Osteosarcoma is the most common primary bone cancer, manifesting in the distal femur with hallmarks such as pain and bone swelling. (Beird, 2022) Clinically, osteosarcoma patients generally receive a limb salvage surgery followed by a chemotherapy regime, improving the 5-year survival rate to 54 %. (Smeland, 2019) Among chemotherapy drugs, doxorubicin (DOX) has been one of the most effective agents in first-line osteosarcoma treatment during the past serval decades, either applied alone or in a combination manner. (Ferrari and Serra, 2015, Bramwell, 1997) However, current treatments have long been stagnating with challenges persisting in patients with metastatic and recurrent osteosarcoma. (Gill and Gorlick, 2021)Therefore, the osteosarcoma treatment requires more sophisticated strategies.

Modulation of tumour microenvironment (TME) has emerged as a promising strategy in cancer treatment, especially in the aspect of immunotherapy which elicits durable antitumour effects to treat metastatic and recurrent cancer. As the most abundant stromal cells in the TME, tumour-associated macrophages (TAMs) play a critical role in cancer therapy. (Ngambenjawong et al., 2017) TAMs participate in various processes, such as tumorigenesis, metastasis, leukocyte infiltration, and immunosuppression. (Mantovani et al., 2022, Cook and Hagemann, 2013) TAMs can polarize into anti-tumourgenic (M1-like) and pro-tumourigenic (M2-like) phenotypes and exert opposite influences on tumour progression. M1 activation is featured with upregulation of markers including CD86, MHC-II, iNOS, and CD68, (Minami, 2018, Chen, 2019, Khabipov, 2019, Jamiyan et al., 2020) which are associated with macrophage functions such as antigen-presenting, inflammation induction, and phagocytosis. Canonical M1 activation is induced by interferon-gamma (IFNγ) or lipopolysaccharide treatment. (Yunna et al., 2020) In addition, some osteoclast inhibitors applied in bone research possess prominent effects in both cancer treatment and macrophage remodelling. (Coxon et al., 2006, La-Beck et al., 2021) For example, zoledronic acid (ZA) could promote M1 macrophage via TLR4 signalling activation and repressed M2 macrophage via Wnt/β-catenin pathway suppression. (Zhu, 2019, Lv, 2020) ZA also exhibits other cell growth inhibition effects such as induction of tumour apoptosis, inhibition of metastasis, and prevention of angiogenesis. (Wang et al., 2020) However, the anticancer application of ZA was hindered by the rapid metabolization in vivo and induction of osteonecrosis of the jaw under high dosage. (Mönkkönen, 2007, Van Poznak, 2021, Lescaille, 2014) Thus, attempts should be made to improve the bioavailability of ZA.

Aluminium hydroxide has been approved for clinical applications over several decades, suggesting its biosafety in vivo. (Lambrecht et al., 2009, Gupta, 1998) With relatively high doses, aluminium hydroxide with nano-size exerted less pathological toxicity than the bulk form. (Hamza et al., 2022) A wide range of aluminium hydroxide nanoparticles has been employed as adjuvants to enhance the immune responses of vaccines. (Hou, 2020, Meena, 2022) Moreover, aluminium hydroxide exerted advantages for drug delivery, such as its intrinsic antacid for neutralizing the pH in tumour microenvironment, equilibrium of extracellular and intracellular ion concentrations, and affinity to immune cells. (Patel, 2023, Danielsson and Eriksson, 2021) As reported, aluminium hydroxide exerted selective adsorbent for many chemicals when it was fabricated with a pseudoboehmite structure and in the form of crumpled assembled nanosheets. (Lerner, 2018) Besides, aluminium can form layered double hydroxide (LDH) with the existence of various covalent elements. (Yu et al., 2017) These aluminium-based LDH were in gibbsite structure with a stack sheet-like morphology. (Guo et al., 2010) Aluminium-based LDHs are featured with a positive surface charge and a high payload to numerous drugs. (Li et al., 2014, Senapati, 2016, Shirin et al., 2021).

Here we employed aluminium hydroxide nanosheets (nAl) as platform to integrate DOX and ZA for osteosarcoma treatment. In this work, a rapid and facile approach was used to fabricate layered nAl, which was inspired by the synthesis of layered double hydroxide nanosheets. (Ladewig et al., 2009, Gu et al., 2015) Firstly, the nAl was prepared under optimized conditions and subsequentially loaded with ZA and DOX to create a nanodrug (nAl/ZD). Next, nAl/ZD was employed to evaluate the cell-killing ability using the LM8 osteosarcoma cell line and the influence on macrophage phenotypes using RAW 264.7 cells. Finally, we investigated the performance of nAl/ZD in a co-culture model.