Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disorder that affects more than 1% population in the world.1 RA patients without proper treatments may suffer from permanent disability and mortality and are at a high risk of cardiovascular disease and lymphoma.2–3 Currently, RA cannot be fully cured.4 Conventional medications for RA with glucocorticoids, nonsteroidal anti-inflammatory drugs, and disease-modifying anti-rheumatic drugs can only relieve the inflammation and swelling to a certain extent. In addition, the long-term therapy with these drugs is often accompanied by various side effects.
It is generally believed that the development of RA involves three stages; initiation phase (arthritis induction), pre-arthritis phase (mild symptom) and a peak phase (fully-developed arthritis).5 According to these stages of RA, the treatment begins prior to the arthritis induction is referred as a prophylactic therapy. The treatment starts before the mild symptom is described as a pre-arthritis therapy. The treatment after the fully developed arthritis is called as a therapeutic intervention. Recent studies have shown that the treatment started at the pre-arthritis stages can prevent the progression of RA to a full-blown condition, resulting in the improved efficacy in preventing disease chronicity.5–6 Therefore, the blocking of RA developments at the early stage of inflammation with efficacious and safe agents presents a promising strategy in the therapy of RA.
Ferulic acid (FA), an active component extracted from Chinese medicine herbs,7 has been commercially used as a food ingredient due to its strong antioxidant properties.8 FA also shows excellent anti-inflammatory properties in some animal models. For example, it was reported that FA was able to promote lymphatic drainages from inflamed joints and eventually reduced the joint inflammation and cartilage erosion in established arthritic models.9 After being incubated with hydrogen peroxide-stimulated chondrocytes, FA was able to reduce the level of various pro-inflammatory cytokines such as IL-1β, TNF-α, and MMPs.10 Furthermore, FA is also known as a powerful scavenger of free radicals,11 which is highly involved in the pathogenesis of RA.12 However, the application of FA as an anti-inflammatory drug is hindered by its instability and short half-life in vivo.13
Nanomedicine has been widely used as a promising therapeutic strategy for improving the bioavailability of drugs, prolonging the circulation time of drugs in vivo, and facilitating the passive targeting of drugs to inflamed tissues.14–19 Biomolecules including peptides and proteins have been extensively used as building blocks to form versatile nanocarriers for drug delivery.20–23 Compared to proteins, peptides are more attractive in form nanocarriers because they have lower immunogenicity and higher stability, which might avoid the unwanted immune response in vivo.24 Diphenylalanine (Phe-Phe) motifs are a short peptide, which is able to self-assemble into various nanostructures, depending on the condition under which the self-assembly occurs.25–28 Previously, Alves and coworkers exploited the application of Phe-Phe microtubes formed by the self-assembly of L-diphenylalanine as a drug delivery carrier for the sustained release of therapeutic compounds in vitro.29 Li and coworkers formed positively charged Phe-Phe nanoparticles with the diameter of ~ 500 nm by using oligomeric glutaraldehyde as a cross-linker of cationic NH2-Phe-Phe-NH2, which showed the improved anti-tumor effect in vitro.30 However, the relatively large size and positive charge of the Phe-Phe nanoparticles may hinder their applications in vivo. It was reported that nanoparticles with large sizes (≥ 500 nm) would undergo the rapid clearance in the blood31 and were unfavorable for passive targeting delivery.32 Furthermore, cationic nanoparticles potentially interacted with negatively charged cell membranes or proteins in circulation, subsequently resulting in the damage of cells and the aggregation of proteins.33–34
In this paper, we report the synthesis of PEGylated Phe-Phe nanoparticles with the diameter of ~ 170 nm by using glutaraldehyde (GTA) as a cross-linker of diphenylalanine NH2-Phe-Phe-COOH and poly (ethylene glycol) methyl ether amine (PEG5k-NH2). We find that the PEGylation endows the stability and circulation time of Phe-Phe nanoparticles in vivo. The cytotoxicity and cellular uptake of PEGylated Phe-Phe nanoparticles are tested. In vivo behaviors of PEGylated Phe-Phe nanoparticles including pharmacokinetics, biodistribution, and pharmacodynamics are systemically investigated. Finally, we study the therapeutic effect of FA-loaded PEGylated Phe-Phe nanoparticles for the treatment of RA at both the pre-arthritis and the fully established stages.