Gum acacia stabilized silver nanoparticles based nano-cargo for enhanced anti-arthritic potentials of hesperidin in adjuvant induced arthritic rats

Abstract

Nanomedicines anticipate drug delivery to inflamed tissues in rheumatoid arthritis (RA) with greater efficacy and lesser side effects. This study investigates the anti-arthritic potentials of Hesperidin (HP) loaded in gum acacia (GA) stabilized green silver nanoparticles (AgNPs). Synthesized GA-AgNPs were characterized through UV–vis spectrophotometer, zetasizer and atomic force microscope (AFM). The HP and its loaded NPs were tested for RA in Complete Freund’s adjuvant (CFA) induced arthritis model. GA-AgNPs were found in nano-range size with negative charge, spherical shape and loaded increased HP amount. HP loaded GA-AgNPs showed minimal arthritic score exhibiting mild to moderate tissue swelling, reduced degenerative changes along with mild articular changes. Histopathological analysis revealed comparatively lesser influx of inflammatory cells and diminished granulamatous inflammation in ankle joints tissues in the presence of HP loaded GA-AgNPs. RT-PCR revealed that HP loaded GA-AgNPs significantly reduced the TLRs mRNA expression. Results validate GA stabilized green AgNPs as stable nano-cargos for targeted delivery of HP for restoring the progression of RA.

Keywords:

• Gum acacia
• silver nanoparticles
• hesperidin
• arthritis
• mRNA expression

Introduction

RA is an autoimmune disease with constant prevalence rate of about 0.5–1.0% worldwide and characterized by chronic inflammation of joints associated with massive infiltration of activated immune cells and cartilage or bone destruction [1]. The pathogenesis of RA remains complicated, however, several mechanistic studies explained that influx of activated inflammatory T cells, B cells and macrophages release various pro-inflammatory cytokines and prostanoids that contribute to severe joint destruction and tissue damage [2,3].

Toll-like receptors (TLRs) is a pattern recognition receptor that bridges innate and adaptive immune systems and is considered important factor in the development of arthritis. Activation of antigen-presenting cells (APC) by microbial or non-microbial antigens via TLRs result in production of pro-inflammatory cytokines, chemokines and destructive enzymes representing RA characteristic features [4]. TLRs involvement in RA progression is supported by their distinct expression pattern in synovial cells, blood and splenocytes of patients and animals with RA [5,6]. Mycobacterium tuberculosis ligands, including lipoproteins and glycolipids, are recognized by APCs through TLRs, resulting in aggravation of inflammatory mediator production [7–9]. Targeting TLRs may modulate inflammation in RA and development of therapeutics that inhibit the TLRs or components of their signaling cascade may allow for greater specificity than existing conventional therapies including non-steroidal anti-inflammatory drugs (NSAIDs) and biological agents such as cytokine blockers.

Nanomedicines have revolutionized pharmaceutical industry for targeted responses and improved efficacy of drugs in a variety of diseases. The smaller size and enhanced surface area of nano-carriers allow selective delivery of drugs to the desired site of inflammation with minimal side effects on normal tissues. Nano-carriers also enhance clinical efficacy of drugs through enhancing their aqueous solubility and protection from enzymatic degradation [10]. Recently, metal NPs have got greater scientific interests for therapeutic and diagnostic applications. Their physicochemical and biological properties validate their effectiveness in drug delivery [11]. Among metal NPs, AgNPs are preferred for constructing drug nano-carriers systems due to their optical activity and surface-enhancing properties. Moreover, they are able to deliver the loaded contents inside the cells through their improved cross-membrane transport. They also exercise higher control over the release of their surface-tethered drugs [12].

AgNPs are commonly synthesized by using reducing agents associated with adverse biological effects. Green chemistry is getting wider attention for fabrication of metal NPs. It uses inexpensive and safe methods for synthesis of metal NPs, thus eliminating the use of toxic materials. Using inactivated plants gums, tissues or other parts as reducing/stabilizing agents for metal NPs has been best alternative to synthetic toxic reducing molecules [13]. GA is a water-soluble polysaccharide produced by Acacia Seyal and Senegal. It is slightly acidic or neutral complex polysaccharide with gum and some potassium, calcium and magnesium ions. It is extensively used in food, cosmetic and pharmaceutical industries. It is used as emulsifying, stabilizing and thickening agent in pharmaceutical preparations [14,15]. Using biodegradable GA as reducing/stabilizing agent for the synthesis of AgNPs would be an innovative idea for designing drug nano-carrier system.

HP, a well-established anti-inflammatory molecule, is known to inhibit both acute and chronic phases of inflammation via inhibiting cytokine production in adjuvant arthritis in rats [16]. It has also demonstrated inhibition of collagen-induced arthritis most likely through free radicals scavenging and reduction in cellular infiltration [17]. In a recent study, HP derivative showed a unique mechanism against adjuvant induced arthritis via inhibiting fibroblast-like-synoviocyte proliferation interfering with Wnt/β-catenin signaling pathways [18]. Interestingly, this pathway showed relationship with the TLR (TLR-2 and TLR-4) signaling during M. tuberculosis lung infection [19]. It has been reported that intake of glucose or a high-fat-high-carbohydrate (HFHC) meal induces an increase in inflammation and oxidative stress in circulating mononuclear cells (MNCs) of normal-weight subjects. Flavonoids have been reported for lowering HFHC meal-induced inflammation and oxidative stress with decreased TLR expression in normal individuals [20]. These findings prompted us to further investigate the molecular mechanism of TLRs targeting by HP in adjuvant induced arthritis model. Though HP is cost-effective and unique anti-arthritic flavonoid, its multiple physicochemical properties have affected its pharmacological versatility. It is less stable against gastric pH and enzymes, thus its therapeutic efficacy decreases distinctly. Moreover, its poor aqueous solubility leads to its lower absorption and hence results in its inferior therapeutic effects [21]. Designing of an efficient delivery system for enhancing its therapeutic efficacy would be an innovative strategy.

The current study is aimed at designing GA stabilized green AgNPs for loading and delivering of HP for RA targeted therapy. CFA-induced arthritic rat model was implemented to evaluate the therapeutic effect of HP and its loaded AgNPs against cartilage degradation and bone destruction associated with RA. The treatment also illustrated potent inhibitory capacity for intervening rheumatic mechanism through preventing the cellular infiltration into the inflamed synovium and interfere the expression of TLR-2 and TLR-4 immune receptors.

Materials and methods

Materials

HPLC grade solvents were purchased from Sigma Aldrich, Germany. GA was obtained from local market. AgNO₃, HP, Complete Freund’s adjuvant (CFA), Dexamethasone and Indomethacin were obtained from Sigma Aldrich, Germany. Invitrogen TRIzol reagent, cDNA synthesis kit and Syber Green Master Mix were purchased from Thermo Fisher (Waltham, MA).

Synthesis of GA reduced/stabilized green AgNPs (GA-AgNPs)

GA was dissolved in double distilled water, filtered for removal of impurities and freeze dried. GA stock solution (6 mg/mL) was prepared by dissolving its specific quantity in specific volume of deionized water. Initially, various concentrations of GA (3–6 mg/mL) were mixed with AgNO₃ (9 mg/mL) in 1:1 v/v ratio and stirred magnetically at 60 °C. The change in color from light yellow to dark yellow indicated the synthesis of GA reduced/stabilized GA-AgNPs. The GA-AgNPs synthesis was further confirmed by observing the characteristic surface plasmon resonance (SPR) peak through UV-visible spectrophotometer (Shimadzu, UV-240, Hitachi U-3200). GA-AgNPs were also evaluated for the effects of various concentrations of GA and AgNO₃.

HP loading in GA-AgNPs

GA-AgNPs solution was subjected to centrifugation at 10,000 rpm for 30 min and GA-AgNPs were collected. The collected GA-AgNPs were re-dispersed in double distilled water (20 mL) and were added 10 mg of HP, thus giving 0.5 mg/mL final concentration of HP in formulation. The mixture was stirred magnetically at 200 rpm for 24 h. HP loaded NPs (GA-AgNPs-HP) were collected through centrifugation at 10,000 rpm for 30 min.

Anti-arthritis activity

Animals

Female Wistar rats (150–200 g) were obtained and housed in Department of Laboratory Animal Science (LAS), Dow University of Health Sciences (DUHS) Karachi, Pakistan. The animals were kept at 22 ± 2 °C with control humidity (50 ± 10%), 12 h light and dark cycle and fed with standard diet and water ad libitum. The ethical guidelines of Association for Assessment and Accreditation of laboratory Animal Care International (AAALAC) were followed for the animals handling. The study was approved by the institutional review board for animal research and ethics committee of DUHS (Ref: AR. IRB-06/DUHS/Approval/2016/04).

CFA induced arthritis

Arthritis was induced as previously described [22], with modification [23]. In each group, rats (n = 3–5) were injected with 0.1 mL of CFA suspension (1 mg/mL of heat-killed M. tuberculosis in paraffin oil) into the right hind paw. Arthritic control group received only sub-planter injection of CFA, while different treatment groups were administered orally: HP (25 mg/kg), GA-AgNPs-HP (1 mg/kg), GA-AgNPs (1 mg/kg), Indomethacin (Indo 5 mg/kg) and Dexamethasone (Dexa 0.5 mg/kg), respectively, on daily basis for 14 days.

Radiographic (X-ray) analysis

For X-ray assessment, the animals were anesthetized by intraperitoneal injection of 40 mg/kg sodium thiopental and images of the right ankle joint were taken at 10 mA, 48 kV and 0.25 s of exposure using X-ray machine (GE Radiography System). During the imaging, animals were placed on X-ray plate and radiographed at a 25 cm focus to film distance for the evaluation of soft tissue swelling, narrowing of the joint space (loss of cartilage), destruction of bone (erosions) and articular surface regularity [24].

qPCR for mRNA expression of TLR-2 and TLR-4 in arthritic rats

Rat spleens were isolated from the experimental groups and minced into tiny piece. Total RNA was extracted by using Trizol method [25], and purity of total RNA was calculated by Colibri Micro volume Spectrophotometer (Titertek Berthold) with the ratio of A260:A280. Total RNA (1.0 µg) from each sample was reverse-transcribed into cDNA by using cDNA synthesis kit. Gene expression of TLR-2, TLR-4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat spleen tissues was measured via quantitative real-time PCR using a SYBR Green master mix Kit and Rotor Gene Q Real-Time PCR Detection System. The primers sequences are as follows: TLR-2: forward 5′-GGAGACTCTGGAAGCAGGTG-3′ and reverse 5′-CGCCTAAGAGCAGGATCAAC-3′, TLR-4: forward 5′-TCAAGGCTTTTCCATCCAAC-3′ and reverse 5′-TGCTCAGACATGGCAGTTTC-3′, GAPDH: forward 5′-GGAAAGCTGTGGCGTGATTGG-3′ and reverse 5′-GTAGGCCATGAGGTCCACCA-3′. PCR was carried out as follows: 1 cycle of 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s [26]. The PCR efficiency was examined by serially diluting template cDNA and the melting curve data were collected to check the PCR specificity. Results were analyzed using comparative CT (2^−ΔΔCT) method normalizing to internal control GAPDH expression for each sample.

Statistical analysis

Values were expressed as means ± SEM and analyzed using the Statistical Package of Social Sciences (SPSS) program version 17. One-way and repeated measure Analysis of Variance (ANOVA) followed by post-hoc Tukey HSD test was implemented to measure the intergroup variation. A p < .05 value was considered statistically significant.

Results and discussion

Synthesis of GA-AgNPs

Biocompatible biopolymer GA was exploited as reducing as well as stabilizing agent for the green synthesis of AgNPs. Green synthesis of metal NPs with such food grade biopolymers has been ideal for constructing nano-drug delivery cargos as they expose their multiple functional moieties on NPs surfaces, thus providing effective drug loading. Moreover, fabrication of NPs with biopolymers gives them elevated stability against various biological environments [13]. Being a powerful analytical tool, UV–visible spectrophotometer was used for the initial characterization of GA-AgNPs synthesis. GA-AgNPs revealed well behaved absorption pattern in the UV–Visible spectral analysis. A characteristic SPR peak at 421 nm with absorption intensity of 1.23 was observed for 4 mg/mL concentration of GA when mixed with 9 mg/mL of AgNO₃ solution in 1:1 v/v ratio (Figure 1(A)). The characteristic SPR for our synthesized GA-AgNPs is accordance with previously published reports for the synthesis of GA AgNPs [27]. The increased absorption behavior of synthesized GA-AgNPs can be attributed to SPR that results from the electron coherent oscillation conduction band due to electromagnetic field [28]. Increasing GA concentration above 4 mg/mL caused a deceasing effect on SPR peak. Similar effect was observed for GA concentration below 4 mg/mL. The effects of various Ag concentrations were also investigated on the synthesis of GA-AgNPs. Increasing or decreasing Ag concentrations beyond 9 mg/mL decreased the characteristic SPR peak (Figure 1(B)).

Figure 1. UV–visible spectra showing effect of (A) GA concentration and (B) AgNO₃ concentration on the synthesis of GA-AgNPs, (C) and (D) show stability studies of GA-AgNPs against salt and plasma, respectively, while (E) and (F) shows AFM images of GA-AgNPs and GA-AgNPs-HP, respectively.

Stability studies

Any nano-system intended for in vivo drug delivery should be enough stable to withstand the degrading effects of blood plasma and higher salt concentrations. GA-AgNPs demonstrated increased stability upon their incubation with various salt concentrations (Figure 1(C)). Slight increase in the intensity of SPR peak with little broadness and no shift was observed for 2 mM concentration. In case of incubation with blood plasma, GA-AgNPs characteristic peak decreased with increasing incubation time without any shift as shown in Figure 1(D). This shows that plasma proteins had little degradation effects on GA-AgNPs. The synthesized NPs revealed stability in a similar manner as reported for other gum based metal NPs [29]. Results of stability studies confirm that GA-AgNPs can withstand the harsh biological environment and can be effectively used for loading and delivering drugs in animal’s model.

Characterization

Size, PDI, zeta potential and surface morphology

Size is an important parameter for nano-drug delivery systems as it affects the in vivo biological performance as well as in vitro stability. Drug delivery systems in nano-range enhance the in vivo performance of their loaded drugs for extended period of time as smaller particles are capable of avoiding rapid clearance. Moreover, they reveal lesser toxicities as compared to their larger size counterparts [30]. GA-AgNPs revealed to be 81.45 ± 2.07 nm in size with PDI value of 0.31 ± 0.01. HP loaded GA-AgNPs-HP revealed an average size of 155.50 ± 1.56 nm with a PDI value of 0.34 ± 0.02. The size of synthesized GA-AgNPs seems relatively larger as compared to other reported AgNPs stabilized with GA. Smaller particle size was achieved in other reported methods for fabrication of GA stabilized AgNPs due to high input of energy either in form of microwaves or elevated heat [31,32]. The homogenous size population of GA-AgNPs is in compliance with reported AgNPs stabilized with same biopolymer [27]. Comparatively larger size and increased PDI of GA-AgNPs-HP can be due to loading of HP that get unevenly distributed on NPs surfaces. Similarly, GA-AgNPs and GA-AgNPs-HP revealed −9.32 ± 0.47 mV and −16.34 ± 0.18 mV zeta potential respectively as shown in Table 1. Drug delivery systems with higher surface negativity are preferred as it enhances their physical stability and in vivo performance. The surface negativity of both the NPs can be better attributed to the anionic nature of GA. GA-AgNPs-HP showed higher surface negativity in comparison with GA-AgNPs. This can be due to negatively charged functional groups of HP, thus indicating the successful loading of HP in NPs. AFM revealed both the NPs to be spherical in shape as shown in Figure 1(E,F).

Table 1. Characterization of GA-AgNPs and GA-AgNPs-HP.

Formulation	Average size (nm)	PDI	Zeta potential (mV)	% Drug loading efficiency
GA-AgNPs	81.45 ± 2.07	0.31 ± 0.01	−9.32 ± 0.47	–
GA-AgNPs-HP	155.50 ± 1.56	0.34 ± 0.02	−16.34 ± 0.18	73.66 ± 4.37

FT-IR analysis

FT-IR spectrum of GA shows its characteristic peaks at 3419.20 and 2927.60 cm⁻¹ for stretching vibration of N–H and C–H groups respectively. The peak at 1612.60 cm⁻¹ is assigned to the amide band associated with stretching vibration of the C = O and C–N groups. Similarly, peaks at 1489.0 and 1073.10 cm⁻¹ correspond to stretching vibration of C–N and –O– groups, respectively, as shown in Figure 2(A). FT-IR spectrum of GA-AgNPs reveals peaks at 3418.40 and 2924.50 cm⁻¹ corresponding to stretching vibration of N–H and C–H groups of GA, respectively. Similarly, peak at 1604.30 cm⁻¹corresponds to C = O and C–N groups of GA. The peaks at 1499.10 and 1039.0 cm⁻¹ in GA-AgNPs FT-IR spectrum correspond to stretching vibration of N–H and C–H groups of GA, respectively, as shown in Figure 2(A). The changes in peaks from 1612.60 to 1604.30 cm⁻¹, from 1489.0 to 1499.1 cm⁻¹ and from 1073.10 to 1039.0 cm⁻¹ indicate that C = O, C–N and –O– groups of GA are actively involved in reduction/stabilization of GA-AgNPs.

Figure 2. FT-IR spectra of (A) GA and GA-AgNPs and (B) HP and GA-AgNPS-HP.

FT-IR spectrum of HP shows its characteristic peaks at 3467.40 and 2924.50 cm⁻¹ for –OH stretching and –CH functional groups, respectively. Similarly, peak for C = O stretching appears at 1645.60 cm⁻¹ while peaks for aromatic C = C appear at 1515.80 and 1444.30 cm⁻¹ as shown in Figure 2(B). FT-IR spectrum of GA-AgNPs-HP shows all the characteristic peaks of HP at their respective places. This indicates that there is no formation of any new bond between the loaded HP and excipients of GA-AgNPs-HP, confirming the chemical stability of HP.

Drug loading efficiency

Enhanced drug loading efficiency of nano-drug systems ensures delivery of increased amount of drugs to the target sites and thus results in increased therapeutic efficacy of loaded drugs. Moreover, it also ensures the drug release from the nano-systems in a controlled manner, maintaining their minimum therapeutic level for extended time [13,33]. GA-AgNPs-HP loaded enhanced amount of HP, i.e. 73.66 ± 4.37% as shown in Table 1. The increased loading of HP in GA-AgNPs-HP can be due to the presence of GA on the surfaces of NPs as reported for other such biopolymers used as reducing/stabilizing agents for metal based nano-drug delivery systems. Such biopolymers expose their multiple functional groups and cause increased amount of drug loading [29,34].

Anti-arthritic effect of HP and its NPs

Adjuvant-induced arthritis model is preferred for evaluation of therapeutic molecules due to its short testing duration, easy measurement and sensitivity to CFA [35]. The progression of disease was marked by the swelling of the knee joints followed by the metatarsal and interpharangeal joints. A slight increase in the paw volume was observed in the normal control group over a period of 14 days which might be due to an increase in normal body weight. The arthritic control group exhibited a clear sign of clinical inflammation strated after 4 h of CFA induction which reached to extreme intensity on day 3 with persistant score uptil 14 days, demonstrating mild changes in inflammation.

As shown in Figure 3, the arthritic control rat confirmed the maximum arthritic index compared to normal control. However, treatment with HP at 25 mg/kg and its loaded GA-AgNPs-HP at 1 mg/kg significantly diminished the arthritic score induced by CFA from 4 to 1 in time dependent manner. Mild reduction in arthritic score in treatment with GA-AgNPs at 1 mg/kg was also observed. The results were comparable with the refernce drugs Indomethacin (5 mg/kg) and Dexamethasone (0.5 mg/kg).

Figure 3. Effect of Indo (5 mg/kg), Dexa (0.5 mg/kg), HP (25 mg/kg), GA-AgNPs-HP (1 mg/kg) and GA-AgNPs (1 mg/kg), on arthritic score in CFA induced arthritic rats. The bar diagram represents arthritic score (y-axis) measured on alternate days (x-axis) in different treatment groups (n = 3–5 rats per group). Asterisks indicate the significance difference at *p < .05; **p < .01 and ***p < .001 with respect to control.

Results were further validated through measurement of paw swelling at different time intervals. Treatment groups demonstrated time dependent decline in paw thickness induced by CFA. At day 14, CFA induced arthritic rats showed significant increase in paw thickness (9.2 mm), whereas HP (25 mg/kg), GA-AgNPs-HP (1 mg/kg), Dexamethasone (0.5mg/kg) and Indomethacin (5 mg/kg) suppressed inflammation by >40% inhibition comparing arthritic control as depicted in Figure 4. However, nano-carrier GA-AgNPs (1 mg/kg) could represent only 13% decline in paw swelling.

Figure 4. Effect of HP (25 mg/kg), GA-AgNPs-HP (1 mg/kg), GA-AgNPs (1 mg/kg), Indo (5 mg/kg), Dexa (0.5 mg/kg) on paw swelling in CFA induced arthritic rats. The ray diagram represents paw thickness in mm (y-axis) measured on alternate days (x-axis) in different treatment groups (n = 3–5 rats per group). Asterisks indicate the significance difference at *p < .05; **p < .01 and ***p < .001 with respect to control.

Radiographic analysis of anti-arthritic effect of HP and its NPs

The radiographic images of ankle joints of all groups of rats are shown in Figure 5 and indicate that adjuvant treated rats developed severe soft tissue swelling, irregular joint spaces, excessive bone destruction and joint space reduction (Figure 5(B)) as campared to the normal control group (Figure 5(A)). Whereas, Indomethacin (5 mg/kg) treated rats revealed minimal degenarative changes in ankle joint and moderate swelling in surrounding tissues (Figure 5(C)). However, rats treated with HP 25 mg/kg and its loaded GA-AgNPs-HP 1 mg/kg exhibited mild to moderate tissue swelling, moderate degenerative changes along with mild articular changes (Figure 5(D,E)), conversely, carrier GA-AgNPs has shown no significant anti-arthritic effect (Figure 5(F)). These findings confirm that HP and its loaded GA-AgNPs-HP can effectively restore the disease progression in arthritic cases.

Figure 5. Radiographic images (X-ray) of right hind paw in CFA induced arthritic rats. (A) Normal control, (B) arthritic control, (C) Indo 5 mg/kg, (D) Dexa 0.5 mg/kg, (E) HP 25 mg/kg, (F) GA-AgNPs-HP 1 mg/kg and (G) GA-AgNPs 1 mg/kg. The arrows indicate degenerative changes in ankle joint, swelling around the soft tissues and articular changes.

Effect of HP and its NPs on soft/bone tissue of ankle joint of rats

Histologic assessments were performed for evaluating the progression of disease in ankle joint of CFA induced arthritic rats at the end of the treatment with HP and its loaded GA-AgNPs-HP (as given in supplement material file). Hematoxylin and eosin staining revealed that ankles from arthritic control group rat showed marked cellular infiltration, mixed acute and chronic inflammatory cells with granuloma composed of epithelioid macrophages and pannus formation (grade 4) as compared with the normal control group. However, rats treated with HP 25 mg/kg and its loaded GA-AgNPs-HP 1 mg/kg exhibited mild to moderate inflammation along with moderate chronic infiltrates (grade 2). While Indomethacin 5 mg/kg and Dexamethasone 0.5 mg/kg significantly reduced the total histologic scores and showed moderate granulamatous inflammation along with variable amount of inflamatory infiltrates with no pannus and cartilage damage as shown in Figure 6.

Figure 6. Microphotograph of rat ankle tissue at magnification 40×, 100× and 400×. Normal control (Row I) panel b represents sclerotic bone with arrow head indicating osteocytes. While in panel c, normal soft tissue arrow on both sides indicate muscles arrow head indicating blood vessels. In (row II) panel b of control tissue arrow head indicating granuloma composed of epithelioid macrophages, arrow on both side indicating fibro collagenous tissue, inflamed by mixed acute and chronic inflammatory cells and line shows thick and congested blood vessels along with granulation tissue. At places granuloma with epithelioid macrophages are seen in panel c. Indo 5 mg/kg (row III) panel b shows moderate inflammation while in panel c arrow head indicate thick and congested blood vessels along with granulation tissue. Dexa 0.5 mg/kg (row IV) panel b shows moderate inflammation while in panel c arrow head indicate thick and congested blood vessels along with granulation tissue. HP 25 mg/kg treatment (row V) shows moderate inflammation underline tissue exhibited granulomatous inflammation along with moderate chronic infiltrates and congested dilation blood vessels. Bony trabeculae identified surrounded by moderate chronic infiltrates and no pannus and cartilage damage seen.GA-AgNPs-HP1 mg/kg treatment (row VI) shows mild inflammation, arrow head indicating moderate cell infiltration. Nano-carriers GA-AgNPs 1 mg/kg (row VII) exhibited severe inflammation and increased of cell infiltration.

Real time analysis of TLR-2 and TLR-4 expressions

To better understand the therapeutic role of HP and its loaded GA-AgNPs-HP against TLRs (TLR-2 and TLR-4) expression in RA pathogenesis, Syber green real time PCR was performed using spleen tissues. Interestingly, it was found that expressions of TLR-2 and TLR-4 genes were highly up-regulated in arthritic control group as compared with normal rat, while HP, GA-AgNPs-HP, Indomethacin and Dexamethasone group showed a significant decrease in expression of TLR-2 and TLR-4 (p < .05) when compared to the arthritic animals. TLR-2 expression was reduced up to 70 and 87% in HP and GA-AgNPs-HP treated groups, respectively whereas the TLR-4 expression was found to be reduced by 86 and 98% in HP and GA-AgNPs-HP treated groups. The treatment with GA-AgNPs indicated non-significant alteration in TLR-2/4 expressions. The result was amazingly comparable with reference drugs Indomethacin (96.7 and 98%) and Dexamethasone (81 and 45%) against TLR-2 and -4 genes, respectively (Figure 7).

Figure 7. mRNA expression of TLR-2 and TLR-4 represented by bar diagram indicates relative expression of TLR-2 and TLR-4 gene normalized with housekeeping GAPDH gene on y-axis and treatment with Indo (5 mg/kg), Dexa (0.5 mg/kg), HP (25 mg/kg) and GA-AgNPs-HP (1 mg/kg) on x-axis. The data calculated using 2^−ΔΔCt indicate significant increase in TLR-2 and TLR-4 expressions in arthritic control as compare to normal control, whereas treatment reduced the expression comparatively. Asterisks indicate the significance difference at *p < .05; **p < .01 and ***p < .001 with respect to control.

Findings demonstrate that HP and its GA loaded AgNPs possess inhibitory effect on RA condition in CFA-induced rats. Treatment of HP and GA-AgNPs-HP significantly reduced paw edema, arthritic score, bone destruction and cellular infiltration in ankle joints with appreciably preserved joint architecture. It is first to report that HP interferes with arthritic phenomenon via suppressing mRNA expression of TLR-2 and TLR-4 in adjuvant induced arthritis, additionally HP NPs showed higher release and potency at lower dose than that of pure compound.

CFA-induced rat arthritic model was chosen in the present investigation as it resembles human RA clinical features [36] and is extensively used for preclinical testing of several compounds including NSAIDs [37]. CFA contains heat killed mycobacterium recognized by APCs through TLRs, resulting in the secretion of inflammatory cytokines and chemokines in the synovium. Moreover, TLRs may also control multiple features of the immunopathology of RA [7,38]. Both TLR-2 and TLR-4 were found to be overexpressed in the splenocytes of CFA-induced arthritic animals, while TLR-2 and -4 deficient mice model showed decreased severity of arthritis [39,40]. Additionally, overexpression of TLRs in synovial tissue and fibroblasts in arthritic patients demonstrated their significant role in RA pathogenesis [41]. In recent era, several new compounds targeting TLRs for treating inflammatory diseases are now undergoing preclinical and clinical evaluation [26]. HP was previously reported for its anti-rheumatic effect on CFA-induced arthritis through reduction in synoviocytes proliferation, polyarthritis index and cytokines expression [16,42,43]. Currently available drugs for the treatment of arthritis are NSAIDs and glucocorticoids that temporarily alleviate rheumatic pain but do not protect against tissue injury and joint destruction [44–46]. To discover more effective and less toxic anti-arthritic therapy, anti-CD20, anti-IL-6 receptor monoclonal antibodies and TNFα blockers have been evaluated in RA patients [47–51]. However, due to drug resistance, poor response and increase risk of malignancies demand the discovery of new drugs with favorable safety and tolerability profile for the treatment of arthritis.

Nanotechnology offers promising targeted delivery of therapeutic agents to the desired site of inflammation in controlled or sustained manner due to their small size and their effective interaction with biological system [52]. Considering the anti-arthritic potential of HP; we developed its GA reduced/stabilized AgNPs formulation for its better therapeutic activity through accurate delivery to target site. It was observed that 1 mg/kg of GA-AgNPs-HP showed significant reduction in paw thickness along with minimal arthritic score (Figures 3 and 4), decreased bone damage, tissue degeneration and bone erosion (Figure 5). It also significantly reduced cellular infiltration and granulamatous inflammation in ankle joints tissues (Figure 6), whereas HP (25 mg/kg) produced the anti-arthritic activity comparable to the reference standards i.e., Indomethacin (5 mg/kg) and Dexamethasone (0.5 mg/kg). In previous studies, HP exerted the anti-inflammatory and anti-arthritic activity up to 200 mg/kg [17,42].

Spleen is an important lymphoid organ containing immune cells including macrophages, dendritic and natural killer cells that express TLRs abundantly and is considered as interesting target for underlying mechanistic studies of arthritis. The development of therapeutics to inhibit the TLRs or components of their signaling cascades may represent a way to control rheumatic inflammation with greater specificity [17,26,53]. Effect of HP against TLR signaling has never been reported and to best of our knowledge, we are going to report this for the first time. Current evidence showed an amazing anti-arthritic effect of and its GA loaded AgNPs with more than 80% inhibitory activity against TLR-2 and TLR-4 expression (Figure 7). The unique mechanism of targeting TLR-2 and -4 signaling for RA treatment through NPs is well justified from the current study. GA and other biopolymers stabilized metal NPs have been reported for targeted delivery of drugs and as contrast agents [54,55], thus justifying drug targeting capabilities of GA-AgNPs. Results confirm that the designed delivery system improved stability, solubility, delivery and therapeutic activity of HP. This can be attributed to the multiple dimensions of the current nano-carrier system including nano-range size, surface negativity and enhanced membrane crossing potentials. AgNPs are easily taken by cells due to their interactions with cell membrane proteins, thus delivering the loaded contents in increased concentration to the target sites.

Conclusion

HP is naturally occurring flavonoid with good anti-arthritic potentials, but its lower aqueous solubility and instability reduce its clinical efficacy. HP loaded GA stabilized green AgNPs were successfully synthesized and examined against arthritic phenomenon intruding TLR-2 and TLR-4 mechanism. The results supported that nano-formulation exponentially increased the effectiveness of pure compound and can be a promising future therapeutic agent for arthritis.

Disclosure statement

All the authors declare no conflict of interests.