NIR-Laser-Controlled Drug Release from DOX/IR-780-Loaded Temperature-Sensitive-Liposomes for Chemo-Photothermal Synergistic Tumor Therapy

Abstract

NIR laser-induced photothermal therapy (PTT) through near-infrared agents has demonstrated the great potential in solid tumor ablation. However, the nonuniform heat distribution over tumors from PTT makes it insufficient to kill all tumor cells, resulting in tumor recurrence and inferior outcomes. To improve the tumor treatment efficacy, it is highly desirable to develop the combinational treatment of PTT with other modalities, especially with chemotherapeutic agents. Here we report a smart 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. Histological analysis further revealed much more apoptotic cells, confirming the advantageous anti-tumor effect of DITSL over DTSL or ITSL. Our study provides a promising strategy to realize chemo-photothermal synergistic combination therapy for breast tumors.

Keywords: Breast tumors.; Doxorubicin; IR-780; Photothermal therapy; Temperature-sensitive-liposome.

Figure 1

Synthesis and characterization of DITSL. (A) Schematic diagram of preparation of DITSL. (B) The size distribution of DITSL by dynamic light scattering. Inset shows a photograph of ITSL (green), DTSL (red), DITSL (brown) solution. (C) UV-vis absorbance spectra of the dispersions containing DTSL (red), ITSL (green) or DITSL (brown). (D) The short-term drug release effects of DITSL incubated at 37 °C in PBS buffer (pH 7.4) or in the presence of 30%, 50% or 90% fetal calf serum. Data points are an average of three replicates.

Figure 2

The photothermal effects of DTSL, ITSL or DITSL and heating-induced DOX release from DITSL. (A) The temperature profiles of PBS, DTSL, ITSL and DITSL dispersions under the irradiation of 808 nm laser. (B) The peak temperature maps were recorded with an infrared thermal imaging camera. (C) Schematic diagram of DOX release from DITSL under NIR-laser irradiation. The liposome membrane temperature would increase when the NIR-laser irradiation was applied. Destruction of liposome membrane occurs when the liposome membrane temperature achieves 42 °C. (D) The cumulative release of DOX from DITSL at 37 °C, 42 °C or 50 °C. (E)The cumulative release of DOX from DTSL or DITSL under the NIR-laser irradiation at a power intensity of 0.8 W/cm² for different time (0-5min). The cumulative release of DOX from DITSL without irradiation was also examined as a control. (F) The effects of irradiation-nonirradiation sequence number of 0 time, 1 time, 2 times or 3 times on the released DOX from DITSL with 808 nm laser (0.8 W/cm², 2-min). Data represents the mean ± standard deviation of triplicate measurements.

Figure 3

The in vitro cellular uptake. (A) Confocal fluorescence images displayed cellular localization of DOX and IR-780 after 3 h incubation with DTSL, ITSL or DITSL. Blue represented the fluorescence of DAPI, green represented the fluorescence of DOX and red represented the fluorescence of IR-780 (Scale bar, 25 μm).

Figure 4

NIR laser-induced drug release from DITSL in 4T1 cells. (A) CLSM images of 4T1 cells which contained DITSL before and after laser irradiation treatment or incubation with 42 °C or 37 °C pre-warmed PBS for 5 min. Nuclei were stained with DAPI (blue), and green was the fluorescence of DOX. (B) Quantitative analysis of fluorescence intensities of DOX before and after treatments. Scale bar = 20 μm.

Figure 5

Cell viability after chemo-photothermal treatment. (A) Quantitative evaluation of cell survivals incubated with PBS, empty liposomes, DTSL, ITSL and DITSL with or without laser irradiation (0.6 W/cm², 5 min), showing much lower cell viability in the cells receiving DITSL plus laser irradiation. (B) Increasing the laser intensity (0.8 W/cm², 5 min) resulted in significantly lower cell viability in these cells incubated with ITSL or DITSL. (C) Fluorescence images of 4T1 cells after chemo-photothermal treatment. Viable cells were stained green with calcein-AM, and dead/later apoptosis cells were stained red with PI. Scale bar = 25 μm. The data was shown as mean ± SD; (*) P < 0.05, (**) P < 0.01.

Figure 6

The anti-tumor efficacy in vivo. (A) The temperature change profiles of the irradiated tumors received with PBS, ITSL or DITSL from 0-5 min. (B) The representative infrared photothermal images of the tumors after laser irradiation. (C) The tumor growth curves of mice receiving PBS, only laser irradiation, DTSL, ITSL, DITSL without laser irradiation, ITSL+ laser or DITSL+ laser. Combined treatment of DITSL+ laser produced better synergistic anti-tumor effect and no tumor recurrence was noted. (D) Survival curve of the tumor-bearing mice after different treatments. (E) Representative images of these tumors after treatments.

Figure 7

H&E and TUNEL staining. H&E staining and TUNEL staining of non-treated or treated tumors after 48 h. The apoptotic cells were stained in brown by TUNEL assay. Scale bar = 25 μm.

Figure 8

In vivo tumor blood vessels detection. (A) In vivo contrast-enhanced ultrasound images of these tumors treated with PBS, only laser irradiation, DTSL, ITSL, DITSL without laser irradiation, ITSL+ laser or DITSL+ laser. The circles were region of interest for these treated tumors. Scale bar = 0.5 cm. (B) Immunofluorescence staining of CD31 proteins in these tumor sections at 48 h after treatments. CD31-positive cells were stained green. Scale bar = 250 μm.