The profound enhancement of cancer chemotherapy involves the use of synergistically active chemotherapeutic combinations. The optimum therapeutic doses would decrease when compared to a single anticancer drug-based approaches with minimized side effects and enhanced therapeutic outcome. Methotrexate (MTX) is an anti-folate anticancer drug that inhibits the synthesis of purines (adnenine and guanine). Therefore, MTX inhibits the DNA synthesis (Osborn et al., 1958, Prasad et al., 2016, Subramanian and Kaufman, 1978). Intriguigly, MTX is a potent anticancer drug against breast cancer (BC). In order to enhance MTX efficiecny and mitigate its advserse effects, its concominant use with other anticancer agents such as tamoxifen, cyclophosamide and remarkbly, with 5-flourouracil (Fu) has been reported in literature (Bonadonna et al., 1995, Colleoni et al., 2002, Crivellari et al., 2000). Fu is a flourinated pyrimidine derivative that exerts its cytotoxicity through binding to thymidylate synthatase. So, Fu interferes and prevents pyrimidine synthesis and as a result, it prevents the RNA and DNA replication (Heidelberger et al., 1957; Longley et al., 2003). When MTX is administered prior to Fu, the intracellular concentration of Fu increases up to four-fold because MTX inhibits the purine synthesis and elevates 5-phosphoribosyl 1-pyrophosphate (PRPP) concentration. PRPP elevated concentration plays a crucial role in the binding of Fu to its target; thymidylate synthetase (TYMS) (Benz et al., 1982, Cadman et al., 2016, Herrmann et al., 2019). This would never happen upon the co-administration of both drugs simultaneously (Benz et al., 1982, Cadman et al., 1981). Consequently, the sequential administration of MTX followed by Fu is a major enhancing factor for their synergism. In literature, it was previously reported that BC pretreatment with MTX followed by Fu administration improved the clinical results of refractory BC in addition to the pain relief (Tisman and Wu, 1980). Also, it was mentioned that at least 4 h time interval is sufficient to enhance the synergism between the sequentially administered MTX and Fu (Herrmann et al., 2019, Sobrero et al., 2005).

The incorporation of the synergically active drugs into multi-compartments nanocarrier addresses an anticancer drug delivery system (DDS) with expected improved efficiency relative to the free drugs administration. Moreover, because MTX and Fu are sequentially synergistic, the nanocarrier should confer sequential release of MTX followed by Fu.

Porous nanomaterials as mesoporous silica nanoparticles (MSN) and nano metal organic frameworks (NMOF) represent efficient nanocarriers for the DDSs development with high loading capacity. MSN showed a great potential to act as efficient anticancer-DDS as the mesopores of MSN confer large surface area that enables MSN to act as cargo carriers with high capacity. Consequently, MSN-based DDSs are reported to advantageously overcome the P-glycoprotein resistance by elevating the intracellular drug’s concentration (Gao et al., 2011). Also, MSN are characterized by their low density and tunable properties as the pores’ sizes, and remarkably, by their biocompatibility (Chen et al., 2010, Chen et al., 2011, Liu et al., 2011). Zeolitic imidazolate framework-8 nono metal organic frameworks (ZIF-8 NMOF) is a zinc-based NMOF that is used extensively to develop DDSs. The pores are uniform and controllable and thus, provide a large surface area that enables the high loading of either hydrophilic (Soares and Nailton, 2012) or hydrophobic drugs (Sun et al., 2012, Zhuang et al., 2014). Although metals are known to represent toxic components to the body, some of them are essential metals to the body functions as the zinc that is present in multiple body organs (3 mg in the whole body) (Jackson, 1989). Consequently, ZIF-8 NMOF are classified as biocompatible when tested against several cancer types, including BC cells (Marcus et al., 2018). Moreover, ZIF-8 NMOF displays an accelerated degradation in the acidic medium thus, provides a cancer-specific drug release platform (Zheng et al. 2013; H. Zheng, Y. Zhang, L. Liu, W. Wan, P. Guo, A. M. Nystrom 2015; Q. Wang et al. 2020).

BC is a prime lethal cancer affecting females worldwide (Deo et al., 2022). Globally, the reported recent cases were more than 2 million patients and about 6.9 % of all-cancer related mortality (Sung et al., 2021). Despite the availability of multiple treatment approaches, the emergence of drug-resistance especially against sole anticancer treatment protocols provoked the search for synergistic drugs’ combinations to eradicate the cancer cells through multiple pathways and concomitantly, overcome the treatment failure threat due to the inevitable side effects (Waks and Winer, 2019).

Hence, in this work, a pH-triggered sequential drug release of MTX followed by Fu was attained by constructing the core–shell nanocarrier; MSN@NMOF (Scheme 1). The nanocarrier was validated by several characterization techniques. MSN represented the core and the acid-labile NMOF; ZIF-8 was the shell, and they were loaded by Fu and MTX, respectively to attain the formulation; Fu-MSN@MTX-NMOF. The in-vitro and in-vivo efficiencies of MTX and Fu either as free drugs or loaded into the core–shell nanocarrier; Fu-MSN@MTX-NMOF were tested by MCF-7 cell line and Ehrlich ascites (EAC)-bearing animal model, respectively. First, the cytotoxic effect against MCF-7 cells of the individual free drugs (MTX and Fu) was investigated. Then, the cytotoxicity of the developed nanoformulation; Fu-MSN@MTX-NMOF was tested at three time-intervals to reveal the sequential release and synergism between MTX and Fu. Flowcytometric analysis of the drug-loaded core–shell nanoformulation; Fu-MSN@MTX-NMOF was conducted to understand the mechanism and kinetics of its cytotoxic effect. Moreover, in-vivo assessment by Ehrlich ascites carcinoma (EAC) mice model involved the testing of the drug-loaded core–shell nanoformulation (Fu-MSN@MTX-NMOF) and compared to the administration of the free drugs (MTX and Fu), spaced by a certain time interval, as positive control.