Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that primarily cause widespread inflammatory responses and severe damage of synovial joints (Chaudhari et al., 2016). Current clinical medications including non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (GCs) and disease-modifying anti-rheumatic drugs (DMARDs) have been widely used in RA management. NSAIDs (such as ibuprofen) and GCs such as (dexamethasone) can rapidly relieve joint pain and inflammatory response. However, they are unable to alter the RA course and cannot be used for long-term therapy (Wang et al., 2021, Wang and Sun, 2017). By contrast, DMARDs can effectively stop the disease development and achieve favorable anti-rheumatic effect. Methotrexate (MTX), as the most used DMARDs, has been recognized as the first-line drug for RA treatment (Friedman and Cronstein, 2019). The anti-rheumatic mechanism of MTX mainly involves the inhibition of various inflammatory signaling pathway and suppression of immune activation (Cronstein and Aune, 2020). Despite the excellent anti-rheumatic efficacy, RA patients receiving high dose or long-term administration of MTX are often at high risk of causing hepatic damage (Ali et al., 2017). As a chronic and incurable inflammatory disorder, the management of RA requires long-term drug therapy, which can inevitably result in liver damage.

Nanomedicines can selectively deliver MTX to inflamed sites via passive or active targeting mechanism and reduce unwanted drug accumulation in liver, thereby improving drug bioavailability and reducing non-targeted distribution-associated toxicity (Liu et al., 2019, Mirchandani et al., 2022). Nevertheless, numerous researches have demonstrated that most nanomedicines still tend to largely accumulate in liver due to the presence of mononuclear phagocyte system (MPS) (Mirkasymov et al., 2021). In addition, liver plays a key role in the metabolism and disposition of drug formulation (Cornu et al., 2020). Therefore, the large accumulation of MTX in liver can hardly be avoid. Currently, MTX-induced liver damage remains a huge challenge in the long-term management of RA.

MTX-induced liver toxicity mainly originates from the oxidative stress-induced damage and activation of apoptosis-related signaling pathway (Ezhilarasan, 2021, Mehrzadi et al., 2018). The utilization of hepatoprotective agents that can prevent oxidative damage and hepatocyte from apoptosis might be an attractive strategy to decrease the MTX-induced liver toxicity (Tao et al., 2018). Glycyrrhizinic acid (GA), the main bioactive ingredient of licorice, has been extensively reported to possess good anti-oxidant, anti-inflammatory and hepatoprotective effect (Huo et al., 2020). GA has often been added to traditional Chinese medicine formulas to improve the therapeutic efficacy and reduce toxicity, and there is a well-known theory named “ten prescriptions nine grass”, indicating the frequent application of GA (Cai et al., 2019). On the other hand, GA has been demonstrated to inhibit the expression of P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1), which are the main drug transporters for MTX (Yasunari et al., 2023). Based on the pharmacological activities of GA, the combination of GA with MTX might achieve synergistic anti-inflammatory efficacy.

As MTX and GA have distinct in vivo pharmacokinetics profile, biodistribution tropism and metabolism behaviors, they might exert pharmacological activities at different sites and time periods (Battaglia et al., 2011, Krähenbühl et al., 1994). For example, the plasma half-life of GA is more than 10 h after intravenous injection, and GA mainly distribute in liver and bile (Krähenbühl et al., 1994). In contrast, intravenously injected MTX has a plasma half-life less than 1 h and tends to accumulate in kidney and liver (Battaglia et al., 2011). Therefore, the direct administration of free MTX and GA might hardly achieve synergistic effect. To achieve enhanced therapeutic efficacy and reduced hepatotoxicity simultaneously, it is necessary to co-encapsulate MTX and GA in one drug carrier to coordinate their in vivo fates. A variety of versatile co-delivery systems have been developed previously. Zhao et al. reported an attractive cyclodextrins (CDs)-polymer co-delivery carrier which can easily achieve ratiometric dose manipulation by simply varying the proportion of CD-polymer conjugates (Chen et al., 2017). Besides, Zeolitic imidazolate framework-8 with large cargo-loading space and high surface area was also utilized as a co-delivery system to fulfill synergistical therapeutic outcome (Meng et al., 2023). For the treatment of chronic inflammatory diseases, drug carriers should have good biocompatibility and low immunogenicity. Based on these principle, we herein designed an albumin-based nanocarrier to co-deliver MTX and GA to arthritic joints. As reported, albumin nanoparticles have high affinity to SPARC (secreted protein acidic and rich in cysteine) and gp60, which are overexpressed in inflamed sites (Li et al., 2023, Liu et al., 2019). Other than that, the elevated metabolism level of inflammatory cells in arthritic joints exhibited increased demand for albumin assumption. Hence, albumin nanoparticles can achieve selective accumulation in inflamed sites via multiple mechanisms. In our study, we assumed the dual drug-loaded albumin nanoparticles (HSN/MTX/GA) can preferentially distribute in inflamed joints, where GA can decrease the MTX efflux and extend MTX retention by inhibiting P-gp and MRP level, thereby exerting synergistic therapeutic effect. When HSN/MTX/GA distributes in liver, GA is expected to reverse the MTX-induced liver damage by activating anti-oxidant defense Nrf2/HO-1 signaling and preventing apoptosis-related Bcl-2/Bax signaling (Scheme 1).

Here, we fabricated an albumin nanoparticle to co-deliver MTX and GA (HSN/MTX/GA) for enhanced therapeutic efficacy in RA model and reduced liver damage caused by MTX. We systemically explored the physicochemical property of HSN/MTX/GA and evaluated its in vivo performance including biodistribution and pharmacokinetics. When MTX and GA were co-encapsulated in one drug nanocarrier, drug-drug interaction would inevitably occur, which can finally affect the in vivo performance of therapeutic drugs. To the best of our knowledge, how GA affect the in vivo pharmacokinetics and distribution of MTX remained unexplored. Our research not only provided a facile approach to effectively treat RA with attenuated liver toxicity caused by MTX, but also verified the mechanism behind the enhanced therapeutic efficacy and reduced hepatotoxicity. Our therapeutic strategy offers important knowledge for drug combination and holds potential in the long-term management of RA.