Bisphosphonates (BPs) are derivatives of inorganic pyrophosphate, characterized by a highly stable phosphorus‑carbon‑phosphorus bond (P-C-P), giving them a high affinity for the hydroxyapatite in bones. BPs have been clinically used in the treatment of osteoporosis and other diseases. Based on different types of side chains connected with the carbon skeleton, BPs can be classified into two classes, non‑nitrogen-containing BPs (non-N-BPs) and nitrogen-containing BPs (N-BPs), with the latter (e.g. risedronate, zoledronate, etc.) exhibiting up to 10,000 folds higher anti-resorptive potency than the former [1,2]. BPs can also coordinate with metals (e.g. Mg2+, Zn2+, and Fe2+, etc.) to form particles [3,4] and can be incorporated into hydrogels, endowing the hydrogels with unique properties like conductivity and tunability [4]. Meanwhile, metal-organic frameworks (MOFs), porous solids with ordered structures, have attractive prospects for strategic applications, especially in biomedicine, due to their biocompatibility [5].

The repurposing of BPs, especially N-BPs, as therapeutic agents in the immune system has recently attracted attention due to their known safety profiles. Studies have shown that zoledronate in combination with a toll-like receptor 3 ligand (peptide/polyriboinosinic: polyribocytidylic acid, polyIC) results in antitumor effects and increased antigen-specific CD8+ T cell responses [6]. With the use of zoledronate, γδ T cells [7], NK cells [8], and DC cells [9] were activated and expanded, which have cytotoxic effects against a variety of solid tumor cell lines. Meanwhile, it has been reported that BPs target B cells directly and enhance antibody responses [10]. Furthermore, zoledronate has been found to have actions on other organs like the lungs and can boost immune responses to infections [11]. It has been previously reported that an N-BPs-modified zinc‑aluminum hybrid adjuvant was used to provide protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [12]. Also, BPs, particularly N-BPs, have potential as immunostimulants against SARS-CoV-2 and may play critical roles in enhancing immune responses [13]. The molecular mechanism of N-BPs as adjuvant includes mevalonate pathway inhibition, resulting in the inhibition of protein prenylation and antigen retention [14]. Additionally, caspase-1-mediated cytokine maturation [8] and miR-21/PTEN/Akt signaling [15] have been identified as crucial mechanisms in response to BPs.

In addition to the inherent activity of compound, their size and morphologies properties can also impact their regulatory effects on the immune system [16]. Incorporating active compounds into micro- or nano-scale particulate salt precipitants has become a common strategy in the development of novel adjuvants. This approach offers several benefits, such as improved target selectively, bioavailability, and sustained release. A strategy based on well-established aluminum salts is therefore promising due to its low-cost production, safety, and excellent adsorption capacity for antigens. AS04™ adjuvant is a combination of aluminum salt and monophosphoryl lipid A (MPL), which is a toll-like receptor 4 ligand. Similarly, structure-modified mannans, which is a C-type lectin receptor ligand, formulated with aluminum salt, generate broad immunity and protect against viral infections [17]. And CpG-ODN, a toll-like receptor 9 ligand, is also a classic example of a varicella-zoster virus (VZV) vaccine termed LZ901™ from Luzhu Biotech which is undergoing clinical experimentation. The phosphonate SMIPs, a toll-like receptor 7 ligand, were designed to facilitate the stable and efficient adsorption of aluminum salt [18]. It is essential to note that all these immunostimulants come in the form of aluminum salts-bound form via the phosphate group. However, BPs, with two phosphate groups, combined with aluminum salts and their potential as vaccine adjuvants have not yet been thoroughly investigated.

In this study, we used risedronate and added it to aluminum salts or other metal salts to compare their adjuvant activity. The distribution of antigen and risedronate in innate immune cells, as well as the activation and proliferation of immune cells at different times in the injection sites were compared. Using single-cell RNA sequencing, the function of particulate risedronate in the process of antigen presentation was investigated. In addition, we analyzed the kinetics of antigen draining to lymph nodes (LNs) and the germinal center response in LNs. As a vaccine adjuvant, particulate risedronate formulated three model proteins to immunized animals and the antibody levels, T responses, and protective efficacy were evaluated.