Study on the diagnostic value of folic acid-targeted nano-microbubbles in breast microcarcinoma

Study on the diagnostic value of folic acid-targeted nano-microbubbles in breast microcarcinoma | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Study on the diagnostic value of folic acid-targeted nano-microbubbles in breast microcarcinoma Nianyu Xue, Zhenyu Shen, Zhenbin Xu, Shengmin Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2603569/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The prognosis of breast cancer is significantly correlated with its early detection. It is difficult to detect breast microcarcinomas less than or equal to 5 mm by imaging examination. Methods Folic acid-targeted nanobubbles (FA-TNBs) were prepared by the thin-film hydration method. Cytotoxicity and cellular uptake assays were used to examine FA-TNBs’ biological toxicity to cells, targeting them to breast cancer cells. In addition, by constructing a mouse 4T1 tumor model and evaluating the targeting and imaging effects of FA-TNBs, the diagnostic value of these microbubbles for breast microcarcinomas (diameter ≤ 5 mm) was evaluated. Results The average particle size of the fabricated FA-TNBs was 244 ± 21 nm. In vitro cell uptake experiments showed that breast cancer 4T1 cells take up more FA-TNBs than non-targeted nanobubbles (N-TNBs) (p 90%. In vivo imaging of the mouse 4T1 tumor model showed that compared with the N-TNBs group, the FA-TNBs group took a shorter time to peak (20 s, 40 s, p < 0.05), had a higher peak intensity (38.3 ± 1.5 dB, 31.7 ± 1.5 dB, p < 0.05), and the extinction time was shorter (180 s, 120 s, p < 0.05). After FA-NB injection, there were no apparent abnormalities in the tissue sections or hematological examinations of the mice’s vital organs. Conclusions The prepared FA-TNBs had good water solubility, safety, biocompatibility, and enhancement of ultrasound imaging. It had a good imaging effect on mouse breast cancer tumors with a diameter of 5 mm and showed apparent active targeting. FA-TNBs may become a new and practical ultrasound contrast agent for the early detection of breast cancer. Folic acid breast cancer microbubble ultrasonography 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 Introduction Breast cancer has become the most common malignant tumor in women, accounting for about 30% of female cancers [ 1 ] . The prognosis of breast cancer is significantly correlated with its early detection. Early breast cancer has a higher survival rate than advanced disease, and it allows for breast-conserving surgery to be performed [ 2 ] . Although imaging (including magnetic resonance (MR), mammography, and ultrasonography) plays a significant role in breast cancer detection, it remains difficult to detect breast cancers less than or equal to 5 mm with this method of examination [ 3 ] . Currently, the ultrasound contrast agent most often in clinical use only flows within the blood vessels and does not enter the interstitial space. Due to a lack of targeting, the identification of breast microcarcinoma is poor [ 4 , 5 ] . The further improvement of clinical breast microcarcinoma diagnosis has become problematic. In recent years, targeting nanobubbles has become a research focus in the early diagnosis of tumors [ 6 , 7 ] . Studies have shown that the positive expression rate of folate receptor alpha (FR-α) in breast cancer is relatively high [ 8 ] . In addition, FR-α expressed in malignant tumor tissues retains its cell surface distribution and ability to bind folic acid. These features allow for the use of tumor cell FR-α as a selectively targeted imaging agent fabrication method [ 9 , 10 ] . Currently, targeted contrast agents are primarily used to focus on tumor blood vessels for benign and malignant identification and treatment [ 11 ] . In contrast, malignant tumors do not depend on neovascularization when they do not exceed 5 mm [ 12 ] . Therefore, we chose liposome nanobubbles combined with folic acid to make targeted nanobubbles and aimed to study their diagnostic value in breast microcarcinoma (tumor diameter less than or equal to 5 mm). Materials And Methods Materials DSPE-PEG(2000), DSPE-PEG(2000)-FA, and DSPE-PEG(2000)-FICT were purchased from Aiwei Tuo (Shanghai) Co., Ltd., and C3H8 gas was purchased from Wula (Hangzhou) Chemical Co., Ltd. In addition, chloroform, agar powder, sodium chloride, potassium chloride, and dipotassium hydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd. DMEM (high glucose), the cell culture medium, and fetal bovine serum were purchased from Gibco (USA). Furthermore, dimethyl sulfoxide, penicillin-streptomycin double antibody, the Dil staining kit, the CCK-8 kit, and DAPI dye were purchased from Biyuntian Biotechnology Co., Ltd. Lastly, 0.25% trypsin and PBS7.4 solution were purchased from Biosharp Saiguo Biotechnology Company. Cells And Animals The 4T1 mouse breast cancer cells were provided by the Shanghai Chinese Academy of Sciences Cell Bank, and the culture medium was configured as DMEM high glucose medium + fetal bovine serum + penicillin-streptomycin double-antibody. Fifteen female Balb/c mice aged 6–8 weeks (provided by Viton Lever Laboratory Animal Center) were reared for one week before the experiment to a body weight of about 16–18 g. The animal experiments were approved by the Association of Animal Experiment Ethics and Ethics of Ningbo University. Preparation Of Nanobubbles Liposome nanobubbles were prepared by the thin film hydration method. The mass ratio of about DPPC:DSPE-PEG(2000)-FA:cholesterol was 10:4:1 (mg/mg/mg), and the materials were dissolved in 3 ml of chloroform. Then, the organic solvent was rotary evaporated to form a lipid film on the bottom of an eggplant-shaped bottle, which was hydrated after cooling to room temperature and continuously mixed. The resulting emulsion was further processed using an ultrasonic cell disintegrator before being filled with perfluoropropane gas. It was rapidly oscillated using an amalgam blender to obtain nano-microbubbles, which were stored in a 4°C refrigerator. All these operations were conducted away from the light. The folic acid-targeted nano-microbubbles (FA-TNBs) were prepared by adding a certain amount of DSPE-PEG-FA in the first step, with the remaining steps continuing as above. Finally, non-targeted nanobubbles (N-TNBs) and FA-TNBs were obtained. The FA-TNBs were diluted to an appropriate concentration and measured on a nanoparticle-sized Zeta potential analyzer (Nano-ZS, Malvern, England) to obtain the particle size, dispersion coefficient, and Zeta potential. Lastly, the FA-TNB and N-TNB morphologies were detected via scanning electron microscopy (SEM, S4800, Hitachi, Tokyo, Japan). Cell Culture The 4T1 cells were cultured on a DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin and placed in an incubator (temperature: 37°C, CO2 volume fraction: 5%) to promote cell growth. The cell culture media were replaced every two to three days, depending on the growth of the tumor cells. Cytotoxicity And Uptake Assays Cytotoxicity experiments were performed using the CCK-8 method. The 4T1 cells grown in the log phase were cultured on a 96-well plate, and 100 ul was added to each well. The plate was incubated in a constant-temperature cell incubator at 37°C for 24 h, and the culture medium was then aspirated. In addition, 100 ul of FA-TNB-containing culture medium was added to each well, and after 24 hours of incubation, 10 ul of CCK-8 was added to each well. Next, the culture medium was incubated at 37°C for one to two hours, and the viability of 4T1 cells was evaluated using a microplate reader. Next, the Dil-labeled FA-TNBs and NTNBs were prepared. In a 6-well plate, 2 mL of complete DMEM medium containing 4T1 cells was inoculated into each well and incubated for 24 hours in a constant temperature cell incubator at 37°C. Then, the culture medium was aspirated, washed twice with 1 mL of PBS solution, and an appropriate amount of DMSO was added to cover the bottom of the cells. Next, the plate was incubated for 15 min in a constant temperature cell incubator, the DMSO was aspirated, and the medium was washed twice with PBS solution. Next, an appropriate amount of DAPI staining solution was added to each well to cover the bottom; the plate was placed in an incubator for 15 minutes, removed, and washed twice with PBS solution. Next, DMEM medium containing a certain amount of FA-TNBs and NTBs was added to the six-well plate, and the process was repeated three times. After being placed in a constant temperature incubator for four hours, the culture medium was aspirated and washed three times with PBS solution. Finally, a small amount of PBS solution was added to the wells for easy observation. The FA-TNBs-Dil or NTBs-Dil entered the 4T1 cells or combined with the cell membrane. Under the FV-1000 confocal microscope (Japan), with 358 nm excitation light, the nucleus presented blue. However, FA-TNBs-Dil or NTBs-Dil appeared orange-red with an excitation light of 549 nm. To further validate the cellular uptake experiments, we performed flow cytometry to obtain quantitative data for comparison. The experimental steps were as follows: fabrication of FA-NTBs and NTBs was conducted with FICT fluorescent dyes. In a 12-well plate, 1 mL of DMEM medium containing 4T1 was inoculated into each well and incubated in a constant temperature cell incubator at 37°C for 24 hours. Then, the culture medium was aspirated, washed twice with PBS solution, and 1 mL of DMEM medium containing a certain amount of FA-NTBs-FICT or NTBs-FICT was added and incubated with the cells for 12 hours. Finally, three wells of cells were set as a blank group, and only ordinary DMEM medium without microvesicles was added. After adding 0.5 mL of trypsin for 7 minutes, 1 mL of culture medium was added, and the mixture was placed in a centrifuge tube for centrifugation (1500 rpm/min, 3 min). Then, the supernatant was discarded, and the cells were remixed with 0.5 mL of PBS solution. The number of nanovesicles entering the cells was determined by measuring the of FA-NTBs-FICT or NTBs-FICT by flow cytometry. In vitro imaging The nano-microbubbles were prepared in a specific concentration of PBS suspension with a pH of 7.4, injected into the holes of the agar block, and ultrasonic imaging was performed using a Mindray M9 portable ultrasonic imager. The ultrasonic signals of PBS buffer and FA-NTBs contrast agent in the agar block holes were tested, and the enhancement intensity of the contrast agent was quantitatively analyzed. In vivo imaging experiments in animals Murine breast cancer 4T1 cells were selected to construct this experimental animal model. The specific method was as follows: about 1.5 million breast cancer 4T1 cells (concentration: 1 million cells/100 µL) were inoculated into the back of the upper right thigh of each mouse. Tumor growth in the mice was observed daily, and tumor size was measured using digital calipers. After the tumor on the right mouse thigh grew to a diameter of about 5 mm, six similar mice were selected and divided into two groups. A total of 200 ul of FA-TNBs and NTBs were injected intratumorally, and the M9 (Minray, China) portable ultrasound machine (L12–4S linear array probe, frequency: 4–12 MHZ, mechanical index: 0.12) was used for intertumoral injection. First, the tumor surface was gently touched for CEUS imaging. Images were collected at 10, 20, 30, 120, 180, and 300 s after injection. Then, 200 ul of NTBs and FA-TNBs were injected from the tail vein of each mouse for contrast-enhanced ultrasound imaging. The other methods were the same as above. Biosafety Testing Six healthy BLab/c mice were selected and randomly divided into a blank control group and a nano-microbubble group. PBS buffer and FA-TNBs 200 ul were injected into the tail vein, respectively. The mice were sacrificed after two weeks of regular feeding, and organ tissue section analysis was performed on five major organs (heart, liver, spleen, lung, and kidney). In addition, the blood routine of the mice was analyzed simultaneously. Statistical analysis The experimental data were analyzed by SPSS 18.0 statistics, and the comparison between the two groups in the cell experiment was analyzed by the independent samples t-test. Notably, p < 0.05 was considered statistically significant. Results Characterization of nanobubbles The FA-TNB particle size was about 244 ± 21 nm (n = 3). The PDI was 0.28 ± 0.07 (n = 3). In addition, the zeta potential was 6.04 ± 0.67 mv (n = 3) (Fig. 1 ). After standing for 10 mins, 30 mins, and one hour, the particle size increased slightly, but the differences were not significant. With the increase in particle size after six hours, the PDI also began to increase significantly. When measured again after standing for 24 h, the particle size increased more significantly (Fig. 2 ). Notably, an automated cell counter showed a microvesicle concentration of approximately 1*10^7/ml (Fig. 3 ). The TEM images showed that the FA-TNB morphology was almost spherical and well-dispersed in the solution. A slightly brighter gas filled the liposomes (Fig. 4 ). Notably, most FA-TNBs were below 500 nm in diameter. Cell Uptake Assay Under the confocal microscope, the cell nuclei were stained with DAPI staining solution. They showed a blue-violet color, whereas the DIL-loaded liposomes combined with the cell membrane stained the cell membrane red. After rinsing with PBS solution, the red fluorescence in the FA-TNBs group was significantly greater than in the N-TNBs group, and a significant amount of red fluorescence was bound around the cells (Fig. 5 ). Compared with the two groups, the red fluorescence intensity inside the FA-TNB cells was also significant. This indicated that FA-TNBs could target and bind to the 4T1 cells, and the volume of microvesicles entering the cell interior was also higher. This was very beneficial for further imaging. As seen from the flow cytometry data, the FA-TNBs group had higher channel targeting and FICT fluorescence values in cells than the N-TNBs group (Fig. 6 ). This showed that compared with the NBS group, the FA-TNBs group had higher targeted binding to the 4T1 cells. Hence, the number of microvesicles entering the 4T1 cells was also higher. Cytotoxicity Assay When the concentration of DDPC was 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, and 10 mg/ml, there was no significant inhibitory effect on the growth of 4T1 cells. In this concentration range, cell viability was 90% greater (Fig. 7 ). However, the DPPC concentration used for animal and in vitro imaging was less than 10 mg/ml. Therefore, the nanobubbles we prepared were of a material with better biocompatibility and had no significant effect on cell viability. In vitro imaging of nanobubbles After injection of PBS buffer solution as a blank control, the imaging of nanobubbles showed no enhancement (Fig. 8 ). In contrast, FA-TNBs showed enhanced performance (Fig. 8 ). The time enhancement curve analysis demonstrated that the enhancement intensity of the PBS solution fluctuated around 20 dB, and that of FA-TNBs around 44 dB. Compared with the PBS solution, the enhancement intensity of FA-TNBs was significantly higher. After five mins of observation, FA-TNBs still showed strong enhancement, proving that the prepared nanobubbles had an excellent development effect and could be further used for in vivo experimental development. In vivo imaging After intertumoral injection of FA-TNBs and N-TNBs, both showed strong angiographic findings, but the regression rate of FA-TNBs was slower than that of N-TNBs (Fig. 9 ). After 200 ul of FA-TNBs and N-TNBs were injected into the tail vein (Fig. 10 ), both began to enhance within 15 s of injection, but the contrast enhancement in the FA-TNBs group was significant. The FA-TNBs group peaked at about 20 s and the N-TNBs group at about 40 s. In addition, the peak intensity was 38.3 ± 1.5 dB in the FA-TNBs group (n = 3) and 31.7 ± 1.5 in the N-TNBs group dB (n = 3) (p < 0.05). At 120 s, the N-TNBs group showed significant regression performance, whereas the FA-TNBs group still had strong enhancement performance. At 180 s, the FA-TNBs group gradually began to subside, while most of the N-TNBs group had subsided (p < 0.05). Biosafety Test Results Compared with the control group, there were no detectable pathological manifestations of tissue damage, such as necrosis, inflammation, hemorrhage, or pulmonary fibrosis (Fig. 11 ). Except for PL T, there was no significant difference in routine blood between the control and FA-TNBs groups (Fig. 12 ). Discussion Characterization of nanobubbles In this study, we improved the process of preparing nanoliposomes by thin film hydration. In addition, we found that increasing the negative pressure more uniformly every few minutes during rotary evaporation and finally stabilizing at the negative target pressure contributed to smooth, uniform lipid film formation at the bottom of the eggplant-shaped flask. Hydration with water bath ultrasonic hydration followed by ultrasonic pulverization for an appropriate period can significantly reduce liposome particle size and PDI. The FA-TNBs particle size we prepared was 244 ± 21 nm, while the truncated diameter of the tumor neovascular gap was about 380–780 nm [ 13 ] . Therefore, FA-TNBs should be able to pass through the tumor vascular gap and enter the tumor tissue. Cellular Experiments CCK-8 experiments demonstrated that FA-TNBs did not inhibit the growth of 4T1 cells, proving that they are safe, reliable, and biologically sound. The FA-TNBs group in the uptake experiment showed significantly higher intracellular fluorescent values and selectivity for the channels corresponding to the FITC fluorescent dye compared to the N-TNBs group, demonstrating the targeting of FA-TNBs to breast cancer 4T1 cells. At the same time, the entry into 4T1 cells showed a significant advantage represented by our FA-TNBs. This finding offers the possibility of a targeted diagnosis of microscopic breast cancer. In vitro imaging of nanobubbles Our FA-TNBs had a similar strong contrast intensity to SonoVue (current microbubble contrast agents in clinical use) in vitro and lasted for several minutes. Both have significantly better contrast performance than the PBS solution. Although SonoVue is a micron-sized microbubble, the scattering ability of a single microbubble is stronger for ultrasound, but the microbubble concentration of the nanoliposomal microbubble is much higher at a similar liposome concentration, so our targeted microbubble was slightly stronger than SonoVue in the overall in vitro contrast intensity. This suggested that nanoscale liposomal microbubbles had diagnostic value and potential in the clinic. In vivo imaging The biosafety of the microbubbles used in vivo must be guaranteed. Therefore, we injected a sufficient volume of microbubbles into the mice and verified the safety of the microbubbles using vital tissue sections and hematological assays. The imaging performance of the microbubbles in vivo was observed by intra-tumoral and tail vein injection in mice, respectively. We verified that our microbubbles had good imaging performance in vivo by both injection methods and that the targeting FA-TNBs were significantly better than N-TNBs for tumors. In addition, in vivo experiments demonstrated the active targeting of our FA-TNBs to 5-mm mouse breast cancer tumors. In conclusion, the targeting and imaging effects of FA-TNBs in this study were verified through in vitro assays and cellular experiments. In addition, in mouse 5-mm tumor imaging, the FA-TNB group showed ultrasound imaging superiority compared with the N-TNBs group and had particular active targeting effects for tumor exactness. These findings are promising for the early diagnosis of microscopic breast cancer. Declarations This study is reported in accordance with ARRIVE guidelines. The animal experiments were approved by the Association of Animal Experiment Ethics and Ethics of Ningbo University. All methods were performed in accordance with the relevant guidelines and regulations. Consent for publication: Not applicable. Availability of data and materials: All data generated or analysed during this study are included in this published article. Competing interests: The authors declare that they have no competing interests. Funding: The research was supported by the Zhejiang Provincial Science and Technology Department (grand number BY23H180018). Author Contributions Statement: All authors wrote and designed the main manuscript text, and reviewed the manuscript. Nianyu Xue and Zhenyu Shen is responsible for data collection and compilation. All authors are responsible for the analysis and interpretation of data and the final approval of manuscript. Acknowledgements: Not applicable. References Siegel R L, Miller K D and Jemal A. Cancer statistics, 2020. CA Cancer J Clin, 2020, 70(1): 7-30. Montemurro F, Nuzzolese I, and Ponzone R. Neoadjuvant or adjuvant chemotherapy in early breast cancer? . Expert Opin Pharmacother, 2020, 21(9): 1071-1082. Mu Q, Wang H, Zhang M. Nanoparticles for imaging and treatment of metastatic breast cancer. Expert Opin Drug Deliv. 2017 Jan;14(1):123-136. Du M, et al. Ultrasound Responsive Magnetic Mesoporous Silica Nanoparticle-Loaded Microbubbles for Efficient Gene Delivery. ACS Biomater Sci Eng, 2020, 6(5): 2904-2912. Rengeng L, et al. Sonodynamic therapy, a treatment developing from photodynamic therapy. Photodiagnosis Photodyn Ther, 2017,19: 159-166. Chen JS, et al. Functionalized nanoparticles with targeted antibody to enhance imaging of breast cancer in vivo[J]. Journal of Nanobiotechnology, 2020, 18(1):127-134. Fan X, et al. Ultrasonic Nanobubbles Carrying Anti-PSMA Nanobody: Construction and Application in Prostate Cancer-Targeted Imaging[J]. PLoS One, 2015, 10(6): 214-223. Hartmann LC, Keeney GL, Lingle WL, et al. Folate receptor overexpression is associated with poor outcome in breast cancer. Int J Cancer 2007; 121(5): 938-42. John F, Ross, et al. Differential regulation of folate receptor isoforms in normal and malignant tissues In vivo and in established cell lines.Physiologic and Clinical Implications,1993,12(2):2432-2441. Christopher P, et al. Delivery of macromolecules into living cells: A method that exploits folate receptor endocytosis.Cell Biology,1991, (88):5572-5576. Bitterer F, Hornung M, Platz Batista da Silva N, Schlitt HJ, Stroszczynski C, Wege AK, Jung EM. In vivo detection of breast cancer liver metastases in humanized tumour mice using tumour specific contrast agent BR55®. Clin Hemorheol Microcirc. 2020;76(4):559-572. Zhao C, Wang X, Zhao Y, Li Z, Lin S, Wei Y, Yang H. A novel xenograft model in zebrafish for high-resolution investigating dynamics of neovascularization in tumors. PLoS One. 2011;6(7):e21768. Wallez Y, Huber P. Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis. Biochim Biophys Acta. 2008 Mar;1778(3):794-809. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About 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-2603569","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":179591777,"identity":"63999f4e-8fe7-4c43-b413-18e4905d2380","order_by":0,"name":"Nianyu Xue","email":"","orcid":"","institution":"The First Affiliated Hospital of Ningbo University(Ningbo First Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nianyu","middleName":"","lastName":"Xue","suffix":""},{"id":179591778,"identity":"c6e9a7d5-e219-484e-bb2c-3550abdae2b0","order_by":1,"name":"Zhenyu Shen","email":"","orcid":"","institution":"The First Affiliated Hospital of Ningbo University(Ningbo First Hospital)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Shen","suffix":""},{"id":179591779,"identity":"c477c9c1-67cb-4075-8031-12daf26f0d2c","order_by":2,"name":"Zhenbin Xu","email":"","orcid":"","institution":"Ningbo University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenbin","middleName":"","lastName":"Xu","suffix":""},{"id":179591780,"identity":"2047376b-536c-4177-b715-19bdff1600b2","order_by":3,"name":"Shengmin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNvbmAwcSeGzk+IEM4rTw8RxLPPBAJs1YsudYAnFa5CRylA8+sDmcaHAjx4BIhzHkMBxIyGFOMDiQ8/HGGwY7Od0GglrOAv1yhi1P8sDZzZZzGJKNzQ4Q0sLYl3AgsYenmO9g7zZpHoYDidsIamHmMTiQ+E8iseEwzzMitbABtSTwGCROOMbDRqQWHrYEoJYEYCCzGVvOMSDCL/LzHx/++IPnvxy//OOHN95U2MkR1IICJHiIjBpkLaTqGAWjYBSMghEBAN5+RiMqmlZNAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Ningbo University(Ningbo First Hospital)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shengmin","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-02-19 06:14:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2603569/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2603569/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":33709119,"identity":"6d6868b5-0e89-480d-9d7a-77673f17053c","added_by":"auto","created_at":"2023-03-02 20:24:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":367740,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size and potential diagram of FA-TNBs\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/6c33d979429ee05430c3ad4c.png"},{"id":33708405,"identity":"74936661-aa40-4ecf-abac-0a4a1dfc9929","added_by":"auto","created_at":"2023-03-02 20:16:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31119,"visible":true,"origin":"","legend":"\u003cp\u003eChange of FA-TNBs particle size after standing for 24 hours\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/ec7742d0f7500f9dd3b39edf.png"},{"id":33709416,"identity":"ea4f4ac1-fc4e-43e2-8dd9-c826caac610f","added_by":"auto","created_at":"2023-03-02 20:40:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":554686,"visible":true,"origin":"","legend":"\u003cp\u003eFA-TNBs diagram under automatic cell counter (Thermo Fisher)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/07177f46ff8cc4b5409bf96e.png"},{"id":33709117,"identity":"7185db7f-ad06-4cc5-ac58-361aeeac38bf","added_by":"auto","created_at":"2023-03-02 20:24:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1558611,"visible":true,"origin":"","legend":"\u003cp\u003eImage under FA-NBs Transmission Electron Microscopy (TEM)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/273b79bc210d08d2ba3f96b1.png"},{"id":33708409,"identity":"7e41bd43-ce68-4b67-a937-1a461155fa5b","added_by":"auto","created_at":"2023-03-02 20:16:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2002021,"visible":true,"origin":"","legend":"\u003cp\u003e(A, B) confocal images of N-TNBs-DIL co-cultured with 4T1 cells after DAPI staining. (C, D) confocal images of FA-TNBs-DIL co-cultured with 4T1 cells after DAPI staining\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/b92d8a1ef1b3a603f07f4323.png"},{"id":33708412,"identity":"0ad29224-652b-465c-a216-9683346f09b5","added_by":"auto","created_at":"2023-03-02 20:16:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":202581,"visible":true,"origin":"","legend":"\u003cp\u003e(A) In the blank group after N-TNBs FITC and FA -TNBs FITC were co-cultured with 4T1 cells for 12 hours, 4T1 cells were targeted to the FITC fluorescence channel on flow cytometry. (B) In the blank group, after N-TNBs FITC and FA-TNBs FITC were co-cultured with 4T1 cells for 12 hours, the fluorescence intensity of nano microbubbles in their cells was measured by flow cytometry (n = 3, **p＜0.05，***p \u0026lt; 0.001, **** p \u0026lt; 0.0001) .\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/2ca09e92ab51aa57ca03ee5d.png"},{"id":33709370,"identity":"817cb10a-4318-43ad-930f-58fe99f36027","added_by":"auto","created_at":"2023-03-02 20:32:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":27850,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival rate of 4T1 cells at different concentrations of DPPC by FA-TNBs\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/d749a698c476436019315431.png"},{"id":33709122,"identity":"79d98fcf-5194-42cc-8b4a-b4306e376ca9","added_by":"auto","created_at":"2023-03-02 20:24:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1829501,"visible":true,"origin":"","legend":"\u003cp\u003eA: Contrast-enhanced ultrasonography of PBS solution in agar block model; B: Contrast-enhanced ultrasonography of FA-TNBs solution in the agar block model.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/d619e03f51b01d1906aad202.png"},{"id":33709371,"identity":"56c39dc7-df4d-46b6-a96b-4222d3819090","added_by":"auto","created_at":"2023-03-02 20:32:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1007493,"visible":true,"origin":"","legend":"\u003cp\u003eThe contrast images at each period after injection of 200 ul of FA-TNBs or N-TNBs solution into the 5 mm tumor on the mice’s backs\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/15ac72749bb21440e065628c.png"},{"id":33709373,"identity":"e0ab73ca-a2dc-436a-b301-0adfd0104946","added_by":"auto","created_at":"2023-03-02 20:32:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2517553,"visible":true,"origin":"","legend":"\u003cp\u003eThe contrast images of the 5 mm tumor on the mice’s backs at each time period after tail vein injection of 200 ul FA-TNBs and N-TNBs\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/88813e2041d384063cd2db5d.png"},{"id":33708416,"identity":"081f8245-2b8d-4833-9936-ac42fb17e3d9","added_by":"auto","created_at":"2023-03-02 20:16:04","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":7146493,"visible":true,"origin":"","legend":"\u003cp\u003eImportant mouse tissue sections injected with 200 ul FA-TNBs after two weeks of natural life (200X)\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/0e1730b7776966dc0c4e1842.png"},{"id":33708415,"identity":"653854ba-ccc3-4a9b-b9d5-b0643440ddd7","added_by":"auto","created_at":"2023-03-02 20:16:04","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":102845,"visible":true,"origin":"","legend":"\u003cp\u003eHematological examination of mice injected with 200 ul FA-TNBs after two weeks of natural life\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/ae0f9933ebe9f5fea94d4980.png"},{"id":35245409,"identity":"abc19c19-108b-4af8-b3ec-5c05197cf6ae","added_by":"auto","created_at":"2023-04-04 05:59:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8712676,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2603569/v1/90186756-9c2e-489b-9fd8-9430ca7e8c3c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the diagnostic value of folic acid-targeted nano-microbubbles in breast microcarcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer has become the most common malignant tumor in women, accounting for about 30% of female cancers\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The prognosis of breast cancer is significantly correlated with its early detection. Early breast cancer has a higher survival rate than advanced disease, and it allows for breast-conserving surgery to be performed\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Although imaging (including magnetic resonance (MR), mammography, and ultrasonography) plays a significant role in breast cancer detection, it remains difficult to detect breast cancers less than or equal to 5 mm with this method of examination\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, the ultrasound contrast agent most often in clinical use only flows within the blood vessels and does not enter the interstitial space. Due to a lack of targeting, the identification of breast microcarcinoma is poor\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The further improvement of clinical breast microcarcinoma diagnosis has become problematic. In recent years, targeting nanobubbles has become a research focus in the early diagnosis of tumors\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudies have shown that the positive expression rate of folate receptor alpha (FR-α) in breast cancer is relatively high\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In addition, FR-α expressed in malignant tumor tissues retains its cell surface distribution and ability to bind folic acid. These features allow for the use of tumor cell FR-α as a selectively targeted imaging agent fabrication method \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, targeted contrast agents are primarily used to focus on tumor blood vessels for benign and malignant identification and treatment \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In contrast, malignant tumors do not depend on neovascularization when they do not exceed 5 mm\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therefore, we chose liposome nanobubbles combined with folic acid to make targeted nanobubbles and aimed to study their diagnostic value in breast microcarcinoma (tumor diameter less than or equal to 5 mm).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eDSPE-PEG(2000), DSPE-PEG(2000)-FA, and DSPE-PEG(2000)-FICT were purchased from Aiwei Tuo (Shanghai) Co., Ltd., and C3H8 gas was purchased from Wula (Hangzhou) Chemical Co., Ltd. In addition, chloroform, agar powder, sodium chloride, potassium chloride, and dipotassium hydrogen phosphate were purchased from Sinopharm Chemical Reagent Co., Ltd. DMEM (high glucose), the cell culture medium, and fetal bovine serum were purchased from Gibco (USA). Furthermore, dimethyl sulfoxide, penicillin-streptomycin double antibody, the Dil staining kit, the CCK-8 kit, and DAPI dye were purchased from Biyuntian Biotechnology Co., Ltd. Lastly, 0.25% trypsin and PBS7.4 solution were purchased from Biosharp Saiguo Biotechnology Company.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCells And Animals\u003c/h3\u003e\n\u003cp\u003eThe 4T1 mouse breast cancer cells were provided by the Shanghai Chinese Academy of Sciences Cell Bank, and the culture medium was configured as DMEM high glucose medium\u0026thinsp;+\u0026thinsp;fetal bovine serum\u0026thinsp;+\u0026thinsp;penicillin-streptomycin double-antibody. Fifteen female Balb/c mice aged 6\u0026ndash;8 weeks (provided by Viton Lever Laboratory Animal Center) were reared for one week before the experiment to a body weight of about 16\u0026ndash;18 g. The animal experiments were approved by the Association of Animal Experiment Ethics and Ethics of Ningbo University.\u003c/p\u003e\n\u003ch3\u003ePreparation Of Nanobubbles\u003c/h3\u003e\n\u003cp\u003eLiposome nanobubbles were prepared by the thin film hydration method. The mass ratio of about DPPC:DSPE-PEG(2000)-FA:cholesterol was 10:4:1 (mg/mg/mg), and the materials were dissolved in 3 ml of chloroform. Then, the organic solvent was rotary evaporated to form a lipid film on the bottom of an eggplant-shaped bottle, which was hydrated after cooling to room temperature and continuously mixed. The resulting emulsion was further processed using an ultrasonic cell disintegrator before being filled with perfluoropropane gas. It was rapidly oscillated using an amalgam blender to obtain nano-microbubbles, which were stored in a 4\u0026deg;C refrigerator. All these operations were conducted away from the light. The folic acid-targeted nano-microbubbles (FA-TNBs) were prepared by adding a certain amount of DSPE-PEG-FA in the first step, with the remaining steps continuing as above. Finally, non-targeted nanobubbles (N-TNBs) and FA-TNBs were obtained.\u003c/p\u003e \u003cp\u003eThe FA-TNBs were diluted to an appropriate concentration and measured on a nanoparticle-sized Zeta potential analyzer (Nano-ZS, Malvern, England) to obtain the particle size, dispersion coefficient, and Zeta potential. Lastly, the FA-TNB and N-TNB morphologies were detected via scanning electron microscopy (SEM, S4800, Hitachi, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eThe 4T1 cells were cultured on a DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin and placed in an incubator (temperature: 37\u0026deg;C, CO2 volume fraction: 5%) to promote cell growth. The cell culture media were replaced every two to three days, depending on the growth of the tumor cells.\u003c/p\u003e\n\u003ch3\u003eCytotoxicity And Uptake Assays\u003c/h3\u003e\n\u003cp\u003eCytotoxicity experiments were performed using the CCK-8 method. The 4T1 cells grown in the log phase were cultured on a 96-well plate, and 100 ul was added to each well. The plate was incubated in a constant-temperature cell incubator at 37\u0026deg;C for 24 h, and the culture medium was then aspirated. In addition, 100 ul of FA-TNB-containing culture medium was added to each well, and after 24 hours of incubation, 10 ul of CCK-8 was added to each well. Next, the culture medium was incubated at 37\u0026deg;C for one to two hours, and the viability of 4T1 cells was evaluated using a microplate reader.\u003c/p\u003e \u003cp\u003eNext, the Dil-labeled FA-TNBs and NTNBs were prepared. In a 6-well plate, 2 mL of complete DMEM medium containing 4T1 cells was inoculated into each well and incubated for 24 hours in a constant temperature cell incubator at 37\u0026deg;C. Then, the culture medium was aspirated, washed twice with 1 mL of PBS solution, and an appropriate amount of DMSO was added to cover the bottom of the cells. Next, the plate was incubated for 15 min in a constant temperature cell incubator, the DMSO was aspirated, and the medium was washed twice with PBS solution.\u003c/p\u003e \u003cp\u003eNext, an appropriate amount of DAPI staining solution was added to each well to cover the bottom; the plate was placed in an incubator for 15 minutes, removed, and washed twice with PBS solution. Next, DMEM medium containing a certain amount of FA-TNBs and NTBs was added to the six-well plate, and the process was repeated three times. After being placed in a constant temperature incubator for four hours, the culture medium was aspirated and washed three times with PBS solution. Finally, a small amount of PBS solution was added to the wells for easy observation.\u003c/p\u003e \u003cp\u003eThe FA-TNBs-Dil or NTBs-Dil entered the 4T1 cells or combined with the cell membrane. Under the FV-1000 confocal microscope (Japan), with 358 nm excitation light, the nucleus presented blue. However, FA-TNBs-Dil or NTBs-Dil appeared orange-red with an excitation light of 549 nm.\u003c/p\u003e \u003cp\u003eTo further validate the cellular uptake experiments, we performed flow cytometry to obtain quantitative data for comparison. The experimental steps were as follows: fabrication of FA-NTBs and NTBs was conducted with FICT fluorescent dyes. In a 12-well plate, 1 mL of DMEM medium containing 4T1 was inoculated into each well and incubated in a constant temperature cell incubator at 37\u0026deg;C for 24 hours. Then, the culture medium was aspirated, washed twice with PBS solution, and 1 mL of DMEM medium containing a certain amount of FA-NTBs-FICT or NTBs-FICT was added and incubated with the cells for 12 hours. Finally, three wells of cells were set as a blank group, and only ordinary DMEM medium without microvesicles was added.\u003c/p\u003e \u003cp\u003eAfter adding 0.5 mL of trypsin for 7 minutes, 1 mL of culture medium was added, and the mixture was placed in a centrifuge tube for centrifugation (1500 rpm/min, 3 min). Then, the supernatant was discarded, and the cells were remixed with 0.5 mL of PBS solution. The number of nanovesicles entering the cells was determined by measuring the of FA-NTBs-FICT or NTBs-FICT by flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003eimaging\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe nano-microbubbles were prepared in a specific concentration of PBS suspension with a pH of 7.4, injected into the holes of the agar block, and ultrasonic imaging was performed using a Mindray M9 portable ultrasonic imager. The ultrasonic signals of PBS buffer and FA-NTBs contrast agent in the agar block holes were tested, and the enhancement intensity of the contrast agent was quantitatively analyzed.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003eimaging experiments in animals\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMurine breast cancer 4T1 cells were selected to construct this experimental animal model. The specific method was as follows: about 1.5\u0026nbsp;million breast cancer 4T1 cells (concentration: 1\u0026nbsp;million cells/100 \u0026micro;L) were inoculated into the back of the upper right thigh of each mouse. Tumor growth in the mice was observed daily, and tumor size was measured using digital calipers.\u003c/p\u003e \u003cp\u003eAfter the tumor on the right mouse thigh grew to a diameter of about 5 mm, six similar mice were selected and divided into two groups. A total of 200 ul of FA-TNBs and NTBs were injected intratumorally, and the M9 (Minray, China) portable ultrasound machine (L12\u0026ndash;4S linear array probe, frequency: 4\u0026ndash;12 MHZ, mechanical index: 0.12) was used for intertumoral injection. First, the tumor surface was gently touched for CEUS imaging. Images were collected at 10, 20, 30, 120, 180, and 300 s after injection. Then, 200 ul of NTBs and FA-TNBs were injected from the tail vein of each mouse for contrast-enhanced ultrasound imaging. The other methods were the same as above.\u003c/p\u003e\n\u003ch3\u003eBiosafety Testing\u003c/h3\u003e\n\u003cp\u003eSix healthy BLab/c mice were selected and randomly divided into a blank control group and a nano-microbubble group. PBS buffer and FA-TNBs 200 ul were injected into the tail vein, respectively. The mice were sacrificed after two weeks of regular feeding, and organ tissue section analysis was performed on five major organs (heart, liver, spleen, lung, and kidney). In addition, the blood routine of the mice was analyzed simultaneously.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were analyzed by SPSS 18.0 statistics, and the comparison between the two groups in the cell experiment was analyzed by the independent samples t-test. Notably, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of nanobubbles\u003c/h2\u003e \u003cp\u003eThe FA-TNB particle size was about 244\u0026thinsp;\u0026plusmn;\u0026thinsp;21 nm (n\u0026thinsp;=\u0026thinsp;3). The PDI was 0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (n\u0026thinsp;=\u0026thinsp;3). In addition, the zeta potential was 6.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 mv (n\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After standing for 10 mins, 30 mins, and one hour, the particle size increased slightly, but the differences were not significant. With the increase in particle size after six hours, the PDI also began to increase significantly. When measured again after standing for 24 h, the particle size increased more significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, an automated cell counter showed a microvesicle concentration of approximately 1*10^7/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe TEM images showed that the FA-TNB morphology was almost spherical and well-dispersed in the solution. A slightly brighter gas filled the liposomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, most FA-TNBs were below 500 nm in diameter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Uptake Assay\u003c/h3\u003e\n\u003cp\u003eUnder the confocal microscope, the cell nuclei were stained with DAPI staining solution. They showed a blue-violet color, whereas the DIL-loaded liposomes combined with the cell membrane stained the cell membrane red. After rinsing with PBS solution, the red fluorescence in the FA-TNBs group was significantly greater than in the N-TNBs group, and a significant amount of red fluorescence was bound around the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared with the two groups, the red fluorescence intensity inside the FA-TNB cells was also significant. This indicated that FA-TNBs could target and bind to the 4T1 cells, and the volume of microvesicles entering the cell interior was also higher. This was very beneficial for further imaging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs seen from the flow cytometry data, the FA-TNBs group had higher channel targeting and FICT fluorescence values in cells than the N-TNBs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This showed that compared with the NBS group, the FA-TNBs group had higher targeted binding to the 4T1 cells. Hence, the number of microvesicles entering the 4T1 cells was also higher.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCytotoxicity Assay\u003c/h3\u003e\n\u003cp\u003eWhen the concentration of DDPC was 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, and 10 mg/ml, there was no significant inhibitory effect on the growth of 4T1 cells. In this concentration range, cell viability was 90% greater (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, the DPPC concentration used for animal and \u003cem\u003ein vitro\u003c/em\u003e imaging was less than 10 mg/ml. Therefore, the nanobubbles we prepared were of a material with better biocompatibility and had no significant effect on cell viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003eimaging of nanobubbles\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter injection of PBS buffer solution as a blank control, the imaging of nanobubbles showed no enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In contrast, FA-TNBs showed enhanced performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The time enhancement curve analysis demonstrated that the enhancement intensity of the PBS solution fluctuated around 20 dB, and that of FA-TNBs around 44 dB. Compared with the PBS solution, the enhancement intensity of FA-TNBs was significantly higher. After five mins of observation, FA-TNBs still showed strong enhancement, proving that the prepared nanobubbles had an excellent development effect and could be further used for \u003cem\u003ein vivo\u003c/em\u003e experimental development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003eimaging\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter intertumoral injection of FA-TNBs and N-TNBs, both showed strong angiographic findings, but the regression rate of FA-TNBs was slower than that of N-TNBs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 200 ul of FA-TNBs and N-TNBs were injected into the tail vein (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e), both began to enhance within 15 s of injection, but the contrast enhancement in the FA-TNBs group was significant. The FA-TNBs group peaked at about 20 s and the N-TNBs group at about 40 s. In addition, the peak intensity was 38.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 dB in the FA-TNBs group (n\u0026thinsp;=\u0026thinsp;3) and 31.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 in the N-TNBs group dB (n\u0026thinsp;=\u0026thinsp;3) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 120 s, the N-TNBs group showed significant regression performance, whereas the FA-TNBs group still had strong enhancement performance. At 180 s, the FA-TNBs group gradually began to subside, while most of the N-TNBs group had subsided (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eBiosafety Test Results\u003c/h3\u003e\n\u003cp\u003eCompared with the control group, there were no detectable pathological manifestations of tissue damage, such as necrosis, inflammation, hemorrhage, or pulmonary fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). Except for PL T, there was no significant difference in routine blood between the control and FA-TNBs groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of nanobubbles\u003c/h2\u003e \u003cp\u003eIn this study, we improved the process of preparing nanoliposomes by thin film hydration. In addition, we found that increasing the negative pressure more uniformly every few minutes during rotary evaporation and finally stabilizing at the negative target pressure contributed to smooth, uniform lipid film formation at the bottom of the eggplant-shaped flask. Hydration with water bath ultrasonic hydration followed by ultrasonic pulverization for an appropriate period can significantly reduce liposome particle size and PDI. The FA-TNBs particle size we prepared was 244\u0026thinsp;\u0026plusmn;\u0026thinsp;21 nm, while the truncated diameter of the tumor neovascular gap was about 380\u0026ndash;780 nm\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, FA-TNBs should be able to pass through the tumor vascular gap and enter the tumor tissue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCellular Experiments\u003c/h3\u003e\n\u003cp\u003eCCK-8 experiments demonstrated that FA-TNBs did not inhibit the growth of 4T1 cells, proving that they are safe, reliable, and biologically sound. The FA-TNBs group in the uptake experiment showed significantly higher intracellular fluorescent values and selectivity for the channels corresponding to the FITC fluorescent dye compared to the N-TNBs group, demonstrating the targeting of FA-TNBs to breast cancer 4T1 cells. At the same time, the entry into 4T1 cells showed a significant advantage represented by our FA-TNBs. This finding offers the possibility of a targeted diagnosis of microscopic breast cancer.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vitro\u003c/span\u003e \u003cb\u003eimaging of nanobubbles\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur FA-TNBs had a similar strong contrast intensity to SonoVue (current microbubble contrast agents in clinical use) \u003cem\u003ein vitro\u003c/em\u003e and lasted for several minutes. Both have significantly better contrast performance than the PBS solution. Although SonoVue is a micron-sized microbubble, the scattering ability of a single microbubble is stronger for ultrasound, but the microbubble concentration of the nanoliposomal microbubble is much higher at a similar liposome concentration, so our targeted microbubble was slightly stronger than SonoVue in the overall \u003cem\u003ein vitro\u003c/em\u003e contrast intensity. This suggested that nanoscale liposomal microbubbles had diagnostic value and potential in the clinic.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003eimaging\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe biosafety of the microbubbles used \u003cem\u003ein vivo\u003c/em\u003e must be guaranteed. Therefore, we injected a sufficient volume of microbubbles into the mice and verified the safety of the microbubbles using vital tissue sections and hematological assays. The imaging performance of the microbubbles \u003cem\u003ein vivo\u003c/em\u003e was observed by intra-tumoral and tail vein injection in mice, respectively. We verified that our microbubbles had good imaging performance \u003cem\u003ein vivo\u003c/em\u003e by both injection methods and that the targeting FA-TNBs were significantly better than N-TNBs for tumors. In addition, \u003cem\u003ein vivo\u003c/em\u003e experiments demonstrated the active targeting of our FA-TNBs to 5-mm mouse breast cancer tumors.\u003c/p\u003e \u003cp\u003eIn conclusion, the targeting and imaging effects of FA-TNBs in this study were verified through \u003cem\u003ein vitro\u003c/em\u003e assays and cellular experiments. In addition, in mouse 5-mm tumor imaging, the FA-TNB group showed ultrasound imaging superiority compared with the N-TNBs group and had particular active targeting effects for tumor exactness. These findings are promising for the early diagnosis of microscopic breast cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study is reported in accordance with ARRIVE guidelines. The animal experiments were approved by the Association of Animal Experiment Ethics and Ethics of Ningbo University. All methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: All data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: The research was supported by the Zhejiang Provincial Science and Technology Department (grand number BY23H180018).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions Statement: All authors wrote and designed the main manuscript text, and reviewed the manuscript. Nianyu Xue and Zhenyu Shen is responsible for data collection and compilation. All authors are responsible for the analysis and interpretation of data and the final approval of manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel R L, Miller K D and Jemal A. Cancer statistics, 2020. CA Cancer J Clin, 2020, 70(1): 7-30.\u003c/li\u003e\n\u003cli\u003eMontemurro F, Nuzzolese I, and Ponzone R. Neoadjuvant or adjuvant chemotherapy in early breast cancer? . Expert Opin Pharmacother, 2020, 21(9): 1071-1082.\u003c/li\u003e\n\u003cli\u003eMu Q, Wang H, Zhang M. Nanoparticles for imaging and treatment of metastatic breast cancer. Expert Opin Drug Deliv. 2017 Jan;14(1):123-136.\u003c/li\u003e\n\u003cli\u003eDu M, et al. Ultrasound Responsive Magnetic Mesoporous Silica Nanoparticle-Loaded Microbubbles for Efficient Gene Delivery. ACS Biomater Sci Eng, 2020, 6(5): 2904-2912.\u003c/li\u003e\n\u003cli\u003eRengeng L, et al. Sonodynamic therapy, a treatment developing from photodynamic therapy. Photodiagnosis Photodyn Ther, 2017,19: 159-166.\u003c/li\u003e\n\u003cli\u003eChen JS, et al. Functionalized nanoparticles with targeted antibody to enhance imaging of breast cancer in vivo[J]. Journal of Nanobiotechnology, 2020, 18(1):127-134.\u003c/li\u003e\n\u003cli\u003eFan X, et al. Ultrasonic Nanobubbles Carrying Anti-PSMA Nanobody: Construction and Application in Prostate Cancer-Targeted Imaging[J]. PLoS One, 2015, 10(6): 214-223.\u003c/li\u003e\n\u003cli\u003eHartmann LC, Keeney GL, Lingle WL, et al. Folate receptor overexpression is associated with poor outcome in breast cancer. Int J Cancer 2007; 121(5): 938-42.\u003c/li\u003e\n\u003cli\u003eJohn F, Ross, et al. Differential regulation of folate receptor isoforms in normal and malignant tissues In vivo and in established cell lines.Physiologic and Clinical Implications,1993,12(2):2432-2441.\u003c/li\u003e\n\u003cli\u003eChristopher P, et al. Delivery of macromolecules into living cells: A method that exploits folate receptor endocytosis.Cell Biology,1991, (88):5572-5576.\u003c/li\u003e\n\u003cli\u003eBitterer F, Hornung M, Platz Batista da Silva N, Schlitt HJ, Stroszczynski C, Wege AK, Jung EM. In vivo detection of breast cancer liver metastases in humanized tumour mice using tumour specific contrast agent BR55\u0026reg;. Clin Hemorheol Microcirc. 2020;76(4):559-572.\u003c/li\u003e\n\u003cli\u003eZhao C, Wang X, Zhao Y, Li Z, Lin S, Wei Y, Yang H. A novel xenograft model in zebrafish for high-resolution investigating dynamics of neovascularization in tumors. PLoS One. 2011;6(7):e21768.\u003c/li\u003e\n\u003cli\u003eWallez Y, Huber P. Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis. Biochim Biophys Acta. 2008 Mar;1778(3):794-809.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Folic acid, breast cancer, microbubble, ultrasonography","lastPublishedDoi":"10.21203/rs.3.rs-2603569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2603569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe prognosis of breast cancer is significantly correlated with its early detection. It is difficult to detect breast microcarcinomas less than or equal to 5 mm by imaging examination.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFolic acid-targeted nanobubbles (FA-TNBs) were prepared by the thin-film hydration method. Cytotoxicity and cellular uptake assays were used to examine FA-TNBs\u0026rsquo; biological toxicity to cells, targeting them to breast cancer cells. In addition, by constructing a mouse 4T1 tumor model and evaluating the targeting and imaging effects of FA-TNBs, the diagnostic value of these microbubbles for breast microcarcinomas (diameter\u0026thinsp;\u0026le;\u0026thinsp;5 mm) was evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe average particle size of the fabricated FA-TNBs was 244\u0026thinsp;\u0026plusmn;\u0026thinsp;21 nm. \u003cem\u003eIn vitro\u003c/em\u003e cell uptake experiments showed that breast cancer 4T1 cells take up more FA-TNBs than non-targeted nanobubbles (N-TNBs) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the cytotoxicity experiment, the survival rate of 4T1 cells under each FA-NBs concentration was \u0026gt;\u0026thinsp;90%. \u003cem\u003eIn vivo\u003c/em\u003e imaging of the mouse 4T1 tumor model showed that compared with the N-TNBs group, the FA-TNBs group took a shorter time to peak (20 s, 40 s, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), had a higher peak intensity (38.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 dB, 31.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 dB, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the extinction time was shorter (180 s, 120 s, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After FA-NB injection, there were no apparent abnormalities in the tissue sections or hematological examinations of the mice\u0026rsquo;s vital organs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe prepared FA-TNBs had good water solubility, safety, biocompatibility, and enhancement of ultrasound imaging. It had a good imaging effect on mouse breast cancer tumors with a diameter of 5 mm and showed apparent active targeting. FA-TNBs may become a new and practical ultrasound contrast agent for the early detection of breast cancer.\u003c/p\u003e","manuscriptTitle":"Study on the diagnostic value of folic acid-targeted nano-microbubbles in breast microcarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-02 20:15:59","doi":"10.21203/rs.3.rs-2603569/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"47821c4a-31e5-4fa6-9dd1-498080f73276","owner":[],"postedDate":"March 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-04-04T05:59:29+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-02 20:15:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2603569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2603569","identity":"rs-2603569","version":["v1"]},"buildId":"qrqWcyKcyGKEg00RQ_lqb","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}