Recent approaches for targeted drug delivery in rheumatoid arthritis diagnosis and treatment

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease with complex pathology characterized by inflammation of joints, devastation of the synovium, pannus formation, bones and cartilage destruction and often is associated with persistent arthritic pain, swelling, stiffness and work disability. In conventional RA therapy, because of short biological half-life, poor bioavailability, high and frequent dosing is required. Thereby, these anti-RA medications, which unable to selectively target affected zone, may cause severe side effects in extra-articular tissues. Today, nanotechnology has emerged as promising tool in the development of novel drug delivery systems for the treatment and diagnosis of intractable diseases such as RA. Active targeting in RA nanomedicine has also been introduced a successful way for facilitating specific uptake of therapeutic agents by the disease cells. In this review, it is attempted to describe various targeted drug delivery systems (localized and receptor-based) used for RA diagnosis and therapy. Then, we highlight recent developments related to various non-viral gene delivery systems for RA gene therapy

Keywords:

• Gene therapy
• liposomes
• rheumatoid arthritis
• nanoparticles
• active targeting
• folic acid
• hyaluronic acid
• magnetic nanoparticles

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory and destructive arthropathy affecting joints, surrounding connective tissue of skin, bones and muscles. It caused inflammation of joints, synovial hyperplasia, pannus formation, bones and cartilage destruction and often is associated with persistent arthritic pain, swelling, stiffness and work disability. RA occurs worldwide affecting 1% of adult population in Europe and USA and 0.5% in other geographic area [1]. The incidence of RA in women is higher than in man with a ratio of approximately 3:1 [2]. Infiltration and activation of various populations of inflammatory cells like CD₄ helper T cells, B cells, dendritic cells, macrophages and mast cells along with release of matrix metalloproteinase and cytokines such as tumor necrosis factor (TNF), IL1, IL6 have a main contribution in development of RA [3]. Serious angiogenesis and migration of leukocytes from the blood cells into synovial tissue are also seen [4]. The goal treatment of RA is to relieve pain and inflammation and improve or maintain joint function. Rapid diagnosis and initiation of treatment before irreversible joint damage is required to maintain productive and normal active life. The conventional drugs used in the treatment of RA are non-steroidal anti-inflammatory drugs (NSAIDs) and steroids that control symptoms and disease-modifying anti-rheumatic drugs (DMARDs) which prevent joint damage. DMARDs include antimalarials (chloroquine, hydroxychloroquine), sulfasalazine, gold compounds (auranofin, sodium aurothiomalate), penicillamine and immunosuppressants (methotrexate, adalimumab, anakinra, azathioprine, cyclosporin, cyclophosphamide, etanercept, infliximab and leflunomide) [5]. It is thought that most DMARDs inhibit the release or activity of cytokines involved in maintaining the inflammatory process, although other actions may also contribute. Indiscriminate distribution of most of these agents by systemic administration and lake of specificity to the organs/tissues affected RA, may be resulted in extra-articular adverse effects. In addition, because of short half-life and insufficient concentration of therapeutic agents in site of action, high and frequent dosing is required which may be resulted in severe side effects as well as high cost [6]. To overcome these limitations, development of new agents or therapeutic strategies has been proposed. The best example of new therapeutic strategies is using new drug delivery systems such as nanoparticles (NPs) [7]. Recently NPs have been proven effective for specific delivery to the site of inflammation. Since NPs allow specific delivery of therapeutic agents through loose vasculature in inflamed areas like enhanced permeation and retention effect in tumour targeting drug delivery. Encapsulation of the drug in the NPs can modify drug pharmacokinetic and bio-distribution and allow specific drug delivery to site of action. Moreover, certain cell receptors are overexpressed on cells involved in pathogenesis of RA; hence, active targeting is possible by binding special ligands on the surface of NPs directed to receptors expressed on the target disease cells [8]. In the previous review, the application of nanotechnology in drug delivery to RA was discussed [9]. The main objective of this review is to describe various targeted drug delivery systems (localized and receptor-based) used for RA diagnosis and therapy. Some representatives of targeted delivery systems for RA are listed in Table 1.

Table 1. Summary of targeted delivery systems for RA to increase targeting effect.

Targeting agent	Receptor	Delivery systems	Drug	References
Folic acid	Folate receptors	PLGA nanoparticles	Dexamethasone phosphate	[10]
		Liposome	Methotrexate	[11]
		Poly(amidoamide) dendrimers	Methotrexate	[12]
		Poly(amidoamide) dendrimers	Indomethacin	[13]
		Albumin	Etoricoxib	[14]
		Dextran	Methotrexate	[15]
		Chitosan	IL-1Ra DNA	[53]
Hyaluronic acid	CD44 receptor	Hyaluronic acid – 5β-Cholanic acid	γ-Secretase inhibitors	[16]
		Biomineral-installed hyaluronan nanoparticles	Methotrexate	[17]
		Ionic complex systems based on hyaluronic acid and PEGylated TRAIL	TRAIL	[18]
		HA- 1,2-distearoylphosphatidylethanolamine	Triamcinolone	[34]
Dextran sulfate	Macrophage scavenger receptor class A	Dextran sulphate –polycaprolactone	Cy5.5	[19]
		Dextran sulphate -5β-cholanic acid	Methotrexate	[20]
RGD	Integrin receptor	Albumin	SB202190	[23]
		PEG-Liposomes	Dexamethasone phosphate	[24]
		Liposomes	CP or prednisolone	[25]
		Gold half shell PLGA NPs	Methotrexate	[41]
		PLGA gold/iron/gold half-shell NPs	Methotrexate	[48]
HAP 1		PTD-(KLAK)2 conjugate	(KLAK)2	[27]
		Liposomes	CP or prednisolone	[28]
Vasoactive intestinal peptide	VIP receptors	DSPE-PEG3400	Camptothecin	[29]
CD 163 monoclonal antibody	Haemoglobin scavenger receptor CD163	Liposome	Doxorubicin Murine interleukin-10	[30]
Tuftsin	Fc and neuropilin-1(NP-1) receptors	Alginate NPs	Plasmid DNA	[58]

Ligand-based active targeting

Folic acid

Activated macrophages greatly influence in initiation, maintenance and pathogenesis of this disease by producing several proinflammatory cytokine such as TNF-α, IL-1, IL-6, chemokines, prostaglandins, metalloproteinases and reactive oxygen species (ROS). Folate (FA) receptors β are highly overexpressed on these activated macrophages but are limited in normal tissues. Therefore, FA conjugate was considered as a therapeutic to target drug to this population of pathologic cells. So, many studies on macrophage-targeted therapy have made. As an example, Cao et al. [10] encapsulated dexamethasone phosphate (DEXP) in PLGA NPs conjugated with FA. It was shown FA targeted PLGA NPs significantly increased cellular uptake of DEXP in activated macrophages and reduced the production of pro-inflammatory cytokines (IL-6 and TNF-α) and nitric oxide (NO) from activated macrophages but cellular uptake of targeted and non-targeted ones was low in normal macrophages. Methotrexate (MTX) is an FA anti-metabolite available to treat psoriasis, cancer, Crohn’s disease, multiple sclerosis and RA. MTX as a DMARD is the first choice in RA patients either alone or in combination with other biologics. MTX inhibit production of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-15 and IL-18 and delays joint destruction during inflammation. However, long-term administration of MTX results in serious side effects including gastrointestinal distress, bone marrow suppression, loss of appetite and hepatotoxicity. To improve efficacy and safety of MTX, Nogueira et al. [11] developed MTX loaded liposome targeted by FA using SP-DS3 peptide linker for application in rheumatoid arthritis. The in vitro result showed FA targeting liposomes increased the specific internalization in activated macrophages expressing FA receptor-β (THP-1 retrovirally transformed to overexpress FA receptor) compared to cells with negligible FA receptor expression (THP-1 macrophages and Jurkat T cells). In vivo study in mice bearing arthritis showed that FA targeted liposomes strongly accumulated in joints and is markedly superior in preventing the onset of arthritis in collagen-induced arthritis (CIA) model. Because of the high affinity of MTX to bind to FA receptor β, in other study, Qi et al. [12] compared FA targeted poly(amidoamide) (PAMAM) dendrimers-MTX conjugate and PAMAM dendrimers-MTX conjugate for inflammatory arthritis treatment. The FA-MTX-PAMAM conjugate showed dose-dependent binding and specific uptake in LPS activated NR8383 cells and peritoneal macrophages. However, PAMAM-MTX conjugate was binded to the macrophages even higher than that of PAMAM-FA-MTX. This finding was explained with aggregation of dendrimers with many planar molecules due to pie stacking between the molecules on different dendrimers. Since FA is planar, the addition of FA caused the molecules to aggregate more and reduced cell binding. In vivo studies in an adjuvant-induced inflammatory arthritis model demonstrated the potential of the PAMAM–MTX conjugate in suppressing arthritis and reducing the spleen toxicity of free MTX. All results indicated MTX might serve as both a targeting ligand and therapeutic moiety. In another study, Chandrasekar et al. [13] developed anionic pegylated PAMAM dendrimers with a different number of FA-PEG moiety for specific delivery of indomethacin to inflammatory region. Pharmacokinetic studies showed a higher area under the curve (AUC), longer mean residence time (MRT) and t_½ for both targeted and non-targeted NPs when compared to free drug. However, formulation with higher FA-PEG arms exhibited shorter blood circulation time in plasma because PEG terminated anionic dendrimers have less surface interaction and hence had longer blood circulation. But, in FA-PEG-dendrimers conjugate, amino (NH₂) groups of FA ligand projected outwards and easily accessible at the surface and possibly has more surface interaction. Tissue distribution studies in arthritic rats indicated better drug targeting efficiency and higher relative exposure of FA-PEG conjugates in the inflammatory region compared non-targeted conjugates. The FA as targeting ligand was also used in albumin NPs to increase the specific delivery of etoricoxib (ETX) in rat carrageenan induced arteritis models [14]. The albumin NPs were prepared by desolvation method and FA was conjugated at the surface of NPs using bifunctional crosslinker1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). FA conjugated NPs showed remarkable success in delivering ETX in tissue sac of inflamed joint as compared to free drug and non-targeted NPs due to strong affinity of FA conjugated ETX NPs towards activated macrophages cells. Analysis of drug distribution showed longer circulation property and maximum residence time of FA-ETX-NP as compared to ETX and ETX-NP. In addition, the FA-NPs resulted in a high concentration of the drug in an inflamed joint probably due to preferred uptake by mononuclear phagocytic system and reduced entry of drug in non-targeted organ. This result showed the targeting properties of NPs to increase the therapeutic effectiveness and reduce the dose of drug. In another study, Yang et al. [15] prepared FA targeted dextran (Dex)-MTX conjugate to target activated macrophages (activated RAW 264.7 cell line). The average diameter of Dex-MTX conjugate and FA/Dex-MTX nanoparticles were 82 and 95 nm, respectively. These FA targeted Dex-MTX conjugate exhibited higher cellular uptake and a little higher toxicity compared to Dex-MTX conjugate in RAW 264.7 cells with overexpressed FA receptor. The strongest fluorescence intensity signal was seen in the affected joints of CIA mice treated with FITC-Dex-g-MTX/FA compared with mice injected with FITC-MTX and FITC-Dex-g-MTX (Figure 1). The in vivo studies showed after intravenous (IV) administration of Dex-g-MTX, FA/Dex-g-MTX and free MTX in the CIA mice, FA/Dex-g-MTX showed most potent inflammatory inhibition indicating active targeting properties of Dex-g-MTX/FA.

Figure 1. Fluorescence images of inflamed joints (a) and fluorescence intensity detected from affected joints of CIA mice treated with administration of Dex-MTX, FA/Dex-MTX, free MTX and normal saline intravenously (Reproduced with permission from Journal of Materials Chemistry (b) [15]).

Hyaluronic acid

Hyaluronic acid (HA) is a natural polysaccharide has attracted much attention as a targeted drug delivery because it is biocompatible and binds to the CD44 receptor, which is over-expressed in cancer cells and synovial lymphocytes, macrophages and fibroblasts of RA patients. Among these cells, activated macrophages are rich in the inflamed joints of RA, and contribute to joint destruction. In addition, cartilage protective and lubricant effects of HA was reported in synovial fluid [16]. HA-based nanoparticulate systems were developed for targeted drug delivery in RA. Notch receptors (Notch 1–4) and their ligands (Jagged 1–2, Delta 1,3,4) are responsible for inflammation and angiogenesis in RA patients and are overexpressed in synovial tissue of these patients. γ-Secretase inhibitors block the γ-secretase-mediated cleavage of notch receptors after notch–ligand interaction, Therefore, they prevent the release of notch intracellular domain into nucleus and transcription of Notch target genes. Heo et al. [16] prepared polymeric micelles using HA-5β-cholanic acid (CA) for specific delivery of γ-secretase inhibitors (DAPT). In vitro study showed more cellular uptake of Cy5.5-labelled HA-NPs in activated macrophages as compared to non-activated macrophages. In addition, strong NIR signals were detected in the inflamed joints of the CIA model mice after IV administration of Cy5.5-labelled HA-NPs but no significant signals were observed in the wild-type mice. In CIA model, unlike free DAPT, DAPT loaded HA-CA NPs resulted in high therapeutic efficacy indicating high target ability of these NPs in RA. In a separate study, Alam’s group [17] developed PEGylated nanomicellar system composed of HA-CA copolymer (P-HANP) as carrier to improve target selectivity of MTX into the arthritic tissues. To improve bio-stability of NPs in physiological pH and the controlled release of MTX into acidic microenvironment of inflamed target tissues, calcium phosphate as ionizable minerals was precipitated on the shell of NPs (MP-HANP). In vitro release study revealed that MTX release was pH dependent and increased as pH of medium decreased due to demineralization of NPs’s shells in response to environmental pH change. In vitro uptake study in macrophages demonstrated in addition to CD44, stabilin-2 and RHAMM receptors are also involved in uptake of MP-HANP via receptor-mediated endocytosis. In vivo study using CIA, mice showed the therapeutic efficacy of free MTX and MP-HANP-MTX was not significantly different at a low dose (3 mg/kg). Increasing dose of free MTX (50 mg/kg) improved efficacy in CIA mice but death of all mice happened within 2 weeks of treatment. In contrast, MTX loaded MP-HANPs (50 mg/kg) ameliorated inflammatory arthritis with remarkable safety. TNF-related apoptosis-inducing ligand (TRAIL), a member of TNF family, has attracted a great attention for treatment of RA by inducing apoptosis of activated lymphocytes and synoviocytes through DR 5-mediated signalling pathways. To increase half-life of TRAIL, a long-acting delivery, ionic complex systems based on HA and PEGylated TRAIL, for treatment of RA was developed [18]. They demonstrated that ionic complexation of TRAIL with HA did not affect the biological activity of TRAIL and improved its pharmacokinetics. The in vivo biodistribution and diffusion kinetics study indicated PEG-TRAIL is released from PEG-TRAIL/HA complex in a sustained manner after subcutaneous injection into the mice. In addition, using an experimental rodent RA model, PEG-TRAIL/HA formulations showed outstanding therapeutic effect due to a combination of PEG-TRAIL’s immunomodulatory functions with its sustained systemic delivery modality.

Dextran sulphate

Dextran sulphate (DS) is a polyanionic polymer made up of 1 → 6 linked α-d-glucopyranosyl units. The application of DS has been widely investigated in the pharmaceutical study due to interesting features such as biodegradability, biocompatibility, non-toxicity, non-immunogenicity and the potential for chemical modification. Since DS is a typical ligand for macrophage scavenger receptor class A (SR-A), Kim et al. [19] developed nanomicellar system composed of hydrophilic DS as the targeting ligand and hydrophobic polycaprolactone (PCL) as the hydrophobic segment via click chemistry method (Figure 2). The nanomicelles were spherical in shape with an average diameter of 200 nm and zeta potential of −9.8 mV. High targetability of DS-b-PCL nanomicelles for the inflamed joint of CIA mice was demonstrated in vivo using live animal imaging. During the whole period of study, Cy5.5-labelled DS-b-PCL NPs created much stronger fluorescent signals in legs of CIA mice, compared to those of wild-type mice after IV administration. Overall, this study demonstrated the potential of DS-b-PCL NPs as the constituents of diagnostic and therapeutic formulations for RA. In another approach, Heo et al. [20] incorporated MTX into nano micelles composed of DS-CA copolymer (DSNPs) and demonstrated specific uptake of DSNPs in LPS activated RAW264.7 macrophages via SR-A-mediated endocytosis. The IV administration of MTX loaded DSNPs in CIA mice showed improved clinical outcomes, including arthritis indices and paw thickness compared to free MTX presumably due to the accumulation of DSNPs in the inflamed joints via the leaky vasculature and more cellular uptake owing to the SR-A-mediated, active targeting mechanism of DSNPs.

Figure 2. Schematic illustration of targeting mechanism of DS-b-PCL nanoparticles as the constituents of diagnostic and therapeutic formulations for RA (Reproduced with permission from Royal Society of Chemistry [19]).

Mannose

Mannose receptors (MR) are highly effective endocytic receptors involved in the recognized glycosylated molecules with mannose terminus and endocytose or phagocytose ligand. MR are highly expressed on mature CD11b⁺F4/80⁺macrophages in synovial tissues of mice with CIA. Moreover, its expression is at a modest level on osteoclasts. Put et al. [21] showed ^99mTC labelled nanobodies against MR were able for visualize and quantifying inflammation in joint of CIA mice using SPECT/micro CT imaging. This result implied that MR might be a potential target in RA treatment.

Peptide-based active targeting

Angiogenesis is the development of new blood vessels. It is an essential process in body’s physiology and normal function such as embryogenesis, menstrual cycle and wound healing but it is a focus of intensive research because of its main role in the pathogenesis of several diseases such as cancer, inflammatory diseases like psoriasis, RA and diabetic retinopathy [22]. New blood vessels provide nutrient and oxygen to inflammatory cells and facilitate their infiltration into inflamed tissue. Endothelial cells have an essential role in angiogenesis and are important target cells for the treatment of angiogenesis. Integrins are considered as a target molecular for vascular targeting strategies because they are specifically overexpressed on angiogenic endothelial cells. It has been shown that RGD (Arg-Gly-Asp) sequence containing peptides interact especially with high affinity to integrin receptors and are considered as a special ligand for targeted drug delivery in endothelial cells [23]. Proinflammatory proteins like TNFα and IL-1β activate endothelial cells via the induction of p38MAP kinase and NFκB. The activated endothelial cells produce chemokines (IL-8, IL-6) which along with P-selectin, E-selectin, VCAM, ICAM cause leukocyte infiltration and mediate vascular remodelling. P38MAP kinase inhibitors are considered promising candidates for the treatment of chronic inflammation. However, their clinical uses are limited due to unacceptable side effects. Therefore, local delivery of the inhibitor to endothelial cells may overcome this problem. In a study conducted by Temming et al. [23], SB202190, an inhibitor of P38MAP kinase was attached through platinum-based linker to albumin. Then, RGD targeting ligand was attached to SB202190-albumin via bivalent 3.5 kDa PEG linkers to develop a therapeutic agent that inhibit inflammatory signalling pathways without affecting the viability of endothelial cells. The experiment demonstrated that RGD-PEG-SB202190-albumin inhibited the TNFα induced activation of HUVEC and secretion of proinflammatory cytokines, both at gene expression and protein levels but inhibitions was lower than free SB202190. This was explained with different uptake mechanisms and intracellular levels of RGD-PEG-SB202190-albumin and free SB202190. However, the authors of this study believed that this conjugate would improve the pharmacokinetic free SB202190 in vivo. Corticosteroids suppress inflammation and decrease adhesion of immune cells through down-regulate the expression of cellular adhesion molecules, cytokines and growth factors on endothelial cells. Additionally, they exert significant anti-angiogenic effects. So, corticosteroids are considered in vascular endothelial cells modulation. However, their use is limited because of considerable systemic toxicity and short systemic half-life. In another approach, Koning et al. [24] evaluated DEXP containing RGD peptide targeted liposomes in rat LPS induced arthritis. RGD-PEG-Liposomes showed high uptake in HUVECs. In vivo studies in a rat model of arthritis suggested 3-fold higher accumulation of RGD-PEG-Liposomes at the site of inflammation in comparison with RAD-PEG-Liposomes or PEG-Liposomes. The efficacy of RGD-DEXP-PEG-Liposomes was greater than either non-RGD-targeted DEXP-PEG-Liposomes or empty RGD-PEG-Liposomes in inhibiting the disease development and also strongly reduced the peak levels of arthritis severity during the entire course. Gerlag et al. [25] synthesized RGD coupled pro-apoptotic hepta peptide dimer, D(KLAKLAK)2 for targeting and destroying new blood vessels in inflamed joints. It was shown that after systemic administration in CIA rats, RGD-targeted peptide enters cells that express αvβ3 integrin, significantly decreased severity of arthritis and increased apoptosis of synovial blood vessels. Fibroblast derived type B synoviocytes (FLS) are mesenchymal cells characterized by the expression of UDP-glucose-6-dehydrogenase and CD 55 and have a key function in the maintenance of joint fluid volume and secretion of lubricating molecules such as hyaluronan into joint space. Activated FLS may produce increased amount of cytokines, chemokine and matrix-degrading enzymes, which can initiate joint damage and bone destruction. Mi et al. [26] showed ability of cationic peptides named protein transduction domain (PTD) to deliver apoptotic peptide (KLAK)2 in rabbit FLS but due to lack of tissue specificity in vivo, in further study [27], PTD-(KLAK)2 conjugate was linked to HAP 1 which able to deliver therapeutic agents specifically in proliferating synovial cells. HAP 1-PTD-KLAK2 caused remarkable synovial cell apoptosis in rabbit knee model of arthritis. Synovial cell apoptosis resulted in reduction of inflammation by inducing expression of anti-inflammatory cytokines and down-modulation of proinflammatory cytokines in resident macrophages and dendritic cells. As consequence inflammation and synovitis reduced. CP (GLRILLLKV) is a short peptide and effective immune suppressant that played the main role in the decrease of inflammation in adjuvant-induced arthritis rat model. Vanniasinghe et al. [28] encapsulated CP or prednisolone in liposomes. Then, liposomes were conjugated to RGD or HAP-1, which are expressed at high frequency on endothelial cells and FLS, respectively. A significant increase in accumulation of RGD or HAP targeted liposomes were seen in the affected joint of arthritis rat. Both CP or prednisolone loaded targeted liposomes were more effective in ameliorating arthritis compared to non-targeted ones but HAP targeted liposome containing CP or prednisolone increase significantly survival probability in arthritis rats compared to RGD ones. This study showed the ability of the novel FLS sequence to target liposomal drug delivery and suggests a substitute therapeutic approach for the treatment of arthritis. Vasoactive intestinal peptide (VIP) receptors are also overexpressed on key effector cells involved in RA such as activated T-lymphocytes, macrophages. Koo et al. [29] developed sterically stabilized micelles (SSM) composed of DSPE-PEG₃₄₀₀ as nanocarriers for camptothecin (CPT-SSM) in which VIP was conjugated as targeting ligand. It was shown that VIP-CPT-SSM performs anti-arthritic effects at lower CPT doses compared with CPT-SSM and CPT alone without systemic cytotoxicity. These results supported by additional observation that VIP-CPT-SSM reduced T-lymphocytes, macrophages and synoviocytes in significantly faster rate and greater number as compared with CPT-SSM and free CPT.

Antibody-based active targeting

One successful targeting strategy for RA could be coupling of NPs with antibodies with ability to recognize antigens on the surface of activated macrophages. CD163 is haemoglobin scavenger receptors overexpressed on the tissue-resident macrophages, inflammatory macrophages, TAMs and pro-angiogenic TIE2+ monocytes. In a study conducted by Ezerdot [30], efforts have been made to the development of long-acting liposomes targeting CD 163 receptors by hydrophobic linkage of CD 163 monoclonal antibody. Both flow cytometry and confocal microscopy confirmed specific uptake of CD163 targeted long-acting liposomes in CD163 expressing cells. High cytotoxicity was seen in CD163⁺ monocytes exposed to CD163 targeted liposomal doxorubicin compared to lymphocyte population confirmed the efficacy of CD163 targeted liposomes targeting macrophages.

Localized drug delivery approaches

Intra-articular (IA) injection is a method that physicians may use to treat inflamed joints when a small number of joints are affected, or for joints that do not respond to systemic medications. It has the advantage of increase local drug concentration at site of action, decreasing drug dose and minimize typical side effects of these drugs. In addition, IA administration would also facilitate the high efficient delivery of drugs with low oral bioavailability, including the delivery of recombinant proteins, therapeutic genes and inhibitory RNAs. Various glucocorticoid and HA formulations are currently available on the market for IA treatment options for RA. The main problems with this method are rapid clearance of injected agent from joints region, which decrease drug concentration at site of action so that frequent injections are required leading to infection, inflammatory events, joint disability and post-injection flare. The IA mean elimination half-lives of anti-inflammatory drugs after oral administration is about 1–5 h [31]. To increase drug retention time, reduction of drug clearance from joints and also increase patient compliance, different kind of controlled release delivery systems including microspheres, liposomes, NPs and hydrogels were developed. These sustained release systems for IA injection conserved effectively within the joint cavity, uptaked or kidnapped by synovial cells in the synovium [32]. Thakkar et al. [33] prepared celecoxib loaded solid lipid nanoparticles (SLN) by a hot melt homogenization technique. Faster clearance of drug from the joints and higher distribution of drug in liver, spleen, and kidney was seen after IA injection of celecoxib solution when compared with celecoxib loaded SLN. Rapid equilibration between the synovial fluid and plasma was the main reason for faster clearance of the drug solution from the joint and release of appreciable levels of drug into the systemic circulation. Retention of SLN in the inflamed joints was explained with their phagocytosis by macrophages. In addition, IA injection of celecoxib solution would result in the sudden exposure of the cartilage to high concentrations of celecoxib that could harm the cartilage. However, IA injection of SLN containing celecoxib led to the exposure of the cartilage to a lower concentration of the drug because of its slow-release behaviour. In another study, Saadat et al. [34] prepared triamcinolone loaded HA-1,2-distearoylphosphatidylethanolamine polymeric micelles by dialysis method. The micelles had a mean particle size of 186 nm. In vivo real-time imaging analysis showed encapsulation of triamcinolone increased retention time of drug in joint region in comparison to free drug that was explained with active uptake of drug loaded HA-based micelles by specific synovial cells that overexpressed HA receptors. Because of higher clearance of triamcinolone suspension, the peak plasma concentration of triamcinolone was found higher after IA administration of triamcinolone suspension as compared to micellar triamcinolone. In another approach [35], I.A. injections of chitosan/HA nanocomplex containing calcitonin in mice exposed to K/BxN serum resulted in a superior reduction in swollen joint diameter and cell infiltration compared to free calcitonin due to relative contributions of both HA and calcitonin in the resolution of inflammation. To increase therapeutic efficacy of indomethacin, Zhang et al. [36] encapsulated indomethacin in nano polymeric micelle based on amphiphilic polyphosphazene. Complete Freund adjuvant (CFA) induced ankle arthritis model was used in this study to evaluate the long-term therapeutic effect of drug-loaded micelles. IA administration of single dose of indomethacin-loaded micelles produced a sustained therapeutic efficacy with marked reduction of the knee swelling in adjuvant arthritis model compared to free drug at the same dose. The long-term therapeutic effect of indomethacin-loaded micelles was explained with more sustained release in the inflamed site probably due to low pH in rheumatic joints. Oral administration of multiple doses of indomethacin was also effective. However, the significant formation of gastrointestinal ulceration limits its application. Clodronate (CLO) is a bisphosphonate drug used in the treatment of osteoporosis, prevention and reduction of bone loss. Moreover, CLO has extra skeletal biological effects, such as anti-inflammatory and anti-arthritic activity, in both animal models and humans. However, its application is limited due to its poor bioavailability (1–2%), short half-life (around 2–3 h). To increase the retention of the CLO in the joint, improve its therapeutic index and decreasing the risk of the occurrence of side effects, Russo et al. [37] prepared chitosan-CLO NPs embedded in poloxamer gel for IA administration. In vitro release study showed incorporation of chitosan-CLO NPs in gel resulted in a decreased release rate compared with free CLO and chitosan-CLO NPs. In vitro study using LPS-stimulated THP1 cells showed that treatment with CLO increased pro-inflammatory markers (IL-1 and IL-8) even higher than the expression measured in non-pre-treated LPS-stimulated cells. In contrast, treatment with chitosan-CLO NPs reduced expression of IL-1 and IL-8 mRNA levels 2- and 10-fold, respectively, in comparison with non-pre-treated cultures. These results indicated the protective role of the NPs which inhibit the pro-inflammatory interaction between CLO and LPS on the THP1 cell line.

Gold nanoparticles

Gold nanoparticles (AuNPs) have been widely used for various biomedical applications thanks to excellent unique properties including biocompatibility, ease of synthesis, a simple modification of surface, tunable optical properties. AuNPs are being used as a therapeutic agent to treat several diseases such as RA. AuNPs have been shown to have an anti-angiogenic effect by binding vascular endothelial growth factor (VEGF) which plays a main role in the of RA pathobiology. In addition, AuNPs are a powerful antioxidant and can react with ROS. AuNPs also inhibit the receptor activator of nuclear factor_κB ligand (RANKL)-induced osteoclast formation, which results in the bone and cartilage erosion. AuNPs have become attractive as a nanoprobe and contrast agent for selectively detecting a target molecule and diagnosing the progress of RA. Gomes et al. [38] synthesized MTX conjugated gold nanoparticles to examine the anti-arthritic effect in vivo. The result of this study also indicated that MTX conjugated with AuNPs decreased both anti-inflammatory and pro-inflammatory cytokines level more effectively compared to free MTX in arthritis animal model. In Lee study [39], a novel RA treatment system using HA_AuNPs/Tocilizumab (TCZ) complex that can simultaneously inhibit both VEGF and Interleukin 6 receptor (IL-6R) was developed. IL-6 is a pleiotropic cytokine with a critical role in joint inflammation and pathogenesis of RA. IL-6 stimulates neutrophil migration, osteoclast maturation and VEGF-stimulated pannus proliferation resulted in synovitis and joint destruction. TCZ is a monoclonal antibody against the IL-6R and mainly used as an immunosuppressive drug for treatment of RA. HA coating increased the stability AuNPs and reduced its non-specific binding to serum proteins in the body. In vivo study using CIA animal models indicated that HA-AuNPs/TCZ complex has a better therapeutic effect than TCZ and HA-AuNPs, possibly because of the long-term synergistic dual effect by simultaneously target VEGF and IL-6R (Figure 3). Nanomaterials such as gold nanoshells and AuNPs also can absorb NIR light and produce local cytotoxic heat upon irradiation and releases its payload at the site of interest. Lima et al. [40] investigated the application of theranostic NPs in the treatment of RA. In this study, Pegylated-PLGA containing MTX and AuNPs (MTXPEG-PLGA-Au) were developed to achieve thermo-responsive controlled drug delivery. In vitro study using THP1 monocytes and differentiated macrophages showed the presence of AuNPs improved the cytotoxic effect of MTX loaded PEG-PLGA NPs. Anti-inflammatory activity study showed AuNPs pronounced anti-inflammatory effect of MTX and MTX-loaded PEG-PLGA-Au nanospheres suppressed secretion of pro-inflammatory cytokines in both THP1 monocytes and differentiated macrophages more than free MTX and AuNPs. In other study, Lee et al. [41] investigated RGD targeted gold half shell NPs containing MTX loaded PLGA NPs to simultaneously produce localized heat under NIR and deliver drug to site of inflammation (Figure 4). The average size of NPs ranged between 100 and 115 nm. The burst release under NIR light showed the MTX release from NPs can be controlled by NIR light (Figure 5). The NPs selectively accumulate in the inflamed joints after IV administration. The accumulation of RGD-MTX-PLGA-AuNPs in inflamed paws was higher than MTX-PLGA-Au NPs. In vivo study in CIA mice demonstrated that greater therapeutic efficacy of RGD-MTX-PLGA-AuNPs combined with NIR in a smaller dosage of MTX compared to conventional therapy with MTX. High levels of ROS and hyaluronidase are expressed in RA and highly metastatic tumour resulting in degradation of HA in the extracellular matrix. So that, for in vivo imaging of RA and tumour, Lee et al. [42], developed NIR fluorescence (NIRF) dye-labelled HA immobilized Au nanoprobes with a high degree of specificity for hyaluronidase. Enhancement level of fluorescence signal in arthritic joints and surrounding area of tumour as a result of liberating cleaved HA fragment demonstrated the applicability of HA immobilized Au nanoprobes as a sensitive molecular imaging probe for the local detection of HA degrading disease condition such as arthritic inflammation and tumour.

Figure 3. Mean clinical scores for arthritis paws treated with (a) PBS, (b) HA-AuNPs, (c) TCZ and (d) HA-AuNPs/TCZ complex (Reproduced with permission from American Chemical Society [39]).

Figure 4. (a) Schematic depicting preparation of RGD-MTX-PLGA-AuNPs. (b) Application of RGD-MTX-PLGA-AuNPs for in vivo RA treatment (Reproduced with permission from American Chemical Society [41]).

Figure 5. In vitro release of MTX from RGD-MTX-PLGA-AuNPs with or without NIR exposure (Reproduced with permission from American Chemical Society [41]).

Magnetic nanoparticles

Magnetic nanoparticles (MNPs) are a major class of nanoscale materials with the potential to revolutionize current clinical diagnostic and therapeutic techniques. Early researches in this field were dated back several decades and the recent surge of interest in nanotechnology has significantly expanded the breadth and depth of research into MNPs. For example, superparamagnetic iron oxide nanoparticles (SPIONs) are attractive materials that have been widely used in medicine for diagnostic imaging and therapeutic applications. Due to their unique physical properties and ability to function at the cellular and molecular level of biological interactions, this kind of materials are being actively assessed as the next generation of magnetic resonance imaging (MRI) contrast agents and as carriers for targeted drug delivery [43]. MNPs have been designed with dual functionality as imaging agents and drug carriers [43]. These advantages could be useful in RA drug delivery and diagnosis. MNPs retained in the inflamed joints using an external magnetic field might prolong the local release of an anti-inflammatory drug. Preclinical studies revealed some of the shortcomings of magnetic drug targeting technology, such as poor penetration depth and diffusion of the released drug from the disease site. These limitations can be overcome by active targeting through attachment of high affinity ligands onto the MNP surface [44]. With the ability to utilize simultaneously magnetic drug targeting and specific targeting of disease biomarkers, therapeutic agents are remotely directed to the disease site. This led to reduce dosage and the deleterious side effects associated with non-specific uptake of drugs by healthy tissue. Other significant challenge associated with the application of these MNPs is their behaviour in vivo such as bio-distribution and vascular penetration of MNPs [43]. The physicochemical properties of NPs directly affect their subsequent pharmacokinetics and biodistribution. To increase the effectiveness of MNPs, improve their “stealthiness” and increase their blood circulation time to maximize the likelihood of reaching targeted tissues, several techniques including reducing size and grafting polymers have been used. For example, SPIONs with particle size below 9 nm show higher blood vessels penetration efficiency than larger NPs [45]. In addition, functionalization of MNPs with amino groups, silica, polymers, various surfactants or other organic compounds is usually performed in order to achieve better physical and chemical properties. Polymers are usually used for modification of MNPs because they offer the advantage of controlled and sustained drug release systems with high biocompatibility and reduction in systemic toxicity [46]. Moreover, the core/shell structure of MNPs has the advantages of good dispersion, high stability against oxidation and an appreciable amount of drug can be loaded into the polymer shell. Furthermore, lots of functional groups from polymers on the surface can be used for further functionalization to obtain various properties. Several studies were shown a combination of photothermal therapy, chemotherapy and targeted delivery has the synergistic effects for the treatment of cancer [47] and RA [48]. So targeted MNPs can be used along with hyperthermia agent, and drugs can be loaded into MNPs for targeted therapy. In this context, Kim et al. [48] coupled RGD with MTX loaded PLGA gold [Au]/iron [Fe]/gold [Au] half-shell NPs to maintain a high MTX concentration in the inflamed joint spaces. NIR irradiation leads to local heat generation due to the NIR resonance of Au half-shells and accelerates MTX release from PLGA NPs. Furthermore, the Fe half-shell layer enables magnetic delivery of the NPs to the target region using a magnetic field, and their retention can be prolonged under an external magnetic field. These NPs were injected IV into CIA mice. The NIR absorbance and MRI revealed NPs accumulation in the inflamed paws, which was enhanced under the external magnetic field. Compared with conventional treatment, this magnetic targeted chemophotothermal treatment provided superior therapeutic efficacy with only 0.05% the dosage of MTX. In another study, Dai et al. [45] coated SPION with Dex and glucose as MRI contrast agents to improve the stability of the MNPs during blood circulation. The balance between hydrodynamic diameter and superparamagnetic properties of the iron oxide colloid crystals were important parameters that were achieved by optimization of glucose content during the iron oxide crystal growth stage. Then, FA was conjugated to glucose and Dex coated SPION (Glu-Dex-SPION) to improve detection of the inflammatory site of arthritis. After IV injection of FA-Glu-Dex-SPION in adjuvant-induced arthritis (AIA) rats, MRI showed significant differences between synovium and surrounding tissues over a long period of time [24 h] compared with the non-targeted Glu-Dex-SPION. These results illustrated that Glu-Dex-SPION decorated with FA is an excellent agent for the enhancement of contrast in the inflamed joints of a rat model of RA. FA coupled Glu-Dex-SPION particles were also used for evaluating long-term response to anti-rheumatic drugs. This system was more sensitive than non-targeted SPION for detecting the outcome of COX-2 inhibitor treatment. Thus, these NPs have potential as a contrast agent for measuring treatment response in RA. In last year, Chen et al. [49] prepared a targeted NP platform for labelling and following T cell in rodent CIA model. In this study, the PEG-coated SPION NPs (IOPC) were carboxylated and then conjugated with the monoclonal anti-CD3 antibody (IOPC-CD3) against CD3 on the surface of T cells. Immunohistochemical and MRI study revealed MNPs accumulated in the inflamed region and these NPs could effectively act as a contrast agent to imaging of RA model. To target and detect FA receptors overexpressing macrophages in the inflamed knee joints, Periyathambi et al. [50] prepared fibrin-based MNPs coupled with FA (FA-MFNPs). The average particle size of prepared NPs was about 18 nm and their shape was spherical. In vitro studies showed successful internalization of FA-MFNPs into the Raw 264.7 macrophage cells. The MRI results showed enhanced contrast of imaging after administration of FA-MFNPs in comparison with MFNPs. Thus, this system could be used as a suitable contrast agent for RA diagnosis. For radiation synovectomy, Vimalnath and his coworker [51] utilized agglomerated Holmium-166 (¹⁶⁶Ho) labelled MNPs for treatment of knee joints arthritis. ¹⁶⁶Ho is nuclear reactor and has suitable nuclear decay properties. It also is available in adequate quantity at affordable cost. The radiolabelled NPs displayed admirable in vitro stability. Biodistribution studies and whole-body radio-luminescence imaging in normal Wistar rats in different time after the loco-regional administration into one of the knee joint cavities showed main part of labelled NPs (98%) is retained within the joint cavity even after 48 h and their spread was minor in blood and other tissues. This finding was due to no leakage of injected labelled NPs from the joint cavity of the animals. The result of this study suggested that this radiochemical formulation could have potential for utility in RA treatment. During the inflammatory process, endothelial selectin (E-selectin) and cell adhesion molecule are overexpressed on leukocytes and endothelial cells and mediate recruitment of granulocyte, monocyte and T lymphocyte in an inflamed site. E-selectin was used as an early marker of inflammation. Tetrasaccharide, sialyl Lewis X (sLeX) is natural ligand of E-selectin. It is used to detect inflammation along with MRI and electron paramagnetic resonance (EPR). Ultra-small particles of iron oxide (USPIO) are molecular markers that are phagocytosed by macrophages during inflammation. To increase selectively and specificity, MR contrast agents were modified with SLeX. The result showed USPIO-PEG-sLex had long intravascular persistence time and was selectively accumulated in inflamed joints. In addition high and prolonged contrast were provided in MRI studies indicating, these targeted MRI contrast agents could efficiently identify inflamed joints [52].

Gene therapy in RA

In recent years, biological therapeutic agents including genes have had huge advances in RA treatment. There are many studies investigated gene therapy in RA treatment and majority of them get some successes. These genes block production and activity of some inflammatory agents involved in RA pathogenesis or express proteins that play role in blocking production of inflammatory agents. For example, transfer of IL-1Ra cDNA to arthritic joints with retroviral vectors has already been accomplished in two clinical trials and the results showed that it could be effective in reducing joint damage in RA and osteoarthritis models [53]. RNA interference (RNAi) and small interfering RNA (siRNA) are sequence of specific genes, which silence or downregulate target protein in the cytoplasm of cells. Discovery of these genes has given a new therapeutic vision to the cure of some diseases and genetic disorders such as RA [54]. Silencing or downregulation of genes involved in inflammation could be beneficial treatment for RA. Notch1 and TNF-α are examples of proteins involved in RA which silencing or downregulation of them could be advantageous in RA therapy [55,56]. TNF-α plays a pivotal role in the induction of chronic inflammation. Notch signalling pathways also have a main role in progression of RA with the development of vascular, cell–cell communication. Notch is also a critical vascular network-remodelling agent. It was demonstrated that effective transfer of siRNA against Notch1 and TNF-α could inhibit the progress of RA [55]. Controlling of interleukin (IL) genes family expression also could be a candidate for treating RA because of its overexpression in the inflammatory site in RA. IL-1 receptor antagonist (IL-1Ra) is an IL-1 inhibitor and act as a receptor antagonist, which blocks IL-1 signalling pathway [53]. The positive effect of IL-1Ra in controlling inflammation and symptoms of RA in animal models and in clinical practice was demonstrated [53]. The main challenge in gene therapy has been delivery of genes (DNA or siRNA) to specific cells to regulate gene expression. In the best way, therapeutic gene must be protected against degradation by nucleases, entered intact in nucleus of targeted cells after bypassing the plasma and nucleus membrane without side effects on normal tissue. Carriers which are utilized for gene delivery could divide into two main categories: viral vectors and non-viral vectors. Viral vectors are good candidates to carry therapeutic genes due to their high transfection efficiency and long-term expression [53,54]. But, there are some shortcoming such as oncogenic effects and immunological reactions which limit viral vectors usage as a transfer system for human gene therapy [53,54]. Therefore, non-viral vectors have got a lot of attention in researches due to safety and cost concerns. Non-viral gene vector systems have additional advantages over viral vectors. These kinds of vectors are inexpensive, non-infectious, relatively non-toxic, non-immunogenic, stable in storage, easy to produce in large quantities and have ability to accommodate a large quantity of DNA plasmids [53]. The application of nanotechnology in gene delivery is offering a possibility to increase biological therapeutic efficacy, especially when coupled with a targeting agent. Although, there are not only many studies about targeted delivery of genes using nanoparticulate systems in RA therapy but also available studies revealed that NP-mediated gene delivery is more effective, safe and acceptable method compared with naked and viral vector gene delivery systems. In the following discussion, we mentioned some of these systems. As described above, FA receptors-β are overexpressed on activated synovial macrophages present in RA and allows the use of FA as a potential ligand for targeted gene therapy in RA. In this context, Fernandes et al. [54] conjugated low MW chitosan with FA and investigated its efficacy as a vector for siRNA delivery in vitro. Results demonstrated M_W of chitosan has a major influence on physicochemical properties and biological effect of siRNA carrier complex. It was found that very low M_W of chitosan (below 10 kDa) formed unstable complex with siRNA due to the weak electrostatic interaction between siRNA and chitosan. However, chitosan (25 and 50 kDa) completely absorbed siRNA and at chitosan/siRNA weight ratio: 50/1, NPs formed with particle size of 220 nm. In addition, FA-chitosan-siRNA NPs improved gene silencing effect compared with chitosan-siRNA due to more cellular uptake and more transfection efficiency of FA-chitosan-siRNA NPs in FA receptor-positive HeLa and OV-3 cells. In another approach, Fernandes et al. [53] examined the anti-inflammatory and bone protective effects of FA-targeted chitosan NPs containing IL-1Ra DNA in AIA rats. It was revealed that FA-chitosan-IL-1Ra DNA NPs have a better toxicity profile on rat primary osteoblasts compared with naked DNA or chitosan-DNA NPs. The result showed that after the injections of both chitosan-DNA and FA-chitosan-DNA NPs, IL-1Ra was detected in the serum of rats as long as 10 days due to lasting, sustain release of IL-1Ra in the serum. In vivo study in AIA rats showed that although FA-chitosan-IL-1Ra DNA NPs, chitosan-IL-1Ra DNA NPs and naked IL-1Ra DNA reduced elevated level of IL-1 β, but this decrease was statistically significant just for FA-chitosan-IL-1Ra DNA NPs compared with non-treated AIA rats. To study arthritis score improvements, paw diameter values determined and it was found that paw diameter significantly decreased in AIA rats treated with FA-chitosan-IL-1Ra DNA NPs, chitosan-IL-1Ra DNA NPs and naked IL-1Ra DNA compared with non-treated AIA rats but the decrease in treatment groups was not significantly different. NF-κB signalling pathway has a key role in the inflammatory response of macrophages and lymphocytes in many inflammatory diseases such as RA. Consequently, inhibition of this pathway can be effective in RA treatment. In this context, Zhou et al. [57] designed stable self-assembly melittin-derived cationic amphipathic peptide-siRNA nanocomplex that targeted the NF-κB signalling subunit p65, which is a major transcriptional controller of the canonical NF-κB pathway. The nanocomplex had a mean size of 50 nm, which was small enough to rapidly diffuse into inflamed site, where it is retained. In vivo study in antibody-mediated inflammatory arthritis model showed p5RHH-Cy5.5-labelled scrambled siRNA increased fluorescence intensity in inflamed joints and prolonged signal for at least 7 h. In addition, p5RHH-Cy5.5-labelled scrambled siRNA was detected in the kidneys 2 h after injection, and the signals keep up to 24 h. However, free Cy5.5-labelled scrambled siRNA in an equivalent dose were not detected in any organ after 2 h. This result indicated that nanocomplex protected siRNA from rapid clearance and degradation by nucleases. Histologic study demonstrated p5RHH-p65 siRNA nanocomplexes effectively suppressed inflammatory cytokine expression and cellular influx into the joints, protected bone from erosions and preserved cartilage integrity compared with free p65 siRNA, p5RHH siRNA complexes and HBSS control. The p5RHH-p65 siRNA nanocomplexes potently suppressed early inflammatory arthritis without affecting p65 expression in off-target organs such as liver, spleen, brain and kidneys. These results indicated that this nanocomplex (NF-κB p5RHH-p65) was effective to protect the siRNA from serum deactivation and delivered functional oligonucleotides effectively to selected inflammatory targets. In another investigation, Lee et al. [56] designed a nanocomplex of polymerized siRNA (poly-siRNA) against TNF-α with thiolated glycol chitosan (TGC) polymers for the treatment of RA. TGC was employed to achieve high gene loading and stable complex through formation of disulphide band between carrier and gene. The average diameter of prepared nanocomplex was about 370 nm. In vitro cell culture study showed that TGC-poly-TNF-α siRNA nanocomplexs specifically were taken up in LPS activated RAW 264.7 cells overexpressed scavenger receptors but not in non-activated cells. In contrast to free poly-TNF-α siRNA, the inhibitory effect of TGC-poly-TNF-α siRNA nanocomplexs on TNF-α gene expression in RAW 264.7 cells was approximately similar to poly-siRNA/Lipofectamine complex indicating gene silencing efficiency of TGC-poly-TNF-α siRNA nanocomplexs. In vivo study in CIA mice showed poly-TNF-α siRNA TGC NPs, not only suppress paw swelling but also prevent progressive joint destruction, which was comparable with MTX. Kim et al. [55] also investigated anti-inflammatory effect of TGC nanocomplex containing poly-siRNA against Notch1 (TGC-poly-Notch1 siRNA) in CIA mice model. The Notch1 siRNA was successfully encapsulated in TGC NPs in aqueous condition. In vitro cellular uptake and gene silencing efficacy of TGC-poly-Notch1 siRNA in RAW 264.7 macrophage cells were confirmed using confocal microscopy and real-time PCR. In vivo study in CIA mice revealed TGC-poly-Notch1 siRNA greatly accumulated in the arthritic joins and inhibited effectively progression of inflammation, bone erosion and cartilage damage. These NPs carried out their function in animals without undesirable severe toxicity and it seems they have potential in RA treatment.

Alginate, a natural anionic polymer, has been widely used for biomedical applications because of its biocompatibility, low toxicity, low cost and easy manipulation. Alginate is capable to form cross-linked nanogels in the presence of multivalent cations such as Ca²⁺ and is extensively used as non-viral vector for gene delivery. For RA treatment, Jain and his coworker [58] used external gelation method to prepare alginate NPs containing murine interleukin-10 (mIL-10) plasmid DNA. Then to increase macrophage-mediated phagocytosis, NPs were coupled with tuftsin (TKPR) peptide sequence and transfection efficiency was assessed in J774A.1 macrophages. In vitro study showed alginate NPs was capable to trap plasmid DNA and protected it from degradation by DNase. The group mentioned rapid internalization of tuftsin-modified alginate NPs in J774A.1 macrophages (15 min) which was explained with specific interactions of tuftsin construct with the cell surface receptors and subsequent receptor-mediated phagocytosis. But, unmodified and scrambled peptide modified NPs were taken up at longer time (1 h) due to non-specific uptake. ELISA and qualitative analysis by fluorescence microscopy also confirmed efficient nuclear uptake and transfection of tuftsin-modified alginate NPs thanks to the combination of active targeted delivery and a non-condensing calcium alginate matrix that maintains the stability of the payload in the phago/lysosomal compartments and the supercoiled structure of the released plasmid in the cell. In addition, tuftsin modified alginate NPs containing mIL-10 plasmid DNA increased mIL-10 expression at protein level and decreased the level of TNF-α more effectively compared with Lipofectin and scrambled modified NPs in J774A.1. This result confirmed the efficacy of tuftsin conjugated alginate NPs containing mIL-10 plasmid DNA in RA treatment.

Cationic liposome which able to condense negatively charged DNA also is an appropriate non-viral vector for gene delivery in RA therapy. For example, Komano et al. [59] evaluated the efficacy of siRNA against TNF-α encapsulated in cationic liposome named wrapsome (siRNA/WS) in CIA mice. The cationic liposome had cationic lipid bilayer composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE)). After IV administration of Cy5-siRNA/WS, fluorescence stereoscopic microscopy and flow cytometry study showed higher fluorescence intensity in a synovial cell of arthritic joints compared to non-arthritic sites, splenocytes, bone marrow cells and peripheral blood leukocytes. The significant decreases in TNF-α mRNA level in the arthritis joints of CIA mice treated with siRNA/WS was confirmed with real-time reverse transcriptase-PCR. Consequently, siRNA/WS decreased and delayed the incidence of arthritis over those treated compared with control siRNA/WS. In conclusion, WS allowed efficient delivery of siRNA to arthritic joints and has beneficial effects in RA treatment by silencing the expression of TNF-α.

Conclusions

RA is chronic inflammatory diseases, which can be controlled with currently available therapies, but these treatments are accompanying with significant side effects. Therefore, development of a new therapeutic strategy for RA treatment is essential. Nanomedicines play a key role in the efficient delivery of therapeutic compounds for RA through its targeted and controlled drug delivering potential. NPs have potential to increase specific accumulation in inflamed tissue either by passive targeting through EPR mechanism, or active targeting using cell-specific targeting ligand or external stimulating factors such magnetic field. The authors of the present review article sought to describe various targeted drug delivery systems used for improving the RA diagnosis and therapy. According to literature, such nanomedicines have the potential to reduce the current limitations in the diagnosis and treatment of RA. Nevertheless, more comprehensive studies should be conducted to prove their clinical efficacy in RA therapy.

Disclosure statement

No potential conflict of interest was reported by the authors.