DOX/IR780-Loaded Liposomes For Chemo-Photothermal Therapy In Breast Cancer

BackgroundBreast cancer is one of the most common malignancies in women, breast cancer therapies are mostly focused on surgery, radiotherapy and chemotherapy. Hyperthermia is a form of medical treatment in which heat is used to treat diseases such as cancers and peripheral infections to normalize the body and relieve the depression. During the local heating or hyperthermia, cells undergo irreversible damage due to the denaturation of proteins and the disruption of the cell membrane. Photothermal therapy (PTT) is an attractive technique for solid tumor ablation, by which light energy absorbed by near-infrared (NIR) photothermal agents can convert into thermal energy and produce local hyperthermia, ultimately leading to irreversible cell injury (tumor apoptosis and coagulative necrosis). NIR laser-induced photothermal therapy (PTT) offers several obvious advantages, including the minimized invasiveness, increased preservation of surrounding tissues, the short recovery times and the reduced complication rates, as well as intra-procedural monitoring by visualization However, the nonuniform heat distribution over tumors makes it insufficient to kill all tumor cells, resulting in incomplete ablation, tumor recurrence and inferior outcomes. Therefore, the combined treatments are required to further improve the therapeutic efficacy from NIR-PTT.Here we report a DOX/IR-780-loaded temperature-sensitive-liposome (DITSL) which can achieve NIR-laser-controlled drug release for chemo-photothermal synergistic tumor therapy. In this system, the liposoluble IR-780 was incorporated into the temperature-sensitive lipid bilayer and the soluble chemotherapeutic doxorubicin (DOX) was encapsulated in the hydrophilic core. The resulting DITSL is proved to be physiologically stable and can provide a fast and laser irradiation-controllable DOX release in the PBS and cellular conditions. We further employed this nanoparticle for tumor treatment, demonstrating significantly higher tumor inhibition efficacy than that of DOX-loaded temperature-sensitive-liposome (DTSL) or IR780-loaded temperature-sensitive-liposome (ITSL) in the in vitro cells and in vivo animals. Our study provides a promising strategy to realize chemo-photothermal synergistic combination therapy for breast tumors.Part One Preparation and characterization of DOX/IR-780-loaded tempera ture-sensitive-liposomeObjective To prepare and characterize DOX/IR-780-loaded temperature-sensitive-liposomes and explore the feasibility of DITSL for chemo-photothermal synergistic tumor therapy.Materials and methodsExperimental materials:1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethy lene glycol)-2000] (DSPE-PEG2000); Doxorubicin; IR-780; Cell Counting Kit-8 (CCK-8); Annex in V-FITC Apoptosis Kit; Calcein-AM/PI Double Stain Kit; Fetal bovine serum (FBS); Trypsin EDTA solution; Dulbecco’s Modified Eagle’s Medium (DMEM)Experiment instruments and equipments:Laica TCS SP5 laser confocal microscopy, BD FACSCantoII flow cytometry, laser device, SynergyTM4 multimode plate reader, Thermostatically controlled dry bath; Malvern Zetasizer; Infrared imaging camera. Methods:Temperature-sensitive-liposomes (TSL) were prepared by the established thin film hydration method. Size, surface charge and size distribution of all liposomes were determined using a dynamic light scattering detector Malvern Zetasizer (Nano ZS, Malvern, USA) at 25℃. The absorption spectra of DTSL, ITSL, and DITSL were obtained using Lambda25 UV/vis spectrometer. Temperature-dependent release and laser-triggered drug release profile of DOX from DITSL was detected by fluorometry.Results1. DITSL were prepared by the thin film hydration method. Due to the difference ofsolubility, the hydrophobic IR-780 would be encapsulated into the lipid bilayer of DITSL and the hydrophilic DOX would exist in the core of temperature-sensitive-liposomes (TSL)2. The average particle size of DITSL was approximately 138.98± 8.74 nm, with a polydispersity index (PDI) of0.32± 0.02. ITSL, DTSL and empty liposomes had smaller particle size, with 94.05± 1.74 nm,84.55± 7.41 nm and 77.53± 4.44, respectively. The zeta potential of these particles appeared negative.The encapsulation efficiencies of all liposomes were over 90%. The absorption curve of DITSL appeared two obvious peaks. The onewas located at 793 nm which belongs to the characteristic absorption of IR-780 and another at 492 nm which belongs to the absorption of DOX.3. With the laser irradiation at 0.8 W/cm2 for 5 min, the temperature of DITSL and ITSL increased quickly, while the PBS and DTSL only increased to 9.3℃ and 9.7℃.4. DITSL displayed a clear temperature-dependent DOX release profile upon exposure to hyperthermia (42℃ or 50℃) but not to body temperature (37℃). DITSL without laser irradiation showed slow drug release. The rate of drug release from DITSL increases as the laser irradiation time, In contrast, DTSL without IR-780 did not show NIR-laser-induced drug release.Conclusion DITSL displayed a clear temperature-dependent DOX release profile upon exposure to hyperthermia; also, DITSL can present a completely controlled drug release platform laser irradiation.Part Two In vitro Inhibition Effect of Chemo-photothermal TherapyObjective To study the inhibition effect of chemo-photothermal therapy with DOX/IR-780-loaded temperature-sensitive-liposomes on 4T1 cells.Materials and methodsExperimental materials:DPPC; MPPC; DSPE-PEG2000; Doxorubicin; IR-780; DAPI; Fetal bovine serum (FBS); Trypsin EDTA solution; Dulbecco’s Modified Eagle’s Medium (DMEM); Cell Counting Kit-8 (CCK-8); Calcein-AM/PI Double Stain Kit.Experiment instruments and equipments:Leica TCS SP5 laser confocal microscopy; laser device; SynergyTM4 multimode plate reader.MethodsTo test 4T1 tumor cells uptake the IR-780 iodide at different concentrations of DITSL for different incubation time, flow cytometry experiments were conducted, and subcellular localization of drugs in 4T1 cells laser scanning was analyzed by confocal laser scanning microscope (CLSM). To test the effect of chemo-photothermal synergistic tumor therapy on viability of tumor cells through CCK-8 testing, different laser intensities (0.6 W/cm2; 0.8 W/cm2) were used. To visually observe the photothermal or chemo-photothermal therapeutic efficacy, the 4T1 cells were treated and then stained with Calcein-AM/PI double staining Kit, followed by observation under microscope.Results1. Results were demonstrated that there were dose-and time-dependence for cellular uptake. CLSM images showed that DOX mainly localized in the cell nucleus in 4T1 cells, while IR-780 was homogenously distributed in the cytoplasm in 4T1 cells.2. TSL carriers and ITSL had no cytotoxicity to the tumor cells when compared with PBS control. Chemotherapy with DTSL showed an obvious cytotoxicity, with 59.4% cell viability when these cells received or did not received with laser irradiation. PPT with ITSL under a 0.6 W/cm2 power density laser showed significant cytotoxicity with 43.6% cell viability, much lower than that of the cells incubated with the same ITSL dose without laser irradiation. Notably, the treatment with DITSL and laser irradiation induced the strongest cytotoxicity, with significantly lower viability than that of the cells received with DL, ITSL+laser irradiation or DITSL without laser irradiation.3. The results demonstrated that the treatment with DITSL and laser irradiation induced significant tumor cell apoptosis/necrosis, which is in accordance with the results of cell viability.Conclusion DITSL can be efficiently uptaken by 4T1 cells. DITSL plus laser irradiation could inhibit the activity of 4T1 cells and induce tumor cells apoptosis/necrosis.Part three Inhibition Effect of Chemo-photothermal Therapy on 4T1 Transplantation TumorObjective To study inhibition effect of chemo-photothermal therapy with DOX/IR-780-loaded temperature-sensitive-liposomes on 4T1 transplantation tumor and further explore its mechanisms.Materials and methodsExperimental materials:DPPC; MPPC; DSPE-PEG2000; Doxorubicin; IR-780; Fetal bovine serum (FBS); Trypsin EDTA solution; Dulbecco’s Modified Eagle’s Medium (DMEM); rat anti-mouse CD31 antibody; in situ Cell Death Detection Kit (TUNEL); DAPI; DAB kit;H&E solution. Female BALB/c mice, weighing about 20 g (6-8 weeks old), were obtained from Guangdong Medical Experimental Animal Center (Guangzhou, China).Experiment instruments and equipments:Leica TCS SP5 laser confocal microscopy; Infrared imaging camera; Laser device; Philip IU22 ultrasound machine. The Paraffin Section MachineMethods:Mammary carcinoma 4T1 cells were injected in the subcutaneous abdomen of Balb/c mice to establish 4T1 tumor-bearing mice models, and the mice were randomly divided into four groups:(1) Control group (0.1 ml saline was injected); (2) Laser group (irradiated by laser for 5 min,0.8W/cm2); (3) Chemotherapy group (20μg DTSL was injected); (4) ITSL group (injected 20μg ITSL, then irradiated by laser for 5 min,0.8W/cm2); (5) PTT group (injected 20μg ITSL, then irradiated by laser for 5 min,0.8W/cm2); (6) DITSL group (injected 20μg ITSL); (7) chemo-photothermal therapy group (injected 20μg DITSL, then irradiated by laser for 5 min,0.8W/cm2). To explore the inhibition effects of DITSL, changes of the tumor volume and weight were observed after laser irradiation. Post-treatment survival situation of mice was also recorded. Tumor tissues were stained by H&E to observe histomorphology and TUNEL experiment were conducted to detect the tissue apoptosis.Mice bearing tumor underwent contrast-enhanced ultrasound after chemo-photothermal treatments. B-mode ultrasound was further used to analyze the tumor volume and echo signal. CD31 staining was used to observe the structure and quantities of tumor vessel.Resluts1.48 hours after treatments, a significantly increased degree of cell necrosis could be observed in the tumors receiving DITSL and laser irradiation. TUNEL-positive cells were significantly more in the tumors treated with chemo-photothermal therapy, compared with other groups.2. For long-term therapy in vivo, mice received with DTSL and ITSL plus laser irradiation showed a tumor growth inhibition, whereas the chemo-photothermal therapy group showed the strongest inhibition of tumor (P<0.01). Survival rate of the tumor-bearing mice after chemo-photothermal therapy is 100%. By contrast, there were no mice survived 30days after injected PBS.3. CEUS showed decreased blood perfusion in tumors receiving DITSL plus laser irradiation. Correspondingly, the reduction of tumor volume and ultrasonic echo signals were also observed by B-mode ultrasound.4. Immunofluorescence staining results revealed CD31-positive cells was significantly decreased after combination therapy, which were correlated with ultrasonographic examinations.Conclusion Chemo-photothermal therapy showed strong anti-tumor efficacy in vivo, manifested as the reduction of tumor volume, which are confirmed by the traditional caliper measurements and tissue staining.CEUS can be used to evaluate the efficacy of chemo-photothermal therapy based on DOX/IR-780-loaded temperature-sensitive-liposomes for breast cancer according to the blood perfusion.