Cysteine-responsive, cyano-functionalized acenaphthopyrazine derivative for tumor microenvironment modulation-based chemotherapy sensitization and side effect reduction

Abstract

Drug resistance and serious side effects are persistent obstacles in chemotherapy. Tumor microenvironment modulation is an emerging strategy to sensitize chemotherapy; however, the relevant side effects caused by chemotherapeutic drugs remain non-negligible. Herein, we constructed a cysteine-reactive, cyano-functionalized acenaphthopyrazine derivative for cisplatin sensitization and side effect reduction by regulating the tumor microenvironment. The developed cyano-functionalized acenaphthopyrazine derivative exhibited appropriate reactivity toward cysteine via an addition reaction. The incorporation of the cyano group not only improved the cellular uptake efficiency of cisplatin but also suppressed the drug inactivation behavior of tumor cells by reducing the expression of GSH within tumor cells. Moreover, selective inhibition of tumor cells was achieved due to the differing GSH dependence between normal and tumor cells. Most importantly, in vivo experiments revealed that the combination of the cyano-functionalized acenaphthopyrazine derivative with cisplatin could efficiently reduce liver and kidney damage during treatment. Our results demonstrated that cysteine consumption could serve as a general strategy for chemotherapy sensitization.

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1. Introduction

Chemotherapy, as the most commonly used treatment strategy, has been widely employed to treat various cancers based on the use of powerful chemicals to kill fast-growing cells; however, it often encounters the obstacle of drug resistance and serious side effects, which frequently result in treatment failure.¹ Take cis-diammineplatinum dichloride as an example; cisplatin is one of the first metal-based chemotherapeutic drugs used to treat solid tumors and hematologic malignancies. However, the molecular mechanisms of altered cellular accumulation, increased DNA repair and enhanced drug inactivation result in the development of resistance to cisplatin.² There is also another clinical impediment of cisplatin-based chemotherapy. To improve treatment efficacy and sensitize chemotherapy, diverse strategies have been established, including reducing drug resistance-related proteins,^3–5 regulating the cell death process⁶ and modulating the tumor microenvironment (TME). Among these strategies, modulation of the tumor microenvironment has emerged as an attractive approach to combat tumor drug resistance, as the tumor microenvironment plays a pivotal role in the development of drug resistance.⁷

To date, many approaches have been developed to regulate the TME for chemotherapy sensitization.^8,9 Targeting abnormal characteristics, such as acidosis, hypoxia, nutrient depletion, and elevated glutathione (GSH)/reactive oxygen species (ROS) levels, strategies such as improving oxygen concentration,¹⁰ neutralizing tumor pH,¹¹ triggering ER stress⁹ and downregulating intracellular GSH expression^12–15 have been established to efficiently improve antitumor efficacy. Although regulating the TME allows for successful sensitization of chemotherapeutic drugs, the side effects caused by chemotherapy still need to be addressed due to its relatively low specificity.

Many bioorthogonal reactions based on the interaction between cyano groups and cysteine have been developed for the specific labeling of terminal cysteine (Cys) residues in biomacromolecules.^16,17 A landmark study by Rao and colleagues in 2009 introduced a biocompatible condensation reaction between the cyano group of 2-cyanobenzothiazole (CBT) and the 1,2-aminothiol group of Cys. This reaction demonstrated a remarkable second-order reaction rate constant of 9.19 M⁻¹ s⁻¹ and was successfully applied to protein labeling both in vitro and on surfaces.¹⁸ Based on this foundational work, various electrophilic aromatic nitriles have been developed to target proteins with N-terminal cysteine residues, e.g., 2-cyanoquinoline, 2-cyanopyridine,¹⁹ 2-cyanopyrimidine,¹⁶ 2-((alkylthio)(aryl)methylene)malononitrile,²⁰ 2,6-dicyanopyridine²¹ and 2-cyanobenzoxazoles,²² among others. It has been demonstrated that enhancing electron delocalization through fused aromatic rings or incorporating heteroatoms with strong electron-withdrawing capabilities into the aromatic core significantly improves the reactivity of these nitriles.^16,22 Cyano-functionalized acenaphthopyrazine (CNAP) is a particularly promising scaffold due to its potent electron-withdrawing properties and extended conjugation. These features not only make CNAP an exceptional acceptor building block for thermally activated delayed fluorescence (TADF) materials, enabling pure near-infrared emission,²³ but also suggest that it may exhibit superior reactivity with Cys compared to previously reported aromatic nitriles. The combination of its strong electron-deficient nature and extended π-conjugated framework positions CNAP as a highly reactive candidate for bioorthogonal applications.

Based on the above design concept, we presented a novel and efficient chemotherapy sensitizer, 4-(4-(phenylamino)phenyl)acenaphtho[1,2-b]pyrazine-3,8,9-tricarbonitrile (PZNA) (Scheme 1). This compound integrated a strongly electron-deficient acenaphtho[1,2-b]pyrazine-3,8,9-tricarbonitrile (APTC) core with a diphenylamine (DPA) moiety. The APTC scaffold was strategically selected for its high reactivity in bioorthogonal reactions with Cys, leveraging its robust electron-withdrawing properties. The conjugation with DPA further enhanced the electronic delocalization across the molecule, thereby augmenting its reactivity. The PZNA NPs were constructed to sensitize cisplatin treatment and reduce the side effects by decreasing the expression of intracellular GSH. Cellular component analysis revealed that the incorporation of the cyano group into the acenaphthopyrazine derivative enhanced the cellular uptake efficiency of cisplatin. Furthermore, benefiting from the depletion of Cys, the expression of intracellular GSH was efficiently reduced, which allowed for the suppression of drug inactivation and enhancement of therapeutic efficacy. In addition, selective ablation of tumor cells was achieved due to the differential GSH-dependent behavior of normal and tumor cells. In vivo observations verified the desirable tumor inhibition ability of the combined treatment, and the serious damage caused by DDP to the liver and kidneys was significantly reduced by the incorporation of the cyano-functionalized acenaphthopyrazine derivative, demonstrating its high potential for chemotherapy sensitization in the clinic.

Scheme 1 Illustration of PZNA NPs treated with sensitizing cisplatin and reducing side effects by lowering intracellular GSH expression.

2. Experimental section

2.1. Preparation of PZNA NPs

PZNA (1 mg) and DSPE-PEG₂₀₀₀ (2 mg) were fully dissolved in THF (1 mL). The mixture was quickly injected into 15 mL of deionized water under an ultrasonic crusher at 25% power for 4 min, and then stirred at room temperature for two days. The crude NPs were further filtered through a membrane filter (diameter = 0.22 μm) for further use.

2.2. Cell cultures and imaging

4T1 cells were cultured in DMEM medium supplemented with 10% FBS and antibiotics (100 units per mL penicillin and 100 μg mL⁻¹ streptomycin) in a humidified incubator with 5% CO₂ at 37 °C.

4T1 cells were seeded in confocal dishes at 37 °C. The cells were pretreated with OA (50 μM mL⁻¹) for 12 h, washed with PBS, and then co-incubated with PZNA NPs (20 μM mL⁻¹) for 12 h. Afterward, the cells were rinsed with PBS, stained with 1 μL of the lipid droplet probe BODIPY for 30 min, and gently washed with PBS. Finally, the cells were imaged with CLSM.

2.3. Intracellular GSH evaluation

4T1 cells were seeded in confocal dishes at 37 °C. After incubation with PZNA NPs (20 μM mL⁻¹) for 0, 2, and 7 h, the cells were stained with 18 μM mL⁻¹ ThiolTracker™ Violet for 30 min. Finally, the cells were gently washed with PBS and observed using CLSM.

2.4. Cytotoxicity test

4T1 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per mL. Cytotoxicity studies of PZNA NPs were first performed. Cells were incubated with different concentrations of PZNA NPs in culture medium for 12 h, followed by incubation with MTT (0.5 mg mL⁻¹) for 4 h. Finally, the absorbance of the product was measured at 490 nm, and the relative cell viability was calculated. For sensitization evaluation, cells were treated with fresh medium containing PZNA NPs (8 μM mL⁻¹) for 10 h. Then, cisplatin was added to the culture medium at different concentrations (0.25, 0.5, 1, 5, 10, 15, 20 and 25 μg mL⁻¹) and incubated for an additional 14 h. Finally, the culture medium was replaced with MTT (0.5 mg mL⁻¹) in DMEM and incubated for another 4 h. The absorbance of the product was measured at 490 nm, and the relative cell viability was calculated.

2.5. Live/dead cell imaging assay

4T1 cells (1 × 10⁵ cells per well) were seeded into confocal dishes and cultured for 24 h. Then, the 4T1 cells were treated with different conditions: (1) blank; (2) PZNA NPs; (3) DDP; (4) DDP + PZNA NPs. The concentrations of PZNA NPs and DDP were 15 μM mL⁻¹ and 10 μg mL⁻¹, respectively. After 36 hours, cells were co-incubated with calcein-AM (green, living) and propidium iodide (red, dead) for 30 min. Finally, the cells were directly imaged using CLSM.

2.6. Flow cytometry assay

4T1 cells were cultured in 60 mm cell dishes for 24 h. After incubation with FBS-free medium, NPs (10 μM mL⁻¹, 24 h), DDP (10 μg mL⁻¹, 24 h) and DDP + NPs, the supernatants were collected into centrifuge tubes. The adherent cells were digested with trypsin, and the digestion was subsequently terminated using medium in the centrifuge tubes. After centrifugation, the cells were gently resuspended in PBS and counted. Fifty thousand resuspended cells were centrifuged and co-stained with Annexin V-FITC/PI. Flow cytometry data were analyzed using FlowJo v10.

2.7. ROS generation measurement in vitro

4T1 cells were seeded into confocal imaging trays at 37 °C. Cells were separately treated with DDP (8 μg mL⁻¹), PZNA NPs (10 μM mL⁻¹) or PZNA NPs + DDP for 12 h. Subsequently, cells were stained with 10 μM mL⁻¹ DCFH for 30 min. After washing 3 times with PBS, the cells were observed using CLSM.

2.8. Intracellular mitochondrial membrane potential (ΔΨ_m)

Cells were treated with different conditions: FBS-free medium, DDP (5 μg mL⁻¹, 24 h), PZNA NPs (10 μM mL⁻¹, 12 h) and DDP + PZNA NPs. For the co-treatment group, cells were pre-incubated with NPs for 12 h and then incubated with DDP for 24 h. Finally, the cells were stained with JC-1 (3 μg mL⁻¹) for 30 min, rinsed with PBS and observed using CLSM.

2.9. RNA-seq and data analysis

4T1 cells (5 × 10⁶) were cultured in 60 mm cell culture dishes and treated with PZNA NPs (30 μM mL⁻¹) for 12 h. Cells treated with culture medium were used as the control. Total RNA from the samples was extracted and purified using Trizol (Thermo Fisher, 15596018) following the manufacturer's protocol. The obtained transcriptome sequencing count data were analyzed using the RNASeqStat2 package. After filtering out genes with expression levels of 0, differentially expressed genes were identified using the DESeq2 differential analysis algorithm, followed by genome enrichment analysis (GSEA) of 50 pathways (Hallmarks) associated with tumor. The threshold criteria for filtering differentially expressed genes (DEGs) were set as fold change >2 (i.e., absolute value of log₂ FC > 2) and adjusted p-value <0.05.

2.10. Immunofluorescence assay

First, 4T1 cells were treated with different conditions: (1) FBS-free culture medium; (2) PZNA NPs; (3) DDP; (4) DDP + PZNA NPs. The concentrations of PZNA NPs and DDP were 15 μM mL⁻¹ and 8 μg mL⁻¹, respectively. After 24 h of treatment, the cells were washed twice with PBS. Subsequently, the cells were stained with Hoechst 33342 for 20 min. Then, cells were fixed with 4% paraformaldehyde for 15 min at 4 °C, and blocked with protein-free rapid closure solution for 1 h. The cells were incubated overnight at 4 °C with an anti-calreticulin antibody, followed by further incubation at room temperature for 2 h with an Alexa Fluor 488 secondary antibody. Afterward, the cells were subjected to CLSM analysis.

2.11. Antitumor efficacy of PZNA NPs in vivo

Female BALB/c mice (7–8 weeks old) were purchased from China Boryxin Biotechnology Co. (Hunan, China). All animal procedures were approved by the Animal Ethics Committee of The University of South China according to the regulations for the administration of affairs concerning experimental animals (Hunan Province, China); approval number: permission 121. The animals were in normal health and immunization status. The mice were housed in SPF-grade animal rooms at 20–26 °C, 40–70% humidity, under a 12/12 h light/dark cycle, with 5 mice per cage and free access to food and water.

The mice were subcutaneously injecting with 4T1 cells (1 × 10⁷ cells) into the right hips using PBS buffer. When the tumor size reached approximately 5 to 7 mm, the 4T1 cell-bearing mice were divided into 5 groups for intravenous injection of the corresponding agents. The mice in the PBS and PZNA NPs (2 mM mL⁻¹) groups were injected with 100 μL of PBS or PZNA NPs via the tail vein. The mice in the DDP group were injected with DDP (2 mg kg⁻¹) via the tail vein.

I: Sterilized PBS (n = 10), II: PZNA NPs (n = 10), III: DDP (n = 10), IV: PZNA + DDP (n = 10), V: Blank (n = 10).

Tumor volumes (V) were calculated according to the following formula:

V = (length_tumor × width_tumor²) ÷ 2

2.12. Hemolysis assay

Hemolysis assays were conducted to assess the in vivo toxicity of PZNA NPs. Briefly, red blood cells were harvested from healthy BALB/c mice and centrifuged at 2500 rpm for 5 min, and the serum was removed using a syringe. The cells were washed five times with 1× PBS solution and then suspended in 1× PBS solution to prepare a 5% erythrocyte suspension. The diluted red blood cell suspension (0.2 mL) was then mixed with 0.8 mL of various samples: PBS as a negative control, ultrapure water as a positive control and different concentrations of PZNA NPs in PBS solution (final PZNA NPs concentrations of 0.5, 1, 2, 4, 8, 16, 32 and 64 μM mL⁻¹). Subsequently, the mixtures were shaken at 37 °C for 2 h. Finally, the mixtures were centrifuged at 3000 rpm for 5 min to precipitate erythrocytes. The absorbance at 540 nm was recorded using a microplate reader. Hemolysis rate (HR) was calculated as:

HR = (O.D._samples − O.D._negative) ÷ (O.D._positive − O.D._negative)

2.13. In vivo biosafety evaluation of NPs

Tumor volume and body weight were monitored every three days throughout the treatment period. After 16 days of treatment, blood samples were collected from each mouse following anesthesia with 1.2% isoflurane gas, and were used for routine and biochemical analyses. Finally, mice were decapitated after deep anesthesia with 5% isoflurane, and major organs (heart, liver, spleen, lungs and kidneys) and tumors from mice in different treatment groups were collected for H&E staining. In addition, tumor samples were collected for TUNEL staining. Mice survival was independently assessed at 31 days.

2.14. Statistical analysis

Most data were obtained from at least three independent experiments. Quantifications were performed in a blinded manner. Statistical analysis was performed with Origin 2021 and GraphPad Prism 9.5.

3. Results and discussion

3.1. Synthesis and characterization of PZNA NPs

The synthetic route for PZNA is illustrated in Scheme S1 (ESI†), with detailed procedures and characterization data provided in the ESI† (Fig. S1–S5). Briefly, the target compound was synthesized via a four-step process comprising Suzuki coupling, cyanation, nitrosation and final cyclocondensation. To gain insights into the chemical structure of PZNA, density functional theory (DFT) calculations were performed using Gaussian 09 at the B3LYP/6-31G(d) level. As shown in Fig. 1(a), the optimized geometry revealed a twisted conformation with a dihedral angle of 51.6° between the APTC and DPNA fragments, indicating its potential to inhibit molecular close packing. Frontier molecular orbital analysis showed that the highest occupied molecular orbital (HOMO) was predominantly localized on the DPNA unit, while the lowest unoccupied molecular orbital (LUMO) was concentrated on the APTC unit, with partial overlap on the adjacent phenyl and naphthalene segments (Fig. 1(b)). This distribution underscored the charge-transfer character of PZNA. Electrostatic potential (ESP) mapping further highlighted nucleophilic regions (blue) on the cyano and nitroso groups, as well as the pyrazine nitrogen atoms, while electrophilic regions (red) were primarily distributed across the conjugated backbone and peripheral phenyl fragments (Fig. 1(c)). Notably, the ESP surface minima (−36.73 kcal mol⁻¹) were located near the nitrogen atom of the cyano group at the 9-position of APTC, suggesting enhanced reactivity of this group in bioorthogonal conjugation reactions with cysteine residues.²⁴

Fig. 1 Characterization of the photosensitizer. (a) Optimized molecular geometries. (b) Frontier molecular orbitals from density functional theory (DFT). (c) Electrostatic potential map. (d) Mass spectrum of the product. (e) Reactions between PZNA and Cys. (f) Absorption and emission spectra of PZNA in toluene (10⁻⁵ M). (g) Zeta potential of PZNA nanoparticles.

To further elucidate the reaction mechanism, high-resolution mass spectrometry (HRMS) was conducted. As shown in Fig. 1(d), an exact molecular ion at 551.1301 m/z was detected, which could be readily assigned to the addition product of compound 2. Combined with the ESP mapping results, we speculated that PZNA would react with Cys via an addition reaction (Fig. 1(e)). We then investigated the photophysical properties of PZNA. As shown in Fig. 1(f), a pronounced charge-transfer character was observed, with absorption at approximately 510 nm and a broad emission centered at 651 nm, consistent with theoretical predictions. To enhance solubility for biological applications, PZNA was encapsulated using DSPE-PEG₂₀₀₀, forming nanoparticles with a zeta potential of −27.3 mV (Fig. 1(g)), which could reduce non-specific interactions with biomacromolecules. These results demonstrated the potential of PZNA-based nanoparticles for both in vivo and in vitro studies.

3.2. In vitro anti-tumor ability

Given the desirable reactivity of PZNA with Cys, we first assessed its affinity and binding behavior toward cells. In this work, 4T1 cells were selected as a representative tumor cell line. The 4T1 cells were initially treated with PZNA NPs and subsequently characterized by confocal laser scanning microscopy (CLSM). The results showed that the nanoparticles could effectively cross the cell membrane, and further colocalization analysis showed good enrichment with the lipid droplet probe BODIPY, with a Pearson correlation coefficient (PCC) of 0.65 (Fig. 2(a)). Given the favorable extracellular response of PZNA NPs to Cys, we assessed changes in intracellular GSH levels after treatment with PZNA NPs, since Cys is a critical precursor for GSH synthesis. As shown in Fig. 2(b) and (c), GSH levels in tumor cells were monitored using the GSH probe ThiolTracker. The results indicated that the GSH level in 4T1 cells decreased substantially with extended incubation time of the NPs. We proposed that the reaction of PZNA NPs with Cys inhibited the synthesis of intracellular GSH. Depletion of Cys and inhibition of GSH biosynthesis in cells may lead to dysregulation of intracellular redox homeostasis, resulting in the massive accumulation of ROS and ultimately leading to cell death. However, this strategy could not efficiently sensitize DPP against drug-resistant tumor cell lines owing to the limited GSH-consuming ability (Fig. S6, ESI†). These results demonstrated that PZNA presented high promise for therapeutic applications.

Fig. 2 Intracellular GSH evaluation. (a) Imaging of co-staining with the lipid droplet probe BODIPY after 12 h of co-incubation of 4T1 cells with PZNA NPs (20 μM mL⁻¹), following OA pre-treatment (50 μM mL⁻¹, 12 h). Scale bar: 10 μm. (b) Glutathione levels in 4T1 cells from different treatment groups after incubation with PZNA NPs for varying times, co-stained with ThiolTracker (18 μM mL⁻¹, 30 min). Scale bar: 10 μm. (c) Changes in average GSH fluorescence after different treatments.

To validate this hypothesis, we investigated the toxicity of PZNA NPs in different cell lines using the classical MTT assay, compared their therapeutic effects on various cells and further explored their sensitizing effects on chemotherapy. cis-Platinum (DDP) was chosen as a representative chemotherapeutic agent in this work. The results showed that below a concentration of 15 μM mL⁻¹, PZNA NPs caused minimal harm to both normal cells (L929) and tumor cells (4T1) (Fig. S7, ESI†). We then explored the efficacy of DDP with and without pretreatment with 8 μM mL⁻¹ PZNA NPs. Only weak cytotoxicity was detected in both normal cells, with and without treatment with PZNA NPs, which was attributed to the relatively low concentration of Cys in the normal cell lines (Fig. 3(a)). Consistent with expectations, the inhibitory effect of DDP (25 μg mL⁻¹) on 4T1 cells was significantly enhanced after treatment with PZNA NPs (Fig. 3(b)), suggesting that PZNA NPs are excellent synergists for sensitizing chemotherapy, with good inhibitory selectivity for tumor cells.

Fig. 3 In vitro tumor inhibition characterization. Cell viability of L929 cells (a) and 4T1 (b) cells after pretreatment with and without PZNA NPs for 10 h, followed by incubation with different concentrations of DDP for 14 h (n = 6). (c) Live/dead cell imaging of 4T1 cells after 36 h treatment with FBS-free medium, PZNA NPs (15 μM mL⁻¹), DDP (10 μg mL⁻¹) or DDP + PZNA NPs; calcein AM (green, living) and propidium iodide (red, dead). Scale bar: 20 μm. (d) Flow cytometry analysis of 4T1 cells after treatment with FBS-free medium, PZNA NPs (10 μM mL⁻¹, 24 h), DDP (10 μg mL⁻¹, 24 h) and DDP + PZNA NPs. (e) Morphological changes of 4T1 cells revealed by scanning electron microscopy. The 4T1 cells were treated with PZNA NPs (10 μM mL⁻¹), DDP (8 μg mL⁻¹) and DDP + PZNA NPs for 12 h. Scale bar: 5 μm. (f) Generation of ROS in 4T1 cells in different treatment groups by co-stained with DCFH after incubation with FBS-free medium, DDP (8 μg mL⁻¹, 12 h), PZNA NPs (10 μM mL⁻¹, 12 h) and DDP + PZNA NPs. Scale bar: 20 μm. (g) Mitochondrial membrane potential changes in 4T1 cells from different treatment groups, co-stained with JC-1 (3 μg mL⁻¹, 30 min) after incubation with FBS-free medium, DDP (5 μg mL⁻¹, 24 h), PZNA NPs (10 μg mL⁻¹, 12 h) and DDP + PZNA NPs. Scale bar: 20 μm. Data are expressed as mean ± S.D. (standard deviation).

To further clarify the ablation effect, we next used calcein AM (AM) and propidium iodide (PI) to visualize the live–dead 4T1 cells under different treatments. As shown in Fig. 3(c), bright green fluorescence (live cells) was observed in both blank and DDP-treated groups, indicating the negligible killing effect of DDP under these conditions. However, intense red fluorescence (dead cells) was detected in tumor cells treated with PZNA NPs, which was further enhanced in the presence of DDP, consistent with the previous MTT observation.

To reveal the sensitization mechanism, inductively coupled plasma-mass spectrometry (ICP-MS) was utilized to explore the cellular uptake of DDP in the absence and presence of PZNA NPs. The results showed that, compared with the group treated with DDP alone, the intracellular content of Pt in 4T1 cells was increased by approximately 3.23 μg L⁻¹ with the combined treatment of PZNA NPs (Fig. S8, ESI†), suggesting that PZNA NPs facilitated the cellular uptake of DDP and further improved the therapeutic effect. The combined results verified the reliable sensitization behavior of PZNA towards chemotherapy.

3.3. In vitro anti-tumor mechanisms

To reveal the killing mechanism, flow cytometric analysis was employed to investigate the cytotoxicity of the combined treatments. The results showed that both apoptosis and necrosis were present in the groups with different treatments. However, the percentage of necrotic cells was 56.4% in the group treated with both PZNA and DDP (Fig. 3(d)), which was much higher than that in the PZNA-treated group (11.5%) and the DDP-treated group (0.67%), suggesting that the combined treatment induced tumor cell death via necrosis. Moreover, scanning electron microscopy (SEM) was used to observe the changes in 4T1 cells with different treatments. As shown in Fig. 3(e), filamentous pseudopods were clearly visible on the cell membrane in the blank group, whereas the cell membrane surface appeared smoother, accompanied by a reduction in filamentous pseudopods in the DDP, PZNA NPs and DDP + PZNA NPs groups. It is well known that filamentous pseudopods play important roles in perception, migration and cell-to-cell communication processes. Thus, we predicted that the treatment with PZNA and DDP would disrupt the filamentous pseudopods and further restrict the “communication” and migration behavior of 4T1 cells, ultimately limiting their proliferation and leading to cell necrosis.

Furthermore, 2′,7′-dichlorofluorescein diacetate (DCFH) was utilized to evaluate the expression of intracellular reactive oxygen species (ROS) in 4T1 cells with different treatments. As shown in Fig. 3(f), only weak signals of DCFH was detected in DDP-treated cells. Intense green fluorescence was observed in both PZNA NPs-treated and combined treatment groups, indicating efficient generation of ROS under these conditions. ROS mainly originate from mitochondria and are closely related to mitochondrial oxidative respiration, further triggering cell death. Thus, the evaluation of the mitochondrial membrane potential in 4T1 cells with different treatments was conducted. As shown in Fig. 3(g), compared with the blank and DDP-treated group, the red fluorescence (JC-1 aggregates) was significantly weakened after PZNA NP treatment, which was further decreased after the combined treatment, suggesting the loss of membrane potential and mitochondrial destruction. As a result, the above findings showed that the synergistic treatment of PZNA NPs with DDP could lead to the generation of intracellular ROS and mitochondrial destruction, which further caused cell necrosis and enabled the ablation of tumor cells in vitro.

To gain more insight into the variation in gene expression associated with synergistic treatment based on PZNA NPs, we employed RNA sequencing (RNA-seq) analysis to explore the effects of PZNA NPs on gene transcription. Differential analysis of gene expression was conducted for the blank and PZNA NPs groups, with three independent replicates performed for each group. Pearson's correlation coefficients ranged from 0.95 to 1, suggesting a high degree of reproducibility (Fig. S9, ESI†). Heatmaps were generated to demonstrate the profiles of differentially expressed genes (14 682 genes) in cells from both the blank and PZNA NPs-treated groups (Fig. 4(a)). Compared to the blank group, 201 genes were upregulated, and 703 genes showed downregulation after PZNA NP treatment (Fig. 4(b)). Furthermore, gene set enrichment analysis (GSEA) showed that PZNA NP treatment upregulated signaling pathways related to the G2M/DNA damage checkpoint, glycolysis, fatty acid metabolism and spindle formation (Fig. 4(d)–(g)). It downregulated signaling pathways related to myelocytomatosis oncogene (MYC) and tumor necrosis factor-α (TNFA) (Fig. 4(h) and (i)). The integrative outcomes indicated that tumor cell necrosis induced by PZNA NPs was closely related to cellular replication cycle-related pathways, substance metabolism and downregulation of intracellular proto-oncogenes.

Fig. 4 RNA-sequencing and pathway enrichment analysis in PZNA NPs-treated 4T1 cells (n = 3). (a) Heat map showing differentially expressed genes in Blank and PZNA NPs samples. (b) Volcano plot of differentially expressed RNAs. (c) GSEA enrichment analysis of significantly altered genes after PZNA NP treatment. (d)–(i) GSEA analysis of significantly enriched pathways in PZNA NPs-treated cells. Data are expressed as mean ± S.D. (standard deviation).

3.4. Evaluating the antitumor efficacy in vivo

Given the good synergistic therapeutic properties of PZNA NPs, we further investigated their in vivo inhibitory effect on solid tumors. As shown in Fig. 5(a), the tumor model was established approximately 7 days after the injection of 4T1 cells into the right hip of mice, which were then randomly divided into four groups. When the tumor volume reached 90 mm³, different treatment groups were injected via the tail vein, and a fifth group of five mice was left untreated as the blank group. To explore the potential of immunotherapy, we studied the expression of biomarkers related to immunogenic cell death (ICD) with different treatments. As shown in Fig. 5(b), relatively weak expression of calreticulin (CRT, green fluorescence) was observed in DDP-treated tumor cells, which was significantly improved upon PZNA NPs and combined treatment. Moreover, bright green fluorescence related to high mobility group box 1 protein (HMGB1), initially concentrated in the nucleus in the blank group, was redistributed to the cytoplasm and plasma membrane with only slight changes in intensity following combined treatment with DDP and PZNA NPs (Fig. S10, ESI†). These results revealed that treatment with PZNA NPs and combination therapy could trigger the expression of CRT and HMGB1, further inducing ICD in the treated tumor cells and activating the immune response, highlighting the potential for immune therapy. Then, the in vivo anti-tumor activity was evaluated. The mice were subsequently injected with PBS, DDP and DDP + PZNA NPs via the tail vein every three days. Tumors and organs from 5 mice were dissected for hematoxylin–eosin staining (HE staining) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) on day 16 (after the fifth round of administration), while the remaining mice were observed and monitored until day 30, and survival curves were plotted. As shown in Fig. 5(c), compared to the blank group, the body weight of mice treated with PZNA NPs did not vary significantly, demonstrating that PZNA NPs did not cause any damage to the mice. However, an obvious decrease in body weight was observed in mice treated with both DDP and the combined treatments, verifying the intense side effects of DDP. Meanwhile, compared to the PBS-treated mice, tumor volumes were suppressed in PZNA NPs-treated group, with further inhibition observed in the combined treatment group (Fig. 5(d) and (e)). Although the DDP-treated group showed the most efficient inhibition effect, the mice in this group showed the earliest death on day 15, accompanied by a significant reduction in body weight (Fig. 5(d) and (f)). Moreover, a significant increase in BUN expression was detected in DDP-treated mice, indicating liver damage and verifying the serious side effects of DDP treatment (Fig. 5(g)). Meanwhile, mice in the DDP + PZNA NPs combined treatment group remained alive even after 30 days of treatment, suggesting that the synergistic treatment could ameliorate the toxic side effects of DDP and prolong the survival time of the mice. In addition, the expression of ALB, ALP and BUN in both the PZNA NPs and combined treatment groups was similar to that of PBS-treated mice (Fig. 5(g)–(i)), demonstrating high biosafety. The hemolysis assay was also conducted to verify the good biocompatibility of PZNA NPs (Fig. 5(j)). The results showed that PZNA NPs caused minimal erythrocyte destruction in mice. It is worth noting that, compared with the HE-stained kidney sections of the other four groups of mice, the glomeruli in the renal pulp of DDP-treated mice were extensively damaged, which we speculated to result in a significant increase in urea nitrogen (Fig. S11 and S12, ESI†).

Fig. 5 In vivo tumor inhibition observation. (a) Treatment schedule for the in vivo anti-tumor study. (b) CLSM images of 4T1 cells with immunofluorescence staining of CRT (green fluorescence) and Hoechst 33342 (blue fluorescence) after different treatments. Scale bar: 20 μm. (c) Body weight of 4T1-tumor-bearing mice under different treatments (n = 5). (d) Relative primary tumor volume changes of 4T1-tumor-bearing mice over 16 days of different treatments (n = 5). (e) Representative images of tumors resected from 4T1 tumor-bearing mice at the end of 16 days of treatment: (I) PBS, (II) DDP (2 mg kg⁻¹), (III) PZNA NPs and (IV) DDP + PZNA NPs (n = 5). (f) Survival curves of 4T1-tumor-bearing mice over 30 days of treatment (n = 5). (g)–(i) Blood biochemistry markers of hepatic and renal function in 4T1-tumor-bearing mice after 16 days of treatment (n = 5). (j) Hemolysis assays of PZNA NPs (n = 3). (k) Flow cytometric analysis of splenic lymphocyte immunophenotyping in mice from different treatment groups after 16 days of treatment (n = 3). (l) TUNEL staining images of tumor tissues on day 16 after different treatments. Scale bar: 50 μm. Data are expressed as mean ± S.D. (standard deviation), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Benefiting from the successful induction of ICD in vitro, we also investigated the immunotherapeutic behavior based on the developed strategies. The maturation of dendritic cells (DCs) and the activation of immune cells were evaluated and analyzed. Different types of T cells in the spleen were investigated by exploring the relevant biomarkers of CD3, CD4 and CD8 via flow cytometry. Herein, CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes, acting as T-helper lymphocytes and T-suppressor cells, play significant roles in the immune system. As shown in Fig. 5(k), compared to the control group, the populations of CD3⁺CD4⁺ T lymphocytes increased from 52.8% to 75.81% after PZNA NP treatment, while the percentage of CD3⁺CD8⁺ T lymphocytes decreased from 33.2% to 19.91%. In contrast, only minimal variation was observed in both CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes in DDP-treated mice. However, upon combined treatment with DDP and PNZA NPs, the population of CD3⁺CD4⁺ T lymphocytes decreased from 52.8% to 34.0%, while the population of CD3⁺CD8⁺ T lymphocytes significantly increased from 33.2% to 50.9%. Thus, the incorporation of PNZA NPs treatment could provide significantly synergetic effect to enhance the therapeutic efficacy of DDP by activating the immune response in the treated mice.

Based on the desirable therapeutic efficacy, TUNEL staining was conducted to reveal the detailed mechanism of cell death in the solid tumor following the combined treatments (Fig. 5(l)). The results showed that the combined treatment of DDP with PNZA NPs resulted in more pronounced apoptosis in tumor tissues compared to the individual treatments of DDP and PNZA NPs. The combined results demonstrated that the synergistic treatment with PZNA NPs not only significantly enhanced the therapeutic effect but also reduced the side effects of DDP, presenting high promise for practical applications in clinical treatment.

4. Conclusion

In conclusion, PZNA bearing promising reactivity with Cys, was designed and prepared for chemotherapy sensitization applications by depleting intracellular GSH. The synergistic treatment of PZNA NPs with DDP effectively exerted anti-tumor effects both in vitro and in vivo by inducing cell necrosis, while greatly ameliorating the toxic side effects of DDP, such as renal tissue destruction and body weight loss, thereby prolonging the survival time of mice.

Author contributions

Hanyi Gao: writing – review & editing, writing – original draft, visualization, validation, software, resources, methodology, investigation, formal analysis, data curation. Yiliang Qin: writing – original draft, visualization, validation, software, methodology. Jiayi Li and Shuhong Xiong: resources, investigation, validation, visualization. Rong Sun: resources, visualization. Xia He: data curation, visualization. Yaxin Wu: resources. Ying Tian: funding acquisition, supervision. Yi Yuan: writing review & editing, funding acquisition, formal analysis, conceptualization. Rong Hu: writing – review & editing, supervision, project administration, funding acquisition, formal analysis, conceptualization.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article or its ESI.†

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by the Hunan Provincial Natural Science Foundation of China (2024RC3206, 2021JJ304060, LINC00475, 2023JJ40552), the Scientific Research Fund of the Hunan Provincial Education Department (22A0287) and the Open Fund of the Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates, Guangzhou 510640, China (South China University of Technology) (2023B1212060003).

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Footnotes

† Electronic supplementary information (ESI) available: The synthetic route for PZNA is illustrated in Scheme S1, with detailed procedures and characterization data provided in Fig. S1–S5. The biological part was shown in Fig. S6–S12. See DOI: https://doi.org/10.1039/d5qm00229j

‡ These authors contributed equally to the work.

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