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

doi:10.21203/rs.3.rs-2603569/v1

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

https://doi.org/10.21203/rs.3.rs-2603569/v1

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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 < 0.001). In the cytotoxicity experiment, the survival rate of 4T1 cells under each FA-NBs concentration was > 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

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

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.

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).

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.

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.

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No competing interests reported.

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Version 1

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You are reading this latest preprint version

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

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Materials

Cells And Animals

Preparation Of Nanobubbles

Cell Culture

Cytotoxicity And Uptake Assays

Biosafety Testing

Statistical analysis

Results

Characterization of nanobubbles

Cell Uptake Assay

Cytotoxicity Assay

Biosafety Test Results

Discussion

Characterization of nanobubbles

Cellular Experiments

Declarations

References

Additional Declarations

Status:

Version 1