Folate-targeted nanoparticles show efficacy in the treatment of inflammatory arthritis.

Folate receptors (FR) comprise a family of glycosylphophatidylinositol (GPI)-anchored, high affinity receptors for folic acid (FA), and are the products of at least four different genes; FRα, β, γ, and δ (1, 2). Although much research has focused on the folate receptor-α (FRα) as a target for therapy and imaging in oncology, several recent studies have used the FRβ as a therapeutic target on macrophages in inflammatory diseases (3–5). In rheumatoid arthritis (RA), for example, FRβ has been shown to be specifically expressed on activated macrophages in inflamed joints, thus providing a target for the delivery of both therapeutics and imaging agents (6–9). Studies have demonstrated the specific internalization/accumulation of radionuclide-conjugated FA in inflamed joints of mice, rats, dogs, horses and even humans (3). The feasibility of using the FRβ to mediate the specific delivery of molecules is based on several unique biological characteristics of the FR. First, expression of the FRβ is specific to the myelomonocytic/macrophage lineages of hematopoietic cells, while there is negligible expression of this molecule on other blood cells such as lymphocytes, granulocytes or erythrocytes (9, 10). Second, elevated levels of functional glycosylated FRβ are expressed only on activated macrophages involved in inflammatory responses, but not on quiescent resident macrophages (5, 11). In addition, the FRα is known to be preferentially expressed at the apical (luminal) side (e.g., the urine side in kidney tubules and the air sac side of the lungs) of normal tissues; therefore it is possible that there will be minimal FRα binding of circulating folate-targeted drugs in normal tissues (12). Finally, although free FA and MTX can access normal cells through the ubiquitously expressed reduced folate carrier (RFC), uptake through this channel does not occur when FA or MTX are conjugated to a macromolecule like a nanoparticle. These findings together suggest that activated macrophages are the only predominant normal cells that could specifically bind and internalize folate targeted nanoparticle drugs from circulation through the FRβ. Methotrexate (MTX) is an “antifolate” drug that competes with FA for binding to the dihydrofolate reductase enzyme and blocks the one-carbon transfer reaction used in the biosynthesis of nucleotides. This inhibits DNA formation and results in reduced cell proliferation and induction of apoptosis. For over two decades MTX has been employed as a disease-modifying anti-rheumatic drug (DMARD) for the treatment of RA, either as a single agent or in combination with biologics such as TNF inhibitors (13, 14). The mechanisms by which MTX suppresses arthritis are complex and may extend beyond the inhibition of DNA synthesis in inflammatory cells. It may include increased release of adenosine, altered production of cytokines, formation of reactive oxygen species, T cell apoptosis, and suppression of B cell responses (4, 15, 16). Several studies have suggested that at least one mechanism for MTX activity in RA is through anti-proliferative effects on activated macrophages following specific uptake into these cells via the FRβ (4, 5, 9, 10, 13, 15, 17–19). Unfortunately, about one-third of RA patients respond poorly to MTX while another third develop toxic side effects. Patients with increased side effects may have polymorphisms in proteins such as RFC and 5,10-methylenetetrahydrofolate reductase involved in the transport and metabolism of free MTX (20–22). Since the side effects of the MTX are the result of its action on normal cells, either preventing the uptake of MTX in normal cells or specifically targeting MTX to inflammatory cells independent of the RFC may prevent these problems. Poly(amidoamine) (PAMAM) dendrimers have been extensively employed as a macromolecular delivery platform and have several attractive properties for this purpose (Figure 1). These properties include uniformity, biocompatibility, defined structure and the capability for chemically coupling multiple molecular entities to primary surface amino groups (23–26). Many biological functions of molecules coupled to the dendrimer’s surface are retained with a variety of molecules including hormones such as FA (27, 28), peptides (29), antibodies (30), chemotherapeutic drugs (28, 31), or an apoptosis-sensing fluorochrome (32). Importantly, prior studies from our laboratory have shown that dendrimers bearing FA can target FRα-expressing xenograft tumors in mice and increase the therapeutic index of carried MTX more than 10-fold (31). In another report, the anti-inflammatory drug indomethacin (IM) encapsulated in a generation 4 dendrimer-FA conjugate has been shown to preferentially target inflamed tissues in rats, presumably by targeting FRβ in macrophages (33). In the present study, we investigated using FA-bearing G5 dendrimers to target macrophages in vitro and deliver MTX as an anti-inflammatory agent in a collagen-induced arthritis model in rats. Figure 1 Schematic representation of the structures and synthesis of different generations of PAMAM dendrimers, starting from the ethylene diamine (EDA) core, and that of the functionalized dendrimer conjugate G5-FA-MTX. The details of the synthesis steps and ...