This new strategy integrating both internal and external triggers offers a powerful tool for potentially effective cancer treatment. To the best of our knowledge, this is the first report on the imaging and therapeutic effect of an enzyme-activated light-triggered pro-prodrug nanosystem.

2. Experimental

2.1 Materials

The m-dihydroxybenzene, ethyl 4-chloroacetoacetate, methanesulfonic acid, trimethylhydroquinone (TMHQ), methyl 3,3-dimethylacrylate, N-bromosuccinimide (NBS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Reagent and used without further purification. The 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG 2000) was provided by Thermo-Scientific. DT-diaphorase (lyophilized powder, recombinant, expressed in E. coli) and β-nicotinamide adenine dinucleotide reduced disodium salt (NADH) were purchased from Sigma-Aldrich. Deionized water was used in the experiments. Dichloromethane (DCM) and N,N-dimethylformamide (DMF) were dried with CaH₂ and distilled under a nitrogen atmosphere. Other solvents used in this study were analytical grade reagents and were used without further purification.

2.2 Pro-prodrug synthesis

2.3 Fabrication of liposomal nanoparticles (LIP-DT-COU-MTX, the pro-prodrug nanosystem)

The liposomal nanoparticles were prepared by a solvent evaporation method. DT-COU-MTX (8 mg) was dissolved in 200 μL of DMSO and DSPE-PEG 2000 (8 mg) was dissolved in 1.8 mL of THF. The solutions were added to ice water under sonication for 30 min for emulsification. After the solution was stirred at room temperature for 24 hours, the solvent was removed under vacuum. Next, the residual solution was subjected to one stage of dialysis against water (2 L) using cellulose membrane with a 1000 molecular weight cutoff. Liposomes were obtained by ultracentrifugation at 12000 rpm for 10 min at 4 °C. Finally, the collected nanoparticles were stored at 4 °C for use.

Determination of the drug loading rate is based on measurement of the absorption spectra. The lyophilized DT-COU-MTX-loaded nanoparticles were dissolved in 1 mL of DMSO and subsequently tested for absorbance at 380 nm. We define the drug loading rate (DLR) as follows:

DLR = (mass of loaded DT-COU-MTX)/(mass of nanoparticles) × 100%

The determined DLR was 49.6%.

2.4 Cell culture

A549 cells (human non-small cell lung cancer cells), HeLa cells (human cervical cancer cells) and L929 cells (murine aneuploid fibrosarcoma cells) were used for all in vitro cell-based studies. A549 and L929 cells were cultured using RPMI 1640 medium, while HeLa cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM). Both media contained 10% FBS (Fetal Bovine Serum), and 1% penicillin and streptomycin (GIBCO). The medium was replaced at regular intervals (1 to 2 days). All three cell lines were incubated in an environment with a temperature of 37 °C and a carbon dioxide concentration of 5%.

2.5 Cell imaging

The three cell lines (A549, L929 and HeLa) were cultured 24 hours later and seeded into a six-well plate containing polylysine-coated cell culture glass slides. When the cell density was 50–70%, the medium was replaced and washed with PBS, followed by treatment with LIP-DT-COU-MTX for 1 h or 2 h. The glass slides were then taken out and washed with PBS, followed by fluorescence imaging experiments. Fluorescence images were obtained using an Olympus IX71 inverted fluorescence microscope with a DP72 color CCD.

2.6 Fluorescence generation of LIP-DT-COU-MTX measured by flow cytometry

A549 and HeLa cells were seeded in 6-well plates at a density of 1.0 × 10⁶ cells per well for 12 h at 37 °C. Cells were exposed to LIP-DT-COU-MTX (20 μg mL⁻¹) and incubated for 1 h and 2 h, respectively. The cells were then washed with PBS and harvested after trypsinization and centrifugation. Approximately 10000 cells were analysed using a BD CS6 flow cytometer equipped with a 370 nm argon laser.

2.7 Cell viability assay

Cells (5000 cells per well) were seeded in 96-well plates. After 24 h of incubation at 37 °C, cells were washed with PBS buffer, and then the PBS was replaced with fresh medium containing a series of LIP-DT-COU-MTX samples from 0.2 to 70 μg mL⁻¹ or MTX from 0.1 to 30 μg mL⁻¹ for 2 h at 37 °C; the cells were then placed under light irradiation for 30 min (LED lamp, with a wavelength range of 400–450 nm at a power of 10 mW cm⁻², blue light), and afterwards the cells were further incubated for 48 h. The experiments involving dicoumarol (DIC) inhibition were conducted as follows: first, the cells were pretreated with DIC (20 μM) for 4 h and then incubated with different concentrations of LIP-DT-COU-MTX (0.2 to 70 μg mL⁻¹) under the same conditions. In addition, to evaluate the toxicity of DIC, the cells were only incubated with DIC (20 μM) for 48 h. For the photocytotoxicity experiment, the cells were irradiated with visible light for 30 min (LED lamp, with a wavelength range of 400–450 nm at a power of 10 mW cm⁻², blue light). After irradiation, the cells were again incubated for 48 h. For the dark cytotoxicity assay, the cells were incubated with LIP-DT-COU-MTX without irradiation. After incubation for 48 h, the cells were washed three times with PBS buffer and treated with DMEM or RPMI 1640 medium containing 0.5 mg mL⁻¹ MTT for another 4 h. The resulting formazan crystal was dissolved in 150 μL of DMSO after carefully removing the medium, and the absorbance was recorded at 570 nm. The cell viability assays were performed using a Thermo MK3 ELISA plate reader. The independent experiments performed in six replicates were used to obtain the statistical mean and standard deviation.

2.8 Apoptosis analysis by annexin V-FITC and propidium iodide (PI) double staining

A549, HeLa and L929 cells were seeded in 6-well plates at a density of 1.0 × 10⁶ cells per well for 24 h at 37 °C. When the A549 and L929 cell densities reached 80–90% of confluence, the medium was replaced with 70 μg mL⁻¹ of LIP-DT-COU-MTX or 20 μg mL⁻¹ of MTX for 2 h, the cells were placed under light irradiation for 30 min (LED lamp, with a wavelength range of 400–450 nm at a power of 10 mW cm⁻², blue light), and afterwards the cells were further incubated for 9.5 h. To evaluate the effects of the nanosystem level, the A549 and HeLa cells were incubated with different concentrations of LIP-DT-COU-MTX (10 μg mL⁻¹ to 100 μg mL⁻¹) for 2 h, the cells were placed under light irradiation for 30 min (LED lamp, with a wavelength range of 400–450 nm at a power of 10 mW cm⁻², blue light), and afterwards the cells were further incubated for 9.5 h. The experiments involving dicoumarol (DIC) inhibition were conducted as follows: the cells were first pretreated with DIC (20 μM) for 4 h and then incubated with LIP-DT-COU-MTX (70 μg mL⁻¹) under the same conditions (under the same light irradiation conditions). Finally, the cells were washed with PBS and harvested after trypsinization and centrifugation at 2000 rpm for 10 min. Subsequently, the cells were collected and resuspended in 1 mL PBS and then mixed with annexin V binding buffer (400 μL) containing 5 μL annexin V-FITC and 10 μL PI. Finally, flow cytometry analyses were performed using a BD Accuri C6 flow cytometer, and the data were analyzed using the BD Biosciences software.

2.9 Measurements

¹H NMR spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer. All chemical shifts are reported in ppm using the peak of residual proton signals of TMS as an internal reference. Mass spectra were obtained on a Bruker Esquire HCT Plus mass spectrometer. High-resolution mass spectra were obtained on a Sciex ×500R mass spectrometer. UV-vis spectra were recorded on a Hitachi U-3010 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. Fluorescence images were obtained using an Olympus IX 71 with a DP72 color CCD. HPLC analyses were performed on an Agilent 1260 system. The two-photon experiments were performed by irradiating with a fs mode-locked Ti:sapphire laser system (output beam ≈ 120 fs duration and 1 kHz repetition rate). Flow cytometry was recorded on a BD CS6 flow cytometer. The particle size and distribution were determined based on a dynamic light scattering (DLS) mechanism on a Malvern Nano-ZS90 particle size analyzer at a fixed angle of 90° at 25 °C. Transmission electron microscopy (TEM) experiments were carried out by mounting a drop (∼15 μL) of the solution on a carbon-coated copper grid and observing with a JEM-2010HR transmission electron microscope.

3. Results and discussion

3.1 Synthesis of the pro-prodrug

The synthesis route for the pro-prodrug (DT-COU-MTX, 6) is shown in Scheme 2. Compounds 1–4 were synthesized according to previously reported methods,^75–77 and 7-hydroxy-4-(hydroxymethyl)-2H-chromen-2-one was allowed to react with quinone propionic acid to yield compound 5. The pro-prodrug (DT-COU-MTX, 6) was obtained by esterification between MTX and compound 5. Subsequently, under dark conditions, DT-COU-MTX reacted with DT-diaphorase to yield the prodrug (HO-COU-MTX, 7), as shown in Fig. S1 (ESI†). The structures of all the intermediates and the final product were confirmed by ¹H nuclear magnetic resonance (¹H NMR) and high-resolution mass spectrometry (HR-MS) (Fig. S2–S15, ESI†).

3.2 Fluorescent response of DT-COU-MTX toward DT-diaphorase

To determine whether DT-COU-MTX is a highly specific senor for DT-diaphorase, we investigated its spectral properties under simulated physiological conditions. First, absorption and fluorescence spectra for DT-COU-MTX solutions were recorded and are shown in Fig. S16 (ESI†) and Fig. 1A, which indicate that both DT-COU-MTX and MTX show strong absorption bands at 320 and 380 nm. Fluorescence spectra were recorded at 37 °C in phosphate buffer (pH 7.4, 10 mM) in the presence of cofactor NADH (100 μM), as shown in Fig. 1A–C. As shown in Fig. 1A, HO-COU-MTX exhibits strong fluorescence at 475 nm, and DT-COU-MTX shows insignificant fluorescence as a result of the PET effect due to the existence of the quinone propionic acid moiety. In the presence of 20 μg mL⁻¹ DT-diaphorase, strong fluorescence (a 50-fold increase in fluorescence intensity) at 475 nm can be observed and this spectrum is similar to that of HO-COU-MTX. When HO-COU-MTX was irradiated with blue light for up to 40 min, a blue-shift in the fluorescence band could be observed and a new emission peak appeared at about 465 nm, corresponding to the emission of the released coumarin (compound 2, Fig. 1A and Fig. S17, ESI†). As for evaluating the enzymatic properties of DT-COU-MTX in vitro, the concentration and time dependence of the HO-COU-MTX fluorescence spectra were also recorded, as shown in Fig. 1B and C. As the incubation time increased, the fluorescence at 475 nm increased steadily and reached a plateau within 120 min, as shown in Fig. 1B, which is about 50-fold its original intensity. On the other hand, with gradual addition of DT-diaphorase (0 μg mL⁻¹ to 40 μg mL⁻¹) to the solution of DT-COU-MTX, the fluorescence intensity at 475 nm increased until it reached a saturation point at 20 μg mL⁻¹ (Fig. 1C). These results indicate that the fluorescence of DT-COU-MTX is restored in the presence of DT-diaphorase, and HO-COU-MTX undergoes further photolysis to produce coumarin and MTX.

3.3 Drug release studies in vitro

In order to further explore the release of MTX from DT-COU-MTX in the presence of DT-diaphorase under irradiation, high-performance liquid chromatography (HPLC, one-photon irradiation) was employed and high-resolution MS spectra (HR-MS, two-photon irradiation) were recorded as shown in Fig. 1D and Fig. 2A–C, respectively. As shown in Fig. 1D, upon irradiation (one-photon), MTX is gradually released from HO-COU-MTX, and the release rate reaches a maximum within an irradiation time of 40 min, up to about 78%, while DT-COU-MTX is quite stable under irradiation with no release of MTX. In addition, the irradiation time for DT-COU-MTX was extended to 48 h, and the release rate remained negligible as shown in Fig. S18 (ESI†). This confirms that the PET effect can effectively lock the photochemical reactions and that DT-COU-MTX is stable to irradiation in the absence of DT-diaphorase. As shown in Fig. 2A and B, in the presence of DT-diaphorase, DT-COU-MTX, compound 3 (quinone propionic acid will be reduced by DT-diaphorase to lactone, with the same structure as compound 3), and HO-COU-MTX give rise to peaks at 1.80, 2.27, and 1.16 min, respectively, in the HPLC chromatogram. Under visible light irradiation (one-photon), HO-COU-MTX, coumarin, and MTX give rise to peaks at 1.26, 1.58 and 1.06 min, respectively, in the HPLC chromatogram. The coumarinyl can be activated by two-photon near-infrared (NIR) irradiation at 800 nm. Compared to one-photon irradiation, two-photon irradiation is more beneficial for biological applications, as it can ensure deeper tissue penetration with less photo-induced damage.^78–80 Moreover, we further evaluated the drug release resulting from the two-photon process by HR-MS. DT-COU-MTX was reacted with DT-diaphorase and then underwent irradiation using a Coherent Legend Elite Ti:sapphire regenerative amplifier system as a femtosecond laser source at 800 nm; afterwards, a small aliquot was subjected to HR-MS analysis. Ionic peaks corresponding to MTX ([HR-MS-H]⁻ = 453.1630), coumarin ([HR-MS-H]⁻ = 191.0344), and HO-COU-MTX ([HR-MS-H]⁻ = 627.1954) can be observed in the HR-MS spectra. The above experimental results show that DT-COU-MTX can detect the level of DT-diaphorase via the fluorescence emission intensity and further release MTX under irradiation. On the other hand, the light-triggered controllability of drug release from DT-COU-MTX upon enzyme activation has been demonstrated using HPLC analysis as shown in Fig. S19 (ESI†). These results indicate that HO-COU-MTX (the product from the reaction between DT-COU-MTX and the enzyme DT-diaphorase) can serve as a fluorescence probe for the DT-diaphorase level and release the anticancer drug MTX by one-photon or two-photon irradiation.

3.4 Anti-interference performance of DT-COU-MTX

In order to investigate the potential application of DT-COU-MTX in biological systems, selectivity experiments were performed with DT-diaphorase and some potential interferents (such as metal ions, amino acids, biological reductants, and enzymes), and the results are depicted in Fig. 3A. Upon incubating with DT-diaphorase or possible interferents for 2 h, a remarkable enhancement of fluorescence at 475 nm can be observed in the presence of DT-diaphorase, while no obvious fluorescence change can be detected with the potential interferents. The results demonstrate that DT-COU-MTX exhibits good selectivity for DT-diaphorase under physiological conditions. In addition, the pH effect on the reaction of DT-COU-MTX and DT-diaphorase was investigated, as shown in Fig. 3B. In the absence of DT-diaphorase, the fluorescence intensities at 475 nm are weak and remain stable over the pH range from 3 to 9, whereas in the presence of DT-diaphorase and its cofactor, an obvious increase in fluorescence intensity can be detected, and the values remain fairly stable within the pH range 3–9. The results of this experiment demonstrate that DT-COU-MTX may be a potentially potent tool for the effective detection of DT-diaphorase and release of anticancer drugs in a physiological environment.

3.5 Imaging and pro-apoptotic effects in DT-diaphorase overexpressing cells

Subsequently, we explored the imaging effect of DT-COU-MTX in DT-diaphorase overexpressing cell lines (A549 cells and HeLa cells) and normal cell lines without overexpression of DT-diaphorase (L929 cells) by using a fluorescence microscope. In order to improve aqueous solubility or dispersibility and enhance cellular uptake of the MTX-based pro-prodrug,^81–85 we loaded DT-COU-MTX into the liposome carrier, forming a pro-prodrug nanosystem (termed as LIP-DT-COU-MTX). The particle size distribution (DLS) and TEM images of LIP-DT-COU-MTX are shown in Fig. S20 (ESI†). We then evaluated the performance of LIP-DT-COU-MTX in cells, and the results are presented in Fig. 4A and B. For A549 cells, upon 1 h of incubation with LIP-DT-COU-MTX (20 μg mL⁻¹), intracellular blue fluorescence can be observed, and after 2 h of incubation, the fluorescence became very bright, as shown in Fig. 4A. The intracellular imaging of HeLa cells is similar to that of A549 cells, as shown in Fig. S21 (ESI†). To determine the effect of DT-diaphorase on LIP-DT-COU-MTX, A549 cells were pretreated with dicoumarol (a DT-diaphorase inhibitor), and the fluorescence in the cells disappeared (Fig. 4B). Moreover, for the L929 cells, in which DT-diaphorase is not overexpressed, incubation with the pro-prodrug nanoparticles for 2 h cannot induce detectable blue fluorescence under similar experimental conditions, as shown in Fig. 4B. The capability of LIP-DT-COU-MTX for DT-diaphorase detection in cells was further confirmed by flow cytometry analysis, as shown in Fig. S22 (ESI†). The above results demonstrate that this nanosystem can effectively discriminate cancer cells from normal cells and serve as a theranostic agent for cancer cells.

We next performed MTT assays for DT-diaphorase overexpressing cancer cell lines (HeLa and A549) and normal cell lines (L929) to evaluate the efficacy of LIP-DT-COU-MTX with respect to cell viability (Fig. 5A). Upon light irradiation, compared to the high viability of L929, LIP-DT-COU-MTX shows strong cytotoxicity towards HeLa and A549 cells; the IC₅₀ (concentration inhibiting cell growth to 50% of control) value was determined as 32.66 μg mL⁻¹ for HeLa cells and 20.07 μg mL⁻¹ for A549 cells. LIP-DT-COU-MTX had a half inhibitory concentration of much greater than 70 μg mL⁻¹ for L929 cells. MTT assays were also performed for A549, HeLa, and L929 cells co-treated with dicoumarol (DIC, an inhibitor of DT-diaphorase) and then treated with LIP-DT-COU-MTX under the same conditions, and the results are presented in Fig. 5A. Upon pretreatment with DIC, the cells with DT-diaphorase overexpression (A549 cells and HeLa cells) and normal cells (L929 cells) without enzyme overexpression all exhibit high viabilities (over 80% at 70 μg mL⁻¹ LIP-DT-COU-MTX). In the control experiment, the IC₅₀ values of MTX for HeLa cells and A549 cells were 9.31 and 5.22 μg mL⁻¹, respectively (Fig. S23, ESI†). The results show that the half-inhibitory concentration of LIP-DT-COU-MTX for cancer cells has the same order of magnitude as that of MTX. Moreover, as shown in Fig. S24 (ESI†), the cell viability for the three cell lines upon exposure to blue light irradiation but without treatment with LIP-DT-COU-MTX was also measured, and the blue light alone leads to insignificant cytotoxicity. These results reveal that the cytotoxicity of LIP-DT-COU-MTX derives from the light-triggered drug release after enzymatic reaction. In order to further verify the effect of irradiation on drug release, three groups of cells were treated with LIP-DT-COU-MTX without irradiation as a control, as shown in Fig. S25 (ESI†). In the absence of light irradiation, the cell viabilities of the three groups of cells were relatively high. In addition, the DIC cytotoxicity experiments show that DIC (20 μM) leads to insignificant cytotoxicity (Fig. S26, ESI†). These results prove that LIP-DT-COU-MTX only produces a therapeutic effect when DT-diaphorase and phototrigger conditions are available at the same time.

Furthermore, the pro-apoptotic effect of LIP-DT-COU-MTX towards DT-diaphorase-overexpressing cells with light irradiation was evaluated by flow cytometry studies. As shown in Fig. 5B, the apoptotic percentage (including early and late apoptosis, Q4 and Q1 in Fig. 5B) is 9.0% after incubation of LIP-DT-COU-MTX with normal cells (L929 cells); however, for the unmodified MTX, the apoptotic percentage reaches 36.4%, which is threefold higher than that with the nanosystem. As for A549 cells, the apoptotic percentage reaches 55.5% and 68.7% upon treatment with LIP-DT-COU-MTX and MTX. As shown in Fig. 5C, we also investigated the concentration dependence of LIP-DT-COU-MTX with respect to pro-apoptotic efficiency towards A549 cells by flow cytometry. The subsequent addition of LIP-DT-COU-MTX (10 μg mL⁻¹ to 100 μg mL⁻¹) can induce a remarkable increase in the apoptotic percentage of A549 cells (17.5% to 73.8%). Similar results were obtained for the same experiment with HeLa cells, as shown in Fig. S27 (ESI†). As shown in Fig. S28 (ESI†), the results from the experiment involving DIC inhibition show that the apoptotic percentage of the cells pretreated with DIC (20 μM) is lower (approximately 5% at 70 μg mL⁻¹ LIP-DT-COU-MTX), but for those treated with LIP-DT-COU-MTX alone, it is higher; this is consistent with the results of MTT experiments. All these flow cytometry results have proven that LIP-DT-COU-MTX has potent therapeutic effects on cancer cells with overexpression of DT-diaphorase with visible light irradiation.

The above results demonstrate that this nanosystem can serve as a DT-diaphorase probe to discriminate tumor cells from normal cells, and it only exhibits strong cytotoxicity for DT-diaphorase overexpressing cells. Moreover, this nanosystem has the advantage of releasing anti-cancer drugs under two-photon irradiation. Hence, it could be developed as a helpful tool for imaging and therapy.

4. Conclusions

In summary, we successfully synthesized an enzyme-activated and light-triggered pro-prodrug nanosystem for detection/imaging and therapy. This pro-prodrug has several advantageous features: first, the PET effect by quinone propionate can quench the fluorescence and block the photolysis of coumarin, which improves the stability and controllability of the prodrugs and avoids premature release of the anticancer drugs; second, the fluorescence of the prodrug (HO-COU-MTX) can discriminate the DT-diaphorase overexpressing tumor cells from aerobic healthy cells, and then controllable release of the anticancer drug (MTX) by one or two-photon activation can be achieved; third, the pro-prodrug was also encapsulated in liposomes to enhance biocompatibility and improve aqueous solubility. All the experiments show that the pro-prodrug only releases MTX via the sequential controls of DT-diaphorase and light irradiation. This strategy may offer an approach for developing a light-triggered pro-prodrug using an enzyme as the activator.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We acknowledge the financial support by NSFC (21574044, 21474031 and 51673066), the Science and Technology Planning Project of Guangzhou (Project No. 201607020015), the Science and Technology Planning Project of Guangdong (Project No. 2014A010105009), and the Natural Science Foundation of Guangdong Province (2016A030312002).

Notes and references

1. J. Z. Zhang, P. Bonnitcha, E. Wexselblatt, A. V. Klein, Y. Najajreh, D. Gibson and T. W. Hambley, Chem. – Eur. J., 2013, 19, 1672–1676 CrossRef CAS PubMed .
2. C. Shen, B. D. W. Harris, L. J. Dawson, K. A. Charles, T. W. Hambley and E. J. New, Chem. Commun., 2015, 51, 6312–6314 RSC .
3. Q. Chen, H. Ke, Z. Dai and Z. Liu, Biomaterials, 2015, 73, 214–230 CrossRef CAS PubMed .
4. S. Guragain, B. P. Bastakoti, V. Malgras, K. Nakashima and Y. Yamauchi, Chem. – Eur. J., 2015, 21, 13164–13174 CrossRef CAS PubMed .
5. R. Kumar, W. S. Shin, K. Sunwoo, W. Y. Kim, S. Koo, S. Bhuniya and J. S. Kim, Chem. Soc. Rev., 2015, 44, 6670–6683 RSC .
6. D. L. Wang, T. Y. Zhao, X. Y. Zhu, D. Y. Yana and W. X. Wang, Chem. Soc. Rev., 2015, 44, 4023–4071 RSC .
7. D. Y. Q. Wong, C. H. F. Yeo and W. H. Ang, Angew. Chem., Int. Ed., 2014, 53, 6752–6756 CrossRef CAS PubMed .
8. J. Q. Wang, X. R. Sun, W. W. Mao, W. L. Sun, J. B. Tang, M. H. Sui, Y. Q. Shen and Z. W. Gu, Adv. Mater., 2013, 25, 3670–3676 CrossRef CAS PubMed .
9. Y. Liu, Q. Pei, L. Chen, Z. Li and Z. Xie, J. Mater. Chem. B, 2016, 4, 2332–2337 RSC .
10. S. Santra, C. Kaittanis, O. J. Santiesteban and J. M. Perez, Cell-Specific, J. Am. Chem. Soc., 2011, 133, 16680–16688 CrossRef CAS PubMed .
11. M. Z. Ye, X. H. Wang, J. B. Tang, Z. Q. Guo, Y. Q. Shen, H. Tian and W. H. Zhu, Chem. Sci., 2016, 7, 4958–4965 RSC .
12. H. Zhang, X. Zhu, Y. Ji, X. Jiao, Q. Chen, L. Hou, H. Zhang and Z. Z. Zhang, J. Mater. Chem. B, 2015, 3, 6310–6326 RSC .
13. X. M. Wu, X. R. Sun, Z. Q. Guo, J. B. Tang, Y. Q. Shen, T. D. James, H. Tian and W. H. Zhu, J. Am. Chem. Soc., 2014, 136, 3579–3588 CrossRef CAS PubMed .
14. Y. N. Zhong, J. Zhang, R. Cheng, C. Deng, F. H. Meng, F. Xie and Z. Y. Zhong, J. Controlled Release, 2015, 205, 144–154 CrossRef CAS PubMed .
15. H. H. Xiao, H. Q. Song, Q. Yang, H. D. Cai, R. G. Qi, L. Yan, S. Liu, Y. H. Zheng, Y. B. Huang, T. J. Liu and X. B. Jing, Biomaterials, 2012, 33, 6507–6519 CrossRef CAS PubMed .
16. X. Zhang, Y. C. Yan, Y. D. Hang, J. Wang, J. L. Hua and H. Tian, Chem. Commun., 2017, 53, 5760–5763 RSC .
17. S. Banerjee, S. Kar, J. M. Perez and S. Santra, J. Phys. Chem. C, 2009, 113, 9659–9663 CAS .
18. Z. Su, Y. Liang, Y. Yao, T. Wang and N. Zhang, J. Mater. Chem. B, 2016, 4, 1122–1133 RSC .
19. Y. N. Zhong, K. Goltsche, L. Cheng, F. Xie, F. H. Meng, C. Deng, Z. Y. Zhong and R. Haag, Biomaterials, 2016, 84, 250–261 CrossRef CAS PubMed .
20. H. Wang, G. Liu, S. Dong, J. Xiong, Z. Dua and X. Cheng, J. Mater. Chem. B, 2015, 3, 7401–7407 RSC .
21. Q. K. Qi, Y. Li, X. Y. Yan, F. L. Zhang, S. Jiang, J. Su, B. Xu, X. Q. Fu, L. K. Sun and W. J. Tian, Polym. Chem., 2016, 7, 5273–5280 RSC .
22. J. Q. Fan, F. Zeng, S. Z. Wu and X. D. Wang, Biomacromolecules, 2012, 13, 4126–4137 CrossRef CAS PubMed .
23. H. H. Xiao, R. G. Qi, T. Li, S. G. Awuah, Y. R. Zheng, W. Wei, X. Kang, H. Q. Song, Y. H. Wang, Y. J. Yu, M. A. Bird, X. B. Jing, M. B. Yaffe, M. J. Birrer and P. P. Ghoroghchian, J. Am. Chem. Soc., 2017, 139, 3033–3044 CrossRef CAS PubMed .
24. R. J. Dong, S. P. Ravinathan, L. Z. Xue, N. Li, Y. J. Zhang, L. Z. Zhou, C. X. Cao and X. Y. Zhu, Chem. Commun., 2016, 52, 7950–7953 RSC .
25. Q. Li, Y. Wen, X. You, F. Zhang, V. Shah, X. Chen, D. Tong, X. Wei, L. Yin and J. Wu, J. Mater. Chem. B, 2016, 4, 4675–4682 RSC .
26. C. Perez, J. P. Monserrat, Y. Chen and S. M. Cohen, Chem. Commun., 2015, 51, 7116–7119 RSC .
27. K. Y. Liu, H. M. Shang, X. Q. Kong, M. G. Ren, J. Y. Wang, Y. Liu and W. Y. Lin, Biomaterials, 2016, 100, 162–171 CrossRef CAS PubMed .
28. H. Deng, X. Zhao, J. Liu, L. Deng, J. Zhang, J. Liu and A. J. Dong, J. Mater. Chem. B, 2015, 3, 9397–9408 RSC .
29. X. Liu, J. J. Xiang, D. C. Zhu, L. M. Jiang, Z. X. Zhou, J. B. Tang, X. G. Liu, Y. Z. Huang and Y. Q. Shen, Adv. Mater., 2016, 28, 1743–1752 CrossRef CAS PubMed .
30. J. Q. Wang, X. R. Sun, W. W. Mao, W. L. Sun, J. B. Tang, M. H. Sui, Y. Q. Shen and Z. W. Gu, Adv. Mater., 2013, 25, 3670–3676 CrossRef CAS PubMed .
31. H. Zhang, X. Zhu, Y. Ji, X. Jiao, Q. Chen, L. Hou, H. Zhang and Z. Z. Zhang, J. Mater. Chem. B, 2015, 3, 6310–6326 RSC .
32. K. Han, J. Zhu, S. Wang, Z. Li, S. Cheng and X. Z. Zhang, J. Mater. Chem. B, 2015, 3, 8065–8069 RSC .
33. B. W. Li, P. L. Liu, D. H. Yan, F. Zeng and S. Z. Wu, J. Mater. Chem. B, 2017, 5, 2635–2643 RSC .
34. S. Y. Li, L. H. Liu, L. Rong, W. X. Qiu, H. Z. Jia, B. Li, F. Li and X. Z. Zhang, Adv. Funct. Mater., 2015, 25, 7317–7326 CrossRef CAS .
35. J. Hu, L. Liu, Z. Li, R. X. Zhuo and X. Z. Zhang, J. Mater. Chem. B, 2016, 4, 1932–1940 RSC .
36. E. Secret, K. Crannell, S. Kelly, M. Villancio-Wolter and M. S. Andrew, J. Mater. Chem. B, 2015, 3, 5629–5634 RSC .
37. S. Thamphiwatana, W. Gao, D. Pornpattananankul, Q. Zhang, V. Fu, J. Y. Li, J. M. Li, M. Obonyo and L. F. Zhang, J. Mater. Chem. B, 2014, 2, 8201–8207 RSC .
38. Y. Wu, S. Huang, F. Zeng, J. Wang, C. Yu, J. Huang, H. Xie and S. Z. Wu, Chem. Commun., 2015, 51, 12791–12794 RSC .
39. Y. Wu, J. Wang, F. Zeng, S. Huang, J. Huang, H. Xie, C. Yu and S. Z. Wu, ACS Appl. Mater. Interfaces, 2016, 8, 1511–1519 CAS .
40. X. R. Song, N. Goswami, H. H. Yang and J. P. Xie, Analyst, 2016, 141, 3126–3140 RSC .
41. X. Yuan, Y. Q. Tay, X. Y. Dou, Z. T. Luo, D. T. Leong and J. P. Xie, Anal. Chem., 2013, 85, 1913–1919 CrossRef CAS PubMed .
42. A. K. Renfrew, N. S. Bryce and T. Hambley, Chem. – Eur. J., 2015, 21, 15224–15234 CrossRef CAS PubMed .
43. M. M. Dcona, D. Mitra, R. W. Goehe, D. A. Gewirtz, D. A. Lebman and M. C. T. Hartman, Chem. Commun., 2012, 48, 4755–4757 RSC .
44. J. E. Sheldon, M. M. Dcona, C. E. Lyons, J. C. Hackett and M. C. T. Hartman, Org. Biomol. Chem., 2016, 14, 40–49 CAS .
45. M. M. Dcona, J. E. Sheldon, D. Mitra and M. C. T. Hartman, Bioorg. Med. Chem. Lett., 2017, 27, 466–469 CrossRef CAS PubMed .
46. W. P. Feng, C. Y. Gao, W. Liu, H. H. Ren, C. Wang, K. Ge, S. H. Li, G. Q. Zhou, H. Y. Li, S. X. Wang, G. Jia, Z. H. Li and J. C. Zhang, Chem. Commun., 2016, 52, 9434–9437 RSC .
47. S. Trombino, T. Ferrarelli and R. Cassano, J. Funct. Biomater., 2014, 5, 99–110 CrossRef PubMed .
48. Q. N. Lin, Z. M. Du, Y. L. Yang, Q. Fang, C. Y. Bao, Y. Yang and L. Y. Zhu, Chem. – Eur. J., 2014, 20, 16314–16319 CrossRef CAS PubMed .
49. Q. N. Lin, C. Y. Bao, Y. L. Yang, Q. N. Liang, D. S. Zhang, S. Y. Cheng and L. Y. Zhu, Adv. Mater., 2013, 25, 1981–1986 CrossRef CAS PubMed .
50. S. Karthik, B. N. P. Kumar, M. Gangopadhyay, M. Mandal and N. D. P. Singh, J. Mater. Chem. B, 2015, 3, 728–732 RSC .
51. Q. N. Lin, C. Y. Bao, S. Y. Cheng, Y. L. Yang, W. Ji and L. Y. Zhu, J. Am. Chem. Soc., 2012, 134, 5052–5055 CrossRef CAS PubMed .
52. S. Biswas, J. Das, S. Barman, S. S. Shah, M. Gangopadhyay, T. K. Maiti and N. D. P. Singh, Sens. Actuators, B, 2017, 244, 327–333 CrossRef CAS .
53. G. Tedeschi, S. Chen and V. Massey, J. Biol. Chem., 1995, 270, 1198–1204 CrossRef CAS PubMed .
54. R. M. Phillips, M. A. Naylor, M. Jaffar, S. W. Doughty, S. A. Everett, A. G. Breen, G. A. Choudry and I. J. Stratford, J. Med. Chem., 1999, 42, 4071–4080 CrossRef CAS PubMed .
55. C. Flader, J. W. Liu and R. F. Borch, J. Med. Chem., 2000, 43, 3157–3167 CrossRef CAS PubMed .
56. S. Danson, T. H. Ward, J. Butler and M. Ranson, Cancer Treat. Rev., 2004, 30, 437–449 CrossRef CAS PubMed .
57. P. Y. Gasdaska, G. Powis, P. Hyman and H. Fisher, Cancer Res., 1993, 53, 5458–5461 CAS .
58. V. Fagan, S. Bonham, M. P. Carty and F. Aldabbagh, Org. Biomol. Chem., 2010, 8, 3149–3156 CAS .
59. C. Flader, J. W. Liu and R. F. Borch, J. Med. Chem., 2000, 43, 3157–3167 CrossRef CAS PubMed .
60. V. Fagan, S. Bonham, M. P. Carty and F. Aldabbagh, Org. Biomol. Chem., 2010, 8, 3149–3156 CAS .
61. T. Fryatt, H. I. Pettersson, W. T. Gardipee, K. C. Bray, S. J. Green, A. M. Z. Slawin, H. D. Beallb and C. J. Moody, Bioorg. Med. Chem., 2004, 12, 1667–1687 CrossRef CAS PubMed .
62. B. H. Huang, A. Desai, S. Z. Tang, T. P. Thomas and J. R. Baker, Org. Lett., 2010, 12, 1384–1387 CrossRef CAS PubMed .
63. P. L. Liu, J. S. Xu, D. H. Yan, P. S. Zhang, F. Zeng, B. W. Li and S. Z. Wu, Chem. Commun., 2015, 51, 9567–9570 RSC .
64. B. W. Li, P. L. Liu, D. H. Yan, F. Zeng and S. Z. Wu, J. Mater. Chem. B, 2017, 5, 2635–2643 RSC .
65. J. Kralovec, G. Spencer, A. H. Blair, M. Mammen, M. Singh and T. Ghose, J. Med. Chem., 1989, 32, 2426–2431 CrossRef CAS PubMed .
66. J. Ren, Z. J. Fang, L. Yao, F. Z. Dahmani, L. F. Yin, J. P. Zhou and J. Yao, Int. J. Pharm., 2015, 487, 177–186 CrossRef CAS PubMed .
67. H. Q. Dong, C. Y. Dong, W. J. Xia, Y. Y. Li and T. B. Ren, MedChemComm, 2014, 5, 147–152 RSC .
68. W. H. Wei, M. Fountain, D. Magda, Z. Wang, P. Lecane, N. Mesfin, D. Miles and J. L. Sessler, Org. Biomol. Chem., 2005, 3, 3290–3296 CAS .
69. M. A. Santos, E. A. Enyedy, E. Nuti, A. Rossello, N. I. Krupenko and S. A. Krupenko, Bioorg. Med. Chem., 2007, 15, 1266–1274 CrossRef CAS PubMed .
70. M. P. Carrasco, E. A. Enyedy, N. I. Krupenko, S. A. Krupenko, E. Nuti, T. Tuccinardi, S. Santamaria and A. Rossello, J. Med. Chem., 2011, 7, 265–274 CrossRef CAS .
71. J. M. Shin, S. H. Kim, T. Thambi, D. G. You, J. Jeon, J. O. Lee, B. Y. Chung, D. G. Jo and J. H. Park, Chem. Commun., 2014, 50, 7632–7635 RSC .
72. S. Wang, S. G. Zhang, J. Q. Liu, Z. Y. Liu, L. Su, H. J. Wang and J. Chang, ACS Appl. Mater. Interfaces, 2014, 6, 10706–10713 CAS .
73. B. W. Li, P. L. Liu, H. Wu, X. Xie, Z. L. Chen, F. Zeng and S. Z. Wu, Biomaterials, 2017, 138, 57–68 CrossRef CAS PubMed .
74. M. W. Tibbitt, J. E. Dahlman and R. Langer, J. Am. Chem. Soc., 2016, 138, 704–717 CrossRef CAS PubMed .
75. W. Ji, G. F. Liu, F. Wang, Z. Zhu and C. L. Feng, Chem. Commun., 2016, 52, 12574–12577 RSC .
76. X. W. Ye, Y. C. Zheng, Y. C. Duan, M. M. Wang, B. Yu, J. L. Ren, J. L. Ma, E. Zhang and H. M. Liu, MedChemComm, 2014, 5, 650–654 RSC .
77. P. L. Liu, J. S. Xu, D. H. Yan, P. S. Zhang, F. Zeng, B. W. Li and S. Z. Wu, Chem. Commun., 2015, 51, 9567–9570 RSC .
78. Y. T. Gao, G. X. Feng, T. Jiang, C. C. Goh, L. G. Ng, B. Liu, B. Li, L. Yang, J. L. Hua and H. Tian, Adv. Funct. Mater., 2015, 25, 2857–2866 CrossRef CAS .
79. B. Xu, J. B. Zhang, H. H. Fang, S. Q. Ma, Q. D. Chen, H. B. Sun, C. Lm and W. J. Tian, Polym. Chem., 2014, 5, 479–488 RSC .
80. B. J. Xu, M. Y. Xie, J. J. He, B. Xu, Z. G. Chi, W. J. Tian, L. Jiang, F. L. Zhao, S. W. Liu, Y. Zhang, Z. Z. Xu and J. R. Xu, Chem. Commun., 2013, 49, 273–275 RSC .
81. D. Y. Chen, Z. T. Luo, N. J. Li, J. Y. Lee, J. P. Xie and J. M. Lu, Adv. Funct. Mater., 2013, 23, 4324–4331 CrossRef CAS .
82. S. Santra and J. M. Perez, Biomacromolecules, 2011, 12, 3917–3927 CrossRef CAS PubMed .
83. S. Santra, C. Kaittanis and J. M. Perez, Mol. Pharmaceutics, 2010, 7, 1209–1222 CrossRef CAS PubMed .
84. Y. Z. Wang, W. Fan, X. Dai, U. Katragadda, D. Mckinley, Q. Teng and C. Tan, Mol. Pharmaceutics, 2014, 11, 1140–1150 CrossRef CAS PubMed .
85. B. S. Pattni, V. V. Chupin and V. P. Torchilin, Chem. Rev., 2015, 115, 10938–10966 CrossRef CAS PubMed .

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Footnotes

† Electronic supplementary information (ESI) available: 1H NMR spectra, mass spectra, absorption spectra, fluorescence spectra, HPLC chromatogram, flow cytometry profiles and cell viabilities. See DOI: 10.1039/c7tb01989k

‡ These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2018