Artificial Cells, Nanomedicine, and Biotechnology

An International Journal

ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: https://www.tandfonline.com/loi/ianb20

Gum acacia stabilized silver nanoparticles based

nano-cargo for enhanced anti-arthritic potentials
of hesperidin in adjuvant induced arthritic rats

Komal Rao, Sabahat Aziz, Talat Roome, Anam Razzak, Bushra Sikandar,
Khawar Saeed Jamali, Muhammad Imran, Tooba Jabri & Muhammad Raza
Shah

To cite this article: Komal Rao, Sabahat Aziz, Talat Roome, Anam Razzak, Bushra Sikandar,
Khawar Saeed Jamali, Muhammad Imran, Tooba Jabri & Muhammad Raza Shah (2018) Gum
acacia stabilized silver nanoparticles based nano-cargo for enhanced anti-arthritic potentials of
hesperidin in adjuvant induced arthritic rats, Artificial Cells, Nanomedicine, and Biotechnology,
46:sup1, 597-607, DOI: 10.1080/21691401.2018.1431653

To link to this article: https://doi.org/10.1080/21691401.2018.1431653

View supplementary material Published online: 30 Jan 2018.

Submit your article to this journal Article views: 626

View related articles View Crossmark data

Citing articles: 8 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=ianb20
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
2018, VOL. 46, NO. S1, S597–S607
https://doi.org/10.1080/21691401.2018.1431653

Gum acacia stabilized silver nanoparticles based nano-cargo for enhanced

anti-arthritic potentials of hesperidin in adjuvant induced arthritic rats
Komal Raoa, Sabahat Azizb,c, Talat Roomeb,c, Anam Razzakb,c, Bushra Sikandard, Khawar Saeed Jamalie,
Muhammad Imrana, Tooba Jabria and Muhammad Raza Shaha
a
HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, Karachi University, Karachi, Pakistan;
b
Molecular Pathology Section, Department of Pathology, Dow International Medical College, Dow Diagnostic Reference and Research
Laboratory, Dow University of Health Sciences, Karachi, Pakistan; cDepartment of Laboratory Animal Sciences, Dow International Medical
College, Dow University of Health Sciences, Karachi, Pakistan; dHistopathology Section, Department of Pathology, Dow Diagnostic Reference
and Research Laboratory, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan; eDepartment of Surgery,
Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan

ABSTRACT ARTICLE HISTORY

Nanomedicines anticipate drug delivery to inflamed tissues in rheumatoid arthritis (RA) with greater effi- Received 13 December 2017
cacy and lesser side effects. This study investigates the anti-arthritic potentials of Hesperidin (HP) loaded Revised 17 January 2018
in gum acacia (GA) stabilized green silver nanoparticles (AgNPs). Synthesized GA-AgNPs were character- Accepted 18 January 2018
ized through UV–vis spectrophotometer, zetasizer and atomic force microscope (AFM). The HP and its
KEYWORDS
loaded NPs were tested for RA in Complete Freund’s adjuvant (CFA) induced arthritis model. GA-AgNPs Gum acacia; silver
were found in nano-range size with negative charge, spherical shape and loaded increased HP amount. nanoparticles; hesperidin;
HP loaded GA-AgNPs showed minimal arthritic score exhibiting mild to moderate tissue swelling, arthritis; mRNA expression
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 sig-
nificantly 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.

Introduction inflammation in RA and development of therapeutics that

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, sev-
eral 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 con-
sidered 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
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 indus-
try 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 nor-
mal tissues. Nano-carriers also enhance clinical efficacy of
drugs through enhancing their aqueous solubility and protec-
tion from enzymatic degradation [10]. Recently, metal NPs
have got greater scientific interests for therapeutic and diag-
nostic 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 sur-
face-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

CONTACT Muhammad Raza Shah raza.shah@iccs.edu HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
Karachi University, Karachi 74200, Pakistan
Supplemental data for this article can be accessed here.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
S598 K. RAO ET AL.
getting wider attention for fabrication of metal NPs. It uses inex-
pensive and safe methods for synthesis of metal NPs, thus elimi-
nating 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 mole-
cules [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 inflamma-
tion 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 pro-
liferation interfering with Wnt/b-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 mono-
nuclear cells (MNCs) of normal-weight subjects. Flavonoids
have been reported for lowering HFHC meal-induced inflam-
mation and oxidative stress with decreased TLR expression in
normal individuals [20]. These findings prompted us to fur-
ther 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 associ-
ated with RA. The treatment also illustrated potent inhibitory
capacity for intervening rheumatic mechanism through pre-
venting 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 indi-
cated 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 vari-
ous 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 col-
lected 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 institu-
tional 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 modi-
fication [23]. In each group, rats (n ¼ 3–5) were injected with
0.1 mL of CFA suspension (1 mg/mL of heat-killed M. tubercu-
losis in paraffin oil) into the right hind paw. Arthritic control
group received only sub-planter injection of CFA, while differ-
ent 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 intra-
peritoneal 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 mg) from each
sample was reverse-transcribed into cDNA by using cDNA
synthesis kit. Gene expression of TLR-2, TLR-4 and glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) in rat spleen tis-
sues 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 50 -GGAGACTCTGGAAGCAGGTG-30 and reverse
50 -CGCCTAAGAGCAGGATCAAC-30 , TLR-4: forward 50 -TCAAGGC
TTTTCCATCCAAC-30 and reverse 50 -TGCTCAGACATGGCA
GTTTC-30 , GAPDH: forward 50 -GGAAAGCTGTGGCGTGATTGG-30
and reverse 50 -GTAGGCCATGAGGTCCACCA-30 . 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 (2DDCT) 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 ver-
sion 17. One-way and repeated measure Analysis of Variance
(ANOVA) followed by post-hoc Tukey HSD test was imple-
mented 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 biopoly-
mers has been ideal for constructing nano-drug delivery
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S599
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 sta-
bility 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 characteris-
tic SPR for our synthesized GA-AgNPs is accordance with pre-
viously 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 coher-
ent 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)).
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 demon-
strated increased stability upon their incubation with various
salt concentrations (Figure 1(C)). Slight increase in the inten-
sity 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 deg-
radation 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 sys-
tems 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 toxic-
ities 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 com-
pared to other reported AgNPs stabilized with GA. Smaller
particle size was achieved in other reported methods for
S600 K. RAO ET AL.

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.

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 compli-
ance 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 dis-
tributed 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 sur-
face 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
GA-AgNPs 81.45 ± 2.07 0.31 ± 0.01
GA-AgNPs-HP 155.50 ± 1.56 0.34 ± 0.02
Figure 2. FT-IR spectra of (A) GA and GA-AgNPs and (B) HP and GA-AgNPS-HP.
FT-IR analysis
FT-IR spectrum of GA shows its characteristic peaks at
3419.20 and 2927.60 cm1 for stretching vibration of N–H
and C–H groups respectively. The peak at 1612.60 cm1 is
assigned to the amide band associated with stretching vibra-
tion of the C ¼ O and C–N groups. Similarly, peaks at 1489.0
and 1073.10 cm1 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 cm1 corresponding to stretching vibration of N–H
and C–H groups of GA, respectively. Similarly, peak at
1604.30 cm1corresponds to C ¼ O and C–N groups of GA.
The peaks at 1499.10 and 1039.0 cm1 in GA-AgNPs FT-IR
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S601
Zeta potential (mV) % Drug loading efficiency
9.32 ± 0.47 –
16.34 ± 0.18 73.66 ± 4.37
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 cm1, from 1489.0
to 1499.1 cm1 and from 1073.10 to 1039.0 cm1 indicate
that C ¼ O, C–N and –O– groups of GA are actively involved
in reduction/stabilization of GA-AgNPs.
FT-IR spectrum of HP shows its characteristic peaks at
3467.40 and 2924.50 cm1 for –OH stretching and –CH func-
tional groups, respectively. Similarly, peak for C ¼ O stretching
appears at 1645.60 cm1 while peaks for aromatic C ¼ C
appear at 1515.80 and 1444.30 cm1 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
S602 K. RAO ET AL.
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 ¼ 3–5 rats per group). Asterisks indicate
the significance difference at p < .05; p < .01 and p < .001 with respect to control.
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 reduc-
ing/stabilizing agents for metal based nano-drug delivery sys-
tems. 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 progres-
sion 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 nor-
mal control group over a period of 14 days which might be
due to an increase in normal body weight. The arthritic con-
trol 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, demon-
strating 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).
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 (0.5
mg/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.
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 min-
imal degenarative changes in ankle joint and moderate swel-
ling 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 degen-
erative 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.
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
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S603

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 ¼ 3–5 rats per group). Asterisks indicate
the significance difference at p < .05; p < .01 and p < .001 with respect to control.

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.

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 signifi-
cantly reduced the total histologic scores and showed moder-
ate granulamatous inflammation along with variable amount
of inflamatory infiltrates with no pannus and cartilage dam-
age as shown in Figure 6.
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)
S604 K. RAO ET AL.

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 indi-
cating 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.

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-signifi-
cant 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).
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

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 2DDCt
indicate significant increase in TLR-2 and TLR-4 expressions in arthritic control as compare to normal control, whereas treatment reduced the expression compara-
tively. Asterisks indicate the significance difference at p < .05; p < .01 and p < .001 with respect to control.
edema, arthritic score, bone destruction and cellular infiltra-
tion in ankle joints with appreciably preserved joint architec-
ture. 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 com-
pounds including NSAIDs [37]. CFA contains heat killed myco-
bacterium 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 com-
pounds 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 prolifer-
ation, polyarthritis index and cytokines expression [16,42,43].
Currently available drugs for the treatment of arthritis are
NSAIDs and glucocorticoids that temporarily alleviate rheum-
atic 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 TNFa blockers have been evaluated in RA
patients [47–51]. However, due to drug resistance, poor
response and increase risk of malignancies demand the dis-
covery 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
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S605
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 signifi-
cant 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) pro-
duced 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-inflam-
matory 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 arth-
ritis. 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, deliv-
ery 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
S606 K. RAO ET AL.
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.
References
[1] Jones G, Nash P, Hall S. Advances in rheumatoid arthritis. Med J
Aust. 2017;206:221–224.
[2] Al-Zifzaf DS, El Bakry SA, Mamdouh R, et al. FoxP3þ T regulatory
cells in rheumatoid arthritis and the imbalance of the Treg/TH17
cytokine axis. Egypt Rheumatol. 2015;37:7–15.
[3] Hu Y, Cheng W, Cai W, et al. Advances in research on animal
models of rheumatoid arthritis. Clin Rheumatol. 2013;32:161–165.
[4] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol.
2004;4:499–511.
[5] Iwahashi M, Yamamura M, Aita T, et al. Expression of toll-like
receptor 2 on CD16þ blood monocytes and synovial tissue mac-
rophages in rheumatoid arthritis. Arthritis Rheumatol. 2004;50:
1457–1467.
[6] Zhu W, Meng L, Jiang C, et al. Arthritis is associated with T-cell-
induced upregulation of Toll-like receptor 3 on synovial fibro-
blasts. Arthritis Res Ther. 2011;13:R103.
[7] Asea A, Rehli M, Kabingu E, et al. Novel signal transduction path-
way utilized by extracellular HSP70: role of toll-like receptor (TLR)
2 and TLR4. J Biol Chem. 2002;277:15028–15034.
[8] Schaefer L, Babelova A, Kiss E, et al. The matrix component bigly-
can is proinflammatory and signals through Toll-like receptors 4
and 2 in macrophages. J Clin Invest. 2005;115:2223.
[9] Krutzik SR, Modlin RL. The role of Toll-like receptors in combating
mycobacteria. Semin Immunol. 2004;16:35–41.
[10] Chang EH, Harford JB, Eaton MA, et al. Nanomedicine: past, pre-
sent and future – a global perspective. Biochem Biophys Res
Commun. 2015;468:511–517.
[11] Indana MK, Gangapuram BR, Dadigala R, et al. A novel green syn-
thesis and characterization of silver nanoparticles using gum
tragacanth and evaluation of their potential catalytic reduction
activities with methylene blue and Congo red dyes. J Anal Sci
Technol. 2016;7:19.
[12] Brown PK, Qureshi AT, Moll AN, et al. Silver nanoscale antisense
drug delivery system for photoactivated gene silencing.
ACS Nano. 2013;7:2948–2959.
[13] Rao K, Imran M, Jabri T, et al. Gum tragacanth stabilized green
gold nanoparticles as cargos for Naringin loading: a morpho-
logical investigation through AFM. Carbohydr Polym. 2017;174:
243–252.
[14] Grein A, da Silva BC, Wendel CF, et al. Structural characterization
and emulsifying properties of polysaccharides of Acacia mearnsii
de Wild gum. Carbohydr Polym. 2013;92:312–320.
[15] Upadhyay RK. Nutritional, therapeutic, and pharmaceutical poten-
tial of plant gums: a review. Int J Green Pharm. 2017;11:S30–S48.
[16] Li R, Li J, Cai L, et al. Suppression of adjuvant arthritis by hesperi-
din in rats and its mechanisms. J Pharm Pharmacol. 2008;60:
221–228.
[17] Umar S, Kumar A, Sajad M, et al. Hesperidin inhibits collagen-
induced arthritis possibly through suppression of free radical load
and reduction in neutrophil activation and infiltration. Rheumatol
Int. 2013;33:657–663.
[18] Liu Y, Sun Z, Xu D, et al. Hesperidin derivative-11 inhibits fibro-
blast-like synoviocytes proliferation by activating Secreted frizzled-
related protein 2 in adjuvant arthritis rats. Eur J Pharmacol.
2017;794:173–183.
[19] Baizabal-Aguirre VM. Cross-talk mechanisms of Wnt/beta-catenin
signaling components with TLR-activated signaling molecules in
the inflammatory response. Front Immunol. 2017;8:1396.
[20] Ghanim H, Sia CL, Upadhyay M, et al. Orange juice neutralizes the
proinflammatory effect of a high-fat, high-carbohydrate meal and
prevents endotoxin increase and Toll-like receptor expression.
Am J Clin Nutr. 2010;91:940–949.
[21] Sansone F, Rossi A, Del Gaudio P, et al. Hesperidin gastroresistant
microparticles by spray-drying: preparation, characterization, and
dissolution profiles. AAPS Pharmscitech. 2009;10:391–401.
[22] Pearson CM. Development of arthritis, periarthritis and periostitis
in rats given adjuvants. Proc Soc Exp Biol Med. 1956;91:95–101.
[23] Quan L-d, Thiele GM, Tian J, et al. The development of novel
therapies for rheumatoid arthritis. Expert Opin Ther Pat.
2008;18:723–738.
[24] Dewangan AK, Perumal Y, Pavurala N, et al. Preparation, character-
ization and anti-inflammatory effects of curcumin loaded carboxy-
methyl cellulose acetate butyrate nanoparticles on adjuvant
induced arthritis in rats. J Drug Deliv Sci Technol. 2017;41:
269–279.
[25] Laukova M, Vargovic P, Rokytova I, et al. Repeated stress exagger-
ates lipopolysaccharide-induced inflammatory response in the rat
spleen. Cell Mol Neurobiol. 2018;38:195–208.
[26] Perveen K, Hanif F, Jawed H, et al. N-(2-hydroxy phenyl) aceta-
mide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4)
in adjuvant-induced arthritic rats. Mol Cell Biochem. 2014;394:
67–75.
[27] Thakur M, Pandey S, Mewada A, et al. Understanding the stability
of silver nanoparticles bio-fabricated using Acacia arabica (Babool
gum) and its hostile effect on microorganisms. Spectrochim Acta
A Mol Biomol Spectrosc. 2013;109:344–347.
[28] Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation
of antimicrobials. Biotechnol Adv. 2009;27:76–83.
[29] Pooja D, Panyaram S, Kulhari H, et al. Xanthan gum stabilized
gold nanoparticles: characterization, biocompatibility, stability and
cytotoxicity. Carbohydr Polym. 2014;110:1–9.
[30] Imran M, Shah MR, Ullah F, et al. Double-tailed acyl glycoside nio-
somal nanocarrier for enhanced oral bioavailability of Cefixime.
Artif Cells Nanomedicine Biotechnol. 2016;45:1440–1451.
[31] Akele ML, Assefa AG, Alle M. Microwave-assisted green synthesis
of silver nanoparticles by using gum acacia: synthesis, character-
ization and catalytic activity studies. Int J Green Chem Bioprocess.
2015;5:21–27.
[32] Venkatesham M, Ayodhya D, Madhusudhan A, et al. Synthesis of
stable silver nanoparticles using gum acacia as reducing and sta-
bilizing agent and study of its microbial properties: a novel green
approach. Int J Green Nanotechnol. 2012;4:199–206.
[33] Singh R, Lillard JW. Nanoparticle-based targeted drug delivery.
Exp Mol Pathol. 2009;86:215–223.
[34] Dhar S, Reddy EM, Shiras A, et al. Natural gum reduced/stabilized
gold nanoparticles for drug delivery formulations. Chemistry.
2008;14:10244–10250.
[35] Bendele A. Animal models of rheumatoid arthritis. J Musculoskelet
Neuronal Interact. 2001;1:377–385.
[36] Bendele A. Animal models of osteoarthritis. J Musculoskelet
Neuronal Interact. 2001;1:363–376.
[37] Bendele A, Mccomb J, Gould T, et al. Animal models of arthritis:
relevance to human disease. Toxicol Pathol. 1999;27:134–142.

[38] Coulombe F, Divangahi M, Veyrier F, et al. Increased NOD2-
mediated recognition of N-glycolyl muramyl dipeptide. J Exp Med.
2009;206:1709–1716.
[39] Kanagawa H, Niki Y, Kobayashi T, et al. Mycobacterium tubercu-
losis promotes arthritis development through toll-like receptor 2.
J Bone Miner Metab. 2015;33:135–141.
[40] Hu F, Li Y, Zheng L, et al. Toll-like receptors expressed by synovial
fibroblasts perpetuate Th1 and th17 cell responses in rheumatoid
arthritis. PLoS One. 2014;9:e100266.
[41] Brentano F, Kyburz D, Gay S. Toll-like receptors and rheumatoid
arthritis. In: McCoy, CE, O'Neill, editors. Toll-like receptors:
methods and protocols. New York: Humana Press; 2009.
p. 329–343.
[42] Guardia T, Rotelli AE, Juarez AO, et al. Anti-inflammatory proper-
ties of plant flavonoids. Effects of rutin, quercetin and hesperidin
on adjuvant arthritis in rat. Farmaco. 2001;56:683–687.
[43] Kawaguchi K, Maruyama H, Kometani T, et al. Suppression of col-
lagen-induced arthritis by oral administration of the citrus flavon-
oid hesperidin. Planta Med. 2006;72:477–479.
[44] Bua S, Di Cesare Mannelli L, Vullo D, et al. Design and synthesis
of novel nonsteroidal anti-inflammatory drugs and carbonic anhy-
drase inhibitors hybrids (NSAIDs–CAIs) for the treatment of
rheumatoid arthritis. J Med Chem. 2017;60:1159–1170.
[45] Bickham K, Kivitz AJ, Mehta A, et al. Evaluation of two doses of
etoricoxib, a COX-2 selective non-steroidal anti-inflammatory drug
(NSAID), in the treatment of Rheumatoid Arthritis in a double-
blind, randomized controlled trial. BMC Musculoskelet Disord.
2016;17:331.
[46] Newman N, Ling R. Acetabular bone destruction related to non-
steroidal anti-inflammatory drugs. Lancet. 1985;2:11–14.
[47] Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, et al. Active
but transient improvement of endothelial function in
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S607
rheumatoid arthritis patients undergoing long-term treatment
with anti-tumor necrosis factor a antibody. Arthritis Rheum.
2004;51:447–450.
[48] Bathon JM, Martin RW, Fleischmann RM, et al. A comparison of
etanercept and methotrexate in patients with early rheumatoid
arthritis. N Engl J Med. 2000;343:1586–1593.
[49] Breedveld FC, Weisman MH, Kavanaugh AF, et al. The PREMIER
study: a multicenter, randomized, double-blind clinical trial of
combination therapy with adalimumab plus methotrexate versus
methotrexate alone or adalimumab alone in patients with early,
aggressive rheumatoid arthritis who had not had previous metho-
trexate treatment. Arthritis Rheum. 2006;54:26–37.
[50] Caporali R, Caprioli M, Bobbio-Pallavicini F, et al. Long term treat-
ment of rheumatoid arthritis with rituximab. Autoimmun Rev.
2009;8:591–594.
[51] Castillo J, Milani C, Mendez-Allwood D. Ofatumumab, a second-
generation anti-CD20 monoclonal antibody, for the treatment of
lymphoproliferative and autoimmune disorders. Expert Opin
Investig Drugs. 2009;18:491–500.
[52] Chen G, Roy I, Yang C, et al. Nanochemistry and nanomedicine for
nanoparticle-based diagnostics and therapy. Chem Rev.
2016;116:2826–2885.
[53] O'neill LA. Primer: toll-like receptor signaling pathways – what do
rheumatologists need to know? Nat Clin Pract Rheumatol. 2008;4:
319–327.
[54] Kattumuri V, Katti K, Bhaskaran S, et al. Gum arabic as a phyto-
chemical construct for the stabilization of gold nanoparticles: in
vivo pharmacokinetics and X-ray-contrast-imaging studies. Small.
2007;3:333–341.
[55] Ganeshkumar M, Ponrasu T, Raja MD, et al. Green synthesis of pul-
lulan stabilized gold nanoparticles for cancer targeted drug delivery.
Spectrochim Acta AMol Biomol Spectrosc. 2014;130:64–71.