Ultrasound-responsive nanoparticles for nitric oxide release to inhibit the growth of breast cancer

Ultrasound-responsive nanoparticles for nitric oxide release to inhibit the growth of breast cancer | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ultrasound-responsive nanoparticles for nitric oxide release to inhibit the growth of breast cancer Haiyan Yang, Guangrong Zheng, GuoChen Li, Jincui Chen, Licui Qi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5186273/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Cancer Cell International → Version 1 posted 18 You are reading this latest preprint version Abstract Gas therapy represents a promising strategy for cancer treatment, with nitric oxide (NO) therapy showing particular potential in tumor therapy. However, ensuring sufficient production of NO remains a significant challenge. In this study, we successfully constructed ultrasound-responsive nanoparticles, which consisted of poly (D, L-lactide-co-glycolic acid) (PLGA) nanoparticles, natural L-arginine (LA), and superparamagnetic iron oxide nanoparticles (SPIO, Fe 3 O 4 NPs), denote as Fe 3 O 4 -LA-PLGA NPs. The Fe 3 O 4 -LA-PLGA NPs exhibited effective therapeutic effects both in vitro and in vivo , particularly in NO-assisted antitumor gas therapy and dual-modality imaging properties. Upon exposure to ultrasound irradiation, LA and Fe 3 O 4 NPs were rapidly released from the PLGA NPs. It was demonstrated that LA could spontaneously react with hydrogen peroxide (H 2 O 2 ) present in the tumor microenvironment to generate NO for gas therapy. Concurrently, Fe 3 O 4 NPs could rapidly react with H 2 O 2 to produce a substantial quantity of reactive oxygen species (ROS), which can oxidize LA to further facilitate the release of NO. In conclusion, the proposed ultrasound-responsive NO delivery platform exhibits significant potential in effectively inhibiting the growth of breast cancer. Breast cancer ultrasound nitric oxide gas therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Breast cancer represents the primary cause of cancer-related mortality among women worldwide [ 1 – 3 ]. While traditional treatment modalities, such as surgery, chemotherapy, and radiotherapy, can be efficacious in certain cases, they do not constitute a comprehensive solution[ 4 – 6 ]. The overall survival rate for patients with breast cancer remains low due to the aggressive nature of the disease and its poor prognosis [ 7 – 9 ]. Therefore, it is imperative to develop new therapeutic strategies for patients with breast cancer. In recent years, gas therapy has emerged as a promising therapeutic modality for tumors, attracting significant attention due to its high efficacy and favorable biological safety profile [ 10 – 12 ]. It has been extensively investigated in numerous anti-tumor applications, including gas-induced tumor cell killing, enhanced radiation therapy (RT) sensitization [ 13 , 14 ], chemotherapy [ 15 – 17 ], photothermal therapy (PTT) [ 18 , 19 ], photodynamic therapy (PDT) [ 20 ], and sonodynamic therapy (SDT) [ 21 ]. Nitric oxide (NO) is a prominent therapeutic gas in cancer therapy. It has been demonstrated to inhibit the growth of tumor cells through various mechanisms, including mitochondrial inhibition, DNA damage, nitrosylation of enzymes, and inhibition of cellular respiration. These effects have been observed at high concentrations (> 1µM), resulting in an excellent anti-tumor effect [ 22 – 24 ]. However, it is unfortunate that low NO concentrations can even promote tumor cell growth [ 25 , 26 ]. Thus, enhancing NO concentration is a crucial aspect of effective NO gas therapy. The direct utilization of free NO gas is limited by its short half-life and vulnerability to diverse biological substances ( e.g ., glutathione (GSH), hemoglobin, superoxide, and molecular oxygen) [ 24 ]. Therefore, numerous versatile nanoplatforms loaded with NO donors or NO-releasing molecules have been explored to deliver NO in a straightforward and precise manner. A plethora of NO-releasing donors e.g ., N, N′-di-sec-Butyl-N, N′-dinitroso-1,4-phenylenediamine (BNN6), S-nitrosothiols (SNO), S-nitroso glutathione (GSNO), and Rusen black salt (RBS)) have been explored to release NO under the external stimuli, including light (UV–vis/near-infrared laser), X-ray, and ultrasound [ 27 – 32 ]. However, previous NO-releasing donors have been found to be unsafe for biological applications due to their toxic production. Therefore, it is urgent to develop a biosafe and efficient strategy to achieve high capacity and controllable NO release. L-Arginine (LA) is a naturally occurring NO-releasing donor with high biocompatibility and the capacity to produce NO in the presence of inducible NO synthase [ 33 , 34 ]. Furthermore, LA can spontaneously release NO when reacting with hydrogen peroxide (H 2 O 2 ) [ 35 ], which is commonly abundant in tumor cells compared to normal cells [ 36 ]. In normal conditions, the release amount and speed of LA and H 2 O 2 is relatively slow. Our previous study demonstrated that the release of NO can be rapid and in large quantities over a short period of time when the presence of superparamagnetic iron oxide nanoparticles (SPIO, Fe 3 O 4 NPs) act as a catalyst [ 37 ]. It is well established that H 2 O 2 can react with Fe 2+ to produce reactive oxygen species (ROS) containing hydroxyl radicals (•OH) and hydroxide ions (OH − ), which is known as the Fenton reaction [ 38 , 39 ]. The greater the quantity of ROS generated, the more effectively it can oxidize LA to generate more NO[ 40 ]. Moreover, Fe 3 O 4 NPs, functioning as an "optical absorber", can generate noninvasive photoacoustic (PA) imaging contrast agent enhancement when stimulated by laser irradiation at specific wavelengths. Herein, in this study, we successfully constructed ultrasound-responsive nanoparticles for the rapid and controlled release of NO, which has been shown to inhibit breast cancer by utilizing NO as the antitumor therapeutic gas. Poly-lactide-co-glycolide (PLGA) is a nanosized polymeric material with high biodegradability and biocompatibility that has been approved by the Food and Drug Administration (FDA) for medical applications. We rationally designed and synthesized a versatile PLGA nanoparticle, encapsulating the natural LA as a NO donor in the core and Fe 3 O 4 NPs packed in the shell as an ultrasound-responsive therapeutic agent (denoted as Fe 3 O 4 -LA-PLGA NPs). The Fe 3 O 4 -LA-PLGA NPs demonstrated efficacious therapeutic effects through NO-assisted antitumor gas therapy. As illustrated in Scheme 1 , Fe 3 O 4 -LA-PLGA NPs can accumulate into the tumor region via the enhanced permeability and retention (EPR) effect after intravenous injection. The distribution of Fe 3 O 4 -LA-PLGA NPs can be monitored via dual-modality imaging, including fluorescence (FL) and photoacoustic (PA) imaging. Upon exposure to ultrasound irradiation, LA and Fe 3 O 4 NPs were released rapidly from the PLGA NPs, thereby initiating a cascade of reactions. LA can spontaneously react with H 2 O 2 present in the tumor microenvironment to generate NO for gas therapy. Simultaneously, the integrated Fe 3 O 4 NPs can rapidly react with H 2 O 2 to produce a substantial quantity of reactive oxygen species (ROS). Moreover, the generated ROS can oxidize LA to facilitate the release of NO. The results of this study suggest that ultrasound-responsive nanoparticles have significant potential as a delivery platform for NO, with the potential to inhibit cancer growth. 2 Materials and Methods PLGA-PEG-COOH (50: 50, MW: 15 000) was purchased from Daigang BIO Engineer Ltd, Co. (Shan Dong, China). Poly (vinyl alcohol) (PVA) was purchased from Sigma-Aldrich Chemical Co., Ltd., (St. Louis, MO, USA). Trichloromethane (CHCl 3 ) was purchased from Chongqing East Chemical Industry Ltd, Co. (China). L-Arginine was purchased from Sigma-Aldrich (USA), and iron oxide nanoparticles (10 nm, 25 mg mL − 1 ) were purchased from Ocean Nanotech Co. Ltd (USA). The cell counting kit (CCK-8) and calcein & propidium iodide (PI) apoptosis assay kit were purchased from Dojindo Laboratories (Kumamoto, Japan). 3-Amino,4-aminomethyl-2',7'-difluorescein, diacetate (DAF-FM DA), a nitric oxide assay kit (Griess assay kit), 4,6-diamidino-2-phenylindole (DAPI), and 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR) were purchased from Beyotime Biotechnology Co., Ltd (China). 2.1 Preparation of Fe 3 O 4 -LA-PLGA NPs The Fe 3 O 4 -LA-PLGA NPs were prepared via the ultrasound double emulsion (water/oil/water, W/O/W) method. First, 150 mg PLGA-PEG-COOH (50: 50, MW: 15 000) and 10 mg Fe 3 O 4 NPs were completely dissolved in 2 mL of CHCl 3 . Then, 40 mg LA was added, and the mixture was sonicated with an ultrasonic cell crusher at 105 W for 2 minutes to obtain the W/O emulsion; subsequently, 4 mL of PVA solutions (w/v = 4%) was added, and the mixture was emulsified again at 105 W for 5 minutes to obtain the W/O/W emulsion. Next, 8 mL of isopropyl alcohol solution (w/v = 2%) was added, and the mixture was stirred to volatilize the CHCl 3 . Finally, the Fe 3 O 4 -LA-PLGA NPs were collected following centrifugation (10 000 rpm, 10 min) and stored at 4°C for subsequent utilization. The fluorescent Fe 3 O 4 -LA-PLGA NPs were prepared by a similar procedure, with DiR added to the mixture. 2.2 Characterization of Fe 3 O 4 -LA-PLGA NPs Transmission electron microscopy (TEM, Hitachi 7500, Tokyo, Japan) was employed to observe the structure of Fe 3 O 4 -LA-PLGA NPs. The Nano ZS90 Zetasizer (Malvern Panalytical, Ltd., Malvern, UK) was employed to ascertain the size distribution and Zeta potential of Fe 3 O 4 -LA-PLGA NPs. Fluorescence (FL) images were acquired using the LB983 imaging system (Berthold Technologies GmbH & Co. KG, Germany). Photoacoustic (PA) images were obtained using the Vevo LAZR PA Imaging System (Visual Sonics Inc., Toronto, Canada). Confocal laser scanning microscope (CLSM) images were captured using a Nikon optical microscope (Japan). The flow cytometry assay was employed by Beckman Coulter (American). 2.3 LA loading content, Encapsulation Efficiency, and NO release determination The ultraviolet-visible spectrophotometer (UV-vis) (Shimadzu UV 2600, Kyoto, Japan) was employed to generate the standard curve for LA, and the LA loading efficiency and encapsulation were quantified and evaluated in accordance with the following equations: Loading efficiency (%) = (total LA - unbound LA)/total LA × 100%; Encapsulation (%) = (total LA - unbound LA)/total nanoparticles × 100% The NO release was quantified using the Griess assay kit. In brief, Fe 3 O 4 -LA-PLGA NPs, Fe 3 O 4 -PLGA NPs, and LA-PLGA NPs (containing 10 mg PLGA, respectively) were dispersed in 1 mL PBS and mixed with an excess of H 2 O 2 (50 µM). Subsequently, the mixture was irradiated with or without ultrasound (Chongqing Haifu Technology, China) at a frequency of 200 kHz and a power density of 2 W for a duration of 200 S. The NO release was quantified by means of a Griess assay kit at various time points (1 min, 2 min, 3 min, 5 min, 10 min, 20 min, and 30 min) following ultrasound irradiation. 2.4 Cell culture and MDA-MB-231 tumor-bearing mouse model The human breast cancer MDA-MB-231 cells and Human umbilical vein endothelial cells (HUVECs) were obtained from the Chinese Academy of Sciences Cell Bank (China). The cells were cultured at 37°C with 5% CO 2 in DMEM, which was supplemented with 10% FBS and 1% streptomycin/penicillin. A total of 0.1 mL of PBS solution (containing 1 × 10 6 MDA-MB-231 cells) was injected subcutaneously into the left flank of female BALB/c nude mice (weighing between18 and 20 g) to establish the tumor model. The tumor volume was calculated according to the formula [0.5 × length × (width) 2 ]. The biosafety of Fe 3 O 4 -LA-PLGA NPs was evaluated in BALB/c mice. All animal care and use procedures were reviewed and approved by the Animal Ethics Committee of Chongqing Medical University. 2.5 In vitro cytotoxicity assay To assess the cytotoxicity and growth inhibition of Fe 3 O 4 -LA-PLGA NPs in vitro , we conducted a series of co-incubation experiments involving different concentrations of NPs (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 mg/mL) and MDA-MB-231 cells (or HUVECs) at 37°C, with or without ultrasound irradiation. The cell viability was assessed by the cell counting kit-8 (CCK-8) assay (n = 5). Furthermore, the viable and dead cells were co-stained with calcein-AM and propidium iodide (PI), and their distribution was observed by confocal laser scanning microscopy (CLSM). 2.6 Ultrasound-responsive intracellular NO release To assess the impact of ultrasound irradiation on intracellular NO release, MDA-MB-231 cells (1 × 10 5 ) were randomly allocated to eight experimental groups: control, Fe 3 O 4 -PLGA NPs, LA-PLGA NPs, Fe 3 O 4 -LA-PLGA NPs, ultrasound irradiation (US), Fe 3 O 4 -PLGA NPs + US, LA-PLGA NPs + US, and Fe 3 O 4 -LA-PLGA NPs + US. Cells in the first and fifth groups were treated with phosphate buffered saline (PBS), respectively. The remaining groups were treated with Fe 3 O 4 -PLGA NPs, LA-PLGA NPs, and Fe 3 O 4 -LA-PLGA NPs (containing 5 mg PLGA), respectively. After a co-incubation period of 4 hours, the cells in the sixth to eighth groups were exposed to ultrasound irradiation. The DAF-FM DA NO assay kit was then added an incubated for a further 30 minutes. The NO release was then qualitatively determined by CLSM. 2.7 The dual‑modality imaging of Fe 3 O 4 -LA-PLGA NPs For FL imaging, DiR (Ex/Em:748 nm/780 nm)-labeled Fe 3 O 4 -LA-PLGA NPs (containing 5 mg PLGA) were intravenously injected into MDA-MB-231 tumor-bearing nude mice, FL images were acquired at pre-injection,1 h, 6 h, 24 h, and 48 h post-injection using the LB983 imaging system. Tumors and major organs were extracted for FL imaging and the corresponding FL intensity was measured. For PA imaging, Fe 3 O 4 -LA-PLGA NPs (containing 5 mg PLGA) were administered intravenously to MDA-MB-231 tumor-bearing mice. PA images were obtained at pre-injection, 1 h, 6 h, 24 h, and 48 h post-injection. 2.8 Biosafety of Fe 3 O 4 -LA-PLGA NPs Female BALB/c mice (18–20 g) were randomly divided into five groups (n = 5 per group), including the control group, the 1-day group, the 3-day group, the 7-day group, and the 14-day group. The control group was intravenously injected with 0.2 mL PBS, and the other groups was intravenously injected with 0.2 mL Fe 3 O 4 -LA-PLGA NPs, respectively. Blood samples were collected one day after injection (control group) and at 1,3, 7, and14 days post-injection (Fe 3 O 4 -LA-PLGA NPs) for biochemical examinations. The major organs (heart, liver, spleen, lungs, and kidneys) were stained with hematoxylin-eosin staining (H&E) for histological analysis at the corresponding time point. 2.9 Therapeutic Efficacy of Fe 3 O 4 -LA-PLGA NPs To assess the therapeutic efficacy of Fe 3 O 4 -LA-PLGA NPs, the MDA-MB-231 tumor-bearing nude mice were randomly divided into four groups (five mice per group). The first and second groups of mice were intravenously injected with PBS, setting as the control group and the “US” group, respectively. The second group was also treated with ultrasound irradiation. The third and fourth groups of mice were injected with Fe 3 O 4 -LA-PLGA NPs (containing 5 mg PLGA), which was set as the “NPs” group and the “NPs + US” group, respectively. The fourth group was also treated with ultrasound irradiation. For the two groups subjected to ultrasound irradiation, the tumor sites were irradiated with ultrasound at a frequency of 200 kHz and a power density of 2 W for a duration of 200 S at 24 hours post-injection. Tumor volumes and body weights were measured from day 0 to day 11 after the corresponding treatments. On the 11th day following the administration of the respective treatments, the mice were euthanized, and the tumor tissues were harvested for histological analysis. This involved the use of H&E and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to observe apoptosis. 2.10 Statistical Analysis All data are expressed as mean ± standard deviation. The statistical analysis was conducted using the SPSS 21.0 program. Paired t -tests and one-way analysis of variance were employed to analyze the data ( p < 0.05 was considered statistically significant). 3 Results and Discussion 3.1 Characterization of Fe 3 O 4 -LA-PLGA NPs The synthesis of Fe 3 O 4 -LA-PLGA NPs is illustrated in Scheme 1a , which depicts the formation of an ultrasonic double emulsion. This method is well established for the encapsulation of both hydrophobic and hydrophilic drugs and bioactive compounds[ 41 ]. The transmission electron microscopy (TEM) image indicates that the Fe 3 O 4 NPs were successfully incorporated into the spherical shell, exhibiting a high degree of contrast (Fig. 1 A). The obtained Fe 3 O 4 -LA-PLGA NPs exhibited a highly uniform size, with a diameter of 302.4 ± 4.3 nm, which is slightly larger than that of Fe 3 O 4 -PLGA NPs (298.6 ± 3.1 nm), LA-PLGA NPs (295.3 ± 2.7 nm), and PLGA NPs (293.9 ± 2.6 nm) (Fig. 1 B). The zeta potential of the NPs was found to be -22.9 ± 2.3 mV, -20.7 ± 3.7 mV, -21.1 ± 4.3 mV, and − 31.7 ± 2.4 mV, respectively (Fig. 1 C). Moreover, the Fe 3 O 4 -LA-PLGA NPs exhibited excellent stability in a relatively uniform size (Fig. 1 D) and zeta potentials (Fig. 1 E) over 7 days. Subsequently, the drug loading efficiencies (DL%) and encapsulation (DE%) of LA were quantified by UV spectroscopy, in accordance with the standard curve of LA at 210 nm (Fig. 1 F). The drugs DL% and DE% were determined to be high at 24.5 wt % and 81.6%, respectively. The high loading capacity of drugs suggests that the FDA-approved PLGA polymers have significant potential as a promising nanocarrier for drug delivery. Moreover, the loading efficiency of Fe 3 O 4 NPs in the Fe 3 O 4 -LA-PLGA NPs was calculated to be 85.0 wt%, indicating that PLGA-based NPs possess high encapsulation efficiency. To assess the NO release by various PLGA NPs in the presence of H 2 O 2 (50 µM) with and without ultrasound irradiation, a standard curve of NO was constructed via the Griess assay (Fig. 1 G). The results demonstrated that Fe 3 O 4 -LA-PLGA NPs and LA-PLGA NPs exhibited enhanced NO release in response to ultrasound irradiation. The NO release from Fe 3 O 4 -LA-PLGA NPs was significantly accelerated compared to LA-PLGA NPs in the presence of H 2 O 2 (50 µM) under ultrasound irradiation (Fig. 1 H), suggesting that ultrasound could accelerate the burst of the PLGA-based NPs, especially the catalytic effect of Fe 3 O 4 NPs. 3.2 Biosafety of Fe 3 O 4 -LA-PLGA NPs The cytotoxicity of three different NPs (LA-PLGA NPs, Fe 3 O 4 -PLGA NPs, and Fe 3 O 4 -LA-PLGA NPs) was initially evaluated in MDA-MB-231 cells and a non-tumor cell line (HUVECs). The viability of MDA-MB-231 cells and HUVECs demonstrated a high cell survival rate even when various NPs were administered at the maximum concentration of 10 mg/mL without the application of ultrasound irradiation (Fig. 2 A and B), indicating the favorable biocompatibility of the NPs. However, when exposed to ultrasound irradiation, the viability of MDA-MB-231 cells was significantly reduced, particularly when co-incubated with Fe 3 O 4 -LA-PLGA NPs (Fig. 2 C), suggesting that ultrasound-triggered cytotoxicity due to the release of NO. Moreover, a series of in vivo safety tests were conducted on the Fe 3 O 4 -LA-PLGA NPs, including blood cell analysis and biochemical examination. The blood biochemical parameters were not significantly different from the control group at any time point (Fig. 2 D), thereby demonstrating the high biosafety of Fe 3 O 4 -LA-PLGA NPs. Furthermore, no significant damage was observed in major organs (heart, liver, spleen, lung, and kidney) based on H&E staining (Fig. 2 E), indicating the excellent biocompatibility of the Fe 3 O 4 -LA-PLGA NPs. 3.3 Ultrasound-responsive intracellular NO release A high concentration of NO is a critical factor in the efficacy of cancer therapy [ 42 ]. The intracellular NO release from Fe 3 O 4 -LA-PLGA NPs triggered by ultrasound was monitored using the NO-specific fluorescent probe (DAF-FM DA), which rapidly reacts with NO to produce benzotriazole and exhibits strong green fluorescence [ 43 ]. As shown in Fig. 3 A, the most notable fluorescence was observed in MDA-MB-231 cells treated with Fe 3 O 4 -LA-PLGA NPs and ultrasound irradiation, followed by MDA-MB-231 cells treated with LA-PLGA NPs and ultrasound irradiation. In contrast, only a minimal fluorescence signal was detected in the control, ultrasound-only, Fe 3 O 4 -PLGA NPs only, and Fe 3 O 4 -LA-PLGA NPs only groups, indicating the occurrence of ultrasound-responsive NO release. The quantitative analysis of average fluorescence intensity also showed the same results (Fig. 3 B; *** p < 0.001). Furthermore, calcein-AM and propidium iodide (PI) were utilized for co-staining viable and dead MDA-MB-231 cells to evaluate the NO-mediated cell viability. As anticipated, the majority of the dead cells (red fluorescence) were observed in the group treated with Fe 3 O 4 -LA-PLGA NPs and ultrasound irradiation (Fig. 3 C), indicating the highest percentage of dead MDA-MB-231 cells. The quantitative analysis of the percentage of red zones in the images also showed the same results (Fig. 3 D; *** p < 0.001). The aforementioned results confirm that the ultrasound-triggered NO release from the reaction between Fe 3 O 4 -LA-PLGA NPs and H 2 O 2 present in the tumor is an effective process. 3.4 Dual‑modality imaging of Fe 3 O 4 -LA-PLGA NPs The combination of PA imaging and FL imaging can compensate for the deficiencies of single imaging and provide comprehensive diagnosis and treatment information with high sensitivity and high resolution. As shown in Fig. 4 A and 4 B, the PA signals in the tumor region exhibited a gradual increase and reached a peak at 24 h post-intravenous injection of Fe 3 O 4 -LA-PLGA NPs, indicating the accumulation of Fe 3 O 4 -LA-PLGA NPs at the tumor site via the EPR effect. Additionally, FL imaging demonstrated the dynamic distribution of Fe 3 O 4 -LA-PLGA NPs in vivo , with a trend that aligned with that observed in PA imaging (Fig. 4 C). The FL intensity also reached a peak 24 hours after intravenous injection (Fig. 4 D), indicating that the Fe 3 O 4 -LA-PLGA NPs were predominantly accumulated in the tumor region at this time point, which is crucial for subsequent in vivo treatment. Furthermore, the tumor tissues and excised organs were analyzed at 48 hours post-injection to confirm the distribution of Fe 3 O 4 -LA-PLGA NPs. As shown by ex vivo imaging (Fig. 4 E) and quantitative analysis (Fig. 4 F), the highest FL intensity was observed in the liver, likely due to the phagocytosis by the reticuloendothelial system. These findings demonstrate that Fe 3 O 4 -LA-PLGA NPs exhibit dual-modality PA and FL imaging capabilities, facilitating precise diagnosis and effective treatment. 3.5 Therapeutic Effects of Fe 3 O 4 -LA-PLGA NPs Encouraged by the evident ultrasound-responsive cytotoxicity of tumor cells, we undertook an evaluation of the in vivo antitumor efficacy of Fe 3 O 4 -LA-PLGA NPs through post-systemic administration. The MDA-MB-231 tumor-bearing nude mice were randomly divided into four groups and treated with the following interventions: (1) PBS as the control group, (2) ultrasound irradiation only (US), (3) Fe 3 O 4 -LA-PLGA NPs (containing 5 mg PLGA, with the same dose used for the subsequent groups, NPs), and (4) Fe 3 O 4 -LA-PLGA NPs + ultrasound irradiation (NPs + US). Tumor volumes and body weights were monitored throughout the experiment. Tumor volume was normalized with relative tumor volumes ( V/V 0 ). As shown in Fig. 5 A and 5 B, mice treated with PBS, “US”, and “NPs” exhibited a negligible therapeutic effect on tumor growth, while mice treated with “NPs + US” exhibited a significant inhibition of tumor growth compared to those of other groups (** p < 0.01), suggesting that enhanced ultrasound-responsive NO release induced therapeutic effects. The mice’s body weights during the therapeutic period revealed minimal loss (Fig. 5 C), indicating that Fe 3 O 4 -LA-PLGA NPs do not induce acute toxicity. Furthermore, histological examination of tumor tissues via hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated the efficacy of NO-induced therapeutic effects (Fig. 5 D). As shown by H&E staining of tumor tissues, mice treated with “NPs + US” exhibited numerous deformed nuclei (karyopyknosis, karyorrhexis, and karyolysis), indicative of severe tumor cell necrosis. Furthermore, the TUNEL assay revealed a greater number of apoptotic cells in the tumor tissues of the treated group compared to the other control groups. 4 Conclusions In conclusion, the ultrasound-responsive nanoplatform, consisting of PLGA, Fe 3 O 4 , and LA was successfully constructed for the rapid and controlled release of NO, which inhibits the growth of breast cancer. Compared with other traditional stimulus-responsive NO-delivery nanoplatforms, our resultant nanoplatform demonstrated several advantages, including: i) an ultrasound-responsive strategy for PLGA NPs loaded with LA and Fe 3 O 4 NPs; ii) a cascade amplification reaction for a rapid and large amount of NO production; and iii) real-time imaging monitoring via PA and FL dual‑modality imaging; and iv) effective therapeutic effects by NO-assisted antitumor gas therapy. Declarations The authors declare no conflict of interest. Ethical Statement The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The animal study protocol was approved by the Ethics Committee of Chongqing Medical University (IACUC-CQMU-2023-10060). Author Contribution Haiyan Yang: Conceptualization, Writing–original draft. Guangrong Zheng: Methodology, Investigation. Guochen Li: Visualization, Software. Jincui Chen, Yong Luo: Methodology, Project administration. Licui Qi: Data curation. Tengfei Ke: Formal analysis, Data curation. Jie Xiong: Writing – review & editing. Xiaojuan Ji: Project administration.All authors reviewed the manuscript Acknowledgements This work was supported by the Natural Science Foundation of Chongqing (CSTB2024NSCQ-MSX0102), Chongqing Medical Youth Top-notch Talent (YXQN202446), Chongqing Postdoctoral Science Foundation (2023CQBSHTB1005 and CSTB2023NSCQ-BHX0035), Chongqing women and Children General Project Fund (2020FY108), and Graduate Innovation Foundation of Kunming Medical University (2024S122 and 2024B013). Data availability No datasets were generated or analyzed during the current study. References Chatterji S, Krzoska E, Thoroughgood CW, Saganty J, Liu P, Elsberger B, Abu-Eid R, Speirs V. Defining genomic, transcriptomic, proteomic, epigenetic, and phenotypic biomarkers with prognostic capability in male breast cancer: a systematic review. Lancet Oncol. 2023;24(2):e74–85. Tsai CJ, Yang JT, Shaverdian N, Patel J, Shepherd AF, Eng J, Guttmann D, Yeh R, Gelblum DY, Namakydoust A, et al. 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Intelligent Albumin–MnO 2 Nanoparticles as pH-/H 2 O 2 -Responsive Dissociable Nanocarriers to Modulate Tumor Hypoxia for Effective Combination Therapy. Adv Mater. 2016;28(33):7129–36. Yang H, Jiang F, Zhang L, Wang L, Luo Y, Li N, Guo Y, Wang Q, Zou J. Multifunctional l-arginine-based magnetic nanoparticles for multiple-synergistic tumor therapy. Biomater Sci. 2021;9(6):2230–43. Huo M, Wang L, Chen Y, Shi J. Tumor-selective catalytic nanomedicine by nanocatalyst delivery. Nat Commun. 2017;8(1):357. Li WP, Su CH, Chang YC, Lin YJ, Yeh CS. Ultrasound-Induced Reactive Oxygen Species Mediated Therapy and Imaging Using a Fenton Reaction Activable Polymersome. ACS Nano. 2016;10(2):2017–27. Zhang K, Xu H, Jia X, Chen Y, Ma M, Sun L, Chen H. Ultrasound-Triggered Nitric Oxide Release Platform Based on Energy Transformation for Targeted Inhibition of Pancreatic Tumor. ACS Nano. 2016;10(12):10816–28. Yang D, Chen Q, Zhang M, Feng G, Sun D, Lin L, Jing X. 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Cite Share Download PDF Status: Published Journal Publication published 28 Dec, 2024 Read the published version in Cancer Cell International → Version 1 posted Editorial decision: Revision requested 19 Nov, 2024 Reviews received at journal 08 Nov, 2024 Reviews received at journal 07 Nov, 2024 Reviews received at journal 06 Nov, 2024 Reviews received at journal 04 Nov, 2024 Reviewers agreed at journal 04 Nov, 2024 Reviews received at journal 03 Nov, 2024 Reviews received at journal 31 Oct, 2024 Reviewers agreed at journal 29 Oct, 2024 Reviewers agreed at journal 29 Oct, 2024 Reviewers agreed at journal 28 Oct, 2024 Reviewers agreed at journal 28 Oct, 2024 Reviewers agreed at journal 28 Oct, 2024 Reviewers agreed at journal 28 Oct, 2024 Reviewers invited by journal 28 Oct, 2024 Editor assigned by journal 28 Oct, 2024 Submission checks completed at journal 03 Oct, 2024 First submitted to journal 01 Oct, 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 Advisory Board 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-5186273","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":379911147,"identity":"da23100b-f885-43be-916f-39adf5a73fb2","order_by":0,"name":"Haiyan Yang","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Yang","suffix":""},{"id":379911148,"identity":"1f89f4eb-1db9-4dec-92a2-984b1ce2806b","order_by":1,"name":"Guangrong Zheng","email":"","orcid":"","institution":"Yan'an Hospital of Kunming City, Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guangrong","middleName":"","lastName":"Zheng","suffix":""},{"id":379911149,"identity":"51c6ec0f-ace7-46a5-b9ca-45fd1d8f0487","order_by":2,"name":"GuoChen Li","email":"","orcid":"","institution":"Yan'an Hospital of Kunming City, Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"GuoChen","middleName":"","lastName":"Li","suffix":""},{"id":379911150,"identity":"5e201f5b-3e4f-4452-b156-592d8fe1f975","order_by":3,"name":"Jincui Chen","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Jincui","middleName":"","lastName":"Chen","suffix":""},{"id":379911151,"identity":"08d183e5-016b-4590-a84d-9ce6cd132f25","order_by":4,"name":"Licui Qi","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Licui","middleName":"","lastName":"Qi","suffix":""},{"id":379911152,"identity":"71bceacf-83d3-40ef-ab38-d573c11b8a82","order_by":5,"name":"Yong Luo","email":"","orcid":"","institution":"The People’s Hospital of Chongqing Liang Jiang New Area","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Luo","suffix":""},{"id":379911153,"identity":"d95ad7fd-b6c4-4d64-9b5c-78e59f6f3f56","order_by":6,"name":"Tengfei Ke","email":"","orcid":"","institution":"Yunnan Cancer Hospital (The Third Affiliated Hospital of Kunming Medical University, Peking University Cancer Hospital Yunnan Campus)","correspondingAuthor":false,"prefix":"","firstName":"Tengfei","middleName":"","lastName":"Ke","suffix":""},{"id":379911154,"identity":"95fc4a80-9a10-4e80-a0db-bda8180026a8","order_by":7,"name":"Jie Xiong","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Xiong","suffix":""},{"id":379911155,"identity":"17b28ca9-0079-49ac-8471-5cb4a979f470","order_by":8,"name":"Xiaojuan Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACxmbGhgMSFTZQLhsRWpjbmw8esDiTRoIW9p5jyQcqWw6ToIV3Ro7BgZsN5+X5rp0xYPhQdpiBf3YDfi2SQC0HZ+64bTjzdo4B44xzhxkk7hzAr8UQqOWw5JnbCQZALcy8bYcZDCQS8GuxvwHU8rftHETLX2K0MPYcSzgg2XYAooWRKC3tzQcOSJxJBvolreBgz7l0HokbhLQ0MzZ/kKiwk+e7nbzxwY8yazn+GQS0IMABMGLgIVY9RMsoGAWjYBSMAqwAAKuLTTDpiThfAAAAAElFTkSuQmCC","orcid":"","institution":"Chongqing University","correspondingAuthor":true,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2024-10-01 09:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5186273/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5186273/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12935-024-03627-4","type":"published","date":"2024-12-28T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70917354,"identity":"dc7032b3-50c1-4d9e-98ef-f663aa00c47c","added_by":"auto","created_at":"2024-12-09 08:19:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1412663,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. (\u003cstrong\u003eA\u003c/strong\u003e) TEM images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. (\u003cstrong\u003eB\u003c/strong\u003e) The hydrodynamic diameter and (\u003cstrong\u003eC\u003c/strong\u003e) zeta potential of PLGA NPs, LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e -PLGA NPs, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (n=3). (\u003cstrong\u003eD\u003c/strong\u003e) The hydrodynamic diameter and (\u003cstrong\u003eE\u003c/strong\u003e) zeta potential of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs over an extended period of time (n=3). (\u003cstrong\u003eF\u003c/strong\u003e) Standard curve of LA as a function of mass concentration via the UV-vis spectrometer. (\u003cstrong\u003eG\u003c/strong\u003e) Standard curve of NO via the Griess assay. (\u003cstrong\u003eH\u003c/strong\u003e) Release profiles of NO from various NPs with or without ultrasound irradiation, respectively (n=3).\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5186273/v1/c020d07016b5999446145783.png"},{"id":70917356,"identity":"af5a5ba7-122d-4144-a1c2-bbaf9dde4ca0","added_by":"auto","created_at":"2024-12-09 08:19:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4363095,"visible":true,"origin":"","legend":"\u003cp\u003eBiosafety evaluation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. (\u003cstrong\u003eA\u003c/strong\u003e) The viability of MDA-MB-231 cells following incubation with distinct NPs (LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs) for 24 hours (n = 5). (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eThe viability of HUVECs following incubation with different NPs (LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs) for 24 hours (n = 5). (\u003cstrong\u003eC\u003c/strong\u003e) The viability of MDA-MB-231 cells following incubation with different NPs (LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs) for 4 hours after ultrasound irradiation (n = 5, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ns: not significant). (\u003cstrong\u003eD\u003c/strong\u003e) Hematological assay of BALB/c mice of the control group and the experimental groups at the corresponding time point (n=5, ns: not significant). (\u003cstrong\u003eE\u003c/strong\u003e) H\u0026amp;E staining of major organs (heart, liver, spleen, lung, and kidney) in the control group and the experimental groups at 3,7, and 14 days post-intravenous injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (Scale bar: 50 μm).\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5186273/v1/b64438ef522646066f4f465d.png"},{"id":70917358,"identity":"f238c0c4-15ff-414a-aa6f-47be4657d6a6","added_by":"auto","created_at":"2024-12-09 08:19:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5551676,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular NO release and \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity assay. (\u003cstrong\u003eA\u003c/strong\u003e) Representative CLSM images of DAF-FM DA-stained cells (Scale bar:100 μm). (\u003cstrong\u003eB\u003c/strong\u003e) The average NO fluorescence intensity in tumor cells was quantified (n=3, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ns: not significant). (\u003cstrong\u003eC\u003c/strong\u003e) MDA-MB-231 cells were stained with calcein-AM and PI following different treatments. (Scale bar: 100 μm). (\u003cstrong\u003eD\u003c/strong\u003e) The percentage of red zones in the images was quantified (n=3, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5186273/v1/38a50ee2fb2954cb1dbac1ea.png"},{"id":70919227,"identity":"54e39bdf-1064-4cc5-9d76-f76d58705fe0","added_by":"auto","created_at":"2024-12-09 08:27:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":384293,"visible":true,"origin":"","legend":"\u003cp\u003eDual‑modality imaging of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. (\u003cstrong\u003eA\u003c/strong\u003e) PA images of tumors following intravenous injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs at various time points. (\u003cstrong\u003eB\u003c/strong\u003e) Quantitative analysis of PA intensity (n = 3). (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e FL imaging of tumor bearing mice at pre-injection, 1 h, 6 h, 24 h, and 48 h post-injection. (\u003cstrong\u003eD\u003c/strong\u003e) Quantitative analysis of FL intensity (n = 3). (\u003cstrong\u003eE\u003c/strong\u003e) The biodistribution of DiR-labeled Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs in the tumor and major organs at 48 hours post-injection is presented. (\u003cstrong\u003eF\u003c/strong\u003e) Quantitative analysis of FL intensity in tumor and major organs (n = 3).\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5186273/v1/dfb1772ecf78b44acd063a13.png"},{"id":70919779,"identity":"9d239752-9574-4d37-aba1-f8df5f173d4c","added_by":"auto","created_at":"2024-12-09 08:35:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1127458,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of \u003cem\u003ein vivo\u003c/em\u003e antitumor effects of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. (\u003cstrong\u003eA\u003c/strong\u003e) Digital images of tumor-bearing mice were obtained at the 11-day of different treatments. (\u003cstrong\u003eB\u003c/strong\u003e) Tumor growth trajectories of tumor-bearing mice post various treatments (n=5). (\u003cstrong\u003eC\u003c/strong\u003e) Body weight measurements of tumor-bearing mice across different treatment groups (n=5). (\u003cstrong\u003eD\u003c/strong\u003e) H\u0026amp;E staining and TUNEL staining of tumor sections from tumor-bearing mice subjected to various treatments (Scale bar: 50\u0026nbsp;µm)\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5186273/v1/59993ddc5177fa05613e1ea0.png"},{"id":72641136,"identity":"150a0bc9-8b1e-44df-86a5-48f657a03174","added_by":"auto","created_at":"2024-12-30 16:11:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10927541,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5186273/v1/b9bbc9c1-6d0d-4bab-a3f7-d092ae7832bd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ultrasound-responsive nanoparticles for nitric oxide release to inhibit the growth of breast cancer","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBreast cancer represents the primary cause of cancer-related mortality among women worldwide [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. While traditional treatment modalities, such as surgery, chemotherapy, and radiotherapy, can be efficacious in certain cases, they do not constitute a comprehensive solution[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The overall survival rate for patients with breast cancer remains low due to the aggressive nature of the disease and its poor prognosis [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it is imperative to develop new therapeutic strategies for patients with breast cancer. In recent years, gas therapy has emerged as a promising therapeutic modality for tumors, attracting significant attention due to its high efficacy and favorable biological safety profile [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been extensively investigated in numerous anti-tumor applications, including gas-induced tumor cell killing, enhanced radiation therapy (RT) sensitization [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], chemotherapy [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], photothermal therapy (PTT) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], photodynamic therapy (PDT) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and sonodynamic therapy (SDT) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nitric oxide (NO) is a prominent therapeutic gas in cancer therapy. It has been demonstrated to inhibit the growth of tumor cells through various mechanisms, including mitochondrial inhibition, DNA damage, nitrosylation of enzymes, and inhibition of cellular respiration. These effects have been observed at high concentrations (\u0026gt;\u0026thinsp;1\u0026micro;M), resulting in an excellent anti-tumor effect [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, it is unfortunate that low NO concentrations can even promote tumor cell growth [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, enhancing NO concentration is a crucial aspect of effective NO gas therapy.\u003c/p\u003e \u003cp\u003eThe direct utilization of free NO gas is limited by its short half-life and vulnerability to diverse biological substances (\u003cem\u003ee.g\u003c/em\u003e., glutathione (GSH), hemoglobin, superoxide, and molecular oxygen) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, numerous versatile nanoplatforms loaded with NO donors or NO-releasing molecules have been explored to deliver NO in a straightforward and precise manner. A plethora of NO-releasing donors \u003cem\u003ee.g\u003c/em\u003e., N, N\u0026prime;-di-sec-Butyl-N, N\u0026prime;-dinitroso-1,4-phenylenediamine (BNN6), S-nitrosothiols (SNO), S-nitroso glutathione (GSNO), and Rusen black salt (RBS)) have been explored to release NO under the external stimuli, including light (UV\u0026ndash;vis/near-infrared laser), X-ray, and ultrasound [\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, previous NO-releasing donors have been found to be unsafe for biological applications due to their toxic production. Therefore, it is urgent to develop a biosafe and efficient strategy to achieve high capacity and controllable NO release.\u003c/p\u003e \u003cp\u003eL-Arginine (LA) is a naturally occurring NO-releasing donor with high biocompatibility and the capacity to produce NO in the presence of inducible NO synthase [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, LA can spontaneously release NO when reacting with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which is commonly abundant in tumor cells compared to normal cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In normal conditions, the release amount and speed of LA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is relatively slow. Our previous study demonstrated that the release of NO can be rapid and in large quantities over a short period of time when the presence of superparamagnetic iron oxide nanoparticles (SPIO, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs) act as a catalyst [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is well established that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can react with Fe\u003csup\u003e2+\u003c/sup\u003e to produce reactive oxygen species (ROS) containing hydroxyl radicals (\u0026bull;OH) and hydroxide ions (OH\u003csup\u003e\u0026minus;\u003c/sup\u003e), which is known as the Fenton reaction [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The greater the quantity of ROS generated, the more effectively it can oxidize LA to generate more NO[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs, functioning as an \"optical absorber\", can generate noninvasive photoacoustic (PA) imaging contrast agent enhancement when stimulated by laser irradiation at specific wavelengths.\u003c/p\u003e \u003cp\u003eHerein, in this study, we successfully constructed ultrasound-responsive nanoparticles for the rapid and controlled release of NO, which has been shown to inhibit breast cancer by utilizing NO as the antitumor therapeutic gas. Poly-lactide-co-glycolide (PLGA) is a nanosized polymeric material with high biodegradability and biocompatibility that has been approved by the Food and Drug Administration (FDA) for medical applications. We rationally designed and synthesized a versatile PLGA nanoparticle, encapsulating the natural LA as a NO donor in the core and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs packed in the shell as an ultrasound-responsive therapeutic agent (denoted as Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs). The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs demonstrated efficacious therapeutic effects through NO-assisted antitumor gas therapy. As illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs can accumulate into the tumor region via the enhanced permeability and retention (EPR) effect after intravenous injection. The distribution of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs can be monitored via dual-modality imaging, including fluorescence (FL) and photoacoustic (PA) imaging. Upon exposure to ultrasound irradiation, LA and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were released rapidly from the PLGA NPs, thereby initiating a cascade of reactions. LA can spontaneously react with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e present in the tumor microenvironment to generate NO for gas therapy. Simultaneously, the integrated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs can rapidly react with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce a substantial quantity of reactive oxygen species (ROS). Moreover, the generated ROS can oxidize LA to facilitate the release of NO. The results of this study suggest that ultrasound-responsive nanoparticles have significant potential as a delivery platform for NO, with the potential to inhibit cancer growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003ePLGA-PEG-COOH (50: 50, MW: 15 000) was purchased from Daigang BIO Engineer Ltd, Co. (Shan Dong, China). Poly (vinyl alcohol) (PVA) was purchased from Sigma-Aldrich Chemical Co., Ltd., (St. Louis, MO, USA). Trichloromethane (CHCl\u003csub\u003e3\u003c/sub\u003e) was purchased from Chongqing East Chemical Industry Ltd, Co. (China). L-Arginine was purchased from Sigma-Aldrich (USA), and iron oxide nanoparticles (10 nm, 25 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were purchased from Ocean Nanotech Co. Ltd (USA). The cell counting kit (CCK-8) and calcein \u0026amp; propidium iodide (PI) apoptosis assay kit were purchased from Dojindo Laboratories (Kumamoto, Japan). 3-Amino,4-aminomethyl-2',7'-difluorescein, diacetate (DAF-FM DA), a nitric oxide assay kit (Griess assay kit), 4,6-diamidino-2-phenylindole (DAPI), and 1,1\u0026rsquo;-dioctadecyl-3,3,3\u0026rsquo;,3\u0026rsquo;-tetramethylindotricarbocyanine iodide (DiR) were purchased from Beyotime Biotechnology Co., Ltd (China).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eThe Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs were prepared via the ultrasound double emulsion (water/oil/water, W/O/W) method. First, 150 mg PLGA-PEG-COOH (50: 50, MW: 15 000) and 10 mg Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were completely dissolved in 2 mL of CHCl\u003csub\u003e3\u003c/sub\u003e. Then, 40 mg LA was added, and the mixture was sonicated with an ultrasonic cell crusher at 105 W for 2 minutes to obtain the W/O emulsion; subsequently, 4 mL of PVA solutions (w/v\u0026thinsp;=\u0026thinsp;4%) was added, and the mixture was emulsified again at 105 W for 5 minutes to obtain the W/O/W emulsion. Next, 8 mL of isopropyl alcohol solution (w/v\u0026thinsp;=\u0026thinsp;2%) was added, and the mixture was stirred to volatilize the CHCl\u003csub\u003e3\u003c/sub\u003e. Finally, the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs were collected following centrifugation (10 000 rpm, 10 min) and stored at 4\u0026deg;C for subsequent utilization. The fluorescent Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs were prepared by a similar procedure, with DiR added to the mixture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM, Hitachi 7500, Tokyo, Japan) was employed to observe the structure of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. The Nano ZS90 Zetasizer (Malvern Panalytical, Ltd., Malvern, UK) was employed to ascertain the size distribution and Zeta potential of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. Fluorescence (FL) images were acquired using the LB983 imaging system (Berthold Technologies GmbH \u0026amp; Co. KG, Germany). Photoacoustic (PA) images were obtained using the Vevo LAZR PA Imaging System (Visual Sonics Inc., Toronto, Canada). Confocal laser scanning microscope (CLSM) images were captured using a Nikon optical microscope (Japan). The flow cytometry assay was employed by Beckman Coulter (American).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 LA loading content, Encapsulation Efficiency, and NO release determination\u003c/h2\u003e \u003cp\u003eThe ultraviolet-visible spectrophotometer (UV-vis) (Shimadzu UV 2600, Kyoto, Japan) was employed to generate the standard curve for LA, and the LA loading efficiency and encapsulation were quantified and evaluated in accordance with the following equations:\u003c/p\u003e \u003cp\u003eLoading efficiency (%) = (total LA - unbound LA)/total LA \u0026times; 100%;\u003c/p\u003e \u003cp\u003eEncapsulation (%) = (total LA - unbound LA)/total nanoparticles \u0026times; 100%\u003c/p\u003e \u003cp\u003eThe NO release was quantified using the Griess assay kit. In brief, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e -PLGA NPs, and LA-PLGA NPs (containing 10 mg PLGA, respectively) were dispersed in 1 mL PBS and mixed with an excess of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (50 \u0026micro;M). Subsequently, the mixture was irradiated with or without ultrasound (Chongqing Haifu Technology, China) at a frequency of 200 kHz and a power density of 2 W for a duration of 200 S. The NO release was quantified by means of a Griess assay kit at various time points (1 min, 2 min, 3 min, 5 min, 10 min, 20 min, and 30 min) following ultrasound irradiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell culture and MDA-MB-231 tumor-bearing mouse model\u003c/h2\u003e \u003cp\u003eThe human breast cancer MDA-MB-231 cells and Human umbilical vein endothelial cells (HUVECs) were obtained from the Chinese Academy of Sciences Cell Bank (China). The cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in DMEM, which was supplemented with 10% FBS and 1% streptomycin/penicillin. A total of 0.1 mL of PBS solution (containing 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e MDA-MB-231 cells) was injected subcutaneously into the left flank of female BALB/c nude mice (weighing between18 and 20 g) to establish the tumor model. The tumor volume was calculated according to the formula [0.5 \u0026times; length \u0026times; (width)\u003csup\u003e2\u003c/sup\u003e]. The biosafety of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs was evaluated in BALB/c mice. All animal care and use procedures were reviewed and approved by the Animal Ethics Committee of Chongqing Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity assay\u003c/h2\u003e \u003cp\u003eTo assess the cytotoxicity and growth inhibition of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs \u003cem\u003ein vitro\u003c/em\u003e, we conducted a series of co-incubation experiments involving different concentrations of NPs (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 mg/mL) and MDA-MB-231 cells (or HUVECs) at 37\u0026deg;C, with or without ultrasound irradiation. The cell viability was assessed by the cell counting kit-8 (CCK-8) assay (n\u0026thinsp;=\u0026thinsp;5). Furthermore, the viable and dead cells were co-stained with calcein-AM and propidium iodide (PI), and their distribution was observed by confocal laser scanning microscopy (CLSM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Ultrasound-responsive intracellular NO release\u003c/h2\u003e \u003cp\u003eTo assess the impact of ultrasound irradiation on intracellular NO release, MDA-MB-231 cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) were randomly allocated to eight experimental groups: control, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs, LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs, ultrasound irradiation (US), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs\u0026thinsp;+\u0026thinsp;US, LA-PLGA NPs\u0026thinsp;+\u0026thinsp;US, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u0026thinsp;+\u0026thinsp;US. Cells in the first and fifth groups were treated with phosphate buffered saline (PBS), respectively. The remaining groups were treated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs, LA-PLGA NPs, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (containing 5 mg PLGA), respectively. After a co-incubation period of 4 hours, the cells in the sixth to eighth groups were exposed to ultrasound irradiation. The DAF-FM DA NO assay kit was then added an incubated for a further 30 minutes. The NO release was then qualitatively determined by CLSM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 The dual‑modality imaging of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eFor FL imaging, DiR (Ex/Em:748 nm/780 nm)-labeled Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (containing 5 mg PLGA) were intravenously injected into MDA-MB-231 tumor-bearing nude mice, FL images were acquired at pre-injection,1 h, 6 h, 24 h, and 48 h post-injection using the LB983 imaging system. Tumors and major organs were extracted for FL imaging and the corresponding FL intensity was measured.\u003c/p\u003e \u003cp\u003eFor PA imaging, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (containing 5 mg PLGA) were administered intravenously to MDA-MB-231 tumor-bearing mice. PA images were obtained at pre-injection, 1 h, 6 h, 24 h, and 48 h post-injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Biosafety of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice (18\u0026ndash;20 g) were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;5 per group), including the control group, the 1-day group, the 3-day group, the 7-day group, and the 14-day group. The control group was intravenously injected with 0.2 mL PBS, and the other groups was intravenously injected with 0.2 mL Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs, respectively. Blood samples were collected one day after injection (control group) and at 1,3, 7, and14 days post-injection (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs) for biochemical examinations. The major organs (heart, liver, spleen, lungs, and kidneys) were stained with hematoxylin-eosin staining (H\u0026amp;E) for histological analysis at the corresponding time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Therapeutic Efficacy of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eTo assess the therapeutic efficacy of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs, the MDA-MB-231 tumor-bearing nude mice were randomly divided into four groups (five mice per group). The first and second groups of mice were intravenously injected with PBS, setting as the control group and the \u0026ldquo;US\u0026rdquo; group, respectively. The second group was also treated with ultrasound irradiation. The third and fourth groups of mice were injected with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (containing 5 mg PLGA), which was set as the \u0026ldquo;NPs\u0026rdquo; group and the \u0026ldquo;NPs\u0026thinsp;+\u0026thinsp;US\u0026rdquo; group, respectively. The fourth group was also treated with ultrasound irradiation. For the two groups subjected to ultrasound irradiation, the tumor sites were irradiated with ultrasound at a frequency of 200 kHz and a power density of 2 W for a duration of 200 S at 24 hours post-injection. Tumor volumes and body weights were measured from day 0 to day 11 after the corresponding treatments. On the 11th day following the administration of the respective treatments, the mice were euthanized, and the tumor tissues were harvested for histological analysis. This involved the use of H\u0026amp;E and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to observe apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The statistical analysis was conducted using the SPSS 21.0 program. Paired \u003cem\u003et\u003c/em\u003e-tests and one-way analysis of variance were employed to analyze the data (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eThe synthesis of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs is illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1a\u003c/span\u003e, which depicts the formation of an ultrasonic double emulsion. This method is well established for the encapsulation of both hydrophobic and hydrophilic drugs and bioactive compounds[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The transmission electron microscopy (TEM) image indicates that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were successfully incorporated into the spherical shell, exhibiting a high degree of contrast (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The obtained Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs exhibited a highly uniform size, with a diameter of 302.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 nm, which is slightly larger than that of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-PLGA NPs (298.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 nm), LA-PLGA NPs (295.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 nm), and PLGA NPs (293.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The zeta potential of the NPs was found to be -22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 mV, -20.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 mV, -21.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 mV, and \u0026minus;\u0026thinsp;31.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 mV, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs exhibited excellent stability in a relatively uniform size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and zeta potentials (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) over 7 days. Subsequently, the drug loading efficiencies (DL%) and encapsulation (DE%) of LA were quantified by UV spectroscopy, in accordance with the standard curve of LA at 210 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The drugs DL% and DE% were determined to be high at 24.5 wt % and 81.6%, respectively. The high loading capacity of drugs suggests that the FDA-approved PLGA polymers have significant potential as a promising nanocarrier for drug delivery. Moreover, the loading efficiency of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs in the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs was calculated to be 85.0 wt%, indicating that PLGA-based NPs possess high encapsulation efficiency. To assess the NO release by various PLGA NPs in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (50 \u0026micro;M) with and without ultrasound irradiation, a standard curve of NO was constructed via the Griess assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The results demonstrated that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs and LA-PLGA NPs exhibited enhanced NO release in response to ultrasound irradiation. The NO release from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs was significantly accelerated compared to LA-PLGA NPs in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (50 \u0026micro;M) under ultrasound irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), suggesting that ultrasound could accelerate the burst of the PLGA-based NPs, especially the catalytic effect of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biosafety of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of three different NPs (LA-PLGA NPs, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e -PLGA NPs, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs) was initially evaluated in MDA-MB-231 cells and a non-tumor cell line (HUVECs). The viability of MDA-MB-231 cells and HUVECs demonstrated a high cell survival rate even when various NPs were administered at the maximum concentration of 10 mg/mL without the application of ultrasound irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B), indicating the favorable biocompatibility of the NPs. However, when exposed to ultrasound irradiation, the viability of MDA-MB-231 cells was significantly reduced, particularly when co-incubated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that ultrasound-triggered cytotoxicity due to the release of NO.\u003c/p\u003e \u003cp\u003eMoreover, a series of \u003cem\u003ein vivo\u003c/em\u003e safety tests were conducted on the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs, including blood cell analysis and biochemical examination. The blood biochemical parameters were not significantly different from the control group at any time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), thereby demonstrating the high biosafety of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. Furthermore, no significant damage was observed in major organs (heart, liver, spleen, lung, and kidney) based on H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), indicating the excellent biocompatibility of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Ultrasound-responsive intracellular NO release\u003c/h2\u003e \u003cp\u003eA high concentration of NO is a critical factor in the efficacy of cancer therapy [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The intracellular NO release from Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs triggered by ultrasound was monitored using the NO-specific fluorescent probe (DAF-FM DA), which rapidly reacts with NO to produce benzotriazole and exhibits strong green fluorescence [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the most notable fluorescence was observed in MDA-MB-231 cells treated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs and ultrasound irradiation, followed by MDA-MB-231 cells treated with LA-PLGA NPs and ultrasound irradiation. In contrast, only a minimal fluorescence signal was detected in the control, ultrasound-only, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e -PLGA NPs only, and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs only groups, indicating the occurrence of ultrasound-responsive NO release. The quantitative analysis of average fluorescence intensity also showed the same results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, calcein-AM and propidium iodide (PI) were utilized for co-staining viable and dead MDA-MB-231 cells to evaluate the NO-mediated cell viability. As anticipated, the majority of the dead cells (red fluorescence) were observed in the group treated with Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs and ultrasound irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating the highest percentage of dead MDA-MB-231 cells. The quantitative analysis of the percentage of red zones in the images also showed the same results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The aforementioned results confirm that the ultrasound-triggered NO release from the reaction between Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e present in the tumor is an effective process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Dual‑modality imaging of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eThe combination of PA imaging and FL imaging can compensate for the deficiencies of single imaging and provide comprehensive diagnosis and treatment information with high sensitivity and high resolution. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, the PA signals in the tumor region exhibited a gradual increase and reached a peak at 24 h post-intravenous injection of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs, indicating the accumulation of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs at the tumor site via the EPR effect. Additionally, FL imaging demonstrated the dynamic distribution of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs \u003cem\u003ein vivo\u003c/em\u003e, with a trend that aligned with that observed in PA imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The FL intensity also reached a peak 24 hours after intravenous injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating that the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs were predominantly accumulated in the tumor region at this time point, which is crucial for subsequent \u003cem\u003ein vivo\u003c/em\u003e treatment. Furthermore, the tumor tissues and excised organs were analyzed at 48 hours post-injection to confirm the distribution of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. As shown by \u003cem\u003eex vivo\u003c/em\u003e imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), the highest FL intensity was observed in the liver, likely due to the phagocytosis by the reticuloendothelial system. These findings demonstrate that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs exhibit dual-modality PA and FL imaging capabilities, facilitating precise diagnosis and effective treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Therapeutic Effects of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u003c/h2\u003e \u003cp\u003eEncouraged by the evident ultrasound-responsive cytotoxicity of tumor cells, we undertook an evaluation of the \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs through post-systemic administration. The MDA-MB-231 tumor-bearing nude mice were randomly divided into four groups and treated with the following interventions: (1) PBS as the control group, (2) ultrasound irradiation only (US), (3) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs (containing 5 mg PLGA, with the same dose used for the subsequent groups, NPs), and (4) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs\u0026thinsp;+\u0026thinsp;ultrasound irradiation (NPs\u0026thinsp;+\u0026thinsp;US). Tumor volumes and body weights were monitored throughout the experiment. Tumor volume was normalized with relative tumor volumes (\u003cem\u003eV/V\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, mice treated with PBS, \u0026ldquo;US\u0026rdquo;, and \u0026ldquo;NPs\u0026rdquo; exhibited a negligible therapeutic effect on tumor growth, while mice treated with \u0026ldquo;NPs\u0026thinsp;+\u0026thinsp;US\u0026rdquo; exhibited a significant inhibition of tumor growth compared to those of other groups (**\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that enhanced ultrasound-responsive NO release induced therapeutic effects. The mice\u0026rsquo;s body weights during the therapeutic period revealed minimal loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating that Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs do not induce acute toxicity. Furthermore, histological examination of tumor tissues via hematoxylin and eosin (H\u0026amp;E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay demonstrated the efficacy of NO-induced therapeutic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). As shown by H\u0026amp;E staining of tumor tissues, mice treated with \u0026ldquo;NPs\u0026thinsp;+\u0026thinsp;US\u0026rdquo; exhibited numerous deformed nuclei (karyopyknosis, karyorrhexis, and karyolysis), indicative of severe tumor cell necrosis. Furthermore, the TUNEL assay revealed a greater number of apoptotic cells in the tumor tissues of the treated group compared to the other control groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn conclusion, the ultrasound-responsive nanoplatform, consisting of PLGA, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, and LA was successfully constructed for the rapid and controlled release of NO, which inhibits the growth of breast cancer. Compared with other traditional stimulus-responsive NO-delivery nanoplatforms, our resultant nanoplatform demonstrated several advantages, including: i) an ultrasound-responsive strategy for PLGA NPs loaded with LA and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs; ii) a cascade amplification reaction for a rapid and large amount of NO production; and iii) real-time imaging monitoring via PA and FL dual‑modality imaging; and iv) effective therapeutic effects by NO-assisted antitumor gas therapy.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthical Statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The animal study protocol was approved by the Ethics Committee of Chongqing Medical University (IACUC-CQMU-2023-10060).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHaiyan Yang: Conceptualization, Writing\u0026ndash;original draft. Guangrong Zheng: Methodology, Investigation. Guochen Li: Visualization, Software. Jincui Chen, Yong Luo: Methodology, Project administration. Licui Qi: Data curation. Tengfei Ke: Formal analysis, Data curation. Jie Xiong: Writing \u0026ndash; review \u0026amp; editing. Xiaojuan Ji: Project administration.All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Chongqing (CSTB2024NSCQ-MSX0102), Chongqing Medical Youth Top-notch Talent (YXQN202446), Chongqing Postdoctoral Science Foundation (2023CQBSHTB1005 and CSTB2023NSCQ-BHX0035), Chongqing women and Children General Project Fund (2020FY108), and Graduate Innovation Foundation of Kunming Medical University (2024S122 and 2024B013).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChatterji S, Krzoska E, Thoroughgood CW, Saganty J, Liu P, Elsberger B, Abu-Eid R, Speirs V. 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Bioac Mater. 2022;18:91\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, ultrasound, nitric oxide, gas therapy","lastPublishedDoi":"10.21203/rs.3.rs-5186273/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5186273/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGas therapy represents a promising strategy for cancer treatment, with nitric oxide (NO) therapy showing particular potential in tumor therapy. However, ensuring sufficient production of NO remains a significant challenge. In this study, we successfully constructed ultrasound-responsive nanoparticles, which consisted of poly (D, L-lactide-co-glycolic acid) (PLGA) nanoparticles, natural L-arginine (LA), and superparamagnetic iron oxide nanoparticles (SPIO, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs), denote as Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs. The Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e-LA-PLGA NPs exhibited effective therapeutic effects both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, particularly in NO-assisted antitumor gas therapy and dual-modality imaging properties. Upon exposure to ultrasound irradiation, LA and Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs were rapidly released from the PLGA NPs. It was demonstrated that LA could spontaneously react with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) present in the tumor microenvironment to generate NO for gas therapy. Concurrently, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs could rapidly react with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to produce a substantial quantity of reactive oxygen species (ROS), which can oxidize LA to further facilitate the release of NO. In conclusion, the proposed ultrasound-responsive NO delivery platform exhibits significant potential in effectively inhibiting the growth of breast cancer.\u003c/p\u003e","manuscriptTitle":"Ultrasound-responsive nanoparticles for nitric oxide release to inhibit the growth of breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 08:19:40","doi":"10.21203/rs.3.rs-5186273/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-19T09:41:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-08T08:27:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T13:25:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T00:37:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-04T13:13:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271127119938205959419285180694142671108","date":"2024-11-04T05:24:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-03T08:30:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-01T01:59:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59502401222842748584026820433224615462","date":"2024-10-29T06:40:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216803970001131861913699673352507823739","date":"2024-10-29T06:01:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197015044999475828281132949939716290371","date":"2024-10-29T01:14:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67702802846227900276164082308815993651","date":"2024-10-28T23:16:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171652920755417570457785074442023601912","date":"2024-10-28T15:42:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51969052592380079506787096113187312846","date":"2024-10-28T15:42:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-28T15:34:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-28T15:09:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-03T04:02:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2024-10-01T09:40:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd2f7c32-cd06-4720-8d86-b5898fbfb0f4","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:06:05+00:00","versionOfRecord":{"articleIdentity":"rs-5186273","link":"https://doi.org/10.1186/s12935-024-03627-4","journal":{"identity":"cancer-cell-international","isVorOnly":false,"title":"Cancer Cell International"},"publishedOn":"2024-12-28 15:57:11","publishedOnDateReadable":"December 28th, 2024"},"versionCreatedAt":"2024-12-09 08:19:40","video":"","vorDoi":"10.1186/s12935-024-03627-4","vorDoiUrl":"https://doi.org/10.1186/s12935-024-03627-4","workflowStages":[]},"version":"v1","identity":"rs-5186273","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5186273","identity":"rs-5186273","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}