Effect of folic acid-targeted triptolide-loaded microbubbles combined with ultrasound on apoptosis of human MCF-7 breast cancer xenografts in nude mice

Introduction

Relevant surveys indicate a consistent rise in the incidence and mortality rates of female breast cancer, making it one of the leading causes of death among women (1). In clinical practice, chemotherapy and radiotherapy are commonly employed for tumor treatment; however, the prognosis often remains unsatisfactory. Tumor recurrence and metastasis are frequent occurrences, resulting in low 5-year survival rates (2). Although drug chemotherapy effectively inhibits tumor growth, prolonged usage leads to increased drug resistance in patients. Furthermore, most chemotherapy drugs lack specificity and exert strong toxic effects on normal organs and tissues of patients, with some even causing more harm than tumors themselves (3,4). Therefore, achieving precise targeted therapy for breast cancer cells is crucial.

The emergence and development of ultrasound molecular imaging has provided a novel direction for the precise treatment of tumors. It enables the integration of diagnosis and treatment on a single platform, facilitating real-time dynamic observation of lesion areas while meeting the targeted drug delivery needs of medical professionals (5). Ultrasonic microbubbles have garnered significant attention in the field of targeted drug delivery due to their convenient preparation, excellent biocompatibility, and non-viral carrier structure (6). Targeted ultrasound microbubbles represent an effective drug delivery platform due to their versatile biocompatible shells. These shells can be engineered to incorporate therapeutic agents, typically via attachment to the lipid membrane rather than encapsulation within the gaseous core. Furthermore, the shell surface presents abundant functional groups for conjugation with various targeting ligands, such as folic acid (FA), enabling active accumulation at tumor sites. This strategy enhances local drug concentration through a dual mechanism: the passive enhanced permeability and retention (EPR) effect (7), which permits nanoscale and microscale structures to extravasate through the leaky vasculature of tumors, and active ligand-receptor targeting. Upon reaching the target site, ultrasound-triggered destruction of the microbubbles facilitates localized drug release, which simultaneously improves ultrasound imaging contrast and therapeutic efficacy (8,9).

In recent years, the anti-tumor therapy of traditional Chinese medicine has garnered significant attention due to its favorable therapeutic effect and minimal side effects, making it a burgeoning research focus in the field of anti-tumor (10-12). Triptolide (TPL) is a crucial constituent of Tripterygium wilfordii, a traditional Chinese medicine with diverse pharmacological effects, including anti-inflammatory, and antioxidant properties (13-15). Numerous studies have demonstrated the potent broad-spectrum antitumor activity of TPL, particularly its remarkable ability to regulate tumor cell cycle and induce apoptosis in breast cancer cells (16-19). However, its molecular structure as an epoxide diterpene lactone results in limited water solubility and significant toxicity to human liver, kidney, gastrointestinal tract, hematopoietic system, etc., thereby restricting its clinical application (20). To address these challenges, Zheng et al. (21) employed cationic liposomes as carriers for encapsulating TPL while modifying hyaluronic acid (HA) to facilitate liposomal targeting towards CD44 receptors overexpressed in breast cancer cells. This approach effectively reduces direct contact between TPL and bodily fluids, thereby mitigating stimulation and biological toxicity. Luo et al. (22) utilized a novel temperature-responsive polymer to further reduce the toxicity of TPL.

Building upon these advanced drug delivery approaches, in this study, we propose an alternative strategy using ultrasound-mediated targeted therapy. Microbubbles, which are gas-filled microparticles, serve as excellent ultrasound contrast agents due to their strong acoustic impedance mismatch with surrounding tissues. When injected intravenously, they significantly enhance ultrasound imaging by efficiently reflecting sound waves, allowing real-time visualization of vascular structures and tissue perfusion. The folate receptor (FR) represents an ideal target for breast cancer therapy, as it is overexpressed on cancer cells while minimally expressed in normal tissues (23). FA, with its high affinity for FR, small molecular size, and ease of conjugation, provides an excellent targeting moiety for drug delivery systems (24,25). This study integrates these elements by developing novel FA-targeted TPL-loaded ultrasound microbubbles (FA-TLUM). We hypothesize that these dual-functional agents will not only enhance ultrasound contrast for tumor detection but also enable targeted drug delivery through ultrasound-triggered microbubble destruction, thereby improving therapeutic precision while reducing systemic toxicity. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1374/rc).

Methods

Preparation of TLUM

The DPPC (5 mg), DSPE (2 mg), and TPL (1 mg) were completely dissolved in 10 mL of trichloromethane, followed by the evaporation of the organic solvent using a rotary evaporator until a lipid film was fully formed. Subsequently, the mixture was hydrated with PBS and glycerol, and then replaced with perfluoropropane gas. After being placed in a mercury mixer for 40 s, the upper foam was discarded after standing for 3 min to obtain TLUM.

Preparation of FA-TLUM

FA-TLUM was prepared using DPPC (5 mg), DSPE-PEG2000-folate (2 mg) instead of DSPE, and TPL (1 mg), following the same procedure as described above.

Characterization of FA-TLUM

After absorbing a small amount of FA-TLUM solution and diluting it 10-fold with deionized water, the 50 µL sample was aspirated using a pipette gun and deposited onto the copper grid for 5 min. Following sample drying, field emission transmission electron microscopy was employed to observe the microbubble morphology, followed by particle diameter evaluation using Nano measure 1.2 software. A small quantity of FA-TLUM solution was absorbed and diluted in deionized water before being transferred to a specialized colorimetric dish for analysis on the instrument. The exterior of the dish was wiped clean prior to insertion into the laser particle size tester, which utilized dynamic light scattering method to determine hydration dynamic size and Zeta potential of the microbubbles.

Detection of drug loading and encapsulation efficiency

The drug loading and inclusion efficiency of TPL in TLUM and FA-TLUM were determined using ultraviolet spectrophotometry. TPL was dissolved in methanol and prepared into sample solutions with varying concentration gradients. The absorbance at 218 nm was measured for each concentration solution. TLUM and FA-TLUM were centrifuged at low speed, and the upper foam and bottom precipitate were dissolved in methanol for measurement of absorbance values. Drug loading and encapsulation efficiency were calculated by combining the fitting standard curve.

Detection of stability

The TLUM and FA-TLUM solutions were aliquoted into Eppendorf tubes and stored at 4 ℃. After storage for 1 hour, 24 hours, 3 days, 5 days, and 7 days, respectively, the microbubbles were removed from the tubes and coated onto slides. Microscopic examination was conducted to observe any morphological changes in the microbubbles. Additionally, the particle size of the microbubbles was measured using a laser particle size analyzer, while their concentration was determined using a counting plate.

Identification of folate connectivity on the FA-TLUM surface

The TLUM and FA-TLUM were separately placed in Eppendorf tubes, followed by the addition of 2 µL FA monoclonal antibody. The mixture was then incubated overnight at 4 ℃ in a refrigerator. Subsequently, the samples were centrifuged at low speed (800 r/min for 3 min) and washed three times with PBS to eliminate excess primary antibodies. Next, 1 µL FITC-labeled rabbit anti-mouse IgG secondary antibody was added and incubated for 1 h at room temperature in the absence of light. Excess secondary antibodies were removed through low-speed centrifugation and further washed three times with PBS. Finally, the diluted FA-TLUM was observed using an inverted fluorescence microscope, while the fluorescence intensity of FA-TLUM before and after adding the primary antibody was measured using flow cytometry.

Detection of FA-TLUM targeting ability of human breast cancer MCF-7 cells in vitro

The homo sapiens MCF-7 cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were seeded in six-well plates at a density of 1×10⁵ cells/well during the logarithmic growth phase and cultured for 24 hours to prepare MCF-7 cell slides. Subsequently, 100 µL of TLUM and FA-TLUM were added to each well, followed by the addition of 2 mL of culture medium. The culture was continued for 30 min. Afterward, the slides were carefully removed and washed three times with PBS. Finally, the cell slides were immersed in fresh culture medium, and the binding of both groups of microbubbles to MCF-7 cells was observed under a light microscope.

Cell toxicity assays

The MCF-7 cells were harvested during the logarithmic growth phase and when they reached 80% confluency, the original medium was discarded. Subsequently, the cells were washed three times with PBS and then digested with trypsin. After low-speed centrifugation (800 r/min, 5 min), the cell pellet was resuspended in complete medium to obtain a cell suspension. The cells were seeded at a density of 8,000 cells/well in a 96-well culture plate. Following transfer to a cell incubator, they were cultured for an additional 24 hours. Different concentrations of TLUM and FA-TLUM were added to the MCF-7 cells for continued culture, with concentration gradients set at 0, 20, 40, 60, 80, and 100 µg/mL, respectively. After another 24 hours of incubation period, each well of the plate received an addition of Cell Counting Kit-8 (CCK-8) solution (10 µL) followed by further incubation for four hours. The resulting orange methylene dye dissolved in each well was measured using a microplate reader after gentle vibration. Absorbance at a wavelength of 450 nm was recorded by the microplate reader to calculate the cell survival rate.

Extracorporeal ultrasound imaging

The imaging performance of FA-TLUM in vitro was evaluated using a Philips iU 22 (Philips Healthcare, Amsterdam, The Netherlands), and the samples were divided into a control group and an experimental group. The control group was supplemented with degassed water, while the experimental group received varying concentrations of FA-TLUM (50, 100, 150, and 200 µg/mL). Each sample was introduced into a specialized rubber model coated with coupler on its surface. Ultrasonic imaging was conducted using a color Doppler ultrasonic diagnostic system, and the resulting images were recorded and saved.

Establishment of experimental animal model

Twenty 6-week-old female BALB/c nude mice weighing 18–20 g were supplied by the Experimental Animal Center of Qiqihar Medical University for the study. They were maintained under specific pathogen free (SPF) conditions at 23±2 ℃ and a 12-hour light-dark cycle. Food and water are provided freely. The inclusion criteria required that the nude mice be in good health prior to the start of the experiment. Animals were removed from the study if they exhibited signs of serious illness, infection, unsuccessful tumor implantation, or any other issue that could interfere with the ongoing conduct of the experiment. All animal experiments were performed under a project license (No. 2024-AE251) granted by the Animal Ethics Committee of The Second Affiliated Hospital of Qiqihar Medical University, in compliance with institutional guidelines for the care and use of laboratory animals. A protocol was prepared before the study without registration. The MCF-7 cells in logarithmic growth phase were washed with PBS and subsequently digested using trypsin. The digestion process was terminated by adding complete medium. The cells were then collected and centrifuged at a low speed of 800 r/min for 5 min. Following cell counting, the concentration was adjusted to 5×10⁶ cells/mL, and a 100 µL suspension of MCF-7 cells was injected subcutaneously under the armpit of each nude mouse. Tumor growth and the condition of the nude mice were monitored daily after inoculation. Tumor inoculations and measurements were performed at the same time of day to control circadian effects.

In vivo ultrasound experiments

In vivo ultrasound experiments, mice were randomly divided into two groups (blinded to experimenters during allocation): the targeted group (injected with FA-TLUM solution) and the non-targeted group (injected with TLUM solution), each consisting of 6 nude mice. An insulin needle was used to administer a 200 µL material dose via the tail vein. Ultrasound was employed to record the dynamic imaging of microbubble injection. The perfusion characteristics of breast cancer were compared between FA-TLUM and TLUM, and software analysis was conducted on contrast-enhanced ultrasound (CEUS) parameters including peak intensity (PI) and time to peak (TTP).

Hematoxylin and eosin (HE) staining

The targeted group and the non-targeted group were administered microbubble injections, while the control group received a tail vein injection of 0.9% normal saline solution. After one week, the animal models from all three groups were euthanized under anesthesia, and their main organs (heart, liver, spleen, lung, kidney) were excised and fixed in 4% paraformaldehyde. Subsequently, paraffin sections were prepared following standard procedures including deparaffinization and hydration steps. HE staining kit was used for tissue staining followed by dehydration, clearing treatment, and sealing. Finally, morphological changes in the tissues were observed using an inverted microscope.

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay

The MCF-7 cells were seeded at a density of 8,000 cells/well in 96-well plates and incubated for 24 hours. Subsequently, the old medium was discarded and replaced with fresh medium. Various concentrations (5, 10, 20, 40, 80, 120 nmol/L) of TPL were added to the cells for further incubation. At time points of 24, 36, and 48 hours, respectively, each well in the plate was supplemented with a solution containing MTT (20 µL). After removing the liquid phase from each well, dimethyl sulfoxide (200 µL) was added to dissolve the resulting crystals by shaking on a microplate oscillator for a duration of 10 min. The culture plate was then transferred to a microplate reader, where the detection wavelength was set at 570 nm to measure the optical density (OD) value.

CCK-8 assay

The experiment was divided into five groups: control group (Con), TPL group (TPL), TPL combined with ultrasound group (TPL + US), TPL-loaded microbubbles combined with ultrasound group (TLUM + US), and FA-targeted TPL-loaded microbubbles combined with ultrasound group (FA-TLUM + US). In the administration group, the concentration of TPL was 35 nmol/L, while in the combined ultrasound group, ultrasonic radiation was applied with a mechanical index (MI) of 1 and an irradiation time of 1 minute.

MCF-7 cells were seeded in 96-well plates at a density of 8,000 cells per well and cultured for 24 hours. The old medium was discarded and replaced with fresh medium. According to the aforementioned grouping, the treatment factors of each respective group were added to their corresponding wells on a 96-well plate, followed by continued culture for another 24 hours. Subsequently, an additional volume of CCK-8 solution measuring at a quantity of 10 µL per well was added and incubated for a duration of four hours. Finally, absorbance at a wavelength of 450 nm was measured using a microplate reader.

Flow cytometry

The MCF-7 cells from each group were harvested and trypsinized, followed by low-speed centrifugation (1,000 r/min, 5 min) to pellet the cells. The supernatant was carefully aspirated, PBS buffer was added to resuspend the cells, and they were transferred into a centrifuge tube. Subsequently, the cells were once again pelleted by low-speed centrifugation and the supernatant was removed. Then, 1 mL of ice-cold 70% ethanol was gently added for fixation and mixed well before being moved to a refrigerator at 4 ℃ for 2 hours. After fixation, the cells underwent another round of low-speed centrifugation to discard the supernatant. PBS buffer was then added to resuspend the cells which were subsequently subjected to centrifugation and precipitation. Finally, each tube of cell sample received an addition of 0.5 mL propidium iodide staining solution followed by incubation at room temperature in darkness for 30 min. Flow cytometry analysis was employed to assess changes in cell cycle.

The MCF-7 cells in each group were harvested, trypsinized without EDTA, collected into a centrifuge tube, and centrifuged at 800 r/min for 5 min. Then, they were washed twice with PBS buffer by aspiration and rinsing, followed by another centrifugation at 2,000 r/min for 5 min to collect the cells. After discarding the PBS buffer, the cells were resuspended in 500 µL of 1 × Annexin V Buffer. Subsequently, each well was gently mixed with 5 µL of Annexin V-FITC and 5 µL propidium iodide before incubating the cells at room temperature in the dark for 10 min. Finally, cell apoptosis was assessed.

Real-time quantitative polymerase chain reaction (RT-qPCR)

The MCF-7 cells from each group were collected, and total RNA was extracted using the TRIzol kit (Invitrogen, Carlsbad, CA, USA). The RNA concentration was measured using a microspectrophotometer. Reverse transcription of RNA into cDNA was performed using the TaKaRa PrimeScriptrm RT reagent Kit with gDNA Eraser (Takara Bio, Kusatsu, Shiga, Japan). PCR procedures were carried out using the TaKaRa TB Green Premix Ex Taq™ II kit. The PCR amplification conditions consisted of an initial denaturation at 95 ℃ for 30 s, followed by 40 cycles of PCR reaction at 95 ℃ for 30 s and 60 ℃ for 31 s. The relative quantitative analysis of messenger RNA (mRNA) expression levels of Caspase-3, Bax, Bcl-2, and Survivin was conducted using the 2^-ΔΔCt method.

Western blot

The MCF-7 cells from each group were harvested and fully covered with a mixture of RIPA and PMSF. The cells were then lysed on ice for 30 min. After centrifugation (4 ℃, 13,000 r/min, 10 min), the supernatant was collected as total protein. The protein concentration in the samples was determined using the BCA protein concentration determination manual. Equal amounts of proteins were loaded onto SDS-PAGE, transferred to a membrane, and blocked at room temperature for 1 hour. Subsequently, primary antibodies Caspase-3 (Abcam, Cambridge, UK, ab32351), Bax (Abcam, ab32503), Bcl-2 (Abcam, ab32124), Survivin (Abcam, ab76424), β-actin (Abcam, ab8226) were added and incubated overnight at 4 ℃. Following PBST washing steps, corresponding secondary antibodies were added and incubated in the dark at room temperature for 1 hour before being washed three times with PBST. The PVDF membrane containing proteins was placed face up in a gel imaging instrument, where pre-prepared ECL hypersensitive luminescent solution was uniformly applied to the membrane surface. Exposure imaging was performed, pictures were saved, and ImageJ software was used for gray analysis.

Statistical analysis

The statistical analysis was performed using SPSS 26.0 software. Data are mean ± standard deviation (SD) of three biologically independent experiments (each performed in triplicate technical replicates). One-way analysis of variance (ANOVA) was conducted to analyze the differences among multiple groups, and pairwise comparisons were examined using t-tests.

Results

Characterization and detection of ultrasonic microbubbles

Under transmission electron microscopy, the FA-TLUM exhibited a spherical shape with a smooth surface, uniform dispersion, and regular morphology (Figure 1A). The diameter of FA-TLUM was 1.36±0.03 µm, consistent with the particle size measured by hydrodynamic method. It carried a negative charge (Figure 1B). The absorbance of different TPL concentrations was measured in the experiment and showed good linear correlation after linear fitting using Origin software (R²=0.99873) (Figure 1C,1D). TLUM had a drug loading of 11.27%±0.52% and encapsulation efficiency of 78.87%±3.61%, while FA-TLUM had a drug loading of 10.85%±0.48% and encapsulation efficiency of 75.94%±3.36%. After being stored at 4 ℃ for 1 hour, 24 hours, 3 days, 5 days, and 7 days, the morphology of both microbubbles remained unchanged significantly, exhibiting spherical shape with excellent dispersion. The particle size range also showed no significant alteration; however, there was a slight decrease in microbubble concentration with prolonged storage time (Figure 1E).

Figure 1 Characterization and detection of ultrasonic microbubbles. (A) TEM image of FA-TLUM. (B) Hydrated particle size and potential image of FA-TLUM. (C) Ultraviolet absorbance curve of TPL at different wavelengths. (D) Linear fitting function image of TPL ultraviolet absorbance varying with concentration. (E) Observation of microbubble morphology under light microscope: (a,d) after 1 hour storage at 4 ℃; (b,e) after 3 days storage at 4 ℃; (c,f) after 7 days storage at 4 ℃. FA-TLUM, folic acid-TLUM; TEM, transmission electron microscopy; TLUM, triptolide-loaded ultrasound microbubble; TPL, triptolide.

Detection of the targeting ability of FA-TLUM on human breast cancer MCF-7 cells

Under the inverted fluorescence microscope, no green fluorescence was observed surrounding TLUM, whereas evident and uniformly distributed green fluorescence was observed around FA-TLUM (Figure 2A). Subsequent flow cytometry analysis revealed a significant rightward shift in the fluorescence curve following incubation with primary and secondary antibodies compared to pre-incubation levels (Figure 2B). Light microscope observation after co-incubation of microbubbles with MCF-7 cells demonstrated a firm connection between FA-TLUM and MCF-7 cells, predominantly localized around the cells, while minimal or negligible adhesion was observed between TLUM and MCF-7 cells (Figure 2C).

Figure 2 Detection of the targeting ability of FA-TLUM on human breast cancer MCF-7 cells. (A) Observe the connection of folic acid to the surface of (a,b) TLUM and (c,d) FA-TLUM under light microscope (left) and fluorescence microscope (right). (B) Detection of fluorescence intensity of FA-TLUM before and after incubation by flow cytometry. (C) Observation of the binding of human breast cancer MCF-7 cells with (a) TLUM and (b) FA-TLUM under light microscope. FA-TLUM, folic acid-TLUM; TLUM, triptolide-loaded ultrasound microbubble.

Detection of microbubble toxicity in vivo and in vitro

After incubating MCF-7 cells with varying concentrations of TLUM and FA-TLUM for 24 hours, the results of cell viability assessment revealed no statistically significant impact on cell survival by both microbubbles, indicating their excellent safety profile (Figure 3A). Following HE staining of tissue sections, examination of heart, liver, spleen, lung, and kidney in animal tumor models from the control group, targeted group, and non-targeted group demonstrated unaltered histomorphology across all three groups without any signs of acute or chronic inflammation or other pathological changes (Figure 3B), further confirming the favorable safety profile exhibited by these two types of microbubbles.

Figure 3 Detection of microbubble toxicity in vivo and in vitro. (A) Cell viability of MCF-7 cells after treatment with TLUM and FA-TLUM. (B) Hematoxylin and eosin staining of the heart, liver, spleen, lung and kidneys from animal tumor model: (a) control group injected with normal saline; (b) non-targeted group injected with TLUM; (c) targeted group injected with FA-TLUM. FA-TLUM, folic acid-TLUM; TLUM, triptolide-loaded ultrasound microbubble.

Ultrasound imaging of microbubbles in vitro and in vivo

In the in vitro imaging experiment of ultrasonic microbubbles, the control group injected with degassed water exhibited no echo signal, while the experimental groups injected with varying concentrations of FA-TLUM demonstrated detectable echo signals. Moreover, the intensity of these echo signals gradually increased with higher concentration gradients. Notably, a robust echo signal was observed when the concentration of FA-TLUM reached 150 and 200 µg/mL (Figure 4A). The contrast imaging analysis of TLUM and FA-TLUM in breast cancer xenografts in nude mice revealed rapid uptake and clearance within the tumor tissue. The peak contrast intensity for both types of microbubbles occurred around 40 s before rapidly declining. During angiography, the tumor capsule was clearly visualized while some areas of liquefaction necrosis remained unfilled. Statistical analysis indicated that the FA-TLUM group had a higher PI compared to the TLUM group, and also exhibited an earlier TTP (Figure 4B-4D).

Figure 4 Ultrasound imaging of microbubbles in vitro and in vivo. (A) Ultrasound imaging in vitro. The control group was injected with degassed water; Group 1 to Group 4 were injected with FA-TLUM at concentrations of 50, 100, 150, and 200 μg/mL, respectively. (B) Contrast images of microbubbles in vivo: (a,c) imaging at 5 s; (b,d) imaging at 40 s. (C) Peak intensity of contrast imaging. (D) Time to peak of contrast imaging. *, P<0.05; **, P<0.01. FA-TLUM, folic acid-TLUM; TLUM, triptolide-loaded ultrasound microbubble.

Effects of microbubbles combined with ultrasound on proliferation, cycle and apoptosis of breast cancer cells

The MTT assay results demonstrated that various concentrations of TPL effectively suppressed the proliferation of MCF-7 cells, a human breast cancer cell line. Specifically, within a specific concentration range, higher concentrations of TPL exhibited stronger inhibitory effects on MCF-7 cell proliferation. Moreover, prolonged exposure to TPL resulted in more pronounced inhibition. These findings indicate a clear dose-dependent and time-dependent relationship between TPL and its inhibitory effect on MCF-7 cell proliferation (Figure 5A). Additionally, CCK-8 results revealed significant cytotoxicity in the TPL group, TPL + US group, TLUM + US group, and FA-TLUM + US group compared to the control group. Furthermore, flow cytometry analysis showed an increased proportion of cells in S phase and an elevated apoptosis rate in these treatment groups compared to the control group. Notably, among all treatment groups examined, the FA-TLUM + US combination exhibited not only the most potent cytotoxic effect but also had the highest proportion of cells in S phase and apoptosis rate (Figure 5B-5F). Collectively, these results demonstrate that combining FA-TLUM with ultrasound significantly enhances its anti-tumor efficacy.

Figure 5 Effects of microbubbles combined with ultrasound on proliferation, cycle and apoptosis of breast cancer cells. (A) Inhibitory effect of TPL on the proliferation of MCF-7 cells. (B) Toxic effect of each treatment group on MCF-7 cells. (C) Cycle diagram of MCF-7 cells for each treatment group. (D) Distribution of MCF-7 cell cycle in each treatment group. (E) Apoptosis diagram of MCF-7 cells in each treatment group. (F) Apoptosis of MCF-7 cells in each treatment group. *, P<0.05; **, P<0.01. CON, control; FA-TLUM, folic acid-TLUM; OD, optical density; TLUM, triptolide-loaded ultrasound microbubble; TPL, triptolide; US, ultrasound.

The effect of microbubble combined with ultrasound on the mRNA and protein expression of Caspase-3, Bax, Bcl-2, and Survivin in breast cancer cells

After treating MCF-7 cells with various treatment factors and culturing them for 24 hours, the mRNA expression levels of Caspase-3, Bax, Bcl-2, and Survivin were assessed using real-time fluorescence quantitative PCR. The results demonstrated that the FA-TLUM + US group exhibited significantly higher mRNA expression levels of Caspase-3 and Bax compared to the other groups, while the mRNA expression levels of Bcl-2 and Survivin were significantly lower (Figure 6A-6D). Furthermore, Western blot analysis was conducted to determine the protein expression levels of Caspase-3, Bax, Bcl-2, and Survivin. The findings revealed that the FA-TLUM + US group displayed significantly higher protein expression levels of Caspase-3 and Bax in comparison to other groups; however, the protein expression levels of Bcl-2 and Survivin were notably lower (Figure 6E-6J). These results indicate that combining FA-TLUM with ultrasound can effectively enhance apoptosis in breast cancer cells.

Figure 6 The effect of microbubble combined with ultrasound on the mRNA and protein expression of Caspase-3, Bax, Bcl-2 and Survivin in breast cancer cells. (A-D) Relative mRNA expression levels of Caspase-3, Bax, Bcl-2 and Survivin in MCF-7 cells of each treatment group. (E,F) Western blot result image. (G-J) Relative protein expression of Caspase-3, Bax, Bcl-2 and Survivin in MCF-7 cells of each treatment group. *, P<0.05; **, P<0.01. CON, control; FA-TLUM, folic acid-TLUM; TLUM, triptolide-loaded ultrasound microbubble; TPL, triptolide; US, ultrasound.

Discussion

In this study, we loaded TPL within the phospholipid bilayer of liposomes as carriers and prepared ultrasonic microbubbles using perfluoropropane as an agent. This not only enhanced the water solubility of TPL but also enabled real-time monitoring of microbubble distribution and location through second harmonic imaging via ultrasound equipment. The prepared microbubbles exhibited predominantly uniform particle sizes ranging from 1 to 2 µm with good stability and a zeta potential of approximately −16.55 mV, meeting the expected requirements for drug loading efficiency and encapsulation.

In recent years, targeted drug delivery technology has emerged as a prominent area of research. Particularly in the treatment of breast cancer, the overexpression characteristics of FR in malignant tumors have garnered significant attention. Moreover, this receptor is highly conserved in normal tissues and its overexpression often correlates with poor prognosis (26). FA, being a natural small molecular substance, offers advantages such as rapid target reach, excellent penetration ability, minimal immune response and easy modification compared to macromolecular substances. Furthermore, folate exhibits high affinity towards FRs, thereby presenting immense potential for targeted delivery applications (27). Building upon this background information, our study further explores a method to modify FA on microbubble surfaces for precise targeted delivery of breast cancer cells. We successfully confirmed the coupling of folate onto microbubble surfaces through immunofluorescence assay and flow semi-quantitative analysis. Additionally, our in vitro targeting experiments demonstrated the exceptional targeting capability of these FA-modified microbubbles; thus laying a strong foundation for subsequent targeted therapy.

The cytotoxicity test demonstrated the biosafety of both TLUM and FA-TLUM microbubbles, which was further confirmed by the histological examination of tissue sections. During the ultrasound imaging experiment, FA-TLUM exhibited favorable echo signal characteristics, with an increase in signal intensity as its concentration gradually increased. Both FA-TLUM and TLUM demonstrated rapid contrast enhancement and clearance in nude mice. However, compared to TLUM, FA-TLUM displayed a significantly higher PI and an earlier TTP. These findings suggest that FA-TLUM possesses enhanced tumor tissue penetration capability, thereby substantially improving the sensitivity and specificity of ultrasound imaging. These results provide robust experimental evidence for the future development of FR-based targeted delivery systems and ultrasound imaging techniques.

The potential of folate-modified targeted drug-loaded microbubbles combined with ultrasound in the progression of breast cancer cells is a promising area for research. Ultrasound can increase cell membrane permeability and promote drug internalization, leading to higher drug concentrations in target tissues (28,29). Targeted drug-loaded microbubbles modified with FA can accurately localize to breast cancer cells under ultrasound guidance, improving cell membrane permeability, enhancing drug delivery efficiency, and ultimately achieving precise treatment of breast cancer (30). We further investigated the anti-tumor effect of microbubbles combined with ultrasound on MCF-7 cells in vitro. Our results showed that TPL significantly inhibited MCF-7 cell proliferation in a dose-dependent and time-dependent manner, indicating its antitumor activity. Moreover, FA-TLUM + US significantly enhanced cytotoxicity against MCF-7 cells by arresting them in S phase and promoting apoptosis. This finding strongly suggests that targeted drug-loaded microbubbles modified with FA could more efficiently deliver TPL to MCF-7 cells, thereby enhancing its antitumor effect. Notably, there was no significant difference between the apoptosis rate of the TPL group and that of the TPL + US group; thus suggesting that ultrasound must be used together with drug-loaded microbubbles to exert an antitumor effect. This result further confirms that the anti-tumor efficacy of FA-TLUM is mediated by ultrasound-triggered microbubble destruction, a well-established mechanism in ultrasound-mediated drug delivery (31), which enables targeted release of TPL at the tumor site. We speculate that this enhanced efficacy may be attributed to inertial cavitation. This physical phenomenon occurs when microbubbles, upon exposure to an ultrasonic field, undergo rapid collapse. The implosion generates localized mechanical forces, including microjets and shock waves, which can significantly enhance cell membrane permeability and promote the intracellular delivery of the released TPL (32-34).

The innovation of this study lies in the successful combination of FA-modified targeted drug-loaded microbubbles with ultrasound targeted fragmentation technology, which enables precise targeting of breast cancer cells. This innovative approach not only enhances therapeutic efficacy but also minimizes side effects, offering novel ideas and methods for cancer treatment. Despite the remarkable results obtained from this study, there are still several limitations that need to be addressed. Although the targeting and therapeutic effect of FA-TLUM have been demonstrated in this study, further investigation is required to elucidate its mechanism of action. Future studies could focus on exploring the interaction between FA-TLUM and cells as well as investigating the specific effects of ultrasound-targeted fragmentation techniques on drug release and apoptosis.

Conclusions

In summary, this study successfully prepared FA-TLUM and confirmed their excellent morphology, particle size distribution, stability, and targeting ability. The results of in vitro and in vivo cell experiments and animal experiments demonstrated the favorable safety profile of FA-TLUM. Furthermore, the combination of FA-TLUM with ultrasound targeted fragmentation technology exhibited enhanced efficacy in promoting apoptosis of human breast cancer MCF-7 cells, thereby offering novel insights and approaches for targeted therapy against breast cancer.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1374/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1374/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1374/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1374/coif). The authors have no conflicts of interest to declare.

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. All animal experiments were performed under a project license (No. 2024-AE251) granted by the Animal Ethics Committee of The Second Affiliated Hospital of Qiqihar Medical University, in compliance with institutional guidelines for the care and use of laboratory animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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