Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS), which is lymphocyte-dependent. The most prominent factors in the progression of the disease and patient morbidity are axonal loss and functional disability which are the result of demyelination and formation of myelin sheaths by oligodendrocytes, the infiltration of T cell subsets, expression of MHC-class II and co-stimulatory molecules on activated microglia, proinflammatory cytokine secretion, and cell-mediated damage either through CD8+ T cells or by macrophages and microglia (Attfield et al., 2022, Dhaiban et al., 2021, Namiecinska et al., 2020). In recent decades, several disease-modifying treatments (DMTs) for MS have been emerged, which are associated with a reduction in deterioration and disease severity with a possible reduction of disability progression. The first generation of these DMTs are injectable immunomodulatory drugs, namely interferon-β and glatiramer acetate (GA), which are the pivot and the first line of therapy in the treatment of patients with relapsing–remitting MS (Ciriello et al., 2018, Edinger and Habibi, 2023, Lebrun-Frenay et al., 2019) GA can affect MS at various levels of the immune response by binding to MHC Class II molecules. The major important mechanism of this drug is activation of GA‐specific T regulatory and T suppressor cells and their entrance to the CNS which results in the secretion of Th2 suppressive cytokines and also anti-inflammatory and neuroprotective cytokines (Karandikar et al., 2002). GA can also modify the immune system by induction and activation of peripheral T-cell suppressors (Afzal et al., 2013).

GA, a random copolymer of four amino acids (alanine, lysine, glutamic acid, and tyrosine) with myelin basic protein (MBP) mimicry structure (Song et al., 2019), is an effective drug in this regard.

However, the adherence of patients to this treatment is low, due to the poor pharmacokinetics of this therapeutic and existence of undesirable effects like post-injection site-reactions (namely swelling, redness, itching, and lump), that lead to treatment failure and poor long-term prognosis (Anderson et al., 2010, Cohen et al., 2015). After subcutaneous administration of this drug, a large proportion of the drug is hydrolyzed locally into fragments with low molecular weights, consisting of small oligopeptides and free amino acids, and some fragments of GA may also be detected as GA-reactive antibodies (Carter and Keating, 2010, Song et al., 2020). Another fraction of this drug is absorbed through the capillaries to the systemic circulation rather than being absorbed by lymphatic capillaries (to be processed by macrophages), and excreted through glomerular filtration due to its lower molecular weight (5–9 kDa) than 16–20 kDa (Song et al., 2019, Trevaskis et al., 2015), which is also smaller than the pores (with 30–50 kDa cut-off) between the endothelial cells of capillaries (Ruggiero et al., 2010, Song et al., 2019). Only a small portion of the injected GA enters the lymphatic system and reaches the lymph nodes as the site of GA action for further being phagocyted by macrophages as the most prominent antigen-processing cells (APCs) involved in MS management by GA (Song et al., 2019). Thus, the delivery of GA may not be efficient and sufficient for MS management.

By incorporating this drug into nano-carriers, such as liposomes, some benefits could be achieved, such as protection of the drug from hydrolysis at the injection site as well as less systemic absorption through blood capillaries (Filipczak et al., 2020). Additionally, a higher rate of GA delivery by liposomal formulation to APCs (mostly macrophages) could be attained through GA liposomal encapsulation by rectifying GA size (Zahednezhad et al., 2019). In fact, nanoparticles with the size of <200 nm could directly enter the lymphatic vessels and be delivered to the APCs in the lymph nodes; and nanoparticles with the size of 200–1000 nm are unable to enter the lymphatic vessels but retained in the administration site, then taken up by APCs and delivered to the nearby lymph nodes for further processing (Hoshyar et al., 2016). Therefore, by controlling the size of liposomes under 1000 nm, it is possible to attain efficient delivery to the APCs.

Several studies have been conducted on encapsulation of GA in nanocarriers up to now. In one study, incorporation of GA in nanolipodendrosomes for treatment of muscular dystrophy showed high efficacy in drug protection, direction, internalization, and targeting (Afzal et al., 2013). In addition, poly(amidoamine) for altering the architecture of the drug (Song et al., 2020), and nano-sized electrostatic aggregation of GA and hyaluronic acid for the purpose of more persistence in the lymph nodes (Song et al., 2019) are among the strategies in the field of nanotechnology-based drug delivery systems for GA.

By liposomal encapsulation, several problems could be covered simultaneously. The size, charge, and shape could be manipulated for alternations in drug’s pharmacokinetics and targeting moieties or functionalization could be applied for active targeting (Rahiman et al., 2021, Song et al., 2020).

In addition, these liposomes enter the lymphatic system due to their size (Oussoren and Storm, 2001) and deliver GA to antigen-presenting cells (APCs) (Longbrake and Cross, 2016) with high efficiency and stimulate humoral immunity as the desirable type of immunity for the amelioration of MS (Longbrake and Cross, 2016). Shimizu et al. reported that liposomal formulation of doxorubicin (as a cytotoxic agent) and autoantigenic myelin oligodendrocyte glycoprotein (MOG) led to suppression of immune cell invasion and eradication of autoantigen-recognizable T cells (Shimizu et al., 2021).

By using liposomal GA delivery, the necessity of daily drug administration is reduced and GA could be protected from degradation and become long-acting. Furthermore, the impediments of GA delivery to lymph nodes could be rectified due to the absorption of liposomes by the lymphatic vessels in the subcutaneously injected site (Oussoren and Storm, 2001) and the higher rate of their phagocytosis by macrophages in the lymphatic system (Song et al., 2019).

Experimental autoimmune encephalomyelitis (EAE) is the animal model of MS that recapitulates key histopathological features of this disease and is widely applicable for simulating autoimmune demyelination and inflammation of the CNS (Dedoni et al., 2023, Smith, 2021). This model is characterized by the activation of autoreactive myelin antigen-specific CD4+ T cells and their expansion within the peripheral lymphoid tissue (Attfield et al., 2022, Yi et al., 2022). Myelin antigen-specific CD4+ T cells would also become activated in a MHC-II restricted manner in the perivascular space of the CNS vasculature (Kawakami et al., 2005, Rahiman et al., 2022a). These procedures also lead to further activation of CNS T cells and production of effector chemokines, cytokines, and also attraction of immune cells like granulocytes and macrophages into the CNS mediating demyelination and tissue inflammation (Faissner et al., 2019). Both MS and its murine model (EAE) are believed to be primarily autoantigen-specific and mediated by Th1 and Th17 (Milovanovic et al., 2020, Van Kaer et al., 2019).

In the current study, we have prepared novel liposomal formulation of GA with the aim of enhancing the efficacy of GA through its higher delivery to macrophages and also prolongation of the administration intervals of this drug. Subsequently, we have evaluated the effects of the liposomal form of GA in the immunomodulation, remyelination processes, and behavioral improvement in the MOG35-55-induced mouse model of MS to be compared in terms of higher efficacy to free GA.