Redox‐Responsive Dual Drug Delivery Nanosystem Suppresses Cancer Repopulation by Abrogating Doxorubicin‐Promoted Cancer Stemness, Metastasis, and Drug Resistance

Abstract Chemotherapy is a major therapeutic option for cancer patients. However, its effectiveness is challenged by chemodrugs' intrinsic pathological interactions with residual cancer cells. While inducing cancer cell death, chemodrugs enhance cancer stemness, invasiveness, and drug resistance of remaining cancer cells through upregulating cyclooxygenase‐2/prostaglandin‐E2 (COX‐2/PGE2) signaling, therefore facilitating cancer repopulation and relapse. Toward tumor eradication, it is necessary to improve chemotherapy by abrogating these chemotherapy‐induced effects. Herein, redox‐responsive, celecoxib‐modified mesoporous silica nanoparticles with poly(β‐cyclodextrin) wrapping (MSCPs) for sealing doxorubicin (DOX) are synthesized. Celecoxib, an FDA‐approved COX‐2 inhibitor, is employed as a structural and functional element to confer MSCPs with redox‐responsiveness and COX‐2/PGE2 inhibitory activity. MSCPs efficiently codeliver DOX and celecoxib into the tumor location, minimizing systemic toxicity. Importantly, through blocking chemotherapy‐activated COX‐2/PGE2 signaling, MSCPs drastically enhance DOX's antitumor activity by suppressing enhancement of cancer stemness and invasiveness as well as drug resistance induced by DOX‐based chemotherapy in vitro. This is also remarkably achieved in three preclinical tumor models in vivo. DOX‐loaded MSCPs effectively inhibit tumor repopulation by blocking COX‐2/PGE2 signaling, which eliminates DOX‐induced expansion of cancer stem‐like cells, distant metastasis, and acquired drug resistance. Thus, this drug delivery nanosystem is capable of effectively suppressing tumor repopulation and has potential clinical translational value.


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Synthesis of celecoxib succinamidic acid (CEL): Briefly, celecoxib (1.0 g, 2.6 mmol), succinic anhydride (1.3 g, 13 mmol) and triethylamine (0.5 mL) were dissolved in dichloromethane (50 mL), and stirred at room temperature for 24 h. After reaction, the solvent was removed under vacuum and the crude product was purified by column chromatography using the mixture of n-hexane and ethyl acetate (3:1) as eluent. The product was characterized by 1 H NMR ( Figure S1).
Synthesis of MSNs-SS-Bz: MSNs-SS-NH 2 (0.2 g) were dispersed in anhydrous DMF (30 mL) with DIPEA (0.5 mL), then benzoyl chloride (200 L in 5 mL DMF) was added dropwise to the dispersion in an ice bath and stirred at room temperature for 12 h. The MSNs-SS-Bz were obtained by washing and drying.
Doxorubicin loading and PCD wrapping: The silica nanoparticles (MSNs-SS-CEL and MSNs-SS-Bz, 200 mg) were dispersed in phosphate buffered saline (PBS, pH 7.4, 100 mL), respectively. Subsequently, doxorubicin solution (50 mg, in 10 mL water) was added and the suspension was stirred for 24 h. Then, poly( -cyclodextrin) (PCD, 300 mg) was added and the suspension was stirred for another 24 h. Next, the DOX-loaded nanoparticles were washed (DOX@MSCPs and DOX@MSBPs) with PBS and water, and obtained by lyophilization.
The PCD wrapped MSNs without DOX (MSCPs and MSBPs) were prepared with the same method without DOX loading procedure. The DOX loading contents of DOX@MSCPs and DOX@MSBPs were determined using a UV-Vis spectrophotometer (Perkin-Elmer Lambda Bio-40 UV/Vis spectrometer) at 480 nm.
Quantitative real-time PCR: Total mRNA in cells or tumor tissues was extracted using Trizol reagent (Invitrogen, USA) and reversed into cDNA using Hiscript II reverse transcriptase (Vazyme Biotech, China). qPCR was performed on ABI StepOne Plus Detector System (ABI, USA) according to the instructions with AceQ qPCR SYBR® Green Master Mix (Vazyme Biotech, China). Relative expressions of genes were normalized using the house-keeping gene GAPDH as a reference. The primers were presented in Table S1.
Western blot analysis: Western blot protein analysis was carried out according to our previous study. [2] Briefly, cells or tissues were washed with PBS and lysed by RIPA Lysis Buffer (Beyotime, China; 50 L, with protease inhibitor). After sonication in the ice bath, the supernatants containing proteins were acquired by centrifugation under 4 o C. Followed by protein measurement by BCA kit, and normalized to uniform concentration, the supernatants were mixed with loading buffer and boiled for 10 min. After electrophoresis in 10% SDS-PAGE gel, the proteins were transferred to NC membranes, followed by primary monoclonal antibody treatment (P-gp, Oct-3/4, Nanog, Notch-3, COX-1, COX-2, GAPDH), then incubated with horseradish peroxidase-coupled secondary antibody. The protein was measured followed by the BeyoECL Plus (Beyotime, China) reagent instructions.
Hemolysis assay: As the previous study, [3] the rabbit red blood cells (RBCs) suspension S5 (2% in PBS, v/v) was mixed with of MSCPs (100 L in PBS), and shaken at 37 o C for 5 h.
Next, the samples were centrifuged to separate the supernatants. The absorbance of supernatants at 545 nm was determined using a UV-Vis spectrophotometer (SHIMADZU UV-1800, Japan).