Synovial fibroblast‐targeting liposomes encapsulating an NF‐κB‐blocking peptide ameliorates zymosan‐induced synovial inflammation

Abstract Synovial fibroblasts (SFs) play a crucial role in the inflammatory process of rheumatoid arthritis (RA). The highly activated NF‐κB signal in SFs is responsible for most of the synovial inflammation associated with this disease. In this study, we have developed an SF‐targeting liposomal system that encapsulates the NF‐κB‐blocking peptide (NBD peptide) HAP‐lipo/NBD. HAP‐lipo/NBDs demonstrated efficient SF‐specific targeting in vitro and in vivo. Our study also showed a significant inhibitory effect of HAP‐lipo/NBD on NF‐κB activation, inflammatory cytokine release and SF migration capability after zymosan stimulation. Furthermore, the systemic administration of HAP‐lipo/NBDs significantly inhibited synovial inflammation and improved the pathological scores of arthritis induced by zymosan. Thus, these results suggest that an SF‐targeting NF‐κB‐blocking strategy is a potential approach for the development of alternative, targeted anti‐RA therapies.


| INTRODUCTION
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammation in the synovium and the destruction of cartilage and bone. 1 One of the most striking features of RA inflammation is the hyperplasia of synovial fibroblasts (SFs) in the synovial lining. In healthy joints, SFs secrete synovial fluid and extracellular matrix and provide structure to the joint. 2 However, SFs transform into principal effector cells in RA due to their ability to degrade the extracellular matrix, to provide chemoattractant cytokines and to activate parenchymal cells and infiltrating immunocytes. [2][3][4] The behaviour of SFs is regulated by multiple intracellular pathways and involves interferon regulatory factors, activator protein-1, mitogen-activated protein kinase and the nuclear factor-kappa B (NF-jB). 2,5 The highly activated NF-jB signal in RA is responsible for the pathological process of RA. 6 NF-jB regulates not only proinflammatory genes such as TNF-a, IL-6 and IL-8 but also the transcription of adhesion molecule-1 and matrix-degrading enzymes (MMP-3, MMP-9, etc.). 7,8 Moreover, NF-jB provides a key survival signal that suppresses apoptosis in SFs. 2 Therapeutic agents targeting NF-jB have exhibited various degrees of effectiveness in arthritis. However, few of these compounds are SF-specific, and some deleterious effects have been reported. 9 Therefore, the development of compounds that target SFs may complement current therapies and avoid major side effects. 10 Nanoparticles hold significant The first three authors contributed equally. promise for resolving this challenge as they can be functionalized to confer specific targeting to their encapsulated therapeutic agents. 11,12 The peptide HAP-1 (SFsHQFARATLAS) has demonstrated specificity for SFs. 13 Therefore, delivering nanoparticles coated with HAP-1 may target NF-jB inhibition to inflamed joints and reduce systemic toxicity. Currently, multiple steps of NF-jB activation can be targeted (IKKs, IjBa or p65/p50 subunit) with various available approaches, that is small molecule peptides or nucleic acids. 14,15 The NEMO-binding domain (NBD) peptide is a classic NF-jB inhibitor that can specifically bind to the NEMO domain and interfere with IjB kinase (IKK) complex formation. 16 Therefore, we hypothesize that liposomes coated with HAP-1 and loaded with the NBD peptide (HAP-lipo/NBD) may be able to target and inhibit NF-jB in SFs of the inflamed synovium, thereby alleviating arthritis. In this study, we describe an SF-specific liposome with inhibitory activity against NF-jB and evaluate the therapeutic potential of this nanoparticle in the treatment of inflammatory arthritis.

| Cell culture
Synovial fibroblasts were obtained from the synovial tissue of patients undergoing total keen arthroplasty who met the American College of Rheumatology classification criteria for RA. 17 Informed consent was obtained from the patients, and the experiments in this study were carried out according to the World Medical Association Declaration of Helsinki. Isolated synovial tissues were digested, and single-cell suspensions were obtained as previously described. 18 The cells were cultured at 37°C with 5% CO 2 in DMEM supplemented with 2 mmol/L L-glutamine, 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin. All the experiments were conducted using synoviocyte cultures from the fourth to seventh passage. The HAP-1 and NBD peptides were purified to more than 95% purity using high-pressure liquid chromatography. 19 The HAP-lipo/NBD nanoparticles were prepared according to previously described methods. 20 HAP-1 peptides were attached to the distal end of the DSPE-

| Electrophoretic mobility shift assays (EMSA)
The NF-jB probe (5 0 -AGTTGAGGGGACTTTCCCAGGC-3 0 ) was labelled with [c-32P] ATP by T4 polynucleotide kinase (Promega) and was purified. Nuclear extract from SFs (20 lg) was mixed with 2 lL of binding buffer and kept on ice for 10 minutes. Then, the labelled probe (0.0175 pmol) was added to the mixture and incubated at room temperature for 30 minutes. A portion of the mixture (20 lL) was loaded onto a 4% native polyacrylamide gel prepared in 0.59 Tris-borate-EDTA and was electrophoresed for 2.5 hours. After being dried, the gel was subjected to autoradiography (Bio-Rad, USA).

| Western blot analysis
The SFs were treated with HAP-lipo/NBDs (NBD peptide concentration at 0.05 mg/mL) for 2 hours and then stimulated with zymosan (0.1 mg/mL) for 45 minutes. The whole cell lysates were separated on SDS-PAGE gels and then transferred to a PVDF membrane. The blots were usually incubated at 4°C overnight with the primary antibodies anti-IjBa (1:500 dilution) and anti-p-IjBa (1:200) (Santa Cruz, USA). The blots were then incubated with a secondary labelled antirabbit IgG antibody (1:1000) at room temperature for 2-5 hours.
Finally, the proteins were visualized by radiography after reacting with an enhanced chemiluminescence reagent (GE Healthcare, UK).
Each blot shown in the figures represents the findings from at least three similar independent experiments.

| NF-jB DNA-binding activity
The binding of NF-jB to DNA was measured in nuclear extracts with the TransAM NF-jB p65 and p50 Assay Kits (Active Motif, Japan). Nuclear extracts were collected using a Nuclear Extract Kit (Active Motif). Proteins were quantified using the BCA method and subjected to an ELISA-based NF-jB p65 or p50 transfactor assay.
All the procedures were performed according to the manufacturer's instructions.

| Cell proliferation assays
The cytotoxicity of HAP-lipo/NBDs was assessed using a water-soluble tetrazolium assay (WST-1; Roch Diagnostics, Germany). SFs (5 9 10 4 cells/well) were incubated with various concentrations of HAP-lipo/NBD for 24 hours. The cells were exposed to WST-1 for 3 hours, and then, the absorbance values were measured at 450 nm.

| Histology
Rat knee synovial tissues harvested from dissected knees were fixed in 4% paraformaldehyde, stained with X-gal and then fixed in 10% formalin. The formalin-fixed tissues were embedded in paraffin, sliced into 5-mm sections, stained with X-gal and counterstained with eosin.

| Enzyme-linked immunosorbent assay (ELISA)
The expression levels of IL-6 and IL-8 were determined in the harvested supernatants using corresponding ELISA kits. The synovium of the knee joint was collected, and the expression levels of IL-1b, IL-6, MMP9, MMP2 and IL-8 in the synovial lysates were measured using commercially available ELISA kits (Boster, Wuhan, China) according to the manufacturer's instructions. All samples were assayed in duplicate.

| Statistical analysis
Data are expressed as the means AE SD. Differences between multiple groups were evaluated by ANOVA, followed by Dunnett's post hoc test or Tukey comparisons. For analyzing the swelling of the knee joints, repeated measure ANOVA was used to determine statistical significance. The results were considered statistically significant at P < .05.

| Characteristics of the HAP-lipo/NBD
The targeting polypeptide HAP-1 (SFHQFARATLAS) was synthesized and covalently conjugated to liposomal nanoparticles encapsulating NBD peptides ( Figure 1A). The zeta potential of the HAP-lipo/NBD was 34.6 AE 7.06 ( Figure 1B). Analysis of the HAP-lipo/NBD nanoparticles by TEM ( Figure 1D) revealed that they had a uniform spherical shape with a diameter of 97.26 AE 33.6 nm ( Figure 1C).

| SF-specific targeting of HAP-lipo/NBD in vitro and in vivo
We proceeded to investigate the in vitro targeting and transmembrane capacity of HAP-lipo/NBDs. Figure 2A  Next, the effect of HAP-lipo/NBD (NBD at 0.05 mg/mL) on NF-jB activity was evaluated in zymosan-activated SFs. Figure 3C shows that the induction of IjBa phosphorylation was readily detected in cytoplasmic extracts prepared from SFs stimulated with zymosan compared with that of unstimulated SFs. Treatment with HAP-lipo/NBD (NBD at 0.05 mg/mL) for 6 hours significantly inhibited the phosphorylation of IjBa. Zymosan-induced NF-jB p65 and p50 DNA binding were reduced significantly after HAPlipo/NBD (NBD at 0.05 mg/mL) treatment for 4 hours ( Figure 3D).

| DISCUSSION
Synovial fibroblasts in the synovial lining play a crucial role in producing both cytokines that perpetuate inflammation and proteases that contribute to cartilage destruction. 21,22 Moreover, increased NF-jB activity has been linked to the pathological behaviour of SFs. 2 Therefore, NF-jB in SFs is a potential target for the development of agents that treat synovial inflammation. 15 In this study, we demonstrated that the inhibitory effect of HAP-lipo/NBDs on synovial inflammation is through the targeted suppression of NF-jB activation in SFs.
Liposomal nanoparticles have emerged as one of the optimal drug carriers for treating RA. 23 | 2453 spleen, augmenting liposome localization at the inflamed site through the enhanced permeability and retention effect. 11 A previous study showed that PEG-coated liposomes, due to their small size (90-100 nm), are optimal for penetrating inflamed arthritic tissue, resulting in greater therapeutic efficacy. 25 Importantly, liposomal nanoparticle surfaces can be modified to achieve the selective delivery of encapsulated drug to specific target cells in RA. 11 Therefore, modified liposomes that target SFs could potentially provide an effective treatment strategy for controlling synovial inflammation.
Increased NF-jB activity has been linked to various inflammatory disorders. 26 In RA, NF-jB is overexpressed in the inflamed synovium.
NF-jB activation is observed in SFs, and both p50 and p65 are highly expressed in the cells of the synovial lining. IKKb is the primary signalling molecule responsible for the pro-inflammatory cytokine-induced IjBa phosphorylation and the subsequent activation of the classical NF-jB complexes containing the p50 and p65 subunits. 27 Thus, IKKb has emerged as an attractive target due to its ability to regulate inflammatory responses in SFs. 2 The NBD peptide has been shown to block the association of NEMO with the IKK complex, which can inhibit both NF-jB DNA-binding activity and target gene expression. 28 Therefore, the activity of SFs may be efficiently inhibited by the NBD peptide. Our in vitro study demonstrated that HAP-lipo/NBDs effectively blocked IjBa activation.
Blocking IKK activity and IjBa phosphorylation with HAP-lipo/NBDs also reduced the transcriptional activation of NF-jB. The effective inhibition of NF-jB-mediated DNA-binding activity was further proven in our experiment. Moreover, the expression levels of genes downstream of NF-jB, such as IL-6 and IL-8, were also inhibited by HAP-lipo/NBD treatment. These data suggest that it is possible to use this therapy for treating synovial inflammation in vivo.
Several NF-jB inhibitors have been used to treat inflammatory arthritis. However, non-specific NF-jB inhibition may lead to side effects that decrease the drug's therapeutic efficacy. 29 Glucocorticoids, the most common anti-RA drugs, are considered to be the most powerful non-specific inhibitors of NF-jB but lead to osteoporosis and dysfunction of the hypothalamic-pituitary-adrenal axis. 30 Therefore, there is a clear need for anti-inflammatory agents that are cell specific and have a lower potential toxicity. To achieve these goals, the "active targeting" technique can be employed. The  lipo/NBDs did not lead to significant toxicity, which is possibly due to its cell-specific targeting and lower dose of therapeutic peptide.
These data suggest that HAP-lipo/NBD is an SF-targeting liposome that incorporates NBD peptides, a potential therapeutic agent for ameliorating synovial inflammation.
Zymosan-induced arthritis exhibits the same characteristics as RA, with pro-inflammatory cytokines, inflammatory cell infiltration and hyperplasia being extensively involved in the pathogenesis of this disease. 33,34 In the inflamed joint, SFs are major cytokine producers. The  35 NF-jB plays a key role in the regulation of inflammatory genes in SFs. 7 In this study, HAP-lipo/NBDs suppressed the zymosan-induced synovial inflammation, as demonstrated by reduced levels of NF-jB targets, including IL-6, IL-8 and IL-1b. The resulting decrease in inflammatory cytokines further reduced the lymphocyte and neutrophil infiltration into the synovium and synovial cavity. MMP-9 is an important mediator that leads to RA synovial fibroblast survival, proliferation, migration and invasion. 36 Thus, the inhibited production of MMP-9 in the in vivo study suggests that HAP-lipo/NBD is an effective agent for disrupting the SF-mediated bone-harming behaviour. 37 Furthermore, the pathological analysis showed that HAP-lipo/NBDs effectively inhibit cell hyperplasia in the synovial lining. The suppressed SF hyperplasia may be attributed to the disruption of inflammatory networks.
In summary, our study shows that HAP-lipo/NBDs, SF-targeting liposomes that incorporate an NF-jB blocking peptide, inhibit NF-jB activity in SFs and efficiently attenuate the zymosan-induced synovium inflammation. These results suggest that the SF-targeting, NF-jB blocking strategy is a potential approach for the development of targeted inhibitors as alternative anti-arthritic therapies.