Accurate programmed multifunctional nano-missiles for self-promoted deep delivery and synergistic cascade tumor therapy: Tactfully collaborating chemosynthesis with tumor microenvironment remodeling

Rationale: Triple-negative breast cancer (TNBC) is considered one of the highest-risk subtypes of breast cancer and has dismal prognosis. The management of aggressive TNBC remains a formidable challenge. Tumor microenvironment (TME), with the unique features, which can serve as the “soil” for the growth and survival of tumor cells (the “seeds”), plays an important regulatory role in the occurrence, proliferation and metastasis of tumors. Catalytic tumor therapy, which can destroy the homeostasis of TME, affect the occurrence and progress of tumors in an all-round way and further magnify chemotherapy, is a quite potential tactic for TNBC-treatment. Methods: Herein, accurate programmed multifunctional cascade nano-missiles (GOx+L-Arg-NM/PTX-NM) composed of novel intelligent all-in-one “nano-rocket” (the drug delivery system) and “ammunitions” (the therapeutic agents) are innovatively constructed by mimicking the functionalities of military precision-guided missiles. Ammunitions can be precisely and effectively transported to the core region of TNBC (the “battlefield”) by organic modification on the surface of nano-rocket via chemical means. Once successfully internalized by TNBC cells, the nano-missiles can automatically trigger relevant cascade reactions without external stimulation, prominently disrupt the homeostasis of TME, and produce a “bomb-like” attack on tumors, further promoting the chemotherapy. Results: Both in vitro and in vivo investigations indicated that the innovative nano-missiles could deliver ammunitions to the core area of TNBC to the utmost extent, dramatically ablate tumor and restrain tumor metastasis via orchestrated multimodal synergistic starvation/oxidation/gas/chemotherapy. Conclusion: The well-designed multifunctional nano-missiles may emerge as a new paradigm to suppress the malignant proliferation and metastasis of TNBC, offering a promising approach for the next generation cancer therapy.

Then, triethylene glycol (747 mg, 4.97 mmol) in dichloromethane (1 mL) was added slowly. After stirring for another 5h at room temperature, the mixture was washed with 1 mol/L HCl and extracted three times with dichloromethane. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography to afford compound 2 as a yellowish oily (582.1 mg, 76.3%). 1
Then, PEG3350 (7.13 g, 2.13 mmol) in dichloromethane (5 mL) was added slowly. After stirring for another 16h at room temperature, the mixture was washed with 1 mol/L HCl and extracted three times with dichloromethane. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by recrystallization (DCM/Et2O) to afford compound 16 as a white solid (5.85 g, 90.1%). M.p. 72.6-74.2 o C. To a solution of 4-formylbenzoic acid (407 mg, 2.7 mmol) in dichloromethane (20 mL) was added DCC (741.6 mg, 3.6 mmol) and DMAP (44.0 mg, 0.36 mmol), and the reaction was stirred at -5 o C for 30 min. Then, compound 16 (6.45 g, 1.8 mmol) in dichloromethane (5 mL) was added slowly. After stirring for another 24h at room temperature, the mixture was cooled at -20 o C for 5h and filtered through a Buchner funnel. The filtrate was washed with 1 mol/L HCl and extracted three times with dichloromethane. The combined organic layers were washed with water and brine, dried To a solution of compound 12 (100 mg, 0.094 mmol) and compound 17 (448.8 mg, 0.141 mmol) in the mixed solvent of DMF (3 mL) and water (0.3 mL) was added CuI (5.3 mg, 0.028 mmol) and sodium ascorbate (11.1 mg, 0.056 mmol). After stirring for another 16h at room temperature under the protection of argon, the mixture was concentrated and purified by silica gel column chromatography to afford compound 18 as a yellowish oily (220.4 mg, 55.2%).

Synthesis of compound 20.
To a solution of compound 13 (1.0 g, 4.50 mmol) in dichloromethane (36 mL) was added EDCI (1.3 g, 6.75 mmol), DMAP (830 mg, 6.75 mmol) and DIPEA (1.16 g, 9.0 mmol), and the reaction was stirred at -5 o C for 30 min. Then, compound 19 (1.56 g, 3.0 mmol) in dichloromethane (3 mL) was added slowly. After stirring for another 5h at room temperature, the mixture was washed with 1 mol/L HCl and extracted three times with dichloromethane. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography to afford compound 20 as a yellowish oily (1.84 g, 84.7%). 1  The solution of compound 20 (1.15 g, 1.59 mmol) in dichloromethane (2 mL) was stirred at 0 o C for 5min, Then, triethylsilane (462.4 mg, 3.98 mmol) in dichloromethane (1 mL) and trifluoroacetic acid (7 mL) was added slowly. After stirring for another 2h at room temperature, the mixture was concentrated and purified by silica gel column chromatography to afford compound 21 as a colorless transparent oil (808.9 mg, 76.3%). 1  To a solution of compound 21 (250 mg, 0.36 mmol) in dichloromethane (3 mL) was added DCC (155 mg, 0.72 mmol) and DMAP (9.5 mg, 0.07 mmol), and the reaction was stirred at -5 o C for 20 min. Then, compound 6 (216 mg, 0.54 mmol) in dichloromethane (0.5 mL) was added slowly. After stirring for another 2h at room temperature, the mixture was washed with 1 mol/L HCl and extracted three times with dichloromethane. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel
Then, triethylene glycol (396 mg, 2.63 mmol) in dichloromethane (1 mL) was added slowly. After stirring for another 5h at room temperature, the mixture was washed with 1 mol/L HCl and extracted three times with dichloromethane. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography to afford compound 29 as a yellowish oily (304.5 mg, 78.5%). was hydrolyzed, indicating that the alkyl-imine bond (AIm) was not stable under normal physiological conditions of blood circulation and could not be used as acid-sensitive bond in guidance unit.
After Bio3-PEG-PIm was oscillated at pH 7.4 for 24 hours, there was still no new signal peak in the corresponding hydrogen spectrum (Figure S2A), indicating that this ligand remained stable for a long time under normal physiological conditions of blood circulation. In contrast, about 18.7% (0.20/(0.20+0.87)×100%) of Bio3-PEG-PIm was hydrolyzed at pH 6.5 for 2 h (Figure S2B), indicating that phenyl-imine bond (PIm) was slowly broken under weakly acidic conditions in the tumor microenvironment.  Similarly, no new signal peak appeared in the hydrogen spectrum of Bio3-PEG-Hz after shock at pH 7.4 for 24h (Figure S3A). In contrast, about 14.3% (0.15/(0.15+0.90)×100%) of Bio3-PEG-Hz was hydrolyzed at pH 6.5 for 2h (Figure S3B), indicating that the hydrazone bond (Hz) fractured slowly under weakly acidic conditions in tumor microenvironment, and the rate was slower than that of Bio3-PEG-PIm.
All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Sichuan University, and approved by the Animal Ethics Committee of Sichuan University.