Enhanced Breast Cancer Therapy Using Multifunctional Lipid- Coated Nanoparticles Combining Curcumin Chemotherapy and Nitric Oxide Gas Delivery

Enhanced Breast Cancer Therapy Using Multifunctional Lipid- Coated Nanoparticles Combining Curcumin Chemotherapy and Nitric Oxide Gas Delivery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced Breast Cancer Therapy Using Multifunctional Lipid- Coated Nanoparticles Combining Curcumin Chemotherapy and Nitric Oxide Gas Delivery Zhirong Yan, Peihan Xiao, Peng Ji, Rongjian Su, Zhenkun Ren, Li Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4210778/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The limitations of conventional treatment modalities for cancer, particularly breast cancer, have promoted the need to develop safer drug delivery systems (DDS). Chemotherapy combined with gas therapy has emerged as an attractive cancer treatment strategy. In this study, curcumin (Cur) loaded amorphous calcium carbonate nanoparticles (Cur-CaCO 3 ) were prepared by gas diffusion reaction. A "one-step" ethanol injection method was then used to prepare lipid-coated calcium carbonate nanoparticles (Cur-CaCO 3 @LA-Lip) loaded with L-arginine (LA) to achieve a combination of chemotherapy and NO to enhance the antitumor effect. The prepared Cur-CaCO 3 @LA-Lip was characterized and evaluated by transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-vis spectrometry, Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD) and differential scanning calorimetry (DSC). TEM observed that the Cur-CaCO 3 @LA-Lip nanoparticles were subspherical and had a distinct lipid layer covering the periphery. FTIR, XRD, and DSC indicated the successful synthesis of Cur-CaCO 3 @LA-Lip. Cur-CaCO 3 @LA-Lip exhibited a large drug loading capacity of 8.89% and 3.1% for Cur and LA, respectively, effectively preventing drug leakage. Furthermore, in vitro and in vivo, Cur-CaCO 3 @LA-Lip nanoparticles exhibited Cur sustained-release, high cellular uptake, high tumor accumulation, good biocompatibility, robust cytotoxicity, and antitumor efficacy. Biological sciences/Cancer/Cancer therapy Health sciences/Oncology/Cancer/Breast cancer Health sciences/Oncology/Cancer/Cancer therapy Physical sciences/Materials science/Biomaterials/Drug delivery Amorphous calcium carbonate breast cancer chemo-gas therapy biocompatible Drug delivery system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 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 overtaken lung cancer as the world's leading cancer for the first time 1 . As countries worldwide continue to study the pathogenesis, therapeutic drugs, prognosis, recurrence, and other aspects of breast cancer, the knowledge system related to the disease is also constantly improving. Although it is not yet possible to achieve a complete cure for breast cancer patients, the survival rate and quality of life of breast cancer patients are improving compared to the past, which undoubtedly has a positive significance for the clinical treatment of this disease 2 . So far, surgery in combination with chemotherapeutic drugs is still the primary tool throughout breast cancer treatment. Still, chemotherapeutic drugs with strong toxic side effects and drug resistance have seriously affected the survival quality and clinical efficacy of patients 3 , 4 . Therefore, it is essential to find low-toxic and highly effective drug candidates for breast cancer treatment, and it has become a research trend to discover highly effective inhibitors from natural products for developing anti-breast cancer drugs. 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 the treatment of inflammation, neurodegenerative diseases, cardiovascular system diseases, and malignant tumors 5 , 6 . Cur inhibits several malignancies and has shown promise in treating breast cancer 7 , 8 . 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 9 , 10 . As a single treatment, it is often ineffective and has highly toxic side effects, significantly limiting its clinical application 11 . 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 12 , 13 . Emerging gas therapy is considered a "green" treatment paradigm with negligible side effects 14 . 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 response 15 . Especially in the field of cancer therapy, nitric oxide not only kills cancer cells directly through nitrosylation of mitochondria and DNA at high concentrations (> 1 mm) but also enhances the efficacy of other therapeutic approaches such as chemotherapy, photodynamic therapy (PDT) and ultrasound (US) therapy 16 . L-arginine (LA) is a natural nitrogen donor with good biocompatibility and can be oxidized by H 2 O 2 to generate NO. LA in the H 2 O 2 -rich tumor microenvironment is expected to generate large amounts of intratumoral NO for gas therapy 17 , 18 . 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 therapy 19 . Amorphous calcium carbonate (CaCO 3 ) 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 carriers 20 . CaCO 3 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 properties 21 . Herein, we prepared a lipid (Lip)-encapsulated CaCO 3 nanoparticle for loading LA and Cur to construct multifunctional nanoparticles (Cur-CaCO 3 @LA-Lip, Scheme 1 ). Cur-CaCO 3 @LA-Lip was characterized by TEM, DLS, FTIR, XRD, and DSC, and its drug loading and release properties were evaluated. In addition, the pharmacodynamics, safety, and cellular uptake of Cur-CaCO 3 @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. Results and Discussion Construction and characterization of Cur-CaCO 3 @LA-Lip As shown in Fig. 1 , CaCl 2 was dissolved in ethanol and placed in a closed vessel along with (NH 4 ) 2 CO 3 . (NH 4 ) 2 CO 3 gradually decomposed volatile NH 3 and CO 2 diffused into the ethanol and dissolved to form CO 3 2− and NH 4+ . Under the alkaline condition formed by NH 4+ , CO 3 2− and Ca 2+ dissolved in ethanol reacted to form amorphous calcium carbonate cores, and due to the low water content of the reaction system (< 2%), the calcium carbonate cores did not undergo crystalline transformation during the growth process and CaCO 3 nanoparticles were finally obtained. The gas diffusion reaction using organic solvent as a medium was shown to be a simple and feasible method for CaCO 3 preparation 20 . Surface Morphology and Drug Loading (DL) of Cur-CaCO 3 @LA-Lip The morphology of Cur-CaCO 3 @LA-Lip observed by TEM is shown in Fig. 2 A. Cur-CaCO 3 @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. Cur-CaCO 3 @LA-Lip shows good solubility and dispersion in water, while free Cur is almost insoluble, indicating that Cur-CaCO 3 @LA-Lip formulation could significantly improve the solubility of Cur (Fig. 2 B). The hydrated particle size of Cur-CaCO 3 @LA-Lip determined by the Malvern particle size analyzer was (160.63 ± 6.96) 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-CaCO 3 @LA-Lip exhibited a large drug loading capacity of 8.89% and 3.1% for Cur and LA. UV-vis absorption of Cur-CaCO 3 @LA-Lip To confirm the co-incorporation of CaCO 3 @Lip with Cur and LA, we first investigated the absorbance of Cur, LA, CaCO 3 , Cur-CaCO 3 , and Cur-CaCO 3 @LA-Lip in UV-visible spectra in deionized water. The results are shown in Fig. 3 , and the maximum absorbance of Cur, Cur-CaCO 3, and Cur-CaCO 3 @LA-Lip was found to be around 440 nm, indicating that Cur and LA were successfully encapsulated in CaCO 3 @Lip. UV-vis spectra showed that Cur-CaCO 3 @LA-Lip was successfully prepared, similar to previous reports 20 , 21 . XRD of Cur-CaCO 3 @LA-Lip The results are presented in Fig. 4 . 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 CaCO 3 , and the characteristic crystal peaks of the drug are still evident, but the intensity is weakened. In contrast, in the formulations Cur-CaCO 3 and Cur-CaCO 3 @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-CaCO 3 and CaCO 3 @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-CaCO 3 @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 phase 22 . FTIR of Cur-CaCO 3 @LA-Lip FTIR was used to study the nature of the molecular interactions occurring within the nanoparticles (Fig. 5 ). FTIR first verified the prepared CaCO 3 . 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 CaCO 3 . 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 CaCO 3 . 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 CaCO 3 23 . For curcumin, the peak at ~ 3202 cm - 1 is attributed to the stretching of the -OH group on the benzene ring, the peak at ~ 1510 cm - 1 is attributed to the vibration of the C-C and C-O groups, and the peak at ~ 1275 cm - 1 is the stretching vibration of the enol C-O. The characteristic peaks of Cur and the carrier were still present in the physical mixture, indicating that the interaction of Cur with CaCO 3 did not occur. The characteristic peaks of Cur hardly appear in the spectra of Cur-CaCO 3 and Cur-CaCO 3 @LA-Lip nanoparticles, which confirms that Cur was successfully encapsulated in the nanoparticles 24 . To confirm the presence of the amorphous phase, it is also necessary to measure the molecular vibrations of the carbonate in it by infrared spectroscopy. All calcium carbonate phases can be distinguished by measuring the infrared absorption bands of carbonate. All phases of calcium carbonate have their characteristic absorption bands. In general, the absorption bands of carbonate can be divided into parts: the symmetric stretching vibration is at 1080 cm - 1 ; the out-of-plane bending absorption is at about 870 cm - 1 ; the asymmetric stretching vibration is at about 1400 cm - 1 ; and the in-plane bending is at 700 cm - 1 . For amorphous calcium carbonate, the in-plane bending vibration is broadened. It almost disappears, the out-of-plane bending vibration moves to 866 cm - 1 , and the asymmetric vibration peak splits into two parts at about 1420 cm - 1 and 1470 cm - 1 , respectively 25 . Figure 5 shows the characteristic absorption peaks of amorphous calcium carbonate at about 876 cm - 1 and the split peaks at about 1405 cm - 1 and 1466 cm - 1 , respectively. These results further indicate that the sample is amorphous calcium carbonate. DSC of Cur-CaCO 3 @LA-Lip The DSC results (Fig. 6 ) showed that the Cur and the physical mixture had a crystal heat absorption peak near 184°C, indicating the presence of Cur in crystalline form. The CaCO 3 carrier has no heat absorption peak near 184°C, while in Cur-CaCO 3 and Cur-CaCO 3 @LA-Lip, the characteristic peak of the melting point of Cur disappears, indicating that Cur exists in the amorphous form. TGA of Cur-CaCO 3 @LA-Lip The TGA results of CaCO 3 and CaCO 3 @Lip are shown in Fig. 7 . The figure shows that the mass loss of CaCO 3 and CaCO 3 @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 CaCO 3 and CaCO 3 @Lip carriers, and no degradation of macromolecules occurs. When the temperature rises to 150–400°C, the mass loss of CaCO 3 and CaCO 3 @Lip starts to accelerate, and the increase in loss is due to the gradual decomposition of the CaCO 3 matrix. When the temperature is 400°C, the residual masses of CaCO 3 and CaCO 3 @Lip were 67.14% and 56.3%, respectively; this is mainly because the Lip in CaCO 3 @Lip also undergoes thermal decomposition, resulting in a higher final weight loss than CaCO 3 . In conclusion, CaCO 3 and CaCO 3 @Lip carriers have good thermal stability. In Vitro Release Studies The in vitro release curve (Fig. 8 ) shows that the main release phase of Cur solution occurs within 8 hours, reaching 67.47% after 8 hours and 74.74% after 24 hours. The cumulative release of Cur-CaCO 3 @LA-Lip within 8 h only got 55.44%. In comparison, the cumulative release rate after 24 h reached 86.89%, indicating that Cur-CaCO 3 @LA-Lip exhibits good anti-surge release and sustained-release properties and can significantly improve the aqueous solubility of Cur, which helps to enhance the bioavailability of Cur. Assessment of NO generation in cells According to the results of the confocal fluorescence microscopy images in Fig. 9 , cells treated with Cur-CaCO 3 @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 cells 26 . The above results suggest that Cur-CaCO 3 @LA-Lip in the internal tumor microenvironment is expected to exert combined anti-tumor effects through NO treatment with chemotherapy. In vitro antitumor activity evaluation To determine the cytotoxicity of Cur-CaCO 3 @LA-Lip, in this experiment, different concentrations of Cur, Cur-CaCO 3, and Cur-CaCO 3 @LA-Lip were applied to the tumor cells for different times, and their inhibitory effects on cell proliferation were determined. The results are shown in Fig. 10 ; the cell proliferation inhibitory ability of Cur-CaCO 3 @LA-Lip was enhanced with the increase of Cur concentration and incubation time within 48 hours. The Cur-CaCO 3 @LA-Lip group showed stronger cytotoxicity than the free Cur group with increasing Cur concentration and incubation time. The corresponding IC 50 for 24 h were 121.07 ± 34.70 µg/mL (Cur) and 62.07 ± 10.45 µg/mL (Cur-CaCO 3 @LA-Lip), and the corresponding IC 50 for 48 h were 63.01 ± 4.36 µg/mL (Cur) and 42.35 ± 7.32 µg/mL (Cur-CaCO 3 @LA-Lip). The IC 50 values of Cur-CaCO 3 @LA-Lip and free Cur groups were significantly different (p < 0.05) at both 24 h and 48 h of incubation, which can be explained by the superior cellular uptake efficiency provided by the nanocarriers. The cytotoxicity of the Cur-CaCO 3 @LA-Lip group was much better than that of the Cur-CaCO 3 group due to the combination of chemotherapy and NO. Chemotherapy is the primary drug therapy in the clinical treatment of breast cancer, and gas therapy is an essential tool for adjuvant tumor treatment. Still, both are not ideal as a single treatment modality applied to breast cancer treatment. Since the mechanism of the cytotoxic effect of chemotherapeutic drugs and the means of NO production by gas therapy leading to cell death are very different, the construction of a nano-delivery system to co-deliver NO donors and chemotherapeutic drugs to tumor tissues is expected to be of significant research and application value to achieve efficient and low-toxic tumor combination therapy by complementing both chemotherapy and gas therapy 27 . Cellular uptake The cellular uptake behavior of CaCO 3 and Cur-CaCO 3 @Lip underlies their intracellular fate and is closely related to their biological function 28 . The uptake effects were analyzed using inverted fluorescence microscopy. Figure 11 A shows the fluorescence inverted microscopy of the free Cur, CaCO 3 , and Cur-CaCO 3 @Lip groups with 4T1 cells incubated for 2 h and 4 h, respectively, and Fig. 11 B shows the mean fluorescence intensity (MFI) of Cur quantified by Image J. The results show that the intracellular fluorescence intensity of Cur-CaCO 3 @Lip at 2 h and 4 h was significantly higher than that of free Cur and CaCO 3 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 endocytosis 29 . 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. Effectiveness and analysis of in vivo anti-tumor therapy To test whether Cur-CaCO 3 @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. 12 A). The body weight of the mice was monitored during treatment, and the results are shown in Fig. 12 B. As can be seen from the figure, the body weights of the mice 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-CaCO 3 @LA-Lip NPs. Figures 12 C, 12 D, and 12 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-CaCO 3 group was significantly reduced, mainly due to the anti-tumor effect of the CaCO 3 carrier, which enhances the passive transport of Cur. Compared to the Cur-CaCO 3 group, the tumor volume in the Cur-CaCO 3 @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. 13 F. The red color in the section is the cytoplasm, and the dark purple color is the nucleus 30 , 31 . Compared to the control group, tumor necrosis was observed in the Cur, Cur-CaCO 3 , and Cur-CaCO 3 @LA-Lip groups, especially in the Cur-CaCO 3 @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 tissue 32 . The Cur-CaCO 3 @LA-Lip group had the largest area of tumor necrosis after treatment and the best anti-tumor effect. Preliminary biosafety evaluation The hemolysis rates of the carrier materials CaCO 3 and CaCO 3 @Lip were investigated by in vitro erythrocyte hemolysis assay, and the results are shown in Fig. 13 A. The hemolysis rates of CaCO 3 and CaCO 3 @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 administration 33 . To further confirm the in vivo safety of the Cur-CaCO 3 @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. 13 B. There were no significant pathological changes in the major organs of the mice in the Cur-CaCO 3 @LA-Lip treatment group, indicating that Cur-CaCO 3 @LA-Lip has good biocompatibility and does not cause substantial damage to the mice. The above results demonstrate that Cur-CaCO 3 @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. Material and Methods Materials Curcumin (Cur), anhydrous calcium chloride (CaCl 2 ), ammonium carbonate, ammonium chloride (NH 4 Cl), and L-arginine were purchased from Shanghai McLean Biochemical Technology Co., Ltd., China. Lecithin was purchased from Shanghai Tai Wei Pharmaceutical Co. MTT Assay kit was purchased from China Biyuntian Biotechnology Co., Ltd. All the water used was double distilled water. Cell lines and animals Mouse-derived breast cancer 4T1 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ and 5% CO 2 in a humidified environment 34 , 35 . The animals in this work were 6- to 8-week-old healthy female mice (18–22 g, BALB/c) purchased from the Nanjing Qinglongshan Experimental Animal Center (Nanjing, China, animal certification number was 202336964). Design and construction of Cur-CaCO 3 @LA-Lip Synthesis of CaCO 3 CaCO 3 nanoparticles were prepared by a vapor diffusion reaction according to a previous report with minor modifications 20 , 36 . Briefly, 600 mg of CaCl 2 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 (NH 4 ) 2 CO 3 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 CaCO 3 , which was rinsed several times with absolute ethanol. The CaCO 3 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-CaCO 3 120 mg of Cur was weighed in a round bottom flask, dissolved in 50 mL of anhydrous ethanol, and 600 mg of CaCl 2 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 (NH 4 ) 2 CO 3 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-CaCO 3 . Preparation of Cur-CaCO 3 @LA-Lip Cur-CaCO 3 @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-CaCO 3 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-CaCO 3 @LA-Lip were centrifugated and dried via vacuum freeze for further use. Characterization of Cur-CaCO 3 @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-CaCO 3 @LA-Lip solution with distilled water and placing it in a Malvern particle size analyzer. A proper amount of Cur-CaCO 3 @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. UV-vis absorbance spectra analysis The appropriate amounts of Cur, LA, CaCO 3 , Cur-CaCO 3 , and Cur-CaCO 3 @LA-Lip were weighed and diluted with proper quantities of deionized water. The UV-vis between 250–600 nm spectra of substances in deionized water were obtained by UV-vis spectrometry. X-ray powder diffraction analysis (XRD) A small amount of Cur API, CaCO 3 , physical mixture, Cur-CaCO 3 , and Cur-CaCO 3 @LA-Lip was analyzed by X-ray powder diffraction with a scanning range of 5°~ 90°, and the scan rate was 1°/min, tube pressure 50 kV, tube current 200 mA, and X-ray diffraction patterns were obtained. Fourier transform infrared spectroscopy (FTIR) analysis The appropriate amounts of Cur API, CaCO 3 , physical mixture, Cur-CaCO 3 , CaCO 3 @Lip, and Cur-CaCO 3 @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. Differential scanning calorimetry (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 30 ~ 200°C and a heating rate of 10°C/min. Thermogravimetric analyzer (TGA) Thermal stability of CaCO 3 and CaCO 3 @Lip were evaluated by TGA. A small amount of CaCO 3 and CaCO 3 @Lip samples was taken and warmed to 400 ℃ under N 2 protection with a warming rate of 10 ℃/min to determine the weight loss curves. Drug loading (DL) Weigh 20–30 mg of Cur-CaCO 3 and Cur-CaCO 3 @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-CaCO 3 @LA-Lip at pH 5.0 was investigated by dialysis method, using saline containing 40% ethanol as the release medium at 37 ℃ and 150 r/min. The appropriate amounts of Lip@Cur-CaCO 3 and Cur API (both with 10 mg of Cur) were weighed precisely, added to equal quantities of release medium and mixed, then put into pretreated dialysis bags (MWCO: 12 kDa) in 200 mL of release medium, and 3 mL were sampled at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h, respectively, and promptly 3 mL of blank medium was replenished, and the absorbance at the corresponding time was measured by UV-Vis spectrophotometer to determine the content, calculate the cumulative release rate of Cur, and plot the in vitro release curve. Assessment of NO generation in cells To examine the extent of NO production in cells, treated cells were stained using the NO probe DAF-DA 37 . Briefly, 4T1 cells were seeded in six-well plates at 1 ×10 5 cells/well density and incubated for 24 h. After that, LA, Cur-CaCO 3 @LA-Lip (LA content of 250 µg) was added to 2 mL of medium, which was added to the wells and incubated for 24 hours. 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 agents 18 . Briefly, 4T1 cells were inoculated in 96-well plates at 1 × 10 4 cells/well density and incubated for 24 h. Cells were treated with three preparations: Cur solution, Cur-CaCO 3 , and Cur-CaCO 3 @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 (IC 50 ) was calculated accordingly. Cellular uptake study Each well was inoculated with 1×10 6 4T1 cells into a 12-well cell culture plate. After 24 h of incubation, the cell culture plate was removed and washed with PBS 38 . After repeated washing several times, Cur-CaCO 3 and Cur-CaCO 3 @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 × 10 6 cells. After anesthesia, 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 mm 3 in size. In vivo antitumor experiments Twenty tumor-bearing mice were randomly divided into four groups, namely the PBS group, Cur group, Cur-CaCO 3 group, and Cur-CaCO 3 @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 d 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 CaCO 3 and CaCO 3 @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 CaCO 3 and CaCO 3 @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 control 39 . 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: Hemolysis rate = (A sample - A nc )/(A pc - A nc ) 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 by 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-CaCO 3 ) by vapor diffusion reaction. Then lipid-encapsulated calcium carbonate nanoparticles (Cur-CaCO 3 @LA-Lip) loaded with L-arginine (LA) were prepared by a "one-step" ethanol injection method. It was demonstrated by TEM, UV-vis, FTIR, XRD, DSC, and TGA techniques that Cur-CaCO 3 @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 H 2 O 2 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 CaCO 3 nanoparticles as promising nanoplatforms to guide the preparation of various innovative nano-combined drug delivery systems for potentially treating diseases such as breast cancer. Declarations 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). Acknowledgement This research was funded by the China Medical Hand-in-Hand Project Committee Beijing Medical Award Foundation Research Project (No. YXJL-2021-1093-0668), Major Projects of Liaoning Provincial Department of Education (JYTZD201912), Zhejiang Province Selected Funding for Postdoctoral Research Projects (ZJ2023083), Zhejiang Provincial Natural Science Foundation of China (LQ24H300001), and Youth project of basic scientific research project of Liaoning Provincial Department of Education (LJKQZ20222376). Author contributions statement 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. 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Schematic diagram about the fabrication of Cur-CaCO 3 @LA-Lip NPs and anticancer mechanism of combined therapies based on chemotherapy and gas therapy. Cite Share Download PDF Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Jun, 2024 Reviews received at journal 10 Jun, 2024 Reviews received at journal 07 Jun, 2024 Reviewers agreed at journal 30 May, 2024 Reviewers agreed at journal 27 May, 2024 Reviewers invited by journal 21 May, 2024 Editor assigned by journal 14 Apr, 2024 Editor invited by journal 11 Apr, 2024 Submission checks completed at journal 11 Apr, 2024 First submitted to journal 03 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4210778","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":291275328,"identity":"a1c5e44b-b67a-4e14-85f7-84d0438df758","order_by":0,"name":"Zhirong Yan","email":"","orcid":"","institution":"Fujian Maternity and Child Health Hospital, Fujian Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhirong","middleName":"","lastName":"Yan","suffix":""},{"id":291275329,"identity":"88774a72-f7ef-40fa-be80-c907c7c5dd9b","order_by":1,"name":"Peihan Xiao","email":"","orcid":"","institution":"Fujian Maternity and Child Health Hospital, Fujian Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peihan","middleName":"","lastName":"Xiao","suffix":""},{"id":291275330,"identity":"17d3f3e1-f72e-4c0f-8a6a-e6af0c0c7c4b","order_by":2,"name":"Peng Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACxmYwJSHHxt984MCHH8RrsTHmkziWeHBmD/GWpSXOY8gxPszBRoRa5nbeg48Lfh1mbGM48+EwAw+DPL/YAUIO40s2ntl3mJmNuXfD4QILBsOZsxMIaeExk+btOczGxnB2w+EZPAwJBrcJazH/DdTCw8aQ8wBEEqXFjJnnR5oEUAsDsVr4kqV5G2wM2CSOGQADWYKwXwz7zx78zPNHon5+f/PjDx9+2MjzSxPS0sADtKoNzpfArxwE5IFRwcDwh7DCUTAKRsEoGMEAACd+Q8Tj72xjAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Ji","suffix":""},{"id":291275331,"identity":"1cf01c8f-25c5-4976-ae41-ed56017e3269","order_by":3,"name":"Rongjian Su","email":"","orcid":"","institution":"Jinzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rongjian","middleName":"","lastName":"Su","suffix":""},{"id":291275332,"identity":"2be9c382-889a-45e9-888e-958215064460","order_by":4,"name":"Zhenkun Ren","email":"","orcid":"","institution":"Jinzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenkun","middleName":"","lastName":"Ren","suffix":""},{"id":291275333,"identity":"8de68a3f-33e4-4738-8419-f5572fcea61c","order_by":5,"name":"Li Xu","email":"","orcid":"","institution":"Liaoning Vocational College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Xu","suffix":""},{"id":291275334,"identity":"97a6d87f-8af1-4624-a0e3-cf23bd42b91e","order_by":6,"name":"Xun Qiu","email":"","orcid":"","institution":"The Second Hospital of Dalian Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xun","middleName":"","lastName":"Qiu","suffix":""},{"id":291275335,"identity":"e107de64-be67-4adb-ae02-2accdf3fd562","order_by":7,"name":"Dan Li","email":"","orcid":"","institution":"The Second Hospital of Dalian Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-04-03 07:36:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4210778/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4210778/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-69229-2","type":"published","date":"2024-08-05T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54997021,"identity":"ccacc602-f332-45f6-b99b-8cce143c6363","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127352,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of CaCO\u003csub\u003e3\u003c/sub\u003e nanoparticles prepared by vapor diffusion method\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/b62ec8eac7a03c6f415dda77.jpg"},{"id":54997900,"identity":"0d79540b-b36e-4d68-a215-93159f8d36d1","added_by":"auto","created_at":"2024-04-19 18:16:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":268940,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Transmission electron micrographs of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip. (B) The appearances of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/742d103c7598c31508b1da7b.jpg"},{"id":54997026,"identity":"f1050c04-426a-4858-822e-4833092610ab","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":219270,"visible":true,"origin":"","legend":"\u003cp\u003eUV-vis absorbance spectra analysis.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/f3d9031d19afeeb5a087fe4c.jpg"},{"id":54998285,"identity":"2793f678-1014-412d-bd39-8e2aa4c45c05","added_by":"auto","created_at":"2024-04-19 18:24:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":163419,"visible":true,"origin":"","legend":"\u003cp\u003eThe XRD of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/223f628615f0bd8f46d7ce56.jpg"},{"id":54997027,"identity":"ac0b4552-ffad-47db-b1ed-47f6cdcac646","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169135,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transforms infrared spectroscopy.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/9aef37d6f35b5527fa73a8b9.jpg"},{"id":54997897,"identity":"b6231cc4-5640-4f73-b7ae-35c072c8cdff","added_by":"auto","created_at":"2024-04-19 18:16:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":141514,"visible":true,"origin":"","legend":"\u003cp\u003eDSC curves of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip.\u003c/p\u003e","description":"","filename":"floatimage7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/4bf7866c7872e3655cf7d7ec.jpg"},{"id":54997031,"identity":"00ef7356-7ba4-4e46-903c-39dbdfd98a6f","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":112642,"visible":true,"origin":"","legend":"\u003cp\u003eThe TGA of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip.\u003c/p\u003e","description":"","filename":"floatimage8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/6bcd9b622abab918f82aaee6.jpg"},{"id":54997025,"identity":"8943ee64-774c-44c8-b8c5-428733a514dd","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":108897,"visible":true,"origin":"","legend":"\u003cp\u003eCur release behavior of Cur API and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip.\u003c/p\u003e","description":"","filename":"floatimage9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/252cc3b0cb857245d2f63227.jpg"},{"id":54998284,"identity":"4b5999e9-c3c0-47a3-aadd-b546f43943a4","added_by":"auto","created_at":"2024-04-19 18:24:34","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":70599,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal fluorescence images of fluorescence intensity were obtained from cells without treatment and those treated with LA and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip. Cells were probed by DAF-DA to assess the presence of NO.\u003c/p\u003e","description":"","filename":"floatimage10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/0bdbe395ed9f6648f073cd20.jpg"},{"id":54997028,"identity":"3ad872c7-e737-4db1-987a-f4d0cb2b58fc","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":140986,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of Cur, Cur-CaCO\u003csub\u003e3,\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip measured by MTT assay in 4T1 after 24 h and 48 h incubation. Summary of the corresponding half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e). Significance levels are shown as *p \u0026lt; 0.05. Error bars indicate ± SD (n = 3).\u003c/p\u003e","description":"","filename":"floatimage11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/aece1e5bc7be38545805d789.jpg"},{"id":54997029,"identity":"f4246293-804f-441c-a83f-9e5f7a66226a","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":154830,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular uptake of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@Lip in 4T1 cells (A) and Quantification of the fluorescence intensity of 4T1 cells (B).\u003c/p\u003e","description":"","filename":"floatimage12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/937b82c867044c457b280fd3.jpg"},{"id":54997032,"identity":"27daf18a-75d7-43d3-9328-fd6109b488d4","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":345081,"visible":true,"origin":"","legend":"\u003cp\u003eCur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip exhibited the strongest in vivo antitumor efficacy. (A) Treatment scheme of PBS, Cur, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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\u0026amp;E.\u003c/p\u003e","description":"","filename":"floatimage13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/d499c064fb1ff16bc0a3d3fe.jpg"},{"id":54997901,"identity":"d7f4ab87-342d-47b5-a380-eb74da89deba","added_by":"auto","created_at":"2024-04-19 18:16:34","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":387182,"visible":true,"origin":"","legend":"\u003cp\u003ePreliminary biosafety evaluation of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip. (A) Hemolysis rate of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip carriers. (B) H\u0026amp;E staining of tissue sections of major organs (heart, liver, spleen, lung, and kidney) from the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip treated group.\u003c/p\u003e","description":"","filename":"floatimage14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/33e1d1107fdb88c7b8fc80b6.jpg"},{"id":62298518,"identity":"59bb6e5f-4398-42b4-b929-5ed8bb7aebde","added_by":"auto","created_at":"2024-08-12 16:14:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3274404,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/e8ab591d-c0a9-4026-bc81-4d974fe15608.pdf"},{"id":54997019,"identity":"3418208a-955a-4ce4-a426-42ed2365d7a4","added_by":"auto","created_at":"2024-04-19 18:08:34","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":339594,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic diagram about the fabrication of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip NPs and anticancer mechanism of combined therapies based on chemotherapy and gas therapy.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4210778/v1/0378fb94bccfc829e0a3f6be.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Breast Cancer Therapy Using Multifunctional Lipid- Coated Nanoparticles Combining Curcumin Chemotherapy and Nitric Oxide Gas Delivery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording 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 overtaken lung cancer as the world's leading cancer for the first time \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As countries worldwide continue to study the pathogenesis, therapeutic drugs, prognosis, recurrence, and other aspects of breast cancer, the knowledge system related to the disease is also constantly improving. Although it is not yet possible to achieve a complete cure for breast cancer patients, the survival rate and quality of life of breast cancer patients are improving compared to the past, which undoubtedly has a positive significance for the clinical treatment of this disease \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. So far, surgery in combination with chemotherapeutic drugs is still the primary tool throughout breast cancer treatment. Still, chemotherapeutic drugs with strong toxic side effects and drug resistance have seriously affected the survival quality and clinical efficacy of patients \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, it is essential to find low-toxic and highly effective drug candidates for breast cancer treatment, and it has become a research trend to discover highly effective inhibitors from natural products for developing anti-breast cancer drugs.\u003c/p\u003e \u003cp\u003eCurcumin (Cur), the main active ingredient of the rhizome of turmeric, has anti-tumor, antioxidant, and anti-inflammatory effects and plays a vital role in the treatment of inflammation, neurodegenerative diseases, cardiovascular system diseases, and malignant tumors \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Cur inhibits several malignancies and has shown promise in treating breast cancer \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. 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 \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As a single treatment, it is often ineffective and has highly toxic side effects, significantly limiting its clinical application \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. 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 \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEmerging gas therapy is considered a \"green\" treatment paradigm with negligible side effects \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. 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 response \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Especially in the field of cancer therapy, nitric oxide not only kills cancer cells directly through nitrosylation of mitochondria and DNA at high concentrations (\u0026gt;\u0026thinsp;1 mm) but also enhances the efficacy of other therapeutic approaches such as chemotherapy, photodynamic therapy (PDT) and ultrasound (US) therapy \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. L-arginine (LA) is a natural nitrogen donor with good biocompatibility and can be oxidized by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate NO. LA in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-rich tumor microenvironment is expected to generate large amounts of intratumoral NO for gas therapy \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, combining NO treatment with Cur chemotherapy has a high potential to improve antitumor efficacy.\u003c/p\u003e\u003cp\u003eCalcium 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 therapy \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Amorphous calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) 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 carriers \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. CaCO\u003csub\u003e3\u003c/sub\u003e 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 properties \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Herein, we prepared a lipid (Lip)-encapsulated CaCO\u003csub\u003e3\u003c/sub\u003e nanoparticle for loading LA and Cur to construct multifunctional nanoparticles (Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip, Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip was characterized by TEM, DLS, FTIR, XRD, and DSC, and its drug loading and release properties were evaluated. In addition, the pharmacodynamics, safety, and cellular uptake of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003ch2\u003eConstruction and characterization of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e was dissolved in ethanol and placed in a closed vessel along with (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e gradually decomposed volatile NH\u003csub\u003e3\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e diffused into the ethanol and dissolved to form CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and NH\u003csup\u003e4+\u003c/sup\u003e. Under the alkaline condition formed by NH\u003csup\u003e4+\u003c/sup\u003e, CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e dissolved in ethanol reacted to form amorphous calcium carbonate cores, and due to the low water content of the reaction system (\u0026lt;\u0026thinsp;2%), the calcium carbonate cores did not undergo crystalline transformation during the growth process and CaCO\u003csub\u003e3\u003c/sub\u003e nanoparticles were finally obtained. The gas diffusion reaction using organic solvent as a medium was shown to be a simple and feasible method for CaCO\u003csub\u003e3\u003c/sub\u003e preparation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eSurface Morphology and Drug Loading (DL) of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe morphology of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip observed by TEM is shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip is regular and sphere-like with a distinct bilayer structure of Lip at the periphery, and the particle size is 50\u0026thinsp;~\u0026thinsp;150 nm. Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip shows good solubility and dispersion in water, while free Cur is almost insoluble, indicating that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip formulation could significantly improve the solubility of Cur (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). The hydrated particle size of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip determined by the Malvern particle size analyzer was (160.63\u0026thinsp;\u0026plusmn;\u0026thinsp;6.96) nm with a PDI of 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 and a \u0026zeta;-potential of (-6.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50) mV. Experiments were performed in triplicate. Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip exhibited a large drug loading capacity of 8.89% and 3.1% for Cur and LA.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eUV-vis absorption of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo confirm the co-incorporation of CaCO\u003csub\u003e3\u003c/sub\u003e@Lip with Cur and LA, we first investigated the absorbance of Cur, LA, CaCO\u003csub\u003e3\u003c/sub\u003e, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip in UV-visible spectra in deionized water. The results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, and the maximum absorbance of Cur, Cur-CaCO\u003csub\u003e3,\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip was found to be around 440 nm, indicating that Cur and LA were successfully encapsulated in CaCO\u003csub\u003e3\u003c/sub\u003e@Lip. UV-vis spectra showed that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip was successfully prepared, similar to previous reports \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eXRD of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe results are presented in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. It can be seen that the Cur API has several strong crystal diffraction peaks between 5\u0026deg; and 30\u0026deg;, indicating the presence of Cur in crystalline form. The physical mixture is a simple mixture of Cur and CaCO\u003csub\u003e3\u003c/sub\u003e, and the characteristic crystal peaks of the drug are still evident, but the intensity is weakened. In contrast, in the formulations Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip, the characteristic peaks of Cur at 5\u0026deg;~25\u0026deg; 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-CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@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-CaCO\u003csub\u003e3\u003c/sub\u003e@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 phase \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eFTIR of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eFTIR was used to study the nature of the molecular interactions occurring within the nanoparticles (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). FTIR first verified the prepared CaCO\u003csub\u003e3\u003c/sub\u003e. The absorption peaks at around 876 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and 1088 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e are attributed to out-of-plane bending and symmetric stretching in the non-centrosymmetric structure of CaCO\u003csub\u003e3\u003c/sub\u003e. The cleavage peak at 1405 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e is attributed to the asymmetric stretching of carbonate ions. These absorption peaks are considered to be the characteristic absorption peaks of CaCO\u003csub\u003e3\u003c/sub\u003e. On the other hand, the absorption peak at 1635 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and the broad absorption near 3000 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e are attributed to the vibrational structure of water molecules in CaCO\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e23\u003c/sup\u003e. For curcumin, the peak at ~\u0026thinsp;3202 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e is attributed to the stretching of the -OH group on the benzene ring, the peak at ~\u0026thinsp;1510 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e is attributed to the vibration of the C-C and C-O groups, and the peak at ~\u0026thinsp;1275 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e is the stretching vibration of the enol C-O. The characteristic peaks of Cur and the carrier were still present in the physical mixture, indicating that the interaction of Cur with CaCO\u003csub\u003e3\u003c/sub\u003e did not occur. The characteristic peaks of Cur hardly appear in the spectra of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip nanoparticles, which confirms that Cur was successfully encapsulated in the nanoparticles \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTo confirm the presence of the amorphous phase, it is also necessary to measure the molecular vibrations of the carbonate in it by infrared spectroscopy. All calcium carbonate phases can be distinguished by measuring the infrared absorption bands of carbonate. All phases of calcium carbonate have their characteristic absorption bands. In general, the absorption bands of carbonate can be divided into parts: the symmetric stretching vibration is at 1080 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e; the out-of-plane bending absorption is at about 870 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e; the asymmetric stretching vibration is at about 1400 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e; and the in-plane bending is at 700 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. For amorphous calcium carbonate, the in-plane bending vibration is broadened. It almost disappears, the out-of-plane bending vibration moves to 866 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and the asymmetric vibration peak splits into two parts at about 1420 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and 1470 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, respectively \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the characteristic absorption peaks of amorphous calcium carbonate at about 876 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and the split peaks at about 1405 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and 1466 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, respectively. These results further indicate that the sample is amorphous calcium carbonate.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eDSC of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe DSC results (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) showed that the Cur and the physical mixture had a crystal heat absorption peak near 184\u0026deg;C, indicating the presence of Cur in crystalline form. The CaCO\u003csub\u003e3\u003c/sub\u003e carrier has no heat absorption peak near 184\u0026deg;C, while in Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip, the characteristic peak of the melting point of Cur disappears, indicating that Cur exists in the amorphous form.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eTGA of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe TGA results of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip are shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. The figure shows that the mass loss of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip decreases at a rate of about 5% and 7%, respectively, when the temperature is below 150\u0026deg;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 CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip carriers, and no degradation of macromolecules occurs. When the temperature rises to 150\u0026ndash;400\u0026deg;C, the mass loss of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip starts to accelerate, and the increase in loss is due to the gradual decomposition of the CaCO\u003csub\u003e3\u003c/sub\u003e matrix. When the temperature is 400\u0026deg;C, the residual masses of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip were 67.14% and 56.3%, respectively; this is mainly because the Lip in CaCO\u003csub\u003e3\u003c/sub\u003e@Lip also undergoes thermal decomposition, resulting in a higher final weight loss than CaCO\u003csub\u003e3\u003c/sub\u003e. In conclusion, CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip carriers have good thermal stability.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eIn Vitro Release Studies\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe in vitro release curve (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) shows that the main release phase of Cur solution occurs within 8 hours, reaching 67.47% after 8 hours and 74.74% after 24 hours. The cumulative release of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip within 8 h only got 55.44%. In comparison, the cumulative release rate after 24 h reached 86.89%, indicating that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip exhibits good anti-surge release and sustained-release properties and can significantly improve the aqueous solubility of Cur, which helps to enhance the bioavailability of Cur.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eAssessment of NO generation in cells\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAccording to the results of the confocal fluorescence microscopy images in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, cells treated with Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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 cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The above results suggest that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip in the internal tumor microenvironment is expected to exert combined anti-tumor effects through NO treatment with chemotherapy.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eIn vitro antitumor activity evaluation\u003c/h2\u003e\n \u003cp\u003eTo determine the cytotoxicity of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip, in this experiment, different concentrations of Cur, Cur-CaCO\u003csub\u003e3,\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip were applied to the tumor cells for different times, and their inhibitory effects on cell proliferation were determined. The results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e; the cell proliferation inhibitory ability of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip was enhanced with the increase of Cur concentration and incubation time within 48 hours. The Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip group showed stronger cytotoxicity than the free Cur group with increasing Cur concentration and incubation time. The corresponding IC\u003csub\u003e50\u003c/sub\u003e for 24 h were 121.07\u0026thinsp;\u0026plusmn;\u0026thinsp;34.70 \u0026micro;g/mL (Cur) and 62.07\u0026thinsp;\u0026plusmn;\u0026thinsp;10.45 \u0026micro;g/mL (Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip), and the corresponding IC\u003csub\u003e50\u003c/sub\u003e for 48 h were 63.01\u0026thinsp;\u0026plusmn;\u0026thinsp;4.36 \u0026micro;g/mL (Cur) and 42.35\u0026thinsp;\u0026plusmn;\u0026thinsp;7.32 \u0026micro;g/mL (Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip). The IC\u003csub\u003e50\u003c/sub\u003e values of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip and free Cur groups were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at both 24 h and 48 h of incubation, which can be explained by the superior cellular uptake efficiency provided by the nanocarriers. The cytotoxicity of the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip group was much better than that of the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e group due to the combination of chemotherapy and NO. Chemotherapy is the primary drug therapy in the clinical treatment of breast cancer, and gas therapy is an essential tool for adjuvant tumor treatment. Still, both are not ideal as a single treatment modality applied to breast cancer treatment. Since the mechanism of the cytotoxic effect of chemotherapeutic drugs and the means of NO production by gas therapy leading to cell death are very different, the construction of a nano-delivery system to co-deliver NO donors and chemotherapeutic drugs to tumor tissues is expected to be of significant research and application value to achieve efficient and low-toxic tumor combination therapy by complementing both chemotherapy and gas therapy \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003ch2\u003eCellular uptake\u003c/h2\u003e\n \u003cp\u003eThe cellular uptake behavior of CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@Lip underlies their intracellular fate and is closely related to their biological function \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The uptake effects were analyzed using inverted fluorescence microscopy. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA shows the fluorescence inverted microscopy of the free Cur, CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@Lip groups with 4T1 cells incubated for 2 h and 4 h, respectively, and Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB shows the mean fluorescence intensity (MFI) of Cur quantified by Image J. The results show that the intracellular fluorescence intensity of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@Lip at 2 h and 4 h was significantly higher than that of free Cur and CaCO\u003csub\u003e3\u003c/sub\u003e 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 endocytosis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e\n \u003ch2\u003eEffectiveness and analysis of in vivo anti-tumor therapy\u003c/h2\u003e\n \u003cp\u003eTo test whether Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eA). The body weight of the mice was monitored during treatment, and the results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB. As can be seen from the figure, the body weights of the mice 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-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip NPs. Figures \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eD, and \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eE 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-CaCO\u003csub\u003e3\u003c/sub\u003e group was significantly reduced, mainly due to the anti-tumor effect of the CaCO\u003csub\u003e3\u003c/sub\u003e carrier, which enhances the passive transport of Cur. Compared to the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e group, the tumor volume in the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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\u0026amp;E staining of tumor tissue from each group of mice are shown in Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003eF. The red color in the section is the cytoplasm, and the dark purple color is the nucleus \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Compared to the control group, tumor necrosis was observed in the Cur, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip groups, especially in the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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 tissue \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip group had the largest area of tumor necrosis after treatment and the best anti-tumor effect.\u003c/p\u003e\n \u003ch2\u003ePreliminary biosafety evaluation\u003c/h2\u003e\n \u003cp\u003eThe hemolysis rates of the carrier materials CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip were investigated by in vitro erythrocyte hemolysis assay, and the results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003eA. The hemolysis rates of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@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 administration \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. To further confirm the in vivo safety of the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip formulation, we performed an H\u0026amp;E staining analysis on the mice\u0026apos;s major organs (heart, liver, spleen, lung, and kidney), as shown in Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003eB. There were no significant pathological changes in the major organs of the mice in the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip treatment group, indicating that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip has good biocompatibility and does not cause substantial damage to the mice. The above results demonstrate that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Material and Methods","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCurcumin (Cur), anhydrous calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e), ammonium carbonate, ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl), and L-arginine were purchased from Shanghai McLean Biochemical Technology Co., Ltd., China. Lecithin was purchased from Shanghai Tai Wei Pharmaceutical Co. MTT Assay kit was purchased from China Biyuntian Biotechnology Co., Ltd. All the water used was double distilled water.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eCell lines and animals\u003c/h2\u003e\n\u003cp\u003eMouse-derived breast cancer 4T1 cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified environment \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe animals in this work were 6- to 8-week-old healthy female mice (18\u0026ndash;22 g, BALB/c) purchased from the Nanjing Qinglongshan Experimental Animal Center (Nanjing, China, animal certification number was 202336964).\u003c/p\u003e\n\u003cp\u003eDesign and construction of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eSynthesis of CaCO\u003csub\u003e3\u003c/sub\u003e\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCaCO\u003csub\u003e3\u003c/sub\u003e nanoparticles were prepared by a vapor diffusion reaction according to a previous report with minor modifications \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, 600 mg of CaCl\u003csub\u003e2\u003c/sub\u003e was dissolved in 200 \u0026micro;L 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 (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e 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 CaCO\u003csub\u003e3\u003c/sub\u003e, which was rinsed several times with absolute ethanol. The CaCO\u003csub\u003e3\u003c/sub\u003e 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.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e120 mg of Cur was weighed in a round bottom flask, dissolved in 50 mL of anhydrous ethanol, and 600 mg of CaCl\u003csub\u003e2\u003c/sub\u003e was dissolved in 200 \u0026micro;L 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 (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e 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-CaCO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCur-CaCO\u003csub\u003e3\u003c/sub\u003e@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-CaCO\u003csub\u003e3\u003c/sub\u003e 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-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip were centrifugated and dried via vacuum freeze for further use.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eCharacterization of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMorphology, particle size, zeta potential\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe nanoparticle size, polydispersity coefficient (PDI), and surface zeta potential were determined by diluting an appropriate amount of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip solution with distilled water and placing it in a Malvern particle size analyzer. A proper amount of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eUV-vis absorbance spectra analysis\u003c/h2\u003e\n \u003cp\u003eThe appropriate amounts of Cur, LA, CaCO\u003csub\u003e3\u003c/sub\u003e, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip were weighed and diluted with proper quantities of deionized water. The UV-vis between 250\u0026ndash;600 nm spectra of substances in deionized water were obtained by UV-vis spectrometry.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eX-ray powder diffraction analysis (XRD)\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eA small amount of Cur API, CaCO\u003csub\u003e3\u003c/sub\u003e, physical mixture, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip was analyzed by X-ray powder diffraction with a scanning range of 5\u0026deg;~ 90\u0026deg;, and the scan rate was 1\u0026deg;/min, tube pressure 50 kV, tube current 200 mA, and X-ray diffraction patterns were obtained.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eFourier transform infrared spectroscopy (FTIR) analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe appropriate amounts of Cur API, CaCO\u003csub\u003e3\u003c/sub\u003e, physical mixture, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, CaCO\u003csub\u003e3\u003c/sub\u003e@Lip, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e with a resolution of 4 cm\u003csup\u003e-\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, respectively.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eDifferential scanning calorimetry (DSC) analysis\u003c/h2\u003e\n \u003cp\u003eThe 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 30\u0026thinsp;~\u0026thinsp;200\u0026deg;C and a heating rate of 10\u0026deg;C/min.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eThermogravimetric analyzer (TGA)\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThermal stability of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip were evaluated by TGA. A small amount of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@Lip samples was taken and warmed to 400 ℃ under N\u003csub\u003e2\u003c/sub\u003e protection with a warming rate of 10 ℃/min to determine the weight loss curves.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eDrug loading (DL)\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eWeigh 20\u0026ndash;30 mg of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eDrug release properties\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe release behavior of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip at pH 5.0 was investigated by dialysis method, using saline containing 40% ethanol as the release medium at 37 ℃ and 150 r/min. The appropriate amounts of Lip@Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur API (both with 10 mg of Cur) were weighed precisely, added to equal quantities of release medium and mixed, then put into pretreated dialysis bags (MWCO: 12 kDa) in 200 mL of release medium, and 3 mL were sampled at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h, respectively, and promptly 3 mL of blank medium was replenished, and the absorbance at the corresponding time was measured by UV-Vis spectrophotometer to determine the content, calculate the cumulative release rate of Cur, and plot the in vitro release curve.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eAssessment of NO generation in cells\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eTo examine the extent of NO production in cells, treated cells were stained using the NO probe DAF-DA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Briefly, 4T1 cells were seeded in six-well plates at 1 \u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well density and incubated for 24 h. After that, LA, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip (LA content of 250 \u0026micro;g) was added to 2 mL of medium, which was added to the wells and incubated for 24 hours. The wells were washed thrice with serum-free DMEM and stained with DAF-DA (10 \u0026micro;M) for 30 min. Fluorescent images of intracellular NO were captured on a fluorescent microscope.\u003c/p\u003e\n \u003c/div\u003e\n \u003ch2\u003eIn vitro cell viability assay\u003c/h2\u003e\n \u003cp\u003eThe relative cell viability was measured using an MTT assay to assess the anticancer effect of the agents \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Briefly, 4T1 cells were inoculated in 96-well plates at 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well density and incubated for 24 h. Cells were treated with three preparations: Cur solution, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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 \u0026micro;L of MTT (5 mg/ mL) was added. Next, the medium was removed, and 150 \u0026micro;L 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) \u0026times; 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 (IC\u003csub\u003e50\u003c/sub\u003e) was calculated accordingly.\u003c/p\u003e\n \u003ch2\u003eCellular uptake study\u003c/h2\u003e\n \u003cp\u003eEach well was inoculated with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e 4T1 cells into a 12-well cell culture plate. After 24 h of incubation, the cell culture plate was removed and washed with PBS \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. After repeated washing several times, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip (equivalent Cur concentration, 50 \u0026micro;g/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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eEstablishment of 4T1 in situ breast cancer tumor-bearing mouse model\u003c/h2\u003e\n \u003cp\u003eThe 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 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells. After anesthesia, 6-8-week-old female Balb/c mice were injected with 200 \u0026micro;L 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 mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e in size.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eIn vivo antitumor experiments\u003c/h2\u003e\n \u003cp\u003eTwenty tumor-bearing mice were randomly divided into four groups, namely the PBS group, Cur group, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e group, and Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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 d 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.\u003c/p\u003e\n \u003ch2\u003eBiocompatibility evaluation\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe in vitro hemocompatibility of CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@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 CaCO\u003csub\u003e3\u003c/sub\u003e and CaCO\u003csub\u003e3\u003c/sub\u003e@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 control \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. 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:\u003c/p\u003e\n \u003cp\u003eHemolysis rate = (A\u003csub\u003esample\u003c/sub\u003e - A\u003csub\u003enc\u003c/sub\u003e)/(A\u003csub\u003epc\u003c/sub\u003e - A\u003csub\u003enc\u003c/sub\u003e)\u003c/p\u003e\n \u003cp\u003eNotes: A\u003csub\u003esample\u003c/sub\u003e is the sample group A value, A\u003csub\u003enc\u003c/sub\u003e is the negative control A value, and A\u003csub\u003epc\u003c/sub\u003e is a positive control A value.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eAll the experiments were conducted independently three more times. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed by GraphPad Prism software version 9.0. Multiple comparisons were assessed by the one-way or two-way ANOVA test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe 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-CaCO\u003csub\u003e3\u003c/sub\u003e) by vapor diffusion reaction. Then lipid-encapsulated calcium carbonate nanoparticles (Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip) loaded with L-arginine (LA) were prepared by a \"one-step\" ethanol injection method. It was demonstrated by TEM, UV-vis, FTIR, XRD, DSC, and TGA techniques that Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@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 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 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 CaCO\u003csub\u003e3\u003c/sub\u003e nanoparticles as promising nanoplatforms to guide the preparation of various innovative nano-combined drug delivery systems for potentially treating diseases such as breast cancer.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protocols and procedures related to the sampling, care, and management of animals were approved\u0026nbsp;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\u0026nbsp;study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by\u0026nbsp;the China Medical Hand-in-Hand Project Committee Beijing Medical Award Foundation Research Project (No. YXJL-2021-1093-0668), Major Projects of Liaoning Provincial Department of Education (JYTZD201912),\u0026nbsp;Zhejiang Province Selected Funding for Postdoctoral Research Projects (ZJ2023083), Zhejiang Provincial Natural Science Foundation of China (LQ24H300001), and Youth project of basic scientific research project of Liaoning Provincial Department of Education (LJKQZ20222376).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhirong 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H. \u003cem\u003eet al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Biomedical and environmental sciences: BES 35, 657\u0026ndash;662, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3967/bes2022.086\u003c/span\u003e\u003cspan address=\"10.3967/bes2022.086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amorphous calcium carbonate, breast cancer, chemo-gas therapy, biocompatible, Drug delivery system","lastPublishedDoi":"10.21203/rs.3.rs-4210778/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4210778/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe limitations of conventional treatment modalities for cancer, particularly breast cancer, have promoted the need to develop safer drug delivery systems (DDS). Chemotherapy combined with gas therapy has emerged as an attractive cancer treatment strategy. In this study, curcumin (Cur) loaded amorphous calcium carbonate nanoparticles (Cur-CaCO\u003csub\u003e3\u003c/sub\u003e) were prepared by gas diffusion reaction. A \"one-step\" ethanol injection method was then used to prepare lipid-coated calcium carbonate nanoparticles (Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip) loaded with L-arginine (LA) to achieve a combination of chemotherapy and NO to enhance the antitumor effect. The prepared Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip was characterized and evaluated by transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-vis spectrometry, Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD) and differential scanning calorimetry (DSC). TEM observed that the Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip nanoparticles were subspherical and had a distinct lipid layer covering the periphery. FTIR, XRD, and DSC indicated the successful synthesis of Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip. Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip exhibited a large drug loading capacity of 8.89% and 3.1% for Cur and LA, respectively, effectively preventing drug leakage. Furthermore, in vitro and in vivo, Cur-CaCO\u003csub\u003e3\u003c/sub\u003e@LA-Lip nanoparticles exhibited Cur sustained-release, high cellular uptake, high tumor accumulation, good biocompatibility, robust cytotoxicity, and antitumor efficacy.\u003c/p\u003e","manuscriptTitle":"Enhanced Breast Cancer Therapy Using Multifunctional Lipid- Coated Nanoparticles Combining Curcumin Chemotherapy and Nitric Oxide Gas Delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 18:08:29","doi":"10.21203/rs.3.rs-4210778/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-11T13:18:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-10T15:28:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-07T19:04:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4301850576701163255640709449207564363","date":"2024-05-30T20:01:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12323359668499662795328682723617412486","date":"2024-05-28T03:13:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-21T18:08:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-14T16:35:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-11T12:41:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-11T10:34:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-03T07:35:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1105aaa5-1583-4e8c-84a5-d25e9cab0a83","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30678859,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":30678860,"name":"Health sciences/Oncology/Cancer/Breast cancer"},{"id":30678861,"name":"Health sciences/Oncology/Cancer/Cancer therapy"},{"id":30678862,"name":"Physical sciences/Materials science/Biomaterials/Drug delivery"}],"tags":[],"updatedAt":"2024-08-12T16:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-4210778","link":"https://doi.org/10.1038/s41598-024-69229-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-05 15:58:01","publishedOnDateReadable":"August 5th, 2024"},"versionCreatedAt":"2024-04-19 18:08:29","video":"","vorDoi":"10.1038/s41598-024-69229-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-69229-2","workflowStages":[]},"version":"v1","identity":"rs-4210778","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4210778","identity":"rs-4210778","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}