The "Trinity" comprehensively regulates the tumor microenvironment of lipid-coated CaCO3@CuO2-watermelon nanoparticles induces "cuproptosis" in HCC

doi:10.21203/rs.3.rs-3364972/v1

Download PDF

Research Article

The "Trinity" comprehensively regulates the tumor microenvironment of lipid-coated CaCO3@CuO2-watermelon nanoparticles induces "cuproptosis" in HCC

https://doi.org/10.21203/rs.3.rs-3364972/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Tumor cell death induced by "cuproptosis" is a novel form of tumor death that differs from apoptosis induced by chemotherapy. It is expected to emerge as a new approach for cancer treatment. In this study, our focus was on exploiting the characteristic of "cuproptosis" which necessitates increased aerobic respiration to induce tumor cell death.

Methods

To achieve this, we developed a novel drug delivery system using a CaCO₃@CuO₂ lipid coating (CaCO₃@CuO₂@L). This system aimed to comprehensively modulate the tumor microenvironment and trigger "cuproptosis" in hepatocellular carcinoma (HCC) through the interaction between copper ions and peroxides.

Results

Experimental results revealed that the CaCO₃@CuO₂@L exhibited a distinct watermelon shape, with CuO₂ evenly distributed within the CaCO₃ nanoparticles. The nanoparticles had an average size of approximately 191 nm. In vitro studies demonstrated that the nanoparticles released CuO₂ in a slightly acidic environment while simultaneously elevating pH levels, reducing glutathione (GSH), and increasing oxygen production. Within liver cancer cells, the CaCO₃@CuO₂@L effectively regulated the acidity, GSH levels, and oxygen-depleted microenvironment through the "trinity" mechanism, ultimately inducing "cuproptosis" in HCC. Furthermore, in mouse models with transplanted tumors and orthotopic liver cancer tumors, the CaCO₃@CuO₂@L significantly suppressed tumor growth.

Conclusions

By triggering "cuproptosis" in HCC, this study offers valuable insights for developing a comprehensive treatment approach for HCC. Ultimately, this research may pave the way for the clinical implementation of the drug delivery system based on "cuproptosis" in liver cancer treatment.

drug delivery system

Bmi1

hepatocellular carcinoma

cancer stem cell

tumor microenvironment

Hepatocellular carcinoma (HCC) is a common malignant tumor of the digestive system[1]. Despite various treatment methods, the prognosis for HCC remains generally poor. It has been confirmed that the anaerobic microenvironment is closely associated with the progression and poor prognosis of HCC. Hypoxia can promote apoptosis resistance, invasion, and metastasis of tumor cells, with hypoxia inducer (HIF-1α) serving as a key regulatory factor in hypoxia[2]. The hypoxia observed in HCC primarily arises from an imbalance between abnormal vascularization, leading to insufficient oxygen supply, and the high oxygen consumption of tumor cells. Elevated expression of the HIF-1α in tumor cells is strongly correlated with tumor proliferation, invasion, metastasis, and neovascularization[3, 4]. Furthermore, increased expression of HIF-1α can upregulate glucose transporters and their downstream proteins, enhancing the efflux of therapeutic drugs and inducing resistance to chemotherapy in tumor cells[5, 6]. Additionally, the oxygen deficiency in liver cancer leads to an imbalance in the REDOX state of tumor tissues and cells. In this regard, the association between abnormal liver cancer and glutathione (GSH) metabolism is highly significant[7]. GSH, serving as the main reducing ligand, is most abundant in the cytoplasm of tumor cells (2–10 mmol·L-1), far exceeding its extracellular concentration (2–20 µmol·L-1)[8, 9]. Moreover, the concentration of GSH in tumor tissues is approximately four times higher than that in normal tissues[10]. The high concentration of GSH in tumor cells forms a complex with metal chemotherapy drugs, reducing their entry into the cell nucleus and hindering DNA binding[11]. Additionally, GSH is more readily pumped out of the cell by multidrug-resistant proteins MRP1 and MRP2, significantly diminishing the effective concentration of intracellular cisplatin and enhancing drug resistance in HCC[12, 13]. The development of a tumor microenvironment (TME) caused by anaerobic glycolysis, characterized by a low extracellular pH and high cytoplasmic pH, is one of the main features of tumor tissue[14, 15]. This TME is an important factor inducing tumor occurrence, metastasis, and drug resistance[16]. In comparison to normal tissue, tumor tissue exhibits increased production of acidic products, intramembrane transport, and clearance barriers, resulting in the formation of an acidic extracellular microenvironment within tumors[14, 17]. Thus, targeting the TME of hypoxia, high GSH concentration, and the acidic microenvironment in tumor tissues and cells are crucial for HCC therapy[18–21].

Over the past year, the concept of "cuproptosis" and its related mechanisms have garnered significant attention. In 2022, Todd R. Golub and Peter Tsvetkov's team published an article titled "Copper induces cell death by targeting lipoylated tricarboxylic acid cycle (TCA) proteins" in Science[22]. This article introduced the concept of "cuproptosis," which quickly became a prominent research topic. The research team elucidated the mechanism behind "cuproptosis," a novel form of copper-dependent cell death that occurs through the direct binding of copper ions in mitochondrial respiration to the fatty acylated components of the TCA. This binding leads to the aggregation of fatty acylated proteins, down-regulation of iron-sulfur tuftin, activation of protein toxic stress, and ultimately, induction of tumor cell death. This study revealed that copper-dependent cell death is a distinct mode of cell death that differs from apoptosis. Even when the apoptosis pathway is blocked, copper ions can still induce cell death through an alternative pathway. Therefore, "cuproptosis" may prove effective against tumor cells that are resistant to apoptosis or DNA damage repair, such as chemotherapy-resistant cells[23, 24]. Thus, understanding the mechanism of "cuproptosis" can provide insights into a novel approach for HCC treatment. Furthermore, the mechanism of "cuproptosis" directly involves the tricarboxylic acid cycle in cellular mitochondrial respiration[25]. A high concentration of GSH, as a complex with copper ions, inhibits the occurrence of "cuproptosis"[26]. Consequently, the presence of high GSH levels and a hypoxic TME hinders the activation of "cuproptosis" in tumor cells. Therefore, investigating whether the effects of "cuproptosis" can be enhanced by providing peroxide oxygen supply to overcome the high concentration of GSH and hypoxic TME in tumors is of great significance. Currently, various copper ions and chelating agent carriers have been employed in preclinical and clinical tumor treatments[24]. Examples include the copper ionocarrier disulfiram (DSF) for glioblastoma, ATN-224 for breast cancer, and Elesclomol for melanoma, all of which are in Phase II clinical trials. However, the abnormal accumulation of copper in the body can harm an individual's overall health and well-being. Therefore, it is crucial to carefully administer copper ions within a specified therapeutic range. By utilizing a drug delivery system (DDS) to deliver more copper selectively to tumor cells, it is possible to achieve targeted tumor destruction while minimizing the toxic side effects of excess copper on the body[27]. In conclusion, the interaction between copper ions and peroxides in the tumor microenvironment presents an opportunity to overcome various adverse factors associated with "cuproptosis" activation in HCC. Through the clever design of a DDS based on peroxides and copper ions, a novel approach to liver cancer treatment can be explored.

In our previous studies, we have reported various DDS targeting tumor hypoxia, utilizing peroxide nanoparticles[28, 29]. These systems have demonstrated effective regulation of tumor hypoxia, high concentrations of GSH, and acidic microenvironments. Building on this research, the current study aims to design and prepare a novel DDS using watermelon-type lipid-coated CaCO₃@CuO₂ nanoparticles (CaCO₃@CuO₂@L) through the reversed-phase microemulsion method. This preparation method is well-established, straightforward, and reliable. Over the years, we have utilized the reversed-phase microemulsion method to develop various DDS for investigating tumor hypoxia. Both in vivo and in vitro studies have demonstrated promising results. Firstly, the CaCO₃@CuO₂@L, prepared using the reversed-phase microemulsion method, exhibited uniformity and stability, with an average particle size of approximately 200 nm. Secondly, in vitro experiments revealed that CuO₂ nanoparticles can decompose within the tumor acidic TME, generating O₂, Cu²⁺, and OH⁻. Consequently, a continuous oxygen supply is generated, GSH levels are reduced, and pH is regulated. In HepG2 cell lines, the uptake of CaCO₃@CuO₂@L was effective. Furthermore, the nanoparticles successfully regulated hypoxia, reduced GSH levels, and increased pH in HepG2 cells. Additionally, CaCO₃@CuO₂@L effectively inhibited the growth of HepG2 cells, regulated oxygen deficiency, and activated pathways associated with "cuproptosis." Finally, the ability of CaCO₃@CuO₂@L to suppress liver cancer growth was demonstrated in vivo using orthotopic HCC and transplanted tumor models. In summary, this study builds upon the "trinity" concept of CaCO₃@CuO₂@L to comprehensively regulate the tumor microenvironment in HCC. By reconstructing the tumor microenvironment and inducing "cuproptosis", the growth of liver cancer is effectively inhibited, representing a promising approach for HCC treatment.

Chemicals and Reagents

All solvents and reagents were used as received without further purification. mPEG2000-silane, DOTAP, TritonX-100, hexanol, and cyclohexane were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture

The human tumor cell line HepG2 was obtained from the China Center for Type Culture Collection at Wuhan University (Wuhan, China). HepG2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% streptomycin, 1% penicillin, and 10% fetal bovine serum (FBS) in a 5% CO₂ incubator at 37°C. Hypoxia conditions were cultured in an anaerobic incubator.

Preparation of CuO₂/PVP nanoparticles

PVP (250 mg) was dissolved in an aqueous solution containing CuCl₂·2H₂O (2.5 mL, 0.01 M), followed by NaOH (2.5 mL, 0.02 M) and H2O2 (30%, 100 µL). After stirring for 30 min, the PVP-coated copper peroxide nanoparticles (referred to as CuO₂@PVP) were collected by centrifugation at 13,000 rpm for 20 min and washed with water three times. They were then dispersed in 1 mL of NaOH (0.1 M) aqueous solution.

Preparation of lipo-coated CaCO₃@CuO₂ hybrid watermelon-shaped Nanoparticles (CaCO₃@CuO₂@L)

First, two copies of a 4 mL inverse microemulsion were prepared using Triton X-100: Hexanol: Cyclohexane: H2O at a ratio of 1.02:1.04:3.94:0.3 (v/v) and stirred for 5 minutes until uniform. Subsequently, 200 µL of 200 mM CaCl₂ or 200 µL of 200 mM CuO₂@PVP and 200 µL of 200 mM Na₂CO₃ were added to the respective inverse microemulsion. To the microemulsion containing the CuO₂@PVP precursor, DOPA (100 µL, 20 mM) was added. The mixture was stirred for 20 minutes. The two types of emulsions were then mixed and reacted for 24 hours. After that, 16.0 mL of ethanol was added to the microemulsion, and the mixture was centrifuged at 12,000 × g for at least 15 minutes to remove the cyclohexane and surfactants. After being extensively washed with ethanol 2–3 times, the pellets were re-dispersed in 3.0 mL of chloroform to obtain CaCO₃@CuO₂ nanoparticles. To prepare CaCO₃@CuO₂@L, 28 mg of DOTAP, 21.3 mg of cholesterol, and 12.6 mg of mPEG-DSPE (molar ratio 40:55:5) were dispersed in 5.0 mL of chloroform. After the chloroform was removed by rotary evaporation, the residual lipids were dispersed in 2.0 mL of ddH₂O to generate CaCO₃@CuO₂@L.

Drug loading determination

The concentration of Cu was determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) after lysing the sample in a Hydrofluoric acid (HF) solution.

Nanoparticle characterization

The particle size of the nanoparticles was measured in their aqueous solution using Zeta PALS (Zeta Potential Analyzer, Brookhaven Instruments Corporation, Austin, TX) following the manufacturer's instructions. All measurements were conducted at room temperature. Each parameter was measured three times, and the average values and standard deviations were calculated. The shape of the nanoparticles was analyzed using transmission electron microscopy (TEM, JEOL 1010, Japan). For TEM analysis at 100 kV, the powder sample of nanoparticles was dispersed in ethanol and dried on a copper grid.

In vitro acid-responsiveness release of Cu under simulated conditions: To examine the acid-responsiveness, one milliliter of CaCO₃@CuO₂@L was placed inside a dialysis bag with a molecular weight cut-off of 2000 Da. The dialysis bag was then immersed in 20 mL of PBS containing 0.5% Tween 80 at different pH values (pH 6.5, 6.8, and 7.4) and maintained at a temperature of 37°C. The released Cu was quantified using ICP-MS.

In vitro simulated regulation of oxygen production, REDOX (GSH) and acidic microenvironments

Simulated oxygen release in vitro: 1 mL of CaCO₃@CuO₂@L nanoparticles were added to 8 mL of distilled water and stirred. The dissolved oxygen concentration was measured at 37°C using a DOG 3082 dissolved oxygen meter.

Simulated oxidation of GSH in vitro: The content of GSH was detected using the sulfhydryl reagent DTNB, and the absorbance was determined using UV-VIS spectroscopy.

In vitro simulation of "acid reduction": The pH was monitored at 37°C using a pH meter (PH-25 pH meter).

Intracellular uptake of nanoparticles

To investigate the intracellular uptake of nanoparticles, HepG2 cells were cultured in 24-well plates and treated with different groups (equivalent to 2.5 µg/ml cisplatin) for 8 hours. After treatment, the cells were digested with 70% HNO3 and diluted in water to achieve a final acid concentration of 2%. The concentration of Cu in the solution was determined using ICP-MS. To detect the fluorescence of CaCO₃@CuO₂@L (labeled with FITC), the nanoparticles were incubated with the cells for 8 hours. After adsorption, the medium was replaced with fresh medium, and the fluorescence was observed under a fluorescence microscope.

Cytotoxicity determination

To assess the cytotoxicity of the drugs, tumor cells were cultured in 96-well plates and treated with different concentrations of the respective drugs. After a 24-hour incubation period, cell survival was measured using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer's instructions. Cell viability was calculated based on the assay results.

Western Blotting Analysis

Following treatment with different formulations, HepG2 cells were collected and lysed using a cell lysis solution. The resulting lysates were subjected to high-speed centrifugation (12,000 rpm), and the supernatant containing the proteins was collected. Separation of the proteins was performed using a 10% polyacrylamide gel. Subsequently, the separated proteins were transferred onto a PVDF membrane, which was then blocked using 5% skim milk for 1 hour to prevent non-specific binding. Monoclonal antibodies against specific proteins were used for immunoblotting. The antibodies used included HIF-1α (1:1000, Cell Signaling, Danvers, MA, USA), FDX1 (1:1000, Cell Signaling), LIAS (1:1000, Cell Signaling), and Tubulin (1:1000, Abcam). The membrane was incubated with the primary antibodies overnight at a specified dilution. Following incubation, the membrane was washed three times with TBST (PBS with 0.1% Tween-20) to remove unbound antibodies. Then, the membrane was incubated with the appropriate secondary antibody for 1 hour. After another round of washing with TBST, an enhanced chemiluminescence system (Perkin Elmer, Waltham, MA, USA) was used for image development and visualization of the protein bands.

Mice

C57BL/6J mice (4–6 weeks old) were obtained from Beijing Huafukang Bioscience Technology Co., Ltd. (Beijing, China). The C57BL/6J mice were housed in filter-topped cages with standard rodent chow and water available ad libitum, under a 12-hour light/dark cycle. The experimental protocol was approved by the Committee on Ethical Animal Experiment at Huazhong University of Science and Technology.

Primary HCC Mouse Model

To establish the primary HCC mouse model, a double overexpression gene of Akt/Ras was generated to induce primary HCC, and the anti-tumor effect of different drugs was evaluated. All gene constructs including hyperactive transposase (pCMV/SB), AKT (myr-AKT/pT3EF1α), and Ras (pCaggs-RasV12) were provided by Professor Xin Chen (University of California at San Francisco). The GenElute Endotoxin-free Plasmid Maxiprep Kit (Sigma, USA) was used to purify the plasmids before injecting them into the mice.

The high-pressure injection technique via the tail vein was performed following the protocol described in previous reports. Briefly, C57BL/6J mice were injected with 2 ml of saline containing NRasV12/pT2-CAGGS (5 µg), myr-AKT/pT3EF1α (5 µg), and pCMV/SB (0.4 µg) within 5–10 seconds via the tail vein. After approximately six weeks, HCC tumors were formed in the liver due to the up-regulation of AKT/Ras gene expression. The presence of tumors was confirmed by bimanual palpation and dissection.

Mice were anesthetized using isoflurane gas (2% concentration) and received an intraperitoneal injection of D-firefly luciferin (240 mg/kg) (Gold Biotechnology, St. Louis, MO). The mice were then placed inside the chamber of a Xenogen IVIS 200 imaging system. Bioluminescence images were acquired under isoflurane anesthesia using 1-minute exposures, starting 10 minutes after the injection of D-luciferin. The obtained images were quantitatively analyzed using Living Image version 4.3.1 image analysis software (Caliper Life Sciences, Hopkinton, MA). Total bioluminescence measurements (expressed in photon/s) were quantified over a contour drawn around the brain and coronal slices. The experiment was repeated two to three times using independent animals. All in vivo images were scaled to a maximum intensity of 1×10⁵ photons/s/cm²/sr.

HepG2 xenograft Mice Model

The xenograft model was established by injecting 2.5 × 10⁶ HepG2 cells into the front armpit of nude mice, and the tumor volume was allowed to grow to 50–100 mm³. Subsequently, different drugs (equivalent to 1.5 mg/kg Cu) were administered via tail vein injection for a total of four times, with intervals of every other day.

Statistical Analysis

Student's t-test (using SPSS Software, Chicago, IL) was used for comparisons between two groups. One-way ANOVA (using GraphPad Prism 5.0, San Diego, CA, USA) with Dunnett's post-test was used to compare multiple groups.

Preparation and Characterization of CaCO₃@CuO₂@L

The reversed-phase microemulsion method has proven to be an excellent technique for the preparation of drug delivery systems. It allows for the formation of nanoscale microreactors and precise control over nanoparticle morphology, resulting in drug delivery systems with controllable particle size, good dispersion, and lower impurity content. In this study, PVP/CuO₂ nanoparticles with a particle size of approximately 5–10 nm were obtained using PVP as the encapsulating agent (Fig. 1A). Subsequently, the CaCO₃@CuO₂@L drug delivery system was prepared using the reversed-phase microemulsion method (Fig. 1B). TEM images demonstrated a heterogenous structure in CaCO₃@CuO₂@L, with small-sized CuO₂ nanoparticles evenly distributed within the CaCO₃ nanoparticles, resembling watermelon seeds (Fig. 1C and 1D). The overall structure of CaCO₃@CuO₂@L exhibited a watermelon-like appearance (Fig. 1E). The particle size and uniformity of the drug delivery system were analyzed using dynamic light scattering (DLS) detection. The average particle sizes of CuO₂, CaCO₃, CaCO₃@CuO₂, and CaCO₃@CuO₂@L were measured to be 5 nm, 41 nm, 97 nm, and 191 nm, respectively (Fig. 1F). Furthermore, to assess the stability of the obtained CaCO₃@CuO₂ and CaCO₃@CuO₂@L nanoparticles, a 10% fetal bovine serum (FBS) solution was used to mimic blood conditions. The results showed that the sizes and polydispersity index (PDI) of CaCO₃@CuO₂@L remained relatively stable within 240 hours compared to CaCO₃@CuO₂@L, indicating that the lipid coating greatly enhances the stability of the nanoparticles (Fig. 1G).

To investigate the acidity-responsive properties of CaCO₃@CuO₂@L nanoparticles, they were dispersed in buffers with varying pH values. The release behavior of Cu²⁺ was evaluated, and the results demonstrated that only 5.7% of Cu²⁺ was released after 150 minutes of incubation in a pH 7.4 buffer. In a pH 6.8 buffer, 16.9% of Cu²⁺ was released at 5 minutes, and the release increased to 37.6% at 150 minutes. Furthermore, in a pH 6.5 buffer, the release rate and cumulative release amount were further enhanced, with 19.4% of Cu²⁺ released at 5 minutes and 53.6% released at 150 minutes (Fig. 1H). These findings indicate that the release behavior of Cu²⁺ from CaCO₃@CuO₂@L nanoparticles is pH-dependent, with a faster and greater release observed at lower pH values. According to the hypothesis, CuO₂ can decompose into O₂, Cu²⁺, and OH- in the acidic TME, thereby regulating the three components of the TME. To simulate this ability, the oxygen production capacity of CaCO₃@CuO₂@L was measured in vitro under simulated acidic conditions. The results demonstrated that CaCO₃@CuO₂@L continuously released oxygen, reaching its peak at 5 hours (Fig. 1I). Additionally, the pH-regulating ability of CaCO₃@CuO₂@L was evaluated, and it was found that the nanoparticles were capable of increasing the pH from 6.8 to 9 in an acidic buffer (Fig. 1J). During the process of H₂O₂ production from CaCO₃@CuO₂@L in a slightly acidic environment, a high concentration of hydroxyl radicals can be generated, leading to the oxidation of GSH and a reduction in its concentration. Moreover, Cu²⁺ plays an enhanced role in this process, as it promotes the rapid generation of high concentrations of H₂O₂ through catalysis. The results also demonstrated that CaCO₃@CuO₂@L can consume 1 mM of GSH in an acidic environment within 6 hours (Fig. 1K). These findings indicate that CaCO₃@CuO₂@L exhibits the ability to continuously produce oxygen, oxidize GSH, and regulate pH in vitro simulations.

The Uptake and TME regulation of CaCO₃@CuO₂@L in HCC cells

To assess the uptake of nanoparticles in HepG2 cells, we treated the cells with saline, CaCO₃@L, Copper ion carrier (Elesclomol-Cu), and CaCO₃@CuO₂@L, and measured the concentration of Cu using ICP-MS. The results indicated that the relative concentration of Cu in HepG2 cells incubated with saline, CaCO₃@L, Elesclomol-Cu, and CaCO₃@CuO₂@L was 1, 1.03, 4.9, and 5.8, respectively (Fig. 2A). These findings demonstrated that after an 8-hour incubation of HepG2 cells with CaCO₃@CuO₂@L, the intracellular Cu concentration significantly increased, with CaCO₃@CuO₂@L delivering Cu ions at a higher intensity compared to Elesclomol-Cu. Additionally, upon an 8-hour incubation of FITC-labeled CaCO₃@CuO₂@L, clear green fluorescence was observed in HepG2 cells, indicating the ingestion of CaCO₃@CuO₂@L by the cells (Fig. 2B). These results provide evidence that CaCO₃@CuO₂@L nanoparticles can effectively be taken up by HepG2 cells.

In our in vitro simulation experiments, CaCO₃@CuO₂@L nanoparticles exhibited sustained strong oxygen production capacity. However, can this capacity be further improved in HCC cells? To validate this, we utilized the anoxic probe [Ru(dpp)₃]Cl₂ (red fluorescence) technique in HCC cell lines. The results from fluorescence microscopy demonstrated that treatment with 0.3 µg/ml of CaCO₃@CuO₂@L significantly improved the hypoxic state of HepG2 cells under hypoxic conditions (Fig. 2C and 2D). In contrast, the fluorescence intensity in the CaCO₃@L and Elesclomol-Cu groups was similar to that of the oxygen-depleted blank control group, indicating their inability to improve the oxygen-depleted status of HepG2 cells (Fig. 2E and 2F). These findings suggest that CaCO₃@CuO₂@L nanoparticles can regulate the oxygen-deficient state of HCC cells at the cellular level by virtue of their continuous oxygen production capacity. Next, we employed the BBcellProbe® P01 intracellular pH fluorescence probe to examine changes in intracellular pH in HepG2 cells. The probe exhibits higher fluorescence intensity under alkaline conditions and lower intensity under acidic conditions. Fluorescence microscopy results showed that the pH of HepG2 cells cultured under anaerobic conditions was lower than that of normal cultured HepG2 cells. Moreover, the intracellular fluorescence intensity after treatment with CaCO₃@CuO₂@L was stronger than that in the other control groups (Fig. 2E and 2G). These results indicate that CaCO₃@CuO₂@L can improve the pH level of tumor cells, thereby enhancing the cellular "pantothenic acid" environment. Furthermore, we employed a GSH detection kit to assess changes in intracellular GSH levels after treatment in different drug administration groups. The results revealed that the GSH concentration in HepG2 cells cultured under anaerobic conditions was significantly higher compared to normal HepG2 cells. Notably, the GSH concentration in oxygen-depleted HepG2 cells treated with CaCO₃@CuO₂@L was significantly decreased compared to the other control groups (Fig. 2H). These findings suggest that CaCO₃@CuO₂@L can reduce the concentration of GSH in HCC cells at the cellular level. Taken together, these results indicate that CaCO₃@CuO₂@L nanoparticles can simultaneously regulate hypoxia, high GSH levels, and the pantothenic acid microenvironment in HCC cells.

Anti-tumor effects and mechanism of induced cuproptosis of CaCO3@CuO2@L in HCC cells

Subsequently, we assessed the inhibitory effect of CaCO₃@L, Elesclomol-Cu, and CaCO₃@CuO₂@L on HepG2 cells under normoxia and hypoxia conditions (21% and 1% O2). The inhibitory tendencies of Elesclomol-Cu and CaCO₃@CuO₂@L on HepG2 cells were similar under normoxia conditions, respectively (Fig. 3A). Under hypoxia conditions, Elesclomol-Cu significantly inhibited the growth of HepG2 cells. However, CaCO₃@CuO₂@L also exhibited a significant inhibitory effect on HepG2 cells (Fig. 3B). The cytotoxicity of Elesclomol-Cu and CaCO₃@CuO₂@L demonstrated a dose-dependent effect. These results suggest that the inhibition of the cuproptosis pathway under hypoxia conditions was verified, and the inhibitory effect of cuproptosis induced by hypoxia could be effectively activated by CaCO₃@CuO₂@L. To verify the mechanism by which cuproptosis is activated by CaCO₃@CuO₂@L under hypoxia conditions, we examined the expression of HIF-1α and "cuproptosis"-related proteins in HCC cells treated with CaCO₃@CuO₂@L under hypoxia conditions. Firstly, we evaluated the expression of HIF-1α through western blot analysis. The results indicated that compared to the normoxia condition, the expression of HIF-1α protein was significantly increased under hypoxia conditions, and the expression of HIF-1α protein was significantly decreased after CaCO₃@CuO₂@L treatment (Fig. 3C and 3D). Additionally, the expression of key proteins FDX1 and LIAS, related to the "cuproptosis" pathway, significantly decreased after CaCO₃@CuO₂@L treatment, while their expression did not change after Elesclomol-Cu treatment (Fig. 3C and 3E). This result also verified that "cuproptosis" in HCC cells was inhibited under hypoxia conditions. The "cuproptosis" pathway could be reactivated, and the proliferation of HCC cells could be inhibited by regulating the hypoxic microenvironment of HCC cells using CaCO₃@CuO₂@L.

Distributions of CaCO₃@CuO₂@L in primary HCC model mice

The Akt gene induced adipogenesis, while the Ras gene induced liver cell mitosis. These two genes synergistically contributed to the development of HCC after a 2-week period (Fig. 4A). To determine whether CaCO₃@CuO₂@L can enter HCC tissues, we examined the distribution of CaCO₃@CuO₂@L in mice with a primary HCC model following drug administration. The Akt/Ras plasmid transfection plasmid is embedded with the luciferase gene, which produces fluorescence when it binds to luciferin. We loaded luciferin during the preparation of CaCO₃@CuO₂@L, and using an in vivo imaging system, we found that free luciferin was injected into the tail vein for 0.5 h and bound to luciferase in liver HCC cells, producing strong fluorescence. However, it was quickly eliminated thereafter (Fig. 4B). Compared to free fluorescein, the fluorescence intensity of CaCO₃@CuO₂@L fluorescein gradually increased after 0.5 h of injection, reached its peak at 12 h, and remained strong until 48 h (Fig. 4C). Ex vivo observation of isolated organs at 12 and 48 h post-injection was consistent with the in vivo imaging results, showing a high distribution of CaCO₃@CuO₂@L in liver tissues (Fig. 4D and 4E). Additionally, CaCO₃@CuO₂@L particles exhibited apparent accumulation in the liver, potentially favoring their efficacy in HCC treatment. We then detected the distribution of copper following CaCO₃@CuO₂@L injection. At 3, 12, and 48 h after injection, the concentration of copper in the liver gradually increased with time and reached a concentration of 27.6 ug/g tissue at 12 h, which was much higher than the concentration of copper in other tissues (Fig. 4F and 4G). After 48 h, the concentration of copper in the liver still reached 20.8 ug/g tissue concentration (Fig. 4H). These results suggest an excellent liver targeting effect of CaCO₃@CuO₂@L and its long circulating property. Collectively, these results demonstrate that CaCO₃@CuO₂@L can efficiently deliver copper into HCC tissues.

CaCO₃@CuO₂@L suppress tumor growth in genedrives primary liver cancer model in mice

Following the formation of tumors, saline, CaCO₃@L, Copper ion carrier (Elesclomol-Cu), and CaCO₃@CuO₂@L were administered via tail vein injection five times per week. The growth of HCC was monitored at regular intervals using a small animal imaging system (Fig. 5A). Multiple screenings and detection of tumor formation time showed that in situ HCC could stably form 2 weeks after the high-pressure injection of the oncogenic plasmid. Therefore, drug administration started 2 weeks after the plasmid injection (Fig. 5B). Compared with saline and CaCO₃@L, the fluorescence intensity of the Elesclomol-Cu group after treatment was weaker, indicating that Elesclomol-Cu had a certain inhibitory effect on HCC but was significantly weaker than the CaCO₃@CuO₂@L group (Fig. 5B, 5C, and 5D). In the CaCO₃@CuO₂@L group, the fluorescence indicating HCC growth almost disappeared two weeks after injection, indicating that CaCO₃@CuO₂@L had the strongest inhibitory effect on HCC growth (Fig. 5E). To further verify the influence of drug therapy on HCC, mouse livers were dissected after administration for comparison, and various indexes of liver and HCC tissues were measured. The phenotype of mouse livers showed that the liver surface in the CaCO₃@CuO₂@L treatment group was smooth, indicating a better prognosis (Fig. 5F).

The weight of mice in the control and CaCO₃@L groups declined significantly after 8 days of treatment. Due to the rapid growth of HCC, which causes abnormal metabolism of liver function, the mice exhibited noticeable weight loss. However, the Elesclomol-Cu group experienced weight loss after injection, which may indicate some toxic side effects. In contrast, the weight of mice in the CaCO₃@CuO₂@L group remained relatively stable throughout the treatment period. This observation suggests that CaCO₃@CuO₂@L could improve the treatment prognosis and exhibit mild toxicity towards primary HCC (Fig. 5G). The maximum tumor size in the CaCO₃@CuO₂@L group was significantly smaller than that in the other groups (Fig. 5H). The liver weight ratio, which represents the prognosis after drug therapy, was the smallest in the CaCO₃@CuO₂@L group (0.019) compared to the control (0.126), CaCO₃@L (0.199), and Elesclomol-Cu (0.076) treatment groups (Fig. 5I). The number of tumor nodules in the CaCO₃@CuO₂@L group (2 nodules) was significantly lower than that in the control (26), CaCO₃@L (26), and Elesclomol-Cu (12) groups (Fig. 5J). Collectively, these data suggest that CaCO₃@CuO₂@L significantly inhibited HCC growth and generated an enhanced anti-tumor effect in this mouse model.

CaCO₃@CuO₂@L Suppresses Tumor Growth in HepG2 Xenograft Models in Mice

The anti-tumor effect of saline, CaCO₃@L, Elesclomol-Cu, and CaCO₃@CuO₂@L was further evaluated in a HepG2 xenograft nude mice model. Drug treatment was initiated with five doses once the tumor growth reached 50–100 mm³. The mice were euthanized, and tumor samples were collected on the final day (Fig. 6A). The results showed that the CaCO₃@CuO₂@L group (312 mm³) had smaller tumors compared to the control group (1716 mm³), CaCO₃@L group (1689 mm³), and Elesclomol-Cu group (1401 mm³) on day 20, and it showed the minimum tumor weight (Fig. 6B, C, and D). Compared with other groups, the tumor growth curve of nude mice in the CaCO₃@CuO₂@L group was gentler, indicating a better effect of inhibiting tumor growth. Notably, CaCO₃@CuO₂@L demonstrated a significant inhibition of tumor growth (Fig. 6E). These data suggest that CaCO₃@CuO₂@L treatment can inhibit tumor growth in HepG2 xenograft mice and enhance therapeutic efficacy in these mouse models.

The body weight curve of the Elesclomol-Cu group showed a sharp decrease (Fig. 6F), while the other groups did not exhibit this phenomenon. This indicates that compared to Elesclomol-Cu, the drug delivery system has fewer toxic and side effects. The excised tumors were further fixed and dissected for terminal deoxynucleotidyl transferase dUTP nick-end labeling (Ki67), which confirmed that the CaCO₃@CuO₂@L group inhibited tumor proliferation (Fig. 6G). Finally, the expression of HIF-1α was detected, and the results showed that the expression in the CaCO₃@CuO₂@L group was significantly lower than that in other groups (Fig. 6G).

It has been established that FDX1 and protein lipid acylation play crucial roles in cell death induced by copper ionophones[30]. Excessive copper levels promote the aggregation and functional loss of fatty acylated proteins, leading to the destabilization of iron-sulfur clusters and subsequent protein toxic stress, ultimately resulting in cell death[31, 32]. FDX1 serves as an upstream regulator of protein lipid acylation, and its loss leads to the complete loss of protein lipid acylation function[33]. Consequently, pyruvate and α-ketoglutaric acid accumulate while succinic acid is depleted within cells, indicating that the loss of protein lipid acylation function hampers the progression of the tricarboxylic acid (TCA) cycle[22]. This process distinguishes cuproptosis from existing cell death methods. In cuproptosis, the accumulation of copper ions within cells is the main process. These copper ions directly bind to the fatty acylated components of the TCA cycle, causing the aggregation and dysregulation of these proteins[25, 34]. As a result, the TCA cycle is disrupted, leading to protein toxic stress and subsequent cell death.

Tumor transplantation models have become a widely used tool for in vivo anti-tumor studies. However, it is important to consider the distinctions between subcutaneous and primary tumors. Firstly, subcutaneous tumors develop in an environment that lacks supportive factors such as sufficient oxygen supply, angiogenesis, and nutrients for mesenchymal or fibroblastic cells. Secondly, the use of immune-deficient mice in subcutaneous tumor models fails to accurately simulate the body's typical immune environment. Lastly, xenograft tumors solely consist of tumor cells and lack matrix components that may impact drug metabolism. In our investigation, we implemented an in situ HCC model based on the Akt/Ras plasmid, which was constructed via high-pressure injection[35]. Currently, animal transplantation tumor tests remain the most commonly used model for screening anti-tumor drugs and conducting research. Most of the drugs used in tumor chemotherapy have been discovered through this method. The advantages of this model include the ability to inoculate a group of animals with the same amount of tumor cells or cell-free filtrate (viral tumor), resulting in a more consistent growth rate, fewer individual differences, and a near 100% inoculation survival rate[36]. In this study, we applied the above two animal models to comprehensively verify the anti-tumor effect of nanoparticles in vivo, and the anti-swelling effect of nanoparticles assessed by the two methods was consistent.

In conclusion, we have reported a novel nanomaterial, CaCO₃@CuO₂@L, capable of inducing cuproptosis and synergistic therapy for HCC. This lipopathic watermelon-type nano drug delivery system demonstrates excellent stability in vitro and uniform distribution of ketone peroxide within the calcium carbonate nanomaterial. Upon entering HCC tissue, CaCO₃@CuO₂@L releases smaller particle size CuO₂ nanoparticles in the acidic tumor microenvironment. These nanoparticles undergo chemical reactions mediated by copper peroxide, resulting in the release of oxygen to regulate tumor hypoxia, the modulation of high concentrations of GSH and the "pantothenic acid" microenvironment, and the release of copper ions, ultimately inducing "cuproptosis" in HCC cells. By targeting multiple aspects of HCC biology, including aerobic respiration via the TCA cycle and high GSH concentrations, this nanoparticle exhibits a "trinity" effect to overcome factors that inhibit the activation of "cuproptosis" in HCC. Both in vitro and in vivo studies have demonstrated that this nanoparticle can effectively regulate the HCC microenvironment, induce cuproptosis, and inhibit the proliferation and growth of HCC. We anticipate that our study will serve as a catalyst for the design and synthesis of cuproptosis-inducing nanoplatforms with enhanced efficacy for cancer therapy.

HCC: hepatocellular carcinoma;

GSH: glutathione;

HIF-1α: hypoxia inducer;

TME: tumor microenvironment;

TCA: tricarboxylic acid cycle;

DDS: drug delivery system;

Acknowledgements

We gratefully acknowledge the assistance provided by the Analytical and Testing Center of HUST, the Centre for Materials Research and Analysis at Wuhan University of Technology, as well as Wuhan Youdu Biotechnology Co., Ltd and Shouzheng Pharma Biotechnology Co., Ltd. The scheme was created with BioRender.

Competing Interests

The authors have declared that no competing interest exists.

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the principles and procedures prescribed by the Ethics Committee of Huazhong University of Science and Technology.

Consent for publication

All authors agree to be published.

Availability of data and materials

All data generated or analyzed during this study are included in this manuscript.

Competing interests

The authors have declared that no competing interest exists.

Funding

This study was supported by the National Natural Science Foundation of China (grants 81703446 and 81673368) and the National Postdoctoral Program for Innovative Talents (grant BX201600057).

Authors' information

Weijie Li, Yong Liu and Han Wang contributed equally to this article.

Authors' contributions:

WL, YL and HW contribution was conception, design, writing and performed the experiment of nanoparticle synthesis study. ML has performed the experiment of cancer stem cells study. BL, YL has performed the experiment of in vitro evaluation study. FW has performed the experiment of nanoparticle characterization study. YX*, YL* and TY* as the corresponding author designed the whole subject and coordinated the resources of all parties. All authors read and approved the final manuscript.

Authors and Affiliations:

Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Weijie Li

School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, P. R. China;

Han Wang, Meijing Li, Yan Sun, Lijie Zhong, Bin Li, Yong Liu, Fei Wang, Tan Yang

Orthopedics Department,Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;

Yong Xu

Department of Pharmacy, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, P. R. China;

Yongji Lai

Corresponding authors:

Correspondence to Yong Xu, Yongji Lai or Tan Yang.

Zhou SL, Yin D, Hu ZQ, Luo CB, Zhou ZJ, Xin HY, et al. A Positive Feedback Loop Between Cancer Stem-Like Cells and Tumor-Associated Neutrophils Controls Hepatocellular Carcinoma Progression. Hepatology. 2019; 70: 1214-30.http://doi.org/10.1002/hep.30630
Zhao B, Ke K, Wang Y, Wang F, Shi Y, Zheng X, et al. HIF-1alpha and HDAC1 mediated regulation of FAM99A-miR92a signaling contributes to hypoxia induced HCC metastasis. Signal Transduct Target Ther. 2020; 5: 118.http://doi.org/10.1038/s41392-020-00223-6
Li Q, Ni Y, Zhang L, Jiang R, Xu J, Yang H, et al. HIF-1alpha-induced expression of m6A reader YTHDF1 drives hypoxia-induced autophagy and malignancy of hepatocellular carcinoma by promoting ATG2A and ATG14 translation. Signal Transduct Target Ther. 2021; 6: 76.http://doi.org/10.1038/s41392-020-00453-8
Chen Z, Han F, Du Y, Shi H, Zhou W. Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions. Signal Transduct Target Ther. 2023; 8: 70.http://doi.org/10.1038/s41392-023-01332-8
Li JQ, Wu X, Gan L, Yang XL, Miao ZH. Hypoxia induces universal but differential drug resistance and impairs anticancer mechanisms of 5-fluorouracil in hepatoma cells. Acta Pharmacol Sin. 2017; 38: 1642-54.http://doi.org/10.1038/aps.2017.79
Wang X, Mao J, Zhou T, Chen X, Tu H, Ma J, et al. Hypoxia-induced myeloid derived growth factor promotes hepatocellular carcinoma progression through remodeling tumor microenvironment. Theranostics. 2021; 11: 209-21.http://doi.org/10.7150/thno.49327
Tang H, Li C, Zhang Y, Zheng H, Cheng Y, Zhu J, et al. Targeted Manganese doped silica nano GSH-cleaner for treatment of Liver Cancer by destroying the intracellular redox homeostasis. Theranostics. 2020; 10: 9865-87.http://doi.org/10.7150/thno.46771
Allocati N, Masulli M, Di Ilio C, Federici L. Glutathione transferases: substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases. Oncogenesis. 2018; 7: 8.http://doi.org/10.1038/s41389-017-0025-3
Mo S, Zhang X, Hameed S, Zhou Y, Dai Z. Glutathione-responsive disassembly of disulfide dicyanine for tumor imaging with reduction in background signal intensity. Theranostics. 2020; 10: 2130-40.http://doi.org/10.7150/thno.39673
Franco R, Cidlowski JA. Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ. 2009; 16: 1303-14.http://doi.org/10.1038/cdd.2009.107
Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov. 2009; 8: 579-91.http://doi.org/10.1038/nrd2803
Cole SP. Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future. Annu Rev Pharmacol Toxicol. 2014; 54: 95-117.http://doi.org/10.1146/annurev-pharmtox-011613-135959
Bakos E, Evers R, Sinko E, Varadi A, Borst P, Sarkadi B. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol. 2000; 57: 760-8.http://doi.org/10.1124/mol.57.4.760
Uthaman S, Huh KM, Park IK. Tumor microenvironment-responsive nanoparticles for cancer theragnostic applications. Biomater Res. 2018; 22: 22.http://doi.org/10.1186/s40824-018-0132-z
Kim M, Lee NK, Wang CJ, Lim J, Byun MJ, Kim TH, et al. Reprogramming the tumor microenvironment with biotechnology. Biomater Res. 2023; 27: 5.http://doi.org/10.1186/s40824-023-00343-4
Jin HS, Choi DS, Ko M, Kim D, Lee DH, Lee S, et al. Extracellular pH modulating injectable gel for enhancing immune checkpoint inhibitor therapy. J Control Release. 2019; 315: 65-75.http://doi.org/10.1016/j.jconrel.2019.10.041
Webb BA, Chimenti M, Jacobson MP, Barber DL. Dysregulated pH: a perfect storm for cancer progression. Nat Rev Cancer. 2011; 11: 671-7.http://doi.org/10.1038/nrc3110
Fu LH, Wan Y, Qi C, He J, Li C, Yang C, et al. Nanocatalytic Theranostics with Glutathione Depletion and Enhanced Reactive Oxygen Species Generation for Efficient Cancer Therapy. Adv Mater. 2021; 33: e2006892.http://doi.org/10.1002/adma.202006892
An L, Cao M, Zhang X, Lin J, Tian Q, Yang S. pH and Glutathione Synergistically Triggered Release and Self-Assembly of Au Nanospheres for Tumor Theranostics. ACS Appl Mater Interfaces. 2020; 12: 8050-61.http://doi.org/10.1021/acsami.0c00302
Huang J, Huang Y, Xue Z, Zeng S. Tumor microenvironment responsive hollow mesoporous Co(9)S(8)@MnO(2)-ICG/DOX intelligent nanoplatform for synergistically enhanced tumor multimodal therapy. Biomaterials. 2020; 262: 120346.http://doi.org/10.1016/j.biomaterials.2020.120346
Wang L, Xia J, Fan H, Hou M, Wang H, Wang X, et al. A tumor microenvironment responsive nanosystem for chemodynamic/chemical synergistic theranostics of colorectal cancer. Theranostics. 2021; 11: 8909-25.http://doi.org/10.7150/thno.61651
Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022; 375: 1254-61.http://doi.org/10.1126/science.abf0529
Xie J, Yang Y, Gao Y, He J. Cuproptosis: mechanisms and links with cancers. Mol Cancer. 2023; 22: 46.http://doi.org/10.1186/s12943-023-01732-y
Ge EJ, Bush AI, Casini A, Cobine PA, Cross JR, DeNicola GM, et al. Connecting copper and cancer: from transition metal signalling to metalloplasia. Nat Rev Cancer. 2022; 22: 102-13.http://doi.org/10.1038/s41568-021-00417-2
Chen L, Min J, Wang F. Copper homeostasis and cuproptosis in health and disease. Signal Transduct Target Ther. 2022; 7: 378.http://doi.org/10.1038/s41392-022-01229-y
Xiong C, Ling H, Hao Q, Zhou X. Cuproptosis: p53-regulated metabolic cell death? Cell Death Differ. 2023; 30: 876-84.http://doi.org/10.1038/s41418-023-01125-0
Xu Y, Liu SY, Zeng L, Ma H, Zhang Y, Yang H, et al. An Enzyme-Engineered Nonporous Copper(I) Coordination Polymer Nanoplatform for Cuproptosis-Based Synergistic Cancer Therapy. Adv Mater. 2022; 34: e2204733.http://doi.org/10.1002/adma.202204733
Zhang X, He C, Chen Y, Chen C, Yan R, Fan T, et al. Cyclic reactions-mediated self-supply of H(2)O(2) and O(2) for cooperative chemodynamic/starvation cancer therapy. Biomaterials. 2021; 275: 120987.http://doi.org/10.1016/j.biomaterials.2021.120987
Yang T, Zhao P, Rong Z, Li B, Xue H, You J, et al. Anti-tumor Efficiency of Lipid-coated Cisplatin Nanoparticles Co-loaded with MicroRNA-375. Theranostics. 2016; 6: 142-54.http://doi.org/10.7150/thno.13130
Zulkifli M, Spelbring AN, Zhang Y, Soma S, Chen S, Li L, et al. FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery. Proc Natl Acad Sci U S A. 2023; 120: e2216722120.http://doi.org/10.1073/pnas.2216722120
Wang Y, Zhang L, Zhou F. Cuproptosis: a new form of programmed cell death. Cell Mol Immunol. 2022; 19: 867-8.http://doi.org/10.1038/s41423-022-00866-1
Xue Q, Kang R, Klionsky DJ, Tang D, Liu J, Chen X. Copper metabolism in cell death and autophagy. Autophagy. 2023; 19: 2175-95.http://doi.org/10.1080/15548627.2023.2200554
Sheftel AD, Stehling O, Pierik AJ, Elsasser HP, Muhlenhoff U, Webert H, et al. Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis. Proc Natl Acad Sci U S A. 2010; 107: 11775-80.http://doi.org/10.1073/pnas.1004250107
Li SR, Bu LL, Cai L. Cuproptosis: lipoylated TCA cycle proteins-mediated novel cell death pathway. Signal Transduct Target Ther. 2022; 7: 158.http://doi.org/10.1038/s41392-022-01014-x
Shang R, Song X, Wang P, Zhou Y, Lu X, Wang J, et al. Cabozantinib-based combination therapy for the treatment of hepatocellular carcinoma. Gut. 2021; 70: 1746-57.http://doi.org/10.1136/gutjnl-2020-320716
Song Y, Lu Q, Jiang D, Lan X. Validation and utility of HepG2 xenograft model for hepatocellular carcinoma. Eur J Nucl Med Mol Imaging. 2023; 50: 639-41.http://doi.org/10.1007/s00259-022-06043-w

Scheme is available in the Supplementary Files section.

Scheme.jpg
Scheme. Schematic illustration of CaCO₃@CuO₂@L remodeled induction of HCC TME and induced “cuproptosis” mechanism.

Download PDF

Reviewers invited by journal
19 Nov, 2023
Editor assigned by journal
21 Sep, 2023
First submitted to journal
20 Sep, 2023

You are reading this latest preprint version

The "Trinity" comprehensively regulates the tumor microenvironment of lipid-coated CaCO3@CuO2-watermelon nanoparticles induces "cuproptosis" in HCC

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Chemicals and Reagents

Cell Culture

Preparation of CuO2/PVP nanoparticles

Preparation of lipo-coated CaCO3@CuO2 hybrid watermelon-shaped Nanoparticles (CaCO3@CuO2@L)

Drug loading determination

Nanoparticle characterization

In vitro simulated regulation of oxygen production, REDOX (GSH) and acidic microenvironments

Intracellular uptake of nanoparticles

Cytotoxicity determination

Western Blotting Analysis

Mice

Primary HCC Mouse Model

HepG2 xenograft Mice Model

Statistical Analysis

Results

Preparation and Characterization of CaCO3@CuO2@L

The Uptake and TME regulation of CaCO3@CuO2@L in HCC cells

Anti-tumor effects and mechanism of induced cuproptosis of CaCO3@CuO2@L in HCC cells

Distributions of CaCO3@CuO2@L in primary HCC model mice

CaCO3@CuO2@L suppress tumor growth in genedrives primary liver cancer model in mice

CaCO3@CuO2@L Suppresses Tumor Growth in HepG2 Xenograft Models in Mice

Discussion

Conclusion

Abbreviations

Declarations

References

Scheme

Supplementary Files

Status:

Version 1

Preparation of CuO₂/PVP nanoparticles

Preparation of lipo-coated CaCO₃@CuO₂ hybrid watermelon-shaped Nanoparticles (CaCO₃@CuO₂@L)

Preparation and Characterization of CaCO₃@CuO₂@L

The Uptake and TME regulation of CaCO₃@CuO₂@L in HCC cells

Distributions of CaCO₃@CuO₂@L in primary HCC model mice

CaCO₃@CuO₂@L suppress tumor growth in genedrives primary liver cancer model in mice

CaCO₃@CuO₂@L Suppresses Tumor Growth in HepG2 Xenograft Models in Mice