Anticancer efficacy and molecular therapeutic mechanism of thymoquinone-loaded calcium carbonate nanoparticles combined with 5-fluorouracil against colon cancer

Colon cancer ranks as the third most prevalent cancer worldwide, contributing to significant morbidity and mortality. The adverse side effects and chemoresistance associated with the primary chemotherapeutic agent, 5-fluorouracil (5-FU), have necessitated the exploration of improved therapeutic strategies. Thymoquinone (TQ) derived from Nigella sativa, a bioactive compound with established anti-colon cancer properties, presents a promising adjunct to 5-FU. The encapsulation of chemotherapeutic agents and phytochemicals into nano delivery systems represents a pivotal advancement, enhancing solubility and bioavailability and minimizing toxic side effects. This study aimed to develop cockle shell-derived calcium carbonate nanoparticles (CaCO3np) as a delivery system for 5-FU and TQ and to evaluate the therapeutic potential of these formulations in combination therapies for colon cancer. Using a mechanical ball milling method, porous CaCO3np was synthesized, and the formulations of 5-FU-loaded CaCO3np (5FU-CaCO3np) and TQ-loaded CaCO3np (TQ-CaCO3np) were optimized by using a high-speed homogenizer for efficient loading of 5-FU and TQ. The resulting nanoparticles were characterized by their high purity, stable aragonite phase, and particle size ranging from 80 to 140 nm. The encapsulation process preserved the chemical integrity of both 5-FU and TQ, and the nanoparticles demonstrated pronounced biocompatibility and pH-sensitive sustained release profiles. In vitro studies revealed that TQ-CaCO3np combined with 5-FU exhibited a superior synergistic effect compared to the reverse combination (5FU- CaCO3np combined with TQ). This combination effectively inhibited CT26 colon cancer cell proliferation, induced apoptosis, caused G0/G1 cell cycle arrest, and suppressed the growth of CT26 spheroids. In vivo studies further validated the enhanced anticancer efficacy of the TQ-CaCO3np and 5-FU combination in CT26 tumour-bearing mic, demonstrating significant tumour growth inhibition and reduced systemic toxicity of 5-FU. Proteomic and metabolomic analyses provided insights into the molecular mechanisms underlying the observed synergy, identifying the key pathways (biosynthesis of amino acids, 2-oxocarboxylic acid metabolism, and HIF-1 signalling pathway) and a series of key targets (Aco2, Gapdh, Psph, Pdha1, Pdhb, Rps6, alanine, glutamate, proline, glutamine, threonine, adenosine 5'-triphosphate, lactate, leucine, and isoleucine) modulated by the combination therapy. Overall, this study demonstrated that TQ-CaCO3np, with its excellent biocompatibility, pH sensitivity, and sustained release properties, holds significant promise in enhancing the efficacy of 5-FU. These findings pave the way for further clinical applications, offering a novel and potentially effective approach to colon cancer treatment.

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