*4.2.1 Folic acid (FA) based active targeting*

Activated macrophages overexpress folate receptor β(FR-β) in the arthritic joints [73]. Owing to post-translational modifications, the folate expressed on neutrophils

*Smart Drug-Delivery Systems in the Treatment of Rheumatoid Arthritis: Current, Future… DOI: http://dx.doi.org/10.5772/intechopen.99641*

are incapable of binding to FR-β [74]. Alternatively, a functional FR-β has been identified on the activated macrophages having nanomolar affinity for folate. Hence, the FR-β receptor emerges as a useful target in various diseases including RA, osteoarthritis [75], systemic lupus erythematosus [76], atherosclerosis [77] and Crohn's disease [78]. Macrophages are key players of RA pathogenesis, as they secrete pro-inflammatory cytokines, metalloproteinases, ROS and prostaglandins. Folate is an attractive option for ease of surface modification, hydrophilicity, and stability in different solvents. Methotrexate (MTX) encapsulated folate-conjugated glycol chitosan (MFGCN) have been targeted to inflamed joint in adjuvant-induced arthritic rat model [79]. Likewise, surface of MTX-loaded liposomes was decorated with folate and were evaluated both *in vitro* and *in vivo* (DBA/1 J mice strain) [80, 81], and targeted against the FR-β on activated macrophages. Clinical efficacy of MTX in the DBA/1 J arthritic mice indicated significant expression of CD73. Since, low doses were required, this nanoformulation was economical and cost-effective. MTX loaded dendrimer nanoparticles with folic acid (FA) surface functionalization were reported to reduce progression of disease [82]. Further, authors suggested a ~ 7.5-fold increase in the maximum-tolerated dose of the MTX, when given as the formulation compared to the free MTX.

#### *4.2.2 Hyaluronic acid (HA) based active targeting*

CD44 is a glycoprotein, overexpressed on the surface of activated macrophages, present in the inflamed joints of RA. CD44 can be exploited as a prospective target in RA treatment. HA, a biocompatible natural polymer has ben explored as a ligand that effectively binds to CD44 receptor. HA coated hydroxyapatite NPs (HA-NPs) encapsulating methotrexate (MTX) and teriflunomide (TEF) - (HYA-HAMT-NP) were reported for RA treatment [83]. Results suggested that HYA-HAMT-NP could emerge as an effective delivery vehicle to circumvent hepatotoxicity caused by drugs in RA.

#### *4.2.3 Anti-angiogenesis*

Hypoxia in a critical factor in inflamed synovium that triggers neo-vascularization from existing vessels termed as angiogenesis [84]. The neo-vascularization preserves the chronic inflammatory state by engaging cells to the inflammatory site, provides nutrition and oxygen to the multiplying cells. Additionally, the enlarged surface of endothelium triggers secretion of adhesion molecules, cytokines and stimulates neutrophil infiltration as well as synovial membrane into the cartilage, causing cartilage destruction and bone erosion [85]. Promising therapies based on angiogenesis are emerging for RA therapy, where VEGF and integrins are the therapeutic targets.

#### *4.2.3.1 Vascular endothelial growth factor (VEGF)*

Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific angiogenic factor principally secreted by SFs in the pannus. In angiogenesis, VEGF triggers multiplication and migration of endothelial cells. Further, it enhanced blood vascular permeability, stimulates maturation and maintenance of the neovessels [86]. TNF-α and IL-1, the pro-inflammatory cytokines induce the SFs and other cells to secrete VEGF, and VEGF is overexpressed at the inflamed joint owing to angiogenesis [87]. Therefore, VEGF and VEGF receptor inhibition can be an attractive strategy for RA treatment as it may effectively decrease inflammation by inhibiting angiogenesis.

#### *4.2.3.2 Vasoactive intestinal peptide (VIP)*

Vasoactive intestinal peptide (VIP) is a neuropeptide of the central and peripheral nervous system that has vasodilatory, anti-proliferative, anti-inflammatory, cell protective agent and broncho-dilatory role. The activity of VIP binds to high affinity VIP receptors, that are overexpressed on T-lymphocytes and several inflammatory cells. VIP inhibits the secretion of pro-inflammatory cytokines to act as anti-inflammatory molecule. It also promotes the secretion of anti-inflammatory molecules by stimulated innate cells [88]. Proliferating synoviocytes and activated macrophages overexpress VIP receptors in inflamed RA. Therefore, VIP receptor specific ligands can be conjugates to nanoparticles to specifically target the diseased site. Therefore, VIP can be exploited as a therapeutic agent for active targeting to RA joint.

#### *4.2.3.3 Integrins*

Integrins are the biogenic markers of endothelium undergoing angiogenesis and play a vital effector role in it. Integrin alpha-V-beta 3 (αvβ3 integrin), also referred to as vitronectin receptor are overexpressed on osteoclasts and activated macrophages of the inflamed synovium. Integrin receptor promotes angiogenesis, helps in osteoclast-mediated bone resorption, and induces pathological neo-vascularization [89]. Inhibition of αvβ3 integrin activity stimulates endothelial cell apoptosis, thereby inhibiting angiogenesis [90]. Hence, αvβ3 is considered a reliable maker for targeted delivery to RA patients.

#### *4.2.3.4 E-selectin*

E- Selectin is a glycoprotein that is associated with leukocyte rolling and adhesion and is expressed on vascular endothelium of the inflamed synovium, and promotes angiogenesis [91]. The inflammatory cytokines maintain its upregulated expression in the inflamed tissue. Therefore, expression of e-selectin can be a useful molecular target for RA therapy. Therefore, e-selectin serves as yet another attractive strategy for active targeting of the chosen delivery of drug to the diseased RA joint [92].

## **5. Nanotherapeutics**

The assembly of stimuli-sensitive nanoparticles necessities the usage of biocompatible constituents, that can undergo supra-molecular changes in conformation, a hydrolytic cleavage, and precise protonation, etc. Polymers have maximum suitability and has been widely explored class of materials that have incredible potential. Polymers may be of natural or synthetic origin. The flexibility of the polymer sources and its ability for synthesis of various combinations of polymers has facilitated manipulation of the polymer sensitivity to specific stimuli within a narrow range [93]. Nanoparticles could be synthesized by lipid, metals and polymers. NPs decoy pro-inflammatory molecules like cytokines and ROS and sometimes osteoclast differentiation factors. Moreover, surface modification of NPs with target moiety is a extensive application in site specific drug delivery by enhancing the bioavailability of drug and reducing non-target side effects [94].

#### **5.1 Polymer-drug conjugate (PDC)**

PDC based DDS has been proposed by Ringsdrof in 1975 [95], in which a low molecular weight drug, targeting moiety and solubilizer are attached to polymeric *Smart Drug-Delivery Systems in the Treatment of Rheumatoid Arthritis: Current, Future… DOI: http://dx.doi.org/10.5772/intechopen.99641*

backbone covalently *via* bioresponsive linkers. PDC improves drug bioavailability, reduces drug toxicity, and are less toxic in nature, can easily be fabricated in regulated sizes that escape through renal filtration, exhibit increased retention time in blood circulation [96]. PDCs under investigation include N-(2-hydroxypropyl) methacrylamide, poly(vinylpyrrolidone) (PVP), hyaluronic acid (HA) and poly (ethylene glycol) (PEG) [11, 97]. N-(2-hydroxypropyl) methacrylamide (HPMA) shows improved biodegradability, biocompatibility and increased efficacy in treating RA [11, 97]. HPMA-Dex conjugates were administered intravenously into CIA induced RA model, that resulted in accumulation at the inflamed joints [98]. PEG has been used for its hydrophilicity and biocompatible properties. PEG-DEX have been developed *via* acid-labile hydrazine bond and given to arthritic rats leading to decrease in joint inflammation [99]. Recently, FDA approved two PEGylated proteins for the treatment of RA: Pegloticase (Krystexxa®) and certolizumab pegol (CIMZIA®). Although, PEG attributes stealth properties to the NPs, there are some concerns regarding PEG conjugates that include low biodegradability and the possibility to elicit an anti-PEG IgM antibody [100].

#### **5.2 Nanoparticles**

Nanoparticles are solid colloidal particles with unique physico-chemical properties such as ultra-small size, surface charge, large surface area to mass ratio Unlike polymer-drug conjugates, NPs allow encapsulation/absorption/entrapment of drug without modification. The high reactivity, diffusivity, solubility, toxicity, immunogenicity and drug release characteristics can be manipulated to make efficient delivery system. Polymeric, liposomes, micelles and metallic nanoparticles are the most commonly used nanoparticles [101].

#### *5.2.1 Biopolymeric nanoparticles*

The biodegradable backbone in biopolymeric NPs protects the drug from *in vitro* and *in vivo* degradation. Alginate, Gelatin, Pectin, Chitosan, are natural biopolymers that are highly investigated as they are biocompatible and biodegradable. Chitosan is polycationic in nature, that allows surface modification with ease and is a natural mucoadhesive. Kumar et al. reported Chitosan nanoparticles encapsulating Dexamethasone (DEX) and Methotrexate (MTX) for their *in vitro* efficacy in RAW264.7 cells and *in vivo* efficacy in arthritic rat model. Results convincingly indicated reduced toxicity and high efficacy in arthritic model [28]. The main drawback of chitosan is insoluble in alkaline and neutral medium due to absence of free amino group, but due to protonation of free amino group in acidic medium, there is enhanced solubility.

Glycol chitosan has enhanced water solubility and functional groups for further chemical modifications making it better suited as a potent drug carrier [102]. Glycol-chitosan nanoparticles(GCNPs) are biocompatible, pH responsive and biodegradable. Methotrexate (MTX) encapsulated folate-conjugated glycol chitosan (MFGCN) have been reported to target the overexpressed folate receptors β (FR-β) on activated macrophages in the inflamed joint in adjuvant-induced arthritic rat model. **Figure 4** gives a pictorial description of MFGCN that reduced the arthritic index, improved the antioxidant response and decreases pro-inflammatory cytokines and suggesting its potential in targeting activated macrophages of synovium [79].

#### *5.2.2 Gold nanoparticles(GNPs)*

GNPs can be surface functionalized through covalent bonding, by cationic polymers or physical or ionic absorption [103], functional groups like e.g. thiol, amine, and carboxyl groups that are reactive [104]. GNPs were strategically planted

#### **Figure 4.**

*Pictorial representation of utilization of folate functionalized methotrexate loaded glycol chitosan nanoparticles (MFGCN) in treating CIA rats targeting inflammation and ROS in rat serum post 21 days of treatment (n = 6). [A] MFGCN development and active targeting of M1 macrophages in the inflammatory synovium. [B] LPO and LDH activity [C] panel showing antioxidant potential (activity quantification of GST, GSH, GP and GR) [D] quantification of TNF-*α*, IL-4, IL-1*β*, IL-10 and IL-17 [E] Representative H&E staining images of study groups comparing healthy and arthritic control groups with the treatment groups(n = 6). Data was analyzed by one-way ANOVA \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.*

in macrophages to target thioredoxin reductase to evaluate its antiangiogenic impact by binding to the vascular endothelial growth factor (VEGF) [105]. Lee et al. suggested MTX encapsulated RGD-attached gold half-shell NP system for RA treatment [106]. On irradiation with near-infrared (NIR), these GNPs delivered MTX to the inflamed joints, maximizing its efficacy with minimal side effects. GNPs were modified physically with Tocilizumab(TCZ) and chemically altered with an end-group thiolated hyaluronate(HA). This complex of HA-GNP-TCZ indicated a synergistic effect for its dual-functional effect on VEGF and IL-6R (receptors for IL-6) in RA treatment [107]. GNPs may block the RANKL induced osteoclast formation which leads to cartilage and bone destruction [108].

## *5.2.3 Liposomes*

Liposomes are bilayered lipids with an aqueous core. Both hydrophobic as well as hydrophilic drugs can be encapsulated within phospholipids and the water phase cavity, making them SDDS [109]. Particle size determines the extent of accumulation at the synovium, with maximum accumulation of liposomes reported with size <100 nm diameter [110]. Therapeutic efficacy is limited due to rapid clearance from circulation *via* the RES in the liver/spleen. Surface modification by PEG enhances its hydrophilicity, makes them sterically stabilized, circumvent rapid clearance, and enhance the retention time in blood circulation [110]. Corvo et al. reported enhanced circulation and accumulation of PEG-coated SOD entrapped liposomes (mean diameter ~ 0.11 μm) at the arthritic joints [111]. Considering the dynamic microenvironment of the diseased synovium, liposomal surface can be modified with ligands, antibodies/antibody fragments or for site-specific delivery of encapsulated cargo. Recently, dexamethasone encapsulated PEG-liposome treated arthritic rats indicated accumulation of liposomes, down regulation of pro-inflammatory cytokines along with reduced inflammation of the arthritic joints [112]. Liposome tagged with a peptide sequence (CKPFDRALC-called ART-2 ligand) encapsulating DEX significantly inhibited RA progression [113].

*Smart Drug-Delivery Systems in the Treatment of Rheumatoid Arthritis: Current, Future… DOI: http://dx.doi.org/10.5772/intechopen.99641*

#### *5.2.4 Micelles*

Micelles can be synthesized in small size with narrow size distribution from amphiphilic molecules that self-assemble into NPs in aqueous solution with a distinct hydrophobic cavity and an exterior hydrophilic surface. This makes them apt for intravenous injection and targeted delivery into the inflamed synovium as a consequence of extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration (ELVIS) [114]. Wang et al. reported self-assembled micelles with an amphiphilic copolymer PEG-poly-e-caprolactone (PEG-PCL), which displayed ELVIS in inflamed joints [115], but the nonbiodegradable backbone of synthetic polymers caused non-specific accumulation in liver. Bader et al. [116] developed micelles from polysialic acid (PSA)-the hydrophilic polymer and synthesized micelles by altering it with N-decylamine (DA) and PCL, that formed the hydrophobic fragment. Prolonged circulation was observed with these micelles that accumulated passively at the inflamed tissue. PSA-DA micelles exhibited *in-vitro* cytotoxicity towards a synovial fibroblast cell line, the PSA-PCL micelles displayed negligible *in-vivo* cytotoxicity [117]. Further modifications were reported to improve the blood kinetic profile of the micelles. Core-cross-linked micelles were developed based on copolymer PEG-b-poly [N-(2-hydroxypropyl) methacrylamide-lactate] for targeted delivery of glucocorticoids to the RA affected joints. Here, dexamethasone was modified by methacrylated linkers *via* ester bonds, and covalently encapsulated within the polymeric structure, leading to tailorable release of dexamethasone [118]. Targeted delivery of aqueous-synthesized MTX-PEI@HA NPs to mitigate inflammatory arthritis was reported [114]. Li et al. formulated pH-sensitive micelles by conjugating hydrophobic prednisolone to PEG-derivative that confers acid-sensitive sites for attachment by forming hydrazone bonds [119]. Dual-drug loading of nimesulide and MTX in RGD-modified micelles to target αvβ3-integrin validated efficient site-specific delivery, decreased angiogenesis with minimal dose of nimesulide and MTX [120].
