Curcumin mediates glutathione depletion via metal–organic framework nanocarriers to enhance cisplatin chemosensitivity on esophageal cancer

Abstract

Cisplatin (CDDP) is the primary drug used in the initial treatment of esophageal cancer (EC). However, its side effects and resistance can limit its effectiveness in clinical therapy. Curcumin (Cur)-mediated glutathione (GSH) depletion can reverse resistance, enhance the chemosensitivity of CDDP, and further improve the efficacy of platinum-containing chemotherapy in the treatment of esophageal cancer. However, it is also faced with problems of poor water solubility and low bioavailability in vivo, which severely hinders cancer treatments. In order to address these issues, we developed a novel nanotherapeutic system called CDCZA, combining Cur/CDDP/Cu/ZIF8@Au to enhance chemotherapy through GSH depletion and chemodynamic therapy through self-produced H₂O₂. Cu and CDDP were precisely co-loaded into Cu/ZIF8 nanoparticles using a one-pot method, then ultra-small gold nanoparticles mimicking glucose oxidase (Au nanoparticles) were embedded in the outer shell to create the CDCZA nano system. The released Cur could notably decrease intracellular GSH content and thus improve the chemosensitivity of CDDP, resulting in severe cellular apoptosis. And the Au nanoparticles effectively enabled chemodynamic therapy enhancement by accelerating the depletion of β-D-glucose into H₂O₂. As a result, the CDCZA nanoparticles showed increased tumor accumulation and improved antitumor effectiveness in a model of EC. Taken together, this work provides a new idea for the clinical design of efficient treatment reagents for EC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-024-04168-5.

Introduction

Esophageal cancer (EC) is the eighth most common cancer worldwide [1]. This deadly illness results in over 400,000 fatalities annually. Two primary histopathologic cell categories, esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), exhibit distinct geographic patterns. In Western countries, EAC is the prevailing subtype and one of the cancers that is growing most quickly. On the other hand, ESCC is the most common type of EC worldwide, being most prevalent in eastern Asia and some regions of Africa. Patients diagnosed with EC typically have a low five-year survival rate of around 15%, with the majority of cases being unresectable or metastatic upon presentation [2, 3].

Cisplatin (CDDP), a type of chemotherapy containing platinum, is frequently administered to patients with inoperable ESCC in medical practice. Nevertheless, CDDP is intrinsically a harmful substance when used in medical settings, leading to symptoms such as queasiness, throwing up, damage to the ears, and kidney damage. Furthermore, the treatment efficacy is diminished due to the occurrence of platinum resistance. Elevated levels of thiol-containing species, like glutathione (GSH), are a major factor in the development of resistance to platinum-based chemotherapy due to cytoplasmic detoxification [4, 5]. Reducing the amount of GSH in cells can greatly increase the sensitivity of cancer cells to CDDP. Curcumin (Cur), a natural polyphenol with anticancer properties, is considered safe and well-tolerated, even when administered at high levels [6]. Increasing evidence suggests that Curcumin can effectively reduce intracellular GSH levels in different cancer cells [7]. Given the inverse relationship between GSH levels and tumor sensitivity to chemotherapy, it is plausible that Curcumin-induced GSH depletion can enhance the effectiveness of Cisplatin-based treatment for EC. However, the high hydrophobicity and poor bioavailability limit the further application of Cur. Currently, several drug delivery platforms for curcumin (Cur) have been reported; however, these systems typically target a single signaling pathway and fail to address the complexity and multifaceted nature of the tumor microenvironment. Consequently, a combination of multiple therapeutic strategies could yield more effective treatment outcomes. Therefore, it is essential to design intelligent nano delivery platforms to enhance the bioavailability of Cur and, at the same time, leverage combined therapeutic approaches to better address the complex tumor microenvironment in vivo.

Fenton chemistry-based chemodynamic therapy (CDT) is a novel approach for treating cancer. The Fenton reaction, facilitated by metal ions, or a similar reaction, can trigger apoptosis in cancer cells by transforming naturally occurring H₂O₂ into harmful •OH molecules [8]. Although H₂O₂ in tumor cells is as high as 50–100 μM, this concentration is insufficient to achieve satisfactory CDT therapeutic effect [9, 10]. Recent studies have found that ultra-small Au nanoparticles with a glucose oxidase-like function can generate H₂O₂ by promoting glucose in tumors and thus increase the concentration of H₂O₂ in tumors [11, 12]. Compared with natural glucose oxidase, Au nanozyme avoids the problems of poor biological stability, high cost and complex synthesis and purification of biological enzymes, so it has a wide application prospect. Furthermore, the presence of high levels of GSH in cancer cells acts as an internal antioxidant, effectively neutralizing the extremely reactive •OH generated by CDT. This significantly boosts the cancer cells’ ability to withstand oxidative stress and diminishes the effectiveness of CDT.

Here, we have successfully designed and prepared a metal–organic framework nanoplatform co-loaded with CDDP and Cur with Fenton-like effects, which combines chemotherapy and CDT in a nanostructure. In Fig. 1A, Cur/CDDP@Cu/ZIF8 (CDCZ) nanoparticles were synthesized using a one-pot method, with ultra-small Au nanoparticles added on the surface to create Cur/CDDP@Cu/ZIF8@Au (CDCZA) nanoparticles. This nanosystem, which aims to improve the therapeutic effect of EC, has the following advantages: (1) Au nanoparticles catalyze excess glucose in tumor microenvironment to produce H₂O₂, providing sufficient H₂O₂ source for Cu⁺-mediated CDT and enhancing CDT effect; (2) ZIF8 nanoparticles would collapse under acidic conditions, promoting the release of Cu²⁺, CDDP and Cur; (3) The released Cu²⁺ and Cur both have the ability to consume GSH, which can enhance the chemosensitivity of CDDP and enhance the chemotherapy effect; (4) Consumption of GSH also enhances the efficacy of CDT, leading to apoptosis of tumor cells (Fig. 1B). Our study combined the enhanced chemotherapy and CDT to achieve a safe and effective treatment of EC.

Fig. 1.

A Schematic illustration for the synthesis of CDCZA nanoparticles. B The scheme of the sequential catalytic-therapeutic mechanism of CDCZA for the esophageal cancer treatment

Materials and methods

Materials

Copper nitrate trihydrate, zinc nitrate hexahydrate, 2-methylimidazole, polyvinyl pyrrolidone K30 (PVP-K30), chloroauric acid (HAuCl4), sodium borohydride (NaBH4), CDDP, methylene blue (MB), glucose, and 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) were all obtained from Aladdin Chemistry Co. Ltd. These chemicals are of high purity and were used as purchased without any additional purification steps. Sigma-Aldrich provided high glucose DMEM, 1% PS, 0.25% trypsin–EDTA, and PBS. Gibco Invitrogen supplied the fetal bovine serum (FBS).

Synthesis of Cu/ZIF8, Cur/CDDP/Cu/ZIF8 (CDCZ) and Cur/CDDP/Cu/ZIF8@Au (CDCZA) nanoparticles

Cu/ZIF8 nanoparticles were prepared using a procedure that had been described in a previous publication [13]. To summarize, a solution was prepared by dissolving 223.1 mg of zinc nitrate hexahydrate and 60.4 mg of copper nitrate trihydrate in 11.3 mL of methanol, while 660 mg of 2-methylimidazole was dissolved in an equal volume of methanol. Under stirring, the solution of 2-methylimidazole was added quickly into the mixed solution of zinc nitrate and copper nitrate. Following a 30-min stir at ambient temperature, the mixture was spun at 12,000 revolutions per minute for 5 min and rinsed with methanol to collect the Cu/ZIF8 nanoparticles. In order to load curcumin and cisplatin into Cu/ZIF8 nanoparticles, we made some changes to the above synthesis method according to the literature [6]. 5 mL of deionized water was used to dissolve 112 mg of zinc nitrate hexahydrate and 30.2 mg of copper nitrate trihydrate; meanwhile, 10 mL methanol was used to dissolve 330 mg of 2-methylimidazole, 5 mg of curcumin, and 5 mg of cisplatin. Subsequently, the aqueous solution containing metal ions was added to the methanol solution. Following a 5-min stir at ambient temperature, the solution was centrifuged at 12,000 rpm for 5 min and then rinsed with methanol to produce the CDCZ nanoparticles. The surface reduction method was used to form Au nanoparticles on the surface of CDCZ nanoparticles [12]. Following the addition of curcumin and cisplatin, the solution underwent three washes with methanol containing PVP-K30 (10 mg/mL) to eliminate any remaining drugs and impurities, before being dispersed in 20 mL of methanol. One milliliter of a solution containing 7 mg of HAuCl4 per milliliter was introduced into the methanol solution mentioned above, and then stirred while placed in an ice bath for a duration of one hour. Next, 2 ml of sodium borohydride solution with a concentration of 4 mg per milliliter was introduced to the previously combined mixture, followed by stirring for a duration of three hours. Following the reaction, the solution underwent centrifugation at a speed of 12,000 rpm for a duration of 5 min and was subsequently rinsed with methanol in order to acquire the CDCZA nanoparticles composed of Cur/CDDP@Cu/ZIF8@Au. The whole experiment was carried out under dark condition.

Characterizations

XRD patterns were obtained using a D8 advance instrument from Bruker in Germany. Samples were analyzed for their sizes and shapes using a transmission electron microscope (TEM) with an accelerating voltage of 200 kV (JEM-2100, JEOL, Japan). The Hitachi U-2900 Spectrophotometer was used to measure UV–visible absorption spectra. The Zetasizer Nano Z from Malvern in the UK was chosen for zeta potential measurement.

Detection of extracellular depletion of GSH and generation of •OH

UV–Vis spectroscopy was employed to identify the reduction of GSH outside the cell. We added GSH solution (10 mM, 30 μL) and ZIF8 or Cu/ZIF8 solution into PBS solution (pH 5.6, 10 mM) to 3 mL. After incubating the mixtures at 37 degrees Celsius for 1 h, DTNB solution (2 mg/mL, 30 μL) was added and the absorbance was measured using UV–Vis spectroscopy to assess the production of extracellular hydroxyl radicals through the degradation of MB. To the 2 mL of PBS solution with a pH of 5.6 and a concentration of 10 mM, we introduced 20 μL of MB solution at a concentration of 1 mg/mL, 20 μL of H₂O₂ solution at a concentration of 1 M, and either ZIF8 or Cu/ZIF8 solution. Following incubation at 37 degrees Celsius for one hour, the solutions underwent centrifugation and the absorbance was determined using UV–Vis spectroscopy.

Cell culture

The Eca-109 cell line of ESCC was grown in DMEM with high glucose, 10% fetal bovine serum, and 1% penicillin–streptomycin solution at 37 °C in a 5% carbon dioxide humidified environment.

Cytotoxicity assay

Eca-109 cells were seeded in a 96-well plate with a concentration of 10 × 10³ cells per well. After 24 h, the cells were treated with CDDP, Cur, Cur + CDDP, and CDCZA for another 24 h. Following this, 100 µl of DMEM (minus FBS) with 10% CCK8 was introduced into every well. Following a 4-h incubation period at 37 degrees Celsius, the optical density at 450 nm was determined with a spectrophotometer in the standard ELISA format. The trial was conducted thrice, and the viability of cells was determined by applying the formula: Cell viability = (average absorbance of experimental wells—average absorbance of control wells with medium) / (average absorbance of untreated wells—average absorbance of control wells with medium) × 100%. Hydrochloric acid was used to lower the pH of DMEM from 7.4 to 6.5 in order to mimic the acidic conditions found in solid tumors’ extracellular microenvironment. Next, the old culture media were exchanged with new medium (with a pH of either 7.4 or 6.5) that included CDCZA NPs at different levels of 20, 40, 60, 80, and 100 μM. After further incubation for 24 h, the cytotoxicity of CDCZA to Eca-109 cells were tested similarly as mentioned above.

Cellular uptake of CDCZA NPs

Quantitative cellular uptake experiments were conducted using flow cytometry (BD FACS Aria II, USA) at various concentrations and time intervals. For the analysis of cellular uptake over time, Eca-109 cells were placed in 6-well plates with a density of 1 × 10⁵ cells per well and incubated for 24 h. Subsequently, the culture media were exchanged with RhB-labeled CDCZA in DMEM (1 mL, 40 μM). Following co-incubation for various time points including 0, 30 min, 1 h, 2 h, 4 h, and 6 h, the cells were collected and suspended in PBS for analysis of uptake using flow cytometry. In experiments testing the effect of different doses, Eca-109 cells were placed in plates with a density of 1 × 10⁵ cells per dish and allowed to grow for 24 h. CDCZA NPs at varying concentrations (0, 10, 20, and 40 μM) were then introduced into the dishes and left to interact with the cells for 6 h at 37 °C. Cell samples were collected and analyzed using flow cytometry after a 6-h incubation period.

Generation and detection of intracellular reactive oxygen species (ROS)

The production of ROS within the cells by CDCZA NPs was measured with the ROS Assay Kit utilizing DCFH-DA (2,7-dichlorodihydrofluorescein diacetate). Eca-109 cells were seeded onto confocal culture plates. After 24 h, the culture media were replaced with 1 mL of acidified DMEM (pH 6.5) for the CZ, CZ + CDDP, CDCZ, and CDCZA groups in DMEM. The cells which were not incubated with CDCZA NPs in dark as the control group. Following a 4-h incubation period, the solution was replaced with DCFH (at a final concentration of 1 × 10^–6 M) and allowed to incubate for 30 min. Subsequently, the cells were washed thrice with PBS before assessing the level of intracellular ROS using a confocal laser scanning microscope (CLSM, Zeiss Axio-Imager LSM-880) to detect the fluorescence of DCF (excitation = 488 nm, emission = 525 nm).

GSH Depleting with CDCZA NPs

Cellular levels of GSH were monitored and analyzed with the ThiolTracker Violet GSH detection reagent from Invitrogen/Molecular Probes in accordance with the manufacturer’s instructions. Eca-109 cells were seeded into confocal culture dishes, at the density of 10 × 10⁵ cells per well and cultured until adherent. Various treatments were applied to cells, such as PBS, CUR, CDDP, CUR + CDDP, and CDCZA in acidified DMEM (pH 6.5) for 4 h. Afterward, the cells were rinsed with PBS and then exposed to Dulbecco’s PBS with 10 µM ThiolTracker Violet for 30 min at 37 °C. Replace the ThiolTracker™ Violet dye working solution with PBS, and the level of intracellular GSH was evaluated with CLSM as mentioned above. ThiolTracker™ Violet dye has an estimated excitation and emission wavelength of 404/526 nm.

Cell apoptosis measurement

Cell apoptosis in Eca-109 cells was assessed using a flow cytometric assay with Annexin V-FITC and PI. Eca-109 cells were plated in 6-well dishes with a concentration of 1 × 10⁵ cells per well and left to attach overnight. They were then exposed to Cur, CDDP, Cur + CDDP, CDCZA in DMEM (pH 6.5). After co-incubation for 6 h, the cells were collected by centrifugation. Cells were stained with the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Biotechnology, Inc., China) for 20 min prior to flow cytometry analysis.

Animal models

Female Balb/c mice (4–5 weeks) were purchased from Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and bred in a sterilized, specific pathogen-free (SPF) Lab of Tongji University. Next, the Eca-109 cells were administered into the right sides of five-week-old female BALB/c mice (1 × 10⁶ cells). All animal experiments followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals.

In vivo fluorescence imaging

Fluorescence imaging was conducted when the tumors reached a volume of 100–200 mm³, with 100 µL of RhB-labeled CDCZA solution injected intravenously into the mice. Following this, the mice were sedated and examined using a small animal imaging device (NightOWL LB 983 IN VIVO) at 1, 2, 8, 12, and 24 h after injection. An excitation wavelength of 640 nm was used to collect the fluorescence emission at 650 nm. To examine the distribution of the composites in tissues, mice were euthanized 24 h after injection, and key organs were harvested for imaging outside of the body.

Treatment of tumors within a living organism

Mice with Eca-109 tumors were divided into various groups (each containing five mice) at random to receive treatments of PBS, Cur, CDDP, Cur + CDDP, CDCZA, or CDCZA/NAC. Cur and CDDP were administered at doses of 0.59 and 2 mg/kg, respectively. NAC (10 mg/kg) was intraperitoneally administrated 1 h before CDCZA NPs treatment. Mice were given intravenous injections six times, with measurements taken every other day for tumor volume and body weight. The caliper was used to measure the size of the tumor, with tumor volume calculated using the formula V = L × W2 /2, where L and W represented the length and width of the tumor. On the 14th day, the rats were euthanized, followed by the removal and weighing of the tumors. Simultaneously, the main organs (heart, liver, spleen, lung, and kidney) were also obtained and utilized for hematoxylin and eosin (H&E) analysis.

Immunohistochemistry and TUNEL

The growth was treated using 4% paraformaldehyde and then enclosed in paraffin before being sliced into 5 μm segments for immunohistochemical analysis of Caspase-3, BAX, Bcl-2, γ-H2AX, and PARP. Cellular apoptosis was assessed by staining the tumor section with a TUNEL assay kit following the manufacturer’s instructions.

Hematology and Histopathology Examination

The murine blood was acquired from each group (n = 3) at the end of the experiment. After centrifugation at 4 °C for 20 min, the plasma was collected for blood biochemical analysis. The concentrations of aspartate aminotransferase (AST), alkaline phosphatase (ALP), and alanine amino transferase (ALT) were determined to evaluate liver function. The concentrations of blood urea nitrogen (BUN) and creatinine (CRE) levels were determined to evaluate renal function. The whole blood was also collected for white blood cells, red blood cells, and platelets analysis.

Statistical analysis

Quantitative data are expressed as mean ± s.d. Statistical analyses were performed using either independent t-tests for comparing two groups or one-way analysis of variance (ANOVA) for comparing three or more groups. *P < 0.05 (significant), **P < 0.01 (moderately significant) and ***P < 0.001 (highly significant).

Results and discussion

Characterizations and properties

Cu-doped ZIF8 nanoparticles were prepared through a straightforward solvothermal process as described in a prior investigation [14]. Figure 2A and D showed SEM and TEM images of Cu/ZIF8 (CZ), respectively. The clear rhomboidal dodecahedron structure of CZ was discovered, aligning with the common shape of ZIF8 described in previous studies. Following treatment with Cur and CDDP, the SEM and TEM images of CDCZ displayed uniformly distributed nanoparticles with the initial shape and an average size of 125 nm, as depicted in Fig. 2B and E. To create the ultra-small Au nanoparticles, HAuCl4 was added to the methanol solution of CDCZ in an ice-water bath with stirring, then NaBH4 was added to convert Au (III) to Au (0) [12]. The SEM image revealed that the particle size of CDCZA nanoparticles increased slightly compared to that of CDCZ nanoparticles, with an average particle size of 143 nm (Fig. 2C). And this particle size distribution facilitates passive targeting and aggregation at the tumor site (< 200 nm). Figure 2F demonstrates the remarkable dispersal of CDCZA, with clear evidence of the effective attachment of Au nanoparticles (approximately 2 nm) on the CDCZ surface. Figure S1 exhibited the photographs of the prepared ZIF8, CZ, CDCZ and CDCZA solutions. It could be clearly seen that the solution turned from milky white, pink, orange to ginger, which proved the successful preparation of the materials in each step. Additionally, DLS was used to confirm alterations in the hydrodynamic dimensions and zeta potential of CZ, CDCZ, and CDZA nanoparticles. There was no obvious size increase for CDCZ nanoparticles, 171.01 ± 34.57 nm (PDI = 0.068), as compared to CZ nanoparticles, 174.55 ± 38.62 nm (PDI = 0.05). After surface modification of Au nanoparticles on the surface of CDCZ, the size increased significantly to 219.39 ± 54.41 nm (PDI = 0.089), which was consistent with TEM image results (Figure S2A-C). The CDCZA nanoparticles showed a significant positive zeta potential of approximately 22.7 mV (Figure S2D), aiding in the absorption of nanoparticles by cancer cells and ultimately improving the effectiveness of the anticancer treatment.

Fig. 2.

The SEM images of CZ (A), CDCZ (B) and CDCZA (C) nanoparticles. Scale bar: 1 μm. The TEM images of CZ (D), CDCZ (E) and CDCZA (F) nanoparticles. Scale bar: 50 nm

The EDX spectrum showed that the CZ nanoparticles contained Cu, Zn, N, O, and C elements, with Cu and Zn making up approximately 4.38% and 26.47% by weight, respectively (Figure S3A). Furthermore, Pt and Au elements were found to exist in the energy spectrum of CDCZA (Figure S3B), representing the successful loading of CDDP and modification of Au nanoparticles, respectively. Figure 3A shows that the XRD pattern of CZ (red line) closely matched the crystal structure data of ZIF8 as described in the literature [15]. They are all body centered cubic lattices, and the peaks at 7.3, 12.7, 18.0 and 26.7 correspond to the crystal planes of (011), (112), (222) and (134) of ZIF8, respectively. In addition, the XRD pattern of CDCZ (blue line) was no different from that of CZ, indicating that the loading of Cur and CDDP would not destroy the structural stability of CZ and thus maintained high crystallinity. After surface modification of Au nanoparticles, the characteristic diffraction peak still appeared in the spectra of CDCZA (orange line), but the intensity of the diffraction peak decreased, which might be because Au nanoparticles had certain destructive effect on the crystal structure.

Fig. 3.

A The XRD patterns of CZ, CDCZ and CDCZA; B The Cur and CDDP cumulative release at different pH (pH 7.4 and 5.6); C Degradation of DTNB by unreacted GSH after treatments with ZIF8 and CZ; The concentration of CZ was 321 μg/mL, and the content of Cu was 30 μg/mg CZ; D Degradation of DTNB by unreacted GSH after treatments with various concentrations of CZ

Figure S4 displayed the UV–Vis absorption spectra of CZ, Cur, and CDCZ. The findings indicated that CZ did not exhibit any distinctive absorption between 300–800 nm. However, the introduction of Cur into CZ resulted in the emergence of a peak around 420 nm in the CDCZ spectra, suggesting the successful encapsulation of Cur within the CZ structure. The drug loading content of Cur was calculated to be approximately 11.4% based on a UV–Vis spectrum method (Figure S5). The pH-responsive release characteristics of CDCZA were examined by conducting in vitro release tests in PBS at pH levels of 5.6 and 7.4. A pH of 7.4 was used to simulate neutral physiological conditions, while a pH of 5.6 was selected to mimic the acidic environment of endosomes in tumor cells. As shown in the Fig. 3B, CDCZA nanocomposites showed obvious sustained release behavior at pH 7.4 and 5.6, especially at the early release stage. After 10 h in vitro release, CDCZA nanocomposites immersed in pH 5.6 released about 21% of Cur, while CDCZA nanocomposites immersed in pH 7.4 released only about 5.8% Cur during the same time period. The rate of release was higher at pH 5.6 compared to pH 7.4, possibly because the crystal structure of ZIF8 breaks down in acidic environments, causing drugs to be released quickly. After immersing for 48 h at pH 5.6, the cumulative released amount of Cur from the drug carrier reached 27%. Thus, the CDCZA nanocomposite exhibited pH-sensitive release characteristics, potentially improving its efficacy against tumor cells while minimizing harm to healthy tissue cells.

GSH consumption and hydroxyl radical (•OH) production

GSH overexpressed in tumor cells can reduce Cu²⁺ in Cu/ZIF8 to Cu⁺, which acts as a potent Fenton-like reagent capable of generating abundant •OH when combined with H₂O₂. We wondered whether the synthesized CZ nanoparticles had the ability for GSH depletion. After being exposed to an abundance of GSH, the CZ nanoparticles were tested for remaining GSH using DTNB, a type of indicator for sulfhydryl (-SH) groups. In Fig. 3C, it is evident that the absorption peak at 412 nm in the control group and ZIF8 treated solution is significantly stronger than in the GSH solution after the addition of CZ nanoparticles, suggesting a noticeable reduction in GSH concentration. Furthermore, the level of GSH showed a correlation with the dosage of CZ in a manner that depended on the dose (Fig. 3D and S6). Subsequently, the breakdown of methylene blue (MB) was employed to assess the production of hydroxyl radicals. MB can be degraded into a colorless solution by •OH, accompanied with the decrease of absorption intensity at 660 nm. The absorption peak intensity of MB remained nearly unchanged when exposed to H₂O₂ + GSH + ZIF8, as shown in Figure S7A, compared to H₂O₂ + GSH solution. The presence of MB, H₂O₂, GSH, and CZ resulted in a noticeable decrease, indicating the production of •OH and implying the involvement of GSH in the Fenton-like reaction of CZ. CDCZA is coated with Au nanoparticles that have the ability to act as glucose oxidase, converting glucose into gluconic acid and H₂O₂ when O₂ is present. The resulting H₂O₂ can then be used as a precursor for CDT. The functionality resembling glucose oxidase of Au nanoparticles was assessed by quantifying the amount of oxygen in solution with a dissolved oxygen meter. Figure S7B shows a clear decrease in dissolved oxygen content over time in the CZ treated solution compared to the control group. This indicates the glucose oxidase-like activity of CDCZA, which could potentially increase H₂O₂ concentration in tumor cells to improve CDT effectiveness.

Cellular uptake and intracellular detection of ROS

Determining the cellular endocytosis activity of the CDCZA nanoparticles was crucial prior to conducting the in vitro anticancer study. After 6 h of coculturing, Eca-109 cells were able to engulf RhB-decorated CDCZA nanoparticles, as illustrated in Fig. 4A, emitting a vibrant red fluorescence from the RhB. Moreover, the intracellular red fluorescence intensified as the concentration rose, suggesting a higher absorption of nanomaterial by the cells. Flow cytometry was used to quantitatively characterize the fluorescence intensity in order to confirm the uptake of CDCZA nanoparticles by cells. The study revealed that the brightness of RhB in the cells exposed to 40 μg/mL RhB-CDCZA was 1.3, 3.7, and 7.4 times higher than those treated with 20 μg/mL, 10 μg/mL, and 0 μg/mL (Figure S8A), indicating that the absorption of nanoparticles depended on the concentration. Furthermore, the absorption of CDCZA nanoparticles by Eca-109 cells increased over time, as shown by flow cytometry analysis (Figure S8B). DCFH-DA was utilized as a probe to assess the generation of intracellular reactive oxygen species (ROS) to verify the potential of Cu²⁺-mediated Fenton-like reactions in CDCZA to induce ROS production. DCFH-DA could be oxidized by intracellular •OH into 2, 7-dichlorofluorescein, which was observed as green fluorescence under confocal laser scanning microscope (CLSM). It was observed that after 4 h of co-incubation with CZ, Eca-109 cells showed weak green fluorescence, indicating that there was less •OH generation (Fig. 4B). When CZ and a certain dose of CDDP were co-cultured with cells, the green fluorescence was enhanced, which was due to the capability of CDDP to produce H₂O₂ and enhance the CDT effect. In addition, we found that the cells co-incubated with CDCZA nanoparticles showed the strongest green fluorescence, indicating that there was a large amount of •OH production in the cells, which was caused by the synergy of Cu²⁺-mediated Fenton-like reaction and Au nanoparticles-mediated H₂O₂ generation as glucose-like oxidase. Quantitative analysis of fluorescence intensity in CLSM images revealed that CDCZA-treated cells exhibited 1.2 times higher fluorescence intensity compared to CDCZ-cultured cells (Fig. 4C), indicating the potential of CDCZA as an effective CDT reagent.

Fig. 4.

A The CLSM images of Eca-109 cells cultured with different concentrations of RhB-CDCZA (10、20 and 40 μg/mL) at 6 h. The scale bar was 50 μm. B The CLSM images and C quantification of intracellular ROS in Eca-109 cells treated with CZ、CZ + CDDP、CDCZ or CDCZA for 4 h. ROS level was stained by DCFH-DA (green). The scale bar was 25 μm. (**P < 0.01, ***P < 0.001). D The CLSM images and E quantification of intracellular GSH in Eca-109 cells treated with CDCZA/NAC、Cur、CDDP、Cur + CDDP or CDCZA for 4 h. GSH level was stained by ThiolTracker Violet (green). The scale bar was 25 μm. (***P < 0.001)

Intracellular GSH detection

Tumor cells exhibit high levels of thiols, particularly GSH, which are crucial for detoxifying CDDP. The effective combination of CDDP and thiols compounds would weaken its own chemosensitivity, thus reducing the efficacy of chemotherapy. Consuming GSH can significantly enhance the responsiveness of tumor cells to CDDP, as demonstrated in studies [16–18]. To investigate the effect of CDCZA-mediated GSH depletion, Thiol TrackerTM Violet dye was used to monitor and analyze intracellular GSH levels after different treatments. Thiol TrackerTM Violet is a stable intracellular thiols probe that shows green fluorescence when combined with intracellular thiols. As shown in Fig. 4D, the green fluorescence was significantly reduced in cells co-cultured with Cur and CDDP compared with the free CDDP treatment group, suggesting that Cur could reduce GSH content in Eca-109 cells. In addition, we observed the lowest intracellular green fluorescence intensity in CDCZA-treated cells, showing that CDCZA nanoparticles could effectively achieve intracellular GSH depletion, which might be the result of the effective cellular uptake of CDCZA nanoparticles, and the released Cur/Cu²⁺-mediated GSH consumption. Next, the images were analyzed to measure the level of fluorescence intensity (Fig. 4E). The free CDDP treatment group exhibited fluorescence intensity 5.4 and 11.0 times higher than the combined Cur + CDDP and CDCZA-treated group, respectively, indicating the effective depletion of GSH in Eca-109 cancer cells by CDCZA nanoparticles. The impact of intracellular GSH on the cytotoxicity caused by various drugs was assessed using the cell counting kit-8 (CCK-8) assay (Figure S9). CDCZA nanoparticles treatment showed the highest cytotoxicity in cells that did not receive N-acetylcysteine (NAC), a precursor of GSH, which is promising. Preconditioning with NAC significantly decreased the toxicity of CDDP, Cur + CDDP, and CDCZA nanoparticles, highlighting the importance of GSH depletion in sensitizing CDDP. Together, these results strongly suggested that the consumption of GSH is essential for enhancing CDDP chemosensitivity, which also explained the superior therapeutic effect of CDCZA nanoparticles in vitro.

In vitro assessment of cell toxicity and combined treatment

The cytotoxicity of different formulations was studied using CCK-8 assay. Figure 5A demonstrates that the combination of Cur and CDDP resulted in higher cytotoxicity to Eca-10⁹ cells compared to individual doses of Cur and CDDP due to the depletion of intracellular GSH and the synergistic killing effect. And CDCZA-treatment resulted in the highest cell inhibition rate, which was the synergy of Cu²⁺-mediated enhanced CDT and CDDP-mediated enhanced chemotherapy. Furthermore, it was discovered that the viability of cells decreased progressively as the concentration increased, with 40% of cells treated with CDCZA still perishing even at the lowest concentration of 2.5 μg/mL. At a concentration of 40 μg/mL, the cells treated with Cur, CDDP, Cur + CDDP, and CDCZA had survival rates of 23.7%, 20.2%, 15.9%, and 8.24%, respectively. This suggests that CDCZA exhibited potent cytotoxicity against tumor cells. To directly observe the combined healing impact of CDCZA, the effectiveness against cancer was confirmed through a live/dead cell staining experiment. Live cells emitted green fluorescence while dead cells showed red fluorescence. As exhibited in Fig. 5B, free Cur and CDDP-treated group showed certain red fluorescence, while Cur + CDDP group showed a greater proportion of red fluorescence, because of the ability of Cur to enhance the chemosensitivity of CDDP. The CDCZA group exhibited the highest red/green fluorescence ratio, suggesting that CDCZA may have the most potent anticancer properties, aligning with the cytotoxicity findings. Conversely, we observed minimal dead cells in the CDCZA-treated group that had been pre-treated with NAC, resulting in no discernible difference from the PBS group. This clearly indicates the significant role of intracellular GSH in the eradication of tumor cells by CDCZA. CDDP was found to induce cell apoptosis as its fundamental pharmacodynamic effect [16]. Quantitative analysis of apoptosis induced by CDCZA was assessed using flow cytometry after co-staining with Annexin V-FITC/PI (Fig. 5C). The findings indicated that CDDP triggered a moderate level of cell death in Eca-109 cells, resulting in an apoptosis rate of 17.96%. In contrast, the combination of Cur and CDDP increased the rate of apoptosis to 26.34%. CDCZA had the highest apoptotic rate at 45.3%, while the group treated with NAC had the lowest apoptosis proportion at 3.82% among all experimental groups.

Fig. 5.

A Cytotoxicity of Eca-109 cells treated with Cur、CDDP、Cur + CDDP or CDCZA for 24 h. (n = 3, **P < 0.01, ***P < 0.001). B Detection of live/dead cells after treatments with CDCZA/NAC、Cur、CDDP、Cur + CDDP or CDCZA. Live and dead cells were stained with calcein-AM (green) and PI (red), respectively. The scale bar was 75 μm. C Apoptosis study of Eca-109 cells treated with CDCZA/NAC、Cur、CDDP、Cur + CDDP or CDCZA for 6 h analyzed by flow cytometry. D Western blot analysis of Bcl-2, BAX and Caspase-3 after treatments with CDCZA/NAC、Cur、CDDP、Cur + CDDP or CDCZA for 12 h. E Statistical analysis of the Bcl-2 protein, BAX protein and Caspase-3 protein expression normalized to control

Numerous studies indicate that upon cellular entry, CDDP interacts with DNA, its primary biological target, to create Pt–DNA complexes. Subsequently, p53 (pro-apoptotic gene) will be activated in Pt–DNA complex mediated DNA damage, and apoptosis induced by activated p53 is in turn repressed by overexpressed Bcl-2 gene, which in turn indirectly inhibits Bcl-2 expression through transcriptional activation of BAX, a pro-apoptotic factor. Simply put, p53 disturbs the equilibrium of pro-apoptotic and anti-apoptotic controllers, leading to the activation of the apoptotic executor Caspase-3, ultimately facilitating cell apoptosis. Western blotting was employed to analyze the levels of apoptotic proteins Bcl-2, BAX, and Caspase-3 following various treatments in order to gain a deeper insight into the process of CDCZA nanoparticles-induced apoptosis. In Fig. 5D, E, it was observed that the levels of the anti-apoptotic protein Bcl-2 decreased slightly following CDDP treatment, while the co-treatment with Cur and CDDP significantly suppressed the expression of Bcl-2. The CDCZA therapy resulted in a substantial decrease in Bcl-2 levels, almost reaching zero, which is promising. It is worth mentioning that the levels of BAX and Caspase-3 increased moderately following CDDP treatment, indicating a slight rise in cell apoptosis. Nevertheless, the combination of Cur and CDDP resulted in a greater upregulation of these proteins associated with apoptosis, indicating that Cur enhanced the effect of CDDP on inducing cell death. CDCZA treatment significantly increased the levels of BAX and Caspase-3, suggesting that it could enhance tumor cell sensitivity by suppressing the expression of the anti-apoptotic protein Bcl-2. This disruption of mitochondrial function and activation of apoptotic pathways ultimately boosts the anti-cancer efficacy. Together, these results demonstrated that the synthesized CDCZA nanomaterial could effectively induce cell apoptosis and kill Eca-109 cancer cells by combining the enhanced CDT and chemotherapy.

In vivo biodistribution of CDCZA

The superior CDT efficacy and therapeutic potential of CDCZA nanoparticles in vitro motivated us to evaluate the performance in vivo. Studying the biological distribution of nanoparticles was of great significance for potential clinical research and application. In order to show the distribution of nanoparticles in mice with Eca-109 tumors, nanoparticles labeled with RhB-CDCZA were prepared initially. Figure 6A demonstrates that there was no detectable fluorescence signal at the tumor site 24 h after the intravenous administration of free RhB, suggesting that free RhB did not accumulate specifically at the tumor site. Conversely, the tumor site exhibited fluorescence signal 2 h post-injection of RhB-CDCZA, which then increased steadily over time, peaking at 12 h post-injection (Fig. 6B). The intense fluorescence detected at the tumor location 24 h after injection suggested that RhB-CDCZA could efficiently gather in tumor tissues due to the enhanced permeability and retention (EPR) effect of nanomaterials, and maintain a prolonged presence in the tumor region. Following a 24-h period post-injection, the mice were euthanized and the tumors, along with the major organs (heart, liver, spleen, lung, and kidney) from various treatment groups, were gathered for subsequent in vitro fluorescence imaging. As shown in Fig. 6C and D, the fluorescence signal of free RhB was mainly distributed in liver and kidney, while RhB-CDCZA was mainly distributed in tumor, liver and kidney, indicating that the designed nanoparticles were mainly metabolized by liver and kidney. These results suggested that RhB-CDCZA could accumulate preferentially and effectively in tumor sites for a long time by means of EPR effect and passive targeting, which had certain guiding significance for subsequent anti-tumor studies in vivo.

Fig. 6.

A In vivo fluorescence images of Eca-109 tumor-bearing mice injected with free RhB and RhB-CDCZA nanoparticles at different time points (1、2、8、12 and 24 h). B The corresponding mean fluorescence intensity (MFI) analysis of tumor in the mice injected with RhB-CDCZA nanoparticles at different time points. C Ex vivo fluorescence images of major organs (heart、liver、spleen、lung and kidney) as well as tumors of Eca-109 tumor-bearing mice injected with free RhB and RhB-CDCZA nanoparticles at 24 h. D The MFI analysis of major organs as well as tumors of Eca-109 tumor-bearing mice injected with free RhB and RhB-CDCZA nanoparticles. (n = 3, ***P < 0.001)

In vivo antitumor efficiency

Based on the collaborative eradication of cancer cells in a laboratory setting and the specific accumulation of tumors in living organisms, we proceeded to investigate the anti-cancer characteristics of CDCZA nanoparticles in mice with Eca-109 tumors. When the tumor volume increased to 200–300 mm3, mice with similar size were selected and randomly divided into 6 groups (n = 5): saline blank control group, CDCZA/NAC, Cur, CDDP, Cur + CDDP and CDCZA administration group, in which the dose of Cur and CDDP was 2.5 mg/kg and 2 mg/kg, respectively. The dosage of Cur + CDDP was equivalent to the individual dosages of Cur and CDDP. The dose of CDCZA group was 22 mg/kg. Caudal vein administration was adopted, and the treated mice were given the second treatment every other day. Throughout the 12-day therapy, the tumor size and weight of the mice in every group were documented every second day. After 14 days, the mice were put down and the tumors, as well as important organs, were gathered for further pathological examination (Fig. 7A). The tumor in PBS group grew rapidly throughout the treatment period, while the tumors of the other drug administration groups had varying degrees of tumor growth inhibition (Fig. 7B). Compared with the free Cur and CDDP group, Cur + CDDP exhibited more effective tumor inhibition. Unsurprisingly, the tumor growth was most significantly inhibited in the CDCZA group. Cell experiments had demonstrated that increased intracellular GSH significantly reduced the toxicity of CDCZA nanoparticles to Eca-109 cells, so the CDCZA/NAC group showed limited inhibition of tumor growth. The mice were euthanized on the 14th day, and the tumors were removed, photographed, and weighed. As shown in Fig. 7C and D, the tumor volume in the CDCZA group was the smallest, significantly smaller than the other five groups, and the average tumor weight was consistent with the tumor volume measurement results. These results verified the excellent antitumor effect of CDCZA nanomaterial in Eca-109 tumor-bearing mouse.

Fig. 7.

A Schematic illustration of establishment of the esophageal cancer model and therapy with CDCZA. B The variation of tumor volume in the Eca-109-tumor-bearing mice injected with PBS、CDCZA/NAC、Cur、CDDP、Cur + CDDP and CDCZA during 12 days. C Photographs of excised tumors from each group after treatments. D Mean tumor weight of each group after 12 days treatment. (n = 5, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Histopathological examination was performed on tumor sections to uncover the molecular mechanism behind the impressive antitumor effectiveness of CDCZA. Hematoxylin and eosin (H&E) staining results showed that CDCZA treatment caused the most obvious nuclear pyrosis, fragmentation and nucleolysis, and normal morphology of cell membrane and nucleus could not be seen (Fig. 8A). Furthermore, the CDCZA group exhibited the highest level of apoptosis, as indicated by the highest percentage of TUNEL-positive cells (Fig. 8B). Immunohistochemistry was performed on tumor tissues from each group to identify the presence of apoptosis-related proteins including Caspase-3, BAX, PARP-1, and γ-H2AX.As shown in Fig. 9 and S10, the expression of apoptosis related proteins were significantly upregulated in the CDCZA group. After CDCZA treatment, the tumor tissues showed the most notable reduction in the anti-apoptotic protein Bcl-2. All of the above results demonstrated that CDCZA nanoparticles combined with the enhanced chemotherapy/CDT could produce the excellent anticancer effect from the molecular perspective, providing a new method for the clinical treatment of esophageal cancer.

Fig. 8.

A The H&E and B the TUNEL staining images of tumor tissues from each group. The scale bar was 50 μm

Fig. 9.

Immunohistochemical staining of Caspase-3, BAX, Bcl-2, PARP-1 and γ-H2X in tumor tissues of different treatment groups. The scale bar was 50 μm

Safety evaluations

An ideal drug nanocarrier should have both satisfactory therapeutic effects and negligible adverse reactions. Hence, assessing the adverse reactions and possible overall toxicity of CDCZA nanoparticles is essential while undergoing treatment. As shown in Figure S11, the body weight of mice in the CDDP treatment group began to decrease on day 6, which might be caused by the side effect of injected CDDP on normal tissues of mice. Throughout the treatment period, the mice in the CDCZA group experienced a consistent increase in body weight, rising from 19.6 g on day 0 to 22.4 g on day 12, suggesting that the CDCZA treatment did not result in notable systemic side effects for the mice. Furthermore, the H&E staining results showed no apparent signs of biological harm in the heart, liver, spleen, lungs, and kidneys of the mice that received CDCZA treatment (see Figure S12).The quantity of white blood cells (WBC) and neutrophils (NEUT) in the bloodstream of mice administered with CDDP and Cur + CDDP fell significantly below the typical levels (Figure S13), as a result of CDDP’s capacity to diminish the count of WBC and NEUT, subsequently diminishing the mice’s immunity to external viruses. Additionally, the concentrations of blood urea nitrogen (BUN) and creatinine (CREA) in the blood of mice exposed to CDDP and Cur⁺ CDDP were notably elevated (Figure S14), indicating the nephrotoxic effects of CDDP. On the other hand, there was no significant difference in blood biochemical parameters and hematology data between the PBS group and the CDCZA group, indicating that CDCZA nanoparticles exhibited excellent biocompatibility both in vivo and in vitro, suggesting a promising future for clinical applications.

Conclusions

To summarize, we have found that the CDCZA nanoparticles, which are highly biocompatible, can serve as an effective nanotheranostic agent for esophageal cancer therapy by combining CDT and chemotherapy. Successful synthesis of hybrid CDCZA nanoparticles involved attaching ultra-small Au nanoparticles to the surface of Cur/CDDP/Cu/ZIF8 nanoparticles. After efficient internalization of CDCZA nanoparticles by Eca-109 tumor cells, the released Cur could notably decrease intracellular GSH content and thus improve the chemosensitivity of CDDP, resulting in severe cellular apoptosis. Due to the increased CDT from self-supplied H₂O₂ along with the enhanced chemotherapy by CDDP, CDCZA nanoparticles effectively suppressed tumor growth in Eca-109 tumor-bearing mice with minimal harm to healthy tissues. This nanosystem could offer a fresh approach to developing highly effective therapeutic agents for treating esophageal cancer in clinical settings.

Author contribution

Y.S. designed the study and developed the methodology and K.Y. and J.S. and Y.W. and M.J. conducted the experiments and analyzed the data and contributed equally to the investigation and writed of the review and editing sections and L.Q. provided the resources and supervised the project, L.Z and J.C. contributed to the conceptualization and methodology, supervised the research, and were responsible for securing funding for the project. All authors reviewed the manuscript.

Data availability

The data are available in the main manuscript, supplementary information files, and from the corresponding authors upon reasonable request.

Declarations

Ethics approval

The authors obtained authorization to perform the tests on animal from the Experimental animal Center of Jiangsu Medical Vocational and Technical College.

Competing interests

The authors declare that they have no competing interests.