Mitochondrion-targeted supramolecular “nano-boat” simultaneously inhibiting dual energy metabolism for tumor selective and synergistic chemo-radiotherapy

Rationale: Tumor energy metabolism has been a well-appreciated target of cancer therapy; however, the metabolism change of cancer cells between oxidative phosphorylation and glycolysis poses a challenge to the above. In this study, we constructed an innovative mitochondrion-targeted supramolecular “nano-boat” based on peptide self-assembly for tumor combined chemo-radiotherapy by simultaneously inhibiting the dual energy metabolism. Methods: A lipophilic self-assembled peptide and a positively charged cyclen were integrated to fabricate a brand new mitochondrion-targeted nano-platform for the first time. The indices of mitochondrial dysfunction including mitochondrial membrane potential, apoptosis proteins expression and ultrastructure change were evaluated using a JC-1 probe, western blotting and biological transmission electron microscopy, respectively. Energy metabolism assays were conducted on a Seahorse XF24 system by detecting the oxygen consumption rate and the glycolytic proton efflux rate. The radio-sensitization effect was investigated by colony formation, the comet assay, and γ-H2AX staining. Results: The supramolecular “nano-boat” could selectively kill cancer cells by much higher enrichment and reactive oxygen species generation than those in normal cells. In the cancer cells treated with the supramolecular “nano-boat”, the dysfunctional morphological changes of the mitochondrial ultrastructure including swelling and pyknosis were evidently observed, and the endogenous mitochondrial apoptosis pathway was effectively triggered by abundant of cytochrome C leaking out. Concurrently, the dual metabolic pathways of glycolysis and oxidative phosphorylation were severely inhibited. More importantly, the supramolecular “nano-boat” displayed an excellent radio-sensitization effect with a sensitization enhancement ratio value as high as 2.58, and hence, in vivo efficiently combining radiotherapy yielded an enhanced chemo-radiotherapy effect. Conclusion: Our study demonstrated that the rationally designed peptide-based “nano-boat” could efficiently induce cancer cell apoptosis by the energy metabolism inhibition involving multiple pathways, which may provide the motivation for designing novel and universal mitochondria-targeted drug delivery systems for cancer therapy.

(iii) Synthesis of compound 3. Compound 2 (14.2 g, 26.1 mmol) was dissolved in 30 mL THF, then 30 mL 2N aqueous sodium hydroxide was added. The resulting mixture was stirred at 60 ℃ overnight, and then was adjusted pH to 6 using 1 N hydrochloric acid after THF was removed under reduced pressure. The resulting reaction mixture was extracted 3 times with ethyl acetate to obtain the organic layer, which was washed with saturated NaCl solution, dried with anhydrous Na 2 SO 4 , concentrated under vacuum. The crude product was dissolved in 50 mL DMF, then HBTU (7.7 g, 20.2 mmol), N-Hydroxysuccinimide (2.3 g, 20.2 mmol) were added, stirred overnight at room temperature.  Figure S1 Synthesis routes of the active ester of cyclen derivatives.

Synthesis of LND-GFFYK-cyclen (LND-Pep-cyclen)
The synthesis routes of LND-Pep-cyclen was shown in Figure S2. Firstly, LND-Pep was prepared by standard solid phase peptide synthesis (SPPS) using the corresponding N-Fmoc protected amino acids with side chains properly protected and 2-chlorotrityl chloride resin. LND was regarded as an amino acid as well, and was directly added into the solid phase reaction. During the condensation reaction, HBTU and DIEA were used as coupling agent and catalyst, respectively. The final product of LND-GFFYK (LND-Pep) was cleaved from the resin using a mixture of TFA (19 mL), TIS (0.5 mL), and H 2 O (0.5 mL) for 30 min. The reaction mixture was concentrated under reduced pressure, followed by adding diethylether to obtain the precipitate.
After drying, the crude product was used for next step without further purification.
LND-Pep (200 mg, crude product), compound 3 (300 mg, crude product), DIEA (0.8 mL), was dissolved in DMF, stirred overnight at room temperature. The solvent evaporated to dryness, then re-dissolved in TFA/DCM (95/5) and stirred 3 hours followed by concentrated under vacuum. The crude product was purified by HPLC and then frozen dried to obtain pure product of LND-Pep-cyclen. NBD-Pep-cyclen was synthesized using the same method.

Synthesis of NBD-GFFYK-cyclen (NBD-Pep-cyclen)
Firstly, the compound of NBD derivative was synthesized according to the following method: under the protection of nitrogen, β-alanine (98 mg, 1.1 mmol) and Na 2 CO 3 were added to the mixed solution of methanol and water, and 5 mL 4-chloro-7-nitrobenzo-2-oxa-1 (200 mg, 1 mmol) was slowly injected into the solution. After stirred at room temperature for 5 hours, methanol was removed by vacuum rotary evaporation, and the pH value of the remaining solution was adjusted to 1~ 2 with hydrochloric acid. Next, the crude product was extracted twice with anhydrous ether, and then extracted with dichloromethane twice. After the organic solvent phase was collected and concentrated in vacuum, the crude product could be directly used for the solid phase reaction to obtain the peptide of NBD-GFFYK (NBD-Pep). The final product of NBD-GFFYK-cyclen (NBD-Pep-cyclen) was yielded using the same synthesis method of LND-Pep-cyclen.

Figure S3
Synthesis routes of the peptide derivatives of NBD-GFFYK-cyclen.

Preparation of TEM samples
5 μL sample solution (1 mg/mL) was dropped onto a carbon-coated copper grid for 5 min, and the redundant liquid was gently absorbed from the edge of the grid using a filter paper. Then the sample was stained with 2% uranyl acetate solution for another 5 min in the dark. After removing the redundant dye solution, the carbon-coated copper grids were dried at room temperature for further TEM observation.
Compounds of 200 μM was diluted with ddH 2 O to 1 mL before measurement. Each sample was detected in triplicate.

Circular dichroism (CD) spectra
The

Mito-ROS detection
The experimental procedures were similar to those of ROS detection. The difference was that the MCF-7 cells were firstly exposed to 10 μM different treatments, and stained with 5 μM MitoSOX Red Mitochondrial Superoxide Indicator (Invitrogen, M36008) at 37 °C for 10 min.

Bio-compatibility evaluation
Six-week-old BALB/c mice were randomly assigned to two groups and intravenously administrated with 200 μL of LND-Pep-cyclen nanofibers (20 mg/kg) and PBS at 0 day, 7 day, 14 day, 21 day. All the mice were sacrificed at the last time point, followed by collecting the whole body blood for the hematology and blood biochemical detection.

Figure S17
Cell viabilities of MCF-7 cells after 12 h incubation with different treatments at different concentrations.

Figure S18
Colony formation photographs of MCF-7 cells treated with the indicated formulations (10 μM) for 12 h, and then exposed to γ-ray with different doses.

Figure S19
Confocal fluorescence microscopy images of Mito-ROS in MCF-7 cells after different treatments at the concentration of 10 μM for 12 h.Scale bar: 50 μm.

Figure S20
Body weight variation of the mice in different groups.

Figure S21
The in vivo tumor inhibition rate of different treatments.

Figure S22
H&E stained tissue sections from the heart, liver, spleen, lung, and kidney of the representative mice in different groups.