Effect of Folic Acid Conjugated Silver Nanoparticles in Treatment of RA Like Adjuvant Arthritis in Rats


 
 Nano medicine has become one of the promising research areas, opening new horizons in disease diagnosis and treatment. Recent years have witnessed a surge in the development of Nano medicine for combating rheumatoid arthritis (RA), the most common autoimmune arthritis. RA is characterized by progressive inflammation and persistent synovitis, leading to joint destruction, functional incapability, and ultimately disability. Although there has been a tremendous evolution in disease assessment and treatment, many patients still fail to attain remission. Therefore, developing new drugs that specifically target inflamed joints and simultaneously attenuate other possible damages to healthy tissues is indispensable. This study was done to evaluate the potential of folic acid conjugated silver nanoparticles (FA-AgNPs) as RA therapy.
 
 
 
 In the CFA-arthritic rat model, FA-AgNPs & methotrexate were administered for 8 consecutive weeks. Therapeutic efficacy was evaluated by measuring paw volume, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels. For safety concerns, complete blood picture (CBC), liver and renal function tests were evaluated. Joints histological assessment was also carried out.
 
 
 
 FA-AgNPs significantly reduced paw volume, paw weight, ESR, CRP, RF, TNF-α, and IL-6 levels compared with arthritic non-treated rats, demonstrating good anti-inflammatory activity. Likewise, histology of tarsal joints depicted comparatively lesser inflammatory cellular infiltration and diminished cartilage erosions. Methotrexate displayed comparable results. In contrast to methotrexate, FA-AgNPs showed normal CBC & significantly improved liver and renal function tests.
 
 
 
 FA-AgNPs exhibited substantial anti-arthritic activity. This notable anti-arthritic potential of FA-AgNPs was as good as the current standard treatment of methotrexate (MTX) with higher biosafety.



Introduction
Rheumatoid arthritis (RA) represents most common in ammatory arthritis [1]. It is characterized by polyarticular joint involvement with subsequent bone and cartilage in ammation and erosions [2]. It has multifactorial pathogenesis [3]. Several studies have identi ed that activated macrophages play a crucial role in the occurrence and perpetuation of RA [4], therefore, they may be considered as the primary targets in RA management [5].
While the available treatment options can alleviate the symptoms and slow arthritis progression, high frequent doses and long-term treatments are often required which inevitably cause undesirable systemic side-effects due to inability to selectively target the in ammatory areas [6]. Limitations of the current therapies have driven the development of nanomaterial-based devices in RA treatment.
Recently, AgNPs have been used as vehicles to deliver anti-in ammatory drugs for RA treatment [7] and interestingly it was indicated that AgNPs on its own showed anti-in ammatory activity [8,9]. Therefore, it is suggested that AgNPs can be used as effective nontoxic therapeutic agent for RA.
Moreover, as cells involved in RA pathogenesis undergo series of alterations such as overexpression of speci c surface receptors or transformation of phenotype, nanocarriers conjugation with targeted ligand would promote targeted drug delivery to in amed tissues [10]. Many reports have shown that folate receptors (FRβ) are overexpressed on activated macrophages that present in abundance at sites of in ammation [11,12]. These FRs of activated macrophages have been utilized as a target for nanomedicine diagnostic and therapeutic agents [13].

Aim Of Work
This work was done to Evaluate the potential of FA conjugated AgNPs as a DMARD therapy for RA.

Drugs and Chemical
Silver nanoparticles (<100 nm particle size), folic acid powder and Freund's adjuvant (Each mL contains 1 mg Mycobacterium tuberculosis, heat killed and dried, 0.85 mL para n oil and 0.15 mL mannide monooleate) purchased from Sigma-Aldrich. Methotrexate purchased from Mylan SAS. Serum TNF-α and IL-6 ELISA kit purchased from from Bioassay technology laboratory of Jiaxing Korain Biotech Company.
3.2. Preparation of folic acid conjugated silver nanoparticle 176.5 mg of folic acid and 76.6 mg ethylene dichloride (EDC) were dissolved in 40 ml DMSO (dimethyl sulfoxide) -water medium (1 : 1) at room temperature by 3 h. Then 130 mg Ag nanoparticles were added to that mixture solution with continuous stirring at room temperature for 12 h. The nanoparticles were collected by centrifugation at 5000 rpm. The product was dried in vacuum at room temperature. The functionalized material was yellowish in color.

Doses determination
Animal equivalent dose calculated using the following formula: Animal dose (mg/kg) = Human effective dose (HED) (mg/kg) conversion factor The HED of MTX in arthritis (7.5 mg/60 kg body weight/week) was used for calculation of rat dose and the conversion factor was taken as 6.17. The animal dose of MTX was calculated to be 0.7 mg/kg/week.
Rat dose of FA-AgNPs was calculated to be 84.8 μg/kg/day.

Animals
Thirty-eight female Sprague Dawley Albino rats weighing of 200±10 gm purchased from the animal house of El-Nile Company for Pharmaceuticals. The animals were housed under standard laboratory conditions of temperature (24-28˚C), average humidity (55 ± 5%), a 12/12h light/dark cycles. The animals were allowed free access to water and were fed a standard rodent pellet diet. All the animals were acclimatized for 7 days prior to commencement of the procedure.

Induction of arthritis
Systemic arthritis was induced in 32 rats by subcutaneous injection of 0.4 ml divided in three doses [one dose every four days] of Complete Freund's Adjuvant (CFA) into footpad of right hind paw [14].

Experimental design
Rats were randomly allocated into four groups and drugs were administrated on day 12 with the onset of arthritis. At rst, they were divided into 2 groups: Group I: Normal healthy control group of six rats.
Group II: Arthritic 32 rats, in which adjuvant arthritis was induced via CFA injection; then they were subdivided randomly into 3 groups: Group IIa: Arthritic control group of twelve rats received only saline (as Placebo).
Group IIb: methotrexate RA-treated group of ten rats. Received methotrexate in a dose of 0.7 mg/kg/week; IP, for 2 months.

Measurement of paw volume
Hind paw edema volume was measured using a digital vernier caliper (the difference between the final volume minus the initial volume results in paw total volume).

Measurement of biochemical parameters
At the end of the study, blood samples were collected for estimation of CBC, ESR, CRP, serum ALT, AST, urea and creatinine.

Measurement of proin ammatory cytokines
Serum TNF-α and IL-6 were estimated using ELISA kit according to the manufacturer's instructions.

Histopathological analysis
All the 38 rats have been sacri ced after ether inhalation anesthesia. The hind paw was amputated proximal to ankle joint and xed in formalin 10% followed by decalci cation in 5% nitric acid. Then we intersected the hind paw in a mid-sagittal plane and transverse plane. After intersection the tissues were processed and embedded in para n blocks to cut into 4-5 μm thick and stained with hematoxylin and eosin (H&E) for microscopic evaluation.

Statistical analysis
All data are expressed as mean ± SEM. The data were subjected for statistical analysis using GraphPad Prism 7.0 (CA, USA). The statistical signi cance of difference between various groups was tested by onefactor analysis of variance (ANOVA) followed by Tukey's multiple comparison test. P values ≤ 0.05 were considered statistically signi cant.

Paw volume
Compared to the control group, the CFA injection was accompanied with obvious swelling and signi cant increase in paw volume (P ≤ 0.001). Compared to arthritic non-treated group, the deleterious effect of paw swelling associated with CFA was inhibited by treatment with either FA-AgNPs or MTX (P ≤ 0.001), while by comparing effect of FA-AgNPs with effect of MTX on paw volume there was non-signi cant difference as p value is > 0.05 (Table 1).
Paw weight CFA injection resulted in a signi cant increase (P ≤ 0.001) in paw weight when compared to healthy control group. Compared to arthritic non-treated group, treatment with FA-AgNPs or MTX showed a signi cant decrease (P ≤ 0.01) in the paw weight (Table 1).

Body weight
The arthritic non-treated group recorded a signi cant reduction (P ≤ 0.01) in the percentage change of body weight compared with healthy control group. However, FA-AgNPs or MTX treatment showed a signi cant improvement in weight gain when compared to arthritic non-treated rats (P ≤ 0.05) ( Table 1).

Acute phase reactants
CFA injection resulted in signi cant increase (P ≤ 0.001) in ESR and CRP levels when compared to healthy control group. Treatment with FA-AgNPs or MTX showed signi cant decrease (P ≤ 0.001) in both ESR and CRP levels when compared to arthritic non-treated group (Figure 1).

Serum TNF-α and IL-6
Serum levels of TNF-α and IL-6 exhibited signi cant elevation in CFA rats compared to healthy controls (P ≤ 0.001), suggesting the model was successfully established. MTX treatment induced signi cant decrease (P ≤ 0.01) in serum TNF-α and IL-6 as well as FA-AgNPs treatment (P ≤ 0.001) when compared to the arthritic non-treated group. Results of FA-AgNPs regarding serum TNF-α and IL-6 levels were insigni cantly better than MTX as P value is > 0.05. (Figure 2 (Table 2).

Liver function tests
Compared to normal control group, serum ALT and AST levels of arthritic rats were signi cantly elevated (P ≤ 0.001). MTX group showed non-signi cant increase in the serum ALT and AST levels more than nontreated arthritic group (P > 0.05). However, the FA-AgNPs group showed a signi cant decrease in these enzymes than that in non-treated arthritic and MTX groups. (P ≤ 0.01) ( Table 3).

Renal function tests
Compared to normal control group, serum urea (P ≤ 0.001) and creatinine (P ≤ 0.01) levels of arthritic rats were signi cantly increased. Compared with non-treated arthritic group, MTX group showed nonsigni cant increase in serum urea and creatinine (P > 0.05). However, FA-AgNPs treatment resulted in a signi cant decrease in the serum urea (P ≤ 0.001) and creatinine (P ≤ 0.05) levels when compared to non-treated arthritic and MTX groups. (P ≤ 0.01) ( Table 3).

Histopathological analysis
Histopathological assessment of tarsal joints revealed that CFA induced signs of in ammatory in ltration, synovial hyperplasia, partial cartilage and bone destruction compared to normal control group. Furthermore, histopathological scoring of the nding revealed that, FA-AgNPs and MTX treatment produced tarsal joints protection by reducing the levels of cellular in ltration, hyperplasia and cartilage destruction compared to the arthritic non-treated rats (Figure 3).

Discussion
Over last decades, the interest in silver as a therapeutic agent has been rekindled because of the accessibility of silver nanoparticles [15].The current study elucidated the e cacy and safety of the FA-AgNPs in treating CFA RA model in rats.
The study revealed that CFA injection induced redness and swelling in the paws, as evidenced by signi cant elevation in paw volume (P ≤ 0.001). This was in agreement with Li et al. [16] who reported signi cant elevation of paw volume (P < 0.05) following CFA injection.
The therapeutic e cacy was measured by treating the CFA arthritic rats with FA-AgNPs daily for two months and MTX was chosen as a comparative clinical standard treatment. Compared to the non-treated arthritic rats, the rats subjected to FA-AgNPs and MTX showed obvious improvement in the degree of swelling as well as much less redness (P ≤ 0.001). This is supported by Kedi et al., [17] who reported that oral administration of AgNPs induced signi cant (P < 0.001) inhibition of paw edema when compared to arthritic group.
Pro-in ammatory cytokines and acute phase reactants were measured as another index to evaluate the therapeutic e cacy of both compounds. Our results showed signi cant elevation (P ≤ 0.001) of serum TNF-α and IL-6 as well as high ESR and CRP levels in the arthritic group of rats. This is in agreement with Ahsan et al. [18] who reported signi cant rise (P < 0.001) in the mRNA expression of TNF-α and IL-6 following CFA injection in rat model. Furthermore, arthritic rats showed conspicuous rise in the levels of ESR and CRP. FA-AgNPs resulted in remarkable decline (P ≤ 0.001) in the cytokines demonstrating a good anti-in ammatory activity. This was in agreement with Yang et al. [19] who studied the e cacy of FA-AgNPs as targeted treatment for RA on collagen-induced arthritis mice model and reported signi cant reduction in levels of TNF-α and IL-6 as well as in ESR and CRP titer with FA-AgNPs and MTX demonstrating good anti-in ammatory activity (P ≤ 0.001).
Moreover, histopathological analysis of hind limb revealed that non-treated arthritic rats showed cellular in ltration, synovial hyperplasia, partial cartilage and bone destruction compared to normal control.
Treatment with FA-AgNPs and MTX showed an obvious improvement with less cellular in ltration, hyperplasia and milder cartilage destruction compared to the non-treated arthritic rats. This is in agreement with Mani et al., [20] who reported amelioration of histopathological features of CFA with AgNPs and methotrexate treatment.
Generally, body weight usually re ects the health condition of the treated rats. The arthritic group of rats showed slow gradual weight gain that is signi cantly lower than weight gain in the normal control group. FA-AgNPs signi cantly ameliorated disease effect on body weight gain with signi cant increase (P ≤ 0.05) in percentage change of body weight almost similar to the normal control group, suggesting a well tolerance of the rats toward FA-AgNPs at the therapeutically e cacious dose. Similarly, MTX treatment signi cantly improves (P ≤ 0.05) weight gain compared with arthritic non-treated group.
For the safety concerns, the hepatotoxicity was evaluated by measuring serum ALT and AST levels and the nephrotoxicity was tested by measuring serum urea and creatinine. Compared to normal rats, all these parameters were not changed after FA-AgNPs treatment (P > 0.05), in contrast with MTX group, in which a signi cant increase of ALT, AST, urea and creatinine levels were noticed (P ≤ 0.01).
As for the hematological parameters, such as RBC, Hb, WBC and platelets, they were at normal level when compared to normal rats (P > 0.05), indicating no signi cant risk associated with FA-AgNPs treatment. Counterwise, MTX group showed a signi cant decrease in these parameters compared to normal control (P ≤ 0.01).
We can explain what happens in that folic acid receptors (FR) are overexpressed on activated M1 macrophages, which are abundant at areas of in ammation (21). These receptors have been employed as a therapeutic target for folic acid linked nanosilver. Because normal macrophages and neutrophils rarely express folate receptors, FA-AgNP targets active macrophages other than healthy macrophages (22). In reaction to intracellular glutathione (GSH), FA-AgNPs dissolved and released Ag+, causing M1 macrophage death and reactive oxygen species (ROS) scavenging, allowing M2 macrophages to repolarize. FA-AgNPs were gradually removed from the body, primarily by faeces, with no evidence of tissue buildup, and no evidence of long-term harm. Furthermore, AgNP were found to inhibit folate reductase enzymes (23)

Conclusion And Recommendations
FA-AgNPs effectively suppressed the pathological condition in CFA-induced arthritic rats. The notable anti-arthritic potential of FA-AgNPs was as good as the current standard treatment of MTX with higher biosafety. Therefore, we can envisage that FA-AgNPs would be a promising clinical therapeutic modality for RA therapy. We recommend further studies on larger groups using different doses for longer durations to establish the e cacy of FA-AgNP as a treatment of RA and other autoimmune diseases.

Declarations
Competing interests: The author con rms that there are no any potential con icts of interest.
Contributorship: I con rm hereby that the manuscript has not been submitted or is not simultaneously being submitted elsewhere, is not at the time of submission under consideration by another journal or other publication, and that no portion of the data has been or will be published elsewhere while the manuscript is under review by the journal, unless rejected or withdrawn by the author.
Data sharing statement: Also we con rm that no portion of the data has been or will be published elsewhere while the manuscript is under review by the journal.
Funding, grant/award info: I con rm also that there are not any nancial support or other bene ts from commercial sources for the work reported on in the manuscript, or any other nancial interests that I may have, which could create a potential con ict of interest or the appearance of a con ict of interest with regard to the work.   Data are presented as means ± SEM, n = 6. a Signi cantly different from healthy control group at (P ≤ 0.001); b Signi cantly different from RA group at (P ≤ 0.01); c Signi cantly different from MTX group at (P ≤ 0.01) Figure 1 Effect on acute phase reactants Effect on serum cytokines.