Journal of Carcinogenesis & Mutagenesis

Growth Suppression of Triple-Negative Breast Cancer Cells by the Photodynamic Therapy Effect of Hybrid Liposomes Containing Indocyanine Green

Masaki Okumura, Hinano Otsuka, Junna Takai, Koichi Goto, Yoko Matsumoto and Hideaki Ichihara^* Department of Applied Life Sciences, Sojo University, Kumamoto, Japan
^*Correspondence: Hideaki Ichihara, Department of Applied Life Sciences, Sojo University, Kumamoto, Japan, Email:

Received: 25-Apr-2025, Manuscript No. JCM-25-28789; Editor assigned: 28-Apr-2025, Pre QC No. JCM-25-28789 (PQ); Reviewed: 12-May-2025, QC No. JCM-25-28789; Revised: 19-May-2025, Manuscript No. JCM-25-28789 (R); Published: 26-May-2025, DOI: 10.35248/2157-2518.25.16.471

Keywords

Hybrid liposome; Growth suppression; Photodynamic therapy; Breast cancer; in vitro

Introduction

Breast cancer has a high incidence of cancer in women by site, and the number of deaths has continued to increase in recent years [1]. Chemotherapy is used to treat cancer, but many anticancer drugs are known to affect normal cells and cause serious side effects. Therefore, the development of anticancer drugs without side effects is an important issue. Focusing particularly on breast cancer for which surgical treatment is preferred, we focused on Photodynamic Therapy (PDT), a method of treating disease using active oxygen generated by irradiating photosensitive materials with excitation light [2-4].

Hybrid Liposomes (HL) can be prepared solely by sonicating vesicular and micellar molecules in a buffer solution [5-6]. HL, composed of L-α-Dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (n) dodecyl ether (C₁₂(EO)_n, n=21-25), has been reported to exhibit strong inhibitory effects on the growth of various cancer cells in vitro [7-12], in vivo [13-23], and in clinical trials [24], as well as to induce apoptosis. Specifically, HL fuses with and accumulates in the membranes of cancer cells, inducing apoptosis via mitochondrial events and caspase activation [9-10].

In this study, HL (HL/ICG) containing Indocyanine Green (ICG), a photosensitive substance, was newly created, and its therapeutic effects on mouse breast cancer (4T1-Luc) cells [25-27], a triple-negative breast cancer cell line, were investigated in vitro for application to Photodynamic Therapy (PDT).

Materials and Methods

Preparation of HL/ICG

Hybrid liposomes (90 mol% DMPC/10 mol% C₁₂(EO)₂₅: HL) were prepared by weighing phospholipid (DMPC) and surfactant (C₁₂(EO)₂₅) at a ratio of 9:1, irradiating them with ultrasonic waves (1min/mL, 45°C) in a 5% glucose solution using an ultrasonic cleaner (ASU-10, AS ONE, 240W) to obtain a uniform and stable membrane, and sterilizing the mixture by filtration through a 0.20μm filter. Storage was carried out at room temperature (25°C).

Hybrid liposomes containing indocyanine green (89 mol% DMPC/10 mol% C₁₂(EO)₂₅/1 mol% ICG: HL/ICG) were prepared in the same manner as HL by weighing phospholipids, surfactants, and ICG. Storage was carried out at 4°C in consideration of fading of ICG.

Measurement of membrane size

The membrane diameters of HL and HL/ICG were measured by the dynamic light scattering method using a light scattering photometer. A 633 nm oscillation line of a He-Ne laser was used as a light source at an output power of 10mW, and measurements were performed at a scattering angle of 165°. The resulting Diffusion coefficient (D) was substituted into the Stokes-Einstein equation (1) to obtain the membrane diameter (d_hy).

d_hy = κT / (3πηD) • • • • (Equation 1)

Where κ is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent.

Cell culture

The cells used in this study were 4T1-Luc cells (RIKEN, Ibaraki, Japan), a mouse triple-negative breast cancer cell line expressing luciferase [25-27]. The cells were cultured in complete medium consisting of RPMI-1640 (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Science Production, Sandy, UK). Cultures were maintained at 37°C in a humidified incubator with 5% CO₂ and 95% humidity.

Cell viability measurement after PDT treatment

4T1-Luc cells were seeded (5.0 × 10⁴ cells/mL) in 96-well plates, and HL/ICG was added after 24 hours of preculture. Measurements were performed using the WST-8 assay ([2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]; Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). Irradiation (100mW/cm², 1min, 3min) was applied 24 hours after HL/ICG addition. WST-8 solution was added 24 hours after irradiation and absorbance was measured 3 hours later. The viability was determined from the ratio of absorbance (A_mean/A_Control) at the time of sample addition (A_mean) to absorbance of control (A_Control).

Viability (%) = (A_mean / A_Control) × 100 • • • (Equation 2)

Observation of HL/ICG accumulation in 4T1-Luc cells

4T1-Luc cells were seeded in 60mm dishes (1.0 × 10⁵ cells/mL) and pre-cultured for 24 hours. Subsequently, HL/ICG was added to a final concentration of 100μM (HL: IC₅₀ = 159μM), and D-luciferin potassium salt (Bio Medical Science Inc., Tokyo, Japan) adjusted to 50 mM with PBS (-) was added to a final concentration of 200μM, and the culture was carried out for 48 hours. Observations were performed 48 hours later using fluorescence microscopy (DAPI/Ex: 345nm, Em: 455nm, CY7/Ex: 710nm, Em: 775nm, EVOS fl, Life Technologies, CA. USA).

Imaging Analysis of ROS generated by PDT of HL/ICG in 4T1-Luc cells

4T1-Luc cells were seeded (1.0 × 10⁵ cells/mL) in 35mm Glass Bottom dishes and, after 24 hours of pre-culture, HL/ICG (150μM) was added. Cell ROX Green (1μM, Invitrogen, MA, USA) was added 24 hours after addition, and irradiation (1 min, 3 min) was performed 45 minutes later using an excitation light source. After irradiation, they were fixed in 10% neutral buffered formalin solution, and 20 minutes later, they were washed 2 times with PBS (-). For the negative control, NAC (200μM, N-acetylcysteine, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) was added, TBHP (200μM, Tert-butyl hydroperoxide, Sigma Aldrich, MA, USA) was added after 1 hour, and Cell ROX Green was added after 30 minutes. They were fixed in 10% neutral buffered formalin solution and washed 2 times with PBS (-) after 20 minutes. Reactive oxygen was observed using a confocal microscope. For the positive control, TBHP (200μM) was added, followed 30 minutes later by the addition of Cell ROX Green. After 20 minutes, the cells were fixed in 10% neutral buffered formalin and washed twice with PBS (-). ROS generation were visualized using a confocal laser scanning microscope (TCS-SP; Leica Microsystems, Berlin, Germany).

Measurement of ROS generated by PDT with HL/ICG in 4T1-Luc cells using flow cytometry

4T1-Luc cells were seeded (1.0 × 10⁵ cells/mL) in 60 mm dishes and, after 24 hours of pre-culture, HL/ICG (150μM) was added. After 24 hours, Cell ROX Green (1μM) was added and 1 hour later irradiation (1min, 3min) was performed using an excitation light source. After irradiation, they were washed with PBS (-), fixed with 10% neutral buffered formalin solution, and washed with PBS (-) 2 times after 20 minutes. NAC (50μM) was added to the Negative Control, TBHP (1000μM) was added to both the Positive Control and the Negative Control after incubation for 1 hour, and Cell ROX Green (100μM) was added after 30 minutes. After 1 hour, they were washed with PBS (-), fixed with 10% neutral buffered formalin solution, and washed with PBS (-) twice after 20 minutes. Active oxygen was measured using a flow cytometer (CytoFLEX, Beckman Coulter, CA, USA).

Statistical analysis

Data were analyzed using a Student’s t-test. All results are presented as the mean ± Standard Error (SE). A p-value of less than 0.05 was considered statistically significant.

Results and Discussion

Physical properties in membrane of HL/ICG

The membrane diameter (d_hy) of HL/ICG and DMPC/ICG was measured by the dynamic light scattering method. The results are shown in Figure 1. The membrane diameter immediately after preparation of HL was about 40 nm, and the film diameter of about 50 nm or less was stably maintained for 1 month from the first day after preparation. The membrane diameter of HL/ICG was 13.7 nm immediately after preparation, and the film diameter of about 10 nm was maintained for 1 month thereafter. The membrane diameter of DMPC/ICG was 252 nm immediately after preparation, and the film diameter of about 200 nm was maintained for 1 month thereafter. These results indicate that HL/ICG can form a film of 100 nm or less for a long time which can avoid the Reticular Endothelial System (RES) [28-31].

Figure 1: Time course of dhy change for HL, DMPC/ICG and HL/ICG in 5% glucose solution at 25°C. Data represent the mean ±S.E. DMPC=30mM.

Photodynamic anti-cancer effect of HL/ICG on 4T1-Luc cells

We investigated the inhibitory effect of HL/ICG on the proliferation of 4T1-Luc cells via Photodynamic Therapy (PDT) using a high-power laser module. The results are shown in Figure 2. The survival rates of HL at 150μM were 56.9%, 56.4%, and 55.6% under non-irradiated conditions, after 1 min, and after 3 min of irradiation, respectively. The survival rates of HL/ICG were 55.8% without irradiation, 49.4% after 1 min, and 43.9% after 3 min. In the case of HL/ICG, the survival rate decreased as the irradiation time increased from 1 min to 3 min. In contrast, the survival rate of HL remained unchanged with irradiation. These results indicate that HL/ICG exerts an inhibitory effect on proliferation through PDT.

Figure 2: Viability of 4T1-Luc cells irradiated with an 808nm laser after adding HL/ICG for 24 hours. Data represent the mean ± S.E DMPC=150μM. Note: *p<0.05(vs. nonirradiated HL/ICG), **p<0.05 (vs. 1-min irradiated HL/ICG)

Accumulation of HL/ICG in 4T1-Luc cells

HL/ICG (100μM) was added to 4T1-Luc cells seeded (1.0 × 10⁵ cells/mL) in 60 mm dish and cultured for 24 and 48 hours. The cells were then washed with PBS (-), followed by addition of D-luciferin potassium salt (200μM), and the fluorescence accumulation of HL/ICG was confirmed using an optical microscope. Results are shown in Figure 3. HL/ICG was observed using CY7 (Ex: 710 nm, Em: 775 nm) wavelength. The fluorescence was stronger in the 48-hour incubation than in the 24-hour incubation. The observation using D-luciferin potassium salt at the DAPI (Ex: 345 nm, Em: 445 nm) wavelength showed that cells incubated for 24 and 48 hours emitted light. These results indicate that HL/ICG accumulates in 4T1-Luc cells because the emission sites of D-luciferin potassium salt and the fluorescent sites of HL/ICG appear to overlap.

Figure 3: Fluorescence micrographs of breast cancer (4T1- Luc) cells after the treatment with D-Luciferin potassium salt or HL/ICG for 24 and 48 hours. Scale bar: 400μm

Observation of ROS generation by HL/ICG in 4T1-Luc cells with PDT using confocal laser microscopy

The generation of Reactive Oxygen Species (ROS) associated with PDT of HL/ICG on 4T1-Luc cells in vitro was observed using confocal laser microscopy. The results are shown in Figure 4. Cell ROX Green was used as an active oxygen detection agent. Strong fluorescence of Cell ROX Green was observed by adding HL/ICG and irradiating with excitation light for 3 min. On the other hand, Cell ROX Green fluorescence was not observed in Control and ICG alone, and Cell ROX Green fluorescence was observed in HL and HL/ICG. In HL, there was no difference in the fluorescence of Cell ROX Green with or without irradiation with excitation light. These results indicate that active oxygen is generated by irradiating HL/ICG accumulated in 4T1-Luc cells with excitation light.

Figure 4: Fluorescence micrographs of breast cancer (4T1- Luc) cells irradiated with near-infrared using Cell ROX Green after the treatment with HL/ICG. Scale bar: 50μm.

Increase of ROS content in PDT against 4T1-Luc cells by HL/ICG in vitro

The generation of ROS associated with PDT of HL/ICG to 4T1-Luc cells in vitro was measured using a flow cytometer. The results are shown in Figure 5. Cell ROX Green was used as the ROS detection agent. The fluorescence of Cell ROX Green increased when 4T1-Luc cells treated with HL/ICG were irradiated with excitation light for 3 min. In addition, there was no difference in the fluorescence of Cell ROX Green in the presence of Control or HL, while the fluorescence of ICG alone increased with irradiation time. These results indicate that the amount of ROS in 4T1-Luc cells increases with the addition of HL/ICG and the time of irradiation with excitation light.

Figure 5: Fluorescence intensity of breast cancer (4T1-Luc) cells irradiated with near-infrared using Cell ROX Green after the treatment with HL/ICG for 24 hours. Data represent the mean±S.E. a:p<0.05 (vs. Control 1 min), b:p<0.05 (vs. Control 3 min), c:p<0.05 (vs. ICG 3 min), d:p<0.05 (vs. HL 3 min).

Conclusion

We demonstrated for the first time that Photodynamic Therapy (PDT) with HL/ICG effectively suppresses the growth of triplenegative breast cancer 4T1-Luc cells in vitro. A clear HL and HL/ ICG solution with a hydrodynamic diameter of 100 nm was maintained for 4 weeks. A time-dependent growth inhibitory effect of HL/ICG on 4T1-Luc cells was observed during PDT. The co-localization of luciferin luminescence and HL/ICG fluorescence indicated that HL/ICG accumulates in 4T1-Luc cells. Flow cytometric and confocal laser microscopic analysis revealed that ROS are generated during PDT of 4T1-Luc cells with HL/ICG, suggesting that HL/ICG-mediated PDT could serve as a potential therapeutic strategy for breast cancer in the future.

Conflict of Interest

No potential conflict of interest was reported by the authors.

Acknowledgements

We would like to thank Editage (www.editage.jp) for English language editing.

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