Mitochondria-Responsive Drug Release along with Heat Shock Mediated by Multifunctional Glycolipid Micelles for Precise Cancer Chemo-Phototherapy

Abstract

Responsive drug release in tumor mitochondria is a pre-requisite for mitochondria-targeted drug delivery systems to improve the efficacy of this promising therapeutic modality. To this end, a photothermal stimulation strategy for mitochondria-responsive drug release along with heat shock is developed to maximize the antitumor effects with minimal side effects. Methods: This strategy relies on mitochondrial-targeted delivery of doxorubicin (DOX) through a photothermal and lipophilic agent IR-780 iodide (IR780)-modified glycolipid conjugates (CSOSA), which can synergistically triggers high-level reactive oxygen species (ROS) to kill tumor cells. Results: Specifically, upon laser irradiation, the photothermal conversion by IR780-CSOSA can not only weaken the hydrophobic interaction between the core of micelles and DOX and trigger unexpected micelle swelling to release DOX in mitochondria for the amplification of ROS, but also induce mitochondria-specific heat shock to promote the fast evolution of ROS at the same locus to eradicate cancer cells in a more effective way. Furthermore, IR780-CSOSA micelles may independently realize the real-time diagnosis and imaging on multiple tumor models. Deep penetration into tumors by IR780-CSOSA/DOX micelles can be manipulated under laser irradiation. Conclusion: Such multifunctional IR780-CSOSA/DOX micelles with integration of mitochondria-responsive drug release and heat shock are demonstrated to be superior to the non-mitochondria-responsive therapy. This study opens up new avenues for the future cancer diagnosis and treatment.

Keywords: chemotherapy; glycolipid micelles; mitochondria-responsive drug release; mitochondrial heat shock; photothermal therapy.

Figure 1

The NIR-triggered drug delivery system with mitochondria-responsive drug release and heat shock capabilities. IR780-CSOSA/DOX micelles selectively targeted into tumor mitochondria and realized photothermal conversion upon NIR-laser irradiation, which photothermally triggered drug release and heat shock in mitochondria, resulting in domino effect on ROS burst for cell apoptosis to perform maximized antitumor efficacy.

Figure 2

Characterization of IR780-CSOSA micelles. (A) Synthetic route of IR780-CSOSA. (B) 1H NMR spectra of IR780, CSOSA and IR780-CSOSA. The characteristic peaks are pointed out and magnified (upper). (C) UV-vis spectra of IR780, CSOSA and IR780-CSOSA solution. (D) Photothermal effect of IR780-CSOSA and free IR780 under laser irradiation (808nm, 1W/cm²) for 5 min. (E) In vitro NIR-triggered drug release profiles. IR780-CSOSA/DOX and CSOSA/DOX solutions after incubated in pH 6.8 PBS for 4 h were irradiated with or without laser (808 nm, 1 W/cm²) for 3 min. (n = 3). (F) Size changes of IR780-CSOSA and IR780-CSOSA/DOX micelles at different temperatures by Zetasizer. (G) Transmission electron microscope (TEM) images of IR780-CSOSA and IR780-CSOSA/DOX micelles at 37 °C and 56 °C.

Figure 3

Cellular uptake and mitochondrial co-localization of IR780-CSOSA micelles in MCF-7 cells. (A) MCF-7 cells were incubated with FITC-labeled CSOSA or IR780-CSOSA micelles (green) for 4 h observed by CLSM. Yellow spots in the merged pictures denoted the co-localization of the micelles within mitochondrial compartments. Red: Mitotracker red. (B) The line scanning profiles of CSOSA micelles and mitochondria in the corresponding confocal images in A. (C) The line scanning profiles of IR780-CSOSA micelles and mitochondria in the corresponding confocal images in A.

Figure 4

NIR-triggered drug release inside mitochondria in MCF-7 cells. (A) MCF-7 cells were treated with CSOSA/DOX or IR780-CSOSA/DOX (equivalent DOX: 1.5 µg/mL) for 4 h. Next, the culture medium were replaced with fresh medium to remove the uninternalized nanoparticles and irradiated with or without laser (808 nm, 1 W/cm²) for 3 min, then continuously incubated for 4 h to observe the NIR-triggered DOX release in mitochondria by CLSM. White dotted lines denoted the corresponding magnified area. Green: Mitotracker green. Red: DOX. Yellow spots in the merged pictures denoted the co-localization of released DOX within mitochondrial compartments. (B) The mean fluorescence intensity (MFI) analysis of released DOX in the corresponding confocal images in Figure S7. (C) The co-localization coefficient of released DOX and mitochondria in the corresponding confocal images in Figure S7.

Figure 5

In vitro chemo-photothermal therapy efficiency. The cytotoxicity of different formulations against MCF-7 cells with or without laser irradiation at different DOX concentrations (A: equivalent DOX: 3 μg/mL, B: equivalent DOX: 0.5 /mL) for 48 h. (n = 3) (C) ROS generation in MCF-7 cells after treatment with different formulations with or without laser irradiation determined by flow cytometry. (D) Quantitative analysis on ROS levels. (E) Expression of HSP70 in MCF-7 cells was tested by western blot (CN: control without laser irradiation; CL: control with laser irradiation for 3min; I0: treatment with IR780-CSOSA/DOX without laser irradiation; I1, I2, and I3 represent treatments with IR780-CSOSA/DOX for 1, 2, and 3 min of laser irradiation, respectively. (F) Expression of apoptosis related proteins in MCF-7 cells treated with different formulations under laser irradiation. 0: control, 1: DOX, 2: CSOSA/DOX, 3: IR780-CSOSA, 4: IR780-CSOSA/DOX. Cytochrome c, cleaved caspase-3 and cleaved caspase-9 were tested by western blot. The conditions of laser irradiation are all at 808 nm with 1 W/cm² for 3 min. ***P < 0.001, **P < 0.01.

Figure 6

In vivo fluorescent and photothermal imaging. (A) In vivo NIR fluorescence imaging in MCF-7 or 4T1 or H22 tumor models of IR780-CSOSA micelles after intravenous injection for 4, 12, 24 and 48 h., respectively. (B) Infrared thermal images of MCF-7 tumor-bearing mice intravenously injected with saline, CSOSA/DOX, IR780-CSOSA/DOX during 5 min of laser irradiation (808 nm, 0.5 W/cm²), respectively.

Figure 7

The evaluation on chemo-photothermal therapy of IR780-CSOSA/DOX micelles in vivo. (A) In vitro penetration of CSOSA/DOX and IR780-CSOSA/DOX micelles into the MCTS irradiated with or without laser (808 nm, 1 W/cm², 3 min). Z-stack images using CLSM were obtained from the top to the equatorial plane of MCTS. Scale bar: 100 μm. (B) In vivo penetration of micelles into the tumors of MCF-7 tumor-bearing nude mice. The frozen tumor sections were observed using CLSM. Scale bar: 20 μm. (C) Tumor volumes of MCF-7 tumor-xenografted mice in different groups with or without laser irradiation (808 nm, 0.5 W/cm², 3 min) (equivalent DOX dosage: 2 mg/kg). (mean ± SD, n = 6). (D) Body weight variation. (mean ± SD, n = 6). (E) Survival rates.

Figure 8

In vivo immunohistochemical analysis in MCF-7 xenograft tumors treated with IR780-CSOSA/DOX micelles-mediated chemo-photothermal therapy. (A) HSP70 protein expression (brown) in saline and IR780-CSOSA/DOX with or without laser groups. Scale bar: 100 μm. (B) The average OD value of HSP70 levels. (n = 3). (C) Under laser irradiation, the induction of apoptosis by staining with cleaved caspase-3 antibody (brown). Cell proliferation evaluation by Ki67 staining (brown). Tumor blood vessel staining with CD 31 antibody (brown). CD 8⁺ T cells detected by immunofluorescent staining (green). Scale bar: 100 μm. (D) Under laser irradiation, the quantitative analysis of cleaved caspase-3, Ki67, CD31 and CD8 levels. (n = 3). The conditions of laser irradiation are all at 808 nm with 0.5 W/cm² for 3 min. ***P < 0.001, *P < 0.05