Enhanced breast cancer therapy using multifunctional lipid-coated nanoparticles combining curcumin chemotherapy and nitric oxide gas delivery

Abstract

The limitations associated with conventional cancer treatment modalities, particularly for breast cancer, underscore the imperative for developing safer and more productive drug delivery systems. A promising strategy that has emerged is the combination of chemotherapy with gas therapy. We synthesized curcumin-loaded amorphous calcium carbonate nanoparticles (Cur-CaCO₃) via a gas diffusion reaction in the present study. Subsequently, a "one-step" ethanol injection method was employed to fabricate lipid-coated calcium carbonate nanoparticles (Cur-CaCO₃@LA-Lip) loaded with L-arginine, aimed at harnessing the synergistic effects of chemotherapy and nitric oxide to enhance antitumor efficacy. Transmission electron microscopy analysis revealed that Cur-CaCO₃@LA-Lip nanoparticles were subspherical with a distinct lipid layer encapsulating the periphery. Fourier transform infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry results confirmed the successful synthesis of Cur-CaCO₃@LA-Lip. The nanoparticles exhibited significant drug loading capacities of 8.89% for curcumin and 3.1% for L-arginine. In vitro and in vivo assessments demonstrated that Cur-CaCO₃@LA-Lip nanoparticles facilitated sustained release of curcumin and exhibited high cellular uptake, substantial tumor accumulation, and excellent biocompatibility. Additionally, the nanoparticles showed robust cytotoxicity and potent antitumor efficacy, suggesting their potential as a formidable candidate for breast cancer therapy.

Introduction

According to the latest global cancer burden data for 2020, published by the WHO's International Agency for Research on Cancer (IARC), new breast cancer cases have surpassed lung cancer as the most common cancer worldwide for the first time¹. Breast cancer, a complex and heterogeneous disease, is currently managed through traditional modalities such as surgery, radiotherapy, and chemotherapy, complemented by endocrine and targeted therapies². Despite their widespread use, these treatment strategies present significant limitations. Surgical intervention can effectively reduce tumor burden by excising visible tumor tissue but is ineffective against metastatic foci and carries risks of high recurrence and postoperative complications. Radiotherapy and chemotherapy, while capable of eradicating tumor cells extensively and at multiple sites, also inflict damage on normal tissues, leading to various side effects, drug resistance, and adverse reactions, thereby reducing clinical satisfaction among patients³. In recent years, the therapeutic potential of natural compounds in cancer treatment has garnered considerable recognition. Natural drugs generally exhibit lower toxicity compared to conventional chemotherapeutic agents, highlighting their promise as low toxicity and effective candidates for breast cancer treatment, thus offering significant clinical benefits.

Curcumin (Cur), the main active ingredient of the rhizome of turmeric, has anti-tumor, antioxidant, and anti-inflammatory effects and plays a vital role in treating inflammation, neurodegenerative diseases, cardiovascular system diseases, and malignant tumors^4,5. Cur inhibits several malignancies and has shown promise in treating breast cancer^6,7. However, Cur suffers from poor water solubility, easy conversion to glucuronide aldehyde and sulfonic acid complexes in the intestinal tract, rapid metabolism, short half-life, non-specific distribution in vivo, and susceptibility to drug resistance^8,9. As a single treatment, it is often ineffective and has highly toxic side effects, significantly limiting its clinical application¹⁰. Currently, a large number of nano-delivery systems have been developed and applied in the delivery of Cur, which not only improves the solubility of Cur and its targeting and bioavailability but also provides an opportunity for the combined application of Cur with other therapeutic tools, which is expected to play an essential role in breast cancer treatment^11,12.

Emerging gas therapy is considered a "green" treatment paradigm with negligible side effects¹³. Among the family of gas transmitters, nitric oxide (NO) is a gaseous transmitter that plays a vital role in various physiological and pathological processes, such as cardiovascular homeostasis, neurotransmission, and immune response¹⁴. Especially in cancer therapy, nitric oxide kills cancer cells directly through nitrosylation of mitochondria and DNA at high concentrations¹⁴. It enhances the efficacy of other therapeutic approaches such as chemotherapy, photodynamic therapy (PDT), and ultrasound (US) therapy¹⁵. L-arginine (LA) is a natural nitrogen donor with good biocompatibility and can be oxidized by H₂O₂ to generate NO. LA in the H₂O₂-rich tumor microenvironment is expected to generate large amounts of intratumoral NO for gas therapy^16,17. Therefore, combining NO treatment with Cur chemotherapy has a high potential to improve antitumor efficacy.

Calcium carbonate is a biomaterial with a wide range of biomedical applications. Although many crystalline calcium carbonate nanoparticle-based drug delivery systems have been developed, their slow-release properties do not meet the need for controlled release in oncology therapy¹⁸. Amorphous calcium carbonate (CaCO₃) has the general properties of calcium carbonate, such as excellent biocompatibility, bioactivity, and biodegradability, and is easy to synthesize and store. It also undergoes a rapid crystalline transformation in contact with water, so it can quickly release its drug load, which can effectively solve the shortcomings of slow drug release of calcium carbonate carriers¹⁹. CaCO₃ has been widely used for tumor-targeted payload delivery because of its multiple loading capabilities for different payloads, including biomolecules, small molecules, ions, and pH-dependent dissociation properties²⁰. Herein, we prepared a lipid (Lip)-encapsulated CaCO₃ nanoparticle for loading LA and Cur to construct multifunctional nanoparticles (Cur-CaCO₃@LA-Lip, Fig. 1). Cur-CaCO₃@LA-Lip was characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), and differential scanning calorimetry (DSC), and its drug loading and release properties were evaluated. In addition, the pharmacodynamics, safety, and cellular uptake of Cur-CaCO₃@LA-Lipd were assessed at the cellular and animal levels. Thus, this study provides a new paradigm for the expansion of chemo-gas-mediated treatment of breast cancer.

Figure 1.

Schematic diagram about the fabrication of Cur-CaCO₃@LA-Lip NPs and anticancer mechanism of combined therapies based on chemotherapy and gas therapy.

Results and discussion

Construction and characterization of Cur-CaCO₃@LA-Lip

As shown in Fig. 2a, CaCl₂ was dissolved in ethanol and placed in a closed vessel with (NH₄)₂CO₃. (NH₄)₂CO₃ gradually decomposed volatile NH₃ and CO₂ diffused into the ethanol and dissolved to form CO₃²⁻ and NH⁴⁺. Under the alkaline condition formed by NH⁴⁺, CO₃²⁻ and Ca²⁺ dissolved in ethanol reacted to form amorphous calcium carbonate cores, and due to the low water content of the reaction system, the calcium carbonate cores did not undergo a crystalline transformation during the growth process, and CaCO₃ nanoparticles were finally obtained. The gas diffusion reaction using organic solvent as a medium was shown to be a simple and feasible method for CaCO₃ preparation¹⁹. As shown in Fig. 2b, Cur-CaCO₃@LA-Lip shows good solubility and dispersion in water, while free Cur is almost insoluble, indicating that Cur-CaCO₃@LA-Lip formulation could significantly improve the solubility of Cur. The morphology of Cur-CaCO₃@LA-Lip observed by TEM is shown in Fig. 2c. Cur-CaCO₃@LA-Lip is regular and sphere-like with a distinct bilayer structure of Lip at the periphery, and the particle size is 50–150 nm. The hydrated particle size of Cur-CaCO₃@LA-Lip determined by the Malvern particle size analyzer was (160.63 ± 6.96, Fig. 2d) nm with a PDI of 0.28 ± 0.01 and a ζ-potential of (−6.16 ± 0.50) mV. Experiments were performed in triplicate. Cur-CaCO₃@LA-Lip exhibited a large drug loading capacity of 8.89% and 3.1% for Cur and LA.

Figure 2.

(a) Schematic diagram of CaCO₃ nanoparticles prepared by vapor diffusion method, (b) the appearances of Cur-CaCO₃@LA-Lip, (c) transmission electron micrographs of Cur-CaCO₃@LA-Lip, (d) the hydrated particle size of Cur-CaCO₃@LA-Lip by DLS.

XRD of Cur-CaCO₃@LA-Lip

The results are presented in Fig. 3. It can be seen that the Cur API has several strong crystal diffraction peaks between 5° and 30°, indicating the presence of Cur in crystalline form. The physical mixture is a simple mixture of Cur and CaCO₃, and the characteristic crystal peaks of the drug are still evident, but the intensity is weakened. In contrast, in the formulations, Cur-CaCO₃ and Cur-CaCO₃@LA-Lip, the characteristic peaks of Cur at 5°–25° were significantly weakened or mostly disappeared. The diffraction peaks were reduced and decreased compared with those of the physical mixture, indicating that Cur in Cur-CaCO₃ and CaCO₃@LA-Lip may exist partly in the microcrystalline state and mainly in the amorphous form, which provides a basis for its dissolution improvement. In addition, the amorphous phase exhibits no specific diffraction spikes, and Cur-CaCO₃@LA-Lip does not show any sharp pinacoidal diffraction peaks on the X-ray diffractograms but only broad peaks that are not pinacoidal, suggesting that the sample exists predominantly as an amorphous phase²¹.

Figure 3.

The XRD of Cur-CaCO₃@LA-Lip. The XRD data were analysed using Jade 6.5 software and the characteristic diffraction peaks were 8.836°, 12.098°, 18.587°, 23.022°, 24.503°, 29.406°, 35.966°, 39.402°, 43.146°, 47.490°, and 48.513°, with values of (001), (002), (003), (012), (004), (104), (110), (113), (202), (018), and (116), respectively.

FTIR of Cur-CaCO₃@LA-Lip

FTIR was used to study the nature of the molecular interactions occurring within the nanoparticles (Fig. 4). The absorption peaks at around 876 cm⁻¹ and 1088 cm⁻¹ are attributed to out-of-plane bending and symmetric stretching in the non-centrosymmetric structure of CaCO₃. The cleavage peak at 1405 cm⁻¹ is attributed to the asymmetric stretching of carbonate ions. These absorption peaks are considered to be the characteristic absorption peaks of CaCO₃. On the other hand, the absorption peak at 1635 cm⁻¹ and the broad absorption near 3000 cm⁻¹ are attributed to the vibrational structure of water molecules in CaCO₃²². This result is consistent with previously reported results, suggesting successful preparation of CaCO₃¹⁹.

Figure 4.

Fourier transforms infrared spectroscopy.

The interaction types and mechanisms within the multi-component delivery system were analyzed using Fourier Transform Infrared (FTIR) spectroscopy. As illustrated in Fig. 4, Cur exhibited characteristic absorption peaks at approximately 3510 cm⁻¹ (phenol O–H stretching vibration), 1628 cm⁻¹ and 1510 cm⁻¹ (combined C=O and C=C vibrations of benzene rings), 1270 cm⁻¹ (aromatic hydrocarbon C–O stretching vibration), and 1154 cm⁻¹ and 1026 cm⁻¹ (C–O–C stretching vibrations)^23,24. Notably, upon embedding Cur in CaCO₃@LA-Lip, several characteristic peaks were absent in the spectrum of Cur-CaCO₃@LA-Lip. These included the phenolic hydroxyl group stretching vibration (~ 3510 cm⁻¹) and the peak arising from the overlap of the C=C ring and C=O vibrations in the enol structure (~ 1508 cm⁻¹), among others. Additionally, the intensities of the characteristic peaks at approximately 1154 cm⁻¹ and 1026 cm⁻¹ were significantly reduced. These observations suggest that Cur is primarily encapsulated within the CaCO₃ cavities, with minimal free Cur adsorbed on the nanoparticle surface. The limitation of stretching and bending vibrations of chemical bonds in Cur following hydrogen bonding or hydrophobic interactions with the carrier leads to the disappearance of most characteristic peaks of Cur in the nano preparation²⁵. This observation confirms the successful encapsulation of Cur within the nanocarrier^24,26,27.

DSC of Cur-CaCO₃@LA-Lip

The DSC results in Fig. 5 indicate that the CaCO₃ support is predominantly amorphous. Cur exhibits a distinct endothermic peak at approximately 184 °C, corresponding to its melting point, signifying its crystalline nature. This endothermic peak is also observed in the physical mixture, maintaining its position at around 184 °C. However, in both the Cur-CaCO₃ and Cur-CaCO₃@LA-Lip formulations, the characteristic melting point peak of Cur is absent. This absence indicates a transformation from the crystalline to the amorphous form following nanoparticle preparation, consistent with the XRD results. These findings suggest a potential improvement in the solubility of Cur^28,29.

Figure 5.

DSC curves of Cur-CaCO₃ and Cur-CaCO₃@LA-Lip.

TGA of Cur-CaCO₃@LA-Lip

The TGA results of CaCO₃ and CaCO₃@Lip are shown in Fig. 6. The figure shows that the mass loss of CaCO₃ and CaCO₃@Lip decreases at a rate of about 5% and 7%, respectively, when the temperature is below 150 °C. This difference is not significant, mainly because the mass loss at this stage is primarily the loss of water molecules from the surface and pore channels of CaCO₃ and CaCO₃@Lip carriers, and no degradation of macromolecules occurs. When the temperature rises to 150–400 °C, the mass loss of CaCO₃ and CaCO₃@Lip starts to accelerate, and the increase in loss is due to the gradual decomposition of the CaCO₃ matrix. When the temperature is 400 °C, the residual masses of CaCO₃ and CaCO₃@Lip were 67.14% and 56.3%, respectively; this is mainly because the Lip in CaCO₃@Lip also undergoes thermal decomposition, resulting in a higher final weight loss than CaCO₃. The CaCO₃ and CaCO₃@Lip weight changes in the TGA were attributed to the breakdown of the encapsulated Lip, which was calculated to have an encapsulated Lip content of 10.84 wt %. In conclusion, CaCO₃ and CaCO₃@Lip carriers have good thermal stability.

Figure 6.

The TGA of CaCO₃ and CaCO₃@Lip.

In vitro stability and release studies

Dynamic dialysis was employed to investigate the in vitro release kinetics of Cur-CaCO₃@LA-Lip. A dialysis bag with a molecular weight cut-off of 8000–14,000 Da was utilized, which permitted the passage of free curcumin but not Cur-CaCO₃@LA-Lip³⁰. Consequently, only curcumin released from the nanoparticles could traverse into the dissolution medium. The results are illustrated in Fig. 7a. The free curcumin solution exhibited rapid release within the first 2 h, achieving near-equilibrium between the internal and external dialysis environments at 4 h, with a cumulative release rate of 88.15 ± 3.15%. Conversely, Cur-CaCO₃@LA-Lip did not reach equilibrium within the 24-h testing period, demonstrating a sustained release profile with a cumulative release rate of 70.11 ± 4.14%. The markedly slower release rate of Cur-CaCO₃@LA-Lip relative to free curcumin can be attributed to the encapsulation of curcumin within nanoparticles and the presence of liposomal bilayers on the surface, which impede the immediate release of curcumin and extend its release duration³¹.

Figure 7.

(a) Cur release behavior of Cur API and Cur-CaCO₃@LA-Lip. (b) Stability test for Cur and Cur-CaCO₃@LA-Lip at 4 ℃ and 37 ℃.

The stability of nanocarriers critically affects their delivery efficiency, and this study aims to predict the in vivo stability of nanocarriers in PBS under simulated physiological conditions³². As illustrated in Fig. 7b, at 4 ℃, the Cur content in both the free Cur and Cur-CaCO₃@LA-Lip groups gradually decreased, remaining above 95% throughout the study period. However, at 37 ℃, the degradation rate of free Cur was markedly higher compared to the Cur-CaCO₃@LA-Lip formulation. These findings indicate that Cur-CaCO₃@LA-Lip significantly enhances the stability of Cur, suggesting its potential for improved in vivo therapeutic efficacy.

Assessment of NO generation in cells

According to the fluorescence microscopy images in Fig. 8, cells treated with Cur-CaCO₃@LA-Lip showed the strongest fluorescence signal in the tested preparations, indicating that a large amount of NO was produced in the cells. The weak signal from cells treated with LA only did not differ from the signal from untreated cells, which can be attributed to the endogenous NO already present in the cells³³. The above results suggest that Cur-CaCO₃@LA-Lip in the internal tumor microenvironment is expected to exert combined anti-tumor effects through NO treatment with chemotherapy.

Figure 8.

Representative fluorescence images of fluorescence intensity were obtained from cells without treatment and those treated with LA and Cur-CaCO₃@LA-Lip. Cells were probed by DAF-DA to assess the presence of NO.

In vitro antitumor activity evaluation

To assess the cytotoxicity of Cur-CaCO₃@LA-Lip, various concentrations of Cur, Cur-CaCO₃, and Cur-CaCO₃@LA-Lip were administered to 4T1 tumor cells for different durations, and their inhibitory effects on cell proliferation were evaluated. As shown in Fig. 9, the inhibitory effect of Cur-CaCO₃@LA-Lip on cell proliferation increased with higher Cur concentrations and extended incubation times up to 48 h. The Cur-CaCO₃@LA-Lip group exhibited more cytotoxicity than free Cur under these conditions. Specifically, the IC₅₀ values for the 24-h incubation were 273.3 μg/mL for Cur and 99.0 μg/mL for Cur-CaCO₃@LA-Lip, while the IC₅₀ values for the 48-h incubation were 93.17 μg/mL for Cur and 54.0 μg/mL for Cur-CaCO₃@LA-Lip. The IC₅₀ values of the Cur-CaCO₃@LA-Lip group were much higher than those of the free Cur group at 24 h and 48 h, which can be attributed to the improved cellular uptake efficiency of the nanocarriers.

Figure 9.

Cytotoxicity of Cur, Cur-CaCO_3, and Cur-CaCO₃@LA-Lip measured by MTT assay in 4T1 after 24 h (a) and 48 h (b) incubation. (c) The corresponding half maximal inhibitory concentration (IC₅₀) of (a,b). Error bars indicate ± SD (n = 3).

Furthermore, the combination index (CI) for the synergistic anti-tumor effect of Cur and LA was calculated based on the in vitro cytotoxicity assays for 4T1 cell proliferation inhibition over 48 h³⁴. The CI value for Cur-CaCO₃@LA-Lip was 0.785. According to the criteria established by Soriano et al., a CI between 0.9 and 1.1 indicates an additive effect, 0.8 ≤ CI < 0.9 indicates a weak synergistic effect, 0.6 ≤ CI < 0.8 indicates a synergistic moderate impact, 0.4 ≤ CI < 0.6 indicates a high synergistic effect, and 0.2 ≤ CI < 0.4 indicates a strong synergistic effect³⁵. Therefore, the Cur-CaCO₃@LA-Lip formulation developed in this study demonstrates a synergistic moderate impact, supporting the potential for combining chemotherapy and gas therapy and laying a foundation for effective in vivo combination therapy for tumors.

Cellular uptake

The cellular uptake behavior of CaCO₃ and Cur-CaCO₃@Lip underlies their intracellular fate and is closely related to their biological function³⁶. The uptake effects were analyzed using fluorescence microscopy. Figure 10a shows the fluorescence inverted microscopy of the free Cur, CaCO₃, and Cur-CaCO₃@Lip groups with 4T1 cells incubated for 2 h and 4 h, respectively, and Fig. 10b shows the mean fluorescence intensity (MFI) of Cur quantified by Image J. The results show that the intracellular fluorescence intensity of Cur-CaCO₃@Lip at 2 h and 4 h was significantly higher than that of free Cur and Cur-CaCO₃ groups, indicating that lipid wrapping can dramatically enhance the cellular uptake of nanoparticles, which may be due to the similar bilayer of lipid and cell membrane and good cellular biocompatibility, which can significantly improve the efficiency of nanoparticle endocytosis³⁷. The pattern of data obtained from the 4 h incubation was the same as that of the 2 h incubation. Still, the qualitative observation of fluorescence by inverted microscopy showed that the green fluorescence of Cur was stronger compared to the 2 h incubation, indicating that the uptake of Cur is time-dependent, with more uptake and eventually saturation with increasing time.

Figure 10.

(a) Intracellular uptake of Cur-CaCO₃ and Cur-CaCO₃@Lip in 4T1 cells. (b) Quantification of the fluorescence intensity of 4T1 cells.

Effectiveness and analysis of in vivo anti-tumor therapy

To test whether Cur-CaCO₃@LA-Lip could be used to achieve an effective combination therapy, its anti-tumor properties were evaluated in a 4T1 mouse model of breast cancer (Fig. 11a). The body weight of the mice was monitored during treatment. As seen from Fig. 11b, the mice's body weights in the different treatment groups did not change significantly. They were all within a reasonable range without significant weight loss, indicating the low side effects of Cur-CaCO₃@LA-Lip NPs. Figure 11c–e show the mean tumor weight results, photographs of tumors obtained from each group, and curves of changes in tumor volume in mice during treatment, respectively. Compared with the PBS group, the tumor growth of mice in the Cur group was slightly inhibited, mainly because Cur is not a chemotherapeutic agent such as adriamycin, which has no significant anti-tumor effect. The tumor size of mice in the Cur-CaCO₃ group was significantly reduced, mainly due to the anti-tumor effect of the CaCO₃ carrier, which enhances the passive transport of Cur. Compared to the Cur-CaCO₃ group, the tumor volume in the Cur-CaCO₃@LA-Lip group was significantly suppressed due to the relatively high level of NO produced by LA, which can act as a cytotoxic and apoptosis-inducing agent for tumor treatment, thus enhancing the efficacy of Cur chemotherapy. Representative micrographs of H&E staining of tumor tissue from each group of mice are shown in Fig. 11f. The red color in the section is the cytoplasm, and the dark purple color is the nucleus^38,39. Compared to the control group, tumor necrosis was observed in the Cur, Cur-CaCO₃, and Cur-CaCO₃@LA-Lip groups, especially in the Cur-CaCO₃@LA-Lip group. The primary manifestation of tumor necrosis was the loss of nuclei, and a small amount of inflammatory cell infiltration was seen at the edge of the necrotic tumor tissue⁴⁰. The Cur-CaCO₃@LA-Lip group had the largest area of tumor necrosis after treatment and the best anti-tumor effect.

Figure 11.

Cur-CaCO₃@LA-Lip exhibited the strongest in vivo antitumor efficacy. (a) Treatment scheme of PBS, Cur, Cur-CaCO₃, and Cur-CaCO₃@LA-Lip. (b) Body change of 4T1-tumor bearing mice posts different treatments. (c) Tumor weights 12 days after the end of treatment. (d) Photographs of tumors were obtained from each group. (e) Tumor growth curve with different treatments. (f) Optical photographs of tumor sections stained with H&E.

Preliminary biosafety evaluation

The hemolysis rates of the carrier materials CaCO₃ and CaCO₃@Lip were investigated by in vitro erythrocyte hemolysis assay, and the results are shown in Fig. 12a. The hemolysis rates of CaCO₃ and CaCO₃@Lip increased slightly with an increasing mass concentration in the experimentally set mass concentration range. Still, they were both below 5%, indicating that they have good biosafety for intravenous administration⁴¹. To further confirm the in vivo safety of the Cur-CaCO₃@LA-Lip formulation, we performed an H&E staining analysis on the mice's major organs (heart, liver, spleen, lung, and kidney), as shown in Fig. 12b. There were no significant pathological changes in the major organs of the mice in the Cur-CaCO₃@LA-Lip treatment group, indicating that Cur-CaCO₃@LA-Lip has good biocompatibility and does not cause substantial damage to the mice. The above results demonstrate that Cur-CaCO₃@LA-Lip can achieve highly effective and low-toxicity therapeutic effects, reflecting the advantages and potential of LA combined with Cur for treating breast cancer.

Figure 12.

Preliminary biosafety evaluation of Cur-CaCO₃@LA-Lip. (a) Hemolysis rate of CaCO₃ and CaCO₃@Lip carriers. (b) H&E staining of tissue sections of major organs (heart, liver, spleen, lung, and kidney) from the Cur-CaCO₃@LA-Lip treated group.

Material and methods

Materials

Curcumin (Cur), anhydrous calcium chloride (CaCl₂), ammonium carbonate, ammonium chloride (NH₄Cl), and l-arginine were purchased from Shanghai McLean Biochemical Technology Co., Ltd., China. Lecithin was purchased from Shanghai Tai Wei Pharmaceutical Co. The MTT Assay kit was purchased from China Biyuntian Biotechnology Co., Ltd. All the water used was double-distilled.

Cell lines and animals

Mouse-derived breast cancer 4T1 cells were purchased from the Chinese Academy of Sciences cell bank (Shanghai, China). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ and 5% CO₂ in a humidified environment^42,43. The animals in this work were 6- to 8-week-old healthy female mice (18–22 g, BALB/c) purchased from Liaoning Changsheng Biotechnology Co., Ltd (Benxi, China).

Design and construction of Cur-CaCO₃@LA-Lip

Synthesis of CaCO₃

CaCO₃ nanoparticles were prepared by a vapor diffusion reaction according to a previous report with minor modifications^19,44. Briefly, 600 mg of CaCl₂ was dissolved in 200 μL of deionized water, diluted with 50 mL of anhydrous ethanol, and placed in a round-bottomed flask sealed with a sealing film. After leaving some air holes in the sealing film, the round bottom flask and the glass flask containing (NH₄)₂CO₃ were placed in a desiccator at 25 ℃, and the vapor diffusion reaction was carried out in a desiccator. After a predetermined time interval (20 h, i.e., a large amount of precipitation), the anhydrous ethanol in the round-bottomed flask was centrifuged (10 min at 8000 rpm) to obtain a white precipitate of CaCO₃, which was rinsed several times with absolute ethanol. The CaCO₃ was redispersed with an appropriate amount of anhydrous ethanol, then dispersed using a probe sonicator (400 W, 2 s operation, 3 s stop, 20 sonications), and prepared for use.

Preparation of Cur-CaCO₃

120 mg of Cur was weighed in a round bottom flask, dissolved in 50 mL of anhydrous ethanol, and 600 mg of CaCl₂ was dissolved in 200 μL of deionized water, mixed with Cur in a round bottom flask, and the flask was sealed with a sealing film. After leaving a few air holes in the sealing film, the round bottom flask and the glass flask containing (NH₄)₂CO₃ were placed in a desiccator, and the vapor diffusion reaction was carried out in a desiccator. After a predetermined time interval (20 h, i.e., a large amount of precipitation), the anhydrous ethanol in the round-bottom flask was centrifuged (8000 rpm for 10 min) to obtain a white precipitate of Cur-CaCO₃.

Preparation of Cur-CaCO₃@LA-Lip

Cur-CaCO₃@LA-Lip was prepared by ethanol injection method. 35 mg of LA was first dissolved by ultrasonication with 15 mL of deionized water. Next, 40 mg of soy lecithin, 10 mg of cholesterol, and 15 mg of Cur-CaCO₃ were weighed in the ratio of 40:10:15 and then dissolved by ultrasonication with 5 mL of anhydrous ethanol and injected into 15 mL of deionized water at a time with rapid stirring for 30 min at 40 ℃. Finally, the resulting Cur-CaCO₃@LA-Lip were centrifugated and dried via vacuum freeze for further use.

Characterization of Cur-CaCO₃@LA-Lip

Morphology, particle size, zeta potential

The nanoparticle size, polydispersity coefficient (PDI), and surface zeta potential were determined by diluting an appropriate amount of Cur-CaCO₃@LA-Lip solution with distilled water and placing it in a Malvern particle size analyzer. A proper amount of Cur-CaCO₃@LA-Lip suspension was dropped on the copper mesh, stained with 2.0% phosphotungstic acid, and observed by transmission electron microscope (TEM) for its morphology.

X-ray powder diffraction analysis (XRD)

A small amount of Cur API, CaCO₃, physical mixture, Cur-CaCO₃, and Cur-CaCO₃@LA-Lip was analyzed by X-ray powder diffraction with a scanning range of 5°–90°. The scan rate was 1°/min, tube pressure 50 kV, tube current 200 mA. The XRD data were analysed and processed using Jade 6.5 software and X-ray diffraction patterns were obtained.

Fourier transform infrared spectroscopy (FTIR) analysis

The appropriate amounts of Cur API, CaCO₃, physical mixture, Cur-CaCO₃, CaCO₃@Lip, and Cur-CaCO₃@LA-Lip were weighed and diluted with proper quantities of spectrally pure potassium bromide, respectively, and then pressed into tablets after grinding uniformly and analyzed by FTIR at 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹, respectively.

DSC analysis

The sample was weighed precisely (about 6 mg) and placed in a tapped aluminum crucible with alumina as the reference material and nitrogen as the protective gas at a temperature range of 40–240 °C and a heating rate of 10 °C/min.

Thermogravimetric analyzer (TGA)

TGA evaluated the thermal stability of CaCO₃ and CaCO₃@Lip. A small amount of CaCO₃ and CaCO₃@Lip samples was taken and warmed to 400 ℃ under N₂ protection with a warming rate of 10 ℃/min to determine the weight loss curves.

Drug loading (DL)

Weigh 20–30 mg of Cur-CaCO₃ and Cur-CaCO₃@LA-Lip, respectively, add 2 mL of 2 mol/L NH4Cl solutions to break the emulsion, then add 5 mL of pH 5.0 release medium, sonicate and dissolve, take the supernatant, dilute it a certain number of times and measure its absorbance, and repeat the measurement three times.

Drug release properties

The release behavior of Cur-CaCO₃@LA-Lip at pH 5.0 was investigated by dialysis using PBS solution containing 0.5% Tween-80 and 25% (v/v) ethanol as release medium at 37 °C and 100 rpm^30,45. Appropriate amounts of Lip@Cur-CaCO₃ and Cur API (each containing 2 mg Cur) were accurately weighed and mixed in equal amounts of release medium, then packed into pretreated dialysis bags (MWCO 8000–14,000 Da) in 150 mL of release medium, and 2 mL of the samples were dialyzed at 0.25, 0. 5, 1, 2, 4, 8, 12 and 24 h and immediately replenished with 2 mL of blank medium, the absorbance at each time point was measured using a UV–visible spectrophotometer, the content was determined, the cumulative release rate of Cur was calculated and the in vitro release curve was plotted.

In vitro stability

The stability of Cur-CaCO₃@LA-Lip was assessed by dispersing a measured quantity of Cur-CaCO₃@LA-Lip and free Cur in phosphate-buffered saline (PBS)⁴⁶. Stability experiments were conducted at 4 ℃ and 37 ℃ over 10 days. The absorbance of the formulation samples was recorded at regular intervals, and the Cur content was subsequently calculated. Each measurement was performed in triplicate to ensure accuracy and reproducibility.

Assessment of NO generation in cells

To examine the extent of NO production in cells, treated cells were stained using the NO probe DAF-DA⁴⁷. Briefly, 4T1 cells were seeded in six-well plates at 1 × 10⁵ cells/well density and incubated for 24 h. After that, LA, Cur-CaCO₃@LA-Lip (LA content of 250 μg) was added to 2 mL of medium, which was added to the wells and incubated for 24 h. The wells were washed thrice with serum-free DMEM and stained with DAF-DA (10 μM) for 30 min. Fluorescent images of intracellular NO were captured on a fluorescent microscope.

In vitro cell viability assay

The relative cell viability was measured using an MTT assay to assess the anticancer effect of the agents¹⁷. Briefly, 4T1 cells were inoculated in 96-well plates at 1 × 10⁴ cells/well density and incubated for 24 h. Cells were treated with three preparations: Cur solution, Cur-CaCO₃, and Cur-CaCO₃@LA-Lip. After 24 h and 48 h of incubation, the medium in each well was replaced with an equal volume of fresh medium, and 20 μL of MTT (5 mg/ mL) was added. Next, the medium was removed, and 150 μL of DMSO was added to each well to dissolve MTT. The absorbance wavelength at 570 nm was measured using an enzyme marker. Cytotoxicity was assessed using the following formula: OD (sample)/OD (control) × 100%, where OD (control) and OD (sample) indicate the absorbance value at 570 nm in the presence or absence of the sample, respectively. The half-maximal inhibitory concentration (IC₅₀) was calculated accordingly.

Cellular uptake study

Each well was inoculated with 1 × 10⁶ 4T1 cells into a 12-well cell culture plate. After 24 h of incubation, the cell culture plate was removed and washed with PBS⁴⁸. After repeated washing several times, Cur-CaCO₃ and Cur-CaCO₃@LA-Lip (equivalent Cur concentration, 50 μg/mL) were added to the wells, and the free Cur group was used as the control group and incubated for another 2 h and 4 h two times. Then, the drug-containing medium was aspirated from the culture plate, and PBS was added to each well for washing. After repeated washing several times, 4% (w/v) paraformaldehyde solution was added to each well for fixation and incubated for 20 min. After reaching the point, the paraformaldehyde solution was discarded, the cells were washed thrice with cold PBS, and the cell plate was placed on an inverted fluorescent microscope for imaging.

Establishment of 4T1 in situ breast cancer tumor-bearing mouse model

The mouse breast cancer 4T1 cell line was harvested at logarithmic growth time, resuspended in pre-chilled sterile PBS, and diluted to a concentration of 1 × 10⁶ cells. After anesthesia (2% isoflurane), 6–8-week-old female Balb/c mice were injected with 200 µL of the above cell suspension under the fat pad of the penultimate breast to establish a tumor-bearing mouse model. After inoculation, mice were observed and monitored daily for survival and tumor growth, and tumors were used for subsequent experiments when they were approximately 100 mm³ in size.

In vivo antitumor experiments

Twenty tumor-bearing mice were randomly divided into four groups, namely the PBS group, Cur group, Cur-CaCO₃ group, and Cur-CaCO₃@LA-Lip group, with five mice in each group. PBS and various preparations were injected into the mice by tail vein injection at 2 mg/kg of Cur once every 2 d for 6 consecutive doses for 12 d. The length and width of the tumors and the weight of the mice in each group were measured before and every 2 days after treatment. On day 12, the mice were killed, and the major organs (heart, liver, spleen, lungs, and kidneys) and tumors were removed, weighed, and photographed for preservation.

Biocompatibility evaluation

The in vitro hemocompatibility of CaCO₃ and CaCO₃@Lip carriers was investigated using an erythrocyte hemolysis assay to evaluate its safety for intravenous drug delivery preliminarily. Briefly, fresh murine blood was taken and treated with saline to prepare a 2% erythrocyte suspension. Different CaCO₃ and CaCO₃@Lip suspension concentrations were mixed with erythrocyte suspension in equal volumes and incubated for 2 h at 37 ℃ with distilled water as the positive control and saline as the negative control⁴⁹. The supernatant was centrifuged at 2500 r/min for 10 min, and its absorbance (A) value was measured at 545 nm, and the hemolysis rate was calculated according to the following equation:

$$\text{Hemolysis rate}=\left({\text{A}}_{\text{sample}}-{\text{A}}_{\text{nc}}\right)/\left({\text{A}}_{\text{pc}}-{\text{A}}_{\text{nc}}\right)$$

Notes: A_sample is the sample group A value, A_nc is the negative control A value, and A_pc is a positive control A value.

Statistical analysis

All the experiments were conducted independently three more times. Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software version 9.0. Multiple comparisons were assessed by the one-way or two-way ANOVA test. p < 0.05 was considered statistically significant.

Conclusions

The combination of chemotherapy and gas therapy has emerged as an attractive strategy for cancer treatment. This study prepared curcumin (Cur)-loaded amorphous calcium carbonate nanoparticles (Cur-CaCO₃) by vapor diffusion reaction. Then lipid-encapsulated calcium carbonate nanoparticles (Cur-CaCO₃@LA-Lip) loaded with L-arginine (LA) were prepared by a "one-step" ethanol injection method. It was demonstrated by TEM, FTIR, XRD, DSC, and TGA techniques that Cur-CaCO₃@LA-Lip was successfully constructed with good stability. Ultimately this formulation, on the one hand, Cur can kill cancer cells through chemotherapy. On the other hand, the acidic H₂O₂ generated by the tumor microenvironment can accelerate the oxidation of LA to enhance NO gas treatment and achieve the combination of chemotherapy and NO to enhance the anti-4T1 tumor effect. In conclusion, this work highlights our lipid-encapsulated CaCO₃ nanoparticles as promising nanoplatforms to guide the preparation of various innovative nano-combined drug delivery systems for potentially treating diseases such as breast cancer.

Ethics approval and consent to participate

All protocols and procedures related to the sampling, care, and management of animals were approved by the Jinzhou Medical University Animal Ethics Committee. All experiments and samplings were carried out in accordance with ethical and biosafety protocols approved by Hospital guidelines. Besides, this study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). All surgical procedures were performed under anesthesia (2% isoflurane). Method of euthanasia of mice: Cervical dislocation. Procedure: The operator held the tail of the nude mouse with the right hand, placed the mouse on the experimental table, held the head and neck of the mouse with the left hand, and applied force to pull the tail backward and upwards with the right hand. When the operator feels the spine separate, the animal dies immediately.

Author contributions

Zhirong Yan, Peihan Xiao and Peng Ji conceived the experiments, discussed the results, and wrote the main manuscript text. Rongjian Su, Zhenkun Ren, Li Xu and conducted the experiments, Xun Qiu and Dan Li analyzed the results. All authors reviewed the manuscript.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon request.

Competing interests

The authors declare no competing interests.