- Email: [email protected]
Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic Wistar rats
Accepted Manuscript Title: Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic Wistar rats Author: R. Jeyadevi T. Sivasudha A. Rameshkumar D. Arul Ananth G. Smilin Bell Aseervatham K. Kumaresan L. Dinesh Kumar S. Jagadeeswari R. Renganathan PII: DOI: Reference:
S0927-7765(13)00501-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2013.07.065 COLSUB 5945
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
18-2-2013 27-7-2013 30-7-2013
Please cite this article as: R. Jeyadevi, T. Sivasudha, A. Rameshkumar, D.A. Ananth, G.S.B. Aseervatham, K. Kumaresan, L.D. Kumar, S. Jagadeeswari, R. Renganathan, Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic Wistar rats, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.07.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped
2
cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic Wistar
3
rats
4
R. Jeyadevi a, T. Sivasudha a,*, A. Rameshkumar a,b, D. Arul Ananth a, G. Smilin Bell
5
Aseervatham a , K. Kumaresan c, L. Dinesh Kumar d, S. Jagadeeswari e, R. Renganathan e
6 7
a
8 9 10 11 12 13
b
14
cr
ip t
1
us
Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli - 620024; Tamil Nadu, India Pesticide Residue Analysis Laboratory, Regional Plant Quarantine Station, Chennai - 600027, Tamil Nadu, India. Department of Histopathology, SRM institute for Medical Sciences, Chennai- 600026, Tamil Nadu, India.
d
Department of Biotechnology, Bharathidasan University , Tiruchirappalli - 620024; Tamil Nadu, India.
e
School of Chemistry, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India
M
an
C
15
d
* Corresponding author
te
16
Dr. T. Sivasudha
18
Department of Environmental Biotechnology
Ac ce p
17
Bharathidasan University
19
Tiruchirappalli – 620 024,
20
Tamilnadu,
21
India
22
Tel: 091-0431-2407088
23
Fax: 091-0431-2407045
Email ID: [email protected]
24 25 26 27
1
Page 1 of 35
ABSTRACT
29
In this present study, we investigated thio glycolic acid-capped cadmium telluride
30
quantum dots (TGA-CdTe QDs) as nano carrier to study the antiarthritic activity of
31
quercetin on adjuvant induced arthritic Wistar rats. The free radical scavenging activity of
32
QDs-QE complex was evaluated by 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid
33
(ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Nitric oxide (NO) and superoxide anion
34
scavenging assays. Fifteen days after adjuvant induction, arthritic rats received QDs-QE
35
complex orally at the dose of 0.2 and 0.4 mg/kg daily for 3 weeks. Diclofenac sodium
36
(DF) was used as a reference drug. Administration of QDs-QE complex showed a
37
significant reduction in inflammation and improvement in cartilage regeneration.
38
Treatment with QDs-QE complex significantly (P < 0.05) reduced the expressions lipid
39
peroxidation and showed significant (P < 0.05) increase in activities of antioxidant
40
enzymes such as superoxide dismutase (SOD), reduced glutathione (GSH), glutathione
41
peroxidase (GPx) catalase (CAT) levels in paw tissue.
42
rheumatoid factor (RF), red blood cells (RBC) and white blood cells (WBC) count and
43
erythrocyte sedimentation rate (ESR) of experimental animals were also estimated.
44
Histology of hind limb tissue in experimental groups confirmed the complete cartilage
45
regeneration in arthritis induced rats treated with QDs-QE complex. Based on our
46
findings, we suggest that the QDs act as nano carrier for the drugs used in the treatment of
47
various degenerative diseases.
48
Keywords: Quercetin, Quantum dots, Fluorescence quenching, Nanocarrier, Anti arthritic,
49
Radiology.
C-reactive protein (CRP),
Ac ce p
te
d
M
an
us
cr
ip t
28
50 51 2
Page 2 of 35
1. Introduction Rheumatic arthritis is one of the commonest inflammatory conditions in developing
54
countries. RA is characterized by severe pain, swelling and destruction of cartilage and
55
bone associated with impaired joint function [1]. It is a systemic autoimmune disease
56
which mainly involves the peripheral synovial joints that causes chronic inflammation
57
and profound tissue destruction in affected patients. The autoimmune characteristic of RA
58
is supported by the presence of circulating rheumatoid factor (RF), C-reactive proteins
59
and other endogenous proteins which can be detected in the serum of arthritic patients [2].
60
Currently synthetic drugs form a major line of treatment in the management of arthritis.
61
The conventional drug treatment of RA consists of analgesic, non-steroidal anti
62
inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs) and
63
corticosteroids [3]. NSAIDs elicit their effects by inhibiting COX-2 activity and blocking
64
the downstream production of prostaglandins and offer effective therapies for RA.
65
However, besides their high cost, prolonged use of many of these drugs is associated with
66
severe adverse reactions such as gastric and duodenal ulcers, complications in the small
67
te
d
M
an
us
cr
ip t
53
Ac ce p
52
intestine and colon can occur, which cause colitis, bleeding, perforation, stricture, and chronic problems such as iron deficiency anemia and protein loss and toxicity [4]. A
68
report also states that, NSAIDs treatment enhances joint destruction in arthritis and
69
inhibits glycosaminoglycan synthesis [5]. Recently, there has been an increasing interest
70
in natural food for scavenging the free radicals because of their wide acceptance.
71 72 73
More than 4,000 natural flavonoids are distributed in the plant which has been commonly
74
consumed as foods or medicinal herbs by humans since ancient time. Flavonoids have
75
gained a great deal of interests due to their extensive biological properties such as
76
antioxidant, anti proliferative and anti-inflammatory activities. Since oxidative stress is 3
Page 3 of 35
implicated in the pathogenesis of chronic inflammatory diseases, modulating the cellular
78
redox status by strengthening the endogenous antioxidant defenses might be an effective
79
mechanism of disease prevention. In this regard, dietary polyphenolics generally known
80
for their antioxidant property by neutralizing the formation of excess of reactive oxygen
81
species, which is considered to be a key event in the pathogenesis of rheumatoid arthritis
82
[6]. Quercetin (QE) is widely distributed in frequently consumed foods including apples,
83
berries, onion, tea, red wine, nuts, seeds, and vegetables of the human diet [7]. QE
84
reported to have many beneficial effects, including cardiovascular protection, anticancer
85
activity, antiulcer activity, anti-allergic activity, cataract prevention, antiviral activity and
86
anti-inflammatory activity [8, 9]. QE is an excellent free radical scavenging compound
87
and reported to reduce the risk for oxidative stress related chronic diseases like arthritis,
88
inflammation and diabetes [10].
M
an
us
cr
ip t
77
In modern biotechnology, drug delivery systems are being developed using
90
nanotechnology. Currently, drug delivery systems are a rapidly growing technology within
91
the areas of nanocarriers and photolabelling. Quantum dots (QDs) are a novel class of
92
inorganic fluorophore which are gaining widespread recognition as a result of their
93
exceptional photophysical properties [11]. Quantum dots (QDs) are nanometer-scale
94
semiconductor crystals composed of groups II–VI or III–V elements, and are defined as
95
particles with physical dimensions smaller than the exciton Bohr radius. Semiconductors
96
nanoparticles (often called QDs) have attracted considerable attention in recent years due
97
to their wide range of applications in the field of photovoltaic devices [12, 13] light-
98
emitting diodes (LEDs) [14], fluorescence labelling [15], cell imaging [16] etc. Among
99
various available quantum dots CdTe QDs have been widely studied as luminescence
100
probes and sensors [17, 18]. Recently, Jhonsi et al. [19] investigated the photoinduced
101
interaction between water-soluble CdTe QDs (Cadmium telluride quantum dots) and
Ac ce p
te
d
89
4
Page 4 of 35
certain antioxidants. They found that the antioxidants quench the fluorescence of TGA-
103
CdTe QDs (Thio glycolic acid - CdTe QDs) through complex formation and trapping the
104
holes of QDs. QDs are also being explored as tools for site-specific gene and drug
105
delivery and are among the most promising candidates for a variety of information and
106
visual technologies; they are currently used for the creation of advance flat-panel LED
107
(light-emitting diode) displays and may be employed for ultrahigh-density data storage
108
and quantum information processing [14]. Our previous study has demonstrated the anti
109
microbial activity of M. emerginata using thioglycolic acid-capped cadmium telluride
110
quantum dots as a fluorescent probe [20]. Thus, in the present investigation, we have
111
examined the enhancement of anti arthritic effect of QE using TGA-CdTe QDs through
112
the analysis of hematological parameters, histology and radiological images of paw in
113
adjuvant induced arthritic Wistar rats.
114
2. Materials and methods
115
2.1 Drugs and chemicals
116
All the chemicals used in the study including the solvents were of analytical grade.
117
Complete Freund’s adjuvant (CFA), ABTS, DPPH, butylated hydroxytoluene (BHT), 3, 3-
118
diaminobenzidine tetrahydrochloride and quercetin were purchased from Sigma Chemical
119
Co. (St. Louis, MO, USA). Sulphanilamide, napththylenediamine dichloride, phosphoric
120
acid, nitroblue tetrazolium, reduced nicotinamide adenine dinucleotide, and phenazine
121
methosulfate were purchased from Merck Chemical Supplies (Damstadt, Germany). Thio
122
glycolic acid (TGA), CdCl2. 2.5 H2O (99.99 %), tellurium powder (99.997 %) and sodium
123
borohydride (95 %) were purchased from Sigma Aldrich (St. Louis, MO, USA).
Ac ce p
te
d
M
an
us
cr
ip t
102
124
5
Page 5 of 35
125
Double distilled water was used for preparing solutions. All measurements were
127
performed at ambient temperature.
128
2.2. Synthesis and characterization of TGA capped CdTe Quantum dots
ip t
126
CdTe quantum dots were synthesized by using the following method as reported
130
earlier [19]. Typically, an aqueous solution of Cd2+ ion (1.25 × 10-2 M) and TGA (3 × 10-2
131
M) was prepared and pH was adjusted to 8-9. Then NaHTe was added under nitrogen
132
atmosphere. In our experiments, the typical molar ratio of Cd2+:NaHTe:TGA used was
133
1:0.2:2.4. The resulting products were precipitated by acetone and superfluous of TGA
134
and Cd2+ that did not participate in the reaction was removed by centrifugation at 4000
135
rpm for 5 min. The resultant precipitate was re-dispersed in water and re-precipitated by
136
acetone for more than two times, then kept at 4 °C in dark for further use. The colloidal
137
solution can be kept for three months at room temperature without any obvious
138
aggregation.
Ac ce p
te
d
M
an
us
cr
129
The prepared TGA capped CdTe quantum dots were characterized by Steady state
139 140
measurements. The particle size of prepared QDs was calculated from the absorption
141
maximum (Eq.1).
D = (9.8127 x 10−7) λ3 − (1.7147 x 10−3) λ2 + (1.0064) λ − 194.84
142
(1)
143
where D (nm) is the particle size of QDs and λ (nm) is the wavelength of first excitonic
144
absorption peak of the corresponding QDs. The particle size of the prepared QDs is 2.96
145
nm and the concentration (0.4×10-7 M) was calculated from Lambert’s Beer law.
6
Page 6 of 35
2.2.1 Particle size, zeta potential measurement and SEM analysis
147
The size and the zeta potential of the CdTe quantum dots were determined using dynamic
148
light scattering (DLS) and microelectrophoretic method, respectively (Zetasizer Nano
149
Series-ZS90, Malvern Instruments). The measurements were conducted at 25 °C. The
150
obtained values of the size were equal to an average of the three subsequent runs with 10
151
measurements, while the zeta potential was calculated as an average from the three runs
152
with 20 measurements [21].
us
cr
ip t
146
The morphology of the samples was examined using a scanning electron microscope
154
(SEM) (Hitachi S-4160; FESEM). The samples were fixed with two sequential
155
incubations with 4 % paraformaldehyde solution and with 2.5 % of glutaraldehyde
156
solution for 30 min at 4oC following the samples were dehydrated with ethanol gradient,
157
dried overnight and coated with gold film before SEM observation [22].
M
an
153
2.2.2. Preparation of QE with QDs TGA-CdTe
te
159
d
158
Freshly prepared QDs (1 ×10−6 M) were titrated by adding the appropriate amountof
161
QE (1:1 v/v) and kept in an ultrasonic bath for 25 min to improve binding. After shaking
162
for 1 h, the QDs-QE complex were then ultra centrifuged at 35000 rpm and washed with
163
PBS (pH 7.4) twice to remove unbound QDs. The mixture was then vortexed well to form
164
a homogeneous dispersion, and stored in refrigerator at 4 °C for further analyses [20]. In
165
general, the cadmium ions on the particle surface can coordinate with both -SH and -
166
COOH groups. Quercetin contains 5 -OH groups which strongly anchor to the surface of
167
quantum dots compared to -SH in thioglycolic acid.
168
2.2.3. Characterization of QDs
169
Drug loading efficiency
Ac ce p
160
7
Page 7 of 35
The drug loading and the drug entrapping efficiency of QDs was determined by the
171
UV absorbance at 365 nm using a UV-visible spectrophotometer [23, 24]. The entrapped
172
QE content in the QDs was calculated from the mass of the incorporated drug using the
173
following equation.
cr
174
ip t
170
us
175
Drug encapsulation efficiency
181
encapsulated QE to the mass of the QE used for complex preparation using the following
182
equation:
an
176 177 178 179 180
te
d
M
The drug encapsulation efficiency was defined as the ratio of the mass of the
183
Ac ce p
184
A certain weight of QE was dissolved in water to obtain standard quercetin solution with
185 186
the concentration ranging from 10 to 60 µg/ml. The absorbance was read at 365 nm in the
187
UV spectrophotometer to obtain the linear equation which in turn used to calculate the
188
correlation coefficient [25].
189
2.2.4. In vitro antioxidant studies
190 191
The in vitro antioxidant activity of QDs-QE complex was determined by their ability
192
to scavenge DPPH, ABTS [26], NO and superoxide anion radicals [27]. Percentage
193
inhibition was expressed in terms of IC50 value calculated by linear regression method.
194
BHT was used as reference standard. 8
Page 8 of 35
195
2.3. In vivo studies
196
2.3.1. Animals Female albino Wistar rats (150-200 g) were obtained and housed in poly-acrylic
198
cages and maintained under standard laboratory conditions (temperature 24-28 ºC, relative
199
humidity 60-70 % and 12 h dark-light cycles). Animals were fed with commercial rat feed
200
(Sai Durga feeds and food stocks, Chennai, India) and water, ad libitum. All the animals
201
were acclimatized to laboratory conditions for 7 days before commencement of the
202
experiment. Experimental protocols were approved by the Committee for the Purpose of
203
Control and Supervision of Experiments on Animals (CPCSEA), Chennai, Tamil Nadu,
204
India (BDU/IAEC/2012/26/28.03.2012).
206
M
205
an
us
cr
ip t
197
2.3.2. Biocompatability of QE and QDs
Quercetin, reported to be non toxic to in vivo model up to the concentration of 200
208
mg/kg b.w. by Naovarat Tarasub et al. [28]. A study about quantum dots, revealed that
209
QDs is non toxic in vivo model up to the concentration of 15.0 nmol over short-term (< 7
210
days) and long-term (> 80 days) periods [29]. The concentration of QE and QDs used in
211
this study were lesser than the above mentioned reports.
Ac ce p
te
d
207
212 213
2.3.3. Anti-arthritic studies
Animals were divided into seven groups of six each. Group 1 consisted of control
214 215
rats, all other groups were comprised of arthritic rats. Test groups were injected with 0.1
216
mL of CFA intradermally. Group 2: disease control, Group 3: treated with standard drug
217
diclofenac sodium (DF) (0.3 mg/kg b.wt/day p.o.). While Group 4 and Group 5 treated
218
with QDs-QE complex at a dose of 0.2 mg/kg b.wt/day p.o. and 0.4 mg/kg b.wt/day p.o.
219
Group 6: treated with 1 mL of QDs (1x10-6 M) and Group 7: treated with QE at the 9
Page 9 of 35
220
concentration of 5 mg/kg respectively, from day 15th to 35th of induction of adjuvant
221
induced arthritis. Animals were monitored for the development of clinical arthritis using a
222
vernier caliper [30]. On the 35th day, animals were anesthetized and the hind legs of the experimental
224
animals of different groups were analyzed by X-ray radiography and examined for the soft
225
tissue swelling, bony erosions and narrowing of the spaces between joints and then
226
animals were sacrificed; blood samples and paw tissue were collected and used for
227
biochemical estimations. Histopathological studies were done in hind limb joint tissues.
228
Tissues were fixed in formalin, decalcified, embedded in paraffin blocks and sections
229
stained with hematoxylin and eosin (H & E).
230
2.3.4. Biochemical estimations
M
an
us
cr
ip t
223
The paw tissue homogenates (10 %, w/v) prepared in phosphate buffered saline
232
(PBS containing 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4 and 1.76 mM
233
KH2PO4 in 1000 mL distilled water pH 7.4) were used for antioxidant studies such as lipid
234
peroxidation [31], SOD [32], CAT [33], GPx [34] and GSH [35] activities. Blood samples
235
were subjected to evaluate the biochemical parameters like white blood corpuscles count,
236
ESR, anti CRP and RF were done using commercial reagent kits and were performed
237
according to the manufacturer’s (Nice Chemicals [P] Ltd. Cochin, Kerala, India)
238
instructions.
239
2.4. Statistical analysis
Ac ce p
te
d
231
The statistical analyses were performed using SPSS (Statistical Package for Social
240 241
Science, SPSS Inc., Chicago, IL, USA). Significance of each group was verified with one-
242
way analysis of variance (ANOVA) followed by Duncan post hoc test (P ≤ 0.05). Values
243
obtained are means of six replicate determinations ± standard deviation.
244
3. Results
10
Page 10 of 35
245
3.1. Absorption studies The UV-visible absorption spectra of QDs in aqueous solution and with QE were
247
shown in Fig. 1. It has been observed that while increasing the concentration of QE (12–60
248
μg in 2.5 mL), the QDs absorbance also increase. Moreover, we could find there is no shift
249
and new peak formed. This may be due to QE is adsorbed to the surface of QDs. This
250
reveals the formation of ground state complex between QDs and QE.
251
3.2. Fluorescence studies
us
cr
ip t
246
Fluorescence quenching measurements have been widely used to analyze the various
253
type interactions between the molecules [36, 37]. This method helps to understand QDs
254
binding mechanisms with QE and provide clues to the nature of the binding phenomenon.
255
Fig. 3. shows the emission spectrum of QDs excited at 500 nm in the absence and
256
presence of QE. The emission intensity of QDs is quenched by raising the concentration of
257
quercetin (12– 60 μg in 2.5 mL), i.e fluorescence quenching. Further, we recorded the
258
emission spectrum of QE (to the highest concentration 60 μg in 2.5 mL) at the same
259
excitation wavelength of QDs, it is transparent at this excitation. From this observation,
260
we confirmed that the observed quenching is mainly due to interaction of QDs with QE
261
and no inner filter effect or reabsorption. Hence the binding constant for this type of
262
interaction was calculated using fluorescence quenching data by this method
Ac ce p
te
d
M
an
252
1 1 + = 0 (F -F) (F -F') 0
263
1 K(F -F')[Q] 0
(4)
264
where K is the binding constant, F0 is the initial fluorescence intensity of QDs, F′ is the
265
fluorescence intensity of QE adsorbed QDs and F is the observed fluorescence intensity at
266
its maximum. The plot of 1/(F0-F) versus 1/[Q] gives a straight line (Fig. 2) and from the
11
Page 11 of 35
267
slope the calculated binding constant is found to be 4.27 × 105 M-1. There is a good linear
268
relationship between 1/(F0-F) and the reciprocal concentration of dyes.
269
3.3. Particle size, zeta potential measurement and SEM analysis The size distribution of QDs in colloidal solution was found to be 185 nm. A negative
271
zeta potential of about −30.2 mV was observed in the present study that pose ideal surface
272
charge. The size and surface charge of the QDs determinethe biological activity. From the
273
Zeta potential measurements, the particle size distribution of the QDs was found to be 16
274
nm (Fig. 3a, b). FE-SEM images of compound loaded quantum dots were found to be
275
spherical in shape with regular arrangement (Fig 3c). The picture shows the magnification
276
image of uniformally arranged QDs.
277
3.4. Charecterization of QDs
M
an
us
cr
ip t
270
The absorbance values of standard QE solution were determined at 365 nm. It was
279
calculated using the following equation. Amount of QE and encapsulation efficiency was
280
calculated using the obtained equation,
(4)
Ac ce p
Y = 0.045X + 0.417
te
d
278
281 282
Where Y is the absorbance values, and X is QE concentration. The calibration curve for
283
the absorbance of QE was linear over the range of standard concentration of QE (10 – 60
284
µg/ml) with a correlation coefficient of R2 = 0.995. The drug entrapped in the QDs and
285
the QE loading efficiency was calculated using the equation 2, 3 and 5. Therefore, the
286
encapsulation efficiency of drug loaded QDs-QE was 6.38% and the drug loading
287
efficiency was 55.26%.
288 289
3.5. Mechanism of quenching
12
Page 12 of 35
Upon excitation of CdTe QDs, it would result in the promotion of electron from its
291
valence band to the conduction band. This results in the formation of a positively charged
292
hole in its valence band and a free electron in the conduction band of QDs. In the absence
293
of QE, the formed electron and hole would recombine which results in emission of
294
fluorescence. On introducing hole acceptors (i.e. QE) to the solution of QDs the electron
295
hole recombination process will be prevented which causes decrease in the emission
296
intensity of QDs (i.e.) fluorescence quenching.
297
3.6. In vitro antioxidant studies
us
cr
ip t
290
The percentage inhibition of ABTS and DPPH free radicals by QDs-QE complex
299
are shown in Fig.4a and Fig. 4b respectively. QDs-QE complex significantly inhibited the
300
ABTS and DPPH radicals in a dose-dependent manner with the IC50 values of 60 and 61
301
µg/mL, respectively. Fig. 5a, shows the scavenging activity of nitric oxide by QDs-QE
302
complex in a concentration dependent manner. The IC50 value of QDs-QE complex, QE
303
and BHT to scavenge the nitric oxide radical is 40 µg/mL, 42 µg/mL and 45 µg/mL
304
respectively. Fig. 5b shows the superoxide radical scavenging capacity of QDs-QE
305
complex measured by the PMS-NADH superoxide generating system. QDs-QE complex
306
was found to be an effective scavenger of superoxide anion radicals in a dose-dependent
307
manner with an IC50 value of 20 µg/mL and thus can prevent the formation of ROS.
Ac ce p
te
d
M
an
298
308 309
3.7. CFA induced rat paw edema
310
There was a significant increase in rat paw volume in CFA injected arthritic
311
control rats when compared to the normal control rats. QDs-QE complex treatment at the
312
dose of 0.2 mg/kg and 0.4 mg/kg body weight showed significant reduction in rat paw
313
edema volume when compared with the arthritic group (Data not shown).
314 13
Page 13 of 35
315
3.8. Protective effect of QDs-QE complex on antioxidant enzymes There was an increased level of LPO and a decreased antioxidant enzyme activity
317
in Group 2 rats. The declined antioxidant enzyme activity is responsible for the increased
318
lipid peroxidation measured as thiobarbituric acid reacting substance (TBARS), which
319
causes loss of membrane fluidity, membrane integrity, and finally loss of cell functions
320
[38]. This peroxidative damage to membranes results in the leakage of enzymes, and
321
metabolites to circulation. Elevated LPO saturates the level of free radicals which
322
sequentially inhibit activities of SOD, CAT, GSH and GPx. Group 6 (1 mL of 1x10-6 M
323
QDs administered) does not show any significant protective effect against CFA induced
324
arthritis, whereas, administration of QE up to 4 mg/kg did not show any protective effect
325
against arthritic induced animals. However, QE at 5 mg/kg showed mild protective effect
326
which is comparable to Group 4 animals. QDs-QE complex attenuates their levels
327
significantly (P ≤ 0.05) in paw tissue comparable to that of reference standard drug treated
328
group (Table 1).
te
329
3.9. Haematological parameters
Ac ce p
330
d
M
an
us
cr
ip t
316
The changes in hematological parameters in adjuvant induced arthritic rats were
331 332
shown in Table 2. There was an increase in WBC count, ESR, anti CRP and RF of
333
arthritic rats, when compared with the control rats. The treatment with QE and QDs-QE
334
complex in a dose dependent manner had significantly brought back the altered
335
hematological changes in adjuvant induced arthritic animals to normal levels.
336 337
3.10. Radiographic changes
338
X-ray radiographs of the different treatment group animal paws taken on 25th day.
339
Adjuvant treated rats had developed definite joint space narrowing of the joints, diffused
14
Page 14 of 35
soft tissue swelling in digits, cystic enlargement of bone and extensive erosions produced
341
narrowing or pseudo widening of all joint spaces (Fig. 6b). The standard drug diclofenac
342
sodium treated group shows no bony destruction and swelling of the joint (Fig. 6e).
343
Treatment with QDs-QE complex for 25 days have shown significant prevention against
344
bony destruction by showing less soft tissue swelling and narrowing of joint spaces in a
345
dose dependant manner (Fig. 6c and d).
cr
ip t
340
347
3.11. Effects of QE treatment on histological findings
us
346
To evaluate the anti-inflammatory effects of QDs-QE complex, samples of the ankle
349
joints from each experimental group were examined by H&E staining. Control group
350
shown normal lobular architecture (Fig. 7a). CFA administered group shown thinning of
351
cartilage plates, severe bone erosion, extensive infiltration of inflammatory cells into the
352
synovial cavity, pannus formation (Fig. 7b). QDs-QE complex treated group at 0.2 mg/kg
353
shown mild improvement cartilage regeneration, moderate edema formation and cellular
354
infiltration (Fig. 7c). QDs-QE complex treated group at 0.4 mg/kg rats showed complete
355
cartilage regeneration. Absence of inflammation and inflammatory cells in synovial region
356
proved the anti-arthritic potential of QDs-QE complex (Fig. 7d). DF (positive drug)
357
treated group exhibited almost normal histology (Fig. 7e). QD treated group shows almost
358
similar observations of CFA treated group with extensive infiltration of inflammatory cells
359
into the synovial cavity (Fig. 7f). QE at 5 mg/kg showed mild improvement in cartilage
360
regeneration, moderate edema formation and cellular infiltration (Fig. 7g)
Ac ce p
te
d
M
an
348
361 362
4. Discussion
363
In general, QE is the highly consumed flavonoid and the richest sources of QE are tea,
364
onions and apples. The best described property of QE is its capacity to act as antioxidant,
15
Page 15 of 35
thus protecting the body against reactive oxygen species (ROS). Because of the high
366
reactivity of the hydroxyl group of the QE, reactive oxygen species are made
367
inactiveReactive oxygen species at the site of inflammation are reported to be involved in
368
the pathogenesis of RA [26]. In recent years, many intense research works are focused on
369
an electron transfer between molecular adsorbents and semiconductor nanonmaterial due
370
to large number of practical applications. Semiconductor QDs have gained a lot of
371
research interest in the last decade due to their existing size and shape dependent
372
properties, photo bleaching threshold, good chemical stability, relatively narrow and
373
symmetric luminescence bands [39-41]. The zeta potential of a nanoparticle is commonly
374
used to characterize the surface charge property of nanoparticles. Generally, the thiol
375
group of TGA will bind on the surface of QDs while COO− will be present as new surface
376
groups. The presence of negatively charged COO− surface groups was confirmed by the
377
observation of zeta potential in the negative region. In our CdTe QDs, we observed a
378
negative zeta potential around −30.2mV which confirms the presence of negatively
379
charged COO− surface groups. The difference between the calculated size from absorption
380
measurements and particle size from zeta potential measurement is mainly because the
381
latter gives the diameter of the particle along with any ligands attached to it, and solvent
382
molecules which may be strongly associated with it. In addition, compared with organic
383
dyes and fluorescent proteins, QDs offer several advantages, such as a narrow, symmetric
384
emission from visible to IR wavelengths and photo-chemical stability. QDs possess
385
dimensional similarities to biological macromolecules, such as nucleic acids and proteins
386
thus, QDs were used to study their interactions with various biomolecules [20]. Recently,
387
Jhonsi et al. [42] reported on the photo-induced interaction between water-soluble CdTe
388
quantum dots and certain antioxidants. The antioxidants quench the fluorescence of TGA-
389
CdTe QDs through complex formation, and the quenching results from antioxidants
Ac ce p
te
d
M
an
us
cr
ip t
365
16
Page 16 of 35
390
trapping the holes of the QDs. Moreover QDs also reported to be stable in the digestive
391
tract of Wistar rats [43]. Pearson and Wood (1959) [44] reported that, rats immunized with Complete
393
Freund’s adjuvant containing Mycobacterium tuberculosis develop arthritis. Nevertheless,
394
their mode of action is still not completely understood. Adjuvant-induced arthritis is a
395
widely used arthritic model for testing and developing anti-arthritic and anti-inflammatory
396
agents. After 10 to 14 days of CFA injection, animals develop arthritis. Adjuvant arthritis
397
animal model is very similar to human rheumatoid arthritis, resembles to pathological and
398
serological changes, including the inflammatory mediators. In adjuvant-induced arthritis,
399
rat model develops chronic swelling and pain in multiple joints with release of cytokines
400
from inflammatory cells, culminating in erosion of cartilage and bone destruction causing
401
severe disability [45]. The mechanisms of CFA induced arthritis may involve prolongation
402
of the presence of antigens at the site of injection or more effective transport of the
403
antigens to the lymphatic system and to the lungs, where the adjuvant promotes the
404
accumulation of cells concerned with the immune response or by excessive production of
405
ROS.
Ac ce p
te
d
M
an
us
cr
ip t
392
Increase in WBC count has been suggested to be one of the characteristic
406 407
diagnoses of arthritis. In our present study, CFA-induced arthritic animals showed
408
elevated WBC level. QDs-QE complex treatment significantly decreased WBC, revealing
409
its beneficial role against arthritis. RF has been regarded as the main serologic marker in
410
inflammatory arthritis [46]. RF is an auto antibody directed against the Fc portion of IgG.
411
RF and IgG join to form immune complexes that contribute to the progress of rheumatoid
412
arthritis. In recent years, C-reactive protein has also been identified as important predictors
413
both for diagnosis and prognosis of rheumatoid arthritis. C-reactive protein is a member of
414
the pentraxin family of proteins. CRP is secreted by the liver in response to a variety of 17
Page 17 of 35
inflammatory cytokines. An elevated CRP level can provide support for the presence of an
416
inflammatory disease, like rheumatoid arthritis [47]. Our findings represent high levels of
417
RF and CRP in arthritic rat serum. Dose-response reductions of these factors have been
418
observed in the arthritic rats treated with QE reveals its protective effect against arthritis.
419
The ESR count which significantly increased in arthritic control group has been
420
remarkably decreased by QDs-QE complex and standard drug diclofenac sodium, thus
421
justifying significant role of QDs-QE complex in arthritic conditions. Our previus study,
422
demonstrated that, Quercetin at a higher concentration of 5 mg/kg b.w shows protective
423
effect against CFA induced arthritis in Wistar rats. Treatment with QDs alone to arthritic
424
rats showed no effect. So, it was suggested that QDs act as nano carrier of the active
425
compound QE and results in better anti arthritic property even at lower concentration (0.4
426
mg/kg b.w.). ROS in arthritis is not surprising since oxidative stress or reactive oxygen
427
species serve as mediators of tissue damage.
428
synovial fluid and induce depolymerization of hyaluronic acid which in turn leads to a loss
429
of viscosity in the joints [48]. The cartilage destruction by ROS in arthritic rats treated
430
with QDs-QE complex has strongly inhibited through increase in the levels of endogenous
431
enzymatic antioxidants, such as SOD, CAT, GPx and non-enzymatic antioxidant GSH.
432
The conventional drug treatment of arthritis elicits their effects by inhibiting COX-2
433
activity and blocking the downstream production of prostaglandins. The possible
434
mechanism of QDs-QE complex to exert anti arthritic activity might be through
435
neutralization of various free radicals generated at the site of inflammation and by
436
inhibiting the production of COX-2 enzyme (Fig. 8).
437
5. Conclusion
The reactive oxygen species degrade
Ac ce p
te
d
M
an
us
cr
ip t
415
438
To conclude, our results suggest that QDs-QE complex showed antioxidant and anti-
439
arthritic effects. Absorbance and fluorescence quenching of QDs-QE was successfully 18
Page 18 of 35
investigated. The anti-inflammatory effects of QDs-QE complex may be related to free
441
radical quenching via increase in the activities of antioxidant enzymes and by inhibiting
442
the expression of inflammatory mediators. QE alone showed anti arthritic potential only at
443
the higher concentration, where as the QDs-QE complex exhibited anti arthritic activity
444
even at a lower concentration. Furthermore, this study reveals that using QDs as nano
445
carrier of QE exhibited enhanced anti arthritic effect even at a lower concentration of the
446
drug. These findings prove the promising effect of thioglycolic acid-capped cadmium
447
telluride quantum dots as a nano carrier to enhance the potential of anti arthritic drugs in
448
rheumatic complications. Further studies are in progress to better understand the
449
mechanism of action of QDs-QE complex, responsible for enhanced anti arthritic effect at
450
lower concentration of the drug.
M
451
Acknowledgment
d
452
an
us
cr
ip t
440
We thank the University Grants Commission (UGC), New Delhi, India for the
454
financial support. S. Jagadeeswari (Ref. No: 039680/E15/2011 Dt: 17.02.2011) thanks
455
UGC-BSR for her Research Fellowship in Science for Meritorious Students. We thank
456
DST–FIST and UGC-Non SAP for providing other instrumental facilities.
Ac ce p
te
453
457 458
References
459
[1] M. Feldmann, F.M. Brennan, R.N. Maini, Cell 85 (1996) 307-310.
460
[2] A.K. Andersson, C. Li, F.M. Brennan, Arthritis. Res. Ther. 10 (2008) 204-212.
461
[3] A.A. Schuna, C. Megeff, Am. J. Health-Syst. Pharm. 57 (2000) 225-234.
462
[4] S. Sharma, D. Sahu, H.R. Das, D. Sharma, Food Chem. Toxicol. 49 (2011) 33953406.
463
19
Page 19 of 35
[5] C.Z. Manon, T.N. de Boer, J.A.V. Roon, J.W.J. Bijlsma, F.P.J.G. Lafeber, S.C Mastbergen, Arthritis. Res. Ther. 13 (2011) 239.
465
[6] M.K. Park, J.S. Park, M.L. Cho, H.J. Oh, Y.J. Heo, Y.J. Woo, Y.M. Heo, M.J. Park,
468
[7] I. Erlund, Review of flavonoids quercetin, hesperetin, and naringenin. Nutr. Res. 24 (2004) 551-874.
469 470
ip t
H.S. Park, S.H. Park, H.Y. Kim, J.K. Immunol. Lett. 124 (2009) 102-110.
467
[8]
cr
466
C.V. de Whalley, S.M. Rankin, J.R. Hoult, W. Jessup, D.S. Leake, Biochem. Pharm. 39 (1990) 1743-750.
471
us
464
[9]
C. Bronner, Y. Landry, Agents and Actions 16 (1985) 147-151.
473
[10] T.N. Kaul, E.Jr. Middleton, P.L. Ogra, J. Med. Virol 15 (1985) 71-79.
474
[11] Q. Xiao, S. Huang, W. Sua, P. Li, J. Ma, F. Luo, J. Chen, Y Liub, Colloids Surf. B: Biointerfaces 102 (2013) 76-82.
475
M
an
472
[12] S. Coe, W.K. Woo, M.G. Bawendi, V. Bulovic, Nature 420 (2002) 800-803.
477
[13] K. Barnham, J.L. Marques, J. Hassard, P. O’Brien, Appl. Phys. Lett. 76 (2000) 11971199.
478
te
d
476
[14] V.L. Colvin, M.C. Schlamp, A.P. Alivisatos, Nature 370 (1994) 354-357.
480
[15] D. Gerion , W.J. Parak, S.C. Williams, D. Zanchet, C.M. Micheel, A.P. Alivisatos, J.
Ac ce p
479
Am. Chem. Soc. 124 (2002) 7070-7074.
481 482
[16] C.L. Wang, H. Zhang, J.H. Zhang, M.J. Li, H.Z. Sun, B. Yang, J. Phys. Chem. C. 111 (2007) 2465-2469.
483 484
[17] A.S. Susha, A.M. Javier, W.J. Parak, A.L. Rogach, Colloids Surf. A: Physicochem. Eng. Aspects 281 (2006) 40-43.
485 486
[18] J.P. Yuan, W.W. Guo, E.K, Wang, Anal. Chem. 80 (2008) 1141-1145.
487
[19] M.A. Jhonsi, R. Renganathan, J. Coll. Int. Sci. 344 (2010) 596-602.
20
Page 20 of 35
488
[20] A. Rameshkumar, T. Sivasudha, R. Jeyadevi, B. Sangeetha, D. Arul Ananth, G.
489
Smilin Bell Aseervatham, N. Nagarajan, R. Renganathan, A. Kathiravan, Colloids
490
Surf. B: Biointerfaces 101 (2013) 74-82. [21] M. Adamczak, M. Krok, E. Pamuła, U. Posadowska, K. Szczepanowicz, J. Barbasz,
ip t
491
P. Warszyński, Colloids and Surf. B: Biointerfaces. 110 (2013) 1-7.
492
[22] G. Ciofani, L. Ricotti, C. Canale, D. Alessandro, S. Berrettini, B. Mazzolai, V.
cr
493
Mattoli, Colloids and Surf. B: Biointerfaces. 102 (2013) 312-320.
494
[23] K.H. Huang, Z.H. Zhu, J.H. Liu, et al., Chin J Cancer. 24 (2005) 1023-1026.
496
[24] E.K. Park, S.B. Lee, Y.M. Lee, Biomaterials 26 (2005) 1053-1061.
497
[25] T. Musumeci, C.A. Ventura, I. Giannone, et al. Int J Pharm 325 (2006)172-179.
498
[26] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, J.M. Harnly, L.Z. Lin, J. Funct. Foods 5 (2013) 289-298.
499
(2013) 115-126.
501 502
[28]
Ac ce p
[29] T.S. Hauck, R.E. Anderson, H.C. Fischer, S. Newbigging, W.C. Chan, Small. 6(1) 2010 138-144. doi: 10.1002/smll.200900626.
505 506
N. Tarasub, C. Tarasub, Watcharaporn Devakul Na Ayutthaya, T. Suramana. (2000). http://www.med.tu.ac.th/medJournal/TUmed74/TuM7_4page321.pdf.
503 504
d
[27] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, L. Dineshkumar, Inflamm. Res. 62
te
500
M
an
us
495
[30] S.C. Ridge, K.M. Ferguson, N. Rath, J. Galivan, J.H. Freisheim, A.L. Oronsky, S.S. Kerwar, J. Rheumatol. 15 (1988) 1193-1197.
507 508
[31] A.J. Buege, S.D. Aust, Meth. Enzymol. 52 (1978) 302-310.
509
[32] J.M. McCord, I. Fridovich, J. Biochem. 244 (1969) 6049-6056.
510
[33] H. Aebi, Meth. Enzymol. 105(1984) 121-126.
511
[34] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.J. Hofmen, W.G. Hoekstra, Science. 179 (1973) 588-590.
512
21
Page 21 of 35
513
[35] M.S. Moron, J.N. De Pierre, V. Mannervik, Biochim. Biophys. Acta. 582 (1979) 6768.
514
[36] H. Wu, H. Wang, L. Xue, X. Li, J. Colloid Interface Sci. 353 (2011) 476-481.
516
[37] Shown I, Ujihara M, Imae T, J. Colloid Interface Sci. 352 (2010) 232-237.
517
[38] B. Halliwell, J.M.C. Gutteridge, 1989. Free Radicals in Biology and Medicine, 2nd
cr
edition. Clarendon Press, Oxford.
518
[39] A. Kumaria, S.K. Yadava, Y. B. Pakadeb, B. Singhc, S.C. Yadava, Colloids Surf. B: Biointerfaces 80 (2010) 184-192.
520
us
519
ip t
515
[40] M. Kakran, N.G. Sahoo, L. Li, Colloids Surf. B: Biointerfaces 88 (2011) 121-130.
522
[41] K.E. Sapsford, T. Pons, I.L. Medintz, H. Mattoussi, Sensors 6 (2006) 925-953.
523
[42] M.A. Jhonsi, E. Vaishnavi, R. Suganya, A. Kathiravan, R. Renganathan, Adv. Sci. Lett. 4 (2011) 1–6.
524
[43] Y.F. Loginova, S.V. Dezhurov, V.V. Zherdeva, N.I. Kazachkina, M.S. Wakstein,
d
525
M
an
521
A.P. Savitsky, Biochem Biophys Res Commun. 419 (2012) 54-9.
te
526
[44] C.M. Pearson, F.D. Wood, Arthritis. Rheum. 2 (1959) 440-459.
528
[45] S. Singh, D.K. Majumdar, Int. J. Pharmacol 34(3) (1996) 218-222.
529
[46] D.L. Scott, Rheumatology (Oxford) 39 (2000) 24-29.
530
[47] G. Sindhu, M. Ratheesh, G.L. Shyni, B. Nambisan, A. Helen, Int. Immunopharmacol.
Ac ce p
527
12 (2012) 205-211.
531 532
[48] F.C. Arnett, S.M. Edworthy, D.A. Bloch, D.J. McShane, J.F. Fries, N.S. Cooper,
533
L.A. Healey, S.R. Kaplan, M.H. Liang, H.S. Luthra, Arthritis. Rheum. 31 (1988) 315-324.
534 535 536 537
22
Page 22 of 35
537
Table 1 Protective effect of QDs-QE complex on TBARS, GSH and antioxidant enzymes
538
in paw tissue sample of arthritic rats
539
±
0.31b
2.7
Group 2 (Disease Control)
1.5
±
±
1.9
±
±
±
0.34b
2.35b
2.4± 1.03a
21.42
±
±
0.90c
te
d
29.15
31.68
1.4
Ac ce p
Group 5 ( QDs-QE complex 0.4 mg/kg b.w)
22.92 1.23a
1.63b
1.02b
b.w)
35.15
M
0.2 mg/kg
20.72 0.79d
0.11b
Group 4 ( QDs-QE complex
±
0.62a
1.82a Group 3 (DF)
36.26
CAT****
ip t
1.5
SOD***
±
±
5.79
15.44
Gpx***** ±
0.43a
cr
Group 1 (Normal Control)
GSH**
us
TBARS*
±
3.34
1.19c
0.41b
20.54±
5.58
1.06a
0.87a
18.01±
4.16
1.35b
0.86a
23.40±
5.68
1.15a
0.56a
14.16±
3.89
0.58c
0.75b
16.01±
4.16
1.35b
0.76a
an
Groups
27.19 0.36a
±
17.57 0.79c
±
26.91 1.16a
±
22.36 1.58b
±
26.82 1.05a
Group 6 (QDs alone )
Group 7 (QE 5 mg/kg)
1.8 1.02b
±
1.36d
±
18.12 1.23c
± 28.15 0.90c
±
±
21.36 1.57b
540 541
23
Page 23 of 35
542 data are expressed as mean ± SD (n = 6)
543
mean values with different superscripts are significantly different from each other as revealed by Duncan post hoc
544
ip t
test (P < 0.05). * mmol/mg protein
546
cr
** µg of reduced glutathione/mg protein
547
*** U/mg of protein
548
**** µmol of H O utilized/min/mg of protein 2 2
549
us
545
***** µmoles of GSH oxidized/min/mg protein
an
550 551
M
552
Table 2 Protective effect of QDs-QE complex on hematological parameters of adjuvant
554
induced arthritic rats
te
d
553
Ac ce p
555
RBC
WBC
(millions/mm3)
(thousands/mm3)
Group 1 (Normal Control)
5.58 ± 0.65a
7.80 ± 0.38cd
3.60 ± 0.51c
Group 2 (Disease Control)
4.09 ± 0.22b
15.25 ± 0.58a
11.80 ± 1.05a
Group 3 (DF)
5.77 ± 0.63a
7.22 ± 0.78b
4.47 ± 0.85b
Group 4 (QDs-QE complex 0.2 mg/kg
4.87 ± 0.35a
10.02 ± 0.40b
7.40 ± 0.76b
Groups
ESR (mm/hr)
b.w)
24
Page 24 of 35
5.78 ± 1.0 8a
7.15 ± 0.77d
3.34 ± 0.90c
b.w)
4.00 ± 1.03b
16.25 ± 0.25a
10.96 ± 1.71a
Group 6 (QDs alone)
4.67 ± 0.35a
9.02 ± 0.40b
6.40 ± 0.76b
ip t
Group 5 (QDs-QE complex 0.4 mg/kg
us
cr
Group 7 (QE 5 mg/kg)
556
mean values with different superscripts are significantly different from each other as revealed by duncan post hoc test (P <
558 0.05).
M
559
an
data are expressed as mean ± SD (n = 6)
557
560
Ac ce p
te
d
561
25
Page 25 of 35
Figure captions
562
Fig. 1. Absorption spectrum of TGA-CdTe QDs (100 μl in 2.5 ml) in the absence and
563
presence of Quercetin (12 μg-60 μg in 2.5 ml) in water. The arrow indicates absorbance
564
increase with increasing concentration of Quercetin.
565
Fig. 2. Emission spectrum of TGA-CdTe QDs (30 ul in 2.5 ml) in the absence and
566
presence of Quercetin (12 μg − 60 μg in 2.5 ml) in aqueous solution. The arrow indicates
567
the intensity decreases with increasing concentration of Quercetin. Blue color peak is the
568
emission spectrum of Quercetin (60 μg in 2.5 ml) in water. Inserted figure represents the
569
binding constant for CdTe QDs with Quercetin.
570
Fig. 3. Direct size measurement of nanocarrier particles (quantum dots) through Dynamic
571
light scattering and SEM analysis (a) Typical particle size distribution of QDs (b) Zeta
572
potential of QDs (c) Scanning electron microscopy (SEM) image of QDs
573
Fig. 4. (a) ABTS.+ radical scavenging activity and (b) DPPH radical scavenging activity of
574
QDs-QE complex compared with butylated hydroxytoluene (BHT). Each value is
575
expressed as the mean ± standard deviation (n=3).
576
Fig. 5. (a) Nitric oxide (NO) radical scavenging activity and (b) Superoxide radical
577
scavenging activity of QDs-QE complex compared with butylated hydroxytoluene (BHT).
578
Each value is expressed as the mean ± standard deviation (n=3).
Ac ce p
te
d
M
an
us
cr
ip t
561
579
Fig. 6. Radiology images of hind limb of experimental rats. (a) Control rat shows normal
580
architecture of hock joint. (b) CFA treated- the soft tissues of the hind paw have become
581
swollen, degradation of joint capsules and cartilage. (c & d) Treatment with QDs-QE
582
complex has shown significant prevention against bony destruction by showing less soft
583
tissue swelling and narrowing of joint spaces in a dose dependant manner. (e).The 26
Page 26 of 35
standard drug diclofenac sodium treated group shows no bony destruction or swelling of
585
the joint. Arrows indicate the swelling and reduced edema of soft tissues in experimental
586
groups.
587
Fig. 7. To evaluate the anti-inflammatory effects of QDs-QE complex, samples of the
588
ankle joints from each experimental group were examined by H&E staining. (a) Control
589
group shown normal lobular architecture (X-40). (b) CFA administered group shown
590
thinning of cartilage plates, extensive infiltration of inflammatory cells into the synovial
591
cavity (X-40). (c) QDs-QE complex treated group at 0.2 mg/kg shows infiltration of
592
inflammatory cells, mild improvement cartilage regeneration (X-40). (d) QDs-QE
593
complex treated group at 0.4 mg/kg rats showed complete cartilage regeneration. Absence
594
of inflammation and inflammatory cells in synovial region proved the anti-arthritic
595
potential of QDs-QE complex (X-40). (e) DF (positive drug) treated group exhibited
596
reduction in inflammation and almost normal histology (X-40). Arrows in control, DF
597
(positive drug) and
598
degradation and regeneration. (f).QDs treated group shows almost similar observations of
599
CFA treated group with extensive infiltration of inflammatory cells into the synovial
d
M
an
us
cr
ip t
584
Ac ce p
te
QDs-QE complex (0.4 mg/kg) treated groups indicates cartilage
cavity. (e).QE treated group exhibited regenerated cartilage and reduced the
600
inflammation.
601
Fig. 8. Schematic representation of the possible mechanism of antiarthritic potential of
602
the QDs-QE complex against CFA induced arthritis.
603 604
27
Page 27 of 35
604
an
us
cr
ip t
605
M
606 607
d
Fig. 1
0.025
te
800
1/(F0 -F)
600
Intensity
0.02
60 μg in 2.5 ml
Ac ce p
700
0 μg in 2.5 ml
500
y = 0.0159x + 0.0068 R² = 0.9969
0.015 0.01 0.005 0.1 0.3 0.5 0.7 0.9 1.1
400
1/[Q] . 10-6 M
300 200 100 0
540
560
580
600
620
640
660
Wavelength (nm)
608
Fig. 2
609
28
Page 28 of 35
610 611 612
(b)
M
an
us
cr
ip t
(a)
613 614
Ac ce p
te
d
(c)
615 616 617 618
Fig. 3
619 620 621 29
Page 29 of 35
Ac ce p
te
d
M
an
us
cr
ip t
622 623
Fig. 4
30
Page 30 of 35
Ac ce p
te
d
M
an
us
cr
ip t
624 625
Fig. 5
31
Page 31 of 35
an
us
cr
ip t
M
626
d
627 628
te
Fig. 6
Ac ce p
629 630 631 632 633 634 635 636
32
Page 32 of 35
Ac ce p
te
d
M
an
us
cr
ip t
637 638 639 640
Fig. 7
33
Page 33 of 35
M
an
us
cr
ip t
d
641 642
te
Fig. 8
Ac ce p
643 644 645 646
34
Page 34 of 35
Highlights
647
Enhancement of antiarthritic effect of quercetin(QE) using quantum dots (QDs) as nanocarrier
648
Quantum dots enhances the efficacy of drug delivery
649
QDs‐QE complex has brought back the hematological changes in arthritic animals
650
Cartilage regeneration in arthritic rats treated with QDs‐QE complex was observed
cr
te
d
M
an
us
Ac ce p
651
ip t
646
35
Page 35 of 35
S0927-7765(13)00501-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2013.07.065 COLSUB 5945
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
18-2-2013 27-7-2013 30-7-2013
Please cite this article as: R. Jeyadevi, T. Sivasudha, A. Rameshkumar, D.A. Ananth, G.S.B. Aseervatham, K. Kumaresan, L.D. Kumar, S. Jagadeeswari, R. Renganathan, Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic Wistar rats, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.07.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhancement of anti arthritic effect of quercetin using thioglycolic acid-capped
2
cadmium telluride quantum dots as nanocarrier in adjuvant induced arthritic Wistar
3
rats
4
R. Jeyadevi a, T. Sivasudha a,*, A. Rameshkumar a,b, D. Arul Ananth a, G. Smilin Bell
5
Aseervatham a , K. Kumaresan c, L. Dinesh Kumar d, S. Jagadeeswari e, R. Renganathan e
6 7
a
8 9 10 11 12 13
b
14
cr
ip t
1
us
Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli - 620024; Tamil Nadu, India Pesticide Residue Analysis Laboratory, Regional Plant Quarantine Station, Chennai - 600027, Tamil Nadu, India. Department of Histopathology, SRM institute for Medical Sciences, Chennai- 600026, Tamil Nadu, India.
d
Department of Biotechnology, Bharathidasan University , Tiruchirappalli - 620024; Tamil Nadu, India.
e
School of Chemistry, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India
M
an
C
15
d
* Corresponding author
te
16
Dr. T. Sivasudha
18
Department of Environmental Biotechnology
Ac ce p
17
Bharathidasan University
19
Tiruchirappalli – 620 024,
20
Tamilnadu,
21
India
22
Tel: 091-0431-2407088
23
Fax: 091-0431-2407045
Email ID: [email protected]
24 25 26 27
1
Page 1 of 35
ABSTRACT
29
In this present study, we investigated thio glycolic acid-capped cadmium telluride
30
quantum dots (TGA-CdTe QDs) as nano carrier to study the antiarthritic activity of
31
quercetin on adjuvant induced arthritic Wistar rats. The free radical scavenging activity of
32
QDs-QE complex was evaluated by 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonic acid
33
(ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Nitric oxide (NO) and superoxide anion
34
scavenging assays. Fifteen days after adjuvant induction, arthritic rats received QDs-QE
35
complex orally at the dose of 0.2 and 0.4 mg/kg daily for 3 weeks. Diclofenac sodium
36
(DF) was used as a reference drug. Administration of QDs-QE complex showed a
37
significant reduction in inflammation and improvement in cartilage regeneration.
38
Treatment with QDs-QE complex significantly (P < 0.05) reduced the expressions lipid
39
peroxidation and showed significant (P < 0.05) increase in activities of antioxidant
40
enzymes such as superoxide dismutase (SOD), reduced glutathione (GSH), glutathione
41
peroxidase (GPx) catalase (CAT) levels in paw tissue.
42
rheumatoid factor (RF), red blood cells (RBC) and white blood cells (WBC) count and
43
erythrocyte sedimentation rate (ESR) of experimental animals were also estimated.
44
Histology of hind limb tissue in experimental groups confirmed the complete cartilage
45
regeneration in arthritis induced rats treated with QDs-QE complex. Based on our
46
findings, we suggest that the QDs act as nano carrier for the drugs used in the treatment of
47
various degenerative diseases.
48
Keywords: Quercetin, Quantum dots, Fluorescence quenching, Nanocarrier, Anti arthritic,
49
Radiology.
C-reactive protein (CRP),
Ac ce p
te
d
M
an
us
cr
ip t
28
50 51 2
Page 2 of 35
1. Introduction Rheumatic arthritis is one of the commonest inflammatory conditions in developing
54
countries. RA is characterized by severe pain, swelling and destruction of cartilage and
55
bone associated with impaired joint function [1]. It is a systemic autoimmune disease
56
which mainly involves the peripheral synovial joints that causes chronic inflammation
57
and profound tissue destruction in affected patients. The autoimmune characteristic of RA
58
is supported by the presence of circulating rheumatoid factor (RF), C-reactive proteins
59
and other endogenous proteins which can be detected in the serum of arthritic patients [2].
60
Currently synthetic drugs form a major line of treatment in the management of arthritis.
61
The conventional drug treatment of RA consists of analgesic, non-steroidal anti
62
inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs) and
63
corticosteroids [3]. NSAIDs elicit their effects by inhibiting COX-2 activity and blocking
64
the downstream production of prostaglandins and offer effective therapies for RA.
65
However, besides their high cost, prolonged use of many of these drugs is associated with
66
severe adverse reactions such as gastric and duodenal ulcers, complications in the small
67
te
d
M
an
us
cr
ip t
53
Ac ce p
52
intestine and colon can occur, which cause colitis, bleeding, perforation, stricture, and chronic problems such as iron deficiency anemia and protein loss and toxicity [4]. A
68
report also states that, NSAIDs treatment enhances joint destruction in arthritis and
69
inhibits glycosaminoglycan synthesis [5]. Recently, there has been an increasing interest
70
in natural food for scavenging the free radicals because of their wide acceptance.
71 72 73
More than 4,000 natural flavonoids are distributed in the plant which has been commonly
74
consumed as foods or medicinal herbs by humans since ancient time. Flavonoids have
75
gained a great deal of interests due to their extensive biological properties such as
76
antioxidant, anti proliferative and anti-inflammatory activities. Since oxidative stress is 3
Page 3 of 35
implicated in the pathogenesis of chronic inflammatory diseases, modulating the cellular
78
redox status by strengthening the endogenous antioxidant defenses might be an effective
79
mechanism of disease prevention. In this regard, dietary polyphenolics generally known
80
for their antioxidant property by neutralizing the formation of excess of reactive oxygen
81
species, which is considered to be a key event in the pathogenesis of rheumatoid arthritis
82
[6]. Quercetin (QE) is widely distributed in frequently consumed foods including apples,
83
berries, onion, tea, red wine, nuts, seeds, and vegetables of the human diet [7]. QE
84
reported to have many beneficial effects, including cardiovascular protection, anticancer
85
activity, antiulcer activity, anti-allergic activity, cataract prevention, antiviral activity and
86
anti-inflammatory activity [8, 9]. QE is an excellent free radical scavenging compound
87
and reported to reduce the risk for oxidative stress related chronic diseases like arthritis,
88
inflammation and diabetes [10].
M
an
us
cr
ip t
77
In modern biotechnology, drug delivery systems are being developed using
90
nanotechnology. Currently, drug delivery systems are a rapidly growing technology within
91
the areas of nanocarriers and photolabelling. Quantum dots (QDs) are a novel class of
92
inorganic fluorophore which are gaining widespread recognition as a result of their
93
exceptional photophysical properties [11]. Quantum dots (QDs) are nanometer-scale
94
semiconductor crystals composed of groups II–VI or III–V elements, and are defined as
95
particles with physical dimensions smaller than the exciton Bohr radius. Semiconductors
96
nanoparticles (often called QDs) have attracted considerable attention in recent years due
97
to their wide range of applications in the field of photovoltaic devices [12, 13] light-
98
emitting diodes (LEDs) [14], fluorescence labelling [15], cell imaging [16] etc. Among
99
various available quantum dots CdTe QDs have been widely studied as luminescence
100
probes and sensors [17, 18]. Recently, Jhonsi et al. [19] investigated the photoinduced
101
interaction between water-soluble CdTe QDs (Cadmium telluride quantum dots) and
Ac ce p
te
d
89
4
Page 4 of 35
certain antioxidants. They found that the antioxidants quench the fluorescence of TGA-
103
CdTe QDs (Thio glycolic acid - CdTe QDs) through complex formation and trapping the
104
holes of QDs. QDs are also being explored as tools for site-specific gene and drug
105
delivery and are among the most promising candidates for a variety of information and
106
visual technologies; they are currently used for the creation of advance flat-panel LED
107
(light-emitting diode) displays and may be employed for ultrahigh-density data storage
108
and quantum information processing [14]. Our previous study has demonstrated the anti
109
microbial activity of M. emerginata using thioglycolic acid-capped cadmium telluride
110
quantum dots as a fluorescent probe [20]. Thus, in the present investigation, we have
111
examined the enhancement of anti arthritic effect of QE using TGA-CdTe QDs through
112
the analysis of hematological parameters, histology and radiological images of paw in
113
adjuvant induced arthritic Wistar rats.
114
2. Materials and methods
115
2.1 Drugs and chemicals
116
All the chemicals used in the study including the solvents were of analytical grade.
117
Complete Freund’s adjuvant (CFA), ABTS, DPPH, butylated hydroxytoluene (BHT), 3, 3-
118
diaminobenzidine tetrahydrochloride and quercetin were purchased from Sigma Chemical
119
Co. (St. Louis, MO, USA). Sulphanilamide, napththylenediamine dichloride, phosphoric
120
acid, nitroblue tetrazolium, reduced nicotinamide adenine dinucleotide, and phenazine
121
methosulfate were purchased from Merck Chemical Supplies (Damstadt, Germany). Thio
122
glycolic acid (TGA), CdCl2. 2.5 H2O (99.99 %), tellurium powder (99.997 %) and sodium
123
borohydride (95 %) were purchased from Sigma Aldrich (St. Louis, MO, USA).
Ac ce p
te
d
M
an
us
cr
ip t
102
124
5
Page 5 of 35
125
Double distilled water was used for preparing solutions. All measurements were
127
performed at ambient temperature.
128
2.2. Synthesis and characterization of TGA capped CdTe Quantum dots
ip t
126
CdTe quantum dots were synthesized by using the following method as reported
130
earlier [19]. Typically, an aqueous solution of Cd2+ ion (1.25 × 10-2 M) and TGA (3 × 10-2
131
M) was prepared and pH was adjusted to 8-9. Then NaHTe was added under nitrogen
132
atmosphere. In our experiments, the typical molar ratio of Cd2+:NaHTe:TGA used was
133
1:0.2:2.4. The resulting products were precipitated by acetone and superfluous of TGA
134
and Cd2+ that did not participate in the reaction was removed by centrifugation at 4000
135
rpm for 5 min. The resultant precipitate was re-dispersed in water and re-precipitated by
136
acetone for more than two times, then kept at 4 °C in dark for further use. The colloidal
137
solution can be kept for three months at room temperature without any obvious
138
aggregation.
Ac ce p
te
d
M
an
us
cr
129
The prepared TGA capped CdTe quantum dots were characterized by Steady state
139 140
measurements. The particle size of prepared QDs was calculated from the absorption
141
maximum (Eq.1).
D = (9.8127 x 10−7) λ3 − (1.7147 x 10−3) λ2 + (1.0064) λ − 194.84
142
(1)
143
where D (nm) is the particle size of QDs and λ (nm) is the wavelength of first excitonic
144
absorption peak of the corresponding QDs. The particle size of the prepared QDs is 2.96
145
nm and the concentration (0.4×10-7 M) was calculated from Lambert’s Beer law.
6
Page 6 of 35
2.2.1 Particle size, zeta potential measurement and SEM analysis
147
The size and the zeta potential of the CdTe quantum dots were determined using dynamic
148
light scattering (DLS) and microelectrophoretic method, respectively (Zetasizer Nano
149
Series-ZS90, Malvern Instruments). The measurements were conducted at 25 °C. The
150
obtained values of the size were equal to an average of the three subsequent runs with 10
151
measurements, while the zeta potential was calculated as an average from the three runs
152
with 20 measurements [21].
us
cr
ip t
146
The morphology of the samples was examined using a scanning electron microscope
154
(SEM) (Hitachi S-4160; FESEM). The samples were fixed with two sequential
155
incubations with 4 % paraformaldehyde solution and with 2.5 % of glutaraldehyde
156
solution for 30 min at 4oC following the samples were dehydrated with ethanol gradient,
157
dried overnight and coated with gold film before SEM observation [22].
M
an
153
2.2.2. Preparation of QE with QDs TGA-CdTe
te
159
d
158
Freshly prepared QDs (1 ×10−6 M) were titrated by adding the appropriate amountof
161
QE (1:1 v/v) and kept in an ultrasonic bath for 25 min to improve binding. After shaking
162
for 1 h, the QDs-QE complex were then ultra centrifuged at 35000 rpm and washed with
163
PBS (pH 7.4) twice to remove unbound QDs. The mixture was then vortexed well to form
164
a homogeneous dispersion, and stored in refrigerator at 4 °C for further analyses [20]. In
165
general, the cadmium ions on the particle surface can coordinate with both -SH and -
166
COOH groups. Quercetin contains 5 -OH groups which strongly anchor to the surface of
167
quantum dots compared to -SH in thioglycolic acid.
168
2.2.3. Characterization of QDs
169
Drug loading efficiency
Ac ce p
160
7
Page 7 of 35
The drug loading and the drug entrapping efficiency of QDs was determined by the
171
UV absorbance at 365 nm using a UV-visible spectrophotometer [23, 24]. The entrapped
172
QE content in the QDs was calculated from the mass of the incorporated drug using the
173
following equation.
cr
174
ip t
170
us
175
Drug encapsulation efficiency
181
encapsulated QE to the mass of the QE used for complex preparation using the following
182
equation:
an
176 177 178 179 180
te
d
M
The drug encapsulation efficiency was defined as the ratio of the mass of the
183
Ac ce p
184
A certain weight of QE was dissolved in water to obtain standard quercetin solution with
185 186
the concentration ranging from 10 to 60 µg/ml. The absorbance was read at 365 nm in the
187
UV spectrophotometer to obtain the linear equation which in turn used to calculate the
188
correlation coefficient [25].
189
2.2.4. In vitro antioxidant studies
190 191
The in vitro antioxidant activity of QDs-QE complex was determined by their ability
192
to scavenge DPPH, ABTS [26], NO and superoxide anion radicals [27]. Percentage
193
inhibition was expressed in terms of IC50 value calculated by linear regression method.
194
BHT was used as reference standard. 8
Page 8 of 35
195
2.3. In vivo studies
196
2.3.1. Animals Female albino Wistar rats (150-200 g) were obtained and housed in poly-acrylic
198
cages and maintained under standard laboratory conditions (temperature 24-28 ºC, relative
199
humidity 60-70 % and 12 h dark-light cycles). Animals were fed with commercial rat feed
200
(Sai Durga feeds and food stocks, Chennai, India) and water, ad libitum. All the animals
201
were acclimatized to laboratory conditions for 7 days before commencement of the
202
experiment. Experimental protocols were approved by the Committee for the Purpose of
203
Control and Supervision of Experiments on Animals (CPCSEA), Chennai, Tamil Nadu,
204
India (BDU/IAEC/2012/26/28.03.2012).
206
M
205
an
us
cr
ip t
197
2.3.2. Biocompatability of QE and QDs
Quercetin, reported to be non toxic to in vivo model up to the concentration of 200
208
mg/kg b.w. by Naovarat Tarasub et al. [28]. A study about quantum dots, revealed that
209
QDs is non toxic in vivo model up to the concentration of 15.0 nmol over short-term (< 7
210
days) and long-term (> 80 days) periods [29]. The concentration of QE and QDs used in
211
this study were lesser than the above mentioned reports.
Ac ce p
te
d
207
212 213
2.3.3. Anti-arthritic studies
Animals were divided into seven groups of six each. Group 1 consisted of control
214 215
rats, all other groups were comprised of arthritic rats. Test groups were injected with 0.1
216
mL of CFA intradermally. Group 2: disease control, Group 3: treated with standard drug
217
diclofenac sodium (DF) (0.3 mg/kg b.wt/day p.o.). While Group 4 and Group 5 treated
218
with QDs-QE complex at a dose of 0.2 mg/kg b.wt/day p.o. and 0.4 mg/kg b.wt/day p.o.
219
Group 6: treated with 1 mL of QDs (1x10-6 M) and Group 7: treated with QE at the 9
Page 9 of 35
220
concentration of 5 mg/kg respectively, from day 15th to 35th of induction of adjuvant
221
induced arthritis. Animals were monitored for the development of clinical arthritis using a
222
vernier caliper [30]. On the 35th day, animals were anesthetized and the hind legs of the experimental
224
animals of different groups were analyzed by X-ray radiography and examined for the soft
225
tissue swelling, bony erosions and narrowing of the spaces between joints and then
226
animals were sacrificed; blood samples and paw tissue were collected and used for
227
biochemical estimations. Histopathological studies were done in hind limb joint tissues.
228
Tissues were fixed in formalin, decalcified, embedded in paraffin blocks and sections
229
stained with hematoxylin and eosin (H & E).
230
2.3.4. Biochemical estimations
M
an
us
cr
ip t
223
The paw tissue homogenates (10 %, w/v) prepared in phosphate buffered saline
232
(PBS containing 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4 and 1.76 mM
233
KH2PO4 in 1000 mL distilled water pH 7.4) were used for antioxidant studies such as lipid
234
peroxidation [31], SOD [32], CAT [33], GPx [34] and GSH [35] activities. Blood samples
235
were subjected to evaluate the biochemical parameters like white blood corpuscles count,
236
ESR, anti CRP and RF were done using commercial reagent kits and were performed
237
according to the manufacturer’s (Nice Chemicals [P] Ltd. Cochin, Kerala, India)
238
instructions.
239
2.4. Statistical analysis
Ac ce p
te
d
231
The statistical analyses were performed using SPSS (Statistical Package for Social
240 241
Science, SPSS Inc., Chicago, IL, USA). Significance of each group was verified with one-
242
way analysis of variance (ANOVA) followed by Duncan post hoc test (P ≤ 0.05). Values
243
obtained are means of six replicate determinations ± standard deviation.
244
3. Results
10
Page 10 of 35
245
3.1. Absorption studies The UV-visible absorption spectra of QDs in aqueous solution and with QE were
247
shown in Fig. 1. It has been observed that while increasing the concentration of QE (12–60
248
μg in 2.5 mL), the QDs absorbance also increase. Moreover, we could find there is no shift
249
and new peak formed. This may be due to QE is adsorbed to the surface of QDs. This
250
reveals the formation of ground state complex between QDs and QE.
251
3.2. Fluorescence studies
us
cr
ip t
246
Fluorescence quenching measurements have been widely used to analyze the various
253
type interactions between the molecules [36, 37]. This method helps to understand QDs
254
binding mechanisms with QE and provide clues to the nature of the binding phenomenon.
255
Fig. 3. shows the emission spectrum of QDs excited at 500 nm in the absence and
256
presence of QE. The emission intensity of QDs is quenched by raising the concentration of
257
quercetin (12– 60 μg in 2.5 mL), i.e fluorescence quenching. Further, we recorded the
258
emission spectrum of QE (to the highest concentration 60 μg in 2.5 mL) at the same
259
excitation wavelength of QDs, it is transparent at this excitation. From this observation,
260
we confirmed that the observed quenching is mainly due to interaction of QDs with QE
261
and no inner filter effect or reabsorption. Hence the binding constant for this type of
262
interaction was calculated using fluorescence quenching data by this method
Ac ce p
te
d
M
an
252
1 1 + = 0 (F -F) (F -F') 0
263
1 K(F -F')[Q] 0
(4)
264
where K is the binding constant, F0 is the initial fluorescence intensity of QDs, F′ is the
265
fluorescence intensity of QE adsorbed QDs and F is the observed fluorescence intensity at
266
its maximum. The plot of 1/(F0-F) versus 1/[Q] gives a straight line (Fig. 2) and from the
11
Page 11 of 35
267
slope the calculated binding constant is found to be 4.27 × 105 M-1. There is a good linear
268
relationship between 1/(F0-F) and the reciprocal concentration of dyes.
269
3.3. Particle size, zeta potential measurement and SEM analysis The size distribution of QDs in colloidal solution was found to be 185 nm. A negative
271
zeta potential of about −30.2 mV was observed in the present study that pose ideal surface
272
charge. The size and surface charge of the QDs determinethe biological activity. From the
273
Zeta potential measurements, the particle size distribution of the QDs was found to be 16
274
nm (Fig. 3a, b). FE-SEM images of compound loaded quantum dots were found to be
275
spherical in shape with regular arrangement (Fig 3c). The picture shows the magnification
276
image of uniformally arranged QDs.
277
3.4. Charecterization of QDs
M
an
us
cr
ip t
270
The absorbance values of standard QE solution were determined at 365 nm. It was
279
calculated using the following equation. Amount of QE and encapsulation efficiency was
280
calculated using the obtained equation,
(4)
Ac ce p
Y = 0.045X + 0.417
te
d
278
281 282
Where Y is the absorbance values, and X is QE concentration. The calibration curve for
283
the absorbance of QE was linear over the range of standard concentration of QE (10 – 60
284
µg/ml) with a correlation coefficient of R2 = 0.995. The drug entrapped in the QDs and
285
the QE loading efficiency was calculated using the equation 2, 3 and 5. Therefore, the
286
encapsulation efficiency of drug loaded QDs-QE was 6.38% and the drug loading
287
efficiency was 55.26%.
288 289
3.5. Mechanism of quenching
12
Page 12 of 35
Upon excitation of CdTe QDs, it would result in the promotion of electron from its
291
valence band to the conduction band. This results in the formation of a positively charged
292
hole in its valence band and a free electron in the conduction band of QDs. In the absence
293
of QE, the formed electron and hole would recombine which results in emission of
294
fluorescence. On introducing hole acceptors (i.e. QE) to the solution of QDs the electron
295
hole recombination process will be prevented which causes decrease in the emission
296
intensity of QDs (i.e.) fluorescence quenching.
297
3.6. In vitro antioxidant studies
us
cr
ip t
290
The percentage inhibition of ABTS and DPPH free radicals by QDs-QE complex
299
are shown in Fig.4a and Fig. 4b respectively. QDs-QE complex significantly inhibited the
300
ABTS and DPPH radicals in a dose-dependent manner with the IC50 values of 60 and 61
301
µg/mL, respectively. Fig. 5a, shows the scavenging activity of nitric oxide by QDs-QE
302
complex in a concentration dependent manner. The IC50 value of QDs-QE complex, QE
303
and BHT to scavenge the nitric oxide radical is 40 µg/mL, 42 µg/mL and 45 µg/mL
304
respectively. Fig. 5b shows the superoxide radical scavenging capacity of QDs-QE
305
complex measured by the PMS-NADH superoxide generating system. QDs-QE complex
306
was found to be an effective scavenger of superoxide anion radicals in a dose-dependent
307
manner with an IC50 value of 20 µg/mL and thus can prevent the formation of ROS.
Ac ce p
te
d
M
an
298
308 309
3.7. CFA induced rat paw edema
310
There was a significant increase in rat paw volume in CFA injected arthritic
311
control rats when compared to the normal control rats. QDs-QE complex treatment at the
312
dose of 0.2 mg/kg and 0.4 mg/kg body weight showed significant reduction in rat paw
313
edema volume when compared with the arthritic group (Data not shown).
314 13
Page 13 of 35
315
3.8. Protective effect of QDs-QE complex on antioxidant enzymes There was an increased level of LPO and a decreased antioxidant enzyme activity
317
in Group 2 rats. The declined antioxidant enzyme activity is responsible for the increased
318
lipid peroxidation measured as thiobarbituric acid reacting substance (TBARS), which
319
causes loss of membrane fluidity, membrane integrity, and finally loss of cell functions
320
[38]. This peroxidative damage to membranes results in the leakage of enzymes, and
321
metabolites to circulation. Elevated LPO saturates the level of free radicals which
322
sequentially inhibit activities of SOD, CAT, GSH and GPx. Group 6 (1 mL of 1x10-6 M
323
QDs administered) does not show any significant protective effect against CFA induced
324
arthritis, whereas, administration of QE up to 4 mg/kg did not show any protective effect
325
against arthritic induced animals. However, QE at 5 mg/kg showed mild protective effect
326
which is comparable to Group 4 animals. QDs-QE complex attenuates their levels
327
significantly (P ≤ 0.05) in paw tissue comparable to that of reference standard drug treated
328
group (Table 1).
te
329
3.9. Haematological parameters
Ac ce p
330
d
M
an
us
cr
ip t
316
The changes in hematological parameters in adjuvant induced arthritic rats were
331 332
shown in Table 2. There was an increase in WBC count, ESR, anti CRP and RF of
333
arthritic rats, when compared with the control rats. The treatment with QE and QDs-QE
334
complex in a dose dependent manner had significantly brought back the altered
335
hematological changes in adjuvant induced arthritic animals to normal levels.
336 337
3.10. Radiographic changes
338
X-ray radiographs of the different treatment group animal paws taken on 25th day.
339
Adjuvant treated rats had developed definite joint space narrowing of the joints, diffused
14
Page 14 of 35
soft tissue swelling in digits, cystic enlargement of bone and extensive erosions produced
341
narrowing or pseudo widening of all joint spaces (Fig. 6b). The standard drug diclofenac
342
sodium treated group shows no bony destruction and swelling of the joint (Fig. 6e).
343
Treatment with QDs-QE complex for 25 days have shown significant prevention against
344
bony destruction by showing less soft tissue swelling and narrowing of joint spaces in a
345
dose dependant manner (Fig. 6c and d).
cr
ip t
340
347
3.11. Effects of QE treatment on histological findings
us
346
To evaluate the anti-inflammatory effects of QDs-QE complex, samples of the ankle
349
joints from each experimental group were examined by H&E staining. Control group
350
shown normal lobular architecture (Fig. 7a). CFA administered group shown thinning of
351
cartilage plates, severe bone erosion, extensive infiltration of inflammatory cells into the
352
synovial cavity, pannus formation (Fig. 7b). QDs-QE complex treated group at 0.2 mg/kg
353
shown mild improvement cartilage regeneration, moderate edema formation and cellular
354
infiltration (Fig. 7c). QDs-QE complex treated group at 0.4 mg/kg rats showed complete
355
cartilage regeneration. Absence of inflammation and inflammatory cells in synovial region
356
proved the anti-arthritic potential of QDs-QE complex (Fig. 7d). DF (positive drug)
357
treated group exhibited almost normal histology (Fig. 7e). QD treated group shows almost
358
similar observations of CFA treated group with extensive infiltration of inflammatory cells
359
into the synovial cavity (Fig. 7f). QE at 5 mg/kg showed mild improvement in cartilage
360
regeneration, moderate edema formation and cellular infiltration (Fig. 7g)
Ac ce p
te
d
M
an
348
361 362
4. Discussion
363
In general, QE is the highly consumed flavonoid and the richest sources of QE are tea,
364
onions and apples. The best described property of QE is its capacity to act as antioxidant,
15
Page 15 of 35
thus protecting the body against reactive oxygen species (ROS). Because of the high
366
reactivity of the hydroxyl group of the QE, reactive oxygen species are made
367
inactiveReactive oxygen species at the site of inflammation are reported to be involved in
368
the pathogenesis of RA [26]. In recent years, many intense research works are focused on
369
an electron transfer between molecular adsorbents and semiconductor nanonmaterial due
370
to large number of practical applications. Semiconductor QDs have gained a lot of
371
research interest in the last decade due to their existing size and shape dependent
372
properties, photo bleaching threshold, good chemical stability, relatively narrow and
373
symmetric luminescence bands [39-41]. The zeta potential of a nanoparticle is commonly
374
used to characterize the surface charge property of nanoparticles. Generally, the thiol
375
group of TGA will bind on the surface of QDs while COO− will be present as new surface
376
groups. The presence of negatively charged COO− surface groups was confirmed by the
377
observation of zeta potential in the negative region. In our CdTe QDs, we observed a
378
negative zeta potential around −30.2mV which confirms the presence of negatively
379
charged COO− surface groups. The difference between the calculated size from absorption
380
measurements and particle size from zeta potential measurement is mainly because the
381
latter gives the diameter of the particle along with any ligands attached to it, and solvent
382
molecules which may be strongly associated with it. In addition, compared with organic
383
dyes and fluorescent proteins, QDs offer several advantages, such as a narrow, symmetric
384
emission from visible to IR wavelengths and photo-chemical stability. QDs possess
385
dimensional similarities to biological macromolecules, such as nucleic acids and proteins
386
thus, QDs were used to study their interactions with various biomolecules [20]. Recently,
387
Jhonsi et al. [42] reported on the photo-induced interaction between water-soluble CdTe
388
quantum dots and certain antioxidants. The antioxidants quench the fluorescence of TGA-
389
CdTe QDs through complex formation, and the quenching results from antioxidants
Ac ce p
te
d
M
an
us
cr
ip t
365
16
Page 16 of 35
390
trapping the holes of the QDs. Moreover QDs also reported to be stable in the digestive
391
tract of Wistar rats [43]. Pearson and Wood (1959) [44] reported that, rats immunized with Complete
393
Freund’s adjuvant containing Mycobacterium tuberculosis develop arthritis. Nevertheless,
394
their mode of action is still not completely understood. Adjuvant-induced arthritis is a
395
widely used arthritic model for testing and developing anti-arthritic and anti-inflammatory
396
agents. After 10 to 14 days of CFA injection, animals develop arthritis. Adjuvant arthritis
397
animal model is very similar to human rheumatoid arthritis, resembles to pathological and
398
serological changes, including the inflammatory mediators. In adjuvant-induced arthritis,
399
rat model develops chronic swelling and pain in multiple joints with release of cytokines
400
from inflammatory cells, culminating in erosion of cartilage and bone destruction causing
401
severe disability [45]. The mechanisms of CFA induced arthritis may involve prolongation
402
of the presence of antigens at the site of injection or more effective transport of the
403
antigens to the lymphatic system and to the lungs, where the adjuvant promotes the
404
accumulation of cells concerned with the immune response or by excessive production of
405
ROS.
Ac ce p
te
d
M
an
us
cr
ip t
392
Increase in WBC count has been suggested to be one of the characteristic
406 407
diagnoses of arthritis. In our present study, CFA-induced arthritic animals showed
408
elevated WBC level. QDs-QE complex treatment significantly decreased WBC, revealing
409
its beneficial role against arthritis. RF has been regarded as the main serologic marker in
410
inflammatory arthritis [46]. RF is an auto antibody directed against the Fc portion of IgG.
411
RF and IgG join to form immune complexes that contribute to the progress of rheumatoid
412
arthritis. In recent years, C-reactive protein has also been identified as important predictors
413
both for diagnosis and prognosis of rheumatoid arthritis. C-reactive protein is a member of
414
the pentraxin family of proteins. CRP is secreted by the liver in response to a variety of 17
Page 17 of 35
inflammatory cytokines. An elevated CRP level can provide support for the presence of an
416
inflammatory disease, like rheumatoid arthritis [47]. Our findings represent high levels of
417
RF and CRP in arthritic rat serum. Dose-response reductions of these factors have been
418
observed in the arthritic rats treated with QE reveals its protective effect against arthritis.
419
The ESR count which significantly increased in arthritic control group has been
420
remarkably decreased by QDs-QE complex and standard drug diclofenac sodium, thus
421
justifying significant role of QDs-QE complex in arthritic conditions. Our previus study,
422
demonstrated that, Quercetin at a higher concentration of 5 mg/kg b.w shows protective
423
effect against CFA induced arthritis in Wistar rats. Treatment with QDs alone to arthritic
424
rats showed no effect. So, it was suggested that QDs act as nano carrier of the active
425
compound QE and results in better anti arthritic property even at lower concentration (0.4
426
mg/kg b.w.). ROS in arthritis is not surprising since oxidative stress or reactive oxygen
427
species serve as mediators of tissue damage.
428
synovial fluid and induce depolymerization of hyaluronic acid which in turn leads to a loss
429
of viscosity in the joints [48]. The cartilage destruction by ROS in arthritic rats treated
430
with QDs-QE complex has strongly inhibited through increase in the levels of endogenous
431
enzymatic antioxidants, such as SOD, CAT, GPx and non-enzymatic antioxidant GSH.
432
The conventional drug treatment of arthritis elicits their effects by inhibiting COX-2
433
activity and blocking the downstream production of prostaglandins. The possible
434
mechanism of QDs-QE complex to exert anti arthritic activity might be through
435
neutralization of various free radicals generated at the site of inflammation and by
436
inhibiting the production of COX-2 enzyme (Fig. 8).
437
5. Conclusion
The reactive oxygen species degrade
Ac ce p
te
d
M
an
us
cr
ip t
415
438
To conclude, our results suggest that QDs-QE complex showed antioxidant and anti-
439
arthritic effects. Absorbance and fluorescence quenching of QDs-QE was successfully 18
Page 18 of 35
investigated. The anti-inflammatory effects of QDs-QE complex may be related to free
441
radical quenching via increase in the activities of antioxidant enzymes and by inhibiting
442
the expression of inflammatory mediators. QE alone showed anti arthritic potential only at
443
the higher concentration, where as the QDs-QE complex exhibited anti arthritic activity
444
even at a lower concentration. Furthermore, this study reveals that using QDs as nano
445
carrier of QE exhibited enhanced anti arthritic effect even at a lower concentration of the
446
drug. These findings prove the promising effect of thioglycolic acid-capped cadmium
447
telluride quantum dots as a nano carrier to enhance the potential of anti arthritic drugs in
448
rheumatic complications. Further studies are in progress to better understand the
449
mechanism of action of QDs-QE complex, responsible for enhanced anti arthritic effect at
450
lower concentration of the drug.
M
451
Acknowledgment
d
452
an
us
cr
ip t
440
We thank the University Grants Commission (UGC), New Delhi, India for the
454
financial support. S. Jagadeeswari (Ref. No: 039680/E15/2011 Dt: 17.02.2011) thanks
455
UGC-BSR for her Research Fellowship in Science for Meritorious Students. We thank
456
DST–FIST and UGC-Non SAP for providing other instrumental facilities.
Ac ce p
te
453
457 458
References
459
[1] M. Feldmann, F.M. Brennan, R.N. Maini, Cell 85 (1996) 307-310.
460
[2] A.K. Andersson, C. Li, F.M. Brennan, Arthritis. Res. Ther. 10 (2008) 204-212.
461
[3] A.A. Schuna, C. Megeff, Am. J. Health-Syst. Pharm. 57 (2000) 225-234.
462
[4] S. Sharma, D. Sahu, H.R. Das, D. Sharma, Food Chem. Toxicol. 49 (2011) 33953406.
463
19
Page 19 of 35
[5] C.Z. Manon, T.N. de Boer, J.A.V. Roon, J.W.J. Bijlsma, F.P.J.G. Lafeber, S.C Mastbergen, Arthritis. Res. Ther. 13 (2011) 239.
465
[6] M.K. Park, J.S. Park, M.L. Cho, H.J. Oh, Y.J. Heo, Y.J. Woo, Y.M. Heo, M.J. Park,
468
[7] I. Erlund, Review of flavonoids quercetin, hesperetin, and naringenin. Nutr. Res. 24 (2004) 551-874.
469 470
ip t
H.S. Park, S.H. Park, H.Y. Kim, J.K. Immunol. Lett. 124 (2009) 102-110.
467
[8]
cr
466
C.V. de Whalley, S.M. Rankin, J.R. Hoult, W. Jessup, D.S. Leake, Biochem. Pharm. 39 (1990) 1743-750.
471
us
464
[9]
C. Bronner, Y. Landry, Agents and Actions 16 (1985) 147-151.
473
[10] T.N. Kaul, E.Jr. Middleton, P.L. Ogra, J. Med. Virol 15 (1985) 71-79.
474
[11] Q. Xiao, S. Huang, W. Sua, P. Li, J. Ma, F. Luo, J. Chen, Y Liub, Colloids Surf. B: Biointerfaces 102 (2013) 76-82.
475
M
an
472
[12] S. Coe, W.K. Woo, M.G. Bawendi, V. Bulovic, Nature 420 (2002) 800-803.
477
[13] K. Barnham, J.L. Marques, J. Hassard, P. O’Brien, Appl. Phys. Lett. 76 (2000) 11971199.
478
te
d
476
[14] V.L. Colvin, M.C. Schlamp, A.P. Alivisatos, Nature 370 (1994) 354-357.
480
[15] D. Gerion , W.J. Parak, S.C. Williams, D. Zanchet, C.M. Micheel, A.P. Alivisatos, J.
Ac ce p
479
Am. Chem. Soc. 124 (2002) 7070-7074.
481 482
[16] C.L. Wang, H. Zhang, J.H. Zhang, M.J. Li, H.Z. Sun, B. Yang, J. Phys. Chem. C. 111 (2007) 2465-2469.
483 484
[17] A.S. Susha, A.M. Javier, W.J. Parak, A.L. Rogach, Colloids Surf. A: Physicochem. Eng. Aspects 281 (2006) 40-43.
485 486
[18] J.P. Yuan, W.W. Guo, E.K, Wang, Anal. Chem. 80 (2008) 1141-1145.
487
[19] M.A. Jhonsi, R. Renganathan, J. Coll. Int. Sci. 344 (2010) 596-602.
20
Page 20 of 35
488
[20] A. Rameshkumar, T. Sivasudha, R. Jeyadevi, B. Sangeetha, D. Arul Ananth, G.
489
Smilin Bell Aseervatham, N. Nagarajan, R. Renganathan, A. Kathiravan, Colloids
490
Surf. B: Biointerfaces 101 (2013) 74-82. [21] M. Adamczak, M. Krok, E. Pamuła, U. Posadowska, K. Szczepanowicz, J. Barbasz,
ip t
491
P. Warszyński, Colloids and Surf. B: Biointerfaces. 110 (2013) 1-7.
492
[22] G. Ciofani, L. Ricotti, C. Canale, D. Alessandro, S. Berrettini, B. Mazzolai, V.
cr
493
Mattoli, Colloids and Surf. B: Biointerfaces. 102 (2013) 312-320.
494
[23] K.H. Huang, Z.H. Zhu, J.H. Liu, et al., Chin J Cancer. 24 (2005) 1023-1026.
496
[24] E.K. Park, S.B. Lee, Y.M. Lee, Biomaterials 26 (2005) 1053-1061.
497
[25] T. Musumeci, C.A. Ventura, I. Giannone, et al. Int J Pharm 325 (2006)172-179.
498
[26] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, J.M. Harnly, L.Z. Lin, J. Funct. Foods 5 (2013) 289-298.
499
(2013) 115-126.
501 502
[28]
Ac ce p
[29] T.S. Hauck, R.E. Anderson, H.C. Fischer, S. Newbigging, W.C. Chan, Small. 6(1) 2010 138-144. doi: 10.1002/smll.200900626.
505 506
N. Tarasub, C. Tarasub, Watcharaporn Devakul Na Ayutthaya, T. Suramana. (2000). http://www.med.tu.ac.th/medJournal/TUmed74/TuM7_4page321.pdf.
503 504
d
[27] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, L. Dineshkumar, Inflamm. Res. 62
te
500
M
an
us
495
[30] S.C. Ridge, K.M. Ferguson, N. Rath, J. Galivan, J.H. Freisheim, A.L. Oronsky, S.S. Kerwar, J. Rheumatol. 15 (1988) 1193-1197.
507 508
[31] A.J. Buege, S.D. Aust, Meth. Enzymol. 52 (1978) 302-310.
509
[32] J.M. McCord, I. Fridovich, J. Biochem. 244 (1969) 6049-6056.
510
[33] H. Aebi, Meth. Enzymol. 105(1984) 121-126.
511
[34] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.J. Hofmen, W.G. Hoekstra, Science. 179 (1973) 588-590.
512
21
Page 21 of 35
513
[35] M.S. Moron, J.N. De Pierre, V. Mannervik, Biochim. Biophys. Acta. 582 (1979) 6768.
514
[36] H. Wu, H. Wang, L. Xue, X. Li, J. Colloid Interface Sci. 353 (2011) 476-481.
516
[37] Shown I, Ujihara M, Imae T, J. Colloid Interface Sci. 352 (2010) 232-237.
517
[38] B. Halliwell, J.M.C. Gutteridge, 1989. Free Radicals in Biology and Medicine, 2nd
cr
edition. Clarendon Press, Oxford.
518
[39] A. Kumaria, S.K. Yadava, Y. B. Pakadeb, B. Singhc, S.C. Yadava, Colloids Surf. B: Biointerfaces 80 (2010) 184-192.
520
us
519
ip t
515
[40] M. Kakran, N.G. Sahoo, L. Li, Colloids Surf. B: Biointerfaces 88 (2011) 121-130.
522
[41] K.E. Sapsford, T. Pons, I.L. Medintz, H. Mattoussi, Sensors 6 (2006) 925-953.
523
[42] M.A. Jhonsi, E. Vaishnavi, R. Suganya, A. Kathiravan, R. Renganathan, Adv. Sci. Lett. 4 (2011) 1–6.
524
[43] Y.F. Loginova, S.V. Dezhurov, V.V. Zherdeva, N.I. Kazachkina, M.S. Wakstein,
d
525
M
an
521
A.P. Savitsky, Biochem Biophys Res Commun. 419 (2012) 54-9.
te
526
[44] C.M. Pearson, F.D. Wood, Arthritis. Rheum. 2 (1959) 440-459.
528
[45] S. Singh, D.K. Majumdar, Int. J. Pharmacol 34(3) (1996) 218-222.
529
[46] D.L. Scott, Rheumatology (Oxford) 39 (2000) 24-29.
530
[47] G. Sindhu, M. Ratheesh, G.L. Shyni, B. Nambisan, A. Helen, Int. Immunopharmacol.
Ac ce p
527
12 (2012) 205-211.
531 532
[48] F.C. Arnett, S.M. Edworthy, D.A. Bloch, D.J. McShane, J.F. Fries, N.S. Cooper,
533
L.A. Healey, S.R. Kaplan, M.H. Liang, H.S. Luthra, Arthritis. Rheum. 31 (1988) 315-324.
534 535 536 537
22
Page 22 of 35
537
Table 1 Protective effect of QDs-QE complex on TBARS, GSH and antioxidant enzymes
538
in paw tissue sample of arthritic rats
539
±
0.31b
2.7
Group 2 (Disease Control)
1.5
±
±
1.9
±
±
±
0.34b
2.35b
2.4± 1.03a
21.42
±
±
0.90c
te
d
29.15
31.68
1.4
Ac ce p
Group 5 ( QDs-QE complex 0.4 mg/kg b.w)
22.92 1.23a
1.63b
1.02b
b.w)
35.15
M
0.2 mg/kg
20.72 0.79d
0.11b
Group 4 ( QDs-QE complex
±
0.62a
1.82a Group 3 (DF)
36.26
CAT****
ip t
1.5
SOD***
±
±
5.79
15.44
Gpx***** ±
0.43a
cr
Group 1 (Normal Control)
GSH**
us
TBARS*
±
3.34
1.19c
0.41b
20.54±
5.58
1.06a
0.87a
18.01±
4.16
1.35b
0.86a
23.40±
5.68
1.15a
0.56a
14.16±
3.89
0.58c
0.75b
16.01±
4.16
1.35b
0.76a
an
Groups
27.19 0.36a
±
17.57 0.79c
±
26.91 1.16a
±
22.36 1.58b
±
26.82 1.05a
Group 6 (QDs alone )
Group 7 (QE 5 mg/kg)
1.8 1.02b
±
1.36d
±
18.12 1.23c
± 28.15 0.90c
±
±
21.36 1.57b
540 541
23
Page 23 of 35
542 data are expressed as mean ± SD (n = 6)
543
mean values with different superscripts are significantly different from each other as revealed by Duncan post hoc
544
ip t
test (P < 0.05). * mmol/mg protein
546
cr
** µg of reduced glutathione/mg protein
547
*** U/mg of protein
548
**** µmol of H O utilized/min/mg of protein 2 2
549
us
545
***** µmoles of GSH oxidized/min/mg protein
an
550 551
M
552
Table 2 Protective effect of QDs-QE complex on hematological parameters of adjuvant
554
induced arthritic rats
te
d
553
Ac ce p
555
RBC
WBC
(millions/mm3)
(thousands/mm3)
Group 1 (Normal Control)
5.58 ± 0.65a
7.80 ± 0.38cd
3.60 ± 0.51c
Group 2 (Disease Control)
4.09 ± 0.22b
15.25 ± 0.58a
11.80 ± 1.05a
Group 3 (DF)
5.77 ± 0.63a
7.22 ± 0.78b
4.47 ± 0.85b
Group 4 (QDs-QE complex 0.2 mg/kg
4.87 ± 0.35a
10.02 ± 0.40b
7.40 ± 0.76b
Groups
ESR (mm/hr)
b.w)
24
Page 24 of 35
5.78 ± 1.0 8a
7.15 ± 0.77d
3.34 ± 0.90c
b.w)
4.00 ± 1.03b
16.25 ± 0.25a
10.96 ± 1.71a
Group 6 (QDs alone)
4.67 ± 0.35a
9.02 ± 0.40b
6.40 ± 0.76b
ip t
Group 5 (QDs-QE complex 0.4 mg/kg
us
cr
Group 7 (QE 5 mg/kg)
556
mean values with different superscripts are significantly different from each other as revealed by duncan post hoc test (P <
558 0.05).
M
559
an
data are expressed as mean ± SD (n = 6)
557
560
Ac ce p
te
d
561
25
Page 25 of 35
Figure captions
562
Fig. 1. Absorption spectrum of TGA-CdTe QDs (100 μl in 2.5 ml) in the absence and
563
presence of Quercetin (12 μg-60 μg in 2.5 ml) in water. The arrow indicates absorbance
564
increase with increasing concentration of Quercetin.
565
Fig. 2. Emission spectrum of TGA-CdTe QDs (30 ul in 2.5 ml) in the absence and
566
presence of Quercetin (12 μg − 60 μg in 2.5 ml) in aqueous solution. The arrow indicates
567
the intensity decreases with increasing concentration of Quercetin. Blue color peak is the
568
emission spectrum of Quercetin (60 μg in 2.5 ml) in water. Inserted figure represents the
569
binding constant for CdTe QDs with Quercetin.
570
Fig. 3. Direct size measurement of nanocarrier particles (quantum dots) through Dynamic
571
light scattering and SEM analysis (a) Typical particle size distribution of QDs (b) Zeta
572
potential of QDs (c) Scanning electron microscopy (SEM) image of QDs
573
Fig. 4. (a) ABTS.+ radical scavenging activity and (b) DPPH radical scavenging activity of
574
QDs-QE complex compared with butylated hydroxytoluene (BHT). Each value is
575
expressed as the mean ± standard deviation (n=3).
576
Fig. 5. (a) Nitric oxide (NO) radical scavenging activity and (b) Superoxide radical
577
scavenging activity of QDs-QE complex compared with butylated hydroxytoluene (BHT).
578
Each value is expressed as the mean ± standard deviation (n=3).
Ac ce p
te
d
M
an
us
cr
ip t
561
579
Fig. 6. Radiology images of hind limb of experimental rats. (a) Control rat shows normal
580
architecture of hock joint. (b) CFA treated- the soft tissues of the hind paw have become
581
swollen, degradation of joint capsules and cartilage. (c & d) Treatment with QDs-QE
582
complex has shown significant prevention against bony destruction by showing less soft
583
tissue swelling and narrowing of joint spaces in a dose dependant manner. (e).The 26
Page 26 of 35
standard drug diclofenac sodium treated group shows no bony destruction or swelling of
585
the joint. Arrows indicate the swelling and reduced edema of soft tissues in experimental
586
groups.
587
Fig. 7. To evaluate the anti-inflammatory effects of QDs-QE complex, samples of the
588
ankle joints from each experimental group were examined by H&E staining. (a) Control
589
group shown normal lobular architecture (X-40). (b) CFA administered group shown
590
thinning of cartilage plates, extensive infiltration of inflammatory cells into the synovial
591
cavity (X-40). (c) QDs-QE complex treated group at 0.2 mg/kg shows infiltration of
592
inflammatory cells, mild improvement cartilage regeneration (X-40). (d) QDs-QE
593
complex treated group at 0.4 mg/kg rats showed complete cartilage regeneration. Absence
594
of inflammation and inflammatory cells in synovial region proved the anti-arthritic
595
potential of QDs-QE complex (X-40). (e) DF (positive drug) treated group exhibited
596
reduction in inflammation and almost normal histology (X-40). Arrows in control, DF
597
(positive drug) and
598
degradation and regeneration. (f).QDs treated group shows almost similar observations of
599
CFA treated group with extensive infiltration of inflammatory cells into the synovial
d
M
an
us
cr
ip t
584
Ac ce p
te
QDs-QE complex (0.4 mg/kg) treated groups indicates cartilage
cavity. (e).QE treated group exhibited regenerated cartilage and reduced the
600
inflammation.
601
Fig. 8. Schematic representation of the possible mechanism of antiarthritic potential of
602
the QDs-QE complex against CFA induced arthritis.
603 604
27
Page 27 of 35
604
an
us
cr
ip t
605
M
606 607
d
Fig. 1
0.025
te
800
1/(F0 -F)
600
Intensity
0.02
60 μg in 2.5 ml
Ac ce p
700
0 μg in 2.5 ml
500
y = 0.0159x + 0.0068 R² = 0.9969
0.015 0.01 0.005 0.1 0.3 0.5 0.7 0.9 1.1
400
1/[Q] . 10-6 M
300 200 100 0
540
560
580
600
620
640
660
Wavelength (nm)
608
Fig. 2
609
28
Page 28 of 35
610 611 612
(b)
M
an
us
cr
ip t
(a)
613 614
Ac ce p
te
d
(c)
615 616 617 618
Fig. 3
619 620 621 29
Page 29 of 35
Ac ce p
te
d
M
an
us
cr
ip t
622 623
Fig. 4
30
Page 30 of 35
Ac ce p
te
d
M
an
us
cr
ip t
624 625
Fig. 5
31
Page 31 of 35
an
us
cr
ip t
M
626
d
627 628
te
Fig. 6
Ac ce p
629 630 631 632 633 634 635 636
32
Page 32 of 35
Ac ce p
te
d
M
an
us
cr
ip t
637 638 639 640
Fig. 7
33
Page 33 of 35
M
an
us
cr
ip t
d
641 642
te
Fig. 8
Ac ce p
643 644 645 646
34
Page 34 of 35
Highlights
647
Enhancement of antiarthritic effect of quercetin(QE) using quantum dots (QDs) as nanocarrier
648
Quantum dots enhances the efficacy of drug delivery
649
QDs‐QE complex has brought back the hematological changes in arthritic animals
650
Cartilage regeneration in arthritic rats treated with QDs‐QE complex was observed
cr
te
d
M
an
us
Ac ce p
651
ip t
646
35
Page 35 of 35