2.2. In vitro cytotoxicity

To evaluate the cytotoxicity of these two fluorescent prodrugs, we evaluated their antiproliferative activity against a panel of human cancer cell lines (HCT-116, A549, MCF-7, HeLa, and U87), and one normal cell line (HUVEC) using the CCK-8 assays, with the anti-cancer drug ANF as the control. The results, presented as IC₅₀ values, are given in Table 1, based on three parallel experiments. The IC₅₀ value of AcKLP against U87 cells was determined to be 2.26 μM, demonstrating similar antitumor activity to ANF (IC₅₀, 3.10 μM). For other cancer types, the cell viability of AcKLP was slightly lower than that of ANF-treated cells, which might be attributed to the incomplete cleavage of the prodrug in these cells. Notably, AcKLP exhibited a significantly reduced cytotoxic effect towards normal HUVEC (IC₅₀ > 100 μM) compared to ANF (IC₅₀, 0.80 μM), with the cancer selectivity index (CSI) values for AcKLP and ANF being >44.25 and 0.25, respectively. In contrast, the IC₅₀ values of the prodrug PhTLP for both cancer and normal cells were similar, indicating that the cysteine-responsive prodrug PhTLP lacked selective cytotoxicity towards cancer cells. Taken together, these findings suggest that AcKLP can selectively target enzyme-positive cells and is therefore suitable for the treatment of GBM. Based on these results, our fluorescent prodrug AcKLP was subjected to additional biological assays.

2.3. In vitro drug release studies

To investigate the activation of the prodrug AcKLP, we used fluorescence imaging in live U87 cells. As shown in Fig. 2, red fluorescence was observed in live cells and increased over 6 hours, confirming the release of ANF. Moreover, the behavior of drug release was validated and confirmed using LC-MS assays (Fig. S5^†). Co-localized fluorescence imaging revealed that the fluorescence signal generated by the prodrug co-localized with lysosomes (Fig. S6^†), suggesting that the prodrug primarily targets lysosomes and releases ANF after being activated therein, which may be due to CTSL, a lysosomal protease, being involved in the activation process. Besides, when U87 cells were incubated with AcKLP in the presence of a histone deacetylase inhibitor (SAHA) or a cathepsin L inhibitor (E64D), the fluorescence signal intensities were attenuated (Fig. S7^†). These findings indicate that the release of ANF from AcKLP occurs in cancer cells as a result of the combined actions of histone deacetylases and cathepsin L. To evaluate its selective release in tumor cells, we further incubated the prodrug AcKLP in normal cells (HUVEC), and the results indicated that normal cells treated with AcKLP exhibited only weak fluorescence (Fig. S8^†). In addition, the cellular uptake of AcKLP was analyzed by HPLC. As shown in Fig. S9,^† lysates of U87 cells treated with AcKLP for 6 h confirmed that AcKLP could be effectively converted into ANF. In marked contrast, no ANF product was detected in the lysates of HUVEC cells. These observations confirmed the lysosomal targeting and activation process of AcKLP, providing insight into its potential effectiveness as a prodrug for cancer treatment.

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Fig. 2

Confocal microscopy images of U87 cells treated with 5 μM AcKLP for different times (λ_ex = 405 nm, λ_em = 550–650 nm). All the data represent the average of three independent experiments with an error bar of ±standard deviation (SD). The fluorescence intensity of control was set as 1. Statistical analyses were performed with a two-tailed Student's t-test with unequal variance, *p-value < 0.05, ***p-value < 0.001.

2.4. Anticancer mechanism of fluorescent prodrug AcKLP

Autophagy, a lysosome-dependent cellular degradation process responds to a variety of environmental and cellular stresses. Given that the prodrug AcKLP primarily targets lysosomes and releases ANF, we thus investigated whether AcKLP induces autophagy. The microtubule-associated protein 1A/1B-light chain 3 (LC3)-phosphatidylethanolamine conjugate (LC3-II), formed by the conjugation of the cytosolic form of LC3 (LC3-I) to phosphatidylethanolamine, is recruited to autophagosomal membranes and serves as a specific biomarker of autophagy. We first examined the expression of LC3 by immunofluorescence. As shown in Fig. 3A and B, red fluorescence was observed, and it increased in a concentration-dependent manner, indicating that the prodrug AcKLP induced a high expression level of LC3 and the formation of autophagosomes. The ratio of LC3-II/LC3-I has been verified as a specific marker of autophagy. To further confirm the ability of AcKLP to induce autophagy, we performed a Western blot analysis. As illustrated in Fig. 3C and S10,^† a concentration-dependent increase in the conversion of LC3-I to LC3-II was observed in AcKLP-treated cells. In addition, the STAT3 signaling pathway is implicated in many aspects of the autophagic process. The protein expression levels were thus detected to determine whether AcKLP could inhibit the STAT3 pathway. As shown in Fig. 3D and S11,^† the STAT3 expression and phosphorylation were inhibited after treatment with AcKLP in U87 cells for 24 h. To further validate the mechanism of AcKLP, the expression of the classical receptor of autophagy, the p62 protein, was examined. The results show that the content of p62 was downregulated in the AcKLP group, while the ANF group exhibited no significant change compared to the blank group (Fig. S12^†). Certainly, AcKLP has apoptosis-inducing effect to some degree owning to the partial transferring of the activated ANF from the lysosome to the nucleus (Fig. S13^†). To better understand the location of activated ANF, we incubated ANF or AcKLP with U87 cells for different durations and performed co-localization confocal imaging with nuclear dyes (Hoechst 33342). Analysis of cell images indicated that in the ANF group, most ANF fluorescence was observed in the nucleus, and the AcKLP group showed some degree of colocalization of red and blue fluorescence at 12 h and 24 h, suggesting that the activated ANF is transferred from the lysosome to the nucleus (Fig. S14^†). Taken together, these results suggest that AcKLP induces autophagic cell death in U87 cells, highlighting its potential as an effective anticancer agent through the modulation of autophagy and STAT3 signaling pathways, which differs from its precursor ANF.

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Fig. 3

(A) Fluorescence imaging of U87 cells treated with different concentrations of AcKLP, stained for the LC-3 antibody. The LC-3 channel is shown in red, λ_ex = 561 nm, λ_em = 570–680 nm. (B) Quantification of relative fluorescence intensities in (A). The fluorescence intensity of cells treated with 0 μM AcKLP was set as 1. Data are presented as mean ± S.D., n = 3. **p-value < 0.01, ***p-value < 0.001. (C) Expression levels of LC3-I and LC3-II and (D) expression levels of STAT3 and p-STAT3 in U87 cells treated with the indicated doses of AcKLP for 24 hours, as determined by western blotting. β-Actin was used as a protein loading control.

2.5. Cytotoxicity in 3D multicellular tumor spheroids

To further validate the anti-tumor effect of AcKLP, we used three-dimensional (3D) multicellular tumor spheroids (MCTSs). These spheroids, which typically exhibit oxygen gradients at diameters ranging from 300 to 500 μm and central hypoxia, serve as a model to simulate solid tumors.²⁸ The performance of the AcKLP treatment was then evaluated. As shown in Fig. 4A and B, after 4 days of incubation with 20 μM AcKLP or 2 μM doxorubicin (DOX), the spheroids began to disintegrate and lose their original structure, indicating that AcKLP effectively disrupted the integrity of the tumor spheroids. Furthermore, staining of the spheroids with propidium iodide (PI) revealed that the dead cells in the AcKLP-treated group emitted red fluorescence (Fig. 4A). These results indicated that AcKLP inhibits the growth of U87 cells in a three-dimensional tumor model, demonstrating its potential efficacy in more complex tumor microenvironmental systems.

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Fig. 4

(A) Representative pictures of 3D U87 tumor spheroids treated with ANF (20 μM), AcKLP (20 μM) and DOX (2 μM) at different times. The blue color (Hoechst 33342, λ_ex = 405 nm, λ_em = 420–500 nm) marks the healthy cells, and the red color (propidium iodide, λ_ex = 561 nm, λ_em = 600–640 nm) indicates the dead cells. (B) The volume changes of 3D tumor spheroids under different conditions with an extended incubation time from 0 to 4 days. Error bars: S.D., n = 3.

4.2. Synthesis of fluorescent prodrugs

4.3. Fluorescence analysis

Solutions of AcKLP (10 μM) and ANF (10 μM) in PBS solution (10 mM, pH 7.4, 1% DMSO) were analyzed using a UV spectrophotometer and a fluorescence spectrometer. The response of the probe PhTLP (10 μM) to Cys in PBS buffer (10 mM, pH 7.4, 1% DMSO, and 1 mM CTAB) was measured by both fluorescence and UV spectrophotometry, including UV absorption, excitation, and emission spectra.

4.4. Stability study

The stability of AcKLP was assessed using UV-vis spectroscopy. A solution of AcKLP (10 μM) in PBS solution (10 mM, pH 7.4, 1% DMSO) was prepared, and its absorbance was recorded at different time intervals over a 48 hour period at 25 °C.

4.5. Cell culture

Cell lines (HCT116, A549, MCF-7, HeLa, HUVEC, and U87) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco Company, USA), supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS). The cultures were maintained in a 5% CO₂ atmosphere at 37 °C.

4.6. Cell viability assay

The cytotoxicity of the fluorescent prodrugs AcKLP and PhTLP was evaluated using the Cell Counting Kit-8 (CCK-8) assay in both normal and tumor cells. Cells were seeded in 96-well plates at a density of 5000 cells per well and incubated in a water-saturated incubator with 5% CO₂ at 37 °C for 12 hours. Subsequently, the prodrugs AcKLP and PhTLP were added at varying concentrations (0.01, 0.1, 1, 10, and 100 μM). After an additional 48 h of incubation, 100 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 4 h at 37 °C. Afterward, the cell viability was determined by measuring the absorbance at 450 nm using a microplate reader. Each concentration was tested in triplicate wells.

4.7. Cellular confocal imaging

To verify the responsiveness of prodrug AcKLP, U87 cells were subjected to fluorescence signal analysis after different treatments. The U87 cells were seeded into 12-well plates at a density of 2 × 10⁴ cells per well and incubated for 12 hours. Following this, the cells were incubated with AcKLP for 0, 1, 2, 4, and 6 h. After incubation, the cells were washed three times with PBS and imaged using a Carl Zeiss LSM 880 confocal fluorescence microscope. Fluorescence signals from different treatments were quantitatively analyzed using ImageJ software.

4.8. Immunofluorescence assays

U87 cells in the logarithmic growth phase were plated into 12-well plates (2 × 10⁴ cells per well) and incubated for 12 h. The cells were then treated with various concentrations of the prodrug AcKLP (1 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM) for another 24 h. After treatment, the cells were washed three times with PBS buffer and fixed with 3.7% formaldehyde for 15 min at room temperature. Afterward, the cells were washed again with PBS, permeabilized with 0.2% Triton X-100 for 15 min, and incubated with the anti-LC3 rabbit polyclonal antibody overnight. Then, the working buffer was discarded, and the cells were washed twice with PBS and incubated with FITC-labeled goat-anti-rabbit lgG for 1 h in the dark. After washing three times with PBS, the cells were stained with DAPI for 5 min. Finally, the samples were analyzed using confocal fluorescence microscopy. DAPI staining (blue) was observed with an excitation wavelength of 405 nm and an emission range of 430–490 nm, while the LC3 channel (red) was observed with an excitation wavelength of 561 nm and an emission range of 570–680 nm.

4.9. Western blot analysis

U87 cells were treated with various concentrations of the probe AcKLP (1, 2.5, 5, 7.5, and 10 μM) and then lysed in RIPA buffer (Beyotime, Shanghai, China) on ice for 30 minutes. Then, the mixture was centrifuged at 13000g for 15 min at 4 °C, and the supernatant was collected. Protein concentrations were determined using the BCA protein assay kit (EpiZyme, Shanghai, China). Proteins were separated by SDS-PAGE (Bio-Rad) according to the molecular weight of the target proteins and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk at room temperature for 1 hour and then incubated with primary antibodies overnight at 4 °C. After washing four times with 1× TBST, membranes were incubated with peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG) at room temperature for 1 hour. Signals were detected using an enhanced chemiluminescence reagent (Thermo Scientific, Rockford, IL, USA) and visualized with a Tanon-4600SF imaging system. The protein levels were normalized to β-actin and quantified using ImageJ software. Antibodies were sourced as follows: LC-3 and Stat3 from Proteintech, Inc., USA; Phospho-Stat3 (Try705) from Cell Signaling Technology Inc., USA.

4.10. 3D multicellular tumor spheroid culture

U87 cells were seeded into a U-shaped 96-well plate at a density of 6 × 10⁴ cells per well and cultured at 37 °C with 5% CO₂ for 5 days to form a 3D multicellular tumor spheroid with a radius of 500 μm. The cell culture medium was replaced with fresh medium every two days.

4.11. 3D multicellular tumor spheroid imaging

Five-day-old multicellular tumor spheroids (MCTSs) were treated with ANF (20 μM), AcKLP (20 μM), and DOX (2 μM). The spheroid growth was monitored using an inverted fluorescence microscope (Brightfield, Olympus, RVL2-K, Echo). After four days, the MCTSs were stained with 5 μL of propidium iodide (PI) and 5 μL of Hoechst 33342 for 30 min at 37 °C and then imaged using an inverted fluorescence microscope (Hoechst 33342, λ_ex = 405 nm, λ_em = 420–500 nm; propidium iodide, λ_ex = 561 nm, λ_em = 600–640 nm).

This work was financially supported by the National Science Fund for Excellent Young Scholars (No. 22222704) and the National Natural Science Foundation of China (22477061, 22074065, and 22307006). Y. Q. also thanks the 2023 Study Abroad Program for Young Backbone Teachers and Nanjing Normal University for their support. T. D. J. wishes to thank the University of Bath and the Open Research Fund of the School of Chemistry and Chemical Engineering, Henan Normal University (2020ZD01) for their support.